Pierre J. Mallorga

N-desmethylclozapine, an allosteric agonist at muscarinic 1 receptor, potentiates N-methyl-d-aspartate receptor activity

The molecular and neuronal substrates conferring on clozapine its unique and superior efficacy in the treatment of schizophrenia remain elusive. The interaction of clozapine with many G protein-coupled receptors is well documented but less is known about its biologically active metabolite, N-desmethylclozapine. Recent clinical and preclinical evidences of the antipsychotic activity of the muscarinic agonist xanomeline prompted us to investigate the effects of N-desmethylclozapine on cloned human M1-M5 muscarinic receptors. N-desmethylclozapine preferentially bound to M1 muscarinic receptors with an IC50 of 55 nM and was a more potent partial agonist (EC50, 115 nM and 50% of acetylcholine response) at this receptor than clozapine. Furthermore, pharmacological and site-directed mutagenesis studies suggested that N-desmethylclozapine preferentially activated M1 receptors by interacting with a site that does not fully overlap with the acetylcholine orthosteric site. As hypofunction of N-methyl-d-aspartate (NMDA) receptor-driven neuronal ensembles has been implicated in psychotic disorders, the neuronal activity of N-desmethylclozapine was electrophysiologically investigated in hippocampal rat brain slices. N-desmethylclozapine was shown to dose-dependently potentiate NMDA receptor currents in CA1 pyramidal cells by 53% at 100 nM, an effect largely mediated by activation of muscarinic receptors. Altogether, our observations provide direct evidence that the brain penetrant metabolite N-desmethylclozapine is a potent, allosteric agonist at human M1 receptors and is able to potentiate hippocampal NMDA receptor currents through M1 receptor activation. These observations raise the possibility that N-desmethylclozapine contributes to clozapines clinical activity in schizophrenics through modulation of both muscarinic and glutamatergic neurotransmission.

Ontogenetic changes in GABA modulation of brain benzodiazepine binding

Abstract The ontogenesis of the [3H]diazepam binding sites and their modulation by gamma-aminobutyric acid has been studied in rat brain cortex. Specific binding of [3H]diazepam (calculated in fmole per mg of wet weight of tissue) is 2% of adult binding level, when measured at 16 days of gestation, 20% at birth and reaches the adult levels approximately 3 weeks after birth. An increase in [3H]diazepam binding after the in vitro addition of GABA to the extensively washed rat brain membrane preparation was observed at each stage of development and was due to a change in affinity without significant alteration in the total number of [3H]diazepam binding sites. Furthermore, the gamma-aminobutyric acid effect was found to decrease with age.

Interaction of diphenylhydantoin and benzodiazepines in the CNS.

Using extracellular unit recording and microiontophoretic techniques, the anticonvulsant diphenylhydantoin (DPH) was found to increase the physiological efficacy of the benzodiazepines. This increased biological effect could be correlated with an enhanced specific binding of benzodiazepines measured in vivo following pretreatment of rats with DPH. The increased binding of benzodiazepines is due to an increase in the total number of benzodiazepine binding sites without an alteration in the affinity of these sites for [3H]diazepam. The data show that the effects of DPH on benzodiazepine binding are qualitatively different and independent from the effects of gamma-amino-butyric acid. Based on the dose-responsive relationship between benzodiazepine binding effects and the anticonvulsant activity of DPH and reports of other convulsant, anticonvulsant compounds which alter benzodiazepine binding, it is suggested that the benzodiazepine binding site may be relevant to convulsant-anticonvulsant activity.

