Hypoxia-activated Prodrug Evofosfamide Research Articles

Influence of the Hypoxia-Activated Prodrug Evofosfamide (TH-302) on Glycolytic Metabolism of Canine Glioma: A Potential Improvement in Cancer Metabolism

The transcription factor hypoxia-inducible factor 1α (HIF-1α) drives metabolic reprogramming in gliomas (GLs) under hypoxic conditions, promoting glycolysis for tumor development. Evofosfamide (EVO) releases a DNA-alkylating agent within hypoxic regions, indicating that it may serve as a hypoxia-targeted therapy. The aim of this study was to investigate the glycolytic metabolism and antitumor effects of EVO in a canine GL model. Our clinical data showed that overall survival was significantly decreased in GL dog patients with higher HIF-1α expression compared to that of those with lower HIF-1α expression, and there was a positive correlation between HIF-1α and pyruvate dehydrogenase kinase 1 (PDK1) expression, suggesting that glycolytic activity under hypoxia conditions may contribute to poor outcomes in canine GL. Our glycolysis assay tests showed that the glycolytic ATP level was higher than the mitochondrial ATP level in three types of canine GL cell lines by activating the HIF-1 signal pathway under hypoxia conditions, resulting in an overall increase in total cellular ATP production. However, treatment with EVO inhibited the glycolytic ATP level in the GL cell lines under hypoxia conditions by targeting HIF-1α-positive cells, leading to decrease in total cellular ATP production. Our in vivo tests showed that EVO significantly reduced tumor development compared to controls and temozolomide in murine GL models. A metabolic analysis demonstrated that EVO effectively suppressed glycolytic metabolism by eliminating HIF-1α-positive cells, suggesting that it may restore metabolism in canine GLs. The evidence presented here supports the favorable preclinical evaluation of EVO as a potential improvement in cancer metabolism.

Evofosfamide and Gemcitabine Act Synergistically in Pancreatic Cancer Xenografts by Dual Action on Tumor Vasculature and Inhibition of Homologous Recombination DNA Repair.

Aims: Pancreatic ductal adenocarcinomas (PDACs) form hypovascular and hypoxic tumors, which are difficult to treat with current chemotherapy regimens. Gemcitabine (GEM) is often used as a first-line treatment for PDACs but has issues with chemoresistance and penetration in the interior of the tumor. Evofosfamide, a hypoxia-activated prodrug, has been shown to be effective in combination with GEM, although the mechanism of each drug on the other has not been established. We used mouse xenografts from two cell lines (MIA Paca-2 and SU.86.86) with different tumor microenvironmental characteristics to probe the action of each drug on the other. Results: GEM treatment enhanced survival times in mice with SU.86.86 leg xenografts (hazard ratio [HR] = 0.35, p = 0.03) but had no effect on MIA Paca-2 mice (HR = 0.91, 95% confidence interval = 0.37-2.25, p = 0.84). Conversely, evofosfamide did not improve survival times in SU.86.86 mice to a statistically significant degree (HR = 0.57, p = 0.22). Electron paramagnetic resonance imaging showed that oxygenation worsened in MIA Paca-2 tumors when treated with GEM, providing a direct mechanism for the activation of the hypoxia-activated prodrug evofosfamide by GEM. Sublethal amounts of either treatment enhanced the toxicity of other treatment in vitro in SU.86.86 but not in MIA Paca-2. By the biomarker γH2AX, combination treatment increased the number of double-stranded DNA lesions in vitro for SU.86.86 but not MIA Paca-2. Innovation and Conclusion: The synergy between GEM and evofosfamide appears to stem from the dual action of GEMs effect on tumor vasculature and inhibition by GEM of the homologous recombination DNA repair process. Antioxid. Redox Signal. 39, 432-444.

