U.S. patent application number 15/277667 was filed with the patent office on 2017-01-19 for high delivery rates for lipid based drug formulations, and methods of treatment thereof.
The applicant listed for this patent is Insmed, Inc.. Invention is credited to Lawrence T. Boni, Xingong LI, Zhili Li, Vladimir Malinin, Brian S. Miller.
Application Number | 20170014342 15/277667 |
Document ID | / |
Family ID | 38582325 |
Filed Date | 2017-01-19 |
United States Patent
Application |
20170014342 |
Kind Code |
A1 |
Li; Zhili ; et al. |
January 19, 2017 |
HIGH DELIVERY RATES FOR LIPID BASED DRUG FORMULATIONS, AND METHODS
OF TREATMENT THEREOF
Abstract
Provided is a method of preparing lipid based drug formulations
with low lipid/drug ratios using coacervation techniques. Also
provided are methods of delivering such lipid based drug
formulations at high delivery rates, and methods of treating
patients with pulmonary diseases comprising administering such
lipid based drug formulations.
Inventors: |
Li; Zhili; (Kendall Park,
NJ) ; Boni; Lawrence T.; (Monmouth Junction, NJ)
; Miller; Brian S.; (Hamilton, NJ) ; Malinin;
Vladimir; (Plainsboro, NJ) ; LI; Xingong;
(Robbinsville, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Insmed, Inc. |
Bridgewater |
NJ |
US |
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|
Family ID: |
38582325 |
Appl. No.: |
15/277667 |
Filed: |
September 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12983659 |
Jan 3, 2011 |
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15277667 |
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11398859 |
Apr 6, 2006 |
7879351 |
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12983659 |
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11185448 |
Jul 19, 2005 |
7718189 |
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11398859 |
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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: |
A61P 31/06 20180101;
Y02A 50/473 20180101; A61P 43/00 20180101; A61P 31/00 20180101;
A61K 31/7036 20130101; A61K 31/7034 20130101; A61P 11/00 20180101;
Y02A 50/30 20180101; Y02A 50/406 20180101; A61K 9/0078 20130101;
A61K 9/5015 20130101; A61K 9/1617 20130101; A61P 31/04 20180101;
A61K 9/127 20130101; A61K 9/5123 20130101 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 9/127 20060101 A61K009/127; A61K 9/51 20060101
A61K009/51; A61K 31/7036 20060101 A61K031/7036 |
Claims
1.-25. (canceled)
26. A lipid antiinfective formulation comprising an antiinfective
complexed to or encapsulated by a lipid component, wherein the
lipid component is net neutral and comprises a phospholipid and a
sterol, and the lipid component to antiinfective weight ratio in
the formulation is 0.40:1 to 0.49:1.
27. The lipid antiinfective formulation of claim 26, wherein the
phospholipid is dipalmitoylphosphatidylcholine (DPPC).
28. The lipid antiinfective formulation of claim 26, wherein the
sterol is cholesterol.
29. The lipid antiinfective formulation of claim 27, wherein the
sterol is cholesterol.
30. The lipid antiinfective formulation of claim 26, wherein the
lipid component is present as liposomes and the antiinfective is
encapsulated by the liposomes.
31. The lipid antiinfective formulation of claim 27, wherein the
lipid component is present as liposomes and the antiinfective is
encapsulated by the liposomes.
32. The lipid antiinfective formulation of claim 28, wherein the
lipid component is present as liposomes and the antiinfective is
encapsulated by the liposomes.
33. The lipid antiinfective formulation of claim 29, wherein the
lipid component is present as liposomes and the antiinfective is
encapsulated by the liposomes.
34. The lipid antiinfective formulation of claim 26, wherein the
antiinfective is 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, a penicillin, a cephalosporin,
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 a combination thereof.
35. The lipid antiinfective formulation of claim 33, wherein the
antiinfective is 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, a penicillin, a cephalosporin,
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 a combination thereof.
36. The lipid antiinfective formulation of claim 26, wherein the
antiinfective is an aminoglycoside.
37. The lipid antiinfective formulation of claim 27, wherein the
antiinfective is an aminoglycoside.
38. The lipid antiinfective formulation of claim 28, wherein the
antiinfective is an aminoglycoside.
39. The lipid antiinfective formulation of claim 29, wherein the
antiinfective is an aminoglycoside.
40. The lipid antiinfective formulation of claim 30, wherein the
antiinfective is an aminoglycoside.
41. The lipid antiinfective formulation of claim 31, wherein the
antiinfective is an aminoglycoside.
42. The lipid antiinfective formulation of claim 32, wherein the
antiinfective is an aminoglycoside.
43. The lipid antiinfective formulation of claim 33, wherein the
antiinfective is an aminoglycoside.
44. The lipid antiinfective formulation of claim 36, wherein the
aminoglycoside is amikacin.
45. The lipid antiinfective formulation of claim 37, wherein the
aminoglycoside is amikacin.
46. The lipid antiinfective formulation of claim 38, wherein the
aminoglycoside is amikacin.
47. The lipid antiinfective formulation of claim 39, wherein the
aminoglycoside is amikacin.
48. The lipid antiinfective formulation of claim 40, wherein the
aminoglycoside is amikacin.
49. The lipid antiinfective formulation of claim 41, wherein the
aminoglycoside is amikacin.
50. The lipid antiinfective formulation of claim 42, wherein the
aminoglycoside is amikacin.
51. The lipid antiinfective formulation of claim 43, wherein the
aminoglycoside is amikacin.
52. A method of treating a pulmonary infection in a patient in need
thereof, comprising administering to the patient a therapeutically
effective amount of a lipid antiinfective formulation comprising an
antiinfective complexed to or encapsulated by a lipid component,
wherein the lipid component is net neutral and comprises a
phospholipid and a sterol, and the lipid component to antiinfective
weight ratio in the formulation is 0.40:1 to 0.49:1.
