U.S. patent application number 14/676367 was filed with the patent office on 2015-07-23 for concentrated, inhalable antibiotic formulation.
The applicant listed for this patent is ARADIGM CORPORATION. Invention is credited to JAMES BLANCHARD, DAVID C. CIPOLLA.
Application Number | 20150202201 14/676367 |
Document ID | / |
Family ID | 41257232 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150202201 |
Kind Code |
A1 |
CIPOLLA; DAVID C. ; et
al. |
July 23, 2015 |
CONCENTRATED, INHALABLE ANTIBIOTIC FORMULATION
Abstract
A concentrated inhalable formulation of an antibiotic drug such
as ciprofloxacin is disclosed. The antibiotic is formulated with
sodium acetate and liposome which incorporate antibiotic. The
formulation is aerosolized and inhaled for treatment of respiratory
tract infections and other medical conditions.
Inventors: |
CIPOLLA; DAVID C.; (San
Ramon, CA) ; BLANCHARD; JAMES; (El Granada,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARADIGM CORPORATION |
Hayward |
CA |
US |
|
|
Family ID: |
41257232 |
Appl. No.: |
14/676367 |
Filed: |
April 1, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12888275 |
Sep 22, 2010 |
9028864 |
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14676367 |
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12437765 |
May 8, 2009 |
8119156 |
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12888275 |
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12261946 |
Oct 30, 2008 |
8071127 |
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12437765 |
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11876539 |
Oct 22, 2007 |
8268347 |
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12261946 |
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60862753 |
Oct 24, 2006 |
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Current U.S.
Class: |
424/450 ;
514/253.08 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 9/5084 20130101; A61K 9/0043 20130101; A61K 47/22 20130101;
A61K 9/12 20130101; A61K 9/127 20130101; A61K 9/0078 20130101; A61P
31/00 20180101; A61K 9/0073 20130101; A61P 11/08 20180101; A61K
31/496 20130101; A61K 45/06 20130101 |
International
Class: |
A61K 31/496 20060101
A61K031/496; A61K 9/00 20060101 A61K009/00; A61K 9/127 20060101
A61K009/127 |
Claims
1-27. (canceled)
28. An aerosolized composition of particles, comprising: a free,
unencapsulated ciprofloxacin in an aqueous solution with sodium
acetate at a pH of 3.2; and a liposome-encapsulated ciprofloxacin;
wherein the composition is formulated for aerosolized delivery to a
human lung and particles of the aerosol have a diameter in a range
from about 2 microns to about 4 microns; and wherein the free,
unencapsulated ciprofloxacin is in the aqueous solution at a
concentration of about 10 mg/mL or more.
29. A pharmaceutical formulation, comprising: a free,
unencapsulated ciprofloxacin in an aqueous solution with sodium
acetate at a pH of 3.2; and a liposome-encapsulated ciprofloxacin;
wherein the composition is formulated for aerosolized delivery to a
human lung; and wherein the free, unencapsulated ciprofloxacin is
in the aqueous solution at a concentration of about 10 mg/mL or
more.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/888,275, filed Sep. 22, 2010 (now allowed)
which is a continuation of U.S. patent application Ser. No.
12/437,765 filed May 8, 2009, now U.S. Pat. No. 8,119,156, issued
Feb. 21, 2012, which is a continuation-in-part application of U.S.
patent application Ser. No. 12/261,946, filed Oct. 30, 2008, now
U.S. Pat. No. 8,071,127, issued Dec. 6, 2011, which is a
continuation-in-part of application of U.S. patent application Ser.
No. 11/876,539 filed Oct. 22, 2007, now U.S. Pat. No. 8,268,347,
issued Sep. 18, 2012, which claims the benefit of priority to U.S.
Provisional Patent Application Ser. No. 60/862,753 filed Oct. 24,
2006, all of which are incorporated herein by reference in their
entirety noting that the current application controls to the extent
there is any contradiction with any earlier application and to
which applications we claim priority under 35 USC .sctn.120.
FIELD OF THE INVENTION
[0002] The present invention relates to pharmaceutical compositions
for inhalation such as for treating respiratory tract infections
caused by a variety of microorganisms. In particular, the present
invention relates to a highly concentrated, inhalable antibiotic
formulation.
BACKGROUND OF THE INVENTION
[0003] Respiratory tract infections are caused by a variety of
microorganisms. Infections which are persistent have a myriad of
consequences for the health care community including increased
treatment burden and cost, and for the patient in terms of more
invasive treatment paradigms and potential for serious illness or
even death. It would be beneficial if an improved treatment
paradigm were available to provide prophylactic treatment to
prevent susceptible patients from acquiring respiratory tract
infections as well as increasing the rate or effectiveness of
eradicating the infections in patients already infected with the
microorganisms.
[0004] In particular, cystic fibrosis (CF) is one example of a
disease in which patients often acquire persistent or tenacious
respiratory tract infections. CF is a life-threatening genetic
disease affecting approximately 30,000 people in the United States
with a frequency of approximately one in every 2,500 live births
(Fitzsimmons S.C., 1993). The name cystic fibrosis refers to the
characteristic scarring (fibrosis) and cyst formation within the
pancreas, first recognized in the 1930s. About 1,000 new cases of
CF are diagnosed each year. More than 80 percent of patients are
diagnosed by age three; however, nearly 10 percent of newly
diagnosed cases are age 18 or older.
[0005] The primary CF defect is expressed as altered ion transport
via the cystic fibrosis transmembrane conductance regulator (CFTR),
which is the protein regulating cyclic-AMP-mediated chloride
conductance at the apical membranes of secretory epithelia
(Schroeder S A et al., 1995). Specifically, the normal release of
intracellular chloride into extracellular fluids fails to respond
to normal cAMP elevation. This impaired release of chloride results
in the dehydration of surrounding respiratory and intestinal
mucosal linings and impaired sodium reabsorption of the
sudoriferous glands. This mucosal dehydration, coupled with
inflammatory and infective by-products, creates a thick and
tenacious mucus that clogs and damages airways. Prompt, aggressive
treatment of CF symptoms can extend the lives of those with the
disease.
[0006] Although most people without CF have two working copies of
the CFTR gene, only one is needed to prevent cystic fibrosis. CF
develops when neither gene works normally. Therefore, CF is
considered an autosomal recessive disease. There are more than
1,500 different genetic mutations associated with the disease (CFTR
mutation database, 2006), thus making homozygous and heterozygous
screening procedures difficult (Zielenski J et al., 1995). However,
approximately two thirds of the mutations are found to be delta
F508, making it the most common CF mutation (CF Genetic Analysis
Consortium, 1994).
[0007] The ongoing treatment of CF depends upon the stage of the
disease and the organs involved. Clearing mucus from the lungs is
an important part of the daily CF treatment regimen. Chest physical
therapy is one form of airway clearance, and it requires vigorous
percussion (by using cupped hands) on the back and chest to
dislodge the thick mucus from the lungs. Other forms of airway
clearance can be done with the help of mechanical devices used to
stimulate mucus clearance. Other types of treatments include:
Pulmozyme.RTM., an inhaled mucolytic agent shown to reduce the
number of lung infections and improve lung function (Hodson M,
1995); TOBI.RTM. (tobramycin solution for inhalation), an
aerosolized aminoglycoside antibiotic used to treat lung infections
and also shown to improve lung function (Weber A et al., 1994); and
oral azithromycin, a macrolide antibiotic shown to reduce the
number of respiratory exacerbations and the rate of decline of lung
function (Wolter J et al., 2002).
[0008] As discussed above, high rates of colonization and the
challenge of managing Pseudomonas aeruginosa infections in patients
with cystic fibrosis (CF) have necessitated a search for safe and
effective antibiotics. Currently, therapy with an aminoglycoside in
combination with a beta-lactam or a quinolone antibiotic is the
standard. A 96-week series of clinical studies including 520
patients with moderate-to-severe CF showed that patients receiving
inhaled tobramycin spent 25 to 33% fewer days in the hospital and
experienced significant increases in lung function (Moss R B,
2001). These results demonstrate the effectiveness of inhaled
antibiotics to treat CF. However, the development of drug resistant
strains, especially P. aeruginosa, is a major concern with the
long-term delivery of aerosolized antibiotics via inhalation
(LiPuma J J, 2001).
[0009] While azithromycin possesses activity against Staphylococcus
aureus, Haemophilus influenzae, and Streptococcus pneumoniae, it
has no direct activity against Pseudomonas aeruginosa, Burkholderia
cepacia, or other gram-negative non-fermenters (Lode H et al.,
1996). Tobramycin possesses activity against P. aeruginosa;
however, the increase in the number of patients with resistant
isolates on continuous therapy from .about.10% to 80% after 3
months (Smith A L et al., 1989) has led to the intermittent dosing
regimen of 28-days-on followed by 28-days-off therapy. Even on
intermittent inhaled tobramycin therapy, the percentage of patients
with multiresistant P. aeruginosa increased from 14% at baseline to
23% after 18 months of treatment (LiPuma J J, 2001). The
development of a therapeutic regimen that delivers the
anti-infective therapy in a continuous fashion, while still
inhibiting the emergence of resistant isolates, may provide an
improved treatment paradigm. It is noteworthy that chronic P.
aeruginosa airway infections remain the primary cause of morbidity
and mortality in CF patients. When patients experience pulmonary
exacerbations, the use of antipseudomonal therapy, frequently
consisting of a .beta.-lactam and an aminoglycoside, may result in
clinical improvement and a decrease in bacterial burden.
Eradication of the infection, however, is quite rare.
[0010] In CF airways, P. aeruginosa initially has a non-mucoid
phenotype, but ultimately produces mucoid exopolysaccharide and
organizes into a biofilm, which indicates the airway infection has
progressed from acute to chronic. Bacteria in biofilms are very
slow growing due to an anaerobic environment and are inherently
resistant to antimicrobial agents, since sessile cells are much
less susceptible than cells growing planktonically. It has been
reported that biofilm cells are at least 500 times more resistant
to antibacterial agents (Costerton J W et al., 1995). Thus, the
transition to the mucoid phenotype and production of a biofilm
contribute to the persistence of P. aeruginosa in CF patients with
chronic infection by protecting the bacteria from host defenses and
interfering with the delivery of antibiotics to the bacterial
cell.
[0011] Although much effort has been made to improve the care and
treatment of individuals with CF, and the average lifespan has
increased, the median age of survival for people with CF is only to
the late 30s (CF Foundation web site, 2006). Thus, a continuing
need exists for improved formulations of anti-infectives,
especially for administration by inhalation. The present invention
addresses this need.
[0012] Ciprofloxacin is a fluoroquinolone antibiotic that is
indicated for the treatment of lower respiratory tract infections
due to P. aeruginosa, which is common in patients with cystic
fibrosis. Ciprofloxacin is broad spectrum and, in addition to P.
aeruginosa, is active against several other types of gram-negative
and gram-positive bacteria. It acts by inhibition of topoisomerase
II (DNA gyrase) and topoisomerase IV, which are enzymes required
for bacterial replication, transcription, repair, and
recombination. This mechanism of action is different from that for
penicillins, cephalosporins, aminoglycosides, macrolides, and
tetracyclines, and therefore bacteria resistant to these classes of
drugs may be susceptible to ciprofloxacin. Thus, CF patients who
have developed resistance to the aminoglycoside tobramycin (TOBI),
can likely still be treated with ciprofloxacin. There is no known
cross-resistance between ciprofloxacin and other classes of
antimicrobials.
