WO2021090177A1 - Composés pour le traitement d'une infection de la dengue - Google Patents

Composés pour le traitement d'une infection de la dengue Download PDF

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Publication number
WO2021090177A1
WO2021090177A1 PCT/IB2020/060325 IB2020060325W WO2021090177A1 WO 2021090177 A1 WO2021090177 A1 WO 2021090177A1 IB 2020060325 W IB2020060325 W IB 2020060325W WO 2021090177 A1 WO2021090177 A1 WO 2021090177A1
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Prior art keywords
extract
composition
compounds
denv
dengue virus
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PCT/IB2020/060325
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English (en)
Inventor
Ravi Kant RAJPOOT
Ruchi Sood
Altaf LAL
Upasana Arora
Navin Khanna
Sumit Madan
Arshad Hussain KHUROO
Kaushal Nayyar
Shahnaz AKHTAR
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Sun Pharmaceutical Industries Limited
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Publication of WO2021090177A1 publication Critical patent/WO2021090177A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to use of compounds, their pharmaceutical acceptable salts and their derivatives for treatment of Dengue and pharmaceutical compositions thereof. These compounds thereof may be isolated from nature or can be synthesized by chemical synthesis. The process for preparation or isolation or extraction of these compounds and determining the activity of these isolated compounds against serotypes of Dengue virus, is also included in the present disclosure. The disclosure also relates to an extract of Cocculus hirsutus for use in the prevention and treatment of Dengue.
  • Dengue is firmly established as the leading arthropod-borne (arbo) viral disease of humans globally, with a primarily tropical and subtropical distribution that offers a native environment for its vector of transmission, female mosquitoes of the genus Aedes.
  • arbo arthropod-borne
  • the disease is currently endemic in more than a hundred countries in Central and South America, the Caribbean, the Mediterranean, Africa, South East Asia and the Western Pacific region, which places over 2.5 billion people at risk of infection.
  • the causative agent of infection is the enveloped, positive-sense, single-stranded RNA virus Dengue (DENV), which belongs to the Flavivirus genus. Contrary to the other named species of the family Flaviviridae, each of which is monotypic, the Dengue virus has four clearly recognized but very similar serotypes, DENV 1-4, which are distinguished by virus plaque reduction neutralization assays. The existence of multiple DENV serotypes may account for diverse episodes of mild to malignant disease. Diverse Dengue serotypes differ in their facility to cause serious illness. Right now, in order to resist this internationally re- emerging public health concern there is neither a specific anti-Dengue preventive vaccine available nor an efficacious approved therapy. The occurrence of several DENV serotypes in the same vicinity poses a significant risk to local residents (Taylor- Robinson, J Clin Diag Treat. 2018; 1(2): 50-52). Thus, a need exists for an effective preventive and treatment therapy for Dengue.
  • Cocculus hirsutus Linn commonly known as Jal-Jammi (Chopra et ak, 1958) or Broom creeper is found in moderately cool and hot regions of India; particularly Tamil nadu, Bihar and Bengal.
  • the leaves and roots of this plant are largely employed in the Indian traditional medicine for a variety of diseases including, hepatic obstruction, jaundice, bronchitis, diabetes mellitus, anorexia, gonorrhoea, and leprosy.
  • hirsutus is documented for its anti-inflammatory, analgesic, antidiabetic and spermatogenic activities (Goodla et ak, Egyptian Journal of Basic and Applied Sciences, Volume 4, Issue 4, December 2017, Pages 264-269).
  • Previous investigations on various parts of the plant resulted in the isolation of d-trilobine, , dl-coclaurine, b-sitosterol, ginnol, a monomethyl ether of inositol, isotrilobine, coclaurine, magnoflorine, hirsudiol, nonacosan-10-ol, shaheenine, cohirsinine, hirsutine, and cohirsutinine (Satyanarayana et ak, Indian Journal of Pharmaceutical Sciences, 2001, 63(1), 30-35).
  • the present disclosure provides certain compounds synthesized or isolated from the plant, and various extracts from the plants to effectively prevent and treat the Dengue viral disease.
  • the present disclosure is drawn to use of compounds, pharmaceutical acceptable salts thereof or their derivatives and pharmaceutical compositions comprising them for the prevention and treatment of Dengue.
  • These compounds or their derivatives may be isolated from nature or can be synthesized by chemical synthesis.
  • the present disclosure also provides a method of preparation or isolation of these compounds or their derivatives from plant species of Menispermaceae family for eg. Cocculus hirsutus ; and their use against Dengue virus. Also provided is a method of determining the activity of these isolated compounds against different serotypes of Dengue virus thereby identifying these compounds as the biomarkers in the treatment of Dengue.
  • the present disclosure also provides process for isolation or extraction of these compounds from plants like Cocculus hirsutus and determining the activity of these isolated compounds against different serotypes of Dengue virus.
  • the disclosure also provides stable compositions comprising one or more of these compounds which are found to be highly effective for administration to mammalian patients infected by Dengue virus and have not shown any toxic effects at therapeutically effective dosages required for preventive and curative treatment of an infection caused by the virus.
  • the disclosure also provides extracts comprising these compounds and their compositions for treatment and prevention of Dengue virus infection. Further, it provides for a stable pharmaceutical composition comprising a therapeutically effective amount of the said extract for use in the prophylaxis and/or treatment of Dengue virus infection in a mammal.
  • the biological material disclosed in the present disclosure is plant mass of Cocculus hirsutus that was procured from Madhya Pradesh, India.
  • Figure 1 Anti-Dengue activity of C. hirsutus and C. pareira (In-vitro): (a) IC50 values calculated separately for each of the three extracts prepared from both the plants and their geometric mean IC50 values against each of the four DENV (Dengue Virus) serotypes were calculated as reported in the table (b) Graph of % DENV-2 infection observed with each of the six extracts with grey and blue curves representing the three C. pareira and C. hirsutus aerial methanolic extracts, respectively. Dashed horizontal line represents 50% DENV-2 infection value.
  • Figure 2 The anti-Dengue activity of the extracts at various concentrations was evaluated against DENV-1 (magenta curve), DENV-2 (green curve), DENV-3 (blue curve) and DENV-4 (black curve) by flow cytometry based virus inhibition assay
  • % DENV infection relative to virus control achieved is represented graphically for denatured spirit (left panel), hydro-alcohol, 50:50 (middle panel) and aqueous (right panel) aerial (dashed curves) and stem (solid curves) extracts
  • the concentration of extract (pg/ml) that resulted in 50% inhibition of viral infection as compared to virus control represented by horizontal dotted line in panel ‘a’
  • IC50 using Graphpad Prism is shown in the table for all the extracts.
  • Figure 3 AQCH (Aqueous extract of Cocculus hirsutus) inhibits secretion of DENV and its antigen, NS1, in a dose-dependent manner: (a) amount of secreted DENV-1 from days 1-6 through FACS based virus titration assay yielding FlUs/ml, and (b) % inhibition of secretion of viral antigen, NS1, evaluated through commercial ELISA kit on day 6 for all the four DENV serotypes.
  • AQCH Aqueous extract of Cocculus hirsutus
  • Figure 4 Chemical fingerprinting of AQCH: (a) AQCH batch was prepared and its anti -Dengue activity against DENV-1 (magenta curve), DENV-2 (green curve), DENV-3 (blue curve) and DENV -4 (black curve) was confirmed by flow cytometry based virus inhibition assay as represented by graph of % DENV infection and extract concentration. IC50 values corresponding to the concentration of AQCH at which 50% of the DENV infection is inhibited as compared to virus control (represented by dotted horizontal line) has been reported in table in the inset (b) HPLC chemical fingerprinting profile of AQCH with the peaks corresponding to the five identified marker compounds marked (c) Chemical structure of the five marker compounds (1-5).
  • FIG. 5 AQCH preparation method is consistent and robust: Various batches of AQCH were prepared and dried through either of the three different methods viz., rotary vapour drying (RD), vacuum tray drying (VTD) or spray drying (SD). The effect of drying method was evaluated through the assessment of (a) anti-Dengue activity by flow cytometry based virus inhibition assay yielding IC50 values (concentration of the extract required to reduce the DENV infection by 50% as compared to virus control), and (b, c) chemical fingerprinting profile; an overlay HPLC chromatograms of the three batches corresponding to the three drying conditions and a table of retention time of five marker compounds are shown in panels ‘b’ and ‘c’, respectively.
  • RD rotary vapour drying
  • VTD vacuum tray drying
  • SD spray drying
  • FIG. 6 AQCH protects AG129 mice from DENV-2 S221 lethal infection:
  • (a) Schematic representation of the design of experiment using five groups of AG 129 mice (n 6).
  • mice in the remaining two groups were infected with DENV-2 S221 and were dosed either with 25 mg/kg, QID (blue curve) or 8.25 mg/kg, QID (pink curve).
  • DENV-2 S221 infection was given i.v. at a lethal dose of 1.0 X 105 FIUs, while AQCH was dosed orally post infection. All the groups were monitored for (b) survival, (c) morbidity score and (d) body weight change over the next 15 days post-infection. Survival data (panel ‘b’) were analysed by Log -Rank (Mantel-Cox) test for statistical evaluation of level of significance in difference in survival rates.
  • the p value ⁇ 0.05 was considered significant.
  • the Morbidity score in panel ‘c’ was based on 5 point system: 0.5, mild ruffled fur; 1.0, ruffled fur; 1.5, compromised eyes; 2, compromised eyes with hunched back; 2.5, loose stools; 3.0, limited movement; 3.5, no movement/hind leg paralysis; 4.0, euthanized if cumulative score was 5. Body weight in panel ‘d’ was monitored twice a day in the morning and evening, and the mean taken for plotting the graph.
  • Figure 7 Paracetamol does not inhibit the anti-Dengue activity of AQCH.
  • DENV-1 infected Vero cells were treated with various concentrations of AQCH extract (0-25 pg/ml) in absence (black curve) and presence of 1 (orange curve), 10 (magenta curve) and 100 (blue curve) pg/ml of Paracetamol separately.
