U.S. patent application number 14/088133 was filed with the patent office on 2014-08-14 for formulations for the oral administration of therapeutic agents and related methods.
This patent application is currently assigned to THE UNIVERSITY OF BRITISH COLUMBIA. The applicant listed for this patent is The University of British Columbia. Invention is credited to Ellen K. Wasan, Kishor M. Wasan.
Application Number | 20140228308 14/088133 |
Document ID | / |
Family ID | 40074494 |
Filed Date | 2014-08-14 |
United States Patent
Application |
20140228308 |
Kind Code |
A1 |
Wasan; Kishor M. ; et
al. |
August 14, 2014 |
FORMULATIONS FOR THE ORAL ADMINISTRATION OF THERAPEUTIC AGENTS AND
RELATED METHODS
Abstract
Formulations for the oral administration of therapeutic agents,
methods for administering therapeutic agents using the
formulations, and methods for treating conditions and diseases
using the formulations.
Inventors: |
Wasan; Kishor M.; (Richmond,
CA) ; Wasan; Ellen K.; (Richmond, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of British Columbia |
Vancouver |
|
CA |
|
|
Assignee: |
THE UNIVERSITY OF BRITISH
COLUMBIA
Vancouver
CA
|
Family ID: |
40074494 |
Appl. No.: |
14/088133 |
Filed: |
November 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12601676 |
Jun 30, 2010 |
8592382 |
|
|
PCT/CA08/00975 |
May 23, 2008 |
|
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14088133 |
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60940307 |
May 25, 2007 |
|
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60976708 |
Oct 1, 2007 |
|
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61041478 |
Apr 1, 2008 |
|
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Current U.S.
Class: |
514/31 ; 514/399;
514/449; 514/786 |
Current CPC
Class: |
A61P 25/22 20180101;
A61P 9/00 20180101; A61P 37/08 20180101; A61P 31/04 20180101; Y02A
50/30 20180101; A61P 1/08 20180101; Y02A 50/409 20180101; A61K
9/1075 20130101; A61P 29/00 20180101; A61K 47/10 20130101; Y02A
50/414 20180101; A61P 25/06 20180101; A61K 31/685 20130101; A61P
25/20 20180101; A61K 31/23 20130101; A61P 31/00 20180101; A61K
47/24 20130101; A61K 45/06 20130101; A61K 31/4174 20130101; A61P
35/00 20180101; A61K 9/4858 20130101; A61K 31/77 20130101; A61K
31/337 20130101; A61P 31/10 20180101; A61K 31/7048 20130101; A61P
31/12 20180101; A61P 33/04 20180101; A61P 37/06 20180101; A61P
33/02 20180101 |
Class at
Publication: |
514/31 ; 514/786;
514/399; 514/449 |
International
Class: |
A61K 31/7048 20060101
A61K031/7048; A61K 31/337 20060101 A61K031/337; A61K 31/4174
20060101 A61K031/4174; A61K 47/10 20060101 A61K047/10; A61K 47/24
20060101 A61K047/24 |
Claims
1. An amphotericin B formulation, comprising, (a) amphotericin B;
(b) one or more fatty acid glycerol esters; and (c) one or more
polyethylene oxide-containing phospholipids or one or more
polyethylene oxide-containing fatty acid esters.
2. A method for administering amphotericin B, comprising
administering a formulation of claim 1 to a subject in need
thereof.
3. A method for treating an infectious disease treatable by the
administration of amphotericin B, comprising administering to a
subject in need thereof a therapeutically effective amount of an
amphotericin B formulation of claim 1.
4. A formulation for the delivery of a therapeutic agent,
comprising, (a) a therapeutic agent; (b) one or more fatty acid
glycerol esters; and (c) one or more polyethylene oxide-containing
phospholipids or one or more polyethylene oxide-containing fatty
acid esters.
5. A method for administering a therapeutic agent, comprising
administering a formulation of claim 4 to a subject in need of such
agent.
6. A composition for formulating a therapeutic agent, comprising,
(a) one or more fatty acid glycerol esters; and (b) one or more
polyethylene oxide-containing phospholipids or one or more
polyethylene oxide-containing fatty acid esters.
7. A method for formulating a therapeutic agent, comprising
combining a therapeutic agent with a composition of claim 6.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/601,676, filed Jun. 30, 2010, now U.S. Pat. No. 8,592,382,
which claims the benefit of U.S. Provisional Application No.
60/940,307, filed May 25, 2007, U.S. Provisional Application No.
60/976,708, filed Oct. 1, 2007, and U.S. Provisional Application
No. 61/041,478, filed Apr. 1, 2008. Each application is expressly
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Each year in the Indian subcontinent alone, over 500,000
individuals play host to Leishmania donovani, an insidious parasite
that invades macrophages, rapidly infiltrates the vital organs and
ultimately leads to severe infection of the visceral
reticuloendothelial system. Visceral leishmaniasis, also known as
Kala-azar, is most prevalent in the weak and the young within a
population. Left untreated, almost all infected individuals will
die. Visceral leishmaniasis affects over 200 million people from 62
countries. The therapeutic arsenal against Leishmania is limited to
a small number of parenterally administered agents, with daily
injections of pentavalent antimony compound. Although more
expensive than the antimonials, amphotericin B (AmpB) has a 97%
cure rate and no reported resistance. However, drug therapy
involves IV administration over 30-40 days and is associated with
infusion-related side-effects (fever, chills, bone pain,
thrombophlebitis). The dose-limiting toxicity, which may even
affect the ability to achieve a cure, is renal impairment. In
addition, due to the prohibitive cost and difficult route of drug
administration, amphotericin B is failing to reach many
patients.
[0003] In developed nations, disseminated fungal infections such as
candidiasis, histoplasmosis, coccidiosis, and aspergillosis are on
the rise, affecting patients with cancer, organ transplant
recipients, diabetics and those with HIV/AIDS. In these patients,
invasive fungal infections may account for as many as 30% of
deaths. Despite the development of a number of new antifungal
agents, amphotericin B formulated as an IV administered micelle and
liposomal dispersion remains one of the most effective agents in
the treatment of systemic fungal infections. In addition, a variety
of parenteral formulation approaches have been studied for AmpB.
While effective, the limitations of these parenteral formulations
of amphotericin B are the safety issues associated with
administration (infection of the indwelling catheter, patient
chills and shaking due to RBC haemolysis, dose-dependent renal
toxicity), feasibility of administration of parenteral products in
remote locations and high drug cost.
[0004] The development of an effective and safe oral formulation of
amphotericin B that would have significant applications in the
treatment of disseminated fungal infections and would dramatically
expand access to treatment of visceral leishmaniasis. However, the
bioavailability of AmpB is negligible due to low aqueous solubility
and instability at the low pH found in gastric fluid. Such
limitations also apply to a variety of other therapeutic agents for
which oral formulations are desirable.
[0005] A need exists for effective and safe oral formulations of
amphotericin B as well as many other therapeutic agents that
provide for enhanced bioavailability and/or increased stability of
the therapeutic agent of interest the low pH found in gastric
fluid. The present invention seeks to fulfill these needs and
provides further related advantages.
SUMMARY OF THE INVENTION
[0006] The present invention provides compositions for formulating
therapeutic agents, therapeutic agent formulations based on the
compositions, methods for administering therapeutic agents using
the formulations, and methods for treating conditions and diseases
using the formulations.
[0007] In one aspect, the invention provides an amphotericin B
formulation, comprising,
[0008] (a) amphotericin B;
[0009] (b) one or more fatty acid glycerol esters; and
[0010] (c) one or more polyethylene oxide-containing phospholipids
or one or more polyethylene oxide-containing fatty acid esters.
[0011] In one embodiment, amphotericin B is present in the
formulation in an amount from about 0.5 to about 10 mg/mL of the
formulation. In one embodiment, amphotericin B is present in the
formulation in about 5 mg/mL. In another embodiment, amphotericin B
is present in the formulation in about 7 mg/mL.
[0012] In one embodiment, the fatty acid glycerol esters comprise
from about 32 to about 52% by weight fatty acid monoglycerides. In
one embodiment, the fatty acid glycerol esters comprise from about
30 to about 50% by weight fatty acid diglycerides. In one
embodiment, the fatty acid glycerol esters comprise from about 5 to
about 20% by weight fatty acid triglycerides. In one embodiment,
the fatty acid glycerol esters comprise greater than about 60% by
weight oleic acid mono-, di-, and triglycerides.
[0013] In one embodiment, the polyethylene oxide-containing
phospholipids comprise a C8-C22 saturated fatty acid ester of a
phosphatidyl ethanolamine polyethylene glycol salt. In one
embodiment, the polyethylene oxide-containing phospholipids
comprise a distearoylphosphatidyl ethanolamine polyethylene glycol
salt. In one embodiment, the distearoylphosphatidyl ethanolamine
polyethylene glycol salt is selected from the group consisting of a
distearoylphosphatidyl ethanolamine polyethylene glycol 350 salt, a
distearoylphosphatidyl ethanolamine polyethylene glycol 550 salt, a
distearoylphosphatidyl ethanolamine polyethylene glycol 750 salt, a
distearoylphosphatidyl ethanolamine polyethylene glycol 1000 salt,
a distearoylphosphatidyl ethanolamine polyethylene glycol 2000
salt, and mixtures thereof.
[0014] In one embodiment, the distearoylphosphatidyl ethanolamine
polyethylene glycol salt is present in the formulation in an amount
from 1 mM to about 30 mM based on the volume of the formulation. In
one embodiment, the distearoylphosphatidyl ethanolamine
polyethylene glycol salt is an ammonium salt or a sodium salt. In
one embodiment, the polyethylene oxide-containing fatty acid esters
comprise a polyethylene oxide ester of a C8-C22 saturated fatty
acid. In one embodiment, the polyethylene oxide-containing fatty
acid esters comprise a polyethylene oxide ester of a C12-C18
saturated fatty acid. In one embodiment, the polyethylene
oxide-containing fatty acid esters is selected from the group
consisting of lauric acid esters, palmitic acid esters, stearic
acid esters, and mixtures thereof. In one embodiment, the
polyethylene oxide-containing fatty acid esters comprise a
polyethylene oxide having an average molecular weight of from about
750 to about 2000.
[0015] In one embodiment, the ratio of the fatty acid glycerol
esters to polyethylene oxide-containing fatty acid esters is from
about 20:80 to about 80:20 v/v. In one embodiment, the ratio of the
fatty acid glycerol esters to polyethylene oxide-containing fatty
acid esters is about 60:40 v/v.
[0016] In one embodiment, the formulation further comprises
glycerol in an amount less than about 10% by weight.
[0017] In one embodiment, the formulation is a self-emulsifying
drug delivery system.
[0018] In another aspect, the invention provides a method for
administering amphotericin B, comprising administering an
amphotericin B formulation of the invention to a subject in need
thereof. In one embodiment, the formulation is administered orally.
In another embodiment, the formulation is administered
topically.
[0019] In another aspect, the invention provides a method for
treating an infectious disease treatable by the administration of
amphotericin B, comprising administering to a subject in need
thereof a therapeutically effective amount of an amphotericin B
formulation of the invention. In one embodiment, the formulation is
administered orally. In another embodiment, the formulation is
administered topically.
[0020] Diseases treatable by the formulations include fungal
infections, visceral leishmaniasis, cutaneous leishmaniasis, Chagas
disease, Alzheimer's disease, or Febrile neutropenia. Fungal
infections treatable by the formulations include aspergillosis,
blastomycosis, candidiasis, coccidioidomycosis, crytococcosis,
histoplasmosis, mucormycosis, paracoccidioidomycosis, or
sporotrichosis.
[0021] In another aspect, the invention provides a formulation for
the delivery of a therapeutic agent, comprising,
[0022] (a) a therapeutic agent;
[0023] (b) one or more fatty acid glycerol esters; and
[0024] (c) one or more polyethylene oxide-containing phospholipids
or one or more polyethylene oxide-containing fatty acid esters.
[0025] In one embodiment, the therapeutic agent is present in the
formulation in an amount from about 0.1 mg/mL to about 25 mg/mL of
the formulation.
[0026] In certain embodiments, the therapeutic agent is selected
from the group consisting of anticancers, antibiotics, antiviral
drugs, antimycotics, anti-prions, anti-amoebics, non-steroidal
anti-inflammatory drugs, anti-allergics, immunosuppressive agents,
coronary drugs, analgesics, local anesthetics, anxiolytics,
sedatives, hypnotics, migraine relieving agents, drugs against
motion sickness, and anti-emetics.
[0027] In certain embodiments, the therapeutic agent is selected
from the group consisting of tetracycline, doxycycline,
oxytetracycline, chloramphenicol, erythromycin, acyclovir,
idoxuridine, tromantadine, miconazole, ketoconazole, fluconazole,
itraconazole, econazole, griseofulvin, amphotericin B, nystatine,
metronidazole, metronidazole benzoate, tinidazole, indomethacin,
ibuprofen, piroxicam, diclofenac, disodium cromoglycate,
nitroglycerin, isosorbide dinitrate, verapamile, nifedipine,
diltiazem, digoxine, morphine, cyclosporins, buprenorphine,
lidocaine, diazepam, nitrazepam, flurazepam, estazolam,
flunitrazepam, triazolam, alprazolam, midazolam, temazepam
lormetazepam, brotizolam, clobazam, clonazepam, lorazepam,
oxazepam, busiprone, sumatriptan, ergotamine derivatives,
cinnarizine, anti-histamines, ondansetron, tropisetron,
granisetrone, metoclopramide, disulfiram, vitamin K, paclitaxel,
docetaxel, camptothecin, SN38, cisplatin, and carboplatin.
[0028] In one embodiment, the formulation further comprises a
second therapeutic agent.
[0029] In one embodiment, the fatty acid glycerol esters comprise
from about 32 to about 52% by weight fatty acid monoglycerides. In
one embodiment, the fatty acid glycerol esters comprise from about
30 to about 50% by weight fatty acid diglycerides. In one
embodiment, the fatty acid glycerol esters comprise from about 5 to
about 20% by weight fatty acid triglycerides. In one embodiment,
the fatty acid glycerol esters comprise greater than about 60% by
weight oleic acid mono-, di-, and triglycerides.
