RU2121362C1 - The prolonged thrombolytic agent and a method of its preparing - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: invention relates to acylated derivatives of enzymes - 4-(N,N,N-trimethylamino)-cinnamoyl derivatives of fibrinolytic enzymes: urokinase, plasminogen and plasmin tissue activators, activating complexes of plasmin-and plasminogen-streptokinase. Method of preparing these acylated derivatives of enzymes involves acylation of fibrinolytic enzymes by serine residue in an active center with an excess of 4-(N,N,N-trimethylamino)-cinnamic acid 4-nitrophenyl ester. Acylation is carried out at 0-25 C and pH 7.0-8.5 for 10-70 min in the absence of an organic solvent. The obtained acylenzymes are separated from unreacted acylating agent and the formed 4-nitrophenyl ester by gel-filtration or ultrafiltration and protein fractions are lyophilized. The prepared agents can be used for treatment of patients with thromboembolic diseases. EFFECT: improved method of preparing. 2 cl, 1 tbl, 7 ex

Description

 The invention relates to medicine and represents new acylated derivatives of fibrinolytic enzymes for the treatment of thromboembolic diseases.

 Thrombosis and thromboembolism associated with a blockage of blood vessels by a thrombus are one of the most common and serious complications of human cardiovascular diseases, leading to death. Therapy of thromboembolic diseases is carried out by the introduction of thrombolytic agents, which cause the dissolution of intravascular blood clots. Thrombolytic agents include: proteolytic enzymes (plasmin, brinase, etc.), which directly lyse thrombus fibrin, and plasminogen activators (urokinase, streptokinase, tissue plasminogen activator and prourokinase), which convert endogenous plasminogen into a thrombus-laden plasmin.

 In recent decades, clinics mainly used activators of plasminogen activators of the old generation: urokinase, excreted from human urine or from the culture fluid of kidney cells, and streptokinase, which is the secretion product of hemolytic streptococci. However, both urokinase and streptokinase do not have a specific affinity for fibrin. Therefore, both plasminogen activators in high doses necessary to achieve a therapeutic effect are able to convert plasminogen into plasmin not only on the thrombus, but also in circulation. This is associated with their significant side effects on the state of hemostasis, such as the destruction of fibrinogen, depletion of plasminogen and alpha-2-antiplasmin, etc.

 In recent years, the use of a new generation of thrombolytic agents has begun in the treatment of thrombosis. One of them is a tissue-type plasminogen activator, which is obtained from the culture fluid of human melanoma cells [1] and by genetic engineering [2].

 Tissue plasminogen activator has a high affinity for fibrin. However, it, like streptokinase and urokinase, is quickly removed from circulation: the half-life in human plasma of streptokinase is 18 minutes, urokinase is 16 minutes and tissue plasminogen activator is 9 minutes. The high clearance rate from the bloodstream of these three activators is associated with the difficulty of administering their therapeutic dose by many hours of infusion. Long-term treatment of the patient with systemic administration of activators stimulates the fibrinolytic system, resulting in dangerous bleeding due to the destruction of fibrinogen and the patient's predisposition to retrombosis due to depletion of plasminogen.

 Another representative of a new generation of thrombolytic agent is the plasminogen activator complex derivative acylated over the active center • streptokinase - anisoyl-plasminogen • streptokinase activator complex (APSAC, "Eminase", BRL921, Beecham Pharmaceutical, Great Britain) [3], which is becoming more widely used in medical practice. The activator complex reversibly inactivated by the active center retains an affinity for fibrin and remains protected from proteolysis and inactivation by antiplasmin until reactivation. Both in the bloodstream and on the thrombus, APSAC is reactivated at a certain rate, which gives it qualities that distinguish it from other thrombolytic agents: a long duration of action and ease of administration. It can be administered as a bolus, single injection without lowering blood pressure. Compared with all known activators, APSAC has the largest half-life in human plasma: 90 min [4].

 However, the therapeutic dose of APSAC (30 mg, which is equivalent to 1,500,000 I. U. streptokinase) causes plasminogen activation comparable to streptokinase in circulation with the development of plasminemia [5]. The formation of significant quantities of free plasmin in the circulation leads to the proteolysis of fibrinogen, factors V and VIII, a decrease in the level of alpha-2-antiplasmin and other effects. These effects, which threaten the development of hemorrhagic complications, are caused by a rather high reactivation rate of the initially catalytically inert acylactivator complex in the blood. As a result, APSAC is used only in emergency therapy for the treatment of acute myocardial infarction, when early perfusion is preferred, and other side effects of the drug must be neglected. The high frequency of hemorrhagic complications associated with the systemic activating effect of APSAC on fibrinolysis does not allow its use for the treatment of thrombosis of peripheral arteries and veins.

