Targeted therapy in oncology: the agony and ecstasy of personalized medicine

Cloretazine is a new sulfonylhydrazine alkylating agent with antileukemic activity. Phase I studies have shown myelosuppression to be the dose limiting toxicity in both solid tumors and leukemias. A large Phase II study of single agent cloretazine (600 mg/m²) confirmed its activity in patients with relapsed acute myeloid leukemia, and in elderly patients with previously untreated acute myeloid leukemia or myelodysplastic syndrome. It also confirmed the limited nonhematological toxicity, even in elderly patients. Cloretazine can be safely combined with cytarabine, and this combination regimen is currently being tested in a large Phase III study in patients with relapsed acute myeloid leukemia. Cloretazine is a promising new antileukemic agent that may be incorporated into an intensive combination regimen.     

A Phase I and pharmacokinetic study of VNP40101M, a novel sulfonylhydrazine alkylating agent, in patients with refractory leukemia.

PURPOSE: VNP40101M is a novel sulfonylhydrazine alkylating agent with broad antitumor activity in animal models. As alkylating agents are important antileukemia drugs, a Phase I and pharmacokinetic study of VNP40101M was conducted in patients with refractory or relapsed leukemias or poor-risk myelodysplastic syndromes (MDS). EXPERIMENTAL DESIGN: VNP40101M was given as a single i.v. infusion over 15-70 min on day 1. Courses were repeated every 4 weeks according to antileukemic activity. The starting dose of 220 mg/m(2) was escalated by approximately 33% in cohorts of 3-6 patients until a maximum-tolerated dose was established. One additional cohort was treated with the maximum-tolerated dose divided over days 1 and 8. RESULTS: Thirty-eight patients, including 28 with acute myeloid leukemia and 5 with MDS, received 52 courses of treatment. Nondose-limiting, reversible infusion-related toxicities were the most frequent adverse event, occurring in 24 (63%) patients on the first course. Dose escalation was terminated at 708 mg/m(2) for prolonged myelosuppression in 1 of 7 patients, and 600 mg/m(2) was selected as the recommended Phase II dose, with no significant extramedullary toxicity at this dose level. Two patients, 1 with MDS treated with 300 mg/m(2) and 1 with acute myeloid leukemia treated with 600 mg/m(2), achieved complete remission. CONCLUSIONS: VNP40101M had significant antileukemic activity and minimal extramedullary toxicity in patients with relapsed or refractory disease.     

Phase I and pharmacokinetic study of DX-8951f (exatecan mesylate), a hexacyclic camptothecin, on a daily-times-five schedule in patients with advanced leukemia.

PURPOSE: DX-8951f is a novel hexacyclic camptothecin-analogue topoisomerase I inhibitor with both in vitro antileukemic activity and myelosuppression as a dose-limiting toxicity in solid tumor Phase I studies. DX-8951f is active in a human acute myeloid leukemia (AML) severe combined immunodeficient mouse model. In a leukemia Phase I study, we investigated the toxicity profile and pharmacokinetics of DX-8951f in patients with primary refractory or relapsed AML or acute lymphocytic leukemia, myelodysplastic syndromes, or chronic myelogenous leukemia in blastic phase (CML-BP). EXPERIMENTAL DESIGN: DX-8951f was given as an i.v. infusion over 30 min daily for 5 or 7 days. The starting dose was 0.6 mg/m(2)/day for 5 days (3.0 mg/m(2)/course). Courses were given every 3-4 weeks according to toxicity and antileukemic efficacy. RESULTS: Twenty-five patients (AML, 21 patients; myelodysplastic syndrome, 1 patient; acute lymphocytic leukemia, 2 patients; CML-BP, 1 patient) were treated. Stomatitis was the dose-limiting toxicity, occurring in two of two patients treated at 1.35 mg/m(2)/day for 5 days, two of three treated at 1.2 mg/m(2)/day for 5 days, and one of six treated at 0.9 mg/m(2)/day for 7 days. The recommended Phase II dose was 0.9 mg/m(2)/day for 5 days. The pharmacokinetics of DX-8951 was linear and well fit by a two-compartment model. CONCLUSIONS: Phase II studies are warranted to further define the activity of DX-8951f in patients with hematological malignancies.     

