U.S. patent application number 14/881011 was filed with the patent office on 2016-09-15 for platinum aggregates and process for producing the same.
The applicant listed for this patent is Insmed Incorporated. Invention is credited to Lawrence T. BONI, Jin K. Lee, Vladimir MALININ, Brian S. Miller, Fangjun WU.
Application Number | 20160263030 14/881011 |
Document ID | / |
Family ID | 40952714 |
Filed Date | 2016-09-15 |
United States Patent
Application |
20160263030 |
Kind Code |
A1 |
Lee; Jin K. ; et
al. |
September 15, 2016 |
PLATINUM AGGREGATES AND PROCESS FOR PRODUCING THE SAME
Abstract
One aspect of the disclosure relates to a new fom1 of
lipid-complexed active platinum compound, which allows for high
concentrations of platinum compound in the composition. For
example, the concentration of cisplatin in the composition is
higher at room temperature, e.g., about greater than 1.2 mg/mL,
compared to 1 mg/mL in aqueous solution. In one embodiment, the
present invention is directed to a composition comprising a
lipid-complexed active platinum compound, wherein the complex has a
lipid to drug (L/D) ratio of less than about 1 by weight, e.g.
about 0.10 to 1, wherein the lipid-complexed active platinum
compound comprises at least one lipid and at least one active
platinum compound. In other embodiments, wherein lipid-complexed
active platinum compound has an average volume-weighted diameter of
about 0.5 to about 20 microns. In still other embodiments, the
composition further comprises a liposome. The liposome may comprise
at least one lipid, and may further comprise at least one active
platinum compound. The disclosure also relates to a pharmaceutical
formulation comprising a lipid complexed active platinum compound
and a pharmaceutically acceptable carrier or diluent. The
pharmaceutical formulation may be formulated for inhalation or
injection.
Inventors: |
Lee; Jin K.; (Belle Mead,
NJ) ; Miller; Brian S.; (Mercerville, NJ) ;
WU; Fangjun; (Livingston, NJ) ; BONI; Lawrence
T.; (Monmouth Junction, NJ) ; MALININ; Vladimir;
(Plainsboro, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Insmed Incorporated |
Bridgewater |
NJ |
US |
|
|
Family ID: |
40952714 |
Appl. No.: |
14/881011 |
Filed: |
October 12, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12027752 |
Feb 7, 2008 |
9186322 |
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14881011 |
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10634144 |
Aug 4, 2003 |
|
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12027752 |
|
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60400875 |
Aug 2, 2002 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 33/24 20130101;
A61K 47/554 20170801; A61K 47/544 20170801; A61K 9/1277 20130101;
A61P 35/00 20180101; A61K 31/28 20130101; A61K 9/127 20130101; A61K
9/0078 20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 33/24 20060101 A61K033/24 |
Claims
1. A composition comprising a lipid-complexed active platinum
compound, wherein the complex has a lipid to drug ratio less than
about 1 by weight, wherein the lipid-complexed active platinum
compound comprises at least one lipid and at least one active
platinum compound.
2. The composition of claim 1, wherein the lipid to drug ratio is
about 0.10 to about 1 by weight.
3. The composition of claim 2, wherein the lipid to drug ratio is
about 0.10 to about 0.50 by weight.
4. The composition of claim 3, wherein the lipid to drug ratio is
about 0.15 to about 0.45 by weight.
5. The composition of claim 4, wherein the lipid to drug ratio is
about 0.20 to about 0.40 by weight.
6. The composition of claim 5, wherein the lipid to drug ratio is
about 0.2 by weight.
7. The composition of claim 1, wherein lipid-complexed active
platinum compound has an average volume-weighted diameter of about
0.5 to about 20 microns.
8. The composition of claim 7, wherein the average volume-weighted
diameter is about 1 to about 15 microns.
9. The composition of claim 8, wherein the average volume-weighted
diameter is about 2 to 10 microns.
10. The composition of claim 1, wherein the concentration of the
active platinum compound is greater than about 1.2 mg/mL.
11. The composition of claim 10, wherein the active platinum
compound has a concentration of about 1.2 to about 20 mg/mL.
12. The composition of claim 11, wherein the platinum compound has
a concentration of about 1.5 to about 5 mg/mL.
13. The composition of claim 1, further comprising a liposome.
14. The composition of claim 13, wherein the liposome comprises an
active platinum compound.
15. The composition of claim 14, wherein the lipid-complexed active
platinum compound contains about 70 to about 100% of the total
active platinum compound in the composition.
16. The composition of claim 15, wherein in the lipid-complexed
active platinum compound contains about 75 to about 99% of the
total active platinum compound.
17. The composition of claim 16, wherein the lipid-complexed active
platinum compound contains about 80 to about 90% of the total
active platinum compound.
18. The composition of claim 14, wherein the liposome contains
about 0 to about 30% of the total active platinum compound in the
composition.
19. The composition of claim 18, wherein the liposome contains
about 0.5 to about 25% of the total active platinum compound.
20. The composition of claim 19, wherein the liposome contains
about 1 to about 20% of the total platinum compound.
21-44. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/027,752, filed Feb. 7, 2008, now U.S. Pat. No. 9,186,322,
which is a continuation in part of U.S. application Ser. No.
10/634,144, filed Aug. 4, 2003, which claims priority to U.S.
Provisional Application No. 60/400,875, filed Aug. 2, 2002, each of
which are herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Liposomes and lipid complexes have been long recognized as
drug delivery systems which can improve therapeutic and diagnostic
effectiveness of many bioactive agents and contrast agents.
Experiments with a number of different antibiotics and X-ray
contrast agents have shown that better therapeutic activity or
better contrast with a higher level of safety can be achieved by
encapsulating bioactive agents and contrast agents with liposomes
or lipid complexes. Research on liposomes and lipid complexes as
encapsulating systems for bioactive agents has revealed that a
successful development and commercialization of such products
requires reproducible methods of large scale production of lipid
vesicles with suitable characteristics. Consequently, workers have
searched for methods which consistently produce liposomes or lipid
complexes of the required size and concentration, size distribution
and, importantly, entrapping capacity, with flexible lipid
composition requirements. Such methods seek to provide liposomes or
lipid complexes with consistent active substance to lipid ratio
while respecting currently accepted good manufacturing practices
for pharmaceutical products.
[0003] Conventional liposome and lipid complex preparation methods
include a number of steps in which the bilayer-forming components
(for example, phospholipids or mixtures of phospholipids with other
lipids e.g., cholesterol) are dissolved in a volatile organic
solvent or solvent mixture in a round bottom flask followed by
evaporation of the solvent under conditions, such as temperature
and pressure, which will prevent phase separation. Upon solvent
removal a dry lipid mixture, usually in form of a film deposit on
the walls of the reactor, is hydrated with an aqueous medium which
may contain dissolved buffers, salts, conditioning agents and an
active substance to be entrapped. Liposomes or lipid complexes form
in the hydration step such that a proportion of the aqueous medium
becomes encapsulated in the liposomes. The hydration can be
performed with or without energizing the solution by means of
stirring, sonication or micro fluidization or with subsequent
extrusion through one or more filters, such as polycarbonate
filters. The free non-encapsulated active substance can be
separated for recovery and the product is filtered, sterilized,
optionally lyophilized, and packaged.
[0004] Other methods of making liposomes or lipid complexes
involving injection of organic solutions of lipids into an aqueous
medium with continuous removal of solvent, use of spray drying,
lyophilization, microemulsification and micro fluidization, and the
like have been proposed in a number of publications or patents.
Such patents include, for example, U.S. Pat. No. 4,529,561 and U.S.
Pat. No. 4,572,425.
[0005] Cisplatin--cis-diamine-dichloroplatinum (II)--is one of the
more effective antitumor agents used in the systemic treatment of
cancers. This chemotherapeutic drug is highly effective in the
treatment of tumor models in laboratory animals and in human
tumors, such as endometrial, bladder, ovarian and testicular
neoplasms, as well as squamous cell carcinoma of the head and neck
(Sur, et al., 1983 Oncology 40(5): 372-376; Steerenberg, et al.,
1988 Cancer Chemother Pharmacol. 21(4): 299-307). Cisplatin is also
used extensively in the treatment of lung carcinoma, both small
cell lung carcinoma (SCLC) and non-small cell lung carcinoma
(NSCLC) (Schiller et al., 2001 Oncology 61(Suppl 1): 3-13). Other
active platinum compounds (defined below) are useful in cancer
treatment.
