Note: Descriptions are shown in the official language in which they were submitted.
Attorney Docket: 09423.020W01
READY-TO-USE BIVALIRUDIN COMPOSITIONS
REFERENCE TO A SEQUENCE LISTING SUBMITTED
AS A TEXT FILE VIA EFS WEB
[0001] The official copy of the sequence listing is submitted electronically
via EFS-Web as an
ASCII formatted sequence listing with a file named BIVSEQ_ST25.TXT, created on
May 20,
2019, and having a size of 2 kilobytes, and is filed concurrently with the
specification. The
sequence listing contained in this ASCII formatted document is part of the
specification and is
herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present disclosure is generally directed to storage stable, ready-
to-use compositions
comprising bivalirudin 3.0, wherein the compositions exhibit a pH in the range
of about 3.0 to
less than 4.0, or a pH in the range of from 5.0 to about 5.7. These
compositions have shelf lives
of at least 6, at least 9 months, or at least 12 months, when stored at 5 C.
In particular
embodiments, these storage-stable, ready-to-use bivalirudin compositions also
include tonicity-
adjusting agents, stabilizers, and/or buffering agents.
BACKGROUND
[0003] Bivalirudin is a member of the class of direct thrombin inhibitors,
which have
anticoagulant activity, and can thus prevent blood clotting. Direct thrombin
inhibitors disrupt the
activity of both circulating and clot-bound thrombin, a serine protease acting
in the coagulation
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cascade that initiates blood clotting when fibrinogen is converted into
fibrin. Bivalirudin
specifically binds to thrombin's catalytic site and anion-binding exosite.
Bivalirudin has a rapid
onset of action, a short half-life, and is regarded as having a predictable
antithrombic response.
Bivalirudin overcomes many of the risks associated with indirect thrombin
inhibitors, for
example, heparin.
[0004] Bivalirudin has been formulated into a lyophilized drug product, e.g.,
ANGIOMAX
(bivalirudin) for injection. This dosage form must be reconstituted before
administration, e.g.,
by injection such as parenteral injection. This generally requires a number of
steps, including
dissolution (or reconstitution) of the lyophilized drug product in a suitable
liquid for injection,
such as Water for Injection, 0.9% Sodium Chloride Injection, or other fluids
known in the art for
this purpose to yield a concentrated solution. Complete dissolution into the
liquid typically
requires physical manipulation, e.g., swirling or shaking, and can be time
consuming. Careful
dilution of the reconstituted (concentrated) solution must then be performed
with the correct
volume of suitable diluent, such as 0.9% Sodium Chloride Injection or 5%
Dextrose Injection, to
ensure the correct concentration for administration. Incomplete dissolution
during the
reconstitution step and/or incorrect dilution during the subsequent dilution
step can lead to
improper dosing, a significant component of medical error. The use of an
incorrect
reconstitution fluid and/or diluent can also lead to dosing errors. Such
lyophilized dosage forms
have been claimed in, for example, U.S. Patent Nos. 7,582,727 and 7,598,343.
[0005] A further limitation of lyophilized drug products which must be
reconstituted with a
suitable liquid for injection is the relatively short shelf-life which is
associated with such
preparations after reconstitution and/or dilution. These reconstituted drug
products are often
only labelled as usable for only a few hours after reconstitution and/or
dilution. For example,
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reconstituted ANGIOMAX solutions are only stable for up to 24 hours at 2-8 C
and diluted
ANGIOMAX solutions are stable at 2-8 C or room temperature for up to 24
hours.
[00061 Liquid ready-to-use bivalirudin formulations having pH of 4 to less
than 5 are described
and claimed in U.S. Patent Nos. 7,713,928 and 7,803,762 (also published as
U.S. Patent
Publication Serial No. 2011/0046063). The most preferred pH of these
formulations is 4.2-4.5,
which is lower than physiological pH and thus they may not be suitable for
direct infusion.
These formulations are reported to show extensive degradation (37.7%, 51.1%)
between pH 3
and 4 and complete degradation of the composition at pH 5 and 6 after storage
for one month at
25 C.
[0007] There is thus a need for further ready-to-use injectable bivalirudin
compositions having
an appreciable shelf-life, e.g., suitable for storage at 5 C for at least 6
months, or at least 9
months, or at least 12 months and with pH closer to the physiological pH
range.
SUMMARY
100081 In one aspect, the embodiments provided herein describe a ready-to-use
liquid
formulation of bivalirudin (label concentration of 5 mg/mL) with at least 6-,
at least 9- or at least
12-month shelf life at refrigerated conditions (5 3 C, i.e., 2-8 C). These
liquid ready-to-use
formulations have pH of about 3.0 to less than 4.0, or of about 3.25 to about
3.75, or
alternatively, these formulations have pH of 5.0 to about 5.7; each type of
formulation has a
substantially similar shelf life. Additionally, these ready-to-use liquid
bivalirudin compositions
may be packaged as commercial off-the-shelf products, for example, are not
lyophilized, have
not been lyophilized as a step in their preparation (outside circumstances in
which the active
pharmaceutical ingredient may be supplied as a lyophilized material), and have
not been
reconstituted to a liquid form.
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[0009] The formulations also have reduced levels of impurities upon storage,
compared to
previously disclosed formulations. The impurity levels at a particular time
point are determined
by high performance liquid chromatography ("HPLC") at a wavelength of 215 nm
and compared
to an initial time point, for example, the time of manufacture of the
formulation, throughout this
disclosure. For example, in the formulations described herein having pH in a
range of about 3.0
to less than 4.0, or about 3.25 to about 3.75, or in a range of 3.3 to about
3.7, or in a range of
about 3.4 to about 3.6, the amounts of total impurities as compared to an
initial time point as
determined by high performance liquid chromatography ("HPLC") at a wavelength
of 215 nm,
remain well under about 15% after storage at 25 C for 1 month. Similarly, in
the formulations
described herein having pH in a range of 5.0 to about 5.7, or in a range of
from about 5.1 to
about 5.4, or in a range of from about 5.2 to about 5.3, the amounts of total
impurities as
compared to an initial time point are also well under about 15% after storage
at 25 C for 1
month, under about 10%, and under about 7.5%, and under after storage at 5 C
for 12 months.
Also, in the formulations described herein, the amount of total impurities as
compared to an
initial time remains well under about 15%, under about 12%, under about 10%
and under about
5% after storage at 5 C for 12 months, for the formulations having pH having
pH in a range of
about 3.0 to less than 4.0, or about 3.25 to about 3.75, or in a range of
about 3.3 to about 3.7, or
in a range of about 3.4 to about 3.6, or in a range of 5.0 to about 5.7, or in
a range of from about
5.1 to about 5.4, or in a range of from about 5.2 to about 5.3.
[0010] Similarly, in the formulations described herein having pH in a range of
about 3.0 to less
than 4.0, or of about 3.25 to about 3.75, or in a range of about 3.3 to about
3.7, or in a range of
about 3.4 to about 3.6, or having pH in a range of 5.0 to about 5.7, or in a
range of from about
5.1 to about 5.4, or in a range of from about 5.2 to about 5.3, the amounts of
the [9-10]-
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cycloimido-bivalirudin or the [11-12]-cycloimido-bivalirudin impurities as
compared to an initial
timepoint, as determined by high performance liquid chromatography ("HPLC") at
a wavelength
of 215 nm, is kept well below 5% after storage at 25 C for 1 month (see Claim
6 and Claim 7 of
US 7,713,928). For the formulations described herein, having pH in a range of
about 3.0 to less
than 4.0, or of about 3.25 to about 3.75, or in a range of about 3.3 to about
3.7, or in a range of
about 3.4 to about 3.6, or having pH in a range of 5.0 to about 5.7, or in a
range of from about
5.1 to about 5.4, or in a range of from about 5.2 to about 5.3, the amounts of
the [9-10]-
cycloimido-bivalirudin or the [11-12]-cycloimido-bivalirudin impurities as
compared to an initial
time point remain well below about 1.5%, under about 1.0%, under about 0.4%,
under about
0.3%, and under about 0.25% after storage at 25 C for 1 month or at 5 C for
about 12 months.
100111 Also, in the formulations described herein, the amount of [3-20]-
bivalirudin impurity as
compared to an initial time point, as determined by high performance liquid
chromatography
("HPLC") at a wavelength of 215 nm, remains well under about 1.5%, under about
1.0%, under
about 0.6% and under about 0.5% after storage at 5 C for 12 months, for the
samples having pH
in a range of about 3.0 to less than 4.0, or from about 3.25 to about 3.75, or
in a range of 3.3 to
3.7, or in a range of 3.4 to 3.6. Also, in the formulations described herein,
the amount of [3-20]-
bivalirudin impurity as compared to an initial time point remains well under
about 1.5% or under
about 1.0% after storage at 5 C for 12 months, for the samples having pH in a
range from 5.0 to
about 5.7, or in a range of from about 5.1 to about 5.4, or in a range of from
about 5.2 to about
5.3.
[0012] The ready-to-use bivalirudin compositions disclosed herein are
considered storage stable.
[0013] The formulations may contain tonicity-adjusting and/or stabilizing
agents, which can be
selected from the group consisting of, for example, salts such as potassium
chloride, sodium
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chloride, for example 0.9% NaCI; saccharides (e.g., monosaccharides,
disaccharides,
polysaccharides) such as, lactose, trehalose, raffinose, dextrose, maltose,
galactose, sucrose, and
polysucrose, sugar alcohols such as mannitol, for example, 5% D-mannitol,
sorbitol, or xylitol;
polymers such as polygalacturonic acid, polyethylene glycol (PEG),
polyvinylpyrrolidine (PVP),
for example, PEG 300, PEG 400, PEG 3350, PEG 6000, PEG 8000 and the like, for
example
10% w/v PEG 400; amino acids such as lysine, arginine, glycine, methionine,
and other amino
acids; and other materials such as dextran, cyclodextrins (for example,
hydroxypropyl-T-
cyclodextrin, Ficoll, galacturonic acid, and other similar excipients. These
formulations may
also contain further excipients, such as buffering agents, pH-adjusting
agents, preservatives,
osmolality-adjusting agents, and solubilizing agents.
[0014] In some embodiments, an injectable ready-to-use bivalirudin composition
of the
invention comprises:
a. bivalirudin (SEQ ID NO: 1), or salts thereof,
b. one or more pharmaceutically acceptable agent(s) selected from the group
consisting of buffering agents, tonicity-adjusting agents, stabilizing agents,
antioxidants, and pH
adjusting agents, and
c. a pH ranging from (i) 5.0 to about 5.7 or (ii) about 3.0 to less than
4.0,
wherein the percentage of total impurities increases by no more than about 9%
from the time of
manufacture up to 12 months of storage at 5 C or up to 1 month of storage at
25 C as determined
by high performance liquid chromatography at a wavelength of 215 nm.
[0015] In some embodiments, the one or more pharmaceutically acceptable
agent(s) comprises a
tonicity-adjusting agent and/or a stabilizing agent. In some embodiments, the
tonicity-adjusting
agent and/or the stabilizing agent comprises (i) an inorganic chloride, (ii) a
saccharide, sugar
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alcohol or an amino sugar, (iii) an amino acid, (iv) an organic solvent, or
(vi) any combination
of (i)-(iv). In some embodiments, the tonicity-adjusting and/or the
stabilizing agent comprises
polyethylene glycol (PEG), mannitol, sucrose, glycerol, ethanol, sorbitol,
glycine, proline, or any
combination thereof
100161 In some embodiments, the pH of a composition of the invention ranges
from 5.0 or more
to about 5.7. In some embodiments, the one or more pharmaceutically acceptable
agent(s)
comprises a buffering agent having at least one pKa from about 4.0 to about
6.7. In some
embodiments, the buffering agent is selected from the group consisting of
acetate, oxalate,
acrylate, ascorbate, benzoate, caprate, caproate, caprylate, cinnamate,
fumarate, maleate,
phosphate, orthophosphate, laurate, palmitate, propionate, adipate,
cacodylate, malonate,
propionate, hydroxypropionate, fumarate, phthalate, maleate, 3- or 4-
hydroxybutanoate,
butenoate, crotonate, methylmalonate, succinate, malate, tartrate, citrate, 2-
(N-
morpholino)ethanesulfonic acid ("MES"), salts thereof, and any combination
thereof. In some
embodiments, the percentage of Asp9-bivalirudin increases by no more than
about 2% from the
time of manufacture up to 12 months of storage at 5 C or up to 1 month of
storage at 25 C as
determined by high performance liquid chromatography at a wavelength of 215
nm; the
percentage of [3-20]-bivalirudin increases by no more than about 2% from the
time of
manufacture up to 12 months of storage at 5 C or up to 1 month of storage at
25 C as determined
by high performance liquid chromatography at a wavelength of 215 nm; the
percentage of [9-
10]-cycloimido-bivalirudin increases by no more than about 2% from the time of
manufacture up
to 12 months of storage at 5 C or up to 1 month of storage at 25 C as
determined by high
performance liquid chromatography at a wavelength of 215 nm, the percentage of
[11-12]-
cycloimido-bivalirudin increases by no more than about 2% from the time of
manufacture up to
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12 months of storage at 5 C or up to 1 month of storage at 25 C as determined
by high
performance liquid chromatography at a wavelength of 215 nm, and/or the
percentage of total
impurities increases by no more than about 8% from the time of manufacture up
to 12 months of
storage at 5 C or up to 1 month of storage at 25 C as determined by high
performance liquid
chromatography at a wavelength of 215 nm. In some embodiments, the one or more
pharmaceutically acceptable agent(s) comprises (i) sodium acetate and (ii) a
tonicity-adjusting
agent and/or a stabilizing agent selected from the group consisting of
polyethylene glycol (PEG),
mannitol, sucrose, glycerol, ethanol, sorbitol, glycine, proline, and any
combination thereof.
[0017] In some embodiments, a composition of the invention comprises
about Ito about 10 mg/mL bivalirudin,
about 1 mM to about 50 mM sodium acetate,
about 5% to about 12.5% PEG by weight, and
a pH of about 5.25.
[0018] In some embodiments, a composition of the invention comprises
about 2.5 to about 7.5 mg/mL bivalirudin,
about 5 mM to about 30 mM sodium acetate,
about 7.5 to about 10% PEG by weight, and
a pH of about 5.25.
[0019] In some embodiments, a composition of the invention comprises
about 5 mg/mL bivalirudin,
about 6 mM sodium acetate,
about 10% PEG 400 by weight, and
a pH of about 5.25.
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[0020] In some embodiments, a composition of the invention comprises a pH
ranging from about
3.0 to less than 4Ø
[0021] In some embodiments, a composition of the invention comprises
about 1 to about 10 mg/mL bivalirudin,
about 1 mM to about 300 mM glycine,
about 5% to 12.5% PEG by weight, and
a pH of about 3.50.
[0022] In some embodiments, a composition of the invention comprises
about 1 to about 10 mg/mL bivalirudin,
about 5 mM to about 50 mM glycine,
about 7.5% to 10 % PEG by weight, and
[0023] a pH of about 3.50.In some embodiments, a composition of the
invention
comprises
about 5 mg/mL bivalirudin,
about 10 mM glycine,
about 10% PEG by weight, and
a pH of about 3.50.
[0024] In some embodiments, the percentage of Asp9-bivalirudin increases by no
more than
about 0.6% from the time of manufacture up to 12 months of storage at 5 C or
up to 1 month of
storage at 25 C as determined by high performance liquid chromatography at a
wavelength of
215 nm, the percentage of [12-20]-bivalirudin increases by no more than about
4% from the time
of manufacture up to 12 months of storage at 5 C or up to 1 month of storage
at 25 C as
determined by high performance liquid chromatography at a wavelength of 215
nm, the
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percentage of [3-20]-bivalirudin increased by no more than about 1% from the
time of
manufacture up to 12 months of storage at 5 C or up to 1 month of storage at
25 C as determined
by high performance liquid chromatography at a wavelength of 215 nm, the
percentage of [11-
12]-cycloimido-bivalirudin increases by no more than about 2.5% from the time
of manufacture
up to 12 months of storage at 5 C or up to 1 month of storage at 25 C as
determined by high
performance liquid chromatography at a wavelength of 215 nmõ the percentage of
[9-10]-
cycloimido-bivalirudin increases by no more than about 0.5% from the time of
manufacture up
to 12 months of storage at 5 C or up to 1 month of storage at 25 C as
determined by high
performance liquid chromatography at a wavelength of 215 nm, and/or the
percentage of total
impurities increases by no more than about 7.5% from the time of manufacture
up to 12 months
of storage at 5 C or up to 1 month of storage at 25 C as determined by high
performance liquid
chromatography at a wavelength of 215 nm.
100251 In some embodiments, a pharmaceutical composition of the invention
comprises a sterile,
storage stable ready-to-use liquid composition that comprises a
therapeutically effective amount
of bivalirudin, a pharmaceutically acceptable carrier, and less than about 19%
total bivalirudin
impurities, and a sterile ready-to-use container pre-packaged with the storage
stable ready-to-use
liquid composition.
100261 In some embodiments, a composition is a commercial off-the-shelf
pharmaceutical
product. In some embodiments, the liquid composition comprises
about 5 mg/mL bivalirudin,
about 10 mM glycine or about 6mM sodium acetate,
About 10% PEG 400 by weight, and
a pH of about 3.5 or 5.25.
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[0027] In a further aspect, the embodiments provided herein describe methods
of using the
ready-to-use liquid formulations of bivalirudin described herein.
[0028] In some embodiments, a method for inhibiting blood clots in a mammal is
provided
comprising administering a therapeutically effective amount of a composition,
e.g.,
pharmaceutical composition as described herein the mammal in need thereof,
wherein the
administration does not involve reconstitution of a lyophilized bivalirudin
composition. In some
embodiments, the compositions comprise less than about 19% total impurities.
[0029] In another further aspect, the embodiments provided herein describe
methods of
preparing the ready-to-use liquid formulations of bivalirudin described
herein.
DETAILED DESCRIPTION
[0030] Bivalirudin is a synthetically-produced, 20-amino acid peptide with the
following amino
acid sequence, and a molecular weight of 2180 daltons in its free base form:
D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-
Leu
[0031] A commonly used salt is the trifluoroacetate salt hydrate, and as used
herein, the term
"bivalirudin" refers to the peptide comprising SEQ ID NO: 1 and salts thereof.
