U.S. patent application number 17/052070 was filed with the patent office on 2021-04-22 for methods for the manufacture of liposomal drug formulations.
The applicant listed for this patent is Insmed Incorporated, Robert WORSHAM. Invention is credited to Robert WORSHAM.
Application Number | 20210113467 17/052070 |
Document ID | / |
Family ID | 1000005324380 |
Filed Date | 2021-04-22 |
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United States Patent
Application |
20210113467 |
Kind Code |
A1 |
WORSHAM; Robert |
April 22, 2021 |
METHODS FOR THE MANUFACTURE OF LIPOSOMAL DRUG FORMULATIONS
Abstract
Provided herein is a method for the large-scale manufacture of
liposomal drug formulations containing an aminoglycoside such as
amikacin having advantageous lipid/drag characteristics. The method
utilizes a particular relative flow rate ratio of lipid to drug
streams to obtain liposomes with a high aminoglycoside
encapsulation efficiency. The resulting liposomal drug formulations
advantageously comprise an overall lipid-to-drug weight ratio of
less than 1:1.
Inventors: |
WORSHAM; Robert;
(Bridgewater, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WORSHAM; Robert
Insmed Incorporated |
Bridgewater
Bridgewater |
NJ
NJ |
US
US |
|
|
Family ID: |
1000005324380 |
Appl. No.: |
17/052070 |
Filed: |
May 2, 2019 |
PCT Filed: |
May 2, 2019 |
PCT NO: |
PCT/US2019/030404 |
371 Date: |
October 30, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62665564 |
May 2, 2018 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/24 20130101;
A61K 47/14 20130101; A61K 9/1277 20130101; A61K 31/7036
20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 47/24 20060101 A61K047/24; A61K 31/7036 20060101
A61K031/7036; A61K 47/14 20060101 A61K047/14 |
Claims
1. A large-scale method of preparing a liposomal aminoglycoside
formulation comprising a lipid and an aminoglycoside, wherein the
overall lipid-to-drug weight ratio is less than 1:1, the method
comprising: (a) mixing a first stream comprising the lipid with a
second stream comprising the aminoglycoside to form a combined
lipid-aminoglycoside stream, (b) mixing the lipid-aminoglycoside
stream of Step (a) with an aqueous saline solution in a reaction
vessel, and (c) washing the product of Step (b) comprising the
liposomal aminoglycoside formulation to remove unencapsulated
aminoglycoside, wherein the relative flow rate ratio of the second
stream to the first stream is about 1.5:1 to about 2:1.
2. The method of claim 1, wherein the second stream comprises an
aqueous solution of amikacin.
3. The method of claim 1, wherein the first stream comprises an
alcoholic solution of lipid.
4. The method of claim 1, wherein the aqueous saline solution is
added to the reaction vessel via a third stream.
5. The method of claim 4, wherein the third stream is added to the
reaction vessel at the same time as the lipid-aminoglycoside
stream.
6. The method of claim 4, wherein the third stream is added to the
reaction vessel prior to the lipid-aminoglycoside stream.
7. The method of any one of claims 1-6, wherein the flow rate of
the first stream is from about 0.5 kg/min to about 1.5 kg/min, and
the flow rate of the second stream is from about 1 kg/min to about
2 kg/min.
8. The method of any one of claims 1-6, wherein the flow rate of
the first stream is from about 3 kg/min to about 4 kg/min, and the
flow rate of the second stream is from about 5 kg/min to about 7
kg/min.
9. The method of claim 6, wherein the flow rate of the first stream
is from about 0.5 kg/min to about 1.5 kg/min, the flow rate of the
second stream is from about 1 kg/min to about 2 kg/min, and the
flow rate of the third stream is from about 1 L/min to about 2
L/min.
10. The method of claim 6, wherein the flow rate of the first
stream is from about 3 kg/min to about 4 kg/min, the flow rate of
the second stream is from about 5 kg/min to about 7 kg/min, and the
flow rate of the third stream is from about 3 L/min to about 6
L/min.
11. The method of any one of claims 1-10, wherein the aqueous
saline solution is 1.5% sodium chloride.
12. The method of any one of claims 1-11, wherein the
aminoglycoside is a pharmaceutically acceptable salt of the
aminoglycoside.
13. The method any one of claims 1-12, wherein the lipid comprises
a phospholipid.
14. The method of claim 13, wherein the phospholipid is a
phosphatidylcholine.
15. The method of claim 14, wherein the phosphatidylcholine is
dipalmitoylphosphatidylcholine (DPPC).
16. The method of any one of claims 1-15, wherein the lipid
comprises a sterol.
17. The method of claim 16, wherein the sterol is cholesterol.
18. The method of any one of claims 1-17, wherein the lipid
comprises DPPC and cholesterol.
19. The method of any one of claims 1-18, wherein the drug and
lipid streams are each maintained at a temperature from about
30.degree. C. to about 50.degree. C. prior to mixing.
20. The method of claim 19, wherein the drug and lipid streams are
each maintained at a temperature from about 35.degree. C. to about
45.degree. C. prior to mixing.
21. The method of any one of claims 1-20, wherein the temperature
of the combined lipid-aminoglycoside stream is cooled by the
aqueous saline solution in the reaction vessel.
22. The method of any one of claims 1-21, wherein following Step
(b) liposomes are prepared with an aminoglycoside encapsulation
efficiency of at least 40%.
23. The method of any one of claims 1-22, wherein liposomes are
formed in the combined lipid-aminoglycoside stream.
24. The method of any one of claims 1-23, for the preparation of a
liposomal drug formulation where the overall lipid-to-drug weight
ratio is about 0.7:1.
25. The method of any one of claims 1-23, wherein the washing Step
(c) is performed using 1.5% aqueous sodium chloride solution.