[0030] In one embodiment, the polyethylene oxide-containing
phospholipids comprise a C8-C22 saturated fatty acid ester of a
phosphatidyl ethanolamine polyethylene glycol salt. In one
embodiment, the polyethylene oxide-containing phospholipids
comprise a distearoylphosphatidyl ethanolamine polyethylene glycol
salt. In one embodiment, the distearoylphosphatidyl ethanolamine
polyethylene glycol salt is selected from the group consisting of a
distearoylphosphatidyl ethanolamine polyethylene glycol 350 salt, a
distearoylphosphatidyl ethanolamine polyethylene glycol 550 salt, a
distearoylphosphatidyl ethanolamine polyethylene glycol 750 salt, a
distearoylphosphatidyl ethanolamine polyethylene glycol 1000 salt,
a distearoylphosphatidyl ethanolamine polyethylene glycol 2000
salt, and mixtures thereof. In one embodiment, the
distearoylphosphatidyl ethanolamine polyethylene glycol salt is
present in the formulation in an amount from 1 mM to about 30 mM
based on the volume of the formulation. In one embodiment, the
distearoylphosphatidyl ethanolamine polyethylene glycol salt is an
ammonium salt or a sodium salt.
[0031] In one embodiment, the polyethylene oxide-containing fatty
acid esters comprise a polyethylene oxide ester of a C8-C22
saturated fatty acid. In one embodiment, the polyethylene
oxide-containing fatty acid esters comprise a polyethylene oxide
ester of a C12-C18 saturated fatty acid. In one embodiment, the
polyethylene oxide-containing fatty acid esters is selected from
the group consisting of lauric acid esters, palmitic acid esters,
stearic acid esters, and mixtures thereof. In one embodiment, the
polyethylene oxide-containing fatty acid esters comprise a
polyethylene oxide having an average molecular weight of from about
750 to about 2000.
[0032] In one embodiment, the ratio of the fatty acid glycerol
esters to polyethylene oxide-containing fatty acid esters is from
about 20:80 to about 80:20 v/v. In one embodiment, the ratio of the
fatty acid glycerol esters to polyethylene oxide-containing fatty
acid esters is about 60:40 v/v.
[0033] In one embodiment, the formulation further comprises
glycerol in an amount less than about 10% by weight.
[0034] In one embodiment, the formulation is a self-emulsifying
drug delivery system.
[0035] In another aspect, the invention provides a method for
administering a therapeutic agent, comprising administering a
therapeutic agent formulation of the invention to a subject in need
of such agent. In one embodiment, the formulation is administered
orally.
[0036] In another embodiment, the formulation is administered
topically.
[0037] In another aspect, the invention provides a composition for
formulating a therapeutic agent, comprising,
[0038] (a) one or more fatty acid glycerol esters; and
[0039] (b) one or more polyethylene oxide-containing phospholipids
or one or more polyethylene oxide-containing fatty acid esters.
[0040] In one embodiment, the fatty acid glycerol esters comprise
from about 32 to about 52% by weight fatty acid monoglycerides. In
one embodiment, the fatty acid glycerol esters comprise from about
30 to about 50% by weight fatty acid diglycerides. In one
embodiment, the fatty acid glycerol esters comprise from about 5 to
about 20% by weight fatty acid triglycerides. In one embodiment,
the fatty acid glycerol esters comprise greater than about 60% by
weight oleic acid mono-, di-, and triglycerides.
[0041] In one embodiment, the polyethylene oxide-containing
phospholipids comprise a C8-C22 saturated fatty acid ester of a
phosphatidyl ethanolamine polyethylene glycol salt. In one
embodiment, the polyethylene oxide-containing phospholipids
comprise a distearoylphosphatidyl ethanolamine polyethylene glycol
salt. In one embodiment, the distearoylphosphatidyl ethanolamine
polyethylene glycol salt is selected from the group consisting of a
distearoylphosphatidyl ethanolamine polyethylene glycol 350 salt, a
distearoylphosphatidyl ethanolamine polyethylene glycol 550 salt, a
distearoylphosphatidyl ethanolamine polyethylene glycol 750 salt, a
distearoylphosphatidyl ethanolamine polyethylene glycol 1000 salt,
a distearoylphosphatidyl ethanolamine polyethylene glycol 2000
salt, and mixtures thereof.
[0042] In one embodiment, the distearoylphosphatidyl ethanolamine
polyethylene glycol salt is present in the formulation in an amount
from 1 mM to about 30 mM based on the volume of the formulation. In
one embodiment, the distearoylphosphatidyl ethanolamine
polyethylene glycol salt is an ammonium salt or a sodium salt.
[0043] In one embodiment, the polyethylene oxide-containing fatty
acid esters comprise a polyethylene oxide ester of a C8-C22
saturated fatty acid. In one embodiment, the polyethylene
oxide-containing fatty acid esters comprise a polyethylene oxide
ester of a C12-C18 saturated fatty acid. In one embodiment, the
polyethylene oxide-containing fatty acid esters is selected from
the group consisting of lauric acid esters, palmitic acid esters,
stearic acid esters, and mixtures thereof. In one embodiment, the
polyethylene oxide-containing fatty acid esters comprise a
polyethylene oxide having an average molecular weight of from about
750 to about 2000.
[0044] In one embodiment, the composition further comprises
glycerol in an amount less than about 10% by weight.
[0045] In another aspect, the invention provides a method for
formulating a therapeutic agent, comprising combining a therapeutic
agent with a composition of the invention for formulating a
therapeutic agent.
DESCRIPTION OF THE DRAWINGS
[0046] The foregoing aspects and many of the attendant advantages
of this invention will become more readily appreciated as the same
become better understood by reference to the following detailed
description, when taken in conjunction with the accompanying
drawings.
[0047] FIG. 1A illustrates the chemical structure of amphotericin B
(AmpB).
[0048] FIG. 1B illustrates the chemical structure of
distearoylphosphatidyl ethanolamine polyethylene glycol 2000
ammonium salt (DSPE-PEG-2000).
[0049] FIG. 2 compares AmpB concentration (.mu.g/mL) in an
AmpB/PECEOL.RTM. formulation and representative AmpB formulations
of the invention (AmpB/PECEOL.RTM./DSPE-PEG-2000) containing
DSPE-PEG-2000 at concentrations of 5, 10, and 15 mM.
[0050] FIG. 3A compares the UV absorbance spectra over time of
representative AmpB formulations of the invention
(PECEOL.RTM./DSPE-PEG) at various concentrations (0.5-15 .mu.g/ml)
incubated in simulated gastric fluid (SGF).
[0051] FIG. 3B compares standard curves from the data in FIG. 2A
combined using peak height at 407 nm to construct the standard
curves of AmpB absorbance vs. concentration. A different standard
curve was prepared for each formulation where DSPE-PEG molecular
weight varied.
[0052] FIG. 4 compares the stability of AmpB in representative
formulations of the invention (PECEOL.RTM./DSPE-PEG 350, 550, 750,
and 2000) with an AmpB/PECEOL.RTM. formulation at 37.degree. C. in
simulated gastric fluid as a function of time (10, 30, and 120
min).
[0053] FIGS. 5A and 5B compare the stability of AmpB in
representative formulations of the invention (PECEOL.RTM./DSPE-PEG
350, 550, 750, and 2000, designated PEG 350, 550, 750, 2000,
respectively) with an AmpB/PECEOL.RTM. formulation at 37.degree. C.
in fasted-state simulated intestinal fluid (FSSIF) without lecithin
(5A) and with lecithin (5B) as a function of time (10, 30, 60, and
120 min).
[0054] FIG. 6 compares the stability of AmpB in representative
formulations of the invention (PECEOL.RTM./DSPE-PEG 350, 550, 750,
and 2000, designated PEG 350, 550, 750, 2000, respectively) with an
AmpB/PECEOL.RTM. formulation at 37.degree. C. in simulated
intestinal fluid (SIF) with pancreatin enzymes as a function of
time (10, 30, 60, and 120 min).
[0055] FIG. 7 compares Candida albicans concentration (CFU/ml) in
the kidneys of rats infected with Candida albicans and treated with
control, an AmpB/PECEOL.RTM. formulation (10 mg/kg), a
representative AmpB formulation of the invention
(AmpB/PECEOL.RTM./DSPE-PEG-2000, designated AmpB/DSPE-PEG-2000, 10
mg/kg), and intravenous ABELCET.RTM. (designated ABLC, 5
mg/ml).
[0056] FIG. 8 compares Candida albicans concentration (CFU/ml) in
the organs of rats infected with Candida albicans and treated with
control, an AmpB/PECEOL.RTM. formulation (10 mg/kg), a
representative AmpB formulation of the invention
(AmpB/PECEOL.RTM./DSPE-PEG-2000, designated AmpB/DSPE-PEG-2000, 10
mg/kg), and intravenous ABELCET.RTM. (designated ABLC, 5
mg/ml).
[0057] FIG. 9 compares plasma creatinine (mg/dl) in rats infected
with Candida albicans and treated with control, an AmpB/PECEOL.RTM.
formulation (10 mg/kg), a representative AmpB formulation of the
invention (AmpB/PECEOL.RTM./DSPE-PEG-2000, designated
AmpB/DSPE-PEG-2000, 10 mg/kg), and intravenous ABELCET.RTM.
(designated ABLC, 5 mg/ml) (blank, 0 hr, and 48 hr).
[0058] FIGS. 10A, 10B, and 10C compare AmpB concentration (mg/mL)
in representative AmpB formulations of the invention
(AmpB/PECEOL.RTM./GELUCIRE.RTM. 44/14;
AmpB/PECEOL.RTM./GELUCIRE.RTM. 50/13; and
AmpB/PECEOL.RTM./GELUCIRE.RTM. 53/10) at varying ratios of
PECEOL.RTM.:GELUCIRE.RTM. (60:40; 50:50; and 40:60 v/v) at 2, 4 and
24 hrs.
[0059] FIG. 11 compares AmpB concentration (% original
concentration, 5 mg/mL) over time (0, 1, 5, 7, 15, 21, 28, 36, 43,
49, and 56 days) for an AmpB/PECEOL.RTM. formulation (designated
PECEOL.RTM.) and representative AmpB formulations of the invention
(AmpB/PECEOL.RTM.GELUCIRE.RTM. 44/14, 50/50; and
AmpB/PECEOL.RTM./DSPE-PEG-2000, 15 mM DSPE-PEG-2000).
[0060] FIG. 12 compares AmpB concentration (% original
concentration, 5 mg/mL) over time (10, 30, 45, 60, 90, and 120 min)
in simulated gastric fluid (SGF) for an AmpB/PECEOL.RTM.
formulation and representative AmpB formulations of the invention
(AmpB/PECEOL.RTM./GELUCIRE.RTM. 44/14, 50/50; and
AmpB/PECEOL.RTM./DSPE-PEG-2000, 15 mM DSPE-PEG-2000).
[0061] FIG. 13 compares AmpB concentration (% original
concentration, 5 mg/mL) over time (10, 30, 45, 60, 90, 120, and 240
min) in fed-state simulated intestinal fluid (FeSSIF) for an
AmpB/PECEOL.RTM. formulation and a representative AmpB formulation
of the invention (AmpB/PECEOL.RTM./GELUCIRE.RTM. 44/14, 50/50).
[0062] FIG. 14 compares AmpB concentration (% original
concentration, 5 mg/mL) over time (10, 30, 45, 60, 90, 120, and 240
min) in fed-state simulated intestinal fluid (FeSSIF) with enzyme
for an AmpB/PECEOL.RTM. formulation and a representative AmpB
formulation of the invention (AmpB/PECEOL.RTM.GELUCIRE.RTM. 44/14,
50/50).
[0063] FIG. 15 compares AmpB concentration (% original
concentration, 5 mg/mL) over time (10, 30, 45, 60, 90, 120, and 240
min) in fasted-state simulated intestinal fluid (FaSSIF) for an
AmpB/PECEOL.RTM. formulation and a representative AmpB formulation
of the invention (AmpB/PECEOL.RTM.GELUCIRE.RTM. 44/14, 50/50).
[0064] FIG. 16 compares AmpB concentration (% original
concentration, 10 mg/mL) of a representative AmpB formulation of
the invention (AmpB/PECEOL.RTM./DSPE-PEG-2000, 15 mM DSPE-PEG-2000)
after seven days at room temperature and 43.degree. C. (AmpB
measured by UV absorbance of centrifuged samples after specified
time).
DETAILED DESCRIPTION OF THE INVENTION
[0065] The present invention provides compositions for formulating
therapeutic agents. The compositions are effective for solubilizing
therapeutic agents, particularly difficulty soluble therapeutic
agents. The compositions advantageously enhance the bioavailability
of the therapeutic agents. The invention also provides therapeutic
agent formulations based on the compositions that are effective for
the delivery of therapeutic agents, particularly oral
administration of therapeutic agents. Amphotericin B formulations
are used herein as the prototypic example, however, one of skill in
the art will appreciate that such formulations are applicable to a
variety of therapeutic agents. Accordingly, in one aspect, the
invention provides amphotericin B formulations based on the
compositions. The amphotericin B formulations effectively
solubilize amphotericin B providing formulations having increased
amphotericin B concentrations and, at the same time, provide for
enhanced amphotericin B bioavailability.
[0066] Amphotericin B Formulations
[0067] In one aspect, the present invention provides amphotericin B
formulations, methods for making the formulations, methods for
administering amphotericin B using the formulations, and methods
for treating diseases treatable by amphotericin B by administering
the formulations.
[0068] Amphotericin B is an effective antifungal agent, and at
present, is the drug of choice for treating most serious systemic
fungal infections. The drug binds strongly to ergosterol, a major
sterol component of fungal membranes, forming pores in the
membranes causing disruption of the membrane, cell permeability,
and lysis.
[0069] Amphotericin B has had limitations in clinical
administration due to several unfavorable properties. First,
amphotericin B has a strong binding affinity for cholesterol, a
sterol present in most mammalian cell membranes, and therefore is
capable of disrupting host cells. This leads to renal toxicity of
the drug. Second, amphotericin B is not absorbed in the
gastrointestinal tract (GIT) due to its poor solubility and its
sensitivity to the acid environment of the stomach. To overcome
this problem, amphotericin B is used parenterally as liposomal
(AMBISOME.RTM.) or as colloidal dispersion (FUNGIZONE.RTM.,
ABELCET.RTM.) for the treatment of certain systemic fungal
infections (Arikan and Rex, 2001. Lipid-based antifungal agents:
current status. Curr. Pharm. Des. 5, 393-415).