[0032] The term "therapeutically effective amount" as used herein is
interchangeable with
"effective amount" for purposes herein, and is determined by such
considerations as are known
in the art. The amount must be effective to achieve a desired drug-mediated
effect in the treated
subjects suffering from the disease thereof A "therapeutically effective
amount" also includes,
but is not limited to, consideration of appropriate measures selected by those
skilled in the art,
for example, improved survival rate, more rapid recovery, or amelioration,
improvement or
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elimination of symptoms. The term "about" in relation to pH units means plus
or minus 0.2 pH
units.
[0033] The term "storage stable" as used herein means a composition that is
stable in storage as
ready-to-use (e.g., liquid) compositions that are stable in storage. Such
storage stable
compositions refer to those in which the composition maintains bivalirudin in
its active form,
and/or has a limited formation of impurities after manufacture, and/or
maintains the pH within a
selected range (e.g., about 3.0 to less than 4.0, or about 3.25 to about 3.75,
or about 3.3 to about
3.7, or about 3.4 to about 3.6, or 5.0 to about 5.7, or about 5.1 to about 5.4
or about 5.2 to about
5.3) upon storage at a selected temperature for a selected time period.
100341 Peptides containing glutamine and asparagine residues are generally
difficult to stabilize
in liquid drug formulations, due to the multitude of degradative pathways to
which such peptides
are susceptible. Glutamine and especially asparagine residues can undergo
deamidation, which
are catalyzed at neutral and alkaline pH through the formation of a
succinimide intermediate.
This reaction can also occur at acidic pH through direct hydrolysis of an
asparagine residue. It is
thus difficult to avoid this degradation by pH adjustment, and drug products
containing peptides
with these specific amino acids often have been prepared as lyophilizates in
an effort to avoid
such issues by minimizing contact with aqueous media.
100351 In one aspect, the embodiments provided herein describe a ready-to-use
liquid
formulation of bivalirudin (e.g., 5 mg/mL) with at least 6 months, at least 9
or at least 12 months
shelf life at refrigerated conditions (5 3 C, i.e., 2-8 C). These compositions
are storage stable.
A "storage stable" composition refers to a composition that is stable in
storage and refers to
ready-to-use or ready-to-dilute aqueous (e.g., liquid) compositions that are
stable in storage.
Such storage stable compositions refer to those in which the composition
maintains bivalirudin
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in its active form and/or has a limited amount of impurities and/or maintains
the pH within the
desired range upon storage at a selected temperature for a selected time
duration after
manufacture of the formulation. Storage stability also implies lower impurity
levels upon such
storage, as compared to previously disclosed liquid ready-to-use bivalirudin
compositions.
[0036] For example, a composition is considered storage stable under certain
conditions so long
as the composition retains at least 90% of its active bivalirudin and/or
increases in total
impurities remain less than about 10%, 7% or 5% as compared to an initial time
point, as
determined by high performance liquid chromatography ("HPLC") at a wavelength
of 215 nm,
for compositions with pH values between 5.0 and about 5.7, or less than about
10% or 7.5% for
compositions with pH values of about 3.0 to less than 4.0, or from about 3.25
to about 3.75.
[0037] In some embodiments, a composition is considered storage stable so long
as the
composition does not exhibit an increase of more than about 0.6%, or more than
about 0.5%, or
more than about 0.25% Asp9-bivalirudin (SEQ ID NO:3) as compared to an initial
time point, as
determined by high performance liquid chromatography ("HPLC") at a wavelength
of 215 nm
for compositions having pH in the range of about 3.0 to less than 4.0, or
about 3.25 to about
3.75. In some embodiments, a composition is considered storage stable so long
as the
composition does not exhibit an increase of more than about 1.5% or more than
about 1.0%, or
more than about 0.75% or more than about 0.6% Asp9-bivalirudin as compared to
an initial time
point, as determined by high performance liquid chromatography ("HPLC") at a
wavelength of
215 nm for compositions having pH in the range of 5.0 to about 5.7.
[0038] In some embodiments, a composition is considered stable so long as the
composition
does not exhibit increases more than about 1.0% or more than about 0.5% [3-20]-
bivalirudin
(SEQ ID NO:2) as compared to an initial time point, as determined by high
performance liquid
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chromatography ("HPLC") at a wavelength of 215 nm for compositions having pH
in the range
of about 3.0 to less than 4.0, or about 3.25 to about 3.75. In some
embodiments, a composition is
considered stable so long as the composition does not exhibit increases more
than about 2.0%, or
more than1.8%, or more than 1.5% [3-20]-bivalirudin as compared to an initial
time point, as
determined by high performance liquid chromatography ("HPLC") at a wavelength
of 215 nm
for compositions having pH in the range of 5.0 to about 5.7.
[0039] In some embodiments, a composition is considered stable so long as no
individual
unknown impurity makes up more than 2% or more than 1% of the composition. In
some
embodiments, a composition is considered stable so long as the initially
adjusted pH remains
within 1.0 pH unit, or within 0.75 pH units, or within 0.5 pH units, or
within 0.25 pH units.
"Shelf-life" refers to the amount of time a composition remains stable under
certain conditions.
Influence of pH and Stabilizers
[0040] The predominant degradation mechanisms of peptides (such as
deamidation) are known
to be pH dependent. In addition to the pH, the role of various stabilizers was
also investigated
ranging from buffers, saccharides (e.g., sucrose, mannitol, sorbitol), amino
acids (e.g., glycine,
proline), and organic co-solvents (e.g., polyethylene glycol, ethanol,
glycerol). Different buffer
species (e.g., acetate, citrate, citrate-phosphate) and levels of buffers were
investigated to
understand if there was any buffer-induced catalysis.
[0041] Formulations have been identified, for example, ready-to-use
bivalirudin formulations
having a pH of about 3.0 to less than 4.0, or from about 3.25 to about 3.75
acceptable for
pharmaceutical use, and ready-to-use bivalirudin formulations having a pH from
5.0 to around
5.7 acceptable for pharmaceutical use. In addition, various buffers and
concentrations of buffers
were also investigated, as disclosed below.
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Bivalirudin
100421 Bivalirudin can be synthesized by methods that include, but are not
limited to, solid-
phase peptide synthesis, solution-phase peptide synthesis, or a combination of
these techniques
(e.g., U.S. Patent No. 5,196,404, U.S. Patent Application 2007/0093423, and
other references
known to those of skill in the art). These citations are incorporated by
reference in their
entireties.
[0043] The bivalirudin of the ready-to-use compositions described herein may
be the peptide
encoded by SEQ ID NO: 1, or salts thereof. In particular embodiments, the
trifluoroacetate salt
may be employed. The bivalirudin concentrations disclosed herein is the actual
bivalirudin (free
base) content. Bivalirudin may be present in amounts ranging from about 0.01
mg/mL, to about
100 mg/mL, or from about 0.05 to about 50 mg/mL, or between about 0.1 mg/mL
and about 25
mg/m or between about 1.0 mg/mL and about 10 mg/mL, or between about 2.5 mg/mL
and
about 7.5 mg/mL, such as 5.0 mg/mL.
Tonicity-adjusting and Stabilizing Agents
[0044] Tonicity-adjusting agents, as used herein, are excipients which adjust
the tonicity of the
liquid ready-to-use bivalirudin compositions to a desired isotonic range. For
physiologically
acceptable compositions, "isotonic" can include compositions which have
similar tonicities to
human serum. An acceptable range would be approximately 200 to 600 mOsm/kg.
Stabilizers
may also be advantageously in the liquid ready-to-use bivalirudin compositions
described herein.
Particular materials introduced in the disclosed formulations may function as
both a tonicity
agent and a stabilizer. In such instances, such materials may be used at
concentrations higher
than needed for tonicity if their primary purpose is stabilization, or may be
used at concentrations
higher than needed for stabilization if their primary purpose is tonicity
adjustment. Tonicity-
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adjusting agents and/or stabilizing agents for use in the presently disclosed
compositions can
include, but are not limited to, for example, pharmaceutically acceptable
inorganic chlorides,
e.g., potassium chloride, sodium chloride, magnesium chloride or calcium
chloride, for example
0.9% NaCl; saccharides (monosaccharides, disaccharides, polysaccharids, etc.)
such as, e.g.,
mannitol (including D-mannitol), sorbitol, lactose, trehalose, raffinose,
dextrose, maltose,
galactose, sucrose, and polysucrose, for example, 5% D-mannitol; polymers such
as
polygalacturonic acid, polyethylene glycol (PEG), polyvinylpyrrolidine (PVP),
for example,PEG
300, PEG 400, PEG 3350, PEG 6000, PEG 8000 and the like, for example 10% w/v
PEG 400;
amino acids such as lysine, arginine, glycine, methionine, and other amino
acids; and other
materials such as dextran, cyclodextrins (for example,
hydroxypropylmcyclodextrin, Ficoll,
galacturonic acid, and other similar excipients and combinations of these
agents. These agents
may be present in the compositions in amounts of from about 0.25% to about 15%
percent by
weight of the formulation. Specific stabilizers such as proline and
polyethylene glycol can also
be used to reduce degradation rates.
[0045] Further stabilizing excipients which may be employed include: poly
alcohols, including
glycerol, propylene glycol, and others; dielectric strength modifiers,
including ethanol,
polyethylene glycol, propylene glycol, glycerol and others; amino acids,
including for example,
proline, glycine, histidine, methionine and others. Furthermore, as the
formulations disclosed
herein are not designed to undergo lyophilization, agents whose primary
function is
lyoprotection or cryoprotection are generally not employed.
Buffers
[0046] In embodiments disclosed herein, the pH of the ready-to-use bivalirudin
compositions
disclosed herein is in a range of about 3.0 to less than 4.0, or between about
3.25 and about 3.75,
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or in a range of from 3.3 to 3.7, or from 3.4 to 3.6. In other embodiments
described herein, the
pH of the final ready-to-use bivalirudin compositions disclosed herein is in a
range of 5.0 to
about 5.7, or in a range from about 5.1 to about 5.4, or from about 5.2 to
about 5.3. Buffers, as
used herein, are pharmaceutically acceptable reagents or compositions which
can contribute to
maintenance of the pH of the ready-to-use bivalirudin compositions to a
desired pH range, i.e.,
about 3.0 to less than 4.0, or from about 3.25 to about 3.75, or from 3.3 to
3.7 or from 3.4 to 3.6,
or from 5.0 to about 5.7, or from about 5.1 to about 5.4, or from 5.2 to 5.3.
[0047] As used herein, the term "buffer" means excipients having ionizable
groups with pKa
values around the target pH of the bivalirudin compositions, plus or minus
about one pH unit.
For formulations having a pH in a range of about 3.0 to less than 4.0, or
between about 3.25 and
about 3.75, or in a range of from 3.3 to 3.7, or from 3.4 to 3.6, a buffer
should have at least one
pKa of about 2.0 to about 5Ø For formulations having a pH from 5.0 to about
5.7, or from
about 5.1 to about 5.4, or from 5.2 to 5.3, a buffer should have at least one
pKa of about 4.0 to
about 5.57.
[0048] Such buffers which may be suitable for use in the ready-to-use
bivalirudin compositions
disclosed herein include, but are not limited to, for example in the pKa 2.0-
5.0 range: acetate,
acetoacetate, adipate, alginate, ascorbate, aspartate, benzoate, acrylate,
butenoate, glyoxylate,
butanoate, oxobutanoate, 2-, 3- or 4-chlorobutanoate, lactobionate, succinate,
a-lipoic acid,
maleate, bromoacetate, chloroacetate, cyanoacetate, 2- or 3-chloropropionate,
citrate, citrate-
phosphate, bicarbonate, tartrate, glycylglycine, formate, fumarate, glycerate,
glycine, crotonate,
lactate, malate, malonate, methylmalonate, melaminate, oxaloacetate, oxalate,
propionate,
methylpropionate, and 3- or 4-hydroxypropionate, pyridine, piperazine,
phosphate,
pyrophosphate, pyruvate, phthalate, histidine, (bis(2-hydroxyethyl)-imino-
tris(hydroxymethyl)-
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methane) ("bis-TRIS"), trimethylamine oxide, bicarbonate, and other buffers
with pKa in this
range, and their various salts and anhydrous or hydrated forms, or some
combination of such
buffers.
[0049] Such buffers which may be suitable for use in the ready-to-use
bivalirudin compositions
disclosed herein include, but are not limited to, for example in the pKa 4-6.7
range: acetate,
oxalate, acrylate, ascorbate, benzoate, caprate, caproate, caprylate,
cinnamate, fumarate, maleate,
phosphate, orthophosphate, laurate, palmitate, propionate, adipate,
cacodylate, malonate,
propionate, hydroxypropionate, fumarate, phthalate, maleate, 3- or 4-
hydroxybutanoate,
butenoate, crotonate, methylmalonate, succinate, malate, tartrate, citrate, 2-
(N-
morpholino)ethanesulfonic acid ("MES"), and other buffers with pKa in this
range, and their
various salts and anhydrous or hydrated form or some combination of such
buffers. These
buffers may be present in the compositions in amounts of about 1 mM to about
500 mM, or
about 5 mM to about 300 mM. An acetate buffer such as sodium acetate (in its
anhydrous or
hydrated form, e.g., sodium acetate trihydrate) may be used for the higher pH
(5.0 to about 5.7)
formulations disclosed herein.
pH-Adjusting Agents
[0050] The pH of particular embodiments of the final ready-to-use bivalirudin
compositions
disclosed herein is in a range of about 3.0 to less than 4.0, or between about
3.25 and about 3.75,
or from 3.3 to 3.7 or from 3.4 to 3.6. The pH of other particular embodiments
of the final ready-
to-use bivalirudin compositions disclosed herein is in a range of 5.0 to about
5.7, or from about
5.1 to about 5.4, or from 5.2 to 5.3. The ready-to-use bivalirudin
compositions described herein
may employ pH-adjusting agents to adjust the pH of the compositions described
herein to a
target range or value. For example, during the preparation of a ready-to-use
bivalirudin
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composition disclosed herein, bivalirudin may be added to a buffering agent,
with an associated
change in pH of the buffer solution. pH-adjusting agents could be added to
achieve a desired
pH. e.g., about 3.0 to less than 4.0, or from about 3.25 to about 3.75, or
from 3.3 to 3.7, or from
3.4 to 3.6, or from greater than or equal to 5.0 to about 5.7, or from about
5.1 to about 5.4, or
from 5.2 to 5.3. The pH-adjusting agents can include pharmaceutically
acceptable acids, bases
or buffering agents. The pharmaceutically acceptable acids could include, but
are not limited to,
for example, one or more inorganic mineral acids such as hydrochloric,
sulfuric, phosphoric,
nitric and the like; or one or more organic acids, e.g., acetic, succinic,
tartaric ascorbic, citric,
glutamic, benzoic, methanesulphonic, ethanesulphonic, trifluoroacetic and the
like.
100511 The pharmaceutically acceptable bases may be one or more inorganic or
organic bases
(e.g., sodium acetate or the like), including but not limited to, for example,
alkaline carbonate
(e.g., calcium carbonate, sodium carbonate or the like), alkaline bicarbonate
(e.g., sodium
bicarbonate or the like), alkaline earth metal carbonate, alkaline hydroxide
(e.g., lithium
hydroxide, potassium hydroxide, cesium hydroxide, sodium hydroxide or the
like), alkaline earth
metal hydroxide or amine. Salts of pH-adjusting agents may also be used. In
particular
formulations having pH in the range of about 3.0 to less than 4.0, sodium
hydroxide and/or
hydrochloric acid solutions were used to adjust the pH to the desired value,
as needed. In other
particular formulations, for example those having pH in the range of 5.0to
about 5.7, glacial
acetic acid and/or sodium hydroxide solution were used to adjust the pH to the
desired value, as
needed.
Methods of Making Ready-to-Use Bivalirudin Compositions
[0052] The ready-to-use liquid bivalirudin compositions can be prepared by
methods described
herein. The methods comprise mixing one or more tonicity-adjusting and/or
stabilizing agents
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and one or more buffers with bivalirudin to form the ready-to-use bivalirudin
compositions.
Optionally, the methods can also comprise providing pH-adjusting agents to
such mixtures, to
adjust the pH of the ready-to-use bivalirudin compositions if the pH is not
within a desired pH
range, e.g., about 3.0 to less than 4.0, or from about 3.25 to about 3.75, or
from 3.3 to 3.7, or
from 3.4 to 3.6, or from greater than or equal to 5.0 to about 5.7, or from
about 5.1 to about 5.4,
or from 5.2 to 5.3.
[0053] The one or more tonicity-adjusting and/or stabilizing agents can first
be dissolved in
aqueous media prior to mixing with bivalirudin. The tonicity-adjusting and/or
stabilizing agents
can comprise any of the materials discussed herein or known to the person of
ordinary skill in the
art. The one or more tonicity-adjusting and/or stabilizing agents and/or
buffer may be dissolved
in aqueous media, optionally containing one or more buffers, by methods known
in the art. For
example, the tonicity-adjusting and/or stabilizing agents may be dissolved by
adding each agent,
and optionally one or more buffers, to aqueous media (optionally containing
one or more
buffers), by adding the agents, and optionally one or more buffers, to each
other and then mixing
them with aqueous media (optionally containing one or more buffers), by mixing
the one or more
tonicity-adjusting and/or stabilizing agents, and optionally one or more
buffers, and aqueous
media (optionally containing one or more buffers) in a common vessel, or a
combination thereof.
[0054] The one or more tonicity-adjusting and/or stabilizing agents, and
optionally one or more
buffers, can be added simultaneously, individually in a particular order,
individually in no
particular order, all at once or in a particular sequence, including partial
additions of portions of
either or both types of components, followed by continued additions of either
or both types of
components until the desired amounts of components are added, or some
combination thereof.
Reasons for adding tonicity-adjusting and/or stabilizing agents, and
optionally one or more
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buffers, in a given order may include, but are not limited to, preventing some
reaction between
tonicity-adjusting and/or stabilizing agents and/or one or more buffers, which
might be mixed
together directly, stimulating a reaction that may occur when tonicity-
adjusting and/or stabilizing
agents and/or one or more buffers are mixed directly together, maintaining a
given pH,
maintaining a given tonicity, and ease of handing.
100551 The aqueous media can be introduced to a suitable vessel at an elevated
temperature, for
example from about 35 C to about 90 C, or from about 50 C to about 85 C, or
from about 60 C
to about 85 C, for example, about 80 C. The aqueous media can then optionally
be brought to
and held at a temperature, for example, of about 2 C to about 35 C, or from
about 10 C to about
35 C, or from about 15 C to about 30 C, or from about 20 C to about 25 C, for
the mixing
processes described herein.
