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Structure-activity studies of 4-substituted-2,5-dimethoxyphenethylamines led to the discovery of 2,5-dimethoxy-4-thiotrifluoromethylphenethylamines, including CYB210010, a potent and long-acting serotonin 5-HT 2 receptor agonist. CYB210010 exhibited high agonist potency at 5-HT 2A and 5-HT 2C receptors, modest selectivity over 5-HT 2B , 5-HT 1A , 5-HT 6 , and adrenergic α 2A receptors, and lacked activity at monoamine transporters and over 70 other proteins. CYB210010 (0.1-3 mg/kg) elicited a head-twitch response (HTR) and could be administered subchronically at threshold doses without behavioral tolerance. CYB210010 was orally bioavailable in three species, readily and preferentially crossed into the CNS, engaged frontal cortex 5-HT 2A receptors, and increased the expression of genes involved in neuroplasticity in the frontal cortex. CYB210010 represents a new tool molecule for investigating the therapeutic potential of 5-HT 2 receptor activation. In addition, several other compounds with high 5-HT 2A receptor potency, yet with little or no HTR activity, were discovered, providing the groundwork for the development of nonpsychedelic 5-HT 2A receptor ligands.

Nsp1 is one of the first proteins expressed from the SARS-CoV-2 genome and is a major virulence factor for COVID-19. A rapid multiplexed assay for detecting the action of Nsp1 was developed in cultured lung cells. The assay is based on the acute cytopathic effects induced by Nsp1. Virtual screening was used to stratify compounds that interact with two functional Nsp1 sites: the RNA-binding groove and C-terminal helix-loop-helix region. Experimental screening focused on compounds that could be readily repurposed to treat COVID-19. Multiple synergistic combinations of compounds that significantly inhibited Nsp1 action were identified. Among the most promising combinations are Ponatinib, Rilpivirine, and Montelukast, which together, reversed the toxic effects of Nsp1 to the same extent as null mutations in the Nsp1 gene., (© 2022. The Author(s).)

Purpose: Encenicline (EVP-6124) is a selective α7 nicotinic acetylcholine receptor partial agonist being developed for cognitive impairment in Alzheimer's disease and schizophrenia. We report on 2 single-dose studies to assess the relative bioavailability, pharmacokinetic profile, tolerability, and cognitive effects of encenicline in healthy volunteers., Methods: A single ascending-dose study assessed the safety, tolerability, pharmacokinetic, and pharmacodynamic profiles of encenicline in healthy male volunteers. Subjects received a single 1-, 3.5-, 7-, 20-, 60-, or 180-mg oral solution dose of encenicline or placebo. A second single-dose, randomized, open-label, 3-period, crossover study in healthy male and female subjects compared the relative bioavailability of a 1-mg oral capsule versus a 1-mg oral solution dose of encenicline and evaluated the effects of food and sex on encenicline pharmacokinetic profile., Findings: In the first study, encenicline was well tolerated and dose-proportional increases in C(max) (mean range 0.59-100 ng/mL) and AUC0-∞ (mean range 45.6-8890 ng·h/mL) were observed over a 1- to 180-mg dose range. Procognitive effects on the Digit Symbol Substitution Test were maximal at the 20-mg dose. In the second study, encenicline 1-mg oral capsules and oral solution were bioequivalent and there was no observed food effect on encenicline pharmacokinetic profile with the 90% confidence intervals of the treatment ratios for both comparisons (ie, capsule to solution and fed to fasted) for Cmax and AUC being within 80% to 125%. A 30% to 40% higher encenicline exposure in female subjects than respective values in male subjects was consistent with a 33% higher weight of the male subjects. No clinically relevant safety profile or tolerability effects of encenicline were observed., Implications: Encenicline was well tolerated at single doses up to 180 mg, and doses as low as 1 mg had dose- and time-dependent pharmacodynamic effects on the central nervous system. Oral capsule and solution were bioequivalent and were not affected by food. Although a sex effect on pharmacokinetic profile was observed, it was attributable to weight differences. Clinical Trial Registration at EudraCT: 2006-005623-42 and EudracT: 2008-000029-20., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Electroconvulsive therapy (ECT) remains the treatment of choice for drug-resistant patients with depressive disorders, yet the mechanism for its efficacy remains unknown. Gene transcription changes were measured in the frontal cortex and hippocampus of rats subjected to sham seizures or to 1 or 10 electroconvulsive seizures (ECS), a model of ECT. Among the 3500-4400 RNA sequences detected in each sample, ECS increased by 1.5- to 11-fold or decreased by at least 34% the expression of 120 unique genes. The hippocampus produced more than three times the number of gene changes seen in the cortex, and many hippocampal gene changes persisted with chronic ECS, unlike in the cortex. Among the 120 genes, 77 have not been reported in previous studies of ECS or seizure responses, and 39 were confirmed among 59 studied by quantitative real time PCR. Another 19 genes, 10 previously unreported, changed by <1.5-fold but with very high significance. Multiple genes were identified within distinct pathways, including the BDNF-MAP kinase-cAMP-cAMP response element-binding protein pathway (15 genes), the arachidonic acid pathway (5 genes), and more than 10 genes in each of the immediate-early gene, neurogenesis, and exercise response gene groups. Neurogenesis, neurite outgrowth, and neuronal plasticity associated with BDNF, glutamate, and cAMP-protein kinase A signaling pathways may mediate the antidepressant effects of ECT in humans. These genes, and others that increase only with chronic ECS such as neuropeptide Y and thyrotropin-releasing hormone, may provide novel ways to select drugs for the treatment of depression and mimic the rapid effectiveness of ECT.

Mental illness affects millions of patients and has been shown to have multiple genetic components. The interaction of environmental factors, such as stress, with the expression of genetic susceptibilities has hampered the development of novel and effective drug treatments. A new approach is described that can discover novel classes of drugs for the treatment of the underlying causes of these diseases rather than their symptoms. This approach screens drug candidates according to their ability to alter the expression of multiple genes in a multiparameter high-throughput screening assay (MPHTS SM) that does not require a prori knowledge of the targets of screening assays. Clinical development of drug candidates will be pursued through partnerships with pharmaceutical companies and/or large biotechnology companies.

