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Forodesine, a purine nucleoside phosphorylase inhibitor, displays in vitro activity in chronic lymphocytic leukemia (CLL) cells in presence of dGuo, which is the basis for an ongoing clinical trial in patients with fludarabine-refractory CLL. Initial clinical data indicate forodesine has significant activity on circulating CLL cells, but less activity in clearing CLL cells from tissues such as marrow. In tissue microenvironments, lymphocytes interact with accessory stromal cells that provide survival and drug-resistance signals, which may account for residual disease. Therefore, we investigated the impact of marrow stromal cells (MSCs) on forodesine-induced response in CLL lymphocytes. We demonstrate that spontaneous and forodesine-induced apoptosis of CLL cells was significantly inhibited by human and murine MSCs. Forodesine-promoted dGuo triphosphate (dGTP) accumulation and GTP and ATP depletion in CLL cells was inhibited by MSCs, providing a mechanism for resistance. Also, MSCs rescued CLL cells from forodesine-induced RNA- and protein-synthesis inhibition and stabilized and increased Mcl-1 transcript and protein levels. Conversely, MSC viability was not affected by forodesine and dGuo. Collectively, MSC-induced biochemical changes antagonized forodesine-induced CLL cell apoptosis. This provides a biochemical mechanism for MSC-derived resistance to forodesine and emphasizes the need to move toward combinations with agents that interfere with the microenvironment's protective role for improving current therapeutic efforts.

Forodesine and nelarabine (the pro-drug of ara-G) are 2 nucleoside analogues with promising anti-leukemic activity. To better understand which pediatric patients might benefit from forodesine or nelarabine (ara-G) therapy, we investigated the in vitro sensitivity to these drugs in 96 diagnostic pediatric leukemia patient samples and the mRNA expression levels of different enzymes involved in nucleoside metabolism. Forodesine and ara-G cytotoxicities were higher in T-cell acute lymphoblastic leukemia (T-ALL) samples than in B-cell precursor (BCP)–ALL and acute myeloid leukemia (AML) samples. Resistance to forodesine did not preclude ara-G sensitivity and vice versa, indicating that both drugs rely on different resistance mechanisms. Differences in sensitivity could be partly explained by significantly higher accumulation of intracellular dGTP in forodesine-sensitive samples compared with resistant samples, and higher mRNA levels of dGK but not dCK. The mRNA levels of the transporters ENT1 and ENT2 were higher in ara-G–sensitive than –resistant samples. We conclude that especially T-ALL, but also BCP-ALL, pediatric patients may benefit from forodesine or nelarabine (ara-G) treatment.

Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine, a potent inhibitor of PNP, was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK), we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization, phosphorylation of p53 at Ser15, and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation, changes in mitochondrial membrane potential, and PARP cleavage. Based on these data, a phase 2 clinical trial of forodesine has been initiated for CLL patients.

Abstract 1796 Background: Forodesine is a rationally designed potent inhibitor of purine nucleoside phosphorylase (PNP) that leads to elevation of plasma deoxyguanosine (dGuo) and intracellular accumulation of dGTP levels and then apoptosis mainly in T cells. Oral forodesine has shown clinical activity in patients with cutaneous T-cell lymphoma (CTCL) (M. Duvic et al, ASH 2007). The objective of this phase I study was to evaluate the safety, PK profile, and efficacy of oral forodesine in patients with recurrent or refractory T/NK malignancies in Japan. Methods: An open-label dose-escalation study of forodesine, 100 to 300 mg/body qd for 4 weeks, was conducted to evaluate safety profile (dose-limiting toxicities, DLT), tolerability and PK profile as primary endpoints. Forodesine was administered until disease progression or unacceptable toxicity is observed. Relapsed or refractory T/NK malignancies with PS 0 to 1 and without major organ dysfunction were eligible. Results: Overall, 13 Japanese patients, 8 males and 5 females, with a median age of 69 (range 30–77) years were enrolled in the study: 5 patients in the 100mg cohort, 3 in the 200mg cohort and 5 in the 300mg cohort. Patients’ histopathologic subtypes were as follows: peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) (6 patients), anaplastic large cell lymphoma (ALCL) (3), primary cutaneous ALCL (C-ALCL) (2) and mycosis fungoides (MF) (2). Median stage and prior treatment regimen were IIIA (range IA-IVA) and 2 (range 1, 8), respectively. No DLT was observed and a maximum tolerated dose was never defined. The most common toxicities of grade 2 or less were constipation (39 %), rash (31%), lymphopenia (31 %), neutropenia (23 %), nausea (23 %), peripheral edema (23 %), LDH elevation (23 %) and leukopenia (23 %). The toxicities of grade 3 or greater were lymphopenia (62 %), anemia (15 %), leukopenia (8 %), thrombocytopenia (8 %) and viral infection (8 %). Median baseline, nadir, and last visit lymphocytes counts (1,000/μL) were 0.69 (95% CI: 0.56, 1.18), 0.35 (95% CI: 0.14, 0.60) and 0.60 (95% CI: 0.24, 0.95), respectively. Plasma levels for forodesine showed less than dose-proportional increase in exposure as mean AUC at Day 1 was 1,948 (ng·h/mL) in the 100mg cohort, 4,608 (ng·h/mL) in the 200mg cohort, and 4,596 (ng·h/mL) in the 300mg cohort. The levels for dGuo displayed a similar trend, with mean AUC at Day 1 4,023 (ng·h/mL) in the 100mg cohort, 5,705 (ng·h/mL) in the 200mg cohort, and 6,074 (ng·h/mL) in the 300mg cohort. One patient with ALCL reached complete response (CR) in the 100mg cohort and 2 patients with MF reached partial response in the 200mg cohort. In addition, 4 patients with stable disease (SD) were observed: 1 patient with PTCL-NOS in the 100mg cohort, 1 with C-ALCL in the 200mg cohort and 2 with C-ALCL and PTCL-NOS in the 300mg cohort. As of Aug, 2010, 2 patients with ALCL (CR patient in the 100mg cohort) and PTCL-NOS (SD patient in the 300mg cohort) have continued the treatment for more than 510 days and 290 days, respectively. Conclusion: Oral forodesine was well tolerated at all the dose levels tested with similar PK findings to those in the CTCL study in USA, demonstrating potential efficacy against relapsed or refractory T/NK lymphomas including PTCL for the first time. Based on these promising data, we are planning a phase I /II study of forodesine in patients with relapsed or refractory PTCL. Disclosures: No relevant conflicts of interest to declare.

