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WO1988001175A1 - Co-facteur de prourokinase - Google Patents

Co-facteur de prourokinase Download PDF

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Publication number
WO1988001175A1
WO1988001175A1 PCT/US1987/001945 US8701945W WO8801175A1 WO 1988001175 A1 WO1988001175 A1 WO 1988001175A1 US 8701945 W US8701945 W US 8701945W WO 8801175 A1 WO8801175 A1 WO 8801175A1
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WO
WIPO (PCT)
Prior art keywords
pro
cofactor
fibrin
column
affinity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1987/001945
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English (en)
Inventor
Victor E. Gurewich
Ralph Pannell
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Individual
Original Assignee
Individual
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Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of WO1988001175A1 publication Critical patent/WO1988001175A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator

Definitions

  • This invention relates to the therapeutic lysis of fibrin blood clots (thrombi) in human patients.
  • urokinase (UK), which is believed to be synthesized in vivo as a single-chain, zymogenic form (pro-UK), which can be converted to the more active two-chain form by a specific proteolytic cleavage.
  • Pro-UK is normally administered by infusion for the lysis of coronary thrombi, in a dosage of 30-60 mg/hr for approximately one hour to achieve thrombus lysis, and then administered for approximately three hours in a dosage of 10-20 mg/hr to prevent re-occlusion.
  • the invention features a method of increasing the efficacy of pro-UK-induced thrombolysis in a human patient to whom pro-UK is administered, the method involving administering to the patient, in addition to the pro-UK, a cofactor of pro-UK and UK, the cofactor being characterized in that a) it has a molecular weight of about 50,000; b) it increases the affinity of pro-UK for fibrin; c) it confers, on the UK generated from pro-UK, affinity for fibrin; and d) it confers, on the UK generated from pro-UK, thrombolyti ⁇ activity which is fib in-dependent.
  • the pro-UK cofactor of the invention because of the above-cited characteristics, can enhance the therapeutic efficacy of pro-UK and thus permit lower dosages to be used.
  • the increased binding affinity for fibrin (of which thrombi are composed) conferred on pro-UK and urokinase by the cofactor of the invention can target the pro-UK and its activated derivative more tightly to the thrombus it is administered to dissolve.
  • the cofactor may enhance specificity by reducing non-specific plasminogen activation, and thus deleterious, non-specific effects are reduced.
  • the clearance time (T. /2 ) of the pro-UK cofactor complex may be greater than that of pro-UK alone.
  • the fibrin affinity of pro-UK or UK and cofactor-bound pro-UK (cfb-pro-UK) or UK was compared by affinity chromatography on a fibrin/Celite column prepared by mixing Celite (Fischer Scientific Co.) with 2 percent human fibrinogen (Kabi) in 25 ml buffer (0.05M NaP0 4 , 0.1 M NaCl, ImM EDTA, pH 7.4) and then precipitating a fibrin matrix onto the Celite by slow addition of 100 units of thrombin (Parke Davis) with constant stirring.
  • the fibrin-matrix was packed on a column and washed with 10 volumes of equilibration buffer (0.05 M NaP0 4 , 0.3 M NaCl, 1 M EDTA, pH 7.4).
  • pro-UK or UK purified from human kidney cell culture
  • cfb-pro-UK or UK were loaded on the column, run in equilibration buffer, and fractions collected and assayed for fibrinolytic activity in a standard fibrin plate assay.
  • Cfb-pro-UK or cfb-UK in urine when loaded on the column, bound to a significantly greater degree to the fibrin affinity column than pro-UK alone or UK alone.
  • the thrombolyti ⁇ activity of cfb-pro-UK and of cfb-UK remained bound until an elution buffer containing 0.2M arginine was run on the column.
  • the UK-cofactor complex in concentrated urine is devoid of amidolytic activity.
  • the cofactor of the invention is a naturally occurring compound which can be isolated from human urine or plasma. It is believed to be identical to the Mr ⁇ 50,000 urokinase inhibitor purified from urine by Stump et al. JBC 261:12759-12766, 1986, hereby incorporated by reference.
  • the cofactor can be isolated and produced by itself and then mixed with pro-UK to form a complex which will bind more tightly to fibrin than pro-UK alone.
  • the cofactor can be isolated from urine or plasma by the published method for isolating the inhibitor (Stump et al., id.).
  • the procedure for isolating the compound from urine given in Stump et al. is as follows. Fresh human urine is collected on benzamidine, cooled to 4° C, and adjusted to pH 7.5 with NaOH and cent ifuged at 6000 X g for 30 min at 4° C. The supernatant is applied to a 10 X 15-cm column of zinc chelate-Sepharose at a flow rate of 500 ml/h.
  • the column is washed with 1 liter of 0.3 M NaCL, 0.02 M NaH 2 P0 4 buffer, pH 7.5, containing 5 mM benzamidine, stirred, and washed with an additional 2 liters of the same buffer.
  • the column is then eluted with the same buffer, containing 0.05 M imidazole, and 20-ml fractions are collected.
