Hypoxia-activated Prodrug TH-302 Research Articles

Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition.

BackgroundThe 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.MethodsWe 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.ResultsTH-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.ConclusionsTH-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.

Chemotherapy Rescues Hypoxic Tumor Cells and Induces Their Reoxygenation and Repopulation-An Effect That Is Inhibited by the Hypoxia-Activated Prodrug TH-302.

Chemotherapy targets rapidly proliferating tumor cells, but spares slowly proliferating hypoxic cells. We hypothesized that nutrition of hypoxic cells would improve in intervals between chemotherapy, and that hypoxic cells destined to die without treatment would survive and proliferate. We therefore evaluated repopulation and reoxygenation following chemotherapy, and the effects of the hypoxia-activated prodrug TH-302 on these processes. Tumor-bearing mice were treated with doxorubicin or docetaxel ± TH-302. Pimonidazole (given concurrent with chemotherapy) and EF5 (given 24 to 120 hours later) identified hypoxic cells. Proliferation (Ki67) and oxygen status (EF5 uptake) of formerly hypoxic (pimo positive) cells were quantified by immunohistochemistry. Chronically hypoxic cells had limited proliferation in control tumors. After chemotherapy, we observed reoxygenation and increased proliferation of previously hypoxic cells; these processes were inhibited by TH-302. Chemotherapy leads to paradoxical sparing of hypoxic cells destined to die in solid tumors in absence of treatment, and their reoxygenation and proliferation: TH-302 inhibits these processes.

Efficacy and safety of the hypoxia-activated prodrug TH-302 in combination with gemcitabine and nab-paclitaxel in human tumor xenograft models of pancreatic cancer

Tumors often contain hypoxic regions resistant to chemo- and radiotherapy. TH-302 (T) is an investigational hypoxia-activated prodrug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. This study evaluated the efficacy and safety profile of combining T with gemcitabine (G) and nab-paclitaxel (nP) in human pancreatic ductal adenocarcinoma (PDAC) xenograft models in mice. Antitumor activity of the G + nP + T triplet was assessed and compared with T-alone or the G + nP doublet in the Hs766t, MIA PaCa-2, PANC-1, and BxPC-3 PDAC xenograft models. Efficacy was assessed by tumor growth kinetic analysis. Body weight, blood cell counts, blood chemistry, and the von Frey neuropathy assay were analyzed to evaluate safety profiles. Pharmacodynamic changes after the treatment were determined by immunohistochemistry of cell proliferation, DNA damage, apoptosis, hypoxia, and tumor stroma density. The G + nP + T triplet exhibited enhanced efficacy compared with T-alone or the G + nP doublet. Compared with vehicle (V), G + nP induced body weight loss, reduced neutrophil and lymphocyte counts, increased the levels of liver function parameters, and induced neurotoxicity. However, when T was added to G + nP, there was no statistically increased impairment compared to G + nP. The triplet significantly increased DNA damage, apoptosis, and tumor necrosis. Furthermore, the triplet further inhibited cell proliferation and reduced stroma density and intratumoral hypoxia. The triplet combination of G + nP + T exhibited superior efficacy but additive toxicity was not evident compared to the G + nP doublet in this study. This study provides a translational rationale for combining G, nP, and T in the clinical setting to assess efficacy and safety. A Phase I clinical trial of the triplet combination is currently underway (NCT02047500).

Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302.

BackgroundHypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models.ResultsThree human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized 13C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors.ConclusionsUsing metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s40170-014-0026-z) contains supplementary material, which is available to authorized users.

