DESC:F O R M 2

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

1. TITLE OF THE INVENTION: CXCR4 INHIBITOR IN REVERSING RESISTANCE TO CHEMOPREVENTION AND CHEMOTHERAPY

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 relates to compounds, compositions, methods to prevent, inhibit and/or treat cancer, and methods to promote Cancer Stem Cell (CSC) targeting. In particular, the invention relates to, CXCR4 inhibitor AMD3100/AMD070 in reversing resistance to chemoprevention and chemotherapy by evaluating the inhibitors, and methods for using them in inventive combinations with existing chemopreventive/chemotherapeutic drugs.
.
BACKGROUND OF THE INVENTION
[002] Resistance to chemoprevention and chemotherapy are the major issues in head and neck cancers. Early detection and efficient chemoprevention in oral cancer necessitate identification of molecular markers that can accurately detect susceptible lesions and enable efficient chemoprevention strategies. The concept of a two-step process of cancer development in the oral mucosa, i.e., through the initial development of precursor lesions such as leukoplakia and erythroplakia, is well-established [1, 2].
[003] In addition, multiple challenges are still associated with the detection of lesions susceptible to malignant progression.
[004] At present, chemoprevention, which is the next strategy that can improve survival in addition to early detection, is challenging due to the relapse into progression once the drug is removed [3]. This is suggested to be due to the presence of resistant cells that escape the effects of the drug and thereby leading to a relapse.
[005] Furthermore, resistance to chemotherapy is also a major challenge with patients showing relapse after an initial response to the drugs. Addressing these issues of resistance is a major challenge in head and neck cancer.
[006] In addition, the existing cellular complexity of solid tumors that includes cells with varied tumorigenic and resistance potentials and the inability of drugs to target these different types of cells is considered one of prime reasons underlying these properties. The CSCs (Cancer Stem Cells) have been previously implicated in resistance properties.
[007] In addition, evidences have also proven that CSC niche plays a major role in determining CSC characters of tumorigenicity and drug resistance [4, 5]; emphasizing that a detailed investigation of the CSCs along with its surrounding niche might be critical towards understanding the underlying processes of carcinogenesis/drug resistance [6].
[008] Multiple pathways have been implicated in the epithelial cell-fibroblast cross talk, Notch1, TGFß and SDF-1/CXCR4 [C-X-C chemokine receptor type 4(CXCR4 is normally the receptor for the chemokine SDF-1)] being some of them [7-10]. Among these pathways, the SDF-1/CXCR4 axis has been implicated in the CSC-fibroblast cross talk in head and neck cancer [11, 12].
[009] In addition, SDF-1 has also been associated with poor prognosis in patients diagnosed with head and neck cancer [13-15].
[010] Chemoprevention is probably the only approach that can prevent progression of the oral lesions and thereby improve survival rates of the disease. Several agents including Curcumin, Metformin, retinols, Beta carotene have been investigated in in vitro and in vivo studies as well as in patient clinical trials for their efficacy in preventing progression of oral premalignant lesions [16-25].
[011] At present, a major challenge associated with these studies has been a relapse on removal of the drug; the relapse rate of up to 60% has been documented in the case of trials with beta carotene [26].
[012] In addition, major challenges in currently available chemopreventive strategies include lack of efficacy, toxicities associated with the interventions and relapses following cessation of therapy [27, 28].
[013] The closest known treatment regimen currently used for chemoprevention are,
[014] Individual use of curcumin in chemoprevention: Curcumin has been evaluated for its anti-cancer effects in many carcinomas including melanoma, head and neck, breast, colon, pancreatic, prostate and ovarian cancers [29-31]. In oral cancer, curcumin is known to inhibit tobacco-induced NFkB and COX-2 in oral premalignant and cancer cells [32]. A recent chemoprevention trial in colorectal cancer, patients also showed a decrease in the aberrant crypt foci at high doses of the drug [29].
[015] Metformin in chemoprevention: 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 [33]. Metformin has also been shown to reduce the incidence of premalignant lesions in oral carcinogenesis animal models in a PIK3CA dependent manner [34, 35].
[016] Vitamin A and Beta Carotene: In a study carried out with vitamin A and beta carotene, the drugs lead to an initial response, however, relapse was observed in up to 50-60% of the patients [36].
[017] Retinol Study: 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 [37].
[018] Other chemopreventive drugs in Oral cancer: 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 [38]. 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 [39].
[019] CXCR4 inhibitors in other cancers: A study on pancreatic cancer cells showed an effective block in metastatic potential of CXCR4-expressing pancreatic cells after systematic utilization of CXCR4 inhibitor AMD3100 (AnorMeD, where the two cyclam rings are tethered by an aromatic bridge) [40]. Use of AMD3100 in another study showed growth inhibition and progression of intracranial medulloblastoma and glioblastoma along with increased cellular apoptosis and decreased excessive proliferation of tumor cells in xenograft model [41]. Treatment of AMD3100 for mice bearing intracranial glioblastoma or medulloblastoma showed a significantly decreased tumor burden in mice treated with AMD3100 [42]. Recent studies also show reduced tumor growth in nude mice inoculated with human gastric cancer cells and treated with AMD3100 [43].
