Bromo-isophosphoramide Mustard Research Articles

Phase 2 trial of hypoxia activated evofosfamide (TH302) for treatment of recurrent bevacizumab-refractory glioblastoma

Evofosfamide (Evo or TH302) is a hypoxia-activated prodrug which is reduced leading to the release of alkylating agent bromo-isophosphoramide mustard, which has shown safety and signals of efficacy in a prior phase 1 study in recurrent glioblastoma. We performed a dual center single-arm Phase II study to expand on the safety and efficacy of Evo plus bevacizumab in bevacizumab refractory glioblastoma. 33 patients with bevacizumab refractory GBM received Evo 670 mg/m2 in combination with Bevacizumab 10 mg/kg IV every 2 weeks. Assessments included adverse events, response, and survival. Median age of patients was 47 (range 19–76) and 24 (69%) were male. At the time of study entry, 9 (26%) had ongoing corticosteroid use. ECOG performance status was 0 or 1 in 83% of patients. Patients were mostly heavily pretreated with 77% have three or more prior regimens. A total of 12 patients (36%) suffered grade 3–4 drug associated adverse event (AE); no grade 5 AE were reported. Of the 33 evaluable patients, best response was PR in 3 (9%), SD in 14 (43%), and PD in 16 (48%) with responses confirmed by a second reviewer. Median time to progression of disease was 53 days (95% CI 42–113) and Median time to death was 129 days (95% CI 86–199 days). Progression free survival at 4 months (PFS-4) on Evo-Bev was 31%, which was a statistically significant improvement over the historical rate of 3%. The median overall survival of patients receiving Evo-Bevacizumab was 4.6 months (95% CI 2.9–6.6). The progression free survival of patients on Evo-Bevacizumab met the primary endpoint of progression free survival at 4 months of 31%, although the clinical significance of this may be limited. Given the patient population and Phase II design, these clinical outcomes will need further validation.

Targeting Hypoxia Using Evofosfamide and Companion Hypoxia Imaging of FMISO-PET in Advanced Biliary Tract Cancer.

PurposeHypoxia is widely known as one of the mechanisms of chemoresistance and as an environmental condition which triggers invasion and metastasis of cancer. Evofosfamide is a hypoxia-activated prodrug of the cytotoxin bromo-isophosphoramide mustard conjugated with 2-nitroimidazole. Biliary tract cancer (BTC) is known to contain large hypoxic area. This study evaluated the efficacy and safety of evofosfamide as a second-line treatment of advanced BTC.Materials and MethodsPatients received evofosfamide at a dose of 340 mg/m2 on days 1, 8, and 15 of every 28-day cycle. Primary end-point was progression-free survival (PFS) rate at 4-months (4m-PFSR). Secondary end-points included overall survival (OS), PFS, disease control rate (DCR), metabolic response by 18F-fluorodeoxyglucose positron emission tomography (PET), hypoxic parameters evaluated by 18F-fluoromisonidazole (FMISO) PET and toxicity.ResultsTwenty patients were treated with evofosfamide, with 16 response-evaluable patients. There was no objective response; stable disease was observed in nine patients, with a DCR of 56.25%. 4m-PFSR was 40.6%. Median PFS was 3.60 months (95% confidence interval [CI], 1.68 to 5.52). Median OS was 6.37 months (95% CI, 3.94 to 8.79). Reduction of tumor metabolic activity was observed in eight of 15 patients (53.3%). High baseline hypoxic parameters were associated with poor PFS. Change of hypoxic parameters between pretreatment and post-treatment reflected hypoxic-activated drug response. There was no treatment-related death.ConclusionEvofosfamide as second-line treatment of advanced BTC showed acceptable safety and comparable efficacy to other agents. Changes in volumetric parameters measured with FMISO PET, showing the degree of tumor hypoxia, reflected the response to evofosfamide based on the mode of action.

1384PD - SUNitinib with EVOfosfamide (TH-302) for G1/G2 metastatic pancreatic neuroendocrine tumours (pNETs) naïve for systemic treatment. The SUNEVO phase II trial of the Spanish task force group for neuroendocrine and endocrine tumours (GETNE)

