U.S. patent application number 12/567470 was filed with the patent office on 2010-04-15 for nanoemulsion therapeutic compositions and methods of using the same.
This patent application is currently assigned to NanoBio Corporation. Invention is credited to James R. Baker, JR., Tarek Hamouda, Joyce A. Sutcliffe.
Application Number | 20100092526 12/567470 |
Document ID | / |
Family ID | 41796583 |
Filed Date | 2010-04-15 |
United States Patent
Application |
20100092526 |
Kind Code |
A1 |
Baker, JR.; James R. ; et
al. |
April 15, 2010 |
NANOEMULSION THERAPEUTIC COMPOSITIONS AND METHODS OF USING THE
SAME
Abstract
The present invention relates to therapeutic nanoemulsion
compositions and to methods of utilizing the same. In particular,
nanoemulsion compositions are described herein that find use in the
treatment and/or prevention of infection (e.g., respiratory
infection (e.g., associated with cystic fibrosis)), in burn wound
management, and in immunogenic compositions (e.g., comprising a
Burkholderia antigen) that generate an effective immune response
(e.g., against a bacterial species of the genus Burkholderia) in a
subject administered the immunogenic composition. Compositions and
methods of the present invention find use in, among other things,
clinical (e.g. therapeutic and preventative medicine), industrial,
and research applications.
Inventors: |
Baker, JR.; James R.; (Ann
Arbor, MI) ; Hamouda; Tarek; (Milan, MI) ;
Sutcliffe; Joyce A.; (West Newton, MA) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
NanoBio Corporation
|
Family ID: |
41796583 |
Appl. No.: |
12/567470 |
Filed: |
September 25, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61100559 |
Sep 26, 2008 |
|
|
|
Current U.S.
Class: |
424/400 ;
514/1.1; 514/2.4; 514/40 |
Current CPC
Class: |
A61K 31/047 20130101;
A61K 31/14 20130101; A61K 31/4425 20130101; A61K 31/341 20130101;
A61P 31/04 20180101; A61P 11/00 20180101; A61K 9/1075 20130101;
A61K 45/06 20130101; A61K 31/198 20130101; A61K 31/045
20130101 |
Class at
Publication: |
424/400 ; 514/11;
514/40 |
International
Class: |
A61K 9/107 20060101
A61K009/107; A61K 38/12 20060101 A61K038/12; A61K 31/7036 20060101
A61K031/7036; A61P 31/04 20060101 A61P031/04 |
Claims
1. A method of treating or preventing a respiratory infection in a
subject having Cystic fibrosis (CF), wherein: (a) the method
comprises administering a nanoemulsion to the subject; (b) the
subject is susceptible to or has an infection by one or more
gram-positive or gram-negative bacterial species; (c) the
nanoemulsion comprises: (i) water; (ii) at least one organic
solvent; (iii) at least one surfactant; and (iv) at least one oil;
and (d) wherein the nanoemulsion comprises droplets having an
average diameter of less than about 1000 nm.
2. A method of treating or preventing an infection in a subject
having a burn wound, wherein: (a) the method comprises
administering a nanoemulsion to the subject; (b) the subject is
susceptible to or has an infection by one or more gram-positive or
gram-negative bacterial species; (c) the nanoemulsion comprises:
(i) water; (ii) at least one organic solvent; (iii) at least one
surfactant; and (iv) at least one oil; and (d) wherein the
nanoemulsion comprises droplets having an average diameter of less
than about 1000 nm.
3. A method of treating or preventing an Haemophilus influenzae
infection in a subject wherein: (a) the method comprises
administering a nanoemulsion to the subject having or at risk of
having a Haemophilus influenzae infection; (b) the nanoemulsion
comprises: (i) water; (ii) at least one organic solvent; (iii) at
least one surfactant; and (iv) at least one oil; and (c) wherein
the nanoemulsion comprises droplets having an average diameter of
less than about 1000 nm.
4. The method of claim 1, wherein the respiratory infection is
associated with a bacterial biofilm present in the lungs of the
subject.
5. The method of claim 1, further comprising administering one or
more antibiotics either before, during, or after administration of
the nanoemulsion.
6. The method of claim 5, wherein the administration of a
nanoemulsion and at least one antibiotic is synergistic as defined
by a fractional inhibitory concentration (FIC) index, a fractional
bactericidal concentration (FBC) index, or a combination
thereof.
7. The method of claim 6, wherein the antibiotic is any antibiotic,
for example a polymyxin antibiotic (e.g. colistin) or any
aminoglycoside (e.g. tobramycin).
8. The method of claim 5, wherein the nanoemulsion does not exhibit
any antagonism with the antibiotic.
9. The method of claim 1, wherein the bacterial species is selected
from the group consisting of Staphylococcus spp., Haemophilus spp.,
Pseudomonas spp., Burkholderia spp., Acinetobacter spp,
Stenotrophomonas spp., Escherichia spp., Klebsiella spp., and
Proteus spp.
10. The method of claim 9, wherein the subject is susceptible to or
has an infection by one or more bacterial species selected from the
group consisting of Pseudomonas aeruginosa, B. cenocepacia, A.
baumannii, Stenotrophomonas maltophilia, Staphylococcus aureus, H.
influenzae, E. coli, K. pneumoniae, and Proteus mirabilis.
11. The method of claim 1, wherein the nanoemulsion exhibits
minimal or no toxicity or side effects.
12. The method of claim 1, wherein the minimum inhibitory
concentration (MIC), the minimum bactericidal concentration (MBC),
or a combination thereof for the nanoemulsion demonstrate
bacteriostatic or bactericidal activity for the nanoemulsion.
13. The method of claim 1, wherein one or more bacterial species
may exhibit resistance against one or more antibiotics.
14. The method of claim 1, wherein the bacterial species is
methicillin-resistant Staphylococcus aureus (MRSA).
15. The method of claim 1, wherein the nanoemulsion does not
exhibit resistance.
16. The method of claim 1, wherein the nanoemulsion comprises: (a)
water; (b) ethanol or glycerol; (c) cetylpyridinium chloride (CPC),
or benzalkonium chloride, or alkyl dimethyl benzyl ammonium
chloride (BTC 824); (d) soybean oil; and (e) Poloxamer 407, Tween
80, or Tween 20.
17. The method of claim 16, wherein the nanoemulsion further
comprises EDTA.
18. The method of claim 1, wherein the nanoemulsion is delivered
via inhalation, topically to a mucosal surface, via nebulization,
or via any combination thereof.
19. The method of claim 2, wherein the nanoemulsion is delivered
topically, topically to a mucosal surface, via nebulization, or via
any combination thereof.
20. The method of claim 3, wherein the nanoemulsion is delivered
via inhalation, topically to a mucosal surfaces, via nebulization,
or via any combination thereof.
21. The method of claim 1, wherein: (a) the nanoemulsion droplets
have an average diameter selected from the group consisting of less
than about 950 nm, less than about 900 nm, less than about 850 nm,
less than about 800 nm, less than about 750 nm, less than about 700
nm, less than about 650 nm, less than about 600 nm, less than about
550 nm, less than about 500 nm, less than about 450 nm, less than
about 400 nm, less than about 350 nm, less than about 300 nm, less
than about 250 nm, less than about 200 nm, less than about 150 nm,
less than about 100 nm, greater than about 50 nm, greater than
about 70 nm, greater than about 125 nm, and any combination
thereof; (b) the nanoemulsion droplets have an average diameter
greater than about 125 nm and less than about 300 nm; (c) the
nanoemulsion droplets have an average diameter of from about 300 nm
to about 600 nm; or (d) the nanoemulsion droplets have an average
diameter of from about 150 nm to about 400 nm.
22. The method of claim 1, wherein the nanoemulsion comprises: (a)
an aqueous phase; (b) about 1% oil to about 80% oil, or about 5%
oil to about 80% oil; (c) about 0.1% organic solvent to about 50%
organic solvent, or about 0.1% organic solvent to about 10% organic
solvent; (d) at least one surfactant present in an amount of about
0.001% surfactant to about 10% surfactant; (e) at least one
non-ionic surfactant present in an amount of about 0.1% to about
10%; (f) at least one cationic agent present in an amount of about
0.01% to about 3%; (g) about 0.0005% to about 1% of a chelating
agent; or (h) any combination thereof.
23. The method of claim 1, wherein: (a) the nanoemulsion has a
narrow range of MIC (minimum inhibitory concentration) and MBC
(minimum bactericidal concentrations) values; (b) the MIC and MBC
for the nanoemulsion differ by less than or equal to four-fold,
meaning that the nanoemulsion is bactericidal; (c) the MIC and MBC
for the nanoemulsion differ by greater than four-fold, meaning that
the nanoemulsion is bacteriostatic; (d) any combination
thereof.
24. The method of claim 1, wherein the nanoemulsion is stable: (a)
at about 40.degree. C. and about 75% relative humidity for a time
period selected from the group consisting of up to about 1 month,
up to about 3 months, up to about 6 months, up to about 12 months,
up to about 18 months, up to about 2 years, up to about 2.5 years,
and up to about 3 years; (b) at about 25.degree. C. and about 60%
relative humidity for a time period selected from the group
consisting of up to about 1 month, up to about 3 months, up to
about 6 months, up to about 12 months, up to about 18 months, up to
about 2 years, up to about 2.5 years, up to about 3 years, up to
about 3.5 years, up to about 4 years, up to about 4.5 years, and up
to about 5 years; (c) at about 4.degree. C. for a time period
selected from the group consisting of up to about 1 month, up to
about 3 months, up to about 6 months, up to about 12 months, up to
about 18 months, up to about 2 years, up to about 2.5 years, up to
about 3 years, up to about 3.5 years, up to about 4 years, up to
about 4.5 years, up to about 5 years, up to about 5.5 years, up to
about 6 years, up to about 6.5 years, and up to about 7 years; or
(d) any combination thereof.
25. The method of claim 1, wherein the organic solvent: (a) is
selected from the group consisting of a C.sub.1-C.sub.12 alcohol,
diol, triol, dialkyl phosphate, tri-alkyl phosphate, and
combinations thereof; (b) is selected from the group consisting of
a nonpolar solvent, a polar solvent, a protic solvent, an aprotic
solvent, semi-synthetic derivatives thereof, and combinations
thereof; (c) is selected from the group consisting of tri-n-butyl
phosphate, ethanol, methanol, isopropyl alcohol, glycerol, medium
chain triglycerides, diethyl ether, ethyl acetate, acetone,
dimethyl sulfoxide (DMSO), acetic acid, n-butanol, butylene glycol,
perfumers alcohols, isopropanol, n-propanol, formic acid, propylene
glycols, glycerol, sorbitol, industrial methylated spirit,
triacetin, hexane, benzene, toluene, diethyl ether, chloroform,
1,4-dixoane, tetrahydrofuran, dichloromethane, acetone,
acetonitrile, dimethylformamide, dimethyl sulfoxide, formic acid,
semi-synthetic derivatives thereof, and any combination thereof;
and (d) any combination thereof.
26. The method of claim 1, wherein the oil is: (a) any cosmetically
or pharmaceutically acceptable oil; (b) non-volatile; (c) selected
from the group consisting of animal oil, vegetable oil, natural
oil, synthetic oil, hydrocarbon oils, silicone oils, and
semi-synthetic derivatives thereof; (d) selected from the group
consisting of mineral oil, squalene oil, flavor oils, silicon oil,
essential oils, water insoluble vitamins, Isopropyl stearate, Butyl
stearate, Octyl palmitate, Cetyl palmitate, Tridecyl behenate,
Diisopropyl adipate, Dioctyl sebacate, Menthyl anthranhilate, Cetyl
octanoate, Octyl salicylate, Isopropyl myristate, neopentyl glycol
dicarpate cetols, Ceraphyls.RTM., Decyl oleate, diisopropyl
adipate, C.sub.12-15 alkyl lactates, Cetyl lactate, Lauryl lactate,
Isostearyl neopentanoate, Myristyl lactate, Isocetyl stearoyl
stearate, Octyldodecyl stearoyl stearate, Hydrocarbon oils,
Isoparaffin, Fluid paraffins, Isododecane, Petrolatum, Argan oil,
Canola oil, Chile oil, Coconut oil, corn oil, Cottonseed oil,
Flaxseed oil, Grape seed oil, Mustard oil, Olive oil, Palm oil,
Palm kernel oil, Peanut oil, Pine seed oil, Poppy seed oil, Pumpkin
seed oil, Rice bran oil, Safflower oil, Tea oil, Truffle oil,
Vegetable oil, Apricot (kernel) oil, Jojoba oil (simmondsia
chinensis seed oil), Grapeseed oil, Macadamia oil, Wheat germ oil,
Almond oil, Rapeseed oil, Gourd oil, Soybean oil, Sesame oil,
Hazelnut oil, Maize oil, Sunflower oil, Hemp oil, Bois oil, Kuki
nut oil, Avocado oil, Walnut oil, Fish oil, berry oil, allspice
oil, juniper oil, seed oil, almond seed oil, anise seed oil, celery
seed oil, cumin seed oil, nutmeg seed oil, leaf oil, basil leaf
oil, bay leaf oil, cinnamon leaf oil, common sage leaf oil,
eucalyptus leaf oil, lemon grass leaf oil, melaleuca leaf oil,
oregano leaf oil, patchouli leaf oil, peppermint leaf oil, pine
needle oil, rosemary leaf oil, spearmint leaf oil, tea tree leaf
oil, thyme leaf oil, wintergreen leaf oil, flower oil, chamomile
oil, clary sage oil, clove oil, geranium flower oil, hyssop flower
oil, jasmine flower oil, lavender flower oil, manuka flower oil,
Marhoram flower oil, orange flower oil, rose flower oil,
ylang-ylang flower oil, Bark oil, cassia Bark oil, cinnamon bark
oil, sassafras Bark oil, Wood oil, camphor wood oil, cedar wood
oil, rosewood oil, sandalwood oil), rhizome (ginger) wood oil,
resin oil, frankincense oil, myrrh oil, peel oil, bergamot peel
oil, grapefruit peel oil, lemon peel oil, lime peel oil, orange
peel oil, tangerine peel oil, root oil, valerian oil, Oleic acid,
Linoleic acid, Oleyl alcohol, Isostearyl alcohol, semi-synthetic
derivatives thereof, and combinations thereof; or (d) any
combination thereof.
27. The method of claim 1, wherein the nanoemulsion comprises a
volatile oil and wherein: (a) the volatile oil is the organic
solvent; (b) the volatile oil is present in addition to an organic
solvent; (c) the volatile oil is a terpene, monoterpene,
sesquiterpene, carminative, azulene, semi-synthetic derivatives
thereof, or combinations thereof; (d) the volatile oil is selected
from the group consisting of a terpene, monoterpene, sesquiterpene,
carminative, azulene, menthol, camphor, thujone, thymol, nerol,
linalool, limonene, geraniol, perillyl alcohol, nerolidol,
farnesol, ylangene, bisabolol, farnesene, ascaridole, chenopodium
oil, citronellal, citral, citronellol, chamazulene, yarrow,
guaiazulene, chamomile, semi-synthetic derivatives thereof, and
combinations thereof; or (e) the nanoemulsion comprises a silicone
component and the volatile oil present in the silicone component is
different than the oil in the oil phase; (f) the nanoemulsion
comprises a silicone component and the silicone component comprises
at least one volatile silicone oil, wherein the volatile silicone
oil can be the sole oil in the silicone component or it can be
combined with other silicone and non-silicone oils, and wherein the
other oils can be volatile or non-volatile; (g) the nanoemulsion
comprises a silicone component and the silicone component is
selected from the group consisting of methylphenylpolysiloxane,
simethicone, dimethicone, phenyltrimethicone (or an organomodified
version thereof), alkylated derivatives of polymeric silicones,
cetyl dimethicone, lauryl trimethicone, hydroxylated derivatives of
polymeric silicones, such as dimethiconol, volatile silicone oils,
cyclic and linear silicones, cyclomethicone, derivatives of
cyclomethicone, hexamethylcyclotrisiloxane,
octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane,
volatile linear dimethylpolysiloxanes, isohexadecane, isoeicosane,
isotetracosane, polyisobutene, isooctane, isododecane,
semi-synthetic derivatives thereof, and combinations thereof; or
(h) a combination thereof.
28. The method of claim 1 further comprising: (a) a chelating
agent; (b) at least one preservative; (c) at least one pH adjuster;
(d) at least one buffer; (e) at least one antibiotic agent; or (f)
any combination thereof.
29. The method of claim 28, wherein: (a) the chelating agent (i) is
present in an amount of about 0.0005% to about 1%; (ii) the
chelating agent is selected from the group consisting of
ethylenediamine, ethylenediaminetetraacetic acid, and dimercaprol;
(iii) the chelating agent is ethylenediaminetetraacetic acid; or
(iv) any combination thereof; (b) the preservative is selected from
the group consisting of cetylpyridinium chloride, benzalkonium
chloride, benzyl alcohol, chlorhexidine, imidazolidinyl urea,
phenol, potassium sorbate, benzoic acid, bronopol, chlorocresol,
paraben esters, phenoxyethanol, sorbic acid, alpha-tocophernol,
ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole,
butylated hydroxytoluene, sodium ascorbate,
Bis(p-chlorophenyldiguanido) hexane,
3-(-4-chloropheoxy)-propane-1,2-diol, Methyl and
methylchloroisothiazolinone, sodium metabisulphite, citric acid,
edetic acid, chlorphenesin (3-(-4-chloropheoxy)-propane-1,2-diol),
Kathon CG (methyl and methylchloroisothiazolinone), parabens
(methyl, ethyl, propyl, butyl hydrobenzoates), phenoxyethanol
(2-phenoxyethanol), Phenonip (phenoxyethanol, methyl, ethyl, butyl,
propyl parabens), Phenoroc (phenoxyethanol 0.73%, methyl paraben
0.2%, propyl paraben 0.07%), Liquipar Oil (isopropyl, isobutyl,
butylparabens), Liquipar PE (70% phenoxyethanol, 30% liquipar oil),
Nipaguard MPA (benzyl alcohol (70%), methyl & propyl parabens),
Nipaguard MPS (propylene glycol, methyl & propyl parabens),
Nipasept (methyl, ethyl and propyl parabens), Nipastat (methyl,
butyl, ethyl and propyel parabens), Elestab 388 (phenoxyethanol in
propylene glycol plus chlorphenesin and methylparaben), Killitol
(7.5% chlorphenesin and 7.5% methyl parabens), semi-synthetic
derivatives thereof, and combinations thereof; (c) the pH adjuster
is selected from the group consisting of diethyanolamine, lactic
acid, monoethanolamine, triethylanolamine, sodium hydroxide, sodium
phosphate, semi-synthetic derivatives thereof, and combinations
thereof; (d) the buffer is selected from the group consisting of
2-Amino-2-methyl-1,3-propanediol, 2-Amino-2-methyl-1-propanol,
L-(+)-Tartaric acid, ACES, ADA, Acetic acid, Ammonium acetate
solution, Ammonium bicarbonate, Ammonium citrate dibasic, Ammonium
formate, Ammonium oxalate monohydrate, Ammonium phosphate dibasic,
Ammonium phosphate monobasic, Ammonium sodium phosphate dibasic
tetrahydrate, Ammonium sulfate solution, Ammonium tartrate dibasic,
BES buffered saline, BES, BICINE, BIS-TRIS, Bicarbonate buffer
solution, Boric acid, CAPS, CHES, Calcium acetate hydrate, Calcium
carbonate, Calcium citrate tribasic tetrahydrate, Citrate
Concentrated Solution, Citric acid, hydrous, Diethanolamine, EPPS,
Ethylenediaminetetraacetic acid disodium salt dihydrate, Formic
acid solution, Gly-Gly-Gly, Gly-Gly, Glycine, HEPES, Imidazole,
Lipoprotein Refolding Buffer, Lithium acetate dihydrate, Lithium
citrate tribasic tetrahydrate, MES hydrate, MES monohydrate, MES
solution, MOPS, Magnesium acetate solution, Magnesium acetate
tetrahydrate, Magnesium citrate tribasic nonahydrate, Magnesium
formate solution, Magnesium phosphate dibasic trihydrate, Oxalic
acid dihydrate, PIPES, Phosphate buffered saline, piperazine,
Potassium D-tartrate monobasic, Potassium acetate, Potassium
bicarbonate, Potassium carbonate, Potassium chloride, Potassium
citrate monobasic, Potassium citrate tribasic solution, Potassium
formate, Potassium oxalate monohydrate, Potassium phosphate
dibasic, Potassium phosphate dibasic, for molecular biology,
anhydrous, Potassium phosphate monobasic, Potassium phosphate
monobasic, Potassium phosphate tribasic monohydrate, Potassium
phthalate monobasic, Potassium sodium tartrate, Potassium sodium
tartrate tetrahydrate, Potassium tetraborate tetrahydrate,
Potassium tetraoxalate dihydrate, Propionic acid, STE buffer, STET
buffer, Sodium 5,5-diethylbarbiturate, Sodium acetate, Sodium
acetate trihydrate, Sodium bicarbonate, Sodium bitartrate
monohydrate, Sodium carbonate decahydrate, Sodium carbonate, Sodium
citrate monobasic, Sodium citrate tribasic dihydrate, Sodium
formate solution, Sodium oxalate, Sodium phosphate dibasic
dihydrate, Sodium phosphate dibasic dodecahydrate, Sodium phosphate
dibasic solution, Sodium phosphate monobasic dihydrate, Sodium
phosphate monobasic monohydrate, Sodium phosphate monobasic
solution, Sodium pyrophosphate dibasic, Sodium pyrophosphate
tetrabasic decahydrate, Sodium tartrate dibasic dihydrate, Sodium
tartrate dibasic solution, Sodium tetraborate decahydrate, TAPS,
TES, TM buffer solution, TNT buffer solution, TRIS Glycine buffer,
TRIS acetate--EDTA buffer solution, TRIS buffered saline, TRIS
glycine SDS buffer solution, TRIS phosphate EDTA buffer solution,
Tricine, Triethanolamine, Triethylamine, Triethylammonium acetate
buffer, Triethylammonium phosphate solution, Trimethylammonium
acetate solution, Trimethylammonium phosphate solution, Tris-EDTA
buffer solution, Trizma.RTM. acetate, Trizma.RTM. base, Trizma.RTM.
carbonate, Trizma.RTM. hydrochloride, Trizma.RTM. maleate, or any
combination thereof; (e) the antibiotic agent is selected from the
group consisting of Aminoglycosides, Ansamycins, Carbacephems,
Carbapenems, Cephalosporins, Glycopeptides, Macrolides,
Monobactams, Penicillins, Polypeptides, Polymyxins, Quinolones,
Sulfonamides, Tetracyclines, and new classes of antibiotic agents;
or (f) any combination thereof.
30. The method of claim 1, wherein: (a) the surfactant is selected
from the group consisting of ethoxylated nonylphenol comprising 9
to 10 units of ethyleneglycol, ethoxylated undecanol comprising 8
units of ethyleneglycol, polyoxyethylene (20) sorbitan monolaurate,
polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20)
sorbitan monostearate, polyoxyethylene (20) sorbitan monooleate,
sorbitan monolaurate, sorbitan monopalmitate, sorbitan
monostearate, sorbitan monooleate, ethoxylated hydrogenated ricin
oils, sodium laurylsulfate, a diblock copolymer of ethyleneoxyde
and propyleneoxyde, Ethylene Oxide-Propylene Oxide Block
Copolymers, and tetra-functional block copolymers based on ethylene
oxide and propylene oxide, Glyceryl monoesters, Glyceryl caprate,
Glyceryl caprylate, Glyceryl cocate, Glyceryl erucate, Glyceryl
hydroxysterate, Glyceryl isostearate, Glyceryl lanolate, Glyceryl
laurate, Glyceryl linolate, Glyceryl myristate, Glyceryl oleate,
Glyceryl PABA, Glyceryl palmitate, Glyceryl ricinoleate, Glyceryl
stearate, Glyceryl thighlycolate, Glyceryl dilaurate, Glyceryl
dioleate, Glyceryl dimyristate, Glyceryl disterate, Glyceryl
sesuioleate, Glyceryl stearate lactate, Polyoxyethylene
cetyl/stearyl ether, Polyoxyethylene cholesterol ether,
Polyoxyethylene laurate or dilaurate, Polyoxyethylene stearate or
distearate, polyoxyethylene fatty ethers, Polyoxyethylene lauryl
ether, Polyoxyethylene stearyl ether, polyoxyethylene myristyl
ether, a steroid, Cholesterol, Betasitosterol, Bisabolol, fatty
acid esters of alcohols, isopropyl myristate, Aliphati-isopropyl
n-butyrate, Isopropyl n-hexanoate, Isopropyl n-decanoate,
Isoproppyl palmitate, Octyldodecyl myristate, alkoxylated alcohols,
alkoxylated acids, alkoxylated amides, alkoxylated sugar
derivatives, alkoxylated derivatives of natural oils and waxes,
polyoxyethylene polyoxypropylene block copolymers, nonoxynol-14,
PEG-8 laurate, PEG-6 Cocoamide, PEG-20 methylglucose
sesquistearate, PEG40 lanolin, PEG-40 castor oil, PEG-40
hydrogenated castor oil, polyoxyethylene fatty ethers, glyceryl
diesters, polyoxyethylene stearyl ether, polyoxyethylene myristyl
ether, and polyoxyethylene lauryl ether, glyceryl dilaurate,
glyceryl dimystate, glyceryl distearate, semi-synthetic derivatives
thereof, and mixtures thereof; (b) the surfactant is a non-ionic
lipid selected from the group consisting of glyceryl laurate,
glyceryl myristate, glyceryl dilaurate, glyceryl dimyristate,
semi-synthetic derivatives thereof, and mixtures thereof; (c) the
surfactant is a polyoxyethylene fatty ether having a
polyoxyethylene head group ranging from about 2 to about 100
groups; (d) the surfactant is an alkoxylated alcohol having the
structure shown in formula I below:
R.sub.5--(OCH.sub.2CH.sub.2).sub.y--OH Formula I wherein R.sub.5 is
a branched or unbranched alkyl group having from about 6 to about
22 carbon atoms and y is between about 4 and about 100, and
preferably, between about 10 and about 100; (e) the surfactant is
an alkoxylated alcohol which is an ethoxylated derivative of
lanolin alcohol; (f) the surfactant is nonionic and is selected
from the group consisting of nonoxynol-9, an ethoxylated
surfactant, an alcohol ethoxylated, an alkyl phenol ethoxylated, a
fatty acid ethoxylated, a monoalkaolamide ethoxylated, a sorbitan
ester ethoxylated, a fatty amino ethoxylated, an ethylene
oxide-propylene oxide copolymer, Bis(polyethylene glycol
bis[imidazoyl carbonyl]), Brij.RTM.35, Brij.RTM. 56, Brij.RTM. 72,
Brij.RTM. 76, Brij.RTM. 92V, Brij.RTM. 97, Brij.RTM. 58P,
Cremophor.RTM. EL, Decaethylene glycol monododecyl ether,
N-Decanoyl-N-methylglucamine, n-Decyl alpha-D-glucopyranoside,
Decyl beta-D-maltopyranoside, n-Dodecanoyl-N-methylglucamide,
n-Dodecyl alpha-D-maltoside, n-Dodecyl beta-D-maltoside,
Heptaethylene glycol monodecyl ether, Heptaethylene glycol
monotetradecyl ether, Heptaethylene glycol monododecyl ether,
n-Hexadecyl beta-D-maltoside, Hexaethylene glycol monododecyl
ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol
monooctadecyl ether, Hexaethylene glycol monotetradecyl ether,
Igepal CA-630,
Methyl-6-O--(N-heptylcarbamoyl)-alpha-D-glucopyranoside,
Nonaethylene glycol monododecyl ether,
N-Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether,
Octaethylene glycol monododecyl ether, Octaethylene glycol
monohexadecyl ether, Octaethylene glycol monooctadecyl ether,
Octaethylene glycol monotetradecyl ether,
Octyl-beta-D-glucopyranoside, Pentaethylene glycol monodecyl ether,
Pentaethylene glycol monododecyl ether, Pentaethylene glycol
monohexadecyl ether, Pentaethylene glycol monohexyl ether,
Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol
monooctyl ether, Polyethylene glycol diglycidyl ether, Polyethylene
glycol ether W-1, Polyoxyethylene 10 tridecyl ether,
Polyoxyethylene 100 stearate, Polyoxyethylene 20 isohexadecyl
ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate,
Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate,
Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene 25
propylene glycol stearate, Saponin from Quillaja bark, Span.RTM.
20, Span.RTM. 40, Span.RTM. 60, Span.RTM. 65, Span.RTM. 80,
Span.RTM. 85, Tergitol, Tergitol Type 15-S-12, Tergitol Type
15-S-30, Tergitol Type 15-S-5, Tergitol Type 15-S-7, Tergitol Type
15-S-9, Tergitol Type NP-10, Tergitol Type NP-4, Tergitol Type
NP-40, Tergitol Type NP-7, Tergitol Type NP-9, Tergitol Type
TMN-10, Tergitol Type TMN-6, Tetradecyl-beta-D-maltoside,
Tetraethylene glycol monodecyl ether, Tetraethylene glycol
monododecyl ether, Tetraethylene glycol monotetradecyl ether,
Triethylene glycol monodecyl ether, Triethylene glycol monododecyl
ether, Triethylene glycol monohexadecyl ether, Triethylene glycol
monooctyl ether, Triethylene glycol monotetradecyl ether, Triton
CF-21, Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-5M,
Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, Triton
X-15, Triton X-151, Triton X-200, Triton X-207, Triton X-114,
Triton X-165, Triton X-305, Triton X-405, Triton X-45, Triton
X-705-70, TWEEN.RTM. 20, TWEEN.RTM. 21, TWEEN.RTM. 40, TWEEN.RTM.
60, TWEEN.RTM. 61, TWEEN.RTM. 65, TWEEN.RTM. 80, TWEEN.RTM. 81,
TWEEN.RTM. 85, Tyloxapol, n-Undecyl beta-D-glucopyranoside,
Poloxamer 101, Poloxamer 105, Poloxamer 108, Poloxamer 122,
Poloxamer 123, Poloxamer 124, Poloxamer 181, Poloxamer 182,
Poloxamer 183, Poloxamer 184, Poloxamer 185, Poloxamer 188,
Poloxamer 212, Poloxamer 215, Poloxamer 217, Poloxamer 231,
Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238,
Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 331,
Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338,
Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer 407,
Poloxamer 105 Benzoate, Poloxamer 182, Dibenzoate, semi-synthetic
derivatives thereof, and combinations thereof; (g) the surfactant
is cationic and is selected from the group consisting of a
quarternary ammonium compound, an alkyl trimethyl ammonium chloride
compound, a dialkyl dimethyl ammonium chloride compound,
Benzalkonium chloride, Benzyldimethylhexadecylammonium chloride,
Benzyldimethyltetradecylammonium chloride,
Benzyldodecyldimethylammonium bromide, Benzyltrimethylammonium
tetrachloroiodate, Cetylpyridinium chloride,
Dimethyldioctadecylammonium bromide, Dodecylethyldimethylammonium
bromide, Dodecyltrimethylammonium bromide,
Ethylhexadecyldimethylammonium bromide, Girard's reagent T,
Hexadecyltrimethylammonium bromide,
N,N',N'-Polyoxyethylene(10)-N-tallow-1,3-diaminopropane, Thonzonium
bromide, Trimethyl(tetradecyl)ammonium bromide,
1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, 1-Decanaminium,
N-decyl-N,N-dimethyl-, chloride, Didecyl dimethyl ammonium
chloride, 2-(2-(p-(Diisobutyl)cresosxy)ethoxy)ethyl dimethyl benzyl
ammonium chloride, 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl
dimethyl benzyl ammonium chloride, Alkyl 1 or 3
benzyl-1-(2-hydroxethyl)-2-imidazolinium chloride, Alkyl
bis(2-hydroxyethyl)benzyl ammonium chloride, Alkyl demethyl benzyl
ammonium chloride, Alkyl dimethyl 3,4-dichlorobenzyl ammonium
chloride (100% C12), Alkyl dimethyl 3,4-dichlorobenzyl ammonium
chloride (50% C14, 40% C12, 10% C16), Alkyl dimethyl
3,4-dichlorobenzyl ammonium chloride (55% C14, 23% C12, 20% C16),
Alkyl dimethyl benzyl ammonium chloride, Alkyl dimethyl benzyl
ammonium chloride (100% C14), Alkyl dimethyl benzyl ammonium
chloride (100% C16), Alkyl dimethyl benzyl ammonium chloride (41%
C14, 28% C12), Alkyl dimethyl benzyl ammonium chloride (47% C12,
18% C14), Alkyl dimethyl benzyl ammonium chloride (55% C16, 20%
C14), Alkyl dimethyl benzyl ammonium chloride (58% C14, 28% C16),
Alkyl dimethyl benzyl ammonium chloride (60% C14, 25% C12), Alkyl
dimethyl benzyl ammonium chloride (61% C11, 23% C14), Alkyl
dimethyl benzyl ammonium chloride (61% C12, 23% C14), Alkyl
dimethyl benzyl ammonium chloride (65% C12, 25% C14), Alkyl
dimethyl benzyl ammonium chloride (67% C12, 24% C14), Alkyl
dimethyl benzyl ammonium chloride (67% C12, 25% C14), Alkyl
dimethyl benzyl ammonium chloride (90% C14, 5% C12), Alkyl dimethyl
benzyl ammonium chloride (93% C14, 4% C12), Alkyl dimethyl benzyl
ammonium chloride (95% C16, 5% C18), Alkyl didecyl dimethyl
ammonium chloride, Alkyl dimethyl benzyl ammonium chloride
(C12-16), Alkyl dimethyl benzyl ammonium chloride (C12-18), dialkyl
dimethyl benzyl ammonium chloride, Alkyl dimethyl dimethybenzyl
ammonium chloride, Alkyl dimethyl ethyl ammonium bromide (90% C14,
5% C16, 5% C12), Alkyl dimethyl ethyl ammonium bromide (mixed alkyl
and alkenyl groups as in the fatty acids of soybean oil), Alkyl
dimethyl ethylbenzyl ammonium chloride, Alkyl dimethyl ethylbenzyl
ammonium chloride (60% C14), Alkyl dimethyl isopropylbenzyl
ammonium chloride (50% C12, 30% C14, 17% C16, 3% C18), Alkyl
trimethyl ammonium chloride (58% C18, 40% C16, 1% C14, 1% C12),
Alkyl trimethyl ammonium chloride (90% C18, 10% C16),
Alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18),
Di-(C8-10)-alkyl dimethyl ammonium chlorides, Dialkyl dimethyl
ammonium chloride, Dialkyl methyl benzyl ammonium chloride, Didecyl
dimethyl ammonium chloride, Diisodecyl dimethyl ammonium chloride,
Dioctyl dimethyl ammonium chloride, Dodecyl bis(2-hydroxyethyl)
octyl hydrogen ammonium chloride, Dodecyl dimethyl benzyl ammonium
chloride, Dodecylcarbamoyl methyl dimethyl benzyl ammonium
chloride, Heptadecyl hydroxyethylimidazolinium chloride,
Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, Myristalkonium
chloride (and) Quat RNIUM 14, N,N-Dimethyl-2-hydroxypropylammonium
chloride polymer, n-Tetradecyl dimethyl benzyl ammonium chloride
monohydrate, Octyl decyl dimethyl ammonium chloride, Octyl dodecyl
dimethyl ammonium chloride, Octyphenoxyethoxyethyl dimethyl benzyl
ammonium chloride, Oxydiethylenebis(alkyl dimethyl ammonium
chloride), Trimethoxysily propyl dimethyl octadecyl ammonium
chloride, Trimethoxysilyl quats, Trimethyl dodecylbenzyl ammonium
chloride, semi-synthetic derivatives thereof, and combinations
thereof; (h) the surfactant is anionic and is selected from the
group consisting of a carboxylate, a sulphate, a sulphonate, a
phosphate, Chenodeoxycholic acid, Chenodeoxycholic acid sodium
salt, Cholic acid, ox or sheep bile, Dehydrocholic acid,
Deoxycholic acid, Deoxycholic acid methyl ester, Digitonin,
Digitoxigenin, N,N-Dimethyldodecylamine N-oxide, Docusate sodium
salt, Glycochenodeoxycholic acid sodium salt, Glycocholic acid
hydrate, synthetic, Glycocholic acid sodium salt hydrate,
synthetic, Glycodeoxycholic acid monohydrate, Glycodeoxycholic acid
sodium salt, Glycolithocholic acid 3-sulfate disodium salt,
Glycolithocholic acid ethyl ester, N-Lauroylsarcosine sodium salt,
N-Lauroylsarcosine solution, Lithium dodecyl sulfate, Lugol
solution, Niaproof 4, Type 4,1-Octanesulfonic acid sodium salt,
Sodium 1-butanesulfonate, Sodium 1-decanesulfonate, Sodium
1-dodecanesulfonate, Sodium 1-heptanesulfonate anhydrous, Sodium
1-nonanesulfonate, Sodium 1-propanesulfonate monohydrate, Sodium
2-bromoethanesulfonate, Sodium cholate hydrate, Sodium choleate,
Sodium deoxycholate, Sodium deoxycholate monohydrate, Sodium
dodecyl sulfate, Sodium hexanesulfonate anhydrous, Sodium octyl
sulfate, Sodium pentanesulfonate anhydrous, Sodium taurocholate,
Taurochenodeoxycholic acid sodium salt, Taurodeoxycholic acid
sodium salt monohydrate, Taurohyodeoxycholic acid sodium salt
hydrate, Taurolithocholic acid 3-sulfate disodium salt,
Tauroursodeoxycholic acid sodium salt, Trizma.RTM. dodecyl sulfate,
Ursodeoxycholic acid, semi-synthetic derivatives thereof, and
combinations thereof; (i) the surfactant is zwitterionic and is
selected from the group consisting of an N-alkyl betaine, lauryl
amindo propyl dimethyl betaine, an alkyl dimethyl glycinate, an
N-alkyl amino propionate, CHAPS (minimum 98%), CHAPSO (minimum
98%), 3-(Decyldimethylammonio)propanesulfonate inner salt,
3-(Dodecyldimethylammonio)propanesulfonate inner salt,
3-(N,N-Dimethylmyristylammonio)propanesulfonate,
3-(N,N-Dimethyloctadecylammonio)propanesulfonate,
3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt,
3-(N,N-Dimethylpalmitylammonio)propanesulfonate, semi-synthetic
derivatives thereof, and combinations thereof; (j) the surfactant
is polymeric and the polymeric surfactant is selected from the
group consisting of a graft copolymer of a poly(methyl
methacrylate) backbone with at least one polyethylene oxide (PEO)
side chain, polyhydroxystearic acid, an alkoxylated alkyl phenol
formaldehyde condensate, a polyalkylene glycol modified polyester
with fatty acid hydrophobes, a polyester, semi-synthetic
derivatives thereof, and combinations thereof; or (k) any
combination thereof.
31. The method of claim 1, wherein the nanoemulsion: (a) comprises
at least one cationic surfactant; (b) comprises a cationic
surfactant which is either cetylpyridinium chloride, or
benzalkonium chloride, or alkyl dimethyl benzyl ammonium chloride
(BTC 824), or a combination thereof; (c) comprises a cationic
surfactant, and wherein the concentration of the cationic
surfactant is less than about 5.0% and greater than about 0.001%;
(d) comprises a cationic surfactant, and wherein the concentration
of the cationic surfactant is selected from the group consisting of
less than about 5%, less than about 4.5%, less than about 4.0%,
less than about 3.5%, less than about 3.0%, less than about 2.5%,
less than about 2.0%, less than about 1.5%, less than about 1.0%,
less than about 0.90%, less than about 0.80%, less than about
0.70%, less than about 0.60%, less than about 0.50%, less than
about 0.40%, less than about 0.30%, less than about 0.20%, less
than about 0.10%, greater than about 0.001%, greater than about
0.002%, greater than about 0.003%, greater than about 0.004%,
greater than about 0.005%, greater than about 0.006%, greater than
about 0.007%, greater than about 0.008%, greater than about 0.009%,
and greater than about 0.010%; or (e) any combination thereof.
32. The method of claim 1, wherein: (a) the nanoemulsion comprises
at least one cationic surfactant and at least one non-cationic
surfactant; (b) the nanoemulsion comprises at least one cationic
surfactant and at least one non-cationic surfactant, wherein the
non-cationic surfactant is a nonionic surfactant; (c) the
nanoemulsion comprises at least one cationic surfactant and at
least one non-cationic surfactant, wherein the non-cationic
surfactant is a polysorbate nonionic surfactant; (d) the
nanoemulsion comprises at least one cationic surfactant and at
least one non-cationic surfactant, wherein the non-cationic
surfactant is a nonionic surfactant, and the non-ionic surfactant
is present in a concentration of about 0.05% to about 10%, about
0.05% to about 7.0%, about 0.1% to about 7%, or about 0.5% to about
4%; (e) the nanoemulsion comprises at least one cationic surfactant
and at least one a nonionic surfactant, wherein the cationic
surfactant is present in a concentration of about 0.05% to about 3%
or about 0.01% to about 3%; or (f) any combination thereof.
33. The method of claim 1, wherein the water is present in
Phosphate Buffered Saline (PBS).
34. The method of claim 1, wherein the nanoemulsion is not absorbed
systemically in the human subject, or very little of the
nanoemulsion is absorbed systemically in the human subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/100,559 filed Sep. 26, 2008, the entire
disclosure of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to therapeutic nanoemulsion
compositions and to methods of utilizing the same. In particular,
nanoemulsion compositions are described herein that find use in the
treatment and/or prevention of infection (e.g., respiratory
infection (e.g., associated with cystic fibrosis)), in burn wound
management, and in immunogenic compositions (e.g., comprising a
Burkholderia antigen) that generate an effective immune response
(e.g., against a bacterial species of the genus Burkholderia) in a
subject administered the immunogenic composition. Compositions and
methods of the present invention find use in, among other things,
clinical (e.g. therapeutic and preventative medicine), industrial,
and research applications.
BACKGROUND OF THE INVENTION
[0003] Bacterial infection caused by opportunistic and/or
pathogenic bacteria (e.g., Burkholderia cepacia, Staphylococcus
aureus and Pseudomonas aeruginosa) is a major problem in both the
developed and undeveloped portions of the world. For example,
certain types of individuals are prone to respiratory infection
(e.g., by bacteria (e.g., opportunistic bacteria), viruses, fungi
and/or parasites) including the immunocompromised, elderly, cancer
chemotherapy patients, individuals suffering from asthma,
individual suffering from genetically inherited disease (e.g.,
cystic fibrosis) and virally infected individuals (e.g., infected
with influenza virus, respiratory syncytial virus (RSV), adenovirus
and/or human immunodeficiency virus). Similarly, wounds (e.g., burn
wounds) present on a subject provide an ideal location for
bacterial growth and survival.
[0004] As the use of conventional pharmaceutical antibiotics has
increased for medical, veterinary and agricultural purposes, there
has been a concurrent emergence of antibiotic-resistant strains of
pathogenic bacteria.
[0005] A need exists to develop alternative strategies of
antibacterial treatment. For example, there exists a need for new
compositions and methods of treating or preventing bacterial
infection (e.g., bacteremia) caused by strains of bacteria
unsusceptible to current forms of antibacterial treatments.
SUMMARY OF THE INVENTION
[0006] The present invention is directed to a method of treating or
preventing a respiratory infection in a subject having Cystic
fibrosis (CF). The method comprises administering a nanoemulsion to
the subject, wherein the subject is susceptible to or has an
infection by one or more gram-positive or gram-negative bacterial
species. The nanoemulsion comprises water, at least one organic
solvent, at least one surfactant, and at least one oil. In
addition, the nanoemulsion comprises droplets having an average
diameter of less than about 1000 nm. In one embodiment of the
invention, the respiratory infection is associated with a bacterial
biofilm present in the lungs of the subject.
[0007] In another embodiment of the invention, described is a
method of treating or preventing an infection in a subject having a
burn wound. The method comprises administering a nanoemulsion to
the subject, wherein the subject is susceptible to or has an
infection by one or more gram-positive or gram-negative bacterial
species. The nanoemulsion comprises water, at least one organic
solvent, at least one surfactant, and at least one oil. In
addition, the nanoemulsion comprises droplets having an average
diameter of less than about 1000 nm.
[0008] For methods directed to subjects having a or susceptible to
a gram-negative or gram-positive bacterial infection, the bacteria
can be any known gram-negative or gram-positive bacteria. In
another embodiment of the invention, the bacterial species is
selected from the group consisting of Staphylococcus spp.,
Haemophilus spp., Pseudomonas spp., Burkholderia spp.,
Acinetobacter spp, Stenotrophomonas spp., Escherichia spp.,
Klebsiella spp., and Proteus spp. The bacterial species can also be
selected from the group consisting of Pseudomonas aeruginosa, B.
cenocepacia, A. baumannii, Stenotrophomonas maltophilia,
Staphylococcus aureus, H. influenzae, E. coli, K. pneumoniae, and
Proteus mirabilis.
[0009] In yet another embodiment of the invention, described is a
method of treating or preventing an Haemophilus influenzae
infection in a non-CF subject. The method comprises administering a
nanoemulsion to the subject having or at risk of having a
Haemophilus influenzae infection. The nanoemulsion comprises water,
at least one organic solvent, at least one surfactant, and at least
one oil. In addition, the nanoemulsion comprises droplets having an
average diameter of less than about 1000 nm.
[0010] All methods described herein may further comprise
administering one or more antibiotics either before, during, or
after administration of the nanoemulsion. In one embodiment, the
administration of a nanoemulsion and at least one antibiotic is
synergistic. The "synergy" may be as defined by a fractional
inhibitory concentration (FIC) index, a fractional bactericidal
concentration (FBC) index, or a combination thereof. Methods
comprising administering an antibiotic encompass the use of any
antibiotic. Exemplary antibiotics include, but are not limited to,
a polymyxin antibiotic (e.g. colistin) or any aminoglycoside (e.g.
tobramycin). Preferably, in the methods of the invention utilizing
an antibiotic, the nanoemulsion does not exhibit any antagonism
with the antibiotic.
[0011] For all methods described herein, preferably the
nanoemulsion exhibits minimal or no toxicity or side effects. In
addition, preferably the nanoemulsion does not exhibit resistance.
In another embodiment applicable to all methods described herein,
the minimum inhibitory concentration (MIC), the minimum
bactericidal concentration (MBC), or a combination thereof for the
nanoemulsion demonstrate bacteriostatic or bactericidal activity
for the nanoemulsion.
[0012] For all methods described herein, one or more bacterial
species may exhibit resistance against one or more antibiotics. For
example, the bacterial species can be methicillin-resistant
Staphylococcus aureus (MRSA).
[0013] In one embodiment of the methods of the invention, the
nanoemulsion is not absorbed systemically in the human subject, or
very little of the nanoemulsion is absorbed systemically in the
human subject.
[0014] In the methods of the invention, the nanoemulsion can be
delivered using any pharmaceutically acceptable means. For example,
the nanoemulsion can be delivered via inhalation, topically,
topically to a mucosal surface, via nebulization, or via any
combination thereof.
[0015] The invention also encompasses compositions useful in the
methods of the invention. Exemplary compositions include, but are
not limited to, nanoemulsions comprising: (a) water; (b) ethanol or
glycerol; (c) cetylpyridinium chloride (CPC), or benzalkonium
chloride, or alkyl dimethyl benzyl ammonium chloride (BTC 824); (d)
soybean oil; and (e) Poloxamer 407, Tween 80, or Tween 20. In a
further embodiment, the nanoemulsion can additionally comprise
EDTA.
[0016] The foregoing general description and following brief
description of the drawings and the detailed description are
exemplary and explanatory and are intended to provide further
explanation of the invention as claimed. Other objects, advantages,
and novel features will be readily apparent to those skilled in the
art from the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of necessary fee.
[0018] FIG. 1 shows killing of Burkholderia cepacia in PBS by 20%
Nanemulsion (NE) alone or with 20 mM EDTA in 10 minutes.
[0019] FIG. 2 shows killing of B. cepacia in PBS by 20% NE alone or
with 20 mM EDTA in 20 minutes.
[0020] FIG. 3 shows killing of B. cepacia in PBS by 20% NE alone or
with 20 mM EDTA in 40 minutes.
[0021] FIG. 4 shows (A) killing of B. cepacia in PBS by 20% NE
alone or with 20 mM EDTA in 40 minutes; (B) killing of B. cepacia
in PBS by 20% NE alone or with 20 mM EDTA in 60 minutes; and (C)
killing of B. cepacia in PBS by 20% NE alone or with 20 mM EDTA in
60 minutes.
[0022] FIG. 5 shows killing of B. cepacia in hypertonic saline (6%
NaCl) at 15 and 30 Minutes by NE alone and NE with EDTA.
[0023] FIG. 6 shows killing of B. cepacia and P. aeruginosa in 7%
NaCl within 15 min.
[0024] FIG. 7 shows killing of B. cepacia and P. aeruginosa
(Mixed-culture) in 7% NaCl within 15 minutes.
[0025] FIG. 8 shows a microtiter serial dilution MIC assay. Numbers
above the plate indicate the concentration (.mu.g/ml CPC) of
P.sub.4075EC added to each column of wells. The bacterial species
added to wells in each row is indicated on the left. The MICs for
these strains (top to bottom) are .ltoreq.15.6 .mu.g/ml 125
.mu.g/ml, and 31.2 .mu.g/ml.
[0026] FIG. 9 shows distribution of P.sub.4075EC MICs for 150
Burkholderia and non-Burkholderia strains
[0027] FIG. 10 shows a time-kill study of P.sub.4075EC activity and
concentration-dependent inhibition of P.sub.4075EC activity by CF
sputum. (A) 10.sup.5 CFU/ml of B. multivorans ATCC 17616 were
exposed to various concentrations of P.sub.4075EC around the MIC
(62.5 .mu.g/ml) and viable bacterial counts were determined at the
times indicated. (B) 10.sup.5 CFU/ml of B. multivorans ATCC 17616
were exposed to various concentrations of P.sub.4075EC in the
presence of three concentrations of CF sputum for 24 hr before
viable bacterial counts were determined. Numbers and symbols in
inset boxes indicate concentrations of P.sub.4075EC (.mu.g/ml CPC)
used.
[0028] FIG. 11 shows in vitro activity of P.sub.4075EC.
[0029] FIG. 12 shows in vitro activity of P.sub.4075EC against
biofilm bacteria and in the presence of CF sputum.
[0030] FIG. 13 shows influence of P.sub.4075EC on B. cepacia, P.
aeruginosa and E. coli individual biofilms.
[0031] FIG. 14 shows influence of P.sub.4075EC on mixed
biofilms.
[0032] FIG. 15 shows influence of P.sub.4075EC on mixed
biofilms.
[0033] FIG. 16 shows influence of P.sub.4075EC on escape colonies
of B. cepacia biofilms.
[0034] FIG. 17 shows serial, two-fold dilutions of P.sub.4075EC in
one embodiment of the invention.
[0035] FIG. 18 shows age specific prevalence of respiratory
infections in CF patients.
[0036] FIG. 19A shows a Fractional Inhibitory Concentration
Index;
[0037] FIG. 19B shows a graph demonstrating the relationship
between antagonism, indifference, and synergy for Antimicrobial A
(micrograms/mL) and Antimicrobial B (micrograms/mL);
[0038] FIGS. 20A-D show figures of electron microscopy. FIG. 2A
shows 0 min, no treatment control; FIG. 2B shows 10 minutes after
treatment; FIG. 2C shows 20 minutes after treatment; and FIG. 2D
shows 30 minutes after treatment.
[0039] FIG. 21 shows topical application of nanoemulsion reduces P.
aeruginosa growth in burn wounds. Male Sprague-Dawley rats received
partial thickness burn wounds. At 8 hours post-injury, animals were
inoculated with 10.sup.6 CFU P. aeruginosa. At 16 and 24 hours
post-burn the animals were treated with topical saline (Control),
placebo (W.sub.205GBA.sub.2ED without benzalkonium chloride),
W.sub.205GBA.sub.2ED (nanoemulsion), or Sulfamylon (mafenide
acetate). At 32 hours, animals were sacrificed, skin samples
obtained and homogenized, plated, and CFUs counted. The scatter
plot represents cultured CFUs for each individual animal. The
median value for each group is plotted as a horizontal line. There
was minimal pathogen growth in 12 out of 23 of the NB-201 treated
animals. A majority of the control (29/32) and placebo (9/12)
animals with burn injury had evidence of wound infection based on a
positive quantitative wound culture with significantly more
bacteria present in the wound than those animals treated with
W.sub.205GBA.sub.2ED. p<0.0001, Kruskal-Wallis test, p<0.05
for saline vs. W.sub.205GBA.sub.2ED, placebo vs.
W.sub.205GBA.sub.2ED, and saline vs. Sulfamylon, Dunn's multiple
comparison test.
[0040] FIG. 22 shows nanoemulsion treatment following burn injury
attenuates dermal proinflammatory cytokine expression. Groups were
sham, burn, and burn with W.sub.205GBA.sub.2ED treatment (n=8-10
per group). A) IL-43 (p=0.02, 1-way ANOVA), B) IL-6 (p=0.8), C)
TNF-.alpha. (p=0.5), D) CINC-1 (p=0.04), E) CINC-3 (p=0.005), and
F) Myeloperoxidase (p=0.07). Burn injured animals treated with
nanoemulsion had decreased levels of IL-1.beta. (1773.+-.516 vs.
5625.+-.1743 pg/mL) and CINC-3 (225.+-.66 vs. 1589.+-.527 pg/mL)
when compared to untreated partial thickness burned animals.
*p<0.05, t-test or Tukey's multiple comparison test.
[0041] FIG. 23 shows nanoemulsion treatment following burn wound
infection with P. aeruginosa attenuates dermal proinflammatory
cytokine expression. All animals received burn injury and groups
were saline, placebo, W.sub.205GBA.sub.2ED, and Sulfamylon (n=10-30
per group). A) IL-1.beta. (p=0.001, 1-way ANOVA), B) IL-6 (p=0.07),
C) TNF-.alpha. (p=0.3), D) CINC-1 (p=0.8), E) CINC-3 (p=0.4), and
F) Myeloperoxidase (p=0.0001). Burn wound infected animals treated
with nanoemulsion had decreased levels of IL-1.beta. (1007.+-.157
vs. 3054.+-.499 pg/mL) and IL-6 (244.+-.51 vs. 485.+-.73 pg/mL).
The nanoemulsion and Sulfamylon treatment groups demonstrated
reduced dermal neutrophil sequestration when compared to saline and
placebo as evidenced on myeloperoxidase assay
(W.sub.205GBA.sub.2ED: 0.09.+-.0.02, Sulfamylon: 0.08.+-.0.02,
Saline: 0.40.+-.0.06, Placebo: 0.45.+-.0.11 .mu.g/mL). *p<0.05,
Tukey's multiple comparison test.
[0042] FIG. 24 shows burn injury upregulates dermal TGF-.beta.
expression and treatment with nanoemulsion in the setting of burn
wound infection decreases the level of TGF-.beta. in the wound.
Dermal levels of the anti-inflammatory cytokines IL-10 and
TGF-.beta. were measured in the burn wound 32 hours after thermal
injury in animals treated with nanoemulsion, and in animals exposed
to bacteria and treated with nanoemulsion or Sulfamylon. A) IL-10
(p=0.4, 1-way ANOVA), and B) TGF-.beta. (p=0.0001). Partial
thickness burn increased the presence of dermal TGF-.beta. as
compared to sham (624.+-.55 vs. 232.+-.17 pg/mL). Treatment of an
infected burn wound with W.sub.205GBA.sub.2ED significantly reduced
the dermal level of TGF-.beta. compared to the untreated burn group
(404.+-.43 vs. 624.+-.55 pg/mL). Sulfamylon treatment did not
suppress the level of IL-10 or TGF-.beta. in the infected burn
wound. *p<0.05, Tukey's multiple comparison test.
[0043] FIG. 25 shows cross sectional histology of burn skin
following infection with P. aeruginosa and treatment with saline or
nanoemulsion. Both views of skin are 32 hours after burn injury. A)
Representative section from saline (control) treated animal
(Hematoxylin and eosin x 40). B) Representative cross-section from
W.sub.205GBA.sub.2ED (nanoemulsion) treated animal (Hematoxylin and
eosin x 40).
[0044] FIG. 26 shows evans blue assay to quantify capillary leak
and tissue edema. Evans blue is a dye that binds to serum albumin
and can be quantitated to determine vascular permeability. Topical
nanoemulsion treatment resulted in less capillary leak following
thermal injury and bacterial inoculation of the wound when compared
to saline treated control animals (1.26.+-.0.05 vs. 1.93.+-.0.24
.mu.g Evans blue/mg tissue, n=8 per group). *p=0.02, t-test.
[0045] FIG. 27 shows photomicrographs of partial thickness burned
skin with fluorescence labeled TUNEL staining to detect hair
follicle cell apoptosis. Skin samples were harvested at 12 hours
post-burn. Treatment was performed at 0 and 8 hours following
thermal injury. All images are at 40.times. magnification.
[0046] FIG. 28 shows TUNEL assay for hair follicle cell apoptosis
following a partial thickness burn injury. Skin samples from sham,
burn+saline (control), burn+placebo (W.sub.205GBA.sub.2ED without
benzalkonium chloride), and burn+W.sub.205GBA.sub.2ED were
sectioned for fluorescein-labeled TUNEL assay. Treatment was
performed at time 0 and 8 hours post injury. No bacterial
inoculation of the burned skin was done in this experiment. There
was a significant reduction in the degree of hair follicle
apoptosis among burn injured and treated animals for tissue samples
harvested at 12 hours post injury (p=0.006, 1-way ANOVA).
Differences were found for sham vs. burn+saline, burn+saline vs.
burn+placebo, and burn+saline vs. burn+W.sub.205GBA.sub.2ED
(*p<0.05, Tukey's multiple comparison test).
[0047] FIG. 29 shows characterization of the OMP preparation. A)
Agarose gel electrophoresis and ethidium bromide-staining. Lane 1.
DNA ladder; Lane 2. Whole Cell lysate (WCL) before separation by
high speed centrifugation; Lane 3. Supernatant following the
100,000.times.g spin (lane B in FIGS. 1B, 1C, & 1D); Lane 4.
Crude OMP preparation (lane C in FIGS. 1B, 1C, & 1D); Lane 5.
Endotoxin (ET) depleted OMP fraction (lane E in FIGS. 1B, 1C, &
1D). Volumetrically-loaded silver stain B) and western blot of OMP
preparation (C-D). Lane A. Protein from the supernatant produced
after the 6000.times.g centrifugation was loaded; Lane B. An equal
volume of the supernatant from the 100,000.times.g centrifugation;
Lane C. Crude OMP (the re-suspended pellet fraction of the
100,000.times.g spin); Lanes D-J. Endotoxin-depleted OMP fractions
(the successive flow-through portions of the endotoxin column);
Lanes K-N. Endotoxin column retentant fractions (the successive
fluid regenerated from the column after the addition of sodium
deoxycholate). C) Western blot probed with serum from mice
immunized with the endotoxin-depleted OMP-NE preparation. D)
Western blot probed with serum from mice immunized with the OMP in
PBS preparation.
[0048] FIG. 30 shows antibody responses against B. cenocepacia OMP.
A) ELISA results of the IgG response in serum post-immunization
with the OMP preparation with or without nanoemulsion. Serum
anti-OMP IgG antibody concentrations are presented as mean of
endpoint titers in individual sera.+-.SEM. * indicates a
statistical difference (p<0.05) in the anti-OMP IgG titers. B)
Mucosal antibodies sIgA and IgG against the OMP after nasal vaccine
with or without nanoemulsion. sIgA and IgG were measured in BAL
solution. The OD levels were normalized to total protein content
within the samples.
