TH-302, a hypoxia-activated prodrug with broad in vivo preclinical combination therapy efficacy: optimization of dosing regimens and schedules.

PURPOSE: Subregional hypoxia is a common feature of tumors and is recognized as a limiting factor for the success of radiotherapy and chemotherapy. TH-302, a hypoxia-activated prodrug selectively targeting hypoxic regions of solid tumors, delivers a cytotoxic warhead to the tumor, while maintaining relatively low systemic toxicity. The antitumor activity, different dosing sequences, and dosing regimens of TH-302 in combination with commonly used conventional chemotherapeutics were investigated in human tumor xenograft models. METHODS: Seven chemotherapeutic drugs (docetaxel, cisplatin, pemetrexed, irinotecan, doxorubicin, gemcitabine, and temozolomide) were tested in combination with TH-302 in eleven human xenograft models, including non-small cell lung cancer (NSCLC), colon cancer, prostate cancer, fibrosarcoma, melanoma, and pancreatic cancer. RESULTS: The antitumor activity of docetaxel, cisplatin, pemetrexed, irinotecan, doxorubicin, gemcitabine, and temozolomide was increased when combined with TH-302 in nine out of eleven models tested. Administration of TH-302 2-8 h prior to the other chemotherapeutics yielded superior efficacy versus other sequences tested. Simultaneous administration of TH-302 and chemotherapeutics increased toxicity versus schedules with dosing separations. In a dosing optimization study, TH-302 administered daily at 50 mg/kg intraperitoneally for 5 days per week in the H460 NSCLC model showed the optimal response with minimal toxicity. CONCLUSIONS: TH-302 enhances the activity of a wide range of conventional anti-neoplastic agents in a broad panel of in vivo xenograft models. These data highlight in vivo effects of schedule and order of drug administration in regimen efficacy and toxicity and have relevance to the design of human regimens incorporating TH-302.

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

PURPOSE: Tumor hypoxia underlies treatment failure and yields a more aggressive, invasive, and metastatic cancer phenotype. TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug of the cytotoxin bromo-isophosphoramide mustard (Br-IPM). The purpose of this study is to characterize the antitumor activity of TH-302 and investigate its selective targeting of the hypoxic cells in human tumor xenograft models. EXPERIMENTAL DESIGN: Antitumor efficacy was assessed by tumor growth kinetics or by clonogenic survival of isolated cells after tumor excision. Hypoxic fractions (HF) were determined by immunohistochemistry and morphometrics of pimonidazole staining. Tumor hypoxia levels were manipulated by exposing animals to different oxygen concentration breathing conditions. The localization and kinetics of TH-302 induced DNA damage was determined by γH2AX immunohistochemistry. RESULTS: TH-302 antitumor activity was dose-dependent and correlated with total drug exposure. Correlation was found between antitumor activity and tumor HF across 11 xenograft models. Tumor-bearing animals breathing 95% 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." CONCLUSIONS: The results show that TH-302 has broad antitumor activity and selectively targets hypoxic tumor tissues.

Potent and highly selective hypoxia-activated achiral phosphoramidate mustards as anticancer drugs.

A series of achiral hypoxia-activated prodrugs were synthesized on the basis of the DNA cross-linking toxin of the prodrug, ifosfamide. The hypoxia-selective cytotoxicity of several of the compounds was improved over previously reported racemic mixtures of chiral bioreductive phosphoramidate prodrugs. Prodrugs activated by 2-nitroimidazole reduction demonstrated up to 400-fold enhanced cytotoxicity toward H460 cells in culture under hypoxia versus their potency under aerobic conditions. Compounds were further assessed for their stability to cytochrome P450 metabolism using a liver microsome assay. The 2-nitroimidazole containing lead compound 3b (TH-302) was selectively potent under hypoxia and stable to liver microsomes. It was active in an in vivo MIA PaCa-2 pancreatic cancer orthotopic xenograft model as a monotherapy and demonstrated dramatic efficacy when used in combination with gemcitabine, extending survival with one of eight animals tumor free at day-44. Compound 3b has emerged as a promising antitumor agent that shows excellent in vivo efficacy and is currently being evaluated in the clinic.

A novel alkylating agent, glufosfamide, enhances the activity of gemcitabine in vitro and in vivo.

Glufosfamide is an alkylating agent consisting of iphosphoramide mustard conjugated to glucose that is currently included in clinical studies of pancreatic cancer. We studied the effects of glufosfamide, in combination with gemcitabine, on in vitro and in vivo models of pancreatic cancer. In proliferation assays, glufosfamide and gemcitabine inhibited the growth of MiaPaCa-2, H766t, and PANC-1 cells, but the combination of the two agents provided greater effects. Apoptosis of MiaPaCa-2 cells, measured by fluorescence-activated cell sorting, was enhanced by the combination of the two drugs, compared to single-agent treatment. Glufosfamide alone inhibited the growth of red fluorescent protein-expressing MiaPaCa-2 tumors in an orthotopic nude mouse model in a dose-dependent manner. Combining glufosfamide (30 mg/kg) with gemcitabine resulted in enhanced inhibition of tumor growth and significantly prolonged survival. Immunohistochemistry of excised tumors revealed that both glufosfamide and gemcitabine increased levels of apoptosis (measured by terminal deoxynucleotidyl transferase-mediated nick end labeling staining) and reduced proliferation (measured by proliferating cell nuclear antigen staining). No effects on microvessel density were observed. These results support the use of the alkylating agent glufosfamide and the DNA synthesis inhibitor gemcitabine, rather than the use of either agent alone, to provide greater benefits and demonstrate that this combination treatment should be useful in the clinical treatment of pancreatic carcinoma.

