A novel adenylyl cyclase-activating serotonin receptor (5-HT7) implicated in the regulation of mammalian circadian rhythms.

We report the cloning and characterization of a novel serotonin receptor, designated as 5-HT7, which is coupled to the stimulation of adenylyl cyclase. 5-HT7 mRNA is expressed discretely throughout the CNS, predominantly in the thalamus and hypothalamus. 5-HT7 has a unique pharmacological profile that redefines agonist and antagonist classification of ligands previously thought to be "selective." The circadian phase of spontaneous neuronal activity of the rat suprachiasmatic nucleus of the hypothalamus advances in response to serotonin ligands with a pharmacological profile consistent exclusively with that of 5-HT7. These findings suggest a physiological role in the regulation of circadian rhythms for one subtype of serotonin receptor, 5-HT7, and provide a pharmacological test to evaluate its role in other neuronal systems.

Human platelet phospholipase A2 activity is responsive in vitro to pH and Ca2+ variations which parallel those occurring after platelet activation in vivo.

Secretion of human platelet dense granule contents in response to epinephrine and other weak agonists requires the prior liberation of membrane-esterified arachidonic acid by a phospholipase A2 enzyme species whose activity is regulated by Na+/H+ exchange (e.g., Sweatt et al. (1986) J. Biol. Chem. 261, 8660-8673 and Banga et al. (1986) Proc. Natl. Acad. Sci. USA 83, (197-9201). Based on our earlier findings in intact platelets, we postulated that the alkalinization of the platelet interior that accompanies accelerated activity of the Na+/H+ antiporter enables the phospholipase A2 enzyme to function at ambient or low concentrations of intraplatelet Ca2+. To test the hypothesis that the Ca2+ dependence of platelet phospholipase A2 activity is influenced by changes in intraplatelet pH that occur following platelet activation, we characterized the Ca2+ dependence of this enzyme as a function of changes in pH (from pH 6.8-8.0), since it is within this range that intraplatelet pH changes occur following platelet activation. Phospholipase A2 enzymatic activity in platelet particulate preparations was detectable in the presence of micromolar concentrations of Ca2+ (EC50 1-2 microM) and plateaued above 10 microM Ca2+. Enzymatic activity measured at 4.8 microM Ca2+ was increased by raising the pH from 5.5 to 8.0 (EC50 7.4), was optimal at pH 8.0 and declined at more alkaline values. Furthermore, increases in pH from pH 6.8 to pH 8.0 not only increased maximal enzymatic activity but also enabled detection of enzymatic activity at lower Ca2+ concentrations. The interdependent regulation of phospholipase A2 activity by changes in pH and Ca2+ suggests that phospholipase A2 could serve to integrate changes in intracellular pH and available Ca2+ that occur subsequent to activation of human platelets by epinephrine and other weak agonists.

Lipid and membrane interactions of neuropeptide Y.

The interactions of neuropeptide Y with dimyristoylphosphatidylcholine and cell membranes were examined by several physical techniques to probe the potential role of its putative C-terminal amphipathic alpha-helix. Neuropeptide Y binding was demonstrated by a rapid release of entrapped 6-carboxyfluorescein and a rapid decrease in the turbidity of dimyristoylphosphatidylcholine liposomes. In addition, an increase in tyrosine fluorescence intensity and an increase in the anisotropy of diphenylhexatriene in dimyristoylphosphatidylcholine liposomes was observed. In isolated, aortic smooth muscle cell membranes, the anisotropy of diphenylhexatriene increased as a function of added neuropeptide Y. The concentration range (low microM) over which neuropeptide Y increases the polarization of diphenylhexatriene in cell membranes is similar to the range in which it inhibits isoproterenol-stimulated cAMP accumulation. This inhibition is not affected by pertussis toxin, nor does neuropeptide Y cause the release of preloaded [3H]adenine from cells into the medium. These data suggest that neuropeptide Y contains an amphipathic alpha-helical region which interacts with lipids in much the same way as the amphipathic alpha-helical regions of the plasma apolipoproteins and that the inhibition of isoproterenol-stimulated cAMP accumulation at low microM concentrations of peptide may be the result of an alteration in the cell membrane bilayer structure.