Phase IB study of sorafenib and evofosfamide in patients with advanced hepatocellular and renal cell carcinomas (NCCTG N1135, Alliance)

Background Sorafenib (Sor) remains a first-line option for hepatocellular carcinoma (HCC) or refractory renal cell carcinomas (RCC). PLC/PRF/5 HCC model showed upregulation of hypoxia with enhanced efficacy when Sor is combined with hypoxia-activated prodrug evofosfamide (Evo). Methods This phase IB 3 + 3 design investigated 3 Evo dose levels (240, 340, 480mg/m2 on days 8, 15, 22), combined with Sor 200mg orally twice daily (po bid) on days 1-28 of a 28-daycycle. Primary objectives included determining maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of Sor + Evo. Results Eighteen patients were enrolled (median age 62.5years; 17 male /1 female; 12 HCC/6 RCC) across three dose levels (DL0: Sor 200mg bid/Evo 240mg/m2 [n = 6], DL1:Sor 200mg bid/Evo 480mg/m2 [n = 5], DL1a: Sor 200mg bid/Evo 340mg/m2 [n = 7]). Two dose-limiting toxicities (DLTs) were reported with Evo 480mg/m2 (grade 3 mucositis, grade 4 hepatic failure). Grade 3 rash DLT was observed in one patient at Evo 240mg/m2. No DLTs were observed at Evo 340mg/m2. MTD and RP2D were established as Sor 200mg/Evo 340mg/m2 and Sor 200/Evo 240mg/m2, respectively. The most common treatment-related adverse events included fatigue, hand-foot syndrome, hypertension, and nausea/vomiting. Two partial responses were observed, one each at DL0 and DL1a.; disease control rate was 55%. Conclusions RP2D was established as sorafenib 200mg bid + Evo 240mg/m2. While preliminary anti-tumor activity was observed, future development must account for advances in immunotherapy in HCC/RCC.

Hypoxia-Activated Prodrug Evofosfamide Treatment in Pancreatic Ductal Adenocarcinoma Xenografts Alters the Tumor Redox Status to Potentiate Radiotherapy.

Aims: In hypoxic tumor microenvironments, the strongly reducing redox environment reduces evofosfamide (TH-302) to release a cytotoxic bromo-isophosphoramide (Br-IPM) moiety. This drug therefore preferentially attacks hypoxic regions in tumors where other standard anticancer treatments such as chemotherapy and radiation therapy are often ineffective. Various combination therapies with evofosfamide have been proposed and tested in preclinical and clinical settings. However, the treatment effect of evofosfamide monotherapy on tumor hypoxia has not been fully understood, partly due to the lack of quantitative methods to assess tumor pO2in vivo. Here, we use quantitative pO2 imaging by electron paramagnetic resonance (EPR) to evaluate the change in tumor hypoxia in response to evofosfamide treatment using two pancreatic ductal adenocarcinoma xenograft models: MIA Paca-2 tumors responding to evofosfamide and Su.86.86 tumors that do not respond. Results: EPR imaging showed that oxygenation improved globally after evofosfamide treatment in hypoxic MIA Paca-2 tumors, in agreement with the ex vivo results obtained from hypoxia staining by pimonidazole and in apparent contrast to the decrease in Ktrans observed in dynamic contrast-enhanced magnetic resonance imaging (DCE MRI). Innovations: The observation that evofosfamide not only kills the hypoxic region of the tumor but also improves oxygenation in the residual tumor regions provides a rationale for combination therapies using radiation and antiproliferatives post evofosfamide for improved outcomes. Conclusion: This study suggests that reoxygenation after evofosfamide treatment is due to decreased oxygen demand rather than improved perfusion. Following the change in pO2 after treatment may therefore yield a way of monitoring treatment response. Antioxid. Redox Signal. 35, 904-915.

Impact of Tumour Hypoxia on Evofosfamide Sensitivity in Head and Neck Squamous Cell Carcinoma Patient-Derived Xenograft Models.