[0006] Like other cancer chemotherapeutic agents, active platinum
compounds such as cisplatin are typically highly toxic. The main
disadvantages of cisplatin are its extreme nephrotoxicity, which is
the main dose-limiting factor, its rapid excretion via the kidneys,
with a circulation half life of only a few minutes, and its strong
affinity to plasma proteins (Freise, et al., 1982 Arch Int
Pharmacodyn Ther. 258(2): 180-192).
[0007] Attempts to minimize the toxicity of active platinum
compounds have included combination chemotherapy, synthesis of
analogues (Prestayko et al., 1979 Cancer Treat Rev. 6(1): 17-39;
Weiss, et al., 1993 Drugs. 46(3): 360-377), immunotherapy and
entrapment in liposomes (Sur, et al., 1983; Weiss, et al., 1993).
It has been reported that antineoplastic agents, including
cisplatin, entrapped in liposomes have a reduced toxicity, relative
to the agent in free form, while retaining antitumor activity
(Steerenberg, et al., 1987; Weiss, et al, 1993).
[0008] Cisplatin, however, is difficult to efficiently entrap in
liposomes or lipid complexes because of its low aqueous solubility,
approximately 1.0 mg/mL at room temperature, and low lipophilicity,
both of which properties contribute to a low cisplatin/lipid
ratio.
[0009] Liposomes and lipid complexes containing cisplatin suffer
from another problem--stability of the composition. In particular,
maintenance of bioactive agent potency and retention of the
bioactive agent in the liposome during storage are recognized
problems (Freise, et al., 1982; Gondal, et al., 1993; Potkul, et
al., 1991 Am J Obstet Gynecol. 164(2): 652-658; Steerenberg, et
al., 1988; Weiss, et al., 1993) and a limited shelf life of
liposomes containing cisplatin, on the order of several weeks at
4.degree. C., has been reported (Gondal, et al., 1993 Eur J Cancer.
29A(11): 1536-1542; Potkul, et al., 1991).
SUMMARY OF THE INVENTION
[0010] Provided, among other things, is a new form of
lipid-complexed active platinum compound, which allows for high
concentrations of platinum compound in a composition. For example,
the concentration of cisplatin in the composition is higher at room
temperature, e.g., about greater than 1.2 mg/mL, compared to 1
mg/mL in aqueous solution. The lipid-complexed active platinum
compound is stable over long periods of time. For example, the
lipid-complexed active platinum compound is stable for more than
one year.
[0011] In one embodiment, the present invention is directed to a
composition comprising a lipid-complexed active platinum compound,
wherein the complex has a lipid to drug (L/D) ratio of less than
about 1 by weight, e.g. about 0.10 to 1, wherein the
lipid-complexed active platinum compound comprises at least one
lipid and at least one active platinum compound.
[0012] In some embodiments, wherein lipid-complexed active platinum
compound has an average volume-weighted diameter of about 0.5 to
about 20 microns.
[0013] In some embodiments, the composition further comprises a
liposome. The liposome may comprise at least one lipid, and may
further comprise at least one active platinum compound.
[0014] In some embodiments, the present invention relates to a
pharmaceutical formulation comprising a lipid complexed active
platinum compound and a pharmaceutically acceptable carrier or
diluent. The pharmaceutical formulation may be formulated for
inhalation or injection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1(A) depicts a graph of the transition temperature for
dissolution and precipitation of cisplatin in aqueous solution as a
function of cisplatin concentration during heating. FIG. 1(B)
depicts a similar graph of transplatin. FIGS. 1(C) and 1(D) depict
graphs of the transition temperature of dissolved-precipitated
cisplatin in aqueous solution as a function of concentration during
cooling of cisplatin and transplatin, respectively.
[0016] FIG. 2 shows the stability of one liter batches of
lipid-complexed cisplatin according to the invention.
[0017] FIGS. 3(A) and 3(B). Cisplatin-rich lipid particulates in
the dense, settled fraction of a composition of the present
invention. Two representative TEM images are shown (A and B).
Samples were prepared as described in the Examples. Large faint
circles in the background are part of the copper plate
structure.
[0018] FIGS. 4(A) and (B) are represented TEM images of cisplatin
crystals taken at magnification 2000.times..
[0019] FIGS. 5(A) and 5(B) are Representative TEM images of the
dense fraction particles from the density gradient of a nebulized
cisplatin lipid complex formulation. Images were taken at
magnification 6300.times. (A) and 8000.times. (B).
[0020] FIGS. 6(A) and (B) are representative TEM images of the
dense fraction particles from the density gradient of a nebulized
cisplatin lipid complex formulation. Images were taken at
magnification 8000.times. (A) and 4000.times. (B).
[0021] FIG. 7 depicts a bar graph showing the effect of
nebulization on the distribution of cisplatin in the light and
dense fractions of a composition of the present invention.
[0022] FIGS. 8(A)-(D) are representative optical micrographs of a
composition of the present invention.
[0023] FIGS. 9(A) and (B) are representative freeze fracture
electron micrographs of a composition of the present invention.
[0024] FIGS. 10(A)-(D) are graphs depicting the particle size
analysis of several batches of a composition of the present
invention.
[0025] FIGS. 11(A) and (B) are graphs depicting the particle size
analysis of two batches of a composition of the present invention
after nebulization.
DETAILED DESCRIPTION OF THE INVENTION
[0026] 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.
[0027] 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.
[0028] The term "bioavailable" is art-recognized and refers to a
form of the subject invention that allows for it, or a portion of
the amount administered, to be absorbed by, incorporated to, or
otherwise physiologically available to a subject or patient to whom
it is administered.
[0029] The terms "comprise" and "comprising" are used in the
inclusive, open sense, meaning that additional elements may be
included.
[0030] The term "including" is used herein to mean "including but
not limited to". "Including" and "including but not limited to" are
used interchangeably.
[0031] 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).
[0032] A "patient," "subject" or "host" to be treated by the
subject method may mean either a human or non-human animal.
[0033] 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.
[0034] The term "treating" is art-recognized and refers to curing
as well as ameliorating at least one symptom of any condition or
disorder.
[0035] "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 injecting the solution into an
aqueous medium containing bioactive agents. Typically a process
compatible solvent is one that can be washed away in a aqueous
process such as dialysis. The composition that is cool/warm cycled
is preferably formed by solvent infusion, with ethanol infusion
being preferred. Alcohols are preferred as solvents. "Ethanol
infusion," a type of solvent infusion, is a process that includes
dissolving one or more lipids in a small, preferably minimal,
amount of ethanol to form a lipid solution and then injecting the
solution into an aqueous medium containing bioactive agents. A
"small" amount of solvent is an amount compatible with forming
liposomes or lipid complexes in the infusion process.
[0036] A "hydrophobic matrix carrying system" is the lipid/solvent
mixture produced by the solvent infusion process described
above.
[0037] The present invention relates to a new form of
lipid-complexed active platinum compound, which allows for high
concentrations of platinum compound in the composition. For
example, the room temperature concentration of cisplatin in the
composition is higher, e.g., greater than 1.2 mg/mL, compared to 1
mg/mL of active platinum compound aqueous solution. In some
embodiments, the lipid complexed active platinum compound comprises
a lipid bilayer, where the lipid bilayer encapsulates or entraps
the platinum compound.
[0038] In some embodiments, the composition comprises a
lipid-complexed active platinum compound, wherein the complex has a
lipid to drug ratio of less than about 1 by weight. For example the
L/D ratio can be about 0.10 to 1 by weight, wherein the
lipid-complexed active platinum compound comprises at least one
lipid and at least one active platinum compound. In some
embodiments, the lipid to drug ratio is about 0.10 to about 0.50 by
weight. In some embodiments, the lipid to drug ratio is about 0.15
to about 0.45 by weight, and in other embodiments, the lipid to
drug ratio is about 0.20 to 0.40 by weight. In some embodiments,
the lipid to drug ratio is about 0.2 by weight.
