Functional Antagonist Activity Research Articles

Synthesis and Biological Evaluation of Benzodioxanylpiperazine Derivatives as Potent Serotonin 5-HT1A Antagonists: The Discovery of Lecozotan

A series of benzodioxanylpiperazine derivatives possessing a 4-aryl amide substituent was prepared and evaluated for 5-HT(1A) affinity and functional antagonist activity in vitro and in vivo. All of the compounds in this series possessed high affinity for the human 5-HT(1A) receptor and many displayed potent antagonist activity in vitro and varying degrees of intrinsic activity in vivo. Compound 11c (Lecozotan) was selected for further development and is currently in clinical trials.

The role of muscarinic receptor antagonism in antipsychotic-induced hippocampal acetylcholine release

Olanzapine and clozapine produce robust increases in hippocampal acetylcholine release during acetylcholinesterase inhibition, while other antipsychotics, including thioridazine, have only small effects. Since thioridazine binds with similar high affinities to muscarinic receptors as olanzapine and clozapine, muscarinic autoreceptor blockade was ruled out as a primary mechanism [Neuropsychopharmacology 26 (2002) 583]. This study compared in vitro binding affinities and functional activities of olanzapine, clozapine, thioridazine, ziprasidone, risperidone, chlorpromazine and scopolamine at muscarinic M 2 receptors with their in vivo potencies to increase acetylcholine release in the rat hippocampus. We found that scopolamine, olanzapine and clozapine, but also high doses of thioridazine and chlorpromazine, markedly increase acetylcholine release. The reduced in vivo potencies of thioridazine and chlorpromazine are consistent with their significantly weaker functional antagonist activity at human muscarinic M 2 receptors, while thioridazine's reduced binding affinity for rat muscarinic M 2 receptors and lower brain exposure, may further contribute to its weak in vivo potency compared to olanzapine. The excellent correlation between in vitro antagonist activities of antipsychotics at muscarinic M 2 receptors and their in vivo potencies to increase acetylcholine release, suggests that olanzapine, clozapine, as well as thioridazine and chlorpromazine, increase acetylcholine release via blockade of terminal muscarinic M 2 autoreceptors.

Medicinal Chemistry of α4β2 Nicotinic Cholinergic Receptor Ligands

The chapter presents a systematic and fairly logical narrative of studies that have been performed over the past few years to understand the medicinal chemistry and neuronal nicotinic cholinergic (nACh) receptor binding of nicotinic ligands. The medicinal chemistry of nicotinic cholinergic ligands suffers from several major problems that are related to multiple subtypes of receptors (making it difficult to determine selectivity), species differences, and efficacy issues. New nicotinic ligands have proliferated at an unprecedented rate over the past decade, but few can be claimed to be truly selective. The binding of relatively few agents has been examined at multiple nACh populations, and even less is known concerning their functional (agonist, partial agonist, and antagonist) activity at each subtype. As a consequence, it is nearly impossible to refer to any of the ligands as being a selective agonist or selective antagonist. A further complication arises from agents that interact with nACh receptors in a non-competitive fashion that might have an indirect influence on binding or functional activity.

Dopamine partial agonists: a new class of antipsychotic.

This review examines the development of dopamine partial agonists as a new class of antipsychotic agents. Partial agonists have a lower intrinsic activity at receptors than full agonists, allowing them to act either as a functional agonist or a functional antagonist, depending on the surrounding levels of naturally occurring neurotransmitter (full agonist). In the absence of a full agonist, partial agonists show functional agonist activity, binding to the receptor to produce a response. In the presence of a full agonist, partial agonists show functional antagonist activity, as receptor binding reduces the response from that seen with the full agonist. A partial agonist at dopamine D(2) receptors therefore offers an attractive option for the treatment of schizophrenia. It should act as a functional antagonist in the mesolimbic dopamine pathway, where excessive dopamine activity is thought to cause positive symptoms, but show functional agonist activity in the mesocortical pathway, where reduced dopamine activity is thought to be associated with negative symptoms and cognitive impairment. In addition, it should avoid the complete blockade of the nigrostriatal or tuberoinfundibular pathways, associated with extrapyramidal symptoms (EPS) and elevated prolactin levels, respectively. Clinical trials with aripiprazole - a new antipsychotic, which shows partial agonist activity at D(2) receptors and serotonin 5-HT(1A) receptors, and antagonist activity 5-HT(2A) receptors - have demonstrated the value of this treatment approach. Aripiprazole produced significant improvements in positive and negative symptoms in short- and long-term studies of patients with schizophrenia or schizoaffective disorder. Improvements occurred rapidly after the start of treatment, and were sustained throughout studies lasting up to 52 weeks. Significantly more patients responded to aripiprazole treatment than to haloperidol in the 52-week study, and aripiprazole-treated patients showed significantly greater improvements in negative and depressive symptoms than those receiving haloperidol. Aripiprazole treatment was well tolerated in both short- and long-term studies, showing a low liability for EPS and hyperprolactinemia, a lack of QTc prolongation, and minimal weight gain or sedation. In conclusion, the findings from clinical studies of aripiprazole show that dopamine partial agonists offer a novel, effective and well-tolerated treatment approach for patients with schizophrenia.