BackgroundSunitinib (SUN) is approved for the treatment of advanced progressive pNETs. Hypoxia induced by SUN could foster the activation of the prodrug evofosfamide (EVO), designed to release the DNA alkylator bromo-isophosphoramide mustard under hypoxic conditions. The SUNEVO trial seeks to exploit the potential synergy of SUN + EVO in advanced pNETs. MethodsPhase-II, single-arm, multicenter trial that included 17 patients (pts) with metastatic G1/G2 pNETS naive for systemic treatment (tx) other than somatostatin analogues (SSA). Tx consisted on EVO 340mg/m2 on days 8, 15 and 22 every 4 weeks (q4w), and sunitinib 37.5mg/day continuously. Objective response rate (ORR) was selected as the primary endpoint and evaluated q8w by RECIST 1.1. ResultsMedian age was 62.4 years, 41.2% had prior SSA, 47% had a Ki-67 >10%, 70.6% had liver involvement. Patients received a median of 5 (1-21) and 4 (0-21) cycles of SUN and EVO, respectively. After a median follow-up time of 15.7 months (m), 23.5% achieved a partial (N=3) or complete response (N=1), 11 pts had stable disease (64.7%) and 1pt (5.3%) had progressive disease as best response. Median time to response was 1.9m (1.4-8.8) and duration of response was 18.5 m (4.2-38.3). Median PFS was 10.3 m (2.6-18.0). Treatment-related adverse events of >G3 or were observed in 52.9% pts, being the most frequent being neutropenia (18.8%), hypertension (12.5%), ALT increase (12.5%), thrombopenia (6.3%), and fatigue (6.3%). 4 pts (26.7%) discontinued at least one drug due toxicity. ConclusionsSUN + EVO is active in pts with advanced pNETs, but the toxicity profile of the combination negatively affects tolerability. Biomarker analyses to select patients most likely to benefit from this therapy are ongoing. Clinical trial identificationEUDRACT Number: 2014-004072-30 Approval date for the trial: 22-January-2015. Editorial acknowledgementMfar Clinical Research S.L. Legal entity responsible for the studyGrupo Español de Tumores Neuroendocrinos y Endocrinos (GETNE). FundingMolecular Templates Inc. and Pfizer Inc. DisclosureE. Grande: Speaker Bureau / Expert testimony, Public speaking and expert: Pfizer, Ipsen, BMS, Eisai, Roche, MSD, Sanofi-Genzyme, Adacap, Novartis, Eusa Pharma, Pierre Fabre, Lexicon, Celgene; Leadership role, Principal Investigator: AstraZeneca, Pfizer, Ipsen, Mtem/Threshold Lexicon; Honoraria (institution), Medical Education Grant: MSD, Roche; Non-remunerated activity/ies, Advasory Board Member: Enets; Non-remunerated activity/ies, Directory Board Member: Getne; Non-remunerated activity/ies, Directory Board Member: Gethi. C. Lopez: Honoraria (self): Pfizer, Ipsen, Roche; Advisory / Consultancy: Pfizer, Ipsen, Roche; Research grant / Funding (self): Pfizer, Ipsen, Roche; Travel / Accommodation / Expenses: Pfizer, Ipsen, Roche; Honoraria (self): Novartis. T. Alonso Gordoa: Research grant / Funding (self): Roche; Travel / Accommodation / Expenses: Sanofi, Pfizer. J. Capdevila: Advisory / Consultancy: Eisai, Bayer, Exelixis, Novartis, Pfizer, Adacap, Merck Serono; Speaker Bureau / Expert testimony: Eisai, Bayer, Exelis, Ipsen, Novartis, Pfizer, Adacap, Merck Serono; Research grant / Funding (self): Eisai, Exelixis, Adacap, AstraZeneca, Novartis; Travel / Accommodation / Expenses: Ipsen, Pfizer, Eisai. A. Teulé: Advisory / Consultancy: Pfizer, Ipsen, Novartis. I. Sevilla: Advisory / Consultancy: Ipsen, Novartis, Pfeizer, Lilly; Travel / Accommodation / Expenses: Ipsen. P. Gajate: Advisory / Consultancy: IPSSEN; Speaker Bureau / Expert testimony: Ipsen; Travel / Accommodation / Expenses: Ipsen. J. Molina-Cerrillo: Speaker Bureau / Expert testimony: Ipsen, Janssen. J. Hernando Cubero: Speaker Bureau / Expert testimony: Eisai, Ipsen, Roche, Angelini Pharma; Travel / Accommodation / Expenses: Ipsen, Novartis, Advanced Accekeratir Applications, Roche, AstraZeneca, Eisai. R. Garcia-Carbonero: Honoraria (self): Ipsen, Roche, Sanofi, BMS, Servier, Novartis, Pfizer, Merck, PharmaMar, Adacap; Honoraria (self), An intermediate Family Member: Merck, PharmaMar, Roche, BMS, AZ, Lilly, Boerhinger, Gilead Sc, Servier, Sysmex; Advisory / Consultancy: Ipsen, Novartis, Pfizer, AAA; Speaker Bureau / Expert testimony: Ipsen, Pfizer; Research grant / Funding (institution): Pfizer, BMS, MSD, AstraZeneca; Travel / Accommodation / Expenses: Roche, Merck. All other authors have declared no conflicts of interest.

An Intratumor Pharmacokinetic/Pharmacodynamic Model for the Hypoxia-Activated Prodrug Evofosfamide (TH-302): Monotherapy Activity is Not Dependent on a Bystander Effect

Tumor hypoxia contributes to resistance to anticancer therapies. Hypoxia-activated prodrugs (HAPs) selectively target hypoxic cells and their activity can extend to well-oxygenated areas of tumors via diffusion of active metabolites. This type of bystander effect has been suggested to be responsible for the single agent activity of the clinical-stage HAP evofosfamide (TH-302) but direct evidence is lacking. To dissect the contribution of bystander effects to TH-302 activity, we implemented a Green's function pharmacokinetic (PK) model to simulate the spatial distribution of O2, TH-302 and its cytotoxic metabolites, bromo-isophosphoramide mustard (Br-IPM) and its dichloro derivative isophosphoramide mustard (IPM), in two digitized tumor microvascular networks. The model was parameterized from literature and experimentally, including measurement of diffusion coefficients of TH-302 and its metabolites in multicellular layer cultures. The latter studies demonstrate that Br-IPM and IPM cannot diffuse significantly from the cells in which they are generated, although evidence was obtained for diffusion of the hydroxylamine metabolite of TH-302. The spatially resolved PK model was linked to a pharmacodynamic (PD) model that describes cell killing probability at each point in the tumor microregion as a function of Br-IPM and IPM exposure. The resulting PK/PD model accurately predicted previously reported monotherapy activity of TH-302 in H460 tumors, without invoking a bystander effect, demonstrating that the notable single agent activity of TH-302 in tumors can be accounted for by significant bioreductive activation of TH-302 even in oxic regions, driven by the high plasma concentrations achievable with this well-tolerated prodrug.