100561 The one or more tonicity-adjusting and/or stabilizing agents and/or the
one or more
buffers can be dissolved or made miscible in aqueous media, optionally
containing one or more
buffers, using mixing technologies and devices known to those of skill in the
art. Examples of
mixing devices may include, but are not limited to, paddle mixers, magnetic
stirring devices,
shakers, re-circulating pumps, homogenizers, and combinations thereof. The
mixing speeds of
any of the mixing steps can be selected from, for example, not less than 50
rpm, or not less than
100 rpm, or not less than 250 rpm, or for example, not less than 200 rpm, for
minimum times of
at least 5 minutes, at least 10 minutes at least 15 minutes, at least 30
minutes, or at least 45
minutes. The dissolution of one or more tonicity-adjusting and/or stabilizing
agents and/or the
one or more buffers may occur under controlled conditions, for example, under
temperature
control or atmospheric control (e.g., under nitrogen or with a particular
humidity) through means
known in the art.
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[0057] Thereafter, bivalirudin (in solution or as a solid, for example, a
lyophilized material) may
be mixed with the dissolved tonicity-adjusting and/or stabilizing agents,
optionally containing
one or more buffers, again using methods known in the art. Bivalirudin can be
added to the
dissolved tonicity-adjusting and/or stabilizing agents and/or the one or more
optional buffers
rapidly or slowly, all at once or in portions, in a constant rate or at
variable rates or a
combination of all these techniques. The bivalirudin can be combined with the
one or more
tonicity-adjusting and/or stabilizing agents and/or the one or more optional
buffers using mixing
technologies and devices known to those of skill in the art. Examples of
mixing devices may
include, but are not limited to, paddle mixers, magnetic stirring devices,
shakers, re-circulating
pumps, homogenizers, and combinations thereof. Mixing techniques can include
those classified
as "efficient," for example those described in U.S. Patent Nos. 7,582,727,
7,598,343, and others.
The dissolution of bivalirudin with the one or more tonicity-adjusting and/or
stabilizing agents
and/or the one or more optional buffers may occur under controlled conditions,
for example,
under temperature control or atmospheric control (e.g., under nitrogen and/or
with a particular
humidity) through means known in the art.
[0058] The bivalirudin could already be dissolved in aqueous medium
(optionally containing one
or more buffers) when it is mixed with the tonicity-adjusting and/or
stabilizing agents and/or one
or more optional buffers. On the other hand, the bivalirudin could be mixed in
its solid form (for
example, a lyophilized form) with the dissolved tonicity-adjusting and/or
stabilizing agents
and/or one or more optional buffers. Alternatively, the bivalirudin can be
mixed with one or
more tonicity-adjusting and/or stabilizing agents prior to dissolution in
aqueous media
(optionally containing one or more buffers), simultaneously with dissolution
in aqueous media
(optionally containing one or more buffers), or a combination thereof. The use
of a lyophilized
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form of bivalirudin in such formulations does not mean that the formulation
itself has been
lyophilized. As set forth herein, the bivalirudin formulations, but not
necessarily the active
pharmaceutical ingredient, are prepared without having been lyophilized at any
stage.
100591 Resulting solutions comprising the dissolved bivalirudin, tonicity-
adjusting and/or
stabilizing agents and optional buffers are ready-to-use bivalirudin
compositions as described
herein. In particular embodiments of the disclosure herein, a desired pH range
for some
embodiments of the liquid ready-to-use bivalirudin compositions is about 3.0
to less than 4.0, or
about 3.25 to about 3.75, or 3.3 to 3.7, or 3.4 to 3.6, and another desired pH
range for other
embodiments of the liquid ready-to-use bivalirudin compositions is greater
than or equal to 5.0 to
about 5.7, or about 5.1 to about 5.4, or 5.2 to 5.3. If the pH of the ready-to-
use bivalirudin
compositions is not in the respective desired range after performing the
methods described
above, one or more pH-adjusting agents can be added to the compositions to
provide a pH in the
desired range. Further, if a specific concentration of bivalirudin is desired
in the ready-to-use
bivalirudin compositions, the concentration can be adjusted, for example, by
addition of aqueous
media to the compositions. Some liquid ready-to-use bivalirudin concentrations
of the
formulations described herein can be 125 mg/25 mL, or 250 mg/50 mL, for
example.
100601 After the ready-to-use bivalirudin compositions are prepared, they can
be sterilized as
desired, using any suitable techniques known in the art. For example, the
aqueous composition
can be made to undergo aseptic filtration using, for example, a suitable
filter such as a 0.2 gm
membrane filter. Sterilization may also or alternatively involve a freeze-thaw
cycle to eliminate
any residual vegetative bacteria.
100611 The ready-to-use liquid bivalirudin compositions can be placed in
containers having a
sterile access port for piercing by hypodermic injection needles, for example,
an intravenous
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solution bag, bottle, stoppered vial, ampoule, pre-filled sterile syringe,
together with instructions
for administration. Also, for example, the formulations can be filled into so-
called "piggy back"
vials that can be pierced directly with the universal port of an IV
administration set and hung to
administer with no preparation steps.
Methods of Using Ready-to-Use Bivalirudin Compositions
[0062] The ready-to-use liquid bivalirudin compositions can be useful in
methods of treating a
patient in need of administration of such bivalirudin compositions. The
methods comprise
administration of a ready-to-use bivalirudin composition comprising
bivalirudin and one or more
tonicity-adjusting and/or stabilizing agents and any optional buffer. The
ready-to-use bivalirudin
can be any ready-to-use bivalirudin composition described herein.
[0063] The ready-to-use bivalirudin composition can be an injectable dosage
form, and they can
be delivered to a patient parenterally. Methods of administering ready-to-use
bivalirudin
compositions parenterally are known in the art. For example, an aqueous
composition can be
delivered intravenously.
[0064] The aqueous composition may be an intravenous bolus dose of between
about 0.25 mg/kg
and about 1.5 mg/kg, or between about 0.5 mg/kg to about 1 mg/kg, or about
0.75 mg/kg. This
may be followed by an infusion of between about 1.25 mg/kg/h and about 2.25
mg/kg/h, or about
1.75 mg/kg/h for the duration of the procedure or treatment protocol. Five
minutes after the bolus
dose is administered, an additional bolus of between about 0.1 mg/kg and about
1 mg/kg, or
about 0.3 mg/kg, may be given if needed.
[0065] The ready-to-use bivalirudin compositions disclosed herein may be
indicated for use as
an anticoagulant, for example, for patients with unstable angina, for example,
those patients with
unstable angina undergoing percutaneous transluminal coronary angioplasty
(PTCA). Such
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compositions may also be indicated for patients that may have, or be at risk
of having, heparin-
induced thrombocytopenia (HIT) and/or heparin-induced thrombocytopenia and
thrombosis
syndrome (HITTS) undergoing percutaneous coronary intervention (PCI). Such
compositions
may also be indicated for patients undergoing PCI with provisional use of
glycoprotein 11b/Illa
(GPI). The ready-to-use bivalirudin compositions disclosed herein may also be
used for the
prevention and/or treatment of venous thromboembolic diseases.
[0066] The ready-to-use bivalirudin compositions described herein can be
administered with
other drug products such as glycoprotein IIb/IIIa inhibitor (see, e.g., Allie
et al., Vasc. Dis.
Manag., 3 (2006) 368-75). Alternatively, the compositions disclosed herein may
be used in
combination with blood thinners, including but not limited to, for example,
coumadin, warfarin
or aspirin.
EXAMPLES
[0067] The examples disclosed herein illustrate some of the embodiments of the
present
disclosure in greater detail, and do not limit the scope of any claims made
herein.
Example 1 ¨ Bivalirudin Assay of the Listed Dru2 (Angiomax )
10068] To understand the impact of the drug preparation procedure on the
actual concentration
of bivalirudin in diluted Angiomax solutions, three lots of the listed drug,
Angiomax were each
prepared by each of the following two methods
A. following the exact procedure specified in the package insert; namely,
withdrawing 5 mL
of the diluent (0.9% Sodium Chloride Injection) from a 50 mL IV bag of the
diluent,
before transferring the entire contents of the reconstituted vial to the bag;
or
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B. adding the contents of the reconstituted vial to accurately measured 45 mL
of diluent
(0.9% Sodium Chloride Injection),
[0069] In both cases, each vial of Angiomax was first reconstituted in 5 mL
of Sterile Water
for Injection as per the package insert, prior to dilution as described above.
The data are
summarized in Table 1.
Table 1: Assay of Angiomax (bivalirudin) for Injection Admixture as a
Function of
Preparation Method
Preparation Method Angiomax (bivalirudin) for Injection, Assay
( /0)
(Nominal =5 mg/mL)
Lot 00103 Lot 00111
Lot 00106
Prepared exactly as per PI 88.6 92.2 88.0 89.6
Prepared in exactly 45 mL of Diluent 98.6 102.8 102.0
102.2
100701 The results show that the bivalirudin concentrations of three
representative lots, when
prepared as per the Angiomax package insert, are consistently below the
nominal concentration
of 5 mg/mL by about 8-12%, thus resulting in significant under-dosing of
patients, resulting in
acute stent thrombosis and/or adverse effects. This is believed to be a direct
result of the overfill
volume in the diluent bag because vials prepared by diluting the content of
the reconstituted vial
in 45 mL of diluent are consistently at the nominal concentration. The liquid
ready-to-use
products described and claimed herein are ready-to-use and supplied at a 5
mg/mL concentration
and require no dilution or additional preparation for use in the PCI
procedure.
Example 2 ¨Stability Testing of Disclosed Bivalirudin Formulations
100711 Exemplary compositions according to the present disclosure were tested
for storage
stability. Bivalirudin was used in 2.5 mg/mL or 5 mg/mL concentrations. For
each exemplary
composition, initial levels of the various degradants were measured, and
samples were generally
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subjected to stability testing at 40 C/75% RH, 2-8 C, and/or 25 C/60% RH.
Table 2 lists
exemplary liquid ready-to-use bivalirudin formulations, sorted by target pH
Table 2. Liquid Bivalirudin Formulations, Sorted by pH
Form. Target Buffer Stabilizer/Tonicity agent
pH
A 2.5 3.8 mg/mL glycine None
2.5 5.25 mg/mL citric acid None
monohydrate/ 2.68
mg/mL dibasic sodium
phosphate
3.0 3.8 mg/mL glycine None
3.0 5.25 mg/mL citric acid None
monohydrate/ 2.68
mg/mL dibasic sodium
phosphate
3.0 0.8 mg/mL glycine 9 mg/mL NaCl
3.0 14.7 mg/mL sodium 9 mg/mL NaCl
citrate dihydrate
3.0 3.8 mg/mL glycine 9 mg/mL NaC1
3.25 3.8 mg/mL glycine 98 mg/mL NaC1
3.5 None 3.8 mg/mL glycine
3.5 5.25 mg/mL citric acid None
monohydrate/ 2.68
mg/mL disbasic
sodium phosphate
3.5 None 9 mg/mL NaCl/3.8 mg/mL
glycine
3.5 None 50 mg/mL mannito1/3.8
mg/mL glycine
3.5 None 9 mg/mL NaC1/3.8 mg/mL
glycine
3.5 None 9 mg/mL NaCl/0.8 mg/mL
glycine
0 3.5 None 100 mg/mL sucrose/3.8
mg/mL glycine
3.5 14.7 mg/mL sodium 9 mg/mL NaC1
citrate dihydrate
3.5 None 6.8 mg/mL sodium acetate
trihydrate/ 9 mg/mL NaCl
3.5 None 150 mg/mL sorbito1/3.8
mg/mL glycine
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Form. Target Buffer Stabilizer/Tonicity agent
PH
3.5 None 22.5 mg/mL glycine
3.5 None 100 mg/mL PEG 400/3.8
mg/mL glycine
3.5 None 150 mg/mL sorbitol;
100 mg/mL PEG 400/3.8
mg/mL glycine
V 3.5 None 28.5 mg/mL L-proline/3.8
mg/mL glycine
3.5 None 75 mg/mL PEG 400/3.8
mg/mL glycine
X 3.5 None 100 mg/mL PEG 400/0.8
mg/mL glycine
3.6 None 20 mg/mL sucrose;
40 mg/mL mannito1/0.8
mg/mL glycine
3.6 None 28.5 mg/mL L-proline
AA 3.6 None 100 mg/mL PEG 400/0.8
mg/mL glycine
BB 3.75 None 9 mg/mL NaCl/3.8 mg/mL
glycine
PP 5.0 0.8 mg/mL sodium 100 mg/mL PEG 400
acetate trihydrate
EE 5.1 3.8 mg/mL sodium 75 mg/mL PEG 400
acetate trihydrate
FF 5.3 3.8 mg/mL sodium 25 mg/mL glycerol
acetate trihydrate
GG 5.3 0.8 mg/mL acetate 100 mg/mL PEG 400
HH 5.25 3.8 mg/mL acetate 75 mg/mL PEG 400
II 5.25 3.8 mg/mL sodium 25 mg/mL glycerol;
acetate trihydrate alcohol
JJ 5.25 3.8 mg/mL sodium 100 mg/mL sucrose
acetate triydrate
KK 5.25 0.8 mg/mL sodium 100 mg/mL PEG 400
acetate trihydrate
QQ 5.5 0.8 mg/mL acetate 100 mg/mL PEG 400
MM 5.75 28 mM acetate 7.5% PEG 400
00 3.6 none 28.5 mg/mL L-proline
*Active is 2.5 mg/mL, rather than 5 mg/mL
[0072] The levels of degradants in the bivalirudin compositions including the
impurity at relative
retention time (RRT) of about 0.49, [12-20]-bivalirudin, [3-20]-bivalirudin,
Asp9-bivalirudin, [9-
10]-cycloimido bivalirudin, [11-12]-cycloimido bivalirudin, and total
impurities were measured
28
CA 3054834 2019-09-10
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at various time points as indicated. Various pH values were investigated, with
a focus on the pH
values between about 3.0 to less than 4.0, or 3.25 and 3.75, and 5.10 and
5.70, which the prior art
teaches away from (see U.S. Patent No. 7,803,762, Table 1). Additionally,
various buffers,
buffer strengths, stabilizers, amino acids and other tonicity-adjusting and/or
stabilizing agents
were used for approximately 50 individual formulations (not all data shown).
For purposes of
testing, stability determinations at 40 C/75% relative humidity (RH) for three
days or 25 C/60%
RH for 1 month were considered roughly equivalent and predictive of stability
at 5 C for 1 year,
based on Arrhenius kinetics and the activation energies of the degradation
pathways involved (Ea
of about 20 to 25 kcal/mol).
100731 For pH values in the range of about 3.0 to less than 4.0, or about 3.25
to about 3.75, and
pH values in the range of 5.0 to about 5.7, representative degradant levels
are expressed as A) in
tables below. The impurities are quantitatively determined by HPLC using very
similar
experimental conditions outlined in Example 21 of US Patent 7,803,762;
however, the
calculation of the percentage of each impurity was performed against a
bivalirudin standard
instead of as percent area under the curve (AUC). Conditions for the HPLC
chromatographic
analysis are as follows:
Method Parameter Condition/ Criteria
0.05 M Sodium Acetate solution in water adjusted to pH 6.5 0.1 with
Solution A
dilute (2M) acetic acid.
Solution B Solution A : Acetonitrile 50:50
Diluent Water
Standard concentration 0.05 mg/mL Bivalirudin
Sample concentration 2.5 mg/mL Bivalirudin
Column Vydac C18 (250 X 4.6mm), 5jim; or equivalent
Flow rate 1.2 mL/min
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Method Parameter Condition/ Criteria
Gradient program
Time(min) Solution - A Solution ¨B
(%v/v) (%v/v)
0.01 90 10
Gradient Program 5 85 15
30 65 35
35 65 35
35.01 90 10
40 90 10
Injection volume 40 pi,
Wavelength 215 nm
Run time 40 minutes
Column oven temperature 40 C
Autosampler temperature 5 C
100741 Degradants are abbreviated as follows, as will be understood by one of
skill in the art,
and with reference to this disclosure: "RRT 0.49" is the impurity at relative
retention time of
about 0.49, a bivalirudin fragment, previously identified in U.S. Patent No.
7,803,762 as [1-11]-
bivalirudin (SEQ ID NO:4), but identified instead as [5-20]-bivalirudin (SEQ
ID NO:6) in some
exemplary formulations of the present disclosure; 112-20] BVN" is the
bivalirudin fragment
comprising residues 12-20 (SEQ ID NO:5); 13-20] BVN" is the bivalirudin
fragment
comprising residues 3-20 (SEQ ID NO;2); "Asp9-BVN" is the bivalirudin
degradant having
aspartic acid as residue 9 (SEQ ID NO:3); "[9-10] BVN" is the 9-10 cycloimido
bivalirudin
intermediate, and "[11-12] BVN" is the 11-12 cycloimido bivalirudin
intermediate (see U.S.
CA 3054834 2019-09-10
Attorney Docket: 09423.020W01
Patent No. 7,803,762). According to the HPLC protocol detailed above, the
relative retention
times of degradants were observed in Table 3 as follows:
Table 3. Bivalirudin Impurities Observed at Approximate Relative Retention
Times
Impurity Approximate RRT
(within +0.02 units)
[12-20] BVN 0.55
[3-20] BVN 0.62
Asp9-BVN 0.91
[9-10] BVN 1.08
[11-12] BVN 1.36
Example 3: Stability Testin2 at 40 C/75% RH
[0075] Table 4 displays stability data of exemplary formulations of this
invention at 40 C and
75% relative humidity after three days or seven days. Initial values (t=0)
determined at the time
of manufacture are listed above the 3-day and 7-day data. Notably, the data
show a suppression
of the known and unknown impurities in the formulations having a pH in the
range of about 3.0
to less than 4.0, or from about 3.25 to about 3.75, and at pH in the range of
from above or equal
to 5.0 to about 5.7. As shown in Table 4, the levels of degradants after
storage for three days at
40 C in the exemplary formulations with pH of about 3.0 to less than 4.0, or
about 3.25 to about
3.75 pH range and the greater than or equal to 5.0 to about 5.7 pH range are
surprisingly and
significantly lower than those reported in U.S. Patent No. 7,803,762, Tables
38-57, pH of 4 to
less than 5 (where for example total impurities ranged from about 6.5% to 7.7%
after three days
at 40 C). As also shown in Table 4, the levels of degradants after storage for
seven days at 40 C
in the exemplary formulations with pH of about 3.0 to less than 4.0, or about
3.25 to about 3.75
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pH range and the greater than or equal to 5 to about 5.7 pH range are
surprisingly lower than
those reported in U.S. Patent No. 7,803,762, Tables 38-57, pH of 4 to less
than 5 (where for
example total impurities ranged from about 12.7% to 14.3% after seven days at
40 C). Multiple
data sets for a given formulation in all tables below (i.e., GI, G2, HI, H2, K
I, K2, K3, Si and
S2) were based on independent analysis of separate batches of the same
formulation.