 The objective of the invention is the creation of a thrombolytic agent that acts for a long time, mainly in the thrombus area, and does not significantly affect the state of fibrinolysis and the content of fibrinogen in the systemic circulation.

где
E - урокиназа, тканевый активатор плазмингена, плазмин, активаторный комплекс плазмин•стрептокиназа, активаторный комплекс плазминоген•стрептокиназа,
где
4-(N,N,N-триметиламино)-циннамоильная группа (4-ТМАЦ-) соединена с серином активного центра фермента.This problem is solved by new compounds, namely 4- (N, N, N, -trimethylamino) -cinnamoyl derivatives of fibrinolytic enzymes of the general formula:

Where
E - urokinase, tissue plasminogen activator, plasmin, plasmin activator complex • streptokinase, plasminogen activator complex • streptokinase,
Where
The 4- (N, N, N-trimethylamino) -cinnamoyl group (4-TMAC-) is connected to the serine of the active center of the enzyme.

4-TMAC-enzymes are reactivated more slowly than their 4-anisoyl derivatives. For example, trans-4-TMAC-plasmin • streptokinase activator complex and 4-anisoyl-plasmin • streptokinase activator complex, obtained as described in example 2, are reactivated in an isotonic aqueous medium (pH 7.4, 37 ^° C) with a half-cycle of reactivation 58 min and 29 min, respectively, and their half-lives in human blood plasma are 136 min and 72 min, respectively.

 The expected therapeutic effect of the use of the proposed acylactivator: greater stability in the bloodstream, a longer duration of thrombolytic action and less side effects compared with APSAC. Due to the low severity of systemic effects, the proposed acylactivator can be used to treat peripheral vascular thrombosis, as well as for the treatment of acute myocardial infarction individually or in combination with streptokinase, depending on the clinical situation.

The method for producing TMAC-derived enzymes is that the fibrinolytic enzyme is acylated at the active center with an excess of 4- (N, N, N-N-trimethylamino) cinnamic acid 4-nitrophenyl ester. Acylation is carried out at a temperature of 0-25 ^o C and a pH of 7.0-8.5 for 10 to 70 minutes in the absence of an organic solvent. Then, the obtained acyl enzyme is separated from the unreacted acylating agent and from the resulting 4-nitrophenol ether by gel filtration, and the protein fractions are lyophilized.

The invention is illustrated by the following examples:
Example No. 1.

Obtaining trans-4-TMAC-plasmin
To 9 ml of a 15 μM solution of human plasmin (M.V. 85000D, λ _max = 280 nm, E $\genfrac{}{}{0ex}{}{\text{I\%}}{\text{Icm}}$ = 17) in 0.1 M Tris-HCI pH 8.5 buffer containing 0.15 M NaCl, add 1 ml of 10 mM solution of trans-4- (N, N, N-trimethylamino) cinnamic acid-4-nitrophenyl ester (C ₁₈ H ₁₉ JN ₂ O ₄ , MB 454D, T _pl = 190 ^o C, λ _max = 278 Nm, ε _max = 2.38 • 10 ⁴ M ^-1 cm ^-1 ) in HCl pH 3.0 and incubated at 25 ^o C for 10 minutes After additional processing of the enzyme, 0.5 ml of the acylating agent for 5 min, the reaction mixture is applied to a Sephadex G-25 column. A 0.075 M NaCl solution (4 ^° C.) was used as eluent. Protein fractions are lyophilized. The yield of white powder is 0.33 g, containing 2.8% acylplasmin. The obtained 4-TMAC-plasmin (M.V. 85 300 D, λ _max = 272 nm) contains 1-5% of the free enzyme impurity and can be reactivated into active plasmin C τ _{I / 2} = 82 min in an isotonic aqueous medium (pH 7.4; 20% glycerol; 37 ^° C).

 Example No. 2.

 Obtaining trans-4-TMAC-plasmin-streptokinase activator complex.