[0049] FIG. 31 shows type of cellular immune responses induced by
nasal OMP-NE vaccine. A) Serum from mice immunized with 5 .mu.g OMP
mixed with either NE (OMP-NE) or with PBS (OMP-PBS) was analyzed
for antibody subtype distribution. The results are presented as
ratio of the specific subclass IgG to the overall IgG titer. *:
indicates statistical difference (p<0.05) between IgG2b and IgG1
subtypes. B) Cytokine profiling of splenocytes of mice immunized
with 5 .mu.g. mixed with either NE (OMP-NE) or with PBS (OMP-PBS)
Data is represented as fold change.+-.SEM comparing OMP-activated
versus non activated splenocytes and is normalized to responses in
non-vaccinated mice. *: indicates statistical difference
(p<0.05) between OMP-NE and OMP in PBS groups.
[0050] FIG. 32 shows identification of protein epitopes and LPS in
OMP preparations. Lane 1. Protein ladder; Lane 2. Crude OMP
preparation; Lane 3. Proteinase K digested crude OMP preparation;
Lane 4. Endotoxin-depleted OMP; Lane 5. Proteinase K digested
endotoxin-depleted OMP. Western blots were probed with serum from
either OMP in PBS or OMP-NE immunized mice or with a monoclonal
anti-Pseudomonas mallei LPS antibody as indicated.
[0051] FIG. 33 shows serum neutralization assay. Percent reduction
of B. cenocepacia or B. multivorans cfu plotted against samples
with naive serum. A statistical difference in B. cenocepacia
neutralizing activity was observed between serum from mice
immunized with OMP-NE and serum from mice immunized with OMP in PBS
formulations (p=9.9.times.10.sup.-5 for 5 ug OMP-NE and 0.03 for 15
ug OMP-NE). B. multivorans cross-neutralizing activity was observed
between serum from mice immunized with OMP-NE and serum from naive
mice (p=0.04). * indicates a statistically significant (p<0.05)
difference in neutralizing activity between OMP-NE and OMP in PBS.
** indicates a statistically significant (p<0.05) difference in
neutralizing activity between OMP-NE vaccinated and naive mice.
[0052] FIG. 34 shows pulmonary and splenic colonization assay. A)
Pulmonary tissue associated and B) Splenic tissue associated cfu
determined at six days following intratracheal challenge of
5.times.10.sup.7 cfu of B. cenocepacia.
DEFINITIONS
[0053] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below:
[0054] As used herein, the term "microorganism" refers to any
species or type of microorganism, including but not limited to,
bacteria, viruses, archaea, fungi, protozoans, mycoplasma, prions,
and parasitic organisms. The term microorganism encompasses both
those organisms that are in and of themselves pathogenic to another
organism (e.g., animals, including humans, and plants) and those
organisms that produce agents that are pathogenic to another
organism, while the organism itself is not directly pathogenic or
infective to the other organism.
[0055] As used herein, the term "pathogen" refers a biological
agent that causes a disease state (e.g., infection, sepsis, etc.)
in a host. "Pathogens" include, but are not limited to, viruses,
bacteria, archaea, fungi, protozoans, mycoplasma, prions, and
parasitic organisms.
[0056] The terms "bacteria" and "bacterium" refer to all
prokaryotic organisms, including those within all of the phyla in
the Kingdom Procaryotae. It is intended that the term encompass all
microorganisms considered to be bacteria including Mycoplasma,
Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of
bacteria are included within this definition including cocci,
bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included
within this term are prokaryotic organisms that are Gram-negative
or Gram-positive. "Gram-negative" and "Gram-positive" refer to
staining patterns with the Gram-staining process, which is well
known in the art. (See e.g., Finegold and Martin, Diagnostic
Microbiology, 6th Ed., CV Mosby St. Louis, pp. 13-15 (1982)).
"Gram-positive bacteria" are bacteria that retain the primary dye
used in the Gram stain, causing the stained cells to generally
appear dark blue to purple under the microscope. "Gram-negative
bacteria" do not retain the primary dye used in the Gram stain, but
are stained by the counterstain. Thus, Gram-negative bacteria
generally appear red. In some embodiments, bacteria are
continuously cultured. In some embodiments, bacteria are uncultured
and existing in their natural environment (e.g., at the site of a
wound or infection) or obtained from patient tissues (e.g., via a
biopsy). Bacteria may exhibit pathological growth or proliferation.
Examples of bacteria include, but are not limited to, bacterial
cells of a genus of bacteria selected from the group comprising
Salmonella, Shigella, Escherichia, Enterobacter, Serratia, Proteus,
Yersinia, Citrobacter, Edwardsiella, Providencia, Klebsiella,
Hafnia, Ewingella, Kluyvera, Morganella, Planococcus,
Stomatococcus, Micrococcus, Staphylococcus, Vibrio, Aeromonas,
Plessiomonas, Haemophilus, Actinobacillus, Pasteurella, Mycoplasma,
Ureaplasma, Rickettsia, Coxiella, Rochalimaea, Ehrlichia,
Streptococcus, Enterococcus, Aerococcus, Gemella, Lactococcus,
Leuconostoc, Pedicoccus, Bacillus, Corynebacterium,
Arcanobacterium, Actinomyces, Rhodococcus, Listeria,
Erysipelothrix, Gardnerella, Neisseria, Campylobacter, Arcobacter,
Wolinella, Helicobacter, Achromobacter, Acinetobacter,
Agrobacterium, Alcaligenes, Chryseomonas, Comamonas, Eikenella,
Flavimonas, Flavobacterium, Moraxella, Oligella, Pseudomonas,
Shewanella, Weeksella, Xanthomonas, Bordetella, Franciesella,
Brucella, Legionella, Afipia, Bartonella, Calymmatobacterium,
Cardiobacterium, Streptobacillus, Spirillum, Peptostreptococcus,
Peptococcus, Sarcinia, Coprococcus, Ruminococcus,
Propionibacterium, Mobiluncus, Bifidobacterium, Eubacterium,
Lactobacillus, Rothia, Clostridium, Bacteroides, Porphyromonas,
Prevotella, Fusobacterium, Bilophila, Leptotrichia, Wolinella,
Acidaminococcus, Megasphaera, Veilonella, Norcardia, Actinomadura,
Norcardiopsis, Streptomyces, Micropolysporas, Thermoactinomycetes,
Mycobacterium, Treponema, Borrelia, Leptospira, and Chlamydiae.
[0057] As used herein, the terms "microorganism" and "microbe"
refer to any species or type of microorganism, including but not
limited to, bacteria, archaea, fungi, protozoans, mycoplasma, and
parasitic organisms.
[0058] As used herein, the term "fungi" is used in reference to
eukaryotic organisms such as molds and yeasts, including dimorphic
fungi.
[0059] As used herein the terms "disease" and "pathologic
condition" are used interchangeably, unless indicated otherwise
herein, to describe a deviation from the condition regarded as
normal or average for members of a species or group (e.g., humans),
and which is detrimental to an affected individual under conditions
that are not inimical to the majority of individuals of that
species or group. Such a deviation can manifest as a state, signs,
and/or symptoms (e.g., diarrhea, nausea, fever, pain, blisters,
boils, rash, immune suppression, inflammation, etc.) that are
associated with any impairment of the normal state of a subject or
of any of its organs or tissues that interrupts or modifies the
performance of normal functions. A disease or pathological
condition may be caused by or result from contact with a
microorganism (e.g., a pathogen or other infective agent (e.g., a
virus or bacteria)), may be responsive to environmental factors
(e.g., malnutrition, industrial hazards, and/or climate), may be
responsive to an inherent defect of the organism (e.g., genetic
anomalies) or to combinations of these and other factors.
[0060] "Respiratory" and "respiration" refer to the process by
which oxygen is taken into the body and carbon dioxide is
discharged, through the bodily system including the nose, throat,
larynx, trachea, bronchi and lungs.
[0061] "Respiratory infection" and "pulmonary infection" refer to
an infection (e.g., bacterial, viral, fungal, etc.) of the
respiratory tract. In humans, the respiratory tract comprises the
upper respiratory tract (e.g., nose, throat or pharynx, and
larynx); the airways (e.g.,: voice box or larynx, windpipe or
trachea, and bronchi); and the lungs (e.g., bronchi, bronchioles,
alveolar ducts, alveolar sacs, and alveoli).
[0062] "Respiratory disease", "pulmonary disease," "respiratory
disorder", "pulmonary disorder," "respiratory condition",
"pulmonary condition," "pulmonary syndrome," and "respiratory
syndrome" refer to any one of several ailments that involve
inflammation and affect a component of the respiratory system
including especially the trachea, bronchi and lungs. Examples of
such ailments include acute alveolar disease, obstructive
respiratory disease (e.g., asthma; bronchitis; and chronic
obstructive pulmonary disease, referred to as COPD), upper airway
disease (e.g., such as otitis media, and rhinitis/sinusitis),
insterstitial lung disease, allergy, and respiratory infection
(e.g., pneumonia, pneyumocystis carinii, and respiratory syncitial
virus (RSV)).
[0063] Specific examples of acute alveolar disease include acute
lung injury (ALI), acute respiratory distress syndrome (ARDS),
meconium aspiration syndrome (MAS) and respiratory distress
syndrome (RDS). ALI is associated with conditions that either
directly or indirectly injure the air sacs of the lung, the
alveoli. ALI is a syndrome of inflammation and increased
permeability of the lungs with an associated breakdown of the
lungs' surfactant layer. The most serious manifestation of ALI is
ARDS. Among the causes of ALI are complications typically
associated with certain major surgeries, mechanical ventilator
induced lung injury (often referred to as VILI), smoke inhalation,
pneumonia, and sepsis.
[0064] The term "subject" as used herein refers to organisms to be
treated by the compositions of the present invention. Such
organisms include animals (domesticated animal species, wild
animals), and humans.
[0065] As used herein, the terms "inactivating," "inactivation" and
grammatical equivalents, when used in reference to a microorganism
refer to the killing, elimination, neutralization and/or reducing
the capacity of the microorganism to infect and/or cause a
pathological response and/or disease in a host.
[0066] As used herein, the term "fusigenic" is intended to refer to
an emulsion that is capable of fusing with the membrane of a
microbial agent (e.g., a bacterium or bacterial spore). Specific
examples of fusigenic emulsions are described herein.
[0067] As used herein, the term "lysogenic" refers to an emulsion
(e.g., a nanoemulsion) that is capable of disrupting the membrane
of a microbial agent (e.g., a virus (e.g., viral envelope) or a
bacterium, bacterial spore, or bacterial biofilm). In preferred
embodiments of the present invention, the presence of a lysogenic
and a fusigenic agent in the same composition produces an enhanced
inactivating effect compared to either agent alone. Methods and
compositions using this improved antimicrobial composition are
described in detail herein.
[0068] The term "nanoemulsion," as used herein, includes
dispersions or droplets, as well as other lipid structures that can
form as a result of hydrophobic forces that drive apolar residues
(i.e., long hydrocarbon chains) away from water and drive polar
head groups toward water, when a water immiscible oily phase is
mixed with an aqueous phase. These other lipid structures include,
but are not limited to, unilamellar, paucilamellar, and
multilamellar lipid vesicles, micelles, and lamellar phases.
[0069] As used herein, the terms "contact," "contacted," "expose,"
and "exposed," when used in reference to a nanoemulsion and a live
microorganism, refer to bringing one or more nanoemulsions into
contact with a microorganism (e.g., a pathogen) such that the
nanoemulsion kill and/or attenuate growth of the microorganism or
pathogenic agent, if present. The present invention is not limited
by the amount or type of nanoemulsion used for microorganism
killing and/or growth attenuation. A variety of nanoemulsion that
find use in the present invention are described herein and
elsewhere (e.g., nanoemulsions described in U.S. Pat. Apps.
20020045667 and 20040043041, and U.S. Pat. Nos. 6,015,832,
6,506,803, 6,635,676, and 6,559,189, each of which is incorporated
herein by reference in its entirety for all purposes). Ratios and
amounts of nanoemulsion are contemplated in the present invention
including, but not limited to, those described herein (e.g., in
Examples 1-4, the Figures associated therewith and FIG. 17).
[0070] As used herein, "about" will be understood by persons of
ordinary skill in the art and will vary to some extent depending
upon the context in which it is used. If there are uses of the term
which are not clear to persons of ordinary skill in the art given
the context in which it is used, "about" will mean up to plus or
minus 10% of the particular term.
[0071] The term "surfactant" refers to any molecule having both a
polar head group, which energetically prefers solvation by water,
and a hydrophobic tail which is not well solvated by water. The
term "cationic surfactant" refers to a surfactant with a cationic
head group. The term "anionic surfactant" refers to a surfactant
with an anionic head group.
[0072] The terms "Hydrophile-Lipophile Balance Index Number" and
"HLB Index Number" refer to an index for correlating the chemical
structure of surfactant molecules with their surface activity. The
HLB Index Number may be calculated by a variety of empirical
formulas as described by Meyers, (Meyers, Surfactant Science and
Technology, VCH Publishers Inc., New York, pp. 231-245 [1992]),
incorporated herein by reference. As used herein, the HLB Index
Number of a surfactant is the HLB Index Number assigned to that
surfactant in McCutcheon's Volume 1: Emulsifiers and Detergents
North American Edition, 1996 (incorporated herein by reference).
The HLB Index Number ranges from 0 to about 70 or more for
commercial surfactants. Hydrophilic surfactants with high
solubility in water and solubilizing properties are at the high end
of the scale, while surfactants with low solubility in water which
are good solubilizers of water in oils are at the low end of the
scale.
[0073] As used herein the term "interaction enhancers" refers to
compounds that act to enhance the interaction of an emulsion with a
microorganism (e.g., with a cell wall of a bacteria (e.g., a Gram
negative bacteria) or with a viral envelope. Contemplated
interaction enhancers include, but are not limited to, chelating
agents (e.g., ethylenediaminetetraacetic acid (EDTA),
ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), and the
like) and certain biological agents (e.g., bovine serum abulmin
(BSA) and the like).
[0074] The terms "buffer" or "buffering agents" refer to materials
which when added to a solution, cause the solution to resist
changes in pH.
[0075] The terms "reducing agent" and "electron donor" refer to a
material that donates electrons to a second material to reduce the
oxidation state of one or more of the second material's atoms.
[0076] The term "monovalent salt" refers to any salt in which the
metal (e.g., Na, K, or Li) has a net 1+ charge in solution (i.e.,
one more proton than electron).
[0077] The term "divalent salt" refers to any salt in which a metal
(e.g., Mg, Ca, or Sr) has a net 2+ charge in solution.
[0078] The terms "chelator" or "chelating agent" refer to any
materials having more than one atom with a lone pair of electrons
that are available to bond to a metal ion.
[0079] The term "solution" refers to an aqueous or non-aqueous
mixture.
[0080] As used herein, the term "effective amount" refers to the
amount of a composition (e.g., a composition comprising a
nanoemulsion) sufficient to effect a beneficial or desired result
(e.g., to treat and/or prevent infection (e.g., through bacterial
cell killing and/or prevention of bacterial cell growth). An
effective amount can be administered in one or more
administrations, applications or dosages and is not intended to be
limited to a particular formulation or administration route.
[0081] As used herein, the term "a composition for inducing an
immune response" refers to a composition that, when administered to
a subject (e.g., once, twice, three times or more (e.g., separated
by weeks, months or years)), stimulates, generates and/or elicits
an immune response in the subject (e.g., resulting in total or
partial immunity to a microorganism (e.g., pathogen) capable of
causing disease). In preferred embodiments of the invention, the
composition comprises a nanoemulsion and an immunogen. In further
preferred embodiments, the composition comprising a nanoemulsion
and an immunogen comprises one or more other compounds or agents
including, but not limited to, therapeutic agents, physiologically
tolerable liquids, gels, carriers, diluents, adjuvants, excipients,
salicylates, steroids, immunosuppressants, immunostimulants,
antibodies, cytokines, antibiotics, binders, fillers,
preservatives, stabilizing agents, emulsifiers, and/or buffers. An
immune response may be an innate (e.g., a non-specific) immune
response or a learned (e.g., acquired) immune response (e.g. that
decreases the infectivity, morbidity, or onset of mortality in a
subject (e.g., caused by exposure to a pathogenic microorganism) or
that prevents infectivity, morbidity, or onset of mortality in a
subject (e.g., caused by exposure to a pathogenic microorganism)).
Thus, in some preferred embodiments, a composition comprising a
nanoemulsion and an immunogen is administered to a subject as a
vaccine (e.g., to prevent or attenuate a disease (e.g., by
providing to the subject total or partial immunity against the
disease or the total or partial attenuation (e.g., suppression) of
a sign, symptom or condition of the disease.
[0082] As used herein, the term "adjuvant" refers to any substance
that can stimulate an immune response (e.g., a mucosal immune
response). Some adjuvants cause activation of a cell of the immune
system (e.g., an adjuvant can cause an immune cell to produce and
secrete a cytokine). Examples of adjuvants that can cause
activation of a cell of the immune system include, but are not
limited to, the nanoemulsion formulations described herein,
saponins purified from the bark of the Q. saponaria tree, such as
QS21 (a glycolipid that elutes in the 21st peak with HPLC
fractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.);
poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus
Research Institute, USA); derivatives of lipopolysaccharides such
as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc.,
Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and
threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine
disaccharide related to lipid A; OM Pharma SA, Meyrin,
Switzerland); cholera toxin (CT), and Leishmania elongation factor
(a purified Leishmania protein; Corixa Corporation, Seattle,
Wash.). Traditional adjuvants are well known in the art and
include, for example, aluminum phosphate or hydroxide salts
("alum"). In some embodiments, immunogenic compositions described
herein are administered with one or more adjuvants (e.g., to skew
the immune response towards a Th1 and/or Th2 type response).
[0083] As used herein, the term "an amount effective to induce an
immune response" (e.g., of a composition for inducing an immune
response), refers to the dosage level required (e.g., when
administered to a subject) to stimulate, generate and/or elicit an
immune response in the subject. An effective amount can be
administered in one or more administrations (e.g., via the same or
different route), applications or dosages and is not intended to be
limited to a particular formulation or administration route.
[0084] As used herein, the term "under conditions such that said
subject generates an immune response" refers to any qualitative or
quantitative induction, generation, and/or stimulation of an immune
response (e.g., innate or acquired).
[0085] As used herein, the term "immune response" refers to any
detectable response by the immune system of a subject. For example,
immune responses include, but are not limited to, an alteration
(e.g., increase) in Toll receptor activation, lymphokine (e.g.,
cytokine (e.g., Th1 or Th2 type cytokines) or chemokine) expression
and/or secretion, macrophage activation, dendritic cell activation,
T cell (e.g., CD4+ or CD8+T cell) activation, NK cell activation,
and/or B cell activation (e.g., antibody generation and/or
secretion). Additional examples of immune responses include binding
of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to
an MHC molecule and induction of a cytotoxic T lymphocyte ("CTL")
response, induction of a B cell response (e.g., antibody
production), and/or T-helper lymphocyte response, and/or a delayed
type hypersensitivity (DTH) response (e.g., against the antigen
from which an immunogenic polypeptide is derived), expansion (e.g.,
growth of a population of cells) of cells of the immune system
(e.g., T cells, B cells (e.g., of any stage of development (e.g.,
plasma cells), and increased processing and presentation of antigen
by antigen presenting cells. An immune response may be to
immunogens that the subject's immune system recognizes as foreign
(e.g., non-self antigens from microorganisms (e.g., pathogens), or
self-antigens recognized as foreign). Thus, it is to be understood
that, as used herein, "immune response" refers to any type of
immune response, including, but not limited to, innate immune
responses (e.g., activation of Toll receptor signaling cascade)
cell-mediated immune responses (e.g., responses mediated by T cells
(e.g., antigen-specific T cells) and non-specific cells of the
immune system) and humoral immune responses (e.g., responses
mediated by B cells (e.g., via generation and secretion of
antibodies into the plasma, lymph, and/or tissue fluids). The term
"immune response" is meant to encompass all aspects of the
capability of a subject's immune system to respond to an antigen
and/or immunogen (e.g., both the initial response to an immunogen
(e.g., a pathogen) as well as acquired (e.g., memory) responses
that are a result of an adaptive immune response).
[0086] As used herein, the term "immunity" refers to protection
from disease (e.g., preventing or attenuating (e.g., suppression
of) a sign, symptom or condition of the disease) upon exposure to a
microorganism (e.g., pathogen) capable of causing the disease.
Immunity can be innate (e.g., non-adaptive (e.g., non-acquired)
immune responses that exist in the absence of a previous exposure
to an antigen) and/or acquired (e.g., immune responses that are
mediated by B and T cells following a previous exposure to antigen
(e.g., that exhibit increased specificity and reactivity to the
antigen)).
[0087] As used herein, the terms "immunogen" and "antigen" are used
interchangeably to refer to an agent (e.g., a microorganism (e.g.,
bacterium, virus or fungus) and/or portion or component thereof
(e.g., a protein antigen)) that is capable of eliciting an immune
response in a subject. In preferred embodiments, immunogens elicit
immunity against the immunogen (e.g., microorganism (e.g., pathogen
or a pathogen product)) when administered in combination with a
nanoemulsion of the present invention. As used herein, the term
Burkholderia antigen refers to a component or product of a bacteria
of the genus Burkholderia that elicits an immune response when
administered to a subject. An antigen may be a component or product
derived from an organism (e.g., bacteria of the genus Burkholderia)
including, but not limited to, polypeptides, peptides, proteins,
nucleic acids, membrane fractions, and polysaccharides.
[0088] As used herein, the term "enhanced immunity" refers to an
increase in the level of adaptive and/or acquired immunity in a
subject to a given immunogen (e.g., microorganism (e.g., pathogen))
following administration of a composition relative to the level of
adaptive and/or acquired immunity in a subject that has not been
administered the composition.
[0089] As used herein, the terms "purified" or "to purify" refer to
the removal of contaminants or undesired compounds from a sample or
composition. As used herein, the term "substantially purified"
refers to the removal of from about 70 to 90%, up to 100%, of the
contaminants or undesired compounds from a sample or
composition.
[0090] As used herein, the terms "administration" and
"administering" refer to the act of giving a drug, prodrug, or
other agent, or therapeutic treatment (e.g., a composition of the
present invention) to a physiological system (e.g., a subject or in
vivo, in vitro, or ex vivo cells, tissues, and organs).
[0091] As used herein, the terms "co-administration" and
"co-administering" refer to the administration of at least two
agent(s) (e.g., a nanoemulsion and one or more other
pharmaceutically acceptable substances (e.g., a second
nanoemulsion)) or therapies to a subject. In some embodiments, the
co-administration of two or more agents or therapies is concurrent.
In other embodiments, a first agent/therapy is administered prior
to a second agent/therapy. In some embodiments, co-administration
can be via the same or different route of administration. Those of
skill in the art understand that the formulations and/or routes of
administration of the various agents or therapies used may vary.
The appropriate dosage for co-administration can be readily
determined by one skilled in the art. In some embodiments, when
agents or therapies are co-administered, the respective agents or
therapies are administered at lower dosages than appropriate for
their administration alone. Thus, co-administration is especially
desirable in embodiments where the co-administration of the agents
or therapies lowers the requisite dosage of a potentially harmful
(e.g., toxic) agent(s), and/or when co-administration of two or
more agents results in sensitization of a subject to beneficial
effects of one of the agents via co-administration of the other
agent. In other embodiments, co-administration is preferable to
treat and/or prevent infection by more than one type of infectious
agent (e.g., bacteria and/or viruses).
[0092] As used herein, the term "topically" refers to application
of a compositions of the present invention (e.g., a composition
comprising a nanoemulsion) to the surface of the skin and/or
mucosal cells and tissues (e.g., alveolar, buccal, lingual,
masticatory, vaginal or nasal mucosa, and other tissues and cells
which line hollow organs or body cavities). Compositions described
herein can be applied using any pharmaceutically acceptable method,
such as for example, intranasal, buccal, sublingual, oral, rectal,
ocular, parenteral (intravenously, intradermally, intramuscularly,
subcutaneously, intracisternally, intraperitoneally), pulmonary,
intravaginal, locally administered, topically administered,
mucosally administered, via an aerosol, or via a buccal or nasal
spray formulation. Further, the nanoemulsion vaccines described
herein can be formulated into any pharmaceutically acceptable
dosage form, such as a liquid dispersion, gel, aerosol, pulmonary
aerosol, nasal aerosol, ointment, cream, semi-solid dosage form,
and a suspension. Further, the composition may be a controlled
release formulation, sustained release formulation, immediate
release formulation, or any combination thereof.
[0093] The terms "pharmaceutically acceptable" or
"pharmacologically acceptable," as used herein, refer to
compositions that do not substantially produce adverse allergic or
immunological reactions when administered to a host (e.g., an
animal or a human). Such formulations include dips, sprays, seed
dressings, stem injections, sprays, and mists. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, wetting agents (e.g., sodium
lauryl sulfate), isotonic and absorption delaying agents,
disintegrants (e.g., potato starch or sodium starch glycolate), and
the like. Examples of carriers, stabilizers and adjuvants have been
described and are known in the art (See e.g., Martin, Remington's
Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa.
(1975), incorporated herein by reference).
[0094] As used herein, the term "pharmaceutically acceptable salt"
refers to any salt (e.g., obtained by reaction with an acid or a
base) of a composition of the present invention that is
physiologically tolerated in the target subject. "Salts" of the
compositions of the present invention may be derived from inorganic
or organic acids and bases. Examples of acids include, but are not
limited to, hydrochloric, hydrobromic, sulfuric, nitric,
perchloric, fumaric, maleic, phosphoric, glycolic, lactic,
salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric,
methanesulfonic, ethanesulfonic, formic, benzoic, malonic,
sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the
like. Other acids, such as oxalic, while not in themselves
pharmaceutically acceptable, may be employed in the preparation of
salts useful as intermediates in obtaining the compositions of the
invention and their pharmaceutically acceptable acid addition
salts.
[0095] Examples of bases include, but are not limited to, alkali
metal (e.g., sodium) hydroxides, alkaline earth metal (e.g.,
magnesium) hydroxides, ammonia, and compounds of formula
NW.sub.4.sup.+, wherein W is C.sub.1-4 alkyl, and the like.
[0096] Examples of salts include, but are not limited to: acetate,
adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide,
2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate,
persulfate, phenylpropionate, picrate, pivalate, propionate,
succinate, tartrate, thiocyanate, tosylate, undecanoate, and the
like. Other examples of salts include anions of the compounds of
the present invention compounded with a suitable cation such as
Na.sup.+, NH.sub.4.sup.+, and NW.sub.4.sup.+ (wherein W is a
C.sub.1-4 alkyl group), and the like. For therapeutic use, salts of
the compounds of the present invention are contemplated as being
pharmaceutically acceptable. However, salts of acids and bases that
are non-pharmaceutically acceptable may also find use, for example,
in the preparation or purification of a pharmaceutically acceptable
compound.
[0097] For therapeutic use, salts of the compositions of the
present invention are contemplated as being pharmaceutically
acceptable. However, salts of acids and bases that are
non-pharmaceutically acceptable may also find use, for example, in
the preparation or purification of a pharmaceutically acceptable
composition.
[0098] As used herein, the terms "at risk for disease" and "at risk
for infection" refer to a subject that is predisposed to
experiencing a particular disease and/or infection. This
predisposition may be genetic (e.g., a particular genetic tendency
to experience the disease, such as heritable disorders), or due to
other factors (e.g., environmental conditions, exposures to
detrimental compounds present in the environment, etc.). Thus, it
is not intended that the present invention be limited to any
particular risk (e.g., a subject may be "at risk for disease"
simply by being exposed to and interacting with other people that
carry a risk of transmitting a pathogen), nor is it intended that
the present invention be limited to any particular disease and/or
infection.
[0099] "Nasal application", as used herein, means applied through
the nose into the nasal or sinus passages or both. The application
may, for example, be done by drops, sprays, mists, coatings or
mixtures thereof applied to the nasal and sinus passages.
[0100] "Pulmonary application" and "pulmonary administration"
refers to any means of applying a composition of the present
invention to the pulmonary system of a subjet. The present
invention is not limited to any particular means of administration.
Indeed, a variety of means are contemplated to be useful for
pulmonary administration including those described herein.
[0101] As used herein, the term "kit" refers to any delivery system
for delivering materials. In the context of the nanoemulsion
compositions of the present invention, such delivery systems
include systems that allow for the storage, transport, or delivery
of the compositions and/or supporting materials (e.g., written
instructions for using the materials, etc.) from one location to
another. For example, kits include one or more enclosures (e.g.,
boxes) containing the relevant nanoemulsions and/or supporting
materials. As used herein, the term "fragmented kit" refers to
delivery systems comprising two or more separate containers that
each contain a subportion of the total kit components. The
containers may be delivered to the intended recipient together or
separately. For example, a first container may contain a
composition comprising a nanoemulsion for a particular use, while a
second container contains a second agent (e.g., an antibiotic or
spray applicator). Indeed, any delivery system comprising two or
more separate containers that each contains a subportion of the
total kit components are included in the term "fragmented kit." In
contrast, a "combined kit" refers to a delivery system containing
all of the components of a composition needed for a particular use
in a single container (e.g., in a single box housing each of the
desired components). The term "kit" includes both fragmented and
combined kits.
DETAILED DESCRIPTION OF THE INVENTION
[0102] The present invention relates to methods and compositions
useful for treating pulmonary infection. In particular, the present
invention provides nanoemulsion compositions and methods of using
the same to treat bacteria associated with biofilms (e.g., found in
pulmonary infections). Compositions and methods of the present
invention find use in, among other things, clinical (e.g.
therapeutic and preventative medicine), industrial, and research
applications.
[0103] Several pathogenic microorganisms initiate infection by
attaching to mucosal epithelial cells lining the gastro-intestinal,
oropharyngeal, respiratory or genito-urinacy tracts. Some
pathogens, such as influenza virus, Bordetella pertussis, or Vibrio
cholerae, remain at or within the mucosal tissue, while others,
such as Salmonella typhi or hepatitis A virus, possess mechanisms
permitting penetration into deeper tissues and spread systemically.
Specific and non-specific defense mechanisms of the mucous
membranes provide first line protection against both types of
pathogen. Non-specific effectors include resident macrophages,
antimicrobial peptides, lactoferrin and lysozyme, extremes of pH,
bile acids, digestive enzymes, mucus, shedding of epithelial cells,
flushing mechanisms (peristalsis, ciliary beating, micturation,
etc.) and competition from local flora. However, successful
pathogens have generally evolved means to survive the non-specific
defenses present at the site they infect and it is the secretory
immune system which plays a major role in protecting against
diseases caused by a number of bacterial and viral pathogens, and
is probably a major effector against pathogens that are restricted
to mucosal surfaces. For organisms that spread systemically, both
local and systemic immune responses are desirable for optimum
immunity.
[0104] As described herein, certain microbes (e.g., bacteria) are
able to thrive when a normal and/or healthy immune system does not
function properly for one reason or another. Cystic fibrosis (CF),
asthma, HIV infection, chemotherapeutic therapy and a host of other
conditions lead to malfunctioning and/or attenuation of immune
responses that would normally function to protect against and clear
microbes capable of causing pathology in a healthy subject.
[0105] For example, Pseudomonas aeruginosa is an opportunistic
pathogen that infects the immunocompromised, elderly, cancer
chemotherapy patients, and individual suffering from CF. In CF lung
disease, P. aeruginosa is trapped in thickened, dehydrated, hypoxic
mucus lining in airway epithelia. Morphologic data suggests that
the airway lumen of CF patients harbor P. aeruginosa biofilms that
are characterized as spherical microcolonies.
[0106] Other types of microbes can cause pathology in an otherwise
healthy host subject. For example, colonization of the respiratory
tract by the Gram-negative coccobacillus Bordetella pertussis
results in whooping cough, also called pertussis, a significant
cause of morbidity and mortality of human infants. Two other
closely-related isolates of Bordetella have also been found in
humans: B. parapertussis and B. bronchiseptica. Molecular genetic
analyses suggest that these three isolates are too closely related
to be classified as separate species. (See Gilchrist. M. J. R.,
1991, "Bordetella", in Manual of Clinical Microbiology, 5th ed.,
Balows, A. et al., eds., American Society for Microbiology,
Washington, D.C.). While B. pertussis differs from B.
bronchiseptica and B. parapertussis in the nature of the toxins it
produces, B. bronchiseptica and B. parapertussis do produce active
toxins (See Hausman, S. Z. et al., 1996, Infect. Immun. 64:
4020-4026), and there is some evidence to indicate that B.
pertussis organisms can covert to the B. parapertussis phenotype
(Gilchrist, M. J. R., 1991, "Bordetella", in Manual of Clinical
Microbiology, 5th ed., Balows, A. et al., eds., American Society
for Microbiology, Washington, D.C.).
[0107] Thus, the present invention provides compositions and
methods for the treatment of respiratory infections. Compositions
and methods of the present invention may be used to treat and/or
prevent respiratory infection (e.g., in cystic fibrosis patients)
caused by one or more of pseudomonas (e.g., P. aeruginosa, P.
paucimobilis, P. putida, P. fluorescens, and P. acidovorans),
staphylococci, Methicillin-resistant Staphylococcus aureus (MRSA),
streptococci (including Streptococcus pneumoniae), Escherichia
coli, Klebsiella, Enterobacter, Serratia, Haemophilus, Yersinia
pestis, Burkholderia pseudomallei, B. cepacia, B. gladioli, B.
multivorans, B. vietnamiensis, Mycobacterium tuberculosis, M. avium
complex (MAC) (M. avium and M. intracellulare), M. kansasii, M.
xenopi, M. marinum, M. ulcerans, or M. fortuitum complex (M.
fortuitum and M. chelonei), Bordetella pertussis, B. parapertussis
and B. bronchiseptica. Furthermore, compositions and methods of the
present invention find use in the treatment and/or prevention of a
host of respiratory infections (e.g., respiratory infections of the
upper respiratory tract (e.g., nose, ears, sinuses, and throat) and
the lower respiratory tract (e.g., trachea, bronchial tubes, and
lungs)). Several examples of microbes that may be treated (e.g.,
killed and/or attenuated in growth (e.g., within the respiratory
tract of a subject)) are provided below.
[0108] Accordingly, in some embodiments, the present invention
provides a composition comprising a nanoemulsion, and methods of
pulmonary administration of the same to prevent and/or treat
respiratory infection. In some embodiments, the nanoemulsion
comprises ethylenediaminetetraacetic acid (EDTA). The present
invention is not limited by the amount of EDTA utilized. In some
embodiments, 0.01-0.1 mM, 0.1-1.0 mM, 1.0-1.5 mM, 1.5-2.5 mM,
2.5-5.0 mM, 5.0-10.0 mM, 10-20 mM, 20-30 mM, or 30-50 mM EDTA is
used. However, the present invention is not limited to this amount
of EDTA. In some embodiments, less than 0.01 mM or more than 50 mM
EDTA is utilized. In some embodiments, the composition is
co-administered with a hypertonic salt (e.g., sodium chloride)
solution (e.g., a 6-7%, 1-3%, 3-6%, 0.1-1%, or more than 7% salt
solution (e.g., NaCl solution)). In some embodiments, the
composition comprises a 20% nanoemulsion solution. In some
embodiments, the composition comprises greater than 20% (e.g., 25%,
30%, or more) nanoemulsion solution. In some embodiments, the
composition comprises less than 20% (e.g., 15%, 10% or less)
nanoemulsion solution. However, the present invention is not
limited to this amount (e.g., percentage) of nanoemulsion. For
example, in some embodiments, a composition comprises less than 10%
nanoemulsion. In some embodiments, a composition comprises more
than 20% nanoemulsion. In some embodiments, the composition
comprises 10 mM EDTA. In some embodiments, the composition
comprises 20 mM EDTA. In some embodiments, a composition of the
present invention comprises any of the nanoemulsions described
herein. In some embodiments, a composition comprising a
nanoemulsion utilized to treat bacteria (e.g., present in pulmonary
space of a subject (e.g., biofilm forming bacteria)) comprises
P.sub.4075EC. In some embodiments, a composition comprising a
nanoemulsion comprises W.sub.805EC.
[0109] Administration of nanoemulsion alone or in combination with
EDTA (e.g., 10-20 mM EDTA) was able to achieve complete killing of
10.sup.6 bacteria in PBS in 60 minutes (See Examples 2-4). In the
presence of hypertonic saline (e.g., 6-7% NaCl), the killing
ability of the nanoemulsion was surprisingly and strikingly
enhanced, achieving complete killing within 15 minutes while in the
presence of 20 mM EDTA (See Example 2). Also, nanoemulsions
comprising a lower concentration of EDTA were able to achieve
complete killing of bacteria in 30 minutes in the presence of
hypertonic saline.
[0110] Thus, in some embodiments, the present invention provides
that a nanoemulsion composition can be used to kill (e.g.,
completely) bacteria over a short time period (e.g., less than 60
minutes, less than 30 minutes, or less than 15 minutes).
[0111] The present invention also demonstrates that compositions of
the present invention are able to eradicate a mixed population of
bacteria. Moreover, toxicity studies performed during the
development of embodiments of the present invention characterized
the nanoemulsion compositions as being safe and causing no
detectable harm to a subject (e.g., no histological changes and/or
detectable pathology (See, e.g., Example 1)).
[0112] As described in Examples 3 and 4, compositions comprising
nanoemulsions of the present invention are able to treat (e.g.,
kill and/or inhibit growth of) bacterial species that are generally
not virulent in healthy persons, but that are opportunists that
cause severe and chronic respiratory tract infections (e.g., in
individuals with cystic fibrosis (CF)). Unremitting infection with
these species results in inflammation and progressive lung disease
that culminates in pulmonary failure, the leading cause of death
for CF patients. Effective therapy of pulmonary infection in CF to
date has been severely limited by the broad spectrum antimicrobial
resistance exhibited by these species, which are among the most
drug-resistant bacteria encountered in human infection. The site of
infection in CF presents another important obstacle to effective
therapy. Infecting bacteria primarily reside within the airway
lumen in sputum, airway epithelial surface fluid, and the bronchial
mucosa (ref). The penetration of systemically delivered
antimicrobials to this infected site is generally poor. Treatment
is further hampered by bacterial biofilm formation, which is
believed to occur in the airways of infected patients, and by the
exceptionally viscous secretions that characterize the CF
respiratory tract.
[0113] Although an understanding of a mechanism is not necessary to
practice the present invention, and the present invention is not
limited to any particular mechanism of action, in some embodiments,
a mechanism of bacterial and/or viral killing utilizing
compositions comprising nanoemulsions of the present invention
involves fusion of the emulsion with microorganism lipid membranes,
leading to rapid osmotic disruption and cell lysis (See, e.g.,
Hamouda and Baker, J Appl Microbiol. 2000 September;
89(3):397-403). Additionally, in some embodiments, electrostatic
attraction (e.g., provided by cationic surface charge of CPC)
overcomes the LPS-mediated resistance of gram-negative bacteria to
neutral and anionic detergents (See, e.g., Hamouda and Baker, J
Appl Microbiol. 2000 September; 89(3):397-403). In some
embodiments, bactericidal activity of a composition comprising a
nanoemulsion (e.g., P.sub.4075EC (e.g., against gram-negative
and/or gram-positive bacteria)) is enhanced by the addition of
EDTA. Although an understanding of a mechanism is not necessary to
practice the present invention, and the present invention is not
limited to any particular mechanism of action, in some embodiments,
EDTA chelates divalent cations that stabilize outer membrane LPS
thereby facilitating interactions with the cationic emulsion, an
interaction that leads to membrane permeabilization and lysis as
well as augmenting transmembrane diffusion of macromolecules (See,
e.g., Rabinovich-Guilatt et al., J Drug Target. 2004;
12(9-10):623-33, Vaara, Microbiol Rev. 1992 September;
56(3):395-411).
[0114] Experiments conducted during development of embodiments of
the present invention identified P.sub.4075EC to be stable after
nebulization in 7% saline using the PARI LC Plus nebulizer.
Inhalation of a single dose of P.sub.4075EC nebulized in 7% saline
was well tolerated by animals. Thus, in some embodiments, the
present invention provides compositions comprising P.sub.4075EC and
hypertonic saline, and methods of using the same (e.g., via
inhalation (e.g., post nebulizing the solution)) for treating one
or more types of pulmonary bacterial infections (e.g., to treat
bacteria and/or bacterial biofilms (e.g., that possess resistance
to conventional treatments) without increasing the already high
treatment burden for CF patients).
[0115] For example, experiments conducted during development of
embodiments of the invention show that compositions comprising
nanoemulsions (e.g., P.sub.4075EC and saline solution) were
effective to treat (e.g., kill and/or inhibit growth of) species
within the Burkholderia cepacia complex (Bcc) (See, e.g., Example
3). Infection with Burkholderia cepacia species is particularly
refractory to antimicrobial therapy and associated with increased
rates of morbidity and mortality in CF. Infection with Bcc is also
regarded by many CF care centers as an absolute contraindication to
lung transplantation. Compositions comprising nanoemulsions (e.g.,
P.sub.4075EC and saline solution) were effective to treat (e.g.,
kill and/or inhibit growth of) B. multivorans and B. cenocepacia
which account for the majority of Bcc infection in CF (See, e.g.,
Reik J Clin Microbiol. 2005 June; 43(6):2926-8). Compositions
comprising nanoemulsions (e.g., P.sub.4075EC and saline solution)
were effective to treat (e.g., kill and/or inhibit growth of) B.
gladioli, which although not a member of the Bcc, is being
recovered with increasing frequency from CF patients. Also shown in
Example 3, compositions comprising nanoemulsions (e.g.,
P.sub.4075EC) were effective to treat (e.g., kill and/or inhibit
growth of) Acinetobacter, which although currently infrequently
recovered in CF, appears to be an emerging pathogen in this patient
population. The great majority (94%) of isolates tested in Example
3 were recovered from cultures of respiratory specimens from
persons with CF. The panel also included one representative isolate
from each of five previously described so-called epidemic lineages,
each of which has been identified as infecting multiple CF
patients. These included the B. cenocepacia ET12 (Johnson et al., J
Clin Microbiol 1994; 32:924-30.), PHDC (Chen et al., J Pediatr
2001; 139:643-9), and Midwest (Coenye and LiPuma, J. Infect. Dis.
185:1454-1462) lineages, as well as the B. multivorans OHBM lineage
(Biddick et al., FEMS Microbiol Lett 2003; 228:57-62) and the B.
dolosa SLC6 lineage (Biddick et al., FEMS Microbiol Lett 2003;
228:57-62). Compositions comprising nanoemulsions (e.g.,
P.sub.4075EC and saline solution) effectively treated (e.g., killed
and/or inhibited growth of) each of these different species.
Moreover, compositions comprising nanoemulsions (e.g.,
P.sub.4075EC) were effective to treat (e.g., kill and/or inhibit
growth of) strains that were found in previous susceptibility
testing to be multi-drug resistant (e.g., defined as resistant to
all antibiotics tested in two of three antibiotic classes: lactams
(including carbapenems), aminoglycosides, and quinolones) as well
as panresistant strains (See Example 3). Genotyping analyses of all
isolates was performed to confirm that each sample treated was a
distinct strain.
[0116] As shown in Example 3, P.sub.4075EC showed very good
activity against the strains tested. With the exception of two B.
cenocepacia strains, all strains in the test panel were inhibited
by a P.sub.4075EC concentration of .ltoreq.125 .mu.g/ml, or a 1:16
dilution of the starting material; 59% of strains were inhibited by
a concentration of .ltoreq.31.2 .mu.g/ml, a 1:64 dilution of
P.sub.4075EC. Multi-drug resistant or panresistant strains did not
show decreased susceptibility to P.sub.4075EC, demonstrating
MIC.sub.90s of 125 .mu.g/ml and 62.5 .mu.g/ml, respectively. In
general, Burkholderia species tended to be slightly less
susceptible to P.sub.4075EC than the other species examined.
Sixteen of the 18 strains requiring the highest MICs were
Burkholderia. Conversely, 33 of the 38 strains with the lowest
tested MIC (15.6 .mu.g/ml) were non-Burkholderia species. No
striking differences in P.sub.4075EC activity was observed among
the 10 Burkholderia species examined, although the two strains
requiring the highest MICs (250 .mu.g/ml and 500 .mu.g/ml) were
both B. cenocepacia. Among the non-Burkholderia species, Ralstonia
strains were most susceptible (MIC.sub.90.ltoreq.5.6 .mu.g/ml)
while Acinetobacter strains were relatively less susceptible
(MIC.sub.90=125 .mu.g/ml). No evidence was found of tolerance to
P.sub.4075EC among a subset of 34 strains for which both MIC and
MBC were determined. P.sub.4075EC killing of planktonically grown
bacteria was time- and concentration-dependent; at a concentration
16 times greater than the MIC, complete killing was achieved within
30 min. Thus, the present invention provides that relatively brief
exposure of bacteria to P.sub.4075EC can be utilized to effect
several log decreases in viable bacteria. Thus, a composition
comprising a nanoemulsion (e.g., P.sub.4075EC) of the present
invention can be utilized individually, or in combination with
other antimicrobials, to kill and/or inhibit growth of
bacteria.
[0117] Experiments were conducted during development of embodiments
of the invention to assess the activity of P.sub.4075EC against
bacteria grown in vitro as biofilms. A relatively strict definition
of in vitro biofilm formation was employed, as described in Example
3, in an effort to provide a stringent test of P.sub.4075EC
activity. 12 strains that met the definition were tested. As shown
in Example 3, the strains represented several species and a range
of susceptibility to P.sub.4075EC based on standard MIC/MBC
testing. Although the MBIC and MBEC of P.sub.4075EC were increased
compared to the respective MIC and MBC for each strain tested
(median four-fold increase in MBIC compared to MIC), all 12 strains
were inhibited or killed by P.sub.4075EC when grown as biofilms.
Only a single strain required undiluted P.sub.4075EC (2000
.mu.g/ml) for eradication of viable biofilm bacteria. Relative
biomass was assessed among the biofilm forming strains
spectrophotometrically with crystal violet staining and no
correlation was observed between biomass and MBIC/MBEC. In fact,
although B. gladioli strain AU10529 produced the greatest biomass
among the 12 strains tested, the MBIC and MBEC of P.sub.4075EC for
this strain were relatively low (both 62.5 .mu.g/ml). Conversely,
B. cenocepacia strain J2315 produced relatively little biomass, yet
required a P.sub.4075EC MBEC of 1000 .mu.g/ml.
[0118] CF sputum, like biofilm, is reported to antagonize the
activity of antibacterial drugs (See, e.g., Hunt et al., Antimicrob
Agents Chemother. 1995 January; 39(1):34-9). Glycoproteins such as
mucin, which compose 2-3% of the dry weight of sputum, and high
molecular weight DNA are present in elevated levels resulting in
exceptionally viscous sputum that provides a physical barrier
protecting bacteria. In addition, these macromolecules bind and
sequester antibiotics while small cationic molecules and the
decreased pH of CF sputum block drug penetration into bacteria and
reduce drug bioactivity. The strategy of increasing drug dosing to
overcome these obstacles is limited by drug toxicity. To assess the
impact of sputum on the antibacterial activity of P.sub.4075EC,
standard planktonic susceptibility testing was repeated for the 12
biofilm-forming strains in the presence of CF sputum. A mixture of
sputum from 15 CF patients was used to avoid inter-patient
variation in macromolecule and high molecular weight DNA
composition, and ionic conditions, and only mechanical shearing was
applied to minimize changes to the native microenvironment (See,
e.g., Grebski et al., Chest. 2001 May; 119(5):1521-5). The activity
of P.sub.4075EC against bacteria suspended in media containing 43%
sputum (the maximum sputum concentration achieved in the test
system) was decreased with bactericidal concentrations 2- to
32-fold greater than the respective planktonic MBCs without sputum.
The sputum-MBCs were identical to (6 of 12) or within one dilution
of (6 of 12) the MBECs obtained with biofilm grown bacteria.
Although the activity of P.sub.4075EC was similarly antagonized by
both CF sputum and biofilm growth, it remained bactericidal for all
the strains tested under both test conditions (See FIG. 10).
[0119] Thus, in some embodiments, the present invention provides
compositions comprising nanoemulsions (e.g., P.sub.4075EC) and
methods of using the same for antimicrobial treatment for infection
due to CF-related opportunistic pathogens. In particular,
nanoemulsion compositions of the present invention are rapidly
bactericidal, and active against bacteria whether grown
planktonically or as a biofilm, or in the presence of CF sputum.
Moreover, compositions comprising nanoemulsions of the present
invention are exceptionally stable, unchanged after nebulization,
and broadly microbicidal. Importantly, the development of
resistance to a nanoemulsion composition of the present invention
has not been observed by any bacterial species examined to date.
Thus, the present invention provides that P.sub.4075EC can be used
effectively as an inhaled antimicrobial.
[0120] The present invention is not limited to treatment of
bacterial biofilms the reside in pulmonary spaces (e.g., within a
subject with CF). Indeed, compositions comprising a nanoemulsion of
the present invention can be utilized as a therapeutic and/or
antimicrobial agent (e.g., to kill and/or inhibit growth of)
bacterial biofilms in any clinical and/or industrial setting.
[0121] Multiple species of bacteria exist that are able to form
biofilms. For example, bacteria that adhere to implanted medical
devices or damaged tissue often encase themselves in a hydrated
matrix of polysaccharide and protein to form biofilm. Biofilms pose
a serious problem for public health because of the increased
resistance of biofilm-associated organisms to antimicrobial agents
and the association of infections with these organisms in patients
with indwelling medical devices or damaged tissue. Antibiotic
resistance of bacteria growing in biofilms contributes to the
persistence and chronic nature of infections such as those
associated with implanted medical devices. The mechanisms of
resistance in biofilms are different from the now familiar
plasmids, transposons, and mutations that confer innate resistance
to individual bacterial cells. In biofilms, resistance seems to
depend on multicellular strategies.
[0122] Biofilms are complex communities of microorganisms attached
to surfaces or associated with interfaces or damaged tissue.
Despite the focus of modern microbiology research on pure culture,
planktonic (free-swimming) bacteria, it is now widely recognized
that most bacteria found in natural, clinical, and industrial
settings persist in association with surfaces as biofilms.
Furthermore, these microbial communities are often composed of
multiple species that interact with each other and their
environment. The determination of biofilm architecture,
particularly the spatial arrangement of microcolonies (clusters of
cells) relative to one another, has profound implications for the
function of these complex communities.
[0123] The biofilm matrix is a dynamic environment in which the
component microbial cells appear to reach homeostasis and are
optimally organized to make use of all available nutrients. The
matrix therefore shows great microheterogeneity, within which
numerous microenvironments can exist. Biofilm formation is believed
to be a two-step process in which the attachment of bacterial cells
to a surface is followed by growth dependent accumulation of
bacteria in multilayered cell clusters. Although exopolysaccharides
provide the matrix framework, a wide range of enzyme activities can
be found within the biofilm, some of which greatly affect
structural integrity and stability.
[0124] More specifically, during the first phase of formation, it
is hypothesized that the fibrinogen and fibronectin of host plasma
cover the surface of a medical implant or damaged tissue and are
identified by constitutively expressed microbial surface
components, which mediate the initial attachment of bacteria to the
surface of the biomaterial or damaged tissue. In the second step, a
specific gene locus in the bacteria cells, called the intracellular
adhesion (ica) locus, activates the adhesion of bacteria cells to
each other, forming the secondary layers of the biofilm. The ica
locus is responsible for the expression of the capsular
polysaccharide operon, which in turn activates polysaccharide
intercellular adhesion (PIA), via the sugar
poly-N-succinylglucosamine (PNSG), a-1,6-linked glucosaminoglycan.
The production of this polysaccharide layer gives the biofilm its
slimy appearance when viewed using electron microscopy.
[0125] Staphylococcus aureus is a highly virulent human pathogen.
Both S. aureus and coagulase-negative staphylococci have emerged as
major nosocomial pathogens associated with biofilm formation on
implanted medical devices and damaged tissue. These organisms are
among the normal carriage flora of human skin and mucous membranes,
making them prevalent complications during and after invasive
surgery or prolonged hospital stays. As bacteria carried on both
healthy and sick people, staphylococci are considered opportunistic
pathogens that invade patients via open wounds and via biomaterial
implants.
[0126] Biofilm infections associated with S. aureus are a
significant cause of morbidity and mortality, particularly in
settings such as hospitals, nursing homes and infirmaries. Patients
at risk include infants, the elderly, the immuno-compromised, the
immuno-suppressed, and those with chronic conditions requiring
frequent hospital stays. Patients with intravascular and other
implanted prosthetic devices are at even greater risk from
staphylococcal infections because of compromised immune systems and
the introduction of foreign bodies, which serve to damage tissue
and/or act as a surface for the formation of biofilms. Such
infections can have chronic, if not fatal, implications.
[0127] The causes of biofilm resistance to antibiotics include the
failure of some antimicrobial agents to penetrate all the layers of
a biofilm, the slow-growth rate of certain biofilm cells that make
them less susceptible to antimicrobial agents requiring active
bacterial growth, and the expression of gene patterns by the
bacterial cells embedded in the biofilm that differ from the genes
expressed in their planktonic (free-swimming) state. These
differences in biofilm-associated bacteria render antimicrobial
agents that work effectively to kill planktonic bacteria
ineffective in killing biofilm-associated bacteria. Often the only
way to treat biofilms (e.g., associated with catheters or
prosthetic devices) is the removal of the contaminated device,
which may require additional surgery and present further risks to
patients.
[0128] Thus, as used herein, biofilms refer to an aggregate of
microorganisms with an extracellular matrix that facilitates
adhesion to, and colonization and growth of the aggregate on a
surface, such as an internal or external tissue or organ. Biofilms
can be comprised of bacteria, fungi, yeast, protozoa, or other
microorganisms. Bacterial biofilms typically display high
resistance to antibiotics, often up to 1,000-times greater
resistance than the same bacteria not growing in a biofilm.
[0129] In some embodiments, compositions and methods of the
invention are utilized to treat (e.g., kill and/or inhibit growth
of) and/or prevent biofilms on and/or within a subject (e.g.,
within the pulmonary system, on internal organs or tissue (e.g.,
the bladder, kidney, heart, middle ear, sinuses, a joint, the eye),
on an external tissue (e.g., the skin), and/or oral surfaces such
as teeth, tongue, oral mucosa, or gums. Compositions and methods of
the invention may be used to treat a biofilm-associated condition
such as a soft-tissue infection, chronic sinusitis, endocarditis,
osteomyelitis, urinary tract infection, chronic bacterial
vaginosis, dental plaque or halitosis, infection of prosthetic
device and/or catheter, bacterial keratitis, or prostatitis.
[0130] As described in Examples 3 and 4, compositions of the
present invention can be utilized to treat (e.g., kill and/or
inhibit growth of) any one or more Gram-positive and Gram-negative
bacterial species. Indeed, compositions and methods of the present
invention can be utilized to kill and/or inhibit growth of a number
of bacterial species including, but not limited to, Staphylococcus
aureus, coagulase negative staphylococci such as Staphylococcus
epidermis, Streptococcus pyogenes (Group A), Streptococcus species
(viridans group), Streptococcus agalactiae (group B), S. bovis,
Streptococcus (anaerobic species), Streptococcus pneumoniae,
Enterococcus species, Bacillus anthracis, Corynebacterium
diptheriae, and Corynebacterium species which are diptheroids
(aerobic and anaerobic), Listeria monocytogenes, Clostridium
tetani, and Clostridium difficile, Escherichia coli, Enterobacter
species, Proteus mirablis, Pseudomonas aeruginosa, Klebsiella
pneumoniae, Salmonella, Shigella, Serratia, Campylobacter jejuni,
Neisseria, Branhamella catarrhalis, and Pasteurella.
[0131] Compositions and methods of the present invention can be
utilized to treat (e.g., kill and/or inhibit growth of) organisms
capable of forming biofilms including, but not limited to,
dermatophytes (e.g, Microsporum species such as Microsporum canis,
Trichophyton species such as Trichophyton rubrum and Trichophyton
mentagrophytes), yeasts (e.g., Candida albicans,
Candidaparapsilosis, Candida glabrata, Candida tropicalis, and
other Candida species including drug resistant Candida species),
Epidermophytonfloccosum, Malasseziafuurfur (Pityropsporon
orbiculare, Pityropsporon ovale) Cryptococcus neoformans,
Aspergillusfumigatus and other Aspergillus species, Zygomycetes
(Rizopus, Mucor), hyalohyphomycosis (Fusarium species),
Paracoccidiodes brasiliensis, Blastmyces dermatitides, Histoplasma
capsulatum, Coccidiodes immitis, Sporothrix schenckii, and
Blastomyces.
[0132] Thus, in some embodiments, the present invention provides a
method for treating a subject possessing a biofilm (e.g.,
possessing an indwelling prosthetic device or catheter, wherein the
indwelling prosthetic device or catheter is in contact with a
biofilm, or wherein the subject has an infection (e.g., respiratory
infection) within which a biofilm resides) comprising administering
to the subject a composition comprising a nanoemulsion (e.g.,
P.sub.4075EC) under conditions such that the biofilm is altered
and/or bacteria residing within the biofilm are killed and/or their
growth is inhibited. In some embodiments, altering the biofilm
comprises eradicating the biofilm. In some embodiments, altering
the biofilm comprises killing bacteria involved in forming the
biofilm. In some embodiments, the bacteria comprise S. aureus, S.
epidermidis, antibiotic resistant bacteria (e.g., methicillin
resistant, vancomycin resistant, etc.), and/or other type of
bacteria described herein. In some embodiments, the composition
comprising a nanoemulsion (e.g., P.sub.4075EC) is co-administered
with one or more antibacterial agents. In some embodiments, the
antibacterial agents are selected from the group comprising, but
not limited to, antibiotics, antibodies, antibacterial enzymes,
peptides, and lanthione-containing molecules. In some embodiments,
the antibiotic interferes with or inhibits cell wall synthesis. In
some embodiments, the antibiotic is selected from the group
including, but not limited to, .beta.-lactams, cephalosporins,
glycopeptides, aminoglycosides, sulfonomides, macrolides, folates,
polypeptides and combinations thereof. In some embodiments, the
antibiotic interferes with protein synthesis (e.g., glycosides,
tetracyclines and streptogramins). The present invention is not
limited by the number of doses of composition comprising
nanoemulsion administered. In some embodiments, multiple doses are
administered on separate days. In some embodiments, the multiple
doses are administered on the same day. In some embodiments, a
composition comprising a nanoemulsion described herein is
administered continuously. In some embodiments, co-administration
with a composition comprising a nanoemulsion permits administering
a lower dose of an antibacterial agent than would be administered
without co-administration of a composition comprising a
nanoemulsion. In some embodiments, the composition comprising a
nanoemulsion described herein is administered using a nebulizer. In
some embodiments, administration is intramuscularly,
subcutaneously, locally, directly into an infected site, directly
onto an indwelling prosthetic device (e.g., a shunt, stent,
scaffold for tissue construction, feeding tube, punctual plug,
artificial joint, pacemaker, artificial valve, etc.) or catheter.
In some embodiments, administration is directly through a
catheter.
[0133] The present invention is not limited by the type of microbe
treated. Indeed a variety of microbial pathogens can be treated
(e.g, killed (e.g., completely killed)) and/or the growth thereof
prevented and/or attenuated in a subject using the compositions and
methods of the present invention including, but not limited to,
bacteria, viruses, and fungi described herein.
[0134] The present invention also provides compositions and methods
for treating (e.g., killing and/or inhibiting growth of) organisms
that heretofore display resistance to a broad spectrum of
antibiotics (e.g., species of the genus Acinetobacter).
[0135] Acinetobacter species are generally considered nonpathogenic
to healthy individuals. However, several species persist in
hospital environments and cause severe, life-threatening infections
in compromised patients (See, e.g., Gerischer U (editor). (2008).
Acinetobacter Molecular Biology, 1st ed., Caister Academic Press).
The spectrum of antibiotic resistances of these organisms together
with their survival capabilities make them a threat to hospitals as
documented by recurring outbreaks both in highly developed
countries and elsewhere. Infections occur in immunocompromised
individuals, and the strain A. baumannii is the second most
commonly isolated nonfermenting bacteria in human specimens.
Acinetobacter is frequently isolated in nosocomial infections and
is especially prevalent in intensive care units, where both
sporadic cases as well as epidemic and endemic occurrence is
common. A. baumannii is a frequent cause of nosocomial pneumonia,
especially of late-onset ventilator associated pneumonia. It can
cause various other infections including skin and wound infections,
bacteremia, and meningitis. A. lwoffi is also causative of
meningitis. A. baumannii can survive on the human skin or dry
surfaces for weeks.
