ANTI-SARS-COV-2 VIRUS AGENT ANTIPROVIR

Abstract

This invention relates to a novel anti-SARS-CoV-2 virus agent Antiprovir to be used for the prevention and treatment of coronavirus disease COVID-19.

The use of a pharmaceutical composition, including Aprotex®, Gordox® and Aerus®, with aprotinin as the active ingredient and excipients, said pharmaceutical composition being anti-SARS-CoV-2 virus drug Antiprovir to be used for the prevention and treatment of coronavirus disease COVID-19.

The drug Antiprovir for the prevention and treatment of coronavirus disease COVID-19, which is a pharmaceutical composition containing 0.1% wt. ÷ 0.2% wt. aprotinin, optionally 0.2 %wt. ÷ 1.0% wt. excipients, and the rest is water for injection.

Claims

1. A pharmaceutical composition for the prevention and treatment of coronavirus disease COVID-19, containing a therapeutically effective amount of aprotinin and excipients.

2. The pharmaceutical composition according to claim 1 containing excipients selected from sodium chloride, sodium hydroxide, benzyl alcohol, lactose, 1,1,1,2-tetrafluoroethane, peppermint leaf oil, ethanol, and glycerol.

3. The pharmaceutical composition according to claims 1-2 in the form of injections.

4. The pharmaceutical composition according to claims 1-2 in the form of spray.

5. A method for the prevention and treatment of SARS-CoV-2 (COVID-19) infections involving the administration to the patient of the pharmaceutical composition according to claims 1-4.

6. The method according to claim 5 involving intravenous therapy.

7. The method according to claim 5 involving spray therapy.

8. The method according to claim 5 involving inhalation therapy.

9. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

Description

PREFERRED EMBODIMENT

[0030] This invention is illustrated by, but not limited to, the following examples.

[0031] Example 1. Lyophilizate for the preparation of Antiprovir intended for COVID-19 intravenous or spray therapy. Aprotinin (1.5 g), sodium chloride (85 g), and benzyl alcohol (100 g) are dissolved in 10 l of water for injection. The resulting solution is poured into suitable vials (10 ml per vial) using the Rota FLR 50 filling machine. Filled vials are sterilized at 120-122° C. at a pressure of 120 kPa for 8 minutes, lyophilized in a Zirbus sublimator under a vacuum of less than 0.3 bar, corked, crimped with aluminum caps, and packed in 10-piece boxes to obtain lyophilizate for the preparation of Antiprovir, 100000 KIU in 10 ml.

[0032] Example 2. Anti-SARS-CoV-2 agent Antiprovir for intravenous therapy of COVID-19. Aprotinin (1.5 g), sodium chloride (85 g), and benzyl alcohol (100 g) are dissolved in 10 l of water for injection. Half of the resulting solution is poured into suitable 1 ml ampoules of neutral glass and the other half, into 10 ml ampoules using the Rota FLR 50 filling machine. Filled ampoules are sterilized at 120-122° C. at a pressure of 120 kPa for 8 minutes, sealed with a gas burner, and packed in 10-piece boxes to obtain an Antiprovir kit of 10000 KIU in 1 ml and 100000 KIU in 10 ml for COVID-19 intravenous therapy.

[0033] Example 3. Anti-SARS-CoV-2 medication Antiprovir for COVID-19 spray therapy. Aprotinin (1.5 g), sodium chloride (57 g), and benzyl alcohol (67 g) are dissolved in 10 l of water for injection. The resulting solution is sterilized at 120-122° C. at a pressure of 120 kPa for 8 minutes and dispensed into appropriate spray cans, 100 ml each (for nose and/or throat), to obtain anti-SARS-CoV-2 viral agent Antiprovir for COVID-19 spray therapy.

[0034] Example 4. Evaluation of the Covid-014 antiviral efficacy of Antiprovir in Vero E6 cell culture against SARS-CoV-2 virus. Antiprovir was studied as Aprotex and Gordox lyophilizates at a concentration of ~53 000 KIU/ml against the SARS-CoV-2 virus. Virus production was evaluated in terms of cytopathic effect in Vero E6 cell culture with real-time PCR confirmation at the Testing Center for Quality Control of Immunobiological Drugs, Gamaleya Federal Research Center for Epidemiology & Microbiology.

