Glunicate (LG 13979) protects the arterial wall from cholesterol-induced atherosclerotic changes in the rabbit without affecting plasma lipids.

Glunicate is evaluated compared to nicotinic acid for effects on aortic atheromatous lesions, lipid parameters and factors involved in thrombosis and haemostasis in rabbits kept on a high-cholesterol diet for 12 weeks, using 2 doses of glunicate (0.17 and 0.69 g/day) and 1 of nicotinic acid (0.6 g/day). Glunicate afforded dose-dependent protection of the arterial wall from atheromatous lesions and from cholesterol and collagen accumulation, while nicotinic acid hardly had any effect. These effects were completely independent of plasma lipid-lowering action, the plasma levels of all lipids being indistinguishable in all cholesterol-fed groups. In addition to inducing the expected changes in the lipid pattern, the atherogenic diet increased platelet aggregation in response to collagen but not to ADP, prolonged the APTT and lowered the plasma fibrinogen levels. Both glunicate and nicotinic acid counteracted the effects of the diet on platelet aggregation and on APTT, but only glunicate normalised the fibrinogen levels. There was no change in PT or in prostacyclin-like activity release from the mesenteric artery after the diet or diet plus drugs.

Molecular determinants of peptide and nonpeptide NK-2 receptor antagonists binding sites of the human tachykinin NK-2 receptor by site-directed mutagenesis.

A series of 14 mutants on nine selected residues of the human tachykinin NK(2) receptor was produced and stably transfected into CHO cells to investigate the binding of the peptide MEN 11420 and the nonpeptide SR 48968 antagonists. The main interactions found for MEN 11420 were with Thr171, Tyr206, Tyr266 and Phe270. In the case of SR 48968 crucial residues were Tyr266 and Tyr289. While some overlapping of the binding sites exists, the binding modes suggested by this study appear not to allow structural correlation, and therefore general SAR, between these two antagonists.

N-3-substituted pyrimidinones as potent, orally active, AT1 selective angiotensin II receptor antagonists.

A novel series of nonpeptide angiotensin II (A II) antagonists containing a pyrimidinone ring which carries a C-linked biphenyltetrazole moiety and a carboxyheteroaryl group on the 3-position have been prepared. Their affinity for the AT1 receptor was determined in a binding assay on rat adrenal cortical membranes. The in vivo antihypertensive properties were tested by evaluating the inhibition of the pressor response to A II followed by iv and id administration. Extensive molecular modeling studies, including comparison of molecular electrostatic potential distributions, conformational analysis, and overlays on a computational pharmacophore model of A II, were used to evaluate structural parameters of the new compounds, in comparison to other known A II antagonists (e.g., DUP-753 and SK&F 108566). According to the modeling studies, the introduction of a (carboxyheteroaryl)methyl moiety at the 3-position of the pyrimidinone ring led to derivatives with increased potency. Methyl 2-[[4-butyl-2-methyl-6-oxo-5-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl ]- 4-yl]methyl]-1-(6H)-pyrimidinyl]methyl]-3-thiophenecarboxylate (3k, LR-B/081), one of the most potent compounds in the series (Ki = 1.4 nM), exhibited a marked antihypertensive activity on oral administration to conscious renal hypertensive rats, with long duration of action. It was selected for clinical evaluation in the treatment of hypertension in man.

Nonpeptide angiotensin II receptor antagonists. Synthesis, in vitro activity, and molecular modeling studies of N-[(heterobiaryl)methyl] imidazoles.

