AU2005200353B2 - Lyophilized HGF preparations - Google Patents

Description

P/00/01 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Lyophilized HGF preparations The following statement is a full description of this invention, including the best method of performing it known to us:

SSPECIFICATION

00 LYOPHILIZED HGF PREPARATIONS rn 5 TECHNICAL FIELD S. The present invention relates to a lyophilized HGF (i Spreparation obtained by lyophilizing a solution containing HGF c1 (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 1 5 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.

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, 2 p agent for gastroduodenal lesion, agent for cerebral and nervous disorder, agent for relieving side effects caused by 00 immunosuppressants, collagen decomposition promoter, agent for on cartilage disorder, agent for arterial disease, agent for lung 0 5 fibroid, agent for liver disease, agent for abnormal blood Cg clotting, agent for hypoproteinemia, wound cure agent, 0 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 1 0 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 Laidopen Patent No. 6-56692, Japanese Laid-open Patent No. 7- 1 5 41429, WO 93/3061, Japanese Laid-open Patent No. 5-213721, etc.).

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).

SGenerally, the protein is not so stable in freezing operation (Protein, Nucleic Acid, Enzyme (Japan), 37(9), 1517, 00 1992). The stabilizer of protein in an aqueous solution is intended to stabilize by mutual action of water molecule and 5 protein. Therefore, in a lyophilized preparation of protein in the absence of water, the stabilizer of protein for an Saqueous solution shows no stabilizing effect in most cases (Protein, Nucleic Acid, Enzyme (Japan), 37(9), 1517, 1992).

On the other hand, nothing has been known about I 0 lyophilized HGF preparation, and it could not expected how far the lyophilized HGF preparation would show physical and biological stability.

The aqueous solution preparation of HGF itself is, when stored at low temperature or room temperature for several 1 5 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 Idw 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 2 5 treatment or animal drugs.

DISCLOSURE OF THE INVENTION SThe invention relates to a lyophilized HGF preparation.

ct This lyophilized HGF preparation may contain a stabilizer such 00 as glycine, alanine, sorbitol, mannitol, and dextran sulfate, Cc or may contain a buffer such as citrate.

M 5 Other invention of the present invention relates to a Cq lyophilized HGF preparation containing stabilizer, sodium O chloride, buffer and surface active agent.

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 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.

1 5 Various methods are known for preparing HGF. For example, HGF can be obtained by extraction and purification from organs liver, spleen, lung, bone marrow, brain, kidney, placenta, etc.), blood cells 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.).

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 c proper host cell to give a transformant, and separating the desired recombinant HGF from the culture supernatant of the 00 C transformant Nature, 342, 440, 1989, Japanese Laid-open SPatent No. 5-111383, Biochem. Biophys. Res. Commun., 163, 967, g 5 1989). The host cell is not specifically limited, and various host cells conventionally used in gene engineering methods can Sbe used, which are, for-example, Escherichia coli, Bacillus CO subtilis, yeast, filamentous fungi, and plant or animal cells.

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.

1 5 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.

In thus obtained HGF, a part of the amino acid sequence of HGF may be deleted or substituted by other amino acid(s), 2 5 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.

QO The "lyophilized HGF preparation" refers to a preparation 00 prepared by lyophilizing an aqueous solution containing HGF by cuse of an ordinary lyophilizing method.

The "stabilizer" includes amino acids glycine, C alanine, etc.), polysaccharides heparin, dextran sulfate, etc.), sugar alcohols 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 1 5 weight of HGF, and more preferably 0.1 to 10 times by weight.

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 redissolving. A preferred range of adding the buffer is, for Sexample, 1 to 100 mM to the amount of water after redissolving.

The "surface active agent" includes, for example, m polysorbate 20, polysorbate 80, pluronic F-68, and g 5 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 1 0 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.

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.

The lyophilized HGF preparation is prepared by Slyophilizing an aqueous solution containing HGF by an ordinary lyophilizing method. For example, HGF is dissolved in a proper solvent (for example, sterilized water, buffer, Cc physiological saline, etc.), filtered through a filter to be c 5 sterilized, and, if necessary, stabilizer, buffer, surface active agent, sodium chloride and others may be added, and the 0 mixture is lyophilized. The preparation of the invention may contain additives necessary for pharmaceutical manufacturing, for example, a dissolving aid, an antioxidant, a pain- 1 0 alleviating agent, an isotonic agent, and the like. The lyophilizing method may comprise three unit operations, for example, a freezing step of cooling and freezing under ordinary pressure, a first drying step of sublimating and drying free water not restrained by solute under reduced 1 5 pressure, and a second drying step of removing the intrinsic adsorbed water and crystal water of solute (Pharm.

Tech. Japan, 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 2 0 content of HGF may be properly adjusted depending on the disease to be treated and route of administration.

The lyophilized preparation is used by adding distilled water for injection and re-dissolving, before use.

2 5 INDUSTRIAL APPLICABILITY The lyophilized HGF preparation of the invention can stabilize HGF, and can be stored for a long period.

EXAMPLES

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 In 10 mM citrate buffer (pH 5.0) containing 300 mM sodium chloride and 0.01% polysorbate 80, HGF was dissolved by 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 in the table shows the temperature is changed.

Table 1 Freezing First drying Second drying step step step Temperature 5 -40 -40 -40 0 0 0 20 Time (hr) 1 10 8 24 1 24 Pressure (mmHg) 760 760 <1 <1 <1 <1 Example 2 Preparation of lyophilized HGF preparation 1 0

(N

SA lyophilized HGF preparation was obtained by using 10 mM citrate buffer (pH 6.0) instead of 10 mM citrate buffer (pH 00 0) in Example 1.