Tumour hypoxia is a marker of poor prognosis and failure of chemoradiotherapy in head and neck squamous cell carcinoma (HNSCC), providing a strategy for therapeutic intervention in this setting. To evaluate the utility of the hypoxia-activated prodrug evofosfamide (TH-302) in HNSCC, we established ten early passage patient-derived xenograft (PDX) models of HNSCC that were characterised by their histopathology, hypoxia status, gene expression, and sensitivity to evofosfamide. All PDX models closely resembled the histology of the patient tumours they were derived from. Pimonidazole-positive tumour hypoxic fractions ranged from 1.7–7.9% in line with reported HNSCC clinical values, while mRNA expression of the Toustrup hypoxia gene signature showed close correlations between PDX and matched patient tumours, together suggesting the PDX models may accurately model clinical tumour hypoxia. Evofosfamide as a single agent (50 mg/kg IP, qd × 5 for three weeks) demonstrated antitumour efficacy that was variable across the PDX models, ranging from complete regressions in one p16-positive PDX model to lack of significant activity in the three most resistant models. Despite all PDX models showing evidence of tumour hypoxia, and hypoxia being essential for activation of evofosfamide, the antitumour activity of evofosfamide only weakly correlated with tumour hypoxia status determined by pimonidazole immunohistochemistry. Other candidate evofosfamide sensitivity genes—MKI67, POR, and SLFN11—did not strongly influence evofosfamide sensitivity in univariate analyses, although a weak significant relationship with MKI67 was observed, while SLFN11 expression was lost in PDX tumours. Overall, these data confirm that evofosfamide has antitumour activity in clinically-relevant PDX tumour models of HNSCC and support further clinical evaluation of this drug in HNSCC patients. Further research is required to identify those factors that, alongside hypoxia, can influence sensitivity to evofosfamide and could act as predictive biomarkers to support its use in precision medicine therapy of HNSCC.

Hypoxia-Targeting Drug Evofosfamide (TH-302) Enhances Sunitinib Activity in Neuroblastoma Xenograft Models

Antiangiogenic therapy has shown promising results in preclinical and clinical trials. However, tumor cells acquire resistance to this therapy by gaining ability to survive and proliferate under hypoxia induced by antiangiogenic therapy. Combining antiangiogenic therapy with hypoxia-activated prodrugs can overcome this limitation. Here, we have tested the combination of antiangiogenic drug sunitinib in combination with hypoxia-activated prodrug evofosfamide in neuroblastoma. In vitro, neuroblastoma cell line SK-N-BE(2) was 40-folds sensitive to evofosfamide under hypoxia compared to normoxia. In IV metastatic model, evofosfamide significantly increased mice survival compared to the vehicle (P=.02). In SK-N-BE(2) subcutaneous xenograft model, we tested two different treatment regimens using 30 mg/kg sunitinib and 50 mg/kg evofosfamide. Here, sunitinib therapy when started along with evofosfamide treatment showed higher efficacy compared to single agents in subcutaneous SK-N-BE(2) xenograft model, whereas sunitinib when started 7 days after evofosfamide treatment did not have any advantage compared to treatment with either single agent. Immunofluorescence of tumor sections revealed higher number of apoptotic cells and hypoxic areas compared to either single agent when both treatments were started together. Treatment with 80 mg/kg sunitinib with 50 mg/kg evofosfamide was significantly superior to single agents in both xenograft and metastatic models. This study confirms the preclinical efficacy of sunitinib and evofosfamide in murine models of aggressive neuroblastoma. Sunitinib enhances the efficacy of evofosfamide by increasing hypoxic areas, and evofosfamide targets hypoxic tumor cells. Consequently, each drug enhances the activity of the other.

Administration of Hypoxia-Activated Prodrug Evofosfamide after Conventional Adjuvant Therapy Enhances Therapeutic Outcome and Targets Cancer-Initiating Cells in Preclinical Models of Colorectal Cancer.

