Volker Hinz

Effect of metrifonate on extracellular brain acetylcholine and object recognition in aged rats.

The effects of metrifonate were investigated in 4-6- and 22-24-month-old rats. Extracellular acetylcholine levels were measured by transversal microdialysis in vivo. Baseline extracellular acetylcholine levels in the cerebral cortex and hippocampus were 42% and 60% lower, respectively, in old than in young rats. Old rats did not discriminate between familiar and novel objects. In old rats, metrifonate (80 mg/kg p.o.) brought about 85% inhibition of cholinesterase activity in the cortex and hippocampus, a 4-fold increase in extracellular acetylcholine levels in the cortex only, and restored object recognition. In young rats, metrifonate caused 75% cholinesterase inhibition in the cerebral cortex and hippocampus, a 2-fold increase in cortical and hippocampal extracellular acetylcholine levels, and no effect on object recognition. The slight cholinesterase inhibition following metrifonate (30 mg/kg) in aged rats had no effect on cortical acetylcholine levels and object recognition. In conclusion, metrifonate may improve the age-associated cholinergic hypofunction and cognitive impairment.

Selective intermediate-/small-conductance calcium-activated potassium channel (KCNN4) blockers are potent and effective therapeutics in experimental brain oedema and traumatic brain injury caused by acute subdural haematoma

Early deterioration and death after brain injury is often the result of oedema in the injured and peri‐lesional tissue. So far, no pharmacotherapy is available that exhibits significant brain oedema‐reducing efficacy in patients. We selected two low molecular weight compounds from different chemical classes, a triazole (1‐[(2‐chlorophenyl)diphenylmethyl]‐1,2,3‐triazole) and a cyclohexadiene (methyl 4‐[4‐chloro‐3‐(trifluoromethyl)phenyl]‐6‐methyl‐3‐oxo‐1,4,7‐tetrahydroisobenzofuran‐5‐carboxylate) to characterize their pharmacological properties on KCNN4 channels (intermediate/small conductance calcium‐activated potassium channel, subfamily N, member 4) in vitro as well as in vivo. In vitro we replaced potassium by rubidium (Rb+) and determined Rb+ fluxes evoked by 10 µm of the calcium ionophore A23187 on C6BU1 rat glioma cells. Compared with known KCNN4 blockers, such as clotrimazole (IC50 = 360 ± 12 nm) and charybdotoxin (IC50 = 3.3 ± 1.9 nm), the triazole and cyclohexadiene were considerably more potent than clotrimazole and displayed similar potencies (IC50 = 12.1 ± 8.8 and 13.3 ± 4.7 nm, respectively). In the rat acute subdural haematoma model, both the triazole and cyclohexadiene displayed reduction of brain water content (−26% at 0.3 mg/kg and −24% at 0.01 mg/kg) and reduction of the intracranial pressure (−46% at 0.1 mg/kg and −60% at 0.003 mg/kg) after 24 h when administered as a 4‐h infusion immediately after brain injury. When infarct volumes were determined after 7 days, the triazole as well as the cyclohexadiene displayed strong neuroprotective efficacy (−52% infarct volume reduction at 1.2 mg/kg and −43% at 0.04 mg/kg, respectively). It is concluded that blockade of KCNN4 channels is a new pharmacological approach to attenuate acute brain damage caused by traumatic brain injury.

Neuroprotective and brain edema-reducing efficacy of the novel cannabinoid receptor agonist BAY 38-7271

BAY 38-7271 is a new high-affinity cannabinoid receptor agonist with strong neuroprotective efficacy in a rat model of traumatic brain injury (acute subdural hematoma, SDH). In the present study we investigated CB1 receptor signal transduction by [35S]GTPgammaS binding in situ and in vitro to assess changes in receptor functionality after SDH. Further, we continued to investigate the neuroprotective properties of BAY 38-7271 in the rat SDH and transient middle cerebral artery occlusion (tMCA-O) model as well as the efficacy with respect to SDH-induced brain edema. [35S]GTPgammaS binding revealed minor attenuation of CB1 receptor functionality on brain membranes from injured hemispheres when compared to non-injured hemispheres or controls. In the rat SDH model, BAY 38-7271 displayed strong neuroprotective efficacy when administered immediately after SDH either as a 1 h (65% infarct volume reduction at 0.1 microg/kg) or short-duration (15 min) infusion (53% at 10 microg/kg). When administered as a 4 h infusion with a 5 h delay after injury, significant neuroprotection was observed (49% at 1.0 microg/kg/h). This was also observed when BAY 38-7271 was administered as a 5 h delayed 15 min short-duration infusion (64% at 3 microg/kg). In addition, the neuroprotective potential of BAY 38-7271 was demonstrated in the rat tMCA-O model, displaying pronounced neuroprotective efficacy in the cerebral cortex (91% at 1 ng/kg/h) and striatum (53% at 10 ng/kg/h). BAY 38-7271 also reduced intracranial pressure (28% at 250 ng/kg/h) and brain water content (20% at 250 ng/kg/h) when determined 24 h post-SDH. Based on these data it is concluded that the neuroprotective efficacy of BAY 38-7271 is mediated by multiple mechanisms triggered by cannabinoid receptors.

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.