Effects of typical and atypical antipsychotics on human glycine transporters

Augmentation strategy in the treatment of schizophrenia with the NMDA receptor co-agonist glycine has demonstrated significant improvement in patient symptoms. Interestingly, the therapeutic efficacy of glycine was more consistent among patients that were not co-administered clozapine suggesting that clozapine modulates glycine levels in brain. Since cerebral glycine concentration in the vicinity of NMDA receptors is thought to be controlled by the glia expressed glycine transporter type 1 (GlyT1), the effects of several typical and atypical antipsychotics on glycine uptake were examined in human placenta choriocarcinoma (JAR) cells expressing human GlyT1a. The selectivity of these compounds was investigated by measuring their inhibitory potency at the closely related glycine transporter type 2 (GlyT2). Typical antipsychotics haloperidol, thioridazine and chlorpromazine non-selectively inhibited [(14)C]glycine uptake mediated by GlyT1a and GlyT2 with potency of 9-21 microM. The atypical antipsychotic, clozapine antagonized glycine transport by human GlyT1a with an IC(50) of 100 microM and was weaker at recombinant GlyT2. Its main metabolites, N-desmethylclozapine and clozapine N-oxide were very weak inhibitors at all glycine transporters. Similarly, olanzapine did not potently block GlyT1a- and GlyT2-mediated uptake. Detailed kinetic analysis of hGlyT1a in the presence and absence of haloperidol and clozapine revealed that both drugs were not competitive inhibitors of glycine uptake. Data also indicated that these compounds did not interact with the Na(+) and Cl(-) sites of hGlyT1a. Our results have revealed the existence of an inhibitory interaction between some antipsychotics and hGlyT1a and raise the possibility that these drugs could interact with GlyT1 function at therapeutic doses.

L-662,583 is a topically effective ocular hypotensive carbonic anhydrase inhibitor in experimental animals

1 L‐662,583 was a potent inhibitor in vitro of purified, human erythrocyte carbonic anhydrase II, possessing an IC50 of 0.7 nm. The IC50 values for MK‐927, acetazolamide and methazolamide were 13.0 nm, 10.8 nm and 21.2 nm, respectively. 2 A 1 h pretreatment with one 50 μl drop of a 0.1% solution of L‐662,583 blocked carbonic anhydrase activity in a homogenate of the iris + ciliary body of albino rabbits by 63%. Similar treatment with 0.1% suspensions of acetazolamide and methazolamide elicited inhibitions of 30% and 20%, respectively. This ex vivo model indirectly assesses the ability of an agent to enter the rabbit eye. 3 Concentrations of L‐662,583 in the cornea, aqueous humour and iris + ciliary body of albino rabbits were determined by h.p.l.c. at predetermined times after the instillation (one drop of 50 μl) of a 2% solution of L‐662,583. Peak levels for cornea (47.4 μg g−1), aqueous humour (4.51 μg ml−1) and iris + ciliary body (9.61 μg g−1) occurred at 0.5, 2 and 1 h after instillation, respectively. 4 The experimentally elevated intraocular pressure of the right eye of rabbits, induced by prior intraocular injection of α‐chymotrypsin, was maximally decreased by 4.5 mmHg, 6.2 mmHg and 9.8 mmHg after the instillation (one drop of 50 μl) of 0.01%, 0.1% and 0.5% solutions of L‐662,583, respectively. All three concentrations lowered intraocular pressure at all time points from 1 h up to and including 5 h, the last recorded time point. The unilateral instillation of L‐662,583 (0.5%) into the contralateral, left eye failed to lower the elevated intraocular pressure of the untreated, right eye. This finding indicates that the site of action of topically applied L‐662,583 in this paradigm is local. The ocular normotensive, albino rabbit was much less susceptible than the ocular hypertensive rabbit to the intraocular pressure lowering effect of topically applied L‐662,583, with a 2% solution maximally decreasing intraocular pressure by 2.3 mmHg. 5 Unilateral ocular hypertension was elicited in the right eye of sedated, cynomolgus monkeys by argon laser‐induced photocoagulation of the trabecular meshwork. The instillation (one drop of 50 μl) of L‐662, 583 (2%) significantly lowered the elevated intraocular pressure of the right eye at all time points from 1 h up to and including 5 h. The maximum decline was 8.3 mmHg at 3 h and this represented a reduction of 23% from the corresponding baseline value of 36.8 mmHg. The intraocular pressure of the hypertensive, right eye was maximally decreased by 4.1 mmHg and 4.8 mmHg after the instillation of 0.5% and 1% solutions of L‐662,583, respectively. Like the rabbit, the normotensive eye of cynomolgus monkeys was more resistant than the hypertensive eye to the ocular hypotensive action of L‐662, 583, as indicated by the inability of 0.5% and 1% solutions of the agent to lower intraocular pressure. L‐662,583 (2%) maximally reduced the intraocular pressure of normotensive monkey eyes by 2.4 mmHg at 2 h. 6 L‐662,583 is structurally different from MK‐927, a carbonic anhydrase inhibitor that lowers the intraocular pressure of glaucoma patients following the instillation of a 2% solution. These preclinical observations indicate that L‐662,583, like MK‐927, is a water‐soluble carbonic anhydrase inhibitor which, on topical administration, lowers intraocular pressure by virtue of an action confined to within the eye.