Synthesis and biological evaluation of analogues of 7-chloro-4,5-dihydro-4- oxo-8-(1,2,4-triazol-4-yl)-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylic acid (TQX-173) as novel selective AMPA receptor antagonists.

In recent papers (Catarzi, D.; et al. J. Med. Chem. 2000, 43, 3824-3826; 2001, 44, 3157-3165) we reported chemical and biological studies on 4,5-dihydro-4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylates (TQXs) bearing different nitrogen-containing heterocycles at position-8. In particular, from these studies it emerged that both the 7-chloro-4,5-dihydro-4-oxo-8-(1,2,4-triazol-4-yl)-1,2,4-triazolo[1,5-a] quinoxaline-2-carboxylic acid TQX-173 (compound B) and its corresponding ethyl ester (compound A) were the most active and selective compounds of this series. In pursuing our investigation on the structure-activity relationships of these TQX derivatives, different electron-withdrawing groups (CF(3), NO(2)) were introduced at position 7 on the TQX ring system, replacing the 7-chloro substituent of B and of other selected 8-heteroaryltriazoloquinoxaline-2-carboxylates previously described. All the newly synthesized compounds were biologically evaluated for their binding at the Gly/NMDA, AMPA, and KA high-affinity receptors. Gly/NMDA binding assays were performed to assess the selectivity of the reported compounds toward the AMPA receptor. Compounds endowed with micromolar binding affinity for the KA high-affinity binding site were also evaluated for their binding at the KA low-affinity receptor. Some selected compounds were also tested for their functional antagonist activity at the AMPA and NMDA receptor-ion channel complex. The results obtained in this study have pointed out that 4,5-dihydro-7-nitro-4-oxo-8-(3-carboxypyrrol-1-yl)-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylic acid (9b) and its corresponding ethyl ester (9a) are the most potent and selective AMPA receptor antagonists reported to date among the TQX series.

Structure–activity relationships of xanthene carboxamides, novel CCR1 receptor antagonists

The structure–activity relationships of xanthene carboxamide derivatives on the CCR1 receptor binding affinity and the functional antagonist activity were described. Previously, we reported a quaternarized xanthen-9-carboxamide 1 as a potent human CCR1 receptor antagonist that was derived from a xanthen-9-carboxamide lead 2a. Further derivatization of 2a focusing on installing an additional substituent into the xanthene ring resulted in the identification of 2b–1 with IC 50 values of 1.8 nM and 13 nM in the binding assay using human CCR1 receptors transfected CHO cells and in the functional assay using U937 cells expressing human CCR1 receptors, respectively.

Insertion of 2-Carboxysuccinate and Tricarballylic Acid Fragments into Cyclic-Pseudopeptides: New Antagonists for the Human Tachykinin NK-2 Receptor

A series of cyclic pseudopeptides were synthesized containing the sequence -Trp-Phe-( d)-PheΨCH 2NH-, the terminal ends of which were bound to 2-carboxy succinate or enantiomerically enriched tricarballylic acid to give the final cyclic structures. These two molecules and their subsequent derivatives were screened for h-NK2 receptor binding and functional antagonist activity on the rabbit urinary bladder.

