JPH0925239A - Therapeutic agent for cerebral vasospasm - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a safe therapeutic agent for cerebral vasospasm, having suppressing actions on cerebral vasospasm and a safe removing agent for a hematoma, having promoting actions on removal of the hematoma after cerebral hemorrhage and causing slight side effects. SOLUTION: The therapeutic agent for cerebral vasospasm and removing agent for a hematoma contain a thrombomodulin-like protein as an active ingredient. The thrombomodulin-like protein can suitably be blended with other usually used ingredients and prepared as an injectable solution, a pernasal absorbent, an oral agent, etc. The daily dose is 0.01-10,000μg/kg, preferably 0.1-1,000μg/kg, expressed in terms of the protein amount of the thrombomodulin- like protein and administered into the cerebral cistern by intracisternal puncture. The thrombomodulin-like protein contains all proteinic substances capable of bonding to thrombin, suppressing the coagulation promoting activities of thrombin and remarkably promoting the protein C activities by the thrombin.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for cerebral vasospasm and a hematoma removing agent after cerebral hemorrhage, which comprises a thrombomodulin (hereinafter abbreviated as TM) -like protein.

[0002]

Cerebral vasospasm is an abnormal contraction reaction of cerebral blood vessels that appears several days after subarachnoid hemorrhage and lasts for 1 to 2 weeks or longer. Rarely, it can be seen in trauma, cerebral artery malformations, and subarachnoid hemorrhage due to surgery for hemorrhagic tumors. It has been suggested that hematoma itself may be the most probable cause of cerebral vasospasm, but there are many unclear points about its body. There are many unclear points regarding the causative agent of cerebral vasospasm, but it has been suggested that endothelin, oxyhemoglobin, thrombin, and lipid peroxide are involved (Kanamaru et al., Japanese clinical practice, 51, 194-201, 19).
93), endothelin antagonists, methylprednisolone which is an inhibitor of lipid peroxidation and steroid hormone derivatives, and heparin which is a thrombin inhibitor [Asada, et al., Advances in Surgery.
Four Celebral Stroke, Springer-Fairlark, Tokyo (Adbances in surgery for cere
bral stroke, Springer-Verlag, Tokyo), 705-
709, 1988], and argatroban (Nakamura et al., 49th abstract of the Japanese Society of Neurosurgery, 199, 1990) have been confirmed to have an inhibitory effect in animal models of cerebral vasospasm. The biggest problem with the treatment of cerebral vasospasm is that its etiology and pathophysiology are not fully understood. Therefore, the treatment method must be symptomatic treatment.
As a method for preventing cerebral vasospasm, since hematoma itself is considered to be the direct cause of cerebral vasospasm, it is said that cerebral vasospasm can be prevented by removing the hematoma as soon as possible.
At this time, since it is difficult and dangerous to surgically remove hematomas, thrombolytic agents such as tissue plasminogen activator and urokinase have been intraventricularly administered. Further, papaverine hydrochloride, calcium blocker and the like are used as vasodilators. Also,
Vascular dilation with balloon catheter is also performed.

TM was discovered by Esmon et al. In 1981 as an anticoagulant present on the surface of vascular endothelial cells.
Then, in 1987, Suzuki et al. Revealed the entire primary structure of the glycoprotein, which binds to thrombin to suppress thrombin's procoagulant action and remarkably promotes activation of protein C by thrombin. Since TM-like protein efficiently suppresses the coagulation system, it has been suggested that TM-like protein is useful for the prevention and treatment of disseminated intravascular coagulation (hereinafter abbreviated as DIC) and various thrombosis.
There has been no report of using a protein like protein as a therapeutic agent for cerebral vasospasm or a hematoma removing agent after cerebral hemorrhage.

