JPH11199508A - Organ failure-treating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an organ failure-preventing/treating agent having a high safety and more effective property, useful in preventing/treating a systemic inflammatory reaction syndrome or multiple organ failure, furthermore effective on a patient not allowed to be dosed with a synthetic protease inhibitor because of its ineffectuality or any side effects. SOLUTION: This agent comprises an urinastatin (referred to as UTI) and thrombomodulin-like protein (referred to as TM-like protein) as an active ingredient. When used in drugs, a TM-like protein is pref. a human-derived one esp. a natural human urinary-derived soluble TM-like protein or a human-derived soluble TM-like protein obtained by a gene recombination. The objective agent is pref. prepared as an injectable agent, esp., freeze-dried product. When injectably dosed, the dose is 4 to 200 mg as a UTI protein or 1 to 200 μg/kg as a TM-like protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to trauma, surgery, burns,
A prophylactic / therapeutic agent for organ failure used for the prevention and treatment of systemic inflammatory response syndrome (hereinafter abbreviated as SIRS) and multiple organ failure (hereinafter abbreviated as MODS) caused by various causes such as infection and radiation exposure, and Specifically, urinastatin (hereinafter abbreviated as UTI) and thrombomodulin (hereinafter, abbreviated as UTI)
TM abbreviated protein) as an active ingredient.

[0002]

2. Description of the Related Art Synthetic protease inhibitors such as gabexate mesylate (FOW, Ono Pharmaceutical Co., Ltd.) and nafamostat mesilate (Fusan, Torii Pharmaceutical Co., Ltd.) have been used to prevent or treat MODS caused by various causes. Is known to have clinical efficacy. However, these effects are not always sufficient, and ineffective cases are often observed, which is a clinical problem.

[0003] UTI is a natural protease inhibitor derived from human urine, and has the same action as the above-mentioned synthetic protease inhibitor. UTI is a highly safe drug that is marketed as Miracled (Mochida Pharmaceutical Co., Ltd.), a therapeutic agent for acute pancreatitis and acute recurrent pancreatitis, and a therapeutic agent for acute circulatory failure.

On the other hand, a TM-like protein is a substance that binds to thrombin and suppresses the procoagulant action of thrombin and remarkably promotes the activation of protein C by thrombin. Since the TM-like protein efficiently suppresses the coagulation system, it has been suggested that the TM-like protein is useful for prevention and treatment of disseminated intravascular coagulation (hereinafter abbreviated as DIC), various thrombosis, and the like. It is also known to have a preventive / therapeutic action for diseases based on organ disorders or a MODS for the SIRS (JP-A-8-231413).
No.).

[0005]

However, as a pharmaceutical composition used for improving organ symptoms, the above-mentioned U.S.P.
There is a need for a drug that is more effective and safer than TI alone or TM-like protein alone. Therefore, the present invention provides SIRS and M generated by various causes.
A pharmaceutical composition used for prevention and treatment of ODS,
Provided is a pharmaceutical composition which is effective even for patients to whom a synthetic protease inhibitor is ineffective or cannot be administered due to side effects, and which is more effective than UTI alone administration or TM-like protein alone administration and has higher safety. That is the task.

[0006]

Means for Solving the Problems In order to achieve the above object, the present invention provides a preventive / therapeutic agent for organ failure characterized by using UTI in combination with a TM-like protein as an active ingredient.

[0007] In particular, as an agent for preventing or treating organ failure,
The present invention provides an embodiment in which the TM-like protein is a soluble TM-like protein, in particular, an embodiment in which the TM-like protein is a human urine-derived soluble TM-like protein or a recombinant human soluble TM-like protein.

[0008] Further, a mode in which UTI and a TM-like protein constituting the agent for preventing or treating organ failure are simultaneously administered;
Both separately administered embodiments are provided.

[0009] Conventionally, UTI
And the effect of the combination of TM and TM-like protein are not known. Rather, UTI has been reported to have no effect on protein C activation by TM (Mimura, Kei et al.
a, K et al. ), Thrombosis and Haemostasis
sis), 62, 382, Abstract 1198, 1989
Year). On the other hand, the present invention relates to the use of a combination of UTI and a TM-like protein to produce SIR caused by various causes.
It is possible to obtain a synergistic preventive and therapeutic effect on S and MODS.

