JPH0640938A - Hgf-containing pharmaceutical preparation - Google Patents

Abstract

PURPOSE:To obtain a medicinal pharmaceutical preparation capable of carrying out prolongation of acting time of HGF(hepatocyte growth factor). CONSTITUTION:The pharmaceutical preparation contains HGF and heparin. Since the HGF-containing pharmaceutical preparation which is effective and long acting at low dosage can reduce administration frequency and amount, relief from patient's pain and reduction of medical expenses can be carried out.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to HGF (Hepatocyto Gr
The present invention relates to a pharmaceutical preparation containing owth factor, hepatocyte growth factor), and more specifically to a pharmaceutical preparation capable of sustaining the action time of HGF.

[0002]

HGF is a physiologically active peptide discovered by Nakamura et al. That has the most potent growth-promoting activity on mature hepatocytes (eg, Biochem. Biophys. Res. C).
ommun., 122 , 1450, 1984, FEBS Letter, 22 , 311, 198
In recent years, mass production has become possible by biotechnological methods (eg Nature, 342 , 440, 1989, Proc. Nat.
l. Acad. Sci. USA, 87 , 3200, 1990, Biochem. Biophy
s. Res. Commun., 172 , 321, 1990, etc.). This factor is expected to be applied not only to hepatitis and cirrhosis but also as a therapeutic / prophylactic agent for nephritis, cancer, etc., as a side effect inhibitor for anticancer agents, and a wound healing agent.

[0003]

As mentioned above, the HGF
Is a substance expected to be used as a drug,
Even if this factor was administered alone, its elimination from the blood within a half-life of several minutes was a major obstacle to the development of this factor as a drug. It is already known that the major organ involved in clearance of this factor from the blood is the liver. This factor is a high-molecular peptide and will be administered to a patient as an injection, for example, but drugs with a short half-life must be administered frequently or continuously as they are. Therefore, the patient will experience greater distress in treatment than long-acting drugs. In addition, if clearance from blood is fast, a large amount of drug must be administered, which leads to a rise in medical costs and a large amount of production of this factor. Although there are attempts to overcome this point by general formulation techniques, they were not designed in accordance with the characteristics of this factor, and sufficient effects have not been obtained. The present invention has been made to solve the above problems, and an object of the present invention is to reduce the clearance from blood and extend the duration of action of this factor while maintaining the biological activity of HGF. It is to provide a formulation capable of

[0004]

[Means for Solving the Problems] In order to solve the above problems, the inventors of the present invention have made earnest studies on sustaining the action time of HGF. As a result, HGF is considered to have a heparin affinity characteristic. It was found that the combined use of heparin, that is, the formation of a complex of HGF and heparin, can reduce the clearance of HGF from the blood. It was also confirmed that the formation of the complex does not hinder the expression of the biological activity of HGF. The present invention has been made based on such findings. That is, the pharmaceutical preparation of the present invention comprises HGF and heparin.

The HGF having the above-mentioned constitution, which is the active ingredient of the present invention, can be prepared by various methods as long as it is purified to the extent that it can be used as a medicine. Various methods are known as methods for preparing HGF, and examples thereof include liver, spleen, lung, bone marrow, brain, kidney, placenta and other organs of mammals such as rats, cows, horses and sheep, platelets, leukocytes. It can be obtained by extraction and purification from blood cells such as blood plasma, plasma and serum. Also, HGF
HGF can also be obtained by culturing a primary culture cell or cell line that produces GF, and separating and purifying from a culture (culture supernatant, culture cell, etc.). Or by genetic engineering techniques, H
The gene encoding GF is incorporated into an appropriate vector, and this is inserted into an appropriate host for transformation, and the desired recombinant HGF can be obtained from the culture of this transformant (for example, Nature, 342). , 440, 1989, etc.). The above-mentioned host cells are not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant or animal cells can be used.

More specifically, as a method for 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 crushed, and S- It can be purified by an ordinary protein purification method such as gel column chromatography using sepharose or heparin sepharose, and HPLC. In addition, an animal cell such as a Chinese hamster ovary (CHO) cell, a mouse C127 cell, etc., which is obtained by incorporating a gene encoding the amino acid sequence of human HGF into a vector such as bovine papilloma virus DNA using a gene recombination method. Transforming monkey COS cells,
It can be obtained from the culture supernatant.

The HGF thus obtained has a part of its amino acid sequence deleted or substituted by another amino acid, a part of another amino acid sequence inserted, or has 1 amino acid at the N-terminal and / or C-terminal. Alternatively, two or more amino acids may be linked, or the sugar chain may be similarly deleted or substituted. Examples of the HGF-similar drug include, for example, JP-A-3-130091 and International Publication WO90 / 106.
The substances described in Japanese Patent Publication No. 51, etc. are mentioned, and these are applicable to the present invention and are included in the scope of the present invention.