[020] At present, there is a need for exploring the molecular basis of Cancer Stem Cells, their interactions with the surrounding niche in carcinogenic progression and in the processes of chemoprevention and chemotherapy.
[021] In addition, there is also a need for development of novel technology for early detection/prognosis, methods to prevent, inhibit and/or treat cancer.
[022] In summary, there is an urgent need in the art to develop drugs in reversing resistance to chemoprevention and chemotherapy.

SUMMARY OF THE INVENTION

[023] According to an exemplary aspect, the present invention relates to compositions, methods to prevent, inhibit and/or treat cancer, and methods to promote Cancer Stem Cell (CSC). In particular, the invention relates to, CXCR4 inhibitor AMD in reversing resistance to chemoprevention and chemotherapy by evaluating the CXCR4 inhibitors, and methods for using the inventive combination, with existing chemopreventive and chemotherapeutic drugs.
[024] More particularly, the invention analyses the correlation between Cancer Stem Cells (CSCs) behaviour and acquired drug resistance in in vitro models. The invention also provides corresponding methods of medical treatment, wherein a therapeutic dose of a CXCR4 antagonist is administered in a pharmacologically acceptable formulation. In summary, the invention provides therapeutic compositions comprising CXCR4 antagonists and pharmacologically acceptable excipients or carriers as adjunct to existing drugs.
[025] More particularly, the invention relates to reversing resistance to chemoprevention and chemotherapy using the CXCR4 inhibitors. A combination of inhibitors with chemo-preventive properties under the effect of the fibroblast niche was tested in 4NQO-derived mice primary cells and human dysplastic cells to evaluate the effect on chemoprevention
[026] According to a further exemplary aspect, the present invention deals with CXCR4 inhibition in drug resistance. Cancer Stem Cells (CSCs) are increasingly being implicated in chemotherapy resistance, inventors have investigated the correlation between CSC behavior and acquired drug resistance in in vitro cell line models. Cell lines resistant to Cisplatin (Cal-27 CisR, Hep-2 CisR) and 5FU (Cal-27 5FUR) with high Resistance Indices (RI) were generated (RI =3) by short-term treatment of Head and Neck Squamous Cell Carcinoma (HNSCC) cell lines with chemotherapeutic drugs (Cisplatin, Docetaxel, 5FU), using a dose-incremental strategy. The effect of fibroblast niche and CXCR4 inhibition is tested in these cell lines.
[027] Yet another exemplary aspect of the present invention, these inhibitors showed synergistic effect of inhibiting the CSC-niche pathways along with the pathways involved in the specific drug activity.
[028] Thus, in one particular embodiment, the study describes the use of these inhibitors in combination to the current chemopreventve drugs in oral cancer. In case of chemotherapy with current platinum/taxol drugs, as mentioned showed an initial response, however high recurrence rates were observed. Inventors suggest that the application of the CSC–Niche inhibtors adjunct to chemotherapeutic/chemopreventive drugs can improve the efficacy of the conventional chemotherapy/chemopreventive drugs.
[029] According to a further exemplary aspect, the present invention deals with a method of treating a human or animal suffering from cancer, wherein the method comprises administering to the patient a CXCR4 antagonist and a chemopreventive/chemotheraputive drug(s), wherein the chemopreventive/chemotheraputive drug(s) acts synergistically with the CXCR4 antagonist, inhibiting the CSC-niche pathways and the pathways involved in chemopreventive/chemotheraputive drug(s), wherein the synergistic combinations are in an amount sufficient to reduce the (a) rate of onset of, and /or (b) reduce the progress of, and/or (c) reverse symptoms of cancer, and/or (d) reversing resistance to chemoprevention and chemotherapy.
[030] According to a further exemplary aspect, the present invention deals CXCR4 antagonist, wherein the CXCR4 antagonist is selected from: (i) AMD3100 and/or AMD070; and /or (ii) an anti-CXCR4 antibody.
[031] According to the embodiments of the invention, the present invention deals a method of treating a human or animal suffering from cancer, wherein the method comprises administering to the patient a CXCR4 inhibitors/antagonist in an amount sufficient to reduce the (a) rate of onset of, and /or (b) reduce the progress of, and/or (c) reverse symptoms of cancer, and/or (d) reversing resistance to chemoprevention and chemotherapy.
[032] According to a further exemplary aspect, the present invention deals a method, wherein the CXCR4 inhibition reduces CSC (Cancer Stem Cell) markers; reduces migration, self-renewal, tumorigenic potential of CSC in vitro; and reversing resistance imparting an increased sensitivity to chemopreventive/chemotheraputive drug(s).
[033] According to a further exemplary aspect, the present invention deals a method, wherein inhibition of one or more cancer drugs or a fragment or a complex thereof or a functional equivalent thereof, prevents, inhibits or ameliorates a pathology or a symptomology of the condition, the method including administration of as therapeutically effective amount of a compound.
[034] According to a further exemplary aspect, the present invention deals a method, wherein the method may increase the sensitivity of the cancer cells to the host immune responses and enhanced stemness induced by CAF-conditioned medium in in vitro human and mice models; reduces immune suppression in the tumor; cancer cell immune recognition is increased within the tumor; and cancer cell growth is inhibited or reduced.