[0039] The lipid-complexed active platinum compound may have an
average volume-weighted diameter of about 0.5 to about 20 microns.
In some embodiments, the average volume-weighted diameter is about
1 to about 15 microns, or about 2 to about 10 microns. In other
embodiments, the average volume-weighted diameter is about 3, 4, 5,
or 6 microns.
[0040] In some embodiments, the concentration of the active
platinum compound in the composition is greater than about 1.2
mg/mL, for example about 1.2 to about 20 mg/mL. In other
embodiments, the concentration of the active platinum compound is
about 1.2 to 10 mg/ML, about 1.5 to about 5 mg/mL, about 2.0 to
about 4 mg/mL, or about 3.0 to 2.5 mg/mL. In other embodiments, the
concentration is about 2, about 3, or about 5 mg/mL.
[0041] In some embodiments, the composition comprising the
lipid-complexed active platinum compound further comprises a
liposome. As explained in greater detail in the examples below, the
liposome comprises at least one lipid. The lipid may be the same as
or different from the lipid in the lipid-complexed active platinum
compound. In some embodiments, the liposome further comprises an
active platinum compound, wherein the active platinum compound can
be the same as or different from the active platinum compound of
the lipid-complexed active platinum compound. The active platinum
compound may be entrapped in the liposome.
[0042] In some embodiments, the liposomes have an average diameter
of about 0.1 to about 1 micron, 0.1 to about 0.5 microns, about 0.2
to about 0.5 microns, or about 0.2 to about 0.3 microns.
[0043] When the lipid composition further comprises a liposome, the
lipid-complexed active platinum compound may contain about 70 to
about 100% of the total active platinum compound in the
composition. In other embodiments, the lipid-complexed active
platinum compound contains about 75 to about 99%, about 75 to about
95%, or about 80 to about 90% of the total active platinum compound
in the composition. In some embodiments, the liposome contains
about 0 to about 30% of the total active platinum compound in the
composition. In other embodiments, the liposome may contain about
0.5 to about 25%, about 1 to about 20%, or about 5 to 10% of the
total active platinum compound.
[0044] When the composition further comprises a liposome, the
lipid-complexed active platinum compound may contain about 0.1 to
about 5% of the total lipid in the composition. In some
embodiments, the lipid-complexed active platinum compound contains
about 0.25 to about 3%, or about 0.5 to about 2% of the total
lipid. In some embodiments, the liposome contains about 75 to about
99.5%, about 80 to about 95%, or about 85 to about 95% of the total
lipid in the composition.
[0045] When present in the composition, the liposome may have a
lipid to active platinum compound ratio of about 100:1 to about
400:1 by weight. In other embodiments, the lipid to active platinum
compound ratio of the liposome is about 200:1 to about 400:1, about
200: to 300:1 about 250:1 to 300:1 or about 250:1 by weight.
[0046] In some embodiments, the composition comprising a
lipid-complexed active platinum compound and a liposome has an
active platinum compound concentration of greater than about 1.2
mg/mL, for example, the concentration may be about 1.2 to about 20
mg/mL, about 1.2 to about 10 mg/mL, about 1.5 to about 5 mg/mL,
about 2.0 to about 4 mg/mL, or about 3.0 to 2.5 mg/mL. In other
embodiments, the concentration is about 2, about 3, or about 5
mg/mL.
[0047] An "active platinum" compound is a compound containing
coordinated platinum and having antineoplastic activity. Additional
active platinum compounds include, for example, carboplatin and
DACH-platinum compounds such as oxaliplatin. In certain
embodiments, the active platinum compounds in the composition is
selected from the group consisting of cisplatin, carboplatin,
oxaliplatin, iproplatin, tetraplatin, transplatin, JM118
(cis-amminedichloro(cyclohexylamine)platinum(II)), JM 149
(cis-amminedichloro(cyclohexylamine)-trans-dihydroxoplatinum(IV)),
JM216 (bis-acetato-cis-amminedichloro(cyclohexylamine)platinum(IV))
and JM335
(trans-amminedichloro(cyclohexylamine)dihydroxoplatinum(IV)). In
some embodiments, the active platinum compound is cisplatin.
[0048] In certain embodiments, the active platinum compound is
selected from the group consisting of cisplatin, carboplatin,
oxaliplatin, iproplatin, tetraplatin, transplatin, JM118
(cis-amminedichloro(cyclohexylamine)platinum(II)), JM 149
(cis-amminedichloro(cyclohexylamine)-trans-dihydroxoplatinum(IV)),
JM216 (bis-acetato-cis-amminedichloro(cyclohexylamine)platinum(IV))
and JM335
(trans-amminedichloro(cyclohexylamine)dihydroxoplatinum(IV)). In
some embodiments, the active platinum compound is cisplatin,
transplatin, carboplatin, or oxaliplatin, while in other
embodiments, the active platinum compound is cisplatin.
[0049] The lipids used in the present invention can be synthetic,
semi-synthetic or naturally-occurring lipids, including
phospholipids, tocopherols, sterols, fatty acids, glycolipids,
negatively-charged lipids, cationic lipids. In terms of
phospholipids, they can include such lipids as egg
phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg
phosphatidylinositol (EPI), egg phosphatidylserine (EPS),
phosphatidylethanolamine (EPE), and phosphatidic acid (EPA); the
soya counterparts, soy phosphatidylcholine
[0050] (SPC); SPG, SPS, SPI, SPE, and SPA; the hydrogenated egg and
soya counterparts (e.g., HEPC, HSPC), stearically modified
phosphatidylethanolamines, cholesterol derivatives, carotinoids,
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 may be
made up of fatty acids of different chain lengths and different
degrees of unsaturation. In particular, the compositions of the
formulations can include DPPC, a major constituent of
naturally-occurring lung surfactant. Other examples include
dimyristoylphosphatidycholine (DMPC) and
dimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidcholine
(DPPC and dipalmitoylphosphatidylglycerol (DPPG)
distearoylphosphatidylcholine (DSPC and
distearoylphosphatidylglycerol (DSPG),
dioleylphosphatidyl-ethanolamine (DOPE) and mixed phospholipids
like palmitoylstearoylphosphatidyl-choline (PSPC) and
palmitoylstearolphosphatidylglycerol (PSPG), triacylglycerol,
diacylglycerol, seranide, sphingosine, sphingomyelin and single
acylated phospholipids like mono-oleoyl-phosphatidylethanolarnine
(MOPE).
[0051] In some embodiments, the lipid complexed active platinum
compound comprises a neutral phospholipid, such as a phosphatidyl
choline. In other embodiments, the phosphatidyl choline is
DPPC.
[0052] In some embodiments, the lipid complexed active platinum
compound further comprises a sterol. In some embodiments, the
sterol is cholesterol.
[0053] Negatively charged lipids include PGs, PAs, PSs and PIs. In
some embodiments, the lipid complexed active platinum compound does
not comprise a phosphatidyl serine (PS). In some embodiments, the
lipid-complexed active platinum compound does not comprise a PG,
PA, PS or PI. In other embodiments, the lipid-complexed active
platinum compound is substantially free of negatively charged
phospholipids. In some embodiments, the lipid-complexed active
platinum compound does not comprise any negatively charged
phospholipids.
[0054] In some embodiments, the lipid complexed active platinum
compound comprises DPPC and cholesterol in a ratio of about 1:1 to
about 5:1 by weight. In other embodiments, the lipid complexed
active platinum compound comprises DPPC and cholesterol in a ratio
of about 2:1 to about 4:1 by weight. In some embodiments, the lipid
complexed active platinum compound comprises DPPC and cholesterol
in a ratio of about 2.25:1 by weight.
[0055] Another aspect of the invention relates to pharmaceutical
formulations comprising any one of the aforementioned compositions
and a pharmaceutically acceptable carrier or diluent. The
pharmaceutical formulation of the lipid complexed active platinum
compound may be comprised of 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
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. In some embodiments, the pharmaceutical formulation
is adapted for inhalation by or injection into a patient.