In C 5 Example 3 C\ Preparation of lyophilized HGF preparation SA lyophilized HGF preparation was obtained by using 10 mM phosphate buffer (pH 6.0) instead of 10 mM citrate buffer (pH in Example 1.

1 0 Example 4 Preparation of lyophilized HGF preparation A lyophilized HGF preparation was obtained by using 10 mM phosphate buffer (pH 7.0) instead of 10 mM citrate buffer (pH 1 5 5.0) in Example 1.

Example Preparation of lyophilized HGF preparation In 10 mM citrate buffer (pH 5) containing 300 mM sodium chloride and 0.01% polysorbate 80, HGF was dissolved by 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 SPreparation of lyophilized HGF preparation ct A lyophilized HGF preparation was obtained by using 00 alanine instead of glycine in Example 0C 5 Example 7 Preparation of lyophilized HGF preparation 0 In 10 mM citrate buffer (pH 5) containing 300 mM sodium chloride and 0.01% polysorbate 80, HGF was dissolved by mg/ml. In succession, sorbitol was dissolved by 200 mg/ml, 1 0 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 In 10 mM citrate buffer (pH 6) containing 300 mM sodium chloride and 0.01% polysorbate 80, HGF was dissolved by mg/ml. In succession, dextran sulfate was dissolved by 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 12 SA lyophilized HGF preparation was obtained in the same ct manner as in Example 1, except by using 10 mM citrate buffer 00 (pH 6.0) instead of 10 mM citrate buffer (pH and on regulating HGF concentration at 10 mg/ml.

In 8,1 C Test example 1 O Effects of lyophilizing process on biological activity of HGF 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 redissolving, which suggests that HGF is usable as a lyophilized preparation.

Measuring method of biological activity Hepatocytes obtained by liver perfusion of male Wistar rats were purified, and, after confirming the cell survival rate, seeded on a plate at x10 4 /well. After pre-incubation for 20 hours in 5% carbon dioxide incubator, HGF sample and standard sample were added After further preincubation for 24 hours in 5% carbon dioxide incubator, 3

H-

thymidine] was added to label for 2 hours. Cells were 13 Scollected by a cell harvester, and the amount of 3 H] taken into cells was measured. Results of measurement were verified 00 by a parallel line calibration method, and the specific cr activity to the standard sample was determined.

c Table 2 0 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 1 5 Properties -after dissolving lyophilized preparation Lyophilized preparations prepared in Examples were stored for 1 month at -40'C, 25C, and 50C, 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 or 25C, the preparations. of all Examples were stable in the properties. When stored at 50C, the preparation in Example 1 was turbid immediately after dissolving, but preparations of Examples 5, 6 and 7 were stable in properties.

14 p a Table 3 O0 Properties after dissolving lyophilized preparations (stored for 1 month) Cc 5 Preparation Properties -40 C 25C* 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 i.n lyophilized preparations Lyophilized preparations prepared in Examples 1, 5, 6 and 7 were stored for 1 month or 2 months at -40"C, 25C, 40'C, and 1 5 50C, 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.

2 5 Measuring method of polymer content The concentration of HGF was diluted to 2 mg/ml, and was measured in the following conditions by the gel filtration 1 method.

Column TOSOH TSK G-3000SW XL (00.78x30 cm) Flow velocity 0.5 ml/min Detection OD 280 Temperature Carrier 10 mM Tris, 150 mM NaC1, 0.05% SDS, pH Application 20 gl Retention time of polymer 13.0 min Retention time of HGF 14.4 min Table 4 Polymer content/HGF content in lyophilized preparations stored for 1 month 25C 40' 0 1 5 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% Table Polymer content/HGF content in lyophilized preparations stored for 2 months 25*C 40C 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% 1 6 STest example 4 00 Effects of dextran sulfate on polymer production The lyophilized preparation prepared in Example 8 was C stored for 1 month at 50'C, and the ratio of polymer content and HGF content contained in the lyophilized preparations were C measured. The measuring method was same as in Test example 3.

OAs 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.

1 5 Table 6 Polymer content/HGF content of lyophilized preparations Before start of After storage for storage 1 month at Example 8 2.46% 9.45% Example 9 1.78% 14.01% Test example Changes of biological activity of lyophilized preparations Lyophilized preparations prepared in Examples 1, 5, 6 and 7 were stored for 1 month or 2 months at -40'C, 40'C, 50°C and 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 redissolving 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 60C, a slightly lowering tendency was noted in the biological activity, but when stored at 50C or lower temperature, there was almost no change in the biological activity in th.e preparations of any Example, and the biological activity was stable.

Table 7 Biological activity of lyophilized preparations stored for 1 month (specific activity) -40' 40C 50C 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 Table 8 Biological activity of lyophilized preparations stored for 2 months (specific activity) 40C 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 005086395 18 00 C As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

Ss Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment, or any form of suggestion, CI that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could CI reasonably be expected to be ascertained, understood and regarded 0Lo as relevant by a person skilled in the art.

Claims (6)

1. A lyophilized preparation, which comprises hepatocyte growth factor as a biologically active ingredient, dextran sulfate as a stabilizing agent, a citrate buffer and a surface active agent which is polysorbate. Ci

2. The lyophilized preparation according to claim 1, wherein the preparation further comprises sodium chloride.

3. Use of dextran sulfate as a stabilizing agent for manufacturing a lyophilized hepatocyte growth factor LO preparation containing hepatocyte growth factor, a citrate buffer and a surface active agent which is polysorbate.

4. Use of dextran sulfate according to claim 3, wherein the preparation further comprises sodium chloride.

A lyophilized preparation according to claim 1 or 2 substantially as hereinbefore described.

6. Use of a dextran sulfate according to claim 3 or 4 substantially as hereinbefore described.