FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
EXTRACT OF COCCULUS HIRSUTUS FOR TREATMENT OF COVID-19
SUN PHARMACEUTICAL INDUSTRIES LIMITED
Corporate Office: Sun House, CTS No. 201 B/1, Western Express Highway, Goregaon (E), Mumbai-400063, Maharashtra, India.
A Company incorporated under the Companies Act, 1956.
The following specification particularly describes the invention and the manner in which it is to be performed:
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EXTRACT OF COCCULUS HIRSUTUS FOR TREATMENT OF COVID-19
CROSS REFERENCE
The present patent application claims the benefit of the priority date of Indian Provisional Patent Application No. 202021018013 filed on April 27, 2020. 5
FIELD OF THE INVENTION
The present disclosure relates to a composition comprising an extract of Cocculus hirsutus and pharmaceutical compositions comprising the extract for use in the prophylactic and curative treatment of an infection caused by a Coronavirus, more particularly, the novel Coronavirus strain known as SARS-CoV-2. 10
BACKGROUND OF THE INVENTION
Coronavirus disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Associated CoronaVirus-2 (SARS-CoV-2), a highly contagious and novel virus belonging to the Coronoviridae family which has caused a global pandemic raising worldwide health concerns. The elderly and people with underlying health conditions are 15 more susceptible to infection and prone to serious outcomes, which may be associated with acute respiratory distress syndrome (ARDS) and cytokine storm. SARS-CoV-2 appears to spread easily in the human population. Many healthcare workers have been infected, and more clusters of cases are being detected with each passing day. According to the World Health Organization (WHO), COVID-19 Situation dashboard data of March 20 29, 2020, there were 638,146 confirmed cases infected with SARS-CoV-2 with 30,105 deaths, By September 24, 2020, the pandemic had grown to 31,798,308 confirmed cases of SARS-CoV-2 infections, including 973,653 deaths globally. Scientists around the world are working tirelessly to understand the transmission mechanisms, understand the clinical spectrum of disease, and develop new diagnostics and preventive and therapeutic 25 strategies. Current therapeutic strategies for SARS-CoV-2 infections are only supportive and not sufficient to control this pandemic. The unprecedented rapidity of spread of this outbreak presents a critical need for an effective treatment against COVID-19 that can relieve the signs and symptoms, reduce the severity of common symptoms, prevent the development of severe complications, reduce the virus load from an infected patient so 30 that other supportive therapies can work more effectively in severely ill patients.
SUMMARY OF THE INVENTION
The present disclosure provides a purified extract of a plant of Menispermeaceae family and its pharmaceutical compositions for use in the prophylactic and curative
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treatment of an infection caused by SARS-CoV-2. The present disclosure also provides for a method of reducing viral load in the treatment of an infection caused by SARS-CoV-2 virus by administering the said extract or its pharmaceutical composition to a mammal in need thereof. Further, the present disclosure provides for a stable pharmaceutical composition comprising a therapeutically effective amount of the extract for use in 5 prophylactic and curative treatment of SARS-CoV-2 virus infection in a mammal. The present disclosure further provides a process for the preparation of said extract. The novel and inventive composition as provided by the present disclosure have been found to highly effective for administration to human patients infected by SARS-CoV-2 virus and have not shown any toxic effects at therapeutically effective dosages required for prophylactic 10 and curative treatment of an infection caused by SARS-CoV-2 virus.
SOURCE OF BIOLOGICAL MATERIAL
The biological material disclosed in the present disclosure is plant mass of Cocculus hirsutus that was procured from Madhya Pradesh, India.
DETAILED DESCRIPTION OF THE INVENTION 15
The terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. As used herein, the term “about” is meant to account for variations due to any experimental errors which may be commonly accepted in the analytical chemistry field. All measurements reported herein are understood to be modified by the term “about,” whether or not the term 20 is explicitly used, unless explicitly stated otherwise. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present 25 disclosure; other suitable methods and materials known in the art can also be used. The materials, methods and examples are illustrative only and not intended to be limiting by any means. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict, the present specification, including definitions, will control. 30
The present disclosure provides for an extract of a plant of Menispermeaceae family for prophylactic and curative treatment of SARS-CoV-2 virus infection. Preferably the plant is from genus Cocculus. More preferably, the plant is Cocculus hirsutus. It is a perennial climber and reaches 2 to 3 m above ground. The present disclosure describes a purified
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extract and a pharmaceutical composition of such extract from Cocculus hirsutus which was found to be effective against SARS-CoV-2 virus infection when tested in clinical studies with human patients. Applicant found that the composition was safe and showed minimal to no side effects during such treatment with the composition.
The present disclosure provides an extract of Cocculus hirsutus and/or a 5 pharmaceutical composition thereof for use in the prevention and treatment of an infection caused by SARS-CoV-2. The disclosed extracts and compositions reduce viral load during the treatment and provides clinical improvement in the signs and symptoms associated with SARS-CoV-2 virus infection.
The terms “extract” or “composite extract” as used herein, refers to a product of an 10 extraction of one or more components obtained from plant of family Menispermaceae, particularly of genus Cocculus, more particularly from Cocculus hirsutus in any concentration comprising one or more of Sinococuline, Magnoflorine, Makisterone-A or 20-Hydroxyecdysone, or a combination thereof. In some embodiments, one or more components of Cocculus hirsutus is removed during the extraction, e.g., the plant mass is 15 removed, and other components in the extract are separated into and concentrated in the extract. Thus, the present disclosure provides for an extract which is a new composition comprising compounds and components in a concentration and ratio that is not found in nature. Additionally, the present disclosure provides for an extract which is free from one or more contaminants, which may, in some embodiments, cause side effects and/or reduce 20 efficacy. In some embodiments, the extracts as described herein exhibit properties not found or appreciated in the Cocculus hirsutus plant, e.g., increased stability, increased effectiveness against SARS-CoV-2, increased bioavailability, increased solubility, reduced side-effects, etc. The extract may be a purified extract or a crude extract. The purified extract is an extract that is substantially free of pesticide residues, aflatoxins or any microbial 25 impurities. The purified extract may be an extract enriched for any one of the Sinococuline, Magnoflorine, Makisterone-A or 20-Hydroxyecdysone up to 85%, preferably 90%, more preferably 95% and even more preferably up to 99% w/w of the said extract. The purified extract may also be enriched in such a way that at least two of Sinococuline, Magnoflorine, Makisterone-A or 20-Hydroxyecdysone comprise up to 85%, preferably 90%, more 30 preferably 95% and even more preferably up to 99% w/w of the said extract. The extract may be enriched for any one or more of the Sinococuline, Magnoflorine, Makisterone-A or 20-Hydroxyecdysone in varying ratios of (0.5 to 85):(0.05 to 75):(0.01 to 80):(0.05 to 70). The said enrichment may be carried out any time before, during or after purification by
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methods commonly known in the art for such enrichment, such as by way of repeated purifications and/or fractionations using a combination of solvents and other conditions. The extract may be present in the form of a liquid, semisolid, solid powder, solid cake, gel, paste, dispersion, solution or a distillate. In one embodiment, the extract is a dried extract.
The term “SARS-CoV-2” or “COVID-19” or “COVID” or “novel coronavirus” are 5 used interchangeably throughout the specification, and refer to the novel coronavirus related to the Sever Acute Respiratory Syndrome (SARS) virus, and named by International Committee on Taxonomy of Viruses (ICTV) as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
The term “treatment” as used within the context of the present disclosure, is meant 10 to include therapeutic treatment, including prophylactic or preventive and curative treatments. For example, the term treatment may include administration of an extract or composition according to present disclosure or in combination with any Standard of Care prior to or following the onset of symptoms thereby preventing or removing signs and symptoms of the disease or disorder or as caused by SARS-CoV-2. As another example, 15 administration of a substance or a pharmaceutical composition prior to or after clinical manifestation of SARS-CoV-2 virus infection to prevent or combat the signs and symptoms and/or complications and disorders associated with SARS-CoV-2 virus infection comprises “treatment” of the disease. Further, administration of the extract or composition according to present disclosure or in combination with any Standard of Care prior to or after onset or 20 after clinical symptoms and/or complications have developed where administration affects clinical parameters of the disease or disorder and perhaps amelioration of the disease, comprises “treatment” of the SARS-CoV-2 infection. In one embodiment, treatment of a subject comprises inducing and maintaining remission of SARS-CoV-2 virus infection in a subject. In another embodiment, treatment of SARS-CoV-2 virus infection in a subject 25 comprises maintaining remission of SARS-CoV-2 virus in a subject.
The term “clinical improvement” as used throughout the specification, refers to the improvement in one or more clinical signs and symptoms or one or more primary and secondary endpoints for the determination of effectiveness of the anti- SARS-CoV-2 therapy in patients enrolled for treatment of SARS-CoV-2 infection. Such improvement 30 parameters include one or more of:
i. A patient meeting discharge criteria; or
ii. At least one point improvement (from the time of enrolment) in disease severity rating on an ordinal scale, wherein the scale is as follows:
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1 - not hospitalized with resumption of normal activities;
2 - not hospitalized, but unable to resume normal activities;
3 - hospitalized, not requiring supplemental oxygen;
4 - hospitalized, requiring supplemental oxygen;
5 - hospitalized, requiring nasal high-flow oxygen therapy or noninvasive 5 mechanical ventilation, or both; and
6 - hospitalized, requiring ECMO (Extracorporeal Membrane Oxygenation) or invasive mechanical ventilation, or both.
In a preferred embodiment, the clinical improvement is characterized by at least two point improvement (from the time of enrolment) in disease severity rating on the said 10 ordinal scale.
The hospital discharge criteria as in i) above may be one or both of resolution of symptoms and radiological improvement with a documented virological clearance in 2 samples at least 24 hours apart.
One or more of secondary endpoints in addition to the above parameters may 15 include:
- the time to normalization of fever without use of antipyretics in last 24 hours;
- time to alleviation of cough;
- time to first negative SARS-CoV-2 RT-PCR in upper or lower respiratory tract specimen; 20
- duration (days) of supplemental oxygen therapy;
- proportion of patients showing deterioration of clinical condition as assessed by at least 1 point worsening on ordinal scale (non-invasive ventilation, mechanical ventilation, ECMO or death)
- duration (days) of hospitalization; 25
- safety evaluation, as measured by Adverse Events (AEs), Adverse Reactions (ARs), Serious AEs (SAEs), Serious ARs (SARs).
In one embodiment, the extract according to present disclosure is an extract of a plant from Menispermeaceae family. In one aspect, the extract according to present disclosure is an extract of a plant of genus Cocculus of Menispermeaceae family. In a 30 preferred aspect, the extract according to present disclosure is an extract of Cocculus hirsutus.
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In another embodiment, the extract is a purified extract comprising Sinococuline not less than 0.5 % w/w, Magnoflorine not less than 0.05% w/w, 20-Hydroxyecdysone not less than 0.01%, Makisterone-A not less than 0.01% w/w, or a combination thereof.
In a preferred embodiment, the extract is a purified extract comprising Sinococuline not less than 1.0 % w/w, Magnoflorine not less than 0.1% w/w, 20-5 Hydroxyecdysone not less than 0.1%, Makisterone-A not less than 0.05% w/w, or a combination thereof
In a more preferred embodiment, the extract is a purified extract comprising Sinococuline from about 0.5% w/w to about 85% w/w, preferably about 0.5% w/w to about 60% w/w, more preferably about 0.5% w/w to about 40% w/wand even more 10 preferably about 0.5% w/w to about 20% w/w; Magnoflorine from about 0.05% w/w to about 75% w/w, preferably about 0.05% w/w to about 65% w/w, more preferably about 0.05% w/w to about 45% w/w and even more preferably about 0.05% w/w to about 25% w/w; Makisterone-A from about 0.01% w/w to about 80% w/w, preferably about 0.01% w/w to about 60% w/w, more preferably about 0.01% w/w to about 40% w/w and even 15 more preferably about 0.01% w/w to about 20% w/w; 20-Hydroxyecdysone from about 0.05% w/w to about 70% w/w, preferably about 0.05% w/w to about 50% w/w, more preferably about 0.05% w/w to about 30% w/w and even more preferably about 0.05% w/w to about 15% w/w or a combination thereof.
In one of the preferred embodiments, the present provides an extract of a plant of 20 Menispermaceae family, wherein the extract comprises therapeutically effective amount of one or more of Sinococuline from about 0.5% w/w to about 15% w/w, Magnoflorine about 0.05% w/w to about 3% w/w, Makisterone-A about 0.01% w/w to about 3% w/w, 20-Hydroxyecdysone about 0.05% w/w to about 3% w/w.
In another embodiment, the extract is a purified extract comprising Sinococuline 25 not less than 1.0 % w/w, Magnoflorine not less than 0.1% w/w, 20-Hydroxyecdysone not less than 0.1%, Makisterone-A not less than 0.05% w/w of the said extract, or a combination thereof.
In yet another embodiment, the extract is a purified extract enriched for any one of the Sinococuline, Magnoflorine, Makisterone-A or 20-Hydroxyecdysone up to 85%, 30 preferably 90%, more preferably 95% and even more preferably up to 99% w/w of the said extract.
In another embodiment, the extract is a purified extract enriched in such a way that at least two of Sinococuline, Magnoflorine, Makisterone-A or 20-Hydroxyecdysone
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comprise up to 85%, preferably 90%, more preferably 95% and even more preferably up to 99% w/w of the said extract.
In one embodiment, the present disclosure provides an extract of Cocculus hirsutus for use in the prophylactic and curative treatment of infection caused by SARS-CoV-2 virus.
In another embodiment, the present disclosure provides a pharmaceutical 5 composition comprising an extract of Cocculus hirsutus for use in prophylactic and curative treatment of infections caused by SARS-CoV-2 virus.
In one aspect, the pharmaceutical composition according to the present disclosure is an oral dosage form.
In a preferred embodiment the oral dosage form is selected from powder, pellets, 10 granules, spheroids, mini-tablets, caplets, tablets, sachet or a capsule comprising such powder, pellets, granules, spheroids, min-tablets or caplets, or a liquids selected from solutions, suspensions, emulsions, syrups, linctuses, elixirs or drops.
In another aspect, the present disclosure provides a pharmaceutical composition of the purified extract of Cocculus hirsutus for non-oral administration. 15
In yet another embodiment, the present disclosure provides a pharmaceutical composition comprising an extract of Cocculus hirsutus administration of which to a patient suffering from SARS-CoV-2 viral infection provides clinical improvement of the signs and symptoms of the said infection.
In one embodiment, the present disclosure provides a stable pharmaceutical 20 composition comprising an extract of Cocculus hirsutus for clinical improvement of the signs and symptoms of SARS-CoV-2 virus infection.
In another embodiment, the present disclosure provides a pharmaceutical composition comprising an extract of Cocculus hirsutus for reducing the SARS-CoV-2 viral load in a patient suffering from such viral infection. 25
In one embodiment, the present disclosure provides a stable, pharmaceutical composition comprising therapeutically effective amount of Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone, or a combination thereof. In a preferred embodiment, the stable pharmaceutical composition comprises a therapeutically effective amount of Sinococuline. In another embodiment, the pharmaceutical composition 30 comprises a therapeutically effective amount of Sinococuline and Magnoflorine.
In one embodiment, the present disclosure provides a stable, pharmaceutical composition comprising an extract of Cocculus hirsutus comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone, or a combination thereof. In a
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preferred embodiment, the said extract is enriched with Sinococuline or Magnoflorine or both.
In another embodiment, the present disclosure provides a stable, pharmaceutical composition comprising an extract of Cocculus hirsutus comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof for 5 reducing viral load in a mammal in need thereof.
In yet another embodiment, the present disclosure provides a stable, anti-COVID pharmaceutical composition comprising an extract of Cocculus hirsutus comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof for reducing SARS-CoV-2 viral load in a mammal in need thereof. 10
In another embodiment, the extract or pharmaceutical composition according to present disclosure provides clinical improvement, the said clinical improvement is characterized by any one or more of the below parameters: (i) reduction in duration of supplemental oxygen requirement; (ii) reduction in duration of ECMO or mechanical ventilation; (iii) reduction in time to alleviation of cough; (iv) reduction in time to 15 normalization of fever without use of antipyretics; (v) reduction in duration of hospitalization; or (vi) reduction in time to first negative SARS-CoV-2 RT-PCR in upper or lower respiratory tract specimen.
In a further embodiment, administration of the extract or pharmaceutical composition according to present disclosure to a patient infected with SARS-CoV-2 virus 20 infection provides clinical improvement in signs and symptoms, wherein the clinical improvement is characterized by a patient meeting at least one point, preferably at least two point improvement in disease severity rating on an ordinal scale after administration of the composition to the subject, the said ordinal scale defined as (i) not hospitalized with resumption of normal activities; (ii) not hospitalized, but unable to resume normal 25 activities; (iii) hospitalized, not requiring supplemental oxygen; (iv) hospitalized, requiring supplemental oxygen; (v) hospitalized, requiring nasal high-flow oxygen therapy or noninvasive mechanical ventilation, or both; or(vi) hospitalized, requiring ECMO or invasive mechanical ventilation, or both.
