Indian Patents. 250374:A GENE SIGNATURE FOR MONITORING CANCER PATIENTS TREATED WITH A COMBINATION OF GEMCITABINE AND P276-00

Title of Invention	A GENE SIGNATURE FOR MONITORING CANCER PATIENTS TREATED WITH A COMBINATION OF GEMCITABINE AND P276-00
Abstract	Novel synergistic combination of gemcitabine with P276-00 or PI 446A and their use in the treatment of cancer are disclosed. The invention further describes gene signatures comprising gene markers used to monitor the drug response in a subject treated with the said combination.

Full Text	THE PATENTS ACT, 1970 (39 of 1970) PROVISIONAL SPECIFICATION (See Section 10, Rule 13) TITLE NOVEL SYNERGISTIC COMBINATION OF GEMCITABINE WITH P276-00 OR P1446A IN TREATMENT OF CANCER" APPLICANT PIRAMAL LIFE SCIENCES LIMITED, 1 Nirlon Complex, Off Western Expressway, Goregaon (E), Mumbai 400 063. The following specification describes the invention Novel synergistic combination of Gemcitabine with P276-00 or P1446A in treatment of cancer Field of Invention The present invention relates to novel synergistic combinations of Gemcitabine with P276-00 or P1446A and their use in the treatment of cancer. It also relates to a gene signature comprising drug response markers used to monitor the drug response in a subject treated with the said combinations. Background It is increasingly being realized that effective treatment of cancer not only requires efficient drugs but also optimal use of specific combination of anti-cancer drugs. During the past 25 years about 10% to 90% cure rate has been achieved in case of acute lymphoblastic leukemia with the optimization and combination of existing drugs. Combination therapy is thus increasingly being looked at as a better alternative to monotherapy in the treatment of cancer. The sequential or simultaneous use of two or more agents is termed as combination therapy. Various combinations of chemotherapeutic agents have been discussed in prior art. WO2004041308 reveals a combination of CDK inhibitor with Gemcitabine for use in the treatment of cancer. Although the disclosure suggests additive effect, a synergistic effect in using the combination is not very clear. The CDK inhibitor preferably used in the said disclosure is rosovitine. US7294332 describes a combination of temozolomide and. IFN a in the treatment of malignant melanoma. Gene expression signature for a set of 14 predictor genes is used as a measure of efficacy in patients suffering from colorectal cancer and receiving combination therapy of leucovorin, fluorouracil, and irinotecan (FOLFIRI). The accuracy of prediction in this study was 95% and could be used as decision tool to assist oncologist in selecting colorectal patients for FOLFIRI chemotherapy (Rio et al, J of Clin Oncol 2007; 25 (7); 773-780) In another study, Li et al, demonstrated that in prostate cancer, docetaxel and estramustine combination treatment directly and indirectly caused changes in the expression of many genes that are critically involved in the control of cell proliferation, apoptosis, transcription, translation, oncogenesis, angiogenesis, metastasis, and drug resistance (Mol Cancer Ther 2005; 4(3): 389-98). A report by Cheok et al provides molecular insights into the synergy of drug combination for purine antagonist mercaptopurine and dihydrofolate reductase inhibitor methotrexate in leukemia patients. Microarray-based gene expression profiling provided unique gene signatures for combination suggesting the effect on molecular targets that are beyond individual drug (Nature Genetics 2003; 34:85-90). The present invention is based on the observation that Gemcitabine in combination with P276-00 or P1446A shows marked synergistic effect as compared to the independent use of these agents. Gemcitabine (2\ 2'-difluorodeoxycytidine) is currently being marketed as Gemzar by Eli Lilly. It is a nucleoside analogue of deoxycytidine, which was first disclosed in US4808614 and US5464826. Gemcitabine has been indicated as the first-line therapy for locally advanced (nonresectable Stage II or III) or metastatic (Stage IV) adenocarcinoma of the pancreas. Gemcitabine has also been indicated as a second-line therapy for patients who have previously been treated with fluorouracil. Cyclin-dependant kinase (CDK), a class of genes involved in cell cycle pathways are emerging targets in cancer drug discovery. Several CDK inhibitors are currently undergoing clinical evaluation either as a single agent or in combination with other anticancer drugs. Flavones are series of novel compounds that exhibit significant specific activity against CDKs especially CDK4. P276-00 is a flavone compound with a potent anti-CDK4 activity which is described in US7271193 which is herein incorporated by reference in its entirety. CDK4 catalyzes the phosphorylation and inhibition of retinoblastoma (RB) protein, which is a negative regulator of cell cycle. Phosphorylation of RB leads to uncontrolled cell proliferation and induces tumorigenesis in cells. P276-00 was found to be more selective for CDK4-D1, CDKl-B, and CDK9 -Tl, as compared with other CDKs, and less selective for non-CDK kinases. It showed potent antiproliferative effects against various human cancer cell lines as demonstrated by both in vitro and in vivo experimental conditions. It is also observed that P276-00 induced a significant down-regulation of cyclin Dl and CDK4 and a decrease in CDK4 -specific pRB Ser(780) phosphorylation. The compound also induced apoptosis in human promyelocytic leukemia (HL-60) cells, as evidenced by the induction of caspase-3 and DNA ladder studies. P1446A-05 is a (+)-trans~3-[2[(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride (Molecular Formula: C22H19CIF3NO5.HCI)), Its mode of action reveals that it is a novel, potent inhibitor of CDK4-D1, CDK4-B and CDK9-T and is described in US7271193 which is incorporated herein by reference in its entirety. The present invention provides novel synergistic combination of Gemcitabine with P276-00 or PI446A. The disclosure also provides an unique gene signature comprising gene markers for monitoring drug response in patients undergoing cancer treatment with the said combination. Summary The present invention relates to a novel pharmaceutical compositions comprising combination of Gemcitabine with P276-00 or P1446A. The said combinations are used in the treatment of cancer. It further provides a gene signature comprising markers used to monitor the drug response in a subject administered with the aforementioned combination compositions. In one aspect, the invention provides a pharmaceutical composition comprising a combination of Gemcitabine and P276-00. This combination shows a synergistic effect as compared to the individual use of the agents. In another aspect, the invention provides a pharmaceutical composition comprising a combination of Gemcitabine and P1446A. The said combination demonstrates a synergistic effect as compared to the individual use of the agents. In yet another aspect, the invention provides a method of treating cancer using the combinations of Gemcitabine with P276-00 or P1446A. The types of cancer treated with these combinations are pancreatic cancer, lung cancer, colorectal carcinoma and head and neck cancer. The invention also provides a kit containing the aforementioned compositions. The invention further provides a gene signature comprising at least two markers for monitoring the drug response in a patient administered with the aforementioned compositions. The gene signature for the combination of Gemicitabine and P276-00 comprises at least two markers selected from the group consisting of SNX7, FA38A, DNAI1, RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2BO. Furthermore, in patients administered with the combination of Gemcitabine and P276-00, up regulation is observed in drug response markers SNX7, FA38A or DNAI1 and down regulation is observed in RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 or HIST1H2BO In another aspect, the invention provides a gene signature for monitoring drug response in a subject administered with the combination of Gemcitabine and P1446A comprising at least two markers selected from the group consisting of P21, REV3L, FGF5, PTK7, POLH, P27 and SSTR2. Upon administration of the combination Gemcitabine and P1446A, there is upregulation of the said markers. In yet another aspect, the invention provides a method of monitoring the drug response in a patient suffering from cancer and treated with a combination of Gemcitabine and P276-00, comprising detection of gene signature with at least two markers selected from the group consisting of SNX7, FA38A, DNAI1, RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2BO. The invention also provides a method a method of monitoring the drug response in a patient suffering from cancer and treated with a combination of Gemcitabine and P276-00, comprising detection of gene signature with at least two markers selected from the group consisting of P21, REV3L, FGF5, PTK7, POLH, P27 and SSTR2. Brief description of figures Fig 1: Effect of sequentially administered combination of Gemcitabine with P276-00 on Panc-1 cells using propidium iodide based fluorescence cytotoxicity assay. The red arrow marks the synergistic effect of the combination Gemcitabine (0.1 nM) and P276-00 (60nM). Fig 2: Effect of sequentially administered combination of Gemcitabine with P276-00 on Panc-1 cells using Flow cytometry. Fig 3: Effect of sequentially administered combination of Gemcitabine with PI446A on Panc-1 cells using CCK-8 cytotoxicity assay. Detailed description of the invention The invention is based on the observation that a novel combination of Gemcitabine with P276-00 or PI446A shows synergistic effect as compared to the compounds given individually. In one embodiment, the combination of Gemcitabine and P276-00 is disclosed. A combination of Gemcitabine and PI446A is also further described. These combinations show a synergistic effect compared to the agents given individually. This synergistic effect observed forms the basis of the invention. Gemcitabine (2'-deoxy-2', 2'-difluorocytidine) is a nucleoside analogue of deoxycytidine. It exhibits cell phase specificity, primarily killing cells undergoing DNA synthesis (S-phase) and also blocking the progression of cells through the Gl/S-phase boundary. Gemcitabine is metabolised intracellularly by nucleoside kinases to the active diphosphate (dFdCDP) and triphosphate (dFdCTP) nucleosides. Its cytotoxic effect can be attributed to the inhibition of DNA synthesis as a result of the combined actions of the diphosphate and triphosphate nucleosides. More specifically, Gemcitabine diphosphate inhibits ribonucleotide reductase, which is responsible for catalysing the reactions that generate the deoxynucleoside triphosphates for DNA synthesis. Inhibition of this enzyme by the diphosphate nucleoside causes a reduction in deoxynucleotide concentrations, for example dCTP. Furthermore, Gemcitabine triphosphate competes with dCTP for incorporation into DNA. The subsequent reduction in the intracellular concentration of dCTP enhances the incorporation of Gemcitabine triphosphate into DNA (self potentiation). Gemcitabine exhibits antitumour activity, particularly