JP2000344681A - Hgf-containing medicine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicine having extremely suppressed manifestation of nephrotoxicity in comparison with the case of continuous administration, capable of sufficiently developing a desired pharmacodynamic effect by administering a hepatocyte growth factor while providing a fixed medicine withdrawal period. SOLUTION: This medicine comprises a hepatocyte growth factor as-an active ingredient and is intermittently administered while providing a medicine withdrawal period enough for alleviating or eliminating nephrotoxicity derived from the hepatocyte growth factor preferably for >=one day at least once in an administration period. For example, preferably the hepatocyte growth factor is a protein factor having chemical and physical properties of 76,000-92,000 estimated molecular weight under nonreduction conditions, hepatocyte growth activity, strong affinity for heparin, deactivation of the hepatocyte growth activity by heat treatment at 80 deg.C for 10 minutes and deactivation of the hepatocyte growth activity by digestion treatment with trypsin and digestion treatment with chymotrypsin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a medicament containing hepatocyte growth factor as an active ingredient.

[0002]

2. Description of the Related Art Hepatocyte growt
h factor: Hereinafter, it may be abbreviated as "HGF" in this specification. ) Is a protein having hepatocyte proliferation activity, its existence is known in various animal species, and a protein having a different amino acid sequence has been reported. Human hepatocyte growth factor (hereinafter sometimes abbreviated as "hHGF" in the present specification) is found in plasma of a patient with fulminant hepatitis by Daikohara et al. (JP-A-63-22526). Kitamura et al. Disclosed the amino acid sequence of hHGF protein and the sequence of the gene (cDNA) encoding it (JP-A-3-72883). Furthermore, as a method for producing hHGF protein and a transformant using this cDNA have been reported (Japanese Patent Application Laid-Open No. 3-285693), mass production of hHGF protein has become possible, and application to pharmaceuticals is expected.

[0003] hHGF is a kind of glycoprotein and has a molecular weight of about 80 to 90 KDa in a non-reduced state and about 52 in a reduced state.
It is a heterodimer consisting of an α subunit of ~ 56 KDa and a β subunit of about 30 to 36 KDa. hHGF is not only active as a hepatocyte growth factor, but also as a scatter factor (scat factor).
ter factor (SF) activity, renal tubular epithelial cell growth factor activity, damaged tissue repair factor activity, vascular endothelial cell growth factor activity, etc. It is expected to be developed as a medicament such as an agent for treating disorders, an agent for promoting hair growth, an agent for treating wounds, and an agent for treating antitumor. However, HGF is known to have toxicity to kidney glomeruli, and there is a need to develop a method for avoiding nephrotoxicity when clinically applying HGF.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a means for reducing the nephrotoxicity of HGF. The present inventors have conducted intensive studies on the mechanism of the occurrence of nephrotoxicity induced by HGF administration. As a result, administration of HGF with a drug holiday of at least one day or more compared to the case of daily administration of HGF It has been found that the occurrence of nephrotoxicity is remarkably suppressed, and that the desired drug effect is sufficiently exhibited. The present invention has been completed based on the above findings.

[0005] That is, the present invention relates to a medicament containing hepatocyte growth factor as an active ingredient, wherein a drug suspension period during administration is sufficient to reduce or eliminate nephrotoxicity derived from hepatocyte growth factor. At least one is provided for intermittent administration. The drug holiday is usually one or more days, preferably one day, and typically the drug can be administered every other day. The medicament of the present invention is characterized in that it can reduce nephrotoxicity derived from hepatic parenchymal cell growth factor, in particular, toxicity to glomeruli of the kidney, and can sufficiently exhibit desired drug effects. According to the present invention, there is provided the use of hepatocyte growth factor for the manufacture of the above medicament.

In another aspect, the present invention provides a method of using the hepatocyte growth factor for producing the above-mentioned medicament, and a method of administering the hepatocyte growth factor which has reduced or eliminated renal toxicity. And a method for intermittently administering at least one drug holiday during the administration period to reduce or eliminate nephrotoxicity caused by the above. In still another aspect, the present invention provides a medicament comprising a hepatocyte growth factor as an active ingredient, which is administered with a drug holiday period sufficient to reduce or eliminate nephrotoxicity derived from the hepatocyte growth factor. A dosing schedule in which at least one hepatocyte growth factor is provided intermittently during the period; a medicament containing a hepatocyte growth factor as an active ingredient for use in the above-mentioned dosing schedule;
Use of a medicament comprising hepatocyte growth factor as an active ingredient for the above-mentioned administration schedule is provided.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The type of HGF used as an active ingredient of the medicament of the present invention is not particularly limited. For example, H
Natural HGF may be isolated from bodily fluids and tissues derived from mammals such as humans and rats known to contain GF, or cells that spontaneously produce HGF. Recombinant HGF obtained by introducing a growth factor cDNA into cells may be used. Hosts that produce recombinant HGF include Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant cells, insect cells, animal cells, and the like.
Specific examples include recombinant HGF, for example, the above-mentioned mammal-derived placenta, liver tissue and blood of liver injury patients, fibroblast cell lines such as MRC-5 cells and IMR-9 cells, or
Examples include those obtained from a production strain or the like in which an expression vector containing cDNA encoding hHGF is introduced into a host such as CHO cells in accordance with the method described in Japanese Patent No. 285693.