Abstract 1397 Introduction: Forodesine is a potent, specific transition-state inhibitor of the purine salvage pathway enzyme PNP. It induces apoptosis in primary CLL cells through accumulation of deoxyguanosine triphosphate. Unlike other nucleoside analogues, forodesine is not phosphorylated, is not incorporated into DNA and is not cytotoxic. Previous phase 1 and 2 studies showed clinical activity in T-cell acute lymphoblastic leukemia (i.v. forodesine) and cutaneous T-cell lymphoma (i.v. and oral). In a single institute exploratory study 5 of 13 (38%) CLL patients demonstrated anti-leukemic effects (decrease in peripheral blood malignant lymphocyte counts) with oral forodesine (200mg QD). However none of the patients achieved remission. Forodesine is absorbed via a saturable mechanism, and in a clinical pharmacology study in normal subjects, 200mg twice-daily (BID) dosing was found to partially overcome the exposure limits from once daily dosing; the 200mg BID regimen was used in the current study. Methods and Results: This is a multicenter phase II open label study. Eligible CLL patients had either failed at least 1 prior therapy (progressive disease [PD] or refractory) or were treatment-naïve provided age was >65 or ECOG 2 or 3, or unable to tolerate standard therapy. Among the 26 patients enrolled 1 was deemed ineligible (Waldenstrom's). The median age was 70 (48-92) years, 65% male and 77% had Rai Stage III/IV disease. The median duration from time to CLL diagnosis was 92.7 (8.4-243.5) months. Features of high-risk disease were del (17p) (n=6), ZAP70+ (n=13), and CD38+ (n=15). All patients had relapsed or refractory CLL with a median of 2 (1-21) prior therapies including fludarabine (76%), alkylating agents (72%), anti-CD20 monoclonal antibodies (72%) and experimental agents (36%). Interim analysis was conducted when 13 patients had been observed for ≥ 6 months: maximum duration of continuous BID forodesine therapy was 364 days. At the date of data cut-off, 20 were evaluable for response (5 were not evaluable because they had received < 8 weeks of therapy), and 12 were still on study. The revised NCI-WG (1996) criteria were used for response assessment. Partial remission was noted in 3 (15%) patients. Additionally 12 (48%) patients demonstrated stabilization of disease. PR onset was noted at 2, 6 and 6 months of therapy. Three patients discontinued due to adverse events (AEs). Serious AEs were reported in 18 patients. Individual SAEs reports included 7 cases of febrile neutropenia, 7 cases of non-neutropenic infections, pneumonia in 4, and herpes zoster in 1. Conclusion: Oral forodesine 200mg BID given continuously was generally safe and well-tolerated in previously-treated CLL patients. The overall response rate was 15%. These safety and efficacy results are particularly intriguing given the relapsed / refractory CLL population studied, which included elderly and poor performance status patients. Furthermore, we observed that, despite being a purine analog, forodesine was well tolerated with continuous daily dosing. This is particularly of interest when considering combination regimens with this new agent. Further studies of optimized dosing regimens of PNP inhibitors in CLL are warranted. These studies could include evaluation of forodesine in combination with other antileukemic agents with different mechanisms of action, in patients intolerant of or ineligible for aggressive conventional chemotherapeutic regimens, or maintenance treatment after conventional therapy. Updated results including ≥6 months follow up for all patients in the Phase II study will be presented at the meeting. Disclosures: Lyman: BioCryst Pharmaceuticals, Inc.: Employment, Equity Ownership. Cantey-Kiser:PharPoint Research, Inc.: Employment. Sheridan:BioCryst Pharmaceuticals, Inc.: Employment, Equity Ownership.