Molecular approach to hypothalamic rhythms: isolation of novel indoleamine receptor genes.

We have utilized polymerase chain reaction with primers corresponding to conserved amino acid sequences within membrane-spanning regions of known serotonin receptors to identify clones of four putative new indoleamine receptors. We have determined complete amino acid sequences of these four receptors, which fall into three subfamilies; two of these subfamilies are novel. The sites of expression within the brain have been determined for each of the genes. Expression in mammalian cells demonstrates that each new protein is a receptor for serotonin and that each has a distinct pharmacology when compared to known receptors. Two of the new receptors are coupled to cyclic adenosine monophosphate, one negatively (Gi) and one positively (Gs). The latter is a candidate for the serotonin receptor that mediates phase advances in circadian rhythms of the suprachiasmatic nucleus.

2,4-Dihydro-3H-1,2,4-triazol-3-ones as anticonvulsant agents.

A series of 5-aryl-2,4-dihydro-3H-1,2,4-triazol-3-ones was evaluated for anticonvulsant activity. In general the members of this series were prepared by the alkaline cyclization of 1-aroyl-4-alkylsemicarbazides. The resulting 2-unsubstituted 3H-1,2,4-triazol-3-ones were then alkylated, yielding 2,4-dialkyl-3H-1,2,4-triazol-3-ones. Approximately one-third of the compounds examined exhibited activity against both maximal electroshock- and pentylenetetrazole-induced seizures in mice. Receptor-binding studies suggest that this activity was not a consequence of activity at either benzodiazepine or NMDA-type glutamate receptors. From this series, compound 45 was selected for further evaluation where it was also found to be active against 3-mercaptopropionic acid, bicuculline, and quinolinic acid induced seizures in mice. In addition, 45 also protected gerbils from hippocampal neuronal degeneration produced by either hypoxia or intrastriatal quinolinic acid injection.

3-(2-Carboxyindol-3-yl)propionic acid-based antagonists of the N-methyl-D-aspartic acid receptor associated glycine binding site.

A series of substituted 3-(2-carboxyindol-3-yl)propionic acids was synthesized and tested as antagonists for the strychnine-insensitive glycine binding site of the NMDA receptor. Chlorine, and other small electron-withdrawing substituents in the 4- and 6-positions of the indole ring, greatly enhanced binding and selectivity for the glycine site over the glutamate site of the NMDA receptor; one of the most potent compounds is 3-(4,6-dichloro-2-carboxyindol-3-yl)propionic acid (IC50 = 170 nM; greater than 2100-fold selective for glycine). The importance of a heteroatom NH and the enhancing effect of the propionic acid side chain were demonstrated and are consistent with previous results which suggest the presence of a pocket on the receptor which can accept an acidic side chain. Substitution of a sulfur at C3 led to the most potent compound 3-[(carboxymethyl)thio]-2-carboxy-4,6-dichloroindole (IC50 = 100 nM).

Binding of 5H-dibenzo[b,e][1,4]diazepine and chiral 5H-dibenzo[a,d]cycloheptene analogues of clozapine to dopamine and serotonin receptors.

