JPH10158190A - Hgf medicine preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicine preparation comprising a protein having hepatocyte proliferation activity, capable of enhancing efficacy of HGF in vivo. SOLUTION: This medicine composition comprises HGF and a substance having bondability to a heparin-like substance on the surface of a hepatocyte. The substance (e.g. protamine) having bondability to the heparin-like substance on the surface of a hepatocyte can enhance the efficacy of HGF in vivo by actions of blocking the heparin-like substance on the surface of a hepatocyte and delaying clearance in vivo of HGF. Consequently, a dose of HGF can be reduced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a pharmaceutical preparation containing HGF. More specifically, the present invention relates to a pharmaceutical preparation capable of enhancing the efficacy of HGF in vivo.

[0002]

2. Description of the Related Art HGF is a protein having an activity of proliferating hepatocytes, and it has been reported that it has a different amino acid sequence. HGF, TCF, SF and the like are used as its name. In the present invention, these known proteins having hepatocyte proliferation activity are collectively referred to as HGF. HGF
Are physiologically active peptides that exhibit various pharmacological actions. For their pharmacological actions, see, for example, Experimental Medicine Vol. 10, No. 3
(Extra number) 330-339 (1992). Because of its pharmacological action, HGF is a therapeutic agent for liver cirrhosis, a therapeutic agent for renal disease, an epithelial cell proliferation promoter, an anticancer agent, an anticancer agent for cancer therapy, a therapeutic agent for lung injury, a therapeutic agent for gastric and duodenal damage, a therapeutic agent for cranial nerve disorders, Inhibitors of side effects, Collagen degradation accelerator, Cartilage disorder treatment, Arterial disease treatment, Pulmonary fibrosis treatment, Liver disease treatment, Blood coagulation disorder treatment, Plasma low protein treatment,
Wound healing agent, neuropathy ameliorating agent, hematopoietic stem cell increasing agent, hair growth promoting agent and the like (JP-A-4-18028, JP-A-4-49246, EP 492614, JP-A-6-25010, WO 93 / 88
JP-A-6-172207, JP-A-7-89869, JP-A-6-40934, WO 94/2165, JP-A-6-4
Developments as 0935, JP-A-6-56692, JP-A-7-41429, WO 93/3061 and JP-A-5-213721 are expected.

[0003]

One of the problems in using HGF as a medicine is that clearance in vivo is fast. For example, when HGF is intravenously injected, it rapidly disappears from plasma, and according to the analysis of the present inventors, the initial phase half-life is about 4 minutes. Therefore, in viv
To obtain a medicinal effect in o, an extremely high dose (several tens of μg / k
(g to several mg / kg) at present. This may cause unexpected side effects, and raises the cost of treatment using HGF. Therefore, when clinically applying HGF, it is necessary to construct some kind of Drug delivery system (DDS) and to devise so as to obtain the efficacy of HGF such as a liver regeneration effect at a lower dose.

By the way, the present inventors have studied the clearance mechanism of HGF, but the organ of clearance of HGF is mainly the liver and the like, and the receptor-mediated uptake (receptor-mediat) through the HGF receptor on the cell surface.
Non-specific elimination via ed endocytosis) and heparin-like substances (matrix) is considered to be the main mechanism. Therefore, the present inventors thought that if the disappearance by the latter mechanism could be avoided, the clearance of HGF could be delayed and the dose could be reduced, and the heparin-like substance on the cell surface was previously bound to the substance by binding to the substance. A method of blocking with a substance having the following was studied. As a result, it was found that the effect of HGF can be enhanced by employing the method. The present invention has been made based on such findings, and an object of the present invention is to provide an HGF-containing preparation capable of enhancing the effect of HGF in a living body.