[0013] Despite its attractive antimicrobial properties,
ciprofloxacin does produce bothersome side effects, such as GI
intolerance (vomiting, diarrhea, abdominal discomfort), as well as
dizziness, insomnia, irritability and increased levels of anxiety.
There is a clear need for improved treatment regimes that can be
used chronically, without resulting in these debilitating side
effects.
[0014] Delivering ciprofloxacin as an inhaled aerosol has the
potential to address these concerns by compartmentalizing the
delivery and action of the drug in the respiratory tract, which is
the primary site of infection.
[0015] Currently there is no aerosolized form of ciprofloxacin with
regulatory approval for human use, capable of targeting antibiotic
delivery direct to the area of primary infection. In part this is
because the poor solubility and bitterness of the drug have
inhibited development of a formulation suitable for inhalation.
Furthermore, the tissue distribution of ciprofloxacin is so rapid
that the drug residence time in the lung is too short to provide
additional therapeutic benefit over drug administered by oral or IV
routes.
[0016] Phospholipid vehicles as drug delivery systems were
rediscovered as "liposomes" in 1965 (Bangham et al., 1965). The
therapeutic properties of many active pharmaceutical ingredients
(APIs) can be improved by incorporation into liposomal drug
delivery systems. The general term liposome covers a wide variety
of structures, but generally all are composed of one or more lipid
bilayers enclosing an aqueous space in which drugs can be
encapsulated. The liposomes applied in this program are known in
the drug delivery field as large unilamellar vesicles (LUV), which
are the preferred liposomal structures for IV drug
administration.
[0017] Liposome encapsulation improves biopharmaceutical
characteristics through a number of mechanisms including altered
drug pharmacokinetics and biodistribution, sustained drug release
from the carrier, enhanced delivery to disease sites, and
protection of the active drug species from degradation. Liposome
formulations of the anticancer agents doxorubicin
(Myocet.RTM./Evacet.RTM., Doxyl.RTM./Caelyx.RTM.), daunorubicin
(DaunoXome.RTM.) the anti-fungal agent amphotericin B
(Abelcet.RTM., AmBisome.RTM., Amphotec.RTM.) and a benzoporphyrin
(Visudyne.RTM.) are examples of successful products introduced into
the US, European and Japanese markets over the last decade.
Furthermore, a number of second-generation products have been in
late-stage clinical trials, including Inex's vincristine sulphate
liposomes injection (VSLI). The proven safety and efficacy of
lipid-based carriers make them attractive candidates for the
formulation of pharmaceuticals.
[0018] Therefore, in comparison to the current ciprofloxacin
formulations, a liposomal ciprofloxacin aerosol formulation should
offer several benefits: 1) higher drug concentrations, 2) increased
drug residence time via sustained release at the site of infection,
3) decreased side effects, 4) increased palatability, 5) better
penetration into the bacteria, and 6) better penetration into the
cells infected by bacteria. It has previously been shown that
inhalation of liposome-encapsulated fluoroquinolone antibiotics may
be effective in treatment of lung infections. In a mouse model of
F. tularensis liposomal encapsulated fluoroquinolone antibiotics
were shown to be superior to the free or unencapsulated
fluoroquinolone by increasing survival (CA2,215,716, CA2,174,803,
and CA2,101,241).
[0019] Another application, EP1083881B1, describes liposomes
containing a drug-conjugate comprising a quinolone compound
covalently attached to an amino acid. Yet another application, U.S.
Publication No. 20004142026, also describes the use of
liposome-encapsulated antibiotics and the potential for
administration of a lower dose of a liposome-encapsulated
anti-infective, by a factor of 10 or 100, than for the free
unencapsulated anti-infective.
[0020] It has also been reported that the presence of
sub-inhibitory concentrations of antibiotic agents within the
depths of the biofilm will provide selective pressures for the
development of more resistant phenotypes (Gilbert P et al., 1997).
This may be partly due to the failure of the antibiotics to
penetrate the glycocalyx adequately.
SUMMARY OF THE INVENTION
[0021] An aspect of the invention is an aerosolized, bi-phasic,
formulation which is aerosolized to create a bi-phasic aerosol of
inhaled droplets or particles. The droplets or particles comprise a
free drug (e.g., an anti-infective compound) in which drug is not
encapsulated and which may be ciprofloxacin. The particles further
comprise a liposome which encapsulates a drug such as an
anti-infective compound which also may be ciprofloxin. The free and
liposome encapsulated drug are included within a pharmaceutically
acceptable excipient which is formulated for aerosolized delivery.
The particles may further include an additional therapeutic agent
which may be free and/or in a liposome and which can be any
pharmaceutically active drug which is different from the first
drug.
[0022] The formulation and the resulting particles created when the
formulation is aerosolized are comprised of a pharmaceutically
acceptable carrier, free drug, and drug encapsulated within
liposomes. In some situations the pharmaceutically acceptable
carrier can be completely eliminated such as when the free drug is
in a liquid state. However, the carrier is generally necessary to
provide a solvent for the free drug and that solvent may be water,
ethanol, a combination of water and ethanol or other useful
solvents. The percentage of solvent in the formulation may vary
from 0% to 90% but is generally kept at a level which is
sufficiently high to maintain the drug in solution at the pH of the
formulation. That level will vary from drug to drug and vary as the
pH varies. The carrier can be present in the formulation in an
amount of 10%, 20%, 30%, 40%, 50%, 60% etc. or more or any
incremental amounts there in between.
[0023] The formulation includes the drug in two different forms.
First, the drug is in a free form which is either liquid or
dissolved in a solvent. Second, the drug is encapsulated in
liposomes. The ratio of the free drug to the drug encapsulated in
liposomes can vary. Generally, the free drug makes up 5%, 10%, 20%,
30%, etc. up to 80% of the formulation. The drug present within the
liposome makes up the remaining percentage of drug present in the
formulation. Thus, drug present in the liposomes can be present in
the amount of from 20% up to 95% of the total drug present in the
formulation.
[0024] The formulation may have a pH of 7.4.+-.20%. In some aspects
of the invention the formulation is prepared at a relatively low pH
such as 5.4 and allowed to adjust to a pH of about 7.4 after it is
delivered. Alternatively, the formulation can be formulated at a
high pH of about 9.4 and allowed to adjust downward to 7.4 after
administration.
[0025] The formulation includes liposomes which have the
encapsulated pharmaceutically active drug therein which liposomes
are designed to provide for controlled release of the drug.
Controlled release of this aspect in the invention indicates that
the drug may be released in an amount of about 0.1% to 100% per
hour over a period of time of 1-24 hours or 0.5% to 20% per hour
over a period of time of 1-12 hours, or alternatively, releases
about 2% to 10% per hour over a period of time of about 1 to 6
hours. Incremental amounts in terms of the percentage of the drug
and the number of hours which are between the ranges provided above
in half percentage amounts and half hour amounts and other
incremental amounts are intended to be encompassed by the present
invention.
[0026] One aspect of the invention is a formulation comprising
liposomes which are delivered via an aerosol to the lungs of a
human patient, the liposomes comprising free and encapsulated
ciprofloxacin or other anti-infective agent. The liposomes may be
unilamellar or multilamellar, and may be bioadhesive, containing a
molecule such as hyaluronic acid. At least one therapeutic agent in
addition to the free and liposome-encapsulated anti-infective may
also be included in the composition. That therapeutic agent may be
free drug or encapsulated drug present with a pharmaceutically
acceptable carrier useful for direct inhalation into human lungs.
The other drugs may include enzymes to reduce the viscoelasticity
of the mucus such as DNase or other mucolytic agents, chemicals to
upregulate the chloride ion channel or increase flow of ions across
the cells, including lantibiotics such as duramycin, agents to
promote hydration or mucociliary clearance including epithelial
sodium channel (ENaC) inhibitors or P2Y2 agonists such as
denufosol, elastase inhibitors including Alpha-1 antitrypsin (AAT),
bronchodilators, steroids, N-acetylcysteine, interferon gamma,
interferon alpha, agents that enhance the activity of the
antibiotic against biofilm bacteria such as sodium salicylate
(Polonio R E et al., 2001), or antibiotics known to those skilled
in the art. Inflammation and constriction of the airways are also
associated with cystic fibrosis and its treatment. Accordingly,
bronchodilators, such as .beta..sub.2-adrenergic receptor agonists
and antimuscarinics, and anti-inflammatory agents, including
inhaled corticosteroids, non-steroidal anti-inflammatories,
leukotriene receptor antagonists or synthesis inhibitors, and
others, may also be combined with an anti-infective.
[0027] A further aspect of the invention is a method for treating
cystic fibrosis in a patient, the method comprising administering a
formulation comprising the anti-infective; e.g., ciprofloxacin,
encapsulated in liposomes to the patient. The formulation is
preferably administered by inhalation to the patient.
[0028] According to another aspect of the present invention, a
formulation comprising both a free and encapsulated anti-infective
provides an initially high therapeutic level of the anti-infective
in the lungs to overcome the barrier to eradicate the difficult to
treat biofilm bacteria, while maintaining a sustained release of
anti-infective over time. While some aspects of biofilm resistance
are poorly understood, the dominant mechanisms are thought to be
related to: (i) modified nutrient environments and suppression of
growth rate within the biofilm; (ii) direct interactions between
the exopolymer matrices, and their constituents, and
antimicrobials, affecting diffusion and availability; and (iii) the
development of biofilm/attachment-specific phenotypes (Gilbert P et
al., 1997). The intent of the immediate-release anti-infective;
e.g., ciprofloxacin, is thus to rapidly increase the antibiotic
concentration in the lung to therapeutic levels around the
difficult to eradicate biofilm bacteria to address the challenges
of lower diffusion rate of antibiotic to and within the biofilm.
The sustained-release anti-infective; e.g., ciprofloxacin, serves
to maintain a therapeutic level of antibiotic in the lung thereby
providing continued therapy over a longer time frame, increasing
efficacy, reducing the frequency of administration, and reducing
the potential for resistant colonies to form. The high levels of
free antibiotic may also serve to attenuate the cytotoxic response
to the presence of liposomes (i.e., particles) in the lung and thus
reduce the influx of activated macrophages.
[0029] According to another aspect of the present invention, the
immediate release of high levels of an anti-infective may allow
enhanced penetration of the glycocalyx. The sustained release of
the anti-infective may ensure that the anti-infective agent never
falls below the sub-inhibitory concentration and so reduces the
likelihood of forming resistance to the anti-infective.
[0030] Although ciprofloxacin is a particularly useful
anti-infective in this invention, there is no desire to limit this
invention to ciprofloxacin. Other antibiotics or anti-infectives
can be used such as those selected from the group consisting of: an
aminoglycoside, a tetracycline, a sulfonamide, p-aminobenzoic acid,
a diaminopyrimidine, a quinolone, a.beta.-lactam, a.beta.-lactam
and a.beta.-lactamase inhibitor, chloraphenicol, a macrolide,
penicillins, cephalosporins, corticosteroid, prostaglandin,
linomycin, clindamycin, spectinomycin, polymyxin B, colistin,
vancomycin, bacitracin, isoniazid, rifampin, ethambutol,
ethionamide, aminosalicylic acid, cycloserine, capreomycin, a
sulfone, clofazimine, thalidomide, a polyene antifungal,
flucytosine, imidazole, triazole, griseofulvin, terconazole,
butoconazole ciclopirax, ciclopirox olamine, haloprogin,
tolnaftate, naftifine, terbinafine, or any combination thereof.
