U.S. patent application number 13/655498 was filed with the patent office on 2013-02-14 for liquid formulations of bendamustine.
The applicant listed for this patent is Anthony S. Drager, Rachel Y. LaBell, Piyush R. Patel. Invention is credited to Anthony S. Drager, Rachel Y. LaBell, Piyush R. Patel.
Application Number | 20130041004 13/655498 |
Document ID | / |
Family ID | 41559533 |
Filed Date | 2013-02-14 |
United States Patent
Application |
20130041004 |
Kind Code |
A1 |
Drager; Anthony S. ; et
al. |
February 14, 2013 |
Liquid Formulations Of Bendamustine
Abstract
Stable liquid formulations of bendamustine, and pharmaceutically
acceptable salts thereof, and polar aprotic solvents, are
described.
Inventors: |
Drager; Anthony S.;
(Thorndale, PA) ; LaBell; Rachel Y.; (Coatesville,
PA) ; Patel; Piyush R.; (Wallingford, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Drager; Anthony S.
LaBell; Rachel Y.
Patel; Piyush R. |
Thorndale
Coatesville
Wallingford |
PA
PA
PA |
US
US
US |
|
|
Family ID: |
41559533 |
Appl. No.: |
13/655498 |
Filed: |
October 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13362430 |
Jan 31, 2012 |
8344006 |
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13655498 |
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13048325 |
Mar 15, 2011 |
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13362430 |
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PCT/US09/58023 |
Sep 23, 2009 |
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13048325 |
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61100074 |
Sep 25, 2008 |
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Current U.S.
Class: |
514/394 |
Current CPC
Class: |
A61K 31/4184 20130101;
A61K 47/22 20130101; A61K 9/08 20130101; A61K 47/20 20130101; A61K
47/40 20130101; A61P 35/02 20180101; A61K 47/10 20130101; A61K
9/0019 20130101; A61K 47/18 20130101; A61P 35/00 20180101 |
Class at
Publication: |
514/394 |
International
Class: |
A61K 31/4184 20060101
A61K031/4184; A61P 35/02 20060101 A61P035/02; A61P 35/00 20060101
A61P035/00 |
Claims
1. A stable, liquid, pharmaceutical formulation comprising
bendamustine, or a pharmaceutically acceptable salt thereof, and a
polar aprotic solvent, wherein said formulation is suitable for
injection into a patient following dilution with a pharmaceutically
acceptable diluent.
2. The formulation of claim 1, wherein the polar aprotic solvent is
1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone,
dimethylacetamide, dimethyl sulfoxide, acetone, tetrahydrofuran,
1,4-dioxane, acetonitrile, dimethyl formamide, propylene carbonate,
or a mixture thereof.
3. The formulation of claim 2, wherein the polar aprotic solvent is
dimethylacetamide.
4. The formulation of claim 1, further comprising a non-aqueous
polar protic solvent.
5. The formulation of claim 4, comprising between about 30% and
about 70%, by volume of the formulation, of the nonaqueous polar
protic solvent.
6. The formulation of claim 4, wherein the non-aqueous polar protic
solvent is an alcohol, a polyalkylene glycol, an amide, or a
mixture thereof.
7. The formulation of claim 4, wherein the non-aqueous polar protic
solvent is an alcohol.
8. The formulation of claim 7, wherein the alcohol is a
cyclodextrin.
9. The formulation of claim 4, wherein the non-aqueous polar protic
solvent is propylene glycol.
10. The formulation of claim 4, wherein the polar aprotic solvent
is dimethylacetamide.
11. The formulation of claim 10, wherein the nonaqueous polar
protic solvent is propylene glycol.
12. The formulation of claim 11, comprising about 66% (v/v) of the
dimethylacetamide and about 34% (v/v) of the propylene glycol.
13. The formulation of claim 12, comprising from about 5 mg/ml to
about 120 mg/mL of bendamustine, or a pharmaceutically acceptable
salt thereof.
14. The formulation of claim 13, comprising about 100 mg/mL of
bendamustine, or a pharmaceutically acceptable salt thereof.
15. The formulation of claim 1, comprising from about 5 mg/ml to
about 200 mg/mL of bendamustine, or a pharmaceutically acceptable
salt thereof.
16. The formulation of claim 1, comprising from about 5 mg/ml to
about 120 mg/mL of bendamustine, or a pharmaceutically acceptable
salt thereof.
