THERAPEUTIC AGENTS CONTAINING CANNABIS FLAVONOID DERIVATIVES TARGETING KINASES, SIRTUINS AND ONCOGENIC AGENTS FOR THE TREATMENT OF MULTIPLE MYELOMA, LYMPHOMA, AND LEUKEMIA

Abstract

A cannabis-based flavonoid pharmaceutical composition for treatment of multiple myeloma, lymphoma, and leukemia, the composition including any one or more selected from among the group of Cannflavin A, Cannflavin B, Cannflavin C, FBL-03G (Caflanone), Chiysoeriol, Cosmosiin, Flavocannabiside and their derivatives selected from among the group of Geraldol, Rhamnetin, Isorhamnetin, Rhamnazin, or their synthases

Claims

1. A cannabis-based flavonoid pharmaceutical composition for the prevention and treatment of multiple myeloma, lymphoma, and leukemia having a general flavone backbone (2-phenyl-1,4-benzopyrone) chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00004## wherein R1, R3, R5, R6, R7 and R10 may be none, 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, R2, R4, R8 and R9 may be any one or more substituents selected from said group, and A and B may be linked by either a single or double bond.

2. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00005##

3. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00006##

4. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00007##

5. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00008##

6. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00009##

7. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00010##

8. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00011##

9. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00012##

10. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00013##

11. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00014##

12. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00015##

13. A cannabis-based flavonoid pharmaceutical composition for the prevention and treatment of multiple myeloma, lymphoma, and leukemia having a general flavone backbone (2-phenyl-1,4-benzopyrone) chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00016## R2 may be one or more substituents selected from the group consisting of a hydrogen molecule (H), a hydroxide molecule (OH), or an oxygen molecule (0); R3 may be none, or one or more hydrogen molecules (H), R4 may be none, or any one or more substituents selected from the group consisting of a hydroxide molecule (OH) or a nitrogen dioxide molecule (NO2); R5 and R8 may be none, or a hydroxide molecule (OH); R7 may be none, or the amine NH2; R9 may be none, or any one or more substituents selected from the group consisting of a hydroxide molecule (OH) or an oxygen molecule (0); and A and B may be be linked by either a single or double bond.

14. The cannabis-based flavonoid pharmaceutical composition according to claim 13, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00017##

15. The cannabis-based flavonoid pharmaceutical composition according to claim 13, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00018##

16. The cannabis-based flavonoid pharmaceutical composition according to claim 13, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00019##

17. The cannabis-based flavonoid pharmaceutical composition according to claim 13, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof: ##STR00020##

18. A method of treating multiple myeloma, lymphoma, and leukemia, the method comprising administering the general composition of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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:

[0022] FIG. 1A is an illustration of the general cannabis-based flavonoid pharmaceutical compositions according to the present invention.

[0023] FIG. 1B is an illustration of the specific family of cannabis-based flavonoid pharmaceutical compositions best-suited to be therapeutic targets for the cancer AML.

[0024] FIG. 2 is a flow diagram illustrating a suitable method for isolating the specific cannabis-based flavonoid pharmaceutical compositions from raw plant material.

[0025] FIG. 3 is a process diagram illustrating a suitable synthesis approach.

[0026] FIG. 4 is an illustration of the specific isolated cannabis-based flavonoid pharmaceutical compositions including Flavone, Flavanone and Flavanol isolates and their synthetic derivatives, according to the present invention.

[0027] FIG. 5 is a graphical illustration of the results of the kinase inhibition by cannabis flavonoids and derivatives presented in Table 1 and Table 4

[0028] FIG. 6 is a graphical illustration of the results of the anticancer activity presented in Table 3 (Hela cells at A and CMK cells at B) and Table 4 (MV4-11, MOLT14 and AML29).

[0029] FIG. 7 is a graphical illustration of the results of the anticancer activity in mice bearing pancreatic cancer treated with FBL-03B.

[0030] FIG. 8 is a graphical illustration of the results of the anticancer activity in mice bearing acute myeloid leukemia harboring FLT3 mutation.

[0031] FIG. 9 is a graphical illustration of the results of the anticancer activity in mice bearing pancreatic cancer treated with Caflanone (FBL-03G)

[0032] FIG. 10 presents the mechanism of inhibition of FLT3 and FLT3-ITD by FBLGS-70.