[0070] However, intravenous injection and infusion of amphotericin
B have significant disadvantages. First, the intravenous injection
and infusion of amphotericin B has been associated with
considerable fluctuation of drug concentrations in the blood and
side effects such as nephrotoxicity (Muller et al., 2000,
Nanosuspensions for the formulation of poorly soluble
drugs-rationale for development and what we can expect for the
future. In: Nielloud, F., Marti-Mestres, G. (Eds.), Pharmaceutical
emulsions and suspensions. Plenum Press/Marcel Dekker, New York,
pp. 383-408). Second, in addition to the high cost, the injection
and infusion formulation of amphotericin B have also presented low
compliance and technical problems with administration in endemic
countries.
[0071] In one embodiment, the present invention overcomes these
disadvantages by providing an amphotericin B formulation that can
be administered orally. The oral amphotericin B formulations of the
invention can be expected to improve patient compliance and to
improve pharmacokinetics of the drug and to increase the
amphotericin B absorption in GI track.
[0072] Amphotericin B is an antimycotic polyene antibiotic obtained
from Streptomyces nodosus M4575. Amphotericin B is designated
chemically as
[1R-(1R*,3S*,5R*,6R*,9R*,11R*,15S*,16R*,17R*,18S*,19E,21E,23E,25E,27E,29E-
,31E,33R*,35S*,36R*,37S,)]-33-[(3-amino-3,6-dideoxy-.beta.-D-mannopyranosy-
l)oxy]1,3,5,6,9,11,17,37-octahydroxy-15,16,18-trimethyl-13-oxo-14,39-dioxa-
bicyclo-[33.3.1]nonatriaconta-19,21,23,25,27,29,31-heptaene-36-carboxylic
acid. The chemical structure of amphotericin B is shown in FIG. 1A.
Crystalline amphotericin B is insoluble in water.
[0073] In one aspect, the present invention provides amphotericin B
formulations. The amphotericin formulations of the invention
include
[0074] (a) amphotericin B;
[0075] (b) one or more fatty acid glycerol esters; and
[0076] (c) one or more polyethylene oxide-containing phospholipids
or one or more polyethylene oxide-containing fatty acid esters.
[0077] In representative formulations, amphotericin B is present in
an amount from about 0.5 to about 10 mg/mL of the formulation. In
one embodiment, amphotericin B or pharmaceutically acceptable salt
thereof is present in the formulation in about 5 mg/mL. In one
embodiment, amphotericin B or its pharmaceutically acceptable salt
thereof is present in the formulation in about 7 mg/mL.
[0078] The amphotericin B formulations include one or more fatty
acid glycerol esters, and typically, a mixture of fatty acid
glycerol esters. As used herein the term "fatty acid glycerol
esters" refers to esters formed between glycerol and one or more
fatty acids including mono-, di-, and tri-esters (i.e.,
glycerides). Suitable fatty acids include saturated and unsaturated
fatty acids having from eight (8) to twenty-two (22) carbons atoms
(i.e., C8-C22 fatty acids). In certain embodiments, suitable fatty
acids include C12-C18 fatty acids.
[0079] The fatty acid glycerol esters useful in the formulations
can be provided by commercially available sources. A representative
source for the fatty acid glycerol esters is a mixture of mono-,
di-, and triesters commercially available as PECEOL.RTM.
(Gattefosse, Saint Priest Cedex, France), commonly referred to as
"glyceryl oleate" or "glyceryl monooleate." When PECEOL.RTM. is
used as the source of fatty acid glycerol esters in the
formulations, the fatty acid glycerol esters comprise from about 32
to about 52% by weight fatty acid monoglycerides, from about 30 to
about 50% by weight fatty acid diglycerides, and from about 5 to
about 20% by weight fatty acid triglycerides. The fatty acid
glycerol esters comprise greater than about 60% by weight oleic
acid (C18:1) mono-, di-, and triglycerides. Other fatty acid
glycerol esters include esters of palmitic acid (C16) (less than
about 12%), stearic acid (C18) (less than about 6%), linoleic acid
(C18:2) (less than about 35%), linolenic aid (C18:3) (less than
about 2%), arachidic acid (C20) (less than about 2%), and
eicosenoic acid (C20:1) (less than about 2%). PECEOL.RTM. can also
include free glycerol (typically about 1%). In one embodiment, the
fatty acid glycerol esters comprise about 44% by weight fatty acid
monoglycerides, about 45% by weight fatty acid diglycerides, and
about 9% by weight fatty acid triglycerides, and the fatty acid
glycerol esters comprise about 78% by weight oleic acid (C18:1)
mono-, di-, and triglycerides. Other fatty acid glycerol esters
include esters of palmitic acid (C16) (about 4%), stearic acid
(C18) (about 2%), linoleic acid (C18:2) (about 12%), linolenic aid
(C18:3) (less than 1%), arachidic acid (C20) (less than 1%), and
eicosenoic acid (C20:1) (less than 1%).
[0080] In certain embodiments, the formulations of the invention
can include glycerol in an amount less than about 10% by
weight.
[0081] Amphotericin B Formulations: Polyethylene Oxide-Containing
Phospholipids (DSPE-PEGs).
[0082] The amphotericin B formulations include one or more
polyethoxylated lipids. In one embodiment, the polyethoxylated
lipids are polyethylene oxide-containing phospholipids, or a
mixture of polyethylene oxide-containing phospholipids. In another
embodiment, the polyethoxylated lipids are polyethylene
oxide-containing fatty acid esters, or a mixture of polyethylene
oxide-containing fatty acid esters.
[0083] Accordingly, in one embodiment, the amphotericin B
formulations of the invention include
[0084] (a) amphotericin B;
[0085] (b) one or more fatty acid glycerol esters; and
[0086] (c) one or more polyethylene oxide-containing
phospholipids.
[0087] As used herein, the term "polyethylene oxide-containing
phospholipid" refers to a phospholipid that includes a polyethylene
oxide group (i.e., polyethylene glycol group) covalently coupled to
the phospholipid, typically through a carbamate or an ester bond.
Phospholipids are derived from glycerol and can include a phosphate
ester group and two fatty acid ester groups. Suitable fatty acids
include saturated and unsaturated fatty acids having from eight (8)
to twenty-two (22) carbons atoms (i.e., C8-C22 fatty acids). In
certain embodiments, suitable fatty acids include saturated C12-C18
fatty acids. Representative polyethylene oxide-containing
phospholipids include C8-C22 saturated fatty acid esters of a
phosphatidyl ethanolamine polyethylene glycol salt. In certain
embodiments, suitable fatty acids include saturated C12-C18 fatty
acids.
[0088] The molecular weight of the polyethylene oxide group of the
polyethylene oxide-containing phospholipid can be varied to
optimize the solubility of the therapeutic agent (e.g.,
amphotericin B) in the formulation. Representative average
molecular weights for the polyethylene oxide groups can be from
about 200 to about 5000 (e.g., PEG 200 to PEG 5000).
[0089] In one embodiment, the polyethylene oxide-containing
phospholipids are distearoyl phosphatidyl ethanolamine polyethylene
glycol salts. Representative distearoylphosphatidyl ethanolamine
polyethylene glycol salts include distearoylphosphatidyl
ethanolamine polyethylene glycol 350 (DSPE-PEG-350) salts,
distearoylphosphatidyl ethanolamine polyethylene glycol 550
(DSPE-PEG-550) salts, distearoylphosphatidyl ethanolamine
polyethylene glycol 750 (DSPE-PEG-750) salts,
distearoylphosphatidyl ethanolamine polyethylene glycol 1000
(DSPE-PEG-1000) salts, distearoylphosphatidyl ethanolamine
polyethylene glycol 1500 (DSPE-PEG-1500) salts, and
distearoylphosphatidyl ethanolamine polyethylene glycol 2000
(DSPE-PEG-2000) salts. Mixtures can also be used. For the
distearoylphosphatidyl ethanolamine polyethylene glycol salts
above, the number (e.g., 350, 550, 750, 1000, and 2000) designates
the average molecular weight of the polyethylene oxide group. The
abbreviations for these salts used herein is provided in
parentheses above.
[0090] Suitable distearoylphosphatidyl ethanolamine polyethylene
glycol salts include ammonium and sodium salts.
[0091] The chemical structure of distearoylphosphatidyl
ethanolamine polyethylene glycol 2000 (DSPE-PEG-2000) ammonium salt
is illustrated in FIG. 1B. Referring to FIG. 1B, the polyethylene
oxide-containing phospholipid includes a phosphate ester group and
two fatty acid ester (stearate) groups, and a polyethylene oxide
group covalently coupled to the amino group of the phosphatidyl
ethanolamine through a carbamate bond.
[0092] As noted above, the polyethylene oxide-containing
phospholipid affects the ability of the formulation to solubilize a
therapeutic agent. In general, the greater the amount of
polyethylene oxide-containing phospholipid, the greater the
solubilizing capacity of the formulation for difficulty soluble
therapeutic agents. The polyethylene oxide-containing phospholipid
can be present in the formulation in an amount from about 1 mM to
about 30 mM based on the volume of the formulation. In certain
embodiments, the distearoylphosphatidyl ethanolamine polyethylene
glycol salt is present in the formulation in an amount from 1 mM to
about 30 mM based on the volume of the formulation. In one
embodiment, the distearoylphosphatidyl ethanolamine polyethylene
glycol salt is present in the formulation in about 15 mM based on
the volume of the formulation.
[0093] FIG. 2 compares amphotericin B concentration (.mu.g/mL) in
an AmpB/PECEOL.RTM. formulation (containing no polyethylene
oxide-containing phospholipids or polyethylene oxide-containing
fatty acid esters) and representative AmpB formulations of the
invention (AmpB/PECEOL.RTM./DSPE-PEG-2000) containing DSPE-PEG-2000
at concentrations of 5, 10, and 15 mM. AmpB measured by UV
absorbance of centrifuged samples after 24 hrs at 45.degree. C.
[0094] In one embodiment, the amphotericin B formulations of the
invention include
[0095] (a) amphotericin B;
[0096] (b) oleic acid glycerol esters; and
[0097] (c) a distearoylphosphatidyl ethanolamine polyethylene
glycol salt.
[0098] In one embodiment, the amphotericin B formulation of the
invention includes amphotericin B, PECEOL.RTM., and a
distearoylphosphatidyl ethanolamine polyethylene glycol salt. In
this embodiment, the distearoylphosphatidyl ethanolamine
polyethylene glycol salt is present in an amount up to about 30
mM.
[0099] The preparation and characterization of representative
amphotericin B formulations of the invention that include
polyethylene oxide-containing phospholipids is described in Example
1.
[0100] The amphotericin B formulations that include polyethylene
oxide-containing phospholipids include amphotericin B that is both
partially solubilized (dissolved) and present as solid particles to
provide a fine solid dispersion. Dispersion of the formulation in
aqueous media provides a nano-/microemulsion having emulsion
droplets that range in size from about 50 nm to about 5 .mu.m.
[0101] Polyethylene glycol molecular weight had no clear effect on
the emulsion droplet size in simulated intestinal fluid (Table 3)
following mixing over a period of 2 h at 37.degree. C. Submicron
mean diameters were observed in the range of 300-600 nm with a
fairly wide polydispersity. A bimodal particle size distribution
was also generated, with a small subpopulation (about 20%) centered
in submicron range (150-300 nm) and another centered in the 1-2
.mu.m range (about 80%). AmpB in PECEOL.RTM. alone also formed
droplets of similar size and distribution in simulated intestinal
fluid.
[0102] To determine their effectiveness as orally administered
formulations, the stability of representative amphotericin B
formulations of the invention was evaluated in simulated gastric
fluid. FIG. 3A compares the UV absorbance spectra over time of
representative AmpB formulations of the invention
(PECEOL.RTM./DSPE-PEG) at various concentrations (0.5-15 .mu.g/ml)
incubated in simulated gastric fluid (SGF). There is no change in
the peak height or peak ratio at any concentration as a function of
incubation time up to 60 min. FIG. 3B compares standard curves from
the data in FIG. 2A combined using peak height at 407 nm to
construct the standard curves of AmpB absorbance vs. concentration.
A different standard curve was prepared for each formulation where
DSPE-PEG molecular weight varied. FIG. 4 compares the stability of
AmpB in representative formulations of the invention
(PECEOL.RTM./DSPE-PEG 350, 550, 750, and 2000) with an
AmpB/PECEOL.RTM. formulation at 37.degree. C. in simulated gastric
fluid as a function of time (10, 30, and 120 min). Data represent
the mean.+-.SD of three independent experiments, each of which was
performed in triplicate. Each of the evaluated representative
formulations of the invention demonstrated stability in simulated
gastric fluid over the time period evaluated.
[0103] The stability of representative amphotericin B formulations
of the invention was also evaluated in fasted-state simulated
intestinal fluid (FSSIF) without lecithin and with lecithin, and in
simulated intestinal fluid with pancreatin enzymes. FIGS. 5A and 5B
compare the stability of AmpB in representative formulations of the
invention (PECEOL.RTM./DSPE-PEG 350, 550, 750, and 2000, designated
PEG 350, 550, 750, 2000, respectively) with an AmpB/PECEOL.RTM.
formulation at 37.degree. C. in fasted-state simulated intestinal
fluid (FSSIF) without lecithin (5A) and with lecithin (5B) as a
function of time (10, 30, 60, and 120 min). Data represent the
mean.+-.SD of three independent experiments, each of which was
performed in triplicate. FIG. 6 compares the stability of AmpB in
representative formulations of the invention (PECEOL.RTM./DSPE-PEG
350, 550, 750, and 2000, designated PEG 350, 550, 750, 2000,
respectively) with an AmpB/PECEOL.RTM. formulation at 37.degree. C.
in simulated intestinal fluid (SIF) with pancreatin enzymes as a
function of time (10, 30, 60, and 120 min) Data represent the
mean.+-.SD of three independent experiments, each of which was
performed in triplicate. Each of the evaluated representative
formulations of the invention demonstrated stability in the
simulated intestinal fluids over the time period evaluated.
[0104] The stability of the representative amphotericin B
formulations in the GI fluids demonstrates their suitability as
orally administered formulations.
