John G. Curd

Selective Tumor Hypoxia Targeting by Hypoxia-Activated Prodrug TH-302 Inhibits Tumor Growth in Preclinical Models of Cancer

Purpose: Tumor hypoxia underlies treatment failure and yields a more aggressive, invasive, and metastatic cancer phenotype. TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug of the cytotoxin bromo-isophosphoramide mustard (Br-IPM). The purpose of this study is to characterize the antitumor activity of TH-302 and investigate its selective targeting of the hypoxic cells in human tumor xenograft models. Experimental Design: Antitumor efficacy was assessed by tumor growth kinetics or by clonogenic survival of isolated cells after tumor excision. Hypoxic fractions (HF) were determined by immunohistochemistry and morphometrics of pimonidazole staining. Tumor hypoxia levels were manipulated by exposing animals to different oxygen concentration breathing conditions. The localization and kinetics of TH-302 induced DNA damage was determined by γH2AX immunohistochemistry. Results: TH-302 antitumor activity was dose-dependent and correlated with total drug exposure. Correlation was found between antitumor activity and tumor HF across 11 xenograft models. Tumor-bearing animals breathing 95% O2 exhibited attenuated TH-302 efficacy, with whereas those breathing 10% O2 exhibited enhanced TH-302 efficacy, both compared with air (21% O2) breathing. TH-302 treatment resulted in a reduction in the volume of the HF 48 hours after dosing and a corresponding increase in the necrotic fraction. TH-302 induced DNA damage as measured by γH2AX was initially only present in the hypoxic regions and then radiated to the entire tumor in a time-dependent manner, consistent with TH-302 having a “bystander effect.” Conclusions: The results show that TH-302 has broad antitumor activity and selectively targets hypoxic tumor tissues. Clin Cancer Res; 18(3); 758–70. ©2011 AACR.

Abstract B185: Bench to bedside experience with TH‐302: A tumor‐selective hypoxia‐activated prodrug as a promising treatment for prostate cancer

Hypoxia is a defining feature of solid tumors, including prostate cancer. Metastases also exhibit subregional hypoxia and micrometastases may be severely hypoxic. Hypoxic cells are known to be resistant to standard chemotherapies and thus play a role in treatment failure and disease relapse. TH‐302, a tumor‐selective hypoxia‐activated prodrug of the cytotoxic warhead bromo‐isophosphoramide mustard (Br‐IPM), is currently in clinical development for the treatment of advanced solid tumors. We explored the preclinical efficacy of TH‐302 in both in vitro and in vivo models of prostate cancer. Based on these translational results, we identified prostate cancer as an indication for further clinical investigations. Preclinically, TH‐302 demonstrated hypoxia‐selective cytotoxicity in the human prostate cancer cell lines PC‐3, LNCaP, and DU‐145. We explored in vivo prostate cancer efficacy in PC‐3 xenograft models that included (1) an ectopic subcutaneous model, (2) an orthotopic model, and (3) a model of bone and soft tissue prostate cancer metastases. TH‐302 demonstrated efficacy both as monotherapy and in combinations with taxanes in all three settings. The combination of TH‐302 and docetaxel in the disseminated prostate cancer metastases model demonstrated 100% complete responses in the 8 of 10 animals surviving to the end of the study at approximately 8 weeks. The TH‐302 Phase 1 dose escalation clinical trial established the monotherapy maximum tolerated dose (MTD) of TH‐302 dosed weekly for 3 weeks of a 4 week cycle at 575 mg/m2. Mucosal and skin toxicity were dose limiting, while hematologic toxicity was minimal. Evidence of anti‐tumor activity was observed including four RECIST responses in patients with small cell lung cancer and metastatic melanoma. In the ongoing Phase 1/2 combination therapy clinical trials of TH‐302, the MTD of TH‐302 was established at 340 mg/m2 when dosed at days 1 and 8 with docetaxel 75 mg/m2 on day 1 every 3 weeks. Neutropenia was dose‐limiting. In the first two patients with metastatic castration‐resistant prostate cancer (CRPC) treated with the combination of TH‐302 and docetaxel, PSA responses (a > 50% reduction from baseline) have been observed along with a marked reduction in cancer‐related pain in one of the patients. An additional 12 CRPC patients will be enrolled. These translational studies and early clinical findings indicate that the addition of TH‐302 to docetaxel may be a promising new treatment option for prostate cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B185.

Abstract 535: Hypoxia-dependent in vivo activity of the hypoxia-activated prodrug (HAP) TH-302

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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 5054: Hypoxia-activated prodrug TH-302 enhances antitumor activity of antiangiogenics in preclinical models