[035] According to a further exemplary aspect, the present invention deals the CXCL4 inhibitors/antagonist, wherein the inhibitors can be in various forms such as combination, alone, with excipients, diluents, carriers, humectants, capsules, tablets, injectables (intravenous, muscular, hypodermal), oral, topical, in combination with polynucleotides, other biologicals, for targeted delivery, powders, suspensions, gels, hydrogels, logenges, in combination with cryo, radiation therapies, binders, for curing, palliative care in humans, animals and such other organisms wherein the said compositions can be employed to relieve them of the various ailments that can be controlled by modulating the CXCR4 and/or chemopreventive/chemotheraputive drug(s) pathways.
[036] In yet another embodiment, the possible uses of this invention include:
[037] Utilization of inhibitor/s to improve the efficacy of chemopreventive drugs
[038] Treatment of Oral potentially malignant lesions for long term chemoprevention
[039] Use as an adjunct to cancer treatment towards reversing resistance.
[040] In yet another embodiment, the best way to practice the invention is to prescribe the inhibitor/s along with the present chemoprevention/chemotherapy drugs. These inhibitor/s may be available as oral version which may be used for treatment.
[041] According to a further exemplary aspect of the present invention, AMD3100 was initially dissolved in water whereas AMD070 was dissolved in DMSO. The different concentrations used in the study were prepared freshly in complete medium or conditioned medium and added to the cells at specified time points.
[042] In yet another embodiment, these inhibitor/s along with present chemopreventive and chemotherapeutic drugs in cancer are tested in vitro
[043] In summary, the present invention deals with compositions, methods to prevent, inhibit progression of cancer and/or treatment of cancer, and methods to promote cancer stem cell targeting. In particular, the invention relates to, CXCR4 inhibitor in reversing resistance to chemoprevention and chemotherapy, which was assessed by the cyotoxicity assay. The invention also relates to methods for using the inventive combination. The invention analyses the correlation between Cancer Stem Cells (CSCs) behavior and acquired drug resistance in in vitro cell line models.
[044] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[045] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.
[046] FIG. 1(A) illustrates the cytotoxicity assay of Curcumin treated cells showing increased cell death with higher concentrations of drug.
[047] FIG. 1(B) illustrates the expression profiling of Curcumin treated cells showing down regulation of NF-?B and CSC markers at high concentrations of Curcumin drug.
[048] FIG. 2(A) illustrates transcriptomic profiling of CSC markers in (a) HaCaT and (b) DOK cells.
[049] FIG. 2(B) illustrates (a) representative images showing migration differences at each time point along with migratory potential of (b) HaCaT and (c) DOK cells.
[050] FIG. 2(C) illustrates transcriptomic profiling of CSC markers in DysMSCTR16 cells.
[051] FIG. 2(D) illustrates (a) representative images showing migration differences at each time point along with migratory potential of (b) DysMSCTR16 cells. Statistical significance is indicated (*p<0.05, **p<0.005, ***p<0.0005). All the migration images were taken at a magnification of 40X and the scale bars represented.
[052] FIG. 3(A) illustrates transcriptomic profiling of CXCR4 in (a) human cell lines and (b) mice primary cells along with protein profiling of CXCR4 in (c) human cell lines by western blot.
[053] FIG. 3(B) illustrates expression profiling of CSC markers under the effect of exogenous SDF1 treatment (0-50ng/ml) in (a) HaCaT and (b) DOK cells. Statistical significance is indicated (***p<0.0005).
[054] FIG. 3(C) illustrates cytotoxicity assay under the effect of (cancer associated fibroblast) CAF-conditioned medium and different concentrations of curcumin in (a) HaCaT (0-500µM of Curcumin) and (b) DOK cells (0-50µM of curcumin).
[055] FIG. 3(D) illustrates cytotoxicity assay under the effect of CAF-conditioned medium and different concentrations of curcumin (0-150 µM of curcumin) in DysMSCTR16 cells.
[056] FIG. 4(A) illustrates treatment schedule for CXCR4 inhibitor/s
[057] FIG. 4(B) illustrates expression profiling of CSC markers under the effect of CAF-conditioned medium in the presence/absence of CXCR4 inhibitor/s (AMD3100 and AMD070) in (a) HaCaT and (b) DOK cells. The inhibitors were used in three concentrations of 10, 20 and 30 µM.
[058] FIG. 4(C) illustrates expression profiling of CSC markers under the effect of CAF-conditioned medium in the presence/absence of CXCR4 inhibitor/s (AMD3100 and AMD070) in DysMSCTR16 cells. Statistical significance is indicated (*p<0.05, **p<0.005, ***p<0.0005).
[059] FIG. 5(A) illustrates (a) representative images showing migration differences at each time point under the effect of CAF-conditioned medium in the presence/absence of CXCR4 inhibitor/s (AMD3100 and AMD070) in HaCaT cells along with the (b) graph representing the statistically significant difference in migratory potential in HaCaT cells.
[060] FIG. 5(B) (a) illustrates representative images showing migration differences at each time point under the effect of CAF-conditioned medium in the presence/absence of CXCR4 inhibitor/s (AMD3100 and AMD070) in DOK cells along with the (b) graph representing the the statistically significant difference in migratory potential in DOK cells. Statistical significance is indicated (***p<0.0005). All the migration images were taken at a magnification of 40X and the scale bars represented.