against ovarian, pancreatic and lung cancers. Gemcitabine finds use in the first-line therapy for locally advanced (nonresectable Stage II or III) or metastatic (Stage IV) adenocarcinoma of the pancreas. Patients previously treated with fluorouracil use Gemcitabine as a second-line therapeutic agent. P276-00 as disclosed in US7271193 is flaovone compound with ant-CDK4 activity. . The compound finds use in antiproliferative therapies for diseases characterized by excessive cell growth such as cancers, cardiovascular abnormalities, nephrological disorders, psoriasis, Alzheimer's disease, immunological disorders involving unwanted proliferation of leukocytes, restenosis and other proliferative smooth muscle disorders, viral infections, and mycotic infections. P1446A-05(+)-trans-3-[2[(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride. It is a novel, potent inhibitor of CDK4-D1, CDK4-B and CDK9-T. Pre-clinical data from human cancer cell lines has shown that P1446A-05 halts transcriptional elongation, promotes apoptosis and arrests cell cycle progression at Gl and G2 phases of cell cycle. In vitro studies with a variety of cancer cell lines suggest that PI446A effectively inhibits proliferation of and induces cytotoxicity in both cisplatin sensitive and resistant cells without any significant cytotoxicity to normal human peripheral blood mononuclear cells. P1446A-05 induces significant down regulation of Cyclin Dl and CDK4 specific retinoblastoma protein (pRb) ser780 phosphorylation, induced p53 and reduced the levels of the anti-apoptotic protein Bcl-2. P1446A-05 also demonstrated good oral bioavailability in the preclinical studies. Upon oral administration, it demonstrated significant tumor growth inhibition in xenograft models of colon cancer (HCT-116, SW-480) and non-small cell lung cancer (H-460) in SCID mice. By virtue of its specific action against CDK4-D1, CDK1-B and CDK9-T, P1446A-05 has the potential to have potent effects on cell cycle arrest while avoiding the unwanted effects associated with more non-specific CDK inhibitors. In another embodiment of the invention is provided a method of treating cancer by administering combinations of Gemcitabine with P276-00 or PI446A. The types of cancer treated with the said combinations include but are not limited to pancreatic cancer, lung cancer, colorectal carcinoma and head and neck cancers. In a preferred embodiment, the combinations are used to treat pancreatic cancer. In yet another embodiment, the invention provides a dosage range of the combinations used to administer to the patients suffering from cancer. The dosage varies between a final concentration range of 0.1 nM-30 nM for Gemcitabine and 60- 960 nM for P276-00. In case of Gemcitabine with PI446A, the dosage varies between a final concentration of 3 nM-10000 nM for Gemcitabine andl nM-10000 nM for PI446A. Various possible combinations of the agents in the aforementioned dosage range are used in the invention. The invention also provides a method of administering the said combinations. The method involves administering the combinations either simultaneously or sequentially. The method of administration preferably is sequential wherein Gemicitabine is first administered followed by the administration of P276-00 or P1446A. Gemcitabine is administered as a 30 min bolus infusion while P276-00 is administered intravenously or as a bolus infusion for 30 mins. PI446A is administered orally. In one embodiment, the present invention provides a dosing schedule of the combinations. The combination of Gemcitabine with P276-00 is administered in the following steps; Gemcitabine is first administered for 0-24 hrs followed by sequential administration of P276-00 for 24-96 hrs. Gemcitabine with P1446A is administered in the following steps; Gemcitabine is first administered for 0-24 hrs followed by sequential administration of P1446A for 24-72 hrs. In another embodiment of the invention is provided a kit comprising of Gemcitabine with P276-00 or PI446A. The combinations in a kit provide convenience to the end user. The kit also comprises an insert detailing the dosing regime for the administration of the combinations. The present invention further provides a gene signature for monitoring the drug response in a patient who has been administered the combinations of the invention. Method of monitoring the drug response following administration of Gemcitabine and P276-00 or Gemcitabine and PI446A is also encompassed in the invention. Microarray-based gene signatures provide valuable information regarding the cellular function and can be effectively used in determining the drug response in patients subsequent to exposure to combination of therapeutic agents. (Nature Genetics 2003; 34:85-90). The gene signature of the present invention used to monitor the drug response in a patient administered with the combination of Gemcitabine and P276-00 comprises of at least two markers selected from the group consisting of SNX7, FA38A, DNAI1, RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2BO. Amongst these markers SNX7, FA38A or DNAI1 are up regulated while RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2B0 are down regulated in patients administered with the said combination. The above described drug response markers are detailed below: SNX7: This gene encodes a member of the sorting nexin family. Sorting nexins are proteins involved in protein trafficking in the endosomes and the vesicular micro-tubular structures in the cytoplasm. Two isoforms of the sorting nexin family (SNX1 & SNX2) have been demonstrated to play a functional role in localization of the endogenous EGFR in the endosomes of colon cancer cells. Deletion mutants for SNX1 have proved to down-regulate the endogenous EGFR expression. Although there is no current evidence identified for SNX7, our data could prove the involvement of SNX7 in pancreatic cancer disease biology. DNAI1: The inner- and outer-arm dyneins, which bridge between the doublet microtubules in axonemes, are the force-generating proteins responsible for the sliding movement in axonemes. The intermediate and light chains, thought to form the base of the dynein arm, help mediate attachment and may also participate in regulating dynein activity. This gene encodes an intermediate chain dynein, belonging to the large family of motor proteins. Mutations in this gene result in abnormal ciliary ultrastructure and function associated with primary ciliary dyskinesia (PCD) and Kartagener syndrome. RRM2: Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from ribonucleotides. It is composed of 2 non-identical subunits, proteins Ml and M2. Synthesis of M2 is regulated in a cell-cycle dependent fashion. Activity of this enzyme, which catalyses conversion of ribonucleotide 5'-diphosphates to their 2'-deoxynucleotides, is modulated by levels of its M2 subunit (RRM2). Here we show that RRM2 overexpression is associated with Gemcitabine chemo resistance in pancreatic adenocarcinoma cells, and that suppression of RRM2 expression using RNA interference mediated by small interfering RNA (siRNA) enhances Gemcitabine-induced cytotoxicity in vitro. CDK8: The protein encoded by this gene is a member of the cyclin-dependent protein kinase (CDK) family and are known to be important regulators of cell cycle progression. This kinase and its regulatory subunit cyclin C are components of the RNA polymerase II holoenzyme complex, which phosphorylates the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. This kinase has also been shown to regulate transcription by targeting the CDK7/cyclin H subunits of the general transcription initiation factor IIH (TFIIH), thus providing a link between the 'Mediator-like' protein complexes and the basal transcription machinery. Furthermore, increased binding of CDK8 to p53 target genes correlates positively with transcriptional strength and CDK8 functions as a coactivator within the p53 transcriptional program. DLG5: This gene encodes a member of the family of discs large (DLG) homologs, a subset of the membrane-associated guanylate kinase (MAGUK) super family. The protein encoded by this gene localizes to the plasma membrane and cytoplasm, and interacts with components of adherens junctions and the cytoskeleton. It is proposed to function in the transmission of extracellular signals to the cytoskeleton and in the maintenance of epithelial cell structure. FGF5: The protein encoded by this gene is a member of the fibroblast growth factor (FGF) family. FGF family members possess broad mitogenic and cell survival activities, and are involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. This gene was identified as an oncogene, which confers transforming potential when transfected into mammalian cells. MKKS: This gene encodes a protein with sequence similarity to the chaperonin family. The encoded protein may have a role in protein processing in limb, cardiac and reproductive system development. Mutations in this gene have been observed in patients with Bardet-Biedl syndrome type 6 and McKusick-Kaufman syndrome. PPIL4: This gene is a member of the cyclophilin family of peptidylprolyl isomerases. The cyclophilins are a highly conserved family, members of which play an important role in protein folding, immunosuppression by cyclosporin A, and infection of HIV-1 virions. HELLS: This gene encodes a lymphoid-specific helicase. Other helicases function in processes involving DNA strand separation, including replication, repair, recombination, and transcription. This protein is thought to be involved with cell proliferation and may play a role in leukemogenesis. ID1: The protein encoded by this gene is a helix-loop-helix (HLH) protein that can form heterodimers with members of the basic HLH family of transcription factors. The encoded protein has no DNA binding activity and therefore can inhibit the DNA binding and transcriptional activation ability of basic HLH proteins with which it interacts. This protein may play a role in cell growth, senescence, and differentiation. ID-1 is suggested as an oncogene and is reported to promote cell proliferation, invasion, and survival in several types of human cancer cells through multiple signaling pathways. DICER1: This gene encodes a protein possessing an RNA helicase motif containing a DEXH box in its amino terminus and an RNA motif in the carboxy terminus. The encoded protein functions as a ribonuclease and is required by the RNA interference and small temporal RNA (stRNA) pathways to produce the active small RNA component that represses gene expression. TMPRSS3: This gene encodes a protein that belongs to the serine protease family. The encoded protein contains a serine protease domain, a transmembrane domain, a LDL receptor-like domain, and a scavenger receptor cysteine-rich domain. Serine proteases are known to be involved in a variety of biological processes, whose malfunction often leads to human diseases and disorders. This gene was identified by its association with both congenital and childhood onset autosomal recessive deafness. This gene was also identified as a tumor associated gene that is overexpressed in ovarian tumors. HIST1H2BO: Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. The linker