Note: Descriptions are shown in the official language in which they were submitted.
CA 02733732 2011-03-02
Attorney Docket No. 056274-4052
FORMULATIONS OF CANFOSFAMIDE AND THEIR PREPARATION
1. Cross-reference to related application
[0001] This application is a continuation-in-part of U.S. Application No.
12/511,245, filed on 29
July 2009, which claims the priority under 35 U.S.C. 119(e) of US Provisional
Application No.
61/092,580, filed 28 August 2008, the entire disclosure of which is
incorporated herein by reference.
II. Background of the invention
A. Field of the invention
[0002] This invention relates to stable lyophilized formulations of
canfosfamide and their
preparation.
B. Description of the related art
[0003] McIntyre and Castaner, "Canfosfamide Hydrochloride", Drugs Fut.,
29(10), 985-991
(2004), describe canfosfamide hydrochloride (USAN), a phosphorodiamidate
anticancer agent
activated by glutathione S-transferase PI-1. The article also identifies
canfosfamide hydrochloride
with the codes TER-286 and TLK-286.
[0004] Morgan et al., "Tumor Efficacy and Bone Marrow-sparing Properties of
TER286, a
Cytotoxin Activated by Glutathione S-Transferase", Cancer Research, 58(12),
2568-2575 (1998)
describe preclinical studies on canfosfamide hydrochloride, including animal
studies. According to
the article, "for some studies, it was prepared in 60 mM sodium citrate for
i.v. delivery."
[0005] Tew et al., "TLK-286: a novel glutathione S-transferase-activated
prodrug", Expert Opin.
Investig. Drugs, 14(8), 1047-1054 (2005), note that "The formulation of TLK-
286 in vials
containing 265 mg of the sterile lyophilized drug permits solubilization in
water for injection to a
concentration of 50 mg/ml, followed by dilution to the appropriate dose in 5%
dextrose for
injection."
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[0006] The disclosures of the documents referred to in this application are
incorporated into this
application by reference.
[0007] Canfosfamide is a very reactive alkylating agent. Given its reactivity,
stable formulations
of this compounds have been difficult to develop. Aqueous solutions form only
in a narrow range of
pH in which the drug is soluble. However, such aqueous formulations are stable
for perhaps 6 to 12
hours before decomposition products are observed. Moreover, the reactivity of
canfosfamide has
made it difficult to find formulation excipients which are stable with the
drug.
III. Summary of the invention
[0008] This invention is directed to the surprising discovery that otherwise
unstable aqueous
formulations of cansfosfamide or its salt can be lyophilized and maintained as
a stable formulation
for exceptionally long periods of time rendering the lyophilized formulation
suitable for therapeutic
utility. One of the most commonly used salts of canfosfamide is the
hydrochloride salt which is
exemplified here. However, this invention also provides the formulations using
other acid salts of
canfosfamide such as canfosfamide hydrobromide and the like.
[0009] Accordingly, in one embodiment, this invention provides a stable
lyophilized formulation
consisting essentially of canfosfamide hydrochloride and sodium citrate,
wherein the said
formulation is stable for therapeutic utility. Preferably, the composition has
an osmolality of no
more than 400 mM. In another preferred embodiment, the stable lyophilized
formulation is prepared
without the addition of any lyophilization aids.
[0010] In another embodiment, this invention provides a stable lyophilized
formulation consisting
of canfosfamide hydrochloride and sodium citrate, wherein the said formulation
is stable for
therapeutic utility, and the said formulation is a product of lyophilization
of (50 5) mg/mL
canfosfamide hydrochloride and (100 10) mM aqueous sodium citrate buffer at a
pH of 4.6 0.2.
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[0011] In another embodiment, this invention provides a method of preparing a
stable lyophilized
formulation consisting essentially of canfosfamide hydrochloride and sodium
citrate, wherein the
said formulation is stable for therapeutic utility, wherein the method
comprises:
(a) preparing a sodium citrate buffer of a pH of 6.5 0.1;
(b) dissolving canfosfamide hydrochloride in the sodium citrate buffer of step
(a) to provide a
solution;
(c) adjusting the pH of the solution of step (b) when pH is > 4.8 to provide a
resulting solution of
pH of 4.6 0.2 by addition of a compatible acid selected from citric acid and
HCI; and
(d) lyophilizing the resulting solution of step (c) to provide the said
formulation.
