THERAPEUTIC FLAVONOID BASED ANTIVIRAL AGENTS

Abstract

The world is plagued with several viruses some of which have prevention and treatment tools available, while every so often a new strain will show up without sensitivity to existing drugs. The present invention provides plant-based flavonoid pharmaceutical compositions for inhibition of kinases, particularly phosphatidylinositol-4-kinases (PI4Kiiiβ), AAK1, BIKE, GAK and other transcription factors required for viral entry, replication and survival, and consequent for prevention and treatment of RNA viruses including but not limited to adenoviruses, alphaviruses, coronaviruses, enteroviruses, flaviviruses, hepatitis, herpes, influenza viruses, measles, picornaviruses, vesicular stomatitis and associated disorders. A method for synthesizing the flavonoids and formulation into therapeutic products are also disclosed.

Claims

1. A method for the treatment of a patient in need thereof by administering to said patient a compound having a general chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00003## wherein, R1-R10 may be any one or more substituents selected from the group consisting of a hydrogen molecule (H), a hydroxide molecule (OH), a methyl group comprising one carbon atom bonded to three hydrogen atoms (CH3), an alkoxy group (O—CH3), a carboxyl group (COOH), chlorine (Cl), Bromine (Br), Fluorine (F), Glutamic acid (Glu), and any salts or derivatives of the foregoing, and A and B may be linked by either a single or double bond.

2. The method according to claim 1 for the treatment of a patients having an RNA virus.

3. The method according to claim 2 for the treatment of a patient having viral hepatitis.

4. The method according to claim 1 for the treatment of a patient with coronaviruses

5. The method according to claim 1 for the treatment of a patient having influenza viruses.

6. The method of claim 1, wherein said compound is administered in a concentration within a range of from 0.1 to 500 mg between 1-6 times per day.

7. An extract of the Vemonia acuminata and cannabis plant having a general chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00004## wherein, R1-R10 may be any one or more substituents selected from the group consisting of a hydrogen molecule (H), a hydroxide molecule (OH), a methyl group comprising one carbon atom bonded to three hydrogen atoms (CH3), an alkoxy group (O—CH3), a carboxyl group (COOH), chlorine (Cl), Bromine (Br), Fluorine (F), Glutamic acid (Glu), and any salts or derivatives of the foregoing, and A and B may be linked by either a single or double bond.

8. The extract of claim 7, derived from said Vemonia acuminata plant by supercritical fluid extraction.

9. A method for the treatment of a patient in need thereof by administering to said patient a compound having the general chemical structure of claim 7.

10. The method of claim 9, wherein said extract is administered in a concentration within a range of from 0.1 to 500 mg between 1-6 times per day.

11. The method of claim 10, wherein said extract is administered in a formulation comprising a carrier, said carrier being selected from the group consisting of: lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl benzoate, propyl benzoate, talc, magnesium stearate, and mineral and vegetable oils.

12. The method of claim 11, wherein said extract is administered in a concentration within a range of from 0.1 to 500 mg between 1-6 times per day.

13. The method of claim 12, wherein said extract is administered in a form selected from the group consisting of: powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and suppositories.

14. The method of claim 13, wherein said extract is administered in a formulation comprising a carrier, said carrier being selected from the group consisting of: lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl benzoate, propyl benzoate, talc, magnesium stearate, and mineral oil.

15. A method of treating viral hepatitis, the method comprising administering the extract of claim 6.

16. The method of claim 15, wherein said extract is administered in a concentration within a range of from 0.1 to 500 mg between 1-6 times per day.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Other objects, features, and advantages of the present invention will become more apparent from the following detailed description of the preferred embodiments and certain modifications thereof when taken together with the accompanying drawings in which:

[0033] FIG. 1 is an illustration of the general plant-based flavonoid pharmaceutical compositions according to the present invention.

[0034] FIG. 2 is the structure of the specific plant-based flavonoid pharmaceutical composition.

[0035] FIG. 3 is a graphical illustration of how the kinase inhibition assay works.

[0036] FIG. 4. Is a block diagram of a suitable isolation scheme.

[0037] FIG. 5 is a process diagram illustrating a suitable synthesis approach.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Reference will now be made in detail to preferred embodiment of the present invention, examples of which are illustrated in the accompanying drawing.

[0039] The present invention is a group of plant-based flavonoid pharmaceutical compositions isolated from a supercritical fluid extract (SFE) of Vemonia acuminate and Cannabis sativa, from the Blue Mountains of Jamaica, and useful for the prevention and treatment of RNA viruses including but not limited to viral Coronaviruses, Chikigunya, Dengue, Ebola, hepatitis, HIV, influenza, picomavirus, Zika.