[0031] These and other objects, advantages, and features of the
invention will become apparent to those persons skilled in the art
upon reading the details of the formulations and methodology as
more fully described below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Aspects and embodiments of the invention are best understood
from the following detailed description when read in conjunction
with the accompanying drawings. It is emphasized that, according to
common practice, the various features of the drawings are not
to-scale. On the contrary, the dimensions of the various features
are arbitrarily expanded or reduced for clarity. Included in the
drawings are the following figures:
[0033] FIG. 1 is a manufacturing flow chart of liposomal
ciprofloxacin for inhalation (HSPC/Chol--10 L Batch).
[0034] FIG. 2 is a graph showing the cumulative survival rate of
mice following infection with P. aeruginosa-laden agarose beads on
Day 0. Mice were treated intranasally once daily starting on Day 2
and ending on Day 9 with the liposomal formulation of ciprofloxacin
(drug) at one of three different concentrations (100%, open
diamond; 33%, closed square; or 10%, open triangle). Diluent was
used as a control (closed circle). Surviving mice were sacrificed
on Day 10.
[0035] FIG. 3 is a bar graph showing increases in levels of IL-1
following 14 days of inhalation of either control (Saline), low
dose liposomal ciprofloxacin (Low), high dose liposomal
ciprofloxacin (High), combination of free and liposomal
ciprofloxacin (Mixture) and free ciprofloxacin (Free). The dark
bars on the graph show the levels all return to baseline values
after the 28 day recovery evaluation.
DETAILED DESCRIPTION OF THE INVENTION
[0036] Before the present method of formulating
ciprofloxacin-encapsulated liposomes and delivery of such for
prevention and/or treatment of cystic fibrosis and other medical
conditions, and devices and formulations used in connection with
such are described, it is to be understood that this invention is
not limited to the particular methodology, devices and formulations
described, as such methods, devices and formulations may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims.
[0037] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0038] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0039] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a formulation" includes a plurality of such
formulations and reference to "the method" includes reference to
one or more methods and equivalents thereof known to those skilled
in the art, and so forth.
[0040] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
[0041] As used herein, anti-infective refers to agents that act
against infections, such as bacterial, viral, fungal,
mycobacterial, or protozoal infections.
[0042] Anti-infectives covered by the invention include but are not
limited to quinolones (such as nalidixic acid, cinoxacin,
ciprofloxacin and norfloxacin and the like), sulfonamides (e.g.,
sulfanilamide, sulfadiazine, sulfamethaoxazole, sulfisoxazole,
sulfacetamide, and the like), aminoglycosides (e.g., streptomycin,
gentamicin, tobramycin, amikacin, netilmicin, kanamycin, and the
like), tetracyclines (such as chlortetracycline, oxytetracycline,
methacycline, doxycycline, minocycline and the like),
para-aminobenzoic acid, diaminopyrimidines (such as trimethoprim,
often used in conjunction with sulfamethoxazole, pyrazinamide, 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, cefinetazole, 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 E.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. Anti-infectives
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 and pharmaceutically acceptable salts thereof and
combinations thereof. Discussion and the examples are directed
primarily toward ciprofloxacin but the scope of the application is
not intended to be limited to this anti-infective. Combinations of
drugs can be used.
[0043] Bronchodilators covered by the invention include but are not
limited to .beta..sub.2-adrenergic receptor agonists (such as
albuterol, bambuterol, salbutamol, salmeterol, formoterol,
arformoterol, levosalbutamol, procaterol, indacaterol, carmoterol,
milveterol, procaterol, terbutaline, and the like), and
antimuscarinics (such as trospium, ipratropium, glycopyrronium,
aclidinium, and the like). Combinations of drugs may be used.
[0044] Anti-inflammatories covered by the invention include but are
not limited to inhaled corticosteroids (such as beclometasone,
budesonide, ciclesonide, fluticasone, etiprednol, mometasone, and
the like), leukotriene receptor antagonists and leukotriene
synthesis inhibitors (such as montelukast, zileuton, ibudilast,
zafirlukast, pranlukast, amelubant, tipelukast, and the like),
cyclooxygenase inhibitors (such as ibuprofen, ketoprofen,
ketorolac, indometacin, naproxen, zaltoprofen, lornoxicam,
meloxicam, celecoxib, lumiracoxib, etoricoxib, piroxicam,
ampiroxicam, cinnoxicam, diclofenac, felbinac, lornoxicam,
mesalazine, triflusal, tinoridine, iguratimod, pamicogrel, and the
like). Combinations of drugs may be used.
[0045] As used herein, "Formulation" refers to the
liposome-encapsulated anti-infective, with any excipients or
additional active ingredients, either as a dry powder or suspended
or dissolved in a liquid.
[0046] The terms "subject," "individual," "patient," and "host" are
used interchangeably herein and refer to any vertebrate,
particularly any mammal and most particularly including human
subjects, farm animals, and mammalian pets. The subject may be, but
is not necessarily under the care of a health care professional
such as a doctor.
[0047] A "stable" formulation is one in which the protein or enzyme
therein essentially retains its physical and chemical stability and
integrity upon storage and exposure to relatively high
temperatures. Various analytical techniques for measuring peptide
stability are available in the art and are reviewed in Peptide and
Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker,
Inc., New York, N.Y., Pubs. (1991), and Jones, A. (1993) Adv. Drug
Delivery Rev. 10:29-90. Stability can be measured at a selected
temperature for a selected time period.
[0048] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, horses,
cats, cows, etc. Preferably, the mammal is human.
[0049] A "disorder" is any condition that would benefit from
treatment with the claimed methods and compositions.
INVENTION IN GENERAL
[0050] Ciprofloxacin is a well-established and extensively utilized
broad-spectrum fluoroquinolone antibiotic that is indicated for the
treatment of lower respiratory tract infections due to P.
aeruginosa, which is common in patients with cystic fibrosis. The
primary advantage of inhaled antimicrobials is that they target
antibiotic delivery to the area of primary infection and bypass
GI-related side effects; however, the poor solubility and
bitterness of the drug have limited development of a formulation
suitable for inhalation. Furthermore, the rapid tissue distribution
of ciprofloxacin means a short drug residence time in the lung thus
limiting therapeutic benefit over oral or IV drug administration. A
liposome-encapsulated formulation of ciprofloxacin decreases the
limitations and improves management of pulmonary infections due to
P. aeruginosa pulmonary infections in patients with CF through
improved biopharmaceutical characteristics and mechanisms such as
altered drug PK and biodistribution, sustained drug release from
the carrier, enhanced delivery to disease sites, and protection of
the active drug species from degradation.
[0051] Preclinical studies demonstrate the efficacy of liposomal
ciprofloxacin in a biofilm inhibitory concentration assay using
clinical isolates of Pseudomonas aeruginosa, a CF mouse model of P.
aeruginosa lung infection and in a mouse model of Francisella
tularensis lung infection. Safety pharmacology studies of
respiration in rats and dogs of inhaled liposomal ciprofloxacin
found no marked effects upon respiratory parameters. These studies
showed that the inhalation of levels of "free" ciprofloxacin
together with the liposomal ciprofloxacin formulation allowed the
dosing of greater amounts of liposomal ciprofloxacin without
observation of focal macrophage accumulation or increases in the
proinflammatory cytokine IL-1.beta. in the lung fluid. This finding
is more generally applicable to other liposomal formulations
delivered to the lung. These results are also applicable to
pharmaceutical preparations or formulations which otherwise may
result in increases in macrophage accumulation in the lung or the
increase in proinflammatory cytokines in the lung in response to
the therapy.
[0052] The invention includes a formulation that combines
ciprofloxacin (or a different immune blunting agent; e.g.,
zithromax) with another drug; e.g., liposomal ciprofloxacin,
delivered via the inhalation route. The liposomal encapsulated
ciprofloxacin may be substituted with an antibiotic other than
ciprofloxacin and may be formulated without using liposomes. The
other drug does not have to be an antibiotic and may be any drug
that is believed to have some beneficial properties when delivered
to the lung.
[0053] The invention is not limited to the treatment of CF
patients. In fact, there are many patients and indications for
which this therapy may be beneficial, including bronchiectasis,
pneumonia, and other lung infections. This treatment paradigm would
also apply to other lung diseases including COPD, asthma, pulmonary
hypertension and others in which a formulation of free
ciprofloxacin is delivered in combination with another drug to
allow higher dosing of the other drug or safer administration of
the other drug.
[0054] The invention also relates to the use of inhaled free
ciprofloxacin (or a different immune blunting agent; e.g.,
zithromax) in combination with other drugs given via inhalation.
These other drugs may include a nucleotide sequence which may be
incorporated into a suitable delivery vector such as a plasmid or
viral vector. The other drug may be a therapeutic nucleotide
sequence (DNA, RNA, siRNA), enzymes to reduce the viscoelasticity
of the mucus such as DNase and other mucolytic agents, chemicals to
upregulate the chloride ion channel or increase flow of ions across
the cells, nicotine, P2Y2 agonists, elastase inhibitors including
Alpha-1 antitrypsin (AAT), N-acetylcysteine, antibiotics and
cationic peptides, such as lantibiotics, and specifically
duramycin, short-acting bronchodilators (e.g., .beta.2-adrenergic
receptor agonists like albuterol or indacaterol), M3 muscarinic
antagonists (e.g., ipatropium bromide), K+-channel openers,
long-acting bronchodilators (e.g., formoterol, salmeterol),
steroids (e.g., budesonide, fluticasone, triamcinolone,
beclomethasone, ciclesonide, etc.), xanthines, leukotriene
antagonists (e.g., montelukast sodium), phosphodiesterase 4
inhibitors, adenosine receptor antagonists, other miscellaneous
anti-inflammatories (e.g., Syk kinase inhibitors (AVE-0950),
tryptase inhibitors (AVE-8923 & AVE-5638), tachykinin
antagonists (AVE-5883), inducible nitric oxide synthase inhibitors
(GW-274150) and others), transcription factor decoys, TLR-9
agonists, antisense oligonucleotides, siRNA, DNA, CGRP, lidocaine,
inverse .beta.2-agonists, anti-infective oxidative therapies,
cytokine modulators (e.g., CCR3 receptor antagonists (GSK-766994,
DPC-168, AZD-3778), TNF-.alpha. production inhibitors (LMP-160
& YS-TH2), and IL-4 antagonists (AVE-0309)), small molecule
inhibitors of IgE, cell adhesion molecule (CAM) inhibitors, small
molecules targeting the VLA4 receptor or integrin .alpha.4.beta.1
(e.g., R-411, PS-460644, DW-908e, & CDP-323), immunomodulators
including those that block T-cell signaling by inhibition of
calcineurin (Tacrolimus), heparin neutralizers (Talactoferrin
alfa), cytosolic PLA2 inhibitors (Efipladib), or combinations
thereof. The delivery of the combination products may be achieved
by combining the drugs into one stable formulation, or providing
the drugs in separate containers to be combined at the time of
administration or alternatively by sequentially delivering the
products.