[0056] The process for producing this active platinum compound
formulation can comprise mixing an active platinum compound with an
appropriate hydrophobic matrix and subjecting the mixture to one or
more cycles of establishing two separate temperatures. For example,
the process comprises the steps of: (a) combining an active
platinum compound and a hydrophobic matrix carrying system; (b)
establishing the mixture at a first temperature; and (c) thereafter
establishing the mixture at a second temperature, which second
temperature is cooler than the first temperature. Step (b) is
typically effected with heating, while step (c) is typically
effected with cooling. In alternative embodiments, the cycles are
counted beginning with the cooler step, transitioning to the warmer
step, and cycling the two steps. The process can comprise
sequentially repeating the steps (b) and (c) for a total of two or
three or more cycles. The active platinum compound solution can be
produced by dissolving active platinum compound in a saline
solution to form a platinum solution. The hydrophobic matrix
carrying system favorably comprises liposome or lipid
complex-forming lipids. The process for making a platinum aggregate
can further comprise, after all of steps (b) and steps (c) have
been completed: (d) removing un-entrapped active platinum compound
by filtering through a membrane having a molecular weight cut-off
selected to retain desired liposomes or lipid complexes and adding
a liposome or lipid complex compatible liquid to wash out
un-entrapped active platinum compound.
[0057] Cisplatin, for example, forms large crystalline aggregates
in aqueous solution with a crystal diameter of greater than a few
microns. In the presence of an amphipathic matrix system, such as a
lipid bilayer, small cisplatin aggregates form. For example, the
aggregates may be formed in the hydrocarbon core region of a lipid
bilayer or be formed such that a lipid bilayer surrounds the
aggregate. During the warming cycle of the process, it is believed
that cisplatin is returned to solution at a greater rate in aqueous
regions of the process mixture than in the bilayers. As a result of
applying more than one cool/warm cycle, cisplatin accumulates
further in the core region of the lipid bilayers or within the
lipid bilayer. Without limiting the invention to the proposed
theory, experimentation indicates that the cisplatin aggregates
cause the immediate surroundings of the interfacial bilayer region
to be more hydrophobic and compact. This results in a high level of
entrapment of active platinum compound as cooling and warming
cycles are repeated.
[0058] The resulting formulation has a markedly high entrapment
percentage. The entrapment has been shown, in some cases, to reach
almost 92%. This amount is far higher than the most efficient
entrapment expected from a conventional aqueous entrapment which is
approximately 2-10% entrapment. This efficiency of the present
invention is demonstrated in example 3.
[0059] In one embodiment, the process comprises combining the
bioactive agent with a hydrophobic matrix carrying system and
cycling the solution between a warmer and a cooler temperature.
Preferably the cycling is performed more than one time. More
preferably the step is performed two or more times, or three or
more times. The cooler temperature portion of cycle can, for
example, use a temperature of about 35.degree. C. or less,
25.degree. C. or less, 20.degree. C. or less, 15.degree. C. or
less, 10.degree. C. or less, or 5.degree. C. or less. In some
embodiments, the temperature is about -25.degree. Celsius to about
35.degree. Celsius, about -5 to about 25.degree. C., about -5 and
about 20.degree. C., about -5 and about 10.degree. C., about -5 and
about 5.degree. C., or about 1 and about 5.degree. C.
[0060] In some embodiments, the warming step temperature is
50.degree. Celsius or higher. The temperatures can also be selected
to be below and above the transition temperature for a lipid in the
lipid composition. In some embodiments the step of warming
comprises warming the reaction vessel to about 4 to about
70.degree. Celsius, about 45 and to about 55.degree. Celsius. The
above temperature ranges are particularly preferred for use with
lipid compositions comprising predominantly diphosphatidycholine
(DPPC) and cholesterol.
[0061] For manufacturing convenience, and to be sure the desired
temperature is established, the cooler and warmer steps can be
maintained for a period of time, such as approximately form 5 to
300 minutes or 30 to 60 minutes.
[0062] Another way to consider the temperature cycling is in terms
of the temperature differential between the warming and cooling
steps of the cycle. This temperature differential can be, for
example, about 25.degree. Celsius or more, such as a differential
from about 25 to about 70.degree. Celsius, or a differential of
about 40 to about 55.degree. Celsius.
[0063] The temperatures of the warming and cooling steps are
selected on the basis of increasing entrapment of active platinum
compound. Without being limited by any particular theory, it is
believed that it is useful to select an upper temperature effective
substantially increase the solubility of active platinum compound
in the process mixture. During repetitive cooling/heating,
bioactive agents are solubilized and crystallized repetitively. As
soluble drug is cooled, some portion enters complexes with the
lipid while the remainder precipitates. On subsequent heating,
unencapsulated bioactive agent that is crystallized becomes soluble
again. Importantly, active platinum compound that has been
encapsulated in the lipid complex substantially stays in the lipid
complex during the heating and cooling cycling (e.g. it leaks at
such a slow rate that no appreciable amount leaves the lipid
complex during the heating phase of this process).
[0064] For example, as the temperature is increased during the
warming step of the cycle, the active platinum compound, such as
cisplatin, dissolves. During the cooling step, the cisplatin in the
aqueous phase precipitates out of solution to a greater extent that
the cisplatin associated with the lipid bilayers, thereby
increasing the amount of lipid-associated cisplatin with each
heating and cooling cycle. Additionally, solubility of cisplatin is
highly temperature-dependent. For example, FIG. 1A depicts a graph
of the transition temperature of dissolved-precipitated cisplatin
in aqueous solution as a function of cisplatin concentration during
heating (where the cisplatin is initially in precipitated). FIG. 1C
depicts a graph of the transition temperature at which aqueous
cisplatin is precipitated during cooling (where the cisplatin is
initially dissolved). Lowering 15.degree. in temperature of a
cisplatin solution decreases the soluble concentration by about
50%. In other words, solubility limiting concentration increases
with increasing temperature by about 3% per degree increase in
temperature of aqueous cisplatin. In addition, the aggregate
(crystal)-to-monomer transition temperature (solubilizing
temperature) is higher than the monomer-to-aggregate (crystal)
transition temperature (crystallizing temperature) by about 15 to
20.degree. C.
[0065] Similar graphs for another active platinum compound,
transplatin, are depicted in FIGS. 1B and 1D, which show solubility
properties similar to cisplatin. Transplatin solubility is poorer
than cisplatin, but it is also temperature-dependent. Lowering the
temperature by about 15.degree. C. decreases the soluble
concentration of transplatin by about 50%. The aggregate
(crystal)-to-monomer transition temperature (solubilizing
temperature) is higher than the monomer-to-aggregate (crystal)
transition temperature (crystallizing temperature) by about 20 to
30.degree. C.
[0066] Experimental results strongly indicate that the physical
state of cisplatin is solid (aggregates) or lipid bound since the
concentration of cisplatin is much higher than the solubility
limit. Results further indicate that process does not require
freezing the compositions, but that cooling to temperature higher
than the freezing point of water is effective. Results further
indicated that an entrapment efficiency achieved by 3-cycles was
similar to that achieved by 6-cycles of cooling and warming cycles,
which indicated that 3 cycles of temperature treatment was
sufficient to achieve high levels of active platinum compound
entrapment.
[0067] Results further indicate that the process can be scaled-up
while increasing process efficiency in entrapping cisplatin. Thus,
the invention further provides processes that are conducted to
provide an amount adapted for total administration (in appropriate
smaller volume increments) of 200 or more mLs, 400 or more mLs, or
800 or more mLs. All else being the same, it is believed that the
larger production volumes generally achieve increased efficiency
over smaller scale processes. While such volume is that appropriate
for administration, it will be recognized that the volume can be
reduced for storage.
[0068] Results further indicate that the lipid-complexed cisplatin
made by the method of the invention can retain entrapped cisplatin
with minimal leakage for over one year. This is a further
demonstration of the uniqueness in the formulation, indicating that
the cisplatin is bound within the liposome structure and not free
to readily leak out.
[0069] The process of the present invention may further comprise
separating the components of the product of the aforementioned
process. For example, in some embodiments, the process provides
both the aforementioned lipid-complexed active platinum compound
and the aforementioned liposome. In certain embodiments, the
portion of the product comprising the lipid-complexed active
platinum compound, referred to herein as "the heavy fraction" may
be separated from the portion comprising the liposome, referred to
herein as "the light fraction." Methods of separating include
allowing the heavy product to settle over a period of time, or
centrifuging the product.
