Transgenic Mouse Model of Pancreatic Cancer
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PreviousworkhasshownthatvitaminE d-tocotrienol(VEDT)prolongssurvivalanddelaysprogressionof pancreatic cancer in theLSL-Kras G12D/þ ;Pdx-1-Cremouse model of pancreatic cancer. However,the effect of VEDT alone or in combination with gemcitabine in the more aggressive LSL-Kras G12D/þ ;LSL-Trp53 R172H/þ ; Pdx-1-Cre (KPC) mouse model is unknown. Here, we studied the effects of VEDT and the combination of VEDT and gemcitabine in the KPC mice. KPC mice were randomized into four groups: (i) vehicle [olive oil, 1.0 mL/kg per os twice a day and PBS 1.0 mL/kg intrapertoneally (i.p.) twice a week], (ii) gemcitabine (100 mg/kg i.p. twice a week), (iii) VEDT (200 mg/kg per os twice a day), and (iv) gemcitabine þ VEDT. Mice received treatment until they displayed symptoms of impending death from pancreatic cancer, at which pointanimalswereeuthanized.At16weeks,survivalwas10%inthevehiclegroup,30%inthegemcitabine group, 70% in the VEDT group (P < 0.01), and 90% in the VEDT combined with gemcitabine group (P < 0.05). VEDT alone and combined with gemcitabine resulted in reversal of epithelial-to-mesenchymal transition in tumors. Biomarkers of apoptosis (plasma CK18), PARP1 cleavage, and Bax expression were more greatly induced in tumors subjected to combined treatment versus individual treatment. Combined treatment induced cell-cycle inhibitors (p27 Kip1 and p21 Cip1 ) and inhibited VEGF, vascularity (CD31), and oncogenic signaling (pAKT, pMEK, and pERK) greater than individual drugs. No significant differences in body weight gain between drug treatment and control mice were observed. These results strongly support further investigation of VEDT alone and in combination with gemcitabine for pancreatic cancer prevention and treatment. Cancer Prev Res; 6(10); 1074–83. � 2013 AACR.Cite
VEP nucleosides bypass two mechanisms of tumor resistance: nucleoside transport and kinase downregulation. Isoforms of VE have shown activity against solid and hematologic tumors. Gemcitabine was conjugated at the 5' position to either δ-tocopherol-MP (NUC050) or δ-tocotrienol-MP (NUC052). NUC050 has been demonstrated to deliver gemcitabine-MP intracellularly. Its half-life IV in mice is 3.9 compared to 0.28 hours for gemcitabine (European J Cancer. 2016. 61(Suppl. 1):S119). When tumors in nude mice reached 32 to 75 mg mm3 (day 4) treatment was initiated with gemcitabine (120 mg/kg IP q3dx9), NUC050 or NUC052 (both 40 mg/kg qwkx4) and compared to saline control (SC). Gemcitabine inhibited tumor growth but was not tolerated. NUC050 resulted in inhibition to tumor growth on days 11-31 (p<0.05), with a nadir of -73% compared to SC. Median survival was 25.5 days (SC) vs 33 days (NUC050) ((hazard ratio) HR=0.24, p=0.017). NUC052 had the dose increased to 50 mg/kg after 2 doses. NUC052 resulted in inhibition to tumor growth on days 14-27 (p<0.05), with a nadir of -45%, and median survival was 34 days (HR=0.27, p=0.033). NUC050 and NUC052 have been shown to be safe and effective in a NSCLC xenograft. Studies have been initiated in a pancreatic cancer xenograft.
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Objective To investigate the enhanced effect of gemcitabine by capsaicin on T3M4 cell xenograft on athymic.Methods The models of T3M4 cell xenograft on athymic mouse were established and randomized to four groups with intraperitoneal (IP) injection of different drugs (group N 0.9% sodium chloride), group G(gemcitabine,125 mg/kg),group C (capsaicin 20 mg/kg) and group G+C (gemcitabine 80 mg/kg and capsaicin 20 mg/kg in combination).The drugs were injected once every 3 days,8 times in all.The mice were sacrificed 1 week after the last injection.The tumor volume and tumor weight were measured during the drug therapy.Immunohistochemistry(IHC) was performed to detect the expression of NF-κB,P-gp and Ki-67,RT-PCR was performed to detect the expression of NF-κB and MDR1 mRNA.Results One week after the last administration,the mean tumor volume and tumor weight in group G+C were significantly decreased compared to the other groups.IHC analysis showed the expression of NF-κB and P-pg were down regulated in C and G+C groups compared to the other two groups.The expression of Ki-67 was also down regulated significantly in G+C group compared to the other groups.RT-PCR analysis showed the expression of NF-κB and MDR1 mRNA in C and G+C groups were down regulated significantly compared to the other groups.Conclusions Capsaicin can enhance the anti-tumor effect of gemcitabine on pancreatic cancer xenograft.Downregulation of NF-κB and MDR1 is possibly one of the mechanisms.