A phase I-II trial of escalating doses of mitoxantrone with fixed doses of cytarabine plus fludarabine as salvage therapy for patients with acute leukemia and the blastic phase of chronic myelogenous leukemia

BACKGROUND: Cytarabine is an essential drug for inducing remission of acute myelogenous leukemia, and it is also one the most effective drugs used as salvage therapy for patients with all types of relapsed acute leukemia. Nevertheless, there is considerable room for improvement in the treatment of patients with relapsed leukemia in terms of both the reinduction rate and the duration of response. Fludarabine has been shown to augment responses to cytarabine, possibly by increasing the intracellular concentrations of the active metabolite cytarabine triphosphate. Higher-than-standard doses of mitoxantrone have been shown to augment responses to cytarabine, possibly by increasing the DNA strand breaks induced by topoisomerase II; these strand breaks cannot be effectively repaired in the presence of cytarabine triphosphate. This preliminary study was designed to determine whether moderately high doses of mitoxantrone could be added to the combination of fludarabine and cytarabine in an attempt to improve the combination's antileukemic efficacy. METHODS: Fifty-five adults with relapsed or refractory acute leukemia or the blastic phase of chronic myelogenous leukemia (CML) received salvage therapy with the FLAM regimen, which consisted of fludarabine, cytarabine, and increasing doses of mitoxantrone. RESULTS: Even with doses of mitoxantrone escalated to as much as 60 mg/m(2) over 4 days, dose-limiting toxicity was not observed. Overall, the complete response rate was 27.3% (15 of 55 patients, including 4 of 17 with acute myelogenous leukemia [AML], 4 of 12 with acute lymphocytic leukemia [ALL], and 7 of 26 with the blastic phase of CML). The median time to complete response was 42 days. Toxicity other than myelosuppression was manifested primarily as hyperbilirubinemia, which was reversible in all cases. Poor performance status and undifferentiated blastic phase of CML were poor prognostic factors for response to FLAM. CONCLUSIONS: The FLAM regimen is an active salvage regimen and should be formally evaluated in Phase II studies of patients with AML, ALL, and the myeloid and lymphoid blastic phases of CML.     

A phase II study of cloretazine (VNP40101M), a novel sulfonylhydrazine alkylating agent, in patients with very high risk relapsed acute myeloid leukemia

Cloretazine® (VNP40101M) is a sulfonylhydrazine alkylating agent with significant anti-leukemia activity. A multicenter phase II study of cloretazine was conducted in patients with first relapse of acute myeloid leukemia (AML) following an initial complete remission (CR) of less than 12 months. Cloretazine was given as a single intravenous infusion at a dose of 600 mg/m². Fifty-three patients (median age 62 years (18–84), 41 of 44 (93%) evaluable with intermediate or high risk cytogenetics, 32 (60%) with initial CR durations ≤6 months) were treated on study. Two patients (4%) achieved a second CR. Five (9%) patients died within 30 days of receiving cloretazine therapy. Median overall survival (2.3 months) in the study cohort was directly comparable to that of 233 matched patients treated with other single agents. The study cloretazine regimen had minimal activity in a very high risk subset of patients with relapsed AML.     