[0008] HGF includes precursor proteins such as proteins having a signal sequence, and modified proteins in which some amino acids have been substituted, deleted and / or inserted within a range that does not impair the activity of proliferating hepatocytes. Alternatively, a variant in which saccharides are deleted or substituted may be used. As a variant,
For example, JP-A-2-288899, WO90 / 10651, JP-A-3-30091, JP-A-3-255096, JP-A-4-30000, Nature, 342, 440-443 (1989) and the like. Those described may be mentioned.

The HGF preferably used as the active ingredient of the medicament of the present invention includes protein factors having the following physicochemical properties. HGF is preferably derived from humans, and particularly preferred HGFs include those represented by the amino acid sequences described in JP-A-3-72883 and JP-A-4-89499. 1) Estimated molecular weight by SDS-PAGE (under non-reducing conditions) is about 76,000-92,000; 2) Hepatocyte proliferating activity; and 3) Strong affinity for heparin . Further, preferred HGF, in addition to the above physicochemical properties, 4) loses its activity by heat treatment at 80 ° C. for 10 minutes, and 5) loses its activity by digestion with trypsin and chymotrypsin. To be active.

HGF is a scatter factor.
r; SF) activity, renal tubular epithelial cell growth factor activity, damaged tissue repair factor activity, vascular endothelial cell growth factor activity, etc. Therapeutic agent, cranial nerve disorder treatment agent, hair growth promoter,
It is useful as a drug for wound treatment, antitumor treatment, low protein plasma treatment, and the like. However, an administration method in which administration at least once a day is continued every day throughout the administration period (daily administration) may cause nephrotoxicity, and the glomeruli of the kidney may be caused by HGF-induced lesions (enlargement of glomerular epithelium). / Degeneration, adhesions, crescent formation, glomerular proliferative lesions).

[0011] The medicament of the present invention is used in an administration regimen of intermittently administering hepatocyte growth factor with a drug holiday in order to reduce or eliminate nephrotoxicity derived from hepatocyte growth factor. It is administered intermittently with a sufficient drug holiday to reduce or eliminate nephrotoxicity due to growth factors. The medicament of the present invention is characterized in that it can reduce nephrotoxicity derived from hepatocyte growth factor, in particular, toxicity to glomeruli of the kidney, and can sufficiently exhibit desired drug effects. The above-mentioned withdrawal period should be at least one during the administration period.
The drug holiday is usually at least one day or more, preferably one day. For example, a one-day drug holiday may be provided during the administration period in which daily administration is performed. Typically, the medicament of the present invention can be administered every other day. The dose may be appropriately adjusted depending on the age, sex, symptoms, body weight, etc. of the patient.
kg body weight to 10 mg / kg body weight, more preferably 10 to 1000 μg / kg
It is administered in a range of weight.

As the medicament of the present invention, a conventionally known medicinal composition containing HGF as an active ingredient can be used. For example, a drug in parenteral administration such as an injection in the form of an aqueous solution, a drop, or a lyophilized preparation can be suitably used. For preparations of HGF, see, for example, WO 90/10651
These are described in Japanese Patent Application Laid-Open Nos. Hei 6-247872 and Hei 9-25241. Further, for the purpose of reducing nephrotoxicity, a composition to which a sulfated polysaccharide such as dextran sulfate described in JP-A-10-324638 is added is also preferable. As the sulfated polysaccharide, those having a high binding affinity to HGF (for example, a Kd value of less than 1.0 × 10 ⁻⁸ ) are suitable, and particularly preferred sulfated polysaccharides have a high binding affinity to HGF. Heparin. The above-mentioned sulfated polysaccharide can be administered as a separate preparation from the drug of the present invention containing HGF in the above-mentioned administration schedule. Toxicity can be further reduced or avoided.