  • the % DENV-1 infection achieved under these conditions was evaluated in a flow cytometry based virus inhibition assay, which is depicted in the graph on the left panel. Concentration of AQCH that led to 50% reduction in DENV-1 infection as compared to virus control was calculated separately for each condition as its corresponding IC50 and is depicted in the table on the right panel.
  • Figure 8 Isolation procedure of marker compounds 1-5 from AQCH.
  • Figure 9 Schematic representation of the overall in vivo protective efficacy studies performed with AQCH.
  • Figure 10 Effect of AQCH dose and dosing regimen against lethal primary DENV infection
  • BID twice a day
  • TID thrice a day
  • QID blue curves
  • mice were monitored for another 10 days for their (b) survival, (c) morbidity and (d) body weight change.
  • Un-infected no infected with virus nor fed with AQCH; represented by magenta
  • Virus-infected, V infected with virus but not fed with AQCH; represented by green curves
  • Only AQCH not infected with virus but fed with AQCH at 25 mg/kg QID; black curves
  • Not significant (ns) and single star (*) in panel ‘b’ denotes their statistical differences between the same dosing schedule of different doses.
  • FIG. 11 Effect of AQCH dose and dosing regimen against secondary DENV infection:
  • IC injected AG 129 mice were orally fed with either AQCH at the indicated higher (solid curve) and lower (dashed curve) doses, or only methylcellulose (marked as IC; grey curve) for 5 days in BID (orange), TID (purple) and QID (blue) dosing regimens.
  • Figure 12 Prophylactic effect of AQCH in secondary Dengue disease AG129 mice
  • AQCH BID range curve
  • DENV-2 S221-4G2 ICs range curve
  • Mice were monitored for another 12 days for (b) survival, (c) morbidity score and (d) change in body weight.
  • Un-infected no infected with virus nor fed with AQCH; represented by magenta curve
  • Virus-infected V (infected with sub-lethal dose of virus but not fed with AQCH; represented by green curves)
  • IC inoculated with IC but not fed with AQCH; grey curves
  • FIG. 13 Delayed treatment effects of AQCH against primary and secondary DENV infection:
  • Post inoculation mice were orally fed for 5 days with AQCH at 25 mg/kg QID with a delay in initiation of treatment. For one group the treatment was delayed by 6 hr (solid blue curve) and for the other by 12 hr (dashed blue curve). The mice were monitored for additional 10 days for survival.
  • Figure 15 Inhibition of intestinal vascular leakage and reduction in cytokine storm by AQCH in secondary DENV infection.
  • mice of each group were euthanized on day 4, perfused them with sterile PBS and luminal content was flushed again with PBS for the identification of visual intestinal vascular leakage; representative images of small intestine of each group are shown in (b).
  • the 50 mg of small intestines from ‘b’ were prepared for the determination of TNF-a (c) and IL-6 (d) by ELISA.
  • the present disclosure is related to use of compounds, their pharmaceutical acceptable salts or their derivatives for the prevention or treatment of Dengue infection.
  • the compounds according to present disclosure comprise compounds of structural formulae as provided below:
  • the compounds according to the present disclosure for use in the prevention or treatment of Dengue infection are prepared synthetically, semi-synthetically, or are isolated from various plant parts of plant, through various extraction and isolation methods as disclosed in the present disclosure.
  • the compound according to present disclosure are isolated from an extract of a plant from the Menispermeaceae family.
  • the extract according to present disclosure is an extract of a plant of genus Cocculus of Menispermeaceae family.
  • the extract according to present disclosure is an extract of Cocculus hirsutus. It is a perennial climber and reaches 2 to 3 m above ground.
  • the present disclosure describes an extract of Cocculus hirsutus, isolated compound therefrom and pharmaceutical composition thereof which were found to be effective against all serotypes of Dengue virus DENV-1, DENV-2, DENV-3 and DENV-4, when tested in-vitro.
  • the present disclosure provides compound isolated from an extract of Cocculus hirsutus and/or a pharmaceutical composition thereof for use in the prevention and treatment of an infection caused by Dengue virus.
  • the disclosed compounds and compositions reduce viral load during the treatment and provides effective remediation against Dengue virus.
  • these compounds or derivatives thereof may be obtained from natural sources or can be synthesized chemically.
  • the compounds may be isolated from plant species.
  • the plant species is Cocculus hirsutus.
  • the commonly owned Indian application IN201821046412 discloses use of extract of Cocculus hirsutus, it was not known as to which constituents or combinations thereof are required for its activity against Dengue virus.
  • Present inventors have identified and isolated these compounds from extract of Cocculus hirsutus.
  • the extract is an aqueous, organic, and alcoholic or hydroalcoholic extract. More preferably, the extract is an aqueous extract.
  • the present disclosure provides a method of isolation of compounds such as Sinococuline, Magnoflorine, Makisterone A and 20-hydroxyecdysone or derivatives from Cocculus hirsutus for the treatment of Dengue virus infection.
  • the present disclosure provides a method of isolation of Sinococuline, Magnoflorine, Makisterone A and 20-hydroxyecdysone from the extract of Cocculus hirsutus for the treatment of Dengue virus infection.
  • isolation of compound or “isolated compound” means a compound that has been substantially separated from or strengthened relative to other compounds that occur with it in nature.
  • the isolated compound is typically at least about 80%, at least about 90%, at least about 98%, or at least about 99% pure by weight.
  • present disclosure is also intended to encompass diastereomers and their racemic and resolved enantiomerically pure forms and pharmaceutically acceptable salts thereof.
  • the present disclosure provides extract of Cocculus hirsutus for the treatment of Dengue virus infection.
  • extract refers to the extract obtained from plant of family Menispermaceae, particularly of genus Cocculus, more particularly from Cocculus hirsutus.
  • one or more components of Cocculus hirsutus is removed during the extraction, e.g., the plant mass is removed, and other components in the extract are separated into and concentrated in the extract.
  • the present disclosure provides for an extract which is used as such or used for isolation of the compound according to present disclosure and new composition comprising the extract or one or more compounds in a concentration and ratio that is not found in nature.
  • the present disclosure provides for an extract which is free from one or more contaminants, which maybe efficiently used in treatment of Dengue virus infection or can be used for isolation of the compounds, Sinococuline, Magnoflorine, Makisterone A or 20-hydroxyecdysone or pharmaceutically acceptable salts or derivatives thereof.
  • the extract also as described herein exhibits properties not found or appreciated in the Cocculus hirsutus plant, e.g., increased stability, increased effectiveness against Dengue virus, increased bioavailability, increased solubility, reduced side-effects, etc.
  • the extract may be present in one or more physical forms, e.g., in form of a liquid, semisolid, solid powder, solid cake, gel, paste, dispersion, solution or a distillate.
  • the extract used for isolation according to present disclosure can be a purified extract or a crude extract.
  • the extract is a purified extract, i.e., the extract is purified by removing one or more contaminants after the extract is obtain from the plant.
  • the extract can be purified to remove one or more contaminants using methods for, e.g., filtration, precipitation, crystallization, chromatography, additional solvent extractions, etc.
  • the extract is a purified, dried extract.
  • the extract comprises one or more of Sinococuline, Magnoflorine, Makisterone-A or 20-Hydroxyecdysone, or a combination thereof
  • the isolates from the extract comprises one or more compounds selected from Sinococuline, Magnoflorine, Makisterone-A or 20-Hydroxyecdysone, or a combination thereof in any concentration.
  • the extract can be optionally used to isolate one particular compound selected from Sinococuline, Magnoflorine, Makisterone-A or 20- Hydroxyecdysone.
  • the extract can be optionally used to isolate two compound selected from Sinococuline, Magnoflorine, Makisterone-A or 20- Hydroxyecdysone. In some embodiments, the extract can be optionally used to isolate three compound selected from Sinococuline, Magnoflorine, Makisterone-A or 20- Hydroxyecdysone. In some embodiments, the extract can be optionally be used to isolate all four compounds selected from Sinococuline, Magnoflorine, Makisterone-A or 20- Hydroxyecdysone .
  • 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.
  • the extraction is a solid/liquid separation operation: e.g., a plant is placed in contact with a fluid (a solvent).
  • the plant components of interest are solubilised into solution with the solvent. The solution thus obtained is the desired extract.
  • 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.
  • the extraction includes a fdtration, precipitation, crystallization, concentration, or centrifugation step.
  • extraction may result in the extract which is very suitable for isolation of one or more of Sinococuline, Magnoflorine, 20-Hydroxyecdysone or Makisterone-A in higher yield.
  • 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 solvent.
  • the extraction is an alcoholic extraction, e.g., a C1-C4 alcohol extraction, a hydroalcoholic extraction, or an aqueous extraction.
  • the extract is an aqueous extract.
  • the solvents in the extract may be removed completely by evaporation to obtain a dried extract, e.g., an extract with less than 5% water, less than 4% water, less than 3% water, less than 2% water, or less than 1% water.
  • the dried extract may be lyophilized, e.g., to form a powder, which can then be used for isolation of desired compound according to present disclosure that may then be formed into a dosage form, e.g., a capsule, of suitable size or compressed into a dosage form suitable for oral administration, e.g., tablets, with or without pharmaceutically acceptable excipients.
  • the extract may be used as such in liquid or semisolid form without further drying, or with reduced drying, for isolation of the desired compound according to present disclosure.
  • the extract is a purified extract.
  • the extract is a crude extract.
  • the extract is an enriched extract which is can be suitably used for isolation of a particular compound selected from Sinococuline, Magnoflorine, Makisterone-A or 20-Hydroxyecdysone in significantly higher yield.
  • 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.
  • 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 evaporation to obtain a dried extract.
  • the dried extract may be lyophilized, e.g., to form a powder.
  • Such extract can be used for isolation of the desired compound according to present disclosure.
