WO1993014199A1 - A pump-1 catalyzed method of making low molecular weight urokinase-type plasminogen activator - Google Patents
A pump-1 catalyzed method of making low molecular weight urokinase-type plasminogen activator Download PDFInfo
- Publication number
- WO1993014199A1 WO1993014199A1 PCT/US1993/000146 US9300146W WO9314199A1 WO 1993014199 A1 WO1993014199 A1 WO 1993014199A1 US 9300146 W US9300146 W US 9300146W WO 9314199 A1 WO9314199 A1 WO 9314199A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- molecular weight
- urokinase
- low molecular
- plasminogen activator
- type plasminogen
- Prior art date
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- 210000002700 urine Anatomy 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 1
Classifications
-
- 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
- C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to a method of preparing low molecular weight urokinase-type plasminogen activator comprising reacting high molecular weight urokinase-type plasminogen activator with a catalytic amount of a metalloproteinase known as pump-1.
- Urokinase is a proteolytic enzyme that acts as a plasminogen activator by catalyzing the conversion of plasminogen to plas in. Because of its thrombolytic activity, urokinase has clinical use in the treatment of coronary artery thrombosis, pulmonary embolism and other thromboembolic diseases..
- Urokinase is a two-peptide-chain (chain A and chain B) serine protease of about 411 amino acid residues having an apparent molecular weight of about 53,000 Daltons (53 kD) .
- urokinase comprises a growth factor domain and a kringle domain.
- Urokinase is formed from a 53 kD, single-chain precursor molecule (zymogen) designated pro-urokinas . Formation of urokinase from pro- urokinase occurs by a plasmin catalyzed cleavage of pro- urokinase between a lysine amino acid residue located at position 158.
- the 53 kD forms of urokinase and pro-urokinase are referred to generally as high-molecular-weight urokinase and pro-urokinase, respectively.
- Both urokinase and pro-urokinase can also be isolated in a low molecular weight form.
- Two different low molecular weight forms of urokinase have been characterized.
- One form, isolated from human urine, has an A-chain amino-terminal lysine (Lys) residue and is produced by cleavage of the Lys(135)-Lys(136) bond of high molecular weight-urokinase by either a slow reaction with plasmin or urokinase autolysis.
- Lys A-chain amino-terminal lysine
- a second low molecular weight form has been isolated from cell culture and has an amino-terminal leucine residue.
- This second low molecular weight form of urokinase is formed from high molecular weight urokinase or high molecular weight pro-urokinase, respectively, via cleavage of the glutamic acid (Glu)143-leucine (Leu)144 bond of the high molecular weight forms.
- Glu glutamic acid
- Leu Leu
- the enzyme responsible for synthesis of this second form has not previously been identified.
- a two-chain urokinase having an A-chain amino- terminal beginning with Leu(144) is the active principle in the thro bolytic enzyme (Abbokinase ® ) produced by Abbott Laboratories.
- Abbokinase ® A low molecular weight pro- urokinase with an amino-terminus of Leu(144) has also been isolated from culture of the lung adenocarcinoma cell line CALU-3.
- Pump-1 is a member of a family of enzymes that catalyze the proteolytic degradation of the fibrous structural proteins of the extra-cellular matrix and, thus, are known as "matrix etalloproteinases".
- Pump-1 has been identified and isolated from cancer cells and shown to catalyze the proteolytic cleavage of casein, fibronectin, type IV collagen and several types of gelatin. See, e.g. , Quantin et al.. Biochemistry. 28:5327 (1989) and Miyazaki et al. , Cancer Research, 50:7758 (1990).
- the present invention is directed to the use of pump-1 to cleave pro-urokinase or urokinase at the Glu (143)-Leu (144) bond to form low molecular weight pro- urokinase or low molecular weight urokinase.
- the present invention relates to a method of making low molecular weight urokinase-type plasminogen activator comprising the steps of:
- the high molecular weight urokinase-type plasminogen activator is selected from high molecular weight urokinase or high molecular weight pro-urokinase.
- the high molecular weight urokinase-type plasminogen activator is high molecular weight pro-urokinase
- the low molecular weight urokinase-type plasminogen activator is low molecular weight pro-urokinase and the method further comprises the steps of: (c) admixing the low molecular weight pro- urokinase of step (b) with a catalytic amount of plasmin to form a second reaction mixture; and
- the pump-1 used in the method of the present invention is a zymogen form of pump-1.
- the liquid medium contains a catalytic amount of a suitable metalloproteinase such as p-aminophenylmercuric acetate or oxidized glutathione.
- the liquid medium used in the method of the present invention is preferably an aqueous buffer having a pH value of from about 7.5 to about 8.5 and containing pump-1 stimulating amounts of calcium and zinc ions and inhibitory amounts of a plasmin inhibitor and a urokinase inhibitor.
- a preferred plasmin inhibitor is aprotinin and a preferred urokinase inhibitor is amiloride.
- the present invention relates to low molecular weight urokinase-type plasminogen activator prepared by the method of this invention.
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising an effective amount of the low molecular weight urokinase-type plasminogen activator prepared by the method of this invention.
- the present invention relates to a method of thrombolysis or preventing thrombosis in a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of low molecular weight urokinase-type plasminogen activator prepared by the method of this invention.
- the present invention also relates to a method of increasing the conversion of plasminogen to plasmin in a patient in need of such conversion comprising administering to said patient a therapeutically effective amount of low molecular weight urokinase-type plasminogen activator prepared by the method of this invention.
- the present invention relates to a method of making low molecular weight urokinase-type plasminogen activator. That method involves cleaving high molecular weight urokinase-type plasminogen activator at the Glu(143)-Leu(144) bond of that high molecular weight urokinase-type plasminogen activator. Cleaving of the Glu(143)-Leu(144) bond is accomplished with the metalloproteinase known as putative metalloproteinase-1, referred to herein as pump-1.
- putative metalloproteinase-1 putative metalloproteinase-1
- urokinase-type plasminogen activator means pro-urokinase or urokinase whether made, in part or in whole, by recombinant, cell culture or other means.
- high molecular weight urokinase-type plasminogen activator is admixed in a liquid medium with a catalytic amount of pump-1 to form a reaction mixture and that reaction mixture is maintained under biological reaction conditions for a time period sufficient for the pump-1 to catalyze cleavage of the Glu(143)-Leu(144) bond of the high molecular weight urokinase-type plasminogen activator and form low molecular weight urokinase-type plasminogen activator.
