U.S. patent application number 08/981846 was filed with the patent office on 2001-12-13 for lyophilized hgf preparations.
Invention is credited to HIGASHIO, KANJI, KUMAZAWA, EITARO, TANAKA, KATSUMI.
Application Number | 20010051604 08/981846 |
Document ID | / |
Family ID | 16400759 |
Filed Date | 2001-12-13 |
United States Patent
Application |
20010051604 |
Kind Code |
A1 |
TANAKA, KATSUMI ; et
al. |
December 13, 2001 |
LYOPHILIZED HGF PREPARATIONS
Abstract
The invention relates to a lyophilized HGF preparation prepared
by lyophilizing an aqueous solution containing HGF, and a
lyophilized HGF preparation containing a stabilizer, sodium
chloride, a buffer, and/or a surface active agent. According to the
invention, HGF can be stabilized, and it can be stored for a long
period.
Inventors: |
TANAKA, KATSUMI;
(TAKATSUKI-SHI, JP) ; HIGASHIO, KANJI;
(KAWAGOE-SHI, JP) ; KUMAZAWA, EITARO;
(KAWACHI-GUN, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
16400759 |
Appl. No.: |
08/981846 |
Filed: |
May 22, 1998 |
PCT Filed: |
July 8, 1996 |
PCT NO: |
PCT/JP96/01898 |
Current U.S.
Class: |
514/11.4 ;
530/350 |
Current CPC
Class: |
A61K 9/19 20130101; A61K
9/0019 20130101; A61K 47/36 20130101; A61P 35/00 20180101; A61K
47/02 20130101; A61P 1/16 20180101; A61K 47/26 20130101; A61K
47/183 20130101; A61P 13/02 20180101; A61K 47/12 20130101; A61K
38/1833 20130101; A61P 15/00 20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/12 ;
530/350 |
International
Class: |
A61K 038/16; C07K
014/435 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 11, 1995 |
JP |
199018 |
Claims
1. A lyophilized HGF preparation.
2. The lyophilized HGF preparation of claim 1, wherein the
preparation contains a stabilizer.
3. The lyophilized HGF preparation of claim 2, wherein the
stabilizer is glycine, alanine, sorbitol, mannitol, or dextran
sulfate.
4. The lyophilized HGF preparation of any one of claims 1 to 3,
wherein the preparation contains a buffer.
5. The lyophilized HGF preparation of claim 4, wherein the buffer
is a citrate buffer.
6. A lyophilized HGF preparation which contains a stabilizer,
sodium chloride, a buffer, and a surface active agent.
Description
TECHNICAL FIELD
[0001] The present invention relates to a lyophilized HGF
preparation obtained by lyophilizing a solution containing HGF
(hepatocyte growth factor). More particular, it relates to the
lyophilized HGF preparation containing at least one of stabilizer,
sodium chloride, buffer or surface active agent. The invention
hence presents a stabilized preparation of HGF that can be stored
for a long period.
BACKGROUND ART
[0002] HGF is a protein that enhances proliferation of liver
parenchyma cells, and proteins having different amino acid
sequences have been reported, and are known in the names of HGF,
TCF, SCF, etc. In the invention, these known proteins having
hepatocyte growth activity are collectively called HGF. HGF is a
physiological active peptide showing various pharmacological
actions, and its pharmacological actions are reported, for example,
in Experimental Medicine (Japan), Vol. 10, No. 3 (extra issue),
330-339 (1992). Owing to its pharmacological actions, HGF is
expected to be developed as agent for liver cirrhosis, agent for
kidney disease, epithelial cell growth promoter, carcinostatic
agent, side effect inhibitor for cancer therapy, agent for lung
disorder, agent for gastroduodenal lesion, agent for cerebral and
nervous disorder, agent for relieving side effects caused by
immunosuppressants, collagen decomposition promoter, agent for
cartilage disorder, agent for arterial disease, agent for lung
fibroid, agent for liver disease, agent for abnormal blood
clotting, agent for hypoproteinemia, wound cure agent, improving
agent for nervous disorder, hematopoietic stem cell promoter, hair
growth promoter, etc. (Japanese Laid-open Patent No. 4-18028,
Japanese Laid-open Patent No. 4-49246, EP No. 492614, Japanese
Laid-open Patent No. 6-25010, WO 93/8821, Japanese Laid-open Patent
No. 6-172207, Japanese Laid-open Patent No. 7-89869, Japanese
Laid-open Patent No. 6-40934, WO 94/2165, Japanese Laid-open Patent
No. 6-40935, Japanese Laid-open Patent No. 6-56692, Japanese
Laid-open Patent No. 7-41429, WO 93/3061, Japanese Laid-open Patent
No. 5-213721, etc. ).