53. The method of claim 52, wherein the administering occurs via
nebulizing the lipid antiinfective formulation at a rate of 10 to
25 mg/min.
54. The method of claim 52, wherein the lipid component is present
as liposomes and the antiinfective is encapsulated by the
liposomes.
55. The method of claim 52, wherein the phospholipid is
dipalmitoylphosphatidylcholine (DPPC).
56. The method of claim 53, wherein the phospholipid is
dipalmitoylphosphatidylcholine (DPPC).
57. The method of claim 54, wherein the phospholipid is
dipalmitoylphosphatidylcholine (DPPC).
58. The method of claim 55, wherein the sterol is cholesterol.
59. The method of claim 56, wherein the sterol is cholesterol.
60. The method of claim 57, wherein the sterol is cholesterol.
61. The method of claim 58, wherein the pulmonary infection is a
Pseudomonas infection.
62. The method of claim 59, wherein the pulmonary infection is a
Pseudomonas infection.
63. The method of claim 60, wherein the pulmonary infection is a
Pseudomonas infection.
64. The method of claim 58, wherein the pulmonary infection is a
mycobacterial infection.
65. The method of claim 59, wherein the pulmonary infection is a
mycobacterial infection.
66. The method of claim 60, wherein the pulmonary infection is a
mycobacterial infection.
67. The method of claim 52, wherein the antiinfective is 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, a
penicillin, a cephalosporin, 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 a combination
thereof.
68. The method of claim 54, wherein the antiinfective is 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, a
penicillin, a cephalosporin, 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 a combination
thereof.
69. The method of claim 60, wherein the antiinfective is 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, a
penicillin, a cephalosporin, 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 a combination
thereof.
70. The method of claim 67, wherein the antiinfective is an
aminoglycoside.
71. The method of claim 68, wherein the antiinfective is an
aminoglycoside.
72. The method of claim 69, wherein the antiinfective is an
aminoglycoside.
73. The method of claim 70, wherein the aminoglycoside is
amikacin.
74. The method of claim 71, wherein the aminoglycoside is
amikacin.
75. The method of claim 72, wherein the aminoglycoside is amikacin.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 11/185,448, filed Jul. 19, 2005, which is a
continuation-in-part of U.S. patent application Ser. No.
11/023,971, filed Dec. 28, 2004, which is a continuation-in-part of
U.S. patent application Ser. No. 10/696,389, filed Oct. 29, 2003,
which claims the benefit of U.S. Provisional Patent Application
Ser. No. 60/421,923, filed Oct. 29, 2002.
BACKGROUND OF THE INVENTION
[0002] Certain sustained release technology suitable for
administration by inhalation employs lipid based formulations such
as liposomes 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.
[0003] For a lipid based 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 lipid 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
[0004] It is an object of the present invention to provide lipid
based drug formulations with low lipid to drug ratios.
[0005] It is also an object of the present invention to provide a
method of preparing lipid based drug formulations with low lipid to
drug ratios.
[0006] It is also an object of the present invention to provide a
method of delivering lipid based drug formulations at high delivery
rates as measured by mg/min of the drug.
[0007] It is also an object of the present invention to provide a
method of treating a patient for a pulmonary infection comprising
administering to the patient in need thereof a therapeutically
effective amount of a lipid based drug formulation comprising a low
L/D ratio wherein the drug is an antiinfective.
[0008] The subject invention results from the realization that
lipid based drug formulations with low L/D ratios are achieved by
preparing them using coacervation techniques.
[0009] Via methods disclosed herein, liposomes of modest size
(<1 .mu.m) comprising entrapped drug at L/D weight ratios of
typically about 0.40-0.49:1 are 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 drug
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 L/D ratios. The solutions in which the liposomes form
have a given drug concentration. The concentration of drug inside
the liposomes should be about the same concentration as in the
solution. However, internal drug concentrations are calculated at
least about 3.times. greater. It has now been discovered that this
phenomenon can be explained by the formation of a drug coacervate
which initiates lipid bilayer formation around the drug
coacervate.
In part the present invention relates to a method of preparing a
lipid based active agent formulation comprising mixing a lipid and
an active agent with a coacervate. In a further embodiment, the
coacervate is formed prior to mixing with the lipid. In a further
embodiment, the coacervate is formed during mixing with a lipid. In
a further embodiment, the coacervate is formed after mixing with a
lipid. In a further embodiment, the coacervate is a coacervate of
the active agent. In a further embodiment, the coacervate is a
coacervate of a third component other that the lipid and active
agent. In a further embodiment, the third component comprises a
counter ion capable of exchanging with the active agent.
[0010] In a further embodiment, the third component is a charged
polymer. In a further embodiment, the charged polymer is an
acrylate and the counter ion is an ammonium counter ion. In a
further embodiment, the active agent is added after mixing the
lipid with the coacervate and the active agent exchanges with the
counter ion.
[0011] In a further embodiment, the third component is an ion
capable of complexing with the active agent. In a further
embodiment, the ion is a metal ion. In a further embodiment, the
metal ion is Mg.sup.2+. In a further embodiment, the active agent
is added after mixing the lipid with the coacervate and the active
agent coordinates to the ion.
[0012] In a further embodiment, the lipid is added as a solution
with an organic solvent. In a further embodiment, the lipid is
added as an aqueous micellar suspension with a surfactant. In a
further embodiment, the lipid is induced to precipitate by diluting
the micellar suspension with an aqueous solution to below the
critical micellar concentration (CMC) of the surfactant.
[0013] In a further embodiment, the lipid is induced to precipitate
by changing the pH.
