U.S. patent application number 17/197867 was filed with the patent office on 2022-01-20 for sustained release of antiinfectives.
The applicant listed for this patent is Insmed Incorporated. Invention is credited to Lawrence T. BONI, Brian S. MILLER.
Application Number | 20220016150 17/197867 |
Document ID | / |
Family ID | 1000005880290 |
Filed Date | 2022-01-20 |
United States Patent
Application |
20220016150 |
Kind Code |
A1 |
BONI; Lawrence T. ; et
al. |
January 20, 2022 |
SUSTAINED RELEASE OF ANTIINFECTIVES
Abstract
Provided, among other things, is a method of treating or
ameliorating pulmonary infection in a cystic fibrosis patient
comprising pulmonary administration of an effective amount of a
liposomal/complexed antiinfective to the patient, wherein the (i)
administrated amount is 50% or less of the comparative free drug
amount, or (ii) the dosing is once a day or less, or (iii)
both.
Inventors: |
BONI; Lawrence T.; (Monmouth
Junction, NJ) ; MILLER; Brian S.; (Mercerville,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Insmed Incorporated |
Bridgewater |
NJ |
US |
|
|
Family ID: |
1000005880290 |
Appl. No.: |
17/197867 |
Filed: |
March 10, 2021 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/496 20130101;
A61K 31/7036 20130101; A61K 31/407 20130101; Y10S 977/906 20130101;
A61P 31/00 20180101; A61K 9/0078 20130101; A61K 9/0073 20130101;
Y02A 50/30 20180101; A61K 9/127 20130101; Y10S 977/773 20130101;
Y10S 977/907 20130101; A61K 31/545 20130101 |
International
Class: |
A61K 31/7036 20060101
A61K031/7036; A61K 9/00 20060101 A61K009/00; A61K 9/127 20060101
A61K009/127; A61K 31/407 20060101 A61K031/407; A61K 31/545 20060101
A61K031/545; A61P 31/00 20060101 A61P031/00; A61K 31/496 20060101
A61K031/496 |
Claims
1. A method of treating or ameliorating pulmonary infection in a
cystic fibrosis patient comprising pulmonary administration of an
effective amount of a liposomal/complexed antiinfective to the
patient, wherein the (i) administrated amount is 50% or less of the
comparative free drug amount, or (ii) the dosing is once a day or
less, or (iii) both.
2.-25. (canceled)
Description
[0001] This application claims the priority of U.S. Provisional
Application 60/421,923, filed Oct. 29, 2002.
[0002] Certain sustained release technology suitable, for example,
for administration by inhalation employs liposomes and lipid
complexes to provide prolonged therapeutic effect of drug in the
lung and systemically by sustained release and the ability to
target and enhance the uptake of drug into sites of disease. The
present invention comprises a liposomal antiinfective, and methods
for treatment of pulmonary infections in cystic fibrosis (CF)
patients using liposomal or lipid-complexed antiinfective.
Unexpectedly, treatments with the new formulation require a
significantly lower dosage than that known to have efficacy in the
art.
[0003] As reported in Goodman and Gilman's The Pharmaceutical Basis
of Therapeutics, Eighth Edition, "Since the incidence of
nephrotoxicity and ototoxicity is related to the concentration to
which an aminoglycoside accumulates, it is critical to reduce the
maintenance dosage of these drugs in patients with impaired renal
function." Since aminoglycosides can produce vestibular or auditory
dysfunction and nephrotoxicity regardless of a patient's
impairments, it is important generally to reduce maintenance
dosages. The present invention provides dramatic reductions in
maintenance dosages.
[0004] CF patients have thick mucous and/or sputum secretions in
the lungs, frequent consequential infections, and biofilms
resulting from bacterial colonizations. All these fluids and
materials create barriers to effectively targeting infections with
antiinfectives. The present invention overcomes these barriers, and
even allows reduced dosing (in amount or frequency), thereby
reducing the drug load on patients.
[0005] For lung infections generally, the dosing schedule provided
by the invention provides a means of reducing drug load.
SUMMARY OF THE INVENTION
[0006] Provided, among other things, is a method of treating or
ameliorating pulmonary infection in a cystic fibrosis patient
comprising pulmonary administration of an effective amount of a
liposomal/complexed antiinfective to the patient, wherein the (i)
administrated amount is 50% or less of the comparative free drug
amount, or (ii) the dosing is once a day or less, or (iii)
both.
[0007] Also provided is a method of treating or ameliorating
pulmonary infection in an animal comprising pulmonary
administration of an effective amount of a liposomal/complexed
antiinfective to the patient, wherein the (i) administrated amount
is 50% or less of the comparative free drug amount, and (ii) the
dosing is once every two days or less.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1: Cross sectional diagram of the sputum/biofilm seen
in patients with cystic fibrosis.
[0009] FIG. 2: Graphical representation of the targeting and depot
effect of the drug of the present invention.
[0010] FIGS. 3 and 4: Graphical representations of bacteriology of
amikacin in various forms.
[0011] FIG. 5: Graphical representation of sustained release for
liposomal/complexed amikacin and tobramycin.
[0012] FIG. 6: Data on free or complexed ciprofloxacin.
[0013] FIG. 7: Graphical representation of drug residence in the
lung given various dosing schedules.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present application discloses a method of treating or
ameliorating pulmonary infections, such as in cystic fibrosis
patients, comprising administration of antiinfective (such as
antibiotic) encapsulated in lipid-based particles.
[0015] Antiinfectives are agents that act against infections, such
as bacterial, mycobacterial, fungal, viral or protozoal
infections.