[0004]

To date, vasodilators such as papaverine hydrochloride and calcium blocker have been used for the treatment of cerebral vasospasm. However, since papaverine hydrochloride is easily decomposed in the liver, its half-life is short,
Intravenous administration is also ineffective for improving cerebral blood flow because it also dilates peripheral blood vessels throughout the body to cause hypotension. In addition, the administration of calcium antagonists should be carried out prophylactically or at the beginning of the onset of spasm, and it is difficult to expect its effect after organic changes occur in the blood vessel wall. In addition, a tissue plasminogen activator, a thrombolytic agent such as urokinase, is administered into the cistern for the purpose of removing hematoma.
Hemorrhagic side effects such as hemorrhage within the brain parenchyma, exacerbation of subarachnoid hemorrhage, and epidural hemorrhage are feared (Yamada et al., Cranial nerve, 4
5, 809-817, 1993). As described above, regarding the treatment of cerebral vasospasm associated with subarachnoid hemorrhage and the removal of hematoma after cerebral hemorrhage, there is no known therapeutic agent or removal agent that has a reliable and long-lasting effect. The development of safe therapeutic agents and elimination agents with mild side effects is an important issue.

Further, it is generally contraindicated to systemically administer an anticoagulant to a patient with cerebral hemorrhage.
Therefore, it is an important issue to develop a safe drug and a safe administration method.

[0006]

[Means for Solving the Problems] In the present specification, a TM-like protein suppresses cerebral vasospasm and promotes elimination of hematoma during the study of physiological action of TM-like protein on cerebral blood vessels. After discovering this, further intensive research was conducted to complete the present invention.

Hereinafter, the present invention will be described in detail. A first aspect of the present invention is a therapeutic agent for cerebral vasospasm containing a TM-like protein as an active ingredient. A second aspect of the present invention is a hematoma removing agent after cerebral hemorrhage, which comprises a TM-like protein as an active ingredient.

As used herein, "therapeutic agent for cerebral vasospasm"
The term includes not only a therapeutic agent after the occurrence of cerebral vasospasm but also a prophylactic therapeutic agent that is administered in the acute phase after cerebral hemorrhage to suppress the occurrence of cerebral vasospasm. The term "TM-like protein" means TM and peptides having a partial amino acid sequence necessary for biological or immunological activity of TM or analogs thereof. That is, T used in the present invention
The M-like protein includes all proteinaceous substances that bind to thrombin to suppress the thrombin procoagulant action and remarkably promote thrombin-induced protein C activation. Therefore, it may be either a natural type or a genetically engineered type, and may be a modified type or a chimeric type obtained by a genetic engineering technique. When using as a medicine
Preferably, a human-derived TM-like protein is desired. Also,
Human-derived TM is membrane-bound, and those having a full-length amino acid sequence have low solubility in water. Therefore, in the present invention, a highly soluble TM-like protein having no full-length amino acid sequence (hereinafter, abbreviated as a soluble TM-like protein), such as a natural human urine-derived soluble TM-like protein or a recombinant DNA Human soluble TM-like proteins are more preferred. It should be noted that various Ts having a partial structure of the amino acid sequence of TM produced by using the gene recombination technology and the like.
M-like proteins having the effect of the present invention as a therapeutic agent for cerebral vasospasm or a hematoma removing agent are also included in the present invention.

Regarding such a TM-type protein as a natural type, for example, TM derived from human placenta and soluble T derived from human urine disclosed in JP-A-60-199819.
The M-like protein has a molecular weight of 55,000 to 5 in a non-reducing state disclosed in JP-A-3-86900.
8,000 and 60,000 to 65,000 soluble TM-like proteins and a molecular weight of 72,000 ± in the non-reducing state disclosed in JP-A-3-218399.
3,000 and 79,000 ± 3,000 soluble T
M-like proteins are mentioned. For genetically modified types,
For example, as a recombinant human soluble TM-like protein,
A soluble TM-like protein containing at least the amino acid sequence 345 to 462 from the amino terminus disclosed in JP-A No. 1-6219 and at least 4-44 from the amino terminus disclosed in WO92 / 00325.
An example is a soluble TM-like protein containing the 6th amino acid sequence. Further, a soluble TM-like protein which may contain a part of the amino acid sequence of human tissue plasminogen activator, etc. disclosed in JP-A-4-505554, is disclosed in WO91 / 04276. Soluble TM-like protein having a sugar chain containing chondroitin and / or chondroitin sulfate and JP-A-6-2794
The soluble TM-like protein to which a sequence containing an acidic amino acid sequence derived from bovine TM is added, which is disclosed in Japanese Patent Application No. 97, is mentioned.