17. The formulation of claim 1, comprising about 100 mg/mL of
bendamustine, or a pharmaceutically acceptable salt thereof.
18. The formulation of claim 1, wherein the bendamustine is
bendamustine hydrochloride.
19. The formulation of claim 1, further comprising at least one
pharmaceutically acceptable excipient or diluent.
20. The formulation of claim 4, further comprising at least one
pharmaceutically acceptable excipient or diluent.
21. The formulation of claim 1, further comprising an antioxidant,
a surfactant, a lipid, a filler, an organic acid, a hydrophilic
polymer, a complexing agent, a preservative, or a combination
thereof.
22. A method of treating cancer comprising providing a liquid,
pharmaceutical formulation of claim 1; diluting the liquid
pharmaceutical formulation with a pharmaceutically acceptable
injectable diluent to form an injectable pharmaceutical
preparation; administering the injectable pharmaceutical
preparation to a patient in need of treatment for cancer.
23. The method of claim 22, wherein the liquid pharmaceutical
formulation comprises the polar aprotic solvent
dimethylacetamide.
24. The method of claim 23, wherein the liquid pharmaceutical
formulation comprises the polar aprotic solvent dimethylacetamide
and further comprises the nonaqueous polar protic solvent propylene
glycol
25. The method of claim 24, wherein the liquid pharmaceutical
formulation comprises about 66% (v/v) of the dimethylacetamide and
about 34% (v/v) of the propylene glycol.
26. The method of claim 25, wherein the liquid pharmaceutical
formulation comprises from about 5 mg/ml to about 120 mg/mL of
bendamustine, or a pharmaceutically acceptable salt thereof.
27. The method of claim 26, wherein the liquid pharmaceutical
formulation comprises about 100 mg/mL of bendamustine, or a
pharmaceutically acceptable salt thereof.
28. The method of claim 22, wherein the pharmaceutically acceptable
injectable diluent is 0.9% sodium chloride.
29. The method of claim 22, wherein the cancer is chronic
lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma,
multiple myeloma, or breast cancer.
30. The method of claim 29, wherein the cancer is chronic
lymphocytic leukemia or non-Hodgkin's lymphoma.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/362,430, filed Jan. 31, 2012, which is a continuation of
U.S. application Ser. No. 13/048,325, filed Mar. 15, 2011, now
abandoned, which is a continuation of International Application No.
PCT/US2009/58023, filed Sep. 23, 2009, which claims the benefit of
U.S. Provisional Application No. 61/100,074, filed Sep. 25, 2008,
the entireties of which are incorporated by reference herein.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to liquid formulations of
bendamustine, and the pharmaceutical salts thereof.
BACKGROUND OF THE INVENTION
[0003] Bendamustine,
(4-{5-[bis(2-chloroethyl)amino]-1-methyl-2-benzimidazolyl}butyric
acid
##STR00001##
is an atypical structure with a benzimidazole ring, which structure
includes an active nitrogen mustard. Bendamustine was initially
synthesized in 1963 in the German Democratic Republic and was
available from 1971 to 1992 in that location under the name
Cytostasan.RTM.. Since that time, it has been marketed in Germany
under the tradename Ribomustin.RTM.. It is currently available for
use in the United States under the tradename Treanda.RTM.
(Cephalon, Inc., Frazer, Pa.). It has been widely used to treat
chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's
lymphoma, multiple myeloma, and breast cancer.
[0004] Like other nitrogen mustards, bendamustine hydrolyzes in
aqueous solution, with the major degradant being the primary
alcohol HP1 (See U.S. application Ser. No. 11/330,868, the entirety
of which is incorporated herein):
##STR00002##
[0005] In light of its instability in aqueous solution,
bendamustine is currently supplied as a lyophilized powder for
injection. Just prior to its infusion, the medical practitioner
reconstitutes the powder with Sterile Water for Injection.
Reconstitution should yield a clear, colorless to pale yellow
solution and the powder should completely dissolve in about 5
minutes. If particulate matter is observed, the reconstituted
product should not be used and should be discarded. The
reconstituted product is then transferred to a 0.9% Sodium Chloride
Injection infusion bag within 30 minutes of reconstitution. This
admixture should be a clear and colorless to slightly yellow
solution. If the admixture comprises particulate matter or is
discolored, it should be discarded and a fresh sample prepared.