26. The method of claim 25, wherein the washing Step (c) is
repeated, and the product concentrated to provide a liposomal drug
formulation with the aminoglycoside present at a concentration of
from about 60 g/L to about 80 g/L.
27. The method of any one of claims 1-26, wherein the
aminoglycoside is arbekacin, astromicin, capreomycin, dibekacin,
framycetin, gentamicin, hygromycin B, isepamicin, kanamycin,
neomycin, netilmicin, paromomycin, rhodestreptomycin, ribostamycin,
sisomicin, spectinomycin, streptomycin, tobramycin, verdamicin, or
a combination thereof.
28. The method of any one of claims 1-26, wherein the
aminoglycoside is AC4437, amikacin, apramycin, arbekacin,
astromicin, bekanamycin, boholmycin, brulamycin, capreomycin,
dibekacin, dactimicin, etimicin, framycetin, gentamicin, H107,
hygromycin, hygromycin B, inosamycin, K-4619, isepamicin, KA-5685,
kanamycin, neomycin, netilmicin, paromomycm, plazomicin,
ribostamycin, sisomicm, rhodestreptomycin, sorbistin,
spectinomycin, sporaricin, streptomycin, tobramycin, verdamicin,
vertilmicin, or a combination thereof.
29. The method of any one of claims 1-26, wherein the
aminoglycoside is amikacin.
30. The method of claim 29, wherein the amikacin is amikacin
sulfate.
31. A liposomal drug formulation manufactured by the method of any
one of claims 1-30.
32. The liposomal drug formulation of claim 31, wherein the
aminoglycoside is present at a concentration from about 60 g/L to
about 80 g/L.
33. The liposomal drug formulation of claim 31, wherein amikacin is
present at a concentration of about 70 g/L.
34. The liposomal drug formulation of any one of claims 31-33,
wherein the lipid is present at a concentration from about 40 g/L
to about 60 g/L.
35. The liposomal drug formulation of claim 34, wherein the lipid
is present at a concentration of about 50 g/L.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority from U.S. Provisional
Application Ser. No. 62/665,564, filed May 2, 2018, the disclosure
of which is incorporated by reference herein in its entirety for
all purposes.
BACKGROUND OF THE INVENTION
[0002] Liposomal drug formulations enable the ability to target and
enhance the uptake of active agents at specific sites of disease.
Such formulations have been developed to treat various pulmonary
disorders, including those caused by pulmonary infections, where
their characteristics make them an ideal choice for the inhalation
delivery of anti-infective agents.
[0003] One such anti-infective agent, amikacin, has been packaged
in liposomes, and has been studied in multiple clinical trials in
adult patients for the treatment of refractory nontuberculous
mycobacterial (NTM) lung disease cause by Mycobacterium avium
complex (MAC). In a recent Phase 3 study of the amikacin liposome
inhalation suspension (ALIS), it was demonstrated that the addition
of ALIS to guideline based therapy (GBT) eliminated evidence of NTM
lung disease caused by MAC in sputum by month 6 in 29% of patients,
compared to 9% of patients on GBT alone.
[0004] Although liposomes containing a relatively high amikacin to
lipid ratio have been prepared at the bench scale, it is well-known
that it is not a routine matter to scale up such processes to
produce, at the commercial manufacturing scale, liposomal
formulations where parameters such as drug concentration, amount of
lipid in the formulation, lipid-to-drug ratio, captured volume,
drug leakage, viscosity, and particle size are consistently
maintained within specification for clinical and/or commercial
use.
[0005] The present invention addresses the need for a repeatable
large-scale process for preparing liposomes containing an
aminoglycoside antibiotic such as amikacin, and having a high
aminoglycoside-to-lipid weight ratio (and in turn, a low
lipid-to-aminoglycoside weight ratio) and superior encapsulation
efficiency.
SUMMARY OF THE INVENTION
[0006] In one aspect, the present invention provides a method for
the large-scale manufacture of a liposomal aminoglycoside
formulation comprising a lipid and an aminoglycoside (e.g.,
amikacin), with a high aminoglycoside loading relative to the lipid
concentration (i.e., a high relative weight ratio of
aminoglycoside-to-lipid). In particular, the
lipid-to-aminoglycoside (e.g., amikacin) weight ratio (also
referred to as the "L/D weight ratio") in the liposomal suspension
prepared according to method of the present invention is less than
1:1 upon completion of the process, for example between about 0.5:1
and about 0.9:1. In one embodiment, the lipid-to-aminoglycoside
weight ratio of the liposomal suspension prepared according to the
method of the present invention is about 0.7:1
(lipid:aminoglycoside) upon completion of the manufacturing
process.
[0007] In another aspect, the present invention relates to a method
for the large-scale manufacture of a liposomal drug formulation
comprising a lipid and aminoglycoside (e.g., amikacin), wherein the
aminoglycoside is contained within the liposome with a high
encapsulation efficiency (e.g., an encapsulation efficiency of at
least about 40% prior to washing to remove free aminoglycoside from
the formulation).
[0008] In one embodiment, the method comprises mixing a first
stream comprising a lipid with a second stream comprising an
aminoglycoside to form a combined lipid-aminoglycoside stream. The
combined lipid-aminoglycoside stream comprises liposomally
encapsulated aminoglycoside, which in one embodiment, is formed at
the intersection of the lipid stream and the aminoglycoside stream.
In a further embodiment, the lipid-aminoglycoside stream is mixed
with an aqueous saline solution in a reaction vessel (see, e.g.,
FIG. 1). In one embodiment, the aminoglycoside is present in an
aqueous solution prior to the mixing step. In another embodiment,
the lipid is present in an alcoholic solution, e.g., an ethanolic
solution, prior to the mixing step. In a further embodiment, the
lipid comprises a phospholipid and cholesterol. In one embodiment,
the relative flow rate ratio of the second stream comprising
aminoglycoside to the first stream comprising a lipid is about
1.5:1 (aminoglycoside stream:lipid stream) to about 2:1
(aminoglycoside stream:lipid stream). In a further embodiment, the
lipid comprises dipalmitoylphosphatidylcholine (DPPC) and
cholesterol.
