Abstract TH-302 is a hypoxia-activated prodrug currently in clinical trials for the treatment of cancer. TH-302 releases the DNA cross-linking bromo-isophosphoramide mustard under hypoxic conditions. When DNA damage occurs, a signal transduction cascade is activated in response to the damage and transmits signals to the downstream effectors that connect with the cell cycle machinery. Checkpoint kinase 1 (Chk1) is a vital link between the upstream sensors of the DNA damage checkpoints and the cell cycle effectors. Generally, cell cycle progression is interrupted at the stage where the cell was when injured to give the cell time to repair the damage by activating DNA damage response and repair pathways. Due to its integral role in maintaining genomic integrity, Chk1 has been considered an attractive molecular target for inhibition to treat cancer. In recent years, Chk1 inhibitors have been studied for use in combination with DNA damaging anticancer agents that cause S and G2/M arrest in attempts to increase the efficacy of cancer treatment while sparing normal cells. We have shown that TH-302 induces G2/M arrest at low concentrations and a pan-cell cycle arrest at high concentrations. We hypothesized that the pharmacological inhibition of Chk1 kinase activity could potentiate the efficacy of TH-302. To investigate this possibility, we tested Chk1 inhibitors PF477736, AZD7762 and LY2603618 in combination with TH-302 in HeLa cervical, HT-29 colon, and H460 NSCLC cell lines. Employing an in vitro proliferation assay, we show that TH-302 activity is greatly enhanced (15 to 50 fold) by the addition of the Chk1 inhibitors in the two p53-deficient HeLa and HT29. In contrast, TH-302 activity is not affected by the presence of the Chk1 inhibitors in the p53 wild-type H460. The differential effects on TH-302 activity in combination with the Chk1 inhibitors were confirmed using clonogenic survival assays. These results are consistent with published studies showing p53 status playing a critical role in the activity of Chk1 inhibitors in combination with other DNA damaging agents. We hypothesized that TH-302 induced cell-cycle arrest at the G2/M phase may be mediated by activation of Chk1 and prevention of the activation of downstream Cdc2 kinase activity. We show that Chk1 inhibitors can abrogate TH-302-induced G2 checkpoint arrest in HeLa cells. Since Chk1 affects Cdc2 phosphorylation, we also evaluated the status of Cdc2 phosphorylation in response to the Chk1 inhibitor alone, TH-302 alone, or the combination of Chk1 inhibitor and TH-302. The results demonstrate that TH-302 alone, but not Chk1 inhibitor alone, increases Cdc2-Y15 phosphorylation due to the induced G2/M arrest. In the combination study, the addition of the Chk1 inhibitor blocks the TH-302-induced increase of Cdc-Y15 phosphorylation. Taken together, the results suggest a novel approach for the treatment of cancer combining Chk1 inhibitors with the tumor-hypoxia targeted TH-302. 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 1784. doi:1538-7445.AM2012-1784
Abstract Hypoxia is a defining feature of solid tumors, associated with treatment failure, poor prognosis, cancer relapse and increased metastatic potential. Tumor-specific hypoxia-targeted therapies have been pursued, including hypoxia-activated prodrugs (HAPs). TH-302, a HAP of the cytotoxic warhead bromo-isophosphoramide mustard (Br-IPM), was designed with the goal of high hypoxia selectivity, in vivo efficacy, and an acceptable safety profile. TH-302 has been previously shown to be hypoxia-selective in human tumor cell line in vitro cytotoxicity assays. Here, using a panel of human tumor xenograft models, we demonstrate selective hypoxic-compartment targeting of TH-302 in vivo. Eleven different xenograft models were employed to investigate the antitumor activity of TH-302. The hypoxic fraction (HF) of the tumors was assessed with the hypoxia-specific stain pimonidazole and semi-quantitative morphometrics. Tumor growth inhibition (TGI) was the read-out of antitumor activity. Localization of TH-302-induced DNA damage was by γH2AX immunohistochemistry. TH-302 demonstrated antitumor activity in nine of eleven xenograft models, including NSCLC, SCLC, melanoma, pancreatic, colon, fibrosarcoma and prostate cancer. The two xenografts in which TH-302 showed lower efficacy (SU.86.86 pancreatic and 786-O RCC) were highly vascular, well-perfused and exhibited very small hypoxic fractions. The relative antitumor activity between the xenograft models significantly correlated with the magnitude of the tumor hypoxia. In an efficacy study employing H460 NSCLC xenografts, the tumor-bearing animals were exposed to different oxygen concentrations (10%, 21% or 95%) using controlled atmospheric chambers. Oxygen concentration control began ½ hour before dosing and continued for 2 hr after dosing. A regimen of daily dosing for 2 weeks (Qdx5 x2wk) was employed. TGI was 77% in the 10%, 68% in the 21%, and 56% in the 95% oxygen groups. To assess TH-302 effects on hypoxic compartments, we measured the change of HF in the tumors after a single dose of TH-302. 