U.S. patent application number 15/792166 was filed with the patent office on 2018-04-19 for sustained release of antiinfectives.
The applicant listed for this patent is INSMED INCORPORATED. Invention is credited to Lawrence T. BONI, Xingong LI, Vladimir MALININ, Brian S. MILLER.
Application Number | 20180104345 15/792166 |
Document ID | / |
Family ID | 37669512 |
Filed Date | 2018-04-19 |
United States Patent
Application |
20180104345 |
Kind Code |
A1 |
BONI; Lawrence T. ; et
al. |
April 19, 2018 |
SUSTAINED RELEASE OF ANTIINFECTIVES
Abstract
Provided are lipid antiinfective formulations substantially free
of anionic lipids with a lipid to antiinfective ratio is about 1:1
to about 4:1, and a mean average diameter of less than about 1
.mu.m. Also provided is a method of preparing a lipid antiinfective
formulation comprising an infusion process. Also provided are lipid
antiinfective formulations wherein the lipid to drug ratio is about
1:1 or less, about 0.75:1 or less, or about 0.50:1 or less prepared
by an in line fusion process. The present invention also relates to
a method of treating a patient with a pulmonary infection
comprising administering to the patient a therapeutically effective
amount of a lipid antiinfective formulation of the present
invention. The present invention also relates to a method of
treating a patient for cystic fibrosis comprising administering to
the patient a therapeutically effective amount of a lipid
antiinfective formulation of the present invention.
Inventors: |
BONI; Lawrence T.; (Monmouth
Junction, NJ) ; MILLER; Brian S.; (Mercerville,
NJ) ; MALININ; Vladimir; (Plainsboro, NJ) ;
LI; Xingong; (Robbinsville, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSMED INCORPORATED |
BRIDGEWATER |
NJ |
US |
|
|
Family ID: |
37669512 |
Appl. No.: |
15/792166 |
Filed: |
October 24, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14319018 |
Jun 30, 2014 |
9827317 |
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15792166 |
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12748756 |
Mar 29, 2010 |
8802137 |
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14319018 |
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11185448 |
Jul 19, 2005 |
7718189 |
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12748756 |
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11023971 |
Dec 28, 2004 |
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11185448 |
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10696389 |
Oct 29, 2003 |
7544369 |
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11023971 |
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60421923 |
Oct 29, 2002 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 31/00 20180101; A61K 31/545 20130101; A61P 31/04 20180101;
A61K 31/407 20130101; A61K 31/704 20130101; A61P 31/12 20180101;
Y02A 50/473 20180101; A61K 31/7036 20130101; A61P 31/10 20180101;
A61K 9/0078 20130101; A61K 9/1277 20130101; A61K 31/4709 20130101;
Y02A 50/30 20180101; A61K 31/496 20130101; A61K 47/28 20130101;
A61K 9/127 20130101; A61P 11/00 20180101 |
International
Class: |
A61K 47/28 20060101
A61K047/28; A61K 9/127 20060101 A61K009/127; A61K 31/407 20060101
A61K031/407; A61K 31/4709 20060101 A61K031/4709; A61K 9/00 20060101
A61K009/00; A61K 31/545 20060101 A61K031/545; A61K 31/704 20060101
A61K031/704; A61K 31/7036 20060101 A61K031/7036; A61K 45/06
20060101 A61K045/06; A61K 31/496 20060101 A61K031/496 |
Claims
1-92. (canceled)
93. A method of treating a mycobacterial pulmonary infection in a
patient in need thereof, comprising, nebulizing a therapeutically
effective amount of a liposomal aminoglycoside composition
comprising amikacin, or a pharmaceutically acceptable salt thereof,
encapsulated in a liposome having a lipid bilayer comprising a
neutral phospholipid and cholesterol, wherein the weight ratio of
lipid to the aminoglycoside, or the pharmaceutically acceptable
salt thereof, in the composition is 0.75:1 or less, to form a
nebulized spray, and administering the nebulized spray to the
patient.
94. The method of claim 93, wherein the amikacin, or
pharmaceutically acceptable salt thereof is amikacin sulfate.
95. The method of claim 93, wherein the neutral phospholipid is a
phosphatidylcholine.
96. The method of claim 94, wherein the neutral phospholipid is a
phosphatidylcholine.
97. The method of claim 95, wherein the phosphatidylcholine is
dipalmitoylphosphatidylcholine (DPPC).
98. The method of claim 96, wherein the phosphatidylcholine is
dipalmitoylphosphatidylcholine (DPPC).
99. The method of claim 93, wherein the mycobacterial infection is
Mycobacterium tuberculosis, Mycobacterium avium complex (MAC)
(Mycobacterium avium and Mycobacterium intracellulare),
Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium
marinum, Mycobacterium ulcerans, or Mycobacterium fortuitum complex
(M. fortuitum and M. chelonae).
100. The method of claim 94, wherein the mycobacterial infection is
Mycobacterium tuberculosis, Mycobacterium avium complex (MAC)
(Mycobacterium avium and Mycobacterium intracellulare),
Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium
marinum, Mycobacterium ulcerans, or Mycobacterium fortuitum complex
(M. fortuitum and M. chelonae).
101. The method of claim 95, wherein the mycobacterial infection is
Mycobacterium tuberculosis, Mycobacterium avium complex (MAC)
(Mycobacterium avium and Mycobacterium intracellulare),
Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium
marinum, Mycobacterium ulcerans, or Mycobacterium fortuitum complex
(M. fortuitum and M. chelonae).
102. The method of claim 96, wherein the mycobacterial infection is
Mycobacterium tuberculosis, Mycobacterium avium complex (MAC)
(Mycobacterium avium and Mycobacterium intracellulare),
Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium
marinum, Mycobacterium ulcerans, or Mycobacterium fortuitum complex
(M. fortuitum and M. chelonae).
103. The method of claim 97, wherein the mycobacterial infection is
Mycobacterium tuberculosis, Mycobacterium avium complex (MAC)
(Mycobacterium avium and Mycobacterium intracellulare),
Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium
marinum, Mycobacterium ulcerans, or Mycobacterium fortuitum complex
(M. fortuitum and M. chelonae).
104. The method of claim 98, wherein the mycobacterial infection is
Mycobacterium tuberculosis, Mycobacterium avium complex (MAC)
(Mycobacterium avium and Mycobacterium intracellulare),
Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium
marinum, Mycobacterium ulcerans, or Mycobacterium fortuitum complex
(M. fortuitum and M. chelonae).
105. The method of claim 99, wherein the mycobacterial infection is
Mycobacterium avium complex (MAC) (Mycobacterium avium and
Mycobacterium intracellulare).
106. The method of claim 100, wherein the mycobacterial infection
is Mycobacterium avium complex (MAC) (Mycobacterium avium and
Mycobacterium intracellulare).
107. The method of claim 101, wherein the mycobacterial infection
is Mycobacterium avium complex (MAC) (Mycobacterium avium and
Mycobacterium intracellulare).
108. The method of claim 102, wherein the mycobacterial infection
is Mycobacterium avium complex (MAC) (Mycobacterium avium and
Mycobacterium intracellulare).
109. The method of claim 103, wherein the mycobacterial infection
is Mycobacterium avium complex (MAC) (Mycobacterium avium and
Mycobacterium intracellulare).
110. The method of claim 104, wherein the mycobacterial infection
is Mycobacterium avium complex (MAC) (Mycobacterium avium and
Mycobacterium intracellulare).
111. The method of claim 93, wherein the liposome has a mean
diameter of about 1 um to about 1.0 um.
112. The method of claim 111, wherein the liposome has a mean
diameter of about 0.2 .mu.m to about 0.5 .mu.m.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/319,018, filed Jun. 30, 2014, which is a
continuation of U.S. patent application Ser. No. 12/748,756, filed
Mar. 29, 2010, now U.S. Pat. No. 8,802,137, which is a continuation
of U.S. patent application Ser. No. 11/185,448, filed Jul. 19,
2005, now U.S. Pat. No. 7,718,189, which is a continuation-in-part
of U.S. patent application Ser. No. 11/023,971, filed Dec. 28,
2004, now abandoned, which is a continuation-in-part of U.S. patent
application Ser. No. 10/696,389, filed Oct. 29, 2003, now U.S. Pat.
No. 7,544,369, which claims the benefit of priority from U.S.
Provisional Patent Application Ser. No. 60/421,923, filed Oct. 29,
2002, each of which is hereby incorporated by reference in its
entirety.
INTRODUCTION
[0002] Certain sustained release technology suitable for
administration by inhalation employs liposomes and lipid complexes
to provide prolonged therapeutic effect of drug in the lung and
systemically by sustained release and the ability to target and
enhance the uptake of drug into sites of disease. The present
invention comprises a liposomal antiinfective, and methods for
treatment of pulmonary infections using liposomal or
lipid-complexed antiinfective.
[0003] As reported in Goodman and Gilman's The Pharmaceutical Basis
of Therapeutics, Eighth Edition, "Since the incidence of
nephrotoxicity and ototoxicity is related to the concentration to
which an aminoglycoside accumulates, it is critical to reduce the
maintenance dosage of these drugs in patients with impaired renal
function." Since aminoglycosides can produce vestibular or auditory
dysfunction and nephrotoxicity regardless of a patient's
impairments, it is important generally to reduce maintenance
dosages. The present invention provides dramatic reductions in
toxicity thus allowing higher doses than usual.
[0004] Cystic fibrosis (CF) patients have thick mucous and/or
sputum secretions in the lungs, frequent consequential infections,
and biofilms resulting from bacterial colonizations. All these
fluids and materials create barriers to effectively targeting
infections with antiinfectives. The present invention overcomes
these barriers, and even allows reduced dosing (in amount or
frequency), thereby reducing the drug load on patients. For lung
infections generally, the dosing schedule provided by the invention
provides a means of reducing drug load.
[0005] For a liposomal drug delivery system, it is often desirable
to lower the lipid-to-drug (L/D) ratio as much as possible to
minimize the lipid load to avoid saturation effects in the body.
For lung delivery by inhalation, this may be particularly true
because for chronic use, dosing of liposomes could outpace
clearance thus limiting the administration and thus effectiveness
of the drug product. A lower L/D ratio would allow more drug to be
given before the dosing/clearance threshold is met.
SUMMARY OF INVENTION
[0006] Via infusion methods disclosed herein, liposomes
substantially free of anionic lipids of modest size (<1 .mu.m)
that entrap antiinfectives at a lipid/antiinfective weight ratio of
typically about 4:1 to about 0.5:1 have been created. The captured
volumes of liposomes have been measured, and from these numbers one
is able to calculate what the theoretical entrapment should be if
the antiinfective behaved as an ideal solute (i.e., does not
interact with the liposome membrane but entraps ideally along with
water). From this comparison, entrapment numbers that are
3-5.times. higher than expected are observed, indicating that a
special interaction is occurring that allows greater than expected
entrapment, and lower than expected lipid/antiinfective ratios. The
solution in which the liposomes form contains a concentration of
antiinfective, the concentration of antiinfective inside the
liposomes should be about the same concentration as in the
solution. However, the internal antiinfective concentration is
calculated to be at least about 3.times. higher.
[0007] In part, the present invention features a liposomal
antiinfective formulation comprising a lipid formulation and an
antiinfective, wherein the lipid formulation is substantially free
of anionic lipids, and wherein the weight ratio of lipid to
antiinfective is about 4:1 to about 1:1. In certain embodiments,
the weight ratio of lipid to antiinfective is about 3:1 to about
1:1, 2:1 to about 1:1, or about 1:1.
[0008] In another embodiment, the present invention relates to a
lipid formulation comprising an antiinfective wherein the lipid to
antiinfective ratio is about 1:1 or less, about 0.75:1 or less, or
about 0.5:1 or less.
[0009] In certain embodiments, the lipid antiinfective formulation
comprises a liposome having a mean diameter of about 0.2 .mu.m to
about 1.0 .mu.m. In certain other embodiments, the mean diameter is
about 0.2 .mu.m to about 0.5 .mu.m. In certain other embodiments,
the mean diameter is about 0.2 .mu.m to about 0.3 .mu.m.
[0010] In certain embodiments, the antiinfective can be any
antiinfective commonly known in the art. In certain embodiments,
the antiinfective can be an aminoglycoside including, but not
limited to, amikacin, tobramycin, or gentamicin, or a
pharmaceutically acceptable salt thereof.
[0011] In certain embodiments, the lipid formulation comprises a
neutral lipid. In certain embodiments, the lipid formulation is
free of anionic lipids. In certain other embodiments, the lipid is
a phospholipid, including but not limited to, a phosphatidyl
choline such as dipalmitoylphosphatidyl choline or
dioleoylphosphatidyl choline; or the lipid can be a steroid such as
a sterol, including, but not limited to, cholesterol; or the lipid
can be a combination thereof.
[0012] In part, the present invention features a method of
preparing the lipid antiinfective formulation described above
comprising infusing an aqueous or alcoholic solution or mixture of
the antiinfective with a lipid-alcohol solution or mixture at a
temperature below the phase transition of at least one of the lipid
components of the neutral lipid, wherein infusing is done from
above. In certain embodiments, the alcohol is ethanol.
[0013] In certain embodiments, the concentration of the
lipid-alcohol solution or mixture is about 10 to about 30 mg/mL. In
certain embodiments, the concentration of the antiinfective aqueous
or alcoholic solution or mixture is about 20 to about 70 mg/mL. In
certain embodiments, the concentration of the neutral lipid-alcohol
solution or mixture is about 10 to about 30 mg/mL, and the
concentration of the antiinfective aqueous or alcoholic solution or
mixture is about 20 to about 70 mg/mL. However, one of ordinary
skill in the art will appreciate that concentrations may vary or
otherwise be optimized depending on the lipid and/or antiinfective
involved.
[0014] In certain embodiments, the present invention relates to the
aforementioned lipid formulation, wherein the antiinfective is
selected from the following: 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, 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 combination
thereof. In certain embodiments, the present invention relates to
the aforementioned lipid formulation, wherein the antiinfective is
an aminoglycoside. In a further embodiment, the antiinfective is an
aminoglycoside selected from the following: amikacin, gentamicin,
or tobramycin. In a further embodiment, the antiinfective is
amikacin. In a further embodiment, the antiinfective is gentamicin.
In a further embodiment, the antiinfective is tobramycin.
[0015] In certain embodiments, the present invention relates to the
aforementioned lipid formulation, wherein the lipid formulation is
a liposome.
[0016] In certain embodiments, the present invention relates to the
aforementioned lipid formulation, wherein the lipid formulation
comprises a phospholipid. In certain embodiments, the lipid
formulation comprises a steroid. In certain embodiments, the lipid
formulation comprises a sterol. In certain embodiments, the lipid
formulation comprises dipalmitoylphosphatidylcholine (DPPC). In
certain embodiments, the lipid formulation comprises cholesterol.
In certain embodiments, the lipid formulation comprises a
phospholipid and a steroid. In certain embodiments, the lipid
formulation comprises a phospholipid and a sterol. In certain
embodiments, the lipid formulation comprises DPPC and cholesterol.
In certain embodiments, the present invention relates to the
aforementioned formulation, wherein the lipid formulation comprises
DPPC, dioleoylphosphatidylcholine (DOPC), and cholesterol.
[0017] In certain embodiments, the present invention relates to the
aforementioned formulation, wherein the lipid formulation comprises
DPPC and cholesterol in a mole ratio of about 20:1, 10:1, 5:1, 2:1,
or 1:1.