A Research on the Mechanism of TH-302 on Hypoxic Condition and TH-302 Combined with Radiation Applied in Gastrointestinal Tumors

Tumors often contain hypoxic regions resistant to chemo- and radiotherapy. TH-302 is an investigational hypoxia-activated prodrug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. TH-302 mainly acts on hypoxic condition instead of normal oxygen condition. There have been some phase II clinical trials of TH-302 in soft tissue sarcoma and pancreatic cancer, it shows that TH-302 has good effect on these tumors. Radiation is less sensitive to tumor cells on hypoxic conditon. This study is to investigate the effect of TH-302 combined with radiation in gastrointestinal tumors and the mechanism of TH-302 on hypoxic condition. HCT116 cells, HT29 cells (both are intestinal cancer cells) and MKN28 cells (gastric cancer cells) are chosen for the following experiments. Firstly, 48h and 72h IC50s of TH-302 both on normal oxygen condition (O2%=21%) and hypoxic condition (O2%=1.3%) are obtained through drug cytotoxicity tests. Then tumor cells on hypoxic condition are dealt with TH-302 and radiation, CCK8 of the cells are tested. What’s more, cells are treated with TH302 (24h) on normal oxygen condition (O2%=21%) and hypoxic condition (O2%=1.3%) respectively. Cell apoptosis is examined using Flow Cytometry. At last, expression of HIF-1α protein related to hypoxia mechanism is examined through Western Blot and expression of HIF-1α gene is examined through qPCR. TH-302 is sensitive to tumor cells on hypoxic condition and has lower IC50.The 48h IC50 of TH-302 in HCT116 cells on normal oxygen and hypoxic condition is 2.8*10ˆ8nmol/L and 2,074nmol/L respectively.In HT29 and MKN28 cells the data is 118,400 nmol/L vs 40,790 nmol/L and 29,143 nmol/L vs 1,473 nmol/L.TH-302 combined with radiation on hypoxic condition has more effect than radiation and TH-302 alone. The figure will be shown in the final paper. Tumor cells dealt with TH-302 have more apoptosis on hypoxic condition than normal oxygen condition. In HCT116 +/+, HCT116 -/-, MKN28 cells, the apoptosis ratio is 11.3% vs 0.9%, 17.0% vs 4.3% and 73.8% vs 16.5%. There is not much difference of HIF-1 gene expression between hypoxia and normal oxygen condition. ON hypoxic condition, tumor cells have higher HIF-1 protein expression level. TH-302 is sensitive to tumor cells on hypoxic condition. TH-302 can be used as a radiosensitizer in gastrointestinal tumors. The mechanism of how TH-302 works on hypoxic condition has relationship with apoptosis.

Cellular pharmacology of evofosfamide (TH-302): A critical re-evaluation of its bystander effects

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug with proven efficacy against hypoxic cells in preclinical tumour models. TH-302 is designed to release the DNA crosslinking agent bromo-isophosphoramide mustard (Br-IPM) when reduced in hypoxic tissue. Br-IPM is considered to diffuse locally from hypoxic regions, eliciting additional tumour cell killing, but the latter ‘bystander effect’ has not been demonstrated directly. Previous studies with multicellular co-cultures that included cells expressing the E. coli nitroreductase NfsA as a model TH-302 reductase have provided clear evidence of a bystander effect (which we confirm in the present study). However, NfsA is an oxygen-insensitive two-electron reductase that is not expected to generate the nitro radical intermediate that has been demonstrated to fragment to release Br-IPM. Here, we use mass spectrometry methods to characterise TH-302 metabolites generated by one-electron reduction (steady-state radiolysis by ionising radiation and cellular metabolism under hypoxia, including HCT116 cells that overexpress P450 oxidoreductase, POR) or by NfsA expressed in HCT116 cells under oxic conditions, and investigate the stability and cytotoxicity of these products. Br-IPM is shown to have very low cytotoxic potency when added to extracellular culture medium and to be rapidly converted to other hydrophilic products including dichloro-isophosphoramide mustard (IPM). Only traces of Br-IPM or IPM were detected in the extracellular medium when generated by cellular metabolism of TH-302. We identify, in NfsA-expressing cells, the hydroxylamine metabolite of TH-302, and downstream products resulting from rearrangement or hydration of the imidazole ring, and demonstrate that formation of these candidate bystander effect mediators is suppressed by hypoxia. This characterisation of the cellular pharmacology of TH-302 implies that bystander effects from hypoxic activation of TH-302 are unlikely to contribute to its anticancer activity.

Radiotherapy Synergizes with the Hypoxia-Activated Prodrug Evofosfamide: In Vitro and In Vivo Studies.

Evofosfamide (TH-302) is a hypoxia-activated prodrug (HAP) that releases the DNA-damaging bromo-isophosphoramide mustard (Br-IPM) moiety selectively under hypoxic conditions. Since solid tumors are known to have hypoxic regions, HAPs in combination with chemotherapy or radiotherapy (XRT) will be beneficial. We tested the oxygen dependence of release kinetics of Br-IPM using electron paramagnetic resonance (EPR) with spin trapping by monitoring redox cycling of the nitroimidazole moiety of TH-302, and oxygen dependence of TH-302 on in vitro cytotoxicity at different levels of hypoxia was also examined. Two tumor implants (SCCVII and HT29) in mice were studied. TH-302 fragmentation to release Br-IPM was noticed at oxygen levels <76 mmHg, which increased with higher levels of hypoxia. Enhanced cellular cytotoxicity was also observed at oxygen levels <76 mmHg. In vivo pO2 imaging in the two tumor implants showed that the SCCVII tumor implant had higher level of hypoxia compared with the HT29 xenograft. TH-302 as a monotherapy in vivo showed modest effects in SCCVII implants and minimal effects in HT29 xenografts, whereas TH-302 in combination with ionizing radiation showed significant benefit in both tumor models. We examined the kinetics of redox cycling versus fragmentation of TH-302. The combination of oxygen-dependent XRT with TH-302 is effective even in tumors with significant hypoxia. Imaging studies identifying the magnitude of hypoxia in tumors indicated that the responsiveness to TH-302 and the antitumor effect of TH-302 were enhanced by combining with XRT in both the TH-302-sensitive SCCVII tumor and -resistant HT29 tumor. Antioxid. Redox Signal. 28, 131-140.

