Claims: , Description:FIELD OF THE INVENTION
The present invention relates in general to medical and pharmaceutical preparation of haemocoagulase for human use, animal use and analytical purposes and more particularly to a method of preparation and testing of haemocoagulase that degrades spike proteins which are present on (SARS) covid viruses.

BACKGROUND OF THE INVENTION
Coronavirus disease 2019 (covid-19) or severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a novel coronavirus strain disease, is rapidly spreading worldwide. This novel strain is highly transmittable by aerosol and severe disease has been reported in up to 16% of hospitalized cases. More than 10 million cases have been confirmed with 0.5 million deaths and the number of deaths is constantly increasing. Covid-19 hospitalized patients, especially those suffering from severe respiratory or systemic manifestations, fall under the spectrum of the acutely ill medical population, which is at increased venous thromboembolism risk. Thrombotic complications seem to emerge as an important issue in patients infected with covid-19. Preliminary reports on covid-19 patients' clinical and laboratory findings include thrombocytopenia, elevated d-dimer, prolonged prothrombin time, and disseminated intravascular coagulation. Fibrin/fibrinogen degradation products (fdp) are the result of the breakdown of fibrinogen (primary fibrinogenolysis) and fibrin (fibrinolysis). The d-dimer detects breakdown products that are only generated by the action of plasmin on stabilized fibrin. Both these values are significantly increased in covid-19 patients suggesting heightened hemostasis in the infected patients. Excessive utilization of coagulation factors may develop in to disseminated intravascular coagulation.
Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses of ~30?kb. They infect a wide variety of host species. They are largely divided into four genera; a, ß, ? and d based on their genomic structure. a and ß coronaviruses infect only mammals. Human coronaviruses such as 229e and nl63 are responsible for common cold and cough and belong to a coronavirus. In contrast, SARS-cov, middle ast respiratory syndrome coronavirus (MERS-cov) and SARS-cov-2 are classified to ß coronaviruses.
The life cycle of the virus with the host consists of the following 5 steps: attachment, penetration, biosynthesis, maturation and release. Once viruses bind to host receptors (attachment), they enter host cells through endocytosis or membrane fusion (penetration). Once viral contents are released inside the host cells, viral RNA enters the nucleus for replication. Viral MRNA is used to make viral proteins (biosynthesis). Then, new viral particles are made (maturation) and released. Coronaviruses consist of four structural proteins; spike (s), membrane (m), envelop (e) and nucleocapsid (n). Spike is composed of a transmembrane trimetric glycoprotein protruding from the viral surface, which determines the diversity of coronaviruses and host tropism. Spike comprises two functional subunits; s1 subunit is responsible for binding to the host cell receptor and s2 subunit is for the fusion of the viral and cellular membranes. All the available and ongoing researches target at preventing the contraction of the virus in the form of vaccine, but availability of curative methods are very few to none.
To overcome the above mentioned problem in curing the patients efficiently once they have caught the viral disease, the present invention, proposes the effect of haemocoagulase (Atrolase®) on sars-cov-2 proteins, as snake venom consists of several proteins with known and unknown functions as well as well characterized proteins such as serine protease and metallo-protease. These could have deleterious effects on viral proteins and hence inactivate the virus. With this invention we can ensure that the attachment and transmission phase of the virus can be eliminated thus preventing the reproduction of the virus.

OBJECT OF THE INVENTION
The main object of the present invention is to ensure that the spike proteins of the viruses are either cleaved, degraded or destroyed partially or completely thereby ensuring that the virus loses its ability to perform its life cycle phases of attachment and transmission.

Another object of the present invention is to provide a method of preparation of haemocoagulase that is safe, with high efficacy and stable with a long shelf life.

Another object of the present invention is to provide knowledge of the science behind the degradation of the spike protein and the pseudo virus.

SUMMARY OF THE INVENTION

The above mentioned objectives are achieved according to the present invention by method of preparation and testing of haemocoagulase disclosed herein.

The haemocoagulase such as Atrolase® [Manufactured by Juggat Pharma a division of Jagdale Industries Private Limited, #782, 1st Phase 15th Cross, J.P. Nagar, Bengaluru - 560 0 78] is approved for use by the regulatory authority to control capillary bleeding and wound healing. The haemocoagulase contains a complex group of isolated, purified and standardized group of enzymes and proteases from the bothrops species of snakes. Haemocoagulase in its raw material form has a crystalline powder like consistency. It has a thrombin like action,i.e. it converts the fibrinogen to a fibrin clot. The mode of action is very specific like a lock and key mechanism. It has not shown to alter any other physiological or pathological parameters in humans and as well as animals. It has also shown use in analysis of clotting and bleeding time in humans. The haemocoagulase is known to be highly specific in its mode of action and is evaluated against the spike proteins of covid 19. It is also well tolerated at higher doses which was one of the criteria for choice of the present invention and it also demonstrated total degradation of spike proteins in a time and dose dependent manner, compared to no other molecules and preparations.

BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing features of this invention, as well as the invention itself may be more fully understood from the following description of the drawings in which:
Figure 1 illustrates the page image of 1stbatch of haemocoagulase (Left), concentration of the proteins (right).
Figure 2 illustrates Spike protein incubated with haemocoagulase at 37°C for 1 hour and visualized on 10% PAGE.
Figure 3 illustrates spike and haemocoagulase PAGE.
Figure 4 illustrates 10ug of BSA incubated with 10ug of haemocoagulase at 37°C for hour and visualized on 8% PAGE.
Figure 5 illustrates Western blot analysis of spike protein treated with haemocoagulase at 37°C for 1 hour.

