DESC:F O R M 2

THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
[See section 10 and rule 13]

1. TITLE OF THE INVENTION: CURCUMIN AND METFORMIN IN PREVENTION OF HEAD AND NECK CANCER

2. APPLICANT (A) Mazumdar Shaw Medical Foundation

(B) Mazumdar Shaw Medical Foundation
A-Block, 8th Floor, Mazumdar Shaw Medical Centre,
#258/A, Narayana Health City, Bommasandra, Bangalore, Karnataka, India, 560099

3. NATIONALITY (C) INDIA

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
TECHNICAL FIELD OF THE INVENTION
001 The present invention is in the technical field of cancer prevention and therapeutics, in particular, the invention relates to the use of Curcumin and Metformin in chemoprevention of cancers, including head and neck cancers.
BACKGROUND OF THE INVENTION
002 Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide with cancers of the oral cavity (OSCC) being the most widespread; accounting for about 5,00,000 cases worldwide per year [1, 2].
003 In addition, the disease shows a stepwise clinical and histological progression in most patients, with the occurrence of premalignant lesions (PML) such as leukoplakia and erythroplakia preceding the development of carcinoma (FIG 1).
004 However, the percentage of transformation of the PML varies with no particular method to identify the susceptible lesions. The current mode of treatment is surgical excision for the lesions that are dysplastic histologically.
005 Multiple chemo-preventive agents including retinols, beta carotene and curcumin have been tested in oral cancer [3-6], in an effort to stall the stepwise progression of the disease. However, lack of efficacy, toxicities associated with the interventions and relapses following cessation of therapy [4, 7] have been major challenges in these chemo-preventive strategies. In a study carried out with vitamin A and beta carotene, tumor relapse was observed in up to 50-60% of the patients [8].
006 In a study carried out with 13-cis retinoic acid alone, the patients showed reduction of leukoplakia in the beginning, however, relapse was observed after cessation of the drug though there was very less or no toxicity observed in the patients [9].
007 The other chemopreventive drugs include vitamin E (a-tocopherol), Nonsteroidal anti-inflammatory drugs (NSAIDs), a-Interferon, Polyphenols (green tea) and Protease inhibitors (soy). In a study carried out with 13-cis retinoic acid, retinyl palmitate and beta-carotene showed that low-dose of 13-cis retinoic acid was not well tolerated for long-term oral cancer prevention and better-tolerated retinyl palmitate alone was ineffective [10]. A phase II study of 400 IU of a-tocopherol administered twice daily showed a 46% clinical response and a 21% histologic response after 24 weeks of treatment [11].
008 Curcumin is known to suppress the expression of NFkB and its regulated targets (CYCLIN D1, VEGF, COX-2, c-myc, Bcl-2) involved in carcinogenesis and angiogenesis (bFGF, VEGF, angiopoetins). Curcumin has been evaluated for its anti-cancer effects in many carcinomas including head, neck and colon cancers [12-14]. In oral cancer, curcumin is known to inhibit tobacco-induced NFkB and COX-2 in oral premalignant and cancer cells
009 Further, in vitro studies have elucidated the inhibitory role of the drug on the translational machinery by suppressing the phosphorylation of 4E-BP1, eIF4G and eIF4B, with the effect being higher in oral cancer cells as compared to the normal epithelial cells [16]. A recent chemoprevention trial in colorectal cancer patients also showed a decrease in the aberrant crypt foci at high doses of the drug [12]; inventor’s own study indicated a clinical and pathological benefit of curcumin in prevention of oral premalignant lesion progression [17]. Additionally, curcumin is also known to be well-tolerated in humans with phase I studies revealing no toxic effects even with doses of 8000 mg/day [12-14,18].