Abstract 3766 Poster Board III-702 Inhibitors of the purine metabolism show promising results in the treatment of lymphatic malignancies due to their suppressive effects on lymphogenesis. Their first representative, Pentostatin (Pento), an inhibitor of the deoxyadenosine deaminase, has been in clinical use for several decades. However, early clinical trials with higher dose ranges of the drug reported unforseen severe myelotoxic effects. Recently, Forodesine (Foro), a novel inhibitor of the nucleoside phosphorylase (PNP) has been introduced and is currently deployed in clinical phase I/II trials for the treatment of acute lymphatic leukemia (ALL). In order to systematically evaluate the myelotoxic effects of Pento and Foro, we have now examined their influence on the proliferation and differentiation of primitive and lineage committed hematopoietic progenitor cells (HPCs). In vitro dose/effect-curves for Foro, Pento, and Cytarabine (AraC) were generated for the leukemic cell line jurkat by 48 hours of co-incubation with the compounds. Adequate cytotoxic effects, measured in the XTT assay and by flow cytometric analysis, were observed in clinically relevant dose ranges. For the following studies, an equivalent IC60 dose of each chemotherapeutic agent was selected and CD34+ HPCs from either bone marrow, mobilized peripheral blood, or umbilical cord blood were incubated with the compounds for 48 hours. Subsequently, the rate of vital cells was determined by flow cytometry after stainig with Annexin-V and Propidium Iodide. Compared to the untreated control, the lowest amount of vital CD34+ cells was found in AraC-treated samples (30%); Foro and Pento yielded more vital cells (66% vs 61%). The combination of Foro and Pento unexpectedly had the least toxic effect on CD34+ cells (72%; n=5; p Disclosures: Schubert: Mundipharma Int. LTD: Research Funding.

Abstract 2369 Poster Board II-346 Background: 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. Aims: 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. Methods: 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. Results: 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. Conclusion: 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. Disclosures: 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.

Mantle cell lymphoma (MCL) represents a subtype of B-cell lymphoma associated with a very unfavourable clinical outcome. Currently no therapy can be considered as standard, and new therapeutic approaches are needed. Forodesine is a potent inhibitor of purine nucleoside phosphorylase (PNP), whose major role is to catalyze the cleavage of inosine, deoxyinosine guanosine, and deoxyguanosine (dGuo) to their corresponding base and sugar 1-phosphate by phosphorolysis. In the presence of deoxycytidine kinase, PNP inhibition leads to an increase in the concentration of dGuo triphosphate (dGTP), followed by inhibition of DNA synthesis and cell death by apoptosis. When combined with dGuo, forodesine has been shown to have in vitro cytotoxic activity on T-cell (T-ALL, T-PLL) and on B-cell malignancies (CLL, B-ALL), and Phase I/II trials are on going in CLL and CTCL patients. Here, we report the first data on in vitro activity of forodesine in MCL. Primary MCL cells, derived from six patients, were exposed to forodesine (0, 2, 20 μM) in combination with dGuo (0, 10, 20 μM), for 48 hrs. Cells were cultured in X-VIVO 10 medium (Cambrex) with 10% FBS. Cell viability was assessed by flow cytometry with the Annexin V - propidium iodide assay. Four patient samples (67%) showed an increase in the number of Annexin V positive cells ranging from 1.9 to 5.3 times compared to untreated cells. The effect was larger for 20 μM forodesine compared with 2 μM. There was no effect of dGuo alone and only a minimal effect of increasing dGuo concentration from 10 μM to 20 μM. Cell lines did not appear to be ideal models to evaluate the efficacy of forodesine in vitro. Three established MCL cell lines (Granta-519, Rec, JeKo1) were treated with escalating doses of forodesine, but the results were not reproducible, while the same cells showed expected IC50 values between 25–30 μM when exposed to bendamustine for 72 hrs. In conclusion, the in vitro data reported here with 4/6 MCL patients primary samples sensitive to forodesine and the results from various groups on other T- and B-cell malignancies suggest that clinical trials of forodesine in MCL may be warranted.