  • alcoholic extract includes any alcohol-based extract, for example, methanolic, ethanolic, n-propanolic, isopropanolic, n-butanolic, iso-butanolic or t-butanolic extract of Cocculus hirsutus.
  • hydroalcoholic extract includes an extract prepared by using a mixture of alcohol and purified water.
  • a hydroalcoholic extract may also include an extract prepared in denatured spirit with other organic solvents.
  • Alcohols can include, e.g., a C1-C4 alcohol such as 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 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.
  • aqueous extract includes a purified water extract of Cocculus hirsutus.
  • the extracts of Cocculus hirsutus can include, but are not limited to, (a) the extracts obtained by extraction of plant mass of Cocculus hirsutus with one or more solvents (e.g., aqueous, alcohol or hydro alcohol), and (b) the fractions obtained by partitioning of the extracts with one or more solvents.
  • 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.
  • Such extract can then be suitably used for isolation of compounds according to present disclosure selected from Sinococuline, Magnoflorine, Makisterone-A or 20-Hydroxyecdysone or pharmaceutically acceptable salts or derivatives thereof.
  • the plant components of interest/extract of the present disclosure are solubilised into solution with the solvent.
  • the solution thus obtained is the desired reservoir for isolation of compounds according to present disclosure.
  • the solvent will eventually be eliminated to arrive at the isolated compound.
  • Separation operations can include mechanical means, e.g., homogenization, chemical means, e.g., acid, alcohol, or aqueous solubilization, and heating means.
  • the extraction and isolation includes a fdtration, precipitation, crystallization, concentration, or centrifugation step.
  • extraction steps may result in the isolation of one or more of Sinococuline, Magnoflorine, 20- Hydroxyecdysone or Makisterone-A in high yield.
  • the solvents for extraction and isolation 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.
  • 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, hex
  • the solvents for partitioning of the components in extract 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.
  • water 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
  • various 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.
  • plant mass of Cocculus hirsutus refers to the whole plant, which includes aerial parts, for example, fruits, flowers, leaves, branches, stem bark, stems, seeds or heartwood, and roots.
  • plant mass of Cocculus hirsutus refers to stem of Cocculus hirsutus.
  • the compound according to present disclosure can be directly isolated from plant mass following extraction using suitable solvent without drying of the extract.
  • the extract may be enriched and standardized with respect to marker compounds, for eg., Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or combination thereof prior to isolation of a particular extract.
  • enriched extract may be used for the isolation of the respective marker compound in higher yield using respective solvent for the marker compound(s).
  • the solvent may be any of the solvent as disclosed above.
  • the extract according to present disclosure is useful both to be directly administrated to a mammal and used in the preparation of a pharmaceutical compositions, or can be used for isolation of compound according to present disclosure.
  • the dose of the extract may be in the range of approximately O.Olmg/kg to approximately 1500mg/kg body weight, particularly in the range of approximately 0.05mg/kg to approximately 1200mg/kg body weight, more particularly in the range of approximately 0.1 mg/kg to approximately 500mg/kg body weight, more particularly in the range of approximately lmg/kg to approximately 150mg/kg body weight.
  • the dose of the extract may be in the range of approximately 2mg/kg to approximately 70mg/kg body weight.
  • the composite extract or its composition may be administered once, twice, thrice or four times a day.
  • the extract of Cocculus hirsutus comprises one or more constituents selected from the group consisting of flavonoids, lignans, steroids and alkaloids or combinations thereof.
  • the extract of Cocculus hirsutus comprises Sinococuline, Magnoflorine, Makisterone or 20-hydroxyecdysone as one of the constituents.
  • the extract of Cocculus hirsutus comprises Magnoflorine in an amount of 0.1% to 1% of the total weight of extract in the composition.
  • the extract of Cocculus hirsutus comprises Magnoflorine in an amount of 0.45% of the total weight of extract in the composition.
  • the composite extract of Cocculus hirsutus comprises quercetin as one of the flavanoids.
  • mamal refers to all mammals including humans.
  • Mammals include, by way of example only, humans, non-human primates, cows, dogs, cats, goats, sheep pigs, rats, mice and rabbits.
  • the present disclosure provides method for prevention and treatment of Dengue infection by administering a therapeutically effective amount of one or more compounds selected from Sinococuline, Magnoflorine, Makisterone A or 20- hydroxyecdysone or combinations or pharmaceutical acceptable salts or derivatives thereof to the mammal.
  • the therapeutically effective amount of the compounds for the treatment of Dengue can be determined by the skilled artisan, e.g., by observation of the patient and physician, including the patient's overall health, the response to the therapy, and the like.
  • the present disclosure provides method for prevention and treatment of Dengue infection by administering a therapeutically effective amount of Sinococuline.
  • the present disclosure provides methods for prevention and treatment of Dengue infection by administering a therapeutically effective amount of Magnoflorine. In another aspect of the embodiment, the present disclosure provides method for prevention and treatment of Dengue infection by administering a therapeutically effective amount of Makisterone A. In another aspect of the embodiment, the present disclosure provides method for prevention and treatment of Dengue infection by administering an effective amount of 20-hydroxyecdysone.
  • terapéuticaally effective amount and “effective amount” are used interchangeably herein and can be referred to as an administered compound (which can be referred to as an agent, a pharmaceutical agent or a drug and is included in the composition or pharmaceutical composition) which is sufficient to carry out the intended use, including but not limited to the treatment of Dengue virus infection or the treatment of a disease or condition related to Dengue virus infection.
  • an effective amount of the compound according to present disclosure reduces one or more of the symptoms, or symptoms of the disease being treated, and / or the amount is associated with the risk that the host being treated has or develops, such as, a fever, pain, headache, joint pain, or other disease symptom is prevented to some extent.
  • the term also applies to a dose that elicits a specific response in the target cell.
  • the specific dose will depend on the particular compound selected, the regimen to follow, whether to administer in combination with other compounds, the timing of administration, the tissue to which it is administered, and the body delivery system in which it is carried.
  • the present disclosure provides method for prevention and treatment of Dengue infection by administering an effective amount of one or more compounds selected from compound of Formulae I-IV or combinations or derivatives thereof, wherein the co-administration of two or more of these compounds results in a synergistic effect against the Dengue virus.
  • the co-administration of Sinococuline (Formula-I) and Magnoflorine (Formula-II) results in a synergistic effect against the Dengue virus.
  • the co-administration of Sinococuline and Makisterone A (Formula-Ill) results in a synergistic effect against the Dengue virus.
  • the co-administration of Sinococuline and 20-hydroxyecdysone results in a synergistic effect against the Dengue virus.
  • the co-administration of Magnoflorine and Makisterone A results in a synergistic effect against the Dengue virus.
  • the co-administration of Magnoflorine and 20-hydroxyecdysone results in a synergistic effect against the Dengue virus.
  • the co-administration of Makisterone A and 20- hydroxyecdysone results in a synergistic effect against the Dengue virus.
  • the co-administration of Sinococuline, Magnoflorine and Makisterone A results in a synergistic effect against the Dengue virus.
  • the co-administration of Sinococuline, Magnoflorine and 20-hydroxyecdysone results in a synergistic effect against the Dengue virus.
  • the co administration of Sinococuline, Makisterone A and 20 -hydroxy ecdysoone results in a synergistic effect against the Dengue virus.
  • the co administration of Magnoflorine, Makisterone A and 20-hydroxyecdysoone results in a synergistic effect against the Dengue virus.
  • Derivatives herein refers to a chemically or biologically modified version of the compounds described above, i.e., structurally similar to and (actually or theoretically) derivable from that parent compound.
  • a hydrogen may be substituted with a halogen, or a hydroxyl group ( — OH) may be replaced with a carboxylic acid moiety ( — COOH) or to esters and amides.
  • the term “derivative” also includes conjugates, and pro-drugs of a parent compound (i.e., chemically modified derivatives which can be converted into the original compound under physiological conditions).
  • the term “derivative” is also used to describe all solvates, for example hydrates or adducts (e.g., adducts with alcohols), active metabolites, and salts of the compounds.
  • co-administration refers to administration of two or more compounds to a mammal.
  • the two or more compounds may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions.
  • Each of the two or more compounds may be administered through the same or different routes of administration.
  • Co administration encompasses administration in parallel or sequentially.
  • the term "synergistic" herein refers to a combination of two or more compounds of the disclosure, which, when taken together, is more effective than the additive effects of the individual compounds.
  • the present disclosure provides a pharmaceutical composition for use in the treatment of Dengue virus infection in mammals comprising Sinococuline, Magnoflorine, Makisterone A or 20-hydroxyecdysone or their derivatives or combinations thereof.
  • the pharmaceutical compositions according to the disclosure are those suitable for enteral, such as oral or rectal, transdermal, topical, and parenteral administration to mammals, including humans.
  • the present disclosure provides an oral pharmaceutical composition for the treatment of Dengue virus infection comprising Sinococuline, Magnoflorine, Makisterone A or 20-hydroxyecdysone or their derivatives or combinations thereof, and one or more pharmaceutically acceptable excipients.
  • the oral pharmaceutical composition can be in the form of powder, pellets, granules, mini-tablets, tablets, capsules or liquid, including but not limited to solutions, suspensions, emulsions or syrups.
  • 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.
  • the present disclosure is an oral pharmaceutical composition for the treatment of Dengue virus infection comprising Sinococuline.
  • the present disclosure provides an oral pharmaceutical composition for the treatment of Dengue virus infection comprising Magnoflorine.
  • the present disclosure provides an oral pharmaceutical composition for the treatment of Dengue virus infection comprising Makisterone A.
  • the present disclosure provides an oral pharmaceutical composition for the treatment of Dengue virus infection comprising 20-hydroxyecdysone.
  • the present disclosure provide for method for prevention and treatment of Dengue infection by administering an effective amount of a pharmaceutical composition of one or more compounds selected from Sinococuline, Magnoflorine, Makisterone A or 20-hydroxyecdysone or combinations or pharmaceutical acceptable salt or derivatives thereof.
  • the pharmaceutical compositions are as described above.