In one embodiment, the extract or pharmaceutical composition according to 30 present disclosure provides clinical improvement in signs and symptoms of SARS-CoV-2 virus infection in a patient. In another embodiment the extract or pharmaceutical composition according to present disclosure provides clinical improvement characterized by radiological improvement with a documented virological clearance in 2 samples tested at least 24 hours
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apart. In another embodiment the extract or pharmaceutical composition according to present disclosure provides clinical improvement characterized by time to normalization of fever without use of one or more antipyretics in last 24 hours. In another embodiment the extract or pharmaceutical composition according to present disclosure provides clinical improvement is further characterized by a negative SARS-CoV-2 RT-PCR of an upper or 5 lower respiratory tract specimen.
In another embodiment, the pharmaceutical composition comprises 25 mg to 1000 mg of the said extract. In one embodiment, the pharmaceutical composition according to the present disclosure for use in treatment of SARS-CoV-2 virus infection is administered to a patient in need thereof as an oral dose of 600 mg twice daily, 400 mg thrice daily, 300 10 mg four times daily or 200 mg six times daily. In an alternative embodiment, the said composition is administered as an 800 mg oral dose twice daily, 400 mg oral dose four times daily or 200 mg oral dose eight times daily. In yet another embodiment, the said composition is administered as 500 mg oral dose once, twice, thrice or four times daily. A pediatric dose may be selected from one fourth, one third, half or a two third of the adult 15 total daily dose. The said dose can be administered as a single dosage form or as multiple dosage forms equating to the required dose.
In yet another embodiment, the present disclosure provides a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof wherein the 20 composition when administered to a patient in need thereof reduces viral load and improves the clinical signs and symptoms of SARS-CoV-2 infection.
In one embodiment the present disclosure provides a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof for use in resolution of 25 clinical signs and symptoms of SARS-CoV-2 viral infection in a patient, wherein the resolution is characterized by radiological improvement with a documented virological clearance in 2 samples at least 24 hours apart.
In another embodiment the present disclosure provides a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, 30 Makisterone-A, 20-Hydroxyecdysone or a combination thereof for use in resolution of clinical signs and symptoms of SARS CoV-2 infection in a patient wherein the resolution is characterized by time to normalization of fever without use of one or more antipyretics in last 24 hours.
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In yet another embodiment the present disclosure provides a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof for eradicating the SARS-CoV-2 viral infection from a patient infected by such virus wherein the eradication is characterized by a negative SARS-CoV-2 RT-PCR of an upper or lower respiratory tract 5 specimen from such patient. In a further embodiment the present disclosure provides a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof for clinical improvement of the signs and symptoms of SARS-CoV-2 viral infection in a patient by administering the extract in an oral dose of 600 mg twice daily, 400 mg thrice daily, 300 10 mg four times daily or 200 mg six times daily. The said dose can be administered as a single dosage form or as multiple dosage forms equating to the required dose.
In one embedment the present disclosure provides a stable, pharmaceutical composition comprising an extract of Cocculus hirsutus comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof for 15 improving the clinical symptoms of SARS-CoV-2 infection, wherein the symptoms include cough, fever, shortness of breath, fatigue, expectoration, Myalgia, Rhinorrhea, Sore throat, diarrhea, Loss of smell (anosmia) or loss of taste (ageusia) preceding the onset of respiratory symptoms.
In one embodiment the stable, pharmaceutical composition according to present 20 disclosure comprises a therapeutically effective amount of a Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A and 20-Hydroxyecdysone or a combination thereof, for alleviating the symptoms of SARS-CoV-2 viral infection in a human.
In another embodiment the stable, pharmaceutical composition according to 25 present disclosure comprises a therapeutically effective amount of Cocculus hirsutus extract for improving the clinical signs and symptoms of a human patient infected with SARS-CoV-2 virus, wherein the improvement in clinical symptoms is characterized by any one or more of the measures selected from: reduction in duration of supplemental oxygen requirement; reduction in duration of mechanical ventilation; reduction in time to alleviation of cough; 30 reduction in time to normalization of fever without use of antipyretics; reduction in duration of hospitalization; or reduction in time to first negative SARS-CoV-2 RT-PCR in upper or lower respiratory tract specimen.
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In another embodiment, the present disclosure provides a method of treatment of SARS-CoV-2 virus infection by administering the extract of Cocculus hirsutus in range of about 2 mg/kg to about 150 mg/kg body weight. In a preferred embodiment, the said extract is a purified extract comprising one or more of Sinococuline, Magnoflorine, 20-Hydroxyecdysone or Makisterone-A. In an alternative embodiment, the said extract is a 5 crude extract of Cocculus hirsutus.
In an embodiment, the present disclosure provides stable pharmaceutical composition comprising a therapeutically effective amount of an extract of Cocculus hirsutus comprising Magnoflorine not less than 0.1% w/w, Sinococuline not less than 1.0% w/w, 20-Hydroxyecdysone not less than 0.1% w/w and Makisterone-A not less than 10 0.05% w/w of the said extract for treating SARS-CoV-2 viral infection by administering the composition to a patient in need thereof. The said composition may also be used alleviating the symptoms of SARS-CoV-2 virus infection in a patient.
In one embodiment, oral administration of the pharmaceutical composition according to the present disclosure provides: 15
a) maximum plasma concentration (Cmax) of Sinococuline at day 1 after a single oral dose administration of 100 mg of the said extract from about 1.4 ng/mL to about 110.0 ng/mL, after a single oral dose administration of 200 mg of the said extract from about 4.2 ng/mL to about 135.0 ng/mL, after a single oral dose administration of 400 mg of the said extract from about 8.0 20 ng/mL to about 205.0 ng/mL; or after a single oral dose administration of 600 mg of the said extract from about 14.0 ng/mL to about 240.0 ng/mL; or after a single oral dose administration of 800 mg of the said extract from about 13.0 ng/mL to about 255.0 ng/mL; or
b) AUC0-24 of Sinococuline at day 1 after a TID oral dose administration of 25 100 mg of the said extract from about 20.0 h*ng/mL to about 575.0 h*ng/mL, after a TID oral dose administration of 200 mg of the said extract from about 88.0 h*ng/mL to about 1250.0 h*ng/mL, after a TID oral dose administration of 400 mg of the said extract from about 135.0 h*ng/mL to about 2360.0 h*ng/mL; or after a TID oral dose administration of 600 mg 30 of the said extract from about 263 h*ng/mL to about 2435.0 h*ng/mL; or after a TID oral dose administration of 800 mg of the said extract from about 226.0 h*ng/mL to about 3150.0 h*ng/mL.
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In yet another embodiment, oral administration of the stable pharmaceutical composition according to the present disclosure provides:
a) maximum plasma concentration (Cmax) of Sinococuline at day 10 after a single oral dose administration of 100 mg of the said extract from about 5.0 ng/mL to about 90.0 ng/mL, after a single daily oral 5 dose administration of 200 mg of the said extract from about 13.5 ng/mL to about 300.0 ng/mL, after a single daily oral dose administration of 400 mg of the said extract from about 20.0 ng/mL to about 500.0 ng/mL; or after a single daily oral dose administration of 600 mg of the said extract from about 29.0 ng/mL to about 604.0 10 ng/mL; or after a single daily oral dose administration of 800 mg of the said extract from about 35.05 ng/mL to about 705.0 ng/mL; or
b) AUC0-24 of Sinococuline at day 10 of a single daily oral dose administration of 100 mg of the said extract from about 67.0 h*ng/mL to about 1090.0 h*ng/mL, after a single daily oral dose 15 administration of 200 mg of the said extract from about 148.0 h*ng/mL to about 2515.0 h*ng/mL, at a single daily oral dose administration of 400 mg of the said extract from about 251.0 h*ng/mL to about 5075.0 h*ng/mL; or at a single daily oral dose administration of 600 mg of the said extract from about 390.0 20 h*ng/mL to about 5205.0 h*ng/mL; or at a single daily oral dose administration of 800 mg of the said extract from about 474.0 h*ng/mL to about 5730.0 h*ng/mL;
c) Trough plasma levels at steady state (Cτ,ss) of Sinococuline from day 3-10 ranging from about 2.8 to about 31.0 ng/mL at a thrice 25 daily oral dose administration of 100-400 mg of the said extract, wherein the steady state is achieved at around day 3.
In one of the preferred embodiments the present disclosure provides a stable pharmaceutical composition comprising an extract of Cocculus hirsutus comprising Sinococuline, wherein an area under the plasma concentration time curve to infinity 30 (AUC∞) of Sinococuline is about 109 h*ng/ml to about 520 h*ng/ml after oral administration of a dose of 100 mg of the extract to a human subject at day 10.
In another embodiment the present disclosure provides a stable pharmaceutical composition comprising 400 mg extract of Cocculus hirsutus, wherein single oral
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administration of the said composition to a human subject provides maximum plasma concentration (Cmax) of Sinococuline at day 1 from about 8.0 ng/mL to about 205.0 ng/mL, or AUC0-24 of Sinococuline at day 1 from about 135.0 h*ng/mL to about 2360.0 h*ng/mL.
In yet another embodiment the present disclosure provides a stable pharmaceutical composition comprising 600 mg extract of Cocculus hirsutus, wherein single oral 5 administration of the said composition to a human subject provides maximum plasma concentration (Cmax) of Sinococuline at day 1 from about 140.0 ng/mL to about 240.0 ng/mL, or AUC0-24 of Sinococuline at day 1 from about 263.0 h*ng/mL to about 2435.0 h*ng/mL.
In one embodiment, the present disclosure provides a composition that is 10 bioequivalent to the said composition as described in above embodiments. The term “bioequivalent” as used in the present specification means the absence of a significant difference in the rate and extent to which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives becomes available at the site of drug action when administered at the same molar dose under similar conditions in an 15 appropriately designed study. Two compositions can be considered as “bioequivalent” if the 90% Confidence Interval of the relative mean Cmax and AUC of the test to reference is within 70% to 130% or 74% to 124% or 75% to 125% or 80.00% to 125.00%.
The term "AUC" as used herein, means area under the plasma concentration-time curve, as calculated by the trapezoidal rule over the complete dosing interval, e.g., 24-hour 20 interval. The term "Cmax" as it is used herein is the highest plasma concentration of the drug attained within the dosing interval. The term "AUC(0-t)" or “AUC0-24” as used herein means the area under the plasma concentration-time curve using linear trapezoidal summation from time zero to time t post-dose, where t is the time of the last measurable concentration Ct), for e.g., 24 hours. The term "AUC(0-∞)" or “AUC∞” as used herein 25 means the area under the plasma concentration-time curve from time 0 to infinity, AUC(0-∞) = AUC(0-t) + Ct/Kel. (Kel is the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration vs. time curve. The term “trough levels” as (Cτ,ss), used herein refers to lowest concentration reached by a drug before administration of next dose. 30
In some embodiments, the term “extraction” refers to the separation and removal of one or more components of Cocculus hirsutus, e.g., plant solids (e.g., fibers, cellulose, etc.) extracted from one or more fluids in the plant. In some embodiments, the extraction is a solid/liquid separation operation: e.g., a plant is placed in contact with a fluid (a solvent).
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In some embodiments, the plant components of interest are solubilised into solution with the solvent. The solution thus obtained is the desired extract. In some embodiments, the solvent will eventually be eliminated to arrive at the extract. Separation operations can include mechanical means, e.g., homogenization, chemical means, e.g., acid, alcohol, or aqueous solubilization, and heating means. In some embodiments, the extraction includes 5 a filtration, precipitation, crystallization, concentration, or centrifugation step. In a preferred embodiment, extraction may result into preferentially enriching the said extract with one or more of Sinococuline, Magnoflorine, 20-Hydroxyecdysone or Makisterone-A.
In another embodiment, the extract of the present disclosure is an aqueous extract or an organic solvent extract, wherein the organic solvent is a polar or non-polar organic 10 solvent. In an aspect of the embodiment, the extraction is an alcoholic extraction, e.g., a C1-C4 alcohol extraction, a hydroalcoholic extraction, or an aqueous extraction. In some embodiment, carious parts of Cocculus hirsutus, can be used, e.g., the extraction can be performed from stem or other parts of the plant, such as aerial parts or roots. In one aspect the extract is an aqueous extract. The solvents in the extract may be removed completely 15 by evaporation to obtain a dried extract. The dried extract may be lyophilized to form a powder, which can then be filled into a capsule of suitable size or compressed into tablets with or without pharmaceutically acceptable excipients. In a related embodiment, the extract may be used as such in liquid or semisolid form without further drying along with a suitable pharmaceutically acceptable carrier for administration. In one embodiment, the 20 extract is a purified extract. In another embodiment, the extract is a crude extract.
In another aspect of the above embodiment, the extract is an alcoholic extract, or a hydro-alcoholic extract from stem or other parts of the plant, such as aerial parts or roots. In one embodiment, the extract will be derived from wet parts of the plant to arrive at an aqueous extract. The solvents in the extract may be removed completely, e.g., by 25 evaporation to obtain a dried extract. The dried extract may be lyophilized to form a powder, which can then be filled into a vial or a capsule of suitable size or compressed into tablets with or without pharmaceutically acceptable excipients. In a related embodiment, the extract may be used as such in liquid or semisolid form without further drying along with a suitable pharmaceutically acceptable carrier for administration. 30
The term “alcoholic extract,” as used herein, includes any alcohol-based extract, for example, methanolic, ethanolic, n-propanolic, isopropanolic, n-butanolic, iso-butanolic or t-butanolic extract of Cocculus hirsutus.
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The term “hydroalcoholic extract,” as used herein, includes an extract prepared by using a mixture of alcohol and purified water. It may also include an extract prepared in denatured spirit with other organic solvents. Examples of alcohols are methanol, ethanol, n-propanol, isopropanol, n-butanol, iso-butanol, and t-butanol. The ratio of alcohol to water in the hydroalcoholic extract may be in the ratio of 99:1 to 1:99, or 95:5 to 5:95, or 5 90:10 to 10:90, or 80:20 to 20:80, or 70:30 to 30:70, or 60:40 to 40:60, or a 1:1 mixture of alcohol and purified water.
The term “aqueous extract,” as used herein, includes a purified water extract of Cocculus hirsutus, also abbreviated as AQCH (Aqueous extract of Cocculus hirsutus).
The extracts of Cocculus hirsutus include (a) the extracts obtained by extraction of 10 plant mass of Cocculus hirsutus with one or more solvents, and (b) the fractions obtained by partitioning of the extracts with one or more solvents. In a preferred embodiment, the extracts of Cocculus hirsutus include (a) the extracts obtained by extraction of stem of Cocculus hirsutus with purified water, and (b) the fractions obtained by partitioning of the extracts with one or more solvents. 15
The solvents for extraction may be, for example, water; alcohols, for example, methanol, ethanol, propanol, isopropanol or butanol; ketones, for example, acetone or methyl isobutyl ketone; esters, for example, methyl acetate or ethyl acetate; halogenated hydrocarbons, for example, chloroform, dichloromethane or ethylene dichloride; petroleum fractions, for example, hexane, petroleum ether or heptane; or mixture(s) thereof. 20
The solvents for partitioning may be, for example, water; petroleum fractions, for example, hexane, petroleum ether or heptane; halogenated hydrocarbons, for example, chloroform, dichloromethane or ethylene dichloride; esters, for example, ethyl acetate or methyl acetate; ketones, for example, acetone or methyl isobutyl ketone; alcohols, for example, butanol; ethers, for example, diethyl ether; or mixture(s) thereof. 25
The term “plant mass of Cocculus hirsutus,” as used herein, refers to the whole plant, which includes aerial parts, for example, fruits, flowers, leaves, branches, stem bark, stems, seeds or heartwood, and roots. In a preferred embodiment, the “plant mass of Cocculus hirsutus” refers to stem of Cocculus hirsutus.
In yet another embodiment, the present disclosure provides enriched fraction of the 30 extract for use in treatment of SARS-CoV-2 infection in mammals. The extract may be enriched with respect to any of the flavonoid, alkaloids, steroids for e.g., Alkaloids like Sinococuline and Magnoflorine, steroids like Makisterone-A and 20-hydroxyecdysne or a combination thereof. In one aspect the extract may be enriched and standardized with
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respect to marker compounds, for e.g., Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or combination thereof. The enriched extract may be prepared by using the respective solvent for the marker compound(s). The solvent may be any of the solvent as disclosed above.
In another embodiment, the present disclosure provides a composite extract of 5 Cocculus hirsutus to reduce the viral load at an early stage in the treatment of COVID-19 infection in mammals.
In one embodiment, the present disclosure provides extract of Cocculus hirsutus or pharmaceutical composition thereof for treatment of mild to moderate COVID-19 infection. In yet another embodiment the present disclosure provides extract of Cocculus hirsutus or 10 pharmaceutical composition thereof for treatment of moderate to severe COVID-19 infection or SARS-CoV-2 infection. In another embodiment, the present disclosure provides a pharmaceutical composition comprising an extract of Cocculus hirsutus for clinical improvement of the signs and symptoms of SARS-CoV-2 virus infection in a patient.
The patient with clinical severity of SARS-CoV-2 infection is categorized as mild, 15 moderate or severe, wherein:
1) Mild infection is said to be with clinical presentation as patients with uncomplicated upper respiratory tract infection, may have mild symptoms such as fever, cough, sore throat, nasal congestion, malaise, headache and clinical parameters like no evidence of breathlessness or hypoxia; 20
2) Moderate infection is said to be with pneumonia with no signs of sever disease and clinical parameters in adults with presence of clinical features of dyspnea and or hypoxia, fever, cough, including SpO2 ≤93% on room air, Respiratory Rate more or equal to 24 per minute and in case of child with presence of clinical features of dyspnea and or hypoxia, fever, cough, 25 including SpO2 <94% (range 90-94%) on room air, Respiratory Rate more or equal to 24 per minute; Fast breathing (in breaths/min): < 2 months: ≥ 60; 2-11 months: ≥ 50; 1-5 years: ≥ 40.