With the aim of explaining the influence of the structural changes on the biphenylic moiety on the activity, a series of N-[(heterobiaryl)methyl]imidazoles (I), constructed on the model of DuPont compounds by replacing either the central or terminal phenyl ring with a heteroaromatic one, such as furan, thiophene, thiazole, and pyridine, was synthesized. Compared to the reference DuPont compound (EXP-7711), all the heterobiaryl derivatives showed a reduced potency both in receptor binding (rat adrenal capsular membranes) and in the functional assay (angiotensin II-induced contraction of rabbit aorta strips). The lower activity was justified by the extensive molecular modeling studies, which took into consideration the conformational and electrostatic features of several heterobiaryl derivatives. On the basis of the results obtained, it was hypothesized that the central aromatic ring of the biarylic portion works as a spacer, orienting in the right way the terminal phenyl ring, whose electronic distribution is, instead, crucial to its fitting well with a lipophilic pocket at the receptor site.

4-Diazinyl- and 4-pyridinylimidazoles: potent angiotensin II antagonists. A study of their activity and computational characterization.

A series of N-[biphenylyl(tetrazolyl)methyl]-2-butylimidazoles containing variously substituted diazine or pyridine moieties either as their free bases or N-oxide derivatives attached to the 4-position of the imidazole ring was synthesized and tested for interaction with the AT1 receptors of rat adrenal cortex membranes (receptor binding assay). Some compounds were then chosen for further evaluation in vivo in the A II-induced pressor response in conscious normotensive rats. The most potent in the AT1 binding assay were found to be compounds in which the diazine or pyridine ring nitrogen is adjacent to the point of attachment between the two heteroaromatic rings such as 2-butyl-4-(3,6-dimethylpyrazin-2-yl)-1-[[2'-(1H-tetrazol-5-y l)-biphenyl-4- yl]methyl]-1H-imidazole (3b) or 2-butyl-4-[5-(methoxycarbonyl)pyrid-2-yl]-1-[[2'-(1H-tetrazol++ +-5- yl)biphenyl-4-yl]methyl]-1H-imidazole (6c). The binding affinities and oral activities of the pyridine N-oxide imidazoles in which a stabilizing group ortho to the pyridine ring nitrogen is present were markedly improved as in 2-butyl-4-[(3-methoxycarbonyl)-6-methyl-N-oxopyridin-2-yl]-1-[[2'- (1H- tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-imidazole 31b. Molecular modeling studies were carried out to determine the molecular electrostatic potential values of related model systems and to correlate their receptor interaction energies with the observed activities of our compounds.

1,2-Cyclomethylenecarboxylic monoamide hydroxamic derivatives. A novel class of non-amino acid angiotensin converting enzyme inhibitors.

A series of monoamidic derivatives of cis- and trans-1,2-cyclohexanedicarboxylic and 1,2-cyclopentanedicarboxylic acids bearing either a carboxylic, sulfhydrylic, or hydroxamic group in the side chain were synthesized and evaluated in vitro for their inhibitory activity against angiotensin converting enzyme. The compounds were designed as potential ACE inhibitors of novel structure, assuming that a monoamidic residue of an 1,2-cyclomethylenedicarboxylic acid could be an alternative structure to the acylproline moiety, the carboxyl-terminal portion common to various ACE inhibitors. The most active compounds were found in the hydroxamic derivatives of cyclohexane series; within this series of derivatives a marked increase of potency was caused by alkylation of the amidic nitrogen with a methyl or ethyl group. Therefore enantiomers of the selected hydroxamic derivatives of cis- and trans-1,2-cyclohexanedicarboxylic acid were prepared by two different chiral synthetic routes and evaluated in vitro for their ACE inhibitor potencies. The active enantiomers both of the cis series (21a, 21c) and trans series (16b, 16d) were found to have all R configuration at the C-2 and R or S configuration at the C-1, while in the classical ACE inhibitors S configuration at the terminal carboxylate (corresponding to the C-1 of our compounds) is strictly required for activity. The most potent compound of the series was (1S,2R)-cis-2[[[2-(hydroxyamino)-2-oxoethyl]methylamino]carbonyl] cyclohexanecarboxylic acid (21a) with an IC50 value of 7.0 nM compared with the value of 3.0 nM for captopril. Further 21a was shown to be highly selective and competitive ACE inhibitor. These results indicate that this non-amino acid structure of inhibitors meets the ACE active site requirements for the binding. The binding compatibility of the most active compounds with a model of ACE active site was evaluated by molecular modeling techniques.