010 Metformin, on the other hand, acts through the mammalian Target of Rapamycin (mTOR) pathway. This anti-diabetic drug is known to activate the liver kinase B1 (LKB1)–5' AMP-activated protein kinase (AMPK) signaling pathway [19-21] and inhibit the mTOR pathway, thereby leading to reduction in cell proliferation [22-25]. Metformin induces cell cycle arrest at G0/G1 phase and apoptosis in oral cancer cells through the down regulation of Cyclin D1, CDK4, 6 and the anti-apoptotic Bcl-2/Bcl-xL [26]. Metformin has also been shown to reduce the incidence of premalignant lesions in oral carcinogenesis animal models in a PIK3CA dependent manner [27, 28]. Clinical evidence was further provided by studies wherein HNSCC patients taking metformin had lower incidences of the disease, low rates of recurrence and metastasis [29]. Importantly, as with curcumin, the tolerability and safety of metformin in humans has also been well documented [30]. The primary targeted pathways by curcumin and metformin, NFkB and PI3K/mTOR are interconnected, with recent evidence indicating that mTOR can lead to activation of NFkB via stimulation of IKK [31].
011 Recent evidences have also shown a Cancer stem cell (CSC)-based mechanism of action by these drugs. Studies have revealed a CIC-based induction of cancers in 4NQO animal models [32, 33]. The pathways targeted by curcumin and metformin, NF?B and PI3K/mTOR pathways, have recently been implicated in cancer stem cell (CSC) activity; activation of STAT3 and NFkB has revealed to regulate the Notch pathway in glioblastoma stem cells [34]. PI3K/mTOR pathway is known to contribute towards CSC maintenance, its inhibition leading to decreased expression of CSC markers [35].
012 At present, chemotherapeutic treatments are used for reduction of tumor mass in cancers. However, the disease often relapses. This phenomenon is explained through cancer stem cell hypothesis, which suggests that tumors contain a small number of tumor-forming, self-renewing, cancer stem cells within a population of non-tumor-forming cancer cells [36, 37] These cancer stem cells are resistant to well-defined chemotherapy, and they can regenerate all the cell types in the tumor through their stem cell-like property.
013 In the present scenario, there are challenges to have agents that can provide effective and long term chemoprevention.
014 Furthermore, there is also need for agents that can provide effective prevention for second primary tumors.
015 In summary, existing cancer therapies have challenges in terms of chemoprevention, tumor relapse, and effective targeting of cancer stem cells. There is also a need for treatment of oral potentially malignant lesions.
016 Accordingly, there is also a need to use strategies that combine different drugs, targeting multiple signaling pathways for effective cancer prevention and therapy.

SUMMARY OF THE INVENTION

017 According to an exemplary aspect, the present invention discloses usage of curcumin and metformin in the field of chemoprevention in cancer, more particularly in head, neck and oral cancer.
018 Yet another exemplary feature of the present invention is that, the use of above mentioned combination of drugs in a pre-clinical carcinogenesis model has provided benefit at the clinical and the pathological level toward chemoprevention of the oral premalignant lesions. In the 4NQO induced animal model, the combination of drugs resulted in a lower number of lesions, reduced tumor volume and higher survival benefit as compared to the animals treated with the drugs individually. Additionally, a pathological benefit was observed in a subset of the animals, indicating that this combination of drugs has synergistic effect, and resulted in a higher response.
019 According to a further exemplary aspect of the present invention, combination of these two said agents has synergistic effect in inhibiting oral carcinogenic progression.
020 Yet another exemplary aspect of the present invention, the combination of both curcumin and metformin give synergistic response in vivo animal study when compared to individual treatment. The overall survival of combination of these two said agents was also found to better when compared to individual treatment.
021 According to a further exemplary aspect of the present invention, the possible uses of this invention include, utilization of the combination of drugs for chemoprevention, prevention for second primary tumors, use as a chemotherapeutic agent for patients with oral cancer.
022 According to a further exemplary aspect of the present invention, these combination of drugs (curucmin and metformin) can be prescribed for patients who are detected with oral cancer at early detection camps, dental clinics and tertiary clinics.
023 Yet another exemplary aspect of the present invention, the use of this combination of drugs in a pre-clinical carcinogenesis model has provided benefit at the clinical and the pathological level toward chemoprevention of the oral premalignant lesions.
