Purine nucleoside analogues for the treatment of hematological malignancies: pharmacology and clinical applications

The purine nucleoside analogues (PNAs), fludarabine (FA), 2-CdA (2-chlorodeoxyadenosine, 2-CdA) and pentostatin (2'-deoxycoformycin, DCF) represent a group of cytotoxic agents with high activity in lymphoid and myeloid malignancies. PNAs share similar chemical structure and mechanism of action. Several mechanisms could be responsible for their cytotoxicity both in proliferating and quiescent cells, such as inhibition of DNA synthesis, inhibition of DNA repair and accumulation of DNA strand breaks. Induction of apoptosis through the mitochondrial pathway, direct binding to apoptosome or modulation of p53 expression all lead to apoptosis, which is the main end-point of PNA action. However, individual PNAs exhibit significant differences, especially in their interaction with enzymes involved in adenosine and deoxyadenosine metabolism. Synergistic interactions between PNAs and other cytotoxic agents (alkylating agents, anthracycline antitumor antibiotics, cytarabine, monoclonal antibodies) have been demonstrated in both preclinical and clinical studies. PNAs are highly effective in chronic lymphoid leukemias and low grade B- and T-cell non-Hodgkin's lymphomas, including Waldenstr?m's macroglobulinemia. DCF and 2-CdA are currently the drugs of choice in hairy cell leukemia. Moreover, clinical studies have confirmed the efficacy of PNAs alone or in combination protocols in the treatment of acute myeloid leukemia and myelodysplastic syndromes. Finally, PNAs, especially FA, play an important role in non-myeloablative conditioning regimens for allogenic stem cell transplantation in high-risk patients. The toxicity profiles of PNAs are similar for all agents and consist mainly of dose-limiting myelotoxicity and prolonged immunosuppression. Three other compounds: clofarabine, nelarabine and immucillin-H are currently being evaluated clinically.     

Purine nucleoside analogs in the treatment of rarer chronic lymphoid leukemias

Purine nucleoside analogues (PNA) are the cytotoxic agents highly active in the treatment of indolent lymphoid malignancies. These drugs have chemical structure similar to adenosine or deoxyadenosine. PNAs are characterized by a similar mechanism of cytotoxicity both in proliferating and quiescent cells, such as inhibition of DNA synthesis, inhibition of DNA repair and accumulation of DNA strand breaks. In addition, PNAs induce apoptosis which is the end-point of their action. Older PNAs, pentostatin (DCF; 2'- deoxycoformycin), cladribine (2-CdA; 2-chloro-2'-deoxyadenosine) and fludarabine (2-fluoro-9-(-D-arabinosyl)-adenine) were approved by Food and Drug Administration (FDA) for the treatment of hematological malignancies. In addition three novel PNAs: clofarabine (CAFdA), nelarabine (ara-G) and forodesine (immucillin H, BCX-1777) have been synthesized and introduced into preclinical studies and clinical trials. This review summarizes current knowledge on the mechanism of action and pharmacokinetic properties of older and new PNAs. Clinical activity and toxicity of PNAs, especially in hairy cell leukemia (HCL), hairy cell leukemia variant (HCL-V), prolymphocytic leukemia (PLL) and other rarer chronic lymphoid leukemias, are also presented. 2-CdA and DCF, introduced in the 1980s, changed radically the treatment modality, inducing complete and durable responses in the majority of patients with HCL. In contrast, the results of the treatment of HCL-V with PNA are rather poor. There are also several reports indicating activity of PNAs in PLL and large granular lymphocyte leukemia. Clofarabine, nelarabine and forodesine need further investigation in rarer lymphid leukemias, to better define their status in the treatment of these disorders.     

AT-9283, a small-molecule multi-targeted kinase inhibitor for the potential treatment of cancer

AT-9283 has been identified and developed by Astex Therapeutics via structure-based optimization of a ligand-efficient pyrazole-benzimidazole fragment. AT-9283 inhibits several important kinases, including the Aurora kinase A, Aurora kinase B, Janus kinase (Jak)2, Jak3 and Abl kinase. Studies using multiple solid tumor and leukemia cell lines have demonstrated the ability of AT-9283 to inhibit growth and survival of tumor cells, and the direct inhibition of these kinases has been demonstrated in cell-based systems. The in vivo antitumor activity of AT-9283 has also been demonstrated in human tumor xenograft models. Based on these preclinical studies, several clinical trials have been conducted in patients with hematological malignancies, such as leukemias, myelodysplastic syndrome, myeloproliferative disease, chronic myeloid leukemia, lymphomas and multiple myeloma, and also in patients with solid tumors. Although phase II clinical trials have not been completed, AT-9283 demonstrated good safety and efficacy in phase I clinical trials. Thus, AT-9283 has potential as a therapeutic agent in several patient populations through its different inhibitory activities.     