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Abstract Introduction: Heparan sulphate proteoglycans (HSPGs) play a central role in tumor progression and metastasis by presenting and modulating growth factors, cytokines, and other soluble factors. A novel heparin sulphate mimetic (M402), engineered from heparin to have low anti-coagulant activity, has shown promising anti-tumor efficacy in several pre-clinical tumor models. This study was designed to probe the efficacy and mechanism of action of M402 in a genetically engineered mouse (GEM) model for pancreatic cancer. Methods: Mice that spontaneously develop pancreatic cancer (LSL-KRASG12D/+; Trp53 LSL-R172H/flox; pdx-CRE) were treated with twice weekly i.p. doses of saline or gemcitabine (50 mg/kg) starting at Day 30, or with saline or M402 (40 mg/kg/day) administered by a subcutaneous osmotic minipump from Day 30-90, or with a combination of gemcitabine plus M402. Results: Treatment with M402 alone did not prolong survival and gemcitabine alone showed only a modest improvement in survival; however the combination of M402 and gemcitabine significantly improved survival. Moreover, mice treated with the combination of M402 and gemcitabine showed a substantially lower incidence of metastasis. RT-qPCR analysis revealed that M402 treated mice had significantly lower levels of TGF-alpha mRNA than the saline control group, which is corroborated by a corresponding decrease in tumor cell proliferation. Immunohistochemical analysis revealed that M402 treated mice developed reduced areas of epithelial-to-mesenchymal transition (EMT), as defined by negative staining for E-Cadherin, strongly positive staining for vimentin and positive nuclear staining for SNAIL. As there is a direct link between EGFR activation and the nuclear localisation of SNAIL we propose that M402 affects gemcitabine sensitivity and metastasis formation by reducing the expression of TGF-alpha. Discussion: M402 increased the anti-tumor efficacy of gemcitabine in a GEM model for pancreatic cancer resulting in increased survival and, interestingly, decreased incidence of metastasis. One potential mechanism is that the observed reduction in EMT may be due to the reduced expression of TGF-alpha, a cognate ligand for EGFR. These data suggest that the EGFR pathway is active in KRAS mutant tumors. Overall, these results provide a rationale for investigating the clinical use of M402 in combination with gemcitabine in the treatment of human pancreatic cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-43.
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Abstract Background: Pancreatic cancer, an aggressive and lethal cancer is the fourth leading cause of cancer-related deaths in the United States. Gemcitabine is currently the standard therapeutic agent for advanced pancreatic cancer, but has limited efficacy due to chemoresistance and dose escalation toxicity. We have shown that natural vitamin E δ-tocotrienol is the most bioactive tocotrienols against pancreatic cancer in vitro using pancreatic cancer cells lines as well as in vivo using xenograft models. In this study we evaluated the combination of δ-tocotrienol and gemcitabine on survival, tumor growth, apoptosis, angiogenesis and oncogenic signaling in a transgenic mouse model of pancreatic cancer Methods: Offspring of LSLKRASG12D x PDX-1-Cre x Trp53R172H intercrosses (triple positive by genotyping) were randomized to 4 groups: 1) Vehicle (olive oil, 1.0 ml/kg x 2/ day, PO and PBS 1.0 ml/kg x 2/week, IP), 2) Gemcitabine (100 mg/kg, x 2/week, IP), 3) δ-tocotrienol (200 mg/kg x 2/day, PO) and 4) Gemcitabine + δ-tocotrienol. The treatment was started at the age of 5 weeks and continued for 12 weeks. The survival of the mice in three groups was plotted by Kaplan-Meir graph. The weights of mice were recorded every week and the tumor weights were recorded at the end of the study. The apoptosis markers in plasma and pancreatic tumor, epithelial to mesenchymal transition (EMT), angiogenesis and kras/p53 signaling markers were determined by ELISA, Western blotting and immunostaining. Results: No significant difference in food intake and body weight gain between drug treatment and control groups was observed. δ-tocotrienol treatment alone significantly increased overall survival compared to vehicle (P<0.01). Gemcitabine slightly increased survival whereas combination of two drugs significantly enhanced survival (P<0.05). The tumor weights were more