FI67300B - FOER REQUIREMENTS FOR FRAMSTAELLNING AV IN VIVO FIBRINOLYTIC ENZYMDERIVAT FOER ANVAENDNING VID BEHANDLING VENTROMBOS - Google Patents

Description

r-, ADVERTISING PUBLICATION

jSK w ^ 11 utlAggningsskkift 6 7 300

Jgxfe C pjj Fr eySnnetljr 11 03 1935 ^ ^ (51) Kv.Hu/lm.CL3 A 61 K 37/48, C 12 N 9/68 FINLAND — FINLAND (21) rttMdMM-piiMMAn ^ 793661 (22) H «fc > mhHht — AmBtaUnf ^ n 22.11.79 (23) AttMpttv · —GHdghatadag 22.11.79 (41) TvHmHkiMlwt —BBrltoWaatMt 23.05.81 PManttl · and registry administrators ............ ._. , * (441 MlhctvUalpMK »] · tmnUuflnl — i pvm. - PManfe · och rcfisterityrtltMi 'AmBkM odi« LakrNM »pvMfcsnrf 30.11.84 (32) (33) (31) tyr *" * rm * »** (71) Beecham Group Limited, Brentford, Middlesex, England-England (GB) (72) Richard Anthony Godwin Smith, Dorking, Surrey, Engl anti-Engl and (GB) (74) Berggren Oy Ab (54) In vivo method for the preparation of fibrinolytic enzyme derivatives is intended for use in the treatment of venous thrombosis -Förfarande för framstal1 Ning av in vivo fibrinolytiska enzymderivat for avvandning vid behandling ventrombos

The present invention relates to a process for the in vivo preparation of fibrinolytic enzyme derivatives for use in the treatment of venous thrombosis.

A venous thrombosis is made up of blood components, forming a solid mass or plug in the vein. Such plugs or blockages can move all or part of the vein from one location to another.

If this happens, such a blockage or part of it is called an embolus. Collectively, the conditions associated with blockages and emboli are termed thromboembolic diseases or disorders, and together they are the leading cause of serious illness and death in the West.

Today, there are two different groups of therapeutic agents that have been proposed for use in the treatment of vascular thrombosis, namely, anticoagulants and thrombolytic agents. Today, coagulation inhibitors are the most commonly used therapeutic agents, with haperin (generally considered the most effective) and dihydroxycoumarin ("Warfarin") being the most widely used. The main disadvantage of its coagulant inhibitors is that they do not have a direct disintegrating effect on the occlusion and, as a result, have little utility in the treatment of acute thrombotic conditions. Second, because they slow down the coagulation system, the use of such substances is hampered by uncontrolled bleeding, which carries a significant risk of risk.

Thrombolytic agents currently proposed for use in the treatment of vascular thrombosis or thrombosis fall into two distinct groups. First, there are proteolytic enzymes capable of degrading fibrin directly. Second, there are enzyme activators that activate the lytic pathway in the body, releasing plasmin that breaks down the blockage. Examples of proteolytic enzymes that have been proposed for use as thrombolytic agents include, for example, brinase, papain, ocrase, trypsin, and plasmin. However, these enzymes are highly toxic because their intravenous administration can cause unregulated proteolytic degradation in vivo.

For example, plasmin breaks down fibrinogen and other coagulation factors, causing bleeding syndrome. In addition, such agents can cause a decrease in plasminogen, thus causing a thrombotic condition. For these reasons, these types of enzymes are very rarely used as therapeutic agents.

The two substances generally considered to be most useful in thrombolytic therapy are streptokinase and urokinase. They are activators and their potency is based on converting plasminogen to plasmin. Strepto kinase is a product secreted by hemolytic streptococcus and can be produced inexpensively in large quantities. Urokinase is only available in very small amounts and is very expensive. Although both of these agents are capable of dissolving even large blockages in deep blood vessels, the intense fibrinolytic state they cause tends to cause bleeding, which in turn is a major hazard. Thus, their use is limited to patients with severe thrombotic thrombotic obstruction or pulmonary embolism. In addition, because the activators stimulate the natural lytic system, their systemic administration can result in a reduction in plasminogen, which contributes to the patient's thrombotic state if treatment is continued. To some extent, this effect can be avoided by using streptokinase-plasminogen combinations. However, this compromise does not satisfactorily solve the therapeutic problem of fibrinolysis because the systemic formation of plasmin by such combinations tends to impair the coagulating and degrading regulatory mechanism that prevails in blood vessels. In addition, nothing can be done with these combinations to control uncontrolled bleeding.

It has now been found that it is possible to avoid the main disadvantages of known thrombolytic agents by using certain enzymes which can be reversibly deactivated. It has been found that certain enzymes, such as plasmin or the streptokinase / plasminogen activator complex, have a certain selectivity for fibrin, especially in vivo. Although the complete structural requirements and properties of such systems have not been determined, an experiment has been designed to detect enzymes with fibrin specificity in vivo. In the following, enzymes that are positive in this experiment are referred to as "in vivo fibrinolytic enzymes".

It has been found that derivatives of these enzymes can be prepared in which the catalytic activity is masked while maintaining their affinity for fibrin, but which are hydrolyzed in an aqueous medium to release the active enzyme.

The present invention thus provides a method for the in vivo production of a fibrinolytic enzyme derivative which, when administered systemically over a period of no more than 5 hours to a rabbit with a radiolabeled occlusion located in the lower vena cava, raises the blood radioactivity level at least twice the background death of the animal, and wherein the catalytic site, 67300, which is essential for fibrinolytic activity, is protected by a group that can be removed by hydrolysis such that the pseudo-first order rate constant of hydrolysis with this derivative is in the range of 10 .mu.m to 10 .mu.m in an isotonic aqueous medium at pH. 7.4 at 37 ° C, characterized in that the in vivo fibrinolytic enzyme is reacted with a compound EF, where E is an attachment group which is a selective catalyst and F is a group capable of shifting from the attachment group to the active site, provided that the derivative is not (i) p-guanidinobenzoyl-human plasmin or (ii) p-guanidinobenzoyl-streptokinase-plasminogen activator complex.

