JP2004269394A - Pulmonary hypertension-preventing and treating medicine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new pulmonary hypertension-preventing and treating medicine. <P>SOLUTION: A hepatocyte growth factor (HGF) can exhibit organ-protecting actions such as an anti-fiberization action, an anti-apoptosis action and a neovascularization action. The pulmonary hypertension-preventing and treating medicine contains the HGF capable of exhibiting the actions as an active ingredient. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel agent for preventing or treating pulmonary hypertension.
[0002]
[Prior art]
Pulmonary hypertension (PH) is a condition in which pulmonary vascular resistance and pulmonary arterial pressure are elevated, which disrupts the ventilation-perfusion relationship. PH is typically characterized by elevated blood pressure in the pulmonary circulation (systolic ≥30 mmHg and diastolic ≥12 mmHg). There are two subsets of pulmonary hypertension: primary (idiopathic or "unknown") and secondary. The most common causes of secondary pulmonary hypertension, whose secondary forms are much more widespread, are heart disease and lung disease. Regardless of the underlying cause of pulmonary hypertension, the resistant (pre-capillary) vessels of the lungs undergo anatomical changes that contribute to the development of pulmonary hypertension. Pulmonary arterial hypertension secondary to acquired heart disease begins with left ventricular impairment, which causes alveolar hypertension followed by pulmonary arterial hypertension.
[0003]
Primary pulmonary hypertension is a disease with a poor prognosis, and cardiopulmonary transplantation is currently the only effective treatment. However, cardiopulmonary transplantation has few donors and has considerable problems as a practical treatment method.
[0004]
It is known that in primary pulmonary hypertension, inflammation of the pulmonary artery (inflammatory reaction mainly by monocytes / macrophages) occurs in the early stage, followed by an increase in pulmonary vascular resistance due to thickening of the media and hypertrophy of the right ventricle. Have been. MCP-1 (Monocyte chemoattractant protein-1: macrophage chemotactic factor) belongs to the CC chemokine family, and is known to be frequently expressed in lesions of arteriosclerosis (such as atherosclerosis). (Takeya, M. et. Al., Hum. Pathol. 24: 534-539 (1993); Yla-Hertutuala, S. et. Al., Proc. Natl. Acad. Sci. USA, 88: 5252-5257. (1991)). A prophylactic / therapeutic agent for primary pulmonary hypertension, which includes an MCP-1 antagonistic mutein having an action of suppressing the action of MCP-1, has been disclosed (Patent Document 1).
However, development of a new drug for preventing or treating primary pulmonary hypertension is desired.
[0005]
Hepatocyte growth factor (hereinafter referred to as "HGF") was discovered as a potent growth-promoting factor for mature hepatocytes, and its gene was cloned (Biochem Biophys Res Commun, Vol. 163, p967 (1989), Nature). , Vol.342, p440 (1989)).
Subsequent studies have revealed that HGF is also involved in the wound healing of kidney, lung, stomach, duodenum, skin, etc. in vivo, and for the receptor for HGF, the c-Met proto-oncogene It has been clarified that it encodes a receptor (Non-Patent Documents 1 and 2).
At present, HGF is considered to be a factor that acts on many tissue repairs and organ regeneration through the receptor (Non-Patent Documents 3 and 4).
An agent for inducing a decrease in bax and / or an increase in bcl-2 or an agent for suppressing progression of apoptosis, which contains HGF as an active ingredient, has been disclosed (Patent Document 2). Furthermore, a therapeutic agent for myocardial infarction or angina containing HGF as an active ingredient has been disclosed (Patent Document 3).