[0016] Antiinfectives covered by the invention include but are not
limited to aminoglycosides (e.g., streptomycin, gentamicin,
tobramycin, amikacin, netilmicin, kanamycin, and the like),
tetracyclines (such as chlortetracycline, oxytetracycline,
methacycline, doxycycline, minocycline and the like), sulfonamides
(e.g., sulfanilamide, sulfadiazine, sulfamethaoxazole,
sulfisoxazole, sulfacetamide, and the like), para-aminobenzoic
acid, diaminopyrimidines (such as trimethoprim, often used in
conjunction with sulfamethoxazole, pyrazinamide, and the like),
quinolones (such as nalidixic acid, cinoxacin, ciprofloxacin and
norfloxacin and the like), penicillins (such as penicillin G,
penicillin V, ampicillin, amoxicillin, bacampicillin,
carbenicillin, carbenicillin indanyl, ticarcillin, azlocillin,
mezlocillin, piperacillin, and the like), penicillinase resistant
penicillin (such as methicillin, oxacillin, cloxacillin,
dicloxacillin, nafcillin and the like), first generation
cephalosporins (such as cefadroxil, cephalexin, cephradine,
cephalothin, cephapirin, cefazolin, and the like), second
generation cephalosporins (such as cefaclor, cefamandole,
cefonicid, cefoxitin, cefotetan, cefuroxime, cefuroxime axetil;
cefmetazole, cefprozil, loracarbef, ceforanide, and the like),
third generation cephalosporins (such as cefepime, cefoperazone,
cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefixime,
cefpodoxime, ceftibuten, and the like), other beta-lactams (such as
imipenem, meropenem, aztreonam, clavulanic acid, sulbactam,
tazobactam, and the like), beta-lactamase inhibitors (such as
clavulanic acid), chloramphenicol, macrolides (such as
erythromycin, azithromycin, clarithromycin, and the like),
lincomycin, clindamycin, spectinomycin, polymyxin B, polymixins
(such as polymyxin A, B, C, D, E.sub.1 (colistin A), or B.sub.2,
colistin B or C, and the like) colistin, vancomycin, bacitracin,
isoniazid, rifampin, ethambutol, ethionamide, aminosalicylic acid,
cycloserine, capreomycin, sulfones (such as dapsone, sulfoxone
sodium, and the like), clofazimine, thalidomide, or any other
antibacterial agent that can be lipid encapsulated. Antiinfectives
can include antifungal agents, including polyene antifungals (such
as amphotericin B, nystatin, natamycin, and the like), flucytosine,
imidazoles (such as miconazole, clotrimazole, econazole,
ketoconazole, and the like), triazoles (such as itraconazole,
fluconazole, and the like), griseofulvin, terconazole, butoconazole
ciclopirax, ciclopirox olamine, haloprogin, tolnaftate, naftifine,
terbinafine, or any other antifungal that can be lipid encapsulated
or complexed. Discussion and the examples are directed primarily
toward amikacin but the scope of the application is not intended to
be limited to this antiinfective. Combinations of drugs can be
used.
[0017] Particularly preferred antiinfectives include the
aminoglycosides, the quinolones, the polyene antifungals and the
polymyxins.
[0018] Among the pulmonary infections (such as in cystic fibrosis
patients) that can be treated with the methods of the invention are
pseudomonas (e.g., P. aeruginosa, P. paucimobilis, P. putida, P.
fluorescens, and P. acidovorans), staphylococcal,
Methicillin-resistant Staphylococcus aureus (MRSA), streptococcal
(including by 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) infections.
[0019] In one preferred embodiment the present invention comprises
a method of treatment comprising administration of
liposomal/complexed amikacin.
[0020] The "liposomal or lipid-complexed" antiinfective, or
"liposomal/complexed" antiinfective, or "Lip-antiinfective," or
"Lip-An" discussed herein is any form of antiinfective composition
where at least about 1% by weight of the antiinfective is
associated with the lipid either as part of a complex with the
lipid, or as a liposome where the antibiotic may be in the aqueous
phase or the hydrophobic bilayer phase or at the interfacial
headgroup region of the liposomal bilayer. Preferably, at least
about 5%, or at least about 10%, or at least about 20%, or at least
about 25%, is so associated. Association is measured by separation
through a filter where lipid and lipid-associated drug is retained
and free drug is in the filtrate.
[0021] Treatment with liposomal/complexed antiinfective requires a
notably lower dosage than prior known treatments. In one preferred
embodiment less than 100 mg per day of an aminoglycoside is
administered to humans. In another preferred embodiment
approximately 30 to 50 mg is administered every other day or every
third day. It is expected that dosages can be correspondingly
lowered for other species as compared to the dosage recommended for
antiinfective that is not liposomal or lipid-complexed. This is an
unexpectedly low dosage.
[0022] Where no specific dosage is provided below, the preferred
dosage of the invention is 50% or less, 35% or less, 20% or less,
or 10% or less, of the minimum free drug (which of course can be a
salt) amount that is effective, if delivered to the lungs via a
nebulizer, to reduce the CFU count in the lungs by one order of
magnitude over the course of a 14-day treatment. The comparative
free drug amount is the cumulative amount that would be used in the
dosing period applied with the drug administration of the
invention. The comparative minimum free drug defined in this
paragraph is a "comparative free drug amount."
[0023] The non-CF treating embodiments of the invention can be used
with any animal, though preferably with humans. Relative amounts in
a given animal are measured with respect to such animal.
[0024] The dosing schedule is preferably once a day or less. In
preferred embodiments, the dosing schedule is once every other day,
every third day, every week, or less. For example, the dosing
schedule can be every other day or less, using 50% or less of the
comparative free drug amount. Or, for example, the dosing can be
daily using 35% or less of the comparative free drug amount.
[0025] To treat the infections of the invention, an effective
amount of a pharmaceutical compound will be recognized by
clinicians but includes an amount effective to treat, reduce,
ameliorate, eliminate or prevent one or more symptoms of the
disease sought to be treated or the condition sought to be avoided
or treated, or to otherwise produce a clinically recognizable
change in the pathology of the disease or condition. Amelioration
includes reducing the incidence or severity of infections in
animals treated prophylactically. In certain embodiments, the
effective amount is one effective to treat or ameliorate after
symptoms of lung infection have arisen. In certain other
embodiments, the effective amount is one effective to treat or
ameliorate the average incidence or severity of infections in
animals treated prophylactically (as measured by statistical
studies).
[0026] Liposome or other lipid based delivery systems can be
administered for inhalation either as a nebulized spray, powder, or
aerosol, or by intrathecal administration. Inhalation
administrations are preferred. The overall result is a less
frequent administration and an enhanced therapeutic index compared
to free drug or parenteral form of the drug. Liposomes or lipid
complexes are particularly advantageous due to their ability to
protect the drug while being compatible with the lung lining or
lung surfactant.