The assay used to determine fibrinolytic activity in vivo was performed by forming a radiolabeled occlusion in the inferior vena cava of the rabbit being tested. The test substance is added to the blood at a point where it must pass through the heart to ensure that it is thoroughly mixed with the fallow blood. Non-specific lytic enzymes affect blood proteins and cause death before any breakdown of the blockage can be detected. Enzymes with a certain degree of specificity for fibrin break down the occlusion at a dose lower than the lethal dose. Decomposition can be seen as the release of radioactive particles into the bloodstream.

The term "in vivo fibrinolytic enzyme" as used in this specification refers to an enzyme that, when administered systematically for no more than 5 hours to a rabbit with a radiolabeled occlusion located in the lower hollow vein, raises the blood radioactivity level to at least twice the background level. without initiating the death of the animal.

One way to perform this experiment is as follows:

A New Zealand white rabbit (2.5-3.5 kg) is anesthetized and three cannulas are inserted into the anterior artery of the head (referred to herein as cannula 1), the left cardiac artery (referred to as cannula 2), and the left external ileum (referred to as cannula 3). The rabbit is then subjected to an abdominal opening to expose the inferior vena cava near its junction with the left renal vein. A portion of the inferior vena cava is separated by occluding the ligament near this site and fitting the clamp to the inferior vena cava at a location even closer to its junction with the left external ileum. A radiolabeled block is then created by injecting 50 μl of a mixture of radioiodinated human fibrinogen (100 μl) and rabbit thermoplastin (150 μl) into an isolated intravenous site. The amount of 50 is determined by evaluation and is an amount between a minimum amount that can be accurately measured and a maximum amount that can be injected without unduly disturbing the physiological conditions in the isolated part of the vein. When measuring the radioactivity released into the bloodstream, it is important that the amount of radioactivity injected intravenously is not less than 0.25 yUC, and an amount in the range of 0.25-1.0 is generally suitable. The swab is fitted around the injection needle at its puncture site through the wall of the lower hollow vein during injection so as to absorb leakage at the injection site and leaked radioactivity. Another 50 μl portion of the fibrinogen-thromboplastin mixture (blank) is introduced into a counting ampoule and its γ-radiation level is determined either by a liquid scintillation counter or by performing a direct γ-radiation counting using sodium iodide crystal technique. This test gives the amount of radioactivity injected into the animal. The first blockage is held in place long enough to collect any leak at the beginning (5 min is usually sufficient) and then replaced with a second swab. The first sheath is then transferred to a counting ampoule and the level of γ-radiation is measured. Cannula 3 is moved to the above-mentioned clamp, and 10 minutes after injection of the fibrinogen-thromboplastin mixture, a heparin solution (0.5 ml; 500 μU / ml) is administered via cannula 3. The purpose of heparin injection is to limit the amount of clotting in order to prevent the accumulation of significant amounts of unlabeled endogenous rabbit fibrin in the test block. The reason for this is that if significant amounts of unlabelled material were to clog, its degradation could go unnoticed because unlabeled degradation products would be released into the bloodstream. This error could become noticeable if, for example, a blockage marked with a radiating substance were encapsulated inside an unlabeled material.

The dressing is then partially opened so that the lower hollow compression is reduced to between 40 and 60% of its normal diameter.

____ - · τ ~ ___ 67300 Such shrinkage is required to intentionally prevent the jam from moving away. The clamp is removed. The heparin solution (1.0 ml; 500 ml) is administered via cannula 2 to anticoagulate the animal and prevent the formation of blockages, in addition to what is necessary to perform the experiment. Any persistent bleeding sites are then examined and stopped using a thrombin-saturated pad. A blood sample (1.8 ml) is taken through cannula 2 15 min after injection of radioactive material and anticoagulated by adding trisodium citrate buffer (0.2 ml; 3.8% w / v). The diluted blood sample (PQ) is examined for γ-radiation using liquid scintillation counting or direct γ-counting using the sodium iodide crystal method. This gives the radioactivity of the blood. The blockage is then washed by infusing saline (4.0 ml; 0.2 ml min 1) through cannula 3 to flush out any exogenous radioactive material into the bloodstream and to prevent growth of the blockage by washing away any thrombogenic agents. A second blood sample (P- ^ q) is taken in citrate buffer as described above and radioactivity is determined as described above 10 minutes after the PQ time. This calculation is performed to check that 1 the father's radioactivity has not been leached into the bloodstream, thus giving an indication of the stability of the blockage.

Twenty minutes after the PQ time, the saline infusion is stopped, an additional blood sample is taken (time t = o) and the test enzyme infusion is started. The amount of radiation from a blood sample t = o is a measure of background radiation.

The enzyme dose is given by systematic infusion and 2 ml blood samples are taken every 30 minutes for 6 hours from the start of the infusion. The radioactivity of each sample is measured by direct γ counting using the sodium iodide crystal method or liquid scintillation counting, and free plasmin activity is measured ex vivo.

The dose to be administered to the test animal in order to obtain a positive result is determined experimentally and therefore until this activity is obtained without causing death.

67300

As a guide to obtain this usable dose, it was found that a dose of human plasmin in the range of 1 x 10 5 x 10 6 moles of active catalytic sites per kg of experimental animal gave a positive result in this experiment. Similarly, the streptokinase / plasminogen activator complex -10-8 is positive in the range of 1 x 10 to 1 x 10 moles / kg.

Thus, the proteolytic active concentration, if unknown, is determined either by chromogenic or fluorogenic titration or by determining the molecular weight and specific activity of an analytically pure sample of the enzyme.

The dose is selected experimentally in the same manner as described above. 20% of the selected dose is given at the beginning of the experiment and the remainder within 2 hours. If no activity can be detected, the experiment is repeated by increasing the doses until the activity is detected or until the toxicity limit is exceeded. The infusion period can be extended up to 5 hours.