Table 4 - Stability Testing of Exemplary Formulations Performed at 40 C
Formulation Time RRT 1.12-201- 13-201- Asp9- 19-101- 111-121-
Total
(pH) Point 0.49 BVN BVN BVN BVN BVN
Initial 0.02 0.06 0.01 0.04 0.01 0.00
0.25
A
3 days 1.37 2.25 0.18 0.64 0.17 0.39
5.65
(pH 2.5)
7 days 2.90 5.41 0.38 1.30 0.38 0.85
12.98
Initial 0.01 0.05 0.01 0.03 0.01 0.00
0.16
B
3 days 1.34 2.07 0.27 0.37 0.15 0.55
5.18
(pH 2.5)
7 days 2.83 5.14 0.58 0.69 0.36 1.21
12.08
Initial 0.01 0.05 0.01 0.03 0.01 0.00
0.26
C
3 days 1.19 1.96 0.23 0.20 0.16 0.63
4.72
(pH 3.0)
7 days 2.62 4.64 0.51 0.35 0.35 1.43
10.72
Initial 0.01 0.04 0.01 0.03 0.01 '
0.00 0.17
D
3 days 1.21 1.85 0.48 0.28 0.18 0.88
5.50
(pH 3.0)
7 days 2.56 4.72 0.99 0.47 0.40 1.88
12.35
Initial 0.01 0.04 0.01 0.02 0.05 0.01
0.23
E
3 days 1.46 2.51 0.25 0.32 0.22 0.69
6.06
(pH 3.0)
7 days 3.06 4.67 0.43 0.49 0.37 1.20
10.72
Initial 0.01 0.03 0.01 0.03 0.06 0.02
0.24
F
3 days 1.45 2.40 0.65 0.36 0.23 1.06
7.39
(pH 3.0)
7 days 2.47 4.63 1.05 0.60 0.40 1.80
12.97
Initial 0.03 0.04 0.02 0.03 0.07 0.02
0.37
G1
_
3 days 1.24 1.70 0.19 0.33 0.13 0.43
4.42
(pH 3.0)
7 days 2.32 3.73 0.36 0.58 0.28 0.93
9.12
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Formulation Time RRT 112-201- 13-201- Asp9- 19-101- 111-121-
Total
(pH) Point 0.49 BVN BVN B'VN BVN BVN
Initial 0.05 0.10 0.02 0.07 0.05 0.01
0.44
G2
3 days 1.16 1.67 0.22 0.28 0.16 0.55
4.21
(pH 3.0)
7 days 2.17 3.58 0.41 0.40 0.30 1.16
8.76
Initial 0.01 0.05 0.02 0.04 0.07 0.03
0.27
H2
3 days 0.86 1.46 0.23 0.12 0.16 0.72
3.87
(pH 3.25)
7 days 2.04 3.42 0.53 0.22 0.33 1.62
9.14
Initial 0.03 0.04 0.02 0.03 0.07 0.01
0.33
H1
3 days 1.07 1.58 0.22 0.21 0.16 0.56
4.12
(pH 3.25)
7 days 2.09 3.42 0.42 0.31 0.29 1.24
8.41
Initial 0.01 0.05 0.02 0.03 0.01 0.03
0.30
I
3 days 0.88 1.63 0.33 0.11 0.22 0.95
4.45
(pH 3.5)
7 days 2.02 3.94 0.73 0.17 0.44 2.13
10.38
Initial 0.01 0.04 0.01 0.03 0.01 0.00
0.16
J
3 days 1.14 1.76 0.46 0.20 0.10 0.92
5.09
(pH 3.5)
7 days 1.45 4.41 0.95 0.35 0.42 . 2.04
11.73
Initial 0.01 0.04 0.02 0.02 0.06 0.02
0.25
K1
3 days 0.88 1.66 0.34 0.09 0.22 1.03
4.64
(pH 3.5)
7 days 2.00 3.84 0.74 0.15 0.45 2.24
10.28
Initial 0.03 0.04 0.02 0.03 0.06 0.02
0.33
K4
3 days 0.78 1.31 0.30 0.12 0.20 0.87
3.91
(pH 3.5)
7 days 1.53 2.76 0.59 0.14 0.37 1.85
7.77
Initial 0.04 0.10 0.02 0.07 0.04 0.01
0.44
K5
3 days 0.85 1.41 0.29 0.17 ' 0.20
0.84 3.96
(pH 3.5)
7 days 1.64 2.97 0.56 0.18 0.38 1.77
8.08
Initial 0.01 0.04 0.01 0.02 0.06 0.02
0.25
L
3 days 0.90 1.74 0.32 0.09 0.20 0.97
4.74
(pH 3.5)
7 days 2.06 4.00 0.69 0.15 0.41 2.09
10.55
Initial 0.01 0.04 0.01 0.02 0.07 0.02
0.25
M
3 days 0.88 1.67 0.33 0.09 0.21 1.01
4.61
(pH 3.5)
7 days 2.06 3.93 0.75 0.16 0.47 2.22
10.53
N Initial 0.01 0.04 0.01 0.02 0.05 0.02
0.25
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Formulation Time RRT 112-201- 13-201- Asp9- 19-101- 111-121-
Total
(pH) Point 0.49 BVN BVN BVN BVN BVN
(pH 3.5) 3 days 0.82 1.59 0.32 0.08 0.22 0.99
4.44
7 days 1.94 3.74 0.74 0.15 0.48 2.19
10.15
Initial 0.01 0.04 0.01 0.02 0.05 0.02
0.24
0
3 days 0.94 1.91 0.43 0.10 0.32 1.26
5.64
(pH 3.5)
7 days 1.75 3.44 0.76 0.12 0.44 2.20
9.87
Initial 0.01 0.04 0.01 0.02 0.06 0.02
0.24
P
3 days 1.15 1.97 0.81 0.13 0.29 1.48
6.78
(pH 3.5)
7 days 1.95 3.66 1.42 0.22 0.48 2.54
12.30
Initial 0.01 0.04 0.01 0.03 0.06 0.02
0.25
Q
3 days 0.85 1.73 1.12 0.09 0.33 1.82
6.80
(pH 3.5)
7 days 1.56 3.45 1.94 0.14 0.52 3.00
11.96
Initial 0.01 0.05 0.02 0.03 0.07 0.01
0.31
R
3 days 0.71 1.23 0.27 0.09 0.16 0.78
3.71
(pH 3.5)
7 days 1.42 2.66 0.54 0.13 0.31 1.70
7.70
S2 Initial 0.02 0.06 0.02 0.04 0.05 0.02
0.32
(pH 3.5) 7 days 2.04 3.81 1.00 0.18 0.52 2.64
11.07
Initial 0.01 0.04 0.02 0.03 0.07 0.01
0.27
S1
3 days 0.64 1.18 0.35 0.11 0.22 0.95
3.69
(pH 3.5)
7 days 1.26 2.56 0.69 0.11 0.40 2.04
7.63
Initial 0.05 0.13 0.02 0.04 0.06 0.12
0.57
T
3 days 0.56 1.11 0.30 0.08 0.19 0.87
3.57
(pH 3.5)
7 days 1.16 2.29 0.63 0.12 0.35 1.70
7.14
Initial 0.05 0.12 0.03 0.04 0.05 0.12
0.68
U
3 days 0.47 0.97 0.28 0.07 0.17 0.80
3.48
(pH 3.5)
7 days 1.00 2.04 0.59 0.11 0.31 1.62
6.93
V Initial 0.02 0.07 0.02 0.03 0.04 0.04
0.31
(pH 3.5) 7 days 1.72 3.56 1.03 0.17 0.54 2.75
10.75
W Initial 0.02 0.06 0.01 0.03 0.05 0.02
0.33
(pH 3.5) 7 days 1.71 3.41 0.90 0.14 0.48 2.31
9.98
BB Initial 0.03 ' 0.04 0.02 0.03 0.08 0.01
0.37
(pH 3.75) 3 days 0.58 1.13 0.40 0.09 0.25 1.08
3.81
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Formulation Time RRT 112-201- [3-20]- Asp9- 19-101- 111-121-
Total
(pH) Point 0.49 BVN BVN BVN BVN BVN
7 days 1.12 2.38 0.80 0.11 0.46 2.25
7.72
00 Initial 0.01 0.05 0.02 0.03 0.05 0.02 0.27
(pH 3.8) 7 days 1.00 2.81 1.28 0.15 0.60 2.96
9.93
EE Initial 0.00 0.04 0.03 0.03 0.04 0.02 0.28
(pH 5.10) 7 days 0.69 0.58 3.79 1.05 1.88 1.80
11.54
1111-1 Initial 0.00 0.03 0.03 0.03 0.05 0.02 0.31
(pH 5.25) 7 days 0.76 0.49 3.99 1.35 2.05 1.60
12.02
II Initial 0.00 0.04 0.04 0.04 0.06 0.02
0.34
(pH 5.25) 7 days 0.74 0.44 3.92 1.66 2.22 1.40
12.51
JJ Initial 0.01 0.04 0.04 0.04 0.06 0.02 0.33
(pH 5.25) 7 days 0.64 0.47 3.74 1.63 2.25 1.42
11.92
MM Initial 0.00 0.04 0.04 0.03 0.09 0.01 0.34
(pH 5.75) 7 days 1.01 0.17 4.54 4.52 3.01 0.64
16.35
Example 4: Stability Testing at 25 C
[0076] Table 5a displays stability data of exemplary formulations of this
invention at 25 C and
65% relative humidity after one month. Initial values (t=0) determined at the
time of
manufacture are listed above the 1-month data. As previously noted, stability
data after 1 month
at 25 C are roughly predictive of stability at 5 C after 12 months.
Table 5a - Stability Testing of Exemplary Formulations Performed at 25 C
Formulation Time RRT 112-201- 13-201- Asp9- 19-101- 111-121-
Total
(pH) Point 0.49 BVN BVN BVN BVN BVN
A Initial 0.02 0.06 0.01 0.04 0.01 0.00
0.25
(pH 2.5) 1 M 1.98 3.33 0.20 1.11 0.23 0.62 9.26
B Initial 0.01 0.05 0.01 0.03 0.01 0.00
0.16
_
CA 3054834 2019-09-10
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Formulation Time RRT 112-201- 13-201- Asp9- [9-101- 111-121-
Total
(pH) Point 0.49 BVN BVN BVN BVN BVN
(pH 2.5) 1 M 2.26 3.60 0.29 0.62 0.22 0.92
9.42
C Initial 0.01 0.05 0.01 0.03 0.01 0.00 0.26
(pH 3.0) 1 M 1.77 2.83 0.27 0.30 0.22 1.13
7.30
D Initial 0.01 0.04 0.01 0.03 0.01 0.00 0.17
_
(pH 3.0) 1 M 1.76 2.94 0.52 0.45 0.25 1.49
8.62
E Initial 0.01 0.04 0.01 0.02 0.05 0.01 0.23
_
(pH 3.0) 1 M 1.72 2.80 ' 0.24 0.48 0.20 0.91
7.01
-
F Initial 0.01 0.03 0.01 0.03 0.06 0.02 0.24
_
(pH 3.0) 1 M 1.71 2.94 0.55 0.56 0.26 1.45
8.63
G1 Initial 0.03 0.04 0.02 0.03 0.07 0.02 0.37
_
(pH 3.0) 1 M 1.82 2.99 0.24 0.61 0.21 0.87
7.58
G2 Initial 0.05 0.10 0.02 0.07 0.05 0.01 0.44
(p113.0) 1 M 1.68 2.77 0.27 0.46 0.24 1.07
7.14
HI Initial 0.03 0.04 0.02 0.03 0.07 0.01 0.33
-
(pH 3.25) 1 M 1.66 2.69 0.28 0.37 0.15 1.14
6.90
H2 Initial 0.01 0.05 0.02 0.04 0.07 0.03 0.27 .
(p113.25) 1 M 1.64 2.79 0.35 0.20 0.27 1.50
7.31
I Initial 0.01 0.05 0.02 0.03 0.01 0.03 0.30
_
(pH 3.5) 1 M ' 1.42 2.42 0.38 0.14 0.28
1.65 7.02
7
J Initial 0.01 0.04 0.01 ' 0.03 0.01 0.00
0.16
(pH 3.5) 1 M 1.69 2.73 0.50 0.33 0.25 1.57
8.12
K1 Initial 0.01 0.04 0.02 0.02 0.06 0.02 0.25
_
(pH 3.5) 1 M 1.30 2.25 0.38 0.16 0.28 1.62
6.40
K4 Initial 0.03 0.04 0.02 0.03 0.06 0.02 0.33
(pH 3.5) 1 M 1.20 2.19 0.39 0.16 0.29 1.69
6.35
KS Initial 0.04 0.10 0.02 0.07 0.04 0.01 0.44
(pH 3.5) 1 M 1.24 2.28 0.37 0.20 0.30 1.59
6.43
L Initial 0.01 0.04 0.01 0.02 0.06 0.02 0.25
(pH 3.5) 1 M 1.36 2.38 0.37 0.17 0.26 1.53
6.82
M Initial 0.01 0.04 0.01 0.02 0.07 0.02 0.25
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Formulation Time RRT 112-201- 13-201- Asp9- 19-101- 111-121-
Total
(pH) Point 0.49 BVN BVN BVN BVN BVN
(pH 3.5) 1 M 1.47 2.32 0.39 0.17 0.31 1.64
6.56
N Initial 0.01 0.04 0.01 0.02 0.05
0.02 0.25
(pH 3.5) 1 M 1.32 2.31 0.38 0.17 0.29 1.63
6.52
O Initial 0.01 0.04 0.01 0.02 0.05
0.02 0.24
(pH 3.5) 1 M 1.14 2.12 0.40 0.16 0.22 1.66
6.23
P Initial 0.01 0.04 0.01 0.02 0.06
0.02 0.24
(pH 3.5) 1 M 1.46 2.24 0.76 0.22 0.29 2.03
8.00
Q Initial 0.01 0.04 0.01 0.03 0.06 0.02
0.25
(pH 3.5) 1 M 1.13 2.05 1.00 0.19 0.32 2.36
7.67
R Initial 0.01 0.05 0.02 0.03 0.07 0.01
0.31
(pH 3.5) 1 M 1.08 2.02 0.34 0.14 0.23 1.49
6.34
SI Initial 0.01 0.04 0.02 0.03 0.07 0.01
0.27
(pH 3.5) 1 M 0.97 1.96 0.44 0.13 0.31 1.80
5.96
S2 Initial 0.02 0.06 0.02 0.04 0.05 0.02
0.32
(pH 3.5) 1 M 1.12 1.96 0.45 0.15 0.29 1.79
6.13
T Initial 0.05 0.13 ' 0.02 0.04 0.06 0.12
0.57
(pH 3.5) 1 M 0.92 1.83 0.42 0.10 0.28 1.57
5.63
U Initial 0.05 0.12 0.03 0.04 0.05
0.12 0.68
(pH 3.5) 1 M 0.78 1.55 0.37 0.10 0.23 1.45
5.47
/ Initial 0.02 0.07 0.02 0.03 0.04
0.04 0.31
(pH 3.5) 1 M 0.98 1.90 0.45 0.15 0.29 1.76
5.88
W Initial 0.02 0.06 0.01 0.03 0.05 0.02
0.33
(pH 3.5) 1 M 1.00 1.83 0.40 0.12 0.20 1.56
5.55
X Initial 0.03 0.09 0.02 0.07 0.06 0.05
0.43
(pH 3.5) 1 M (U) 0.97 1.83 0.47 0.13 0.30 1.72
5.93
1 M(I) 0.98 1.82 0.46 0.13 0.30 1.72
5.93
Y Initial 0.03 0.07 0.04 0.05 0.09 0.04
0.45
(pH 3.6) 1 M 0.81 1.64 0.44 0.10 0.30 1.80
5.69
Z Initial 0.02 0.08 0.03 0.05 0.08 0.08
0.42
(pH 3.6) 1 M 0.77 1.69 0.49 0.11 0.33 1.90
5.71
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Formulation Time FtRT 112-201- 13-201- Asp9- 19-101- 111-121-
Total
(PH) Point 0.49 BVN BVN BVN BVN BVN
AA Initial 0.00 0.04 0.02 0.05 0.12 0.01
0.36
(pH 3.6) 1 M 0.76 1.52 0.43 0.10 0.27 1.56
4.97
BB Initial 0.03 0.04 0.02 0.03 0.08 0.01
0.37
(pH 3.75) 1 M 0.89 1.87 0.53 0.12 0.36 2.04
6.26
00 Initial 0.01 0.05 0.02 0.03 0.05 0.02
0.27
(pH 3.8) 1 M 0.60 1.51 0.55 0.11 0.35 1.96
5.56
EE Initial 0.00 0.04 0.03 0.03 0.04 0.02
0.28
(pH 5.10) 1 M 0.14 0.32 1.69 0.46 1.07 1.28
5.57
I-111 Initial 0.00 0.03 0.03 0.03 0.05 0.02
0.31
(pH 5.25) 1 M 0.16 0.28 1.81 0.61 1.21 1.15
5.90
II Initial 0.00 0.04 0.04 0.04 0.06 0.02
0.34
(pH 5.25) 1 M 0.16 0.25 1.80 0.74 1.30 1.02
6.02
JJ Initial 0.01 0.04 0.04 0.04 0.06 0.02
0.33
(pH 5.25) 1 M 0.13 0.26 1.64 0.72 1.30 1.00
5.72
KK Initial 0.00 0.05 0.02 0.04 0.05 0.01
0.27
(pH 5.25) 1 M (U) 0.03 0.30 1.90 0.62 1.18 1.21
6.11
1 M (I) 0.03 0.32 1.99 0.65 1.23 1.26
6.36
FF Initial 0.00 0.04 0.06 0.05 0.09 ' 0.04
0.44
(pH 5.3) 1 M 0.14 0.23 1.75 0.97 1.47 0.89
6.18
GG Initial 0.00 0.05 0.03 0.06 0.05 0.02
0.30
(pH 5.3) 1 M 0.02 0.26 1.78 0.54 1.14 1.16
5.77
MM Initial 0.00 0.04 0.04 0.03 0.09 0.01
0.34
(pH 5.75) 1 M 0.21 0.11 2.15 2.11 1.97 0.52
7.90
100771 Notably, the 25 C stability data also show a suppression of the known
and unknown
impurities in the formulations having a pH in the range of about 3.0 to less
than 4.0, or from
about 3.25 to about 3.75, and at pH in the range of from above or equal to 5.0
to about 5.7. As
shown in Table 5a, the levels of degradants after storage for one month at 25
C in the exemplary
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formulations with pH of about 3.0 to less than 4.0, or about 3.25 to about
3.75 pH range and the
greater than or equal to 5.0 to about 5.7 pH range are surprisingly and
significantly lower than
those reported in U.S. Patent No. 7,803,762, Table 1, (where extensive
degradation ranging from
37.7% and 51.1% total impurities was observed at pH 3 to 4, respectively, and
complete
degradation was observed at both pH 5 and pH 6). As also shown in Table 5a,
the levels of
degradants after storage for 1 month at 25 C in many of the exemplary
formulations with pH of
about 3.0 to less than 4.0, or about 3.25 to about 3.75 pH range and the
greater than or equal to
5.0 to about 5.7 pH range are lower than those reported in U.S. Patent No.
