Effect of aromatic amino acid substitutions in the 3-position of cyclic β-casomorphin analogues on μ-opioid agonist/δ-opioid antagonist properties*

The β-casomorphin-5 analog H-Tyr-c[-D-Orn-2-Nal-D-Pro-Gly-] (2-Nal = 2-naphthylalanine) was the first reported cyclic opioid peptide with mixed μ agonist/δ antagonist properties [R. Schmidt et al. (1994) J. Med. Chem. 37 , 1136-1144]. The 2-Na1³ residue in this peptide was replaced with benzothienylalanine (Bta) (3), His(Bz1) (4), Tyr(Bz1) (5), 4′-benzoylphenylalanine (Bpa) (6), 4′-benzylphenylalanine (Bzp) (7), thyrnine (Thy) (8), thyroxine (Thx) (9), 4′-biphenylalanine (Bip) (10), 4′-biphenylglycine (Bpg) (12) and 3,3-diphenylalanine (Dip) (14), and the in vitro opioid activity profiles of the resulting compounds were determined in μ and δ receptor-representative binding assays and bioassays. Analogues 3, 12 and 14 were full agonists in the μ receptor-representative guinea-pig ileum (GPI) assay and also were agonists in the δ receptor-representative mouse vas deferens (MVD) assay. The agonist effects of the latter compounds in the MVD assay were antagonized by the highly selective δ antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP), indicating that they were triggered by δ receptor activation. The Bzp³- and Bip³-containing peptides 7 and 10 turned out to be μ antagonists against the μ selective agonist H-Tyr-D-Ala-Phe-Phe-NH₂, in the GPI assay. The other analogues were weak partial μ agonists which displayed remarkably decreased μ receptor affinity as compared to parent peptide 1. Compounds 4-10 were found to be δ antagonists in the MVD assay. Analogues 4 and 9 exhibited δ antagonist potency similar to that of parent peptide 1, while compounds 5-8 and 10 showed 3-12-fold higher δ antagonist potency against DPDPE and deltorphin I and, in most cases, increased δ receptor affinity. These results indicate that the & delta; receptor tolerates bulky aromatic side chains in the 3-position of cyclic β-casomorphin analogs with either δ agonist or δ antagonist properties. However, these compounds displayed drastically reduced μ receptor affinity in nearly all cases. © Munksgaard 1996.     

Structure-activity studies of analogs of β, γ-methylene-ATP at P2X-purinoceptors in the rabbit ear central artery

Structure-activity studies using naphthylmethyl analogs of β, γ-methylene-ATP were conducted at the P_2X-purinoceptor that mediates contraction of the rabbit ear central artery by ATP, α, β-m-ATP. On the adenine base, substitution at the C²-position (WRC-0440) increased the agonist potency 2-fold and substitution at the C⁸-position (WRC-0431) did not change agonist potency, and both compounds had the same maximal response as β, γ-m-ATP, whereas substitution at the N⁶-position (WRC-0416) abolished activity. On the D-ribose sugar, substitution on the 2′-hydroxyl generated a partial agonist (WRC-0479), which had a maximal effect of only 39% of that of β-γ-m-ATP. Attempts to substitute the 3′-hydroxyls by naphthylmethyl failed, but substitution by p-methoxybenzyl (WRC-0617) did not change potency or the maximal response. Cyclic substitution of both the 2′- and 3′-hydroxyls by naphthylmethylidine (WRC-0498) had no effect on the agonist potency or the maximal response relative to β-γ-m-ATP. On the β, γ-methylenetriphosphonate chain, substitution on the methylene linkage by naphthylmethyl (WRC-0433) had no effect on agonist potency, but the maximal response increased to 122% that of β-γ-ATP. However, the contractile response to WRC-0433 was not desensitized by α, β-γ-ATP (contractile responses to all other agonists were abolished by α-β-γ-ATP pretreatment), but was blocked by the α₁ antagonist prazosin (10^?6 M). WRC-0433 appears to act at a prejunctional site that mediates ATP-induced release of norepinephrine. Purine nucleotides with substituents at the 2′-position of the ribose sugar could provide a lead to the generation of P_2X-purinoceptor antagonists. © 1995 Wiley-Liss, Inc.     