Discovery of a novel CCR3 selective antagonist

In searching for a novel CCR3 receptor antagonist, we designed a library that included a variety of carboxamide derivatives based on the structure of our potent antagonists for human CCR1 and CCR3 receptors, and screened the new compounds for inhibitory activity against 125I-Eotaxin binding to human CCR3 receptors expressed in CHO cells. Among them, two 2-(benzothiazolethio)acetamide derivatives ( 1a and 2a) showed binding affinities with IC 50 values of 750 and 1000 nM, respectively, for human CCR3 receptors. These compounds ( 1a and 2a) also possessed weak binding affinities for human CCR1 receptors. We selected 1a as a lead compound for derivatization to improve in vitro potency and selectivity for CCR3 over CCR1 receptors. Derivatization of 1a by incorporating substituents into each benzene ring of the benzothiazole and piperidine side chain resulted in the discovery of a compound ( 1b) exhibiting 820-fold selectivity for CCR3 receptors (IC 50=2.3 nM) over CCR1 receptors (IC 50=1900 nM). This compound ( 1b) also showed potent functional antagonist activity for inhibiting Eotaxin (IC 50=27 nM)- or RANTES (IC 50=13 nM)-induced Ca 2+ increases in eosinophils.

Evidence that ranolazine behaves as a weak β 1 - and β 2 -adrenoceptor antagonist in the rat cardiovascular system

The clinical anti-anginal effectiveness of ranolazine is currently being evaluated. However, the mechanism of its anti-ischaemic action is still unclear. The aim of this work was to establish whether ranolazine exerts functional beta-adrenoceptor antagonist activity in the rat cardiovascular system. Radioligand binding studies were performed in rat hearts and guinea-pig lungs for beta1- and beta2-adrenoceptor affinity, respectively. Ranolazine had micromolar affinity for both beta,- and beta2-adrenoceptors (pKi5.8 and 6.3, respectively). Developed tension was measured in isolated rat left atria (electrically driven at 4 Hz) and cumulative concentration/response curves to (+/-)isoprenaline (0.01-1,000 nM) constructed. Ranolazine (0.32-10 microM) surmountably but weakly antagonised isoprenaline-induced positive inotropic responses, with an apparent pA2 of 5.85 (5.69-6.00) and a slope of -0.74 (-0.70 to -0.77). In bivagotomised, atropinised pithed rats, ranolazine per se evoked marked bradycardia at doses above 10 mg/kg i.v. (maximum variation at 80 mg/kg -125+/-15 bpm, n=6, P<0.001) by a mechanism apparently unrelated to blockade of beta1- or beta2-adrenoceptors. Cumulative incremental doses of (+/-)isoprenaline (0.63 ng/kg to 0.16 mg/kg i.v.) administered to pithed rats induced concomitant depressor and chronotropic responses. Animals received either vehicle (saline 0.9% i.v., n=12), atenolol (0.04-2.5 mg/kg i.v., n=6 per dose), ICI 118551 (0.01-0.63 mg/kg i.v., n=6 or 7 per dose), (+/-)propranolol (0.01-0.63 mg/kg i.v., n=6 per dose) or ranolazine (2.5-80 mg/kg i.v., n=6 or 7 per dose) 10 min prior to isoprenaline. Ranolazine dose-dependently and competitively antagonised isoprenaline-induced decreases in diastolic arterial pressure (DAP, dose ratio 12.2 with 80 mg/kg ranolazine) and increases in heart rate (HR, dose ratio 20.3 with 80 mg/kg ranolazine). Collectively, these results demonstrate that ranolazine behaves as a weak beta1- and beta2-adrenoceptor antagonist in the rat cardiovascular system.