Purpose: Cancer-initiating cells (C-IC) have been described in multiple cancer types, including colorectal cancer. C-ICs are defined by their capacity to self-renew, thereby driving tumor growth. C-ICs were initially thought to be static entities; however, recent studies have determined these cells to be dynamic and influenced by microenvironmental cues such as hypoxia. If hypoxia drives the formation of C-ICs, then therapeutic targeting of hypoxia could represent a novel means to target C-ICs.Experimental Design: Patient-derived colorectal cancer xenografts were treated with evofosfamide, a hypoxia-activated prodrug (HAP), in combination with 5-fluorouracil (5-FU) or chemoradiotherapy (5-FU and radiation; CRT). Treatment groups included both concurrent and sequential dosing regimens. Effects on the colorectal cancer-initiating cell (CC-IC) fraction were assessed by serial passage in vivo limiting dilution assays. FAZA-PET imaging was utilized as a noninvasive method to assess intratumoral hypoxia.Results: Hypoxia was sufficient to drive the formation of CC-ICs and colorectal cancer cells surviving conventional therapy were more hypoxic and C-IC-like. Using a novel approach to combination therapy, we show that sequential treatment with 5-FU or CRT followed by evofosfamide not only inhibits tumor growth of xenografts compared with 5-FU or CRT alone, but also significantly decreases the CC-IC fraction. Furthermore, noninvasive FAZA-PET hypoxia imaging was predictive of a tumor's response to evofosfamide.Conclusions: Our data demonstrate a novel means to target the CC-IC fraction by adding a HAP sequentially after conventional adjuvant therapy, as well as the use of FAZA-PET as a biomarker for hypoxia to identify tumors that will benefit most from this approach. Clin Cancer Res; 24(9); 2116-27. ©2018 AACR.

MEK inhibition leads to BRCA2 downregulation and sensitization to DNA damaging agents in pancreas and ovarian cancer models.

Targeting the DNA damage response (DDR) in tumors with defective DNA repair is a clinically successful strategy. The RAS/RAF/MEK/ERK signalling pathway is frequently deregulated in human cancers. In this study, we explored the effects of MEK inhibition on the homologous recombination pathway and explored the potential for combination therapy of MEK inhibitors with DDR inhibitors and a hypoxia-activated prodrug.We studied effects of combining pimasertib, a selective allosteric inhibitor of MEK1/2, with olaparib, a small molecule inhibitor of poly (adenosine diphosphate [ADP]-ribose) polymerases (PARP), and with the hypoxia-activated prodrug evofosfamide in ovarian and pancreatic cancer cell lines. Apoptosis was assessed by Caspase 3/7 assay and protein expression was detected by immunoblotting. DNA damage response was monitored with γH2AX and RAD51 immunofluorescence staining. In vivo antitumor activity of pimasertib with evofosfamide were assessed in pancreatic cancer xenografts.We found that BRCA2 protein expression was downregulated following pimasertib treatment under hypoxic conditions. This translated into reduced homologous recombination repair demonstrated by levels of RAD51 foci. MEK inhibition was sufficient to induce formation of γH2AX foci, suggesting that inhibition of this pathway would impair DNA repair. When combined with olaparib or evofosfamide, pimasertib treatment enhanced DNA damage and increased apoptosis. The combination of pimasertib with evofosfamide demonstrated increased anti-tumor activity in BRCA wild-type Mia-PaCa-2 xenograft model, but not in the BRCA mutated BxPC3 model.Our data suggest that targeted MEK inhibition leads to impaired homologous recombination DNA damage repair and increased PARP inhibition sensitivity in BRCA-2 proficient cancers.

A Combination of Radiation and the Hypoxia-Activated Prodrug Evofosfamide (TH-302) is Efficacious against a Human Orthotopic Pancreatic Tumor Model