Schizophrenia is a major health problem that affects 2 million individuals in the United States. Antipsychotics offer considerable symptomatic relief and, although commonly discovered by screening with single biological targets, most interact with multiple receptors and signaling pathways. Considerable evidence from family and twin studies demonstrates genetic components and multiple chromosomal regions associated with schizophrenia. The polygenic nature of schizophrenia and multiple mechanisms for most effective agents indicate the need for broader approaches to target identification. Gene expression profiling of post-mortem human brain tissue simultaneously reveals the expression of many thousands of genes. A comparison of tissue from normals and patients provides a 'disease signature' of aberrantly expressed genes. 'Drug signatures' are the gene expression changes of cultured human or animal neurons treated with psychiatric drugs, and from animals chronically treated with these drugs. A selection of genes from disease and drug signatures can create a set of targets whose changes may better predict disease and its treatment by effective agents. This multi-parameter high throughput screening (MPHTS(SM)) approach evaluates the mRNA expression pattern of cultured cells exposed to candidate compounds. Compounds that normalize genes altered in schizophrenia may better address its underlying causes. Drugs that mimic gene expression changes that are consistently altered by effective antipsychotic agents provide a drug improvement strategy if efficacy is enhanced or side effects are attenuated.

The massive effort to sequence the human, mouse, rat, nematode (Caenorhabditis elegans), fruit fly (Drosophila), zebra fish, yeast (Saccharomyces cerevisiae), fungal (Candida albicans and Aspergillus fumigatus) and several bacterial genomes has produced a flood of sequence data. Of the more than 100,000 human genes and thousands from other organisms, many partial sequences and several completed microbial genomes are available in both public and private databases. However, elucidation of function has been achieved for only a very small portion and an even smaller percentage have been validated as drug targets. Many companies interested in identifying new drug targets also see this bounty of opportunity as a major challenge. The raw sequence data say little about the importance of the gene and nothing about its potential as a target for drug discovery. Since 1994, a new term, 'functional genomics', has entered our lexicon. Functional genomics, which in effect is 'high-throughput biology', was originally focused on understanding gene function by studying the genes of simpler organisms, such as the nematode, C. elegans. As the genes from a number of organisms are highly conserved across species, it is believed that studying these basic systems can yield valuable insights for drug companies interested in targeting therapeutics for the higher organisms. More recently, the approach to functional genomics has expanded to include study of gene function in organisms to be targeted for therapeutic intervention. This new approach was the theme of the Functional Genomics Conference: From Identifying Proteins to Faster Drug Discovery held in Washington DC on March 10 and 11, 1998. The organisers (NMHCC) hoped that the breadth of the conference topics would reflect the complexities of the modern drug discovery process and covered technologies from gene chips, bioinformatics, disease models, protein discovery and expression, target validation, high-throughput screening for genes of unknown function, to integration of the drug discovery process. The two day conference placed emphasis on cutting edge technology solutions and the development of high-throughput tools to address the emerging opportunities in genome-based drug discovery.

Some new phenothiazines have been synthesized on the basis of previous studies. The anticancer activity of "half-mustard type" phenothiazines was investigated on sixty different cancer cell lines in vitro. The percentage of growth (PG), 50% inhibition of growth (GI50), the tumor growth inhibition (TGI) and the concentration required for 50% lethality of cells (IC50) were examined and calculated in the presence of various (from 10(-4) to 10(-8) M) concentrations of phenothiazine alkylurea derivatives. The following cell lines were involved in the study: 6 leukemia, 9 non-small-cell lung cancer, 7 colon cancer, 6 central nervous system cancer, 8 melanoma, 6 ovarian cancer, 8 renal cancer, 2 prostate and 8 breast cancer cell lines. The antileukemic activity of four chloroethyl-substituted phenothiazine-alkylureas was shown by considerable growth inhibition, in the 10(-5) M range, of the six different leukemia cell lines. The 50% inhibition of growth was nearly the same for the four compounds on all cell lines. Tumor growth inhibition (TGI) and IC50 value to cells varied from -4.0 to -4.66. The two derivatives with the butylene bridge were more effective than propylene linked compounds against the CCRP-CEM, HL60 (TB), K-562 and MOLT-4 cell lines. However, the anti-leukemic activity of the derivatives was nearly the same for RPMT 8226 and SR cell lines. The substituent at the 2- position of phenothiazine ring and the length of the linker between the side chain nitrogen and the phenothiazine ring system are apparently important for antileukemic activity. Four of the 9 non-small-cell lung cancer cell lines were sensitive, while the other 5 cell lines were not. The compounds had a slight growth inhibitory effect on colon cell carcinoma and melanoma cells in which case the butylene linker seemed to be more effective than the propylene linker. At the same time, all of the compounds were weak or mostly inactive on cancer cells from the central nervous system. One ovarian cancer line of the 6, the IGROVI was sensitive to butylurea phenothiazines, however, the other five were not sensitive at all. The difference in the sensitivity of various renal cell carcinomas was significant: 5 lines were not sensitive, three of them (786-0, RXF-393 and TK-10) were sensitive to only butylene-substituted phenothiazine-ureas, propylene substitution resulted in ineffective compounds. The compounds were not able to inhibit the 2 prostate and 4 breast cancer cell lines, even at 10(-4) M. It was interesting that propylene-linked ureas were more effective than butylene-linked derivatives on MCF-7, but butylene-linked derivatives were more effective than propylene-linked compounds on MDA MB-231 and MDA-N. In addition, MDA MB 435 was more sensitive to the trifluoromethyl derivatives than the compounds without this substituent. Since the phthalimido-alkyl phenothiazines were not active at the first level of prescreen, these compounds were omitted from this study. The drug sensitivity of some cancer cell lines was not uniform for the different groups, therefore we postulate that the resistance can be related to some kind of (existing) drug-efflux mechanism. Apparently, the tumor specificity of phenothiazine alkylureas is more related to the leukemia specificity of alkylureas than to any CNS or lung specificity of phenothiazines.