histone, HI, interacts with linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures. This gene is intronless and encodes a member of the histone H2B family. Transcripts from this gene lack polyA tails but instead contain a palindromic termination element. This gene is found in the small histone gene cluster on chromosome 6p22-p21.3. Drug response in patients administered with the combination of Gemcitabine and P1446A is monitored using a gene signature comprising at least two markers selected from the group consisting of P21, REV3L, FGF5, PTK7, POLH, P27, and SSTR2. Expression of these markers is upregulated upon administration of Gemcitabine and P1446A. These gene markers are herein further described: P21: This gene encodes a potent cyclin-dependent kinase inhibitor. The encoded protein binds to and inhibits the activity of cyclin-CDK2 or -CDK4 complexes, and thus functions as a regulator of cell cycle progression at Gl phase of the cell cycle. The expression of this gene is tightly controlled by the tumor suppressor protein p53, through which this protein mediates the p53-dependent cell cycle Gl phase arrest in response to a variety of stress stimuli. This protein can interact with proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory factor, and plays a regulatory role in S phase DNA replication and DNA damage repair. REV1: This gene encodes a protein with similarity to the S. cerevisiae mutagenesis protein Revl. The Revl proteins contain a BRCT domain, which is important in protein-protein interactions. A suggested role for the human Revl-like protein is as a scaffold that recruits DNA polymerases involved in translesion synthesis (TLS) of damaged DNA. PTK7: Receptor protein tyrosine kinases transduce extracellular signals across the cell membrane. A subgroup of these kinases lack detectable catalytic tyrosine kinase activity but retain roles in signal transduction. The protein encoded by this gene is a member of this subgroup of tyrosine kinases and may function as a cell adhesion molecule. This gene is thought to be expressed in colon carcinomas but not in normal colon, and therefore may be a marker for or may be involved in tumor progression. P27: This gene encodes a cyclin-dependent kinase inhibitor, which shares a limited similarity with CDK inhibitor CDKNlA/p21. The encoded protein binds to and prevents the activation of cyclin E-CDK2 or cyclin D-CDK4 complexes, and thus controls the cell cycle progression at Gl. The degradation of this protein, which is triggered by its CDK dependent phosphorylation and subsequent ubiquitination by SCF complexes, is required for the cellular transition from quiescence to the proliferative state. SSTR2: Somatostatin acts at multiple sites to inhibit the release of many hormones and other secretory proteins. The biologic effects of somatostatin are probably mediated by a family of G protein-coupled receptors that are expressed in a tissue-specific manner. SSTR2 is a member of the superfamily of receptors having seven transmembrane segments and is expressed in highest levels in cerebrum and kidney. Introduction of the SSTR2 gene, the expression of which is frequently lost in human pancreatic adenocarcinoma, exerts its anti-angiogenic effects by down-regulating the expression of the factors, which are involved in tumor angiogenesis and metastasis, suggesting SSTR2 gene transfer as a promising strategy of gene therapy for pancreatic cancer. In a yet another embodiment the invention provides a method of monitoring the drug response in a patient administered with the combination of Gemcitabine and P276-00. The method involves detection of the gene signature wherein SNX7, FA38A or DNAI1 are up regulated and RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2BO are down regulated. In a further embodiment a method of monitoring the drug response in a patient administered with the combination of Gemcitabine and PI446A is provided. The method involves detection of the gene signature wherein up regulation of markers P21, REV3L, FGF5, PTK7, POLH, P27, and SSTR2 is observed. Examples Example 1. Propidium iodide-based fluorescence cytotoxicity assay The combination of P276-00 and Gemcitabine was screened for its synergistic effect in Panc-1 cells using propidium iodide based fluorescence cytotoxicity assay. Test System The Panc-1 cell line was procured from ATCC (American Tissue type Culture Collection), USA. Catalog number: CRL-1469 and the frozen vial were stored in liquid nitrogen container. Propidium iodide dye was procured from Sigma-Aldrich, USA. Catalog number: P-4170-100 mg and it was stored in 2-8°C. Method to perform the assay Panc-1 cells were seeded at a density of 2000 cells/well, in a 200 uL in tissue culture grade 96 well plate and allowed to recover for 24 hrs in a humidified 5% ± 0.2 CO2 incubator at 37 °C ± 0.5 °C. After 24 hrs, 1 uL of 200 X (200 times higher than required concentration is denoted as 200 X) compound (dissolved in neat DMSO), as per Table 1 and 2 was added to the wells. The final DMSO concentration was 0.5% in wells. The plate was incubated for 24hrs in humidified 5% + 0.2 CO2 incubator at 37 ± 0.5 °C. After 24 hrs the Minimum Essential Medium (MEM) from the Gemcitabine treated wells was removed and washed two times with fresh MEM and followed by addition of 200 uL of fresh MEM per well. 1 uL of 200 X (200 times higher than required concentration is denoted as 200 X) compound (dissolved in neat DMSO) was then added as per the Table 1 and 2 designs. The final DMSO concentration was 0.5% in wells which also served as the vehicle control for the study. After 72 hrs the plate was removed from CO2 incubator and spent MEM aspirated from the wells and supplemented with fresh MEM. This was followed by addition of 25 uL of propidium iodide (50 fig/ml in medium) per well. The same plate was frozen at -80°C for 24 hrs and then thawed and allowed to come to room temperature. The fluorescence at a wavelength of 530nM excitation and 620nM emission was read. The percent cytotoxicity was calculated using the following formula Percent (Reading of Control -Reading of Treated cells] X 100 Cytotoxicity = Reading of Control Results The propidium iodide based cytotoxicity assay showed that Gemcitabine (0.1 nM) ICio for 24 hrs followed by P276-00 (60 nM) ICio for 72 hrs showed synergistic toxicity of 78% (Fig.l, Table 2). In vitro testing showed that these two agents produce minimal cytotoxicity at ICio alone (Table 1) but on combination at ICio, they displayed additional antiproliferative /cytotoxicity effects across a range of dosages (Table 2). The synergistic effect of gemcitabine and P276-00 combination not only make cancer cells more susceptible but further facilitate use lower doses of drugs in patients. Table 1: Effect of independent administration of Gemcitabine and P276-00 on Panc-1 cells Concentration (nM) Fluorescence Unit % Coefficient of Variance % Cytotoxicity in Panc-1 cells Gemcitabine 0.1 nM 31455 4.844 10 60 nM P276-00 24558 3.168 27 120 nM P276-00 22768 9.355 33 240 nM P276-00 22212 4.860 35 480 nM P276-00 18831 6.630 45 960 nM P276-00 15416 6.76 58 Control 34599 3.829 0 Table 2. Effect of the sequentially administered combination of Gemcitabine with P276-00 on Panc-1 cells Gemcitabine concentration 0.1 nM for 24 hrs + P276-00 at various concentrations Fluorescence unit % Coefficient of Variance % Cytotoxicity 60 nM P276-00 for 72 hrs 12147 3.413 78 120nMP276-00for72hrs 10384 4.320 82 240 nM P276-00 for 72 hrs 11851 2.885 79 480 nM P276-00 for 72 hrs 11796 3.390 79 960 nM P276-00 for 72 hrs 9654 2.069 83 Control 55847 6.010 0 Example 2. Cell cycle analysis by DNA content The synergistic effect Gemcitabine with P276-00 on Panc-1 cancer cells was analyzed using propidium iodide based cell cycle analysis where total population of cells was sorted in to sub GO, Gl, S and G2/M populations according to total fluorescent intensity. Dosage P276-00 at a final concentration of 60 nM, 240 nM and 480 nM and Gemcitabine at a final concentration of 1 nM, 3 nM and 10 nM were analyzed in single dose and in all possible combinations of the dose range for the Gemcitabine and P276-00. Cell cycle analysis using Flow cytometry Methodology Panc-1 cells were treated with Gemcitabine for 0 to 24 hrs. After 24 hrs the cells were washed two times with plain MEM. Fresh MEM with 10% serum (2 mL/well) was added to the wells, followed by treatment with P276-00 for 24 hrs to 96 hrs. After 96 hrs the treated cells were harvested, fixed, stained with propidium iodide and analyzed for specific cell arrest using DNA content analysis by flow cytometry. Adherent cells were trypsinized and washed with PBS or HBSS without Ca++ & Mg++ containing EDTA pH 8.0 and BSA. They were further isolated, centrifuged and total number of cells were counted and recorded. The pellet of cells was resuspended in ice-cold PBS and a uniform suspension was prepared. Cold ethanol was slowly added to the suspension while vortexing it. The cells were then fixed at 4°C overnight. The cells were then taken in a conical tube and centrifuged to remove the ethanol. The pellet was resuspended in PBS and Calf serum after vortexing and washing it in the same. Propidium iodide and boiled RNase S was added to it and incubated at 37° C for 30 mins. Analysis was carried out using a flow cytometer (Becton Dickinson FACSCalibur, a 4-color, dual-laser benchtop flow cytometer). Results The results of the cell cycle analysis revealed that control cells showed 5.5 % population of cells in sub GO (apoptotic cells) (Fig 2). Gemcitabine 1 nM treated cells showed 16.85% population of cells in subGO and P276-00 60 nM treated cells showed 17.6 % population of cells in subGO. Interestingly, when cells were exposed to the combination of Gemcitabine (1 nM) for 24 hr followed by P276-00 (60 nM, 240 nM and 480 nM) for 72 hr, they showed an increase of 59.5%, 51.6% and 61.3 respectively in subGO population (apoptosis). These results further substantiate the synergistic activity displayed by Gemcitabine with P276-00. Example 3. Microarray analysis to detect gene expression signatures associated with the in vitro synergistic effect of the combination therapy of Gemcitabine with P276-00 in Panc-1 cells The goal of the microarray analysis was to evaluate putative gene expression signatures associated with the in vitro synergistic effect of the combination therapy of Gemcitabine with P276-00 in Panc-1 cells. The time points (3 hrs, 6 hrs, 12 hrs) were chosen to establish early molecular changes in the transcription profile for probable biomarkers and mechanism of action for the combination therapy. Methodology Sample processing and RNA extraction Approx 20 million cells were lysed in Trizol Reagent (Invitrogen Corp, USA) and passed through a 21-gauge needle for 5-10 times before RNA extraction was performed. Total RNA extraction was performed using the RNeasy Mini kit (Qiagen, USA) according to the manufacturer's protocol. The final total RNA was eluted in nuclease-free water and the concentration/purity was determined by Nanodrop spectrophotometer (Thermo Fisher Scientific, Delaware, USA). cDNA generation and labeling 20 ug of total RNA was reverse transcribed using amino allyl dUTP and oligo dT, the