Differences between α-Adrenergic and β-Adrenergic Inotropic Effects in Rat Heart Papillary Muscles

Abstract: α-And β-adrenergic inotropic effects have been shown to be qualitatively different. In order to further characterize these differences we compared the mechanical responses to α- and β-adrenoceptor stimulation, respectively, in electrically driven left ventricular papillary muscles from rat heart. The muscles were stimulated by either isoprenaline (β-adrenoceptor stimulation), phenylephrine in the presence of propranolol (α-adrenoceptor stimulation) or phenylephrine alone (combined α- and β-adrenoceptor stimulation). Isometric tension (T), rate of rise and decline of tension (first derivative = T′) and rate of transition from tension rise to tension decline (negative part of second derivative = T″) were recorded. These recordings disclosed qualitative differences between the α- and β-inotropic response both in dose-response and time course experiments. Maximal β-adrenoceptor stimulation caused a small increase in T_max (18%), intermediate increases in T′_max (45%) and T′_min (68%) and a considerable increase in T″_min (145%) (“β-type” effect). Maximal α-adrenoceptor stimulation increased all qualities by about the same degree (23–24%) (“α-type” effect). While β-adrenoceptor stimulation gave a dose-dependent and pronounced increase in the ratio T″_min/T′_max (relaxation-onset index), α-adrenoceptor stimulation decreased it to subcontrol values and phenylephrine alone gave a small dose-dependent increase at higher doses. The time course of the α-adrenoceptor stimulation was characterized by a transient decrease in all qualities followed by an increase which reached maximum at 4–5 min. β-Adrenoceptor stimulation gave a monophasic response which reached maximum after 1–2 min. Phenylephrine alone gave mainly an “α-type” effect although T″_min increased significantly more in the absence than in the presence of propranolol and T″_min/T′_max showed a small increase which developed slowly. Thus β-adrenoceptor stimulation activated relaxation compared to contraction by a higher degree than did α-adrenoceptor stimulation. This probably reflects different mechanisms of action. While the α-effect may rely primarily on an increased calcium influx, the β-effect probably is the final result of several subcellular effects of cyclic AMP.     

Ferulidilol: A vasodilatory and antioxidant adrenoceptor and calcium entry blocker,with ancillary β2‐agonist activity

Intravenous injection of ferulidilol (0.5, 1.0, 1.5 mg kg⁻¹) produced dose‐dependent hypotensive and bradycardia responses in pentobarbital‐anesthetized Wistar rats. Ferulidilol competitively antagonized (‐)isoprenaline‐induced positive inotropic and chronotropic effects of the atria and tracheal relaxation responses on isolated guinea pig tissues. The parallel shift to the right of the concentration–response curve of (‐)isoprenaline suggested that ferulidilol was a β‐adrenoceptor antagonist. The apparent pA₂ values were 8.04 ± 0.09 for the right atria, 8.03 ± 0.15 for the left atria, and 7.51 ± 0.06 for the trachea, respectively. Ferulidilol was more potent than labetalol. In thoracic aorta experiments, ferulidilol also produced a competitive antagonism of norepinephrine‐ and CaCl₂‐induced contraction with pA₂ and pKCa⁻¹ values of 7.05 ± 0.03 and 6.04 ± 0.05, respectively. Ferulidilol produced cumulative relaxation responses on isolated tracheal strips from reserpine‐treated guinea pigs. The effects were competitively antagonized by ICI 118,551 (10⁻⁸–10⁻⁶ M), a relatively selective β₂‐adrenoceptor antagonist. The results implied that ferulidilol had partial β₂‐agonist activity. In the radioligand binding assay, ferulidilol produced dose‐dependent inhibition of [³H]CGP‐12177 binding to rat ventricle and lung membranes with K_i values of 3.40 and 17.94 nM, respectively. In addition, ferulidilol also antagonized [³H]prazosin and [³H]nitrendipine binding to rat brain membrane with K_i values of 32.48 and 305.01 nM, respectively. These results further confirmed the α/β and calcium entry blocking activities of ferulidilol described in functional studies. Furthermore, ferulidilol (10⁻⁸–10⁻⁵ M] inhibited lipid peroxidation induced by Fe²⁺ and ascorbic acid, indicating that it possesses the antioxidant activity inherent in ferulic acid. Our results demonstrate that ferulidilol is a new generation α/β‐adrenoceptor blocker with ancillary calcium entry blockade, partial β₂‐agonist activities and additional antioxidant effects. Drug Dev. Res. 47:77–89, 1999. © 1999 Wiley‐Liss, Inc.     