[0009] In one embodiment, the aqueous saline solution is added to
the reaction vessel via a third stream. In a further embodiment,
the third stream is added to the reaction vessel at the same time
as the combined lipid-aminoglycoside stream. In another embodiment,
the third stream is added to the reaction vessel prior to the
addition of the combined lipid-aminoglycoside stream to the
reaction vessel. In another embodiment, the aqueous saline solution
is at about room temperature prior to entering the reaction vessel.
In one embodiment, the aqueous saline solution is about 1.5%
aqueous sodium chloride.
[0010] In one particular aspect, the present invention provides a
method for the large-scale manufacture of a liposomal drug
formulation comprising a lipid and aminoglycoside (e.g., amikacin),
wherein the aminoglycoside is encapsulated within or complexed with
the liposome, prior to a washing step, at an encapsulation
efficiency of at least 40%. Following the washing step, which in
one embodiment, is carried out via tangential flow filtration, the
weight ratio of lipid-to-aminoglycoside in the liposomal
aminoglycoside formulation is less than 1:1, for example between
about 0.5:1 and about 0.9:1 (e.g., about 0.7:1). In one embodiment
of this method, the aminoglycoside is amikacin. In a further
embodiment, the amikacin is present as amikacin sulfate.
[0011] In one embodiment, a first stream comprising a lipid is
mixed with a second stream comprising an aminoglycoside to form a
combined lipid-aminoglycoside stream (e.g., a lipid-amikacin
stream) comprising liposomal aminoglycoside. In one embodiment, the
liposomal aminoglycoside formulation is formed at the intersection
of the two streams, i.e., upon formation of the combined
lipid-aminoglycoside stream. In a further embodiment, the flow rate
of the first stream comprising a lipid is from about 0.5 kg/min to
about 1.5 kg/min and the flow rate of the second stream comprising
aminoglycoside is from about 1 kg/min to about 2 kg/min. In a
further embodiment, the flow rate of the first stream comprising a
lipid is from about 3 kg/min to about 4 kg/min and the flow rate of
the second stream comprising the aminoglycoside is from about 5
kg/min to about 7 kg/min. In another embodiment, the relative flow
rate ratio of the second stream comprising aminoglycoside to the
first stream comprising a lipid is about 1.5:1 (aminoglycoside
stream flow rate:lipid stream flow rate) to about 2:1
(aminoglycoside stream flow rate:lipid stream flow rate). In yet
another embodiment, the lipid comprises
dipalmitoylphosphatidylcholine (DPPC) and cholesterol.
[0012] In one embodiment, the method for the large-scale
manufacture of a liposomal drug formulation comprises mixing a
first stream comprising a lipid with a second stream comprising
aminoglycoside to form a combined lipid-aminoglycoside stream, and
adding the combined lipid-aminoglycoside stream to a vessel
comprising an aqueous saline solution. The aqueous saline solution,
in one embodiment, is added to the reaction vessel via a third
stream (see, e.g., FIG. 1).
[0013] In a further embodiment, when the flow rate of the first
stream comprising a lipid is from about 0.5 kg/min to about 1.5
kg/min and the flow rate of the second stream comprising
aminoglycoside is from about 1 kg/min to about 2 kg/min, the flow
rate of the third stream is from about 0.5 L/min and about 2.0
L/min, for example, from about 1.0 L/min to about 2.0 L/min, e.g.
from about 1.0 L/min to about 1.5 L/min, including about 1.25
L/min. In another embodiment, when the flow rate of the first
stream comprising a lipid is from about 3 kg/min to about 4 kg/min
and the flow rate of the second stream comprising aminoglycoside is
from about 5 kg/min to about 7 kg/min, the flow rate of the third
stream is from about 3 L/min and about 6 L/min, for example, from
about 4 L/min to about 6 L/min, e.g. from about 4.5 L/min to about
5.5 L/min, including about 5 L/min.
[0014] As used herein, except where specifically stated otherwise,
the term "aminoglycoside" is intended to include the aminoglycoside
free base and any pharmaceutically acceptable salt thereof. For
example, the term "amikacin" is intended to include amikacin free
base and any pharmaceutically acceptable salt thereof (e.g.,
amikacin sulfate).
[0015] In one embodiment, the method for the large-scale
manufacture of a liposomal aminoglycoside (e.g. amikacin)
formulation comprises mixing a first stream comprising a lipid
comprising a phospholipid with a second stream comprising
aminoglycoside (e.g. amikacin) to form a combined
lipid-aminoglycoside stream. In a further embodiment, the
lipid-aminoglycoside stream is mixed with an aqueous saline
solution in a reaction vessel. In one embodiment, the phospholipid
is a phosphatidylcholine. Ina further embodiment, the
phosphatidylcholine is DPPC. In another embodiment, the lipid
comprises a phospholipid and a sterol. In a further embodiment, the
sterol is cholesterol. In one embodiment, the lipid comprises DPPC
and cholesterol.
[0016] In one embodiment, the method for the large-scale
manufacture of a liposomal aminoglycoside formulation comprises
mixing a first stream comprising a lipid with a second stream
comprising aminoglycoside, wherein the first stream is mixed with
the second stream to form a combined lipid-aminoglycoside stream.