7,803,762, for
formulations of pH of 4 to less than 5 (where for example total impurities
ranged from about
5.1% to 9.3% after 1 month at 25 C).
Example 5: Long Term Stability Testing at 2-8 C
[0078] The long-term stability testing under refrigerated conditions (5 C for
up to 18 months) is
presented in Table 5b. The standard data presentation is initial (time equal
zero) and, 3-, 6-, 9-,
12-, or 18-month data, presented in a row in each data cell. If other or
further months are
reported for sampling, this is indicated in the left column below the
formulation information.
The initial values (at time zero) are always presented first.
Table 5b - Stability Testing of Exemplary Formulations Performed at 5 C
Formulation RRT 112-201- 13-201- Asp9- 19-101- 111-121-
Time Point
Total
(pH) 0.49 BVN BVN BVN BVN BVN
A Initial 0.02 0.06 0.01 0.04 0.01 0.00
0.25
(pH 2.5) 3M 0.43 0.78 0.02 0.36 0.06 0.17
2.24
Initial 0.01 0.05 0.01 0.03 0.01 0.00
0.26
(pH 3.0) 3M 0.37 0.66 0.06 0.11 0.06 0.33
1.81
6 M 0.85 1.33 0.10 0.22 0.12 0.67
3.49
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Formulation RRT 112-201- 13-201- Asp9- [9-101- 11.1-121-
Time Point
Total
(pH) 0.49 BVN BVN BVN BVN BVN
9M 1.35 2.10 0.16 0.29 0.17 1.06 5.52
E Initial 0.01 0.04 0.01 0.02 0.05 0.01 0.23
(pH 3.0) 3 M ' 0.43 0.65 0.04 0.14 0.10
0.27 1.78
HI Initial 0.03 0.04 0.02 0.03 0.07 0.01 0.33
(pH 3.25) 3 M 0.39 0.67 0.06 0.13 0.07 0.38
1.99
H2 Initial 0.01 0.05 0.02 0.04 0.07 0.03 0.27
(pH 3.25) 3M 0.39 0.65 0.08 0.11 0.08 0.49
2.03
I Initial
' 0.01 0.05 0.02 0.03 0.01 0.03 0.30
(pH 3.5) 3M 0.32 0.59 0.08 0.06 0.08 0.49
1.80
6M 0.67 1.15 0.14 0.12 0.15 1.00 3.45
9M 1.04 1.73 0.21 0.14 0.21 1.52 5.07
K1 Initial 0.01 0.04 0.02 0.02 0.06 0.02
0.25
(pH 3.5) 3M 0.31 0.53 0.06 0.06 0.11 0.47
1.65
6M 0.59 1.00 0.14 0.09 0.21 0.93 3.23
9M 0.88 1.45 0.19 0.11 0.21 1.30 4.40
K4 ' Initial 0.03 0.04 0.02 0.03 0.06 0.02
0.33
(pH 3.5) 3M 0.31 0.56 0.09 0.07 0.09 0.56
1.95
6 M 0.04 0.56 0.15 0.09 0.14 1.03 3.18
L Initial 0.01 0.04 0.01 0.02 0.06
0.02 0.25
(pH 3.5) 3 M 0.33 0.58 0.08 0.06 0.09 0.52
1.92
6M 0.62 1.06 0.12 0.10 0.20 0.91 3.37
9M 0.91 1.50 0.17 0.11 0.15 1.26 4.72
12 M 1.24 2.37 0.27 0.21 0.23 1.90 7.16
M Initial 0.01 0.04 0.01 0.02 0.07 0.02
0.25
(pH 3.5) 6M 0.59 1.02 0.13 0.10 0.21 0.93
3.21
N Initial 0.01 0.04 0.01 0.02 0.05
0.02 0.25
(pH 3.5) 6M 0.58 1.00 0.13 0.09 0.22 0.91
3.20
12M 1.18 2.22 0.26 0.23 0.28 1.91 6.48
O Initial 0.01 0.04 0.01 0.02 0.05
0.02 0.24
(pH 3.5) 3M 0.27 0.49 0.06 0.04 0.07 0.51
1.56
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Formulation RRT 112-201- 13-201- Asp9- 19-101- 111-121-
Time Point Total
(PH) 0.49 BVN BVN BVN BVN BVN
6M 0.53 0.96 0.14 0.08 0.20 0.97 3.19
P Initial 0.01 0.04 0.01 0.02 0.06
0.02 0.24
(pH 3.5) 6M 0.61 0.79 0.23 0.13 0.25 1.21
3.69
12 M 1.32 2.08 0.55 0.28 0.30 2.42 7.67
Q Initial 0.01 0.04 0.01 0.03 0.06 0.02
0.25
(pH 3.5) 6M 0.58 1.03 0.39 0.11 0.17 1.44
3.96
12 M 1.12 1.87 0.75 0.17 0.30 2.69 7.26
R Initial 0.01 0.05 0.02 0.03 0.07 0.01
0.31
(pH 3.5) 6M 0.05 0.43 0.13 0.09 0.12 0.91
3.71
Si Initial 0.01 0.04 0.02 0.03 0.07
0.01 0.27
(pH 3.5)
3 M 0.37 0.66 0.09 0.07 0.09 0.59 2.07
S2 Initial 0.02 0.06 0.02 0.04 0.05
0.02 0.32
(pH 3.5) 3M 0.29 0.52 0.12 0.07 0.10 0.59
1.92
,
M 21 days 0.55 0.93 0.16 0.08 0.15 1.09
3.18
9M 1.03 1.72 0.26 0.18 0.25 1.78 6.17 '
_
T Initial 0.05 0.13 0.02 0.04 0.06
0.12 0.57
(pH 3.5) 3 M 0.25 0.45 0.09 0.07 0.09 0.52
1.66
6M 0.22 0.01 0.15 0.08 0.13 0.88 2.41
9M 0.69 1.25 0.23 0.10 0.19 1.42 4.21
U Initial 0.05 0.12 0.03 0.04 0.05
0.12 0.68
(pH 3.5) 3M 0.21 0.39 0.09 0.06 0.07 0.47
1.55
4 M 0.26 0.50 0.09 0.06 0.08 0.60 2.09
6M 0.15 0.66 0.13 0.07 0.10 0.79 2.69
9M 0.00 0.95 0.20 0.10 0.12 1.28 4.48
/ Initial 0.02 0.07 0.02 0.03 0.04
0.04 0.31
(pH 3.5) 3 M 0.29 0.58 0.12 0.08 0.10 0.62
1.95
5 M 21 days 0.55 1.04 0.15 0.05 0.12 1.14
3.25
,
9 M 0.76 1.54 0.26 0.18 0.24 1.75 5.03
W Initial 0.02 0.06 0.01 0.03 0.05
' 0.02 0.33
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Formulation RRT 112-201- 13-201- Asp9- [9-10]- [11-12]-
Time Point
Total
(pH) 0.49 BVN BVN BVN BVN BVN
(pH 3.5) 3M 0.30 0.52 0.10 0.07 0.09 0.55 2.05
M21 days 0.47 0.82 0.13 0.07 0.11 0.89 2.72
9M 0.76 1.47 0.23 0.18 0.21 1.54 4.69
12 M 0.85 1.46 0.26 0.14 0.20 0.79 4.09
15 M 1.26 2.18 0.36 0.16 0.31 2.25 7.06
18 M 0.08 2.76 0.47 0.17 0.37 2.72 8.68
X Initial 0.03 0.09 0.02 0.07 0.06 0.05 0.43
(pH 3.5) 3 M (U) 0.28 0.54 0.11 0.08 0.10 0.61
1.80
6 M (U) 0.47 0.87 0.18 0.09 0.15 1.02
3.04
9 M (U) 0.03 1.37 0.26 0.10 0.22 1.58
4.62
12 M(U) 0.06 1.84 0.34 0.15 0.30 2.05
6.26
3 M(l) 0.27 0.52 0.11 0.07 0.09 0.60
1.75
6 M (I) 0.47 0.90 0.18 0.09 0.15 1.04
3.10
9 M (I) 0.03 1.37 0.25 0.10 0.21 1.57
4.61
.
.
12 M (I) 0.06 1.82 0.34 0.15 0.29 2.04
6.24
Y Initial 0.03 0.07 0.04 0.05 0.09 0.04
0.45
(pH 3.6) 3M 0.22 0.47 0.11 0.11 0.11 0.63 1.83
Z Initial 0.02 0.08 0.03 0.05 0.08 0.08
0.42
(pH 3.6) 3M 0.20 0.50 0.12 0.09 0.11 0.67 1.81
AA Initial 0.00 ' 0.04 0.02 0.05 0.12
0.01 0.36
(pH 3.6) 3M 0.20 0.44 0.07 0.10 0.11 0.55 1.67
,
5 M 08 days 0.02 0.66 0.14 0.08 0.13 0.84
2.43
9M 0.66 1.19 0.24 0.11 0.22 1.45 4.17
12M 0.01 1.65 0.34 0.12 0.23 1.93 5.52
BB Initial 0.03 0.04 0.02 0.03 0.08 0.01
0.37
(pH 3.75) 3M 0.23 0.48 0.11 0.06 0.11 0.69 1.88
9 M 0.68 1.28 0.28 0.09 0.25 1.79 4.77
00 Initial 0.01 0.05 0.02 0.03 0.05 0.02
0.27
(pH 3.8) 3M 0.17 0.48 0.13 0.06 0.12 0.69 1.80
5 M21 days 0.33 0.74 0.19 0.06 0.22 1.17
3.01
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Formulation RRT 112-201- 13-201- Asp9- [9-10]- 111-121-
Time Point
Total
(pH) 0.49 BVN
BVN BVN BVN BVN
9 M 0.51 1.22 0.32 0.14 0.13 1.86
4.41
Initial - 0.05 BQL 0.04 0.04 BQL
0.12
PP
3 M - 0.15 0.30 0.07 0.27 0.60
1.43
(pH 5.0)
6M - 0.25 0.59 0.16 0.49 1.12
2.79
EE Initial 0.00 0.04 0.03 0.03 0.04 0.02
0.28
(pH 5.10) 3M 0.02 0.12 0.37 0.10 0.38 0.51
1.76
M 21 days 0.02 0.18 0.60 0.16 0.58 0.82
2.67
9M 0.04 0.27 1.00 0.39 0.82 1.19
4.07
12 M 0.03 0.28 1.09 0.47 0.75 0.55
4.09
M 0.05 0.39 1.46 0.72 1.03 1.46
5.77
18 M 0.02 0.50 1.83 0.95 1.13 1.64
6.92
HH Initial 0.00 0.03 0.03 0.03 0.05 0.02
0.31
(pH 5.25) 3M 0.02 0.09 0.41 0.11 0.44 0.46
1.88
5 M21 days 0.02 0.16 0.65 0.19 0.80 0.76
2.95
9M 0.05 0.20 1.07 0.51 0.92 1.05
4.21
II Initial 0.00 0.04 0.04 0.04 0.06 0.02
0.34
(pH 5.25) 3 M 0.02 0.09 0.39 0.13 0.48 0.41
1.79
5 M 21 days 0.02 0.14 0.61 0.23 0.68 0.62
2.60
9M 0.07 0.22 1.06 0.63 1.00 0.94
4.26
JJ Initial 0.01 0.04 0.04 0.04 0.06 0.02
0.33
(pH 5.25) 3 M 0.02 0.09 0.36 0.13 ' 0.50 0.40
1.85
Initial 0.00 0.05 0.02 0.04 0.05 0.01
0.27
KK 3 M (U) 0.01 0.12 0.43 0.12 0.41 0.50
1.71
(pH 5.25) 6 M (U) 0.00 0.17 0.70 0.24 0.63 0.77
2.80
9 M (U) 0.04 0.24 1.06 0.44 0.85 1.08
4.12
12 M (U) 0.11 0.32 1.40 0.70 1.03 1.26
5.33
15 M (U) 0.11 0.34 1.55 0.79 1.08 1.35
5.81
18 M (U) 0.13 0.41 1.77 1.05 1.11 1.46
6.52
3 M (I) 0.01 0.12 0.42 0.12 0.37 0.49
1.63
6 M (I) 0.00 0.17 0.70 0.24 0.63 0.77
2.80
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Formulation RRT 112-201- 13-201- Asp9- 19-101- 111-121-
Time Point Total
(pH) 0.49 BVN BVN BVN BVN BVN
9 M (I) 0.03 0.24 1.07 0.44 0.85 1.08
4.14
12 M (I) 0.11 0.32 1.38 0.68 1.02 1.26
5.28
15 M (I) 0.11 0.34 1.54 0.79 1.06 1.46
5.77
18 M (I) 0.13 0.41 1.77 1.05 1.10 1.46
6.51
FF Initial 0.00 0.04 0.06 0.05 0.09 0.04
0.44
(pH 5.3) 3M 0.02 0.07 0.40 0.21 0.57 0.37
1.85
GG Initial 0.00 0.05 0.03 0.06 0.05 0.02 0.30
(pH 5.3) 3M 0.00 0.10 0.37 0.12 0.46 0.45
1.61
M 09 days 0.02 0.13 0.56 0.19 0.56 0.63 2.35
9M 0.02 0.21 0.95 0.41 0.81 0.97 3.82
12 M 0.07 0.28 1.32 0.61 0.95 1.24 4.94
,
Initial - 0.05 BQL 0.04 0.04 BQL 0.12
QQ ..
3 M - 0.09 0.40 0.14 0.49 0.39 1.57
(pH 5.50)
6M - 0.14 0.79 0.38 0.79 0.72 3.01
MM Initial 0.00 0.04 0.04 0.03 0.09
0.01 0.34
(pH 5.75) 3 M 0.02 0.05 0.50 0.33 0.85 0.23 2.25
[0079] For example, the increase in the amount of total impurities as compared
to an initial time
point, as determined by high performance liquid chromatography ("HPLC") at a
wavelength of
215 nm, remains well under about 10%, under about 7.5% and under about 6.5%
after storage at
5 C for 12 months, for the samples having pH in the range of about 3.0 to less
than 4.0, or about
3.25 to about 3.75. Also, the increase in the amount of total impurities as
compared to an initial
time point remains well under about 8%, under about 7.5%, under about 6.5% and
under about
5% after storage at 5 C for 12 months, for the samples having pH in the range
of 5.0 to about
5.7.
[0080] Moreover, the increase in Asp9-bivalirudin % and [9-10]-cycloimido-
bivalirudin %, as
determined by high performance liquid chromatography (HPLC) at 215 nm, as
compared to an
44
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initial value (for example, at the time of manufacture of the formulation)
remains well under
about 1.0%, under about 0.5%, under about 0.4%, under about 0.3%, and under
about 0.25%
after storage at 5 C for 12 months, for the samples having pH in the range of
about 3.0 to less
than 4.0, or about 3.25 to about 3.75. The increase in [3-20]-bivalirudin %
and [9-10]-
cycloimido-bivalirudin, as determined by high performance liquid
chromatography (HPLC) at
215 nm, as compared to an initial value remains well under about well under
about 1.0%, or
under about 0.5% after storage at 5 C for 12 months. The increase in [12-20]-
bivalirudin and
[11-12]-cycloimido-bivalirudin as compared to an initial value remains well
under about well
under about 3%, or under about 2.5%, or under about 2.0% after storage at 5 C
for 12 months.
100811 The increase in the amount of Asp9-bivalirudin % area under the curve
from an initial
value remains well under about 1.5%, under about 1.0%, under about 0.75% and
under about
0.6% after storage at 5 C for 12 months, for the samples having pH in the
range of 5.0 to about
5.7. Also, the increase in the amount of the [3-20]-bivalirudin impurity as
compared to an initial
time point remains well under about 1.8%, or under 1.5% after storage at 5 C
for 12 months, for
the samples having pH in the range of 5.0 to about 5.7. Also, the increases in
the amount of the
[11-12]-cycloimido-bivalirudin and the [9-10]-cycloimido-bivalirudin impurity
as compared to
an initial time point remains well under about 2.0%, or under 1.6%, after
storage at 5 C for 12
months, for the samples having pH in the range of 5.0 to about 5.7.
100821 These formulations described and taught herein provide storage stable,
ready-to-use
bivalirudin formulations. The formulations described herein and characterized
in Table 5b show
high stability over 6 months, or 9 months, or 12 months or more at 2-8 C.
Example 6: pH Variation at pH of about 2.5 to 3.5
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100831 Data presented in Tables 6a and 6b shows an investigation into the
influence of pH on the
stability (5 C for 3 months; and 25 C/60% RH, respectively) of various
bivalirudin formulations
with 50 mM glycine, in the about 2.5 to about 3.5 pH range. The initial values
are reported first
in each data cell of Table 6a and Table 6b, followed by the available
stability data for each
formulation.