- the formed low molecular weight urokinase- type plasminogen activator is low molecular weight urokinase.
- the formed low molecular weight urokinase- type plasminogen activator is low molecular weight pro- urokinase.
- Low molecular weight urokinase can also be made by "the method of this invention using high molecular weight pro-urokinase as the high molecular weight urokinase- type plasminogen activator substrate for pump-1.
- high molecular weight pro- urokinase is admixed in a liquid medium with a catalytic amount of pump-1 to form a reaction mixture and that reaction mixture is maintained under biological reaction conditions for a time period sufficient for the pump-1 to catalyze cleavage of the Glu(143)-Leu(144) bond of the high molecular weight pro-urokinase and form low molecular weight pro-urokinase.
- the formed low molecular weight pro-urokinase is then admixed with a catalytic amount of plasmin to form a second reaction mixture and that second reaction mixture is maintained under biological reaction conditions for a time period sufficient for the plasmin to cleave the low molecular weight pro-urokinase and form low molecular weight urokinase.
- the high molecular weight forms of urokinase and pro- urokinase used as substrates for pump-1 can be isolated from kidney cells [Stump et al. , J. Biol. Chem. , 261:1274 (1980)] or obtained in recombinant form from host cells that express those proteins. Lo et al.
- the high molecular weight substrates are purified prior to use in the present method.
- purification is accomplished by standard protein purification methods well known to those of skill in the art.
- Exemplary purification methods are ammonium sulfate precipitation, chromatographic separation, gel filtration and electrophoresis.
- Pump-1 used in the method of the present invention is isolated and purified from cells or tissues known to contain that enzyme or obtained in recombinant form from host cells that express pump-1. See, e.g. , Miyazaki et al.. Cancer Research. 50:7758 (1990) and Quantin et al.. Biochemistry. 28:5327 (1989).
- pump-1 can be isolated from a serum-free conditioned medium of a culture of the human rectal carcinoma cell line Car-1.
- Pump-1 is isolated and purified from the conditioned media using ammonium sulfate precipitation, molecular sieve column chromatography, anion-exchange chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) . Miyazaki et al., Cancer Research, 50:7758 (1990) .
- recombinant pump-1 or its zymogen can be obtained from Cos cells transformed with an expression vector containing a DNA sequence encoding pump-1 or its zymogen.
- Suitable expression vectors are pKCR3 and pPROTA. Quantin et al., Biochemistry. 28:5327 (1989) .
- pump-1 is isolated and partially purified from human kidney cells (See Example 1 hereinafter) .
- human kidney cells HEK
- HEK human kidney cells
- Pump-1 is extracted from the culture medium by loading conditioned culture media (clarified by centrifugation and adjusted to pH 7.0) onto a cation exchange resin such as a column of beaded agarose that had been pre-equilibrated with 0.05 M MOPS (3-[N- Morpholino] propanesulfonic acid)/ ⁇ .l M NaCl/0.01% Tween-80 (polyoxyethylenesorbitan)/pH 7.0. After loading, the column is washed three times with the same buffer and the proteins are eluted with a gradient of the same buffer and 0.05 M MOPS/1.0 M NaCl/0.01 Tween- 80/pH 7.5.
- a cation exchange resin such as a column of beaded agarose that had been pre-equilibrated with 0.05 M MOPS (3-[N- Morpholino] propanesulfonic acid)/ ⁇ .l M NaCl/0.01% Tween-80 (polyoxyethylenesorbitan)/pH 7.0.
- Aprotinin (TrasylolTM, Bayer, FRG) is added to the column fractions, and fractions with enzyme activity are pooled, concentrated by ultrafiltration, and dialyzed against 50 mM TRIS HCl/0.1 M NaCl/0.01% Tween-80/pH 7.5. The dialyzed material is applied to a column of zinc- chelated sepharose and the proteins eluted with a gradient of 0-10 mM imidazole in that same dialysis buffer.
- Fractions containing enzyme activity are pooled, concentrated, and applied to a column of cross-linked dextran. Using this method, pump-1 is purified over 1000 fold from the starting culture media.
- the enzyme activity is the property of a soluble protease, as it remains in the supernatant upon centrifugation, and it appears in the lower molecular weight fraction eluted from an ion- exchange chromatography column.
- the pump-1 used in the method of the present invention does not have to be completely purified.
- pump-1 can be extracted from tissue or culture media as a precursor or zymogen. That pump-1 zymogen can also be used in the method of the present invention.
- the zymogen form of pump-1 is used, however, it is necessary to include a catalytic amount of a suitable metalloproteinase activator in the liquid medium.
- exemplary metalloproteinase activators are p_- aminophenylmercuric acetate and oxidized glutathione.
- Pump-1 is present in a catalytic amount.
- catalytic amount means that amount of pump-i at least sufficient to catalyze, in a non-rate-limiting manner, the conversion of high molecular weight pro- urokinase or urokinase to the respective low molecular weight forms of pro-urokinase or urokinase.
- the catalytic amount of a pump-1 varies according to the purity of pump-1, the concentration of high molecular weight substrate (pro-urokinase or urokinase) as well as to reaction conditions such as temperature, time and pH value. Means for determining the catalytic amount of a pump-1 under preselected substrate concentrations and reaction conditions are well known to those of skill in the art.
- Pump-1 concentration is typically expressed in activity Units.
- One activity Unit catalyzes the formation of l ⁇ mol of low molecular weight product at a given temperature (typically 37°C) and pH value (typically about 7.5 to about 8.5) per minute.
- 10 Units of pump-l is a catalytic amount of that enzyme where 10 ⁇ mols of high molecular weight substrate are converted to 10 ⁇ mols of low molecular weight product in one minute at a temperature of 37°C and a pH value of from 7.5 to about 8.5.
- Admixing comprises mixing each listed ingredient with each of the other ingredients in a suitable aqueous solvent to form a reaction mixture. The reaction mixture is then maintained under biological reaction conditions of temperature, pH value, solvent osmolality, ionic composition and ambient atmosphere for a period of time sufficient to cleave the high molecular weight substrate.
- Temperature can range from about 15°C to about 0°C. Preferably temperature is from about 30°C to about 40°C and, more preferably about 37°C.
- the pH value can range from about 6.0 to about 11.0.
- the pH value is from about 6.5 to about 8.5 and, more preferably about 7.5 to about 8.50.
