PHARMACEUTICALLY ACTIVE COMPOUNDS

Abstract: A derivative of a fibrinolytically active glycoprotein in which at least part of the carbohydrate portion of the protein is absent or has been degraded, characterised in that the catalytic site essential for fibrinolytic activity is blocked by a group which is removable by hydrolysis at a rate such that the pseudo-first order rate constant for hydrolyses is in the range 10<-6> sec <-1> to 10<-3> sec <-1> in isotonic aqueous media at pH 7.4 at 34<o>C. The derivative is useful in treating thrombosis.

Description: PHARMACEUTICALLY ACTIVE COMPOUNDS This invention relates to enzyme derivatives for use in the treatment of thrombotic diseases. European Published Patent Application No. 0,009,879 discloses derivatives of in vivo fibrinolytic enzymes which are useful therapeutic agents for treating venous thrombosis. The derivatives are characterised by the active catalytic site on the enzymes being blocked by a group which is removable by hydrolysis such that the pseudo-first order rate constant for hydrolysis is in the range lO^sec"" 1 to 10~ 3 sec" 1 . It has now been found that a particular class of enzymes, namely fibrinolytically active glycoproteins, can be modified by removal or degradation of the carbohydrate portion of the protein and subsequently blocked by a removable group, to produce derivatives having surprisingly slow physiological clearance rates and prolonged ^11 vivo stability. " Z X OMFI σ CU n H- o B 0 H- fD ft ►< % CO c o 3 * P» oi P> B P- 3 0 0 n tΩ C P» 3 H- L H » 3 O • The pseudo-first order rate constant is determined by hydrolysing the glycoprotein derivative under physiological conditions ie. in isotonic aqueous media at pH 7.4 and at 37°C. At regular intervals aliquots are withdrawn and incubated with a chromogenic or fluorogenic protease substrate such as S-2444 for urokinase or S-2288 for tissue activators and the rate of conversion of the substrate measured. ' The hydrolysis is followed until such time as the rate of conversion of substrate reaches a maximum. The rate constant k is then calculated by plotting: log e (1- At /Amax)against t where A max is the maximum rate at which an aliquot converts substrate and A t is the rate at which an aliquot converts substrate at time t. The derivatives of this invention may be prepared by, (a) modifying -a fibrinolytically active glycoprotein to remove or degrade carbohydrate, and then (b) subjecting the thus modified glycoprotein to direct or inverse blocking as described in Published European Patent Application No. 0 009 879. Alternatively, step (b) of the preparation can be carried out first, and the resulting blocked glycoprotein can then be subjected to the modification process of step (a). Step (a) of the above preparation may be carried out in various ways, but preferred methods are as follows: i) Chemical degradation of carbohydrate units utilising, for example, sodium periodate. This method of degradation is based on published procedures (Biochem. Biophys. Res. Commun. , 57, 55, 1974). ii) Enzymatic treatment of glycoproteins with a mixture of exoglycosidases, selected for example from β-galactosidase, α-mannosidase, α-fucosidase, • β-N-acetylglucosaminidase or neuraminidase, or one or more endoglycosidases, such as endoglycosidase D (from Diplococcus pneumoniae) or endoglycosidase H (from Streptomyces plicatus). Optionally, the glycosidases may be immobilised on insoluble carriers to avoid contamination of the modified glycoprotein. iii) In the case of melanoma plasminogen activator, preparation from cultured melanoma cells grown in the presence of an inhibitor of protein glycosylation, such as tunicamycin. iv) Preparation by recombinant DNA techniques in bacterial systems. This can be carried out by cloning the genetic information for the glycoproteins into prokaryotes. Step (b) of the preparation is preferably carried out by reacting the modified glycoprotein from step (a) with a blocking agent AB in which A is a locating group which locates the agent in the catalytic site, and B is an acyl group. Preferably B is a 2- or 4-aminobenzoyl group otionally substituted in the aromatic ring by an electron donating moiety. O PI Preferred examples of the group A include 4-amidinophenyl, 2-nitro 4-amidinophenyl and 4-acetamidinophenyl or structurally similar substituted phenyl groups containing a positively charged moiety in the 3- or 4- position. Preferred agents AB are 4-aminophenyl-4"- aminobenzoate and 4-aminophenyl-2 * -aminobenzoate. When step.