  • Fractions containing protein are pooled, adjusted to pH 7.5 with 1 M HCl, and applied directly at a flow rate of 20 ml/h to a 2 X 6.5-cm column of concanavalin A-Sepharose equilibrated with 0.15 M NaCl, 0.02 M NaH 2 P0 4 buffer, pH 7.5, containing 5 mM benzamidine.
  • the column is washed first with 100 ml of equilibration buffer and then with equilibration buffer without benzamidine until the absorbance at 280 nm is below 0.05 (5 to 10 column volumes).
  • the column is then eluted with 0.15 M NaCl, 0.02 M NaH 2 P0 4 buffer, pH 7.5, containing 0.5 M methyl- ⁇ -D-glucopyranoside at a flow rate of 10 ml/h, collecting fractions of 5 ml.
  • Fractions containing protein are pooled and dialyzed against 0.02 M NaCl, 0.02M aH 2 P0 4 buffer, pH 6.8, to a conductance equal to that of the dialysis buffer.
  • the dialyzed sample is then applied to a 0.9 X 13-cm column of SP-Sephadex C-50, equilibrated with dialysis buffer, at a flow rate of 5 ml/h.
  • the column is washed with the same buffer until the absorbance at 280 nm is less than 0.05 and then a linear 60-ml NaCl gradient from Q.02 to 0.5 M is applied.
  • Two-ml fractions, containing the inhibitor/cofactor compound, as measured by ELISA, are pooled and concentrated to 2 ml by dialysis against solid PEG 20,000. This sample is dialyzed against 1 liter of 0.3 M NaCl, 0.02 M NaH 2 P0 4 buffer, pH 7.5, and applied to a 1.5 X 90-cm column of Sephadex G-100 superfine at a flow rate of 4 ml/h. Two-ml fractions are assayed for inhibitor/cofactor, and enriched fractions are pooled.
  • the first step chro atography of cooled, pH-adjusted, human urine on zinc chelate-Sepharose results in 75% absorption of inhibitor/cofactor. Elution with imidazole results in a recovery of 50% of the original protein with a 30-fold volume reduction and a 10-fold purification. Direct application of this eluate to concanavalin A-Sepharose gives 90% absorption of inhibitor/cofactor whereas free urokinase is not bound. Elution with 0.5 M methyl- ⁇ -D-glucopyranoside results in a recovery of 43 percent of the original starting inhibitor/cofactor, with an additional 4-fold purification and 10-fold volume reduction.
  • the cofactor and pro-UK are administered together, either as a complex or separately so that the complex forms in vivo, to dissolve thrombi in patients who have recently suffered a thromboembolic event (e.g., coronary thrombi, deep vein thrombi, pulmonary emboli, and cerebral and peripheral vascular thrombi).
  • Pro-UK and the cofactor are admixed with a pharmaceutically acceptable carrier substance, e.g., saline, and administered intravenously.
  • a pharmaceutically acceptable carrier substance e.g., saline
  • lyophilized pro-UK and a molar equivalent of lyophilized cofactor are mixed together after solubilization in saline and placed in the chamber of a syringe, which is used to inject the resulting cfb-pro-UK bolus into the patient intravenously.
  • lyophilized pro-UK and a molar equivalent of lyophilized cofactor dissolved in saline are infused at the same time, together or via separate infusion lines, intravenously over a period of about 1 hour, followed by intravenous infusion of about 5 mg/hr pro-UK and a molar equivalent of cofactor over a period of about three more hours.
  • Example 4 For infusion treatment for the slow lysis of deep vein thrombi, about 2-5 mg/hr of lyophilized pro-UK and a molar equivalent of lyophilized pro-UK dissolved in saline are infused, at the same time, together or via separate infusion lines, intravenously over a period of about 12-24 hours.
  • Example 5 For infusion treatment for the slow lysis of deep vein thrombi, about 2-5 mg/hr of lyophilized pro-UK and a molar equivalent of lyophilized pro-UK dissolved in saline are infused, at the same time, together or via separate infusion lines, intravenously over a period of about 12-24 hours.
  • Example 5 For infusion treatment for the slow lysis of deep vein thrombi, about 2-5 mg/hr of lyophilized pro-UK and a molar equivalent of lyophilized pro-UK dissolved in saline are infused, at the same time, together or via separate infusion lines, intravenously over a period of
  • Pro-UK and cofactor in a 1:1 molar ratio are dissolved together in saline and the solution then lyophilized to provide lyophilized cfb-pro-UK which is easily transported and stored prior to reconstitution, with saline, and use.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Procédé permettant d'accroître l'efficacité d'une thrombolyse induite par une prourokinase chez un patient auquel une prourokinase est administrée. Ce procédé consiste à administrer au patient, en plus de la prourokinase, un co-facteur de prourokinase et d'urokinase, le co-facteur se caractérisant en ce que a) il présente un poids moléculaire de 50 000 environ; b) il accroît l'affinité de la prourokinase pour la fibrine; c) il confère à l'urokinase produite par la prourokinase une affinité pour la fibrine; d) il confère à l'urokinase produite par la prourokinase une activité thrombolytique dépendant de la fibrine.
PCT/US1987/001945 1986-08-12 1987-08-11 Co-facteur de prourokinase Ceased WO1988001175A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US89588486A 1986-08-12 1986-08-12
US895,884 1986-08-12
US883787A 1987-01-30 1987-01-30
US008,837 1987-01-30