[061] FIG. 6 illustrates (a) representative images showing migration differences at each time point under the effect of CAF-conditioned medium in the presence/absence of CXCR4 inhibitor/s (AMD3100 and AMD070) in DysMSCTR16 cells along with the (b) graph representing the the statistically significant difference in migratory potential in DysMSCTR16 cells. Statistical significance is indicated (*p<0.05, **p<0.005). All the migration images were taken at a magnification of 40X and the scale bars represented.
[062] FIG. 7(A) illustrates cytotoxicity assay in HaCaT cells showing cell viability in the presence of CXCR4 inhibitor/s, (a) AMD3100 and (b) AMD070), and under the effect of CAF-conditioned medium and Curcumin. The cells show increased viability in presence of conditioned media which is abrogated by CXCR4 inhibition.
[063] FIG. 7(B) illustrates cytotoxicity assay in DOK cells showing cell viability in the presence of CXCR4 inhibitor/s, (a) AMD3100 and (b) AMD070, and under the effect of CAF-conditioned medium and Curcumin. The cells show increased viability in presence of conditioned media which is abrogated by CXCR4 inhibition
[064] FIG. 7(C) illustrates cytotoxicity assay in DysMSCTR16 cells showing cell viability in the presence of CXCR4 inhibitor/s, (a) AMD3100 and (b) AMD070, and under the effect of CAF-conditioned medium and Curcumin. The cells show increased viability in presence of conditioned media which is abrogated by CXCR4 inhibition.
[065] FIG. 8 illustrates the CXCR4 expression status in Cal-27 and in its Cisplatin, Docetaxol and 5-FU resistant sub-lines by expression profiling (A) and by FACS analysis (B). Increased expression of CSC markers and MDR genes under the effect of exogenous rSDF-1 alpha induction on Cal-27 (C) and Cisplatin resistant sub-line (D) is also shown.
[066] FIG. 9A illustrates effect of cancer associated fibroblast (CAF) conditioned media in Cal-27 cells. CAF induced expression of CXCR4 in Cal-27 (Cal-27CM) by FACS
[067] FIG 9B showed increased expression of CSCs and MDR genes in Cal-27 cell lines by qPCR under the effect of CAF-Conditioned media. Statistical significance is indicated (*=p>0.05, 0.005, 0.0005).
[068] FIG 9C showed increased expression of CSCs by FACS profiling. Statistical significance is indicated (*=p>0.05, 0.005, 0.0005).
[069] FIG 9D The capacity of colony formation increased under effect of CAF-conditioned media as shown in the representative images (a) and graphical representation indicating statistical significance. Statistical significance is indicated (*=p>0.05, 0.005, 0.0005).
[070] FIG 9E indicated a similar result obtained with spheroid formation (E) with representative microscopic images (a) and graphical representation (b). Statistical significance is indicated (*=p>0.05, 0.005, 0.0005).
[071] FIG 9F showed a statistically significant increase in migration potential increase in CM-treated cells. Statistical significance is indicated (*=p>0.05, 0.005, 0.0005).
[072] FIG 9G showed increased resistance to cisplatin (G) was also observed under the effect of CM. Statistical significance is indicated (*=p>0.05, 0.005, 0.0005).
[073] FIG. 10A illustrated effect of AMD3100-dependent inhibition of CXCR4 receptor as seen in reduced surface expression of CXCR4. Data were statistically significant (*=p>0.05, 0.005, 0.0005).
[074] FIG 10B showed reduced migration capacity in Cal-27 cells when treated with AMD3100. Data were statistically significant (*=p>0.05, 0.005, 0.0005).
[075] FIG 10C showed reduced migration in Cal-27CM (C) when treated with AMD3100. Data were statistically significant (*=p>0.05, 0.005, 0.0005)
[076] FIG 10D indicates reduced spheroid formation capacity when treated with AMD3100. Data were statistically significant (*=p>0.05, 0.005, 0.0005).
[077] FIG 10E shows the increased sensitivity to Cisplatin observed in Cal-27CM-AMD3100 cells as compared to parental counterpart. Data were statistically significant (*=p>0.05, 0.005, 0.0005).
[078] 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
[079] 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.
[080] 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.
[081] 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.
[082] 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.
[083] 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.
[084] Example embodiments of the present invention are described with reference to the accompanying figures.
[085] 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.
[086] Definitions
[087] The following terms are used as defined below throughout this application, unless otherwise indicated.
[088] The terms “tumour” or “tumour tissue” refer to an abnormal mass of tissue which results from uncontrolled cell division. A tumour or tumour tissue comprises “tumour cells” which are neoplastic cells with anomalous growth properties and no functional bodily function. Tumours, tumour cells and tumour tissue can be benign or malignant.
[089] The phrase "differentially present" refers to differences in the quantity of the marker present in a sample taken from patients as compared to a control subject. A biomarker can be differentially present in terms of frequency, quantity or both.
[090] "Diagnostic" means identifying a pathologic condition.