IV. Detailed description of the invention
A. Definitions
[0012] "Consisting of' is a term of limitation and it excludes any element,
step, or ingredient not
specified in the claim. "Consisting essentially of' is a term of limitation
and means to include the
presence of stated components, groups, steps, and the like and to exclude the
presence of others that
materially alter the basic characteristics of what is being claimed or
described. Thus a formulation
consisting essentially of canfosfamide hydrochloride contains canfosfamide
hydrochloride and not
another active ingredient or another salt of canfosfamide except for
incidental impurities.
"Comprising" is a term of inclusion and not of limitation and means to include
the presence of stated
components, groups, steps, and the like but not to exclude the presence or
addition of other
components, groups, steps, and the like. Unless the context clearly requires
otherwise, the singular
includes the plural; so that, for example, "a sodium salt of citric acid"
includes both a single such salt
and two or more such salts.
[0013] A "sodium citrate buffer" is a solution consisting essentially of a
sodium salt of citric acid,
and optionally citric acid, dissolved in water, typically at a stated pH. The
amount of citric acid
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depends, of course, on the pH of the aqueous solution. Thus, when used herein,
the term "sodium
citrate buffer" is meant to contain both sodium citrate and citric acid.
[0014] A "lyophilized formulation" refers to a formulation resulted from
freeze-drying of an
aqueous solution.
[0015] A "lyophilization aid" refers to one or more additives that help in
assisting the
lyophilization process. "Lyophilization aids" as per this invention include
any one of the following:
lubricants, anhydrous lactose, matrix forming agents, and the like.
[0016] A "reconstituted formulation" refers to a formulation resulted from
adding water (for
example, sterile water) or an aqueous solvent to a solid composition in an
amount to dissolve the
composition. In one embodiment, the solid composition is a lyophilized
formulation.
[0017] An "injectable formulation" refers to a formulation that is suitable
for parenteral
administration, e.g., subcutaneous, intravenous, intramuscular, or
intraperitoneal administration.
[0018] A formulation "stable for therapeutic utility" refers to canfosfamide
formulation that over a
period of at least 6 months and preferably at least 2 years does not contain
more than 2 weight %
vinyl sulfone as a decomposition product.
[0019] Because the formulation of this invention contains salts dissolved in
water, it will
necessarily contain dissociated ions as well as non-ionized species.
Accordingly, describing the
formulation as consisting essentially of canfosfamide hydrochloride in sodium
citrate buffer does not
describe the association of canfosfamide with hydrochloric acid, or a
particular state of ionization of
the canfosfamide (which has an amine and two carboxylate groups), or the
association of sodium
ions with citrate/hydrogen citrate/dihydrogen citrate ions, or other
associations or ionization states,
but rather that the formulation contains the product of the dissolution of the
stated ingredients at the
stated concentration in water at the stated pH.
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B. Brief description of the drawings
[0020] Figure 1 illustrates a plot of assay value of lyophilized canfosfamide
hydrochloride
formulation versus time.
[0021] Figure 2 illustrates a plot of percent total degradation impurities in
lyophilized
canfosfamide hydrochloride formulation versus time.
C. The canfosfamide formulation
[0022] A targeted approach to the treatment of solid tumors involves the
development of potent
alkylating agents activated by glutathione S-transferase P 1-1 (GST P 1-1).
The novel nitrogen
mustard prodrug canfosfamide, commonly used as its hydrochloride salt, is one
such compound that
is useful as a powerful antitumor agent in the treatment of cancer. However,
canfosfamide has a
limited shelf-life at room temperature as it degenerates quickly to form vinyl
sulfone as a major
degradation product. Nonetheless, canfosfamide needs to be administered at
high doses, for
example, at 960 mg/m2 or about 1.5 grams in a single administration, for the
treatment of cancer.
Thus, there is a need for a stable formulation having a high canfosfamide
concentration for
intravenous administration and a convenient method for the attending clinician
to prepare such a
stable formulation when needed.