[0033] FIG. 11 presents the computer aided drug design study of FBLGS-70 and FBLGS-81 docking interaction with the crystal structure of FLT3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Reference will now be made in detail to preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

[0035] An oncogene is herein defined as a gene that has the potential to cause cancer. In tumor cells, oncogenes are often mutated or expressed at high levels. Certain cancers overexpress certain oncogenic factors including kinases, sirtuins, bromodomains, matrix metalloproteinases and histone deacetylases. Thus, these particular oncogenic factors are identified as useful therapeutic targets for purposes of the present invention. Given the foregoing targets, some of the cancers that can be treated by use of cannabis flavonoids based on the inhibition of these therapeutic targets include brain, breast, colon, renal, liver, lung, lymphoma, pancreatic, pigmented villonodular synovitis, prostate, leukemia, melanoma, tenosynovial giant cell tumor as well as any other cancers (solid, soft or heamatological) that overexpress the oncogenic factors inhibited by the identified cannabis based flavonoids.

[0036] The present invention is a group of cannabis-based flavonoid pharmaceutical compositions selected from among the group of Apigenin, Caflanone, Cannflavin A, Cannflavin B, Cannflavin C, Chrysoeriol, Cosmosiin, Flavocannabiside, Kaempferol, Luteolin, Myricetin, Orientin, Isoorientin (Homoorientin), Quercetin, (+)-Taxifolin, Vitexin, and Isovitexin and their derivatives including geraldol, rhamnetin, isorhamnetin, rhamnazin, useful for the prevention and treatment of certain cancers by targeting kinases, sirtuins, bromodomains, matrix metalloproteinases and histone deacetylases which have been identified to be useful therapeutic targets for said cancers.

[0037] Some of the cancers that can be treated by use of cannabis flavonoids based on the inhibition of these therapeutic targets include brain, breast, colon, renal, liver, lung, lymphoma, pancreatic, pigmented villonodular synovitis, prostate, leukemia, melanoma, multiple myeloma, tenosynovial giant cell tumor as well as any other cancers (solid, soft or heamatological) that overexpress the oncogenic factors inhibited by the cannabis flavonoids identified under this invention.

[0038] The cannabis-based flavonoid pharmaceutical composition for the prevention and treatment of cancers has the structure of the general formula shown below (see also FIG. 1), or a pharmaceutically acceptable salt thereof

##STR00002##

[0039] wherein,

[0040] 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. A and B may be linked by either a single or double bond.

[0041] As described below the present inventors have tested candidates from the general grouping on various cancer cell lines and were further able to identify the most potent family of candidates as therapeutic targets for the particular cancer AML, the family being selective FLT3, FLT3-ITD and FLT-3 D835Y inhibitors with low toxicity and safety screen issues during in vitro profiling. This family of AML-therapeutic targets likewise has the flavone backbone (2-phenyl-1,4-benzopyrone) chemical structure, in this case more defined as shown below (see also FIG. 1B), or any pharmaceutically acceptable salt thereof:

##STR00003## [0042] R2 may be one or more substituents selected from the group consisting of a hydrogen molecule (H), a hydroxide molecule (OH), or an oxygen molecule (0); [0043] R3 may be none, or one or more hydrogen molecules (H), [0044] R4 may be none, or any one or more substituents selected from the group consisting of a hydroxide molecule (OH) or a nitrogen dioxide molecule (NO2); [0045] R5 and R8 may be none, or a hydroxide molecule (OH); [0046] R7 may be none, or the amine NH2; [0047] R9 may be none, or any one or more substituents selected from the group consisting of a hydroxide molecule (OH) or an oxygen molecule (0); [0048] and A and B may each be either a single or double bond.

[0049] From the family of AML-therapeutic targets the inventors isolated one particular flavonoid molecule (FBLGS-70 described below) that proved to be a first-in-class selective FLT3, FLT3-ITD and FLT-3 D835Y inhibitor with no detectable toxicity or safety screen issues. The molecule FBLGS-70 exhibited significant in-vivo efficacy on MV4-11 AML. Thus, FBLGS-70, FBL-03G and derivatives were synthesized, and structure activity relationship studies were conducted. From these the inventors identified FBLGS-81, an isomer of FBLGS-70 also described below, as one of the most potent candidate to advance for further development against AML.