  • the present disclosure provides method for prevention and treatment of Dengue infection by administering an effective amount of a pharmaceutical composition comprising Sinococuline.
  • the present disclosure provides method for prevention and treatment of Dengue infection by administering an effective amount of a pharmaceutical composition comprising Magnoflorine.
  • the present disclosure provides method for prevention and treatment of Dengue infection by administering an effective amount of a pharmaceutical composition comprising Makisterone A. In another aspect of the embodiment, the present disclosure provides method for prevention and treatment of Dengue infection by administering an effective amount of a pharmaceutical composition comprising 20-hydroxyecdysone.
  • the present disclosure provides compounds such as Sinococuline, Magnoflorine, Makisterone A or 20-hydroxyecdysone or pharmaceutical acceptable salts or derivatives or combinations thereof, for use in the treatment of Dengue virus infection in mammals, wherein the compounds exhibit a platelet protective effect.
  • the present disclosure provides compounds such as Sinococuline, Magnoflorine, Makisterone A and 20-hydroxyecdysone or derivatives or combinations thereof, to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals.
  • the present disclosure provides compounds such as Sinococuline, Magnoflorine, Makisterone A and 20-hydroxyecdysone or pharmaceutical acceptable salts or derivatives or combinations thereof, to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals, wherein the compounds exhibits an platelet protective effect.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more compounds selected from Sinococuline, Magnoflorine, Makisterone A and 20-hydroxyecdysone or pharmaceutical acceptable salts or derivatives or combinations thereof, and one or more pharmaceutically acceptable excipients for use in the treatment of Dengue virus infection in mammals, wherein the compounds exhibit a platelet protective effect.
  • the pharmaceutical composition is an oral pharmaceutical composition.
  • the present disclosure provides a pharmaceutical composition comprising Sinococuline, Magnoflorine, Makisterone A, 20-hydroxyecdysone or their derivatives or combinations thereof, and one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals.
  • the pharmaceutical composition is an oral pharmaceutical composition.
  • the present disclosure provides a pharmaceutical composition comprising Sinococuline, and one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals.
  • the present disclosure provides a pharmaceutical composition comprising Magnoflorine, and one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals.
  • the present disclosure provides a pharmaceutical composition comprising Makisterone A, and one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals.
  • the present disclosure provides a pharmaceutical composition comprising 20-hydroxyecdysone, and one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals.
  • the present disclosure provides a pharmaceutical composition comprising Sinococuline, Magnoflorine, Makisterone A, 20-hydroxyecdysone or their derivatives or combinations thereof, and one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals, wherein the compounds exhibits a platelet protective effect.
  • the pharmaceutical composition is an oral pharmaceutical composition.
  • the present disclosure provides a pharmaceutical composition comprising Sinococuline, and one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals, wherein the compounds exhibits a platelet protective effect.
  • the present disclosure provides a pharmaceutical composition comprising Magnoflorine, and one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals, wherein the compounds exhibits a platelet protective effect.
  • the present disclosure provides a pharmaceutical composition comprising Makisterone A, and one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals, wherein the compounds exhibits a platelet protective effect.
  • the present disclosure provides a pharmaceutical composition comprising 20-hydroxyecdysone, and one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals, wherein the compounds exhibits a platelet protective effect.
  • the present disclosure provides a pharmaceutical composition comprising Sinococuline, Magnoflorine, Makisterone A and 20-hydroxyecdysone or derivatives or combinations thereof, wherein the compounds exhibits an inhibitory activity against one or all of Dengue virus serotypes selected from DENV-1, DENV-2, DENV-3 and DENV-4.
  • the compounds exhibit an inhibitory activity against all of Dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4.
  • the pharmaceutical composition is an oral pharmaceutical composition.
  • the present disclosure provides a pharmaceutical composition comprising Sinococuline, wherein it exhibits an inhibitory activity against one or all of Dengue virus serotypes selected from DENV-1, DENV-2, DENV-3 and DENV-4.
  • the present disclosure provides a pharmaceutical composition comprising Magnoflorine, wherein it exhibits an inhibitory activity against one or all of Dengue virus serotypes selected from DENV-1, DENV-2, DENV-3 and DENV-4.
  • the present disclosure provides a pharmaceutical composition comprising Makisterone A, wherein it exhibits an inhibitory activity against one or all of Dengue virus serotypes selected from DENV-1, DENV-2, DENV-3 and DENV-4.
  • the present disclosure provides a pharmaceutical composition comprising 20-hydroxyecdysone, wherein it exhibits an inhibitory activity against one or all of Dengue virus serotypes selected from DENV-1, DENV-2, DENV-3 and DENV-4.
  • the present disclosure relates to use of these compounds and their derivatives as biomarkers in Dengue treatment.
  • these compounds and their derivatives are used as biomarkers in Dengue treatment.
  • the levels of the compounds of the disclosure in a given treatment may therefore be used to predict the effectiveness of the treatment.
  • Different pharmacokinetic parameters like concentration of the compounds or derivatives in the serum or plasma of the Dengue patient, clearance, half-life etc.
  • the present disclosure provides a compound selected from Formula I-IV :
  • Formula-Ill Formula-IV or a pharmaceutically acceptable salt thereof, for use in the treatment of Dengue virus infection.
  • the present disclosure provides a method of treatment of Dengue virus infection, the said method comprising administering to a mammal in need thereof, a therapeutically effective amount of one or more compound selected from Formula I-IV.
  • the said method of treatment is selected from a prophylactic treatment or a curative treatment in a mammal.
  • the compounds according to present disclosure or method of treatment of Dengue virus infection is effective against one or more serotypes of Dengue virus selected from DENV-1, DENV-2, DENV-3 and DENV-4.
  • the said treatment is characterized by a reduction in the titer of Dengue virus in a tissue sample of the mammal.
  • the compound for use or method of treatment according to present disclosure wherein the said compound is a synthetic compound, a semi-synthetic compound or a compound isolated from an extract of the plant Cocculus hirsutus.
  • the present disclosure provides a method for inhibiting the growth and/or proliferation of Dengue virus in a mammal by administering a therapeutically effective amount of one or more compounds selected from Formula I-IV.
  • the method of treatment according to present disclosure wherein the inhibiting the growth and/or proliferation of Dengue virus is determined by measuring the platelet protective effect.
  • the method of treatment according to present disclosure wherein the inhibiting the growth and/or proliferation of Dengue virus takes place in vitro or in vivo.
  • the disclosure provides stable pharmaceutical composition comprising a therapeutically effective amount of one or more compounds selected from Formula I-IV, wherein the said composition exhibits a platelet protective effect.
  • the present disclosure provides stable pharmaceutical composition comprising a therapeutically effective amount of two or more compounds selected from Formula I-IV.
  • the present disclosure provides compounds selected from structure of Formulas I-IV or a pharmaceutically acceptable salt thereof, for treatment or prevention of Dengue virus infection.
  • the compounds according to present disclosure are used in in treatment of Dengue virus infection in a mammal.
  • the compounds of Formula I-IV according to present disclosure may be given in a composition containing one or more of the said compounds.
  • the present disclosure provides a stable pharmaceutically composition for treatment or prevention of Dengue virus infection, comprising extract of Cocculus hirsutus comprising at least a 400% increase in concentration of one or more of the compounds of Formula I to IV relative to the Cocculus hirsutus.
  • extract of Cocculus hirsutus comprising at least a 400% increase in concentration of one or more of the compounds of Formula I to IV relative to the Cocculus hirsutus.
  • about 95% of the fiber has been removed from the said extract
  • about 95% of the lignin has been removed from the said extract.
  • about 95% of the tannin has been removed from the said extract.
  • the said pharmaceutically acceptable extract comprises therapeutically effective amount of one or more of the compounds of formula I-IV.
  • the said pharmaceutically acceptable extract is formed by an aqueous extraction.
  • the said pharmaceutically acceptable extract according to present disclosure comprises compounds of formula I-IV and the said compounds are stable at 25 °C at 75% relative humidity for at least 3 months.
  • the said extract has less than 5% water.
  • the said extract is in form of a solid oral dosage form.
  • the present disclosure provides a pharmaceutically composition comprising one or more compounds of formula I-IV, wherein the one or more compounds are stable at 25°C and 75% relative humidity for at least 3 months.
  • the present disclosure provides a pharmaceutically composition
  • a pharmaceutically composition comprising one or more compounds of formula I-IV and a pharmaceutically acceptable excipient, wherein the one or more compounds are stable at 25 °C and 75% relative humidity for at least 3 months.
  • the said composition has less than 5% water.
  • the said composition is a solid oral dosage form.
  • the present disclosure provides a method of treatment of Dengue virus infection, the said method comprising administering to a mammal in need thereof, a therapeutically effective amount of one or more compounds of formula I to IV, or the pharmaceutically acceptable extract according to present disclosure, or the pharmaceutical composition comprising compounds I-IV or extract as presently disclosed.
  • the said method treatment is selected from a prophylactic treatment and a curative treatment.
  • the said method of treatment of Dengue virus infection wherein the infection is caused by one or more serotypes of Dengue virus selected from DENV-1, DENV-2, DENV-3 and DENV-4.
  • the said method of treatment is characterized by reduced titer of Dengue virus in a tissue sample of the mammal.
  • the present disclosure provides a method for inhibiting the growth and/or proliferation of Dengue virus in a mammal, the said method comprising administering a therapeutically effective amount of one or more compounds of formula I-IV according to present disclosure, the pharmaceutically acceptable extract of present disclosure, or the pharmaceutical composition comprising one or more compound or extract according to present disclosure.
  • the present disclosure provides A method of providing a platelet protective effect to a subject in need thereof, the said method comprising administering a therapeutically effective amount of one or more compounds of formula I-IV according to present disclosure, the pharmaceutically acceptable extract of present disclosure, or the pharmaceutical composition comprising one or more compound or extract according to present disclosure.