Human umbilical vein smooth muscle cells as a model to study thrombin generation and function: effect of thrombin inhibitors.

The aim of the present work was to study how human umbilical vein smooth muscle cells (HUVSMC) can initiate the coagulation process and to investigate the responses of these cells to thrombin. Exposure of HUVSMC to recalcified human plasma led to a time-dependent production of thrombin, measured both as amidolytic activity and as release of fibrinopeptide A. Thrombin activity was dose-dependently reduced by an anti-human tissue factor antibody (76 +/- 3% at 10 micrograms/ml) and by inhibitors like heparin, rec-hirudin, hirulog 1, Napap and hirunorm, a novel hirudin-like thrombin inhibitor (IC50 = 2 +/- 0.4, 8 +/- 1, 130 +/- 22, 199 +/- 29 and 68 +/- 8 nM, respectively). The release of fibrinopeptide A was similarly prevented (IC50 = 14 +/- 1, 132 +/- 25 and 50 +/- 8 nM for rec-hirudin, Napap and hirunorm, respectively). Exogenously added thrombin increased thymidine incorporation into HUVSMC to 240 +/- 30% of basal (EC50 = 0.49 +/- 0.09 nM) and thrombin inhibitors blocked this effect (IC50 = 10 +/- 3, 37 +/- 17, 343 +/- 165 and 1402 +/- 758 nM for rec-hirudin, hirunorm, Napap and hirulog-1, respectively). Also recalcified human plasma was mitogenic for HUVSMC and its effect was mainly due to endogenously generated thrombin, as shown by the use of thrombin inhibitors. In conclusion, HUVSMC are capable of initiating the extrinsic coagulation cascade, leading to the formation of thrombin which promotes clotting and stimulates DNA synthesis. Thrombin inhibitors prevent both coagulative and cellular effects of thrombin.

Comparison of the cardiovascular and neural activity of endothelin-1, -2, -3 and respective proendothelins: effects of phosphoramidon and thiorphan.

