Combination of HDAC inhibitors and statins for treatment of pancreatic cancer

Abstract

The present invention relates to combinations of HDAC inhibitors and statins for the treatment of pancreatic cancer. Preferably, the present invention relates to a combination of valproic acid (VPA) or any salt thereof and simvastatin (SIM). The combination of the present invention synergistically improves the anti-proliferative and apoptotic promoting effects of conventional chemotherapy, such as gemcitabine / paclitaxel.

Description

Technical Field

[0001] The present invention relates to a combination of an HDAC inhibitor and a statin for treating pancreatic cancer. Preferably, the present invention relates to a combination of valproic acid (VPA) or any salt thereof and simvastatin (SIM). The combination of the present invention synergistically improves the anti-proliferative and pro-apoptotic effects of conventional chemotherapy such as gemcitabine / paclitaxel. Background Art

[0002] Despite continued advances in cancer therapy, the prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) is extremely poor [Lambert et al. Semin Oncol, 2021], indicating an urgent need for new treatments for this disease. In this regard, repurposing approved non-oncology drugs may be an attractive strategy to provide effective treatment options that are easily translated into early clinical trials.

[0003] Valproic acid (VPA) is a universal, low-cost anticonvulsant with histone deacetylase (HDAC) inhibitory activity. Its anticancer properties have been demonstrated in tumor models including PDAC monotherapy [Luo D et al. Carcinogenesis, 2020] or in combination with gemcitabine [Lin T et al. JECCR, 2019]. The authors of the present invention have recently demonstrated the potential of HDAC inhibitors in sensitizing PDAC cells to gemcitabine / abraxane dual treatment [Roca MS et al. JECCR, 2022]. We are currently exploring the use of VPA plus traditional chemotherapy in clinical studies of solid tumors to fully confirm the feasibility and safety of this approach [Avallone A et al. BMC cancer, 2016; Budillon A et al. Ann One, 2018].

[0004] Statins were developed as lipid-lowering drugs by inhibiting HMG-CoA reductase, and their direct antitumor effects as monotherapy or in combination with chemotherapy and targeted therapy have been further demonstrated in different tumor models including pancreatic cancer [Gupta V et al., Cancer Lett 2018].

[0005] Recently, the authors of the present invention demonstrated the preclinical synergistic antitumor interaction of VPA and the cholesterol-lowering agent simvastatin in a metastatic prostate cancer model, as well as the ability of combined treatment to sensitize prostate cancer cells to docetaxel and restore docetaxel resistance. Mechanistically, this effect is related to the ability of the combination approach to target the cancer stem cell compartment by inhibiting the oncogene YAP [Iannelli F et al. JECCR, 2020]. The present invention reports for the first time data on the synergistic antitumor effect of the VPA / SIM combination in a pancreatic cancer model, demonstrating that the combination approach enhances GEM / NP (gemcitabine / albumin-bound-paclitaxel) dual treatment in both in vitro and in vivo tumor models. In addition, the present invention provides evidence that the mechanism of synergistic antitumor interaction depends at least in part on the reversal of the epithelial-mesenchymal (EMT) transition mediated by VPA / SIM-mediated TGF-β. Summary of the invention

[0006] The present invention relates to a combination comprising at least one HDAC inhibitor and at least one statin for use alone or in combination with at least one other anticancer agent in the treatment of pancreatic cancer.

[0007] Preferably, at least one HDAC inhibitor is selected from valproic acid or a salt thereof, panobinostat, vorinostat, entinostat or mocetinostat, and the statin is selected from simvastatin, atorvastatin, lovastatin; more preferably, the at least one HDAC inhibitor is valproic acid and the statin is simvastatin.

[0008] Preferably, at least one HDAC inhibitor is selected from valproic acid or a salt thereof, panobinostat, vorinostat, entinostat or moxistat, and the statin is selected from simvastatin, atorvastatin, lovastatin; more preferably, the at least one HDAC inhibitor is valproic acid and the statin is simvastatin.

[0009] In a preferred embodiment, the combination comprises at least one HDAC inhibitor and a statin, and the combination is used in subjects who are responsive, resistant, or have developed resistance to first-line therapy, preferably, the first-line therapy comprises the administration of gemcitabine and / or nab-paclitaxel.

[0010] In a preferred embodiment, the combination comprises at least one HDAC inhibitor and one statin, the combination being used at a ratio of at least one HDAC inhibitor and one statin at about a 50:50 cytotoxicity ratio. As used herein, the 50:50 cytotoxicity ratio indicates that the two classes of drugs are used at equivalent doses.

[0011] In other preferred embodiments, the present invention relates to a combination comprising at least one HDAC inhibitor and at least one statin for use alone or in combination with other anticancer agents in the treatment of pancreatic cancer, wherein the at least one HDAC inhibitor is selected from:

[0012] - valproic acid at a concentration ranging from about 16 mM to about 0.5 mM; and / or

[0013] - Vorinostat at a concentration ranging from about 16 μM to about 0.125 μM; and / or

[0014] - Panobinostat at a concentration ranging from about 160 nM to about 2.5 nM; and / or

[0015] - entinostat at a concentration ranging from about 16 μM to about 0.125 μM;

[0016] And wherein the at least one statin is selected from:

[0017] - simvastatin at a concentration ranging from about 8 μM to about 0.06 μM; and / or

[0018] - atorvastatin at a concentration ranging from about 8 μM to about 0.06 μM; and / or

[0019] - Lovastatin at a concentration ranging from about 8 μM to about 0.06 μM.

[0020] In the clinical setting, both valproic acid and simvastatin are orally bioavailable drugs tested at doses within the range of their non-cancer approved indications that were preclinically effective in combination therapy with both drugs plus chemotherapy.

[0021] In other embodiments, the present invention relates to a combination consisting of an HDAC inhibitor and a statin, which is used alone or in combination with at least one other anticancer agent for the treatment of pancreatic cancer, as described above; preferably, the HDAC inhibitor is selected from valproic acid or a salt thereof, panobinostat, vorinostat, entinostat or moxistat, and the statin is selected from simvastatin, atorvastatin, lovastatin; more preferably, the HDAC inhibitor is valproic acid and the statin is simvastatin.

[0022] In other preferred embodiments, the combination of the present invention is used together with one or more other anticancer agents selected from the group consisting of: Btk tyrosine kinase inhibitors, Erbb2 tyrosine kinase receptor inhibitors; Erbb4 tyrosine kinase receptor inhibitors, mTOR inhibitors, thymidylate synthase inhibitors, EGFR tyrosine kinase receptor inhibitors, epidermal growth factor antagonists, Fyn tyrosine kinase inhibitors, KIT tyrosine kinase inhibitors, Lyn tyrosine kinase inhibitors, NK cell receptor modulators, PDGF receptor antagonists, PARP inhibitors, poly ADP ribose polymerase inhibitors, poly ADP ribose polymerase 1 inhibitors, poly ADP ribose polymerase 2 inhibitors, poly ADP ribose polymerase 3 inhibitors, galactosyltransferase regulators, dihydropyrimidine dehydrogenase inhibitors, orotic acid phosphoribosyltransferase inhibitors, telomerase regulators, mucin 1 inhibitors, mucin inhibitors, secretin agonists, TNF-related apoptosis-inducing ligand regulators, IL 17 gene stimulators, interleukin 17E ligands, neurokinin receptor agonists, cyclin G1 inhibitors, checkpoint inhibitors, PD-1 inhibitors, PD-L1 inhibitors, CTLA4 inhibitors, topoisomerase I inhibitors, Alk-5 protein kinase inhibitors, connective tissue growth factor ligand inhibitors, notch-2 receptor antagonists, notch-3 receptor antagonists, hyaluronidase stimulators, MEK-1 protein kinase inhibitors; MEK-2 protein kinase inhibitors, GM-CSF receptor modulators; TNFα ligand modulators, mesothelin modulators, asparaginase stimulators stimulators, caspase-3 stimulators; caspase-9 stimulators, PKN3 gene inhibitors, sonic hedgehog inhibitors; smoothened receptor antagonists, AKT1 gene inhibitors, DHFR inhibitors, thymidine kinase stimulators, CD29 regulators, fibronectin regulators, interleukin-2 ligands, serine protease inhibitors, D40LG gene stimulators; TNFSF9 gene stimulators, 2-ketoglutarate dehydrogenase inhibitors, TGF-β type II receptor antagonists, Erbb3 tyrosine kinase receptor inhibitors, cholecystokinin CCK2 receptor antagonists, Wilms tumor protein modulators, Ras GTPase modulators, histone deacetylase inhibitors, cyclin-dependent kinase 4 inhibitor A modulators, estrogen receptor β modulators, 4-IBB inhibitors, 4-1BBL inhibitors, PD-L2 inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, BTLA inhibitors, HVEM inhibitors, TIM3 inhibitors, GAL9 inhibitors, LAG3 inhibitors, VISTA inhibitors, KIR inhibitors, 2B4 inhibitors, CD160 inhibitors and CD66e modulators, or a combination thereof.

[0023] Preferably, the anticancer agent is selected from baviximab, IMM-101, CAP1-6D, Rexin-G, genistein, CVac, MM-D37K, PCI-27483, TG-01, LOAd-703, CPI-613, umomostat, CRS-207, NovaCaps, trametinib, Atu-027, sonidegib, GRASP A, trabedersen, nastorazepide, Vaccell, oregovomab, istiratumab, refametinib, regorafenib, lapatinib, selumetinib, rucaparib, pelareorep, tarextumab, PEGylated hyaluronidase, varlitinib, aglatimagene besadenovec, GBS-01, GI-4000, WF-10, galunisertib, afatinib, RX-0201, FG-3019, pertuzumab, DCVax-Direct, selinexor, glufosfamide, virulizin, 90Y) tettrium (90Y) clivatuzumab tetraxetan, brivudine, nimotuzumab, algenpantucel-L, tegafur + gimeracil + oteracil potassium + calcium folinate, olaparib, ibrutinib, pirarubicin, Rh-Apo2L, tertomotide, tegafur + gimeracil + oteracil potassium, tegafur + gimeracil + oteracil potassiumpotassium), masitinib, Rexin-G, mitomycin, erlotinib, adriamycin, dexamethasone, vincristine, cyclophosphamide, topotecan, taxol, interferon, platinum derivatives, taxane, paclitaxel, vinca alkaloids, vinblastine, anthracyclines, doxorubicin, epipodophyllotoxins, etoposide, cisplatin, rapamycin, methotrexate, actinomycin D, dolastatin 10, colchicine colchicine, emetine, trimetrexate, metoprine, cyclosporine, daunorubicin, teniposide, amphotericin, alkylating agents, chlorambucil, 5-fluorouracil, campthothecin, metronidazole, imatinib (Gleevec), panitumumab (panitumumab) tumumab), abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, AZD9291, active BCG (BCGLive), bevacuzimab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, camptothecin, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cladribine, clofarabine, cyclophosphamide, cytarabine, dactinomycin, darbepoetin alfa alfa), daunorubicin, denileukin, dexrazoxane, docetaxel, doxorubicin (neutral), doxorubicin hydrochloride, dromostanolone propionate, epirubicin, epoetin alfa, estramustine, etoposide phosphate, etoposide, exemestane, filgrastim, floxuridine fludarabine, fulvestrant, gefitinib, gemcitabine, gemtuzumab, goserelin acetate acetate, histrelinacetate, hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib mesylate, interferon α-2a, interferon α-2b, irinotecan, lenalidomide, letrozole, folinic acid, leuprolide acetateacetate), levamisole, lomustine, progesterone acetate, melphalan, mercaptopurine, 6-MP, mesna, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nelarabine, nofetumomab, Opp oprelvekin, oxaliplatin, paclitaxel, palifermin, pamidronate, pegademase, pegademase, pegademase, pemetrexed disodium, pentostatin, pipobroman, plicamycin, porfimer sodium sodium), procarbazine, quinacrine, rasburicase, rituximab, rociletinib, sargramostim, sorafenib, streptozocin, sunitinib maleate, talc, tamoxifen, temozolomide, teniposide, VM-26, testolactone, thioguanine, 6-TG, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, ATRA, uracil mustard mustard), valrubicin, vinblastine, vincristine, vinorelbine, zoledronate, zoledronic acidacid), pembrolizumab, nivolumab, IBL308, mDX-400, BGB-108, MEDI-0680, SHR-1210, PF-06801591, PDR-001, GB-226, STI-1110, durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-170, STI-1014, FOLFIRINOX, and KY-1003, and combinations thereof. Even more preferably, the other anticancer agent is at least one of the following: paclitaxel (taxol), gemcitabine, albumin-bound paclitaxel, cisplatin (cisplatin), capecitabine (capecitabine), irinotecan (irinotecan) and a combination thereof, more preferably a combination of paclitaxel (taxol) and gemcitabine or a combination of albumin-bound paclitaxel and gemcitabine or a combination of gemcitabine, albumin-bound paclitaxel, cisplatin (cisplatin), capecitabine (capecitabine).