[0035] Vero E6 cells were placed in 96-well culture flat-bottomed plates (12000 cells/well) in 100.0 .Math.l of freshly prepared complete culture medium (CM). The cells were cultured for 24 h at 37° C., 5% CO.sub.2. To prepare a two-fold dilution of the test drug at a concentration of 106,400 U/ml, the contents of 8 vials was serially dissolved in 1 ml of the reaction medium (RM). The resulting 106,400 KIU/ml solution was kept for 2 hours at 2-8° C. to control the drug’s solubility. CM was removed from the plates, and the cells were washed with RM medium and filled with 100 .Math.l of each dilution of test substance. Each dilution point was tested in 3 wells (in triplicate). In addition, the drug was added to virus-free control wells in order to evaluate potential cytotoxic effects and keep a research record. Pure RM was added to the cell control wells. To infect cells, a suspension of SARS-CoV-2 virus (passage 4) with an infection activity of 10.sup.6 TCID.sub.50/1 ml for Vero E6 cells was prepared. A series of 10-fold virus dilutions were made: 10.sup.-1 and up to 10.sup.-6. The suspension was diluted by successive transfer to test tubes with the necessary amount of corresponding RM: 900 .Math.l of RM and 100 .Math.l of virus suspension in each tube. To prepare a virus suspension at a concentration of 1000 TCID.sub.50/ml, 10 .Math.l of the suspension was taken from the stock virus suspension at a concentration of 1×10.sup.6TCID.sub.50/ml and placed in 10.0 ml of RM. To prepare a virus suspension at a concentration of 10000 TCID.sub.50/ml, 100 .Math.l of the suspension was taken from the stock virus suspension at a concentration of 1×10.sup.6TCID.sub.50/ml and placed in 9.9 ml of RM. The dilutions of the virus suspension were added to the cells following their 2-hour incubation with the dilution of the tested drug and then co-incubated them for 96 hours. The virus (100 .Math.l) at concentrations of 1000 and 10000 TCID.sub.50 /ml (100 and 1000 TCID.sub.50 per well, respectively) was added to wells 100 TCID.sub.50 and 1000 TCID.sub.50 in RM medium. RM (100 .Math.l) was added to each cell control well. Plates with cells were incubated for 96 hours at 37° C., 5% CO.sub.2 until the virus in the viral control fully manifested the cytopathic effect in the expected range. The antiviral activity of the samples was evaluated visually under a microscope 96 hours after infection by the inhibition of the Cytopathic Effect (CPE) of the virus in Vero E6 cell culture. The study culminated in a report on the inhibition of the cytopathic effect of the virus in Vero E6 cell culture upon exposure to the drug: complete inhibition (CPE absence in 3 wells out of 3), incomplete inhibition (CPE presence in 1 or 2 wells out of 3), absence of inhibition (CPE presence in 3 wells out of 3). Outcome: Antiprovir at a concentration of ~53 000 KIU/ml (or ~10 000KIU/well) completely suppressed the cytopathic effect of the virus at a dose of 100 TCID.sub.50/well and partially suppressed said effect at a dose of 1000 TCID.sub.50/well.

[0036] The efficacy of anti-SARS-CoV-2 virus action of the drug in the studied wells was determined in real time using the polymerase chain reaction (PCR) method. From three wells of drug dilution for 0 TCID.sub.50, 100 TCID.sub.50 and 1000 TCID.sub.50 of the virus, supernatant was removed and used for RNA isolation in parallel with positive and negative control of the isolation stage. The isolated RNA was used for real-time PCR. The presence of viral RNA was evaluated in terms of threshold cycle (Ct). The outcome is as follows: PCR data showed complete inhibition of SARS-CoV-2 virus reproduction at Antiprovir concentration of ~53,000 KIU/ml and confirmed high anti-SARS-CoV-2 virus activity of Antiprovir.

INDUSTRIAL APPLICABILITY

[0037] This invention can be used in medicine and veterinary.