Anticancer efficacy of the hypoxia‐activated prodrug evofosfamide is enhanced in combination with proapoptotic receptor agonists against osteosarcoma

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia also leads to treatment opportunities as demonstrated by the development of compounds that target regions of hypoxia within tumors. Evofosfamide is a hypoxia‐activated prodrug that is created by linking the hypoxia‐seeking 2‐nitroimidazole moiety to the cytotoxic bromo‐isophosphoramide mustard (Br‐IPM). When evofosfamide is delivered to hypoxic regions of tumors, the DNA cross‐linking toxin, Br‐IPM, is released leading to cell death. This study assessed the anticancer efficacy of evofosfamide in combination with the Proapoptotic Receptor Agonists (PARAs) dulanermin and drozitumab against human osteosarcoma in vitro and in an intratibial murine model of osteosarcoma. Under hypoxic conditions in vitro, evofosfamide cooperated with dulanermin and drozitumab, resulting in the potentiation of cytotoxicity to osteosarcoma cells. In contrast, under the same conditions, primary human osteoblasts were resistant to treatment. Animals transplanted with osteosarcoma cells directly into their tibiae developed mixed osteosclerotic/osteolytic bone lesions and consequently developed lung metastases 3 weeks post cancer cell transplantation. Tumor burden in the bone was reduced by evofosfamide treatment alone and in combination with drozitumab and prevented osteosarcoma‐induced bone destruction while also reducing the growth of pulmonary metastases. These results suggest that evofosfamide may be an attractive therapeutic agent, with strong anticancer activity alone or in combination with either drozitumab or dulanermin against osteosarcoma.

Abstract 169: Preclinical efficacy and sensitivity determinants of evofosfamide in molecularly defined models of head and neck squamous cell carcinoma

Abstract Tumor hypoxia is prevalent in head and neck squamous cell carcinoma (HNSCC), where it limits radiotherapy outcomes. Hypoxia-activated prodrugs (HAPs) have been developed to target hypoxic regions of tumors. These agents undergo oxygen-sensitive reductive activation, thereby delivering cytotoxic species within hypoxic cells. This study investigated the efficacy and sensitivity determinants of the clinical-stage HAP evofosfamide (TH-302) using molecularly-characterized models of HNSCC. We deployed a collection of 27 HPV-negative HNSCC cell lines derived from lesions of varying TNM stages and primary, nodal or recurrent sites. The collection was characterized for gene expression by RNA-seq, from which somatic variants were also called. Their transcriptomic features were investigated in the context of pan-cancer TCGA data by hierarchical clustering. The potency and hypoxic selectivity of 3 HAPs - evofosfamide, PR-104A and SN30000 - were assessed by antiproliferative assay in 22 lines and compared to bromo-isophosphoramide mustard (Br-IPM), cisplatin and 5-FU. The antitumor activity of evofosfamide (50 mg/kg qdx5 for 2-3 cycles with or without a single 10 Gy dose of radiation on day 5 of cycle 1) was evaluated in HNSCC xenografts in addition to a PDX isolated from an SCC of the glottic larynx. The hypoxic fraction at baseline and after 5 days of treatment was quantified by pimonidazole staining. Genetic modifiers of sensitivity to evofosfamide and its cytotoxic metabolite Br-IPM were explored through whole-genome CRISPR-Cas9 screens using the GeCKO v2 library. High-throughput screens with a custom shRNA pool were performed in one HNSCC and two pancreatic ductal adenocarcinoma cell lines to identify reductases responsible for the activation of evofosfamide in hypoxic cells. Evofosfamide was more potent and more selective for hypoxic HNSCC cells in vitro than PR-104A or SN30000. Cell line sensitivity to evofosfamide was correlated with Br-IPM and cisplatin but not with PR-104A, SN30000 or 5-FU, indicating distinct sensitivity determinants. Evidence of antitumor activity with evofosfamide was observed in vivo. CRISPR screens identified potential evofosfamide sensitivity genes that were reproducibly enriched following drug exposure. Reductase-focused RNA interference screens defined a cluster of sensitivity genes that mapped to mitochondrial electron transport, whereas shRNA’s targeted against presumed activating enzymes such as POR were not enriched. Concentration-dependent oxidation of cytochrome a and decreased respiration was observed in cells exposed to evofosfamide, suggesting reduction by mitochondrial complexes. This study provides a rationale for the clinical evaluation of evofosfamide with radiotherapy in genetically defined subsets of HNSCC patients. Citation Format: Francis W. Hunter, Avik Shome, Dan Li, Way W. Wong, Peter Tsai, Nooriyah Poonawala, Purvi M. Kakadiya, Troy M. Ketelä, Maria K. Kondratyev, Courtney R. Lynch, Tet-Woo Lee, Khanh B. Tran, Jules B. Devaux, Rachel Zussman, Cho R. Hong, Dennis Kee, Andrew M. Macann, Anthony J. Hickey, Stefan K. Bohlander, Cristin G. Print, William R. Wilson, Bradly G. Wouters, Stephen M. Jamieson. Preclinical efficacy and sensitivity determinants of evofosfamide in molecularly defined models of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 169. doi:10.1158/1538-7445.AM2017-169

Doxorubicin plus evofosfamide versus doxorubicin alone in locally advanced, unresectable or metastatic soft-tissue sarcoma (TH CR-406/SARC021): an international, multicentre, open-label, randomised phase 3 trial