The TM-like protein is a DIC therapeutic agent currently under clinical development, and it is expected to have high safety as a pharmaceutical with few side effects such as bleeding tendency associated with treatment with heparin (Yoshitaka Hosaka). 56th Annual Meeting of the Japanese Society of Hematology, 791 titles, 1994). In addition, it has been confirmed that TM-like proteins have a long half-life in vivo and exhibit favorable pharmacokinetics as pharmaceuticals. TM used in the present invention
Such a protein has an action of suppressing cerebral vasospasm, and further has an action of promoting the removal of hematoma. Therefore, it can be used as a therapeutic agent for cerebral vasospasm or a hematoma removing agent after cerebral hemorrhage, for example, after subarachnoid hemorrhage.

The formulation of the present invention need only contain a TM-like protein as an active ingredient, and can be produced by any known pharmaceutical production method. The TM-like protein is a suitable carrier or medium generally used as a drug, for example, sterile water or physiological saline, vegetable oil, mineral oil, higher alcohol, higher fatty acid, harmless organic solvent, etc., and if necessary. Excipients, colorants, emulsifiers, suspensions, surfactants, solubilizers, anti-adsorption agents, stabilizers, preservatives, humectants, antioxidants, buffers, isotonic agents, soothing agents Injectables suitable for effective administration to living bodies, nasal absorbents, pharmaceutical preparations such as oral preparations, preferably injectables, can be prepared by appropriately combining them with agents and the like. Some are known. Injectable preparations can be provided, for example, in the form of a lyophilized product, a solution for injection, or a form enclosed in an osmotic pump.

The formulation of the present invention can be administered by intraarterial injection, intravenous injection or intracisternal injection. For example, the amount of TM-like protein is about 0.01 to 10 per day.
000 μg / kg, more preferably about 0.1-10
A dose of 00 μg / kg is administered. Further, the TM-like protein which is the active ingredient of the present invention is used as a liposome-forming substance (JP-A-60-243022 or JP-A-64-85).
It is also possible to administer it as a liposome after encapsulating it in a liposome (see the description of Japanese Patent Publication No. 920). Alternatively, it can be administered continuously by enclosing it in an osmotic pump or the like and placing it in a living body. Further, as a dosage form of the sustained-release preparation, the preparation may be surgically implanted at the subarachnoid hemorrhage site, for example, in the cistern.

The preparation of the present invention can be administered by intraarterial injection, intravenous injection, or intracisternal injection, but from the viewpoint that a more direct effect can be expected, it is administered by intracisternal puncture into the intracisternal. Preferably.

(Acquisition Example) The present invention will be described in more detail with reference to acquisition examples of TM-like protein, but the TM-like protein used in the present invention is not limited thereto.

(Example 1 of Obtaining Soluble TM-Like Protein) Production of Soluble TM-Like Protein Derived from Human Urine It was prepared according to the method described in JP-A-3-218399. That is, 100 L of raw urine was filtered through an acrylic fiber to adsorb and remove urokinase in urine, and the urine passing through was desalted and concentrated using an ultrafiltration membrane. Then, it was successively purified using a DEAE Cellulose (manufactured by Whatman) column, a DIP-thrombin-agarose chromatography and a Sephacryl S-300 (manufactured by Pharmacia Fine Chemicals) column, and an active fraction was collected (hereinafter referred to as UT).
(Abbreviated as M0).

