Purine Nucleoside Phosphorylase Inhibition as a Novel Therapeutic Approach for B-Cell Lymphoid Malignancies

Purine Nucleoside Phosphorylase Inhibition as a Novel Therapeutic Approach for B-Cell Lymphoid Malignancies Richard R. Furmana and Dieter Hoelzerb Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of ribonucleosides and 2=-deoxyribonucleosides to their respective bases. Endogenous PNP deficiency leads to specific T-cell immunodeficiency, a genetic disease that has prompted the development of PNP inhibitors as potential therapies for T-cell–mediated diseases. PNP inhibition leads to the elevation of 2=-deoxyguanosine levels and accumulation of intracellular deoxyguanosine 5=-triphosphate, inducing cellular apoptosis. Forodesine is a highly potent, orally active, rationally designed PNP inhibitor that has shown activity in preclinical studies with malignant cells and clinical utility against T-cell acute lymphoblastic leukemia and cutaneous T-cell lymphoma. Additional preliminary findings support its use for the management of some B-cell malignancies. Semin Oncol 34(Suppl 5):S29-S34 © 2007 Elsevier Inc. All rights reserved.

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n the purine salvage pathway, purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine nucleosides to their corresponding bases and ribose1-phosphate sugar. There are approximately 40 individuals described with an inherited deficiency of PNP; they show a selective depletion of T cells to levels between 1% and 3% of normal.1 This observation led investigators to speculate that PNP inhibitors might show promise as treatments for T-cell malignancies and T-cell–mediated diseases. Several 9-benzyl-9-deazaguanine–related compounds have been developed as potential PNP inhibitors.2 One such agent, forodesine, has been shown to be a highly potent, orally bioavailable PNP inhibitor with in vitro and clinical activity against T-cell malignancies.3 We review here the mechanism of action of forodesine, present an overview of its effects on malignant cells in preclinical models, and describe initial clinical trial results to date in patients with hematologic malignancies. Preliminary results support the use of forodesine for the treatment of patients with certain T- and B-cell malignancies.

aCLL

Research Center, and the Center for Lymphoma and Myeloma, Weill Cornell Medical Center, New York, NY. bKlinklum der JW Goethe Universitat, Frankfurt, Germany. Address reprint requests to Richard R. Furman, MD, Division of Hematology and Oncology, Weill Cornell Medical College, 525 East 68th St, New York, NY 10021.

0093-7754/07/$-see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1053/j.seminoncol.2007.11.004

Preclinical Studies and Mechanism of Action Under normal physiologic conditions, plasma 2=-deoxyguanosine (dGuo) levels are very low to undetectable (ⱕ0.004 ␮mol/L). dGuo does not accumulate because it is rapidly metabolized by PNP into guanine and dexoyribose1-phosphate. In inherited PNP deficiency, or the presence of a PNP inhibitor, plasma dGuo levels rise to 2 to 15 ␮mol/L. dGuo is shunted through an alternative pathway initiated by deoxycytidine kinase (dCK) and metabolized into deoxyguanosine 5=-triphosphate (dGTP; Fig 1). The accumulation of dGTP results in an imbalance of the cellular deoxynucleoside triphosphate (dNTP) pools and induces cell apoptosis. This process is most pronounced in cells that possess high levels of dCK and low levels of 5=-nucleotidase, the enzyme that catalyzes the reverse reaction of dCK, removing the phosphate from deoxyguanosine monophosphate. The accumulation of dGTP occurs only in the absence of PNP activity because PNP possesses a much higher affinity for dGuo than dCK. Consistent with this mechanism, T cells are the predominantly affected cell type in the absence of PNP activity, as they are characterized by a relatively high level of dCK and low levels of 5=-nucleotidase activity.4 Assessments of individuals with PNP deficiency and in vitro studies indicate that PNP inhibition has to be virtually complete to result in an elevation of plasma dGuo and intracellular dGTP and subsequent cell apoptosis.4 This is demonstrated by individuals S29