[0005]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and its gist is to provide a pharmaceutical preparation comprising HGF and a substance having a binding property to a heparin-like substance on the surface of hepatocytes; The above-mentioned pharmaceutical preparation, wherein the substance having a binding property to the heparin-like substance on the cell surface is a basic protein; the above-mentioned pharmaceutical preparation, wherein the substance having a binding property to the heparin-like substance on the liver cell surface is protamine; The pharmaceutical preparation according to any of the above, wherein the compounding ratio of the substance having a binding property to the cell surface heparin-like substance is 0.5 to 50 times by weight.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION As HGF used in the present invention, those prepared by various methods can be used as long as they are purified to the extent that they can be used as pharmaceuticals. Various methods are known for preparing HGF. For example, it can be extracted and purified from organs such as liver, spleen, lung, bone marrow, brain, kidney, and placenta of mammals such as rats, cows, horses, and sheep, blood cells such as platelets and leukocytes, plasma, and serum. (FEBS Letters, 224 ,
312, 1987, Proc. Natl. Acad. Sci. USA, 86 , 5844, 1
989 etc.). In addition, HGF can also be obtained by culturing primary cultured cells or cell lines that produce HGF, and separating and purifying them from cultures (culture supernatants, cultured cells, etc.). Alternatively, a gene encoding HGF is inserted into an appropriate vector by a genetic engineering technique, inserted into an appropriate host, and transformed, and the desired recombinant HGF can be obtained from the culture supernatant of the transformant. (E.g., Nature, 342 , 44
0, 1989, JP-A-5-111383, Biochem.
ophys. Res. Commun., 163 , 967, 1989, etc.). The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques, for example, Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, silkworm, plant or animal cells, and the like can be used.

More specifically, as a method of extracting and purifying HGF from living tissue, for example, carbon tetrachloride is intraperitoneally administered to a rat, and the liver of a rat in a hepatitis state is excised and pulverized, and S- The protein can be purified by a conventional protein purification method such as gel column chromatography such as Sepharose and heparin Sepharose, and HPLC. In addition, animal cells, such as Chinese hamster ovary (CHO) cells, mouse C127 cells, and the like, using an expression vector in which a gene encoding the amino acid sequence of human HGF is incorporated into a vector such as bovine papilloma virus DNA using a genetic recombination method. Transform monkey COS cells etc.
It can be obtained from the culture supernatant.

[0008] The HGF thus obtained has a part of its amino acid sequence deleted or replaced by another amino acid, or another amino acid sequence partially inserted, as long as it is substantially equivalent to HGF. Alternatively, one or more amino acids may be bound to the N-terminal and / or C-terminal, or the sugar chain may be deleted or substituted in the same manner.

The substance having a binding property to a heparin-like substance on the surface of a hepatocyte (hereinafter, simply referred to as a “binding substance”) used in the present invention is a substance having a binding property to the heparin-like substance. Any substance can be used, and examples thereof include basic proteins. Basic protein means a protein whose isoelectric point is on the alkaline side, for example, histone, protamine and the like,
Particularly, protamine is preferably used.

In the preparation of the present invention, the mixing ratio of HGF to the binding substance is not particularly limited, and is appropriately adjusted according to the disease to which HGF is applied, the type of the binding substance, and the like. For example,
When the binding substance is protamine, the preferred range of the mixing ratio of protamine to HGF is about 0.5 to 50 times by weight, more preferably about 1 to 20 times by weight, and particularly preferably 1.3 to 10 times by weight. It is about weight times.

[0011] The preparation of the present invention contains at least the above-mentioned HGF and a binding substance, and as shown in Examples described below, the binding substance can enhance the effect of HGF in a living body. Can be reduced.
The preparation of the present invention can be used for mammals (eg, bovine,
Horse, pig, sheep, dog, cat, etc.)
Is applied to the treatment and prevention of various diseases that can act.

The preparation of the present invention can be prepared in various preparation forms (for example,
Liquid, solid, capsule, etc.), but is generally made into an injection, inhalant, suppository or oral preparation together with HGF, which is an active ingredient, and a binding substance alone or with a conventional carrier. Is preferred. The injection can be prepared by a conventional method. For example, after dissolving HGF and a binding substance in an appropriate solvent (eg, sterilized water, buffer, physiological saline, etc.), the solution is filtered through a filter or the like. And then filled in a sterile container. The HGF content in the injection is usually 0.0002 to
It is adjusted to about 3 (W / V%), preferably about 0.001 to 2 (W / V%). As oral drugs, for example, tablets, granules, fine granules, powders, soft or hard capsules, solutions, emulsions,
They are formulated into dosage forms such as suspensions and syrups, and these formulations can be prepared according to the usual methods of formulation. Suppositories can also be prepared by a conventional method using a conventional base (for example, cacao butter, laurin butter, glycerogelatin, macrogol, witepsol). Also, an inhalant can be prepared according to conventional means for preparation. The HGF content in the preparation can be appropriately adjusted according to the dosage form, applicable disease and the like.