To 4.5 ml of 11.8 μM human plasminogen solution (M.V. 85000D, λ _max = 280 nm, E $\genfrac{}{}{0ex}{}{\text{I\%}}{\text{Icm}}$ = 17) in 0.05 Tris-HCl buffer pH 8.0 containing 0.15 M NaCl, weighed portion (300 000 IU) of streptokinase (MB 47000 D, λ _max = 280 nm, E $\genfrac{}{}{0ex}{}{\text{I\%}}{\text{Icm}}$ = 10) and the mixture is incubated at 25 ^o C for 15 minutes The progress of the formation of the plasmin streptokinase activator complex is monitored by the hydrolysis rate of 0.6 mM S-225I solution on a spectrophotometer. The activator complex is treated with a solution of trans-4- (N, N, N-trimethylamino) cinnamic acid 4-nitrophenyl ester (final concentration 0.1 mM) for 40 minutes at 25 ^° C. The solution is then subjected to gel filtration and the protein fractions are lyophilized as described in example N 1. The output of a solid powder of 0.25 g containing 2.5% of the target product. The obtained trans-4-TMAC-plasmin • streptokinase activator complex (M.V. 132 300 D, λ _max = 272 nm) is reactivated in an isotonic aqueous medium (pH 7.4; 20% glycerol; 37 ^o C) with τ _{I / 2} = = 58 minutes

 Example No. 3.

 Obtaining a mixture of trans-4-TMAC-plasmin and trans-4-TMAC-plasmin-streptokinase activator complex.

To 3 ml of 25 μM plasminogen in 0.1 M Tris-HCl buffer pH 8.0 containing 0.15 NaCl, 3 ml of streptokinase solution (71,500 IU / ml) was added and incubated at 25 ^° C for 10 min. The resulting mixture of the enzyme and activator complex is treated with 1.3 ml of a 10 mM solution of trans-4- (N, N, N-trimethylamino) cinnamic acid 4-nitrophenyl ester in HCl pH 3.0 for 30-40 minutes at 25 ^° C and then proceed as in example N 1. The yield of solid powder is 0.39 g. A mixture of trans-4-TMAC-plasmin and trans-4-TMAC-plasmin • streptokinase λ _max = 272 nm) contains 2-7% unacylated enzymes and can be reactivated into active enzyme and activator complex for 6 hours in an isotonic aqueous medium (pH 7.4; 20% glycerol; 37 ^o C).

 Example No. 4.

Preparation of cis-4-TMAC-plasmin • streptokinase activator complex
According to the method described in example No. 2, cis-4-TMAC-plasmin-streptokinase activator complex is obtained. As the acylating agent, 4-nitrophenyl ester of cis- (N, N, N-trimethylamino) cinnamic acid λ _max = 265 nm, ε _max = 1.0 • 10 ⁴ M ^-1 cm ^-1 ) in a final concentration of 1 is used mm. The acylation reaction is carried out for 70 minutes Then proceed as described in Example No. 2. The yield of solid powder 0.27 g, containing 1.2% of the target product, cis-4-TMAC-plasmin • streptokinase activator complex (M.V. 132300D, λ _max = 265 nm) contains 10 -15% unacylated complex and can be reactivated in an isotonic aqueous medium (pH 7.4; 20% glycerol; 37 ^o C) for 5 hours.

 Example No. 5.

Obtaining trans-4-TMAC-plasminogen-streptokinase activator complex
To a solution containing streptokinase (57,200 IU / ml) and plasminogen (10 μM) in 0.05 M Tris-HCl buffer pH 7.0, 0.15 M NaCl, a solution of trans-4- (N, N, N-trimethylamino) 4-nitrophenyl ether is added cinnamic acid to a final concentration of 1 mm and incubated for 60 min at 4 ^o C. Then the solution is subjected to gel filtration and protein fractions are lyophilized as described in example No. 1. The output of a solid powder of 0.88 g, containing 2.1% acyl activator, trans-4- • TMATS plasminogen-streptokinase activator complex (132 MV 300D, λ _max = 272 nm) contain 5-10% non-acylated complex and may be eaktivirovan in the active complex in an isotonic aqueous medium (pH 7.4; 20% glycerol; 37 ^o C) c τ _{I / 2} = 58 min.

 Example No. 6.