CAL-101: a phosphatidylinositol-3-kinase p110-delta inhibitor for the treatment of lymphoid malignancies

INTRODUCTION: The management of lymphoid malignancies has greatly evolved in the last decade with the advent of targeted therapies, which have improved response and survival in patients with Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL) and plasma cell myeloma (PCM). The PI3K pathway seems to play a seminal role in the development of lymphoid malignancies. CAL-101 is a highly selective PI3K p110δ inhibitor currently undergoing clinical development. AREAS COVERED: The aims of this review are to summarize our understanding of the PI3K pathway, its role in lymphoid malignancies, the preclinical and clinical experience accumulated with CAL-101, a PI3Kδ inhibitor, and potential areas of future development. EXPERT OPINION: CAL-101 is a novel drug that has shown preclinical activity against CLL, NHL, HL and PCM cells. There is early evidence of clinical efficacy in CLL and indolent NHL. Studies using CAL-101 alone or in combination are also ongoing in PCM, HL and aggressive NHL. However, additional studies are needed to prove CAL-101 is effective and safe as the goals of therapy for patients with lymphoid neoplasms are not only directed towards improving response and cure rates but also prolonging survival without affecting quality of life.     

PCI-32765: a novel Bruton's tyrosine kinase inhibitor for the treatment of lymphoid malignancies

INTRODUCTION: There has been a significant paradigm shift in the manner in which lymphoid malignancies are treated and managed. Treatment has been moving away from conventional chemotherapy and towards targeted therapy. The success of new classes of agents such as monoclonal antibodies, proteasome inhibitors and immunomodulatory derivatives has sparked further searches for novel pathways to inhibit. The Bruton's tyrosine kinase (Btk) pathway is a downstream mediator of the B-cell receptor (BCR) pathway, which is crucial in B-cell production and maintenance, and a potential therapeutic target. AREAS COVERED: This review will summarize the current knowledge of the Btk pathway and its role in lymphoid malignancies. It will also discuss the present data about PCI-32765 in both the preclinical and clinical setting. EXPERT OPINION: PCI-32765 is an oral irreversible Btk inhibitor with high potency and both preclinical and clinical activity in chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL). Phase I studies have demonstrated that it is well tolerated and has an excellent safety profile. Further studies are ongoing as a single agent and in combination with other targeted and conventional therapies. PCI-32765 is a very promising targeted therapy, and the data from these trials will ultimately decide its future role and success.     

Review of amsacrine, an investigational antineoplastic agent

The pharmacology, chemistry, pharmacokinetics, clinical studies, and adverse effects of amsacrine, an investigational antineoplastic agent, are reviewed. Amsacrine's mechanism of action is not clearly understood, although the drug is known to inhibit DNA synthesis. As an investigational NCI "Group C" agent, amsacrine is available to physicians for the treatment of adult patients with refractory acute nonlymphocytic leukemia (ANLL) under an established protocol. Following intravenous administration, amsacrine has a biphasic plasma clearance. It is extensively metabolized by the liver to inactive compounds that are excreted in the bile. Phase I studies indicated that amsacrine was potentially effective in patients with solid tumors and acute leukemias. Patients with solid tumors could tolerate much lower doses of amsacrine than leukemia patients because of dose-limiting bone-marrow suppression in the former. In Phase II studies, amsacrine appeared effective in treating the acute leukemias, with response rates of 31% and 23% for acute lymphocytic leukemia and ANLL, respectively. Patients with other types of cancers have not responded to amsacrine therapy. Frequently occurring adverse effects of amsacrine include leukopenia and thrombocytopenia in patients with solid tumors; nausea, vomiting, and diarrhea; mucositis in patients receiving higher doses (leukemia patients); alopecia; hepatotoxicity; and phlebitis. The clinical usefulness of amsacrine appears limited to treatment of the acute leukemias. Studies of combination therapies that include amsacrine are currently underway and should further define the therapeutic role of amsacrine.     