The antitumor effects of "half-mustard type" phenothiazines were studied on 57 different tumor cell lines, including leukemias, non-small lung cancer, colon, central nervous system, ovarian, renal, breast, and prostate cancer, as well as melanoma cell cultures. Alkyl-urea derivatives of phenothiazines displayed in vitro antitumor activity. The phenothiazine phthalimido derivatives (1-6) were not active on the majority of cancer cell cultures. In contrast, propylureas (9, 11) were active against some leukemia cell types. Only two compounds with the butylene [(CH2)4] linker (10, 12) were active against non-small lung cancer cells. Compounds containing the propylene linker were less effective. On colon cancer lines, tumor cells from the central nervous system and on melanoma cells the same compounds were effective, however, having substituents at the 2-position of phenothiazine seems to be important. Surprisingly, the majority of ovarian cancer cell lines (except one type, IGROVI) and five of eight renal cancer lines were not sensitive to these phenothiazine derivatives. The two butylene linked phenothiazine ureas (10, 12) had moderate antiproliferative action on two renal cancer cell lines. The prostate cancer and some breast cancer cell lines were not sensitive. Nevertheless some breast cancer cell lines were apparently sensitive to CF3-substituted phenothiazine alkylureas. On the basis of these experiments one may postulate that in the case of insensitive cells an mdr-gene encoded multidrug resistance efflux pump is responsible for the resistance. The selectivity or organ cell specificity of the effective phenothiazines will be targeted for improvement in further studies, in order to avoid the general cytotoxic effects of "half mustard type" phenothiazines.

Twelve different "half-mustard type" phenothiazines were newly synthesized and tested on seven AIDS-related lymphoma (ARL) tumor cell lines, one leukemia CCRF-CEM cell culture and five different lymphoma lines; RL, KD-488, AS283, PA682 and SU-DHL-7 cell lines. The alkylene-urea substituted phenothiazines affected the growth and inhibited the growth rate of AIDS-related lymphoma cells. The Cl-substituent at the 2-position was more effective than the CF3 substitution. In AIDS-related leukemia also the compounds with Cl at the 2-position with propylene or butylene linkers, -(CH2)3- and -(CH2)4-, respectively, were more effective than the CF3 substituted compounds. Two of the six phenothiazine-substituted alkyl-urea derivatives, i.e., 1-(2-chloroethyl)-3-(2-chloro-10H-phenothiazin-10-yl)propyl-l-urea (9, GI50 = -5.66, TGI = -5.04) and 1-(2-chloroethyl)- 3-(2-chloro-10H-phenothiazin-10-yl)butyl-1-urea (10, GI50 = -5.61, TGI = -5.12) exhibited antitumor activity for AIDS-related leukemia and five AIDS-related lymphomas. The trifluoromethyl-substituted derivatives were not as effective on AIDS-related tumor cell lines. Apparently, the substituent at the 2-position on the phenothiazine and the alkylene number of the linker attached to the nitrogen of the phenothiazine ring have an important role in the compound's antitumor effects on AIDS-related leukemia and lymphomas.

Glycine receptor antagonists have been proposed to have multiple therapeutic applications, including the treatment of stroke, epilepsy, and anxiety. The present study compared the biochemical and behavioral profiles of two strychnine-insensitive glycine receptor antagonists, MDL 100,458 (3-(benzoylmethylamino)-6-chloro-1H-indole-2- carboxylic acid) and MDL 102,288 (5,7-dichloro-1,4-dihydro-4-[[[4- [(methoxycarbonyl)amino]phenyl]sulfonyl]imino]-2-quinolinecarboxylic acid monohydrate). Both compounds potently inhibited [3H]glycine binding to rat cortical/hippocampal membranes (Ki = 136, 167 nM, respectively) without showing significant activity in 18 other receptor binding assays. In an in vitro functional assay, both compounds completely antagonized N-methyl-D-aspartate (NMDA)-stimulated cGMP accumulation in rat cerebellar slices. However, in contrast to their equipotency in the glycine receptor assay, MDL 100,458 was approximately 6-fold more potent than MDL 102,288 in the cGMP assay (IC50 values = 1.25, 7.8 microM, respectively). Behavioral tests demonstrated that MDL 102,288 and MDL 100,458 exhibited strikingly different in vivo profiles. MDL 100,458 antagonized audiogenic seizures in DBA/2J mice (ED50 = 20.8 mg/kg i.p.), whereas MDL 102,288 was without effect in the dose range tested (ED50 > 300 mg/kg i.p.). Central nervous system penetration did not appear to account for this difference. For example, MDL 102,288 was not active following direct intracerebroventricular administration (ED50 > 16 micrograms; vs. 0.78 microgram for MDL 100,458). In a test of anxiolytic activity, MDL 102,288 reduced separation-induced ultrasonic vocalizations in rat pups (ED50 = 6.3 mg/kg i.p.) whereas MDL 100,458 was only weakly active (ED50 = 80.8 mg/kg i.p.). Furthermore, the anxiolytic effect of MDL 102,288 was selective in that it occurred at doses that did not produce motoric disruption as measured by an inclined-plane test (ED50 > 160 mg/kg; therapeutic index > 25.4). In contrast, the anxiolytic activity of MDL 100,458 was non-selective in that it occurred at doses that also produced motoric disruption (ED50 = 57.7 mg/kg; therapeutic index = 0.7). Thus, two glycine receptor antagonists which have similar in vitro binding profiles as selective ligands for the strychnine-insensitive glycine receptor, demonstrate different in vitro and in vivo functional profiles. The reason for these differences is not clear, though one possibility could be that the compounds may act on different NMDA receptor subtypes. These data support the possibility that different glycine receptor antagonists may have different therapeutic targets.

