1
FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
TITLE OF THE INVENTION
LYOPHILIZED PHARMACEUTICAL COMPOSITIONS OF COPPER
HISTIDINATE
APPLICANT(S):
a) NAME ZYDUS LIFESCIENCES LIMITED
b) NATIONALITY INDIAN
c) ADDRESS ZYDUS CORPORATE PARK, SCHEME NO. 63,
SURVEY NO. 536, KHORAJ (GANDHINAGAR),
NR. VAISHNODEVI CIRCLE, AHMEDABAD,
GANDHINAGAR, GUJARAT, INDIA, 382481
PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which
it has to be performed.
2
FIELD OF THE INVENTION
The present invention relates to lyophilized pharmaceutical compositions of
copper histidinate. The invention also relates to processes for preparing such
compositions.5
BACKGROUND OF THE INVENTION
Copper is an essential nutrient for the body. Together with iron, it enables the
body to form red blood cells. It helps maintain healthy bones, blood vessels,
nerves, and immune function, and it contributes to iron absorption. Copper10
deficiency is rare and causes both hematological and neurological diseases.
Copper deficiency can also result from a rare genetic disorder called Menkes
disease. Researchers have tried to develop various formulations containing copper
to treat copper deficiency.
15
Sherwood et al., Journal of Inherited Metabolic Disease, 12 Suppl., 2, 393-396
(1989), discloses liquid formulation of copper histidinate.
A book “Small molecule therapy for genetic disease” by Cambridge University
press (ISBN 978-0-521-51781-2) in Chapter No. 14 named, “Small copper20
complexes for treatment of acquired and inherited copper deficiency syndromes”,
by Stephen G. Kaler, pages 202-212, discloses a freeze-dried product of copper
histidine.
Basak et al. (Journal of Inorganic Biochemistry, 51 (1-2), page no. 415 (1993)),25
discloses that, in order to circumvent the stability problems associated in solution,
copper-histidine was formulated in freeze-dried form. It also discloses that the
presence of sodium chloride causes aggregations during lyophilization resulting in
a turbid solution when reconstituted with sterile water. It also discloses that, the
freeze-dried copper-histidine with no additives is by far the best as reflected by30
3
the stability studies at different storage conditions. Sarkar et al., The Journal of
Pediatrics, 123 (5), 828-30 (1993), cites Basak et al., and reiterates the same.
Kaler et al., Biochemical and Molecular Medicine, 57, 37-46 (1996), discloses
copper histidine prepared as a freeze-dried product for Menkes disease. It also5
discloses that sterile water was used to dissolve copper histidine prior to freeze-
drying, and 0.9% normal saline was used to resuspend the freeze-dried product.
International (PCT) Publication No. WO/2010/042102 discloses preparation and
administration of copper histidine. It also discloses a process for dissolving copper10
chloride dihydrate, L-histidine, and sodium hydroxide in water to prepare a
solution and freeze drying the solution to obtain freeze- dried product. It also
discloses reconstitution of freeze-dried product with 0.9% sodium chloride
injection.
15
There is still a need for alternate lyophilized pharmaceutical compositions of
copper histidinate having improved stability and meeting other requirements as set
forth in the description below.
SUMMARY OF THE INVENTION20
In one general aspect, the present invention provides a lyophilized pharmaceutical
composition comprising copper histidinate, wherein the lyophilized
pharmaceutical composition, for example, a lyophilized powder or cake, has
improved parameters, such as good aesthetic appearance.
25
Embodiments of the lyophilized pharmaceutical composition may include one or
more of the following features. The composition may further comprise one or
more pharmaceutically acceptable excipients, for example, one or more
lyoprotectants, one or more pH adjusting agents, optionally one or more
antioxidants, optionally one or more buffering agents, and optionally one or more30
preservatives.
4
The lyoprotectant may be mixed with copper histidinate solution to provide a pre-
lyophilization solution and then the solution may be lyophilized to provide a
lyophilized composition comprising copper histidinate and a lyoprotectant.