024 In summary, the present invention is in the technical field of cancer prevention and therapeutics, in particular, the invention relates to the use of curcumin and metformin in chemoprevention of cancers, including head and neck cancers. The use of this said combination of drugs in a pre-clinical carcinogenesis model has provided benefit at the clinical and the pathological level toward chemoprevention of the oral premalignant lesions. In the 4NQO induced animal model, the combination of drugs resulted in a lower number of lesions, reduced tumor volume and higher survival benefit as compared to the animals treated with the drugs individually. Additionally, this said combination of drugs resulted in pathological benefit, indicating synergistic effect, and that resulted in a higher response to cancer chemoprevention and therapy. The present invention also relates to components useful in prevention and treatment of cancer, as well as the compositions, methods, processes, and uses thereof.
025 Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.
026 As will be appreciated by a person skilled in the art the present invention provides a variety of following advantages. Listing of Claims,
027 A method of treating cancer by administering, in combination, at least 2-3 agents that together target at least two cancer-associated pathways selected from the group consisting of angiogenesis, apoptosis, immune surveillance, metabolism and energetics, and combinations thereof.
028 The method of [027], wherein none of the at least 2-3 agents is a chemotherapeutic agent utilized at its maximum tolerated dose, wherein the agents are selected such that each of the at two three pathways is targeted by at least two agents.
029 The method of [027], wherein at least one of the 2-3 agent is a chemotherapeutic agent utilized in chemoprevention of cancers, wherein the cancer is head or neck or oral cancer.
030 A method of treating cancer by administering to a subject suffering from cancer at least 2-3 agents selected from the group consisting of curcumin, metformin and equivalents thereof, wherein each of the agents are administered in doses well below the maximum tolerated dose for the agent.
031 The method of [030], wherein equivalents of Curcumin are other agents that inhibit the signal transduction pathway of PI3K/Akt, MAPK, and NF-?B activation, and the Sonic Hedgehog (Shh) signalling, wherein equivalents of Curcumin are other agents that down-regulate epidermal growth factor receptors (EGFR and erbB2), Insulin-like growth factor type-1 receptor (IFG-1R), sonic hedgehog (SHH)/GLIs) and Wnt/b-catenin and PARP, IKK, EGFR, JNK, MAPK, 5-LOX, STATs, IL-6, COX-2, and MMPs.
032 The method of [030], wherein equivalents of Metformin are other agents that affect cellular metabolism through oxidative stress, anaerobic glycolysis, Warburg effect and COX-2 activation, wherein equivalents of Metformin are other agents that suppress tumor angiogenesis by inhibiting HIF-la stabilization under hypoxia and VEGF mRNA expression, wherein equivalents of Metformin are other agents that suppress tumor angiogenesis by inhibiting HIF-la stabilization under hypoxia and VEGF mRNA expression.
033 The method of [030], wherein equivalents of Metformin are other agents that inhibit cell proliferation and induce apoptosis in cancer cells in vitro by simultaneously suppressing the COX-2/PGE2, p300/NF-?B, and PI3K/Akt/signaling and activating the Apaf-1/caspase-dependent apoptotic pathway.
034 The method of [027], wherein at least some of the at least 2-3 agents are administered according to a continuous regimen, which method comprises continuing administration after one or more symptoms or features of the cancer have resolved, wherein the at least 2-3 agents are administered as an adjunct to agents used for chemoprevention.
035 The method of [030], wherein the subject is suffering from cancer, wherein the cancer is Stage premalignant or early stage for chemoprevention of dysplastic progression and II, Stage III, or Stage IV cancers for second primary prevention, or late-stage cancer, or metastasized cancer, or solid tumors, or cancer that has relapsed after treatment with another therapeutic modality, or tumors of epithelial origin, or cancer selected from the group consisting of epithelial cell cancers, sarcomas, and blood cancers (leukemias or lymphomas), or adenocarcinoma, or cancer selected from the group consisting of breast cancer, lung cancer, ovarian cancer, pancreatic cancer, etc
036 A composition comprising two or more compounds, wherein each of said two compounds is capable of regulating a specific metabolic pathway or intracellular signalling pathway or intercellular signalling pathway implicated in the progression of cancer in an individual suffering from cancer, and wherein at least two such pathways are addressed.