The correlation between the clinical activity of antipsychotic agents and their affinity for the D2 dopamine receptor has been the mainstay of the hypothesis that schizophrenia is due to excessive dopaminergic function. More recently, the unique clinical profile of the atypical antipsychotic clozapine has been proposed to involve actions on additional receptor systems. In particular, the high affinity of clozapine for the 5HT2A receptor subtype has been suggested to contribute to its reduced side-effect liability, greater efficacy and its activity in therapy-resistant schizophrenia. We have used the highly selective 5-HT2A antagonist MDL 100,907 to explore the contribution of 5-HT2A receptor blockade to antipsychotic activity. Biochemical, electrophysiological and behavioral studies reveal that selective 5HT2A receptor antagonists have the preclinical profile of an atypical antipsychotic. The limited clinical evidence available also suggests that compounds producing 5-HT2A receptor blockade are effective, in particular, against the negative symptoms of schizophrenia.

Based on mechanistic understandings, molecular modeling and extensive quantitative structure-activity relationships, appropriately substituted haloallylamine derivatives were designed as potential mechanism-based inhibitors of MAO and/or SSAO. Potent inhibition of MAO-B and SSAO occurred with fluoroallylamines whereas chloroallylamines, such as MDL 72274A ((E)-2-phenyl-3-chloroallylamine hydrochloride), were selective and potent inhibitors of SSAO. MDL 72974A (E)-2-(4-fluorophenethyl)-3-fluoroallylamine hydrochloride is a potent (IC50 = 10(-9) M) inhibitor of both MAO-B and SSAO, with 190-fold lower affinity for MAO-A. In clinical studies, oral doses as low as 100 micrograms produced substantial inhibition of platelet MAO-B. Essentially complete inhibition occurred at 1 mg with the effect lasting 6-10 days. One or 4 mg MDL 72974A given daily for 28 days to 40 Parkinson's patients treated with L-dopa produced statistically significant reductions in the Unified Parkinson's Disease Rating Scale. MAO-B inhibitors, such as MDL 72974A and L-deprenyl, offer the potential of being neuroprotective in Parkinson's Disease and other neurogenerative disorders. Concommitant inhibition of SSAO may provide additional, but as yet unproven, advantages over pure inhibitors of MAO-B.

The bioprecursor amino acid MDL 72394 is decarboxylated by aromatic L-amino acid decarboxylase (AADC) to liberate MDL 72392, an irreversible selective MAO-A inhibitor. Pretreatment with the AADC inhibitor carbidopa, which does not penetrate the brain-blood barrier, prevents the liberation of the MAO-A inhibitor outside the brain and results in exclusive inhibition of brain MAO-A. We found that systemic administration of MDL 72394 (0.5 mg/kg, i.p.) stimulated rat pineal melatonin biosynthesis. Carbidopa, in a dose-dependent manner, attenuated or completely prevented MDL-induced stimulation of melatonin biosynthesis in the pineal gland located outside the blood-brain-barrier.

MDL 72,974A [(E)-2-(4-fluorophenethyl)-3-fluoroallylamine, hydrochloride] was designed to be a selective, mechanism-based irreversible inhibitor of monoamine oxidase type B (MAO-B). The compound is a potent, selective MAO-B inhibitor in vitro and in vivo. In vitro studies revealed an IC50 value (MAO-B) of 3.6 nM with 189-fold selectivity compared to MAO-A. In rats, profound inhibition of MAO-B was achieved after a single oral dose with an ED50 of 0.18 mg/kg; a dose 44 times this amount was required to inhibit MAO-A by 50%. Selectivity was maintained following chronic dosing. MDL 72,974A had minimal sympathomimetic effects and did not potentiate the cardiovascular effects of tyramine, even at 50 times the MAO-B inhibiting dose. This inhibitor was equally effective and well-tolerated in man. In human volunteers, potent inhibition of platelet MAO-B activity was observed at submilligram doses (ED50 = 90 micrograms) following a single oral dose. Upon multiple oral doses of 100 micrograms, as much as 80% of MAO-B could be inhibited. In phase II studies, MDL 72,974A is proving to be a useful adjunct to conventional therapy. Patients (250) with Parkinson's disease, treated once daily with either 1 or 4 mg, together with L-Dopa and a decarboxylase inhibitor (MadoparR or SinemetR), saw significant improvements in symptoms compared with those on standard therapy without the inhibitor.

Several lines of evidence have suggested a link between serotonergic and dopaminergic systems in the brain. The interpretation of much of these early data needs careful reevaluation in light of the recent understanding of the plethora of serotonin receptor subtypes, their distribution in the brain and the new findings with more selective serotonin antagonists. Electrophysiological, biochemical and behavioral evidence obtained using highly selective antagonists of the 5-HT2 or 5-HT3 receptor subtypes, MDL 100,907 or MDL 73,147EF, respectively, supports the thesis that serotonin modulates the dopaminergic system. This modulation is most evident when the dopaminergic system has been activated.

Disrupted glycinergic inhibition in the brainstem and spinal cord may contribute to some of the alterations in reflex control seen in patients with spastic disorders. MDL 27,531, which acts functionally like a glycine agonist in its capacity to selectively reverse seizures produced by the glycine antagonist strychnine, was evaluated in a model of spinal injury-induced reflex dysfunction. Rats recovering chronically from complete spinal cord transections exhibited intermittent contractions of the paralyzed hindlimbs, as measured with an automated apparatus. MDL 27,531 selectively decreased these hindlimb contractions, as did the clinically demonstrated antispastic agent clonidine. In its therapeutic dose range, clonidine, but not MDL 27,531, produced ataxia in non-transected rats. These data suggest that MDL 27,531 may be a useful therapeutic agent for the treatment of dysfunctions of reflex control seen in spastic disorders of spinal origin, with potentially fewer side effects than are seen with existing drug therapies.