[0027] The present invention includes methods for treatment of
pulmonary gram-negative infections. One usefully treated infection
is chronic pseudomonal infection in CF patients. Known treatments
of lung infections (such as in CF patients) with amikacin generally
comprise administering approximately 200-600 mg of amikacin or
tobramycin per day via inhalation. The present invention allows for
treatment by administering, in one preferred embodiment, 100 mg or
less of amikacin per day (or normalized to 100 mg per day or less
if dosing less frequent). In yet another embodiment administration
of 60 mg or less of amikacin every day is performed. And in still
another embodiment administration of approximately 30 to 50 mg not
more than once every 2 days is performed. The most preferred
embodiment comprises administration of approximately 30 to 50 mg
every other day or every third day.
[0028] Known treatments of lung infections with tobramycin
generally comprise administering 300 mg, twice a day, in adults and
children 6 years of age or older. The present invention allows for
treatment by administering, in one preferred embodiment, 100 mg or
less of tobramycin per day. In yet another embodiment
administration of 60 mg or less of tobramycin every day is
performed. And in still another embodiment administration of
approximately 30 to 50 mg not more than once every 2 days is
performed. The most preferred embodiment comprises administration
of approximately 30 to 50 mg every other day or every third
day.
[0029] The lipids used in the compositions of the present invention
can be synthetic, semi-synthetic or naturally-occurring lipids,
including phospholipids, tocopherols, steroids, fatty acids,
glycoproteins such as albumin, negatively-charged lipids and
cationic lipids. Phosholipids include egg phosphatidylcholine
(EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol
(EPI), egg phosphatidylserine (EPS), phosphatidylethanolamine
(EPE), and egg phosphatidic acid (EPA); the soya counterparts, soy
phosphatidylcholine (SPC); SPG, SPS, SPI, SPE, and SPA; the
hydrogenated egg and soya counterparts (e.g., HEPC, HSPC), other
phospholipids made up of ester linkages of fatty acids in the 2 and
3 of glycerol positions containing chains of 12 to 26 carbon atoms
and different head groups in the 1 position of glycerol that
include choline, glycerol, inositol, serine, ethanolamine, as well
as the corresponding phosphatidic acids. The chains on these fatty
acids can be saturated or unsaturated, and the phospholipid can be
made up of fatty acids of different chain lengths and different
degrees of unsaturation. In particular, the compositions of the
formulations can include dipalmitoylphosphatidylcholine (DPPC), a
major constituent of naturally-occurring lung surfactant as well as
dioleoylphosphatidylcholine (DOPC). Other examples include
dimyristoylphosphatidylcholine (DMPC) and
dimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidcholine
(DPPC) and dipalmitoylphosphatidylglycerol (DPPG)
distearoylphosphatidylcholine (DSPC) and
distearoylphosphatidylglycerol (DSPG),
dioleylphosphatidylethanolamine (DOPE) and mixed phospholipids like
palmitoylstearoylphosphatidylcholine (PSPC) and
palmitoylstearoylphosphatidylglycerol (PSPG), triacylglycerol,
diacylglycerol, seranide, sphingosine, sphingomyelin and single
acylated phospholipids like mono-oleoyl-phosphatidylethanolamine
(MOPE).
[0030] The lipids used can include ammonium salts of fatty acids,
phospholipids and glycerides, steroids, phosphatidylglycerols
(PGs), phosphatidic acids (PAs), phosphotidylcholines (PCs),
phosphatidylinositols (Pis) and the phosphatidylserines (PSs). The
fatty acids include fatty acids of carbon chain lengths of 12 to 26
carbon atoms that are either saturated or unsaturated. Some
specific examples include: myristylamine, palmitylamine,
laurylamine and stearylamine, dilauroyl ethylphosphocholine (DLEP),
dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl
ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine
(DSEP), N-(2, 3-di-(9
(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride
(DOTMA) and 1, 2-bis(oleoyloxy)-3-(trimethylammonio)propane
(DOTAP). Examples of steroids include cholesterol and ergosterol.
Examples of PGs, PAs, PIs, PCs and PSs include DMPG, DPPG, DSPG,
DMPA, DPPA, DSPA, DMPI, DPPI, DSPI, DMPS, DPPS and DSPS, DSPC,
DPPC, DMPC, DOPC, egg PC.
[0031] Liposomes or lipid complexes composed of
phosphatidylcholines, such as DPPC, aid in the uptake by the cells
in the lung such as the alveolar macrophages and helps to sustain
release of the antiinfective agent in the lung (Gonzales-Rothi et
al. (1991)). The negatively charged lipids such as the PGs, PAs,
PSs and PIs, in addition to reducing particle aggregation, can play
a role in the sustained release characteristics of the inhalation
formulation as well as in the transport of the formulation across
the lung (transcytosis) for systemic uptake. The sterol compounds
are believed to affect the release and leakage characteristics of
the formulation.
[0032] Liposomes are completely closed lipid bilayer membranes
containing an entrapped aqueous volume. Liposomes can be
unilamellar vesicles (possessing a single membrane bilayer) or
multilamellar vesicles (onion-like structures characterized by
multiple membrane bilayers, each separated from the next by an
aqueous layer). The bilayer is composed of two lipid monolayers
having a hydrophobic "tail" region and a hydrophilic "head" region.
The structure of the membrane bilayer is such that the hydrophobic
(nonpolar) "tails" of the lipid monolayers orient toward the center
of the bilayer while the hydrophilic "heads" orient towards the
aqueous phase. Lipid complexes are associations between lipid and
the antiinfective agent that is being incorporated. This
association can be covalent, ionic, electrostatic, noncovalent, or
steric. These complexes are non-liposomal and are incapable of
entrapping additional water soluble solutes. Examples of such
complexes include lipid complexes of amphotericin B (Janoff et al.,
Proc. Nat Acad. Sci., 85:6122 6126, 1988) and cardiolipin complexed
with doxorubicin.
[0033] A lipid clathrate is a three-dimensional, cage-like
structure employing one or more lipids wherein the structure
entraps a bioactive agent. Such clathrates are included in the
scope of the present invention.
[0034] Proliposomes are formulations that can become liposomes or
lipid complexes upon coming in contact with an aqueous liquid.
Agitation or other mixing can be necessary. Such proliposomes are
included in the scope of the present invention.
[0035] Liposomes can be produced by a variety of methods (for
example, see, Bally, Cullis et al., Biotechnol Adv. 5(1):194,
1987). Banghant's procedure (J. Mol. Biol., J Mol Biol.