The combined lipid-aminoglycoside stream comprises liposomal
aminoglycoside. The liposomal aminoglycoside in one embodiment, is
formed upon mixing the first stream and second stream, e.g., at the
intersection of the two streams. In a further embodiment, the
combined lipid-aminoglycoside stream is added to a reaction vessel
and mixed with an aqueous saline solution. In a further embodiment,
the aminoglycoside stream and the lipid stream are each maintained
at a temperature from about 30.degree. C. to about 50.degree. C.
prior to mixing. In a further embodiment, the aminoglycoside and
lipid streams are each maintained at a temperature of from about
35.degree. C. to about 45.degree. C., for example from about
38.degree. C. to about 42.degree. C. prior to mixing. In one
embodiment, the combined lipid-aminoglycoside stream is cooled upon
entering the reaction vessel. In another embodiment, the combined
lipid-aminoglycoside stream is cooled by the aqueous saline
solution in the reaction vessel. In one embodiment, the reaction
vessel is maintained at a temperature from about 25.degree. C. and
about 40.degree. C., e.g., from about 27.degree. C. to about
35.degree. C. In another embodiment, the reaction vessel is
maintained at a temperature of about 30.degree. C. In another
embodiment, the reaction vessel is maintained at a temperature of
about 33.degree. C.
[0017] In another aspect of the invention, a liposomal
aminoglycoside formulation is manufactured on a large-scale
according to a method provided herein. In one embodiment, the
concentration of aminoglycoside (e.g. amikacin) present in the
liposomal drug formulation so prepared is about 10 g/L or greater,
for example from about 50 g/L to about 100 g/L, including about 60
g/L to about 80 g/L and about 65 g/L to about 75 g/L (e.g., about
20 g/L, about 30 g/L, 40 about g/L, about 50 g/L, about 60 g/L,
about 70 g/L or about 80 g/L). In a further embodiment, the
concentration of lipid present in the liposomal drug formulation so
prepared is from about 10 g/L to about 100 g/L, including about 20
g/L to about 80 g/L and about 40 g/L to about 60 g/L (e.g. about 50
g/L). In another embodiment, the L/D ratio of a liposomal drug
formulation manufactured on a large-scale according to a method
provided herein is less than 1:1, for example between about 0.5:1
and about 0.8:1 (e.g. about 0.7:1).
[0018] In another embodiment, the liposomal drug formulation
manufactured on a large-scale according to a method provided herein
comprises liposome particles with a mean particle size (i.e. a mean
diameter) of from about 200 nm to about 500 nm, for example from
about 200 nm to about 400 nm (e.g. from about 250 nm to about 350
nm).
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 depicting one embodiment of the invention for
preparing a liposomal aminoglycoside formulation.
[0020] FIG. 2 shows the effect of relative lipid/amikacin flow
rates on the resulting L/D ratio of various liposomal amikacin
formulations.
DETAILED DESCRIPTION OF THE INVENTION
[0021] In one aspect, the invention described herein relates to a
method for manufacturing a liposomal aminoglycoside formulation on
a large-scale. In one embodiment, the method comprises mixing a
first stream comprising a lipid (also referred to herein as a
"lipid stream") with a second stream comprising an aminoglycoside
such as amikacin (also referred to herein as a "drug stream") to
form a combined lipid-aminoglycoside stream, and the
lipid-aminoglycoside stream is mixed with an aqueous saline
solution in a reaction vessel. In some embodiments, the aqueous
saline solution enters the reaction vessel via a third stream.
[0022] The mixing of the lipid and drug streams is effected such
that a turbulent flow results when forming the combined
lipid-aminoglycoside stream. A turbulent flow is conveniently
achieved using an appropriate T-shaped or Y-shaped infusion module
for "in-line" mixing of the lipid and drug streams.
[0023] The term "large-scale" means the use of at least about 5 kg
aminoglycoside base starting material in the drug stream
(calculated to at least about 5 kg aminoglycoside base if a
pharmaceutically acceptable salt is used). In one embodiment, about
5 kg to about 50 kg aminoglycoside base starting material is used,
for example about 5 kg to about 35 kg aminoglycoside base starting
material. In one embodiment, at least about 8 kg aminoglycoside
base starting material is used. In another embodiment, at least
about 30 kg aminoglycoside base starting material is used. In one
embodiment, the aminoglycoside is amikacin (e.g. amikacin
sulfate).
[0024] The aminoglycoside used in the methods provided herein can
be present as a pharmaceutically acceptable salt or as the free
base. As provided above, in one embodiment, the aminoglycoside is
amikacin, e.g., amikacin sulfate.
[0025] In another embodiment, the aminoglycoside is amikacin,
apramycin, arbekacin, astromicin, capreomycin, dibekacin,
framycetin, gentamicin, hygromycin B, isepamicin, kanamycin,
neomycin, netilmicin, paromomycin, rhodestreptomycin, ribostamycin,
sisomicin, spectinomycin, streptomycin, tobramycin, verdamicin, or
a combination thereof.
[0026] In yet another embodiment, the aminoglycoside is AC4437,
amikacin, apramycin, arbekacin, astromicin, bekanamycin,
boholmycin, brulamycin, capreomycin, dibekacin, dactimicin,
etimicin, framycetin, gentamicin, H107, hygromycin, hygromycin B,
inosamycin, K-4619, isepamicin, KA-5685, kanamycin, neomycin,
netilmicin, paromomycm, plazomicin, ribostamycin, sisomicm,
rhodestreptomycin, sorbistin, spectinomycin, sporaricin,
streptomycin, tobramycin, verdamicin, vertilmicin, or a combination
thereof.
[0027] A "pharmaceutically acceptable salt" includes both acid and
base addition salts. A pharmaceutically acceptable addition salt
refers to those salts which retain the biological effectiveness and
properties of the free bases, which are not biologically or
otherwise undesirable, and which are formed with inorganic acids
such as, but are not limited to, hydrochloric acid (HCl),
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and
the like, and organic acids such as, but not limited to, acetic
acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic
acid, aspartic acid, benzenesulfonic acid, benzoic acid,
4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,
capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic
acid, citric acid, cyclamic acid, dodecylsulfuric acid,
ethane-1,2-disulfonic acid, ethanesulfonic acid,
2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric
acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic
acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid,
glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric
acid, lactic acid (e.g., as lactate), lactobionic acid, lauric
acid, maleic acid, malic acid, malonic acid, mandelic acid,
methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid,
naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic
acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic
acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic
acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic
acid, acetic acid (e.g., as acetate), tartaric acid, thiocyanic
acid, p-toluenesulfonic acid, trifluoroacetic acid (TFA),
undecylenic acid, and the like. In one embodiment, the
pharmaceutically acceptable salt is HCl, TFA, lactate or acetate.