[0136] Since the start of the Iraq War, over 700 U.S. soldiers have
been infected or colonized by A. baumannii. Four civilians
undergoing treatment for serious illnesses at Walter Reed Army
Medical Center in Washington, D.C., contracted A. baumannii
infections and died. At Landstuhl Regional Medical Center, a U.S.
military hospital in Germany, another civilian under treatment, a
63-year-old German woman, contracted the same strain of A.
baumannii infecting troops in the facility and also died.
[0137] Acinetobacter species are innately resistant to many classes
of antibiotics, including penicillin, chloramphenicol, and often
aminoglycosides. Resistance to fluoroquinolones has been reported
during therapy and this has also resulted in increased resistance
to other drug classes mediated through active drug efflux. A
dramatic increase in antibiotic resistance in Acinetobacter strains
has been reported by the CDC and the carbapenems are recognized as
the gold-standard and/or treatment of last resort. An increase in
resistance to the carbapenems leaves very little treatment option
although there has been some success reported with polymyxin B.
Acinetobacter species are unusual in that they are sensitive to
sulbactam; sulbactam is most commonly used to inhibit bacterial
beta-lactamase, but this is an example of the antibacterial
property of sulbactam itself.
[0138] Thus, in some embodiments, compositions and methods of the
present invention are utilized to treat (e.g., kill and/or inhibit
growth of) bacteria of the Acinetobacter species (e.g.,
individually or in combination with other treatments (e.g.,
carbapenems, polymyxin B, and/or sulbactam)).
Cystic Fibrosis
[0139] Cystic fibrosis (CF) is a life-threatening disorder that
causes severe lung damage due to a defective transmembrane protein
called CFTR responsible for the balance of electrolytes. Thick
mucus forms plugging the tubes, ducts and passageways in the lungs.
This environment is ideal for opportunistic bacteria to establish
biofilm communities, leading to respiratory infections.
Systemically-administered antibiotics can decrease the frequency
and severity of exacerbations; however, the bacteria are never be
completely eradicated from the airways and the lungs. Nebulized
antibiotics are used, but resistance emergence and/or colonization
of different resistant species is a major concern. Cystic fibrosis
(CF) results in the functional impairment of innate respiratory
defense mechanisms, providing an environment for colonization of
pathogenic bacterial species such as Staphylococcus aureus and
Haemophilus influenzae, and a number of opportunistic species such
as Pseudomonas aeruginosa, Achromobacter xylosoxidans,
Stenotrophomonas maltophilia, Ralstonia spp., Pandoraea spp., and
the Burkholderia cepacia complex (Bcc) species (See, e.g., LiPuma
et al., (2009) Antimicrob Agents Chemother 53, 249-255). The Bcc
comprises a group of at least 17 phylogenetically related
saprophytic gram-negative bacilli, most of which can form biofilm
(See, e.g., Al Bakri et al., 2004, Journal of Applied Microbiology
96, 455-463; Eberl and Tummler, 2004, International Journal of
Medical Microbiology 294, 123-131; LiPuma et al., (2009) Antimicrob
Agents Chemother 53, 249-255; and Tomlin et al., 2004, Journal of
Microbiological Methods 57, 95-106). They are particularly
difficult to treat and are associated with increased rates of
morbidity and mortality in CF patients. They also are among the
most antimicrobial-resistant bacterial species encountered in human
infections (See, e.g., LiPuma et al, 2005, Curr Opin Pulm Med 11,
528-533; LiPuma et al., (2009) Antimicrob Agents Chemother 53,
249-255). Once established, the infection and associated
inflammation are rarely eliminated, resulting in progressive lung
disease ending in pulmonary failure and death (See, e.g., LiPuma et
al., (2009) Antimicrob Agents Chemother 53, 249-255; Saiman and
Seigel, 2003, Infect Control Hosp Epidemiol 24, S6-S52).
[0140] The present invention is directed to a novel broad-spectrum
antimicrobial nanoemulsion (NE) and uses thereof. The NE kills
pathogens by interacting with their membranes. This physical
kill-on-contact mechanism significantly reduces any concerns about
resistance. The NE is formulated from pharmaceutically approved
safe ingredients.
[0141] In Example 8 below, the minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC) of a against
four genera of problematic bacteria in CF patients was evaluated:
Pseudomonas, Burkholderia, Acinetobacter and Stenotrophomonas.
Example 8 also describes evaluating potential synergy between the
NE and other antimicrobials. P. aeruginosa, B. cenocepacia, A.
baumannii and S. maltophilia, which were evaluated in Example 8,
are important respiratory pathogens implicated in acute
exacerbations of cystic fibrosis patients. In one aspect of the
invention, the activity of a nanoemulsion is defined herein in
terms of its minimum inhibitory concentration (MIC), and/or minimum
bactericidal concentration (MBC), both in comparison to
traditionally used antimicrobial medicines
[0142] The results described in Example 8 show that the
MIC.sub.90/MBC.sub.90 values for the NE tested were 8/64 .mu.g/ml
for P. aeruginosa, 64/>514 .mu.g/ml for B. cenocepacia, 8/64
.mu.g/ml for A. baumannii and 8/32 .mu.g/ml for S. maltophilia.
Colistin had MIC.sub.90/MBC.sub.90 values of 2/8, >32/>32,
1/>16 and >32/>32 for P. aeruginosa, B. cenocepacia, A.
baumannii and S. maltophilia, respectively. Cefepime, imipenem,
levofloxacin and tobramycin had MIC.sub.90/MBC.sub.90 values of
.gtoreq.32/.gtoreq.32, .gtoreq.32/>32, 16/16 and >32/>32
.mu.g/ml, respectively, against all strains. These results are
significant as in contrast to conventional drugs such as colistin,
cefepime, imipenem, levofloxacin and tobramycin used to treat CF
and which can exhibit significant side effects and potential
toxicity with increasing dosage, NE are completely non-toxic.
[0143] Also evaluated in Example 8 below was synergy data.
Specifically, also evaluated was the fractional inhibitory
concentration (FIC) index when in combination with another
antimicrobial and the fractional bactericidal concentration (FBC)
index when in combination with another antimicrobial. The FIC and
the FBC are calculated to make the judgment on synergy, antagonism
or indifference of the nanoemulsion in combination. For this
determination, ten strains of Burkholderia, Stenotrophomonas and 10
strains of Acinetobacter were tested to determine a shift in MIC
when P.sub.4075EC+EDTA was in combination with either colistin or
tobramycin, two traditional antimicrobials used in the lungs of CF
patients to treat chronic lung infections. The results showed that
the NE texted (P.sub.4075EC+EDTA) in combination with colistin was
found to be synergistic for 90% (in terms of the FIC) and 70% (in
terms of the FBC) of the Stenotrophomonas strains, but indifferent,
only 20% synergy in by the FIC and 0% by the FBC, when in
combination with tobramycin. For the Acinetobacter strains,
P.sub.4075EC+EDTA in combination with colistin was found to be
indifferent, only 20% synergy in by the FIC and 0% by the FBC, as
well as when in combination with tobramycin, only 10% synergy in by
the FIC and 10% by the FBC. For the Burkholderia strains,
P.sub.4075EC+EDTA in combination with colistin was found to be
indifferent, only 30% synergy in by the FIC and 10% by the FBC, but
when in combination with tobramycin, 50% synergy in by the FIC and
20% by the FBC.
[0144] Finally, Example 8 generated a short time-kill curve to
produce samples to be used in pictures using electron microscopy
for a strain of Burkholderia to demonstrate the physical
kill-on-contact mechanism of action. Time-kill resulted in an
overall 4.44 log reduction in cfu/ml from the untreated beginning
to the 30 minute time point. Each 10 minute time point had between
1-2 log reduction as follows: from the untreated to the 10 minute
point there was a 1.40 log reduction, from the 10 to 20 minute time
points there was a 1.91 log reduction and from the 20 to 30 minute
time points there was a 1.13 log reduction. See FIGS. 2A-2D.
[0145] The data herein, such as that presented in Example 8,
demonstrates that nanoemulsions, such as the tested P.sub.4075EC,
are effective against strains that are multidrug-resistant,
including colistin-resistant isolates of Burkholderia and
Stenotrophomonas. None of the described lipid A modifications in
Pseudomonas species impacted the MIC/MBCs with P.sub.4075EC. No
evidence of antagonism with two major antibiotics, colistin and
tobramycin, was observed and in the case of the Stenotrophomonas,
synergy was evident. This is valuable because the treatment of
patients with CF should not need their normal antibiotic regime
suspended in order to use the nanoemulsion, complicating their
treatment programs. The SEM images demonstrate the kill on contact
mechanism, here in this case a Gram negative bacterium with a
reputation of having a tough outer membrane. Further studies are
ongoing to investigate the nebulization of P.sub.4075EC for the
treatment of pulmonary infection in cystic fibrosis patients.
[0146] While therapeutic progress has been made in preserving
functional pulmonary physiology in CF patients by managing
nutrition and mucosal secretions, little progress has been made in
therapeutic interventions for the prevention and management of Bcc
infections. For example, at present, no effective vaccines against
Bcc are available. Mucosal vaccine development for Bcc has been
limited in part due to the lack of an identified protective antigen
and the lack of effective mucosal adjuvants (See, e.g., Davis,
2001, Advanced Drug Delivery Reviews 51, 21-42).
[0147] As shown in Example 10 below, the present invention provides
immunogenic compositions comprising a nanoemulsion and Burkholderia
antigen (e.g., Burkholderia outer membrane protein (OMP)) that,
when administered to a subject, induces immunity (e.g., protective
immunity) in the subject against bacteria from the genus
Burkholderia (e.g., B. cenocepacia, B. multivorans or others
species associated with respiratory infection). In some
embodiments, the present invention provides all or a portion of an
OMP (e.g., isolated, purified, and/or recombinant OMPs)) from a
bacteria of the genus Burkholderia (See, e.g., Example 10, Table
25) that is utilized in an immunogenic composition comprising a
nanoemulsion (e.g., for administration to a subject (e.g., to
induce immunity to a bacteria of the genus Burkholderia in the
subject)). As shown in Example 10, the present invention provides
that when administered to a subject, an immunogenic composition
comprising a nanoemulsion and Burkholderia OMP induces both mucosal
and systemic anti-OMP antibodies. Also shown in Example 10, the
present invention provides that when administered to a subject, an
immunogenic composition comprising a nanoemulsion and an OMP from a
specific strain of Burkholderia (e.g., B. cenocepacia) induces both
mucosal and systemic anti-OMP antibodies that neutralize bacteria
of the specific species from which the OMP is derived (e.g., B.
cenocepacia) and cross-neutralize bacteria from one or more other
species of bacteria (e.g., of the genus Burkholderia).
[0148] Example 10 shows that a single nasal immunization with
OMP-NE produces a robust immune response characterized by rapid
induction of antigen-specific T cell responses and secretory
antibody responses, as documented by induction of IFN-.gamma., IL-2
and mucosal sIgA and IgG, and that the immune response can be
subsequently boosted. Mice immunized with OMP-NE displayed a
dramatic decrease in K56-1 colony forming units (cfu) in the lungs
when compared with non-immunized controls. Pulmonary bacterial
clearance was significantly enhanced in OMP-NE vaccinated mice
which showed fewer B. cenocepacia organisms in the spleens as
compared to non-vaccinated groups. Thus, the present invention
provides that mucosal OMP-NE administration produced protective
immunity and reduced the likelihood of sepsis in subjects.
[0149] Example 10 also provides data identifying a 17 KDa OmpA-like
protein and immune responses associated with exposure to the
protein. The 17 KDa lipoprotein fractions were better recognized by
antibodies contained in serum from OMP-NE vaccinated mice (See,
e.g., Example 10, FIG. 29C). Thus, the present invention provides
that immunogenic compositions comprising epitopes contained within
the 17 KDa OmpA-like proteins generate protective antibodies (e.g.,
against one or more species of Burkholderia) in a subject
administered the immunogenic composition.
[0150] Significant differences between OMP-NE and OMP in PBS
responses were observed. For example, immunization with OMP in the
absence of nanoemulsion resulted in relatively high titers of IgG,
but were consistently lower (13 to 30 fold) than those induced by
OMP-NE immunogenic compositions. Immunization with OMP in the
absence of nanoemulsion failed to respond to a booster
immunization. The response induced with OMP plus nanoemulsion was
primarily directed toward specific proteins contained within the
preparation, whereas OMP in the absence of nanoemulsion appears to
have elicited immune responses to the intrinsic endotoxin
content.
[0151] Accordingly, the present invention provides methods and
compositions for the stimulation of immune responses. In
particular, the present invention provides immunogenic nanoemulsion
compositions and methods of using the same for the induction of
immune responses (e.g., innate and/or adaptive immune responses
(e.g., for generation of host immunity against a bacterial species
of the genus Burkholderia (e.g., B. cenocepacia, B. multivorans,
etc.))). Compositions and methods of the present invention find use
in, among other things, clinical (e.g. therapeutic and preventative
medicine (e.g., vaccination)) and research applications.
[0152] In some embodiments, the present invention provides
nanoemulsion adjuvants and compositions comprising the same (e.g.,
vaccines) for the stimulation of immune responses (e.g., immunity)
against a bacterial species of the genus Burkholderia (e.g., B.
cenocepacia, B. multivorans, etc.). In some embodiments, the
present invention provides nanoemulsion adjuvant compositions that
stimulate and/or elicit immune responses (e.g., innate immune
responses and/or adaptive/acquired immune responses) when
administered to a subject (e.g., a human or other mammalian
subject)). In some embodiments, the present invention provides
nanoemulsion adjuvant compositions comprising one or a plurality of
Burkholderia (e.g., B. cenocepacia, B. multivorans, etc.) antigens
(e.g., Burkholderia components isolated and/or purified, and/or
recombinant Burkholderia proteins (e.g., OMPs)). The present
invention is not limited to any particular nanoemulsion or
Burkholderia antigen. Exemplary immunogenic compositions (e.g.,
vaccine compositions) and methods of administering the compositions
are described in more detail below.
[0153] In some embodiments, the present invention provides an
immunogenic composition comprising a nanoemulsion and one or more
Burkholderia antigens (e.g., B. cepacia antigens). In some
embodiments, the present invention provides a method of inducing an
immune response to Burkholderia (e.g., B. cepacia) in a subject
comprising: providing a subject and an immunogenic composition
comprising a nanoemulsion and an immunogen, wherein the immunogen
comprises a Burkholderia (e.g., Burkholderia cepacia) antigen and
administering the composition to the subject under conditions such
that the subject generates a Burkholderia (e.g., Burkholderia
cepacia) specific immune response. The present invention is not
limited by the route chosen for administration of a composition of
the present invention. In some preferred embodiments, administering
the immunogenic composition comprises contacting a mucosal surface
of the subject with the composition. In some embodiments, the
mucosal surface comprises nasal mucosa. In some embodiments,
inducing an immune response induces immunity to Burkholderia (e.g.,
Burkholderia cepacia) in the subject.
[0154] Experiments were conducted during development of embodiments
of the invention to determine if a composition comprising a
nanoemulsion (NE) and Burkholderia antigen could be utilized to
generate an immune response in a subject. Nasal immunization with a
whole cell Streptococcus pneumoniae antigen (WCPAg) mixed with
nanoemulsion was performed and shown to induce an IgG response in a
host subject and the ability to eradicate upper respiratory
colonization of S. pneumoniae.
[0155] In particular, as described in Example 10, the present
invention provides immunogenic compositions comprising a
nanoemulsion and Burkholderia antigen (e.g., Burkholderia outer
membrane protein (OMP)) that, when administered to a subject,
induces immunity (e.g., protective immunity) in the subject against
bacteria from the genus Burkholderia (e.g., B. cenocepacia, B.
multivorans or others species associated with respiratory
infection). Accordingly, in some embodiments, the present invention
provides that administration (e.g., nasal administration) of a
composition comprising nanoemulsion and Burkholderia antigen (e.g.,
OMP antigen (e.g., a 17 KDa protein comprising an amino acid
sequence selected from SEQ ID NOs. 1-16)) to a subject produces
immunity toward Burkholderia in the subject thereby protecting the
subject against Burkholderia infection (e.g., associated with a
respiratory infection).
[0156] The present invention is not limited by the type of bacteria
of the genus Burkholderia utilized in the immunogenic compositions
and methods of using the same of the invention. In some
embodiments, the bacteria is a pathogen. In some embodiments, the
pathogen is a Burkholderia species responsible for respiratory or
respiratory associated infection. A variety of Burkholderia species
find use in the compositions and methods of the invention
including, but not limited to, B. cenocepacia, B. dolosa, B.
multivorans, B. ambifaria, B. vietnamiensis, B. ubonensis, B.
thailandensis, B. graminis, B. oklahomensis, B. pseudomallei, B.
xenovorans, B. phytofirmans, B. phymatum, R. metallidurans, R.
eutropha, R. solanacearum.
[0157] In some embodiments, the bacteria of the genus Burkholderia
is B. cepecia. In some embodiments, an immunogenic composition
comprising a nanoemulsion and an Omp-A like protein comprises an
Omp-A like protein from a Burkholderia species including, but not
limited to, B. cenocepacia, B. dolosa, B. multivorans, B.
ambifaria, B. vietnamiensis, B. ubonensis, B. thailandensis, B.
graminis, B. oklahomensis, B. pseudomallei, B. xenovorans, B.
phytofirmans, B. phymatum, R. metallidurans, R. eutropha, R.
solanacearum. In some embodiments, an immunogenic composition
comprising a nanoemulsion and an Omp-A like protein comprises an
Omp-A like protein (e.g., isolated, purified, and/or recombinant
Omp-A like protein) comprising an amino acid sequence identified in
SEQ ID NOs.: 1-16. In some embodiments, an immunogenic composition
comprising a nanoemulsion and an Omp-A like protein comprises an
Omp-A like protein (e.g., isolated, purified, and/or recombinant
Omp-A like protein) comprising an amino acid sequence of SEQ ID NO.
1.
[0158] In some embodiments, an immunogenic composition comprising a
nanoemulsion and Burkholderia antigen comprises antigens (e.g.,
polysaccharide, protein, killed whole cells (e.g., conjugated or
non-conjugated antigens)), wherein the antigens are derived from
multiple (e.g., at least 2, 3, 5, 7, 10, 15, 20 or more) serotypes
of Burkholderia. The number of Burkholderia antigens utilized can
range from 2 different serotypes to about 20 different serotypes.
In some embodiments, an immunogenic composition comprising a
nanoemulsion and Burkholderia antigen may comprise Burkholderia
antigen (e.g., whole cell, polysaccharide, Omp-A protein, other
protein, etc.) from every known and/or isolated Burkholderia
serotype.
[0159] In some embodiments, an immunogenic composition comprising a
nanoemulsion and Burkholderia (e.g., B. cepacia) antigen comprises
one, two or more different types of carrier protein (e.g., that act
as carriers for proteins, saccharides, etc.). For example, in one
embodiment, two or more different saccharides or proteins may be
conjugated to the same carrier protein, either to the same molecule
of carrier protein or to different molecules of the same carrier
protein. Carrier proteins may be TT, DT, CRM197, fragment C of TT,
PhtD, PhtBE or PhtDE fusions (particularly those described in WO
01/98334 and WO 03/54007), detoxified pneumolysin and protein D. In
some embodiments, a carrier protein present in a composition
comprising a nanoemulsion and Burkholderia (e.g., B. cenocepacia)
antigen is a member of the polyhistidine triad family (Pht)
proteins, fragments or fusion proteins thereof. The PhtA, PhtB,
PhtD or PhtE proteins may have an amino acid sequence sharing 80%,
85%, 90%, 95%, 98%, 99% or 100% identity with a sequence disclosed
in WO 00/37105 or WO 00/39299 (e.g. with amino acid sequence 1-838
or 21-838 of SEQ ID NO: 4 of WO 00/37105 for PhtD). For example,
fusion proteins are composed of full length or fragments of 2, 3 or
4 of PhtA, PhtB, PhtD, PhtE. Examples of fusion proteins are
PhtA/B, PhtA/D, PhtA/E, PhtB/A, PhtB/D, PhtB/E. PhtD/A. PhtD/B,
PhtD/E, PhtE/A, PhtE/B and PhtE/D, wherein the proteins are linked
with the first mentioned at the N-terminus (see for example
WO01/98334). Carriers may comprise histidine triad motif(s) and/or
coiled coil regions. A histidine triad motif is the portion of
polypeptide that has the sequence HxxHxH where H is histidine and x
is an amino acid other than histidine. A coiled coil region is a
region predicted by "Coils" algorithm Lupus, A et al (1991) Science
252; 1162-1164.
[0160] Examples of carrier proteins which may be used in the
present invention are DT (Diphtheria toxoid), TT (tetanus toxoid)
or fragment C of TT, DT CRM197 (a DT mutant) other DT point
mutants, such as CRM176, CRM228, CRM 45 (Uchida et al J. Biol.
Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 and
CRM107 and other mutations described by Nicholls and Youle in
Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc,
1992; deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala
158 to Gly and other mutations disclosed in U.S. Pat. No. 4,709,017
or U.S. Pat. No. 4,950,740; mutation of at least one or more
residues Lys 516, Lys 526, Phe 530 and/or Lys 534 and other
mutations disclosed in U.S. Pat. No. 5,917,017 or U.S. Pat. No.
6,455,673; or fragment disclosed in U.S. Pat. No. 5,843,711,
pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63;
2706-13) including ply detoxified in some fashion for example
dPLY-GMBS (WO 04081515, PCT/EP2005/010258) or dPLY-formol, PhtX,
including PhtA, PhtB, PhtD, PhtE and fusions of Pht proteins for
example PhtDE fusions, PhtBE fusions (WO 01/98334 and WO 03/54007),
(Pht A-E are described in more detail below) OMPC (meningococcal
outer membrane protein--usually extracted from N. meningitidis
serogroup B--EP0372501), PorB (from N. meningitidis), PD
(Haemophilus influenzae protein D--see, e.g., EP 0 594 610 B), or
immunologically functional equivalents thereof, synthetic peptides
(EP0378881, EP0427347), heat shock proteins (WO 93/17712, WO
94/03208), pertussis proteins (WO 98/58668, EP0471177), cytokines,
lymphokines, growth factors or hormones (WO 91/01146), artificial
proteins comprising multiple human CD4+ T cell epitopes from
various pathogen derived antigens (Falugi et al (2001) Eur J
Immunol 31; 3816-3824) such as N19 protein (Baraldoi et al (2004)
Infect Immun 72; 4884-7) pneumococcal surface protein PspA (WO
02/091998), iron uptake proteins (WO 01/72337), toxin A or B of C.
difficile (WO 00/61761).
Generation of Antibodies
[0161] An immunogenic composition comprising a nanoemulsion and
Burkholderia (e.g., B. cenocepacia) antigen can be used to immunize
a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or
human, to produce polyclonal antibodies. If desired, a Burkholderia
(e.g., B. cenocepacia) antigen can be conjugated to a carrier
protein, such as bovine serum albumin, thyroglobulin, keyhole
limpet hemocyanin or other carrier described herein. Depending on
the host species, various adjuvants can be used to increase the
immunological response. Such adjuvants include, but are not limited
to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and
surface active substances (e.g. lysolecithin, pluronic polyols,
polyanions, peptides, nanoemulsions described herein, keyhole
limpet hemocyanin, and dinitrophenol). Among adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum
are especially useful.
[0162] Monoclonal antibodies that specifically bind to a
Burkholderia (e.g., B. cepacia) antigen can be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These techniques include, but
are not limited to, the hybridoma technique, the human B cell
hybridoma technique, and the EBV hybridoma technique (See, e.g.,
Kohler et al., Nature 256, 495 497, 1985; Kozbor et al., J.
Immunol. Methods 81, 3142, 1985; Cote et al., Proc. Natl. Acad.
Sci. 80, 2026 2030, 1983; Cole et al., Mol. Cell. Biol. 62, 109
120, 1984).
[0163] In addition, techniques developed for the production of
"chimeric antibodies," the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity, can be used (See, e.g.,
Morrison et al., Proc. Natl. Acad. Sci. 81, 68516855, 1984;
Neuberger et al., Nature 312, 604 608, 1984; Takeda et al., Nature
314, 452 454, 1985). Monoclonal and other antibodies also can be
"humanized" to prevent a patient from mounting an immune response
against the antibody when it is used therapeutically. Such
antibodies may be sufficiently similar in sequence to human
antibodies to be used directly in therapy or may require alteration
of a few key residues. Sequence differences between rodent
antibodies and human sequences can be minimized by replacing
residues which differ from those in the human sequences by site
directed mutagenesis of individual residues or by grating of entire
complementarity determining regions.
[0164] Alternatively, humanized antibodies can be produced using
recombinant methods, as described below. Antibodies which
specifically bind to a particular antigen can contain antigen
binding sites which are either partially or fully humanized, as
disclosed in U.S. Pat. No. 5,565,332.
[0165] Alternatively, techniques described for the production of
single chain antibodies can be adapted using methods known in the
art to produce single chain antibodies which specifically bind to a
particular antigen. Antibodies with related specificity, but of
distinct idiotypic composition, can be generated by chain shuffling
from random combinatorial immunoglobin libraries (See, e.g.,
Burton, Proc. Natl. Acad. Sci. 88, 11120 23, 1991).
[0166] Single-chain antibodies also can be constructed using a DNA
amplification method, such as PCR, using hybridoma cDNA as a
template (See, e.g., Thirion et al., 1996, Eur. J. Cancer Prey. 5,
507-11). Single-chain antibodies can be mono- or bispecific, and
can be bivalent or tetravalent. Construction of tetravalent,
bispecific single-chain antibodies is taught, for example, in
Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63.
Construction of bivalent, bispecific single-chain antibodies is
taught, for example, in Mallender & Voss, 1994, J. Biol. Chem.
269, 199-206.
[0167] A nucleotide sequence encoding a single-chain antibody can
be constructed using manual or automated nucleotide synthesis,
cloned into an expression construct using standard recombinant DNA
methods, and introduced into a cell to express the coding sequence,
as described below. Alternatively, single-chain antibodies can be
produced directly using, for example, filamentous phage technology
(See, e.g., Verhaar et al., 1995, Int. J. Cancer 61, 497-501;
Nicholls et al., 1993, J. Immunol. Meth. 165, 81-91).
[0168] Antibodies which specifically bind to a particular antigen
also can be produced by inducing in vivo production in the
lymphocyte population or by screening immunoglobulin libraries or
panels of highly specific binding reagents as disclosed in the
literature (See, e.g., Orlandi et al., Proc. Natl. Acad. Sci. 86,
3833 3837, 1989; Winter et al., Nature 349, 293 299, 1991).
[0169] Chimeric antibodies can be constructed as disclosed in WO
93/03151. Binding proteins which are derived from immunoglobulins
and which are multivalent and multispecific, such as the
"diabodies" described in WO 94/13804, also can be prepared.
Antibodies can be purified by methods well known in the art. For
example, antibodies can be affinity purified by passage over a
column to which the relevant antigen is bound. The bound antibodies
can then be eluted from the column using a buffer with a high salt
concentration.
Burn Wound Management
[0170] Contemporary burn wound management involves early
debridement and reconstruction of non-viable skin coupled with
provision of supportive care and topical antimicrobial dressing
changes to partial thickness burn wounds. The goal of modern burn
wound care is to provide an optimal environment for epidermal
renewal. Restoration of skin integrity takes place via regrowth of
keratinocytes from preserved hair follicles or transfer of split
thickness skin grafts harvested from non-burn regions. During the
period of epidermal renewal it is important to avoid further injury
to the skin, abrogate burn wound progression, and minimize
secondary complications such as wound infection. Early excision of
full-thickness burn eschar, immediate skin grafting, and treatment
of remaining open or partial thickness areas of burn wound with
topical antimicrobial agents is the most effective way of
minimizing burn wound colonization and invasive wound infection.
(See, e.g., Bessey, Wound care. In Herndon D N, ed: Total Burn Care
3.sup.rd edition. Philadelphia, Pa.: Elsevier Inc., 2007, pp
127-135.). Popular topical antimicrobial agents include silver
sulfadiazine (SILVADENE), mafenide acetate (SULFAMYLON), and
colloidal silver impregnated dressings (ACTICOAT, SILVERLON). Each
of these agents has potential limitations such as variable ability
to penetrate eschar, uneven efficacy against both Gram-negative and
Gram-positive bacteria, and potential toxicity to host immune cells
(See, e.g., Steinstraesser et al., Antimicrob Agents Chemother
46(6):1837-1844, 2002).
[0171] Accordingly, the present invention provides nanoemulsion
compositions and methods of using the same for the treatment of
burn wounds. For example, as shown in Example 9, the present
invention provides nanoemulsion compositions and methods of using
the same to reduce, attenuate and/or prevent bacterial growth in a
burn wound. The present invention also provides nanoemulsion
compositions that reduce wound inflammation following burn injury.
Although an understanding of a mechanism of action is not needed to
practice the present invention, and the present invention is not
limited to any particular mechanism of action, in some embodiments,
a nanoemulsion composition that is applied to a wound following
burn injury is able to penetrate more deeply and uniformly into a
burn wound (e.g., thereby decreasing and/or inhibiting bacterial
growth and/or inflammation at the site of the wound). As shown in
Example 9, the present invention provides a method of reducing,
inhibiting and/or eliminating bacterial growth in a burn wound
comprising providing a burn wound and a nanoemulsion and
administering the nanoemulsion to the burn wound under conditions
that bacterial growth is reduced, inhibited and/or eliminated. In
some embodiments, a nanoemulsion composition described herein is
combined with one or more antimicrobial drugs for administration to
a burn wound to minimize bacterial growth at the burn wound site.
The present invention is not limited to any particular
antimicrobial drug. Indeed, any antimicrobial drug that inhibits
bacterial growth known to those in the art can be utilized in
combination with a nanoemulsion composition described herein.
[0172] In addition to local effects, severe dermal burns are known
to induce the systemic inflammatory response syndrome (SIRS), which
results in a high-risk of end-organ dysfunction (See, e.g., Barton
et al., J Burn Care Rehabil 18(1):1-9, 1997). Increased vascular
permeability and systemic capillary leak as a consequence of SIRS
following burn injury creates seepage of plasma into interstitial
tissue throughout the body. This tissue edema and intravascular
hypovolemia is responsible for a host of undesired clinical
problems such as shock, pulmonary dysfunction, abdominal or
extremity compartment syndrome, and cardiac failure.
[0173] As shown in Example 9 below, nanoemulsion compositions
described herein can be administered to a burn wound to treat
(e.g., reduce, attenuate and/or prevent) inflammation, tissue edema
and/or intravascular hypovolemia at the site of a burn wound. In
some embodiments, reducing inflammation, tissue edema and/or
intravascular hypovolemia at the site of a burn wound reduces the
occurrence of shock, pulmonary dysfunction, abdominal or extremity
compartment syndrome, and/or cardiac failure. In some embodiments,
a nanoemulsion composition described herein is used in combination
with (e.g., is co-administered with) one or more anti-inflammatory
drugs to minimize early burn wound inflammation and tissue edema.
The present invention is not limited to any particular
anti-inflammatory drug. Indeed, any anti-inflammatory drug that
minimizes early burn wound inflammation and tissue edema can be
utilized in combination with a nanoemulsion composition described
herein.
[0174] In some embodiments, a nanoemulsion of the invention (e.g.,
W.sub.205GBA.sub.2ED) is administered to a burn wound to prevent,
attenuate and/or eradicate bacterial growth (e.g., Staphylococcus
aureus, P. aeruginosa, or other bacteria) within a partial
thickness burn wound. Example 9 shows that reduction in microbial
infection was coupled with generation of lower levels of local
dermal pro-inflammatory cytokines and evidence of reduced
neutrophil sequestration into the burn wound. This decrease in burn
wound bacterial growth and inflammation also produced less
capillary leak in the early post-thermal injury time-period. Having
the ability to clinically reduce capillary leak and tissue edema in
the immediate post-burn time-period provides a lesser need for
large volume crystalloid fluid resuscitation and a reduction in the
associated sequela of physiologic volume overload, pulmonary
dysfunction, and abdominal compartment syndrome.
[0175] Skin that is damaged by thermal injury loses its ability to
protect the host against infection from both the loss of physical
barrier function and the secondary immunosuppression caused by the
thermal injury. Moreover, increased production of TGF-.beta. and
IL-10 during the post-burn period can result in immunosuppression.
(See, e.g., Lyons et al., Arch Surg 134(12):1317-1323, 1999; Varedi
et al., Shock 16(5):380-382, 2001). It has been established that
treatment of burn injured animals with anti-TGF-.beta. can improve
local and systemic clearance of P. aeruginosa (See, e.g., Huang et
al., J Burn Care Res 27(5):682-687, 2006). Inhibition of TGF-.beta.
also results in increased survival following bacterial challenge.
As shown in Example 9, a significant elevation of TGF-(3, but not
IL-10 was observed in the skin following partial thickness burn
injury. However, topical nanoemulsion application (e.g., 10%
W.sub.205GBA.sub.2ED) to the burn wound inoculated with bacteria
resulted in a reduction of the level of TGF-.beta. when compared to
the untreated burn wound.
[0176] Onset of a bacterial infection within a burn wound can delay
or even reverse the tissue healing process (See, e.g.,
Steinstraesser et al., Crit. Care Med 29(7):1431-1437, 2001).
Topical antimicrobial therapy is used to reduce the microbial load
in the burn wound and reduce this risk of infection. Current
topical agents include silver nitrate (AgNO.sub.3), silver
sulfadiazine, mafenide acetate, and nanocrystalline impregnated
silver dressings. Silver nitrate is limited in its use because of
the problem it creates from contact staining and its limited
antifungal activity. Silver sulfadiazine is the mainstay of topical
burn antimicrobial treatment. It is bactericidal against P.
aeruginosa and other Gram-negative enteric bacteria. Resistance to
Silvadene by some of these organisms has emerged (See, e.g., Silver
et al., J Ind Microbiol Biotechnol 33(7):627-634, 2006). The agent
has limited antifungal activity, but can be used in conjunction
with nystatin. Silvadene has no real ability to penetrate burn
eschar and sometimes leads to leukopenia which requires conversion
to another topical agent. The use of mafenide acetate is narrowed
by the fact that it is bacteriostatic against select organisms, it
has limited activity against Gram-positive bacteria such as
Staphylococcus aureus, and that its use over a large surface area
can lead to a metabolic acidosis because of its metabolism into a
carbonic anhydrase inhibitor. The nanocrystalline silver dressings
have the broadest activity against burn wound pathogens of the
current agents available. They have a modest ability to penetrate
eschar and can be left in place for many days (See, e.g., Church et
al., Clin Microbiol Rev 19(2):403-434, 2006). SB 202190, an
inhibitor of activated p38 MAPK, can substantially reduce the
dermal inflammation generated in burn wounds (See, e.g., Arbabi et
al., Shock. 26(2):201-209, 2006). Thermal injury initiates dermal
inflammatory and pro-apoptotic cell signaling.
[0177] As shown in Example 9, topical application of nanoemulsion
(e.g., W.sub.205GBA.sub.2ED resulted in reduced hair follicle cell
apoptosis within the dermis of burned skin. Thus, in some
embodiments, the present invention provides nanoemulsion
compositions that can be utilized to reduce, when administered to a
burn wound, conversion of the partial thickness burn wound within
the "zone of stasis" to regions of full thickness burn.
[0178] In patients without evidence of inhalational injury, the
burn wound itself is the primary source triggering the systemic
inflammatory response via generation of pro-inflammatory cytokines
and sequestration of neutrophils into the burn wound (See, e.g.,
Hansbrough et al., J Surg Res 61(1):17-22, 1996; Piccolo et al.,
Inflammation 23(4):371-385, 1999; Till et al., J Clin Invest
69(5):1126-1135, 1982). Topical application of a p38 MAPK inhibitor
can control the source of inflammation at the level of the dermis,
resulting in lower levels of pro-inflammatory mediators, reduced
neutrophil sequestration and microvascular damage, and less
epithelial apoptosis in burn wound hair follicle cells (See, e.g.,
Ipaktchi et al., Shock. 26(2):201-209, 2006). Dermal source control
of inflammation also reduces bacterial growth and attenuates the
systemic inflammatory response resulting in less acute lung injury
and cardiac dysfunction following partial thickness burn injury in
a rodent model. Accordingly, in some embodiments, a nanoemulsion of
the invention is utilized (e.g., administered) alone or in
combination with an anti-inflammatory and/or antimicrobial agent to
reduce local dermal inflammation and risk of infection within burn
wounds (e.g., early burn wounds, partial thickness wounds, full
thickness wounds or other burn wounds). The present invention is
not limited by the type of anti-inflammatory agent and/or
antimicrobial utilized for co-administration with a nanoemulsion
described herein. Indeed, a variety of anti-inflammatory agents
and/or antimicrobial agents can be used including, but not limited
to, silver nitrate (AgNO.sub.3), silver sulfadiazine, mafenide
acetate, nanocrystalline impregnated silver dressings, p38 MAPK
inhibitor (e.g., SB 202190), or another anti-inflammatory or
antimicrobial agent described herein.
[0179] In some embodiments, when a nanoemulsion of the invention is
administered to a burn wound, the nanoemulsion can be administered
(e.g., to a subject (e.g., to a burn or wound surface)) by multiple
methods, including, but not limited to, direct use or being
suspended in a solution (e.g., colloidal solution) and applied to a
surface (e.g., a surface comprising bacteria (e.g., pathogenic
bacteria) or susceptible to bacterial invasion); being sprayed onto
a surface using a spray applicator; being mixed with fibrin glue
and applied (e.g., sprayed) onto a surface (e.g., skin burn or
wound); being impregnated onto a wound dressing or bandage and
applying the bandage to a surface (e.g., an infection or burn
wound); being applied by a controlled-release mechanism; or being
impregnated on one or both sides of an acellular biological matrix
that is then placed on a surface (e.g., skin burn or wound) thereby
protecting at both the wound and graft interfaces. In some
embodiments, the invention provides a pharmaceutical composition
containing (a) a composition comprising a nanoemulsion (e.g.,
W.sub.205GBA.sub.2ED); and (b) one or more other agents (e.g., an
antibiotic). Examples of other types of antibiotics include, but
are not limited to, almecillin, amdinocillin, amikacin,
amoxicillin, amphomycin, amphotericin B, ampicillin, azacitidine,
azaserine, azithromycin, azlocillin, aztreonam, bacampicillin,
bacitracin, benzyl penicilloyl-polylysine, bleomycin, candicidin,
capreomycin, carbenicillin, cefaclor, cefadroxil, cefamandole,
cefazoline, cefdinir, cefepime, cefixime, cefinenoxime,
cefinetazole, cefodizime, cefonicid, cefoperazone, ceforanide,
cefotaxime, cefotetan, cefotiam, cefoxitin, cefpiramide,
cefpodoxime, cefprozil, cefsulodin, ceftazidime, ceftibuten,
ceftizoxime, ceftriaxone, cefuroxime, cephacetrile, cephalexin,
cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine,
chloramphenicol, chlortetracycline, cilastatin, cinnamycin,
ciprofloxacin, clarithromycin, clavulanic acid, clindamycin,
clioquinol, cloxacillin, colistimethate, colistin, cyclacillin,
cycloserine, cyclosporine, cyclo-(Leu-Pro), dactinomycin,
dalbavancin, dalfopristin, daptomycin, daunorubicin,
demeclocycline, detorubicin, dicloxacillin, dihydrostreptomycin,
dirithromycin, doxorubicin, doxycycline, epirubicin, erythromycin,
eveminomycin, floxacillin, fosfomycin, fusidic acid, gemifloxacin,
gentamycin, gramicidin, griseofulvin, hetacillin, idarubicin,
imipenem, iseganan, ivermectin, kanamycin, laspartomycin,
linezolid, linocomycin, loracarbef, magainin, meclocycline,
meropenem, methacycline, methicillin, mezlocillin, minocycline,
mitomycin, moenomycin, moxalactam, moxifloxacin, mycophenolic acid,
nafcillin, natamycin, neomycin, netilmicin, niphimycin,
nitrofurantoin, novobiocin, oleandomycin, oritavancin, oxacillin,
oxytetracycline, paromomycin, penicillamine, penicillin G,
penicillin V, phenethicillin, piperacillin, plicamycin, polymyxin
B, pristinamycin, quinupristin, rifabutin, rifampin, rifamycin,
rolitetracycline, sisomicin, spectrinomycin, streptomycin,
streptozocin, sulbactam, sultamicillin, tacrolimus, tazobactam,
teicoplanin, telithromycin, tetracycline, ticarcillin, tigecycline,
tobramycin, troleandomycin, tunicamycin, tyrthricin, vancomycin,
vidarabine, viomycin, virginiamycin, BMS-284,756, L-749,345,
ER-35,786, S-4661, L-786,392, MC-02479, Pep5, RP 59500, and
TD-6424. In some embodiments, two or more combined agents (e.g., a
composition comprising a nanoemulsion and an antibiotic) may be
used together or sequentially. In some embodiments, an antibiotic
may comprise bacteriocins, type A lantibiotics, type B
lantibiotics, liposidomycins, mureidomycins, alanoylcholines,
quinolines, eveminomycins, glycylcyclines, carbapenems,
cephalosporins, streptogramins, oxazolidonones, tetracyclines,
cyclothialidines, bioxalomycins, cationic peptides, and/or
protegrins. In some embodiments, the composition comprises
lysostaphin.
[0180] The present invention is not limited by the type of
nanoemulsion utilized (e.g., for respiratory administration,
administration to a burn wound and/or for use in an immunogenic
composition for induction of protective immune responses). Indeed,
a variety of nanoemulsion compositions are contemplated to be
useful in the present invention.
[0181] For example, in some embodiments, a nanoemulsion comprises
(i) an aqueous phase; (ii) an oil phase; and at least one
additional compound. In some embodiments of the present invention,
these additional compounds are admixed into either the aqueous or
oil phases of the composition. In other embodiments, these
additional compounds are admixed into a composition of previously
emulsified oil and aqueous phases. In certain of these embodiments,
one or more additional compounds are admixed into an existing
emulsion composition immediately prior to its use. In other
embodiments, one or more additional compounds are admixed into an
existing emulsion composition prior to the compositions immediate
use.
[0182] Additional compounds suitable for use in a nanoemulsion of
the present invention include, but are not limited to, one or more
organic, and more particularly, organic phosphate based solvents,
surfactants and detergents, cationic halogen containing compounds,
germination enhancers, interaction enhancers, food additives (e.g.,
flavorings, sweeteners, bulking agents, and the like) and
pharmaceutically acceptable compounds. Certain exemplary
embodiments of the various compounds contemplated for use in the
compositions of the present invention are presented below. Unless
described otherwise, nanoemulsions are described in undiluted
form.
Stability on Storage and after Application of the Nanoemulsions of
the Invention
Storage Stability
[0183] The nanoemulsions of the invention can be stable at about
40.degree. C. and about 75% relative humidity for a time period of
at least up to about 1 month, at least up to about 3 months, at
least up to about 6 months, at least up to about 12 months, at
least up to about 18 months, at least up to about 2 years, at least
up to about 2.5 years, or at least up to about 3 years.
[0184] In another embodiment of the invention, the nanoemulsions of
the invention can be stable at about 25.degree. C. and about 60%
relative humidity for a time period of at least up to about 1
month, at least up to about 3 months, at least up to about 6
months, at least up to about 12 months, at least up to about 18
months, at least up to about 2 years, at least up to about 2.5
years, or at least up to about 3 years, at least up to about 3.5
years, at least up to about 4 years, at least up to about 4.5
years, or at least up to about 5 years.
[0185] Further, the nanoemulsions of the invention can be stable at
about 4.degree. C. for a time period of at least up to about 1
month, at least up to about 3 months, at least up to about 6
months, at least up to about 12 months, at least up to about 18
months, at least up to about 2 years, at least up to about 2.5
years, at least up to about 3 years, at least up to about 3.5
years, at least up to about 4 years, at least up to about 4.5
years, at least up to about 5 years, at least up to about 5.5
years, at least up to about 6 years, at least up to about 6.5
years, or at least up to about 7 years.
Stability Upon Application
[0186] The nanoemulsions of the invention are stable upon
application, as surprisingly the nanoemulsions do not lose their
physical structure upon application. Microscopic examination of
skin surface following application of a nanoemulsion according to
the invention demonstrates the physical integrity of the
nanoemulsions of the invention. This physical integrity may result
in the desired absorption observed with the nanoemulsions of the
invention.
Nanoemulsions
[0187] The term "nanoemulsion", as defined herein, refers to a
dispersion or droplet or any other lipid structure. Typical lipid
structures contemplated in the invention include, but are not
limited to, unilamellar, paucilamellar and multilamellar lipid
vesicles, micelles and lamellar phases.
[0188] The nanoemulsion of the present invention comprises droplets
having an average diameter size of less than about 1,000 nm, less
than about 950 nm, less than about 900 nm, less than about 850 nm,
less than about 800 nm, less than about 750 nm, less than about 700
nm, less than about 650 nm, less than about 600 nm, less than about
550 nm, less than about 500 nm, less than about 450 nm, less than
about 400 nm, less than about 350 nm, less than about 300 nm, less
than about 250 nm, less than about 200 nm, less than about 150 nm,
or any combination thereof. In one embodiment, the droplets have an
average diameter size greater than about 125 nm and less than or
equal to about 300 nm. In a different embodiment, the droplets have
an average diameter size greater than about 50 nm or greater than
about 70 nm, and less than or equal to about 125 nm. In other
embodiments of the invention, the nanoemulsion droplets have an
average diameter of from about 300 nm to about 600 nm; or the
nanoemulsion droplets have an average diameter of from about 150 nm
to about 400 nm.
[0189] In one embodiment of the invention, the nanoemulsion has a
narrow range of MIC (minimum inhibitory concentration) and MBC
(minimum bactericidal concentrations) values. In another
embodiment, the MIC and MBC for the nanoemulsion differ by less
than or equal to four-fold, meaning that the nanoemulsion is
bactericidal. In addition, the MIC and MBC for the nanoemulsion may
differ by greater than four-fold, meaning that the nanoemulsion is
bacteriostatic.
[0190] In one embodiment of the invention, the nanoemulsion
comprises: (a) an aqueous phase; (b) about 1% oil to about 80% oil;
(c) about 0.1% organic solvent to about 50% organic solvent; (d)
about 0.001% surfactant or detergent to about 10% surfactant or
detergent; (e) about 0.0005% to about 1.0% of a chelating agent; or
(e) any combination thereof. In another embodiment of the
invention, the nanoemulsion comprises: (a) about 10% oil to about
80% oil; (b) about 1% organic solvent to about 50% organic solvent;
(c) at least one non-ionic surfactant present in an amount of about
0.1% to about 10%; (d) at least one cationic agent present in an
amount of about 0.01% to about 3%; or any combination thereof.
[0191] In another embodiment, the nanoemulsion comprises a cationic
surfactant which is either cetylpyridinium chloride (CPC) or
benzalkonium chloride, or alkyl dimethyl benzyl ammonium chloride
(BTC 824), or combination thereof. The cationic surfactant may have
a concentration in the nanoemulsion of less than about 5.0% and
greater than about 0.001%, or further, may have a concentration of
less than about 5%, less than about 4.5%, less than about 4.0%,
less than about 3.5%, less than about 3.0%, less than about 2.5%,
less than about 2.0%, less than about 1.5%, less than about 1.0%,
less than about 0.90%, less than about 0.80%, less than about
0.70%, less than about 0.60%, less than about 0.50%, less than
about 0.40%, less than about 0.30%, less than about 0.20%, less
than about 0.10%, greater than about 0.001%, greater than about
0.002%, greater than about 0.003%, greater than about 0.004%,
greater than about 0.005%, greater than about 0.006%, greater than
about 0.007%, greater than about 0.008%, greater than about 0.009%,
and greater than about 0.010%.
[0192] In a further embodiment, the nanoemulsion comprises a
non-ionic surfactant, and may have a concentration of about 0.01%
to about 10.0%, or about 0.1% to about 3% of a non-ionic
surfactant, such as a polysorbate.
[0193] In yet other embodiments of the invention, the nanoemulsion:
(a) comprises at least one cationic surfactant; (b) comprises a
cationic surfactant which is either cetylpyridinium chloride or
benzalkonium chloride, or alkyl dimethyl benzyl ammonium chloride
(BTC 824), or combination thereof; (c) comprises a cationic
surfactant, and wherein the concentration of the cationic
surfactant is less than about 5.0% and greater than about 0.001%;
(d) comprises a cationic surfactant, and wherein the concentration
of the cationic surfactant is selected from the group consisting of
less than about 5%, less than about 4.5%, less than about 4.0%,
less than about 3.5%, less than about 3.0%, less than about 2.5%,
less than about 2.0%, less than about 1.5%, less than about 1.0%,
less than about 0.90%, less than about 0.80%, less than about
0.70%, less than about 0.60%, less than about 0.50%, less than
about 0.40%, less than about 0.30%, less than about 0.20%, less
than about 0.10%, greater than about 0.001%, greater than about
0.002%, greater than about 0.003%, greater than about 0.004%,
greater than about 0.005%, greater than about 0.006%, greater than
about 0.007%, greater than about 0.008%, greater than about 0.009%,
and greater than about 0.010%; or (e) any combination thereof. In
yet other embodiments, (a) the nanoemulsion comprises at least one
cationic surfactant and at least one non-cationic surfactant; (b)
the nanoemulsion comprises at least one cationic surfactant and at
least one non-cationic surfactant, wherein the non-cationic
surfactant is a nonionic surfactant; (c) the nanoemulsion comprises
at least one cationic surfactant and at least one non-cationic
surfactant, wherein the non-cationic surfactant is a polysorbate
nonionic surfactant; (d) the nanoemulsion comprises at least one
cationic surfactant and at least one non-cationic surfactant,
wherein the non-cationic surfactant is a nonionic surfactant, and
the non-ionic surfactant is present in a concentration of about
0.05% to about 10%, about 0.05% to about 7.0%, about 0.1% to about
7%, or about 0.5% to about 5%; (e) the nanoemulsion comprises at
least one cationic surfactant and at least one a nonionic
surfactant, wherein the cationic surfactant is present in a
concentration of about 0.05% to about 2% or about 0.01% to about
2%; or (f) any combination thereof.
[0194] In other embodiments, the nanoemulsion comprises: (a) water;
(b) ethanol or glycerol (glycerine), or a combination thereof; (c)
either cetylpyridinium chloride (CPC), or benzalkonium chloride, or
alkyl dimethyl benzyl ammonium chloride (BTC 824), or a combination
thereof; (c) soybean oil; and (e) Poloxamer 407, Tween 80, or Tween
20. The nanoemulsion can further comprise EDTA.
[0195] These quantities of each component present in the
nanoemulsion refer to a therapeutic nanoemulsion, and not to a
nanoemulsion to be tested in vitro. This is significant, as
nanoemulsions tested in vitro generally have lower concentrations
of oil, organic solvent, surfactant or detergent, and (if present)
chelating agent than that present in a nanoemulsion intended for
therapeutic use, e.g., topical use. This is because in vitro
studies do not require the nanoemulsion droplets to traverse the
skin. For topical, aerosol, intradermal etc. use, the
concentrations of the components must be higher to result in a
therapeutic nanoemulsion. However, the relative quantities of each
component used in a nanoemulsion tested in vitro are applicable to
a nanoemulsion to be used therapeutically and, therefore, in vitro
quantities can be scaled up to prepare a therapeutic composition,
and in vitro data is predictive of topical application success.
[0196] 1. Aqueous Phase
[0197] The aqueous phase can comprise any type of aqueous phase
including, but not limited to, water (e.g., H.sub.2O, distilled
water, tap water) and solutions (e.g., phosphate buffered saline
(PBS) solution). In certain embodiments, the aqueous phase
comprises water at a pH of about 4 to 10, preferably about 6 to 8.
The water can be deionized (hereinafter "DiH.sub.2O"). In some
embodiments the aqueous phase comprises phosphate buffered saline
(PBS). The aqueous phase may further be sterile and pyrogen
free.
[0198] 2. Organic Solvents
[0199] Organic solvents in the nanoemulsions of the invention
include, but are not limited to, C.sub.1-C.sub.12 alcohol, diol,
triol, dialkyl phosphate, tri-alkyl phosphate, such as tri-n-butyl
phosphate, semi-synthetic derivatives thereof, and combinations
thereof. In one aspect of the invention, the organic solvent is an
alcohol chosen from a nonpolar solvent, a polar solvent, a protic
solvent, or an aprotic solvent.
[0200] Suitable organic solvents for the nanoemulsion include, but
are not limited to, ethanol, methanol, isopropyl alcohol, glycerol,
medium chain triglycerides, diethyl ether, ethyl acetate, acetone,
dimethyl sulfoxide (DMSO), acetic acid, n-butanol, butylene glycol,
perfumers alcohols, isopropanol, n-propanol, formic acid, propylene
glycols, glycerol, sorbitol, industrial methylated spirit,
triacetin, hexane, benzene, toluene, diethyl ether, chloroform,
1,4-dixoane, tetrahydrofuran, dichloromethane, acetone,
acetonitrile, dimethylformamide, dimethyl sulfoxide, formic acid,
semi-synthetic derivatives thereof, and any combination
thereof.
[0201] 3. Oil Phase
[0202] The oil in the nanoemulsion of the invention can be any
cosmetically or pharmaceutically acceptable oil. The oil can be
volatile or non-volatile, and may be chosen from animal oil,
vegetable oil, natural oil, synthetic oil, hydrocarbon oils,
silicone oils, semi-synthetic derivatives thereof, and combinations
thereof.
[0203] Suitable oils include, but are not limited to, mineral oil,
squalene oil, flavor oils, silicon oil, essential oils, water
insoluble vitamins, Isopropyl stearate, Butyl stearate, Octyl
palmitate, Cetyl palmitate, Tridecyl behenate, Diisopropyl adipate,
Dioctyl sebacate, Menthyl anthranhilate, Cetyl octanoate, Octyl
salicylate, Isopropyl myristate, neopentyl glycol dicarpate cetols,
Ceraphyls.RTM., Decyl oleate, diisopropyl adipate, C.sub.12-15
alkyl lactates, Cetyl lactate, Lauryl lactate, Isostearyl
neopentanoate, Myristyl lactate, Isocetyl stearoyl stearate,
Octyldodecyl stearoyl stearate, Hydrocarbon oils, Isoparaffin,
Fluid paraffins, Isododecane, Petrolatum, Argan oil, Canola oil,
Chile oil, Coconut oil, corn oil, Cottonseed oil, Flaxseed oil,
Grape seed oil, Mustard oil, Olive oil, Palm oil, Palm kernel oil,
Peanut oil, Pine seed oil, Poppy seed oil, Pumpkin seed oil, Rice
bran oil, Safflower oil, Tea oil, Truffle oil, Vegetable oil,
Apricot (kernel) oil, Jojoba oil (simmondsia chinensis seed oil),
Grapeseed oil, Macadamia oil, Wheat germ oil, Almond oil, Rapeseed
oil, Gourd oil, Soybean oil, Sesame oil, Hazelnut oil, Maize oil,
Sunflower oil, Hemp oil, Bois oil, Kuki nut oil, Avocado oil,
Walnut oil, Fish oil, berry oil, allspice oil, juniper oil, seed
oil, almond seed oil, anise seed oil, celery seed oil, cumin seed
oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil,
cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemon
grass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli
leaf oil, peppermint leaf oil, pine needle oil, rosemary leaf oil,
spearmint leaf oil, tea tree leaf oil, thyme leaf oil, wintergreen
leaf oil, flower oil, chamomile oil, clary sage oil, clove oil,
geranium flower oil, hyssop flower oil, jasmine flower oil,
lavender flower oil, manuka flower oil, Marhoram flower oil, orange
flower oil, rose flower oil, ylang-ylang flower oil, Bark oil,
cassia Bark oil, cinnamon bark oil, sassafras Bark oil, Wood oil,
camphor wood oil, cedar wood oil, rosewood oil, sandalwood oil),
rhizome (ginger) wood oil, resin oil, frankincense oil, myrrh oil,
peel oil, bergamot peel oil, grapefruit peel oil, lemon peel oil,
lime peel oil, orange peel oil, tangerine peel oil, root oil,
valerian oil, Oleic acid, Linoleic acid, Oleyl alcohol, Isostearyl
alcohol, semi-synthetic derivatives thereof, and any combinations
thereof.
[0204] The oil may further comprise a silicone component, such as a
volatile silicone component, which can be the sole oil in the
silicone component or can be combined with other silicone and
non-silicone, volatile and non-volatile oils. Suitable silicone
components include, but are not limited to,
methylphenylpolysiloxane, simethicone, dimethicone,
phenyltrimethicone (or an organomodified version thereof),
alkylated derivatives of polymeric silicones, cetyl dimethicone,
lauryl trimethicone, hydroxylated derivatives of polymeric
silicones, such as dimethiconol, volatile silicone oils, cyclic and
linear silicones, cyclomethicone, derivatives of cyclomethicone,
hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane,
decamethylcyclopentasiloxane, volatile linear
dimethylpolysiloxanes, isohexadecane, isoeicosane, isotetracosane,
polyisobutene, isooctane, isododecane, semi-synthetic derivatives
thereof, and combinations thereof.
[0205] The volatile oil can be the organic solvent, or the volatile
oil can be present in addition to an organic solvent. Suitable
volatile oils include, but are not limited to, a terpene,
monoterpene, sesquiterpene, carminative, azulene, menthol, camphor,
thujone, thymol, nerol, linalool, limonene, geraniol, perillyl
alcohol, nerolidol, farnesol, ylangene, bisabolol, farnesene,
ascaridole, chenopodium oil, citronellal, citral, citronellol,
chamazulene, yarrow, guaiazulene, chamomile, semi-synthetic
derivatives, or combinations thereof.
[0206] In one aspect of the invention, the volatile oil in the
silicone component is different than the oil in the oil phase.
[0207] 4. Surfactants/Detergents
[0208] The surfactant or detergent in the nanoemulsion of the
invention can be a pharmaceutically acceptable ionic surfactant, a
pharmaceutically acceptable nonionic surfactant, a pharmaceutically
acceptable cationic surfactant, a pharmaceutically acceptable
anionic surfactant, or a pharmaceutically acceptable zwitterionic
surfactant.
[0209] Exemplary useful surfactants are described in Applied
Surfactants: Principles and Applications. Tharwat F. Tadros,
Copyright 8 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 3-527-30629-3), which is specifically incorporated by
reference.
[0210] Further, the surfactant can be a pharmaceutically acceptable
ionic polymeric surfactant, a pharmaceutically acceptable nonionic
polymeric surfactant, a pharmaceutically acceptable cationic
polymeric surfactant, a pharmaceutically acceptable anionic
polymeric surfactant, or a pharmaceutically acceptable zwitterionic
polymeric surfactant. Examples of polymeric surfactants include,
but are not limited to, a graft copolymer of a poly(methyl
methacrylate) backbone with multiple (at least one) polyethylene
oxide (PEO) side chain, polyhydroxystearic acid, an alkoxylated
alkyl phenol formaldehyde condensate, a polyalkylene glycol
modified polyester with fatty acid hydrophobes, a polyester,
semi-synthetic derivatives thereof, or combinations thereof.
[0211] Surface active agents or surfactants, are amphipathic
molecules that consist of a non-polar hydrophobic portion, usually
a straight or branched hydrocarbon or fluorocarbon chain containing
8-18 carbon atoms, attached to a polar or ionic hydrophilic
portion. The hydrophilic portion can be nonionic, ionic or
zwitterionic. The hydrocarbon chain interacts weakly with the water
molecules in an aqueous environment, whereas the polar or ionic
head group interacts strongly with water molecules via dipole or
iondipole interactions. Based on the nature of the hydrophilic
group, surfactants are classified into anionic, cationic,
zwitterionic, nonionic and polymeric surfactants.