The N-methyl-D-aspartate (NMDA)-preferring glutamate receptor subtype possesses, in addition to the recognition site for glutamate, a binding site for glycine. We report here on the pharmacological properties of 3-(4,6-dichloro-2-carboxyindol-3-yl)-propionic acid (MDL 29,951) and 4-carboxymethylamino-5,7-dichloroquinoline-2-carboxylic acid (MDL 100,748), two novel glycine antagonists of NMDA receptor activation in vitro and in vivo. We have measured in parallel the effects of two previously described glycine antagonists, 7-chlorokynurenic acid and 5,7-dichlorokynurenic acid. All were potent inhibitors of [3H]glycine binding. Ki values (microM) were 0.36 (7-chlorokynurenic acid), 0.08 (5,7-dichlorokynurenic acid), 0.07 (MDL 100,748) and 0.14 (MDL 29,951). MDL 100,748 and MDL 29,951 were approximately 2000-fold selective for the glycine binding site relative to the glutamate recognition sites. All four compounds completely inhibited the use-dependent binding of [3H]N-[1-(2-thienyl) cyclohexyl]-piperidine and were noncompetitive, glycine-reversible inhibitors of both NMDA-induced biochemical and electrophysiological responses in brain slice preparations. A competitive interaction with the glycine binding site was also evident in that MDL 29,951 and MDL 100,748 produced parallel rightward shifts in the glycine requirement for demonstration of NMDA-stimulated elevations in cytosolic calcium in cultured neuronal preparations. The glycine antagonists were potent anticonvulsants after their i.c.v. administration to audiogenic seizure-susceptible DBA/2J mice. Because the compounds chosen encompass a variety of chemical structures, the results indicate that glycine is required for NMDA receptor activation and that bioavailable glycine antagonists may form the basis of a novel therapy for epilepsy.

A strychnine-insensitive glycine binding site is located on the N-methyl-D-aspartate (NMDA)-preferring glutamate receptor complex. Kynurenic acid analogs are antagonists at this binding site. A derivative of kynurenic acid, 5,7-dichlorokynurenic acid (5,7-DCKA) was radiolabeled with 3H and used to study antagonist binding to the glycine recognition site. This ligand ( [3H]5,7-DCKA) showed high affinity (Kd = 69 nM), saturable (Bmax = 14.5 pmol/mg protein) binding to rat brain membranes. A variety of agonists and antagonists inhibited the binding of [3H]5,7-DCKA and [3H]glycine in a similar fashion (r = 0.93). In addition, glutamate site agonists and antagonists exerted opposite allosteric effects on [3H]5,7-DCKA binding suggesting that [3H]5,7-DCKA preferentially binds to the agonist-activated conformation of the receptor.

Pre-weaning rat pups emit ultrasonic vocalizations when removed from the litter. These 'separation-induced vocalizations' (SIV) are suppressed by classical benzodiazepine anxiolytics and by non-benzodiazepine anxiolytics which lack muscle relaxant and sedative properties. The present study used the SIV model to assess potential anxiolytic properties of compounds which target different sites associated with the NMDA receptor complex. Comparison was made to drugs which affect benzodiazepine or serotonin (5-HT) receptors. Muscle relaxant potential was assessed using 'TIP' (time on an inclined plane), the amount of time a pup was able to retain its position on a steeply inclined surface. Mephenesin, a centrally acting muscle relaxant, significantly suppressed TIP but not SIV. The benzodiazepine agonist diazepam suppressed both SIV and TIP, whereas the 5-HT1A partial agonists, buspirone and MDL 73,005EF, suppressed SIV without affecting TIP. The 5-HT2 antagonist MDL 11,939 suppressed TIP but not SIV, whereas neither measure was affected by the 5-HT3 antagonist MDL 73,147EF. SIV was suppressed by NMDA antagonists including those acting at the glutamate recognition site (D,L-amino-phosphonovaleric acid (AP5) and MDL 100,453) or at the ion channel (MK-801), or by the strychnine-insensitive glycine antagonist 5,7-dichlorokynurenic acid (5,7-DCKA). TIP was suppressed even more potently by AP5, MDL 100,453 and MK-801, whereas 5,7-DCKA was inactive on this measure. Thus, antagonists acting at different sites present on the glutamate recognition site exhibit potential anxiolytic activity, but the glycine antagonist was unusual in its lack of prominent muscle relaxant side effects.

5,7-Dichlorokynurenic acid (5,7-DCKA), one of the most potent excitatory amino acid receptor antagonists yet described, binds to a strychnine-insensitive glycine binding site located on the N-methyl-D-aspartate (NMDA) receptor complex (Ki = 79 nM versus [3H]glycine). 5,7-DCKA (10 microM) antagonized the ability of NMDA to stimulate the binding of the radiolabeled ion channel blocker N-[3H][1-(2-thienyl)cyclohexyl]-piperidine ([3]TCP). Glycine was able to overcome this effect and in the presence of 5,7-DCKA enhanced [3H]TCP binding to antagonist-free levels. 5,7-DCKA completely and noncompetitively antagonized several NMDA receptor-mediated biochemical and electrophysiological responses. Thus, micromolar concentrations of 5,7-DCKA inhibited NMDA-stimulated elevation of cytosolic calcium in cultured hippocampal neurons, cGMP accumulation in cerebellar slices, and norepinephrine release from hippocampal slices. The glycine antagonist could also block the action of synaptically released agonist, as shown by its ability to inhibit the increase in the magnitude of the population spike that follows tetanic stimulation of the hippocampus in vitro (long term potentiation). Inclusion of glycine or D-serine prevented all these effects of the antagonist. 5,7-DCKA was a potent anticonvulsant when administered intracerebroventricularly to mice. As in the in vitro experiments, the dose-response curve for the antagonist was shifted rightward in a parallel fashion when D-serine was coinjected. This spectrum of activity displayed by a compound acting at the glycine binding site suggests that the therapeutic utility of glycine antagonists will be similar to those proposed for other types of glutamate receptor antagonists.

Three polypeptide neurotoxins (curtatoxins) were isolated from the venom of the spider Hololena curta by reverse-phase high performance liquid chromatography, gel permeation, and ion-exchange chromatography. The purified toxins induced an immediate paralysis in the cricket Acheta domestica that resulted in desiccation and death of the insect within 24-48 h (LD50 congruent to 4-20 micrograms/g); this toxic effect is consistent with irreversible presynaptic neuromuscular blockade. Curtatoxins are a class of sequence-related, cysteine-rich, carboxyl-terminal amidated polypeptides of 36 to 38 amino acid residues. The cysteine residues are conserved at identical sequence positions among these polypeptides and form 4 intramolecular disulfide bonds. Hydropathy calculations show that the toxins have an internal hydrophobic region flanked by hydrophilic and oppositely charged amino- and carboxyl-terminal ends. By analogy to other cysteine-rich arthropod venom proteins, the folded structure of the curtatoxins is likely important for their target specificity and mode of action at the neuromuscular junction.