2038 Poster Board II-15 Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with elevated deoxyguanosine (dGuo) plasma levels. DGuo is converted into dGTP inducing apoptosis in T-cells and this provides the rationale for the development of deoxyguanosine analogues as a potential treatment option for T-cell malignancies. Forodesine (BCX-1777; BioCryst-Mundipharma) is an efficient blocker of PNP activity, thereby boosting the conversion of dGuo into dGTP and raising intracellular dGTP levels. AraG (9-b-D-arabinofuranosyl-guanine) is a compound that is resistant to PNP-mediated degradation that is efficiently converted into AraGTP. AraGTP becomes incorporated in the DNA, blocking DNA synthesis and promoting apoptosis. In a phase II clinical trial, the AraG prodrug Nelarabine enforced a complete remission rate of 55% for pediatric T-ALL patients at 1st relapse. (Berg, JCO 2005). Clinical data of Forodesine treatment in pediatric ALL patients are not yet available. As tested on primary pediatric acute lymphoblastic leukemia (ALL) patient samples (4 T-ALL, 2 BCP-ALL), 1μM of Forodesine is sufficient to completely block PNP and abolish rapid dGuo degradation resulting in a median 7.9 (range 0.5-378) fold raise of intracellular dGTP levels. Accumulation of dGTP is comparable for T-ALL (n=31) and BCP-ALL (n=11) patient samples. This reflects equal intrinsic ability of salvage nucleotide synthesis for both T-ALL and BCP-ALL cells. Cytotoxic effect of Forodesine was tested on primary leukemia cells from newly diagnosed pediatric ALL patients in-vitro by incubating cells with Forodesine (1μM) in the presence of increasing concentrations of dGuo (0.001-50μM). In accordance with selective T-cell toxicity, T-ALL cells were more sensitive to Forodesine/dGuo treatment (median T-ALL LC50 value: 1.1μM dGuo/1μM Forodesine, n=27, p=0.001) compared to BCP-ALL cells, which had a median LC50 value of 8.8μM dGuo/1μM Forodesine (n=30). All patients that responded demonstrated dGTP accumulation (1.5-222.1 fold), although the raise of dGTP levels did not correlate with Forodesine/dGuo toxicity (r2= 0.10, p=0.22). Studying in-vitro responsiveness to AraG, T-ALL cells were more sensitive compared to BCP-ALL cells (p=0.0002) with a median AraG LC50 value of 20.5μM for T-ALL samples (n=24) versus 48.3μM for BCP-ALL samples (n=20). Remarkably, TELAML1 positive BCP-ALL cases were insensitive to AraG treatment (median LC50 value >50μM, n=9). No correlation was identified between in-vitro Forodesine/dGuo and AraG cytotoxicities (r2=0.05, p=0.29). Most patient samples that displayed AraG resistance still responded to Forodesine/dGuo treatment. This may be explained by the fact that the uptake of both drugs may be facilitated by different transporters. Using RQ-PCR we could demonstrate that AraG toxicity, in contrast to Forodesine, was significantly associated with ENT1 (equilibrative nucleoside transporter 1) expression levels (p=0.008), which was previously identified as strong predictor for AraC cytotoxicity in pediatric ALL (Stam RW. et al., Blood 2003). AraG cytotoxicity strongly correlated with AraC cytotoxicity (r2=0.71, p

Background: Forodesine is a rationally designed, potent inhibitor of purine nucleoside phosphorylase (PNP) that leads to intracellular accumulation of dGTP and then apoptosis. Intravenous forodesine has demonstrated activity in treatment of cutaneous T-cell lymphoma (CTCL) and served as the basis for the design of an oral forodesine Phase I/II trial Methods: An open label dose escalation study of oral forodesine (40 mg/m2 to 320 mg/m2 QD) for 4 weeks was performed to determine the maximum tolerated dose (MTD) and/or the optimal biologic dose (OBD) based on PK, and PNP inhibition as evidenced by elevation of plasma deoxyguanosine (dGuo) levels. Additional subjects were accrued at the optimal dose (80 mg/m2) to further assess clinical safety and efficacy. Patients with previously treated, refractory CTCL with stage IB disease or higher were eligible. The primary efficacy endpoint (objective response rate [ORR]) for this analysis was defined as at least a 50% decrease in modified severity-weighted assessment tool (mSWAT) from baseline maintained for at least 28 days. Only subjects who had at least 6 months follow-up as of March 1, 2007 were included in this analysis. Results: Although an MTD was not reached, based on plateau of the AUC versus dose plot at and above 80 mg/m2, and the same observation for plasma dGuo versus dose, 80 mg/m2 was judged as an OBD. The 36 subjects treated at 80 mg/m2 are the main subject of this report. Median age was 61.6 years (range 28.4–81.1) and 67% were males and were exposed to a median of 3 prior systemic therapies (range 0, 8). The ORR using mSWAT was 39% (14/36) with a median duration of response of 127 days (25%–75%, 71 - NA). Response by stage was: IB 3/9, IIA 1/1, IIB 3/5, III 4/12, IVA 2/5, IVB 1/4. Median time to response was 42 days (25%–75%, 29–58). The median time on treatment was 131 days (range 1, 479) with 6 subjects remaining on treatment. For subjects with Sezary Syndrome (n=20, defined by ISCL B2 classification), the ORR by mSWAT was 40%, and 65% by erythroderma score. More than a 50% reduction in Sezary cells (detected by flow cytometry) was observed in 9/20 (45%) subjects with SS. For all 56 forodesine-treated subjects, the only grade 3 or higher non-laboratory adverse events (without regard to attribution and observed in at least 2 subjects) were diarrhea, acute renal failure (not related), cellulitis, and rash (2 subjects each). The only grade 3 or higher related non-laboratory AEs were vertigo, diarrhea, generalized edema, and pneumonia (1 each). For laboratory events, a single grade 3 elevation for each of the following liver-related parameters was noted: AST, ALT, bilirubin, and alkaline phosphatase. There were no grade 3 or higher elevations of creatinine. Grade 3 or higher lymphopenia and low CD4 counts were observed in 71% and 31% of subjects and these rates were similar across dose groups. Median baseline, nadir, and last visit lymphocytes counts (1000/mm3) were 0.8 (95%CI: 0.0, 6.0), 0.2 (95% CI: 0.0, 0.8), and 0.6 (95% CI: 0.0, 2.9) respectively. Hematopoietic toxicity was limited to 1 episode of grade 3 neutropenia, and 1 episode of grade 3 anemia. Conclusion: Oral forodesine demonstrates clinical activity in subjects with refractory CTCL, including those with SS, with minimal toxicity to date.