Publications (1)

Publication Number Publication Date
WO1988001175A1 true WO1988001175A1 (fr) 1988-02-25

Family

ID=26678686

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1987/001945 Ceased WO1988001175A1 (fr) 1986-08-12 1987-08-11 Co-facteur de prourokinase

Country Status (2)

Country Link
EP (1) EP0281579A4 (fr)
WO (1) WO1988001175A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4029767A (en) * 1971-09-24 1977-06-14 Choay S.A. Pharmaceutical compositions of stable urokinase-heparin complexes and methods for use thereof
US4106992A (en) * 1971-09-24 1978-08-15 Choay S.A. Purification of urokinase

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61111684A (ja) * 1984-11-05 1986-05-29 Terumo Corp 精製されたウロキナ−ゼ複合体の製造方法
EP0208785B1 (fr) * 1985-01-14 1992-12-30 Shiseido Company Limited Complexe d'urokinase fibrinophile et procede de preparation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4029767A (en) * 1971-09-24 1977-06-14 Choay S.A. Pharmaceutical compositions of stable urokinase-heparin complexes and methods for use thereof
US4106992A (en) * 1971-09-24 1978-08-15 Choay S.A. Purification of urokinase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP0281579A4 *
The Journal of Biological Chemistry, Volume 261, No. 27, 25 September 1986, DAVID C. STUMP et al, "Purification and Characterization of a Novel Inhibitor of Urokinase from Human Urine" pages 12759-12766. *

Also Published As

Publication number Publication date
EP0281579A1 (fr) 1988-09-14
EP0281579A4 (fr) 1989-01-19

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