Substitution of aromatic and nonaromatic amino acids for the Phe3 residue in the δ-selective opioid peptide deltorphin I: Effects on binding affinity and selectivity

Deltorphins I and II (Tyr-D-Ala-Phe-Asp-Val-Val-Gly NH₂ and Tyr-D-Ala-Phe-Glu-Val-Val-Gly NH₂) display a high degree of 6-opioid receptor selectivity. Since they lack the intervening Gly3 residue found between the Tyr and Phe aromatic moieties in pentapeptide enkephalins, deltorphins I and II resemble a previously described series of cyclic tetrapep-tides based on Tyr-c[D-Cys-Phe-D-Pen] (JOM-13). With the goal of development of structure-activity relationships for deltorphins and comparison with that of the cyclic tetrapep-tides, ten analogs of deltorphin I were synthesized in which Phe³ was replaced with specific aromatic and nonaromatic amino acids with varying physicochemical properties. Results indicated that analogs containing the bicyclic aromatic amino acids 3-(l-naphthyl)-L-alanine [1-Nal; K_i(μ) = 767 nM, K_i(§) = 7.70 nM], 3-(2-naphthyl)-L-alanine [2-Nal; K_i(μ)= 1910 nM, K_i(§) = 49.2 nM], tryptophan [K_i(μ)= 1250 nM, K_i(§) = 23.9nM], and 3-(3-benzothienyl)-L-alanine [Bth; K_i(μ)= 112nM, K_i(§) = 3.36 nM] were fairly well tolerated at μ- and §-receptors, though affinity was compromised to varying degrees relative to deltorphin I. Shortening the Phe side chain by incorporation of phenylglycine (Pgl) was detrimental to both μ (K_i= 4710 nM) and § (K_i= 15.6 nM) binding, while extension of the side chain with homophenylalanine (Hfe) enhanced μ binding (K_i= 67.8 nM), leaving § affinity unaffected (K_i= 2.64 nM). Substitution with nonaromatic amino acids valine and isoleucine led expectedly to poor opioid binding [K_i(μ) =≥ 10000 nM for each, K_i(§) = 160 and 94.7 nM, respectively], while peptides containing cyclohexylalanine (Cha) and leucine surprisingly retained affinity at both μ (K_i= 322 and 1240 nM, respectively) and § (K_i= 10.5 and 12.4 nM, respectively) sites. In general, these trends mirror those observed for similar modification in Tyr-c[D-Cys-Phe-D-Pen].     

Synthesis and evaluation of conformationally restricted pyridino N‐alkylated nicotine analogs as nicotinic acetylcholine receptor antagonists