[0003] Preparations of HGF are disclosed in WO 90/10651 and
Japanese Laid-open Patent No. 6-247872. This publication of WO
90/10651 discloses a deletion type HGF (dLeHGF) deleting five
residues of amino acid from HGF, and it is named TCFII. This
specification shows that HGF is stabilized by albumin, human serum,
gelatin, sorbitol, mannitol, xylitol, etc. But, it relates to
aqueous solution preparations, and HGF is stabilized in an aqueous
solution. The publication of Japanese Laid-open Patent No. 6-247872
unveils a preparation having HGF contained at high concentration by
coexistence of basic amino acids and HGF (TCF).
[0004] Generally, the protein is not so stable in freezing
operation (Protein, Nucleic Acid, Enzyme (Japan), 37(9), 1517,
1992). The stabilizer of protein in an aqueous solution is intended
to stabilize by mutual action of water molecule and protein.
Therefore, in a lyophilized preparation of protein in the absence
of water, the stabilizer of protein for an aqueous solution shows
no stabilizing effect in most cases (Protein, Nucleic Acid, Enzyme
(Japan), 37(9), 1517, 1992).
[0005] On the other hand, nothing has been known about lyophilized
HGF preparation, and it could not expected how far the lyophilized
HGF preparation would show physical and biological stability.
[0006] The aqueous solution preparation of HGF itself is, when
stored at low temperature or room temperature for several days,
changed in properties, showing aggregation, turbidity and gelation,
and forms variants and polymers, and it is low in physical
stability and is lowered in biological activity, and hence it is
low in stability of biological activity and is not a stable
preparation suited long-term storage. It has been a fatal point for
development of HGF as medicines or animal drugs in a form of
injection preparation. The invention solves the above-mentioned
problems. That is, it is an object of the invention to present a
stable preparation which can store for a long period as medicines
for medical treatment or animal drugs.
DISCLOSURE OF THE INVENTION
[0007] The invention relates to a lyophilized HGF preparation. This
lyophilized HGF preparation may contain a stabilizer such as
glycine, alanine, sorbitol, mannitol, and dextran sulfate, or may
contain a buffer such as citrate.
[0008] Other invention of the present invention relates to a
lyophilized HGF preparation containing stabilizer, sodium chloride,
buffer and surface active agent.
[0009] In the lyophilized HGF preparation of the invention, HGF is
stabilized and can be stored for a long period.
THE BEST MODE FOR CARRYING OUT THE INVENTION
[0010] As HGF used in the present invention, there can be used one
which prepared by various methods if it is purified to an extent
that it can be used as a medicine.
[0011] Various methods are known for preparing HGF. For example,
HGF can be obtained by extraction and purification from organs
(e.g. liver, spleen, lung, bone marrow, brain, kidney, placenta,
etc.), blood cells (e.g. platelet, leucocyte, etc.), serum and
plasma of mammals such as rat, cow, horse, sheep and the like (see
FEBS Letters, 224, 312, 1987; Proc. Natl. Acad. Sci. USA, 86, 5844,
1989, etc.).
[0012] Also, it is possible to obtain HGF by cultivation of primary
culture cells or cell lines producing HGF, followed by separation
and purification from the culture product (e.g. culture
supernatant, cultured cell, etc.). Further, HGF can be obtained by
gene engineering method which comprises cloning the gene coding HGF
with a proper vector, inserting it into a proper host cell to give
a transformant, and separating the desired recombinant HGF from the
culture supernatant of the transformant (e.g. Nature, 342, 440,
1989, Japanese Laid-open Patent No. 5-111383, Biochem. Biophys.