Copper histidinate present in the pre-lyophilization solution may be formed in-situ5
upon mixing of copper(II) chloride dihydrate solution and histidine solution.
In another general aspect, the present invention provides a process for preparing a
lyophilized pharmaceutical composition of copper histidinate, wherein the process
comprises the steps of:10
(a) preparing copper(II) chloride dihydrate solution,
(b) preparing histidine solution,
(c) mixing the solutions obtained at steps (a) and (b),
(d) adding one or more lyoprotectants to the solution obtained at step (c),
(e) adding one or more pH adjusting agents to have a pH of solution to about 7.0 -15
7.5, and
(f) performing lyophilization (freeze drying) of the solution obtained at step (e).
In another general aspect, the present invention provides a glass vial/SiO2
vial/quartz vial, a glass ampoule, a glass bottle, a plastic bottle, a plastic bag, a20
glass/plastic pre-filled syringe, or a glass/plastic single/dual chamber cartridge
containing a lyophilized pharmaceutical composition comprising copper
histidinate and one or more lyoprotectants.
In another general aspect, the present invention provides a method of treating25
Menkes disease in a patient in need thereof, comprising administering to the
patient a solution comprising copper histidinate, one or more pharmaceutically
acceptable excipients, and a vehicle, wherein the administration is through a
subcutaneous route.
30
5
The details of one or more embodiments of the invention are set forth in the
description below. Other features, objects and advantages of the invention will be
apparent from the description.
BRIEF DESCRIPTION OF THE FIGURES5
Figure 1 depicts physical stability of blue color powder without any lyophilization
defect of composition of the invention
Figure 2 depicts blue color solid/powder with one or more of the lyophilization
defects of composition of comparative example
10
DETAILED DESCRIPTION OF THE INVENTION
The inventors of the present invention have found that when a lyoprotectant, for
example, sodium chloride, is used for preparing a lyophilized pharmaceutical
composition of copper histidinate, the composition having improved parameters15
can be obtained. The lyophilized pharmaceutical composition, for example, a
lyophilized powder or cake, having improved parameters, such as good aesthetic
appearance (lyophilized powder or cake essentially free from any aggregates,
lumps, and/or lyophilization defects, viz., melt back, collapsed cake, shrinkage of
cake, slanted cake, etc.) can be obtained.20
The inventors have also found that when the lyophilized pharmaceutical
composition is mixed with sterile water for injection, a clear solution
(reconstituted) having improved physical parameters can be obtained. This also
allows usage of sterile water for injection as a reconstitution vehicle and avoids25
dependency on saline solution as a reconstitution vehicle. The reconstituted
solution having improved physical parameters, such as solution being clear (not
hazy or turbid / without any visible particles / free of any particles) can be
obtained. Such a clear solution being essentially free from any particulate matter
is suitable for administration through a parenteral route to a human being in need30
thereof, for example, a patient in need thereof.
6
In one embodiment, the present invention provides a lyophilized pharmaceutical
composition comprising copper histidinate and one or more pharmaceutically
acceptable excipients (additives). The lyophilized pharmaceutical composition of
the invention is a lyophilized powder or cake for reconstitution which is5
essentially free from any lyophilization defects, for example, melt back, collapsed
cake, shrinkage of cake, slanted cake. The lyophilized powder or cake is
essentially free from any aggregates, lumps, and/or lyophilization defects.
Examples of pharmaceutically acceptable excipients may include, but not limited10
to, lyoprotectants, pH adjusting agents, antioxidants, buffering agents, and
preservatives.
Examples of suitable lyoprotectants may include, but are not limited to, sodium
chloride, mannitol, sucrose, dextrose, lactose, and cyclodextrin derivatives15
(hydroxypropyl-beta-cyclodextrin, sulphobutylether-beta-cyclodextrin, etc.), or
any combination thereof. For example, sodium chloride is a suitable lyoprotectant.