resulting cDNA was indirectly labeled with Cy3/Cy5 dyes (GE Healthcare, USA) using a fluorescent labeling kit (Promega Biosciences, USA). The concentration and dye incorporation of the labeled products were determined by Nanodrop spectrophotometer (Thermo Fisher Scientific, Delaware, USA). Equal amounts of labeled cDNA were denatured at 95° C for 5 mins, cooled and microfuged at 13200 rpm for 5 mins before hybridization. Array fabrication and hybridization 70-mer transcript oligonucleotides representing 36,480 human genes was purchased from Operon Biotechnologies (Germany) and spotted onto aminosilane coated glass slides (Arraylt, USA) by contact printing using a Omnigrid (OG100) spotter (Genomic Solutions Inc, USA). Post printing, the slides were cross-linked with UV light at 650 mJ/sec2, vacuumed, sealed and stored in the dark. The slides were pre-processed for background reduction using Pronto (Promega Life sciences, USA) pre-hyb kit as per the manufacturer's instructions. Hybridization was carried out in the GeneTAC Hybstation (Genomic Solutions Inc, USA) for 18 hrs. The hybridization protocol is as follows: 6 hrs at 42° C (with agitation), 6 hrs at 35° C (with agitation), and 6 hrs at 30 C (with agitation). Post hybridization, the arrays were washed according to the manufacturer's protocol. The slides were dried by centrifugation at 1600 rpm for 5 mins. Scanning and image analysis Post drying, the slides were scanned in a laser scanner (Genomic Solutions, USA) for Cy3 and Cy5 detection. The PMT settings were adjusted using the auto-exposure feature of the scanner. The TIFF files thus generated were exported to GeneTAC Integrator software (Genomic Solutions, USA) for image analysis. The resultant CSV file containing the annotated genome and the corresponding ratios were further used for data analysis. Results The combination of Gemcitabine and P276-00 used for the treatment in Panc-1 cell line induces a dynamic gene expression change with respect to compound action, time of dosing and the concentrations. The major gene families that have been dys-regulated by combination treatment are the proteins of the serine-threonine kinase family (Table 3). The IC50 doses also up regulate the inflammatory pathways downstream of TNFR family without the induction of NFKBI indicating the inhibition of the pro-survival pathways in Panc-1 cell line. Table 3: Gene expression values following drug exposure in Panc-1 cell lines GeneID QenelVame _, •-.■ t •, ;-.-,\ , tGeni!cit»lyne. E276 .Combination HIST1H2BO Histone l,H2BO -0.71 -0.39 -3.28 TMPRSS3 Transmembrane protease, serine 3 0.85 0.33 -1.92 DICER1 Dicer 1, Dcr-1 homolog -0.16 0.14 -1.74 IDl Inhibitor of DNA binding 1 -0.60 0.18 -1.24 SLC19A2 Solute carrier family 19 member 2 0.37 0.97 -1.02 PPIL4 Peptidylprolyl isomerase -like 4 0.36 0.30 -1.04 HELLS Helicase, lymphoid-specific 0.40 0.62 -1.05 MKKS McKusick-Kaufman syndrome -0.02 -0.08 -1.86 FGF5 Fibroblast growth factor 5 0.28 0.23 -0.49 DLG5 Discs, large homolog 5 -0.19 0.25 -0.46 CDK8 Cyclin-dependent kinase 8 -0.03 0.16 -0.61 RRM2 Ribonucleotide reductase M2 0.69 -0.14 -0.94 DNAI1 Dynein, 0.03 -0.20 2.02 FA38A FA38A -0.70 -0.93 1.96 SNX7 Sorting nexin 7 0.27 -0.12 1.93 *Numerical value indicates log value of intensities as obtained by microarray experiments. Values close to zero indicate normal expression, negative value denotes down regulation and positive value indicates up-regulation of gene expression. Example 4. CCK-8 based cytotoxicity assay The combination of Gemcitabine with PI446A was screened for its synergistic effect in Panc-1 by CCK-8 (Cell counting kit-8) based cytotoxicity assay. CCK-8 (Cell counting kit-8) based cytotoxicity assay is a sensitive nonradioactive colorimetric assay for determining the number of viable cells in cell proliferation and cytotoxicity assays. WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H tetrazolium, monosodium salt) is bioreduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells. Test System The Panc-1 cell line was procured from ATCC (American Tissue type Culture Collection USA, catalog number: CRL-1469) and the frozen vial was stored in liquid nitrogen container. Methodology Gemcitabine at final concentrations of 1 nM, 10 nM and 100 nM and PI446A at a final concentration of 3 nM, 10 nM, 30 nM, 100 nM and 300 nM were analyzed in single dose and in all possible combinations of the dose range for the two drugs mentioned above. Panc-1 cells were seeded at a density of 3000 cells/well in a 96 well plate and allowed to recover for 24 hrs in a CO2 incubator at 37 C. Subsequently, compounds (Gemcitabine and/or P1446A) were added to the plates. The final DMSO concentration was 0.5% in wells. Plates were incubated for 24, 48, and 72 hrs. At the end of incubation CCK-8 was added and kept at 37°C for 3 hrs following which absorbance was measured at 450nM. Results Combination of Gemcitabine and PI 446A was observed to exhibit synergistic effect at a concentration of 10 nM and 100 nM respectively. Gemcitabine at 10 nM showed cytotoxicity of 31.47% while P1446A at 100 nM, showed cytotoxicity of 29.6%. However when used as a combination of Gemcitabine 10 nM for 24 hrs, followed by incubation with P1446A 100 nM for 72 hrs, an increase in cytotoxicity to 90.5% was noted, which is at least 29% more cytotoxicity than the additive effect of the two agents suggesting a marked synergistic effect of the combination (Table 4). Gemcitabine and PI446A were also found to be more synergistic at the 10 nM and 300 nM respectively. Gemcitabine at 10 nM showed cytotoxicity of 31.47% and P1446A at 300 nM, showed cytotoxicity of 31.05%. However when used as a combination of Gemcitabine 10 nM for 24 hrs, followed by PI446A 300 nM for 72 hrs an increase in cytotoxicity to the extent of about 95% was noted, which is 33% more cytotoxicity than the additive effect suggesting a significant synergy of the combination drug (Table 4). Table 4. Effect of combination of Gemcitabine with P1446A in Panc-1 cells Concentration in nM % Cytotoxicity 24 hrs 48 hrs 72 hrs GemlOnM 21.997 27.571 31.471 P1446A 30 nM 29.825 14.163 23.33 P1446A100nM 26.525 24.596 29.603 P1446A 300 nM 34.36 28.636 31.075 GemlO nM+ P1446A/ 30 nM 63.099 73.640 83.655 Geml0nM+P1446A/100 nM 73.935 75.136 90.527 Geml0nM+P1446A/300 nM 82.788 84.482 94.720 Gem= Gemcitabine Example 5. Gene expression profile using RTQ-PCR The objective of the experiment was to evaluate the synergistic effect of drug combination (Gemcitabine with PI 446 A) as compared to individual use of the same. The synergistic effect of the compound or drug combination in the Panc-1 cell line was measured in terms of gene expression and expressed as fold changes as compared to the cell control with no drug treatment. Methodology Cell lines treated with drugs (Gemcitabine, P1446A or both) were used for total RNA isolation using a commercial RNA extraction kit (Qiagen Corporation, Germany). The first-strand cDNA was synthesized from total RNA using first strand cDNA synthesis kit from Invitrogen Corporation (California, USA). This is followed by real time quantitative polymerase chain reaction (RTQ PCR) using gene specific primers and standard thermal program of initial denaturation at 95°C for 5 mins and 40 cycles of 95°C for 10 seconds, followed by 60°C for 30 seconds (Realplex PCR machine from Eppendorf, Germany). Quantitative measurement of products made during PCR cycles was normalized against a housekeeping gene (Actin) and used to measure the gene expression as fold changes as compared to respective control. Results Gene markers, which showed up regulation in response to combination therapy of Gemcitabine with P1446A, are listed in Table 7. Table 7. Gene expression profile for cancer markers in Panel cell line after exposure to a Gemcitabine, P1446A and combination of both. Genes Gene Name Gemcitabine P1446A Gemcitabine + P1446A P21 Cyclin-dependent kinase inhibitor 1A 2.87 6.04 7.94 REV3L REV3-like 0.59 1.04 2.35 FGF5 Fibroblast growth factor 5 1.42 2.76 4.63 PTK7 PTK7 protein tyrosine kinase 7 0.96 -0.19 4.00 POLH Polymerase (DNA directed), eta 0.51 0.10 1.40 P27 Cyclin-dependent kinase inhibitor IB -0.38 0.70 2.61 SSTR2 Somatostatin receptor 2 -1.52 0.71 7.38 Claims We claim: 1. A pharmaceutical composition comprising a combination of Gemcitabine and P276-00. 2. A pharmaceutical composition comprising a combination of Gemcitabine and P1446A. 3. A method of treating cancer in a subject, the said method comprising administering the combination of claim 1 or claim 2. 4. The method of claim 3, wherein the said cancer is selected from a group comprising of pancreatic cancer, lung cancer, colorectal carcinoma and head and neck cancer. 5. The method of claim 3, wherein the said cancer is pancreatic cancer. 6. The method of claim 3, where the combination is administered simultaneously or sequentially. 7. A kit comprising the pharmaceutical composition as claimed in claim 1 and 2. 8. A gene signature comprising at least two markers for monitoring drug response in patients administered with the composition as claimed in claim 1. 9. The gene signature of claim 8, wherein the said drug response markers are selected from the group consisting of SNX7, FA38A, DNAI1, RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2BO. 10. The gene signature of claim 9, wherein the said drug response markers SNX7, FA38A or DNAI1 are up regulated in patients administered with the composition as claimed in claim 1. 11. The gene signature of claim 9, wherein the said markers RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 or HIST1H2BO are down regulated in patients administered with the composition as claimed in claim 1. 12. A gene signature comprising at least two drug response markers for monitoring drug response in patients administered with the composition as claimed in claim 2. 13. The gene signature of claim 12, wherein the drug response markers are selected from the group consisting of P21, REV3L, FGF5, PTK7, POLH, P27 and SSTR2. 14. The gene signature of claim 12, wherein the said markers P21, REV3L, FGF5, PTK7, POLH, P27 and SSTR2 are up regulated in patients administered with the composition as claimed in claim 2. 15. A method of monitoring drug response in a patient suffering from cancer treated with a combination of Gemcitabine and P276-00, comprising detection of a gene signature with at least two drug response markers. 16. The method of claim 15, wherein the said drug response markers are selected from the group consisting of SNX7, FA38A, DNAI1, RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2BO. 17. A method of monitoring drug response in a patient suffering from cancer treated with a combination of Gemcitabine and P1446A, comprising detection of a gene signature with at least two drug response markers. 18. The method of claim 17, wherein the said drug response markers are selected from the group consisting of P21, REV3L, FGF5, PTK7, POLH, P27 and SSTR2. Abstract Novel synergistic combination of gemcitabine with P276-00 or PI 446A and their use in the treatment of cancer are disclosed. The invention further describes gene signatures comprising gene markers used to monitor the drug response in a subject treated with the said combination.