- the pH value is maintained by buffers in the aqueous solvent. The selection of a buffer is based on the ability of the buffer to maintain pH value at the desired level. Where the pH value is about 8.0, a preferred buffer is TRIS.
- the buffer is also designed to contain cofactors necessary for pump-1 activity.
- the buffer contains zinc and calcium ions, both of which ions are known to stimulate pump-1 catalytic activity.
- calcium and zinc ions are present in the form of anionic salts such as CaCl 2 or ZnCl 2 .
- the concentration of those ions can range from about 0.1 to about 1.0 M.
- the buffer is preferably devoid of EDTA, EGTA and other chelators that bind calcium or zinc.
- the aqueous buffer preferably contains inhibitors of plasmin activity as well as inhibitors of urokinase activity.
- a preferred plasmin inhibitor is aprotinin and a preferred urokinase inhibitor is amiloride. The concentrations of such inhibitors is dependent upon the concentrations of high molecular weight substrate and pump-1 in the reaction mixture.
- aprotinin is preferably present at a concentration of about 10 KlU/ml and amiloride is present at a concentration of about 1 mM.
- the reaction mixture is maintained for time period sufficient for pump-1 to cleave the Glu(143)-Leu(144) bond of the high molecular weight urokinase or pro- urokinase substrate.
- the time period varies with the biological reaction conditions as well as the concentrations of the substrate and pump-1 used.
- the time period ranges from about 1 to about 120 hours and, preferably from about 12 to about 96 hours.
- the method of the present invention preferably further comprises isolating the formed low molecular weight urokinase or pro-urokinase.
- Isolating comprises recovering the formed compound from the reaction mixture.
- Means for recovering the formed urokinase compound include gel filtration, column chromatography, paper chromatography, affinity chromatography, extraction, precipitation and the like.
- the recovered low molecular weight pro-urokinase product may be converted to low molecular weight urokinase. That conversion is accomplished by admixing the recovered low molecular weight pro- urokinase with a catalytic amount of plasmin to form a second reaction mixture and maintaining that second reaction mixture under biological reaction conditions for a time period sufficient for the plasmin to catalyze the conversion of low molecular weight pro-urokinase to low molecular weight urokinase.
- the formed low molecular weight urokinase is preferably recovered from the second reaction mixture using the same recovery methods as set forth above.
- the low molecular weight urokinase-type plasminogen activators formed by the method of the present invention have use as therapeutic agents in the treatment of thromboembolic disorders.
- both low molecular weight pro-urokinase and low molecular weight urokinase have utility as thrombolytic agents as well as agents that prevent thrombosis.
- the use of those compounds parallels the use of currently available formulations of urokinase such as ABBOKINASE ® , available from Abbott Laboratories. Physicians Desk Reference, 45 th Edition, published by E.R. Barnhart, Medical Economics Data (1991) .
- the present invention contemplates a method of thrombolysis or a method of preventing blood clot formation comprising administering to that patient a therapeutically effective amount of low molecular weight urokinase-type plasminogen activator (low molecular weight urokinase or low molecular weight pro-urokinase) prepared by the pump-1 catalyzed synthetic method of this invention.
- Low molecular weight urokinase can be prepared from either high molecular weight pro-urokinase or high molecular weight urokinase as disclosed hereinbefore.
- a therapeutically effective amount of the low molecular weight urokinase- type plasminogen activator used in the methods of the present invention can be calculated from a knowledge of the activity (Units/ g) of that low molecular weight urokinase-type plasminogen activator and the known effective doses of currently available form of urokinase such as ABBOKINASE*.
- a priming dose of about 4,400 International Units per kilogram of body weight (4,400 IU/ kg) of ABBOKINASE ® is administered in a volume of about 15 over a time period of about 10 minutes. This priming dose is followed by a continuous infusion at a rate of about 4,400 IU/kg/hour in a volume of about 15 ml/hour.
- urokinase is used to treat coronary artery thrombosis
- a dose of about 6,000 IU/minute is infused into the occluded artery at a rate of 4 ml/minute for a time period of about 120 minutes.
- Low molecular weight urokinase-type plasminogen activators catalyze the conversion of plasminogen to plasmin.
- the present invention further contemplates a method of increasing the conversion of plasminogen to plasmin in a patient in need of such conversion comprising administering to said patient a therapeutically effective amount of low molecular weight urokinase-type activator by the pump-1 catalyzed synthetic method of this invention.
- compositions of the present invention comprise physiologically acceptable carriers and low molecular weight urokinase-type plasminogen activators prepared by the pump-1 catalyzed method of this invention.
- the present invention thus, further contemplates low molecular weight urokinase-type plasminogen activator formulated into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are collectively referred to herein as carriers, for parenteral injection.
- compositions suitable for such administration may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
- suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like) , suitable mixtures thereof.
- compositions may also contain adjuvants such as preserving, bulking and stabilizing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid> and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like.
- the compositions can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
- compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration.
- the selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
- Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the required daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
- HEK Human kidney cells
- Proteins were eluted from the column by running a linear gradient comprised of 1 liter equilibration buffer and 1 liter of 50 mM MOPS/1 M NaCl/0.01% Tween- 80/pH 7.5. Fifteen ml fractions were collected in culture tubes and aprotinin (an inhibitor of trypsin and other similar proteinases) was added to each tube at a final concentration of 20 KlU/ l. Those fractions showing enzyme activity (fractions 60 - 85) were pooled and concentrated by ultrafiltration (A icon stirred cell, PM-10 membrane) .
- the concentrated fractions were then dialyzed against 20 mM Tris HC1/500 mM NaCl/pH 7.5 and the dialyzed fractions loaded onto a 2.2 x 30 cm column of Cibacron- blue agarose (Sigma Chem. Co.), which had been equilibrated with the dialysis buffer.
- the agarose column was washed with 100 ml of the equilibration bu fer and proteins were eluted by the application of a linear gradient of 500 ml of the equilibration buffer and 500 ml of 20 mM Tris HCl/1 M NaCl/pH 7.5.
- the recovered protein was found to be active in converting high molecular weight urokinase to low molecular weight urokinase on an analytical scale.
- Example 2 Production of low molecular weight pro-urokinase fLeu (144)-Leu(411-1.
- Proteins were isolated from the reaction mixture using a column of Sephadex G-75, which was equilibrated and developed using the same buffer. Two major products were identified. One product was identified as low molecular weight pro-urokinase [Leu(144-Leu(411) ] . The other major product was identified as an amino-terminal fragment [Ser(l)-Glu(143) ]) of pro-urokinase. Identification was based on analysis of molecular weight and amino terminal amino acid residue sequences.