Examples of enzymes having in vivo fibrinolytic activity as defined above include urokinase, streptokinase-plasminogen activator complex, vascular activator, especially human vascular activator, and plasmins, especially mammalian, bovine, e.g. , monkey and human plasmin.

The most preferred enzymes that can be used in accordance with the invention are human plasmin and the streptokinase / human plasminogen activator complex. It has been found that it is highly advantageous to form a protected derivative, especially an acyl derivative, using a mixture containing human plasmin and a streptokinase / human plasminogen activator complex. Such a mixture is conveniently prepared by activating human plasminogen with streptokinase in a conventional manner without removing all of the activator complex.

It has been found that the protected enzymes used in the compositions of the invention have the following advantages over the use of the corresponding free enzymes: 3 67300 a) they are more potent than the free enzyme; (b) they have a longer biological activity; c) they can be administered as a single, often large intravenous dose because of their relatively low toxicity, whereas in current therapy, enzymes, activators and activator complexes must be administered by continuous intravenous infusions over several hours or days.

Enzymes, such as plasmins, can be prepared by known methods, as disclosed, e.g., in B.A.K. Chibber et al., Methods in Enzymol, Vol. 34, pp. 424-432; K. C. Robbins and K. Summaria, Methods in Enzymol, Vol 45, pp. 257-273. Alternatively, plasminogen can be separated by the methods set forth in British Patent Publication Nos. 1013507, 1065467, 1078141 and 1096953, and can then be converted to plasmin by conventional methods.

The human vascular activator can be isolated by the method of D.S. Pepper et al., Progress in Chem. Fibrinolysis and Thrombolysis, 1978, 3, 91-98, Raven Press New York. The streptokinase-plasminogen activator complex has been described by D.K. McClintock and P.H. Bell, Bio chem.

Biophys Res. Comm. 43, 694-702, 1971. Urokinase can be isolated from human urine as described by T. Macaig et al., Methods in Enzymol, Vol. 34, pp. 451-459, and G.H. Barlow, Methods in Enzymol, Vol. 45, pp. 239-245, Academic Press.

The most important feature of the catalytic site protecting group is that it must be able to be removed by hydrolysis at a rate at which the pseudo-first order rate constant of the hydrolysis is not less than _6 -1 —3- * l than 10 sec nor greater than 10 ~ sec '. This rate constant of -5 -3 -1 is preferably in the range of 10 to 10 sec.

Derivatives with a pseudo-first order rate constant greater than 10 ~ sec release excessive amounts of free enzyme before acting on fibrin. Derivatives with a pseudo-first-order rate constant of less than 10 6 sec release the enzyme too slowly to have clinical use.

67300

The compositions of the invention may be used as either prophylactic or therapeutic agents. For prophylactic purposes, a derivative which has a low rate of hydrolysis and consequently a long half-life is more preferable. The pseudo-first order rate constant for the hydrolysis of derivatives suitable for this purpose is in the range of 5 x 10-5-10 ^ sec 1 and the half-life is 3.5-16 hours. For therapeutic purposes, a faster hydrolyzable derivative is more preferred, i. one having a hydrolysis pseudo-first--4 -5 -] rate constant in the range of 5 x 10 to 7 x 10 sec, corresponding to a half-life of approximately 30 minutes to 2 hours.

The pseudo-first-order rate constant is determined by hydrolyzing the enzyme derivative under physiological conditions, i. in an isotonic aqueous medium at pH 7.4 and 37 ° C. Samples are taken at regular intervals and incubated with a chromogenic or fluorogenic protease substrate such as S-2251 (H-D-Val-Leu-Lys-p-nitroanilide 2HCl) and the rate of substrate change is measured.

The hydrolysis is therefore monitored until the rate of change of the substrate reaches a maximum. The rate constant k is then calculated from the formula: lo? E U-At / Ä) t max where Amaks is the maximum rate at which the substrate changes in the sample and At is the rate at which the substrate changes at time t.

The exact structure of the protecting group used in the derivatives of the invention depends in part on the nature of the enzyme selected and the purpose for which the derivative is used.

In the case of, for example, plasmin and streptokinase / plasminogen activator, the catalytic site essential for proteolytic activity comprises a serine residue having a free hydroxyl group. This site is usually protected by acyl groups such as benzoyl, substituted benzoyl, acryloyl or substituted ·· * · '? · 10 67300 with acryloyl groups. The pseudo-first order rate constant for the hydrolysis of each particular substituted benzoyl enzyme derivative can be determined from the Hammett cjr = value of each substituent as long as the pseudo-first order rate constant of two or more substituted benzoyl derivatives is not determined, unless between the substituent in question and the enzyme.

It is generally accepted that the Hammett values of the meta- and para-substituents (crm and σ ') give a rate of hydrolysis with satisfactory accuracy. In addition, the values of <jm and cf ^ can be summed with sufficient accuracy to calculate the kinetic properties of other substituted benzoyl groups having more than one substituent. The Hammett values of the ortho substituents (δ) cannot be summed with the same accuracy as the ° m ÖL values due to steric effects.

ir

However, when the ortho substituent is small and results in a negligible steric effect, i.e. in the case of fluorine, methyl and methoxy, the value of σ 'may be used only within the generally accepted error estimate or may be added to the value of öjfj, and / or σ' to calculate the reaction rates.

Within these limits, a substituted benzoyl group having one or more substituents on the phenyl ring, especially a meta and / or para substituent, wherein the sum of Hammett σ 'values in the range of 0.1 and -1.1, can be used according to the invention to block human plasmin. A substituted benzoyl group having one or more substituents on the phenyl ring, especially a meta and / or para substituent such that the sum of Hammett CT values is between -0.9 and +0.3, can be used according to the invention to block the catalytic site of porcine plasmin .

•

Suitable benzoyl and substituted benzoyl groups include benzoyl optionally substituted with halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkanoyloxy, C 1-6 alkanoylamino (RCONH-). Examples are benzoyl, p-fluorobenzoyl, o-, m- or p-toluoyl, o-, m- or p-methoxybenzoyl (i.e. anisovyl), o-, m- or p-ethoxybenzyl.