The lyophilized pharmaceutical composition of the invention, upon mixing with
sterile water for injection, provides a clear solution, wherein the sodium chloride
is in a concentration of between about 1 mg/mL and about 18 mg/mL. The20
lyoprotectant may be mixed with copper histidinate solution to provide a pre-
lyophilization solution and then the solution is lyophilized to provide a
lyophilized composition comprising copper histidinate and a lyoprotectant.
Copper histidinate present in the pre-lyophilization solution may be formed in-situ
upon mixing of copper(II) chloride dihydrate solution and L-histidine solution.25
The lyophilized powder or cake of the present invention comprising
lyoprotectants have good aesthetic appearance, for example, essentially free from
any aggregates, lumps, and/or lyophilization defects, viz., melt back, collapsed
cake, shrinkage of cake, slanted cake, etc. The presence of lyoprotectant decreases
the collapse temperature, for example, presence of sodium chloride decreases the30
collapse temperature from about -70 0 C to about -27 0 C and allows efficient
7
lyophilization. Additionally, the lyophilized powder or cake of the invention
comprising sodium chloride upon reconstitution with suitable reconstitution
vehicle, for example, sterile water for injection, provides a clear solution (not hazy
or turbid / without any visible particles / free of any particles) which remains clear
after storage for 24 hours at 100°C. The lyophilized powder or cake of the5
invention comprising pharmaceutically acceptable excipient / lyoprotectant /
sodium chloride may have higher surface area compared to that of the lyophilized
powder or cake without such excipient / lyoprotectant / sodium chloride.
The term “about” as used herein, refers to encompass +/- 20%, 15%, 10%, 5%,10
2%, 1%, 0.5%, or 0.25% of the numerical value of the number with which it is
being used.
Examples of suitable pH adjusting agents may include, but are not limited to,
sodium hydroxide, potassium hydroxide, hydrochloric acid, and phosphoric acid,15
or any combination thereof. Sodium hydroxide present in a lyophilized
pharmaceutical composition is in a sufficient amount to produce such a clear
solution upon mixing with sterile water for injection which has a pH value of
between about 6.0 and about 8.0, for example, between about 7.0 and about 7.5.
20
Examples of suitable antioxidants may include, but are not limited to,
monothioglycerol, ascorbic acid, l-cysteine, sodium sulfite, sodium bisulfite,
disodium edetate, butylated hydroxytoluene (BHT), butylated hydroxyanisole
(BHA), propyl gallate, sodium ascorbate, erythorbic acid, potassium metabisulfite,
propionic acid, sodium formaldehyde sulphoxylate, reduced glutathione, thiourea,25
n-acetylcysteine, methionine, alkyl gallate, including propyl gallate, vitamin E, or
other tocopherol analogs, including tocopherol acetate or TPGS, or any
combination thereof.
Examples of suitable buffering agents may include, but are not limited to, acetate30
buffer (e.g. sodium acetate and acetic acid etc.), phosphate buffer (e.g. monobasic
8
sodium phosphate, dibasic sodium phosphate etc.), citrate buffer (e.g. anhydrous
citric acid and trisodium citrate dihydrate etc.), carbonate buffer, lactate buffer,
glycine buffer, borate buffer (boric acid/potassium chloride), tris buffer,
tromethamine buffer, or any combination thereof.
5
Examples of suitable preservatives may include, but are not limited to,
chlorobutanol, benzalkonium chloride, methyl paraben, propyl paraben, benzoic
acid, sodium benzoate, sorbic acid, benzethonium chloride, cetyl pyridinium
chloride, benzyl bromide, phenylmercury nitrate, phenylmercury acetate,
thiomersal, merthiolate, chlorhexidine, phenylethyl alcohol, quaternary10
ammonium chloride, sodium benzoate, sodium propionate, or any combination
thereof.