  • the isolated compounds of formula I-IV, Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone, respectively, are present in the composition according to present disclosure in the following weight percentages:
  • the present disclosure provides stable pharmaceutical compositions comprising one or more compound selected from Sinococuline, Magnoflorine, Makisterone-A, and 20-Hydroxyecdysone in the following ratios (by weight):
  • the composition can comprise two marker compounds, for e.g., Sinococuline, and Magnoflorine, in the following ratios (by weight):
  • the extract can comprise two marker compounds, for e.g., Sinococuline, and Makisterone-A, in the following ratios (by weight):
  • the extract can comprise two marker compounds, for e.g., Sinococuline, and 20-Hydroxyecdysone in the following ratios (by weight):
  • the extract can comprise two marker compounds, for e.g., Magnoflorine and Makisterone-A in the following ratios (by weight):
  • the extract can comprise two marker compounds, for e.g., Magnoflorine, and 20-Hydroxyecdysone in the following ratios (by weight):
  • the extract can comprise two marker compounds, for e.g., Makisterone-A and 20-Hydroxyecdysone in the following ratios (by weight):
  • the compounds of Formulae I-IV according to present disclosure are present in the composition in a concentration range of 0.1-100% w/w and in combination composition they are present in varying ratios of (0.5 to 99):(0.05 to 99):(0.01 to 99):(0.05 to 99).
  • the one or more compounds according to present disclosure can be administered as such in a therapeutically effective amount with or without suitable vehicle for treatment or prevention of Dengue virus infection.
  • the one or more compounds according to present disclosure can be administered in therapeutically effective amount in suitable dosage form with one or more pharmaceutically acceptable excipient for treatment or prevention of Dengue virus infection.
  • the disclosure provides a pharmaceutical composition comprising one or more compounds as disclosed for use in prophylactic and curative treatment of infections caused by Dengue virus.
  • the pharmaceutical composition according to the present disclosure is an oral dosage form selected from powder, pellets, granules, spheroids, mini-tablets, caplets, tablets, or capsules.
  • the present disclosure provides a pharmaceutical composition of the compounds according to present disclosure for non-oral administration.
  • the present disclosure provides a pharmaceutical composition for use in the prevention and treatment of Dengue virus infection in mammals comprising an extract of Cocculus hirsutus and one or more pharmaceutically acceptable excipients.
  • the present disclosure provides a stable pharmaceutical composition comprising a therapeutically effective amount of an extract of Cocculus hirsutus or one or more isolated compounds therefrom for use in the prevention and treatment of Dengue virus infection in a mammal, wherein the composition when administered to a mammal in need thereof reduces the viral load.
  • the stable pharmaceutical compositions of the present disclosure further comprise one or more pharmaceutically acceptable excipients.
  • pharmaceutical composition includes any composition that can effectively deliver the compound according to present disclosure to the desired site of action to treat or prevent Dengue virus infection.
  • the composition can be delivered by any suitable route of administration, such as oral, nasal, pulmonary, transdermal, or rectal.
  • 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 fdled, with pharmaceutically acceptable excipients, into a capsule of suitable size.
  • 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.
  • the pharmaceutical composition comprising compound according to present disclosure is an oral dosage form selected from powder, pellets, granules, spheroids, mini-tablets, caplets, tablets, or capsules.
  • the pharmaceutical composition comprising one or more compound 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.
  • the product can be stored at room temperature for a shelf life of 6 months to 2 years. This is believed to be surprising as the composition comprises of isolated compounds or an extract comprising flavonoids, alkaloids such as Magnoflorine, lignans etc.
  • the stability of the composition can be assessed by determining some parameters like, assay of one or more marker substance on storage .
  • the marker is a compound of Formula II.
  • Loss on drying (LOD) and disintegration time (in tablet composition) upon storage can serve as a measure of stability of composition.
  • LOD Loss on drying
  • disintegration time in tablet composition
  • a “stable pharmaceutical composition” as used herein refers to a composition 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.
  • the product can be stored at room temperature for a shelf life of 6 months to 2 years.
  • the composition of the present disclosure when stored for 6 months in an HDPE bottle at accelerated condition of 40°C/75% RH, showed no degradation of a compound of Formula II content, LOD was found to be within the range of 2-5%w/w and there was no change in the disintegration time.
  • the pharmaceutical composition comprising one or more compound according to the present disclosure comprises one or more of the pharmaceutically acceptable excipients selected from diluents, binders, disintegrants, lubricants, glidants, polymers, flavoring agents, surfactants, preservatives, antioxidants, buffers, and tonicity modifying agents.
  • pharmaceutically acceptable excipients includes diluents, binders, disintegrants, lubricants, glidants, polymers, flavoring agents, surfactants, preservatives, antioxidants, buffers, and tonicity modifying agents.
  • 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 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, 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.
  • a water-soluble starch for example, pregelatinized starch
  • a polysaccharide for example, agar, gum acacia, dextri
  • 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 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 combinations thereof.
  • cinnamon oil 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 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 long-chain fatty acid ester such as a polyoxyethylene sorbitan monolaurate, for example, a polysorbate; amphoteric surfactants, for example, a glycine compound such as do
  • 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, 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; dipolar aprotic solvents; methylene chloride; acetone; 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.
  • a diluent may be used as binder in particular concentration.
  • the pharmaceutically acceptable excipient in the composition of the instant disclosure includes microcrystalline cellulose, anhydrous lactose, croscarmellose sodium, colloidal silicon dioxide and magnesium stearate.
  • the present disclosure provides a stable pharmaceutical composition comprising a therapeutically effective amount of one or more compounds according to present disclosure for use in the treatment of Dengue virus infection in a mammal, wherein the composition when administered to a mammal in need thereof reduces the viral load.
  • the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds according to present disclosure with one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of Dengue virus infection in mammals.
  • the composition is a stable pharmaceutical composition. More preferably, the composition is a stable oral pharmaceutical composition.
  • the present disclosure provides a stable pharmaceutical composition comprising a therapeutically effective amount of one or more compounds according to present disclosure for use in the prevention of Dengue virus infection in a mammal.
  • treatment is meant to include therapeutic treatment, including prophylactic or preventive and curative treatments.
  • the term treatment may include administration of a therapeutically effective amount of one or more compounds according to present disclosure or compositions thereof or in combination with any other 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 Dengue virus infection.
  • administration of a compound or a pharmaceutical composition prior to or after clinical manifestation of Dengue virus infection to prevent or combat the signs and symptoms and/or complications and disorders associated with Dengue virus infection comprises “treatment” of the disease.
  • treatment of a subject comprises inducing and maintaining remission of Dengue virus infection in a subject.
  • treatment of Dengue virus infection in a subject comprises maintaining remission of Dengue virus in a subject.
  • the present disclosure provides a method for treatment of Dengue virus infection in mammals comprising administering compounds of Formula I, II, III or IV or pharmaceutical acceptable salts or their derivatives or combinations thereof.
  • the present disclosure provides a method for prevention of Dengue virus infection in mammals comprising administering compounds of Formula I, II, III or IV or pharmaceutical acceptable salts or their derivatives or combinations thereof.
  • the present disclosure provides a method for treatment of Dengue virus infection in mammals comprising administering compounds of Formula I, II, III or IV or pharmaceutical acceptable salts or their derivatives or combinations thereof, wherein the compounds exhibits a platelet protective effect.
  • the present disclosure provides a method for reducing the viral load at an early stage in the treatment of Dengue virus infection in mammals comprising administering compounds of Formula I, II, III or IV or pharmaceutical acceptable salts or derivatives or combinations thereof.
  • the present disclosure provides a method for reducing the viral load at an early stage in the treatment of Dengue virus infection in mammals comprising administering compounds of Formula I, II, III or IV or pharmaceutical acceptable salts or derivatives or combinations thereof, wherein the compounds exhibit a platelet protective effect.
  • the appropriate dosages for the compounds or derivatives or combinations thereof or extract for the treatment of Dengue can be determined by the skilled artisan, e.g., by observation of the patient and physician, including the patient's overall health, the response to the therapy, and the like.
  • the present disclosure provides a method of treating Dengue virus infection in a mammal comprising administering a pharmaceutical composition comprising a administering therapeutic effective amount of an extract of Cocculus hirsutus to a mammal in need thereof, wherein the extract reduces the viral load.
  • the present disclosure provides a method of treating Dengue 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 exhibits a platelet protective effect. It was found that when administered to winstar rats at a dose of 600 or 1200 mg/kg/day by oral gavage for 14 days a higher platelet count was observed.
  • the present disclosure provides a method of treating
  • Dengue virus infection in a mammal comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof, wherein the extract exhibits a platelet protective effect.
  • the present disclosure provides a method of treating Dengue 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 wherein the extract further exhibits a platelet protective effect.
  • the present disclosure provides a method of treating Dengue virus infection in a mammal comprising administering a composite extract of Cocculus hirsutus to a mammal in need thereof, wherein the extract reduces the viral load, not only from the serum but also from tissues such as but not limited to intestine, and wherein the extract further exhibits a platelet protective effect.
  • the present disclosure provides a method for reducing the viral load at an early stage in the treatment of Dengue virus infection in mammals comprising administering a pharmaceutical composition comprising a therapeutically effective amount of an extract of Cocculus hirsutus to a mammal in need thereof, wherein the extract exhibits a platelet protective effect.
  • the present disclosure provides a method for reducing the viral load at an early stage in the treatment of Dengue virus infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof, wherein the extract exhibits a platelet protective effect.
  • the present disclosure provides a method of prophylaxis and/or treating both primary and secondary Dengue virus infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof.
  • the present disclosure provides a method of reducing the cytokine load in both primary and secondary Dengue virus infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof.
  • the present disclosure provides an oral composition comprising a composite extract of Cocculus hirsutus, wherein the said composition exhibits an inhibitory activity with IC50 value ranging from about 1 to 100 pg/ml as determined using FACS based assay, against Dengue virus selected from DENV-1, DENV-2, DENV-3 and DENV-4.