CLAG方案治疗复发难治急性髓系白血病的疗效观察

目的:评价CLAG方案（2-CdA+Ara-C+G-CSF）治疗复发难治急性髓系白血病的疗效及安全性。方法:回顾性分析我中心2015年6月至2016年8月应用CLAG方案治疗的12例复发难治急性髓系白血病患者。结果:12例患者均系复发难治急性髓系白血病,根据NCCN急性髓系白血病指南（2017年第1版）细胞遗传学及分子生物学标记,进行危险度分级,其中预后良好组3例,预后中等组5例,预后不良组4例。所有患者均给予1疗程CLAG方案化疗,其中8例（72.7%）达到完全缓解（CR）,2例（18.2%）达到部分缓解（PR）,总有效率（OR）90.9%。所有患者均出现Ⅲ-Ⅳ级血液学毒性,主要毒副反应为粒细胞缺乏及血小板减少所导致的感染和出血,其中肺部感染8例（72.7%）,侵袭性真菌病5例（45.5%）,革兰阴性杆菌败血症2例（18.2%）。6例患者（54.5%）发生Ⅲ-Ⅳ级出血,1例因弥漫性肺泡出血早期死亡。化疗所致恶心呕吐、肝肾毒性、口腔黏膜炎等非血液学毒性均为Ⅰ-Ⅱ级。结论:CLAG方案治疗复发难治性急性髓系白血病有效率较高。化疗所致骨髓抑制较重,但合并感染、出血可控制,非血液学毒性轻微,安全性较好,可作为复发难治急性髓系白血病挽救性治疗的首选方案。     

High-dose cytosine arabinoside and etoposide in the treatment of relapsed or refractory adult leukemia

Thirteen patients with leukemia were treated with a combination of cytosine arabinoside (ara-C) (3 g/m2 by 1-h infusion every 12 h for 12 doses) and etoposide (100 mg/m2 daily over 1 h for 3 doses). Toxicity of the regimen consisted of severe hematologic suppression, moderate abdominal colic with vomiting and diarrhea, and occasionally severe central nervous system (CNS) toxicity. Two patients received the regimen as consolidation for acute myelogenous leukemia in remission. Of the remaining 11 patients with chronic myeloid leukemia (CML)-blast crises or relapsed/refractory acute myeloid leukemia (AML), nine patients (82%) obtained CR (or chronic phase) and two patients obtained partial remission (PR). High-dose ara-C and etoposide is an effective but toxic regiment for the treatment of relapsed or refractory myeloid leukemias.     

Trials with gemtuzumab ozogamicin (Mylotarg) combined with chemotherapy regimens in acute myeloid leukemia

Gemtuzumab ozogamicin (Mylotarg) is an immunoconjugate composed of a recombinant humanized murine anti-CD33 antibody linked to calicheamicin, a potent cytotoxic agent. The aim of this review is to summarize ongoing trials with gemtuzumab ozogamicin in combination with chemotherapy in acute myeloid leukemia (AML) patients. The studies include determination of safety and efficacy of gemtuzumab ozogamicin in combination with chemotherapy in previously untreated as well as relapsed and refractory AML patients. These studies also determine gemtuzumab ozogamicin's activity in patients with other CD33+ neoplastic diseases such as myelodysplastic syndrome, acute promyelocytic leukemia, chronic myeloid leukemia, and certain subsets of acute lymphocytic leukemia. Moreover, trials are exploring the use of gemtuzumab ozogamicin with novel targeted agents such as Bcl-2 antisense molecules. Gemtuzumab ozogamicin is associated with an acceptable toxicity profile as a single agent; however, the incidence of veno-occlusive disease remains a concern with the use of gemtuzumab ozogamicin in combination with chemotherapy.     

Gemtuzumab ozogamicin in the treatment of acute myeloid leukemia

Options for treatment of poor-prognosis or relapsed acute myeloid leukemia (AML) remain limited. Gemtuzumab ozogamicin (Mylotarg, Wyeth-Ayerst, Philadelphia, PA) is an immunoconjugate composed of recombinant humanized murine anti-CD33 antibody linked to calicheamicin, a potent cytotoxic agent. Phase II trials have shown the efficacy of gemtuzumab ozogamicin in the treatment of relapsed AML. Trials exploring this agent in other CD33+ hematologic malignancies and in combination with other agents for AML are ongoing. Gemtuzumab ozogamicin is associated with acceptable toxicity as a single agent. However, the incidence of veno-occlusive disease of the liver remains a concern when this agent is used in combination with chemotherapy or in the context of hematopoietic stem cell transplant.     