1. Oral doses of 10-100 mg/kg of BRL 13776 lowered the blood pressure of both deoxycorticosterone acetate (DOCA)/NaCl-treated hypertensive rats and untreated normotensive rats. 2. BRL 13776 (100 mg/kg, orally) also reduced the blood pressure of renal hypertensive cats (cellophane perinephritis model). 3. No tolerance developed to the blood-pressure lowering action of BRL 13776 when an oral daily dose of 100 mg/kg was administered repeatedly for up to 15 days to hypertensive rats and cats. 4. The fall in blood pressure to BRL 13776 in rats was associated with a reduction of tissue catecholamines. 5. The catecholamine depletion occurred in all the peripheral tissues examined but in the brain was restricted to certain regions, these being the hind-brain on single dosing and the hind-brain, hypothalamus and mid-brain on repeated dosing. Catecholamine levels in the cerebral hemispheres were not affected by either single or repeated doses of BRL 13776. 6. BRL 13776 caused some reduction of the 5-hydroxytryptamine content of the heart but not of whole brain or any brain region. 7. Neither single doses (up to 900 mg/kg orally) nor repeated doses (100-300 mg/kg orally) of BRL 13776 produced any significant behavioural effects in animals. 8. BRL 13776 is a new type of agent to display both antihypertensive and monoamine-depleting properties. The reduction of noradrenaline in certain brain regions may be a cause of the antihypertensive response but depletion in the periphery could contribute in a major or minor way. The differential action on noradrenaline in the brain together with the lack of effect on 5-hydroxytryptamine might also explain the apparent absence of behavioural effects.

MDL 72,974, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine, was designed to be a selective inhibitor of monoamine oxidase type B (MAO-B). In vitro, the compound inhibits rat brain mitochondrial MAO in a concentration and time-dependent fashion and shows marked selectivity for the B form (IC50 = 680 and 3.6 nM for MAO-A and MAO-B, respectively). After oral administration to rats, the compound shows preferential inhibition of brain MAO-B with ED50 values of 8 and 0.18 mg/kg p.o. for the A and B forms, respectively. Selectivity is retained on repeat dosing. MDL 72,974 did not significantly potentiate the cardiovascular effects of intraduodenually-administered tyramine in anaesthetized rats and had only minor indirect sympathomimatic effects in the pithed rat. At MAO-B selective doses the neurotoxic effect of MPTP in mice was blocked.

Rats maintained on a tryptophan deficient diet had reduced brain and serum tryptophan and brain 5-hydroxyindolacetic acid levels compared to controls. 5-Hydroxytryptophan and L-tryptophan administered to these deficient rats in a combination (5:95) produced a greater elevation of indolamines and tryptophan in the brain than either amino acid alone. In rats maintained on a normal diet the urinary output of 3-hydroxykynurenine was considerably reduced by treatment with the combination of amino acids as compared to tryptophan treatment. 5-Hydroxytryptophan reduced the induction of kynurenine synthesis in the liver produced by tryptophan, implying that it is capable of inhibiting the enzyme tryptophan pyrrolase in vivo. It is suggested that the possession by 5-hydroxytryptophan of tryptophan pyrrolase inhibitory properties may make the administration of the combination a better treatment of depressed patients exhibiting an indolamine deficit than either amino acid alone.

We investigated the mechanism of the antihypertensive action of DL-alpha-monofluoromethyldopa (MFMD), an irreversible inhibitor of L-aromatic amino acid decarboxylase, in spontaneously hypertensive rats (SHR). MFMD, 25 mg/kg i.p. daily for 3 days, reduced mean arterial blood pressure by 47 +/- 11 mm Hg (n = 6; p less than 0.001). The effect was associated with marked attenuation of the responses to stimulation of the whole sympathetic outflow in pithed preparations and with substantial reductions in the norepinephrine concentrations of hearts and portal veins. Similar functional and biochemical deficits were produced in age-matched, Wistar--Kyoto, normotensive rats, but blood pressure in these animals fell only by an average of 8 +/- 4 mm Hg (n = 5; NS). Blood pressure of SHR, lowered by MFMD, was restored to pretreatment levels by infusion with dopamine (0.1 mg/kg/min for 5 min), and there was a concomitant return towards normal of both the peripheral sympathetic transmitter stores and the response to stimulation of the nerves supplying the cardiovascular system. Neither the dopamine nor the norepinephrine concentrations of the brain, depleted by treatment with MFMD, were altered following infusion of dopamine. These results provide direct evidence that attenuation of sympathetic function arising from depletion of the peripheral transmitter store is the mechanism by which MFMD lowers blood pressure in SHR. They further lend strong support to the view that hyperactivity of the sympathetic nervous system plays a primary role in the maintenance of the elevated blood pressure in the genetically hypertensive rat.

Unilateral intrastriatal injection of various substances induces a characteristic dyskinetic syndrome in rats. These substances include picrotoxin as well as a series of irreversible GABA-transaminase inhibitors. Using the degree of enzyme inhibition in various brain areas as a measure of drug distribution following intrastriatal administration of gamma-acetylenic GABA and gamma-vinyl GABA, there was found considerable retrodiffusion via the needle tract to the overlying cortex. Topical application of gamma-acetylenic GABA and gamma-vinyl GABA to the cortical surface overlying the striatum produced a high incidence of identical dyskinesias without any evidence of diffusion of drugs to the striatum. The cortically induced movements could be duplicated by picrotoxin application to a defined cortical area. These findings suggest that interference with gabaergic function in the striatum is not necessary for the production of the dyskinetic syndrome and that this syndrome may be a cortically induced phenomenon.