[0023] In one embodiment, this invention provides stable lyophilized
formulations and convenient
methods that allow preparation of these formulations suitable for intravenous
administration to a
patient in a needed amount at the time canfosfamide is to be administered.
a. Aqueous Solubility
[0024] It has been discovered that aqueous solubility of canfosfamide
hydrochloride is strongly
dependent on the pH of the solvents. It is poorly soluble in water at about 2
mg/mL and is more
soluble at high pH and strengths of the buffers. Canfosfamide hydrochloride is
soluble at pH above
4.2 and it has limited stability at pH above pH 5Ø The compounding of the
drug product and final
pH is therefore in a narrow range. Due to the acidic nature of canfosfamide
hydrochloride, its
dissolution into a pH 6.5 buffered solution reduces the pH. A buffer system
which could keep the
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final solution pH between 4.3 and 5.0 is therefore essential for the
dissolution and for the stability of
the in process bulk solution used for formulating the product. Different
buffer systems (citrate,
succinate, histidine acetate, phosphate) were studied and citrate buffer
system showed highest
solubility. For example, the solubility of canfosfamide hydrochloride can be
as high as 174 mg/mL
in 0.2 M pH 5.5 citrate buffer. Thus the aqueous solubility of canfosfamide
hydrochloride is
dependent on the pH and strengths of the buffers.
[0025] Due to the presence of two carboxylic acid groups and one equivalent of
hydrochloric acid,
canfosfamide hydrochloride has high acidity and dissolution of canfosfamide
hydrochloride lowers
the pH of the solution significantly even in the presence of a buffer system.
For example, when
canfosfamide hydrochloride is dissolved into a 0.1 M (100 mM) pH 6.5 citrate
buffer, the pH can be
reduced to below 5. Once the solution pH is reduced to below pH 4.2, the
solubility of canfosfamide
hydrochloride is dramatically reduced and drug oils out.
[0026] However, canfosfamide is unstable in alkaline environment. High pH
buffer or even high
local pH of the aqueous solvent leads to degradation of canfosfamide. Thus,
back titrating of
canfosfamide hydrochloride with a low pH solution would result in significant
degradation of
canfosfamide hydrochloride. For example, when a 50 mg/mL canfosfamide
hydrochloride in a pH
4.3 citrate buffer was titrated with a 0.1 N and a 1.0 N NaOH solution to pH
4.5, the content of a
degradation product, vinyl sulfone, was increased from 0.64% to 1.3% and 6.5%
from 0.1 N and 1.0
N NaOH titrated solutions respectively. Based on the discovery that
canfosfamide hydrochloride is
soluble at pH above 4.2 and such aqueous formulations have sufficient
stability at a pH below 5,
albeit limited to less than that required for a suitable shelf-life, a buffer
system which could keep the
pH of the final solution at between 4.3 to 5.0 is therefore essential for the
dissolution and for the
stability of the bulk solution used for formulating lyophilized canfosfamide
hydrochloride. Thus, a
high concentration of buffer system which can provide a high buffering
capacity is desired.
[0027] The solubility of canfosfamide hydrochloride depends on the
concentration and initial pH
of buffer system. Higher buffer concentration and higher initial pH dissolves
greater amounts of
canfosfamide hydrochloride. A 0.1 M citrate buffer at pH 6.5 could dissolve
approximately 100
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mg/mL canfosfamide hydrochloride. When 50 mg/mL canfosfamide hydrochloride is
dissolved in
this buffer system, the pH is about 4.6. The solution has an osmolality of
about 400 mM. A higher
strength citrate buffer could dissolve more canfosfamide hydrochloride, but it
also results in higher
osmolality which could make freeze drying challenging and decrease the
stability of a lyophilized
product. A high osmolality buffer at pH 4.6 could also cause infusion
irritation.
[00281 Accordingly, there are numerous and divergent constraints on the
formation of a
lyophilized canfosfamide hydrochloride formulation and each of these
constraints must be met in
order to provide for a pharmaceutically acceptable formulation.
b. Formulation Critical Parameters
[00291 The following items are some critical issues for the compounding of the
drug product.
= If the solution pH is higher than target pH, it can be titrated with 1.0 N
HCL to the target pH.
If the pH is too low, it will not be possible to back titrate with NaOH due to
the sensibility of
canfosfamide hydrochloride to alkaline environment.
