Phase 2 and pharmacodynamic study of oral forodesine in patients with advanced, fludarabine-treated chronic lymphocytic leukemia

Abstract

Forodesine is a new and potent purine nucleoside phosphorylase (PNP) inhibitor. Patients with chronic lymphocytic leukemia (CLL) with primary resistance to fludarabine-based therapy or with progressive disease were eligible for oral forodesine (200 mg/d) for up to 24 weeks. Eight patients with median lymphocyte count of 35.9 x 10(9)/L and median serum beta2 microglobulin level of 6.45 mg/L were treated. Six had Rai stage III to IV and were previously heavily treated (median prior therapy = 5). Two had transient decrease in lymphocyte count to normal, whereas in 5, disease progressed. Adverse events were mild. Steady-state level of forodesine ranged from 200 to 1300 nM and did not reach desired 2 microM level. PNP inhibition ranged from 57% to 89% and steady-state 2'-deoxyguanosine (dGuo) concentration median was 1.8 microM. Intracellular deoxyguanosine triphosphate (dGTP) increase was very modest, from median of 6 microM to 10 microM. Compared with in vivo, in vitro incubations of CLL lymphocytes with 10 or 20 microM dGuo and forodesine (2 microM) resulted in accumulation of higher levels of dGTP (40-250 microM) which resulted in increase in apoptosis. Forodesine has biologic activity in CLL; pharmacodynamic parameters suggest that an alternate dosing schedule and/or higher doses to achieve greater intracellular dGTP may be beneficial in this patient population.

Trial registration: ClinicalTrials.gov NCT00289549.

Figure 1

Role of PNP in purine pathway. This mammalian enzyme is involved in phosphorolysis of substrates such as inosine/deoxyinosine, xanthosine/deoxyxanthosine, and guanosine/deoxyguanosine. With these conversions, bases such as hypoxanthine, xanthine, and guanine, respectively, are formed.

Figure 2

PNP inhibition in red blood cells during therapy. Red blood cells from patients (all 8 patients; except patient no. 6) were isolated from whole blood, starting day 0 to day 4 (n = 7) and day 27 (n = 5), and the cells were processed to determine the level of PNP and its inhibition after start of therapy as described in “Measurement of PNP inhibition.” Data were fitted with the use of the nonlinear rectangular hyperbola. PNP assay was done in quadruplicate, and data are mean ± SD. The average specific activity of PNP was 3.7 mU/min where 1 mU is defined as the amount of PNP crude lysate that catalyzes the phosphorolysis of 1μmole of inosine per minute under standard assay condition.

Figure 3

Plasma forodesine and dGuo levels during therapy. Whole blood was collected from all 8 patients at indicated time points, and plasma was separated. Forodesine (A) and dGuo (B) levels in each sample were determined with the use of a tandem mass spectrometry liquid chromatography as described in “Measurement of plasma dGuo and forodesine.” Each symbol represents a patient. The correlation between plasma forodesine and plasma dGuo (C) was evaluated by plotting data from panels A and B, and linear regression analysis was performed.

Figure 4

Accumulation of dGTP and induction of cell death during therapy. Blood samples were collected from all patients (except patient no. 1) at the indicated time points before and after start of therapy, and CLL lymphocytes were isolated. After methanol extraction, cells were processed to measure dGTP by DNA polymerase assay (A) and cell death by annexin binding procedure (B) as described in sections on measurement of dNTP pool and cell viability.

Figure 5

In vitro accumulation of dGTP and induction of cell death. Primary CLL cells from 6 patients were incubated without drug or with 2μM forodesine and 20μM dGuo for 24 hours, and the nucleotides were extracted, and dGTP was measured by DNA polymerase assay in untreated () or forodesine and dGuo–treated () cells (A). Data from Figure 4A are plotted with data from Figure 5A to compare in vivo () and in vitro () accumulation of dGTP (B). In the same cells, cell death was analyzed at 24 hours by Annexin positivity procedure as described in “Measurement of cell viability” in untreated () and forodesine and dGuo–treated () cells (C).