[0014] In part the present invention relates to a method of
preparing a lipid based drug formulation comprising mixing a lipid
with a drug coacervate. In a further embodiment the lipid is
dissolved in an organic solvent forming a lipid solution, and the
drug coacervate forms from mixing an aqueous solution of the drug
with the lipid solution. In a further embodiment the lipid solution
and aqueous drug solution are mixed from two separate streams in an
inline fashion. In a further embodiment the two streams enter a Y
or T-connector prior to mixing in line. In a further embodiment a
third stream of water or salt water is added to dilute the
resulting lipid and drug mixture. In a further embodiment the
organic solvent is ethanol.
[0015] In a further embodiment, the present invention relates to
the aforementioned method wherein the lipid solution is added at a
rate of 1 L/min and the aqueous drug solution is added at a rate of
1.5 L/min. In a further embodiment the lipid solution is added at a
rate of 1 L/min, the aqueous drug solution is added at a rate of
1.5 L/min, and the water or salt wafer is added at a rate of 1
L/min.
[0016] In a further embodiment, the present invention relates to
the aforementioned method wherein the lipid is a mixture of a
phospholipid and a sterol. In a further embodiment the phospholipid
is dipalmitoylphosphatidylcholine (DPPC) and the sterol is
cholesterol. In a further embodiment the DPPC:cholesterol ratio is
2:1 by weight. In a further embodiment the lipid solution is at 20
mg/ml and the aqueous drug solution is at 75 mg/ml.
[0017] In a further embodiment, the present invention relates to
the aforementioned method wherein the drug is an antiinfective. In
a further embodiment 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, penicillins, cephalosporins,
corticosteroid, prostaglandin, linomycin, clindamycin,
spectinomycin, polymyxin B, colistin, vancomycin, bacitracin,
isoniazid, rifampin, ethambutol, ethionamide, aminosalicylic acid,
cycloserine, capreomycin, a sulfone, clofazimine, thalidomide, a
polyene antifungal, flucytosine, imidazole, triazole, griseofulvin,
terconazole, butoconazole ciclopirax, ciclopirox olamine,
haloprogin, tolnaftate, naftifine, terbinafine, or combination
thereof. In a further embodiment the antiinfective is an
aminoglycoside. In a further embodiment the aminoglycoside is
amikacin. In a further embodiment the aminoglycoside is tobramicin.
In a further embodiment the aminoglycoside is gentamicin.
[0018] In another embodiment the present invention relates to a
lipid based drug formulation wherein the lipid to drug ratio is
0.40-0.49:1 by weight. In a further embodiment the lipid based
formulation is a liposome. In a further embodiment, the drug is an
antiinfective. In a further embodiment 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, penicillins, cephalosporins,
corticosteroid, prostaglandin, linomycin, clindamycin,
spectinomycin, polymyxin B, colistin, vancomycin, bacitracin,
isoniazid, rifampin, ethambutol, ethionamide, aminosalicylic acid,
cycloserine, capreomycin, a sulfone, clofazimine, thalidomide, a
polyene antifungal, flucytosine, imidazole, triazole, griseofulvin,
terconazole, butoconazole ciclopirax, ciclopirox olamine,
haloprogin, tolnaftate, naftifine, terbinafine, or combination
thereof. In a further embodiment the antiinfective is an
aminoglycoside. In a further embodiment the aminoglycoside is
amikacin. In a further embodiment the aminoglycoside is tobramycin.
In a further embodiment the aminoglycoside is gentamicin.
[0019] In a further embodiment the lipid comprises a mixture of a
phospholipid and a sterol. In a further embodiment the phospholipid
is DPPC and the sterol is cholesterol. In a further embodiment the
DPPC and the cholesterol is in a 2:1 ratio by weight.
[0020] In another embodiment, the present invention relates to a
method of delivering a lipid based drug formulation at a rate of 10
to 25 mg/min of drug comprising nebulizing the aforementioned lipid
based drug formulations of the present invention using a compressor
pressure of 20 to 40 psi. In a further embodiment, the entrapped
drug retention of greater than 45%. In a further embodiment the
lipid based drug formulation has an L/D ratio of less than
0.49.
[0021] In another embodiment, the present invention relates to a
method of treating a patient for a pulmonary infection comprising
administering to the patient a therapeutically effective amount of
the aforementioned lipid based drug formulations. In a further
embodiment the lipid based drug formulation has an L/D ratio of
less than 0.49. In a further embodiment the pulmonary infection is
a pseudomonas, P. aeruginosa, P. paucimobilis, P. putida, P.
fluorescens, and P. acidavorans, staphylococcal,
Methicillinresistant Staphylococcus aureus (MRSA), streptococcal.
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, M. intracellulare, M. kansasii, M. xenopi, M. marinum, M.
ulcerans, M. fortuitum complex, M. fortuitum, or M. chelonei
infection.
[0022] In another embodiment, the present invention relates to a
method of treating a patient for a pulmonary infection caused by
cystic fibrosis (CF) comprising administering to the patient a
therapeutically effective amount of the aforementioned lipid based
drug formulations.
[0023] 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
[0024] FIG. 1 depicts the cross sectional diagram of the
sputum/biofilm seen in patients with cystic fibrosis.
[0025] FIG. 2 depicts the graphical representation of the targeting
and depot effect of the drug of the present invention.
[0026] FIGS. 3 and 4 depict graphical representations of
bacteriology of amikacin in various forms.
[0027] FIG. 5 depicts a graphical representation of sustained
release for liposomal/complexed amikacin and tobramycin.
[0028] FIG. 6 depicts data on free or complexed ciprofloxacin.
[0029] FIG. 7 depicts a graphical representation of drug residence
in the lung given various dosing schedules.
[0030] FIG. 8 depicts graphically the two-stream in-line infusion
process of preparing liposomal antiinfective formulations. The flow
rates depicted are non-limiting examples of flow rates subject to
change as the need requires. Also, a third NaCl solution line is
depicted but this may be absent or deliver just water.
[0031] 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.
[0032] FIG. 10 depicts a graph of drug delivery rate v. L/D ratio
showing that the lower the L/D ratio, the higher the drug delivery
rate that can be achieved.