13(1):238-52, 1965) produces ordinary multilamellar vesicles
(MLVs). Lenk et al. (U.S. Pat. Nos. 4,522,803, 5,030,453 and
5,169,637), Fountain et al. (U.S. Pat. No. 4,588,578) and Cullis et
al. (U.S. Pat. No. 4,975,282) disclose methods for producing
multilamellar liposomes having substantially equal interlamellar
solute distribution in each of their aqueous compartments.
Paphadjopoulos et al., U.S. Pat. No. 4,235,871, discloses
preparation of oligolamellar liposomes by reverse phase
evaporation.
[0036] Unilamellar vesicles can be produced from MLVs by a number
of techniques, for example, the extrusion of Cullis et al. (U.S.
Pat. No. 5,008,050) and Loughrey et al. (U.S. Pat. No. 5,059,421).
Sonication and homogenization can be used to produce smaller
unilamellar liposomes from larger liposomes (see, for example,
Paphadjopoulos et al., Biochim. Biophys. Acta., 135:624-638, 1967;
Deamer, U.S. Pat. No. 4,515,736; and Chapman et al., Liposome
Technol., 1984, pp. 1-18).
[0037] The original liposome preparation of Bangham et al. (J. Mol.
Biol., 1965, 13:238-252) involves suspending phospholipids in an
organic solvent which is then evaporated to dryness leaving a
phospholipid film on the reaction vessel. Next, an appropriate
amount of aqueous phase is added, the mixture is allowed to
"swell", and the resulting liposomes which consist of multilamellar
vesicles (MLVs) are dispersed by mechanical means. This preparation
provides the basis for the development of the small sonicated
unilamellar vesicles described by Papahadjopoulos et al. (Biochim.
Biophys, Acta., 1967, 135:624-638), and large unilamellar
vesicles.
[0038] Techniques for producing large unilamellar vesicles (LUVs),
such as, reverse phase evaporation, infusion procedures, and
detergent dilution, can be used to produce liposomes. A review of
these and other methods for producing liposomes can be found in the
text Liposomes, Marc Ostro, ed., Marcel Dekker, Inc., New York,
1983, Chapter 1, the pertinent portions of which are incorporated
herein by reference. See also Szoka, Jr. et al., (1980, Ann, Rev.
Biophys. Bioeng., 9:467), the pertinent portions of which are also
incorporated herein by reference.
[0039] Other techniques that are used to prepare vesicles include
those that form reverse-phase evaporation vesicles (REV),
Papahadjopoulos et al., U.S. Pat. No. 4,235,871. Another class of
liposomes that can be used are those characterized as having
substantially equal lamellar solute distribution. This class of
liposomes is denominated as stable plurilamellar vesicles (SPLV) as
defined in U.S. Pat. No. 4,522,803 to Lenk, et al. and includes
monophasic vesicles as described in U.S. Pat. No. 4,588,578 to
Fountain, et al. and frozen and thawed multilamellar vesicles
(FATMLV) as described above.
[0040] A variety of sterols and their water soluble derivatives
such as cholesterol hemisuccinate have been used to form liposomes;
see specifically Janoff et al., U.S. Pat. No. 4,721,612, issued
Jan. 26, 1988, entitled "Steroidal Liposomes." Mayhew et al,
described a method for reducing the toxicity of antibacterial
agents and antiviral agents by encapsulating them in liposomes
comprising alpha-tocopherol and certain derivatives thereof. Also,
a variety of tocopherols and their water soluble derivatives have
been used to form liposomes, see Janoff et al., U.S. Pat. No.
5,041,278.
[0041] A process for forming liposomes or lipid complexes involves
a "solvent infusion" process. This is a process that includes
dissolving one or more lipids in a small, preferably minimal,
amount of a process compatible solvent to form a lipid suspension
or solution (preferably a solution) and then injecting the solution
into an aqueous medium containing bioactive agents. Typically a
process compatible solvent is one that can be washed away in a
aqueous process such as dialysis. The composition that is cool/warm
cycled is preferably formed by solvent infusion, with ethanol
infusion being preferred. Alcohols are preferred as solvents.
"Ethanol infusion," a type of solvent infusion, is a process that
includes dissolving one or more lipids in a small, preferably
minimal, amount of ethanol to form a lipid solution and then
injecting the solution into an aqueous medium containing bioactive
agents. A "small" amount of solvent is an amount compatible with
forming liposomes or lipid complexes in the infusion process. Such
processes are described in Lee et al., U.S. patent application Ser.
No. 10/634,144, filed Aug. 4, 2003, Pilkiewicz et al, U.S. patent
application Ser. No. 10/383,173, filed Mar. 5, 2003, and Boni et
al., U.S. patent application Ser. No. 10/383,004, filed Mar. 5,
2003, which applications are hereby incorporated by reference in
their entirety.
[0042] Liposome or lipid complex sizing can be accomplished by a
number of methods, such as extrusion, sonication and homogenization
techniques which are well known, and readily practiced, by
ordinarily skilled artisans. Extrusion involves passing liposomes,
under pressure, one or more times through filters having defined
pore sizes. The filters are generally made of polycarbonate, but
the filters may be made of any durable material which does not
interact with the liposomes and which is sufficiently strong to
allow extrusion under sufficient pressure. Preferred filters
include "straight through" filters because they generally can
withstand the higher pressure of the preferred extrusion processes
of the present invention. "Tortuous path" filters may also be used.
Extrusion can also use asymmetric filters, such as AnotecO.RTM.
filters, which involves extruding liposomes through a branched-pore
type aluminum oxide porous filter. 031 Liposomes or lipid complexes
can also be size reduced by sonication, which employs sonic energy
to disrupt or shear liposomes, which will spontaneously reform into
smaller liposomes. Sonication is conducted by immersing a glass
tube containing the liposome suspension into the sonic epicenter
produced in a bath-type sonicator. Alternatively, a probe type
sonicator may be used in which the sonic energy is generated by
vibration of a titanium probe in direct contact with the liposome
suspension. Homogenization and milling apparatii, such as the
Gifford Wood homogenizer, Polytron.TM. or Microfluidizer.RTM., can
also be used to break down larger liposomes or lipid complexes into
smaller liposomes or lipid complexes.