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bine, cytarabine, clofarabine). First, only forodesine and pentostatin, an adenosine deaminase inhibitor, are transition-state analogues. As a result of the great specificity for their target enzymes, they are able to achieve sufficient inhibition of their target enzymes at lower concentrations, increasing their therapeutic window. Second, forodesine is not phosphorylated intracellularly. As a result, forodesine does not become integrated into newly synthesized DNA, which results in termination of DNA chain synthesis. This property enhances the cell specificity of forodesine as an anti-proliferative agent and minimizes myelosuppression. Several strategies are being considered to enhance the effectiveness of forodesine. Combination therapy with other agents, such as corticosteroids, cyclophosphamide, and other nucleoside analogues, may, by the actions of these drugs, increase the imbalance in dNTP pools and further promote apoptosis. Alternatively, cell death caused by cytotoxic agents may increase intracellular dGuo, further enhancing the generation of intracellular dGTP. Forodesine has been studied in vitro in freshly isolated malignant cells and several cancer cell lines.4 Given that chronic lymphocytic leukemia (CLL) B cells are known to possess high dCK activity,5 Balakrishnan et al6 investigated forodesine in vitro with leukemia cells isolated from 12 patients with CLL. Incubating lymphocytes with forodesine and dGuo led to an accumulation of intracellular dGTP. No effect on any other deoxynucleotides was seen (Fig 3). Most importantly, the dGTP accumulation led to p53 stabilization and p21 activation in the leukemia cells, followed by the induction of apoptosis, demonstrated by poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) cleavage, loss of mitochondrial membrane potential, and caspase activation. The caspase activation was most pronounced in the samples that showed the greatest increase in dGTP levels.

Figure 1 Mechanism of T-cell inhibition by inhibition of PNP. dCK, deoxycytidine kinase; dGMP, 2=-deoxyguanosine monophosphate; dGTP, deoxyguanosine 5=-triphosphate; dGuo, 2=-deoxyguanosine; dNTP, deoxynucleoside triphosphate; PNP, purine nucleoside phosphorylase.

with T-cell lymphopenia secondary to PNP deficiency possessing PNP activity levels of ⬍5% of normal.1 The parents of these individuals often possess PNP activity levels of 30% to 50% of normal and demonstrate no elevation of dGuo levels or T-cell lymphopenia. In vivo, normal cell turnover is the source of dGuo. In conditions with high cell turnover, such as acute leukemia, the rate of dGuo production is considerably higher. For in vitro studies, exogenous dGuo must be added to the system. Forodesine is a potent, transition-state analogue inhibitor of PNP. Transition-state inhibitors show higher affinity and specificity for their target enzymes than non–transition-state analogues. Structurally, forodesine belongs to the class of nucleoside analogues (Fig 2). Two important features distinguish the pharmacology of forodesine from the other nucleoside analogues (fludarabine, cladribine, pentostatin, nelara-

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Figure 2 Chemical structures of the purine nucleoside analogues.

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Phase I studies. In a phase I dose-escalation trial, 15 patients with relapsed or refractory hematologic malignancies were treated with forodesine.7 Forodesine was administered intravenously (IV) over 30 minutes, followed by a second dose 24 hours later, and then seven subsequent doses given every 12 hours, for a total of nine doses over 5 days. A second course of therapy could be given after a 2-week break. The first dose level of forodesine tested was 40 mg/m2; this was escalated by 50% to 60, 90, and 135 mg/m2 for subsequent cohorts of three patients each. PNP was sufficiently inhibited at the lowest dose level as demonstrated by elevated levels of plasma dGuo (Fig 4). Increasing the dose to 60 mg/m2 did not result in any additional inhibition of PNP or further increases in dGuo levels, a result that was expected based on studies of PNP-deficient individuals.1 Forodesine was remarkably well tolerated in all 15 patients, without the occurrence of any dose-limiting toxicities. Six of the 15 patients showed clinical benefit, including four of the five patients with B-cell acute lymphoblastic leukemia (B-ALL). The data from these patients with B-ALL indicate that forodesine has activity against B-ALL. All five B-ALL patients showed decreased numbers of their peripheral blood leukemic cells from approximately day 3 of treatment through the

first week off therapy. The leukemic cells quickly returned to the peripheral blood thereafter. Based on these observations and pharmacokinetic data that showed a serum half-life for forodesine of 8 hours and a half-life of the drug-enzyme complex of 5 days, it was apparent that once-daily dosing of a longer duration would be required for maximal benefit. Three of the five responding patients with B-ALL continued therapy with once-daily dosing of forodesine for an additional 6 weeks on a compassionate-use protocol. All three patients completed the 6 weeks of therapy without any significant toxicity. One patient, treated with forodesine at 135 mg/m2, experienced a complete response (CR) at the end of treatment. The two other patients, who were treated with forodesine 90 and 135 mg/m2, showed clearance of peripheral blood blasts and recovery of normal hematopoiesis, but had persistent numbers of blast cells in the bone marrow. In all patients, plasma dGuo levels rose, as

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Clinical Trials

Figure 4 Inhibition of PNP and subsequent generation of high plasma dGuo levels following the administration of forodesine 40 mg/m2. dGuo, 2=-deoxyguanosine; PNP, purine nucleoside phosphorylase. (Reprinted with permission from Blood [Furman et al].7)