Obtaining trans-4-TMAC activator plasminogen tissue type
To 1 ml of a solution of tissue plasminogen activator (M.V. 66000D; λ _max = 280 nm) containing 1 mg / ml of the enzyme, 0.4 ml of 0.1 M Tris-HCl buffer pH 7.0 containing 0.15 M NaCl and 0.2 ml a stock solution of 4-nitrophenyl ester of trans-4- (N, N, N-trimethylamino) cinnamic acid (10 mM) and incubated for 10 min at 25 ^o C. After repeated processing of the enzyme 0.1 ml acylating agent for 3-4 min, solution was subjected to gel filtration on Sephadex G-25 at 4 ^o C. The protein fractions were frozen and stored at -40 ^o C, trans-4-TMATS-tissue-type plasminogen activator (M.V.66 300D, λ _max = 272 nm) ozhet be reactivated to active enzyme in an isotonic aqueous medium (pH 7.4; 20% glycerol; 37 ^o C ^o).

 Example No. 7.

Obtaining trans-4-TMAC-urokinase
To 1 ml (25,000 IU / ml) of urokinase solution (M.V. 54000D, λ _max = 280 nm, E $\genfrac{}{}{0ex}{}{\text{I\%}}{\text{Icm}}$ = 13.6) in a 0.05 M Tris-HCl pH 8.5 buffer, a solution of trans-4- (N, N, N-trimethylamino) cinnamic acid 4-nitrophenyl ester is added to a final concentration of 1 mM and incubated at 25 ^° C for 30-40 min The course of acylation is monitored by the decrease in the esterase activity of the enzyme over time, measuring the hydrolysis rate of a 6 mM solution of acetyl-glycyl-lysine methyl ester under the influence of aliquots on the pH-stat. The resulting acyl enzyme is isolated as described in Example No. 6. The obtained trans-4-TMAC-urokinase (M.V. 54300D; λ _max = 272 nm) contains 1-4% unacylated enzyme and can be reactivated in an isotonic aqueous medium (pH 7.4; 20% glycerol; 37 ^o C) C τ _{I / 2} = 18 min.

 Example N 8.

Fibrinolytic activity of acyl activators
A solution of bovine thrombin is added to 0.9 ml of human blood plasma to a concentration of 4 U / ml, incubated for 1 - 1.5 hours at 25 ^o C and placed over a clot of 0.5 ml of human plasma. 10 μl of a solution of streptokinase, an activator complex of plasmin • streptokinase, 4-trans-TMAC plasmin • streptokinase of an activator complex or 4-anisoyl-plasmin • streptokinase of an activator complex obtained according to Example No. 2 is added to a plasma above a clot to a final concentration of 41 μM, incubated at 37 ^o C and measure the decrease in the height of the column of fibrin clot (Δl) from time to time. The fibrinolysis rate is defined as the slope of the dependences Δl on Δt. The rate of fibrinolysis of plasma clots immersed in plasma, trans-4-TMAC-plasmin-streptokinase activator complex is significantly higher than streptokinase or the free complex plasmin • streptokinase, and slightly lower than 4-anisoyl-plasmin • streptokinase activator complex obtained according to example N 2 (table).

Sources of information
1. Collen DJ, Rijken D., Matsuo O.// Eur.Pat.Appl., 1981, Int.Cl. C 12 N 9/64, A 61 K 37/54.

 2. Pennica D., Holmes W.E., Kohr W.J., Harkins R.N., Vehar G.A. et al. // Cloning and expression of human tissue-type plasminogen activator cDNA in E. Coli. Nature, 1983, V.301, P.214-221.

 3. Smith R. A. G. // Eur. Pat. Appl., 1980, N 0009879, Int. Cl. A 61 K 37/48, C 12 N 9/68.

 4. Ferres H. // Preclinical pharmacological evaluation of anisoylated plasminogen-streptokinase activator complex. Drugs, V.33, (Suppl.3), P.33-50.

 5. Collen D. // Coronary thrombolysis: Streptokinase or recombinant tissue-type plasminogen activator? Annals of Internal Medicine, 1990, V.112, P. 529-538.

Claims (2)

1. 4- (N, N, N-trimethylamino) -cinnamoyl derivative of fibrinolytic enzymes of the General formula

where E is urokinase, tissue plasminogen activator, plasmin, plasmin streptokinase activator complex, plasminogen streptokinase activator complex,
as a prolonged thrombolytic agent.  2. The method for producing the compound according to claim 1, wherein the fibrinolytic enzyme is acylated at the active site serine with an excess of 4- (N, N, N-trimethylamino) cinnamic acid 4-nitrophenyl ether isomers, followed by isolation target product by gel filtration or ultrafiltration.

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