In one embodiment, the pharmaceutically acceptable salt is a
sulfate salt, e.g., amikacin sulfate.
[0028] "Liposomal aminoglycoside formulation" refers to a
lipid-aminoglycoside formulation wherein the lipid is in the form
of a liposome and the aminoglycoside is encapsulated by the
liposome bilayer, or complexed with the liposome bilayer. Liposomes
are completely closed lipid bilayer membranes containing an
entrapped aqueous volume. Liposomes may 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) or a combination
thereof. 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.
[0029] In one embodiment, the lipid-aminoglycoside formulation is
manufactured via a method comprising a two-stream infusion process.
In one embodiment, the method comprises mixing a first lipid stream
with a second aminoglycoside stream in a T-shaped infusion module
or Y-shaped infusion module. The terms "T-shaped infusion module,"
and "Y-shaped infusion module" as used herein, refer to a T-shaped
or Y-shaped chamber in which two or more streams are combined, for
example, in which a lipid stream and a drug stream are combined to
form a single lipid-aminoglycoside stream. See, e.g., the diagram
at FIG. 1. It will be appreciated that the infusion module will
have a bore size appropriate for the required rate of the lipid and
drug streams used. Examples of suitable bore sizes include, but are
not limited to, 3/16'' and 3/8''.
[0030] In one embodiment, the first stream (lipid stream) comprises
an alcoholic (e.g., ethanolic) lipid solution. In one embodiment,
the second stream (aminoglycoside stream) comprises an aqueous
aminoglycoside solution (e.g., aqueous amikacin solution). In one
embodiment, the first and second streams are mixed to form a
combined lipid-aminoglycoside stream. In one embodiment, the first
and second streams each enter the infusion module and the first and
second streams are mixed in the infusion module. In a further
embodiment, the combined lipid-aminoglycoside stream exits the
infusion module and subsequently enters the reaction vessel. See
FIG. 1.
[0031] In one embodiment, the combined lipid-aminoglycoside stream
is mixed with an aqueous saline solution in a reaction vessel,
e.g., the same reaction vessel that the combined
lipid-aminoglycoside stream enters after exiting the infusion
module. In one embodiment, the aqueous saline solution comprises
about 0.5-2% aqueous sodium chloride solution (e.g., about 1.5%).
In one embodiment, the saline solution is added to the reaction
vessel prior to the combined lipid-aminoglycoside stream. In
another embodiment, the saline solution is added to the reaction
vessel at or about the same time as the combined
lipid-aminoglycoside stream. In a further embodiment, the saline
solution is added to the reaction vessel via a third stream. Thus,
in some embodiments, the lipid-aminoglycoside formulation is
manufactured via a method comprising a 3-stream infusion process.
In some embodiments, the third stream is added to the reaction
vessel separately from the combined lipid-aminoglycoside
stream.
[0032] In one embodiment, the combined lipid-aminoglycoside stream
comprises liposomal aminoglycoside, (e.g. amikacin), wherein the
encapsulation efficiency of the aminoglycoside within the liposomes
(or complexed to the liposomes) is at least about 40%.
"Encapsulation efficiency", as used herein, refers to the amount of
aminoglycoside encapsulated or complexed with liposomes prior to a
filtration step, e.g., tangential flow filtration of the liposomal
aminoglycoside formulation to remove free aminoglycoside. For
example, an encapsulation efficiency of between about 400% and
about 70% (e.g., from about 45% to about 55%) can be achieved by
mixing the lipid and aminoglycoside (e.g. amikacin) streams
according to the method of this invention as herein described.
[0033] In one embodiment, the aminoglycoside stream and the lipid
stream are each maintained at a temperature from about 30.degree.
C. to about 50.degree. C. prior to mixing the two streams. In a
further embodiment, the aminoglycoside and lipid streams are each
maintained at a temperature of from about 35.degree. C. to about
45.degree. C., for example from about 38.degree. C. to about
42.degree. C. prior to mixing. In another embodiment, the
combination of the lipid and aminoglycoside solutions exhibits
exothermal behavior. The temperature of the combined
lipid-aminoglycoside stream, in one embodiment, is from about
40.degree. C. to 55.degree. C. In a further embodiment, the
temperature of the combined lipid-aminoglycoside stream is from
about 45.degree. C. to about 50.degree. C. In another embodiment,
the combined lipid-aminoglycoside stream is mixed with an aqueous
saline solution in a reaction vessel, wherein the aqueous saline
solution is maintained at room temperature prior to mixing with the
combined lipid-aminoglycoside stream. In another embodiment, an
aqueous saline solution is added to the reaction vessel via a third
stream, wherein the third stream is maintained at room temperature
prior to mixing with the combined lipid-aminoglycoside stream. In
one embodiment, the combined lipid-aminoglycoside stream is cooled
upon entering the reaction vessel. In another embodiment, the
combined lipid-aminoglycoside stream is cooled by the aqueous
saline solution in the reaction vessel. In another embodiment, the
combined lipid-aminoglycoside stream is cooled upon entering the
reaction vessel. In one embodiment, the reaction vessel is
maintained at a temperature from about 25.degree. C. and about
40.degree. C., e.g., from about 27.degree. C. to about 35.degree.