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Figure 3 Accumulation of intracellular 5=-dNTP levels following the incubation of lymphocytes together with forodesine and dGuo. Primary CLL cells from 11 patients were incubated with 2 ␮mol/L forodesine and 10 ␮mol/L dGuo at 4 and 8 hours. The nucleotides in the leukemia cells were extracted by 60% methanol and the dNTPs were measured by DNA polymerase assay. The data for accumulation of the four dNTPs are plotted. dATP, deoxyadenosine triphosphate; dCTP, deoxycytidine triphosphate; dGTP, 2=-deoxyguanosine triphosphate; dNTP, deoxynucleoside triphosphate; dTTP, deoxythymidine triphosphate. (This research was originally published in Blood. Balakrishnan, K, et al. Forodesine, an inhibitor of purine nucleoside phosphorylase, induces apoptosis in chronic lymphocytic leukemia cells. Blood 2006;108:2392-2398. © the American Society of Hematology.)

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Figure 5 Pharmacodynamic effects following the administration of forodesine 90 mg/m2. dGTP, deoxyguanosine 5=-triphosphate; dGuo, 2=-deoxyguanosine; WBC, white blood cell.

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Patients treated, n Patients on whom data were available Gender, male/female Age, median, yr (range) Prior therapies, median, n (range)

20 17 13/7 31 (6–81) 4 (1–19)

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did intracellular dGTP levels, coincident with the decrease in circulating white blood cells. An example of one patient who demonstrated this is shown (Fig 5). The choice of adding dexamethasone to the regimens of patients not responding to single-agent forodesine was also based on previous clinical experience. One patient in the phase I trial developed bilateral tonsillar abscesses on day 10 of treatment with single-agent forodesine. This patient had not yet shown a reduction in the number of circulating leukemia cells. Dexamethasone 10 mg twice daily was initiated for the tonsillar abscesses to protect the patient’s airway. Although the patient had not responded to previous treatment with high-dose dexamethasone therapy, by day 3 of combination therapy the patient cleared all of the peripheral blood blasts. In summary, this phase I trial showed that forodesine was well tolerated and safe and that there were no dose-limiting toxicities identified at the doses studied. Additionally, forodesine did not impair normal granulocytic, erythrocytic, and megakaryocytic hematopoiesis. The therapeutic dose was identified to be 40 mg/m2 administered IV once daily, based on the biological endpoint of dGuo elevation, well below the maximum dose tested. Phase II B-cell studies. There are currently four ongoing phase II clinical trials investigating the efficacy of forodesine in patients with T-cell ALL (T-ALL), B-ALL, CLL, and cutaneous T-cell lymphoma. This section will discuss the B-ALL studies. (The T-ALL and cutaneous T-cell lymphoma clinical trials are discussed elsewhere in this supplement.) Based on the benefits seen with forodesine treatment in the patients with B-ALL in the phase I study, a multicenter, phase II study of forodesine was conducted in 20 patients with relapsed or refractory B-ALL (Table 1).8 Patients received a single daily infusion of forodesine 80 mg/m2 for 5 consecutive days every week for 4 weeks. Patients showing clinical benefit were eligible to continue with additional cycles of forodesine. Dexamethasone 20 mg/day was added to the regimens of patients not achieving a response by week 4 of forodesine therapy. The dose of 80 mg/m2 was chosen for the B-ALL study because of concerns that patients with B-ALL might require a higher dose of forodesine than that used in patients with T-ALL, given the differences in the inherent sensitivities between B and T cells to alterations in nucleotide metabolism. Interim results are available for 17 patients, 11 of whom have completed the first 4-week treatment cycle. The peripheral blood blast count was elevated at baseline in six of 17 patients. All six patients showed a decrease of ⱖ50% in pe-

