Sasson Cohen

Psychotomimetics as anticholinergic agents—I: 1-Cyclohexylpiperidine derivatives: Anticholinesterase activity and antagonistic activity to acetylcholine

Abstract Phencyclidine (sernyl ® , 1-[(1 -phenylcyclohexyl)piperidinel]) and ten of its derivatives, known for their psychotomimetic activity, are potent competitive inhibitors of butyry-lcholinesterase and acetylcholinesterase. The drugs also protect the enzyme against inactivation by an organophosphate (sarin), presumably by their direct interaction with the active site. In addition to the acetylcholine-like structural factors identifiable in these molecules, the cyclohexyl moiety is considered necessary for the interaction. The drugs are also competitive antagonists of acetylcholine in perfused organs (guinea-pig ileum, frog rectus abdominis) and in the eye of three mammals. This peripheral activity is three orders of magnitudes less potent than that of atropine. Antiacetylcholine activity in the central nervous system was studied through the antidotal effect of THA (tacrine), before and after injection of the drugs. The correlation between the anticholinesterase activity and the CNS activity as well as the structural relation of the drugs to agonists and antagonists of the cholinergic system is discussed

Studies with rasagiline, a MAO-B inhibitor, in experimental focal ischemia in the rat

Summary. Rasagiline, as the mesylate salt (TVP-1012), is a selective, potent, non-reversible MAO-B inhibitor of the propargylamine type. Current cellular and whole animal studies suggested a potential for neuroprotection by rasagiline. Rasagiline in repeat ip doses of 1–3 mg/kg within 16 h, or by sustained iv infusion to maintain a 3-h steady-state at corresponding levels, improved the outcome of permanent middle cerebral artery occlusion (MCAO) in the rat. In five independent studies using different protocols, rasagiline improved neurological severity score (NSS) with respect to saline from a high of 8.96 ± 2.18 (n = 94) at 24 h, and 7.64 ± 2.52 (n ± 49) at 48 h, to a low of 7.13 ± 2.32 (n = 88) at 24 h, and 4.99 ± 2.31 (n = 68) at 48 h. Under the same conditions, there was a decrease in the volume of necrotic brain region determined at 48 h by triphenyl tetrazolium chloride (TTC), from a high of 240 ± 66 (n = 54) to a low of 176 ± 77 mm3 (n = 55); and by MRI scan at 48 h, from a high of 297 ± 62 (n = 25), to a low of 209 ± 63 mm3 (n = 28). Improvement in NSS was more obvious at 48 h post MCAO, at the higher dose, when timing of drug administration was within the interval −30 min to 3 h from MCAO. A 3-h iv infusion of rasagiline caused a maximal reduction in infarct volume of about 49% of control. The (S)enantiomer of rasagiline TVP-1022, not a MAO inhibitor, was less effective, but still significantly different from saline, NSS at 48 h 5.6 ± 2.5 (n = 24) vs. 7.5 ± 2.5 (n = 24), infarct volume 200 ± 64 (n = 24) vs. 240 ± 55 mm3 (n = 24). Selegiline (n = 19) at corresponding ip doses was not different from saline. Dizocilpine decreased infarct volume from 277 ± 65 (n = 20) to 203 ± 52 mm3 (n = 21) but could not improve NSS at 24 or 48 h. In this model, rasagiline could have exerted a neuroprotective effect independent of MAO inhibition.

Sparing by Rasagiline (Tvp-1012) of Cholinergic Functions and Behavior in the Postnatal Anoxia Rat

Rasagiline (N-propargyl-1(R)aminoindan) is a selective and potent MAO-B inhibitor currently under development as the mesylate salt (TVP-1012) for the treatment of various neurologic disorders. Preliminary work in adult and senescent rats, either normal or hypoxia-lesioned, showed that chronic rasagiline treatment improved performance in memory and learning tasks, suggesting some beneficial effect on central cholinergic function. We have now used the postnatal anoxia-lesioned rat as a model of cholinergic dysfunction. In the neonatal rat, anoxia strongly affects the cholinergic system, which has not yet reached full maturation at this state of life. Rasagiline mesylate was administered from day 1 to completion of the study (day 60), first through nursing mother milk until weaning (day 21), then in drinking water, at the rate of 0.5 mg/kg/day. Drug access to the CNS was verified by analysis of MAO activity in brain (at 21 days). Treatment improved the juvenile hyperactivity syndrome associated with anoxia (at day 28). It improved performance in the passive avoidance test to normal control level (at day 40). It improved spatial memory performance in the Morris water maze to normal control level (at day 50). The untreated anoxia group failed in these tasks and was significantly inferior to either the normal control and rasagiline-treated anoxia groups. Determination of ChAT activity in the caudate and hippocampus of rats from each of these groups gave the following results (pmol ACh/mg protein/min). Caudate: normal control, 588 +/- 56; anoxia, 398 +/- 54; rasagiline-treated anoxia, 536 +/- 35. Hippocampus: normal control, 380 +/- 31; anoxia, 275 +/- 47; rasagiline-treated anoxia, 325 +/- 35. Results are mean +/- SD from each of seven to nine different donors in a group. Thus, improvement in memory and learning tasks of the rasagiline-treated anoxia group finds correspondence in the activity of the cholinergic marker ChAT in two brain regions that have prominent cholinergic innervation.