In one embodiment, the present invention provides a lyophilized pharmaceutical
composition comprising copper histidinate, one or more lyoprotectants, and one or15
more pH adjusting agents. The lyophilized pharmaceutical composition may
comprise copper histidinate, sodium chloride, and sodium hydroxide. The
lyophilized pharmaceutical composition in each vial may comprise copper
histidinate in an amount of between about 0.500 mg and about 10.000 mg, for
example, between about 0.725 mg and about 6.100 mg, sodium chloride in an20
amount of between about 1 mg and about 18 mg, and sodium hydroxide in a
sufficient amount to adjust the pH between about 7.0 and about 7.5.
In another embodiment, the lyophilized pharmaceutical composition comprising
copper histidinate, sodium chloride, and sodium hydroxide, wherein the25
composition contains sodium chloride in an amount of between about 1 mg and
about 18 mg, for example, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9, mg, 10
mg, 11, mg, 12 mg, 13, mg, 14 mg, 15 mg, 16 mg, or 17 mg.
In another embodiment, the lyophilized pharmaceutical composition comprising30
copper histidinate, sodium chloride, and sodium hydroxide provides a weight ratio
9
of copper histidinate to sodium chloride of between about 1:0.1 and about 1:7.0,
for example, between about 1:0.3 and about 1:6.3. The lyophilized pharmaceutical
composition retains at least about 90 % of the potency of copper histidinate (%
assay) in the pharmaceutical composition, as determined by Ultraviolet–visible
spectroscopy, after storage for 1 month or more, for example, 6 months, 125
months, 18 months, 24 months, or 36 months at 50 0 C and 80 %RH, at 40 0 C and
75 %RH, at controlled room temperature (between about 20 0 C and about 25 0 C)
and 60 %RH, at 2 0 C to 8 0 C, or at -15 0 C to -20 0 C. The lyophilized
pharmaceutical composition can be supplied at storage condition either at 2 0 C to
8 0 C or at controlled room temperature.10
In another embodiment, the lyophilized pharmaceutical composition comprising
copper histidinate, sodium chloride, and sodium hydroxide has % water content
(% by weight of the composition) not more than 4% immediately after its
manufacturing (initial) as well as during its storage at various conditions. For15
example, the % water content (% by weight of the composition) is not more than
about 4 %, for example, not more than 3 %, after storage of the lyophilized
pharmaceutical composition for 1 month or more, for example, 6 months, 12
months, 18 months, 24 months, or 36 months at 40 0 C and 75 %RH, at 25 0 C and
60 %RH, or at 2 0 C to 8 0 C. The % water content (% by weight of the20
composition) of the lyophilized pharmaceutical composition after storage for 6
months at 25 0
C and 60 %RH do not change / increase more than 5.0 %, for
example, 4.0 %, 3.0 %, or 2.0 %, compared to that of the composition at an initial
level. The % water content (% by weight of the composition) of the lyophilized
pharmaceutical composition after storage for 6 months at 2 0 C to 8 0 C does not25
change / increase more than 5.0 %, for example, 4.0 %, 3.0 %, or 2.0 %, compared
to that of the composition at an initial level. The determination of % water content
can be performed by Karl Fischer Coulometer.
In one embodiment, the present invention provides a clear solution (reconstituted)30
upon mixing of the lyophilized pharmaceutical composition comprising copper
10
histidinate, sodium chloride, and sodium hydroxide with sterile water for
injection. The clear solution is provided in less than 60 seconds, for example, 45
seconds, 30 seconds, 15 seconds, or 10 seconds, after the mixing. The clear
solution comprises copper histidinate in a concentration of about 2.9 mg/mL,
sodium chloride in a concentration of about 9 mg/mL, and sodium hydroxide in a5
sufficient amount to provide pH of between about 7.0 and about 7.5, which can be
administered to a human being in need thereof without any further dilution. The
administration can be through a subcutaneous injection route with a dose volume
of about 0.5 mL of the clear solution (2.9 mg/mL of copper histidinate).