Res. Commun., 163, 967, 1989). The host cell is not specifically
limited, and various host cells conventionally used in gene
engineering methods can be used, which are, for example,
Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, and
plant or animal cells.
[0013] More specifically, the method of extracting and purifying
HGF from live tissues is, for example, to administer carbon
tetrachloride to a rat intraperitoneally, remove a liver from the
rat with hepatitis, grind it, and purify by the ordinary protein
purifying technique such as gel column chromatography using
S-Sepharose and heparin Sepharose, HPLC and the like.
[0014] Further, by the gene engineering method, the gene coding the
amino acid sequence of human HGF is cloned into a vector such as
bovine papilloma virus DNA and the like to obtain an expression
vector, and by using this expression vector, animals cells such as
Chinese hamster ovary (CHO) cells, mouse C127 cells, monkey COS
cells and the like are transformed, and HGF can be obtained from
the culture supernatant of the transformants.
[0015] In thus obtained HGF, a part of the amino acid sequence of
HGF may be deleted or substituted by other amino acid(s), another
amino acid sequence may be inserted, one or more amino acids may be
bonded to the N-terminal and/or C-terminal, or saccharide chain(s)
may likewise be deleted or substituted, providing it has
substantially the same effect as HGF.
[0016] The "lyophilized HGF preparation" refers to a preparation
prepared by lyophilizing an aqueous solution containing HGF by use
of an ordinary lyophilizing method.
[0017] The "stabilizer" includes amino acids (e.g. glycine,
alanine, etc.), polysaccharides (e.g. heparin, dextran sulfate,
etc.), sugar alcohols (e.g. sorbitol, mannitol, etc.) and the like,
and two or more types thereof may be used simultaneously. The
lyophilized HGF preparation prepared by adding the stabilizer is a
preparation further increased in storage stability of HGF.
Preferred stabilizers are glycine, alanine, sorbitol, mannitol, and
dextran sulfate. For example, a preferred adding amount of glycine,
alanine, sorbitol or mannitol is 0.01 to 100 times by weight of the
weight of HGF, and more preferably 0.1 to 10 times by weight.
[0018] The "buffer" includes, for example, phosphate buffer and
citrate buffer. The buffer acts to adjust the pH of the aqueous
solution after re-dissolving, and keep the solubility of HGF. That
is, for example, in the case of the recombinant HGF used in
Examples, the solubility of HGF varies with the pH, and the
solubility is about 0.1 to 5.0 mg/ml around pH 7, but the
solubility is over 20 mg/ml around pH 5, and therefore it is
preferred to keep the pH around 5.0 to 6.0. A preferred buffer is a
citrate buffer, and more preferably sodium citrate buffer is used.
This citrate buffer also contributes to stabilization of HGF in an
aqueous solution after re-dissolving. A preferred range of adding
the buffer is, for example, 1 to 100 mM to the amount of water
after re-dissolving.
[0019] The "surface active agent" includes, for example,
polysorbate 20, polysorbate 80, pluronic F-68, and polyethylene
glycol, and two or more types thereof may be used simultaneously. A
particularly preferred surface active agent is polysorbate 80. It
is known that HGF is likely to be adsorbed on a container material
such as glass and resin. Therefore, by adding a surface active
agent, adsorption of HGF to after re-dissolving to the container is
prevented. A preferred range of adding amount of surface active
agent is 0.001 to 2.0% by weight, for example, to the weight of
water after re-dissolving.
[0020] The "sodium chloride" acts to keep solubility of HGF. That
is, for example, in the case of recombinant HGF used in Examples,
the solubility is enhanced by adding sodium chloride, and the
solubility is notably increased in particular at 300 mM or more
(Japanese Laid-open Patent No. 6-247872). An amount of addition of
sodium chloride is limited by the osmotic pressure ratio, but it
may be an amount showing an osmotic pressure ratio of injection
preparation for general use. In particular, the osmotic pressure
ratio is preferred to be 1 to 2 which is permitted as the osmotic
pressure ratio of injection for medical treatment or animal drug,
and it is preferred to add, for example, by 150 to 300 mM to the
amount of water after re-dissolving.