[0043] The resulting liposomes/complexes can be separated into
homogeneous populations using methods well known in the art; such
as tangential flow filtration. In this procedure, a heterogeneously
sized population of liposomes or lipid complexes is passed through
tangential flow filters, thereby resulting in a liposome population
with an upper and/or lower size limit. When two filters of
differing sizes, that is, having different pore diameters, are
employed, liposomes smaller than the first pore diameter pass
through the filter. This filtrate can the be subject to tangential
flow filtration through a second filter, having a smaller pore size
than the first filter. The retentate of this filter is a
liposomal/complex population having upper and lower size limits
defined by the pore sizes of the first and second filters,
respectively.
[0044] Mayer et al. found that the problems associated with
efficient entrapment of lipophilic ionizable bioactive agents such
as antineoplastic agents, for example, anthracyclines or vinca
alkaloids, can be alleviated by employing transmembrane ion
gradients. Aside from inducing greater uptake, such transmembrane
gradients can also act to increase antiinfective retention in the
liposomes/complexes.
[0045] Liposomal/complexed antiinfective has a sustained
antiinfective effect and lower toxicity allowing less frequent
administration and an enhanced therapeutic index. In preclinical
animal studies and in comparison to inhaled Tobramycin
(not-liposomal or lipid-complexed) at the equivalent dose level,
liposomal/complexed amikacin was shown to have, during the time
period shortly after administration to over 24 hours later, drug
levels in the lung that ranged from two to several hundred times
that of Tobramycin. Additionally, liposomal/complexed amikacin
maintained these levels for well over 24 hours. In an animal model
designed to mimic the pseudombnas infection seen in CF patients,
liposomal/complexed amikacin was shown to significantly eliminate
the infection in the animals' lungs when compared to free
aminoglycosides.
[0046] Lung surfactant allows for the expansion and compression of
the lungs during breathing. This is accomplished by coating the
lung with a combination of lipid and protein. The lipid is
presented as a monolayer with the hydrophobic chains directed
outward. The lipid represents 80% of the lung surfactant, the
majority of the lipid being phosphatidylcholine, 50% of which is
dipalmitoyl phosphatidylcholine (DPPC) (Veldhuizen et al, 1998).
The surfactant proteins (SP) that are present function to maintain
structure and facilitate both expansion and compression of the lung
surfactant as occurs during breathing. Of these, SP-B and SP-C
specifically have lytic behavior and can lyse liposomes (Hagwood et
al., 1998; Johansson, 1998). This lytic behavior is believed to
facilitate the gradual break-up of liposomes followed, by their
release of internal contents allowing for a depot effect. This
break-up of liposomes occurs naturally as evidenced by the
spontaneous unraveling of lamellar bodies ejected by exocytosis
(Ikegami & Jobe, 1998) In addition to becoming assimilated
within the lung surfactant, liposomes can be directly ingested by
macrophages through phagocytosis (Couveur et al., 1991;
Gonzales-Roth et al., 1991; Swenson et al, 1991). Uptake of
liposomes by alveolar macrophages is another means by which drugs
can be delivered to the diseased site.
[0047] The lipids preferably used to form either liposomes or lipid
complexes for inhalation use are common to the endogenous lipids
found in the lung surfactant. Liposomes are composed of bilayers
that entrap the desired pharmaceutical. These can be configured as
multilamellar vesicles of concentric bilayers with the
pharmaceutical trapped within either the lipid of the different
layers or the aqueous space between the layers. The present
invention unique processes to create unique liposomes and
lipid/drug complexes. Both the processes and the product of these
processes are part of the present invention.
[0048] The lipid to drug ratio using the process of the present
invention is preferably less than 3 to 1. And more preferably the
lipid to drug ratio is less than 2.5 to 1. Further the percentage
of free antiinfective, present after the product is dialyzed for a
particular duration, is decreased.
[0049] All processes described herein can be easily adapted for
large scale, aseptic manufacture. The final liposome size can be
adjusted by modifying the lipid composition, concentration,
excipients, and processing parameters.
[0050] An obstacle to treating infectious diseases such as
Pseudomonas aeruginosa, the leading cause of chronic illness in
cystic fibrosis patients is drug penetration within the
sputum/biofilm barrier on epithelial cells (FIG. 1). In FIG. 1, the
donut shapes represent liposomal/complexed antiinfective, the "+"
symbol represents free antiinfective, the "-" symbol mucin,
alginate and DNA, and the solid bar symbol represents Pseudomonas
aeruginosa. This barrier is composed of both colonized and
planktonic P. aeruginosa embedded in alginate or exopolysaccharides
from bacteria, as well as DNA from damaged leukocytes, and mucin
from lung epithelial cells, all possessing a net negative charge
(Costerton, et al., 1999). This negative charge binds up and
prevent penetration of positively charged drugs such as
aminoglycosides, rendering them biologically ineffective (Mendelman
et al., 1985). Entrapment of antiinfectives within liposomes or
lipid complexes could shield or partially shield the antiinfectives
from non-specific binding to the sputum/biofilm, allowing for
liposomes or lipid complexes (with entrapped aminoglycoside) to
penetrate (FIG. 1).
[0051] Amikacin has been shown to have a high degree of resistance
to bacterial enzymes, thus providing a greater percent of
susceptible clinical isolates than found for other aminoglycosides
including tobramycin and gentamicin (Price et al., 1976). In
particular, P. aeruginosa isolates are far more sensitive to
amikacin than other aminoglycosides while exhibiting no
cross-resistance (Damaso et al., 1976).
[0052] The sustained release and depot effect of
liposomal/complexed amikacin is clearly seen in FIG. 2. In this
study rats were given tobramycin via intratracheal and intravenous
administration: The rats were also given liposomal/complexed
amikacin intratracheally at the same dose (4 mg/rat). The data show
that it is only with the liposomal/complexed amikacin that a
sustained release and depot effect is achieved. In fact, 24 hours
after dosing, only liposomal/complexed amikacin shows significant
levels of the drug in the animal's lungs, while both tobramycin
formulations revealed negligible levels, primarily due, it is
believed to rapid systemic absorption. This greater than a
hundred-fold increase of aminoglycoside in the lung for
liposomal/complexed antiinfective supports the idea of a sustained
release liposomal/complexed antiinfective that can be taken
significantly less often than the currently approved TOBI.TM.
formulation (Chiron Corporation, Ameryville, Calif.).