Table 6a. Stability Testing pH Variation, 3-month (repeated) Data at 5 C
Formulation R. R T [12-201- [3-201- Asp9- 19-
101- 111-12]--
Time Point
Total
(PH) 0.49 BVN BVN BVN BVN BVN
A Initial 0.02 0.06 0.01 0.04
0.01 ' 0.00 0.25
(pH 2.5) 3M 0.43 0.78 0.02 0.36 0.06 ' 0.17
2.24
C Initial 0.01 0.05 0.01 0.03 0.01
0.00 0.26
(pH 3.0) 3M 0.37 0.66 0.06 0.11 0.06 0.33
1.81
6 M 0.85 1.33 0.10 0.22 0.12 0.67 3.49
9M 1.35 2.10 0.16 0.29 0.17 ' 1.06 5.52
I Initial 0.01 0.05 0.02 0.03 0.01
0.03 0.30
(pH 3.5) 3 M 0.32 0.59 0.08 0.06 0.08 0.49
1.80
6 M 0.67 1.15 0.14 0.12 0.15 1.00 3.45
9M 1.04 1.73 0.21 0.14 0.21 1.52 5.07
Table 6b. Stability Testing pH Variation, 1-month Data at 25 C/60% RH
Formulation Time RRT [12-20)- 13-201- Asp9- 19-101- [11-12J-
Total
(pH) Point 0.49 BVN BVN BVN BVN BVN
A Initial 0.02 0.06 0.01 0.04 0.01
0.00 0.25
(pH 2.5) 1 M 1.98 3.33 0.20 1.11 0.23 0.62
9.26
C Initial 0.01 0.05 0.01 0.03 0.01
0.00 0.26
(pH 3.0) 1 M 1.77 2.83 0.27 0.30 0.22 1.13
7.30
I Initial 0.01 0.05 0.02 0.03 0.01
0.03 0.30
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Formulation Time RRT [12-201- 13-201- Asp9- 19-101- 111-121-
Total
(PH) Point 0.49 BVN BVN BVN BVN BVN
(pH 3.5) 1 M 1.42 2.42 0.38 0.14 0.28 1.65
7.02
[0084] As can be seen from the data in Tables 6a and 6b, the pH value of about
3.0 to about 3.5
provides an acceptable environment to effectively stabilize liquid bivalirudin
formulations,
contrary to the data reported in U.S. Patent No. 7,803,762, Table 1.
Example 7: Effect of Buffer at pH of about 2.5 to 3.5
100851 Although glycine was used in the formulations of Example 6, it only
provides a buffering
effect in the pH range of about + 1 unit about its pKat (2.34). Table 7
presents the results of an
investigation into the influence of buffer type on the stability (25 C/60% RH
for 1 month) of
various bivalirudin formulations, using a citrate:phosphate buffer (25mM
citrate; 15m1V1
phosphate) that provides buffering over the full target pH range of about 2.5
to about 3.5 pH
range (pKai of 3.15 and 2.15 respectively). The initial values are reported
first in each data cell
of Table 7, followed by the available stability data for each formulation.
Table 7. Stability Testing Buffer Variation, 1-month Data at 25 C/60% RH
Formulation Time RRT 112-201- 13-201- Asp9- [9-101- 111-121-
Total
(pH) Point 0.49 BVN BVN BVN BVN BVN
B Initial 0.01 0.05 0.01 0.03 0.01 0.00
0.16
(pH 2.5) 1 M 2.26 3.60 0.29 0.62 0.22 0.92
9.42
D Initial 0.01 0.04 0.01 0.03 0.01 0.00
0.17
(pH 3.0) 1 M 1.76 2.94 0.52 0,45 0.25 1.49
8.62
J Initial 0.01 0.04 0.01 0.03 0.01 0.00
0.16
(pH 3.5) 1 M 1.69 2.73 0.50 0.33 0.25 1.57
8.12
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[0086] As can be seen from the data in Table 7, the citrate-phosphate buffer
provides an
acceptable environment for the effective stabilization of liquid bivalirudin
formulations, although
the glycine formulations of Example 6 are slightly better at pH 3.0 and pH
3.5.
Example 8: Tonicity Agent Variation at pH 3.5
[0087] Tables 8a and 8b display the results of an investigation into the
influence of various
tonicity agents on the refrigerated stability (5 C for 3 months, and 25 C/60%
RH, respectively)
of various bivalirudin formulations at about pH 3.5. The initial values (t=0)
are reported first in
each data cell of Tables 8a and 8b, and the following values were from data
points taken at
various times (1-, 3-, 6-, 9- or 12-months).
Table 8a. Stability Testing, tonicity agent variation, various month data at 5
C
Formulatio RRT [12-20]- [3-201- Asp9- [9-10]- 111-121-
Time Point
Total
n (pH) 0.49 BVN BVN BVN BVN BVN
K1 Initial 0.01 0.04 0.02 0.02 0.06 0.02 0.25
(pH 3.5) 3M 0.31 0.53 0.06 0.06 0.11 0.47 1.65
6 M 0.59 1.00 0.14 0.09 0.21 0.93 3.23
9M 0.88 1.45 0.19 0.11 0.21 1.30 4.40
K4 Initial 0.03 0.04 0.02 0.03 0.06 0.02 0.33
(pH 3.5) 3 M 0.31 0.56 0.09 0.07 0.09 0.56 1.95
6M 0.04 0.56 0.15 0.09 0.14 1.03 3.18
Initial 0.01 0.04 0.01 0.02 0.06 0.02 0.25
(pH 3.5) 3 M 0.33 0.58 0.08 0.06 0.09 0.52 1.92
6 M 0.62 1.06 0.12 0.10 0.20 0.91 3.37
9M 0.91 1.50 0.17 0.11 0.15 1.26 4.72
12 M 1.24 2.37 0.27 0.21 0.23 1.90 7.16
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Table 8b. Stability Testing, tonicity agent variation, 1-month data at 25
C/60% RH
Formulation Time RRT 112-201- 13-201- Asp9- [9-101- [11-121-
Total
.
(pH) Point 0.49 BVN BVN BVN BVN BVN
Kl Initial 0.01 0.04 0.02 0.02 0.06 0.02
0.25
(pH 3.5) 1 M 1.30 2.25 0.38 0.16 0.28 1.62
6.40
K4 Initial 0.03 0.04 0.02 0.03 0.06 0.02
0.33
(pH 3.5) 1 M 1.20 2.19 0.39 0.16 0.29 1.69
6.35
K5 Initial 0.04 0.10 0.02 0.07 0.04 0.01
0.44
(pH 3.5) 1 M 1.24 2.28 0.37 0.20 0.30 1.59
6.43
L Initial 0.01 0.04 0.01 0.02 0.06 0.02
0.25
(pH 3.5) 1 M 1.36 2.38 0.37 0.17 0.26 1.53
6.82
100881 As can be seen from the data in Tables 8a and 8b, the tonicity agents
chosen are useful to
stabilize liquid bivalirudin formulations, as measured at pH of about 3.5.
Example 9: pH Variation with Stabilizer/Tonicity Agent
[0089] Data presented in Tables 9a and 9b shows an investigation into the
influence of pH on the
stability (at 5 C for various numbers of months, and at 25 C/60% RH for one
month,
respectively) of various bivalirudin formulations having 0.9% NaC1 as tonicity
agent, in the
about 2.5 to about 3.5 pH range, and having 7.5% PEG 400 as a
stabilizer/tonicity agent, and at
28 mM buffer strength (3.8 mg/mL sodium acetate tiihydrate) in the 5.0 up to
about 5.7 pH
range. The initial values are reported first in each data cell of Tables 9a
and 9b, and the other
values were from data points of various months, as reported in Tables 9a and
9b.
Table 9a. pH variation with Stabilizer/Tonicity Agent at 5 C
Formulation RRT 112-201- [3-201- Asp9- 19-101- 111-121-
Time Point Total
(PH) 0.49 BVN BVN BVN BVN BVN
HI Initial 0.03 0.04 0.02 0.03 0.07 0.01 0.33
49
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Formulation RRT [12-201- 13-201- Asp9- 19-101- 111-121-
Time Point
Total
(PH) 0.49 BVN BVN BVN BVN BVN
(pH 3.25) 3 M 0.39 0.67 0.06 0.13 0.07 0.38 1.99
H2 Initial 0.01 0.05 0.02 0.04 0.07 0.03 0.27
(pH 3.25) 3M 0.39 0.65 0.08 0.11 0.08 0.49 2.03
K1 Initial 0.01 0.04 0.02 0.02 0.06 0.02 0.25
(pH 3.5) 3M 0.31 0.53 0.06 0.06 0.11 0.47 1.65
6 M 0.59 1.00 0.14 0.09 0.21 0.93 3.23
9M 0.88 1.45 0.19 0.11 0.21 1.30 4.40
K4 Initial 0.03 0.04 0.02 0.03 0.06 0.02 0.33
(pH 3.5) 3M 0.31 0.56 0.09 0.07 0.09 - 0.56
1.95
6 M 0.04 0.56 0.15 0.09 0.14 1.03 3.18
BB Initial 0.03 0.04 0.02 0.03 0.08 0.01
0.37
(pH 3.75) 3M 0.23 0.48 0.11 0.06 0.11 0.69 1.88
6M 5.08 3.36 0.28 0.10 0.16 1.20 11.11
9 M 0.68 1.28 0.28 0.09 0.25 1.79 4.77
CC Initial 0.02 0.04 0.02 0.03 0.07 0.02 0.34
(pH 4.0) 3M 0.16 0.36 0.12 0.05 0.14 0.68 1.69
6M 1.87 1.09 0.06 0.05 0.24 1.25 5.10
9 M 0.47 0.96 0.32 0.09 0.33 1.80 4.32
EE Initial 0.00 0.04 0.03 0.03 0.04 0.02
0.28
(pH 5.10) 3 M 0.02 0.12 0.37 0.10 0.38 0.51 1.76
M 21 days 0.02 0.18 0.60 0.16 0.58 0.82 2.67
9M 0.04 0.27 1.00 0.39 0.82 1.19 4.07
12 M 0.03 0.28 1.09 0.47 0.75 0.55 4.09
15 M 0.05 0.39 1.46 0.72 1.03 1.46 5.77
18 M 0.02 0.50 1.83 0.95 1.13 1.64 6.92
1-11-1 Initial 0.00 0.03 0.03 0.03 0.05 0.02
0.31
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Formulation RRT [12-20J- 13-201- Asp9- 19-101- 111-121-
Time Point Total
(PH) 0.49 BVN BVN BVN BVN BVN
(pH 5.25) 3M 0.02 0.09 0.41 0.11 0.44 0.46 1.88
M 21 days 0.02 0.16 0.65 0.19 ' 0.80 0.76 2.95
9 M 0.05 0.20 1.07 0.51 0.92 1.05 4.21
MM Initial 0.00 0.04 0.04 0.03 0.09 0.01
0.34
(pH 5.75) 3 M 0.02 0.05 0.50 0.33 0.85 0.23 2.25
Table 9b. pH variation with Stabilizer/Tonicity Agent at 25 C/60% RH
Formulation Time RRT 112-201- 13-201- Asp9- [9-101- 111-121-
Total
(PH) Point 0.49 BVN BVN BVN BVN BVN
G1 Initial 0.03 0.04 0.02 0.03 0.07
0.02 0.37
(pH 3.0) 1 M 1.82 2.99 0.24 0.61 0.21 0.87
7.58 -
G2 Initial 0.05 0.10 0.02 0.07 0.05
0.01 0.44 _
(pH 3.0) 1 M 1.68 2.77 0.27 0.46 0.24 1.07
7.14
H1 Initial 0.03 0.04 0.02 0.03 0.07
0.01 0.33
(pH 3.25) 1 M 1.66 2.69 0.28 0.37 0.15 1.14
6.90
H2 Initial 0.01 0.05 0.02 0.04 0.07
0.03 0.27 _
(pH 3.25) 1 M 1.64 2.79 0.35 0.20 0.27 1.50
7.31 _
K1 Initial 0.01 0.04 0.02 0.02 0.06
0.02 0.25 _
_
(pH 3.5) 1 M 1.30 2.25 0.38 0.16 0.28 1.62
6.40
K4 Initial 0.03 0.04 0.02 0.03 0.06
0.02 0.33
(pH 3.5) 1 M 1.20 2.19 0.39 0.16 0.29 1.69
6.35 '
K.5 Initial 0.04 0.10 0.02 0.07 0.04
0.01 0.44
(pH 3.5) 1 M 1.24 2.28 0.37 0.20 0.30 1.59
6.43
BB Initial 0.03 0.04 0.02 0.03 0.08
0.01 0.37
(pH 3.75) 1 M 0.89 1.87 0.53 0.12 0.36 2.04
6.26
CC Initial 0.02 0.04 0.02 0.03 0.07
0.02 0.34
(pH 4.0) 1 M 0.57 1.39 0.61 0.12 0.49 2.10
5.81
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Formulation Time RRT 112-201- 13-201- Asp9- 19-101- [11-121-
Total
(PH) Point 0.49 BVN BVN BVN BVN BVN
CC Initial 0.04 0.10 0.03 0.07 0.05
0.02 0.46
(pH 4.0) 1 M 0.71 1.63 0.54 0.15 0.44 2.05
5.99
EE Initial 0.00 0.04 0.03 0.03 0.04
0.02 0.28
(pH 5.10) 1 M 0.14 0.32 1.69 0.46 1.07 1.28
5.57
HH Initial 0.00 0.03 0.03 0.03 0.05
0.02 0.31
(pH 5.25) 1M 0.16 0.28 1.81 0.61 1.21 1.15
5.90
MM Initial 0.00 0.04 0.04 0.03 0.09
0.01 0.34
(pH 5.75) 1 M 0.21 0.11 2.15 2.11 1.97 0.52
7.90
100901 As can be seen from the data in Tables 9a and 9b, the compositions
having pH in these
ranges with a suitable tonicity agent and/stabilizer provide an acceptable
environment to stabilize
liquid bivalirudin formulations.
Example 10: 10 mM Glvcine Formulations at pH 3.0 to 3.5
[0091] Data presented in Tables 10a and 10b shows an investigation into the
influence of a lower
concentration of glycine (10 mM glycine) on the stability (5 C for 3-,6-, or
12-months, and 1
month at 25 C/60% RH, respectively) of various bivalirudin formulations in the
about 3.0 to
about 3.5 pH range. The initial values are reported first in each data cell of
Tables 10a and 10b,
and the following values were from the other time points.
Table 10a. 10 mM Glvcine at pH 3.0 and 3.5, with 0.9% NaC1 at 5 C
Formulation RRT 112-201- 13-201- Asp9- 19-101- 111-121-
Time Point Total
(PH) 0.49 BVN BVN BVN BVN BVN
E Initial 0.01 0.04 0.01 0.02 0.05 0.01
0.23
(pH 3.0) 3M 0.43 0.65 0.04 0.14 0.10 0.27 1.78
N Initial 0.01 0.04 0.01 0.02 0.05 0.02
0.25
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Formulation RRT 112-201- 13-201- Asp9- 19-101- 111-121-
Time Point
Total
(PH) 0.49 BVN BVN BVN BVN BVN
(pH 3.5) 6M 0.58 1.00 0.13 0.09 0.22 0.91
3.20
12 M 1.18 2.22 0.26 0.23 0.28 1.91 6.48
Table 10b. 10 mM Glvcine at pH 3.0 and 3.5, with 0.9% NaC1 at 25 C/60% RH
Formulation Time RRT 112-201- 13-201- Asp9- 19-101- [11-121-
Total
(PH) Point 0.49 BVN BVN BVN BVN BVN
E Initial 0.01 0.04 0.01 0.02 0.05
0.01 0.23
(pH 3.0) 1 M 1.72 2.80 0.24 0.48 0.20 0.91
7.01
N Initial 0.01 0.04 0.01 0.02 0.05
0.02 0.25
(pH 3.5) 1 M 1.32 2.31 0.38 0.17 0.29 1.63
6.52
100921 As can be seen from the data in Tables 10a and 10b, the 10 mM glycine
at pH in the
range of about 3.0 to less than 4.0, or about 3.25 to about 3.5 provides an
acceptable environment
to effectively stabilize liquid bivalirudin formulations.
Example 11: Stabilizer Variation at about pH 3.5
100931 Data presented in Tables lla and 11 b shows an investigation into the
influence of
stabilizer on the stability (5 C for various months, and 1 month at 25 C/60%
RH, respectively)
of various bivalirudin formulations with 10 mM or 50 mM glycine in the about
3.5 to about 3.6
pH range. The initial (t=0) values are reported first in each data cell of
Tables ha and 1 lb, and
the values following the initial value were from data points taken at various
time points as
indicated in the next row.