[0021] The lyophilized HGF preparation is prepared by lyophilizing
an aqueous solution containing HGF by an ordinary lyophilizing
method. For example, HGF is dissolved in a proper solvent (for
example, sterilized water, buffer, physiological saline, etc.),
filtered through a filter to be sterilized, and, if necessary,
stabilizer, buffer, surface active agent, sodium chloride and
others may be added, and the mixture is lyophilized. The
preparation of the invention may contain additives necessary for
pharmaceutical manufacturing, for example, a dissolving aid, an
antioxidant, a pain-alleviating agent, an isotonic agent, and the
like. The lyophilizing method may comprise three unit operations,
for example, (1) a freezing step of cooling and freezing under
ordinary pressure, (2) a first drying step of sublimating and
drying free water not restrained by solute under reduced pressure,
and (3) a second drying step of removing the intrinsic adsorbed
water and crystal water of solute (Pharm. Tech. Japan, 8(1), 75-87,
1992). HGF is very stable when preparing a solution, when
lyophilizing, and in an aqueous solution by re-dissolving the
lyophilized preparation. The content of HGF may be properly
adjusted depending on the disease to be treated and route of
administration.
[0022] The lyophilized preparation is used by adding distilled
water for injection and re-dissolving, before use.
INDUSTRIAL APPLICABILITY
[0023] The lyophilized HGF preparation of the invention can
stabilize HGF, and can be stored for a long period.
EXAMPLES
[0024] The invention is further described by presenting Examples,
but it must be noted that the invention is not limited to these
Examples alone. In the Examples, dLeHGF (five-amino acid depletion
type HGF, also known as TCFII) disclosed in the publication of WO
90/10651 was used.
Example 1
Preparation of Lyophilized HGF Preparation
[0025] In 10 mM citrate buffer (pH 5.0) containing 300 mM sodium
chloride and 0.01% polysorbate 80, HGF was dissolved by 20 mg/ml,
and an aqueous solution of HGF was obtained aseptically. After
adjusting the pH of the aqueous solution, it was aseptically
charged into a vial, and lyophilized in the condition as shown in
Table 1, and a lyophilized HGF preparation was obtained. The arrow
mark (.fwdarw.) in the table shows the temperature is changed.
1 TABLE 1 Freezing First drying Second drying step step step
Temperature (.degree. C.) 5 .fwdarw. -40 -40 -40 .fwdarw. 0 0 0
.fwdarw. 20 20 Time (hr) 1 10 8 24 1 24 Pressure (mmHg) 760 760
<1 <1 <1 <1
Example 2
Preparation of Lyophilized HGF Preparation
[0026] A lyophilized HGF preparation was obtained by using 10 mM
citrate buffer (pH 6.0) instead of 10 mM citrate buffer (pH 5.0) in
Example 1.
Example 3
Preparation of Lyophilized HGF Preparation
[0027] A lyophilized HGF preparation was obtained by using 10 mM
phosphate buffer (pH 6.0) instead of 10 mM citrate buffer (pH 5.0)
in Example 1.
Example 4
Preparation of Lyophilized HGF Preparation
[0028] A lyophilized HGF preparation was obtained by using 10 mM
phosphate buffer (pH 7.0) instead of 10 mM citrate buffer (pH 5.0)
in Example 1.
Example 5
Preparation of Lyophilized HGF Preparation
[0029] In 10 mM citrate buffer (pH 5) containing 300 mM sodium
chloride and 0.01% polysorbate 80, HGF was dissolved by 20 mg/ml.
In succession, glycine was dissolved by 50 mg/ml, and a dissolved
solution of HGF was obtained aseptically. After adjusting the pH of
the dissolved solution, it was aseptically charged into a vial, and
lyophilized in the same condition as in Example 1 and a lyophilized
HGF preparation was obtained.
Example 6
Preparation of Lyophilized HGF Preparation
[0030] A lyophilized HGF preparation was obtained by using alanine
instead of glycine in Example 5.
Example 7
Preparation of Lyophilized HGF Preparation
[0031] In 10 mM citrate buffer (pH 5) containing 300 mM sodium
chloride and 0.01% polysorbate 80, HGF was dissolved by 20 mg/ml.