[091] The terms "detection", "detecting" and the like, may be used in the context of detecting markers or biomarkers.
[092] A "test amount" of a marker refers to an amount of a marker present in a sample being tested. A test amount can be either in absolute amount (e.g., µg/ml) or a relative amount (e.g., relative intensity of signals).
[093] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. "Polypeptide," "peptide" and "protein” can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
[094] "Detectable moiety" or a "label" refers to spectroscopic, photochemical, biochemical, immunochemical, or chemical means of detection of a composition. For example, labels may include 32P, 35S, fluorescent dyes, biotin-streptavidin, dioxigenin, haptens, electron-dense reagents and enzymes. The detectable moiety generates a measurable signal that can quantify the amount of bound detectable moiety in a sample. Quantitation of the signal is done by scintillation counting, densitometry, or flow cytometry.
[095] "Antibody” refers to a polypeptide ligand encoded by an immunoglobulin gene(s), which specifically binds and recognizes an epitope.
[096] The terms "subject", "patient" or "individual" generally refer to a human or mammals. "Sample" refers to a polynucleotide, antibodies fragments, polypeptides, peptides, genomic DNA, RNA, or cDNA, polypeptides, a cell, a tissue, and derivatives thereof may comprise a bodily fluid or a soluble cell preparation, or culture media, a chromosome, an organelle, or membrane isolated or extracted from a cell.
[097] Inventors addressed the needs for reversing resistance to chemoprevention and chemotherapy by evaluating the CXCR4 inhibitors. A combination of inhibitorswith chemo-preventive properties, along with existing chemopreventive drugswas tested initially in 4NQO-derived mice dysplastic primary cells under the effect of the fibroblast niche. Similar experiments were carried out in drug resistant cells for evaluating effect on chemotherapy. The details of the experiments performed to assess the activity of the chemopreventive/chemotherapy drugs in the presence of inhibitors and the niche are as given below:
[098] EXPERIMENTAL METHODS:
[099] Treatment of cells with Curcumin alone: The16th week cells derived from 4NQO-induced mice with severe dysplastic lesions, were initially treated with different concentration of Curcumin (50µM, 100µM and 150µM) to assess the viability of the cells in the presence of the drug through cytotoxicity assay (MTT) and then expression profiling of CSC markers were carried out in the same cells. Cytotoxicity assay revealed higher cell death (~56%) with increased concentration of Curcumin (150µM) (FIG 1A). Expression profiling of these cells showed down regulation of NF-?B and CSC markers (CD44 and Notch1) with high drug concentrations (FIG 1B).
[0100] Enhanced stemness under the effect of CAF-conditioned medium in in vitro human and mice models: Transcriptomic profiling of CSC markers indicated that HaCaT cells showed up regulation of CD133 (>5 fold) and NOTCH1 (>10 fold) at higher 1:5 (5 parts of conditioned medium) dilution of the conditioned media, along with up regulation of CD44 (1.2 fold) (FIG 2A(a)) DOK cells showed >1.5 fold up regulation in both CD44 and Notch1 at 1:5 dilution along with up regulation of SOX-2 (24 fold) as compared to untreated controls (FIG 2A(b)). All further co-culture experiments were performed with 1:5 concentration of the conditioned medium in both HaCaT and DOK cells. Evaluation of the migratory capacity (FIG 2B (a))under the effect of the conditioned media showed that at 6th hour, HaCaT cells showed 52% wound closure, while DOK showed 74% wound closure as compared to untreated cells (HaCaT: 37%; DOK: 49%). At 12th hour, treated HaCaT (p=0.0001) and DOK (p=0.0102) cells showed complete wound closure as compared to their control cells (HaCaT: 49% and DOK: 87%) (FIG 2B (b) and (c)). A comparison further indicated an induction of migratory potential in HaCaT that was similar to DOK, emphasizing that CAFs can induce CSC properties in non-neoplastic cell lines.
[0101] In the mice cell lines, profiling of the CSC markers indicated a high expression of CD44 (4.25 fold), CD133 (1.6 fold) and SOX-2 (1.3 fold) in DysMSCTR16 cells treated with previously characterized (unpublished data) FibroMSCTR12 (mCAFs)-conditioned medium as compared to untreated control (FIG 2C). The mice primary cells when assessed for their migratory potential under the effect of conditioned medium (FIG 2D (a)) indicated that at 6th hour, DysMSCTR6 showed 80% (p=0.0003) wound closure while, DysMSCTR14 and DysMSCTR16 showed 89% (p=0.0023) and 92% (p=0.0105) wound closure respectively as compared to their respective control cells (DysMSCTR6: 12%, DysMSCTR14: 31% and DysMSCTR16: 48%). At 12th hour, all the conditioned media treated cells showed 100% wound closure that was significantly different from the control cells (DysMSCTR6: 30%, p=0.0021; DysMSCTR14: 53%, p=0.0287; DysMSCTR16: 70%, p=0.0056) Representative results of 16th week are shown (FIG 2D (b).