[0018] In certain embodiments, the present invention relates to the
aforementioned formulation, wherein the lipid formulation comprises
DPPC, DOPC, and cholesterol in a mole ratio of about
5-20:1-20:0.5-1.
[0019] In certain embodiments, the present invention relates to the
aforementioned lipid formulation, wherein the lipid formulation is
a liposome and the antiinfective is amikacin.
[0020] In certain embodiments, the present invention relates to the
aforementioned lipid formulation, wherein the lipid formulation is
a liposome, the antiinfective is amikacin, and the lipid
formulation comprises a phospholipid and a sterol.
[0021] In certain embodiments, the present invention relates to the
aforementioned lipid formulation, wherein the lipid formulation is
a liposome, the antiinfective is amikacin, and the lipid
formulation comprises a DPPC and a cholesterol.
[0022] In another embodiment, the present invention relates a
method of preparing a lipid formulation comprising an antiinfective
comprising: mixing a stream of a lipid solution or mixture, with a
stream of an antiinfective solution or mixture, wherein the two
streams are mixed in line. In certain embodiments, the two streams
enter a Y-connector prior to mixing in line.
[0023] In certain embodiments, the present invention relates to the
aforementioned method, wherein the stream of a lipid solution or
mixture, and the stream of an antiinfective solution or mixture are
mixed at a total flow rate of about 700 to about 900 mL/min. In
certain embodiments, the stream of a lipid solution or mixture, and
the stream of an antiinfective solution or mixture are mixed at a
total flow rate of about 800 mL/min. In certain embodiments, the
stream of a lipid solution or mixture is added at a flow rate of
about 200 to about 400 mL/min. In certain embodiments, the stream
of a lipid solution or mixture is added at a flow rate of about 300
mL/min. In certain embodiments, the stream of an antiinfective
solution or mixture is added at a flow rate of about 400 to about
600 mL/min. In certain embodiments, the stream of an antiinfective
solution or mixture is added at a flow rate of about 500 mL/min. In
certain embodiments, the stream of a lipid solution or mixture is
added at a flow rate of about 300 mL/min, and the stream of an
antiinfective solution or mixture is added at a flow rate of about
500 mL/min.
[0024] In certain embodiments, the present invention relates to the
aforementioned method, wherein the temperature of the combined
streams is about 30-40.degree. C. In certain embodiments, the
temperature of the lipid solution or mixture is about 30.degree.
C., and the temperature of the antiinfective solution or mixture is
about 30.degree. C. In certain embodiments, the temperature of the
lipid solution or mixture is about 50.degree. C., and the
temperature of the antiinfective solution or mixture is room
temperature.
[0025] In certain embodiments, the present invention relates to the
aforementioned method, wherein the method of preparing a lipid
formulation comprising an antinfective further comprises the step
of diluting the combined streams with water at least about 20
seconds after mixing.
[0026] In certain embodiments, the present invention relates to the
aforementioned method, wherein the concentration of the
antiinfective solution or mixture is about 30 to about 50 mg/mL. In
certain embodiments, the concentration of the antiinfective
solution or mixture is about 40 to about 50 mg/mL.
[0027] In certain embodiments, the present invention relates to the
aforementioned method, wherein the stream of a lipid solution or
mixture is added at a flow rate of about 300 mi./min, and the
stream of an antiinfective solution or mixture is added at a flow
rate of about 500 mL/min; the temperature of the combined streams
is about 30-40.degree. C.; the combined streams are diluted with
water at least about 20 seconds after mixing; and the concentration
of the antiinfective solution or mixture is about 40 to about 50
mg/mL.
[0028] In certain embodiments, the present invention relates to the
aforementioned method, wherein the solutions or mixtures are
aqueous or alcoholic. In certain embodiments, the present invention
relates to the aforementioned method, wherein the lipid formulation
is a liposome.
[0029] In certain embodiments, the present invention relates to the
aforementioned method, wherein the antiinfective is selected from
the following: 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, 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 combination
thereof. In certain embodiments, the antiinfective is an
aminoglycoside. In certain embodiments, the antiinfective is an
aminoglycoside selected from the following: amikacin, gentamicin,
or tobramycin. In certain embodiments, the antiinfective is
amikacin. In certain embodiments, the antiinfective is gentamicin.
In certain embodiments, the antiinfective is tobramycin.
[0030] In certain embodiments, the present invention relates to the
aforementioned method, wherein the lipid comprises a phospholipid.
In certain embodiments, the lipid comprises a steroid. In certain
embodiments, the lipid comprises a sterol. In certain embodiments,
the lipid comprises DPPC. In certain embodiments, the lipid
comprises cholesterol. In certain embodiments the lipid comprises a
phospholipid and a sterol. In certain embodiments, the lipid
comprises DPPC and cholesterol.
[0031] In certain embodiments, the present invention relates to the
aforementioned method, wherein the lipid formulation is a liposome
and the antiinfective is amikacin.
[0032] In certain embodiments, the present invention relates to the
aforementioned method, wherein the lipid formulation is a liposome,
the antiinfective is amikacin, and the lipid comprises a
phospholipid and a sterol.
[0033] In certain embodiments, the present invention relates to the
aforementioned method, wherein the lipid formulation is a liposome,
the antiinfective is amikacin, and the lipid comprises DPPC and
cholesterol.
[0034] In certain embodiments, the present invention relates to the
aforementioned method, wherein the lipid formulation has a lipid to
antiinfective ratio of about 1:1 or less.
[0035] In certain embodiments, the present invention relates to the
aforementioned method, wherein the lipid formulation has a lipid to
antiinfective ratio of about 0.75:1 or less.
[0036] In certain embodiments, the present invention relates to the
aforementioned method, wherein the lipid formulation has a lipid to
antiinfective ratio of about 0.5:1 or less.
[0037] In certain embodiments, the present invention relates to the
aforementioned method, wherein the lipid formulation is a liposome,
the antiinfective is amikacin, the lipid comprises DPPC and
cholesterol, and the lipid to antiinfective ratio is about 1:1 or
less.
[0038] In another embodiment, the present invention relates to a
method of treating pulmonary infections in a patient in need
thereof comprising administering to the patient a therapeutically
effective amount of a liposomal antiinfective formulation
comprising a lipid formulation and an antiinfective, wherein the
dosage of antiinfective is about 100 mg/day or less. In a further
embodiment, the dosage amount of antiinfective is about 30 mg to
about 50 mg every other day.
[0039] In a further embodiment, the dosage amount of antiinfective
is about 30 mg to about 50 mg every third day.
[0040] In another embodiment, the present invention relates to the
aforementioned method of treating, wherein the liposome has a mean
diameter of about 0.2 .mu.m to about 1.0 .mu.m. In a further
embodiment, the liposome has a mean diameter of about 0.2 .mu.m to
about 0.5 .mu.m, or about 0.2 .mu.m to about 0.3 .mu.m.
[0041] In another embodiment, the present invention relates to the
aforementioned method of treating, wherein the pulmonary infection
is a result of cystic fibrosis.
[0042] In another embodiment, the present invention relates to the
aforementioned method of treating, wherein the weight ratio of
lipid to antiinfective is about 4:1 to about 0.5:1, about 3:1 to
about 0.5:1, about 2:1 to about 0.5:1, or about 1:1 to about
0.5:1.
[0043] In another embodiment, the present invention relates to the
aforementioned method of treating, wherein the antiinfective is
selected from the following: 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, penicillins, cephalosporins, a macrolide,
linomycin, clindamycin, coricosteroids, prostaglandin,
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 combination
thereof. In another embodiment, the antiinfective is an
aminoglycoside. In another embodiment, the antiinfective is
amikacin.
[0044] In another embodiment, the present invention relates to the
aforementioned method of treating, wherein the lipid formulation
comprises neutral lipids. In another embodiment, the lipids that
make up the lipid formulation are all neutral lipids. In another
embodiment, the liposome is free of anionic lipids. In another
embodiment, the lipid formulation comprises a phospholipid. In
another embodiment, the lipid formulation comprises a sterol. In
another embodiment, the lipid formulation comprises DPPC and
cholesterol.
[0045] In another embodiment, the present invention relates to the
aforementioned method of treating, wherein the antiinfective is
amikacin, and the lipid formulation comprises DPPC and
cholesterol.
[0046] In another embodiment, the present invention relates to the
aforementioned method of treating, wherein the antiinfective is
amikacin, the weight ratio of lipid to antiinfective is about 4:1
to about 1:1, and the lipid formulation comprises DPPC and
cholesterol. In a further embodiment, the weight ratio is about 3:1
to about 1:1, 2:1 to about 1:1, or about 1:1.
[0047] In another embodiment, the present invention relates to the
aforementioned method of treating, wherein the antiinfective is
amikacin, the weight ratio of lipid to antiinfective is about 4:1
to about 1:1, the lipid formulation comprises DPPC and cholesterol,
and the pulmonary infection is a result of cystic fibrosis. In a
further embodiment, the weight ratio is about 3:1 to about 1:1, 2:1
to about 1:1, or about 1:1.
[0048] In another embodiment, the present invention relates to the
aforementioned method of treating, wherein the antiinfective is
amikacin, the weight ratio of lipid to antiinfective is about 4:1
to about 0.5:1, the lipid formulation comprises DPPC and
cholesterol, and the liposome has a mean diameter of about 0.1
.mu.m to about 0.5 .mu.m. In a further embodiment, the mean
diameter is about 0.2 .mu.m to about 0.4 .mu.m, or about 0.2 .mu.m
to about 0.3 .mu.m.
[0049] In another embodiment, the present invention relates to the
aforementioned method of treating, wherein the antiinfective is
amikacin, the weight ratio of lipid to antiinfective is about 4:1
to about 0.5:1, the lipid formulation comprises DPPC and
cholesterol, the pulmonary infection is the result of cystic
fibrosis, and the liposome has a mean diameter of about 0.1 .mu.m
to about 1.0 .mu.m. In a further embodiment, the mean diameter is
about 0.2 .mu.m to about 0.5 .mu.m, or about 0.2 .mu.m to about 0.3
.mu.m.
[0050] These embodiments of the present invention, other
embodiments, and their features and characteristics, will be
apparent from the description, drawings and claims that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 depicts the cross sectional diagram of the
sputumfbiofilm seen in patients with cystic fibrosis.
[0052] FIG. 2 depicts the graphical representation of the targeting
and depot effect of the drug of the present invention.
[0053] FIGS. 3 and 4 depict graphical representations of
bacteriology of amikacin in various forms.
[0054] FIG. 5 depicts a graphical representation of sustained
release for liposomal/complexed amikacin and tobramycin.
[0055] FIG. 6 depicts data on free or complexed ciprofloxacin.
[0056] FIG. 7 depicts a graphical representation of drug residence
in the lung given various dosing schedules.
[0057] FIG. 8 depicts graphically the two-stream in-line infusion
process of preparing liposomal antiinfective formulations.
[0058] FIG. 9 depicts miscibility of amikacin sulfate with
ethanol/water. Lines represent maximal amikacin concentration
(base) miscible with ethanol solution at room temperature (RT) and
40.degree. C. At higher concentrations amikacin forms a separate
liquid phase (coacervates), which later precipitates as crystals.
Vertical lines show ethanol concentration in the lipid/amikacin
infusion mixture (300/500 parts) and after adding water 200
parts.
DETAILED DESCRIPTION
[0059] The present invention discloses a lipid formulation
comprising an antiinfective wherein the size and lipid to drug
ratios are smaller than previously known. The present invention
also discloses a method of preparing these lipid formulations.
1. Definitions
[0060] For convenience, before further description of the present
invention, certain terms employed in the specification, examples
and appended claims are collected here. These definitions should be
read in light of the remainder of the disclosure and understood as
by a person of skill in the art. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood by a person of ordinary skill in the art.
[0061] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0062] The term "bioavailable" is art-recognized and refers to a
form of the subject invention that allows for it, or a portion of
the amount administered, to be absorbed by, incorporated to, or
otherwise physiologically available to a subject or patient to whom
it is administered.
[0063] The terms "comprise" and "comprising" are used in the
inclusive, open sense, meaning that additional elements may be
included.
[0064] The terms "encapsulated" and "encapsulating" are refers to
adsorption of antiinfectives on the surface of lipid based
formulation, association of antiinfectives in the interstitial
region of bilayers or between two monolayers, capture of
antiinfectives in the space between two bilayers, or capture of
antiinfectives in the space surrounded by the inner most bilayer or
monolayer.
[0065] The term "including" is used herein to mean "including but
not limited to". "Including" and "including but not limited to" are
used interchangeably.
[0066] The term "lipid antiinfective formulation," or
"Lip-antiinfective," or "Lip-An" discussed herein is any form of
antiinfective composition where at least about 1% by weight of the
antiinfective is associated with the lipid either as part of a
complex with the lipid, or as a liposome where the antibiotic may
be in the aqueous phase or the hydrophobic bilayer phase or at the
interfacial headgroup region of the liposomal bilayer. Preferably,
at least about 5%, or at least about 10%, or at least about 20%, or
at least about 25%, can be so associated. Association can be
measured by separation through a filter where lipid and
lipid-associated antiinfective is retained and free antiinfective
is in the filtrate. A "liposomal antiinfective formulation" is a
lipid antiinfective formulation wherein the lipid formulation is
the form of a liposome.
[0067] The term "mammal" is known in the art, and exemplary mammals
include humans, primates, bovines, porcines, canines, felines, and
rodents (e.g., mice and rats).
[0068] A "patient," "subject" or "host" to be treated by the
subject method may mean either a human or non-human animal.
[0069] The term "pharmaceutically-acceptable salts" is
art-recognized and refers to the relatively non-toxic, inorganic
and organic acid addition salts of compounds, including, for
example, those contained in compositions of the present
invention.
[0070] The term "solvent infusion" is a process that includes
dissolving one or more lipids in a small, preferably minimal,
amount of a process compatible solvent to form a lipid suspension
or solution (preferably a solution) and then adding the solution to
an aqueous medium containing bioactive agents. Typically a process
compatible solvent is one that can be washed away in a aqueous
process such as dialysis. The composition that is cool/warm cycled
is preferably formed by solvent infusion, with ethanol infusion
being preferred. Alcohols are preferred as solvents. "Ethanol
infusion," a type of solvent infusion, is a process that includes
dissolving one or more lipids in a small, preferably minimal,
amount of ethanol to form a lipid solution and then adding the
solution to an aqueous medium containing bioactive agents. A
"small" amount of solvent is an amount compatible with forming
liposomes or lipid complexes in the infusion process. The term
"solvent infusion" may also include an in-line infusion process
where two streams of formulation components are first mixed
in-line.
[0071] The term "substantially free" is art recognized and refers
to a trivial amount or less.
[0072] The term "therapeutic agent" is art-recognized and refers to
any chemical moiety that is a biologically, physiologically, or
pharmacologically active substance that acts locally or
systemically in a subject. Examples of therapeutic agents, also
referred to as "drugs", are described in well-known literature
references such as the Merck Index, the Physicians Desk Reference,
and The Pharmacological Basis of Therapeutics, and they include,
without limitation, medicaments; vitamins; mineral supplements;
substances used for the treatment, prevention, diagnosis, cure or
mitigation of a disease or illness; substances which affect the
structure or function of the body; or pro-drugs, which become
biologically active or more active after they have been placed in a
physiological environment.
[0073] The phrase "therapeutically effective amount" as used herein
means that amount of a compound, material, or composition
comprising a lipid antiinfective formulation according to the
present invention which is effective for producing some desired
therapeutic effect by inhibiting pulmonary infections.