[0050] FIG. 2 is a flow diagram illustrating a suitable method for isolating the specific cannabis-based flavonoid pharmaceutical compositions from raw plant material.

[0051] From the general family, it was found that the most preferred flavonoid molecules for use in combatting AML are primarily FBLGS-81, FBLGS-70, and FBL-03G, all Type 1 selective tyrosine kinase inhibitors with potent activity against FLT3, FLT3-ITD and FLT3-D835Y kinases.

[0052] FIG. 3 shows a molecular illustration of the specific isolated cannabis-based flavonoid pharmaceutical compositions of the present invention including Flavone, Flavanone and Flavanol isolates and their synthetic derivatives, complete with a process diagram illustrating a suitable synthesis approach for each.

[0053] A method for the prevention and treatment of multiple myeloma, lymphoma, and leukemia using the specific cannabis-based flavonoid pharmaceutical compositions above is also disclosed. Administration may be by oral or intravenous injections. The flavonoid derivatives of the general formula (FIG. 1) 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.

[0054] The composition may be formulated for oral dosage such as powders, granules, tablets, capsules, suspensions, emulsions, syrups 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, mineral oil, castor oil and polyethylene glycol.

[0055] Bioactivity

[0056] Bioactivity of the above-described compounds were verified and is presented in Tables 1, 2, 3 and 4 below:

TABLE-US-00001 TABLE 1 FBL- FBL- FBL- FBL-03G Kinase 03A 03B FBL-03C 03D (FIG. 2) IC.sub.50 (nM) Aurora A 730 12 >30000 1090 BIKE >20000 >20000 21 >30000 81 CK2a 740 768 58 >30000 38 CK2a2 350 477 19 >30000 9.7 c-Kit(Y823D) >20000 244 >20000 >30000 84 c-Kit(D820Y) >20000 1280 >20000 >30000 113 DRAK2 >20000 <1000 980 >30000 1400 DYRK1/DYRK1A >20000 <1000 620 >30000 36 DYRK1B >20000 1670 6400 >30000 22.8 EFGR(L858R, >20000 >1000 680 >30000 >1000 T7790M) EPHB6 >20000 >1000 270 >30000 >1000 FGR >20000 224 >20000 >30000 880 FLT3 >20000 41.9 >20000 >30000 44 FLT3(D835Y) >20000 12.7 >20000 >30000 45 FLT3(D835V) 220 <1000 190 >30000 12.5 FLT3(ITD) >20000 57 >20000 >30000 38.2 FLT4(VEGFR3) 330 9.3 >20000 >30000 4220 FMS/CSF1R 1500 199 1200 >30000 4 JAK1 — — — — 644 JAK2 — — — — 1850 JAK3 >20000 >1000 780 >30000 5.58 KIT 350 <1000 >1000 >30000 <1000 KIT(L576P) 180 <1000 >1000 >30000 <1000 KIT(V559D) 200 <1000 >1000 >30000 <1000 MELK >20000 232 >1000 >30000 88.9 MEK5 140 >1000 84 >30000 190 MNK2 — — — — 549 PASK >20000 2060 >20000 >30000 116 PDGFRa — 982 — >30000 698 PDGFRa(T6741) — 0.92 >1000 >30000 1670 PDGFRB 330 1160 >1000 >30000 3310 PIK3CA(1800L) >20000 780 >30000 125 PIK4CB 670 >1000 >30000 136 PIM-1 >20000 >1000 >30000 78 PIM-3 >20000 173 >1000 >30000 35 PIP5K1A >20000 >10000 360 >30000 320 RIOK1 >20000 >10000 340 >30000 >20000 RIOK3 >20000 >10000 280 >30000 >20000 SIK2 >20000 >10000 >1000 >30000 63 SRPK1 >20000 >10000 300 >30000 >10000 SRC — — — — 1700 c-SRC — — — — 1100 TNIK >20000 152 >1000 >30000 115 TXK — — — — 164 Yes/yes1 — — — — 294