[0053] Moreover, the presence of a sputum/biofilm prevents the
penetration of the free aminoglycosides due to binding of the
antiinfectives to its surface (FIG. 1). Therefore, doses in excess
of 1000 .mu.g of tobramycin/gram of lung tissue are needed to show
a therapeutic effect in CF patients. This is overcome with
liposomal/complexed amikacin. Thus, the therapeutic level of drug
is maintained for a longer period of time in the
liposomal/complexed formulations of amikacin compared to free
tobramycin. This facilitation of binding and penetration could also
be a means by which liposomal/complexed amikacin could
significantly reduce bacterial resistance commonly seen to develop
when antibacterials are present in vivo at levels below the minimum
inhibitory concentration.
[0054] The pharmacokinetics of amikacin was determined in rats
following intratracheal (IT) administration of either free
tobramycin or liposomal/complexed amikacin. These data were
compared to the distribution obtained in the lungs following a tail
vein injection of free tobramycin. In all cases a dose of 4 mg/rat
was administered. As can be seen in FIG. 2, a much larger
deposition of aminoglycoside can be delivered by TT compared to
injection. The depot effect of liposomal/complexed antiinfective
technology is also demonstrated in that in comparison to tobramycin
given either IT or IV, a greater than a hundred-fold increase in
drug for liposomal/complexed amikacin still remains in the lungs
twenty-four hours following administration. Thus, the therapeutic
level of drug is maintained for a longer period of time in the
liposomal formulations of amikacin compared to free tobramycin.
[0055] The binding of aminoglycosides to sputum of CF patients is a
concern, particularly if this binding reduces the bioactivity of
the antiinfective (Hunt et al., 1995). To determine whether
liposomal/complexed amikacin can retain biological activity over a
prolonged period of time, normal rats were administered
liposomal/complexed amikacin by intratracheal instillation. This
was followed by its removal at 2 or 24 hours via a bronchial
alveolar lavage (BAL) to determine biological activity. Samples
were concentrated by ultrafiltration followed by filtration (0.2
micron) to remove contaminating lung microbes. Amikacin
concentration was determined employing a TUX instrument and
biological activity determined using a Mueller Hinton broth
dilution assay (Pseudomonas aeruginosa). The results are shown in
the following Table I:
TABLE-US-00001 time amakacin in BAL amakacin in filtrate MIC
(hours) (microgram/mL) (microgram/mL) (.mu.g/mL) 2 160 119 1.9 24
73 32 4.0
[0056] As shown by the above table, the recovered filtered
liposomal/complexed amikacin was capable of killing P. aeruginosa
in a Mueller Hinton broth assay even after 24 hours with an MIC of
4. At 2 hours an MIC of 2 was obtained, which is similar to that
obtained for the filtered liposomal/complexed amikacin stock. Thus,
the liposomal/complexed amikacin was still active following 24
hours in the lung. At 24 hours free tobramycin at the same dose was
undetectable in a BAL. This indicates that not only is the
liposomal/complexed antiinfective formulation retained in the lung,
but it is also freely available to penetrate a sputum/biofilm over
time. These data combined with the facts as evident in FIG. 2 and
Table II (below), that liposomal/complexed amikacin releases the
free antiinfective over time while maintaining high levels of the
antiinfective in the lungs, supports the rationale that this system
may yield a sustained antiinfective effect over time. This effect
should prove significant in reducing both the bio-burden of the
Pseudomonas and the development of resistance due to trough levels
of antiinfective.
[0057] As an in vitro demonstration of slow release of
liposomal/complexed amikacin and its sustained antiinfective
effect, the formulation was incubated in sputum from patients with
Chronic Obstructive Pulmonary Disease (COPD) containing PAO1 mucoid
Pseudomonas. The liposomal/complexed amikacin was also incubated in
alginate containing PAO1 mucoid Pseudomonas. In both cases
sustained and enhanced killing of the pseudomonas over time was
observed, as shown in Table II:
TABLE-US-00002 In Vitro Sputum/Alginate Assay (% survival of PA01
Mucoid Pseudomonas) Incubation time at 37.degree. C. Amikacin conc.
1 h 3 h 6 h 24 h (microgram/mL) Lip-An-15 Sputum 81 15 22 <1 8
Lip-An-15 Alginate 100 59 1 <1 10
[0058] Classical kill curves are not applicable for
liposomal/complexed antiinfective technology because the liposomal
formulations exhibit a slow release of antiinfective with an
enhanced antiinfective effect. The liposome/complex protects the
amikacin from the sputum and/or alginate until its release. In
time, complete killing is observed, consistent with slow release
sustained antiinfective effect model with no interference or
inactivation of antiinfective.
[0059] The efficacy of liposomal/complexed amikacin formulations
was studied using a model for chronic pulmonary infection (Cash et
al., 1979) where P. aeruginosa, embedded in an agarose bead matrix,
was instilled in the trachea of rats. This mucoid Pseudomonas
animal model was developed to resemble the Pseudomonas infections
seen in CF patients. Some of the clinical correlates to CF include:
a similar lung pathology; the development of immune complex
disorders; and a conversion to the mucoid phenotype by P.
aeruginosa strains (Cantin and Woods, 1999), Rat lungs were
infected with over 10.sup.7 CFUs of a mucoid Pseudomonas (strain
PAO1) taken from a CF patient isolate, and subsequently treated
with (a) free aminoglycoside, (b) the lipid vehicle alone as
non-drug control, and (c) liposomal/complexed amikacin. In
addition, formulations were first screened on the ability to kill
in vitro P. aeruginosa on modified Kirby-Bauer plates.
[0060] Various liposomal/complexed amikacin formulations were
tested based on either different lipid compositions or
manufacturing parameters resulting in different killing zones in in
vitro experiments. This experiment was designed to determine the
increase in efficacy obtained with liposomal/complexed
aminoglycoside over free aminoglycoside. Blank control lipid
compositions, two different liposomal/complexed amikacin
formulations and free amikacin and free Tobramycin at the same
aminoglycoside concentrations as the liposomal/complexed
antiinfective formulations were compared. In addition, a 10 fold
higher dose of free amikacin and a 10 fold higher dose of free
tobramycin were also given. Dosing was IT daily over seven days.
Results (FIG. 3) indicate that liposomal/complexed amikacin in the
two formulations (differing in lipid composition) revealed a
significant reduction in CFU levels and were better at reducing
CFUs than free amikacin or free tobramycin at 10-fold
higher-dosages. In the Figure, Lip-An-14 is DPPC/Chol/DOPC/DOPG
(42:45:4:9) and 10 mg/nil amikacin, Lip-An-15 is DDPC/Chol (1:1)
also at 10 mg/ml. All lipid-lipid and lipid-drug ratios herein are
weight to weight.