In succession, sorbitol was dissolved by 200 mg/ml, and a dissolved
solution of HGF was obtained aseptically. After adjusting the pH of
the dissolved solution, it was aseptically charged into a vial, and
lyophilized in the same condition as in Example 1 and a lyophilized
HGF preparation was obtained.
Example 8
Preparation of Lyophilized HGF Preparation
[0032] In 10 mM citrate buffer (pH 6) containing 300 mM sodium
chloride and 0.01% polysorbate 80, HGF was dissolved by 10 mg/ml.
In succession, dextran sulfate was dissolved by 50 mg/ml, the pH
was adjusted, and a dissolved solution of HGF was obtained. It was
then charged into a vial, and lyophilized in the same condition as
in Example 1 and a lyophilized HGF preparation was obtained.
Example 9
Preparation of Lyophilized HGF Preparation
[0033] A lyophilized HGF preparation was obtained in the same
manner as in Example 1, except by using 10 mM citrate buffer (pH
6.0) instead of 10 mM citrate buffer (pH 5.0), and regulating HGF
concentration at 10 mg/ml.
Test Example 1
Effects of Lyophilizing Process on Biological Activity of HGF
[0034] To observe changes in biological activity of HGF in the
lyophilizing process, using HGF aqueous solution before
lyophilization and HGF aqueous solution re-dissolved directly after
lyophilization in Example 1, the biological activity of HGF was
measured (the measuring method of biological activity is shown
below). The results are shown in Table 2. Since the specific
activity was not changed before and after lyophilization, it is
shown that the biological activity of HGF is not inactivated by the
lyophilizing process and re-dissolving, which suggests that HGF is
usable as a lyophilized preparation.
Measuring Method of Biological Activity
[0035] Hepatocytes obtained by liver perfusion of male Wistar rats
were purified, and, after confirming the cell survival rate, seeded
on a plate at 1.times.10.sup.4/well. After pre-incubation for 20
hours in 5% carbon dioxide incubator, HGF sample and standard
sample were added (n=3). After further pre-incubation for 24 hours
in 5% carbon dioxide incubator, [.sup.3H-thymidine] was added to
label for 2 hours. Cells were collected by a cell harvester, and
the amount of [.sup.3H] taken into cells was measured. Results of
measurement were verified by a parallel line calibration method,
and the specific activity to the standard sample was
determined.
2TABLE 2 Biological activity before and after lyophilization Sample
Specific activity Solution preparation before 0.89 lyophilization
Lyophilized preparation immediately 0.94 after re-dissolving
Test Example 2
Properties after Dissolving Lyophilized Preparation
[0036] Lyophilized preparations prepared in Examples were stored
for 1 month at -40.degree. C., 25.degree. C., and 50.degree. C.,
and dissolved, and properties of the dissolved preparations were
observed visually. The lyophilized preparation was dissolved by
using purified water. Results are shown in Table 3. When stored at
-40.degree. C. or 25.degree. C., the preparations of all Examples
were stable in the properties. When stored at 50.degree. C., the
preparation in Example 1 was turbid immediately after dissolving,
but preparations of Examples 5, 6 and 7 were stable in
properties.
3TABLE 3 Properties after dissolving lyophilized preparations
(stored for 1 month) Properties Preparation -40.degree. C.
25.degree. C. 50.degree. C. Example 1 Clear Clear Turbid Example 5
Clear Clear Clear Example 6 Clear Clear Clear Example 7 Clear Clear
Clear
Test Example 3
Polymer Content Changes in Lyophilized Preparations
[0037] Lyophilized preparations prepared in Examples 1, 5, 6 and 7
were stored for 1 month or 2 months at -40.degree. C., 25.degree.
C., 40.degree. C., and 50.degree. C., and the ratio of polymer
content and HGF content contained in the lyophilized preparations
were measured. The measuring method is the gel filtration method as
explained below. Results are shown in Table 4 and Table 5.
Regardless of the storage temperature, a polymer production was low
in the preparations of all Examples, and the preparations were
stable physically. In particular, the polymer production was
extremely small in the preparations of Examples 5, 6 and 7, and the
preparations were stable physically.
Measuring Method of Polymer Content
[0038] The concentration of HGF was diluted to 2 mg/ml, and was
measured in the following conditions by the gel filtration
method.