As the heparin which is another component of the present invention, any one can be used as long as it is purified to such an extent that it can be used as a medicine, and its origin (eg, bovine, porcine etc.) is also particularly limited. Not done. The molecular weight of heparin used is also not particularly limited, and any of high molecular weight heparin, low molecular weight heparin and mixtures thereof can be used. The usage ratio of heparin to HGF is about 0.01 to 50 mg of heparin per 1 pmol of HGF. Heparin usage is 0.01
If it is less than mg, a sufficient HGF clearance-lowering effect may not be exhibited, and if it exceeds 50 mg, there is no problem, but the effect can be exhibited with the amount up to that time, so there is little need to add more than that amount.

The object of the present invention is achieved by administering a pre-prepared preparation containing HGF and heparin, or preparing and administering a preparation containing HGF and heparin at the time of use. Applicable symptoms include treatment / prevention of hepatitis, cirrhosis, nephritis, cancer, etc., suppression of side effects of anticancer agents, promotion of wound healing, etc.

The formulations of the present invention can be formulated into various formulations (eg,
Solution, solid formulation, capsule, etc.), but generally only HGF and heparin which are active ingredients, or an injection together with them and a conventional carrier, or an external preparation together with a conventional carrier. . The injection can be prepared by a conventional method, for example, HGF and heparin are dissolved in a suitable solvent (eg, sterilized water, buffer solution, physiological saline, etc.), and then sterilized by filtering with a filter or the like. Then, it can be prepared by filling an aseptic container. The HGF content in the injection is usually adjusted to about 0.0002 to 0.2 (W / V%), preferably about 0.001 to 0.1 (W / V%), and the heparin content is appropriately adjusted according to the HGF content. Also,
The external preparation is formulated into, for example, an ointment-like, gel-like, liquid-like dosage form, and the HGF content in the preparation can be appropriately adjusted according to the disease to which the external preparation is applied, the application site and the like. Upon formulation, a stabilizer is preferably added,
Examples of the stabilizer include albumin, globulin,
Gelatin, mannitol, glucose, dextran,
Examples thereof include ethylene glycol. Furthermore, the formulation of the present invention may contain additives necessary for formulation, for example, excipients, solubilizers, antioxidants, soothing agents, isotonic agents and the like. In the case of a liquid preparation, it is desirable to remove water by freezing, or freeze-drying, and then save. The lyophilized preparation is used by re-dissolving it by adding distilled water for injection or the like at the time of use.

The formulation of the present invention can be administered by an appropriate administration route depending on the form of the formulation. For example, it can be administered in the form of an injection such as vein, artery, subcutaneous or intramuscular. The dose is appropriately adjusted depending on the patient's symptoms, age, body weight, etc., but is usually 0.01 mg to 100 m as HGF.
g, and it is appropriate to administer this once or several times a day.

[0012]

EXAMPLES The present invention will be described in detail below based on examples and test examples, but the present invention is not limited to these examples. In the examples described below, a review by Nakamura et al. (For example, Critical Reviews in Oncogenesis, 3 , 27
-54, 1992, Metabolism 28 , 599-608, 1991, etc.) was used, but the type 2 and type 3 HGF already have activity equivalent to that of the type 1 HGF. Are known, type 2 and type 3
Also, it is clear that the same effect can be obtained by using each type of derivative.

Example 1 A solution containing 1 mg of HGF, 4 g of heparin, 1 g of mannitol and 10 mg of polysorbate 80 in 100 ml of physiological saline was aseptically prepared, and 1 ml of each was aseptically dispensed into a vial, and the solution was prepared according to a conventional method. Lyophilization was performed to obtain a freeze-dried preparation.

Example 2 pH 7.4 containing 0.15M NaCl and 0.01% polysorbate 80
HGF 1 mg, heparin 4 g in 100 ml of 0.02 M phosphate buffer
And an aqueous solution containing 100 mg of human serum albumin was aseptically prepared, and 1 ml each was aseptically dispensed into a vial and freeze-dried according to a conventional method to obtain a freeze-dried preparation.

