Use of prourokinase and its variants against coagulopathy caused by viruses

Abstract

A method for prophylaxis and/or treatment of coagulopathy in a subject caused by a viral infection is provided. The method includes administering prourokinase or its variant; the viral infection is caused by a coronavirus.

Claims

1. A method for prophylaxis and/or treatment of coagulopathy caused by a coronavirus infection in a subject, the method comprising administering to the subject in need prourokinase or a variant thereof.

2. The method according to claim 1, wherein for the prophylaxis of coagulopathy, the prourokinase is administered by an administration route and dosage selected from the group consisting of: Intravenous drip: 1 to 5 miu/hr, continuous administration for 24 hours a day, or 6-hour to 7-hour administrations each followed by a 1-hour to 2-hour break, for consecutive 1 to 7 days; or Intravenous drip: 1 to 5 miu/hr, continuous administration for 24 hours a day, or 6-hour to 7-hour administrations each followed by a 1-hour to 2-hour break, for consecutive 1 to 7 days, combined with one or more anticoagulant drugs.

3. The method according to claim 1, wherein for the treatment of coagulopathy, the prourokinase is administered by an administration route and dosage selected from the group consisting of: Intravenous drip: 5 to 8 miu/hr, until thrombus disappears; or Intravenous drip: 5 to 8 miu/hr, plus TNK-tissue plasminogen activator (TNK-tPA intravenous bolus once every 1 to 3 hours with 1 to 25 mg each time, until the thrombus disappears; or Intravenous drip: 5 to 8 miu/hr, plus TNK-tPA intravenous catheter administration at the site of thrombus, once every 1 to 3 hours with 1 to 25 mg each time, until the thrombus disappears.

4. The method according to claim 1, wherein the prourokinase or the variant thereof is in form of a pharmaceutical formulation.

5. The method according to claim 1, wherein the coagulopathy caused by the coronavirus infection is selected from the group consisting of disseminated intravascular coagulation, ST-segment elevation myocardial infarction (STEMI), ischemic stroke (IS), pulmonary embolism (PE), deep vein thrombosis (DVT) and venous thromboembolism (VTE).

6. The method according to claim 1, wherein the coronavirus is coronavirus SARS-CoV-2.

7. The method according to claim 4, wherein the pharmaceutical formulation further comprising an injectable formulation.

8. The method according to claim 7, wherein the pharmaceutical formulation further comprising a powder injection in form of a powder.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic representation of the primary structure of human prourokinase.

(2) FIG. 2 shows the process of diagnosis and anticoagulation treatment of Covid-19 patients with hypercoagulability.

(3) FIG. 3-1 is the experimental results of the stability (inertness) of prourokinase in human plasma.

(4) FIG. 3-2 is the experimental results of the stability (inertness) of prourokinase variant M5 in human plasma.

(5) FIG. 3-3 is the experimental results of stability (inertness) of prourokinase variant LMW-prouk in human plasma.

(6) FIG. 4-1 shows the experimental results of the in vitro human plasma clot-dissolving effect of prourokinase.

(7) FIG. 4-2 shows the experimental results of the in vitro human plasma clot-dissolving effect of prourokinase variant M5.

(8) FIG. 4-3 shows the experimental results of the in vitro human plasma clot-dissolving effect of prourokinase variant LMW-prouk.

EXPERIMENTAL EXAMPLES

(9) The following specific experimental examples are used to illustrate the technical solutions of the present invention, but the scope of protection of the present invention is not limited thereto.

(10) The contents of the experimental examples in this specification are only exemplary embodiments of the inventive concept. The scope of protection of the present invention should not be construed as limited to the specific embodiments given in the experimental examples, but also extends to the equivalent technical means that can be conceived by a person skilled in the art based on the inventive concept. Although the embodiments of the present invention are described below, the present invention is not limited to the specific embodiments and application fields, and the following specific embodiments are only illustrating and guiding rather than limiting. In light of this specification and without departing from the scope of protection of the claims of the present invention, a person ordinarily skilled in the art can make many other forms, which all fall within the scope of protection of the present invention.

(11) All the quantitative tests in the following experimental examples are each repeated three times, and the data is given as the average value of the three repeated tests or as the average±standard deviation.

Experiment 1—the Study on the Stability (Inertness) of Prourokinase (Prouk) and its Variants in Human Plasma

(12) Method: Prouk (0.5-2 μg/mL) or M5 (6-12 μg/mL) or LMW-prouk (0.25-1 μg/mL) was added to 1.0 mL citrated plasma, and incubated at 37° C. for 7 days. A sample was taken every day, to which 0.2 mL aprotinin (10,000 kallikrein inhibitor units/mL) was added to stop the reaction, and then the residual fibrinogen in the plasma was measured and compared with a baseline.

(13) Results: After incubation in citrated plasma at 37° C. for 7 days, prouk remained inert at a concentration of 1 μg/mL, but was capable of inducing degradation of fibrinogen when its concentration reached 2 μg/mL (see FIG. 3-1); M5 was inert until a concentration limit of 8 μg/mL, and caused degradation of fibrinogen when it reached 10 μg/mL (see FIG. 3-2); and LMW-prouk was inert until a limit of 0.5 μg/mL, and caused degradation of fibrinogen when it reached 1 μg/mL (see FIG. 3-3).

Experiment 2—the Study on In Vitro Clots Lysis in Human Plasma by Prouk and its Variants

(14) Method: .sup.125I-labeled fibrinogen clots were prepared with 0.2 mL plasma, and put in 4 mL plasma, to which prouk (0.5-1.5 μg/mL) or M5 (0.5-5 μg/mL) or LMW-prouk (0.25-1 μg)/mL) was added, and co-incubated at 37° C. to examine the lysis of blood clots. The experiment ended when the blood clots in the highest-concentration group were completely dissolved, and the lysis at other concentrations was also examined.