[0105] AmpB in PECEOL.RTM. was stabilized and its solubility
enhanced 50-fold by the incorporation of 15 mM DSPE-PEG, where the
PEG mean molecular weight was varied between 350 and 2000. Drug
stability in stomach and intestine is critical for promoting drug
absorption in the GI tract. AmpB is well known to be more soluble
but relatively unstable at low pH, therefore any protection
afforded by the lipid components of the formulation could be a
significant benefit toward increasing the oral bioavailability of
AmpB. It is also important to know if the lipidic vehicles
influenced the superaggregation state of AmpB, which has been
previously been shown to influence drug solubility as well as in
vivo activity. The UV spectral pattern of AmpB in the lipidic
vehicles described herein was consistent with monomeric AmpB before
and after incubation in simulated gastric or intestinal fluids (see
FIG. 3A). No UV spectral pattern change was noted upon ambient
temperature storage (21.degree. C.) over a period of 4 weeks either
(data not shown). However, interactions between the AmpB and the
lipid components in the undiluted formulation (in the absence of
the assay solvent) or following oral absorption in vivo may be
different.
[0106] Stability of representative formulations of the invention in
simulated gastric fluid over 2 h was excellent, with surprisingly
little variability between formulations prepared with the various
DSPE-PEGs or with only PECEOL.RTM. (see FIGS. 4-6). All showed a
translucent appearance with no precipitate appearing. More
variation in stability was observed in simulated fasted-state
intestinal fluid containing bile salts (see FIGS. 5A and 5B). The
emulsification properties of the bile salts, lecithin and
phospholipase in pancreatin could influence formulation stability
and therefore drug stability was evaluated in simulated intestinal
fluids containing these components. Lecithin would likely be
incorporated into the lipid mixture when including in the simulated
intestinal fluid, which had the potential to either improve the
association of amphotericin B with the lipid excipients or to
exclude it. The presence of lecithin, however, made no appreciable
difference in the rate or extent of degradation or in the rank
order of degradation at the end of 2 h (see FIG. 5B). Clearly,
DSPE-PEG 350 containing formulations provided less drug stability
than those containing the longer-chain PEGs. In the absence of
lecithin, submicron particle size analysis did not show a
significant population below 50 nm that would be consistent with
DSPE-PEG micelles, e.g., if DSPE-PEG350 had self-associated into a
separate micelle population (see FIG. 5A). Furthermore, there was
no significant effect on particle size due to the presence of
DSPE-PEG or polyethylene glycol molecular weight, suggesting that
the emulsification properties are largely derived from the
PECEOL.RTM. component. However, the possibility that a small
fraction of micelles exists in equilibrium with the lipid
excipient/drug mixture in the simulated intestinal fluid cannot be
excluded for any of the DSPE-PEG formulations, however, it does not
appear to be a major component. Therefore, it is possible that the
improved stability of AmpB in PECEOL.RTM./DSPE-PEG of higher
molecular weight may be related to the surface properties of the
emulsion droplets themselves, in spite of the lack of a direct
relationship to particle size distribution, such that the
hydrophilic polyethylene glycol chains may orient to the water
interface while the PECEOL.RTM./AmpB fraction would remain in the
inner oil phase and thereby sequestering and protecting the AmpB
from degradation. Thus, there was a trend to increased stability
for PEG molecular weight 750 and 2000 compared to 350 and 550. The
interactions between AmpB and the PECEOL.RTM./DSPE-PEG resulted in
a UV spectral shape consistent with monomeric AmpB rather than
aggregated AmpB (FIG. 3A).
[0107] Amphotericin B Formulations: Polyethylene Oxide-Containing
Fatty Acid Esters.
[0108] As noted above, the amphotericin B formulations include one
or more polyethoxylated lipids such as polyethylene
oxide-containing phospholipids or one or more polyethylene
oxide-containing fatty acid esters, and typically, a mixture of
polyethylene oxide-containing phospholipids or a mixture of
polyethylene oxide-containing fatty acid esters.
[0109] Accordingly, in one embodiment, the amphotericin B
formulations of the invention include
[0110] (a) amphotericin B;
[0111] (b) one or more fatty acid glycerol esters; and
[0112] (c) one or more polyethylene oxide-containing fatty acid
esters.
[0113] As used herein, the term "polyethylene oxide-containing
fatty acid ester" refers to a fatty acid ester that includes a
polyethylene oxide group (i.e., polyethylene glycol group)
covalently coupled to the fatty acid through an ester bond.
Polyethylene oxide-containing fatty acid esters include mono- and
di-fatty acid esters of polyethylene glycol. Suitable polyethylene
oxide-containing fatty acid esters are derived from fatty acids
including saturated and unsaturated fatty acids having from eight
(8) to twenty-two (22) carbons atoms (i.e., a polyethylene oxide
ester of a C8-C22 fatty acid). In certain embodiments, suitable
polyethylene oxide-containing fatty acid esters are derived from
fatty acids including saturated and unsaturated fatty acids having
from twelve (12) to eighteen (18) carbons atoms (i.e., a
polyethylene oxide ester of a C12-C18 fatty acid). Representative
polyethylene oxide-containing fatty acid esters include saturated
C8-C22 fatty acid esters. In certain embodiments, suitable
polyethylene oxide-containing fatty acid esters include saturated
C12-C18 fatty acids.
[0114] The molecular weight of the polyethylene oxide group of the
polyethylene oxide-containing fatty acid ester can be varied to
optimize the solubility of the therapeutic agent (e.g.,
amphotericin B) in the formulation. Representative average
molecular weights for the polyethylene oxide groups can be from
about 350 to about 2000. In one embodiment, the average molecular
weight for the polyethylene oxide group is about 1500.
[0115] In this embodiment, the amphotericin B formulations include
one or more polyethylene oxide-containing fatty acid esters, and
typically, a mixture of polyethylene oxide-containing fatty acid
esters (mono- and di-fatty acid esters of polyethylene glycol).
[0116] The polyethylene oxide-containing fatty acid esters useful
in the formulations can be provided by commercially available
sources. Representative polyethylene oxide-containing fatty acid
esters (mixtures of mono- and diesters) are commercially available
under the designation GELUCIRE.RTM. (Gattefosse, Saint Priest
Cedex, France). Suitable polyethylene oxide-containing fatty acid
esters can be provided by GELUCIRE.RTM. 44/14, GELUCIRE.RTM. 50/13,
and GELUCIRE.RTM. 53/10. The numerals in these designations refer
to the melting point and hydrophilic/lipophilic balance (HLB) of
these materials, respectively.
[0117] GELUCIRE.RTM. 44/14, GELUCIRE.RTM. 50/13, and GELUCIRE.RTM.
53/10 are mixtures of (a) mono-, di-, and triesters of glycerol
(glycerides) and (b) mono- and diesters of polyethylene glycol
(macrogols). The GELUCIRES can also include free polyethylene
glycol (e.g., PEG 1500).
[0118] Lauric acid (C12) is the predominant fatty acid component of
the glycerides and polyethylene glycol esters in GELUCIRE.RTM.
44/14. GELUCIRE.RTM. 44/14 is referred to as a mixture of glyceryl
dilaurate (lauric acid diester with glycerol) and PEG dilaurate
(lauric acid diester with polyethylene glycol), and is commonly
known as PEG-32 glyceryl laurate (Gattefosse) lauroyl macrogol-32
glycerides EP, or lauroyl polyoxylglycerides USP/NF. GELUCIRE.RTM.
44/14 is produced by the reaction of hydrogenated palm kernel oil
with polyethylene glycol (average molecular weight 1500).
GELUCIRE.RTM. 44/14 includes about 20% mono-, di- and,
triglycerides, about 72% mono- and di-fatty acid esters of
polyethylene glycol 1500, and about 8% polyethylene glycol
1500.
[0119] GELUCIRE.RTM. 44/14 includes lauric acid (C12) esters (30 to
50%), myristic acid (C14) esters (5 to 25%), palmitic acid (C16)
esters (4 to 25%), stearic acid (C18) esters (5 to 35%), caprylic
acid (C8) esters (less than 15%), and capric acid (C10) esters
(less than 12%). GELUCIRE.RTM. 44/14 may also include free glycerol
(typically less than about 1%). In a representative formulation,
GELUCIRE.RTM. 44/14 includes lauric acid (C12) esters (about 47%),
myristic acid (C14) esters (about 18%), palmitic acid (C16) esters
(about 10%), stearic acid (C18) esters (about 11%), caprylic acid
(C8) esters (about 8%), and capric acid (C10) esters (about
12%).
[0120] Palmitic acid (C16) (40-50%) and stearic acid (C18) (48-58%)
are the predominant fatty acid components of the glycerides and
polyethylene glycol esters in GELUCIRE.RTM. 50/13. GELUCIRE.RTM.
50/13 is known as PEG-32 glyceryl palmitostearate (Gattefosse),
stearoyl macrogolglycerides EP, or stearoyl polyoxylglycerides
USP/NF). GELUCIRE.RTM. 50/13 includes palmitic acid (C16) esters
(40 to 50%), stearic acid (C18) esters (48 to 58%) (stearic and
palmitic acid esters greater than about 90%), lauric acid (C12)
esters (less than 5%), myristic acid (C14) esters (less than 5%),
caprylic acid (C8) esters (less than 3%), and capric acid (C10)
esters (less than 3%). GELUCIRE.RTM. 50/13 may also include free
glycerol (typically less than about 1%). In a representative
formulation, GELUCIRE.RTM. 50/13 includes palmitic acid (C16)
esters (about 43%), stearic acid (C18) esters (about 54%) (stearic
and palmitic acid esters about 97%), lauric acid (C12) esters (less
than 1%), myristic acid (C14) esters (about 1%), caprylic acid (C8)
esters (less than 1%), and capric acid (C10) esters (less than
1%)
[0121] Stearic acid (C18) is the predominant fatty acid component
of the glycerides and polyethylene glycol esters in GELUCIRE.RTM.
53/10. GELUCIRE.RTM. 53/10 is known as PEG-32 glyceryl stearate
(Gattefosse).
[0122] In one embodiment, the polyethylene oxide-containing fatty
acid ester is a lauric acid ester, a palmitic acid ester, or a
stearic acid ester (i.e., mono- and di-lauric acid esters of
polyethylene glycol, mono- and di-palmitic acid esters of
polyethylene glycol, mono- and di-stearic acid esters of
polyethylene glycol). Mixtures of these esters can also be
used.
[0123] For embodiments that include polyethylene oxide-containing
fatty acid esters, the ratio of the fatty acid glycerol esters to
polyethylene oxide-containing fatty acid esters is from about 20:80
to about 80:20 v/v. In one embodiment, the ratio of the fatty acid
glycerol esters to polyethylene oxide-containing fatty acid esters
is about 30:70 v/v. In one embodiment, the ratio of the fatty acid
glycerol esters to polyethylene oxide-containing fatty acid esters
is about 40:60 v/v. In one embodiment, the ratio of the fatty acid
glycerol esters to polyethylene oxide-containing fatty acid esters
is about 50:50 v/v. In one embodiment, the ratio of the fatty acid
glycerol esters to polyethylene oxide-containing fatty acid esters
is about 60:40 v/v. In one embodiment, the ratio of the fatty acid
glycerol esters to polyethylene oxide-containing fatty acid esters
is about 70:30 v/v.
[0124] In one embodiment, the amphotericin B formulations of the
invention include
[0125] (a) amphotericin B;
[0126] (b) oleic acid glycerol esters; and
[0127] (c) lauric acid esters of polyethylene glycol.
[0128] In another embodiment, the amphotericin B formulations of
the invention include
[0129] (a) amphotericin B;
[0130] (b) oleic acid glycerol esters; and
[0131] (c) palmitic and stearic acid esters of polyethylene
glycol.
[0132] In a further embodiment, the amphotericin B formulations of
the invention include
[0133] (a) amphotericin B;
[0134] (b) oleic acid glycerol esters; and
[0135] (c) stearic acid esters of polyethylene glycol.
[0136] In one embodiment, the amphotericin B formulation of the
invention includes amphotericin B, PECEOL.RTM., and GELUCIRE.RTM.
44/14. In another embodiment, the amphotericin B formulation of the
invention includes amphotericin B, PECEOL.RTM., and GELUCIRE.RTM.
50/13. In a further embodiment, the amphotericin B formulation of
the invention includes amphotericin B, PECEOL.RTM., and
GELUCIRE.RTM. 53/10. In these embodiments, the ratio of PECEOL.RTM.
to GELUCIRE.RTM. can be from 20:80 to 80:20 (e.g., 20:80, 30:70;
40:60; 50:50; 60:40; 70:30; and 80:20).
[0137] The preparation and characterization of representative
amphotericin B formulations of the invention that include
polyethylene oxide-containing fatty acid esters is described in
Example 1.
[0138] The amphotericin B formulations that include polyethylene
oxide-containing fatty acid esters include amphotericin B that is
both partially solubilized (dissolved) and present as solid
particles to provide a fine solid dispersion. Dispersion of the
formulations in aqueous media provides a nano-/microemulsion.
[0139] The preliminary SEDDS formulations of amphotericin B (see
Example 1) did produce self-emulsification and a small droplet size
upon dispersion into physiological saline. The dispersion
properties of the CAPTEX.RTM. 355-based formulations were similar
to those based on mixtures of PECEOL.RTM./GELUCIRE.RTM. 44/14 or
50/13, generating multiple subpopulations of emulsion droplets in
the submicron or 1 .mu.m range (see Tables 1 and 2). This particle
size would be appropriate for dispersing the drug in the GI tract
to best facilitate absorption.
[0140] The solubility of representative amphotericin B formulations
of that include polyethylene oxide-containing fatty acid esters is
illustrated in FIGS. 10A-10C. FIGS. 10A, 10B, and 10C compare AmpB
concentration (mg/mL) in representative AmpB formulations of the
invention (AmpB/PECEOL.RTM.GELUCIRE.RTM. 44/14;
AmpB/PECEOL.RTM./GELUCIRE.RTM. 50/13; and
AmpB/PECEOL.RTM./GELUCIRE.RTM. 53/10) at varying ratios of
PECEOL.RTM.:GELUCIRE.RTM. (60:40; 50:50; and 40:60 v/v) at 2, 4 and
24 hrs (AmpB measured by UV absorbance of centrifuged samples after
specified time at 45.degree. C.). FIG. 11 compares AmpB
concentration (% original concentration, 5 mg/mL) over time (0, 1,
5, 7, 15, 21, 28, 36, 43, 49, and 56 days) for an AmpB/PECEOL.RTM.
formulation (designated PECEOL.RTM.) and representative AmpB
formulations of the invention (AmpB/PECEOL.RTM./GELUCIRE.RTM.