[0006] The reconstitution of the bendamustine lyophilized powder is
time consuming and cumbersome. Moreover, lyophilization of solids
on a commercial scale requires specialized equipment and incurs
significant expense. As such, formulations of bendamustine that do
not require lyophilization and/or reconstitution are needed.
[0007] Solutions of bendamustine hydrochloride in anhydrous
propylene glycol, prepared under an inert gas atmosphere, have been
reported (GDR Patent 159289). It was reported that analysis of
these solutions using thin-layer chromatography, eluting with
butanol/acetic acid/water (4:1:5) and detection with Dragendorff
reagent and UV (360 nm) did not suggest any decomposition.
Curiously, however, commercial development of propylene glycol
formulations have heretofore not been reported. Thus, improved
liquid formulations of bendamustine are still needed.
SUMMARY OF THE INVENTION
[0008] The present invention is directed to liquid pharmaceutical
formulations comprising bendamustine, or a pharmaceutically
acceptable salt or prodrug thereof, and a polar aprotic solvent.
Certain preferred embodiments include liquid pharmaceutical
formulations comprising bendamustine, or a pharmaceutically
acceptable salt or prodrug thereof, a polar aprotic solvent, and a
non-aqueous polar protic solvent. Methods of making and using the
formulations of the present invention are also described, as are
methods of treating cancer using the claimed formulations.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a graph of a stability analysis of bendamustine in
various solvents at 25.degree. C.
[0010] FIG. 2 is a graph of a stability analysis of bendamustine in
various solvents at 5.degree. C.
[0011] FIG. 3 is a graph of bendamustine purity, over time, in 99%
propylene glycol, at 5.degree. C. and at 25.degree. C.
[0012] FIG. 4 shows the mean+standard deviation
concentration-versus-time profiles of bendamustine in male
Cynomolgus monkeys (N=4) administered single 3 mg/kg bolus
intravenous doses of bendamustine hydrochloride in 3 different
formulations.
DETAILED DESCRIPTION OF THE INVENTION
[0013] Stable, liquid formulations of bendamustine have been
discovered and are reported herein.
[0014] Experiments to produce commercially viable propylene glycol
preparations have been performed. Unfortunately, the results
described in GDR Patent 159289 were not reproducible. Solutions of
bendamustine in 99% propylene glycol degraded to non-bendamustine
products over a time equivalent to commercial storage. Two of the
impurities were identified as propylene glycol esters of
bendamustine. As such, a 100% propylene glycol commercial
formulation of bendamustine is not feasible for pharmaceutical
purposes.
[0015] It has been determined that pharmaceutically acceptable
liquid formulations of bendamustine, and the pharmaceutically
acceptable salts thereof, in particular the hydrochloride salt, can
be prepared by combining bendamustine, or the pharmaceutically
acceptable salt thereof, with a polar aprotic solvent or mixture of
polar aprotic solvents. Polar, aprotic solvents are known in the
art and include, for example, 1-methyl-2-pyrrolidone,
1,3-dimethyl-2-imidazolidinone, dimethylacetamide, dimethyl
sulfoxide, acetone, tetrahydrofuran, 1,4-dioxane, acetonitrile,
dimethyl formamide, propylene carbonate. See also, e.g., Florence
Mottu, et al. Organic solvents for pharmaceutical parenterals and
embolic liquids: A review of toxicity data, PDA J. Pharma. Sci.
& Tech. vol 54, no. 6, 456-469 (November-December 2000).
Particularly preferred polar aprotic solvents include
dimethylacetamide, dimethyl sulfoxide, and mixtures thereof.
[0016] Without wishing to be held to any particular theory, it is
believed that polar, aprotic solvents are sufficiently
non-nucleophilic towards bendamustine such that polar aprotic
solvent-bendamustine adducts do not form over the course of typical
commercial storage conditions. Typical commercial storage
conditions include time periods of, for example, about 30 days,
about 90 days, about 180 days, and about 365 days (about 1 month,
about 3 months, about 6 months, and about 1 year). Typical
commercial storage conditions also include temperatures of about
23.degree. C. (ambient room temperature) and refrigerated
temperatures below ambient room temperature, for example, about
5.degree. C. Preferably, the liquid formulations of the present
invention are stored at refrigerated temperatures.
[0017] It has also been discovered that stable formulations of
bendamustine can be obtained by mixing a polar aprotic solvent, or
a mixture of polar aprotic solvents, with a non-aqueous polar
protic solvent or mixture of nonaqueous polar protic solvents.
