Patent Application
20180074045
The present disclosure relates to novel means and methods for high throughput screening of cancer cells. More particularly the current invention pertains to a method for high throughput screening (HTS) for identifying an analyte with a measurable effect on cells and a system thereof.
Cannabinoids include phytocannabinoids, endogenous endocannabinoids, and synthetic cannabinoids. More than 60 phytocannabinoids have been identified within the Cannabis plant. Cannabinoids elicit their pharmacological activities through cannabinoid receptor type 1 (CB1) and type 2 (CB2), two G-protein coupled receptors (GPCR) in the endocannabinoid signaling pathway. These receptors share 44% amino acid identity and a distinct yet similar binding profile for cannabinoids. CB1 receptors are found predominantly in the central and peripheral nervous systems and suppress neuronal excitability and transmitter release, leading to hypothermia, sedation, euphoria, and altered mental status. CB2 receptors are found at higher levels in the peripheral nervous system, gastrointestinal system and immune tissues.
Multiple sclerosis, neuropathic pain, cancer, atherosclerosis, stroke, myocardial infarction, hypertension, glaucoma, obesity/metabolic syndrome and osteoporosis are some of the diseases in which alterations in the cannabinoid pathway have been demonstrated. Since these diseases are found to be multifactorial, variations in expression and pharmacological cannabinoid receptor binding could be harnessed to elicit a therapeutic effect. Therefore, a defined botanical extract may better achieve this therapeutic goal than a single synthetic compound, as the multiple components could elicit a synergistic effect.
Cannabinoids are not yet approved for the treatment of cancer, although their anti-tumor effects have been known for over 30 years. Evidences exist that cannabinoids may have anti-cancer activity. This was noted in lung adenocarcinoma models in the 1970s and subsequent studies have demonstrated tumor growth inhibition in vitro and in vivo in glioblastoma, breast, prostate, thyroid, colon, skin, pancreatic, leukemia and lymphoma cancer cell models. The exact mechanism by which this anti-tumor effect occurs may involve suppression of proliferative cell signaling pathways, inhibition of angiogenesis and cell migration and induction of apoptosis and/or induction of autophagy.
A wide spectrum of cancer cells and mouse tumor models have been employed to evaluate the antitumor efficacy and the mechanisms of action of cannabinoids, supported by findings that the endocannabinoid system may be altered during various malignant and non-malignant disease states. Significant levels of cannabinoid receptors are found in prostate, breast, leukemia, melanoma, and thyroid cell lines, as well as colorectal and hepatocellular carcinoma tissue specimens. Of particular significance is the fact that in prostate cancer cell lines, the expression of both CB1 and CB2 is elevated compared to normal prostate cells. Similarly, in lymphoma and breast cancer tissue, as well as some derived cell lines, CB1 and CB2 are overexpressed.
It is reported that isolated compounds, which are then made or refined into synthetic drugs, are much more toxic than their plant sources. They produce effects of more rapid onset, greater intensity, and shorter duration. It is reported that they fail to reproduce the desirable effects of plants they come from.
Chemotherapy is an example of the attempt to cure a disease by producing a condition in the body that does not allow the disease to live or thrive. However, this therapy is unnatural and highly poisonous, and therefore, harmful.
Since cancer is a deadly disease people are whiling to suffer from harsh side effects, however, there is no biological link between the potency of therapy and its toxicity.
Personalized Medicine (PM) is a novel approach that proposes the customization of therapy being tailored to the individual patient. There are over 200 different known cancers and the genetic divergence among humans makes it nearly impossible to find one remedy for a group of people.
In view of the above, there is still a long felt and unmet need for novel therapeutic strategies for treating multifunctional diseases such as cancer, especially using botanical extracts.
It is therefore one object of the present invention to disclose a method for high throughput screening (HTS) for identifying an analyte with a measurable effect on cells, said method comprises steps of: (a) providing an array comprising a plurality of cell samples; (b) providing at least one analyte to be tested; (c) contacting said cell samples with said analyte; and (d) detecting a signal indicative of said measurable effect on cells, wherein alteration of said signal over time measured on said cell sample relative to a control sample, is indicative of said measurable effect of said analyte on said cell sample.
It is another object of the present invention to disclose the method as defined above, wherein said analyst is selected from the group consisting of cannabinoid-type, cannabinoid derivative, cannabis extract or fraction thereof, non cannabinoid-type constituent, product, compound, molecule or substance and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said measurable effect on cells is selected from the group consisting of physiological, genetic, biochemical, structural and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said measurable effect on cells is selected from the group consisting of: anti proliferative, regenerative, anti inflammatory, anti mitotic, differentiative, anti metastatic, anti angiogenic, apoptotic, cytotoxic, cytopathic and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said measurable effect on cells is an effect on a biological parameter selected from the group consisting of: proliferation, migration, absorbance, adherence, apoptosis, necrosis, autophagy, cytotoxicity, cell size, motility, cell cycle and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said cancer cells are selected from the group consisting of: breast, ovarian, colon/rectum, prostate, melanoma, head and neck, pancreatic, osteosarcoma, gastric, glioma, glioblastoma, neuroblastoma, leukemia, adenocarcinoma, adrenal, anal, bile duct, bladder, bone, brain/CNS, cervical, endometrial, esophagus, eye, gastrointestinal, kidney, leukemia, liver, lung, lymphoma, multiple myeloma, nasal cavity and paranasal sinus, nasopharyngeal, non-hodgkin lymphoma, oral cavity, oropharyngeal, osteosarcoma, ovarian, pancreatic, penile, pituitary, retinoblastoma, rhabdomyosarcoma, salivary gland, sarcoma, skin, small intestine, stomach, testicular, thymus, thyroid, uterine sarcoma, vaginal and vulvar and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said cell samples are selected from the group consisting of: xenografts, allografts, cell lines, biopsy cells and a combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said cell lines are cancer cell lines selected from the group consisting of: central nervous system, bone, prostate, stomach, urinary tract, ovary, haematopoietic and lymphoid tissue, kidney, thyroid, skin, soft tissue, salivary gland, ovary, lung, pleura, liver, endometrium, pancreas, breast, upper aerodigestive tract, large intestine, autonomic ganglia, oesophagus, biliary tract, small intestine, autonomic ganglia and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said cell samples are selected from the group consisting of: human cell lines, animal cell lines and xenografts.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said cell samples are selected from the group consisting of: cancer cells, stem cells, neuronal cells, cardiomyocyte cells, somatic cells germ cells, normal cells, and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said signal is selected from the group consisting of: optic, luminescent, fluorescent, immunological, cell count, radioactive, non radioactive isotopic, electrical and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said measurable effect on cells is an effect on the expression level of a cancer marker selected from the group consisting of: ALK gene, Alpha-fetoprotein (AFP), Beta-2-microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), BCR-ABL fusion gene, BRAF mutation V600E, CA15-3/CA27.29, CA19-9, CA-125, Calcitonin, Carcinoembryonic antigen (CEA), CD20, Chromogranin A (CgA), Chromosomes 3, 7, 17, and 9p21, Cytokeratin fragments 21-1, EGFR mutation, Estrogen receptor (ER)/progesterone receptor (PR), Fibrin/fibrinogen, HE4, HER2/neu, Immunoglobulins, KIT, KRAS mutation, Lactate dehydrogenase, Nuclear matrix protein 22, Prostate-specific antigen (PSA), Thyroglobulin, Urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), 5-Protein signature (Ova1), 21-Gene signature (Oncotype DX), 70-Gene signature (Mammaprint) and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said analyte is extracted from cannabis; said cannabis is selected from a group consisting of: Cannabis sativa, Cannabis indica, Cannabis ruderalis, and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said cannabinoid-type is selected from the group consisting of: Cannabigerol (CBG) type, Cannabichromene (CBC) type, Cannabidiol (CBD) type, Δ9-Tetrahydrocannabinol (THC) type, Δ8-THC type, Cannabicyclol (CBL) type, Cannabielsoin (CBE) type, Cannabinol (CBN) and Cannabinodiol (CBND) types, Cannabitriol (CBT) type, cannabinoids with miscellaneous types and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said cannabinoid-type is further selected from the group consisting of: Tetrahydrocannabidiol (THC) or a derivative thereof, cannabidiol (CBD) or a derivative thereof, CBG (Cannabigerol), CBC (Cannabichromene), CBL (Cannabicyclol), CBV (Cannabivarin), THCV (Tetrahydrocannabivarin), CBDV (Cannabidivarin), CBCV (Cannabichromevarin), CBGV (Cannabigerovarin), CBGM (Cannabigerol Monomethyl Ether) and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said THC or a derivative thereof is selected from the group consisting of THC, THCV, THCA, THCVA, Delta-9-tetrahydrocannabinol (Δ9-THC) and delta-8-tetrahydrocannabinol (Δ8-THC) and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said cannabidiol (CBD) or a derivative thereof is selected from the group consisting of CBD, CBDV, CBDA and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said non cannabinoid-type constituent, product, compound, molecule or substance is selected from the group consisting of: terpenoids, hydrocarbons, essential oil derived from cannabis, nitrogen-containing compounds, carbohydrates, flavonoids, fatty acids, amino acids, proteins, glycoproteins, enzymes, sugars and related compounds, noncannabinoid phenols, simple alcohols, aldehydes, ketones, acids, esters, lactones, steroids, terpenes, phytosterols such as campesterol, ergosterol, E-sitosterol, and stigmasterol, vitamins such as vitamin A and vitamin K, pigments such as carotene and xanthophylls, elements such as Na, K, Ca, Mg, Fe, Cu, Mn, Zn and Hg and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said analyte is derived from a source selected from the group consisting of body of humans and animals, extracted from plants, synthetic, and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said HTS is selected from the group consisting of: microtiter plate, automatic colony pickers, uHTS or ultra-high-throughput screening, 3D tumor spheroid analysis method for HTS drug discovery, Celigo Imaging Cytometer, automation systems, a carousel system to store assay plates for high storage capacity and high speed access, integrated robot system, readout or detection, data-collection process and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said analyte provides a synergistic effect with respect to said measurable effect on cells as compared to the effect provided by conventional antitumor or anti-inflammatory therapies administered separately.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said analyte provides a contra indicatory effect with respect to antitumor or anti-inflammatory activity as compared to the effect provided by conventional antitumor or anti-inflammatory therapies administered separately.
It is a further object of the present invention to disclose a system for high throughput screening (HTS) for identifying an analyte with a measurable effect on cells, said system comprises: (a) an array comprising a plurality of cell samples; (b) at least one analyte to be tested; and (c) means for detecting a signal indicative of said measurable effect on cells, wherein alteration of said signal over time measured on said cell sample relative to a control sample, is indicative of said measurable effect of said analyte on said cell sample.
It is a further object of the present invention to disclose the system as defined above, wherein said analyst is selected from the group consisting of cannabinoid-type, cannabinoid derivative, cannabis extract or fraction thereof, non cannabinoid-type constituent, product, compound, molecule or substance and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said measurable effect on cells is selected from the group consisting of physiological, genetic, biochemical, structural and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said measurable effect on cells is selected from the group consisting of: anti proliferative, regenerative, anti inflammatory, anti mitotic, differentiative, anti metastatic, anti angiogenic, apoptotic, cytotoxic, cytopathic and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said measurable effect on cells is an effect on a biological parameter selected from the group consisting of: proliferation, migration, absorbance, adherence, apoptosis, necrosis, autophagy, cytotoxicity, cell size, motility, cell cycle and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said cancer cells are selected from the group consisting of: breast, ovarian, colon/rectum, prostate, melanoma, head and neck, pancreatic, osteosarcoma, gastric, glioma, glioblastoma, neuroblastoma, leukemia, adenocarcinoma, adrenal, anal, bile duct, bladder, bone, brain/CNS, cervical, endometrial, esophagus, eye, gastrointestinal, kidney, leukemia, liver, lung, lymphoma, multiple myeloma, nasal cavity and paranasal sinus, nasopharyngeal, non-hodgkin lymphoma, oral cavity, oropharyngeal, osteosarcoma, ovarian, pancreatic, penile, pituitary, retinoblastoma, rhabdomyosarcoma, salivary gland, sarcoma, skin, small intestine, stomach, testicular, thymus, thyroid, uterine sarcoma, vaginal and vulvar and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said cell samples are selected from the group consisting of: xenografts, allografts, cell lines, biopsy cells and a combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said cell lines are cancer cell lines selected from the group consisting of: central nervous system, bone, prostate, stomach, urinary tract, ovary, haematopoietic and lymphoid tissue, kidney, thyroid, skin, soft tissue, salivary gland, ovary, lung, pleura, liver, endometrium, pancreas, breast, upper aerodigestive tract, large intestine, autonomic ganglia, oesophagus, biliary tract, small intestine, autonomic ganglia and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said cell samples are selected from the group consisting of: human cell lines, animal cell lines and xenografts.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said cell samples are selected from the group consisting of: cancer cells, stem cells, neuronal cells, cardiomyocyte cells, somatic cells germ cells, normal cells, and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said signal is selected from the group consisting of: optic, luminescent, fluorescent, immunological, cell count, radioactive, non radioactive isotopic, electrical and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said measurable effect on cells is an effect on the expression level of a cancer marker selected from the group consisting of: ALK gene, Alpha-fetoprotein (AFP), Beta-2-microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), BCR-ABL fusion gene, BRAF mutation V600E, CA15-3/CA27.29, CA19-9, CA-125, Calcitonin, Carcinoembryonic antigen (CEA), CD20, Chromogranin A (CgA), Chromosomes 3, 7, 17, and 9p21, Cytokeratin fragments 21-1, EGFR mutation, Estrogen receptor (ER)/progesterone receptor (PR), Fibrin/fibrinogen, HE4, HER2/neu, Immunoglobulins, KIT, KRAS mutation, Lactate dehydrogenase, Nuclear matrix protein 22, Prostate-specific antigen (PSA), Thyroglobulin, Urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), 5-Protein signature (Ova1), 21-Gene signature (Oncotype DX), 70-Gene signature (Mammaprint) and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said analyte is extracted from cannabis; said cannabis is selected from a group consisting of: Cannabis sativa, Cannabis indica, Cannabis ruderalis, and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said cannabinoid-type is selected from the group consisting of: Cannabigerol (CBG) type, Cannabichromene (CBC) type, Cannabidiol (CBD) type, Δ9-Tetrahydrocannabinol (THC) type, Δ8-THC type, Cannabicyclol (CBL) type, Cannabielsoin (CBE) type, Cannabinol (CBN) and Cannabinodiol (CBND) types, Cannabitriol (CBT) type, cannabinoids with miscellaneous types and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said cannabinoid-type is further selected from the group consisting of: Tetrahydrocannabidiol (THC) or a derivative thereof, cannabidiol (CBD) or a derivative thereof, CBG (Cannabigerol), CBC (Cannabichromene), CBL (Cannabicyclol), CBV (Cannabivarin), THCV (Tetrahydrocannabivarin), CBDV (Cannabidivarin), CBCV (Cannabichromevarin), CBGV (Cannabigerovarin), CBGM (Cannabigerol Monomethyl Ether) and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said THC or a derivative thereof is selected from the group consisting of THC, THCV, THCA, THCVA, Delta-9-tetrahydrocannabinol (Δ9-THC) and delta-8-tetrahydrocannabinol (Δ8-THC) and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said cannabidiol (CBD) or a derivative thereof is selected from the group consisting of CBD, CBDV, CBDA and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said non cannabinoid-type constituent, product, compound, molecule or substance is selected from the group consisting of: terpenoids, hydrocarbons, essential oil derived from cannabis, nitrogen-containing compounds, carbohydrates, flavonoids, fatty acids, amino acids, proteins, glycoproteins, enzymes, sugars and related compounds, noncannabinoid phenols, simple alcohols, aldehydes, ketones, acids, esters, lactones, steroids, terpenes, phytosterols such as campesterol, ergosterol, E-sitosterol, and stigmasterol, vitamins such as vitamin A and vitamin K, pigments such as carotene and xanthophylls, elements such as Na, K, Ca, Mg, Fe, Cu, Mn, Zn and Hg and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said analyte is derived from a source selected from the group consisting of body of humans and animals, extracted from plants, synthetic, and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said HTS is selected from the group consisting of: microtiter plate, automatic colony pickers, uHTS or ultra-high-throughput screening, 3D tumor spheroid analysis method for HTS drug discovery, Celigo Imaging Cytometer, automation systems, a carousel system to store assay plates for high storage capacity and high speed access, integrated robot system, readout or detection, data-collection process and any combination thereof.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said analyte provides a synergistic effect with respect to said measurable effect on cells as compared to the effect provided by conventional antitumor or anti-inflammatory therapies administered separately.