3) Severe is said to be with:
- Sever Pneumonia, clinical parameters with clinical signs of Pneumonia 30 plus one of the following; respiratory rate >30 breaths/min, severe respiratory distress, SpO2 <90% on room air and in case of child with cough or difficulty in breathing, plus at least one of the following: central cyanosis or SpO2 <90%; severe respiratory distress (e.g. grunting, chest
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in- drawing); signs of pneumonia with any of the following danger signs: inability to breastfeed or drink, lethargy or unconsciousness, or convulsions. Other signs of pneumonia may be present: chest in drawing, fast breathing (in breaths/min): <2 months ≥60; 2– 11 months ≥50; 1–5 years ≥40; The diagnosis is clinical; chest imaging can 5 exclude complications;
- Acute respiratory distress syndrome, clinical parameters at onset: new or worsening respiratory symptoms within 1 week of known clinical consult; chest imaging (Chest X ray and portable) bed side lung ultrasound): bilateral opacities, not fully explained by effusions, lobar 10 or lung collapse, or nodules; Origin of Pulmonary infiltrates: respiratory failure not fully explained by cardiac failure or fluid overload. Need objective assessment (e.g. echocardiography) to exclude hydrostatic cause of infiltrates/ oedema if no risk factor present; Oxygenation impairment in adults: Mild ARDS: 200 mmHg < PaO2/FiO2 ≤ 300 15 mmHg (with PEEP or CPAP ≥5 cm H2O) Moderate ARDS: 100 mmHg < PaO2/FiO2 ≤200 mmHg with PEEP ≥5 cm H2O), Severe ARDS: PaO2/FiO2 ≤ 100 mmHg with PEEP ≥5 cm H2O), When PaO2 is not available, SpO2/FiO2 ≤315 suggests ARDS (including in non- ventilated patients); Oxygenation impairment in Children Note Oxygenation Index 20 (OI) and OSI (Oxygen Saturation Index); Use OI when available. If PaO2 not available, wean FiO2 to maintain SpO2 <_97% to calculate OSI or SpO2/FiO2 ratio: using SpO2); Bi-level (NIV or CPAP) ≥5 cm H2O via full face mask: PaO2/FiO2 ≤ 300 mmHg or SpO2/FiO2 ≤264; Mild ARDS (invasively ventilated): 4 ≤ OI < 8 or 5 ≤ OSI < 7.5; 25 Moderate ARDS (invasively ventilated): 8 ≤ OI < 16 or 7.5 ≤OSI < 12.3; Severe ARDS (invasively ventilated): OI ≥ 16 or OSI ≥ 12.3;
- Sepsis, clinical parameters in adults as acute life-threatening organ dysfunction caused by a dysregulated host response to suspected or proven infection. Signs of organ dysfunction include: altered mental 30 status, difficult or fast breathing, low oxygen saturation, reduced urine output, fast heart rate, weak pulse, cold extremities or low blood pressure, skin mottling, or laboratory evidence of coagulopathy, thrombocytopenia, acidosis, high lactate or hyperbilirubinemia and in
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Children: suspected or proven infection and ≥2 age based Systemic Inflammatory Response Syndrome (SIRS) criteria, of which one must be abnormal temperature or white blood cell count;
- Septic shock, clinical parameters in adult with persisting hypotension despite volume resuscitation, requiring vasopressors to maintain MAP 5 ≥65 mmHg and serum lactate level > 2 mmol/L; And in children: any hypotension (SBP <5th centile or >2 SD below normal for age) or 2- 3 of the following: altered mental state; bradycardia or tachycardia (HR <90 bpm or >160 bpm in infants and HR <70 bpm or >150 bpm in children); prolonged capillary refill (>2 sec) or weak pulse; tachypnea; 10 mottled or cool skin or petechial or purpuric rash; high lactate; reduced urine output ; hyperthermia or hypothermia.
The extract of Cocculus hirsutus according to the present disclosure was surprisingly found to be useful both to be directly administrated to a mammal and to be used in the preparation of a pharmaceutical composition, with the dose in the range of 15 approximately 0.05 mg/kg to approximately 1500 mg/kg body weight, particularly in the range of approximately 0.1 mg/kg to approximately 1200 mg/kg body weight, more particularly in the range of approximately 1 mg/kg to approximately 500 mg/kg body weight, more particularly in the range of approximately 2mg/kg to approximately 150 mg/kg body weight. The composite extract or its composition may be administered once, 20 twice, thrice or four times a day in patients with mild to severe COVID-19 infection, preferably mild to moderate COVID-19 infection with Standard of Care therapy.
The term “Standard of Care” or “SOC” as used herein the specification, refers to standard or routine care administered to patient by a health care professional for the treatment of viral infections, in particular SARS-CoV-2, including, but not limited to, 25 medication for fever, inflammation, antitussives, adequate nutrition, hypoxia including antipyretics, methyl prednisolone, dexamethasone, hydroxychloroquine, ventilation, oxygen support therapy or any other medication or treatment as instructed in an institutional protocol of various Clinical setup depending upon patients conditions and need of the therapy. 30
In one embodiment, the pharmaceutical the composition according to the present disclosure comprises a therapeutically effective amount of the extract of Cocculus hirsutus. In on embodiment the composition according to the present disclosure is administered to a patient or mammal in need thereof in a dosage range of about 25 mg to about 1000 mg for
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once, twice or thrice or four times daily. The composition may be administered in a dosage range of about 25 mg to about 1000 mg per day, such as for example 25mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg 950 mg or 1000 mg for once, twice or thrice a day. In one embodiment the composition according to present disclosure 5 is administered at a dose of 400 mg, 600 mg or 800 mg for once, twice or thrice daily or four times daily. In one embodiment wherein the extract or the a composition according to present disclosure is administered to a patient in need thereof as 600 mg twice daily, 400 mg thrice daily, 300 mg four times daily or 200 mg six times daily. In another embodiment the extract or the composition is administered to a patient in need thereof as 10 600 mg twice daily, 400 mg thrice daily, 300 mg four times daily or 200 mg six times daily, wherein the said dose can be administered as single dosage form or as multiple dosage forms equating to the required dose i.e., two 100 mg strengths can be combined to form a single 200 mg dosage or a 300 mg and a 100 mg strengths can be combined to give a single administration of 400 mg or one or more 100 mg strengths can be combined to 15 form a higher dose.
In an embodiment the present disclosure provides pharmaceutical composition of Cocculus hirsutus extract, wherein the composition comprises about 10-80% of the extract by weight of the tablet. In another embodiment the present disclosure provides pharmaceutical composition of Cocculus hirsutus extract, wherein the composition 20 comprises about 15-70% of the extract by weight of the tablet.
The term "mammal" herein refers to all mammals, by way of example only, humans, non-human primates, cows, dogs, cats, goats, sheep pigs, rats, mice and rabbits.
In another embodiment, the present disclosure provides a pharmaceutical composition for use in the prevention and treatment of SARS-CoV-2 virus infection in 25 mammals comprising a composite extract of Cocculus hirsutus and one or more pharmaceutically acceptable excipients. In one aspect of the above embodiment, the present disclosure provides a stable pharmaceutical composition comprising a therapeutically effective amount of a composite extract of Cocculus hirsutus for use in the prevention and treatment of SARS-CoV-2 in a mammal, wherein the composition when 30 administered to a mammal in need thereof reduces the viral load.
The stable pharmaceutical composition of the present disclosure further comprises one or more pharmaceutically acceptable excipient. The term “pharmaceutical composition,” as used herein, includes any composition that can effectively deliver the
21
extracts of Cocculus hirsutus to the desired site of action to treat or prevent SARS-CoV-2 infection.
The extract and pharmaceutical compositions described herein can be administered via various modes of delivery, e.g., oral delivery, enteral/gastrointestinal delivery, parenteral delivery, intravenous delivery, topical delivery, rectal delivery, vaginal 5 delivery, ophthalmic delivery, transmucosal delivery, nasal, pulmonary, or transdermal. The pharmaceutical composition includes one or more pharmaceutically acceptable excipients. The oral pharmaceutical composition can be in the form of powder, pellets, granules, spheroids, mini-tablets, caplets, tablets, or capsules. The powder can be in the form of a lyophilized powder filled, with pharmaceutically acceptable excipients, into a 10 capsule of suitable size. Preferably, the pharmaceutical composition is in the form of a tablet. The oral pharmaceutical composition can be present in the form of liquid, including but not limited to solutions, suspensions, emulsions or syrups.
In another embodiment the pharmaceutical composition comprising extract according to present disclosure is an oral dosage form selected from powder, pellets, 15 granules, spheroids, mini-tablets, caplets, tablets, or capsules. In an aspect the pharmaceutical composition comprising extract according to present disclosure is storage stable at accelerated condition of 40± 2°C/75±5% RH, long term storage condition of 30±2°C/75±5% RH or at 25±2°C/75±5% RH for at least 3 months. In an aspect the pharmaceutical composition comprising extract according to present disclosure comprises 20 one or more of the pharmaceutically acceptable excipients selected from diluents, binders, disintegrants, lubricants, glidants, polymers, flavoring agents, surfactants, solvents, suspending agents, stabilizers, preservatives, antioxidants, buffers, and tonicity modifying agents.
A “stable pharmaceutical composition” as used herein refers to a composition 25 which is stable over extended period of time on storage as assessed from the content of one or more impurities in the composition as described in standard textbooks. The stable pharmaceutical composition of the present disclosure were found to be stable for at least 3 months at accelerated condition of 40± 2°C/75±5% RH; and for at least 3 months at long term storage condition of 30±2°C/75±5% RH and at 25±2°C/75±5% RH. Alternatively, 30 the product can be stored at room temperature for a shelf life of 6 months to 2 years. Surprisingly, the composition was stable despite the presence of flavonoids, alkaloids, lignans, etc., as constituents in the aqueous extract which together may be difficult to formulate and may not be stable during storage.
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A “therapeutically effective amount” as used herein refers to an amount of the extract of the disclosure sufficient to provide a benefit in the treatment or prevention of COVID-19, to delay or minimize symptoms associated with the infection or to cure or ameliorate the infection or cause thereof. In particular, a therapeutically effective amount means an amount sufficient to provide a therapeutic benefit in-vivo. 5
The term “pharmaceutically acceptable excipients,” as used herein, includes diluents, binders, disintegrants, lubricants, glidants, polymers, flavoring agents, surfactants, preservatives, antioxidants, buffers, and tonicity modifying agents.
In another embodiment the pharmaceutical composition comprising extract according to present disclosure comprises intragranular excipients in a concentration range 10 of about 8% to about 45% by weight of the composition and extragranular excipients in a concentration range of about 6% to about 38% by weight of the composition. In an aspect the pharmaceutical composition comprising extract according to present disclosure comprises diluent in a concentration range of about 5% to about 50% by weight of the composition and disintegrant in a concentration range of about 2% to about 33% by 15 weight of the composition. In an aspect the pharmaceutical composition comprising extract according to present disclosure comprises microcrystalline cellulose about 6% to about 42% by weight of the composition, anhydrous lactose about 0.5% to about 28% by weight of the composition, croscarmellose sodium about 2% to about 3% by weight of the composition, colloidal silicon dioxide about 0.05% to about 12% by weight of the 20 composition and magnesium stearate about 0.05% to about 14% by weight of the composition.
Non-limiting examples of diluents include microcrystalline cellulose, powdered cellulose, starch, pregelatinised starch, dextrates, lactitol, fructose, sugar compressible, sugar confectioners, dextrose, anhydrous lactose, calcium phosphate-dibasic, calcium 25 phosphate-tribasic, calcium sulfate, and mixtures thereof.
Non-limiting examples of binders include a water-soluble starch, for example, pregelatinized starch; a polysaccharide, for example, agar, gum acacia, dextrin, sodium alginate, tragacanth gum, xanthan gum, hyaluronic acid, pectin, or sodium chondroitin sulfate; a synthetic polymer, for example, polyvinylpyrrolidone, polyvinyl alcohol, 30 carboxyvinyl polymer, polyacrylic acid-series polymer, polylactic acid, or polyethylene glycol; a cellulose ether, for example, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, or hydroxypropyl methyl cellulose; and mixtures thereof.
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Non-limiting examples of disintegrants include calcium carbonate, carboxymethyl cellulose or a salt thereof, for example, croscarmellose sodium, crosslinked povidone, low-substituted hydroxypropyl cellulose, and sodium starch glycolate.
Non-limiting examples of lubricants/glidants include talc, magnesium stearate, hydrogenated vegetable oils, sodium stearyl fumarate, calcium stearate, colloidal silicon 5 dioxide, Aerosil®, stearic acid, sodium lauryl sulphate, sodium benzoate, polyethylene glycol, hydrogenated castor oil, sucrose esters of fatty acids, microcrystalline wax, yellow beeswax, white beeswax, and mixtures thereof.
Non-limiting examples of flavoring agents include synthetic flavor oils and flavoring aromatics; natural oils or extracts from plants, leaves, flowers, and fruits; and 10 combinations thereof. These may include cinnamon oil, oil of wintergreen, peppermint oils, bay oil, anise oil, eucalyptus, thyme oil, vanilla, citrus oil, including lemon, orange, lime, and grapefruit, and fruit essences including apple, banana, grape, pear, peach, strawberry, raspberry, cherry, plum, pineapple, and apricot.
Non-limiting examples of surfactants include anionic surfactants, for example, a 15 sulfonic acid or a salt thereof such as benzenesulfonic acid, dodecylbenzenesulfonic acid, or dodecanesulfonic acid; an alkyl sulfate, for example, sodium dodecyl sulfate or sodium lauryl sulfate; cationic surfactants, for example, a tetraalkylammonium salt such as a tetraalkylammonium halide, benzethonium chloride, benzalkonium chloride, or cetylpyridinium chloride; a nonionic surfactant, for example, a (poly) oxyethylene sorbitan 20 long-chain fatty acid ester such as a polyoxyethylene sorbitan monolaurate, for example, a polysorbate; amphoteric surfactants, for example, a glycine compound such as dodecyl-di-(aminoethyl)glycin, a betaine compound such as betaine or dimethyldodecylcarboxybetaine, and a phosphatidic acid derivative such as lecithin; polymeric surfactants, for example, a polyoxyethylene polyoxypropylene glycol such as 25 Pluronic® or poloxamer; and mixtures thereof.
Non-limiting examples of buffers include phosphate buffers such as dihydrogen sodium phosphate, citrate buffers such as sodium citrate, meglumine, tri(hydroxymethyl) aminomethane, and mixtures thereof.
Non-limiting examples of tonicity modifying agents include sodium chloride, 30 mannitol, dextrose, glucose, lactose, sucrose, and mixtures thereof.
Non-limiting examples of solvents for the preparation of the pharmaceutical composition include water; water miscible organic solvents, for example, isopropyl alcohol or ethanol; protic solvent or dipolar aprotic solvents; methylene chloride; acetone;
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polyethylene glycol; polyethylene glycol ether; polyethylene glycol derivatives of a mono- or di-glyceride; buffers; organic solvents; and combinations thereof.
The pharmaceutical excipient as used in the present disclosure can be used interchangeably for various roles in the pharmaceutical composition, and are not limited by their application as widely known. For example a diluent may be used as binder in 5 particular concentration.
In a preferred embodiment, the pharmaceutically acceptable excipient in the composition of the disclosure includes microcrystalline cellulose, anhydrous lactose, croscarmellose sodium, colloidal silicon dioxide and magnesium stearate.
In another embodiment, the present disclosure provides a stable pharmaceutical 10 composition comprising a therapeutically effective amount of a composite extract of Cocculus hirsutus for use in the treatment of SARS-CoV-2 infection in a mammal, wherein the composition when administered to a mammal in need thereof reduces the viral load.
In a further embodiment, the present disclosure provides a pharmaceutical 15 composition comprising a composite extract of Cocculus hirsutus and one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of SARS-CoV-2 infection in mammals. Preferably, the composition is a stable pharmaceutical composition. More preferably, the composition is a stable oral pharmaceutical composition. 20
In yet another embodiment, the present disclosure provides a stable pharmaceutical composition comprising a therapeutically effective amount of a composite extract of Cocculus hirsutus for use in the treatment of SARS-CoV-2 infection in a mammal, wherein the composition when administered to a mammal in need thereof is effective in a delayed treatment onset. 25
In a further embodiment, the present disclosure provides a stable pharmaceutical composition comprising a therapeutically effective amount of an extract of Cocculus hirsutus for use in the prevention of SARS-CoV-2 virus infection in a mammal.
In one embodiment, the extract of Cocculus hirsutus comprises one or more constituents selected from the group consisting of flavonoids, lignans and alkaloids or 30 combinations thereof. Preferably, the extract of Cocculus hirsutus comprises Magnoflorine as one of the alkaloids. More preferably, the composite extract of Cocculus hirsutus comprises Magnoflorine in an amount of 0.1% to 1% of the total weight of extract in the composition. In a preferred embodiment, the composite extract of Cocculus hirsutus
25
comprises Magnoflorine in an amount of 0.45% of the total weight of extract in the composition.