Evofosfamide is a hypoxia-activated prodrug of bromo-isophosphoramide mustard. We aimed to assess the benefit of adding evofosfamide to doxorubicin as first-line therapy for advanced soft-tissue sarcomas. We did this international, open-label, randomised, phase 3, multicentre trial (TH CR-406/SARC021) at 81 academic or community investigational sites in 13 countries. Eligible patients were aged 15 years or older with a diagnosis of an advanced unresectable or metastatic soft-tissue sarcoma, of intermediate or high grade, for which no standard curative therapy was available, an Eastern Cooperative Oncology Group performance status of 0-1, and measurable disease by Response Evaluation Criteria in Solid Tumors version 1.1. Patients were randomly assigned (1:1) to receive doxorubicin alone (75 mg/m2 via bolus injection administered over 5-20 min or continuous intravenous infusion for 6-96 h on day 1 of every 21-day cycle for up to six cycles) or doxorubicin (given via the same dose procedure) plus evofosfamide (300 mg/m2 intravenously for 30-60 min on days 1 and 8 of every 21-day cycle for up to six cycles). After six cycles of treatment, patients in the single-drug doxorubicin group were followed up expectantly whereas patients with stable or responsive disease in the combination group were allowed to continue with evofosfamide monotherapy until documented disease progression. A web-based central randomisation with block sizes of two and four was stratified by extent of disease, doxorubicin administration method, and previous systemic therapy. Patients and investigators were not masked to treatment assignment. The primary endpoint was overall survival, analysed in the intention-to-treat population. Safety analyses were done in all patients who received any amount of study drug. This study was registered with ClinicalTrials.gov, number NCT01440088. Between Sept 26, 2011, and Jan 22, 2014, 640 patients were enrolled and randomly assigned to a treatment group (317 to doxorubicin plus evofosfamide and 323 to doxorubicin alone), all of whom were included in the intention-to-treat analysis. The overall survival endpoint was not reached (hazard ratio 1·06, 95% CI 0·88-1·29; p=0·527), with a median overall survival of 18·4 months (95% CI 15·6-22·1) with doxorubicin plus evofosfamide versus 19·0 months (16·2-22·4) with doxorubicin alone. The most common grade 3 or worse adverse events in both groups were haematological, including anaemia (150 [48%] of 313 patients in the doxorubicin plus evofosfamide group vs 65 [21%] of 308 in the doxorubicin group), neutropenia (47 [15%] vs 92 [30%]), febrile neutropenia (57 [18%] vs 34 [11%]), leucopenia (22 [7%] vs 17 [6%]), decreased neutrophil count (31 [10%] vs 41 [13%]), and decreased white blood cell count (39 [13%] vs 33 [11%]). Grade 3-4 thrombocytopenia was more common in the combination group (45 [14%]) than in the doxorubicin alone group (four [1%]), as was grade 3-4 stomatitis (26 [8%] vs seven [2%]). Serious adverse events were reported in 145 (46%) of 313 patients in the combination group and 99 (32%) of 308 in the doxorubicin alone group. Five (2%) patients died from treatment-related causes in the combination group (sepsis [n=2], septic shock [n=1], congestive cardiac failure [n=1], and unknown cause [n=1]) versus one (<1%) patient in the doxorubicin alone group (lactic acidosis [n=1]). The addition of evofosfamide to doxorubicin as first-line therapy did not improve overall survival compared with single-drug doxorubicin in patients with locally advanced, unresectable, or metastatic soft-tissue sarcomas and so this combination cannot be recommended in this setting. Threshold Pharmaceuticals.

Glioblastoma multiforme: Effect of hypoxia and hypoxia inducible factors on therapeutic approaches.

Central nervous system-based cancers have a much higher mortality rate with the 2016 estimates at 6.4 for incidence and 4.3 for deaths per 100,000 individuals. Grade IV astrocytomas, known as glioblastomas are highly aggressive and show a high proliferation index, diffused infiltration, angiogenesis, microvascular proliferation and pleomorphic vessels, resistance to apoptosis, and pseudopalisading necrosis. Extensive hypoxic regions in glioblastomas contribute to the highly malignant phenotype of these tumors. Hypoxic regions of glioblastoma exacerbate the prognosis and clinical outcomes of the patients as hypoxic tumor cells are resistant to chemo- and radiation therapy and are also protected by the malfunctional vasculature that developed due to hypoxia. Predominantly, hypoxia-inducible factor-1α, vascular endothelial growth factor (VEGF)/VEGF receptor, transforming growth factor-β, epidermal growth factor receptor and PI3 kinase/Akt signaling systems are involved in tumor progression and growth. Glioblastomas are predominantly glycolytic and hypoxia-induced factors are useful in the metabolic reprogramming of these tumors. Abnormal vessel formation is crucial in generating pseudopalisading necrosis regions that protect cancer stem cells residing in that region from therapeutic agents and this facilitates the cancer stem cell niche to expand and contribute to cell proliferation and tumor growth. Therapeutic approaches that target hypoxia-induced factors, such as use of the monoclonal antibody against VEGF, bevacizumab, have been useful only in stabilizing the disease but failed to increase overall survival. Hypoxia-activated TH-302, a nitroimidazole prodrug of cytotoxin bromo-isophosphoramide mustard, appears to be more attractive due to its better beneficial effects in glioblastoma patients. A better understanding of the hypoxia-mediated protection of glioblastoma cells is required for developing more effective therapeutics.

Evofosfamide (TH-302) in combination with gemcitabine in previously untreated patients with metastatic or locally advanced unresectable pancreatic ductal adenocarcinoma: Primary analysis of the randomized, double-blind phase III MAESTRO study.