[0074] The term "treating" is art-recognized and refers to curing
as well as ameliorating at least one symptom of any condition or
disease. The term "treating" also refers to prophylactic treating
which acts to defend against or prevent a condition or disease.
2. Antiinfectives
[0075] Antiinfectives are agents that act against infections, such
as bacterial, mycobacterial, fungal, viral or protozoal infections.
Antiinfectives covered by the invention include but are not limited
to aminoglycosides (e.g., streptomycin, gentamicin, tobramycin,
amikacin, netilmicin, kanamycin, and the like), tetracyclines (such
as chlortetracycline, oxytetracycline, methacycline, doxycycline,
minocycline and the like), sulfonamides (e.g., sulfanilamide,
sulfadiazine, sulfamethaoxazole, sulfisoxazole, sulfacetamide, and
the like), paraaminobenzoic acid, diaminopyrimidines (such as
trimethoprim, often used in conjunction with sulfamethoxazole,
pyrazinamide, and the like), quinolones (such as nalidixic acid,
cinoxacin, ciprofloxacin and norfloxacin and the like), penicillins
(such as penicillin G, penicillin V, ampicillin, amoxicillin,
bacampicillin, carbenicillin, carbenicillin indanyl, ticarcillin,
azlocillin, mezlocillin, piperacillin, and the like), penicillinase
resistant penicillin (such as methicillin, oxacillin, cloxacillin,
dicloxacillin, nafcillin and the like), first generation
cephalosporins (such as cefadroxil, cephalexin, cephradine,
cephalothin, cephapirin, cefazolin, and the like), second
generation cephalosporins (such as cefaclor, cefamandole,
cefonicid, cefoxitin, cefotetan, cefuroxime, cefuroxime axetil;
cefmetazole, cefprozil, loracarbef, ceforanide, and the like),
third generation cephalosporins (such as cefepime, cefoperazone,
cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefixime,
cefpodoxime, ceftibuten, and the like), other beta-lactams (such as
imipenem, meropenem, aztreonam, clavulanic acid, sulbactam,
tazobactam, and the like), betalactamase inhibitors (such as
clavulanic acid), chlorampheriicol, macrolides (such as
erythromycin, azithromycin, clarithromycin, and the like),
lincomycin, clindamycin, spectinomycin, polymyxin B, polymixins
(such as polymyxin A, B, C, D, E1(colistin A), or E2, colistin B or
C, and the like) colistin, vancomycin, bacitracin, isoniazid,
rifampin, ethambutol, ethionamide, aminosalicylic acid,
cycloserine, capreomycin, sulfones (such as dapsone, sulfoxone
sodium, and the like), clofazimine, thalidomide, or any other
antibacterial agent that can be lipid encapsulated. Antiinfectives
can include antifungal agents, including polyene antifungals (such
as amphotericin B, nystatin, natamycin, and the like), flucytosine,
imidazoles (such as n-ticonazole, clotrimazole, econazole,
ketoconazole, and the like), triazoles (such as itraconazole,
fluconazole, and the like), griseofulvin, terconazole, butoconazole
ciclopirax, ciclopirox olamine, haloprogin, tolnaftate, naftifine,
terbinafine, or any other antifungal that can be lipid encapsulated
or complexed. Discussion and the examples are directed primarily
toward amikacin but the scope of the application is not intended to
be limited to this antiinfective. Combinations of drugs can be
used.
[0076] Particularly preferred antiinfectives include the
aminoglycosides, the quinolones, the polyene antifungals and the
polymyxins.
[0077] Also included as suitable antiinfectives used in the lipid
antiinfective formulations of the present invention are
pharmaceutically acceptable addition salts and complexes of
antiinfectives. In cases wherein the compounds may have one or more
chiral centers, unless specified, the present invention comprises
each unique racemic compound, as well as each unique nonracemic
compound.
[0078] In cases in which the antiinfectives have unsaturated
carbon-carbon double bonds, both the cis (Z) and trans (E) isomers
are within the scope of this invention. In cases wherein the
antiinfectives may exist in tautomeric forms, such as keto-enol
tautomers, such as
##STR00001##
each tautomeric form is contemplated as being included within this
invention, whether existing in equilibrium or locked in one form by
appropriate substitution with R'. The meaning of any substituent at
any one occurrence is independent of its meaning, or any other
substituent's meaning, at any other occurrence.
[0079] Also included as suitable antiinfectives used in the lipid
antiinfective formulations of the present invention are prodrugs of
the platinum compounds. Prodrugs are considered to be any
covalently bonded carriers which release the active parent compound
in vivo.
3. Pulmonary Infections
[0080] Among the pulmonary infections (such as in cystic fibrosis
patients) that can be treated with the methods of the invention are
Pseudomonas (e.g., P. aeruginosa, P. paucimobilis, P. putida, P.
fluorescens, and P. acidovorans), staphylococcal,
Methicillinresistant Staphylococcus aureus (MRSA), streptococcal
(including by Streptococcus pneumoniae), Escherichia coli,
Klebsiella, Enterobacter, Serratia, Haemophilus, Yersinia pesos,
Burkholderia pseudomallei, B. cepacia, B. gladioli, B. multivorans,
B. vietnamiensis, Mycobacterium tuberculosis, M. avium complex
(MAC) (M. avium and M. intracellulare), M. kansasii, M. xenopi, M.
marinum, M. ulcerans, or M. fortuitum complex (M. fortuitum and M.
chelonei) infections.
4. Methods of Treatment
[0081] In one embodiment the present invention comprises a method
of treatment comprising administration of a therapeutically
effective amount of a lipid antiinfective formulation.
[0082] Where no specific dosage is provided below, the preferred
dosage of the invention is 50% or less, 35% or less, 20% or less,
or 10% or less, of the minimum free drug (which of course can be a
salt) amount that is effective, if delivered to the lungs via a
nebulizer, to reduce the CFU count in the lungs by one order of
magnitude over the course of a 14-day treatment. The comparative
free drug amount is the cumulative amount that would be used in the
dosing period applied with the drug administration of the
invention. The comparative minimum free drug defined in this
paragraph is a "comparative free drug amount."
[0083] The non-CF treating embodiments of the invention can be used
with any animal, though preferably with humans. Relative amounts in
a given animal are measured with respect to such animal.
[0084] The dosing schedule is preferably once a day or less. In
preferred embodiments, the dosing schedule is once every other day,
every third day, every week, or less. For example, the dosing
schedule can be every other day or less, using 50% or less of the
comparative free drug amount. Or, for example, the dosing can be
daily using 35% or less of the comparative free drug amount. See
FIGS. 3 and 4 for animal data showing that the lipid antiinfective
formulations of the present invention are more efficacious than the
free drug.
[0085] To treat infections, the effective amount of the
antiinfective will be recognized by clinicians but includes an
amount effective to treat, reduce, ameliorate, eliminate or prevent
one or more symptoms of the disease sought to be treated or the
condition sought to be avoided or treated, or to otherwise produce
a clinically recognizable change in the pathology of the disease or
condition. Amelioration includes reducing the incidence or severity
of infections in animals treated prophylactically. In certain
embodiments, the effective amount is one effective to treat or
ameliorate after symptoms of lung infection have arisen. In certain
other embodiments, the effective amount is one effective to treat
or ameliorate the average incidence or severity of infections in
animals treated prophylactically (as measured by statistical
studies).
[0086] Liposome or other lipid delivery systems can be administered
for inhalation either as a nebulized spray, powder, or aerosol, or
by intrathecal administration. Inhalation administrations are
preferred. The overall result is a less frequent administration and
an enhanced therapeutic index compared to free drug or parenteral
form of the drug. Liposomes or other lipid formulations are
particularly advantageous due to their ability to protect the drug
while being compatible with the lung lining or lung surfactant.
[0087] The present invention includes methods for treatment of
pulmonary gram-negative infections. One usefully treated infection
is chronic pseudomonal infection in CF patients. Known treatments
of lung infections (such as in CF patients) with aminoglycoside
generally comprise administering approximately 200-600 mg of
amikacin or tobramycin per day via inhalation. The present
invention allows for treatment by administering, in one preferred
embodiment, 100 mg or less of amikacin per day (or normalized to
100 mg per day or less if dosing less frequent). In yet another
embodiment, administration of 60 mg or less of amikacin every day
is performed. And in still another embodiment administration of
approximately 30 to 50 mg not more than once every 2 days is
performed. The most preferred embodiment comprises administration
of approximately 30 to 50 mg every other day or every third
day.
5. Lipids and Liposomes
[0088] The lipids used in the compositions of the present invention
can be synthetic, semi-synthetic or naturally-occurring lipids,
including phospholipids, tocopherols, steroids, fatty acids,
glycoproteins such as albumin, anionic lipids and cationic lipids.
The lipids may be anionic, cationic, or neutral. In one embodiment,
the lipid formulation is substantially free of anionic lipids. In
one embodiment, the lipid formulation comprises only neutral
lipids. In another embodiment, the lipid formulation is free of
anionic lipids. In another embodiment, the lipid is a phospholipid.
Phosholipids include egg phosphatidylcholine (EPC), egg
phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI), egg
phosphatidylserine (EPS), phosphatidylethanolamine (EPE), and egg
phosphatidic acid (EPA); the soya counterparts, soy
phosphatidylcholine (SPC); SPG, SPS, SPI, SPE, and SPA; the
hydrogenated egg and soya counterparts (e.g., HEPC, HSPC), other
phospholipids made up of ester linkages of fatty acids in the 2 and
3 of glycerol positions containing chains of 12 to 26 carbon atoms
and different head groups in the 1 position of glycerol that
include choline, glycerol, inositol, serine, ethanolamine, as well
as the corresponding phosphatidic acids. The chains on these fatty
acids can be saturated or unsaturated, and the phospholipid can be
made up of fatty acids of different chain lengths and different
degrees of unsaturation. In particular, the compositions of the
formulations can include dipalmitoylphosphatidylcholine (DPPC), a
major constituent of naturally-occurring lung surfactant as well as
dioleoylphosphatidylcholine (DOPC). Other examples include
dimyristoylphosphatidylcholine (DMPC) and
dimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidcholine
(DPPC) and dipalmitoylphosphatidylglycerol (DPPG) di
stearoylphosphatidylcholine (DSPC) and di
stearoylphosphatidylglycerol (DSPG),
dioleylphosphatidylethanolamine (DOPE) and mixed phospholipids like
palmitoylstearoylphosphatidylcholine (PSPC) and
palmitoylstearoylphosphatidylglycerol (PSPG), driacylglycerol,
diacylglycerol, seranide, sphingosine, sphingomyelin and single
acylated phospholipids like mono-oleoyl-phosphatidylethanol amine
(MOPE).
[0089] The lipids used can include ammonium salts of fatty acids,
phospholipids and glycerides, steroids, phosphatidylglycerols
(PGs), phosphatidic acids (PAs), phosphotidylcholines (PCs),
phosphatidylinositols (PIs) and the phosphatidylserines (PSs). The
fatty acids include fatty acids of carbon chain lengths of 12 to 26
carbon atoms that are either saturated or unsaturated. Some
specific examples include: myristylamine, palmitylamine,
laurylamine and stearylamine, dilauroyl ethylphosphocholine (DLEP),
dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl
ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine
(DSEP),
N-(2,3-di-(9(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium
chloride (DOTMA) and 1,
2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP). Examples of
steroids include cholesterol and ergosterol. Examples of PGs, PAs,
PIs, PCs and PSs include DMPG, DPPG, DSPG, DMPA, DPPA, DSPA, DMPI,
DPPI, DSPI, DMPS, DPPS and DSPS, DSPC, DPPG, DMPC, DOPC, egg
PC.
[0090] Liposomes or lipid antiinfective formulations composed of
phosphatidylcholines, such as DPPC, aid in the uptake by the cells
in the lung such as the alveolar macrophages and helps to sustain
release of the antiinfective agent in the lung (Gonzales-Rothi et
al. (1991)). The negatively charged lipids such as the PGs, PAs,
PSs and PIs, in addition to reducing particle aggregation, can play
a role in the sustained release characteristics of the inhalation
formulation as well as in the transport of the formulation across
the lung (transcytosis) for systemic uptake. The sterol compounds
are believed to affect the release and leakage characteristics of
the formulation.
[0091] Liposomes are completely closed lipid bilayer membranes
containing an entrapped aqueous volume. Liposomes can be
unilamellar vesicles (possessing a single membrane bilayer) or
multilamellar vesicles (onion-like structures characterized by
multiple membrane bilayers, each separated from the next by an
aqueous layer). The bilayer is composed of two lipid monolayers
having a hydrophobic "tail" region and a hydrophilic "head" region.
The structure of the membrane bilayer is such that the hydrophobic
(nonpolar) "tails" of the lipid monolayers orient toward the center
of the bilayer while the hydrophilic "heads" orient towards the
aqueous phase. Lipid antiinfective formulations are associations
lipid and the antiinfective agent. This association can be
covalent, ionic, electrostatic, noncovalent, or steric. These
complexes are non-liposomal and are incapable of entrapping
additional water soluble solutes. Examples of such complexes
include lipid complexes of amphotencin B (Janoff et al., Proc. Nat
Acad. Sci., 85:6122 6126, 1988) and cardiolipin complexed with
doxorubicin.
[0092] A lipid clathrate is a three-dimensional, cage-like
structure employing one or more lipids wherein the structure
entraps a bioactive agent. Such clathrates are included in the
scope of the present invention.
[0093] Proliposomes are formulations that can become liposomes or
lipid complexes upon corning in contact with an aqueous liquid.
Agitation or other mixing can be necessary. Such proliposomes are
included in the scope of the present invention.
[0094] Liposomes can be produced by a variety of methods (for
example, see, Bally, Cullis et al., Biotechnol Adv. 5(1):194,
1987). Bangham's procedure (J. Mol. Biol., J Mol Biol.
13(1):238-52, 1965) produces ordinary multilamellar vesicles
(MLVs). Lenk et al. (U.S. Pat. Nos. 4,522,803, 5,030,453 and
5,169,637), Fountain et al. (U.S. Pat. No. 4,588,578) and Cullis et
al. (U.S. Pat. No. 4,975,282) disclose methods for producing
multilamellar liposomes having substantially equal interlamellar
solute distribution in each of their aqueous compartments.
Paphadjopoulos et al., U.S. Pat. No. 4,235,871, discloses
preparation of oligolamellar liposomes by reverse phase
evaporation.
[0095] Unilamellar vesicles can be produced from MLVs by a number
of techniques, for example, the extrusion of Cullis et al. (U.S.
Pat. No. 5,008,050) and Loughrey et al. (U.S. Pat. No. 5,059,421).
Sonication and homogenization can be used to produce smaller
unilamellar liposomes from larger liposomes (see, for example,
Paphadjopoulos et al., Biochim. Biophys. Acta., 135:624-638, 1967;
Deamer, U.S. Pat. No. 4,515,736; and Chapman et al., Liposome
Technol., 1984, pp. 1-18).
[0096] The original liposome preparation of Bangham et al. (J. Mol.
Biol., 1965, 13:238-252) involves suspending phospholipids in an
organic solvent which is then evaporated to dryness leaving a
phospholipid film on the reaction vessel. Next, an appropriate
amount of aqueous phase is added, the mixture is allowed to
"swell", and the resulting liposomes which consist of multilamellar
vesicles (MLVs) are dispersed by mechanical means. This preparation
provides the basis for the development of the small sonicated
unilamellar vesicles described by Papahadjopoulos et al. (Biochim.