This study was designed to investigate the effect of single-dose radiation therapy (RT) in combination with evofosfamide (TH-302), a hypoxia-activated prodrug, in a pre-clinical model of pancreatic cancer. AsPC1 tumors were implanted orthotopically in the pancreas of nude mice. Tumors were treated with 15 Gy of RT, using a 1 cm diameter field, and delivered as a continuous arc. Image-guidance to center the field on the tumor was based on CT imaging with intraperitoneal contrast. Evofosfamide (100 mg/kg, i.p.) was administered 3 hours before RT. Tumor volumes were measured using ultrasound, and regrowth curves were plotted. Tumor hypoxia and cell proliferation were measured using pimonidazole and the thymidine analog EdU, respectively. In vitro clonogenic assays were performed. Tumors were shown to contain substantial areas of hypoxia, as calculated by percent pimonidazole staining. Evofosfamide was active in these tumors, as demonstrated by a significant reduction in uptake of the thymidine analog EdU. This effect was visible in oxygenated tissue, consistent with the previously reported bystander effects of evofosfamide. RT produced significant regrowth delay, as did evofosfamide. The combination of both agents produced a growth delay that was at least equal to the sum of the two treatments given separately. The improvement in tumor response when evofosfamide is combined with RT supports the hypothesis that hypoxia is a cause of radioresistance in high dose RT for pancreatic cancer. Assessing the efficacy and safety of stereotactic radiation treatment and evofosfamide is warranted in patients with locally advanced pancreatic cancer.

The hypoxia-activated prodrug evofosfamide in combination with multiple regimens of radiotherapy.

The promising treatment combination of ionizing radiation (IR) with a hypoxia-activated prodrug (HAP) is based on biological cooperation. Here we investigated the hypoxia-activated prodrug evofosfamide in combination with different treatment regimens of IR against lung A549- and head&neck UT-SCC-14-derived tumor xenografts. DNA damage-related endpoints and clonogenic cell survival of A549 and UT-SCC-14 carcinoma cells were probed under normoxia and hypoxia.Evofosfamide (TH-302) induced DNA-damage and a dose-dependent antiproliferative response in A549 cells on cellular pretreatment under hypoxia, and supra-additively reduced clonogenic survival in combination with IR. Concomitant treatment of A549-derived tumor xenografts with evofosfamide and fractionated irradiation induced the strongest treatment response in comparison to the corresponding neoadjuvant and adjuvant regimens. Adjuvant evofosfamide was more potent than concomitant and neoadjuvant evofosfamide when combined with a single high dose of IR. Hypoxic UT-SCC-14 cells and tumor xenografts thereof were resistant to evofosfamide alone and in combination with IR, most probably due to reduced P450 oxidoreductase expression, which might act as major predictive determinant of sensitivity to HAPs.In conclusion, evofosfamide with IR is a potent combined treatment modality against hypoxic tumors. However, the efficacy and the therapeutic outcome of this combined treatment modality is, as indicated here in preclinical tumor models, dependent on scheduling parameters and tumor type, which is most probably related to the status of respective HAP-activating oxidoreductases. Further biomarker development is necessary for the launch of successful clinical trials.

Final Results of a Phase 1/2 Open-Label Study to Assess the Safety, Tolerability and Preliminary Efficacy of Evofosfamide, a Hypoxia-Activated Prodrug, and Dexamethasone with or without Bortezomib in Subjects with Relapsed/Refractory Multiple Myeloma

Final Results of a Phase 1/2 Open-Label Study to Assess the Safety, Tolerability and Preliminary Efficacy of Evofosfamide, a Hypoxia-Activated Prodrug, and Dexamethasone with or without Bortezomib in Subjects with Relapsed/Refractory Multiple Myeloma

Hypoxia Imaging With PET Correlates With Antitumor Activity of the Hypoxia-Activated Prodrug Evofosfamide (TH-302) in Rodent Glioma Models.

High-grade gliomas are often characterized by hypoxia, which is associated with both poor long-term prognosis and therapy resistance. The adverse role hypoxia plays in treatment resistance and disease progression has led to the development of hypoxia imaging methods and hypoxia-targeted treatments. Here, we determined the tumor hypoxia and vascular perfusion characteristics of 2 rat orthotopic glioma models using 18-fluoromisonidozole positron emission tomography. In addition, we determined tumor response to the hypoxia-activated prodrug evofosfamide (TH-302) in these rat glioma models. C6 tumors exhibited more hypoxia and were less perfused than 9L tumors. On the basis of these differences in their tumor hypoxic burden, treatment with evofosfamide resulted in 4- and 2-fold decreases in tumor growth rates of C6 and 9L tumors, respectively. This work shows that imaging methods sensitive to tumor hypoxia and perfusion are able to predict response to hypoxia-targeted agents. This has implications for improved patient selection, particularly in clinical trials, for treatment with hypoxia-activated cytotoxic prodrugs, such as evofosfamide.