At the time of formulation, a stabilizer is preferably added, and examples of the stabilizer include albumin, globulin, gelatin, alanine, glycine, mannitol, glucose, dextran, sorbitol, ethylene glycol and the like. Further, the preparation of the present invention may contain additives necessary for preparation, for example, excipients, solubilizing agents, antioxidants, soothing agents, isotonic agents and the like. In the case of a liquid preparation, it is desirable to preserve by freezing or freeze-drying to remove water. The lyophilized preparation is used after reconstitution by adding distilled water for injection or the like at the time of use.

The preparation of the present invention can be administered by an appropriate administration route depending on the form of the preparation. For example, it can be administered in the form of an injection to vein, artery, subcutaneous, intramuscular, and the like. The dose is appropriately adjusted according to the patient's disease, symptoms, age, body weight, etc., for example, for adults,
Usually 0.01 mg to 500 mg as HGF, preferably 0.05 mg to 1
It is appropriate to administer the dose once or several times a day.

[0015]

According to the pharmaceutical preparation of the present invention, the effect of HGF in vivo can be enhanced. Therefore, H
Since the dose of GF can be reduced, side effects can be prevented and the cost of a therapeutic method using HGF can be reduced.

[0016]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the following examples, H
As the GF, dLeHGF (5-amino acid deleted HGF) described in JP-A-5-111383 was used. In addition, as a liver disease model animal, male Wistar rats were subjected to ANIT (α-naphthyl isothiocyanate) treatment or 30% partial hepatectomy and subjected to experiments. The administration schedule of HGF is as follows.
Minute, and thereafter 8, 22, 32, 46, 56, 70, 80, 9
Four hours later, they were administered once by intravenous injection. The dose of protamine shown in the figure (or the text) was intravenously injected in advance 10 minutes before the administration of HGF. Note that, unlike the present embodiment, H
GF and protamine can be administered simultaneously.

Example 1 Effect of protamine on liver repair effect of HGF (in vivo)
o) HGF and / or protamine were administered to ANIT-treated rats, and the degree of liver regeneration was examined by a labeling index. The results are shown in FIG. In the figure, Pr
o represents protamine (the same applies to the following figures). As shown in FIG. 1 (A), HGF was added to ANIT-treated rats.
(300 μg / kg), the labeling index of hepatocytes increased about 2 times more than that of the ANIT-treated rats (Control (+)) to which HGF was not administered (Control (−) in the figure). ) Means rats that have not been treated with ANIT or HGF). Here, HG
When protamine is administered 10 minutes before F administration, the labeling index increases in a protamine dose-dependent manner,
The effect was maximal at 1.6 mg / kg. At this time, the labeling index was actually 5 times or more that of HGF alone. HG by protamine
To show that the enhancement of F activity is not specific to the model of liver injury called ANIT treatment, a similar study was performed using 30% partially hepatectomized rats. The result is shown in FIG. As shown in FIG. 1 (B), the result was approximately AN
It was the same as in the case of the IT treatment. Protamine alone had no effect in any of the liver injury models.

Example 2 Measurement of Serum Levels of Hepatocyte Marker Enzymes, etc. In the ANIT-treated rat of Example 1, hepatic cells were tested to confirm that protamine also enhances the effect of HGF on hepatocyte repair. Serum levels of cell marker enzymes were examined. The measurement of hepatocyte marker enzymes and the like was performed according to a conventional method. The results are shown in FIG. 2 (BIL, GPT,
LAP) and FIG. 3 (ALT, γ-GTP). FIG.
And 3, BIL (bilirubin), GP
Serum levels of T, LAP, ALP, γ-GTP are H
Almost untreated rats with GF (300 μg / kg) alone
(Control (-)) level. Therefore, although the enhancement of the liver repair effect by protamine treatment was hard to observe, the effect of γ-GTP was stronger than that of HGF alone. No significant effect was observed with protamine alone for the levels of these marker enzymes and the like. From the above, it was proved that protamine enhances the liver regeneration-promoting action and the liver damage repairing action of HGF.

Example 3 Time course of labeling index after ANIT treatment Protamine only (1.6 mg / kg), HGF only (3
00 μg / kg) or protamine (1.6 mg / kg)
With respect to the ANIT-treated rats to which + HGF (300 μg / kg) was administered, the time course of the labeling index after the ANIT treatment was observed. FIG. 4 shows the results.
As shown in FIG. 4, in the case of protamine + HGF, a clear peak of liver proliferation was observed after 48 hours, whereas H
No significant effect was observed with GF alone.
That is, it was revealed that protamine increases the hepatic proliferative action of HGF at any time after the occurrence of liver injury.