Biophys, Acta., 1967, 135:624-638), and large unilamellar
vesicles.
[0097] Techniques for producing large unilamellar vesicles (LUVs),
such as, reverse phase evaporation, infusion procedures, and
detergent dilution, can be used to produce liposomes. A review of
these and other methods for producing liposomes can be found in the
text Liposomes, Marc Ostro, ed., Marcel Dekker, Inc., New York,
1983, Chapter 1, the pertinent portions of which are incorporated
herein by reference. See also Szoka, Jr. et al., (1980, Ann. Rev.
Biophys. Bioeng., 9:467), the pertinent portions of which are also
incorporated herein by reference.
[0098] Other techniques that are used to prepare vesicles include
those that form reverse-phase evaporation vesicles (REV),
Papahadjopoulos et al., U.S. Pat. No. 4,235,871. Another class of
liposomes that can be used are those characterized as having
substantially equal lamellar solute distribution. This class of
liposomes is denominated as stable plurilamellar vesicles (SPLV) as
defined in U.S. Pat. No. 4,522,803 to Lenk, et al. and includes
monophasic vesicles as described in U.S. Pat. No. 4,588,578 to
Fountain, et al. and frozen and thawed multilamellar vesicles
(FATMLV) as described above.
[0099] A variety of sterols and their water soluble derivatives
such as cholesterol hemisuccinate have been used to form liposomes;
see specifically Janoff et al., U.S. Pat. No. 4,721,612, issued
Jan. 26, 1988, entitled "Steroidal Liposomes." Mayhew et al,
described a method for reducing the toxicity of antibacterial
agents and antiviral agents by encapsulating them in liposomes
comprising alpha-tocopherol and certain derivatives thereof. Also,
a variety of tocopherols and their water soluble derivatives have
been used to form liposomes, see Janoff et al., U.S. Pat. No.
5,041,278.
6. Methods of Preparation
[0100] A process for forming liposomes or lipid antiinfective
formulations involves a "solvent infusion" process. This is a
process that includes dissolving one or more lipids in a small,
preferably minimal, amount of a process compatible solvent to form
a lipid suspension or solution (preferably a solution) and then
infusing the solution into an aqueous medium containing the
antiinfective. Typically a process compatible solvent is one that
can be washed away in a aqueous process such as dialysis or
diafiltration. "Ethanol infusion," a type of solvent infusion, is a
process that includes dissolving one or more lipids in a small,
preferably minimal, amount of ethanol to form a lipid solution and
then infusing the solution into an aqueous medium containing the
antiinfective. A "small" amount of solvent is an amount compatible
with forming liposomes or lipid complexes in the infusion process.
Such processes are described in Lee et al., U.S. patent application
Ser. No. 10/634,144, filed Aug. 4, 2003, Pilkiewicz et al, U.S.
patent application Ser. No. 10/383,173, filed Mar. 5, 2003, and
Boni et al., U.S. patent application Ser. No. 10/383,004, filed
Mar. 5, 2003, which applications are hereby incorporated by
reference in their entirety.
[0101] The step of infusing the lipid-alcohol solution into the
aqueous or alcoholic solution or mixture containing the
antiinfective can be performed above or below the surface of the
aqueous or alcoholic solution or mixture containing the
antiinfective. Preferably, the step is performed above the surface
of the solution or mixture.
[0102] Liposomes can also be prepared by the methods disclosed in
copending U.S. patent application Ser. No. 10/383,004, filed Mar.
5, 2003; Ser. No. 10/634,144, filed Aug. 4, 2003; Ser. No.
10/224,293, filed Aug. 20, 2002; and Ser. No. 10/696,389, filed
Oct. 29, 2003, the specifications of which are incorporated herein
in their entirety.
[0103] Liposome or lipid formulation sizing can be accomplished by
a number of methods, such as extrusion, sonication and
homogenization techniques which are well known, and readily
practiced, by ordinarily skilled artisans. Extrusion involves
passing liposomes, under pressure, one or more times through
filters having defined pore sizes. The filters are generally made
of polycarbonate, but the filters may be made of any durable
material which does not interact with the liposomes and which is
sufficiently strong to allow extrusion under sufficient pressure.
Preferred filters include "straight through" filters because they
generally can withstand the higher pressure of the preferred
extrusion processes of the present invention. "Tortuous path"
filters may also be used. Extrusion can also use asymmetric
filters, such as Anopore.TM. filters, which involves extruding
liposomes through a branched-pore type aluminum oxide porous
filter.
[0104] Liposomes or lipid formulations can also be size reduced by
sonication, which employs sonic energy to disrupt or shear
liposomes, which will spontaneously reform into smaller liposomes.
Sonication is conducted by immersing a glass tube containing the
liposome suspension into the sonic epicenter produced in a
bath-type sonicator. Alternatively, a probe type sonicator may be
used in which the sonic energy is generated by vibration of a
titanium probe in direct contact with the liposome suspension.
Homogenization and milling apparatii, such as the Gifford Wood
homogenizer, Polytron.TM. or Microfluidizer, can also be used to
break down larger liposomes or lipid formulations into smaller
liposomes or lipid formulations.
[0105] The resulting liposomal formulations can be separated into
homogeneous populations using methods well known in the art; such
as tangential flow filtration. In this procedure, a heterogeneously
sized population of liposomes or lipid formulations is passed
through tangential flow filters, thereby resulting in a liposome
population with an upper and/or lower size limit. When two filters
of differing sizes, that is, having different pore diameters, are
employed, liposomes smaller than the first pore diameter pass
through the filter. This filtrate can the be subject to tangential
flow filtration through a second filter, having a smaller pore size
than the first filter. The retentate of this filter is a
liposomal/complexed population having upper and lower size limits
defined by the pore sizes of the first and second filters,
respectively.
[0106] Mayer et al. found that the problems associated with
efficient entrapment of lipophilic ionizable bioactive agents such
as antineoplastic agents, for example, anthracyclines or vinca
alkaloids, can be alleviated by employing transmembrane ion
gradients. Aside from inducing greater uptake, such transmembrane
gradients can also act to increase antiinfective retention in the
liposomal formulation.
[0107] Lipid antiinfective formulations have a sustained
antiinfective effect and lower toxicity allowing less frequent
administration and an enhanced therapeutic index. In preclinical
animal studies and in comparison to inhaled tobramycin
(not-liposomal or lipid-based) at the equivalent dose level,
liposomal amikacin was shown to have, during the time period
shortly after administration to over 24 hours later, drug levels in
the lung that ranged from two to several hundred times that of
tobramycin. Additionally, liposomal amikacin maintained these
levels for well over 24 hours. In an animal model designed to mimic
the pseudomonas infection seen in CF patients, liposomal amikacin
was shown to significantly eliminate the infection in the animals'
lungs when compared to free aminoglycosides.
[0108] Lung surfactant allows for the expansion and compression of
the lungs during breathing. This is accomplished by coating the
lung with a combination of lipid and protein. The lipid is
presented as a monolayer with the hydrophobic chains directed
outward. The lipid represents 80% of the lung surfactant, the
majority of the lipid being phosphatidylcholine, 50% of which is
dipalmitoyl phosphatidylcholine (DPPC) (Veldhuizen et al, 1998).
The surfactant proteins (SP) that are present function to maintain
structure and facilitate both expansion and compression of the lung
surfactant as occurs during breathing. Of these, SP-B and SP-C
specifically have lytic behavior and can lyse liposomes (Hagwood et
al., 1998; Johansson, 1998). This lytic behavior could facilitate
the gradual break-up of liposomes. Liposomes can also be directly
ingested by macrophages through phagocytosis (Couveur et al., 1991;
Gonzales-Roth et al., 1991; Swenson et al, 1991). Uptake of
liposomes by alveolar macrophages is another means by which drugs
can be delivered to the diseased site.
[0109] The lipids preferably used to form either liposomal or lipid
formulations for inhalation are common to the endogenous lipids
found in the lung surfactant. Liposomes are composed of bilayers
that entrap the desired pharmaceutical. These can be configured as
multilamellar vesicles of concentric bilayers with the
pharmaceutical trapped within either the lipid of the different
layers or the aqueous space between the layers. The present
invention utilizes unique processes to create unique liposomal or
lipid antiinfective formulations. Both the processes and the
product of these processes are part of the present invention.
[0110] 6.1 In-Line Infusion Method
[0111] In one particularly preferred embodiment, the liposomal
antiinfective formulations of the present invention are prepared by
an in-line infusion method where a stream of lipid solution is
mixed with a stream of antiinfective solution in-line. For example,
the two solutions may be mixed in-line inside a mixing tube
preceded by a Y-connector as depicted in FIG. 8. In this way, the
in-line infusion method differs from the infusion method described
above, where the lipid solution is infused as a stream into a bulk
of antiinfective solution. Surprisingly, this infusion method
results in lower lipid to drug ratios and higher encapsulation
efficiencies. The process may be further improved by optimizing
parameters such as flow rate, temperature, antiinfective
concentration, and salt addition after infusion step.
[0112] 6.1.a Effect of Flow Rates
[0113] Individual flow rates were varied while keeping the total
flow rate at 800 mL/min. To do so, two separate pumps were used set
at different pumping rates. The mixed solutions were infused for 10
s into a beaker containing NaCl solution such that the final NaCl
concentration was 1.5% and the final ethanol concentration did not
exceed 30%. After mixing, a 1 mL aliquot was run though a Sephadex
G-75 gel filtration column to separate free amikacin from
encapsulated. A 1 mL fraction with highest density (determined by
visual turbidity) was collected for further analysis. The results
are presented in Table 1. Increasing the lipid/amikacin flow rate
ratio resulted in an almost constant L/D until 300/500 mL/min. With
further increase of lipid rate, L/D started to increase and
particle size also started getting larger. At the same time, higher
lipid flow rates gave better amikacin recovery (encapsulation
efficiency) as more lipid mass was added.
TABLE-US-00001 TABLE 1 Effect of flow rates on amikacin
encapsulation.* Flow rates AMK AMK mL/min total AMK Lipid VOL
Recovery Batch AMK Lipid mg/mL free % mg/mL L/D Size % 1 600 200
1.38 5.3 1.25 0.91 289 14.7 2 550 250 1.80 5.1 1.90 1.06 305 17.2 3
500 300 2.18 5.2 2.29 1.05 314 22.8 4 450 350 1.27 5.8 1.47 1.16
388 26.8 5 400 400 1.05 6.1 1.69 1.61 471 24.9 *Lipid and amikacin
solutions were kept at 40.degree. C. Amikacin stock solution was 50
mg/mL. NaCl 10% solution was added before infusion to obtain final
1.5%. Infusion time was set at 10 s. Mixing tube 10 cm; 6-element
in-line mixer positioned at 0 cm.
[0114] Batch 3 with the lipid/amikacin flow rates of 300/500 mL/min
showed the best L/D and particle size, combined with reasonably
high amikacin recovery. Thus it was decided to use these flow rates
for all further experiments.
[0115] In order to reproduce the results at chosen conditions a
fully washed batch (batch 6) using diafiltration was prepared as
presented in Table 2. NaCl 10% solution was added into the beaker
prior to infusion to make the final concentration 2% (as compared
to 1.5% in the batches in Table 1). The resulting L/D (1.71) was
not as good as in batch 3 in Table 1 and the particle size was
higher. This may be due to an adverse effect of high NaCl
concentration contacting liposomes in the early stages of liposome
formation. Samples separated (washed) using gel-filtration columns
tend to have better L/D than ones washed by diafiltration. This may
have to do with the different degree of stress liposomes
experience, or simply samples separated on the gel filtration
column contained a fraction of liposomes with better L/D which does
not represent the whole population.
TABLE-US-00002 TABLE 2 Summary of the fully washed batches. Process
parameters varied were: temperatures, amikacin stock concentration,
and other (see Table 3 below). All batches were concentrated to
nearly a maximum extent, until the inlet pressure reached 10 PSI.
AMK AMK AMK Size Size Temp, C. stock total free Lipid VOL SD Batch
L/AMK/W mg/mL mg/mL % mg/mL L/D nm % 6 40/40/30 50 36.1 2.7 61.8
1.71 392 43.4 8 50/RT/30 50 48.5 9.6 49.3 1.02 332 32.0 9 50/RT/30
50 41.6 5.1 43.2 1.04 359 34.4 10 50/RT/30 50 53.1 10.2 34.4 0.65
350 28.6 11 50/RT/30 40 20.7 4.8 46.9 2.27 407 35.9 12 50/RT/30 40
81.0 1.9 49.4 0.61 341 33.0 13 50/RT/30 30 68.6 1.7 62.5 0.91 311
22.4 14 50/RT/30 40 79.6 1.6 47.8 0.60 346 37.2 15 50/RT/30 40 71.3
2.0 42.3 0.59 353 33.4 16 30/30/30 40 61.9 6.1 51.5 0.83 369 28.4
17 30/30/30 40 73.8 2.4 57.2 0.77 362 32.6 18 30/30/30 40 74.4 2.3
54.0 0.73 549 61.7 *The 3rd column represents the temperatures of
Lipid and Amikacin solutions just before infusion, and the
temperature during washing (diafiltration). RT = room temperature.
"VOL size" is the volume weighted particle size.
TABLE-US-00003 TABLE 3 Processing conditions for batches 1-18.*
Washing Mixing Mixer NaCl added conditions tube position Stock
Volume Timing to NaCl Batch cm cm % Parts infusion % 1st wash 1-5
10 0 VAR VAR before 1.5 (Seph column) 6 10 0 10 200 before 1.5
diafiltration 7 10 5 10 100 before 1.5 (Seph column) 8 10 5 10 150
during 1.5 diafiltration 9 10 5 10 150 during 1.5 diafiltration 10
10 5 10 100 5' after 1.5 2 .times. dilution 11 10 5 10 150 imm
after 1.5 2 .times. dilution 12 10 5 H2O 180 20'' after 1.5 2
.times. dilution 13 10 5 H2O 180 30'' after 1.5 2 .times. dilution
14 10 5 H2O 180 30'' after 1.5 diafiltration 15 10 5 1.5 180 30''
after 1.5 diafiltration 16 60 NO 0.9 180 during 0.9 diafiltration
17 60 NO 1.5 180 during 1.5 diafiltration 18 60 0 1.5 180 during
1.5 diafiltration *Lipid and amikacin solutions were infused at
rates 300/500 mL/min for 30 s (examples 6-10) or 20 s (examples
11-18). Additional aqueous solution (NaCl or water) was added (as
parts relative to 500 parts amikacin volume).
[0116] 6.1.b Effects of Process Temperature.
[0117] The settings were kept the same as in batch 3 except that
the amount of NaCl solution added was less, making the final
concentration 1.0%. Solution was added again before infusion was
initiated because with the short infusion time it was difficult to
make the addition during infusion. Also, during infusion the
in-line mixer shifted to the end of the mixing tube under the
pressure of the flow. The position of the mixer was 5 cm from the
front end of the tube instead of 0 cm for batch 3. This may be
important, as the L/D ratio obtained at the same temperature
40/40.degree. C. condition in batch 20 was 0.55, almost half of
that in batch 3. On comparing amikacin encapsulation at different
infusion temperatures, one can see that, surprisingly, lower
temperatures gave better L/D. Of the temperatures tested,
lipid/amikacin temperatures 30/30.degree. C. and 50/RT gave similar
L/D ratios of 0.32 and 0.37. Again, as in batches 1-5, the numbers
from these washed samples by gel-filtration were low, perhaps less
than that if the batches had been washed by diafiltration.