Development of a scintillation proximity assay for analysis of Na+/Cl- -dependent neurotransmitter transporter activity.

Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [14C]glycine was time dependent and saturable with a Michaelis-Menten constant (Km) of 27+/-3 microM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, respectively. The [14C]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2+/-1.7 microM and fully inhibited by ALX-5407 (IC50=522 +/-83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors.

Differences in the β-adrenergic responsiveness between high and low passage rat glioma C6 cells

Responsiveness to catecholamines was studied in two different strains of rat glioma C6 cells. The C6 cells of low passage possessed a high capacity to accumulate cyclic AMP in response to (-)-isoproterenol. Cholera toxin was also able to stimulate cyclic AMP accumulation in these cells. High passage C6 cells were unresponsive to (-)-isoproterenol or to cholera toxin except in the presence of a high concentration of phosphodiesterase inhibitor. The affinity of beta-adrenergic receptors on both strains for (-) [3H] dihydroalprenolol was similar; however, C6 low passage possessed several times the number of beta-adrenergic receptors found in C6 high passage. This difference correlated with the difference found in (-)-isoproterenol-stimulated adenylate cyclase between C6 low passage and high passage. The sodium fluoride-stimulated adenylate cyclase was similar in both strains. Cyclic AMP phosphodiesterase activity was 2-3 times higher in homogenates of C6 high passage than in low passage. In intact cells, the rate of breakdown of cyclic AMP was 5-times faster in C6 high passage than in low passage. Thus, differences in beta-adrenergic receptor number and phosphodiesterase activity explain in part the lack of responsiveness of C6 high passage. Our studies indicate that continuous subculturing of rat glioma C6 cells led to complex alterations in the beta-adrenergic receptor-adenylate cyclase system.

Characterization of [3H]forskolin binding sites in the iris-ciliary body of the albino rabbit

[3H]Forskolin binding sites were identified using membranes prepared from the iris-ciliary body of adult, albino rabbits. Scatchard analysis of saturation binding experiments demonstrated that [3H]forskolin bound to a single population of high affinity sites. The Kd and Bmax values were 8.7 +/- 0.9 nM and 119.0 +/- 30.9 fmol/mg prot. using membranes prepared from frozen tissue and 17.0 +/- 6.2 nM and 184.4 +/- 47.2 fmol/mg prot. using fresh tissue. The binding of [3H]forskolin was magnesium-dependent. The Bmax was enhanced by sodium fluoride and Gpp(NH)p, a nonhydrolyzable guanine nucleotide analog. Forskolin was the most potent inhibitor of [3H]forskolin binding; two commercially-available analogs were weaker inhibitors. In an adenylate cyclase assay, there was the same rank order of potency to enhance enzyme activity. Based upon binding affinities, magnesium-dependence, sensitivity to sodium fluoride and Gpp(NH)p, rank order of potencies of analogs and correlation of binding with adenylate cyclase activity, these studies suggest that the [3H]forskolin binding site in the iris-ciliary body is similar to the binding site in other tissues.