Vas Deferens Assay Research Articles

New Insights into the Opioid Analgesic Profile of cis-(-)-N-Normetazocine-derived Ligands.

In this work, we report on the in vitro and in vivo pharmacological properties of LP1 analogs to complete the series of structural modifications aimed to generate compounds with improved analgesia. To do that, the phenyl ring in the N-substituent of our lead compound LP1 was replaced by an electron-rich or electron-deficient ring and linked through a propanamide or butyramide spacer at the basic nitrogen of the (-)-cis-N-normetazocine skeleton. In radioligand binding assays, compounds 3 and 7 were found to display nanomolar binding affinity for the μ opioid receptor (MOR) (Ki = 5.96 ± 0.08 nM and 1.49 ± 0.24 nM, respectively). In the mouse vas deferens (MVD) assay, compound 3 showed an antagonist effect against DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin), a highly selective MOR prototype agonist, whereas compound 7 produced naloxone reversible effect at MOR. Moreover, compound 7, as potent as LP1 and DAMGO at MOR, was able to reduce thermal and inflammatory pain assessed by the mouse tail-flick test and rat paw pressure thresholds (PPTs) measured by a Randall-Selitto test.

Design, synthesis, and pharmacological profiling of cannabinoid 1 receptor allosteric modulators: Preclinical efficacy of C2-group GAT211 congeners for reducing intraocular pressure

Allosteric modulators of cannabinoid 1 receptor (CB1R) show translational promise over orthosteric ligands due to their potential to elicit therapeutic benefit without cannabimimetic side effects. The prototypic 2-phenylindole CB1R allosteric modulator, GAT211 (1), demonstrates preclinical efficacy in various disease models. The limited systematic structure–activity relationship (SAR) data at the C2 position of the indole ring within GAT211 invites the opportunity for further modifications to improve GAT211′s pharmacological profile while serving to amplify and variegate this library of therapeutically attractive agents. These considerations prompted this focused SAR study in which we substituted the GAT211 C2-phenyl ring with heteroaromatic substituents. The synthesized GAT211 analogs were then evaluated in vitro as CB1R allosteric modulators in cAMP and β-arrestin2 assays with CP55,940 as the orthosteric ligand. Furan and thiophene rings (15c-f and 15m) were the best-tolerated substituents at the C2 position of GAT211 for engagement with human CB1R (hCB1R). The SAR around the novel ligands reported allowed direct experimental characterization of the interaction profile of that pharmacophore with its binding domain in functional, human CB1R, thus offering guidance for accessing subsequent-generation hCB1R allosteric modulators as potential therapeutics. The most potent analog, 15d, markedly promoted orthosteric ligand binding to hCB1R. Pharmacological profiling in the GTPγS and mouse vas deferens assays demonstrated that 15d behaves as a CB1R agonist-positive allosteric modulator (ago-PAM), as confirmed electrophysiologically in autoptic neurons. In vivo, 15d was efficacious as a topical agent that significantly reduced intraocular pressure (IOP) in the ocular normotensive murine model of glaucoma. Since elevated IOP is a decisive risk factor for glaucoma and attendant vision loss, our data support the proposition that the 2-phenylindole class of CB1R ago-PAMs has therapeutic potential for glaucoma and other diseases where potentiation of CB1R signaling may be therapeutic.

14-O-Methylmorphine: A Novel Selective Mu-Opioid Receptor Agonist with High Efficacy and Affinity

14-O-methyl (14-O-Me) group in morphine-6-O-sulfate (M6SU) or oxymorphone has been reported to be essential for enhanced affinity, potency and antinociceptive effect of these opioids. Herein we report on the pharmacological properties (potency, affinity and efficacy) of the new compound, 14-O-methylmorphine (14-O-MeM) in in vitro. Additionally, we also investigated the antinociceptive effect of the novel compound, as well as its inhibitory action on gastrointestinal transit in in vivo. The potency and efficacy of test compound were measured by [35S]GTPγS binding, isolated mouse vas deferens (MVD) and rat vas deferens (RVD) assays. The affinity of 14-O-MeM for opioid receptors was assessed by radioligand binding and MVD assays. The antinociceptive and gastrointestinal effects of the novel compound were evaluated in the rat tail-flick test and charcoal meal test, respectively. Morphine, DAMGO, Ile5,6 deltorphin II, deltorphin II and U-69593 were used as reference compounds.14-O-MeM showed higher efficacy (Emax) and potency (EC50) than morphine in MVD, RVD or [35S]GTPγS binding. In addition, 14-O-MeM compared to morphine showed higher affinity for μ-opioid receptor (MOR). In vivo, in rat tail-flick test 14-O-MeM proved to be stronger antinociceptive agent than morphine after peripheral or central administration. Additionally, both compounds inhibited the gastrointestinal peristalsis. However, when the antinociceptive and antitransit doses for each test compound are compared, 14-O-MeM proved to have slightly more favorable pharmacological profile.Our results affirm that 14-O-MeM, an opioid of high efficacy and affinity for MOR can be considered as a novel analgesic agent of potential clinical value.