[0040] The plant-based flavonoid pharmaceutical composition for the prevention and treatment of RNA viruses including but not limited to adenoviruses, alphaviruses, coronaviruses, enteroviruses, flaviviruses, hepatitis, herpes, influenza viruses, measles, picomaviruses, vesicular stomatitis and associated disorders has the structure of the general formula of FIG. 1 or a pharmaceutically acceptable salt thereof.

##STR00002##

[0041] wherein,

[0042] R1-R10 may be any one or more substituents selected from the group consisting of a hydrogen molecule (H), a hydroxide molecule (OH), a methyl group comprising one carbon atom bonded to three hydrogen atoms (CH3), an alkoxy group (O—CH3), a carboxyl group (COOH), chlorine (Cl), Bromine (Br), Fluorine (F), Glutamic acid (Glu), geranyl chain, prenyl chain and any salts or derivatives of the foregoing. A and B may be linked by either a single or double bond.

[0043] The most preferred structure of the synthesized flavonoids presented in FIG. 2.

[0044] In an embodiment, a method for the prevention and treatment of RNA viruses including but not limited to adenoviruses, alphaviruses, coronaviruses, enteroviruses, flaviviruses, hepatitis, herpes, influenza viruses, measles, picornaviruses, vesicular stomatitis and associated disorders using the specific plant-based flavonoid pharmaceutical compositions above is also disclosed. Administration may be by various routes including oral, rectal or intravenous, epidural muscle, subcutaneous, intranasal, intrauterine, or blood vessels in the brain (intracerebroventricular) injections. The flavonoid derivatives of the general and specific formulas (FIGS. 1-2) according to the present invention and a pharmaceutically acceptable salt thereof may be administered in an effective dose, depending on the patient's condition and body weight, extent of disease, drug form, route of administration, and duration, within a range of from 0.1 to 500 mg between 1-6 times a day. Of course, most dosages will be by a carrier. The specific dose level and carrier for patients can be changed according to the patient's weight, age, gender, health status, diet, time of administration, method of administration, rate of excretion, and the severity of disease.

[0045] The composition may be formulated for external topical application, oral dosage such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, suppositories, or in the form of a sterile injectable solution. Acceptable carriers and excipients may comprise lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl benzoate, propyl benzoate, talc, magnesium stearate, polyethylene glycol and mineral and vegetable oils.

[0046] Bioactivity of the above-described compounds have been verified by use of kinase inhibition assays to determine the effect of the flavonoids in the onset and progression of RNA viruses. The inhibition of PI4K kinases in particular has been shown to be a therapeutic target that could block the replication of RNA viruses including but not limited to adenoviruses, alphaviruses, coronaviruses, enteroviruses, flaviviruses, hepatitis, herpes, influenza viruses, measles, picornaviruses, vesicular stomatitis and associated disorders.

[0047] Anti-Hepatitis C Activity

[0048] Huh-7.5 cells are grown in Dulbecco's modified essential media (DMEM), 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (pen-strep), 1% Non-essential amino acids (NEAA) in a 5% CO.sub.2 incubator at 37° C. Huh7.5 cells will be seeded at 1×10.sup.4 cells per well into 96-well plates according to Southern Research Institute standard format. Test article will be serially diluted with DMEM plus 5% FBS. The diluted compound in the amount of 50 μl will be mixed with equal volume of cell culture-derived HCV (HCVcc), then applied to appropriate wells in the plate. Human interferon alpha-2b (rIFNα-2b) is included as a positive control compound. After 72 hr incubation at 37° C., the cells were lysed for measurement of luciferase activity using Renilla Luciferase Assay System (Promega) according to manufacturer's instruction. The number of cells in each well will be determined by CytoTox-1 reagent (Promega). Test articles are tested with 6 serial dilution in triplicate to derive, if applicable, IC.sub.50 and IC.sub.90 (concentration inhibiting HCVcc infectivity by 50% and 90%, respectively), TC.sub.50 (concentration decreasing cell viability by 50%) and SI (selective index: TC.sub.50/IC.sub.50) values.