The advantages of this combination of drugs are following but are not limited to,
037 The drug combination resulted in a clinical response in terms of reduced lesion, reduced tumor volume and increased survival, as observed in the animal model.
038 The combination also resulted in a partial histological response.
039 Both the drugs are tolerated well in patients.
040 The combination can be effective for chemoprevention of premalignant lesions.
041 Combination might also be effective for second primary tumor prevention.
042 Invention also shows that the combination acts on cancer stem cells and thereby providing a more effective chemoprevention.
043 The effect of the combination may also be observed in other sites of HNSCC along with OSCC.

BRIEF DESCRIPTION OF THE DRAWINGS

044 Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.
045 FIG. 1 illustrates the step-wise process of oral carcinogenesis.
046 FIG. 2 illustrates a schematic illustration of development of animal model.
047 FIG. 3 illustrates of histopathology analysis. 3A Carcinogenesis model with corresponding histology; 3B: Clinical (Top panel) and histological (Bottom panel) representation of tissues from different arms post chemo-preventive treatment; 3C: Average tumor volume; 3D: Kaplan Meier Survival Curve; 3E: Average body weight [Statistical significance is indicated (*p<0.05, **p<0.005, ***p<0.0005)].
048 FIG. 4 graphs illustrates the imunohistochemical analysis for CSC markers during carcinogenesis (A); and IHC of targeted pathway markers (B) and CIC markers (C) for chemo-preventive treatment arms [Statistical significance is indicated (*p<0.05, **p<0.005, ***p<0.0005)].
049 FIG. 5 graphs illustrating expression profiling for CSC markers during carcinogenesis (A and B); and for chemo-preventive treatment arms (C and D) [Statistical significance is indicated (*p<0.05, **p<0.005)].
050 FIG. 6 illustrates profiling of CD44+ cells in treated primary cells by FACS analysis (A and B); CSC markers profiling of treated primary cells by qRT-PCR (C) [Statistical significance is indicated (*p<0.05, **p<0.005, ***p<0.0005)].
051 FIG. 7 illustrates wound healing/migration assay of 6th week (A) and 16th week (B) primary treated cells [Statistical significance is indicated (**p<0.005)].
052 FIG. 8 illustrates Colony formation assay in 6th and 16th week primary treated cells [Statistical significance is indicated (**p<0.005)].
053 In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

DETAILED DESCRIPTION OF THE INVENTION
It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.
054 The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
055 As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.
056 The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.
057 All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.
058 The pharmaceutical formulations of the said invention may also include, pharmaceutically acceptable nontoxic carriers or diluents, for animal or human administration.
059 The present invention concerns methods and compounds useful for the protection and treatment of proliferative and/or inflammatory disorders.
060 “Proliferative disease or disorder” means all neoplastic cell growth and proliferation, whether malignant or benign, including all transformed cells and tissues and all cancerous cells and tissues. Proliferative diseases or disorders include, but are not limited to, premalignant or precancerous lesions, abnormal cell growths and/or lesions, benign tumours, malignant tumours, and “cancer.”
061 Additional examples of proliferative diseases and/or disorders include, but are not limited to neoplasms, whether benign or malignant, located in the: liver, pancreas, peritoneum, endocrine glands (adrenal, testicles, ovary, thymus, thyroid, parathyroid, pituitary), eye, head and neck, nervous (central and peripheral), soft tissue, spleen, thoracic, lymphatic system, pelvic, skin, and urogenital tract embodiment. According to a non limiting exemplary aspect of the present invention, the proliferative disorder involves tumour.
062 The terms “tumour” or “tumour tissue” refer to an abnormal mass of tissue which results from excessive cell division. A tumour or tumour tissue comprises “tumour cells” which are neoplastic cells with abnormal growth properties and no useful bodily function. Tumours, tumour tissue and tumour cells may be benign or malignant. A tumour or tumour tissue may also comprise “tumour-associated non-tumour cells”, e.g., vascular cells which form blood vessels to supply the tumour or tumour tissue. Non-tumour cells may be induced to replicate and develop by tumour cells, for example, the induction of angiogenesis in a tumour or tumour tissue. According another non limiting exemplary aspect of the present invention, the proliferative disorder involves malignancy or cancer.