Pharmaceutically acceptable nonaqueous polar protic solvents are
known in the art and include alkyl alcohols, for example, ethanol,
ethylene glycol, propylene glycol, butylene glycol, glycerin,
polysorbates, for example TWEEN 20, TWEEN 40, and TWEEN 80, and
cyclodextrins (such as hydroxypropyl-.beta.-cyclodextrin),
polyalkylene glycols, such as polyethylene glycol, polypropylene
glycol, and polybutylene glycol, and primary amides such as
niacinamide.
[0018] Such formulations will typically comprise 90% or less, by
volume of the formulation, of the nonaqueous polar protic solvent.
In other preferred embodiments, formulations will comprise between
about 20% and about 85%, by volume of the formulation, of the
nonaqueous polar protic solvent. In still other embodiments,
formulations will comprise between about 30% and about 70%, by
volume of the formulation, of the nonaqueous polar protic solvent.
In most preferred embodiments, formulations will comprise about
80%, about 67% or about 34%, by volume of the formulation, of the
nonaqueous polar protic solvent.
[0019] Alternatively, formulations of the present invention will
comprise 10 moles per liter, or less, of the nonaqueous polar
protic solvent. Preferably, formulations of the present invention
will comprise between about 4 moles per liter to about 9.5 moles
per liter, of the nonaqueous polar protic solvent. In certain
embodiments, formulations will comprise about 9.1 moles per liter
of the nonaqueous polar protic solvent. In other embodiments,
formulations will comprise about 4.6 moles per liter, of the
nonaqueous polar protic solvent.
[0020] While not wishing to be held to any particular theory, it is
believed that while nonaqueous polar protic solvents are of
sufficient nucleophilicity to form potentially undesirable polar
protic solvent-bendamustine adducts, such adducts will not form
during typical commercial storage if the concentration of the polar
protic solvent is kept within the scope of the present
invention.
[0021] Liquid formulations of the present invention are stable over
the course of a typical commercial storage period. As used herein,
"stable" is defined as no more than about a 10% loss of
bendamustine under typical commercial storage conditions.
Preferably, formulations of the present inventions will have no
more than about a 10% loss of bendamustine, more preferably, no
more than about a 5% loss of bendamustine, under typical commercial
storage conditions.
[0022] Bendamustine converts to non-bendamustine products (i.e.,
"degrades") upon exposure to certain nucleophiles, for example,
water and alkyene glycols such as propylene glycol. Exposure of
bendamustine to water can produce "HP1," which is undesirable.
##STR00003##
Another undesirable compound that bendamustine can convert to over
time is "BM1 dimer."
##STR00004##
Still another undesirable compound that bendamustine can convert to
over time is "DCE."
##STR00005##
Upon exposure to an alkylene glycol, for example, propylene glycol,
esters of bendamustine can form, e.g., PG-1 and PG-2.
##STR00006##
[0023] In preferred embodiments of the present invention, analysis
of formulations of the present invention will exhibit 1.50% or less
of DCE, as determined by HPLC analysis, after about 1 year (about
365 days) at about 5.degree. C. More preferably, the formulations
will exhibit 1.0% or less of DCE, as determined by HPLC analysis,
after about 1 year (about 365 days) at about 5.degree. C. Even more
preferably, the formulations will exhibit 0.5% or less of DCE, as
determined by HPLC analysis, after about 1 year (about 365 days) at
about 5.degree. C. Most preferably, the formulations will exhibit
about 0.1% or less of DCE, as determined by HPLC analysis, after
about 1 year (about 365 days) at about 5.degree. C.
[0024] In other embodiments of the present invention, analysis of
the formulations will exhibit about 0.4% or less of HP1, as
determined by HPLC analysis, after about 1 year (about 365 days) at
about 5.degree. C. Preferably, the formulations will exhibit about
0.10% or less of HP1, as determined by HPLC analysis, after about 1
year (about 365 days) at about 5.degree. C.
[0025] In certain other embodiments of the present invention,
analysis of the formulations will exhibit about 0.70% or less of
BM1 dimer, as determined by HPLC analysis, after about 1 year
(about 365 days) at about 5.degree. C. Preferably, the formulations
will exhibit about 0.30% or less of dimer, as determined by HPLC
analysis, after about 1 year (about 365 days) at about 5.degree. C.