II

11 6730 0 soyl, 2,4-dimethoxybenzoyl, 3,4-dimethylbenzoyl, 4-butylbenzoyl, 3-methyl-4-methoxybenzoyl, o-acetoxybenzoyl (i.e. acetylsalicyloyl) and p-acetamidobenzoyl. Another aromatic group is naphthoyl.

An exception to this general rule is when the benzoyl group contains a basic group, e.g. an amino, dimethylamino or guanidino group. The rate of hydrolysis of such derivatives is always ten times lower than the calculated value.

Examples of such derivatives are p-guanidino-Bent-soyl-human and porcine plasmin.

Another set of acyl groups that can be used to block human and porcine plasmins according to the invention are aryloyl and substituted aryloyl, especially cinnamoyl and substituted cinnamoyl groups having one or more substituents, especially meta- and / or para-substituents, in which The sum of the Hammett (^ values) is between -1.0 and +0.15 and is also subject to the above restrictions.

Suitable substituted acryloyl groups include, for example, C 1-8 alkylacryloyl, furylacryloyl, cinnamoyl and C.

J.— O

alkyl-cinnamoyl. Specific examples of these are 3,3-dimethylacryloyl, 3- (2-furyl) acryloyl, cinnamoyl and p-methylcinnamoyl.

The compositions of the invention are formulated by conventional methods into pharmaceutical compositions intended for intravenous administration to humans.

Conventional compositions for intravenous administration include solutions of a sterile derivative in sterile isotonic, aqueous buffer. If necessary, the mixture may also contain a solubility enhancer to keep the derivative in solution, and a local anesthetic such as lignocaine to reduce pain at the injection site. Typically, the enzyme derivative is administered in unit dosage form, e.g., as a dry powder or anhydrous concentrate, in a hermetically sealed container, such as an ampoule or bottle, showing the amount of enzyme in units of activity, and the time during which the free enzyme is released. If the derivative is administered by infusion, the derivative is marketed in an infusion bottle containing sterile, pharmaceutical, water for injections. If the derivative is administered by injection, the derivative is marketed in an ampoule which also contains sterile water for injection. The mixture to be injected or infused is formed by mixing the ingredients together before administration.

The amount of product to be administered depends on the degree of fibrinolysis required and the rate at which this is desired, the severity of the thromboembolic condition, and the location and size of the occlusion. For example, a patient with a pulmonary embolism or large life-threatening ascending femoral artery occlusion requires rapid administration of the active material. On the other hand, if desired, to prevent occlusion after surgery required small amount of slow acting material. The exact dose and route of administration to be used must be determined by the attending physician, taking into account the nature and circumstances of the disease. In general, however, a patient treated for a moderate thrombus will usually receive a daily dose of 1 to 20 mg per kg of body weight, either by injection of eight doses or by infusion.

The following literature references describe the preparation of two enzyme derivatives: 1. p-guanidinobenzoyl-human plasmin, T. Chase and E. Shaw, Biochem. 8, No. 5, 2212-2224, 1969.

2. p-guanidinobenzoyl-streptokinase-plasminogen activator complex, D.K. McClintock and P.H. Bell, Biochem. Biophys. Res. Comm. 43, 694-702, 1971.

13 67300

Recently, these two derivatives have been prepared on the basis of titrations of the active sites of the above-mentioned enzymes. The separation and characterization of these derivatives has never been reported. In addition, these enzyme derivatives have never been found to be useful fibrinolytic agents.

The derivatives of the invention can be prepared using reverse protection. As described above, EF is used in this method.

Examples of group E when the enzyme to be protected is plasmin or a streptokinase / plasminogen activator are p-amidinophenyl and p-acetamidinophenyl, or structurally similarly substituted phenyl groups containing a positively charged group in the meta or para position.

Examples of inverse protective agents are: p-amidinophenyl p'-fluorobenzoate, p-amidinophenyl p'-toluate, p-amidinophenyl p'-anisate, p-amidinophenyl benzoate, p-amidinophenyl cinnamate, p-amidinophenyl p1 methyl cinnamate, p-amidinophenyl 3- (2-furyl) acrylate, p-amidinophenyl 2-naphthoate, p-amidinophenyl-3,3-dimethylacrylate, p-amidinophenyl-4-butylbenzoate, p-amidinophenyl-2 , 4-dimethoxybenzoate, p-amidinophenylacetylsalicylate, p-amidinophenyl-4-ethoxybenzoate, p-acetamidinophenyl p1-anisate, p-amidinophenyl-o-toluate, p-amidinophenyl-o-anisate, p-amidinophen , 4-dimethylbenzoate, p-amidinophenyl-3-methyl-4-methoxybenzoate and p-amidinophenyl-4-acetamidobenzoate.

The inverse protection reactions are performed in an aqueous medium in a pH range that is not detrimental to the enzyme, preservative or product, e.g. in the pH range of 4-8 and preferably in the range of 5.0-7.5.

The reaction is usually carried out using equimolar amounts of 14,67,300 enzyme and a preservative, but an excess of a preservative may also be used. It is also preferred to carry out the reaction in dilute solution, i.e. less than 10 molar with respect to the enzyme and less than 10 molar with respect to the preservative. In general, the reaction is not carried out in a solution in which the concentration of the enzyme or preservative is less than 10 .mu.mol.

The protection reaction must be carried out at reasonable temperatures, i.e. at or below room temperature, or more specifically at a temperature below 10 ° C, but above the freezing point of the reaction medium.

The reaction time depends on the protecting reagent used, the temperature at which the reaction is carried out and the enzyme chosen for protection. The optimum time under certain conditions can be selected by monitoring the decrease in enzymatic activity by incubating batches of samples with a chromogenic or fluorogenic substrate.

After completion of the reaction, the derivative is purified by conventional methods, e.g., dialysis, affinity chromatography, or ultrafiltration, and then recovered from the aqueous medium by conventional methods, such as freeze-drying. If necessary, the product can be treated, e.g., by sterilization for intravenous administration to humans.