[0006]
[Patent Document 1]
JP 2002-47203 A [Patent Document 2]
JP-A-11-246434 [Patent Document 3]
JP-A-11-246433 [Non-Patent Document 1]
Science, Vol. 251, p802-804 (1991)
[Non-patent document 2]
Oncogene, Vol. 6, p501-504 (1991)
[Non-Patent Document 3]
Experimental Medicine Vol. 10, p144-153 (1992)
[Non-patent document 4]
Atherosclerosis Vol. 23, p683-688 (1996)
[0007]
【task to solve】
An object of the present invention is to provide a new agent for preventing and treating pulmonary hypertension. Specifically, an object of the present invention is to provide a prophylactic / therapeutic agent for pulmonary hypertension containing HGF as an active ingredient.
[0008]
[Means to solve the problem]
The present inventors have paid attention to the fact that HGF can exert an organ protective effect such as an anti-fibrotic action, an anti-apoptotic action, and an angiogenic action, and as a result of intensive studies, administration of HGF is effective for pulmonary hypertension. I found that.
[0009]
That is, the present invention
1. An agent for preventing and treating pulmonary hypertension comprising hepatocyte growth factor as an active ingredient,
2. The agent for preventing / treating pulmonary hypertension according to item 1, wherein the pulmonary hypertension is primary pulmonary hypertension,
3. A method for treating pulmonary hypertension by using the agent for preventing or treating pulmonary hypertension according to the above 1 or 2.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
The HGF contained in the prophylactic / therapeutic agent of the present invention is a known substance, and as long as it is purified to the extent that it can be used as a medicine, it can be prepared by various methods.
As a method for producing HGF, for example, the HGF can be obtained by culturing primary cultured cells or cell lines that produce HGF, and separating and purifying it from a culture supernatant or the like. Alternatively, a gene encoding HGF may be inserted into an appropriate vector by genetic engineering techniques, inserted into an appropriate host, and transformed to obtain a desired recombinant HGF from the culture supernatant of the transformant. (See, for example, Biochem. Biophys. Res. Commun. Vol. 175, p660 (1991)).
The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques, for example, Escherichia coli, yeast, baculovirus (arthropod polyhedrosis virus) -insect cells or animal cells, and the like can be used. it can.
[0011]
The transfectant into which the recombinant vector has been introduced is cultured in a medium suitable for each. The medium contains a carbon source, a nitrogen source, inorganic substances, vitamins, serum, drugs used for resistance screening and the like necessary for growth of the transformant. Specifically, when the host of the transformant is Escherichia coli, for example, an LB medium (manufactured by Nacalai Tesque) or the like, and when the host is a yeast, a YPD medium (Genetic Engineering, 1, 117, Plenum Press (1979)). When the host is an insect cell or an animal cell, examples thereof include a Ham-12 medium, a MEM medium, a DMEM medium, and an RPMI1640 medium (manufactured by SIGMA) containing 20% or less of fetal calf serum. Further, the culture temperature, CO ₂ concentration, and culture time can be appropriately selected depending on the host, the recombinant vector, and the like. Further, ventilation and stirring are performed as necessary. Any medium may be used as long as the introduced host grows under a medium composition or culture conditions other than those described above and the protein encoded by the inserted HGF polynucleotide is produced.
[0012]
For example, when the host is a cell, a method of recovering the introduced culture cultured in this manner is, for example, a method of separating the culture by centrifugation or the like, and then collecting the cell as a cell body or a culture supernatant. Can be As a method for extracting the recombinant protein from the recovered cell body, a commonly used method known per se can be mentioned.
[0013]
HGF can also be prepared by a known cell-free protein synthesis system or the like. Specific examples of the cell extract used in the cell-free protein synthesis system include known cells such as microorganisms such as Escherichia coli, embryos of plant seeds, and cell extracts such as rabbit reticulocytes. These can be used commercially, or a method known per se, specifically, an Escherichia coli extract is described in Pratt, J .; M. et al. J., Transcription and Tranlation, Hames, 179-209, B.C. D. & Higgins, S.W. J. , Et. Al. , IRL Press, Oxford (1984).
[0014]
The HGF thus obtained may have one or more amino acids in its amino acid sequence substituted, deleted and / or added, as long as it has substantially the same action as natural HGF. May be substituted, deleted and / or added.
