JP2000219633A - Prophylactic and therapeutic agent for ischemic nephropathy - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a prophylactic and therapeutic agent for the ischemic nephropathy, which is caused by renal ischemia and re-perfusion that are observed in the kidney transplantation and the septic shock by using a specific protein. SOLUTION: This prophylactic and therapeutic agent includes protein C (abbreviated to PC thereafter) or activated protein C (abbreviated to APC). In one embodiment, PC is a serine protease precursor distributing in the blood plasma in a concentration of 4 μg/ml and is activated by the limited digestion with the thrombin that has formed a complex with thrombomodulin distributing in the vascular endothelial cells and converted to the APC having the serine protease activity. In an embodiment, PC is purified by the affinity chromatography with the anti-PC antibody from the human plasma, the purified PC is activated with human thrombin and purified through the cationic chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a new use of plasma proteins. More specifically, the present invention relates to a medicament for ischemic renal injury that occurs during organ transplantation or various shocks. More specifically, the present invention relates to a prophylactic / therapeutic agent for ischemic renal injury containing PC or APC as an active ingredient.

[0002]

2. Description of the Prior Art The kidneys are located on the left and right sides of the retroperitoneum at the height of the eleventh thoracic vertebra and the second lumbar vertebra. The right kidney is 1/2 lumbar lower than the left kidney. The size is 1
0 × 5 × 3 cm, weight is 100-130 g. The parenchyma is divided into the cortex and the medulla, and the cortex has a glomerular appearance due to the presence of glomeruli, and the medulla has a linear system-like appearance due to Helen loops running in parallel. The functional units of the kidney are called nephrons, and each nephron is formed from glomeruli and tubules. A glomerulus is a collection of capillaries that have a spherical shape with a diameter of about 200 μm.
Here, blood filtration is performed. Substances having a molecular weight of 7,000 or less are freely permeated, and as the molecular weight increases, the filtration rate decreases, and substances having a molecular weight of 50,000 or more are not filtered. Tubules are formed from proximal tubules, loops of Henle, distal tubules, and collecting tubules, where reabsorption of necessary substances from glomerular filtrate and secretion of unnecessary substances are performed.
There are about 1 million nephrons in one kidney. The main function of the kidney is urine production by this nephron, but urine production has an important significance not only in the excretion of waste products but also in regulating water electrolyte metabolism (maintaining the internal environment) . The kidney controls not only urine production but also blood pressure regulation and bone marrow erythrocyte production, and also performs metabolic functions such as hormone inactivation, vitamin D activation and gluconeogenesis.

[0003] Acute renal failure is, in a broad sense, defined as a syndrome that results in a rapid extinction of the glomerular filtration capacity for various reasons, resulting in azotemia. According to this definition, not only renal renal failure but also early prerenal renal failure and renal renal failure without renal parenchyma fall into the category of acute renal failure.
On the other hand, glomerular filtration rate (GRF) is rapidly reduced by renal ischemia and / or nephrotoxic substances, and a pathological condition that causes hypernitremia and abnormalities in renal parenchyma is referred to as acute renal failure in a narrow sense. In this case, even if the cause is removed, the GRF is not immediately restored to normal. Acute renal failure due to renal ischemia occurs when renal ischemia lasts for a long period of time, such as severe heart failure, severe fluid loss, hemorrhagic shock, septic shock, renal infarction, aortic or renal artery surgery.

[0004] The prognosis of ischemic acute renal failure is poor and its mortality is high, so that prevention before treatment is considered important. Treatment includes administration of loop diuretics and dopamine, but there is no established specific treatment.

[0005]

Means for Solving the Problems, Constitution of the Invention The inventors of the present invention have conducted intensive studies to develop a prophylactic / therapeutic agent for ischemic renal injury, and as a result, surprisingly, no attempt has been made so far. PC or APC can be used to prevent or prevent ischemic kidney injury by actual in vivo administration in model animals.
They have found that they can be sufficiently applied to humans and other animals as therapeutic agents, and have completed the present invention based on this finding.

[0006] PC is a serine protease precursor present in plasma at a concentration of 4 µg / ml, and is activated by limited degradation by thrombin complexed with thrombomodulin present on vascular endothelial cells. Is converted to APC having PC is a light chain composed of a Gla domain and an EGF-like domain (molecular weight: about 21,
000) and a heavy chain (molecular weight of about 41,000) comprising an activating peptide and a catalytic domain are disulfide-linked double-chain glycoproteins (Proc. Natl. Acad.
Sci. USA, vol. 82, p. 4673-467.
7, 1985). Activation of PC to APC depends on A of heavy chain.
This is achieved by subjecting the rg12-Leu13 bond to limited degradation and releasing an activation peptide consisting of 12 amino acids.

APC selectively degrades and inactivates the activated factor V or VIII of the blood coagulation system on cell membrane phospholipids to deactivate it, thereby exerting a strong anticoagulant effect (Bioch).
chemistry, vol. 16, p. 5824-583.
1, 1977; J. Biol. Chem., Vol. 25.
8, p. 1914-1920, 1982). Also, APC
Neutralizes plasminogen activator inhibitor (PAI) derived from vascular endothelial cells or platelets (Proc. Natl. Acad. Sci.
USA, vol. 82, p. 1121-1125, 198.
5) and TAFI (Thrombin A) as an antifibrinolytic factor.
Activable Fibrinolysis In
It is thought that the inhibition of activation of H.hibitor is involved in the enhancement of the fibrinolytic system (Blood,
vol.88, p.2093-2100, 1996). Furthermore, APC has been shown to be effective in sepsis models in which other anticoagulants are ineffective (J. Clin. In
vest., vol. 79, pp. 918-925, 198.
7) Also, it has an action of suppressing cytokine production from leukocytes (Am. J. Physiol., P.
L197-L202, 1997), suggesting that APC has an anti-inflammatory effect.