5H-Dibenzo[b,e][1,4]diazepine, dibenz[b,f]oxepin, and 5H-dibenzo[a,d]cycloheptene analogues of clozapine [8-chloro-11-(4-methylpiperazino)-5H- dibenzo[b,e][1,4]diazepine] were evaluated for their binding affinity to dopamine D-1, D-2, and D-4 and serotonin S-2A (5-HT2A), S-2C (5-HT2C) and S-3 (5-HT3) receptors. The diazepine analogues display selective binding to the dopamine D-4 and serotonin S-2A receptors similar to that of clozapine, but none has a dopamine D-4 selectivity (Ki for the dopamine D-2A receptor/Ki for the dopamine D-4 receptor) greater than that of clozapine. All of the oxepin analogues also show substantial binding to the dopamine D-4 and serotonin S-2A receptors with 10-(4-methylpiperazino)dibenz[b,f]oxepin having a dopamine D-4 selectivity greater than that of clozapine. Some of the 5H-dibenzo-[a,d]cycloheptene analogues also show strong binding to both the dopamine D-4 and serotonin S-2A receptors, 5-methyl-10-(4-methylpiperazino)-5H-dibenzo[a,d]cycloheptene having a dopamine D-4 selectivity of 7.8 as compared to 10 for clozapine but a serotonin S-2A selectivity (Ki for the dopamine D-2 receptor/Ki for the serotonin S-2A receptor) of 2.0 as compared to 28 for clozapine. The serotonin S-2A selectivity of 2-chloro-10-(4-methylpiperazino)-5H-dibenzo[a,d]-cycloheptene++ + is 200. As an extension of these studies, chiral 5-substitute 10-(1,2,3,6-tetrahydro-1-methyl-4-pyridinyl)-5H-dibenzo[a,d]cyclohept ene analogues show a substantial enantiospecificity toward dopamine and serotonin receptor subtypes, (R)-(-)-5-methyl compound having a 2-fold higher dopamine D-4 selectivity than its (S)-(+) enantiomer as the result of enhanced binding to the dopamine D-4 receptor rather than diminished binding to the dopamine D-2 receptor. (pRa,pSb)-(+)-5-(2-Propylidene)-10-(1,2,3,6-tetrahydro-1-met hyl- 4-pyridinyl)-5H-dibenzo[a,d]cycloheptene is 17 times more active in binding to the dopamine D-4 receptor than is its pSa,pRb enantiomer while being only 1.5 times more active in binding to the dopamine D-2 receptor.

Binding of 5H-dibenzo[a,d]cycloheptene and dibenz[b,f]oxepin analogues of clozapine to dopamine and serotonin receptors.

Series of 5,11-dicarbo- and 11-carbo-5-oxy-10-(1-alkyl-1,2,3,6-tetrahydro-4 pyridinyl) analogues and a 11-carbo-5-oxy-10-(1-methyl-4-piperidinyl) analogue of the atypical antipsychotic agent clozapine were prepared and tested for binding to the dopamine D-2L and D-4 and serotonin S-2A and S-2C receptors. Some of these analogues were found to have dopamine D-2L and D-4 and serotonin S-2A and S-2C receptor binding activities as high as or higher than those of clozapine, indicating that neither the diazepine structure nor the piperazine ring present in clozapine is essential for high antidopamine activity and or for high dopamine D-4 selectivity (Ki for the dopamine D-2L receptor/Ki for the dopamine D-4 receptor). Increasing in the effective size of the alkyl substituent at the tertiary amine nitrogen atom in the 1,2,3,6-tetrahydro-4-pyridinyl moiety in the 5H-dibenzo[a,d]cycloheptene series reduces the affinity for the dopamine D-4 receptor, but in the dibenz[b,f]oxepin series, no significant change in binding affinity to the dopamine D-4 receptor was observed. Equal or slightly higher affinity for the serotonin S-2A and S-2C receptors was observed for the 10-(1-ethyl-1,2,3,6-tetrahydro-4- pyridinyl) analogues in both series, but for the 10-[1,2,3,6-tetrahydro-1-(2-propenyl)-4- pyridinyl] analogues, any favourable steric factor is overshadowed by an unfavorable electronic effect as a result of change in the basicity of the tertiary amino group in the pyridinyl moiety. Replacement of three of the four nitrogen atoms in clozapine with three carbon or two carbon atoms and an oxygen atom and removal of the chlorine atoms gives 10-(1,2,3,6-tetrahydro-1- methyl-4-pyridinyl)dibenzo[a,d]cycloheptene and 10-(1-methyl-4-piperidinyl)dibenz[b,f]oxepin, each having twice the binding activity to the dopamine D-4 receptor as does clozapine and a dopamine D-4 selectivity equal to that of clozapine.