Abstract A51: Chemotherapy rescues hypoxic tumor cells and induces reoxygenation and repopulation - an effect that is inhibited by the hypoxia-activated pro-drug TH-302

Abstract Introduction: Hypoxia is associated with tumor treatment failure. Tumor cells closest to blood vessels are well nourished and rapidly proliferating but hypoxic cells located farther away are slowly-proliferating. Chemotherapy targets rapidly proliferating cells and therefore may spare hypoxic cells because of poor drug distribution to them. Intervals between administrations of chemotherapy allow normal tissues to recover but might also allow re-oxygenation and resumed proliferation of formerly hypoxic cells due to a better supply of nutrients to them. Tumor cell repopulation following radiation therapy is well-documented; but repopulation following chemotherapy and its effect on hypoxia has not been studied. Hypoxia activated pro-drugs (i.e. TH-302) may inhibit tumor repopulation by killing hypoxic cells. Here we evaluate repopulation following chemotherapy and the effect of TH-302 on changes in hypoxia over time in human tumor xenografts. Methods: Human breast (MCF7) tumor bearing mice were administered two specific markers of hypoxia (pimonidazole [pimo] and EF5). Hypoxia-labeled tumor cells were recognized in tumor sections (in relation to DioC7+ve functional tumor blood vessels) using immunohistochemistry. Proliferating cells were identified by an antibody to Ki67. Mice were treated with pimo and: saline, doxorubicin, TH-302 or TH-302 + doxorubicin; EF5 was given after a variable interval of 24, 48, 72, 96 or 120 hours and tumors collected. Changes in the hypoxia, proliferation and oxygen status of formerly hypoxic (pimo+ve) cells were quantified by their Ki67 status and uptake of EF5 as a function of time. Results: Following doxorubicin treatment, the proportion of hypoxic cells in the entire tumor decreased from 1.5% (pimo+ve) prior to injection to 0.7% (EF5+ve) at 24 hours. The percentage of pimo+ve formerly hypoxic cells that are no longer hypoxic (i.e. EF5 – ve) at 24 hours was 90% after doxorubicin compared to 27% in controls, indicating rescue of previous hypoxic cells that would have died in the absence of treatment. Proliferation of these pimo+ve cells that were cycling (Ki67+ve) increased from 7% to 15.0% at 24 hours and then slowly decreased. TH-302 + doxorubicin combination inhibited repopulation by reducing Ki-67 to 1%. Conclusions: Chemotherapy (doxorubicin) treatment paradoxically contributes to tumor treatment failure (tumor cell repopulation) at all time points investigated by causing formerly hypoxic cells to become oxygenated and proliferate above those levels found in the untreated control tumors. TH-302 treatment led to increased DNA damage and inhibited hypoxic cell proliferation and tumor repopulation, thereby increasing chemotherapeutic outcome. Supported by a research grant from the Canadian Institutes for Health Research. Citation Format: Jasdeep K. Saggar, Ian F. Tannock. Chemotherapy rescues hypoxic tumor cells and induces reoxygenation and repopulation - an effect that is inhibited by the hypoxia-activated pro-drug TH-302. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A51. doi:10.1158/1538-7445.CHTME14-A51

Evaluation of the "steal" phenomenon on the efficacy of hypoxia activated prodrug TH-302 in pancreatic cancer.

Pancreatic ductal adenocarcinomas are desmoplastic and hypoxic, both of which are associated with poor prognosis. Hypoxia-activated prodrugs (HAPs) are specifically activated in hypoxic environments to release cytotoxic or cytostatic effectors. TH-302 is a HAP that is currently being evaluated in a Phase III clinical trial in pancreatic cancer. Using animal models, we show that tumor hypoxia can be exacerbated using a vasodilator, hydralazine, improving TH-302 efficacy. Hydralazine reduces tumor blood flow through the “steal” phenomenon, in which atonal immature tumor vasculature fails to dilate in coordination with normal vasculature. We show that MIA PaCa-2 tumors exhibit a “steal” effect in response to hydralazine, resulting in decreased tumor blood flow and subsequent tumor pH reduction. The effect is not observed in SU.86.86 tumors with mature tumor vasculature, as measured by CD31 and smooth muscle actin (SMA) immunohistochemistry staining. Combination therapy of hydralazine and TH-302 resulted in a reduction in MIA PaCa-2 tumor volume growth after 18 days of treatment. These studies support a combination mechanism of action for TH-302 with a vasodilator that transiently increases tumor hypoxia.