= Due to the acidity of the drug substance, the rate of dissolution of drug
substance influences
the pH of the compounding solution. A quick dissolution could reduce the pH
faster and
therefore reduce the time of exposure of the drug to high pH. A study of 50 mL
batch size
showed that dissolution at 5 C was slower than at room temperature (45
minutes versus 25
minutes). The solution was then analyzed by HPLC. The vinyl sulfone generated
at 5 C
and room temperature was not significantly different.
[00301 An additional study of two liter batch size was conducted. Canfosfamide
hydrochloride
was dissolved into three batches of pH 6.5, 0.1 M citrate buffer and monitored
at different
temperatures; 5 C, 20 C and 40 C. After 80 minutes, the first batch at 5
C, minor particles were
observed. In the second batch at 20 C (room temperature), the drug was
totally dissolved after 20
minutes. The amount of vinyl sulfone generated at 5 C and room temperature
was not significantly
different. The third batch consisted of adding drug at room temperature and
allowing the
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temperature to increase to 40 C over 90 minutes. The vinyl sulfone was higher
than the two
previous batches. These experiments support controlling the compounding
procedure at room
temperature until all drug is dissolved and filtered.
[0031] Canfosfamide hydrochloride is very hydrophilic. It forms lumps with a
hydrate layer once
in contact with the citrate buffer. This layer prevents further penetration of
water into the lumps and
slows down the dissolution process. A vigorous agitation using a homogenizer
minimizes the lump
formation and shortens the mixing time.
[0032] The formulation comprising (50 5) mg/mL canfosfamide hydrochloride in
(100 10) mM
aqueous sodium citrate buffer at pH 4.6 0.2 remained relatively stable with
total impurities
increased to about 2% after 6 months at 5 C. At 25 C and 40 C, however, the
total impurities
increased by 20% and 70% respectively after three months.
[0033] Due to the instability of canfosfamide hydrochloride in aqueous
solution for an adequate
shelf-life, lyophilization of the aqueous formulation was considered. While a
higher concentration,
high strength citrate buffer could dissolve more canfosfamide hydrochloride,
it will also result in a
higher osmolality which could make freeze drying challenging and decrease the
stability of a
lyophilized product. A 50 mg/mL solution of canfosfamide hydrochloride in a
100 mM citrate
buffer has an osmolality of about 400 mM, which is close to the limit in
performing satisfactory
lyophilization to obtain acceptable product. Further, a high osmolality buffer
at pH 4.6 could also
cause infusion irritation when administered to a patient.
[0034] Thus, this invention provides a delicate formulation that achieves high
concentration, high
stability of canfosfamide and minimizes osmolality by using a 100 nM citrate
buffer with an initial
pH of about 6.5.
[0035] In one embodiment, this invention provides a stable lyophilized
formulation consisting
essentially of canfosfamide hydrochloride and sodium citrate, wherein the said
formulation is stable
for therapeutic utility. In a preferred embodiment, the composition has an
osmolality of less than
400 mM which permits its therapeutic use for intravenous injection.
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[0036] Further to minimize osmolality, it is preferred that no bulk agent is
added to the
formulation. In this regard, it is surprising that canfosfamide hydrochloride
(5% w/w) and citrate
buffer (3% w/w) act as adequate bulking agents for the lyophilized product.
The citrate buffer not
only acts as a bulking agent but long term stability tests on the lyophilized
product indicates that it
has a preservative effect as well. For example, table 1 shows the stability of
the formulation:
Table 1. Stability of Aqueous versus lyophilized formulation at different
temperatures
Composition Stability at 5 C Stability at room temperature (25 C)
Aqueous formulation 6 months 7 days
Lyophilized product At least 6 years About 6 months
[0037] When compared to the aqueous formulation, the lyophilized product when
stored at 5 C
has adequate stability of more than 6 years. We believe that the significant
increase in stability is
unrelated to reaction kinetics because the temperature differential would not
support such a vast
difference. Hence, we believe that the citrate is acting as a preservative. It
was a surprising and
unexpected result that the lyophilized formulation would have such a superior
stability (>12 times)
as compared to the aqueous formulation, particularly as the lyophilized
product preferably does not
include any lyophilization aids. In some embodiments, no other additives
(e.g., anti-oxidants,
stabilizers, or chelating agents) are needed.