TABLE-US-00004 TABLE 4 Effect of temperature on amikacin
encapsulation.* Temperature, C. AMK AMK VOL mL/min total free Lipid
Size Batch Lipid AMK mg/mL % mg/mL L/D nm 19 30 30 4.88 2.8 1.54
0.32 278 20 40 40 3.62 1.5 1.98 0.55 335 21 50 50 3.50 1.8 2.74
0.78 309 22 50 RT 5.27 2.9 1.93 0.37 342 *Lipid and amikacin
solutions were infused at rates 300/500 mL/min for 10 s. Amikacin
stock solution was 50 mg/mL. NaCl 10% solution was added before
infusion to obtain a final 1.0% concentration. Mixing tube 10 cm,
6-element in-line mixer positioned at 5 cm.
[0118] In separate experiments it was found that mixing of 90%
ethanol and water at either 30.degree. C. and 30.degree. C. or
50.degree. C. and 22.degree. C., respectively, resulted in a
similar final temperature of nearly 36.degree. C. This suggests
that the temperature of the final mixture rather than that of the
individual components is important for amikacin encapsulation. The
temperatures 50.degree. C./RT were used in examples 6-15. In
examples 16-18 temperatures of 30.degree. C. and 30.degree. C. for
the two streams were used with comparable results, although a
little less amikacin encapsulation was observed.
[0119] 6.1.c Effect of Post-Infusion Addition of Aqueous Volume
[0120] Attention was next focused on the steps of NaCl solution
addition and the washing process. Process parameters were varied in
various directions. Right after the infusion step at flow rates
300/500, ethanol concentration in the mixture reaches 34%. Amikacin
has limited solubility at this concentration (see FIG. 9).
[0121] If one starts with 50 mg/mL amikacin stock, then after
mixing with the lipid solution there will be more than 30 mg/mL
total amikacin where at least half (15 mg/mL) is free amikacin,
assuming 50% encapsulation efficiency. This is higher than the
solubility limit at 34% ethanol. One possible solution to this
problem is to add more water to the vessel with the lipid/amikacin
mixture, thus reducing both ethanol and amikacin concentration. For
example, adding 200 parts of water (or NaCl solution) to 800 parts
of lipid/amikacin would reduce ethanol to 27% (FIG. 9). This makes
amikacin soluble at 15 mg/mL or even higher depending on
temperature.
[0122] In addition, adding NaCl may stabilize osmotic conditions.
When liposomes are formed and amikacin is encapsulated at an
internal concentration of 200-300 mg/mL, there is only .about.15
mg/mL or so of amikacin not encapsulated. In the absence of saline
this would create an osmotic imbalance, which in turn might lead to
leakage of amikacin. Adding 150 parts of 10% NaCl to 800 parts of
lipid/amikacin will result in about 1.5% NaCl final concentration
(outside liposomes).
[0123] A number of batches were generated where different amounts
of NaCl solution (or water in some batches) were added at different
times relative to the infusion event (see Table 5, compiled from
Tables 2 and 3). From the table a general trend can be seen,
leading to the following conclusions: [0124] Some time interval
between infusion and addition of the aqueous volume is required to
obtain lower L/D (if a short mixing tube is used). Of batches 6-15,
those with an interval 20 s or longer had lower L/D. One possible
explanation is that liposomes are not completely formed immediately
after mixing of the streams. When a longer mixing tube is used
(batches 16-18), which allows for a longer mixing time, the time
interval is not required. [0125] Adding a high concentration NaCl
solution to balance osmolality does not actually help retain
amikacin. In fact, adding pure water at an appropriate time
interval resulted in the lowest L/D and total amikacin
concentration. [0126] Adding 100 parts NaCl 10% (batch 9) 5 min
after infusion gave a competitive L/D ratio but did not give as
good a total amikacin concentration. It may be that NaCl, when
present at early stages with relatively high ethanol
concentrations, leads to increased aggregation and viscosity.
TABLE-US-00005 [0126] TABLE 5 Role of aqueous volume and NaCl
concentration added to the lipid/amikacin mixture to adjust ethanol
concentration. Not all the variables shown; see Tables 2 and 3. AMK
NaCl added AMK Size stock Stock Volume Timing to total VOL Batch
mg/ML % parts infusion mg/mL L/D nm 6 50 10 200 before 36.1 1.71
392 8 50 10 150 during 48.5 1.02 332 9 50 10 150 during 41.6 1.04
359 10 50 10 100 5' after 53.1 0.65 350 11 40 10 150 imm after 20.7
2.27 407 12 40 H2O 180 20'' after 81.0 0.61 341 13 30 H2O 180 30''
after 68.6 0.91 311 14 40 H2O 180 30'' after 79.6 0.60 346 15 40
1.5 180 30'' after 71.3 0.59 353 16 40 0.9 180 during 61.9 0.83 369
17 40 1.5 180 during 73.8 0.77 362 18 40 1.5 180 during 74.4 0.73
549
[0127] 6.1.d Effect of Antiinfective Stock Solution
[0128] Previously it was found that using 50 mg/mL amikacin stock
solution produced the best entrapment. Reducing the amikacin stock
concentration to 40 mg/mL increased L/D when used in conventional
processes. With the two-stream in-line infusion process, ethanol
concentration reaches higher levels, so the current 50 mg/mL
amikacin may not be the optimal concentration.
[0129] Table 6 summarizes the effect of using various amikacin
stock concentrations. 40 mg/mL delivered comparable or better 1-0
values, and even improved amikacin recovery. Using less amikacin
relative to a constant amount of lipid, and providing a similar
1/D, resulted in a higher percent encapsulation (batch 12). Further
decrease of amikacin stock concentration to 30 mg/mL resulted in a
slightly increased L/D, although recovery was still impressive
(batch 13).
TABLE-US-00006 TABLE 6 Amikacin stock concentration can be reduced
while improving efficiency. Amikacin recovery is calculated based
on L/D obtained and assumed 100% lipid recovery. AMK AMK AMK Size
AMK stock total free Lipid VOL Recovery Batch mg/mL mg/ML % mg/mL
L/D nm % 10 50 53.1 10.2 34.4 0.65 350 37.0 12 40 81.0 1.9 49.4
0.61 341 51.2 13 30 68.6 1.7 62.5 0.91 311 45.7 14 40 79.6 1.6 47.8
0.60 346 52.0
[0130] Reducing amikacin stock concentration has another
implication. It reduces the concentration of free amikacin in a
post-infusion lipid/amikacin mixture, allowing it to remain soluble
at higher ethanol concentration. Assuming that lipid and amikacin
are mixed at 300/500 ratio, amikacin stock is 50 mg/mL, and
encapsulation efficiency is 37%, then initial free amikacin would
be .about.20 mg/mL. Similarly, 40 mg/mL amikacin stock with 52%
encapsulation would result in .about.12 mg/mL free amikacin. 30
mg/mL amikacin stock with 46% encapsulation would result in
.about.10 mg/mL free amikacin.
7. Lipid to Drug Ratio
[0131] There are several ways to increase the entrapment of
antiinfectives (e.g. aminoglycosides such as amikacin, tobramycin,
gentamicin) in liposomes. One way is to make very large liposomes
(>1 .mu.m) where the entrapped volume per amount of lipid is
large. This approach to achieve a smaller L/D ratio is not
practical for inhalation (nebulization) of liposomes because 1)
shear stress during nebulization tends to rupture liposomes in a
size dependent manner where larger liposomes (>0.5 .mu.m) suffer
greater release and 2) the smaller droplet sizes necessary for good
lung deposition are themselves less than about .about.3 .mu.m. So
for inhalation, it is desirable to keep the liposome size as small
as possible to avoid too much release. Currently, the mean diameter
for the liposomes disclosed herein is less than about 0.4 .mu.m
(see Table 4).
[0132] Another approach to decrease L/D is to use negatively
charged lipids. The aminoglycosides listed above are highly
positively charged with 4 to 5 amines per compound. Usually sulfate
salts of these aminoglycosides are used in therapeutic
formulations. Along with the multi-cationic character comes strong
binding to negatively charged liposomes. This results in greater
entrapment during liposome formation. The purpose of antiinfective
formulations is to provide sustained release to the lung
environment. Rapid clearance of the liposomes by macrophage uptake
would run counter to this. It has been well documented that
negatively charged liposomes experience a much higher degree of
uptake by macrophages than neutral liposomes. Therefore, it is
desirable to use neutral liposomes.
[0133] One group of technologies that allow very high drug
entrapment into small liposomes is based on gradient loading where
a pH gradient, ammonium sulfate gradient, or Mg-sulfate gradient
are used to load amine-drugs into liposomes: see U.S. Pat. Nos.
5,578,320 5,736,155 5,837,279 5,922,350 (pH gradient); U.S. Pat.
Nos. 5,837,282 5,785,987 (Mg-sulfate gradient); and U.S. Pat. No.
5,316,771 (ammonium sulfate gradient). These techniques only work
for membrane permeable amines (mono-amines where neutral form is
permeable like doxorubicin and daunorubicin). Gradient loading will
not work for the certain antiinfectives such as aminoglycosides as
they are impermeable (too large and too highly charged).
[0134] All processes described herein can be easily adapted for
large scale, aseptic manufacture. The final liposome size can be
adjusted by modifying the lipid composition, concentration,
excipients, and processing parameters.
[0135] The lipid to drug ratio using the processes of the present
invention is about 4:1 to about 1:1. In another embodiment, the
lipid to drug ratio is about 3:1 to about 1:1, 2:1 to about 1:1,
about 1:1 or less, about 0.75:1 or less, or about 0.5:1 or less.
Further the percentage of free antiinfective, present after the
product is dialyzed for a particular duration, is decreased.
8. Results
[0136] 8.1 Biofilm Barriers of Pulmonary Infections
[0137] An obstacle to treating infectious diseases such as
Pseudomonas aeruginosa, the leading cause of chronic illness in
cystic fibrosis patients is drug penetration within the
sputum/biofilm barrier on epithelial cells (FIG. 1). In FIG. 1, the
donut shapes represent a liposomal antiinfective formulation, the
"+" symbol represents free antiinfective, the "- symbol mucin,
alginate and DNA, and the solid bar symbol represents Pseudomonas
aeruginosa. This barrier is composed of both colonized and
planktonic P. aeruginosa embedded in alginate or exopolysaccharides
from bacteria, as well as DNA from damaged leukocytes, and mucin
from lung epithelial cells, all possessing a net negative charge
(Costerton, et al., 1999). This negative charge binds up and
prevents penetration of positively charged drugs such as
aminoglycosides, rendering them biologically ineffective (Mendelman
et al., 1985). Entrapment of antiinfectives within liposomal or
lipid formulations could shield or partially shield the
antiinfectives from non-specific binding to the sputum/biofilm,
allowing for liposomal or lipid formulations (with entrapped
aminoglycoside) to penetrate (FIG. 1).
[0138] Amikacin has been shown to have a high degree of resistance
to bacterial enzymes, thus providing a greater percent of
susceptible clinical isolates than found for other aminoglycosides
including tobramycin and gentamicin (Price et al., 1976). In
particular, P. aeruginosa isolates are far more sensitive to
amikacin than other aminoglycosides while exhibiting no
cross-resistance (Damaso et al., 1976).
[0139] The sustained release and depot effect of liposomal
formulations of amikacin is clearly seen in FIG. 2. In this study
rats were given tobramycin via intratracheal and intravenous
administration. The rats were also given liposomal formulations of
amikacin intratracheally at the same dose (4 mg/rat). The data show
that it is only with the liposomal formulation of amikacin that a
sustained release and depot effect is achieved. In fact, 24 hours
after dosing, only liposomal formulations of amikacin show
significant levels of the drug in the animal's lungs, while both
tobramycin formulations revealed negligible levels, primarily due,
it is believed to rapid systemic absorption. This greater than a
hundred-fold increase of aminoglycoside in the lung for liposomal
antiinfective formulations supports the idea of a sustained release
liposomal formulation antiinfective that can be taken significantly
less often than the currently approved TOBI.RTM. formulation (a
tobramycin inhalation solution made by the Chiron Corporation,
Ameryville, Calif.).
[0140] Moreover, the presence of a sputum/biofilm prevents the
penetration of the free aminoglycosides due to binding of the
antiinfectives to its surface (FIG. 1). Therefore, doses in excess
of 1,000 gm of tobramycin/gram of lung tissue are needed to show a
therapeutic effect in CF patients. This is overcome with liposomal
formulations of amikacin. Thus, the therapeutic level of drug is
maintained for a longer period of time in the liposomal
formulations of amikacin compared to free tobramycin. This
facilitation of binding and penetration could also be a means by
which liposomal formulations of amikacin could significantly reduce
bacterial resistance commonly seen to develop when antibacterials
are present in vivo at levels below the minimum inhibitory
concentration.
[0141] 8.2 Pharmacokinetics
[0142] The pharmacokinetics of amikacin was determined in rats
following intratracheal (IT) administration of either free
tobramycin or liposomal formulations of amikacin. These data were
compared to the distribution obtained in the lungs following a tail
vein injection of free tobramycin. In all cases a dose of 4 mg/rat
was administered. As can be seen in FIG. 2, a much larger
deposition of aminoglycoside can be delivered by IT compared to
injection. The depot effect of liposomal antiinfective technology
is also demonstrated in that in comparison to tobramycin given
either IT or IV, a greater than a hundred-fold increase in drug for
liposomal formulations of amikacin still remains in the lungs
twenty-four hours following administration. Thus, the therapeutic
level of drug is maintained for a longer period of time in the
liposomal formulations of amikacin compared to free tobramycin.
[0143] The binding of aminoglycosides to sputum of CF patients is a
concern, particularly if this binding reduces the bioactivity of
the antiinfective (Hunt et al., 1995). To determine whether
liposomal formulations of amikacin can retain biological activity
over a prolonged period of time, normal rats were administered
liposomal formulations of amikacin by intratracheal instillation.
This was followed by its removal at 2 or 24 hours via a bronchial
alveolar lavage (BAL) to determine biological activity. Samples
were concentrated by ultrafiltration followed by filtration (0.2
micron) to remove contaminating lung microbes. Amikacin
concentration was determined employing a TDX instrument and
biological activity determined using a Mueller Hinton broth
dilution assay (Pseudomonas aeruginosa). The results are shown in
Table 7.
TABLE-US-00007 TABLE 7 Results showing that liposomal formulations
of amikacin retain biological activity over a prolonged period of
time. Time amikacin in BAL amikacin in filtrate MIC (hours)
(.mu.g/mL) (.mu.g/mL) (.mu.g/mL) 2 160 119 1.9 24 73 32 4.0
[0144] As shown by the above table, the recovered filtered
liposomal formulation of amikacin was capable of killing P.
aeruginosa in a Mueller Hinton broth assay even after 24 hours with
an MIC of 4. At 2 hours an MIC of 2 was obtained, which is similar
to that obtained for the filtered liposomal/complexed amikacin
stock. Thus, the liposomal formulation of amikacin was still active
following 24 hours in the lung. At 24 hours free tobramycin at the
same dose was undetectable in a BAL. This indicates that not only
is the liposomal antiinfective formulation retained in the lung,
but it is also freely available to penetrate a sputum/biofilm over
time. These data combined with the facts as evident in FIG. 2 and
Table 9 (below), that liposomal formulations of amikacin release
the free antiinfective over time while maintaining high levels of
the antiinfective in the lungs, supports the rationale that this
system may yield a sustained antiinfective effect over time. This
effect should prove significant in reducing both the bio-burden of
the Pseudomonas and the development of resistance due to trough
levels of antiinfective.
