Development of Forodesine Hydrochloride (FH), an Inhibitor of Purine Nucleoside Phosphorylase, for Patients with Chronic Lymphocytic Leukemia (CLL).

Abstract

Mammalian purine nucleoside phosphorylase (PNP) catalyzes the cleavage of inosine, deoxyinosine, guanosine, and deoxyguanosine (dGuo) to their corresponding base and sugar 1-phosphate by phosphorolysis. PNP deficiency in humans produces a relatively selective depletion of T cells without much effect on normal B cells. FH, a potent inhibitor of PNP, was designed based on the transition-state analog structure stabilized by the enzyme. Previous studies demonstrated that FH in the presence of dGuo inhibits the proliferation of T-lymphocytes (Kicska et al. PNAS 2001). Based on these observations, we conducted a phase I clinical trial of FH in patients with advanced T-cell malignancies. Significant antileukemic activity was correlated with an increase in plasma FH (median 5 μM) and dGuo (median 14 μM), and an accumulation of intracellular dGuo triphosphate (dGTP) (Gandhi et al, Blood, in press, 2005). High accumulation of dGTP in T-cells may be dependant on activity of deoxynucleoside kinases. Because B-CLL cells have high activity of deoxycytidine kinase, we hypothesized that they would be sensitive to FH. This postulate was tested in primary CLL lymphocytes during in vitro investigations. Lymphocytes from patients with CLL were incubated in vitro with FH (2 μM) in the presence of 10 μM dGuo. Lymphocytes from 3 of 4 patients showed an elevation in the intracellular dGTP levels to a median 30-fold at 8 hr, without any effect on other deoxynucleotides. This increase in dGTP was associated with phosphorylation of p53 at ser15, stabilization of p53, and an increase in p21 protein. The dGTP accumulation was related to induction of apoptosis measured by activation of caspase 8, 9, and 3 and cleavage of PARP. Incubation with either FH or dGuo alone did not result in dGTP accumulation or cell death suggesting that PNP inhibition by FH and phosphorylation of dGuo to dGTP are essential for CLL cell death. Based on these encouraging results, availability of oral formulation and to validate these in vitro data during clinical trial, a phase II study of FH in patients with advanced, fludarabine-refractory CLL has been initiated. FH is administered orally at a dose of 200 mg/day for 7 days each week for 4 weeks (cycle 1). Patients are evaluated after 1 full cycle of therapy and in the absence of serious side effects, or disease progression, they continue the treatment for up to 5 more cycles. Laboratory endpoints such as level of FH and dGuo in plasma, PNP activity, dGTP levels in cells, will be measured and correlated with cytoreduction. It is postulated that a high kinase/low nucleotidase activity leading to the accumulation of intracellular dGTP in target CLL cells and apoptosis (akin to what has been seen in T-cells) will result in response to therapy. This is the first trial of a PNP inhibitor for treatment of B-CLL.