  • said extract and composition is used in the treatment of Dengue virus infection caused by serotypes selected from DENV-1, DENV-2, DENV-3 and DENV-4 or combinations thereof.
  • the Dengue virus infection is caused by serotypes selected from DENV-1 and/or DENV-2 or combinations thereof.
  • the Dengue virus infection is caused by serotypes selected from DENV-1 and/or DENV-3 or combinations thereof. In another aspect of the embodiment, the Dengue virus infection is caused by serotypes selected from DENV-1 and/or DENV-4 or combinations thereof. In another aspect of the embodiment, the Dengue virus infection is caused by serotypes selected from DENV-2 and/or DENV-3 or combinations thereof. In another aspect of the embodiment, the Dengue virus infection is caused by serotypes selected from DENV-2 and/or DENV-4 or combinations thereof. In another aspect of the embodiment, the Dengue virus infection is caused by serotypes selected from DENV-3 and/or DENV-4 or combinations thereof.
  • the extract of Cocculus hirsutus is not known in the prior art for its inhibitory activity against different serotypes of Dengue virus.
  • FNT FACS based Neutralization Test
  • a composite extract of Cocculus hirsutus was found to be very efficacious in inhibiting DENV4 serotype. It is known that recovery from infection by one Dengue serotype provides lifelong immunity against that particular serotype. However, cross-immunity to the other serotypes after recovery is only partial and temporary. Subsequent infections (secondary infection) by other serotypes increase the risk of developing severe Dengue because of antibody dependent enhancement (ADE).
  • ADE antibody dependent enhancement
  • the present compounds provide advantage over treatments known in the art which are effective only against a particular serotype, as it was surprisingly found that the composite extract of Cocculus hirsutus and the composition of the present disclosure were active against all serotypes Dengue virus DENV-1, DENV-2, DENV-3 and DENV-4.
  • the composite extract of Cocculus hirsutus and the composition of the disclosure exhibits an inhibitory activity with IC50 value ranging from about 1 to 20 pg/ml as determined using FACS based assay, against all the Dengue virus serotypes DENV-1, DENV- 2, DENV-3 and DENV-4.
  • the extract and composition were effective against both primary and secondary Dengue virus infection.
  • the present inventors have found that the extract, the isolated compounds from the extract and their synthetic equivalents and the composition comprising them when tested in animal model of primary and secondary Dengue infection were found to exhibit protection in both primary and secondary Dengue in AG 129 mouse model.
  • AG 129 is an immune-compromised mouse model (IFN a/b and g receptors deficient) in which DENV infection can be established to exhibit increased vascular leakage, viremia, and inflammatory cytokines.
  • IFN a/b and g receptors deficient immune-compromised mouse model
  • ADE Antibody Dependent Enhancement
  • the present inventors analyzed the effect of different doses, dosing schedules and delayed treatment of AQCH in both the primary and secondary Dengue infections in AG129 mice.
  • the ability of AQCH was also assessed as a prophylactic model in secondary Dengue AG 129 mice.
  • Serum viremia and small intestinal pathologies, viremia, vascular leakage and pro-inflammatory cytokines, which are considered as hallmarks of Dengue infection were also examined in secondary Dengue disease AG129 model.
  • the composition comprising quercetin as a constituent exhibits an IC50 value ranging from about 2 to 10 pg/ml against DENV-1. In another aspect of the embodiment, the composition comprising quercetin exhibits an IC50 value ranging from about 1 to 10 pg/ml against DENV-2. In another aspect, the composition comprising quercetin exhibits an IC50 value ranging from about 5 to 15 pg/ml against DENV-3. In another aspect, the composition comprising quercetin exhibits an IC50 value ranging from about 5 to 20 pg/ml against DENV-4.
  • the present disclosure provides a method of preventing vascular leakage in severe Dengue infection due to primary or secondary Dengue infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof.
  • mice orally fed with the extract or the compositions of the instant disclosure exhibited lower vascular leakage (in a dose-dependent manner) as compared to untreated group, which corroborates that the extract to inhibit DENV infection.
  • the present disclosure provides a method of inhibiting the secretion of cytokines in severe Dengue infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof.
  • the extract and the compositions were also found to be effective in inhibiting the secretion of cytokines in severe Dengue infection.
  • Pro-inflammatory cytokines like TNF-a and IFN-g are produced in excessive amounts which lead to disease progression and vascular leakage.
  • the extract or the composition of the disclosure inhibited the secretion of cytokines in the small intestine.
  • Small intestines of AG 129 mice challenged with DENV-2 S221-4G2 Immune Complex (IC) and fed with the extract or the composition of the disclosure possessed lesser amounts of TNFa and IL-6.
  • the present disclosure provides a method of treatment of Dengue 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.
  • the extract and composition of the disclosure when administered after 6 to 12 hours of establishing primary Dengue infection in a AG 129 mice model, it was found that the delay in treatment did not affect the protective efficacy of the extract or the composition.
  • the present disclosure provides a method of prevention of Dengue virus infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof.
  • the extract and the composition of the present disclosure when administered pre-infection were found to be show protective efficacy against secondary Dengue infection when tested in an in-vivo study in AG 129 mouse model.
  • the extract and composition of the present disclosure were found to be safe and didn’t show any toxic effect when administered in a therapeutically effective dose to the mammal in need thereof. After repeated oral administration at dose levels of 100, 300, 600 or 1200 mg/kg/day by oral gavage in wistar rats for 14 days the hematological, gross and histological changes were determined. At these doses a higher platelet count was observed. Based on these results, NOAEL in Wistar rats, was established at 300 mg/kg/day. Thus the extract was found to be safe and non-toxic for human use at the maximum dose of 50mg/kg/day.
  • the extract may be co-administered with one or more additional therapeutic agents.
  • the composition of the disclosure may further comprise one or more additional therapeutic agents.
  • the one or more additional therapeutic agents may be selected from anti-pyretic/analgesic compounds such as NSAIDs like Paracetamol, other anti-viral drugs etc.
  • co-administration 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. Each of the extract or additional therapeutic agents may be administered through the same or different routes of administration. Co-administration encompasses administration in parallel or sequentially.
  • the extract or the isolated compounds 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 delivery, ophthalmic delivery, transmucosal delivery, etc.
  • the extract and pharmaceutical compositions described herein can be administered via oral delivery.
  • the pharmaceutical composition comprises 0.1 mg to 100 mg of the active (isolated compound or the extract).
  • the pharmaceutical composition according to the present disclosure for use in treatment of Dengue virus infection is administered to a patient in need thereof as an oral dose of 600 mg twice daily, 400 mg thrice daily, 300 mg four times daily or 200 mg six times daily.
  • the 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.
  • the 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 total daily dose.
  • the present disclosure provides a 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 a pharmaceutical composition of Cocculus hirsutus extract, wherein the composition comprises about 15-70% of the extract by weight of the tablet.
  • the present disclosure provides a method for treating Dengue virus infection by administering to a patient in need thereof a stable, composition comprising a therapeutically effective amount of an extract of Cocculus hirsutus or isolated compound thereof selected from a compound of Formula II (Magnoflorine) 0.1 - 200 pg/ml, a compound of Formula I (Sinococuline) 0.1 - 5 pg/ml, a compound of Formula IV (20-Hydroxyecdysone) >500 pg/ml and a compound of Formula III (Makisterone-A) 1 - 5 pg/ml.
  • the present disclosure provides use of a pharmaceutical composition
  • a pharmaceutical composition comprising one or more compound according to present disclosure selected from Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof in method for treating Dengue virus infection by administering the composition to a patient in need thereof, wherein the composition reduces the viral load.
  • the present disclosure provides use of a pharmaceutical composition comprising one or more compound according to present disclosure selected from Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof in a method for prevention of Dengue virus infection in a patient.
  • the present disclosure provides use of a pharmaceutical composition comprising one or more compound according to present disclosure selected from Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof for a method of treatment of Dengue virus infection in mammals comprising administering the composition to the mammals in need thereof, wherein the composition provides a platelet protective effect.
  • the present disclosure provides use of a pharmaceutical composition comprising one or more compound according to present disclosure selected from Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof in a method of prevention of Dengue virus infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof.
  • the present disclosure provides use of one or more compounds according to present disclosure for manufacture of a medicament useful for treatment of Dengue virus infection in a patient
  • the one or more compound according to present disclosure is isolated from an extract.
  • the extract is prepared by a process comprising 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, 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.
  • the extraction of the plant mass of Cocculus hirsutus is done at a temperature in the range of about 50° to about 100°C. In another aspect of the above embodiment, the extraction of the plant mass of Cocculus hirsutus is done at a temperature of about 80° to about 85°C. In another aspect of the above embodiment, the extraction of the plant mass of Cocculus hirsutus is done at a temperature of about 60° to 65°C. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 40° to about 95°C.
  • the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 40° to about 45°C. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 45° to about 50°C. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 55° to about 65°C. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 90° to about 95°C. In yet another aspect, the plant mass can be extracted from a dry part or a wet part of the plant.
  • the isolated compounds are obtained from a dried extract from Cocculus hirsutus, wherein the dried extract is obtained by: 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, 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 25°C for at least one week.
  • the dried extract according to present disclosure has a water concentration of not more than 5% w/w.
  • the dried extract according to present disclosure has a C1-C4 alcohol concentration of not more than 10,000 ppm.
  • the present disclosure provides 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; d) fdtering the extract and collecting the fdtrate; e) optionally fdtering the residue at least once with solvent to obtain fdtrate; and f) concentrating the fdtrate from and optionally drying to obtain said extract.
  • the present disclosure provides a process of preparation of tablet composition comprising one or more compounds selected from Sinococuline, Magnoflorine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof for use in the treatment of Dengue virus infection, the process comprising the steps of: i. sifting the one or more compounds and blending with pharmaceutically acceptable excipients; ii. lubricating the blend obtained and compressing into tablets and iii. film coating the tablets.
  • the present disclosure provides a process of preparation of tablet composition comprising one or more compounds selected from a compound of Formula I, II, III and IV or a combination thereof for use in the treatment of Dengue virus infection, the process comprising the steps of: i. blending one or more compounds 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.