193 Background: Pancreatic ductal adenocarcinoma (PDAC) is invariably diagnosed at an advanced stage and has poor clinical outcome. Hypoxia is a significant prognostic factor in PDAC progression and is associated with poor prognosis. Evofosfamide (Evo, previously known as TH-302) is a hypoxia-activated prodrug of bromo-isophosphoramide mustard (Br-IPM) that is preferentially activated under hypoxic conditions. The addition of Evo to gemcitabine (Gem) significantly improved progression-free survival (PFS) in a randomized phase II trial in advanced PDAC (NCT01144455). Methods: MAESTRO is an international, randomized, double-blind, placebo-controlled phase III trial of Evo/Gem vs Placebo/Gem in patients (pts) with measurable, locally advanced unresectable or metastatic PDAC (NCT01746979). Evo and Gem were administered intravenously at a dose of 340 mg/m2 and 1,000 mg/m2, respectively, on days 1, 8, and 15 of a 28-day cycle. Treatment continued until disease progression. Key eligibility criteria included ECOG PS 0/1 and no neoadjuvant or adjuvant chemotherapy &lt;6 months prior to entry. The primary endpoint was overall survival (OS) with the study designed to detect a HR of 0.75 with 90% power. Secondary endpoints included PFS and objective response rate (ORR), employing a hierarchical testing procedure with a 2-sided α=0.05 at each level. Results: A total of 693 pts were randomized to treatment with Evo/Gem (n=xxx) or Placebo/Gem (n=yyy). Baseline pt characteristics were similar between treatment arms. The OS HR was X.XX (95% CI: Y.YY, Z.ZZ; p=A.AAA). Median OS was AA.A months (m) for Evo/Gem vs BB.B m for Placebo/Gem. Median PFS was C.C m and D.D m, respectively (HR E.EE [95% CI: F.FF, G.GG; p = H.HHH). ORR was JJ.J% with Evo/Gem vs KK.K% with Placebo/Gem (p = L.LLL). Grade ≥3 adverse events (AEs) occurring in &gt;5% of pts in treated with Evo/Gem were: TBC. Conclusions: The data from the MAESTRO trial will make an important contribution to our understanding of PDAC treatment. Clinical trial information: NCT01746979.

Anticancer efficacy of the hypoxia-activated prodrug evofosfamide (TH-302) in osteolytic breast cancer murine models.

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia offers treatment opportunities, exemplified by the development of compounds that target hypoxic regions within tumors. Evofosfamide (TH‐302) is a prodrug created by the conjugation of 2‐nitroimidazole to bromo‐isophosphoramide mustard (Br‐IPM). When evofosfamide is delivered to hypoxic regions, the DNA cross‐linking effector, Br‐IPM, is released. This study assessed the cytotoxic activity of evofosfamide in vitro and its antitumor activity against osteolytic breast cancer either alone or in combination with paclitaxel in vivo. A panel of human breast cancer cell lines were treated with evofosfamide under hypoxia and assessed for cell viability. Osteolytic MDA‐MB‐231‐TXSA cells were transplanted into the mammary fat pad, or into tibiae of mice, allowed to establish and treated with evofosfamide, paclitaxel, or both. Tumor burden was monitored using bioluminescence, and cancer‐induced bone destruction was measured using micro‐CT. In vitro, evofosfamide was selectively cytotoxic under hypoxic conditions. In vivo evofosfamide was tumor suppressive as a single agent and cooperated with paclitaxel to reduce mammary tumor growth. Breast cancer cells transplanted into the tibiae of mice developed osteolytic lesions. In contrast, treatment with evofosfamide or paclitaxel resulted in a significant delay in tumor growth and an overall reduction in tumor burden in bone, whereas combined treatment resulted in a significantly greater reduction in tumor burden in the tibia of mice. Evofosfamide cooperates with paclitaxel and exhibits potent tumor suppressive activity against breast cancer growth in the mammary gland and in bone.

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 2424: Association between Chk1 inhibitor AZD7762-mediated modulation of pharmacodynamic biomarkers and potentiation of hypoxia-activated prodrug TH-302 antitumor efficacy in a human tumor xenograft model

Abstract Background: TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug of the DNA cross-linker bromo-isophosphoramide mustard (Br-IPM). Checkpoint kinase 1 (Chk1) plays a key role in the activation of the DNA damage checkpoint in the cell cycle. We investigated the association between Chk1 inhibitor AZD7762′s-mediated modulation of select downstream pharmacodynamic (PD) biomarkers and potentiation of TH-302 efficacy in the HT29 xenograft model. Material and methods: HT29 ectopic human colorectal xenograft model was employed. Six animals per group were treated with Vehicle (V), TH-302 (T) at 150 mg/kg on Day 1, AZD7762 (A) at 25 mg/kg on Day 1 and 2, or T + A. Two dosing schedules were used in the combination: 1), A was given first, and 4 h and 24 h later, T and A was dosed, respectively (ATA); and 2), T was given first, and 4 h and 24 h later, A was dosed (TAA). The PD biomarkers were evaluated by immunohistochemistry (IHC) on the samples collected on Day 3. In a separate efficacy study, ten animals per group were treated with V, T at 100 mg/kg on Day 1, 4/wk x 2wks, A at 20 mg/kg on Day 1, 2, 4 and 5/wk x 2wks, ATA or TAA combination. Antitumor activity was assessed by tumor growth inhibition (TGI). Results: The biomarker study showed phosphorylated Chk1 (Ser345), γH2AX-positive cells (DNA damage), and Caspase-3 positive cells (apoptosis), were significantly increased in ATA, compared with V, A, T or TAA (p&amp;lt;0.05). ATA-induced DNA damage occurred in 38% cells, which were mainly cancer cells (vs. 3%, 8%, 14% and 24% in V, A, T, and TAA, respectively). 12% of the cells underwent apoptosis after ATA, which was observed mainly in endothelial cells and activated fibroblasts of the tumor stroma. Tumor hypoxia was characterized with pimonidazole IHC and the endogenous marker CA-IX, which showed a strong co-localization of staining. The tumoral hypoxic fraction was reduced after ATA or TAA. In the efficacy study, T or A monotherapy did not show any effect. However, the efficacy was significant in the combination groups, in which ATA yielded TGI of 67%, compared to the TAA of 48%. The dosing schedule dependent antitumor activity was consistent with the PD biomarker observation. Maximal body weight loss in ATA was 1% vs. 6% in TAA. Conclusion: The results suggest that the Chk1 inhibitor A potentiates the efficacy of T in a dose schedule-dependent manner, consistent with dose schedule dependent modulations of PD biomarkers. The detailed mechanistic analysis indicates that the sensitization is due to both disruption of the Chk1 mediated DNA damage checkpoint of the cell cycle and the induction of apoptosis. These studies provide additional support to the preclinical translational rationale for combining TH-302 with Chk1 inhibitors in the clinical trial setting. Citation Format: Jessica D. Sun, Fanying Meng, Qian Liu, Dharmendra Ahluwalia, Wenwu Li, Yan Wang, Deepthi Bhupathi, Charles P. Hart. Association between Chk1 inhibitor AZD7762-mediated modulation of pharmacodynamic biomarkers and potentiation of hypoxia-activated prodrug TH-302 antitumor efficacy in a human tumor xenograft model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2424. doi:10.1158/1538-7445.AM2015-2424