(b) is the first step in the overall process, the reaction is conveniently carried out by treating the unmodified glycoprotein directly with the blocking agent. The blocking reactions are preferably carried out in aqueous media at a pH range which is not detrimental to the glycoprotein, blocking agent or product, eg between pH 5 and 9 and preferably at a pH in the range 6.0 to 8.0. The reaction is generally carried out using a molar excess of blocking agent, but equi-molar equivalents may also be employed. It is also preferred to carry out the reaction in dilute solution, ie less than 10~3 molar with respect to glycoprotein and less than 10"2 molar with respect to blocking agent. Generally the reaction will not be carried out in a solution where the concentration of glycoprotein or blocking agent is less than 10""7 molar. The blocking reaction should be carried out at moderate temperatures, ie room temperature or below, and more particularly less then 10°C but greater than the freezing point of the reaction medium. ^ \ S E OMPI The blocking agent AB wherein A is 4-amidinophenyl, may itself be prepared by reacting 4-amidinophenol with an N-protected 2- or 4-aminobenzoic acid, optionally substituted in the aromatic ring by an electron donating moiety, and subseqently deprotecting the resulting product. A suitable protecting group is the tertiary-butoxy- carbonyl (BOC) group. The reaction. is preferably carried out in a tertiary organic base, such as pyridine, and in the presence of a condensation promoting agent such as dicyclohexyl carbodiimide. If desired, the condensation reaction may also be carried out without prior protection of the amino group. The N-protection of the a inobenzoic acid material is preferably carried out by treating the material with ditertiary butyl dicarbonate. The de-protection of the product is suitably carried out by treating the product with TFA (trifϊuoroacetic acid), preferably at room temperature. The preparation of the blocking agent AB is described in detail in published European Patent Application No. 0091240. The derivative of this invention is preferably administered as a pharmaceutical compostion. Accordingly the present invention also provides a pharmaceutical composition comprising the derivative of the invention in combination with a pharmaceutically acceptable carrier. The compositions according to the invention may be formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to human beings. Typically compositions for intravenous administration are solutions of the sterile derivative in sterile isotonic aqueous buffer. Where necessary the composition may also include a solubilising agent to keep the derivative in solution and a local anaesthetic such as lignocaine to ease pain at the site of injection. Generally, the derivative will be supplied in unit dosage form for example,as a dry powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of glycoprotein in activity units, as well as an indication of the time within which the free, modified protein will be liberated. Where the derivative is to be administered by infusion, the derivative will be dispensed with an infusion bottle containing sterile pharmaceutical grade 'Water for Injection' . • Where the derivative is to be administered by injection the derivative is dispensed with an ampoule of sterile water for injection. The injectable or infusable composition will be made up by mixing the ingredients prior to administration. The quantity of material administered will depend upon the amount of fibrinolysis required and the speed with which it is required, the seriousness of the thromboembolic condition and position and size of the clot. The precise dose to be employed and mode of administration must per force in view of the nature of the complaint be decided according to the circumstances -ifi OKMPI by the physician supervising treatment. However, in general, a patient being treated for a mature thrombus will generally receive a daily dose of from 0.10 to 2.0 mg/kg-1 of body weight either by injection in up to five doses or by infusion. Accordingly, in