  • the present disclosure provides a process of preparation of tablet composition comprising one or more compounds selected from a compound of Formula I, II, III and IV or a combination thereof for use in the treatment of Dengue virus infection, the process comprising the steps of: i. blending one or more compounds with pharmaceutically acceptable excipients and compacting the mixture; ii. milling the compacts and blending with extragranular excipients; iii. lubricating the blend and compressing into tablets and iv. film coating the tablets.
  • the present disclosure provides a process of preparation of tablet composition comprising of Cocculus hirsutus extract for use in the treatment of Dengue virus infection, the process comprising the steps of: i. blending one or more compounds with pharmaceutically acceptable excipients and compacting the mixture; ii. milling the compacts and blending with extragranular excipients; iii. lubricating the blend and compressing into tablets and iv. fdm coating the tablets.
  • one or more compounds according to present disclosure may also be formulated into any other suitable oral dosage forms like powders, pellets, granules, spheroids, mini-tablets, caplets, sachets, etc.
  • the one or more compounds may be co administered simultaneously or sequentially with one or more additional therapeutic agents.
  • the 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 which may provide symptomatic relief from the conditions, for examples antibiotics, antipyretic and analgesic drugs.
  • Example 1 Extract preparation:
  • BRMs botanical raw materials
  • C. pariera accesion No. RRLH-23148
  • C hirsutus accesion No. RRLH-23152
  • RRLH Janaki Ammal Herbarium
  • BRM of C. hirsutus collected from the same area was procured from the local vendor and duly identified samples of C. hirsutus i.e., aerial (Accession Nos.
  • CDR-4037, and CDR- 4038) and stem (Accession Nos. CDR-4061, CDR-4064, CDR-4065, and CDR-4078) parts were submitted to the Crude Drug Repository (CDR) at CSIR-IIIM, Jammu. BRMs were dried in shade and pulverized. The ground plant masses of C. pareira and/or C. hirsutus were separately charged into an extractor at ambient temperature. The extracting solvent (hydro alcohol, 50:50/ denatured Spirit/ water) was added and the mixture was heated to reflux temperature for about 3 hr. The extracted mass was fdtered, collected and stored in a container. The extraction process was repeated from the residue plant mass two more times. All three filtered extracts were combined concentrated and further dried either using rotary vapour, tray under vacuum or spray drying. The yield obtained was 7-14 % depending on the part of the plant and extracting solvent used.
  • CDR Crude Drug Repository
  • Example 1-A Preparation of 95:5 Ethanol : purified water extract of Cocculus hirsutus
  • Example 1-B Preparation of 1 : 1 Ethanol : purified water extract of Cocculus hirsutus
  • Example 1-C Preparation of an aqueous extract of Cocculus hirsutus
  • ambient temperature includes a temperature ranging from about 18°C to about 25°C.
  • Example 2 Isolation of compounds from Cocculus hirsutus:
  • Coniferyl (SP-A-01) was obtained in pure form fractions based on TLC profile.
  • aqueous layer (B) was basified with ammonium hydroxide (NH 4 OH) solution, and subsequently extracted with CHCb, resulted in alkaloid rich fraction (El) and aqueous layer (FI).
  • the CHCb layer (El) was further processed for separation of a compound of Formula I (Sinococuline) through column chromatography eluted with CHCb-McOH.
  • the Aqueous layer (FI) was powdered using lyophilization and dissolved in methanol.
  • the methanol dissolved portion was further purified by column chromatography, eluted with a gradient of CHCb -Me OH, and concentrated, giving fifty fractions.
  • Two UV active spots were visualized on TLC.
  • This mixture containing two UV active compounds were further subjected to RP-HPLC (using a gradient solvent system of McOH/FhO) to obtain a compound of Formula IV (20-hydroxyecdysone) and Formula III (Makisterone A).
  • Example 3 In vitro and in vivo DENV inhibition assays
  • Vero cell line was purchased from American Type Cell Culture (ATCC), Virginia, USA. This monkey kidney cell line was maintained using Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% AFBS, in a 10% CO2 humidified incubator at 37 °C. WHO reference DENV strains DENV-1 (WP 74), DENV -2 (S16803), DENV-3 (CH53489), and DENV -4 (TVP-360) were received from Dr. Aravinda de Silva’s lab, University of North Carolina (UNC), USA. These viruses were cultured in C6/36 cells procured from ATCC, Virginia, USA.
  • DMEM Modified Eagle medium
  • AFBS AFBS
  • WHO reference DENV strains DENV-1 (WP 74), DENV -2 (S16803), DENV-3 (CH53489), and DENV -4 (TVP-360) were received from Dr. Aravinda de Silva’s lab, University of North Carolina (UNC), USA. These viruses were cultured in C6/
  • the Vero cells were infected with DENVs, and post-infection the cells were incubated in media containing extract at various concentrations for 46 hr. Post-incubation cells were fixed, permeabilized and stained with Alexa fluor labelled anti-Dengue mAh, 2H2, reactive to all the four DENV serotypes, which were read in a flow cytometer to determine the percentage of DENV infected cells. This was used to calculate the extract concentration at which 50% of the DENV infection was inhibited (IC50).
  • Flow cytometry based virus inhibition assay Vero cells were seeded in a 96- well plate (20,000-25,000 cells/well) in 200 m ⁇ DMEM + 10% AFBS and incubated for 24 hr in an incubator adjusted at 37 °C and 10% CO2. Next day, cells were infected with 100 m ⁇ of DENV-1, -2, -3, and -4 dilutions to yield 10% infection in DMEM + 0.5% AFBS (dilution media).
  • test substance AQCH extract/isolated compounds
  • the media was aspirated from the top of the cells and washed with PBS.
  • Cells were trypsinised and transferred to a 96 well U bottom plate. After transfer, the cells were centrifuged at 1500 rpm for 5 mins and supernatant was aspirated. Cells were washed with PBS again and then fixed with 4% para-formaldehyde for 20 mins. Cells were centrifuged at 2500 rpm for 5 mins and supernatant was aspirated. Cells were washed twice with permeabilization or perm buffer and blocked with 1% normal mouse sera (prepared in perm buffer) for 30 mins.
  • Alexa-488 labeled 2H2 mAb was added to stain the cells for DENVs and the cells were incubated for 1 hr at 37 °C with gentle shaking. Post-incubation, the cells were centrifuged at 2500 rpm for 5 mins and the supernatant aspirated. Cells were washed twice with perm buffer and re suspended in 100 m ⁇ of PBS. The above processed cells were analysed through a BD FACS Verse flow cytometer and 5000 cells were counted per well. Data was analyzed through FlowJo software to determine the relative percentage of infected cells for each test substance concentration with respect to virus only control group. The 50% inhibitory concentration (IC50) of the test substance was determined as the concentration that inhibited 50% of Dengue virus infection with respect to vims control, calculated using non-linear regression analysis of GraphPad Prism software.
  • IC50 50% inhibitory concentration
  • Example 4 Selection of aqueous extract of stem of C. hirsutus for further evaluation
  • Example 5 NS1 ELISA assay & Extracellular viral estimation
  • NS1 Assay Vero cells were seeded in a 48 well plate (40,000 cells/well) in 500 m ⁇ DMEM + 10% AFBS. Next day, cells were infected with DENV- 1, -2, -3, and -4 at 0.1 MOI. After 2 hr of infection, the media was aspirated and the cell monolayer was overlaid with different concentrations of extracts (100, 50, 25, and 12.5 pg/ml) in vims infection media (DMEM + 0.5% AFBS). Wells that did not receive any infection but only vims infection media treatment served as negative control.
  • Extracellular viral estimation In this assay, culture supernatant of DENV- infected cells were collected and the vims was titrated in both the AQCH treated and untreated wells. Briefly, Vero cells were seeded and infected in six 96 well plates as detailed in flow cytometry based vims inhibition assay. After 2 hr of infection, the vims infection media was aspirated and the monolayer was overlaid with 200 m ⁇ of different concentrations of extracts (100, 50, 25, and 12.5 mg/ml) in vims infection media. Post 24 hr of infection, one plate was harvested each day for the next 6 days and the supernatant was transferred to a U-bottom 96- well plate.
  • Example 6 In-vitro evaluation of anti-Dengue activity of compounds (Sinococuline, Magnoflorine, Makisterone A, and 20-hydroxyecdysone) through FACS based Neutralization Test (FNT)
  • the above processed cells were analyzed through flow cytometer and cells counted. Data obtained was analyzed (through FlowJo software) to determine the relative percentage of infected cells for each inhibitor concentration with respect to virus only (without any inhibitor treatment) control group.
  • the IC50 of all compounds was determined as the concentration that inhibited 50% of Dengue virus infection, calculated using GraphPad Prism software.
  • the IC50 value of Sinococuline, Magnoflorine, Makisterone A, and 20-hydroxyecdysone for DENV-4 serotype is as provided in Table 1 below.
  • Table 1 Tabular representation of IC50 (pg/ml) of marker compounds against Dengue virus serotype 4 (DENV-4) as examined through FNT.
  • Example 7 AQCH chemical fingerprinting instrumentation and characterization methodology
  • 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 a 0.45 pm filter for HPLC fingerprinting.
  • the HPLC fingerprinting was performed on a RP18e Purospher- STAR (Hibar) (250 c 4.6 mm; 5 pm) column.
  • the mobile phase containing a buffer (0.1% formic acid in water) and 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 pi 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.
  • SI Fig For the isolation (SI Fig), 500 g of AQCH was suspended in distilled water and partitioned between ethyl acetate (A) and H20 (B). The aqueous layer (B) was basified with NH40H solution (pH 9) and then extracted with chloroform, CHC13.