Reverse protective agents

EF

wherein E is p-amidinophenoxy and F is an acyl group, can be prepared by acylation of p-hydroxybenzamidine or a salt thereof with an acylation derivative

FX

wherein F is as defined above and X is hydroxyl or an activated acylation derivative thereof, optionally in the presence of a catalyst.

Examples of activated acylation derivatives are acyl-II 67300 chloride and bromide. These derivatives can be prepared by conventional methods.

Suitable catalysts for this process include, for example, tertiary organic bases such as pyridine and scavengers such as dicyclohexylcarbodiimide.

The acylation reaction is usually carried out in a polar organic solvent which is inert to the reagents and the product. Examples of suitable solvents are N, N-dimethylformamide and dimethyl sulfoxide. Alternatively, when the catalyst is a liquid, such as in the case of pyridine, the reaction can be carried out without a solvent.

The reaction is generally carried out at reasonable temperatures, i.e. at a temperature below 70 ° C and generally below 40 ° C, and ambient temperature is most suitable.

The reaction time to complete the reaction depends on the specific reagents used, the solvent and the temperature at which the reaction is carried out. This can be determined by monitoring the reaction, for example, by thin layer chromatography.

When the reaction is complete, the product is recovered and purified by conventional methods.

Reverse shielding agents in which F is a substituted benzoyl group are novel and form an embodiment of the invention.

The following examples illustrate the invention:

Examples 1-18 illustrate the preparation of inverse preservatives.

Example 1

Benzoyl-appo-p-amidinophenyl ester · HCl p-Hydroxybenzamidine · HCl (0.172 g) was dissolved in dry pyridine (1.0 ml) and a solution of benzoyl chloride (0.141 g) in pyridine (1.0 ml) was added dropwise. The mixture 67300 was stirred for 1 h at ambient temperature and allowed to stand for 4 days at ambient temperature. The product was evaporated to near dryness, triturated with dry diethyl ether and the solid recrystallized from 2-propanol / diethyl ether 2: 1 v / v (ca. 3 ml). Yield 0.223 g, m.p. 160 ° C.

N.M.R. δ (dimethyl sulfoxide d 2): 9.65. Double-stripe. 4H. Interchangeable with D20. Amidine H .: about 8.0 Multiline.

9H. Phenyl and amidinophenyl.

The compounds of Examples 2-10 were prepared in the same manner as in Example 1.

Example 2

Trans-cinnamic acid p-amidinophenyl ester · HCl Sp. 192 ° C.

N.M.R. δ (dimethyl sulfoxide d 2): 9.55 double line 4H. Interchangeable with D20. Amidine H .: 7.95. Multi stripe.

6H. Amidinophenyl and acryloyl H .: 7.10. Multi-line 5H.

Phenyl H .: 6.96. Double line (J: 16 Hz) 1H. Acryloyl H.

Example 3 p-Anisic acid p1-amidinophenyl ester · HCl

Recrystallized from 2-propanol. Sp. 225 ° C.

£ N.M.R. δ (dimethyl sulfoxide d): 9.50. Double line 4H.

8.05: four line (J: 6 Hz) 4H

Amidinophenyl H .: 7.38. Four stripe. (J: 9 Hz) 4H. p-anisoyl H .: 3.90.

One line 3H. Methoxy H.

Example 4 3,3-Dimethylacrylic acid p-amidinophenyl ester hydrochloride

Recrystallized from water. Sp. 173 ° C. N.M.R. δ (dimethyl sulfoxide d®). 9.50 double band 4H interchangeable with O 2 O, amidine H; 7.95 and 7.37 quad band (J = 9 Hz) 4H, amidinophenyl H; 5.96 broad double band (J = 1 Hz) 1H, acryloyl H; 2.09 single line 3H, methyl H; 1.90 single line 3H, methyl H.

Example 5 4-Butyl-benzoic acid p-amidinophenyl ester 1-hydrochloride 17 67300

Recrystallized from isopropanol / water (1: 4 v / v). Sp. 190 ° C. N.M.R. δ (dimethyl sulfoxide d 2). 9.55 broad double band 4H interchangeable with D 2 O, amidine H. 7.6-8.1 overlapping four-band 8H with amidinophenyl + benzoyl H. 1.30 single band 9H t-butyl H.

Example 6 2,4-Dimethoxybenzoic acid p-amidinophenyl ester hydrochloride__

Recrystallized from methanol, m.p. 213-214 ° C. N.M.R. δ (dimethyl sulfoxide d®): 9.48 double band interchangeable with D 2 O, amidine H; 8.01 and 7.50 AA1BB 'four-band (J: 8Hz) 4H, amidinophenyl H; 8.03 and 6.75 multiline 3H, 2.4 substituted phenyl H; 3.90 single line _6H, 2 x methoxy H.

Example 7

Acetylsalicylic acid p-amidinophenyl ester hydrochloride Recrystallized from isopropanol, m.p. 109-111 ° C.

N.M.R. δ (dimethyl sulfoxide d 2): 9.62 broad double band 4H interchangeable with D 2 O, amidine H; 8.12 and 7.55 AA'BB 'four-line (J: 9 Hz) εH, amidinophenyl H; 2.26 single line 3H, acetyl H.

Example 8 4-Ethoxybenzoic acid p-amidinophenyl ester hydrochloride Recrystallized from isopropanol, m.p. 211-212 ° C.

N.M.R. δ (dimethyl sulfoxide d 2): 9.60 broad double band 4H interchangeable with D 2 O, amidine H; 8.10 and 7.56 AA'BB 'four-band (J: 9 Hz) 4H amidinophenyl H; 8.13 and 7.15 ΑΑ'ΒΒ 'four-line (J: 9 Hz) H, 4-ethoxyphenyl H; 4.19 four-band 2H, -OCHqCH 2; 9.38 triple band 3H, -OCH 2 CH 2.