Forodesine (BCX-1777) is a potent purine nucleoside phosphorylase (PNP) inhibitor. PNP inhibition results in elevation of plasma 2′-deoxyguanosine (dGuo) and intracellular accumulation of deoxyguanosine triphosphate (dGTP), which in turn affects deoxynucleotide-triphosphate pools and induces cell death. It has been reported that forodesine exerts a cytotoxic effect in cells from CLL probably due to high deoxycytidine kinase (dCK) activity in these cells. dCK is the primary enzyme for the conversion of dGuo to dGMP (dGuo monophosphate), which is then converted to dGTP. Several markers such as ZAP-70 and p53 status (17p deletions) identify CLL patients with a different biological and clinical behaviour. High ZAP-70 expression levels are associated with poorer overall survival and shorter time to disease progression, whereas p53 alterations convey drug resistance and short survival. We analyzed the in vitro cytotoxic effect of forodesine in primary cells from 29 patients with CLL, 11 of them carrying 17p deletions. Forodesine (2 μM) and dGuo (10–20 μM) induced apoptosis at 24–48 hours in CLL cells (56.7±14.3% of mean cytotoxicity in respect to control). As per the individual cytotoxic effect, this was higher than 60% in 17 cases (58.6%), 40–60% in 8 cases (27,5%), and lower than 40% in 4 cases (13.8%). No significant differences were observed between CLL cells with low levels of ZAP-70 (11 cases; 61.85±11.2% mean cytotoxicity) and CLL cells with high levels of ZAP-70 (16 cases; 55.7±16.8% mean cytotoxicity). Cases with 17p deletion showed good response to forodesine (11 cases; 58.5± 20% of mean cytoxicity vs. 18 CLL cases with no 17p deletion, 55.2±10.3 of mean cytotoxicity). Next, we analyzed the effect of combination of forodesine with fludarabine (3.75–7.5 μM) or bendamustine (10–25 μM). A significant synergistic effect (Chou Talalay Combination Index CI

Background: Forodesine is a potent, rationally designed purine nucleoside phosphorylase (PNP) inhibitor that has shown activity in acute lymphoblastic leukemia (ALL). Although the drug was thought to be most active in T-cell malignancies, preclinical data and results of a phase I/II trial showed clinical activity in B-lineage ALL (B-ALL). Methods: Twelve patients with relapsed/refractory B-ALL were enrolled (median age of 10 adults, 48.5 yr; median age of 2 children, 10 yr). Three patients had relapsed after allogeneic bone marrow transplantation (BMT), and 9 patients relapsed after a median of two prior regimens. The patients were treated with a 30-minute infusion of forodesine, 80 mg/m2 for 5 days/wk for a 4-week period. If the patient showed clinical improvement at 4 weeks, the drug was continued for additional cycles. Results: Two of 12 patients achieved a complete response. One of these patients had relapsed after allogeneic BMT and restored complete donor chimerism after 16 weeks of forodesine treatment. As restoration of donor chimerism was achieved, the patient developed hepatic graft-versus-host disease (GVHD) and was treated simultaneously with dexamethasone beginning at 4 weeks; this was tapered off and restarted when GVHD again worsened. The second patient failed one prior regimen and had no leukemic blasts in the bone marrow after 4 weeks of treatment. Both these patients continue to receive therapy (5+ and 2+ months respectively). There were no patients with a partial response. Four patients presented with high white blood cell (WBC) counts; each demonstrated an initial decrease in WBCs without an increase in neutrophils while they were receiving forodesine but then developed persistent pancytopenia with residual bone marrow disease at 4 weeks. Dexamethasone was added to the treatment of 3 nonresponders, with no clinical benefit. Three patients with Ph+ B-ALL were treated on the study. Two rapidly progressed on the medication and were taken off the study before the end of 2 weeks; one had persistent pancytopenia and bone pain with residual marrow disease at 4 weeks and was taken off the study. Adverse events included increases in liver transaminase (grade ≥ 2, 2 patients) and alkaline phosphatase levels (grade 2,3, 2 patients), hyperkalemia (grade 2,3, 2 patients), and hypomagnesemia (grade 1, 1 patient). Two patients with increased liver transaminase levels underwent liver biopsies: 1 showed leukemic infiltration and the other GVHD. Patient accrual continues for this study. Conclusions: Forodesine is a safe, well-tolerated drug with preliminary evidence of activity as a single agent in B-ALL.