In another aspect of the embodiment, the extract of Cocculus hirsutus comprises quercetin as one of the flavonoids. In another aspect of the embodiment, the extract of Cocculus hirsutus comprises Sinococuline as one of the alkaloid. 5
In another embodiment, the present disclosure provides an extract of a plant of Menispermeaceae family, wherein the extract comprises Sinococuline from about 0.5% w/w to about 15% w/w, Magnoflorine from about 0.05% w/w to about 3% w/w, Makisterone-A from about 0.01% w/w to about 3% w/w, 20-Hydroxyecdysone from about 0.05% w/w to about 3% w/w., or a combination thereof. In a preferred embodiment, the 10 extract comprises Sinococuline not less than 1.0 % w/w, Magnoflorine not less than 0.1% w/w, 20-Hydroxyecdysone not less than 0.05%, Makisterone not less than 0.05% w/w, or a combination thereof.
In an aspect the composition of the present disclosure improves the clinical symptoms of human patient infected with SARS-CoV-2 virus, wherein the improvement 15 in clinical symptoms is characterized by any one or more of the measures selected from: reduction in duration of supplemental oxygen requirement; reduction in duration of mechanical ventilation; reduction in time to alleviation of cough; reduction in time to normalization of fever without use of antipyretics; reduction in duration of hospitalization; or reduction in time to first negative SARS-CoV-2 RT-PCR in upper or lower respiratory 20 tract specimen.
In a further embodiment, the extract and composition of the present disclosure were surprisingly found to be safe and didn’t show any toxic effect when administered in a therapeutically effective dose to the mammal in need thereof.
In yet another embodiment the pharmaceutical composition according to present 25 disclosure comprising the Cocculus hirsutus extract were found to be safe and with minimal adverse events in the dosage range of about 25 mg to about 1000 mg once, twice or thrice or four times daily, more preferably in the range of about 200 to about 600 mg once, twice or thrice or four times daily.
In one embodiment, the present disclosure provides a method of treating SARS-30 CoV-2 virus infection in a mammal comprising administering a pharmaceutical composition comprising a therapeutic effective amount of an extract of Cocculus hirsutus to a mammal in need thereof, wherein the extract reduces the viral load and improves the clinical signs and symptoms of infection.
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In yet another embodiment, the present disclosure provides a method for reducing the viral load at an early stage in the treatment of SARS-CoV-2 virus infection in mammals comprising administering an extract of Cocculus hirsutus to the mammals in need thereof.
In another embodiment, the present disclosure provides a method of treating SARS-CoV-2 virus infection in mammals comprising administering an extract of 5 Cocculus hirsutus to the mammals in need thereof. In one embodiment, the present disclosure provides a method for reducing the SARS-CoV-2 viral load in a patient suffering from such viral infection.
In another embodiment the present disclosure provides a method for improving clinical signs and symptoms of SARS-CoV-2 viral infection by administering a 10 pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof wherein the composition when administered to a patient in need thereof reduces the SARS-CoV-2 viral load.
In another embodiment the present disclosure provides a method for resolution of 15 clinical signs and symptoms of SARS-CoV-2 viral infection in a patient, by administering a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof, wherein the resolution is characterized by radiological improvement with a documented virological clearance in 2 samples at least 24 hours apart. 20
In another embodiment the present disclosure provides a method for resolution of clinical signs and symptoms of SARS-CoV-2 viral infection in a patient, by administering a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof, wherein the resolution is characterized by time to normalization of fever without 25 use of one or more antipyretics in last 24 hours.
In another embodiment the present disclosure provides a method for eradicating the SARS-CoV-2 viral infection in a patient, by administering a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone, 20-Hydroxyecdysone or a combination thereof, wherein the 30 eradication is determined by a negative SARS-CoV-2 RT-PCR of an upper or lower respiratory tract specimen from such patient.
In another embodiment the present disclosure provides a method for clinical improvement of the signs and symptoms of SARS-CoV-2 viral infection in a patient, by
27
administering a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof, in a dosage range of 100-800 mg once, twice, thrice or four times daily.
The clinical signs and symptoms as used herein include one or more symptoms comprising cough, fever, shortness of breath, fatigue, expectoration, Myalgia, Rhinorrhea, 5 Sore throat, diarrhea, Loss of smell (anosmia) or loss of taste (ageusia) preceding the onset of respiratory symptoms, duration of supplemental oxygen requirement, duration of mechanical ventilation, duration of hospitalization, time to first negative SARS-CoV-2 RT-PCR in upper or lower respiratory tract specimen.
In an embodiment the present disclosure provides a method for treating SARS-10 CoV-2 viral infection by administering to a patient in need thereof a stable, anti-COVID composition comprising a therapeutically effective amount of an extract of Cocculus hirsutus comprising Magnoflorine not less than 0.1% w/w, Sinococuline not less than 1.0% w/w, 20-Hydroxyecdysone not less than 0.1% w/w and Makisterone-A not less than 0.05% w/w for alleviating the symptoms of SARS-CoV-2 virus infection in a human patient. 15
In a further embodiment, the present disclosure provides a method of inhibiting the secretion of cytokines in SARS-CoV-2 infection in mammals comprising administering an extract of Cocculus hirsutus to the mammals in need thereof.
In yet another embodiment, the present disclosure provides a method of treatment of SARS-CoV-2 virus infection in mammals comprising administering a composite extract 20 of Cocculus hirsutus to the mammals in need thereof, wherein the extract is effective in a delayed treatment onset.
In yet another embodiment, the present disclosure provides a method of prevention of SARS-CoV-2 virus infection in mammals comprising administering an extract of Cocculus hirsutus to the mammals in need thereof. 25
In one embodiment, the present disclosure provides use of a pharmaceutical composition comprising a therapeutic effective amount of an extract of Cocculus hirsutus for reducing the viral load in a method of treating SARS-CoV-2 virus infection in a mammal.
In yet another embodiment, the present disclosure provides use of an extract of 30 Cocculus hirsutus for a method of reducing the viral load at an early stage in the treatment of SARS-CoV-2 virus infection in mammals by administering the said composition.
In another embodiment, the present disclosure provides use of an extract of Cocculus hirsutus for administering to a mammal for treatment of SARS-CoV-2 virus infection,
28
wherein the composition reduces the SARS-CoV-2 viral load in the patient suffering from such viral infection.
In another embodiment, the method of treatment according to present disclosure comprise administration of the extract or the pharmaceutical composition according to present disclosure in an oral dosage form and is administered once, twice, thrice or four 5 times a day to a mammal in need thereof. In an aspect in the method of treatment, the treatment period is of 7-28 days, optionally extendible up to 60 days or up to 90 days or up to 180 days.
In another embodiment the present disclosure provides use of a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, 10 Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof in method for improving clinical signs and symptoms of SARS-CoV-2 viral infection by administering the composition to a patient in need thereof, wherein the composition reduces the SARS-CoV-2 viral load.
In another embodiment the present disclosure provides use of a pharmaceutical 15 composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof in a method for resolution of clinical signs and symptoms of SARS-CoV-2 viral infection in a patient, wherein the resolution is characterized by radiological improvement with a documented virological clearance in 2 samples at least 24 hours apart. 20
In another embodiment the present disclosure provides use of a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof in a method for resolution of clinical signs and symptoms of SARS-CoV-2 viral infection in a patient, wherein the resolution is characterized by time to normalization of fever without use of 25 one or more antipyretics in last 24 hours.
In another embodiment the present disclosure provides use of a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof in a method for eradication the SARS-CoV-2 viral infection in a patient, by administering, wherein the 30 eradication is determined by a negative SARS-CoV-2 RT-PCR of an upper or lower respiratory tract specimen from such patient.
In another embodiment the present disclosure provides use of a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine,
29
Makisterone-A, 20-Hydroxyecdysone or a combination thereof in a method for clinical improvement of the signs and symptoms of SARS-CoV-2 viral infection in a patient, by administering, in a dosage range of 100-800 mg once, twice or thrice daily. In another embodiment the dosage range for the composition is in the range of 200-600 mg once, twice or thrice daily. In another embodiment the dosage range for the composition is in the 5 range of 400-600 mg once, twice or thrice daily. In an aspect to composition alleviates the symptoms of SARS-CoV-2 infection within a period of 7-28 days, optionally extendible up to 60 days, up to 90 days or up to 180 days.
The clinical signs and symptoms as used herein include one or more symptoms comprising cough, fever, shortness of breath, duration of supplemental oxygen 10 requirement, duration of mechanical ventilation, duration of hospitalization, time to first negative SARS-CoV-2 RT-PCR in upper or lower respiratory tract specimen.
In a further embodiment, the present disclosure provides use of a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof in a method 15 of inhibiting the secretion of cytokines in SARS-CoV-2 infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof.
In another embodiment, the present disclosure provides use of a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof for a 20 method of treatment of SARS-CoV-2 virus infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof, wherein the extract is effective in a delayed treatment onset.
In yet another embodiment, the present disclosure provides use of a pharmaceutical composition comprising Cocculus hirsutus extract comprising Sinococuline, 25 Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof in a method of prevention of SARS-CoV-2 virus infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof.
In another embodiment the present disclosure provides use of the extract according to present disclosure for manufacture of a medicament useful for clinical improvement of 30 the signs and symptoms of SARS-Cov-2 virus infection in a patient
The extract is prepared by a process comprises of extracting the plant mass of Cocculus hirsutus with one or more solvents, concentrating the extract, and drying the extract, or extracting the plant mass of Cocculus hirsutus with one or more solvents,
30
concentrating the extract, adding water and partitioning the extract with one or more solvents, and drying the extract, or extracting the plant mass of Cocculus hirsutus with one or more solvents, concentrating the extract, extracting the extract with one or more solvents, and drying the extract. In another aspect of the above embodiment, the extraction of the plant mass of Cocculus hirsutus is done at a temperature in the range of about 50º to 5 about 100ºC. In another aspect of the above embodiment, the extraction of the plant mass of Cocculus hirsutus is done at a temperature of about 80º to about 85ºC. In another aspect of the above embodiment, the extraction of the plant mass of Cocculus hirsutus is done at a temperature of about 60º to 65ºC. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 40º to about 10 95ºC. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 40º to about 45ºC. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 45º to about 50ºC. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 55º to 15 about 65ºC. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 90º to about 95ºC. In yet another aspect, the plant mass can be extracted from a dry part or a wet part of the plant.
In an embodiment the present disclosure provides a dried extract from Cocculus hirsutus, wherein the dried extract is obtained by: a) performing an C1-C4 alcohol 20 extraction or an aqueous extraction, whereby the alcohol extraction or an aqueous extraction uses heat, thereby forming a liquid phase and a solid phase; b) separating the liquid phase from the solid phase, c) drying the liquid phase to obtain dried extract from Cocculus hirsutus, whereby the extract comprises less than 1% of the solid mass of the Cocculus hirsutus plant, whereby the dried extract is stable at 25oC for at least one week. 25 In an aspect the dried extract according to present disclosure has a water concentration of not more than 5% w/w. In another aspect the dried extract according to present disclosure has a C1-C4 alcohol concentration of not more than 10,000 ppm.
In another embodiment the present disclosure provides a process for preparation of an extract of Cocculus hirsutus comprising: a) collecting dry or wet part of plant mass 30 of Cocculus hirsutus; b) charging the plant mass into an extractor and adding solvent for extraction; c) heating the reaction mixture to obtain an extract; d) filtering the extract and collecting the filtrate; e) optionally filtering the residue at least once with solvent to obtain filtrate; f) concentrating the filtrate from any one of the above steps, and optionally drying
31
to obtain said extract; g) optionally enriching the said extract with one or more of Sinococuline, Magnoflorine, 20-Hydroxyecdysone or Makisterone-A
In yet another embodiment, the present disclosure provides a process of preparation of tablet composition of Cocculus hirsutus extract for use in the treatment of SARS-CoV-2 infection, the process comprising the steps of: 5
i. sifting the extract and blending with pharmaceutically acceptable excipients;
ii. lubricating the blend and compressing into tablets and
iii. film coating the tablets.
In yet another embodiment, the present disclosure provides a process of preparation of tablet composition of Cocculus hirsutus extract for use in the treatment of 10 SARS-CoV-2 infection, the process comprising the steps of:
i. blending the extract with pharmaceutically acceptable excipients;
ii. granulating the blend with a solvent;
iii. lubricating and compressing the blend into tablets and
iv. film coating the tablets. 15
In a further embodiment, the present disclosure provides a process of preparation of tablet composition of Cocculus hirsutus extract for use in the treatment of SARS-CoV-2 infection, the process comprising the steps of:
i. blending the extract with pharmaceutically acceptable excipients and compacting the mixture; 20
ii. milling the compacts and blending with extragranular excipients;
iii. lubricating the blend and compressing into tablets and
iv. film coating the tablets.
Although the above embodiments are related to tablet composition, however the composite extract of Cocculus hirsutus may also be formulated into any other suitable oral 25 dosage forms like powder, pellets, granules, spheroids, mini-tablets, caplets, tablets, sachet or a capsule comprising such powder, pellets, granules, spheroids, min-tablets or caplets or liquids selected from solution, suspensions, emulsions, linctuses, elixirs, drops or syrups.
In further related embodiment, the extract may be co-administered simultaneously or sequentially with one or more additional therapeutic agents. In another embodiment, the 30 composition of the disclosure may further comprise one or more additional therapeutic agents. The one or more additional therapeutic agents may be selected from related antiviral therapies or compounds such as which may provide symptomatic relief from the conditions, for examples antipyretic and analgesic drugs.
32
The term "co-administration" herein refers to administration of one or more additional therapeutic agents with the extract to a mammal. The extract and additional therapeutic agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions or may include a Standard of Care therapy as defined in various Global COVID guidelines. Each of the extract or additional therapeutic agents 5 may be administered through the same or different routes of administration simultaneously of sequentially.
EXAMPLES
The following examples include only exemplary embodiments to illustrate the 10 practice of this disclosure. It will be evident to those skilled in the art that the disclosure is not limited to the details of the following illustrative examples and that the present disclosure may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive. 15
Example 1: Preparation of 95:5 Ethanol: purified water extract of Cocculus hirsutus
The plant mass of Cocculus hirsutus (1kg) was charged into an extractor at ambient temperature*. A mixture of ethanol and purified water (95:5; 6L) was added and the reaction mixture was heated at a temperature of 60-65ºC for about 3 hours. The extracted 20 mass was filtered, collected and stored in a container. The extraction and filtration steps were repeated twice with a mixture of ethanol and purified water (95:5; 3L). The three filtered extracts were combined and concentrated to the maximum possible extent under reduced pressure at a low temperature. The resulting extract was decanted into stainless steel trays, and then dried under vacuum at 45-50ºC until ethanol content was not more 25 than 10000 ppm and the moisture content was not more than 5%. The dried extract was cooled to about 20-25ºC and unloaded at controlled humidity (RH NMT 40%).
Yield obtained = 90 g to 120 g
Example 2: Preparation of 1:1 Ethanol:purified water extract of Cocculus hirsutus 30
The plant mass of Cocculus hirsutus (1kg) was charged into an extractor at ambient temperature*. A mixture of ethanol and purified water (1:1; 6L) was added and the reaction mixture was heated at a temperature of 60-65ºC for about 3 hours. The extracted mass was filtered, collected and stored in a container. The extraction and filtration steps were repeated
33
twice with ethanol and purified water (1:1, 3L). The three filtered extracts were combined and concentrated to the maximum possible extent under reduced pressure at a low temperature. The resulting extract was decanted into stainless steel trays, and then dried under vacuum at 45-50ºC until the ethanol content was not more than 10000 ppm and the moisture content was not more than 5%. The dried extract was cooled to about 20-25ºC 5 and unloaded at controlled humidity (RH NMT 40%).
Yield obtained = 80 g to 120g
Example 3: Preparation of an aqueous extract of Cocculus hirsutus
The plant mass of Cocculus hirsutus (1kg) was charged into an extractor at ambient 10 temperature*. Purified water (6L) was added and the reaction mixture was heated at a temperature of 60-65ºC for about 3 hours. The extracted mass was filtered, collected and stored in a container. The extraction and filtration steps were repeated twice with purified water (3L). The three filtered extracts were combined and concentrated to the maximum possible extent under reduced pressure at a low temperature. The resulting extract was 15 decanted into stainless steel trays, and then dried under vacuum at 45-50ºC until the ethanol content was not more than 10000 ppm and the moisture content was not more than 5%. The dried extract was cooled to about 20-25ºC and unloaded at controlled humidity (RH NMT 40%).
Yield obtained = 80 g to 120g 20
*The term "ambient temperature" as used herein, includes a temperature ranging from about 18°C to about 25°C.
Example 4: Preparation of Tablets from the extract of Cocculus hirsutus using direct compression 25
S/N
Ingredients
Quantity in mg/tablet
(1)
(2)
(3)
(4)
1
Extract (from Examples 1, 2 or 3)
1000.0
1000.0
1000.0
1000.0
2
Magnesium aluminium trisilicate
300.0
300.0
300.0
300.0
3
Lactose monohydrate
90.0
-
-
-
4
Dicalcium Phosphate
-
90.0
-
-
5
Starch Pregelatinised
-
-
90.0
-
6
Calcium silicate
-
-
-
90.0
7
Microcrystalline cellulose
40.0
40.0
40.0
40.0
34
8
Colloidal silicon dioxide
20.0
20.0
20.0
20.0
9
Croscarmellose
40.0
40.0
40.0
40.0
10
Magnesium stearate
10.0
10.0
10.0
10.0
Core tab wt.