DESCRIPTION OF THE INVENTION

The preferred embodiment of the present invention herein describes a method of preparation and testing of haemocoagulase to ensure that the spike proteins of the viruses are either cleaved, degraded or destroyed partially or completely, thereby ensuring that the virus loses its ability to perform its life cycle phases of attachment and transmission.

Haemocoagulase such as Atrolase® is a haemocoagulant and wound healer which is approved for use by the regulatory authority to control capillary bleeding. Haemocoagulase contains a complex group of isolated, purified and standardized group of enzymes and proteases from the Crude Coagulative Enzyme (CCE) of the bothrops species of snakes.

In the present invention, the effect of haemocoagulase on sars-cov-2 proteins was envisaged as snake venom consists of several proteins with known and unknown functions as well as characterized proteins such as serine protease and metallo-protease. These could have deleterious effects on viral proteins and hence inactivate the virus. The present invention ensures that the attachment and transmission phase of the virus can be eliminated thus preventing the reproduction of the virus.

In the present invention, Angiotensin converting enzyme 2 (ace2) is identified as a functional receptor for SARS-cov. Structural and functional analysis showed that the spike for sars-cov-2 also bound to ace2. Ace2 expression is high in lung, heart, ileum, kidney and bladder. In lung, ace2 is highly expressed on lung epithelial cells. Following the binding of sars-cov-2 to the host protein, the spike protein undergoes protease cleavage.

A two-step sequential protease cleavage to activate spike protein of SARS-cov and mers-cov was proposed as a model, consisting of cleavage at the s1/s2 cleavage site for priming and a cleavage for activation at the s'2 site, a position adjacent to a fusion peptide within the s2 subunit. After the cleavage at the s1/s2 cleavage site, s1 and s2 subunits remain non-covalently bound and the distal s1 subunit contributes to the stabilization of the membrane anchored s2 subunit at the prefusion state. Subsequent cleavage at the s'2 site presumably activates the spike for membrane fusion via irreversible, conformational changes. The characteristics unique to sars-cov-2 among corona viruses is the existence of furin cleavage site (“RPPA” sequence) at the s1/s2 site. The s1/s2 site of sars-cov-2 is entirely subjected to cleavage proteases such as transmembrane protease serine 2 (tmprss2) and cathepsin l, and the ubiquitous expression of furin likely makes this virus very pathogenic.

Result of invitro study:
Effect of haemocoagulase on spike protein in vitro: Purified trimeric spike protein was treated with Atrolase® (haemocoagulase). The reaction was stopped and analyzed by using sds-page gel electrophoresis. As controls, haemocoagulase was boiled to inactivate the proteins and in another reaction, haemocoagulase was treated with EDTA solution. Simultaneously, bovine serum albumin and fibrinogen was treated under similar conditions with haemocoagulase to test for the specificity. Unlike the specific cleavage of fibrinogen, haemocoagulase did not breakdown bovine serum albumin. At the same time, haemocoagulase completely degraded spike protein. The breakdown of the protein was in a dose and time dependent manner.

Impact of Haemocoagulase [Atrolase] on SARS-CoV-2 proteins and coagulation effects

Dilution of Haemocoagulase
1. As seen in figure 1, the lyophilized proteins were dissolved in PBS at a concentration of 10mg/mL and tapped at an interval of 2-3 mins for 15 mins at room temperature.
2. The solution was centrifuged at 3000 rpm for 5 mins at 4°C and supernatant containing dissolved proteins were transferred to a fresh tube.
3. The proteins were loaded and visualized in Polyacrylamide Gel (PAGE) and concentration was determined using NanoDrop at 280 nm absorbance.

SDS PAGE and Western Blot of Recombinant Spike and Haemocoagulase
1. 500ng of recombinant spike protein was incubated with 10ug of coagulase and haemocoagulase in 1.5mL microfuge tube and made upto 20uL with sterile PBS at 37°C for 1 hour.
2. 6x Gel loading dye containing 1% SDS was added in the mixture and incubated at 95°C for 5 mins to denature the proteins.
3. The entire volume of the proteins were loaded in 8% PAGE.
4. Silver Staining was used to visualize the gel.
5. This experiment was repeated 3 times separately.

As seen in Figure 2, Spike protein was incubated with and haemocoagulase at 37°C for 1 hour and visualized on 10% PAGE. Spike protein degradation was observed in lanes and 6, confirming proteolytic effects of Atrolase.

As seen in Figure 3, Spike and haemocoagulase PAGE, haemocoagulase was heat inactivated at 95°C for 15 mins and incubated with spike at 37°C for 1 hour (Lane 4), proteolytic activity was not observed. Similarly, spike protein was incubated with haemocoagulase in presence of 1mM EDTA at 37°C for 1-hour, proteolytic activity was not observed (Lane 6). Spike protein degradation was observed when incubated with active haemocoagulase at 37°C for 1 hour (Lane 4).

A seen in Figure 4, 10ug of BSA was incubated with 10ug of haemocoagulase at 37°C for hour and visualized on 8% PAGE. Considerable BSA degradation was not observed after treating with CCE.

As seen in Figure 5, Western blot analysis of spike protein treated with haemocoagulase at 37°C for 1 hour. Anti-His-tag primary antibody and anti-mouse secondary antibody conjugated with peroxide was used to stain the spike protein. Spike proteins were not present in Lanes 5 and 6 (treated with haemocoagulase, respectively), suggesting total proteolytic degradation of spike protein by the haemocoagulase.

The above and other features of the present invention will be more clearly described in the complete specification which will be filed pursuant to the present provisional specification.