I. Generation of Liposomes Containing Ciprofloxacin
[0055] According to aspects of the instant invention, a method is
provided for formulating ciprofloxacin and other anti-infectives by
encapsulating these drugs in liposomes. Composed of
naturally-occurring materials which are biocompatible and
biodegradable, liposomes are used to encapsulate biologically
active materials for a variety of purposes. Having a variety of
layers, sizes, surface charges and compositions, numerous
procedures for liposomal preparation and for drug encapsulation
within them have been developed, some of which have been scaled up
to industrial levels. Liposomes can be designed to act as sustained
release drug depots and, in certain applications, aid drug access
across cell membranes.
[0056] The sustained release property of the liposomes may be
regulated by the nature of the lipid membrane and by the inclusion
of other excipients in the composition of the liposomes. Extensive
research in liposome technology over many years has yielded a
reasonable prediction of the rate of drug release based on the
composition of the liposome formulation. The rate of drug release
is primarily dependent on the nature of the phospholipids, e.g.
hydrogenated (--H) or unhydrogenated (--G), or the
phospholipid/cholesterol ratio (the higher this ratio, the faster
the rate of release), the hydrophilic/lipophilic properties of the
active ingredients and by the method of liposome manufacturing.
[0057] Methods for making bioadhesive liposomes can be found, for
example, in U.S. Pat. No. 5,401,511 which is incorporated herein by
reference in its entirety along with the patents and publications
cited therein which describe liposomes and methods of making
liposomes. In recent years, successful attempts have been made to
bind different substances to liposomes. For example, binding of
chymotrypsin to liposomes has been studied as a model for binding
substances to liposomal surfaces. Recognizing substances, including
antibodies, glycoproteins and lectins, have been bound to liposomal
surfaces in an attempt to confer target specificity to the
liposomes.
[0058] The number and surface density of the discrete sites on the
liposomal surfaces for covalent bonding are dictated by the
liposome formulation and the liposome type. The liposomal surfaces
also have sites for noncovalent association. Covalent binding is
preferred as noncovalent binding might result in dissociation of
the recognizing substances from the liposomes at the site of
administration since the liposomes and the bioadhesive counterparts
of the target site (that is, the bioadhesive matter) compete for
the recognizing substances. Such dissociation would reverse the
administered modified liposomes into regular, non-modified
liposomes, thereby defeating the purpose of administration of the
modified liposomes.
[0059] To form covalent conjugates of recognizing substances and
liposomes, crosslinking reagents have been studied for
effectiveness and biocompatibility. Once such reagent is
glutaraldehyde (GAD). Through the complex chemistry of crosslinking
by GAD, linkage of the amine residues of the recognizing substances
and liposomes is established.
[0060] The crosslinking reagents can include glutaraldehyde (GAD)
and a water soluble carbodiimide, preferably,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The
recognizing substances include gelatin, collagen, and hyaluronic
acid (HA). Following these methodologies, recognizing substances
may be utilized as an adhesive or glue to attach the liposomes onto
a target area, such as the lung.
[0061] Thus, while not essential to the instant invention, the use
of such bioadhesive liposomes, particularly those having hyaluronic
acid as the bioadhesive ligand, will potentially increase residence
time in pulmonary sites, and reduce mucociliary clearance and
macrophage uptake.
[0062] In general, ciprofloxacin is preferably used in the
formulations of the instant invention, although other antibiotics
or anti-infectives known to those skilled in the art may be
used.
[0063] Multilamellar vesicles (MLV) are prepared according to
techniques well known in the art. Briefly, in an embodiment, lipids
are weighed and dissolved in a suitable organic solvent (such as
chloroform or chloroform-methanol mixtures). The organic solvent is
evaporated to complete dryness in a rotary evaporator, under low
pressure, and at a temperature range of about 37-40.degree. C.
Following evaporation, the ciprofloxacin solution is added to the
dry lipid film. The system is vigorously mixed, then incubated for
about two hours in, for example, a shaker bath at a temperature
range appropriate for the lipid composition. The preparation is
then preferably buffered, for example, by adding about a one tenth
volume of ten-fold concentrated phosphate buffered saline (PBS), of
pH 7.4.
[0064] In an embodiment, MLV generated as described above serve as
the source material for acidic unilamellar vesicles (ULV). For
example, MLV are prepared as described above and subjected to
extrusion in a device such as, for example, that manufactured by
Lipex Biomembranes, Inc. (Vancouver, British Columbia). Extrusion
is performed through a series of membranes with
progressively-smaller pore sizes, such as, for example, starting
with pore sizes in the range of 0.8 to 1.0 .mu.m (one to two
extrusion cycles per pore size) and ending at the pore size range
selected according to the desired liposome size (e.g., about seven
cycles of extrusion at the final pore size).
[0065] Exemplary liposome compositions and methods of making them
are disclosed in U.S. Pat. Nos. 6,890,555; 6,855,296; 6,770,291;
6,759,057; 6,623,671; 6,534,018; 6,355,267; 6,316,024; 6,221,385
and 6,197,333 all of which are incorporated herein by reference.
The liposomes of the invention may be multilamellar, unilamellar,
or any configuration known such as described in the above patents.
The liposomes of the instant invention are preferably made from
biocompatible lipids. In general, the size of the liposomes
generated is over a wide range depending on mode of delivery, e.g.
1 nm to 10 .mu.m or 20 nm to 1 .mu.m or about 100 nm in diameter
.+-.20% for pulmonary delivery.
II. Pharmaceutical Formulation of Ciprofloxacin-containing
Liposomes
[0066] In a preferred embodiment, the liposome-encapsulated
ciprofloxacin is administered to a patient in an aerosol inhalation
device but could be administered by the IV route. In some
embodiments, ciprofloxacin is encapsulated in the liposomes in
combination with other pharmaceuticals that are also encapsulated.
In some embodiments, ciprofloxacin is encapsulated in the liposomes
in combination with other pharmaceuticals that are not
encapsulated. In some embodiments, the liposomes are administered
in combination with ciprofloxacin that is not encapsulated, with
pharmaceuticals that are not encapsulated, or various combinations
thereof.
[0067] Formulations of the invention can include liposomes
containing ciprofloxacin in combination with an amount of alveolar
surfactant protein effective to enhance the transport of the
liposomes across the pulmonary surface and into the circulatory
system of the patient. U.S. Pat. No. 5,006,343, issued Apr. 9,
1991, which is incorporated herein by reference, disclosed
liposomes and formulations of liposomes used in intrapulmonary
delivery. The formulations and methodology disclosed in U.S. Pat.
No. 5,006,343 can be adapted for the application of ciprofloxacin
and may be included within the delivery device of the present
invention in order to provide for effective treatments of cystic
fibrosis patients.
[0068] Regardless of the form of the drug formulation, it is
preferable to create droplets or particles for inhalation in the
range of about 0.5 .mu.m to 12 .mu.m, preferably 1 .mu.m to 6
.mu.m, and more preferably about 2-4 .mu.m. By creating inhaled
particles which have a relatively narrow range of size, it is
possible to further increase the efficiency of the drug delivery
system and improve the repeatability of the dosing. Thus, it is
preferable that the particles not only have a size in the range of
0.5 .mu.m to 12 .mu.m or 2 .mu.m to 6 .mu.m or about 3-4 .mu.m but
that the mean particle size be within a narrow range so that 80% or
more of the particles being delivered to a patient have a particle
diameter which is within .+-.20% of the average particle size,
preferably .+-.10% and more preferably .+-.5% of the average
particle size.
[0069] The formulations of the invention may be administered to a
patient using a disposable package and portable, hand-held,
battery-powered device, such as the AERx device (U.S. Pat. No.
5,823,178, Aradigm, Hayward, Calif.). Alternatively, the
formulations of the instant invention may be carried out using a
mechanical (non-electronic) device. Other inhalation devices may be
used to deliver the formulations including conventional jet
nebulizers, ultrasonic nebulizers, soft mist inhalers, dry powder
inhalers (DPIs), metered dose inhalers (MDIs), condensation aerosol
generators, and other systems. The proportion of free ciprofloxacin
to encapsulated ciprofloxacin was shown to remain constant after
nebulization; i.e., there was no damage to the liposomes during
nebulization that would result in premature release of a portion of
the encapsulated antibiotic. This finding is unexpected based upon
prior literature reports (Niven R W and Schreier H, 1990) but
ensures that the animal or human inhaling the aerosol will get a
reproducible proportion of free to encapsulated drug depositing
throughout the lung.
[0070] An aerosol may be created by forcing drug through pores of a
membrane which pores have a size in the range of about 0.25 to 6
microns (U.S. Pat. No. 5,823,178). When the pores have this size
the particles which escape through the pores to create the aerosol
will have a diameter in the range of 0.5 to 12 microns. Drug
particles may be released with an air flow intended to keep the
particles within this size range. The creation of small particles
may be facilitated by the use of the vibration device which
provides a vibration frequency in the range of about 800 to about
4000 kilohertz. Those skilled in the art will recognize that some
adjustments can be made in the parameters such as the size of the
pores from which drug is released, vibration frequency, pressure,
and other parameters based on the density and viscosity of the
formulation keeping in mind that an object of some embodiments is
to provide aerosolized particles having a diameter in the range of
about 0.5 to 12 microns.
[0071] The liposome formulation may be a low viscosity liquid
formulation. The viscosity of the drug by itself or in combination
with a carrier should be sufficiently low so that the formulation
can be forced out of openings to form an aerosol, e.g., using 20 to
200 psi to form an aerosol preferably having a particle size in the
range of about 0.5 to 12 microns.
[0072] In an embodiment, a low boiling point, highly volatile
propellant is combined with the liposomes of the invention and a
pharmaceutically acceptable excipient. The liposomes may be
provided as a suspension or dry powder in the propellant, or, in
another embodiment, the liposomes are dissolved in solution within
the propellant. Both of these formulations may be readily included
within a container which has a valve as its only opening. Since the
propellant is highly volatile, i.e. has a low boiling point, the
contents of the container will be under pressure.
[0073] In accordance with another formulation, the
ciprofloxacin-containing liposomes are provided as a dry powder by
itself, and in accordance with still another formulation, the
ciprofloxacin-containing liposomes are provided in a solution
formulation. The dry powder may be directly inhaled by allowing
inhalation only at the same measured inspiratory flow rate and
inspiratory volume for each delivery. The powder may be dissolved
in an aqueous solvent to create a solution which is moved through a
porous membrane to create an aerosol for inhalation. Any
formulation which makes it possible to produce aerosolized forms of
ciprofloxacin-containing liposomes which can be inhaled and
delivered to a patient via the intrapulmonary route may be used in
connection with the present invention. Specific information
regarding formulations which can be used in connection with
aerosolized delivery devices are described within Remington's
Pharmaceutical Sciences, A. R. Gennaro editor (latest edition) Mack
Publishing Company. Regarding insulin formulations, it is also
useful to note the findings of Sciarra et al., (1976). When low
boiling point propellants are used, the propellants are held within
a pressurized canister of the device and maintained in a liquid
state. When the valve is actuated, the propellant is released and
forces the active ingredient from the canister along with the
propellant. The propellant will "flash" upon exposure to the
surrounding atmosphere, i.e., the propellant immediately
evaporates. The flashing occurs so rapidly that it is essentially
pure active ingredient which is actually delivered to the lungs of
the patient.