Fludarabine: a review of its use in non-Hodgkin's lymphoma

Fludarabine (Fludara), a purine nucleoside analogue, has been extensively evaluated in the treatment of a number of lymphoproliferative malignancies, including various types of non-Hodgkin's lymphoma. Clinical studies have shown that fludarabine (alone, and particularly as a component of combination therapy) can result in high overall and complete response in adults with various types of non-Hodgkin's lymphoma, including follicular lymphoma. As mono- or combination therapy, intravenous fludarabine is as effective as several other standard treatment regimens in treatment-naive patients and is also effective in patients with recurrent or refractory disease. The efficacy of fludarabine therapy is improved with the use of rituximab, as part of the initial therapeutic regimen or as maintenance therapy, and deserves consideration. The once-daily oral formulation was effective in the treatment of patients with relapsed indolent B-cell non-Hodgkin's lymphoma; however, further studies are required to confirm its role and establish its efficacy relative to that of standard treatment in this patient population. Fludarabine has generally acceptable tolerability; however, it is associated with haematological adverse events, including myelosuppression. Fludarabine, therefore, provides a highly effective first- or second-line option in the treatment of non-Hodgkin's lymphoma.     

Oral fludarabine

Fludarabine is an antimetabolite antineoplastic agent used in the treatment of various haematological malignancies, particularly B-cell chronic lymphocytic leukaemia (CLL). An oral formulation of fludarabine has recently become available in the majority of European countries for the treatment of patients with relapsed or refractory B-cell CLL after initial treatment with an alkylating agent-based regimen. It is the first oral formulation of a purine analogue available for clinical use in B-cell CLL. Pharmacokinetic studies evaluating the bioavailability of oral fludarabine indicate that an oral dose of 40 mg/m2/day would provide similar systemic drug exposure to the standard intravenous dose of 25 mg/m2/day. A phase II study evaluated the clinical efficacy of six to eight cycles of oral fludarabine 40 mg/m2/day for 5 days of each 28-day cycle in 78 patients with previously treated B-cell CLL. Depending on the criteria used, the overall response rate was 46.2% (20.5% complete response [CR], 25.6% partial response [PR]) or 51.3% (17.9% CR, 33.3% PR). These results were similar to the 48% overall response rate reported in a similar historical control group treated with intravenous fludarabine. Myelosuppression (WHO grade 3 or 4) was the most frequently reported adverse effect with oral fludarabine therapy. Other common adverse effects included infection and gastrointestinal disturbances, although these were usually of mild to moderate severity (WHO grade 1 or 2). Overall, the tolerability profile of oral fludarabine is similar to that of the intravenous formulation.     

Activity of CHS 828 in primary cultures of human hematological and solid tumors in vitro.

CHS 828 is a pyridyl cyanoguanidine that has shown promising preclinical anticancer activity against various experimental tumor models and is presently being tested in a phase II trial in man. In the present study the fluorometric microculture cytotoxicity assay was used for in vitro evaluation of CHS 828 activity in primary cell cultures from hematological and solid tumors. In total, 156 samples from various diagnoses were tested with 72-h continuous drug exposure. CHS 828 showed high relative in vitro activity against tumor cells from chronic lymphocytic leukemia as well as from acute leukemia and high-grade lymphoma. Activity was also observed in several solid tumor cell samples, although the group as a whole appeared less responsive. CHS 828 was significantly more active against hematological malignancies compared to normal lymphocytes. Correlation analysis with standard drugs revealed low to moderate correlation coefficients. The results show that CHS 828 has potent antitumor activity against primary cultures of human tumor cells from patients and might have a unique mechanism of action.     

PCI-32765: a novel Bruton's tyrosine kinase inhibitor for the treatment of lymphoid malignancies

Introduction: There has been a significant paradigm shift in the manner in which lymphoid malignancies are treated and managed. Treatment has been moving away from conventional chemotherapy and towards targeted therapy. The success of new classes of agents such as monoclonal antibodies, proteasome inhibitors and immunomodulatory derivatives has sparked further searches for novel pathways to inhibit. The Bruton's tyrosine kinase (Btk) pathway is a downstream mediator of the B-cell receptor (BCR) pathway, which is crucial in B-cell production and maintenance, and a potential therapeutic target.

Areas covered: This review will summarize the current knowledge of the Btk pathway and its role in lymphoid malignancies. It will also discuss the present data about PCI-32765 in both the preclinical and clinical setting.