[0024] In other preferred embodiments, the combination as defined above is used to treat pancreatic cancer selected from the group consisting of pancreatic adenocarcinoma, unresectable pancreatic cancer, locally advanced pancreatic cancer, borderline resectable pancreatic cancer, locally advanced pancreatic ductal adenocarcinoma, borderline resectable pancreatic ductal adenocarcinoma, metastatic pancreatic cancer, chemotherapy-resistant pancreatic cancer, pancreatic ductal adenocarcinoma, squamous pancreatic cancer, pancreatic progenitor cells, immunogenic pancreatic cancer, abnormally differentiated endocrine-exocrine (ADEX) tumors, exocrine pancreatic cancer, pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, mucinous cystic neoplasms, mucinous pancreatic cancer, adenosquamous carcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with osteoclast-like giant cells, pancreatic cystic tumors, islet cell tumors, pancreatic endocrine tumors or pancreatic neuroendocrine tumors.

[0025] The present invention further relates to a combination comprising or consisting of at least one HDAC inhibitor and one statin as defined above, wherein the at least one HDAC inhibitor and the one statin are administered in a single dosage unit or are administered separately, preferably, the single dosage unit comprises at least one pharmaceutically acceptable excipient, preferably, the single dosage unit or the separate dosage formulations are in the form of an oral, parenteral and / or topical dosage form. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will now be described with reference to the following drawings.

[0027] Figure 1. Characterization of pancreatic cancer cell lines. A. Basal mRNA levels of EMT markers vimentin and E-cadherin were determined by qRT-PCR in PDAC cell lines L3.6pl, BxPC3, COLO357, PANC28, PANCI, ASPC1 and MIAPACA2, in normal pancreatic cells HPDE cells, and in 2 established PDX-derived primary cells KPC ID11 and KPC ID95. B. Basal protein expression analysis shows that EMT markers in PDAC cell lines L3.6pl, BxPC3, COLO357, PANC28, PANCI, ASPC1 and MIAPACA2, in normal pancreatic cells HPDE cells, and in 2 established cell lines KPC ID11 and KPC ID95 derived from C57 / BL6 mice were determined by Western blotting. After 48 hours of cell growth, cell lysates were prepared. Proteins were separated by SDS-PAGE and transferred to PVDF membranes using standard protocols. Membranes were probed with specific antibodies and Ponceau Red as a total protein loading control. Data represent representative results of at least three independent experiments.

[0028] Figure 2 .Synergy of statin / HDACi combination in PDAC cell lines. The synergy of statin / HDACi combination on PDAC cell lines was analyzed according to the T-CChou and P.Talalay method. PANC28, PANCI, ASPC1, BxPC3, MIAPACA2, L3.6pl, KPC ID11 and KPC ID95 were plated as described (see Materials and Methods) and treated with single drugs or combinations (50:50 ratio) for 96 hours. Cell viability was assessed using sulforhodamine B colorimetric assay and combination index (CI). CI indicates the advantage of drug combination and is mathematically calculated using Calcusyn software (Biosoft, Cambridge, UK). Specifically, CI < 0.8, CI < 0.9, CI = 0.9-1.1 and CI > 1.1 indicate strong synergy, synergy, additivity or antagonism, respectively (Terranova-Barberio M, J Exp Clin Cancer Res. 2017; 36 (l): 177). Data represent the combined results of three independent experiments (mean ± DS); Abbreviations: VPA: valproic acid; SIM: simvastatin; PAN: panobinostat; VOR: vorinostat; MS-275: entinostat; ATOR: atorvastatin; LOV: lovastatin. Grayscale coding is used to highlight antagonism, additivity, synergy and strong synergy.

[0029] Figure 3. VPA plus simvastatin has antiproliferative and pro-apoptotic effects on PDAC cell lines. A. Synergistic inhibition of colony formation by VPA / SIM in ASPC1, PANC28, PANCI and BxPC3 cell lines. Cells were plated at very low concentrations (50-200 cells / well) and treated with VPA and SIM alone and in combination at IC10, IC25 after 24 hours, the values ​​are reported in the figure. Upper part: representative images of one experiment; lower part: bar graphs represent the absorbance values ​​of colonies after SRB colorimetric assay. Statistical significance was determined by two-tailed, unpaired Sigmund T test (*P<0.05; **P<0.01). B.-C. Synergistic inhibition of microtissue formation by VPA and SIM alone and in combination. 500 cancer cells (red cells - marked by cell tracker) and 1500 normal fibroblasts were plated into each well and treated with their respective IC25 after 24 hours. Representative images of OperaPhenix confocal microscope (B). Images represent the number of viable cells in 3D cell cultures quantified based on ATP content. Results (±SD) were obtained from a single experiment performed in triplicate. Statistical significance was determined by a two-tailed, unpaired Student's T-test (*P<0.05; **P<0.01) (C). D. Western blot analysis of PARP1 cleavage expression in PDAC cells that were untreated or treated with IC25 and IC50 of VPA and / or SIM for 24, 48, and 72 hours. P-actin expression was used as a loading control.

[0030] Figure 4 Apoptotic / necrotic effects of VPA / SIM treatment on PC cell lines. Apoptosis was assessed by flow cytometry after Annexin V-FITC staining in PANCI, MDA-PANC28, ASPC1, and BXPC3 cells that were untreated or treated with VPA and / or SIM for 48 or 72 hours at an IC25, calculated as a drug concentration that inhibits cell proliferation by 25% compared to untreated control cells. Specifically, cells were seeded into 96-well plates and 24 hours later VPA and SIM were administered in increasing doses starting from 16 mM and 80 mM, respectively. After 96 hours, cell viability and IC25 were assessed using a sulforhodamine B colorimetric assay.

[0031] Figure 5. The combination of low-dose VPA / SIM plus gemcitabine / paclitaxel (taxol) induces synergistic cell growth inhibition and apoptosis in monolayer and spheroid cultures of PDAC cells. A. Cells were treated for 96 hours with increasing concentrations of GEM / TAX alone or with a fixed low dose of VPA / SIM (VPA 0.5mM and SIM 0.625μM). The antiproliferative effect of all PDAC cell lines was evaluated by sulforhodamine B colorimetric assay (see Methods) and expressed as a percentage of control. B PANCI, PANC28 and ASPC1 cells that were untreated or treated with low doses of VPA / SIM and / or GEM / TAX (VPA 0.5mM, SIM 0.625μM, GEM 25nM and TAX 1.25nM) for 24 hours were evaluated by caspase 3 / 7 activity assay, and apoptosis was evaluated by luminescence assay. C. Expression of cleaved PARP in PANCI, PANC28 and ASPC1 cell lines untreated or treated with VPA / SIM and / or GEM / TAX for 24 hours was evaluated by western blotting, and p-actin was used as a loading control. DE Anti-tumor effect of VPA / SIM plus GEM / TAX in 3D spheroid system. Specifically, PANCI (D) and ASPC1 (E) were inoculated into spheroid culture medium of low attachment support to form first generation spheroids. After 48 hours, the spheroids were disaggregated and plated under three different conditions: adhesion conditions and testing cell viability (left figure), forming the second generation in spheroid culture medium of low attachment support, and testing 3D cell viability (middle figure) and cell apoptosis induction (right figure). Specifically, cells were inoculated in spheroid culture medium of low attachment support to form first generation spheroids for 48 hours, then disaggregated and plated again under adhesion conditions, and 24 hours after inoculation, cells were treated with VPA / SIM and / or GEM / TAX at specified doses for 96 hours. The anti-proliferative effect (cell viability-SRB, left figure) of all PDAC cell lines was evaluated by sulforhodamine B colorimetric test. Cells (40,000 / mL) were seeded in the sphere culture medium of low attachment support to form the first generation spheres for 48 hours, then disaggregated and plated again in low attachment support to form the second generation spheres, and synchronously untreated or treated with specified doses of VPA / SIM and / or GEM / TAX for 72 hours. Spheroid viability was evaluated by luminescence test (middle figure). For induction of cell apoptosis, cells (40,000 / mL) were seeded in the sphere culture medium of low attachment support to form the first generation spheres for 48 hours, then disaggregated and plated again in low attachment support to form the second generation spheres, and synchronously untreated or treated with specified doses of VPA / SIM and / or GEM / TAX for 24 hours. Cell apoptosis was evaluated by caspase 3 / 7 activity test.F. VPA and SIM in combination with chemotherapy produce a synergistic inhibitory effect on induced colony formation in PANCI, ASPC1, PANC28 and BxPC3 cell lines. Cells were plated at very low concentrations and treated 24 hours later with IC10 of VPA and SIM alone and / or in combination with chemotherapy. Representative images of one experiment.