1. In the anaesthetized, ganglion-blocked rat, intravenous boluses of endothelin-1, endothelin-2 and endothelin-3 induced a transient hypotensive effect followed by a potent long lasting pressor response (ED50 mmHg: 0.72 +/- 0.05, 1.8 +/- 0.2 and 2.7 +/- 0.3 nmol kg-1, respectively). The maximal effect for the three peptides was of a similar order of magnitude (delta MAP: 84 to 89 mmHg). Neither of these effects was influenced by phosphoramidon or thiorphan (10 mg kg-1, i.v.). 2. Intravenously administered big-endothelin-1 and -2 induced a transient (1-2 min) hypotension followed by a potent long lasting (> 25 min) vasopressor effect (ED50 mmHg: 1.8 +/- 0.2 and 6.7 +/- 0.4 nmol kg-1, respectively), with a similar maximal activity (delta MAP: 85 +/- 4 and 81 +/- 2.4 mmHg, respectively). The onset of the big-endothelin-1 vasopressor effect was more rapid (5-6 min) than that of big-endothelin-2 (10-13 min). Big-endothelin-3 was found to induce only a potent, long lasting (> 35 min) hypertension, with a maximal effect of 75 +/- 4.6 mmHg at 10 nmol kg-1 and an ED50 mmHg of 6.5 +/- 0.4 nmol kg-1. The onset of this effect was much slower (20-25 min) than that of the other proendothelins. Pressor responses induced by big-endothelin-1, -2 and -3 (3, 15 and 10 nmol kg-1, respectively) were markedly reduced (60, 80 and 100%) in the presence of phosphoramidon (10 mg kg-1, i.v.). Thiorphan (10 mg kg-1, i.v.) did not inhibit the effects of big-endothelin-1, -2 and -3. 3. In the electrically stimulated rat vas deferens, endothelin-I and -2 were found to be equipotent enhancers of the twitch response (EC100%: 4.0 +/- 0.4 nm and 7.9 +/- 4.8 nm, respectively), both about 3-4 fold as active as endothelin-3 (EC100%: 19 +/- 2.5 nM). Endothelin-1 and -3 showed a comparable maximalstimulatory effect (Emax: 296 +/- 30 and 262 +/- 24%) while endothelin-2 was less active (Emax: 194 +/- 30%).4. Big-endothelin-l and -2 were potent enhancers of the twitch response too (EC 100,%: 10.0 +/- 2.6 nM and 21.6 +/- 3.2 nM, respectively), with a comparable maximal stimulatory effect (Emax: 254 +/- 22 and 264 +/-24%). Big-endothelin-3 was found to be less potent (EC,100%: 275 +/- 21 nM), but retained a marked potentiating effect (Emax: 200 +/- 38%). Phosphoramidon, but not thiorphan, concentration-dependently(10 and 100 microM) reduced big-endothelin-1 (58 and 86% respectively) and big-endothelin-2 (21 and 56%)-mediated responses. Conversely, the big-endothelin-3 effect was reduced by phosphoramidon only at 100 microM (-70%), while thiorphan acts concentration-dependently (31 and 71% at 10 and 100 microM respectively); thus, in the rat vas deferens, big-endothelin-I and -2 were as potent as their corresponding endothelins, while big-endothelin-3 was about 20 times less potent than endothelin-3.5. The increasing effect of endothelin-2 (194 +/- 30% over baseline) was significantly enhanced by either 10 microM phosphoramidon (277 +/- 42%) or thiorphan (318 +/- 15%). The endothelin-I and endothelin-3-mediated twitch enhancement was not affected by the two protease inhibitors (10 microM).6. These results suggest that in vivo big-endothelin-1, -2 and -3, are processed through a similar phosphoramid on-sensitive enzymatic pathway although with different apparent affinity. This enzymatic process is probably attributable to a neutral endoprotease, distinct from neutral-endopeptidase 24.11(NEP). On the other hand, a NEP-like enzymatic activity may be involved, in the rat vas deferens, in the activation of big-endothelin-3 to endothelin-3 and in the metabolism of endothelin-2, but not of endothelin-I or endothelin-3.

Pharmacology of LR-B/081, a new highly potent, selective and orally active, nonpeptide angiotensin II AT1 receptor antagonist.