- Pro-urokinase or Recombinant Urokinase (high molecular weight two-chain enzyme) .
- High molecular weight recombinant pro-urokinase or high molecular weight urokinase (2mg/ml) was incubated with various concentrations of partially purified pump- 1, prepared in accordance with the procedures of Example 1, in 50 mM Tris HCl/100 mM NaCl/0.01% Tween-80/pH 7.5 for 17 hr at 37 C.
- the concentration of pump-l was varied over a 600-fold range to assess its activity in conversion of the high molecular weight substrates to their corresponding low molecular weight forms.
- the reaction of high molecular weight pro-urokinase with pump-l produced two single-chain products: (a) an amino terminal fragment of urokinase [Ser(l) to Glu(143) ] comprised of the growth factor and kringle domains; and (b) low molecular weight pro-urokinase [Leu(144) to Leu(411) ] .
- the formed low molecular weight pro-urokinase can be further cleaved by plasmin to form low molecular weight urokinase.
- the pump-l catalyzed cleavage of the Glu(143)- Leu(144) bond of high molecular weight pro-urokinase or high molecular weight urokinase occurs with high site- specificity, as the only significant other reaction observed is a slow hydrolysis of the Glu(3)-Leu(4) bond. This side reaction can be minimized by monitoring the time course of cleavage of high molecular weight pro- urokinase by pump-l.
- Pump-l cleaves either natural or recombinant high molecular weight pro-urokinase or high molecular weight urokinase into two fragments.
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Abstract
The present invention provides a method of making low molecular weight urokinase-type plasminogen activator comprising cleaving high molecular weight urokinase-type plasminogen activator with the metalloproteinase pump-1. Pharmaceutical compositions and methods of thrombolysis or preventing blood clot formation using that low molecular weight urokinase-type plasminogen activator are also provided.
Description
A PUMP-1 CATALYZED METHOD OF MAKING LOW MOLECULAR WEIGHT UROKINASE-TYPE PLASMINOGEN ACTIVATOR
Description
Technical Field of the Invention
The present invention relates to a method of preparing low molecular weight urokinase-type plasminogen activator comprising reacting high molecular weight urokinase-type plasminogen activator with a catalytic amount of a metalloproteinase known as pump-1.
Background of the Invention Urokinase is a proteolytic enzyme that acts as a plasminogen activator by catalyzing the conversion of plasminogen to plas in. Because of its thrombolytic activity, urokinase has clinical use in the treatment of coronary artery thrombosis, pulmonary embolism and other thromboembolic diseases..
Urokinase is a two-peptide-chain (chain A and chain B) serine protease of about 411 amino acid residues having an apparent molecular weight of about 53,000 Daltons (53 kD) . In addition to a serine protease domain, urokinase comprises a growth factor domain and a kringle domain. Urokinase is formed from a 53 kD, single-chain precursor molecule (zymogen) designated pro-urokinas . Formation of urokinase from pro- urokinase occurs by a plasmin catalyzed cleavage of pro- urokinase between a lysine amino acid residue located at position 158. and an isoleucine residue located at position 159 of pro-urokinase. The 53 kD forms of urokinase and pro-urokinase are referred to generally as high-molecular-weight urokinase and pro-urokinase, respectively.
Both urokinase and pro-urokinase can also be isolated in a low molecular weight form. Two different low
molecular weight forms of urokinase have been characterized. One form, isolated from human urine, has an A-chain amino-terminal lysine (Lys) residue and is produced by cleavage of the Lys(135)-Lys(136) bond of high molecular weight-urokinase by either a slow reaction with plasmin or urokinase autolysis. See, e.g.. Gunzler et al., Hoppe-Seγler's Z Physiol. Chem.. 363:122 (1982); Barlow et al., Thromb. Res.. 23:541 (1981); and Stoppelli et al., Proc. Nat'l. Acad. Sci. USA. 82:4939 (1985).
A second low molecular weight form has been isolated from cell culture and has an amino-terminal leucine residue. This second low molecular weight form of urokinase is formed from high molecular weight urokinase or high molecular weight pro-urokinase, respectively, via cleavage of the glutamic acid (Glu)143-leucine (Leu)144 bond of the high molecular weight forms. See, e.g. Hommandberg et al., Biochim. Biophvs. Actaf 1038 (1990); and Stump et al. , J. Biol. Chem.. 261:17120 (1986) . The enzyme responsible for synthesis of this second form has not previously been identified.
A two-chain urokinase having an A-chain amino- terminal beginning with Leu(144) is the active principle in the thro bolytic enzyme (Abbokinase®) produced by Abbott Laboratories. A low molecular weight pro- urokinase with an amino-terminus of Leu(144) has also been isolated from culture of the lung adenocarcinoma cell line CALU-3. Stump et al. , J. Biol. Chem. , 261:17120 (1986). Pump-1 is a member of a family of enzymes that catalyze the proteolytic degradation of the fibrous structural proteins of the extra-cellular matrix and, thus, are known as "matrix etalloproteinases". Pump-1 has been identified and isolated from cancer cells and shown to catalyze the proteolytic cleavage of casein,
fibronectin, type IV collagen and several types of gelatin. See, e.g. , Quantin et al.. Biochemistry. 28:5327 (1989) and Miyazaki et al. , Cancer Research, 50:7758 (1990). The present invention is directed to the use of pump-1 to cleave pro-urokinase or urokinase at the Glu (143)-Leu (144) bond to form low molecular weight pro- urokinase or low molecular weight urokinase.
Brief Summary of the Invention
In one aspect, the present invention relates to a method of making low molecular weight urokinase-type plasminogen activator comprising the steps of:
(a) admixing high molecular weight urokinase- type plasminogen activator in a liquid medium with a catalytic amount of pump-1 to form an a reaction mixture; and
(b) maintaining said reaction mixture under biological reaction conditions for a time period sufficient for said pump-1 to cleave the Glu(143)-
Leu(144) bond of said high molecular weight urokinase- type plasminogen activator and form said low molecular weight urokinase-type plasminogen activator.
In a preferred embodiment, the high molecular weight urokinase-type plasminogen activator is selected from high molecular weight urokinase or high molecular weight pro-urokinase.