In most preferred embodiments, the formulations will exhibit about
0.10% or less of BM1 dimer, as determined by HPLC analysis, after
about 1 year (about 365 days) at about 5.degree. C.
[0026] In those embodiments of the present invention comprising
alkylene glycol as the nonaqueous polar protic solvent, analysis of
those formulations will exhibit 1.5% or less of alkylene glycol
esters of bendamustine, as determined by HPLC analysis, after about
1 year (about 365 days) at about 5.degree. C. For example, in those
embodiments comprising propylene glycol, analysis of those
formulations will exhibit 1.5% or less of propylene glycol esters
PG-1 and PG-2, as determined by HPLC analysis, after about 1 year
(about 365 days) at about 5.degree. C.
[0027] Analysis of the liquid formulations of the present invention
can be performed using techniques known in the art, including, for
example, HPLC, gas chromatography, and NMR. After exposure to
typical commercial storage conditions, analysis of the formulations
of the present invention will indicate that the formulation
contains no less than about 90% of the amount of bendamustine
present prior to exposure to the storage conditions. Preferably,
analysis will indicate that the formulation contains no less than
about 95% of the amount of bendamustine present prior to exposure
to the storage conditions.
[0028] In preferred embodiments of the present invention, analysis
of the formulations of the present invention will indicate that the
formulation contains no less than about 90% of the amount of
bendamustine present prior to exposure to storage conditions that
include temperatures of about 5.degree. C. and time periods of
about 30 days (about 1 month) to about 365 days (about 1 year).
Preferably, analysis of the formulations of the present invention
will indicate that the formulation contains no less than about 90%
of the amount of bendamustine present prior to exposure to storage
conditions that include temperatures of about 5.degree. C. and time
periods of about 30 days (about 1 month), about 90 days (about 3
months), and about 180 days (about 6 months). Preferably, analysis
will indicate that the formulation contains no less than about 95%
of the amount of bendamustine present prior to exposure to storage
conditions that include temperatures of about 5.degree. C. and time
periods of about 30 days (about 1 month) to about 365 days (about 1
year). More preferably, analysis will indicate that the formulation
contains no less than about 95% of the amount of bendamustine
present prior to exposure to storage conditions that include
temperatures of about 5.degree. C. and time periods of about 30
days (about 1 month), about 90 days (about 3 months), and about 180
days (about 6 months).
[0029] Formulations of the present invention can comprise
pharmaceutically useful concentrations of bendamustine, or a
pharmaceutically acceptable salt thereof. Useful concentrations
include concentrations ranging from about 5 mg/mL to about 200
mg/mL. Preferably, the concentration of bendamustine, or a
pharmaceutically acceptable salt thereof, ranges from about 5 mg/mL
to about 120 mg/mL. Preferred concentrations include about 5 mg/mL,
about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL,
about 50 mg/mL, about 60 mg/mL, about 100 mg/mL and about 200 mg/mL
of bendamustine, or a pharmaceutically acceptable salt thereof.
Greater than 200 mg/ml of bendamustine, or a pharmaceutically
acceptable salt thereof, for example, greater than about 300 mg/mL,
are also within the scope of the present invention, as are
saturated solutions of bendamustine, or a pharmaceutically
acceptable salt thereof.
[0030] As used herein, the term "about" is defined as .+-.10%,
preferably .+-.5%,
[0031] In addition to comprising a polar aprotic solvent, or
mixture of polar aprotic solvents, and optionally, a nonaqueous
polar protic solvent, or mixture of solvents, formulations of the
present invention may further comprise other pharmaceutically
acceptable excipients. Pharmaceutically acceptable excipients are
known in the art and include, for example, antioxidants (e.g.,
tocopherol (Vitamin E), ascorbic acid, methyl paraben,
butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), and propyl
gallate), surfactants, (e.g., polysorbates (TWEEN 20, TWEEN 40,
TWEEN 80)), lipids (e.g., dimyristoylphophatidylcholine (DMPC),
Dimyristoylphosphatidylglycerol (DMPG),
distearoylphophatidylglycerol (DSPG), fillers (e.g., mannitol),
organic acids (e.g., citric acid, lactic acid, benzoic acid),
hydrophilic polymers (e.g., polyethylene glycols (PEG 300, PEG
400), complexing agents (e.g., niacinamide, nicotinic acid,
creatine, cyclodextrins), and preservatives (e.g., benzyl
alcohol).