Expert opinion: PCI-32765 is an oral irreversible Btk inhibitor with high potency and both preclinical and clinical activity in chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL). Phase I studies have demonstrated that it is well tolerated and has an excellent safety profile. Further studies are ongoing as a single agent and in combination with other targeted and conventional therapies. PCI-32765 is a very promising targeted therapy, and the data from these trials will ultimately decide its future role and success.     

Computer assisted structure-activity correlations. Evaluation of benzo(de)isoqinoline-1,3-diones and related compounds as antitumor agents

Computer assisted evaluations of benzo(de)isoquinoline-1,3-diones and related compounds screened for antitumor activity against P388 lymphocytic leukemia and L1210 lymphoid leukemia are presented. Two important features necessary for good anticancer activity are the nature of the imide side-chain and the type of substituent on the aromatic portion. Based on these considerations 1H-benzo(de)isoquinoline-1,3(2H)dione,5-amino-2-(2-dimethyl-aminoethyl) (NSC 308847) has been selected for preclinical toxicology studies.     

New nucleoside analogs for patients with hematological malignancies

INTRODUCTION: In the last few years, several new purine and pyrimidine nucleoside analogs have been synthesized and made available for both preclinical studies and clinical trials. AREAS COVERED: This article summarizes recent achievements in the mechanism of action, pharmacological properties and clinical activity and toxicity as well as the emerging role of newer purine and pyrimidine nucleoside analogs potentially active in lymphoid and myeloid malignancies. A literature review was conducted from the MEDLINE database PubMed for articles in English. Publications from 2000 to October 2010 were scrutinized. The search terms used were clofarabine, nelarabine, forodesine, 8-chloroadenosine, LMP-420, azacitidine, decitabine, sapacitabine, troxacitabine, thiarabine and zebularine in conjunction with hematologic malignancies, leukemia and lymphoma. Conference proceedings from the previous 5 years of the American Society of Hematology, European Hematology Association, and American Society of Clinical Oncology were searched manually. Additional relevant publications were obtained by reviewing the references from the chosen articles. EXPERT OPINION: Several new nucleoside analogs are currently under investigation in preclinical and clinical studies concerning hematological malignancies. Clofarabine, nelarabine, azacitidine and decitabine have been recently approved for the treatment of leukemias and/or myelodysplastic syndromes. Other agents including forodesine, 8-chloroadenosine, LMP-420, sapacitabine, troxacitabine, thiarabine and zebularine seem to be promising for the treatment of lymphoid and myeloid malignancies. However, definitive data from ongoing and future clinical trials will aid in better defining their status in the treatment of hematological disorders.     

Spirogermanium: A new investigational drug of novel structure and lack of bone marrow toxicity

Spirogermanium (NSC 192965) is a new metallic investigational anticancer drug of novel heterocyclic structure. Although its mode of action has not been fully elucidated, it appears that spirogermanium is not a phase or cell cycle specific drug and inhibits DNA, RNA and protein synthesis, the protein synthesis being the most susceptible to this agent. Spirogermanium has shown cytotoxic activity in vitro against several human tumor cell lines at concentrations (1 micrograms/ml) that were also found toxic to the cultured rat neurons. Although spirogermanium has no effect on normal bone marrow colony forming cells in mice, dogs, or man, it has revealed cytotoxic activity in vitro against human myeloid leukemia cell line K 562 at clinically achievable concentrations. These in vitro findings, indicating selective cytotoxic activity against leukemic cells suggest this drug as a candidate for clinical studies in acute and chronic leukemias. Spirogermanium has revealed activity in vivo against intraperitoneally implanted Walker 256 sarcoma, 13762 mammary adenocarcinoma, and 11095 prostatic carcinoma in rats, but no antitumor activity in vivo was found in the murine tumors used in the past by the NCI screen (L 1210 and P 388 leukemia, B 16 melanoma, Lewis lung carcinoma). Spirogermanium is remarkable for its lack of bone marrow toxicity confirmed in preclinical toxicology and clinical studies; moderate, predictable, and reversible CNS toxicity is dose-limiting. Activity in malignant lymphoma, ovarian cancer, breast cancer, large bowel cancer, and prostatic cancer was reported in the clinical studies. The drug is currently under clinical investigation against the wide spectrum of solid tumors and malignant lymphomas. The dose of 80-120 mg/m2, given by 60' infusion three times a week, is currently used and tolerated in Phase II clinical studies. The recently introduced five days continuous infusion schedule has been also under clinical investigation and the doses of 250-300 mg/m2/day are recommended for Phase II studies. Of interest are results reported in this paper of spirogermanium in vitro preferential activity against the resistant strains of Plasmodium falciparum at clinically achievable concentrations suggesting this drug as a possible new antimalarial agent of novel structure.     