[0102] SDF1 and CXCR4 profiling in the tumor cells/CAF conditioned media: Expression profiling by qPCR indicated an increase in overall expression of CXCR4 in the DOK cells (~11 fold) as compared to HaCaT (FIG 3A (a)) and in mice cell lines, DysMSCTR16 (~8 fold) showed increased expression of CXCR4 as compared to 0th week mice tissue sample (FIG 3A (b)). The protein profiling of CXCR4 using western blot technique also showed high total CXCR4 protein in conditioned medium treated samples (FIG 3A (c)). The presence of SDF1, when quantified by ELISA, showed that the conditioned medium from hCAFs (after 48-hour culture) had 40pg/ml, while mCAFs showed higher concentration of SDF-1 (430pg/ml), the levels remained unchanged after a 48-hour storage period.
[0103] Further, the human cells when treated exogenously with high concentrations (10, 25, 50ng/ml) of recombinant hSDF1 showed an increased CSC profile similar to the effect of the conditioned media. In HaCaT cells, ALDH1A1 (4.5-7fold), CD133 (33-46 fold) SOX-2 (7-19 fold) showed upregulation (FIG 3B (a)), while DOK cells showed high expression of ALDH1A1 (10-86 fold), CD133 (1.3-47 fold), SOX-2 (24-230 fold), NOTCH1 (>1000 fold) and CD44 (~2 fold) (FIG 3B (b)).
[0104] CAF-conditioned medium increases resistance to Curcumin: Cytotoxic assays carried out to evaluate the CAF-conditioned medium effect on Curcumin sensitivity indicated that HaCaT cells, when cultured in conditioned media (1:5) showed a 20% increase in cell viability; the IC50 increasing by 1.8fold (365µM vs 648µM) (FIG 3C (a)). In DOK cells, comparatively a more sensitive cell line, a 30-50% increase in cell viability (FIG 3C (b)) was observed with the IC50 increasing by 3.3fold (33µM vs 111µM). A similar assessment of the effect of the conditioned media in DysMSCTR16 cells showed 50% increase in cell viability and 3.26 fold increase in the IC50 (150µM vs 489µM), albeit at higher Curcumin concentrations (100-150µM) (FIG 3D).
[0105] Inhibition of SDF1-CXCR4 pathway leads to down regulation of CSC properties: The schematic treatment schedule of the CXCR4 inhibitor/s along with conditioned medium and curcumin is represented in FIG 4A. In HaCaT cells, the enrichment of CSC profile observed under the effect of the conditioned media was abrogated by the CXCR4 inhibitors; the qPCR based expression profiling of HaCaT cells showed down regulation of ALDH1A1, CD44, CD133, NOTCH1 in the presence of AMD3100 (p<0.05) (FIG 4B(a) Profiling of DOK cells also showed a similar effect with CD44, CD133 and SOX-2 being downregulated on addition of both the inhibitors (p<0.05) (FIG 4B (b)). In DysMSCTR16 cells, which showed a CAF-media induced upregulation of all the markers, treatment with both CXCR4 inhibitors led to the abrogation of this effect. ALDH1A1, CD44, CD133, NOTCH-1 and SOX-2 were downregulated (p<0.05); CD44 showing maximum down regulation (FIG 4C).
[0106] Assessment of migratory potential in HaCaT cells (FIG 5A (a) indicated that the effect of the drug (AMD3100 or AMD070; 10-30ug) could be observed at 6th hour itself, wherein the percentage wound closure was reduced by ~20% in the presence of the drug (52% vs 30%), comparable to the control cells (37%). At 12th hour, the difference was significant (p=0.0001), wherein the drugs inhibited the wound closure by ~50% compared to the conditioned media treated cells (100% vs 41-48%). This was also comparable to the closure observed in the control cells (49%) (FIG 5A (b)). An assessment in DOK cells showed that at 6th hour, the wound closure was inhibited by ~30% (75% vs 33-48%) under the effect of either of the inhibitors. At 12th hour, 100% (p=0.0102) wound closure was observed in conditioned medium alone, while it was reduced to 86-89% at different concentrations of CXCR4 inhibitors. At both the time points, the effect of the inhibitors reduced the migratory potential of the cells to that of the controls (6th hour: 49%; 12th hour: 87%) (FIG 5B (a) & (b)).
[0107] In the mice DysMSCTR16 cells, at 6th hour, wound closure was reduced by 40% (92% vs 47-48%) on treatment with different concentrations of CXCR4 inhibitors, comparable to the control cells (48%) (p<0.05). At 12th hour, wherein the conditioned medium treated cells showed complete wound closure, only ~70% wound closure was observed at different concentrations of CXCR4 inhibitors (p=0.005), which was similar to the control cells (70%) (FIG 6 (a) & (b)).
[0108] Inhibition of SDF1-CXCR4 pathway sensitizes curcumin drug treatment: The inhibition of SDF1-CXCR4 pathway on resistance to curcumin (IC50) was evaluated by cytotoxicity assay using CXCR4 inhibitors, AMD3100 and AMD070. In HaCaT cells, the resistance-inducing effect of the CAF-conditioned media to Curcumin (cell viability 93%) was abrogated under the effect of the inhibitors (cell viability: ~40-50%). Comparison of cell viability indicated that the cells were rendered more sensitive to Curcumin in the presence of AMD3100 (FIG 7A (a)) or AMD070 (FIG 7A (b)) as opposed to Curcumin alone (40-50% vs 67%). There was no difference between the two inhibitors or between the different concentrations (FIG 7A). In DOK cells, the increased resistance (viability: ~73%) to Curcumin observed under the effect of CAF-conditioned medium, was significantly reversed on treatment with the CXCR4 inhibitors (viability: ~42-50%) (FIG 7B (a) & (b)). This result was replicated in the mice cell line DysMSCTR16, wherein reversal of resistance to Curcumin was documented by the decrease in cell viability when cells in CAF-conditioned media, were treated with Curcumin and AMD inhibitors (43-53%) as compared to Curcumin alone (~73%) (FIG 7C (a) & (b)).