The muscarinic cholinergic receptors in the posterior hypothalamus of hypertensive and normotensive rats

The density of [3H]quinuclidin-3-yl benzilate ([3H]QNB) binding sites in the posterior hypothalamus was determined in spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats at the ages of 1, 3, 11 and 50 weeks. In SHR, even at the early age of 1 week which is prehypertensive, the values obtained were 1.5 times greater than those of age-matched WKY rats. The values of the equilibrium dissociation constant (KD) did not differ between SHR and WKY rats of the same age. In the pons medulla, however, the density of [3H]QNB binding sites was not different between the two strains of rats of matching age. Isolation-induced hypertension in adult Wistar rats and an increase in the density of [3H]QNB binding sites in the posterior hypothalamus were observed to arise concomitantly. A hypothesis is offered whereby a relative increase in ACh receptor sites in the posterior hypothalamus is a primary cause of hypertension in the models considered.

Rasagiline and its (S) enantiomer increase survival and prevent stroke in salt-loaded stroke-prone spontaneously hypertensive rats

Summary. The aim of this study was to determine whether chronic treatment with the selective MAO-B inhibitor rasagiline, N-propargyl-1-(R)-aminoindan (R-PAI), can prevent or delay stroke or improve its outcome in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP). The S-enantiomer of rasagiline, S-PAI a much weaker MAO inhibitor was included in the study in order to expose a possible contribution from MAO inhibition to any beneficial effect by R-PAI. SHRSP were isolated, fed rat stroke prone diet and given 1% NaCl instead of water. Drugs were administered in the drinking fluid for 84 days. Rats were grouped as follows: (1) Untreated control; (2) R-PAI 1 mg/kg/day; (3) R-PAI 3 mg/kg/day; (4) S-PAI 3 mg/kg/day; (5) S-PAI 6 mg/kg/day. Survival, stroke frequency and neurological severity score following stroke were determined. R-PAI at 3 mg/kg/day significantly increased cumulative survival from 56.09 ± 1.77 days in the untreated to 73.6 ± 2.22 days in the R-PAI treated; S-PAI at 6 mg/kg/day to 78.61 ± 2.05 (censored at 84 days). In these groups stroke was delayed, its incidence was decreased and its outcome was less severe than in the control group. Proteinurea observed in the untreated rats and in both lower dose groups of R-PAI and S-PAI was absent in the higher dose groups of both drugs. Histological examination of the brains and kidneys showed that the effects on stroke and survival were associated with decreased infarcts and hemorrhages in the brains and decreased tubular nephropathy and glomerulopathy. The time-dependent rise in systolic blood pressure in the untreated rats was significantly attenuated only in the R-PAI group at 3 mg/kg/day but not in the S-PAI group. There were no significant effects on heart rate. MAO-B activity in the brain was 95% blocked by both doses of R-PAI but only 62% by S-PAI at 6 mg/kg/day. In salt-loaded SHRSP chronic therapy with either R-PAI or S-PAI prevented stroke and severe vascular lesions in the kidney. When stroke did occur its neurological outcome was less severe. The drugs were equipotent when they were given at a ratio of 1 : 2. The mechanism has yet to be elucidated. The differential effects of the drugs on blood pressure and MAO inhibition rule out either effect as the sole explanation.

A new probe for heterogeneity in muscarinic receptors: 2-methyl-spiro-(1, 3-dioxolane-4, 3')-quinuclidine.

The title compound (MSDQ) is a new muscarinic agonist related to 3-acetoxyquinuclidine (3-AcQ) but also of a highly rigid structure. In view of this, it may constitute a probe for the detection of heterogeneity among muscarinic receptors. Indeed, equipotent molar ratios (EPMR) for ACh, 3-AcQ and MSDQ were as follows: guinea pig ileum, 1 : 14 : 240; vasodepressor effect in the cat, 1 : 6 : 188. But EPMR for 3-AcQ and MSDQ as stimulants of the superior cervical ganglion in the cat were 1 : 1 and for the induction of tremors in mice, 9 : 5. No such subtle differences in receptor specificity were detected when the probe used was the 2, 2-diphenyl analogue (DiPSDQ) of MSDQ which was a powerful competitive antagonist in all systems, more potent than atropine, but with a CNS/PNS activity of 1.1 compared to 26 for atropine. In view of this, the use of potent antagonists as probes for muscarinic receptor heterogeneity is questionable.