ripheral blood blast counts. Two of 17 patients achieved CR (no evidence of circulating blasts or extramedullary disease, M1 bone marrow with ⱕ5% blasts, and recovery of peripheral blood cell counts). One of these patients had not achieved a CR after six induction regimens and underwent a sibling allogeneic transplant but relapsed within 90 days of transplantation. Forodesine was initiated and blood cell counts began to normalize within the first month of treatment. During the fourth week of forodesine therapy, the patient developed markedly abnormal liver function tests. A liver biopsy showed graft-versus-host disease, for which high-dose dexamethasone was initiated, followed by tacrolimus and sirolimus. The liver function tests normalized, and, after 3 months of forodesine, the patient demonstrated normal blood and bone marrow and 100% donor chimerism. The patient subsequently did well, but the B-ALL ultimately relapsed 8 months later while receiving forodesine after requiring additional therapy for graft-versus-host disease. The second patient, who had relapsed after one prior therapy, achieved a CR after 4 weeks of forodesine therapy. He continues to receive forodesine with no evidence of residual leukemia, but he does have myelodysplasia, which had been present before the first relapse and which necessitates twice-monthly transfusions. After several months of receiving IV forodesine both patients were switched to the oral formulation. Plasma dGuo levels were determined for nine patients. Forodesine treatment resulted in increases in dGuo levels in all patients, increasing from ⱕ0.004 ␮mol/L before treatment to a median of 7.5 ␮mol/L (range, 2.3 to 45.0 ␮mol/L) with forodesine treatment. Leukemic cells from seven patients with circulating blasts were incubated ex vivo with forodesine (1 ␮mol/L) and dGuo (10 ␮mol/L). Forodesine led to an elevation of dGTP in all cases, with a median fold increase of 17.2 (range, 2.7 to 60.0 fold; Fig 6).8 It is interesting to note the uniformity of dGTP elevation in the leukemic cells ex vivo, which did not correspond to responses in

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Table 1 Demographic Variables From the Study by Ritchie et al of Patients With Relapsed or Refractory B-ALL

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Figure 6 Elevation of dGTP levels following the ex vivo incubation of B-ALL cells with forodesine and dGuo from seven patients with circulating blasts. Intracellular dGTP levels increased by a median of 17.2 fold over baseline. dGTP, deoxyguanosine 5=-triphosphate; dGuo, 2=-deoxyguanosine. (Data from Ritchie et al.8)

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Figure 7 Protocol being developed by the EWALL for the evaluation of new drugs against Philadelphia chromosome– negative ALL in elderly patients. R, randomization; cons, consolidation; IV, intravenous.

all of those patients. It will be important to determine if the lack of in vivo responses in all patients was the result of a failure of dGTP to become elevated in vivo in B-ALL or if the elevation of dGTP was not sufficient to induce apoptosis of B-ALL cells. Overall, forodesine was safe and well tolerated; most adverse events were grade I or II. Adverse events occurring in ⱖ 20% of patients, without regard to grade or causality, were insomnia (six patients; 30%), nausea (six patients; 30%), arthralgia (five patients; 25%), cough (five patients; 25%), diarrhea (five patients; 25%), fatigue (five patients; 25%), headache (five patients; 25%), pruritus (five patients; 25%), pyrexia (five patients; 25%), asthenia (four patients; 20%), constipation (four patients; 20%), febrile neutropenia (four patients; 20%), hypotension (four patients; 20%), and peripheral edema (four patients; 20%).8 An interim analysis of this phase II trial showed that forodesine treatment was well tolerated and that forodesine may show clinical activity as a single agent against B-ALL. Furthermore, the ex vivo accumulation of dGTP by leukemic cells in the presence of forodesine and dGuo supports the idea of using forodesine as therapy for B-ALL. Based on this preliminary evidence of clinical activity of forodesine in B-cell disease, combined with the previously discussed in vitro studies showing a marked accumulation of dGTP and induction of apoptosis in CLL cells treated with forodesine plus dGuo, a phase II study of oral forodesine has been initiated in patients with CLL.

Future Directions Additional studies of forodesine in patients with B-cell malignancies are clearly warranted. Currently, the European Working Group for Adult ALL (EWALL) is developing a unified protocol for evaluating new drugs against elderly patients with Philadelphia chromosome–negative ALL (Fig 7).

The study will involve 30 patients in each arm randomized to receive standard chemotherapy; standard chemotherapy plus forodesine 160 mg/m2 IV, followed by forodesine 300 mg/ day orally; or standard chemotherapy plus forodesine 270 mg/m2 IV, followed by oral forodesine 300 mg twice daily. A second strategy is being developed by Ritchie et al,8 focusing on combining forodesine with agents that are better tolerated, but still effective, for elderly patients with B-ALL. This study involves the combination of vincristine, prednisone, and forodesine at a dose of 300 mg/day orally. These patients are unable to tolerate aggressive consolidation secondary to their age and performance status. This study builds on the observation that it is possible to induce remissions of short duration in elderly patients with ALL using vincristine plus prednisone. As a single agent, forodesine appears to control B-ALL proliferation better than it induces apoptosis. The hope is that this combination will induce remissions that will be maintained with continued forodesine therapy. Despite promising advances in drug development and collaborative efforts of investigators to implement well-designed clinical trials, many issues remain to be resolved. One of the most important relates to evaluating molecularly targeted therapies with novel mechanisms of action. These agents might produce clinical benefit without yielding responses based on the current criteria. While forodesine is clearly able to induce CRs in patients with T-ALL, its role in B-ALL is less clear. Drugs such as forodesine, which might induce “stasis” of leukemia, could prolong survival and improve quality of life without necessarily yielding a response. Our current methodologies and regulatory requirements do not lend themselves to the easy development of these types of drugs. Thus, with regard to the development of forodesine for B-cell malignancies, the identification of the optimal end points, the significance of molecular CRs, the role of stem cell transplantation, and the inclusion of subgroups of patients with ALL