(Example 2 of Obtaining Soluble TM-Like Protein) Production of Recombinant Human Soluble TM-Like Protein It was produced according to the method of WO92 / 00325.
That is, the amino acid sequence at the amino terminus is converted to Ala using DNA picked from a human placenta cDNA library.
A vector that expresses a soluble TM-like protein consisting of 498 amino acids, which is -Pro-Ala-, was prepared and inserted into CHO cells, followed by gene amplification to obtain a high expression strain. The culture fluid of this highly expressing strain was purified by a DIP-thrombin-agarose column and gel filtration to obtain two kinds of TM-like proteins (RTM1, RTM2) having the same amino acid sequence but different molecular weights.

(Experimental Example) The effect will be specifically described below with reference to an experimental example in order to explain the present invention in detail, but the present invention is not limited thereto.

(Experimental Example 1: Effect of TM-like protein on cerebral vasospasm) 2 ml of autologous arterial blood was injected into a rabbit by percutaneous transcatheter puncture to prepare a subarachnoid hemorrhage model. 1
After a lapse of time or 6 hours, the test drug was administered again by cisternal puncture. Angiography was performed on the first day, the third day, and the fifth day, and the changes in the diameter of the basilar artery with time were compared with the diameter before making the subarachnoid hemorrhage model being 100%. At the end of the observation, the animals were sacrificed, and the subarachnoid hematoma remaining on the ventral side of the brain stem was observed.
The effect of the test drug was evaluated by classifying basilar artery caliber and residual subarachnoid hematoma into 5 grades of Grade 0 (no residual) to 4 (high residual). The results are shown in FIG. 1 and Table 1.

As is clear from FIG. 1, UTM0 almost completely suppressed basilar artery spasm. Further, the same action is exhibited for TM-like proteins other than UTM0.

Table 1 ───────────────────────────── Group Grade Subarachnoid Hematoma Residual Grade ────────── ──────────────────── Untreated group (control group) 3.1 UTM0 1 hour after administration 0.5 UTM0 6 hours after administration 1.2 ──── ───────────────────────── As is clear from Table 1, UTM0 was observed to have a promoting effect on the disappearance of subarachnoid hematomas. Further, the same action is exhibited for TM-like proteins other than UTM0.

[0021]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

(Example 1) UTM0 5 mg Purified gelatin 50 mg Sodium phosphate 34.8 mg Sodium chloride 81.8 mg Mannitol 25 mg The above components were dissolved in 10 ml of distilled water for injection, and after sterile filtration, 1.0 ml sterile vials each. And then lyophilized to prepare a preparation for injection.

Example 2 UTM0 5 mg Albumin 20 mg Sodium Phosphate 34.8 mg Sodium Chloride 81.8 mg Mannitol 25 mg The above components were dissolved in 10 ml of distilled water for injection, and after sterile filtration, 1.0 ml each was placed in a sterile vial. The mixture was dispensed and freeze-dried to prepare a preparation for injection.

(Example 3) RTM1 10 mg Purified gelatin 20 mg Sodium phosphate 34.8 mg Sodium chloride 81.8 mg Mannitol 25 mg The above components were dissolved in 10 ml of distilled water for injection, and after sterile filtration, 1.0 ml sterile vials each. And then lyophilized to prepare a preparation for injection.

[0025]

The therapeutic agent of the present invention containing the TM-like protein as an active ingredient has an action of suppressing cerebral vasospasm and further has an action of promoting the removal of hematoma. Therefore, it can be used as a therapeutic agent for cerebral vasospasm or a cerebral hemorrhage, for example, a subarachnoid hemorrhage and a later hematoma removing agent. Also, the TM of the present invention
The like protein is expected to have fewer side effects than existing drugs, and can be a safe therapeutic agent for cerebral vasospasm and a hematoma removing agent after cerebral hemorrhage.

[Brief description of drawings]

FIG. 1 is a graph showing the effect of TM-like protein (UTM0) used in the present invention to suppress basilar artery caliber contraction.

Claims (2)

[Claims] 1. A therapeutic agent for cerebral vasospasm containing a thrombomodulin-like protein as an active ingredient. 2. A hematoma removing agent after cerebral hemorrhage, which comprises a thrombomodulin-like protein as an active ingredient.