1500.0
1500.0
1500.0
1500.0
11
Opadry Green
45.0
45.0
45.0
45.0
12
Purified water
Qs.
Qs.
Qs.
Qs.
Total weight of coated tablet
1545.0
1545.0
1545.0
1545.0
MANUFACTURING PROCEDURE:
1. The extract was passed through sieve # 10 mesh (2 mm);
2. Step 1 material along with Magnesium aluminium trisilicate was sifted through # 14 mesh (1.4 mm); 5
3. Lactose monohydrate, Dicalcium Phosphate, Starch Pregelatinised and Calcium silicate were sifted through #36 mesh (420 μ);
4. Microcrystalline cellulose, Colloidal silicon dioxide and Croscarmellose were passed through # 25 mesh(600 μ);
5. The material from steps 3 and 4 were mixed in a blender with the step 2 material; 10
6. The blend obtained from step 5 was lubricated with magnesium stearate and compressed into tablets;
7. Opadry green was dispersed in purified water to prepare a dispersion;
8. The compressed tablets from step 6 were coated with the dispersion of step 7.
15
Example 5: Preparation of Tablets from the extract of Cocculus hirsutus using wet granulation technique
S/N
Ingredients
Quantity in mg/tablet
(1)
(2)
(3)
(4)
Part (a) :Intra granular part
1
Extract (from Examples 1, 2 or 3)
1000.0
1000.0
1000.0
1000.0
2
Magnesium aluminium trisilicate
250.0
250.0
250.0
250.0
3
Methanol
Qs.
Qs.
Qs.
Qs.
Total
1250.0
1250.0
1250.0
1250.0
Part (b):Extra granular part
4
Magnesium aluminium trisilicate
40.0
40.0
40.0
40.0
5
Lactose monohydrate
90.0
-
-
-
35
6
Dicalcium Phosphate
-
90.0
-
-
7
Starch Pregelatinised
-
-
90.0
-
8
Calcium silicate
-
-
-
90.0
9
Microcrystalline cellulose
40.0
40.0
40.0
40.0
10
Colloidal silicon dioxide
20.0
20.0
20.0
20.0
11
Croscarmellose
40.0
40.0
40.0
40.0
12
Magnesium Stearate
10.0
10.0
10.0
10.0
Core tab wt.
1490.0
1490.0
1490.0
1490.0
13
Opadry Green
44.7.0
44.7
44.7
44.7
14
Purified water
Qs.
Qs.
Qs.
Qs.
Total weight of coated tablet
1534.7
1534.7
1534.7
1534.7
MANUFACTURING PROCEDURE:
1. The extract was passed through sieve # 10 mesh (2mm);
2. Magnesium aluminium trisilicate was sifted through sieve #36 mesh(420 μ);
3. The material from step 1 and step 2 was granulated with methanol and dried; 5
4. The dried material from step 3 was passed through 16 mesh (1 mm);
5. Magnesium aluminium trisilicate, Lactose monohydrate, Dicalcium Phosphate, Starch Pregelatinised and Calcium silicate were sifted through #36 mesh (420 μ);
6. Microcrystalline cellulose, Colloidal silicon dioxide and Croscarmellose were passed through # 25 mesh (600 μ); 10
7. The material from steps 5 and 6 were mixed in a blender along with step 4 material;
8. The blend obtained from step 7 was lubricated with magnesium stearate and compressed into tablets;
9. Opadry green was dispersed in purified water to prepare a dispersion;
10. The compressed tablets from step 8 were coated with dispersion of step 9. 15
Example 6: Preparation of Tablets from the extract of Cocculus hirsutus using dry granulation technique
S/N
Ingredients
Quantity in mg/tablet
(1)
(2)
(3)
(4)
Part (a) :Intra granular part
1
Extract (from Examples 1, 2 or 3)
1000.0
1000.0
1000.0
1000.0
2
Magnesium aluminium trisilicate
300.0
300.0
300.0
300.0
36
3
Magnesium stearate
15.0
15.0
15.0
15.0
Total
1315.0
1315.0
1315.0
1315.0
Part (b) Extra granular part
4
Lactose monohydrate
30.0
-
-
-
5
Dicalcium Phosphate
-
30.0
-
-
6
Starch Pregelatinised
-
30.0
-
7
Calcium silicate
-
-
30.0
8
Microcrystalline cellulose 100.0 100.0 100.0 100.0
9
Colloidal silicon dioxide 10.0 10.0 10.0 10.0
10
Croscarmellose 30.0 30.0 30.0 30.0
11
Magnesium stearate 15.0 15.0 15.0 15.0
Core tab wt.
1500.0
1500.0
1500.0
1500.0
12
Opadry Green
45.0
45.0
45.0
45.0
13
Purified water
Qs.
Qs.
Qs.
Qs.
Total weight of coated tablet
1545.0
1545.0
1545.0
1545.0
MANUFACTURING PROCEDURE:
1. The extract was passed through # 10 mesh (2 mm);
2. The step 1 material was sifted along with Magnesium aluminium trisilicate through # 14 mesh (1.4 mm); 5
3. Magnesium stearate was sifted through #36 mesh (420 μ) and mixed with step 2 material in a blender;
4. The blended material was compacted using roll compactor;
5. The compacts obtained from step 4 were milled;
6. The extra granular excipients were sifted and blended with magnesium stearate to 10 obtain a lubricated blend;
7. The lubricated blend of step 6 was compressed into tablets;
8. Opadry green was dispersed in purified water to prepare a dispersion;
9. The compressed tablets from step 7 were coated with dispersion of step 8.
15
Example 7: Various strengths of the extract based composition were prepared following the below manufacturing method:
1. Sifted Cocculus hirsutus aqueous extract, microcrystalline cellulose, anhydrous lactose, croscarmellose sodium and colloidal silicon dioxide through a suitable sieve in the range of ASTM # 10 sieve to # 60 sieve; 20
37
2. Blended the step 1 material in a blender for 5-20 minutes;
3. Sifted lubricant through a suitable sieve and blended with the material of step 2 for 2-10 minutes;
4. Compacted material of step 3 and passed the compacts through upper granulation screen followed by lower granulation screen followed by sifting over suitable sieve 5 until the desired percentage of granules were obtained;
5. Blended the material above the sieve and below the sieve to get the intragranular part;
6. Sifted a second set of excipients (extragranular part) as in step 1 through suitable sieve, and then blended them with step 5 material;
7. Sifted lubricant through suitable sieve and blended with step 6 material followed by 10 compression of the blend to get sufficient strength;
8. Dispersed the coating material in solvent and stirred well to get uniform dispersion followed by coating of the tablets
- The Extract used in below examples is an aqueous extract of Cocculus hirsutus.
15
Example-7.1: Preparation of tablets of strength 25 mg, 50 mg, 100 mg and 300 mg: S/N Ingredients Quantity in mg/tablet 25 mg 25 mg 25 mg 50 mg 50 mg 100 mg 100 mg 100 mg 300 mg 300 mg 300 mg
1
Extract
25.0
25.0
25.0
50.0
50.0
100.0
100.0
100.0
300.0
300.0
300.0
2
Microcrystalline cellulose
-
47.0
37.0
27.0
7.0
14.0
8.2
3.0
24.6
9.0
42.0
3
Anhydrous Lactose
41.0
60.0
29.0
35.0
29.0
6.0
3.5
10.5
-
18.0
4
Croscarmellose sodium
11.1
14.0
19.0
24.0
23.0
6.0
11.3
8.5
33.9
25.5
18.0 5 Lactose monohydrate 1.9 - - - - - - - - - -
6
Colloidal silicon Dioxide
0.9
2.0
1.0
2.0
1.0
1.0
2.0
2.1
6.0
6.3
3.0
7
Magnesium stearate
1.1
2.0
1.8
2.0
1.8
0.7
3.0
1.1
9.0
3.3
2.1 8 Starch Pre-gelatinised 22.5 - 12.0 - 12.0 - - 6.0 - 18.0 - 9 Dicalcium Phosphate - - - 10.0 - - - 2.5 - 7.5 - 10 Calcium silicate 8.7 - - - - - - 5.5 - 16.5 -
11
Microcrystalline cellulose
23.3
-
11.8
-
11.8
12.0
13.6
9.5
40.8
28.5
60.0
12
Croscarmellose
12.5
-
12.4
-
12.4
8.0
5.0
9.1
15.0
27.3
40.0
38
13
Colloidal silicon dioxide
2.0
-
1.0
-
1.0
1.0
2.2
1.5
6.6
4.5
5.0
14
Magnesium Stearate
-
-
-
-
1.1
1.3
1.2
1.2
3.6
3.6
6.5
Core tab wt.
150.0
150.0
150.0
150.0
150.0
150.0
150.0
150.0
450.0
450.0
450.0
15
Opadry Green
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
18.0
18.0
18.0
16
Purified water
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Total wt. of coated tablet
156.0
156.0
156.0
156.0
156.0
156.0
156.0
156.0
468.0
468.0
468.0
Example-7.2: Preparation of tablets of strength 400 mg and 500 mg: S/N Ingredients Quantity in mg/tablet 400 mg 400 mg 400 mg 400 mg 400 mg 400 mg 500 mg 500 mg 500 mg
1
Extract
400.0
400.0
400.0
400.0
400.0
400.0
500.0
500.0
500.0
2
Microcrystalline cellulose
-
37.0
63.0
56.0
33.0
12.0
41.0
15.0
70.0
3
Anhydrous Lactose
30.5
37.0
-
24.0
14.0
17.5
-
30.0
4
Croscarmellose sodium
30.5
30.0
44.0
24.0
45.0
35.0
56.5
42.5
30.0 5 Lactose monohydrate 9.0 - - - - - - - -
6
Colloidal silicon Dioxide
8.0
4.0
6.0
4.0
8.0
9.7
10.0
10.5
5.0
7
Magnesium stearate
4.0
3.0
8.0
2.8
12.0
4.5
15.0
5.5
3.5 8 Starch Pre-gelatinised 33.0 - 27.0 - - 24.0 - 30.0 - 9 Dicalcium Phosphate - 13.0 - - - 8.5 - 12.5 - 10 Calcium silicate - - - - - 22.0 - 27.5 -
11
Microcrystalline cellulose
54.0
46.8
32.8
48.0
54.4
39.0
68.0
47.5
60.0
12
Croscarmellose
23.5
23.0
16.0
32.0
20.0
35.0
25.0
45.5
40.0
13
Colloidal silicon dioxide
6.6
3.0
2.0
4.0
8.6
6.0
11.0
7.5
5.0
14
Magnesium Stearate
0.9
3.2
1.2
5.2
5.0
4.3
6.0
6
6.5
Core tab wt.
600.0
600.0
600.0
600.0
600.0
600.0
750.0
750.0
750.0
15
Opadry Green
24.0
24.0
24.0
24.0
24.0
24.0
30.0
30.0
30.0
16
Purified water
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Total wt. of coated tablet
624.0
624.0
624.0
624.0
624.0
624.0
780.0
780.0
780.0
5
39
Example-7.3: Preparation of tablets of strength 600 mg and 800 mg: S/N Ingredients Quantity in mg/tablet 600 mg 600 mg 600 mg 600 mg 600 mg 600 mg 800 mg 800 mg 800 mg
1
Extract
600.0
600.0
600.0
600.0
600.0
600.0
800.0
800.0
800.0
2
Microcrystalline cellulose
20
84.0
63
106.0
43
65
66.0
112.0
126.0
3
Anhydrous Lactose
55
36.0
-
14.0
34.0
-
28.0
48.0
-
4
Croscarmellose sodium
46
36.0
48
14.0
42.0
45.6
90.0
48.0
88.0 5 Lactose monohydrate 19.0 - 11.0 29.0 - 67 - - -
6
Colloidal silicon Dioxide
8.0
6.0
6.0
8.0
11.0
5.7
16.0
16.0
12.0
7
Magnesium stearate
7.0
4.2
9.0
0.8
6.0
4.9
24.0
5.6
16.0 8 Starch Pre-gelatinised 13.5 - - - - 9.5 - - 54 9 Dicalcium Phosphate 23.0 - 27.0 - - - - - - 10 Calcium silicate - - 19 7.0 - 12.0 - - -
11
Microcrystalline cellulose
64.0
72.0
82.8
59.0
114.4
49.0
108.8
96.0
65.6
12
Croscarmellose
36.5
48.0
26.2
52.0
43.0
29.0
40.0
64.0
32.0
13
Colloidal silicon dioxide
6.6
6.0
3.9
4.0
3.9
7.0
17.2
8.0
4.0
14
Magnesium Stearate
1.4
7.8
4.1
6.2
3.1
5.3
10.0
10.4
2.4
Core tab wt.
900.0
900.0
900.0
900.0
900.0
900.0
1200.0
1200.0
1200.0
15
Opadry Green
36.0
36.0
36.0
36.0
36.0
36.0
48.0
48.0
48.0
16
Purified water
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Qs.
Total wt. of coated tablet
936.0
936.0
936.0
936.0
936.0
936.0
1248.0
1248.0
1248.0
Example 7.4: Stability data of AQCH and AQCH tablets with respect to content of Magnoflorine as analytical marker
Strength
EXTRACT AQCH
Condition
40°C/75%RH
30°C/75%RH
25°C/60%RH
Station
1M
2M
3M
6M
3M
6M
3M
6M
Assay: Magnoflorine content (%w/w)
0.37
0.37
0.37
0.38
0.43
0.38
Not Performed
Strength
100 mg
Condition
40°C/75%RH
30°C/75%RH
25°C/60%RH
Station
Initial
1M
2M
3M
6M
3M
6M
3M
6M
Assay: Magnoflorine content (%w/w)
0.37
0.37
0.37
0.38
0.43
0.38
0.42
Not Performed
LOD (%W/W)
4.0
2.9
2.8
6.7
2.7
5.9
2.7
40
Strength
300 mg
Condition
40°C/75%RH
30°C/75%RH
25°C/60%RH
Station
Initial
1M
2M
3M
6M
3M
6M
3M
6M
Assay: Magnoflorine content (%w/w)
0.40
0.41
0.40
0.41
0.45
0.41
0.46
0.41
0.45
LOD (%W/W)
3.4
2.9
2.9
6.7
3.2
5.4
2.7
5.6
2.7
Strength
500 mg
Condition
40°C/75%RH
30°C/75%RH
25°C/60%RH
Station
Initial
1M
2M
3M
6M
3M
6M
3M
6M
Assay: Magnoflorine content (%w/w)
0.40
0.40
0.40
0.41
0.45
0.41
0.46
0.41
0.46
LOD (%W/W)
3.9
3.6
3.5
5.0
3.8
4.8
3.4
4.5
3.4
Based on the stability data it was found that there no significant change in the Magnoflorine content of AQCH and AQCH tablets of all the three strengths (100, 300 and 500 mg) under the conditions tested up to 6 months. The long-term stability study is on-going and samples will be evaluated up to 3 years of storage. This data demonstrates the 5 feasibility of formulating AQCH into a stable tablet dosage form, which is believed to be advantageous for clinical evaluation.
Example 8: Biological activity
Evaluation of the efficacy of the pharmaceutical composition comprising aqueous 10 extract of Cocculus hirsutus in treatment of SARS-CoV-2 infected patients:
Patients were screened in an initial screening following assessments conducted during a patient visit:
Written Informed Consent; Demographic Information; Inclusion/ Exclusion Criteria; Medical and surgical History; Physical Examination; Vital Signs (pulse rate, respiratory 15 rate, systolic and diastolic pressure, oxygen saturation by SaO2 or SpO2); Clinical Symptoms Assessment; 12- lead ECG; Body Temperature (oral); RT-PCR for COVID viral load; Arterial Blood Gas; Hematology (Absolute neutrophil count, Total bilirubin and Random Blood Sugar will be done only at screening); Biochemistry (ALT/AST, S. creatinine, BUN, ALP); Exploratory markers (D-dimer, CRP, LDH and Serum ferritin); 20 Urinalysis (R/M); Chest X-Ray/ CT Scan; Urine pregnancy test (In case of a female subject).
41
During Hospital stay: (Day 1-10), the following assessments and activities were done:
Hospitalization/admission to study facility; Physical Examination; Vital Signs (Vital signs (pulse rate, respiratory rate, systolic and diastolic pressure, oxygen saturation by SaO2 or SpO2) within 10 minutes before first dosing on day 1 and then every 24 hours (± 10 minutes) until discharge and as clinically indicated); Clinical Symptoms Assessment; 12- 5 lead ECG (were be performed on Day 5 and 10) (For patients taking HCQ it was done every day till HCQ is given; ECG on rest of the days was done as per given schedule); Body Temperature (Body temperature was measured every 4-6 hourly till fever subsides, within 30 minutes before first dosing on day 1 and then every 24 hours (± 10 minutes) until day of discharge, and as clinically indicated); RT-PCR for SARS-CoV-2 viral load* (was 10 performed on Day 3, 7 and 10), Arterial Blood Gas (was performed on Day 1, 3, 5, 7 and 10); IP administration; Hematology (was performed on Day 3, 5 and 7); Biochemistry (ALT/AST, S. creatinine, BUN, ALP) (was performed on Day 7); Exploratory markers** (D-dimer, CRP, LDH and Serum ferritin) (was performed on Day 3 and 7); Chest X-Ray/ CT Scan (was performed on Day 7; On day 3, imaging was done only if lung involvement was seen 15 on screening); Adverse events; Concomitant Medications. Also during the hospital stay if RT-PCR results came negative for SARS-CoV-2 viral load, then another RT-PCR after 24 hours was performed
Day of discharge can be day 14 or later as per patient’s clinical condition and includes the following evaluation: 20
Physical Examination; Vital Signs (pulse rate, respiratory rate, systolic and diastolic pressure, oxygen saturation by SaO2 or SpO2) every 24 hours (± 10 minutes) until discharge, and as clinically indicated; Clinical Symptoms Assessment; 12- lead ECG (For patients taking HCQ was done every day till HCQ is given. ECG on rest of the days was done as per given schedule); Body Temperature (Body temperature was measured every 4-25 6 hourly till fever subsides, every 24 hours (± 10 minutes) until day of discharge, and as clinically indicated); RT-PCR for SARS-CoV-2 viral load (was repeated on Day 14 if patient was discharged based on 2 negative RT-PCR test results before day 14); Arterial Blood Gas; Hematology; Biochemistry (ALT/AST, S. creatinine, BUN, ALP); Exploratory markers* (D-dimer, CRP, LDH and Serum ferritin); Chest X-RAY/ CT Scan; 30 Adverse events; and Concomitant Medications.