[0032] Also within the scope of the invention are methods of
treating diseases, such as, for example, chronic lymphocytic
leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple
myeloma, or breast cancer, with a pharmaceutical formulation of the
present invention. These methods comprise administering to the
patient a therapeutically effective amount of a preparation
prepared from a pharmaceutical formulation of the present
invention. The term "therapeutically effective amount," as used
herein, refers to the amount determined to be required to produce
the physiological effect intended and associated with a given drug,
as measured according to established pharmacokinetic methods and
techniques, for the given administration route. Appropriate and
specific therapeutically effective amounts can be readily
determined by the attending diagnostician, as one skilled in the
art, by the use of conventional techniques. The effective dose will
vary depending upon a number of factors, including the type and
extent of progression of the disease or disorder, the overall
health status of the particular patient, the relative biological
efficacy of the compound selected, the formulation of the active
agent with appropriate excipients, and the route of
administration.
[0033] The liquid formulations of bendamustine described herein are
intended to be administered via injection, for example, they may be
administered subcutaneously, intracutaneously, intravenously,
intramuscularly, intra-articularly, intrasynovially,
intrasternally, intrathecally, intralesionally, intracranially or
via infusion. In a typical preparation, the volume of the liquid
formulation of the present invention needed for the required dose
can be aseptically withdrawn and transferred to an infusion bag of
0.9% Sodium Chloride (or other pharmaceutically acceptable
intravenous solution) for injection. After transfer, the contents
of the infusion bag are thoroughly mixed. Administration by
intravenous infusion is typically provided over a time period of
from about 30 to about 60 minutes. Previously described lyophilized
formulations of bendamustine required reconstitution of the
lyophilized bendamustine prior to mixture with the acceptable
intravenous solution before infusion.
[0034] It is envisioned that the pharmaceutical formulations and
preparations of the present invention can be administered in
combination with one or more anti-neoplastic agents where the
anti-neoplastic agent is given prior to, concurrently with, or
subsequent to the administration of the formulation or preparation
of the present invention. Pharmaceutically acceptable
anti-neoplastic agents are known in the art. Preferred
anti-neoplastic agents are those disclosed in co-pending U.S.
application Ser. No. 11/330,868, filed Jan. 12, 2006, the entirety
of which is incorporated herein by reference.
EXAMPLES
Solubility and Stability of Bendamustine Hydrochloride in Polar
Aprotic Solvents
[0035] Equilibrium solubility was determined for solvents including
1-methyl-2-pyrrolidone (NMP), 1,3-dimethyl-2-imidazolidinone (DMI),
dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), acetone,
tetrahydrofuran (THF), dimethylformamide (DMF), and propylene
carbonate (PC). The solubility of bendamustine hydrochloride was
also determined for two solutions, 25 mg/mL niacinamide in DMA and
66% DMA/34% propylene glycol (PG). A saturated solution of
bendamustine hydrochloride was made in triplicate for each solvent
or solution and mixed on a Lab-Quake with gentle mixing and low
shear for 3 days at room temperature. A sample of each suspension
was put into a microcentrifuge tube and spun at 10,000 rpm for 5
min on an Eppendorf microcentrifuge. The supernatant was removed
and put into a clean vial. Each solution was diluted with sample
solvent: 50% NMP/50% 0.1% trifluoroacetic acid in water. A reverse
phase method for bendamustine hydrochloride was used to determine
the concentration of each sample calculated from a standard.
Analysis was performed within 18 hours of preparation of the
diluted sample. The solubilities are listed in Table I below. Each
value is an average of three samples.
TABLE-US-00001 TABLE I Sample* % Purity Assay (mg/mL) NMP 99.1
104.0 DMI 98.5 75.8 DMSO 99.5 311.7 DMF 99.6 71.8 66% DMA/34% PG
99.5 110.1 DMA 99.4 56.2 PC 98.7 7.7 Niacinamide/DMA 99.2 61.3
*acetone and THF have no measurable solubility of bendamustine.
[0036] The three replicates were combined and mixed well and then
pipetted into amber HPLC vials and placed in stability chambers at
25.degree. C. and 5.degree. C. All the samples were clear and
colorless except for the DMI sample which was clear and yellow. The
25.degree. C. stability leveled out from about 180 days (about 6
months) to about 365 days (about 12 months, about 1 year). At
5.degree. C., all solutions had a purity greater than 90%. The
analysis of stability samples can be seen in the graphs of FIGS. 1
and 2.