Background: Forodesine, a potent, rationally designed purine nucleoside phosphorylase inhibitor, has shown clinical activity in T-cell leukemia following IV administration. This phase II study was undertaken to evaluate further the efficacy and safety of IV forodesine in patients with relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL). Methods: In this open-label, single-arm, multicenter, ongoing study, patients received forodesine, 40 mg/m2 IV, 5 days each week (1 cycle) for a total of 6 cycles. The dose was escalated to 90 mg/m2 in patients who were not responding after cycle 2. The primary end point was objective response (OR), which included complete response (CR), CR with incomplete recovery of peripheral blood cell counts (CRi), and partial response (PR). Results: In total, 34 T-ALL patients (median age, 31 yr; range, 3–76 yr) with relapsed/refractory T-ALL (median, 3 prior treatments) received forodesine IV. The OR rate was 32.4% (11 patients; 7 [20.6%] CR, 0 CRi [0%] and 4 [11.8%] PR). Patient demographics and response for the 7 CR patients are shown in the Table. One CR patient has continued to receive treatment for almost 1 year. Two patients with CR proceeded to hematopoietic stem cell transplantation following remission. The restoration of normal hematopoiesis observed in patients while receiving therapy indicates a specificity of forodesine for leukemic cell populations. A total of 18 patients were dose escalated to 90 mg/m2 of forodesine after cycle 2 due to a lack of response. None of these patients demonstrated an improved response with dose escalation. The most frequently reported possibly drug-related AEs without regard to grade were nausea (22%), headache (16%), thrombocytopenia (14%), leukopenia (14%), asthenia (14%), and anemia (11%). Possibly drug-related AEs of grade 3 and higher occurring in ≥2 patients were thrombocytopenia (3 patients; 8%) and leukopenia and lymphopenia (2 patients each; 5%). Conclusions: IV forodesine is effective as a single agent in the treatment of patients with relapsed or refractory T-ALL and is safe and well tolerated. Additionally, 40 mg/m2 has been confirmed as the dose for subsequent phase II development in T-ALL. T-ALL Patients With Complete Response Patient No. No. of Previous Treatments Age/Gender Relapsed/Refractory Weeks of Tx Time to CR Time to Progression(Days) Overall Survival (Days) #Patient died in CR due to a cardiovascular event *Patient discontinued treatment and then progressed †Patient underwent transplantation following remission 1# 9 23/M Relapsed 9 2 weeks 77 77 2 1 60/M Relapsed 31 6 weeks 238 238+ 3* 4 28/F Relapsed 10 2 weeks 119 459+ 4 2 3/F Relapsed 51 2 weeks 398+ 398+ 5† 2 20/F Refractory 7 6 weeks 207 224 6 2 76/M Refractory 12 2 weeks 91 91+ 7† 3 27/M Refractory 6 2 weeks 180+ 180+

Background: Forodesine is a potent, rationally designed purine nucleoside phosphorylase (PNP) inhibitor that elevates plasma 2′-deoxyguanosine (dGuo) and intracellular deoxyguanosine triphosphate (dGTP) levels, leading to T-cell apoptosis. It has shown promising clinical activity in patients with T-cell malignancies. Here we describe our experience with three patients with refractory or relapsed T-cell acute lymphocytic leukemia (T-ALL) who received IV forodesine before or after allogeneic transplant (pts. treated within an ongoing study from BioCryst Pharmaceuticals, Inc, Birmingham, Alabama, USA; ClinicalTrials.gov Identifier: NCT00095381). Methods: Forodesine was given at 40 mg/m2 for 5 days per week (1 cycle) for ≥6 cycles. Patients 1 and 2 received forodesine at 443 and 161 days after allogeneic hematopoietic stem cell transplantation (HSCT), respectively. Patient 3 received forodesine until 4 days before conditioning therapy prior to allogeneic HSCT. Results: Patient 1 with T-ALL was transplanted in the 2nd CR (10/10 HLA-identical sibling donor), after conditioning therapy and developed extensive wide-spread nodal relapse. After 2 weeks of forodesine, he had a very good partial response. The patient developed new-onset chronic graft-versus-host disease (GVHD) of the oral mucosa and lung during treatment. After 6 cycles, treatment was stopped and corticosteroids were given for the GVHD. This patient has been in CR3 (minimal residual disease [MRD]-negative by molecular analysis and complete donor cell chimerism) for >6 months and is no longer receiving immunosuppressives. Patient 2 is a 3-year-old girl with T-ALL who, following two induction failures, proceeded to uncomplicated 6/6 unrelated marrow transplantation in CR1. She had a bone marrow relapse 5 months after transplantation and was started on forodesine on study. After 2 weeks of treatment she achieved a CR, was MRD-negative by flow cytometry, and had 100% donor chimerism. By the middle of week 3 of treatment, she developed GVHD of the liver and was treated with cyclosporine and prednisone. Twice-weekly forodesine was restarted, and all laboratory parameters returned to normal and repeat bone marrow showed continuous CR2. This patient received forodesine twice weekly for 9 additional months and remains in CR with 100% donor chimerism for >12 months (she is no longer receiving forodesine). Patient 3, who had refractory T-ALL and disease progression shortly after relapse therapy with cladribine, cytarabine, and V16, achieved stable remission after 2 weeks of forodesine and completed 6 weeks of treatment. He then received conditioning therapy followed by an HSCT from a 10/10 HLA-identical sibling donor, and remains in CR2. No drug-specific adverse events of grade 2 or higher were seen in these patients. Conclusions: These encouraging experiences in patients with relapsed and refractory T-ALL suggest that forodesine monotherapy can be effective before and after allogeneic HSCT with minimal toxicity and without affecting potential graft-versus-leukemia effects. Forodesine Treatment in Patients before and after Allogeneic HSCT Patient No. Age/Gender Diagnosis HSCT (prior/post forodesine Tx) Response Disease Status 1 28/M T-ALL, relapse Prior PR CR ongoing 215+ days 2 3/F T-ALL, relapse Prior CR CR ongoing 398 + days 3 27/M T-ALL, refractory Post CR CR ongoing 180+ days