rBPI(10-193) is secreted by CHO cells and retains the activity of rBPI21.

rBPI23, a recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI), kills Gram-negative bacteria and neutralizes endotoxin. rBPI21, a variant in which cysteine 132 is changed to alanine, retains the activities of rBPI23. Analysis of certain purified rBPI21 preparations revealed that some of the molecules had lost nine amino acids from the amino terminus. To explore the effect of this modification on structure and activity, we cloned and expressed a variant of rBPI21, designated rBPI(10-193), which lacks the first nine amino acids. A monoclonal antibody believed to recognize the amino terminus of rBPI21 cross-reacted with rBPI21, but not with rBPI(10-193) or full length recombinant BPI (rBPI). These results demonstrated that the antibody recognizes the first nine amino acids of rBPI21 and that this region of the holoprotein (rBPI) is inaccessible to the antibody (as suggested by the known 3-D structure). Purified rBPI(10-193) and rBPI21 were similarly potent in in vitro assays measuring bactericidal, endotoxin binding and neutralization activities. In a mouse model of lethal bacteremia, rBPI(10-193) and rBPI21 were similarly potent whereas in a mouse endotoxin challenge model, rBPI(10-193) appeared to be at least 2-fold more potent than rBPI21. In conscious rats, a rapid bolus dose of 40 mg/kg of rBPI21 caused a significant transient decrease in blood pressure while the same dose of rBPI(10-193) caused no blood pressure decrease. We conclude from these studies that the first nine amino acids of rBPI21 are not essential for the anti-infective and endotoxin-neutralizing activities of BPI.

In vitro and in vivo pharmacology and pharmacokinetics of a human engineered monoclonal antibody to epithelial cell adhesion molecule.

ING-1(heMAb), a Human Engineered monoclonal antibody to epithelial cell adhesion molecule (Ep-CAM), was evaluated for its in vitro and in vivo activity. The dissociation constant of ING-1(heMAb) for binding to Ep-CAM on HT-29 human colon tumor cells was 2 to 5 nM, similar to chimeric ING-1. In antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays, ING-1(heMAb) caused a concentration-dependent lysis of BT-20 breast, MCF-7 breast, HT-29 colon, and CACO-2 colon tumor cells, with maximum cytolysis at approximately 1 microg/ml. After an intravenous injection in rats, plasma ING-1(heMAb) levels declined with an alpha half-life of 8 to 11 hours, and a beta half-life of 20 days, typical of an IgG in a species without the target for ING-1. In nude mice with human HT-29 colon tumors, plasma ING-1(heMAb) levels declined more rapidly than in non-tumor-bearing mice, suggesting an enhanced clearance via the tumor-associated human Ep-CAM. In nude mice, intravenous treatments with ING-1(heMAb) twice a week for 3 weeks significantly suppressed the growth of human HT-29 colon and PC-3 prostate tumors in a dose-dependent manner, with 1.0 mg/kg providing the greatest benefit. These results indicate that Human Engineered ING-1(heMAb) is a high-affinity antibody with potent in vitro activity that targets and suppresses the growth of human tumors in vivo.

ING-1(heMAb), a monoclonal antibody to epithelial cell adhesion molecule, inhibits tumor metastases in a murine cancer model.

ING-1(heMAb), a human-engineered monoclonal antibody (MAb) that specifically targets the epithelial cell adhesion molecule (Ep-CAM), kills adenocarcinoma cells in vitro and inhibits tumor growth in vivo. In the current study, we evaluated the efficacy of ING-1(heMAb) in a murine model of cancer metastases. Mice received intravenous dosing of 1 mg/kg ING-1(heMAb), twice a week, starting on day 2 or day 5. A negative control group received 1 mg/kg human immunoglobulin G with the same dose frequency starting on day 2. A positive control group received weekly 100 mg/kg 5-flurouracil/leucovorin starting on day 2. ING-1(heMAb)/day 2 treatment significantly reduced both the number of visible tumor nodules in body cavities (P <.01) and the number of metastases on lung surfaces (P <.005). The treatment also resulted in a 91% reduction of micrometastases in lung tissues (P <.0001). Delaying ING-1(heMAb) treatment until day 5 caused 54% reduction in micrometastases (P <.005). Our results indicate that a number of parameters, including treatment starting day, dose level, and dose frequency, are critical in achieving the optimal efficacy of ING-1(heMAb). We conclude that ING-1(heMAb) effectively reduced tumor metastases in a murine cancer model. Immunotherapy with ING-1(heMAb) may be beneficial in treating human metastatic diseases.