Previous work has shown that quaternization of the pyridine‐N atom of S‐(–)‐nicotine (NIC) affords compounds such as N‐n‐octylnicotinium iodide (NONI) and N‐n‐decylnicotinium iodide (NDNI) that act as competitive nicotinic acetylcholine receptor (nAChR) antagonists at α3β2* and α4β2* subtypes, respectively. To ascertain the rotameric preference about the C3‐C2′ bond of NONI and NDNI for interaction with several nAChR subtypes, two classes of bridged analogs representing extreme rotameric conformations (syn and anti) of NONI and NDNI were synthesized. NIC‐evoked [³H]dopamine ([³H]DA) release from superfused rat striatal slices was used to determine the activity of the analogs at the α3β2* nAChR. [³H]NIC and [³H]methyllycaconitine ([³H]MLA) binding to rat brain membranes were used to determine affinity for α4β2* and α7* nAChRs, respectively. With the exception of BCDD (IC₅₀ value = 1,580 nM), all analogs potently and selectively inhibited NIC‐evoked [³H]DA release (IC₅₀ values = 30–660 nM), indicating antagonism of α3β2* nAChRs. None of the analogs inhibited either [³H]NIC or [³H]MLA binding, indicating a lack of interaction with α4β2* and α7* nAChR subtypes. Interestingly, the C_{10 N}‐alkyl chain analogs, ACD and BCD, had negligible affinity for the α4β2* subtype compared to the high affinity exhibited by NDNI, suggesting that the α4β2* subtype does not recognize the unique stereochemistry of these conformationally restricted analogs. Thus, conformational restriction of N‐n‐alkylnicotinium iodides eliminated inhibitory activity at α4β2* nAChRs, but more importantly afforded high affinity and selectivity for α3β2* nAChRs. Conformational restriction of N‐n‐alkyl analogs of NIC appears to be a viable approach for the development of α3β2*‐selective nAChR antagonists. Drug Dev. Res. 55:172–186, 2002. © 2002 Wiley‐Liss, Inc.     

Qualitative Differences between the Inotropic Responses in Rat Papillary Muscles to α-Adrenoceptor and β-Adrenoceptor Stimulation by both Noradrenaline and Adrenaline

Abstract: Mammalian heart has obviously both α- and β-adrenergic inotropic mechanisms, and stimulation of these two mechanisms by appropriate agonists lead to qualitatively different inotropic responses. This difference can serve to recognize the two adrenergic inotropic effects. In order to explore if the endogenous catecholamines, noradrenaline and adrenaline, could activate both these mechanisms, and if so, to characterize them qualitatively and quantitatively, the mechanical responses in electrically driven rat left ventricular papillary muscles were examined in the absence or presence of appropriate receptor blockade. Isometric tension (T), rate of rise and decline of tension (first derivative = T) and rate of transition from tension rise to tension decline (negative part of second derivative=T) were recorded. α-Adrenergic and β-adrenergic inotropic effects were demonstrated both for noradrenaline and adrenaline. Maximal β-adrenoceptor stimulation (agonist in the presence of an α-adrenoceptor blocker) caused a small increase in T_max, intermediate increases in T'_max and T'_min, and a considerable increase in T_min (β-type effect). Maximal α-adrenoceptor stimulation (agonist in the presence of a β-adrenoceptor blocker) increased all parts of the contraction-relaxation cycle by about the same degree (T_minT_minT'_maxT_max α-type effect). While β-adrenoceptor stimulation gave a dose dependent and pronounced increase in the ratio T_min/T_max (relaxation-onset index), α-adrenoceptor stimulation decreased it to subcontrol values. The time course of the response to the α-adrenoceptor stimulation was characterized by a transient decrease in all qualities followed by an increase which reached maximum at 4–5 min. β-Adrenoceptor stimulation gave a monophasic inotropic response which developed in the course of 1–2 min. Both agents alone gave a monophasic response with the characteristics of a β-type effect (i.e. relative maximal increase of T_minT_minT_maxT_max), and a marked increase in the relaxation-onset index (T_min/T_max). Thus the β-adrenergic inotropic component was the dominating one when the amines were used alone. The two different response patterns probably reflect a dual mechanism of action of the endogenous catecholamines: the β-adrenergic component which is dependent upon an increase in cyclic AMP levels and the α-adrenergic component which is independent on cyclic AMP.     