According to the present invention, SIRS and MOD
It is possible to reduce the dose of each of UTI and TM-like protein required to obtain a predetermined preventive / therapeutic effect on S, and it is expected that side effects are reduced and safety is further improved. .

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

The prophylactic / therapeutic agent for organ failure according to the present invention is characterized in that UTI and TM-like protein are used in combination as active ingredients.

[0013] Here, the UTI can be obtained by a known method, for example, the method of Proksch (The Journal of Laboratory and Clinical Methods).
ry and Clinical Medicine),
79, 491-499, 1972) or the method of Sumi et al. (Journal of Biochemistry (J
own of Biochemistry), 83
Vol. 141-147, 1978). In addition, commercially available products can also be used.

UTI is a human-derived substance and has been confirmed to be highly safe as a drug.

On the other hand, in the present invention, "TM-like protein"
Means peptides having the amino acid sequence of TM and a portion required for the biological or immunological activity of TM or analogs thereof. That is, the TM-like protein used in the present invention includes all proteinaceous substances that bind to thrombin to suppress the procoagulant action of thrombin and remarkably promote protein C activation by thrombin. Therefore, either a natural type or a product produced by genetic engineering may be used, and a modified type or chimeric type obtained by genetic engineering techniques may be used.

In the case of a pharmaceutical, a human-derived TM-like protein (hereinafter abbreviated as human TM-like protein) is preferable. In addition, human-derived TM is membrane-bound, and those having a full-length amino acid sequence have low solubility in water. Therefore, the present invention does not have a full-length amino acid sequence,
A highly soluble TM-like protein (hereinafter abbreviated as a soluble TM-like protein), for example, a natural human urine-derived soluble TM-like protein or a recombinant human soluble TM-like protein is more preferable. In addition, various TM-like proteins having a partial structure of the amino acid sequence of TM, which are produced by using genetic recombination technology, also bind to thrombin to suppress the procoagulant action of thrombin and activate protein C by thrombin. Those having an effect of remarkably accelerating are included in the present invention.

[0017] Such TM-like proteins are naturally-occurring, for example, the TM derived from human placenta and the 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 protein, Journal of Biochemistry (Journal of Biochemistry)
y), 113, 433-440, 1993, having a molecular weight of 55,000 and 6 in the non-reduced state.
000 soluble TM-like protein and European
Journal of Biochemistry (Europe
an Journal of Biochemistry
y) Soluble TM-like proteins having a molecular weight of 57,000 and 63,000 in a non-reducing state described in Vol. 221, pp. 1079-1087, 1994.

As for the recombinant type, for example, as a recombinant human soluble TM-like protein,
No. 6219 discloses a soluble TM-like protein containing at least the amino acid sequence at 345-462 from the amino terminus and WO92 / 00325 contains at least the amino acid sequence at 4-446 from the amino terminus. And soluble TM-like proteins.

Also, Japanese Patent Application Laid-Open Nos. Hei 2-255699, Hei 3-2590084 and Hei 5-213
No. 508150, a TM-like protein disclosed in Japanese Patent Application Laid-Open No. 998/1992, which partially lacks the constituent amino acids of TM.
Publication, WO93 / 15755, WO93 / 25
No. 675 or Japanese Patent Application Laid-Open No. 5-310787 discloses a TM-like protein in which a part of the protein is substituted with another amino acid. For example, those in which oxidation is prevented by substituting methionine with another amino acid (Japanese Unexamined Patent Publication No. 5-508150), those in which degradation by proteolytic enzymes is prevented by modifying the amino acid sequence, (WO93 / 15755) Publication).