a further aspect of the invention there is provided a method of treating thrombotic diseases, which comprises administering to the sufferer an effective non-toxic amount of the derivative of the invention. The following Methods and Examples illustrate the invention. OVrl Method a) Assay of fibrinolytic activity in the bloodstream of rabbits Male New Zealand White rabbits (2.5-3.0kg) were anaesthetised with 0.1 ml/kg s.c. Fentanyl/Fluanisone ('Hypnorm', Janssen Pharmaceuticals, Belgium) and anaesthesia maintained with the same dose at hourly intervals. An ear was shaved and the agent under test (2.0-3.0 ml) injected in the lateral ear vein. Blood samples (2.0ml) were removed from the central ear vessel at the following intervals: (pre- ose), iπmediately post-dose and 10, 20, 30, 45, 60, 90, 120, and 180 minutes post-dose. 1.8ml of blood was added to 0.2ml 3.8% w/v trisodium citrate and centrifuged to obtain plasma. Platelet-poor plasma (0.5ml) was added to cold water (9.0ml) , mixed and 1% v/v acetic acid in water (0.1ml) added. After mixing, the acidified plasma was allowed to stand on ice for 30 min and then centrifuged (c. 2,000 g/4°C/10 min) to obtai a euglobulin precipitate) . The precipitate was dissolved in 0.1 M triethar-Olamine. HC1 pH 8.0 (0.2 ml) and 25λ of this solution applied to fibrin plates. Fibrin plates were prepared from 0.4% w/v human fibrinogen (KabiVitrum, Sweden) in 0.05 M sodium barbitone 0.45% w/v NaCl pH 7.4 (10ml) on 10 x 10 cm square plastic dishes, clotting with bovine throiribin (c. 10 NIH units, Parke-Davis, UK) . Plates were incubated at 26°C for 16-18 hr (occasionally longer if zones of lysis did not develop adequately) and stained with aqueous Bromophenol Blue. Zones of lysis were measured with Vernier calipers and the area calculated from the mean of perpendicular diameters. OMPI b) Assay of fibrinolytic activity in the bloodstream of rats Male Sprague-Dawley rats (300-400 g) were anaesthetized with pentobarbitone sodium (60 mg/kg i.p.). One carotid artery was cannulated for collection of blood samples. One femoral vein was cannulated for injection of heparn (50 U/kg) and coπpound under test. Approximately 5 min after heparinization, a pre-dose blood sample (0.8 ml) was taken and mixed with 0.1 volumes 129 irM trisodiu citrate. The compound under test was then injected (1 ml kg) over 10s. Further blood sairples were taken exactly 1, 2, 4, 8, 16, 30 and 60 min later. Heparin treatment (50 U/kg) was repeated after # the 30 min saπple to maintain cannula patency. All citrated blood samples were kept on ice until the erd of each experiment, then centrifuged at 1700 g for 15 min at 4 to obtain plasma. The euglohulin fraction was precipitated by adding 0.1 ml each plasma to 1.82 L ice-cold 0.011% (v/v) acetic acid in water. After 30 min standing in ice, all tubes were centrifuged at 1700 g for 15 min at 4°. The supernatants were poured away, the inner walls of each tube carefully wiped dry and each precipitate redissolved in 0.4 ml 0.1 M triethanolamine HC1 buffer, pH 8.0, containing 0.05% (w/v) sodium azide. Aliquots (20 yl) were then .applied to fibrin plates in quadruplicate. Fibrin plates were prepared from 0.4% (w/v) human fibrinogen (Kabi, Grade L, Flow Laboratories, Scotland) dissolved in 0.029 M barbitone in 125 irM NaCl, pH 7.4, pipetted.(9 ml) into 10 x 10 cm square plastic dishes (Sterilin) and clotted by rapid lT-i-xing with 0.3 ml bovine thrombin (50 NUi units/ml, Parke-Davis, UK). Plates were incubated at 37° for 18-24h usually, but longer if required, and stained with aqueous hrxxco phenol blue. Ptor each lysis zone, two diameters perpendicular to each other were ireasured using Vernier calipers. All diameters for each sample were averaged, and this mean converted to fibrinolytic activity by reference to a calibration curve. ■ The latter was obtained by adding .. known amounts of the coπpound under test-to a stock of plasma pooled from at least ten rats. These standards were processed using the same methods and at the same time as the experimental sairples. To construct the calibration curve, diameters (mm) were plotted against log 1Q concentration of compound. The plasma concentration of coirpound in each experimental saπple was expressed as a percentage of that expected on the basis of the dose