[0049] Results of the inhibition of HCVcc are indicated in the table below:

TABLE-US-00001 Compound Test Concentration EC.sub.50 CC.sub.50 SI (CC.sub.50/EC.sub.50) rIFNa-2b 10 IU/mL 0.63 >10.0 >15.9 FBL-02 100 μg/mL 1.37 4.18 3.05

[0050] Anti Coronavirus Activity

[0051] This test is for initial screening of potentially antiviral compounds. The antiviral activity of the compound is evaluated based on the ability of the compound to prevent virus from causing viral CPE in mammalian cell culture. Different dilutions of test compound are evaluated, and the effective antiviral concentration determined by regression analysis. The toxicity of the test compound is determined in parallel. CPE is determined by microscopic observation of cell culture monolayers as well as uptake of neutral red dye. Cell line, Vero 76, was obtained from American Type Culture Collection (ATCC; Manassas, Va., USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). The HCoV-OC43 (VR-1558) strain was obtained from the ATCC. Vero-76 cells were treated with multiple concentrations of each compound then infected with virus, and the 50% effective concentration (EC.sub.50), the 50% cytotoxic concentration (IC.sub.50), and the selective index (SI) for each compound were calculated. The results of the anti coronavirus study are presented in the table below.

TABLE-US-00002 hCoV-OC43 (beta) Concentration (μM) Compound EC.sub.50 CC.sub.50 SI (CC.sub.50/EC.sub.50) Reference 0.24 >100 >420 FBL-03A 3.2 4.8 1.5 FBL-03C 2.7 3.5 1.3 FBL-03G 0.42 2.9 6.9 FBLGS70 3.1 55.0 18 FBLGS71 6.8 >100 >15

[0052] Kinase Inhibition Assay

[0053] In vitro profiling of lipid and tyrosine kinases was accomplished using the “HotSpot” assay platform. Briefly, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer; 20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO. Compounds were delivered into the reaction, followed ˜20 min later by addition of a mixture of ATP (Sigma) and 33P ATP (PerkinElmer) to a final concentration of 10 μM. Reactions were carried out at 25° C. for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper (e.g., Whatman Ashless Filter Paper). Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme, kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. IC.sub.50 values and curve fits were obtained using Prism™ (by GraphPad Software). Kinome tree representations were prepared using Kinome Mapper.

[0054] To determine the kd values, competition binding assays were established, authenticated and executed as described previously (Fabian et al., 2005, Karaman et al., 2008). For most assays, kinases were fused to T7 phage strains (Fabian et al. 2005) and for the other assays, kinases were produced in HEK-293 cells after which they were tagged with DNA for quantitative PCR detection (data not shown). In general, full-length constructs were used for small, single domain kinases, and catalytic domain constructs for large multi-domain kinases. The binding assays utilized streptavidin-coated magnetic beads treated with biotinylated small molecule ligands for 30 minutes at room temperature which generated affinity resins for the kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 40× stocks in 100% DMSO and diluted directly into the assay (Final DMSO concentration=2.5%). All reactions were performed in polypropylene 384-well plates in a final volume of 0.04 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by quantitative PCR. A graphical illustration of the kinase interaction process is presented below. Kd values were determined using a standard dose response curve using the hill equation. Curves were fitted using a non-linear least square fit with the Levenberg-Marquardt algorithm.

[0055] FIG. 3 is a graphical illustration of how the foregoing assay works.

[0056] Percent Control (% Ctrl)

[0057] The compound(s) were screened at 10 μM and results for primary screen binding interactions are reported as ‘% Ctrl’, where lower numbers indicate stronger hits in the matrix.

[00001]$\%\mathrm{Ctrl}C\mathrm{alculation}\left(\frac{\mathrm{test}\mathrm{compound}\mathrm{signal}-\mathrm{positive}\mathrm{control}\mathrm{signal}}{\mathrm{negative}\mathrm{control}\mathrm{signal}-\mathrm{positive}\mathrm{control}\mathrm{signal}}\right)\times 100$

test compound=compound submitted by Environmental Health Foundation
negative control=DMSO (100% Ctrl)
positive control=control compound (0% Ctrl)

[0058] Results of the inhibition of 12 lipid kinases by flavonoids are shown in the table below:

TABLE-US-00003 Compound IC50 (μM) Kinase: FBL-02 FBLGS70 FBLGS71 FBL-03C FBL-03G FBL-03G-M1 FBLGS81 AAK1 ND ND ND ND 0.004 0.047 ND ABL2 ND ND 0.926 ND 0.274 ND ND BIKE ND ND ND ND 0.258 0.870 ND CSNK2A1 ND 0.118 0.802 ND 0.020 0.370 0.137 CSNK2A2 ND 0.071 0.617 ND 0.004 0.280 0.060 GAK ND ND ND ND 0.008 0.189 ND MNK2 ND 0.148 — ND 0.549 1.0  0.023 PI3Kb 2.27 21.2   ND 0.928 0.136 ND ND

[0059] A method for isolating the specific flavonoid pharmaceutical compositions from raw plant material is also disclosed. The isolation was realized according to the scheme shown in FIG. 4.