1. The pharmacological profile of LR-B/081, (methyl 2-[[4-butyl-2-methyl- 6-oxo-5-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-1(6H)- pyrimidinyl]methyl]-3-thiophenecarboxylate), a novel antagonist at the angiotensin II (AII) AT1-receptor, was studied in vitro and in vivo. 2. In rabbit aortic strips incubated with LR-B/081 (1-1,000 nM), the concentration-response curve to AII was displaced to the right in a nonparallel fashion and the maximal contraction was progressively reduced, indicating that the compound is an insurmountable antagonist in this preparation (apparent pKB = 9.50 +/- 0.23). However, the interaction of LR-B/081 with AII receptors was found to be reversible, since the maximal response to AII was restored by coincubation with losartan, a surmountable AII AT1-antagonist. Contractions elicited by KCl or phenylephrine were not affected by 10 microM LR-B/081. 3. In rat isolated perfused kidney, LR-B/081 and losartan antagonized the AII-induced vasoconstriction [IC50 (95% confidence limits) = 17(13-24) and 39(32-54) nM, respectively]. The LR-B/081 antagonism was incompletely reversed by excess AII, while losartan was fully displaced. The IC50 values of LR-B/081 and losartan obtained against vasoconstriction induced by endothelin-1 and noradrenaline were two orders of magnitude higher. 4. In pithed rats, the intravenous administration of LR-B/081 (0.2-2 mumol kg-1) dose-dependently shifted to the right in a nonparallel fashion the dose-pressor response curve to AII. The maximal pressor response to AII was reduced by LR-B/081 in a dose-dependent fashion. The coadministration of losartan induced a progressive recovery of the maximal pressor response to All, indicating that in vivo the interaction of LR-B/081 with All receptors is reversible. LR-B/081 at 6 micromol kg-1, i.v. also did not affect the vasopressor response induced by noradrenaline in the pithed rat.5. In conscious normotensive rats, single oral administration of LR-B/081 at 6 micromol kg-1 markedly inhibited the All-induced pressor response; the inhibition lasted more than 24 h.6. In conscious renal hypertensive rats, intravenous LR-B/081 appeared as potent as losartan (ED40mmHg(95% confidence limits) = 0.50(0.36-0.70) and 0.86(0.57-1.3) micromol kg-1, respectively). A single intravenous(2 micromol kg-1) or oral (6 micromol kg-1) administration of LR-B/081 induced a marked fall in blood pressure which lasted for at least 12 h.7. In conscious spontaneously hypertensive rats, LR-B/081 at 20 micromol kg-1 , p.o., induced a marked and sustained fall in blood pressure. The duration of the antihypertensive effect was longer than 12 h.Heart rate was not modified by LR-B/081 treatment. Repeated oral administration of 17 micromol kg-1LR-B/081 for 16 days did not result in the development of tolerance.8 These results demonstrate that LR-B/081 is a potent, selective and orally active antagonist of All at the AT1-receptor subtype, which markedly lowers the blood-pressure in conscious renal and spontaneously hypertensive rats.

MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist.

1. The pharmacological profile was studied of MEN 11420, or cyclo[[Asn(beta-D-GlcNAc)-Asp-Trp-Phe-Dap-Leu]cyclo(2beta-5beta )], a glycosylated derivative of the potent, selective, conformationally-constrained tachykinin NK2 receptor antagonist MEN 10627 (cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2beta-5beta)). 2. MEN 11420 competitively bound with high affinity to the human NK2 receptor stably transfected in CHO cells, displacing radiolabelled [125I]-neurokinin A and [3H]-SR 48968 with Ki values of 2.5+/-0.7 nM (n = 6) and 2.6+/-0.4 nM (n = 3), respectively. 3. MEN 11420 showed negligible binding affinity (pIC50 < 6) at 50 different receptors (including tachykinin NK1 and NK3 receptors) and ion channels. 4. In the rabbit isolated pulmonary artery and rat urinary bladder MEN 11420 potently and competitively antagonized tachykinin NK2 receptor-mediated contractions (pK(B) = 8.6+/-0.07, n = 10, and 9.0+/-0.04, n = 12; Schild plot slope = -1.06 (95% c.l. = -1.3; -0.8) and -1.17 (95% c.l. = -1.3; -1.0), respectively). MEN 11420 produced an insurmountable antagonism at NK2 receptors in the hamster trachea and mouse urinary bladder. However, in both preparations, the effect of MEN 11420 was reverted by washout and an apparent pK(B) of 10.2+/-0.14, n = 9, and 9.8+/-0.15, n = 9, was calculated in the hamster trachea and mouse urinary bladder, respectively. 5. MEN 11420 showed low affinity (pK(B) < 6) at guinea-pig and rat tachykinin NK1 (guinea-pig ileum and rat urinary bladder) and NK3 (guinea-pig ileum and rat portal vein) receptors. On the whole, the affinities (potency and selectivity) showed by MEN 11420 for different tachykinin receptors, measured either in binding or in functional bioassays, were similar to those shown by the parent compound, MEN 10627. 6. The in vivo antagonism of the contractions produced by [betaAla8]neurokinin A(4-10) (1 nmol kg(-1)) was observed after intravenous (dose range: 1-10 nmol kg(-1)), intranasal (3-10 nmol kg(-1)), intrarectal (30-100 nmol kg(-1)) and intraduodenal (100-300 nmol kg(-1)) administration of MEN 11420. MEN 11420 was more potent (about 10 fold) and longer lasting than its parent compound MEN 10627, possibly due to a greater metabolic stability. 7. A dose of MEN 11420 (100 nmol kg(-1), i.v.), that produced potent and long lasting inhibition of the contraction of the rat urinary bladder induced by challenge with the NK2 selective receptor agonist [betaAla8]neurokinin A(4-10) (10-300 nmol kg(-1)), was without effect on the responses produced by the NK1 receptor selective agonist [Sar9]substance P sulphone (1-10 nmol kg(-1)). 8. These findings indicate that MEN 11420 is a potent and selective tachykinin NK2 receptor antagonist. The introduction of a sugar moiety did not produce major changes in the affinity profile of this antagonist as compared to MEN 10627, but markedly improved its in vivo potency and duration of action. With these characteristics, MEN 11420 is a suitable candidate for studying the pathophysiological significance of tachykinin NK2 receptors in humans.