Full Text

THE PATENTS ACT, 1970 (39 of 1970)
PROVISIONAL SPECIFICATION (See Section 10, Rule 13)
TITLE
NOVEL SYNERGISTIC COMBINATION OF GEMCITABINE WITH P276-00 OR P1446A IN TREATMENT OF CANCER"
APPLICANT
PIRAMAL LIFE SCIENCES LIMITED,
1 Nirlon Complex, Off Western Expressway,
Goregaon (E), Mumbai 400 063.
The following specification describes the invention

Novel synergistic combination of Gemcitabine with P276-00 or P1446A in treatment of cancer
Field of Invention
The present invention relates to novel synergistic combinations of Gemcitabine with P276-00 or P1446A and their use in the treatment of cancer. It also relates to a gene signature comprising drug response markers used to monitor the drug response in a subject treated with the said combinations.
Background
It is increasingly being realized that effective treatment of cancer not only requires efficient drugs but also optimal use of specific combination of anti-cancer drugs. During the past 25 years about 10% to 90% cure rate has been achieved in case of acute lymphoblastic leukemia with the optimization and combination of existing drugs. Combination therapy is thus increasingly being looked at as a better alternative to monotherapy in the treatment of cancer. The sequential or simultaneous use of two or more agents is termed as combination therapy.
Various combinations of chemotherapeutic agents have been discussed in prior art. WO2004041308 reveals a combination of CDK inhibitor with Gemcitabine for use in the treatment of cancer. Although the disclosure suggests additive effect, a synergistic effect in using the combination is not very clear. The CDK inhibitor preferably used in the said disclosure is rosovitine. US7294332 describes a combination of temozolomide and. IFN a in the treatment of malignant melanoma.
Gene expression signature for a set of 14 predictor genes is used as a measure of efficacy in patients suffering from colorectal cancer and receiving combination therapy of leucovorin, fluorouracil, and irinotecan (FOLFIRI). The accuracy of prediction in this study was 95% and could be used as decision tool to assist oncologist in selecting colorectal patients for FOLFIRI chemotherapy (Rio et al, J of Clin Oncol 2007; 25 (7); 773-780)

In another study, Li et al, demonstrated that in prostate cancer, docetaxel and estramustine combination treatment directly and indirectly caused changes in the expression of many genes that are critically involved in the control of cell proliferation, apoptosis, transcription, translation, oncogenesis, angiogenesis, metastasis, and drug resistance (Mol Cancer Ther 2005; 4(3): 389-98). A report by Cheok et al provides molecular insights into the synergy of drug combination for purine antagonist mercaptopurine and dihydrofolate reductase inhibitor methotrexate in leukemia patients. Microarray-based gene expression profiling provided unique gene signatures for combination suggesting the effect on molecular targets that are beyond individual drug (Nature Genetics 2003; 34:85-90).
The present invention is based on the observation that Gemcitabine in combination with P276-00 or P1446A shows marked synergistic effect as compared to the independent use of these agents. Gemcitabine (2\ 2'-difluorodeoxycytidine) is currently being marketed as Gemzar by Eli Lilly. It is a nucleoside analogue of deoxycytidine, which was first disclosed in US4808614 and US5464826. Gemcitabine has been indicated as the first-line therapy for locally advanced (nonresectable Stage II or III) or metastatic (Stage IV) adenocarcinoma of the pancreas. Gemcitabine has also been indicated as a second-line therapy for patients who have previously been treated with fluorouracil.
Cyclin-dependant kinase (CDK), a class of genes involved in cell cycle pathways are emerging targets in cancer drug discovery. Several CDK inhibitors are currently undergoing clinical evaluation either as a single agent or in combination with other anticancer drugs. Flavones are series of novel compounds that exhibit significant specific activity against CDKs especially CDK4. P276-00 is a flavone compound with a potent anti-CDK4 activity which is described in US7271193 which is herein incorporated by reference in its entirety. CDK4 catalyzes the phosphorylation and inhibition of retinoblastoma (RB) protein, which is a negative regulator of cell cycle. Phosphorylation of RB leads to uncontrolled cell proliferation and induces tumorigenesis in cells. P276-00 was found to be more selective for CDK4-D1, CDKl-B, and CDK9 -Tl, as compared with other CDKs, and less selective for non-CDK kinases. It showed potent

antiproliferative effects against various human cancer cell lines as demonstrated by both in vitro and in vivo experimental conditions. It is also observed that P276-00 induced a significant down-regulation of cyclin Dl and CDK4 and a decrease in CDK4 -specific pRB Ser(780) phosphorylation. The compound also induced apoptosis in human promyelocytic leukemia (HL-60) cells, as evidenced by the induction of caspase-3 and DNA ladder studies.
P1446A-05 is a (+)-trans~3-[2[(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride (Molecular Formula: C22H19CIF3NO5.HCI)), Its mode of action reveals that it is a novel, potent inhibitor of CDK4-D1, CDK4-B and CDK9-T and is described in US7271193 which is incorporated herein by reference in its entirety.
The present invention provides novel synergistic combination of Gemcitabine with P276-00 or PI446A. The disclosure also provides an unique gene signature comprising gene markers for monitoring drug response in patients undergoing cancer treatment with the said combination.
Summary
The present invention relates to a novel pharmaceutical compositions comprising combination of Gemcitabine with P276-00 or P1446A. The said combinations are used in the treatment of cancer. It further provides a gene signature comprising markers used to monitor the drug response in a subject administered with the aforementioned combination compositions.
In one aspect, the invention provides a pharmaceutical composition comprising a combination of Gemcitabine and P276-00. This combination shows a synergistic effect as compared to the individual use of the agents.

In another aspect, the invention provides a pharmaceutical composition comprising a combination of Gemcitabine and P1446A. The said combination demonstrates a synergistic effect as compared to the individual use of the agents.
In yet another aspect, the invention provides a method of treating cancer using the combinations of Gemcitabine with P276-00 or P1446A. The types of cancer treated with these combinations are pancreatic cancer, lung cancer, colorectal carcinoma and head and neck cancer.
The invention also provides a kit containing the aforementioned compositions.
The invention further provides a gene signature comprising at least two markers for monitoring the drug response in a patient administered with the aforementioned compositions.
The gene signature for the combination of Gemicitabine and P276-00 comprises at least two markers selected from the group consisting of SNX7, FA38A, DNAI1, RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2BO. Furthermore, in patients administered with the combination of Gemcitabine and P276-00, up regulation is observed in drug response markers SNX7, FA38A or DNAI1 and down regulation is observed in RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 or HIST1H2BO
In another aspect, the invention provides a gene signature for monitoring drug response in a subject administered with the combination of Gemcitabine and P1446A comprising at least two markers selected from the group consisting of P21, REV3L, FGF5, PTK7, POLH, P27 and SSTR2. Upon administration of the combination Gemcitabine and P1446A, there is upregulation of the said markers.

In yet another aspect, the invention provides a method of monitoring the drug response in a patient suffering from cancer and treated with a combination of Gemcitabine and P276-00, comprising detection of gene signature with at least two markers selected from the group consisting of SNX7, FA38A, DNAI1, RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2BO.
The invention also provides a method a method of monitoring the drug response in a patient suffering from cancer and treated with a combination of Gemcitabine and P276-00, comprising detection of gene signature with at least two markers selected from the group consisting of P21, REV3L, FGF5, PTK7, POLH, P27 and SSTR2.
Brief description of figures
Fig 1: Effect of sequentially administered combination of Gemcitabine with P276-00 on Panc-1 cells using propidium iodide based fluorescence cytotoxicity assay. The red arrow marks the synergistic effect of the combination Gemcitabine (0.1 nM) and P276-00 (60nM).
Fig 2: Effect of sequentially administered combination of Gemcitabine with P276-00 on Panc-1 cells using Flow cytometry.
Fig 3: Effect of sequentially administered combination of Gemcitabine with PI446A on Panc-1 cells using CCK-8 cytotoxicity assay.
Detailed description of the invention
The invention is based on the observation that a novel combination of Gemcitabine with
P276-00 or PI446A shows synergistic effect as compared to the compounds given
individually.
In one embodiment, the combination of Gemcitabine and P276-00 is disclosed. A
combination of Gemcitabine and PI446A is also further described. These combinations
show a synergistic effect compared to the agents given individually. This synergistic
effect observed forms the basis of the invention.

Gemcitabine (2'-deoxy-2', 2'-difluorocytidine) is a nucleoside analogue of deoxycytidine. It exhibits cell phase specificity, primarily killing cells undergoing DNA synthesis (S-phase) and also blocking the progression of cells through the Gl/S-phase boundary. Gemcitabine is metabolised intracellularly by nucleoside kinases to the active diphosphate (dFdCDP) and triphosphate (dFdCTP) nucleosides. Its cytotoxic effect can be attributed to the inhibition of DNA synthesis as a result of the combined actions of the diphosphate and triphosphate nucleosides. More specifically, Gemcitabine diphosphate inhibits ribonucleotide reductase, which is responsible for catalysing the reactions that generate the deoxynucleoside triphosphates for DNA synthesis. Inhibition of this enzyme by the diphosphate nucleoside causes a reduction in deoxynucleotide concentrations, for example dCTP. Furthermore, Gemcitabine triphosphate competes with dCTP for incorporation into DNA. The subsequent reduction in the intracellular concentration of dCTP enhances the incorporation of Gemcitabine triphosphate into DNA (self potentiation). Gemcitabine exhibits antitumour activity, particularly against ovarian, pancreatic and lung cancers. Gemcitabine finds use in the first-line therapy for locally advanced (nonresectable Stage II or III) or metastatic (Stage IV) adenocarcinoma of the pancreas. Patients previously treated with fluorouracil use Gemcitabine as a second-line therapeutic agent.
P276-00 as disclosed in US7271193 is flaovone compound with ant-CDK4 activity. . The compound finds use in antiproliferative therapies for diseases characterized by excessive cell growth such as cancers, cardiovascular abnormalities, nephrological disorders, psoriasis, Alzheimer's disease, immunological disorders involving unwanted proliferation of leukocytes, restenosis and other proliferative smooth muscle disorders, viral infections, and mycotic infections.
P1446A-05(+)-trans-3-[2[(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride. It is a novel, potent inhibitor of CDK4-D1, CDK4-B and CDK9-T. Pre-clinical data from human