In yet another preferred embodiment, the high molecular weight urokinase-type plasminogen activator is high molecular weight pro-urokinase, the low molecular weight urokinase-type plasminogen activator is low molecular weight pro-urokinase and the method further comprises the steps of:
(c) admixing the low molecular weight pro- urokinase of step (b) with a catalytic amount of plasmin to form a second reaction mixture; and
(d) maintaining the second reaction mixture under biological reaction conditions for a time period sufficient for said plasmin to cleave the low molecular weight pro-urokinase and form low molecular weight urokinase.
In another embodiment, the pump-1 used in the method of the present invention is a zymogen form of pump-1.
Where such a zymogen form of pump-1 is used, the liquid medium contains a catalytic amount of a suitable metalloproteinase such as p-aminophenylmercuric acetate or oxidized glutathione. The liquid medium used in the method of the present invention is preferably an aqueous buffer having a pH value of from about 7.5 to about 8.5 and containing pump-1 stimulating amounts of calcium and zinc ions and inhibitory amounts of a plasmin inhibitor and a urokinase inhibitor. A preferred plasmin inhibitor is aprotinin and a preferred urokinase inhibitor is amiloride.
In another aspect, the present invention relates to low molecular weight urokinase-type plasminogen activator prepared by the method of this invention.
In yet another aspect, the present invention relates to a pharmaceutical composition comprising an effective amount of the low molecular weight urokinase-type plasminogen activator prepared by the method of this invention.
In a further aspect, the present invention relates to a method of thrombolysis or preventing thrombosis in a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of low molecular weight urokinase-type
plasminogen activator prepared by the method of this invention.
The present invention also relates to a method of increasing the conversion of plasminogen to plasmin in a patient in need of such conversion comprising administering to said patient a therapeutically effective amount of low molecular weight urokinase-type plasminogen activator prepared by the method of this invention.
Detailed Description of the Invention Synthetic Methods
In one aspect, the present invention relates to a method of making low molecular weight urokinase-type plasminogen activator. That method involves cleaving high molecular weight urokinase-type plasminogen activator at the Glu(143)-Leu(144) bond of that high molecular weight urokinase-type plasminogen activator. Cleaving of the Glu(143)-Leu(144) bond is accomplished with the metalloproteinase known as putative metalloproteinase-1, referred to herein as pump-1.
As used herein, the term urokinase-type plasminogen activator" means pro-urokinase or urokinase whether made, in part or in whole, by recombinant, cell culture or other means.
According to the method of the present invention, high molecular weight urokinase-type plasminogen activator is admixed in a liquid medium with a catalytic amount of pump-1 to form a reaction mixture and that reaction mixture is maintained under biological reaction conditions for a time period sufficient for the pump-1 to catalyze cleavage of the Glu(143)-Leu(144) bond of the high molecular weight urokinase-type plasminogen activator and form low molecular weight urokinase-type plasminogen activator.
Where the high molecular weight urokinase-type plasminogen activator is high molecular weight urokinase, the formed low molecular weight urokinase- type plasminogen activator is low molecular weight urokinase.
Where the high molecular weight urokinase-type plasminogen activator is high molecular weight pro- urokinase, the formed low molecular weight urokinase- type plasminogen activator is low molecular weight pro- urokinase.
Low molecular weight urokinase can also be made by" the method of this invention using high molecular weight pro-urokinase as the high molecular weight urokinase- type plasminogen activator substrate for pump-1. In accordance with that method, high molecular weight pro- urokinase is admixed in a liquid medium with a catalytic amount of pump-1 to form a reaction mixture and that reaction mixture is maintained under biological reaction conditions for a time period sufficient for the pump-1 to catalyze cleavage of the Glu(143)-Leu(144) bond of the high molecular weight pro-urokinase and form low molecular weight pro-urokinase. The formed low molecular weight pro-urokinase is then admixed with a catalytic amount of plasmin to form a second reaction mixture and that second reaction mixture is maintained under biological reaction conditions for a time period sufficient for the plasmin to cleave the low molecular weight pro-urokinase and form low molecular weight urokinase. The high molecular weight forms of urokinase and pro- urokinase used as substrates for pump-1 can be isolated from kidney cells [Stump et al. , J. Biol. Chem. , 261:1274 (1980)] or obtained in recombinant form from host cells that express those proteins. Lo et al. , Biochem, Biophys, Acta. , 1088:217 (1991).
Preferably, the high molecular weight substrates are purified prior to use in the present method. Such purification is accomplished by standard protein purification methods well known to those of skill in the art. Exemplary purification methods are ammonium sulfate precipitation, chromatographic separation, gel filtration and electrophoresis.
Pump-1 used in the method of the present invention is isolated and purified from cells or tissues known to contain that enzyme or obtained in recombinant form from host cells that express pump-1. See, e.g. , Miyazaki et al.. Cancer Research. 50:7758 (1990) and Quantin et al.. Biochemistry. 28:5327 (1989).
By way of example, pump-1 can be isolated from a serum-free conditioned medium of a culture of the human rectal carcinoma cell line Car-1. Pump-1 is isolated and purified from the conditioned media using ammonium sulfate precipitation, molecular sieve column chromatography, anion-exchange chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) . Miyazaki et al., Cancer Research, 50:7758 (1990) .
By way of further example, recombinant pump-1 or its zymogen can be obtained from Cos cells transformed with an expression vector containing a DNA sequence encoding pump-1 or its zymogen. Suitable expression vectors are pKCR3 and pPROTA. Quantin et al., Biochemistry. 28:5327 (1989) .
In a preferred embodiment, pump-1 is isolated and partially purified from human kidney cells (See Example 1 hereinafter) . Briefly, human kidney cells (HEK) are cultured in roller bottles in a suitable medium.
Pump-1 is extracted from the culture medium by loading conditioned culture media (clarified by centrifugation and adjusted to pH 7.0) onto a cation
exchange resin such as a column of beaded agarose that had been pre-equilibrated with 0.05 M MOPS (3-[N- Morpholino] propanesulfonic acid)/θ.l M NaCl/0.01% Tween-80 (polyoxyethylenesorbitan)/pH 7.0. After loading, the column is washed three times with the same buffer and the proteins are eluted with a gradient of the same buffer and 0.05 M MOPS/1.0 M NaCl/0.01 Tween- 80/pH 7.5.