III. Dosing Regimens
[0074] Based on the above, it will be understood by those skilled
in the art that a plurality of different treatments and means of
administration can be used to treat a single patient. Thus,
patients already receiving such medications, for example, as
intravenous ciprofloxacin or antibiotics, etc., may benefit from
inhalation of the formulations of the present invention. Some
patients may receive only ciprofloxacin-containing liposome
formulations by inhalation. Such patients may have symptoms of
cystic fibrosis, be diagnosed as having lung infections, or have
symptoms of a medical condition, which symptoms may benefit from
administration to the patient of an antibiotic such as
ciprofloxacin. The formulations of the invention may also be used
diagnostically. In an embodiment, for example, a patient may
receive a dose of a formulation of the invention as part of a
procedure to diagnose lung infections, wherein one of more of the
patient's symptoms improves in response to the formulation.
[0075] A patient will typically receive a dose of about 0.01 to 10
mg/kg/day of ciprofloxacin .+-.20% or .+-.10%. This dose will
typically be administered by at least one, preferably several
"puffs" from the aerosol device. The total dose per day is
preferably administered at least once per day, but may be divided
into two or more doses per day. Some patients may benefit from a
period of "loading" the patient with ciprofloxacin with a higher
dose or more frequent administration over a period of days or
weeks, followed by a reduced or maintenance dose. As cystic
fibrosis is typically a chronic condition, patients are expected to
receive such therapy over a prolonged period of time.
[0076] It has previously been shown that inhalation of
liposome-encapsulated fluoroquinolone antibiotics may be effective
in treatment of lung infections and were shown to be superior to
the free or unencapsulated fluoroquinolone in a mouse model of F.
tularensis (CA 2,215,716, CA 2,174,803 and CA 2,101,241). However,
they did not anticipate the potential benefit of combining the free
and encapsulated fluoroquinolone antibiotics to treat those lung
infections. According to one aspect of the present invention, high
concentrations of an antibiotic are delivered immediately while
also providing a sustained release of the therapeutic over hours or
a day.
[0077] Another application, EP1083881B1, describes liposomes
containing a drug-conjugate comprising a quinolone compound
covalently attached to an amino acid. That application does not
foresee the requirement to have both an immediate release and
sustained release component to treat those lung infections.
[0078] Another application, US 2000142026, also describes the use
of liposome-encapsulated antibiotics. That application discusses
the potential for administration of a lower dose of a
liposome-encapsulated antibiotic, by a factor of 10 or 100, than
for the free unencapsulated antibiotic. However, they did not
anticipate the benefit of combining both free and encapsulated
antibiotic to provide an initially high therapeutic level of the
antibiotic in the lungs to overcome the barrier to eradicating the
difficult to treat biofilm bacteria.
[0079] Other applications describing the use of liposomes
containing anti-infectives to treat cystic fibrosis, US 20060073198
and US 20040142026 show efficacy for a lower dose liposomal
formulation compared to the use of the free drug alone.
[0080] Thus, as discussed above, the formulations according to some
aspects of the invention include free or non-encapsulated
ciprofloxacin in combination with the liposome-encapsulated
ciprofloxacin. Such formulations may provide an immediate benefit
with the free ciprofloxacin resulting in a rapid increase in the
antibiotic concentration in the lung fluid surrounding the
bacterial colonies or biofilm and reducing their viability,
followed by a sustained benefit from the encapsulated ciprofloxacin
which continues to kill the bacteria or decrease its ability to
reproduce, or reducing the possibility of antibiotic resistant
colonies arising. The skilled practitioner will understand that the
relative advantages of the formulations of the invention in
treating medical conditions on a patient-by-patient basis.
[0081] It has long been known that inhalation of fine particulate
matter (PM) from environmental and occupational sources can be
associated with an increase in respiratory symptoms including
cough, wheeze, shortness of breath (Koren 1995), reduction in lung
function (Pope et al., 1995), exacerbation of asthma (Choudhury et
al., 1997) and COPD (Abbey et al, 1995), and even increased
premature mortality. Nurkiewicz et al (2004) have shown that
pulmonary exposure to fine PM impairs endothelium dependent
dilation in systemic arterioles and more recently that ultrafine PM
was more toxic by virtue of its increased surface area (2008). In
those studies in rats, the principal histopathologic alterations in
the lung consisted of particle accumulation within alveolar
macrophages, the presence of anuclear macrophages, and an intimate
association between particle-laden alveolar macrophages and the
alveolar wall. In addition to the particle accumulation,
macrophages containing particles were frequently intimately
associated with the alveolar wall a location, suggestive of, but
not diagnostic of, macrophage activation. These results are
consistent with long standing data that shows that particle
delivery to the lung results in an increase in macrophages in rats
(Yevich, 1965). The alveolar macrophages are phagocytic and provide
an important defense mechanism against inhaled pathogens and
clearing inhaled dust from the alveoli. However, if the level of
exposure is too high, the macrophages can become sufficiently
stimulated resulting in the secretion of mediators that cause
inflammatory changes in the bronchoalveolar region.
[0082] The implication of these studies is that any particulate
containing formulation, e.g., liposomes containing an
anti-infective, may result in an increased particle load in the
lung, stimulation of macrophages and the potential to cause
inflammation and lung damage. Thus, it is important that the
"particulate" formulation is safe and does not produce an excessive
inflammatory response in the lung. As we describe in some aspects
of the invention, the presence of free ciprofloxacin, or any free
antibiotic with similar anti-inflammatory properties, in the
formulation, in addition to providing an immediate high level of
antibiotic to treat the lung infection, may also provide a
mechanism to attenuate the lung's response to the inhaled liposome
or particulate containing formulation; i.e., reduce macrophage
influx and other cytokines.
IV. Other Formulations or Carriers
[0083] Although liposomes have been primarily described as the
vehicle to provide encapsulation of the therapeutic and thus the
sustained release effect, there is no intention to limit the
formulation to liposomal formulations. A combination of immediate
and sustained release formulations or carriers of an anti-infective
in the lung may be achieved via a multitude of ways including
microspheres, polymers, gels, emulsions, particulates or
suspensions, either singly or in combination. Some formulations or
carriers may have properties that result in closer association with
the biofilm matrix and these may prove more advantageous with
respect to increasing the therapeutic levels of the anti-infective
proximal to the biofilm bacteria.
Viscous Controlled Release Formulations
[0084] An example of a sustained release polymer formulation is the
use of poly(ortho esters) as the vehicle. For example, see U.S.
Pat. Nos. 4,304,767, 4,957,998, 5,968,543 and WO 02/092661 as well
as Adv. Polymer Sci., 107, 41-92 (1993) and references therein.
Viscosities of these controlled release polymers were reported to
be in the 1,500 cP range (see Biomaterials, 23, 2002, 4397-4404).
Considerably higher forces were required for higher molecular
weight polymers (see Adv. Drug Del Reviews, 53, 2001, 45-73).
Novel Liposomes
[0085] Various long-circulating liposomes have been prepared by
incorporating glycolipids or other amphiphilic molecules into the
lipid bilayer of conventional liposomes. Vasopressin entrapped in
PEGylated long-circulating liposomes even remained bioactive one
month after intravenous injection (Woodle et al., 1992).
[0086] A new approach, rather than using unilamellar or
multilamellar liposomes, is based on the DEPOFOAM system. These
multivesicular liposomes (1-100 .mu.m) contain multiple
non-concentric internal aqueous compartments and lead to an
increase in the encapsulation efficiency. After subcutaneous
injection, the release of encapsulated peptide and protein was
shown to be prolonged up to 7 days for Depolnsulin and up to 3
weeks for the DepoLeuprolide.RTM. formulation (Ye, Q et al.,
2000).
[0087] The company Novosom AG has patented a novel liposome-based
depot system for proteins and peptides. The Cagicles.RTM. depots
are produced by a two step method: first, proteins are dissolved in
an aqueous medium and then added to solutions of membrane-forming
substances, which are selected such that the resulting membrane
enters into a reversible mutual reaction with the protein. This
mild-condition process enables to increase the encapsulation rate
over 30% of incorporated protein. Furthermore, a one month
sustained protein release was feasible after subcutaneous or
intramuscular injection of the Cagicles.RTM. depots (Panzner, S.,
Novosom A G, Application No. 2000-EP11079, WO 2001034115 (2000)).
These studies have proven the basic applicability of liposomes. The
solubility benefits of liposomes are well known and reported.
Lipid Nanoparticles and Microspheres
[0088] Solid lipid nanoparticles (SLNs) represent a colloidal
carrier system mainly based on triglycerides. Due to their
hydrophobic nature and their small size, SLNs may be more
appropriate for incorporation of lipophilic drugs, which can be
easily dissolved in the melted mixture. For instance, only small
quantities of lysozyme can be incorporated into various lipids
(Almeida et al., 1997). Solid lipid nanoparticles own potential for
the encapsulation of drugs with a low solubility (e.g. paclitaxel),
for the application of surface-modified SLNs in drug targeting, or
maybe for the use as adjuvant for vaccines. Furthermore, it can be
hypothesised that SLNs can be applied for oral drug delivery in the
form of aqueous dispersions or that they can alternatively be used
as additives in traditional dosage forms such as tablets, capsules
or pellets.
[0089] U.S. Pat. No. 6,277,413 describes a biodegradable
microsphere having a matrix, the matrix comprising at least one
type of biodegradable polymer, and at least one type of lipid; and
a physiologically active substance which is releasable from the
biodegradable microsphere.
Lipid Crystals
[0090] EP 0767,656B 1 describes a pharmaceutical composition, which
is glycerol-ester based and contains diacyl glycerol as well as
phospholipid(s), or a polar group containing water, glycerol,
ethylene glycol or propylene glycol. The proportions between the
components are adjusted to form an L2 phase or a liquid crystalline
phase, with the biological material being dispersed or dissolved in
the L2 or liquid crystalline phase.
Oil Suspensions
[0091] Generally, the viscosity of oily media is considerably
higher than the viscosity of an aqueous phase such as buffer.
Therefore, drug release can be prolonged by implementing oil
suspensions. In addition, the viscosity of the oily carrier may be
further increased by the addition of gelling agents such as
aluminum monostearate--thus enabling the control of process
parameters like drug solubility and drug transfer rate. A further
important aspect using oils as drug carrier refers to the
distribution coefficient of compounds in the oily medium and the
surrounding tissue. A lipophilic drug with a high distribution
coefficient will primarily accumulate in the oily medium resulting
in further deceleration of effective drug actions.
[0092] For several years, various peptides and proteins have been
dispersed in oils to engineer sustained-release formulations.
Nestor et al. patented as early as 1979 the development of
long-acting injectable depot formulations for super-agonist
analogues of luteinizing hormone-releasing hormone (LH-RH),
applying oils such as peanut oil or sesame oil and a gelling agent
such as aluminum stearate (Nestor et al., Syntex Inc., U.S. Pat.