[0145] As an in vitro demonstration of slow release of liposomal
formulation of amikacin and its sustained antiinfective effect, the
formulation was incubated in sputum from patients with Chronic
Obstructive Pulmonary Disease (COPD) containing PAOI mucoid
Pseudomonas. The liposomal formulation of amikacin was also
incubated in alginate containing PAO1 mucoid Pseudomonas. In both
cases sustained and enhanced killing of the Pseudomonas over time
was observed, as shown in Table 8.
TABLE-US-00008 TABLE 8 In vitro killing of Pseudomonas over time.
In vitro Sputum/Alginate Assay (% survival of PA01 Mucoid
Pseudomonas) Incubation time at 37.degree. C. Amikacin conc. 1 h 3
h 6 h 24 (.mu.g/mL) Lip-An-15 Sputum 81 15 22 <1 8 Lip-An-15
Alginate 100 59 1 <1 10
Classical kill curves are not applicable for liposomal
antiinfective formulation technology because the liposomal
formulations exhibit a slow release of antiinfective with an
enhanced antiinfective effect. The liposomal formulation protects
the amikacin from the sputum and/or alginate until its release. In
time, complete killing is observed, consistent with slow release
sustained antiinfective effect model with no interference or
inactivation of antiinfective.
[0146] The efficacy of liposomal amikacin formulations was studied
using a model for chronic pulmonary infection (Cash et al., 1979)
where P. aeruginosa, embedded in an agarose bead matrix, was
instilled in the trachea of rats. This mucoid Pseudomonas animal
model was developed to resemble the Pseudomonas infections seen in
CF patients. Some of the clinical correlates to CF include: a
similar lung pathology; the development of immune complex
disorders; and a conversion to the mucoid phenotype by P.
aeruginosa strains (Cantin and Woods, 1999). Rat lungs were
infected with over 10.sup.7 CFUs of a mucoid Pseudomonas (strain
PAO1) taken from a CF patient isolate, and subsequently treated
with (a) free aminoglycoside, (b) the lipid vehicle alone as
non-drug control, and (c) liposomal amikacin formulation. In
addition, formulations were first screened on the ability to kill
in vitro P. aeruginosa on modified Kirby-Bauer plates.
[0147] Various liposomal amikacin formulations were tested based on
either different lipid compositions or manufacturing parameters
resulting in different killing zones in in vitro experiments. This
experiment was designed to determine the increase in efficacy
obtained with liposomal aminoglycoside formulations over free
aminoglycoside. Blank control lipid compositions, two different
liposomal amikacin formulations and free amikacin and free
Tobramycin at the same aminoglycoside concentrations as the
liposomal antiinfective formulations were compared. In addition, a
10 fold higher dose of free amikacin and a 10 fold higher dose of
free tobramycin were also given. Dosing was IT daily over seven
days. Results (FIG. 3) indicate that liposomal amikacin in the two
formulations (differing in lipid composition) revealed a
significant reduction in CFU levels and were better at reducing
CFUs than free amikacin or free tobramycin at 10-fold
higher-dosages. In FIG. 3, Lip-An-14 is DPPC/Chol/DOPC/DOPG
(42:45:4:9) and 10 mg/mL amikacin, Lip-An-15 is DDPC/Chol (1:1)
also at 10 mg/mL. All lipid-lipid and lipid-drug ratios herein are
weight to weight.
[0148] The next experiment (FIG. 4) was designed to demonstrate the
slow release and sustained antiinfective capabilities of liposomal
amikacin formulations. The dosing was every other day for 14 days,
as opposed to every day for seven days as in the previous
experiments. Results indicate that liposomal amikacin in the two
formulations (differing in lipid composition) had a 10 to 100 times
more potent (greater ability to reduce CFU levels) than free
amikacin or free tobramycin. A daily human dose of 600 mg TOBI.RTM.
(a tobramycin inhalation solution made by the Chiron Corporation,
Ameryville, Calif.), or about 375 mg/m.sup.2, corresponds to a
daily rat dose of 9.4 mg. Thus the data can be directly correlated
to a 10 to 100 fold improvement in human efficacy. It should be
noted that a two-log reduction is the best that can be observed in
this model. A 100-fold reduction in P. aeruginosa in sputum assays
has been correlated with improved pulmonary function (Ramsey et
al., 1993). The sustained release of the liposomal amikacin
formulations indicate that a lower dose and/or less frequent dosing
can be employed to obtain a greater reduction in bacterial growth
than can be obtained with free aminoglycoside.
[0149] The efficacy of liposomal amikacin formulation was studied
in a model for chronic pulmonary infection where P. aeruginosa was
embedded in an agarose bead matrix that was instilled via the
trachea of Sprague/Dawley rats. Three days later free amikacin or
liposomal amikacin was dosed every day (FIG. 3) or every other day
(FIG. 4) at 1 mg/rat or 10 mg/rat of the given aminoglycoside or 1
mg/rat liposomal amikacin, as well as with blank liposomes (lipid
vehicle) as the control, with five rats per group.
[0150] The homogenized rat lungs (frozen) following the 14 day
experiment were analyzed for aminoglycoside content and activity.
The clinical chemical assay was performed using a TDX instrument
while the bioassay was performed by measuring inhibition zones on
agar plates embedded with Bacillus subtilis. The results are shown
in Table 9:
TABLE-US-00009 TABLE 9 Results from liposomal amikacin formulation
treated rat lungs infected with P. aeruginosa. Bioassay Clinical
Assay Formulation (microgram/mL) (microgram/mL) Lip-An-14 (1
mg/rat) 9.5 9.1 Lip-An-15 (1 mg/rat) 21.5 18.4 Free amikacin (10
mg/rat) nd 2.0 Free tobramycin (10 mg/rat) nd 1.4
Drug weights are for the drug normalized to the absence of any salt
form.
[0151] The Table 10 results indicate that aminoglycoside is present
and active for both liposomal antiinfective formulations, while
little can be detected for the free aminoglycoside even at the
10-fold higher dose. These further results establish the sustained
release characteristics of liposomal antiinfective formulations,
and also confirm that that antiinfective which remains is still
active. Of the above formulations only the free tobramycin (0.1
microgram/mL) exhibited any detectable levels of aminoglycoside in
the kidneys.
[0152] The sustained release and depot effect of liposomal amikacin
formulation is further demonstrated in FIG. 5. Rats were given a
chronic pulmonary infection where P. aeruginosa was embedded in an
agarose bead matrix that was instilled via the trachea, using the
same beads employed in the efficacy studies. The rats were then
given free tobramycin or liposomal amikacin (formulation Lip-An-14)
via intratracheal administration at the same dose (2 mg/rat). The
data, measured in microgram antiinfective per gram lung tissue over
time, show that liposomal antiinfective exhibits a sustained
release and depot effect while free tobramycin revealed negligible
levels in the lungs by 24 hours, primarily due it is believed to
rapid systemic absorption. This greater than a hundred-fold
increase of antiinfective in the lung for liposomal amikacin
formulations in an infected rat supports the idea of a sustained
release liposomal antiinfective that can be taken significantly
less often than the currently approved TOBI.RTM. formulation (a
tobramycin inhalation solution made by the Chiron Corporation,
Ameryville, Calif.).
[0153] The pharmacokinetics of amikacin was determined in rats
following intratracheal (IT) administration of either free
tobramycin or liposomal amikacin. A dose of 2 mg/rat was
administered. The depot effect of liposomal antiinfective
technology is demonstrated in that in comparison to free tobramycin
given IT, a greater than a hundred-fold increase in drug for
liposomal amikacin still remains in the infected lungs twenty-four
hours following administration. Thus, the therapeutic level of drug
is maintained for a longer period of time in the liposomal
formulations compared to free tobramycin.
[0154] FIG. 7 shows remarkable residence time and accumulation of
effective amounts of antiinfective in the lungs, a result that
establishes that relatively infrequent dosings can be used. Each
dose is 4 hr. by inhalation (in rat, 3 rats per group, as above) of
nebulized liposomal amikacin formulations (DPPC/Chol., 1:1) at 15
mg/mL amikacin. Dosing was at either day one; day one, three and
five; or day one, two, three, four and five. Rats providing a given
data bar were sacrificed after the respective dosing of the data
bar. The formulation is made as in the Example.
[0155] Similar anti-infectives can be utilized for the treatment of
intracellular infections like pulmonary anthrax and tularemia. In
pulmonary anthrax the anthrax spores reach the alveoli in an
aerosol. The inhaled spores are ingested by pulmonary macrophages
in the alveoli and carried to the regional tracheobronchial lymph
nodes or mediastinal lymph nodes via the lymphatics (Pile et al.,
1998; Gleiser et al., 1968). The macrophage is central in the both
the infective pathway and is the major contributor of host
self-destruction in systemic (inhalation) anthrax. In addition to
its attributes of sustained release and targeting, liposomal
antiinfective formulation technology can enhance cellular uptake
and can use alveolar macrophages and lung epithelial cells in drug
targeting and delivery. The possession of these characteristics is
believed to facilitate the treatment of these intracellular
infections, which infections occur in the lungs and are transported
by macrophages. More importantly, these characteristics should make
the antiinfective more effective in that the liposomal
antiinfective should be phagocytized by the very cells containing
the disease. The antiinfective would be released intracellularly in
a targeted manner, thereby attacking the infection before it is
disseminated. The encapsulated drug can be an already approved
pharmaceutical like ciprofloxacin, tetracycline, erthyromycin or
amikacin. Liposomal ciprofloxacin formulations have been
developed.
[0156] In a study, this compound was administered to mice and
compared to both free ciprofloxacin administered intratracheally
and free ciprofloxacin administered orally, with all three
compounds given at the same dose (FIG. 6). The dose for each mouse
was 15 mg/kg, with three mice per group. Liposomal ciprofloxacin
was in DPPC/Cholesterol (9:1), at 3 mg/mL ciprofloxacin, with the
formulation produced as in the Example. The lipid to drug ratio was
12.5:1 by weight. In comparison to orally administered
ciprofloxacin, liposomal ciprofloxacin was present in the mice
lungs at amounts over two orders of magnitude higher than free
ciprofloxacin. Moreover, only liposomal ciprofloxacin showed levels
of drug in the lung after 24 hours, while the orally administered
drug was undetectable in less than two hours. This data supports
the use of liposomal ciprofloxacin formulations and other
antiinfectives like aminoglycosides, tetracyclines and macrolides
for the treatment and for the prophylactic prevention of
intracellular diseases used by bioterrorists.
[0157] 8.3 Liposome Parameters
[0158] The lipids to be employed are dissolved in ethanol to form a
lipid-ethanol solution. The lipid-ethanol solution is infused in an
aqueous or ethanolic solution containing the molecule of the
bioactive agent to be entrapped. The lipids spontaneously form
vesicles.
[0159] Table 10 discloses liposomal antiinfective formulation
parameters where the lipids are DPPC and cholesterol.
TABLE-US-00010 TABLE 10 Additional liposomal antiinfective
formulation parameters. Amikacin Liposomes (DPPC/Chol) % of Total
Liposome [Total [Total Amikacin Mean Amikacin] Lipid]* that is LID
Diameter Batch # mg/mL mg/mL Entrapped (w/w)** (.mu.m) 1 14.7 44.8
96.7 3.2 -- 2 21.4 71.3 98.1 3.4 0.36 3 18.5 46.6 90.2 2.8 0.27 4
9.4 40.6 95.0 4.5 0.34 5 15.8 52.3 97.7 3.4 0.27 6 20.7 31.8 95.5
1.6 0.25 7 20.6 40.0 98.6 2.0 0.25 8 19.9 40.7 98.3 2.1 0.28 9 20.9
40.5 98.1 2.0 0.28 *DPPC/Cholesterol liposomes where the DPPC/Chol
mole ratio is approximately 1:1. **Only the entrapped amount of
amikacin was considered in calculating L/D.
[0160] Further information on forming liposomal antiinfective
formulations can be found in PCT/US03/06847, filed Mar. 5, 2003,
which is incorporated herein by reference in its entirety.
[0161] Entrapped volume is a basic characteristic of a liposomal
formulation and is determined as the volume of intraliposomal
aqueous phase per unit of lipid. It is generally expressed in the
units of .mu.liters/.mu.mole. One often assumes that when liposomes
are formed the concentration of the solute inside liposomes is
equal to that outside in the bulk solution. A higher entrapped
volume then would lead to higher drug/lipid ratio, i.e., a higher
overall drug concentration for the final formulation.
[0162] In formulating liposomal amikacin, however, it has been
found that the actual drug/lipid ratio that can be produced was
more than 3-fold higher that one would expect based on the
entrapped volume. Table 11 shows the results for 4 different sample
preparations of lipid antiinfective formulations (see Example 2 in
the Exemplification section).
Table 11. Amikacin Loading into Liposomes Prepared by Different
Methods.
TABLE-US-00011 TABLE 11 Amikacin loading into liposomes prepared by
different methods. Sample # Measured Parameter 1 2 3 4 Lipids
concentration (mg/mL) 35.1 39.5 50.4 45.0 AMK concentration (mg/mL)
19.9 20.7 10.5 5.0 Actual Lipid/Drug (w/w) 1.8 1.9 4.8 9.0
Entrapped volume (ul/umole) 2.4 2.5 2.9 1.6 Expected Lipid/Drug
(w/w) 5.6 6.0 4.1 8.1 Expected/Actual L/D ratio 3.19 3.17 0.85 0.90
Liposome Size (urn) 0.230 0.217 4.65 3.96
Samples 1 and 2 were made by the ethanol infusion procedure
disclosed herein, and Samples 3 and 4 were made by liposome
formation techniques known in the art.
[0163] Concentrations of amikacin were measured by
immuno-fluorescent assay using INNOFLUO Seradyn reagent set on TDx
analyzer. Lipids were measured by reverse-phase HPLC using C-8
column and Light scattering detector.
[0164] Liposomal volume (volume occupied by liposomes per unit of
total volume) in samples #1-3 was determined by measuring the
concentration of the fluorescent probe (Sulforhodamine 101 or
Carboxyfluorescein) in the total volume and in the filtrate volume
of the formulation obtained by centrifugation in CentriSart
filtering device. Probe concentration in the filtrate is higher
that the average one due to exclusion of the probe from the volume
occupied by liposomes.
[0165] In sample #4, liposomal volume was determined by measuring
the concentration of Potassium ion in a sample after adding fixed
amount of it, 250 ul of KCl (V.sub.add) into 10 mL liposomal
suspension (V.sub.o). Samples were then centrifuged 30 min at 4000
rpm and a supernatant was taken to measure potassium ion (K) by
Cole-Parmer potassium-sensitive electrode. Potassium concentration
measured was always higher than expected due to exclusion of
potassium ions from the volume occupied by liposomes. In the
control, an equal amount of KCl was added into 10 mL saline
solution. Potassium concentration in control K.sub.c was
measured.
[0166] Aqueous and liposomal volumes were than estimated as:
v a = K c K ( V 0 + V add ) - V add , v L = 1 - v a .
##EQU00001##
[0167] Knowing the liposomal volume and the lipid concentration one
can determine the entrapped volume:
v ent = v L - L w L m , ##EQU00002##
where L.sub.w and L.sub.m are the weight and molar lipid
concentrations, respectively. Lipid density is assumed to be close
to 1 mg/mL. Consequently, one can estimate expected Lipid/Drug
ratio that the sample would have if the drug was distributed
ideally in the aqueous spaces inside and outside liposomes:
( L D ) ex = L w D 0 ( v L - L w ) = M L D 0 v ent , ,
##EQU00003##
where D.sub.o is the bulk concentration of the drug during liposome
formation, M.sub.L is the average molecular weight of lipids.