Aprotinin (Trasylol™, Bayer, FRG) is added to the column fractions, and fractions with enzyme activity are pooled, concentrated by ultrafiltration, and dialyzed against 50 mM TRIS HCl/0.1 M NaCl/0.01% Tween-80/pH 7.5. The dialyzed material is applied to a column of zinc- chelated sepharose and the proteins eluted with a gradient of 0-10 mM imidazole in that same dialysis buffer.
Fractions containing enzyme activity are pooled, concentrated, and applied to a column of cross-linked dextran. Using this method, pump-1 is purified over 1000 fold from the starting culture media. The enzyme activity is the property of a soluble protease, as it remains in the supernatant upon centrifugation, and it appears in the lower molecular weight fraction eluted from an ion- exchange chromatography column.
The pump-1 used in the method of the present invention does not have to be completely purified. By way of example, pump-1 can be extracted from tissue or culture media as a precursor or zymogen. That pump-1 zymogen can also be used in the method of the present invention. Where the zymogen form of pump-1 is used, however, it is necessary to include a catalytic amount of a suitable metalloproteinase activator in the liquid medium. Exemplary metalloproteinase activators are p_- aminophenylmercuric acetate and oxidized glutathione.
Pump-1 is present in a catalytic amount. As used herein, "catalytic amount" means that amount of pump-i at least sufficient to catalyze, in a non-rate-limiting manner, the conversion of high molecular weight pro- urokinase or urokinase to the respective low molecular weight forms of pro-urokinase or urokinase. The catalytic amount of a pump-1 varies according to the purity of pump-1, the concentration of high molecular weight substrate (pro-urokinase or urokinase) as well as to reaction conditions such as temperature, time and pH value. Means for determining the catalytic amount of a pump-1 under preselected substrate concentrations and reaction conditions are well known to those of skill in the art. Pump-1 concentration is typically expressed in activity Units. One activity Unit catalyzes the formation of lμmol of low molecular weight product at a given temperature (typically 37°C) and pH value (typically about 7.5 to about 8.5) per minute. Thus, 10 Units of pump-l is a catalytic amount of that enzyme where 10 μmols of high molecular weight substrate are converted to 10 μmols of low molecular weight product in one minute at a temperature of 37°C and a pH value of from 7.5 to about 8.5. Admixing comprises mixing each listed ingredient with each of the other ingredients in a suitable aqueous solvent to form a reaction mixture. The reaction mixture is then maintained under biological reaction conditions of temperature, pH value, solvent osmolality, ionic composition and ambient atmosphere for a period of time sufficient to cleave the high molecular weight substrate.
The selection of particular conditions depends primarily upon the amount of low molecular weight pro- urokinase or urokinase to be formed. Temperature can
range from about 15°C to about 0°C. Preferably temperature is from about 30°C to about 40°C and, more preferably about 37°C.
The pH value can range from about 6.0 to about 11.0. Preferably, the pH value is from about 6.5 to about 8.5 and, more preferably about 7.5 to about 8.50. The pH value is maintained by buffers in the aqueous solvent. The selection of a buffer is based on the ability of the buffer to maintain pH value at the desired level. Where the pH value is about 8.0, a preferred buffer is TRIS. The buffer is also designed to contain cofactors necessary for pump-1 activity. In this regard, the buffer contains zinc and calcium ions, both of which ions are known to stimulate pump-1 catalytic activity. Preferably, calcium and zinc ions are present in the form of anionic salts such as CaCl2 or ZnCl2. The concentration of those ions can range from about 0.1 to about 1.0 M. The buffer is preferably devoid of EDTA, EGTA and other chelators that bind calcium or zinc. The aqueous buffer preferably contains inhibitors of plasmin activity as well as inhibitors of urokinase activity. A preferred plasmin inhibitor is aprotinin and a preferred urokinase inhibitor is amiloride. The concentrations of such inhibitors is dependent upon the concentrations of high molecular weight substrate and pump-1 in the reaction mixture. Where the concentration of pro-urokinase is about 175 mg/ml and the concentration of partially purified pump-1 is about 3.5 mg/ml, aprotinin is preferably present at a concentration of about 10 KlU/ml and amiloride is present at a concentration of about 1 mM.
The reaction mixture is maintained for time period sufficient for pump-1 to cleave the Glu(143)-Leu(144) bond of the high molecular weight urokinase or pro- urokinase substrate. The time period varies with the
biological reaction conditions as well as the concentrations of the substrate and pump-1 used. Preferably, the time period ranges from about 1 to about 120 hours and, preferably from about 12 to about 96 hours.
The method of the present invention preferably further comprises isolating the formed low molecular weight urokinase or pro-urokinase. Isolating comprises recovering the formed compound from the reaction mixture. Means for recovering the formed urokinase compound include gel filtration, column chromatography, paper chromatography, affinity chromatography, extraction, precipitation and the like.
Where high molecular weight pro-urokinase is used as the substrate, the recovered low molecular weight pro- urokinase product may be converted to low molecular weight urokinase. That conversion is accomplished by admixing the recovered low molecular weight pro- urokinase with a catalytic amount of plasmin to form a second reaction mixture and maintaining that second reaction mixture under biological reaction conditions for a time period sufficient for the plasmin to catalyze the conversion of low molecular weight pro-urokinase to low molecular weight urokinase. The formed low molecular weight urokinase is preferably recovered from the second reaction mixture using the same recovery methods as set forth above.
Therapeutic Methods The low molecular weight urokinase-type plasminogen activators formed by the method of the present invention have use as therapeutic agents in the treatment of thromboembolic disorders. In particular, both low molecular weight pro-urokinase and low molecular weight urokinase have utility as thrombolytic agents as well as
agents that prevent thrombosis. The use of those compounds parallels the use of currently available formulations of urokinase such as ABBOKINASE®, available from Abbott Laboratories. Physicians Desk Reference, 45th Edition, published by E.R. Barnhart, Medical Economics Data (1991) .
Thus, the present invention contemplates a method of thrombolysis or a method of preventing blood clot formation comprising administering to that patient a therapeutically effective amount of low molecular weight urokinase-type plasminogen activator (low molecular weight urokinase or low molecular weight pro-urokinase) prepared by the pump-1 catalyzed synthetic method of this invention. Low molecular weight urokinase can be prepared from either high molecular weight pro-urokinase or high molecular weight urokinase as disclosed hereinbefore.