No. 4,256,737 (1979)).
Hydrogels
[0093] Thermoreversible hydrogels are of great interest in drug
delivery. These include thermosensitive gel materials including
poly(ethylene glycol)/poly(propylene glycol) block copolymers
(poloxamers), poly(ethylene glycol)/poly(butylenes glycol) block
copolymers, poloxamer-g-poly(acrylic acid) and copolymers of
Nisopropylacrylamide that exhibit a sol-to-gel transition in
aqueous solutions. Diblock copolymers of poly(ethylene oxide) (PEG)
and poly(lactic acid) (PLA), and triblock copolymers of
PEG-PLGA-PEG are also used as alternative hydrogels that would
provide biodegradable and injectable drug-delivery systems under
physiological conditions. Some natural polymers including gelatin,
agarose, amylase, amylopectin, cellulose derivatives, carrageenans,
and gellan, exhibit thermoreversible gelation behavior. Some
cellulose derivatives of natural polymers, such as methyl cellulose
and hydroxypropyl cellulose, exhibit reverse thermogelation
behavior (gelation at elevated temperatures). Viscosity of these
hydrogels is a concern for parenteral delivery. Viscosity of these
hydrogels can be extremely high at low shear rates (Thorgeirsdottir
T O et al., 2005). Poly hydroxyl methacralate is extensively used
in hydrogel formulations (Peppas et al., 2000). U.S. Pat. No.
6,602,952 describes a polymeric structure comprising a
multifunctional poly(alkylene oxide), such as a poly(ethylene
glycol) derivative, covalently cross-linked to a polymer selected
from the group consisting of chitosan and conjugates of chitosan
and a monofunctional poly(alkylene oxide), such as methoxy
poly(ethylene glycol). In aqueous media, the polymeric structure
forms a hydrogel.
Depot Formulations
[0094] Implantable drug delivery devices provide an attractive
therapeutic tool for treatment of a variety of diseases and
conditions, especially when a sustained release effect is also
added to the therapy. Various implantable drug delivery devices
have been developed, and are based upon different mechanisms to
accomplish movement of drug from a reservoir to the treatment site.
U.S. Pat. No. 4,938,763 discloses a method for forming an implant
in situ by dissolving a non-reactive, water insoluble thermoplastic
polymer in a biocompatible, water soluble solvent to form a liquid,
placing the liquid within the body, and allowing the solvent to
dissipate to produce a solid implant. U.S. Pat. No. 5,747,058
describes a composition for the controlled release of substances
that includes a non-polymeric non-water soluble high-viscosity
liquid carrier material of viscosity of at least 5,000 cP at body
temperature that does not crystallize neat under ambient or
physiological conditions.
Delivery of Macromolecules
[0095] The addition of a protein or peptide drug to the
anti-infective formulation may provide additional therapeutic
benefit. A previously discussed example includes Pulmozyme rhDNase
which is approved in the treatment of CF. While some macromolecules
may be delivered at a low dose or at relatively low concentrations,
for others it may be necessary to deliver at high concentrations.
Protein formulations at high concentrations may have physical
properties that impact the ability to easily deliver the protein
drug. U.S. Pat. No. 6,541,606 describes protein crystals or crystal
formulations that are encapsulated within a matrix comprising a
polymeric carrier to form a composition. The formulations and
compositions enhance preservation of the native biologically active
tertiary structure of the proteins and create a reservoir which can
slowly release active protein where and when it is needed.
Conjugated Systems
[0096] Polymer carrier systems may have certain advantages over
non-polymeric carriers in terms of avoiding uptake by macrophages.
Because liposomes are spherical vesicles made of phospholipids are
particles, they get taken up by macrophages. High levels can be
found in the liver and spleen, even when the liposomes are given
"stealth" characteristics by coating them with PEG. Antibodies,
meanwhile, have the disadvantage that most receptors on tumor cells
are also present on normal cells, making it hard to find ones that
are unique to cancer.
[0097] In contrast, water-soluble polymers allow working with a
single molecule rather than a large particle. To avoid the liver
and spleen, uncharged hydrophilic polymers, such as PEG and
N-(2-hydroxypropyl) methacrylamide can be used. When these polymers
are hydrated, they can circulate in the blood for periods of up to
about 24 hours (C&E News, Volume 80, Number 34, 39-47).
[0098] Examples of other conjugated systems include PEGylation.
PEGylation decreases the rate of clearance from the bloodstream by
increasing the apparent molecular weight of the molecule. Up to a
certain size, the rate of glomerular filtration of proteins is
inversely proportional to the size of the protein. Decreased
clearance can lead to increased efficiency over the non-PEGylated
material (Conforti et al. 1987 and Katre et al. 1987). The
conjugation could be either in-vitro or in-vivo.
[0099] WO2005034909A2 describes a hyperbranched polymer attached to
a core and a biologically active moiety. The biologically active
moiety is attached to the core by means of a substantially
non-enzymatically cleavable linker L. The composition can be used
to deliver the biologically active moiety to its target.
[0100] U.S. Pat. No. 6,946,134 describes therapeutic proteins fused
to albumin or fragments or variants of albumin, that exhibit
extended shelf-life and/or extended or therapeutic activity in
solution. The role of albumin as a carrier molecule and its inert
nature are desirable properties for use as a carrier and
transporter of polypeptides in vivo. The use of albumin as a
component of an albumin fusion protein as a carrier for various
proteins has been suggested in WO93/15199, WO93/15200, and
EP413622. The use of N-terminal fragments of HA for fusions to
polypeptides has also been proposed (EP399666).
[0101] U.S. Pat. No. 5,367,051 describes fullerene-functionalized
amine-containing polymers and polymerizable monomers characterized
by high temperature stability, i.e., capable of withstanding a
temperature of at least about 300.degree. C., when in polymerized
form. The fullerene groups are bonded to the polymers through the
amine groups on the polymer.
[0102] WO Patent No. 2005073383 describes novel heterodimeric
fusion proteins comprising a first polypeptide including an alpha
subunit of FSH (aFSH) linked directly or indirectly to a binding
partner of neonatal Fc receptor (FcRn) and a second polypeptide
including a beta subunit of FSH (BFSH) linked directly or
indirectly to an FcRn binding partner. The conjugated polypeptide
has increased half-life and bioavailability as compared to
traditional forms of FSH therapy.
Dendrimers
[0103] Dendrimers are well-defined polymeric structures. Dendrimers
are based on repeating hyperbranched structures emanating from a
central core (U.S. Pat. No. 4,507,466). Typical dendrimers are
based on polyamidoamine (PAMAM), polyethylene imine (PEI),
polypropylene imine or polylysine. These synthetic macromolecules
are assembled in a stepwise fashion, with each reaction cycle
adding another layer of branches (dubbed "generation"). Dendrimers
are synthetically accessed by stepwise, divergent "bottom-up" or
convergent "top-down" synthesis. Central structural component is
the core unit from which hyperbranched dendrimers extend in a
radially symmetric fashion. The core may provide at least two
reactive groups for dendrimer conjugation, it may also be of
heterofunctional nature and protecting groups may be used. In the
latter case, the dendrimer may be assembled, and a guest compound
may be subsequently conjugated to an anilin core by means of
orthogonal chemistries (WO88/01180). The core and dendrimers form
the interior or backbone of a dendrimer. As a consquence of the
spherical symmetry supported by sterical crowding, the terminal
groups of the hyperbranches are defining the exterior. In higher
generation dendrimers, the terminal branches form rather dense
shells and flexible internal voids have been discovered. It is
understood, that for a given dendrimer these cavities are filled up
by backfolded end groups and tightly coordinated solvent molecules.
Dendrimers are related to micelles, similarly well suited to
complex hydrophobic compounds. But in contrast they exhibit higher
structural order because of their monomolecular nature and the
absence of a dynamic equilibrium of various species. Synthetic
compounds can only diffuse into dendrimers if certain structural
requirement such as conformational rigidity and flatness as well as
charge distribution such as affinity to tertiary amines are met.
Various apolar compounds such as pyrene or naphthalene have been
encapsulated in dendrimers.
[0104] In U.S. Pat. No. 5,714,166 and WO95/24221, dendrimer-protein
conjugates are revealed. PAMAM dendrimers of G4 are covalently
coupled through their terminal functional groups to insulin,
fluorescently labeled insulin, avidin, monoclonal antibodies and
bradykinin. The reactive groups used for conjugation are only
present at the surface of the dendrimers, and therefore any
covalent adduct generated by the leached method will be associated
with the dendrimer exterior.
[0105] PAMAM dendrimers contain free amine groups on their surfaces
and readily associate with DNA through electrostatic
interactions.
[0106] WO01/07469 details water-soluble polypeptide dendrimers
constituted of ornithin and glycine amino acids. The patent
application also teaches the noncovalent encapsulation of an
oligosaccharide, heparin, by dendrimerization of the dendrimer core
in presence of heparin under mild conditions. The oligosaccharide
is released from the dendrimer by light-induced cleavage of
W-labile bonds within the dendritic backbone. The core structure
used here was tris(2-maleimidoethyl) amine. Other Polymeric
Systems
[0107] The use of heparin, dextran and methyl methacralate in a
biomimetric approach was evaluated in the development of drug
carriers escaping early capture by phagocytosis (Passirani et al.,
1998).
[0108] The synthesis of hybrid block and graft copolymers of
polyphosphazenes and polystyrene is a way to combine the attributes
of both polymers and generate new properties. Many of the valuable
properties of the respective phosphazene and styrene homopolymers
can be combined without sacrificing the overall solid state or
solution properties of both polystyrene and polyphosphazene
polymers. U.S. Pat. No. 6,392,008 describes such compositions of
polyphosphazene-containing polymers.
[0109] U.S. Pat. No. 5,176,907 describes biocompatible and
biodegradable poly(phosphoester-urethanes), compositions comprising
the poly(phosphoester-urethanes), and methods of use as a drug
delivery device and an implant.
V. Combination Therapies
[0110] Liposome formulations of the invention may be administered
concurrently with other drugs as described here. For example, the
liposomes of the invention may be used along with drugs such as
DNase, a mucolytic agent, chemicals that upregulate the chloride
ion channel or increase flow of ions across the epithelial surface
of cells, a bronchodilator, a steroid, a P2Y2 agonist, an elastase
inhibitor such as Alpha-1 antitrypsin (AAT), N-acetylcysteine,
agents that enhance the activity of the antibiotic against biofilm
bacteria such as sodium salicylate, interferon gamma, interferon
alpha, or a fluoroquinolone selected from the group consisting of
amifloxacin, cinoxacin, ciprofloxacin, danofloxacin, difloxacin,
enoxacin, enrofloxacin, fleroxacin, irloxacin, lomefloxacin,
miloxacin, norfloxacin, ofloxacin, pefloxacin, rosoxacin,
rufloxacin, sarafloxacin, sparfloxacin, temafloxacin and
tosufloxacin or an antibiotic selected from the group of
tobramycin, colistin, azithromycin, amikacin, cefaclor (Ceclor),
aztreonam, amoxicillin, ceftazidime, cephalexin (Keflex),
gentamicin, vancomycin, imipenem, doripenem, piperacillin,
minocycline, or erythromycin.