[0033] FIG. 11 depicts a ternary phase diagram of amikacin
sulfate-water-ethanol system.
[0034] FIG. 12 depicts the effect of ionic strength and pH on
ethanol-induced coacervation of BSA. A sample of BSA at 10 mg/mL in
an optical cuvette was titrated with a flow of degassed ethanol
under constant stirring. Light scattering signal was measured at
the right angle at 600 nm wavelength using PTI fluorimeter (Photon
Technology International, NJ). Temperature was fixed at 25 C.
[0035] FIG. 13 depicts the effect of MgCl.sub.2 on ethanol induced
coacervation of BSA. EtOH.sub.crit is the concentration of ethanol
at the onset of increase in light scattering. BSA 10 mg/mL was
dissolved in NaCl 10 mM at pH 7.0.
[0036] FIG. 14 depicts the effect of low molecular weight (MW 800)
polycation Polyethylenimine (PEI) on ethanol induced coacervation
of BSA. BSA 10 mg/mL was dissolved in NaCl 10 mM at pH 7.0.
DETAILED DESCRIPTION
[0037] The present invention discloses a lipid drug formulation
prepared by forming a drug coacervate which induces lipid bilayer
formation around the drug. The method results in low lipid to drug
ratios for the resulting lipid drug formulation and inner drug
concentrations that are 3 to 5.times. higher than the external drug
concentration used. The present invention also discloses a method
of preparing these lipid formulations using coacervation
techniques, high delivery rates for administering these lipid drug
formulations via nebulization, and methods of treating pulmonary
infections comprising administering these lipid drug formulations
to a patient in need thereof.
1. Definitions
[0038] 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.
[0039] 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.
[0040] The term "bioavailable" is an-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.
[0041] The terms "comprise" and "comprising" are used in the
inclusive, open sense, meaning that additional elements may be
included.
[0042] The term "drug" is an-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 drugs, also referred to as
"therapeutic agents", 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, antiinfectives, 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.
[0043] The terms "encapsulated" and "encapsulating" are refers to
adsorption of drugs on the surface of lipid based formulation,
association of drugs in the interstitial region of bilayers or
between two monolayers, capture of drugs in the space between two
bilayers, or capture of drugs in the space surrounded by the inner
most bilayer or monolayer.
[0044] The term "including" is used herein to mean "including but
not limited to". "Including" and "including but not limited to" are
used interchangeably.
[0045] 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.
[0046] 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).
[0047] A "patient," "subject" or "host" to be treated by the
subject method may mean either a human or non-human animal.
[0048] 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.
[0049] 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 mixed in-line.
[0050] The term "substantially free" is art recognized and refers
to a trivial amount or less.
[0051] The term "surfactant" as used herein refers to a compound
which lowers the surface tension of water by adsorbing at the
air-water interface. Many surfactants can assemble in the bulk
solution into aggregates that are known as micelles. The
concentration at which surfactants begin to form micelles is known
as the "critical micelle concentration" or CMC. Lipids useful for
the current application may also be surfactants with extremely low
CMC. Micelle-forming surfactants useful for the current application
should have a CMC higher than the CMC of the lipid. At
concentrations above CMC the micelle-forming surfactants can form
mixed micelles composed of surfactants and lipid molecules. Upon
dilution below CMC, micelle-forming surfactants will dissociate
into a true solution thus leaving lipid molecules exposed to the
aqueous medium. This leads to spontaneous precipitation of lipids,
preferably in a form of bilayers.
[0052] The phrase "therapeutically effective amount" as used herein
means that amount of a compound, material, or composition
comprising a lipid drug formulation according to the present
invention which is effective for producing some desired therapeutic
effect by inhibiting pulmonary infections.
[0053] 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. Coacervation
[0054] Coacervation in its simplest form can be thought of as a
heaping together. In more technical terms, coacervation is the
separation into two liquid phases in colloidal systems. The phase
more concentrated in the colloid component (drug) is called the
coacervate, and the other phase is the equilibrium solution.
[0055] The term colloidal refers to a state of subdivision,
implying that the molecules or polymolecular particles dispersed in
a medium have at least in one direction a dimension roughly between
1 nm and 1 .mu.m, or that in a system discontinuities are found at
distances of that order. IUPAC Compendium of Chemical Terminology
1972, 31, 605.
[0056] A solution of macromolecules is a simple and the most common
Colloid system. Small molecules also can form association colloids
as reversible aggregates. An association colloid is a reversible
chemical combination due to weak chemical bonding forces wherein up
to hundreds of molecules or ions aggregate to form colloidal
structures with sizes of from about 1 to about 2000 nanometers or
larger.
[0057] Current classification of coacervation phenomenon is based
on the mechanism driving the separation of two phases. Gander B,
Blanco-Prieto M. J., Thomasin C, Wandrey Ch. and Hunkeler D.,
Coacervation/Phase Separation. In: Encyclopedia of Pharmaceutical
Technology, Vol. 1, Swarbrick J, Boylan J. C., Eds., Marcel Dekker.