Background: Forodesine is a potent, rationally designed purine nucleoside phosphorylase (PNP) inhibitor that is orally bioavailable. IV forodesine has shown clinical activity in patients with cutaneous T-cell lymphoma (CTCL). The objective of this phase I/II study was to evaluate the safety, PK profile, and efficacy of oral forodesine in patients with refractory CTCL. Methods: An open-label dose-escalation study of fordesine, 40 to 320 mg/m2 qd for 4 weeks, was undertaken to evaluate the safety and PK profile of oral forodesine, followed by an investigation of the expansion of the optimal biologic dose (dose with maximum PNP inhibition and elevation of plasma deoxyguanosine levels) to assess efficacy. Previously treated, refractory CTCL patients with stage IB or greater disease were eligible for participation. The primary end point was objective response (OR) = complete response [CR] plus partial response [PR], as measured by the severity-weighted assessment tool (SWAT; see Figure) and physicians’ global assessment (PGA) Results: Overall, 37 patients were treated, including 14 patients in the dose-escalation portion of the study. No dose-limiting toxicities were observed with target doses of ≤320 mg/m2; thus, a maximum tolerated dose was never defined. Based on PK/PD results, an 80 mg/m2 qd dose was identified as the optimal biologic dose, and this ongoing study was expanded to enroll a total of 28 patients (median age, 64 yr; range, 28–81 yr). The OR rate was 53.6% (15/28 patients; 2 [7.1%] with a CR and 13 [46.4%] with a PR). The proportion of patients with stage IIB or greater disease was 19/28 (67.9%). Response in this population was seen in 10 (52.6%) of the 19 patients (1 [5.3%] with a CR and 9 [47.4%] with a PR). The percent change in SWAT score from baseline as a function of time demonstrated a progressive decrease over a 24-week period. To-date, 12 (42.9%) of 28 patients have completed ≥4 months of treatment. Safety data were recorded for all 37 patients treated with forodesine. The most common adverse events classified as grade 2 or less, without regard to causality, were nausea (30%), dizziness (22%), pruritus (22%), fatigue (19%), headache (19%), peripheral edema (19%), and pyrexia (16%). The only adverse event classified as grade 3 or greater, without regard to causality, and occurring in at least two patients, was lymphopenia, which was observed in two patients (5%). Conclusions: Oral forodesine is effective in the treatment of refractory CTCL patients and has an encouraging safety profile. SWAT SCORE: Percent Change from Baseline SWAT SCORE: Percent Change from Baseline

Suppression of T-cell immunity seen in children with inherited purine nucleoside phosphorylase (PNP) deficiency supports the potential application of PNP inhibitors for the therapy of T-cell malignancies. Forodesine (FodosineTM) is a specific PNP inhibitor that raises plasma 2′-deoxyguanosine (dGuo) and intracellular dGTP levels, leading to T-cell apoptosis. IV administration of forodesine has shown activity in cutaneous T-cell lymphoma (CTCL) patients. To determine the safety, pharmacokinetics and pharmacodynamics of oral forodesine in patients with refractory CTCL, as well as preliminary evidence of efficacy, five cohorts of patients (≥ 3patients per cohort) were evaluated sequentially, receiving forodesine at 40, 80, 160, 320, and 480 mg/m2 once per day for 28 days. At the end of the treatment cycle (Day 28), all patients with stable or improved disease could enter a long-term, follow-up period. To date, 12 patients have been treated, 11 completing the 28-day cycle. Forodesine is orally absorbed (40%–50%), mean terminal half-life of 12 to 20 h. dGuo levels were elevated in all patients (0.8–2.8 μM, predose levels ≤ 0.004 μM). Forodesine was generally safe and well tolerated in this population. Safety data is available for first 7 patients. No serious adverse events possibly related to the drug occurred. The most common adverse event possibly drug related was nausea (2 patients). Of the 11 patients completing the treatment cycle, 9 began long-term treatment. Of these 9 patients, 4 have received >3 months of treatment, while 1 patient continues to receive for >8 months. Efficacy data are available for 8 patients: 2 with partial response, 3 with minor response, 2 with progressive disease, and 1 with stable disease (Table). Forodesine showed preliminary evidence of clinical activity in refractory CTCL patients. Additional clinical and pharmacodynamic data will be presented. Clinical and Pharmacodynamic Activity of Oral Forodesine In CTCL Patients Patient Diagnosis Dose (mg/m2) Treatment Plasma dGuo (Cmax, M) μ Clinical Response PR = partial response; MR = minor response; SD = stable disease; PD = progressive disease; N/A = not available 2001 Sezary syndrome (IVA) 40 Completed 0.6 PR 3001 Mycosis fungoides (IVB) 40 Completed 1.7 PD 3002 Mycosis fungoides (IIB) 40 Completed 1.2 MR 3003 Mycosis fungoides (IB) 80 Completed 1.2 MR 3004 Sezary syndrome (IVA) 80 Completed 1.0 MR 3005 Sezary syndrome 80 Completed 1.6 PD 2002 IIA 80 Completed 1.1 PR 2003 III 80 Completed 1.7 N/A 3006 Mycosis fungoides/Sezary syndrome (IVB) 160 Completed 2.8 SD 4001 III 320 Completed 1.8 N/A 1001 Mycosis fungoides (IB) 320 Completed 1.3 N/A 3012 Mycosis fungoides/Sezary syndrome (IVA vs B) 320 Not Completed N/A Not evaluable