A phase II trial to assess the activity and safety of the hypoxia-activated prodrug evofosfamide (TH-302) in combination with sunitinib in patients with disseminated grade 1 and 2 pancreatic neuroendocrine tumors (pNET) as a first-line approach: The GETNE-1408 trial.

TPS479 Background: Pancreatic-NETs are highly vascular tumors. The anti-angiogenetic sunitinib was approved in advanced pNETs based on prolongation of progression-free survival. Upregulation of several proangiogenic factors that reflects intratumor hypoxia conditions might drive resistance to sunitinib in pNETs. Evofosfamide is a prodrug that under hypoxic conditions preferentially releases a brominated version of isophosphoramide mustard and has shown activity in cell lines from neural crest derived tumors such as melanoma and glioblastoma/astrocytoma. We hypothesize that evofosfamide may have activity in the pathologic hypoxic conditions present in the tumor environment of neuroendocrine tumors inducing responses that may be consolidated and prolonged with sunitinib in patients with advanced pNETs that are naïve for systemic treatment. Methods: This is a prospective, non randomized, open-label, phase II study that is being conducted in 10 university sites belonging to the Spanish Task Force Group for NETs (GETNE) in Spain. Patients with histologically proven diagnosis of progressive unresectable or metastatic pNET with Ki67 < 20% and grade 1 or 2 will receive sunitinib orally at 37.5 mg PO daily on days 1 to 28 of a 28-day cycle (4 weeks) plus evofosfamide administered at 340 mg/m2 by IV infusion over 30-60 minutes on Days 8, 15 and 22 of a 28-day cycle (4 weeks). Hypoxia-related tumor markers, single nucleotide polymorphisms related to activity and metabolism of antiangiogenic agents (VEGFR2, VEGFR3, PDGFR-α, VEGF-A, IL8, CYP3A4, CYP3A5, ABCB1-2) will be correlated with clinical outcome. In this study it is planned to include 43 patientsbased on a two-stage Simon’s phase II design (α = 0.05, β = 80%). If the trial shows 8 responses or more among 43 patients, the treatment will be considered for further investigation. EudraCT number: 2014-004072-30 Clinical trial information: NCT02402062.

Comparison of hypoxia-activated prodrug evofosfamide (TH-302) and ifosfamide in preclinical non-small cell lung cancer models

ABSTRACTEvofosfamide (TH-302) is a hypoxia-activated prodrug of the cytotoxin bromo-isophosphoramide. In hypoxic conditions Br-IPM is released and alkylates DNA. Ifosfamide is a chloro-isophosphoramide prodrug activated by hepatic Cytochrome P450 enzymes. Both compounds are used for the treatment of cancer. Ifosfamide has been approved by the FDA while evofosfamide is currently in the late stage of clinical development. The purpose of this study is to compare efficacy and safety profile of evofosfamide and ifosfamide in preclinical non-small cell lung cancer H460 xenograft models. Immunocompetent CD-1 mice and H460 tumor-bearing immunocompromised nude mice were used to investigate the safety profile. The efficacy of evofosfamide or ifosfamide, alone, and in combination with docetaxel or sunitinib was compared in ectopic and intrapleural othortopic H460 xenograft models in animals exposed to ambient air or different oxygen concentration breathing conditions. At an equal body weight loss level, evofosfamide showed greater or comparable efficacy in both ectopic and orthotopic H460 xenograft models. Evofosfamide, but not ifosfamide, exhibited controlled oxygen concentration breathing condition-dependent antitumor activity. However, at an equal body weight loss level, ifosfamide yielded severe hematologic toxicity when compared to evofosfamide, both in monotherapy and in combination with docetaxel. At an equal hematoxicity level, evofosfamide showed superior antitumor activity. These results indicate that evofosfamide shows superior or comparable efficacy and a favorable safety profile when compared to ifosfamide in preclinical human lung carcinoma models. This finding is consistent with multiple clinical trials of evofosfamide as a single agent, or in combination therapy, which demonstrated both anti-tumor activity and safety profile without severe myelosuppression.