[0168] As one can see, actual L/D ratios for samples #1 and #2 (1.8
and 1.9) are consistently lower than one would expect from even
distribution of amikacin (5.6 and 6.0), while LTD's for samples #3
and #4 are closer to theoretical values.
[0169] A similar comparison was made between 2 sample preparations
of a lipid antiinfective formulations where gentamicin sulfate was
the antiinfective (see Example 3 in the Exemplification section).
The data in Table 12 indicate that the method disclosed herein
provides unexpectedly high entrapment of gentamicin. In both
samples #5 and #6, the actual Lipid/Drug ratios were almost twice
the theoretically expected value.
TABLE-US-00012 TABLE 12 Gentamicin loading into liposomes prepared
by different methods. Sample # Measured Parameter 5 6 Lipids
concentration (mg/mL) 44.8 41.8 Drug concentration (mg/mL) 14.2
14.9 Actual Lipid/Drug (w/w) 3.2 2.8 Entrapped volume (ul/umole)
2.3 2.7 Expected Lipid/Drug (w/w) 5.7 5.4 Expected/Actual L/D ratio
1.82 1.92 Liposome Size (urn) 0.226 0.236
[0170] 8.4 Drug Release Mediated by P. aeruginosa Infection
[0171] Release of drug in an active form in the vicinity of the
infections is an important aspect of the action of liposomal drug
formulation of the present invention. The potential for such
targeted release was tested by monitoring the release of drug upon
incubation with sputum from a CF patient, release in the lungs of
rats pre-inoculated with P. aeruginosa, as well as the activity of
against cultures of P. aeruginosa.
[0172] The release of amikacin by direct incubation of a culture of
P. aeruginosa with a liposomal amikacin formulation of the present
invention was previously discussed. To further investigate this
phenomenon, a liposomal amikacin formulation was incubated with a
preparation of sputum from a cystic fibrosis patient with P.
aeruginosa infection. Expectorated sputum was liquefied with bovine
DNase I and alginate lyase for 2 hr. at 37.degree. C. A liposomal
amikacin formulation or soluble amikacin (1 mg/mL amikacin) was
mixed 1:1: with liquefied sputum or control and incubated at
37.degree. C. with gentle shaking. Aliquots were analyzed for
amikacin concentration by Abbott TDx Analyzer. Intact liposomes
were lysed in a separate aliquot of each sample using a detergent,
1% Triton X-100. Supernatants from each sample were used for
analysis. Over the period of 48 hours, (80-90%) of the amikacin was
released in a time-dependent manner from the lipid composition
under these conditions, indicating that drug release may occur at
the sites of infection in the CF lung.
[0173] Release of free drug from liposomes in vivo was compared for
rats that had been instilled with agar beads containing P.
aeruginosa (3.5.times.10.sup.4 CFU/rat) versus those that had not.
Three days after bead instillation, rats were allowed to inhale
liposomal amikacin formulations of the present invention (approx. 6
mg/kg daily dose) every day (no bacteria group) or every other day
for 14 days (group instilled with beads). 24 hours after the last
treatment, the total amikacin and free amikacin were measured as
described above. In rats that had received bacteria, an average of
approximately 50-70% of the detected amikacin was in the free form,
i.e. released from the liposome. In the rats that had not received
bacteria approximately 20-25% of the drug was in free form. These
data strongly suggest that release of free amikacin from the
liposome may be mediated by the presence of P. aeruginosa in
vivo.
[0174] An in vitro test of release and activity was performed under
conditions similar to the pharmacokinetics in the lung, where it
has been previously shown that free antibiotic is cleared on the
time scale of a few hours. Free amikacin or a liposomal amikacin
formulation was incubated with P. aeruginosa PA01
(.about.10.sup.8/mL) in sterile 0.5 mL Slide-A-Lyzer cartridges at
varying drug concentrations. Free drug dialyzes out of the
cartridges on the time scale of hours under these conditions. After
24 hrs., the samples were withdrawn from the cartridges and plated
to measure CFU. In the preliminary experiments free amikacin only
slightly reduced the CFU of these samples, while a two log
reduction of CFUs was observed for amikacin comprising lipid
compositions at the same amikacin concentration (50 .mu.g/mL).
These data suggest that amikacin is indeed released in an active
form in the presence of bacteria and that the slow release afforded
by the formulation makes more effective use of the drug.
[0175] The interaction of the liposomal amikacin formulations of
the present invention with P. aeruginosa or its virulence factors
leads to release of amikacin possibly directing release to the site
of infection. When amikacin is released it is active against P.
aeruginosa, and the slow release in the vicinity of the bacteria
may have an advantage over the non-specific distribution and rapid
clearance of inhaled free drug.
[0176] 8.5 Effect of Inhaled Liposomal Drug Formulations on the
Function of Alveolar Macrophages
[0177] The liposomal amikacin formulations of the present invention
are in one embodiment a nanoscale (200-300 nm)
liposome-encapsulated form of amikacin that is formulated to treat
chronic P. aeruginosa infections in cystic fibrosis patients. It is
designed for inhalation with sustained release of amikacin in the
lung. Because alveolar macrophages are known to avidly take up
particles in this size range, the effect of the liposomal
formulations on these cells is of particular interest. The basal
and stimulated functions of rat alveolar macrophages obtained by
lavage were studied with and without administration of liposomal
amikacin formulations and compared to various controls.
[0178] Aerosols of the liposomal amikacin formulations, amikacin,
placebo liposomes and saline were generated with a PARI LC Star
nebulizer and inhaled by CD IGS female rats in a nose-only
inhalation chamber. Inhalation therapy was conducted for 4 hr for
14 consecutive days, such that the estimated daily lung dose of
total lipid was approximately 12 mg/kg for the liposomal amikacin
group and 11 mg/kg for the placebo liposome group. Half the rats
were euthanized on day 15. The remaining rats were euthanized on
day 43. Bronchial alveolar lavage fluid (BALF) was collected from
each rat and stored at -80.degree. C. for subsequent assay of
nitric oxide (as represented by total nitrates) and tumor necrosis
factor alpha (TNF-.alpha.). The cells from the BALF were collected
by centrifugation, counted and cultured in medium with and without
lipopolysaccharide (LPS) for 24 hr. The supernatants from these
cultures were collected by centrifugation and assayed for nitric
oxide and TNF-.alpha.. The phagocytic function of BAL macrophages
((10.sup.6)/mL) was tested by measuring the overnight uptake of
opsonized fluorescent microspheres (0.2 .mu.m, 2
(10.sup.9)/mL).
[0179] Inhalation of the liposomal amikacin formulation, empty
liposomes, soluble amikacin, or saline for 14 consecutive days did
not produce a significant acute or delayed inflammatory response in
the lungs of rats as evident by levels of nitric oxide (nitrates)
and TNF-.alpha. in BALF which were insignificantly different from
controls, although there was an early trend toward higher NO levels
in all groups receiving inhalants, including controls. The total
recovery of cells was insignificantly different in all groups with
an early trend toward more polymorphonuclear leukocytes in all
groups receiving inhalants. Rat alveolar macrophages had normal
functions after exposure to the aerosols of the above test articles
despite the fact that they appeared enlarged on day 15 in groups
inhaling liposomes. The concentrations of nitrates and TNF-.alpha.
detected upon culturing of alveolar macrophages in medium on day 15
or 43 of the study were insignificantly different from controls.
The macrophages responded normally when stimulated by LPS,
producing substantial concentrations of nitric oxide (20-40
nmol/10.sup.6 cells) and TNF-.alpha. (5-20 ng/10.sup.6 cells).
These macrophages also had normal phagocytic functions, as shown by
identical uptake of fluorescent beads compared to untreated
controls.
[0180] Inhalation of the liposomal amikacin formulations for 14
consecutive days did not substantially affect the function of
alveolar macrophages in terms of phagocytosis of opsonized beads,
production of inflammatory mediators TNF and NO.
9. Dosages
[0181] The dosage of any compositions of the present invention will
vary depending on the symptoms, age and body weight of the patient,
the nature and severity of the disorder to be treated or prevented,
the route of administration, and the form of the subject
composition. Any of the subject formulations may be administered in
a single dose or in divided doses. Dosages for the compositions of
the present invention may be readily determined by techniques known
to those of skill in the art or as taught herein.
[0182] In certain embodiments, the dosage of the subject compounds
will generally be in the range of about 0.01 ng to about 10 g per
kg body weight, specifically in the range of about 1 ng to about
0.1 g per kg, and more specifically in the range of about 100 ng to
about 50 mg per kg.
[0183] An effective dose or amount, and any possible affects on the
timing of administration of the formulation, may need to be
identified for any particular composition of the present invention.
This may be accomplished by routine experiment as described herein,
using one or more groups of animals (preferably at least 5 animals
per group), or in human trials if appropriate. The effectiveness of
any subject composition and method of treatment or prevention may
be assessed by administering the composition and assessing the
effect of the administration by measuring one or more applicable
indices, and comparing the post-treatment values of these indices
to the values of the same indices prior to treatment.
[0184] The precise time of administration and amount of any
particular subject composition that will yield the most effective
treatment in a given patient will depend upon the activity,
pharmacokinetics, and bioavailability of a subject composition,
physiological condition of the patient (including age, sex, disease
type and stage, general physical condition, responsiveness to a
given dosage and type of medication), route of administration, and
the like. The guidelines presented herein may be used to optimize
the treatment, e.g., determining the optimum time and/or amount of
administration, which will require no more than routine
experimentation consisting of monitoring the subject and adjusting
the dosage and/or timing.
[0185] While the subject is being treated, the health of the
patient may be monitored by measuring one or more of the relevant
indices at predetermined times during the treatment period.
Treatment, including composition, amounts, times of administration
and formulation, may be optimized according to the results of such
monitoring. The patient may be periodically reevaluated to
determine the extent of improvement by measuring the same
parameters. Adjustments to the amount(s) of subject composition
administered and possibly to the time of administration may be made
based on these reevaluations.
[0186] Treatment may be initiated with smaller dosages which are
less than the optimum dose of the compound. Thereafter, the dosage
may be increased by small increments until the optimum therapeutic
effect is attained.
[0187] The use of the subject compositions may reduce the required
dosage for any individual agent contained in the compositions
(e.g., the antiinfective) because the onset and duration of effect
of the different agents may be complimentary.
[0188] Toxicity and therapeutic efficacy of subject compositions
may be determined by standard pharmaceutical procedures in cell
cultures or experimental animals, e.g., for determining the
LD.sub.50 and the ED.sub.50.
[0189] The data obtained from the cell culture assays and animal
studies may be used in formulating a range of dosage for use in
humans. The dosage of any subject composition lies preferably
within a range of circulating concentrations that include the
ED.sub.50 with little or no toxicity. The dosage may vary within
this range depending upon the dosage form employed and the route of
administration utilized. For compositions of the present invention,
the therapeutically effective dose may be estimated initially from
cell culture assays.
10. Formulation
[0190] The lipid antiinfective formulations of the present
invention may comprise an aqueous dispersion of liposomes. The
formulation may contain lipid excipients to form the liposomes, and
salts/buffers to provide the appropriate osmolarity and pH. The
formulation may comprise a pharmaceutical excipient. The
pharmaceutical excipient may be a liquid, diluent, solvent or
encapsulating material, involved in carrying or transporting any
subject composition or component thereof from one organ, or portion
of the body, to another organ, or portion of the body. Each
excipient must be "acceptable" in the sense of being compatible
with the subject composition and its components and not injurious
to the patient. Suitable excipients include trehalose, raffinose,
mannitol, sucrose, leucine, trileucine, and calcium chloride.
Examples of other suitable excipients include (1) sugars, such as
lactose, and glucose; (2) starches, such as corn starch and potato
starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol, and
polyethylene glycol; (12) esters, such as ethyl oleate and ethyl
laurate; (13) agar; (14) buffering agents, such as magnesium
hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
EXEMPLIFICATION
Example 1
[0191] The following is a detailed description of the manufacture
of 150 mL of Liposomal/complexed amikacin.
Total Intial Volume=1.5 L
[0192] Ethanol Content=23.5% (v/v) Lipid Composition: DPPC/Chol
(1:1 mole ratio) Intial [Lipid]=7.6 mg/mL Intial [amikacin
sulfate]=57.3 mg/mL Final product Volume=150 mL
[0193] I) Compounding and Infusion:
[0194] 7.47 g DPPC and 3.93 g Cholesterol were dissolved directly
in 352.5 mL ethanol in a 50 C water bath. 85.95 g amikacin sulfate
was dissolved directly in 1147.5 mL PBS buffer. The solution is
then titrated with ION NaOH or KOH to bring the pH to approximately
6.8.
[0195] 352.5 mL ethanol/lipid was added or infused to the 1147.5 mL
amikacin/buffer to give a total initial volume of 1.5 L. The
ethanol/lipid was pumped @ -30 mL/min (also called infusion rate)
with a peristaltic pump into the amikacin/buffer solution which was
being rapidly stirred at 150 RPM in a reaction vessel on a stir
plate at room temperature
[0196] The product was stirred at room temperature for 20-30
minutes.
[0197] II) Diafiltration or "Washing" Step:
[0198] The mixing vessel was hooked up to a peristaltic pump and
diafiltration cartridge. The diafiltration cartridge is a hollow
membrane fiber with a molecular weight cut-off of 500 kilodaltons.
The product was pumped from the reaction vessel through the
diafiltration cartridge and then back into the mixing vessel at
room temperature. A back pressure of approximately 7 psi is created
throughout the cartridge. Free amikacin and ethanol was forced
through the hollow fiber membrane by the back pressure leaving the
liposomal amikacin (product) behind. The product was washed 8 times
at room temperature. Fresh PBS buffer was added (via another
peristaltic pump) to the reaction vessel to compensate for the
permeate removal and to keep a constant product volume.
[0199] The product was concentrated.
Example 2
[0200] High Liposomal Entrapment of Amikacin.
[0201] Four samples of lipid antiinfective formulations were
prepared at various lipid and antiinfective concentrations
according to the following procedures.
[0202] Sample #1. Amikacin sulfate 1.72 kg was dissolved in 23
Liters saline solution (0.9% NaCl) and pH was adjusted to 6.5 by
adding necessary amount NaOH. Lipids -98.2 g DPPC and 51.8 g
Cholesterol were dissolved in 7 liters ethanol. Liposomes were
formed by infusion of lipid solution into amikacin solution at a
rate of .about.600 mL/min and under constant stirring. Resulting
suspension was then washed to remove ethanol and un-entrapped
amikacin by diafiltration using an Amersham Hollow Fiber cartridge
500 kD pore size. The suspension was concentrated to a final volume
of -3.5 L.
[0203] Sample #2. The procedure was similar to that for sample #1
with all material quantities scaled down 100 fold. Amikacin sulfate
17.2 g was dissolved in 230 mL saline solution (0.9% NaCl) and pH
was adjusted to 6.6 by adding necessary amount NaOH. Lipids -0.982
g DPPC and 0.518 g Cholesterol were dissolved in 70 mL ethanol.
Liposomes were formed by infusion of the lipid solution into the
amikacin solution at a rate of .about.300 mL/min and under constant
stirring. The resulting suspension was then washed to remove
ethanol and un-entrapped amikacin by diafiltration using an
Amersham Hollow Fiber cartridge. The suspension was concentrated to
a final volume of .about.35 mL.