In vitro drug resistance in B cell chronic lymphocytic leukemia: a comparison with acute myelocytic and acute lymphocytic leukemia

The aim of the study was to evaluate cellular drug resistance in B cell chronic lymphocytic leukemia (B-CLL) in vitro, and compare it with that in acute myelocytic leukemia (AML) and acute lymphocytic leukemia (ALL). In vitro drug resistance was analyzed by the fluorometric microculture cytotoxicity assay (FMCA) in all samples from patients with leukemia sent to our laboratory between 1992 and 2001. Up to 14 standard drugs were evaluated in samples from 66 patients with B-CLL, 212 patients with AML and 80 patients with ALL. B-CLL cells were found to be more sensitive than cells from both AML and ALL to cytarabine, cladribine, fludarabine, doxorubicin, idarubicin, vincristine and cyclophosphamide (p<0.05). No difference in cellular drug resistance was found between B-CLL and ALL cells for prednisolone, whereas AML cells were more resistant (p<0.0001). In B-CLL, cells from patients who had received previous chemotherapy were more resistant to almost all tested drugs as compared to cells from treatment-naive patients. In AML and ALL, in vitro drug resistance was not related to previous chemotherapy. For all drugs, there was a good agreement between the activity in vitro and the known clinical disease-specific activity. The study also demonstrated an acquired cellular drug resistance in B-CLL, but not in the acute leukemias.     

Novel monoclonal antibodies for the treatment of chronic lymphocytic leukemia

For many years, alkylating agents and purine nucleoside analogs (PNA) have been considered the drug of choice for treatment of chronic lymphocytic leukemia (CLL). More recently the introduction of monoclonal antibodies (mAb), especially rituximab directed against CD20 and alemtuzumab directed against CD52, has renewed interest in CLL therapy. Over the last few years, several new mAbs directed against lymphoid cells have been developed and investigated in preclinical studies and clinical trials. Some of them are highly active in CLL. New mAbs directed against CD20 include human mAb ofatumumab (HuMax CD20), IMMU-106 (hA20) which has a >90% humanized framework and GA-101, a novel third - generation fully humanized and optimized mAb. These agents are highly cytotoxic against B-cell lymphoid cells and are evaluated in CLL. Lumiliximab (anti-CD23 mAb) is a genetically engineered macaque-human immunoglobulin (Ig) A1. This antibody showed high activity and good tolerability in phase I clinical trial and is evaluated in phase I/II clinical trials as a single agent and in combination. Epratuzumab is a humanized anti-CD22 mAb currently used in clinical trials for treatment of non-Hodgkin lymphoma and autoimmune disorders. Further studies are needed to elucidate the role of this agent in CLL. Apolizumab (HU1D10) is a humanized IgG1 antibody specific for a polymorphic determinant found on the HLA-DRbeta chain. Preclinical and early clinical studies suggest that this mAb has some activity in CLL. HCD122 (CHIR-12.12) and SGN-40 are anti-CD40 mAbs which induce cytotoxicity against CLL cells. Phase I study has shown a favorable safety profile and some activity of HCD122 in pretreated CLL patients. Immunotoxins, especially BL22, LMP-2 and denileukin diftitox, are also being evaluated in lymphoid malignancies and seem to be active in CLL. Finally, antiangiogenic mAbs, especially bevacimzumab, have a potential therapeutic role in this disease. In this review, new mAbs, potentially useful in CLL are presented.     

Clinical pharmacokinetics of nucleoside analogues: focus on haematological malignancies