TABLE-US-00002 TABLE 2 Activity FBL-03A FBL-03B FBL-03C FBL-03D FBL-03G SIRT IC.sub.50 (μM) SIRT-1 19.00 27.40 39.50 — — SIRT-2 2.57 10.80 14.00 2.38 24.10 SIRT-3 94.90 77.00 65.40 — 66.40 SIRT-5 123.00 104.00 132.00 — 974.00 Bromodomain IC.sub.50 (μM) BRD2 NT 9.52 NT NT 12.00 BRD3 NT 7.05 NT NT 8.69 BRD4 NT 10.40 NT NT 6.14 Matrix metallo- proteinase IC.sub.50 (μM) MMP-2 NT 115.00 NT NT 6.64 MMP-3 NT — NT NT 66.30 MMP-7 NT 17.52 NT NT 3.35 MMP-9 NT — NT NT 85.40 IC.sub.50 (μM) BCL-2 NT — NT NT 2.49 BCL-XL NT — NT NT —

TABLE-US-00003 TABLE 3 Isolation And Synthesis Cell Line FBL-03A FBL-03B FBL-03C FBL-03D FBL-03G IC.sub.50 (μM) A498 (Kidney) 17 NT 14 NT NT A549 (Lung) 17 NT 9.4 NT NT CFPAC-1 12 17 12 NT 14.32 (Pancreatic) CMK NT 11.60 NT NT 1.78 (leukemia) COLO-205 27 NT 17 NT NT (Colon) DLD-1 (Colon) 15 NT 13 NT NT HC-1 NT 29.70 NT NT 5.00 (Leukemia) HeLa (cervical) NT 10.40 NT NT 2.53 IGROV-1 29 15 NT NT (Ovarian) KMS-11 NT NT NT NT 0.0647 (Multiple myeloma) MCF-7 (Breast) 17 NT 12 NT NT MiaPaca-2 16 NT 9.5 NT NT (Pancreatic) MOLT-4 NT 13.20 NT NT 20.00 (Leukemia) MV4-11 NT 1.43 NT NT 0.0386 (Leukemia) MM.1S NT NT NT NT 0.95 (Multiple Myeloma) NCI-H69 16 11 18 NT 9.5 (Small lung) PC-3 (Prostate) 26 NT 20 NT NT RL 5.9 NT 12 NT NT (Lymphoma) SNU-16 NT 15.00 NT NT 4.09 (Stomach) U2-OS (Bone) NT 19.40 NT NT 8.70 UACC-62 27 NT 14 NT NT (Melanoma) U87 (Glioma) 34.00 6.20 12.50 NT 5.46

[0057] A method for isolating the specific cannabis-based flavonoid pharmaceutical compositions from raw plant material is also disclosed. The isolation was realized according to the scheme shown in FIG. 2.

[0058] At step 10 an appropriate amount of plant biomass is collected. For present purposes, Cannabis sativa plants were collected by hand. See, Radwan, M. M., ElSohly, M. A., Slade, D., Ahmed, S. A., Wilson, L., El-Alfy, A T., Khan, I. A., Ross, S. A., “Non-Cannabinoid Constituents From A High Potency Cannabis Sativa Variety”, Phytochemistry 69, 2627-2633 (2008) and Radwan, M. M., Ross, S. A., Slade, D., Ahmed, S. A., Zulfiqar, F., ElSohly, M. A., “Isolation And Characterization Of New Cannabis Constituents From A High Potency Variety”, Planta Med. 74, 267-272 (2008). The collected plant material was air dried under shade and pulverized into powder.

[0059] 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.

[0060] 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/CH.sub.2Cl.sub.2/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.

[0061] At step 40, bioactivity of the extracts were verified by an anticancer cell proliferation assay as described above. This identified the bioactive flavonoids from all the supercritical fluid extracts (SFE). As reported previously, the identified cannabis-based flavonoid extracts showed activity against several cancer cell lines including brain, breast, Kaposi sarcoma, leukemia, lung, melanoma, tenosynovial giant cell tumor ovarian, pancreatic, colon and prostate cancer.

[0062] At step 50 Nuclear Magnetic Resonance Spectroscopy and mass spectrometry (NMR/MS) was performed and the interpreted spectra were consistent with cannabis-based flavonoid compositions as identified above, as illustrated in step 60.