[0013] The form of the medicament of the present invention is not particularly limited, but is generally desirably in a form suitable for parenteral administration, and can preferably be prepared as an ampoule for injection or a lyophilized powder for injection (vial). . For the preparation of the composition or the preparation, an appropriate means available in the art can be used. Pharmaceutical additives can be appropriately selected by those skilled in the art according to the form of the formulation, for example, lyophilized powder for injection is
An effective amount of the purified HGF is dissolved in, for example, a diluent such as distilled water, physiological saline, or aqueous glucose solution, and if necessary, excipients such as carboxymethylcellulose and sodium alginate, polyethylene glycol, dextran sodium sulfate. , Amino acids, stabilizers such as human serum albumin, preservatives such as benzyl alcohol, benzalkonium chloride, phenol, soothing agents such as glucose, calcium gluconate, procaine hydrochloride, hydrochloric acid,
PH adjusters such as acetic acid, citric acid and sodium hydroxide,
It can be prepared by adding a solubilizing agent such as sodium chloride, a stabilizer such as amino acids, an isotonic agent such as sodium chloride, and the like, and freeze-drying according to a conventional method.

The ampoule for injection is prepared by dissolving an effective amount of the HGF in a diluent such as distilled water, physiological saline, Ringer's solution and the like, and if necessary, a solubilizing agent such as sodium salicylate or mannitol; Buffering agents such as sodium citrate and glycerin; isotonic agents such as glucose, added sugars and sodium chloride; stabilizing agents, preservatives; soothing agents; pH adjusting agents; The solution can be prepared by adding a pharmaceutical additive such as a stabilizer such as amino acids, and sterilizing the resulting solution by usual heat sterilization, sterile filtration, or the like. When used as an external preparation, it can be used in a dosage form as described in JP-A-5-213721 and JP-A-5-279230.

When the medicament of the present invention is prepared as a lyophilized preparation, it preferably contains HGF, a stabilizing agent, sodium chloride, and a buffer, and contains hepatocyte growth factor at a concentration of 5 mg / mL or less. And it is preferable to prepare an aqueous solution having a desired pH and osmotic pressure as an injection. Such a formulation may contain HGF at 5 mg /
It is preferable to obtain the aqueous solution containing the solution at a concentration of mL or less by freeze-drying. As a stabilizer, for example, arginine,
A stabilizer selected from the group consisting of lysine, histidine, glutamine, proline, glutamic acid, aspartic acid, and pharmacologically acceptable salts thereof can be used, and a phosphate can be used as a buffer. An aqueous solution prepared from such a lyophilized preparation has excellent storage stability, and has a desirable pH (for example, in the range of 6.0 to 7.0) as an injection and an isotonic ratio between the living body and the osmotic pressure ratio of the injection. Is preferred for implementing the above dosing regimen since it has an osmotic ratio (1-2) that is acceptable as

[0016]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to the following Examples. In the following examples, recombinant human HGF produced according to the method described in JP-A-3-285693 was used as HGF.

Example 1 Effect of Intermittent Administration of HGF on DMN (Dimethylnitrosamine) Liver Cirrhosis Rats <Animals Used> Wistar male rats 6 weeks old were used. <Group composition> Group A: no treatment (N = 10) Group B: DMN treatment, control of group C (N = 13) Group C: DMN treatment + HGF 0.03 mg / kg daily administration (N = 15) Group D: DMN Treatment, control of groups E and F (N = 12) Group E: DMN treatment, intermittent administration of HGF 0.03 mg / kg (N = 15) Group F: DMN treatment, intermittent administration of HGF 0.1 mg / kg (N = 13)

<Experimental method> Treatment on animals: 6-week-old rats (about 170 g) were treated with 1% DM.
N solution (diluted with physiological saline) at 1 ml / kg for 3 consecutive days /
Administered intraperitoneally for 4 weeks per week. Group A was similarly administered with saline. HGF is diluted buffer (0.14 MN
aCl, 0.005% Tween80, 10 mM phosphate buffer pH7.
4) Dissolve so as to be 0.03 mg / ml (groups C and E) or 0.1 mg / ml (group F) in group C, daily for 27 days, group E and F every other day 3 days / week for 4 weeks , 1 ml / kg body weight, once a day, via the tail vein. Groups A, B and D were similarly administered only the dilution buffer. After the administration for 27 days, blood was collected from the descending vena cava under fasting conditions. Serum albumin concentration measurement: For albumin serum, albumin B-Test Wako (Wako Pure Chemical Industries, Bromcresol Green Method) was used.

<Results> FIG. 1 shows the results of the serum albumin concentration. Serum albumin is one of the clinically important indicators of liver function, and is known to decrease in cirrhosis patients. The serum albumin concentration was significantly lower in the DMN control group B than in group A due to DMN treatment, and significantly increased in group C administered daily with HGF as compared to group B. The serum albumin concentration of group D, which was a control of the intermittent administration group, was significantly lower than that of group A. However, in groups E and F to which HGF was intermittently administered at 0.03 and 0.1 mg / kg, respectively, D There was a dose-dependent increase compared to the group. In particular, the improvement was significantly improved in the F group.