2002, p. 481-497). They include: [0058] 1. Coacervation induced by
partial desolvation. This in turn can involve a binary system of a
solvent and a polymer, where coacervation inducing factors are
temperature or pH. Or it can be a ternary system including a
solvent, a polymer, and a coacervating agent (nonsolvent for the
polymer or electrolyte (salt)). This type of Coacervation is often
called Simple Coacervation. Classical example of Simple
Coacervation is coacervation of gelatin solution by adding alcohol
(nonsolvent for gelatin). Other nonsolvents useful to induce
coacervation in aqueous systems may include propanol, isopropanol,
ecetone, dioxane. When electrolytes are used for polymer
desolvation, the phenomenon is called salting-out. In aqueous
systems the ability of ions to cause dehydration follows the
Hofmeister or lyotropic series
NH4.sup.+<K.sup.+<Na.sup.+<Ca.sup.2+<Mg.sup.2+<Al.sup.3+
for cations, and Cl.sup.-<SO4.sup.2-<tartrate.sup.2-,
phosphate.sup.2-<citrate.sup.3-, in order of increasing
salting-out capacity. [0059] 2. Coacervation induced by
Polymer-Polymer repulsion. In this type, the second polymer added
to the solution of the first polymer induces phase separation with
the 1.sup.st polymer being in the coacervate phase suspended in a
phase of the 2.sup.nd polymer. An example of Polymer-Polymer
repulsion is PLA coacervation in dichloromethane solvent induced by
silicone oil. [0060] 3. Coacervation induced by non-covalent
polymer cross-linking ("Complex Coacervation"). The cross-linking
agent can be a polymer of opposite charge to the coacervating
polymer, or di- or trivalent counter-ion to the polymer, such as
Ca.sup.2+, Mg.sup.2+, Al.sup.3+, Zn.sup.2+, Tartrate.sup.2- and
others. Typical polymers used in complex coacervation include:
polyanions Alginate, Carrageenan, Carboxymethylcellulose,
Chondroitin sulfate, Cellulose sulfate, Gellan, Hyaluronic acid,
Poly(acrylic acid), Xanthan; polycations Chitosan,
Poly(diallyldimethylammonium chloride), Poly(L-lysine),
Poly(vinylamine). In general, polyanion-polycation interaction is
controlled by a number of parameters, such as charge density, type
of ionic group, chain architecture. In addition, pH, ionic
strength, concentrations, temperature influence the complex
formation.
[0061] Obviously, a combination of the listed above types can be
used to control coacervation. Particularly, nonsolvent addition in
combination with cross-linking agents, or nonsolvent and desalting
agents.
[0062] FIG. 10 depicts a ternary phase diagram for an amikacin
sulfate-water-ethanol system. The two-phase area under the binodial
curve is a zone where the system separates into two phases, a
coacervate phase and an equilibrium phase. The area above the
binodial curve is a zone where single liquid phase system of
amikacin sulfate dissolved in water-ethanol mixture exists. When 3
parts of amikacin sulfate solution in water at 70 mg/mL (point 1)
is mixed with 2 parts of ethanol, the resulting mixture has
composition (point 2), which spontaneously separates into two
phases: coacervate phase rich in amikacin (point C) and equilibrium
phase pour in amikacin (point E). Coacervate phase comprises only
about 4.5% of total volume and originally forms as small droplets
suspended in equilibrium phase. If lipids are present in
surrounding solution when coacervates are just formed, they can
spontaneously form bilayers around those droplets. During
manufacturing it is often desired to limit exposure of the product
to high ethanol concentration. For examples, another 3 parts of
saline or buffer can be added consequently to the mixture, which
shifts composition to the single-phase zone (point 3). Since at
that point liposomes are already formed encapsulating majority of
coacervate phase material, amikacin will stay encapsulated inside
the liposomes.
[0063] It is key that the methods and lipid formulations of the
present invention are not prepared passively, i.e encapsulation is
not earned out by equilibrium alone. Coacervate formation leads to
higher internal drug concentrations relative to external drug
concentrations and lower L/A ratios.
3. Drug
[0064] The drug coacervate can conceivably occur with any type of
drug. Preferably, the drug is a water soluble antiinfective.
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), chloramphenicol, 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.
[0065] Particularly preferred antiinfectives include the
aminoglycosides, the quinolones, the polyene antifungals and the
polymyxins. Particularly preferred aminoglycosides include
amikacin, gentamicin, and tobramycin.
[0066] Also included as suitable antiinfectives used in the lipid
drug formulations of the present invention are pharmaceutically
acceptable addition salts and complexes of drugs. 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.
[0067] In cases in which the drugs 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 drugs 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.
[0068] Also included as suitable drugs used in the lipid
antiinfective formulations of the present invention are prodrugs of
the drug compounds. Prodrugs are considered to be any covalently
bonded carriers which release the active parent compound in
vivo.
4. Pulmonary Infections
[0069] The lipid drug formulations of the present invention with
their low L/D ratios are particularly useful for treating pulmonary
infections when the drug is an antiinfective. 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.
5. Methods of Treatment
[0070] In one embodiment the present invention comprises a method
of treating a patient for a pulmonary infection comprising
administering to the patient in need thereof a therapeutically
effective amount of a lipid drug formulation with a low L/D ratio
wherein the drug is an antiinfective.
[0071] 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."
[0072] 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.
[0073] 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 lipid antiinfective
formulations are more efficacious than the free drug.
[0074] 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).
[0075] 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.
[0076] 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.
6. Lipids and Liposomes
[0077] 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 phosphatidyl inositol (EPI), egg
phosphatidylserine (EPS), phosphatidylethanolamine (EPE), and egg
phosphatidic acid (EPA); the soya counterparts, soy
phosphatidylcholine (SPC); SPG, SPS, SPI, SPE, and SPA; the
hydrogenated egg and soya counterparts (e.g., HEPC, HSPC), other
phospholipids made up of ester linkages of fatty acids in the 2 and
3 of glycerol positions containing chains of 12 to 26 carbon atoms
and different head groups in the 1 position of glycerol that
include choline, glycerol, inositol, serine, ethanolamine, as well
as the corresponding phosphatidic acids. The chains on these fatty
acids can be saturated or unsaturated, and the phospholipid can be
made up of fatty acids of different chain lengths and different
degrees of unsaturation. In particular, the compositions of the
formulations can include dipalmitoylphosphatidylcholine (DPPC), a
major constituent of naturally-occurring lung surfactant as well as
dioleoylphosphatidylcholine (DOPC). Other examples include
dimyristoylphosphatidylcholine (DMPC) and
dimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidcholine
(DPPC) and dipalmitoylphosphatidylglycerol (DPPG)
distearoylphosphatidylcholine (DSPC) and
distearoylphosphatidylglycerol (DSPG),
dioleylphosphatidylethanolamine (DOPE) and mixed phospholipids like
palmitoylstearoylphosphatidylcholine (PSPC) and
palmitoylstearoylphosphatidylglycerol (PSPG), driacylglycerol,
diacylglycerol, seranide, sphingosine, sphingomyelin and single
acylated phospholipids like mono-oleoyl-phosphatidylethanol amine
(MOPE).