Determination of a therapeutically effective amount is to be made by skilled medical professionals using currently available guidelines. Physicians Desk
Reference, 45th Edition, published by E.R. Barnhart, Medical Economics Data (1991) . A therapeutically effective amount of the low molecular weight urokinase- type plasminogen activator used in the methods of the present invention can be calculated from a knowledge of the activity (Units/ g) of that low molecular weight urokinase-type plasminogen activator and the known effective doses of currently available form of urokinase such as ABBOKINASE*. By way of example, to treat pulmonary embolism, a priming dose of about 4,400 International Units per kilogram of body weight (4,400 IU/ kg) of ABBOKINASE® is administered in a volume of about 15 over a time period of about 10 minutes. This priming dose is followed by a
continuous infusion at a rate of about 4,400 IU/kg/hour in a volume of about 15 ml/hour.
Where urokinase is used to treat coronary artery thrombosis, a dose of about 6,000 IU/minute is infused into the occluded artery at a rate of 4 ml/minute for a time period of about 120 minutes. Physicians Desk Reference, 45th Edition, published by E.R. Barnhart, Medical Economics Data (1991) .
Low molecular weight urokinase-type plasminogen activators catalyze the conversion of plasminogen to plasmin. In this regard, the present invention further contemplates a method of increasing the conversion of plasminogen to plasmin in a patient in need of such conversion comprising administering to said patient a therapeutically effective amount of low molecular weight urokinase-type activator by the pump-1 catalyzed synthetic method of this invention.
Compositions Pharmaceutical compositions of the present invention comprise physiologically acceptable carriers and low molecular weight urokinase-type plasminogen activators prepared by the pump-1 catalyzed method of this invention. The present invention, thus, further contemplates low molecular weight urokinase-type plasminogen activator formulated into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are collectively referred to herein as carriers, for parenteral injection.
The compositions can generally be administered to humans and animals intravenously, intraarterially or by catheter. Compositions suitable for such administration may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or
emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like) , suitable mixtures thereof.
These compositions may also contain adjuvants such as preserving, bulking and stabilizing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid> and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. The compositions can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
Actual dosage levels of the active ingredient in the compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration. The selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the required daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of
administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
The following examples further illustrate the invention and are not to be construed as limiting of the specification and claims in any way.
EXAMPLES
Example l: Preparation of Pump-1.
Human kidney cells (HEK) were cultured in roller bottles for 21 days to a state of confluency. Eighteen liters of 21 day harvest culture medium was clarified by centrifugation and the pH adjusted to between 6.5 and 7.0. The clarified media was pumped onto a 4.4 x 39 cm column of beaded agarose (S-Sepharose, Sigma Chem. Co., St. Louis, MO) at a flow rate of 400 ml/hour. The S- sepharose had been previously equilibrated with an equilibration buffer comprising 50 mM MOPS/100 mM NaCl/O.01% Tween-80/pH 7.0. After loading the column, the column was washed with 2 liters of the same equilibration buffer.
Proteins were eluted from the column by running a linear gradient comprised of 1 liter equilibration buffer and 1 liter of 50 mM MOPS/1 M NaCl/0.01% Tween- 80/pH 7.5. Fifteen ml fractions were collected in culture tubes and aprotinin (an inhibitor of trypsin and other similar proteinases) was added to each tube at a final concentration of 20 KlU/ l. Those fractions showing enzyme activity (fractions 60 - 85) were pooled and concentrated by ultrafiltration (A icon stirred cell, PM-10 membrane) .
The concentrated fractions were then dialyzed against 20 mM Tris HC1/500 mM NaCl/pH 7.5 and the dialyzed fractions loaded onto a 2.2 x 30 cm column of Cibacron-
blue agarose (Sigma Chem. Co.), which had been equilibrated with the dialysis buffer. The agarose column was washed with 100 ml of the equilibration bu fer and proteins were eluted by the application of a linear gradient of 500 ml of the equilibration buffer and 500 ml of 20 mM Tris HCl/1 M NaCl/pH 7.5. Eleven ml fractions were collected and those with enzyme activity (fractions 50-90) were pooled and concentrated by ultrafiltration (Amicon stirred cell, PM-10 membrane) . The concentrated fractions from the Cibacron-blue agarose column (0.1 mL) were acidified by the addition of 10 μL of 10 percent (v/v) trifluoroacetic acid (TFA) and injected onto an analytical high performance liquid chromatography (HPLC) column (VYDAC C-4, Cat. # 214TP54) that had been pre-equilibrated and washed with 80% water/20% acetonitrile (0.1 percent trifluoroacetic acid) at 1 ml/min. Proteins were eluted from the column with a gradient of increasing acetonitrile concentration, with the final conditions being 50% acetonitrile/50% water (0.1% trifluoroacetic acid).
A major peak of protein was eluted from the column and concentrated by evaporation in vacuo. That protein corresponded to the zymogen form of "pump-l" as demonstrated by its molecular weight (29,000 daltons) and its amino-terminal amino acid residue sequence. Quantin et al.. Biochemistry. 28:5327 (1989).
Where aprotinin was omitted from the fractions collected upon elution of the S-Sepharose column and the Cibacron-blue agarose and C-4 HPLC columns were performed as described above, a second major protein component eluted from the C-4 column slightly before the "pump-l" zymogen. That second protein had a molecular weight of about 20,000 daltons and corresponded to an active form of "pump-l" as determined from its amino-
terminal amino acid residue sequence. Quantin et al., Biochemistry, 28:5327 (1989).
The recovered protein was found to be active in converting high molecular weight urokinase to low molecular weight urokinase on an analytical scale.
Example 2: Production of low molecular weight pro-urokinase fLeu (144)-Leu(411-1.
Ten mg of recombinant high molecular weight pro- urokinase [obtained from a culture of murine hybridoma cells as described by Lo et al., Biochim. Biophys. Acta. 1088:217 (1991)] was dissolved in 2 ml 50 mM Tris HCl/100 mM NaCl/0.01% Tween-80/pH 8.0 and added to 0.2 ml of 10 mM amiloride, 0.1 ml 20 mM CaCl2, 0.02 ml of 10,000 KIU/mL aprotinin, and 0.22 ml of partially purified "pump-l" prepared in accordance with the procedures of Example 1 to form a reaction mixture. The reaction mixture was maintained at 37 C for 4 days at which point the cleavage of recombinant pro-urokinase was >90% complete.