Hypoxia-activated pro-drug TH-302 exhibits potent tumor suppressive activity and cooperates with chemotherapy against osteosarcoma

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, tumor hypoxia also offers treatment opportunities, exemplified by the development compounds that target hypoxic regions within tumors. TH-302 is a pro-drug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide (Br-IPM). When TH-302 is delivered to regions of hypoxia, Br-IPM, the DNA cross linking toxin, is released. In this study we assessed the cytotoxic activity of TH-302 against osteosarcoma cells in vitro and evaluated its anticancer efficacy as a single agent, and in combination with doxorubicin, in an orthotopic mouse model of human osteosarcoma (OS). In vitro, TH-302 was potently cytotoxic to osteosarcoma cells selectively under hypoxic conditions, whereas primary normal human osteoblasts were protected. Animals transplanted with OS cells directly into their tibiae and left untreated developed mixed osteolytic/osteosclerotic bone lesions and subsequently developed lung metastases. TH-302 reduced tumor burden in bone and cooperated with doxorubicin to protect bone from osteosarcoma induced bone destruction, while it also reduced lung metastases. TH-302 may therefore be an attractive therapeutic agent with strong activity as a single agent and in combination with chemotherapy against OS.

Pyruvate induces transient tumor hypoxia by enhancing mitochondrial oxygen consumption and potentiates the anti-tumor effect of a hypoxia-activated prodrug TH-302.

BackgroundTH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302.Methodology/ResultsThe hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2), with minimal effect under aerobic conditions (21% O2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500–1500 mm3. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼550 mm3), significantly delayed tumor growth.Conclusions/SignificanceOur in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.

Activity of the hypoxia‐activated pro‐drug TH‐302 in hypoxic and perivascular regions of solid tumors and its potential to enhance therapeutic effects of chemotherapy

Many chemotherapy drugs have poor therapeutic activity in regions distant from tumor blood vessels because of poor tissue penetration and low cytotoxic activity against slowly-proliferating cells. The hypoxia-activated pro-drug TH-302 may have selective toxicity for hypoxic and neighboring cells in tumors. Here we characterize the spatial distribution and ability of TH-302 to selectively target hypoxic regions and complement the effect of doxorubicin and docetaxel by modifying biomarker distribution. Athymic nude mice bearing human breast MCF-7 or prostate PC-3 tumors were treated with doxorubicin or docetaxel respectively and TH-302 alone or in combination. Biomarkers of drug effect including γH2aX (a marker of DNA damage), cleaved caspase-3 or -6 (markers of apoptosis) and reduction in Ki-67 (a marker of cell proliferation) were quantified in tumor sections in relation to functional blood vessels (recognized by DiOC7) and hypoxia (recognized by EF5) using immunohistochemistry. γH2aX expression at 10 min and cleaved caspase-3 or -6 at 24 hr after doxorubicin or docetaxel decreased with increasing distance from tumor blood vessels, with minimal expression in hypoxic regions; maximum reduction in Ki67 levels was observed in regions closest to vasculature at 24 hr. TH-302 induced maximal cell damage in hypoxic and neighboring regions, but was also active in tumor regions closer to blood vessels. TH-302 given 4 hr before doxorubicin or docetaxel increased DNA damage and apoptosis throughout the tumor compared to chemotherapy alone. When given with doxorubicin or docetaxel, TH-302 complements and enhances anticancer effects in both perivascular and hypoxic regions but also increases toxicity.