Example 9 O-Toluic acid p-amidinophenyl ester hydrochloride Crystallized twice from isopropanol / diethyl ether. Sp. 157-159 ° C. N.M.R. δ (dimethyl sulfoxide d6): broad double band 4H interchangeable with D 2 O, amidine H; 8.20 and 7.62 ΑΑ'ΒΒ 'four-band 4H, amidinophenyl Hj 7.3-8.2 multi-line _H, 2-methylphenyl Hj 2.11 single band 3H, methyl H.

18 67300

Example 10 (a) 2- (4-Hydroxyphenyl) -acetamidine hydrochloride p-Hydroxybenzyl cyanide (5.0 g) was dissolved in absolute ethanol (50 ml) and the solution was saturated with dry HCl gas for 4 hours at ambient temperature. After standing at 4 ° C for 72 hours, dry diethyl ether (200 ml) and light petroleum (100 ml) were added and white crystals were allowed to form for 2 hours. The solid was filtered off and reacted immediately with saturated ethanolammonia (300 ml) for 3 hours at ambient temperature. The reaction mixture was evaporated to dryness on a steam bath and the residue was recrystallized from ethanol. Yield 4.36 g, m.p. 248 ° C (decomposes). N.M.R. δ (dimethyl sulfoxide d 6) 9.0-9.8 enveloped jjH interchangeable with D 2 O, amidine H, hydroxyl H. 6.85 + 7.37 AA'BB 'four-band 4H (J: 9 Hz) phenyl B; 3.70 single line 2H, benzyl H.

(b) 4-Methoxybenzoic acid p- (formphenidinomethyl) phenyl ester________

Recrystallized from isopropanol / diethyl ether.

Sp. 174-176 ° C. N.M.R. δ (dimethyl sulfoxide d 2): 9.42 broad double band 4 H interchangeable with D 2 O 2, amidine H; 8.15 and 6.29 ΑΑ'ΒΒ 'four-band (J: 8 Hz) ε H, 4-methoxyphenyl H; 7.69 and 7.13 ΑΑ'ΒΒ 'four-band (J: 8 Hz) 4H, amidinophenyl H; 3.90 single line 3H, methoxy H.

Example 11 p-Toluene-p1-amidinophenyl ester · HCl p-Hydroxybenzamidine HCl (1.72 g) and p-toluic acid (1.36 g) were dissolved in a mixture of dry pyridine (5.0 ml) and dimethyl sulfoxide (10.0 g). ml) and stirred with N, N-dicyclohexylcarbodiimide (2.1 g) for 72 hours at ambient temperature. The product was filtered and the filtrate was precipitated with dry diethyl ether (200 ml). The resulting oil crystallized after precipitation and was recrystallized from 2-propanol / 1,4-dioxane 1: 1 v / v. Sp. 164 ° C.

N.M.R. δ (dimethyl sulfoxide d 2): 9.6 wide double band interchangeable with D 2 O. 4H. Amidine H .: 8.15 four-band (J: c. 4Hz) 4H. Amidinophenyl H .: 7.60 four-band (J: c. 9Hz) 4H. Benzoyl H .: 2.44 one line 3H. Methyl H. Examples

II

19 67300 12-18 were prepared by the method described in Example 11.

Example 12 p-Fluorobenzoic acid p'-amidinophenyl ester perchlorate The oil precipitated with ether was recrystallized from 5% w / v perchloric acid (10 ml), m.p. 180 ° C (decomposes). N.M.R. 6 (dimethyl sulfoxide d 2). : 9.3 double line 4H. Interchangeable with D20. Amidine H .: 8.25 four-stripe (J: c.

5 Hz) 5H. 3.5 H in benzoyl .: 7.8 overlapping quadrants 6H. Amidinophenyl H and 2.6 H in benzoyl.

Example 13 3- (2-Furyl) -acrylic acid p-amidinophenyl ester perchlorate ________

Coupling was performed in pyridine. The filtrate was evaporated to near dryness and the residue was recrystallized from dilute perchloric acid. Light plates, m.p. 157 ° C. N.M.R. 6 (dimethyl sulfoxide d 6): 9.35 double band interchangeable with δ 20 4H, amidine H: 7.7 quadrature (J: c. 8 Hz) 4H amidinophenyl H .: 7.9 double band (J: c. 3 Hz) 1H 5-furyl H.: 7.85 / 7.40 double band (J: 20 Hz) 1H 2-acryloyl H.: 7.10 double band (J: c. 3 Hz) 1H. 3-furyl H .: 6.70 multiline 1H: 4-furyl H .: 6.44 double band (J: 15 Hz) 1H 3-acryloyl H.

Example 14 p-Methyl-trans-cinnamic acid p-amidinophenyl ester perchlorate ____

Separated in the same manner as 3- (2-furyl) -acrylic acid ester. Sp. 155 ° C.

N.M.R. 6 (dimethyl sulfoxide d6): 9.15 double band interchangeable with D 2 O. 4H. Amidine H .: 7.2-8.2 multiline 9H. Amidinophenyl and phenyl H .: 6.82 double band (J: 16 Hz) 1H 2-acryloyl H.: 6.44 double band (J: 16 Hz) 1H. Acryloyl H .: 2.42 double band (J: 2 Hz) 3H. Methyl H.

Example 15 O-Anisic acid p-amidinophenyl ester hydrochloride Recrystallized from isopropanol. Sp. 149 ° C. N.M.R.

. _, f 20 67300 6 (dimethyl sulfoxide d6) 9.50 broad band 4H interchangeable with D 2 O, amidine H; 7.50 and 7.95 irregular polyline M1, amidinophenyl H and 2-H; 7.0-7.5 multiline 3H 3,4,5 benzoyl H; 3.86 single line 3H, methoxy H.

Example 16 3,4-Dimethyl-benzoic acid p-amidinophenyl ester hydrochloride ______

Recrystallized from isopropanol / brine, m.p. 107 ° C. N.M.R. δ (dimethyl sulfoxide d 2) 9.56 broad band 4h interchangeable with D 2 O, amidine H; 7.50-7.95 irregular triple bands 7H amidinophenyl and benzoyl H. 2.30 single band 6H, methyl H.

