Clinical application of proteasome inhibitors in the treatment of hematologic malignancies such as myeloma and lymphoma is restricted in part by the necessity of frequent IV administration and would be improved by oral (PO) administration. Selective inhibitors of the protease subunits of the 20S proteasome can be generated from peptidyl aldehydes, boronates, vinyl sulfones, and epoxyketones. Many of these peptide based proteasome inhibitors are cell permeant and capable of systemic proteasome inhibition upon intravenous (IV) administration to experimental animals such as mice and rats. In the cases of the peptidyl boronate bortezomib (Velcadeā„¢) and the epoxyketone PR-171, proteasome inhibition can be achieved in patients with IV administration. However, systemic exposure following PO administration of these inhibitors may be limited by several factors including gastric pH, gastric and intestinal peptidases, efflux pumps, biliary excretion and intestinal and hepatic metabolic activities. We have tested over 80 peptide epoxyketones with potent (IC50 <100 nM) in vitro inhibitory activity against the chymotrypsin-like activity of the 20S proteasome for bioavailability following PO administration in mice. Systemic exposure was monitored by measuring chymotrypsin-like inhibition in a number of tissues and an approximation of the relative bioavailability for selected compounds was measured by comparing the pharmacodynamics of IV and PO administration using a range of doses. These compounds were also tested in vitro for solubility, intestinal cell permeability, intestinal and hepatic metabolism, and sensitivity to the multidrug resistance protein 1 (MDR1) efflux pump in order to determine which properties were associated with oral bioavailability. We have found that oral bioavailability in mice is associated with increased intrinsic solubility and metabolic stability and reduced MDR1 sensitivity. Proteasome inhibition following PO administration is rapid, resulting in maximal proteasome inhibition within 15 minutes. Rapid absorption and clearance of selected compounds was also confirmed in mice and rats by pharmacokinetic analysis. Repeated oral administration was well tolerated at doses that resulted in significant (>80%) proteasome inhibition in most tissues. The anti-tumor efficacy of these orally bioavailable proteasome inhibitors are being assessed in both human tumor xenograft and mouse syngeneic models. The results from these studies will enable further pre-clinical development of potent, orally bioavailable proteasome inhibitors for the treatment of malignant diseases.

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