significantly decreased in the combination group (p<0.001) than in each drug alone groups (p<0.02). Gemcitabine and δ-tocotrienol alone and in combination decreased EMT (E-cadherin to vimentin) in tumor tissues. Gemcitabine and δ-tocotrienol combination profoundly induced apoptosis (increased CK18 in plasma), PARP1 cleavage, and Bax expression in tumor tissues than either drug alone. δ-tocotrienol was more effective than gemcitabine in the induction of cyclin-dependent kinase inhibitors (p27Kip1 and p21Cip1), inhibition of angiogenesis (VEGF), vascularity (CD31) and Kras down stream signaling (pAKT. pMEK and pERK). However the combination exerted synergistic effect on the above parameters. Conclusion: δ-tocotrienol synergizes with gemcitabine to inhibit pancreatic tumor growth through inhibition of angiogenesis, cell cycle, Kras signaling and induction of apoptosis in transgenic mouse model of pancreatic cancer. Citation Format: Kazim Husain, Barbara Centeno, Marta Perez, Chen Dung-Tsa, Said M. Sebti, Mokenge P. Malafa. Natural vitamin E delta-tocotrienol combined with gemcitabine prolongs the survival, induces apoptosis, inhibits tumor growth, angiogenesis and oncogenic signaling in a transgenic mouse model of pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2070. doi:10.1158/1538-7445.AM2013-2070
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Abstract We have previously reported inhibition of cell migration and proliferation induced by difluoromethylornithine (DMFO) and metformin (Met) combination in human melanoma and colon cancer cells. We further investigated the mechanism of cell death with the combination treatment in melanoma and colon cancer cells. The in vivo effect of the combination in melanoma was also determined. Human colon cancer (HCT 116, HT 29) and melanoma (MEL1861, SK-23) were treated with Met, DFMO, or combination. Western blotting was performed to determine the expression of AMP kinase, mTOR, p70S6K and 4E-BP1. Apoptosis was measured by Annexin V assay. Autophagy was determined by fluorescence microscopy staining for LC3A/B and Western blot analysis of LC3A/B and Beclin-1 expression. For in vivo evaluation, SK-23 cells were injected SQ into BALB/c nu mice. After tumor nodule was established, 4 groups of 6 mice : group 1 - IP injections of vehicle and normal drinking water, group 2 - 2% (w/v) DFMO in drinking water; group 3 - IP Met (250 mg/kg/day), group 4 - DFMO plus Met. Mice body weight and tumor volume were measured every 3 days. Tumor weight was measured on Day 20 at necropsy. For comparison between groups, the student's t test was used and p< 0.05 was considered to be significant. DFMO and Met induced apoptosis in colon cancer and melanoma cells in a dose- and time-dependent manner. Significant increase in apoptosis was noted in the combined treatment group compared with either one alone. Increased number of autolysosomes was observed in the combination group under fluorescence microscopy. Up-regulation of Beclin-1 and LC3A/B expression were also increased with combination treatment compared with either treatment alone. Increased expression of phosphor-AMPK, decreases expression of p70S6 and 4EBP1 were observed with the combination treatment. Both DFMO and Met alone have significant in vivo anti-proliferative effect on human melanoma cells compared with control. However, the anti-proliferative effect of the combination treatment was significantly better than either regimen alone (p< 0.001). Average SK-23 tumor weight was 100 mg for control group compared with 45 mg for group 2 (p<0.05), 35mg for group 3 (p< 0.05), and 25mg for the combination group (p< 0.001). Our findings suggest that combination of DFMO and Met induced cancer cell death via apoptosis and autophagy though activation of AMPK pathway and suppression of the Akt/mTOR signaling pathway. DFMO and Met combination can have significant anti-tumor effect in vivo. Citation Format: Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. Induction of autophagy and apoptosis with polyamine synthesis inhibition and metformin in human melanoma and colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1418. doi:10.1158/1538-7445.AM2014-1418