The synthetic cathinone psychostimulant α‐PPP antagonizes serotonin 5‐HT2A receptors: In vitro and in vivo evidence

Synthetic cathinones (SCs) are β‐keto analogs of amphetamines. Like amphetamines, SCs target monoamine transporters; however, unusual neuropsychiatric symptoms have been associated with abuse of some SCs, suggesting SCs might possess additional pharmacological properties. We performed radioligand competition binding assays to assess the affinities of nine SCs at human 5‐HT_2A receptors (5‐HT_2AR) and muscarinic M₁ receptors (M₁R) transiently expressed in HEK293 cells. None of the SCs exhibited affinity at M₁R (minimal displacement of [~K_d] [³H]scopolamine up to 10 μM). However, two SCs, α‐pyrrolidinopropiophenone (α‐PPP) and 4‐methyl‐α‐PPP, had low μM K_i values at 5‐HT_2AR. In 5‐HT_2AR–phosphoinositide hydrolysis assays, α‐PPP and 4‐methyl‐α‐PPP displayed inverse agonist activity. We further assessed the 5‐HT_2AR functional activity of α‐PPP, and observed it competitively antagonized 5‐HT_2AR signaling stimulated by the 5‐HT₂R agonist (±)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI; K_b = 851 nM). To assess in vivo 5‐HT_2AR activity, we examined the effects of α‐PPP on the DOI‐elicited head‐twitch response (HTR) in mice. α‐PPP dose‐dependently blocked the HTR with maximal suppression at 10 mg/kg (P < 0.0001), which is a moderate dose used in studies investigating psychostimulant properties of α‐PPP. To corroborate a 5‐HT_2AR mechanism, we also tested 3,4‐methylenedioxy‐α‐PPP (MDPPP) and 3‐bromomethcathinone (3‐BMC), SCs that we observed had 5‐HT_2AR K_is > 10 μM. Neither MDPPP nor 3‐BMC, at 10 mg/kg doses, attenuated the DOI HTR. Our results suggest α‐PPP has antagonist interactions at 5‐HT_2AR in vitro that may translate at physiologically‐relevant doses in vivo. Considering 5‐HT_2AR antagonism has been shown to mitigate effects of psychostimulants, this property may contribute to α‐PPPs unpopularity compared to other monoamine transporter inhibitors.     

Synthesis of [18F]3‐[1‐(3‐fluoropropyl)‐(S)‐pyrrolidin‐2‐ylmethoxy]pyridine ([18F]NicFP): a potential α4β2 nicotinic acetylcholine receptor radioligand for PET

Nicotinic acetylcholine receptors are widely distributed throughout the human brain and are believed to play a role in several neurological and psychiatric disorders. In order to identify an effective PET radioligand for in vivo assessment of the α4β2 subtype of nicotinic receptor, we synthesized [¹⁸F]3‐[1‐(3‐fluoropropyl)‐(S)‐pyrrolidin‐2‐ylmethoxy]pyridine (NicFP). The in vitro K_D of NicFP was determined to be 1.1 nM, and the log P value obtained by HPLC analysis of the unlabelled standard was found to be 2.2. The radiosynthesis of [¹⁸F]NicFP was carried out by a nucleophilic substitution reaction of anhydrous [¹⁸F]fluoride and the corresponding mesylate precursor. After purification by HPLC, the radiochemical yield was determined to be 11.3±2.1% and the specific activity was 0.47±0.18 Ci/μmol (EOS, n = 3). The time of synthesis and purification was 99±2 min. The final product was prepared as a sterile saline solution suitable for in vivo use. Copyright © 2003 John Wiley & Sons, Ltd.     