[0061] The next experiment (FIG. 4) was designed to demonstrate the
slow release and sustained antiinfective capabilities of
liposomal/complexed amikacin. The dosing was every other day for 14
days, as opposed to every day for seven days as in the previous
experiments. Results indicate that liposomal/complexed amikacin in
the two formulations (differing in lipid composition) had a 10 to
100 times more potent (greater ability to reduce CFU levels) than
free amikacin or free tobramycin. A daily human dose of 600 mg
TOBI.RTM. (or about 375 mg/m2) corresponds to a daily rat dose of
9.4 mg. Thus the data can be directly correlated to a 10 to 100
fold improvement in human efficacy. It should be noted that a
two-log reduction is the best that can be observed in this model. A
100-fold reduction in P. aeruginosa in sputum assays has been
correlated with improved pulmonary function (Ramsey et al., 1993).
The sustained release of the liposomal/complexed amikacin
formulations indicate that a lower dose and/or less frequent dosing
can be employed to obtain a greater reduction in bacterial growth
than can be obtained with free aminoglycoside.
[0062] The efficacy of liposomal/complexed amikacin was studied in
a model for chronic pulmonary infection where P. aeruginosa was
embedded in an agarose bead matrix that was instilled via the
trachea of Sprague/Dawley rats. Three days later free amikacin or
liposomal/complexed amikacin was dosed every day (FIG. 3) or every
other day (FIG. 4) at 1 mg/rat or 10 mg/rat of the given
aminoglycoside or 1 mg/rat liposomal/complexed amikacin, as well as
with blank liposomes (lipid vehicle) as the control, with five rats
per group.
[0063] The homogenized rat lungs (frozen) following the 14 day
experiment were analyzed for aminoglycoside content and activity.
The clinical chemical assay was performed using a TDX instrument
while the bioassay was performed by measuring inhibition zones on
agar plates embedded with Bacillus subtilis.
[0064] The results are shown in Table III:
TABLE-US-00003 Bioassay Clinical Assay Formulation (microgram/mL)
(microgram/mL) Lip-An-14 at 10 mg/mL 9.5 9.1 Lip-An-15 at 10 mg/mL
21.5 18.4 Free amikacin at 100 mg/mL nd 2.0 Free tobramycin at 100
mg/mL nd 1.4
Drug weights are for the drug normalized to the absence of any salt
form.
[0065] The Table III results indicate that aminoglycoside is
present and active for both liposomal/complexed antiinfective
formulations, while little can be detected for the free
aminoglycoside even at the 10-fold higher dose. These further
results establish the sustained release characteristics of
liposomal/complexed antiinfective, and also confirm that that
antiinfective which remains is still active. Of the above
formulations only the free tobramycin (0.1 microgram/nil) exhibited
any detectable levels of aminoglycoside in the kidneys.
[0066] The sustained release and depot effect of
liposomal/complexed amikacin is further demonstrated in FIG. 5.
Rats were given a chronic pulmonary infection where P. aeruginosa
was embedded in an agarose bead matrix that was instilled via the
trachea, using the same beads employed in the efficacy studies. The
rats were then given free tobramycin or liposomal/complexed
amikacin (formulation Lip-An-14) via intratracheal administration
at the same dose (2 mg/rat). The data, measured in microgram
antiinfective per gram lung tissue over time, show that
liposomal/complexed antiinfective exhibits a sustained release and
depot effect while free tobramycin revealed negligible levels in
the lungs by 24 hours, primarily due it is believed to rapid
systemic absorption. This greater than a hundred-fold increase of
antiinfective in the lung for liposomal/complexed amikacin in an
infected rat supports the idea of a sustained release
liposomal/complexed antiinfective that can be taken significantly
less often than the currently approved TOBI.TM. formulation.
[0067] The pharmacokinetics of amikacin was determined in rats
following intratracheal (IT) administration of either free
tobramycin or liposomal/complexed amikacin. A dose of 2 mg/rat was
administered. The depot effect of liposomal/complexed antiinfective
technology is demonstrated in that in comparison to free tobramycin
given IT, a greater than a hundred-fold increase in drug for
liposomal/complexed amikacin still remains in the infected lungs
twenty-four hours following administration. Thus, the therapeutic
level of drug is maintained for a longer period of time in the
liposomal formulations compared to free tobramycin.
[0068] FIG. 7 shows remarkable residence time and accumulation of
effective amounts of antiinfective in the lungs, a result that
establishes that relatively infrequent dosings can be used. Each
dose is 4 hr. by inhalation (in rat, 3 rats per group, as above) of
nebulized liposomal amikacin (DPPC/Chol., 1:1) at 15 mg/ml
amikacin. Dosing was at either day one; day one, three and five; or
day one, two, three, four and five. Rats providing a given data bar
were sacrificed after the respective dosing of the data bar. The
formulation is made as in the Example.
[0069] Similar anti-infectives can be utilized for the treatment of
intracellular infections like pulmonary anthrax and tularemia. In
pulmonary anthrax the anthrax spores reach the alveoli in an
aerosol. The inhaled spores are ingested by pulmonary macrophages
in the alveoli and carried to the regional tracheobronchial lymph
nodes or mediastinal lymph nodes via the lymphatics (Pile et al.,
1998; Gleiser et al., 1968). The macrophage is central in the both
the infective pathway and is the major contributor of host
self-destruction in systemic (inhalation) anthrax. In addition to
its attributes of sustained release and targeting,
liposomal/complexed antiinfective technology can enhance cellular
uptake and can use alveolar macrophages and lung epithelial cells
in drug targeting and delivery. The possession of these
characteristics is believed to facilitate the treatment of these
intracellular infections, which infections occur in the lungs and
are transported by macrophages. More importantly; these
characteristics should make the antiinfective more effective in
that the liposomal/complexed antiinfective should be phagocytized
by the very cells containing the disease. The antiinfective would
be released intracellularly in a targeted manner, thereby attacking
the infection before it is disseminated. The encapsulated drug can
be an already approved pharmaceutical like ciprofloxacin,
tetracycline, erthyromycin or amikacin. Liposomal/complexed
ciprofloxacin has been developed.