In Vitro Opioid Receptor Assays

Although opioid analgesics have been used for centuries, identification of opioid receptors and the ability of an opioid receptor antagonist to block natural pain processes prompted a search for endogenous opioid peptides. In vitro models were needed to characterize opioid activity in biological samples. The longitudinal muscle/myenteric plexus (LM/MP) of the guinea pig ileum was the classical in vitro assay system, but the development of the mouse vas deferens (MVD) assay provided another important model that could be employed. Both assays entail electrical stimulation of intramural nerves to produce muscle contractions of the target organ. The robust contractions of the LM/MP are inhibited by µ and κ opioid receptor agonists, while the more labile contractions of the MVD are inhibited by µ, κ, and δ opioid receptor agonists. These in vitro assay systems are useful for evaluating biological activity of unknown substances and studying the properties of drug tolerance and both are described in this unit.

Cyclic enkephalin-deltorphin hybrids containing a carbonyl bridge: structure and opioid activity.

Six hybrid N-ureidoethylamides of octapeptides in which an N-terminal cyclic structure related to enkephalin was elongated by a C-terminal fragment of deltorphin were synthesized on MBHA resin. The synthetic procedure involved deprotection of Boc groups with HCl/dioxane and cleavage of the peptide resin with 45 % TFA in DCM. d-Lys and d-Orn were incorporated in position 2, and Lys, Orn, Dab, or Dap in position 5. The side chains of the dibasic amino function were protected with the Fmoc group. This protection was removed by treatment with 55 % piperidine in DMF, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate. Using various combinations of dibasic amino acids, peptides containing a 17-, 18-, 19- or 20-membered ring structure were obtained. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse opioid activities were observed, depending on the size of the ring. Extension of the enkephalin sequence at the C-terminus by a deltorphin fragment resulted in a change of receptor selectivity in favor of the δ receptor. The conformational propensities of selected peptides were determined using the EDMC method in conjunction with data derived from NMR experiments carried out in water. This approach allowed proper examination of the dynamical behavior of these small peptides. The results were compared with those obtained earlier with corresponding N-(ureidoethyl)pentapeptide amides.

Structure-activity Study of Endomorphins Analogs with C- terminal Substitution

Aims: To further wonder the influence of C-terminal residues on the pharmacological4 activities. Methods: The in vitro and in vivo opioid activities of C-terminal substitution analogs [L-Tic] EM1 and [L-Tic] EM2 were investigated using radioligand binding assay, guinea pig ileum (GPI) assay, mouse vas deferens (MVD) assay, systemic arterial pressure (SAP) assay and tail-flick test. Results: Our data showed that the analogs produced a higher δ-opioid affinity but low colon-opioid affinity, dose-dependent but reduced analgesic activities and cardiovascular effect comparing with those of EMs. Moreover, these effects induced by the analogs can be inhibited by naloxone, indicating an opioid mechanism. Conclusion: These results provided suggestive evidences that the substitution of C-terminal residue may play an important role in the regulation of opioid affinities and pharmacological activities.

Structural and pharmacological analysis of O-2050, a putative neutral cannabinoid CB1 receptor antagonist

Rimonabant, the prototypic antagonist of cannabinoid CB1 receptors, has been reported to have inverse agonist properties at higher concentrations, which may complicate its use as a tool for mechanistic evaluation of cannabinoid pharmacology. Consequently, recent synthesis efforts have concentrated on discovery of a neutral antagonist using a variety of structural templates. The purpose of this study was to evaluate the pharmacological properties of the putative neutral cannabinoid CB1 receptor antagonist O-2050, a sulfonamide side chain analog of Δ8-tetrahydrocannabinol. O-2050 and related sulfonamide cannabinoids exhibited good affinity for both cannabinoid CB1 and CB2 receptors. While the other sulfonamide analogs produced cannabinoid agonist effects in vivo (e.g., activity suppression, antinociception, and hypothermia), O-2050 stimulated activity and was inactive in the other two tests. O-2050 also decreased food intake in mice, an effect that was reminiscent of that produced by rimonabant. Unlike rimonabant, however, O-2050 did not block the effects of cannabinoid agonists in vivo, even when administered i.c.v. In contrast, O-2050 antagonized the in vitro effects of cannabinoid agonists in [35S]GTPγS and mouse vas deferens assays without having activity on its own in either assay. Further evaluation revealed that O-2050 fully and dose-dependently substituted for Δ9-tetrahydrocannabinol in a mouse drug discrimination procedure (a cannabinoid agonist effect) and that it inhibited forskolin-stimulated cyclic AMP signaling with a maximum efficacy of approximately half that of the full agonist CP55,940 [(-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol]. Together, these results suggest that O-2050 is not a viable candidate for classification as a neutral cannabinoid CB1 receptor antagonist.