Inherited deficiency of purine nucleoside phosphorylase (PNP) is associated with a profound loss of T-cells. This rare clinical condition provides a model for developing specific inhibitors of PNP as therapy for T-cell malignancies. Forodesine is a specific PNP inhibitor that raises plasma 2′-deoxyguanosine (dGuo) and intracellular dGTP levels, leading to T-cell apoptosis. The objective of this study was to determine the efficacy and safety of forodesine in patients with relapsed or refractory T-cell leukemia. Patients received IV forodesine 40 mg/m2 5 days each week (1 cycle) for a total of 6 cycles (8 cycles if dose escalation needed after cycle 2). To date, 21 patients have been treated, 19 of which are T-ALL. Results are based on available data from 13 T-ALL patients. Median number of prior regimens was 4 (range: 1–6). Overall response rate was 38%, including 3 patients with complete response (CR) (23%) and 2 patients with partial response (PR) (15%). Three patients had stable disease and 5 patients had disease progression. Time to progression (TTP) for the 3 CR patients was 80, 119, and 160+ days. One CR patient continues to receive treatment. TTP for the 2 PR patients was 89 and 40 days. Plasma dGuo was elevated in all patients (range: 3.4 to 88.5 μM; predose levels ≤0.004 μM). Treatment was well tolerated. One serious adverse event possibly drug related was CMV pneumonitis in 1 patient. The most common adverse events possibly drug related were nausea (33%), headache (26%), anemia (20%), thrombocytopenia (20%), and weakness (20%). In 1 patient, after 2 doses of forodesine, WBC count dropped dramatically, requiring interruption of treatment for 2 weeks because of tumor lysis syndrome (Figure). After 2 cycles of treatment, the patient achieved a CR. By week 3, platelets and neutrophils recovered, indicating a specificity of forodesine for leukemic cell populations. These study results show that forodesine is active, with minimal toxicity, as a single agent in relapsed or refractory T-cell leukemia. Additional clinical efficacy and safety data will be presented. Effect of Forodesine on Blood Components in One T-All Patient Effect of Forodesine on Blood Components in One T-All Patient

Forodesine is a potent, specific transition-state analog inhibitor of PNP with clinical activity in T-cell malignancies. Pharmacodynamic studies support that this anti-leukemic effect is mediated through the accumulation of plasma 2′-deoxyguanosine (dGuo) and intracellular Dgtp. In vitro studies indicated that B-cell acute lymphoblastic leukemia (B-ALL) cells can also accumulate Dgtp. These preclinical data led to a phase I/II multicenter dose-escalation study to evaluate forodesine in pts with various hematologic malignancies. 15 pts were treated with forodesine, including 6 with B-ALL. Forodesine was given via a 30-min IV infusion followed 24 hrs later by doses every 12 hrs for a total of 9 doses. A second course could be repeated after a 2-week break. Doses were escalated by 50% to pts grouped in cohorts of 3 (starting dose: 40 mg/m2). There was a rapid rise in plasma dGuo and a maximum PNP inhibition was achieved at 40 mg/m2 (Fig 1). 7/15 treated pts (2 T-cell malignancies and 5 B-ALL), including 5/6 treated B-ALL pts, demonstrated a hematologic benefit, defined as a decrease in tumor burden. 3 of the responding B-ALL pts were further treated with 6 courses of forodesine on a compassionate use protocol. One pt treated at 135 mg/m2 demonstrated a complete response with a decrease in bone marrow blast cells from 22% to 5% at the end of therapy. The other 2 pts, treated with 90 mg/m2 and 135 mg/m2 forodesine respectively, showed dramatic improvement in their CBC despite the persistence of bone marrow blasts. The dramatic fall in WBC count was accompanied in each pt by a rise in plasma dGuo (Cmax: 1.9–7.1 μM) and intracellular Dgtp (380–800 pmoles/107 cells vs 25–40 pmoles/107 cells pre-treatment), The response of the pt treated with 90 mg/m2 forodesine is shown in Fig 2. The absolute neutrophil count (ANC) improved from ~0 at pre-treatment to 1800 cells/mm3 at Day 18. To-date forodesine has been safe and well-tolerated at all dose levels tested with no dose-limiting toxicities. Clinical activity has been seen in T- and B-ALL. It is interesting to note the restoration of normal hematopoiesis, indicating the specificity of forodesine for leukemic cell populations and supporting that forodesine represents an important breakthrough in the development of less toxic ALL therapy. This encouraging clinical activity has led to the initiation of a phase II trial in pts with B-ALL. Additional clinical and pharmacodynamic results will be presented.