[0063] Synthesis

[0064] Given the known structure of the general formula of FIG. 1 and the isolate of FIG. 2, a method for synthesizing the same becomes possible. The bioactive cannabis-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.

[0065] FIG. 3 is a process diagram illustrating a suitable synthesis approach for the cannflavins. The 2′,4′,6′-Trihydroxyacetophenone was the major starting material and the synthesis was carried out using art known to the industry with modifications yielded the flavonoid backbone, which contains two phenyl rings for the cannflavins. 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, Flavone and Flavanol as identified above and as shown in step 60 (FIG. 3). The specific Flavone, Flavanone and Flavanol isolates are shown in step FIG. 4.

[0066] For background see Minassi, A., Giana, A., Ech-Chahad, A., & Appendino, G. “A regiodivergent synthesis of ring A C-prenylflavones”, Organic Letters 10(11), 2267-2270 (2008). 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”,

[0067] European Journal Of Organic Chemistry, 449-462. doi:10.1002/ejoc.201101228. PMC 3412359. PMID 22876166 (2012). Borsari, Chiara, et al. “Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs.” Journal of Medicinal Chemistry 59.16 (2016): 7598-7616. Pandurangan, N. “A new synthesis for acacetin, chiysoeriol, diosmetin, tricin and other hydroxylated flavones by modified baker-venkataraman transformation.” Letters in Organic Chemistry 11.3 (2014): 225-229. Wang, Q. Synthesis of citrus bioactive polymethoxyflavonoids and flavonoid glucosides. Chinese journal of organic chemistry, 2010, 30, 11, 1682-1688.

[0068] Bioactivity Assays

[0069] Cannabis flavonoids and their analogs were subjected to kinase inhibition assay. The compounds were first screened at a single concentration of 10 μM in the primary assay. Compounds inhibiting at least 70% of specific kinases were subjected to further screening to determine kd/IC.sub.50 values. To determine the kd or IC.sub.50 values, competition binding assays were established, authenticated and executed as described previously. Fabian et al., “A Small Molecule-Kinase Interaction Map For Clinical Kinase Inhibitors.”, Nat Biotechnol, 23(3):329-36, Epub (2005). See also, Karaman et al., “A Quantitative Analysis Of Kinase Inhibitor Selectivity”, Nat. Biotechnol. January, 26(1):127-32. doi: 10.1038/nbt1358 (2008). For most assays, kinases were fused to T7 phage strains (Fabian, supra) and for the other assays, kinases were produced in HEK-293 cells after which they were tagged with DNA for quantitative PCR detection. 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. An illustration of the kinase interaction process is presented below. Kd/IC.sub.50 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.

[0070] Percent Control (% Ctrl)

[0071] 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]$\%\mathrm{Ctrl}\mathrm{Calculation}\left(\frac{\mathrm{test}\mathrm{compound}\mathrm{signal}-\mathrm{positive}\mathrm{control}\mathrm{signal}}{\mathrm{negative}\mathrm{control}\mathrm{signal}-\mathrm{positive}\mathrm{control}\mathrm{signal}}\right)\times 100$

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

[0072] The results of the kinase inhibition by cannflavins and flavonoid derivatives are presented in Table 1 and Table 4 (below) and in FIG. 5 and FIG. 10.

TABLE-US-00004 TABLE 4 Acute Myeloid Leukemia Kinases Acute Myeloid Normal FLT3- FLT3- Leukemia cell lines Bone Code FLT3 ITD D835Y MV4-11 MOLM-14 THP-1 marrow IC.sub.50 (μM) FBLGS-70 0.011 0.006 0.021 1.25 1.7 >100 >100 FBLGS-71 0.127 0.079 0.022 3.26 3.68 >100 >100 FBLGS-73 0.152 0.046 0.049 4.53 4.86 >100 >100 FBLGS-74 16.6 9.09 8.58 >100 >100 >100 >100 FBLGS-75 0.220 0.091 0.076 4.35 4.57 >100 >100 FBLGS-76 0.87 0.75 0.68 1.9 2.3 >100 >100 FBLGS-77 1.32 0.799 0.577 7.21 7.34 >100 >100 FBLGS-78 0.307 0.345 0.149 5.62 5.73 >100 >100 FBLGS-79 0.025 0.084 0.013 3.41 4.84 >100 >100 FBLGS-80 0.060 0.084 0.027 5.86 6.12 >100 >100 FBLGS-81 0.0019 0.0038 0.0009 0.9 0.7 >100 >100 FBLGS-82 17.4 12.1 11.0 >10 >10 >100 >100 FBLGS-83 0.578 0.180 0.082 1.6 2.35 13.0 NT FBLGS-84 0.822 0.453 0.194 6.3 7.42 NA >100 FBLGS-85 0.050 0.057 0.022 0.9 1.5 >100 >100 FBLGS-86 0.074 0.045 0.043 2.2 3.1 >100 >100 Midostaurin 0.0004 0.001 0.00009 0.008 0.022 >100 NT

