JP3537844B2 - Preventive and therapeutic agent for cerebral infarction and / or cerebral hemorrhage - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a preventive and therapeutic agent for cerebral infarction and cerebral hemorrhage containing haptoglobin.

[0002]

2. Description of the Related Art Stroke is a general term for cerebrovascular disorders such as cerebral hemorrhage, cerebral infarction and subarachnoid hemorrhage. At present, these cerebrovascular disorders are the third leading cause of death in Japan after cancer and heart disease. Previously, hypertensive cerebral hemorrhage predominantly occupied the stroke form, but recently severe cases have decreased significantly and cerebral infarction has increased relatively.
At present, the aging of stroke patients is progressing, and an increase in elderly people suffering from sequelae and vascular dementia patients is becoming a social problem. Although hypertension, arrhythmia, hyperlipidemia, and diabetes have been pointed out as risk factors for stroke, the mechanism by which stroke occurs is still unknown. Antiplatelet drugs, anticoagulants, microcirculation improvers,
A thrombolytic agent or the like is used, and a surgical operation is performed on a serious patient. In the chronic phase, antiplatelet therapy, anticoagulation therapy, cerebral circulation improvement therapy, brain metabolic activation therapy, etc. are performed,
To prevent recurrence, risk factors such as hypertension have been removed.

[0003] Haptoglobin is present in plasma at about 30-190 mg / dl.
Is a glycoprotein present at a concentration of Regarding the physiological action of haptoglobin, metabolism and immunosuppressive action by binding to oxyhemoglobin have been reported. In particular, haptoglobin is used as a remedy for hemolytic kidney injury and as a hemoglobin remover at the time of forming an external circuit since it is involved in oxyhemoglobin metabolism. Furthermore, the correlation between serotype of haptoglobin and systemic arterial blood pressure, that haptoglobin is an endothelial cell-derived vasorelaxant,
Involvement in the metabolism of hemoglobin, an inhibitor of (EDRF) [Cardiovascuul Research (Cardiovascu
lar Research), Vol. 20, pp. 549-556, (1986)], It is useful to know the disappearance of haptoglobin in plasma in preeclampsia for prognosis [LANCET,
Volume 336, 1030-1032 (1990); LANCET, Volume 336, 159
4 (1990)]. Haptoglobin has the above-mentioned physiological effects and is known to cause quantitative fluctuations in various disease states. However, at present, there is no report on preventive treatment of cerebral infarction and cerebral hemorrhage by administration of haptoglobin.

[0004]

Since a cerebrovascular disorder causes various sequelae after the onset, it is very important to reduce the neurological symptoms after the onset to return the patient to social life. Conventionally, treatment by the above-mentioned therapy has been performed, but it would be more desirable if we could clarify the unknown intrinsic defense mechanism that the living body originally uses for healing and use the physiologically active substance involved in the treatment for the treatment. Needless to say. Furthermore, if the onset of stroke in a patient having the above-mentioned risk factor can be predicted by any method, appropriate treatment can be performed early to prevent the onset, and such a method is desired.

[0005]

Means for Solving the Problems The present inventors have found a new fact that haptoglobin in plasma disappears from plasma around the time of the onset of cerebral hemorrhage and cerebral infarction. Found to prevent the onset of stroke and to improve the neurological symptoms caused by stroke. Furthermore, the present inventors have found that quantification of haptoglobin in plasma is extremely effective in the prediction and prognosis of cerebral infarction and cerebral hemorrhage, and completed the present invention.

That is, the present invention provides a preventive and therapeutic agent for cerebral infarction and cerebral hemorrhage containing haptoglobin as an active ingredient. The haptoglobin used in the present invention may be any of mammals such as human, monkey, pig, cow, rat and the like, but it is preferable to use one derived from an animal to be administered. Further, it may be a natural type or a recombinant type produced by using genetic engineering, or may be a mutein obtained by modifying them or a peptide fragment having haptoglobin activity. Natural human haptoglobin can be obtained from a plasma by a conventional method using an ethanol fractionation method from plasma [J.
E. Cohn et al., Journal of the American Chemical
Society (J. Am. Chem. Soc., Vol. 68, p. 459 (194
It is prepared from fraction IV obtained in 6)]. Further, affinity chromatography in which hemoglobin is immobilized
[Biochemical and Biophysical Research Communication (Biochem. Biophys. Res. Commu
n.) 52, 774-778 (1973)]. Hp 1-1, Hp 2-1,
There are three serotypes, Hp 2-2 and Hp 2-2. For example, the abundance in Japanese is 6.47%, 35.2%, 5
Although it is 7.4%, in the present invention, various serotypes may be used alone or as a mixture in various ratios. The prophylactic / therapeutic agent of the present invention may be used in combination with a known prophylactic / therapeutic agent for cerebral infarction and cerebral hemorrhage.

