The past year has seen progress in studies of CAI which include in vitro and in vivo effects, mechanism of action, and resistance to CAI. The preclinical pharmacology and CAI toxicity studies were completed with the approval of the Investigational New Drug application by the FDA, and initiation of the Phase I clinical study. Our laboratory has refined and mastered the HPLC technique for measuring CAI plasma levels and is providing patient pharmacokinetic data. the first patients studied had CAI plasma levels in the lower end of the effective in vitro range. Structure- activity studies have shown that dehalogenation of the hydrophobic tail completely abrogates the CAI-induced inhibition of signal transduction, proliferation, and experimental metastasis, whereas, modifications of the triazole head group has no effect. Over the past year, CAI-drug resistant A2058 human melanoma, OVCAR3 human ovarian cancer, and CHOm5 cell lines have been developed. Studies have been initiated to investigate this drug resistant phenotype. A functional endogenous muscarinic receptor has been identified on the A2058 cells. Activation of this receptor mediates reversal of the transformed phenotype, stimulates arachidonic acid release, and calcium influx. Stimulated calcium influx and internal release are inhibited by CAI treatment. Molecular and biologic techniques are in use to identify and clone this receptor. The project to identify and clone the CAI binding site has been initiated; tools for this task have been developed which include production of a 14C-labelled compound and anti-CAI antibodies for expression cloning, and use of the resistant/wild type paired cell lines for protein and gene isolation. Collaborative studies have identified in vitro myelosuppressive effects of CAI in mouse bone marrow cultures, and mixed responses of human astrocytoma and gliomamultiforme cell lines to incubation with CAI.