10
As used herein, the term “clear solution” means a solution which is essentially
free from any visible particles / particulate matter that can be observed on visual
inspection and shall comply with the following limit for sub-visible particulate
matters: NMT 6000 particles/container of NMT 25 μm size and NMT 600
particles/container of NMT 10 μm size, as determined by laser light scattering15
microscopy. The clear solution may provide % transmittance, when measured at
650 nm, not less than 95%, for example, not less than 96%, not less than 97%, not
less than 98%, not less than 99%, not less than 99.5%, not less than 99.6%, not
less than 99.7% or not less than 99.8%.The clear solution may provide the value
of absorbance not more than 1 AU, for example, not more then 0.75, 0.5, 0.4, 0.3,20
0.2, 0.1 or 0.05.
The clear solution of the invention may provide osmolality value of between
about 300 mOsmol/kg and about 380 mOsmol/kg.
25
The clear solution (reconstituted solution) of the invention is suitable for
administration through various parenteral routes, for example, intramuscular route
(intradeltoid or intragluteal), subcutaneous route, or intravenous route.
In one embodiment, the lyophilized pharmaceutical composition comprising30
copper histidinate, sodium chloride, and sodium hydroxide may retain at least 90
11
% of the copper histidinate (% assay), as determined by Ultraviolet–visible
spectroscopy, after storage for more than 1 month, for example, 3 months, 6
months, 12 months, 18 months, 24 months, 30 months, or 36 months, at 40 0 C and
75 %RH, controlled room temperature and 60 %RH, 2 0 C to 8 0 C, or at -15 0 C to -
20 0 C .5
The lyophilized pharmaceutical composition does not contain total impurities
more than 2.0 %, for example, 1.5 %, 1.0 %, or 0.5 %, by weight of copper
histidinate, as determined by HPLC, after storage for 6 months, 12 months, 18
months, 24 months, 30 months, or 36 months at 40 0 C and 75 %RH, controlled10
room temperature and 60 %RH, 2 0 C to 8 0 C, or at -15 0 C to -20°C. The
lyophilized pharmaceutical composition does not contain 2-Oxo-L-Histidine more
than 0.5 %, for example, 0.2 %, or 0.1 %, by weight of copper histidinate, as
determined by HPLC, after storage for 6 months, 12 months, 18 months, 24
months, 30 months, or 36 months at 40 0 C and 75 %RH, controlled room15
temperature and 60 %RH, 2 0 C to 8 0 C, or at -15 0
C to -20°C.
In one embodiment, the reconstituted solution of the present invention remains
clear immediately after preparing it as well as after its storage for 1 month at 25
0 C and 60 %RH and/or after its storage for 2 months at 2 0 C - 8 0 C.20
In one embodiment, the present invention provides a process for preparing a
lyophilized pharmaceutical composition comprising copper histidinate, sodium
chloride, and sodium hydroxide, the process comprising the steps of:
(a) dissolving copper(II) chloride dihydrate in water, for example, sterile water for25
injection, to obtain copper(II) chloride dihydrate solution,
(b) dissolving L-histidine in sterile water for injection to obtain a histidine
solution,
(c) mixing the solutions obtained at steps (a) and (b),
(d) adding a lyoprotectant to the solution obtained at step (c),30
(e) adding a pH adjusting agent to have a solution of pH 7.0 - 7.5,
12
(f) filtering the solution obtained at step (e) using aseptic filtration,
(g) filling the solution obtained at step (f) in vials, and
(h) performing lyophilization of the vials obtained at step (g).
In another embodiment, the present invention provides a process for preparing a5
lyophilized pharmaceutical composition comprising copper histidinate, sodium
chloride, and sodium hydroxide, the process comprising the steps of:
(a) dissolving lyophilized copper histidinate in sterile water for injection,
(b) adding a lyoprotectant to the solution obtained at step (a),
(c) adding a pH adjusting agent to have a solution of pH 7.0 - 7.5,10
(d) filtering the solution obtained at step (c) using aseptic filtration,
(e) filling the solution obtained at step (d) in vials, and
(f) performing lyophilization of the vials obtained at step (e).
