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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6456—Plasminogen activators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• 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 10 -6 sec -1 to 10 -3 sec -1 .
• 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 in vivo stability.
• the term 'fibrinolytically active glycoprotein' means any glycoprotein which demonstrates jLn. vivo fibrinolytic activity as defined in the above mentioned published European Patent Application, and includes glycoproteins which are obtainable from mammalian urine, blood or tissues or by recombinant DNA methods and which can activate plasminogen. Examples of these include melanoma plasminogen activator, the extraction of which is described in Published European Patent Application No, 41766, and urokinase.
• 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 to sec ⁇ "l in isotonic aqueous media at pH 7.4 at 37oc.
• Preferred glycoproteins are tissue activators, such as melanoma plasminogen activator, and urokinase, and preferably substantially all the carbohydrate in the glycoprotein is removed or degraded.
• Suitable blocking groups are those described in Published European Patent Application No. 0 009 879, and preferred groups are acyl groups, particularly optionally substituted benzoyl groups.
• the most preferred benzoyl groups are those substituted with a basic moiety, such as amino or guanidino.
• 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.
• 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,
• 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).
• glycosidases selected for example from ⁇ -galactosidase, ⁇ -mannosidase, ⁇ -fucosidase, ⁇ -N-acetylglucosaminidase or neuraminidase, or one or more endoglycosidases, such as endoglycosidase D (from Diplococcus pneumoniae) or endoglycosidase H (from Streptomyces plicatus).
• the glycosidases may be immobilised on insoluble carriers to avoid contamination of the modified glycoprotein.
• melanoma plasminogen activator preparation from cultured melanoma cells grown in the presence of an inhibitor of protein glycosylation, such as tunicamycin.
• Step (b) of the preparation is preferably carried out by reacting the modified glycoprotein from step (a) with a blocking agent
• A is a locating group which locates the agent in the catalytic site
• B is an acyl group.
• B is a 2- or 4-aminobenzoyl group otionally substituted in the aromatic ring by an electron donating moiety.
• 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.
• 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.
• A is 4-amidinophenyl
• a suitable protecting group is the tertiary-butoxycarbonyl (BOC) group.
• BOC tertiary-butoxycarbonyl
• 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 aminobenzoic 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 (trif ⁇ uoroacetic acid), preferably at room temperature.
• the derivative of this invention is preferably administered as a pharmaceutical compostion.
• the present invention also provides a pharmaceutical composition
• 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.
• compositions for intravenous administration are solutions of the sterile derivative in sterile isotonic aqueous buffer.
• 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.
• 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.
• the derivative will be dispensed with an infusion bottle containing sterile pharmaceutical grade 'Water for Injection'.
• 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 by the physician supervising treatment.
• 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.
• a method of treating thrombotic diseases which comprises administering to the sufferer an effective non-toxic amount of the derivative of the invention.
• pre-dose i ⁇ mediately post-dose and 10, 20, 30, 45, 60, 90, 120, and 180 minutes post-dose.
• 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. b) Assay of fibrinolytic activity in the bloodstream of rats
• mice 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 heparin (50 U/kg) and compound under test. Approximately 5 min after heparinization, a pre-dose blood sample (0.8 ml) was taken and mixed with 0.1 volumes 129 mM trisodium citrate. The compound under test was then injected (1 ml/kg) over 10s. Further blood samples were taken exactly 1, 2, 4, 8, 16, 30 and 60 min later.
• Heparin treatment 50 U/kg was repeated after the 30 min sample to maintain cannula patency. All citrated blood samples were kept on ice until the end of each experiment, then centrifuged at 1700 g for 15 min at 4o to obtain plasma. The euglohulin fraction was precipitated by adding 0.1 ml each plasma to 1.82 mL 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°.
• 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 mM NaCl, pH 7.4, pipetted (9 ml) into 10 x 10 cm square plastic dishes (Sterilin) and clotted by rapid mixing with 0.3 ml bovine thrombin (50 NIH units/ml, Parke-Davis, UK) . Plates were incubated at 37° for 18-24h usually, but longer if required, and stained with aqueous bromophenol blue. For each lysis zone, two diameters perpendicular to each other were ireasured using Vernier calipers.
• Urokinase (Abbokinase, Abbott Laboratories, USA) with a molecular weight of approximately 33,000 daltons (2 vials, 5 x 10 5 international units) was dissolved in 0.1 M sodium phosphate, 0.145 M NaCl, 0.01% Tween 80, pH 6.0 (1.0 ml). The solution was gel filtered into the same buffer in order to remove excipient mannitol. A small column (PD 10, Pharmacia, Sweden) of Sephadex 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 for 70 min on ice in the dark.
• the reaction mixture was quenched with 0.2 ml glycerol for 30 min on ice, followed by neat ethanolamine (2.5 ⁇ l).
• 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 mM in 0.1 M triethanolamine. HCl pH 8.0, 25°C) and the recovery of amidolytic activity was 37.5%.
• This eluate (1.5 ml) was treated with 4-aminobenzoic acid 4'-amidinophenyl ester HCl (15 ⁇ l of a 100 mM solution in dimethyl sulphoxide) for 30 min at ambient temperature (c. 25oC).
• the amidolytic activity of the product indicated that the enzyme was at least 98% acylated.
• 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/POLUK). 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.
• Example 2 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.
• Urokinase (Abbokinase, Abbott Laboratories, USA, 2.5 x 10 5 international units) was dissolved in 0.1 M triethanolamine HCl pH 8.0 (1.0 ml) and treated with 0.5 mM 4-aminobenzoic acid 4'-amidihophenyl ester HCl 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 M sodium phosphate 0.145 M NaCl 0.01% 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.
• 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 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 acid 4'- amidinophenyl ester. HCl for lh at 0°C. Amidolytic assay indicated essentially complete acylation and the preparation was stored at -40oC.
• 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 4oC for 1 hr. Enzyme activity was measured using substrate S-2288 (Kabi Vitrum, Sweden) under the conditions described for S-2444.
• TPA Human tissue plasminogen activator
• 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'-aminobenzoic acid HCl (50 mM in dimethylsulphoxide) added to a final concentration of 1.0 mM.
• acylation was essentially complete and excess acylating agent was removed by gel filtration as 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 25oC and measuring the activity using S-2288.

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• 1983
  • 1983-11-10 EP EP19830903560 patent/EP0125269A1/de not_active Withdrawn
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