[0039] Column: TOSOH TSK G-3000SW XL (.phi.0.78.times.30 cm)
[0040] Flow velocity: 0.5 ml/min
[0041] Detection: OD 280
[0042] Temperature: 25.degree. C.
[0043] Carrier: 10 mM Tris, 150 mM NaCl, 0.05% SDS, pH 7.0
[0044] Application: 20 .mu.l
[0045] Retention time of polymer: 13.0 min
[0046] Retention time of HGF: 14.4 min
4TABLE 4 Polymer content/HGF content in lyophilized preparations
stored for 1 month -40.degree. C. 25.degree. C. 40.degree. C.
50.degree. C. Example 1 1.07% 1.59% 2.76% 6.17% Example 5 0.92%
1.39% 1.83% 4.09% Example 6 0.93% 1.54% 1.81% 2.90% Example 7 0.90%
1.35% 2.57% 6.64%
[0047]
5TABLE 5 Polymer content/HGF content in lyophilized preparations
stored for 2 months -40.degree. C. 25.degree. C. 40.degree. C.
50.degree. C. Example 1 0.92% 1.44% 3.91% 12.23% Example 5 0.88%
1.21% 2.49% 7.49% Example 6 0.85% 1.10% 1.96% 5.76%
Test Example 4
Effects of Dextran Sulfate on Polymer Production
[0048] The lyophilized preparation prepared in Example 8 was stored
for 1 month at 50.degree. C., and the ratio of polymer content and
HGF content contained in the lyophilized preparations were
measured. The measuring method was same as in Test example 3. As a
comparative example, the lyophilized preparation of Example 9
prepared in the same composition and method except that dextran
sulfate was not contained was used and tested similarly. The
results are shown in Table 6. As shown in Table 6, by adding
dextran sulfate, it has been found that the polymer production was
low even if stored at high temperature, and that the stability is
enhanced.
6TABLE 6 Polymer content/HGF content of lyophilized preparations
Before start of After storage for storage 1 month at 50.degree. C.
Example 8 2.46% 9.45% Example 9 1.78% 14.01%
Test Example 5
Changes of Biological Activity of Lyophilized Preparations
[0049] Lyophilized p reparations prepared in Examples 1, 5, 6 and 7
were stored for 1 month or 2 months at -40.degree. C., 40.degree.
C., 50.degree. C. and 60.degree. C., and the biological activity of
the aqueous solution after re-dissolving the lyophilized
preparations was measured by the biological activity measuring
method shown in Test example 1. The results are shown in Table 7
and Table 8. The initial values of biological activity of aqueous
solutions after re-dissolving the preparations in Examples 1, 5, 6
and 7 were respectively 1.01.+-.0.25, 0.91.+-.0.18, 0.88.+-.0.05,
and 1.03.+-.0.04. When stored at 60.degree. C., a slightly lowering
tendency was noted in the biological activity, but when stored at
50.degree. C. or lower temperature, there was almost no change in
the biological activity in the preparations of any Example, and the
biological activity was stable.
7TABLE 7 Biological activity of lyophilized preparations stored for
1 month (specific activity) -40.degree. C. 40.degree. C. 50.degree.
C. 60.degree. C. Example 1 0.96 .+-. 0.13 0.92 .+-. 0.13 0.81 .+-.
0.07 0.54 .+-. 0.05 Example 5 0.80 .+-. 0.14 0.99 .+-. 0.10 0.80
.+-. 0.16 0.72 .+-. 0.03 Example 6 0.92 .+-. 0.14 1.02 .+-. 0.06
0.94 .+-. 0.08 0.78 .+-. 0.03 Example 7 0.92 .+-. 0.02 0.97 .+-.
0.04 0.83 .+-. 0.06 --
[0050]
8TABLE 8 Biological activity of lyophilized preparations stored for
2 months (specific activity) -40.degree. C. 40.degree. C.
60.degree. C. Example 1 1.14 .+-. 0.14 0.98 .+-. 0.01 0.46 .+-.
0.09 Example 5 0.95 .+-. 0.05 0.84 .+-. 0.09 0.57 .+-. 0.01 Example
6 1.11 .+-. 0.14 1.09 .+-. 0.03 0.52 .+-. 0.02
* * * * *