[0073] Inhibition of Sirtuins, matrix metalloproteinase, bromodomains was also confirmed using standard protocols and the results are present in Table 2.

[0074] Bioactivity of the above-described compounds has been verified by an anticancer cell proliferation assay using the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazoliol-1, 3-benzene disulfonate) colorimetric assay by Roche Life Sciences®. Anticancer activity was tested against several standard cancer cell lines including brain, breast, Kaposi sarcoma, leukemia, lung, melanoma, tenosynovial giant cell tumor, ovarian, pancreatic, colon and prostate cancer. Cells were trypsinized and plated into 96 well plates in 50 μl of media and incubated overnight. The next day approximately 18 hours after plating, 50 μl of media containing the required flavonoid-based pharmaceutical composition was added per well. Cells were plated at a density so that 72 hours post drug addition, the cells will be in log phase (500-2000 cells/well). The compounds and extracts were solubilized in Dimethyl sulfoxide (DMSO). The cells are allowed to proliferate for 72 hours 37° C. in humidified atmosphere of 5% CO.sub.2. The experiment is terminated using WST-1 (Roche®) 10 μl per well and absorbance is read at 450 nm/690 nm. The effect of drugs on growth is assessed as percent of cell viability. The IC.sub.50 values were determined from the extract dose versus control growth curves using Graphpad Prism® software. All experiments were carried out in duplicate and the mean results determined.

[0075] The results of the anticancer activity are presented in Tables 3 and 4 (above) and in FIG. 6, Hela cells shown at (A) and CMK cells at (B) and MV4-11 and MOLT-14 acute myeloid leukemia cells. To demonstrate a proof of concept in vivo, human pancreatic cancer xenograft CFPAC-1 cells implanted on scid mice were treated with FBL-03B and FBL-03G and demonstrated significant inhibition of tumor compared to the control. Caflanone (FBL-03G) has a flavone backbone (2-phenyl-1,4-benzopyrone) and the chemical structure shown in FIG. 4. The results of the anti-pancreatic cancer activity in mice are presented in FIG. 7 and FIG. 8. To further demonstrate activity in-vivo, mice infected with acute myeloid leukemia cells harboring the FLT-3 mutation were treated with FBLGS-70 and its derivatives. The results of the anti-acute myeloid leukemia activity of FBLGS-70 and its derivatives are shown in FIG. 9.

[0076] It should now be apparent that the above-described invention provides a pharmaceutical composition for the prevention and treatment of disease with specific cannabis-based flavonoid compounds selected from among the groups of Caflanone, Cannflavin A, Cannflavin B, Cannflavin C, Chrysoeriol, Cosmosiin, Flavocannabiside and their derivatives selected from among the group of Geraldol, Rhamnetin, Isorhamnetin, Rhamnazin, a method for the prevention and treatment of disease using the specific cannabis-based flavonoid pharmaceutical compositions, a method for isolating the cannabis-based flavonoid pharmaceutical compositions from raw plant material, and a method for synthesizing said specific cannabis-based flavonoid pharmaceutical compositions.

[0077] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the claims. In addition, as one of ordinary skill in the art would appreciate, any dimensions shown in the drawings or described in the specification are merely exemplary, and can vary depending on the desired application of the invention. Many variations and modifications of the embodiments described herein will be obvious to one of ordinary skill in the art in light of the above disclosure. The scope of the invention is to be defined only by the claims, and by their equivalents.