Table ha. Stabilizer Variation at about pH 3.5 at 5 C
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Formulation RRT [12-201- [3-20]- Asp9- 19-101- 111-121-
Time Point
Total
(PH) 0.49 BVN BVN BVN BVN BVN
0 Initial 0.01 0.04 0.01 0.02 0.05 0.02 0.24
(pH 3.5) 3 M 0.27 0.49 0.06 0.04 0.07 0.51 1.56
6M 0.53 0.96 0.14 0.08 0.20 0.97 3.19
Initial 0.01 0.05 0.02 0.03 0.07 0.01 0.31
(pH 3.5) 6M 0.05 0.43 0.13 0.09 0.12 0.91 3.71
Initial 0.05 0.13 0.02 0.04 0.06 0.12 0.57
(pH 3.5) 3 M 0.25 0.45 0.09 0.07 0.09 0.52 1.66
6M 0.22 0.01 0.15 0.08 0.13 0.88 2.41
9M 0.69 1.25 0.23 0.10 0.19 1.42 4.21
Initial 0.05 0.12 0.03 0.04 0.05 0.12 0.68
(pH 3.5) 3M 0.21 0.39 0.09 0.06 0.07 0.47 1.55
4 M 0.26 0.50 0.09 0.06 0.08 0.60 2.09
6M 0.15 0.66 0.13 0.07 0.10 0.79 2.69
9M 0.00 0.95 0.20 0.10 0.12 1.28 4.48
Initial 0.02 0.06 0.01 0.03 0.05 0.02 0.33
(pH 3.5) 3M 0.30 0.52 0.10 0.07 0.09 0.55 2.05
M21 days 0.47 0.82 0.13 0.07 0.11 0.89 2.72
9 M 0.76 1.47 0.23 0.18 0.21 1.54 4.69
12 M 0.85 1.46 0.26 0.14 0.20 0.79 4.09
M 1.26 2.18 0.36 0.16 0.31 2.25 7.06
18 M 0.08 2.76 0.47 0.17 0.37 2.72 8.68
Initial 0.03 0.07 0.04 0.05 0.09 0.04 0.45
(pH 3.6) 3M 0.22 0.47 0.11 0.11 0.11 0.63 1.83
AA Initial 0.00 0.04 0.02 0.05 0.12 0.01 0.36
(pH 3.6) 3M 0.20 0.44 0.07 0.10 0.11 0.55 1.67
5 M08 days 0.02 0.66 0.14 0.08 0.13 0.84 2.43
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Formulation
Time Point RRT 112-201- 13-201- Asp9- 19-
101- 111-121-
(PH) 0.49 BVN BVN BVN BVN BVN Total
9M 0.66 1.19 0.24 0.11 0.22 1.45 4.17
12 M 0.01 1.65 0.34 0.12 0.23 1.93 5.52
Table 11b. Stabilizer Variation at about pH 3.5 at 25 C/60% RH
Formulation Time RRT 112-201- 13-201- Asp9- 19-101- 111-121-
Total
(PH) Point 0.49 BVN BVN BVN BVN BVN
O Initial 0.01 0.04 0.01 0.02 0.05
0.02 0.24
(pH 3.5) 1 M 1.14 2.12 0.40 0.16 0.22 1.66
6.23
R Initial 0.01 0.05 0.02 0.03 0.07
0.01 0.31
(pH 3.5) 1 M 1.08 2.02 0.34 0.14 0.23 1.49
6.34
T Initial 0.05 0.13 0.02 0.04 0.06
0.12 0.57
(pH 3.5) 1 M 0.92 1.83 0.42 0.10 0.28 1.57
5.63
U Initial 0.05 0.12 0.03 0.04 0.05
0.12 0.68
(pH 3.5) 1 M 0.78 1.55 0.37 0.10 0.23 1.45
5.47
/ Initial 0.02 0.07 0.02 0.03 0.04
0.04 0.31
(pH 3.5) 1 M 0.98 1.90 0.45 0.15 0.29 1.76
5.88
W Initial 0.02 0.06 0.01 0.03 0.05
0.02 0.33
(pH 3.5) 1 M 1.00 1.83 0.40 0.12 0.20 1.56
5.55
Y Initial 0.03 0.07 0.04 0.05 0.09
0.04 0.45
(pH 3.6) 1 M 0.81 1.64 0.44 0.10 0.30 1.80
5.69 '
Z Initial 0.02 0.08 0.03 0.05 0.08
0.08 0.42
(pH 3.6) 1 M 0.77 1.69 0.49 0.11 0.33 1.90
5.71
AA Initial 0.00 0.04 0.02 0.05 0.12
0.01 0.36
(pH 3.6) 1 M 0.76 1.52 0.43 0.10 0.27 ' 1.56
4.97
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100941 As can be seen from the data in Tables lla and 11b, the various
stabilizers provide an
acceptable environment to effectively stabilize liquid bivalirudin
formulations in this pH range.
Example 12: Stabilizer Variation at about pH 5.25
[0095] Data presented in Tables 12a and 12 b shows an investigation into the
influence of
stabilizer on the stability (5 C for various months, and 1 month at 25 C/60%
RH, respectively)
of various biyalirudin formulations with 6 or 28 mM buffer strength (acetate)
in the pH range of
about 5.25 to about 5.3. The initial values (t=0) are reported first in each
data cell of Tables 12a
and 12b, and the values following the initial value were from data points
taken at various time
points as indicated in the second column.
Table 12a. Stabilizer Variation at about pH 5.25 at 5 C
Formulation RRT [12-20]- 13-201- Asp9- 19-101- 111-121-
Time Point
Total
(PH) 0.49 BVN BVN BVN BVN BVN
RH Initial 0.00 0.03 0.03 0.03 0.05 0.02
0.31
(pH 5.25) 3M 0.02 0.09 0.41 0.11 0.44 0.46
1.88
M21 days 0.02 0.16 0.65 0.19 0.80 0.76 2.95
9M 0.05 0.20 1.07 0.51 0.92 1.05
4.21
II Initial 0.00 0.04 0.04 0.04 0.06 0.02
0.34
(pH 5.25) 3 M 0.02 0.09 0.39 0.13 0.48 0.41
1.79
5 M21 days 0.02 0.14 0.61 0.23 0.68 0.62
2.60
9M 0.07 0.22 1.06 0.63 1.00 0.94
4.26
JJ Initial 0.01 0.04 0.04 0.04 0.06 0.02
0.33
(pH 5.25) 3 M 0.02 0.09 0.36 0.13 0.50 0.40
1.85
FF Initial 0.00 0.04 0.06 0.05 0.09 0.04
0.44
(pH 5.3) 3 M 0.02 0.07 0.40 0.21 0.57 0.37
1.85
GG Initial 0.00 0.05 0.03 0.06 0.05 0.02
0.30
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Formulation RRT 112-201- 13-201- Asp9- ' [9-10]- [11-12]-
Time Point Total
(PH) 0.49 BVN BVN BVN BVN BVN
(pH 5.3) 3M 0.00 0.10 0.37 0.12 0.46 0.45 1.61
M09 days 0.02 0.13 0.56 0.19 0.56 0.63 2.35
9 M 0.02 0.21 0.95 0.41 ' 0.81 0.97 3.82
12 M 0.07 0.28 1.32 0.61 0.95 1.24 4.94
Table 12b. Stabilizer Variation at about pH 5.25 at 25 C/60% RH
Formulation Time RRT 112-201- 13-201- Asp9- [9-10]- [11-121-
Total
(PH) Point 0.49 BVN BVN BVN B'VN BVN
HEI Initial 0.00 0.03 0.03 0.03 0.05
0.02 0.31
(pH 5.25) IM 0.16 0.28 1.81 0.61 1.21 1.15
5.90
II Initial 0.00 0.04 0.04 0.04 0.06
0.02 0.34
(pH 5.25) 1 M 0.16 0.25 1.80 0.01 1.30 '
1.02 ' ' 6.02
JJ Initial 0.01 0.04 0.04 0.04 0.06
0.02 0.33
(pH 5.25) 1 M 0.13 0.26 1.64 0.72 1.30 1.00
5.72
FF Initial 0.00 0.04 0.06 0.05 0.09
0.04 0.44
(pH 5.3) 1 M 0.14 0.23 1.75 0.97 1.47 0.89
6.18
GG Initial 0.00 0.05 0.03 0.06 0.05
0.02 0.30
(pH 5.3) 1 M 0.02 0.26 1.78 0.54 1.14 1.16
5.77
[0096] As can be seen from the data in Tables 12a and 12b, the various
stabilizers provide an
acceptable environment to effectively stabilize liquid bivalirudin
formulations in this pH range.
Example 13: Amino Acids as Stabilizers/Tonicity Agents
[0097] Data presented in Tables 13a and 13b shows an investigation into the
influence of amino
acids on the stability (5 C for various months, and 1 month at 25 C/60% RH,
respectively) of
various bivalirudin formulations with 50 or 300 mM glycine, in the pH range of
about 3.5 to
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about 3.8. The initial values (t=0) are reported first in each data cell of
Tables 13a and 13b, and
the values following the initial value were from data points taken at various
time points as
indicated in the second column.
Table 13a. Stability at 5 C - Amino Acid Variation
Formulation RRT [12-201- 13-201- Asp9- 19-101- 111-121-
Time Point Total
(PH) 0.49 BVN BVN BVN BVN BVN
Si Initial 0.01 0.04 0.02 0.03 0.07 0.01 0.27
(pH 3.5) 3 M 0.37 0.66 0.09 0.07 0.09 0.59 2.07
S2 Initial 0.02 0.06 0.02 0.04 0.05 0.02 0.32
(pH 3.5) 3 M 0.29 0.52 0.12 0.07 0.10 0.59 1.92
M 21 days 0.55 0.93 0.16 0.08 0.15 1.09 3.18
9M 1.03 1.72 0.26 0.18 0.25 1.78 6.17
V Initial 0.02 0.07 0.02 0.03 0.04 0.04 0.31
(pH 3.5) 3M 0.29 0.58 0.12 0.08 0.10 0.62 1.95
5 M21 days 0.55 1.04 0.15 0.05 0.12 1.14 3.25
9M 0.76 1.54 0.26 0.18 0.24 1.75 5.03
Initial 0.02 0.08 0.03 0.05 0.08 0.08 0.42
(pH 3.6) 3M 0.20 0.50 0.12 0.09 0.11 0.67 1.81
00 Initial 0.01 0.05 0.02 0.03 0.05 0.02 0.27
(pH 3.8) 3M 0.17 0.48 0.13 0.06 0.12 0.69 1.80
5 M 21 days 0.33 0.74 0.19 0.06 0.22 1.17 3.01
9M 0.51 1.22 0.32 0.14 0.13 1.86 4.41
Table 13b. Stability at 25 C/60% RH - Amino Acid Variation
Formulation Time RRT [12-201- 13-201- Asp9- 19-101- 111-121-
Taal
(PH) Point 0.49 BVN BVN BVN MTN BVN
Si Initial 0.01 0.04 0.02 0.03 0.07 0.01 0.27
(pH 3.5) 1 M 0.97 1.96 0.44 0.13 0.31 1.80
5.96
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Formulation Time RRT 112-201- 13-201- Asp9- 19-101- 111-121-
Total
(PH) Point 0.49 BVN BVN BVN BVN BVN
_
S2 Initial 0.02 0.06 0.02 0.04 0.05 0.02
0.32
(pH 3.5) 1 M 1.12 1.96 0.45 0.15 0.29 1.79
6.13
V Initial 0.02 0.07 0.02 0.03 0.04 0.04
0.31
_
(pH 3.5) 1 M 0.98 1.90 0.45 0.15 0.29 1.76
5.88
Z Initial 0.02 0.08 0.03 0.05 0.08 0.08
0.42
_
(pH 3.6) 1 M 0.77 1.69 0.49 0.11 0.33 1.90
5.71
_
AA Initial 0.00 0.04 0.02 0.05 0.12 0.01
0.36
(pH 3.6) 1 M . 0.76 1.52 0.43 0.10 0.27 1.56
4.97
00 Initial 0.01 0.05 0.02 0.03 0.05 0.02
0.27
(pH 3.8) 1 M 0.60 1.51 0.55 . 0.11 0.35 1.96
5.56
[0098] As can be seen from the data in Tables 13a and 13b, the various amino
acids provide an
acceptable environment to effectively stabilize liquid bivalirudin
formulations in this pH range.
Example 14: Long Term Stability Data, Specific Embodiments
[0099] Table 14a provides data for long term stability (5 C) of preferred
embodiments of this
invention for up to 18 months, and Table 14b provides stability data after 1
month at 25 C/60%
RH. In all cases, the impurity profile after 1 month under accelerated
conditions is predictive of
the long-term data at the 12-month time point.
Table 14a: Long Term Stability Data for Exemplified Embodiments of the Present
Disclosure
Formulation RRT [12-201- 13-201- Asp9- [9-101- [11-121-
Time Point Total
(PH) 0.49 BVN BVN BVN BVN BVN
X Initial 0.03 0.09 0.02 0.07 0.06 0.05 0.43
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Formulation RRT [12-201- 13-201- Asp9- 19-101- 111-121-
Time Point
Total
(PH) 0.49 BVN BVN BVN BVN BVN
(pH 3.5) 3 M (U) 0.28 0.54 0.11 0.08 0.10 0.61 1.80
6 M (U) 0.47 0.87 0.18 0.09 0.15 1.02 3.04
9 M (U) 0.03 1.37 0.26 0.10 0.22 1.58 4.62
12 M(U) 0.06 1.84 0.34 0.15 0.30 2.05 6.26
3 M (I) 0.27 0.52 0.11 0.07 0.09 0.60 1.75
6 M (I) 0.47 0.90 0.18 0.09 0.15 1.04 3.10
9 M (I) 0.03 1.37 0.25 0.10 0.21 1.57 4.61
12 M (I) 0.06 1.82 0.34 0.15 0.29 2.04 6.24
AA Initial 0.00 0.04 0.02 0.05 0.12 0.01 0.36
(pH 3.6) 3M 0.20 0.44 0.07 0.10 0.11 0.55 1.67
M08 days 0.02 0.66 0.14 0.08 0.13 0.84 2.43
9M 0.66 1.19 0.24 0.11 0.22 1.45 4.17
12 M 0.01 1.65 0.34 0.12 0.23 1.93 5.52
Initial 0.00 0.05 0.02 0.04 0.05 0.01 0.27
3 M (U) 0.01 0.12 0.43 0.12 0.41 0.50 1.71
6 M (U) 0.00 0.17 0.70 0.24 0.63 0.77 2.80
KK 9 M (U) 0.04 0.24 1.06 0.44 0.85 1.08 4.12
(pH 5.25) 12 M (U) 0.11 0.32 1.40 0.70 1.03 1.26
5.33
M (U) 0.11 0.34 1.55 0.79 1.08 1.35 5.81
18 M (U) 0.13 0.41 1.77 1.05 1.11 1.46 6.52
3 M (I) 0.01 0.12 0.42 0.12 0.37 0.49 1.63
6 M (I) 0.00 0.17 0.70 0.24 0.63 0.77 2.80
9 M (I) 0.03 0.24 1.07 0.44 0.85 1.08 4.14
12 M (I) 0.11 0.32 1.38 0.68 1.02 1.26 5.28
15 M (I) 0.11 0.34 1.54 0.79 1.06 1.46 5.77
18 M (I) 0.13 0.41 1.77 1.05 1.10 1.46 6.51
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Formulation R. R T 112-201- 13-201- Asp9- 19-
101- 111-121-
Time Point Total
(PH) 0.49 BVN BVN BVN BVN BVN
GG Initial 0.00 0.05 0.03 0.06 0.05 0.02
-- 0.30
(pH 5.3) 3 M 0.00 0.10 0.37 0.12 0.46 0.45 1.61
. 5 M 09 days 0.02 0.13 0.56 0.19 0.56 0.63 2.35
9 M 0.02 0.21 0.95 0.41 0.81 0.97 3.82
12 M 0.07 0.28 1.32 0.61 0.95 1.24 4.94
Table 14b: 25C Stability Data for Exemplified Embodiments of the Present
Disclosure
Formulation Time RRT 112-201- [3-201- Asp9- 19-101- 111-121-
Total
(PH) Point 0.49 BVN BVN BVN BVN BVN
X Initial 0.03 0.09 0.02 0.07 0.06
0.05 0.43
(pH 3.5) 1 M (U) 0.97 1.83 0.47 0.13 0.30
1.72 5.93
1 M (I) 0.98 1.82 0.46 0.13 0.30 1.72 5.93
AA Initial 0.00 0.04 0.02 0.05 0.12
0.01 0.36
(pH 3.6) 1 M 0.76 1.52 0.43 0.10 0.27 1.56
4.97
KK Initial 0.00 0.05 0.02 0.04 0.05
0.01 .. 0.27
(pH 5.25) 1 M (U) 0.03 0.30 1.90 0.62 1.18
1.21 6.11
1 M (I) 0.03 0.32 1.99 0.65 1.23 1.26 -- 6.36
GG Initial 0.00 0.05 0.03 0.06 0.05
0.02 0.30
(pH 5.3) 1 M 0.02 0.26 1.78 0.54 ' 1.14
1.16 5.77
Example 15: Hemolysis Study
101001 Hemolysis studies were performed on Formulation KK to demonstrate that
there is no
incompatibility with whole human blood for the bivalirudin products described
herein. The
relevant details of this investigation are provided in Table 15a.
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Table 15a: Hemolvsis Study Desi2n
Study Parameter Hemolysis Study
Test Matrix Whole human blood with K2EDTA as anticoagulant
Test Concentration 2.14 mg/mL
(Blood:Drug Ratio = 1:0.75)
Incubation Conditions 37 5 C for 30 minutes
Centrifugation Conditions (to 2570g for 5 minutes at ambient temperature
separate plasma after
incubation)
No. of Replicates 3
Test Drug Bivalirudin Injection, 5 mg/mL
Test Item Vehicle Bivalirudin Injection Placebo
Reference Drug Angiomax (bivalirudin) for Injection
Negative Control 0.9% NaCl control or Whole blood control
Positive Control 1% Saponin
Measurement Direct measurement of hemoglobin using an Advia 120
hematology(Hb) analyzer (cyanmethemoglobin method)
0/0 Hemolysis Determination Based on Hb content of the sample as a percent of
the Hb
content of the donor, after adjusting for dilution and
interference from the sample (if any)
10101] Briefly, human whole blood was collected from a single donor (K2EDTA
anticoagulant).
The donor blood was analyzed for hemoglobin content in triplicate using an
Advia 120
hematology analyzer. The dynamic mixing of drug infusate in human whole blood
was
simulated in this in vitro study by incubating the diluted test, reference and
placebo items at a
blood:formulation ratio of 1:0.75, corresponding to a final bivalirudin
concentration of 2.14
mg/mL. Blood was mixed with either bivalirudin for injection as described
herein, Angiomax ,
a bivalirudin placebo, or 0.9% saline. The mixture as incubated at 37 C ( 5 C)
for 30 minutes.
Negative controls containing only blood (whole blood control) or blood mixed
with 0.9% saline
at a blood:saline ratio of 1:0.75 (0.9% NaC1 control) were also prepared. A
positive control
containing blood mixed 1:1 with 1% saponin was also prepared. All control
tubes were
incubated at 37 C ( 5 C) for 30 minutes. Each sample was tested in triplicate.
During the
incubation tubes containing the test and reference item mixed with saline were
prepared to check
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if the test or reference item interfered with the absorbance readings. After
incubation, the blood
and saline tubes were centrifuged at 2570g for 5 minutes at ambient
temperature. The
supernatant was tested for hemoglobin on the Advia 120. The percent hemolysis
of each
sample was calculated based on the hemoglobin content of the supernatant,
expressed as a
percentage of hemoglobin content of the donor sample, after correcting for
dilution and
interference from the sample, if any.
[0102] The following acceptance criteria were used for the study: the 1%
Saponin positive
control must demonstrate a % hemolysis value of >25% for the assay run to be
considered
acceptable; and the two negative controls for hemolysis must demonstrate a %
hemolysis value
of <10% for the assay run to be considered acceptable.
[0103] The following criteria were used to assess hemolytic potential: a
treated sample must
demonstrate a percent hemolysis value of >25% to be definitive as positive for
hemolysis; a
treated sample must demonstrate a percent hemolysis value of <10% to be
definitive as negative
for hemolysis; and a treated sample that demonstrates a percent hemolysis
value of >10% and
<25% will be considered indicative, but not definitive of hemolysis.