Postjunctional α 2C-adrenoceptor contractility in human saphenous vein

The postjunctional α 2-adrenoceptor-mediated contractility was characterized in human saphenous vein derived from coronary artery bypass graft surgery. Human saphenous vein contracted to α 2-adrenoceptor selective agonists BHT-920 (5,6,7,8-Tetrahydro-6-(2-propenyl)-4 H-thiazolo[4,5-d]azepin-2-amine dihydrochloride; p D 2=6.7±0.1) and UK 14,304 (5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline; p D 2=7.2±0.1). BHT-920-induced contractions were inhibited by the α 2-adrenoceptor antagonist yohimbine (17-Hydroxy-yohimban-16-carboxylic acid methyl ester hydrochloride; p A 2=8.7±0.5), but not by the α 1-adrenoceptor antagonist prazosin (1-[4-Amino-6,7-dimethoxy-2-quinazolinyl]-4-[2-furanylcarbonyl]-piperazine hydrochloride; 300 nM). In contrast, prazosin (p K b=7.9±0.2) potently antagonized contractions elicited by the α 1-adrenoceptor agonist phenylephrine (( R)-3-Hydroxy-α-[(methylamino)methyl] benzenemethanol hydrochloride; p D 2=4.9±0.1), indicating that both α 2- and α 1-adrenoceptor evoke human saphenous vein contractions. Functional antagonist activity estimates (p A 2 or p K b) obtained for the α-adrenoceptor antagonists ARC 239 (2-[2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2 H,4 H)-isoquinolindione dihydrochloride), WB 4101 (2-(2,6-Dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) and HV 723 (α-ethyl-3,4,5-trimethoxy-α-(3-((2-(2-methoxyphenoxy) ethyl)amino)propyl)benzeneacetonitrile) against BHT-920-induced human saphenous vein contractions were 7.0±0.6, 8.3±0.6 and 7.7±0.3, respectively. The α 2-adrenoceptor subtype affinities (p K i) obtained in recombinant human α 2A-, α 2B- and α 2C-adrenoceptor competition binding assays were 8.6, 8.3 and 8.6 for yohimbine; 6.3, 8.4 and 7.0 for ARC 239; 8.4, 7.5 and 8.4 for WB 4101 and 7.5, 7.4 and 7.9 for HV 723, respectively. Taken together, the binding and functional antagonist activity estimates obtained in these investigations indicate that α 2C-adrenoceptor is the predominant postjunctional α 2-adrenoceptor subtype in human saphenous vein.

Novel non-peptide lead structures for bradykinin B2-receptor antagonists

A series of new non-peptide Bradykinin (BK) B2-receptor antagonists is reported. These new leads belong to a larger set including both commercially or otherwise available potential candidates found by proprietary database searches using 3D-pharmacophore models as query, and several bis-benzamidino compounds selected from our tryptase-like protease inhibitor library on the basis of topological considerations, derived from the same models. Some of these compounds show functional competitive antagonistic activity, inhibiting in vitro the BK-induced contraction of isolated guinea-pig ileum (GPI) and rat uterus with a pA2 up to 5.3 and 7.0, respectively. They display also binding affinity (IC50 up to 0.56 μM) to the BK B2-receptor in radioligand binding assays on GPI membrane preparations and on human IMR-90 fetal lung fibroblast cells expressing this receptor subtype. Furthermore, the results with the commercially available compounds, in some cases developed as therapeutics, show that the used 3D-pharmacophore model allows to predict to some certainty possible side actions of potential drugs.

Vascular-selective effect of lercanidipine and other 1,4-dihydropyridines in isolated rabbit tissues.

The aim of this study was to characterize the in-vitro vasoselectivity of lercanidipine (in comparison with lacidipine, amlodipine, nitrendipine and felodipine) by evaluating its functional calcium antagonistic activity on rabbit vascular (aorta) and cardiac tissues (heart ventricle). Although incubation with all the compounds tested elicited a concentration-dependent relaxant effect on vascular tissue precontracted with KCl (80 mM), 50% relaxation was reached at different times for each concentration and drug tested. At 10 nM concentration 50% relaxation was reached after 210 min with lercanidipine, 278 min with amlodipine, 135 min with lacidipine, 75 min with nitrendipine and 70 min with felodipine. The onset of the effect was, therefore, similar for lercanidipine, amlodipine and lacidipine, but faster for nitrendipine and felodipine. Similarly, all the compounds tested concentration-dependently reduced the force of cardiac contraction (negative inotropic activity). In this model, the time needed to reach 50% reduction in contractile force was also concentration-dependent, and the ranking order of the speed of onset of the effect (evaluated as the ratio of the IC50 values (the concentrations inhibiting contraction by 50%) calculated after 1 and 4 h incubation) was lacidipine (3.8) > amlodipine (9.6) > felodipine (39) > lercanidipine (68) = nitrendipine (89). The vasoselectivity, expressed as the ratio of the IC50 values obtained on cardiac and vascular tissue, were (for 4 h incubation) 730, 193, 95, 6 and 3 for lercanidipine, lacidipine, amlodipine, felodipine and nitrendipine, respectively, showing that lercanidipine is the most vasoselective of the calcium-antagonists tested. The results show that lercanidipine reduces the inotropic force of the rabbit heart to a lesser extent than do other calcium antagonists, and that this drug had the best heart/vessel selectivity index among the compounds tested at all the times tested.