given and the assuirption of 35 ml plasma/kg body weight for each rat. L " x ample 1 4-aminobenzoylated periodate-oxidised low molecular weight urokinase (AB/POLUK) Urokinase (Abbokinase, Abbott Laboratories, USA) with a molecular, weight of approximately 33,000 daltons (2 vials, 5 x 10 international units) was dissolved in 0.1. sodium phosphate, 0.145 M NaCl, O.OISJ Tween 80, pH 6.0 (1.0 ml). The solution was gel filtered into the same buffer in order to remove excipient annitol. A small column (PD 10, Pharmacia, Sweden) of Sephade G-25 M was used and the protein eluate volume was 3.0 ml. 2.0 ml or this eluate was treated with 0.2 ml of freshly prepared 0.1 M sodium periodate in water Tor 70 min on ice in the dark. The reaction mixture was quenched with 0.2 ml glyccrol Tor 30 min on ice, followed by neat ethanolamine (2.5 ul). The above gel filtration was repeated and the activity of the eluate was measured spectrophotometrically using the chromogenic substrate S-2444 (Kabi Vitrum, Sweden, 0.25 irfl in 0.1 M triethanolamine. HC1 pH 8.0, 25°C) and the recovery of amidolytic activity was 37.5?ό. This eluate (1.5 ml) was treated with 4-aminobenzoic acid 4'-amidinophenyl ester HC1 (15 yl of a 100 mM solution in dimethyl sulphoxide) for 30 min at ambient | temperature (c. 25 C). The amidolytic activity of the product indicated that the enzyme was at least 9855 acylatεd. Samples of AB/P0-UK prepared in this way were given to rabbits and the circulating fibrinolytic activity determined, as described above. Fig. 1 shows the time dependence of fibrin plate lysis following doses of: (1) unmodified urokinase; (2) urokinase modified only by acylation; (3) urokinase modified only by periodate oxidation; . (4) doubly modified urokinase (AP/P0LUK).. Five animals were used in each group. The figure shows that the clearance of at least part of the doubly modified enzyme is appreciably slower than that of either unmodified or singly modified urokinases. . • j ' JΕEX ' OMPI Example 2 Periodate-oxidised 4-aminobenzoylated low molecular weight urokinase (PO/ABLUK) Urokinase (Abbokinase, Abbott Laboratories, USA, 2. .x-10 - international units) was dissolved in 0.1 M triethanolamine HC1 pH 8.0 (1.0 ml) and treated with 0.5 mM 4-aminσbenzoic acid.4'-amidihopheny3 ester HC1 for 4 hr on ice, after which time acylation was essentially complete'as judged by amidolytic assay with S-2444 (see above). The solution was gel filtered as described in Example 1 into 0.1 H sodium phosphate 0.145 M NaCl 0.01SJ Tween 80 pH 6.0 (3.0 ml). 1.0 ml . of this solution was retained and the remaining 20 ml treated with sodium periodate as described in Example 1. After quenching of the reaction "and gel filtration as previously described, a portion of the periodate-treated acyl-enzyme was deacylated (0.1 M disodium hydrogen phosphate pH 8.0, 165 min, 37°C) in comparison with a portion of the retained unoxidised acyl-enzyme. Assay with substrate S-2444 indicated a recovery of approximately 252 of the original enzyme activity after the oxidation procedure. Example 3 -aminobenzoylated periodate oxidised high molecular weight urokinase (AB/P0HUK) Urokinase (Serono Pharmaceuticals, Germany) with a molecular weight of 54,000 daltons (3 x 10 units) was dissolved in 2.5 ml of 0.1 M sodium phosphate, 0.01% Tween 80, pH 6.0 and gel filtered into 3.0 ml of the same buffer as described in Example 1." 2.0 ml of the eluate was treated with sodium periodate as described in Example 1 except that. oxidation was carried out for 90 min on ice..- Quenching and gel s filtration were performed as described in Example 1 and amidolytic assay (S-2444) indicated an activity recovery of 56%. . The final .. . eluate (1.5 ml) was treated with 1.0 mM 4-aminobenzoic βcid 4'- • : . a idinophenyl ester. HC1 for lh at 0°C. Amidolytic ' assay indicated essentially complete " acylation and the preparation was stored at -40 C. Example 4 '• 4-aminobenzoylated periodate-oxidised human ' tissue "plasminogen activator (AB/PO TPA) . • • • * * Human tissue plasminogen activator (TPA) was obtained from the culture filtrate of Bowes melanoma cells and was purified to 70-90% homogeneity by standard chromatographic procedures. The activator was dissolved in 0.1 M sodium phosphate, 0.145 ' M NaCl, 0.01% Tween 80 ' , pH 6.0 to a concentration