Abstract 5333: TH-302 potentiates the antitumor activity of topotecan in neuroblastoma and rhabdomyosarcoma preclinical models

Abstract Background: There is strong evidence that tumor cells in the hypoxic regions cause drug resistance and tumor relapse. Hypoxia targeting agents may make cytotoxic therapy more effective. In this study, we investigated the efficacy of TH-302, a hypoxia-activated prodrug of bromo-isophosphoramide mustard, both alone and in combination with the cytotoxic agent topotecan, in preclinical models of neuroblastoma (NBL) and rhabdomyosarcoma (RMS). Materials and methods: A panel of four NBL and three RMS cell lines was tested in vitro under normoxic or hypoxic conditions to assess the effect of TH-302 on cell proliferation, both as a single agent and in combination with topotecan. In vivo activity was also evaluated in different xenograft models. TH-302 (50 mg/kg) and topotecan (1 mg/kg) were administered daily for 2 consecutive weeks, as single agents or in combination. Animal survival was studied with an NBL (SK-N-BE(2)) metastatic tumor model. Cleaved caspase-3 and the hypoxia staining agent pimonidazole were used as apoptotic and hypoxic markers in vivo. Results: Under normoxic conditions, all tested lines showed an anti-proliferative response to TH-302 with the IC50s ranging from 4.6 to 52μM. When exposed to hypoxia (1% O2) overnight, a dramatic decrease of IC50 was observed, ranging from 0.07 to 2.4μM. Adding topotecan to the TH-302 treatment significantly increased cytotoxicity in all tested tumor cell lines. In both NBL models (SK-N-BE(2) and CHLA-20), single-agent TH-302 treatment delayed tumor growth. Complete tumor regression was observed in the combined treatment group after a two-week treatment period, but all the regressed tumors relapsed after treatment ended. In the CHLA-20 xenograft model, single-agent topotecan also induced complete tumor regression. However, median time to relapse in the combination group was significantly longer than in the topotecan group (43 days vs. 16 days; p&amp;lt;0.01). In the combination group, all relapsed tumors remained responsive to the drug treatment when re-challenged with the original treatment regimen. In the neuroblastoma metastatic model, all three regimens prolonged animal survival, but the combined treatment group showed better survival than single-agent treatments (p&amp;lt;0.01). In a RMS xenograft model, combined treatment was also more effective than single-agent TH-302 or topotecan. Double immunostaining of apoptotic and hypoxic markers showed that TH-302 mostly targeted tumor cells residing at hypoxic regions while topotecan was more effective on tumor cells outside the hypoxic zones. The combined treatment induced tumor cell apoptosis in both normoxic and hypoxic regions. Conclusions: TH-302 showed antitumor effects in neuroblastoma and rhabdomyosarcoma xenograft models. Compared to single-agent TH-302 or topotecan, the combination treatment significantly improved tumor response, delayed tumor relapse, and enhanced animal survival. Citation Format: Libo Zhang, Bing Wu, Paula Marrano, Paul Thorner, Sylvain Baruchel. TH-302 potentiates the antitumor activity of topotecan in neuroblastoma and rhabdomyosarcoma preclinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5333. doi:10.1158/1538-7445.AM2015-5333

Abstract B88: Pharmacodynamic changes from the TH-302, gemcitabine, and nab-paclitaxel triplet combination in a xenograft model of pancreatic cancer