Example 1
[0070] 70 mg DPPC and 28 mg cholesterol was dissolved in 1 mL
ethanol and added to 10 mL of 4 mg/mL cisplatin in 0.9% saline
solution.
[0071] (i) An aliquot (50%) of the sample was treated by 3 cycles
of cooling to 4.degree. C. and warming to 50.degree. C. The
aliquot, in a test tube, was cooled by refrigeration, and heated in
a water bath. The resulting unentrapped cisplatin (free cisplatin)
was washed by dialysis.
[0072] (ii) The remainder of the sample was not treated by
temperature cycles and directly washed by dialysis.
TABLE-US-00001 TABLE 1 Percentage entrapment of cisplatin with and
without cooling and warming cycles. Final Concentration of %
cisplatin, .mu.g/mL Entrapment Lipid-complexed cisplatin without 56
1.4 cooling and warming cycles lipid-complexed cisplatin after 360
9.0 cooling and warming cycles
Example 2
[0073] The rigidity of a membrane bilayer in lipid-complexed
cisplatin prepared with cool/warm cycling ("HLL" cisplatin or
"high-load liposomal" cisplatin) as described in Example 1 was
measured by fluorescence anisotropy of diphenylhexatriene (membrane
probe) inserted in the hydrophobic core region of the bilayer.
[Ref. Jahnig, F., 1979 Proc. Natl. Acad. Sci. USA 76(12): 6361.]
The hydration of the bilayers was gauged by the deuterium isotope
exchange effect on fluorescence intensity of TMA-DPH
(trimethylamine-diphenylhexatriene). [Ref. Ho, C, Slater, S. J.,
and Stubbs, C D., 1995 Biochemistry 34: 6188.]
TABLE-US-00002 TABLE 2 Degree of hydration and rigidity of
liposomes, lipid-complexed cisplatin without cool/warm cycling and
HLL cisplatin. Placebo Lipid-complexed (Liposomes cisplatin without
without cooling & warming HLL cisplatin) cycles cisplatin
Degree bilayer rigidity 0.29 0.29 0.36 Degree of bilayer hydration
1.13 1.15 1.02
Example 3
[0074] 1.0 g DPPC and 0.4 g cholesterol were dissolved in 6 mL of
ethanol. 60 mg of cisplatin was dissolved in 10 mL of 0.9% saline
solution at 65.degree. C. 1 mL of the resultant lipid mixture
solution was added to 10 mL of the resultant cisplatin solution.
The lipid/cisplatin suspension was cooled to approximately
4.degree. C. and held at that temperature for 20 min. and warmed to
50.degree. C. and held at that temperature for 20 min. Ethanol was
removed by bubbling N.sub.2 gas into the suspension during the
warming period. The cooling and warming steps were repeated 5
further times.
TABLE-US-00003 TABLE 3 Entrapment of cisplatin. Concentration of
Total Cisplatin % Cisplatin Drug:Lipid (mg/mL) entrapped (by
weight) HLL Cisplatin 5.8 91.6 1:26
Example 4
[0075] A cisplatin lipid formulation was prepared using
phosphatidylcholine (PC) and cholesterol (in a 57:43 mol ratio).
0.55 mmoles of PC and 0.41 mmoles of cholesterol were dissolved in
2 mL ethanol and added to 20 mL of 4 mg/mL cisplatin solution. An
aliquot (50%) of each sample was treated by 3 cycles of cooling and
warming and then washed by dialysis. Another part of each sample
was directly washed by dialysis. Entrapment was estimated from the
ratio of final concentration and initial concentration.
TABLE-US-00004 TABLE 4 Entrapment and drug to lipid ratios for
cisplatin with various phosphatidylcholines No Cooling and Warming
Cooling and Warming Cisplatin % Drug:Lipid Cisplatin % Drug:Lipid
PC (mg/mL) Entrapment (by weight) (mg/mL) Entrapment (by weight)
DOPC 0.16 4.0 1:142 0.21 5.3 1:108 EggPC 0.09 2.3 1:247 0.12 3.0
1:185 DMPC 0.15 3.8 1:123 0.24 6.0 1:77 DPPC 0.17 4.3 1:115 0.85
21.3 1:23 HSPC 0.11 2.8 1:202 0.23 5.8 1:97 DSPC 0.10 2.5 1:184
0.58 14.5 1:32
Example 5
[0076] A lipid formulation (DPPC: cholesterol in a ratio of 5:2
w/w) was dissolved in ethanol and added to a cisplatin solution.
Part of the formulation was treated by cycles of cooling to
4.degree. Celsius and warming to 55.degree. Celsius cycles while
part was not treated thus. The lipid/cisplatin suspension was then
washed by dialysis.
TABLE-US-00005 TABLE 5 Concentration of cisplatin with and without
cooling and warming cycles. Starting Cooling Total Cisplatin
Concentration & warming concentration Concentration of lipids
cycles of Cisplatin 0.2 mg/mL 1.4 mg/mL No Not Detectable 0.2 mg/mL
1.4 mg/mL Yes Not Detectable 4.0 mg/mL 28 mg/mL No 0.22 mg/mL 4.0
mg/mL 28 mg/mL Yes 0.46 mg/mL
Example 6
Determination of Captured Volume of Cisplatin Vesicles of the
Invention
[0077] The object was to determine the nature of the liposomal
entrapped cisplatin (HLL cisplatin) by determining the
concentration of the entrapped cisplatin within the liposome.
V.sub.total=V.sub.liposome+V.sub.outside
TABLE-US-00006 TABLE 6 [Measurement of V.sub.liposome] Abs at 450
nm [dichromate] V.sub.outside V.sub.liposome HLL Cisplatin 0.874
0.67 mg/mL 1.88 mL 0.12 mL Saline only 0.822 0.60 mg/mL 2 mL 0
mL
[0078] Method: 1) 2 mL HLL Cisplatin prepared as described in
Example 4 was concentrated by centrifugation filter kit. 2) 0.8 mL
of concentrated sample was recovered and 1.2 mL of 1 mg/mL
dichromate was added to recover original volume. 0.8 mL normal
saline+1.2 mL of dichromate was also prepared as a control. 3) Abs
at 450 nm was measured to detect difference in dichromate
concentration. To avoid turbidity from liposome sample, both
samples were filtered by centrifugal filtration.
[0079] Result: 6% of total volume was occupied by liposomes.
V.sub.liposome=1.53 .mu.L/.mu.moles lipid (total lipid 39.3 mM)
Next, V.sub.liposome=V.sub.captured+V.sub.bilayer
[0080] To estimate V.sub.bilayer, the lamellarity of the vesicles
of HLL cisplatin was determined.
Measurement of Lamellarity of HLL Cisplatin Vesicles:
TABLE-US-00007 [0081] Measurement of lamellarity of HLL cisplatin
vesicles: % probe lipid at F.sub.total F.sub.inside outmost
leaflet* Fluorescence intensity 14193 11349 20 *% probe lipid at
outmost leaflet = (F.sub.total - F.sub.inside) .times. 100 /
F.sub.total
[0082] Method: Cisplatin vesicles were prepared with the method of
Example 9, described below (1 liter batch) modified to add 0.5 wt %
fluorescence probe lipid (NBD-PE). This probe lipid distributes
evenly in membrane inside and outside. The ratio of amount of
probes located in outmost membrane layer (surface of liposome) vs.
the rest of probes is determined to estimate how many lipid layers
exist in HLL Cisplatin. The ratio between probes located on
liposome surface and probes located inside liposome was determined
by adding a reducing agent dithionite to quench only surface
probes. Then, total quenching was achieved by rupturing liposome
with detergent.
[0083] Result: Outmost bilayer shell contains 40% of total
lipids.
[0084] Based on geometric calculation, % lipid at outmost bilayer
shell would be 52% and 36% for bi-lamellar and tri-lamellar
vesicles, respectively. Therefore, it was concluded that the
average lamellarity of HLL Cisplatin was three.
[0085] Assuming tri-lamellar vesicles, the ratio of
V.sub.liposome/V.sub.captured was calculated to be 1.2635.