063 Example embodiments of the present invention are described with reference to the accompanying figures.
064 In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
065 This invention describes the advantages of the usage of curcumin and metformin in the field of chemoprevention in oral cancer, including head and neck cancers. The use of this said combination of drugs in a pre-clinical carcinogenesis model has provided benefit at the clinical and the pathological level toward chemoprevention of the oral premalignant lesions. In the 4NQO induced animal model, the combination of drugs resulted in a lower number of lesions, reduced tumor volume and higher survival benefit as compared to the animals treated with the drugs individually.
066 Additionally, this said combination of drugs resulted in pathological benefit, indicating synergistic effect, and that resulted in a higher response to cancer chemoprevention and therapy. The present invention also relates to components useful in prevention and treatment of cancer, as well as the compositions, methods, processes, and uses thereof.

067 The advantages of this combination of drugs are followings but are not limited to,
068 The drug combination resulted in a clinical response in terms of reduced lesion, reduced tumor volume and increased survival, as observed in the animal model.
069 The combination also resulted in a partial histological response.
070 Both the drugs are tolerated well in patients.
071 The combination can be effective for chemoprevention of premalignant lesions
072 Combination might also be effective for second primary tumor prevention.
073 Invention also shows that the combination acts on cancer stem cells and thereby providing a more effective chemoprevention.
074 The effect of the combination may also be observed in other sites of HNSCC along with OSCC.
075 The combination of drugs was tested in a 4NQO-induced oral carcinogenesis model; the animals were randomized into different arms and treated with the drugs, individual and combination. The response in the animals was tested at three levels; clinical, pathological and molecular. The results were analyzed for their statistical significance. The details of the experiment and the results are given below.
076 Treatment of a 4NQO carcinogenesis model with the drugs: The animal model was established using 4-6 weeks C57Bl/6 mice (N=60); the mice were divided into two arms; i) Control Arm (N=10) administered with plain drinking water and ii) Treatment Arm (N=50). The treatment arm was administered with 4NQO (4-nitroquinoline-oxide) in drinking water (50ppm) for a period of 17 weeks. The mice were then taken off the carcinogen and administered the drugs in drinking water.
077 The mice (N=45) were divided into four arms; i) Arm I (N=15) with plain water, ii) Arm II (N=15) with Curcumin (64µg/ml), Arm III (N=15) with Metformin (5mg/ml) and iv) Arm IV (N=15) with a combination of both Curcumin and Metformin (FIG 2). The mice were dissected at every 2nd week (N=5 at each time point) as well as at the end of the study period and the samples were collected for histology, primary culture and molecular analysis of stem cell related pathways.
078 Primary Culture: Tissue samples, decontaminated with Betadine solution, were placed in 10% DMEM+2X Penicillin-Streptomycin (Himedia, India), cut into small pieces (1-2mm in size) and partially trypsinized (1% Trypsin). The samples were then placed on a serrated 60mm culture dish and allowed to stand for 5 minutes to remove excess moisture. The dish was subsequently filled with media and supplemented every second day. Cultures from passage >25 were used in this study.
079 Primary culture cells from the 6th and 16th week tissues were cultured to 80-90% confluence and treated with curcumin, metformin or combination for 48 hours and evaluated for CD44+ population by FACS, expression level of CSC markers, colony formation and migration. The untreated primary cells were used as the calibrator.
080 Results:
081 Clinical Response: Our in vivo animal study has shown that the combination of both curcumin and metformin give better response when compared to individual treatment. Histopathology carried out at 16th week, showed dysplastic changes after which the animals were administered with the drugs (FIG 3A). Average number of lesions per mice (FIG 3B) and average tumor volume (FIG 3C) was reduced in combination arm (p<0.05). The overall survival of combination arm was also found to better when compared to individual treatment (FIG 3D). Toxicity assessment did not show any change in body weight when compared to Control Arm II, though there was a significant difference (p>0.005) when compared to Control Arm I (FIG 3E).