[0032] Fig. 6. VPA / SIM synergizes with gemcitabine / paclitaxel (taxol) to reduce PANCI cell migration ability by targeting TGFβ-induced EMT. AB VPA / SIM combined with GEM / TAX can reduce vimentin and induce E-cadherin expression, which is evaluated by fluorescence confocal microscopy in PANCI cells that are not treated or treated for 48 hours as shown. Cells are stained with vimentin antibodies (secondary anti-Alex Fluor488) and E-cadherin antibodies (secondary anti-Alex Fluor488), and DAPI is used for nucleus detection (blue). Quantitative measurement is performed by Harmony software (PerkinElmer). C. EMT markers vimentin and E-cadherin and pro-apoptotic markers, cleaved PARP and cleaved caspase 3 in the sub-graph cell lines that are not treated or treated for 48 hours with VPA and / or SIM and / or GEM / TAX are evaluated by western blotting. CDK4 and p-actin are used as loading controls. D. Evaluation of vimentin, ZEB1 and CDH1 mRNA expression in PANCI cells untreated or treated with low doses (0.5 mM and 0.625 μM, respectively) of VPA or SIM for 48 hours by RT-PCR; values ​​represent the mean ± SD of technical triplicates. E. The network was generated by Ingenuity Pathway Analysis (IPA) using the search "vimentin (VIM), E-cadherin (CDH1), HMGCR and HDAC". The network genes are visualized by appropriate symbols, which illustrate the functional properties of the corresponding proteins. Each node represents a gene and its direct (represented by a solid line) and indirect (represented by a dotted line) association with other genes. The genes used as input are highlighted in dark gray, while TGFβ in light gray is used as the center. F. Relative mRNA expression of vimentin, ZEB1 and CDH1 expressed in PANCI cells untreated or treated with low doses (0.5 mM and 0.625 μM, respectively) of VPA / SIM, with or without TGFβ (5 ng / ml) stimulation for 8 hours was measured by real-time RT-PCR. G. TGFβ mRNA expression in PANCI cells untreated or treated with low doses (0.5 mM and 0.625 μM, respectively) of VPA or SIM was evaluated by RT-PCR; values ​​represent the mean±SD of technical triplicates.

[0033] Figure 7. VPA / SIM synergistically reduces PANCI cell migration and invasion ability and the effect on the PDAC microenvironment with gemcitabine / paclitaxel (taxol). A. Migration assays were performed to evaluate by wound healing assays. PDAC cells were seeded in 96-well CellCarrier ultra (Parkin Elmer) to 90% confluence. 24 hours after plating, a sterile 10-pL pipette tip was used to longitudinally draw a strip of constant diameter in the confluent monolayer to simulate a wound. The cells were then left untreated or exposed to VPA, SIM and GEM / TAX until the wound was almost completely closed. At the specified time, the wells were photographed with an Opera Phenix microscope (Parkin Elmer) air objective at 20X magnification. Quantitative measurements were performed by determining the distance between the wound edges using Harmony software (Parkin Elmer). B. Invasion assays were performed in Transwell using PVPF filters with an 8 μm pore size and expressed as the percentage of invasive cells after 48 hours of exposure to VPA, SIM alone and in combination with GEM / TAX. C. HPaSteC cells were untreated or treated with VPA and / or SIM for 24 hours. Cells were collected and stained with GFAP-APC for 30 minutes at 4°C to evaluate activated stellate cells and tested by flow cytometry. D. HPaSteC were treated with increasing concentrations of VPA and SIM alone or in combination for 96 hours. The antiproliferative effect was evaluated by sulforhodamine B colorimetric assay (see Methods) and expressed as a percentage of the control. E. HPaSteC cells were treated with conditioned medium from PANCI that had been previously untreated or treated with VPA and / or SIM for 24 hours. HPaSteC were exposed to PANCI conditions for 24 hours, and then cells were collected and stained with GFAP-APC for 30 minutes at 4°C to evaluate activated stellate cells and tested by flow cytometry. F. HPaSteC cells were treated with conditioned medium from PANCI transfected with an empty vector (PANC1_EV), transfected with wild-type YAP (YAPwt) and YAP5SA (constitutively active), untreated or treated with VPA and / or SIM for 24 hours. HPaSteC were exposed to PANCI conditions for 24 hours, and then the cells were collected and stained with GFAP-APC for 30 minutes at 4°C to evaluate activated stellate cells and tested by flow cytometry. G. Western blot analysis of YAP subcellular localization after VPA and / or SIM treatment in PANCI cells. Gamma tubulin and PARP were used as loading controls. H. Microtissues obtained by mixing PDAC cells labeled with green fluorescent probe-cell trackers with human pancreatic stellate cells with red probes (PKH-26 Sigma Aldrich) were cultured in a ULA system (Perkin Elmer).Normal fibroblasts were used as scaffolds for PDAC cell lines at a ratio of 3:1 to obtain 3D microtissue models, either untreated or treated with drugs for 96 hours. The 3D microtissues were kept in an incubator and photographed at 5x magnification using an Opera Phenix microscope (PerkinElmer) air objective, and the average volume was scored using Harmony software.

[0034] Figure 8. In vivo synergistic antitumor effects of valproic acid and simvastatin combined with gemcitabine / albumin-bound-paclitaxel in the Pancl-LUC orthotropic model. A. Schematic diagram of in vivo experiment: Effects of VPA / SIM plus Gem / NP on the growth of orthotopic PDAC xenograft tumors. PANCI cells transfected with LUC (0.5*10 6 cells) were injected directly into the pancreas of athymic mice as described in Materials and Methods. One week after injection, mice were treated with VPA / SIM and / or GEM / NP, a combination of the four drugs, or their vehicle. B. Digital grayscale images of five mice / group on days 5, 12, and 19 (follow-up study), superimposed on pseudo-color images representing the spatial distribution of detected photons emerging from active luciferase. C Tumor volume was quantified as the sum of all detected photons within the tumor area. The measurements of each single mouse at the specified time point were compared with the corresponding values ​​of day 1 (TO). The relative fold increase values ​​are reported in the figure. D Tumor volume was quantified as the sum of all detected photons within the tumor area as the average of each measurement of the four groups at the specified time point. E Body weight was measured twice a week. F. TGF-β serum levels in control mice and treated mice after 2 weeks of treatment were evaluated by Quantikine ELISA TGFβ1 immunoassay (see Materials and Methods). The results are expressed as the mean ± SD of 3 mice. G. Representative sections of orthotopic pancreatic tumor sections stained with Masson's trichrome for IHC. Scale bar 50 μm, magnification 20X. Collagen fibers are stained blue and nuclei are stained black. The left image shows a clear blue color indicating the presence of fibrotic areas within the tumor, quantified as ≥10% fibrosis. While the right image shows a Masson's trichrome stain showing the presence of blue <10% collagen fibers, indicating a decrease in fibrotic tissue. The table shows the number of mice / groups characterized by the presence of fibrosis ≥10% or <10%.

[0035] Figure 9. In vivo synergistic antitumor effect of valproic acid and simvastatin combined with gemcitabine / albumin-bound-paclitaxel in a ectopic model. A. Schematic diagram of the in vivo experiment: the effect of VPA / SIM plus Gem / NP on the growth of PDAC ectopic xenograft tumors. B. Relative tumor volume curves of PANCI xenografts. Mean ± SD of tumor volumes measured at pre-specified time points. C. TGF-β serum levels in control mice and treated mice after 2 weeks of treatment, assessed by Quantikine ELISAT GFβ1 immunoassay (see Materials and Methods). The results are expressed as the mean ± SD of 3 mice. DEF. Serum levels of GOT, GPT and creatinine in mice sacrificed at the end of treatment. DETAILED DESCRIPTION

[0036] In the present invention, it was unexpectedly found that a combination comprising at least one HDAC inhibitor and a statin, preferably a VPA / SIM combination, synergistically improved the antiproliferative and pro-apoptotic effects of conventional chemotherapy of gemcitabine / paclitaxel (taxol) in vitro and in vivo when used at low doses. The combination of the HDAC inhibitor and the statin has anticancer activity and is used to treat pancreatic cancer.

[0037] Mechanistically, VPA / SIM treatment alone or in combination with chemotherapy induced e-cadherin and impaired vimentin and ZEB-1 expression, which was functionally associated with the synergistic inhibition of cell migration and invasion.

[0038] Ingenuity pathway analysis (IPA) highlighted a protein network connecting HDAC and HMGCR (targets of VPA and SIM, respectively) to two major EMT markers. Here, TGFp emerged as a hierarchical dominant network node.

[0039] VPA / SIM inhibited Tgfp transcription and Tgfp-regulated EMT gene expression in PDAC cells. In addition, VPA / SIM treatment impaired YAP nuclear translocation, consistent with data obtained in prostate cancer models [Iannelli F et al. JECCR, 2020],

[0040] The VPA / SIM combination affects the activation of human pancreatic stellate cells (HPaSteC), as shown by impaired expression of glial fibrillary acidic protein (GFAP), but does not affect its proliferation. This effect is directly induced by VPA / SIM treatment, and treatment of HpaSteC with conditioned medium of the PDAC cell line PANCI, which is untreated or treated with VPA / SIM, also induces this effect. In addition, the latest inhibitory effect can be restored by using the same PDAC cell line transfected with a constitutively active YAP oncogene (YAP5SA), confirming its involvement in the crosstalk between PDAC and HpaStec cells. In general, the present invention provides a novel combination strategy based on a combination of HDAC inhibitors and statins, specifically a combination of two safe and generic drugs, which can sensitize the widely used treatment regimens in patients with metastatic PDAC. On this basis, the inventors designed a randomized phase II clinical study using VPA combined with simvastatin and gemcitabine / albumin-bound-paclitaxel or gemcitabine / albumin-bound-paclitaxel / cisplatin / capecitabine in patients with untreated metastatic pancreatic adenocarcinoma, and the patients will begin to be enrolled in the near future (VESPA trial-EudraCT n.2022-004154-63).

[0041] In the clinical setting, both valproic acid and simvastatin are orally bioavailable drugs tested at doses within the range of their non-cancer approved indications that were preclinically effective in combination therapy with both drugs plus chemotherapy.

[0042] The present invention also relates to a method for treating pancreatic cancer, comprising administering at least one HDAC inhibitor and a statin to a subject in need thereof. Preferably, the method comprises administering at least one HDAC inhibitor selected from the following: valproic acid or a salt thereof, panobinostat, vorinostat, entinostat or moxistat, and at least one statin selected from the following: simvastatin, atorvastatin, lovastatin.

[0043] Unless otherwise specified, the technical terms used in this aspect have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. In addition, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms, as long as they are applicable to the context and / or application. For the sake of clarity, various singular / plural arrangements may be explicitly stated herein.

[0044] As used herein, the term "combination" refers to a single composition / formulation comprising an HDAC inhibitor and a statin or their respective pharmaceutically acceptable salts or derivatives; or a kit comprising an HDAC inhibitor and a statin or their respective pharmaceutically acceptable salts or derivatives as separate preparations; or a separate preparation / dosage form in a non-kit form, as long as the effect achieved is commensurate with the intended purpose of the present invention (i.e., for the treatment of pancreatic cancer). The combination comprises one or more pharmaceutically acceptable excipients. Therefore, a separate preparation comprising an HDAC inhibitor and a statin or their respective pharmaceutically acceptable salts or derivatives can be administered simultaneously, or sequentially in any order. Preferably, the HDAC inhibitor is valproic acid or a salt thereof, panobinostat, vorinostat, entinostat or moxistat, and the statin is selected from simvastatin, atorvastatin, lovastatin; more preferably, the HDAC inhibitor is valproic acid and the statin is simvastatin. The use of the expression "at least" or "at least one" indicates the use of one or more elements or ingredients or quantities, which can be seen in the embodiments of the present disclosure to achieve one or more desired purposes or results. Throughout the specification, the word "comprise" or its variations, such as "include" or "contain" or "cover" or "with" or "have" should be understood to mean including the elements, integers or steps, or groups of elements, integers or steps, but not excluding any other elements, integers or steps, or groups of elements, integers or steps. "One embodiment" or "an embodiment" mentioned in the specification means that the specific features, structures or characteristics described in relation to the embodiment may be included in at least one embodiment of the present disclosure. Therefore, the phrases "in one embodiment" or "in one embodiment" appearing in various places in this specification do not necessarily all refer to the same embodiment. It should be understood that, for clarity, certain features of the present disclosure described in the context of a separate embodiment may also be provided in a single embodiment. On the contrary, for the sake of brevity, the various features of the present disclosure described in the context of a single embodiment may also be provided individually or in any suitable subcombination. As used herein, the term "about" encompasses variations of ±10% and more preferably ±5%, as such variations are suitable for practicing the present invention.