Example 2 Effect of Intermittent Administration on Reduction of Nephrotoxicity <Animal> 24 male SD rats (5 weeks old) with good health condition after purchasing SD male rats and quarantine and acclimatization period of 6 days or more
Was used for the test. <Group composition> HGF alone test Control group: untreated animals (n = 4) HGF 1 mg / kg daily administration (n = 10) HGF 1 mg / kg intermittent administration (n = 10) HGF was diluted with buffer (140 mM NaCl) , 0.005% Tween8
(0, 10 mM phosphate buffer, pH 7.5) to give a concentration of 0.2 mg / mL.

<Method of Administration> Rats were administered daily or intermittently (three times / week) into the tail vein of rats at a volume of 5 mL / kg based on the body weight on the day immediately before administration for 2 weeks. The control group was an untreated animal. <Test item> Urinalysis: Animal urine 5 hours after administration at the time of each test under fasting on the day before administration and 2 weeks after administration, creatinine was J
By the affe method, albumin was measured by the EIA method using a rat albumin measurement kit (Panatest A series rat albumin, Panafirm Laboratories Co., Ltd.), and the urinary albumin / creatinine ratio was calculated. Histopathology: Sacrifice under anesthesia the day after final administration
The cells were fixed with 10% neutral formalin solution. Thereafter, a paraffin-embedded pathological specimen was prepared, sectioned, stained with hematoxylin and eosin, and histopathologically evaluated with an optical microscope.

<Results> In the untreated group, no enlargement / degeneration of glomerular epithelial cells was observed as a renal lesion, but HGF 1m
In the g / kg daily administration group, hypertrophy / degeneration of glomerular epithelial cells was observed in 7 out of 10 cases. On the other hand, in the HGF 1 mg / kg intermittent administration group, no hypertrophy / degeneration of glomerular epithelial cells was observed in all 10 cases. In addition, the albumin / creatinine ratio also showed a lower value in the intermittent administration group than in the HGF daily administration group (FIG. 2). These results indicate that intermittent administration of HGF significantly reduces nephrotoxicity derived from HGF as compared to daily administration.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of intermittent administration of HGF on dimethylnitrosamine liver cirrhosis rats. In the figure, NT
Indicates the results of the untreated group, and DMN indicates the results of the dimethylnitrosamine-treated group (no HGF administration).
GF0.1 was the HGF dose 0.03 mg / kg group and 0.
The results for the 1 mg / kg group are shown. ## is p <0.01 for NT group
(Student's t-test); *: p <0.05 compared to DMN group (B) (Student's t-test); Δ: p <0.01 (DMN group (D)) Dunnett
test) indicates a significant difference. daily for daily administration, i
ntermittent indicates the result of intermittent administration.

FIG. 2 is a graph showing an effect of reducing nephrotoxicity by intermittent administration of HGF.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 17/02 A61P 17/02 17/14 17/14 25/28 25/28 35/00 35/00 C07K 14/475 C07K 14/475 (72) Inventor Jiro Sugimoto 1000 Kamoshita-cho, Aoba-ku, Yokohama-shi, Kanagawa Prefecture Inside the Mitsubishi Chemical Research Laboratory (72) Inventor Eiko Mizuno 1000-1000 Kamoshita-cho, Aoba-ku, Yokohama-shi, Kanagawa Mitsubishi (72) Inventor Yoshifumi Uno 1000, Kamoshidacho, Aoba-ku, Yokohama-shi, Kanagawa Prefecture Mitsubishi-Chemical Co., Ltd. (72) Inventor Masae Kakuzari 2-5-2-2 Marunouchi, Chiyoda-ku, Tokyo Mitsubishi Chemical Corporation Pharmaceutical Company F-term (reference) 4C084 AA02 AA03 BA34 CA53 DB62 MA66 NA07 ZA152 ZA512 ZA592 ZA752 ZA812 ZA892 ZA922 ZB26 2 4H045 AA10 CA40 DA01 EA20 FA74

Claims (5)

[Claims] 1. A medicament comprising hepatocyte growth factor as an active ingredient, wherein at least one drug suspension period during administration is sufficient to reduce or eliminate nephrotoxicity derived from hepatocyte growth factor. A drug for intermittent administration. 2. The medicament according to claim 1, wherein the drug holiday is one day or more. 3. A method for administering a medicament containing a hepatocyte growth factor as an active ingredient, wherein a drug suspension period during the administration period is sufficient to reduce or eliminate nephrotoxicity derived from the hepatocyte growth factor. A dosing schedule in which at least one is provided and intermittently administers hepatocyte growth factor. 4. A medicament comprising hepatocyte growth factor as an active ingredient for use in the dosage regimen according to claim 3. 5. Use according to claim 3 for a medicament comprising hepatocyte growth factor as an active ingredient.