C. In another embodiment, the reaction vessel is maintained at a
temperature of about 30.degree. C. In another embodiment, the
reaction vessel is maintained at a temperature of about 33.degree.
C.
[0034] In one embodiment, the lipid component of the liposomal drug
formulation manufactured by the method provided herein comprises
electrically net neutral lipids, positively charged lipids,
negatively charged lipids, or a combination thereof. In another
embodiment, the lipid component comprises electrically net neutral
lipids. In a further embodiment, the lipid component consists
essentially of electrically net neutral lipids. In even a further
embodiment, the lipid is DPPC and cholesterol.
[0035] The lipids used in the manufacture of the liposomal
formulations of the present invention can be synthetic,
semi-synthetic or naturally-occurring lipids, including one or more
of phospholipids, tocopherols, sterols, fatty acids,
negatively-charged lipids and cationic lipids. In one embodiment,
the lipid component consists of electrically neutral lipids, e.g.,
a sterol and a phospholipid.
[0036] In one embodiment, at least one phospholipid is present in
the liposomal drug formulation. In one embodiment, the phospholipid
is phosphatidylcholine (PC), phosphatidylglycerol (PG),
phosphatidylinositol (PI), phosphatidylserine (PS),
phosphatidylethanolamine (PE), phosphatidic acid (PA), soy
phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG), soy
phosphatidylserine (SPS), soy phosphatidylinositol (SPI), soy
phosphatidylethanolamine (SPE), and soy phosphatidic acid (SPA);
hydrogenated egg and soya counterparts (e.g., hydrogenated egg
phosphatidylcholine and hydrogenated soy phosphatidylcholine),
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 carbon chains on these
fatty acids can be saturated or unsaturated, and the phospholipid
may be made up of fatty acids of different chain lengths and
different degrees of unsaturation.
[0037] In one embodiment, the lipid component of the liposomal drug
formulation manufactured by the method provided herein comprises a
phosphatidylcholine. For example, in one embodiment, the lipid
component in the liposomal drug formulation comprises
dipalmitoylphosphatidylcholine (DPPC). In one embodiment, the lipid
component of the liposomal drug formulation comprises DPPC and a
sterol, for example DPPC and cholesterol. Alternatively, the lipid
consists essentially of DPPC and cholesterol, or consists of DPPC
and cholesterol. In a further embodiment, the DPPC and cholesterol
have a molar ratio in the range of from about 19:1
(DPPC:cholesterol) to about 1:1 (DPPC:cholesterol), or from about
9:1 (DPPC:cholesterol) to about 1:1 (DPPC:cholesterol), or from
about 4:1 (DPPC:cholesterol) to about 1:1 (DPPC:cholesterol), or
from about 2:1 (DPPC:cholesterol) to about 1:1 (DPPC:cholesterol).
In even a further embodiment, the DPPC and cholesterol have a molar
ratio of about 2:1 (DPPC:cholesterol).
[0038] Other examples of lipid components of the liposomal drug
formulation manufactured by the method provided herein include, but
are not limited to, dimyristoylphosphatidycholine (DMPC),
dimyristoylphosphatidylglycerol (DMPG),
dipalmitoylphosphatidcholine (DPPC),
dipalmitoylphosphatidylglycerol (DPPG),
distearoylphosphatidylcholine (DSPC),
distearoylphosphatidylglycerol (DSPG),
dioleylphosphatidyl-ethanolamine (DOPE), mixed phospholipids such
as palmitoylstearoylphosphaidyl-choline (PSPC), and single acylated
phospholipids, for example, mono-oleoyl-phosphatidylethanolamine
(MOPE).
[0039] Examples of sterol compounds in the liposomal drug
formulation manufactured by the method provided herein include, but
are not limited to, cholesterol, esters of cholesterol including
cholesterol hemi-succinate, salts of cholesterol including
cholesterol hydrogen sulfate and cholesterol sulfate, ergosterol,
esters of ergosterol including ergosterol hemi-succinate, salts of
ergosterol including ergosterol hydrogen sulfate and ergosterol
sulfate, lanosterol, esters of lanosterol including lanosterol
hemi-succinate, salts of lanosterol including lanosterol hydrogen
sulfate, lanosterol sulfate and tocopherols. The tocopherols
include tocopherols, esters of tocopherols including tocopherol
hemi-succinates, salts of tocopherols including tocopherol hydrogen
sulfates and tocopherol sulfates. The term "sterol compound"
includes sterols, tocopherols and the like. Tocopherols and their
water-soluble derivatives have been used to form liposomes, see,
e.g., PCT Publication No. 87/02219.
[0040] In one embodiment, the concentration of lipid in the first
stream is from about 10 g/L to about 50 g/L, or from about 10 g/L
to about 30 g/L, or from about 15 g/L to about 25 g/L. In one
embodiment, the concentration of lipid in the first stream is about
17 g/L, about 18 g/L, about 19 g/L, about 20 g/L, about 21 g/L,
about 22 g/L, about 23 g/L, about 24 g/L or about 25 g/L. In one
embodiment, the concentration of lipid in the first stream is about
20 g/L
[0041] In one embodiment, the concentration of aminoglycoside in
the second stream (aminoglycoside stream) is from about 10 g/L to
about 100 g/L; or from about 20 g/L to about 70 g/L; or from about
30 g/L to about 60 g/L; or from about 40 g/L to about 50 g/L. In
one embodiment, the concentration of drug in the second stream is
about 4 g/L, about 42 g/L, about 43 g/L, about 44 g/L, about 45
g/L, about 46 g/L, about 47 g/L, about 48 g/L, about 49 g/L or
about 50 g/L. In one embodiment, the concentration of
aminoglycoside in the second stream is about 45 g/L. In a further
embodiment, the aminoglycoside is amikacin.