48 hours after TH-302 treatment, tumor hypoxia in the Hs766t pancreatic cancer xenograft was significantly reduced (11.5% ± 1.4% in vehicle vs. 4.5% ± 0.9% in the TH-302 treatment group, p<0.05). Similar hypoxic volume reductions after TH-302 treatment were also observed in the other models (H82 SCLC, H460 NSCLC and A375 melanoma xenograft models). After TH-302 treatment, DNA damage was assessed by γH2AX and detected first in the pimonidazole-positive hypoxic regions of the tumors and then radiated outward in a time-dependent manner. Taken together, these results demonstrate TH-302 targets the hypoxia compartment selectively in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 535. doi:10.1158/1538-7445.AM2011-535
Abstract TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP). In hypoxic cells the DNA cross-linker bromo-isophosphoramide mustard (Br-IPM) is released. TH-302 demonstrates hypoxia-dependent cytotoxicty in cell-based in vitro assays with human tumor cell lines, efficacy both as monotherapy and combination therapy in human tumor xenografts in nude mice, and is currently in multiple clinical trials for the treatment of cancer, including pancreatic cancer. In the present study, we investigated the antitumor activity and mechanism of action of TH-302 in two pancreatic xenograft models. The Hs766t and SU.86.86 human pancreatic cancer lines reflect two different pancreatic adenocarcinoma cell phenotypes. The Hs766t cells are more mesenchymal, having passed through the epithelial- mesenchymal transition (EMT), while the SU.86.86 cell line retains a more epithelial differentiative state. By employing a histological and immunohistochemical approach, we show that the two cell types form xenograft tumors with distinct microenvironmental features. As characterized by CD31 and Hoechst 33342 staining, SU.86.86 is more highly vascularized and well-perfused than Hs766t. With the exogenous hypoxia marker pimonidazole, a much larger hypoxic volume is present in Hs766t tumors compared with that in SU.86.86 (11.5% ± 1.4% vs. 4.5% ± 0.9%, p<0.001, tumors ~250 mm3). We tested TH-302 and gemcitabine, both as single agents and in combination, in these two pancreatic cancer models with in vitro cell-based models and in vivo tumor xenograft models. In the ectopic xenograft models, TH-302 (75mg/kg, Q3Dx5, ip), gemcitabine (60mg/kg, Q3Dx5, ip) or the combination of TH-302 and gemcitabine was administered to the animals. The TH-302 was given 4 hours before the gemcitabine. The schedule of TH-302 and gemcitabine mirrors the clinical trial schedule where TH-302 and gemcitabine are given on the same day weekly (3 weeks on, 1 week off per cycle). In the Hs766t model, tumor growth inhibition (TGI) of TH-302 alone and gemcitabine alone was 75%, and 45%, respectively. When TH-302 was combined with gemcitabine, the antitumor activity was significantly enhanced, and TGI was 89% (p<0.001 vs. Vehicle and p<0.05 vs. gemcitabine alone). In contrast, gemcitabine alone was highly efficacious in the SU.86.86 xenograft model and showed TGI of 104%, while the antitumor activity of TH-302 monotherapy was modest and TGI was only 28% (the combination TGI was 105%). To characterize specific pharmacodynamic effects of TH-302 treatment on the tumors, we examined Hs766t tumors immunohistochemically 48 hours after a single dose of TH-302 treatment (150mg/kg, ip). The hypoxic volume was significantly reduced from 11.5% (n=9) to 4.6% (n=5) (p<0.01) after TH-302 treatment. The gemcitabine and TH-302 showed no synergy when tested together in vitro. The results support the hypothesis that the two agents (TH-302 and gemcitabine) work in a complementary manner by targeting two distinct tumor compartments and add support for the hypothesis of hypoxic compartment selectivity of TH-302's mechanism of action. Recently published studies have implicated the mesenchymal phenotype of the Hs766t model being associated with gemcitabine resistance. Thus the enhanced anti-tumor efficacy observed with the addition of TH-302 to the gemcitabine regimen in the xenografts may model inter-patient response differences to therapy and lead to biomarker strategies for patient stratification. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A22.