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Abstract Metformin (MET) is the first-line treatment for type 2 diabetes mellitus. Several epidemiological studies have reported anti-cancer effects of MET, including against pancreatic ductal adenocarcinoma (PDAC), which mainly acts through induction of AMP activated protein kinase (AMPK). Gemcitabine (GEM) has become the standard chemotherapy for PDAC but tolerance to GEM has become a burdensome issue. We evaluated the anti-tumor effects of MET for GEM-resistant PDAC in a xenograft mouse model. For this in vivo study, BxG30 (the cell line for GEM-resistant PDCA) was implanted into both flanks of female BALB/c nude mice. Mice were divided into four groups: (i) control (no treatment); (ii) the GEM-treated group (100 mg/kg); (iii) the MET-treated group (600 mg/kg); and (iv) the combined treatment group (G+M). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET was administrated orally once per day. GEM was given by intraperitoneal injection once per week. Compared with the control, the final tumor volumes were decreased significantly only in the G+M group. [SCW1] The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; G+M, 47.2%. The anti-tumor effect of GEM for BxG30 was clearly limited[SCW2] . MET group showed satisfactory anti-tumor effects, but T/C% was <50% only in the G+M group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDAC. The phosphorylation of ribosomal proteins S6 and 4E-BP, important targets of the mammalian target of rapamycin (mTOR) signaling pathway, was surveyed by western blot analysis. Western blot analysis showed inhibition of S6 and 4E-BP phosphorylation by co-incubation with MET, but not with GEM. Hypoxia-inducible factor 1 (HIF-1) is the one of the most important target of mTOR signaling pathway influencing tumor cell to progress, thus the expression level of HIF-1 was evaluated by western blot analysis as well. The results showed significant inhibition of HIF-1 expression by MET treatment, but not by GEM incubated under hypoxia (95%N2, 5%O2). Then the production of VEGF was evaluated by ELISA under hypoxia. The result showed suppression of VEGF production by MET treatment, but not by GEM. Our data showed that MET develops a different anti-tumor effect from GEM by suppression of mTOR-HIF-1 signaling. These results are of great clinical interest and reveal the potential of another anti-tumor agent for treatment of PDAC. Citation Format: Keiichi Suzuki, Osamu Takeuchi, Yukio Suzuki. The mechanism of antitumor effect of metformin forgemcitabine-resistant pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3511.
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Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human malignancies characterized by late clinical presentation, rapid local and metastatic progression and high resistance to conventional therapies. Gemcitabine, an approved treatment for PDAC, has limited clinical benefits. The insulin-like growth factor (IGF) type 1 receptor (IGF-1R) has shown to be expressed in many human cancers including PDAC. The present study evaluated the therapeutic potential of BMS-754807, a small molecule inhibitor of IGF-1R and insulin receptor (IR), alone and in combination with gemcitabine, in experimental PDAC. The human PDAC cell lines were grown in RPMI 1640 medium supplemented with 10% FBS. In vitro cell proliferation and protein expression were measured by WST-1 assay and immunoblotting. Tumor growth and animal survival studies were performed in murine xenografts. Ki67 and TUNEL staining were used to detect intratumoral proliferation and apoptosis. PDAC cell lines AsPC-1, BxPC-3, Mia PaCa-2 and Panc-1 expressed IGF-1R and phospho-IGF-1R proteins. BMS-754807 and gemcitabine inhibited in vitro cell proliferation of PDAC cell lines; the combination of BMS-754807 with gemcitabine had additive effects. IC25 concentrations of BMS-754807 decreased gemcitabine IC50 from 9.7 μM to 75 nM for AsPC-1, from 3 μM to 70 nM for Panc-1, from 72 nM to 16 nM for Mia PaCa-2 and from 28 nM to 16 nM for BxPC-3 cells, respectively. BMS-754807 caused a significant decrease in phospho-IGF-1R and phospho-AKT proteins in AsPC-1 and Panc-1 cells. BMS-754807 and gemcitabine caused an increase in apoptosis-related PARP-1 and caspase-3 cleavage with additive effects in combination. Compared with controls, in vivo net tumor growth inhibition in BMS-754807, gemcitabine and BMS-754807+gemcitabine groups was 59 percent (p=0.05), 35 percent (p=0.02) and 94 percent (p=0.0007), respectively. Effects on intratumoral proliferation and apoptosis after BMS-754807 and gemcitabine treatments corresponded directly with tumor growth inhibition data. BMS-754807 also caused a decrease in phospho-IGF-1R and phospho-AKT in tumor tissue lysates. Median animal survival (Controls: 21 days) after BMS-754807 was 27 days (p=0.03 versus control), after gemcitabine group 28 days (p=0.05 versus control) and in the BMS-754807+gemcitabine combination group 41 days (p=0.007 versus control and p=0.02 versus gemcitabine or BMS-754807 monotherapy). BMS-754807 has strong antitumor activity in experimental PDAC, and it significantly improves gemcitabine response. These results support the potential of BMS-754807-induced mechanisms for clinical PDAC therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2740. doi:1538-7445.AM2012-2740