Abstract Background: Tumors often consist of hypoxic regions which are resistant to chemo- and radiotherapy. TH-302 (T) is an investigational prodrug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. We previously reported that the triplet combination of gemcitabine (G), nab-paclitaxel (nP) and T exhibited superior efficacy, without evidence of additive toxicity, compared to the G and nP doublet combination in preclinical pancreatic ductal adenocarcinoma (PDAC) models. These studies provided a translational rationale for combining G, nP, and T in the clinical setting to assess efficacy and safety, and a Phase 1 trial of G + nP + T in pancreatic cancer is currently open (NCT02047500). We used the PANC-1 PDAC xenograft model to investigate the pharmacodynamic (PD) changes resulting from combining T with G and nP and to explore the mechanism of action and biomarkers. Methods: The PANC-1 ectopic xenograft model was established by s.c. implantation of PANC-1 cells into the flank of nude mice. When the tumor size was 500 mm3, 5 animals in each group were treated with vehicle (V), T alone at 50 mg/kg, ip, G at 60 mg/kg ip + nP at 30 mg/kg, iv, or the triplet combination of G + nP + T, all with a Q3Dx5 regimen. Tumor samples were collected 24 h after the last treatment and were evaluated for hypoxia by immunostaining for pimonidazole (Pimo), and endogenous carbonic anhydrase IX (CA-IX). Tumor cell proliferation was assessed by Ki67, DNA damage by gamma histone 2AX (γH2AX) immunostaining, apoptosis by an in situ TUNEL assay, and tumor stromal density by alpha-smooth muscle actin (α-SMA) immunostaining. In a separate study, PANC-1-bearing animals (10 per group), treated with the same doses and regimens, were used to investigate the anti-tumor activity by quantifying tumor growth kinetics. Results: Two days after the last treatment, tumor size change% in V, T, G + nP and G + nP + T groups was 238%, 130%, -34%, and -51% respectively. The tumor size reduction achieved with the triplet treatment was significant compared with V, T or G + nP (p&amp;lt;0.05). The necrotic region in G + nP + T as measured by H&amp;E staining was increased compared with all other groups. In the non-necrotic region the number of γH2AX-positive cells, as measured by immunostaining, was 31.3/field in G + nP + T, and was significantly increased compared with all other groups (3.6, 19.7 and 19.2/field in the V, T, and G + nP groups, p&amp;lt;0.05). Apoptosis was found in both stromal and tumor cells, and the number of apoptotic cells was significantly increased by the triple combination treatment compared with V, T or G + nP doublet (p&amp;lt;0.05). Ki67-positive cells and thus cell proliferation were significantly reduced after all drug treatments, and to a greater magnitude in the G + nP + T triplet group (p&amp;lt;0.05, compared with all other groups). T alone did not reduce the hypoxic fraction (HF) as measured by morphometrics of the Pimo staining to a significant degree (10.6 ± 1.1% vs. 12.4 ± 2% in V); however, by adding T to the G + nP doublet, the triplet significantly reduced the hypoxic level, with an HF of 2.5 ± 0.5%, compared with 7.1 ± 1.7% in the G + nP group (p&amp;lt;0.05). There was no co-localization between Pimo-positive and CA-IX-positive cells, and the α-SMA staining, and G + nP doublet significantly reduced α-SMA expression, compared with V (p&amp;lt;0.05), but no further reduction was observed in the G + nP + T group. Conclusions: In xenograft model of pancreatic cancer, the triplet combination of G + nP + T exhibits superior efficacy to T alone or G + nP doublet, and is associated with an increased inhibition of tumor cell proliferation, a reduction of the hypoxic fraction, and an enhancement of DNA damage, apoptosis, and necrosis. Citation Format: Jessica D. Sun, Qian Liu, Dharmendra Ahluwalia, Wenwu Li, Yan Wang, Charles P. Hart. Pharmacodynamic changes from the TH-302, gemcitabine, and nab-paclitaxel triplet combination in a xenograft model of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B88.

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.

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).

A phase I dose-escalation trial of TH-302 with nab-paclitaxel and gemcitabine in patients with locally advanced or metastatic pancreatic adenocarcinoma.

TPS505 Background: TH-302 is a hypoxia-activated, nitroimidazole-linked prodrug of bromo-isophosphoramide mustard (Br-IPM), which is released under hypoxic conditions. TH-302 is currently being studied in combination with gemcitabine (G) in a phase III trial in patients (pts) with advanced pancreatic adenocarcinoma (PDAC) (NCT01746979). Encouraging preclinical activity of TH-302 plus nab-paclitaxel (nab-P) and G provides a rationale for further study of this triplet (Sun JD et al, AACR Special Conference on Pancreatic Cancer 2014; abstract #B88). Methods: Clinical trial: NCT02047500. Open-label, phase I, dose-escalation trial of TH-302 plus nab-P and G in pts with previously untreated locally advanced or metastatic PDAC. Dose escalation: 3 + 3 design with one of 3 planned dose levels of TH-302 (170, 240 and 340 mg/m2) on days 1, 8, and 15 of a 28-day cycle, plus nab-P 100–125 mg/m2 followed by G 800–1,000 mg/m2. Expansion cohort (n≥15): TH-302 at the recommended phase II dose (RP2D) with nab-P 100–125 mg/m2 followed by G 800–1,000 mg/m2. Treatment will be continued until disease progression or intolerable toxicity. Primary objectives: To evaluate safety and tolerability, define the maximum tolerated dose and determine the RP2D of TH-302 in combination with nab-P/G. The primary endpoint is no. of pts with dose-limiting toxicities during the first 28 days. Secondary objectives: To evaluate overall safety of the triplet regimen, metabolic response rate using EORTC criteria for 18F-FDG PET, CA19–9 response rate (defined as &gt;50% decline from baseline), and RECIST response rate of TH-302 plus nab-P/G. The pharmacokinetic (PK) profile of TH-302 and Br-IPM in the plasma of pts will also be evaluated, as will the effect of TH-302 on the PK of nab-P, and vice versa. Exploratory analyses include the association of pharmacogenomic markers, such as CYP2C9, BRCA1 and BRCA2, with PK, safety and efficacy. The association of potential hypoxia biomarkers (CA IX, GLUT1, VEGF, osteopontin) with antitumor activity will also be explored. The trial is in progress; 7 of an estimated 48 planned pts have been recruited (trial start Jan 2014, estimate end Aug 2016). Clinical trial information: NCT02047500.