[0045] As used herein, the term "histone deacetylase" or "HDAC" refers to an enzyme that removes acetyl groups from histones. There are 18 known HDACs, which can be divided into four groups. Class I HDAC, including HDAC1-3 and HDAC8. Class II HDAC includes HDAC4-7 and HDAC9-10. Class III HDAC (also known as deacetylase (sirtuin)) includes SIRT1-7. Class IV HDAC has the characteristics of Class I and Class II HDAC, which only includes HDAC11. As used herein, the term "histone deacetylase inhibitor" or "HDAC inhibitor" or "HDACi" refers to a natural or synthetic compound that inhibits the activity of histone deacetylase. According to its selectivity for substrates, there are different categories of HDACi, which are divided into classical HDACi, selective Class I HDACi and selective Class II HDACi. Therefore, "classical HDACi" (also known as pan-HDACi) refers to a natural or non-natural compound with the ability to inhibit histone deacetylase activity independent of the HDAC category. Therefore, classical HDACi is non-selective HDACi. "Non-selective" refers to that the compound suppresses the activity of classical HDAC (i.e., Class I, Class II, and Class IV) with similar efficiency independent of HDAC categories. The example of classical HDACi includes but is not limited to Belinostat (PDX-101), vorinostat (SAHA) and panobinostat (LBH-589). Compared with Class II HDAC (i.e., HDAC4-7, 9 and 10), "selective Class I HDACi" has selectivity to HDAC categories (i.e., HDAC 1-3 and 8). "Selectivity" refers to that the inhibition of selective Class I HDACi to Class I HDAC is at least 5 times higher than Class II HDAC, preferably 10 times higher, more preferably 25 times higher, more preferably 100 times higher. The selectivity of HDACi for Class I or Class II HDAC can be determined according to the previously described method (Kahn et al., 2008). Examples of selective class I HDACi include, but are not limited to, valproic acid (VPA), romidepsin (FK-228) and entinostat (MS-275). Compared to class I HDAC (i.e., HDAC 1-3 and 8), "selective class II HDACi" is selective for class II HDAC (i.e., HDAC 4-7, 9 and 10). "Selectivity" means that the inhibition of class II HDAC by selective class II HDACi is at least 5 times higher than that of class I HDAC, preferably 10 times higher, more preferably 25 times higher, and more preferably 100 times higher. Examples of selective class II HDACi include, but are not limited to, tubacin and MC-1568 (aryloxypropenyl) pyrrolyl hydroxamate). HDAC inhibition mainly relies on the mechanism based on the inhibition of HDAC enzymatic activity, which can be determined by a variety of methods well known to those skilled in the art.Typically, these methods include using a colorimetric HDAC test to assess the lysine deacetylase activity of an HDAC enzyme. Commercial kits for such techniques are available (see, for example, histone deacetylase (HDAC) activity test kits (fluorimetry) purchased from Abeam or Sigma Aldrich). These methods are very suitable for determining the IC50 values ​​of known or suspected HDAC inhibitors. Many HDAC inhibitors are known, and therefore can be synthesized from known starting materials by known methods, can be commercially available, or can be prepared by the method for preparing the corresponding compound in the literature. One class of preferred HDAC inhibitors are the hydroxamic acid inhibitors which are disclosed, for example, in WO 97 / 35990, US-A 5,369,108, US-A 5,608,108, US-A 5,700,811, WO 01 / 18171, WO 98 / 55449, WO 93 / 12075, WO 01 / 49290, WO 02 / 26696, WO 02 / 26703, JP 10182583, WO 99 / 12884, WO 01 / 38322, WO 01 / 70675, WO 02 / 46144, WO 02 / 22577 and WO 02 / 30879. Other HDAC inhibitors that may be included in the combinations of the invention are cyclic peptide inhibitors, reference being made here to, for example, US-A 5,620,953, US-A 5,922,837, WO 01 / 07042, WO 00 / 08048, WO 00 / 21979, WO 99 / 11659, WO 00 / 52033 and WO 02 / 0603. Suitable HDAC inhibitors also include those based on benzamide structures, which are disclosed in Proc.Natl.Acad.Sci.USA (1999), 96:4592-4597, and are also disclosed in EP-A 847 992, US 6,174,905, JP 11269140, JP 11335375, JP11269146, EP 974 576, WO 01 / 38322, WO 01 / 70675 and WO 01 / 34131. HDAC inhibitors can be used in any pharmaceutically acceptable form, including but not limited to their free forms and their pharmaceutically acceptable salts or solvates.

[0046] The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable, preferably non-toxic base or acid (including an inorganic or organic acid or an organic or inorganic base). Such salts are also referred to as acid addition salts and base addition salts. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19. More specifically, examples of suitable HDAC inhibitors according to the present invention include, but are not limited to, the compounds listed in Table 1 below.

[0047] Table 1: Examples of HDAC inhibitors useful in the context of the present invention

[0048]

[0049]

[0050] In a preferred embodiment, the HDAC inhibitor is selected from the group consisting of valproic acid, belinostat (PXD-101), vorinostat (SAHA), entinostat (MS-275), panobinostat (LBH-589), moxistat (MGCD0103), chidamide (HBI-8000), romidepsin (FK-228) and trichostatin A (TSA).

[0051] The chemical name of valproic acid is 2-propyl valproic acid. Divalproex sodium is a stable coordination compound of valproate sodium and valproic acid. As used herein, the term valproic acid includes valproic acid itself, valproate salts such as valproate sodium, divalproex sodium and other valproic acid derivatives.

[0052] Belinostat (also known as PXD-101) is chemically known as (2E)-N-hydroxy-3-[3-(phenylsulfamoyl)phenyl]prop-2-enamide. Vorinostat is currently marketed in the United States under the trade name (Merck Sharp & Dohme Corp) is marketed in an oral administration form. The chemical name of panobinostat (also known as LBH-589) is 2-(E)-N-hydroxy-3-[4[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2-acrylamide. Currently, panobinostat lactate is marketed in the United States under the trade name (Novartis) is marketed in an oral administration form. The chemical name of moxistat (also known as MGCD0103) is N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl]benzamide. The chemical name of cedamide (also known as HBI-8000) is N-(2-amino-5-fluorophenyl)-4-[[[1-oxo-3-(3-pyridinyl)-2-propen-1-yl]amino]methyl]-benzamide. The chemical name of entinostat (also known as MS-275) is N-(2-aminophenyl)-4-N-(pyridin-3-yl)methoxycarbonylamino-methyl]-benzamide. Romidepsin is a natural product isolated from Chromobacterium violaceum by Fujisawa Pharmaceuticals. Romidepsin (also known as FK-228) is a bicyclic depsipeptide [1S,4S,7Z,10S,16E,21R)-7-ethylidene-4,21-bis(1-methylethyl)-2-oxa-12,13-dithio-5,8,20,23-tetraazabicyclo[8.7.6]tricosaccharide-16-ene-3,6,9,19,22-pentanone]. The chemical name of trichostatin-A (TSA) (also known as TSA) is 2,4-heptadienamide, 7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxo-, (2E,4E,6R). TSA is an organic compound that acts as an antifungal antibiotic that selectively inhibits class I and II mammalian histone deacetylases (HDAC) families, but not class III HDACs (i.e., deacetylases). It is a member of a large class of histone deacetylase inhibitors (HDI or HDACI) with a wide range of epigenetic activity. Therefore, TSA has a certain potential as an anticancer drug (Drummond DC et al. (2005) Annu Rev Pharmacol Toxicol. 45: 495-528). The term "treatment / treatment" used herein means reversing, alleviating, inhibiting the progression of the disease or condition to which the term is applicable or preventing the disease or condition to which the term is applicable, or reversing, alleviating, inhibiting one or more symptoms of the disease or condition to which the term is applicable. Referring to "therapeutically effective amount" means an amount that is effective for a reasonable benefit / risk ratio for any medical treatment. However, it should be understood that the total daily dosage will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dosage level for any particular patient in need will depend on a variety of factors, including the patient's age, weight, overall health, sex and diet, administration time, administration route, duration of treatment; drugs combined with factors known in the medical field or used simultaneously.For example, it is well known to those skilled in the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and then gradually increase the dosage until the desired effect is achieved.

[0053] Statins are a class of drugs widely used to treat and prevent cardiovascular disease. Studies have shown that statins can reduce low-density lipoprotein (LDL) cholesterol levels by up to 551% and reduce cardiovascular events by 20-301% (Postmus, 2014). Statins are 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors. HMG CoA reductase is the rate-limiting enzyme in cholesterol synthesis. Statins competitively inhibit HMG CoA reductase activity, reducing cellular cholesterol concentration, thereby activating cell signaling cascades and ultimately activating sterol regulatory element binding protein (SREBP). SREBP is a transcription factor that can upregulate the expression of the gene encoding the LDL receptor. The LDL receptor is responsible for receptor-mediated endocytosis of LDL cholesterol. Therefore, increased LDL receptor expression leads to increased plasma LDL uptake, thereby reducing plasma LDL cholesterol concentration. The best-selling statin is atorvastatin, which is sold under the trade name Lipitor (LIPITOR) and is produced by Pfizer. Lipitor is available in tablet form for oral administration daily, each tablet containing 10, 20, 40 or 80 mg of atorvastatin. In addition to Lipitor, statins are also commercially available as single-ingredient products, such as Lescol (fluvastatin), Mevacor (lovastatin), (extended release lovastatin), (pitavastatin), Pravachol (pravastatin), (rosuvastatin) and (Simvastatin). Statins are also commercially available as combination products, such as Advicor (lovastatin / niacin extended release), (simvastatin / niacin sustained release) and Vytorin (simvastatin / ezetimibe). The statin used in the combination therapy disclosed herein is selected from the following group: simvastatin, atorvastatin, lovastatin, rosuvastatin, fluvastatin, pitavastatin, pravastatin or any combination thereof, preferably simvastatin, atorvastatin, lovastatin, and more preferably simvastatin. According to a study conducted by Jing et al., any of these statins is expected to play a role in this combination. They studied the anticancer effects of different statins and observed that almost all statins showed anticancer activity ("In vitro and in vivo anticancer effects of mevalonate pathway modulation on human cancer cells", Br J Cancer. 2014 Oct 14; 111(8):1562-71).