It is a further object of the present invention to disclose the system as defined in any of the above, wherein said analyte provides a contra indicatory effect with respect to antitumor or anti-inflammatory activity as compared to the effect provided by conventional antitumor or anti-inflammatory therapies administered separately.
It is a further object of the present invention to disclose a non transitory computer readable medium comprising instructions which, when implemented by one or more computers cause the one or more computers to present data concerning a measurable effect on cells of one or more analytes on preselected cell samples by processing data concerning a signal indicative of said measurable effect on cells, wherein alteration of said signal over time measured on said cell sample relative to a control sample, is indicative of said measurable effect of said analyte on said cell sample.
It is a further object of the present invention to disclose a composition comprising therapeutically effective amount of, or an extract comprising essentially therapeutically effective amount of an analyte selected according to method of claim 1, wherein said composition has an antitumor or anti-inflammatory activity or synergistic effect thereof for use in the treatment of a cancer type or inflammatory disease.
It is a further object of the present invention to disclose the composition as defined above, wherein said analyst is selected from the group consisting of cannabinoid-type, cannabinoid derivative, cannabis extract or fraction thereof, non cannabinoid-type constituent, product, compound, molecule or substance and any combination thereof.
It is a further object of the present invention to disclose a method for identifying one or more genetic markers derived from cannabis, wherein said one or more genetic markers correlates with a measurable effect on cells as indicated by the method as defined in any of the above, said method comprises additional steps of correlating said signal with cannabis DNA sequence data.
It is a further object of the present invention to disclose one or more genetic markers derived from cannabis, wherein said one or more genetic markers correlates with a measurable effect on cells as indicated by the method as defined in any of the above, further wherein said signal is correlated with cannabis DNA sequence data.
It is a further object of the present invention to disclose the genetic markers as defined in any of the above, wherein said one or more genetic markers is selected from the group consisting of: variation, mutation or alteration in a genomic loci, a single nucleotide polymorphism (SNP), minisatellites, RFLP (Restriction fragment length polymorphism), SSLP (Simple sequence length polymorphism), AFLP (Amplified fragment length polymorphism), RAPD (Random amplification of polymorphic DNA), VNTR (Variable number tandem repeat), SSR (Simple sequence repeat), microsatellite polymorphism, STR (Short tandem repeat), SFP (Single feature polymorphism), DArT (Diversity Arrays Technology), RAD markers (Restriction site associated DNA markers) nucleotide changes, indel, deletion, duplication, inversion and/or insertion and any combination thereof.
It is a further object of the present invention to disclose a database of analytes, wherein said database comprises data concerning said analyte, correlated with a measurable effect on cells, defined by implementing steps as described in any of the above.
It is a further object of the present invention to disclose a system for high throughput screening (HTS) for identifying an analyte selected from the group consisting of: cannabis extract or a fraction thereof, cannabinoid-type constitute, non cannabinoid-type constitute and any combination thereof, said analyte is indicative of cytotoxic or anti proliferative or anti mitotic or cell growth inhibitory activity in vitro. The system comprises: (a) an array comprising a plurality of cancer cell samples; (b) at least one analyte to be tested, said analyte is selected from the group consisting of: cannabis extract or a fraction thereof, cannabinoid-type constitute, non cannabinoid-type constitute and any combination thereof; and (c) means for detecting a signal indicative of said cytotoxic or anti proliferative or anti mitotic or cell growth inhibitory activity in vitro, wherein alteration of said signal over time measured on said cancer cell sample relative to a control sample, is indicative of said cytotoxic or anti proliferative or anti mitotic or cell growth inhibitory activity in vitro of said analyte on said cancer cell sample.
It is a further object of the present invention to disclose a method for high throughput screening (HTS) for identifying an analyte selected from the group consisting of: cannabis extract or a fraction thereof, cannabinoid-type constitute, non cannabinoid-type constitute and any combination thereof, said analyte is indicative of cytotoxic or anti proliferative or anti mitotic or cell growth inhibitory activity in vitro. The method comprises steps of: (a) providing an array comprising a plurality of cancer cell samples; (b) providing said analyte to be tested, said analyte is selected from the group consisting of: cannabis extract or a fraction thereof, cannabinoid-type constitute, non cannabinoid-type constitute and any combination thereof; (c) contacting said cancer cell samples with said analyte; and (d) detecting a signal indicative of said cytotoxic or anti proliferative or anti mitotic or cell growth inhibitory activity in vitro, wherein alteration of said signal over time measured on said cancer cell sample relative to a control sample, is indicative of said cytotoxic or anti proliferative or anti mitotic or cell growth inhibitory activity in vitro of said analyte on said cancer cell sample.
It is a further object of the present invention to disclose a method for high throughput screening (HTS) for identifying an analyte selected from the group consisting of: cannabis extract or a fraction thereof, cannabinoid-type constitute, non cannabinoid-type constitute and any combination thereof, said analyte is indicative of antitumour activity, said method comprises steps of the method as defined in any of the above, and additionally comprising steps of: (a) transplanting cancer cell xenographs derived from said cancer cell samples into experimental animals; (b) treating said experimental animals with said analyte selected from the group consisting of: cannabis extract or a fraction thereof, cannabinoid-type constitute, non cannabinoid-type constitute and any combination thereof, identified by the method as defined in any of the above, and (c) monitoring tumor growth of said experimental animal.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said experimental animal is nude mice.
It is a further object of the present invention to disclose a protocol useful for identifying a botanical analyte with a measurable effect on cells correlated with a disease. The protocol comprises steps of: (a) providing input data comprising data selected from the group consisting of: parameters of said analyte, parameters of said cells, high throughput screening (HTS) results data for identifying an analyte with a measurable effect on cells as indicated in claim 1, clinical or preclinical data and any combination thereof; (b) processing said data; and (c) presenting output data at an electronic display concerning a measurable effects of said analyte on said cells correlated with a disease, and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined above, wherein said data processing comprises steps selected from the group consisting of: correlating, normalizing, calibrating, factorizing, calculating, statistically analyzing and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said analyte parameters are selected from the group consisting of: analyte source, analyte processing and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said botanical analyte source parameters are selected from the group consisting of: source strain, source genotype, source phenotype, source growth conditions, source harvest conditions, source nutrition, source part or organ and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said source part or organ is selected from the group consisting of: root, stem, leaf, flower, seed and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said botanical analyte processing parameters are selected from the group consisting of: curing, drying, extraction process, decarboxylation and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said extraction process is selected from the group consisting of: butane, CO2 gradients, ethanol, dry ice and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said cell parameters are selected from the group consisting of: cells source, cells treatment and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said cells source is selected from the group consisting of: biopsies, cell lines, xenographs, mutated cells or molecules and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said cells treatment is selected from the group consisting of: cells medium treatment, serum treatment, cells dilution, cell cycle phase and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said measurable effect on cells is selected from the group consisting of proliferation, apoptosis, migration, regeneration, differentiation, angiogenesis, and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said clinical or preclinical data is selected from the group consisting of: administration route of said analyte to a subject, dosages, release form, cancer markers level, tumor size monitoring, metastasis monitoring, survival, quality of life measured according to one or more scales, and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said administration route is selected from the group consisting of: sublingual, oral, intravenous, topical, subcutaneous and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said release form is selected from the group consisting of: slow release, controlled release, sustained release, immediate or rapid release and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said cancer markers are selected from the group consisting of: ALK gene, Alpha-fetoprotein (AFP), Beta-2-microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), BCR-ABL fusion gene, BRAF mutation V600E, CA15-3/CA27.29, CA19-9, CA-125, Calcitonin, Carcinoembryonic antigen (CEA), CD20, Chromogranin A (CgA), Chromosomes 3, 7, 17, and 9p21, Cytokeratin fragments 21-1, EGFR mutation, Estrogen receptor (ER)/progesterone receptor (PR), Fibrin/fibrinogen, HE4, HER2/neu, Immunoglobulins, KIT, KRAS mutation, Lactate dehydrogenase, Nuclear matrix protein 22, Prostate-specific antigen (PSA), Thyroglobulin, Urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), 5-Protein signature (Ova1), 21-Gene signature (Oncotype DX), 70-Gene signature (Mammaprint) and any combination thereof.