Neurotoxicity of dipiperidinoethane due to in vivo conversion to a selective cholinesterase inhibitor.

Dipiperidinoethane (DPE) administration produces seizures and CNS lesions. Here we elucidate the cholinergic origin of DPE toxicity. DPE is both an acetylcholinesterase (AChE) inhibitor and a muscarinic antagonist. This dual action negates most of the toxic effects of the compound in vivo. The neurotoxicity is believed to arise from oxidative conversion to DPE-N-oxide, which selectively inhibits AChE. Cytotoxicity does not involve muscarinic neurons, since binding parameters were unchanged following in vivo exposure.

Binding of the radiolabeled glycine site antagonist [3H]MDL 105,519 to homomeric NMDA-NR1a receptors.

We have characterized the binding of [3H]MDL 105,519 ((E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1 H-indole-2-carboxylic acid), a NMDA receptor glycine recognition site antagonist, to homomeric NMDA subunit 1a (NR 1a) receptors. Chinese hamster ovary cells (CHO-K1) were transfected with the rat NR 1a gene and cell lines stably expressing the receptor were isolated from amongst clones resistant to the neomycin analog G418. Saturation analysis indicated that the radioligand bound to the homomeric receptor with a similar high affinity (Kd = 1.8 nM) to that reported for the native receptor. The binding capacity (Bmax) was 370 fmol/mg protein reflecting approximately 110000 receptors per cell. The radioligand interacted with a single class of binding sites as indicated by linear Scatchard transformation of the saturation data and a unitary Hill slope in competition experiments. Thus, the MDL 105,519 recognition site is present on the NR 1a subunit and has similar radioligand binding properties to the native brain-derived receptor. However, pharmacologic characterization of [3H]MDL 105,519 binding indicated that agonists were weaker competitors at the homometric receptor relative to the native receptors. In contrast, representative of three distinct chemical classes of glycine site antagonists exhibited similar potencies at both types of binding sites.

Activation of Gi protein by peptide structures of the muscarinic M2 receptor second intracellular loop.

The muscarinic M2 receptor that normally couples via Gi to inhibit adenylyl cyclase was made to couple to Gs by exchange of its third intracellular loop for the comparable domain of the beta 2-adrenoceptor. In HeLa cells transfected with the recombinant M2 beta i-3 cDNA, the chimaeric receptor showed carbachol-mediated activation of adenylyl cyclase (EC50 = 73 nM) that was blocked by atropine, but not by propranolol. The chimaeric receptor was shown to mediate a carbachol-stimulated, Bordetella pertussis toxin-sensitive GTPase activity in membranes of transfected HeLa cells. Interestingly, stimulation of adenylyl cyclase by carbachol was 2-fold higher in transfected cells that had been pretreated with pertussis toxin. These data suggested that the M2 beta i-3 receptor was able to couple to both Gi and Gs, and that the ability to recognise and stimulate Gi did not involve the third cytoplasmic loop of M2. We investigated peptide elements taken from the second intracellular loop of the M2 receptor for their ability to mediate activation of Gi and found that a nine amino acid peptide representing the C-terminal sequence, VKRTTKMAG-NH2 (V9G), was capable of inhibiting forskolin-stimulated adenylyl cyclase by up to 18% and could stimulate high affinity GTPase activity of rat brain membranes by 32%. Further, V9G was shown to cause a doubling of the initial rate of [35S]GTP gamma S binding to purified bovine brain Gi/Go in reconstituted phospholipid vesicles. These data identify a domain on the second intracellular loop of the muscarinic M2 receptor that is involved in the selection of a pertussis toxin-sensitive G protein.

Activities of novel aryloxyalkylimidazolines on rat 5-HT2A and 5-HT2C receptors.