[0054] In some embodiments, the therapeutically effective amount of the HDAC inhibitor or valproic acid or a pharmaceutically acceptable salt or derivative thereof ranges from about 1 mg to about 2500 mg per day, preferably from about 100 mg to about 2500 mg, about 200 mg to about 2500 mg, about 300 mg to about 2500 mg, about 400 mg to about 2500 mg, about 500 mg to about 2500 mg, about 600 mg to about 2500 mg, about 700 mg to about 2500 mg, about 800 mg to about 2500 mg, about 900 mg to about 2500 mg, about 1000 mg to about 2500 mg, about 1100 mg to about 2500 mg, or about 1200 mg to about 2500 mg per day. In some embodiments, the therapeutically effective amount of a statin or a pharmaceutically acceptable salt thereof or a derivative thereof is about 1 mg to about 200 mg per day, preferably 1 mg to about 100 mg per day or about 50 mg to about 100 mg per day, including values ​​and ranges therebetween.

[0055] In some embodiments, the foregoing values ​​and ranges are illustrative only. The dosage may be varied depending on many variables, including, for example, the activity of the compound used, the disease, disorder or condition to be treated, the mode of administration, the needs of the individual subject, the severity of the disease, the disease, disorder or condition being treated, and the physician's judgment. The dosage may be adjusted to achieve a desired pharmacokinetic or pharmacodynamic profile, such as a desired or effective blood profile.

[0056] The combination of the present invention also includes other anticancer agents. The other anticancer agent can be a taxane. The term "taxane" can refer to any chemical analogue that exerts its anticancer effect by stabilizing tubulin microtubules involved in cell division. Examples of taxanes that can be combined with HDAC inhibitors and statins according to the present invention include: (2aR, 3aR, 4aR, 6R, 9S, 11S, 12S, 12aR, 12bS)-6,12b-diacetoxy-9-[3(S)-(tert-butoxycarbonylamino)-2(R)-hydroxy-3-phenylpropionyloxy]-12-benzoyloxy-11-hydroxy-8,13,13-trimethyl-2a,3,3a,4,5,6,9,10,II,12,12a,12b-decyl Dihydro-1H-7,11-methylenecyclodecan[3,4]-cyclopropyl[4,5]benz[1,2-b]oxin-5-one dihydrate; Taxol, BMS-184476 (7-methylthiomethylpaclitaxel); BMS-188797; BMS-275183; BMS-188797; BMS-109881; CYC-3204 (penetrant-paclitaxel conjugate); Taxoprexin; DJ-927; docetaxel TM ); Larotexel (XRP9881; RPR-109881A); XRP6258 (RPR112658); Milataxel (MAC-321); MST997; MBT-206; NBT-287; Ortataxel; Protax-3; PG-TXL; PNU-166945; PNU-106258; Orataxel (BAY59-8862; IDN5109; semi-synthetic taxane); TPI-287; Protaxel and MAC-321 (Taxalog). Examples of taxane preparations include conventional preparations of paclitaxel or docetaxel, such as the currently approved Taxol TM and Taxotere TMFormulations; Formulations with biocompatible polymers, particularly proteins such as albumin, more particularly nanoparticle or microparticle formulations of paclitaxel or docetaxel with albumin, such as Abraxane TM or albumin-bound-paclitaxel (described in US5,439,686 and US6,749,868) or albumin-bound-docetaxel (described, for example, in US20080161382, US20070117744 and US20070082838); polymer conjugates, in particular polymer conjugates of paclitaxel or docetaxel, more particularly conjugates of paclitaxel or docetaxel with poly-L-glutamic acid, such as Opaxio (also known as Xyotax, polyglutamic acid paclitaxel, CT-2103 and described, for example, in Li C.; poly(L-glutamic acid)-anticancer drug conjugates (Poly(L-glutamic acid)-anticancer drug conjugates); Adv. 2002; 54; 695-713); docetaxel or a conjugate of docetaxel with a fatty acid, in particular a conjugate of paclitaxel or docetaxel with docosahexaenoic acid (DHA), such as paclitaxel (DHA-paclitaxel, for example described in Bradley MO et al. Tumor targeting by covalent conjugation of a natural fatty acid to paclitaxel); Clin. Cancer Res. 2001; 7: 3229-38); microparticle compositions, such as porous microparticle preparations described in US 6,645,528, for example microparticle preparations of paclitaxel AI-850, which comprise paclitaxel nanoparticles in a porous hydrophilic matrix comprising mainly sugars; and emulsions of paclitaxel or docetaxel in vitamin E, such as Tocosol.

[0057] Other anticancer agents may also be gemcitabine (a broad-spectrum antimetabolite and deoxycytidine analog with antitumor activity) and / or cisplatin (cis-diamine, dichloroplatinum (II), CAS No. 15663-27-1) and / or capecitabine ( Roche).

[0058] As used herein, "pancreatic cancer" includes or refers collectively to different types of pancreatic cancer, including pancreatic adenocarcinoma, unresectable pancreatic cancer, locally advanced pancreatic cancer, borderline resectable pancreatic cancer, locally advanced pancreatic ductal adenocarcinoma, borderline resectable pancreatic ductal adenocarcinoma, metastatic pancreatic cancer, chemotherapy-resistant pancreatic cancer, pancreatic ductal adenocarcinoma, squamous pancreatic cancer, pancreatic cancer progenitors, immunogenic pancreatic cancer, abnormally differentiated endocrine-exocrine (ADEX) tumors, exocrine pancreatic cancer, pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, mucinous cystic neoplasms, mucinous pancreatic adenocarcinoma, adenosquamous carcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with osteoclast-like giant cells, pancreatic cystic tumors, islet cell tumors, pancreatic endocrine tumors, or pancreatic neuroendocrine tumors.

[0059] The combination of the present invention can be formulated into a dosage form containing a pharmaceutically acceptable excipient or carrier. The pharmaceutically acceptable excipient or carrier may include, but is not limited to, at least one of the following: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, human serum albumin, buffer substances, phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, electrolytes, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, wax, polyethylene glycol, starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, dextrose, talc, magnesium carbonate, kaolin; nonionic surfactants, edible oils, physiological saline, antibacterial water, Cremophor EL TM (BASF Corporation, Parsippany, NJ) and phosphate-buffered saline (PBS).

[0060] Materials and methods

[0061] Cell lines

[0062] Human pancreatic cancer cell lines PANCI, ASPC1, BxPC3, L3.6pl, COLO357, MIAPACA2 and hTERT immortalized foreskin fibroblasts B and hTERT were purchased from the American Type Culture Collection (ATCC, Rockville, Maryland, USA). The PANC28 cell line was obtained from the laboratory of Dr. Marsha L. Fraizer and Dr. Douglas B Evans (Frazier, ML et al. International Journal of Pancreatology 19, 31-38-1996). The established PDX-derived primary cells KPC ID 11 and KPC ID95 were gifted by the Bruno Sainz Laboratory in Madrid, Alonso-Nocelo M, Sainz B et al. Gut. February 2023; 72 (2): 345-359). Stem cells (HPastec) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) (Robinson, BK, et al. (2016) Biology Open. Vol. 5). PANCI, ASPC1, L3.6pl, COLO357, MIAPACA2, B JhTERT, PANC28, KPC ID11 and ID95 cell lines were maintained as monolayer cultures under adherent conditions and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g / L glucose, glutamine and non-essential amino acids, supplemented with 10% heat-inactivated fetal bovine serum and penicillin (100 IU / mL)-streptomycin (100 μg / mL). BxPC3 cell line was grown in RPMI (Roswell Park Memorial Institute) supplemented with heat-inactivated 10% fetal bovine serum (FBS, Cambrex, Belgium), 50 units / ml penicillin (Cambrex, Belgium), 500 μg / ml streptomycin (Cambrex, Belgium), and 4 mM glutamine. HPaStec was cultured in Stellate Cell Medium (SteCM). Cultures were maintained at 37°C, 95% air and 5% CO 2 All cell lines were regularly checked for mycoplasma. The cells were authenticated by the short tandem repeats generated by the LGC standard. The PANC1 LUC cell line was stably transduced with RediFect firefly luciferase lentiviral particles (Catalog #CLS960004, PerkinHelmer) by lentiviral infection and selection with puromycin.

[0063] Reagents

[0064] All media, serum, antibiotics and glutamine used for cell culture were from Lonza (Basel, Switzerland). Primary antibodies used for western blotting: Poly-(ADP-ribose)-polymerase (PARP) (#556494), purchased from BD Pharmingen according to the manufacturer's protocol TM YAP (#4912) and vimentin (#5741) were purchased from Cell Signaling Technology (Danvers, MA, USA); P-actin (#ab8227), E-cadherin (#40772) and secondary antibodies were purchased from the following: polyclonal goat anti-rabbit IgG (H+L)-HRP conjugate (#1706515) and polyclonal goat anti-mouse IgG (H+L)-HRP conjugate (#1706516) were purchased from Abcam (Cambridge, UK); polyclonal rabbit anti-goat IgG-HRP conjugate (#sc-2768) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Goat polyclonal secondary antibody mouse IgG-H&L-Alexa 594 (#ab150120). According to the manufacturer's protocol, microtissues were labeled using green fluorescent probe cell tracking (CellTracker, Promega) and HpaStec with red probe (PKH-26, Sigma Aldrich). Stem cell viability was assessed by 3D cell viability assay (ThermoFisher) according to the manufacturer's protocol.

[0065] drug

[0066] Valproic acid (VPA) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Simvastatin (#1693), panobinostat (#1612-25), and atorvastatin (#2278-10) were purchased from Biovision Incorporated (Milpitas, CA, USA). Vorinostat (SAHA) (SML0061) was purchased from Sigma-Aldrich, entinostat (MS-275) (#S1053) was purchased from Selleckchem, and gemcitabine (Accord, Devon, UK) and nab-paclitaxel (Celgene, Milan, Italy) were provided by pharmacies. Recombinant human TGF-β1 (240-B002) was provided by R&D systems. Cell proliferation assays and drug combination studies

[0067] Cell proliferation of untreated cells and cells treated with the drugs as single agents or in combination was measured in 96-well plates. Cell proliferation was measured using a spectrophotometric dye incorporation assay (sulforhodamine B) and the 50% cell inhibitory concentration (IC50) of each drug was calculated as previously described (Di Gennaro et al. Br J Cancer 2010, 103(11): 1680-1691). Drug combination studies were based on concentration-effect curves, which are plots of the fraction of unaffected (surviving) cells versus drug concentration after 96 hours of treatment. Synergy, additivity and antagonism were quantified after assessment of the combination index (CI), which was calculated by the Chou-Talalay equation using CalcuSyn software (Biosoft, Cambridge, UK) - (Chou TC. Cancer Res. 2010 Jan 15;70(2):440-6.), as described elsewhere (Terranova-Barberio M, J Exp Clin Cancer Res. 2017;36(1):177); Bruzzese et al., J Cell Physiol 2011, 226(9):2378-2390). Specifically, CI < 0.8, CI < 0.9, CI = 0.9-1.1 and CI > 1.1 indicate strong synergy, synergy, additivity or antagonism, respectively, calculated as 50%, 75% and 90% of cell killing rate (ED50, ED75 and ED90, respectively) (Terranova-Barberio M, J Exp Clin Cancer Res. 2017; 36 (1): 177). The DRI determines the dose reduction allowed when each drug combination is given, compared with the concentration of a single dose required to achieve the same effect. Clonogenic assay The colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. PANCI, ASPC1 and PANC28 cell lines were seeded in 96-well plates at 50 cells / well, while the BxPC3 cell line was seeded at 100 cells / well. The cells were treated with VPA, SIM (at doses of IC10, IC25 and IC50) alone and the day after the combination treatment. The cells were grown for 10 days. The colonies formed were fixed with TCA (50%) and measured using a spectrophotometric dye incorporation assay (sulforhodamine B or crystal violet). Microtissue formation assay Pancreatic cancer cell lines PANCI, ASPC1, PANC28, BxPC3, and MiaPaca were cultured as microtissues using an ultra-low attachment (ULA) system (PerkinElmer). Cancer cells were labeled with a green fluorescent probe cell tracker (Thermo Fisher) according to the manufacturer's instructions, while fibroblasts were labeled with a red probe (PKH-26, Sigma Aldrich) (only for preliminary experiments to assess tissue formation).Normal fibroblasts or stellate cells isolated from the tumor microenvironment of the PANCI xenograft model were used as scaffolds for PC cell lines at a ratio of 3:1 as described in the literature to obtain 3D microtissue models, where the cell lines were untreated or treated with drugs, treated with VPA, SIM alone or in combination with IC25 and IC50 for 96 hours. The 3D microtissues were kept in an incubator and photographed at 5 times magnification using an Opera Phenix microscope (Parkin Elmer) air objective and / or by using a Cytation 5 multifunctional detector (Biotek). The cells were scored using the 3D cell viability assay (Promega).

