Development of a bioanalytical liquid chromatography method for quantitation of 9-nitrocamptothecin in human plasma.

9-Nitrocamptothecin (9-NC) is an orally administered camptothecin (CPT) that is under evaluation in clinical trials. This compound is not fluorescent, which has hampered development of a sensitive high-performance liquid chromatographic (LC) assay for measurement of drug concentrations in clinical trials. We now report development of an assay that involves reduction of 9-NC to the fluorescent compound 9-aminocamptothecin (9-AC). The method is based on enzymatic reduction of 9-NC using bovine liver S-9 fraction. This method is validated to quantitate 9-NC and 9-AC in patient samples, and yields results comparable to those obtained with an LC/MS method.

Preclinical activity of an i.v. formulation of rubitecan in IDD-P against human solid tumor xenografts.

An i.v. formulation of rubitecan (9-nitrocamptothecin) was evaluated in five human solid tumor xenograft models. Rubitecan in IDD-P, a particulate suspension of the insoluble analog, produced significant tumor growth delay in athymic nude mice bearing A375 melanoma, and MX-1 breast, SKMES non-small-cell lung, Panc-1 pancreatic and HT29 colon carcinomas. The activity of i.v. rubitecan was similar or somewhat superior to those of i.p. regimens with the reference drugs, irinotecan and topotecan. Tumor sensitivity to rubitecan in IDD-P was MX-1>A375>SKMES >Panc-1>HT29. Some complete regression responses were seen with MX-1, A375 and SKMES tumors treated with 2.5 mg/kg on a schedule of two 5-day dosing cycles separated by 2 drug-free days. In nude mice, the MTD of rubitecan in IDD-P lies between 2 and 2.5 mg/kg on this schedule; antitumor efficacy was achieved with doses between 2.5 and 1.25 mg/kg. Dosing with 6.6 mg/kg rubitecan in IDD-P on intermittent schedules (4- or 7-day intervals) was tolerated, but less efficacious, when tested in the A375 model. The good responses obtained with rubitecan in IDD-P suggest it could be used clinically in circumstances where an i.v. formulation offers advantages to oral or aerosol formulations.

Phase I clinical trial of 5-fluoro-pyrimidinone (5FP), an oral prodrug of 5-fluorouracil (5FU).

PURPOSE: 5-Fluoro-Pyrimidinone (5FP) is an oral pro-drug of 5-Fluorouracil (5FU), and is converted to 5FU by hepatic aldehyde oxidase. Preclinically, 5FP demonstrated anti-tumor activity against colon 38 and P 388 leukemia models in mice. Using an accelerated titration trial design with one patient cohorts and initial 100% escalations, a Phase I trial was conducted to determine the maximum tolerated dose (MTD) of 5FP and describe its toxicity and pharmacokinetic profile. PATIENTS AND METHODS: 5FP was administered orally once daily for 5 days every 4 weeks. The initial dose level was 23 mg/m2/d. Using single patient cohorts, escalation proceeded according to accelerated titration 4B design, initially by 100% and subsequently 30-35% escalations (exact escalation determined by pill size) until dose limiting toxicity was observed. A total of 19 patients were enrolled with a median age of 56 years and median performance status of 1. Most patients were heavily pre-treated with chemotherapy, radiation therapy, or both, and patient population included a wide variety of tumor types. RESULTS: Dose escalation proceeded rapidly to 1715 mg/m2/d with the only toxicities observed being nausea and vomiting. The large number of pills necessary at that point required a formulation change, which resulted in appreciable hematologic toxicity. This led to rapid de-escalation of dose in subsequent patients, with the MTD finally being determined to be 625 mg/m2/d. The DLTs observed were grade 4 neutropenia for greater than 5 days and grade 3 anemia. Other toxicities included nausea, vomiting, fatigue, constipation and mucositis. Pharmacology studies confirmed that SFP was converted to 5FU in humans at all dose levels. However, the extent of conversion decreased over the five daily treatments, but returned for the subsequent cycle. The hematologic toxicity was not related to 5FU exposure per course. CONCLUSION: 5FP is a tolerable oral outpatient therapy. Accelerated titration was an efficient way of conducting this phase I trial. The recommended phase 2 dose is 625 mg/m2/d orally for 5 days every 28 days.

LEX032, a novel recombinant serpin, protects the brain after transient focal ischemia.

This investigation examined the effectiveness of a serine protease inhibitor (LEX032) when used as a cerebral protective agent after ischemia. Focal cerebral ischemia in the rat was produced by intravascular occlusion of the middle cerebral artery for a period of 30 min. Just prior to thread withdrawal (i.e., reperfusion), rats received an iv bolus administration of either vehicle or LEX032 (50 mg/kg), an optimal dose chosen based on previous studies. Somatosensory evoked potentials (SSEP's) were monitored prior to, during, and for a period of 60 min after removal of occlusion. The animals were allowed to recover for 24 h after the ischemic insult. Cortical activity in the occluded region, as assessed by SSEPs, returned much sooner in the LEX032-treated animals (10 +/- 6 min) than in the untreated animals (40 +/- 25 min). On a scale ranging from 0 to 3, with three indicating the most severely injured, the LEX032 animals had a significantly better neurologic score (1.0 +/- 0.9) than the untreated animals (2.3 +/- 0.5) 24 h after ischemia. The improved neurobehavior was related to a 55% reduction in brain injury as assessed by TTC staining. LEX032-treated animals had significantly (P < 0.01) smaller infarcts (115 +/- 40 mm3) compared to vehicle-treated animals (263 +/- 13 mm3). In a separate group of animals (n = 6/group), leukocyte infiltration, as evaluated by tissue myeloperoxidase activity (MPO U/g tissue wt), was also significantly (P < 0.05) lower in the LEX032-treated animals (1.4 +/- 0.3) compared to vehicle-treated animals (3.6 +/- 0.7). This data demonstrates that LEX032 reduces brain injury and suggests that serine protease inhibitors may reduce ischemia/reperfusion injury by decreasing leukocyte activation and migration.