Antiangiogenics have revolutionized cancer therapy. Angiogenesis is a critical step in the establishment, growth, and metastasis of solid tumors. However, relative to other cancer drugs, the dramatic delays in disease progression after antiangiogenic therapy are tempered by the smaller differences in overall survival time. Such differences have led to the hypothesis that antiangiogenic therapy increases tumor hypoxia and leads to the emergence of an aggressive cancer phenotype. TH-302, a 2-nitroimidazole triggered hypoxia-activated prodrug, releases bromo-isophosphoramide mustard (Br-IPM), which induces DNA crosslinking in hypoxic cells. TH-302 has been shown in human tumor xenograft models to target the hypoxic compartment selectively and reduce its volume. A series of studies have been conducted to investigate whether TH-302 and antiangiogenic combination have improved efficacy. Xenograft models were established by s.c. implantation of 4 x106 786-O human renal cell carcinoma (RCC) or 1×106 H460 human non-small cell lung cancer (NSCLC) cells into the flanks of nude mice. Tumor hypoxia was detected by pimonidazole immunostaining, and morphometric analysis was performed to determine the hypoxic fraction. When tumor size was approximately 100-150mm3, sunitinib or sorafenib was administered daily. TH-302 administration began one week after antiangiogenics administration. Sunitinib and sorafenib increased hypoxia in both the RCC and NSCLC xenografts in a dose- and time-dependent manner. In the RCC model, sunitinib activity was potentiated with TH-302: TGI increased from 38% with 40mg/kg sunitinib monotherapy to 75% with 50 mg/kg TH-302. In the NSCLC model, sunitinib (20, 40 or 80mg/kg QDx21) activity was potentiated with TH-302 (50mg/kg, QDx5 x2wk) from 78% TGI (sunitinib monotherapy) to 97% TGI with TH-302 combination therapy. In another study, sunitinib was administered for 33 days, and TH-302 was administered for 4 weeks. With this longer dosing regimen, antitumor activity increased. Medium dose sunitinib (40mg/kg) in combination with TH-302 reached 99% TGI and delayed tumor growth to 500mm3 for 29 days. A complementary effect of TH-302 in combination with sorafenib was also observed (94% TGI for combination therapy versus less than 80% TGI for either monotherapy in the NSCLC model). Importantly, body weight loss, a toxicity indicator, was not significantly increased with TH-302 in combination with any of the antiangiogenics in these studies. In summary, TH-302 potentiates the anti-tumor efficacy of sunitinib and sorafenib by selectively targeting the antiangiogenic-induced increase in tumor hypoxia. These studies provide a translational rationale for combining TH-302 with antiangiogenics to increase the treatment benefit in not only the approved indication (RCC) but also other indications, such as NSCLC. 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 5054. doi:10.1158/1538-7445.AM2011-5054

Abstract A22: Hypoxia-dependent antitumor activity of TH-302, a hypoxia-activated prodrug, in preclinical pancreatic xenograft models

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 Citation Information: Clin Cancer Res 2010;16(14 Suppl):A22.

Abstract A42: Antiangiogenic-induced increase in tumor hypoxia in RCC and NSCLC human tumor xenografts and its selective targeting by the hypoxia-activated prodrug TH-302: A model for clinical exploration?

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 Citation Information: Clin Cancer Res 2010;16(14 Suppl):A42.

Abstract 5388: Sequence- and regimen-dependent combination therapy with the hypoxia-activated Prodrug TH-302 and Doxorubicin in a preclinical sarcoma xenograft model

Tumor hypoxia is associated with resistance to standard chemotherapy, cancer relapse, and poor prognosis. TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP). In hypoxic conditions, bromo-isophosphoramide mustard (Br-IPM) is released and induces crosslinking of DNA. Preclinical anti-tumor activity of TH-302 was observed in a variety of in vitro and in vivo models including sarcoma. TH-302 in combination with Doxorubicin (Dox) is being explored for the treatment of soft tissue sarcoma in a Phase 1/2 clinical trial. To help guide clinical design, we conducted a series of translational studies to optimize preclinical dose sequence and regimen of TH-302 in combination with Dox, focusing on both efficacy and safety profiles. Efficacy was investigated in the ectopic xenograft model after s.c. implantation of 1×106 HT1080, human fibrosarcoma cells into the flank of nude mice and allowing tumors to form with a size of approximately 150mm3. TH-302 was given ip at 50mg/kg (QDx5/wk x 2wks) or 100mg/kg (Q7Dx2), while Dox was administered iv at 4mg/kg (Q7Dx2). These are MTD doses. As a monotherapy, 100mg/kg weekly dose of TH-302 showed better efficacy compared with 50mg/kg daily dose (TGI 74.7% vs. 19.8%). This result can be contrasted to previous findings in the H460 NSCLC model, which showed that lower doses with more frequent dosing yielded greater efficacy. When combined with Dox, different synergistic antitumor effects were observed with different dose sequences. TH-302 given 2-4 hours before Dox was more efficacious than the other time intervals tested, e.g. 8 hours or 24hours (TGD500 of 21 days vs. 14 and 12 days, respectively). Effect of dosing schedule on hemotoxicity was performed in CD-1 mice. TH-302 at 100mg/kg ip once, followed by Dox 6mg/kg iv once, were administered to the animals with different dosing schedules. In the monotherapy groups, a significant decrease of total white blood cells was only observed in the Dox group, while both Dox and TH-302 treatment reduced the number of neutrophils. TH-302 did not add to the hemotoxicity in the combination groups. Compared with simultaneous dosing, TH-302 given 4 hours before Dox attenuated both WBC and neutrophil drops (4.4±0.4 vs. 3.5±0.3 in WBC, and 0.8±0.1 vs. 0.6±0.04 in neutrophils). In summary, consistent with previous studies, combination therapy of TH-302 and conventional chemotherapeutics shows a sequence- and regimen-dependent antitumor activity and toxicity profiles. In this sarcoma model, TH-302 given 2 or 4 hours before Dox yielded better efficacy while simultaneous administration of TH-302 and Dox is not supported because of the higher hemotoxicity. 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 5388.