Mitoxantrone for refractory and relapsed acute leukemia

Seventy-seven patients with relapsed or refractory acute leukemia and three with acute blastic chronic myeloid leukemia (CML) were treated in an open Phase II study using mitoxantrone 12 mg/m2 intravenously daily X 5 days. Complete remission (CR) was achieved in 32 of 80 (40%), including 23/45 (52%) with relapsed acute nonlymmphocytic leukemia (ANLL), four of 12 (33%) with relapsed acute lymphocytic leukemia ALL, four of 17 (24%) with ANLL refractory to daunorubicin + cytosine arabinoside, and one of three (33%) with refractory ALL. None of the patients with acute blastic CML achieved CR. Median survival time for all patients was 121 days. Median duration of complete response was 303 days with ten of 32 patients in continuing CR for periods varying from 44+ to 1210+ days. Apart from moderately prolonged hematologic suppression toxicity was mild and subjective side effects were tolerable. Mitoxantrone is an active agent in the treatment of acute leukemia and demonstrates incomplete cross resistance with duanorubicin. Mitoxantrone should be considered for first-line therapy in ANLL.     

Phase I study of cloretazine (VNP40101M), a novel sulfonylhydrazine alkylating agent, combined with cytarabine in patients with refractory leukemia.

PURPOSE: Cloretazine (VNP40101M) is a novel sulfonylhydrazine alkylating agent with significant antileukemia activity. A phase I study of cloretazine combined with cytarabine (1-beta-d-arabinofuranosylcytosine, ara-C) was conducted in patients with refractory disease. DESIGN: Ara-C was given i.v. at a fixed dose of 1.5 gm/m(2)/d by continuous infusion for 4 days (patients ages <65 years at time of diagnosis) or 3 days (patients ages > or =65 years). Cloretazine was given i.v. over 15 to 60 minutes on day 2 at a starting dose of 200 mg/m(2), with escalation in 100 mg/m(2) increments in cohorts of three to six patients until a maximum tolerated dose was established. The DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase (AGT) was measured at baseline. RESULTS: Forty patients, including 32 with acute myeloid leukemia, received 47 courses of treatment. Complete responses were seen at cloretazine dose levels of > or =400 mg/m(2) in 10 of 37 (27%) evaluable patients, and in this patient subset, AGT activity was significantly lower in patients that responded to treatment than in patients who did not (P < or = 0.027). Dose-limiting toxicities (gastrointestinal and myelosuppression) were seen with 500 and 600 mg/m(2) of cloretazine combined with the 4-day ara-C schedule but not seen with the 3-day schedule. CONCLUSION: The recommended cloretazine dose schedule for future studies is 600 mg/m(2) combined with 1.5 gm/m(2)/d continuous infusion of ara-C for 3 days. The cloretazine and ara-C regimen has significant antileukemic activity. AGT activity may be a predictor of response to cloretazine.     

Less intense conditioning with fludarabine, cyclophosphamide, idarubicin and etoposide (FCIE) followed by allogeneic unselected peripheral blood stem cell transplantation in elderly patients with leukemia.