[0060] At step 10 an appropriate amount of plant biomass is collected. For present purposes, Vemonia acuminate, a plant from the Blue Mountains of Jamaica, was collected by hand. The collected plant material was air dried under shade and pulverized into powder.

[0061] At step 20 the powder is subjected to supercritical fluid extraction (SFE) by which carbon dioxide (CO.sup.2) is used for separating one component (the extractant) from another (the matrix). The extract is evaporated to dryness resulting in a green residue.

[0062] At step 30, for experimental purposes, a bioassay-guided fractionation was employed, using a standard protocol to isolate a pure chemical agent from its natural origin. This entailed a step-by-step separation of extracted components based on differences in their physicochemical properties and assessing all their biological activity. The extracted components may, for example, be fractionated by dry column flash chromatography on Si gel using hexane/CH2Cl2/ethyl acetate and mixtures of increasing polarity to yield different fractions. The sample is then degassed by ultra-sonication to yield an insoluble solid, which solid is then filtered. The sample may then be subjected to high performance liquid chromatography (HPLC) using a column Phenomenex Luna™ C18, 5 μm, 2×50 mm; eluent, acetonitrile with 0.05% MeOH to confirm the presence of the various fractions.

[0063] At step 40, bioactivity of the extracts were verified in a kinase inhibition assay as described above. This identified the bioactive flavonoids from all the supercritical fluid extracts (SFE). As reported previously, the identified plant-based flavonoid extracts showed activity against several kinases implicated in the pathogenesis of the prevention and treatment of RNA viruses including but not limited to adenoviruses, alphaviruses, coronaviruses, enteroviruses, flaviviruses, hepatitis, herpes, influenza viruses, measles, picomaviruses, vesicular stomatitis and associated disorders.

[0064] The next step was to identify the plant-based flavonoid constituents responsible for the observed kinase inhibitory activities and to further isolate them.

[0065] At step 50 Nuclear Magnetic Resonance Spectroscopy and mass spectrometry (NMR/MS) was performed and the interpreted spectra were consistent with plant-based flavonoid compositions, as identified above, and as shown in step 60. The bioactive plant-based flavonoid extracts found bioactive for the prevention and treatment of RNA viruses had the structure of the general formula of FIG. 1, and the specific structure of FIG. 2.

[0066] The compounds are designated FBL-02 and FBL-03G, and purity of the compounds were confirmed by HPLC prior to spectroscopic analysis.

[0067] Given the known structure of the general formula of FIG. 1, a method for synthesizing the same becomes possible. The bioactive plant-based flavonoid pharmaceutical composition may be synthesized by the phenylpropanoid metabolic pathway in which the amino acid phenylalanine is used to produce 4-coumaroyl-CoA.

[0068] FIG. 5 is a process diagram illustrating a suitable synthesis approach. The 4-coumaroyl-CoA is combined with malonyl-CoA to yield the flavonoid backbone, which contains two phenyl rings. Conjugate ring-closure of chalcones results in the familiar form of flavonoids, the three-ringed structure of a flavone. The metabolic pathway continues through a series of enzymatic modifications to yield the desired Flavone, Flavanone and Flavanol as identified above, and as shown in step 60. Of course, one skilled in the art will readily understand that other methods for synthesis are possible, such as the asymmetric methods set forth in Nibbs, A E; Scheidt, K A, “Asymmetric Methods for the Synthesis of Flavanones, Chromanones, and Azaflavanones”, European journal of organic chemistry (2012): 449-462. doi:10.1002/ejoc.201101228 (PMC 3412359; PMID 22876166).

[0069] It should now be apparent that the above-described invention provides a pharmaceutical composition for inhibition of phosphatidylinositol-4-kinases and consequent prevention and treatment of RNA viruses including but not limited to adenoviruses, alphaviruses, coronaviruses, enteroviruses, flaviviruses, hepatitis, herpes, influenza viruses, measles, picomaviruses, vesicular stomatitis and associated disorders. The invention also provides a method for isolating the flavonoid pharmaceutical compositions from raw plant material.

[0070] It is to be understood, therefore, that the invention may be practiced otherwise than as specifically set forth in the appended claims.