of 0.5-1.0 mg/ml.. Oxidation with sodium • periodate and subsequent quenching and gel. filtration were carried out as described in Example 1 except.that oxidation was performed at 4 C for 1 hr. Enzyme activity was measured using substrate S-2288 (Kabi Vitrum, Sweden) under the conditions described for S-2444. During oxidation enzyme activity dropped to 70% of initial but returned to 100% of initial after-treatment with glycerol and ethanolamine. The eluate was bought-to pH 8.0 by"addition of 1/10th of the volume of 1.0 M trishydroxyrnethylaminomethane and 4-aminobenzoic.acid 4'-amino- benzoic acid HC1 (50 mM in dime hylsulphoxide) added to a final • * concentration of 1.0 mM. After lh at 0 W C, acylation was essentiall complete and excess acylating agent was removed by gel filtration βs previously described. The potential enzyme activity in the acyl-enzyme was checked by allowing a portion of the final eluate to deacylate at pH 7.5 for 16 hr and 25 C and measuring the activity using S-2288. Samples of AB/P0-TPA prepared in this way were administered to rabbits and the circulating fibrinolytic activity determined as described above. Figure 2 compares the time dependence of lysis ex vivo, following doses of: (1) unmodified TPA; (2) TPA modified only by acylation; (3) TPA modified only, by periodate oxidation and (4) doubly modified TPA. ' The results indicate that clearance of the•doubly modified enzyme is appreciably slower than clearance of the unmodified or singly modified formes. EXAMPLE 5 CLEARANCE OF AB/PO t-PA FROM THE BLOODSTREAM OF RATS The clearance of AB/PO t-PA (prepared as in Example 4) was also studied in rats using a calibrated fibrin plate assay of plasma fibrinolytic activity (see Methods). The results are shown in Figure 3. Unmodified t-PA was cleared rapidly and the plasma concentration/time curve approximated to that expected of two parallel first order processes; one with a half-life of about 1.5 min accounted for about 98% of the administered dose and the second accounted for 2% of the dose and was characterised by a half-life of about 12.5 min. When AB/PO t-PA was administered to rats, approximately 80% of the administered dose was cleared rapidly but about 20% was cleared with a half-life of about 45 min. Thus, the double modification achieved a 10-fold increase in the proportion of enzyme that was slowly cleared and increased the half-life of that proportion 3-4 fold.. OMPI

Claims: CLAIMS 1. A derivative of a fibrinolytically active glycoprotein in which at least part of the carbohydrate portion of the protein is absent or has been degraded, characterised in that the catalytic site essential for fibrinolytic activity is blocked by a group which is removable by hydrolysis at a rate such that the pseudo- first order rate constant for hydrolysis is in the range 10-6 sec"l to 10 _ 3 sec""l in isotonic aqueous media at pH 7.4 at 34θC. 2. A derivative according to claim 1, in which the glycoprotein is tissue plasminogen activator or urokinase. 3. A derivative according to claim 1 or claim 2, in which the blocking group is a substituted benzoyl group. 4. A derivative according to claim 3, in which the blocking group is benzoyl substituted with amino or guanidino. 5. A process of preparing a derivative according to any one " of claims 1 to 4, which comprises, in either order, modifying a fibrinolytically active glycoprotein to remove or degrade carbohydrate, and reacting the glycoprotein with a blocking agent AB in which A is a locating group which locates the agent in the catalytic site, and B is an acyl group. OMPI 6. A pharmaceutical composition comprising a derivative according to any one of claims 1 to 4, together with a pharmaceutically acceptable carrier. 7. A derivative according to any one of claims 1 to 4, for use in treating thrombotic diseases in human or non-human animals. 8. A method for treating thrombotic diseases in human or non-human animals, which comprises treating an animal with ari effective non-toxic amount of a derivative according to any one of claims 1 to 4.

Inventors: ROBINSON JEFFERY HUGH, SMITH RICHARD ANTHONY GODWIN, ROBINSON, JEFFERY, HUGH, SMITH, RICHARD, ANTHONY, GODWIN
Applicants: BEECHAM GROUP PLC, BEECHAM GROUP P.L.C.
Application year: 1983
Main classification: C12N9
IPC codes: A61K38, C12N9