Further, WO 91/04276, WO
No. 91/05803 or JP-A-3-1333
No. 80, a TM-like protein having a sugar chain containing chondroitin and / or chondroitin sulfate,
Bovine T disclosed in JP-A-6-279497
A TM-like protein to which an O-glycoside sugar chain binding site containing an acidic amino acid sequence derived from M is added and a chondroitin sulfate sugar chain is bound, or an O-glycosylation site region disclosed in WO92 / 03149. TM in which the sugar chain is modified or the O-glycosylation site region is deleted
And a TM-like protein containing no chondroitin and / or chondroitin sulfate disclosed in JP-A-4-210700. Furthermore, a TM-like protein which may include a part of an amino acid sequence such as human tissue plasminogen activator disclosed in Japanese Patent Publication No. 4-505554 is cited.

Such a TM-like protein is currently used as a therapeutic agent for DIC in clinical development, and has little side effects such as bleeding tendency associated with treatment with heparin and high safety as a pharmaceutical. Expected (Takahashi, Y et a
l. ), Thrombosis and hemostasis (Thro
mbosis and Haemostasis), 77
Vol. 789-795, 1997).

In the present invention, the term "combination" refers to a plurality of drugs each containing UTI and TM-like protein, which are the active ingredients of the prophylactic / therapeutic agent for organ failure of the present invention, separately.
Simultaneous administration of the multiple agents, sequential administration of the multiple agents, or simultaneous administration of a mixture of UTI and a TM-like protein in the form of a single agent is included. here,
The multiple agents may be a two-agent configuration in which UTI and TM-like protein are each separately contained, and such two agents may include:
It may have a configuration of three or more agents other than UTI or TM-like protein and other agents comprising various optional components described later. Furthermore, the agent itself containing the UTI or TM-like protein may be a plurality of agents containing different optional components and the like. In addition, as the form of the above sequential administration,
For example, the prophylactic / therapeutic agent for organ failure of the present invention is
When the M-like protein is composed of two separate drugs, one of the two drugs containing UTI is administered first, and then the other containing the TM-like protein is administered. Or administering one of the plurality of agents containing the TM-like protein first and then administering the other containing the UTI.

[0023] The method for preparing the prophylactic / therapeutic agent for organ failure according to the present invention includes various known pharmaceutical preparation methods as long as UTI and TM-like protein are used as active ingredients separately or as a mixture thereof. Can be taken.

Therefore, the UTI and the TM-like protein may be prepared separately from each other, or may be prepared as a single agent prepared by mixing them. Saline, vegetable oils, mineral oils, higher alcohols, higher fatty acids, harmless organic solvents, etc., and human serum albumin, excipients, coloring agents, emulsifiers, suspending agents, and purified or genetically manufactured as required. Surfactants, solubilizers, adsorption inhibitors,
Injection, transdermal, suitable for effective administration to a living body, appropriately combined with optional components such as a stabilizer, a preservative, a humectant, an antioxidant, a buffer, a tonicity agent, and a soothing agent. Pharmaceutical preparations such as absorbents, eye drops, nasal absorbents and oral preparations, preferably injections can be prepared. The formulation of the injection can be provided, for example, in the form of a lyophilized product, a solution for injection, or a form enclosed in an osmotic pump, and particularly preferably a lyophilized product.

The method for administering the agent for preventing or treating organ failure of the present invention can be intraarterial injection, intravenous injection, subcutaneous injection, intramuscular injection, etc., and if necessary, intraperitoneal administration, cerebral cistern Internal administration and oral administration can also be used. In this case, the dose may be, for example, about 1 to 1000 mg, preferably 4 to 200 mg per day as a UTI protein amount. The protein amount of the TM-like protein is about 0.01 to 1000 μg / kg per day, preferably about 0.1 to 1000 μg / kg.
-500 µg / kg, more preferably 1-200 µg
/ Kg.

Further, the active ingredients of the present invention, UTI and TM-like protein, were added to a liposome-forming substance (JP-A-60-2).
No. 43022 or JP-A-64-85920) and then administered as liposomes. Alternatively, it can be administered continuously by enclosing it in an osmotic pump or the like and placing it in a living body. In addition, the preparation may be implanted surgically as a sustained release preparation, and as a percutaneous absorption agent, topically administered to a surgical operation site by a method such as a liquid preparation, an ointment, a cream, a gel, and a vap. It can also be applied. In this case, UT
The I concentration can be, for example, about 0.1 to 1000 mg / mL, preferably 1 to 200 mg / mL. TM
The protein concentration is, for example, about 0.001 to 1000 μg /
mL, preferably 0.01 to 500 µg / mL, and more preferably 0.1 to 200 µg / mL.