TABLE-US-00002 TABLE II Impurity profile of certain liquid
formulations of Bendamustine HCl after storage at 5.degree. C. for
about 12 months BM1 DCE HP1 dimer PG-1 PG-2 Formulation (Area %)
(Area %) (Area %) (Area %) (Area %) Niacinamide/ 1.40 0.08 0.06 ND
ND DMA DMA 1.10 0.08 0.05 ND ND 66% DMA/ 0.12 0.08 0.06 1.09 0.27
34% PG DMF 0.07 0.11 0.07 ND ND NMP 0.90 0.10 ND ND ND DMSO 0.04
0.38 0.70 ND ND ND = not detected
Analysis conducted using reverse phase HPLC with 50% NMP/50% 0.1%
trifluoroacetic acid in water as the running solvent.
[0037] As can be seen in FIG. 3, bendamustine (BM1) in 99%
propylene glycol degrades significantly when stored at 25.degree.
C. for less than 100 days. After storage at 5.degree. C. for about
365 days, the purity of the bendamustine is about 80% or less.
Pharmacokinetic Study of Formulations in Monkey
[0038] 4 fasted (18 to 23 hr), drug-naive male cynomolgus monkeys
consecutively received single 3-mg/kg bolus intravenous doses of
bendamustine hydrochloride prepared from 3 different formulations.
The formulations evaluated in the study included:
1) TREANDA (lyophilized mixture of bendamustine hydrochloride and
mannitol; 25 mg (bendamustine hydrochloride) vials; 2) a 66%
dimethylacetamide (DMA)/34% propylene glycol (PG) (w/w) solution
(90 mg (bendamustine hydrochloride)/mL stock); and 3) a 100% DMA
solution (45 mg (bendamustine hydrochloride)/mL stock). The
lyophilized powder and stock solutions of bendamustine
hydrochloride were constituted or diluted with 0.9% saline, as
appropriate, to give solutions of 3 mg bendamustine
hydrochloride/ml, just prior to dose administration. The resulting
solutions were administered as a bolus via a saphenous vein at a
fixed volume of 1.0 mL/kg. There was at least a 7-day washout
period separating successive doses. During all 3 phases of dosing,
blood samples for pharmacokinetic profiling of bendamustine and its
2 active circulating metabolites, .gamma.-hydroxybendamustine (M3)
and N-des-methylbendamustine (M4), were collected via a femoral
vein immediately prior to dosing and at preselected timepoints
through 12 hr postdose. Concentrations of bendamustine, M3 and M4
in plasma samples were determined using a validated
high-performance liquid chromatography method with tandem mass
spectrometric detection (LC-MS/MS) as follows. Bendamustine and the
M3 and M4 metabolites are extracted from plasma by protein
precipitation using acetonitrile. After the extraction, the
aliquoted sample is acidified with 1% formic acid and bendamustine
with an added carbon in the carboxylic acid chain is added as an
internal standard. The samples are evaporated to dryness and the
residue is reconstituted with an acetonitrile/water/formic
acid/ammonium formate mixture. The sample is injected into an HPLC
system with LC/MS/MS detection using a Phenomenex Synergi Max-RP
column with an acetonitrile/water/formic acid/ammonium formate
mobile phase. Pharmacokinetic analyses were performed using
noncompartmental methods.
[0039] After single bolus intravenous doses of bendamustine
hydrochloride to male cynomolgus monkeys, the shapes of the mean
plasma concentration-versus-time profiles of bendamustine were
similar in each of the 3 formulations (See FIG. 4). In all cases,
the highest observed plasma levels of bendamustine were achieved at
0.083 hr postdose (ie, the first sampling time after dose
administration) and subsequent removal of the compound from plasma
occurred in a bi-phasic manner that was characterized by an initial
rapid distribution phase and a somewhat slower terminal phase of
drug elimination. The harmonic mean t.sub.1/2 of the terminal phase
was approximately 0.6 hr for each formulation (See Table III).