[0109] CXCR4 inhibition in Drug resistance
[0110] Chemoresistance leading to disease relapse is one of the major challenges to improve outcome in head and neck cancers. Cancer Stem Cells (CSCs) are increasingly being implicated in chemotherapy resistance, our study investigated the correlation between CSC behavior and acquired drug resistance in in vitro cell line models. Cell lines resistant to Cisplatin (Cal-27 CisR, Hep-2 CisR) and 5FU (Cal-27 5FUR) with high Resistance Indices (RI) were generated (RI =3) by short-term treatment of Head and Neck Squamous Cell Carcinoma (HNSCC) cell lines with chemotherapeutic drugs (Cisplatin, Docetaxel, 5FU), using a dose-incremental strategy. The cell lines (Cal-27 DoxR, Hep-2 DoxR, Hep-2 5FUR) showed low RI, nevertheless had a high cross resistance to Cisplatin/5FU (p<0.05). Cal-27 CisR and DoxR sublines showed enrichment of CD44+ cells (12-14%, p<0.05), while CisR/5FUR showed ALDH1A1+ cell enrichment (4-6%, p<0.05). Increased expression of stem cell markers (CD44, CD133, NOTCH1, ALDH1A1, OCT4, SOX2) in these cell lines, correlated with enhanced spheroid/colony formation, migratory potential and increased in vivo tumor burden (p<0.05). Inhibition of ALDH1A1 in Cal-27 CisR led to down regulation of the CSC markers, reduction in migratory, self-renewal and tumorigenic potential (p<0.05) accompanied by an induction of sensitivity to Cisplatin (p<0.05). As a next step to this study, inventors looked at the effect of CSC-Niche cross talk on CSC properties and resistance. Inventors exposed Cal-27 and Cisplatin resistant cell lines to exogenous SDF-1 and evaluated the effect on CSC properties.
[0111] CXCR4 profiling and SDF-1 alpha activation of CXCR4 pathway induced up-regulation of CSC markers: CXCR4 profiling by qPCR indicated an increased expression of overall CXCR4 (FIG 8A) in the resistant cell lines as compared to the parental, while FACS indicated a reduction in surface expression (FIG 8B) in Cal-27 CisR. Induction of CXCR4 pathway with SDF-1a recombinant protein (10ng/ml), significantly up regulated (p>0.05) the CSC expression (CD44, CD133, ALDH1A1 and Oct41) and MDR genes (MDR1, MRP2, CTR1) among the Cal-27 and Cal-27 CisR cell lines (FIG 8C & D).
[0112] Effect of SDF 1-alpha secreting patient-derived CAF on Cal-27: Flow cytometry analysis of surface CXCR4 expression after induction with CAF-conditioned media indicated a reduction in expression due to competitive binding by SDF-1 ligand (FIG 9A). Indirect co-culturing of Cal-27 with fibroblast condition media (Cal-27CM; 1:1 & 4:1 dilution)by transwell culture showed significant up-regulation (p>0.05) of CSC (CD44, CD133, Aldh1a1, Notch1, Nanog, Sox2, Oct-4) and MDR genes (MDR1, MRP2, Survivin, ABCG2 and ERCC1) (FIG 9 B) ). Assessment of CSC profile by FACS analysis also showed significant increase in expression of CD44+, ALDH1A1+ and double positive cells (FIG 9C). Evaluation of functional properties of cells such as colony formation (FIG 9D (a) & (b)), spheroid formation (FIG 9E (a) & (b)) and migratory capacity (FIG 9F) of Cal-27CM cells were also significantly up-regulated (p>0.05).. The IC50 of cisplatin drug also increased from 7.6µM (in Cal-27) to 10.8µM (RI=1.4) (FIG 9G) indicating an increase in the resistance to Cisplatin in cells treated with CAF-conditioned media.
[0113] AMD3100 based targeting of CXCR4 pathway inhibition: Cal-27 and Cal-27CM cells were treated with AMD3100 (6µM) for 48hours in presence of condition media showed a significant lower surface expression of CXCR4 receptor in Cal-27CM cells p>0.01) (FIG 9A) due to the competitive binding of the inhibitor. Cal-27 (FIG 10B) and Cal-27CM cells (FIG 10C) in presence of AMD3100 treatment also showed a significant reduction in migration rate at 18th and 24th hour (p>0.01) as compared to the untreated cells. Spheroid formation capacity of the cells showed a highly significant reduction (p>0.005) in Cal-27CM cells (FIG 10D). Cell viability assay (MTT) in presence of AMD3100 also showed a significant reduction in resistance to cisplatin by Cal-27CM cells (FIG 10E), the viability of the cells being comparable to the control cells.
