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The growth of the breast cancer line MDA-MB-231 is inhibited by the HIV protease inhibitor ritonavir, with an IC50 of 45 μM. Cell cycle arrest occurs at Go/G1 at the IC50 for proliferation by Hoechst 33342 dye/pyronine flow cytometry and there is induction of apoptosis, measured by propidium iodide/annexin V flow cytometry. Fluorometric analysis of protease inhibition in the MDA-MB-231 line reveals that ritonavir inhibits greater than 90% of calpain activity, but inhibits proteasome activity by only 10-20%, at the IC50 concentration for growth of 45 μM. Surface EGFR levels are down-regulated by 25% at the ritonavir IC90. Phospho-Akt is reduced by 70% after 48 h exposure to ritonavir. Calpain’s physiological inhibitor, calpastatin, also down-regulates phospho-Akt. Pharmacokinetic analysis was used to determine a dosing route and schedule for ritonavir in mice. Gavage did not result in adequate blood levels 1 h following administration and this approach was abandoned. IP injection of 40 mg/kg was found to result in ritonavir Cmax levels of 12-67 μM, measured 1 h following drug administration. The Tween 80 vehicle was well tolerated. A mouse mammary fat pad model was used to test the hypothesis that ritonavir will block the growth of solid tumors in a xenograft model. Tumors were established for 21 days before treatment. Daily ritonavir treatment, 40 mg/kg ip, resulted in arrest of growth of the MDA-MB-231 xenografts, with a plateau of tumor size, 300 + 20 mm3, compared to 800 + 200 mm3 for vehicle, by day 52 of treatment (P=0.002, by Gompertzian curve fitting). The ritonavir treatment was well tolerated and the mouse weights were 23 + 2 g at the beginning of drug treatment and 25 + 2 g on day 52. No effects of ritonavir on angiogenesis or EGFR levels were observed by immunohistochemical (IHC) analysis of tumors harvested at the end of the study. Data on phospho-Akt IHC and dose dense treatment will be presented. These results point to the development of ritonavir for treatment of solid tumors, because tumor growth inhibition is observed at well tolerated ritonavir doses.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]