[0007] The diseases to be treated in the present invention are cerebral infarction and cerebral hemorrhage, and cerebrovascular disorders such as cerebrovascular dementia and cerebral vascular haze caused by them. In addition, it can be used for preventive treatment of cerebral hemorrhage and cerebral infarction not only in human diseases but also in mammals such as dogs and cats. By administering the prophylactic / therapeutic agent of the present invention, the onset of hypertensive cerebral hemorrhage is delayed or reduced, and neurological symptoms due to cerebral infarction and cerebral hemorrhage are reduced and mortality is reduced. It is particularly effective in treating sequelae. The use of this drug for the prevention of cerebral infarction and cerebral hemorrhage may include the use of blood pressure measurement and measurement in individuals or animals with these risk factors, such as those with high blood pressure or those at risk of recurrence of cerebral infarction or cerebral hemorrhage. A blood test including quantification of haptoglobin is performed. If the haptoglobin value is significantly lower than the normal value (about 30 mg / dl or less), haptoglobin is infused intravenously as described below, and thereafter, the blood haptoglobin is traced. Prophylactic effects can be obtained by conducting investigations and adjusting the dosage appropriately. When using this drug for treatment in patients who have developed cerebral hemorrhage or cerebral infarction, perform a blood test including quantification of haptoglobin in an emergency and confirm that the amount of haptoglobin has decreased or disappeared, and as soon as possible And then follow up the blood haptoglobin, adjust the dose appropriately, and administer to normal values. Haptoglobin, which is an active ingredient of the present invention, is an endogenous factor present at a concentration of about 30-190 mg / dl in the plasma of healthy subjects, and is characterized by the fact that side effects are unlikely to appear by administration of this drug. . The mechanism by which haptoglobin alleviates the symptoms of cerebral infarction and cerebral hemorrhage is not always clear,
Considering the physiological activity of the protein, it is presumed that oxyhemoglobin, an inhibitor of endothelial cell-derived vasorelaxant, is removed, cerebrovascular endothelial cells are protected, and cerebral circulation is improved by protecting the kidneys. In addition, experiments using animal models show that haptoglobin in plasma disappears prior to the onset of neurological symptoms of cerebral hemorrhage, and that haptoglobin in plasma disappears during the acute and chronic phases of cerebral infarction and cerebral hemorrhage, and drug administration, etc. Measurement of haptoglobin in plasma is extremely effective in predicting these diseases and diagnosing the prognosis, since haptoglobin reappears when symptoms are alleviated by the treatment of.

[0008] The prophylactic / therapeutic agent for cerebral infarction and cerebral hemorrhage used in the present invention is a pharmaceutical composition (eg, haptoglobin) alone or together with other pharmacologically acceptable carriers, excipients and diluents. For the purpose of preventing or treating cerebral infarction and cerebral hemorrhage in warm-blooded animals (eg, humans, mice, rats, hamsters, rabbits, dogs, cats) as injections, tablets, capsules, solutions, ointments) It can be safely administered parenterally or orally. Above all,
It is preferably administered parenterally, and the parenteral preparation is preferably in the form of, for example, an injection, a solution for injection administration, a frozen product or a lyophilized product.

In preparing a pharmaceutical composition,
According to a known pharmaceutical manufacturing method, pharmaceutically acceptable additives, diluents, excipients and the like are used as required. For example, in the case of an aqueous solution for injection, a solvent such as an aqueous solvent (eg, distilled water), a water-soluble solvent (eg, physiological saline, Ringer's solution), an oily solvent (eg, sesame oil, olive oil),
Or, if desired, a solubilizer (eg, sodium salicylate, sodium acetate), a buffer (eg, sodium citrate, glycerin), an isotonic agent (eg, glucose, invert sugar), a stabilizer (eg, human serum albumin, Polyethylene glycol), preservatives (eg, benzyl alcohol, phenol), and soothing agents (eg, benzalkonium chloride, procaine hydrochloride) and other additives are used in a conventional manner. For example, in order to prepare a solid injection preparation, a diluent (eg, distilled water, physiological saline, glucose), an excipient (eg, carboxymethyl cellulose (CMC), sodium alginate), a preservative (eg, benzyl alcohol,
By mixing benzalkonium chloride, phenol) and soothing agents (glucose, calcium gluconate, procaine hydrochloride) and the like, a solid injection preparation can be produced by conventional means. Further, in formulating, monosaccharides such as glucose, amino acids, various salts, human serum albumin, etc. may be added. In addition, isotonicity agents, pH regulators, soothing agents, preservatives, etc. Thus, a stable and effective preparation of the present invention can be prepared.