Synergistic Induction of Apoptosis in Multiple Myeloma Cells by Bortezomib and Hypoxia-Activated Prodrug TH-302, In Vivo and In Vitro

Recently, we showed that hypoxia is a critical microenvironmental factor in multiple myeloma, and that the hypoxia-activated prodrug TH-302 selectively targets hypoxic multiple myeloma cells and improves multiple disease parameters in vivo. To explore approaches for sensitizing multiple myeloma cells to TH-302, we evaluated in this study the antitumor effect of TH-302 in combination with the clinically used proteasome inhibitor bortezomib. First, we show that TH-302 and bortezomib synergistically induce apoptosis in multiple myeloma cell lines in vitro. Second, we confirm that this synergism is related to the activation of caspase cascades and is mediated by changes of Bcl-2 family proteins. The combination treatment induces enhanced cleavage of caspase-3/8/9 and PARP, and therefore triggers apoptosis and enhances the cleavage of proapoptotic BH3-only protein BAD and BID as well as the antiapoptotic protein Mcl-1. In particular, TH-302 can abrogate the accumulation of antiapoptotic Mcl-1 induced by bortezomib, and decreases the expression of the prosurvival proteins Bcl-2 and Bcl-xL. Furthermore, we found that the induction of the proapoptotic BH3-only proteins PUMA (p53-upregulated modulator of apoptosis) and NOXA is associated with this synergism. In response to the genotoxic and endoplasmic reticulum stresses by TH-302 and bortezomib, the expression of PUMA and NOXA were upregulated in p53-dependent and -independent manners. Finally, in the murine 5T33MMvv model, we showed that the combination of TH-302 and bortezomib can improve multiple disease parameters and significantly prolong the survival of diseased mice. In conclusion, our studies provide a rationale for clinical evaluation of the combination of TH-302 and bortezomib in patients with multiple myeloma.

Highlights of This Issue

c-Myc, as the central hub for cellular signals, is deregulated at multiple levels in almost all human cancers. Although this makes c-Myc an attractive candidate target for cancer therapy, drug development against c-Myc has been challenging. Fan-Minogue and colleagues have identified the new antineoplastic function of an antiparasitic agent, nitazoxanide, through their robust cell-based high throughput screening system by utilizing a c-Myc activation sensor. This new system allows large-scale and fast screening for compounds inhibiting c-Myc, thus potentially accelerating drug development against c-Myc–associated cancer.Aberrant activity of receptor tyrosine kinases has been associated with tumor progression in a wide variety of human malignancies. In the present study, Burbridge and colleagues demonstrate that S49076, a novel, potent inhibitor of MET, AXL/MER, and FGFR1/2/3, has specific targeting and strong antitumor effect in vitro and in vivo. S49076 not only showed synergy with bevacizumab in colon cancer xenograft models but also blocked the growth of bevacizumab-resistant tumors. A phase I clinical trial of S49076 is currently underway in patients with advanced solid tumors.Hypoxia is a critical microenvironmental factor in multiple myeloma. It has been shown previously that the hypoxia-activated prodrug TH-302 selectively targets hypoxic multiple myeloma cells. To further explore the application of TH-302 in multiple myeloma, Hu and colleagues evaluated in this study the antitumor effect of TH-302 in combination with the proteasome inhibitor bortezomib, which is already approved for multiple myeloma. The combination of TH-302 and bortezomib was found to synergistically induce apoptosis in human multiple myeloma cell lines in vitro and prolong the survival of 5T33MMvv multiple myeloma mice in vivo. This study provides a rationale for clinical evaluation of this combination in multiple myeloma patients.Patients with glioblastoma have an exceptionally grim prognosis. Although HER1/EGFR is the most frequently amplified oncogene in this disease, monotargeted agents against this receptor have fallen short of improving the clinical course. In order to increase the efficacy of HER1/EGFR inhibition, Karpel-Massler and colleagues screened rationally identified candidate molecular targets and found that combining NSC23766, an inhibitor of RAC1, with erlotinib results in a synergistic antiproliferative effect on primary and established glioblastoma cells. This novel therapeutic approach might be promising in developing a more efficient treatment for glioblastoma patients.

Abstract 3289: Mechanistic studies of the potentiation of hypoxia-targeted drug TH-302 by co-administration of checkpoint kinase1 (Chk1) inhibitors.