[0104] The results of the hemolysis study, summarized in Table 15b, showed
that no
hemoglobin, and therefore no hemolysis (0% hemolysis), was detected in samples
which had
been treated with Formulation KK (pH 5.5, 10 mM acetate buffer, with 10% PEG
400) described
herein, the reference listed drug, or the negative controls. Hemoglobin was
successfully detected
in samples which had been treated with positive control (saponin, 100%
hemolysis). The
positive control for hemolysis demonstrated a % hemolysis value of >25%, the
two negative
controls demonstrated a % hemolysis value of <10% and therefore the assay run
was considered
acceptable.
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Table 15b: Summary of Hemolvsis Study Results
Corrected Hemoglobin
Treatment % Hemolysis*
Concentration (g/dL)*
Bivalirudin Injection as described herein 0.0
0.0
Angiomax (bivalirudin) for Injection 0.0 0.0 0.0
0.0
Bivalirudin Injection Placebo as described herein 0.0 0.0 0.0
0.0
Negative Control (0.9% Saline) 0.0 0.0 0.0
0.0
Negative Control (whole blood only) 0.0 0.0 0.0
0.0
Positive Control (1% saponin) 7.47 0.06 100.0 0.8
* Based on average (and standard deviation) of three replicates
[0105] The samples treated with Bivalirudin Injection as described herein,
Angiomax , or the
Bivalirudin Injection Placebo as described herein all had % hemolysis values
of <10% and were
=
therefore considered to be definitively negative for hemolysis.
Example 16: Continuous Infusion Repeat Dose Rat Toxicology Study
[0106] The safety of the degradants (impurities) disclosed in the formulation
of the present
invention was established in a GLP-compliant 14-day continuous infusion repeat-
dose toxicity
study in rats. The 14-day repeat-dose toxicity study compared the toxicity of
an intentionally
degraded exemplary formulation of this invention (Test Article) to the
marketed authorized
generic for Angiomax for Injection (Reference Article) - note that the
authorized generic is the
same product as Angiomax but marketed under a different label (in this case
Sandoz). In this
study, a batch of formulation KK was intentionally heat-degraded at 40 C for
14 days to contain
about 19% total impurities and then continuously intravenously infused 24
hours per day for 14
consecutive days ,at dosages of 36, 100 and approximately 258 mg/kg/day
(maximum feasible
dose), containing 6.8 mg/kg/day, 19 mg/kg/day, and 49 mg/kg/day of impurities,
respectively.
The authorized generic for Angiomax was administered similarly at 258
mg/kg/day, which
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contained a dose of less than 2 mg/kg/day of impurities. Table 16a summarizes
the degradant
levels of the Test and Reference Articles used in the study; the Test Article
was tested on Days 1,
7, and 15 of the study, while the Reference Article was tested immediately and
48 hours after
reconstitution and dilution.
Table 16a: Analytical Characterization of Test and Reference Articles used in
the
Toxicology Study
Reference Drug
Test Drug
(Authorized generic of
Impurity (Formulation KK intentionally degraded at
Angiomax for Injection, Lot
40 C for 14 days)
Attribute 00117), after
reconstitution
48 hours after
Day 1 Day 7 Day 15 Initial
reconstitution
RRT 0.49 1.6% 1.7% 1.8% Not detected Not
detected
[12-20]-
1.0% 1.0% 0.95% 0.08% 0.09%
bivalirudin
[3-20]-
5.9% 6.0% 6.1% 0.12% 0.21%
bivalirudin
[Asp9]-
2.4% 2.5% 2.6% 0.14% 0.16%
bivalirudin
19-10]-
cycloimido 2.5% 2.5% 2.5% 0.06% 0.18%
bivalirudin
[11-121-
cycloimido 2.6% 2.6% 2.6% 0.10% 0.11%
bivalirudin
Total
18.7% 19.1% 19.1% 0.60% 0.77%
Impurities
101071 In the study, Sprague-Dawley rats (10/sex/group) were continuously
intravenously
infused for 24 hours a day for 14 consecutive days (Days 1-14) with placebo
(0.9% sodium
chloride), Test Article vehicle (0.8 mg/mL sodium acetate trihydrate and 100
mg/mL
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polyethylene glycol 400, pH 5.25), Reference Article at approximately 258
mg/kg/day or Test
Article at 36, 100 and approximately 258 mg/kg/day. The infusion rate was 2.5
mL/kg/h for all
groups. A separate set of satellite animals (6/sex/group) were administered
the same set of
materials and used for evaluating the toxicokinetics (TK) of bivalirudin at 5
minutes and 24
hours after termination of dosing (3 animals/sex/group/timepoint). See Table
16b for group
allocation. All animals were sacrificed on Day 16, one day following the end
of the 14-day
infusion.
Table 16b: Group Allocations of Toxicology Study
Group Treatment No. of No. of
Bivalirudin Concentration Degradant
Toxicity Toxico- dose
Animals kinetic Dose (mg/mL)
Animals (mg/kg/
(mg/kg/day)
MF M F day)
1 Placebo- 0.9% 10 10 6 6 0 0 0
sodium chloride
2 Test Article 10 10 6 6 0 0 0
Vehicle
3 Test Article 10 10 6 6 36 0.6 6.8
4 Test Article 10 10 6 6 100 1.37 19
Test Article 10 10 6 6 ¨258 ¨4.3 49
6 Comparator- 10 10 6 6 ¨258 ¨4.3 <2
Reference
Article
Results
-Mortalities-
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[0108] A total of seven animals, three Toxicity animals that were treated with
bivalirudin and
three TK animals, were sacrificed moribund or found dead during the study.
With respect to the
bivalirudin-treated Toxicity animals, the premature sacrifices of these
animals, two high-dose
(258 mg/kg/day) Test Article-treated males and one high-dose Reference Article-
treated male,
were attributed to the pharmacology of bivalirudin. These animals displayed
abnormal clinical
signs consisting of decreased activity, hunched posture, abnormal color or
pallor, piloerection,
cold to touch, and/or irregular breathing prior to their sacrifice. In these
three high-dose (258
mg/kg/day) early decedents, the dark material in the gastrointestinal tract
and hemoglobin
crystals in the lung was consistent with hemorrhage, and the pallor of the
skin and organs was
considered to be due to blood loss. These findings were considered associated
with the mode of
action of bivalirudin (direct-acting thrombin inhibitor).
[0109] With respect to the TK animals, two Test Article-treated animals (one
mid-dose and one
high dose) and one high-dose Reference Article-treated animal were found dead
or sacrificed.
All three animals had thin red fluid within the abdominal cavity; these gross
findings are often
seen in unscheduled decedents and therefore could not be attributed
definitively to the mode of
action of bivalirudin (histopathology was not performed).
-Findings in Surviving Animals-
101101 In the animals that survived until the end of the study, there were no
test article-related
effects on clinical observations, body weights, food consumption, hematology,
coagulation,
urinalysis or organ weights. The only clinical chemistry findings were an
approximate 20-30%
decrease in glucose in both high-dose group male animals (groups 5 and 6),
compared to control,
at the end of dosing on Day 15. These decreases were considered non-adverse
due to their
relatively small magnitude.
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101111 Macroscopically, a single Test Article-treated high-dose female had
unilateral red ovarian
cyst, correlating microscopically with hemorrhage and fibrin deposition within
a corpora luteum.
These effects were considered a consequence of the biological activity of
bivalirudin.
[0112] Procedure-related macroscopic and microscopic findings associated with
catheter
placement and maintenance were present at the infusion site in both control
and treated groups at
similar severity and frequency; these findings consisted of vascular changes
of intimal
proliferation/ fibrosis, thrombus formation, and/or inflammatory cell
infiltrates. Finally, 1
female in the low-dose (36 mg/kg/day) Test Article treated animal had
bacterial infection of the
catheter site with systemic spread to lungs, kidneys and skeletal muscle.
These findings were
consistent with prolonged catheter placement and are known effects and
complications of
infusion studies in rodents.
-Toxicokine tics-
[0113] In the surviving TK animals, plasma concentrations of bivalirudin were
quantifiable at
the first sampling time (5 minutes post-dose on Day 15) for all test article-
treated animals except
for one high-dose (258 mg/kg/day) Reference Article-treated female. It is
noted that this animal
was found to have leaking or disconnected infusion line on two occasions
(observed between
Day 8-9 and between Day 12-13), but the test article was presumably
administered from Day 13-
15 and therefore the cause of the no exposure is unknown. At the 24-hour
timepoint only 7 out
of 23 samples (range 50.5 ¨ 159 ng/mL) had bivalirudin concentrations, all
other samples were
below the lower level of quantitation (50 ng/mL). These results are consistent
with the expected
short half-life of bivalirudin.
[0114] Mean plasma concentrations of bivalirudin at 5 minutes post-dose (C5)
increased
approximately proportionately with increasing dose over the dose range 36 to
¨258 mg/kg/day
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on Day 15 (after termination of dosing) (Table 16c). Mean plasma
concentrations of bivalirudin
at C5 were similar in males treated with the high-dose (250 mg/kg/day) of MAIA
Bivalirudin
Injection or Sandoz Bivalirudin, and only slightly lower (approximately 20%
lower) in the
female treated with MAIA Bivalirudin than in the females treated with Sandoz
Bivalirudin.
However, it must be noted that data are available from just one female for the
MAIA product,
due to premature deaths or inability to collect blood.
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Table 16c: Plasma Concentrations of Bivalirudin (C5) Five Minutes Following
End of
Infusion
Bivalirudin dose level C5 (ng/mL) C5/D [(ng/mL)/(mg/kg)]
(mg/kg/day) Day 15 Day 15
Males Females Males Females
36 (Test Article) 961 283 593 88 26.7 16.5
100 (Test Article) 2500 + 200 1800 300 25.0 18.0
¨258 (Test Article)a 7240 120 5360* 28.1 20.8*
¨258 (Reference Article)b 7270** 6700** 28.2** 26.0**
*based on one animal. **based on two animals, 'One high-dose female was found
dead prior to study termination
and no sample could be collected from a second high-dose female animal despite
several attempts. "One high-dose
male animal was sacrificed moribund prior to the end of dosing; data from
female Animal 6633 was not included in
the mean as the value was considered an outlier.
Conclusion
[0115] A 14-day infusion of the Test Article up to 100 mg/kg/day was tolerated
with no test
article effects observed. At the highest dose (258 mg/kg,/day), the Test
Article and Reference
Article caused mortality that was related directly or indirectly to
hemorrhage, which is consistent
with the pharmacology of the product. The effects consisted of dark material
in the
gastrointestinal tract, pallor of the skin and organs, centrilobular necrosis
in the liver and
hemoglobin crystals in the lungs. Hemorrhage was also observed in the ovary in
a 258
mg/kg/day female given Test Article that survived to study termination. In
high-dose (258
mg/kg/day) animals that survived until the end of the study, only a minor, non-
adverse decrease
in glucose was observed in males with both the Test and Reference Articles.
Due to the test
article-related premature deaths, the NOAEL of the Test Article was determined
to be 100
mg/kg/day, which corresponds with a Cmax of 2500 +200 ng/ml in males and 1800
300 ng/mL
in females. Overall, there were no differences noted between rats administered
the Test Article
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and rats administered the Reference Article. Additionally, degradant
impurities of the Test
Article (19%) up to an impurity dose of 49 mg/kg/day intravenously infused for
14 continuous
days did not induce any unexpected toxicities, thereby qualifying their
safety.
Example 17: In-Vitro Pharmacodynamic Study
101161 An in-vitro study was performed to compare the (pharmacodynamic)
effects of an
exemplary ready-to-use liquid formulation of this disclosure (Formulation KK)
at the end of its
shelf-life and the Angiomax (bivalirudin) for Injection, 250 mg/vial) on the
(i) Prothrombin Time
(PT) assay, (ii) the activated Partial Thromboplastin Time (aPTT) assay and
(iii) the Thrombin
Time (TT) assay over the therapeutic concentration range in male and female
plasma samples.
The purpose of the study was to demonstrate that the degradant levels found in
exemplary
formulations of this invention do not adversely affect the efficacy of
bivalirudin in terms of its
anticoagulant (pharmacodynamic) activity.
101171 In this study, a batch of formulation KK was intentionally heat-
degraded at 30 C for 10
days to contain about 7% total impurities (Test Drug) and compared against
Angiomax for
Injection (Reference Drug) for PT, aPTT,a and TT in human plasma. Table 17a
summarizes the
impurity levels in the Test and Reference Drug used in the study.
Table 17a: Impurity Profile of Test and Reference Drug used in the
Pharmacodynamic
Study
Test Drug Reference Drug
Attribute (Formulation KK, (Angiomax for injection,
30 2 C for 10 days) Lot 00111)$
RRT 0.49 0.30% 0.09%
[12-20]-bivalirudin 0.42% 0.15%
[3-20J-bivalirudin 2.17% 0.09%
[Aspl-bivalirudin 0.94% 0.23%
[9-10J-cycloimido bivalirudin 1.17% Not detected
[11-12]-cycloimido bivalirudin 1.64% 0.03%
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Test Drug Reference Drug
Attribute (Formulation KK, (Angiomax for Injection,
30 2 C for 10 days) Lot 00111)I
Total Impurities 7.22% 0.70%
[0118] This study employed a design that is similar to an open controlled 2-
way replicated
crossover study that is traditionally used for the assessment of
bioequivalence between a Test
Drug and a Reference Drug; the study parameters are summarized in Table 17b.
Table 17b: Pharmacodvnamic Study Parameters
Study Parameter Pivotal Study (MAIA-BVN-CLOT-003)
Mat Individual donor plasma (male and female)
("subject"),
rix
platelet-poor
Five concentrations: 1.0, 2.5, 5.0, 10.0, and 20.0 g/mL
aPTT Test Concentrations
bivalirudin
Four concentrations: 5.0, 10.0, 15.0, and 20.0 pig/mL
PT Test Concentrations
bivalirudin
Three concentrations: 0.5, 0.75, and 1.0 vg/mL
TT Test concentrations
bivalirudin
Number of subjects 50 subjects (25 male and 25 female)
90% confidence intervals of the estimated ratio of
geometric mean (RGM) of the parameter between the
Test and the Reference (Test/Reference) will be within
Equivalence criterion
0.90 to 1.11 (or 90.0% to 111.0%), original scale (-0.154
to +0.1054 natural log scale), inclusive for all
coagulation parameters at all tested concentration levels
[0119] The PT, aPTT, and TT measurements were conducted using in-vitro
diagnostic (IVD)
approved instrument applications provided by Siemens Healthcare Inc. for the
BCS XP
coagulation analyzer. Individual donor plasma samples (100 mL) were inspected
visually for
clotting, hemolysis, icterus, and lipemia or excess turbidity; plasma
exhibiting any of these
conditions were excluded from the study. Each plasma subject was used to
prepare plasma
samples spiked with either Test Drug or the Reference Drug at the target
concentrations. A
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blank control (plasma only) and a vehicle control (0.9% sodium chloride
injection) were also
prepared.
[0120] The equivalence of the Test and the Reference products for the
endpoints was tested
using two one-sided tests at the 0.05 level of significance. SAS1 PROC MIXED
procedure with
model statements and variance covariance structure similar to Appendix E of
Statistical
Approaches to Establishing Bioequivalence (FDA Guidance for Industry 2001) was
used to carry
out the Two One-sided Test (TOST) procedure by fitting mixed effects models to
the log-
transformed aPTT, PT, and TT measures. The model had gender and formulation as
fixed
effects. A separate mixed effects model was used to analyze each measure at
each dose using
restricted maximum likelihood estimation, the Kenward and Roger method of
calculating the
degrees of freedom and the variance-covariance structure FA0(2). Formulation
and subjects
within each product were the random and repeated measures, respectively.
[0121] Table 17c summarizes the results of the equivalence analysis for all
plasma samples; the
results for the 90% confidence interval of the ratios of geometric means
between the Test and the
Reference are within are within 98.9% to 103.4% for all coagulation parameters
at all tested
concentration levels for all evaluable samples; thus the results meet the
acceptance criterion of
90% to 111% (-0.1054 to +0.1054 on log scale) for all coagulation parameters
at all tested
concentration levels.
Table 17c: Equivalence Analysis of All Samples from Pharmacodynamic Study
Geometric Mean Clotting Ratio of Geometric Means
Parameter Dose ( g/mL) N Time (seconds) (90% CI)
Test Reference (%)
Ot 50 10.89 10.62 102.5
(102.2, 102.8)
PT 5.0 50 37.12 36.35 102.1
(100.9,103.4)
10.0 50 72.98 71.80 101.6
(100.6, 102.7)
'SAS is a registered trademark of the SAS Institute Inc.. Cary, NC.
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Geometric Mean Clotting Ratio of Geometric Means
Parameter Dose (pg/mL) N Time (seconds) (90% CI)
Test Reference (0/0)
15.0 50 105.01 103.54 101.4
(100.3, 102.5)
20.0 50 134.56 132.19 101.8
(100.8, 102.8)
Ot 50 26.17 25.41 103.0
(102.7, 103.3)
1.0 50 55.33 55.11 100.4
(100.1, 100.7)
2.5 50 70.99 70.41 100.8
(100.4, 101.2)
aPTT
5.0 50 87.25 86.80 100.5
(100.2, 100.9)
10.0 50 108.46 108.46 100.0
(98.9, 101.2)
20.0 50 139.41 138.16 100.9
(100.5, 101.3)
Ot 50 20.74 20.34 102.0
(101.4, 102.6)
0.5 50 251.03 246.63 101.8
(100.7, 102.9)
TT
0.75 50 342.17 338.18 101.2
(100.0, 102.4)
1.0 48* 415.28 407.8 101.8
(100.7, 103.0)
N = number of subjects; t Test and Reference correspond to the blank (plasma
only) and the vehicle
control (0.9% saline); * two subjects yielded no measurable clotting time ("no
clot", clotting time > 500
sec) for all replicates of the test and reference drug samples; thus, clotting
data were available for only 48
subjects
control (0.9% saline)
101221 The results of the in vitro pharmacodynamic study clearly demonstrate
that exemplary
formulations of this disclosure with degradant levels at or above its shelf
life levels are
equivalent to Angiomax for Injection in terms of its anticoagulant activity,
as measured by PT,
aPTT, and TT at all concentrations tested over the therapeutic range.
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