Therefore, the captured volume was:
V captured = V liposome / 1.2635 = 1.53 .mu.L / .mu. moles lipid /
1.2635 = 1.21 .mu.L / .mu. moles lipid = 1.21 .mu.L / .mu. moles
lipid .times. 39.3 mM ( total lipid concentration ) = 47.6 .mu.L /
mL ##EQU00001##
[0086] The captured volume was 47.6 .mu.L per every mL HLL
Cisplatin and 4.76% of total volume. If entrapped cisplatin was
assumed to be in an aqueous compartment of liposomes, its local
cisplatin concentration would be estimated to be 21.0 mg/mL. This
concentration was not only higher than cisplatin solubility limit
at room temperature but more significantly it was much higher than
initial charging concentration (4 mg/mL).
Example 7
Effect of Cooling Temperature on Entrapment Efficiency of HLL
Cisplatin
[0087] The object was to find an optimum cooling temperature for
the highest entrapment of cisplatin and avoid freezing and thawing.
20 mg/mL DPPC, 8 mg/mL cholesterol, and 4 mg/mL cisplatin
suspension was prepared by ethanol infusion. The sample was split
to three equal aliquots which were treated by 6 cycles of cooling
and warming using three different cooling temperatures. After a
treatment of temperature cycles the samples were dialyzed to remove
free cisplatin. The resulting data (Table 7) helps optimize the
manufacturing process.
TABLE-US-00008 TABLE 7 Effect of cooling temperature. Post-infusion
Actual Cooling temperature temperature and warming [Cisplatin] %
treatment of the sample cycles mg/mL Entrapment Dry ice bath Frozen
15 min. cold & 0.34 8.5 (-70.degree. C.) 15 min. warm 6 cycles
Freezer 0.degree. C. 15 min. cold & 0.98 24.5 (-20.degree. C.)
15 min. warm 6 cycles Ice bath 4.degree. C. 15 min. cold & 0.63
15.8 (1.degree. C.) 15 min. warm 6 cycles
Example 8
Effect of Number of Temperature Cycles on Entrapment Efficiency
[0088] To determine an optimum number of temperature cycles for the
most efficient entrapment of cisplatin (Table 8). Samples were
prepared as in the previous example. At cooling the temperature of
samples was 0.degree. C. The temperature cycle was done by 15 min
cooling and 15 min warming. The starting cisplatin concentration
was 4 mg/mL and free cisplatin was removed by dialysis.
TABLE-US-00009 TABLE 8 Effect of Number of Temperature Cycles. Low
Lipids High Lipids Number (7.5 mg/mL DPPC & (12.5 mg/mL DPPC
& of 3 mg/mL cholesterol 5 mg/mL cholesterol cycles [cisplatin]
% Entrapment [cisplatin] % Entrapment 0 0.05 mg/mL 1.3 0.21 mg/mL
5.3 1 0.11 mg/mL 2.8 0.23 mg/mL 5.8 3 0.39 mg/mL 9.8 0.88 mg/mL
22
Example 9
Batch Scale and Process Efficiency
[0089] To determine if the efficiency of entrapment changed upon
changing the size of the batch. The 20 mL batch was prepared as
described in example 4. The 1 L batch was prepared indicated in the
following steps:
1. Four grams of cisplatin were dissolved in 1 Liter of injection
grade 0.9% sodium chloride at 65.degree. C. 2. 20 grams of DPPC and
8 grams of cholesterol were dissolved in 120 mL of absolute ethanol
at 65.degree. C. 3. While mixing the cisplatin solution at 300 rpm
(65.degree. C.), the lipid solution was metered (infused) into the
cisplatin solution at a flow rate of 20 mL/min. 4. After infusion,
cisplatin/lipid dispersion was cooled down to -5.degree. C. to
0.degree. C. using a propylene glycol/water bath and kept for 45
minutes (cooling). 5. The dispersion was warmed up to 50.degree. C.
and maintained for 15 minutes (warming). 6. The cooling/warming
cycle described in steps 4 and 5 was performed for two more times
(three cycles total). 7. The dispersion was washed to remove free
cisplatin by diafiltration. The permeate removing rate was 17-22
mL/min. The dispersion volume (1 L) was maintained constant by
compensating the permeate with a feed of fresh sterile 0.9% sodium
chloride solution.
[0090] The 200 mL batch was made in the same manner but employed
20% of the components. The process efficiency was defined as the
lipid/drug (wt/wt) ratio of initial ingredients divided by the
lipid/drug ratio for the final product (Table 9).
TABLE-US-00010 TABLE 9 Process efficiency. Lipid/drug pre-
Lipid/drug final Process Batch Batch size formation product
efficiency 1 20 mL 4.4 54.5 0.08 2 200 mL 5.85 27.3 0.21 3 200 mL
5.85 37.2 0.16 4 200 mL 5.85 36.9 0.16 5 1 L 5.85 14.4 0.41 6 1 L
7.0 19.2 0.36 7 1 L 7.0 21.2 0.33
Example 10
Stability of Entrapped Lipid-Complexed Cisplatin
[0091] The stability of one liter batches of HLL cisplatin was
monitored in time for the leakage of internal contents. The
resulting data is presented in FIG. 2.
Example 11
Density Characterization of Light and Heavy Fractions
[0092] Samples were prepared as in the previous example. At cooling
the temperature of samples was 0.degree. C. The temperature cycle
was done by 15 min cooling and 15 min warming The starting
cisplatin concentration was 4 mg/mL and free cisplatin was removed
by dialysis.
[0093] Density Gradient Analysis
[0094] Seven different batches of cisplatin lipid complex were used
for these experiments. Density gradients were formed using
Iodixanol (SIGMA (D1556, lot no. 025K1414)) as a dense media and
0.9% NaCl saline solution to keep osmolality close to normal 300
mOsM. First, about 5.5 mL saline was added to the centrifuge tube,
and then the same volume of heavy medium (1:1 mixture of Iodixanol
60% and saline) was layered on the bottom of the tube using a
syringe with a long needle. Gradients were formed using a BioComp
107ip Gradient Master at the settings: time=2:14 min, angle=79.0,
speed=17 rpm, and using the long tube cap. An aliquot of Cisplatin
Lipid Complex samples (1 mL) were placed on the top of the gradient
and centrifuged for 30 min at 30,000 rpm at 20.degree. C. After
centrifugation, the top 0.8-1.0 mL volume of clear liquid was
discarded, and the next 2 mL was collected representing the light
fraction. The light fraction is believed to contain liposomes,
wherein at least some of the liposomes are associated with
cisplatin. There was a detectable amount of free cisplatin in the
light fraction of nebulized samples, which was determined by
filtering through Centricon-30 filtering devices and subtracted
from the total cisplatin.
[0095] The rest of the media was removed, leaving only a small
yellow pellet on the bottom representing the dense (heavy)
fraction, which was subsequently dispersed in 2 mL solution of 75%
n-Propanol, 5% saline, 20% water. Cisplatin in the heavy fraction
was not completely soluble at this point. An aliquot of this
dispersion was taken for cisplatin determination. Another part of
the dispersion (1 mL) was mixed with equal volume of 60% n-Propanol
and centrifuged 5 min at 1000 rpm on an Eppendorf 5810R centrifuge
to settle undissolved cisplatin, and then 1 mL of clear supernatant
was used for HPLC lipid determination.
[0096] Cisplatin Concentration:
[0097] Cisplatin was measured by HPLC by separating cisplatin on
YMC-Pack NH2 column using 90% acetonitrile mobile phase and
measuring absorbance at 305 nm.
[0098] Cisplatin standards and samples were diluted in solution of
75% n-Propanol, 5% saline and 20% water. Standards were used with
cisplatin concentrations of 75, 50, 25, and 10 .mu.g/mL. Cisplatin
peak retention time was usually around 6.4 min.
[0099] Lipid Analysis by HPLC:
[0100] Lipids were analyzed by HPLC as follows: lipids were
separated on a Phenomenex Luna C 8(2) column using binary gradient
mode. Mobile phase A: methanol 70%, acetonitrile 20%, water 10%,
ammonium acetate 0.1%, mobile phase B: methanol 70%, acetonitrile
30%, ammonium acetate 0.07%. Lipid standards and samples were
diluted in a solution of 60% n-Propanol, 40% water. Lipids were
detected by Sedex 55 Evaporative Light Scattering Detector. The
retention time for cholesterol was about 8 min, for DPPC about 10
min.