It is a further object of the present invention to disclose the protocol as defined in any of the above, wherein said one or more scales for assessing quality of life are selected from the group consisting of: pain scale, quality of life scale, functional assessment of cancer therapy scale and any combination thereof.
Exemplary non-limited embodiments of the disclosed subject matter will be described, with reference to the following description of the embodiments, in conjunction with the figures. The figures are generally not shown to scale and any sizes are only meant to be exemplary and not necessarily limiting. Corresponding or like elements are optionally designated by the same numerals or letters.
In the following detailed description of the preferred embodiments, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration specific embodiments in which the invention may be practiced. It is understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. The present invention may be practiced according to the claims without some or all of these specific details. For the purpose of clarity, technical material that is known in the technical fields related to the invention has not been described in detail so that the present invention is not unnecessarily obscured.
The present invention provides a method for high throughput screening (HTS) for identifying an analyte with a measurable effect on cells. The aforementioned method comprises steps of: (a) providing an array comprising a plurality of cell samples; (b) providing at least one analyte to be tested; (c) contacting said cell samples with said analyte and (d) detecting a signal indicative of said measurable effect on cells, wherein alteration of said signal over time measured on said cell sample relative to a control sample, is indicative of said measurable effect of said analyte on said cell sample.
According to a specific aspect, the present invention provides a method for high throughput screening (HTS) for identifying an analyte selected from the group consisting of: cannabis extract or a fraction thereof, cannabinoid-type, non cannabinoid-type and any combination thereof. It is within the scope that the analyte is indicative of cytotoxic or anti proliferative or anti mitotic or cell growth inhibitory activity in vitro. Such a method comprises steps of: (a) providing an array comprising a plurality of cancer cell samples; (b) providing said analyte to be tested, said analyte is selected from the group consisting of: cannabis extract or a fraction thereof, cannabinoid-type, non cannabinoid-type and any combination thereof; (b) contacting said cancer cell samples with said analyte; (c) detecting a signal indicative of said cytotoxic or anti proliferative or anti mitotic or cell growth inhibitory activity in vitro, wherein alteration of said signal over time measured on said cancer cell sample relative to a control sample, is indicative of said cytotoxic or anti proliferative or anti mitotic or cell growth inhibitory activity in vitro of said analyte on said cancer cell sample.
As used herein the term “about” denotes ±25% of the defined amount or measure or value.
The term “high throughput screening” or “HTS” used hereinafter refers to any method for scientific experimentation especially used in the fields of biology and chemistry. The screening facility includes usage of robotics, data processing and control software, liquid handling devices, and sensitive detectors allowing a researcher to quickly conduct millions of chemical, genetic, or pharmacological tests. Through this process one can rapidly identify active compounds or analytes, antibodies, or genes that modulate a particular biomolecular pathway. The results of these experiments provide starting points for drug design and for understanding the interaction or role of a particular biochemical process in biology.
It is herein acknowledged that automation is an important element in HTS's technique. Typically, an integrated robot system consisting of one or more robots transports assay-microplates from station to station for sample and reagent addition, mixing, incubation, and finally readout or detection. An HTS system can usually prepare, incubate, and analyze many samples simultaneously, further speeding the data-collection process. It is further within the scope that the term HTS further relates to uHTS or ultra-high-throughput screening referring to screening in excess of 100,000 compounds per day.
It is further within the scope that additional or HTS methods are used in the present invention such as 3D tumor spheroid analysis method for HTS drug discovery using Celigo Imaging Cytometer, automation systems such as a carousel system to store assay plates for high storage capacity and high speed access and any other HTS system or technique.
The term “analyte” as used hereinafter generally refers to a component, a molecule, a substance or chemical or botanical constituent that is of interest in an analytical procedure. The analytical procedure is designed to measure properties of the analyte.
The term “cannabis” refers hereinafter to a genus of flowering plants that includes three different species, Cannabis sativa, Cannabis indica and Cannabis ruderalis.
It is within the scope that cannabis extract or cannabis concentrates or fractions thereof are used as analytes on cell samples for screening for a measurable effect on cells. Such an extract may include cannabinoid-type compounds or fractions, non-cannabinoid-type compounds or fractions and combinations thereof.
The term “non cannabinoid” or “non cannabinoid-type” as used hereinafter refers to any molecule or compound or constitute which is not a cannabinoid.
The term “Cannabinoids” refer hereinafter to a class of diverse chemical compounds that act on cannabinoid receptors and other signal transduction receptors or proteins on cells that repress or activate neurotransmitter release in the brain, heart, liver, immune system and lungs. These receptor proteins include the endocannabinoids (produced naturally in the body by humans and animals), the phytocannabinoids (found in cannabis and some other plants), and synthetic cannabinoids (manufactured chemically). The most notable cannabinoid is the phytocannabinoid Δ9-tetrahydrocannabinol (THC), the primary psychoactive compound of cannabis. Cannabidiol (CBD) is another major constituent of the plant, representing up to 40% in extracts of the plant resin. There are at least 85 different cannabinoids isolated from cannabis, exhibiting varied effects. Reference is now made to http://www.medicinalgenomics.com/wp-content/uploads/2011/12/Chemical-constituents-of-cannabis.pdf, which is incorporated herein by reference in its entirety, presenting a non limiting list of identified cannabinoids. The current invention includes all cannabinoids, for example, cannabinoids belonging to the following classes or groups:
The term “cannabinoid extract” refers hereinafter to any extract or concentrate derived from the cannabis plant which contains at least one cannabinoid. The cannabinoids may be extracted from the cannabis plant using any one of the many known extraction methods, such as non-hydrocarbons extraction methods and hydrocarbons extraction methods.
The term “cannabinoid fraction” used hereinafter refers to cannabis extract treated by separation or purification or fractionation processes. More particularly it refers to purified or partially purified cannabis extract containing cannabinoid-type portions or elements. In alternative embodiments, cannabinoid fraction may contain synthetic cannabinoids.
The term “cannabidiol” or “CBD” refers hereinafter to one of at least 85 active cannabinoids identified in cannabis Cannabidiol is a major phytocannabinoid, accounting for up to 40% of the plant's extract. CBD is considered to have a wider scope of medical applications than tetrahydrocannabidiol (THC). Cannabidiol has a very low affinity for CB1 and CB2 receptors but acts as an indirect antagonist of their agonists. CBD may potentiate THC's effects by increasing CB1 receptor density or through another CB1-related mechanism. It is also an inverse agonist of CB2 receptors. CBD possesses antiproliferative, pro-apoptotic effects and inhibits cancer cell migration, adhesion and invasion. The term CBD also refers to Cannabidivarin (CBDV) a homolog of cannabidiol (CBD) and to cannabidiolic acid (CBDA).