The present invention will be described below based on test examples. The heparin used in the test is as follows. High molecular weight heparin: Sigma, product number H 7005 [sodium salt, grade II, derived from porcine intestinal mucosa, molecular weight: 25
000-35000 (laser light scattering method), 18000-
23000 (gel filtration method)] Low molecular weight heparin: manufactured by Sigma, product number H5640 (sodium salt, derived from porcine intestinal mucosa, molecular weight: 4000-60
00)

Test Example 1 When rat liver was perfused with HGF-heparin complex
HG of tracer concentration (0.8 pM) labeled with ¹²⁵ I of radioactivity and liver extraction rate ¹²⁵ I that appeared in the effluent
F only, and 0.1 mg / ml with this standard, 1 respectively
Rats were perfused with a single passage of each complex prepared by mixing 50 mg / ml and 3 mg / ml of high molecular weight heparin, followed by incubation at room temperature for 50 minutes (perfusion rate, 12 ml / min). The perfusate is 20% (V / V) bovine red blood cells, 2% (W / V) bovine serum albumin (BSA),
Neutral buffer containing 5 mM glucose [120 mM Na
Cl, 4.8 mM KCl, 1 mM KH ₂ PO ₄ , 1.2
mM MgSO ₄ , 2.2 mM CaCl ₂ , 20 mM
MES (pH 7.4)] was used. The time course of the ratio of TCA-precipitable radioactivity in the hepatic vein to the trichloroacetic acid (TCA) -precipitable radioactivity in the influent (FIG. 1 left side) and its liver extraction rate (FIG. 1 right side) were measured. . In addition,
The liver extraction rate was calculated from (radioactivity in influent-radioactivity in hepatic vein) / (radioactivity in influent) under a steady state. As shown in FIG. 1, when mixed with 1 mg / ml or more of heparin, the liver extraction rate was significantly reduced. In addition, considering that the major organ involved in the clearance of HGF from the blood is the liver, it was suggested that the clearance of HGF from the blood was significantly reduced by heparin even under in vivo conditions.

Test Example 2 Plasma when intravenously injecting ¹²⁵ I-HGF-heparin complex
Time course of TCA-precipitating radioactivity in medium HG with tracer concentration (500 pM) labeled with ¹²⁵ I
F only, and this preparation and 20 mg / ml, 40 respectively
mg / ml, 80 mg / ml high-molecular-weight heparin or 80 mg / ml low-molecular-weight heparin were mixed respectively, and then incubated at room temperature for 50 minutes to prepare each complex, and then to the rat, about 0.25 ml of each complex was prepared from the femoral vein. I made a note. Then, blood is collected from the femoral artery and plasma TCA-
The change in precipitating radiation concentration was measured. Under these conditions, the HGF dose was 0.13 pmol / rat, and the heparin dose was 5 m for high molecular weight heparin.
g / rat, 10 mg / rat, 20 mg / rat, and low molecular weight heparin correspond to 20 mg / rat.
The obtained results are shown in FIG. The plasma concentration obtained as a result was shown normalized by the dose. As shown in FIG. 2, for high molecular weight heparin, 5 mg / rat or more,
With low molecular weight heparin, a marked decrease in HGF clearance from blood was observed at 20 mg / rat.

Test Example 3 Promotion of DNA synthesis by HGF in primary culture hepatocytes (HGF
Effect of contact time with rat ) Rat free hepatocytes (2.5 × 10 ⁵ cells / ml) prepared by collagenase perfusion method were added to a culture dish so that the number of cells per cm ² was 7 × 10 ^4. Get in,
Williams' medium E medium (1 nM insulin, 1 nM dexamethasone, 5% (V / V) calf serum, 30 mg /
1 kanamycin (including monosulfate) was cultured for 24 hours. On the way, 2 hours after the start of culture, the medium was replaced with a fresh one. 24 hours after the start of culture,
iams' medium E medium (containing 1 nM insulin, 1 nM dexamethasone, 5 U / ml aprotinin, 30 mg / l kanamycin monosulfate) and HGF (0-250 pM) at various times (0.3-
After incubating for 28 hours), the cells were washed, the same culture medium was added, and the incubation was continued until a total of 28 hours. Twenty-two hours after the incubation, ¹²⁵ I-labeled deoxyuridine (collection concentration, 0.3 μCi / ml, 0.14 nM) was added together with unlabeled substance (final concentration, 480 nM). 28 hours later (
6 hours after the addition of ¹²⁵ I-deoxyuridine), the amount of ¹²⁵ I-deoxyuridine incorporated was measured to measure DN.
A synthesis was evaluated. The results are shown in Fig. 3. The results are shown with the maximum activity obtained when contacting 90 pM HGF for 28 hours as 100. As is clear from FIG. 3, the longer the contact time between HGF and hepatocytes as the target cells, the more the DNA synthesis increased. This is H
It has been shown that the presence of GF in blood for a longer period of time leads to enhancement of its efficacy.

