Phase I and pharmacological study of cytarabine and tanespimycin in relapsed and refractory acute leukemia

Abstract

Background: In preclinical studies the heat shock protein 90 (Hsp90) inhibitor tanespimycin induced down-regulation of checkpoint kinase 1 (Chk1) and other client proteins as well as increased sensitivity of acute leukemia cells to cytarabine. We report here the results of a phase I and pharmacological study of the cytarabine + tanespimycin combination in adults with recurrent or refractory acute leukemia.

Design and methods: Patients received cytarabine 400 mg/m(2)/day continuously for 5 days and tanespimycin infusions at escalating doses on days 3 and 6. Marrow mononuclear cells harvested before therapy, immediately prior to tanespimycin, and 24 hours later were examined by immunoblotting for Hsp70 and multiple Hsp90 clients.

Results: Twenty-six patients were treated at five dose levels. The maximum tolerated dose was cytarabine 400 mg/m(2)/day for 5 days along with tanespimycin 300 mg/m(2) on days 3 and 6. Treatment-related adverse events included disseminated intravascular coagulation (grades 3 and 5), acute respiratory distress syndrome (grade 4), and myocardial infarction associated with prolonged exposure to tanespimycin and its active metabolite 17-aminogeldanamycin. Among 21 evaluable patients, there were two complete and four partial remissions. Elevations of Hsp70, a marker used to assess Hsp90 inhibition in other studies, were observed in more than 80% of samples harvested 24 hours after tanespimycin, but down-regulation of Chk1 and other Hsp90 client proteins was modest.

Conclusions: Because exposure to potentially effective concentrations occurs only for a brief time in vivo, at clinically tolerable doses tanespimycin has little effect on resistance-mediating client proteins in relapsed leukemia and exhibits limited activity in combination with cytarabine. (Clinicaltrials.gov identifier: NCT00098423).

Figures

Figure 1.

Schematic representation of trial events. After pretreatment bone marrow aspirates were obtained, cytarabine was administered at a dose of 400 mg/m2/day by continuous infusion for 5 days. Approximately 48 h into this infusion, the day 3 bone marrow aspirate was obtained. Patients then received escalating doses of tanespimycin (repeated again on day 6 at the very end of the cytarabine infusion), followed 22±2 h later by the day 4 bone marrow aspirate to assess the impact on client protein expression

Figure 2.

Effects of treatment on client protein levels. (A). After U937 cells had been treated for 24 h with diluent (0.1% DMSO, lane 1) or tanespimycin at 30, 100, 300 or 1000 nM (lanes 2–5), whole cell lysates were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblotting. Numbers on the left are molecular weight markers in kDa. (B-D) Marrow mononuclear cells from patients exposed to tanespimycin in vivo at (B) dose level 3, (C) dose level 4 or (D) dose level 1. In each case whole cell lysates were isolated from marrows harvested prior to treatment (day 0), after 48 h of cytarabine (day 3) and 22±2 h after tanespimycin administration (day 4). Polypeptides from 0.5–5x105 HL-60 cells were included on each gel to provide a standard curve for quantitating signals. Hsp90β and Histone H1 represent two loading controls. After immunoblotting, digitized signals were normalized for histone H1 content and compared to HL-60 cell dilutions. A value of 1.0 indicates the same antigen signal as an equal number of HL-60 cells. In panels (B) and (D), pretreatment marrow mononuclear cells from the same patients were also prospectively exposed ex vivo to diluent (0.1% DMSO), 300 nM cytarabine, 1000 nM cytarabine, 300 nM tanespimycin, or 1000 nM tanespimycin (lanes 1–5, respectively) for 24 h and subjected to immunoblotting. Cells for ex vivo exposure were not available from the patient in panel (C).