This review establishes the pharmacokinetic characteristics of the major nucleoside analogues with cytotoxic activity. Cytarabine, pentostatin, fludarabine, cladribine and gemcitabine are all prodrugs whose plasma pharmacokinetics do not fully reflect their therapeutic activity; after cellular uptake, these compounds undergo phosphorylation by deoxycytidine kinase before their incorporation into DNA results in cell death. Cytarabine is principally active in the S phase of the cell cycle and is most toxic to replicating cells, whereas pentostatin, fludarabine and cladribine are incorporated into DNA during the process in which strand breaks are repaired and are therefore cytotoxic to slowly replicating cells (although the action of pentostatin results from its inhibition of adenosine deaminase). Gemcitabine is unusual in being highly metabolised in solid tumour cells. The cytotoxic activity of pentostatin, fludarabine and cladribine against the clonal cells of lymphoproliferative disorders is accompanied by damage to normal lymphoid cells, which results in significant and long-lasting immunosuppression. Useful interactions between nucleoside analogues have been defined. Cells that are primed by exposure to fludarabine or cladribine exhibit enhanced accumulation of cytarabine triphosphate (the cytotoxic nucleotide of cytarabine) and an improved therapeutic effect against acute myeloid leukaemia and chronic lymphocytic leukaemia can be achieved by clinical schedules that exploit this effect. Combinations of alkylating agents and fludarabine or cladribine are also synergistic in producing significantly enhanced activity against refractory lymphoid malignancies, but at the cost of increased haematological toxicity. Developments in the clinical administration of gemcitabine are concentrating on efforts to extend the duration of exposure to the drug as a means of counteracting its rapid catabolism in the circulation. Future developments with this group of agents will further explore the use of fludarabine-based combination therapies to produce a transient period of myelosuppression and immunosuppression that is sufficient to permit the engraftment of allogeneic haemopoietic stem cells and also exploit the immunological benefits of graft-versus-tumour reactions. In addition, the clinical spectrum of activity of gemcitabine is also being extended by combining the drug with other active chemotherapeutic agents, such as cisplatin, and by early studies of its role as a radiosensitiser.     

Nucleoside analogues in the treatment of haematological malignancies

The nucleoside analogues are a group of antimetabolite cytotoxics which generally have to be metabolised to the equivalent nucleotide before incorporation into DNA. Cytarabine is a well established component of the treatment of acute leukaemias and has its principal action on dividing cells. New formulations include a liposome encapsulated product for intrathecal use and oral cytarabine ocfosfate which may be suitable for long-term outpatient use. Pentostatin acts by causing accumulation of deoxynucleotides and, although active against hairy cell leukaemia, is associated with a poor tolerance profile. Cladribine and fludarabine have substantial activity in the treatment of chronic lymphocytic leukaemia (CLL) and low-grade non-Hodgkin's lymphoma (NHL). Fludarabine is the more thoroughly investigated of the two and is currently being developed in combination therapies for CLL and NHL and also in a combination with cytarabine for acute myeloid leukaemia. Fludarabine's immunosuppressive activity is being exploited in the conditioning of patients for non-myeloablative stem cell transplantation. Gemcitabine is an established agent in the treatment of a number of solid tumours but also has activity in haematological malignancies which might be exploited by the use of extended infusion schedules. Newer agents including nelarabine, clofarabine and troxacitabine are undergoing clinical evaluation and show promising activity.     

Changes in ceramide and sphingomyelin following fludarabine treatment of human chronic B-cell leukemia cells

Fludarabine is used to treat chronic lymphocytic leukemia. Both in vitro and in vivo studies have indicated that apoptosis is an important mode of fludarabine-induced cell death. However, the apoptotic pathways activated are not known. The effects of apoptotic doses of fludarabine on sphingomyelin, ceramide and the production of reactive oxygen species were investigated in the chronic B-cell leukemia lines WSU and JVM-2. Apoptosis, as assessed by an increase in phosphatidylserine externalization, internucleosomal DNA fragmentation and caspase-3-like activity, was evident by 18 h after fludarabine in both cell lines. The general caspase inhibitor t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone (OMe, methyl ester) significantly inhibited apoptosis supporting a role for caspases in fludarabine-induced cell death. A 2.5- to threefold elevation in ceramide levels was observed 6 h after fludarabine treatment. Concomitantly, a decrease in sphingomyelin levels was observed. Fumonisin B1 (an inhibitor of ceramide synthase) pretreatment significantly prevented fludarabine-induced ceramide generation and apoptosis. Conversely, C6-ceramide induced apoptosis in both cell lines. No effect of fludarabine on indices of oxidative stress (dichlorofluorescin oxidation and glutathione disulfide formation) were detected, although partial protection from apoptosis, and prevention of ceramide generation and caspase-3 activation, were achieved with N-acetylcysteine. These findings are consistent with the involvement of caspases and ceramide in fludarabine-induced apoptosis in WSU and JVM-2 cells. Oxidative stress does not appear to be induced by fludarabine, although the protective effects of N-acetylcysteine suggest that thiol redox balance may play a role in the apoptotic pathway.