[0038] In another embodiment, this invention provides a stable lyophilized
formulation consisting
essentially of canfosfamide hydrochloride and sodium citrate, wherein the said
formulation is stable
for therapeutic utility and has a osmolality of less than 400 mM. Preferably,
the lyophilized product
does not comprise a lyophilization aid.
[0039] In a further embodiment, the stable lyophilized formulation is
anhydrous. In another
embodiment, the stable lyophilized formulation is stored in a dry atmosphere.
In a further
embodiment, the dry atmosphere is nitrogen, argon, and the like. In another
embodiment, the stable
lyophilized formulation is stored at a temperature not higher than 8 C.
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c. Stability of lyophilized formulation
[00401 The lyophilized formulation is generally stable at 5 C with no
significant potency,
individual impurities or total impurities change over period having adequate
shelf-life (for example,
about 18 months). Indeed, the data show that when stored at 5 C, the
lyophilized product has a
stability for over at least 6 years. Critically, other properties of the
lyophilized product such as,
appearance, moisture content, pH and reconstitution time were not changed. At
25 C, the
lyophilized formulation was found to be stable for about six months. At 25 C,
the potency of the
lyophilized formulation appears to be reduced over period of 18 months and the
total impurities
increased. The main impurity that increased was vinyl sulfone.
[0041] In another embodiment, this invention provides a stable lyophilized
formulation consisting
of canfosfamide hydrochloride and sodium citrate, wherein the said formulation
has an osmolality of
less than 400 mM, is stable for therapeutic utility, and the said formulation
is a product of
lyophilization of (50 5) mg/mL canfosfamide hydrochloride and (100 10) mM
aqueous sodium
citrate buffer at a pH of 4.6 0.2. In a further embodiment, the stable
lyophilized formulation is
stored in a dry atmosphere at a temperature not higher than 8 C.
[0042] In another embodiment, this invention provides a stable lyophilized
formulation, wherein
the said formulation is a product of lyophilization of 50 mg/mL canfosfamide
hydrochloride, 100
mM sodium citrate dihydrate, and 2.9 mM citric acid monohydrate in water at a
pH of 4.6 0.2,
wherein the stable lyophilized formulation is stored in a dry atmosphere at a
temperature not higher
than 8 C.
[0043] In another embodiment, this invention provides a lyophilized
formulation of canfosfamide
hydrochloride, citric acid and sodium citrate, which formulation is capable of
being reconstituted to
a formulation consisting essentially of (50 5) mg/mL canfosfamide
hydrochloride, (100 10) mM
sodium citrate and water, and having a pH of 4.6 0.2.
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d. Stability of bulk solution
[00441 The stable lyophilized formulation of this invention is preferably
administered via an
injection. Following studies were conducted to check the stability of the bulk
solution.
Stability in Solution at 1 mg/mL - effect of pH
[00451 A stability study was performed by monitoring vinyl sulfone generation.
Canfosfamide
hydrochloride was dissolved at 1 mg/mL into 0.1 M citrated buffer from pH 4.5
to pH 6.5. The
solutions were incubated at 25 C for 24 hours. Vinyl sulfone generation was
monitored. Table 2
shows the results of the study.
Table 2. Area Percent Vinyl Sulfone at Different pH
Percent vinyl sulfone at 25 C
pH 0 hrs 6 hrs 24 hrs
4,5 ND 0.1 0.4
5.0 ND 0.2 1.1
5.5 ND 0.6 2.9
6.0 ND 1.7 8.2
6.5 ND 6.6 24.2
ND = not detected
[00461 The results show that the stability of canfosfamide hydrochloride is pH
dependant. Higher
pH generates more vinyl sulfone. However, the amount of vinyl sulfone produced
at 25 C at pH
between 4.5 and 5.5 over at least a 6 hour period is sufficiently low such
that administration of an
intravenous formulation is permitted.
Stability in Solution of Current Formulation (50 mg/mL)
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[0047] The stability of the bulk solution of 50 mg/mL (at pH 4.6) was
conducted. The results
showed that at 25 C, the purity was relatively unchanged after 1 day. Table 3
shows the bulk
solution stability results.