Metrifonate and dichlorvos: Effects of a single oral administration on cholinesterase activity in rat brain and blood

Cholinesterase activities in rat forebrain, erythrocytes, and plasma were assessed after a single oral administration of metrifonate or dichlorvos. In 3-month-old rats, the dichlorvos (10 mg/kg p.o.)-induced inhibition of cholinesterase reached its peak in brain after 15–45 min and after 10–30 min in erythrocytes and plasma. Cholinesterase activity recovered rapidly after the peak of inhibition, but did not reach control values in brain and erythrocytes within 24 h after drug administration. The recovery of plasma cholinesterase activity, in contrast, was already complete 12 h after dichlorvos treatment. Metrifonate (100 mg/kg p.o.) had qualitatively similar inhibition kinetics as dichlorvos, albeit with a slightly delayed onset. Peak values were attained 45–60 min (brain) and 20–45 min (blood), after drug administration. Apparently complete recovery of cholinesterase activity was noted in both tissues 24 h after treatment. The dose-dependence of drug-induced inhibition of cholinesterase in rat blood and brain was determined at the time of maximal inhibition, i.e., 30 min after dichlorvos treatment and 45 min after metrifonate treatment. The oral ED50 values obtained for dichlorvos were 8 mg/kg for brain and 6 mg/kg for both erythrocyte and plasma cholinesterase. The corresponding oral ED50 values for metrifonate were 10 to 15 times higher, i.e., 90 mg/kg in brain and 80 mg/kg in erythrocytes and plasma. In rats deprived of food for 18 h before drug treatment, the corresponding ED50 values for metrifonate were 60 and 45 mg/kg, respectively, indicating an about two-fold higher sensitivity of fasted rats to metrifonate-induced cholinesterase inhibition compared to non-fasted rats. Compared to 3-month-old rats, 19-month-old rats showed a higher sensitivity towards metrifonate and dichlorvos. At the time of maximal inhibition, there was a strong correlation between the degree of cholinesterase inhibition in brain and blood. These results demonstrate that single oral administration of metrifonate and dichlorvos induces an inhibition of blood and brain cholinesterase in the conscious rat in a dose-dependent and apparently fully reversible manner. While the efficiency of a given dose of inhibitor may vary with the satiety status or age of the animal, the extent of brain ChE inhibition can be estimated from the level of blood ChE activity.

Receptor interaction profile and CNS general pharmacology of metrifonate and its transformation product dichlorvos in rodents

In this study we assessed the possible effects of the putative Alzheimer therapeutic, metrifonate (39, 120, 390 μmol/kg), and its active transformation product, dichlorvos (4.5, 13.6, 45 μmol/kg), on the mammalian central nervous system (CNS). We did this by (1) investigating the receptor interaction profile of the two compounds in a range of in‐vitro radioligand binding assays, and (2) by studying the acute compound effects in rats and mice using a battery of behavioral tests after a single oral administration. Metrifonate and dichlorvos failed to displace various radioligands from their respective receptor binding sites on cell membranes at a concentration of 10 μM. In particular, there was no high‐affinity interaction with muscarinic or nicotinic acetylcholine receptor binding in‐vitro. In the modified Irwin test (rat) both compounds induced transient cholinergic symptoms after oral administration of a single dose of 390 μmol/kg metrifonate and 13.6–45 μmol/kg dichlorvos. The observed symptoms, such as salivation, tremor, and diarrhea, lasted for up to 75 min. In the open field test (rat) metrifonate increased the number of rearings at all doses, whereas dichlorvos had no effect on the parameters tested. Both compounds dose‐dependently reduced the pentylenetetrazole threshold dose in mice. In this test, only the highest dose of metrifonate, but all doses of dichlorvos, caused a significant reduction of the convulsion threshold dose. Metrifonate and dichlorvos did not influence traction ability in mice. Metrifonate and dichlorvos did not influence hexobarbital‐induced anesthesia in mice. Metrifonate induced hypothermia in rats only at the dose of 390 μmol/kg. Dichlorvos did not affect body temperature. No analgesic potential was observed in the hot‐plate test in mice. Furthermore, metrifonate and dichlorvos neither influenced motor coordination nor exhibited any cataleptic potential when administered to rats. Taken together, at cognition‐enhancing doses, metrifonate (39–120 μmol/kg) is safe and well tolerated. The adverse symptoms observed at higher doses, together with the apparent lack of high‐affinity interaction with neurotransmitter receptors in brain tissue and the similar profile of the active transformation product, dichlorvos, support the assumption that these compounds mediate a highly selective activation of the cholinergic system.

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.

Preclinical Pharmacology of Metrifonate: A Promise for Alzheimer Therapy

Among the multiple transmitter deficits which have been described in Alzheimer’s Disease (AD), the degeneration of brain cholinergic cell bodies is the most sensitive, specific and severe, as indicated by the good correlation between the cholinergic pathology and dementia. Therefore, current drug development strategies in AD therapy focus on the enhancement of cholinergic neurotransmission. The most advanced class of compounds in this respect are cholinesterase (ChE) inhibitors which aim to restore the concentration of acetylcholine (ACh) in the synaptic cleft.

The Preclinical Pharmacology of Metrifonate, Along-Acting and Well Tolerated Cholinesterase Inhibitor for Alzheimer Therapy

Progressive degeneration of the cholinergic system is nowadays well recognized as one of the most sensitive and specific hallmarks of Alzheimer’s disease. Numerous approaches to overcome the cholinergic deficit and the resulting impairments in cognitive function have been investigated, including attempts to increase acetylcholine (ACh) synthesis by precursor therapy, replacement of ACh with muscarinic or nicotinic agonists, nerve growth factor therapy to stimulate the outgrowth of cholinergic neurones, and inhibition of ACh breakdown by cholinesterases (ChEs), such as acetyl- and butyryl-ChE. Most progress has been made with the last approach, thanks to the recent discovery of safe and well-tolerated, long-lasting and orally bioavailable ChE inhibitors.