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LB-324 Currently, therapeutic strategies for pancreatic cancer (PC) are very limited and most of the first-line therapies had limited survival benefit. Eicosanoids generated from 5-Lipoxygenase (5-LOX) and cyclooxygenase (COX)- pathways are involved in tumor promotion, progression and metastasis of several cancers, including PC. Thus, inhibition of both COX and 5-LOX pathways may be an important strategy for the prevention and treatment of PC. In this study, we compared the effect of Licofelone (LF), a 5-LOX/ COX-inhibitor, therapeutic efficacy in comparison to standard therapeutic agents, Gemcitabine (GT) and Oxaliplatin (OP) in PC xenografts.
Athymic NCr-nu/nu mice were divided into 4 treatment groups (5/group) and fed AIN-76A diet. LF was administered at 200 ppm in diet, while GT (25mg/Kg BW, twice weekly) and OP (5mg/kg BW, once in 4 weeks) was administered by i.p. All mice were given s.c. injection of BxPC-3 pancreatic cancer cells to initiate xenograft tumors. After 10 weeks of treatment, mice were sacrificed and the final Tumor Volume (TV) and tumor weight were measured. Tumor xenografts were assessed for cell proliferation, apoptosis, expression of COX-2, 5-LOX, and VEGFR-1/2 by western blot and Immunohistochemical (IHC) methods.
None of the experimental agents induced any toxicity or weight loss. Treatmentwith GT significantly suppressed TV (37%) and weight (32.4%), whereas OP did not provide any effect. Interestingly, LF showed a very significant decrease in TV (68%) and weight (64%). Further, tumor xenograft treated with GT and LF showed significant suppression of cell proliferation by PCNA staining (66% and 88%, respectively) and induction of apoptosis by TUNEL method (3-fold and 8-fold, respectively) compared to control. IHC & Western blot analysisof tumors from GT and LF treated mice revealed a significant inhibition of COX-2, 5-LOX, and VEGFR-1/2. In summary, our results are the first to show that dietary LF possesses PC inhibitory properties and had better efficacy than GT.
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Abstract Several epidemiological studies have revealed the anti-cancer effects of metformin (MET), first-line treatment for type-2 diabetes mellitus, including against pancreatic ductal carcinoma (PDC). Gemcitabine (GEM) has become the standard chemotherapy for PDC but tolerance to GEM becomes an important issue. We evaluated the anti-tumor effects of MET for GEM-resistant PDC in a xenograft mouse model. For this in vivo study, wild-type BxPC-3 was implanted into both flanks of female BALB/c nude mice. Mice were divided into four groups: (i) control (without any treatment); (ii) GEM-treated group (100 mg/kg); (iii) MET-treated group (600 mg/kg); and (iv) combined treatment group (G+M). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET was administrated orally every day. GEM was given by intraperitoneal injection every week. In the final tumor volumes, the two groups treated with GEM had significantly reduced tumors compared with control, but there were no differences between control and MET groups. The anti-tumor effect of MET for BxG30 (the cell line for GEM-resistant PDC) was evaluated using the method described above. Tumor volumes were decreased significantly in GEM+MET groups compared with control. The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; GEM+MET, 47.2%. The anti-tumor effect of GEM for BxG30 was limited. The MET group showed satisfactory anti-tumor effects, but T/C% was <50% only in the GEM+MET group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDC. Western blot analysis was performed to confirm mammalian target of rapamycin (mTOR) expression level was surely inhibited. The results revealed that the expression level of mTOR treated with MET was significantly decreased, but no in GEM