Example 4 Effect of Protamine on HGF Clearance
vo) Based on the HGF clearance mechanism described above, it is expected that the administration of protamine will delay the disappearance of HGF from plasma. Therefore, the change in plasma concentration of HGF after intravenous instant administration after no treatment or after protamine pretreatment was measured. The result is shown in FIG. As shown in FIG.
It was shown that the disappearance of GF was slightly delayed by protamine administration. The rats used here were all ANIT
24 hours after the treatment. Kinetic parameters were obtained by kinetic analysis of the plasma concentration transition in FIG. Table 1 shows the results. As shown in Table 1, the systemic clearance (CL total) was reduced to about 40% by protamine administration. The distribution volume (V1, distribution volume of the central compartment; Vdss, steady-state distribution volume) also decreased. These results indicate that protamine blocks cell surface heparin-like substances,
This can be explained if it is considered to be to inhibit the distribution and clearance of HGF there.

[0021]

[Table 1]

As mentioned above, protamine is indeed HGF
, But the degree of the decrease is at most a little over half, and the enhancement of the labeling index by more than 5 times as compared with HGF alone (see FIG. 1) is not at a level that can be explained very much. Therefore, the dose of HGF was increased, and HGF (300 μg / kg) + protamine (1.
The labeling index was measured when 500 μg / kg of HGF alone, which gives an AUC (area under plasma concentration) comparable to that at the time of administration of 6 mg / kg, was administered alone. FIG. 6 shows the result. As shown in FIG. 6, the labeling index obtained did not change much with the administration of 300 μg / kg. Therefore, HG by protamine
It was suggested that the enhancement of the F effect also involves actions other than the increase in the blood retention of HGF.

Formulation Example 1 1 mg of HGF and 6 parts of protamine in 100 ml of physiological saline
mg, 1 g of mannitol and polysorbate 80 10
A solution containing mg was aseptically prepared, dispensed into vials 1 ml at a time, freeze-dried and sealed to obtain a freeze-dried preparation.

Example 2 0.02 M phosphate buffer (containing 0.15 M NaCl and 0.01% polysorbate 80, pH 7.4) 10
An aqueous solution containing 1 mg of HGF, 5 mg of protamine and 100 mg of human serum albumin in 0 ml was aseptically prepared, dispensed into vials 1 ml at a time, freeze-dried and sealed to obtain a freeze-dried preparation.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of protamine on the liver repair effect of HGF in liver disease rats. (A) of the figure shows the case of the ANIT-treated rat, and (B) shows the case of the 30% partially hepatectomized rat.

FIG. 2 shows hepatocyte marker enzymes and the like (BIL, GP) in ANIT-treated rats to which protamine and HGF were administered.
T, LAP) shows serum levels.

FIG. 3 shows hepatocyte marker enzymes and the like (ALT, γ-gamma) in ANIT-treated rats to which protamine and HGF were administered.
FIG. 2 shows the serum levels of GTP).

FIG. 4. Protamine only, HGF only or protamine +
About the ANIT-treated rats to which HGF was administered, ANI
It is a figure which shows the time transition of the labeling index after T process.

FIG. 5 is a graph showing changes in the plasma concentration of HGF after intravenous instantaneous administration without treatment or after protamine pretreatment.

FIG. 6 is a graph showing changes in the labeling index when the dose of HGF is increased.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/00 ADU A61K 37/02 AED AED 37/24 ACV (72) Inventor Tae-gu Guo 1-44, Tomigaya, Shibuya-ku, Tokyo 4-401 (72) Inventor Shunichi Nakamura 4-1 Takamidai, Takatsuki-shi, Osaka (72) Inventor Kunio Matsumoto 5-26-15-603 Onohara Higashi 5-chome, Minoh-shi, Osaka

Claims (4)

[Claims] 1. A pharmaceutical preparation comprising HGF and a substance having a binding property to a heparin-like substance on the surface of a hepatocyte. 2. The pharmaceutical preparation according to claim 1, wherein the substance having a binding property to a heparin-like substance on the surface of a hepatocyte is a basic protein. 3. The pharmaceutical preparation according to claim 1, wherein the substance having a binding property to the heparin-like substance on the surface of hepatocytes is protamine. 4. The compounding ratio of a substance having a binding property to a heparin-like substance on the surface of a hepatocyte to HGF is 0.5 to 5%.
The pharmaceutical preparation according to any one of claims 1 to 3, which is 0 times by weight.