Abstract B174: Complementary activity of the vascular disruption agent BNC105 and the hypoxia-activated prodrug evofosfamide (TH-302) in suppressing the growth of preclinical renal and breast solid tumors

Abstract BNC105 is a vascular disruption agent that selectively disrupts tumor blood vessels resulting in hypoxia and necrosis. Evofosfamide (previously known as TH-302, Evo) is a nitroimidazole-triggered prodrug of bromo-isophosphoramide mustard (Br-IPM), an alkylating agent with DNA cross-linking ability. Evo is reduced by intracellular reductases, and under hypoxic conditions selectively releases Br-IPM. Evo has little activity under normoxic conditions, but is highly cytotoxic under hypoxic conditions. Both BNC105 and Evo are currently being evaluated in Phase 1-3 clinical trials. Our studies sought to establish the potential therapeutic combination of BNC105 and Evo. It was reasoned that tumor hypoxia caused by BNC105 will result in increased intratumoral activation of Evo. A greater localized conversion of Evo to Br-IPM would increase the tumor kill achieved. A dose range-finding study was conducted to define the dose level and administration schedule for the BNC105 + Evo combination. The study assessed the safety of administering these two drugs in combination in three different dosing sequences whereby Evo was administered 1hr prior to, immediately following (concurrently) or 1 hr after BNC105. Each of the drugs was administered weekly for 2 weeks. Evo dose levels examined were 50, 75, 100, and 150 mg/kg, and. BNC105 doses investigated were 10 and 16 mg/kg. The combination was well-tolerated at all dose levels evaluated when the drugs were administered concurrently or when Evo was administered 1hr before BNC105. The anti-tumor efficacy of the combination was assessed in the orthotopic syngeneic RENCA renal cancer model and in the MDA-MB-231 breast cancer xenograft model. BNC105 was used at 10 or 16 mg/kg and Evo at 75 mg/kg. RENCA kidney cancer cells were inoculated under the kidney capsule and tumors were allowed to grow for 11 days prior to commencement of dosing (Day 1). BNC105 and Evo were dosed concurrently on Days 1 and 8. Treatment groups included: vehicle control; BNC105; Evo and BNC105 + Evo. The growth of tumors implanted in the kidneys of mice was significantly impeded by the monotherapies and considerably more pronounced when these two drugs were used as a combination treatment. Mice bearing subcutaneous MDA-MB-231 xenografts were treated with BNC105, Evo, or the BNC105 + Evo combination administered concurrently with the same schedule (dosing on Days 1 and 8 of a 21-day cycle). A total of 3 treatment cycles were administered. Based on tumor growth inhibition, there was a clear advantage in treating MDA-MB-231 tumors with the BNC105 + Evo combination compared to either monotherapy. The combination of 16 mg/kg BNC105 + 75 mg/kg Evo substantially inhibited tumor growth as early as the 1st cycle of treatment (Day 1, 8 treatment). This effect continued through the 2nd and 3rd cycles of treatment with the tumor growth curve remaining relatively static. In both models tested the combination of BNC105 with Evo was found to be more efficacious than either therapy alone. Based on these encouraging data the combination is currently being evaluated in additional models including pancreatic cancer and soft tissue sarcoma. Citation Format: Tina C. Lavranos, Daniel Inglis, Donna Beaumont, Annabell F. Leske, Chloe Hawkins, Michaela Scherer, Charles Hart, Jessica Sun, Brian Elenbaas, Gabriel Kremmidiotis. Complementary activity of the vascular disruption agent BNC105 and the hypoxia-activated prodrug evofosfamide (TH-302) in suppressing the growth of preclinical renal and breast solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B174.