Using transfected NIH 3T3 mouse fibroblast cell lines expressing the rat 5-HT2A and rat 5-HT2C receptor subtypes, and techniques of 2-[125I](+)-iodolysergic acid diethylamide ([125I]LSD) binding and serotonin (5-hydroxytryptamine, 5-HT)-stimulated phosphoinositide hydrolysis, we have characterized a new structural class of 5-HT receptor ligands, the aryloxyalkylimidazolines. These compounds were found to be potent competitors of [125I]LSD binding at both receptor subtypes (Ki approximately 5-200 nM) and to have efficacy ranging from potent competitive antagonists (IC50 approximately 25 nM) to moderately potent full agonists (EC50 approximately 200 nM). Some of these compounds are agonists at both receptor subtypes, while others are 5-HT2C receptor agonists with 5-HT2A receptor antagonist activity. None of the aryloxyalkylimidazolines reported here have 5-HT2A or 5-HT2C receptor selective antagonist activity. Since these compounds are novel structures, we compared them with a variety of reference 5-HT receptor ligands selected from other chemical classes that have previously been studied at 5-HT2A and 5-HT2C receptors in native tissues.

Characterization of cell selectivity of two novel inhibitors of nitric oxide synthesis.

We have assessed the capacity of two novel inhibitors to block cytokine-induced nitric oxide (NO) synthesis by macrophages and vascular smooth muscle cells, as well as NO production by the constitutive enzyme in central nervous system tissue. NG-Cyclopropyl-L-arginine selectively inhibited Ca2+/calmodulin-dependent NO synthesis, with an IC50 of 0.55 microM in brain versus 184 and 258 microM in macrophages and vascular smooth muscle cells, respectively. In contrast, NG-amino-L-homoarginine blocked NO production by all of the cell types examined, with IC50 values ranging from 6.6 to 26 microM. Both inhibitors were active in an in vivo model of endotoxic shock.

Anticonvulsant activity of a novel NMDA/glycine site antagonist, MDL 104,653, against kindled and sound-induced seizures.

MDL 104,653 (3-phenyl-4-hydroxy-7-chloro-quinolin-2(1H)-one), acts as an antagonist at the glycine site of the NMDA receptor. MDL 104,653 protects against sound-induced clonic seizures in DBA/2 mice following intracerebroventricular (ED50 = 19.1 nmol, 30 min), intraperitoneal (i.p.; ED50 = 6.1 mumol/kg, 45 min), or oral (ED50 = 23.0 mumol/kg, 2 h) administration. Optimal protection by MDL 104,653 was observed 15-60 min after i.p. administration, and the therapeutic index, as assessed by rotarod performance, was 4.0 at 45 min after i.p. administration. Fully amygdala-kindled motor seizures in rats were significantly reduced at 15, 30 and 60 min, and the duration of the after-discharge was significantly shortened at 30 min after the i.p. administration of 74 mumol/kg MDL 104,653. A lower dose of MDL 104,653 (37 mumol/kg) had no significant effect on either motor seizures or after-discharge duration. The rate of amygdala kindling was also significantly retarded following the daily administration of 56 mumol/kg MDL 104,653 (1 times daily for 6 days; i.p. 30 min before kindling stimulus).

Characterization of the novel 5-HT3 antagonists MDL 73147EF (dolasetron mesilate) and MDL 74156 in NG108-15 neuroblastoma x glioma cells.

In radioligand binding experiments, MDL 73147EF and MDL 74156 inhibited the binding of [3H]GR65630 to 5-hydroxy-tryptamine3 (5-HT3) binding sites on membranes prepared from NG108-15 neuroblastoma x glioma cells. The calculated dissociation constants (KI) were 20.03 +/- 6.58 and 0.44 +/- 0.18 nM, respectively (means +/- S.E.M., n = 6 and 9, respectively). Application of 5-HT (10-50 microM) to voltage-clamped NG108-15 cells elicited a rapidly desensitizing inward membrane current, characteristic for the activation of 5-HT3 receptors. The 5-HT-induced membrane current was suppressed in a reversible, concentration-dependent manner by MDL 73147EF and MDL 74156EF. The concentrations required to block half the 5-HT response (IC50) were 3.8 and 0.1 nM, respectively. It is concluded that both compounds are potent and reversible antagonists at 5-HT3 receptors in this neuroblastoma cell line.