[0068] Western blotting

[0069] Western blotting was performed according to standard procedures (Terranova-Barberio et al. Oncotarget 2016, 7(7):7715-7731). Images were acquired using Image Quant LAS 500 and intensity was measured by Image Quant TL imaging software (GE Healthcare, IL, USA).

[0070] Flow cytometric analysis of apoptosis

[0071] FACScan flow cytometry analysis was performed on cells treated with the indicated concentrations of VPA and / or SIM as previously reported (Bruzzese et al. Clin Cancer Res 2006, 12(2):617-625). Annexin-V binding was identified by flow cytometry using Annexin-V-FITC staining according to the manufacturer's instructions (Becton Dickinson, San Jose, CA).

[0072] RNA isolation, RT-PCR assay and real-time PCR

[0073] RNA was isolated by Trizol reagent (Invitrogen, CA, USA) as previously described (Terranova-Barberio et al. Oncotarget 2016, 7(7):7715-7731). Real-time PCR was performed using an ABI Prism 7900HT sequence detection system (Applied Biosystems, CA, USA) using specific primers. The relative mRNA expression levels of all genes were calculated using the 2-ΔΔCT method and normalized to the level of β-actin as an endogenous control gene. The probes used were as follows: vimentin (QT00004081), ZEB1 (QT00052899), CDH1 (QT00003451), TGFβ (QT00081186), β-actin (QT01025850), purchased from Qiagen (Valencia, CA, USA).

[0074] Migration and invasion assays

[0075] As previously described (Moreno-Bueno et al., Nat Protoc 2009, 4 (11): 1591-1613), migration was assessed by wound healing assay. In brief, PANCI, PANC28, COLO357, MIAPACA, L3.6pl and ASPC1 cells were seeded in 96-well CellCarrier ultra (Parkin Elmer) to 90% confluence. 24 hours after plating, a strip of constant diameter was longitudinally drawn in the confluent monolayer using a sterile 10-μL pipette tip to simulate a wound. The cells were then untreated or exposed to VPA, SIM and gemcitabine / paclitaxel (taxol) (GEM / TAX) until the wound was almost completely closed. At the specified time, the holes were photographed with an Opera Phenix microscope (Parkin Elmer) air objective lens amplified 20X. The distance between the wound edges was determined by Harmony software (Parkin Elmer) for quantitative measurement. Invasion assay was performed in Transwell using PVPF filter with 8 μm pore size. Briefly, PANCI, PANC28, COLO357, MIAPACA, L3.6pl and ASPC1 5000 cells were stained with cell tracking green (Thermo Fisher Scientific) and inoculated in medium containing 1% FBS in the upper part of Transwell, and 500 μL medium containing 10% FBS was added in the lower part. Cells were treated after 24 hours, and the lower cells were measured by 5 times magnification of the air objective lens of Opera Phenix microscope (Parkin Elmer) and counted by Harmony software (Parkin Elmer) after 48 hours.

[0076] Immunofluorescence assay

[0077] Cells were plated on slides in 24-well plates at 50,000 cells / well and treated with drugs as indicated in the legend. The cells were then fixed in 4% paraformaldehyde (20 minutes at room temperature), blocked with 0.2% PBS / BSA solution (5 minutes at room temperature), and incubated with anti-vimentin primary antibody at 37°C for 1 hour. After washing, the cells were incubated with anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, USA) at 37°C for 30 minutes. At the specified time, the wells were photographed with an Opera Phenix microscope (Parkin Elmer) air objective at 20X magnification. Quantitative measurements were performed by determining the distance between the wound edges using Harmony software (Parkin Elmer).

[0078] Cell fluorescence assay

[0079] PANCI, ASPC1, PANC28, and BxPC3 cell lines were treated with VPA and SIM alone or in combination as reported in the figure legends. Cells were harvested after 24 and 48 hours and stained with BD's Annexin V-FITC for 15 minutes at 4°C to assess apoptotic cells. HPaSteC cells were treated with VPA and SIM alone or in combination and treated with conditioned medium from PANCI at the same doses of VPA and SIM alone or in combination for 24 hours. Cells were harvested after 24 hours and stained with Thermo Fisher's GFAP-APC for 30 minutes at 4°C to assess activated stellate cells.

[0080] In vivo xenograft studies

[0081] In vivo studies were performed in accordance with Directive 2010 / 63 / EU on the protection of Animals used for scientific purposes, which came into force in Italy through Legislative Decree DLGS26 / 2014. For orthogonal xenograft experiments, female athymic nude mice (NCLnu) aged 6 to 8 weeks were purchased from Envigo Laboratories (Huntington, UK). The mice were acclimatized in the animal care facility of the CROM Tumor Research Center in Mercogliano. To generate pancreatic tumors, PANC1 LUC cells were harvested from subconfluent cultures and resuspended in PBS solution. Orthotopic injection of pancreatic cancer cells was performed as previously described (Santoro et al., Mol Cancer Ther 2020, 19(1): 247-257). Briefly, mice were anesthetized with a 3% isoflurane-air mixture. A small incision was made on the left flank and the spleen was removed. Tumor cells (0.5 x 10 6 240 cells in 50 μL PBS) were injected subcapsularly into the pancreatic region just below the spleen. The tumor cell suspension was injected using a 30-gauge needle and a 1 mL disposable syringe. Successful subcapsular intrapancreatic injection of tumor cells was identified by the appearance of vacuoles without intraperitoneal leakage. A single-layer abdominal wound was closed with wound clips (Clay Adams, New Jersey, NJ). The mice tolerated the surgical procedure well and no anesthesia-related deaths occurred. After 1 week, the mice were randomized into four experimental groups (n=5). Mice were treated as follows: (a) vehicle; (b) gemcitabine (25 mg / Kg per week, intraperitoneal injection) and albumin-bound paclitaxel (20 mg / Kg per week, intraperitoneal injection) resuspended in saline solution, 100 μL / dose; (c) VPA (thawed in water and diluted in physiological solution) and SIM (thawed in DMSO and diluted in physiological solution); (d) combination of VPA / SIM plus gemcitabine / albumin-bound paclitaxel. Injections continued for 2 weeks. All mice received drug vehicle. The treatment schematic is shown in Figure 8. Tumor volume was monitored by IVIS imaging (PerkinElmer) and signal intensity (photons / second) was quantified using Living Image software 4.1 (PerkinElmer).

[0082] For heterotopic xenograft experiments, 2*10 6PANCI cells were suspended in 200 μL PBS and injected subcutaneously into the right flank of 6-week-old female balb / c nude mice (Envigo Laboratories) (Huntington, UK). Tumor volume [1 / 2 (length x width 2)] was assessed using a digital caliper twice a week (Monday and Thursday). When the tumor became obvious (7 days after injection), the mice were randomly divided into 8 experimental groups (n=6). The mice were treated as follows: (a) vehicle; (b) gemcitabine (50 mg / Kg per week, intraperitoneal injection) and albumin-bound paclitaxel (10 mg / Kg per week, intraperitoneal injection) resuspended in saline solution, 100 μL / dose; (c) valproic acid (200 mg / Kg, 7 days / week, oral), simvastatin (2 mg / Kg, 7 days / week, oral) resuspended in saline solution, 100 μL / dose; (dg) double and triple combinations. Drug treatment lasted for 22 days. All mice received the drug vehicle. At the end of the treatment, all mice were killed and tumor samples were collected. At the end of the treatment (the 28th day), three mice were killed in each group, and whole blood samples were collected by intracardiac puncture. The blood was centrifuged at 2,500 rpm for 10 minutes to separate serum. Biochemical assessment of glutamate oxaloacetate transaminase (GOT) activity, glutamate pyruvate transaminase (GPT) activity and creatinine level was performed by COBAS analyzer (Roche).

[0083] Quantification of TGF-β in mouse blood

[0084] Serum samples were tested using the TGF-β1 Quantikine Elisa kit (R&D Systems) after acid activation according to the manufacturer's protocol. A standard curve using 31.5-2,000 pg / ml human recombinant TGF-β 1 was generated using the kit reagents and used to calculate TGF-β1 equivalents in mouse serum. Each sample was examined in duplicate. Masson's trichrome staining For direct visualization of collagen fibers and histological assessment of collagen deposition, trichrome staining was performed using the Masson's trichrome staining kit (R&D Systems). Slides were analyzed blindly by a single pathologist (FT).

[0085] Statistical analysis

[0086] All experiments were performed at least three times. Statistical significance was determined by one-way ANOVA test, and p < 0.05 was considered statistically significant. All statistical evaluations were performed using Graph Pad Prism 7.

[0087] result

[0088] Synergistic effects of statin / HDACi combinations in PDAC cell lines: repurposing of VPA and simvastatin as anticancer agents.

[0089] The inventors first evaluated the antiproliferative effects of different statins (simvastatin-SIM, lovastatin-LOV and atorvastatin-ATOR) and HDAC inhibitors (panostat-PAN, vorinostat-VOR, valproic acid-VPA and entinostat-MS-275) on a group of PDAC cell lines (PANC28, PANCI, ASPC1, BxPC3, MIAPACA2, L3.6pl, KPC ID11 and KPC ID95) with different genetic and phenotypic characteristics by SRB colorimetric assay (Table 1). Although the tested cell lines showed different basal proteins and epithelial mesenchymal marker (EMT) transcription levels at the protein and transcript levels (Figure 1A-B), they were almost equally sensitive to the antiproliferative effects of VPA, PAN, VOR and MS-275 at their respective mM, nM, μM concentrations (Table 1).

[0090]

[0091] Table 1 Pancreatic cancer cell model. The antiproliferative effects of HDAC inhibitors and statins were evaluated as half maximal inhibitory concentration (IC50) after 96 h of treatment. Abbreviations: VPA: valproic acid; SIM: simvastatin; PAN: panobinostat; VOR: vorinostat; MS-275: entinostat; ATOR: atorvastatin; LOV: lovastatin.