082 Pathological Response: Histological assessment of the chemoprevention arms indicated that 33% (2/6) animals in the combination arm and 25% (1/4) in the monotherapy arms did not show disease progression.
083 Molecular Response: The molecular response of treatment was assessed with respect to the targeted markers and the CSC markers. The markers were assessed at both the protein and the transcript levels.
084 Immunohistochemical studies: Initially the markers of CSCs (CD44 and NOTCH1) were profiled during the development of carcinogenesis; the tissues from different stages of oral cancer development showed a trend of up regulation of these markers when compared to 0th week (FIG 4A).
085 To assess the effect of treatment, the levels of targeted molecules (NF-?B and pS6) and the CSC markers were assessed in the treatment arms in comparison to the control arm. NF-?B and pS6 showed a down-regulated expression in curcumin and metformin arms respectively (FIG 4B). A trend of down regulation of CD44 and NOTCH1 was observed in treated groups (FIG 4C).
086 Expression profiling of CSC markers (CD44, NOTCH1, JAGGED1, STAT3, CD133 and ALDH1A1) in tissue samples: A similar trend, as observed in IHC, was seen for CD44 and NOTCH1. The other CSC markers (CD133 and ALDH1A1) and down-stream markers (JAGGED1) showed an up regulation during carcinogenesis (0th, 8th, 16th and 25th week) whereas STAT3 did not show difference in regulation across the progression groups.The CIC markers (CD44, NOTCH1, ALDH1A1 and CD133) and down-stream markers (JAGGED1) showed a down regulation in treated samples (curcumin, metformin and combination). STAT3 did not show difference in regulation across the different treatment cohorts (FIG 5)
087 Profiling in primary culture cells: Primary cells developed from tissues of the 6th week (mild dysplastic) and 16th week (severe dysplastic) were used to further evaluate the effect of drugs on CSC behavior by FACS (FIG 6A). The 6th week cells revealed a decrease in CD44+ population when treated with curcumin (46.7%, p=0.0001), metformin (60.4%, p=0.0001) and combination (45.7%, p=0.0001), as compared to the untreated cells (77.4%) (FIG 6B).The isotype control showed no non-specific binding to the cells. The 16th week cells, however, showed a reduced CD44+ population when treated with curcumin (85%, p=0.03), and combination (84.9%, p=0.0001), as compared to the untreated cells (90.3%) (FIG 6B) with the metformin treated cells not showing any difference (90.9%).
088 qPCR profiling showed that CD44, CD133, ALDH1A1, NOTCH1, JAGGED and STAT3 expression was down regulated across all the treatment arms of the cells from mild dysplastic tissue [CD44 (<0.54 fold); CD133 (<0.29 fold), ALDH1A1 (<0.18 fold), NOTCH1 (<0.38 fold) and JAGGED1 (<0.75 fold)] (FIG 6C). Cultures from severe dysplastic tissue, on treatment individually with the drugs, showed a down regulation of CD44 (<0.15), JAGGED1 (< 0.0008 fold), STAT3 (<0.14 fold), ALDH1A1 (0.001 fold) and NOTCH1 (0.116 fold) expression. CD133 expression was not down regulated, indicating a comparative low efficiency of CSC inhibition by curcumin treatment in these cells. The combination treatment arm was not evaluated due to cell death by the end of treatment (FIG 6C).
089 Functional assays in primary culture cells: The wound healing/scratch assay was carried out using two different concentrations of curcumin and metformin in the both the cells. At 24 hours, mild dysplastic cells (6th week) with treatment using high concentrations of 1µM curcumin and 5µM metformin showed ~52% wound closure in parental cells, while the curcumin and combination arms showed 34% (p=0.02) and 28% (p=0.007) closure. The metformin treated showed a closure rate of 54% (p=0.05). At 36 hours, untreated cells and metformin cells showed = 90% closure, the curcumin and combination arm showed 43% and 72% wound closure respectively. Assessment at half the concentrations indicated a non-optimal drug effect, with metformin at 2.5µM being extremely ineffective with the drug treatment showing no specific pattern, although there is an inhibition in all the treatment arms at 36 hours (FIG 7A).