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 4. Discovery of novel frameworks mimicking the active conformation.

In recent articles we reported the identification of a series of 8-[[2, 6-dichloro-3-[N-methyl-N-[(E)-(substituted)acryloylglycyl]amino]++ +benzy l]oxy]-2-methylimidazo[1,2-a]pyridines as the first orally active non-peptide bradykinin (BK) B2 receptor antagonists. Optimization of the terminal glycine part and the imidazo[1,2-a]pyridine moiety led to the discovery of a clinical candidate (5, FR173657). With the aim of completion of the structure-activity relationship (SAR) study, we next investigated the roles of the substituents on the central phenyl ring. The results suggested that the 2,6-dichloro or 2, 6-dimethyl groups may play important roles in regulating the conformations of the 1- and 3-substituents and also may interact with hydrophobic pockets of the B2 receptors. Furthermore, according to the results of a molecular modeling study reported in part 1 of this series, we designed and synthesized a series of sterically constrained analogues by replacing the N-methylamide group with cis-amide-like rigid moieties. We discovered several bioisosteres and chemically proved that the N-methylamide moiety adopts the cis-amide form in the active conformation. Extensive chemical modification led to the identification of a novel class of highly potent and orally active non-peptide B2 antagonists represented by a pyrrole derivative (52a, FR193517). Compound 52a inhibited the specific binding of [3H]BK to recombinant human B2 receptors expressed in Chinese hamster ovary (CHO) cells and guinea pig ileum membrane preparations expressing B2 receptors with IC50s of 0.37 and 0.56 nM, respectively. This compound also displayed excellent in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs at 1 mg/kg by oral administration.

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 3. Discovering bioisosteres of the imidazo[1,2-a] pyridine moiety.

Recently we reported on overcoming the species difference of our first orally active non-peptide bradykinin (BK) B2 receptor antagonists, incorporating an 8-[[3-(N-acylglycyl-N-methylamino)-2, 6-dichlorobenzyl]oxy]-3-halo-2-methylimidazo[1,2-a]pyridine skeleton, leading to identification of the first clinical candidate 4a (FR167344). With this potent new lead compound in hand, we then investigated further refinement of the basic framework by replacement of the imidazo[1,2-a]pyridine moiety and discovered several bioisosteric heterocycles. Extensive optimization of these new heteroaromatic derivatives revealed the detailed structure-activity relationships (SAR) around the imidazo[1, 2-a]pyridine ring and the 2,6-dichlorobenzyl moiety, leading to the discovery of our second clinical candidate 87b (FR173657) which inhibited the specific binding of [3H]BK to recombinant human B2 receptors expressed in Chinese hamster ovary (CHO) cells and guinea pig ileum membrane preparations expressing B2 receptors with IC50's of 1.4 and 0.46 nM, respectively. This compound also displayed excellent in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs with an ED50 value of 0.075 mg/kg by oral administration. Further modifications of the terminal substituents on the pyridine moiety led to a novel pharmacophore and resulted in the identification of 99 (FR184280), whose IC50 value for human B2 receptors (0.51 nM) was comparable to that of the second-generation peptide B2 antagonist Icatibant.

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 2. Overcoming the species difference between guinea pig and man.

Recently we reported the identification of a series of 8-[[3-(N-acylglycyl-N-methylamino)-2, 6-dichlorobenzyl]oxy]-3-halo-2-methylimidazo[1,2-a]pyridines as the first orally active non-peptide bradykinin (BK) B2 receptor antagonists (1-3). These compounds inhibited the specific binding of [3H]BK to guinea pig ileum membrane preparations expressing B2 receptors with nanomolar IC50's and also displayed in vivo functional antagonistic activities against BK-induced bronchoconstriction in guinea pigs at 1 mg/kg by oral administration. However, it was found that their affinities for the B2 receptors in human A-431 cells (human epidermoid carcinoma) were much lower. Intensive modifications of the terminal substituents at the glycine moiety elucidated the structure-activity relationships (SAR) for human B2 receptors, leading to an extended basic framework which incorporated a novel key pharmacophore. Thus, we overcame the species difference and identified the first clinical candidate 18c (FR167344) with IC50's of 0.66 and 1.4 nM for guinea pig ileum and human A-431 cells, respectively. This compound displayed in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs with an ED50 value of 0.17 mg/kg by oral administration. This novel non-peptide B2 antagonist is extremely potent both in vitro and in vivo by oral administration and is expected to be the first member of a new class of drug for the treatment of various inflammatory diseases.

