All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules. To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T. gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E. coli β-lactamase. Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space. Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface. Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules. The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail. Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network. Targeting of secreted proteins to T. gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events.

Cloretazine has significant activity and modest extramedullary toxicity in elderly patients with AML or high-risk MDS. Response rates remain consistent despite increasing age and comorbidity.

Purpose: Cloretazine (VNP40101M) is a novel sulfonylhydrazine alkylating agent with significant antileukemia activity. A phase I study of cloretazine combined with cytarabine (1-h-Darabinofuranosylcytosine, 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 z65 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 z400 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 V 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.Patients with relapsed or refractory leukemia have a poor prognosis, and more effective agents are needed to improve complete remission (CR) rates and durations of response (1, 2). Many novel compounds with unique and targeted mechanisms of action are being introduced into the clinic. Current data suggest that most will require combination with standard cytotoxic agents in therapeutic antileukemia regimens (3 -7). The development of more active, better tolerated cytotoxic agents continues to be important in attempting to improve therapy for patients with leukemia.DNA alkylating agents are an important class of antileukemia agents, sharing the ability to damage DNA and/or impair its replication (5,7,8). Among the class, there is a broad spectrum of antitumor activity and toxicity, attributable to individual biological properties of the agents, including the type of DNA damage, relative specificity of attacking DNA versus other cellular components, the DNA repair mechanisms used by the cell in response to the agent, uptake and distribution of the agent in malignant and normal cells, and the relative susceptibility of the agent to leukemia resistance mechanisms (9 -12). With such diverse mechanisms capable of affecting efficacy and/or tolerability, there is an ongoing search for novel alkylating agents with potential advantages over currently available drugs. This search is particularly important in patients with hematologic malignancies, where a...

PURPOSE An international phase II study of laromustine (VNP40101M), a sulfonylhydrazine alkylating agent, was conducted in patients age 60 years or older with previously untreated poor-risk acute myeloid leukemia (AML). PATIENTS AND METHODS Laromustine 600 mg/m(2) was administered as a single 60-minute intravenous infusion. Patients were age 70 years or older or 60 years or older with at least one additional risk factor-unfavorable AML karyotype, Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 2, and/or cardiac, pulmonary, or hepatic comorbidities. Results Eighty-five patients (median age, 72 years; range, 60 to 87 years) were treated. Poor-risk features included age 70 years or older, 78%; adverse karyotype, 47%; PS of 2, 41%; pulmonary disease, 77%; cardiac disease, 73%; and hepatic disease, 3%. Ninety-six percent of patients had at least two risk factors, and 39% had at least four risk factors. The overall response rate (ORR) was 32%, with 20 patients (23%) achieving complete response (CR) and seven (8%) achieving CR with incomplete platelet recovery (CRp). ORR was 20% in patients with adverse cytogenetics; 32% in those age 70 years or older; 32% in those with PS of 2; 32% in patients with baseline pulmonary dysfunction; 34% in patients with baseline cardiac dysfunction; and 27% in 33 patients with at least four risk factors. Twelve (14%) patients died within 30 days of receiving laromustine therapy. Median overall survival was 3.2 months, with a 1-year survival of 21%; the median duration of survival for those who achieved CR/CRp was 12.4 months, with a 1-year survival of 52%. CONCLUSION Laromustine has significant single-agent activity in elderly patients with poor-risk AML. Adverse events are predominantly myelosuppressive or respiratory. Response rates are consistent across a spectrum of poor-risk features.