Effects of subchronic administration of metrifonate on cholinergic neurotransmission in rats.

The effects of subchronic oral administration of metrifonate, a long-acting cholinesterase (ChE) inhibitor, on cholinergic neurotransmission were assessed in young adult male Wistar rats. Animals were treated twice daily with metrifonate. In a pilot study testing a 100 mg/kg dose of metrifonate for up to 14 days, ChE activity was found to steadily decrease to reach maximum inhibition levels of about 55%, 80% and 35% in brain, erythrocytes and plasma. Steady-state inhibition levels were attained by the 10th day of treatment. When metrifonate-treatment was discontinued, ChE activity in plasma returned to control levels within another day, while erythrocyte and brain ChE activity took more than 2 weeks to recover. In subsequent dose-response studies, metrifonate treatment was given for 3 and 4.5 weeks at doses of 0, 12.5, 25, 50, and 100 mg/kg, to different groups of animals, respectively. Correlation analysis indicted that brain ChE inhibition was more accurately reflected by erythrocyte than by plasma ChE inhibition, although all effects were highly correlated. The changes in ChE activity were not paralleled by changes in other parameters of the cholinergic neurotransmission, such as acetylcholine synthesis rate or acetylcholine receptor binding. It is therefore concluded that repeated administration of metrifonate to rats induces a long-lasting inhibition of ChE activity in a dose-related and predictable manner, which is neither subject to desensitization nor paralleled by counterregulatory downregulation of muscarinic or nicotinic receptor binding sites in brain.

Effects of metrifonate, its transformation product dichlorvos, and other organophosphorus and reference cholinesterase inhibitors on Morris water escape behavior in young-adult rats.

Metrifonate is currently under development as a putative cholinergic Alzheimer therapeutic, because it is a prodrug of the long-acting organophosphate cholinesterase (ChE) inhibitor dichlorvos. The aim of this study was to examine whether the transformation of metrifonate to dichlorvos and the resulting indirect inhibition of ChE are required for its previously documented cognition-enhancing properties in a standard Morris water escape task with intact rats. This was done by investigating whether the cognition-enhancing effects of metrifonate could be mimicked by dichlorvos, by the organophosphorus compounds diisopropylfluorophosphate and paraoxon or by structurally unrelated reference ChE inhibitors, such as tetrahydroaminoacridine, E2020, and physostigmine. Metrifonate, and to a lesser degree dichlorvos, and diisopropylfluorophosphate improved the acquisition of the water escape task, whereas paraoxon did not. The dose-response curves of the organophosphorus compounds were bell-shaped with apparent optimal doses of 10 to 30 mg/kg for metrifonate and 0.03 mg/kg for both dichlorvos and diisopropylfluorophosphate. The reference compounds E2020, physostigmine and tetrahydroaminoacridine did not affect learning and memory in the young-adult rat at doses that had previously been reported to mediate cognitive enhancement in deficiency models. Our results question whether the effect of metrifonate is mediated by inhibition of ChE alone and suggest the involvement of an additional, as yet unknown, mechanism of action.

Metrifonate induces cholinesterase inhibition exclusively via slow release of dichlorvos.

Metrifonate, a long-acting cholinesterase (ChE) inhibitor with very low toxicity in warm-blooded animals, inhibits rat brain and serum cholinesterase (ChE) in vitro through its hydrolytic degradation product, dichlorvos. This conclusion is based on the finding that metrifonate-induced ChE inhibition showed the same pH dependence as its reported dehydrochlorination to dichlorvos. The ChE inhibition induced by dichlorvos was not pH dependent. It was mediated by a competitive drug interaction with the catalytic site of the enzyme, which led to irreversible inhibition within several minutes of incubation. After this time, addition of further substrate to the inhibited enzyme was not able to promote drug dissociation and hence enzyme reactivation. Similar characteristics of inhibition, i.e. interaction with the substrate binding site and time-dependent switch to non-competitive inhibition were observed with the reference compound, physostigmine. However, the physostigmine-induced inhibition of ChE could be readily reversed by further substrate addition. Another reference compound, tetrahydroaminoacridine (THA), also induced a reversible inhibition of rat brain and serum cholinesterase, but with a mechanism of action different from that of both dichlorvos and physostigmine in that enzyme inhibition occurred rapidly upon drug addition at an allosteric site on the enzyme surface. It is suggested that the unique slow release plus the slow inhibition of ChE by dichlorvos is responsible for the lower toxicity of metrifonate compared to that of directly acting ChE inhibitors.

Effects of metrifonate on escape and avoidance learning in young and aged rats.

The present study assessed the effects of the indirect acetylcholinesterase inhibitor metrifonate on learning and memory functions in young (3-month-old) and aged (25-month-old) rats. In the shuttle box, metrifonate at a dose of 12.5mg/kg, p.o., 30min before each of the daily acquisition sessions, improved the acquisition of the active avoidance response, whereas a dose of 25mg/kg did not. Metrifonate, 12.5mg/kg, p.o., administered before each of the daily acquisition sessions, also facilitated the acquisition of the Morris water escape task in both young and aged rats: metrifonate-treated rats swam a shorter distance to reach the escape platform than did the vehicle-treated rats. The 3-month-old rats treated with metrifonate did not show the increase in swimming speed over training observed in vehicle-treated animals; no effects of metrifonate were found on the swimming speed of aged rats. In a probe trial carried out immediately after the fifth daily acquisition session, metrifonate treatment did not affect the bias of the aged rats for the quadrant in which the platform had been positioned during acquisition. It is concluded that metrifonate improves performance during the acquisition phase of two aversively motivated learning and memory tasks at the dose of 12.5mg/kg, p.o.