[0015]
The prophylactic / therapeutic agent of the present invention can be prepared and administered as a pharmaceutical composition of the above-mentioned HGF alone or in combination with an appropriate pharmaceutical additive. The administration form of such a pharmaceutical composition is not particularly limited as long as it is generally used for parenteral administration.For example, an ampoule for injection or a lyophilized powder for injection may be used. it can. Preparation into various formulation forms can be performed according to a conventional method using well-known formulation additives available to those skilled in the art, for example, diluents and additives.
[0016]
For example, a freeze-dried powder for injection is prepared by dissolving an effective amount of the purified HGF in a diluent such as distilled water for injection, physiological saline, or an aqueous glucose solution, and then adding carboxymethyl sepharose or sodium alginate as necessary. Excipients, polyethylene glycol, dextran sulfate sodium, 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, It can be produced by a conventional method by adding a pH adjuster such as hydrochloric acid, acetic acid, citric acid and sodium hydroxide. Further, the ampoule for injection is prepared by dissolving an effective amount of the HGF in a diluent such as distilled water for injection, physiological saline, Ringer's solution, etc., and dissolving aids such as sodium salicylate and mannitol, if necessary, sodium citrate, After adding additives such as buffering agents such as glycerin, glucose, isotonic agents such as added sugars, and the above-mentioned stabilizers, preservatives, soothing agents, pH regulators, etc., usual heat sterilization, sterile filtration, etc. Can be prepared by sterilization. It should be noted that, depending on the type of the active ingredient, the active ingredient may be inactivated in the heat sterilization step, so the sterilization method should be appropriately selected.
[0017]
The medicament of the present invention may contain an active ingredient of another drug having the same or similar pharmacological action as HGF of the present invention. Also, sulfated other saccharides such as heparin and dextran sulfate or derivatives thereof (JP-A-5-301824), which are known to enhance the hepatocyte proliferation action of HGF which is an active ingredient of the medicament of the present invention, and the like. You may mix an active ingredient.
[0018]
The prophylactic / therapeutic agent for pulmonary hypertension of the present invention can be prepared using a pharmaceutically acceptable carrier or the like, in the form of tablets, granules, capsules, solids such as powders, or liquids, suspensions, syrups, emulsions, lemonades. In such a liquid form, the composition can be formulated into a dosage form suitable for oral administration, parenteral administration, inhalation administration or external use, and used as a pharmaceutical preparation. If necessary, auxiliaries, stabilizers, wetting agents and other conventional additives can be added to the above-mentioned preparations, for example, lactose, citric acid, tartaric acid, stearic acid, magnesium stearate, clay, sucrose, corn starch, talc, gelatin, agar. Pectin, peanut oil, olive oil, cocoa oil, ethylene glycol and the like.
[0019]
The dose of the above-mentioned preparation varies depending on the administration route, the kind of disease, the condition of the patient, the weight or age, and also varies depending on the kind of the drug to be applied, but generally about 1 mg to 200 mg per day. Or more can be administered per patient. The average dose of HGF contained as an active ingredient may be about 5 mg to 100 mg and used for prevention and treatment of pulmonary hypertension.
[0020]
Furthermore, the HGF of the present invention can be used as a gene therapy drug for preventing and / or treating pulmonary hypertension. For example, by encapsulating the HGF of the present invention in a lipofection reagent and administering it to a living body, HGF in blood can be maintained at a concentration necessary for prevention and / or treatment of pulmonary hypertension. The administration of the HGF of the present invention depends on the severity of the disease state, the responsiveness of the living body, and the like, and depends on the effectiveness of the prevention and / or treatment or the period until the reduction of the disease state is achieved. It may be performed at an appropriate dose, administration method and frequency.
[0021]
【Example】
Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited to the examples.