cancer cell lines has shown that P1446A-05 halts transcriptional elongation, promotes apoptosis and arrests cell cycle progression at Gl and G2 phases of cell cycle. In vitro studies with a variety of cancer cell lines suggest that PI446A effectively inhibits proliferation of and induces cytotoxicity in both cisplatin sensitive and resistant cells without any significant cytotoxicity to normal human peripheral blood mononuclear cells. P1446A-05 induces significant down regulation of Cyclin Dl and CDK4 specific retinoblastoma protein (pRb) ser780 phosphorylation, induced p53 and reduced the levels of the anti-apoptotic protein Bcl-2. P1446A-05 also demonstrated good oral bioavailability in the preclinical studies. Upon oral administration, it demonstrated significant tumor growth inhibition in xenograft models of colon cancer (HCT-116, SW-480) and non-small cell lung cancer (H-460) in SCID mice. By virtue of its specific action against CDK4-D1, CDK1-B and CDK9-T, P1446A-05 has the potential to have potent effects on cell cycle arrest while avoiding the unwanted effects associated with more non-specific CDK inhibitors.
In another embodiment of the invention is provided a method of treating cancer by administering combinations of Gemcitabine with P276-00 or PI446A. The types of cancer treated with the said combinations include but are not limited to pancreatic cancer, lung cancer, colorectal carcinoma and head and neck cancers. In a preferred embodiment, the combinations are used to treat pancreatic cancer.
In yet another embodiment, the invention provides a dosage range of the combinations used to administer to the patients suffering from cancer. The dosage varies between a final concentration range of 0.1 nM-30 nM for Gemcitabine and 60- 960 nM for P276-00. In case of Gemcitabine with PI446A, the dosage varies between a final concentration of 3 nM-10000 nM for Gemcitabine andl nM-10000 nM for PI446A. Various possible combinations of the agents in the aforementioned dosage range are used in the invention.
The invention also provides a method of administering the said combinations. The method involves administering the combinations either simultaneously or sequentially. The method of administration preferably is sequential wherein Gemicitabine is first

administered followed by the administration of P276-00 or P1446A. Gemcitabine is administered as a 30 min bolus infusion while P276-00 is administered intravenously or as a bolus infusion for 30 mins. PI446A is administered orally.
In one embodiment, the present invention provides a dosing schedule of the combinations. The combination of Gemcitabine with P276-00 is administered in the following steps; Gemcitabine is first administered for 0-24 hrs followed by sequential administration of P276-00 for 24-96 hrs. Gemcitabine with P1446A is administered in the following steps; Gemcitabine is first administered for 0-24 hrs followed by sequential administration of P1446A for 24-72 hrs.
In another embodiment of the invention is provided a kit comprising of Gemcitabine with P276-00 or PI446A. The combinations in a kit provide convenience to the end user. The kit also comprises an insert detailing the dosing regime for the administration of the combinations.
The present invention further provides a gene signature for monitoring the drug response in a patient who has been administered the combinations of the invention. Method of monitoring the drug response following administration of Gemcitabine and P276-00 or Gemcitabine and PI446A is also encompassed in the invention.
Microarray-based gene signatures provide valuable information regarding the cellular function and can be effectively used in determining the drug response in patients subsequent to exposure to combination of therapeutic agents. (Nature Genetics 2003; 34:85-90).
The gene signature of the present invention used to monitor the drug response in a patient administered with the combination of Gemcitabine and P276-00 comprises of at least two markers selected from the group consisting of SNX7, FA38A, DNAI1, RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2BO. Amongst these markers SNX7, FA38A or DNAI1 are up regulated while RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1,

TMPRSS3 and HIST1H2B0 are down regulated in patients administered with the said combination.
The above described drug response markers are detailed below:
SNX7: This gene encodes a member of the sorting nexin family. Sorting nexins are proteins involved in protein trafficking in the endosomes and the vesicular micro-tubular structures in the cytoplasm. Two isoforms of the sorting nexin family (SNX1 & SNX2) have been demonstrated to play a functional role in localization of the endogenous EGFR in the endosomes of colon cancer cells. Deletion mutants for SNX1 have proved to down-regulate the endogenous EGFR expression. Although there is no current evidence identified for SNX7, our data could prove the involvement of SNX7 in pancreatic cancer disease biology.
DNAI1: The inner- and outer-arm dyneins, which bridge between the doublet microtubules in axonemes, are the force-generating proteins responsible for the sliding movement in axonemes. The intermediate and light chains, thought to form the base of the dynein arm, help mediate attachment and may also participate in regulating dynein activity. This gene encodes an intermediate chain dynein, belonging to the large family of motor proteins. Mutations in this gene result in abnormal ciliary ultrastructure and function associated with primary ciliary dyskinesia (PCD) and Kartagener syndrome.
RRM2: Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from ribonucleotides. It is composed of 2 non-identical subunits, proteins Ml and M2. Synthesis of M2 is regulated in a cell-cycle dependent fashion. Activity of this enzyme, which catalyses conversion of ribonucleotide 5'-diphosphates to their 2'-deoxynucleotides, is modulated by levels of its M2 subunit (RRM2). Here we show that RRM2 overexpression is associated with Gemcitabine chemo resistance in pancreatic adenocarcinoma cells, and that suppression of RRM2 expression using RNA interference mediated by small interfering RNA (siRNA) enhances Gemcitabine-induced cytotoxicity in vitro.
CDK8: The protein encoded by this gene is a member of the cyclin-dependent protein kinase (CDK) family and are known to be important regulators of cell cycle progression.

This kinase and its regulatory subunit cyclin C are components of the RNA polymerase II holoenzyme complex, which phosphorylates the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. This kinase has also been shown to regulate transcription by targeting the CDK7/cyclin H subunits of the general transcription initiation factor IIH (TFIIH), thus providing a link between the 'Mediator-like' protein complexes and the basal transcription machinery. Furthermore, increased binding of CDK8 to p53 target genes correlates positively with transcriptional strength and CDK8 functions as a coactivator within the p53 transcriptional program.
DLG5: This gene encodes a member of the family of discs large (DLG) homologs, a subset of the membrane-associated guanylate kinase (MAGUK) super family. The protein encoded by this gene localizes to the plasma membrane and cytoplasm, and interacts with components of adherens junctions and the cytoskeleton. It is proposed to function in the transmission of extracellular signals to the cytoskeleton and in the maintenance of epithelial cell structure.
FGF5: The protein encoded by this gene is a member of the fibroblast growth factor (FGF) family. FGF family members possess broad mitogenic and cell survival activities, and are involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. This gene was identified as an oncogene, which confers transforming potential when transfected into mammalian cells.
MKKS: This gene encodes a protein with sequence similarity to the chaperonin family. The encoded protein may have a role in protein processing in limb, cardiac and reproductive system development. Mutations in this gene have been observed in patients with Bardet-Biedl syndrome type 6 and McKusick-Kaufman syndrome.

PPIL4: This gene is a member of the cyclophilin family of peptidylprolyl isomerases. The cyclophilins are a highly conserved family, members of which play an important role in protein folding, immunosuppression by cyclosporin A, and infection of HIV-1 virions.
HELLS: This gene encodes a lymphoid-specific helicase. Other helicases function in processes involving DNA strand separation, including replication, repair, recombination, and transcription. This protein is thought to be involved with cell proliferation and may play a role in leukemogenesis.
ID1: The protein encoded by this gene is a helix-loop-helix (HLH) protein that can form heterodimers with members of the basic HLH family of transcription factors. The encoded protein has no DNA binding activity and therefore can inhibit the DNA binding and transcriptional activation ability of basic HLH proteins with which it interacts. This protein may play a role in cell growth, senescence, and differentiation. ID-1 is suggested as an oncogene and is reported to promote cell proliferation, invasion, and survival in several types of human cancer cells through multiple signaling pathways.
DICER1: This gene encodes a protein possessing an RNA helicase motif containing a DEXH box in its amino terminus and an RNA motif in the carboxy terminus. The encoded protein functions as a ribonuclease and is required by the RNA interference and small temporal RNA (stRNA) pathways to produce the active small RNA component that represses gene expression.
TMPRSS3: This gene encodes a protein that belongs to the serine protease family. The encoded protein contains a serine protease domain, a transmembrane domain, a LDL receptor-like domain, and a scavenger receptor cysteine-rich domain. Serine proteases are known to be involved in a variety of biological processes, whose malfunction often leads to human diseases and disorders. This gene was identified by its association with both congenital and childhood onset autosomal recessive deafness. This gene was also identified as a tumor associated gene that is overexpressed in ovarian tumors.

HIST1H2BO: Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. The linker histone, HI, interacts with linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures. This gene is intronless and encodes a member of the histone H2B family. Transcripts from this gene lack polyA tails but instead contain a palindromic termination element. This gene is found in the small histone gene cluster on chromosome 6p22-p21.3.
Drug response in patients administered with the combination of Gemcitabine and P1446A is monitored using a gene signature comprising at least two markers selected from the group consisting of P21, REV3L, FGF5, PTK7, POLH, P27, and SSTR2. Expression of these markers is upregulated upon administration of Gemcitabine and P1446A.
These gene markers are herein further described:
P21: This gene encodes a potent cyclin-dependent kinase inhibitor. The encoded protein binds to and inhibits the activity of cyclin-CDK2 or -CDK4 complexes, and thus functions as a regulator of cell cycle progression at Gl phase of the cell cycle. The expression of this gene is tightly controlled by the tumor suppressor protein p53, through which this protein mediates the p53-dependent cell cycle Gl phase arrest in response to a variety of stress stimuli. This protein can interact with proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory factor, and plays a regulatory role in S phase DNA replication and DNA damage repair.
REV1: This gene encodes a protein with similarity to the S. cerevisiae mutagenesis protein Revl. The Revl proteins contain a BRCT domain, which is important in protein-protein interactions. A suggested role for the human Revl-like protein is as a scaffold that recruits DNA polymerases involved in translesion synthesis (TLS) of damaged DNA.

