Development, validation and application of assays to quantify metrifonate and 2,2-dichlorovinyl dimethylphosphate in human body fluids.

Gas chromatographic procedures [GC with electron-capture detection (ECD) and GC-MS] for the quantitative analysis of metrifonate and its active metabolite 2,2-dichlorovinyl dimethylphosphate (DDVP) in human blood and urine were developed, validated, and applied to the analysis of clinical study samples. Analysis of metrifonate involved extraction of acidified blood with ethyl acetate followed by solid-phase clean-up of the organic extract. Acidified urine was extracted with dichloromethane and the residue of evaporated organic phase was reconstituted in toluene. ECD and diethyl analogue of metrifonate internal standard (I.S.) were used for quantitation of metrifonate. The metrifonate lower limit of quantitation (LOQ) was 10.0 microg/l. The DDVP metabolite was chromatographed separately after cyclohexane extraction of acidified blood and urine using d6-DDVP I.S. and MS detection. The LOQ of DDVP was 1 microg/l. Stability studies have confirmed that the matrix should be acidified prior to storage at -20 degrees C or -80 degrees C to inhibit chemical and enzymatic degradation of the analytes and to avoid overestimation of DDVP concentrations. Metrifonate was found to be stable in acidified human blood after 20 months of storage at -20 degrees C and after 23 months of storage at -80 degrees C. Under these conditions DDVP was found to be stable after 12 months of storage. Both assay procedures were cross-validated by different world-wide laboratories and found to be accurate and robust during analyses of clinical study samples.

Safety and tolerability of metrifonate in patients with Alzheimer's disease: results of a maximum tolerated dose study.

Metrifonate, a pro-drug that is transformed non-enzymatically into a potent inhibitor of acetylcholinesterase (AChE), has been used in the tropics for over 30 years for the treatment of schistosomiasis. A pilot study, and Phase I and Phase II studies of metrifonate in Alzheimer's disease (AD) patients conducted prior to the current study showed benign, dose-dependent adverse event profiles consisting primarily of gastrointestinal events, optimal daily dosing with a loading phase (in the absence of a loading dose phase, 6-8 weeks were required to attain steady-state AChE inhibition levels), and an improvement in Alzheimer's Disease Assessment Scale (ADAS) scores. The current open-label study was designed to evaluate the safety and tolerability of relatively high loading doses, followed by lower maintenance doses of metrifonate in the same patient population, and to determine the maximum tolerated dose (MTD) of metrifonate. Accordingly, the first cohort of 8 probable AD patients (per National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association [NINCDS-ADRDA] criteria) were administered once-daily loading doses of 2.5 mg/kg (125-225 mg) for 14 days, followed by 4.0 mg/kg (200-360 mg) for an additional 3 days. These patients were maintained on once-daily doses of 2.0 mg/kg (100-180 mg) for 14 days. AChE inhibition for this cohort ranged from 88% to 94%. On Day 28 of 31, this cohort was discontinued due to moderate to severe side effects in 6 patients; consequently, the second cohort of 8 probable AD patients received a once-daily loading dose of 2.5 mg/kg (125-225 mg) for 14 days followed by a once-daily maintenance dose of 1.5 mg/kg (75-135 mg) for 35 days. This maintenance dose yielded an AChE inhibition level ranging from 89% to 91%. In spite of an AChE inhibition level comparable to that achieved with the higher dose, the reduced dose was associated with a more favorable adverse event profile which was mainly gastrointestinal and musculoskeletal in nature. The maximum tolerated dose was established at 1.5 mg/kg/day (75-135 mg/day) for maintenance dosing in AD patients.

Mutagenic activities in vitro and in vivo of five antischistosomal compounds.

Five antischistosomal compounds--hycanthone, two of its chloroindazole analogs (IA-4 and IA-4 N-oxide), oxamniquine, and metrifonate--were tested for mutagenic activity, using Salmonella typhimurium strains TA 98 and TA 100 under in vitro and in vivo (host-mediated) conditions. In all assay systems hycanthone exhibited by far the highest mutagenic potency. Although oxamniquine and metrifonate had low metagenic activity in vitro and although their administration resulted in urine of low metagenic activity, their host-mediated mutagenic activities on strain TA 100 were fairly high. Confirming earlier studies with a less sensitive Salmonella strain, TA 1535, IA-4 N-oxide was found to be less metagenic than IA-4. Orally administered IA-4 and IA-4-oxide were less mutagenic under in vivo conditions than an equal dose administered intramuscularly. By contrast, the antihistosomal activity of a given dose of each compound was the same, regardless of which of these two routes was used, suggesting that mutagenic and antischistosomal effects are produced by different metabolites. The observations reported in this paper provide additional evidence that mutagenic activities can be dissociated from desired chemotherapeutic effects by suitable structural modifications.