Effects of castration on α1-adrenoceptor subtypes in the rat aorta

The expression of α₁-adrenoceptor subtypes in several tissues is regulated by gonadal hormones. In this study, we investigated whether castration regulates the α₁-adrenoceptor subtypes mediating the contractions of the aorta from male rats to noradrenaline. Noradrenaline induced similar concentration-dependent contractions in the aorta from control and castrated rats. Treatment of the aorta from both control and castrated rats with the α_1B/α_1D-adrenoceptor alkylating agent chloroethylclonidine resulted in ≈1600-fold rightward shift in the concentration–response curves to noradrenaline. The pA₂ values found for WB 4101, benoxathian (α_1A-selective) and BMY 7378 (α_1D-selective) indicate that α_1D-adrenoceptors are involved in the contractions of the aorta from control and castrated rats to noradrenaline. However, there was a 15-fold difference between the pK_B estimated through the lowest effective concentrations of the α_1A-adrenoceptor selective antagonist 5-methyl-urapidil in the aorta from control and castrated rats. The pK_B estimated in aorta from control rats is consistent with the interaction with α_1D-adrenoceptors (7.58±0.06), while that calculated in organs from control rats is consistent with α_1A-adrenoceptors (8.76±0.09). These results suggest that castration induces plasticity in the α₁-adrenoceptor subtypes involved in the contractions of the aorta to noradrenaline.     

Concomitant Glucocorticoid Treatment Prevents the Development of β-Adrenoceptor Desensitization in the Guinea Pig Lung

The aim of the present study was to investigate, whether concomitant administration of the synthetic glucocorticoid betamethasone (BM), theophylline (THEO), or the muscarinic antagonist ipratropium bromide (IPRA) could influence the desensitization-associated decrease of β-adrenoceptors in the guinea pig lung during prolonged in vivo treatment with the β₂-agonist terbutaline (TER). The animals were sacrificed 20 hrs after the last drug dosage and the lung membrane homogenates were prepared for ³H-dihydroalprenolol (³H-DHA) binding in vitro. Treatment with TER 200 µg/kg subcutaneously twice a day for five days decreased by 22% the maximum number of binding sites (B_max) at saturation in comparison with the saline-treated controls. Concomitant administration of BM 2 mg/kg intraperitoneally abolished this effect of TER, whereas THEO 20 mg/kg or IPRA 5 µg/kg failed to modify it. None of the in vivo treatments affected the binding affinity of ³H-DHA. In vitro, TER inhibited in a concentration-dependent manner ³H-DHA binding to the lung membranes of untreated guinea pigs. At high concentrations IPRA, but not THEO or BM, showed some binding to the β-receptors as well. Thus, it is concluded that glucocorticoids may prevent β-adrenoceptor desensitization in the lungs via an indirect mechanism, e.g. inhibition of phospholipase A₂ enzyme.     

Receptor specificity of the 5HT2 receptor antagonist,LY53857

LY53857 is a potent antagonist at vascular 5HT₂ receptors that lacks prominent α₁ or dopamine antagonist activity. The present report investigates the interaction of LY53857 with other receptor mechanisms. LY53857 showed no agonist activity in the guinea pig trachea, guinea pig atria, guinea pig ileum, or rat vas deferens. Furthermore, LY53857 did not antagonize histamine (H₁) or β₂ receptors in the guinea pig trachea, β₁ in the guinea pig atria, muscarinic, or angiotensin I receptors in the guinea pig ileum. However, LY53857 did block α₂ receptors (?log K_B = 6.51) as determined by antagonism of the inhibitory effects of the selective β₂ agonist, UK-14,304, on the twitch response in the guinea pig ileum. LY53857 antagonized β₂ receptors at concentrations approximately 6000-fold higher than necessary to block 5HT₂ receptors (?log K_B = 10.3). Taken in concert, these data support the contention that LY53857 is a highly selective antagonist of 5HT₂ receptors, and that LY53857 is a useful tool with which to probe 5HT₂ receptors and serotonergic mechanisms.     

β-ENDORPHIN: SYNTHESIS AND BIOLOGICAL ACTIVITY OF SHORTENED PEPTIDE CHAINS

Three analogs of β-endorphin have been synthesized by the solid-phase method: β_c-endorphin-(1–5)-(28–31), β_c-endorphin-(6–31) and β_h-endorphin-(1–5)-(16–31). The analgesic activities of these synthetic peptides relative to that of the parent molecule are reported. All three peptides at high doses exhibit either no or much weaker analgesic activity than β-endorphin. These data suggest that the entire β-endorphin molecule is necessary for full in vivo analgesic activity.     