Proteins were isolated from the reaction mixture using a column of Sephadex G-75, which was equilibrated and developed using the same buffer. Two major products were identified. One product was identified as low molecular weight pro-urokinase [Leu(144-Leu(411) ] . The other major product was identified as an amino-terminal fragment [Ser(l)-Glu(143) ]) of pro-urokinase. Identification was based on analysis of molecular weight and amino terminal amino acid residue sequences.
Example 3: Reaction of Pump-l with Recombinant
Pro-urokinase or Recombinant Urokinase (high molecular weight two-chain enzyme) .
High molecular weight recombinant pro-urokinase or high molecular weight urokinase (2mg/ml) was incubated with various concentrations of partially purified pump- 1, prepared in accordance with the procedures of Example 1, in 50 mM Tris HCl/100 mM NaCl/0.01% Tween-80/pH 7.5 for 17 hr at 37 C. The concentration of pump-l was varied over a 600-fold range to assess its activity in conversion of the high molecular weight substrates to their corresponding low molecular weight forms. After the incubation, a sample was analyzed by SDS-PAGE (20% Pharmacia Phast Gel, stained with Coomassie-blue) to - determine the extent of low molecular weight product formation. At all pump-l concentrations tested, the extent of conversion of the high molecular weight substrates to the low molecular weight products was similar (within a factor of 2) , demonstrating that pump-l is equally effective in reaction with both high molecular weight pro-urokinase and high molecular weight urokinase. The reaction of high molecular weight pro-urokinase with pump-l produced two single-chain products: (a) an amino terminal fragment of urokinase [Ser(l) to Glu(143) ] comprised of the growth factor and kringle domains; and (b) low molecular weight pro-urokinase [Leu(144) to Leu(411) ] . The formed low molecular weight pro-urokinase can be further cleaved by plasmin to form low molecular weight urokinase.
The pump-l catalyzed cleavage of the Glu(143)- Leu(144) bond of high molecular weight pro-urokinase or high molecular weight urokinase occurs with high site- specificity, as the only significant other reaction observed is a slow hydrolysis of the Glu(3)-Leu(4) bond. This side reaction can be minimized by monitoring the time course of cleavage of high molecular weight pro- urokinase by pump-l.
Pump-l cleaves either natural or recombinant high molecular weight pro-urokinase or high molecular weight urokinase into two fragments.
The foregoing is intended as illustrative of the present invention but not limiting. Numerous variations and modifications can be affected without departing from the true spirit and scope of the invention.
Claims
1. A method of making low molecular weight urokinase-type plasminogen activator comprising the steps of:
(a) admixing high molecular weight urokinase- type plasminogen activator in a liquid medium with a catalytic amount of pump-l to form an a reaction mixture; and (b) maintaining said reaction mixture under biological reaction conditions for a time period sufficient for said pump-l to cleave the Glu(143)- Leu(144) bond of said high molecular weight urokinase- type plasminogen activator and form said low molecular weight urokinase-type plasminogen activator.
2. The method according to claim 1 wherein said high molecular weight urokinase-type plasminogen activator is selected from high molecular weight urokinase or high molecular weight pro-urokinase.
3. The method according to claim 1 wherein said high molecular weight urokinase-type plasminogen activator is high molecular weight pro-urokinase and said low molecular weight urokinase-type plasminogen activator is low molecular weight pro-urokinase further comprising the steps of:
(c) admixing said low molecular weight pro- urokinase of step (b) with a catalytic amount of plasmin to form a second reaction mixture; and
(d) maintaining said second reaction mixture under biological reaction conditions for a time period sufficient for said plasmin to cleave said low molecular weight pro-urokinase and form low molecular weight urokinase.
4. The method according to claim 1 wherein the pump-l is a zymogen form of pump-l and the liquid medium contains a catalytic amount of a suitable
5 metalloproteinase activator.
_•
5. The method according to claim 1 wherein said liquid medium comprises an aqueous buffer containing pump-l stimulating amounts of calcium and zinc ions and
10 inhibitory amounts of a plasmin inhibitor and a urokinase inhibitor.
6. The method according to claim 5 wherein said plasmin inhibitor is aprotinin and said urokinase
15 inhibitor is amiloride.
7. The method according to claim 5 wherein said aqueous buffer has a pH value of from about 7.5 to about 8.5.
20
8. Low molecular weight urokinase-type plasminogen activator prepared by the method of claim 1.
9. A pharmaceutical composition comprising an
25 effective amount of the low molecular weight urokinase- type plasminogen activator prepared by the method of claim 1.
10. A method of lysing or preventing the formation 30 of blood clots in a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of low molecular weight urokinase-type plasminogen activator prepared by the method of claim 1. 35
11. A method of increasing the conversion of plasminogen to plasmin in a patient in need of such conversion comprising administering to said patient a therapeutically effective amount of low molecular weight urokinase-type plasminogen activator prepared by the method of claim 1.
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| US81971392A | 1992-01-13 | 1992-01-13 | |
| US07/819,713 | 1992-01-13 |
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| Application Number | Title | Priority Date | Filing Date |
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| PCT/US1993/000146 WO1993014199A1 (en) | 1992-01-13 | 1993-01-08 | A pump-1 catalyzed method of making low molecular weight urokinase-type plasminogen activator |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5780265A (en) * | 1995-06-05 | 1998-07-14 | Genentech, Inc. | Kunitz type plasma kallikrein inhibitors |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4992274A (en) * | 1983-12-24 | 1991-02-12 | Beecham Group P.L.C. | Tissue plasminogen activator A-chain/urokinase B-chain hybrid protein |
| US5045315A (en) * | 1985-08-14 | 1991-09-03 | American Home Products Corporation | Process for treating thrombosis by administering poly-kringle plasminogen activator |
| US5126134A (en) * | 1988-09-21 | 1992-06-30 | Ciba-Geigy Corporation | Pharmaceutically active combination |
-
1993
- 1993-01-08 WO PCT/US1993/000146 patent/WO1993014199A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4992274A (en) * | 1983-12-24 | 1991-02-12 | Beecham Group P.L.C. | Tissue plasminogen activator A-chain/urokinase B-chain hybrid protein |
| US5045315A (en) * | 1985-08-14 | 1991-09-03 | American Home Products Corporation | Process for treating thrombosis by administering poly-kringle plasminogen activator |
| US5126134A (en) * | 1988-09-21 | 1992-06-30 | Ciba-Geigy Corporation | Pharmaceutically active combination |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5780265A (en) * | 1995-06-05 | 1998-07-14 | Genentech, Inc. | Kunitz type plasma kallikrein inhibitors |
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