[0040] In addition to the similarities in the shapes of the mean
plasma concentration-versus-time profiles, the 3 formulations were
also similar with respect to bendamustine systemic exposure (i.e.,
C.sub.max and AUC). Specifically, the respective mean values of
C.sub.max and AUC.sub.0-.infin. for bendamustine were 6037 ng/mL
and 2314 nghr/mL for the TREANDA formulation, 7380 ng/mL and 2854
nghr/mL for the 66% DMA/34% PG formulation and 6209 ng/mL and 2372
nghr/mL for the 100% DMA formulation. Plasma clearance (CL) and
volume of distribution (V.sub.z and V.sub.ss) for bendamustine were
also comparable between each of the 3 formulations (See Table III).
In Table III, t.sub.max, hr is given as Median [range], t.sub.1/2,
hr is given as the Harmonic Mean, .lamda..sub.z, hr.sup.-1 is the
slope of line in elimination phase used to calculate half-life, and
MRT.sub.0-.infin. is the mean residence time.
[0041] In summary, the pharmacokinetic profiles of bendamustine, M3
and M4 for the 2 liquid formulations of bendamustine hydrochloride
were qualitatively and quantitatively similar to those obtained for
the TREANDA formulation after single bolus intravenous doses to
monkeys.
[0042] Table III shows the mean+/-Standard Deviation
pharmacokinetic parameters of bendamustine in male Cynomolgus
monkeys (N=4) administered single 3 mg/kg bolus intravenous doses
of bendamustine hydrochloride in the three different
formulations.
TABLE-US-00003 TABLE III Formulation Parameter TREANDA 66% DMA/34%
PG 100% DMA C.sub.0, ng/mL 8664 .+-. 3841 10716 .+-. 2033 8956 .+-.
1965 C.sub.max, ng/mL 6037 .+-. 2456 7380 .+-. 1170 6209 .+-. 1300
t.sub.max, hr 0.083 0.083 0.083 [0.083 for all] [0.083 for all]
[0.083 for all] AUC.sub.0-t, ng.cndot.hr/mL 2313 .+-. 800 2853 .+-.
398 2371 .+-. 535 AUC.sub.0-.infin., ng.cndot.hr/mL 2314 .+-. 800
2854 .+-. 398 2372 .+-. 535 .lamda..sub.z, hr.sup.-1 1.220 .+-.
0.111 1.295 .+-. 0.108 1.092 .+-. 0.219 t.sub.1/2, hr 0.57 0.54
0.63 CL, L/hr/kg 1.27 .+-. 0.40 0.96 .+-. 0.14 1.18 .+-. 0.27
V.sub.z, L/kg 1.04 .+-. 0.36 0.74 .+-. 0.05 1.17 .+-. 0.44
V.sub.ss, L/kg 0.34 .+-. 0.11 0.26 .+-. 0.05 0.30 .+-. 0.04
MRT.sub.0-.infin., hr 0.26 .+-. 0.02 0.27 .+-. 0.02 0.26 .+-.
0.03
In-Use Studies of Formulations
[0043] Admixtures in 0.9% sodium chloride (500 mL bag) were
prepared at a high dose (360 mg bendamustine hydrochloride) and
purity was determined over time at room temperature for up to 8
hours using HPLC, using a Zorbax Bonus-RP column with a gradient
from 93% 0.1% trifluoroacetic acid in water (Mobile Phase A)/7%
0.1% trifluoroacetic acid in acetonitrile (Mobile Phase B) to 10%
Mobile Phase A/90% Mobile Phase B.
[0044] The 66% DMA/34% PG formulation had a concentration of
bendamustine hydrochloride of 90 mg/g, so 4 mL was injected into a
500 mL bag of saline, inverted 10 times and sampled at room
temperature for 8 hours. After 8 hours the purity was 95.4%. This
is within the label requirements for dosing Treanda. This
formulation of the present invention could be used for up to 8
hours at room temperature. By way of contrast, reconstituted
Treanda can only be stored at room temperature for up to 3
hours.
[0045] The 100% DMA formulation had a concentration of 45 mg/g, so
8 mL was injected into a 500 mL bag of saline, inverted 10 times,
and sampled at room temperature for 4 hours. After 4 hours the
purity was 97.9%. This formulation of the present invention could
be used for more than 4 hours at room temperature.
[0046] The comparative Treanda admixture purity was 95.0% after 4
hours at 25.degree. C.
[0047] As those skilled in the art will appreciate, numerous
modifications and variations of the present invention are possible
in view of the above teachings. It is therefore understood that
within the scope of the appended claims, the invention can be
practiced otherwise than as specifically described herein, and the
scope of the invention is intended to encompass all such
variations.
* * * * *