[0212] Suitable surfactants include, but are not limited to,
ethoxylated nonylphenol comprising 9 to 10 units of ethyleneglycol,
ethoxylated undecanol comprising 8 units of ethyleneglycol,
polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20)
sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate,
polyoxyethylene (20) sorbitan monooleate, sorbitan monolaurate,
sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate,
ethoxylated hydrogenated ricin oils, sodium laurylsulfate, a
diblock copolymer of ethyleneoxyde and propyleneoxyde, Ethylene
Oxide-Propylene Oxide Block Copolymers, and tetra-functional block
copolymers based on ethylene oxide and propylene oxide, Glyceryl
monoesters, Glyceryl caprate, Glyceryl caprylate, Glyceryl cocate,
Glyceryl erucate, Glyceryl hydroxysterate, Glyceryl isostearate,
Glyceryl lanolate, Glyceryl laurate, Glyceryl linolate, Glyceryl
myristate, Glyceryl oleate, Glyceryl PABA, Glyceryl palmitate,
Glyceryl ricinoleate, Glyceryl stearate, Glyceryl thighlycolate,
Glyceryl dilaurate, Glyceryl dioleate, Glyceryl dimyristate,
Glyceryl disterate, Glyceryl sesuioleate, Glyceryl stearate
lactate, Polyoxyethylene cetyl/stearyl ether, Polyoxyethylene
cholesterol ether, Polyoxyethylene laurate or dilaurate,
Polyoxyethylene stearate or distearate, polyoxyethylene fatty
ethers, Polyoxyethylene lauryl ether, Polyoxyethylene stearyl
ether, polyoxyethylene myristyl ether, a steroid, Cholesterol,
Betasitosterol, Bisabolol, fatty acid esters of alcohols, isopropyl
myristate, Aliphati-isopropyl n-butyrate, Isopropyl n-hexanoate,
Isopropyl n-decanoate, Isoproppyl palmitate, Octyldodecyl
myristate, alkoxylated alcohols, alkoxylated acids, alkoxylated
amides, alkoxylated sugar derivatives, alkoxylated derivatives of
natural oils and waxes, polyoxyethylene polyoxypropylene block
copolymers, nonoxynol-14, PEG-8 laurate, PEG-6 Cocoamide, PEG-20
methylglucose sesquistearate, PEG40 lanolin, PEG-40 castor oil,
PEG-40 hydrogenated castor oil, polyoxyethylene fatty ethers,
glyceryl diesters, polyoxyethylene stearyl ether, polyoxyethylene
myristyl ether, and polyoxyethylene lauryl ether, glyceryl
dilaurate, glyceryl dimystate, glyceryl distearate, semi-synthetic
derivatives thereof, or mixtures thereof.
[0213] Additional suitable surfactants include, but are not limited
to, non-ionic lipids, such as glyceryl laurate, glyceryl myristate,
glyceryl dilaurate, glyceryl dimyristate, semi-synthetic
derivatives thereof, and mixtures thereof.
[0214] In additional embodiments, the surfactant is a
polyoxyethylene fatty ether having a polyoxyethylene head group
ranging from about 2 to about 100 groups, or an alkoxylated alcohol
having the structure R.sub.5--(OCH.sub.2CH.sub.2).sub.y--OH,
wherein R.sub.5 is a branched or unbranched alkyl group having from
about 6 to about 22 carbon atoms and y is between about 4 and about
100, and preferably, between about 10 and about 100. Preferably,
the alkoxylated alcohol is the species wherein R.sub.5 is a lauryl
group and y has an average value of 23.
[0215] In a different embodiment, the surfactant is an alkoxylated
alcohol which is an ethoxylated derivative of lanolin alcohol.
Preferably, the ethoxylated derivative of lanolin alcohol is
laneth-10, which is the polyethylene glycol ether of lanolin
alcohol with an average ethoxylation value of 10.
[0216] Nonionic surfactants include, but are not limited to, an
ethoxylated surfactant, an alcohol ethoxylated, an alkyl phenol
ethoxylated, a fatty acid ethoxylated, a monoalkaolamide
ethoxylated, a sorbitan ester ethoxylated, a fatty amino
ethoxylated, an ethylene oxide-propylene oxide copolymer,
Bis(polyethylene glycol bis[imidazoyl carbonyl]), nonoxynol-9,
Bis(polyethylene glycol bis[imidazoyl carbonyl]), Brij.RTM. 35,
Brij.RTM. 56, Brij.RTM. 72, Brij.RTM. 76, Brij.RTM. 92V, Brij.RTM.
97, Brij.RTM. 58P, Cremophor.RTM. EL, Decaethylene glycol
monododecyl ether, N-Decanoyl-N-methylglucamine, n-Decyl
alpha-D-glucopyranoside, Decyl beta-D-maltopyranoside,
n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D-maltoside,
n-Dodecyl beta-D-maltoside, n-Dodecyl beta-D-maltoside,
Heptaethylene glycol monodecyl ether, Heptaethylene glycol
monododecyl ether, Heptaethylene glycol monotetradecyl ether,
n-Hexadecyl beta-D-maltoside, Hexaethylene glycol monododecyl
ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol
monooctadecyl ether, Hexaethylene glycol monotetradecyl ether,
Igepal CA-630, Igepal CA-630,
Methyl-6-O-(N-heptylcarbamoyl)-alpha-D-glucopyranoside,
Nonaethylene glycol monododecyl ether,
N--N-Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl
ether, Octaethylene glycol monododecyl ether, Octaethylene glycol
monohexadecyl ether, Octaethylene glycol monooctadecyl ether,
Octaethylene glycol monotetradecyl ether,
Octyl-beta-D-glucopyranoside, Pentaethylene glycol monodecyl ether,
Pentaethylene glycol monododecyl ether, Pentaethylene glycol
monohexadecyl ether, Pentaethylene glycol monohexyl ether,
Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol
monooctyl ether, Polyethylene glycol diglycidyl ether, Polyethylene
glycol ether W-1, Polyoxyethylene 10 tridecyl ether,
Polyoxyethylene 100 stearate, Polyoxyethylene 20 isohexadecyl
ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate,
Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate,
Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene 25
propylene glycol stearate, Saponin from Quillaja bark, Span.RTM.
20, Span.RTM. 40, Span.RTM. 60, Span.RTM. 65, Span.RTM. 80,
Span.RTM. 85, Tergitol, Type 15-S-12, Tergitol, Type 15-S-30,
Tergitol, Type 15-S-5, Tergitol, Type 15-S-7, Tergitol, Type
15-S-9, Tergitol, Type NP-10, Tergitol, Type NP-4, Tergitol, Type
NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Tergitol,
Tergitol, Type TMN-10, Tergitol, Type TMN-6,
Tetradecyl-beta-D-maltoside, Tetraethylene glycol monodecyl ether,
Tetraethylene glycol monododecyl ether, Tetraethylene glycol
monotetradecyl ether, Triethylene glycol monodecyl ether,
Triethylene glycol monododecyl ether, Triethylene glycol
monohexadecyl ether, Triethylene glycol monooctyl ether,
Triethylene glycol monotetradecyl ether, Triton CF-21, Triton
CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, Triton QS-15,
Triton QS-44, Triton X-100, Triton X-102, Triton X-15, Triton
X-151, Triton X-200, Triton X-207, Triton.RTM. X-114, Triton.RTM.
X-165, Triton.RTM. X-305, Triton.RTM. X-405, Triton.RTM. X-45,
Triton.RTM. X-705-70, TWEEN.RTM. 20, TWEEN.RTM. 21, TWEEN.RTM. 40,
TWEEN.RTM. 60, TWEEN.RTM. 61, TWEEN.RTM. 65, TWEEN.RTM. 80,
TWEEN.RTM. 81, TWEEN.RTM. 85, Tyloxapol, n-Undecyl
beta-D-glucopyranoside, semi-synthetic derivatives thereof, or
combinations thereof.
[0217] In addition, the nonionic surfactant can be a poloxamer.
Poloxamers are polymers made of a block of polyoxyethylene,
followed by a block of polyoxypropylene, followed by a block of
polyoxyethylene. The average number of units of polyoxyethylene and
polyoxypropylene varies based on the number associated with the
polymer. For example, the smallest polymer, Poloxamer 101, consists
of a block with an average of 2 units of polyoxyethylene, a block
with an average of 16 units of polyoxypropylene, followed by a
block with an average of 2 units of polyoxyethylene. Poloxamers
range from colorless liquids and pastes to white solids. In
cosmetics and personal care products, Poloxamers are used in the
formulation of skin cleansers, bath products, shampoos, hair
conditioners, mouthwashes, eye makeup remover and other skin and
hair products. Examples of Poloxamers include, but are not limited
to, Poloxamer 101, Poloxamer 105, Poloxamer 108, Poloxamer 122,
Poloxamer 123, Poloxamer 124, Poloxamer 181, Poloxamer 182,
Poloxamer 183, Poloxamer 184, Poloxamer 185, Poloxamer 188,
Poloxamer 212, Poloxamer 215, Poloxamer 217, Poloxamer 231,
Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238,
Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 331,
Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338,
Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer 407,
Poloxamer 105 Benzoate, and Poloxamer 182 Dibenzoate.
[0218] Suitable cationic surfactants include, but are not limited
to, a quarternary ammonium compound, an alkyl trimethyl ammonium
chloride compound, a dialkyl dimethyl ammonium chloride compound, a
cationic halogen-containing compound, such as cetylpyridinium
chloride, Benzalkonium chloride, Benzalkonium chloride,
Benzyldimethylhexadecylammonium chloride,
Benzyldimethyltetradecylammonium chloride,
Benzyldodecyldimethylammonium bromide, Benzyltrimethylammonium
tetrachloroiodate, Dimethyldioctadecylammonium bromide,
Dodecylethyldimethylammonium bromide, Dodecyltrimethylammonium
bromide, Dodecyltrimethylammonium bromide,
Ethylhexadecyldimethylammonium bromide, Girard's reagent T,
Hexadecyltrimethylammonium bromide, Hexadecyltrimethylammonium
bromide, N,N',N'-Polyoxyethylene(10)-N-tallow-1,3-diaminopropane,
Thonzonium bromide, Trimethyl(tetradecyl)ammonium bromide,
1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, 1-Decanaminium,
N-decyl-N,N-dimethyl-, chloride, Didecyl dimethyl ammonium
chloride, 2-(2-(p-(Diisobutyl)cresosxy)ethoxy)ethyl dimethyl benzyl
ammonium chloride, 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl
dimethyl benzyl ammonium chloride, Alkyl 1 or 3
benzyl-1-(2-hydroxethyl)-2-imidazolinium chloride, Alkyl
bis(2-hydroxyethyl)benzyl ammonium chloride, Alkyl demethyl benzyl
ammonium chloride, Alkyl dimethyl 3,4-dichlorobenzyl ammonium
chloride (100% C12), Alkyl dimethyl 3,4-dichlorobenzyl ammonium
chloride (50% C14, 40% C12, 10% C16), Alkyl dimethyl
3,4-dichlorobenzyl ammonium chloride (55% C14, 23% C12, 20% C16),
Alkyl dimethyl benzyl ammonium chloride, Alkyl dimethyl benzyl
ammonium chloride (100% C14), Alkyl dimethyl benzyl ammonium
chloride (100% C16), Alkyl dimethyl benzyl ammonium chloride (41%
C14, 28% C12), Alkyl dimethyl benzyl ammonium chloride (47% C12,
18% C14), Alkyl dimethyl benzyl ammonium chloride (55% C16, 20%
C14), Alkyl dimethyl benzyl ammonium chloride (58% C14, 28% C16),
Alkyl dimethyl benzyl ammonium chloride (60% C14, 25% C12), Alkyl
dimethyl benzyl ammonium chloride (61% C11, 23% C14), Alkyl
dimethyl benzyl ammonium chloride (61% C12, 23% C14), Alkyl
dimethyl benzyl ammonium chloride (65% C12, 25% C14), Alkyl
dimethyl benzyl ammonium chloride (67% C12, 24% C14), Alkyl
dimethyl benzyl ammonium chloride (67% C12, 25% C14), Alkyl
dimethyl benzyl ammonium chloride (90% C14, 5% C12), Alkyl dimethyl
benzyl ammonium chloride (93% C14, 4% C12), Alkyl dimethyl benzyl
ammonium chloride (95% C16, 5% C18), Alkyl didecyl dimethyl
ammonium chloride, Alkyl dimethyl benzyl ammonium chloride
(C12-16), Alkyl dimethyl benzyl ammonium chloride (C12-18), dialkyl
dimethyl benzyl ammonium chloride, Alkyl dimethyl dimethybenzyl
ammonium chloride, Alkyl dimethyl ethyl ammonium bromide (90% C14,
5% C16, 5% C12), Alkyl dimethyl ethyl ammonium bromide (mixed alkyl
and alkenyl groups as in the fatty acids of soybean oil), Alkyl
dimethyl ethylbenzyl ammonium chloride, Alkyl dimethyl ethylbenzyl
ammonium chloride (60% C14), Alkyl dimethyl isopropylbenzyl
ammonium chloride (50% C12, 30% C14, 17% C16, 3% C18), Alkyl
trimethyl ammonium chloride (58% C18, 40% C16, 1% C14, 1% C12),
Alkyl trimethyl ammonium chloride (90% C18, 10% C16),
Alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18),
Di-(C8-10)-alkyl dimethyl ammonium chlorides, Dialkyl dimethyl
ammonium chloride, Dialkyl methyl benzyl ammonium chloride, Didecyl
dimethyl ammonium chloride, Diisodecyl dimethyl ammonium chloride,
Dioctyl dimethyl ammonium chloride, Dodecyl bis(2-hydroxyethyl)
octyl hydrogen ammonium chloride, Dodecyl dimethyl benzyl ammonium
chloride, Dodecylcarbamoyl methyl dimethyl benzyl ammonium
chloride, Heptadecyl hydroxyethylimidazolinium chloride,
Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, Myristalkonium
chloride (and) Quat RNIUM 14, N,N-Dimethyl-2-hydroxypropylammonium
chloride polymer, n-Tetradecyl dimethyl benzyl ammonium chloride
monohydrate, Octyl decyl dimethyl ammonium chloride, Octyl dodecyl
dimethyl ammonium chloride, Octyphenoxyethoxyethyl dimethyl benzyl
ammonium chloride, Oxydiethylenebis(alkyl dimethyl ammonium
chloride), Trimethoxysily propyl dimethyl octadecyl ammonium
chloride, Trimethoxysilyl quats, Trimethyl dodecylbenzyl ammonium
chloride, semi-synthetic derivatives thereof, and combinations
thereof.
[0219] Exemplary cationic halogen-containing compounds include, but
are not limited to, cetylpyridinium halides, cetyltrimethylammonium
halides, cetyldimethylethylammonium halides,
cetyldimethylbenzylammonium halides, cetyltributylphosphonium
halides, dodecyltrimethylammonium halides, or
tetradecyltrimethylammonium halides. In some particular
embodiments, suitable cationic halogen containing compounds
comprise, but are not limited to, cetylpyridinium chloride (CPC),
cetyltrimethylammonium chloride, cetylbenzyldimethylammonium
chloride, cetylpyridinium bromide (CPB), cetyltrimethylammonium
bromide (CTAB), cetyidimethylethylammonium bromide,
cetyltributylphosphonium bromide, dodecyltrimethylammonium bromide,
and tetrad ecyltrimethylammonium bromide. In particularly preferred
embodiments, the cationic halogen containing compound is CPC,
although the compositions of the present invention are not limited
to formulation with a particular cationic containing compound.
[0220] Suitable anionic surfactants include, but are not limited
to, a carboxylate, a sulphate, a sulphonate, a phosphate,
chenodeoxycholic acid, chenodeoxycholic acid sodium salt, cholic
acid, ox or sheep bile, Dehydrocholic acid, Deoxycholic acid,
Deoxycholic acid, Deoxycholic acid methyl ester, Digitonin,
Digitoxigenin, N,N-Dimethyldodecylamine N-oxide, Docusate sodium
salt, Glycochenodeoxycholic acid sodium salt, Glycocholic acid
hydrate, synthetic, Glycocholic acid sodium salt hydrate,
synthetic, Glycodeoxycholic acid monohydrate, Glycodeoxycholic acid
sodium salt, Glycolithocholic acid 3-sulfate disodium salt,
Glycolithocholic acid ethyl ester, N-Lauroylsarcosine sodium salt,
N-Lauroylsarcosine solution, N-Lauroylsarcosine solution, Lithium
dodecyl sulfate, Lithium dodecyl sulfate, Lithium dodecyl sulfate,
Lugol solution, Niaproof 4, Type 4,1-Octanesulfonic acid sodium
salt, Sodium 1-butanesulfonate, Sodium 1-decanesulfonate, Sodium
1-decanesulfonate, Sodium 1-dodecanesulfonate, Sodium
1-heptanesulfonate anhydrous, Sodium 1-heptanesulfonate anhydrous,
Sodium 1-nonanesulfonate, Sodium 1-propanesulfonate monohydrate,
Sodium 2-bromoethanesulfonate, Sodium cholate hydrate, Sodium
choleate, Sodium deoxycholate, Sodium deoxycholate monohydrate,
Sodium dodecyl sulfate, Sodium hexanesulfonate anhydrous, Sodium
octyl sulfate, Sodium pentanesulfonate anhydrous, Sodium
taurocholate, Taurochenodeoxycholic acid sodium salt,
Taurodeoxycholic acid sodium salt monohydrate, Taurohyodeoxycholic
acid sodium salt hydrate, Taurolithocholic acid 3-sulfate disodium
salt, Tauroursodeoxycholic acid sodium salt, Trizma.RTM. dodecyl
sulfate, TWEEN.RTM. 80, Ursodeoxycholic acid, semi-synthetic
derivatives thereof, and combinations thereof.
[0221] Suitable zwitterionic surfactants include, but are not
limited to, an N-alkyl betaine, lauryl amindo propyl dimethyl
betaine, an alkyl dimethyl glycinate, an N-alkyl amino propionate,
CHAPS, minimum 98% (TLC), CHAPS, SigmaUltra, minimum 98% (TLC),
CHAPS, for electrophoresis, minimum 98% (TLC), CHAPSO, minimum 98%,
CHAPSO, SigmaUltra, CHAPSO, for electrophoresis,
3-(Decyldimethylammonio)propanesulfonate inner salt,
3-Dodecyldimethylammonio)propanesulfonate inner salt, SigmaUltra,
3-(Dodecyldimethylammonio)propanesulfonate inner salt,
3-(N,N-Dimethylmyristylammonio)propanesulfonate,
3-(N,N-Dimethyloctadecylammonio)propanesulfonate,
3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt,
3-(N,N-Dimethylpalmitylammonio)propanesulfonate, semi-synthetic
derivatives thereof, and combinations thereof.
[0222] In some embodiments, the nanoemulsion comprises a cationic
surfactant, which can be cetylpyridinium chloride. In other
embodiments of the invention, the nanoemulsion comprises a cationic
surfactant, and the concentration of the cationic surfactant is
less than about 5.0% and greater than about 0.001%. In yet another
embodiment of the invention, the nanoemulsion comprises a cationic
surfactant, and the concentration of the cationic surfactant is
selected from the group consisting of less than about 5%, less than
about 4.5%, less than about 4.0%, less than about 3.5%, less than
about 3.0%, less than about 2.5%, less than about 2.0%, less than
about 1.5%, less than about 1.0%, less than about 0.90%, less than
about 0.80%, less than about 0.70%, less than about 0.60%, less
than about 0.50%, less than about 0.40%, less than about 0.30%,
less than about 0.20%, or less than about 0.10%. Further, the
concentration of the cationic agent in the nanoemulsion is greater
than about 0.002%, greater than about 0.003%, greater than about
0.004%, greater than about 0.005%, greater than about 0.006%,
greater than about 0.007%, greater than about 0.008%, greater than
about 0.009%, greater than about 0.010%, or greater than about
0.001%. In one embodiment, the concentration of the cationic agent
in the nanoemulsion is less than about 5.0% and greater than about
0.001%.
[0223] In another embodiment of the invention, the nanoemulsion
comprises at least one cationic surfactant and at least one
non-cationic surfactant. The non-cationic surfactant is a nonionic
surfactant, such as a polysorbate (Tween), such as polysorbate 80
or polysorbate 20. In one embodiment, the non-ionic surfactant is
present in a concentration of about 0.05% to about 7.0%, or the
non-ionic surfactant is present in a concentration of about 0.5% to
about 4%. In yet another embodiment of the invention, the
nanoemulsion comprises a cationic surfactant present in a
concentration of about 0.01% to about 2%, in combination with a
nonionic surfactant.
[0224] 5. Additional Ingredients
[0225] Additional compounds suitable for use in the nanoemulsions
of the invention include but are not limited to one or more
solvents, such as an organic phosphate-based solvent, bulking
agents, coloring agents, pharmaceutically acceptable excipients, a
preservative, pH adjuster, buffer, chelating agent, etc. The
additional compounds can be admixed into a previously emulsified
nanoemulsion, or the additional compounds can be added to the
original mixture to be emulsified. In certain of these embodiments,
one or more additional compounds are admixed into an existing
nanoemulsion composition immediately prior to its use.
[0226] Suitable preservatives in the nanoemulsions of the invention
include, but are not limited to, cetylpyridinium chloride,
benzalkonium chloride, benzyl alcohol, chlorhexidine,
imidazolidinyl urea, phenol, potassium sorbate, benzoic acid,
bronopol, chlorocresol, paraben esters, phenoxyethanol, sorbic
acid, alpha-tocophernol, ascorbic acid, ascorbyl palmitate,
butylated hydroxyanisole, butylated hydroxytoluene, sodium
ascorbate, sodium metabisulphite, citric acid, edetic acid,
semi-synthetic derivatives thereof, and combinations thereof. Other
suitable preservatives include, but are not limited to, benzyl
alcohol, chlorhexidine (bis(p-chlorophenyldiguanido) hexane),
chlorphenesin (3-(-4-chloropheoxy)-propane-1,2-diol), Kathon CG
(methyl and methylchloroisothiazolinone), parabens (methyl, ethyl,
propyl, butyl hydrobenzoates), phenoxyethanol (2-phenoxyethanol),
sorbic acid (potassium sorbate, sorbic acid), Phenonip
(phenoxyethanol, methyl, ethyl, butyl, propyl parabens), Phenoroc
(phenoxyethanol 0.73%, methyl paraben 0.2%, propyl paraben 0.07%),
Liquipar Oil (isopropyl, isobutyl, butylparabens), Liquipar PE (70%
phenoxyethanol, 30% liquipar oil), Nipaguard MPA (benzyl alcohol
(70%), methyl & propyl parabens), Nipaguard MPS (propylene
glycol, methyl & propyl parabens), Nipasept (methyl, ethyl and
propyl parabens), Nipastat (methyl, butyl, ethyl and propyel
parabens), Elestab 388 (phenoxyethanol in propylene glycol plus
chlorphenesin and methylparaben), and Killitol (7.5% chlorphenesin
and 7.5% methyl parabens).
[0227] The nanoemulsion may further comprise at least one pH
adjuster. Suitable pH adjusters in the nanoemulsion of the
invention include, but are not limited to, diethyanolamine, lactic
acid, monoethanolamine, triethylanolamine, sodium hydroxide, sodium
phosphate, semi-synthetic derivatives thereof, and combinations
thereof.
[0228] In addition, the nanoemulsion can comprise a chelating
agent. In one embodiment of the invention, the chelating agent is
present in an amount of about 0.0005% to about 1.0%. Examples of
chelating agents include, but are not limited to, phytic acid,
polyphosphoric acid, citric acid, gluconic acid, acetic acid,
lactic acid, ethylenediamine, ethylenediaminetetraacetic acid
(EDTA), and dimercaprol, and a preferred chelating agent is
ethylenediaminetetraacetic acid.
[0229] The nanoemulsion can comprise a buffering agent, such as a
pharmaceutically acceptable buffering agent. Examples of buffering
agents include, but are not limited to,
2-Amino-2-methyl-1,3-propanediol, .gtoreq.9.5% (NT),
2-Amino-2-methyl-1-propanol, .gtoreq.99.0% (GC), L-(+)-Tartaric
acid, .gtoreq.99.5% (T), ACES, .gtoreq.99.5% (T), ADA,
.gtoreq.99.0% (T), Acetic acid, .gtoreq.09.5% (GC/T), Acetic acid,
for luminescence, .gtoreq.99.5% (GC/T), Ammonium acetate solution,
for molecular biology, .about.5 M in H.sub.2O, Ammonium acetate,
for luminescence, .gtoreq.99.0% (calc. on dry substance, T),
Ammonium bicarbonate, .gtoreq.99.5% (T), Ammonium citrate dibasic,
.gtoreq.99.0% (T), Ammonium formate solution, 10 M in H.sub.2O,
Ammonium formate, .gtoreq.99.0% (calc. based on dry substance, NT),
Ammonium oxalate monohydrate, .gtoreq.99.5% (RT), Ammonium
phosphate dibasic solution, 2.5 M in H.sub.2O, Ammonium phosphate
dibasic, .gtoreq.99.0% (T), Ammonium phosphate monobasic solution,
2.5 M in H.sub.2O, Ammonium phosphate monobasic, .gtoreq.99.5% (T),
Ammonium sodium phosphate dibasic tetrahydrate, .gtoreq.99.5% (NT),
Ammonium sulfate solution, for molecular biology, 3.2 M in
H.sub.2O, Ammonium tartrate dibasic solution, 2 M in H.sub.2O
(colorless solution at 20.degree. C.), Ammonium tartrate dibasic,
.gtoreq.99.5% (T), BES buffered saline, for molecular biology,
2.times. concentrate, BES, .gtoreq.99.5% (T), BES, for molecular
biology, .gtoreq.99.5% (T), BICINE buffer Solution, for molecular
biology, 1 M in H.sub.2O, BICINE, .gtoreq.99.5% (T), BIS-TRIS,
.gtoreq.99.0% (NT), Bicarbonate buffer solution, >0.1 M
Na.sub.2CO.sub.3, >0.2 M NaHCO.sub.3, Boric acid, .gtoreq.99.5%
(T), Boric acid, for molecular biology, .gtoreq.99.5% (T), CAPS,
99.0% (TLC), CHES, .gtoreq.99.5% (T), Calcium acetate hydrate,
.gtoreq.99.0% (calc. on dried material, KT), Calcium carbonate,
precipitated, .gtoreq.99.0% (KT), Calcium citrate tribasic
tetrahydrate, .gtoreq.98.0% (calc. on dry substance, KT), Citrate
Concentrated Solution, for molecular biology, 1 M in H.sub.2O,
Citric acid, anhydrous, .gtoreq.99.5% (T), Citric acid, for
luminescence, anhydrous, .gtoreq.99.5% (T), Diethanolamine,
.gtoreq.99.5% (GC), EPPS, .gtoreq.99.0% (T),
Ethylenediaminetetraacetic acid disodium salt dihydrate, for
molecular biology, .gtoreq.99.0% (T), Formic acid solution, 1.0 M
in H.sub.2O, Gly-Gly-Gly, .gtoreq.99.0% (NT), Gly-Gly,
.gtoreq.99.5% (NT), Glycine, .gtoreq.99.0% (NT), Glycine, for
luminescence, .gtoreq.99.0% (NT), Glycine, for molecular biology,
.gtoreq.99.0% (NT), HEPES buffered saline, for molecular biology,
2.times. concentrate, HEPES, .gtoreq.99.5% (T), HEPES, for
molecular biology, .gtoreq.99.5% (T), Imidazole buffer Solution, 1
M in H.sub.2O, Imidazole, .gtoreq.99.5% (GC), Imidazole, for
luminescence, .gtoreq.99.5% (GC), Imidazole, for molecular biology,
.gtoreq.99.5% (GC), Lipoprotein Refolding Buffer, Lithium acetate
dihydrate, .gtoreq.99.0% (NT), Lithium citrate tribasic
tetrahydrate, .gtoreq.99.5% (NT), MES hydrate, .gtoreq.99.5% (T),
MES monohydrate, for luminescence, .gtoreq.99.5% (T), MES solution,
for molecular biology, 0.5 M in H.sub.2O, MOPS, .gtoreq.99.5% (T),
MOPS, for luminescence, .gtoreq.99.5% (T), MOPS, for molecular
biology, .gtoreq.99.5% (T), Magnesium acetate solution, for
molecular biology, .about.1 M in H.sub.2O, Magnesium acetate
tetrahydrate, .gtoreq.99.0% (KT), Magnesium citrate tribasic
nonahydrate, .gtoreq.98.0% (calc. based on dry substance, KT),
Magnesium formate solution, 0.5 M in H.sub.2O, Magnesium phosphate
dibasic trihydrate, .gtoreq.98.0% (KT), Neutralization solution for
the in-situ hybridization for in-situ hybridization, for molecular
biology, Oxalic acid dihydrate, .gtoreq.99.5% (RT), PIPES,
.gtoreq.99.5% (T), PIPES, for molecular biology, .gtoreq.99.5% (T),
Phosphate buffered saline, solution (autoclaved), Phosphate
buffered saline, washing buffer for peroxidase conjugates in
Western Blotting, 10.times. concentrate, piperazine, anhydrous,
.gtoreq.99.0% (T), Potassium D-tartrate monobasic, .gtoreq.99.0%
(T), Potassium acetate solution, for molecular biology, Potassium
acetate solution, for molecular biology, 5 M in H.sub.2O, Potassium
acetate solution, for molecular biology, .about.1 M in H.sub.2O,
Potassium acetate, .gtoreq.99.0% (NT), Potassium acetate, for
luminescence, .gtoreq.99.0% (NT), Potassium acetate, for molecular
biology, .gtoreq.99.0% (NT), Potassium bicarbonate, .gtoreq.99.5%
(T), Potassium carbonate, anhydrous, .gtoreq.99.0% (T), Potassium
chloride, .gtoreq.99.5% (AT), Potassium citrate monobasic,
.gtoreq.99.0% (dried material, NT), Potassium citrate tribasic
solution, 1 M in H.sub.2O, Potassium formate solution, 14 M in
H.sub.2O, Potassium formate, .gtoreq.99.5% (NT), Potassium oxalate
monohydrate, .gtoreq.99.0% (RT), Potassium phosphate dibasic,
anhydrous, .gtoreq.99.0% (T), Potassium phosphate dibasic, for
luminescence, anhydrous, .gtoreq.99.0% (T), Potassium phosphate
dibasic, for molecular biology, anhydrous, .gtoreq.99.0% (T),
Potassium phosphate monobasic, anhydrous, .gtoreq.99.5% (T),
Potassium phosphate monobasic, for molecular biology, anhydrous,
.gtoreq.99.5% (T), Potassium phosphate tribasic monohydrate,
.gtoreq.95% (T), Potassium phthalate monobasic, .gtoreq.99.5% (T),
Potassium sodium tartrate solution, 1.5 M in H.sub.2O, Potassium
sodium tartrate tetrahydrate, .gtoreq.99.5% (NT), Potassium
tetraborate tetrahydrate, .gtoreq.99.0% (T), Potassium tetraoxalate
dihydrate, .gtoreq.99.5% (RT), Propionic acid solution, 1.0 M in
H.sub.2O, STE buffer solution, for molecular biology, pH 7.8, STET
buffer solution, for molecular biology, pH 8.0, Sodium
5,5-diethylbarbiturate, .gtoreq.99.5% (NT), Sodium acetate
solution, for molecular biology, .about.3 M in H.sub.2O, Sodium
acetate trihydrate, .gtoreq.99.5% (NT), Sodium acetate, anhydrous,
.gtoreq.99.0% (NT), Sodium acetate, for luminescence, anhydrous,
.gtoreq.99.0% (NT), Sodium acetate, for molecular biology,
anhydrous, .gtoreq.99.0% (NT), Sodium bicarbonate, .gtoreq.99.5%
(T), Sodium bitartrate monohydrate, .gtoreq.99.0% (T), Sodium
carbonate decahydrate, .gtoreq.99.5% (T), Sodium carbonate,
anhydrous, .gtoreq.99.5% (calc. on dry substance, T), Sodium
citrate monobasic, anhydrous, .gtoreq.99.5% (T), Sodium citrate
tribasic dihydrate, .gtoreq.99.0% (NT), Sodium citrate tribasic
dihydrate, for luminescence, .gtoreq.99.0% (NT), Sodium citrate
tribasic dihydrate, for molecular biology, .gtoreq.99.5% (NT),
Sodium formate solution, 8 M in H.sub.2O, Sodium oxalate,
.gtoreq.99.5% (RT), Sodium phosphate dibasic dihydrate,
.gtoreq.99.0% (T), Sodium phosphate dibasic dihydrate, for
luminescence, .gtoreq.99.0% (T), Sodium phosphate dibasic
dihydrate, for molecular biology, .gtoreq.99.0% (T), Sodium
phosphate dibasic dodecahydrate, .gtoreq.99.0% (T), Sodium
phosphate dibasic solution, 0.5 M in H.sub.2O, Sodium phosphate
dibasic, anhydrous, .gtoreq.99.5% (T), Sodium phosphate dibasic,
for molecular biology, .gtoreq.99.5% (T), Sodium phosphate
monobasic dihydrate, .gtoreq.99.0% (T), Sodium phosphate monobasic
dihydrate, for molecular biology, .gtoreq.99.0% (T), Sodium
phosphate monobasic monohydrate, for molecular biology,
.gtoreq.99.5% (T), Sodium phosphate monobasic solution, 5 M in
H.sub.2O, Sodium pyrophosphate dibasic, .gtoreq.99.0% (T), Sodium
pyrophosphate tetrabasic decahydrate, .gtoreq.99.5% (T), Sodium
tartrate dibasic dihydrate, .gtoreq.99.0% (NT), Sodium tartrate
dibasic solution, 1.5 M in H.sub.2O (colorless solution at
20.degree. C.), Sodium tetraborate decahydrate, .gtoreq.99.5% (T),
TAPS, .gtoreq.99.5% (T), TES, .gtoreq.99.5% (calc. based on dry
substance, T), TM buffer solution, for molecular biology, pH 7.4,
TNT buffer solution, for molecular biology, pH 8.0, TRIS Glycine
buffer solution, 10.times. concentrate, TRIS acetate--EDTA buffer
solution, for molecular biology, TRIS buffered saline, 10.times.
concentrate, TRIS glycine SDS buffer solution, for electrophoresis,
10.times. concentrate, TRIS phosphate--EDTA buffer solution, for
molecular biology, concentrate, 10.times. concentrate, Tricine,
.gtoreq.99.5% (NT), Triethanolamine, .gtoreq.99.5% (GC),
Triethylamine, .gtoreq.99.5% (GC), Triethylammonium acetate buffer,
volatile buffer, .about.1.0 M in H.sub.2O, Triethylammonium
phosphate solution, volatile buffer, .about.1.0 M in H.sub.2O,
Trimethylammonium acetate solution, volatile buffer, .about.1.0 M
in H.sub.2O, Trimethylammonium phosphate solution, volatile buffer,
.about.1 M in H.sub.2O, Tris-EDTA buffer solution, for molecular
biology, concentrate, 100.times. concentrate, Tris-EDTA buffer
solution, for molecular biology, pH 7.4, Tris-EDTA buffer solution,
for molecular biology, pH 8.0, Trizma.RTM. acetate, .gtoreq.99.0%
(NT), Trizma.RTM. base, .gtoreq.99.8% (T), Trizma.RTM. base,
.gtoreq.99.8% (T), Trizma.RTM. base, for luminescence,
.gtoreq.99.8% (T), Trizma.RTM. base, for molecular biology,
.gtoreq.99.8% (T), Trizma.RTM. carbonate, .gtoreq.98.5% (T),
Trizma.RTM. hydrochloride buffer solution, for molecular biology,
pH 7.2, Trizma.RTM. hydrochloride buffer solution, for molecular
biology, pH 7.4, Trizma.RTM. hydrochloride buffer solution, for
molecular biology, pH 7.6, Trizma.RTM. hydrochloride buffer
solution, for molecular biology, pH 8.0, Trizma.RTM. hydrochloride,
.gtoreq.99.0% (AT), Trizma.RTM. hydrochloride, for luminescence,
.gtoreq.99.0% (AT), Trizma.RTM. hydrochloride, for molecular
biology, .gtoreq.99.0% (AT), and Trizma.RTM. maleate, .gtoreq.99.5%
(NT).
[0230] The nanoemulsion can comprise one or more emulsifying agents
to aid in the formation of emulsions. Emulsifying agents include
compounds that aggregate at the oil/water interface to form a kind
of continuous membrane that prevents direct contact between two
adjacent droplets. Certain embodiments of the present invention
feature nanoemulsions that may readily be diluted with water to a
desired concentration without impairing their antiviral
properties.
[0231] 6. Active Agents Incorporated into a Nanoemulsion of the
Invention
[0232] In a further embodiment of the invention, a nanoemulsion
comprises an additional active agent, such as an antibiotic or a
palliative agent (such as for burn wound treatment). Addition of
another agent may enhance the therapeutic effectiveness of the
nanoemulsion. The nanoemulsion in and of itself has anti-bacterial
activity and does not need to be combined with another active agent
to obtain therapeutic effectiveness. Any antibacterial (or
antibiotic) agent suitable for treating a bacterial infection can
be incorporated into the topical nanoemulsions of the
invention.
[0233] Examples of such antibiotic agents include, but are not
limited to, aminoglycosides, Ansamycins, Carbacephems, Carbapenems,
Cephalosporins, Glycopeptides, Macrolides, Monobactams,
Penicillins, Polypeptides, Polymyxin, Quinolones, Sulfonamides,
Tetracyclines, and others (e.g., Arsphenamine, Chloramphenicol,
Clindamycin, Lincomycin, Ethambutol, Fosfomycin, Fusidic acid,
Furazolidone, Isoniazid, Linezolid, Metronidazole, Mupirocin,
Nitrofurantoin, Platensimycin, Pyrazinamide,
Quinupristin/Dalfopristin, Rifampicin (Rifampin in US),
Thiamphenicol, Timidazole, Dapsone, and lofazimine).
[0234] Examples of these classes of antibiotics include, but are
not limited to, Amikacin, Gentamicin, Kanamycin, Neomycin,
Netilmicin, Streptomycin, Tobramycin, Paromomycin, Geldanamycin,
Herbimycin, Loracarbef, Ertapenem, Doripenem, Imipenem/Cilastatin,
Meropenem, Cefadroxil, Cefazolin, Cefalotin or Cefalothin,
Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime,
Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime,
Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone,
Cefepime, Ceftobiprole, Teicoplanin, Vancomycin, Azithromycin,
Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin,
Troleandomycin, Telithromycin, Spectinomycin, Aztreonam,
Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin,
Dicloxacillin, Flucloxacillin, Mezlocillin, Meticillin, Nafcillin,
Oxacillin, Penicillin, Piperacillin, Ticarcillin, Bacitracin,
Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin,
Levofloxacin, Lomefloxacin, Moxifloxacin, Norfloxacin, Ofloxacin,
Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide,
Sulfonamidochrysoidine (archaic), Sulfacetamide, Sulfadiazine,
Sulfamethizole, Sulfanilimide (archaic), Sulfasalazine,
Sulfisoxazole, Trimethoprim, rimethoprim-Sulfamethoxazole
(Co-trimoxazole) (TMP-SMX), Demeclocycline, Doxycycline,
Minocycline, Oxytetracycline, and Tetracycline.
[0235] Examples of palliative agents which may be incorporated into
the nanoemulsions of the invention include, but are not limited to,
menthol, camphor, phenol, allantoin, benzocaine, corticosteroids,
phenol, zinc oxide, camphor, pramoxine, dimethicone, meradimate,
octinoxate, octisalate, oxybenzone, dyclonine, alcohols (e.g.,
benzyl alcohol), mineral oil, propylene glycol, titanium dioxide,
silver nitrate (AgNO.sub.3), silver sulfadiazine, mafenide acetate,
nanocrystalline impregnated silver dressings, a p38 MAPK inhibitor,
and magnesium stearate.
D. Pharmaceutical Compositions
[0236] The nanoemulsions of the invention may be formulated into
pharmaceutical compositions that comprise the nanoemulsion in a
therapeutically effective amount and suitable,
pharmaceutically-acceptable excipients for administration to a
human subject in need thereof using any conventional pharmaceutical
method of administration. Such excipients are well known in the
art.
[0237] By the phrase "therapeutically effective amount" it is meant
any amount of the nanoemulsion that is effective in treating
microorganisms by killing or inhibiting the growth of the
microorganisms, causing the microorganisms to lose pathogenicity,
or any combination thereof.
[0238] Exemplary dosage forms may include, but are not limited to,
patches, ointments, creams, emulsions, liquids, lotions, gels,
bioadhesive gels, aerosols, pastes, foams, sunscreens, capsules,
microcapsules, or in the form of an article or carrier, such as a
bandage, insert, syringe-like applicator, pessary, powder, and talc
or other solid.
[0239] The pharmaceutical compositions may be formulated for
immediate release, sustained release, controlled release, delayed
release, or any combinations thereof. In some embodiments, the
formulations may comprise a penetration-enhancing agent for
enhancing penetration of the nanoemulsion through the stratum
corneum and into the epidermis or dermis (i.e., for methods of
treating burn wounds). Suitable penetration-enhancing agents
include, but are not limited to, alcohols such as ethanol,
triglycerides and aloe compositions. The amount of the
penetration-enhancing agent may comprise from about 0.5% to about
40% by weight of the formulation.
[0240] When appropriate, for example when treating burn wounds, the
nanoemulsions of the invention can be applied and/or delivered
utilizing electrophoretic delivery/electrophoresis. Such
transdermal methods, which comprise applying an electrical current,
are well known in the art.
[0241] In another embodiment of the invention, minimal systemic
absorption of the nanoemulsion occurs upon topical administration.
Such minimal systemic exposure can be determined by the detection
of less than 10 ng/mL, less than 8 ng/mL, less than 5 ng/mL, less
than 4 ng/mL, less than 3 ng/mL, or less than 2 ng/mL of the one or
more surfactants present in the nanoemulsion in the plasma of the
subject. Lack of systemic absorption may be monitored, for example,
by measuring the amount of the surfactant, such as the cationic
surfactant, in the plasma of the human subject undergoing
treatment. Amounts of surfactant of equal to or less than about 10
ng/ml in the plasma confirms minimal systemic absorption.
[0242] The pharmaceutical compositions may be applied in a single
administration or in multiple administrations. The pharmaceutical
compositions can be applied for at least one day, at least two days
at least three days at least four days at least 5 days, once a
week, at least twice a week, at least once a day, at least twice a
day, multiple times daily, multiple times weekly, biweekly, at
least once a month, or any combination thereof.
[0243] Following topical or intradermal administration, the
nanoemulsion may be occluded or semi-occluded. Occlusion or
semi-occlusion may be performed by overlaying a bandage, polyolefin
film, article of clothing, impermeable barrier, or semi-impermeable
barrier to the topical preparation.
Exemplary Nanoemulsions
[0244] Several exemplary nanoemulsions are described below,
although the methods of the invention are not limited to the use of
such nanoemulsions. The components and quantity of each can be
varied as described herein in the preparation of other
nanoemulsions. ("CPC" refers to cetylpyridinium chloride, which is
a cationic surfactant present in the nanoemulsions). Compositions
are w/w % unless otherwise noted.
TABLE-US-00001 TABLE 1 Exemplary Nanoemulsions Nanoemulsion
Component Weight Percent W.sub.205EC ED Distilled Water 23.418%
EDTA 0.0745% Cetylpyridinium Chloride 1.068% Tween 20 5.92% Ethanol
6.73% Soybean Oil 62.79% P.sub.4075EC Distilled Water 23.49% CPC
1.068% Poloxamer 407 5.92% Ethanol 6.73% Soybean Oil, NP 62.79%
W.sub.205GBA.sub.2 (v/v %) Distilled Water 20.93% BTC 824 2% Tween
20 5% Glycerine 8% Soybean Oil 64% W.sub.805EC Water 23.490%
Ethanol 6.730% Cetylpyridinium Chloride 1.068% Polysorbate 80
5.920% Refined Soybean Oil 62.790% W.sub.205ECEDL2 Distilled Water
23.418% EDTA 0.0745% Cetylpyridinium Chloride 1.068% Tween 20 5.92%
Ethanol 6.73% Soybean Oil 62.79% W.sub.205GBA.sub.2ED (v/v %)
Distilled Water 20.93% EDTA 0.0745% BTC 824 2% Tween 20 5%
Glycerine 8% Soybean Oil 64%
[0245] The following nanoemulsions have an average particle
(droplet) size of about 300 nm to about 600 nm: W.sub.205EC ED,
P.sub.4075EC, W.sub.205GBA.sub.2, W.sub.805EC, and
W.sub.205GBA.sub.2ED. The W.sub.205ECEDL2, which undergoes high
pressure processing has an average particle (droplet) size of about
150 nm to about 400 nm. The formulations listed in the table above
are "neat" or "concentrated" formulations, meaning that the
formulation intended for therapeutic use can be diluted as
desired.
Methods of Manufacture
[0246] The nanoemulsions of the invention can be formed using
classic emulsion forming techniques. See e.g., U.S. 2004/0043041.
See also U.S. Pat. Nos. 6,015,832, 6,506,803, 6,559,189, 6,635,676,
and US Patent Publication No. 20040043041, all of which are
incorporated by reference. In addition, methods of making emulsions
are described in U.S. Pat. Nos. 5,103,497 and 4,895,452 (herein
incorporated by reference). In an exemplary method, the oil is
mixed with the aqueous phase under relatively high shear forces
(e.g., using high hydraulic and mechanical forces) to obtain a
nanoemulsion comprising oil droplets having an average diameter of
less than about 1000 nm. Some embodiments of the invention employ a
nanoemulsion having an oil phase comprising an alcohol such as
ethanol. The oil and aqueous phases can be blended using any
apparatus capable of producing shear forces sufficient to form an
emulsion, such as French Presses or high shear mixers (e.g., FDA
approved high shear mixers are available, for example, from Admix,
Inc., Manchester, N.H.). Methods of producing such emulsions are
described in U.S. Pat. Nos. 5,103,497 and 4,895,452, herein
incorporated by reference in their entireties.
[0247] In an exemplary embodiment, the nanoemulsions used in the
methods of the invention comprise droplets of an oily discontinuous
phase dispersed in an aqueous continuous phase, such as water. The
nanoemulsions of the invention are stable, and do not decompose
even after long storage periods. Certain nanoemulsions of the
invention are non-toxic and safe when swallowed, inhaled, or
contacted to the skin of a subject.
[0248] The compositions of the invention can be produced in large
quantities and are stable for many months at a broad range of
temperatures. The nanoemulsion can have textures ranging from that
of a semi-solid cream to that of a thin lotion, and can be applied
topically by hand, and can be sprayed onto a surface or
nebulized.
[0249] As stated above, at least a portion of the emulsion may be
in the form of lipid structures including, but not limited to,
unilamellar, multilamellar, and paucliamellar lipid vesicles,
micelles, and lamellar phases.
[0250] The present invention contemplates that many variations of
the described nanoemulsions will be useful in the methods of the
present invention. To determine if a candidate nanoemulsion is
suitable for use with the present invention, three criteria are
analyzed. Using the methods and standards described herein,
candidate emulsions can be easily tested to determine if they are
suitable. First, the desired ingredients are prepared using the
methods described herein, to determine if a nanoemulsion can be
formed. If a nanoemulsion cannot be formed, the candidate is
rejected. Second, the candidate nanoemulsion should form a stable
emulsion. A nanoemulsion is stable if it remains in emulsion form
for a sufficient period to allow its intended use. For example, for
nanoemulsions that are to be stored, shipped, etc., it may be
desired that the nanoemulsion remain in emulsion form for months to
years. Typical nanoemulsions that are relatively unstable, will
lose their form within a day. Third, the candidate nanoemulsion
should have efficacy for its intended use. For example, the
emulsions of the invention should kill or disable microorganisms in
vitro. To determine the suitability of a particular candidate
nanoemulsion against a desired microorganism, the nanoemulsion is
exposed to the microorganism for one or more time periods in a
side-by-side experiment with an appropriate control sample (e.g., a
negative control such as water) and determining if, and to what
degree, the nanoemulsion kills or disables the microorganism.
[0251] The nanoemulsion of the invention can be provided in many
different types of containers and delivery systems. For example, in
some embodiments of the invention, the nanoemulsions are provided
as a liquid, lotion, cream or other solid or semi-solid form. The
nanoemulsions of the invention may be incorporated into hydrogel
formulations.
[0252] The nanoemulsions can be delivered (e.g., to a subject or
customers) in any suitable container. Suitable containers can be
used that provide one or more single use or multi-use dosages of
the nanoemulsion for the desired application. In some embodiments
of the invention, the nanoemulsions are provided in a suspension or
liquid form. Such nanoemulsions can be delivered in any suitable
container including spray bottles (e.g., pressurized spray bottles,
nebulizers).
[0253] Exemplary Methods of Use
[0254] As described in more detail throughout this application, the
present invention is directed to methods of treating and/or
preventing a respiratory infection in a subject having Cystic
fibrosis (CF). In general, the method comprises administering a
nanoemulsion to the subject, wherein the nanoemulsion comprises:
(i) water; (ii) at least one organic solvent; (iii) at least one
surfactant; and (iv) at least one oil; and wherein the nanoemulsion
comprises droplets having an average diameter of less than about
1000 nm. In one embodiment of the invention, the subject is
susceptible to or has an infection by one or more bacterial species
selected from the group consisting of Staphylococcus spp.,
Haemophilus spp., Pseudomonas spp., Burkholderia spp.,
Acinetobacter spp, Stenotrophomonas spp., Escherichia spp.,
Klebsiella spp., and Proteus spp. The nanoemulsion can be delivered
using any pharmaceutically acceptable means, with inhalation being
one example of a useful administration method.
[0255] In yet another embodiment, the invention is directed to a
method of treating or preventing an infection in a subject having a
burn wound, wherein: (a) the method comprises administering a
nanoemulsion to the subject; and (b) the nanoemulsion comprises:
(i) water; (ii) at least one organic solvent; (iii) at least one
surfactant; and (iv) at least one oil; and wherein the nanoemulsion
comprises droplets having an average diameter of less than about
1000 nm. In one embodiment of the invention, the subject is
susceptible to or has an infection by one or more gram-negative or
gram-positive bacterial species. In another embodiment, the
bacterial species are selected from the group consisting of
Staphylococcus spp., Haemophilus spp., Pseudomonas spp.,
Burkholderia spp., Acinetobacter spp, Stenotrophomonas spp.,
Escherichia spp., Klebsiella spp., and Proteus spp. The
nanoemulsion can be delivered using any pharmaceutically acceptable
means, with inhalation, nebulization, and topical application to
mucosal surfaces being examples of useful administration
methods.
[0256] In yet another embodiment, the invention is directed to a
method of treating or preventing an Haemophilus influenzae
infection in a subject wherein: (a) the method comprises
administering a nanoemulsion to the subject having or at risk of
having a Haemophilus influenzae infection; (b) the nanoemulsion
comprises: (i) water; (ii) at least one organic solvent; (iii) at
least one surfactant; and (iv) at least one oil; and (c) wherein
the nanoemulsion comprises droplets having an average diameter of
less than about 1000 nm. The nanoemulsion can be delivered using
any pharmaceutically acceptable means, with inhalation,
nebulization, and delivery to a mucosal surface being examples of
useful administration methods.
[0257] In one embodiment of the invention, the nanoemulsion
exhibits minimal or no toxicity or side effects. Preferably, the
nanoemulsion does not exhibit resistance to bacteria. This
embodiment applies to all methods described herein.
[0258] If the method relates to a respiratory infection, then in
one embodiment the respiratory infection may be associated with a
bacterial biofilm, such as a biofilm present in the lungs of a
subject.
[0259] All of the methods of the invention may further comprise
administering one or more antibiotics either before, during, or
after administration of the nanoemulsion. In yet another
embodiment, one or more antibiotics may be incorporated into a
nanoemulsion. In yet another embodiment of the invention, the
nanoemulsion does not exhibit any antagonism with the
antibiotic.
[0260] In one embodiment of the invention, administration of a
nanoemulsion and at least one antibiotic is synergistic as defined
by a fractional inhibitory concentration (FIC) index, a fractional
bactericidal concentration (FBC) index, or a combination thereof.
This embodiment applies to all methods described herein. Examples
of such antibiotics include, but are not limited to polymyxins
(colistin) and aminoglycosides (tobramycin).
[0261] In yet another embodiment, the methods of the invention may
be used to treat or prevent infection by one or more bacterial
species selected from the group consisting of Pseudomonas
aeruginosa, B. cenocepacia, A. baumannii, Stenotrophomonas
maltophilia, Staphylococcus aureus, H influenzae, E. coli, K.
pneumoniae, and Proteus mirabilis. All other gram positive or gram
negative bacteria are also encompassed by the methods of the
invention.
[0262] In one embodiment, the minimum inhibitory concentration
(MIC), the minimum bactericidal concentration (MBC), or a
combination thereof for the nanoemulsion demonstrate bacteriostatic
or bactericidal activity for the nanoemulsion. This embodiment
applies to all methods described herein.
[0263] In another embodiment of the invention, one or more
bacterial species may exhibit resistance against one or more
antibiotics. For example, the bacterial species can be
methicillin-resistant Staphylococcus aureus (MRSA). This embodiment
applies to all methods described herein.
[0264] The present invention is not limited by the type of subject
administered a composition of the present invention. Each of the
subjects (e.g., susceptible to respiratory infection) described
above may be administered a composition of the present invention.
In addition, the compositions and methods of the present invention
are useful in the treatment of other respiratory diseases and
disorders, such as acute bronchitis, bronchiectasis, pneumonia
(including ventilator-associated pneumonia, nosocomial pneumonia,
viral pneumonia, bacterial pneumonia, mycobacterial pneumonia,
fungal pneumonia, eosinophilic pneumonia, and Pneumocystis carinii
pneumonia), tuberculosis, cystic fibrosis (CF), emphysema radiation
pneumonitis, and respiratory infection associated with inflammation
caused by smoking, pulmonary edema, pneumoconiosis, sarcoidiosis,
silicosis, asbestosis, berylliosis, coal worker's pneumonoconiosis
(CWP), byssinosis, interstitial lung diseases (ILD) such as
idiopathic pulmonary fibrosis, ILD associated with collagen
vascular disorders, systemic lupus erythematosus, rheumatoid
arthritis, ankylosing spondylitis, systemic sclerosis, and
pulmonary inflammation that is a result of or is secondary to
another disorder such as influenza.
[0265] The present invention is not limited by the particular
formulation of a composition comprising a nanoemulsion of the
present invention. Indeed, a composition comprising a nanoemulsion
of the present invention may comprise one or more different agents
in addition to the nanoemulsion. These agents or cofactors include,
but are not limited to, adjuvants, surfactants, additives, buffers,
solubilizers, chelators, oils, salts, therapeutic agents, drugs,
bioactive agents, antibacterials, and antimicrobial agents (e.g.,
antibiotics, antivirals, etc.). In some embodiments, a composition
comprising a nanoemulsion of the present invention comprises an
agent and/or co-factor that enhance the ability of the nanoemulsion
to kill a microbe (e.g., located in the respiratory tract). In some
preferred embodiments, the presence of one or more co-factors or
agents reduces the amount of nanoemulsion required for killing
and/or attenuation of growth of a microbe. The present invention is
not limited by the type of co-factor or agent used in a therapeutic
agent of the present invention.
[0266] In some embodiments, a co-factor or agent used in a
nanoemulsion composition is a bioactive agent. For example, in some
embodiments, the bioactive agent may be a bioactive agent useful in
a cell (e.g., a cell expressing a CFTR). Bioactive agents, as used
herein, include diagnostic agents such as radioactive labels and
fluorescent labels. Bioactive agents also include molecules
affecting the metabolism of a cell (e.g., a cell expressing a
CFTR), including peptides, nucleic acids, and other natural and
synthetic drug molecules. Bioactive agents include, but are not
limited to, adrenergic agent; adrenocortical steroid;
adrenocortical suppressant; alcohol deterrent; aldosterone
antagonist; amino acid; ammonia detoxicant; anabolic; analeptic;
analgesic; androgen; anesthesia, adjunct to; anesthetic; anorectic;
antagonist; anterior pituitary suppressant; anthelmintic; anti-acne
agent; anti-adrenergic; anti-allergic; anti-amebic; anti-androgen;
anti-anemic; anti-anginal; anti-anxiety; anti-arthritic;
anti-asthmatic; anti-atherosclerotic; antibacterial;
anticholelithic; anticholelithogenic; anticholinergic;
anticoagulant; anticoccidal; anticonvulsant; antidepressant;
antidiabetic; antidiarrheal; antidiuretic; antidote; anti-emetic;
anti-epileptic; anti-estrogen; antifibrinolytic; antifungal;
antiglaucoma agent; antihemophilic; antihemorrhagic; antihistamine;
antihyperlipidemia; antihyperlipoproteinemic; antihypertensive;
antihypotensive; anti-infective; anti-infective, topical;
anti-inflammatory; antikeratinizing agent; antimalarial;
antimicrobial; antimigraine; antimitotic; antimycotic,
antinauseant, antineoplastic, antineutropenic, antiobessional
agent; antiparasitic; antiparkinsonian; antiperistaltic,
antipneumocystic; antiproliferative; antiprostatic hypertrophy;
antiprotozoal; antipruritic; antipsychotic; antirheumatic;
antischistosomal; antiseborrheic; antisecretory; antispasmodic;
antithrombotic; antitussive; anti-ulcerative; anti-urolithic;
antiviral; appetite suppressant; benign prostatic hyperplasia
therapy agent; blood glucose regulator; bone resorption inhibitor;
bronchodilator; carbonic anhydrase inhibitor; cardiac depressant;
cardioprotectant; cardiotonic; cardiovascular agent; choleretic;
cholinergic; cholinergic agonist; cholinesterase deactivator;
coccidiostat; cognition adjuvant; cognition enhancer; depressant;
diagnostic aid; diuretic; dopaminergic agent; ectoparasiticide;
emetic; enzyme inhibitor; estrogen; fibrinolytic; fluorescent
agent; free oxygen radical scavenger; gastrointestinal motility
effector; glucocorticoid; gonad-stimulating principle; hair growth
stimulant; hemostatic; histamine H2 receptor antagonists; hormone;
hypocholesterolemic; hypoglycemic; hypolipidemic; hypotensive;
imaging agent; immunizing agent; immunomodulator; immunoregulator;
immunostimulant; immunosuppressant; impotence therapy adjunct;
inhibitor; keratolytic; LHRH agonist; liver disorder treatment;
luteolysin; memory adjuvant; mental performance enhancer; mood
regulator; mucolytic; mucosal protective agent; mydriatic; nasal
decongestant; neuromuscular blocking agent; neuroprotective; NMDA
antagonist; non-hormonal sterol derivative; oxytocic; plasminogen
activator; platelet activating factor antagonist; platelet
aggregation inhibitor; post-stroke and post-head trauma treatment;
potentiator; progestin; prostaglandin; prostate growth inhibitor;
prothyrotropin; psychotropic; pulmonary surface; radioactive agent;
regulator; relaxant; repartitioning agent; scabicide; sclerosing
agent; sedative; sedative-hypnotic; selective adenosine A1
antagonist; serotonin antagonist; serotonin inhibitor; serotonin
receptor antagonist; steroid; stimulant; suppressant; symptomatic
multiple sclerosis; synergist; thyroid hormone; thyroid inhibitor;
thyromimetic; tranquilizer; amyotrophic lateral sclerosis agent;
cerebral ischemia agent; Paget's disease agent; unstable angina
agent; uricosuric; vasoconstrictor; vasodilator; vulnerary; wound
healing agent; xanthine oxidase inhibitor.
