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The prognosis of fludarabine-refractory patients is poor and the current salvage regimens produce low CR rates and are unlikely to improve long-term survival in this patient population. Forodesine, an analog inhibitor of the enzyme purine nucleoside phosphorylase (PNP), leads to rise in plasma dGuo levels followed by accumulation of dGTP in T-cells,that results in DNA breakdown and cell apoptosis. Forodesine has exhibited promising clinical activity in T cell leukemia with complete inhibition of PNP during therapy (Gandhi et al, Blood 106:4253, 2005). Previous in-vitro studies conducted by our group demonstrated that forodesine induces caspase dependent cell death in primary CLL cells by accumulating high intracellular dGTP in CLL cells (Balakrishnan et al, Blood 108:2392, 2006). This provided the rationale to test forodesine in B-CLL.i) to investigate the efficacy (CR + PR) of forodesine in treating patients with advanced, fludarabine-treated CLL, ii) to evaluate the toxicity, duration of response, disease-free survival and overall survival associated with treatment with forodesine, and iii) to correlate PK/PD data of forodesine in CLL with its clinical activity.Patients with primary resistance to fludarabine-based therapy (no CR or PR) or with progressive disease after response to prior fludarabine based regimen were eligible. The forodesine dose was 200 mg oral once daily, administered continuously up to a maximum of 24 weeks. Blood samples were collected on days 1-5 and day 28 and pharmacokinetic and pharmacodynamic parameters were determined. Ex vivo incubation with forodesine and dGuo were also performed in CLL lymphocytes to compare the treatment effects in vivo and ex vivo investigations.8 patients were treated, the median age was 62 years (range 51-68), 7 males, median absolute lymphocyte count 35.85 (range 1.4-156.66) × 109/L, median serum beta 2 microglobulin level was 6.45 (range 3.6-16.3) mg/L. Six patients had Rai stage III-IV disease, the, median number of prior therapy was 5 (range 1-10), 5 patients were fludarabine refractory, 5 were ZAP-70 positive. Seven patients are evaluable for response and toxicity. Two patients had a transient decrease in their absolute lymphocyte count to normal level after the first 4 weeks, but it was short lasting. In the other 5 patients, the WBC counts increased progressively and in 3 patients there was also progression in lymphadenopathies. The toxicities observed were mild and included fatigue, bronchitis, diarrhea and low grade fever. Myelosuppression was transient and included neutropenia thrombocytopenia in 3 patients. One patient had pneumonia.The steady-state level of forodesine, measured on day 2, 3, 4, and 5, ranged between 200-1300 nM (n=8). At these concentrations, PNP inhibition in circulating RBCs, ranged between 57 and 89% (n=8). With this extent of PNP inhibition, steady-state dGuo concentration was a median 1.8 μM (range, 0.56 – 4.4 μM, n=8). The starting level of intracellular dGTP was a median 5 μM (range, 0.9 - 7.8 μM, n = 7) which increased to a median 12.5 μM (range 1.9 - 65 μM, n = 7). The circulating lymphocytes did not show annexin positivity above 10% during the first five days of treatment. To determine if CLL lymphocytes accumulate dGTP at high dGuo (10 and 20 μM) levels, we performed ex vivo incubations. Compared to in vivo, higher levels of dGTP were achieved ex vivo with a 10-50% increase in apoptosis.Oral forodesine showed activity with decrease in peripheral lymphocyte counts in 2 of 7 evaluable patients. However, all patients ultimately progressed while on treatment. Side effects were mild. The ex vivo biologic data and in vivo pharmacodynamic and clinical data suggest that forodesine has biological activity, and that alternative dosing schedules should be explored in future CLL trials.Off Label Use: Forodesine is not approved by FDA for treatment of CLL. Bantia:BioCryst: Employment. Gandhi:Mundipharma: Honoraria, Research Funding; Biocryst: Honoraria, Research Funding. Ravandi:Biocryst: Research Funding.

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.