[0111] The preceding merely illustrates the principles of the
invention. It will be appreciated that those skilled in the art
will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
VI. Method of Treatment
[0112] Until now we have discussed primarily the application of
this invention to treat infections in cystic fibrosis patients.
However, it will be obvious to one skilled in the art that this
invention will have utility and advantages beyond CF. This method
of treatment applies to other disease states which involve
infections of the nasal passages, airways, inner ear, or lungs;
including but not limited to: bronchiectasis, tuberculosis,
pneumonia; including but not limited to ventilator associated
pneumonia, community acquired pneumonia, bronchial pneumonia, lobar
pneumonia; infections by Streptococcus pneumoniae, Chlamydia,
Mycoplasma pneumonia, staphylococci, prophylactive treatment or
prevention for conditions in which infection might arise, e.g.,
intubated or ventilated patients, infections in lung transplant
patient, bronchitis, pertussis (whooping cough), inner ear
infections, streptococal throat infections, inhalation anthrax,
tularemia, or sinusitis.
EXPERIMENTAL
[0113] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor is it intended to represent that the experiment
below is the only experiment performed. Efforts have been made to
ensure accuracy with respect to numbers used (e.g., amounts,
temperature, etc.) but some experimental errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by weight, molecular weight is weight average molecular
weight, temperature is in degrees Centigrade, and pressure is at or
near atmospheric.
Example 1
Manufacture of Encapsulated Ciprofloxacin
[0114] Ciprofloxacin (50 mg/mL) is encapsulated into liposomes
consisting of hydrogenated soy phosphatidyl-choline (HSPC) (70.6
mg/mL), a semi-synthetic fully hydrogenated derivative of natural
soy lecithin (SPC), and cholesterol (29.4 mg/mL). The lipid is
organized in a bilayer, with an average particle size of 75 to 120
nm. The sterile suspension is suspended in an isotonic buffer (25
mM histidine, 145 mM NaCl at pH 6.0, 300 mOsm/kg) and administered
by inhalation. These liposomal ciprofloxacin preparations contained
approximately 1% unencapsulated ciprofloxacin.
[0115] The manufacturing process includes the following steps.
[0116] 1. Preparation of buffers. [0117] 2. Weighing of lipid
components. [0118] 3. Dissolution of lipids in solvent
(tBuOH:EtOH:dH2O/49:49:2). [0119] 4. Mixing of the solvent solution
of lipids with methylamine sulphate buffer (10% v/v solvent) to
form multilamellar vesicles (MLVs) with encapsulated methylamine
sulphate buffer at 30 mg/mL lipid. [0120] 5. Extrusion through four
stacked 80 nm pore size polycarbonate filters to generate large
unilamellar vesicles (LUVs). A second extrusion pass was performed
to generate liposomes with a mean diameter of .about.100 nm [0121]
6. Ultrafiltration to concentrate the liposomes to .about.55 mg/mL
total lipid. [0122] 7. Diafiltration against 10 volumes of buffer
(145 mM NaCl, 5 mM histidine, pH 6.0) to remove ethanol and
generate a transmembrane pH gradient.
[0123] 8. Determination of the lipid concentration by HPLC.
[0124] 9. Heating of the liposome suspension to 50.degree. C. and
slow addition of powdered ciprofloxacin (60% of the total lipid
mass) with stirring. Ciprofloxacin is added incrementally (10% of
mass every 4 minutes over a 40-minute period) and the product
incubated at 50.degree. C. for 20 minutes following addition of the
last aliquot to allow completion of the drug loading process.
[0125] 10. Diafiltration of the ciprofloxacin loaded liposomes
against 3-volumes of 145 mM NaCl, 5 mM acetate, pH 4.0 to remove
unencapsulated ciprofloxacin under conditions in which the free
ciprofloxacin is soluble. [0126] 11. Diafiltration of the
ciprofloxacin loaded liposomes against 5-volumes of 145 mM NaCl, 25
mM histidine, pH 6.0 to remove any remaining unencapsulated
ciprofloxacin, further reducing the residual solvent levels and
exchanging the external buffer for the desired final product
buffer. [0127] 12. Ultrafiltration of the formulation to a
ciprofloxacin concentration of 50 mg/mL (in-process testing
required). [0128] 13. Pre-filtration of the liposomes through
0.45/0.2 .mu.m filter sheets to remove particulates which can clog
sterilizing grade filters. The filters employed are in fact
sterilizing grade filters; however they are employed at elevated
pressures not compatible with their use for sterile filtration.
[0129] 14. Redundant filtration through 0.2 .mu.m sterilizing grade
filters. [0130] 15. Sample vialing and packaging.
[0131] The overall manufacturing scheme is shown in FIG. 1.
Description of Infection Model:
[0132] The ciprofloxacin encapsulated liposomes were evaluated in a
mouse model of P. aeruginosa lung infection. The gut-corrected,
Cftr knockout mice have been shown to have a cystic fibrosis lung
phenotype following infection with P. aeruginosa-laden agarose
beads (van Heeckeren et al., 2004), and have a similar inflammatory
response as the UNC Cftr knockout mice (van Heeckeren et al.,
2004). All of these features make this the strain of choice to
investigate whether the drug has efficacy in a mouse model of
cystic fibrosis lung infection, and not if there is a differential
response between wild type and cystic fibrosis mice. Mice of one
sex, male, were used to eliminate sex as a potential confounder.
All mice were between 6-8 weeks of age and weighed >16 g.
[0133] P. aeruginosa-laden agarose (PA) beads were made and used,
as described previously (van Heeckeren, et al., 1997, van Heeckeren
et al., 2000, van Heeckeren and Schluchter, 2002), with minor
differences. Mice were inoculated with a 1:35 dilution of the
beads, and beads were delivered in mice anesthetized with
isoflurane. This was established to be an LD50 dose, though subtle
differences from experiment to experiment may lead to differential
responses, which is not predictable. That is, in one experiment the
dose is an LD50, but it may be an LD90 in another. Since we are
interested in investigating whether these drugs have clinical
efficacy, we attempted to dose between the LD50 and the LD90 range
in infected CF control mice. Interventional euthanasia was
performed if the mice were moribund (severe delay in the righting
reflex and palpably cold), and a necropsy performed to determine
whether there was overt lung infection. Mice that were sacrificed
were included as if spontaneous death had occurred.
Liposomal Ciprofloxacin Treatments:
[0134] Formulations of liposomal ciprofloxacin or sham (diluent)
(.ltoreq.0.05 ml) were delivered intranasally.
Design of Dose-Ranging Study:
[0135] Three doses were tested: 10%, 33%, and 100% of full strength
(50 mg/ml) ciprofloxacin composed of 99% encapsulated and 1% free
ciprofloxacin, plus the liposomal diluent as a negative control.
The low- and mid-dose were prepared by dilution. On Day 0, mice
were inoculated transtracheally with P. aeruginosa-laden agarose
beads diluted 1:35 in sterile PBS, pH 7.4. On Days 2 through 9,
mice were treated with the drug or diluent sham once daily. On Day
10, mice were sacrificed. The outcome measures included clinical
signs (including coat quality, posture, ability to right themselves
after being placed in lateral recumbency, ambulation), changes from
initial body weight, and survival. At the time of sacrifice, gross
lung pathology was noted, bronchoalveolar lavage (BAL) was
performed using 1 ml sterile PBS, pH 7.4, whole blood, unprocessed
BAL fluid and spleen homogenates were tested for presence or
absence of P. aeruginosa, and BAL cells were enumerated using a
hemacytometer.
Survival Results:
[0136] FIG. 2 shows the cumulative survival rate for each group out
to 10 days reported as a percentage of the number of mice that
survived. At Day 10, the three groups treated with liposomal
ciprofloxacin had greater survival rates than the diluent control
group. There were only 2 deaths in each of the liposomal treatment
groups, whereas there were 6 deaths in the diluent group. The
100%-dose group had the longest survival of all the groups, with
all mice surviving out to Day 5, whereas the other groups all had 2
deaths by this time.
[0137] Intranasal administration (to target the lung) of
liposome-encapsulated ciprofloxacin containing approximately 1%
free ciprofloxacin increased the survival rate of mice with P.
aeruginosa lung infections. Accordingly, inhaled liposomal
ciprofloxacin is efficacious in patients with cystic fibrosis, or
other diseases with lung infections.
[0138] FIG. 2 shows the cumulative survival rate following
infection. Mice were infected with P. aeruginosa-laden agarose
beads on Day 0. Mice were treated intranasally once daily starting
on Day 2 and ending on Day 9 with the liposomal formulation of
ciprofloxacin (drug) at one of three different concentrations
(100%, open diamond; 33%, closed square; or 10%, open triangle).
Diluent was used as a control (closed circle). Surviving mice were
sacrificed on Day 10.
Example 2
Preparation of Unencapsulated Ciprofloxacin
[0139] A solution of unencapsulated, or "free" ciprofloxacin at a
concentration of 30 mg/mL in 10 mM sodium acetate, pH 3.2, was
prepared.
[0140] Manufacture of Encapsulated Ciprofloxacin:
[0141] Ciprofloxacin (50 mg/mL) was encapsulated into liposomes
consisting of hydrogenated soy phosphatidyl-choline (HSPC) (70.6
mg/mL), a semi-synthetic fully hydrogenated derivative of natural
soy lecithin (SPC), and cholesterol (29.4 mg/mL), as described in
Example 1. Characterization of this liposomal formulation indicated
that approximately 1% of the ciprofloxacin was free; that is, it
was not encapsulated within the liposome.
[0142] Description of Infection Model:
[0143] Formulations containing free ciprofloxacin and liposome
encapsulated ciprofloxacin were evaluated in two additional
experiments in a mouse model of P. aeruginosa lung infection as
described in Example 1.
[0144] Design of Dose-Ranging Study:
[0145] One dose of the combination of free and liposomal
ciprofloxacin (0.36 mg/kg free and 0.6 mg/kg liposomal
ciprofloxacin), two doses of liposomal ciprofloxacin (0.6 mg/kg and
1.2 mg/kg) and the liposomal diluent as a negative control were
evaluated in two separate experiments. On Day 0, mice were
inoculated transtracheally with P. aeruginosa-laden agarose beads
diluted 1:35 in sterile PBS, pH 7.4. On Days 2 through 9, mice were
treated with the drug or diluent sham once daily. On Day 10, mice
were sacrificed. The outcome measures included clinical signs
(including coat quality, posture, ability to right themselves after
being placed in lateral recumbency, ambulation), changes from
initial body weight, and survival. At the time of sacrifice, gross
lung pathology was noted, bronchoalveolar lavage (BAL) was
performed using 1 ml sterile PBS, pH 7.4, whole blood, unprocessed
BAL fluid and spleen homogenates were tested for presence or
absence of P. aeruginosa, and BAL cells were enumerated using a
hemacytometer.
[0146] Survival Results:
[0147] Table 1 shows the cumulative survival rate for each group
out to 10 days reported as a percentage of the number of mice that
survived from both studies. At Day 10, all groups treated with a
combination of free and liposomal ciprofloxacin had greater
survival rates than the diluent control group.