[0267] Molecules useful as antimicrobials can be delivered by the
methods and compositions of the invention, such that the
respiratory infection is reduced or eliminated. Antibiotics that
may find use in co-administration with a composition comprising a
nanoemulsion of the present invention include, but are not limited
to, agents or drugs that are bactericidal and/or bacteriostatic
(e.g., inhibiting replication of bacteria or inhibiting synthesis
of bacterial components required for survival of the infecting
organism), including, but not limited to, almecillin, amdinocillin,
amikacin, amoxicillin, amphomycin, amphotericin B, ampicillin,
azacitidine, azaserine, azithromycin, azlocillin, aztreonam,
bacampicillin, bacitracin, benzyl penicilloyl-polylysine,
bleomycin, candicidin, capreomycin, carbenicillin, cefaclor,
cefadroxil, cefamandole, cefazoline, cefdinir, cefepime, cefixime,
cefinenoxime, cefinetazole, cefodizime, cefonicid, cefoperazone,
ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin,
cefpiramide, cefpodoxime, cefprozil, cefsulodin, ceftazidime,
ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cephacetrile,
cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin,
cephradine, chloramphenicol, chlortetracycline, cilastatin,
cinnamycin, ciprofloxacin, clarithromycin, clavulanic acid,
clindamycin, clioquinol, cloxacillin, colistimethate, colistin,
cyclacillin, cycloserine, cyclosporine, cyclo-(Leu-Pro),
dactinomycin, dalbavancin, dalfopristin, daptomycin, daunorubicin,
demeclocycline, detorubicin, dicloxacillin, dihydrostreptomycin,
dirithromycin, doxorubicin, doxycycline, epirubicin, erythromycin,
eveminomycin, floxacillin, fosfomycin, fusidic acid, gemifloxacin,
gentamycin, gramicidin, griseofulvin, hetacillin, idarubicin,
imipenem, iseganan, ivermectin, kanamycin, laspartomycin,
linezolid, linocomycin, loracarbef, magainin, meclocycline,
meropenem, methacycline, methicillin, mezlocillin, minocycline,
mitomycin, moenomycin, moxalactam, moxifloxacin, mycophenolic acid,
nafcillin, natamycin, neomycin, netilmicin, niphimycin,
nitrofurantoin, novobiocin, oleandomycin, oritavancin, oxacillin,
oxytetracycline, paromomycin, penicillamine, penicillin G,
penicillin V, phenethicillin, piperacillin, plicamycin, polymyxin
B, pristinamycin, quinupristin, rifabutin, rifampin, rifamycin,
rolitetracycline, sisomicin, spectrinomycin, streptomycin,
streptozocin, sulbactam, sultamicillin, tacrolimus, tazobactam,
teicoplanin, telithromycin, tetracycline, ticarcillin, tigecycline,
tobramycin, troleandomycin, tunicamycin, tyrthricin, vancomycin,
vidarabine, viomycin, virginiamycin, BMS-284,756, L-749,345,
ER-35,786, S-4661, L-786,392, MC-02479, Pep5, RP 59500, and
TD-6424.
[0268] In some embodiments, a composition comprising a nanoemulsion
of the present invention comprises one or more mucoadhesives (See,
e.g., U.S. Pat. App. No. 20050281843, hereby incorporated by
reference in its entirety). The present invention is not limited by
the type of mucoadhesive utilized. Indeed, a variety of
mucoadhesives are contemplated to be useful in the present
invention including, but not limited to, cross-linked derivatives
of poly(acrylic acid) (e.g., carbopol and polycarbophil), polyvinyl
alcohol, polyvinyl pyrollidone, polysaccharides (e.g., alginate and
chitosan), hydroxypropyl methylcellulose, lectins, fimbrial
proteins, and carboxymethylcellulose. Although an understanding of
the mechanism is not necessary to practice the present invention
and the present invention is not limited to any particular
mechanism of action, in some embodiments, use of a mucoadhesive
(e.g., in a composition comprising a nanoemulsion) enhances killing
and/or attenuation of growth of a microbe (e.g., exposed to a
composition of the present invention) due to an increase in
duration and/or amount of exposure to the nanoemulsion that a
subject and/or microbe experiences when a mucoadhesive is used
compared to the duration and/or amount of exposure to the
nanoemulsion in the absence of using the mucoadhesive.
[0269] In some embodiments, a composition of the present invention
may comprise sterile aqueous preparations. Acceptable vehicles and
solvents include, but are not limited to, water, Ringer's solution,
phosphate buffered saline and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed
mineral or non-mineral oil may be employed including synthetic
mono-ordi-glycerides. In addition, fatty acids such as oleic acid
find use in the preparation of injectables. Carrier formulations
suitable for mucosal, pulmonary, subcutaneous, intramuscular,
intraperitoneal, intravenous, or administration via other routes
may be found in Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, Pa.
[0270] A composition comprising a nanoemulsion of the present
invention can be used therapeutically (e.g., to kill and/or
attenuate growth of an existing infection) or as a prophylactic
(e.g., to prevent microbial growth and/or colonization (e.g., to
prevent signs or symptoms of disease)). A composition comprising a
nanoemulsion of the present invention can be administered to a
subject via a number of different delivery routes and methods.
[0271] For example, the compositions of the present invention can
be administered to a subject (e.g., mucosally or by pulmonary
route) by multiple methods, including, but not limited to: being
suspended in a solution and applied to a surface; being suspended
in a solution and sprayed onto a surface using a spray applicator;
being mixed with a mucoadhesive and applied (e.g., sprayed or
wiped) onto a surface (e.g., mucosal or pulmonary surface); being
placed on or impregnated onto a nasal and/or pulmonary applicator
and applied; being applied by a controlled-release mechanism;
applied using a nebulizer, aerosolized, being applied as a
liposome; or being applied on a polymer.
[0272] In some embodiments, compositions of the present invention
are administered mucosally (e.g., using standard techniques; See,
e.g., Remington: The Science and Practice of Pharmacy, Mack
Publishing Company, Easton, Pa., 19th edition, 1995 (e.g., for
mucosal delivery techniques, including intranasal and pulmonary
techniques), as well as European Publication No. 517,565 and Illum
et al., J. Controlled Rel., 1994, 29:133-141 (e.g., for techniques
of intranasal administration), each of which is hereby incorporated
by reference in its entirety). The present invention is not limited
by the route of administration.
[0273] Methods of intranasal and pulmonary administration are well
known in the art, including the administration of a droplet or
spray form of the nanoemulsion into the nasopharynx of a subject to
be treated. In some embodiments, a nebulized or aerosolized
composition comprising a nanoemulsion is provided. Enteric
formulations such as gastro resistant capsules for oral
administration, suppositories for rectal or vaginal administration
may also form part of this invention. Compositions of the present
invention may also be administered via the oral route. Under these
circumstances, a composition comprising a nanoemulsion may comprise
a pharmaceutically acceptable excipient and/or include alkaline
buffers, or enteric capsules. Formulations for nasal delivery may
include those with dextran or cyclodextran and saponin as an
adjuvant.
[0274] In preferred embodiments, a nanoemulsion of the present
invention is administered via a pulmonary delivery route and/or
means. In some embodiments, an aqueous solution containing the
nanoemulsion is gently and thoroughly mixed to form a solution. The
solution is sterile filtered (e.g., through a 0.2 micron filter)
into a sterile, enclosed vessel. Under sterile conditions, the
solution is passed through an appropriately small orifice to make
droplets (e.g., between 0.1 and 10 microns).
[0275] The particles may be administered using any of a number of
different applicators. Suitable methods for manufacture and
administration are described in the following U.S. Pat. Nos.
6,592,904; 6,518,239; 6,423,344; 6,294,204; 6,051,256 and 5,997,848
to INHALE (now NEKTAR); and U.S. Pat. No. 5,985,309; RE37,053; U.S.
Pat. Nos. 6,436,443; 6,447,753; 6,503,480; and 6,635,283, to
Edwards, et al. (MIT, AIR), each of which is hereby
incorporated
[0276] Thus, in some embodiments, compositions of the present
invention are administered by pulmonary delivery. For example, a
composition of the present invention can be delivered to the lungs
of a subject (e.g., a human) via inhalation (See, e.g., Adjei, et
al. Pharmaceutical Research 1990; 7:565-569; Adjei, et al. Int. J.
Pharmaceutics 1990; 63:135-144; Braquet, et al. J. Cardiovascular
Pharmacology 1989 143-146; Hubbard, et al. (1989) Annals of
Internal Medicine, Vol. III, pp. 206-212; Smith, et al. J. Clin.
Invest. 1989; 84:1145-1146; Oswein, et al. "Aerosolization of
Proteins", 1990; Proceedings of Symposium on Respiratory Drug
Delivery II Keystone, Colo.; Debs, et al. J. Immunol. 1988;
140:3482-3488; and U.S. Pat. No. 5,284,656 to Platz, et al, each of
which are hereby incorporated by reference in its entirety). A
method and composition for pulmonary delivery of drugs for systemic
effect is described in U.S. Pat. No. 5,451,569 to Wong, et al.,
hereby incorporated by reference; See also U.S. Pat. No. 6,651,655
to Licalsi et al., hereby incorporated by reference in its
entirety)). In some embodiments, a composition comprising a
nanoemulsion is administered to a subject by more than one route or
means (e.g., administered via pulmonary route as well as a mucosal
route).
[0277] Further contemplated for use in the practice of this
invention are a wide range of mechanical devices designed for
pulmonary and/or nasal mucosal delivery of pharmaceutical agents
including, but not limited to, nebulizers, metered dose inhalers,
and powder inhalers, all of which are familiar to those skilled in
the art. Some specific examples of commercially available devices
suitable for the practice of this invention are the ULTRAVENT
nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the ACORN II
nebulizer (Marquest Medical Products, Englewood, Colo.); the
VENTOLIN metered dose inhaler (Glaxo Inc., Research Triangle Park,
N.C.); and the SPINHALER powder inhaler (Fisons Corp., Bedford,
Mass.). All such devices require the use of formulations suitable
for dispensing of the therapeutic agent. Typically, each
formulation is specific to the type of device employed and may
involve the use of an appropriate propellant material, in addition
to the usual diluents, adjuvants, surfactants, carriers and/or
other agents useful in therapy. Also, the use of liposomes,
microcapsules or microspheres, inclusion complexes, or other types
of carriers is contemplated.
[0278] Thus, in some embodiments, a composition comprising a
nanoemulsion of the present invention may be used to protect and/or
treat a subject susceptible to, or suffering from, a disease by
means of administering compositions comprising a nanoemulsion by
mucosal, intramuscular, intraperitoneal, intradermal, transdermal,
pulmonary, intravenous, subcutaneous or other route of
administration described herein. Methods of systemic administration
of the nanoemulsion and/or agent co-administered with the
nanoemulsion may include conventional syringes and needles, or
devices designed for ballistic delivery (See, e.g., WO 99/27961,
hereby incorporated by reference), or needleless pressure liquid
jet device (See, e.g., U.S. Pat. No. 4,596,556; U.S. Pat. No.
5,993,412, each of which are hereby incorporated by reference), or
transdermal patches (See, e.g., WO 97/48440; WO 98/28037, each of
which are hereby incorporated by reference). In some embodiments,
the present invention provides a delivery device for systemic
administration, pre-filled with the nanoemulsion composition of the
present invention.
[0279] As described above, the present invention is not limited by
the type of subject administered a composition of the present
invention. Indeed, a wide variety of subjects are contemplated to
be benefited from administration of a composition of the present
invention. In preferred embodiments, the subject is a human. In
some embodiments, human subjects are of any age (e.g., adults,
children, infants, etc.) that have been or are likely to become
exposed to a microorganism. In some embodiments, the human subjects
are subjects that are more likely to receive a direct exposure to
pathogenic microorganisms or that are more likely to display signs
and symptoms of disease after exposure to a pathogen (e.g.,
subjects with CF or asthma, subjects in the armed forces,
government employees, frequent travelers, persons attending or
working in a school or daycare, health care workers, an elderly
person, an immunocompromised person, and emergency service
employees (e.g., police, fire, EMT employees)). In some
embodiments, any one or all members of the general public can be
administered a composition of the present invention (e.g., to
prevent the occurrence or spread of disease). For example, in some
embodiments, compositions and methods of the present invention are
utilized to treat a group of people (e.g., a population of a
region, city, state and/or country) for their own health (e.g., to
prevent or treat disease) and/or to prevent or reduce the risk of
disease spread from animals (e.g., birds, cattle, sheep, pigs,
etc.) to humans. In some embodiments, the subjects are non-human
mammals (e.g., pigs, cattle, goats, horses, sheep, or other
livestock; or mice, rats, rabbits or other animal). In some
embodiments, compositions and methods of the present invention are
utilized in research settings (e.g., with research animals).
[0280] A composition comprising a nanoemulsion of the present
invention can be administered (e.g., to a subject (e.g., via
pulmonary and/or mucosal route) or to microbes (e.g., bacteria
(e.g., opportunistic and/or pathogenic bacteria (e.g., residing on
or within the respiratory system of a subject and/or on or within a
burn wound)))) as a therapeutic or as a prophylactic to prevent
microbial infection. Thus, in some embodiments, the present
invention provides a method of altering microbial (e.g., bacterial
(e.g., opportunistic and/or pathogenic bacterial) growth comprising
administering a composition comprising a nanoemulsion to the
microbes (e.g., bacteria (e.g., opportunistic and/or pathogenic
bacteria). In some embodiments, administration of a composition
comprising a nanoemulsion to the microbes (e.g., bacteria (e.g.,
opportunistic and/or pathogenic bacteria) kills the microbes. In
some embodiments, administration of a composition comprising
nanoemulsion to the microbes (e.g., bacteria (e.g., opportunistic
and/or pathogenic bacteria) inhibits growth of the microbes. It is
contemplated that a composition comprising a nanoemulsion can be
administered to microbes (e.g., bacteria (e.g., opportunistic
and/or pathogenic bacteria (e.g., residing within the respiratory
tract))) via a number of delivery routes and/or mechanisms.
[0281] The compositions of the present invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions. Thus, for example, the compositions
may contain additional, compatible, pharmaceutically-active
materials such as, for example, antipruritics, astringents, local
anesthetics or anti-inflammatory agents, or may contain additional
materials useful in physically formulating various dosage forms of
the compositions of the present invention, such as dyes, flavoring
agents, preservatives, antioxidants, opacifiers, thickening agents
and stabilizers. However, such materials, when added, preferably do
not unduly interfere with the biological activities of the
components of the compositions of the present invention. The
formulations can be sterilized and, if desired, mixed with
auxiliary agents (e.g., lubricants, preservatives, stabilizers,
wetting agents, emulsifiers, salts for influencing osmotic
pressure, buffers, colorings, flavorings and/or aromatic substances
and the like) that do not deleteriously interact with the
nanoemulsion. In some embodiments, nanoemulsion compositions of the
present invention are administered in the form of a
pharmaceutically acceptable salt. When used the salts should be
pharmaceutically acceptable, but non-pharmaceutically acceptable
salts may conveniently be used to prepare pharmaceutically
acceptable salts thereof. Such salts include, but are not limited
to, those prepared from the following acids: hydrochloric,
hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic,
salicylic, p-toluene sulphonic, tartaric, citric, methane
sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and
benzene sulphonic. Also, such salts can be prepared as alkaline
metal or alkaline earth salts, such as sodium, potassium or calcium
salts of the carboxylic acid group.
[0282] Suitable buffering agents include, but are not limited to,
acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3%
w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and
a salt (0.8-2% w/v). Suitable preservatives may include
benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%
w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02%
w/v).
[0283] In some embodiments, a composition comprising a nanoemulsion
is co-administered with one or more antibiotics. For example, one
or more antibiotics may be administered with, before and/or after
administration of a composition comprising a nanoemulsion. The
present invention is not limited by the type of antibiotic
co-administered. Indeed, a variety of antibiotics may be
co-administered including, but not limited to, .beta.-lactam
antibiotics, penicillins (such as natural penicillins,
aminopenicillins, penicillinase-resistant penicillins, carboxy
penicillins, ureido penicillins), cephalosporins (first generation,
second generation, and third generation cephalosporins), and other
.beta.-lactams (such as imipenem, monobactams), .beta.-lactamase
inhibitors, vancomycin, aminoglycosides and spectinomycin,
tetracyclines, chloramphenicol, erythromycin, lincomycin,
clindamycin, rifampin, metronidazole, polymyxins, doxycycline,
quinolones (e.g., ciprofloxacin), sulfonamides, trimethoprim, and
quinolines.
[0284] A wide variety of antimicrobial agents are currently
available for use in treating bacterial, fungal and viral
infections. For a comprehensive treatise on the general classes of
such drugs and their mechanisms of action, the skilled artisan is
referred to Goodman & Gilman's "The Pharmacological Basis of
Therapeutics" Eds. Hardman et al., 9th Edition, Pub. McGraw Hill,
chapters 43 through 50, 1996, (herein incorporated by reference in
its entirety). Generally, these agents include agents that inhibit
cell wall synthesis (e.g., penicillins, cephalosporins,
cycloserine, vancomycin, bacitracin); and the imidazole antifungal
agents (e.g., miconazole, ketoconazole and clotrimazole); agents
that act directly to disrupt the cell membrane of the microorganism
(e.g., detergents such as polmyxin and colistimethate and the
antifungals nystatin and amphotericin B); agents that affect the
ribosomal subunits to inhibit protein synthesis (e.g.,
chloramphenicol, the tetracyclines, erthromycin and clindamycin);
agents that alter protein synthesis and lead to cell death (e.g.,
aminoglycosides); agents that affect nucleic acid metabolism (e.g.,
the rifamycins and the quinolones); the antimetabolites (e.g.,
trimethoprim and sulfonamides); and the nucleic acid analogues such
as zidovudine, gangcyclovir, vidarabine, and acyclovir which act to
inhibit viral enzymes essential for DNA synthesis. Various
combinations of antimicrobials may be employed.
[0285] The present invention also includes methods involving
co-administration of a composition comprising a nanoemulsion with
one or more additional active and/or anti-infective agents. In
co-administration procedures, the agents may be administered
concurrently or sequentially. In one embodiment, the compositions
described herein are administered prior to the other active
agent(s). The pharmaceutical formulations and modes of
administration may be any of those described herein. In addition,
the two or more co-administered agents may each be administered
using different modes (e.g., routes) or different formulations. The
additional agents to be co-administered (e.g., antibiotics, a
second type of nanoemulsion, etc.) can be any of the well-known
agents in the art, including, but not limited to, those that are
currently in clinical use.
[0286] In some embodiments, a composition comprising a nanoemulsion
is administered to a subject via more than one route. For example,
a subject may benefit from receiving mucosal administration (e.g.,
nasal administration or other mucosal routes described herein) and,
additionally, receiving one or more other routes of administration
(e.g., pulmonary administration (e.g., via a nebulizer, inhaler, or
other methods described herein.
[0287] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the compositions, increasing
convenience to the subject and a physician. Many types of release
delivery systems are available and known to those of ordinary skill
in the art. They include polymer based systems such as poly
(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides.
[0288] Microcapsules of the foregoing polymers containing drugs are
described in, for example, U.S. Pat. No. 5,075,109, hereby
incorporated by reference. Delivery systems also include
non-polymer systems that are: lipids including sterols such as
cholesterol, cholesterol esters and fatty acids or neutral fats
such as mono-di- and tri-glycerides; hydrogel release systems;
sylastic systems; peptide based systems; wax coatings; compressed
tablets using conventional binders and excipients; partially fused
implants; and the like. Specific examples include, but are not
limited to: (a) erosional systems in which an agent of the
invention is contained in a form within a matrix such as those
described in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152,
each of which is hereby incorporated by reference and (b)
diffusional systems in which an active component permeates at a
controlled rate from a polymer such as described in U.S. Pat. Nos.
3,854,480, 5,133,974 and 5,407,686, each of which is hereby
incorporated by reference. In addition, pump-based hardware
delivery systems can be used, some of which are adapted for
implantation.
[0289] In preferred embodiments, a composition comprising a
nanoemulsion of the present invention comprises a suitable amount
of the nanoemulsion to kill and/or attenuate growth of microbes
(e.g., pathogenic microbes (e.g., pathogenic bacteria, viruses,
etc.)) in a subject when administered to the subject. The present
invention is not limited by the amount of nanoemulsion used. In
some preferred embodiments, the amount of nanoemulsion in a
composition comprising a nanoemulsion is selected as that amount
which kills and/or attenuates microbial growth without significant,
adverse side effects. The amount will vary depending upon which
specific nanoemulsion(s) is/are employed, and can vary from subject
to subject, depending on a number of factors including, but not
limited to, the species, age and general condition (e.g., health)
of the subject, and the mode of administration. Procedures for
determining the appropriate amount of nanoemulsion administered to
a subject to kill and/or attenuate growth of a microbe (e.g.,
pathogenic microbe (e.g., pathogenic bacteria, viruses, etc.)) in a
subject can be readily determined using known means by one of
ordinary skill in the art.
[0290] In some embodiments, it is expected that each dose (e.g., of
a composition comprising a nanoemulsion (e.g., administered to a
subject to kill and/or attenuate microbial growth)) comprises
10-40% nanoemulsion, in some embodiments, 20% nanoemulsion, in some
embodiments less than 20% (e.g., 15%, 10%, 8%, 5% or less
nanoemulsion), and in some embodiments greater than 20%
nanoemulsion (e.g., 25%, 30%, 35%, 40% or more nanoemulsion). An
optimal amount for a particular administration (e.g., to kill
and/or attenuate microbial growth) can be ascertained by one of
skill in the art using standard studies involving observation of
microbial growth and/or death and other responses in subjects.
[0291] In some embodiments, it is expected that each dose (e.g., of
a composition comprising a nanoemulsion (e.g., administered to a
subject to kill and/or attenuate microbial growth)) is from 0.001
to 40% or more (e.g., 0.001-10%, 0.5-5%, 1-3%, 2%, 6%, 10%, 15%,
20%, 30%, 40% or more) by weight nanoemulsion.
[0292] Similarly, the present invention is not limited by the
duration of time a nanoemulsion is administered to a subject (e.g.,
to kill and/or attenuate microbial growth). In some embodiments, a
nanoemulsion is administered one or more times (e.g. twice, three
times, four times or more) daily. In some embodiments, a
composition comprising a nanoemulsion is administered one or more
times a day until an infection is eradicated or microbial growth
and/or presence has been reduced to a desired level. In some
embodiments, a composition comprising a nanoemulsion of the present
invention is formulated in a concentrated dose that can be diluted
prior to administration to a subject. For example, dilutions of a
concentrated composition may be administered to a subject such that
the subject receives any one or more of the specific dosages
provided herein. In some embodiments, dilution of a concentrated
composition may be made such that a subject is administered (e.g.,
in a single dose) a composition comprising 0.5-50% of the
nanoemulsion present in the concentrated composition. Concentrated
compositions are contemplated to be useful in a setting in which
large numbers of subjects may be administered a composition of the
present invention (e.g., a hospital). In some embodiments, a
composition comprising a nanoemulsion of the present invention
(e.g., a concentrated composition) is stable at room temperature
for more than 1 week, in some embodiments for more than 2 weeks, in
some embodiments for more than 3 weeks, in some embodiments for
more than 4 weeks, in some embodiments for more than 5 weeks, and
in some embodiments for more than 6 weeks.
[0293] Dosage units may be proportionately increased or decreased
based on several factors including, but not limited to, the weight,
age, and health status of the subject. In addition, dosage units
may be increased or decreased for subsequent administrations.
[0294] In some embodiments, a composition comprising a nanoemulsion
is administered to a subject under conditions such that microbes
(e.g., bacteria (e.g., opportunistic and/or pathogenic bacteria))
are killed. In some embodiments, a composition comprising a
nanoemulsion is administered to a subject under conditions such
that microbial (e.g., bacterial (e.g., opportunistic and/or
pathogenic bacterial) growth is prohibited and/or attenuated. In
some embodiments, greater than 90% (e.g., greater than 95%, 98%,
99%, all detectable) of microbes (e.g., bacteria (e.g.,
opportunistic and/or pathogenic bacteria) are killed. In some
embodiments, there is greater than 2 log (e.g., greater than 3 log,
4 log, 5 log, or more) reduction in microbe (e.g., bacteria (e.g.,
opportunistic and/or pathogenic bacteria) presence. In some
embodiments, reduction and/or killing is observed in one hour or
less (e.g., 45 minutes, 30 minutes, 15 minutes, or less). In some
embodiments, reduction and/or killing is observed in 6 hours or
less (e.g., 5 hours, 4, hours, 3 hours, two hours or less than one
hour). In some embodiments, reduction and/or killing is observed in
two days or less following initial treatment (e.g., less than 24
hours, less than 20 hours, 18 hours or less). In some embodiments,
the reduction and/or killing is observed in three days or less,
four days or less, or five days or less.
[0295] A composition comprising a nanoemulsion of the present
invention finds use where the nature of the infectious and/or
disease causing agent (e.g., causing signs, symptoms or indications
of respiratory infection) is known, as well as where the nature of
the infectious and/or disease causing agent is unknown (e.g., in
emerging disease (e.g., of pandemic proportion (e.g., influenza or
other outbreaks of disease))). For example, the present invention
contemplates use of the compositions of the present invention in
treatment of or prevention of infections associated with an
emergent infectious and/or disease causing agent yet to be
identified (e.g., isolated and/or cultured from a diseased person
but without genetic, biochemical or other characterization of the
infectious and/or disease causing agent).
[0296] It is contemplated that the compositions and methods of the
present invention will find use in various settings, including
research settings. For example, compositions and methods of the
present invention also find use in studies of the immune system
(e.g., characterization of adaptive immune responses (e.g.,
protective immune responses (e.g., mucosal or systemic immunity))).
Uses of the compositions and methods provided by the present
invention encompass human and non-human subjects and samples from
those subjects, and also encompass research applications using
these subjects. Compositions and methods of the present invention
are also useful in studying and optimizing nanoemulsions,
immunogens, and other components and for screening for new
components. Thus, it is not intended that the present invention be
limited to any particular subject and/or application setting.
[0297] The formulations can be tested in vivo in a number of animal
models developed for the study of pulmonary, mucosal and other
routes of delivery. As is readily apparent, the compositions of the
present invention are useful for preventing and/or treating a wide
variety of diseases and infections caused by viruses, bacteria,
parasites, and fungi. Not only can the compositions be used
prophylactically or therapeutically, as described above, the
compositions can also be used in order to prepare antibodies, both
polyclonal and monoclonal (e.g., for diagnostic purposes), as well
as for immunopurification of an antigen of interest.
[0298] In some embodiments, the present invention provides a kit
comprising a composition comprising a nanoemulsion. In some
embodiments, the kit further provides a device for administering
the composition. The present invention is not limited by the type
of device included in the kit. In some embodiments, the device is
configured for pulmonary application of the composition of the
present invention (e.g., a nasal inhaler or nasal mister). In some
embodiments, a kit comprises a composition comprising a
nanoemulsion in a concentrated form (e.g., that can be diluted
prior to administration to a subject).
[0299] In some embodiments, all kit components are present within a
single container (e.g., vial or tube). In some embodiments, each
kit component is located in a single container (e.g., vial or
tube). In some embodiments, one or more kit components are located
in a single container (e.g., vial or tube) with other components of
the same kit being located in a separate container (e.g., vial or
tube). In some embodiments, a kit comprises a buffer. In some
embodiments, the kit further comprises instructions for use.
Therapeutic and Prophylactic Use of Immunogenic Compositions
[0300] The present invention provides immunogenic nanoemulsion
compositions and methods of using the same for the induction of
immune responses (e.g., innate and/or adaptive immune responses
(e.g., for generation of host immunity against a bacterial species
of the genus Burkholderia (e.g., B. cenocepacia, B. multivorans,
etc.))). Compositions and methods of the present invention find use
in, among other things, clinical (e.g. therapeutic and preventative
medicine (e.g., vaccination)) and research applications.
[0301] As shown in Example 10, an immunogenic composition (e.g.,
comprising a nanoemulsion and Bukholderia antigen) of the present
invention induces (e.g., when administered to a subject) both
systemic and mucosal immunity. Thus, in some preferred embodiments,
administration of a composition comprising a nanoemulsion and
Burkholderia antigen to a subject results in protection against an
exposure (e.g., a mucosal and/or respiratory exposure) to
Burkholderia. Although an understanding of the mechanism is not
necessary to practice the present invention and the present
invention is not limited to any particular mechanism of action,
mucosal administration (e.g., vaccination) provides protection
against Burkholderia (e.g., Burkholderia cepacia) infection (e.g.,
that initiates at a mucosal surface). Although it has heretofore
proven difficult to stimulate secretory IgA responses and
protection against pathogens that invade at mucosal surfaces (See,
e.g., Mestecky et al, Mucosal Immunology. 3ed edn. (Academic Press,
San Diego, 2005)), in some embodiments, the present invention
provides compositions and methods for stimulating mucosal immunity
(e.g., a protective IgA response) towards a pathogen (e.g.,
pathogenic species of Burkholderia, Haemophilus, Staphylococcus or
other bacterial genus) in a subject.
[0302] In some embodiments, the present invention provides a
composition (e.g., a composition comprising a nanoemulsion and
Burkholderia (e.g., Burkholderia cenocepacia) antigen (e.g., an
immunogenic polypeptide comprising an amino acid sequence of SEQ ID
NO. 1 or SEQ ID NOS. 2-16)) to serve as a mucosal vaccine. In some
embodiments, an immunogenic composition comprising nanoemulsion
(NE) and Burkholderia antigen is produced with NE and killed whole
cell bacteria of the genus Burkholderia (e.g., Burkholderia cepacia
(e.g., killed using nanoemulsion, alcohol (e.g., ethanol), or other
methods), isolated, purified and/or recombinant protein and/or
saccharide component of Burkholderia (e.g., protein/peptide (e.g.,
Burkholderia-derived protein, live-virus-vector-derived protein,
recombinant protein, recombinant denatured protein/antigens, small
peptide segments protein/antigen).
[0303] In some preferred embodiments, the present invention
provides a composition for generating an immune response comprising
a NE and an immunogen (e.g., a purified, isolated or synthetic
Burkholderia protein or derivative, variant, or analogue thereof;
or, one or more species of Burkholderia (e.g., Burkholderia
multivorans (e.g., killed and or inactivated whole cell bacteria).
When administered to a subject, a composition of the present
invention stimulates an immune response against the immunogen
within the subject. Although an understanding of the mechanism is
not necessary to practice the present invention and the present
invention is not limited to any particular mechanism of action, in
some embodiments, generation of an immune response (e.g., resulting
from administration of a composition comprising a nanoemulsion and
an immunogen) provides total or partial immunity to the subject
(e.g., from signs, symptoms or conditions of a disease associated
with Burkholderia infection (e.g., strep throat, meningitis,
bacterial pneumoniae, endocarditis, erysipelas and/or necrotizing
fasciitis)). Without being bound to any specific theory, protection
and/or immunity from disease (e.g., the ability of a subject's
immune system to prevent or attenuate (e.g., suppress) a sign,
symptom or condition of disease) after exposure to an immunogenic
composition of the present invention is due to adaptive (e.g.,
acquired) immune responses (e.g., immune responses mediated by B
and T cells following exposure to a NE comprising an immunogen of
the present invention (e.g., immune responses that exhibit
increased specificity and reactivity towards Burkholderia (e.g.,
Burkholderia cepacia)). Thus, in some embodiments, the compositions
and methods of the present invention are used prophylactically or
therapeutically to prevent or attenuate a sign, symptom or
condition associated with Burkholderia (e.g., Burkholderia
cepacia)).
[0304] In some embodiments, a NE comprising an immunogen (e.g., a
Burkholderia (e.g., Burkholderia cepacia) antigen) is administered
alone. In some embodiments, a composition comprising a NE and an
immunogen (e.g., a Burkholderia (e.g., Burkholderia cepacia)
antigen) comprises one or more other agents (e.g., a
pharmaceutically acceptable carrier, adjuvant, excipient, and the
like). In some embodiments, a composition for stimulating an immune
response of the present invention is administered in a manner to
induce a humoral immune response. In some embodiments, a
composition for stimulating an immune response of the present
invention is administered in a manner to induce a cellular (e.g.,
cytotoxic T lymphocyte) immune response, rather than a humoral
response. In some embodiments, a composition comprising a NE and an
immunogen of the present invention induces both a cellular and
humoral immune response.
[0305] The present invention is not limited by the isotype, strain
or species of Burkholderia (e.g., Burkholderia cepacia) used in a
composition comprising a NE and immunogen. Indeed, each
Burkholderia (e.g., Burkholderia cepacia) family member alone, or
in combination with another family member, may be used to generate
a composition comprising a NE and an immunogen (e.g., used to
generate an immune response) of the present invention. Exemplary
species of Burkholderia are described herein.
[0306] In some embodiments, the Burkholderia (e.g., Burkholderia
cepacia) species utilized is a modified (e.g., genetically modified
(e.g., naturally modified via natural selection or modified using
recombinant genetic techniques)) species that displays greater
pathogenic capacity (e.g., causes more sever Burkholderia--(e.g.,
Burkholderia cepacia)-induced disease (e.g., comprising enhanced
and/or more severe respiratory infection, etc.)). In some
embodiments, any one or more members of the genus Burkholderia is
utilized in an immunoreactive composition of the invention
including but not limited to B. cenocepacia, B. dolosa, B.
multivorans, B. ambifaria, B. vietnamiensis, B. ubonensis, B.
thailandensis, B. graminis, B. oklahomensis, B. pseudomallei, B.
xenovorans, B. phytofirmans, B. phymatum, R. metallidurans, R.
eutropha, R. solanacearum.
[0307] The present invention is not limited by the Burkholderia
strain used. Indeed, a variety of Burkholderia strains are
contemplated to be useful in the present invention including, but
not limited to, classical strains, attenuated strains,
non-replicating strains, modified strains (e.g., genetically or
mechanically modified strains (e.g., to become more or less
virulent)), or other serially diluted strains of Burkholderia. A
composition comprising a NE and immunogen may comprise one or more
strains of Burkholderia cenocepacia and/or other type of
Burkholderia (e.g., Burkholderia multivorans). Additionally, a
composition comprising a NE and immunogen may comprise one or more
strains of Burkholderia and, in addition, one or more strains of a
non-Burkholderia immunogen.
[0308] In some embodiments, the immunogen may comprise one or more
antigens derived from a pathogen (e.g., Burkholderia). For example,
in some embodiments, the immunogen is a purified, recombinant,
synthetic, or otherwise isolated protein (e.g., added to the NE to
generate an immunogenic composition). Similarly, the immunogenic
protein may be a derivative, analogue or otherwise modified (e.g.,
conjugated) form of a protein from a pathogen.
[0309] The present invention is not limited by the particular
formulation of a composition comprising a NE and immunogen of the
present invention. Indeed, a composition comprising a NE and
immunogen of the present invention may comprise one or more
different agents in addition to the NE and immunogen. These agents
or cofactors include, but are not limited to, adjuvants,
surfactants, additives, buffers, solubilizers, chelators, oils,
salts, therapeutic agents, drugs, bioactive agents, antibacterials,
and antimicrobial agents (e.g., antibiotics, antivirals, etc.). In
some embodiments, a composition comprising a NE and immunogen of
the present invention comprises an agent and/or co-factor that
enhance the ability of the immunogen to induce an immune response
(e.g., an adjuvant). In some preferred embodiments, the presence of
one or more co-factors or agents reduces the amount of immunogen
required for induction of an immune response (e.g., a protective
immune response (e.g., protective immunization)). In some
embodiments, the presence of one or more co-factors or agents can
be used to skew the immune response towards a cellular (e.g., T
cell mediated) or humoral (e.g., antibody mediated) immune
response. The present invention is not limited by the type of
co-factor or agent used in a therapeutic agent of the present
invention.
[0310] Adjuvants are described in general in Vaccine Design--the
Subunit and Adjuvant Approach, edited by Powell and Newman, Plenum
Press, New York, 1995. The present invention is not limited by the
type of adjuvant utilized (e.g., for use in a composition (e.g.,
pharmaceutical composition) comprising a NE and immunogen). For
example, in some embodiments, suitable adjuvants include an
aluminum salt such as aluminum hydroxide gel (alum) or aluminum
phosphate. In some embodiments, an adjuvant may be a salt of
calcium, iron or zinc, or may be an insoluble suspension of
acylated tyrosine, or acylated sugars, cationically or anionically
derivatised polysaccharides, or polyphosphazenes.
[0311] In some embodiments, it is preferred that a composition
comprising a NE and immunogen of the present invention comprises
one or more adjuvants that induce a Th1-type response. However, in
other embodiments, it will be preferred that a composition
comprising a NE and immunogen of the present invention comprises
one or more adjuvants that induce a Th2-type response.
[0312] In general, an immune response is generated to an antigen
through the interaction of the antigen with the cells of the immune
system. Immune responses may be broadly categorized into two
categories: humoral and cell mediated immune responses (e.g.,
traditionally characterized by antibody and cellular effector
mechanisms of protection, respectively). These categories of
response have been termed Th1-type responses (cell-mediated
response), and Th2-type immune responses (humoral response).
[0313] Stimulation of an immune response can result from a direct
or indirect response of a cell or component of the immune system to
an intervention (e.g., exposure to an immunogen). Immune responses
can be measured in many ways including activation, proliferation or
differentiation of cells of the immune system (e.g., B cells, T
cells, dendritic cells, APCs, macrophages, NK cells, NKT cells
etc.); up-regulated or down-regulated expression of markers and
cytokines; stimulation of IgA, IgM, or IgG titer; splenomegaly
(including increased spleen cellularity); hyperplasia and mixed
cellular infiltrates in various organs. Other responses, cells, and
components of the immune system that can be assessed with respect
to immune stimulation are known in the art.
[0314] Although an understanding of the mechanism is not necessary
to practice the present invention and the present invention is not
limited to any particular mechanism of action, in some embodiments,
compositions and methods of the present invention induce expression
and secretion of cytokines (e.g., by macrophages, dendritic cells
and CD4+ T cells). Modulation of expression of a particular
cytokine can occur locally or systemically. It is known that
cytokine profiles can determine T cell regulatory and effector
functions in immune responses. In some embodiments, Th1-type
cytokines can be induced, and thus, the immunostimulatory
compositions of the present invention can promote a Th1 type
antigen-specific immune response including cytotoxic T-cells (e.g.,
thereby avoiding unwanted Th2 type immune responses (e.g.,
generation of Th2 type cytokines (e.g., IL-13) involved in
enhancing the severity of disease (e.g., IL-13 induction of mucus
formation))).
[0315] Cytokines play a role in directing the T cell response.
Helper (CD4+) T cells orchestrate the immune response of mammals
through production of soluble factors that act on other immune
system cells, including B and other T cells. Most mature CD4+ T
helper cells express one of two cytokine profiles: Th1 or Th2.
Th1-type CD4+ T cells secrete IL-2, IL-3, IFN-.gamma., GM-CSF and
high levels of TNF-.alpha.. Th2 cells express IL-3, IL-4, IL-5,
IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF-.alpha.. Th1
type cytokines promote both cell-mediated immunity, and humoral
immunity that is characterized by immunoglobulin class switching to
IgG2a in mice and IgG1 in humans. Th1 responses may also be
associated with delayed-type hypersensitivity and autoimmune
disease. Th2 type cytokines induce primarily humoral immunity and
induce class switching to IgG1 and IgE. The antibody isotypes
associated with Th1 responses generally have neutralizing and
opsonizing capabilities whereas those associated with Th2 responses
are associated more with allergic responses.
[0316] Several factors have been shown to influence skewing of an
immune response towards either a Th1 or Th2 type response. The best
characterized regulators are cytokines. IL-12 and IFN-.gamma. are
positive Th1 and negative Th2 regulators. IL-12 promotes
IFN-.gamma. production, and IFN-.gamma. provides positive feedback
for IL-12. IL-4 and IL-10 appear important for the establishment of
the Th2 cytokine profile and to down-regulate Th1 cytokine
production.
[0317] Thus, in preferred embodiments, the present invention
provides a method of stimulating a Th1-type immune response in a
subject comprising administering to a subject a composition
comprising a NE and an immunogen. However, in other embodiments,
the present invention provides a method of stimulating a Th2-type
immune response in a subject (e.g., if balancing of a T cell
mediated response is desired) comprising administering to a subject
a composition comprising a NE and an immunogen. In further
preferred embodiments, adjuvants can be used (e.g., can be
co-administered with a composition of the present invention) to
skew an immune response toward either a Th1 or Th2 type immune
response. For example, adjuvants that induce Th2 or weak Th1
responses include, but are not limited to, alum, saponins, and
SB-As4. Adjuvants that induce Th1 responses include but are not
limited to MPL, MDP, ISCOMS, IL-12, IFN-.gamma., and SB-AS2.
[0318] Several other types of Th1-type immunogens can be used
(e.g., as an adjuvant) in compositions and methods of the present
invention. These include, but are not limited to, the following. In
some embodiments, monophosphoryl lipid A (e.g., in particular
3-de-O-acylated monophosphoryl lipid A (3D-MPL)), is used. 3D-MPL
is a well known adjuvant manufactured by Ribi Immunochem, Montana.
Chemically it is often supplied as a mixture of 3-de-O-acylated
monophosphoryl lipid A with either 4, 5, or 6 acylated chains. In
some embodiments, diphosphoryl lipid A, and 3-O-deacylated variants
thereof are used. Each of these immunogens can be purified and
prepared by methods described in GB 2122204B, hereby incorporated
by reference in its entirety. Other purified and synthetic
lipopolysaccharides have been described (See, e.g., U.S. Pat. No.
6,005,099 and EP 0 729 473; Hilgers et al., 1986, Int. Arch.
Allergy. Immunol., 79(4):392-6; Hilgers et al., 1987, Immunology,
60(1):141-6; and EP 0 549 074, each of which is hereby incorporated
by reference in its entirety). In some embodiments, 3D-MPL is used
in the form of a particulate formulation (e.g., having a small
particle size less than 0.2 .mu.m in diameter, described in EP 0
689 454, hereby incorporated by reference in its entirety).
[0319] In some embodiments, saponins are used as an immunogen
(e.g., Th1-type adjuvant) in a composition of the present
invention. Saponins are well known adjuvants (See, e.g.,
Lacaille-Dubois and Wagner (1996) Phytomedicine vol 2 pp 363-386).
Examples of saponins include Quil A (derived from the bark of the
South American tree Quillaja Saponaria Molina), and fractions
thereof (See, e.g., U.S. Pat. No. 5,057,540; Kensil, Crit. Rev Ther
Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0 362 279, each of
which is hereby incorporated by reference in its entirety). Also
contemplated to be useful in the present invention are the
haemolytic saponins QS7, QS17, and QS21 (HPLC purified fractions of
Quil A; See, e.g., Kensil et al. (1991). J. Immunology 146,
431-437, U.S. Pat. No. 5,057,540; WO 96/33739; WO 96/11711 and EP 0
362 279, each of which is hereby incorporated by reference in its
entirety). Also contemplated to be useful are combinations of QS21
and polysorbate or cyclodextrin (See, e.g., WO 99/10008, hereby
incorporated by reference in its entirety.
[0320] In some embodiments, an immunogenic oligonucleotide
containing unmethylated CpG dinucleotides ("CpG") is used as an
adjuvant in the present invention. CpG is an abbreviation for
cytosine-guanosine dinucleotide motifs present in DNA. CpG is known
in the art as being an adjuvant when administered by both systemic
and mucosal routes (See, e.g., WO 96/02555, EP 468520, Davis et
al., J. Immunol, 1998, 160(2):870-876; McCluskie and Davis, J.
Immunol., 1998, 161(9):4463-6; and U.S. Pat. App. No. 20050238660,
each of which is hereby incorporated by reference in its entirety).
For example, in some embodiments, the immunostimulatory sequence is
Purine-Purine-C-G-pyrimidine-pyrimidine; wherein the CG motif is
not methylated.
[0321] Although an understanding of the mechanism is not necessary
to practice the present invention and the present invention is not
limited to any particular mechanism of action, in some embodiments,
the presence of one or more CpG oligonucleotides activate various
immune subsets including natural killer cells (which produce
IFN-.gamma.) and macrophages. In some embodiments, CpG
oligonucleotides are formulated into a composition of the present
invention for inducing an immune response. In some embodiments, a
free solution of CpG is co-administered together with an antigen
(e.g., present within a NE solution (See, e.g., WO 96/02555; hereby
incorporated by reference). In some embodiments, a CpG
oligonucleotide is covalently conjugated to an antigen (See, e.g.,
WO 98/16247, hereby incorporated by reference), or formulated with
a carrier such as aluminium hydroxide (See, e.g., Brazolot-Millan
et al., Proc. Natl. Acad Sci., USA, 1998, 95(26), 15553-8).
[0322] In some embodiments, adjuvants such as Complete Freunds
Adjuvant and Incomplete Freunds Adjuvant, cytokines (e.g.,
interleukins (e.g., IL-2, IFN-.gamma., IL-4, etc.), macrophage
colony stimulating factor, tumor necrosis factor, etc.), detoxified
mutants of a bacterial ADP-ribosylating toxin such as a cholera
toxin (CT), a pertussis toxin (PT), or an E. Coli heat-labile toxin
(LT), particularly LT-K63 (where lysine is substituted for the
wild-type amino acid at position 63) LT-R72 (where arginine is
substituted for the wild-type amino acid at position 72), CT-S109
(where serine is substituted for the wild-type amino acid at
position 109), and PT-K9/G129 (where lysine is substituted for the
wild-type amino acid at position 9 and glycine substituted at
position 129) (See, e.g., WO93/13202 and WO92/19265, each of which
is hereby incorporated by reference), and other immunogenic
substances (e.g., that enhance the effectiveness of a composition
of the present invention) are used with a composition comprising a
NE and immunogen of the present invention.
[0323] Additional examples of adjuvants that find use in the
present invention include poly(di(carboxylatophenoxy)phosphazene
(PCPP polymer; Virus Research Institute, USA); derivatives of
lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi
ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide
(MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a
glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin,
Switzerland); and Leishmania elongation factor (a purified
Leishmania protein; Corixa Corporation, Seattle, Wash.).
[0324] Adjuvants may be added to a composition comprising a NE and
an immunogen, or, the adjuvant may be formulated with carriers, for
example liposomes, or metallic salts (e.g., aluminium salts (e.g.,
aluminium hydroxide)) prior to combining with or co-administration
with a composition comprising a NE and an immunogen.
[0325] In some embodiments, a composition comprising a NE and an
immunogen comprises a single adjuvant. In other embodiments, a
composition comprising a NE and an immunogen comprises two or more
adjuvants (See, e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO
98/56414; WO 99/12565; WO 99/11241; and WO 94/00153, each of which
is hereby incorporated by reference in its entirety).
[0326] In some embodiments, a composition comprising a NE and an
immunogen of the present invention comprises one or more
mucoadhesives (See, e.g., U.S. Pat. App. No. 20050281843, hereby
incorporated by reference in its entirety). The present invention
is not limited by the type of mucoadhesive utilized. Indeed, a
variety of mucoadhesives are contemplated to be useful in the
present invention including, but not limited to, cross-linked
derivatives of poly(acrylic acid) (e.g., carbopol and
polycarbophil), polyvinyl alcohol, polyvinyl pyrollidone,
polysaccharides (e.g., alginate and chitosan), hydroxypropyl
methylcellulose, lectins, fimbrial proteins, and
carboxymethylcellulose. Although an understanding of the mechanism
is not necessary to practice the present invention and the present
invention is not limited to any particular mechanism of action, in
some embodiments, use of a mucoadhesive (e.g., in a composition
comprising a NE and immunogen) enhances induction of an immune
response in a subject (e.g., administered a composition of the
present invention) due to an increase in duration and/or amount of
exposure to an immunogen that a subject experiences when a
mucoadhesive is used compared to the duration and/or amount of
exposure to an immunogen in the absence of using the
mucoadhesive.
[0327] In some embodiments, a composition of the present invention
may comprise sterile aqueous preparations. Acceptable vehicles and
solvents include, but are not limited to, water, Ringer's solution,
phosphate buffered saline and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed
mineral or non-mineral oil may be employed including synthetic
mono-ordi-glycerides. In addition, fatty acids such as oleic acid
find use in the preparation of injectables. Carrier formulations
suitable for mucosal, subcutaneous, intramuscular, intraperitoneal,
intravenous, or administration via other routes may be found in
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa.
[0328] A composition comprising a NE and an immunogen of the
present invention can be used therapeutically (e.g., to enhance an
immune response) or as a prophylactic (e.g., for immunization
(e.g., to prevent signs or symptoms of disease)). A composition
comprising a NE and an immunogen of the present invention can be
administered to a subject via a number of different delivery routes
and methods.
[0329] For example, an immunogenic compositions of the invention
can be administered to a subject (e.g., mucosally (e.g., nasal
mucosa, vaginal mucosa, etc.)) by multiple methods, including, but
not limited to: being suspended in a solution and applied to a
surface; being suspended in a solution and sprayed onto a surface
using a spray applicator; being mixed with a mucoadhesive and
applied (e.g., sprayed or wiped) onto a surface (e.g., mucosal
surface); being placed on or impregnated onto a nasal and/or
vaginal applicator and applied; being applied by a
controlled-release mechanism; being applied as a liposome; or being
applied on a polymer.
[0330] In some preferred embodiments, immunogenic compositions
described herein are administered mucosally (e.g., using standard
techniques; See, e.g., Remington: The Science and Practice of
Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995
(e.g., for mucosal delivery techniques, including intranasal,
pulmonary, vaginal and rectal techniques), as well as European
Publication No. 517,565 and Illum et al., J. Controlled Rel., 1994,
29:133-141 (e.g., for techniques of intranasal administration),
each of which is hereby incorporated by reference in its entirety).
Alternatively, immunogenic compositions described herein are
administered dermally or transdermally, using standard techniques
(See, e.g., Remington: The Science and Practice of Pharmacy, Mack
Publishing Company, Easton, Pa., 19th edition, 1995). The present
invention is not limited by the route of administration.
[0331] Mucosal vaccination, such as intranasal vaccination, may
induce mucosal immunity not only in the nasal mucosa, but also in
distant mucosal sites such as the genital mucosa (See, e.g.,
Mestecky, Journal of Clinical Immunology, 7:265-276, 1987). In
addition to inducing mucosal immune responses, mucosal vaccination
also induces systemic immunity (See, e.g., Example 10). In some
embodiments, non-parenteral administration (e.g., mucosal
administration of vaccines) provides an efficient and convenient
way to boost systemic immunity (e.g., induced by parenteral or
mucosal vaccination (e.g., in cases where multiple boosts are used
to sustain a vigorous systemic immunity)).
[0332] In some embodiments, a composition comprising a NE and an
immunogen of the present invention may be used to protect or treat
a subject susceptible to, or suffering from, disease by means of
administering a composition of the present invention via a mucosal
route (e.g., an oral/alimentary or nasal route). Alternative
mucosal routes include intravaginal and intra-rectal routes. In
preferred embodiments of the present invention, a nasal route of
administration is used, termed "intranasal administration" or
"intranasal vaccination" herein. Methods of intranasal vaccination
are well known in the art, including the administration of a
droplet or spray form of the vaccine into the nasopharynx of a
subject to be immunized. In some embodiments, a nebulized or
aerosolized composition comprising a NE and immunogen is provided.
Enteric formulations such as gastro resistant capsules for oral
administration, suppositories for rectal or vaginal administration
also form part of this invention. Immunogenic compositions
described herein may also be administered via the oral route. Under
these circumstances, a composition comprising a NE and an immunogen
may comprise a pharmaceutically acceptable excipient and/or include
alkaline buffers, or enteric capsules. Formulations for nasal
delivery may include those with dextran or cyclodextran and saponin
as an adjuvant.
[0333] Immunogenic compositions described herein may also be
administered via a vaginal route. In such cases, a composition
comprising a NE and an immunogen may comprise pharmaceutically
acceptable excipients and/or emulsifiers, polymers (e.g.,
CARBOPOL), and other known stabilizers of vaginal creams and
suppositories. In some embodiments, compositions of the present
invention are administered via a rectal route. In such cases, a
composition comprising a NE and an immunogen may comprise
excipients and/or waxes and polymers known in the art for forming
rectal suppositories.
[0334] In some embodiments, the same route of administration (e.g.,
mucosal administration) is chosen for both a priming and boosting
vaccination. In some embodiments, multiple routes of administration
are utilized (e.g., at the same time, or, alternatively,
sequentially) in order to stimulate an immune response (e.g., using
an immunogenic compositions described herein).
[0335] For example, in some embodiments, a composition comprising a
NE and an immunogen is administered to a mucosal surface of a
subject in either a priming or boosting vaccination regime.
Alternatively, in some embodiments, a composition comprising a NE
and an immunogen is administered systemically in either a priming
or boosting vaccination regime. In some embodiments, a composition
comprising a NE and an immunogen is administered to a subject in a
priming vaccination regimen via mucosal administration and a
boosting regimen via systemic administration. In some embodiments,
a composition comprising a NE and an immunogen is administered to a
subject in a priming vaccination regimen via systemic
administration and a boosting regimen via mucosal administration.
Examples of systemic routes of administration include, but are not
limited to, a parenteral, intramuscular, intradermal, transdermal,
subcutaneous, intraperitoneal or intravenous administration. A
composition comprising a NE and an immunogen may be used for both
prophylactic and therapeutic purposes.
[0336] In some embodiments, immunogenic compositions described
herein are administered by pulmonary delivery. For example,
immunogenic compositions described herein can be delivered to the
lungs of a subject (e.g., a human) via inhalation (e.g., thereby
traversing across the lung epithelial lining to the blood stream
(See, e.g., Adjei, et al. Pharmaceutical Research 1990; 7:565-569;
Adjei, et al. Int. J. Pharmaceutics 1990; 63:135-144; Braquet, et
al. J. Cardiovascular Pharmacology 1989 143-146; Hubbard, et al.
(1989) Annals of Internal Medicine, Vol. III, pp. 206-212; Smith,
et al. J. Clin. Invest. 1989; 84:1145-1146; Oswein, et al.
"Aerosolization of Proteins", 1990; Proceedings of Symposium on
Respiratory Drug Delivery II Keystone, Colo.; Debs, et al. J.
Immunol. 1988; 140:3482-3488; and U.S. Pat. No. 5,284,656 to Platz,
et al, each of which are hereby incorporated by reference in its
entirety). A method and composition for pulmonary delivery of drugs
for systemic effect is described in U.S. Pat. No. 5,451,569 to
Wong, et al., hereby incorporated by reference; See also U.S. Pat.
No. 6,651,655 to Licalsi et al., hereby incorporated by reference
in its entirety)). Further contemplated for use in the practice of
delivery of immunogenic compositions described herein are the wide
range of mechanical devices designed for pulmonary and/or nasal
mucosal delivery of pharmaceutical agents described herein
[0337] Thus, in some embodiments, an immunogenic composition
described herein may be used to protect and/or treat a subject
susceptible to, or suffering from, a disease by means of
administering a compositions comprising a NE and an immunogen by
mucosal, intramuscular, intraperitoneal, intradermal, transdermal,
pulmonary, intravenous, subcutaneous or other route of
administration described herein under conditions that induce an
immunogen-specific immune response in the subject. Methods of
systemic administration of the vaccine preparations may include
conventional syringes and needles, or devices designed for
ballistic delivery of solid vaccines (See, e.g., WO 99/27961,
hereby incorporated by reference), or needleless pressure liquid
jet device (See, e.g., U.S. Pat. No. 4,596,556; U.S. Pat. No.
5,993,412, each of which are hereby incorporated by reference), or
transdermal patches (See, e.g., WO 97/48440; WO 98/28037, each of
which are hereby incorporated by reference). The present invention
may also be used to enhance the immunogenicity of antigens applied
to the skin (transdermal or transcutaneous delivery, See, e.g., WO
98/20734; WO 98/28037, each of which are hereby incorporated by
reference). Thus, in some embodiments, the present invention
provides a delivery device for systemic administration, pre-filled
with the vaccine composition of the present invention.
[0338] The present invention is not limited by the type of subject
administered (e.g., in order to stimulate an immune response (e.g.,
in order to generate protective immunity (e.g., mucosal and/or
systemic immunity))) a composition of the present invention.
Indeed, a wide variety of subjects are contemplated to be benefited
from administration of a composition of the present invention. In
preferred embodiments, the subject is a human. In some embodiments,
human subjects are of any age (e.g., adults, children, infants,
etc.) that have been or are likely to become exposed to a
microorganism (e.g., bacteria of the genus Burkholderia). In some
embodiments, the human subjects are subjects that are more likely
to suffer from opportunistic infection (e.g., a subject with CF or
an immune suppressed subject (e.g., a subject with human
immunodeficiency virus)). In some embodiments, the subjects are
non-human mammals (e.g., pigs, cattle, goats, horses, sheep, or
other livestock; or mice, rats, rabbits or other animal). In some
embodiments, compositions and methods of the present invention are
utilized in research settings (e.g., with research animals). In
some embodiments, the present invention provides a method to elicit
an immune response (e.g., protective immune response) in infants
(e.g., from about 0-2 years old) by administering to the infant a
safe and effective amount of an immunogenic composition of the
invention (e.g., a pediatric vaccine). Further embodiments of the
invention include the provision of the immunogenic Burkholderia
nanoemulsion compositions of the invention for use in medicine and
the use of the Burkholderia nanoemulsion compositions of the
invention in the manufacture of a medicament for the prevention (or
treatment) of disease caused by Burkholderia. In yet another
embodiment, the present invention is provides a method to elicit an
immune response (e.g., a protective immune response) in the elderly
population (e.g., in a subject 50 years or over in age, typically
over 55 years and more generally over 60 years) by administering a
safe and effective amount of an immunogenic composition of the
invention.
[0339] Immunogenic compositions described herein may be formulated
for administration by any route, such as mucosal, oral, topical,
parenteral or other route described herein. The compositions may be
in any one or more different forms including, but not limited to,
tablets, capsules, powders, granules, lozenges, foams, creams or
liquid preparations.
[0340] Topical formulations may be presented as, for instance,
ointments, creams or lotions, foams, and aerosols, and may contain
appropriate conventional additives such as preservatives, solvents
(e.g., to assist penetration), and emollients in ointments and
creams.
[0341] Topical formulations may also include agents that enhance
penetration of the active ingredients through the skin. Exemplary
agents include a binary combination of N-(hydroxyethyl)pyrrolidone
and a cell-envelope disordering compound, a sugar ester in
combination with a sulfoxide or phosphine oxide, and sucrose
monooleate, decyl methyl sulfoxide, and alcohol.
[0342] Other exemplary materials that increase skin penetration
include surfactants or wetting agents including, but not limited
to, polyoxyethylene sorbitan mono-oleoate (Polysorbate 80);
sorbitan mono-oleate (Span 80); p-isooctyl polyoxyethylene-phenol
polymer (Triton WR-1330); polyoxyethylene sorbitan tri-oleate
(Tween 85); dioctyl sodium sulfosuccinate; and sodium sarcosinate
(Sarcosyl NL-97); and other pharmaceutically acceptable
surfactants.
[0343] In certain embodiments of the invention, compositions may
further comprise one or more alcohols, zinc-containing compounds,
emollients, humectants, thickening and/or gelling agents,
neutralizing agents, and surfactants. Water used in the
formulations is preferably deionized water having a neutral pH.
Additional additives in the topical formulations include, but are
not limited to, silicone fluids, dyes, fragrances, pH adjusters,
and vitamins.
[0344] Topical formulations may also contain compatible
conventional carriers, such as cream or ointment bases and ethanol
or oleyl alcohol for lotions. Such carriers may be present as from
about 1% up to about 98% of the formulation. The ointment base can
comprise one or more of petrolatum, mineral oil, ceresin, lanolin
alcohol, panthenol, glycerin, bisabolol, cocoa butter and the
like.
[0345] Immunogenic compositions described herein may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions. Thus, for example, the compositions
may contain additional, compatible, pharmaceutically-active
materials such as, for example, antipuritics, astringents, local
anesthetics or anti-inflammatory agents, or may contain additional
materials useful in physically formulating various dosage forms of
the compositions of the present invention, such as dyes, flavoring
agents, preservatives, antioxidants, opacifiers, thickening agents
and stabilizers. However, such materials, when added, preferably do
not unduly interfere with the biological activities of the
immunogenic compositions described herein. The formulations can be
sterilized and, if desired, mixed with auxiliary agents (e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like) that
do not deleteriously interact with the NE and immunogen of the
formulation. In some embodiments, immunogenic compositions
described herein are administered in the form of a pharmaceutically
acceptable salt. When used the salts should be pharmaceutically
acceptable, but non-pharmaceutically acceptable salts may
conveniently be used to prepare pharmaceutically acceptable salts
thereof. Such salts include, but are not limited to, those prepared
from the following acids: hydrochloric, hydrobromic, sulphuric,
nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic,
tartaric, citric, methane sulphonic, formic, malonic, succinic,
naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts
can be prepared as alkaline metal or alkaline earth salts, such as
sodium, potassium or calcium salts of the carboxylic acid
group.