DL-alpha-monofluoromethyldopa (MFMD) is a potent enzyme-activated irreversible inhibitor of aromatic L-amino acid decarboxylase which, when given to rats or mice at 100 and 250 mg/kg i.p., respectively, causes a linear accumulation of L-DOPA and 5HTP and an exponential decline of noradrenaline, dopamine, and 5HT concentrations in the brain. Rates of change of these parameters can be used to calculate the turnover of the three principle monoamine neurotransmitters. Experiments with haloperidol (1 mg/kg s.c.) and the central 5HT agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (0.25 mg/kg s.c.), have been performed to validate the use of MFMD to measure monoamine turnover. MFMD has several advantages over classical methods for the determination of comparative turnovers using enzyme inhibitors.

MDL 72145, (E)-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine hydrochloride, was designed and synthesised as a potential enzyme-activated irreversible inhibitor of monoamine oxidase (MAO). In vitro, the compound displayed time-dependent pseudo-first-order irreversible inhibitory characteristics with high selectivity for the B form of rat brain mitochondrial MAO. At 10 degrees C the Ki and tau 50 values for the B enzyme were 40 microM and 1.7 min, respectively, while these same kinetic constants for the A enzyme were 131 microM and 14.5 min, respectively. Selective protection against inactivation of the two forms of MAO by MDL 72145 was obtained by preincubating the enzyme with suitable concentrations of the selective A and B substrates, 5-hydroxytryptamine and benzylamine.

The purpose of the experiments reported were to determine whether chronic treatment with either a monoamine oxidase (MAO) inhibitor or an uptake inhibitor would increase extracellular levels of 5-HT in vivo and whether such treatment resulted in a down-regulation of the 5-HT1B-mediated decrease in extracellular levels of 5-HT. Rats were given either saline, (E)-beta-fluoromethyline-m-tyrosine (MDL 72394 0.25 mg/kg p.o.) or amitriptyline (10 mg/kg p.o.) once a day for 21 days. Twenty-four hr after the final injection, dialysis loops were implanted into the frontal cortices of these rats and basal extracellular levels of 5-HT were measured. The effect of the 5-HT1 receptor agonist 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)1H indole succinate (RU 24969 10 mg/kg i.p.) on the extracellular level of 5-HT was then studied. Basal levels of 5-HT in saline-treated rats was found to be 27.9 +/- 3.9 fmol/20 microliters perfusate. Chronic treatment with amitriptyline had no effect on extracellular levels of 5-HT but chronic treatment with MDL 72394 significantly increased extracellular levels of 5-HT. Chronic treatment with either MDL 72394 or amitriptyline had no significant effect on the ability of RU 24969 to reduce extracellular levels of 5-HT. These results suggest that 5-HT1B receptors are not down-regulated in response to chronically raised extracellular levels of 5-HT.

Using reversed-phase high performance liquid chromatography with electrochemical detection it is possible to measure concomitantly the concentration of several monoamines, their metabolites and aminoacid precursors in 100 microliters of rat cerebrospinal fluid. To study the quantitative relationship between CSF and brain, alterations in brain monoamines and monoamine metabolites were effected by treatment with L-DOPA or L-5HTP administered with or without concomitant inhibition of extracerebral aromatic amino acid decarboxylase and by treatment with alpha-monofluoromethyldopa, probenecid, haloperidol, or probenecid plus haloperidol. The concentrations of the monoamine metabolites, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid, and homovanillic acid as well as of the L-DOPA metabolite, 3-methoxy-4-hydroxyphenylalanine in the cerebrospinal fluid were linearly correlated with the concentrations of these metabolites in the brain. However, these correlations need to be interpreted cautiously, since the slopes of the individual regression lines obtained after different pharmacological treatments differed significantly.

The inhibition of semicarbazide-sensitive amine oxidase (SSAO) in rat aorta homogenates by some 2-phenyl-3-haloallylamines has been studied. Derivatives containing a fluorine atom were approximately three times more potent than the corresponding 3-chloroallylamines. These halogen-containing compounds were irreversible inhibitors of SSAO after preincubation with aorta homogenates; kinetic evidence for an initial competitive, reversible interaction (Ki around 0.4-0.6 microM) was found with two compounds (MDL 72145 and 72274). A similar Ki (approx. 0.7 microM) was obtained with 2-phenylallylamine (MDL 72200). However, this compound which lacks a halogen atom was a reversible inhibitor, even after preincubation. The use of a spectrophotometric assay to measure H2O2 production from amine metabolism demonstrated that MDL 72200 was a substrate (Km = 1.4 microM) for SSAO, with a Vmax approximately five times smaller than that of benzylamine (Km = 8.1 microM). Of particular interest in this study is the finding that (E)-2-phenyl-3-chloroallylamine (MDL 72274) is highly selective as an inhibitor of SSAO, compared with MAO-A or B activities, and may be a useful compound for investigating the importance of SSAO in animal tissues.

Rat brain GABA levels were elevated chronically by daily administration of gamma-vinyl GABA, an enzyme-activated, irreversible inhibitor of GABA:2-oxo-glutarate aminotransferase (GABA-T; EC2.6.1.19). Following various periods of drug treatment and withdrawal, the sensitivity of dopamine and GABA receptors in the CNS was determined by biochemical and behavioral evaluations. In contrast to chronic haloperidol treatment, none of the treatment schedules with gamma-vinyl GABA had any significant effect on parameters such as apomorphine induced locomotor activity, [3H] spiperone binding or dopamine-stimulated adenylate cyclase in the corpus striatum; nor did gamma-vinyl GABA treatment affect [3H] GABA binding or GABA-activated [3H] diazepam binding in the cerebral cortex. Moreover, co-administration of gamma-vinyl GABA and haloperidol did not alter the ability of the neuroleptic to induce supersensitivity in the striatal dopaminergic system. Thus, it appears that, in contrast to reported studies using chronic administration of other less specific GABA-T inhibitors such as gamma-acetylenic GABA, amino-oxyacetic acid and isonicotinic acid hydrazide or direct GABA agonists such as THIP (4,5,6,7-tetrahydroisoxazolo (5,4-c-)-pyridin-3-ol) or kojic amine, gamma-vinyl GABA does not alter the sensitivity of the striatal dopaminergic system.