[0078] 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.
[0079] Liposomal 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.
[0080] 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 amphotericin B (Janoff et al., Proc. Nat
Acad. Sci., 85:6122 6126, 1988) and cardiolipin complexed with
doxorubicin.
[0081] 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.
[0082] Proliposomes are formulations that can become liposomes or
lipid complexes upon coming in contact with an aqueous liquid.
Agitation or other mixing can be necessary. Such proliposomes are
included in the scope of the present invention.
7. Methods of Preparation
[0083] The process for forming lipid drug 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
with an aqueous medium containing the drug. 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 with an
aqueous medium containing the drug. A "small" amount of solvent is
an amount compatible with forming liposomes or lipid complexes in
the infusion process. It is key that the conditions for the
infusion process have to lead to coacervate formation. Ultimate
conditions for infusing a given lipid solution with a given aqueous
solution of the active agent have to be determined based on the
Examples presented herein and the effect of various parameters
taught below. Also useful to someone of ordinary skill in the art,
are the techniques for forming coacervates as described in such
references as Bunderberg de Jong, H. G., Kruyt, H. R. Koazevation
(Entmischung in Kolloidalen Systemen), Koll. Zeitsch. 1930, 50(10),
39-48; Gander B, Blanco-Prieto M. J., Thomasin C, Wandrey Ch. and
Hunkeler D., Coacervation/Phase Separation. In: Encyclopedia of
Pharmaceutical Technology, Vol. 1, Swarbrick J, Boylan J. C., Eds.,
Marcel Dekker, 2002, p. 481-497; Newton D. W. Coacervation:
Principles and Applications. In: Polymers for Controlled drug
delivery. Tarcha P. J., Ed., CRC Press. Boca Raton, 1991, 67-81;
Scott P. W., Williams A. C., Barry B. W., Characterization of
complex coacervates of Some Tricyclic Antidepressants and
evaluation of their potential for Enhancing Transdermal Flux. J.
Controlled Release 1996, 41 (3), 215-227; Thomasin C., Merkle H.
P., Gander B. Drug microencapsulation by PLA/PLGA Coacervation in
the Light of Thermodynamics. 2. Parameters determining Microsphere
Formation. J. Pharm Sci. 1998, 87 (30), 269-275; Ball V.,
Winterhalter M., Schwinte P., Lavalle Ph., Voegel J.-C., Schaal P.
Complexation mechanism of Bovine Serum Albumin and Poly(allylamine
hydrochloride). J. Phys. Chem. B. 2002, 106, 2357-2364; Mohanty B.,
Bohidar H. B. Systematic of Alcohol-induced Simple Coacervation in
Aqueous Gelatin Solutions. Biomacromolecules 2003, 4, 1080-1086,
all of which are incorporated herein by reference in their
entirety. Preferably, the step is performed by an in-line infusion
process.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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 drug retention in the liposomal
formulation.
[0088] Lipid drug formulations have a sustained 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.
[0089] 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.
[0090] 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 drug. These can be configured as
multilamellar vesicles of concentric bilayers with the drug 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 drug formulations.
Both the processes and the product of these processes are part of
the present invention.
[0091] In one particularly preferred embodiment, the lipid drug
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 drug solution in-line. For example, the two solutions may
be mixed in-line inside a mixing tube preceded by a Y or
T-connector. In this way, the in-line infusion method creates the
best conditions for forming a drug coacervate. This infusion method
results in lower lipid to drug ratios and higher encapsulation
efficiencies.
[0092] In another particularly preferred embodiment, the lipid drug
formulations of the present invention are prepared by vortexing a
lipid-organic solvent solution with an aqueous drug solution at a
suitable vortexing level.
[0093] Another novel method of preparing the lipid drug
formulations of the present invention involves initially
encapsulating a charged polymer by way of farming a coacervate with
the charged polymer in the presence of a lipid. It is believed that
this technique will lead to low lipid to charged polymer ratios in
the same way that low lipid to drug ratios are obtained. Drug is
introduced into the interior of the lipid formulation via ion
exchange across the lipid membrane between the charged drug and
counter ions of the charged polymer. This technique, not including
coacervation formation, is known as "remote loading." Examples of
remote loading are disclosed in U.S. Pat. Nos. 5,316,771 and
5,192,549, both of which are incorporated herein by reference in
their entirety.
[0094] The processes described above may be further improved by
optimizing parameters such as flow rate, temperature, activation
agent concentration, and salt addition after infusion step. The
following experiments do not necessarily represent the methods of
the present invention as indicated by the higher lipid to drug
ratios. Rather they represent a set of experiments for testing the
effect of the aforementioned parameters. The multiple variables
give one an idea of the novelty behind using coacervation
techniques to form lipid based drug formulations with low L/D
ratios.
7.1. Effect of Flow Rates
[0095] 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 AMK mL/min total free Lipid VOL
Recovery Batch AMK Lipid mg/mL % 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.
[0096] Batch 3 with the lipid/amikacin flow rates of 300/500 mL/min
showed the host L/D and particle size, combined with reasonably
high amikacin recovery. Thus it was decided to use these flow rates
for all further experiments.
[0097] 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.
Temp, C. AMK stock AMK total AMK free Lipid Size VOL Size Batch
L/AMK/W mg/mL mg/mL % mg/mL L/D nm SD % 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 3.sup.rd column represents the temperature
of the Lipid and Amikacin solutions just before infusion, and the
temperature during washing (diafiltration). RT = room temperature.