After obtaining the informed consent, patients were screened by undergoing assessments as per defined protocol and after confirming eligibility, eligible patients were given either
42
purified aqueous extract of Cocculus hirsutus (AQCH) tablets with standard of care or only standard of care, for 10 days treatment period.
The selected patients as per the inclusion and exclusion criteria below were administered tablets of aqueous extract of Cocculus hirsutus 400 mg thrice daily (every 8±1 hours) before meal, preferably at same time, for 10 days. 5
Inclusion Criteria: Subjects were included in the study if they meet all of the following criteria:
1. Provided written informed consent;
2. Male or non-pregnant, non-lactating female aged ≥ 18 and ≤ 75 years;
3. Patients had a body temperature > 37.3oC with cough/shortness of breath; 10
4. Patients with moderate symptoms of COVID-19 had either one of the following criteria:
a. PaO2/FiO2: 200-300, OR
b. Respiratory Rate > 24/min and SAO2/SpO2 ≤93% on room air;
5. Patients with RT-PCR confirmed diagnosis of SARS-CoV-2; 15
6. Patient who was able to take the drug orally and comply with study procedures;
7. Women of childbearing potential must have had a negative urine pregnancy test prior to study entry.
Exclusion Criteria: Patients were deemed ineligible to participate in the study if they 20 fulfilled any of the following criteria:
1. Patients had persistent vomiting (more than three episodes of vomiting in 12 hours, preventing adequate oral hydration) that make it difficult to swallow the drug;
2. Patient had known active hepatitis, tuberculosis and definite bacterial or 25 fungal infections;
3. Patients had altered mental state;
4. Patient had multiple organ failure requiring ICU monitoring and the treatment;
5. Patient had respiratory failure and requiring ventilation; 30
6. Patient had history of retinopathy or macular degeneration;
7. Patient had history of glucose-6-phosphate dehydrogenase (G6PD) deficiency;
43
8. Patient had prolonged QTc-interval at baseline ECG (>450 ms in males or > 470 ms in females);
9. Patient took concomitant medication associated with QTc-interval prolongation, which could not be withdrawn prior to study drug administration; 5
10. Patient had a history of hypersensitivity towards any drug of standard of care;
11. Patient had a history of evidence of chronic interstitial infiltration at imaging;
12. Patient had a history of hospitalization within the past six months for respiratory failure; 10
13. Patient had any concurrent medical condition or uncontrolled, clinically significant systemic disease (e.g., heart failure, COPD, hypertension, liver disease, chronic respiratory failure, chronic kidney disease, diabetes, anaemia etc.) that, in the opinion of the Investigator precluded the subject’s participation in the study or interfered with the interpretation of the study 15 results;
14. Patient’s had a history of serology tests positive for hepatitis B, hepatitis C, or human immunodeficiency virus;
15. Patients who received specific antiviral drugs ritonavir/ lopinavir, or chloroquine, hydroxychloroquine, azithromycin, monoclonal antibodies 20 within 1 week before admission;
16. Patient who had participated in another investigational study within 3 months prior to enrolment in the study;
17. Investigators, study personnel, sponsor’s representatives and their first degree relatives. 25
Efficacy evaluation is based on both clinical and laboratory assessments. Following assessments were conducted:
- Physical examination at screening, everyday till day of discharge, Day of Discharge, Day 17 and as clinically indicated;
- Vital signs (pulse rate, respiratory rate, systolic and diastolic pressure, 30 oxygen saturation by SaO2 or SpO2) at screening, within 10 minutes before first dosing on day 1 and then every 24 hours (± 10 minutes) until discharged (on day 11 or later), at the FU visit on Day 17 and as clinically indicated;
44
- Body temperature (oral) was measured at screening, within 30 minutes before first dosing on day 1 and then every 24 hours (± 10 minutes) until day of discharge (on day 11 or later), at the FU visit on day 17 (or later) and as clinically indicated;
- Blood for evaluation of hematology and biochemistry for liver and renal 5 function test was withdrawn at screening, and subsequently on days 3, 5, 7, 10. Additional hematological and biochemistry estimations were performed until recovery as per the discretion of the investigator.
- RT-PCR for Corona virus titer: At screening, within 30 minutes before first dosing on day 1 and then every 24 hours (± 10 minutes) till day 10 and on 10 Discharge.
- Chest X-Ray/ CT scan – At screening on Day 3, 5, 7, 10 and on Discharge.
Safety was assessed by assessments of recording of Adverse events, vital signs (pulse rate, systolic and diastolic blood pressure (seated), body temperature and respiratory rate), ECG, physical examination, and clinical laboratory investigations (hematology, 15 biochemistry and urinalysis). Adverse events were classified according to their severity based on CTCAE v 5.0 criteria. Any clinically significant abnormal changes from baseline in the concurrent medical condition(s), physical examination and/or laboratory data were recorded as an AE. 12 lead ECG was conducted at screening, 1-hour post-dose on day 1 and repeated after 24 hours (± 10 minutes) until day 10, on day of discharge and as 20 clinically indicated. Any subject who was judged by the treating physician/ PI to be at risk for developing severe disease was managed as per standard of care.
The Primary end points were:
Proportion of patients showing clinical improvement [Time Frame: Day 14] 25
(Clinical improvement was defined as patient meeting discharge criteria OR a 2 point improvement (from time of enrolment) in disease severity rating on the 7-point ordinal scale)
The scale was as follows:
- 1- not hospitalized with resumption of normal activities; 30
- 2- not hospitalized, but unable to resume normal activities;
- 3- hospitalized, not requiring supplemental oxygen;
- 4- hospitalized, requiring supplemental oxygen;
45
- 5- hospitalized, requiring nasal high-flow oxygen therapy or non-invasive mechanical ventilation, or both;
- 6- hospitalized, requiring ECMO or invasive mechanical ventilation, or both; and
- 7- death.
Hospital Discharge Criteria was defined as resolution of symptoms, radiological 5 improvement with a documented virological clearance in 2 samples at least 24 hours apart:
- reduction in viral load in nasopharyngeal swab and percentage of subject achieving Clinical Cure (clinical Cure was defined as negative viral load of the respiratory specimen for two consecutive times when measured on frequency of greater than 10 or equal to one day, improvement in lung image, normal body temperature for more than 3 days, and improvement in clinical manifestation).
The Secondary end points were:
1. Proportion of patients showing clinical improvement [Time Frame: Day 7]
2. Proportion of patients showing clinical improvement [Time Frame: Day 28] 15
3. Time (Days) to clinical improvement [Time Frame: up to 28 days]
Clinical improvement was defined as patient meeting discharge criteria OR a 2 point improvement (from time of enrolment) in disease severity rating on the 7-point ordinal scale The ordinal scale was an assessment of the clinical status at the first assessment of a given study day. The scale was as follows: 20
- 1- not hospitalized with resumption of normal activities;
- 2- not hospitalized, but unable to resume normal activities;
- 3- hospitalized, not requiring supplemental oxygen;
- 4- hospitalized, requiring supplemental oxygen;
- 5- hospitalized, requiring nasal high-flow oxygen therapy or noninvasive 25 mechanical ventilation, or both;
- 6- hospitalized, requiring ECMO or invasive mechanical ventilation, or both; and
- 7- death.
Hospital Discharge Criteria was defined as resolution of symptoms, radiological improvement with a documented virological clearance in 2 samples at least 24 hours 30 apart.
46
4. Time to normalization of fever without use of antipyretics in last 24 hours [Time Frame: up to 28 days]
5. Time to alleviation of cough [Time Frame: up to 28 days]
6. Time to first negative SARS-CoV-2 RT-PCR in in upper or lower respiratory tract specimen [Time Frame: up to 28 days] 5
7. Duration (days) of supplemental oxygen therapy [Time Frame: up to 28 days]
8. Proportion of patients showing deterioration of clinical condition as assessed by at least 1 point worsening on 7 point ordinal scale (non-invasive ventilation, mechanical ventilation, ECMO or death) [Time Frame: up to 28 days]
9. Duration (days) of hospitalization [Time Frame: up to 28 days] 10
10. Number of deaths (All-cause mortality) [Time Frame: up to Day 28]
11. Safety evaluation, as measured by AEs, Adverse Reactions (ARs), SAEs, Serious ARs (SARs) [Time Frame: Throughout the therapy and during follow up]
The viral nucleic acid conversion rate and days from positive to negative (Time frame: within 10 days of admission), Time to clinical improvement as the time to normalization 15 of fever, respiratory rate, oxygen saturation and alleviation of cough, Time to clinical failure defined as time to death, mechanical ventilation or ICU admission, Safety and tolerability of the drug as assessed by treatment emergent adverse events.
Example 8.1: Clinical Trial Data: 20
Based on clinical finding of the test and the outcomes as obtained, the assessment of efficacy was done considering both clinical and laboratory assessment data for both primary and secondary endpoints as defined in study. Proportion of patients showing improvement [Time Frame: Day 7, 14 and 28] (Clinical improvement defined as patient meeting discharge criteria OR a 2 point improvement (from time of enrolment) in disease 25 severity rating on the 7-point ordinal scale)
Efficacy Data: Primary Endpoint
Table I: Proportion of patients showing clinical improvement [Time Frame: Day 14] – PP population:
Measure
AQCH + Standard of Care
(N=85)
Standard of Care
(N=82)
Clinical Improvement
n (%)
82 (96.5)
80 (97.6)
(95% CI)
(90, 99.3)
(91.5, 99.7)
Estimate of difference (95% CI)
-0.0109 (-0.0767, 0.0537)
NA
47
p-value *
0.9999
NA
N: number of subjects in PP population per treatment group.
n: number of subjects showing clinical improvement.
p-value is derived from Chi-square/Fisher’s Exact
Efficacy Data: Secondary Endpoint
Table II: Proportion of patients showing clinical improvement [Time Frame: Day 7] – PP population:
Measure
AQCH + Standard of Care
(N=85)
Standard of Care
(N=82)
Clinical Improvement
n (%)
10 (11.8)
6 (7.3)
(95% CI)
(5.8, 20.6)
(2.7, 15.2)
Estimate of difference (95% CI)
-0.0445 (-0.049, 0.1386)
NA
p-value *
0.3289
NA
N: number of subjects in PP population per treatment group.
n: number of subjects showing clinical improvement.
p-value is derived from Chi-square/ Fisher’s Exact
5
Table III: Proportion of patients showing clinical improvement [Time Frame: Day 28] – PP population:
Measure
AQCH + Standard of Care
(N=85)
Standard of Care
(N=82)
Clinical Improvement
n (%)
85 (100)
82 (100)
(95% CI)
(95.8, 100)
(95.6, 100)
Estimate of difference (95% CI)
0 (-0.0432, 0.0448)
NA
p-value *
NC
NA
N: number of subjects in PP population per treatment group.
n: number of subjects showing clinical improvement.
p-value is derived from Chi-square/ Fisher’s Exact
Table IV: Analysis of Time (Day) to clinical improvement [Time Frame: up to 28 days] – PP population: 10
Measure
AQCH + Standard of Care (N=85)
Standard of Care (N=82)
Hazard Ratio
Estimate
(95% CI)
p-value log rank* test
Clinical Improvement
85
82
1.3046
(0.9576, 1.7772)
0.0223
Median time to clinical improvement (in days)
8
11
95% CI for Median time
(8, 11)
(9, 11)
48
Time to clinical improvement (min, max)
4, 17
4, 15
* Determined by a Cox proportion hazards model with treatment as the only covariate.
n: number of subjects showing clinical improvement.
N: number of subjects in PP population from each treatment group.
Note: The time-to-event variable is defined as the time (days) from the date of first dose of study medication to the date of the event.
Table V: Analysis of Time (Day) to first negative SARS-CoV-2 RT-PCR in upper or lower respiratory tract specimen [Time Frame: up to 28 days] – PP population:
Measure
AQCH + Standard of Care (N=85)
Standard of Care (N=82)
Hazard Ratio
Estimate
(95% CI)
p-value log rank* test
1st negative SARS-CoV-2 RT-PCR
85
82
1.345
(0.9855, 1.8356)
0.0031
Median time to 1st negative SARS-CoV-2 RT-PCR (in days)
7
10
95% CI for Median time
(NC, NC)
(7, 10)
Time to 1st negative SARS-CoV-2 RT-PCR (min, max)
3, 16
3, 14
* Determined by a Cox proportion hazards model with treatment as the only covariate.
n: number of subjects with 1st negative SARS-CoV-2 RT-PCR.
N: number of subjects in PP population from each treatment group.
Note: The time-to-event variable is defined as the time (days) from the date of first dose of study medication to the date of the event.
Table VI: Analysis of Time (Day) to normalization of fever without use of 5 antipyretics in last 24 hours [Time Frame: up to 28 days] – PP population:
Measure
AQCH + Standard of Care (N=85)
Standard of Care (N=82)
Hazard Ratio
Estimate
(95% CI)
p-value log rank* test
Normalization of fever
85
82
1.3252
(0.9757, 1.7999)
0.0325
Median time to Normalization of fever (in days)
6
7
95% CI for Median time
(5, 6)
(6, 8)
Time to Normalization of fever (min, max)
1, 14
1, 14
* Determined by a Cox proportion hazards model with treatment as the only covariate.
n: number of subjects with normalization of fever.
N: number of subjects in PP population from each treatment group.
49
Note: The time-to-event variable is defined as the time (days) from the date of first dose of study medication to the date of the event.
Table VII: Analysis of Time (Day) to alleviation of cough [Time Frame: up to 28 days] – PP population:
Measure
AQCH + Standard of Care (N=70)
Standard of Care (N=65)
Estimate
(95% CI)
p-value log rank* test
Patients with cough at screening
67
61
1.1238
(0.7999, 1.5789)
0.403
Alleviation of cough
70
65
Median time to Alleviation of cough (in days)
5
5
95% CI for Median time
(4, 5)
(5, 6)
Time to Alleviation of cough (min, max)
1, 13
1, 11
* Determined by a Cox proportion hazards model with treatment as the only covariate.
n: number of subjects showing alleviation of cough.
N: number of subjects in PP population from each treatment group.
Note: The time-to-event variable is defined as the time (days) from the date of first dose of study medication to the date of the event.
Table VIII: Summary of duration (Days) of supplemental oxygen therapy [Time 5 Frame: up to 28 days] – PP population:
Statistics
AQCH + Standard of Care (N=105)
Standard of Care
(N=105)
Duration of supplemental oxygen therapy (Days)
n
56
49
Mean
6.6
6.2
SD
4.58
2.60
Median
6.5
7.0
Min, Max
2, 33
2, 11
Q1, Q3
3.5, 8
3, 8
N: number of subjects in PP population for each treatment group.
n: number of subjects with non-missing Duration of supplemental oxygen therapy.
Table IX: Proportion of patients showing deterioration of clinical condition [Time Frame: up to 28 days]–PP population:
Statistics
AQCH + Standard of Care (N=85)
Standard of Care
(N=82)
Duration of clinical condition
n (%)
16 (18.8)
18 (22.0)
50
95% CI
(11.16, 28.76)
(13.56, 32.46)
Estimate of difference (95 % CI)
-0.0313 (-0.1536, 0.091)
NA
p-value*
0.6158
NA
Deterioration of clinical condition as assessed by at least 1 point worsening on 7 point ordinal scale (non-invasive ventilation, mechanical ventilation, ECMO or death)
N: number of subjects in PP population for each treatment group.
n: number of subjects showing deterioration of clinical condition.
95% CI – the 95% confidence interval for the proportion.
*p-value is derived from hi-square / Fisher’s Exact
Table X: Summary of duration (Days) of Hospitalization [Time Frame: up to 28 days] – PP population:
Statistics
AQCH + Standard of Care (N=85)
Standard of Care
(N=82)
Duration of Hospitalization (Days)
n
85
82
Mean
10.8
11.5
SD
2.46
2.09
Median
11.0
12.0
Min, Max
5, 18
7, 16
Q1, Q3
9, 12
10, 13
N: number of subjects in PP population for each treatment group.
n: number of subjects with non-missing Duration of supplemental oxygen therapy.