Example 17 3-Methyl-4-methoxy-benzoic acid p-amidinophenyl ester hydrochloride_________

Recrystallized from isopropanol / 2N HCl (10: 1 v / v). Sp. 210 ° C. N.M.R. δ (dimethyl sulfoxide d®).

9.54 double band 4H interchangeable with D 2 O, amidine H. 8.07 irregular double band 4H. 2-H + 6-H benzoyl, 2H, 6H amidinophenyl; 7.60 double band (J = 9 Hz) 2H, 3H, 5H amidinophenyl; 7.22 double band (J = 9 Hz) -1H 5 H benzoyl; 3.92 single line 311 methoxyl H; 2.26 single line _3H, methyl H.

Example 18 4-Acetamidobenzoic acid p-amidinophenyl ester hydrochloride Recrystallized from isopropanol / water 1: 2 v / v. Sp. 257 ° C. N.M.R. δ (dimethyl sulfoxide d6) 10.62 one line 1H interchangeable with D 2 O, amidine H. 7.90 AA1BB 'four-line _4H superimposed 7.50-7.9 AA' BB 'four-line 4H, benzoyl and amidinophenyl H. 2.09 one lane 3H, acetyl H.

Example 19

Separated lyophilized p-anisoyl-human plasmid Human plasmin (0.15 micromolar) in 20% v / v glycerol / 0.9% w / v saline (4.2 mL) was treated with 50 microliters of 0.1 M p- a solution of amidinophenyl p'-anisate (prepared according to Example 3)

II

67300 in dimethyl sulfoxide at 22 ° C. After 7.5 minutes, an additional 50 microliters of acylating agent was added. The product was dialyzed for 2 hours using 2 L of the glycerol / saline solution described above and then added to a mixture of L-lysine-SEPHAROSE 4B (Pharmacia Fine Chemicals, 23 g wet weight) and 0.1 M triethanolamine hydrochloride buffer, pH 7.0 (20 mL). .

The mixture was incubated for 10 min at 4 ° C and then filtered and the gel was washed with 100 ml of the above buffer. The gel was then mixed with a solution of 0.2 M ε-amino-caproic acid in the above buffer and incubated at 4 ° C for 10 min. After filtration and washing with a further 55 ml of ε-aminocaproic acid solution, the filtrate was dialyzed against two 50 mM ammonium bicarbonate buffer solutions, pH

7.0 (5 L), at 4 ° C for 2 hours. The final recovered product was mixed with "Dextran T 70" (Pharmacia) and lyophilized. 1.98 g of a white solid were obtained. This inactivated material could be reactivated to completely free plasmin in approximately 2 hours under the deacylation conditions mentioned above.

The acylating agents of Examples 1, 2 and 4-18 were also reacted with human plasmin by the method of Example 19 to give the corresponding protected plasmin. When using the acylating agent of Example 10, it is preferable to use a high concentration of the acylating agent or to allow the reaction to proceed for a longer time to ensure a complete reaction. The deacylation rates of the latter compounds are shown in Table 1 on page 26.

22 67300

Example 20

Preparation of trans-cinnamoyl-swine plasmin solution In 10% v / v glycerol-0.9% w / v saline (26 ml), porcine plasmin (1.73 micromolar) was treated with p-amidinophenyl transcinnamate (3.02 mg). suspended in 0.9% w / v brine (1 mL) with stirring for 20 min at 4 ° C. The mixture was then frozen at -20 ° C and stored at this temperature overnight. After melting, the mixture was dialyzed against the above glycerol / saline mixture (2 L) at 4 ° C for 2 hours. The product was stored at 0 ° C until used. The activity of the derivative was less than 0.5% of the activity of the original enzyme and could be reactivated within 2 hours under deacylation conditions.

Example 21 Preparation of a solution of p-anisoyl-streptokinase-human plasminogen activator-ri complex__ 4

Streptokinase (5 x 10 units A.B. Kabi, Stockholm,

Sweden) in 0.9% w / v saline (10.5 mL) was mixed with lys-human plasminogen (0.025 mL; 4.9 nanomolar). The mixture was incubated at 25 ° C for 40 min and then diluted with 0.1 M trishydroxymethylaminomethane hydrochloride, 0.9% w / v saline, 20% v / v glycerol, pH 7.4 (2.0 mL). This solution was treated with 3 portions of 0.1 mM (final concentration) p-amidino-phenyl p'-anisate (prepared as a stock solution in 0.1 M dimethyl sulfoxide) for 3 five minute periods at 25 ° C. The acylated enzyme was mixed with a 33% w / v suspension of L-lysine-SEPHAROSE 4B (2.5 ml) for 10 min at 0 ° C. The suspension was filtered by suction filtration and washed with the above tris / glycerol / saline buffer (100 ml) at 4 ° C. The gel was resuspended in Tris / glycerol / saline buffer (5 ml) further containing 0.1 M aminocaproic acid after 10 minutes at 0 ° C. The gel suspension was clarified by centrifugation at 2 ° C.

II

23 67300 min at 1000 g and the supernatant liquid (4 ml) was dialyzed using Tris / glycerol / saline buffer (2.0 L) at 4 ° C for 2 hours. The resulting solution of the acyl activator complex could be regenerated to the free enzyme at 37 ° C with a pseudo-first order rate constant of -4 -1 for 2.7 x 10 sec. Example 22 Preparation of 3-methyl-p-anisoyl-streptokinase-human plasminogen activator complex solution_ This product was prepared in the same manner as in Example 21 except that the acylation was performed using 2 portions of 0.2 mM (final concentration) 3-methyl-p-anisino-acid-p-amidinophenyl ester and two 15-minute periods at 25 ° C, followed by a 1-hour period at 0 ° C. ° C.

The pseudo-first order deacylation rate constant was 1 x 10-4 sec.