090 In the cells derived from the severe dysplastic tissue (16th week), the cells closed completely by 24 hours and hence the assessment was carried out at 12 and 24hours. At 12 hours, the percentage wound closure ranged from 10-23% in the treatment arms [curcumin: 10% (p=0.03), metformin: 23%, combination: 13% (p=0.003), parental: 16%]. However, at end of 24 hours, the combination arm showed a migration inhibition with percentage wound closure of only 45% (p=0.0001) as compared to complete closure by the other treatment arms. In comparison, treatment with lower concentration of drugs showed no significant effect at 12 hours or 24 hours (FIG 7B).
091 The effect of the drugs was also assessed on the clonogenic property of the cells at both the concentrations. Contrary to the migratory assay, the cells showed inhibition of colony formation at both the concentrations. Treatment with high concentrations showed increased effect in the early stage cultures with 97.5% reduction in clone formation in combination arm (N=1 clone, p<0.0001) as compared to single agent curcumin (75% reduction; N=10, p<0.001). Metformin showed no effect on clone formation (N=39), when compared to untreated control (N=39). Decrease in concentration of the drug was less effective in inhibiting the clone formation with reduction ranging from 25% (N=29 clone, p<0.01) in combination arm, to 18% in curcumin arm (N=32). Metformin arm showed no effect on the colony formation (FIG 8, left panel).
092 In case of the cultures from severe dysplastic tissues, treatment with high concentrations showed 100% reduction of colonies in combination arm (N=0, p<0.0001), as compared to 75% (N=13, p<0.001) in curcumin and 68% (N=16, p<0.001) in metformin arm. In comparison, decreased drug concentrations showed reduction in colony forming capacity in combination (53%; N=24, p<0.001) and curcumin (57%; N=22, p<0.01) arms, with only 14% reduction (N=44) in metformin arm. However, there was no significant synergy observed between the drugs at this low concentrations (FIG 8, right panel).
093 Methods of preparing the combination or formulation
094 Curcumin drug was prepared in Tween 80 and then given to the animals in drinking water. Metformin was prepared in drinking water itself. Curcumin and metformin were given in the concentrations 1g/70g body weight/week and 5mg/ml/week respectively.
095 The possible uses of this invention include but not limited to,
096 Utilization of the combination of drugs for chemoprevention of cancer.
097 Prevention for second primary tumors.
098 Use as an adjunct to cancer treatment.
099 Treatment of oral potentially malignant lesions.
0100 Merely for illustration, only representative number/type of graph, chart, block and sub- block diagrams were shown. Many environments often contain many more block and sub- block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.
0101 According to a non limiting exemplary aspect of the present invention, use of curcumin and metformin in chemoprevention of proliferative and/or inflammatory disorders including cancers, in particular head and neck cancers.
0102 The use of this said combination of drugs in a pre-clinical carcinogenesis model has provided benefit at the clinical and the pathological level toward chemoprevention of the oral premalignant lesions.
0103 Yet another aspect of the present invention, the present invention describes the effective the combination of drugs in the 4NQO induced animal model, resulted in a lower number of lesions, reduced tumor volume and higher survival benefit as compared to the animals treated with the drugs individually.
0104 According to a non limiting exemplary aspect of the present invention, this said combination of drugs resulted in pathological benefit, indicating synergistic effect, and that resulted in a higher response to cancer chemoprevention.
0105 According to a non limiting exemplary aspect, the present invention also relates to components useful in prevention and treatment of cancer, as well as the compositions, methods, processes, and uses thereof.
0106 According to a non limiting exemplary aspect of the present invention, the pharmaceutical formulations of the said invention may be in form of solid, semi-solid, or liquid such as, e.g. tablets, suspension, or the like.