[0042] In one embodiment of the invention, the pH of the
aminoglycoside stream is from 6 to about 7, or from about 6.5 to
about 7.0. In a further embodiment, the pH of the aminoglycoside
stream is about 6.7. The aminoglycoside stream pH may be adjusted
to the appropriate pH using a suitable base, such as an alkali or
alkaline earth metal hydroxide, e.g. sodium hydroxide.
[0043] In another embodiment, the aqueous saline solution comprises
about 0.5% sodium chloride to about 3% sodium chloride, for example
about 0.75%, about 1.0%, about 1.25%, about 1.5%, about 1.75%,
about 2.0%, or about 2.5% sodium chloride. In one embodiment, the
aqueous saline solution comprises about 1.5% sodium chloride.
[0044] In one embodiment, the flow rate of the lipid stream is from
about 0.5 kg/min to about 1.5 kg/min and the flow rate of the
aminoglycoside stream is from about 1 kg/min to about 2 kg/min.
[0045] In a further embodiment, the flow rate of the lipid stream
is from about 3 kg/min to about 4 kg/min and the flow rate of the
drug stream is from about 5 kg/min to about 7 kg/min. In another
embodiment, the relative flow rate ratio of the aminoglycoside
stream to the lipid stream is about 1.5:1 (aminoglycoside stream
flow rate:lipid stream flow rate) to about 2:1 (aminoglycoside
stream flow rate:lipid stream flow rate).
[0046] In one embodiment, when the flow rate of the lipid stream is
from about 0.5 kg/min to about 1.5 kg/min and the flow rate of the
aminoglycoside stream is from about 1 kg/min to about 2 kg/min, the
flow rate of the third stream comprising aqueous saline solution is
from about 0.5 L/min and about 2.0 L/min, for example, from about
1.0 L/min to about 2.0 L/min, e.g., from about 1.0 L/min to about
1.5 L/min, including about 1.25 L/min. In another embodiment, when
the flow rate of the lipid stream is from about 3 kg/min to about 4
kg/min and the flow rate of the aminoglycoside stream is from about
5 kg/min to about 7 kg/min, the flow rate of the third stream
comprising the aqueous saline solution is from about 3 L/min and
about 6 L/min, for example, from about 4 L/min to about 6 L/min,
e.g. from about 4.5 L/min to about 5.5 L/min, including about 5
L/min.
[0047] In one embodiment of the invention, the lipid and
aminoglycoside (e.g., amikacin) solutions are both filtered, for
example through one or more (e.g., two in series) about 0.2 .mu.m
filters, prior to mixing into a combined stream (FIG. 1). Although
FIG. 1 shows two filters in series, it should be noted that this
number can be changed according to the preference of the user of
the method. For example, one to five filters can be used to
initially filter the lipid stream and the aminoglycoside
stream.
[0048] In another embodiment, the aqueous saline solution (e.g.,
1.5% saline solution) is also filtered, for example through one or
more (e.g., two in series) about 0.2 .mu.m filters, prior to mixing
with the lipid-aminoglycoside combined stream in the reaction
vessel. In a further embodiment, the liposomal suspension,
comprising liposomes formed at the intersection of the lipid and
aminoglycoside streams, and/or in the combined lipid-aminoglycoside
stream, is concentrated within the reaction vessel using a
recirculating filtration system such as diafiltration. As provided
above, "encapsulation efficiency", as used herein, refers to the
amount of aminoglycoside encapsulated or complexed with liposomes
prior to a filtration step, e.g., tangential flow filtration of the
liposomal aminoglycoside formulation to remove free aminoglycoside.
For example, an encapsulation efficiency of between about 40% and
about 70% (e.g., from about 45% to about 55%) can be achieved by
mixing the lipid and aminoglycoside (e.g. amikacin) streams
according to the method of this invention as herein described.
[0049] In another embodiment, the resulting concentrated liposomal
suspension is treated (i.e., "washed") with additional aqueous
saline solution (e.g., filtered 1.5% saline solution) and subjected
to further filtration using a recirculating filtration system such
as diafiltration until the liposomal suspension contains an
appropriate final aminoglycoside concentration and substantially
all of the free aminoglycoside is removed. In a further embodiment,
three or more washes (e.g., 3, 4, 5 or 6 washes) are conducted to
achieve the appropriate final aminoglycoside concentration.
[0050] In one embodiment, following washing, the concentration of
aminoglycoside present in the liposomal aminoglycoside formulation
manufactured on a large-scale according to a method provided herein
is about 10 g/L or greater. In a further embodiment, aminoglycoside
is present in the formulation at a concentration of about 20 g/L or
greater. In a further embodiment, aminoglycoside is present in the
formulation at a concentration of about 30 g/L or greater. In a
further embodiment, aminoglycoside is present in the formulation at
a concentration of about 40 g/L or greater. In a further
embodiment, aminoglycoside is present in the formulation at a
concentration of about 50 g/L or greater. In a further embodiment,
aminoglycoside is present in the formulation at a concentration of
about 60 g/L or greater. In a further embodiment, aminoglycoside is
present in the formulation at a concentration of about 70 g/L or
greater. In another embodiment, the aminoglycoside is present in
the formulation at a concentration of from about 10 g/L to about
100 g/L. In a further embodiment, the aminoglycoside is amikacin.
In one embodiment, the aminoglycoside is present in the formulation
at a concentration of from about 50 g/L to about 100 g/L. In a
further embodiment, the aminoglycoside is amikacin. In one
embodiment, the aminoglycoside is present in the formulation at a
concentration of from about 60 g/L to about 80 g/L. In a further
embodiment, the aminoglycoside is amikacin. In yet another
embodiment, the aminoglycoside is present in the formulation at a
concentration from about 65 g/L to about 80 g/L. In a further
embodiment, the aminoglycoside is amikacin. In yet another
embodiment, the aminoglycoside is present in the formulation at a
concentration from about 65 g/L to about 75 g/L. In a further
embodiment, the aminoglycoside is amikacin. In another embodiment,
amikacin is present in the formulation at a concentration of about
70 g/L. In a further embodiment, the aminoglycoside is
amikacin.