Abstract Several published studies have demonstrated an increase in tumor hypoxia after administration of anti-angiogenesis agents that target VEGF signaling (e.g., sunitinib, sorafenib, bevacizumab). We tested the hypothesis that the hypoxia-activated prodrug (HAP) TH- 302, currently in phase 1/2 trials for the treatment of cancer, would exhibit enhanced efficacy in the context of an antiangiogenic- mediated increase in tumor hypoxia and potentiate the antitumor efficacy of the antiangiogenic. To characterize sunitinib-induced effects on tumor vasculature and tumor hypoxia, 786-O (RCC) and H460 (NSCLC) human ectopic tumor xenografts were treated with sunitinib (20 or 40 mg/kg) daily for 5 days and then 72 hours later animals were injected with pimonidazole to label hypoxic cells and Hoechst 33342 to label vascular perfusion. The NSCLC model (H460) exhibits a baseline hypoxic fraction of 7%. Sunitinib induced a dose-dependent increase in tumor hypoxia volume (24 ± 3.2% with 40 mg/kg vs. 7.3 ± 3.8% with Vehicle, p<0.05) and a corresponding decrease in tumor microvasculature. The 786-O RCC model is a well- vascularized tumor as characterized by CD31 and Hoechst staining with a relatively small baseline hypoxic compartment (<5% volume) in the xenograft tumors. Sunitinib also induced a dose-dependent increase in tumor hypoxia volume (17.2 ± 7.1 % with 40 mg/kg vs. 1.4 ± 0.9% with Vehicle, p<0.05) and a corresponding decrease in functional vasculature in the RCC model. We then tested whether TH-302, a 2-nitroimidazole triggered bromo-isophoramide mustard (Br-IPM) DNA crosslinker, could selectively target the antiangiogenic induced increase in tumor hypoxic fraction. H460 NSCLC tumor- bearing animals (~150 mm3 at start of dosing) were randomized into groups of 10 and treated with sunitinib at 20,40, or 80 mg/kg p.o. daily for 3 weeks (QDx21). After one week of sunitinib monotherapy animals began combination therapy with the addition of TH-302 at 50 mg/kg i.p. daily for 5 days on and 2 days off (QDx5) for 2 weeks. Sunitinib was administered 4 hours before TH-302 on days when both agents were given. Control arms included all 3 doses of sunitinib monotherapy, TH-302 monotherapy, and vehicles-only groups. Sunitinib exhibited dose-dependent antitumor efficacy (by TGI, TGD, and conditional survival), as did TH-302 monotherapy. All three combination therapy treatment groups exhibited superior efficacy compared to the corresponding monotherapy groups. A similarly designed experiment was performed in the RCC (786-O) model. in vitro monolayer cell-based profiling of the two cell lines (H460 and 786-O) with sunitinib and TH-302 in combination, under hypoxic or normoxic conditions, did not show any synergism. Taken together, the combination therapy efficacy observed in the xenograft models are consistent with complementary pharmacological effects at the tumor microenvironmental level, rather than cell autonomous effects. Morphometric analysis of TH-302 effects on hypoxic compartment volume in six different xenograft models (H460 intrapleural and H82 SCLC, H460 and Calu-6 NSCLC, and SU.86.86 and Hs766t CaPanc ectopic models) showed selective targeting of the hypoxic compartment resulting in a decrease in relative hypoxic volumes. Analogous experiments testing TH-302 effects on sunitinib-induced increases in tumor hypoxia volume are underway. The results support the hypothesis that the hypoxia-activated prodrug TH-302 may be an effective addition to antiangiogenic-containing chemotherapy regimens. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A42.
Abstract Topoisomerase I targeted camptothecin analogs are currently employed in a number of different anti-cancer therapeutic regimens. Although efficacious, there continue to be active efforts to develop new camptothecin analogs that avoid deficiencies of the current drugs. The extensive historical SAR efforts on camptothecin and its analogs have largely ignored the 14 position. Only the chloro derivative has been reported, and it is inactive, suggesting a lack of tolerance for substitution at that position. We have developed a highly regioselective process for the synthesis of 14-nitro and 14-aminocamptothecin. 14-aminocamptothecin (TH-1320) demonstrated comparable cytotoxic potency relative to the parent camptothecin, and to clinically relevant camptothecin analogs (topotecan, SN-38) when tested on a panel of human cancer cell lines in vitro (H460, PC3, HT29). Unlike most camptothecin analogs in clinical trials, TH-1320 is not a substrate for any of the major clinically relevant efflux pumps, (MDR1, MRP1, and BCRP). Unlike camptothecin, the potency of TH-1320 is minimally affected by the addition of human serum albumin to cell culture media. TH-1320 showed good bioavailability and brain penetration when dosed orally in mice. TH-1320 is tolerated up to 30 mg/kg with daily oral dosing (MTD). An H460 human NSCLC xenograft model in nude mice was produced by s.c. implantation of 1 ×106 cells into the flank of the animals. When tumor size was approximately 150mm3, animals were randomized into groups of 10 mice each for dosing. 30mg/kg of TH-1320 treatment yielded 79% tumor growth inhibition (TGI) and 15 days tumor growth delay (TGD), while the body weight loss was less than 4%, which suggested a good therapeutic index. Two out of 10 animals showed complete response in the study. However, in same model, Topotecan daily dose at 2mg/kg (MTD) showed only 46% TGI. A xenograft model of metastatic ovarian cancer was set up by the intraperitoneal (i.p.) injection of 3×106 IGROV1 human ovarian cancer cells in nude mice. Mice were evaluated twice a week for morbidity and mortality. Kaplan-Meier survival curves were plotted. Vehicle treated animals had a median survival time (MST) of 39 days. TH-1320 treatment significantly prolonged the survival time. MST of 2/3 MTD dose of TH-1320 (20mg/kg, QDx5/wk x 2wks) was 78 days (p<0.05 vs. vehicle by Logrank test), which was similar to the MTD dose of Topotecan (2mg/kg) or Karenitecin (1.5mg/kg) (MST was 72.5 and 71 days, respectively). Given the favorable properties of TH-1320, further effort is warranted. 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 762.
