Therapeutic agents containing cannabis flavonoid derivatives targeting kinases, sirtuins and oncogenic agents for the treatment of cancers

Abstract

An embodiment of the invention provides a cannabis-based flavonoid pharmaceutical composition including any one or more selected, from among the group of Apigenin, Cannflavin. A. Cannflavin B, Cannflavin C, Chrysoeriol, Cosmosiin, Flavocannabiside, Kaempferol, Luteolin, Myricetin, Orientin, Isoorientin (Homoorientin), Quercetin (+)-Taxifolin, Vitexin, and Isovitexin, or their synthases, for the prevention and treatment of certain cancers that can be treated by therapeutically targeting oncogenic factors including kinases, sirtuins, bromodomains, matrix metalloproteinases and BCL-2. 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, pancreatic, prostate, leukemia, melanoma as well as any other cancers that overexpress the oncogenic factors inhibited by the cannabis flavonoids identified herein.

Claims

1. A method of treating brain cancer, comprising administering to a patient in need thereof, a pharmaceutical composition comprising a compound according to the chemical structure shown below, or any pharmaceutically acceptable salt thereof: ##STR00003## wherein: R1 is selected from a group consisting of 3-methyl-2-butenyl and 3,7-dimethyl-2,6-octenyl; R2 and R4-R10 are each selected from a group consisting of hydrogen, hydroxide, a methyl group, a methoxy group, a carboxyl group, chlorine, bromine, fluorine, and glutamic acid; R3 is hydrogen; and the bond between A and B is selected from a group consisting of a single flavanone bond and a double flavone bond.

2. The method of claim 1, wherein the administration to the patient of the composition comprises administering the composition via a route selected from a group consisting of: a topical route, an oral route, and a rectal route.

3. The method of claim 1, wherein the administration to the patient of the composition comprises injecting the composition into a location of the patient's body selected from the group consisting of: a vein, an epidural muscle, a subcutaneous location, an intrauterine location, and an intracerebroventricular location.

4. The method of claim 1, wherein the administration to the patient of the composition comprises: selecting a dose of the composition within a range of from 0.1 to 500 mg; and providing the selected dose of the composition to the patient.

5. The method of claim 1, wherein the administration to the patient of the composition comprises: selecting a dose of the composition; and providing between 1 to 6 of the selected doses of the composition to the patient each day.

6. The method of claim 1, wherein the administration to the patient of the composition comprises: selecting a dose of the composition within a range from 0.1 milligrams (mg) to 500 mg; and providing the selected dose of the composition to the patient.

7. The method of claim 1, wherein the brain cancer comprises at least one selected from a group consisting of medulloblastoma and glioma.

8. The method of claim 1, wherein the composition comprises a carrier substance selected from a 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.

9. The method of claim 1, wherein the composition is formulated in at least one form selected from a group consisting of: a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, an aerosol, a suppository, and an injectable solution.

10. A method of treating brain cancer, comprising administering to a patient in need thereof, a pharmaceutical composition comprising a compound according to the chemical structure shown below, or any pharmaceutically acceptable salt thereof: ##STR00004##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) 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:

(2) FIG. 1 is an illustration of the general cannabis-based flavonoid pharmaceutical compositions according to the present invention.

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

(4) FIG. 3 is a process diagram illustrating a suitable synthesis approach.

(5) FIG. 4 is an illustration of the specific isolated cannabis-based flavonoid pharmaceutical compositions including Flavone, Flavanone, and Flavanol isolates, according to the present invention.

(6) FIG. 5 is a graphical illustration of the results of the kinase inhibition by cannflavins presented in Table 1.

(7) 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).

(8) FIG. 7 is a graphical illustration of the results of the anticancer activity in mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(9) 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.

(10) 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 BCL-2. Thus, these particular oncogenic factors are identified as useful therapeutic targets for purposes of the present invention.

(11) 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, pancreatic, prostate, leukemia, melanoma as well as any other cancers that overexpress the oncogenic factors inhibited.