Biological Characterization of Neurokinin Antagonists Discovered Through Screening of a Combinatorial Library

Recent advances in combinatorial chemistry have resulted in the rapid screening of libraries against biological targets. Another advance in biological screening is the ability to design and utilize novel, automated, nonradioactive assays for targets of pharmaceutical interest. Using encoding technology and europium time-resolved fluorescence, we have designed primary receptor binding assays to define active compounds against the neurokinin-1 and neurokinin-2 receptor subtypes. In addition, a secondary, cell-based, functional assay measuring intracellular calcium flux with calcium sensitive fluorophores was used to determine receptor agonist or antagonist activities. We adapted a cuvette based assay to a CCD camera and digital imaging system that allowed us to demonstrate functional receptor antagonist activity in all 96 wells of a microtiter plate simultaneously. The screening of a 20,000 member library using europium labeled neurokinin ligands resulted in the identification of 43 active compounds for neurokinin-1 and 27 for neurokinin-2. Through medicinal chemistry and structure-activity relationships, a compound was synthesized with balanced dual antagonist activity at both neurokinin receptors with greater than 100-fold less activity against the neurokinin-3 receptor subtype. The structure-activity relationships generated from this initial library can now be used to design a new focused library to improve on neurokinin-1/neurokinin-2 receptor potency and selectivity.

YM022 [( R)-1-[2,3-dihydro-1(2'-methylphenacyl)-2- oxo-5-phenyl-1 H-1,4-benzodiazepin-3- yl]-3-(3-methylphenyl)urea]: An irreversible cholecystokinin type-B receptor antagonist

A functional evaluation of the recently developed cholecystokinin type-B (CCK-B) receptor antagonist YM022 [( R)-1-[2,3-dihydro-1-(2'-methylphenacyl)-2-oxo-5-phenyl-1 H-1,4-benzodiazepin-3-yl]-3-(3-methylphenyl)urea]was undertaken in Chinese hamster ovary cells stably expressing the human CCK-B receptor gene (hCCK-B.CHO). YM022 exhibited high affinity and selectivity for the CCK-B receptor subtype as estimated from [ 125I]CCK8S displacement studies using membranes derived from hCCK-B.CHO and hCCK-A.CHO cells. Functional antagonist activity of YM022 was demonstrated employing CCK-4-stimulated Ca 2+ mobilization in hCCK-B.CHO cells. In the presence of 30 nM YM022, the maximum effect of CCK-4 was suppressed to 48 ± 11% of control, an effect that was accompanied by a modest rightward shift in the CCK-4 concentration-response curve. In contrast, the structurally similar CCK-B receptor antagonist L-365,260 [3R(+)- N-[2,3-dihydro-1-methyl-2-oxo-5-phenyl-1 H-1,4-benzodiazepin-3- yl]- N'-(methylphenyl)] urea; 30 nM-10 μM]produced progressive rightward shifts in the CCK-4 concentration-response curve, with no effect observed on the CCK-4 maximum response. Further characterization using the technique of microphysiometry revealed that the agonist activity of CCK-4 was not restored following washout after exposure to YM022. The antagonist activity of L-365,260, however, was found to be fully reversible in this system. Thus, YM022 behaves as an irreversible antagonist, whilst its structural analogue L-365,260 exhibits properties consistent with a competitive antagonist.