The ratio between the UTI and the TM-like protein may be any ratio as long as it exerts a preventive / therapeutic action on organ failure, and can be varied widely. For example, 1:10 by weight
To 100: 1, preferably 1:20 to 40: 1, more preferably 1: 1 to 10: 1.

Further, the above-mentioned dosage is determined based on the dosage form, administration method,
Number of doses per day, degree of symptoms, weight, age, UT
It can be appropriately increased or decreased depending on the ratio of the dose of I and TM-like protein, conditions such as simultaneous administration or sequential administration.

[0029]

[Examples] Acquisition Examples Hereinafter, the present invention will be described more specifically with reference to examples of obtaining UTI and TM-like protein.
And TM-like proteins are not limited to these.

1-1) Example of obtaining UTI Proksch method (The Journal
Of Laboratory and Clinical Medicine (T
he Journal of Laboratory an
d Clinical Medicine), volume 79, 4
91-499, 1972). Healthy adult urine 6
After concentrating 50 L and dialyzing against deionized water, 1N
Adjust the pH to 7.8 with sodium hydroxide solution, then
UTI was adsorbed by passing through a DEAE cellulose column (20 × 80 cm) equilibrated with 0.05 M Tris-HCl buffer solution (pH 7.8). This column was added to the warm buffer 4
After washing with 0 L, the adsorbed UTI is eluted using the same buffer solution containing 0.3 M sodium chloride, and the eluate is heated at 60 ° C. for 20 minutes to remove the contaminating protease. Inactivate, 16g of crude UTI
I got This crude UTI was adsorbed on a DEAE cellulose column (8 × 60 cm) equilibrated with 0.02 M glycine-hydrochloric acid buffer solution (pH 3.4). The column was washed with 10 L of the same buffer followed by 10 L of the same buffer containing 0.2 M sodium chloride, and then UTI was eluted with 8 L of the same buffer containing 0.4 M sodium chloride. After the eluate was concentrated by ultra-concentration, gel chromatography was performed using a column (10 × 95 cm) packed with Sephadex G-100 using a saline solution as a developing solvent to obtain purified UTI. The specific activity of the UTI obtained here is 2500 u / m
g.

1-2) Example of obtaining soluble TM-like protein derived from human urine 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 passing urine was desalted and concentrated using an ultrafiltration membrane. Next, purification was performed sequentially using a DEAE cellulose (manufactured by Whatman) column, DIP-thrombin-agarose chromatography, and Sephacryl S-300 (manufactured by Pharmacia Fine Chemical Company) to collect an active fraction (hereinafter, U
TM0). This fraction was dialyzed against distilled water overnight and freeze-dried.

1-3) Recombinant human soluble TM
Example of obtaining a similar protein 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 456 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 solution of this high expression strain was purified by DIP-thrombin-agarose column and gel filtration to obtain the desired product.

1-4) Recombinant human soluble TM
Example of obtaining a similar protein 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 solution of this high expression strain was purified by DIP-thrombin-agarose column and gel filtration to obtain the desired product.

2. Combined effect of UTI and TM-like protein A 58-year-old man (weight 49Kg) diagnosed with DIC based on acute gallbladder cholangitis and sepsis was ineffective at 2000mg / day for 2 days with FOY For patients, 300,000 U / day of UTI (Miraclid, Mochida Pharmaceutical Co., Ltd.) and TM-like protein (UTM0)
4900 TMU / day intravenously for 3 days, and TM
The same protein (UTM0) 4900 TMU / day was infused intravenously for 2 days. Before the start of administration and on the day after the end of the administration, observation of organ symptoms, coagulation examination along with biochemical examination, and DIC score judgment (Ministry of Health and Welfare specific disease blood coagulation disorder research group, 198)
8 years). The results are shown in Table 1.

