Purpose: The high molecular weight and binding affinity of trastuzumab, a monoclonal antibody in use for treatment of breast cancers overexpressing human epidermal growth factor receptor type 2 (HER2), in combination with microenvironmental factors, may limit its distribution and efficacy. We assessed and mapped the distribution of systemically given, unlabeled trastuzumab at micrometer resolution in tumor xenografts using immunohistochemistry. Experimental Design: Mice bearing MDA-435/LCC6 HER2 xenografts were given single doses of 4 or 20 mg/kg unlabeled trastuzumab with tumor harvest at various time points thereafter; bound trastuzumab was imaged directly in tumor cryosections using fluorescently tagged antihuman secondary antibodies. Combinations of additional markers, including HER2, 5-bromo-2-deoxyuridine, CD31, DioC 7 (3), desmin, and collagen IV were also mapped on the same tumor sections. Results: Distribution of trastuzumab in MDA-435/LCC6HER2 tumors is found to be heterogeneous, with tumor margins saturating more thoroughly in doses and times analyzed. Considerable intervessel heterogeneity is also seen. For example, in unsaturated tissues, there remain perfused vessels without any trastuzumab in addition to vessels with a few layers of positively stained perivascular cells, in addition to vessels with bound drug up to 150 Am away. This heterogeneity is independent of HER2 expression, microvessel density, and perfusion. A slightly greater proportion of vessels were associated with pericytes in sections with greater trastuzumab saturation, but this would not adequately account for observed heterogeneous trastuzumab distribution. Conclusions: Complete penetration of trastuzumab in tumor tissue was not seen in our study, leaving the possibility that inadequate distribution may represent a mechanism for resistance to trastuzumab.

Tumour hypoxia presents a barrier to conventional chemotherapy and radiation therapy. To combat hypoxic cells a number of hypoxia modifying treatments are currently in development. In this study we assessed the potential for inhibiting DNA double strand break repair in hypoxic cells by targeting DNA-dependent protein kinase (DNA-PK) and we report the synthesis and in vitro efficacy of BCCA621C (1), a hypoxia activated inhibitor of DNA-PK. We found that DNA-PK deficient hypoxic cells are radiosensitive compared to hypoxic DNA-PK proficient cells and that this effect can be observed using both a small molecule inhibitor of DNA-PK, IC86621 (2), as well as with a genetically deficient model cell line. BCCA621C, which is designed to selectively release a DNA-PK inhibitor in hypoxic cells was synthesized and assessed for bioreduction using mouse liver microsomes and NCI-H460 cells. BCCA621C is activated by bioreduction in severely hypoxic NCI-H460 cells and was able to radiosensitize hypoxic NCI-H460 cells with a sensitizer enhancement ratio (SER) of 1.85. No enhancement of radiosensitivity was found to occur with BCCA621C treatment in oxygenated NCI-H460 cells in a range of clinically relevant ionizing radiation doses.

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