Abstract The hypoxia-activated prodrug TH-302 is reduced at its nitroimidazole group and under hypoxic conditions, releases Br-IPM, a bis-alkylating DNA crosslinker. We previously reported that Chk1 inhibitors are TH-302 sensitizers both in vitro and in vivo in the context of p53 checkpoint deficiency. To assess the impact of the Chk1 inhibitor AZD7762 on DNA damage in TH-302 treated cells directly, we used the single-cell electrophoresis-based ‘Comet’ assay. Cells treated with vehicle, TH-302, or AZD7762 exhibited no visible tail moment. In contrast, cells treated with both AZD7762 and TH-302 exhibited a tail moment, indicating the induction of double-strand breaks (DSBs). To determine the effect of AZD7762 on TH-302-induced DNA damage response, γH2AX was evaluated. We demonstrated in p53−/− HT29 cells that either TH-302 or AZD7762 induced γH2AX. However, treatment with both AZD7762 and TH-302 greatly increased γH2AX staining, demonstrating a greater DNA damage response with the combiniaton therapy. Induction of apoptosis was also assessed. TH-302 did not induce apoptosis in HT29, while AZD7762 caused a slight increase in apoptosis. However, treatment with both TH-302 and AZD7762 induced a dramatic increase in apoptosis, indicating that the Chk1 inhibitor AZD7762 sensitizes HT29 cells to apoptosis in response to the DNA damage induced by TH-302. To explore involved signaling pathways, we conducted immunoblot analysis and observed an increase in phospho-histone H3 at serine 10 in the combination treatment group, consistent with DNA damaged cells undergoing mitotic catastrophe. To investigate which DNA repair system is affected by Chk-1 inhibitor-mediated enhancement of TH-302 cytotoxicity, we utilized CHO cell-based DNA repair mutant cell line pairs. Potentiation of TH-302 cytotoxicity by AZD7762 was only observed in cell lines proficient in, and not deficient in, the specific type of DNA repair involved in the repair of TH-302 DNA damage (homology-dependent repair; HDR). To explore the role of HDR in AZD7762-mediated potentiation of TH-302 cytotoxicity further, we investigated the effects of AZD7762 on Rad51 levels. In response to TH-302, Rad51 expression levels were increased. However, combination treatment of cells with AZD7762 and TH-302 led to Rad51 expression levels lower than those observed in control cells. The results suggest that the down-regulation of Rad51-dependent HDR by AZD7762 results in the persistence of unrepaired DNA damage in response to TH-302 and underlies the mechanism of potentiation of TH-302 cytotoxicity by AZD7762. The preclinical data presented in this study support a new approach for the treatment of cancer by combining Chk1 inhibitors with the tumor-hypoxia targeted TH-302. Citation Format: Fanying Meng, Deepthi Bhupathi, Charles P. Hart. Mechanistic studies of the potentiation of hypoxia-targeted drug TH-302 by co-administration of checkpoint kinase1 (Chk1) inhibitors. [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 3289. doi:10.1158/1538-7445.AM2013-3289

Enhancement of the Efficacy of Hypoxia Dependent Cytotoxicity of the Bioreductively Activated Hypoxia-Activated Prodrug TH-302

Enhancement of the Efficacy of Hypoxia Dependent Cytotoxicity of the Bioreductively Activated Hypoxia-Activated Prodrug TH-302

Imaging biomarkers to monitor response to the hypoxia-activated prodrug TH-302 in the MiaPaCa2 flank xenograft model

TH-302, a hypoxia-activated anticancer prodrug, was evaluated for antitumor activity and changes in dynamic contrast-enhanced (DCE) and diffusion-weighted (DW) magnetic resonance imaging (MRI) in a mouse model of pancreatic cancer. TH-302 monotherapy resulted in a significant delay in tumor growth compared to vehicle-treated controls. TH-302 treatment was also associated with a significant decrease in the volume transfer constant (Ktrans) compared to vehicle-treated controls 1 day following the first dose measured using DCE-MRI. This early decrease in Ktrans following the first dose as measured is consistent with selective killing of the hypoxic fraction of cells which are associated with enhanced expression of hypoxia inducible transcription factor-1 alpha that regulates expression of permeability and perfusion factors including vascular endothelial growth factor-A. No changes were observed in DW-MRI following treatment with TH-302, which may indicate that this technique is not sensitive enough to detect changes in small hypoxic fractions of the tumor targeted by TH-302. These results suggest that changes in tumor permeability and/or perfusion may be an early imaging biomarker for response to TH-302 therapy.