Test Example 4 DNA of primary culture hepatocytes with HGF-heparin complex
Synthetic HGF 1 nM or 12.5 nM and heparin (concentration, 0
-100 mg / ml) was preincubated for 50 minutes at room temperature to allow complex formation. 20 μl of this mixed solution
Was added to the cultured hepatocytes in 500 μl of the medium shown in Test Example 3 and incubated for 28 hours. Therefore, the final concentration in the medium was about 40 pM and 500 p with HGF.
M and heparin were added to 0-4 mg / ml.
22 hours after the addition of the HGF-heparin mixed solution, ¹²⁵ I-deoxyuridine was added in the same manner as in Test Example 3, and DN
A synthetic ability was measured. The result is shown in FIG. In addition, in FIG. 4, DNA synthesizing ability in the absence of heparin is 10%.
It was set to 0 and expressed as a ratio (%) to it. As is clear from FIG. 4, a sufficiently high biological activity was exhibited even in the complex with a high concentration of heparin. By the way, the amount of haplin of 10 mg / rat, which is sufficient to reduce the blood clearance of HGF shown in Test Example 2, was found to be equivalent to the volume of circulating plasma (about 10 m
1 / rat) corresponds to approximately 1 mg / ml.

Test Example 5 Plasma when intravenous injection of ¹²⁵ I-HGF followed by intravenous injection of heparin
Time course of TCA-precipitable radioactivity in normal rats and rats treated with carbon tetrachloride (1 with olive oil)
Carbon tetrachloride diluted 0 times was added to the rat peritoneal cavity at 1 ml / 1.
00 g of body weight was administered, and rats were used 24 hours later).
¹²⁵ I-labeled tracer amount (0.13 pmol / rat) of HGF was intravenously injected into the femoral vein, and 11 to 16 minutes later, high molecular weight heparin was administered at various doses (0, 10, 2).
TC in plasma when injected intravenously at 5, 50 mg / rat)
The time course of A-precipitating radioactivity was measured. The blood was collected from the femoral artery. Fig. 5 shows that ¹²⁵ I-in normal rats.
The time course of TCA-precipitating radioactivity in plasma when heparin is intravenously injected 16 minutes after HGF is intravenously injected is shown. In addition, FIG. 6 shows that TCA-precipitable radiation in plasma when ¹²⁵ I-HGF was intravenously injected into normal rats (control) and carbon tetrachloride-treated rats 11 minutes after that, and heparin (25 mg / rat) was intravenously injected. The time course of activity is shown. As shown in FIGS. 5 and 6, it is clear that heparin administration transiently increases the plasma concentration of ¹²⁵ I-HGF. The reason for this is that heparin reduces the clearance of HGF in blood and that it binds to the surface of each tissue.
Both causes of ¹²⁵ I-HGF being removed by heparin and translocating into circulating blood are considered. Here, it is important to note that the same effect of heparin was observed in hepatitis-induced rats treated with carbon tetrachloride as in normal rats.

[0021]

As described above, by mixing HGF with heparin to form a complex, and by adding various commonly used excipients and stabilizers,
It is possible to obtain a long-acting HGF-containing drug which is effective at a low dose. Therefore, according to the present invention, the number of administrations and the amount of administration can be reduced, so that the patient's pain can be alleviated and the medical cost can be reduced.

[Brief description of drawings]

FIG. 1 is a diagram showing the ratio of radioactivity (left side) and the liver extraction rate (right side) that appeared in the effluent when a rat liver was perfused with an HGF-heparin complex.

FIG. 2 is a diagram showing the time course of TCA-precipitating radioactivity in plasma when ¹²⁵ I-HGF-heparin complex is intravenously injected.

FIG. 3 is a diagram showing the influence of contact time with HGF on the DNA synthesis promotion of primary culture hepatocytes by HGF.

FIG. 4 is a diagram showing DNA synthesis of primary culture hepatocytes by HGF-heparin complex. The left figure shows HGF concentration 4
The case of 0 pM and the right figure show the case of HGF concentration of 500 pM.

FIG. 5 is a diagram showing the time course of TCA-precipitating radioactivity in plasma when ¹²⁵ I-HGF was intravenously injected into normal rats, followed by intravenous injection of heparin at 16 minutes.

FIG. 6 TC in plasma when ¹²⁵ I-HGF was intravenously injected to normal rats (control) and carbon tetrachloride-treated rats after 11 minutes and heparin (25 mg / rat) was intravenously injected.
It is a figure which shows the time transition of A precipitation radioactivity.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Yuichi Sugiyama 3-4-10 Nishikubo, Musashino-shi, Tokyo (72) Manabu Hanano 1-24-3 Matsugaoka, Funabashi-shi

Claims (4)

[Claims] 1. A pharmaceutical preparation comprising HGF and heparin. 2. The pharmaceutical preparation according to claim 1, wherein HGF is derived from human or animal tissues or blood components. 3. The pharmaceutical preparation according to claim 1, wherein HGF is produced by genetic recombination. 4. The pharmaceutical preparation according to any one of claims 1 to 4, which is prepared by mixing HGF and heparin at the time of use.