The method for producing APC used in the present invention is not particularly limited. For example, a method for activating PC isolated from human blood or obtained by genetic recombination, It can be produced by a method of isolating APC or a method of directly preparing APC by genetic recombination technology. There is no particular limitation on the method of activating PC to APC. For example, the method can be performed by a method of activating with thrombin separated from blood of humans or cows, or a method of activating with a synthetic peptide.

[0009] The method for producing APC derived from blood includes the following method. For example, a method in which PC purified from human plasma by affinity chromatography using an anti-PC antibody is activated with human thrombin, and then purified using cation chromatography (Blood,
vol. 63, pp. 115-121, 1984) or Kisiel, for the adsorption of Ba citrate from human plasma.
A method of activating PC obtained by purification by the steps of elution, ammonium sulfate fractionation, DEAE-Sephadex column chromatography, dextran sulfate agarose chromatography and polyacrylamide gel electrophoresis to obtain APC (J. Clin. Invest. , vo
1.64, pp. 761-769, 1979) or a commercially available blood coagulation product containing PC was prepared by the method of Tayer et al.
(J. Clin. Invest., Vol. 79, p. 918)
-925, 1987).

As a method for preparing APC using a gene recombination technique, for example, Japanese Patent Application Laid-Open No. 61-20548
No. 7, JP-A No. 1-2338 or JP-A No. 1-8508
No. 4 and the like.

In order to maintain the activity of the APC prepared by the above-described method to a maximum, the APC of the present invention is preferably fresh or, when stored at 4 ° C., within about 5 days after storage. . Alternatively, the APC of the present invention can be lyophilized and stored with a suitable stabilizer, and further, the APC solution can be frozen and stored. In the present invention, APC as an active ingredient and a known suitable excipient are combined, and can be used as a preventive / therapeutic agent for ischemic renal injury of the present invention by a known method. PC or AP
The effective dose of C varies depending on the age, symptom, severity, etc. of the administration subject, and ultimately varies according to the intention of the doctor. For example, generally 1-10m per adult per day
g, and preferably 2 to 6 mg in 1 or 2 divided doses. Optimum administration is a single large dose (bolus) or intravenous infusion.

The subject of administration of the PC or APC-containing preparation of the present invention is not particularly limited as long as it is a patient with ischemic renal disorder. PC as a prophylactic / therapeutic agent for ischemic renal injury
Alternatively, when APC is used, PC or APC can be administered alone, and co-administration with another therapeutic agent can be expected as an effective means for increasing the effect. The PC or APC-containing preparation of the present invention is most effectively administered immediately after a patient suffers from ischemic renal injury or immediately after a surgical operation for kidney transplantation. In addition, since there is no tendency to bleeding as one of the characteristics of APC, administration immediately after the operation does not cause any problem.

APC derived from blood used in the present example
Has been confirmed by a single intravenous dose toxicity test in mice, a repeated intravenous dose toxicity test, a general pharmacology test (effect on the respiratory circulatory system using beagle dogs), a virus inactivation test, etc. I have. In addition, clinical trials for the indication of generalized intravascular blood coagulation syndrome (DIC) have been conducted, and its safety in humans has been confirmed. Hereinafter, the present invention will be described in more detail with reference to examples. However, the examples do not limit the scope of the present invention.

[0014]

EXAMPLES Example 1 (Improvement of renal function by administration of APC) The improvement of ischemic kidney injury by administration of APC was confirmed by the following examples.
This experiment was mainly performed with reference to a method established as a model reflecting ischemic renal injury occurring after transplantation, and is considered to be an optimal evaluation system. Rat
(Wistar, male, body weight: 300-350 g) was anesthetized with pentobarbital, and the kidney was placed in an ischemic state by clamping the right renal pedicle for 60 minutes after the left nephrectomy was performed, followed by reperfusion. The drug containing APC was administered from the tail vein of 0.15 mg / kg rat 30 minutes before ischemia. For comparison, a factor Xa-specific inhibitor, DEGR
Xa (dansyl glutamyl-glycyl-arginyl-chloromethyl k
etone-treated factor Xa) was administered in a similar manner at 3 mg / kg as a drug having the same anticoagulant activity as APC.
BUN (blood urea nitrogen), serum creatinine, TNF (tumor necrosis factor)-
α, MPO (myeloperoxidas
e) Evaluation was made by measuring activity and the like as indices.
In addition, the measuring method of each index followed the usual method widely used by those skilled in the art. Table 1 shows the results. Although renal dysfunction was observed by ischemia-reperfusion (I / R) treatment of rat kidney (Experimental group B), administration of APC could prevent the impairment (Experimental group C). On the other hand, DEGRXa used as an anticoagulant did not show a remarkable effect (Experimental group D). In addition, the numerical value of each group showed a significant difference by the statistical analysis.

[0015]

[Table 1]

Claims (2)

[Claims] 1. A prophylactic / therapeutic agent for ischemic renal disorder, which comprises protein C (hereinafter sometimes referred to as PC) or activated protein C (hereinafter sometimes referred to as APC). 2. The prophylactic / therapeutic agent according to claim 1, which is an ischemic renal disorder caused by renal ischemia / reperfusion observed at the time of kidney transplantation or septic shock.