PTK7: Receptor protein tyrosine kinases transduce extracellular signals across the cell membrane. A subgroup of these kinases lack detectable catalytic tyrosine kinase activity but retain roles in signal transduction. The protein encoded by this gene is a member of this subgroup of tyrosine kinases and may function as a cell adhesion molecule. This gene is thought to be expressed in colon carcinomas but not in normal colon, and therefore may be a marker for or may be involved in tumor progression.
P27: This gene encodes a cyclin-dependent kinase inhibitor, which shares a limited similarity with CDK inhibitor CDKNlA/p21. The encoded protein binds to and prevents the activation of cyclin E-CDK2 or cyclin D-CDK4 complexes, and thus controls the cell cycle progression at Gl. The degradation of this protein, which is triggered by its CDK dependent phosphorylation and subsequent ubiquitination by SCF complexes, is required for the cellular transition from quiescence to the proliferative state.
SSTR2: Somatostatin acts at multiple sites to inhibit the release of many hormones and other secretory proteins. The biologic effects of somatostatin are probably mediated by a family of G protein-coupled receptors that are expressed in a tissue-specific manner. SSTR2 is a member of the superfamily of receptors having seven transmembrane segments and is expressed in highest levels in cerebrum and kidney. Introduction of the SSTR2 gene, the expression of which is frequently lost in human pancreatic adenocarcinoma, exerts its anti-angiogenic effects by down-regulating the expression of the factors, which are involved in tumor angiogenesis and metastasis, suggesting SSTR2 gene transfer as a promising strategy of gene therapy for pancreatic cancer.
In a yet another embodiment the invention provides a method of monitoring the drug response in a patient administered with the combination of Gemcitabine and P276-00. The method involves detection of the gene signature wherein SNX7, FA38A or DNAI1 are up regulated and RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2BO are down regulated.
In a further embodiment a method of monitoring the drug response in a patient administered with the combination of Gemcitabine and PI446A is provided. The method

involves detection of the gene signature wherein up regulation of markers P21, REV3L, FGF5, PTK7, POLH, P27, and SSTR2 is observed.
Examples
Example 1. Propidium iodide-based fluorescence cytotoxicity assay
The combination of P276-00 and Gemcitabine was screened for its synergistic effect in Panc-1 cells using propidium iodide based fluorescence cytotoxicity assay.
Test System
The Panc-1 cell line was procured from ATCC (American Tissue type Culture Collection), USA. Catalog number: CRL-1469 and the frozen vial were stored in liquid nitrogen container. Propidium iodide dye was procured from Sigma-Aldrich, USA. Catalog number: P-4170-100 mg and it was stored in 2-8°C.
Method to perform the assay
Panc-1 cells were seeded at a density of 2000 cells/well, in a 200 uL in tissue culture grade 96 well plate and allowed to recover for 24 hrs in a humidified 5% ± 0.2 CO2 incubator at 37 °C ± 0.5 °C. After 24 hrs, 1 uL of 200 X (200 times higher than required concentration is denoted as 200 X) compound (dissolved in neat DMSO), as per Table 1 and 2 was added to the wells. The final DMSO concentration was 0.5% in wells. The plate was incubated for 24hrs in humidified 5% + 0.2 CO2 incubator at 37 ± 0.5 °C. After 24 hrs the Minimum Essential Medium (MEM) from the Gemcitabine treated wells was removed and washed two times with fresh MEM and followed by addition of 200 uL of fresh MEM per well. 1 uL of 200 X (200 times higher than required concentration is denoted as 200 X) compound (dissolved in neat DMSO) was then added as per the Table 1 and 2 designs. The final DMSO concentration was 0.5% in wells which also served as the vehicle control for the study. After 72 hrs the plate was removed from CO2 incubator and spent MEM aspirated from the wells and supplemented with fresh MEM. This was

followed by addition of 25 uL of propidium iodide (50 fig/ml in medium) per well. The same plate was frozen at -80°C for 24 hrs and then thawed and allowed to come to room temperature. The fluorescence at a wavelength of 530nM excitation and 620nM emission was read.
The percent cytotoxicity was calculated using the following formula
Percent (Reading of Control -Reading of Treated cells]
X 100
Cytotoxicity = Reading of Control
Results
The propidium iodide based cytotoxicity assay showed that Gemcitabine (0.1 nM) ICio for 24 hrs followed by P276-00 (60 nM) ICio for 72 hrs showed synergistic toxicity of 78% (Fig.l, Table 2). In vitro testing showed that these two agents produce minimal cytotoxicity at ICio alone (Table 1) but on combination at ICio, they displayed additional antiproliferative /cytotoxicity effects across a range of dosages (Table 2). The synergistic effect of gemcitabine and P276-00 combination not only make cancer cells more susceptible but further facilitate use lower doses of drugs in patients. Table 1: Effect of independent administration of Gemcitabine and P276-00 on Panc-1 cells

Concentration (nM) Fluorescence Unit % Coefficient of Variance % Cytotoxicity in Panc-1 cells
Gemcitabine 0.1 nM 31455 4.844 10
60 nM P276-00 24558 3.168 27
120 nM P276-00 22768 9.355 33
240 nM P276-00 22212 4.860 35
480 nM P276-00 18831 6.630 45
960 nM P276-00 15416 6.76 58
Control 34599 3.829 0

Table 2. Effect of the sequentially administered combination of Gemcitabine with P276-00 on Panc-1 cells

Gemcitabine concentration 0.1 nM for 24 hrs + P276-00 at various concentrations Fluorescence unit % Coefficient of Variance % Cytotoxicity
60 nM P276-00 for 72 hrs 12147 3.413 78
120nMP276-00for72hrs 10384 4.320 82
240 nM P276-00 for 72 hrs 11851 2.885 79
480 nM P276-00 for 72 hrs 11796 3.390 79
960 nM P276-00 for 72 hrs 9654 2.069 83
Control 55847 6.010 0
Example 2. Cell cycle analysis by DNA content
The synergistic effect Gemcitabine with P276-00 on Panc-1 cancer cells was analyzed using propidium iodide based cell cycle analysis where total population of cells was sorted in to sub GO, Gl, S and G2/M populations according to total fluorescent intensity.
Dosage
P276-00 at a final concentration of 60 nM, 240 nM and 480 nM and Gemcitabine at a final concentration of 1 nM, 3 nM and 10 nM were analyzed in single dose and in all possible combinations of the dose range for the Gemcitabine and P276-00.
Cell cycle analysis using Flow cytometry
Methodology
Panc-1 cells were treated with Gemcitabine for 0 to 24 hrs. After 24 hrs the cells were washed two times with plain MEM. Fresh MEM with 10% serum (2 mL/well) was added to the wells, followed by treatment with P276-00 for 24 hrs to 96 hrs. After 96 hrs the

treated cells were harvested, fixed, stained with propidium iodide and analyzed for specific cell arrest using DNA content analysis by flow cytometry.
Adherent cells were trypsinized and washed with PBS or HBSS without Ca++ & Mg++ containing EDTA pH 8.0 and BSA. They were further isolated, centrifuged and total number of cells were counted and recorded. The pellet of cells was resuspended in ice-cold PBS and a uniform suspension was prepared. Cold ethanol was slowly added to the suspension while vortexing it. The cells were then fixed at 4°C overnight.
The cells were then taken in a conical tube and centrifuged to remove the ethanol. The pellet was resuspended in PBS and Calf serum after vortexing and washing it in the same. Propidium iodide and boiled RNase S was added to it and incubated at 37° C for 30 mins. Analysis was carried out using a flow cytometer (Becton Dickinson FACSCalibur, a 4-color, dual-laser benchtop flow cytometer).
Results
The results of the cell cycle analysis revealed that control cells showed 5.5 % population of cells in sub GO (apoptotic cells) (Fig 2). Gemcitabine 1 nM treated cells showed 16.85% population of cells in subGO and P276-00 60 nM treated cells showed 17.6 % population of cells in subGO. Interestingly, when cells were exposed to the combination of Gemcitabine (1 nM) for 24 hr followed by P276-00 (60 nM, 240 nM and 480 nM) for 72 hr, they showed an increase of 59.5%, 51.6% and 61.3 respectively in subGO population (apoptosis). These results further substantiate the synergistic activity displayed by Gemcitabine with P276-00.
Example 3. Microarray analysis to detect gene expression signatures associated with the in vitro synergistic effect of the combination therapy of Gemcitabine with P276-00 in Panc-1 cells
The goal of the microarray analysis was to evaluate putative gene expression signatures associated with the in vitro synergistic effect of the combination therapy of Gemcitabine with P276-00 in Panc-1 cells.

The time points (3 hrs, 6 hrs, 12 hrs) were chosen to establish early molecular changes in the transcription profile for probable biomarkers and mechanism of action for the combination therapy.
Methodology
Sample processing and RNA extraction
Approx 20 million cells were lysed in Trizol Reagent (Invitrogen Corp, USA) and passed through a 21-gauge needle for 5-10 times before RNA extraction was performed. Total RNA extraction was performed using the RNeasy Mini kit (Qiagen, USA) according to the manufacturer's protocol. The final total RNA was eluted in nuclease-free water and the concentration/purity was determined by Nanodrop spectrophotometer (Thermo Fisher Scientific, Delaware, USA).
cDNA generation and labeling
20 ug of total RNA was reverse transcribed using amino allyl dUTP and oligo dT, the resulting cDNA was indirectly labeled with Cy3/Cy5 dyes (GE Healthcare, USA) using a fluorescent labeling kit (Promega Biosciences, USA). The concentration and dye incorporation of the labeled products were determined by Nanodrop spectrophotometer (Thermo Fisher Scientific, Delaware, USA). Equal amounts of labeled cDNA were denatured at 95° C for 5 mins, cooled and microfuged at 13200 rpm for 5 mins before hybridization.
Array fabrication and hybridization
70-mer transcript oligonucleotides representing 36,480 human genes was purchased from Operon Biotechnologies (Germany) and spotted onto aminosilane coated glass slides (Arraylt, USA) by contact printing using a Omnigrid (OG100) spotter (Genomic Solutions Inc, USA). Post printing, the slides were cross-linked with UV light at 650 mJ/sec2, vacuumed, sealed and stored in the dark.
The slides were pre-processed for background reduction using Pronto (Promega Life sciences, USA) pre-hyb kit as per the manufacturer's instructions. Hybridization was carried out in the GeneTAC Hybstation (Genomic Solutions Inc, USA) for 18 hrs. The hybridization protocol is as follows: 6 hrs at 42° C (with agitation), 6 hrs at 35° C (with

agitation), and 6 hrs at 30 C (with agitation). Post hybridization, the arrays were washed according to the manufacturer's protocol. The slides were dried by centrifugation at 1600 rpm for 5 mins.
Scanning and image analysis
Post drying, the slides were scanned in a laser scanner (Genomic Solutions, USA) for Cy3 and Cy5 detection. The PMT settings were adjusted using the auto-exposure feature of the scanner. The TIFF files thus generated were exported to GeneTAC Integrator software (Genomic Solutions, USA) for image analysis. The resultant CSV file containing the annotated genome and the corresponding ratios were further used for data analysis.
Results
The combination of Gemcitabine and P276-00 used for the treatment in Panc-1 cell line induces a dynamic gene expression change with respect to compound action, time of dosing and the concentrations. The major gene families that have been dys-regulated by combination treatment are the proteins of the serine-threonine kinase family (Table 3). The IC50 doses also up regulate the inflammatory pathways downstream of TNFR family without the induction of NFKBI indicating the inhibition of the pro-survival pathways in Panc-1 cell line.
Table 3: Gene expression values following drug exposure in Panc-1 cell lines