[0114] Targeting the CXCR4 pathway using AMD3100, thus showed a marked reduction in resistance to cisplatin and other function property possibly rendered due to increase in CSC expression. Thus inventors claim that, targeting CSC along with CXCR4 pathway possibly gives an extended effect on conventional therapy by downregulating CSC population.
[0115] AMD3100 was initially dissolved in water whereas AMD070 was dissolved in DMSO. The different concentrations used in the study were prepared freshly in complete medium or conditioned medium and added to the cells at specified time points.
[0116] According to an exemplary aspect of the present invention, the major advantages of these inhibitors are the possible lack of relapse upon cessation of therapy and more effective chemoprevention compared to other currently used drugs due to the synergistic effect of inhibiting the CSC-niche pathways along with the pathways involved in the specific drug activity.
[0117] According to a non-limiting exemplary aspect of the present invention, the present invention describes the use of these inhibitors in combination to chemopreventive drugs in oral cancer. In case of chemotherapy with current platinum/taxol drugs, as mentioned show an initial response, however high recurrence rates are observed. Invention suggests that the application of the CSC–niche inhibtors adjunct to chemotherapeutic drugs can improve the efficacy of the conventional chemotherapy drugs.
[0118] According to a further exemplary aspect of the present invention, the possible uses of this invention include:
[0119] Utilization of inhibitor/s will improve the efficacy of chemopreventive drugs
[0120] Treatment of Oral potentially malignant lesions for long term chemoprevention
[0121] Use as an adjunct to cancer treatment towards reversing resistance.
[0122] According to a further exemplary aspect of the present invention, the best way to practice the invention is to prescribe the inhibitor/s along with the present chemoprevention drugs. These inhibitor/s may be available as oral version which may be used for treatment.
[0123] According to a non-limiting exemplary aspect of the present invention, the invention will be used for the development of drugs. These drugs developed can then be utilized by hospitals/private clinics/dental doctors or the public as such to treat oral cancer.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] This disclosure is not limited to the particular systems, devices and methods described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope.
[0128] 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.
[0129] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof.
[0130] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various aspects. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[000] References
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,CLAIMS:CLAIMS:

I/WE CLAIM:
1. A method of treating a human or animal suffering from cancer, wherein the method comprises administering to the patient a CXCR4 antagonist and a chemopreventive/chemotheraputive drug(s), wherein the chemopreventive/chemotheraputive drug(s) acts synergistically with the CXCR4 antagonist, inhibiting the CSC-niche pathways and the pathways involved in chemopreventive/chemotheraputive drug(s), wherein the synergistic combinations are in an amount sufficient to reduce the (a) rate of onset of, and /or (b) reduce the progress of, and/or (c) reverse symptoms of cancer, and/or (d) reversing resistance to chemoprevention and chemotherapy.
2. The method of claim 1, wherein the CXCR4 antagonist is selected from:
i. AMD3100 and/or AMD070; and /or
ii. an anti-CXCR4 antibody.
3. A method of treating a human or animal suffering from cancer, wherein the method comprises administering to the patient a CXCR4 inhibitors/antagonist in an amount sufficient to reduce the (a) rate of onset of, and /or (b) reduce the progress of, and/or (c) reverse symptoms of cancer, and/or (d) reversing resistance to chemoprevention and chemotherapy.
4. The method of claim 1 and 3, wherein the method decreases CSC (Cancer Stem Cell) markers; reduces migration, self-renewal, tumorigenic potential of CSC; and reverses resistance with increased sensitivity to chemopreventive/chemotherapeutic drug(s).
5. A method of treating or preventing a condition in a human or animal suffering from cancer, wherein inhibition of one or more cancer drugs or a fragment or a complex thereof or a functional equivalent thereof, prevents, inhibits or ameliorates a pathology or a symptomology of the condition, the method including administration of as therapeutically effective amount of a compound according to any one of claims 2.
6. The CXCR4 antagonist according to claim 2 wherein the CXCR4 antagonists are non-limiting examples but preferably include AMD3100 and/or AMD070 and/or anti-CXCR4 antibody and/or a combination thereof.
7. The method of claim 1, wherein the method increases the sensitivity of the cancer cells to the host immune responses and reduce the CAF-conditioned medium-induced CSC enrichment in in vitro human and mice models.
8. The method of claim 1 and 3, wherein cancer cell growth is inhibited or reduced.
9. The method of claim 1 and 3, wherein the tumor is resistant to immunotherapy.
10. The CXCL4 inhibitors/antagonist according to claim 1, wherein the inhibitors can be in various forms such as combination, alone, with excipients, diluents, carriers, humectants, capsules, tablets, injectables (intravenous, muscular, hypodermal), oral, topical, in combination with polynucleotides, other biologicals, for targeted delivery, powders, suspensions, gels, hydrogels, logenges, in combination with cryo, radiation therapies, binders, for curing, palliative care in humans, animals and such other organisms wherein the said compositions can be employed to relieve them of the various ailments that can be controlled by modulating the CXCR4 and/or chemopreventive/chemotheraputive drug(s) pathways.