TABLE-US-00001 TABLE 1 Mean survival per group from two studies in
CF mice with P. aeruginosa lung infection treated with intranasally
instilled ARD-3100, or control Dose % Free Mean Starting Mean Mean
(mg/kg) Ciprofloxacin Number Mortality Survival (%) 0 (Control) N/A
9 6/9 34% 0.6 1 8.5 2.5/8.5 66% 1.2 1 8.5 .sup. 3/8.5 65% 0.96 38
10.5 2.5/10.5 76%
[0148] Conclusion:
[0149] Intranasal administration (to target the lung) of
liposome-encapsulated ciprofloxacin increased the survival rate of
mice with P. aeruginosa lung infections. Inhaled liposomal
ciprofloxacin or combinations of free and liposomal ciprofloxacin
are efficacious in patients with cystic fibrosis, or other diseases
with lung infections.
Example 3
Manufacture of Encapsulated Ciprofloxacin
[0150] Ciprofloxacin (50 mg/mL) was encapsulated into liposomes
consisting of hydrogenated soy phosphatidyl-choline (HSPC) (70.6
mg/mL), a semi-synthetic fully hydrogenated derivative of natural
soy lecithin (SPC), and cholesterol (29.4 mg/mL), as described in
Example 1. Characterization of this liposomal formulation indicated
that approximately 1% of the ciprofloxacin was free; that is, it
was not encapsulated within the liposome.
[0151] Delivery of Combination of Free and Encapsulated
Ciprofloxacin:
[0152] Rather than using a formulation which contains both free and
encapsulated ciprofloxacin, an alternative method is to create the
mixture during the delivery event. For example, the addition of
shear or heat in a controlled fashion may reproducibly result in
some of the liposomes losing their integrity and releasing the
contents of drug that were previously encapsulated within the
liposomes. Studies using the electromechanical AERx system
confirmed the possibility of using this approach. Formulations
containing approximately 99% encapsulated ciprofloxacin were
delivered using the AERx system and the aerosol droplets were
collected. With the temperature controller set at temperatures of
13, 45, 77, 108 and 140.degree. C. the collected aerosol contained
89, 84, 82, 77 and 41 percent encapsulated, respectively.
[0153] The instant invention is shown and described herein in a
manner which is considered to be the most practical and preferred
embodiments. It is recognized, however, that departures may be made
therefrom which are within the scope of the invention and that
obvious modifications will occur to one skilled in the art upon
reading this disclosure.
[0154] While the instant invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
Example 4
Manufacture of Encapsulated Ciprofloxacin
[0155] Ciprofloxacin (50 mg/mL) was encapsulated into liposomes
consisting of hydrogenated soy phosphatidyl-choline (HSPC) (70.6
mg/mL), a semi-synthetic fully hydrogenated derivative of natural
soy lecithin (SPC), and cholesterol (29.4 mg/mL), as described in
Example 1. Characterization of this liposomal formulation indicated
that approximately 1% of the ciprofloxacin was free; that is, it
was not encapsulated within the liposome.
[0156] Manufacture of Unencapsulated (or Free) Ciprofloxacin:
[0157] Under aseptic manufacturing conditions, ciprofloxacin-HCl as
powder was dissolved in 10 mM sodium acetate solution, pH 3.3 and
sterile filtered resulting in a 24 mg/mL solution.
[0158] Delivery of Combination of Free and Encapsulated
Ciprofloxacin:
[0159] In this example the free and encapsulated formulations are
mixed in the nebulizer and delivered as aerosol at the time of
administration. The pH of the resulting mixture is between 4.0 and
5.0. Samples of the delivered aerosol and the solution remaining in
the nebulizer following aerosolization (i.e., the nebulizer
residual) were analyzed to ensure that the mixing and nebulization
process did not degrade the integrity of the liposomes. No loss in
encapsulated ciprofloxacin was observed. This result ensures that a
precise and reproducible amount of free and encapsulated
ciprofloxacin is delivered to the human subject or patient, or
animals during preclinical safety or preclinical efficacy
studies.
[0160] Evaluation of the Free and Encapsulated Ciprofloxcain in a
14-Day Nose-Only Inhalation Toxicology Study in Sprague Dawley
Rats:
[0161] Various ciprofloxacin formulations were evaluated in this 28
day inhalation toxicology study with 28 and 56 day recovery arms.
Exposure to nebulized liposomal ciprofloxacin by nose-only
inhalation for 6 hours per day, 7 days per week for 14 days at a
target pulmonary dose of 8.2 (Low), or 16.5 (High) mg/kg/day
resulted in histopathology findings in the lung (focal macrophage
accumulation, and alveolar wall monocyte infiltration) at the end
of the 14 day exposure period. After a 28-day recovery period,
histopathology findings were still present in the lung (focal
macrophage accumulation, and alveolar wall monocyte infiltration,
Table II).
[0162] Evaluation of the Free and Encapsulated Ciprofloxcain in a
28-Day Nose-Only Inhalation Toxicology Study in Sprague Dawley
Rats:
[0163] Various ciprofloxacin formulations were evaluated in this 28
day inhalation toxicology study with 28 and 56 day recovery arms.
Exposure to nebulized liposomal ciprofloxacin by nose-only
inhalation for 6 hours per day, 7 days per week for 28 days at a
target pulmonary dose of 11 (Low), 21 (Mid), or 31 (High) mg/kg/day
resulted in histopathology findings in the lung (diffuse macrophage
accumulation, focal macrophage accumulation, and alveolar wall
monocyte infiltration) at the end of the 28 day exposure period.
After a 28-day recovery period, histopathology findings were still
present in the lung (focal macrophage accumulation, and alveolar
wall monocyte infiltration, Table II). After a 56 day recovery
period, histopathology findings were still present in the lung for
all three groups exposed to liposomal ciprofloxacin (focal
macrophage accumulation, and alveolar wall monocyte infiltration,
alveolar wall fibrosis and crystal formation in alveolar lumens,
Table III).
TABLE-US-00002 TABLE II Recovery Necropsy (after 28-day recovery)
Group/Mean Lung Dose Low Low High Mid High Mixture Mixture Saline
14-day 28-day 14-day 28-day 28-day 14-day 28 day 0 mg/kg 8.2 mg/kg
11 mg/kg 16.5 mg/kg 21 mg/kg 31 mg/kg 11.5 mg/kg 34 mg/kg Organ
Lesion Sex Incidence (group mean severity score) Lung Focal M 0/5
(0.00)* 2/5 (0.40) 3/5 (0.60) 5/5 (1.40) 5/5 (2.60) 5/5 (2.80) 1/5
(0.20) 3/5 (0.60) macrophage accumulation Focal F 2/5 (0.40) 4/5
(0.80) 5/5 (2.40) 5/5 (1.20) 5/5 (2.60) 5/5 (3.00) 0/5 5/5 (1.60)
macrophage accumulation Diffuse M 0/5 (0.00) 0/5 0/5 (0.00) 0/5 0/5
(0.00) 0/5 (0.00) 0/5 0/5 (0.00) macrophage accumulation Diffuse F
0/5 (0.00) 0/5 0/5 (0.00) 0/5 0/5 (0.00) 0/5 (0.00) 0/5 0/5 (0.00)
macrophage accumulation Monocyte M 0/5 (0.00) 1/5 (0.20) 0/5 (0.00)
0/5 4/5 (1.00) 5/5 (1.20) 0/5 1/5 (0.20) interstitial infiltration,
alveolar wall Monocyte F 0/5 (0.00) 2/5 (0.40) 4/5 (1.00) 3/5
(0.60) 5/5 (1.20) 5/5 (1.40) 0/5 4/5 (0.80) interstitial
infiltration, alveolar wall Larynx Squamous M 0/5 (0.00) 0/5 (0.00)
0/5 (0.00) 0/5 (0.00) 0/5 (0.00) cell hyperplasia- metaplasia
Squamous F 0/4 (0.00) 0/5 (0.00) 0/5 (0.00) 0/5 (0.00) 0/5 (0.00)
cell hyperplasia- metaplasia
TABLE-US-00003 TABLE III Recovery Necropsy (after 56-day recovery)
Group/Mean Lung Dose (Human Dose) Saline Low Mid High Mixture 0
mg/kg 11 mg/kg 21 mg/kg 31 mg/kg 34 mg/kg Organ Lesion Sex
Incidence (group mean severity score) Lung Focal macrophage M 0/6
(0.00)* 1/5 (0.20) 6/6 (2.33) 6/6 (2.00) 0/6 (0.00) accumulation
Focal macrophage F 1/6 (0.17) 4/5 (1.40) 6/6 (2.33) 6/6 (2.83) 1/6
(0.17) accumulation Diffuse macrophage M 0/6 (0.00) 0/5 (0.00) 0/6
(0.00) 0/6 (0.00) 0/6 (0.00) accumulation Diffuse macrophage F 0/6
(0.00) 0/5 (0.00) 0/6 (0.00) 0/6 (0.00) 0/6 (0.00) accumulation
Monocyte interstitial M 0/6 (0.00) 0/5 (0.00) 6/6 (1.50) 5/6 (1.17)
0/6 (0.00) infiltration, alveolar wall Monocyte interstitial F 0/6
(0.00) 2/5 (0.60) 6/6 (1.50) 6/6 (1.67) 0/6 (0.00) infiltration,
alveolar wall Fibrosis, alveolar wall M 0/6 (0.00) 0/5 (0.00) 5/6
(0.83) 4/6 (0.67) 0/6 (0.00) '' F 0/6 (0.00) 2/5 (0.40) 5/6 (0.83)
5/6 (0.83) 0/6 (0.00) Crystals, alveolar M 0/6 (0.00) 0/5 (0.00)
4/6 (1.17) 5/6 (1.17) 0/6 (0.00) lumen Crystals, alveolar F 0/6
(0.00) 2/5 (0.40) 5/6 (1.33) 5/6 (1.33) 0/6 (0.00) lumen Larynx
Squamous cell M 0/6 (0.00) 0/5 (0.00) 0/6 (0.00) 0/6 (0.00) 0/6
(0.00) hyperplasia- metaplasia Squamous cell F 0/6 (0.00) 0/5
(0.00) 0/6 (0.00) 0/6 (0.00) 0/6 (0.00) hyperplasia- metaplasia
[0164] In contrast, a fourth group of rats exposed to a combination
of encapsulated and free ciprofloxacin for a similar treatment
duration experienced a reduction in histopathology findings (Table
II). After the 56 day recovery period, the histopathology findings
for this group of rats was not significantly different from
controls (Table III).
[0165] The explanation for this surprising effect may be that the
presence of free ciprofloxacin in the lung attenuates the release
of cytokines which attract macrophages into the lung. The
concentration of one cytokine marker, IL-1, in the lungs of rats
after 14 days of inhalation treatment increases with increasing
doses of liposomal ciprofloxacin (FIG. 3). The addition of free
ciprofloxacin to the liposomal ciprofloxacin formulation reduces
the levels of IL-1 to values similar to those for the control
animals. These findings suggest that the presence of free
ciprofloxacin down regulates cytokine markers and reduces the
influx of macrophages into the lungs.
[0166] The instant invention is shown and described herein in a
manner which is considered to be the most practical and preferred
embodiments. It is recognized, however, that departures may be made
therefrom which are within the scope of the invention and that
obvious modifications will occur to one skilled in the art upon
reading this disclosure.
[0167] While the instant invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
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