[0051] In a further embodiment, following washing, the
concentration of lipid present in the liposomal drug formulation
manufactured on a large-scale according to a method provided herein
is from about 10 g/L to about 100 g/L, including about 20 g/L to
about 80 g/L and about 40 g/L to about 60 g/L (e.g., about 50
g/L).
[0052] In another embodiment, following diafiltration, the
lipid-to-aminoglycoside weight ratio in a liposomal drug
formulation manufactured on a large-scale according to a method
provided herein is less than 1:1, for example between about 0.5:1
(lipid:aminoglycoside) and about 0.8:1 (lipid:aminoglycoside)
(e.g., about 0.5:1 (lipid:aminoglycoside) or 0.6:1
(lipid:aminoglycoside) or 0.7:1 (lipid:aminoglycoside) or 0.8:1
(lipid:aminoglycoside)). In one embodiment, the
lipid-to-aminoglycoside weight ratio is about 0.7:1
(lipid:aminoglycoside).
[0053] The liposomal aminoglycoside formulation manufactured on a
large-scale according to a method provided herein comprises
liposome particles with a mean particle size (i.e. a mean diameter)
of from about 200 nm to about 500 nm, for example from about 200 nm
to about 400 nm (e.g. from about 250 nm to about 350 nm). The
liposome diameter may be measured using commercially available
light scattering technology, for example by quasi-elastic light
scattering using a Nicomp.TM. 380 submicron particle sizer (Nicomp,
Santa Barbara, Calif. USA).
[0054] The present invention is further illustrated by reference to
the following Examples. However, it should be noted that these
Examples, like the embodiments and aspects described above, are
illustrative and are not to be construed as limiting the scope of
the invention in any way.
EXAMPLES
Example 1: Manufacturing Process and Process Controls for Liposomal
Amikacin
[0055] The manufacture of liposomal amikacin sulfate was conducted
using an aseptic process that involves the preparation of three
sterile solution streams, mixing the lipid and amikacin sulfate
streams at appropriate flow rates via a T-connector infusion
module, collecting the combined lipid-amikacin sulfate streams
containing liposomes with encapsulated amikacin sulfate in a
sterilized diafiltration (reaction) vessel, adding a stream of 1.5%
aqueous sodium chloride at an appropriate flow rate to the
diafiltration vessel, followed by diafiltration (including washing)
and concentration of the resulting liposomal dispersion to form the
final product.
a) Solution Preparation: Sufficient quantities of the following
three solutions were prepared. [0056] Amikacin sulfate solution:
Amikacin sulfate in water for injection (WFI), pH adjusted with
sodium hydroxide to 6.6-6.8. [0057] Lipid solution:
DPPC/cholesterol (2:1 w/w) in ethanol. [0058] 1.5% Sodium chloride
solution: 1.5% Sodium chloride in WFI, pH adjusted to 6.6-6.8. The
solutions must be used within 24 hours of preparation. b)
Infusion/Initial Concentration: The amikacin sulfate solution and
lipid solution were warmed and passed through separate sterilizing
filters before flowing through an in-line T-connector infusion
module at controlled rates of addition. The mixed streams were
collected in a pre-sterilized reactor vessel. Simultaneously, 1.5%
aqueous sodium chloride solution was passed through a sterilizing
filter and introduced as a stream at an appropriate flow rate into
the reactor vessel. At this stage, the solution may be sampled for
the level of amikacin encapsulation. c) Diafiltration:
Diafiltration was conducted. This step functions to remove the
ethanol from the bulk solution and to wash away any "unentrapped"
or free amikacin sulfate. d) Final Concentration: Using an
in-process test result, the bulk solution was concentrated to an
appropriate concentration level of amikacin sulfate. After
concentration is complete, confirmatory tests for concentration of
amikacin sulfate and L/D ratio may be performed. Table 1 describes
experiments (A) and (B), performed according to the general method
of Example 1. In (A), a 3/8'' T-connector infusion module is used.
In (B), a 3/16'' T-connector infusion module is used.
TABLE-US-00001 [0058] TABLE 1 Amikacin Amikacin Amikacin Amikacin
calculated sulfate sulfate Lipid Lipid sulfate free base DPPC
solution solution solution solution L/D concentration weight weight
Cholesterol concentration flow rate concentration flow rate ratio
obtained Ex. (kg) (kg) weight (kg) (g/L) (kg/min) (g/L) (kg/min)
obtained (mg/mL) (A) 30.428 7.33 .+-. 3.67 .+-. 45 5.64 20 3.62
0.72 70 .+-. 3 0.050 0.050 (B) 8.250 1.666 .+-. 0.834 .+-. 45 1.464
20 0.851 0.68 to 70 .+-. 3 0.001 0.001 0.74
[0059] In additional experiments, generally following the process
of Example 1, the lipid and amikacin stream (flow) rates were
varied, and the resulting concentrations of lipid and amikacin in
the liposomal formulations were measured. The L/D ratio for each
experiment was calculated and the results presented in FIG. 2. The
results provide guidance for an optimal relative lipid/amikacin
flow rate to achieve a preferred L/D ratio.
[0060] All, documents, patents, patent applications, publications,
product descriptions, and protocols which are cited throughout this
application are incorporated herein by reference in their
entireties for all purposes.
[0061] The embodiments illustrated and discussed in this
specification are intended only to teach those skilled in the art
the best way known to the inventors to make and use the invention.
Modifications and variation of the above-described embodiments of
the invention are possible without departing from the invention, as
appreciated by those skilled in the art in light of the above
teachings. It is therefore understood that, within the scope of the
claims and their equivalents, the invention may be practiced
otherwise than as specifically described.
* * * * *