Table 3. Area Percent Canfosfamide Hydrochloride Upon Bulk Solution Stability
(data is * 2%)
Time % Purity 25 C
Day 0 92
Day l 89
Day 3 93
Day 7 89
Day 28 84
3 Month 69
r6Month NP*
NP: Not performed
[0048] The results show that the stability of canfosfamide hydrochloride
solution decreases over
time at 25 C. However, percent purity decrease at 25 C is such that
immediate (within 24 hours)
lyophilization is permitted.
[0049] Figure 1, a plot of assay value of lyophilized canfosfamide
hydrochloride formulation (50
mg/mL) versus time, illustrates that canfosfamide formulation is stable at 5
C even after 72 months
as seen by the almost unchanged assay value. Similarly, Figure 2, a plot of
total degradation
impurities percent versus time, illustrates that the amount of impurities in
lyophilized canfosfamide
hydrochloride formulation (50 mg/mL) are about I% even after 72 months.
[0050] In another embodiment, this invention provides a kit for preparing an
aqueous formulation
consisting essentially of (50 5) mg/mL canfosfamide hydrochloride, and (100
10) mM aqueous
sodium citrate having a pH of 4.6 0.2, which kit comprises sterile water and a
lyophilized
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formulation comprising canfosfamide hydrochloride, citric acid and sodium
citrate capable of being
reconstituted to said formulation.
[0051] In another embodiment, the kit is for preparing a formulation
consisting essentially of
(50 5) mg/mL canfosfamide hydrochloride, and (100 10) mM aqueous sodium
citrate having a
pH of 4.6 0.2, which kit comprises sterile water and a lyophilized formulation
of canfosfamide
hydrochloride, citric acid and sodium citrate capable of being reconstituted
to said formulation.
[0052] In another embodiment, this invention provides a reconstituted
formulation of canfosfamide
hydrochloride, which formulation comprises (50 5) mg/mL canfosfamide
hydrochloride in
(100 10) mM aqueous sodium citrate buffer at pH 4.6 0.2.
[0053] In another embodiment, the reconstituted formulation of canfosfamide
consists essentially
of (50 5) mg/mL canfosfamide hydrochloride in (100 10) mM aqueous sodium
citrate buffer at
pH 4.6 0.2.
[0054] In some embodiments, the reconstituted formulation is prepared by a
process comprising
adding water (e.g., sterile water) to the stable lyophilized composition of
this invention.
[0055] In another embodiment, this invention provides an injectable
formulation of canfosfamide
comprising (50 5) mg/mL canfosfamide hydrochloride in (100 10) mM aqueous
sodium citrate
buffer at pH 4.6 0.2, and a diluent so that the final canfosfamide
hydrochloride in the injectable
formulation is about 6-8 mg/mL canfosfamide hydrochloride.
[0056] In some embodiments, this invention provides an injectable formulation
of canfosfamide
consisting essentially of (50 5) mg/mL canfosfamide hydrochloride in (100 10)
mM aqueous
sodium citrate buffer at pH 4.6 0.2, and a diluent so that the final
canfosfamide hydrochloride in the
injectable formulation is about 6-8 mg/mL canfosfamide hydrochloride.
[0057] In some embodiments, the diluent is 5% dextrose aqueous solution.
[0058] In some embodiments, the injectable formulation is for intravenous
administration.
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Methods
[0059] In another embodiment, this invention is a method of preparing the
stable lyophilized
formulations consisting essentially of canfosfamide hydrochloride and sodium
citrate, wherein the
said formulation is stable for therapeutic utility, wherein the method
comprises:
(a) preparing a sodium citrate buffer of a pH of 6.5 0.1;
(b) dissolving canfosfamide hydrochloride in the sodium citrate buffer of step
(a) to provide a
solution;
(c) adjusting the pH of the solution of step (b) when pH is > 4.8 to provide a
resulting solution of
pH of 4.6 0.2 by addition of a compatible acid selected from citric acid and
HC1; and
(d) lyophilizing the resulting solution of step (c) to provide a lyophilized
formulation having an
osmolality of less than 400 mM.
[0060] In a further embodiment, the method does not comprise addition of a
lyophilization aid.
[0061] In another embodiment, the method further comprises storing the
lyophilized formulation
of step (d) in a dry atmosphere at a temperature not higher than 8 C. In a
further embodiment, the
dry atmosphere is nitrogen or argon.