44/14, 50/50; and AmpB/PECEOL.RTM./DSPE-PEG-2000, 15 mM
DSPE-PEG-2000) (AmpB measured by UV absorbance of centrifuged
samples after specified time at 43.degree. C.). Of the formulations
evaluated, the AmpB/PECEOL.RTM./GELUCIRE.RTM. 44/14, 50/50,
formulation shows the greatest stability, up to 21 days.
[0141] To determine their effectiveness as orally administered
formulations, the stability of representative amphotericin B
formulations of the invention was evaluated in simulated gastric
fluid.
[0142] FIG. 12 compares AmpB concentration (% original
concentration, 5 mg/mL) over time (10, 30, 45, 60, 90, and 120 min)
in simulated gastric fluid (SGF) for an AmpB/PECEOL.RTM.
formulation and representative AmpB formulations of the invention
(AmpB/PECEOL.RTM./GELUCIRE.RTM. 44/14, 50/50; and
AmpB/PECEOL.RTM./DSPE-PEG-2000, 15 mM DSPE-PEG-2000) (AmpB measured
by UV absorbance of centrifuged samples after specified time at
37.degree. C. in SGF, 30 mM NaCl at pH 1.2).
[0143] FIG. 13 compares AmpB concentration (% original
concentration, 5 mg/mL) over time (10, 30, 45, 60, 90, 120, and 240
min) in fed-state simulated intestinal fluid (FeSSIF) for an
AmpB/PECEOL.RTM. formulation and a representative AmpB formulation
of the invention (AmpB/PECEOL.RTM./GELUCIRE.RTM. 44/14, 50/50)
(AmpB measured by UV absorbance of centrifuged samples after 4 hrs
in FeSSIF, which contains potassium chloride (15.2 g/L), sodium
taurolaurate (15 mM), egg phosphatidylcholine (3.75 mM), and acetic
acid, adjusted to pH 5.0). The representative formulation of the
invention demonstrates consistent AmpB concentration for up to 2
hours.
[0144] FIG. 14 compares AmpB concentration (% original
concentration, 5 mg/mL) over time (10, 30, 45, 60, 90, 120, and 240
min) in fed-state simulated intestinal fluid (FeSSIF) with enzyme
for an AmpB/PECEOL.RTM. formulation and a representative AmpB
formulation of the invention (AmpB/PECEOL.RTM.)/GELUCIRE.RTM.
44/14, 50/50) (AmpB measured by UV absorbance of centrifuged
samples after 4 hrs in FeSSIF, which contains potassium chloride
(15.2 g/L), sodium taurolaurate (7.5 mM), egg phosphatidylcholine
(2.0 mM), glyceryl monooleate (5.0 mM), sodium oleate (0.8 mM),
pancreatin (1000 u lipase/L), and acetic acid, adjusted to pH 5.8).
The representative formulation of the invention demonstrates
consistent AmpB concentration for up to 2 hours.
[0145] FIG. 15 compares AmpB concentration (% original
concentration, 5 mg/mL) over time (10, 30, 45, 60, 90, 120, and 240
min) in fasted-state simulated intestinal fluid (FaSSIF) for an
AmpB/PECEOL.RTM. formulation and a representative AmpB formulation
of the invention (AmpB/PECEOL.RTM./GELUCIRE.RTM. 44/14, 50/50)
(AmpB measured by UV absorbance of centrifuged samples after 4 hrs
in FaSSIF, which contains potassium chloride (7.7 g/L), dibasic
potassium phosphate (3.9 g/L), sodium taurolaurate (3.0 mM), egg
phosphatidylcholine (0.75 mM), and acetic acid, adjusted to pH
6.5). The representative formulation of the invention demonstrates
consistent AmpB concentration for up to 2 hours.
[0146] Each of the evaluated representative formulations of the
invention demonstrated stability in the simulated fluids over the
time period evaluated. The stability of the representative
amphotericin B formulations in the GI fluids demonstrates their
suitability as orally administered formulations.
[0147] Self-Emulsifying Drug Delivery Systems.
[0148] The amphotericin B formulations of the invention can be
self-emulsifying drug delivery systems. Self-emulsifying drug
delivery systems (SEDDS) are isotropic mixtures of oils,
surfactants, solvents, and co-solvents/surfactants. SEDDS can be
used for the design of formulations in order to improve the oral
absorption of highly lipophilic drug compounds, such as
amphotericin B. When a SEDDS composition is released into the lumen
of the gut, the composition disperses to form a fine emulsion, so
that the drug remains in solution in the gut, avoiding the
dissolution step that frequently limits the rate of absorption of
hydrophobic drugs from the crystalline state. The use of SEDDS
usually leads to improved bioavailability and/or a more consistent
temporal profile of absorption from the gut. A description of
compositions of SEDDS can be found in C. W. Pouton, Advanced Drug
Delivery Reviews 25: 47-58 (1997).
[0149] The amphotericin B formulations of the invention can be
orally administered in soft or hard gelatin capsules and form fine
relatively stable oil-in-water (o/w) emulsions upon aqueous
dilution owing to the gentle agitation of the gastrointestinal
fluids. The efficiency of oral absorption of the drug compound from
the SEDDS depends on many formulation-related parameters, such as
the formulations' components, polarity of the emulsion, droplet
size and charge, all of which in essence determine the
self-emulsification ability. Thus, only very specific
pharmaceutical excipient combinations will lead to efficient
self-emulsifying systems.
[0150] Methods for Administration and Treatment with Amphotericin
B.
[0151] The administration of intravenous AmpB has been limited by
its dose-dependent kidney toxicity that has not been predictable by
monitoring plasma and/or serum drug concentration. A number of
studies have reported that AmpB, solubilized in methanol, is poorly
absorbed from the gastrointestinal (GI) tract and therefore is not
commonly administered orally but intravenously, which can result in
the aforementioned renal toxicity. However, to date, few studies
investigating the development and assessing the antifungal activity
of oral AmpB formulations have been reported.
[0152] The effectiveness of representative amphotericin B
formulations of the invention that include polyethylene
oxide-containing phospholipids in treating fungal infections is
described in Example 2. The effectiveness of these formulations for
treating Aspergillus fumigatus and Candida albicans was
demonstrated in animal studies.
[0153] Treatment of rats infected with Aspergillus fumigatus with
representative amphotericin B formulations of the invention that
include polyethylene oxide-containing phospholipids significantly
decreased total fungal CFU concentrations recovered in all the
organs added together by 80% compared to non-treated controls
(Table 4) without significant changes in plasma creatinine levels
(Table 5). ABELCET.RTM. treatment significantly decreased total
fungal CFU concentrations recovered in all the organs added
together by 88% compared to non-treated controls (Table 4) without
significant changes in plasma creatinine levels (Table 5).
[0154] The results for Candida albicans are similar to those for
Aspergillus fumigatus. Fungal analysis of the kidneys of Candida
albicans-infected rats treated with a representative AmpB
formulation of the invention demonstrate significantly decreased
total fungal CFU concentrations compared to control. FIG. 7
compares Candida albicans concentration (CFU/ml) in the kidneys of
rats infected with Candida albicans and treated with control, an
AmpB/PECEOL.RTM. formulation (10 mg/kg), a representative AmpB
formulation of the invention (AmpB/PECEOL.RTM./DSPE-PEG-2000,
designated AmpB/DSPE-PEG-2000, 10 mg/kg), and intravenous
ABELCET.RTM. (designated ABLC, 5 mg/ml). FIG. 8 compares Candida
albicans concentration (CFU/ml) in the organs of rats infected with
Candida albicans and treated with control, an AmpB/PECEOL.RTM.
formulation (10 mg/kg), a representative AmpB formulation of the
invention (AmpB/PECEOL.RTM./DSPE-PEG-2000, designated
AmpB/DSPE-PEG-2000, 10 mg/kg), and intravenous ABELCET.RTM.
(designated ABLC, 5 mg/ml). The effectiveness of the representative
AmpB formulation in reducing Candida albicans concentration was
comparable to ABELCET.RTM.. Treatment with the representative AmpB
formulation significantly decreased total fungal CFU concentrations
recovered in the kidneys without significant changes in plasma
creatinine levels. FIG. 9 compares plasma creatinine (mg/dl) in
rats infected with Candida albicans and treated with control, an
AmpB/PECEOL.RTM. formulation (10 mg/kg), a representative AmpB
formulation of the invention (AmpB/PECEOL.RTM./DSPE-PEG-2000,
designated AmpB/DSPE-PEG-2000, 10 mg/kg), and intravenous
ABELCET.RTM. (designated ABLC, 5 mg/ml) (blank, 0 hr, and 48 hr).
No renal toxicity was observed as measured by plasma creatine
levels.
[0155] In another aspect, the invention provides a method for
treating an infectious disease treatable by the administration of
amphotericin B. In the method, a therapeutically effective amount
of an amphotericin B formulation of the invention is administered
to a subject in need thereof. In one embodiment, the formulation is
administered orally. In another embodiment, the formulation is
administered topically.
[0156] As used herein, the terms "treating" and "treatment" refer
to reduction in severity and/or frequency of symptoms, elimination
of symptoms and/or underlying cause, reduction in likelihood of the
occurrence of symptoms and/or underlying cause, and improvement or
remediation of damage. Thus, "treating" a patient with an active
agent as provided herein includes prevention of a particular
condition, disease or disorder in a susceptible individual as well
as treatment of a clinically symptomatic individual. As used
herein, "effective amount" refers to an amount covering both
therapeutically effective amounts and prophylactically effective
amounts. As used herein, "therapeutically effective amount" refers
to an amount that is effective to achieve the desired therapeutic
result. A therapeutically effective amount of a given active agent
will typically vary with respect to factors such as the type and
severity of the disorder or disease being treated and the age,
gender, and weight of the patient.
[0157] Infectious diseases treatable by the method and formulations
of the invention include fungal infections (aspergillosis,
blastomycosis, candidiasis, coccidioidomycosis, crytococcosis,
histoplasmosis, mucormycosis, paracoccidioidomycosis, and
sporotrichosis), visceral leishmaniasis, cutaneous leishmaniasis,
Chagas disease, and Febrile neutropenia. Amphotericin B has been
shown to bind to amyloid and prevent the formulation of fibrils.
Amphotericin B has been indicated as useful for the treatment of
Alzheimer's disease. Accordingly, the amphotericin B formulation of
the invention can be used in the treatment of Alzheimer's
disease.
[0158] In summary, in one aspect, the present invention provides
amphotericin B formulations that can be orally administered. The
amphotericin B formulations of the invention provide excellent drug
solubilization, drug stability in simulated gastric and intestinal
fluids, and have significant antifungal activity without the
dose-limiting renal toxicity for which the parenteral formulations
of amphotericin B are well known.
[0159] Therapeutic Agent Formulations
[0160] In another aspect, the present invention provides
formulations for the delivery of therapeutic agents, methods for
making the formulations, and methods for administering the
therapeutic agents using the formulations.
[0161] In one aspect, the invention provides a formulation for the
delivery of a therapeutic agent. The therapeutic agent formulation
includes
[0162] (a) a therapeutic agent;
[0163] (b) one or more fatty acid glycerol esters; and
[0164] (c) one or more polyethoxylated lipids such as one or more
polyethylene oxide-containing phospholipids or one or more
polyethylene oxide-containing fatty acid esters.
[0165] In the therapeutic agent formulation above, the fatty acid
glycerol esters, the polyethylene oxide-containing phospholipids,
and the polyethylene oxide-containing fatty acid esters are as
described above for the amphotericin B formulations. The amounts of
these components in the above therapeutic agent formulation is also
as described above for the amphotericin B formulations. The
therapeutic agent can be present in the formulation in an amount
from about 0.1 mg/mL to about 25 mg/mL of the formulation. In
certain embodiments, the formulations can further include glycerol
in an amount less than about 10% by weight.
[0166] The therapeutic drug formulation of the invention
advantageously solubilizes difficulty soluble therapeutic drugs.
Representative therapeutic agents that can be advantageously
formulated and delivered by the formulation and methods of the
invention include anticancers, antibiotics, antiviral drugs,
antimycotics, anti-prions, anti-amoebics, non-steroidal
anti-inflammatory drugs, anti-allergics, immunosuppressive agents,
coronary drugs, analgesics, local anesthetics, anxiolytics,
sedatives, hypnotics, migraine relieving agents, drugs against
motion sickness, and anti-emetics.
[0167] Specific therapeutic agents that can be advantageously
formulated and delivered by the formulation and methods of the
invention include tetracycline, doxycycline, oxytetracycline,
chloramphenicol, erythromycin, acyclovir, idoxuridine,
tromantadine, miconazole, ketoconazole, fluconazole, itraconazole,
econazole, griseofulvin, amphotericin B, nystatine, metronidazole,
metronidazole benzoate, tinidazole, indomethacin, ibuprofen,
piroxicam, diclofenac, disodium cromoglycate, nitroglycerin,
isosorbide dinitrate, verapamile, nifedipine, diltiazem, digoxine,
morphine, cyclosporins, buprenorphine, lidocaine, diazepam,
nitrazepam, flurazepam, estazolam, flunitrazepam, triazolam,
alprazolam, midazolam, temazepam lormetazepam, brotizolam,
clobazam, clonazepam, lorazepam, oxazepam, busiprone, sumatriptan,
ergotamine derivatives, cinnarizine, anti-histamines, ondansetron,
tropisetron, granisetrone, metoclopramide, disulfiram, vitamin K,
paclitaxel, docetaxel, camptothecin, SN38, cisplatin, and
carboplatin.
[0168] In certain embodiments, the therapeutic agent formulation of
the invention can include a second therapeutic agent.
[0169] The therapeutic agent formulation can be a self-emulsifying
drug delivery system.
[0170] In one embodiment, the therapeutic agent formulation
includes
[0171] (a) a therapeutic agent;
[0172] (b) one or more fatty acid glycerol esters (e.g., oleic acid
glycerol esters); and
[0173] (c) one or more polyethylene oxide-containing phospholipids
(e.g., a distearoylphosphatidyl ethanolamine polyethylene glycol
salt).
[0174] In one embodiment, the therapeutic agent formulation of the
invention includes a therapeutic agent, PECEOL.RTM., and a
distearoylphosphatidyl ethanolamine polyethylene glycol salt. In
this embodiment, the distearoylphosphatidyl ethanolamine
polyethylene glycol salt is present in an amount up to about 30
mM.
[0175] In another embodiment, the therapeutic agent formulation
includes
[0176] (a) a therapeutic agent;
[0177] (b) one or more fatty acid glycerol esters (e.g., oleic acid
glycerol esters); and
[0178] (c) one or more polyethylene oxide-containing fatty acid
esters (e.g., lauric, palmitic and/or stearic acid esters of
polyethylene glycol).