(12) The present invention is a group of cannabis-based flavonoid pharmaceutical compositions selected from among the group of Apigenin, Cannflavin A, Cannflavin B, Cannflavin C, Chrysoeriol, Cosmosiin, Flavocannabiside, Kaempferol, Luteolin, Myricetin, Orientin, Isoorientin (Homoorientin), Quercetin, (+)-Taxifolin, Vitexin, and Isovitexin, useful for the prevention and treatment of certain cancers especially those that can be treated by targeting kinases, sirtuins, bromodomains, matrix metalloproteinases and BCL-2 which have been identified to be useful therapeutic targets for some cancers. 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, pancreatic, prostate, leukemia, melanoma as well as any other cancers that overexpress the oncogenic factors inhibited by the cannabis flavonoids identified under this invention.

(13) 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.

(14) ##STR00002##
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. A and B may each be either a single or double bond.

(15) In an embodiment, a method for the prevention and treatment of cancer using the specific cannabis-based flavonoid pharmaceutical compositions above is also disclosed. Administration may be by various routes including oral, rectal or intravenous, epidural muscle, subcutaneous, intrauterine, or blood vessels in the brain (intracerebroventricular) 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.

(16) 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, and mineral oil.

(17) Bioactivity

(18) Bioactivity of the above-described compounds was verified and is presented in Tables 1, 2, and 3 below:

(19) TABLE-US-00001 TABLE 1 FBL-03A FBL-03B FBL-03C FBL-03D FBL-03G Kinase IC.sub.50 (nM) Aurora A 730 12 >30000 1090 BIKE >20000 >20000 21 >30000 >20000 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 >10000 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 <1000 FLT3(ITD) >20000 57 >20000 >30000 <1000 FLT4(VEGFR3) 330 9.3 >20000 >30000 4220 FMS/CSF1R 1500 199 1200 >30000 4 JAK3 >20000 >1000 780 >30000 >20000 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 105 MEK5 140 >1000 84 >30000 >1000 PASK >20000 2060 >20000 >30000 116 PDGFRa — 982 — >30000 5920 PDGFRa(T674I) — 0.92 >1000 >30000 2360 PDGFRB 330 1160 >1000 >30000 3310 PIK3CA(1800L) >20000 780 >30000 >20000 PIK4CB 670 >1000 >30000 >20000 PIM-1 >20000 >1000 >30000 78 PIM-3 >20000 173 >1000 >30000 35 PIP5K1A >20000 >10000 360 >30000 >20000 RIOK1 >20000 >10000 340 >30000 >20000 RIOK3 >20000 >10000 280 >30000 >20000 SIK2 >20000 >10000 >1000 >30000 >63 SRPK1 >20000 >10000 300 >30000 >10000 TNIK >20000 152 >1000 >30000 115

(20) 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 metalloproteinase 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 —

(21) TABLE-US-00003 TABLE 3 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 (Pancreatic) 12 17   12 NT 14.32  CMK (leukemia) NT 11.60 NT NT 1.78 COLO-205 (Colon) 27 NT 17 NT NT DLD-1 (Colon) 15 NT 13 NT NT HC-1 (Leukemia) NT 29.70 NT NT 5.00 HeLa (cervical) NT 10.40 NT NT 2.53 IGROV-1 (Ovarian) 29 15 NT NT KMS-11 (Multiple NT NT NT NT NT myeloma) MCF-7 (Breast) 17 NT 12 NT NT MiaPaca-2 16 NT   9.5 NT NT (Pancreatic) MOLT-4 (Leukemia) NT 13.20 NT NT 20.00  MV4-11 (Leukemia) NT  1.43 NT NT 3.1  NCI-H69 (Small lung) 16 11   18 NT 9.5  PC-3 (Prostate) 26 NT 20 NT NT RL (Lymphoma)   5.9 NT 12 NT NT SNU-16 (Stomach) NT 15.00 NT NT 4.09 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
Isolation and Synthesis

(22) 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.