We investigated the effects on peripheral sympathetic nervous function of monofluoromethyldopa (MFMD, RMI 71963), a selective, enzyme-activated, irreversible inhibitor of L-aromatic amino acid decarboxylase. Three daily injections of 2.5, 25, or 100 mg/kg MFMD to rats reduced the dopamine and norepinephrine concentrations in the heart and increased those of L-dopa. Sedation was seen with the two highest doses of MFMD. Cardiovascular responses in the pithed rat to stimulation of the whole spinal sympathetic outflow and to injections of tyramine were reduced by treatment with MFMD. Both the reduced sympathetic function and the depleted transmitter store were restored by infusions of dopamine. Norepinephrine depletion and functional inhibition were obtained with smaller doses of MFMD (3, 6, and 10 mg/kg daily) given for longer periods (10 days). Blood pressure in spontaneously hypertensive rats was reduced by treatment with MFMD, 25 mg/kg, daily for 3 days. In vitro responses to nerve stimulation of portal veins declined during incubation with MFMD or alpha-methyl-p-tyrosine; responses of atria were unaffected. Changes in norepinephrine concentrations of portal veins and atria closely paralleled the changes in function. MFMD is the first compound which depletes peripheral norepinephrine concentrations by inhibiting transmitter biosynthesis at the decarboxylase step. Such depletion results in inhibition of sympathetic function in vivo and introduces a novel approach to pharmacological manipulation of the activity of the peripheral sympathetic nervous system.

(E)-beta-Fluoromethylene-m-tyrosine (FMMT) is a dual-enzyme-activated inhibitor of monoamine oxidase (MAO). The compound is not an inhibitor per se but is decarboxylated by aromatic L-amino acid decarboxylase (AADC) to yield a potent enzyme-activated irreversible inhibitor of MAO, (E)-beta-fluoromethylene-m-tyramine, which shows some selectivity for inhibition of MAO type A. Decarboxylation of FMMT was demonstrated in vitro using hog kidney AADC and in vivo in rats by the ability of alpha-monofluoromethyldopa (MFMD), a potent inhibitor of AADC, to prevent MAO inhibition produced by FMMT. In isolated synaptosomes, FMMT was decarboxylated by AADC, and, furthermore, the compound was actively transported into these isolated nerve endings. An active transport into the CNS has also been demonstrated in vivo by performing competition experiments with leucine. To demonstrate that FMMT is preferentially decarboxylated within monoamine nerves of the CNS, the nigrostriatal 3,4-dihydroxyphenylethylamine (dopamine) pathway of rats was unilaterally lesioned with 6-hydroxydopamine or infused with MFMD. Under these conditions, MAO inhibition produced by orally administered FMMT in the striatum ipsilateral to the lesion or infusion was markedly attenuated. Combination of FMMT with an inhibitor of extracerebral AADC, such as carbidopa, protected peripheral organs against the MAO inhibitory effects and concomitantly enhanced MAO inhibition in the CNS. Such combinations had a greatly reduced propensity to augment the cardiovascular effects of intraduodenally administered tyramine, when compared with FMMT given alone or with clorgyline, a selective inhibitor of MAO type A. The results obtained with FMMT suggest the possibility of achieving selective inhibition of MAO within monoamine nerves of the CNS and, further, suggest that combination of FMMT with an inhibitor of extracerebral AADC will reduce the propensity of this inhibitor to produce adverse interactions with tyramine.

MDL 73,147EF (1H-indole-3-carboxylic acid-trans-octahydro-3-oxo-2,6- methano-2H-quinolizin-8-yl-ester methanesulphonate) is a potent and selective 5-HT3 receptor antagonist (pA2 9.8, rabbit heart; pIC50 less than 5, D-2 receptor). The effects of acutely and chronically administered haloperidol and MDL 73,147EF were compared in an electrophysiologic model for antipsychotic activity. Haloperidol, but not MDL 73,147EF, given acutely increased the number of active dopamine neurons in the substantia nigra (A9). Both haloperidol and MDL 73,147EF, given chronically, decreased the number of active ventral tegmental dopamine neurons and the number of active A9 dopamine neurons. The results indicate that MDL 73,147EF may prove useful as an antipsychotic with a unique mechanism of action.

Seventeen 2-aryl-3-haloallylamine derivatives were prepared and evaluated as inhibitors of monoamine oxidase (MAO, EC 1.4.3.4). The synthesis of these compounds was achieved from either alpha-methylstyrene or ring-substituted phenylacetic acid derivatives. With one exception, these 2-arylallylamines were found to be enzyme-activated, irreversible inhibitors of MAO. The most potent inhibitors were ring-substituted derivatives of (E)-2-phenyl-3-fluoroallylamine with IC50 values ranging from 10(-6) to 10(-8) M. Selectivity for the A and B form of MAO was found to depend on the nature of aromatic ring substitution. In general, hydroxyl substitution favored the inactivation of the A form of MAO, while very selective B inhibitors were obtained when the aromatic ring was substituted with a 4-methoxy group. (E)-2-(4-Methoxyphenyl)-3-fluoroallylamine and (E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine proved to be in vitro as selective for the B form of MAO as deprenyl.

The potential of a new, potent, irreversible and selective inhibitor of monoamine oxidase type B, (E)-2-(3,4-dimethoxyphenyl)-3-fluorallyamine (MDL 72145), to augment the effects of L-DOPA in an animal model which reproduces the biochemical defect of Parkinson's disease has been evaluated. In rats bearing unilateral 6-hydroxydopamine lesions of the nigro-striatal dopamine pathways, both MDL 72145 and clorgyline, a selective inhibitor of MAO A, augmented the contralateral turning response to L-DOPA combined with carbidopa. The potential of inhibitors of MAO to interact adversely in the periphery with L-DOPA was investigated in the pithed rat; L-DOPA was given either intravenously or intraduodenally. Clorgyline consistently potentiated L-DOPA when given 18 h before testing. Neither MDL 72145 nor the selective inhibitor of MAO B, L-deprenyl, augmented the cardiovascular effects of intraduodenally administered L-DOPA. The data provide no reason to suppose that MDL 72145 would be very different in clinical use from L-deprenyl which is both effective and well-tolerated as an adjunct to the L-DOPA-based therapy of Parkinson's disease.