(15) Result: prouk at 1.5 μg/mL completely dissolved the blood clots in 4 hours, and lysis at the other concentrations is shown in FIG. 4-1; M5 at 5 μg/mL completely dissolved the blood clots in 3 hours, and lysis at the other concentrations is shown in FIG. 4-2; LMW-prouk at 1 μg/mL completely dissolved the blood clots in 6 hours, and lysis at the other concentrations is shown in FIG. 4-3. The results of LMW-prouk proved that the thrombolytic ability of prourokinase and its variants came from the protease domain.

Experiment 3—the Study on the Safety of Prouk Intravenously Administrated in Monkeys

(16) Method: To study the safety of prouk, a safety test was carried out on cynomolgus monkeys. They were administered by intravenous drip at doses of 0.57 mg/(kg.Math.hr), 0.86 mg/(kg.Math.hr), and 1.15 mg/(kg.Math.hr) for consecutive 10 days, wherein each 6.5-hour administration was followed by a 1.5-hour break. A sample was taken every day to test the blood fibrinogen content, and whether an adverse effect such as bleeding occurred was observed.

(17) Results: The blood fibrinogen content of monkeys in all test groups remained stable during the continuous administration period, and no adverse effect such as bleeding was observed.

Experiment 4—the Pharmacokinetics of Prouk and its Variants

(18) 1. The Pharmacokinetic Study on Prouk

(19) Method: Administrations at 0.29 mg/(kg.Math.hr) or 0.57 mg/(kg.Math.hr) to human bodies were conducted by continuous intravenous drip, and changes in the blood drug concentration were examined.

(20) Results: For the study group administered intravenously at a dose of 0.29 mg/(kg.Math.hr), the maximum blood concentration was reached at 20-30 minutes, and the average maximum blood concentration was 0.68 μg/mL; for the study group administered intravenously at a dose of 0.57 mg/(kg.Math.hr), the maximum blood concentration was reached at 20-30 minutes, and the average maximum blood concentration was 1.43 μg/mL.

(21) 2. The Pharmacokinetic Study on LMW-Prouk

(22) The pharmacokinetic study on LMW-prouk was conducted according to the experimental method for prouk, and the experimental results were basically the same as those for prouk, without a significant difference.

Experiment 5—Clinical Dosing Regime

(23) 1. Eligible people were determined according to the process shown in FIG. 2.

(24) 2. Dosage and administration route of prourokinase

(25) For prophylaxis (no thrombosis in the patient):

(26) 1) Intravenous drip: 3 miu/hr (1-5 miu/hr), 24 hrs, or by 6.5-hr (6 to 7 hr) administrations each followed by a 1.5-hr (1 to 2 hr) break, for 1-7 days.

(27) or

(28) 2) Intravenous drip: 3 miu/hr (1-5 miu/hr), 24 hrs, or by 6.5-hr (6 to 7 hr) administrations each followed by a 1.5-hr (1 to 2 hr) break, for 1-7 days, in combination with administration of low-molecular-weight heparin or other anticoagulant drug(s).

(29) For treatment (with thrombosis):

(30) 1) Intravenous drip: 6 miu/hr (5 to 8 miu/hr) until the thrombus disappears.

(31) or

(32) 2) Intravenous drip: 6 miu/hr (5 to 8 miu/hr), plus TNK-tPA intravenous bolus, once every 1-3 hours, 5 mg (1 to 25 mg) each time, until the thrombus disappears.

(33) or

(34) 3) Intravenous drip: 6 miu/hr (5 to 8 miu/hr), plus TNK-tPA intravenous catheter administration at the site of thrombus, once every 1-3 hours, 5 mg (1 to 25 mg) each time, until the thrombus disappears.

(35) 3. Dosage and administration route of variants of prourokinase

(36) 1) For prophylaxis of coagulopathy caused by viruses, the dose of variants of prourokinase was determined according to its inert concentration in plasma, usually not exceeding 70% of the inert concentration, while the administration route and combination with other drugs were the same as those for the prophylactic use of prourokinase.

(37) 2) For treatment of coagulopathy caused by viruses, the dose of variants of prourokinase required for a thrombolytic therapy was determined according to its effective thrombolytic dose, while the administration route and combination with other drugs were the same as those for the treatment use of prourokinase.

(38) Conclusion: (1) after prophylactic administration to patients infected with the novel coronavirus, the incidence of coagulopathy in the patients was significantly reduced, without adverse effects such as bleeding in patients observed, which fully demonstrates that prourokinase and its variants according to the present invention can effectively prevent coagulopathy caused by the virus, without causing significant adverse effects and with good safety; (2) after therapeutic administration to patients infected with the novel coronavirus, the damage caused by thrombi to the patients was effectively controlled, and the potential risk of death was reduced. In terms of both prophylaxis and treatment, it is fully demonstrated that prourokinase and its variants can be used against viral infections that cause coagulopathy, can reduce the damage and possible sequelae caused by viruses to patients, and can improve the survival of patients.

(39) The examples of the present invention include but are not limited to those described above.

REFERENCES

(40) 1. Ren, Yan, Deng, Zhang, et al., (2020); Extremely High Incidence of Lower Extremity Deep Venous Thrombosis in 48 Patients with Severe COVID-19 in Wuhan, 10.116I/CIRCULATIONAHA.120.047407. 2. ISTH interim guidance on recognition and management of coagulopathy in COVID-19. doi: 10.1111/JTH.14810.