β-Endorphin

A double-headed analog of human β-endorphin (β_h-EP), N, N'-bis (β-endorphinyl)-cystine (II), has been synthesized by the solid-phase method, along with β_h-EP-Cys(CH₂CONH₂)-OH (I) and (Tyr³¹]-β_h-EP (III). Their relative potencies in a radioreceptor-binding assay were: B_h-EP, 100; II, 235; I, 170; and III, 204. In the tail-flick test for analgesic activity their relative potencies were: β_hEP, 100; II, 86; I, 93; and III, 116.     

Conformation—activity correlations for chemotactic tripeptide analogs incorporating dialkyl residues with linear and cyclic alkyl sidechains at position 2

Five stereochemically constrained analogs of the chemotactic tripeptide incorporating l-aminocycloalkane-l-carboxylic acid (Ac_nc) and α, α-dialkylglycines (Deg, diethylglycine; Dpg, N, N-dipropylglycine and Dbg, N, N-dibutylglycine) at position 2 have been synthesized. NMR studies of peptides For-Met-Xxx-Phe-OMe (Xxx = Ac₇c. I: Ac₈c. II: Deg, III; Dpg, IV and Dbg, V; For, formyl) establish that peptides with cycloalkyl residues, I and II, adopt folded β-turn conformations in CDCl₃, and (CD³)₂SO. In contrast, analogs with linear alkyl sidechains, III-V, favour fully extended (C₅) conformations in solution. Peptides I-V exhibit high activity in inducing β-glucosaminidase release from rabbit neutrophils, with ED₅₀ values ranging from 1.4–8.0 × 10–¹¹. M. In human neutrophils the Dxg peptides III-V have ED₅₀ values ranging from 2.3 × 10^?8 to 5.9 × 10^?10 M, with the activity order being V>IV>III. While peptides I-IV are less active than the parent. For-Met-Leu-Phe-OH, in stimulating histamine release from human basophils, the Dbg peptide V is appreciably more potent, suggesting its potential utility as a probe for formyl peptide receptors. © Munksgaard 1996.     

β-Endorphin

Three β_h-EP analogs which show different extents of alteration in analgesic potency by substitution of a single amino acid residue were assayed for their peripheral opioid activity and the binding to opioid μ-receptor to determine the relationships among the opioid activities obtained from different assays. In the guinea pig ileum assay, [Gln⁸]-β_h-EP showed a higher inhibitory activity than the parent peptide. [Tyr³¹]-analog had the same potency as β_h-EP, while [Trp²⁷]-analog retained only one fourth the potency of β_h-EP. Assayed on the vas deferens of the mouse and the rat, all three substituted β_h-EP analogs exhibited a lower potency than their parent peptide. Receptor binding assay using [³H]-dihydromorphine as the primary ligand showed that [Gln⁸]-analog had a binding potency 1.5-fold that of β_h-EP, while the potencies of [Tyr³¹]- and [Trp²⁷]-analogs were not significantly different from that of the parent peptide. No correlation in relative potency was found between vas deferens assays and their μ-receptor binding or analgesic activity. However, the relative potencies of binding to μ-receptor in [Gln⁸]- and [Tyr³¹]-analogs were found to be consistent with those of analgesic and guinea pig ileum assays, whereas the binding to β-EP receptor of all analogs appeared to be related to the charge properties of β-EP molecule.     

Beta-adrenoceptors invloved in inhibition of histamine release from sensitized guinea-pig lung.

Selective β-adrenoceptor agonists and antagonists were used to study the type of β-adrenoceptors involved in inhibiting antigen-induced histamine release from actively sensitized guinea-pig lung. Results obtained with six non-catechol β-adrenergic agonists were compared with those found in guinea-pig atrial (β₁) and tracheal (β₂) preparations. In terms of rank order the relative activities of the compounds differed in the three preparations. Dissociation constants (K_B values) for the cardioselective antagonist H93/26 were assessed using (?)-isoprenaline as an agonist. The K_B value for inhibition of histamine release was significantly different from, and intermediate between, the K_B values obtained in atria and trachea. In the guinea-pig tissues H35/25 was not a selective β-adrenoceptor antagonist; K_B values were not significantly different in the three preparations. The results using the β-adrenoceptor agonists and antagonists suggest that the β-receptors involved in inhibition of antigen-induced histamine release in the guinea-pig lung differ from those found in guinea-pig atria and trachea.     