[0092] We then systematically screened the effects of HDACi / statin combinations in PDAC cell lines by exploring equipotent doses of both drug classes (50:50 cytotoxicity ratio) ( Figure 2 and Table 1). When VPA was associated with any of SIM, LOV or ATOR, all cell lines obtained consistent anti-tumor synergistic effects calculated by low combination index (Cis) at 50% (CI50), 75% (CI75) and 90% (CI90) cell lethality, showing good results. On the other hand, among the other HDACIs tested, the combination of VPA and SIM seemed to be the best performer in all tested PDAC cells. Therefore, for all further experiments, the inventors will focus on the VPA / SIM combination.

[0093] The synergistic effects observed thus far were next evaluated by colony formation assays to determine the effects of VPA and SIM alone or in combination at high (IC25) and low (IC10) doses on PANCI, ASPC1, PANC28, and BxPC3 cells (Figure 3A). In all cell lines, VPA and SIM alone or in combination inhibited colony formation, and a statistically significant enhancement of the antiproliferative effects of the drugs was also observed at low doses when the two drugs were given together (Figure 3A). Since 3D cultures better recapitulate the complexity of tumor growth compared to 2D monolayer conditions, the inventors also tested these combinations on 3D cultures of PANCI, ASPC1, PANC28, and BxPC3 cells. It uses the cell carrier spheroid ULA microplate, which allows cell-based microplate experiments to be performed using a 3D cell culture model and forms consistent round spheroids to obtain highly reproducible imaging data. A 3D microtissue model was obtained using normal fibroblasts as a scaffold for the PDAC cell line at a ratio of 3:1, as described in the literature [Brancato V et al., Biomaterials 2020, 232: 119744.]. Microtissues were obtained after 24 hours, and confocal images showed strong interactions between fibroblasts and PDAC cells, with clear membranes around cell aggregates. The assembled microtissues were treated with VPA and SIM alone or in combination (IC25 of each drug) for 96 hours. The data showed that the VPA / SIM combination resulted in almost complete destruction of the microtissue compared to the control or single treatment (Figure 3B), which was confirmed by microtissue ATP bioluminescence assays, and cell viability was significantly reduced in the combined treatment in all analyzed cell models (Figure 3C).

[0094] Consistent with the above data, there was a significant induction of apoptosis in all PDAC cells treated with the VPA / SIM combination, as indicated by significant PARP cleavage after 24 and 48 h of treatment, especially at the IC50 values ​​of both drugs (Figure 3D). These results were confirmed by cytofluorimetric assessment of Annexin V-positive cells at 48 and 72 h ( Figure 4 ).

[0095] The combination of VPA / SIM plus gemcitabine / paclitaxel (taxol) induced apoptosis and reduced cell growth in monolayer and spheroid cultures of PDAC cells.

[0096] Next, the inventors explored the potential of the VPA / SIM combination to sensitize PDAC cells to the double combination of gemcitabine / paclitaxel (taxol) (GEM / TAX) chemotherapy. A fixed low dose (VPA 0.5mM and SIM 0.625μM) of VPA / SIM combination was studied, while increasing the GEM / TAX concentration (Figure 5A). In all four PDAC cell lines tested, a consistent synergistic antiproliferative effect was obtained when low doses of VPA / SIM were combined with the lowest concentration of GEM / TAX compared to the double combination of GEM / TAX alone. Interestingly, these results were verified in primary PDAC cells (data not shown). In addition, the VPA / SIM / GEM / TAX combination showed a synergistic induction of apoptosis in PANCI, PANC28 and ASPC1 compared to the double combination, with caspase 3 / 7 activation (Figure 5B) and PARP cleavage (Figure 5C) as the measurement index. In order to verify the efficacy of this combination in a more complex model to better reproduce tumor growth, the inventors used a 3D cell self-assembly spheroid system. Specifically, cells were plated in a low-attachment plate in a spheroid culture medium to form a first-generation spheroid, and the cells were treated with VPA and SIM for 24 hours after 48 hours. The spheroids were then disaggregated and plated again to obtain the second generation under three different conditions to highlight different effects. In Figures 5D and 5E, the second generation PANCI and ASPC1 were plated under adhesion conditions, respectively, and treated for 96 hours as shown to study the effects of treatment on cell viability with more invasive characteristics (Figure 5D-E, right side). Then, the inventors evaluated the ability of treatment to prevent / reduce more aggressive tumors in terms of viability (Figure 5D-E, middle) and inducing apoptosis (Figure 5D-E, left side), and the second generation was treated in the presence of drugs for 72 hours and 24 hours in the 3D system, respectively. All 3D culture experiments confirmed that the VPA / SIM combination had a strong inhibitory effect, and the GEM / TAX effect was significantly enhanced, while as a single treatment, the effect was poor. Similarly, we demonstrated (data not shown) the efficacy of the VPA / SIM combination in sensitizing microtissues to GEM / TAX treatment, which were generated by co-culture with stellate cells isolated from the tumor microenvironment of the PANCI xenograft model. Finally, VPA and SIM alone or in combination with chemotherapy inhibited colony formation, and when the four drugs were given together, we observed that the antiproliferative effect of the drugs was also significantly enhanced at low doses (Figure 5F). Valproic acid and simvastatin were combined with gemcitabine / paclitaxel (taxol) to target TGFβ-induced EMT.

[0097] Multiple evidences show that the involvement of EMT program makes PDAC cells more invasive and resistant to apoptosis induced by treatment. Here, the inventors show that the VPA / SIM combination causes changes in EMT markers, in which e-cadherin increases and vimentin levels decrease. Combined with GEM / TAX, and the induction of pro-apoptotic effects assessed by PARP and caspase 3 cleavage, this effect is maintained or further enhanced (Fig. 6A-BC). Consistently, the inventors also observed that the mRNA levels of mesenchymal marker vimentin and e-cadherin repressor zinc finger E-box binding homeobox 1 (ZEB1) were similarly reduced, while e-cadherin (CDH1) mRNA levels increased (Fig. 6D). To further explore the molecular mechanisms behind the synergistic interaction of the VPA / SIM combination with GEM / TAX involved in EMT regulation, we performed an Ingenuity Pathway Analysis (IPA) search for “vimentin and e-cadherin” combined with “HDACs and HMGCR” as targets of VPA and SIM, respectively. As shown in Figure 6E, we revealed a network with direct and indirect relationships connecting all proteins used as inputs, confirming the functional relationship between the targets of our treatment combination and EMT markers. Transforming growth factor β (TGFβ) emerged as a central hierarchical dominant hub in this IPA network, which is consistent with the definition of TGFβ as one of the major drivers of EMT, especially in PDAC. A large number of documents are consistent. It is consistent that "migration of tumor cell lines", "fibrosis" and "invasion of tumor cell lines" are the most significantly enriched functions of the network (Figure 6E). These results prompted us to deepen this relationship and test the hypothesis that the synergistic effect of VPA / SIM and GEM / TAX combination can weaken the EMT induced by TGFβ. In addition, the inventors confirmed that VPA / SIM can prevent the regulation of EMT markers (VIM, ZEB1 and CDH1) induced by TGFβ in PANCI cells after TGFβ stimulation (Figure 6F). They also evaluated the ability of VPA / SIM to directly affect TGFβ at the transcriptional level, indicating that VPA / SIM significantly weakened its transcription (Figure 6G) at a very early time point.

[0098] VPA / SIM synergistically reduces the migration and invasion ability of PANCI cells and their impact on the PDAC microenvironment with gemcitabine / paclitaxel (taxol).

[0099] Functional testing of the effects of mechanical evaluation was performed to evaluate the ability of the combination of the present invention to affect cell migration and invasion. This showed that the combination of VPA / SIM and GEM / TAX significantly inhibited the migration and invasion of a group of PDAC cell lines (Figures 7A-B). Some published evidence shows that activated stellate cells (a group of fibroblasts in the pancreatic stroma) play a key role in the pathogenesis of pancreatic fibrosis (desmoplasia) and pancreatitis (Thomas D. et al., Molecular Cancer 2019). Since these cells, once activated by tumor cell crosstalk, can regulate ECM remodeling, leading to chemotherapy resistance, the inventors decided to test the ability of the VPA / SIM combination to interfere with its activation directly or by regulating cancer cell / fibroblast crosstalk. First, HPaSteC cells were treated with VPA and / or SIM for 24 hours, and it was shown that the treatment was able to impair its activation (measured in the form of G-FAP-positive cells) (Figure 7C) without affecting its proliferation state (Figure 7D). Interestingly, similar results were obtained when HpaSteC was treated with PANC1-conditioned medium from untreated or VPA / SIM-treated PDAC cell lines ( FIG. 7E ), suggesting that our treatment was able to interfere with the crosstalk between PDAC and HpaSteC cells.

[0100] To further investigate the mechanism, we found that the above-mentioned VPA / SIM inhibitory effects were restored using conditioned medium from PANCI cells transfected with a constitutively active YAP oncogene (YAP5SA), confirming its involvement in this mechanism ( Figure 7F ), as well as the impaired translocation of YAP into the nucleus after VPA / SIM treatment ( Figure 7G ).

[0101] Likewise, the inventors demonstrated the efficacy of the VPA / SIM combination in sensitizing GEM / TAX-treated microtissues generated by co-culturing PANCI, ASPC1, and MIAPACA2 cells with human stellate cells HpaSteC. This synergistic antitumor effect was very evident in PANCI and ASPC1 cells, but slightly weaker in MIAPACA cells, and the volume of the microtissues seemed to be reduced to almost the same level after treatment with all combinations (Figure 7G).

[0102] Synergistic antitumor effects of valproic acid and simvastatin combined with gemcitabine / nab-paclitaxel in vivo.

[0103] The synergistic interaction of the proposed combination was confirmed in orthotopic and heterotopic xenograft in vivo models. For an orthotopic model that better reproduces the PDAC tumor microenvironment, the inventors utilized PANC1 LUC cells injected into the mouse pancreas. One week after implantation, mice were randomly assigned to receive subtherapeutic doses of VPA / SIM combination (200 mg / Kg and 2 mg / Kg, respectively, daily intraperitoneal injection for 2 weeks) and GEM / NP (gemcitabine, 25 mg / Kg per week, intraperitoneal injection and albumin-bound-paclitaxel, 20 mg / Kg per week, intraperitoneal injection); a combination of VPA / SIM+GEM / NP, or its vehicle, as shown in Figure 8A. Tumor growth was monitored once a week by photon intensity at specific time points (Figure 8B), and for each group, single mice were measured on days 5, 12, and one week after the end of treatment (follow-up study), and compared with the corresponding values ​​at day 1 (T0) (Figure 8C). Compared with the other groups, all mice in the VPA / SIM+GEM / NP group had significant tumor growth inhibition observed within 5 days after the start of treatment and maintained over time (Figure 8C). Compared with the control group and the single treatment group, the VPA / SIM+GEM / NP combination produced statistically significant tumor growth inhibition, evaluated as the mean of the measurements of each group (Figure 8D). Xenograft mice tolerated the combined treatment well, as shown by weight maintenance (Figure 8E) and the absence of other signs of acute or delayed toxicity. Consistent with the in vitro results, the levels of circulating TGFβ1 in the serum of mice treated with VPA / SIM combined with GEM / NP were significantly reduced (Figure 8F), while fibrosis was reduced, as assessed by Masson's trichrome staining of pancreatic tumor sections. Specifically, four of the six mice / group treated with the VPA / SIM plus GEM / NP combination showed <10% fibrosis, while most of the mice in the control group showed ≥10% fibrotic areas (Figure 8G). Consistently, data not shown demonstrate the ability of the VPA / SIM combination to target pancreatic stellate cells by reducing the levels of their major activation marker, GPAF.