[0010] The dose of the preventive / therapeutic agent for cerebral infarction and cerebral hemorrhage of the present invention includes an amount distributed in a body fluid of a living body, for example, plasma, including a subject suffering from severe symptoms in which haptoglobin is completely eliminated. About 0.1 to 10 mg / ml, more preferably about 0.1 mg / ml for prevention.
An amount of 3-3.0, preferably about 1.0-3.0 mg / ml, and for therapeutic purposes about 0.4-6.0, preferably about 2.0-6.0 mg / ml. It is preferred to administer the volume to be ml. Such dosages include those wherein the amount administered is about 10-2000 mg / kg per body weight. The parenteral administration of the agent for preventing or treating cerebral infarction and cerebral hemorrhage of the present invention includes, for example, intravenous, subcutaneous, intramuscular or transmucosal administration. Routes of transmucosal administration include nasal, buccal or rectal. The prophylactic / therapeutic agent of the present invention is preferably administered such that the drug is present systemically in warm-blooded animals. Therefore, rather than administering directly to the brain,
For example, administration to the circulatory system is preferred. In particular, intravenous administration is preferred. Upon administration of the present preparation, a fixed dose may be administered regularly once to several times a day, or intermittently, for example, about once a week. Intravenous infusion such as intravenous infusion is also effectively used in the clinical administration of this drug.

Reference Example 1 Method for Preparing Human Haptoglobin Fraction obtained from human plasma by ethanol fractionation of corn
Ammonium sulfate fractionation was repeated using IV (fraction IV) under the conditions of saturation of 30-45%, and haptoglobin was purified by QAE Sephadex A50 (trademark) (Pharmacia) column chromatography. Reference Example 2 Preparation method of rat haptoglobin A column prepared by binding bovine hemoglobin to formylcellulofine (manufactured by Seikagaku Corporation) at 7 mg / ml was loaded with rat plasma, and a buffer containing 1 M guanidine hydrochloride was added. After washing the column with 0.1 M glycine hydrochloride buffer (pH
In 2.5), rat haptoglobin was eluted. Eluted fraction
DEAE-5PW ™ (Tosoh) column chromatography
Was purified. Reference Example 3 Haptoglobin Measurement Method Haptoglobin was administered to a rabbit (Japanese white species) according to a conventional method to prepare an antiserum. A specific antibody was prepared by affinity chromatography using a column prepared by immobilizing a haptoglobin standard of an animal species used for the preparation of the antiserum. Horseradish peroxidase was used as a specific antibody in the literature [Immunobiochemical Research, Tokyo Kagaku Dojin,
(1986)] to prepare a secondary antibody.
Haptoglobin was measured by the sandwich ELISA method described in the literature. Reference Example 4 Fluctuation of plasma haptoglobin in an experimental cerebral ischemia model Bilateral common carotid artery of SHRSP (male: 11 weeks old) was occluded with a clip, and the clip was released 15 minutes after the onset of convulsions to release the experimental cerebral ischemia. Blood was produced. The haptoglobin concentration in plasma was measured at a total of three time points before ischemia, immediately before reperfusion, and one hour after reperfusion, and viability was observed one day after reperfusion. The results were divided into surviving cases and dead cases one day after reperfusion, and are shown in FIG. 1 as relative values to values before ischemia.
As is clear from FIG. 1, cerebral ischemia significantly reduced the plasma haptoglobin concentration in all individuals as compared to the pre-ischemic value. In addition, the plasma haptoglobin concentration of an individual who died within one day of reperfusion was measured immediately before and after reperfusion.
At any time point after time, it showed a statistically significantly lower value than the individual that survived one day after reperfusion (t-test p <0.001). These results showed a clear correlation between the severity of neurological symptoms associated with cerebral ischemia and the decrease in plasma haptoglobin concentration.