The objective of this study was to assess toxicity and feasibility of achieving engraftment of allogeneic blood progenitor cells following nonmyeloablative conditioning according to the FCIE protocol (fludarabine 25 mg/m(2)/day, days -7 to -3; cyclophosphamide 200 mg/m(2)/day, days -7 to -3; idarubicin 12 mg/m(2)/day, days -7 to -5; etoposide 250 mg/m(2)/day, days -4 to -3) in elderly patients with leukemia. Eleven patients were included in the study: six patients with acute myeloid leukemia (AML) in complete remission (CR); three patients with refractory or relapsed AML; one patient with chronic myeloid leukemia; one patient with acute lymphoblastic leukemia. The median age of the patients was 62 years. All patients received blood progenitor cells from an HLA-identical sibling with 8.8 x 10(6) CD34(+) cells/kg (median; range 4.7 to 26.2 x 10(6)/kg) and 5.5 x 10(8) CD3(+)cells/kg (median; range 4.5 to 7.9 x 10(8)/kg). Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and three courses of methotrexate. The median duration of white blood cell counts <1 x 10(9)/l was 17 days and of platelet counts <50 x 10(9)/l 20 days. In two patients acute GVHD grade I occurred. Nine of 10 patients analyzed developed mixed chimerism. Of seven patients transplanted in CR, three remained in CR 19 to 31 months after transplantation. Three patients with refractory leukemia did not achieve CR, while the patient with relapsed AML achieved a 3rd CR. After a median follow-up time of 22 months, chronic GVHD was mild and limited. The data from this pilot study in elderly patients with leukemia show that the combination of primarily immunosuppressive (FC) and antileukemic (IE) drugs for nonmyeloablative conditioning has moderate nonhematological toxicity and allows engraftment of allogeneic blood progenitor cells.     

FLAG-IDA regimen (fludarabine,cytarabine, idarubicin and G-CSF) in the treatment of patients with high-risk myeloid malignancies

Forty-five patients with high-risk myeloid malignancies (32 acute myeloid leukemia and 13 high-risk myelodysplastic syndromes) were treated with fludarabine, cytarabine, idarubicin, and G-CSF (FLAG-IDA). Twenty-four (53%) patients achieved complete remission (CR), and five (11%) partial remission. Infection predominantly with pulmonary involvement was the most common regimen-related toxicity. Mucositis (15 patients) and pulmonary toxicity (19 patients) were the most frequently observed non-hematologic side effects. There were four early deaths and 12 patients presented with resistant disease. Overall survival (OS) at 12 months was 40%. The FLAG-IDA regimen shows evident antileukemic activity in patients with high-risk myeloid malignancies with acceptable toxicity.     

Management of Refractory Acute Myeloid Leukemia: Re-induction Therapy or Straight to Transplantation?

Patients with primary resistant and relapsed acute myeloid leukemia (AML) are rarely cured without undergoing allogeneic stem cell transplantation. What is currently debated is whether a trial of re-induction chemotherapy prior to transplantation is beneficial. Data from multiple retrospective analyses have shown that pretreatment variables are useful in predicting response to salvage chemotherapy. For patients unlikely to respond, re-induction attempts may be detrimental, leading to added organ toxicity and possible increased tumor resistance. Allogeneic transplantation in the setting of active disease is the alternative strategy. Multiple studies have demonstrated the feasibility of this approach, but cure rates have been low with the use of traditional transplant approaches. Newer strategies employing allogeneic transplantation earlier in patients with relapsed or refractory AML, as well as the incorporation of novel and effective antileukemic agents into the transplant conditioning regimen, may lead to better outcomes.     

Gemtuzumab ozogamicin, cytosine arabinoside, G-CSF combination (G-AraMy) in the treatment of elderly patients with poor-prognosis acute myeloid leukemia.

BACKGROUND: Gemtuzumab ozogamicin (GO) is effective as single agent in the treatment of acute myeloid leukemia (AML). We evaluated efficacy and safety of a chemotherapy including growth factors, cytarabine, and GO (G-AraMy) in the treatment of poor-prognosis AML in elderly patients. PATIENTS AND METHODS: In three Italian hematology departments from September 2003 to September 2006, 53 elderly patients [median age 69 years (range 65-77)] with untreated or primary refractory/relapsed AML were enrolled on the combination G-AraMy administered according to two consecutive schedules (G-AraMy1 and G-AraMy2), with intensified consolidation in the second. Twenty-three of 53 patients had a secondary acute myeloid leukemia (sAML). RESULTS: The overall response rate was 57%. The most common adverse event was myelosuppression. Seven patients died in induction (13%). No differences for response rate and toxicity profile were observed between untreated and primary resistant/relapsed patients, de novo AML and sAML, and in the two treatment trials. Median disease-free survival and overall survival were 8 months (range 2-23+) and 9 months (range 2-24+). CONCLUSIONS: G-AraMy therapy may be considered an useful treatment approach for poor-risk elderly AML patients, with a complete remission rate comparable to literature data with reduced side-effects, also in a poor-prognosis population.     