Table XI: Number (%) of patients with TEAE(s) by SOC and PT – Safety Population 5
System Organ Class
Preferred Term
AQCH + Standard of Care
(N=105)
Standard of Care
(N=105)
Total
(N=210)
n (%)
events
n (%)
events
n (%)
events
Any Class
12 (11.4)
17
7 (6.7)
9
19 (9.0)
26
Gastrointestinal disorders
1 (1.0)
2
1 (1.0)
1
2 (1.0)
3
Diarrhoea
1 (1.0)
1
0
0
1 (0.5)
1
Vomiting
1 (1.0)
1
1 (1.0)
1
2 (1.0)
2
Hepatobiliary Disorder
1 (1.0)
1
0
0
1 (0.5)
1
Liver Injury
1 (1.0)
1
0
0
1 (0.5)
1
Investigations
7 (6.7)
9
4 (3.8)
5
11 (5.2)
14
Alanine Amino transferase↑
4 (3.8)
4
3 (2.9)
3
7 (3.3)
7
Aspartate Amino transferase↑
2 (1.9)
2
0
0
2 (1.0)
2
Blood Pressure transferase↑
2 (1.9)
2
1 (1.0)
1
3 (1.0)
3
Blood Creatinine transferase↑
1 (1.0)
1
0
0
1 (1.0)
1
Transaminase transferase↑
0
0
1 (1.0)
1
1 (1.0)
1
51
Musculoskeletal and Connective Tissue Disorder
1 (1.0)
1
0
0
1 (0.5)
1
Pain in Extremity
1 (1.0)
1
0
0
1 (0.5)
1
Nervous System Disorders
3 (2.9)
3
1 (1.0)
1
4 (1.9)
4
Headache
2 (1.9)
2
1 (1.0)
1
3 (1.4)
3
Dizziness
1 (1.0)
1
0
0
1 (0.5)
1
Respiratory, Thoracic and Mediastinal Disorders
1 (1.0)
1
2 (1.9)
2
3 (1.4)
3
Acute Respiratory Distress syndrome
1 (1.0)
1
1 (1.0)
1
2 (1.0)
2
Dyspnoea
0
0
1 (1.0)
1
1 (0.5)
1
TEAE: Treatment emergent adverse events, SOC: System organ class, PT: Preferred term
N: number of subjects in safety population for each treatment group.
n (%): number and percentage of patients with at least one TEAE.
Note: i. Events are total no. of adverse events.
ii. Table sorted by SOC alphabetic order and by decreasing frequency of PTs within each SOC in AQCH + Standard of Care treatment arm.
Observation:
Based on Primary and Secondary endpoint as defined in the study, it is evident that numerically higher number of subjects have achieved clinical improvement at Day 7 in AQCH arm as compared to the Standard of Care (SoC). Further there were key 5 statistically significant differences between AQCH and SOC arm observed for following secondary efficacy endpoint:
- Time to clinical improvement ( 8 vs. 11 days)
- Time to first RT-PCR negative ( 7 vs. 10 days)
- Time to normalization of fever ( 6 vs. 7 days ) 10
- Duration of hospitalization ( 10.8 vs. 11.5 days)
Based on clinical outcomes, the pharmaceutical compositions of the extract according to present disclosure are superior and effective in the treatment of patients with COVID-19 with no significant side-effects. The therapy was found to be promising:
- in relieving the clinical symptoms or improving the condition of the patient as 15 defined in the primary and secondary endpoints as:
a. time to clinical improvement of the condition;
b. time to normalization of fever without antipyretics;
c. time to first RT-PCR negative results;
d. duration of hospitalisation; 20
e. duration of oxygen therapy or mechanical ventilation.
52
Example 9: Safety and Pharmacokinetic Study data for the tested samples:
In the study for evaluating safety and tolerability of AQCH Tablet, three cohorts of 100 mg, 200 mg and 400 mg TID dosing in healthy subjects were completed. Based on these data doses of 100 mg, 200 mg and 400 mg TID for 10 days they were found to be safe and tolerable. 5
Sinococuline is identified as biomarker and analyzed. There is linear increase in Cmax and AUC with ascending dose up to 400 mg. The Tmax observed at day 1 and day 10 and across doses is consistent. The Tmax ranged between 1.0 to 1.5 hours across doses.
Maximum concentration observed (Cmax) based on various patient data at dose 100 mg, 200 mg and 400 mg at day 1 is shown below: 10
- maximum plasma concentration (Cmax) of Sinococuline at day 1 after a single oral dose administration of 100 mg of the said extract from about 1.4 ng/mL to about 110.0 ng/mL, after a single oral dose administration of 200 mg of the said extract from about 4.2 ng/mL to about 135 ng/mL, after a single oral dose administration of 400 mg of the said extract from about 8.0 ng/mL to about 205.0 ng/mL; or after 15 a single oral dose administration of 600 mg of the said extract from about 14.0 ng/mL to about 240.0 ng/mL; or after a single oral dose administration of 800 mg of the said extract from about 13.0 ng/mL to about 255.0 ng/mL.
AUC0-24 observed based on various patient data at dose 100 mg, 200 mg and 400 mg at day 1 is shown below: 20
- AUC0-24 of Sinococuline at day 1 after a TID oral dose administration of 100 mg of the said extract from about 20.0 h*ng/mL to about 575.0 h*ng/mL, after a TID oral dose administration of 200 mg of the said extract from about 88.0 h*ng/mL to about 1250.0 h*ng/mL, after a TID oral dose administration of 400 mg of the said extract from about 135.0 h*ng/mL to about 2360.0 h*ng/mL; or after a TID oral 25 dose administration of 600 mg of the said extract from about 263 h*ng/mL to about 2435.0 h*ng/mL; or after a TID oral dose administration of 800 mg of the said extract from about 226.0 h*ng/mL to about 3150.0 h*ng/mL.
Maximum concentration observed (Cmax) based on various patient data at dose 100 mg, 200 mg and 400 mg at day 10 is shown below: 30
- maximum plasma concentration (Cmax) of Sinococuline at day 10 after a single oral dose administration of 100 mg of the said extract from about 5.0 ng/mL to about
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90.0 ng/mL, after a single daily oral dose administration of 200 mg of the said extract from about 13.5 ng/mL to about 300.0 ng/mL, after a single daily oral dose administration of 400 mg of the said extract from about 20.0 ng/mL to about 500.0 ng/mL; or after a single daily oral dose administration of 600 mg of the said extract from about 29.0 ng/mL to about 604.0 ng/mL; or after a single daily oral dose 5 administration of 800 mg of the said extract from about 35.05 ng/mL to about 705.0 ng/mL.
AUC0-24 observed based on various patient data at dose 100 mg, 200 mg and 400 mg at day 10 is shown below:
- AUC0-24 of Sinococuline at day 10 of a single daily oral dose administration of 100 10 mg of the said extract from about 67.0 h*ng/mL to about 1090.0 h*ng/mL, after a single daily oral dose administration of 200 mg of the said extract from about 148.0 h*ng/mL to about 2515.0 h*ng/mL, at a single daily oral dose administration of 400 mg of the said extract from about 251.0 h*ng/mL to about 5075.0 h*ng/mL; or at a single daily oral dose administration of 600 mg of the said extract from about 390.0 15 h*ng/mL to about 5205.0 h*ng/mL; or at a single daily oral dose administration of 800 mg of the said extract from about 474.0 h*ng/mL to about 5730.0 h*ng/mL.
Trough levels at steady state observed based on various patient data at dose 100 mg, 200 mg and 400 mg at day 10 is shown below:
- the trough levels at steady state (Cτ,ss ) of Sinococuline from day 3-10 is ranging 20 from about 2.8 to about 31.0 ng/mL at a thrice daily oral dose administration of 100 to 400 mg of the said extract, wherein the steady state is achieved at around day 3.
Also an area under the plasma concentration time curve to infinity (AUC∞) of Sinococuline is found to be in a range of about 109.0 h*ng/ml to about 520.0 h*ng/ml after oral administration of a dose of 100 mg of the extract to a human subject at day 10. 25
Example 10: AQCH chemical fingerprinting instrumentation and characterisation methodology
Methods: All NMR spectral data were recorded on a Bruker 400 MHz spectrometer. Chemical shifts (δ) were referenced internally to the residual solvent peak (CD3OD: 1H δ 30 3.30, 13C δ 49.0 ppm; CDCl3: 1H 7.26, 13C 77.0 ppm) and the reference point was TMS (δH and δC: 0.00 ppm). HR-ESIMS spectra were recorded on an Agilent 1100 LC-Q-TOF mass
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spectrometer and HRMS-6540-UHD machines. HPLC purifications were performed on Thermo Scientific Dionex UltiMate 3000 HPLC system with UV detector. Column chromatography was performed using silica gel (60-120 and 230-400 mesh); fractions were monitored by TLC, pre-coated silica gel plates 60 F254 (Merck). Spots were visualized by UV light or by spraying with H2SO4-MeOH, anisaldehyde-H2SO4 reagents. 5
For the HPLC fingerprinting, 100 mg of AQCH was transferred in 20 ml volumetric flask and added ~10 ml of diluent with sonication/shaking/stirring for 5-10 minutes to dissolve. The volume was made up with diluent, mixed and filtered through 0.45 μm filter for HPLC fingerprinting. It was performed on RP18e Purospher-STAR (Hibar) (250 × 4.6 mm; 5 μm) column. The mobile phase containing a buffer (0.1% formic acid in water) and 10 acetonitrile was used at the flow rate of 0.65 ml/ min at a column temperature of 30 °C at 254 nm wavelength. Volume of injection was 5 μl and a total run time of the assay was 75 min. A gradient program was used as follows: 0–15 min, 00–05% B; 15–40 min, 05–20% B; 40–55 min, 20–30% B; 55–65 min, 30–60% B; 65-68 min, 60-00% B and 68–75 min, 00% B. For the isolation (S1 Fig), 500 g of AQCH was suspended in distilled water and partitioned 15 between ethyl acetate (A) and H2O (B). The aqueous layer (B) was basified with NH4OH solution (pH 9) and then extracted with chloroform, CHCl3. This resulted in separation of chloroform layer (4.0 g, C) and aqueous layer (D). The CHCl3 layer (C) was further purified through repeated column chromatography in neutral alumina and eluted with a gradient of CHCl3-MeOH (100:0 to 0:100) to obtain Compound 1 as major constituent along 20 with Compound 2. The Aqueous layer (D) was lyophilized (480.5 g) and suspended in methanol. The methanol soluble portion (400.0 g) was purified by column chromatography (silica gel, 100-200 mesh), eluted with a gradient of CHCl3-MeOH (100:0 to 0:100, 500 ml, collected volumes of each fraction), and concentrated, giving fifty fractions (Fr.1-Fr.50) and their composition was monitored by TLC. Those showing similar TLC profiles were 25 grouped into six major fractions (Fr-1a to Fr-5a). Fraction Fr-2a afforded two UV active compounds as crystals. These crystals containing two UV active compounds were further subjected to semi-preparative C18 reversed-phase HPLC {Eclipse 5 μm; 9.4 × 250 mm; 3 ml/min; gradient of water (B)/acetonitrile (A) over 32 min; 100–80% B (5 min), 80–60% B (5 min), 60–50% B (5 min), 50–40% B (5 min), 40-20% B (5 min), 20-45% B (3 min), 45-70% 30 B (2 min) and 70-100% B (2 min); Column oven temperature 25 °C} to give Compounds 3 and 4. The ethyl acetate soluble fraction (A) was subjected to column chromatography (silica gel, 100-200 mesh), eluted with a gradient of CHCl3-MeOH (100:0 to 0:100, 250 ml, collected volumes of each fraction), and concentrated, giving thirty fractions (Fr.51-Fr.80).
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Compound 5 was obtained from fractions Fr. 66-Fr. 70 in pure. All the isolated compounds 1-5 were identified by detailed spectral analysis 1D and 2D NMR, and HRESI-MS data and comparisons with the reported spectral data.
Results: HPLC chromatography was performed on AQCH. This was followed by isolation of five marker compounds using repeated chromatographic methods, which were 5 characterised using advance 1D and 2D NMR spectroscopic and mass analysis. Marker compounds were identified to be Sinococuline (1), Magnoflorine (2), 20-Hydroxyecdysone (3), Makisterone-A (4), and Coniferyl alcohol (5). The physico-chemical data of all the five identified marker compounds are given in Table A.
Table A: Physico-chemical data of identified markers 1-5 in AQCH 10
Sinococuline (1): Pink amorphous powder; 1H NMR (CD3OD, 400 MHz): δ 6.74 (1H, d, J= 8.4 Hz, H-2), 6.53 (1H, d, J= 8.4 Hz, H-1), 4.37 (1H, d, J= 5.6 Hz, H-9), 4.28 (1H, d, J= 2.8 Hz, H-7), 3.85 (1H, ddd, J= 13.3, 3.7 and 3.5 Hz, C-6), 3.82 (3H, s, 3-OCH3), 3.69 (3H, s, 8-OCH3), 3.15 (1H, dd, J= 17.6 and 6.0 Hz, H-10), 2.92 (1H, dd, J= 13.3, 3.7 Hz, H-5), 2.88 (1H, d, J= 17.7 Hz, H-10), 2.63-2.71 (2H, ddd, J= 13.1, 12.5, 3.4 Hz, H-16), 2.16 (1H, t, J= 13.2 Hz, H-5), 2.00 (1H, dd, J= 12.7, 3.4 Hz, H-15), 1.86 (1H, ddd, J= 12.7, 12.5 and 4.7 Hz, H-15); 13C NMR (CD3OD, 100 MHz): δ 147.2 (C-4), 145.9 (C-3), 145.4 (C-8), 131.2 (C-11), 130.2 (C-14), 124.7 (C-12), 119.2 (C-1), 110.5 (C-2), 68.5 (C-6), 66.8 (C-7), 57.4 (3-OCH3), 56.7 (8-OCH3), 46.7 (C-9), 41.0 (C-16), 39.8 (C-13), 38.4 (C-15), 37.2 (C-5), 36.8 (C-10); ESI-MS m/z 334.25 [M+H]+ (calcd for C18H24NO5, 334.25).
Magnoflorine (2): Yellowish brown powder; 1H NMR (400 MHz, CD3OD): δH 6.72 (1H, d, J= 8.0 Hz, H-9), 6.56 (2H, t, J= 4.0 Hz, H-3, H-8), 3.84 (3H, s, OCH3-10), 3.80 (3H, s, OCH3-2), 3.56 (1H, dd, J= 18.0, 5.6 Hz, H-6a), 3.45 (1H, dd, J= 18.0, 5.2 Hz, H-5), 3.31 (3H, s, N-CH3), 3.22 (1H, m, H-4), 3.04 (1H, dd, J= 18.0, 5.2 Hz, H-5), 2.89 (3H, s, N-CH3), 2.74 (1H, dd, J= 20.0, 4.0 Hz, H-4), 2.60 (1H, t, J= 13.2 Hz, H-5); 13C NMR (100MHz, CD3OD): δC 151.9 (C-2), 150.5 (C-10), 149.5 (C-1), 148.6 (C-11), 124.7 (C-7a), 122.4 (C-11b), 122.3 (C-11a), 119.7 (C-6b), 115.6 (C-8), 114.4 (C-3a), 109.5 (C-9), 108.3 (C-3), 70.0 (C-6a), 61.2 (C-5), 55.0 (OCH3), 54.7 (OCH3), 52.6 (N-CH3), 42.2 (N-CH3), 30.5 (C-7), 23.4 (C-4); ESIMS m/z 343.20 [M+H]+ (cal. for C20H24NO4+ m/z 342.20).
20-Hydroxyecdysone (3): White soft crystals; 1H NMR (CD3OD, 400 MHz): δ 5.81 (1H, d, J= 2.6 Hz, H-7), 3.95 (1H, q, H-3α), 3.84 (1H, ddd, J= 12.0, 4.0, 3.2 Hz, H-2α), 3.33 (1H, dd, J= 11.0, 1.7 Hz, H-22), 3.15 (1H, ddd, J= 11.2, 7.0, 2.6 Hz, H-9), 2.39 (1H, dd, J= 9.5, 8.0 Hz, H-17), 2.38 (1H, dd, J= 13.0, 4.5 Hz, H-5), 2.13 (1H, dt, J= 13.0, 13.0, 4.8 Hz, H-12α), 1.99 (1H, H-15α), 1.95 (1H, H-16α), 1.88 (1H, ddd, J= 12.8, 4.6, 2.3 Hz, H-12β), 1.81 (1H, H-11β), 1.79 (2H, H-1α/24a), 1.75 (1H, H-4α), 1.73 (1H, H-16β), 1.70 (1H, H-4β), 1.69 (1H, H-11α), 1.66 (1H, H-23a), 1.60 (1H, H-15β), 1.43 (2H, dd, J= 13.3, 12.3 Hz, H-1β/24b) 1.28 (1H, dddd, J= 13.0, 11.5, 11.0, 4.6 Hz, H-23b) 1.20 (3H, s, 21-CH3), 1.20 (3H, s, 26-CH3), 1.19 (3H, s, 27-CH3), 0.97 (3H, s, 19-CH3), 0.89 (3H, s, 18-CH3); 13C NMR (CD3OD, 100 MHz): δ 206.4 (C-6), 167.9 (C-8), 122.1 (C-7), 85.2 (C-14), 78.4 (C-22), 77.9 (C-20), 71.3 (C-25), 68.7 (C-2), 68.5 (C-3), 51.8 (C-5), 50.5 (C-17), 48.6 (C-13), 42.4 (C-24), 39.3 (C-10), 37.4 (C-1), 35.1 (C-9), 32.8 (C-4), 32.5 (C-12) 31.8 (C-15), 29.7 (C-26), 28.9 (C-27),
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27.4 (C-23), 24.4 (C-19), 21.5 (C-16), 21.5 (C-11), 21.0 (C-21), 18.0 (C-18); HR-MS m/z 481.3161 [M+H]+ (calcd for C27H45O7, 481.3165).