Abstract 3720: Combination treatment with hypoxia-activated prodrug TH-302 and the mTOR inhibitor everolimus results in enhanced antitumor efficacy in preclinical models

Abstract TH-302, a 2-nitroimidazole triggered hypoxia-activated prodrug, releases bromo-isophosphoramide mustard (Br-IPM), which induces DNA crosslinking in hypoxic cells. It shows broad antitumor activity in a number of preclinical models, both as a single agent and in combination with conventional chemotherapeutics. The intracellular kinase mTOR plays a key role in multiple pathways which are important in cancer progression. Everolimus directly targets mTOR and results in reduced tumor cell proliferation, decreased tumor angiogenesis, and an inhibiton of tumor cell metabolism. Here we investigated whether TH-302 in combination with everolimus could improve the efficacy profile versus the agents as monotherapies. Two renal cell carcinoma (RCC) ectopic xenograft models and a neuroblastoma ectopic xenograft model were established by subcutaneous implantation of Caki-1, 786-O, or SK-N-BE(2) cells into the flanks of nude mice. When tumor size was approximately 150mm3, animals were treated with everolimus (5mg/kg, QDx19, oral), TH-302 (50mg/kg, QDx5/wk x 3wks, ip), or both everolimus and TH-302. In the Caki-1 model, 87% Tumor Growth Inhibition (TGI) was observed in the combination group versus 52% TGI from everolimus monotherapy or 16% TGI from TH-302 monotherapy. A pharmacodynamic immunohistochemistry study showed that after 7 days of treatment with everolimus, cell proliferation (by Ki67), microvessel density (by CD31) and phosphorylation of the S6 ribosomal protein (by p-S6) was significantly decreased. In the 786-O model, everolimus alone showed 49% TGI, while TH-302 alone did not demonstrate statistically significant efficacy. However, when TH-302 was combined with everolimus, a TGI of 85% was reached. In the ectopic SK-N-BE(2) neuroblastoma model, TH-302 or everolimus alone demonstrated 45% and 40% TGI, respectively, while the combination therapy yielded 65% TGI. Importantly, body weight loss was very minor (<5%) in all groups tested, indicating no significant added toxicity in the combination groups. In summary, there is additive antitumor activity when TH-302 is combined with everolimus. The efficacy profile of TH-302 in combination with the mTOR inhibitor temsirolimus also shows an additive effect in the combination therapy setting. These studies provide a translational rationale for combining TH-302 with mTOR inhibitors to increase the treatment benefit. 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 3720. doi:1538-7445.AM2012-3720

Abstract 1784: Enhancement of hypoxia-activated prodrug TH-302 activity by Chk1 inhibition

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

AACR annual meeting 2012

A phase 2b trial of the hypoxia-activated cytotoxic prodrug TH-302 showed that patients with advanced, first-line pancreatic cancer lived longer without disease progression when the drug was combined with gemcitabine compared with gemcitabine alone. Mitesh Borad (Scottsdale, AZ, USA) and colleagues randomly assigned 214 patients, including 77% with distant metastases, to receive either gemcitabine, TH-302 240 mg/m2 plus gemcitabine, or TH-302 340 mg/m2 plus gemcitabine intravenously. Progression-free survival (PFS) in the combination groups was 5·6 months versus 3·6 months with gemcitabine (hazard ratio 0·61 [95% CI 0·43–0·87], p=0·005). Median PFS in the higher dose TH-302 group was 6·0 months (hazard ratio 0·58 [95% CI 0·39–0·87], p=0·008). Response rate also showed the same positive trend: 12% for gemcitabine, 17% in the low-dose TH-302 group and 27% in the high-dose TH-302 group. Rash and stomatitis, mostly grades 1 and 2, were significantly greater in the combination groups. Myelosuppression was a dose-limiting toxic effect in the combination groups but did not result in an increase in treatment discontinuation.