[0179] In one embodiment, the therapeutic agent formulation of the
invention includes a therapeutic agent, PECEOL.RTM., and
GELUCIRE.RTM. 44/14. In another embodiment, the formulation
includes a therapeutic agent, PECEOL.RTM., and GELUCIRE.RTM. 50/13.
In a further embodiment, the formulation includes a therapeutic
agent, PECEOL.RTM., and GELUCIRE.RTM. 53/10. In these embodiments,
the ratio of PECEOL.RTM. to GELUCIRE.RTM. can be from 20:80 to
80:20 (e.g., 20:80, 30:70; 40:60; 50:50; 60:40; 70:30; and
80:20).
[0180] The formulation of two representative therapeutic agents,
econazole and docetaxel, is described in Examples 3 and 4,
respectively.
[0181] In another aspect, the invention provides a method for
administering a therapeutic agent. In the method, a therapeutically
effective amount of the therapeutic agent is administered using the
therapeutic agent formulation described above. In one embodiment,
the formulation is administered orally. In another embodiment, the
formulation is administered topically.
[0182] In further aspects, the invention provides methods for
treating conditions and diseases treatable by therapeutic agents
formulated in accordance with the present invention. In the
methods, an effective amount of a therapeutic drug formulation of
the invention is administered to a subject in need thereof. The
methods for treating conditions and diseases use formulations of
the therapeutic agent families and specific therapeutic agents
disclosed herein.
[0183] Therapeutic Drug Carrier
[0184] In a further aspect, the present invention provides
compositions for formulating a therapeutic agent, methods for
making the composition, and methods for formulating a therapeutic
agent for delivery using the composition.
[0185] In one aspect, the invention provides a composition for
formulating a therapeutic agent for delivery. The composition
includes
[0186] (a) one or more fatty acid glycerol esters; and
[0187] (b) one or more polyethoxylated lipids such as one or more
polyethylene oxide-containing phospholipids or one or more
polyethylene oxide-containing fatty acid esters.
[0188] In the composition above, the fatty acid glycerol esters,
the polyethylene oxide-containing phospholipids, and the
polyethylene oxide-containing fatty acid esters are as described
above for the amphotericin B formulations. The amounts of these
components in the above composition is also as described above for
the amphotericin B formulations. In certain embodiments, the
compositions can further include glycerol in an amount less than
about 10% by weight.
[0189] The composition advantageously solubilizes difficulty
soluble therapeutic drugs for their delivery. With the
incorporation of a therapeutic agent, the composition can be
provide a self-emulsifying drug delivery system.
[0190] In one embodiment, the composition includes
[0191] (a) one or more fatty acid glycerol esters (e.g., oleic acid
glycerol esters); and
[0192] (b) one or more polyethylene oxide-containing phospholipids
(e.g., a distearoylphosphatidyl ethanolamine polyethylene glycol
salt).
[0193] In one embodiment, the composition includes PECEOL.RTM. and
a distearoylphosphatidyl ethanolamine polyethylene glycol salt. In
this embodiment, the distearoylphosphatidyl ethanolamine
polyethylene glycol salt is present in an amount up to about 30
mM.
[0194] In another embodiment, the composition includes
[0195] (a) one or more fatty acid glycerol esters (e.g., oleic acid
glycerol esters); and
[0196] (b) one or more polyethylene oxide-containing fatty acid
esters (e.g., lauric, palmitic and/or stearic acid esters of
polyethylene glycol).
[0197] In one embodiment, the composition includes PECEOL.RTM. and
GELUCIRE.RTM. 44/14. In another embodiment, the composition
includes PECEOL.RTM. and GELUCIRE.RTM. 50/13. In a further
embodiment, the composition includes PECEOL.RTM. and GELUCIRE.RTM.
53/10. In these embodiments, the ratio of PECEOL.RTM. to
GELUCIRE.RTM. can be from 20:80 to 80:20 (e.g., 20:80, 30:70;
40:60; 50:50; 60:40; 70:30; and 80:20).
[0198] In another aspect, the invention provides a method for
making a therapeutic agent formulation. In one embodiment of the
method, a therapeutic agent is combined with the composition
described above. In another embodiment of the method, a therapeutic
agent is combined with one of the components of the composition
(e.g., one or more fatty acid glycerol esters) to provide a first
combination followed by combining the first combination with the
other component of the composition (e.g., one or more polyethylene
oxide-containing phospholipids, or one or more polyethylene
oxide-containing fatty acid esters).
[0199] The formulations and compositions of the invention described
herein include (i.e., comprise) the components recited. In certain
embodiments, the formulations and compositions of the invention
include the recited components and other additional components that
do not affect the characteristics of the formulations and
compositions (i.e., the formulations and compositions consist
essentially of the recited components). Additional components that
affect the formulations' and compositions' characteristics include
components such as additional therapeutic agents that
disadvantageously alter or affect therapeutic profile and efficacy
of the formulations or compositions, additional components that
disadvantageously alter or affect the ability of the formulations
and compositions to solubilize the recited therapeutic agent
components, and additional components that disadvantageously alter
or affect the ability of the formulations and compositions to
increase the bioavailability of the recited therapeutic agent
components. In other embodiments, the formulations and compositions
of the invention include only (i.e., consist of) the recited
components.
[0200] The following examples are provide for the purpose of
illustrating, not limiting, the invention.
EXAMPLES
Materials
[0201] The following materials were used as described in the
following examples.
[0202] Amphotericin B (from Streptomyces sp., Calbiochem, >86%
purity) was purchased from EMD Biosciences (San Diego, Calif.) and
used without further purification. Amphotericin B as the
commercially available deoxycholate micelle dispersion
(FUNGIZONE.RTM.) was purchased from Vancouver General Hospital
pharmacy. Phospholipids and poly(ethylene glycol)-lipids were all
from Avanti Polar Lipids (Alabaster, Ala.). HPLC grade solvents
were from Fluka. PECEOL.RTM. (glyceryl oleate), LABRASOL.RTM.
(caprylocaproyl macrogol glycerides) and GELUCIRE.RTM. 44/14,
GELUCIRE.RTM. 50/13, and GELUCIRE.RTM. 53/10 were obtained from
Gattefosse Canada (Mississauga, Ontario). CAPTEX 355.RTM. and
CAPMUL.RTM. were obtained from Abitech. Simulated gastric fluid
(SGF) without enzymes was composed of 30 mM NaCl, titrated to pH
1.2 with 1N HCl. Simulated intestinal fluid with pancreatin enzymes
(SIFe) was prepared according to the US Pharmacopeia method (USP28)
as modified by Vertzoni et al., Dissolution media simulating the
intralumenal composition of the small intestine: physiological
issues and practical aspects, J. Pharmacy and Pharm. 56(4):453-462
(2004), and was composed of 0.2M NaOH, 6.8 g/L of monobasic
potassium phosphate and 10 g/L of pancreatin (Sigma), adjusted to
pH 7.5 with NaOH. Fasted-state simulated intestinal fluid with bile
salts (FaSSIF) (Vertzoni et al.) was composed of 3 mM sodium
taurocholate (Sigma), 3.9 g/L sodium dihydrogen phosphate, 6.2 g/L
NaCl in water, either with or without 0.75 mM lecithin and then
titrated to pH 6.5 with NaOH. Water was purified by a reverse
osmosis system and filtered (0.2 .mu.m) prior to use. All other
chemicals were of reagent grade purchased from SigmaAldrich.
Example 1
The Preparation and Characterization of Representative Amphotericin
B Formulations
[0203] In this example the preparation and characterization of
representative amphotericin formulations of the invention are
described.
[0204] Preparation of Self-Emulsifying Drug Delivery Systems
(SEDDS).
[0205] Amphotericin B (AmpB) was mixed with the SEDDS lipid
vehicles by combining the drug powder with the lipids followed by
mild heating and stirring (45.degree. C. for 1-2 h), protected from
light. Any visible remaining drug particulates were removed by
centrifugation at 10,000.times.g for 15 min.
[0206] Preparation of AmpB/PECEOL.RTM./DSPE-PEG Formulations.
[0207] AmpB was completely dissolved in a mixture of PECEOL.RTM. at
5 mg/mL to which 95% ethanol (1:3 v/v) had been added, as well as
15 mM distearoyl phosphatidylethanolamine (DSPE)-poly(ethylene
glycol).sub.n (PEG) (where n is the average PEG molecular weight,
350, 550, 750 or 2000). AmpB concentration was 3 mg/ml in the
ethanol and 5 mg/mL in the PECEOL.RTM., respectively, to allow for
complete drug solubilization in the initial mixture. The solution
was stirred at 40.degree. C. for 1 h, protected from light, to
dissolve AmpB and lipids, followed by solvent evaporation at
40.degree. C. with under vacuum (65 mbar) over several hours in a
rotary evaporator. Ethanol was considered to be completely removed
by achieving the original weight of the sample containing AmpB,
PECEOL.RTM., and lipids measured immediately prior to the addition
of the ethanol. A translucent yellow mixture without particulates
was formed. No degradation or spectral shape changes of AmpB were
observed following this processing.
[0208] Characterization of amphotericin B stability. Drug
concentrations were measured by reverse-phase HPLC/UV or by UV
spectrophotometry (.lamda.=407 nm). For HPLC analysis of AmpB,
samples were diluted in 20% (v/v) methanol in DMSO and 20 .mu.L
were injected on a Luna 5 .mu.m (2.0.times.150 mm) C18 column
(Phenomenex) at 30.degree. C. The mobile phase was 10 mM sodium
acetate and acetonitrile using a gradient program on a Waters 996
HPLC system, detected by a Waters photodiode array detector
(.lamda.=408 nm). Run time was 13 min and retention time was
approximately 8.5 min. Stability of AmpB against decomposition or
superaggregation upon mild heating of the lipid vehicles during
drug solubilization and upon storage (21.degree. C.) over 14 days
was assessed by UV spectral shift analysis using a Thermoscan
UV/visible spectrophotometer with .lamda.=250-500 nm.
[0209] Solubility, Physical Stability, and Self-Emulsification.
[0210] SEDDS (self-emulsifying drug delivery systems) formulations
had AmpB solubilities ranging from 100-500 .mu.g/mL as measured by
HPLC, compared to negligible solubility in aqueous solution (pH 7).
For the tested self-emulsifying lipid mixtures, 3 mg of AmpB powder
were combined with 0.3 ml (10 mg/mL) of the various lipid
combination and the mixture was stirred in a 1 mL amber glass vial
at 37.degree. C. for 2 h. Following mixing, the samples were
centrifuged at 10,000.times.g for 15 min to remove any remaining
drug particulates. This procedure does not sediment the lipid
components. The samples were then dispersed as a 1:1000 (v/v)
dilution in 150 mM NaCl with vigorous mixing at 37.degree. C. for
30 min.
[0211] The results for CAPTEX.RTM. 355-based AmpB SEDDS are
summarized in Table 1. The results for
PECEOL.RTM./GELUCIRE.RTM.-based AmpB SEDDS are summarized in Table
2.
TABLE-US-00001 TABLE 1 CAPTEX .RTM. 355-based Amphotericin B
Preliminary SEDDS Effective Hydrodynamic Components (% v/v)
Diameter (nm) Subpopulations Tween CAPMUL .RTM. NaH.sub.2PO.sub.4
(Poly-dispersity Diameter range Relative CAPTEX .RTM. 355 80 MCM
(10 mM, pH 4.0) index) (nm) proportion 50 17 31 2 186 (0.232) 49-69
79 196-277 21 53 5 40 2 237 (0.278) 24-44 73 111-242 12 614-1334 15
58 10 30 2 216 (0.258) 44-64 51 154-255 5 541-893 44 63 5 30 2 223
(284).sup. 62-89 19 168-241 6 413-648 75 70 10 18 2 168 (0.215)
50-65 82 199-273 18 50 17 31 2 179 (0.24) 55-71 80 214-297 20
[0212] Particle sizing was performed after mixture was dispersed
1:1000 (v/v) in 150 mM NaCl at 37.degree. C..times.30 min. Relative
proportion is based on the cumulative distribution of particle
sizes.
TABLE-US-00002 TABLE 2 PECEOL .RTM./GELUCIRE .RTM.-based
Amphotericin B SEDDS. Effective Hydrodynamic Sub- Components (%
v/v) Diameter (nm) populations Relative PECEOL .RTM. GELUCIRE .RTM.
(polydispersity) (nm) proportion 44/14 70 30 156 20-43 55 (0.291)
75-132 16 307-621 29 50 50 314 35-62 56 (0.319) 165-332 5 768-1345
59 30 70 252 28-50 63 (0.294) 135-241 7 568-1160 30 50/13 70 30 158
30-47 55 (0.279) 94-168 17 336-599 28 50 50 192 19-22 25 (0.301)
71-145 24 396-704 51 30 70 396 50-99 25 (0.265) 228-527 63
1703-2382 12
[0213] Particle sizing was performed after mixture was dispersed
1:1000 (v/v) in 150 mM NaCl at 37.degree. C..times.30 min. Relative
proportion is based on the cumulative distribution of particle
sizes.
[0214] As shown in Tables 1 and 2, the effective hydrodynamic
diameter was 168-237 nm at equilibrium for CAPTEX.RTM.
355/CAPMUL.RTM. MCM/Tween 80 (Table 1) and 58-396 nm for
PECEOL.RTM./GELUCIRE.RTM. 44/14 (Table 2) but not for formulations
based on soybean oil, PECEOL.RTM./LABRASOL.RTM., or
PECEOL.RTM./GELUCIRE.RTM. 50/13 (>1 .mu.m, data not shown).
Importantly, multiple subpopulations of emulsion droplet size were
observed. For the CAPTEX.RTM. 355-based SEDDS, these subpopulations
included diameters consistent with the size of micelles (e.g. 20-50
nm) and all populations remained in the submicron range (Table 1).
For the PECEOL.RTM./GELUCIRE.RTM. 44/14 mixtures, three
subpopulations were observed, including 20-50 nm, 1000-200 nm range
(minor population) and those closer to 1 .mu.m in diameter. The
proportion of very small and large droplets varied by ratio of
components. In the case of PECEOL.RTM./GELUCIRE.RTM. 50/13, three
subpopulations were also observed with similar droplet size ranges
as with PECEOL.RTM./GELUCIRE.RTM. 44/14, although there was a trend
to slightly larger droplets in the largest diameter subpopulation,
which also increased with increasing proportion of GELUCIRE.RTM.