Abstract A163: The combined treatment modality of a hypoxia-activated prodrug (Evofosfamide) with ionizing radiation

Abstract Hypoxia is a hallmark of many solid malignancies and confers resistance to radiotherapy as well as other treatment regimens. We investigated the combined treatment modality of ionizing radiation (IR) with the clinical stage hypoxia-activated prodrug (HAP; evofosfamide) in vitro and in vivo using mechanistic and efficacy-oriented endpoints. Based on the current insights, the combined treatment modality of IR with this HAP might lead to complementary tumor cell killing. Ionizing radiation will primarily target well-oxygenated tumor cells, while the HAP will primarily kill hypoxic tumor cells. At the same time enhanced, supra-additive cytotoxicity will be expected by the diffusible activated prodrug leading to more complex DNA damage when used in combination with IR. METHODS We investigated the cytotoxic effects of evofosfamide on tumor cell (A549 lung carcinoma and UT-SCC-14 head&neck squamous cell carcinoma, HNSCC) proliferation and clonogenicity in normoxic (21% O2) and hypoxic (0.2% O2) conditions. For in vivo experiments, cells (4×106) were subcutaneously injected on the back of athymic nude mice. Treatment was initiated when tumors reached volume of 300 mm3 +/- 10%. Irradiation was performed with either a fractionated (3×2Gy) or a single high dose regimen (1×10Gy). Evofosfamide was administered i.p. Q3Dx5 (50 mg/kg in saline), control mice were treated i.p. with saline. RESULTS Lung carcinoma A549 cells were more sensitive to evofosfamide when incubated with the compound under hypoxic conditions in comparison to normoxic conditions in a dose- and time-dependent manner. We observed decreased clonogenicity of A549 cells with evofosfamide/IR co-treatment, which suggests that the evofosfamide has a radiosensitizing effect in vitro. Moreover, evofosfamide alone and in combination with IR induced a strong DNA-damage response (γH2AX foci staining) and senescence (β-galactosidase staining) in A549 cells. We performed in vivo, efficacy- oriented experiments with lung A549 and HNSCC UT-SCC-14 xenografts to test different schedules of evofosfamide in combination with fractionated and single high-dose IR (neoadjuvant, concomitant, adjuvant). Combined treatment resulted in a strongly enhanced tumor growth delay when compared to the single treatment regimens in lung A549 xenografts applying the three different scheduling regimens in combination with fractionated and single high dose IR. Interestingly, the evofosfamide did not reduce tumor growth of HNSCC UT-SCC-14 xenografts suggesting that the response to evofosfamide and IR is highly dependent on the tumor type. CONCLUSIONS Enhanced tumor growth delay of lung carcinoma xenografts in response to a combined treatment of evofosfamide with two different regimens of IR suggests a potent complementary effect with IR of this compound in vivo. Further studies to investigate DNA-damage related endpoints in response to evofosfamide alone and in combination with IR in a genetically defined background (BRCA1/2 deficient cells) are ongoing to further understand the mechanism of a combined treatment. These efficacy- and mechanistic-oriented experiments on the preclinical level are highly relevant to launch clinical phase I/II trials combining radiotherapy with this clinical stage HAP. Citation Format: Katarzyna J. Nytko-Karouzakis, Ivo Grgic, Janosch Ott, Martin Pruschy. The combined treatment modality of a hypoxia-activated prodrug (Evofosfamide) with ionizing radiation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A163.

Metabolism of Hypoxia-Activated Prodrug Evofosfamide in Hypoxic and Normoxic Conditions and Its Potential to Enhance the Therapeutic Efficacy of Single-Dose Radiation Therapy in Pancreatic Cancer

Metabolism of Hypoxia-Activated Prodrug Evofosfamide in Hypoxic and Normoxic Conditions and Its Potential to Enhance the Therapeutic Efficacy of Single-Dose Radiation Therapy in Pancreatic Cancer

2312 Japanese phase I trial of hypoxia-activated prodrug evofosfamide (TH-302) as monotherapy in patients with solid tumors or in combination with gemcitabine in patients with advanced pancreatic cancer

2312 Japanese phase I trial of hypoxia-activated prodrug evofosfamide (TH-302) as monotherapy in patients with solid tumors or in combination with gemcitabine in patients with advanced pancreatic cancer