[0346] Suitable buffering agents include, but are not limited to,
acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3%
w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and
a salt (0.8-2% w/v). Suitable preservatives may include
benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%
w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02%
w/v).
[0347] In some embodiments, a composition comprising a NE and an
immunogen is co-administered with one or more antibiotics. For
example, one or more antibiotics may be administered with, before
and/or after administration of a composition comprising a NE and an
immunogen. The present invention is not limited by the type of
antibiotic co-administered. Indeed, a variety of antibiotics may be
co-administered including, but not limited to, .quadrature.-lactam
antibiotics, penicillins (such as natural penicillins,
aminopenicillins, penicillinase-resistant penicillins, carboxy
penicillins, ureido penicillins), cephalosporins (first generation,
second generation, and third generation cephalosporins), and other
.beta.-lactams (such as imipenem, monobactams), .beta.-lactamase
inhibitors, vancomycin, aminoglycosides and spectinomycin,
tetracyclines, chloramphenicol, erythromycin, lincomycin,
clindamycin, rifampin, metronidazole, polymyxins, doxycycline,
quinolones (e.g., ciprofloxacin), sulfonamides, trimethoprim, and
quinolines.
[0348] There are an enormous amount of antimicrobial agents
currently available for use in treating bacterial, fungal and viral
infections. For a comprehensive treatise on the general classes of
such drugs and their mechanisms of action, the skilled artisan is
referred to Goodman & Gilman's "The Pharmacological Basis of
Therapeutics" Eds. Hardman et al., 9th Edition, Pub. McGraw Hill,
chapters 43 through 50, 1996, (herein incorporated by reference in
its entirety). Generally, these agents include agents that inhibit
cell wall synthesis (e.g., penicillins, cephalosporins,
cycloserine, vancomycin, bacitracin); and the imidazole antifungal
agents (e.g., miconazole, ketoconazole and clotrimazole); agents
that act directly to disrupt the cell membrane of the microorganism
(e.g., detergents such as polmyxin and colistimethate and the
antifungals nystatin and amphotericin B); agents that affect the
ribosomal subunits to inhibit protein synthesis (e.g.,
chloramphenicol, the tetracyclines, erythromycin and clindamycin);
agents that alter protein synthesis and lead to cell death (e.g.,
aminoglycosides); agents that affect nucleic acid metabolism (e.g.,
the rifamycins and the quinolones); the antimetabolites (e.g.,
trimethoprim and sulfonamides); and the nucleic acid analogues such
as zidovudine, gangcyclovir, vidarabine, and acyclovir which act to
inhibit viral enzymes essential for DNA synthesis. Various
combinations of antimicrobials may be employed.
[0349] The present invention also includes methods involving
co-administration of a composition comprising a NE and an immunogen
with one or more additional active and/or immunostimulatory agents
(e.g., a composition comprising a NE and a different immunogen, an
antibiotic, anti-oxidant, etc.). Indeed, it is a further aspect of
this invention to provide methods for enhancing prior art
immunostimulatory methods (e.g., immunization methods) and/or
pharmaceutical compositions by co-administering a composition of
the present invention. In co-administration procedures, the agents
may be administered concurrently or sequentially. In one
embodiment, the compositions described herein are administered
prior to the other active agent(s). The pharmaceutical formulations
and modes of administration may be any of those described herein.
In addition, the two or more co-administered agents may each be
administered using different modes (e.g., routes) or different
formulations. The additional agents to be co-administered (e.g.,
antibiotics, adjuvants, etc.) can be any of the well-known agents
in the art, including, but not limited to, those that are currently
in clinical use.
[0350] In some embodiments, a composition comprising a NE and
immunogen is administered to a subject via more than one route. For
example, a subject that would benefit from having a protective
immune response (e.g., immunity) towards a pathogenic microorganism
may benefit from receiving mucosal administration (e.g., nasal
administration or other mucosal routes described herein) and,
additionally, receiving one or more other routes of administration
(e.g., parenteral or pulmonary administration (e.g., via a
nebulizer, inhaler, or other methods described herein). In some
preferred embodiments, administration via mucosal route is
sufficient to induce both mucosal as well as systemic immunity
towards an immunogen or organism from which the immunogen is
derived. In other embodiments, administration via multiple routes
serves to provide both mucosal and systemic immunity. Thus,
although an understanding of the mechanism is not necessary to
practice the present invention and the present invention is not
limited to any particular mechanism of action, in some embodiments,
it is contemplated that a subject administered a composition of the
present invention via multiple routes of administration (e.g.,
immunization (e.g., mucosal as well as airway or parenteral
administration of a composition comprising a NE and immunogen of
the present invention) may have a stronger immune response to an
immunogen than a subject administered a composition via just one
route.
[0351] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the compositions, increasing
convenience to the subject and a physician. Many types of release
delivery systems are available and known to those of ordinary skill
in the art. They include polymer based systems such as
poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109,
hereby incorporated by reference. Delivery systems also include
non-polymer systems that are: lipids including sterols such as
cholesterol, cholesterol esters and fatty acids or neutral fats
such as mono-di- and tri-glycerides; hydrogel release systems;
sylastic systems; peptide based systems; wax coatings; compressed
tablets using conventional binders and excipients; partially fused
implants; and the like. Specific examples include, but are not
limited to: (a) erosional systems in which an agent of the
invention is contained in a form within a matrix such as those
described in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152,
each of which is hereby incorporated by reference and (b)
diffusional systems in which an active component permeates at a
controlled rate from a polymer such as described in U.S. Pat. Nos.
3,854,480, 5,133,974 and 5,407,686, each of which is hereby
incorporated by reference. In addition, pump-based hardware
delivery systems can be used, some of which are adapted for
implantation.
[0352] In preferred embodiments, a composition comprising a NE and
an immunogen of the present invention comprises a suitable amount
of the immunogen to induce an immune response in a subject when
administered to the subject. In preferred embodiments, the immune
response is sufficient to provide the subject protection (e.g.,
immune protection) against a subsequent exposure to the immunogen
or the microorganism (e.g., bacteria of the genus Burkholderia)
from which the immunogen was derived. The present invention is not
limited by the amount of immunogen used. In some preferred
embodiments, the amount of immunogen (e.g., Burkholderia bacteria
(e.g., Burkholderia cepacia) or one or more component parts
thereof) in a composition comprising a NE and immunogen (e.g., for
use as an immunization dose) is selected as that amount which
induces an immunoprotective response without significant, adverse
side effects. The amount will vary depending upon which specific
immunogen or combination thereof is/are employed, and can vary from
subject to subject, depending on a number of factors including, but
not limited to, the species, age and general condition (e.g.,
health) of the subject, and the mode of administration.
[0353] In some embodiments, each dose (e.g., of a composition
comprising a NE and an immunogen (e.g., administered to a subject
to induce an immune response (e.g., a protective immune response
(e.g., protective immunity))) comprises about 0.05-5000 .mu.g of
immunogen (e.g., recombinant and/or purified Burkholderia OMP
protein). In some embodiments, each dose (e.g., of a composition
comprising a NE and an immunogen (e.g., administered to a subject
to induce an immune response (e.g., a protective immune response
(e.g., protective immunity))) comprises about 0.05-5000 .mu.g of
the immunogen (e.g., recombinant and/or purified Burkholderia OMP
protein) and comprises about 0.05-5000 .mu.g of another immunogen
(e.g., recombinant and/or purified protein, adjuvant (e.g., cholera
toxin), etc.). In some embodiments, each dose will comprise 1-500
.mu.g, in some embodiments, each dose will comprise 350-750 .mu.g,
in some embodiments, each dose will comprise 50-200 .mu.g, in some
embodiments, each dose will comprise 25-75 .mu.g of immunogen
(e.g., recombinant and/or purified protein (e.g., Burkholderia
antigen). In some embodiments, each dose comprises an amount of the
immunogen sufficient to generate an immune response. An effective
amount of the immunogen in a dose need not be quantified, as long
as the amount of immunogen generates an immune response in a
subject when administered to the subject.
[0354] In some embodiments, it is expected that each dose (e.g., of
a composition comprising a NE and an immunogen (e.g., administered
to a subject to induce and immune response)) is from 0.001 to 15%
or more (e.g., 0.001-10%, 0.5-5%, 1-3%, 2%, 6%, 10%, 15% or more)
by weight immunogen (e.g., neutralized bacteria, or recombinant
and/or purified protein). In some embodiments, an initial or prime
administration dose contains more immunogen than a subsequent boost
dose
[0355] In some embodiments, a composition comprising a NE and an
immunogen of the present invention is formulated in a concentrated
dose that can be diluted prior to administration to a subject. For
example, dilutions of a concentrated composition may be
administered to a subject such that the subject receives any one or
more of the specific dosages provided herein. In some embodiments,
dilution of a concentrated composition may be made such that a
subject is administered (e.g., in a single dose) a composition
comprising 0.5-50% of the NE and immunogen present in the
concentrated composition. In some preferred embodiments, a subject
is administered in a single dose a composition comprising 1% of the
NE and immunogen present in the concentrated composition.
Concentrated compositions are contemplated to be useful in a
setting in which large numbers of subjects may be administered a
composition of the present invention (e.g., an immunization clinic,
hospital, school, etc.). In some embodiments, a composition
comprising a NE and an immunogen of the present invention (e.g., a
concentrated composition) is stable at room temperature for more
than 1 week, in some embodiments for more than 2 weeks, in some
embodiments for more than 3 weeks, in some embodiments for more
than 4 weeks, in some embodiments for more than 5 weeks, and in
some embodiments for more than 6 weeks.
[0356] Generally, the emulsion compositions of the invention will
comprise at least 0.001% to 100%, preferably 0.01 to 90%, of
emulsion per ml of liquid composition. It is envisioned that the
formulations may comprise about 0.001%, about 0.0025%, about
0.005%, about 0.0075%, about 0.01%, about 0.025%, about 0.05%,
about 0.075%, about 0.1%, about 0.25%, about 0.5%, about 1.0%,
about 2.5%, about 5%, about 7.5%, about 10%, about 12.5%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
50%, about 55%, about 60%, about 65%, about 70%, about 75%, about
80%, about 85%, about 90%, about 95% or about 98% of emulsion per
ml of liquid composition. It should be understood that a range
between any two figures listed above is specifically contemplated
to be encompassed within the metes and bounds of the present
invention. Some variation in dosage will necessarily occur
depending on the condition of the specific pathogen and the subject
being immunized.
[0357] In some embodiments, following an initial administration of
a composition of the present invention (e.g., an initial
vaccination), a subject may receive one or more boost
administrations (e.g., around 2 weeks, around 3 weeks, around 4
weeks, around 5 weeks, around 6 weeks, around 7 weeks, around 8
weeks, around 10 weeks, around 3 months, around 4 months, around 6
months, around 9 months, around 1 year, around 2 years, around 3
years, around 5 years, around 10 years) subsequent to a first,
second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth,
and/or more than tenth administration. Although an understanding of
the mechanism is not necessary to practice the present invention
and the present invention is not limited to any particular
mechanism of action, in some embodiments, reintroduction of an
immunogen in a boost dose enables vigorous systemic immunity in a
subject. The boost can be with the same formulation given for the
primary immune response, or can be with a different formulation
that contains the immunogen. The dosage regimen will also, at least
in part, be determined by the need of the subject and be dependent
on the judgment of a practitioner.
[0358] Dosage units may be proportionately increased or decreased
based on several factors including, but not limited to, the weight,
age, and health status of the subject. In addition, dosage units
may be increased or decreased for subsequent administrations (e.g.,
boost administrations).
[0359] It is contemplated that the compositions and methods of the
present invention will find use in various settings, including
research settings. For example, compositions and methods of the
present invention also find use in studies of the immune system
(e.g., characterization of adaptive immune responses (e.g.,
protective immune responses (e.g., mucosal or systemic immunity))).
Uses of the compositions and methods provided by the present
invention encompass human and non-human subjects and samples from
those subjects, and also encompass research applications using
these subjects. Compositions and methods of the present invention
are also useful in studying and optimizing nanoemulsions,
immunogens, and other components and for screening for new
components. Thus, it is not intended that the present invention be
limited to any particular subject and/or application setting.
[0360] The formulations can be tested in vivo in a number of animal
models developed for the study of mucosal and other routes of
delivery. As is readily apparent, the compositions of the present
invention are useful for preventing and/or treating a wide variety
of diseases and infections caused by viruses, bacteria, parasites,
and fungi, as well as for eliciting an immune response against a
variety of antigens. Not only can the compositions be used
prophylactically or therapeutically, as described above, the
compositions can also be used in order to prepare antibodies, both
polyclonal and monoclonal (e.g., for diagnostic purposes), as well
as for immunopurification of an antigen of interest. If polyclonal
antibodies are desired, a selected mammal, (e.g., mouse, rabbit,
goat, horse, etc.) can be immunized with the compositions of the
present invention. The animal is usually boosted 2-6 weeks later
with one or more--administrations of the antigen. Polyclonal
antisera can then be obtained from the immunized animal and used
according to known procedures (See, e.g., Jurgens et al., J. Chrom.
1985, 348:363-370).
[0361] In some embodiments, the present invention provides a kit
comprising a composition comprising a NE and an immunogen. In some
embodiments, the kit further provides a device for administering
the composition. The present invention is not limited by the type
of device included in the kit. In some embodiments, the device is
configured for nasal application of the composition of the present
invention (e.g., a nasal applicator (e.g., a syringe) or nasal
inhaler or nasal mister). In some embodiments, a kit comprises a
composition comprising a NE and an immunogen in a concentrated form
(e.g., that can be diluted prior to administration to a
subject).
[0362] In some embodiments, all kit components are present within a
single container (e.g., vial or tube). In some embodiments, each
kit component is located in a single container (e.g., vial or
tube). In some embodiments, one or more kit component are located
in a single container (e.g., vial or tube) with other components of
the same kit being located in a separate container (e.g., vial or
tube). In some embodiments, a kit comprises a buffer. In some
embodiments, the kit further comprises instructions for use.
EXPERIMENTAL
[0363] The following examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof.
Example 1
In Vivo Toxicity Studies
[0364] A nanoemulsion composition of the present invention was
tested to determine if pulmonary administration of the composition
to a subject would elicit any histological changes and/or
pathology.
[0365] A solution comprising 20% nanoemulsion (W.sub.805EC) and
0.1% EDTA was administered via a nebulizer. A custom murine nose
only nebulization chamber with PARI LC nebulizer (Midlothian, Va.)
and compressor was used. A 0.9% NaCl solution was used as a
control. Using the nebulizer, mice were administered nebulized
nanoemulsion+EDTA or NaCl solution for 10 minutes. Twenty-four
hours after administration, mice were sacrificed and physical
properties and histology assessed.
[0366] Upon examination, there was an absence of histological
changes indicating the absence of toxicity upon administration of
nebulized nanoemulsion. Physical findings were normal.
Example 2
Killing Assays Utilizing Nanoemulsion Compositions and Bacteria
Found in the Respiratory Tract
[0367] It was determined whether a composition comprising
nanoemulsion (W.sub.805EC) alone or in combination with EDTA and/or
the presence of a hypertonic salt solution would be able to
attenuate growth and/or kill bacteria found in the respiratory
tract.
[0368] An overnight culture of Burkholderia cepacia or Pseudomonas
aeruginosa was started in 6 ml of cation adjusted Mueller Hinton
Broth (MHB) from a frozen bacterial stock at -80.degree. C. The
culture was incubated in a shaking incubator at 37.degree. C. The
following day, bacteria were brought to logarithmic growth phase by
back diluting the overnight culture 1:4 with fresh MHB. Back
diluted culture was incubated at 37.degree. C. in the shaking
incubator until the OD600 reached between 0.40 to 0.45. One ml of
the culture was spun down at 3500 to 4000 rpm for 15 minutes. The
bacterial pellet was resuspended in 1 ml of sterile 2.times.PBS,
12% or 14% saline. Appropriate dilutions were done with the same
solutions to get desired numbers of bacteria in 50 .mu.l (OD600 of
0.5 approximately =10.sup.9 bacteria). Dilutions of the starting
bacterial suspensions were plated onto Luria Bertani (LB) agar
plates to estimate the starting bacterial counts. Double the
concentrations of the desired final nanoemulsion and nanoemulsion
with EDTA concentrations were made with sterile milliQ water. Fifty
micro liters of the nanoemulsion was mixed with 50 .mu.l of the
bacteria and vortexed to mix the reaction. The mixture was
incubated at 37.degree. C. for desired time intervals. Following
incubation, the reaction was diluted with 500 .mu.l of 1.times.PBS
and vortexed to mix the contents. The contents were centrifuged at
4000 rpm for 15 minutes to pellet the bacteria and separate the
nanoemulsion. Supernatant containing the nanoemulsion was removed
and the bacterial pellet resuspended in 1.times.PBS. Undiluted or
dilutions of the resuspended bacterial pellet was plated onto LB
agar plates for colony count. Log killing of the bacteria were
calculated from ratio with the starting numbers.
[0369] The following killing assays were performed:
[0370] Killing of Burkholderia cepacia in PBS by 20% Nanemulsion
(NE) alone or with 20 mM EDTA in 10 minutes (See FIG. 1);
[0371] Killing of B. cepacia in PBS by 20% NE alone or with 20 mM
EDTA within 20 minutes (See FIG. 2);
[0372] Killing of B. cepacia in PBS by 20% NE alone or with 20 mM
EDTA within 40 minutes (See FIG. 3);
[0373] Killing of B. cepacia in PBS by 20% NE alone or with 20 mM
EDTA within 40 or 60 minutes (See FIG. 4);
[0374] Killing of B. cepacia in hypertonic saline (6% NaCl) at 15
and 30 minutes by NE alone and NE with EDTA (See FIG. 5);
[0375] Killing of B. cepacia and P. aeruginosa in 7% NaCl within 15
min (See FIG. 6); and
[0376] Killing of B. cepacia and P. aeruginosa (Mixed-culture) in
7% NaCl within 15 minutes (See FIG. 7).
[0377] Nanoemulsion alone or in combination with EDTA (e.g., 10-20
mM EDTA) was able to achieve complete killing of 10.sup.6 bacteria
in PBS in 60 minutes. In the presence of hypertonic saline (e.g.,
6-7% NaCl), the killing ability of the nanoemulsion was strikingly
enhanced, achieving complete killing within 15 minutes while in the
presence of 20 mM EDTA. Also, nanoemulsions comprising a lower
concentration of EDTA were able to achieve complete killing of
bacteria in 30 minutes in the presence of hypertonic saline. Thus,
the present invention provides that a nanoemulsion composition
comprising EDTA can be used to kill (e.g., completely) bacteria
over a short time period (e.g., <60 minutes). Moreover, the
present invention provides that nanoemulsion with hypertonic saline
and EDTA can be used to kill bacteria over even shorter time
periods (e.g., <30 minutes, <15 minutes) and that the
concentration of EDTA and hypertonic saline solution alter the pace
at which the nanoemulsion is able to eradicate the bacteria. The
present invention also demonstrates that compositions of the
present invention are able to eradicate a mixed population of
bacteria.
Example 3
Nanoemulsion Killing of Cystic Fibrosis (CF) Related Bacteria
Including Multi-Drug Resistant Strains
[0378] Materials and Methods.
[0379] Bacterial strains and culture conditions. One hundred fifty
isolates were analyzed including 75 Burkholderia isolates and 75
isolates belonging to other CF-relevant species, including
Pseudomonas aeruginosa, Achromobacter xylosoxidans,
Stenotrophomonas maltophilia, Acinetobacter species, Pandoraea
species (P. apista, P. pnomenusa, P. pulmonicola, P.
norimburgensis, and P. sputorum), and Ralstonia species (R.
mannitolilytica and R. pickettii). One hundred forty-five clinical
isolates were obtained from the Burkholderia cepacia Research
Laboratory and Repository (BcRLR, University of Michigan, Ann
Arbor, Mich.). These were recovered from 142 individuals between
September 1997 and October 2007 and were referred to the BcRLR from
62 CF treatment centers in the U.S. for analysis. The remaining
five strains included environmental isolates B. multivorans ATCC
17616 (American Type Culture Collection, Manassas, Va.), P.
norimburgensis LMG 18379.sup.T (BCCM/LMG Bacteria Collection,
Laboratorium voor Micrbiologie Gent, Universiteit Gent, Ghent,
Belgium) and B. pyrrocinia HI3642 (BcRLR collection), and the
clinical type strains B. cenocepacia LMG 16656.sup.T (aka J2315)
and P. pulmonicola LMG 18106.sup.T. One hundred thirty four (91%)
of the 147 clinical isolates were recovered from persons with CF;
114 (78%) of these were from sputum culture, with the remainder
from throat swab (n=17), endotracheal suction/tracheal aspiration
(n=5), blood (n=5), bronchial lavage (n=3), and one each from
maxillary sinus, peritoneal cavity, and epiglottis. Forty nine
(33%) of the 150 isolates were defined as multi-drug resistant
(resistant to all drugs tested in two of three antibiotic classes:
lactams including carbapenems, aminoglycosides, and quinolones;
See, e.g., Taccetti et al., Eur J Epidemiol. 1999 January;
15(1):85-8) based on susceptibility testing performed at the
referring microbiology laboratory; 20 (41%) of these were
panresistant. Seventy two (48%) of the remaining isolates were
susceptible to at least one antibiotic, and susceptibility testing
results were unavailable for 29 (19%) isolates. All isolates were
identified to the species level at the BcRLR by polyphasic analyses
using phenotypic and genotypic assays as described (See, e.g., Reik
et al., J Clin Microbiol. 2005 June; 43(6):2926-8). The exception
was Acinetobacter, which was identified only to the genus level due
to lack of definitive species-specific assays. All isolates were
also subjected to repetitive extragenic element-PCR (rep-PCR)
typing using the BOX A1R primer as previously described (See, e.g.,
Coenye et al., J Clin Microbiol. 2002 September; 40(9):3300-7) to
ensure that all 150 isolates included in the test panel were
genotypically distinct. Bacteria were stored at -80.degree. C. in
skim milk or Lauria-Bertani (LB) broth with 15% glycerol and
recovered from frozen stock overnight at 37.degree. C. on
Mueller-Hinton (MH) agar.
[0380] Nanoemulsion. Nanoemulsion P.sub.4075EC, described herein,
was manufactured by NANOBIO Corp. (Ann Arbor, Mich.). P.sub.4075EC
droplets had a mean particle diameter of 400 nm. Surfactants and
food substances utilized in the manufacture of P.sub.4075EC were
`Generally Recognized as Safe` (GRAS) by the FDA, and manufactured
in accordance with Good Manufacturing Practices (GMP). The
concentration of CPC was used as a surrogate for the amount of
P.sub.4075EC used experimentally. P.sub.4075EC was stable for no
less than 12 months at 40.degree. C.
[0381] Susceptibility testing. Because P.sub.4075EC is opaque, the
MICs of this compound for test bacteria were determined by using a
modification of the Clinical and Laboratory Standards Institute
(CLSI)-approved microtiter serial dilution method (See, e.g.,
Clinical and Laboratory Standards Institute. 2006. Approved
standard M7-A7, seventh edition. Clinical and Laboratory Standards
Institute, Wayne, Pa.). P.sub.4075EC was diluted to a concentration
of 2 mg/ml (of CPC) in MH broth supplemented with 7% NaCl and 20 mM
EDTA. Serial two-fold dilutions of this preparation were made in
unsupplemented MH broth and aliquoted into 96-well flat bottom
microtiter plates (100 .mu.l/well). Bacteria from overnight growth
on MH agar were suspended in MH broth to a 0.5 McFarland turbidity
standard (absorbance of 0.08-0.13 at 625 nm), further diluted 1:100
in MH broth, and added (5 .mu.l/well) to the P.sub.4075EC serial
dilution wells. Appropriate controls, including wells with bacteria
but no P.sub.4075EC and wells with P.sub.4075EC dilutions but no
bacteria, were included on each plate. Microtiter plates were
shaken briefly, and 1 .mu.l was removed from wells containing
bacteria but no P.sub.4075EC, diluted in 1 ml of MH broth, plated
on MH agar (100 .mu.l), and incubated for 24-48 h at 37.degree. C.
to determine bacterial concentrations of initial inoculums.
Microtiter plates were then incubated at 37.degree. C. without
shaking. To determine MBCs, 10 .mu.l were removed from each well
after overnight growth, spotted on MH agar, and incubated at
37.degree. C. Colonies were enumerated 24 h later, and MBCs were
recorded as the P.sub.4075EC concentration with a 3 log decrease in
CFU/ml compared to the initial inoculum. To determine MICs, 10
.mu.l of resazurin (R&D SYSTEMS, Minneapolis, Minn.) were added
to each well and microtiter plates were shaken briefly, covered
with foil, and incubated at 37.degree. C. without shaking. Wells
were visually inspected the next day, and the MIC was recorded as
the lowest concentration of P.sub.4075EC remaining a blue color.
MIC results were further quantified by recording fluorescence on a
spectrofluorometer at 560 nm excitation/590 nm emission.
[0382] Biofilm growth and susceptibility testing. To identify
biofilm-forming isolates, bacteria were grown overnight in tryptic
soy broth, adjusted to a 0.5 McFarland turbidity standard, and
further diluted 1:10 in MH broth, and 100 .mu.l were seeded in
triplicate in internal wells of 96-well flat bottom microtiter
plates. Negative control wells without bacteria were included. To
minimize evaporation, the remaining wells were filled with MH
broth, and the plate was wrapped with plastic wrap before
incubating for 48 h at 37.degree. C. without shaking. Wells were
gently washed twice with phosphate buffered saline (PBS; pH 7.4),
dried for 2 h at 37.degree. C., and then stained with 1% crystal
violet in water for 15 min. Stained wells were washed 3 times with
PBS, and crystal violet was solubilized by the addition of absolute
methanol. After incubation for 5 min at room temperature, the
solubilized crystal violet was transferred to a new microtiter
plate and scanned at 590 nm in a spectrophotometer. A
biofilm-forming isolate was defined as one where the average
absorbance of the 3 wells was greater than the average absorbance
of the negative control wells plus 3 standard deviations (See,
e.g., Stepanovic et al., J Microbiol Methods. 2000 April;
40(2):175-9).
[0383] For biofilm susceptibility testing, isolates shown to
produce biofilm by crystal violet testing were grown in triplicate
for 48 h as described above. Wells were gently washed twice with
PBS before addition of P.sub.4075EC, serially diluted as described
for planktonic MIC testing. After overnight incubation at
37.degree. C., wells were again washed twice with PBS, and 100
.mu.l of 10% resazurin in MH broth was added to wells. Plates were
wrapped in plastic, covered with foil, and again incubated
overnight at 37.degree. C. without shaking. Plates were visually
inspected the next day, and the minimum biofilm inhibitory
concentration (MBIC) was recorded as the lowest concentration of
P.sub.4075EC in which the wells were a blue color. To calculate
minimum biofilm eradication concentrations (MBECs), biofilm was
resuspended in the same wells used for MBIC testing by shaking the
microtiter plate and then scraping the sides of the wells with a
pipet tip. Ten .mu.L were removed from each well, spotted on MH
agar plates and incubated at 37.degree. C. After overnight growth,
MBECs were recorded as the lowest P.sub.4075EC concentration
resulting in no growth (See, e.g., Tomlin et al., Can J Microbiol.
2001 October; 47(10):949-54). To confirm initial inoculum
concentrations and to quantify viable bacteria in biofilm grown
cultures, colonies were enumerated from 10-fold serial dilutions of
the 0.5 McFarland inoculating culture and from the untreated
positive control wells with resuspended biofilm for MBEC
calculations.
[0384] Sputum preparation. Expectorated sputum, collected from CF
patients during the course of routine care, was obtained from the
University of Michigan Health System clinical microbiology
laboratory and stored at -80.degree. C. Equal volumes of sputum
from 15 individuals were pooled, mechanically sheared using a
TISSUE MISER homogenizer (FISHER SCIENTIFIC, Pittsburgh, Pa.) at
room temperature for 5 min at maximum speed, and then incubated in
an 80.degree. C. water bath for 20 min. Processed sputum was
divided into 10 ml aliquots, and 100 .mu.l from each aliquot was
plated on MH agar and incubated for 48 h at 37.degree. C. to
confirm sterility. Aliquots were stored at -80.degree. C.
[0385] Results.
[0386] Susceptibility of planktonic bacteria. Due to the opaque
white color of P.sub.4075EC, the standard CLSI-approved microtiter
serial dilution method was modified to include the addition of
resazurin as an indicator of bacterial viability. P.sub.4075EC was
tested in a concentration range of 15.6-2000 .mu.g/ml. MICs were
defined as the lowest concentration of P.sub.4075EC that did not
produce a color change from blue to pink (See FIG. 8). Comparison
of this visual inflection point with fluorometric analysis showed
that 63% of MIC wells had .ltoreq.1% of the metabolic activity of
untreated control wells; 91% had .ltoreq.5% metabolic activity, and
96% had .ltoreq.10% metabolic activity compared to control wells.
The MIC results are shown in FIG. 11. All strains were inhibited by
the concentrations of P.sub.4075EC tested. The MIC.sub.50 for the
entire panel of 150 strains was 31.2 .mu.g/ml; the MIC.sub.90 was
125 .mu.g/ml. Thirty eight strains (25%) were inhibited by the
lowest concentration of P.sub.4075EC tested (15.6 .mu.g/ml), and
only a single strain each required a concentration of 250 .mu.g/ml
and 500 .mu.g/ml for inhibition. P.sub.4075EC was slightly more
active against non-Burkholderia strains (MIC.sub.50, 31.2 .mu.g/ml;
MIC.sub.90 62.5 .mu.g/ml) than Burkholderia strains (MIC.sub.R,
62.5 .mu.g/ml; MIC.sub.90 125 .mu.g/ml) (See FIG. 9). Activity was
comparable across the 10 Burkholderia species tested. No difference
was found in the activity of P.sub.4075EC against multi-drug
resistant Burkholderia strains compared to strains susceptible to
one or more antibiotics.
[0387] To evaluate the bactericidal activity of P.sub.4075EC
against planktonic bacteria, MBCs were determined on a subset of 34
strains including 22 Burkholderia and 12 non-Burkholderia. All MBCs
were within 1 dilution of the respective MICs for this subset of
strains except for a single B. cenocepacia strain that had a MIC of
250 .mu.g/ml and an MBC of 2000 .mu.g/ml. A time-kill study of B.
multivorans ATCC 17616 showed time- and concentration-dependent
killing, with a 99% decrease in bacterial viability within 90 min
at a P.sub.4075EC concentration two times greater than the MIC and
complete killing within 30 min at concentrations eight times
greater than the MIC (See FIG. 10A).
[0388] Susceptibility of bacteria grown in biofilm. To further
assess the activity of P.sub.4075EC, bacteria grown as biofilms
were tested for susceptibility. Crystal violet staining identified
12 biofilm-forming strains from among 25 strains screened from the
test panel. Nine (75%) of the 12 strains showed decreased
susceptibility to P.sub.4075EC, defined as at least a four-fold
increase in the MBIC compared to the MIC, when grown as a biofilm
(See FIG. 12). The median increase in MBIC compared to the
respective MIC of P.sub.4075EC for the strains in this set was
eight-fold. No evidence of tolerance of biofilm bacteria to
P.sub.4075EC was observed; the MBEC was the same as the respective
MBIC for 10 of the 12 strains.
[0389] Susceptibility of bacteria in CF sputum. The 12
biofilm-forming strains were also tested for susceptibility to
P.sub.4075EC in the presence of CF sputum to model more closely the
CF pulmonary microenvironment. The MBCs of P.sub.4075EC for all 12
strains, grown under planktonic conditions, increased in the
presence of 43% sputum (the highest sputum concentration achievable
in the microtiter assay) compared to the respective MBCs under
standard conditions (See FIG. 12). Nevertheless, all strains
remained susceptible to P.sub.4075EC in the presence of sputum. CF
sputum inhibited the activity of P.sub.4075EC against planktonic
bacteria in a concentration dependent manner (See FIG. 10B).
Example 4
Nanoemulsion Killing of Individual and Mixed Bacterial Biofilms
[0390] A composition comprising nanoemulsion P.sub.4075EC, 7%
saline and 20 mM EDTA was tested for its ability to kill bacteria
present in biofilms (individual or mixed).
[0391] Biofilm Growth. Bacterial biofilms on polycarbonate
membranes were formed following the procedure described in Current
Protocols in Microbiology (John Wiley & Sons, Inc., NJ, USA).
Polycarbonate membranes were sterilized by exposing the membrane to
UV light for 10 minutes each side using sterile forceps or by steam
sterilization at 121.degree. C. for 20 minutes. The sterile
membrane was placed on the surface of an agar medium with the shiny
surface facing upwards. Overnight culture of the bacterial strain
of interest was diluted with broth media to an OD.sub.600 of 0.05
and 0.5 .mu.l of the diluted culture placed onto the shiny surface
of the polycarbonate membrane on the agar plate medium. The plate
with the membrane was incubated at 37.degree. C. for 24 hours
following which; the membrane is transferred to a new agar medium
for another 24 hours for biofilm formation.
[0392] Bactericidal Assay for Biofilm Bacteria. Polycarbonate
membrane with the biofilm was immersed in 250 .mu.l of 20%
P.sub.4075EC with 20 mM EDTA and 7% sodium chloride (NaCl) in a 1.5
ml eppendorf tube for 3 hours at 37.degree. C. Seven percent NaCl
served as the negative control. Following incubation, the membrane
was transferred to 15 ml sterile polypropylene tubes containing 10
ml of sterile PBS. One ml of sterile PBS was added to the eppendorf
reaction tubes, vortexed and spun at 3500 rpm for 15 minutes.
Following centrifugation, the supernatant containing the NE was
removed and the bacterial pellet resuspended in their respective 15
ml tube in which the polycarbonate membrane was collected. The 15
ml tube was then vortexed at maximum speed for 3 minutes to get
bacteria from membrane into the PBS. One hundred microliters of
undiluted and diluted treated and control samples were plated onto
LB agar plates for colony count.
[0393] As shown in FIGS. 13-16, 20% P.sub.4075EC with 7% saline and
20 mM EDTA was effective to kill bacteria present in biofilms
comprising a single bacterial strain (See, e.g., FIG. 13, biofilms
comprising a plurality of bacterial strains (See, e.g., FIGS.
14-15), as well as biofilms generated from bacteria that escaped
killing in a first round of treatment (See, e.g., FIG. 16),
attaining between a 4 to 9 log reduction in bacterial numbers.
Example 5
[0394] This example describes the effectiveness of various
nanoemulsion formulations against various gram negative pathogens
that may be associated with CF, wounds, burns and burn patients, or
other environments.
[0395] As noted above, CF is characterized by chronic respiratory
infection that begins early in life with Staphylococcus aureus and
Haemophilus influenzae infections and later colonization with
mucoid strains of Pseudomonas aeruginosa. In CF lung disease, early
colonization with Burkholderia cepacia correlates with a poor
prognosis in CF patients. Patients are often prescribed inhaled
tobramycin to prevent exacerbations of bacteria infections. With
time, patients can become unresponsive to tobramycin
therapy/prophylaxis. New inhaled topical agents that are not
cross-resistant to known antibiotics would be valued as an
alternative to inhaled tobramycin.
[0396] Several topical nanoemulsions were evaluated for
microbiological activity against gram-negative isolates, including
P. aeruginosa and B. cepacia. All 3 nanoemulsions kill quickly in
the presence of an outer membrane permeabilizer such as EDTA. The
nanoemulsions were evaluated against 23 of the 35 gram-negative
isolates for cidal activity, and all were bactericidal (85-95%).
Thus, the one or more of these novel nanoemulsions can be used to
prevent exacerbation of chronic pulmonary infections.
[0397] Methods: MICs and MBCs were determined using CLSI standard
methods. MICs to nanoemulsions were determined in the presence of 5
mM EDTA, a known enhancer of nanoemulsion activity. The addition of
alamar blue, a redeox indicator that yields a colorimetric change
in response to metabolic activity, was used to determine the MICs
of nanoemulsions that are opaque at higher concentrations.
[0398] Emulsion manufacturing: Nanoemulsions W.sub.205EC,
P.sub.4075EC, and W.sub.205GBA.sub.2ED are oil-in-water emulsions
manufactured from ingredients that are Generally Recognized As Safe
(GRAS) with a cationic detergent (cetylpyridinium chloride (CPC) or
benzalkonium chloride (BA)) as active ingredients that have proven
safe for oral human use. The emulsion is formed from highly
purified oil, ethanol, a nonionic surfactant and water. The average
nanoemulsion droplet size was 180 nm for W.sub.205EC and 350 nm for
P.sub.4075EC and W.sub.205GBA.sub.2ED, as measured by dynamic light
scattering using a Malvern Zetasizer Nano3600 (Malvern Instruments
Ltd., Worcestershire, UK). The formulations for W.sub.205EC and
P.sub.4075EC are described above. W.sub.205GBA.sub.2ED (v/v %) is
distilled water (18.93%), TWEEN 20 (5%), Glycerine (8%), Soybean
oil (64%), BTC 824 50% (4%) (Stepan, Northfield, Ill.), and EDTA
(0.0745%). The formulation may be diluted in water (e.g., 60%
W.sub.205GBA.sub.2ED, 40% water) to provide 60%
W.sub.205GBA.sub.2ED. This material may be diluted to a 10%
formulation with 10 mM EDTA (e.g., 76 g water per 24 g 100 mM EDTA
per 20 g 60% W.sub.205GBA.sub.2ED). Thus, in some embodiments, a
formulation comprising oil, water, glycerine, surfactant, and BTC
(n-alkyl dimethyl benzyl ammonium chloride), and/or EDTA is
provided.
[0399] Source of isolates. The source of the clinical isolates were
blood stream or skin and soft tissue isolates collected by JMI
Laboratories in the past 2 years.
[0400] MIC and MBC determinations. MICs of W.sub.205EC.+-.0, 5, 10,
15, and 20 mM EDTA were evaluated in cation-adjusted Mueller-Hinton
broth by microdilution per M7-A7 (2006). MICs for a panel of
gram-negative isolates were determined for the nanoemulsions,
W205EC, P4075EC, and W205GBA2ED in the presence of 5 mM EDTA. EDTA
was used to permeabilize the gram-negative envelope, aiding
infusion of the nanodroplets to the cell membrane, resulting in
lysis. Alamar blue was added to the assay panels 2 hours
post-inoculation for enhanced MIC endpoint detection. MBC values
were assessed for all nanoemulsions and a comparator compound
(fusidicacid) by plating the entire broth content from the MIC well
and from those four doubling dilutions above the MIC for each
selected organism onto blood agar growth media. Quantitative colony
counts were performed on the initial inoculum. The lowest
concentration of antimicrobial agent that killed.gtoreq.9.9% of the
starting test inoculum was defined as the MBC endpoint. See Tables
2, 3, and 4.
TABLE-US-00002 TABLE 2 MIC values of W205EC + EDTA Bacterial
Isolates 0 mM 5 mM 10 mM 15 mM 20 mM K. pneumoniae 64 8-16 8 8 8
24-5A E. coli ATCC 16 1 .ltoreq.0.12 .ltoreq.0.12 .ltoreq.0.12
25922 P. mirabilis 128 .ltoreq.0.12* .ltoreq.0.12 .ltoreq.0.12
.ltoreq.0.12 119-163A A. baumannii 32 .ltoreq.0.12* .ltoreq.0.12
.ltoreq.0.12 .ltoreq.0.12 67-299A P. aeruginosa >256 32-64 32 32
32 ATCC 27853 B. cepacia >256 >256 >256 >256 >256
30-492A *EDTA alone inhibited these strains.
TABLE-US-00003 TABLE 3 Cidality of nanoemulsions against a subset
(23) of gram-negative isolates MBC/ MIC Compound (number of
strains) ratio W.sub.205EC P.sub.4075EC W.sub.205GBA.sub.2ED
Levofloxacin Cidal .uparw. 1 7 10 9 9 2 6 7 7 8 4 4 2 2 1 Static
.dwnarw. 8 1 1 1 >8x 2 1 5 No 3 3 3 growth*
TABLE-US-00004 TABLE 4 Escherichia coli (n = 5).sup.a Klebsiella
pneumoniae (n = 5) % % Antimicrobial susceptible/ susceptible/
agent MIC.sub.50 MIC.sub.90 Range resistant.sup.b MIC.sub.50
MIC.sub.90 Range resistant W.sub.205EC 2 -- 1-2 --/-- 8 -- 4-16
--/-- P.sub.4075EC 4 -- 4 --/-- 8 -- 8-16 --/-- W.sub.205G BA.sub.2
ED 2 -- 2-4 --/-- 4 -- 4-8 --/-- Ceftazidime <=1 --
<=1->16 80.0/20.0 <=1 -- <=1->16 80.0/20.0 Cefepime
<=0.12 -- <=0.12->16 80.0/20.0 <=0.12 --
<=0.12->16 80.0/20.0 Piperacillin/ 2 -- 1-8 100.0/0.0 2 --
1-64 80.0/0.0 tazobactam Imipenem 0.25 -- <=0.12-0.5 .sup.
100.0/0.0 0.25 -- <=0.12-0.25 .sup. 100.0/0.0 Gentamicin <=2
-- <=2->8 80.0/20.0 <=2 -- <=2 100.0/0.0 Tobramycin 0.5
-- 0.25-16 80.0/20.0 0.25 -- 0.25-16 80.0/20.0 Levofloxacin 0.03 --
0.03->8 60.0/40.0 0.06 -- 0.06-8 80.0/20.0 Tetracycline >8 --
4->8 20.0/80.0 <=2 -- <=2-4 .sup. 100.0/0.0 Colistin
<=0.5 -- <=0.5 --/-- <=0.5 -- <=0.5 --/-- Proteus
mirabilis (n = 5) Pseudomonas aeruginosa (n = 5) % % Antimicrobial
susceptible/ susceptible/ agent MIC.sub.50 MIC.sub.90 Range
resistant MIC.sub.50 MIC.sub.90 Range resistant W.sub.205EC 8 --
4-8 --/-- 16 -- 16-32 --/-- P.sub.4075EC 16 -- 8-16 --/-- 64 --
32-64 --/-- W.sub.205G BA.sub.2 ED 8 -- 4-16 --/-- 16 -- 8-16 --/--
Ceftazidime <=1 -- <=1 100.0/0.0 2 -- 2-4 100.0/0.0 Cefepime
<=0.12 -- <=0.12 100.0/0.0 4 -- 1-8 100.0/0.0 Piperacillin/
<=0.5 -- <=0.5-1 .sup. 100.0/0.0 2 -- 2-16 100.0/0.0
tazobactam Imipenem 1 -- <=0.12-2 .sup. 100.0/0.0 1 -- 1->8
80.0/20.0 Gentamicin <=2 -- <=2 100.0/0.0 <=2 -- <=2-4
.sup. 100.0/0.0 Tobramycin 1 -- 0.25-1 100.0/0.0 0.5 -- 0.25-1
100.0/0.0 Levofloxacin 0.12 -- 0.06-2 100.0/0.0 1 -- 0.25-4
80.0/0.0 Tetracycline >8 -- >8 0.0/100.0 8 -- 8->8
0.0/40.0 Colistin >4 -- >4 --/-- 1 -- <=0.5-2 .sup.
100.0/0.0 Acinetobacter baumannii (n = 5).sup.b Stenotrophomonas
maltophilia (n = 5).sup.c % % Antimicrobial susceptible/
susceptible/ agent MIC.sub.50 MIC.sub.90 Range resistant MIC.sub.50
MIC.sub.90 Range resistant W.sub.205EC <=0.12 -- <=0.12-2
.sup. --/-- <=0.12 -- <=0.12 --/-- P.sub.4075EC <=0.12 --
<=0.12-4 .sup. --/-- <=0.12 -- <=0.12 --/-- W.sub.205G
BA.sub.2 ED <=0.12 -- <=0.12-4 .sup. --/-- <=0.12 --
<=0.12 --/-- Ceftazidime 4 -- <=1->16 80.0/20.0 16 --
2->16 40.0/40.0 Cefepime 2 -- <=0.12->16 80.0/20.0 >16
-- 4->16 --/-- Piperacillin/ 2 -- <=0.5->64 80.0/20.0
>64 -- 8->64 --/-- tazobactam Imipenem <=0.12 --
<=0.12->8 .sup. 80.0/20.0 >8 -- 4->8 --/-- Gentamicin
<=2 -- <=2->8 80.0/20.0 >8 -- <=2->8 --/--
Tobramycin 0.5 -- 0.25->16 80.0/20.0 >16 -- 0.5->16 --/--
Levofloxacin 0.25 -- 0.06->8 80.0/20.0 0.5 -- 0.5-8 60.0/20.0
Tetracycline <=2 -- <=2->8 80.0/20.0 >8 -- <=2->8
--/-- Colistin <=0.5 -- <=0.5-2 .sup. 100.0/0.0 <=0.5 --
<=0.5-1 .sup. --/-- Burkholderia cepacia (n = 5) % Antimicrobial
susceptible/ agent MIC.sub.50 MIC.sub.90 Range resistant
W.sub.205EC 256 -- 64->256 --/-- P.sub.4075EC 256 -- 128->256
--/-- W.sub.205G BA.sub.2 ED 128 -- 32->256 --/-- Ceftazidime 2
-- 2-4 100.0/0.0 Cefepime 8 -- 4-16 --/-- Piperacillin/ 4 -- 2-32
--/-- tazobactam Imipenem 8 -- 4-8 --/-- Gentamicin >8 -- >8
--/-- Tobramycin >16 -- >16 --/-- Levofloxacin 2 -- 1-2
100.0/0.0 Tetracycline >8 -- >8 --/-- Colistin -- -- >4
--/--
[0401] Results: All 3 nanoemulsions had activity against
Gram-negative isolates (EDTA alone inhibited 3 and 5 strains of A.
baumannii and S. maltophilia, respectively). This included isolates
that were resistant to comparator agents. MBCs were performed for
23 isolates; W.sub.205EC, P.sub.4075EC, and W.sub.205GBA.sub.2ED
were bactericidal against 85, 95, and 90% of the isolates,
respectively. No cross-resistance to any known antibiotic was
observed for any of the nanoemulsions.
[0402] Conclusions: Nanoemulsions were broadly active against
Gram-negative species, including multidrug-resistant isolates. The
documented MICs were within the range of concentrations achievable
with topical application to skin or mucosal tissues. One or more of
the nanoemulsions may be useful for prophylaxis and/or therapeutic
treatment of chronic pulmonary infections in cystic fibrosis
patients.
Example 6
[0403] The purpose of this example was to determine if
nanoemulsions according to the invention have activity against
Haemophilus influenzae isolates. H. influenzae is an important
respiratory pathogen implicated in acute exacerbations of cystic
fibrosis patients as well as upper and lower respiratory tract
infections for non-CF normal individuals.
[0404] This example determined the minimum inhibitory concentration
of three exemplary nanoemulsions (W.sub.205EC, P.sub.4075EC and
W.sub.205GBA.sub.2) plus comparator drugs against clinical isolates
of H. influenzae. The results, as described in more detail below,
show that P.sub.4075EC and W.sub.205EC had similar MIC ranges of
1.88-3.75 .mu.g/ml. W.sub.205GBA.sub.2 had a MIC range of 8-16
.mu.g/ml. The addition of EDTA at 1 mM to each well containing
nanoemulsion had no effect on the MIC. Concentrations of EDTA
greater than 1 mM inhibited the growth of bacteria, likely reducing
the concentration of cations to levels below those that support
growth. MBC data for the three nanoemulsions+1 mM EDTA were
obtained for seven strains. The MBCs were within 4-fold of the
respective MICs, consistent with each nanoemulsion having
bactericidal activity against H. influenzae isolates.
A. Materials and Methods
[0405] Source of drugs. W.sub.205EC, lot x1151, at a concentration
of 5000 .mu.g/ml (stock concentration) was a pooled sample of
clinical trial material (NB-001), manufactured at Patheon,
Mississauga, Ontario, Canada. P.sub.4075EC, lot X1138, and
W.sub.205GBA.sub.2, lot X1103, were manufactured at NanoBio
Corporation. Comparator compounds, azithromycin, ampicillin,
cefepime and clindamycin were purchased from The United States
Pharmacopeia; catalog numbers were 1046056, 1033000, 1097636,
1136002, respectively. Tetracycline was purchased from Fluka
Biochemika, catalog number 87128. The emulsions were produced by
mixing a water immiscible oil phase with an aqueous phase. The base
formulations are shown in Table 5 below and represent the neat
emulsions, which were further diluted to the desired %.
Compositions are w/w % unless otherwise noted.
TABLE-US-00005 TABLE 5 Nanoemulsions Nanoemulsion Component Weight
Percent (w/w %) W.sub.205EC ED Distilled Water 23.418% EDTA 0.0745%
Cetylpyridinium Chloride 1.068% Tween 20 5.92% Ethanol 6.73%
Soybean Oil 62.79% P.sub.4075EC Distilled Water 23.49% CPC 1.068%
Poloxamer 407 5.92% Ethanol 6.73% Soybean Oil, NP 62.79%
W.sub.205GBA.sub.2 (v/v %) Distilled Water 20.93% BTC 824 2% Tween
20 5% Glycerine 8% Soybean Oil 64%
[0406] Source of bacterial strains. Ten clinical isolates of H.
influenzae were obtained from Case Western Reserve University,
Cleveland, Ohio. The quality control isolate, ATCC 49247, was
obtained from the American Type Culture Collection.
[0407] Susceptibility assays. The susceptibility of H. influenzae
isolates to five commercial antibiotics and three nanoemulsion
formulations was evaluated using the guidelines published by
Clinical Laboratory Standards Institute (Clinical and Laboratory
Standards Institute. Performance Standards for Antimicrobial
Susceptibility and Testing; Seventeenth Informational Supplement.
CLSI document MI00-S17 (ISBN 1-56238-625-5). CLSI, 940 West Valley
Road, Suite 1400, Wayne, Pa. 19087-1988, 2007). The ranges of drug
concentrations tested were as follows: Azithromycin at 0.25-128
.mu.g/ml, Ampicillin at 0.063-32 .mu.g/ml, Cefepime at 0.063-32
.mu.g/ml, Clindamycin at 0.125-64 .mu.g/ml, Tetracycline at
0.125-64 .mu.g/ml, P.sub.4075EC at 0.12-60 .mu.g/ml cetylpyridinium
chloride, W.sub.205EC at 0.12-60 .mu.g/ml cetylpyridinium chloride,
W.sub.205GBA.sub.2 at 0.125-64 .mu.g/ml benzalkonium chloride.
Since nanoemulsions are not a single component, the MICs/MBCs are
expressed as the concentration of the cationic surfactant;
W.sub.205EC and P.sub.4075EC both contain cetylpyridinium chloride
(CPC) as the cationic surfactant, while W.sub.205GBA.sub.2 contains
benzalkonium chloride (BA) as the cationic surfactant. Isolates
were also tested with the same range of nanoemulsion
concentrations+1-5 mM EDTA to see if the latter enhanced the
activity of the nanoemulsion. EDTA concentrations were tested alone
to ensure that chelation of cations by EDTA was sufficient to
inhibit bacterial growth independent of the nanoemulsion.
[0408] Minimum inhibitory concentrations (MIC) were determined
visually as the first totally clear well in the series of the 10
concentrations of each drug. For the nanoemulsions, because they
are intrinsically opaque, the MIC was determined as the first well
observed to have some clearing from the least concentrated to the
most concentrated of the serial dilution. MBC values were assessed
for the nanoemulsions+1 mM EDTA for 7 clinical strains and the ATCC
49257 isolate by plating 10 .mu.l from the well determined to be
the MIC and 4 wells above the MIC onto chocolate blood agar media.
The lowest concentration of antimicrobial agent that
killed.gtoreq.99.9% of the starting test inoculum was defined as
the MBC. A compound or nanoemulsion is defined as bactericidal if
its MBC/MIC ratio was .ltoreq.4.
[0409] Results: Data for the MICs and MBCs are shown in Tables 6-7.
P.sub.4075EC, W.sub.205EC, and W.sub.205GBA.sub.2 were equally
effective antimicrobials in the presence or absence of 1 mM EDTA.
The MIC.sub.90 values for these compounds were 3.75, 3.75, and 16
.mu.g/ml, respectively (Table 6). By MIC.sub.90 values, the
majority of isolates were susceptible to azithromycin, cefepime,
tetracycline and ampicillin, but were resistant to clindamycin and
ampicillin.
[0410] The MBC values were within 4-fold of the respective MICs for
each isolate tested (Table 7); thus each of the nanoemulsions were
bactericidal for H. influenzae, with a range of MBCs for
P.sub.4075EC, W.sub.205EC, and W.sub.205GBA.sub.2 of 1.88-7.5,
1.88-7.5 and 8-16 .mu.g/ml, respectively.
[0411] Conclusions: P.sub.4075EC and W.sub.205EC appeared equally
effective against clinical isolates of H. influenzae. The
benzalkonium chloride formulation, W.sub.205GBA.sub.2, was two- to
four-fold less active than the other nanoemulsions by MIC.sub.90
and MBC values. EDTA at concentrations.gtoreq.2 mM EDTA inhibited
the growth of H. influenzae under these growth conditions. The
addition of EDTA was not necessary to enhance the activity of any
of the nanoemulsions.
TABLE-US-00006 TABLE 6 Susceptibility of H. influenzae clinical
isolates to nanoemulsions and control antibiotics MIC (.mu.g/ml)
P.sub.4075EC + W.sub.205EC + W.sub.205GBA.sub.2 + 1 mM 1 mM 1 mM
Strain P.sub.4075EC.sup.a EDTA W.sub.205EC.sup.a EDTA
W.sub.205GBA.sub.2.sup.b EDTA Azi.sup.c Amp.sup.c Cef.sup.c
Tet.sup.c Cli.sup.c 49247 1.88 1.88 1.88 0.94 8 4 1 4 1 16 8 30
1.88 0.94 1.88 0.94 4 8 2 0.25 0.125 0.5 16 32 1.88 1.88 1.88 3.75
8 4 8 32 0.125 2 8 33 3.75 1.88 3.75 3.75 8 8 1 0.25 0.0625 1 16 34
3.75 3.75 3.75 3.75 8 8 1 >32 0.125 1 16 35 3.75 3.75 7.5 7.5 16
16 1 0.25 0.125 0.5 8 36 3.75 3.75 3.75 3.75 8 16 2 0.125 0.125 1
16 37 1.88 1.88 3.75 3.75 16 16 1 >32 0.125 1 4 38 3.75 3.75
3.75 3.75 16 16 1 0.25 0.0625 0.5 16 41 1.88 0.94 1.88 0.94 8 4 2
>32 0.25 1 8 42 1.88 1.88 1.88 3.75 8 8 1 >32 0.125 1 4 MIC
1.88-3.75 0.94-3.75 1.88-3.75 0.94-7.5 4-16 4-16 1-8 0.25->32
0.0625-1 0.5-16 4-16 range MIC.sub.90 3.75 3.75 3.75 3.75 16 16 2
>32 0.125 2 16 .sup.aMIC value reflects the amount of .mu.g
cetylpyridinium chloride/ml .sup.bMIC values reflects the amount of
.mu.g benzalkonium chloride/ml .sup.cAzi = azithromycin; Amp =
ampicillin; Cef = cefepime; Tet = tetracycline; Cli =
clindamycin
TABLE-US-00007 TABLE 7 Cidality of nanoemulsions against H.
influenzae clinical isolate MBC (.mu.g/ml) P407 5EC + W.sub.205EC +
1 mM W.sub.205GBA.sub.2 + Strain 1 mM EDTA EDTA 1 mM EDTA 49247
1.88 1.88 8 30 1.88 1.88 8 32 3.75 3.75 8 34 7.5 3.75 8 35 7.5 7.5
16 36 7.5 7.5 16 37 3.75 3.75 8 38 7.5 7.5 16
[0412] For Table 8 below, drug concentrations are in .mu.g/ml for
the comparators, as .mu.g cetylpyridinium chloride (CPC)/ml for
W.sub.205EC and P.sub.4075EC and as .mu.g benzalkonium chloride
(BA)/ml for W.sub.205GBA.sub.2. NC is for Negative Control and GC
is for Growth Control. The chart describes the wells of a 96 well
microtiter plate. The rows listed as I, J, K, and L are the rows of
a partial plate used to evaluate the nanoemulsions with different
EDTA concentrations. For each clinical strain and control strain,
the chart below was used for tracking the MIC and MBC data. If the
wells were too opaque to read the MIC ( ), the MBC was relied upon
to provide the MIC. In Table 8, contain plate counts for MBC. The
second number is the plate count of a 10 .mu.l sample from the
microtiter plate used to determine the MBC. The MBC was defined as
.gtoreq.3-log reduction of the inoculum. The darker highlighting
corresponds to MIC.
TABLE-US-00008 TABLE 8 H. influenzae MIC of Nanoemulsions with EDTA
##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005##
##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010##
##STR00011## Highlighted in Green are rows containing plate counts
for the MBC. The second number is the plate count of a 10 .mu.l
sample from the microtiter plate used to determine the MBC. The MBC
was defined as .gtoreq.3-log reduction of the inoculum. Azi =
azithromycin: Amp = ampicillin: Cef = cefepime: Cli = clindamycin:
Tet = tetracycline.
Example 7
[0413] The purpose of this study was to determine the MIC and MBC
of four nanoemulsions (NEs) according to the invention and compare
the performance of the NEs to conventional antibiotics against
MRSA. MRSA is one of the microorganisms that cause lung infections
in young cystic fibrosis patients and is also implicated in
infections of skin and soft tissue, including burn wound
infections. Because of its varied virulence mechanisms, infections
caused by MRSA can be lethal.
[0414] The purpose of this example was to evaluate 4 nanoemulsions
according to the invention for use in the topical treatment of
infectious diseases. The nanoemulsions tested were W.sub.805EC,
W.sub.205ECEDL2, W.sub.205GBA.sub.2ED and P.sub.4075EC. This
example determined the minimum inhibitory concentration (MIC) of
the four nanoemulsions plus comparator drugs against 29 clinical
isolates of methicillin-resistant Staphylococcus aureus (MRSA).
[0415] The results shown below demonstrate that the MIC ranges for
the nanoemulsions were 0.5-2 (W.sub.805EC), 0.5-4
(W.sub.205ECEDL2), 1-4 (W.sub.205GBA.sub.2ED), 1-4 .mu.g/ml
(P.sub.4075EC), respectively. Minimum bactericidal concentration
(MBC) data was also collected and the MBC ranges were 0.5-16
(W.sub.805EC), 1->32 (W.sub.205ECEDL2), 2-16
(W.sub.205GBA.sub.2ED), and 1->16 .mu.g/ml (P.sub.4075EC),
respectively.
A. Methods and Materials
[0416] Source of drugs and isolates. Twelve comparator compounds,
clindamycin (USP #1136002), doxycycline (USP #1226003),
erythromycin (Sigma #E0774), levofloxacin (Sigma #28266), mafenide
acetate (USP #1373008), mupirocin (Sigma #M7694), silvadene (USP
#61260), vancomycin (Sigma #V1764), Oxacillin (Sigma #28221),
sulfamethoxazole (USP #1631001), trimethoprim (USP # 1692505), and
silver nitrate (VWR #VW3462-0) were compared to the four
nanoemulsions. The four nanoemulsions were (a) W.sub.805EC, (b)
W.sub.205ECEDL2, (c) W.sub.205GBA.sub.2ED and (d) P.sub.4075EC. See
Table 10, which provides drug panel templates (with increasing
dilutions across the panel). The emulsions were produced by mixing
a water immiscible oil phase with an aqueous phase. The
W.sub.205ECEDL2 formulation was microfluidized. The base
formulations are shown in Table 9 below and represent the neat
emulsions, which were further diluted to the desired %.
Compositions are w/w % unless otherwise noted.