The lyophilized copper histidinate may be obtained by a process comprising the15
steps of:
(a) dissolving copper(II) chloride dihydrate in water, for example, sterile water for
injection, to obtain copper(II) chloride dihydrate solution,
(b) dissolving L-histidine in water, for example, sterile water for injection, to
obtain a histidine solution,20
(c) mixing the solutions obtained at steps (a) and (b), and
(d) performing lyophilization of the solution obtained at step (c).
The lyophilized pharmaceutical composition may be suitable to undergo a
sterilization process to provide a sterile composition. The examples of suitable25
sterilization process may include, but are not limited to, aseptic membrane
filtration sterilization.
In one embodiment, the lyophilized pharmaceutical composition of the present
invention is supplied/provided in a suitable container (primary packaging30
material), for example, in a glass vial/SiO2 vial/ quartz vial, a glass ampoule, a
13
glass bottle, a plastic bottle, a glass/plastic pre-filled syringe, or a glass/plastic
single/dual chamber cartridge.
In another embodiment, the lyophilized pharmaceutical composition of the present
invention is supplied/provided, in a dual chamber injector device wherein the first5
chamber contains copper histidinate lyophilized powder and the second chamber
contains sterile water for injection. At the time of drug administration to a human
being in need thereof, for example, to a patient in need thereof, mixing of the
content of both the chambers provide a clear solution. The dual chamber injector
device may contain various constituent parts, for example, a glass or plastic barrel,10
a plunger, one or more rubber stoppers, a needle, and a cap.
In one embodiment, the present invention provides a method of treating Menkes
disease comprising administering to a patient in need thereof a solution
comprising copper histidinate, one or more pharmaceutically acceptable15
excipients, and a vehicle, wherein the administration is through a subcutaneous
route. The subcutaneous administration provides 0.5 mL dose of 2.9 mg/mL of
copper histidinate solution (1.45 mg copper histidinate). The solution is a
reconstituted solution provided by reconstitution of the lyophilized
pharmaceutical composition comprising copper histidinate and one or more20
pharmaceutically acceptable excipients (for example, lyoprotectants) in the
vehicle (for example, sterile water for injection). The administration is through a
parenteral route, for example, subcutaneous route, intravenous route, or
intramuscular route.
25
In one embodiment, the lyophilized pharmaceutical composition of the present
invention upon mixing with water, for example, sterile water for injection,
provides a clear solution for administration through parenteral route, wherein
upon administration to a human being in need thereof, for example, a patient in
need thereof, the administration may provide a very mild pain intensity. The30
visual analog scale score (VAS score) is a tool to determine intensity of perceived
14
pain. A human being / patient after administration, for example, after 1 minute of
administration, may indicate his/her perceived pain intensity along a 100 mm
horizontal line, where 0 means “no pain” and 100 means “unbearable pain”. The
administration according to the present invention may provide a VAS score value
below 80, for example, below 50, below 30, below 16, below 10, or 0.5
In another embodiment, the present invention provides methods of treating low
copper levels in blood plasma (copper deficiency) of a human being, for example
a patient, comprising administering to a human being in need thereof, for
example, a patient in need thereof, a solution comprising copper histidinate, one10
or more pharmaceutically acceptable excipients, and a vehicle, wherein the
administration is through parenteral route. The solution is a reconstituted solution
provided by reconstitution of the lyophilized pharmaceutical composition
comprising copper histidinate and one or more pharmaceutically acceptable
excipients (for example, lyoprotectants) in the vehicle (for example, sterile water15
for injection).