ATP-evoked membrane responses inXenopus oocytes

Voltage-clamp technique and intracellular injections of drugs were used to study the adenosine triphosphate (ATP)-evoked depolarizing current response in theXenopus laevis oocytes. The depolarizing current was comprised of a fast transient component (D1) followed by a late long-lasting component (D2). It was carried mainly by Cl− ions. The depolarizing current was better elicited by ATP and ADP than by AMP or adenosine and was not blocked either by theophylline (0.2 mM) or by quinidine sulphate (1 mM). The D2 current was sometimes masked by an ATP-evoked K+ hyperpolarizing current which was blocked by theophylline and mediated via P1 purinoceptors. This study suggests that the oocytes membrane embodies at least two different purinoceptors types, each of these types subserves a different set of ionic channels.

Behavioral differences in the developing rat following postnatal anoxia or postnatally injected AF-64A, a cholinergic neurotoxin

Rat pups were submitted postnatally to one of two procedures: a 25-min exposure to 100% nitrogen or an i.c.v. bilateral injection of AF-64A, 2 nmol contained in 1-microliter saline. Throughout further development of either group, their performance in passive and active avoidance tests and in amphetamine-induced stereotype behavior was followed and compared. Both groups exhibited hyperactivity which persisted until 42 days of age in the anoxia group and beyond 120 days in the AF-64A group. Both groups were equally inferior to controls in the passive avoidance test, but only the anoxia group was inferior to controls in the active avoidance test. Amphetamine-induced stereotype behavior was much less pronounced in the anoxia group relative to AF-64A-treated rats or to controls. The results suggest that the lesion induced by the neurotoxin is more specific and less widespread than the one caused by anoxia.

Further characterization of the slow muscarinic responses in Xenopus oocytes

In immature follicular ocytes of the frogXenopus laevis, application of muscarinic agonists evokes a complex response consisting of a fast and a slow Cl currents (the dominant responses), Cl current fluctuations, and a less prominent slow K current. The characteristics of the slow ACh-evoked potassium current were studied using the two-electrode voltage clamp method, and compared to those of the ACh-evoked Cl currents. In experiments designed to study the K current response separately, without the interference of ACh-evoked Cl currents, the holding potential was set close or equal to Cl equilibrium potential (measured as the reversal potential of the ACh-evoked Cl current). The Cl current responses were studied in cells that had negligible K current response. The dose-response curve of the potassium response followed classical Michaelis-Menten kinetics. The dose-response characteristics of the slow ACh-evoked Cl current displayed a positive cooperativity of at least 3. In spite of this difference, kinetic analysis revealed that these two responses, as well as the fast Cl current response that was characterized earlier (Dascal and Landau 1982), had almost identical apparent equilibrium dissociation constants (0.29–0.39 μM), suggesting involvement of a single receptor class. Both K and Cl currents were reduced (to 32–56% of control) by millimolar concentrations of phosphodiesterase (PDE) inhibitors, theophylline and isobutylmethylxanthine. Elevation of extracellular Ca concentration from 1 to 10 mM doubled the K current; depletion of external Ca caused a partial inhibition of this response. The K current was potentiated by 0.1 μM 4-phorbol 12,13-dibutyrate (PDBu). Ca-dependence of the ACh-evoked K current resembles that of ACh-evoked Cl currents, described earlier, and suggests mediation by a similar mechanism, i.e. mobilization of Ca from intracellular stores. On the other hand, most of the features described here are in a sharp contrast to those reported for adenosine-evoked, cAMP-mediated slow K current. Thus, we suggest that purinergic and muscarinic receptors inXenopus follicular oocytes are coupled to potassium channels through different molecular mechanisms.

Histamine-mediated acetylcholine release in the guinea-pig ileum.

The isometric contraction induced by histamine in guinea-pig ileum longitudinal muscle was biphasic in Krebs ([Ca]: 3.36 mM) but monophasic in Tyrode solution ([Ca]: 1.80 mM). The late phase (histamine: 20-400 nM) was common to the two solutions but the early phase (histamine: 2-20 nM) was observed only in Krebs solution. This early phase could be blocked with atropine (0.01-0.2 microM), morphine (0.1, 1 microM), adenosine (5, 20 microM) and tetrodotoxin (0.3 microM) without affecting the late phase. Washout of morphine or adenosine was fast. Neostigmine (100 nM) greatly potentiated the effect of histamine (4 nM) in the early phase, the muscle undergoing almost maximum contraction but also reversible desensitization to doses of histamine less than or equal to 20 nM for as long as 40 min after washout. Beyond this concentration, the preparation responded to increasing doses of histamine as observed in the late phase. It is concluded that low concentrations of histamine that have no observable direct effect on muscle contractility release acetylcholine in the presence of [Ca] 3.36 mM, the early phase being entirely due to release of endogenous acetylcholine.