(23) 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. (2008). Non-Cannabinoid Constituents From A High Potency Cannabis Sativa Variety. Phytochemistry 69, 2627-2633 and Radwan, M. M., Ross, S. A., Slade, D., Ahmed, S. A., Zulfiqar, F., ElSohly, M. A. (2008). Isolation And Characterization Of New Cannabis Constituents From A High Potency Variety. Planta Med. 74, 267-272. The collected plant material was air dried under shade and pulverized into powder.

(24) 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.

(25) 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.

(26) 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, ovarian, pancreatic, colon and prostate cancer.

(27) 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.

(28) Synthesis

(29) 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.

(30) 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. 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 (FIG. 3). The specific Flavone, Flavanone and Flavanol isolates are shown in step FIG. 5.

(31) For background see Minassi, A., Giana, A., Ech-Chahad, A., & Appendino, G. (2008). A regiodivergent synthesis of ring A C-prenylflavones. Organic Letters 10(11), 2267-2270. 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 (2012). Asymmetric Methods for the Synthesis of Flavanones, Chromanones, and Azaflavnones. European Journal Of Organic Chemistry, 449-462. doi:10.1002/ejoc.201101228.PMC 3412359. PMID 22876166.

(32) Bioactivity Assays

(33) 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. (2005). A Small Molecule-Kinase Interaction Map For Clinical Kinase Inhibitors. Nat. Biotechnol, 23(3):329-36, Epub. See also, Karaman et al. (2008). A Quantitative Analysis Of Kinase Inhibitor Selectivity Nat. Biotechnol. January, 26(1):127-32. doi: 10.1038/nbt1358. 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.

(34) Percent Control (% Ctrl)

(35) 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.

(36) $\%\mathrm{Ctrl}\mathrm{Calculation}\left(\frac{\mathrm{test}\mathrm{compound}\mathrm{signal}-\mathrm{positve}\mathrm{control}\mathrm{signal}}{\mathrm{negative}\mathrm{contol}\mathrm{signal}-\mathrm{positve}\mathrm{control}\mathrm{signal}}\right)\times 100$$\mathrm{where}\text{:}$$\mathrm{test}\mathrm{compound}=\mathrm{compound}\mathrm{submitted}\mathrm{by}\mathrm{Environmental}\mathrm{Health}\mathrm{Foundation}\mathrm{negative}$$\mathrm{control}=\mathrm{DMSO}\left(100\%\mathrm{Ctrl}\right)$$\mathrm{positive}\mathrm{control}=\mathrm{control}\mathrm{compound}\left(0\%\text{/}\mathrm{Ctrl}\right)$

(37) The results of the kinase inhibition by cannflavins are presented in Table 1 (above) and in FIG. 5. Inhibition of Sirtuins, matrix metalloproteinase, bromodomains was also confirmed using standard protocols and the results are present in Table 2.

(38) PDGERb is implicated in a variety of myeloproliferatue disorders and cancers result from translocations that actuate.

(39) PD Flab by fusion with proteins such as TELETV6, H2, CEV14/TRP11, rabaptin 5, and huntington interacting protein 1.

(40) PD Flab is also overexpressed in metastatic medulloblastoma.

(41) PDGERb is involved also in angiogenasis.

(42) 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-tetrazolio]-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, 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.

(43) The results of the anticancer activity are presented in Table 3 (above) and in FIG. 6, Hela cells shown at (A) and CMK cells at (B). 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 demonstrated significant inhibition of tumor compared to the control during 14 days of treatment. The results of the anticancer activity in mice are presented in FIG. 7.

(44) 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 Apigenin, Cannflavin A, Cannflavin B, Cannflavin C, Chrysoeriol, Cosmosiin, Flavocannabiside, Kaempferol, Luteolin, Myricetin, Orientin, Isoorientin (Homoorientin), Quercetin, (+)-Taxifolin, Vitexin, and Isovitexin, 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.

(45) 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.