Evaluations of in vitro metabolism, drug-drug interactions mediated by reversible and time-dependent inhibition of CYPs, and plasma protein binding of MMB4 DMS.

1,1'-Methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) dimethanesulfonate (DMS) is a bisquaternary pyridinium aldoxime that reactivates acetylcholinesterase inhibited by organophosphorus nerve agent. Drug metabolism and plasma protein binding for MMB4 DMS were examined using various techniques and a wide range of species. When (14)C-MMB4 DMS was incubated in liver microsomes, 4-pyridine aldoxime (4-PA) and an additional metabolite were detected in all species tested. Identity of the additional metabolite was postulated to be isonicotinic acid (INA) based on liquid chromatography with a tandem mass spectrometry analysis, which was confirmed by comparison with authentic INA. Formation of INA was dependent on species, with the highest level found in monkey liver microsomes. The MMB4 DMS exhibited reversible inhibition in a concentration-dependent manner toward cytochrome P450 1A2 (CYP1A2), CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in human liver microsomes showing the highest inhibition for CYP2D6. Human recombinant CYPs were used to evaluate inhibitory curves more adequately and determine detailed kinetic constants for reversible inhibition and potential time-dependent inhibition (TDI). The MMB4 DMS exhibited reversible inhibition toward human-recombinant CYP2D6 with an inhibition constant (K i) value of 66.6 µmol/L. Based on the k inact/K I values, MMB4 DMS was found to exhibit the most potent TDI toward CYP2D6. The MMB4 DMS at 5 different concentrations was incubated in plasma for 5 hours using an equilibrium dialysis device. For all species tested, there were no concentration-dependent changes in plasma protein binding, ranging from 10% to 17%. These results suggest that MMB4 was not extensively bound to plasma protein, and there were no overt species-related differences in the extent of MMB4 bound to plasma protein.

Absorption, distribution, metabolism, and excretion of 14C-MMB4 DMS administered intramuscularly to Sprague-Dawley rats and New Zealand White rabbits.

1,1'-Methylenebis[4-[(hydroxyimino)methyl]-pyridinium] dimethanesulfonate (MMB4 DMS) is currently under development for the treatment of chemical warfare organophosphorus nerve agent poisoning. The present study evaluates the absorption, distribution, metabolism, and excretion of (14)C-MMB4 DMS administered intramuscularly to rats and rabbits. The formulated mixture of radiolabeled and nonradiolabeled MMB4 DMS was administered as a single or 7-day repeated dose. Rat doses were 55 or 220 mg/kg (100 µCi/kg), and rabbit doses were 25 or 100 mg/kg (31.25 and 62.5 µCi/kg, respectively). Urine, bile (rats only), feces, blood, and tissues were collected for up to 72 hours. Metabolic profiling using high-performance liquid chromatography with radiodetection was performed on selected urine samples. For both animal species, the majority of the total radioactivity was excreted in the urine (74%-94%) by 72 hours after dosing with greater than 90% of the radioactivity measured in the urine within 8 to 12 hours after dosing. There were no apparent species or dose differences in the urine excretion pattern. The distribution of (14)C-MMB4 DMS-derived radioactivity was rapid and generally reached the highest concentration by the first collection time point (0.25 hours). The tissue-blood concentration ratios were highest at the injection sites and in the kidneys and gastrointestinal tract contents for both the species. Two metabolites of MMB4 DMS were detected in rat and rabbit urine; their structure was confirmed by liquid chromatography with tandem mass spectrometry as 4-pyridine aldoxime and isonicotinic acid (pyridine-4-carboxylic acid).