WO1988001175A1

WO1988001175A1 - Cofactor of prourokinase

Info

Publication number: WO1988001175A1
Application number: PCT/US1987/001945
Authority: WO
Inventors: Victor E. Gurewich; Ralph Pannell
Original assignee: Individual
Current assignee: Individual
Priority date: 1986-08-12
Filing date: 1987-08-11
Publication date: 1988-02-25
Anticipated expiration: 1989-02-12
Also published as: EP0281579A1; EP0281579A4

Abstract

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, thrombolytic activity which is fibrin-dependent.

Description

COFACTOR OF PROUROKINASE

Background of the Invention

This invention relates to the therapeutic lysis of fibrin blood clots (thrombi) in human patients.

Several naturally-occurring enzymes are known to participate in the lysis of thrombi, and have been used therapeutically to lyse thrombi in patients, e.g., coronary patients, in whom life-threatening thrombi have formed. One of these enzymes is 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.

Summary of the Invention In general, 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, thrombolytiσ 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. By the same mechanism, 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.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiment thereof, and from the claims. Description of the Preferred Embodiment

Characterization of the cofactor of the invention was carried out as follows. Increased Fibrin Affinity

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 NaP0₄, 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 NaP0₄, 0.3 M NaCl, 1 M EDTA, pH 7.4). In separate experiments, pro-UK or UK (purified from human kidney cell culture) and 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. (For technique, see P. Brakman, "Fibrinolysis: A Standardized Fibrin Plate Method and a Fibrinolytic Assay of Plasminogen" published by Scheltema and Holkema, Amsterdam, pp. 1-124 (1967)). 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 thrombolytiσ activity of cfb-pro-UK and of cfb-UK remained bound until an elution buffer containing 0.2M arginine was run on the column.

Increase in Amidolytic Activity

The UK-cofactor complex in concentrated urine is devoid of amidolytic activity.

In contrast, when amidolytic (S2444) or plasminogen activity (S2251) was measured in the presence of fibrin, a significant amidolytic activity and plasminogen activating activity was seen. It is believed that the fibrin binding of the complex causes the enzymatic site on UK and on pro-UK to become available to both the synthetic substrate and the natural substrate, plasminogen. Production and Use

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. After 24 liters are applied, the column is washed with 1 liter of 0.3 M NaCL, 0.02 M NaH₂P0₄ 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 NaH₂P0₄ 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 NaH₂P0₄ buffer, pH 7.5, containing 0.5 M methyl-α-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 aH₂P0₄ 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 NaH₂P0₄ 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.

In the above procedure, 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-α-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. When the extensively dialyzed eluate is applied to an SP-Sephadex C-50 column, 90% of the protein passes through the column unbound, while 85% of the inhibitor/cofactor is absorbed. The bound proteins elute in two partially separated peaks, the second of which contains the inhibitor/cofactor. The pooled fractions contain 33% of the original inhibitor/cofactor with a purification factor of 260. Final purification is obtained by gel filtration of a 10-fold concentrate of this pool on Sephadex G-100 superfine. Some of the physical characteristics of the isolated inhibitor compound recited in Stump et al. are as follows. It is a glycoprotein with an apparent Mr of 50,000 under both non-reducing and reducing conditions; its urokinase inhibitory activity is stable for over 1-h at pH 4 but is destroyed within 5 min at pH 2.5; its urokinase inhibitory activity is destroyed during a 1-h incubation with 0.1% sodium dodecyl sulfate; and it has an amino acid composition, upon isolation from urine, as follows (expressed as number of residues /100 amino acid residues) :

Aspartic acid 10

Threonine 5.2

Serine 10

Glutamic acid 12

Proline 4.5

Glycine 6.6

Alanine 6.6

Cysteine 0.6

Valine 4.7

Methionine 2.8

Isoleucine 2.4

Leucine 11

Tyrosine 2.4

Phenylalanine 5.1

Histidine 1.7

Lysine 5.2

Arginine 5.0

Tryptophan not determined

Glucosamine 2.7

Galactosamine 1.3

Referring now to clinical uses to be made of the invention, 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. The following examples are illustrative.

Example 1

For emergency treatment of thrombi by bolus injection, about 5 mg of 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. Example 2

Bolus injection is carried out as in example 1, except that the syringe contains two chambers, one containing cofactor in saline and the other containing pro-UK in saline. Example 3

For infusion treatment for the rapid lysis of coronary thrombi, about 5 mg/hr of 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

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.

Other embodiments are within the following claim.

Claims

01175- 8 -Claim

1. A method of increasing the efficacy of pro-UK-induced thrombolysis in a human patient to whom pro-UK is administered, said method comprising administering to said patient, in addition to said pro-UK, a cofactor of pro-UK and UK, said 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 imparts, on the UK generated from pro-UK, an affinity for fibrin; and d) it imparts, on the UK generated from pro-UK, a thrombolytic activity which is fibrin-dependent.