Example 1 Production of Injection Haptoglobin purified from human plasma by the method described in Reference Example 1 was dialyzed against physiological saline. The dialysate was concentrated to a protein concentration of 3.3%, filtered to remove bacteria, and used as a preparation for injection. Example 2 Prevention of hypertensive cerebral hemorrhage Male stroke-prone spontaneously hypertensive rats (SHRSP) at 10 weeks of age 1
In a system in which 5 animals were given 1% saline as drinking water to induce cerebral hemorrhage, human haptoglobin was intraperitoneally administered at 165 mg / kg / mouse every 24 hours for 2 consecutive weeks. Two weeks later, human haptoglobin in plasma was measured.
Of the three animals with haptoglobin in plasma at concentrations of 1 mg / ml or higher (2 animals), 2 animals did not show any neurological symptoms,
The other one showed only mild neurological symptoms and no deaths. On the other hand, 11 of 12 animals without haptoglobin in their blood showed some neurological symptoms, and 7 animals died. These results suggest that human haptoglobin has a preventive effect on hypertensive cerebral hemorrhage. Example 3 Treatment of Cerebral Infarction Both carotid arteries of SHRSP (male; 11 weeks old) are occluded with a clip, and the clip is released 10 minutes after convulsion to cause experimental cerebral ischemia. Immediately after reperfusion, human haptoglobin was continuously administered intraperitoneally at 165 mg / kg every 6 hours. The human haptoglobin administration group had significantly lower mortality and improved neurological symptoms than the control group. From the above, the therapeutic effect of human haptoglobin on cerebral infarction was suggested. Example 4 Treatment of Cerebral Infarction Bilateral common carotid arteries of SHRSP (male: 11 weeks old) were occluded with clips, and the clips were released 15 minutes after the onset of convulsions, thereby causing experimental cerebral ischemia. Human haptoglobin was intravenously administered at 165 mg / kg twice immediately after reperfusion and 5 hours later. From immediately after reperfusion to 7 days after, neurological symptoms were scored according to the criteria shown in [Table 1].
Further, the mortality rate 7 days after reperfusion was determined. The results are shown in Table 2 and FIG. As is clear from Table 2, the neurological symptoms were improved in the group administered with human haptoglobin as compared with the control group. Further, the reperfusion 7 shown in FIG.
Regarding the mortality rate after day, the control group was 42%, whereas the human haptoglobin administration group was clearly 10%, showing a decrease in the mortality rate. From the above, the therapeutic effect of cerebral infarction by intravenous administration of human haptoglobin became clear.

[0013]

[Table 1]

[0014]

[Table 2]

[0015]

EFFECT OF THE INVENTION The administration of the pharmaceutical preparation containing the haptoglobin of the present invention delays the onset of cerebral hemorrhage, reduces neurological symptoms after hemorrhagic or ischemic cerebrovascular disorder, and decreases mortality. The drug is useful as a prophylactic or therapeutic agent for cerebral infarction and cerebral hemorrhage.

[Brief description of the drawings]

FIG. 1 shows seven cases that survived one day after reperfusion after experimental cerebral infarction using SHRSP (survival group)
And the relative values of the plasma haptoglobin concentration of each individual immediately before reperfusion and one hour after reperfusion with respect to the pre-ischemic value and the average relative value of each group were shown. FIG.

FIG. 2 is a graph showing the mortality rate of a control group and a human haptoglobin administration group 7 days after reperfusion for experimental cerebral infarction using SHRSP.

[Explanation of symbols]

○ indicates the relative concentration of haptoglobin in plasma of each individual in the surviving group (%), ● indicates the relative concentration of haptoglobin in plasma of each individual in the dead group (%), and △ indicates the relative concentration of haptoglobin in plasma of each individual in the surviving group (%), And Δ indicates the relative plasma haptoglobin concentration (%) of each individual in the average of the death group.

──────────────────────────────────────────────────続 き Continued on the front page (56) References KUMANA, C.I. R. , Et al. , Br. J. clin. Pharmacol. , 29, pp. 347-353 (1990) STRUGALSKA, H .; , Et al. , Neurologia, Neurochirurgia i Phychiatria Polska, 16 (5), pp. 569-577 (1966) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 38/00-38/42 A61K 9/00-9/72 A61P 1/00-43/00 BIOSIS (STN) BIOTECHABS (STN) CA (STN) MEDLINE (STN) EMBASE (STN) JMEDPLUS (JOIS) JSTPLUS (JOIS)

Claims (3)

(57) [Claims] 1. A preventive / therapeutic agent for cerebral infarction and / or cerebral hemorrhage comprising haptoglobin. 2. The prophylactic / therapeutic agent according to claim 1, which is an injection. 3. A therapeutic agent for cerebral infarction and / or cerebral hemorrhage, which comprises haptoglobin.