0107 While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.
0108 Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
0109 It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.
0110 The said invention has been sent for publication. Gangotri Siddappa, Safeena Kulsum, Ravindra D R, Vinay V Kumar, Nalini Raju, Sudheendra H V, Nisheena R, Sumsum P Sunny, Tina Jacob, Binu T Kuruvilla, Merina Benny, Benny Antony, Mukund Seshadri, Padma L, Wesley Hicks Jr, Amritha Suresh, Moni A Kuriakose; Curcumin and metformin mediated chemoprevention of oral cancer is associated with inhibition of cancer stem cells; Molecular Carcinogenesis; June 2017 (Accepted).
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,CLAIMS:Claims
I/We Claim,
1. A method of treating cancer by administering, in combination, at least 2-3 agents that together target at least two cancer-associated pathways selected from the group consisting of angiogenesis, apoptosis, immune surveillance, metabolism and energetics, and combinations thereof.
2. The method of claim 1, wherein none of the at least 2-3 agents is a chemotherapeutic agent utilized at its maximum tolerated dose, wherein the agents are selected such that each of the at two three pathways is targeted by at least two agents.
3. The method of claim 1, wherein at least one of the 2-3 agent is a chemotherapeutic agent utilized in chemoprevention of cancers, wherein the cancer is head or neck or oral cancer.
4. A method of treating cancer by administering to a subject suffering from cancer at least 2-3 agents selected from the group consisting of curcumin, metformin and equivalents thereof, wherein each of the agents are administered in doses well below the maximum tolerated dose for the agent.
5. The method of claim 4, wherein equivalents of Curcumin are other agents that inhibit the signal transduction pathway of PI3K/Akt, MAPK, and NF-?B activation, and the Sonic Hedgehog (Shh) signalling, wherein equivalents of Curcumin are other agents that down-regulate epidermal growth factor receptors (EGFR and erbB2), Insulin-like growth factor type-1 receptor (IFG-1R), sonic hedgehog (SHH)/GLIs) and Wnt/b-catenin and PARP, IKK, EGFR, JNK, MAPK, 5-LOX, STATs, IL-6, COX-2, and MMPs.
6. The method of claim 4, wherein equivalents of Metformin are other agents that affect cellular metabolism through oxidative stress, anaerobic glycolysis, Warburg effect and COX-2 activation, wherein equivalents of Metformin are other agents that suppress tumor angiogenesis by inhibiting HIF-la stabilization under hypoxia and VEGF mRNA expression, wherein equivalents of Metformin are other agents that suppress tumor angiogenesis by inhibiting HIF-la stabilization under hypoxia and VEGF mRNA expression.
7. The method of claim 4, wherein equivalents of Metformin are other agents that inhibit cell proliferation and induce apoptosis in cancer cells in vitro by simultaneously suppressing the COX-2/PGE2, p300/NF-?B, and PI3K/Akt/signaling and activating the Apaf-1/caspase-dependent apoptotic pathway.
8. The method of claim 1, wherein at least some of the at least 2-3 agents are administered according to a continuous regimen, which method comprises continuing administration after one or more symptoms or features of the cancer have resolved, wherein the at least 2-3 agents are administered as an adjunct to agents used for chemoprevention.
9. The method of claim 4, wherein the subject is suffering from cancer, wherein the cancer is Stage premalignant or early stage for chemoprevention of dysplastic progression and II, Stage III, or Stage IV cancers for second primary prevention, or late-stage cancer, or metastasized cancer, or solid tumors, or cancer that has relapsed
after treatment with another therapeutic modality, or tumors of epithelial origin, or cancer selected from the group consisting of epithelial cell cancers, sarcomas, and blood cancers (leukemias or lymphomas), or adenocarcinoma, or cancer selected from the group consisting of breast cancer, lung cancer, ovarian cancer, pancreatic cancer, etc
10. A composition comprising two or more compounds, wherein each of said two compounds is capable of regulating a specific metabolic pathway or intracellular signalling pathway or intercellular signalling pathway implicated in the progression of cancer in an individual suffering from cancer, and wherein at least two such pathways are addressed.