Inactivation of brain cortex muscarinic receptors by 4-diphenylacetoxy-1-(2-chloroethyl) piperidine mustard.

We demonstrated in this study that 4-DAMP [4-diphenylacetoxy-1-(2- chloroethyl) piperidine] mustard, which cyclizes to the aziridinium ion, behaved as a non-selective, non-competitive inhibitor of muscarinic receptors in rat brain cortex. It inactivated to the same extent the M1, M2 and M4 muscarinic receptors present in this tissue, as well as receptors accessible or not accessible to quaternary antimuscarinic drugs. Under mild incubation conditions, the muscarinic receptors in a state with super high affinity for agonists (SH receptors) were less affected by preactivated 4-DAMP mustard than the receptors in the states with lower affinity for agonists (H and L receptors).

Defects of tyrosine289phenylalanine mutation on binding and functional properties of the human tachykinin NK2 receptor stably expressed in chinese hamster ovary cells.

A point mutation was made at position 289 in the transmembrane segment 7 of the human tachykinin NK2 receptor to yield a tyrosine/phenylalanine (Tyr/Phe) substitution. Chinese hamster ovary cells stably transfected with the wild-type or Tyr289Phe mutant NK2 receptor both bound neurokinin A (NKA) and the synthetic NK2 receptor-selective agonists, GR 64349 and [betaAla8]NKA(4-10), with high and even affinities. Neurokinin B (NKB) and substance P (SP) also displayed sizeable binding affinities, albeit with lower affinity as compared to NKA. In a functional assay (production of inositol-1,4,5-trisphosphate, IP3), NKA, GR 64349, and [betaAla8]INKA(4-10) stimulated IP3 accumulation via the wild-type and mutant receptors with similar potencies. On the other hand, NKB and SP exhibited a dramatic reduction in their agonist efficacies at the mutant receptor, NKB acting as a partial agonist (maximum effect = 50% of the response to NKA) and SP being totally inactive. The results obtained with phenoxybenzamine inactivation experiments indicated that a large and similar receptor reserve existed for both the wild-type and the mutant receptor. SP, which displayed sizeable binding affinity for the mutant receptor but did not stimulate IP3 accumulation, antagonized the agonist effect of NKA. The antagonist action of SP at the mutant NK2 receptor cannot be ascribed to receptor internalization. The Tyr/Phe replacement at position 289 markedly reduced the binding affinity and antagonist potency of the non-peptide ligand, SR 48968, without affecting the binding affinity and antagonist potency of the bicyclic peptide antagonist MEN 11420. The results indicate that the hydroxyl radical function of Tyr289 in transmembrane segment 7 of the human NK2 receptor is, directly or indirectly, involved in stimulus transduction when the NK2 receptor is occupied by NKB or SP, but not when using NKA or NK2 receptor-selective agonists.