  • the CHC13 layer (C) was further purified through repeated column chromatography in neutral alumina and eluted with a gradient of CHC13-MeOH (100:0 to 0:100) to obtain Compound 1 as major constituent along 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 CHC13-MeOH (100:0 to 0:100, 500 ml, collected volumes of each fraction), and concentrated, giving fifty fractions (Fr.l-Fr.50) and their composition was monitored by TLC. Those showing similar TLC profiles were grouped into six major fractions (Fr-lato Fr-5a). Fraction Fr-2a afforded two UV active compounds as crystals.
  • the ethyl acetate soluble fraction (A) was subjected to column chromatography (silica gel, 100-200 mesh), eluted with a gradient of CHC13-MeOH (100:0 to 0: 100, 250 ml, collected volumes of each fraction), and concentrated, giving thirty fractions (Fr.51-Fr.80).
  • Compound 5 was obtained from fractions Fr. 66-Fr. 70 in pure. All the isolated compounds 1-5 were identified by detailed spectral analysis ID and 2D NMR, and HRESI-MS data and comparisons with the reported spectral data.
  • Example 8 In vivo evaluation of the efficacy of AQCH in AG129 primary Dengue lethal mouse model
  • mice deficient in IFN-a/b and IFN-g receptors were purchased from the B&K Universal, United Kingdom, and housed and bred at International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi.
  • Experimental mice (six per group), 6 to 8 weeks old, were housed in BSL-2 containment facility. They were intravenously injected with a lethal dose (1.0 x 10 5 FIU) of mouse -adapted DENV-2 strain S221.
  • mice were fed orally four times a day (QID) with either 8.25 or 25 mg/kg/dose of AQCH for a period of five days.
  • QID a lethal dose
  • mice groups were monitored for their survival, body weight change and morbidity score for a period of 15 days post-infection. Statistical evaluation of survival score was performed through Log Rank (Mantel Cox) test and p value ⁇ 0.05 was considered to be statistically significant.
  • Morbidity score was based on 5 point system: 0.5, mild ruffled fur; 1.0, ruffled fur; 1.5, compromised eyes; 2, compromised eyes with hunched back; 2.5, loose stools; 3.0, limited movement; 3.5, no movement/hind leg paralysis; 4.0, euthanized if cumulative score was 5. All AQCH doses used for feeding were prepared at once in water with 0.1% methylcelhilose (v/v) and stored at 4 °C. An appropriate volume of doses was pre-incubated at room temperature before feeding to animals. For a QID dosing, mice were fed with AQCH at 4/4/4/12 hr cycle (7 AM, 11 AM, 3 PM and 7 PM on each day for 5 days).
  • Example 9 In vitro evaluation of the interaction of Paracetamol with AQCH
  • Example 10 In vivo evaluation in Primary and Secondary Dengue Disease AG129 Mouse Model
  • mice in ‘Uninfected’ and Only AQCH’ groups are expected to survive through the entire study period. Mice in ‘V’ group succumb to infection in 4-7 days in primary model because of infection being lethal, while in secondary model they survive because of infection being sub-lethal. The sub-lethal dose gets enhanced to lethal dose through 4G2 mediated ADE causing IC inoculated AG 129 mice to succumb to infection in 4-7 days.
  • Figure 9 provides a schematic overview of the experiments performed in primary and secondary Dengue AG 129 mouse models in this study for the in-depth characterization of protective efficacy of AQCH.
  • Secondary Dengue AG129 mice model was established through inoculation of immune complex (IC) of sub-lethal dose of DENV-2 S221 (2x104 FIU) and neutralizing concentration of 4G2 mAh (10 pg).
  • the level of protection conferred by AQCH and delayed treatment effect were examined in similar manner as in primary Dengue AG 129 model.
  • AQCH was orally gavaged 3 days before and after infection with IC in secondary disease mice at 50 mg/kg/dose BID. Mice were monitored for body weight change, disease symptoms and survival.
  • ADE has been implicated in causing severe Dengue.
  • Vascular leakage was examined using Evans blue dye which serves as a marker for albumin extravasation. Leakage was quantitated by colorimetric estimation of the amount of dye absorbed, which was represented as fold increase in OD620 per gm of tissue’s wet weight in comparison to the ‘Un-infected’ control. Vascular leakage was found to have been significantly reduced upon AQCH treatment, both with 25 mg/kg QID ( Figure 15a, solid blue bar) and 8.25 mg/kg QID ( Figure 15a, empty blue bar). Expectedly, the highest level vascular leakage was observed in the IC group of mice ( Figure 15a, grey bar) due to persistence of 4G2 mAb mediated enhanced infection.
  • AQCH was formulated into tablets of different strengths (100 mg, 300 mg, and 500 mg) using the approved excipients.
  • the accelerated and long-term stability of AQCH and AQCH tablets was assessed by exposing them to different conditions (30 ⁇ 2°C / 65 ⁇ 5% RH, and 40 ⁇ 2°C / 75 ⁇ 5% RH).
  • the in vitro anti-Dengue activity and the content of a selected marker compound were evaluated for the stored samples at different time points (1, 2, 3, and 6 months) through flow cytometry based virus inhibition assay and HPLC chromatography, respectively.
  • IC50 Anti-DENV-2 activity of AQCH and AQCH tablets under the stated conditions of storage a Anti -DENV-2 activity is measured as IC50, which corresponds to the concentration at which 50% of the virus is inhibited with respect to virus control
  • Table 3 Stability data of AQCH and AQCH tablets with respect to content of Magnoflorine as analytical marker *nd: not determined; these specific storage conditions were for long-term stability studies and were therefore not sampled on 1 st and 2 nd month of storage.
  • AQCH was found to inhibit the secretion of both DENV and NS1 in a dose-dependent manner, with complete inhibition being observed at 100 and 50 pg/ml extract. This is relevant because DENV load and NS1 have been implicated in Dengue disease pathogenesis in humans. From the HPC profiling of an AQCH batch five marker compounds- Sinococuline, Magnoflorine, 20-Hydroxyecdysone, Makisterone-A and Coniferyl alcohol were also identified (Fig 4).
  • AQCH was also evaluated for its protective efficacy in vivo in the AG129 mouse model (Fig 6), and when fed at 25 mg/kg/dose QID, it was found to provide 100% protection to AG 129 mice that were lethally infected with DENV -2.
  • Paracetamol a standard care drug in treating Dengue, was also found to have no effect on the anti-Dengue activity of AQCH (Fig 7).
  • spray -dried AQCH was formulated into tablets of various strengths, which were found to be stable upon storage (Table 1). It was also examined if AQCH could protect AG 129 mice at 100 mg/kg/day QID in both the infection models. Surprisingly, AQCH could survive the mice upon delayed treatment in both the infection models.

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Abstract

La présente invention concerne un ou plusieurs composés ou leurs sels ou dérivés pharmaceutiquement acceptables pour le traitement prophylactique et/ou curatif d'une infection provoquée par le virus de la Dengue. L'invention concerne également une composition pharmaceutique stable comprenant un ou plusieurs composés ou leurs sels ou dérivés pharmaceutiquement acceptables et des extraits pour le traitement prophylactique et/ou curatif d'une infection provoquée par le virus de la Dengue. La présente invention concerne également une méthode de réduction de la charge virale et/ou d'inhibition de la croissance ou de la prolifération de sérotypes du virus de la Dengue chez un mammifère, ladite méthode comprenant l'administration d'un ou de plusieurs composés ou de leurs sels ou dérivés pharmaceutiquement acceptables ou d'un extrait comprenant de tels composés, en tant que tels ou dans une composition pharmaceutiquement acceptable, à un mammifère en ayant besoin.
PCT/IB2020/060325 2019-11-04 2020-11-03 Composés pour le traitement d'une infection de la dengue WO2021090177A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
DE102007004589A1 (de) * 2007-01-30 2008-07-31 Orlowski, Michael, Dr. Bioresorbierbarer Metallstent mit kontrollierter Resorption
US20110150989A1 (en) * 2009-12-22 2011-06-23 Mallinkckrodt Inc. Methods of Producing Stabilized Solid Dosage Pharmaceutical Compositions Containing Morphinans
EP2775859A1 (fr) * 2011-11-10 2014-09-17 Institut Biophytis SAS Phytoecdysones pour leur utilisation dans la stabilisation du poids après un régime amaigrissant

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DE102007004589A1 (de) * 2007-01-30 2008-07-31 Orlowski, Michael, Dr. Bioresorbierbarer Metallstent mit kontrollierter Resorption
US20110150989A1 (en) * 2009-12-22 2011-06-23 Mallinkckrodt Inc. Methods of Producing Stabilized Solid Dosage Pharmaceutical Compositions Containing Morphinans
EP2775859A1 (fr) * 2011-11-10 2014-09-17 Institut Biophytis SAS Phytoecdysones pour leur utilisation dans la stabilisation du poids après un régime amaigrissant

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CHENG YUAN-BIN ET AL: "Ecdysones fromZoanthusspp.with inhibitory activity against dengue virus 2", BIORGANIC & MEDICINAL CHEMISTRY LETTERS, ELSEVIER, AMSTERDAM , NL, vol. 26, no. 9, 9 March 2016 (2016-03-09), pages 2344 - 2348, XP029500656, ISSN: 0960-894X, DOI: 10.1016/J.BMCL.2016.03.029 *
GOODLA ET AL., EGYPTIAN JOURNAL OF BASIC AND APPLIED SCIENCES, vol. 4, December 2017 (2017-12-01), pages 264 - 269
LEE JIN-CHING; CHANG FANG-RONG; CHEN SHU-RONG; WU YU-HSUAN; HU HAO-CHUN; WU YANG-CHANG; BACKLUND ANDERS; CHENG YUAN-BIN: "Anti-Dengue Virus Constituents from Formosan Zoanthid Palythoa mutuki", MARINE DRUGS, vol. 14, no. 8, 9 August 2016 (2016-08-09), Switzerland, XP009525089, DOI: 10.3390/md14080151 pubmed:27517937 *
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SOO, KUAN-MENG ET AL.: "Meta-analysis of biomarkers for severe Dengue infections", PEERJ, vol. 5, 2017, pages e3589
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