[0204] Sample #3. Liposomes were made by a procedure known as SPLV.
Amikacin sulfate 1.4 g was dissolved in 20 mL saline solution (0.9%
NaCl) making pH 3.3. Lipids, 0.666 g DPPC and 0.333 g Cholesterol
were dissolved in 40 mL dichloromethane. Amikacin and lipid
solutions were mixed together in a 500 mL round flask and briefly
sonicated to form an emulsion. Flask was then connected to a BUCHI
Rotavapor system to remove dichloromethane at low vacuum (-5 inches
Hg) and temperature 50.degree. C. and constant rotation until the
amikacin-lipid mixture formed a gel. When the gel eventually
collapsed, vacuum was gradually increased to -20 inches Hg and
drying continued for 30 more minutes. The final volume of formed
liposomal suspension was 22 mL.
[0205] Sample #4. The procedure was similar to that for sample #3.
Amikacin sulfate 1.3 g was dissolved in 20 mL of saline solution,
and pH was adjusted to 6.5 by adding NaOH. Lipids, 0.583 g DPPC and
0.291 g Cholesterol were dissolved in 35 mL dichloromethane. The
sonication step was skipped. The solvent removal step on the
Rotavapor system was carried out at 40.degree. C. for 2 hr. Final
volume was 20 mL.
Example 3
High Liposomal Entrapment of Gentamicin.
[0206] Sample #5. Gentamicin sulfate 20.0 g was dissolved in 230 mL
saline solution (0.9% NaCl) and pH was adjusted to 6.5 by adding
necessary amount of sulfuric acid. Lipids -0.982 g DPPC and 0.518 g
Cholesterol were dissolved in 70 mL ethanol. Liposomes were formed
by infusion of lipid solution into gentamicin solution at a rate of
.about.500 mL/min and under constant stirring. Un-entrapped
gentamicin and ethanol were removed by diafiltration using an
Amersham Hollow Fiber cartridge. The suspension was concentrated to
a final volume of .about.35 mL.
[0207] Sample #6. The procedure was similar to that for sample #5,
except: Gentamicin sulfate 17.0 g was dissolved in 230 mL
Na.sub.2SO.sub.4 100 mM solution and pH was adjusted to 6.5 by
adding necessary amount of H.sub.2SO.sub.4. Lipids -0.982 g DPPC
and 0.518 g Cholesterol were dissolved in 75 mL ethanol.
Example 4
Entrapment of Other Salt Forms of Amikacin.
[0208] Sample #7. The procedure was similar to that for sample #2
under Example 2. Amikacin base 10.7 g and Citric acid 4.2 g were
dissolved in 230 mL saline solution (0.9% NaCl). pH of resulted
amikacin-citrate solution was 6.2. Lipids -0.982 g DPPC and 0.518 g
Cholesterol were dissolved in 70 mL ethanol. Liposomes were formed
by infusion of lipid solution into amikacin solution at a rate of
.about.500 mL/min and under constant stirring. Un-entrapped
amikacin and ethanol were removed by diafiltration using an
Amersham Hollow Fiber cartridge. The suspension was concentrated to
a final volume of .about.35 mL.
[0209] The actual Lipid/Drug ratio was similar to that for sample
#2 and again lower than expected (Drug entrapment higher than
expected). Considering the fact that the entrapped volume in the
sample #7 was only 1.5 (compared to 2.5 for sample #2), the
Expected/Actual L/D ratio was as high as 5.2. Thus, liposomal
amikacin citrate, like amikacin sulfate, can also be formulated
with high entrapment.
TABLE-US-00013 TABLE 13 Samples 5-7 parameter summary. Sample #
Measured Parameter 5 6 7 Lipids concentration (mg/mL) 44.8 41.8
41.7 Drug concentration (mg/mL) 14.2 14.9 17.8 Actual Lipid/Drug
(w/w) 3.2 2.8 2.3 Entrapped volume (ul/umole) 2.3 2.7 1.5 Expected
Lipid/Drug (w/w) 5.7 5.4 12.2 Expected/Actual L/D ratio 1.82 1.92
5.20 Liposome Size (urn) 0.226 0.236 0.234
Example 5
[0210] Bioavailability of amikacin from inhaled liposomal amikacin
formulations in the rat.
[0211] The rate of release of amikacin from the liposomes was
measured after inhalation by rats and compared to inhaled soluble
amikacin.
[0212] The test items were aerosolized via a Pari LC Star nebulizer
attached to a nose-only inhalation chamber. CD.RTM.IGS rats
received an estimated lung deposited dose of 6 mg/kg of amikacin in
the form of a liposomal formulation or 5 mg/kg of soluble amikacin
as a single dose or dosed daily for 14 consecutive days. Lung or
other tissue was homogenized with a Polytron apparatus. The
kinetics of clearance of amikacin from the lung was examined by
analysis of lung homogenates at varying time points after the
single dose treatment or 1 day and 28 days after administration of
the multiple doses. Amikacin levels were measured by
immunofluorescence polarization on a Abbott TDx.RTM. analyzer in
the absence or presence of 1% Triton X-100, which releases amikacin
from liposomes. Whole lung samples were spiked with liposomes
before homogenization to test the release of amikacin under these
conditions. Free and total amikacin were measure with and without
1% Triton X-100 to assess leakage of drug.
[0213] Liposomal amikacin, spiked into whole lung samples, showed
no significant release of amikacin as a result of tissue
homogenization with the Polytron homogenizer in the absence of this
detergent. However, addition of 1% Triton X-100 led to recovery of
all of the expected drug. Therefore a direct comparison could be
made of the total level of amikacin (with detergent) versus the
freely available levels in lung tissue.
[0214] A high total concentration of amikacin (approx. 500-600
.mu.g/g of lung tissue) was observed immediately after the 6 mg/kg
single dose of the liposomal amikacin, which slowly decreased by
about 50% over a 7 day period. The temporal profile for the release
of free amikacin from these liposomes showed an initial high
concentration of free drug, probably resulting from amikacin
liberated as a result of nebulization. This phase was followed by a
nadir at about 24 hours and a subsequent increase, reaching a
maximum of 279 .mu.g/g at 96 hours after administration. By the end
of the 7 day experiment, a substantial portion of drug remaining in
the lung was in the free form (approximately 50-70%). It appeared
that a small portion of the soluble drug administered by inhalation
also remained for a long period of time in the lung. However, most
of the amikacin administered in soluble form was cleared within
several hours, and the apparent free amikacin AUC over 7 days was
at least 2.times. higher for the liposomal amikacin animals than
for those that received soluble amikacin. Some aspects of this
behavior can be qualitatively modeled with appropriate rate
constants for clearance and slow release of drug from
liposomes.
[0215] After 14 consecutive days of administration (24 hours after
the last dose), more than 20% of the total amikacin in the lungs of
rats that received liposomal amikacin was present as free drug
(approximately 650 .mu.g/g). The total free drug level was even
greater than the amount in rats that inhaled soluble amikacin
(approx. 500 .mu.g/g).
[0216] Free amikacin is released slowly from the liposomes of the
liposomal amikacin formulations in the lungs of healthy animals
over a time scale of days. The free drug that is released has a
relatively long residence time in the lung as seen by a substantial
depot of free drug in the lungs.
Example 6
In-Line Infusion Process
[0217] The essence of the In-Line infusion process is that a stream
of lipid solution is mixed with a stream of antiinfective solution
"in-line" via, for example, a Y-connector which connects to a
length of tubing, termed a mixing tube, where further mixing can
occur. In this regard, this new process differs from the
`conventional` ethanol infusion process, where lipid solution is
infused as a stream into a bulk of amikacin solution.
Amikacin and Lipid Solutions Preparation.
[0218] Amikacin sulfate 12.0 g was dissolved in 200 mL water and pH
was adjusted to 6.5 by adding necessary amounts of 25% NaOH
solution. Lipids, 1.480 g DPPC and 0.520 g cholesterol, were
dissolved in a mixture of 60 mL ethanol and 10 mL water. These
amounts result in a 300 mL batch after infusion at a lipid/amikacin
flow rate of 300/500 mL/min, respectively. Volumes can be
proportionally adjusted for larger scale or if different flow rates
are desired.
[0219] The amikacin solution prepared according to above results in
approximately 40 mg/mL amikacin (per base) solution. The lipid
solution as presented was DPPC/Chol (mole ratio of 60/40) with a
total lipid of approximately 20 mg/mL solution (90% ethanol).
Lipids were heated to .about.40.degree. C. for faster
dissolution.
[0220] The exact amounts needed for a 300 mL batch are: amikacin
150 mL, lipid 90 mL, and 60 mL of additional saline (or water)
which is added after or during infusion to adjust final ethanol
concentration.
Manufacturing Procedure.
[0221] One embodiment of the infusion system is shown in FIG.
8.
[0222] Lipid and Amikacin solutions are mixed in-line using a
Y-connector (ID 3.2 mm, OD 6.4 mm) at flow rates .about.300/500
mL/min (i.e. .about.1/1.67 volume ratio instead of .about.1/3.35 in
the conventional process). A MasterFlex tube US 25 (ID 4.8 mm) was
used to deliver the lipid solution and a L/S 17 tube (ID 6.4 mm)
was used to deliver the amikacin solution. To obtain synchronous
flow rates, two pump heads with one MasterFlex drive were used.
According to the tube cross-section areas, the theoretical flow
rate ratio should be 4.8.sup.2/6.4.sup.2=0.562=1/1.78. When the
pump drive was set to 500 mL/min for Amikacin tube LS 17, the
measured flow rates were .about.300/500=1/1.67.
[0223] Since the lipid solution contains 90% ethanol, the in-line
mixture had .about.34% ethanol. To prevent amikacin precipitation,
NaCl solution can be added after or during infusion at a flow rate
100-200 mL/min (it is assumed that the liposomes are already formed
at this point). Thus the final mixture would have .about.27%
ethanol, of which all free amikacin is expected to be soluble.
[0224] Total liquid infusion flow rate, 800-1000 mL/min, is
comparable to the permeate flow rate when using two big
diafiltration cartridges. This makes it possible to do simultaneous
infusion and concentration by diafiltration.
[0225] The resulting liposome suspension was washed to remove free
amikacin by diafiltration using an Amersham hollow fiber cartridge
UFP-500-C-3MA (membrane area 140 cm.sup.2, fiber ID 0.5 mm). In the
first step, the suspension was concentrated to nearly half of the
original volume (150 mL). Then, during diafiltration to wash, the
suspension was re-circulated and fresh saline solution was fed into
the mixture at a rate of .about.6 mL/min in order to match the
permeate rate and thus maintain a constant volume. Diafiltration
continued until 4 times the suspension volume of the feeding saline
solution was dispensed (i.e., 4*150 mL=600 mL). This
diafiltration/washing procedure will be referred to as 4 "washes".
Finally, the suspension was concentrated (diafiltration without
saline input) to obtain the Final Product at a desired amikacin and
lipid concentration. The recirculation flow rate during the
diafiltration step was .about.350 mL/min, and during the final
concentration step it was gradually reduced to .about.150 mL/min in
order to maintain the inlet pressure below 10 PSI.
REFERENCES
[0226] 1. Veldhuizen, R., Nag, K., Orgeig, S. and Possmayer, F.,
The Role of Lipids in Pulmonary Surfactant, Biochim. Biophys. Acta
1408:90-108 (1998). [0227] 2. Hagwood, S., Derrick, M. and Poulain,
F., Structure and Properties of Surfactant Protein B, Biochim.
Biophys. Acta 1408:150-160 (1998). [0228] 3. Johansson, J.,
Structure and Properties of Surfactant ProteinC, Biochim. Biophys.
Acta 1408:161-172 (1998). [0229] 4. Ikegami, M. and Jobe, A. H.,
Surfactant Protein Metabolism in vivo, Biochim. Biophys. Acta
1408:218-225 (1998). [0230] 5. Couveur, P., Fattel, E. and
Andremont, A., Liposomes and Nanoparticles in the Treatment of
Intracellular Bacterial Infections, Pharm. Res. 8:1079-1085 (1991).
[0231] 6. Gonzales-Rothi, R. J., Casace, J., Straub, L., and
Schreier, H., Liposomes and Pulmonary Alveolar Macrophages:
Functional and Morphologic Interactions, Exp. Lung Res. 17:685-705
(1991). [0232] 7. Swenson, C. E., Pilkiewicz, F. G., and Cynamon,
M. H., Liposomal Aminoglycosides and TLC-65 Aids Patient Care
290-296 (December, 1991). [0233] 8. Costerton, J. W., Stewart, P.
S., and Greenberg, E. P., Bacterial Biofilms: A Common Cause of
Persistent Infections, Science 284:1318-1322 (1999). [0234] 9.
Cash, H. A., Woods, D. E., McCullough, W. G., Johanson, J. R., and
Bass, J. A., A Rat Model of Chronic Respiratory Infection with
Pseudomonas aeruginosa, American Review of Respiratory Disease
119:453-459 (1979). [0235] 10. Cantin, A. M. and Woods, D. E.
Aerosolized Prolastin Suppresses Bacterial Proliferation in a Model
of Chronic Pseudomonas aeruginosa Lung Infection, Am. J. Respir.
Crit. Care Med. 160:1130-1135 (1999). [0236] 11. Ramsey, B. W.,
Dorkin, H. L., Eisenberg, J. D., Gibson, R. L., Harwood, I. R.,
Kravitz, R. M., Efficacy of Aerosolized Tobramycin in Patients with
cystic Fibrosis. New England J. of Med. 328:1740-1746 (1993).
[0237] 12. Mendelman, P. M., Smith, A. L., Levy, J., Weber, A.,
Ramsey, B., Davis, R. L., Aminoglycoside Penetration, Inactivation,
and Efficacy in Cystic Fibrosis Sputum, American Review of
Respiratory Disease 132:761-765 (1985). [0238] 13. Price, K. E.,
DeFuria, M. D., Pursiano, T. A. Amikacin, an aminoglycoside with
marked activity against antibiotic-resistant clinical isolates. J
Infect Dis 134:S249261 (1976). [0239] 14. Damaso, D., Moreno-Lopez,
M., Martinez-Beltran, J., Garcia-Iglesias, M. C. Susceptibility of
current clinical isolates of Pseudomonas aeruginosa and enteric
gram-negative bacilli to Amikacin and other aminoglycoside
antibiotics. J Infect Dis 134:S394-90 (1976). [0240] 15. Pile, J.
C., Malone, J. D., Eitzen, E. M., Friedlander, A. M., Anthrax as a
potential biological warfare agent. Arch. Intern. Med. 158:429-434
(1998). [0241] 16. Gleiser, C. A., Berdjis, C. C., Hartman, H. A.,
& Glouchenour, W. S., Pathology of experimental respiratory
anthrax in Macaca mulatta. Brit. J. Exp. Path., 44:416-426
(1968).
INCORPORATION BY REFERENCE
[0242] Publications and references, including but not limited to
patents and patent applications, cited in this specification are
herein incorporated by reference in their entirety in the entire
portion cited as if each individual publication or reference were
specifically and individually indicated to be incorporated by
reference herein as being fully set forth. Any patent application
to which this application claims priority is also incorporated by
reference herein in the manner described above for publications and
references.
EQUIVALENTS
[0243] While this invention has been described with an emphasis
upon preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations in the preferred devices and
methods may be used and that it is intended that the invention may
be practiced otherwise than as specifically described herein.
Accordingly, this invention includes all modifications encompassed
within the spirit and scope of the invention as defined by the
claims that follow.
* * * * *