The hypoxia-activated prodrug TH-302 is reduced at its nitroimidazole group and selectively under hypoxic conditions releases the DNA cross-linker bromo-isophosphoramide mustard (Br-IPM). Here, we have explored the effect of Chk1 inhibition on TH-302-mediated pharmacological activities. We employed in vitro cell viability, DNA damage, cellular signaling assays and the in vivo HT29 human tumor xenograft model to study the effect of Chk1inhibition on TH-302 antitumor activities. TH-302 cytotoxicity is greatly enhanced by Chk1 inhibition in p53-deficient but not in p53-proficient human cancer cell lines. Chk1 inhibitors reduced TH-302-induced cell cycle arrest via blocking TH-302-induced decrease of phosphorylation of histone H3 and increasing Cdc2-Y15 phosphorylation. Employing the single-cell gel electrophoresis (comet) assay, we observed a potentiation of the TH-302 dependent tail moment. TH-302 induced γH2AX and apoptosis were also increased upon the addition of Chk1 inhibitor. Potentiation of TH-302 cytotoxicity by Chk1 inhibitor was only observed in cell lines proficient in, but not deficient in homology-directed DNA repair. We also show that combination treatment led to lowering of Rad51 expression levels as compared to either agent alone. In vivo data demonstrate that Chk1 inhibitor enhances TH-302 anti-tumor activity in p53 mutant HT-29 human tumor xenografts, supporting the hypothesis that these in vitro results can translate to enhanced in vivo efficacy of the combination. TH-302-mediated in vitro and in vivo anti-tumor activities were greatly enhanced by the addition of Chk1 inhibitors. The preclinical data presented in this study support a new approach for the treatment of p53-deficient hypoxic cancers by combining Chk1 inhibitors with the hypoxia-activated prodrug TH-302.
The present study was to evaluate the space provided for the temporary luting cement, after the application of various coats of die spacers, during the fabrication of provisional crowns and bridges.A total of 50 specimens of dental stone with provisional crowns on all these samples were prepared and were divided into five groups based on the application of various coats of different die spacers. Later these specimens were sectioned buccolingually and were observed using a stereomicroscope under 100X magnification. The images thus obtained were evaluated and noted for the amount of space between the inner surface of the provisional crown and the specimens at five different locations using Image Pro 6.0 Express software and the values were subjected to one-way ANOVA test, and unpaired t-test.There was a significant increase of luting space thickness with various die spacer applications than the specimens of control group.Specimens of double coat applications of silver and gold die spacers showed higher luting cement space than the separating media application specimens.
CD73 (ecto-5'-nucleotidase) has emerged as an attractive target for cancer immunotherapy of many cancers. CD73 catalyzes the hydrolysis of adenosine monophosphate (AMP) into highly immunosuppressive adenosine that plays a critical role in tumor progression. Herein, we report our efforts in developing orally bioavailable and highly potent small-molecule CD73 inhibitors from the reported hit molecule 2 to lead molecule 20 and then finally to compound 49. Compound 49 was able to reverse AMP-mediated suppression of CD8+ T cells and completely inhibited CD73 activity in serum samples from various cancer patients. In preclinical in vivo studies, orally administered 49 showed a robust dose-dependent pharmacokinetic/pharmacodynamic (PK/PD) relationship that correlated with efficacy. Compound 49 also demonstrated the expected immune-mediated antitumor mechanism of action and was efficacious upon oral administration not only as a single agent but also in combination with either chemotherapeutics or checkpoint inhibitor in the mouse tumor model.
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