Rapid down regulation of beta-adrenoceptors by co-administration of desipramine and fluoxetine.

Co-administration of desipramine and fluoxetine resulted in a 27% decline in cerebral cortical beta-adrenoceptor density after four days - a time point at which neither agent alone was effective. After 14 days, desipramine- and desipramine + fluoxetine-treated rats showed decreased receptor levels, with a greater decrement seen with the combined treatment. Fluoxetine, alone, had no affect on beta-adrenoceptor density at any time point examined. These effects are attributable to central serotonergic action since they were prevented by prior treatment with p-chlorophenylalanine. Cyproheptadine, a 5-HT2 antagonist, did not block these effects. Independent administration of fluoxetine and desipramine produced approximately 20% decrement in isoproterenol-stimulated cyclic AMP accumulation after four days of treatment. Co-administration of desipramine and fluoxetine resulted in a 35% decrement in cyclic AMP accumulation which was nearly additive with that produced by either drug alone. Consequently, the combination of a norepinephrine and serotonin uptake inhibitor may be an advantageous and rapid treatment for the alleviation of certain forms of depression.

[3H]5,7-dichlorokynurenic acid, a novel radioligand labels NMDA receptor-associated glycine binding sites.

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.

NMDA receptor complex antagonists have potential anxiolytic effects as measured with separation-induced ultrasonic vocalizations.

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.

The selectivity of MDL 74,721 in models of neurogenic versus vascular components of migraine.

MDL 74,721 (R)-2-(N1,N1-dipropylamino)-8-methylaminosulfonylmethyl-1,2,3,4-te trahydronaphthalene, a sulfonamidotetralin, has been found to exhibit a 10,000-fold greater potency in neurogenic versus vascular models of migraine. Sumatriptan, a relatively pure 5-HT1D/5-HT1B receptor agonist, also showed higher potency versus neurogenic inflammation. However, for sumatriptan the potency difference (100-fold) in the two pathophysiological models was less pronounced than seen for MDL 74,721. The affinity profile of MDL 74,721 at 5-HT1 receptor subtypes may in part explain its ability to differentiate these two physiological responses. MDL 74,721 demonstrated nanomolar affinity for 5-HT1A (12.7 +/- 0.3 nM) and 5-HT1D (41.3 +/- 10.9 nM) but considerably lower affinity for 5-HT1B receptors (> 1000 nM). Serotonin-like activity was seen in in vitro functional assays including inhibition of forskolin-stimulated cAMP accumulation in human 5-HT1D receptor-transfected fibroblasts or eliciting vasoconstriction in isolated human pial arteries. The intrinsic activity (relative to 5 - HT[E(Amax)]) and affinity (pD2) for the human cerebrovascular 5-HT receptors were: 5-HT (100%, 7.51 +/- 0.09), sumatriptan (94%, 6.85 +/- 0.1) and MDL 74,721 (66%, 5.70 +/- 0.23). In anaesthetised cats, treatment with MDL 74,721 resulted in a dose-related reduction in the percentage of carotid flow going through the arteriovenous anastomoses to the lungs, with an ED50 of 0.3 mg/kg i.v., the same as sumatriptan. However, in the guinea-pig neurogenic model, MDL 74,721 inhibited plasma protein extravasation with an ED50 of 0.023 microg/kg compared to 2.5 microg/kg for sumatriptan. MDL 74,721 was also effective in this model (in rats) after oral administration. In conclusion, MDL 74,721 demonstrates a preclinical profile consistent with anti-migraine efficacy. Its marked preference for inhibiting neurogenic inflammation makes this compound a useful tool for assessing the relative contribution of this pathophysiological mechanism to the human disease state.

MDL 100,458 and MDL 102,288: two potent and selective glycine receptor antagonists with different functional profiles.

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.