[0101] Preparation of Samples on Carbon Coated Grids for TEM
Analysis
[0102] About 50 mL of a cisplatin lipid complex batch was allowed
to settle by gravity for at least one week at 4.degree. C. The
white to portion containing mostly light fraction was removed and
the yellowish brown fraction on the bottom was used for TEM
analysis. A small amount of sample (less than 10 microliters) was
placed on the carbon coated grid. The grid was placed on the top of
a piece of filter paper and spun an Eppendorf centrifuge for 1
minute at 500 RPM to remove excess water. Samples were air-dried
for at least one hour before analysis. If centrifugation was done
at too low a speed, water was not removed sufficiently enough, and
the sample remained too thick, even after drying, tending to boil
when exposed to high vacuum inside the TEM microscope.
Centrifugation conditions were adjusted to be gentle enough to
avoid damage to the grid or loss of the sample. Experimentally, it
was found that mild centrifugation at a speed of 200-500.times.g
for 1 min produced samples of good quality with sufficient amount
of particles on the grid, but a minimum volume of water.
[0103] For comparison, samples of cisplatin crystals (not complexed
with a lipid) were prepared by the following procedure: 15 mg
cisplatin was dissolved in 5 mL saline solution by heating to
50.degree. C. to provide a 3 mg/mL cisplatin solution. The
cisplatin solution was briefly sonicated in a bath of room
temperature water. At the first signs of cisplatin precipitation
and cloudiness, a small volume was taken with a pipette and
immediately placed on a carbon coated grid as described in the
preceding paragraph.
[0104] TEM images were obtained on a Zeiss 910 Transmission
Electron Microscope at the Princeton Imaging and Analysis Center,
Princeton University, Princeton, N.J. An accelerating high voltage
of 100 kV was used, and pictures were captured on a charged couple
device (CCD) camera at magnifications of 2000 to 25,000.times.. TEM
images of heavy fraction lipid-complexed cisplatin are shown in
FIGS. 3A-B. The cisplatin-rich particles were electron dense enough
to be seen without staining.
[0105] TEM images of cisplatin crystals which are not complexed
with a lipid are shown in FIGS. 4A-B. The un-complexed cisplatin
crystals appear as dark particles of rectangular shape
approximately 2 microns wide and 10-20 microns long. It is believed
that the cisplatin particles in the lipid-complexed cisplatin are
surrounded by a lipid bilayer, and therefore, cannot grow as large
as "free" cisplatin. Additionally, the cisplatin in the
lipid-complexed cisplatin does not dissolve in saline upon
dilution, further suggesting that the cisplatin is surrounded by a
lipid bilayer.
[0106] Results
[0107] Nine batches of Lipid-cisplatin complex were fractionated on
an Iodixanol density gradient as described in the Methods section.
All nine samples separated into a similarly positioned white band
of light fraction and a yellow pellet of dense fraction. 2 mL of
the light fraction were collected and the rest of the liquid was
removed. The remaining pellet was dispersed in 2 mL of 75%
n-Propanol. Cisplatin and lipid concentrations in each fraction
were measured by HPLC as described. The lipid/cisplatin ratio in
the dense fraction was very high so that both lipid and cisplatin
could not be solubilized in same solvent at high enough
concentration for the lipid analysis. For that reason, the
lipid-cisplatin mixture in 75% n-propanol solution was centrifuged
to remove the insoluble portion of the cisplatin, and the
supernatant was used as is for lipid HPLC analysis, as described in
Methods.
[0108] Results of the density gradient analysis are presented in
Table 10. L/D represents the ratio of lipid to cisplatin by weight.
The percentages presented are with respect to the total cisplatin
or lipid in the formulation. Lower section of the table shows
averages of lipid and cisplatin contents in each fraction derived
from all nine samples tested. Standard deviations (SD) are shown to
demonstrate consistency. These data demonstrate that the majority
of lipid (90.6% on average, +/-3.1%) is in the light fraction,
while only 0.87+/-0.09% lipid is in the dense fraction. The
majority of cisplatin (82.3+/-2.9%) is in the dense fraction, while
only 8.4+/-2.1% is in the light fraction. The lipid to drug ratio
(L/D) calculated for the total sample was an average of 22.7. The
same L/D ratio in separate fractions was as high as 255+/-47 for
the light fraction, and as low as 0.24+/-0.03 for dense
fraction.
TABLE-US-00011 TABLE 10 Distribution of cisplatin and lipids in the
light and dense fractions of Cisplatin Lipid Complex samples Lipid
Lipid Cisplatin Cisplatin Batch mg/mL % total mg/mL % total L/D 8
total 62.2 2.47 25.2 8 light fraction 58.0 93.3 0.20 8.2 285 8
Dense fraction 0.49 0.79 2.01 81.3 0.24 9 total 51.5 2.46 20.9 9
light fraction 47.8 92.7 0.18 7.2 270 9 Dense fraction 0.44 0.85
1.96 79.7 0.22 10 total 55.2 2.57 21.5 10 light fraction 46.7 84.7
0.20 7.7 237 10 Dense fraction 0.47 0.85 2.15 83.6 0.22 11 total
57.3 2.61 22.0 11 light fraction 49.7 86.8 0.15 5.9 326 11 Dense
fraction 0.48 0.84 2.20 84.2 0.22 12 total 57.9 2.57 22.5 12 light
fraction 51.6 89.1 0.18 6.9 290 12 Dense fraction 0.47 0.82 2.17
84.3 0.22 13 total 80.46 3.36 24.0 13 light fraction 73.42 91.26
0.44 13.0 168 13 Dense fraction 0.82 1.01 2.58 76.7 0.32 14 total
71.19 3.41 20.9 14 light fraction 64.82 91.06 0.29 8.7 220 14 Dense
fraction 0.65 0.92 2.76 81.1 0.24 15 total 66.92 2.83 23.7 15 light
fraction 62.35 93.17 0.23 8.0 275 15 Dense fraction 0.66 0.99 2.45
86.5 0.27 16 total 68.5 2.86 23.9 16 light fraction 64.0 93.48 0.28
9.8 228 16 Dense fraction 0.51 0.75 2.40 83.8 0.21 Average 22.7
Light fraction 90.6 8.4 255 .+-.SD 3.1 2.1 47 Dense fraction 0.87
82.3 0.24 .+-.SD 0.09 2.9 0.03
Example 12
Nebulization Study
[0109] Separation of Light and Dense Fractions
[0110] 30 mL of cisplatin lipid complex was mixed with 10 mL
iodixanol 30% in saline. This mixture was in half and 20 mL
portions were layered on the top of another 10 mL iodixanol 30% in
saline using two 50 mL centrifuge tubes. The samples were
centrifuged for 30 minutes at 4000 rpm at 5.degree. C. on an
Eppendorf 5810 centrifuge. Supernatant, containing a mixture of
light and heavy fractions, was discarded. The pellet, containing
the dense fraction of the cisplatin lipid composition, was gently
dispersed in 5 mL of saline. After determining the concentration of
cisplatin, the concentration was adjusted to make the concentration
2.7 mg/mL of cisplatin.
[0111] To obtain the light fraction, the cisplatin lipid complex
batch was allowed to settle by gravity at 5.degree. C. for 1 week.
The top portion of the sample, containing the light fraction, was
collected.
[0112] Nebulization Studies
[0113] TEM Analysis of Nebulized Lipid-Complexed Cisplatin:
[0114] 5 mL of sample was nebulized using a PARI-LC STAR nebulizer,
and the aerosol was collected by a chilled (ice water) impinger.