[0022]
(Example 1) Effect of HGF administration in rat pulmonary hypertension model (production of pulmonary hypertension model)
Six-week old Sprague-Dawley male rats (180-200 g) were used.
The rat was anesthetized by intraperitoneal administration of pentobarbital (50 mg / kg), and then reached intraperitoneally by midline abdominal incision to protect the intestinal tract. An abdominal aorta-inferior aortic shunt was created. The shunt was prepared by blocking the blood flow at the head side and the tail side of the exposed aorta, and then penetrating an 18G injection needle from the artery side to the vein side. One week later, 60 mg / kg subcutaneous administration of monocrotaline (hereinafter referred to as “MCT”) was performed to prepare a pulmonary hypertension model.
[0023]
MCT is a toxic pyrrolidizine alkaloid extracted from the seeds of the plant Crotalaria spestabilis, where monocrotaline pyrrole, which is metabolized by hepatic cytochrome P-450, causes inflammation of the pulmonary blood vessels. A single subcutaneous administration causes pulmonary arteritis, followed by pulmonary hypertension and right ventricular hypertrophy. A pulmonary hypertension model can be prepared only by administering MCT, but a more appropriate disease state model can be prepared by preparing a shunt in advance and adding a hemodynamic change. That is, pathologically, neointimal lesions and plexiform lesions are also recognized, and the model approaches a more primary pulmonary hypertension (primary pulmonary hypertension) model. In fact, it has been reported that hemodynamic changes are important in addition to MCT injury in the formation of neoarterial lesions.
[0024]
(Administration of HGF)
One week after MCT administration, a group to which recombinant human HGF (manufactured by Mitsubishi Pharma Corporation) was administered at 125 μg / kg from the tail vein for 2 weeks continuously for 2 weeks, group H (n = 4), phosphate buffer (PBS) Is designated as group H (n = 4).
[0025]
(Confirmation of effect)
Three weeks after the start of HGF administration, the rats were sacrificed, the heart and lungs were removed, the right ventricle free wall and the left ventricle + septum were weighed, and the right ventricular myocardial weight to left matter + septum weight ratio ( Right ventricle / left ventricle + septum: RV / LV + IVS) and right ventricle wall thickness (RV wall thickness) were determined. In addition, H. in a paraffin-embedded section of the left lung tissue. E. FIG. Histological examination was performed by staining and elastic fiber staining (EVG staining).
In the MCT-administered group, the body weight did not increase as compared with the non-administered group, and on the contrary, the body weight decreased. In addition, the systolic pulmonary artery pressure of normal rats was 25.75 mmHg on average, while the systolic pulmonary artery pressure of group C was 64 mmHg on average.
[0026]
(result)
The right ventricle weight to left ventricular + septal weight ratio and right ventricle wall thickness were significantly reduced in group H compared to group C.
1) Right ventricular myocardial weight to left matter + septal weight ratio (RV / LV + IVS)
H: 0.589 ± 0.040, C: 0.661 ± 0.033; p <0.05,
2) Right wall thickness (PR wall thickness)
H: 0.133 ± 0.011, C: 0.148 ± 0.002 mm; p <0.05
[0027]
In a rat model of pulmonary hypertension, HGF improved right ventricular weight to left ventricular + septal weight ratio, right ventricular wall thickening and pulmonary artery media thickening, suggesting utility in the prevention and treatment of pulmonary hypertension.
[0028]
【The invention's effect】
As described above, administration of the prophylactic / therapeutic agent for pulmonary hypertension containing the hepatocyte growth factor of the present invention as an active ingredient is useful for prevention and treatment of pulmonary hypertension.

Claims (3)

An agent for preventing or treating pulmonary hypertension, comprising a hepatocyte growth factor as an active ingredient. The agent according to claim 1, wherein the pulmonary hypertension is primary pulmonary hypertension. A method for treating pulmonary hypertension by using the agent for preventing or treating pulmonary hypertension according to claim 1 or 2.