Example 23 Preparation of a mixture of p-anisoyl-human plasmin and p-anisoyl-streptokinase-human plasminogen activator complex Lys-human plasminogen (216 mg; 2.56 micromoles) dissolved at a concentration of 6.8 mg / ml in 0.1 M trishydroxymethylaminomethane-hydrogen methane .9 w / v in saline, 20% v / v in glycerol, pH 7.4, was treated with streptokinase (AB Kabi, Sweden, 1.2 x 104 units, approximately 2.67 nanomolar). The mixture was incubated at 25 ° C for 1 hour and added to 33% v / v glycerol. This solution (9.0 mL) was acylated with 2 aliquots of 0.1 mM (final concentration) ρ-amidinophenyl p-anisate over two 15 minute periods at 25 ° C and then dialyzed for 1 hour using 2 liters of the above buffer. The resulting solution had less than 1% of the original plasmin activity and was in a form suitable for use in a rabbit assay system.

Example 24 Stoichiometric reaction of (3H-methoxy) -p-amidinophenyl-p'-anisate with human and porcine plasmins__ (a) Human plasmin - 5

Solutions of human plasmin (2.67 x 10 M, 0.2 ml, 5.34 nano- - τ 24 67300 moles) were reacted with 2 portions of a 2 mM solution of tritiated p-amidinophenyl-p-anisate (specific radioactivity 22.8 mC ^ / m moles) in two 7.5 minute batches at 25 ° C. After addition of a 10% w / v solution of trichloroacetic acid in acetone (1.8 ml), the protein was precipitated and separated by centrifugation at 1500 g for 3 minutes. The protein sphere was washed with two portions of acid / acetone (2 mL) and dissolved in 2 N sodium hydroxide solution (1.0 mL). Liquid scintillation counting of this solution showed that an average of 0.130 μl of Ci was associated with the protein, corresponding to 5.73 nanomoles of anisic acid.

(b) Pig plasmin

A similar experiment using porcine plasmin solution (using 5.29 x 10 M; 10.54 nanomolar) gave an average radiochemical analysis of anisic acid of 9.58 nanomolar.

Example 25 Freeze-dried p-anisoyl streptokinase-human plasminogen activator complex

Streptokinase (9 x 10 units) in 0.9% w / v saline (0.9 ml) was mixed with the lys-human plasminogen gene (0.075 ml; 14.9 nanomolar). The mixture was incubated at 25 ° C for 30 min and then diluted with 0.1 μl of trishydroxymethylaminomethane hydrochloride, pH 7.4 (4.0 ml). This solution was treated with 3 portions of 0.1 mM (final concentration) p-amidinophenyl p-anisate (prepared as a stock solution in 10 mM dimethyl sulfoxide) for three 5 minute periods at 25 ° C. The acylated enzyme was mixed with a 33% wet w / v suspension of L-lysine ("Sepharose 4B") (5.0 mL) for 10 min at 0 ° C. The suspension was filtered by suction filtration and washed with the above-mentioned Tris-HCl buffer (200 ml) at 4 ° C. The gel was resuspended in 0.1 M aminocaproic acid in 50 mM Tris-HCl, pH 7.4 (5.0 mL), and then filtered, washed with three 5 mL portions of the same buffer. The combined eluates were dialyzed against 215% w / v mannitol in Tris-HCl buffer (5.0 mM), pH 7.4, for 2 hours at 4 ° C and then lyophilized to a white powder (0.560 g). This material had an enzyme activity of less than 5% f. *** t · · 25 6 7 3 0 0 of the original activity and could be regenerated in an aqueous medium at 37 ° C.

of hydrolysis

The pseudo-first order rate constants (K) of the enzyme derivatives prepared from the acylating agents described in Examples 1-18 are shown in Table 1 below.

table 1

Deacylation rates of serine-substituted plasmins in the active site, pH 7.4, 37 ° C_

Acyl group Human plasmin Porcine plasmin -14 -14 _ K sec x 10 K sec x 10 p-fluorobenzyl - 3.30

Benzoyl 5.20 1.90 p-toluoyl 2.30 0.74 p-anisoyl 1.10 0.33

Trans-cinnamoyl-4,20 p-methyl-t-cinnamoyl-2,92 3- (2-furyl) -acryloyl 1,33 1,50 o-anisoyl 1,82 p-acetamidobenzoyl 2,70 acetylsalicyloyl 4,70 3.3 - dimethylacryloyl 0.69 3,4-dimethylbenzoyl 0.55 3-methyl-4-methoxybenzoyl 0.70 o-toluoyl 1,71 p-ethoxybenzoyl 1,10 2,4-dimethoxybenzoyl 1,10

Claims (11)

A process for producing a derivative of an in vivo fibrinolytic enzyme, which enzyme, which is administered systemically for no longer than 5 hours to a rabbit with a radiolabeled thrombus located in the lower half, raises the blood's radioactivity level at least twice that of the background level for 6 hours from the beginning of administration without causing death to the animal, and wherein the catalytic site essential for fibrinolytic activity is blocked by a group which can be removed by hydrolysis so that the pseudo-first the rate constant of the order - 6 - for hydrolysis of this derivative is in the range of 10 sec -3 -1 10 sec in an isotonic aqueous medium at pH 7.4 at a temperature of 37 ° C, characterized in that an in vivo fibrinolytic enzyme is reacted with a compound EF where E is a locating group which is selective for the catalytic site, and F is a group having the ability to transfer from free localization the ring group to the active site, provided that the derivative is not (i) p-guanidinobenzoylhuman plasmin or (ii) p-guanidinobenzoyl streptokinase plasminogen activator complex. Process according to Claim 1, characterized in that F is benzoyl or benzoyl substituted by halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkanoyloxy or C 1-6 alkanoylamino. 3. A process according to claim 1, characterized in that E is p-amidinophenyl or p-acetamidinophenyl. A process according to claim 1, characterized in that e is a p-amidinophenoxy group or p-acetamidinophenyl group and F is a benzoyl group, a substituted benzoyl group, an acryloyl group or a substituted cycloyl group.