[0062] The aqueous sodium citrate buffer may be prepared by the dissolution of
a predetermined
amount of one or more sodium citrate salts, and optionally citric acid, for
example trisodium citrate
dihydrate and citric acid monohydrate, in water, followed if necessary by an
adjustment of the pH to
the desired value of 6.5 0.1 by the addition of an acid, such as hydrochloric
acid, or a base, such as
1 M sodium hydroxide.
[0063] In some embodiments, the pH 6.5 of the 0.1 M citrate buffer is achieved
by titrating sodium
citrate solution with citric acid to obtain pH 6.5.
[0064] In some embodiments, the pH 6.5, 0.1 M citrate buffer is prepared by
dissolving a
predetermined amount of sodium citrate and a predetermined amount of citric
acid, followed if
necessary by an adjustment of the pH to the desired value of 6.5 0.1 by the
addition of an acid.
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[0065] The canfosfamide hydrochloride is added to the buffer and stirred to
achieve dissolution.
The addition of the canfosfamide hydrochloride to the buffer will reduce the
pH of the mixture to
approximately 4.6+0.2, or slightly higher; and the pH of the mixture may be
reduced by the addition
of small quantities of acid if it exceeds 4.8. Addition of the canfosfamide
hydrochloride to the
aqueous citrate buffered solution is preferably conducted at room temperature
or below.
[0066] Typically, steps (a) through (c) are performed with the use of less
water than is required to
achieve the desired final concentrations of (50+5) mg/mL canfosfamide
hydrochloride and
(100+10) mM sodium citrate, and as a final step (c') water is added to achieve
the desired final
concentrations and an osmolality of less than 400. For example, when the
aqueous sodium citrate
buffer is prepared by adding solid sodium citrate and then citric acid to
water, the initial amount of
water in which the buffer ingredients are to be dissolved may be (85 - 95)% of
the amount of water
needed to achieve the final concentration, allowing for the use of additional
water to rinse in the
solid ingredients as the formulation is prepared; then, following any
necessary pH adjustment, the
remaining amount of water is added to achieve the desired final
concentrations.
[0067] The resulting formulation may be filtered, such as by filtration
through a 0.45 m filter; and
is then typically sterile filtered, such as by filtration through a 0.2 m
filter, and the formulation
would then be suitable for injection.
[0068] In an embodiment, the formulation steps can be performed under dry
conditions, for
example, under nitrogen purge.
[0069] Then, the formulation is lyophilized for storage (and subsequent
reconstitution before
administration), using methods conventional in the art with the exception that
lyophilization aids are
preferably not employed. Lyophilization as per this invention is illustrated
in the following
Example. For example, the formulation is dispensed into containers of
appropriate size, such as 10
mL or 20 mL, frozen in the containers to substantially below 0 C. Removal of
the water content of
the formulation is achieved under reduced pressure by sublimation. Upon
drying, the pressure and
temperature are increased, and the containers are sealed to provide for the
lyophilized composition.
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[0070] The quantity per container of the formulation will typically be chosen
to permit
reconstitution to the same concentration as the original formulation and
withdrawal of a convenient
amount (e.g. 250 mg or 1 g) of canfosfamide hydrochloride. For example, 5.3 mL
of the formulation
(a 6% overage) may be lyophilized in a 10 mL vial, allowing for convenient
withdrawal of 5.0 mL of
formulation after reconstitution with 5.2 mL water to provide 250 mg of
canfosfamide
hydrochloride; 10.3 mL of the formulation of this invention may be lyophilized
in 20 mL vial to
provide 515 mg of canfosfamide hydrochloride, and 20.6 mL of the formulation
(a 3% overage) may
be lyophilized in a 50 mL vial, allowing for convenient withdrawal of 20.0 mL
of the formulation
after reconstitution to provide 1.0 g of canfosfamide hydrochloride.
Examples
[0071] The following example describes the lyophilized formulations of this
invention, and their
preparation. The mixing and filtration operation were conducted under nitrogen
purge.