R.R. Furman and D. Hoelzer

S34 (B-ALL, T-ALL, Philadelphia chromosome–positive ALL) all deserve thoughtful discussion and analysis. Many questions remain to be investigated for an optimal understanding of the full potential of forodesine. In vitro analyses may be performed to assess the sensitivity of primary ALL cells to forodesine and whether forodesine shows synergistic or additive effects when combined with other agents. Resistance mechanisms can thus be elucidated and, perhaps, ultimately circumvented. Further, it is possible that findings from studies with forodesine and B-ALL may extend to other B-cell malignancies. With its tolerability, forodesine may also be of great use as maintenance therapy, either post-ALL induction or even following bone marrow transplantation.

Summary and Conclusion PNP is the enzyme responsible for the reversible phosphorolysis of ribonucleosides and 2=-deoxyribonucleosides to generate the corresponding bases and ribose-1-phosphate or 2=deoxyribose-1-phosphate. Inherited PNP deficiency leads to T-cell immunodeficiency, suggesting a role for PNP inhibition in T-cell malignancies. The mechanism by which PNP inhibition exerts its anti-cancer effect includes the elevation of dGuo levels and subsequent accumulation of intracellular dGTP, leading to the induction of cellular apoptosis. Forodesine is a highly potent, orally active, rationally designed 9-benzyl-9-deazaguanine compound that inhibits PNP. As predicted by the PNP-deficient patients, forodesine has shown clinical utility against T-cell malignancies. More unexpectedly, preliminary preclinical and clinical findings support its use for the management of some B-cell malignancies. CLL and B-ALL represent the two B-cell malignancies cur-

rently being investigated in phase II studies. While there were patients with B-cell malignancies treated as part of the phase I study, these patients received a very different dosing schedule of forodesine than what is currently being evaluated. Therefore, the full potential of forodesine as a treatment for patients with these other B-cell malignancies is not yet known. Ongoing and planned investigations are seeking to refine the optimal protocol by which PNP inhibition by forodesine can be used as an easily tolerated, safe treatment option for patients with B-cell malignancies.

References 1. Markert ML: Purine nucleoside phosphorylase deficiency. Immunodef Rev 3:45-81, 1991 2. Montgomery JA: Inhibitors of purine nucleoside phosphorylase. Exp Opin Invest Drugs 3:1303-1313, 1994 3. Bantia S, Miller PJ, Parker CD, et al: Purine nucleoside phosphorylase inhibitor BCX-1777 (Immucillin-H)—A novel potent and orally active immunosuppressive agent. Int Immunopharmacol 1:1199-1210, 2001 4. Bantia S, Kilpatrick JM: Purine nucleoside phosphorylase inhibitors in T-cell malignancies. Curr Opin Drug Discov Devel 7:243-247, 2004 5. Kawasaki H, Carrera CJ, Piro LD, et al: Relationship of deoxycytidine kinase and cytoplasmic 5=-nucleotidase to the chemotherapeutic efficacy of 2-chlorodeoxyadenosine. Blood 81:597-601, 1993 6. Balakrishnan K, Nimmanapalli R, Ravandi F, et al: Forodesine, an inhibitor of purine nucleoside phosphorylase, induces apoptosis in chronic lymphocytic leukemia cells. Blood 108:2392-2398, 2006 7. Furman RR, Gandhi VV, Bennett JC, et al: Intravenous forodesine (BCX1777), a novel purine nucleoside phosphorylase (PNP) inhibitor, demonstrates clinical activity in phase I/II studies in patients with B-cell acute lymphoblastic leukemia. Blood 104(suppl 1):750a, 2004 (abstr 2743) 8. Ritchie E, Gore L, Roboz G, et al: Phase II study of forodesine, a PNP inhibitor, in patients with relapsed or refractory B-lineage acute lymphoblastic leukemia. Poster presented at the American Society of Hematology 48th Annual Meeting, Orlando, FL, December 2006.