TABLE-US-00009 TABLE 9 Nanoemulsions Weight Percent Nanoemulsion
Component (w/w %) W.sub.805EC Water 23.490% Ethanol 6.730%
Cetylpyridinium Chloride 1.068% Polysorbate 80 5.920% Refined
Soybean Oil 62.790% W.sub.205ECEDL2 Distilled Water 23.418% EDTA
0.0745% Cetylpyridinium Chloride 1.068% Tween 20 5.92% Ethanol
6.73% Soybean Oil 62.79% W.sub.205GBA.sub.2ED (v/v %) Distilled
Water 20.93% EDTA 0.0745% BTC 824 2% Tween 20 5% Glycerine 8%
Soybean Oil 64% P.sub.4075EC Distilled Water 23.49% CPC 1.068%
Poloxamer 407 5.92% Ethanol 6.73% Soybean Oil, NP 62.79%
TABLE-US-00010 TABLE 10 Drug Panel Templates Drug Panel Templates
Compound in .mu.g/ml (% for AgNO.sub.3) Compound 1 2 3 4 5 6 7 8 9
10 11 12 A Clindamycin 64 32 16 8 4 2 1 0.5 0.25 0.125 NC GC B
Doxycycline 128 64 32 16 8 4 2 1 0.5 0.25 NC GC C Erythromycin 128
64 32 16 8 4 2 1 0.5 0.25 NC GC D Levofloxacin 64 32 16 8 4 2 1 0.5
0.25 0.125 NC GC E Mafenide acetate 128 64 32 16 8 4 2 1 0.5 0.25
NC GC F Mupirocin 64 32 16 8 4 2 1 0.5 0.25 0.125 NC GC G Silvadene
128 64 32 16 8 4 2 1 0.5 0.25 NC GC H Vancomycin 64 32 16 8 4 2 1
0.5 0.25 0.125 NC GC A Oxacillin 16 8 4 2 1 0.5 0.25 0.125 0.0625
0.03125 NC GC B Sulfamethazole 32 16 8 4 2 1 0.5 0.25 0.125 0.0625
NC GC C Trimethoprim 64 32 16 8 4 2 1 0.5 0.25 0.125 NC GC D
AgNO.sub.3 0.01 0.005 0.0025 0.00125 0.00063 0.00031 0.00016
0.00008 0.00004 0.00002 NC GC E P.sub.4075EC 32 16 8 4 2 1 0.5 0.25
0.125 0.0625 NC GC F W.sub.205ECEDL2 32 16 8 4 2 1 0.5 0.25 0.125
0.0625 NC GC G W.sub.205GBA.sub.2ED 64 32 16 8 4 2 1 0.5 0.25 0.125
NC GC H W.sub.805EC 32 16 8 4 2 1 0.5 0.25 0.125 0.0625 NC GC
[0417] Source of bacterial strains. Twenty-nine clinical isolates
of MRSA were obtained from the University of Dentistry and Medicine
of New Jersey. The quality control isolate, ATCC 29213, was
obtained from the American Type Culture Collection. Table 11 below
provides the phenotype/genotype of each strain.
TABLE-US-00011 TABLE 11 Phenotype and genotype of MRSA isolates
Strain Phenotype or Genotype Number MLST spatype spa repeat pattern
SCCmec PFGE PVL 2394 ST59 17 ZI-DI-MI-DI-MI-NI-KI-BI IV USA1000 +
2402 2926 ST5 2 TI-JI-MI-BI-MI-DI-MI-GI-MI-KI II - 9897 ST1 131
UI-JI-JI-FI-KI-BI-PI-EI IV USA400 + 11118 131
UI-JI-JI-FI-KI-BI-PI-EI IV USA400 + 11512 ST36 16
WI-GI-KI-AI-KI-AI-OI-MI-QI-QI-QI II - 11540 ST8 1
YI-HI-GI-FI-MI-BI-QI-BI-LI-OI IV USA300 + 11554 1
YI-HI-GI-FI-MI-BI-QI-BI-LI-OI IV + 13219 2
TI-JI-MI-BI-MI-DI-MI-GI-MI-KI IV - 13367 2
TI-JI-MI-BI-MI-DI-MI-GI-MI-KI II - 13386 2
TI-JI-MI-BI-MI-DI-MI-GI-MI-KI II - 13408 2
TI-JI-MI-BI-MI-DI-MI-GI-MI-KI IV - 13606 2
TI-JI-MI-BI-MI-DI-MI-GI-MI-KI II - 13610 1
YI-HI-GI-FI-MI-BI-QI-BI-LI-OI I - 13643 1
YI-HI-GI-FI-MI-BI-QI-BI-LI-OI IV + 13693 7
YI-HI-GI-CI-MI-BI-QI-BI-LI-OI IV - 13701 2
TI-JI-MI-BI-MI-DI-MI-GI-MI-KI II 13722 2
TI-JI-MI-BI-MI-DI-MI-GI-MI-KI II - 13756 2
TI-JI-MI-BI-MI-DI-MI-GI-MI-KI II - 13759 1
YI-HI-GI-FI-MI-BI-QI-BI-LI-OI IV - 13868 2
TI-JI-MI-BI-MI-DI-MI-GI-MI-KI II - 15337 18 WI-FI-KI-AI-OI-MI-QI II
- 18998 1 YI-HI-GI-FI-MI-BI-QI-BI-LI-OI IV USA300 + (MUP-R) 19001 1
YI-HI-GI-FI-MI-BI-QI-BI-LI-OI IV USA300 + (MUP-R) 19017 1
YI-HI-GI-FI-MI-BI-QI-BI-LI-OI IV USA300 + (MUP-R) 19024 1
YI-HI-GI-FI-MI-BI-QI-BI-LI-OI IV USA300 + (MUP-R) 19047 1
YI-HI-GI-FI-MI-BI-QI-BI-LI-OI IV + (MUP-R) 19069 ST8 1
YI-HI-GI-FI-MI-BI-QI-BI-LI-OI IV USA300 + (MUP-R) 19156 1
YI-HI-GI-FI-MI-BI-QI-BI-LI-OI IV + (MUP-R)
[0418] Susceptibility assays. The susceptibility of MRSA to twelve
traditional antimicrobials and four nanoemulsion formulations was
tested using the guidelines published by the Clinical Laboratory
Standards Institute (Clinical and Laboratory Standard Institute,
"Antimicrobial Susceptibility and Testing; Seventeenth
Informational Supplement. CLSI document M100-S17 (ISBN
1-56238-625-5), CLSI, Wayne, Pa.). The ranges of drug
concentrations were as follows: clindamycin (64-0.125 .mu.g/ml),
doxycycline (128-0.25 .mu.g/ml), erythromycin (128-0.25 .mu.g/ml),
levofloxacin (64-0.125 .mu.g/ml), mafenide acetate (128-0.25
.mu.g/ml), mupirocin (64-0.125 .mu.g/ml), silvadene (128-0.25
.mu.g/ml), vancomycin (64-0.125 .mu.g/m), oxacillin (16-0.03125
.mu.g/ml), sulfamethoxazole (32-0.0625 .mu.g/ml), trimethoprim
(64-125 .mu.g/ml), silver nitrate (0.01-0.00002%), W.sub.805EC
(32-0.0625 .mu.g/ml), W.sub.205ECEDL2 (32-0.0625 .mu.g/ml),
W.sub.205GBA.sub.2ED (64-0.125 .mu.g/ml), and P.sub.4075EC
(32-0.0625 .mu.g/ml).
[0419] Minimum inhibitory concentrations (MIC) were determined
visually as the first totally clear well in the series of the 10
concentrations of each drug. For the nanoemulsions, because they
are intrinsically opaque, the MIC was called as the first well
observed to have some clearing from the least concentrated to the
most concentrated. Minimum bactericidal concentrations (MBC) values
were assessed for all the antimicrobials tested. The lowest
concentration of antimicrobial agent that killed 2: 99.9% of the
starting test inoculum was defined as the MBC. A compound or
nanoemulsion is defined as bactericidal if its MBC/MIC ratio was
.about.4.
[0420] B. Results
[0421] Data for the MICs and MBCs are shown in Tables 11 and 12.
All four nanoemulsions were potent against the clinical isolates of
MRSA, with no differential activity noted for community-acquired
MRSA (SCCmec type IV) or hospital-associated MRSA (SCCmec types
I-III). Further, there appeared to be no cross-resistance of the
nanoemulsions to any of the known antibiotics. The MIC.sub.90
values were 2 .mu.g/ml for P.sub.4075EC, W.sub.205ECEDL2 and
W.sub.805EC. W.sub.205GBA.sub.2ED had a MIC.sub.90 Of 4 .mu.g/ml.
The MIC.sub.90 values indicated that this collection of MRSA was
susceptible to oral antibiotics, doxycyline (MIC.sub.90=4
.mu.g/ml), sulfamethoxazole (MIC.sub.90=4 .mu.g/ml), trimethoprim
(MIC.sub.90=0.5 .mu.g/ml), and vancomycin (MIC.sub.90=1 .mu.g/ml).
Isolates were generally resistant to oral antibiotics levofloxacin
(MIC.sub.90=32 .mu.g/ml), clindamycin (MIC.sub.90=>64 .mu.g/ml)
and erythromycin (MIC.sub.90=>128 .mu.g/ml). Topical antibiotics
that are used in the treatment of burn wound infections, silvadene
and silver nitrate, had MIC.sub.90 values of 32 .mu.g/ml and
0.00063%, respectively. Mafenide acetate (Sulfamylon) is another
topical treatment for burn wound infections, but it was uniformly
inactive against MRSA (MIC.sub.90=>128 .mu.g/ml). Mupirocin is a
topical treatment for skin and soft tissue infections and is also
used to eradicate carriage of MRSA; 31% of the isolates were
resistant to this antibiotic.
[0422] The MBC.sub.90 values for all of the nanoemulsions were
within four-fold of the respective MIC.sub.90 values, indicating
that the nanoemulsions are bactericidal to .about.90% of the
isolates. Among the other 12 antibiotics, only vancomycin was
bactericidal.
TABLE-US-00012 TABLE 12 Susceptibility of MRSA to nanoemulsions and
comparator compounds: MIC analysis MIC (.mu.g/ml) Rank Maf. Order
Clind Dox Eryth Levo Acet Mup Silva Vanco Ox Sulfa 29 >64 4
>128 >64 >128 >64 64 2 >16 >32 28 >64 4
>128 >64 >128 >64 64 1 >16 >32 27 >64 4
>128 32 >128 >64 32 1 >16 4 26 >64 4 >128 32
>128 >64 32 1 >16 4 25 >64 0.25 >128 32 >128
>64 32 1 >16 2 24 >64 0.25 >128 32 >128 >64 32 1
>16 2 23 >64 0.25 >128 32 >128 >64 32 1 >16 2 22
>64 0.25 >128 16 >128 >64 32 1 >16 1 21 >64 0.25
>128 16 >128 16 32 1 >16 1 20 >64 0.25 >128 16
>128 0.25 32 1 >16 1 19 >64 0.25 >128 16 >128 0.25
32 1 >16 1 18 >64 0.25 >128 8 >128 0.25 32 1 >16 1
17 >64 0.25 >128 8 >128 0.25 32 1 >16 1 16 >64 0.25
128 8 >128 0.25 32 1 >16 1 15 >64 0.25 64 8 >128 0.25
32 1 >16 1 14 >64 0.25 64 8 >128 0.25 32 1 >16 1 13
>64 0.25 64 4 >128 0.25 32 1 >16 1 12 0.125 0.25 64 4
>128 0.25 32 1 >16 0.5 11 0.125 0.25 64 4 >128 0.125 32 1
>16 0.5 10 0.125 0.25 64 4 >128 0.125 32 1 >16 0.5 9 0.125
0.25 32 4 >128 0.125 32 1 >16 0.5 8 0.125 0.25 32 2 >128
0.125 32 0.5 >16 0.5 7 0.125 0.25 32 0.5 >128 0.125 32 0.5
>16 0.5 6 0.125 0.25 16 0.25 >128 0.125 16 0.5 >16 0.25 5
0.125 0.25 8 0.25 >128 0.125 8 0.5 >16 0.25 4 0.125 0.25 0.25
0.25 >128 0.125 8 0.5 >16 0.25 3 0.125 0.25 0.25 0.125
>128 0.125 8 0.5 16 0.25 2 0.125 0.25 0.25 0.125 >128 0.125 8
0.5 16 0.25 1 0.125 0.25 0.25 0.125 128 0.125 8 0.5 0.5 0.0625
MIC.sub.50(15) >64 0.25 64 8 >128 0.25 32 1 >16 1
MIC.sub.90(26) >64 4 >128 32 >128 >64 32 1 >16 4 MIC
(.mu.g/ml) Rank Silver W.sub.205EC W.sub.205G Order Trim Nitrate
P.sub.4075EC ED L2 BA.sub.2 ED W.sub.805EC 29 >64 0.00250 4 4 4
2 28 1 0.00125 4 2 4 2 27 1 0.00125 2 2 4 2 26 0.5 0.00626 2 2 4 2
25 0.25 0.00626 2 2 4 2 24 0.125 0.00626 2 2 4 2 23 0.125 0.00626 2
2 2 2 22 0.125 0.00626 2 2 2 1 21 0.125 0.00626 2 2 2 1 20 0.125
0.00626 2 2 2 1 19 0.125 0.00312 2 1 2 1 18 0.125 0.00312 2 1 2 1
17 0.125 0.00312 1 1 2 1 16 0.125 0.00312 1 1 2 1 15 0.125 0.00312
1 1 2 1 14 0.125 0.00312 1 1 2 1 13 0.125 0.00312 1 1 2 1 12 0.125
0.00312 1 1 2 1 11 0.125 0.00312 1 1 2 1 10 0.125 0.00312 1 1 2 1 9
0.125 0.00312 1 1 2 1 8 0.125 0.00312 1 1 2 1 7 0.125 0.00312 1 1 2
1 6 0.125 0.00312 1 1 2 1 5 0.125 0.00312 1 1 2 1 4 0.125 0.00312 1
1 2 1 3 0.125 0.00312 1 1 1 0.5 2 0.125 0.00312 1 0.5 1 0.5 1 0.125
0.00312 1 0.5 1 0.5 MIC.sub.50(15) 0.125 0.00031 1 1 2 1
MIC.sub.90(26) 0.5 0.00063 2 2 4 2 indicates data missing or
illegible when filed
TABLE-US-00013 TABLE 13 Susceptibility of MRSA to nanoemulsions and
comparator compounds: MBC analysis MBC (.mu.g/ml) Maf. Rank Order
Clind Dox Eryth Levo Acet. Mup Silva. Vanco Ox Sulfa 29 >64
>128 >128 >64 >128 >64 >128 4 >16 >32 28
>64 >64 >128 >64 >128 >64 >128 4 >16 >32
27 >64 >64 >128 >64 >128 >64 >128 4 >16
>32 26 >64 >64 >128 >64 >128 >64 >128 2
>16 >32 25 >64 >64 >128 >64 >128 >64
>128 2 >16 >32 24 >64 >4 >128 >64 >128
>64 >128 2 >16 >32 23 >64 >4 >128 64 >128
>64 >128 2 >16 >32 22 >64 >4 >128 64 >128
>64 >128 2 >16 >16 21 >64 >4 >128 >32
>128 >64 >128 2 >16 >16 20 >64 >4 >128 32
>128 >8 >128 2 >16 >16 19 >64 >4 >128 32
>128 >4 >128 1 >16 >16 18 >64 >4 >128 32
>128 >4 >128 1 >16 >16 17 >64 >4 >128 32
>128 >4 >128 1 >16 >16 16 >64 >4 >128 32
>128 >4 >128 1 >16 >16 15 >64 >4 >128 16
>128 >4 >128 1 >16 >16 14 >64 >4 >128 16
>128 >2 128 1 >16 >16 13 >64 >4 >128 16
>128 >2 128 1 >16 >16 12 >2 >4 >128 8 >128
>2 128 1 >16 >8 11 >2 >4 >128 8 >128 >2 128
1 >16 >8 10 >2 >4 >128 8 >128 >2 128 1 >16
>8 9 >2 >4 >128 8 >128 >2 128 1 >16 >8 8
>2 >4 >128 4 >128 >2 128 1 >16 >8 7 >2
>4 >128 1 >128 >2 128 1 >16 >4 6 >2 >4
>128 0.5 >128 >2 64 1 >16 >4 5 >2 >4 64 0.25
>128 >2 64 1 >16 >4 4 >2 >4 >4 0.25 >128
>2 64 1 >16 >4 3 >2 2 >4 0.25 >128 1 64 0.5
>16 >1 2 2 1 >4 0.25 >128 1 64 0.5 >16 1 1 1 1 >4
0.25 >128 1 64 0.5 1 0.0625 MBC.sub.50(15) >64 >4 >128
16 >128 >2 128 1 >16 >16 MBC.sub.90(26) >64 >64
>128 >64 >128 >64 >128 2 >16 >32 MBC
(.mu.g/ml) Silver W.sub.205EC W.sub.205G Rank Order Trim Nitrate
P.sub.4075EC ED L2 BA.sub.2 ED W.sub.805EC 29 >64 0.01 >16
>32 16 16 28 >16 >0.01 16 8 8 4 27 >16 >0.01 16 8 8
4 26 >4 >0.01 8 8 8 4 25 >2 >0.005 8 8 8 4 24 >2
>0.005 8 4 8 4 23 >2 >0.005 4 4 4 4 22 4 0.005 4 4 4 4 21
2 0.0025 4 4 4 4 20 2 0.0025 4 4 4 4 19 2 0.0025 4 4 4 4 18 1
0.0025 4 4 4 2 17 1 0.0025 4 4 4 2 16 0.5 0.0025 4 4 4 2 15 0.5
0.0025 2 4 4 2 14 0.5 0.0025 2 2 4 2 13 0.5 0.0025 2 2 4 2 12 0.5
0.0025 2 2 4 2 11 0.25 0.0025 2 2 4 2 10 0.25 0.0025 2 2 4 2 9 0.25
0.00125 2 2 4 2 8 0.25 0.00125 2 2 4 2 7 0.25 0.00125 2 2 4 2 6
0.25 0.00125 2 2 4 2 5 0.125 0.00125 2 2 4 1 4 0.125 0.00125 2 2 4
1 3 0.125 0.00125 2 2 2 1 2 0.125 0.00125 1 1 2 1 1 0.0125 0.00125
1 1 2 0.5 MBC.sub.50(15) 0.5 0.0025 2 2 4 2 MBC.sub.90(26) >4
>0.01 8 8 8 4 indicates data missing or illegible when filed
C. Conclusions
[0423] W.sub.805EC, W.sub.205ECEDL2, W.sub.205GBA.sub.2ED and
P.sub.4075EC appeared equally effective against clinical isolates
of MRSA, with MIC.sub.90 values of 2 or 4 .mu.g/ml. In addition,
based on the ratio of MBC.sub.90/MIC.sub.90, the four nanoemulsions
were bactericidal. As expected, the MRSA were multidrug-resistant,
emphasizing the need for new agents for treating MRSA infections.
Since nanoemulsions according to the invention also have activity
against serious gram-negative pathogens, such as Pseudomonas
aeruginosa and Burkholderia spp. (LiPuma et al., "In vitro
activities of a novel nanoemulsion against Burkholderia and other
multidrug-resistant cystic fibrosis-associated bacterial species,"
Antimicrob. Agents Chemother., 53:249-255 (2009)), the
nanoemulsions are useful for inhalation treatment/maintenance of
cystic fibrosis patients. In addition, the nanoemulsions are useful
in treating burn wounds to prevent/treat infections of the
pathogenic agents described herein.
Example 8
[0424] The purpose of this example was to determine the minimum
inhibitory concentration (MIC) and the minimum bactericidal
concentration (MBC) of an exemplary nanoemulsion (P.sub.4075EC)
side by side with comparator drugs against clinical isolates of
Pseudomonas aeruginosa, Burkholderia cenocepacia, Acinetobater
baumanni, Stenotrophomonas maltophilia from patients suffering from
cystic fibrosis (CF).
[0425] Summary of Results for MIC and MBC: P.sub.4075EC+EDTA had an
MIC range of <4-16, <4-64, 2-8, <1-16 .mu.g CPC/ml,
respectively. The addition of EDTA at 5 mM for the Pseudomonas and
Burkholderia and 1 mM to Acinetobacter and Stenotrophomonas to each
well containing nanoemulsion improved the MIC. MBC data for the
nanoemulsion+EDTA were obtained for 20 Pseudomonas, 10
Burkholderia, 10 Acinetobacter and 11 Stenotrophomonas. For three
genera of bacteria tested, the data suggested bacteriostatic
activity. The MBCs were within 4-fold of the respective MICs for
the Stenotrophomonas, consistent with the nanoemulsion having
bactericidal activity.
[0426] Summary of Synergy Results: In addition to the standardized
MIC and MBC determinations, checkerboard synergy studies were
conducted to evaluate the potential of P.sub.4075EC+EDTA to
synergize or antagonize traditional antimicrobials commonly used to
treat patients with cystic fibrosis. The fractional inhibitory
concentration (FIC) index and the fractional bactericidal
concentration (FBC) index were determined to judge if a two drug
combination was synergistic, antagonistic or indifferent to one
another. Ten strains of Burkholderia, 10 strains of
Stenotrophomonas and 10 strains of Acinetobacter were tested to
determine a shift in MIC when P.sub.4075EC+EDTA was in combination
with either colistin or tobramycin, two traditional antimicrobials
used in the lungs of CF patients to treat chronic lung infections.
P.sub.4075EC+EDTA in combination with colistin was found to be
synergistic for 90% (in terms of the FIC) and 70% (in terms of the
FBC) of the Stenotrophomonas strains, but indifferent, only 20%
synergy in by the FIC and 0% by the FBC, when in combination with
tobramycin. For the Acinetobacter strains, P.sub.4075EC+EDTA in
combination with colistin was found to be indifferent, only 20%
synergy in by the FIC and 0% by the FBC, as well as when in
combination with tobramycin, only 10% synergy in by the FIC and 10%
by the FBC. For the Burkholderia strains, P.sub.4075EC+EDTA in
combination with colistin was found to be indifferent, only 30%
synergy in by the FIC and 10% by the FBC, but when in combination
with tobramycin, 50% synergy in by the FIC and 20% by the FBC.
A. Materials and Methods
[0427] Multidrug-resistant Gram-negative bacteria isolated from CF
patients were tested in this example. MICs and MBCs were determined
using CLSI guidelines and standard methods M7-A7 and M100-S17.
Depending on the genus, MICs were performed in the presence of 1 or
5 mM EDTA, a permeability enhancer of NE activity. Stenotrophomonas
and Acintetobacter had 1 mMEDTA in the nanoemulsion. Pseudomonas
and Burkholderia had 5 mM EDTA in the nanoemulsion. This was the
case during the standard MIC and MBC determination as well as the
Checkerboard synergy work and time kill study to be described
below. The addition of alamar blue, a redox indicator that yields a
colorimetric change in response to metabolic activity, was used to
determine the MICs of the NE (P.sub.4075EC) because of opacity at
higher concentrations.
[0428] Checkerboard synergy studies were carried out by using the
MIC and MBC data collected, microtiter plates with a combination
for P.sub.4075EC+EDTA and colistin or P.sub.4075EC+EDTA and
tobramycin.
[0429] The Fractional Inhibitory Concentration (FIC) Index (see
e.g., FIG. 19) examines the ratio of the MIC of a single drug when
in combination with another to the MIC of that drug alone. The
reduction of the MIC when a drug is in combination results in a
fraction that is less than one.
X=MIC of Drug in Combination/MIC of Drug along
This ratio is calculated for both drug A and drug B. The fractions
are added together. The summation is compared to the following
ranges:
[0430] Synergism: Sum of FIC for the two drugs.ltoreq.0.5;
[0431] Indifference: Sum of FIC for the two drugs>0.5 to
.ltoreq.4;
[0432] Antagonism: Sum of FIC for the two drugs>4.
[0433] In FIG. 19A, Row D denotes the previously determined MIC
used to chose the flanking concentrations of the two drugs being
combined. In FIG. 19B, the horizontal slash marks on the X and Y
axis indicate typical MIC concentration of each drug alone.
[0434] The time-kill study was done over a brief time curve of 10,
20, 30 minutes including a 0 minute non-treated control was
conducted to fix samples for pictures under the electron
microscope.
B. Source of isolates
[0435] 46 isolates were evaluated, all of which were obtained from
the University of Maryland-Baltimore School of Dentistry. The
isolates received were as follows: 20 Pseudomonas, 11 Burkholderia,
10 Acinetobacter and 5 Stenotrophomonas. The P. aeruginosa isolates
had defined lipid A modifications that have been documented in many
CF patients. 10 additional isolates of Stenotrophomonas were
received from a second source (Eurofins, Virginia). These 10 are
the last on the list and are the isolates with the ages of the
individuals the samples were taken from. The isolates are
summarized in Table 14 below.
TABLE-US-00014 TABLE 14 Pseudomonas aeruginosa, Burkholderia
cenocepacia, Acinetobacter baumannii, Stenotrophomonas maltophilia
isolates Pseudomonas Aminoarabinose modification Isolate Patient
aeruginosa NanoBio number Probable colistin resistance Location
Identifier 1 NB1 - CF Patient A 2 NB2 - CF Patient A 3 NB3 + CF
Patient B 4 NB4 + CF Patient B 5 NB5 + CF Patient C 6 NB6 + CF
Patient C 7 NB7 + CF Patient D 8 NB8 + CF Patient D 9 NB9 - CF
Patient E 10 NB10 - CF Patient E 11 NB11 - CF Patient F 12 NB12 -
CF Patient G 13 NB13 - CF Patient G 14 NB14 - CF Patient H 15 NB15
- CF Patient I 16 NB16 + CF Patient J 17 NB17 - CF Patient K 18
NB18 - CF Patient L 19 NB19 - CF Patient M 20 NB20 + CF Patient N
Burkholderia Isolate Patient cenocepacia NanoBio number Genomovar
Location Identifier 1 NB21 B. c. Genomovar I CF Patient A 2 NB22 B.
c. Genomovar I CF Patient B 3 NB23 B. c. Genomovar III CF Patient C
4 NB24 B. c. Genomovar III CF Patient D 5 NB25 B. c. Genomovar III
CF Patient E 6 NB26 B. c. Genomovar III CF Patient F 7 NB27 B. c.
Genomovar III CF Patient G 8 NB28 B. c. Genomovar IV CF Patient H 9
NB29 B. c. Genomovar IV CF Patient I 10 NB30 B. c. Genomovar IV CF
Patient J 11 NB28b B. vietnamienis Genomovar V Acinetobacter
Isolate Patient baumannii NanoBio number Colistin Susceptibility
Location Identifier 1 NB31 Sensitive wound, intra- Unknown
abdominal 2 NB32 Sensitive abcess Unknown 3 NB33 Resistant
peritoneal fluid Unknown 4 NB34 Sensitive sub-clav blood Unknown 5
NB35 Resistant blood Unknown 6 NB36 Resistant wound Unknown 7 NB37
Sensitive tissue Unknown 8 NB38 Sensitive urine Unknown 9 NB39
Resistant CSF Unknown 10 NB40 Resistant urine Unknown Isolate
Patient Stenotrophomonas NanoBio number Location Identifier 1 NB41
CF Patient A 2 NB42 CF Patient B 3 NB43 CF Patient C 4 NB44 CF
Patient D 5 NB45 CF Patient E Date Stenotrophomonas Isolation
Source maltophilia Short Id. Region Year Description Age 2482274 1
NEW ENGLAND 2006 Sputum 14 2482270 2 EAST NORTH CENTRAL 2007 Sputum
10 2482275 3 MOUNTAIN 2007 Sputum 17 2482269 4 MID ATLANTIC 2008
Sputum 15 2482273 5 SOUTH ATLANTIC 2008 Sputum 17 2482276 6 EAST
SOUTH CENTRAL 2008 Sputum 9 2482267 7 WEST NORTH CENTRAL 2008
Sputum 13 2482271 8 WEST SOUTH CENTRAL 2008 Sputum 4 2482272 9
PACIFIC 2008 Sputum 16 2482268 10 SOUTH ATLANTIC 2008 Sputum 1
C. Source of drugs
[0436] The drugs and the source thereof used in the drug panel
plates are summarized in the table below.
TABLE-US-00015 TABLE 15 Drugs used in drug panel plates. Ingredient
Manufacturer Lot # Cefepime USP H0G278 Colistin USP G-1 Imipenem
USP H0E040 Levofloxacin Sigma 1333515 Ceftazidime USP H Tobramycin
USP L0E077 Ceftoxin USP J0E038 Piperacillin USP H P407-5EC NanoBio
X1138 and X1180
[0437] The composition of the P.sub.407-5EC is provided in Table
16, below. The emulsion was produced by mixing a water immiscible
oil phase with an aqueous phase. The base formulation is below and
represents the neat emulsion, which was further diluted to the
desired %.
TABLE-US-00016 TABLE 16 Lot # A0499 Neat P.sub.407 5EC Ingredient
w/w % Sterile distilled water 23.49 CPC 1.068 Poloxamer 407 5.92
Ethanol 6.73 Soybean oil 62.79
D. Preparation of Drug Concentrations
[0438] The preparation of drug concentration is described below in
Tables 17 and 18. Specifically, Table 17 provides details regarding
the preparation of the 96-well drug panel plates, and Table 18
provides details regarding the preparation of the 96-well
checkerboard synergy plates.
TABLE-US-00017 TABLE 17 Details describing the preparation of the
96-well drug panel plates. Exp. Stock Molecular Concentrations
Volume Required Weight Potency Starting in Concentration Weight
Name & Lot # g/mole (ug/mg) at X ug/mL: Solvent/Diluent mL
(ug/mL) Needed (mg) Cefepime-USP 571.5 865 32 Phos. Buffer 5.00
3200 18.50 Lot H0G278 pH 6 Colistin 1163 629 8 water 10.00 800
12.72 (Polymyxin E)- USP Lot G-1 Imipenem-USP 317.36 929 32 Phos.
Buffer 5.00 3200 17.22 Lot H0E040 pH 7.2 Levofloxacin- 361.37
999.00 16 1/2 volume 10.00 1600 16.02 Sigma 1333515 water, then 0.1
mol/L NaOH dropwise to dissolve Piperacillin- 535.57 984 1024 1/2
volume 2.00 102400 208.13 USP Lot H water, then 0.1 mol/L NaOH
dropwise to dissolve Tobramycin- 467.52 970 32 water 5.00 3200
16.49 USP Lot L0E077 Cefoxitin-USP 449.44 992 256 1/2 volume 2.00
25600 51.61 Lot J0E038 water, then 0.1 mol/L NaOH dropwise to
dissolve Ceftazidime-Lot H 535.57 852 32 Calcium 2.00 3200 7.51
Carbonate Sln 10% of the weight of compound to be dissolved YELLOW
HIGHLIGHT INDICATES POTENCY TAKEN FROM PERCENT PURITY (99% = 999
ug/mg). 1st Dilute 1/2, nine times from stock in solvent 2nd Dilute
1/50 in medium in a respective well of a 12 well trough. 3rd
Transfer 50 uL from each well of the reservoir into the respective
well for each agent at each concentration. Required with or Solvent
Stock Concentration Volume Needed Volume without and Volume in (mM
or of Initial Stock Needed of Nanoemulsion EDTA Volume mL ug/mL)
(mL) broth (mL) X1138 or with 1 mM Water 4.00 512 0.68 3.32 X1180 =
P.sub.4075EC (@ 6 mg/mL) X1138 or with 5 mM Water 4.00 2056 2.74
1.26 X1180 = P.sub.4075EC (@ 6 mg/mL) ** Because of the high
concentration of this test antimicrobial, a 100x stock could not be
made. The calculations provided a 2x stock that is diluted in broth
from the start and through the nine 1/2 dilutions. A 1/50 dilution
at any time is not required because the dilutions are made directly
in broth. X mM EDTA Diluted EDTA in Broth Broth Stock @ 100x volume
Volume Total Volume EDTA Stock 100 800 200 1000 solution starting
500 0 1000 1000 at 500 mM EDTA used to make these dilutions. For
the required concentration of EDTA, a 1/50 dilution of the EDTA
100x stock was made in each well of the 12 well trough containing
nanoemulsion. Only 10 wells of the 12 well trough contain drug, the
last two wells are for broth only which set up the negative and
positive growth controls.
TABLE-US-00018 TABLE 18 Details describing the preparation of the
96-well checkerboard synergy plates. Required with or Solvent Stock
Concentration Volume Needed Volume without and Volume (mM or of
Initial Stock Needed of Nanoemulsion EDTA Volume in mL ug/mL) (mL)
broth (mL) X1180 = P.sub.4075EC with 1 or Broth 4.00 Varied on
plate (@ 6 mg/mL) 5 mM Broth 4.00 for each strain of bacteria
Colistin no Broth 4.00 Varied on plate (Polymyxin E)- for each
strain USP Lot G-1 of bacteria Tobramycin- no Broth 4.00 Varied on
plate USP Lot L0E077 for each strain of bacteria ** Because of the
high concentration of this test antimicrobial, a 100x stock could
not be made. The calculations provided a 4x stock that is diluted
in broth from the start and through the nine 1/2 dilutions.
E. Determination of MICs and MBCs
[0439] In the 96 well microtiter plate, the inoculated wells were
observed after 20 hours of incubation at 35 degrees C. In each row,
there is a range from high to low concentrations of the drug
respective of the row from left to right, each well 1/2 the
concentration of the adjacent well to the left. The first clear
well in the series was called the MIC. This is the concentration of
drug that inhibited the growth of the bacteria from turning the
broth turbid. Because of the nanoemulsions intrinsic turbidity,
alamar blue (CellTiter Blue, Promega) was added for the row
containing nanoemulsion at a rate of 20 uL to 100 uL of culture
volume. The plate was returned to the incubator for one hour and
the first blue well was called the MIC (wells with bacterial growth
turned pink at lower concentrations of nanoemulsion). The starting
inoculum in each well was generally between 2-7.times.10 5 cfu/mL.
The specific concentration for starting inoculum was determined for
each test plate by sampling the inoculum used to inoculate the
microtiter plate at the time it was set up. The exact concentration
of bacteria was used to determine the MBC for each drug in the
panel for each test plate. The MBC is defined as the concentration
of drug that reduced the number of bacteria 99.9%. The MIC is the
landmark used to begin sampling 10 uL/well from the MIC plus four
wells to the left and put on agar plates, one sample per plate. The
agar plates were incubated for 24 hours at 35 degrees C. and
colonies were counted. The resulting colony counts were compared to
the starting concentration of the inoculum to determine which well,
that is which concentration of drug, resulted in a three log drop
in cfu/mL.
F. Checkerboard Synergy Study
[0440] The starting inoculum in each well was generally between
2-7.times.10 5 cfu/mL. The specific concentration for starting
inoculum was determined for each test plate by sampling the
inoculum used to inoculate the microtiter plate at the time it was
set up. Those exact concentrations of bacteria were used to
determine the MBC for each drug in the panel for each test plate.
The MBC is defined as the concentration of drug that reduced the
number of bacteria 99.9%, a three log drop from the original
concentration of bacteria. The MIC is the landmark used to begin
sampling 10 uL/well from the MIC plus all wells through to column
11 to the right and put on agar plates, one sample per plate. The
agar plates were incubated for 24 hours at 35 degrees C. and
colonies were counted. The resulting colony counts were compared to
the starting concentration of the inoculum to determine which well,
that is which concentration of drug, resulted in a three log drop
in cfu/mL.
[0441] In the 96 well microtiter plate, the inoculated wells were
observed after 20 hours of incubation at 35 degrees C. In each row,
there is a range from high to low concentrations of the drug
respective of the row from right to left, each well 1/2 the
concentration of the adjacent well to the left. Typically, but not
always, the rows contained the nanoemulsion, drug A. Going up and
down in the columns is drug B and here is the second drug tested in
combination going high to low from A to G, respectively. Row H did
not contain the second drug for it to act as the control for drug
A. Column 1 did not contain any drug A so that column 1 could be
the control for drug B. Traditionally, first clear well in the
series from right to left in the rows was called the MIC. This is
the concentration of drug that inhibited the growth of the bacteria
from turning the broth turbid, in this case as indicated by a color
change from blue to pink since alamar blue was used to find the
metabolic activity to define the MIC. To each well 20 uL of
CellTiter Blue from Promega was added. That is 20% of the original
culture volume.
[0442] The well with the MIC and the well with the MBC were noted
for each row. All wells noted for the MIC of the two drugs in
combination were compared to the MIC of concentration of drug A and
drug B each acting alone. For example, a well that contained 2
ug/mL of drug A and 8 ug/mL of drug B would have the concentration
of A at 1 ug/mL when in combination divided by 16 ug/mL when acting
alone. This renders a ratio of 0.125. Further in this example, 8
ug/mL of drug B would be divided by 32 ug/mL of B acting alone. The
ratio for drug B in combination:alone is then 0.25. In this example
the FIC index is said to be 0.125 for drug A and 0.25 for drug B,
the sum is 0.325 in this example. The definition of synergy is for
the sum to be equal to 0.5 or less. Indifference is greater than
0.5 to less than or equal to four. Antagonism is greater than four.
See e.g., FIG. 19 for a detailed illustration.
G. Time-Kill Study
[0443] A bacterial suspension of Burkholderia NB 29@O.D. 0.14-0.20
was made up. In a saline tube, 500 uL saline and 500 uL of the
bacterial suspension was mixed for the TO, non-treated control,
then mixed one to one with TA (Trypton-Azolectin) broth with 5 mM
CaCl.sub.2 to be diluted and plated as described below. In a
treatment tube, added one to one the bacterial suspension to the
treatment mix (2.times.P4075EC at 64 ug/mL CPC and 10 mM EDTA) and
after 10, 20 and 30 minutes drew 400 uL at each time point and
mixed with 400 uL TA broth with 5 mM CaCl.sub.2. Next, 100 uL were
drawn to be diluted serially 1/10 in saline to 10.sup.-8. 100 uL
were then drawn from each dilution tube in the series and plated in
triplicate. These plates were incubated at 35.degree. C. overnight
and counted to determine the cfu/mL at each time point.
Simultaneously at the time the sample of untreated or treated
bacteria was added to the neutralization TA solution, a sample of
450 uL was added to 113 uL of glutaraldehyde for each respective
time point.
H. Scanning Electron Microscopy (SEM)
[0444] For scanning electron microscopy (SEM), 113 ul of 10%
aqueous solution of gluteraldehyde in Sorenson's buffer, pH 7.4,
was mixed with 450 uL of the bacterial suspension that underwent
the exposure to nanoemulsion. Mixtures were vortexed and placed at
4.degree. C. for at least 18 hours. Table 19 provides the procedure
for fixing and staining the samples for scanning electron
microscopy.
TABLE-US-00019 TABLE 19 Method for fixing and staining samples for
SEM Step 1 Samples were fixed in 2.5% glutaraldehyde in Sorenson's
buffer, pH 7.4 then agitated on a rotary stirrer at each step 2
Samples were rinsed twice for 15 minutes each in 0.1 Sorensen's
buffer 3 samples were fixed in 1.0% OsO.sub.4 in Sorenson's buffer
4 samples were rinsed twice for 5 minutes each in 0.1 M Sorenson's
buffer 5 Samples were dehydrated for 15 minutes each in each of the
following: 30% EtOH, 50% EtOH, 70% EtOH, 90% EtOH, 100% EtOH, 100%
EtOH 6 Samples were immersed in four, 15 minutes changes of
hexamethyldisilazane (HMDS) 7 Samples were removed following the
fourth change of HMDS and replaced with just enough HMDS to cover
tissue. Samples were allowed to evaporate in the hood overnight 8
Samples were mounted on SEM stubs, using the mixture of Colloidal
graphite and duco cement 9 Samples were placed in a vacuum
desiccator overnight 10 Samples were sputter-coated with gold using
"Polaron" sputter coater 11 Samples were examined on an "Amray 1910
FE" Sacnning Electron Microscope and digitally imaged using
"Xstream" imaging software
I. Results
[0445] a. MIC and MBC Data
[0446] The MIC.sub.90/MBC.sub.90 values for P.sub.4075EC were 8/64
.mu.g/ml for P. aeruginosa, 64/>514 .mu.g/ml for B. cenocepacia,
8/64 .mu.g/ml for A. baumannii and 8/32 .mu.g/ml for S.
maltophilia. Colistin had MIC.sub.90/MBC.sub.90 values of 2/8,
>32/>32, 11>16 and >32/>32 for P. aeruginosa, B.
cenocepacia, A. baumannii and S. maltophilia, respectively.
Cefepime, imipenem, levofloxacin and tobramycin had
MIC.sub.90/MBC.sub.90 values of .gtoreq.32/>32,
.gtoreq.32/>32, 16/16 and >32/>32 .mu.g/ml, respectively,
against all strains.
b. Synergy Data
[0447] Ten strains of Burkholderia, Stenotrophomonas and 10 strains
of Acinetobacter were tested to determine a shift in MIC when
P.sub.4075EC+EDTA was in combination with either colistin or
tobramycin, two traditional antimicrobials used in the lungs of CF
patients to treat chronic lung infections. P.sub.4075EC+EDTA in
combination with colistin was found to be synergistic for 90% (in
terms of the FIC) and 70% (in terms of the FBC) of the
Stenotrophomonas strains, but indifferent, only 20% synergy in by
the FIC and 0% by the FBC, when in combination with tobramycin. For
the Acinetobacter strains, P.sub.4075EC+EDTA in combination with
colistin was found to be indifferent, only 20% synergy in by the
FIC and 0% by the FBC, as well as when in combination with
tobramycin, only 10% synergy in by the FIC and 10% by the FBC. For
the Burkholderia strains, P.sub.4075EC+EDTA in combination with
colistin was found to be indifferent, only 30% synergy in by the
FIC and 10% by the FBC, but when in combination with tobramycin,
50% synergy in by the FIC and 20% by the FBC.
c. Time-Kill Study
[0448] Time-kill resulted in an overall 4.44 log reduction in
cfu/ml from the untreated beginning to the 30 minute time point.
Each 10 minute time point had between 1-2 log reduction as follows:
from the untreated to the 10 minute point there was a 1.40 log
reduction, from the 10 to 20 minute time points there was a 1.91
log reduction and from the to 30 minute time points there was a
1.13 log reduction. See FIGS. 20A-20D.
TABLE-US-00020 TABLE 20 A comparison of MIC (.mu.g/ml) of
P.sub.4075EC and comparator drugs. MIC & MBC 50 & 90 CF
Pseudomonas Isolates All Drug Values in ug/mL Cefepime Tobramycin
Cefoxitin Colistin Levofloxacin Imipenem count Pseudomonas MIC MBC
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC 1 NB1 2 8 1 16 >256
>256 2 >8 0.5 1 1 8 2 NB2 0.13 0.125 0.25 2 >256 >256 1
8 0.5 0.5 2 2 3 NB3 1 2 0.5 4 >256 >256 1 8 0.25 0.5 2 16 4
NB4 1 1 0.5 4 >256 >256 2 4 0.25 0.5 4 8 5 NB5 2 4 0.5 4
>256 >256 2 8 0.5 1 2 16 6 NB6 2 4 0.5 4 >256 >256 2 4
0.5 1 1 2 7 NB7 2 2 1 >16 >256 >256 2 4 0.5 1 4 16 8 NB8 2
2 1 8 >256 >256 2 8 0.5 0.5 4 4 9 NB9 2 2 1 8 >256 >256
2 8 0.5 0.5 4 >32 10 NB10 2 2 0.5 4 >256 >256 2 2 0.5 0.5
4 8 11 NB11 16 32 2 4 >256 >256 1 4 8 16 2 8 12 NB12 4 32 0.5
8 >256 >256 4 8 0.25 1 >32 >32 13 NB13 4 16 1 4 >256
>256 2 8 0.5 2 8 16 14 NB14 32 >32 32 >32 >256 >256
2 4 16 >16 >32 >32 15 NB15 8 >32 0.5 8 >256 >256
2 8 2 4 8 >32 16 NB16 16 16 2 16 >256 >256 2 4 2 8 8 32 17
NB17 >32 >32 16 32 >256 >256 1 4 16 16 >32 >32 18
NB18 >32 >32 16 >32 >256 >256 1 4 4 8 >32 >32
19 NB19 16 32 2 16 >256 >256 2 4 2 4 >32 >32 20 NB20 8
8 0.5 8 >256 >256 2 >8 0.5 1 2 16 MIC 50 Cefepime 2
Tobramycin 1 Cefoxitin >256 Colistin 2 Levofloxacin 0.5 Imipenem
4 MIC 90 32 16 >256 2 8 >32 MBC 50 Cefepime 8 Tobramycin 8
Cefoxitin >256 Colistin 4 Levofloxacin 1 Imipenem 16 MBC 90
>32 >16 >256 8 16 >32 P407-5EC & 5 mM P407-5EC
& Ceftazidime Pipercillin EDTA 0 mM EDTA count Pseudomonas MIC
MBC MIC MBC MIC MBC MIC MBC 1 NB1 n/a n/a 1024 1024 16.0625 64.25
n/a 2 NB2 n/a n/a 128 128 8.03125 32.125 n/a 3 NB3 n/a n/a >1024
>1024 4.015625 16.0625 n/a 4 NB4 n/a n/a 512 1024 4.015625
16.0625 n/a 5 NB5 n/a n/a >1024 >1024 8.03125 32.125 n/a 6
NB6 n/a n/a 1024 >1024 4.015625 16.0625 n/a 7 NB7 n/a n/a 512
1024 4.015625 16.0625 n/a 8 NB8 n/a n/a >1024 >1024 4.015625
8.03125 n/a 9 NB9 n/a n/a 512 >1024 4.015625 64.25 n/a 10 NB10
n/a n/a 256 512 8.03125 64.25 n/a 11 NB11 n/a n/a n/a n/a
<4.015625 16.0625 n/a 12 NB12 256 512 n/a n/a <4.015625
16.0625 514 >2056 13 NB13 512 512 n/a n/a 8.03125 16.0625 514
>2056 14 NB14 1024 >1024 n/a n/a <4.015625 32.156 4.01563
>64.25 15 NB15 256 512 n/a n/a <4.015625 16.0625 1028
>2056 16 NB16 256 >1024 n/a n/a <4.015625 >64.25 2056
>2056 17 NB17 n/a n/a n/a n/a <4.015625 16.0625 n/a 18 NB18
n/a n/a n/a n/a <4.015625 <4.015625 n/a 19 NB19 512 1024 n/a
n/a <4.015625 8.03125 1028 >2056 20 NB20 256 512 n/a n/a
8.03125 8.03125 1028 >2056 MIC 50 Ceftazidime n/a Pipercillin
n/a P407-5EC 4.015625 P407- n/a & 5 mM 5EC & EDTA 0 mM EDTA
MIC 90 n/a n/a 8.03125 n/a MBC 50 Ceftazidime n/a Pipercillin n/a
P407-5EC 16.0625 P407- n/a & 5 mM 5EC & EDTA 0 mM EDTA MBC
90 n/a n/a 64.25 n/a MIC & MBC 50 & 90 CF Burkholderia
Isolates All Drug Values in ug/mL Cefepime Colistin Imipenem
Levofloxacin Ceftazidime count Burkholderia MIC MBC MIC MBC MIC MBC
MIC MBC MIC MBC 1 NB21 32 >32 >32 >32 16 16 2 2 8 >32 2
NB22 >32 >32 >32 >32 16 16 1 4 8 >32 3 NB23 >32
>32 >32 >32 >32 >32 8 >16 >32 >32 4 NB24 32
>32 >32 >32 32 >32 8 16 4 32 5 NB25 >32 >32
>32 >32 32 >32 4 8 16 32 6 NB26 >32 >32 32 >32
>32 >32 8 8 16 32 7 NB27 >32 >32 >32 >32 16
>32 4 8 8 16 8 NB29 8 >32 >32 >32 8 16 1 4 4 >32 9
NB30 8 16 >32 >32 4 4 1 1 2 2 10 NB28b 4 4 >32 >32 0.5
0.5 1 2 16 16 MIC 50 Cefepime 32 Colistin >32 Imipenem 16
Levofloxacin 2 Ceftazidime 8 MIC 90 >32 >32 >32 8 16 MBC
50 Cefepime >32 Colistin >32 Imipenem 32 Levofloxacin 4
Ceftazidime 32 MBC 90 >32 >32 >32 >16 >32 P407-5EC
& 5 mM Tobramycin Cefoxitin EDTA count Burkholderia MIC MBC MIC
MBC MIC MBC 1 NB21 32 >32 256 >256 32.125 514 2 NB22 >32
>32 128 256 32.125 >514 3 NB23 >32 >32 >256 >256
<4.015625 64.25 4 NB24 >32 >32 128 >256 32.125 514 5
NB25 >32 >32 128 256 64.25 257 6 NB26 >32 >32 128 256
<4.01563 64.25 7 NB27 >32 >32 128 >256 32.125 257 8
NB29 >32 >32 256 >256 16.0625 >514 9 NB30 32 32 128 128
<4.015625 >64.25 10 NB28b 1 4 16 16 <4.015625 >64.25
MIC 50 Tobramycin >32 Cefoxitin 128 P407-5EC 16.0625 & 5 mM
EDTA MIC 90 >32 256 64.25 MBC 50 Tobramycin >32 Cefoxitin 256
P407-5EC 128.5 & 5 mM EDTA MBC 90 >32 >256 >514 MIC
& MBC 50 & 90 CF Acinetobacter Isolates All Drug Values in
ug/mL Cefepime Colistin Imipenem Levofloxacin Ceftazidime count
Acinetobacter MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC 1 NB31 16 16
1 1 1 1 16 16 >32 >32 2 NB32 32 >32 1 8 32 32 8 8 >32
>32 3 NB33 >32 >32 1 4 32 >32 8 8 >32 >32 4 NB34
32 32 1 1 32 32 16 16 32 32 5 NB35 32 >32 1 4 8 8 >16 >16
>32 >32 6 NB36 2 2 1 1 0.125 0.125 1 1 2 4 7 NB37 32 >32 1
>16 4 8 16 16 32 32 8 NB38 >32 >32 1 >16 32 >32 8
>16 >32 >32 9 NB39 >32 32 1 8 2 2 16 16 32 >32 10
NB40 >32 >32 1 >16 2 32 >16 >16 >32 >32 MIC 50
Cefepime 32 Colistin 1 Imipenem 4 Levofloxacin 16 Ceftazidime
>32 MIC 90 >32 1 32 >16 >32 MBC 50 Cefepime >32
Colistin 4 Imipenem 8 Levofloxacin 16 Ceftazidime >32 MBC 90
>32 >16 >32 >16 >32 P407-5EC & Tobramycin
Cefoxitin 1 mM EDTA count Acinetobacter MIC MBC MIC MBC MIC MBC 1
NB31 1 1 >256 >256 4 16 2 NB32 1 4 256 256 4 64 3 NB33 2 8
>256 >256 4 64 4 NB34 16 >32 >256 >256 4 8 5 NB35 2
16 >256 >256 8 32 6 NB36 0.5 0.5 128 128 2 16 7 NB37 0.5 2
>256 >256 2 16 8 NB38 1 16 >256 >256 4 64 9 NB39 2 4
>256 >256 2 16 10 NB40 >32 >32 >256 >256 8
>128 MIC 50 Tobramycin 1 Cefoxitin >256 P407- 4 5EC & 1
mM EDTA MIC 90 16 >256 8 MBC 50 Tobramycin 4 Cefoxitin >256
P407- 16 5EC & 1 mM EDTA MBC 90 >32 >256 64 MIC & MBC
50 & 90 CF Stenotrophomonas Isolates All Drug Values in ug/mL
Cefepime Colistin Imipenem Levofloxacin Ceftazidime count
Stenotrophomonas MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC 1 NB41 32
>32 2 32 >32 >32 2 4 >32 >32 2 NB42 >32 >32 2
8 >32 >32 8 16 8 >32 3 NB43 >32 >32 16 >32 >32
>32 2 4 >32 >32 4 NB44 >32 >32 32 >32 >32
>32 8 16 >32 >32 5 NB45 32 >32 8 16 >32 >32 1 2
>32 >32 6 Steno 1 >32 >32 >32 >32 >32 >32 1
4 >32 >32 7 Steno 2 >32 >32 32 >32 32 >32 4 8
>32 >32 8 Steno 3 >32 >32 >32 >32 >32 >32 1
4 >32 >32 9 Steno 4 16 >32 1 4 >32 >32 0.5 2 2 16 10
Steno 6 >32 >32 16 >32 32 32 2 4 32 >32 11 Steno 8
>32 >32 8 32 >32 >32 2 2 16 >32 MIC 50 Cefepime
>32 Colistin 16 Imipenem >32 Levofloxacin 2 Ceftazidime
>32 MIC 90 >32 >32 >32 8 >32 MBC 50 Cefepime >32
Colistin >32 Imipenem >32 Levofloxacin 4 Ceftazidime >32
MBC 90 >32 >32 >32 16 >32 Acinetobacter MIC 50 32 1 4
16 >32 MIC 90 >32 1 32 >16 >32 MBC 50 >32 4 8 16
>32 MBC 90 >32 >16 >32 >16 >32 Stenotrophomonas
MIC 50 >32 16 >32 2 >32 MIC 90 >32 >32 >32 8
>32 MBC 50 >32 >32 >32 4 >32 MBC 90 >32 >32
>32 16 >32 MBC 50 8 4 16 1 Not Tested MBC 90 >32 8 >32
16 Not Tested Burkholderia MIC 50 32 >32 >32 2 8 MIC 90
>32 >32 >32 8 16 MBC 50 >32 >32 32 4 32 MBC 90
>32 >32 >32 >16 >32 P407-5EC & Tobramycin
Cefoxitin 1 mM EDTA count Stenotrophomonas MIC MBC MIC MBC MIC MBC
1 NB41 >32 >32 >256 >256 8 8 2 NB42 32 >32 >256
>256 16 16 3 NB43 >32 >32 >256 >256 8 16 4 NB44
>32 >32 256 >256 <1 16 5 NB45 >32 >32 >256
>256 8 16 6 Steno 1 >32 >32 >256 >256 8 16 7 Steno 2
>32 >32 >256 >256 4 16 8 Steno 3 >32 >32 256
>256 8 32 9 Steno 4 >32 >32 256 256 2 32 10 Steno 6 16 32
128 256 8 32 11 Steno 8 >32 >32 >256 >256 8 16 MIC 50
Tobramycin >32 Cefoxitin >256 P407- 8 5EC & 1 mM EDTA MIC
90 >32 >256 8 MBC 50 Tobramycin >32 Cefoxitin >256
P407- 16 5EC & 1 mM EDTA MBC 90 >32 >256 32 Acinetobacter
MIC 50 1 >256 4 MIC 90 16 >256 8 MBC 50 4 >256 16 MBC 90
>32 >256 64 Stenotrophomonas MIC 50 >32 >256 8 MIC 90
>32 >256 8 MBC 50 >32 >256 16 MBC 90 >32 >256 32
MBC 50 8 >256 16.0625 MBC 90 >16 >256 64.25 Burkholderia
MIC 50 >32 128 16.0625 MIC 90 >32 256 64.25 MBC 50 >32 256
128.5 MBC 90 >32 >256 >514
TABLE-US-00021 TABLE 21 Isolate FIC Synergy FBC Synergy Isolate FIC
Synergy FBC Synergy Checkerboard Synergy Study Results;
Acinetobacter NB31 Colistin 0.83 indifferent 2.44 indifferent NB31
Tobra 0.98 indifferent 1.50 no NB32 Colistin 0.58 indifferent 1.36
indifferent NB32 Tobra 0.88 indifferent 1.79 no NB33 Colistin 0.33
yes 2 indifferent NB33 Tobra 1.27 indifferent 1.75 no NB34 Colistin
0.46 yes 1.10 indifferent NB34 Tobra 0.41 yes 0.24 yes NB35
Colistin 0.72 indifferent 0.97 indifferent NB35 Tobra 1.21
indifferent 1.44 no NB36 Colistin 0.56 indifferent 2 indifferent
NB36 Tobra 3.30 indifferent 2.29 no NB37 Colistin 0.63 indifferent
1.00 indifferent NB37 Tobra 0.75 indifferent 1.72 no NB38 Colistin
0.77 indifferent 0.68 indifferent NB38 Tobra 1.05 indifferent 1.57
no NB39 Colistin 0.72 indifferent 1.22 indifferent NB39 Tobra 3.30
indifferent 3.52 no NB40 Colistin 0.63 indifferent 0.91 indifferent
NB40 Tobra 3.01 indifferent 3.01 no 20% 0% 10% 10% Checkerboard
Synergy Study Results; Stenotrophomonas NB42 Colistin 0.08 yes 1.28
indifferent NB42 Tobra 0.14 yes 2.15 indifferent NB43 Colistin 0.08
yes 0.15 yes NB43 Tobra 1.40 indifferent 2.29 indifferent NB44
Colistin 1.01 indifferent 0.57 indifferent NB44 Tobra 1.5
indifferent 2.86 indifferent NB45 Colistin 0.04 yes 0.45 yes NB45
Tobra 0.97 indifferent 1.50 indifferent Steno 1 Colistin 0.08 yes
0.11 yes Steno 1 Tobra 0.79 indifferent 1.57 indifferent Steno 2
Colistin 0.09 yes 0.04 yes Steno 2 Tobra 0.04 yes 2.23 indifferent
Steno 3 Colistin 0.09 yes 0.14 yes Steno 3 Tobra 0.79 indifferent
1.29 indifferent Steno 4 Colistin 0.46 yes 1.14 indifferent Steno 4
Tobra 1.72 indifferent 1.61 indifferent Steno 6 Colistin 0.09 yes
0.02 yes Steno 6 Tobra 0.59 indifferent 0.89 indifferent Steno 8
Colistin 0.09 yes 0.07 yes Steno 8 Tobra 0.63 indifferent 1.15
indifferent 90% 70% 20% 0% Checkerboard Synergy Study Results;
Burkholderia NB21 Colistin 1.45 indifferent 2.07 indifferent NB21
Tobra 0.27 yes 0.48 yes NB22 Colistin 1.37 indifferent 2.29
indifferent NB22 Tobra 0.35 yes 1.23 indifferent NB23 Colistin 1.01
indifferent 2.00 indifferent NB23 Tobra 1.06 indifferent 1.09
indifferent NB24 Colistin 1.72 indifferent 2.00 indifferent NB24
Tobra 1.31 indifferent 1.36 indifferent NB25 Colistin 2.00
indifferent 3.00 indifferent NB25 Tobra 1.45 indifferent 0.99
indifferent NB26 Colistin 0.56 yes 0.04 yes NB26 Tobra 0.12 yes
0.92 indifferent NB27 Colistin 1.93 indifferent 1.64 indifferent
NB27 Tobra 1.44 indifferent 1.27 indifferent NB28b Colistin 0.29
yes 1.54 indifferent NB28b Tobra 0.54 yes 1.06 indifferent NB29
Colistin 1.61 indifferent 1.68 indifferent NB29 Tobra 0.32 yes 0.07
yes NB30 Colistin 0.02 yes 1.86 indifferent NB30 Tobra 0.27 yes
0.54 yes 30% 10% 50% 30%
TABLE-US-00022 TABLE 22 Summary of Synergy Data with Comparison to
Original MIC/MBC Data of Each Drug Alone P.sub.4075EC &
Stenotrophomonas Colistin Tobramycin 1 mM EDTA MIC 50 16 >32 8
MBC 50 >32 >32 16 MIC 90 >32 >32 8 MBC 90 >32 >32
32 % FIC Synergy 90% 20% % FBC Synergy 70% 0% Synergy No
Interference P.sub.4075EC & Acinetobacter Colistin Tobramycin 1
mM EDTA MIC 50 1 1 4 MBC 50 4 4 16 MIC 90 1 16 8 MBC 90 >16
>32 64 % FIC Synergy 20% 10% % FBC Synergy 0% 10% No
Interference No Interference P.sub.4075EC & Burkholderia
Colistin Tobramycin 5 mM EDTA MIC 50 >32 >32 16 MBC 50 >32
>32 64 MIC 90 >32 >32 128 MBC 90 >32 >32 >514 %
FIC Synergy 30% 50% % FBC Synergy 10% 30% No Interference No
Interference
TABLE-US-00023 TABLE 23 Time Kill with additional 5 mM EDTA
Burkholderia NB29 MIC = 16 Pseudomonas with W205EC at 4 ug/mL + 5
mM EDTA, 1 min resulted in a 2 log drop in count. 32 ug/mL
Burkholderia MIC 50 = 16 ug/mL and 90 = 64 ug/mL P4075EC + 5 mM
EDTA Starting Bacteria at 0.159 O.D. at 625 nm = ----8.24 .times.