Tipifarnib: farnesyl transferase inhibition at a crossroads

Tipifarnib is an oral nonpeptidomimetic farnesyl transferase inhibitor developed to inhibit a variety of farnesylated targets potentially relevant to the therapy of various malignancies. The agent has, thus far, been tested in a wide array of both solid tumors and myeloid malignancies. Phase I trials have demonstrated that tipifarnib is best given in a twice-daily fashion in doses of 600-1200 mg/day to avoid significant neuropathy, fatigue and myelosuppression. Subsequent trials demonstrated that pauses in therapy (with staccato dosing schedules) seem to increase tolerability without a clear decrease in efficacy. Phase II and III trials of tipifarnib as monotherapy for breast, colorectal, lung (both non-small cell and small cell), brain, pancreatic and urothelial cancers have all been disappointing. Combination trials of tipifarnib with cytotoxic, hormonal or biological therapies are ongoing. Tipifarnib has displayed the most interesting activity in the myeloid malignancies of myelodysplastic syndrome, myelofibrosis with myeloid metaplasia and elderly/high-risk acute myeloid leukemia. Overall clinical response rates of approximately 20-30% have been reported in myelodysplastic syndrome and acute myeloid leukemia patients who have few alternative therapeutic options. US FDA approval for tipifarnib awaits results of subsequent Phase III trials of the agent in elderly acute leukemia.     

Tipifarnib: farnesyl transferase inhibition at a crossroads

Tipifarnib is an oral nonpeptidomimetic farnesyl transferase inhibitor developed to inhibit a variety of farnesylated targets potentially relevant to the therapy of various malignancies. The agent has, thus far, been tested in a wide array of both solid tumors and myeloid malignancies. Phase I trials have demonstrated that tipifarnib is best given in a twice-daily fashion in doses of 600–1200 mg/day to avoid significant neuropathy, fatigue and myelosuppression. Subsequent trials demonstrated that pauses in therapy (with staccato dosing schedules) seem to increase tolerability without a clear decrease in efficacy. Phase II and III trials of tipifarnib as monotherapy for breast, colorectal, lung (both non-small cell and small cell), brain, pancreatic and urothelial cancers have all been disappointing. Combination trials of tipifarnib with cytotoxic, hormonal or biological therapies are ongoing. Tipifarnib has displayed the most interesting activity in the myeloid malignancies of myelodysplastic syndrome, myelofibrosis with myeloid metaplasia and elderly/high-risk acute myeloid leukemia. Overall clinical response rates of approximately 20–30% have been reported in myelodysplastic syndrome and acute myeloid leukemia patients who have few alternative therapeutic options. US FDA approval for tipifarnib awaits results of subsequent Phase III trials of the agent in elderly acute leukemia.     

Etoposide, 6-thioguanine and idarubicin, an oral combination regimen (ETI) for the induction treatment of acute leukemia

Twenty patients with advanced acute leukemia (16 acute myeloid leukemia (AML), three myeloid blast crisis (BC) of chronic myeloid leukemia (CML), one acute lymphatic leukemia) were treated with a peroral regimen consisting of etoposide 80 mg/m2 and 6-thioguanine 100 mg/m2 twice daily for 5 days, and idarubicin 15 mg/m2 once daily for 3 days (ETI). Two AML patients were in first relapse. All the other patients with acute leukemia had a later relapse or were refractory to primary or salvage treatment. One to six ETI cycles were given. Four AML patients achieved remission and one patient with BC of CML entered the second chronic phase. Clearing of the blood of leukemic cells was seen in seven additional patients. Infection was the most common complication, gastrointestinal toxicity was not a major problem. In conclusion, peroral ETI treatment has a marked antileukemic effect even in an advanced disease, and the toxicity is moderate and well acceptable.     