Molecular and Cellular Pharmacology of the Hypoxia-Activated Prodrug TH-302

TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) of bromo-isophosphoramide mustard currently undergoing clinical evaluation. Here, we describe broad-spectrum activity, hypoxia-selective activation, and mechanism of action of TH-302. The concentration and time dependence of TH-302 activation was examined as a function of oxygen concentration, with reference to the prototypic HAP tirapazamine, and showed superior oxygen inhibition of cytotoxicity and much improved dose potency relative to tirapazamine. Enhanced TH-302 cytotoxicity under hypoxia was observed across 32 human cancer cell lines. One-electron reductive enzyme dependence was confirmed using cells overexpressing human NADPH:cytochrome P450 oxidoreductase and radiolytic reduction established the single-electron stoichiometry of TH-302 fragmentation (activation). Examining downstream effects of TH-302 activity, we observed hypoxia-dependent induction of γH2AX phosphorylation, DNA cross-linking, and cell-cycle arrest. We used Chinese hamster ovary cell-based DNA repair mutant cell lines and established that lines deficient in homology-dependent repair, but not lines deficient in base excision, nucleotide excision, or nonhomologous end-joining repair, exhibited marked sensitivity to TH-302 under hypoxia. Consistent with this finding, enhanced sensitivity to TH-302 was also observed in lines deficient in BRCA1, BRCA2, and FANCA. Finally, we characterized TH-302 activity in the three-dimensional tumor spheroid and multicellular layer models. TH-302 showed much enhanced potency in H460 spheroids compared with H460 monolayer cells under normoxia. Multicellular layers composed of mixtures of parental HCT116 cells and HCT116 cells engineered to express an oxygen-insensitive bacterial nitroreductase showed that TH-302 exhibits a significant bystander effect.

Selective tumor hypoxia targeting by hypoxia-activated prodrug TH-302 inhibits tumor growth in preclinical models of cancer.

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. 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. 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% O(2) exhibited attenuated TH-302 efficacy, with whereas those breathing 10% O(2) exhibited enhanced TH-302 efficacy, both compared with air (21% O(2)) 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." The results show that TH-302 has broad antitumor activity and selectively targets hypoxic tumor tissues.

A Phase I Study of the Safety and Pharmacokinetics of the Hypoxia-Activated Prodrug TH-302 in Combination with Doxorubicin in Patients with Advanced Soft Tissue Sarcoma

Purpose: The purpose of this study was to determine the dose-limiting toxicities (DLT), maximum tolerated dose (MTD), safety, pharmacokinetics and preliminary activity of TH-302, a hypoxia-activated prodrug, in combination with doxorubicin in patients with advanced soft tissue sarcoma. Patients and Methods: TH-302 was administered intravenously on days 1 and 8 and doxorubicin 75 mg/m² on day 1 (2 h after TH-302) of every 3-week cycle. TH-302 starting dose was 240 mg/m² with a classic 3 + 3 dose escalation. Pharmacokinetics were assessed on days 1 and 8 of cycle 1. Tumor assessments were performed after every second cycle. Results: Sixteen patients enrolled. Prophylactic growth factor support was added due to grade 4 neutropenia. The MTD was 300 mg/m². DLTs at 340 mg/m² were neutropenia-associated infection and grade 4 thrombocytopenia. Common adverse events included fatigue, nausea and skin rash. There was no evidence of pharmacokinetic interaction between TH-302 and doxorubicin. Five of 15 (33%) evaluable patients had a partial response by RECIST (Response Evaluation Criteria in Solid Tumors) criteria. Conclusions: The hematologic toxicity of doxorubicin is increased when combined with TH-302. This can be mitigated by prophylactic growth factor support. Toxicities were manageable and there was evidence of antitumor activity.