[0104] For the ectopic model, PANCI cells were injected into the right flank of mice (2*10 6). One week after implantation, mice were randomly assigned to receive a combination of VPA / SIM (200 mg / Kg and 2 mg / Kg, respectively, daily intraperitoneal injection for 2 weeks) and GEM / NP (gemcitabine, 50 mg / Kg, intraperitoneal injection per week and albumin-bound-paclitaxel, 10 mg / Kg, intraperitoneal injection per week) and a combination, this time, the GEM / NP ratio explored was closer to the ratio used to treat PDAC patients in clinical practice, as shown in the schematic diagram (Figure 9A). Tumor volume was measured by caliper (Figure 9B). In this model, circulating TGF-β levels were also significantly reduced in mice treated with VPA / SIM combined with GEM / NP (Figure 9C). In addition, there were no significant changes in biochemical parameters (GOT, GPT and creatinine) in the serum of untreated mice compared to the treated groups, confirming that the combination did not significantly increase the hepatotoxicity of single treatments (Figures 9E-FG).

Claims

1. A combination comprising at least one HDAC inhibitor and at least one statin for use alone or in combination with at least one other anticancer agent in the treatment of pancreatic cancer.

2. The combination according to claim 1, wherein the at least one HDAC inhibitor is selected from valproic acid or a salt thereof, panobinostat, vorinostat, entinostat or moxistat, and the statin is selected from simvastatin, atorvastatin, lovastatin.

3. The combination according to claim 2, wherein the at least one HDAC inhibitor is valproic acid and the statin is simvastatin.

4. A combination consisting of an HDAC inhibitor and a statin as claimed in any one of the preceding claims, for use alone or in combination with at least one other anticancer agent in the treatment of pancreatic cancer.

5. A combination for use according to any one of the preceding claims, wherein the combination is for use in subjects who are responsive to, or are resistant to, or have developed resistance to first-line therapy.

6. The combination for use according to claim 5, wherein the first-line therapy comprises the administration of gemcitabine and / or nab-paclitaxel.

7. The combination for use according to any one of the preceding claims, wherein the ratio of the at least one HDAC inhibitor and the one statin is about a 50:50 cytotoxic ratio.

8. The combination for use according to any one of the preceding claims, wherein the HDAC inhibitor is selected from: - valproic acid at a concentration ranging from about 16 mM to about 0.5 mM; and / or - Vorinostat at a concentration ranging from about 16 μM to about 0.125 μM; and / or - Panobinostat at a concentration ranging from about 160 nM to about 2.5 nM; and / or - entinostat at a concentration ranging from about 16 μM to about 0.125 μM; And wherein the statin is selected from: - simvastatin at a concentration ranging from about 8 μM to about 0.06 μM; and / or - atorvastatin at a concentration ranging from about 8 μM to about 0.06 μM; and / or - Lovastatin at a concentration ranging from about 8 μM to about 0.06 μM.

9. The combination for use according to any one of the preceding claims, wherein the other anticancer agent is selected from one or more of the following: Btk tyrosine kinase inhibitors, Erbb2 tyrosine kinase receptor inhibitors; Erbb4 tyrosine kinase receptor inhibitors, mTOR inhibitors, thymidylate synthase inhibitors, EGFR tyrosine kinase receptor inhibitors, epidermal growth factor antagonists, Fyn tyrosine kinase inhibitors, KIT tyrosine kinase inhibitors, Lyn tyrosine kinase inhibitors, NK cell receptor modulators, PDGF receptor antagonists, PARP inhibitors, poly ADP ribose polymerase inhibitors, poly ADP ribose polymerase 1 inhibitors, poly ADP ribose polymerase 2 inhibitors, poly ADP ribose polymerase 3 inhibitors, galactosyltransferase modulators, dihydropyrimidine dehydrogenase inhibitors, orotate phosphoribosyltransferase inhibitors, telomerase modulators, mucin inhibitors, secretin agonists, TNF-related apoptosis-inducing ligand modulators, IL 17 gene stimulators, interleukin 17E ligands, neurokinin receptor agonists, cyclin G1 inhibitors, checkpoint inhibitors, PD-1 inhibitors, PD-L1 inhibitors, CTLA4 inhibitors, topoisomerase I inhibitors, Alk-5 protein kinase inhibitors, connective tissue growth factor ligand inhibitors, notch-2 receptor antagonists, notch-3 receptor antagonists, hyaluronidase stimulators, MEK-1 protein kinase inhibitors; MEK-2 protein kinase inhibitors, GM-CSF receptor modulators; TNFα ligand modulators, mesothelin modulators, asparaginase stimulators stimulators, caspase-3 stimulators; caspase-9 stimulators, PKN3 gene inhibitors, sonic hedgehog inhibitors; smoothened receptor antagonists, AKT1 gene inhibitors, DHFR inhibitors, thymidine kinase stimulators, CD29 regulators, fibronectin regulators, interleukin-2 ligands, serine protease inhibitors, D40LG gene stimulators; TNFSF9 gene stimulators, 2-ketoglutarate dehydrogenase inhibitors, TGF-β type II receptor antagonists, Erbb3 tyrosine kinase receptor inhibitors, cholecystokinin CCK2 receptor antagonists, Wilms tumor protein modulators, Ras GTPase modulators, cyclin-dependent kinase 4 inhibitor A modulators, estrogen receptor β modulators, 4-IBB inhibitors, 4-1BBL inhibitors, PD-L2 inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, BTLA inhibitors, HVEM inhibitors, TIM3 inhibitors, GAL9 inhibitors, LAG3 inhibitors, VISTA inhibitors, KIR inhibitors, 2B4 inhibitors, CD 160 inhibitors and CD66e modulators, taxanes or a combination thereof.

10. The combination for use according to any of the preceding claims, wherein the other anticancer agent is selected from the group consisting of baviximab, IMM-101, CAP1-6D, Rexin-G, genistein, CVac, MM-D37K, PCI-27483, TG-01, LO Ad-703, CPL613, umomostat, CRS-207, novacap, trametinib, Atu-027, sonidegi, GRASPA, trabecin, nartocipa, Vaccell, ogavuzumab, estuzumab, refactinib, regorafenib, lapatinib, selumetinib, rucaparib, peraracept, taritozumab, PEGylated hyaluronidase, valitinib, aglatimagene besadenovec, GBS-01, GI-4000, WF-10, golunitib, afatinib, RX-0201, FG-3019, pertuzumab, DCVax-Direct, selinexor, glufosfamide, virulizumab (90Y), tilizumab, brivudine, nimotuzumab, algenpantucel-L, tegafur + gimeracil + oxazolidinone potassium + leucovorin, olaparib, ibrutinib, pirarubicin, Rh-Apo2L, tetomorphine, tegafur + gimeracil pyridamole + oxazide potassium, tegafur + gimeracil + oxazide potassium, masitinib, Rexin-G, mitomycin, erlotinib, doxorubicin, dexamethasone, vincristine, doxorubicin, topotecan, paclitaxel, interferon, platinum derivatives, taxanes, paclitaxel, vinca alkaloids, vinblastine, anthracyclines, doxorubicin, epipodophyllotoxin, etoposide, cisplatin, rapamycin, methotrexate, actinomycin D, Aplysia 10, colchicine, emetine, trimetrexate, chlorpheniramine, cyclosporine, daunorubicin, teniposide, amphotericin , alkylating agents, chlorambucil, 5-fluorouracil, camptothecin, metronidazole, imatinib, panitumumab, abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, hexamethylmelamine, amifostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, AZD9291, active BCG, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, carbendazim, capecitabine (), camptothecin, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cladribine, clofarabine, Doxorubicin, cytarabine, actinomycin D, darbepoetin alfa, daunorubicin, denileukin, dexrazoxane, docetaxel, doxorubicin (neutral), doxorubicin hydrochloride, drostanolone propionate, epirubicin, epoetin alfa, estramustine, etoposide phosphate, etoposide, exemestane, filgrastim, floxuridine fludarabine, fulvestrant, gefitinib, gemcitabine, gemtuzumab, goserelin acetate, histrelin acetate, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa-2a,Interferon α-2b, irinotecan, lenalidomide, letrozole, folinic acid, leuprolide acetate, levamisole, lomustine, progesterone acetate, melphalan, mercaptopurine, 6-MP, mesna, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nelarabine, nofitumomab, oprelleukin, oxaliplatin, paclitaxel, palifermin, par Mildronate, pegaspase, pegaspase, pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin, porfimer sodium, procarbazine, quinacrine, rasburicase, rituximab, rociletinib, sargramostim, sorafenib, streptozotocin, sunitinib maleate, talc, tamoxifen, temozolomide, teniposide, VM-26, testolactone, thioguanine , 6-TG, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, ATRA, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, zoledronic acid, zoledronic acid, pembrolizumab, nivolumab, IB1-308, mDX-400, BGB-108, MEDI-0680, SHR-1210, PF-06801591, PDR-001, GB-226, STI-1110, durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-170, STI-1014, FOLFIRINOX, and KY-1003, and combinations thereof. , 11. The combination for use according to any one of the preceding claims, wherein the other anticancer agent is at least one of paclitaxel, gemcitabine, nab-paclitaxel, cisplatin, capecitabine, irinotecan or a combination thereof.

12. The combination according to claim 11, wherein the other anticancer agent is a combination of paclitaxel and gemcitabine or a combination of nab-paclitaxel and gemcitabine or a combination of gemcitabine, nab-paclitaxel, cisplatin, capecitabine.

13. The combination according to any one of the preceding claims, wherein the pancreatic cancer is selected from the group consisting of pancreatic adenocarcinoma, unresectable pancreatic cancer, locally advanced pancreatic cancer, borderline resectable pancreatic cancer, locally advanced pancreatic ductal adenocarcinoma, borderline resectable pancreatic ductal adenocarcinoma, metastatic pancreatic cancer, chemotherapy-resistant pancreatic cancer, pancreatic ductal adenocarcinoma, squamous pancreatic cancer, pancreatic progenitor cells, immunogenic pancreatic cancer, abnormally differentiated endocrine-exocrine (ADEX) tumors, exocrine pancreatic cancer, pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, mucinous cystic neoplasms, mucinous pancreatic cancer, adenosquamous carcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with osteoclast-like giant cells, pancreatic cystic tumors, islet cell tumors, pancreatic endocrine tumors, or pancreatic neuroendocrine tumors.

14. The combination for use according to any of the preceding claims, wherein the at least one HDAC inhibitor and the one statin are administered in a single dosage unit or are administered separately.

15. The combination for use according to claim 14, wherein said single dosage unit comprises at least one pharmaceutically acceptable excipient.

16. The combination for use according to claim 15 or 16, wherein the single dosage unit or divided dosage formulation is in the form of an oral, parenteral and / or topical dosage form.

Images