The term “Tetrahydrocannabidiol” or “THC” refers hereinafter to the principal psychoactive constituent (or cannabinoid) of the cannabis plant. THC has a partial agonist activity at the cannabinoid receptor CB1, and the CB2 receptor and is known to increase cortisol levels. It is further included within the scope that the term THC further refers to Tetrahydrocannabivarin (THCV or THV) a homologue of tetrahydrocannabinol (THC) and Tetrahydrocannabinolic acid (THCA, 2-COOH-THC), a biosynthetic precursor of tetrahydrocannabinol (THC).
It is noted that cannabinol (CBN), cannabichromene (CBC), the acids (CBDA, CBGA, THCA) and propyl homologues (CBDV, CBGV, THCV) of CBD, cannabigerol (CBG) and THC, and tetrahydrocannabivarin acid (THC-V and THC-VA) are also included as optional active ingredient(s) of the composition or formulation of the present invention.
The cannabis extract or a fraction thereof may comprise noncannabinoid-type constituents selected from the group consisting of: terpenoids, hydrocarbons, essential oil derived from cannabis, nitrogen-containing compounds, carbohydrates, flavonoids, fatty acids, noncannabinoid phenols, simple alcohols, aldehydes, ketones, acids, esters, lactones, phytosterols such as campesterol, ergosterol, E-sitosterol, and stigmasterol, vitamin K, pigments such as carotene and xanthophylls, elements such as Na, K, Ca, Mg, Fe, Cu, Mn, Zn and Hg and any combination thereof. Reference is made to the publication of http://www.medicinalgenomics.com/wp-content/uploads/2011/12/Chemical-constituents-of-cannabis.pdf, which is incorporated herein by reference in its entirety. It is further within the scope that there are 483 different identifiable chemical constituents known to exist in cannabis. The most distinctive and specific class of compounds are the cannabinoids (66 known), that are only known to exist in the cannabis plant. Other constituents of the cannabis plant are: nitrogenous compounds (27 known), amino acids (18), proteins (3), glycoproteins (6), enzymes (2), sugars and related compounds (34), hydrocarbons (50), simple alcohols (7), aldehydes (13), ketones (13), simple acids (21), fatty acids (22), simple esters (12), lactones (1), steroids (11), terpenes (120), non-cannabinoid phenols (25), flavonoids (21), vitamins (1) [Vitamin A], pigments (2), and elements (9). It is further within the scope that http://medicalmarijuana.procon.org/view.answers.php?questionID=000636 is incorporated herein in its entirety.
The term “cannabinoid receptor” refers hereinafter to a class of cell membrane receptors under the G protein-coupled receptor superfamily. There are currently two known subtypes of cannabinoid receptors, termed CB1 and CB2. The CB1 receptor is expressed mainly in the brain, but also in the lungs, liver and kidneys. The CB2 receptor is expressed mainly in the immune system, the digestive system and in hematopoietic cells.
It is further within the scope that cell lines screened by the method and system of the present invention include, but are not limited to the list of cell lines detailed in http://www.broadinstitute.org/ccle/data/browseSamples?actionMethod=pages %2Fsearch %2FsearchResult.xhtml%3AbrowseSamplesBean.checkSkipFirstStep%28%29&conversationPropagation=begin, incorporated herein by reference.
The term “quality of life scale” as used hereinafter refers to scales for assessing quality of life of a patient after treatment. Non limiting examples of such scales include pain scales such as Faces Pain Scale, Wong-Baker FACES Pain Rating Scale, Coloured Analogue Scale, Visual Analog Scale (VAS), Verbal Numerical Rating Scale (VNRS), Verbal Descriptor Scale (VDS) and Brief Pain Inventory; Quality of Life Scale (QOLS); functional assessment of cancer therapy (FACT) scale and any combination thereof.
The term “sustained release dosage form” refers hereinafter to the release of a drug at a predetermined rate in order to maintain a constant drug concentration for a specific period of time with minimum side effects. This can be achieved through a variety of formulations, including liposomes and drug-polymer conjugates. Sustained release's definition is more akin to a “controlled release” rather than “sustained”.
According to certain embodiments, the present invention provides a personalized medicine (PM) based system and method for screening for novel cancer therapies which comprises at least one of the following aspects:
It is therefore one object of the present invention to provide a method and system for screening for nontoxic natural cancer therapy.
Up until now, the common concept of the drug industry is to use isolates, or to synthesize, parts of the whole plant, which, during use by medical practitioners, sometimes produces undesirable side effects in their patients. For instance, the most powerful drug used in cancer chemotherapy was isolated from the plant Madagascar periwinkle. It is an effective agent against breast and lymph cancers. However, its side effects may be debilitating and dangerous. It is therefore noted that the isolation of active molecules from plants may be miscalculated. In certain cases, a single compound could not account for desirable properties of plant extract or a fraction thereof. The assumption that it would be better to treat a disease with a purified compound rather than with the whole plant extract may be misleading.
It is herein acknowledged that it has been shown that using the whole cannabis plant extract may be more effective in treating certain diseases and conditions relative to isolated compounds derived from cannabis. There may be synergistic or additive effects between the various cannabis extract components which will be absent when using isolated compounds or specific combinations thereof.
According to a further aspect, without wishing to be bound by theory, harmonized ratios of active molecules within mixtures of extracts (biological mechanisms) may be the result of co-evolution along with human receptors.
The continues scanning and the building of big data for an “oriented evolution of active ratios” is a further unmet need, realized by the present invention.
Without wishing to be bound by theory, it is a well-established prospect that the state of mind affects the physiology of our body through its balance that is experienced as “health” and its imbalance that is experienced as a “disease”. Most of today's cancer therapies are harmful and toxic regardless of their therapeutic benefit. It is plausible that this phenomenon is related to the cultural way of thinking that a harsh disease is cured by a harsh treatment. However, on real grounds, cancer patients are usually “sick” people with a minor pain that become “treated” patients with unbearable pain. Nausea and weight loss weakens the body immensely and derives the will to live. The pessimistic prophecy of the proximity of death in conjunction with the weakened body is a high barrier to cross to become healthy again. Therefore, empowering the emotional state of cancer patients is translated into the physiological realm. The current invention pertains to providing a treatment that addresses both the psychological state of cancer patients as well as their physiological condition. While cannabinoids or cannabis extracts are screened inter alia for their cytotoxic or anticancer properties it is also their “side effects” that are desirable since they are well known as beneficial for Cancer Related Cachexia and Anorexia Syndrome. They are also known for pain reduction and antidepressant activity. These combined therapeutic benefits make the current invention a potent therapy with minimal undesirable side effects.
Using high screening technology for biological processes such as cell necrosis and apoptosis in cannabinoid treated cancer cell lines and/or cells from tumors derived from patients, preferable natural cannabinoid combinations are herein identified.
It is further within the scope of the present invention to screen for and provide potent extract which is found to halt cancer cells to be administered to patients in a predetermined dosage form or administration rout such as capsule, intravenously or orally.
According to a further aspect, treating a cancer patient with an extract (i.e. cannabis extract or a fraction thereof) identified by the method of the present invention, benefits the following therapeutic prospects:
According to a further aspect of the invention, pharmacological importance of cannabinoids in cancer and other non-malignant diseases is revealed by the current invention.
To address the objectives and aspects disclosed above, the present invention provides a robust procedure of high through output screening (HTS) for the detection of correlations between selected analytes such as cannabinoid ratios or dosages, and anti-tumor activity. According to one embodiment, the present invention uses a growing library of human cancer cell lines tumor cells derived from patients or experimental animals, and creating an enlarged variety of cannabis-based compounds. Examination of the biological activity of these compounds on tumor cells of distinct tissue lineage creates a highly potent therapeutic data.
It is further within the scope that the HTS system is applied on cell lines, for the screening for potent cannabinoid or other natural extracts, with or without the conjunction of standard chemotherapy.
