Evidence for Microtubule Target Engagement in Tumors of Patients Receiving Ixabepilone

Abstract

Purpose: Microtubule-stabilizing agents, such as taxanes, have been shown to be effective anticancer drugs. a-Tubulin, a basic unit of microtubules, can undergo several posttranslational modifications after assembly into stabilized microtubules, including acetylation and detyrosination. These modifications have been observed in cell cultures after exposure to microtubule stabilizers. Our objective was to develop a straightforward and dependable assay to show tubulin target engagement in tumor tissue after treatment of patients with ixabepilone(BMS-247550; Ixempra). Experimental Design: Levels of posttranslationally modified a-tubulin were assessed in lysates of cultured malignant cell lines, as well as in both tumor tissue and peripheral blood mononuclear cells derived from patients before and after treatment with ixabepilone. Modification-specific antibodies permitted quantitative Western blot analysis. Results: In cultured cell lines, the levels of detyrosinated (glu-terminated) and acetylated a-tubulin increased after microtubule stabilization induced by ixabepilone. Ixabepilone treatment also induced a 2-fold to 25-fold increase in detyrosinated a-tubulin levels in 11of 13 serial biopsies and a 2-fold to 100-fold increase in acetylated a-tubulin in 11of 12 serial biopsies obtained from patients receiving ixabepilone. Overall, little or no difference in tubulin modifications were observed between the before and after ixabepilone treatment in lysates from their peripheral blood mononuclear cells at the time point examined. Conclusion: Assessing the levels of detyrosinated and/or acetylated a-tubulin seems to provide a simple and reliable assay to show target engagement by the microtubule-stabilizing agent ixabepilone. Such analyses may provide further understanding of therapeutic success or failure of microtubule-stabilizing agents in cancer therapy.

Preclinical efficacy spectrum and pharmacokinetics of ixabepilone

“…Consistent with its favorable pharmacokinetics and notable preclinical activity in this study, promising clinical activity of ixabepilone has been seen against a wide range of tumor types, including breast [locally advanced (LABC) and metastatic (MBC)], lung, renal, prostate, pancreas, and lymphoma [16][17][18][19][20][21][22][23][24][25][26][27][28]. In several instances, tumors had been heavily pretreated or were resistant to current therapies, including anthracycline-pretreated MBC [25], taxaneresistant MBC [26], and MBC or LABC resistant to an anthracycline, a taxane, and capecitabine, [39] suggesting that the reduced susceptibility of ixabepilone to mechanisms of drug resistance seen in these and other preclinical studies can translate to the clinic.…”

“…Single-agent ixabepilone has shown encouraging antitumor activity in a broad range of tumor types during phase I [12][13][14][15] and phase II [16][17][18][19][20][21][22][23][24][25][26][27][28] clinical trials.…”

A Phase II Clinical Trial of Ixabepilone (Ixempra; BMS-247550; NSC 710428), an Epothilone B Analog, in Patients with Metastatic Renal Cell Carcinoma

“…We have previously reported target engagement with two markers of stable tubulin before and after ixabepilone treatment in cycle 1 (25). As shown in Fig.…”

Novel 2‐Substituted Quinolin‐4‐yl‐benzenesulfonate Derivatives: Synthesis, Antiproliferative Activity, and Inhibition of Cellular Tubulin Polymerization

“…[49] After treatment with the indicated compounds, HeLa-S3 cells were harvested in lysis buffer (100 mm PIPES pH 6.9, 1 mm EGTA, 1 mm MgCl 2 , 30 % glycerol, 0.5 % DMSO, 1 % NP-40, 1 mm GTP, and protease inhibitors). Following centrifugation at 180 000 g at 37 8C for 1 h, the polymerized tubulin fraction (pellet) and the soluble tubulin fraction (supernatant) were separated.…”