In Vitro and In Vivo Vascular Selectivity of Lercanidipine and Its Enantiomers

In this study we evaluated the in vitro and in vivo vascular selectivity of lercanidipine and its enantiomers compared with nifedipine, nitrendipine, and felodipine. The in vitro functional calcium antagonist activity of lercanidipine and its enantiomers was studied in different rat tissues as relaxing potency against tonic contractions induced by preincubation with KCl, and lercanidipine and its enantiomers proved more active in the vascular tissue than in nonvascular tissues. In contrast, nitrendipine exhibited almost the same potency in all tested tissues. In rabbit heart ventricular strips electrically driven, lercanidipine (and its enantiomers) showed a very weak negative inotropic activity, being 857- and 667-fold less potent than felodipine and nitrendipine, respectively. The in vivo effects of i.v. lercanidipine. nitrendipine, and nifedipine were studied in anesthetized dogs catheterized and continuously monitored for systemic blood pressure, heart rate, and the isovolumic contractility index dP/dtmax. At doses producing equal vasodilatation, lercanidipine affected cardiac ventricular contractility much less than nifedipine and nitrendipine. Considering together the in vitro and in vivo data, it can be reasonably stated that lercanidipine displays marked vascular selectivity, causing blood pressure but not cardiac inotropism to be reduced.

P.6.008 - Role of noradrenergic, dopaminergic, serotonergic and opioidergic systems on the effect of neonatal handling in learned helplessness

The dysregulation of arginine vasopressin (AVP) release and activation of vasopressin V1A and V2 receptors may play a role in disease. The in vitro and in vivo pharmacology of RWJ-676070, a potent, balanced antagonist of both the V1A and V2 receptors is described. RWJ-676070 binding and intracellular functional antagonist activity was characterized using cells expressing V1A, V1B or V2 receptors. Its inhibition of V1A receptor-mediated contraction of vascular rings and platelet aggregation was determined. V2 receptor-medated aquaresis was determined in rats, dogs and monkeys. V1A receptor-mediated inhibitory activity was assessed in vivo in a vasopressin-induced hypertension model and in normotensive rats and in two hypertensive rat models. RWJ-676070 inhibited AVP binding to human V1A and V2 receptors (Ki = 1 and 14 nM, respectively). RWJ-676070 inhibited V1A receptor-induced intracellular calcium mobilization and V2 receptor-induced cAMP accumulation with Ki values of 14 nM and 13 nM, respectively. The compound was slightly less potent against rat V1A receptors. RWJ-676070 inhibited V1A receptor-mediated vasoconstriction in rat and dog vascular rings and AVP-induced human platelet aggregation. Dose dependent aquaresis was demonstrated in rats, dogs and monkeys following oral administration. RWJ-676070 inhibited AVP-induced hypertension in rats but had no effect on arterial pressure in normotensive and spontaneously hypertensive rats but did decrease arterial pressure in Dahl, salt-sensitive hypertensive rats. RWJ-676070 is a new, potent antagonist of V1A and V2 receptors that may be useful for treatment of diseases benefiting from balanced inhibition of both V1A and V2 receptors.

Synthesis, characterization, and conformational analysis of the D/L-Tic7 stereoisomers of the bradykinin receptor antagonist D-Arg0[Hyp3,Thi5,D-Tic7,Oic8]bradykinin.

D-Arg0[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HOE-140) is a potent (Ki = 0.11 nM) inhibitor of [3H]bradykinin binding to bradykinin B2 receptors found on human IMR-90 fetal lung fibroblasts. During the synthesis of this compound, we isolated and unambiguously identified the L-Tic7 stereoisomer (WIN 65365), which exhibits a 2000-fold lower binding affinity (Ki = 130 nM) than HOE-140 to the bradykinin receptor. A similar decrease in potency is observed for WIN 65365 inhibition of bradykinin-stimulated 45Ca2+ efflux from IMR-90 cells. Both HOE-140 and WIN 65365 appear to be competitive antagonists at the IMR-90 bradykinin receptor. This is the first documentation of bradykinin binding and functional antagonist activity by a bradykinin peptide analogue with an L amino acid replacing Pro7. In an attempt to rationalize the differences in binding affinities of HOE-140 and WIN 65365, a conformational analysis of the peptides was undertaken using annealed molecular dynamics (AMD). Conformational analysis of HOE-140 reveals a strong preference for the formation of a type II' beta-turn in the carboxy-terminal region. Analogous modeling of WIN 65365 reveals that its conformation is strikingly different from HOE-140 in that the four carboxy-terminal residues of WIN 65365 do not form a beta-turn. These differences in low-energy conformations between the two peptides may lead to a better understanding of the molecular interaction of antagonists with the bradykinin receptor.