It is further within the scope that the HTS system is applied on biopsies derived from patients for the screening for potent cannabinoid or cannabinoid combinations or other natural extracts with the conjunction of chemotherapy according to patients' overall treatment.
It is further within the scope that a learning algorithm to predict cannabinoid ratio or terpens or mixtures or extracts potencies is developed by the present invention.
In another embodiment, a bank of tumor specific highly effective proprietary compounds is provided.
According to a further aspect, the effect of cannabinoids or cannabis extracts or a fraction thereof or other analyte of interest is assessed on cancer cells, stem cells, neurons and cardiomyocyte cells for the development of advanced natural therapeutics.
The antitumor activity of cannabinoids reflected in their potent therapeutic activity against diverse types of cancers is assessed by the system and method of the present invention.
In order to identify active compounds such as cannabinoid-type compound or cannabinoid ratios with an effect on specific types of cancer cells, the present invention provides a screen (i.e. high throughput screen, or HTS), which tests the anti-tumor activity of different cannabis derived fractions. The antitumor activity tests include: anti-proliferative effects (cell cycle arrest), decreased viability and cell death measured by cytotoxicity colorimetric assays such as XTT assays, apoptosis, necrosis, autophagy, as well as anti-angiogenic, anti-migratory, and anti-metastatic assays and possible synergetic effects of cannabinoids with conventional chemotherapeutic drugs that are currently in clinical use.
According to one embodiment, in order to perform the screen, the ImageXpress Micro XLS System is used. The ImageXpress Micro XLS System is a wide-field automated microscope capable of fluorescent, transmitted light, and phase-contrast imaging of fixed- or live cell studies. This state-of-the art system has the capability to collect a fewer images per well. It has a shorter imaging time with a field of view that is three times larger than industry current standards. Moreover, the ImageXpress Micro XLS System can capture >10 million cells/hour in a low-resolution, whole-well, 3-color cell scoring application, or >1 million cells/hour in a high resolution two-color assay which will be used to perform a high-throughput screening (HTS). From this HTS screen parameters of cell size, proliferation, apoptosis and migration are obtained. Additionally, current studies describe that the cannabinoids exerted selective anti-tumor activity in several distinct tumor models. The present invention is capable of rapidly and effectively screening many human and mouse cancer cell lines including: breast, ovarian, colon, prostate, melanoma, head and neck, pancreatic, osteosarcoma, gastric, glioma, glioblastoma, neuroblastoma, leukemia and more. Moreover in certain aspects of the invention, the effect of the tested compound or analyte is simultaneously tested on cancer cells, normal cells, metastatic cells and/or to tumor cells after chemotherapeutic treatment and relapse derived from the same tissue and/or from the same patient.
It is therefore, within the scope of the present invention to provide a method for high throughput screening (HTS) for identifying an analyte with a measurable effect on cells. The aforementioned method comprises steps of: (a) providing an array comprising a plurality of cell samples; (b) providing at least one analyte to be tested; (c) contacting the cell samples with the analyte; and (d) detecting a signal indicative of the measurable effect on cells, wherein alteration of the signal over time measured on the cell sample relative to a control sample, is indicative of the measurable effect of the analyte on the cell sample.
It is further within the scope to disclose the method as defined in any of the above, wherein the analyst is selected from the group consisting of cannabinoid-type, cannabinoid derivative, cannabis extract or fraction thereof, non cannabinoid-type constituent, product, compound, molecule or substance and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the measurable effect on cells is selected from the group consisting of physiological, genetic, biochemical, structural and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the measurable effect on cells is selected from the group consisting of: anti proliferative, regenerative, anti inflammatory, anti mitotic, differentiative, anti metastatic, anti angiogenic, apoptotic, cytotoxic, cytopathic and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the measurable effect on cells is an effect on a biological parameter selected from the group consisting of: proliferation, migration, absorbance, adherence, apoptosis, necrosis, autophagy, cytotoxicity, cell size, motility, cell cycle and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the cancer cells are selected from the group consisting of: breast, ovarian, colon/rectum, prostate, melanoma, head and neck, pancreatic, osteosarcoma, gastric, glioma, glioblastoma, neuroblastoma, leukemia, adenocarcinoma, adrenal, anal, bile duct, bladder, bone, brain/CNS, cervical, endometrial, esophagus, eye, gastrointestinal, kidney, leukemia, liver, lung, lymphoma, multiple myeloma, nasal cavity and paranasal sinus, nasopharyngeal, non-hodgkin lymphoma, oral cavity, oropharyngeal, osteosarcoma, ovarian, pancreatic, penile, pituitary, retinoblastoma, rhabdomyosarcoma, salivary gland, sarcoma, skin, small intestine, stomach, testicular, thymus, thyroid, uterine sarcoma, vaginal and vulvar and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the cell samples are selected from the group consisting of: xenografts, allografts, cell lines, biopsy cells and a combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the cell lines are cancer cell lines selected from the group consisting of: central_nervous_system, bone, prostate, stomach, urinary_tract, ovary, haematopoietic_and_lymphoid_tissue, kidney, thyroid, skin, soft_tissue, salivary_gland, ovary, lung, pleura, liver, endometrium, pancreas, breast, upper_aerodigestive_tract, large_intestine, autonomic_ganglia, oesophagus, biliary_tract, small_intestine, autonomic_ganglia and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the cell samples are selected from the group consisting of: human cell lines, animal cell lines and xenografts.
It is further within the scope to disclose the method as defined in any of the above, wherein the cell samples are selected from the group consisting of: cancer cells, stem cells, neuronal cells, cardiomyocyte cells, somatic cells germ cells, normal cells, and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the signal is selected from the group consisting of: optic, luminescent, fluorescent, immunological, cell count, radioactive, non radioactive isotopic, electrical and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the measurable effect on cells is an effect on the expression level of a cancer marker selected from the group consisting of: ALK gene, Alpha-fetoprotein (AFP), Beta-2-microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), BCR-ABL fusion gene, BRAF mutation V600E, CA15-3/CA27.29, CA19-9, CA-125, Calcitonin, Carcinoembryonic antigen (CEA), CD20, Chromogranin A (CgA), Chromosomes 3, 7, 17, and 9p21, Cytokeratin fragments 21-1, EGFR mutation, Estrogen receptor (ER)/progesterone receptor (PR), Fibrin/fibrinogen, HE4, HER2/neu, Immunoglobulins, KIT, KRAS mutation, Lactate dehydrogenase, Nuclear matrix protein 22, Prostate-specific antigen (PSA), Thyroglobulin, Urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), 5-Protein signature (Ova1), 21-Gene signature (Oncotype DX), 70-Gene signature (Mammaprint) and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the analyte is extracted from cannabis; the cannabis is selected from a group consisting of: Cannabis sativa, Cannabis indica, Cannabis ruderalis, and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the cannabinoid-type is selected from the group consisting of: Cannabigerol (CBG) type, Cannabichromene (CBC) type, Cannabidiol (CBD) type, Δ9-Tetrahydrocannabinol (THC) type, Δ8-THC type, Cannabicyclol (CBL) type, Cannabielsoin (CBE) type, Cannabinol (CBN) and Cannabinodiol (CBND) types, Cannabitriol (CBT) type, cannabinoids with miscellaneous types and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the cannabinoid-type is further selected from the group consisting of: Tetrahydrocannabidiol (THC) or a derivative thereof, cannabidiol (CBD) or a derivative thereof, CBG (Cannabigerol), CBC (Cannabichromene), CBL (Cannabicyclol), CBV (Cannabivarin), THCV (Tetrahydrocannabivarin), CBDV (Cannabidivarin), CBCV (Cannabichromevarin), CBGV (Cannabigerovarin), CBGM (Cannabigerol Monomethyl Ether) and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the THC or a derivative thereof is selected from the group consisting of THC, THCV, THCA, THCVA, Delta-9-tetrahydrocannabinol (Δ9-THC) and delta-8-tetrahydrocannabinol (Δ8-THC) and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the cannabidiol (CBD) or a derivative thereof is selected from the group consisting of CBD, CBDV, CBDA and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the non cannabinoid-type constituent, product, compound, molecule or substance is selected from the group consisting of: terpenoids, hydrocarbons, essential oil derived from cannabis, nitrogen-containing compounds, carbohydrates, flavonoids, fatty acids, amino acids, proteins, glycoproteins, enzymes, sugars and related compounds, noncannabinoid phenols, simple alcohols, aldehydes, ketones, acids, esters, lactones, steroids, terpenes, phytosterols such as campesterol, ergosterol, E-sitosterol, and stigmasterol, vitamins such as vitamin A and vitamin K, pigments such as carotene and xanthophylls, elements such as Na, K, Ca, Mg, Fe, Cu, Mn, Zn and Hg and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the analyte is derived from a source selected from the group consisting of body of humans and animals, extracted from plants, synthetic, and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the HTS is selected from the group consisting of: microtiter plate, automatic colony pickers, uHTS or ultra-high-throughput screening, 3D tumor spheroid analysis method for HTS drug discovery, Celigo Imaging Cytometer, automation systems, a carousel system to store assay plates for high storage capacity and high speed access, integrated robot system, readout or detection, data-collection process and any combination thereof.