Cortical β- and α2- Adrenoceptor Binding,Hypothalamic Noradrenaline and Pineal Melatonin Concentrations Measured at Different Times of the Day after Repeated Treatment of Rats with Imipramine,Zimeldine, Alaproclate and Amiflamine

Abstract: The effect of repeated treatment of rats for 21 days with the monoamine reuptake inhibitors imipramine, zimeldine, alaproclate (in each case 10 μmol/kg b.i.d.) and the reversible monoamine oxidase-A inhibitor amiflamine (3 μmol/kg b.i.d.) on brain noradrenergic mechanisms measured at different times of the day and night was investigated. Imipramine treatment produced a down-regulation of the B_max for ³H-dihydroalprenolol binding to cortical β-adrenoceptors that was not dependent upon the time of day the animals were killed. Zimeldine, on the other hand, reduced both B_max, and K_d of binding for day-time, but not night-time samples. Alaproclate and amiflamine were without effect on the binding. Twenty-four hour mean values for 1 nM ³H-p-aminoclonidine binding to α₂-adrenoceptors were lower for the zimeldine-treated rats than for the saline-treated rats. Pineal melatonin concentrations, which are regulated by β-adrenoceptors, showed a pronounced diurnal rhythm, with the highest concentrations being found at 02:00. At this time point, a lower pineal melatonin content was found after amiflamine treatment, whereas imipramine, zimeldine and alaproclate were without significant effect. The importance of the use of more than one time point and the use of more than one biochemical test for the determination of the effects of repeated antidepressant treatment on central noradrenergic systems measured ex vivo is discussed.     

Solid phase synthesis of human growth hormone-releasing factor analogs containing a bicyclic β-turn dipeptide

Three analogs derived from the N-terminal 29-residue fragment of human growth hormone-releasing factor (hGRF) which contained a bicyclic β-turn dipeptide (BTD) at 7-8,8-9, and 9-10 positions were synthesized by solid phase methodology to ascertain if the β-turns are important for the biological activity of hGRF and also to show the applicability of the BTD unit to solid phase synthesis. All three analogs were obtained in good yield and purity indicating that the BTD unit can be used in the usual condition of solid phase synthesis. The capacity of these analogs to release growth hormone (GH) was tested in an in vitro bioassay using rat anterior pituitary cells. All three BTD-containing analogs showed the same maximal GH secretion with parallel dose-response curves to that of hGRF(1-29)NH₂, except their relative potencies were very low.     

Structure–activity relationship of [Nphe1]‐NC‐(1‐13)‐NH2, a pure and selective nociceptin/orphanin FQ receptor antagonist

Abstract: A series of analogs of the ORL1 receptor antagonist [Nphe¹]‐NC(1‐13)‐NH₂ was prepared and tested for agonistic and antagonistic activities in the mouse vas deferens, a preparation that shows high sensitivity to nociceptin and related peptides. The purpose of this study was to determine the role of the aromatic residue at the N‐terminal for antagonism and eventually identify compounds with improved potency. Results indicated that all 23 compounds are inactive as agonists, and the antagonistic potency of the initial template [Nphe¹]‐NC(1‐13)‐NH₂ is high (pK_B 6.43) compared with those of all other compounds except [(S)(βMe)Nphe¹]NC(1‐13)‐NH₂ (pK_B 6.48). The other 22 compounds can be divided into two groups: 10 show antagonistic potencies (pK_B) ranging from 5.30 to 5.86, whereas the other 12 compounds are inactive. This study clearly shows that the aromatic ring of Nphe is very critical for the interaction with the ORL1 receptor and can not be enlarged or sterically modified without significant loss of antagonistic potency.