[0070] In a study this compound was administered to mice and
compared to both free ciprofloxacin administered intratracheally
and free ciprofloxacin administered orally, with all three
compounds given at the same dose (FIG. 6). The dose for each mouse
was 15 mg/kg, with three mice per group. Liposomal/complexed cipro
was in DPPC/Cholesterol (9:1), at 3 mg/ml cipro, with the
formulation produced as in the Example. The lipid to drug ratio was
12.5:1 by weight. In comparison to orally administered
ciprofloxacin, liposomal/complexed ciprofloxacin was present in the
mice lungs at amounts over two orders of magnitude higher than free
ciprofloxacin; Moreover, only liposomal/complexed ciprofloxacin
showed levels of drug in the lung after 24 hours, while the orally
administered drug was undetectable in less than two hours. This
data supports the use of liposomal/complexed ciprofloxacin and
other antiinfectives like aminoglycosides, tetracyclines and
macrolides for the treatment and for the prophylactic prevention of
intracellular diseases used by bioterrorists.
[0071] One type of process of manufacture of liposomal/complexed
typically comprises ethanol infusion at room temperature, which is
below the phase transition temperature for the lipids used in the
formulation, Liposomes in the form of small unilamellar vesicles
(SUVs) are mixed with an aqueous or ethanolic solution containing
the bioactive agent to be entrapped. Ethanol is infused into this
mixture. The mixture immediately forms either extended sheets of
lipid or multilamellar vesicles (MLVs). The extended sheets of
lipid, if formed, can be induced form MLVs upon removal of ethanol
by either sparging or washing by such methods as centrifugation,
dialysis or diafiltration. The MLVs will typically range in
diameter between approximately 0.1 and approximately 3.0 .mu.m.
[0072] Or, the lipids to be employed are dissolved in ethanol to
form a lipid-ethanol solution. The lipid-ethanol solution is
infused in an aqueous or ethanolic solution containing the molecule
of the bioactive agent to be entrapped. All manipulations are
performed below the phase transition of the lowest melting lipid.
The mixture immediately forms either extended sheets of lipid or
multilamellar vesicles (MLVs)(10). The extended sheets of lipid
will form MLVs upon removal of ethanol by either sparging or
washing by such methods as centrifugation, dialysis or
diafiltration. The MLVs will typically range in diameter from
approximately 0.1 to approximately 3.0 .mu.m.
TABLE-US-00004 Lipids Mol ratio Lipid/amikacin, w/w DPPC -- 1.1
DPPC/DOPG 9:1 1.0 DPPC/DOPG 7:1 3.9 DPPC/DOPG 1:1 2.8 DPPC/DOPG 1:2
2.7 DOPG -- 2.6 DPPC/Cholesterol 19:1 1.0 DPPC/Cholesterol 9:1 1.2
DPPC/Cholesterol 4:1 1.7 DPPC/Cholesterol 13:7 2.1 DPPC/Cholesterol
1:1 2.7 DPPC/DOPC/Cholesterol 8.55:1:.45 2.0 DPPC/DOPC/Cholesterol
6.65:1:.35 3.0 DPPC/DOPC/Cholesterol 19:20:1 2.5
DPPC/DOPG/Cholesterol 8.55:1:.45 3.8 DPPC/DOPG/Cholesterol
6.65:1:.35 4.1 DPPC/DOPG/Cholesterol 19:20:1 4.2
DPPC/DOPC/DOPG/Cholesterol 42:4:9:45 3.7 DPPC/DOPC/DOPG/Cholesterol
59:5:6:30 3.7
[0073] A number of formulations with Amikacin were made by the
method of the Example, as summarized below:
[0074] Further information of forming liposomal/complexed
antiinfective can be found in PCT/US03/06847, filed Mar. 5, 2003,
which is incorporated herein by reference in its entirety.
EXAMPLE
[0075] The following is a detailed description of the manufacture
of 150 mL of
Liposomal/complexed amikacin.
Total Initial Volume=1.5 L
[0076] Ethanol Content=23.5% (v/v) Lipid Composition: DPPC/Chol
(1:1 mole ratio) Initial [Lipid]=7.6 mg/ml Initial [amikacin
sulfate]=57.3 mg/nil Final product Volume=150 mL
[0077] I) Compounding and Infusion:
[0078] 7.47 g DPPC and 3.93 g Cholesterol were dissolved directly
in 352.5 mL ethanol in a 50 C water bath. 85.95 g amikacin sulfate
was dissolved directly in 1147.5 mL PBS buffer. The solution is
then titrated with 10N NaOH or KOH to bring the pH to approximately
6.8.
[0079] 352.5 mL ethanol/lipid was added or infused to the 1147.5 mL
amikacin/buffer to give a total initial volume of 1.5 L. The
ethanol/lipid was pumped @-30 mL/min (also called infusion rate)
with a peristaltic pump into the amikacin/buffer solution which was
being rapidly stirred at 150 RPM in a reaction vessel on a stir
plate at room temperature
[0080] The product was stirred at room temperature for 20-30
minutes.
[0081] II) Diafiltration or "Washing" Step:
[0082] The mixing vessel was hooked up to a peristaltic pump and
diafiltration cartridge. The diafiltration cartridge is a hollow
membrane fiber with a molecular weight cut-off of 500 kilodaltons.
The product was pumped from the reaction vessel through the
diafiltration cartridge and then back into the mixing vessel at
room temperature. A back pressure of approximately 7 psi is created
throughout the cartridge. Free amikacin and ethanol was forced
through the hollow fiber membrane by the back pressure leaving the
liposomal amikacin (product) behind: The product was washed 8 times
at room temperature. Fresh PBS buffer was added (via another
peristaltic pump) to the reaction vessel to compensate for the
permeate removal and to keep a constant product volume.
[0083] The product was concentrated.
[0084] Publications and references, including but not limited to
patents and patent applications, cited in this specification are
herein incorporated by reference in their entirety in the entire
portion cited as if each individual publication or reference were
specifically and individually indicated to be incorporated by
reference herein as being fully set forth. Any patent application
to which this application claims priority is also incorporated by
reference herein in the manner described above for publications and
references.
[0085] While this invention has been described with an emphasis
upon preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations in the preferred devices and
methods may be used and that it is intended that the invention may
be practiced otherwise than as specifically described herein.
Accordingly, this invention includes all modifications encompassed
within the spirit and scope of the invention as defined by the
claims that follow.
REFERENCES
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Susceptibility of current clinical isolates of Pseudomonas
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* * * * *