It is further within the scope to disclose the method as defined in any of the above, wherein the analyte provides a synergistic effect with respect to the measurable effect on cells as compared to the effect provided by conventional antitumor or anti-inflammatory therapies administered separately.
It is further within the scope to disclose the method as defined in any of the above, wherein the analyte provides a contra indicatory effect with respect to antitumor or anti-inflammatory activity as compared to the effect provided by conventional antitumor or anti-inflammatory therapies administered separately.
It is further within the scope to disclose a system for high throughput screening (HTS) for identifying an analyte with a measurable effect on cells. The aforementioned system comprises: (a) an array comprising a plurality of cell samples; (b) at least one analyte to be tested; and (c) means for detecting a signal indicative of the measurable effect on cells, wherein alteration of the signal over time measured on the cell sample relative to a control sample, is indicative of the measurable effect of the analyte on the cell sample.
It is further within the scope to provide a non transitory computer readable medium comprising instructions which, when implemented by one or more computers cause the one or more computers to present data concerning a measurable effect on cells of one or more analytes on preselected cell samples by processing data concerning a signal indicative of the measurable effect on cells, wherein alteration of the signal over time measured on the cell sample relative to a control sample, is indicative of the measurable effect of the analyte on the cell sample.
It is further within the scope to provide a composition comprising therapeutically effective amount of, or an extract comprising essentially therapeutically effective amount of an analyte selected as defined by the method described in any of the above, wherein the composition has an antitumor or anti-inflammatory activity or synergistic effect thereof for use in the treatment of a cancer type or inflammatory disease.
It is further within the scope to provide a method for identifying one or more genetic markers derived from cannabis, wherein the one or more genetic markers correlates with a measurable effect on cells as indicated by the method as defined in any of the above, the method comprises additional steps of correlating the signal with cannabis DNA sequence data.
It is further within the scope to disclose provide one or more genetic markers derived from cannabis, wherein the one or more genetic markers correlates with a measurable effect on cells as indicated by the method as defined in any of the above, further wherein the signal is correlated with cannabis DNA sequence data.
It is further within the scope to disclose the method as defined in any of the above, wherein the one or more genetic markers is selected from the group consisting of: variation, mutation or alteration in a genomic loci, a single nucleotide polymorphism (SNP), minisatellites, RFLP (Restriction fragment length polymorphism), SSLP (Simple sequence length polymorphism), AFLP (Amplified fragment length polymorphism), RAPD (Random amplification of polymorphic DNA), VNTR (Variable number tandem repeat), SSR (Simple sequence repeat), microsatellite polymorphism, STR (Short tandem repeat), SFP (Single feature polymorphism), DArT (Diversity Arrays Technology), RAD markers (Restriction site associated DNA markers) nucleotide changes, indel, deletion, duplication, inversion and/or insertion and any combination thereof.
It is further within the scope to provide a database of analytes, wherein the database comprises data concerning the analyte, correlated with a measurable effect on cells, defined by implementing steps of the method described in any of the above.
It is further within the scope to provide a method for high throughput screening (HTS) for identifying an analyte selected from the group consisting of: cannabis extract or a fraction thereof, cannabinoid-type constitute, non cannabinoid-type constitute and any combination thereof, the analyte is indicative of cytotoxic or anti proliferative or anti mitotic or cell growth inhibitory activity in vitro. The method comprises steps of: (a) providing an array comprising a plurality of cancer cell samples; (b) providing the analyte to be tested, the analyte is selected from the group consisting of: cannabis extract or a fraction thereof, cannabinoid-type constitute, non cannabinoid-type constitute and any combination thereof; (c) contacting the cancer cell samples with the analyte; and (d) detecting a signal indicative of the cytotoxic or anti proliferative or anti mitotic or cell growth inhibitory activity in vitro, wherein alteration of the signal over time measured on the cancer cell sample relative to a control sample, is indicative of the cytotoxic or anti proliferative or anti mitotic or cell growth inhibitory activity in vitro of the analyte on the cancer cell sample.
It is further within the scope to provide a method for high throughput screening (HTS) for identifying an analyte selected from the group consisting of: cannabis extract or a fraction thereof, cannabinoid-type constitute, non cannabinoid-type constitute and any combination thereof, the analyte is indicative of antitumour activity, the method comprises steps of the method as described in any of the above, and additionally comprising steps of: (a) transplanting cancer cell xenographs derived from the cancer cell samples into experimental animals; (b) treating the experimental animals with the analyte selected from the group consisting of: cannabis extract or a fraction thereof, cannabinoid-type constitute, non cannabinoid-type constitute and any combination thereof, identified by the method as defined in any of the above, and (c) monitoring tumor growth of the experimental animal.
It is further within the scope to disclose the method as defined in any of the above, wherein the experimental animal is nude mice.
It is further within the scope to provide a protocol useful for identifying a botanical analyte with a measurable effect on cells correlated with a disease. The aforementioned protocol comprises steps of: (a) providing input data comprising data selected from the group consisting of: parameters of the analyte, parameters of the cells, high throughput screening (HTS) results data for identifying an analyte with a measurable effect on cells as indicated in claim 1, clinical or preclinical data and any combination thereof; (b) processing the data; and (c) presenting output data at an electronic display concerning a measurable effects of the analyte on the cells correlated with a disease, and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the data processing comprises steps selected from the group consisting of: correlating, normalizing, calibrating, factorizing, calculating, statistically analyzing and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the analyte parameters are selected from the group consisting of: analyte source, analyte processing and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the botanical analyte source parameters are selected from the group consisting of: source strain, source genotype, source phenotype, source growth conditions, source harvest conditions, source nutrition, source part or organ and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the source part or organ is selected from the group consisting of: root, stem, leaf, flower, seed and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the botanical analyte processing parameters are selected from the group consisting of: curing, drying, extraction process, decarboxylation and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the extraction process is selected from the group consisting of: butane, CO2 gradients, ethanol, dry ice and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the cell parameters are selected from the group consisting of: cells source, cells treatment and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the cells source is selected from the group consisting of: biopsies, cell lines, xenographs, mutated cells or molecules and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the cells treatment is selected from the group consisting of: cells medium treatment, serum treatment, cells dilution, cell cycle phase and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the measurable effect on cells is selected from the group consisting of proliferation, apoptosis, migration, regeneration, differentiation, angiogenesis, and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the clinical or preclinical data is selected from the group consisting of: administration route of the analyte to a subject, dosages, release form, cancer markers level, tumor size monitoring, metastasis monitoring, survival, quality of life measured according to one or more scales, and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the administration route is selected from the group consisting of: sublingual, oral, intravenous, topical, subcutaneous and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the release form is selected from the group consisting of: slow release, controlled release, sustained release, immediate or rapid release and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the cancer markers are selected from the group consisting of: ALK gene, Alpha-fetoprotein (AFP), Beta-2-microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), BCR-ABL fusion gene, BRAF mutation V600E, CA15-3/CA27.29, CA19-9, CA-125, Calcitonin, Carcinoembryonic antigen (CEA), CD20, Chromogranin A (CgA), Chromosomes 3, 7, 17, and 9p21, Cytokeratin fragments 21-1, EGFR mutation, Estrogen receptor (ER)/progesterone receptor (PR), Fibrin/fibrinogen, HE4, HER2/neu, Immunoglobulins, KIT, KRAS mutation, Lactate dehydrogenase, Nuclear matrix protein 22, Prostate-specific antigen (PSA), Thyroglobulin, Urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), 5-Protein signature (Ova1), 21-Gene signature (Oncotype DX), 70-Gene signature (Mammaprint) and any combination thereof.