10{circumflex over ( )}7 cfu/mL Set 0 min Set Set Set Average
(Saline) 8.24E+07 Average 10 min 3.27E+06 Average 20 min 4.00E+04
Average 30 min 2.99E+03 0 min (Saline) 10 min 20 min 30 min Set A
cfu/mL Set A cfu/mL Set A cfu/mL Set A cfu/mL 2.00E+09 0 2.00E+09 0
2.00E+09 0 2.00E+09 0 2.00E+08 0 2.00E+08 0 2.00E+08 0 2.00E+08 0
2.00E+07 5 1.00E+08 2.00E+07 0 2.00E+07 0 2.00E+07 0 2.00E+06 44
8.80E+07 2.00E+06 0 2.00E+06 0 2.00E+06 0 2.00E+05 128 2.56E+07
2.00E+05 23 4.60E+06 2.00E+05 1 2.00E+05 0 2.00E+04 TNTC 2.00E+04
180 3.60E+06 2.00E+04 1 2.00E+04 2.00E+04 0 2.00E+03 Lawn 2.00E+03
TNTC 2.00E+03 19 3.80E+04 2.00E+03 2 4.00E+03 2.00E+02 Lawn
2.00E+02 TNTC 2.00E+02 151 3.02E+04 2.00E+02 7 1.40E+03 2.00E+01
Lawn 2.00E+01 Lawn 2.00E+01 TNTC 2.00E+01 134 2.68E+03 Average
7.12E+07 Average 4.10E+06 Average 2.94E+04 Average 2.69E+03 Set B
cfu/mL Set B cfu/mL Set B cfu/mL Set B cfu/mL 2.00E+09 0 2.00E+09 0
2.00E+09 0 2.00E+09 0 2.00E+08 0 2.00E+08 0 2.00E+08 0 2.00E+08 0
2.00E+07 5 1.00E+08 2.00E+07 0 2.00E+07 0 2.00E+07 0 2.00E+06 39
7.80E+07 2.00E+06 1 2.00E+06 2.00E+06 0 2.00E+06 0 2.00E+05 234
4.68E+07 2.00E+05 11 2.20E+06 2.00E+05 1 2.00E+05 0 2.00E+04 TNTC
2.00E+04 155 3.10E+06 2.00E+04 6 1.20E+05 2.00E+04 0 2.00E+03 TNTC
2.00E+03 TNTC 2.00E+03 14 2.80E+04 2.00E+03 0 2.00E+02 Lawn
2.00E+02 Lawn 2.00E+02 177 3.54E+04 2.00E+02 18 3.60E+03 2.00E+01
Lawn 2.00E+01 Lawn 2.00E+01 TNTC 2.00E+01 128 2.56E+03 Average
7.49E+07 Average 2.43E+06 Average 6.11E+04 Average 3.08E+03 Set C
cfu/mL Set C cfu/mL Set C cfu/mL Set C cfu/mL 2.00E+09 0 2.00E+09 0
2.00E+09 0 2.00E+09 0 2.00E+08 0 2.00E+08 0 2.00E+08 0 2.00E+08 0
2.00E+07 6 1.20E+08 2.00E+07 1 2.00E+07 0 2.00E+07 0 2.00E+06 41
8.20E+07 2.00E+06 1 2.00E+06 2.00E+06 0 2.00E+06 0 2.00E+05 TNTC
2.00E+05 22 4.40E+06 2.00E+05 0 2.00E+05 0 2.00E+04 TNTC 2.00E+04
172 3.44E+06 2.00E+04 1 2.00E+04 2.00E+04 0 2.00E+03 Lawn 2.00E+03
TNTC 2.00E+03 18 3.60E+04 2.00E+03 2 4.00E+03 2.00E+02 Lawn
2.00E+02 TNTC 2.00E+02 164 3.28E+04 2.00E+02 12 2.40E+03 2.00E+01
Lawn 2.00E+01 Lawn 2.00E+01 TNTC 2.00E+01 161 3.22E+03 Average
1.01E+08 Average 3.28E+06 Average 2.96E+04 Average 3.20E+03
J. Conclusions
[0449] This examples demonstrates that nanoemulsions, such as the
tested P.sub.4075EC, are effective against strains that are
multidrug-resistant, including colistin-resistant isolates of
Burkholderia and Stenotrophomonas. None of the described lipid A
modifications in Pseudomonas species impacted the MIC/MBCs with
P.sub.4075EC. No evidence of antagonism with two major antibiotics,
colistin and tobramycin, was observed and in the case of the
Stenotrophomonas, synergy was evident. This is valuable because the
treatment of patients with CF should not need their normal
antibiotic regime suspended in order to use the nanoemulsion,
complicating their treatment programs. The SEM images demonstrate
the kill on contact mechanism, here in this case a Gram negative
bacterium with a reputation of having a tough outer membrane.
Example 9
Topical Nanoemulsion Therapy Reduces Bacterial Wound Infection and
Inflammation Following Burn Injury
Materials and Methods
[0450] Reagents. Unless otherwise indicated, all reagents were
purchased from Sigma-Aldrich Corp. (St. Louis, Mo.).
[0451] Animals. Male specific pathogen-free Sprague-Dawley rats
(Harlan, Indianapolis, Ind.) weighing approximately 250-300 g were
used in all experiments. Rats were housed in standard cages and
allowed to acclimate to their surroundings for 7 days prior to
being used in experiments. They were kept on a 12 hour light cycle
and provided with unrestricted access to standard rat chow and
water throughout the study. Experiments were performed in
accordance with National Institutes of Health guidelines for care
and use of animals. Approval for the experimental protocol was
obtained from the University of Michigan Animal Care and Use
Committee.
[0452] Burn model. Animals were anesthetized with a 40 mg/kg
intraperitoneal (ip) injection of sodium pentobarbital (Nembutal;
Abbott Laboratories, North Chicago, Ill.). Dorsal hair was closely
clipped and removed using Nair depilatory cream (Church &
Dwight Inc., Princeton, N.J.) resulting in a thorough and uniform
removal of fur. Each rat was placed in an insulated, custom-made
mold, which exposes the dorsal region over 20% of the total body
surface area. The burn surface area as a fraction of total body
surface area was determined using Meeh's formula: body surface area
(cm.sup.2)=9.46.times.(animal weight (g)).sup.2/3 (See Gilpin,
Burns 22(8):607-611, 1996). Partial thickness scald burn injury was
achieved by placing the exposed skin of the rat in a 60.degree. C.
water bath for 27 seconds. Sham burn animals received the same
treatment except they were immersed in room temperature water
(21-24.degree. C.). The burn wound was scrub-debrided with dry
sterile gauze and rinsed with 0.9% sterile NaCl. Each animal was
resuscitated with 4 mL Ringer's lactate/% total body surface area
burn/kg body weight. One half of this fluid volume was given
intraperitoneally and half subcutaneously immediately following the
burn injury. The burn injured skin was left uncovered to air dry.
After drying, an occlusive dressing of sterile TELFA (Kendall Co.,
Tyco Healthcare Group LP, Mansfield, Mass.) and TEGADERM HP (3M
Health Care, St Paul, Minn.) was applied to prevent wound
contamination. During experiments each rat was singly housed and
received 0.01 mg/kg buprenorphine subcutaneously at the time of
burn and at 16 hours for post burn pain control.
[0453] Local wound treatment. Stock nanoemulsion
W.sub.205GBA.sub.2ED was obtained from NanoBio Corporation (Ann
Arbor, Mich.). This nanoemulsion was manufactured by emulsification
of super-refined soybean oil and water with surfactants and alcohol
as described herein. The resultant droplets had a mean particle
diameter of 350 nm. The experimental solution was made by diluting
1 mL of the 60% stock formulation with 4.88 mL sterile saline and
adding 120 .mu.L of 1 M ethylenediaminetetraacetic acid (EDTA)
giving a final concentration of 10% W.sub.205GBA.sub.2ED and 20 mM
EDTA. A placebo nanoemulsion (W.sub.205GBA.sub.2ED placebo)
compound was manufactured in the same manner as
W.sub.205GBA.sub.2ED, but benzalkonium chloride was omitted from
the formulation. 5% Sulfamylon (UDL Laboratories, Inc., Rockford,
Ill.) solution was formulated by mixing 50 g of mafenide acetate
powder in 1 L of 0.9% sterile saline. The control reagent used was
0.9% sterile saline. Experimental groups consisted of sham, burn,
burn+W.sub.205GBA.sub.2ED, burn+bacteria+saline,
burn+bacteria+placebo, burn+bacteria+W.sub.205GBA.sub.2ED, and
burn+bacteria+Sulfamylon. Sixteen hours following burn injury
animals were anesthetized with inhaled isoflurane. The occlusive
dressing and TELFA was removed. Nanoemulsion
(W.sub.205GBA.sub.2ED), placebo, Sulfamylon or sterile saline was
applied in a uniform fashion to the burn wound surface using a
spray bottle. Animals in the sham or burn group received no topical
treatment, but did undergo dressing change under anesthesia. The
burn wound was then redressed with TELFA and a TEGADERM occlusive
dressing. This treatment and dressing change was repeated at 24
hours following burn injury.
[0454] Bacterial culture and inoculation. Pseudomonas aeruginosa
isolated from a human burn patient was previously provided by the
Department of Pathology at the University of Michigan. This
bacterial isolate is sensitive to the topical agent Silvadene and
Sulfamylon. A bacterial inoculum was prepared by thawing an aliquot
(0.5 mL, stored in 50% skim milk at -80.degree. C.) in 40 mL of
Trypticase soy broth (Becton Dickinson, Franklin Lakes, N.J.) and
grown overnight at 37.degree. C. with constant shaking at 275 rpm.
A sample of the resulting stationary-phase culture was transferred
to 35 mL of fresh Trypticase soy broth and incubated for 2.5 hours
to reach the log-phase. This subculture was transferred to a 50 mL
conical polystyrene tube and centrifuged for 10 minutes at
4.degree. C. and 880 g. The bacterial pellet was washed with 0.9%
sterile saline, and resuspended in 10 mL of ice-cold saline. The
optical density of the suspension was measured at 620 nm and
bacterial concentration (colony forming units (CFU)/mL) calculated
using the formula OD.sub.620.times.2.5.times.10.sup.8. The
bacterial suspension was diluted with 0.9% sterile saline to a
final concentration of 1.times.10.sup.6 CFU per 100 .mu.L. Eight
hours following burn injury animals were anesthetized with inhaled
isoflurane. The rats then underwent topical application of
1.times.10.sup.6 CFUs of log-phase Pseudomonas aeruginosa in 100
.mu.L of sterile saline pipetted onto a piece of TELFA in a uniform
fashion followed by coverage with a TEGADERM occlusive
dressing.
[0455] Tissue harvest. Thirty-two hours after thermal injury the
animals were sacrificed and skin tissue samples harvested using
sterile technique. Skin samples were used immediately or frozen in
liquid nitrogen.
[0456] Quantitation of bacterial wound infection. A 100 mg piece of
excised skin tissue was mechanically homogenized in 1 mL of 0.9
NaCl. This homogenate was then further diluted with 9 mL of sterile
saline. Serial dilutions were performed and skin homogenates plated
in triplicate on blood agar plates (Becton Dickinson, Franklin
Lakes, N.J.). Culture plates were incubated for 24 hours at
37.degree. C. and CFUs counted.
[0457] Dermal cytokine analysis (ELISA). A 100 mg sample of dorsal
skin was homogenized in 1 mL of ice-cold lysis buffer consisting of
50 mL of PBS and protease inhibitor (Complete X, Roche,
Indianapolis, Ind.) and 504 of Triton X (Roche). Homogenates were
centrifuged at 3000 g for 5 minutes and the supernatants collected
and stored frozen at -80.degree. C. until use. Rat IL1-.beta.,
IL-6, TNF-.alpha., CINC-1, CINC-3, IL-10 and TGF-.beta. were
measured by sandwich enzyme-linked immunosorbent assay (ELISA)
using antibodies and reagents from R&D Systems, Inc.
(Minneapolis, Minn.). The assay was carried out in 96-well
microplates (Immunoplate Maxisorb, Nunc, Neptune, N.J.) according
to the kit instructions and samples were read using a microplate
reader (Biotek Instruments, Winooski, Vt.) at 450 nm with a
wavelength correction of 540 nm. Cytokine concentrations were
determined using the plate reader software and a 7-point standard
curve. Results were adjusted for previous dilution and expressed as
pg/mL.
[0458] Detection of neutrophil sequestration (Myeloperoxidase
assay). 100 mg of skin tissue was mechanically homogenized in 1 mL
ice cold potassium phosphate buffer consisting of 115 mM monobasic
potassium phosphate (Sigma Aldrich, Milwaukee, Wis.). Homogenates
were centrifuged at 3000 g for 10 min at 4.degree. C., the
supernatants were removed and the pellets were re-suspended in 1 mL
C-TAB buffer consisting of dibasic potassium phosphate,
cetyltrimethylammonium bromide, and acetic acid (Sigma Aldrich,
Milwaukee, Wis.). The suspensions were sonicated (Branson Sonifier
250, Danbury, Conn.) on ice for 40 seconds. Homogenates were
centrifuged at 3000 g for 10 min at 4.degree. C. and the
supernatant collected. Supernatants were incubated in 60.degree. C.
water bath for 2 hours (Shaker Bath, 2568; Form a Scientific,
Marietta, Ohio). Samples were stored at -80.degree. C. until needed
or assayed immediately.
[0459] 20 .mu.L standards (Calbiochem, Gibbstown, N.J.) or samples
were added to a 96-well immunosorbent micro-plates (NUNC,
Rochester, N.Y.), followed by the addition of 155 .mu.L of 20 mM
TMB/DMF consisting of
3,3',5,5'-tetramethylbenzidine/N,N-dimethylformamide in 115 mM
potassium phosphate buffer (Fischer Scientific, Pittsburgh, Pa.) to
each well. The samples were mixed well, after which 20 .mu.L of 3
mM H.sub.2O.sub.2 was rapidly added to each well. The reaction was
stopped immediately by adding 504/well of 0.061 mg/mL Catalase
(Roche, Indianapolis, Ind.). The plates were read using a
microplate reader at 620 nm. Myeloperoxidase (MPO) concentrations
were calculated using a linear standard curve and adjusted for
previous dilution. The final concentrations were expressed as
.mu.g/mL.
[0460] Determination of dermal capillary leak and tissue edema
(Evans blue). Animals were anesthetized 90 minutes before tissue
harvest. 50 mg/kg body weight of 10% Evans blue (Merck KgaA,
Darmstadt, Germany) was injected ip into the burned animal at time
30.5 hours following thermal injury. At the tissue harvest time
point animals were exsanguinated by incision of the inferior vena
cava. Systemic Evans blue was washed out by inserting a 20 G
angiocatheter into the apex of the left ventricle past the aortic
valve and into the ascending aorta. A total of four times the blood
volume (7.46 mL/100 g body weight) of 0.9 NaCl with 100 units/mL
heparin was used to flush the vasculature and administered via a
perfusion pump at a constant flow rate. By the end of the perfusing
period the effluent from the right atrium had turned clear. Dorsal
skin samples were harvested and a 100 mg sample was placed in 4 mL
99.5% formamide in polyethylene tubes. Tubes were placed on a
shaker at room temperature for 48 hours for Evans blue extraction.
Supernatants were collected and the absorbance read on a microplate
reader at 620 nm. Concentrations were calculated from an Evans blue
in formamide standard curve. Results are expressed as micrograms of
Evans blue per mg of skin tissue.
[0461] Histology. Skin samples were fixed in 10% buffered formalin
and embedded in paraffin. Eight .mu.m thick sections were affixed
to slides, deparaffinized, and stained with hematoxylin and eosin
to assess morphologic changes.
[0462] Detection of hair follicle cell apoptosis (TUNEL assay).
Animals were anesthetized and underwent creation of a 20% partial
thickness scald burn wound or sham injury.
[0463] Treatment groups consisted of sham, burn+saline,
burn+placebo, and burn+W.sub.205GBA.sub.2ED. Treatment and dressing
changes were performed at 0 and 8 hours post-burn. No bacterial
infection was created in this experiment. Full-thickness skin
samples were taken from three locations across the entire burn
wound at 12, and 24 hours post thermal injury for determination of
hair follicle cell apoptosis. There were four animals per treatment
group per time sample.
[0464] Apoptosis was detected in situ with fluorescein based
labeling of DNA strand breaks using terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) assay (ApopTag, CHEMICON
International, Inc, Temecula, Calif.). The three fresh skin samples
for each animal were placed in disposable vinyl cryomolds filled
with optimal cutting temperature compound (Sakura Finetek, U.S.A.,
Inc., Torrance, Calif.), and frozen at -80.degree. C. until ready
for use. Frozen embedded skin specimens were cut into 4-mm-thick
serial sections in a cryostat and collected on Superfrost Plus
glass slides (Fisher Scientific, Pittsburgh, Pa.). Sections were
fixed in 1% paraformaldehyde in PBS overnight at 4.degree. C. for
indirect immunofluorescence according to the manufacturer's
instructions. After 2 washes with PBS, sections were post-fixed in
a 2:1 solution of ethanol:acetic acid for 5 minutes at -20.degree.
C. Following 2 additional washes with PBS, sections were incubated
in equilibrium buffer for 5 minutes. Sections were then incubated
with terminal deoxynucleotidyl transferase enzyme in a humidified
chamber for 1 h at 37.degree. C. The reaction was terminated by
rinsing the sections in a stop/wash buffer. Sections were incubated
in a humidified chamber at room temperature with antidigoxigenin
fluorescein (fluorescein isothiocyanate, FITC) for 30 min and
rinsed 3 times in PBS. After aspiration, sections were washed once
with water and coverslips applied using ProLong Gold Antifade
(Molecular Probes, Inc, Eugene, Oreg.), which included 4'
6-diamidino-2-phenylindole dihydrochloride (DAPI)
counterstaining.
[0465] The TUNEL assay slides were blinded to groups, and under the
microscope appropriate hair follicle cells in a randomly chosen
high-power field were identified. Appropriate hair follicles for
analysis were those sectioned in the mid-sagittal or mid-coronal
plane. A total of 3-6 hair follicles were selected from among the
three skin samples present on a slide. Fluorescent-labeled TUNEL
slides were captured digitally at identical time post-labeling to
control fading of fluorescence using an Olympus BX-51 fluorescence
microscope at fixed image capture settings and 40.times.
magnification. Each hair follicle was selected and first digitally
captured by visualizing counterstained nuclei present using the
DAPI excitation/emission channel. Then for each hair follicle
analyzed, the excitation/filter channel was changed to visualize
the fluorescein-labeled TUNEL-positive cells, and images again
digitally captured. Within the captured images a region of interest
(ROI) was digitally defined, set to include only hair follicle
cells and exclude bright fluorescing hair shafts and surrounding
cells (NIH Image J software, NIH, Bethesda, Md.). Fluorescence of
TUNEL-positive cells was quantified, normalized to ROI size, and
expressed as pixels/area fraction, controlling for differences in
ROI size.
[0466] Statistical Methods. Statistical analysis and graphs were
performed using GraphPad Prism 5.0 software (GraphPad Software, La
Jolla, Calif.). Results are presented as mean values.+-.the SEM
unless otherwise noted. Continuous variables were analyzed using an
unpaired two-tailed Student's t-test and/or One-way ANOVA followed
by Tukeys post-test comparisons. The Kruskal-Wallis test with
Dunn's multiple comparisons was used to evaluate differences in
medians for data with a non-parametric distribution. Discrete
variables were compared using Fisher's exact test. Statistical
significance was defined as a p-value<0.05.
Topical Application of Nanoemulsion Reduces P. aeruginosa Growth in
Burn Wounds
[0467] Animals treated with nanoemulsion had a decreased mean
(6.5.times.10.sup.4 vs. 7.9.times.10.sup.7, p=0.07) and median (0
vs. 4.4.times.10.sup.6, p<0.05) number of CFUs of bacteria per
gram of skin tissue when compared to the saline treated controls
(See FIG. 21) A similar reduction in skin bacterial counts was
found for W.sub.205GBA.sub.2ED treated animals vs. those treated
with the W.sub.205GBA.sub.2ED placebo (mean: 6.5.times.10.sup.4 vs.
5.5.times.10.sup.6, p=0.02). When performing quantitative wound
culture on clinical tissue samples a positive result is generally
considered to be growth of organisms at greater than
1.times.10.sup.5 CFUs per g of tissue (See, e.g., Neal et al., J
Burn Care Rehabil 2:35-39, 1981; Taddonio et al., Burns Incl Therm
Inj 14(3):180-184, 1988; Uppal et al., Burns 33(4):460-463, 2007).
Using these criteria, 29 of 32 animals in the control group
exhibited evidence of a positive quantitative wound culture and
only 3 of 23 animals in the nanoemulsion group demonstrated proof
of this level of wound infection (91% vs. 13%, p<0.0001, See
Table 24, below). The Sulfamylon treated animals also demonstrated
a significant reduction in the median wound bacterial level when
compared to the saline controls (3.times.10.sup.4 vs.
4.4.times.10.sup.6, p<0.05). Treatment with nanoemulsion or
Sulfamylon produced a similar reduction in the level of Pseudomonas
cultured from the burn wound when compared to the saline treated
animals. However, there was no statistically significant difference
between the placebo and Sulfamylon groups whereas there was a
difference for the W.sub.205GBA.sub.2ED group compared to the
W.sub.205GBA.sub.2ED placebo.
TABLE-US-00024 TABLE 24 Quantitative Wound Culture Results Saline
Placebo NB-201 Sulfamylon Total Animals, n 32 12 23 10 Animals with
29 (91) 9 (75) 3 (13)* 2 (20)* CFU's/g > 1 .times. 10.sup.5, n
(%) *p < 0.05, vs. saline control, Fishers exact test
Nanoemulsion Treatment Following Burn Injury Attenuates Dermal
Pro-Inflammatory Cytokine Levels
[0468] Scald injury resulting in a partial thickness burn produced
differences in dermal levels of IL-1.beta. and cytokine-induced
neutrophil chemoattractant-3 (CINC-3) within skin homogenates
obtained 32 hours post-injury compared to sham injured animals (See
FIGS. 22A and E). Treatment with W.sub.205GBA.sub.2ED at 16 and 24
hours post-burn reduced the dermal level of these two inflammatory
mediators back down to the baseline (sham) in the absence of
bacterial infection. In experiments where a bacterial wound
infection was not created, a difference in neutrophil sequestration
as measured by myeloperoxidase assay was not observed despite the
rise in the rat CXC chemokine CINC-3 within burned skin. A
difference was found between all three groups (sham, burn, and
burn+W.sub.205GBA.sub.2ED) for CINC-1 (p=0.04, ANOVA), however the
values for intergroup comparison did not reach statistical
significance.
Nanoemulsion Treatment Following Burn Wound Infection with P.
aeruginosa Attenuates Dermal Cytokine Levels and Results in Reduced
Neutrophil Sequestration
[0469] Skin homogenates from the nanoemulsion treated group had
levels of IL-1.beta. and IL-6 that were considerably diminished
when compared to the levels measured in the saline treated animals
(See FIGS. 23A and B). There was no statistically significant
difference seen in the level of TNF-.alpha. between the two
experimental groups of animals (See FIGS. 22 C and D). Treatment of
the infected burn wound with Sulfamylon did not result in any
significant alteration of dermal levels of the measured
proinflammatory cytokines (IL-1.beta., IL-6, TNF-.alpha., CINC-1 or
CINC-3) when compared to controls. Treatment with either
W.sub.205GBA.sub.2ED or Sulfamylon reduced the level of
myeloperoxidase found in the infected burn wound at 32 hours
post-injury. Thus, in some embodiments, treatment with a
nanoemulsion and/or antimicrobial reduces the level of neutrophil
sequestration into a partial thickness burn wound.
[0470] Burn injury caused a rise in the level of the
anti-inflammatory cytokine TGF-.beta., but not IL-10 when compared
to the sham injured animals (See FIG. 24). W.sub.205GBA.sub.2ED
treatment reduced the amount of TGF-.beta. present in the infected
burn wound as compared to the level found in the burn wound alone.
Accordingly, in some embodiments, the present invention provides
that W.sub.205GBA.sub.2ED can be utilized to reduce acute burn
wound dermal inflammation, and in further embodiments, can be
utilized to reduce the eventual immunosuppression created by
thermal injury.
[0471] On histological examination of skin from the saline control
animals, there is loss of most of the epidermis and a diffuse
cellular infiltrate in the subepidermal region, extending into the
lower dermal connective tissue in which collagen fibrils are
separated by the infiltrating leukocytes and edema fluid (See FIG.
25A). At a higher power, the cellular infiltrate between the
collagen bundles consists almost entirely of neutrophils. Edema
fluid causes separation of the collagen fibrils. In FIG. 25B, the
skin was subjected to thermal injury followed by application of P.
aeruginosa after which the nanoemulsion was topically applied to
the burned area. The keratin layers of the epidermis are separating
and some of the keratin has been lost. There is a barely detectable
intradermal presence of neutrophils together with neutrophils that
are adhering to the wall of a venule, which has been longitudinally
sectioned (in the center of the microphotograph). The changes in
this microphotograph are substantially less extensive than those
seen in FIG. 25A.
Quantification of Capillary Leak and Tissue Edema
[0472] Burn wounds are associated with significant levels of
capillary leak. This can lead to depletion of the intravascular
volume and a need for large amounts of intravenous crystalloid
fluid administration. To assess whether therapeutic treatment with
a nanoemulsion reduced capillary leak in conjunction with reducing
inflammation, an Evans blue assay was utilized to measure vascular
permeability. Quantitative measurement of the amount of this dye
leaching out of the blood stream and into the skin tissue revealed
that the nanoemulsion treated animals had less evidence of
post-burn capillary leak and tissue edema than the saline treated
controls (See FIG. 26).
Treatment with Nanoemulsion Reduces Burn Induced Hair-Follicle
Apoptosis
[0473] Dermal apoptosis occurs in the hair follicle cells following
thermal injury. Using a fluorescence labeled TUNEL assay the burn
wounds treated with saline showed evidence of intense FITC-TUNEL
positive cells which appear green (See FIG. 27). The DAPI nuclear
stain allows identification of coronal or sagittally sectioned hair
follicles with the cells staining blue. FITC-TUNEL positive cells
appear green and are representative of apoptotic cells. In the
merged images, the apoptotic hair follicle cells are evident in
slides from the burn+saline animals and these changes are
diminished in the burn+W.sub.205GBA.sub.2ED treated animals.
Counting the pixels of TUNEL positive cells within a hair follicle
region of interest allowed quantification of the reduction in hair
follicle cell apoptosis by treatment with topical nanoemulsion (See
FIG. 28). The saline treated control animals had an increased
amount of TUNEL positive cells when compared to the sham burn
animals. Both the W.sub.205GBA.sub.2ED and placebo treatment
resulted in a decrease in hair follicle cell apoptosis following
partial thickness burn injury in tissue harvested 12 hours
following thermal injury. This difference was not evident in the
dermal skin sampled at 24 hours post-burn. Thus, in some
embodiments, the present invention provides a method of reducing
apoptotic cell death in hair follicles in the early post-burn
period comprising administering a nanoemulsion (e.g.,
W.sub.205GBA.sub.2ED) to a burn wound during an early post-burn
period.
Example 10
Immunogenic Compositions Comprising Nanoemulsion and Burkholderia
Immunogen and Methods of Inducing Protective Immunity Against
Burkholderia Species in a Subject
Materials and Methods
[0474] B. cenocepacia Strain K56-2 Stock Maintenance and Culture.
Burkholderia cenocepacia strain K56-2 was generously provided by
Dr. Pam Sokol (University of Calgary). K56-2 is a clinical isolate
and has been used for Burkholderia molecular microbiology and
genomic studies (See, e.g., Goldberg, 2007 Burkholderia Molecular
Microbiology and Genomics. Gent, Belgium: Taylor & Francis). It
is a representative of the transmissible and virulent B.
cenocepacia ET12 lineage (See, e.g., Johnson et al., 1994, J Clin
Microbiol 32, 924-930; Saijan et al., 2008, Infect Immun 76,
5447-5455). Burkholderia multivorans ATCC 17616 was used for
cross-strain neutralization assays. Both K56-2 and ATCC 17616
strains were stored at -80.degree. C. in Luria-Bertani broth with
15% glycerol and recovered from frozen stock on brain-heart
infusion agar overnight at 37.degree. C.
[0475] B. cenocepacia Outer Membrane Protein (OMP) Preparation. An
overnight culture of K56-2 in brain-heart infusion media was
centrifuged at 3500 rpm for 20 minutes. The cell pellet was washed
several times with PBS (pH 7.4), re-suspended in 1 mM EDTA in PBS
(pH 8.0), then incubated for 30 minutes at room temperature.
Post-incubation, the bacteria were passed several times through a
26-gauge needle (Becton Dickinson) using high pressure. Cell lysis
was achieved with Triton X-100 (Sigma) added to a final
concentration of 2% and incubated for 10 minutes at room
temperature. Mechanical separation was performed with multiple
rounds of sonication (1 minute each). The sonicated lysates were
then centrifuged twice for 10 minutes at 6000.times.g (Beckman
Optima XL-100 k ultracentrifuge, 25.degree. C.) with the
supernatant retained after each spin. Following the second
centrifugation, the supernatant was centrifuged at 100,000.times.g
for 1 hour at 4.degree. C. The resulting pellet was resuspended in
endotoxin-free PBS. The rough OMP preparation was purified
(endotoxin-depleted OMP) with an endotoxin-removal column (Pierce)
according to the manufacturer's instructions. The flow-through
fractions were stored at -80.degree. C. until used.
[0476] OMP Analysis. The protein contained within the OMP
preparation was quantified using BCA Assay (Pierce). Western
blotting and silver staining were performed according to
established protocol as described previously (See, e.g., Makidon et
al., 2008, PLoS ONE 3, e2954). To quantify endotoxin contaminate,
OMP was analyzed using the LAL Kinetic-QCL (Lonza). Endotoxin was
detected at 1.93 endotoxin units/mg in the endotoxin-depleted OMP
preparation. DNA contaminant removal was verified by agarose-EtBr
gel electrophoresis and imaged on a UV table (See FIG. 29A).
Oligonucleotides contaminants were not detected in either the crude
OMP or the endotoxin-depleted OMP preparations.
[0477] OMP Sequencing and Verification.
[0478] Sample Preparation. The protein sample was separated by
SDS-PAGE (See FIG. 29B). The protein band immunostained with the
highest intensity (.about.17 KDa) on western blot (See FIG. 29C)
was excised and subjected to in-gel digestion. In-gel digestion was
performed at the Protein Structure Facility at the University of
Michigan according to procedures by Rosenfeld et al. and Hellman et
al. See, e.g., Hellman et al., 1995, Analytical Biochemistry 224,
451-455; Rosenfeld et al., 1992, Analytical Biochemistry 203,
173-179). A gel slice containing 5-.rho.mol bovine serum albumin
(BSA) was analyzed in parallel as a positive control.
[0479] Mass Spectrometry. HPLC-Electrospray Ionization (ESI) Tandem
Mass Spectrometry (MS/MS) was performed on a Q-tof premier mass
spectrometer (Waters Inc) fitted with a nanospray source (Waters
Inc.). The mass spectrometer was calibrated with a mass accuracy
within 3 ppm. On-line capillary HPLC was performed using a Waters
UPLC system with an Atlantis C18 column (Waters, 100-mm inner
diameter, 100-mm length, 3-mm particle size). Digests were desalted
using an online trapping column (Waters, Symmetry C18, 180-mm inner
diameter, 20-mm length, 5-mm particle size) before being loaded
onto the Atlantis column. A data-dependent tandem mass spectrometry
approach was utilized to identify peptides in which a full scan
spectrum (survey scan) was used, followed by collision-induced
dissociation (CID) mass spectra of all ions in the survey scan with
a peak intensity rising above 20 counts per second. The survey scan
was acquired in V+mode over a mass range of 50-2000 Da with
lockmass correction (Gu-Fibrinopeptide B, (M+H).sup.2+: 785.8426)
and charge state peak selection (2+, 3+ and 4+). The MS/MS scans
were acquired for 5 seconds with collision energy control by charge
state recognition.
[0480] Data Analysis and Bioinformatics. Tandem mass spectra were
acquired using Mass Lynx software (Version 4.1). Raw data files
were first processed for lockmass correction, noise reduction,
centering, and deisotoping using the Protein Lynx Global Server
software Ver. 4.25 (Waters Inc.). The generated peaklist files
containing the fragment mass spectra were subjected to database
searches using the ProteinLynxGlobal Server and Mascot (Matrix
Science Inc., Boston, Mass.) search engines and Swiss Prot and NCBI
databases, as well as the Burkholderia cenocepacia database
downloaded from the Sanger Institute web site.
[0481] Preparation of Nanoemulsion-based OMP (OMP-NE) vaccine.
Nanoemulsion (W.sub.805EC: 5 vol. % of TWEEN 80, 8 vol. % of
ethanol, 1 vol. % of CPC, 64 vol. % of soybean oil) and 22 vol. %
of DiH.sub.2O) was provided by NanoBio Corporation (Ann Arbor,
Mich.), the droplets having a particle diameter of 200-400 nm.
OMP-NE formulations were prepared by vigorously mixing the
endotoxin-depleted OMP preparation (See FIG. 29B, Lane F) with
concentrated NE, using PBS as the diluent. For the intranasal
immunizations, OMP-NEs were formulated to contain either 0.25
.mu.g/mL or 0.75 .mu.g/.mu.L OMPs mixed in 20% (v/v) NE.
[0482] Animals and immunization procedures. Pathogen-free, outbred
CD-1 mice (females 6-8 weeks old) were purchased from Charles River
Laboratories. The mice were housed in accordance to the standards
of the American Association for Accreditation of Laboratory Animal
Care. The use of these mice was approved by the University of
Michigan University Committee on Use and Care of Animals (UCUCA).
The mice (n=10 per group) were vaccinated with two administrations
of either OMP-NE vaccines four weeks apart. Intranasal (i.n.)
immunizations were performed in mice anaesthetized with isoflurane
using the IMPAC6 anesthesia delivery system. The anesthetized
animals were held in a supine position and 20 .mu.L (10 .mu.L/nare)
of OMP-NE vaccine was administered slowly to the nares using a
micropipette tip. Mice were immunized with either 15 .mu.g OMP with
NE, 15 .mu.g OMP in PBS, 5 .mu.g OMP with NE, or 5 .mu.g OMP in
PBS.
[0483] Phlebotomy, bronchoalveolar lavage, and splenocyte
collection. Blood was collected from the saphenous vein every 21
days throughout the duration of the study. The terminal sample was
obtained by cardiac puncture immediately following euthanasia.
Whole blood samples were separated by centrifugation at 3500 rpm
(15 minutes) following coagulation. The serum samples were stored
at -20.degree. C. until analyzed. Bronchoalveolar lavage (BAL)
fluid was obtained from the mice immediately following euthanasia
as described (See Makidon et al., 2008, PLoS ONE 3, e2954). In
vitro measurement of the cytokine response was determined using the
spleens of the vaccinated mice. Spleens were mechanically disrupted
to obtain single-cell splenocyte suspension in PBS. Red blood cells
were lysed with ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM
Na2EDTA) and removed by washing the cell suspension twice in PBS.
The splenocytes were then resuspended in RPMI 1640 medium
supplemented with 2% FBS, 200 nM L-glutamine, and
penicillin/streptomycin (100 U/mL and 100 mg/mL).
[0484] Enzyme-linked immunosorbent assay (ELISA). Serum IgG and
mucosal secretory IgA (sIgA) were determined by ELISA against
endotoxin-depleted OMP. The OMP was prepared at 15 .mu.g/mL in
coating buffer (Sigma) and 100 .mu.L was applied per well to
Polysorb plates (Nunc) for a 4.degree. C. overnight incubation.
Serum was serially diluted in 0.1% BSA/PBS. Either diluted serum or
undiluted BAL was then added to the plates for an overnight
incubation at 4.degree. C. Following a 16 hour incubation, the
plates were washed and an alkaline phosphatase conjugated
anti-mouse IgG (H&L), IgG1, IgG2a, IgG2b, sIgA (a
chain-specific) (Rockland Immunochemicals, Inc) antibody was added
at 1:2000 or 1:1000 in 0.1% BSA/PBS. The plates were read at
OD.sub.405 and end titers based on naive animal levels. Levels of
sIgA were normalized to total protein content.
[0485] Analysis of antigens recognized by serum IgG. In addition to
western blot analysis, serum lipoprotein-specific and
lipopolysaccharide-specific IgG antibodies in serum were measured
using ELISA. Briefly, 96-well Polysorb assay plates (Nunc) were
coated with 0.05 .mu.g/well of crude OMP preparation,
endotoxin-depleted OMP, or LPS (List Biological Laboratories).
Serum was collected from mice immunized with OMP+NE 8 weeks
following prime vaccination. The serum was serially diluted in 0.1%
BSA/PBS and then added to the coated wells. The ELISA procedure was
performed as described supra. To evaluate the immunogenic
contribution of protein and LPS contained within the OMP-based
vaccine, an enzymatic protein digest was performed using 250 .mu.g
of PCR-grade recombinant proteinase K (Roche) in 100 .mu.l of
either the crude OMP preparation or the endotoxin-depleted OMP
preparation. Proteinase K was incubated with the OMP preparations
for 16 hours at room temperature. The protein digestion was
verified using a silver-stained SDS-PAGE. LPS and OMP-protein
specific epitopes were evaluated using western blot analysis
probing with serum from mice vaccinated with either OMP-NE or OMP
in PBS, or probing with an anti-Pseudomonas mallei LPS antibody
(GeneTex).
[0486] Analysis of cytokine expression. Freshly isolated mouse
murine splenocytes were seeded at 4.times.10.sup.6 cells/mL (RPMI
1640, 2% FBS) onto a 24-well plate and incubated for 72 hours with
OMPs (15 .mu.g/mL) or PHA-P mitogen (2 .mu.g/mL) as a control. Cell
culture supernatants were collected and analyzed for the presence
of cytokines using LUMINEX multi-analyte profiling beads (LUMINEX
Corporation, Austin, Tex.), according to the manufacturer's
instructions.
[0487] Measurement of serum antibody neutralization. B. cenocepacia
strain K56-2 and B. multivorans strain ATCC 17616
antibody-complement neutralization activity was assayed using serum
harvested from mice 6 weeks following primary i.n. vaccination.
Twenty-five .mu.L of serum (serially diluted 1.times., 2.times.,
3.times., 4.times., or 5.times.) was added to 25 .mu.L of a
solution containing 1.times.10.sup.3 K56-2 or B. multivorans
supplementing MH medium in microculture plates (Nunc). The plates
were incubated at 37.degree. C. for 48 hours. Controls included 25
.mu.L serum with 25 .mu.L of sterile 1.times.PBS and 50 .mu.L of MH
medium containing 1.times.10.sup.3 bacteria. Following incubation,
the wells were mixed and 10 .mu.L of solution was plated on
Burkholderia selective agar (BCSA) media (See Henry et al., 1997, J
Clin Microbiol 35, 614-619) on polystyrene culture plates
(Fisherbrand) then incubated 72 hours at 37.degree. C. prior to
manual colony forming units (cfu) enumeration.
[0488] Pulmonary bacterial challenge. Bacterial challenge studies
were performed in immunized mice (n=5 per group) or non-vaccinated
control mice (n=10) 10 weeks following primary vaccination using a
modified version of the chronic pulmonary model of B. cepacia
infection described (See Bertot et al., 2007, Infect Immun 75,
2740-2752; Chu et al., 2002 Infect Immun 70, 2715-2720; Chu et al.,
2004, Infect Immun 72, 6142-6147). Briefly, 5.times.10.sup.7 of
K56-2 suspended in 85 .mu.L of PBS was instilled through a
trans-tracheal catheter extending to the bifurcation of the
main-stem bronchi in anesthetized mice. The mice were maintained
for a period of 6 days. Prior to the challenge study, the mice were
non-surgically implanted with programmable temperature transponders
(IPTT-3000, Bio Medic Data Systems, Inc.) for non-invasive
subcutaneous temperature measurement with a handheld portable
scanner (DAS-6002, Bio Medic Data Systems, Inc.). In-life analysis
included monitoring the clinical status of each animal by
evaluating the following metrics: body weight, body temperature,
food consumption, and activity. Immediately following euthanasia,
pulmonary tissues and the spleen were collected in sterile
conditions and each organ was placed into individual containers
containing 1 mL of 1.times.PBS (Mediatec Inc). The tissue was then
homogenized using a Tissue TEAROR (Biospec Products Inc.) for 50
seconds on ice. Serial dilutions (1:10.sup.2 to 1:10.sup.8) of the
homogenate were plated onto separated BCSA plates. All plates were
then incubated for 72 hours at 37.degree. C. prior to manual cfu
enumeration. Whole blood was collected in tubes containing EDTA
(BD) immediately following euthanasia. Anti-coagulated blood was
processed to determine total peripheral lymphocytes and mononuclear
lymphocytes by the Animal Diagnostic Laboratory at the University
of Michigan, using a HEMAVETH 950 hematology analyzer (Drew
Scientific, Inc.) in accordance to manufacturer's
recommendation.
[0489] Statistics. Antibody end-titer results are expressed as
mean.+-.standard error of the mean (SEM) or .+-.standard deviation
(SD). The statistical significance was determined by ANOVA
(analysis of variance) using the Student t and Fisher exact
two-tailed tests. For the bacterial challenge studies, the
distribution of the colonization was highly skewed, and therefore
the data was transformed (natural log transformation) for data
normalization. The normalized mean bacterial response in the lung
and spleen were compared using an independent samples test with a
two sided p-value. p<0.05 was considered to be statistically
significant. All analyses were done with 95% confidence limits.
Identification of Immunoreactive Proteins
[0490] Immunoreactive OMP proteins were identified via silver stain
(See FIG. 29B) and western blot (See FIGS. 29C and D). Major
reactive bands were documented at 62, 45, 17, and 10 KDa in both
cases; however, the intensity of the bands was much higher with
serum from mice immunized with the NE-based vaccine (See FIG. 29C)
as compared to the blot probed with an equivalent serum dilution
from mice immunized with the OMP in PBS preparation (See FIG.
29D).
[0491] The band with the highest intensity was located at .about.17
KDa (See FIG. 29C). For the purpose of identification, the 17 KDa
band was isolated from the gel and identified by MALDI-TOF
analysis. The protein was identified as Burkholderia cepacia outer
membrane lipoprotein A (OmpA) with a MW of 16.396 KDa (See, e.g.,
Ortega et al., 2005 J Bacteriol 187, 1324-1333; Plesa et al., 2004,
J Med Microbiol 53, 389-398). Sequence analysis using National
Center for Biotechnology Information (NCBI) protein BLAST confirmed
that these proteins are present in other closely related
Burkholderia species (See Table 25, below). A high degree of
protein homology (87.9% sequence homology) was identified in
numerous strains of Burkholderia and Ralstonia organisms and highly
conserved amino acids from the OmpA family residues are present in
the 17 KDa protein sequence (See Table 25). Thus, the present
invention identified and provides immunoreactive OMP proteins from
B. cepacia as well as proteins from related strains of Burkholderia
species displaying a high degree of homology to the identified
immunoreactive OMP proteins from B. cepacia (e.g., that find use in
immunogenic compositions comprising a nanoemulsion (e.g., that are
administered to a subject in order generate a specific immune
response in the subject toward the immunogenic composition
comprising nanoemulsion and immunoreactive OMP proteins or
homologues)).
TABLE-US-00025 TABLE 25 Protein sequence alignment of OmpA-like
protein family. IDENTITY ORGANISMS Omp-A LIKE SEQUENCES ALIGNMENT
(%) B. CENOCEPACIA
LDDKANAGAVSTQPSADNVAQVNVDPLNDPNSPLAKRSIYFDFDSYSVKDEYQPLLQQHAQYLKSHP
100 QRHVLIQGNTDERGTSEYNLALGQKRAEAVRRALALLGVADSQMEAVS
LGKEKPLATGHDEASWAQNRRADLVYQQ B. DOLOSA
LDDKANAGAVSTQPSADNVAQVNVDPLNDPNSPLAKRSIYFDFDSYSVKDEYQPLLQQHAQYL 100
KSHPQRHVLIQGNTDERGTSEYNLALGQKRAEAVRRALALLGVADSQMEAVSLGKEKP
LATGHDEASWAQNRRADLVYQQ B. MULTIVORANS
LDDKANAGA+STQPSADNVAQVNVDPLNDPNSPLAKRSIYFDFDSYSVKDEYQPLLQQHAQYLKS
98 HPQRHVLIQGNTDERGTSEYNLALGQKRAEAVRRALALLGVADSQMEAV
SLGKEKPLATGHDEASWAQNRRADLVYQQ B. AMBIFARIA
LDDKANAGAVSTQPSADNVAQVNVDPLNDPNSPLAKRSIYFDFDSYSVKDEYQPL+QQHAQYLKSHP
96 QRHVLIQGNTDERGTSEYNLALGQKRAEAVRRA+ALLGV DSQMEAVS
LGKEKPLA+GHDEASWAQNRRADLVYQQ B. VIETNAMIENSIS
LDDKAN-AGAVSTQPSADNVAQVNVDPLNDPNSPLAKRSIYFDFDSYSVKDEYQPLLQQHAQYLKSH
97 PQRHVLIQGNTDERGTSEYNLALGQKRAEAVRRA+ALLGV DSQMEAVS
LGKEKPLATGHDEASWAQNRRADLVYQQ B. UBONENSIS LD+KANAGA+STQP++DNVAQV
VDPLNDPNSPLAKRSIYFDFDSYSVKDEYQPL+ 92
QQHAQYLKSHPQRHVLIQGNTDERGTSEYNLALGQKRAEAVRRA+ALLGVADSQMEAYSLGKEKPLA
GHDEASWAQNRR+DLVYQQ B.THAILANDENSIS LD+AN-GAVSTQP++NAQV
VDPLNDPNSPLAKRSIYFDFDSYSV+D+YQPLLQ 88
QHAQYLKSHPQRH+LIQGNTDERGTSEYNLALGQKRAEAVRRAL+LLGV DSQMEAVSLGK
EKPLATGHDEASWAQNRRADLVYQQ B. GRAMINIS
LD+AN-GAVSTQP+++VAQVNVDPLNDPNSPLAKRSIYFDFDSYSVKD+ 88
YQPLLQQH+QYLKSHPQRHVLIQGNTDERGTSEYNLALGQKRAEAVRR+L+L+ GV
DSQMEAVSLGKEKPLATGHDE+SWAQNRRADLVYQQ B. OKLAHOMENSIS LD+
AN-GAVSTQP++NVAQV VDPLNDPNSPLAKRSIYFDFDSYSV+D+ 87 YQPLLQQHAQYLK
HPQRH+LIQGNTDERGTSEYNLALGQKRAEAVRRAL+LLGV DSQMEAVSLGKEKPLA
GHDEASWAQNRRADLVYQQ B. PSEUDOMALLEI LD+ AN G-AVSTQP++NVAQV
VDPLNDPNSPLAKRSIYFDFDSYSV+D+ 87
YQPLLQQHAQYLKSHPQRH+LIQGNTDERGTSEYNLALGQKRAEAVRRAL+LLGV D+
QMEAVSLGKEKPLA GHDEASWAQNRRADLVYQQ B. XENOVARANS LD+ AN G-+VS QP+
++VA V VDPLNDPNSPLAKRSIYFDFDSYSVKD+ 86 YQ
LLQQHAQYLKSHPQRH+LIQGNTDERGTSEYNLALGQKRAEAVRR+L+L+GV DSQMEAVSLGKEKP
ATGHDE+SWAQNRRADLVYQQ B. PHYTOFIRMANS LD+ AN-G VS QP+
++VAVNVDPLNDPNSPLAKRSIYFDFDSYSVKD+YQ 86
LLQQHAQYLKSHPQRHVLIQGNTDERGTSEYNLALGQKRAEAVRR+L+L+GV DSQMEAVSLGKEKP
ATGHDE+SWAQNRRADLVYQQ B. PHYMATUM LD+ A- GA QP+ ++VA
VNVDPLNDPNSPLAKRSIYFDFDSYSVKD+ 86
YQPLLQQHAQYLKSHPQRHVLIQGNTDERGTSEYNLALGQKRAEAVRR+L+LLGV DS+
MEAVSLGKEKP ATGHDEASWAQNRRADLVYQQ B. METALLIDURANS LDD++GA- A NVA
V+V-DPLNDPN PLAKRSIYFDFDSYSVK +YQ + 73 LQ H+QYL S+ R
+LIQGNTDERGTSEYNLALGQKRAEAVRR+LA +GV DSQMEAVSLGKEKP
ATGHD+ASWA+NRRAD+VY B. EUTROPHA LDD ANAGA AD-V
V+V-DPLNDPNSPLAKRSIYFDFDSYSVK EYQ+L +HA+YL 73 S+ R
+LIQGNTDERGTSEYNLALGQKRAEAVRR+LA+GV+
DSQMEAVSLGKEKP+TGHDEA+WA+NRRAD+Y B. SOLANACEARUM LDD-K G +T NV
V+V-DL DPNSPLAKRSIYFDFDSY+VK EYQ LL 74 QHA+YL+SH QR
VLIQGNTDERGTSEYNLALGQKRAEAVRRAL+ GV DSQMEAVSLGKEKP ATGHDE
SWAQNRR+D+VY
Intranasal Immunization with OMP-NE Induces Anti-OMP Specific
Antibodies
[0492] The humoral immune responses against the OMP induced by
nasal vaccine with OMP were characterized in vivo in the CD-1 mice.
Intranasal vaccination with either 5 .mu.g or 15 .mu.g OMP
preparations+NE (OMP-NE) resulted in high serum titers of
OMP-specific IgG antibodies of 2.8.times.10.sup.5 and
5.1.times.10.sup.5 at 6 weeks, respectively, following primary
vaccination (See FIG. 30A). OMPs without adjuvant were immunogenic
and resulted in serum anti-OMP IgG titers of 1.9.times.10.sup.4 and
3.8.times.10.sup.4 six weeks following primary i.n. immunization.
However, treatment groups with NE as an adjuvant responded
significantly higher (13 to 30 fold) than groups without adjuvant
at all time points following the boost (p<0.05). Further, mice
immunized with OMP in PBS did not demonstrate a significant boost
effect following the second vaccination (See FIG. 30A).
[0493] To further characterize the OMP-NE vaccine, the OMP-NE
composition's ability to elicit specific antibody production in
bronchiolar secretions was evaluated. Mucosal antibody production
may be important for protection against Burkholderia colonization
since secretory antibodies are thought to be critical effectors in
protection against mucosal respiratory pathogens (See, e.g., Nelson
et al., 1993, J Med Microbiol 39, 39-47). Mucosal immune responses
were evaluated in bronchoalveolar lavage (BAL) fluid of animals
immunized with 5 .mu.g OMP-NE or 5 .mu.g OMP in PBS. Comparable
levels of anti-OMP antibodies in BALs were detected in mice
immunized either with OMP-NE or OMP without NE adjuvant (See FIG.
30B).
OMP-NE Immunization Yields a Balanced Th1/Th2 Cellular Response
[0494] The analysis of the serum IgG subclass was performed to
determine the T helper-type bias of the cellular response. The
pattern of IgG subclass distribution shows that i.n. immunization
with OMP-NE produced increased levels of Th1-type IgG2b versus
Th2-type IgG1 subclass antibodies (p=0.048) (See FIG. 31A). In
comparison, immunization with OMP in PBS produced similar levels of
IgG1 and IgG2b antibodies (See FIG. 31A).
[0495] OMP-specific cellular responses were characterized in
splenocytes obtained 6 weeks following primary vaccination with
OMP-NE. The cells were stimulated with endotoxin-depleted OMP and
then evaluated for specific cytokine production. In splenocytes
collected from OMP-NE vaccinated mice, Th1 cytokines IFN-.gamma.
and IL-2 production increased 22.1 and 18.6-fold respectively over
that of splenocytes from non-vaccinated mice (p=0.01 and 0.003,
respectively) (See FIG. 31B). IFN-.gamma. was equally induced in
splenocytes from mice immunized with OMP in PBS. The Th2 cytokines
IL-4, IL-5, IL-6, and IL-10 were minimally induced
(.ltoreq.5.22-fold increase) in both OMP-NE and OMP immunized mice
in equivalent amounts. The pattern of splenic cytokine expression
and the IgG subclass distribution results indicated a balanced
Th1/Th2 response to OMP-NE vaccination.
Antibody Response is Directed Toward OMP Lipoprotein and LPS
[0496] It has been previously documented that LPS is intrinsically
associated with Burkholderia spp. OMP preparations (See, e.g.,
Bertot et al., 2007, Infect Immun 75, 2740-2752). To investigate
the specificity of immune response and to evaluate the possible
immune enhancing role of endotoxin in B. cencocepacia OMP
preparations, serum from mice immunized with OMP-NE was analyzed
using ELISA with either crude OMP, endotoxin-depleted OMP, or LPS.
The highest IgG reactivity was against the endotoxin-depleted and
crude OMP preparations. The statistical analysis indicated a
significantly higher response in endotoxin-depleted OMP versus LPS
(OD.sub.405=2.37.+-.0.11 (p=0.001)) and crude OMP versus LPS
(OD.sub.405=1.49.+-.0.14 (p=0.01)). However, the serum also
contained significant levels of IgG anti-LPS antibodies
(OD.sub.405=0.59.+-.0.05).
[0497] To clarify the role of endotoxin in the OMP preparation, a
proteolytic digest of the crude OMP and endotoxin-depleted OMP
preparations were performed with proteinase K. The immunogenic
epitopes were determined by western blot analysis comparing serum
antibodies generated in mice vaccinated with OMP in PBS versus from
mice vaccinated with OMP-NE (See FIG. 32). The results clearly
indicated that antibody response was directed mainly toward 62 KDa
and 17 KDa proteins contained within the OMP preparation. This was
confirmed by the complete absence of the protein bands in the
proteolytically digested OMP preparations (See FIG. 32 OMP-NE serum
western blot). Further, when the OMP preparations were probed with
commercial monoclonal anti-LPS antibody, LPS was detected as an
intrinsic part of the OMP preparation. However, the proteolytic
digest diminished the LPS detection but not totally. Overall, the
present invention provides that nasal immunization with OMP-NE
enhances the production of antibodies specific against OMP proteins
(See FIG. 32).
Intranasal Vaccination with OMP-NE Results in Cross-Protective
Neutralizing Serum Antibody
[0498] B. cenocepacia neutralizing antibodies were determined using
a colony reduction assay. The mice intranasally vaccinated with 5
.mu.g or 15 .mu.g OMP-NE produced serum-neutralizing antibodies
that inhibited 92.5% or 98.3% of B. cenocepacia colonies,
respectively, as compared to serum from non-vaccinated mice (See
FIG. 33). The mice vaccinated with OMP lacking adjuvant also
demonstrated a relatively high level of inhibition, resulting in
33.3% and 46.7% (for 5 .mu.g and 15 .mu.g OMP-PBS, respectively)
cfu reduction. The statistical analysis showed that B. cenocepacia
growth was significantly inhibited by serum derived from OMP-NE as
compared to serum from those who were immunized with the OMP
preparation alone (See FIG. 33).
[0499] To evaluate if the OMP-NE can produce cross-reactive
protection from other bacterial strains, serum from mice vaccinated
with 15 .mu.g OMP-NE was also incubated with B. multivorans. The B.
multivorans was selected because it is the most common isolate
cultured from CF patients infected with Bcc organisms (See, e.g.,
Baldwin et al., 2008, J Clin Microbiol 46, 290-295). The serum
inhibited B. multivorans growth by 80.1% and by 49.8% derived from
mice vaccinated with OMP-NE and OMP without adjuvant, respectively,
suggesting significant cross-protective antibodies following
immunization with either OMP-NE or OMP in PBS (See FIG. 33).
Immunization with OMP-NE Protects Against Pulmonary B. cenocepacia
Challenge and Reduces Incidence of Sepsis
[0500] The protective effect of intranasal immunization was further
tested in vivo in a lung infection model. The clearance of B.
cenocepacia from pulmonary tissue was evaluated 6 days following
intra-tracheal inoculation in mice that were intranasally immunized
with the OMP-NE or the OMP without an adjuvant. Vaccination with 15
.mu.g OMP-NE resulted in significantly higher rates of pulmonary
clearance (p=9.2.times.10.sup.-3) as compared to the non-vaccinated
mice (See FIG. 34A). At day 6, the average pulmonary bacterial load
was 22.5.+-.26.2 cfu in mice vaccinated with 15 .mu.g OMP-NE,
compared to 1.28.times.10.sup.6.+-.3.36.times.10.sup.6 cfu per lung
in non-vaccinated mice, representing a greater than 5-log reduction
in the bacterial load.
[0501] Splenic colonization following pulmonary inoculation with B.
cenocepacia was evaluated as a means of assessing sepsis (See FIG.
34B). Vaccination with 15 .mu.g OMP-NE significantly reduced the
incidence of bacteria in the spleens 6 days following the pulmonary
challenge of individual mice from an average of
3.54.times.10.sup.3.+-.6.97.times.10.sup.3 cfu per spleen in
non-vaccinated mice to 2.5.+-.5 cfu per spleen in vaccinates
(p=0.0307).
Intranasal Immunization with OMP-NE Attenuates Systemic Illness
after B. cenocepacia Infection
[0502] B. cenocepacia infection resulted in greater loss of
thermoregulation by day 6 in the non-vaccinated mice than in the
mice immunized with OMP-NE or OMP without the adjuvant. The mean
body temperature 6 days following infection with B. cenocepacia was
significantly lower in non-vaccinated mice (35.4.degree. C..+-.0.7)
as compared to mice vaccinated with 15 .mu.g OMP-NE (36.8.degree.
C..+-.0.31, p=0.008), 5 .mu.g OMP-NE (37.1.degree. C..+-.0.42,
p=0.008), or 5 .mu.g OMP without the adjuvant (36.8.degree.
C..+-.0.61, p=0.01).
[0503] The total peripheral leukocyte counts determined at the time
of sacrifice in the non-vaccinated mice were significantly higher
from the values observed for the mice vaccinated with 15 .mu.g
OMP-NE (p=0.047), 5 .mu.g OMP-NE (p=0.026), or 15 .mu.g OMP in PBS
(p=0.032). The ratio of polymorphonuclear leukocytes to total
peripheral leukocytes were also significantly higher in
non-vaccinated mice (76.0%) compared to mice vaccinated with 15
.mu.g OMP-NE (47.7%, p=0.02), 5 .mu.g OMP-NE (45.6%, p=0.024), or
15 .mu.g OMP in PBS (48.7%, p=0.04) (See Table 26, below). In
combination with the splenic colonization results described above
(See FIG. 39B), the present invention provides that immunization
with OMP-NE resulted in significantly decreased systemic disease
following infection with B. cenocepacia.
TABLE-US-00026 TABLE 26 Ratio of polymorphonuclear leukocytes to
total peripheral leukocytes. Mean Polymorphonuclear Leukocytes
Immunization Concentration % Total Peripheral Group (10.sup.9/L)
Leukocyte Count 5 .mu.g OMP-NE 1.99 .+-. 0.20* 45.6* 5 .mu.g OMP in
PBS 3.23 .+-. 0.29 59.7 15 .mu.g OMP-NE 2.58 .+-. 0.48* 47.7* 15
.mu.g OMP in PBS 2.56 .+-. 0.55* 48.7* Non-Vaccinated 7.29 .+-.
1.35 76.0
[0504] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described compositions and
methods of the invention will be apparent to those skilled in the
art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
for carrying out the invention that are obvious to those skilled in
the relevant fields are intended to be within the scope of the
present invention.
* * * * *