GeneID QenelVame _, •-.■ t •, ;-.-,\ , tGeni!cit»lyne. E276 .Combination
HIST1H2BO Histone l,H2BO -0.71 -0.39 -3.28
TMPRSS3 Transmembrane protease, serine 3 0.85 0.33 -1.92
DICER1 Dicer 1, Dcr-1 homolog -0.16 0.14 -1.74
IDl Inhibitor of DNA binding 1 -0.60 0.18 -1.24
SLC19A2 Solute carrier family 19 member 2 0.37 0.97 -1.02
PPIL4 Peptidylprolyl isomerase -like 4 0.36 0.30 -1.04
HELLS Helicase, lymphoid-specific 0.40 0.62 -1.05
MKKS McKusick-Kaufman syndrome -0.02 -0.08 -1.86

FGF5 Fibroblast growth factor 5 0.28 0.23 -0.49
DLG5 Discs, large homolog 5 -0.19 0.25 -0.46
CDK8 Cyclin-dependent kinase 8 -0.03 0.16 -0.61
RRM2 Ribonucleotide reductase M2 0.69 -0.14 -0.94
DNAI1 Dynein, 0.03 -0.20 2.02
FA38A FA38A -0.70 -0.93 1.96
SNX7 Sorting nexin 7 0.27 -0.12 1.93
*Numerical value indicates log value of intensities as obtained by microarray experiments. Values close to zero indicate normal expression, negative value denotes down regulation and positive value indicates up-regulation of gene expression.
Example 4. CCK-8 based cytotoxicity assay
The combination of Gemcitabine with PI446A was screened for its synergistic effect in Panc-1 by CCK-8 (Cell counting kit-8) based cytotoxicity assay. CCK-8 (Cell counting kit-8) based cytotoxicity assay is a sensitive nonradioactive colorimetric assay for determining the number of viable cells in cell proliferation and cytotoxicity assays. WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H tetrazolium, monosodium salt) is bioreduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells.
Test System
The Panc-1 cell line was procured from ATCC (American Tissue type Culture Collection USA, catalog number: CRL-1469) and the frozen vial was stored in liquid nitrogen container.
Methodology
Gemcitabine at final concentrations of 1 nM, 10 nM and 100 nM and PI446A at a final concentration of 3 nM, 10 nM, 30 nM, 100 nM and 300 nM were analyzed in single dose and in all possible combinations of the dose range for the two drugs mentioned above. Panc-1 cells were seeded at a density of 3000 cells/well in a 96 well plate and allowed to

recover for 24 hrs in a CO2 incubator at 37 C. Subsequently, compounds (Gemcitabine and/or P1446A) were added to the plates. The final DMSO concentration was 0.5% in wells. Plates were incubated for 24, 48, and 72 hrs. At the end of incubation CCK-8 was added and kept at 37°C for 3 hrs following which absorbance was measured at 450nM.
Results
Combination of Gemcitabine and PI 446A was observed to exhibit synergistic effect at a concentration of 10 nM and 100 nM respectively. Gemcitabine at 10 nM showed cytotoxicity of 31.47% while P1446A at 100 nM, showed cytotoxicity of 29.6%. However when used as a combination of Gemcitabine 10 nM for 24 hrs, followed by incubation with P1446A 100 nM for 72 hrs, an increase in cytotoxicity to 90.5% was noted, which is at least 29% more cytotoxicity than the additive effect of the two agents suggesting a marked synergistic effect of the combination (Table 4).
Gemcitabine and PI446A were also found to be more synergistic at the 10 nM and 300 nM respectively. Gemcitabine at 10 nM showed cytotoxicity of 31.47% and P1446A at 300 nM, showed cytotoxicity of 31.05%. However when used as a combination of Gemcitabine 10 nM for 24 hrs, followed by PI446A 300 nM for 72 hrs an increase in cytotoxicity to the extent of about 95% was noted, which is 33% more cytotoxicity than the additive effect suggesting a significant synergy of the combination drug (Table 4).
Table 4. Effect of combination of Gemcitabine with P1446A in Panc-1 cells

Concentration in nM % Cytotoxicity
24 hrs 48 hrs 72 hrs
GemlOnM 21.997 27.571 31.471
P1446A 30 nM 29.825 14.163 23.33
P1446A100nM 26.525 24.596 29.603
P1446A 300 nM 34.36 28.636 31.075
GemlO nM+ P1446A/ 30 nM 63.099 73.640 83.655

Geml0nM+P1446A/100 nM 73.935 75.136 90.527
Geml0nM+P1446A/300 nM 82.788 84.482 94.720
Gem= Gemcitabine
Example 5. Gene expression profile using RTQ-PCR
The objective of the experiment was to evaluate the synergistic effect of drug combination (Gemcitabine with PI 446 A) as compared to individual use of the same. The synergistic effect of the compound or drug combination in the Panc-1 cell line was measured in terms of gene expression and expressed as fold changes as compared to the cell control with no drug treatment.
Methodology
Cell lines treated with drugs (Gemcitabine, P1446A or both) were used for total RNA isolation using a commercial RNA extraction kit (Qiagen Corporation, Germany). The first-strand cDNA was synthesized from total RNA using first strand cDNA synthesis kit from Invitrogen Corporation (California, USA). This is followed by real time quantitative polymerase chain reaction (RTQ PCR) using gene specific primers and standard thermal program of initial denaturation at 95°C for 5 mins and 40 cycles of 95°C for 10 seconds, followed by 60°C for 30 seconds (Realplex PCR machine from Eppendorf, Germany). Quantitative measurement of products made during PCR cycles was normalized against a housekeeping gene (Actin) and used to measure the gene expression as fold changes as compared to respective control.
Results
Gene markers, which showed up regulation in response to combination therapy of Gemcitabine with P1446A, are listed in Table 7.
Table 7. Gene expression profile for cancer markers in Panel cell line after exposure to a Gemcitabine, P1446A and combination of both.

Genes Gene Name Gemcitabine P1446A Gemcitabine + P1446A
P21 Cyclin-dependent kinase inhibitor 1A 2.87 6.04 7.94
REV3L REV3-like 0.59 1.04 2.35
FGF5 Fibroblast growth factor 5 1.42 2.76 4.63
PTK7 PTK7 protein tyrosine kinase 7 0.96 -0.19 4.00
POLH Polymerase (DNA directed), eta 0.51 0.10 1.40
P27 Cyclin-dependent kinase inhibitor IB -0.38 0.70 2.61
SSTR2 Somatostatin receptor 2 -1.52 0.71 7.38

Claims
We claim:
1. A pharmaceutical composition comprising a combination of Gemcitabine and P276-00.
2. A pharmaceutical composition comprising a combination of Gemcitabine and P1446A.
3. A method of treating cancer in a subject, the said method comprising administering the combination of claim 1 or claim 2.
4. The method of claim 3, wherein the said cancer is selected from a group comprising of pancreatic cancer, lung cancer, colorectal carcinoma and head and neck cancer.
5. The method of claim 3, wherein the said cancer is pancreatic cancer.
6. The method of claim 3, where the combination is administered simultaneously or sequentially.
7. A kit comprising the pharmaceutical composition as claimed in claim 1 and 2.
8. A gene signature comprising at least two markers for monitoring drug response in patients administered with the composition as claimed in claim 1.
9. The gene signature of claim 8, wherein the said drug response markers are selected from the group consisting of SNX7, FA38A, DNAI1, RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2BO.
10. The gene signature of claim 9, wherein the said drug response markers SNX7, FA38A or DNAI1 are up regulated in patients administered with the composition as claimed in claim 1.
11. The gene signature of claim 9, wherein the said markers RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 or HIST1H2BO are down regulated in patients administered with the composition as claimed in claim 1.

12. A gene signature comprising at least two drug response markers for monitoring drug response in patients administered with the composition as claimed in claim 2.
13. The gene signature of claim 12, wherein the drug response markers are selected from the group consisting of P21, REV3L, FGF5, PTK7, POLH, P27 and SSTR2.
14. The gene signature of claim 12, wherein the said markers P21, REV3L, FGF5, PTK7, POLH, P27 and SSTR2 are up regulated in patients administered with the composition as claimed in claim 2.
15. A method of monitoring drug response in a patient suffering from cancer treated with a combination of Gemcitabine and P276-00, comprising detection of a gene signature with at least two drug response markers.
16. The method of claim 15, wherein the said drug response markers are selected from the group consisting of SNX7, FA38A, DNAI1, RRM2, CDK8, DLG5, FGF5, MKKS, HELLS, PPIL4, SLC19A2, ID1, DICER1, TMPRSS3 and HIST1H2BO.
17. A method of monitoring drug response in a patient suffering from cancer treated with a combination of Gemcitabine and P1446A, comprising detection of a gene signature with at least two drug response markers.
18. The method of claim 17, wherein the said drug response markers are selected from the group consisting of P21, REV3L, FGF5, PTK7, POLH, P27 and SSTR2.

Abstract
Novel synergistic combination of gemcitabine with P276-00 or PI 446A and their use in the treatment of cancer are disclosed. The invention further describes gene signatures comprising gene markers used to monitor the drug response in a subject treated with the said combination.

A GENE SIGNATURE FOR MONITORING CANCER PATIENTS TREATED WITH A COMBINATION OF GEMCITABINE AND P276-00

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