Role of D-tryptophan for affinity of MEN 10207 tachykinin antagonist at NK2 receptors.

The role of the D-Trp residues in the sequence of the NK2-selective tachykinin antagonist, MEN 10207 (Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Arg-NH2). has been examined by replacement of each D-Trp with either the L-isomer or the residue naturally occurring in the same position of neurokinin A(4-10). The biological activity of the analogues thus obtained has been characterized, with special attention to the selectivity for the three tachykinin receptors and for the two subtypes of the NK2 receptor recently described. We conclude that the simultaneous presence of the three D-Trp residues of MEN 10207 is crucial both for affinity and for selectivity.

Differential effect of dithiothreitol and DuP 753 on angiotensin II receptor subtypes in bovine cerebellar cortex.

Angiotensin II (AII) receptor subtypes were investigated in bovine cerebellar cortical membranes (BCCM) using agonists and antagonists. Differences were observed whether the tissue for the characterization was used fresh or frozen. In fresh BCCM, AII and angiotensin III (AIII) (not selective), p -aminophenylalanine6 -AII ([pNH2Phe6]AII) (AT2 selective), and DuP 753 (AT1 selective) interacted with two sites, at high and low affinity. In the presence of 10 mM dithiothreitol (DTT), the low-affinity site for AIII and [pNH2Phe6]AII and the high-affinity site for DuP 753 were no longer detectable. The same effect was obtained with 1 microM DuP 753, with only the low-affinity component of [pNH2Phe6]AII being resistant to this blocker. In frozen BCCM, DuP 753, PD 123319 (AT2 selective), and sarcosine1, isoleucine8-AII ([Sar1Ile8] AII) yielded monophasic curves. These data confirm the presence of multiple receptors (AT1 and AT2) for AII on BCCM and suggest the possibility that within the AT2 receptors, one subtype, DTT sensitive, may exist. Alternatively, a heterogeneity in the AT1 class, with respect to the sensitivity to DuP753, may be considered.

A review of the design, synthesis and biological activity of the bicyclic hexapeptide tachykinin NK2 antagonist MEN 10627.

We review the reported data on the design, the conformational features and the pharmacological properties of the bicyclic peptide tachykinin NK2 receptor antagonist MEN 10,627 or cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2 beta-5 beta). MEN 10,627 possesses a highly constrained structure characterized by two consecutive beta-turns, as confirmed by the almost coincident results of NMR and X-ray analyses. The compound has been efficiently synthesized by solid-phase methodology using either Boc or Fmoc strategies. It is quite stable to metabolic degradation and is endowed with high affinity and selectivity for NK2 receptor expressed in various species. At the hamster NK2 receptor MEN 10,627 is about 30-fold more potent than the nonpeptide NK2 receptor antagonist, SR 48,968, while the converse is true for the rabbit NK2 receptor. MEN 10,627 and SR 48,968 show comparable affinities for the human NK2 receptor. MEN 10,627 produces a long lasting inhibition of the response to the selective NK2 receptor agonist [beta Ala8]NKA(4-10) in the rat urinary bladder in vivo after intravenous, intranasal and intraduodenal administration. Therefore different administration routes are possible for this compound that overcomes the usual drawbacks for the application of peptides as drugs.

Identification and characterization of functional angiotensin II receptors in human neuroblastoma cells.

The presence of specific AII receptors in 6 different human neuroblastoma cell lines was investigated using binding, cAMP and [Ca2+]i studies. We found high affinity (0.1 nM), low capacity ((1-2).10(3) sites/cell) binding sites for [125I](Sar-1,Ile-8)AII in one half of the cell lines studied. In the positive cell lines mathematical modeling of multiple competition curves among AII and analogs strongly indicated the presence of a homogenous class of sites, i.e., AT1 receptors. The presence of AT1 receptors was further substantiated by AII-induced inhibition of VIP-stimulated cAMP levels and by AII-evoked [Ca2+]i transient. The density of AT1 receptors in neuroblastoma cells was not affected by treatment with pertussis toxin and retinoic acid but was significantly increased by subacute treatment with VIP. In neuroblastoma cells, AII does not stimulate DNA synthesis, suggesting other roles rather than mitogenesis. Neuroblastoma cells represents an interesting model to investigate the function of AII in neural crest derived tissues.