From the impinger, 2.5 mL of the nebulizate was obtained for the
analysis. An aliquot of the nebulizate was filter-centrifuged using
a centricon-YM-30, when necessary, immediately after nebulization
to determine non-encapsulated (free) cisplatin. Nebulization air
pressure was 30 PSI and the flow rate of the aerosol collection
system provided by the pump was 8 L/min. 1 mL of aerosol, collected
in an impinger, was diluted with 2 mL of saline and allowed to
incubate at 4.degree. C. for 20 hours. In parallel, 1 mL of the
original (non-nebulized) formulation was treated the same. After
the heavy fraction settled, the whiter top portion of each sample
was removed and the yellowish portion was dispersed in a solution
of 10% iodixanol in saline. Samples were centrifuged at 4000 RPM
for 20 minutes on an Eppendorf 5810 centrifuge. Supernatants were
discarded and pellets having heavy fraction particles dispersed in
200 microliters saline each, the amount of heavy fraction pellet
obtained from the nebulized formulation was less than the amount
from the original formulation. Samples for TEM were prepared as
described previously. FIG. 5 depicts TEM images of the nebulized
heavy fraction particles and FIG. 6A-B depicts TEM images of the
non-nebulized heavy fraction particles. In both cases, the
particles are dark and dense of rectangular and rhomboidal shape of
average size between 1 and 2 microns. In some instances, bigger and
longer particles of 3 microns and greater in size were present in
the non-nebulized sample (FIG. 8B) compared to the nebulized
sample.
[0115] Density Gradient Analysis of Nebulized Lipid-Complexed
Cisplatin:
[0116] 5 mL of sample was nebulized as described above. Within a
few hours after nebulization, a 1 mL sample was run on the density
gradient system. Visibly all the gradients appeared similar to the
ones of the original samples (non-nebulized). Each fraction was
collected as described above and analyzed for lipid and cisplatin
content.
[0117] The results of density gradient analysis of the samples
after nebulization are presented in Table 11. Similar to the
non-nebulized samples, the majority of lipid (85.2% on average,
.+-.6.1%) was found in the light fraction, while only
0.36%.+-.0.08% lipid was in the dense fraction. The distribution of
cisplatin was 46.0.+-.1.2% in the dense fraction, and 8.5.+-.1.1%
in the light fraction. The L/D ratio was similar to that in the
non-nebulized samples, being 232.+-.15 for the light fraction, and
0.18.+-.0.03 for the dense fraction.
TABLE-US-00012 TABLE 11 Table 10. Distribution of cisplatin and
lipids in the light and dense fractions of Cisplatin Lipid Complex
samples Lipid Lipid Cisplatin Cisplatin Batch mg/mL % total mg/mL %
total L/D 11 total 70.1 3.22 21.8 11 light fraction 55.8 79.7 0.25
7.7 225 11 Dense fraction 0.28 0.40 1.50 46.5 0.19 12 total 61.9
2.80 22.1 12 light fraction 56.8 91.7 0.23 8.1 249 12 Dense
fraction 0.25 0.4 1.25 44.6 0.23 13 total 77.57 2.80 23.76 13 light
fraction 60.98 78.61 0.23 9.6 195.15 13 Dense fraction 0.33 0.43
1.25 44.6 0.23 14 total 71.65 3.17 22.59 14 light fraction 63.46
88.56 0.28 8.9 225.85 14 Dense fraction 0.26 0.37 1.40 44.2 0.19 16
total 77.2 3.00 25.7 16 light fraction 64.9 84.1 0.29 9.8 221 16
Dense fraction 0.21 0.27 1.40 46.8 0.15 Average 23.2 Light fraction
84.5 8.8 223 .+-.SD 5.6 0.9 15 Dense fraction 0.37 45.3 0.19 .+-.SD
0.06 1.2 0.03
[0118] A comparison of density gradient analyses before and after
nebulization is shown in Table 12.
TABLE-US-00013 TABLE 12 Pre and post nebulization gradient
analysis. Cisplatin Lipid Complex Cisplatin % of total Lipid/Drug
ratio Fractions PreNeb PostNeb Change PreNeb PostNeb Change Light
fraction 8.4 8.8 0.4 255 233 -32 SD 2.1 0.9 3.0 47 15 52 Dense
Fraction 82.3 45.3 -37.0 0.24 0.19 -0.05 SD 2.9 1.2 4.1 0.03 0.03
0.06
[0119] FIG. 7 shows a bar graph of the effect of nebulization on
the distribution of cisplatin in the light and dense fractions of
the cisplatin lipid complex. While not being bound by any
particular theory, the above data indicates that a significant
portion of cisp latin leaks out of the dense fraction during
nebulization, but does not leak out of the light fraction to a
significant extent.
[0120] The separated light and dense fractions were nebulized as
described above and then analyzed for total and free cisplatin.
Table 13 shows the results, compared with nebulization of the total
cisplatin lipid complex. The total cisplatin lipid complex sample
lost 45.1% of encapsulated cisplatin during nebulization. The light
fraction was essentially unchanged. The dense fraction lost more
than 70% of its original encapsulated cisplatin. The results
demonstrate that the dense fraction leaks, while the light fraction
essentially does not. Additionally, the dense fraction leaked to a
greater extent when nebulized alone, compared to the total
cisplatin lipid complex sample. While not being bound by any
particular, it is believed that liposomes contained in the light
fraction "protect" the dense particles during nebulization, thereby
reducing cisplatin leakage.
TABLE-US-00014 TABLE 13 Cisplatin Lipid Cisplatin free % Complex
fractions preNeb postNeb Leakage % Total 4.4 49.5 45.1 Light
fraction 65.7 64.7 -1.0 Dense fraction 3.1 74.7 71.6
[0121] The distribution of cisplatin in a the light and dense
fractions are shown in FIG. 7.
Example 13
Comparison of Cisplatin and Transplatin Entrapment
[0122] Entrapment of cisplatin or transplatin in a lipid complex by
repetitive cooling/heating achieves a high drug/lipid ratio as
shown in Table 14.
TABLE-US-00015 TABLE 14 Cisplatin transplatin Starting drug 5 mg/mL
1 mg/mL concentration Lipids (DPPC: 25 mg/mL 5 mg/mL Cholesterol =
7:3 wt) Temperature cycles 6 cycles 6 cycles Final drug 1.4 mg/mL
(1.4% free) 0.3 mg/mL (6.0% free) concentration Recovery % 29% 26%
Drug/Lipid 0.056 0.06
Example 14
Optical Microscopy
[0123] Four batches of the formulation were studied by optical
microscopy. Batches 17-20 are shown in FIGS. 8A-D, respectively.
The optical micrographs depict round particles of about 1 to 2
microns in size, or less, and a number of rod-shaped particles of
about 1-2 microns wide and up to about 10 microns in length.
Example 14
Freeze Fracture Images
[0124] An aliquot of the sample was plated on a copper plate and
then sandwiched with another copper plate. This sample sandwich was
quickly frozen by dipping into liquid propane and transferred to a
copper plate holder, which was immersed in liquid nitrogen. The
holder was transferred into a vacuum-freeze chamber (Balzers
freeze-etching system BAF 400 T), which was at a temperature of
170.degree. C. with a vacuum of 2-5xHr6 bar. Fracturing and
subsequent Pt and carbon coating were carried out at -115.degree.
C. The replica was taken out of the chamber and treated with 2%
nitric acid fix about 4-5 hours, followed by bleaching overnight.
The washed replica was placed on a copper grid for EM observation.
Representative freeze fracture images are shown in Figure
Example 16
Particle Size Analysis
[0125] Samples of the heavy fraction comprising lipid-complexed
cisplatin were diluted in filtered saline (NaCl 0.9%) at a ratio of
1:2000 and analyzed by an AccuSizer Optical Particle Sizer 780
using the following settings: injection loop volume 1 mL, Data
collection time 60 s, Detector LE 400-05 SE summary mode, Minimum
diameter 0.05 microns. The detector used counts only particles 0.5
microns and larger. Four batches were analyzed (batch nos. 17-20),
as shown in FIGS. 10A-D. The distribution plots show relative
volumes occupied by particles of different sizes. `111e particles
in the range of 0.5 to 1 micron represent the right side tail of
the main distribution of the light fraction, the majority of which
has particle sizes less than 5 microns. The plots also show a large
peak at the right from 1 micron to 20 micron, with a median size of
about 8 to 10 microns.
[0126] Batches 17 and 20 were subjected to particle size analysis
after nebulization as well, and the results are shown in FIGS.
11A-B.
EQUIVALENTS
[0127] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *