JP2867905B2 - Pharmaceutical composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a pharmaceutical composition comprising a 1,5-benzothiazepine derivative [I] or a pharmaceutically acceptable salt thereof and a plasminogen activator.

[0002]

2. Description of the Related Art 2- (4-Lower alkylphenyl) -3
-Lower alkanoyloxy (or hydroxy) -5
(2-di-lower alkylaminoethyl) -8-lower alkyl-2,3-dihydro-1,5-benzothiazepine-4
(5H) -one, 2- (4-lower alkylphenyl)-
3-lower alkanoyloxy (or hydroxy) -5
(2-mono lower alkylaminoethyl) -8-lower alkyl-2,3-dihydro-1,5-benzothiazepine-
It is known that 1,5-benzothiazepine derivatives such as 4 (5H) -one have an antihypertensive action, a cerebral / coronary vasodilatory action, and an inhibitory action on platelet aggregation (for example, Japanese Patent Publication 2-2).
No. 8594, Tokuhei 3-74661, etc.).

On the other hand, urokinase (hereinafter, referred to as UK) separated and purified from urine or cultured kidney cells, pro-urokinase (hereinafter, referred to as PUK) in which the amino acid sequence of UK is changed in one place, Staphylococcus aureus (Staff)
staphylokinase (hereinafter referred to as SFK), a neutral protease produced by S. ylococcus aureus, and tissue plasminogen activation found in human or animal tissues and in cell cultures or tumor cell cultures derived from those tissues Plasminogen activator such as factor (hereinafter referred to as t-PA) is known to have thrombolytic action.

[For example, Merck Index (No. 11)
Edition), p.1491, 9394 (1989), etc.].

Among them, t-PA, which has a high affinity for thrombus and excellent thrombolytic ability, is a particularly useful substance for treating cardiovascular diseases such as acute myocardial infarction. However, t-PA
In order to maintain effective blood concentration, the bleeding tendency in the subcutaneous, digestive organs, brain, etc., reocclusion after opening of the responsible coronary artery is observed, and the half-life in human blood is very short, several minutes. Requires a large amount of t-PA administration, and as a result, there is a clinical problem that bleeding tendency is inevitable. In other words, in such a treatment method using a single large-volume injection, t
-The half-life of PA is less than 5 minutes, and most t-PA must be administered by continuous infusion to maintain sufficient t-PA concentration to exert lytic activity in the circulatory system It is believed that there is. [For example, Nilsso
netal. , Scand. J. Haemato
l. , 33, 49-53 (1984)]. Therefore, reduction of the amount of t-PA to be administered and prevention of reocclusion after thrombolysis are important issues in the treatment of cardiovascular diseases.

[0006]

An object of the present invention is to provide a novel pharmaceutical composition useful as a therapeutic agent for thrombosis and / or an agent for preventing or treating reocclusion after thrombotic treatment.

[0007]

As a result of various studies, the present inventors have found that by using a 1,5-benzothiazepine derivative in combination with PA, an extremely excellent thrombolytic action that is not observed when PA alone is administered alone. And / or found to have a preventive effect on reocclusion after thrombolysis.

That is, the present invention provides a compound represented by the general formula [I]:

[0009]

Embedded image

Wherein R ¹ represents a lower alkyl group;
² represents a hydrogen atom or a lower alkanoyl group; R ³ and R ⁴ are the same or different and represent a hydrogen atom or a lower alkyl group; and R ⁵ represents a lower alkyl group).
A pharmaceutical composition comprising a benzothiazepine derivative or a pharmacologically acceptable salt thereof and plasminogen activator (PA), more specifically a therapeutic agent for thrombosis or reocclusion after thrombotic treatment Is a prophylactic / therapeutic agent.

Another aspect of the present invention is a thrombolysis-promoting agent for PA, comprising a 1,5-benzothiazepine derivative [I] or a pharmaceutically acceptable salt thereof as an active ingredient.

Another aspect of the present invention is an agent for preventing reocclusion after use of PA, comprising a 1,5-benzothiazepine derivative [I] or a pharmaceutically acceptable salt thereof as an active ingredient.

In general, thrombin activates platelets,
In addition to causing platelet aggregation, fibrinogen is limitedly degraded and converted to fibrin around the aggregate to form a fibrin network. These are the processes of thrombus formation.

The pharmaceutical composition of the present invention comprises 1,5,
-The benzothiazepine derivative [I] or a pharmacologically acceptable salt thereof, or PA exhibits remarkably excellent dissociation activity of platelet aggregates as compared with the case of using each alone.

For example, as shown in Experimental Example 1 below, thrombin was used to induce platelet aggregation using human platelet-rich plasma (PRP), and a fibrin network was formed. In the case, the 1,5-benzothiazepine derivative [I] or a pharmaceutically acceptable salt thereof alone did not show any change, and t-PA alone showed fibrin dissolution, but the platelet aggregates Almost no dissociation was observed. However, when a 1,5-benzothiazepine derivative [I] or a pharmacologically acceptable salt thereof was used in combination with t-PA, a platelet aggregate dissociation action based on fibrinolysis was observed (these 1,5-benzothiazepine derivatives [I]) were used. T of the azepine derivative [I] or a pharmaceutically acceptable salt thereof
-The effect of promoting PA thrombolysis was observed over time as the change in light transmittance accompanying the dissociation of platelet aggregates was observed. FIG. 1 shows a series of experimental results.

As a result, when t-PA is used alone, a decrease in transmittance due to the dissociation of platelet aggregates is hardly observed when measured for about 10 minutes. On the other hand, 1,5-benzothiazepine derivative [I] or a pharmacologically acceptable salt thereof and t-PA
As a result of the combined use, dissociation of aggregates was observed with the lapse of time from 1 minute after the addition.

In Experimental Example 2 to be described later, a thrombus was formed artificially by inserting a copper coil into the canine femoral artery, and t-
In the thrombus formation / lysis model of dissolving with PA, t-PA and 1,5-benzothiazepine derivative [I]
Alternatively, it was confirmed that the pharmaceutical composition of the present invention prevented reocclusion after thrombolysis by using a pharmaceutically acceptable salt thereof in combination (in Experimental Example 2, these 1,5-benzothiazepines were used). The combined effect of the derivative [I] or a pharmaceutically acceptable salt thereof and t-PA was confirmed by prolonging the time from thrombolysis to reocclusion compared to administration of t-PA alone).

In the present invention, as a specific example of the 1,5-benzothiazepine derivative [I], R ¹ has ¹ to 4 carbon atoms.
Wherein R ² is a hydrogen atom or C 2-5
R ³ and R ⁴ are the same or different and are a hydrogen atom or an alkyl group having 1 to 4 carbon atoms;
Compounds in which ⁵ is an alkyl group having 1 to 4 carbon atoms are exemplified. Among these, preferred compounds include compounds in which R ¹ is a methyl group, R ² is a hydrogen atom or an acetyl group, and R ³ and R ⁴ are the same or different and are a hydrogen atom or a methyl group, and more preferred compounds are , R ¹ is a methyl group, R ² is an acetyl group, R ³ and R ⁴ are methyl groups, R ⁵
Is a methyl group.

1,5-benzothiazepine derivative [I]
Has two asymmetric carbon atoms at the 2- and 3-positions of the benzothiazepine skeleton, and therefore has two stereoisomers (cis, trans isomers) or four optical isomers [(+)-cis, (-)- Cis, (+)-trans, (-)-trans isomerism], and the present invention includes any of these isomers and a mixture thereof, and the (-)-cis isomer is particularly preferred.

In the present invention, the 1,5-benzothiazepine derivative [I] can be used in a free form or a pharmaceutically acceptable salt form thereof.

Pharmaceutically acceptable salts of the compound [I] include, for example, inorganic acid addition salts such as hydrochloride, hydrobromide, sulfate and phosphate, or oxalate and acetate; Organic acid addition salts such as maleate, fumarate, tartrate, methanesulfonate are mentioned.

Examples of PA (plasminogen activator) include UK (urokinase), PUK (prourokinase), SRK (streptokinase), SFK (staphylokinase) and t-PA (tissue plasminogen activator). Factor).

The above PA is UK, PUK, SRK, SF
K or t-PA may be used alone or in combination of two or more. Among them, preferred are t-PA alone or t-PA and UK in combination, and more preferred. The use of t-PA alone is mentioned.

The t-PA may be obtained by extracting from a natural product, or may be obtained from a culture of microorganisms or animal cells artificially prepared by a genetic recombination technique. Often, the invention is not limited by its origin. Specifically, mammals, especially humans
Any biologically active protein corresponding to -PA may be used, and may not be glycosylated. When fully glycosylated, the apparent molecular weight on a polyacrylamide gel is about 70,000 and the isoelectric point is 7.5.
８8.0. The specific activity of t-PA is about 300,000
６００600,000 IU / mg (I
U / mg = international unit / mg, international unit is WHO, Na
Tional Institute for Biol
official Standards and Cont
lol, Holly Hill, Hampstead,
London, NW 3b f6RB, U.S.A. K. Is the activity unit defined by European Patent No. 11
As described in No. 2122, t-PA is single-stranded,
It may be a double strand or a mixture thereof, and may be obtained by any known method. For example, Biochimi
ca Acta, 580, 140-153 (197
9); EP 41766; EP 1
No. 13319, etc. can be obtained from a normal cell line or a tumor cell line. However, for example, EP-A-93619; EP-A-1170
No. 59; EP 117060; EP 173552; EP 174835; EP 178105; EP 2251.
No. 77; EP 225286; WO 86/01538; WO 86/05.
No. 514; it is desirable to obtain t-PA by culturing transformed cells or transfected cells using recombinant DNA technology as described in WO86 / 05807 and the like. In particular, Chinese hamster oocytes (CHO) cells were used for t-PA production and
ullar and Cellular Biolog
It is preferable to induce in the same manner as described in y, 5 (7), 1750-1759 (1985). In this method, the cloned gene is converted to dihydrofolate reductase (dh
It has been introduced into dhfr-CHO cells together with the gene encoding fr). Transformants expressing dhfr were selected in a nucleotide-deficient medium and exposed to high concentrations of methotrexate. Then the dhfr gene and t
-A stable cell line capable of amplifying the PA gene and expressing high levels of t-PA has been obtained. Also Biochimic
a et Biophysica Acta, 580,
140-153 (1979); Biol. Che
m. , 254 (6), 1998-2003 (1979;
ibid, 256 (13), 7035-7041 (19
81); Eur. J. Biochem. , 132,68
1-686 (1983); EP 41766.
No .; EP-A-113319; British Patent No. 21222219, etc., and it is desirable to purify t-PA by any known method. After purification, t-PA is preferably lyophilized as described in GB-A-2176702.

In the pharmaceutical composition of the present invention, when both the 1,5-benzothiazepine derivative [I] or a pharmaceutically acceptable salt thereof and PA are used, the active ingredient is used as a pharmaceutical formulation. Preferably, it is used. In this case, the active ingredients can be used as separate dosage forms which are administered simultaneously or sequentially. If they are to be administered sequentially,
It is preferred to administer the 5-benzothiazepine derivative [I] or a pharmaceutically acceptable salt thereof first, followed by PA. In any case, the second administration of the two preparations should not be so delayed that the combined effect in the prevention / treatment of reocclusion after thrombosis treatment in vivo by the combination of active ingredients is lost. However, it is much more convenient to include both active ingredients together in a single formulation than to use separate formulations.

In general, when the pharmaceutical composition of the present invention is a single combination preparation, it will be administered by an intravascular route such as infusion or single injection. Thus, parenteral formulations are required.

Due to the advantages of transportation and storage during supply,
Preferably, the formulation is provided as a lyophilized dosage form. The lyophilized dosage form can be reconstituted with an appropriate amount of solvent as needed at the time of use.

Examples of the solvent used here include distilled water for injection, physiological saline, 0.01 M to 0.1 M phosphate buffer and the like.

[0029] Lyophilized pharmaceutical compositions for parenteral administration containing t-PA are known in the art. Such techniques include EP-A-41 766; EP-A-93619; EP-A-112122;
EP-A-113319; EP-A-123
No. 304; EP 1 430 81; EP 156 169; WO 86/0110.
No. 4; Japanese Patent Publication No. 57-120523; and Japanese Patent Publication No. 58-
No. 65218. Such a formulation is also suitable for the pharmaceutical composition of the present invention, an agent for treating thrombosis, or an agent for preventing or treating reocclusion after thrombosis treatment.

The combination ratio of these two drugs depends on many factors including, for example, age, body weight, the exact condition requiring treatment, the severity thereof, the route of administration, and the like. It is determined. However, in general, 1,5
-T-PA is 0.0025 to 1 part by weight of the benzothiazepine derivative [I] or a pharmaceutically acceptable salt thereof.
The amount is preferably 2000 parts by weight, particularly preferably 0.0025 to 20 parts by weight when compound [I] is administered orally, and 0.25 to 2000 parts by weight when compound [I] is administered parenterally. In addition, the daily dose is the above-mentioned mixing ratio,
1,5-benzothiazepine derivative [I] or a pharmaceutically acceptable salt thereof is 0.05 to 400 mg, particularly 5 to 400 mg for oral administration, and 0.05 to 4 mg for parenteral administration.
And t-PA is 1 to 100 mg (that is, t-PA
With a specific activity of 600,000 IU / mg from 600,000 to 6
000 IU), and particularly preferably 5 to 70 mg (that is, 3 to 42 million IU with a specific activity of t-PA of 600,000 IU / mg).

When the 1,5-benzothiazepine derivative of the present invention or a pharmaceutically acceptable salt thereof is administered orally, it can be used in the form of a pharmaceutical preparation together with an excipient suitable for oral administration. Suitable excipients in this case include:
For example, starch, lactose, glucose, gelatin, sorbitol, tragacanth gum, polyvinylpyrrolidone, sucrose, corn starch, polyethylene glycol, talc, calcium phosphate, stearic acid, conventional extenders such as magnesium, binders, disintegrants, lubricants and the like. can give. On the other hand, when administered parenterally, it can be used as an injection. Excipients in this case include, for example, distilled water for injection, vegetable oil, propylene glycol and the like. If necessary, a suitable stabilizer, solubilizing agent or buffer may be further contained.

When the PA of the present invention is administered, it may contain a dissolution-promoting substance such as arginine in addition to the main component PA. If necessary, other components such as albumin, mannitol, gelatin, It is optional to include excipients, stabilizers, and the like, such as phosphoric acid and citric acid, which are commonly used in the preparation of pharmaceuticals such as sodium chloride.

The following experimental examples and production examples further illustrate the present invention and do not limit the present invention in any way.

[0034]

[Action]

[Experimental example 1 / dissociation of platelet aggregates] (Method) Nine volumes of blood collected from humans were collected at 3.8% (W /
V) The mixture was mixed with 1 volume of a trisodium citrate aqueous solution, and the mixture was centrifuged to prepare human platelet-rich plasma (PRP). The remaining blood was further centrifuged to prepare platelet-free plasma (PPP). PRP is used as an automatic hemocytometer (CE
LLTAC MEK-5158, manufactured by Nihon Kohden) and used at a concentration of 4.1 × 10 ⁵ cells / mm ^{3 on} average. 25 microliters of physiological saline, PRP200
After stirring the microliter mixture for 2 minutes at 37 ° C,
25 microliters of thrombin (7.5 U / ml) was added to form platelet aggregates. Immediately after formation, t-
28 μl of PA 40 μg / ml was added, and the dissolution of fibrin clots and dissociation of platelet aggregates were measured.

On the other hand, a solution obtained by mixing physiological saline with a solution of the following test compound (A) or (B), which is a platelet aggregation inhibitor, was adjusted to a predetermined concentration, and was added simultaneously with t-PA after the formation of fibrin clots ( 28 μl of the test compound (A) 300 μM and 28 μl of the test compound (B) 100 μM were added). The platelet aggregation dissociation effect was measured by using the change in light transmittance as an index, using the sample containing no t-PA alone as a sample-free control. The transmittance is PRP +
Sample solution: 0%, PPP: 100%.

FIG. 1 shows the results.

[Experimental example 2 / Preventive action of reocclusion after thrombolysis] (Method) Male and female beagle dogs weighing 7.4 to 12.2 kg (each group population 8 to 10) were used in the experiment. Right upper arm vein,
The median mesothelial vein and left jugular vein in the left forearm were exfoliated, and a polyethylene catheter was inserted for medication and blood collection. In order to measure the blood flow, the left and right femoral arteries were separated from the surrounding connective tissue, and then an electromagnetic flowmeter (MFV-3100, manufactured by Nihon Kohden Corporation)
Was attached. When a coil is inserted from the left common carotid artery into the left femoral artery, the blood flow in the left femoral artery decreases immediately. After confirming complete occlusion, 60 minutes later, t-PA was continuously administered at a rate of 12.5 μg / kg / min for 60 minutes through the right upper arm vein (Syringe Infusion).
n Pump, Model 22, Harvard). At the same time, a solution of the test compound (A) or (B) dissolved in a 5% glucose solution from the left forearm mesothelial vein is first added to b
olus, 0.4 ml / kg until the end of the experiment
/ Hr continuously. Administration dose is 0.3mg
After bolus administration at a rate of 0.3 mg / kg, bolus was continuously administered at a rate of 1 mg / kg, or 1 mg / kg was continuously administered at a rate of 1 mg / kg. The control group received the same dose of 5% glucose solution. Thrombolysis was determined by resumption of blood flow in the left femoral artery. 300 after completion of t-PA administration
The time during which there was blood flow during the observation period of minutes (total patency) was measured, and the effect of preventing reocclusion after thrombolysis was observed.

(Analyte Compound) (A) (-)-cis-2- (4-methylphenyl)-
3-acetoxy-5- [2- (dimethylamino) ethyl] -8-methyl-2,3-dihydro-1,5-benzothiazepin-4 (5H) -one maleate (general formula [I] , R ¹ , R ³ , R ⁴ , R ⁵ = CH ₃ , R ²
= CH ₃ CO) (B) (-) - cis-2- (4-methylphenyl) -
3-hydroxy-5- [2- (monomethylamino) ethyl] -8-methyl-2,3-dihydro-1,5-benzothiazepin-4 (5H) -one maleate (general formula [I] Where R ¹ = CH ₃ , R ² , R ³ = H, R ⁴ ,
R ⁵ = CH ₃ ,) The results are shown in Table 1.

[0039]

[Table 1]

In the above experimental system, a catheter-type pressure transducer (Miller
Micro-Tip, Model MPC-500, M
Illar Instruments' catheter was inserted, and the blood pressure was observed (preamplifier, AP-601)
G, manufactured by Nihon Kohden) and a heart rate monitor (A
C-601G, manufactured by Nihon Kohden Corporation. Blood was collected from a polyethylene catheter inserted into the left jugular vein before coil insertion, t-PA administration, thrombolysis and reocclusion or at the end of the experiment, and blood coagulation time was measured according to a modified Lee-White method. At the same time, the back of the earlobe was injured with a Frankel phlebotomy needle (Bunkyo Iriyama Seisakusho Co., Ltd.).
The bleeding time was measured according to the ke method. As a result, blood coagulation time, bleeding time, blood pressure and heart rate were all unaffected.

[Production Example 1 / Production of parenteral administration preparation of t-PA] The parenteral administration preparation of t-PA is alteplase (genetical recombination), [trade name: Glutopa, manufactured by Mitsubishi Chemical Corporation]
Was used. The specific activity is about 600,000 IU / mg.

[Production Example 2/1 Production of parenteral administration preparation of 1,5-benzothiazepine derivative [I] or a pharmaceutically acceptable salt thereof] As a powder according to the production method described in JP-B-2-28594. Obtained and dissolved in neutral physiological saline or 5% glucose solution (manufactured by Otsuka Pharmaceutical Co., Ltd.).

[Production Example 3/1, 5-benzothiazepine derivative [I] or a pharmaceutically acceptable salt thereof and t-PA
Preparation of Parenteral Administration Formulation] The preparations of Production Examples 1 and 2 were diluted with physiological saline or 5% glucose solution to achieve the required dose.

[0044]

As is clear from the above experimental results, the pharmaceutical composition of the present invention comprises a 1,5-benzothiazepine derivative [I] or a pharmaceutically acceptable salt thereof and t-PA alone. As compared with the case where the thrombus is dissolved, a higher platelet aggregate dissociation effect and a reocclusion prevention effect after thrombolysis are exhibited. Further, the pharmaceutical composition of the present invention has low toxicity and high safety as a pharmaceutical.

[0045]

[Brief description of the drawings]

FIG. 1 shows a case where t-PA (to which 28 μl of 40 μg / ml is added) alone, a case where t-PA and a test compound (A) are simultaneously administered (to which 28 μl of 300 μM is added) and t-PA. In the case of simultaneous administration of PA and the test compound (B) (28 μl of 100 μM added), changes in the respective light transmittances are shown.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-60-231669 (JP, A) JP-A-2-255617 (JP, A) The Japan Pharmacists Association, “Newly Revised Diseases and Drugs,” Pharmaceutical Affairs Co., Ltd. Published by Nippo-sha (June 15, 1986) 33-34 Osamu Matsuo, “t-PA and Pro-UK”, interdisciplinary project published (November 15, 1986) 75 (58) Field surveyed (Int.Cl. ⁶ , DB name) A61K 38/46 A61K 31/55 ACB C07D 281/10 CA (STN) REGISTRY (STN)

Claims (8)

(57) [Claims] 1. A compound of the general formula [I] (Wherein, R ¹ represents a lower alkyl group, R ² represents a hydrogen atom or a lower alkanoyl group, R ³ and R ⁴ are the same or different and represent a hydrogen atom or a lower alkyl group, and R ⁵ is a lower alkyl group. 1,5-benzothiazepine derivative or a pharmaceutically acceptable salt thereof with tissue plasminogen activator represented by the representative) (t-PA) thrombosis containing the Osamu
Therapeutic agent and / or an agent for preventing or treating reocclusion after thrombosis treatment . Wherein R ¹ is a methyl group, R ² is a hydrogen atom or an acetyl group, R ³ and R ⁴ are the same or different and each represents a hydrogen atom or a methyl group, R ⁵ is thrombosis according to claim 1, wherein methyl Treatment
And / or a preventive / therapeutic agent for reocclusion after thrombosis treatment . Wherein R ¹ is a methyl group, R ² is an acetyl group, R ³ and R ⁴ are methyl, R ⁵ is according to claim 2, wherein a methyl group
Prevention of thrombosis treatment and / or reocclusion after thrombosis treatment
Therapeutic agent . 4. The treatment for thrombosis according to claim 2, wherein the 1,5-benzothiazepine derivative is a (-)-cis form.
And / or a preventive / therapeutic agent for reocclusion after thrombosis treatment . 5. thrombosis therapeutic agent according to claim 1, 2, 3 also
Is a thrombosis therapeutic agent according to 4 and / or reclosing after thrombosis treatment
Prevention and treatment of obstruction . 6. The therapeutic agent for thrombosis according to claim 1, 2, 3 or 4, which is a preventive / therapeutic agent for reocclusion after thrombosis treatment.
Or a prophylactic / therapeutic agent for reocclusion after thrombosis treatment . 7. A compound of the general formula [I] (Wherein, R ¹ represents a lower alkyl group, R ² represents a hydrogen atom or a lower alkanoyl group, R ³ and R ⁴ are the same or different and represent a hydrogen atom or a lower alkyl group, and R ⁵ is a lower alkyl group. A thrombolysis accelerator for tissue plasminogen activator ( t- PA), comprising a 1,5-benzothiazepine derivative represented by the following formula or a pharmaceutically acceptable salt thereof as an active ingredient: 8. A compound of the general formula [I] (Wherein, R ¹ represents a lower alkyl group, R ² represents a hydrogen atom or a lower alkanoyl group, R ³ and R 4 are the same or different and represent a hydrogen atom or a lower alkyl group, and R ⁵ represents a lower alkyl group. A re-occlusion preventive agent after use of tissue plasminogen activator ( t- PA), which comprises a 1,5-benzothiazepine derivative represented by the following formula) or a pharmaceutically acceptable salt thereof as an active ingredient.