Down-regulation of substance P receptors during colitis induced by trinitrobenzene sulfonic acid in rats.

Neurochemical and functional studies were performed to investigate the role of substance P (SP) during trinitrobenzene sulfonic acid (TNB)-induced colitis. Time course studies showed that tissutal SP-like immunoreactivity levels decreased in acute or chronic phases of the experimental colitis. The affinity of SP was not significantly reduced up to 1 week after TNB-induced colitis but a decreased density of SP binding sites was observed at all times. The subcutaneous administration of neurokinin (NK)1 receptor antagonist RP 67580 (0.1-1 mumol/kg daily x 1 week) did not affect the injury induced by the hapten. These findings suggest that changes in SP seem to be the effect rather than the cause of colitis and differ from those observed in human inflammatory bowel diseases.

Characterization of NK-3 binding sites in rat and guinea pig cortical membranes by the selective ligand [3H]Senktide.

We have used [3H]Senktide, a selective Neurokinin B receptor ligand, for the characterization of NK-3 receptors in rat and guinea pig CNS membranes. Scatchard analysis of saturation binding studies in cerebral cortex membranes indicated that this ligand bound to a single site with apparent high affinity (KD = 4.6 +/- 1.6 and 3.1 +/- 0.37 nM, Bmax = 13.7 +/- 1.6 and 21.8 +/- 2.2 fmol/mg protein in rat and guinea pig membranes, respectively). However, in competition studies with a group of neurokinins and related peptides two different rank orders of affinities were obtained, as follows: NKB greater than [MePhe7]-NKB greater than or equal to Arg0-NKB greater than or equal to Senktide much greater than NKA greater than SP, in rat membranes, and [MePhe7]NKB greater than Senktide = NKB greater than Arg0-NKB much greater than SP greater than NKA, in guinea pig membranes.

Interaction of amyloid beta protein (25-35) with tachykinin receptors.

Amyloid beta protein (25-35) failed to significantly interact with tachykinin NK1 (rat forebrain, guinea-pig ileum) NK2 (rabbit pulmonary artery, hamster trachea) or NK-3 (guinea-pig cortex) receptors, as determined by radioligand binding and functional assays. A weak interaction (Ki 14.8 microM) was detected with NK2 receptors in rat small intestine. It appears unlikely that direct interaction with tachykinin receptors may account for the reported ability of amyloid beta protein (25-35) to affect neuronal survival.

Heterogeneity of tachykinin NK-2 receptors in rabbit, guinea-pig and human smooth muscles.

Recent evidence indicates that the tachykinin NK-2 receptor is heterogenous (subtypes/species variants) and the existence of NK-2A (or 'non-classical') and NK-2B (or 'classical') forms of the NK-2 receptor in mammalian tissues has been proposed. In this study we have compared the affinities of 7 linear octa- and heptapeptide derivatives of neurokinin A (4-10) and that of two cyclic hexapeptides endowed with selective NK-2 receptor antagonist properties in 5 mammalian smooth muscle preparations previously characterized as expressing the NK-2A receptor subtype (rabbit pulmonary artery and bronchus, guinea-pig bronchus, human ileum and colon) and 2 preparations previously characterized as expressing the NK-2B receptor subtype (rat vas deferens and hamster trachea). The results of this comparative study reinforce the concept of two broad categories of preparations expressing pharmacologically distinguishable forms of the tachykinin NK-2 receptor and suggest the possibility of a further heterogeneity within the previously defined NK-2A receptor subtype.