"VOL size" is the volume of weighted particle size.
TABLE-US-00003 TABLE 3 Processing conditions for batches 1-18.*
Mixing Mixer NaCl added tube position Volume Timing to Washing
conditions Batch cm cm Stock % parts infusion NaCl % 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 2x dilution 11 10 5 10 150 imm after 1.5
2x dilution 12 10 5 H2O 180 20'' after 1.5 2x dilution 13 10 5 H2O
180 30'' after 1.5 2x 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).
7.2. Effects of Process Temperature
[0098] 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.* AMK AMK VOL Temperature, C. 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.
[0099] 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.
7.3. Effect of Post-Infusion Addition of Aqueous Volume
[0100] 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).
[0101] 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.
[0102] 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).
[0103] 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: [0104] 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. [0105] 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. [0106] 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 [0106] 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 H.sub.2O 180 20'' after 81.0 0.61 341 13 30 H.sub.2O
180 30'' after 68.6 0.91 311 14 40 H.sub.2O 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
7.4. Effect of Antiinfective Stock Solution
[0107] 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.
[0108] Table 6 summarizes the effect of using various amikacin
stock concentrations. 40 mg/mL delivered comparable or better L/D
values, and even improved amikacin recovery. Using less amikacin
relative to a constant amount of lipid, and providing a similar
L/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
[0109] 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.
8. Lipid to Drug Ratio
[0110] There are several ways to increase the entrapment of drug
(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 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).
[0111] 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.
[0112] 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).
[0113] 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.
[0114] The lipid to drug ratio obtained by the processes of the
present invention is about 0.40 to 0.49:1. Further, the percentage
of free drug, present after the product is dialyzed for a
particular duration, is decreased. When the drug is a macromolecule
such as a protein, the L/D ratio can be as high as about 1.2 which
is low in comparison to the literature (for example, see U.S. Pat.
No. 6,843,942, where encapsulation of recombinant human superoxide
dismutase (rh-SOD) in a DPPC-cholesterol-stearylamine formulation
was prepared with a L/D ratio of .about.5).
9. Drug Delivery Rate
[0115] For a pharmaceutical inhalation therapy, it is essentially
desirable to establish a high delivery rate to achieve a desired
dose in a minimum dosing time. For a liposomal formulation, it is
also desirable to maximize the retention of the entrapped drug
during nebulization. Nebulization flow rate is the volume (or
weight) of the aqueous solution that aerosolizes and flies out of
the nebulizer in a unit of time (e.g. g/min, ml/min). Drug delivery
rate is defined as the amount of drug that gets delivered by the
nebulizer in a unit of time (e.g. mg/min).
[0116] Drug delivery efficacy can be increased by making
improvements in two areas: 1) improvements in the drug delivery
device, an 2) increasing the drug concentration in the drug
formulation. When the drug delivery device is a jet nebulizer, key
factors that affect the drug delivery rate include: model of
nebulizer, i.e. structure of nebulizer, which determines the air
flow rate exit from the device and aerosol droplet size; and air
pressure used to propel the drug formulation. For drug
formulations, drug concentration can be increased by decreasing the
amount of water. By doing this, though, viscosity and surface
tension increase, which has the effect of decreasing the
nebulization rate.
[0117] In order to improve drug delivery rate for a given jet
nebulizer, drug concentration should be increased while keeping
viscosity and sulfate tension relatively constant. By doing this,
the drug delivery rate increases even though the nebulization rate
stays the same according to the formula drug delivery rate
(mg/min)=drug concentration (mg/ml).times.nebulization rate
(ml/min).
[0118] The present invention, in part, is drawn to concentrated
high dose lipid based drug formulations that provide maximal
aerosol output with particle size ranges within the optimal range
of 1-5 .mu.m mass median aerodynamic diameter (MMAD).
[0119] Table 7 presents delivery rate and % drug retention data for
several nebulization trials. Nebulization was carried out by a jet
nebulizer for 20 min at 30 psi compressor pressure.
TABLE-US-00007 TABLE 7 Compositions and characteristics of
liposomal amikacin delivery rates greater than 10 mg/min and
retention of entrapped drug greater than 45%. DPPC/ Flow Delivery
cholesterol [Amikacin] [Lipid] Lipid/ Viscosity Rate Rate Lot (mol
%) Salinity % mg/ml mg/ml Drug (cP) ml/min mg/min % Drug A 50/50
0.9 65.0 53.3 0.82 24.9 0.157 10.2 57.0 B 50/50 3.0 58.7 59.8 1.02
N/D* 0.177 10.4 71.5 C 60/40 1.5 76.8 48.8 0.64 38.5 0.153 11.8
50.6 D 50/50 1.5 81.6 54.6 0.67 35.4 0.164 13.4 52.8 E 50/50 1.5
78.2 55.0 0.70 24.9 0.173 13.5 57.5 F 60/40 1.5 75.5 50.7 0.67 27.5
0.180 13.6 47.4 G 50/50 1.5 80.3 47.8 0.60 14.2 0.170 13.7 68.0 H
50/50 3.0 108.1 69.0 0.64 25.5 0.154 16.6 85.0 I 50/50 3.0 107.0
44.6 0.42 22.1 0.180 19.3 68.1 J 50/50 3.0 117.8 56.4 0.48 31.1
0.175 20.6 65.2 K 50/50 3.0 130.3 54.2 0.42 34.8 0.170 22.2
68.1
[0120] One can see that as the L/D ratio decreases from lots A and
B to the L/D ratios of the present invention obtained in lots I, J,
and K, the concentration of drug also increases. At a nearly
constant flow rate, an increase in drug concentration gives the
highest drug delivery rate of .about.19 to 22 mg/min. These results
are graphed in FIG. 10.
10. Results
10.1. Biofilm Barriers of Pulmonary Infections
[0121] An obstacle to treating infectious diseases such as
Pseudomonas aeruginosa, the leading cause of chronic illness in CF
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).