Makisterone A (4): White crystals; 1H NMR (CD3OD, 400 MHz): δ 5.81 (1H, d, J= 2.6 Hz, H-7), 3.95 (1H, q, H-3α), 3.84 (1H, ddd, J= 12.0, 4.0, 3.2 Hz, H-2α), 3.46 (1H, dd, J= 11.0, 1.7 Hz, H-22), 3.15 (1H, ddd, J= 11.2, 7.0, 2.6 Hz, H-9), 2.39 (1H, dd, J= 9.5, 8.0 Hz, H-17), 2.38 (1H, dd, J= 13.0, 4.5 Hz, H-5), 2.13 (1H, dt, J= 13.0, 13.0, 4.8 Hz, H-12α), 1.99 (1H, H-15α), 1.95 (1H, H-16α), 1.88 (1H, ddd, J= 12.8, 4.6, 2.3 Hz, H-12β), 1.81 (1H, H-11β), 1.79 (2H, H-1α/24a), 1.75 (1H, H-4α), 1.73 (1H, H-16β), 1.70 (1H, H-4β), 1.69 (1H, H-11α), 1.60 (1H, H-23a), 1.57 (1H, H-15β), 1.43 (1H, dd, J= 13.3, 12.3 Hz, H-1β) 1.29 (1H, dddd, J= 13.0, 11.5, 11.0, 4.6 Hz, H-23b), 1.19 (3H, s, 21-CH3), 1.17 (3H, s, 27-CH3), 1.14 (3H, s, 28-CH3), 0.97 (3H, s, 25-CH3), 0.94 (3H, s, 19-CH3), 0.90 (3H, s, 18-CH3); 13C NMR (CD3OD, 100 MHz): δ 204.4 (C-6), 165.9 (C-8), 120.1 (C-7), 83.1 (C-14), 75.9 (C-20), 73.3 (C-22), 71.8 (C-26), 66.6 (C-2), 66.4 (C-3), 49.6 (C-5), 48.2 (C-17), 39.7 (C-10), 37.2 (C-1), 35.3 (C-9), 33.0 (C-12), 32.3 (C-4), 30.9 (C-15), 30.4 (C-13), 29.7 (C-15), 28.7 (C-23), 28.6 (C-24), 25.8 (C-27), 23.8 (C-28), 22.5 (C-19), 19.5 (C-16), 19.3 (C-11), 19.0 (C-21), 16.1 (C-18), 13.2 (C-25); HR-MS m/z 495.3318 [M+H]+ (calcd for C28H47O7, 495.3322).
Coniferyl alcohol (5): White solid; 1H NMR (CD3OD, 400 MHz): δ 7.01 (1H, d, J= 1.8 Hz, H-3), 6.85 (1H, dd, J= 7.9 and 1.8 Hz, H-5), 6.74 (1H, d, J= 7.9 Hz, H-6), 6.50 (1H, d, J= 15.9 Hz, H-7), 6.20 (1H, dt, J= 15.9 and 5.4 Hz, H-8), 4.21 (2H, d, J= 6.1 Hz, H-9), 3.87 (3H, s, 2-OCH3); 13C NMR (CD3OD, 100 MHz): δ 149.2 (C-2), 147.6 (C-1), 132.2 (C-7), 130.7 (C-4), 127.2 (C-8), 121.1 (C-5), 116.3 (C-6), 110.8 (C-3), 64.0 (C-9), 56.5 (C-2-OCH3)
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We Claim:
1. An extract of a plant of Menispermeaceae family comprising therapeutically effective amount of one or more of Sinococuline, Magnoflorine, 20-Hydroxyecdysone or Makisterone-A.
2. The extract as claimed in claim 1 comprising Sinococuline not less than 0.5 % w/w, Magnoflorine not less than 0.05% w/w, 20-Hydroxyecdysone not less than 5 0.01%, Makisterone-A not less than 0.01% w/w, or a combination thereof.
3. The extract as claimed in claim 1 comprising Sinococuline not less than 1.0 % w/w, Magnoflorine not less than 0.1% w/w, 20-Hydroxyecdysone not less than 0.1%, Makisterone-A not less than 0.05% w/w, or a combination thereof.
4. The extract as claimed in claim 1 comprising Sinococuline from about 0.5% w/w 10 to about 85% w/w, Magnoflorine from about 0.05% w/w to about 75% w/w, Makisterone-A from about 0.01% w/w to about 80% w/w, 20-Hydroxyecdysone from about 0.05% w/w to about 70% w/w, or a combination thereof.
5. The extract as claimed in any one of claims 1 to 4, wherein the plant is Cocculus hirsutus. 15
6. A pharmaceutical composition comprising therapeutically effective amount of one or more of Sinococuline, Magnoflorine, 20-Hydroxyecdysone or Makisterone-A.
7. A pharmaceutical composition comprising the extract as claimed in claim 1.
8. The extract as claimed in claim 1 or the pharmaceutical composition as claimed in claim 7 for use in the prophylactic and/or curative treatment of an infection caused 20 by SARS-CoV-2 virus.
9. The extract as claimed in claim 1 or the pharmaceutical composition as claimed in claim 6 comprising Sinococuline, Magnoflorine, 20-Hydroxyecdysine and Makisterone-A, wherein the Sinococuline, Magnoflorine, 20-Hydroxyecdysine and Makisterone-A are in therapeutically effective amount and concentration suitable 25 for the treatment of symptoms associated with a SARS-CoV-2 infection.
10. The pharmaceutical composition as claimed in any one of claims 6 to 9, wherein the composition is an oral dosage form.
11. The pharmaceutical composition as claimed in claim 10, wherein the oral dosage form is selected from powder, pellets, granules, spheroids, mini-tablets, caplets, 30 tablets, sachet or a capsule comprising such powder, pellets, granules, spheroids,
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min-tablets or caplets, or a liquids selected from solutions, suspensions, emulsions, syrups, linctuses, elixirs or drops.
12. The pharmaceutical composition as claimed in any one of claims 6 to 9, wherein the composition is storage stable at temperature and relative humidity conditions of 40±2°C/75±5% RH, 30±2°C/75±5% RH or 25±2°C/75±5% RH for at least 3 5 months.
13. The pharmaceutical composition as claimed in any one of claims 6 to 9, wherein the composition comprises one or more of the pharmaceutically acceptable excipients selected from diluents, binders, disintegrants, lubricants, glidants, polymers, flavoring agents, surfactants, solvents, suspending agents, stabilizers, 10 preservatives, antioxidants, buffers, and tonicity modifying agents.
14. The pharmaceutical composition as claimed in claim 13, wherein the composition comprises intragranular excipients in a concentration range of about 8% to about 45% by weight of the composition and extragranular excipients in a concentration range of about 6% to about 38% by weight of the composition. 15
15. The pharmaceutical composition as claimed in claim 13, wherein the composition comprises diluent in a concentration range of about 5% to about 50% by weight of the composition and disintegrant in a concentration range of about 2% to about 33% by weight of the composition.
16. The pharmaceutical composition as claimed in claim 13, wherein the composition 20 comprises microcrystalline cellulose about 6% to about 42% by weight of the composition, anhydrous lactose about 0.5% to about 28% by weight of the composition, croscarmellose sodium about 2% to about 3% by weight of the composition, colloidal silicon dioxide about 0.05% to about 12% by weight of the composition and magnesium stearate about 0.05% to about 14% by weight of the 25 composition.
17. Use of the extract as claimed in claim 1 or the pharmaceutical composition as claimed in claim 6 for administration to a patient infected with SARS-CoV-2 virus for clinical improvement in signs and symptoms of SARS-CoV-2 infection in such patient. 30
18. The use as claimed in claim 17, wherein the clinical improvement is characterized by radiological improvement with a documented virological clearance in 2 samples tested at least 24 hours apart.
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19. The use as claimed in claim 17, wherein the clinical improvement is characterized by time to normalization of fever without use of one or more antipyretics in last 24 hours.
20. The use as claimed in claim 17, wherein the clinical improvement is optionally further characterized by a negative SARS-CoV-2 RT-PCR of an upper or lower 5 respiratory tract specimen.
21. The pharmaceutical composition as claimed in claim 7, wherein the composition comprises about 25 mg to about 1000 mg of the extract.
22. The use as claimed in claim 16, wherein the clinical improvement in signs and symptoms is characterized by any one or more of the below parameters: 10
i. reduction in duration of supplemental oxygen requirement;
ii. reduction in duration of ECMO or mechanical ventilation;
iii. reduction in time to alleviation of cough;
iv. reduction in time to normalization of fever without use of antipyretics;
v. reduction in duration of hospitalization; or 15
vi. reduction in time to first negative SARS-Cov-2 RT-PCR in upper or lower respiratory tract specimen.
23. The use as claimed in claim 16, wherein the clinical improvement in signs and symptoms is characterized by a patient meeting at least one point improvement in disease severity rating on an ordinal scale after administration of the composition 20 to the subject, wherein the ordinal scale comprises:
i. not hospitalized with resumption of normal activities;
ii. not hospitalized, but unable to resume normal activities;
iii. hospitalized, not requiring supplemental oxygen;
iv. hospitalized, requiring supplemental oxygen; 25
v. hospitalized, requiring nasal high-flow oxygen therapy or noninvasive mechanical ventilation, or both; and
vi. hospitalized, requiring ECMO or invasive mechanical ventilation, or both.
24. A stable pharmaceutical composition comprising an extract of Cocculus hirsutus, wherein oral administration of the said composition to a human subject provides: 30
a) maximum plasma concentration (Cmax) of Sinococuline at day 1 after a single oral dose administration of 100 mg of the said extract from about 1.4 ng/mL to about 110.0 ng/mL, after a single oral dose administration of 200 mg of the said extract from about 4.2 ng/mL to about 135 ng/mL, after a
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single oral dose administration of 400 mg of the said extract from about 8.0 ng/mL to about 205.0 ng/mL; or after a single oral dose administration of 600 mg of the said extract from about 14.0 ng/mL to about 240.0 ng/mL; or after a single oral dose administration of 800 mg of the said extract from about 13.0 ng/mL to about 255.0 ng/mL; or 5
b) AUC0-24 of Sinococuline at day 1 after a TID oral dose administration of 100 mg of the said extract from about 20.0 h*ng/mL to about 575.0 h*ng/mL, after a TID oral dose administration of 200 mg of the said extract from about 88.0 h*ng/mL to about 1250.0 h*ng/mL, after a TID oral dose administration of 400 mg of the said extract from about 135.0 h*ng/mL to 10 about 2360.0 h*ng/mL; or after a TID oral dose administration of 600 mg of the said extract from about 263 h*ng/mL to about 2435.0 h*ng/mL; or after a TID oral dose administration of 800 mg of the said extract from about 226.0 h*ng/mL to about 3150.0 h*ng/mL.
25. A stable pharmaceutical composition comprising an extract of Cocculus hirsutus, 15 wherein oral administration of the said composition to a human subject provides:
a) maximum plasma concentration (Cmax) of Sinococuline at day 10 after a single oral dose administration of 100 mg of the said extract from about 5.0 ng/mL to about 90.0 ng/mL, after a single daily oral dose administration of 200 mg of the said extract from about 13.5 ng/mL to about 300.0 ng/mL, 20 after a single daily oral dose administration of 400 mg of the said extract from about 20.0 ng/mL to about 500.0 ng/mL; or after a single daily oral dose administration of 600 mg of the said extract from about 29.0 ng/mL to about 604.0 ng/mL; or after a single daily oral dose administration of 800 mg of the said extract from about 35.05 ng/mL to about 705.0 ng/mL; or 25
b) AUC0-24 of Sinococuline at day 10 of a single daily oral dose administration of 100 mg of the said extract from about 67.0 h*ng/mL to about 1090.0 h*ng/mL, after a single daily oral dose administration of 200 mg of the said extract from about 148.0 h*ng/mL to about 2515.0 h*ng/mL, at a single daily oral dose administration of 400 mg of the said extract from about 30 251.0 h*ng/mL to about 5075.0 h*ng/mL; or at a single daily oral dose administration of 600 mg of the said extract from about 390.0 h*ng/mL to about 5205.0 h*ng/mL; or at a single daily oral dose administration of 800
61
mg of the said extract from about 474.0 h*ng/mL to about 5730.0 h*ng/mL; or
c) Trough plasma levels at steady state (Cτ,ss) of Sinococuline from day 3-10 ranging from about 2.8 to about 31.0 ng/mL at a thrice daily oral dose administration of 100-400 mg of the said extract, wherein the steady state is 5 achieved at around day 3.
26. A stable pharmaceutical composition comprising 400 mg extract of Cocculus hirsutus, wherein single oral administration of the said composition to a human subject provides maximum plasma concentration (Cmax) of Sinococuline at day 1 from about 8.0 ng/mL to about 205.0 ng/mL, or AUC0-24 of Sinococuline at day 1 10 from about 135.0 h*ng/mL to about 2360.0 h*ng/mL.
27. A stable pharmaceutical composition comprising 600 mg extract of Cocculus hirsutus, wherein single oral administration of the said composition to a human subject provides maximum plasma concentration (Cmax) of Sinococuline at day 1 from about 140.0 ng/mL to about 240.0 ng/mL, or AUC0-24 of Sinococuline at day 15 1 from about 263.0 h*ng/mL to about 2435.0 h*ng/mL.
28. A pharmaceutical composition which is bioequivalent to any one of the compositions as claimed in 24-27.
29. A method of reducing the viral load of SARS-CoV-2 virus in a mammal, comprising administrating a therapeutically effective amount of the extract as 20 claimed in any one of claims 1 to 4 or the pharmaceutical composition as claimed in any one of claims 6 to 16 to a mammal in need thereof.
30. A method of alleviating the clinical signs and symptom of a SARS-CoV-2 virus comprising administering a therapeutically effective amount of the extract as claimed in any one of claims 1 to 4 or the pharmaceutical composition as claimed 25 in any one of claims 6-16 to a mammal in need thereof, wherein the said signs and symptoms are selected from fever, cough, fatigue, shortness of breath, expectoration, myalgia, rhinorrhea, sore throat, diarrhea, loss of smell (anosmia) or loss of taste in a mammal.
31. The method as claimed in claim 29, wherein the viral load of SARS-CoV-2 virus is 30 reduced at an early stage of an administration regimen.
32. The method as claimed in 29 or 30, wherein the therapeutically effective amount is in the range of about 2 mg/kg to about 150 mg/kg body weight of the said extract.
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33. The method as claimed in claim 29 or 30, wherein the said extract is administered to a patient in need thereof as 600 mg dose twice daily, 400 mg dose thrice daily, 300 mg dose four times daily or 200 mg dose six times daily.
34. The method as claimed in claim 33, wherein the said dose can be administered as a single dosage form or as multiple dosage forms equating to the required dose. 5
35. The method as claimed in claim 29 or 30, wherein the said extract is administered to a patient in need thereof for a period of about 7-28 days, optionally extendible up to 90 days.
36. The extract as claimed in claim 1 for use in the manufacture of a medicament for clinical improvement of the signs and symptoms of SARS-CoV-2 virus infection 10 in a patient in need thereof.
37. A process for preparation of an extract of Cocculus hirsutus comprising:
a) collecting dry or wet part of plant mass of Cocculus hirsutus;
b) charging the plant mass into an extractor and adding solvent for extraction;
c) heating the reaction mixture to obtain an extract; 15
d) filtering the extract and collecting the filtrate;
e) optionally filtering the residue at least once with solvent to obtain filtrate;
f) concentrating the filtrate from any one of the above steps, optionally drying to obtain said extract; and
g) optionally enriching the said extract with one or more of Sinococuline, 20 Magnoflorine, 20-Hydroxyecdysone or Makisterone-A.
38. The process as claimed in claim 37, wherein the said plant mass is selected from stem or aerial parts of plants or roots.
39. The process as claimed in claim 37, wherein the said solvent is selected from water, an alcohol, a ketone, an ester, a halogenated hydrocarbon, a petroleum 25 fraction, or a mixture thereof.
40. The process as claimed in claim 37, wherein the heating is done at a temperature in the range of about 50oC to 100oC.
41. The extract as claimed in claim 37, wherein the extract is a purified extract.
42. The extract as claimed in claim 37, wherein the extract is purified by 30
a) performing an C1-C4 alcohol extraction or an aqueous extraction, whereby the alcohol extraction or an aqueous extraction uses heat, thereby forming a liquid phase and a solid phase;
b) separating the liquid phase from the solid phase,
63
c) optionally drying the liquid phase to obtain dried extract from Cocculus hirsutus,
wherein the extract comprises less than 1% of the solid mass of the Cocculus hirsutus plant, and
wherein the purified extract is stable at 25oC for at least one week. 5
43. The extract as claimed in claim 37, wherein the extract has a water concentration of not more than 5% w/w.
44. The extract claimed in claim 37, wherein the extract has a C1-C4 alcohol concentration of not more than 10,000 ppm.
45. The extract as claimed in claim 1, wherein the extract is an aqueous, alcoholic or a 10 hydroalcoholic extract.