50/13 (Table 2). Representative single samples are fully described
in the tables, however, it should be noted that replicate samples
did show consistency in their effective diameters and in the
particle size ranges of the subpopulations. Visual observations
were made regarding miscibility, phase separation and precipitation
over several days at ambient temperature (21.degree. C.). Several
SEDDS formulations remained transparent and homogeneous. For
example, the CAPTEX.RTM.-based SEDDS formulations all generated
semi-transparent mixtures after mixing with 150 mM NaCl that were
homogeneous for all combination ratios of CAPTEX.RTM., Tween 80 and
sodium phosphate (Table 1). Combinations of PECEOL.RTM. and
GELUCIRE.RTM. 44/14 in the range of 70/30 to 30/70 (v/v) generated
a fine emulsion whereas some partial solidification was observed
over 24 h at 21.degree. C. when using PECEOL.RTM. with
GELUCIRE.RTM. 50/13, consistent with the high melting point of
GELUCIRE.RTM. 50/13 (50.degree. C.).
[0215] AmpB Solubility in PECEOL.RTM./DSPE-PEG Formulations and
Emulsification in Fasted-State Simulated Intestinal Fluid.
[0216] The combination of PECEOL.RTM. and DSPE-PEG.sub.n, where
average molecular weight of PEG was varied from 350 to 2000, showed
an even greater solubilization of AmpB (5 mg/mL) compared to the
preliminary SEDDS formulations. At concentrations .gtoreq.10 mg/mL,
some precipitation of AmpB did occur upon standing at ambient
temperature (21.degree. C.) over 24 h. Upon dispersion in SGF at
37.degree. C. at 0.5 mg/mL followed by stirring for 30 min, the
PECEOL.RTM./DSPE-PEG.sub.2000 AmpB formulations generated
translucent emulsions with particle sizes of 300-500 nm and no
visible precipitate. In some cases, there appeared to be two
populations of submicron particles, with some at 100 nm and others
several hundred nm in diameter.
[0217] AmpB Stability in Simulated Gastric and Intestinal
Fluids.
[0218] Amphotericin B in PECEOL.RTM./DSPE-PEG formulations (5
mg/mL) were prepared in triplicate and were incubated in simulated
gastric fluid (SGF) as a 1:10 (v/v) dilution) or in simulated
intestinal fluid prepared with and without lecithin or with enzymes
(as described above) as a 1:50 (v/v) dilution at 37.degree. C. with
vigorous stirring. Incubation times were 0, 10, 30 or 120 min. At
each time point, AmpB concentration was determined by
spectrophotometry using triplicate measures of absorbance (407 nm)
after complete solubilization in 95% ethanol to clarify the
samples, thereby also diluting the samples to the linear range of
the UV assay. Values were normalized to the baseline at 330 nm and
concentrations were calculated based on an amphotericin B standard
curve prepared in each fluid type (r.sup.2>0.99). The linearity
of the standard curve and concentration range of standards prepared
in PECEOL.RTM./DSPE-PEG were not affected by the type of simulated
GI fluid or by incubation time, however, separate triplicate
standard curves were prepared for each formulation containing the
various molecular weights of DSPE-PEG (350, 550, 750, or 2000).
[0219] The chemical stability and aggregation state (monomeric vs.
self-associated) of AmpB was evaluated in USP simulated gastric
fluid as well as fasted-state simulated intestinal fluid with and
without bile salts and pancreatin. As described above, AmpB in
PECEOL.RTM. alone or in PECEOL.RTM./DSPE-PEG formulations (PEG
molecular weight=350, 550, 750, or 2000) was prepared at 5 mg/mL
and incubated in the simulated GI fluids for a total period of 2 h.
At 30 min intervals, the AmpB concentration and UV spectra were
evaluated. AmpB exhibits 5 main spectrophotometric peaks in the UV
range. Peaks 4 and 5 have the greatest amplitude in monomeric AmpB,
whereas there is a left shift when AmpB becomes self-associated.
FIG. 3A shows typical UV spectra of AmpB in PECEOL.RTM./DSPE-PEG
over the linear range of the UV assay, illustrating the
predominance of monomeric AmpB. This pattern was maintained when
PEG molecular weight was varied from 350 to 2000 (data not shown).
The same spectral pattern was also observed following incubation in
SGF, as well as resulting in nearly identical standard curves for
the various AmpB/PECEOL.RTM./DSPE-PEG.sub.n preparations, as shown
in FIG. 3B.
[0220] Regarding chemical stability, the trend was to slightly less
drug stability in formulations prepared with DSPE-PEG 350 or 550
compared to DSPE-PEG 750 or 2000. AmpB alone (e.g., neat powder)
was not soluble in these media and therefore could not be used
properly as a control at comparable concentrations due to the
confounding factors of increased dissolution over time vs.
degradation. AmpB in PECEOL.RTM. alone prepared otherwise the same
way was included as a negative control for the stabilizing effect
of DSPE-PEG in the formulations. AmpB/PECEOL.RTM. showed a trend to
slightly less drug stability in SGF than formulations containing
DSPE-PEG 350 or 2000 as shown in FIG. 4. FIG. 5 shows AmpB
stability in simulated intestinal fluid containing bile salts
either without lecithin (FIG. 5A) or with lecithin (FIG. 5B) is
less for AmpB in PECEOL.RTM. alone or in PECEOL.RTM./DSPE-PEG 350
compared to formulations using the higher PEG molecular
weights.
[0221] FIG. 6 illustrates the stability of AmpB in simulated
intestinal fluid with pancreatin, which contains degradative
enzymes. These data suggest better stability of formulations
containing DSPE-PEG 750 or 2000 compared to 350 or 550 or in
PECEOL.RTM. alone. In evaluating the degradation of AmpB in
PECEOL.RTM. alone, however, is it is important to note that poor
mixing of AmpB/PECEOL.RTM. in the simulated GI fluids was observed;
this formulation tended to float. No changes associated with
conversion of the monomeric form vs. aggregated AmpB, such as a
difference in the height ratios of specific peaks in the UV spectra
or overall pattern, was observed following the full incubation time
in the various media described here (data not shown).
[0222] Particle Size Analysis.
[0223] Particle size analysis by dynamic light scattering (ZetaPALS
instrument, Brookhaven Laboratories, New York, operating at 650 nm)
was used to assess self-emulsification properties. Emulsion droplet
size was measured in physiological saline (150 mM NaCl) following
30 min incubation at 37.degree. C. For PECEOL.RTM./DSPE-PEG AmpB
formulations, the mean diameter was measured at 37.degree. C. every
10 min in preliminary experiments and it was found that the mean
diameter came to equilibrium by 1 h and remained stable. Drug
stability was measured after 2 h, therefore 2 h was the time point
used for reported particle size analysis from samples incubated in
simulated intestinal fluids. Two data analysis modes are available
in the ZetaPALS software (version 3.88), which calculate a weighted
mean effective hydrodynamic diameter based on a lognormal
distribution, and a multimodal distribution to identify
subpopulations centered on two or more mean diameters. Both values
are reported where bimodal or multimodal distributions were
detected, with the proportion of each subpopulation reported based
on the cumulative distribution analysis (ZetaPALS software, version
3.88).
[0224] Varying the DSPE-PEG molecular weight had no clear effect on
the emulsion droplet size in simulated intestinal fluid (Table 3)
following mixing over a period of 2 h at 37.degree. C. Submicron
mean diameters were observed in the range of 300-600 nm with a
fairly wide polydispersity. A bimodal particle size distribution
was also generated, with a small subpopulation centered in
submicron range (150-300 nm) and another centered in the 1-2 .mu.m
range. AmpB in PECEOL.RTM. alone also formed droplets of similar
size and distribution in simulated intestinal fluid. These particle
size measurements were performed in the absence of lecithin, which
formed very large emulsion droplets under the mixing conditions
employed and opacified the samples. The results are presented in
Table 3.
TABLE-US-00003 TABLE 3 PECEOL .RTM./DSPE-PEG Amphotericin B
Formulation Particle Size. Formulation: Effective PECEOL .RTM./
Diameter (nm) Poly- Sub- DSPE-PEGn lognormal dispersity populations
Relative n distribution index (nm) proportion 350 370 0.344 129-186
20 888-1282 80 550 600 0.402 108-171 20 1206-1909 80 750 596 0.395
134-210 18 1245-3400 82 2000 533 0.392 119-200 30 1390-2330 70 AmpB
in 351 0.333 128-194 20 PECEOL .RTM. 738-1120 80 alone
[0225] Particle sizing by dynamic light scattering of AmpB in
PECEOL.RTM./DSPE-PEG, where the molecular weight of PEG was varied
from 350 to 2000, following 2 h incubation simulated fasted-state
intestinal fluid (pH 6.8) at 0.5 mg AmpB/mL. Relative proportion is
based on the cumulative distribution of particle sizes.
Example 2
The Effectiveness of Representative Amphotericin B Formulations in
Treating Fungal Infections: Aspergillus fumigatus and Candida
albicans
[0226] In this example, the effectiveness of representative
amphotericin B formulations of the invention in treating fungal
infections is described. Animal studies were conducted to determine
the effectiveness of representative amphotericin B formulations of
the invention in treating rats infected with Aspergillus fumigatus
or Candida albicans.
[0227] 1. Aspergillus fumigatus
[0228] (2.7-3.3.times.10.sup.7 colony forming units [CFU]) was
injected via the jugular vein; 48 h later male albino
Sprague-Dawley rats (350-400 g) were administered either as a
single oral gavage of monoglyceride-DSPE/PEG2000-based AmpB (10 mg
AmpB/kg; n=7) twice daily for 2 consecutive days, a single
intravenous (i.v.) dose of ABELCET.RTM. (5 mg AmpB/kg; n=4), or
physiologic saline (non-treated controls; n=9) once daily for 2
consecutive days. Organs were harvested at sacrifice (day 3) and
processed (see below). Blood was drawn before inoculation (Blank),
pre-dose (0 hour) and 48 hours after treatment for plasma
creatinine analysis. Male albino Sprague-Dawley rats (350 to 400 g)
were purchased from Charles River Laboratories (Wilmington, Mass.).
The rats were surgically implanted with a port (Access
Technologies) and catheter with access to venous blood by a similar
method used for rabbits. The rats were housed in an animal care
facility with a 12 hour light-dark cycle and controlled temperature
and humidity. The rats were given ad libitum access water and
standard rat chow (Purina Rat Chow) for the duration of the study.
The ports were primed daily with normal saline and heparin to
prevent blockages. The animals were cared for according to
principals promulgated by the Canadian Council on Animal Care and
the University of British Columbia.
[0229] Aspergillus fumigatus Inoculum.
[0230] A. fumigatus were collected from a pool of patients with
either disseminated aspergillosis (BC Centre for Disease Control).
Cultures were grown on Sabouraud dextrose agar for 48 hours at
37.degree. C. Conidia were isolated by washing the agar with
pyrogen free saline. The conidia were suspended by vortexing with
glass beads and diluted with pyrogen free saline to obtain between
2.7 to 3.3.times.10.sup.7 conidia in 300 .mu.l of saline. Conidia
were counted using a hemocytometer and a 100 .mu.l aliquot was
serially diluted and aliquots were plated on sabouraud dextrose
agar for 48 hours at 37.degree. C. to determine the number of
viable conidia and purity of the inoculum. The average percentage
of viable conidia in the inoculum was 62%.+-.19. None of the spore
suspensions were contaminated with any other organism. Rats were
inoculated with 300 .mu.l through the indwelling port 48 hours
before the beginning of treatment to allow for aspergillosis to
develop.
[0231] 2. Candida albicans
[0232] (1-1.35.times.10.sup.6 colony forming units [CFU]) was
injected via the jugular vein; 48 h later male albino
Sprague-Dawley rats (350-400 g) were administered either as a
single oral gavage of monoglyceride-DSPE/PEG2000-based AmpB (10 mg
AmpB/kg; n=7) twice daily for 2 consecutive days, a single
intravenous (i.v.) dose of ABELCET.RTM. (5 mg AmpB/kg; n=3), or
physiologic saline (non-treated controls; n=9) once daily for 2
consecutive days. Organs were harvested at sacrifice (day 3) and
processed (see below). Blood was drawn before inoculation (Blank),
pre-dose (0 hour) and 48 hours after treatment for plasma
creatinine analysis. Male albino Sprague-Dawley rats (350 to 400 g)
were purchased from Charles River Laboratories (Wilmington, Mass.).
The rats were surgically implanted with a port (Access
Technologies) and catheter with access to venous blood by a similar
method used for rabbits. The rats were housed in an animal care
facility with a 12 hour light-dark cycle and controlled temperature
and humidity. The rats were given ad libitum access water and
standard rat chow (Purina Rat Chow) for the duration of the study.
The ports were primed daily with normal saline and heparin to
prevent blockages. The animals were cared for according to
principals promulgated by the Canadian Council on Animal Care and
the University of British Columbia.
[0233] Candida albicans Inoculum.
[0234] Candida albicans were collected from a pool of patients with
either disseminated Candidiasis (BC Centre for Disease Control).
Cultures were grown on Sabouraud dextrose agar for 48 hours at
37.degree. C. Conidia were isolated by washing the agar with
pyrogen free saline. The conidia were suspended by vortexing with
glass beads and diluted with pyrogen free saline to obtain between
2.7 to 3.3.times.10.sup.7 conidia in 300 .mu.l of saline. Conidia
were counted using a hemocytometer and a 100 .mu.l aliquot was
serially diluted and aliquots were plated on sabouraud dextrose
agar for 48 hours at 37.degree. C. to determine the number of
viable conidia and purity of the inoculum. The average percentage
of viable conidia in the inoculum was 62%.+-.19. None of the spore
suspensions were contaminated with any other organism. Rats were
inoculated with 300 .mu.l through the indwelling port 48 hours
before the beginning of treatment to allow for aspergillosis to
develop.
[0235] 3. Animal Methods.
[0236] One-ml whole blood samples were drawn into pediatric
collection tubes (3.6 mg K.sub.2 EDTA) before infection (blank),
pre-dose (0 hour) and 48 hours after treatment (48 hour). All whole
blood samples were mixed by inversion and plasma was separated by
centrifugation (15 minutes, 3000 RPM at 4.degree. C.). Plasma
samples were stored at -20.degree. C. for creatinine analysis.