Mitoxantrone, etoposide, and intermediate-dose cytarabine: an effective and tolerable regimen for the treatment of refractory acute myeloid leukemia

Thirty-two patients with refractory acute myeloid leukemia (AML) received salvage therapy with a single course of mitoxantrone 6 mg/m2 intravenous (IV) bolus, etoposide 80 mg/m2 IV for a period of 1 hour, and cytarabine (Ara-C) 1 g/m2 IV for a period of 6 hours daily for 6 days (MEC). Eighteen patients were primarily resistant to conventional daunorubicin and Ara-C induction treatment; eight patients had relapsed within 6 months from initial remission; six patients had relapsed after a bone marrow transplantation (BMT) procedure. Overall, 21 patients (66%) achieved a complete remission (CR), two (6%) died of infection during induction, and nine (28%) had resistant disease. Age greater than 50 years was the only factor predictive for a significantly lower response rate (P = .03). The median remission duration was 16 weeks; the overall median survival was 36 weeks. Severe myelosuppression was observed in all patients resulting in fever or documented infections in 91% of patients. Nonhematologic toxicity was minimal. We conclude that the MEC regimen has significant antileukemic activity and acceptable toxicity in salvage AML. Its benefit in front-line AML therapy is being investigated.     

The antineoplastic efficacy of the prodrug Cloretazine is produced by the synergistic interaction of carbamoylating and alkylating products of its activation

Cloretazine {1,2-bis(methylsulfonyl)-1-[(2-chloroethyl)-2-(methylamino)carbonyl]hydrazine; VNP40101M; 101M} is a sulfonylhydrazine prodrug that possesses broad spectrum antitumor efficacy against transplanted murine and human tumor models and has shown activity in clinical trials against relapsed or refractory acute myeloid leukemia. Base catalyzed activation of this prodrug generates two different reactive intermediates: chloroethylating species that covalently interact with DNA at the O6-position of guanine residues that progress to a G-C interstrand cross-link, and a carbamoylating agent, methyl isocyanate. Previous findings from this laboratory have provided initial evidence that methyl isocyanate can contribute to the efficacy of Cloretazine by enhancing the cytotoxicity of the generated chloroethylating species. This action may be due in part to inhibition of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT); however, activity in cells devoid of AGT indicates that other actions are involved in the synergistic cytotoxicity. Herein we demonstrate that O6-benzylguanine can also produce synergistic cell kill with the alkylating component of Cloretazine but differs from methyl isocyanate in that the enhancement occurs in AGT-containing cells, but not in cells devoid of AGT. Methyl isocyanate generated by the decomposition of 1,2-bis(methylsulfonyl)-1-[methylaminocarbonyl]hydrazine also acts to enhance the activity of a variety of DNA cross-linking agents, while only producing additive cytotoxicity with methylating agents. Flow cytometric studies using annexin as a marker for apoptosis indicate that in Chinese hamster ovary cells and in human leukemia cells Cloretazine-induced apoptosis is primarily caused by the generated methyl isocyanate. Comet assays designed to detect DNA cross-links in intact cells indicate that the chloroethylating species generated by the activation of Cloretazine produce DNA cross-links, with the co-generated methyl isocyanate increasing the degree of cross-linking produced by the reactive chloroethylating species. These findings provide further evidence that the methyl isocyanate produced by the activation of Cloretazine can be a major contributor to the cytotoxicity produced by this antineoplastic agent.