Example 1
[0072] To prepare a 50 L volume of the formulation (density 1.034 Kg/L), 43.5
Kg of water for
injection (WFI) was added to a jacketed 80 L stainless steel vessel equipped
with a Silverson mixer
with a 10 cm stainless steel blade. This was stirred at 800 r.p.m.; 1470 g
trisodium citrate dihydrate
was added and rinsed in with 1 L of WFI; and stirring was continued for 10
minutes. Citric acid
monohydrate, 30.2 g, was added and rinsed in with 500 mL of WFI; and stirring
was continued at
1320 r.p.m. for a further 10 minutes. The pH of the solution was measured and
adjusted to 6.6 by
the addition of two 20 mL increments of 1 M hydrochloric acid, with 5 minutes
of stirring at
1320 r.p.m. after each addition. Canfosfamide hydrochloride, 2600 g of 96.8%
pure material, was
added and rinsed in with 1500 mL of WFI; and stirring was continued for 20
minutes at 2300 r.p.m.
and 30 minutes at 2200 r.p.m. The pH of the solution was measured as 4.7. A
further 1 Kg of WFI
was added and stirred for 10 minutes at 880 r.p.m., after which the solution
was cooled to 8 C,
giving 50 L of the formulation of this invention, containing 50.3 mg/mL of
canfosfamide
hydrochloride in 103 mM sodium citrate buffer at pH 4.7.
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[0073] The formulation was filtered through a 0.45 m filter and then through
two 0.2 gm filters
(sterile filtration). The filtered formulation was filled into 10 mL type I
glass vials with 5.3 mL fill
volume per vial; a 20 mm butyl rubber lyophilization stopper was placed in the
lyophilization
position on each vial; and the vials were placed onto trays and loaded into
the lyophilizer, with the
lyophilizer shelves at 5 C. Once the lyophilizer was closed, the
lyophilization cycle comprised:
hold for 0.6 hours; decrease shelf temperature to -46 C over 1.7 hours; hold
for 5 hours; decrease
lyophilizer chamber pressure to 13.3 Pa over 0.8 hours; start nitrogen gas
sweep at 12.4 Pa and hold
for 5 hours; increase shelf temperature to -25 C over 1.1 hours; hold for
16.2 hours; increase shelf
temperature to 0 C over 1 hour; hold for 20 hours; increase shelf temperature
to 30 C over
1.7 hours; hold for 15 hours; decrease shelf temperature to 8 C over 0.6
hours; increase lyophilizer
chamber pressure to 69 KPa with nitrogen; stopper the vials; increase
lyophilizer chamber pressure
to atmospheric pressure; open the lyophilizer and unload the stoppered vials.
Each vial contained a
lyophilized formulation of this invention containing 265 mg canfosfamide
hydrochloride and 159 mg
trisodium citrate/citric acid. The vials were sealed with an aluminum seal
with a flip-off
polypropylene cap. When reconstituted with 5.2 mL of WFI, each vial contains
5.3 mL of an
aqueous formulation containing 50.3 mg/mL of canfosfamide hydrochloride in 103
mM sodium
citrate buffer at pH 4.7; allowing convenient withdrawal of 5.0 mL of the
formulation.
[0074] An aqueous formulation containing 50 mg/mL of canfosfamide
hydrochloride may also be
lyophilized in vials of other sizes if a larger unit dose is required, such as
20 mL vials with a fill
volume of 10.3 mL, 50 mL vials with a fill volume of 20.6 mL, giving a
canfosfamide hydrochloride
content of 1030 mg (1 g plus 3% overage per vial). The lyophilization cycle
may require
modification (e.g. lengthening of the cycle time) for larger fill volumes, but
such modification will
be within the capability of a person of ordinary skill in the art having
regard to that skill and this
disclosure.
[0075] For intravenous (i.v.) injection, the reconstituted formulation with 50
mg/mL canfosfamide
was diluted 7 times (1 part to 6 part) with a 250 mL 5% Dextrose Injection
solution in an i.v. bag to
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form an i.v. injectable formulation. Both the reconstituted formulation and
the i.v. injectable
formulation were stable at room temperature for 24 hours.
100761 While this invention has been described in conjunction with specific
embodiments and
examples, it will be apparent to a person of ordinary skill in the art, having
regard to that skill and
this disclosure, that equivalents of the specifically disclosed materials and
methods will also be
applicable to this invention; and such equivalents are intended to be included
within the following
claims.
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