After the collection of the 48 hour blood specimen, the rat was
euthanized with intravenous overdose (1 ml) of EUTHANYL.RTM.,
(sodium pentobarbital 240 mg/ml). Spleen, right kidney, liver,
lung, heart and brain tissue samples were harvested, weighed and
placed in sterile containers. Normal saline was added, 1 ml/g of
specimen and homogenized (Heidolph diax 900). An aliquot of organ
homogenate was stored at room temperature until plating and the
remaining sample was placed at -80.degree. C. until HPLC
analysis.
[0237] The choice of organ colony forming units (CFU) as an
indicator of antifungal activity was based on previously published
work (K. M. Wasan et al., Assessing the antifungal activity and
toxicity profile of Amphotericin B Lipid Complex (ABLC;
ABELCET.RTM.) in combination with Caspofungin in experimental
systemic aspergillosis, Journal of Pharm. Sci. 2004;
93(6):1382-1389). Aliquots of 100 .mu.l full strength organ
homogenate and 1:10 dilution (with sterile saline) were each spread
plated onto Saboraud Dextrose Agar plates in duplicate. After 48 hr
incubation at 37.degree. C., the resulting colonies of A. fumigatus
or C. albicans were counted and averaged over the duplicate plates.
The limit of detection of the assay was 0.1.times.10.sup.2 CFU/ml
homogenate.
[0238] Renal toxicity was indirectly assessed, as previously
described (K. M. Wasan et al., Assessing the antifungal activity
and toxicity profile of Amphotericin B Lipid Complex (ABLC;
ABELCET.RTM.) in combination with Caspofungin in experimental
systemic aspergillosis, Journal of Pharm. Sci. 2004;
93(6):1382-1389), by determining creatinine concentration in plasma
using a commercially available kit (Sigma Chemicals Co.). A
baseline was determined by measuring creatinine concentration in
the blank sample, and was compared to plasma creatinine
concentration in the 0 hour (pre-dose), 48 hour samples. For the
purposes of this study, a 50% or greater increase in plasma
creatinine concentration as compared to baseline was considered to
be a sign of renal toxicity.
[0239] 4. Statistical Analysis.
[0240] The number of CFU's in organs and plasma creatinine
concentrations prior to and following administration of treatment
were compared between each treatment group by analysis of variance
(INSTAT2; GraphPad Inc.). Critical differences were assessed by
Tukey post hoc tests. Serum creatinine values were compared prior
to 48 hours following treatment using repeat measures ANOVA with a
Tukey post hoc test to determine critical differences (Prism 4;
Graphpad Inc.). A difference was considered significant if the
probability of chance explaining the results was reduced to less
than 5% (p<0.05). All data were expressed as a mean.+-.standard
error of the mean.
[0241] Antifungal Activity and Renal Toxicity in Rats Infected with
Aspergillus fumigatus.
[0242] PECEOL.RTM./DSPE/PEG2000-based oral AmpB treatment
significantly decreased total fungal CFU concentrations recovered
in all the organs added together by 80% compared to non-treated
controls (Table 4) without significant changes in plasma creatinine
levels (Table 5). ABELCET.RTM. treatment significantly decreased
total fungal CFU concentrations recovered in all the organs added
together by 88% compared to non-treated controls (Table 4) without
significant changes in plasma creatinine levels (Table 5).
TABLE-US-00004 TABLE 4 Fungal analysis of Aspergillus
fumigatus-infected male Sprague Dawley rats treated with oral
gavage doses of Normal Saline (non-treated control),
Amphotericin-DSPE-PEG200 incorporated into PECEOL .RTM. (10 mg/kg
twice daily x 2 days) or a single intravenous dose of ABELCET .RTM.
(ABLC; 5 mg/kg once daily .times. 2 days). All rats were infected
with 2.9-3.45 .times. 10.sup.7 Viable Colony Forming Units
(CFU)/0.3 ml/rat of Aspergillus fumigatus prior to initiation of
treatment. Treatment Infected Tissues (CFU/ml of homogenized
tissues) Groups Brain Lungs Heart Liver Spleen Kidney All Organs
Non-treated 3538 .+-. 1810 74 .+-. 30 101 .+-. 63 .sup. 308 .+-.
114 1163 .+-. 772 364 .+-. 119 5549 .+-. 2498 Controls (n = 9) ABLC
5 550 .+-. 445.sup.a 10 .+-. 4.sup.a 15 .+-. 3.sup.a 18 .+-.
5.sup.a 88 .+-. 44.sup.a 10 .+-. 0.sup.a 690 .+-. 419.sup.a (n = 4)
AmpB-DSPE- 736 .+-. 186.sup.a 51 .+-. 18 20 .+-. 4 180 .+-. 48 107
.+-. 32.sup.a 44 .+-. 10.sup.a 1139 .+-. 221.sup.a PEG-2000 (n = 7)
.sup.ap < 0.05 vs. non-treated controls using student T-Test;
All Data are presented as mean .+-. SEM. *Note: Previous studies
have shown that AmpB alone does not have measurable accumulation at
the doses used herein. ABLC: Amphotericin B Lipid Complex.
TABLE-US-00005 TABLE 5 Plasma creatinine concentrations before
infection (blank), pre-dose (0 hour) and 48 hours after treatment
(48 hour). Blank 0 48 Creatinine (mg/dl) 0.4 .+-. 0.1 0.5 .+-. 0.1
0.9 .+-. 0.2 Control (n = 9) AmpB/DSPE-PEG2000/PECEOL .RTM. 0.6
.+-. 0.2 0.6 .+-. 0.2 0.5 .+-. 0.1 10 mg/kg (oral) (n = 7) ABLC 5
mg/kg-IV (n = 4) 0.3 .+-. 0.2 0.4 .+-. 0.1 0.5 .+-. 0.1 Data
presented as mean .+-. SEM
[0243] The results for Candida albicans are similar to those for
Aspergillus fumigatus. Fungal analysis of the kidneys of Candida
albicans-infected rats treated with a representative AmpB
formulation of the invention demonstrate significantly decreased
total fungal CFU concentrations compared to control. FIG. 7
compares Candida albicans concentration (CFU/ml) in the kidneys of
rats infected with Candida albicans and treated with control, an
AmpB/PECEOL.RTM. formulation (10 mg/kg), a representative AmpB
formulation of the invention (AmpB/PECEOL.RTM./DSPE-PEG-2000,
designated AmpB/DSPE-PEG-2000, 10 mg/kg), and intravenous
ABELCET.RTM. (designated ABLC, 5 mg/ml). FIG. 8 compares Candida
albicans concentration (CFU/ml) in the organs of rats infected with
Candida albicans and treated with control, an AmpB/PECEOL.RTM.
formulation (10 mg/kg), a representative AmpB formulation of the
invention (AmpB/PECEOL.RTM./DSPE-PEG-2000, designated
AmpB/DSPE-PEG-2000, 10 mg/kg), and intravenous ABELCET.RTM.
(designated ABLC, 5 mg/ml). The effectiveness of the representative
AmpB formulation in reducing Candida albicans concentration was
comparable to ABELCET.RTM.. Treatment with the representative AmpB
formulation significantly decreased total fungal CFU concentrations
recovered in the kidneys without significant changes in plasma
creatinine levels.
[0244] FIG. 9 compares plasma creatinine (mg/dl) in rats infected
with Candida albicans and treated with control, an AmpB/PECEOL.RTM.
formulation (10 mg/kg), a representative AmpB formulation of the
invention (AmpB/PECEOL.RTM./DSPE-PEG-2000, designated
AmpB/DSPE-PEG-2000, 10 mg/kg), and intravenous ABELCET.RTM.
(designated ABLC, 5 mg/ml) (blank, 0 hr, and 48 hr). No renal
toxicity was observed as measured by plasma creatine levels.
Example 3
Representative Econazole Formulation
[0245] In this example, the preparation and characterization of a
representative econazole formulation of the invention is described.
The solubility of econazole in water is <1 mg/mL (19-66.degree.
F.), and the solubility in ethanol is <20 mg/mL.
[0246] Econazole nitrate formulations (10 and 15 mg econazole
nitrate/mL formulation) were prepared by a method similar to the
method for preparing the amphotericin B formulation described in
Example 1. To econazole nitrate (Sigma) and DSPE-PEG2000 (15 mM)
powders was added 45.degree. C. PECEOL.RTM. and the resulting
mixture shaken for 2-4 h at 45.degree. C. in a shaking incubator.
No ethanol was used. Samples were centrifuged in order to visualize
unsolubilized material. The mixture was then evaluated for clarity
over time.
[0247] The product econazole nitrate formulation was incubated at
37.degree. C. for 2 h in simulated gastric fluid (1:100 v/v
dilution), fasted-state simulated intestinal fluid, and fed-state
simulated intestinal fluid with and without pancreatin (all at
1:500 v/v dilution) to evaluate emulsification properties. Emulsion
droplet size was evaluated immediately by dynamic light scattering
(Zetasizer, Malvern Instruments).
[0248] The formulations were evaluated for clarity over time. The
results are summarized in Table 6.
TABLE-US-00006 TABLE 6 Visual appearance of econazole formulations.
Econazole Visual Appearance Formulation Day 0 24 h 4 days 5 days 10
mg/mL Clear Clear Clear Clear >5 days 15 mg/mL Some solids Still
clear Not Not remaining after portion with tested tested
centrifugation solids
[0249] Emulsion droplet size analysis was determined for the
econazole formulation (10 mg/mL) in simulated gastric and
intestinal fluids (SGF, FaSSIF, FeSSIF, and FeSSIFe), with mean
based on peak analysis by volume. Emulsion droplet size in the
table refers to the mean and peak half-width, a range for each
subpopulation. The results are summarized in Table 8.
TABLE-US-00007 TABLE 7 Emulsion droplet size for econazole
formulation. Dilution Lower size Upper size Media factor (nm) (nm)
Mean (nm) SGF 100 30 291 72 .+-. 46 (71%) and 310 .+-. 200 (29%)
FaSSIF 500 59 333 123 .+-. 65 (44%) and 342 .+-. 138 (56%) FeSSIF
500 66 1557 386 .+-. 182 (10%); 726 .+-. 143 (5%) and 2503 .+-. 565
(85%) FeSSIFe 500 510 >5000 650 .+-. 150 (75%) >5 .mu.m (25%)
SGF: simulated gastric fluid FaSSIF: fasted-state simulated
intestinal fluid FeSSIF: fed-state simulated intestinal fluid
FeSSIFe: fed-state simulated intestinal fluid with pancreatic
enzymes (Sigma)
[0250] Composition of simulated gastric/intestinal fluids (1 L)
used in emulsification studies for the econazole formulation.
[0251] SGF (simulated gastric fluid): [0252] Distilled water: 1 L
[0253] Sodium chloride: 30 mM (1740 mg/L) [0254] Hydrochloric acid:
as required to adjust to pH 1.2.
[0255] FaSSIF (fasted-state simulated intestinal fluid): [0256]
Dibasic potassium phosphate: 3.9 g [0257] Distilled water: 1 L
[0258] Sodium taurocholate: 3 mM (1613.04 mg/L) [0259] Egg
phosphatidylcholine: 0.75 mM (570.07 mg/L) [0260] Potassium
chloride: 7.7 g [0261] Hydrochloric acid: as required to adjust pH
to 6.5.
[0262] FeSSIF (fed-state simulated intestinal fluid): [0263]
Distilled water: 1 L [0264] Acetic acid: 8.65 g=9.073 ml [0265]
Sodium taurocholate: 15 mM (8065.2 mg/L) [0266] Egg
phosphatidylcholine: 3.75 mM (2850.34 mg/L) [0267] Potassium
chloride: 15.2 g [0268] Hydrochloric acid or sodium hydroxide: as
required to adjust pH to 5.0.
[0269] FeSSIF with pancreatic enzymes: [0270] Distilled water: 1 L
[0271] Sodium taurocholate: 7.5 mM (4032.6 mg/L) [0272] Egg
phosphatidylcholine: 2.0 mM (1520.18 mg/L) [0273] Glyceryl
monooleate: 5.0 mM (1782.72 mg/L) [0274] Sodium oleate: 0.8 mM
(241.96 mg/L) [0275] Pancreatin: 1000 u lipase [0276] Acetic acid:
9.073 ml [0277] Potassium chloride: 15.2 g [0278] Hydrochloric acid
or sodium hydroxide: as required to adjust pH to 5.8.
Example 4
Representative Docetaxel Formulation
[0279] In this example, the preparation of a representative
docetaxel formulation of the invention is described. The solubility
of docetaxel in water is about 10-25 .mu.g/mL.
[0280] A docetaxel formulation (10 mg docetaxel/mL formulation) was
prepared by combining docetaxel (Fluka) powder with the
DSPE-PEG2000 powder and wetting the combined powders 100% ethanol
to 10% v/v of the final intended volume. The ethanol did not
solubilize the powders. To the wetted powders was added PECEOL.RTM.
that was pre-warmed to 50.degree. C., followed by vortex mixing for
2 min which resulted in a clear solution. Ethanol was not
removed.
[0281] Ethanol was used with docetaxel as a co-solvent in this
formulation to maximize solubility. Docetaxel is poorly water
soluble but has polar regions.
[0282] The formulation was evaluated for clarity over time. The
results are summarized in Table 8.
TABLE-US-00008 TABLE 8 Visual appearance of docetaxel formulations.
Docetaxel Visual Appearance Formulation Day 0 24 h 4 days 5 days 10
mg/mL -- Solidified Solidified Solidified at 4.degree. C. at
4.degree. C. at 4.degree. C. at 4.degree. C. but clear upon but
clear upon but clear upon melting briefly melting briefly melting
briefly 10 mg/mL Clear Clear Clear Clear at 21.degree. C. 10 mg/mL
-- Solidified Solidified Solidified at -20.degree. C. at
-20.degree. C. at -20.degree. C. at -20.degree. C. but clear upon
but clear upon but clear upon melting briefly melting briefly
melting briefly 10 mg/mL -- Clear Clear with Clear with at
50.degree. C. yellow tinged* yellow tinge* *Consistent with color
change observed with PECEOL .RTM. alone at 50.degree. C. for this
length of time.
[0283] Docetaxel formulations of the invention include those as
described above, but that do not include ethanol.
[0284] While illustrative embodiments have been illustrated and
described, it will be appreciated that various changes can be made
therein without departing from the spirit and scope of the
invention.
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