group. Then the expression level of hypoxia-inducible factor 1 (HIF-1) was evaluated by western blot analysis also. The results showed significant inhibition of HIF-1 expression by MET treatment, but not by GEM, again. Our results showed that MET has a partial anti-tumor effect for PDC. Combination therapy has an excellent effect not only for wild-type PDC but also for GEM-resistant PDC. mTOR is well known as an upstream activator of HIF-1 function in cancer cells. That is, the mechanism of anti-tumor effect of MET seems to be, at least partly, the inhibition of HIF-1 through the mTOR regulation. These data suggest that MET could be used to treat PDC. Citation Format: Keiichi Suzuki, Osamu Takeuchi, Yoshiyuki Ishii, Masayoshi Osaku. The mechanism of anti-tumor effect of metformin for gemcitabine-resistant pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2570. doi:10.1158/1538-7445.AM2015-2570
Pancreatic tumor
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Hypoxia is a prevalent feature of solid tumors and is a negative prognostic factor associated with treatment failure and the emergence of a more aggressive, invasive, and metastatic phenotype. Hypoxia in human pancreatic adenocarcinoma (PAC) has been characterized by Eppendorf needle oxygen electrodes, and more recently by the use of the exogenous hypoxia biomarker pimonidazole in patient biopsies. Both methods have demonstrated significant subregional hypoxia. TH-302 (T) is a hypoxia-activated bioreductive prodrug that is selectively activated in regions of severe hypoxia, and upon activation releases the bis-alkylating DNA cross-linker bromoisophosphoramide mustard (Br-IPM). T exhibits hypoxia-selective in vitro cytotoxicity across a wide panel of human cancer cell lines including PAC cell lines and in vivo anti-tumor efficacy in a range of human tumor models including a PAC orthotopic model. T is currently under investigation in multiple oncology clinical trials. A panel of human PAC human tumor xenograft models were established (Hs766t, SU.86.86, BxPc3, MIA PaCa2) and tested with gemcitabine (G) and T both as monotherapies and in combination (G+T) for antitumor efficacy. Select tissue biomarkers, focused on characterizing the magnitude and extent of tumor hypoxia, tumor vascularity, and EMT and G resistance/sensitivity phenotypic markers were assessed by IHC and compared with the corresponding drug efficacy profiles observed in the models. Initial clinical study of G+T combination was a phase 1/2a trial with G administered at its full labeled dose and schedule (1000 mg/m 2 on days 1, 8, and 15 of a 28 day cycle) and T dose escalated starting at 240 mg/m 2 in combination with G with the same schedule (T administered 2 hr before G). An expansion phase 2a portion of the trial explored both 240 and 340 mg/m 2 T in combination with G (n=47 pts). A 21% response rate (RR), median PFS and OS of 5.9 and 8.5 mo, and one-yr survival of >40% was observed. These promising results led to initiation of a randomized controlled Phase 2b trial (G vs. G+T240 vs. G+T340). This trial had 80% power to detect 50% improvement in the primary endpoint of PFS with one-sided alpha of 10%. 214 pts were treated across the three arms (~1:1:1). Primary efficacy endpoint was met with a median PFS of 3.6 mo in G arm vs. 5.6 mo in G+T arms with HR of 0.61 (95%CI: 0.43-0.87; logrank p-value of 0.005). Median PFS in G+T340 was 6.0 mo. RR was 12% in G, 17% in G+T240 and 27% in G+T340. Decreases in circulating PAC biomarker CA19-9 were greater in G+T groups than in the G group and greatest in G+T340. G+T was well-tolerated with skin and mucosal toxicities and myelosuppression the most common TH-302 related AEs with no increase in discontinuations for AE. SAEs were balanced across the three arms. These studies of safety and activity of TH-302 in combination with gemcitabine are consistent with the novel design and hypoxia selective characterization of TH 302. Animal and human studies indicate that TH-302, a tumor hypoxia targeting prodrug, can significantly improve the activity of chemotherapy. Citation Format: Charles P. Hart, Jessica D. Sun, Qian Liu, Dharmendra Ahluwalia, Clarence Eng, David P. Ryan, Mitesh J. Borad, Stew Kroll. Hypoxia-activated prodrug TH-302 in combination with gemcitabine for the treatment of pancreatic adenocarcinoma: Preclinical and clinical studies. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A34.
Hypoxia
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