It is further within the scope to disclose the protocol as defined in any of the above, wherein the one or more scales for assessing quality of life are selected from the group consisting of: pain scale, quality of life scale, functional assessment of cancer therapy scale and any combination thereof.
In order to understand the invention and to see how it may be implemented in practice, a plurality of preferred embodiments will now be described, by way of non-limiting example only, with reference to the following examples.
Dried flowers of six cannabis sativa strains (i.e. CNB1, CNB2, CNB3, CNB4, CNB6, CNB8) were soaked in butane and resin was purged to exclude butane residues from the concentrated oil.
The concentration of 10 cannabinoids (i.e. CBDA, CBGA, CBG, CBD, THCV, CBN, THCA, Δ9THC, Δ8THC and CBC) in the extracts were evaluated in HPLC (see
For preparation of a stock for cell lines, the cannabis extracts were dissolved in DMSO (e.g. about 50 mg/ml) and kept in −20° C. until use.
Reference is now made to non limiting examples of cell cultures used in the present invention:
Examples of human cancer cell lines:
Examples of human non-cancer cell lines (Control):
Reference is now made to a procedure for growing cell lines:
Cell lines were maintained at 37° C. in a humidified atmosphere containing 5% CO2.
All cell lines, except for PC3 and SW480 were routinely grown in phenol red-containing minimum essential medium (DMEM) (Sigma). PC3 and SW480 were grown in phenol red containing RPMI-1640 (Sigma).
Media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, 100 μg/mL of streptomycin and 100 mM L-glutamine
Cells were harvested three days before exposure and seeded (about 50,000-200,000 cells/well) into a 24-well microplate. The medium was replaced in respective culture medium containing 0.5% FBS, and vehicle (DMSO) or cannabis extracts (1-10 μg/ml) were added to the medium for 24-48 hours in duplicates.
The effect of cannabinoid compounds on cell viability was measured using high-content screening analysis. For example, differentiation of subpopulations of cells within the same well was analysed by distinguishing: live versus necrotic cells, early apoptotic cells and late apoptotic cells.
Cells were imaged in ImageXpress micro (see
Reference is now made to non limiting examples of probes used to screen the effect of cannabis extract on tested cell lines:
After predetermined periods of incubation, the cell lines medium was replaced with PBS containing fluorescent probes, including at least one of:
It is noted that the staining pattern resulting from the simultaneous use of these three dyes makes it possible to distinguish normal, apoptotic and dead cell populations by flow cytometry or fluorescence microscopy.
Reference is now made to
Providing a robust procedure and system for high through output screening (HTS) for the detection of correlations between cannabinoid ratios and dosages, and anti-tumor activity. The procedure includes screening of a growing library of human cancer cell lines and/or biopsies by an enlarged variety of cannabis-based compounds or extracts. It is demonstrated by the present invention that the examination of the biological activity of cannabis extracts, fractions and compounds thereof on tumor cell lines or biopsies of distinct tissue lineage, creates a highly potent therapeutic data. In another aspect, the HTS system and method is applied on cell lines for the screening of potent cannabinoids, cannabis fraction or extract, with or without the conjunction of standard chemotherapy. In another embodiment, the HTS system and method is applied on biopsies derived from patients, for the screening of most potent cannabinoid or cannabis extracts or fractions thereof with or without the conjunction of chemotherapy according to patients' overall treatment.
Reference is now made to
Reference is now made to
Reference is now made to
Reference is now made to
The results described above clearly demonstrate that the system and method of the current invention could be used to screen and select for cannabis extracts or fractions thereof with in vitro cytotoxicity towards cancer cells and minimal cytotoxicity towards normal cells. The cannabis extracts or fractions thereof that have been screened in this way can be further investigated for potential anti-tumor activity.
It is clearly shown that the HTS method and system of the present invention could differentiate cannabis plant or other plant extract's potential antitumor effects. It is shown that different strains cause varied apoptotic effects on different cancer cell lines with no effect on normal cells.
It is clear that the aforementioned potentially anti tumour extracts could be used as a basis for novel anti cancer compositions and therapies. Such compositions offer significant improvements to the currently available treatments against cancer in the following aspects:
In this example, the cancer cells lines which have shown in-vitro measurable effect on cells such as apoptosis and/or necrosis effects, as a result of treatment with specific compounds or analytes (e.g. cannabis extracts, see Example 3) are implanted in experimental animal, such as mice. The implanted mice are than treated with the selected analytes (such as extracts or compound or compounds combinations), with or without chemotherapy treatment as means for better evaluation of the effect of the selected analyte on cancer in humans.
Reference is now made to a xenograft tumor assay exemplified protocol (https://www.mcdb.ucla.edu/Research/Arispe/Protocols/Xenograft_Tumor_Assay.pdf is incorporated herein by its entirety). It is noted that modifications of this protocol are included within the scope of the present invention.
1) Determine the number of cells for injection (i.e. 5*106) to determine the number of plates that will require trypsinizing (usually a 100% confluent plate of 100 mm2 will yield at least 2 injections at 5*106 cells/injection)
2) Trypsinize the number of plates to be counted all at once
3) Collect detached cells in 50 ml conical and spin for 4 min at 800 rpm
4) Remove sup and resuspend in 25 ml of SFM for counting
5) Remove three 100 μl aliquots into 3 separate eppendorfs and dilute each 100 μl 1:5 by adding 400u. of SFM, mix well
6) Remove 50 μl of 1:5 dilutions for counting, count each of three dilutions and average the three numbers
7) Determine the conc. of cells in cells/ml by using the following formula:
Average counts*10,000*dilution factor (5)=#cells/ml
8) Determine the volume required to add to achieve final concentration of cells for injection per volume to be injected (i.e. 5*10+ cells/100 μl injection) by first determining the total number of cells in the 25 ml suspension by multiplying the conc. of cells in #cells/ml*25=total number of cells. Then use the following formula
Total # cells/x volume=5*106/100 μl, solve for x=volume to resuspend pellet of cells to achieve desired final concentration (i.e. 5*106 cells/100 μl)
9) Spin down 50 ml conical for 4 min at 800 rpm
10) Discard sup and resuspend the pellet in the previously determined volume from step #8.
11) Draw up each injection/mouse in 1 ml syringes in the tissue culture hood prior to going to the animal facility. Place the separate syringes each containing 100 μl on ice (this step minimized the possibility of the cells settling after being resuspended thus altering the concentration of cells.
12) Anesthetize each mouse with isoflorane inhalent just prior to injection. Be careful not to over anesthetize as the mice will succumb to respiratory depression. Just the right amount is when they just begin to stop moving, remove them from the source of anesthetic, let them breath pure air for a few seconds then place their noses just adjacent to the opening of the 50 ml conical during the injection
Reference is now made to
Reference is now made to
Reference is now made to Table 1 presenting some of the parameters used in the present invention for evaluation of antitumor activity of selected botanical extract or analyte.
Reference is now made to non limiting examples of cancer markers which may be used in the present invention for evaluation of antitumor activity of selected botanical extract or any other analyte.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2016/050471 | 5/4/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62166716 | May 2015 | US |