Abbreviations:
μm: Micrometer
mg: Milligram20
mL: Millilitre
RH: Relative Humidity
°C: Degree Centigrade / Celsius
NMT: Not More Than
q.s.: Quantity Sufficient25
AU: Absorbance Units
mOsm/kg: Milliosmoles Per Kilogram
HPLC: High-performance liquid chromatography
1M: 1 month
2M: 2 months30
15
The invention is further illustrated by the following examples which are provided
to be exemplary of the invention and do not limit the scope of the invention.
While the present invention has been described in terms of its specific
embodiments, certain modifications and equivalents will be apparent to those
skilled in the art and are intended to be included within the scope of the present5
invention.
Example 1:
Table 1:
Sr.
no.
Ingredients Quantity
1 Copper(II) chloride dihydrate 1.345 mg/mL
2 L-Histidine 2.450 mg/mL
3 Sodium chloride 9.000 mg/mL
4 Sodium hydroxide q.s. to pH
5 Sterile Water for Injection q.s. to 1 mL
10
Process:
Solution of Copper(II) chloride dihydrate:
1. Copper(II) chloride dihydrate was added in water for injection.
15
Solution of Histidine:
2. L-Histidine was added in water for injection.
Lyophilized powder / cake of copper histidinate:
3. Solution of copper(II) chloride dihydrate and solution of histidine were mixed.20
4. Sodium chloride was added to the above solution obtained at step 3.
5. pH was adjusted to 7.0 - 7.5 using sodium hydroxide.
6. The solution obtained at step 5 was filtered using aseptic membrane filtration.
7. The solution obtained at step 6 was filled in glass vials.
16
8. The vials obtained at step 7 were lyophilized to obtain lyophilized powder / cake.
Comparative example:
Table 2:
Sr.
no.
Ingredients Quantity
1 Copper(II) chloride dihydrate 1.345 mg/mL
2 L-Histidine 2.450 mg/mL
3 Sodium hydroxide q.s. to pH
4 Sterile Water for Injection q.s. to 1 mL
Process:5
Solution of Copper(II) chloride dihydrate:
1. Copper(II) chloride dihydrate was added in water for injection.
Solution of Histidine:
2. L-Histidine was added in water for injection.10
Lyophilization:
3. Solution of copper(II) chloride dihydrate and solution of histidine were mixed.
4. pH was adjusted to 7.0 - 7.5 using sodium hydroxide.
5. The solution obtained at step 4 was filtered using aseptic membrane filtration.15
6. The solution obtained at step 5 was filled in glass vials.
7. The vials obtained at step 6 were lyophilized.
The lyophilized compositions of Example 1 (step 8) and comparative example
(step 7), were tested for their physical stability, and the results are reported in20
Table 3A below.
Table 3A:
Test With sodium chloride Without sodium
17
chloride
T0 25 0 C/
60
%RH
2-8
0 C
40 0 C/
75
%RH
50
0 C/
80
%RH
T 0 25 0 C/
60
%RH
2-8 0 C
6M 12M 6M 1M 1M 1M 2M
Description * * * * * ** ** ** **
*Blue color powder without any lyophilization defect (melt back, collapsed cake,
shrinkage of cake, slanted cake) as shown in Figure 1.
** Blue color solid/powder with one or more of the lyophilization defects (melt5
back, collapsed cake, shrinkage of cake, slanted cake) as shown in Figure 2.
It is evident from a comparison of the physical stability data of lyophilized
compositions of Example 1 (step 8) and comparative example (step 7), provided
in the above Table 3A, that sodium chloride provides a better physical stability to10
the lyophilized composition, which is free of any lyophilization defects.
The lyophilized composition of Example 1 (step 8) was tested for its chemical
stability, and the results are reported in Table 3B below.