[0122] 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).
[0123] 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.).
[0124] 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.
10.2. Pharmacokinetics
[0125] 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.
[0126] 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 duration (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-00008 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
[0127] 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.
[0128] 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 PAOI mucoid Pseudomonas. In both
cases sustained and enhanced killing of the Pseudomonas over time
was observed, as shown in Table 8.
TABLE-US-00009 TABLE 8 In vitro killing of Pseudomonas over time.
In vitro Sputum/Alginate Assay (% survival of PA01 Mucoid
Pseudomonas) Amikacin Incubation time at 37.degree. C. 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 kilting is observed, consistent with slow release
sustained antiinfective effect model with no interference or
inactivation of antiinfective.
[0129] 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 CPUs 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.
[0130] 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
[0131] 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.
[0132] 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.
[0133] 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-00010 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.
[0134] 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.
[0135] 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.).
[0136] 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.
[0137] 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.
[0138] 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.
[0139] 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. 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.
10.4. Drug Release Mediated by P. Aeruginosa Infection
[0140] 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.
[0141] 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.
[0142] 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 heads). 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.
[0143] 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.
[0144] 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.
10.5. Effect of Inhaled Liposomal Drug Formulations on the Function
of Alveolar Macrophages
[0145] 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.
[0146] Aerosols of the liposomal amikacin formulations, amikacin,
placebo liposomes and saline were generated with a PARI LC Star
nebulizer and inhaled by CD.RTM.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).
[0147] 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 or 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.
[0148] 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.
11. Dosages
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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.
[0156] 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.
[0157] 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.
12. Formulation
[0158] 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
In-Line Infusion Process
[0159] About 20 mg/ml total lipid (DPPC:cholesterol=2:1 by wt) in
ethanol and about 75 mg/ml amikacin sulfate (about 50 mg/ml
amikacin) in water were mixed together into the reactor vessel by
the two-stream in line infusion method. Two solutions were fed into
Y-shaped connector at a rate of about 1.0 L/min and about 1.5
L/min, respectively. During the two-stream infusion, water was
separately added into the reactor vessel at a similar flow rate
(about 1.0 L/min) as the flow rate of lipid solution. The
amikacin-lipid suspension infused into the reactor vessel is
instantaneously diluted by the continuous feed of water. This
additional water helps to seal the membrane by diluting ethanol and
it also reduces viscosity of the suspension, consequently reducing
the inlet pressure of the diafiltration cartridge. After infusion,
the suspension is concentrated by reducing the volume half using
diafiltration. The concentrated suspension is washed by
diafiltration during a fresh supply of 3.0% NaCl solution. The
washed suspension is further concentrated by diafiltration until
the desired total amikacin concentration is achieved. The results
are given in Table 10.
TABLE-US-00011 TABLE 10 Washing Temp Total [amikacin] Total [lipid]
Lot (.degree. C.) mg/ml mg/ml Lipid/Drug I RT* 130.3 54.2 0.42 II
RT* 126.0 57.0 0.45 III 35 130.0 60.9 0.47 *Room temperature
(19~23.degree. C.).
Example 2
Encapsulation of Bovine Serum Albumin (BSA) by Coacervation
Technique
[0160] BSA is a protein having isoelectric point pI=4.9. At pH
above that point, it can be considered as a colloid with a net
negative charge. It has been shown to form complex coacervates with
various polyelectrolytes, such as Poly(allylamine hydrochloride),
which in turn is affected by the medium ionic strength, pH and
temperature. It was found that addition of nonsolvent to albumin
(ethanol) can also induce coacervation. When BSA is dissolved in
water at pH 7.0, and ethanol concentration added exceeds .about.45
wt %, BSA molecules aggregate to form droplets of coacervate phase
thus leading to strong increase in light scattering. Adding NaCl
(increasing ionic strength) results in less ethanol needed to
induce coacervation. Lowering the pH has a similar effect (FIG.
12). Di-valent ions (e.g. Mg.sup.2+) have an even stronger effect
on lowering the critical ethanol concentration required to induce
BSA coacervation (FIG. 13). The most drastic effect was found when
the low molecular weight polycation PEI was added to the BSA
solution (FIG. 14). Thus, 0.05 mg/mL of PEI in molar terms is
.about.60 .mu.M concentration, which represents only about 1
molecule PEI per 3 molecules of BSA.
[0161] To encapsulate BSA into liposomes, a BSA aqueous solution at
10 mg/ml in 20 mM NaCl, pH 5.5 was used. A lipid solution was
prepared separately at a concentration of 10 mg/mL and a molar
ratio DPPC/DPPG/Cholesterol of 60:5:40 in 95% ethanol. All
solutions were preheated to 30'' C. The lipid solution (0.4 mL) was
added by pipette into a 1 mL BSA solution in a test tube and
immediately vortexed to ensure complete mixing. 20 seconds later
0.6 mL of 5% sucrose solution was added and vortex tug repeated. To
determine BSA encapsulation, 0.8 mL of the resulting liposome
suspension was placed on 5-20% sucrose gradient and centrifuged 30
min at 30,000 RPM. The loaded liposomes formed a pellet heavier
than 20% sucrose. The pellet was collected and quantified for
lipids and BSA. Lipids were measured by reverse-phase HPLC and BSA
was measured by fluorescence (excitation 280 nm, emission 320 nm).
It was found that the pellet contained 1.6 mg lipid and 1.3 mg BSA
thus giving L/D ratio of 1.2 which is lower than what is normally
seen for proteins (for example, see U.S. Pat. No. 6,843,942, where
encapsulation of recombinant human superoxide dismutase (rh-SOD) in
a DPPC-cholesterol-stearylamine formulation was prepared with a L/D
ratio of .about.5).
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INCORPORATION BY REFERENCE
[0178] 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
[0179] 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.
* * * * *