[0035]

[Table 1] Item Before start of administration Organ symptom the day after the end of administration +-Serum creatinine 0.90.5 (mg / dl) Serum GOT (IU / L) 317 23 Serum GPT (IU / L) 304 95 Urine protein ++-Bleeding symptom + -DIC score 83

As shown in Table 1, the combined use of UTI and the TM-like protein improved the function test values of the kidney and liver, and markedly improved organ symptoms. Also,
Hemorrhagic symptoms and DIC scores also improved. On the other hand, no side effects were observed.

3. Formulation Examples Specific embodiments of the present invention will be described below with reference to formulation examples, but the present invention is not limited thereto.

(Example 1) UTI (obtained in acquisition example 1-1)
Dissolve 4.0 g in 200 mL of saline and filter aseptically using a membrane filter. The filtrate was filled into a sterilized glass container in a volume of 1 mL, and freeze-dried by a conventional method to obtain a freeze-dried preparation.

This freeze-dried agent is used together with a TM-like protein-containing agent separately prepared in an arbitrary dosage form.

Example 2 UTM0 (obtained in Acquisition Example 1-2) 10 mg Maltose 100 mg Purified gelatin 100 mg Pluronic F68 10 mg Disodium hydrogen phosphate dodecahydrate 0.77 mg Sodium dihydrogen phosphate dihydrate Product 0.18 mg Sodium chloride 81.8 mg The above-mentioned components were dissolved in 10 mL of distilled water for injection, sterile filtered, dispensed 1.0 mL each in a sterile vial, and lyophilized to prepare a formulation for injection.

This preparation for injection is used together with a UTI-containing agent separately prepared in an arbitrary dosage form.

Example 3 UTI / TM Combination UTI (obtained in Acquisition Example 1-1) 200 mg UTM0 (obtained in Acquisition Example 1-2) 5 mg Purified gelatin 50 mg Sodium phosphate 34.8 mg Sodium chloride 81 0.8 mg Mannitol 25 mg The above components were dissolved in 10 mL of distilled water for injection, sterile-filtered, dispensed 1.0 mL each in a sterile vial, and lyophilized to prepare a formulation for injection.

Example 4 UTI / TM Combination UTI (obtained in Acquisition Example 1-1) 200 mg UTM0 (obtained in Acquisition Example 1-2) 10 mg Maltose 100 mg Purified gelatin 100 mg Pluronic F68 10 mg Dihydrogen phosphate Sodium dodecahydrate 0.77 mg Sodium dihydrogen phosphate dihydrate 0.18 mg Sodium chloride 81.8 mg The above components were dissolved in 10 mL of distilled water for injection, sterile filtered, and then sterile vials were added 1.0 mL each. , And lyophilized to prepare a preparation for injection.

[0044]

According to the agent for preventing or treating organ failure of the present invention, since UTI and TM-like protein are used in combination, SIRS caused by various causes such as trauma, surgery, burns, infection, and radiation exposure, and the subsequent occurrence of SIRS occur. The preventive and therapeutic effects of MODS are synergistically obtained as compared to the case where UTI and TM-like protein are used alone. In addition, the dose can be reduced, side effects are reduced, and safety is further improved.

Claims (7)

[Claims] (1) An agent for preventing or treating organ failure, comprising a combination of ulinastatin and a thrombomodulin-like protein as active ingredients. 2. The preventive or therapeutic agent for organ failure according to claim 1, wherein the thrombomodulin-like protein is a soluble thrombomodulin-like protein. 3. The preventive / therapeutic agent for organ failure according to claim 2, wherein the thrombomodulin-like protein is a human urine-derived soluble thrombomodulin-like protein. 4. The agent according to claim 2, wherein the thrombomodulin-like protein is a recombinant human soluble thrombomodulin-like protein. 5. The method according to claim 1, which comprises a single or a plurality of preparations.
4. The preventive or therapeutic agent for organ failure according to any one of 4. 6. The preventive or therapeutic agent for organ failure according to claim 1, wherein ulinastatin and thrombomodulin-like protein are administered simultaneously. 7. The preventive / therapeutic agent for organ failure according to claim 1, wherein ulinastatin and thrombomodulin-like protein are administered separately.