15
Table 3B:
Test at 6M stability 2-8 0 C 25 0 C/ 60
%RH
40 0 C/ 75
%RH
% Assay (By Ultraviolet–visible
spectroscopy)
113.90 112.00 113.00
2-Oxo-L-Histidine BQL BQL BQL
Total impurities 0.0 0.0 0.0
18
BQL = Below Quantification Limit
2-Oxo-L-Histidine: 2-amino-3-(2-oxo-2,3-dihydro-1H-imidazol-4-yl) propanoic
acid
5
The lyophilized compositions of Example 1 (step 8) and comparative example
(step 7), were tested for % water content (% by weight of the composition) and
the results are reported in Table 3C below, as determined by Karl Fischer
Coulometer (Instrument: Metrohm 831 KF coulometer).
10
Table 3C:
Composition Storage / time
point
Water
content (%)
Water content (%)
change compared to
initial
With sodium
chloride
T0 - 0.89 0
25 0 C/ 60
%RH
6M 2.78 1.89
2-8 0 C 6M 2.65 1.76
40 0 C/ 75
%RH
6M 2.84 1.95
Without sodium
chloride
T0 - 1.89 0
25 0 C/ 60
%RH
6M 9.01 7.12
2-8 0 C 6M 7.86 5.97
40 0 C/ 75
%RH
6M 9.29 7.40
It is evident from a comparison of the % water content data for lyophilized
compositions of Example 1 (step 8) and comparative example (step 7), provided
in the above Table 3C, that (i) % water content (% by weight of the composition)15
of the lyophilized composition containing sodium chloride is not more than 4%
19
immediately after its manufacturing (initial) as well as during its storage under
various temperature/humidity conditions, and (ii) % water content (% by weight
of the composition) of the lyophilized composition containing sodium chloride,
after storage for 6 months at various conditions do not change / increase more
than 5.0 % compared to that of the composition at an initial level.5
The lyophilized compositions of Example 1 (step 8) and comparative example
(step 7), were mixed separately with a sufficient quantity of sterile water for
injection to prepare two separate reconstituted solutions having a concentration of
copper histidinate at about 2.9 mg/mL, and such reconstituted solutions were10
tested for physical and chemical parameters, and the results are reported in Table
3D below.
Table 3D: Reconstituted solution results (Reconstitution vehicle: Sterile
Water for injection)15
Test With sodium
chloride
Without sodium chloride
Storage condition 25 0 C / 60 %RH 25 0 C / 60 %RH
1M 1M
Description
Clear blue solution
with no visible
particle
Blue color solution with brown
tint, slightly hazy solution /
with particles
pH 7.3 7.5
Osmolality 326 38
% Assay (By
Ultraviolet–visible
spectroscopy)
96.8 91.9
Storage condition 100 0C 100 0C
24 hours 24 hours
Description Clear blue solution Blue color solution with brown
20
with no visible
particle
tint, slightly hazy solution /
with particles
It is evident from a comparison of the description for reconstituted solutions as
reported in Table 3D above, that sodium chloride provides stability to the
reconstituted solution.
5
21
We Claim:
1. A lyophilized pharmaceutical composition comprising copper histidinate and one
or more pharmaceutically acceptable excipients.
5
2. The lyophilized pharmaceutical composition according to claim 1, wherein the
excipient is a lyoprotectant.
3. The lyophilized pharmaceutical composition according to claim 2, wherein the
lyoprotectant is sodium chloride.10
4. The lyophilized pharmaceutical composition according to claim 1, wherein the
excipient is a pH adjusting agent.
5. The lyophilized pharmaceutical composition according to claim 4, wherein the pH15
adjusting agent is sodium hydroxide.
6. The lyophilized pharmaceutical composition according to claim 1, wherein the
composition comprises about 0.500 mg to about 10.000 mg of copper histidinate.
20
7. The lyophilized pharmaceutical composition according to claim 3, wherein the
sodium chloride is present in an amount of from about 1 mg to about 18 mg.
8. The lyophilized pharmaceutical composition according to claim 1, wherein the
composition is a lyophilized powder or cake.25
9. The lyophilized pharmaceutical composition according to claim 8, wherein the
lyophilized powder or cake is essentially free from any aggregates, lumps, and/or
lyophilization defects.
30