Enkephalin derivative, cyclo[Nε,Nβ-carbonyl-d-Lys2, Dap5] enkephalinamide (cUENK6), induces a highly potent antinociception in rats

The aim of the study was to evaluate whether the newly synthesized analog of enkephalin, cyclo[N(epsilon),N(beta)-carbonyl-D-Lys(2), Dap(5)] enkephalinamide (cUENK6), a highly potent mu- (guinea pig ileum assay) and delta-receptors (mouse vas deferens assay) ligand, induces an antinociceptive effect in the hot-plate test and tail-immersion test after intracerebroventricular administration. Our study indicated that this peptide at the dose of 0.25 nmol produced comparable but at the dose of 0.5 nmol stronger than morphine (13 nmol), antinociceptive effect in both tests. Furthermore, rats with developed tolerance to morphine indicated cross-tolerance to antinociceptive effects of cUENK6. The antinociceptive effects of cUENK6 and morphine were inhibited by non-selective opioid receptor antagonist--naloxone. More detailed study indicated that the delta-opioid receptor antagonist - naltrindole very strongly and, to the lower extent, mu-opioid antagonist - beta-funaltrexamine (beta-FNA), inhibited antinociceptive effect of cUENK6 in the tail-immersion test. Nor-binaltorphimine (nor-BNI), a kappa-opioid receptor antagonist, did not influence this effect. These data suggest the dominant role of delta-opioid receptors as compared with mu-receptors in mediation antinociceptive effect of cUENK6. Furthermore, we found that cUENK6 is much more effective in inhibiting pain in the hot-plate (ED(50)=0.0792 nmol) than in the tail-immersion (ED(50)=0.3526 nmol) test. However, cUENK6 at the antinociceptive doses induced hypolocomotion, and although this effect is observed after administration of opioid agonists in rats as a one phase of their biphasic action (inhibition followed by activation), in our study it was not naloxone-reversible. Therefore, our study suggests that not only opioid receptors may be involved in behavioral effects of cUENK6.

N‐(ureidoethyl)amides of cyclic enkephalin analogs

Novel N-(ureidoethyl)amides of cyclic enkephalin analogs have been synthesized. The p-nitrophenyl carbamate of 1-Boc-1,2-diaminoethane was coupled with 4-methylbenzhydrylamine (MBHA) resin. The Boc group was removed by treatment with HCl/dioxane, and the peptide chain was assembled using Boc strategy. For deprotection of amino function, HCl/dioxane was used. D-Lys or D-Orn were incorporated in position 2, and the side chains of Lys, Orn, Dab, or Dap in position 5 were protected with Fmoc group. Side chain protection was removed by treatment with 55% piperidine in DMF, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate to form a urea bridge. The peptide was cleaved from the resin by treatment with 45% TFA in DCM. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Divers opioid activities were observed, depending on the size of the ring. In comparison with [Leu(5)]enkephalin, all peptides were more active in the GPI assay (between 125 and 12 times), and some of them were also more potent in the MVD assay. The conformational propensities of each peptide were determined using the EDMC method in conjunction with NMR experiments. This approach allows treating the dynamical behavior of small peptides properly. The results were compared with those obtained previously for corresponding nonsubstituted amides and are in agreement with the biologically active conformation proposed by us earlier.

Spectroscopic analysis of [Trp3]-β-casomorphin analogs Comparative structure conformation-activity studies

A series of [3-tryptophan]-beta-casomorphin-5([Trp3]-beta-CM-5) analogs were investigated by circular dichroism (CD) and fluorescence spectroscopy to explore their structure-conformation properties in solution. In addition, the comparative opioid activities of these compounds were evaluated using the in vitro guinea pig ileum (GPI) and mouse vas deferens (MVD) assays. Specifically, the pentapeptide sequence of [Trp3]-beta-CM-5, H-Tyr-Pro-Trp-Pro-Gly-OH (I) was modified at Pro-2 and Pro-4 by D-Pro substitutions to provide two diastereometric analogs, [Trp3-D-Pro-4]-beta-CM-5 (II) and [D-Pro2,4,Trp3]-beta-CM-5 (III). In the GPI and MVD assays, beta-CM-5 effected IC50 values of 1.3 microM and 8.9 microM, respectively, which confirmed its known mu/delta-selectivity on these two peripheral opioid receptor subtypes. The potencies of compounds I, II, and III were 0.2, 2.0, and less than 0.005 relative to beta-CM-5 on the GPI assay. Compounds I and II exhibited pronounced mu/delta-selectivities (greater than 18.9- and 12.4-fold respectively), whereas compound III was essentially inactive in both the GPI and MVD assays. CD studies of beta-CM-5 and its [Trp3]-beta-CM-5 analogs showed striking differences in their near-UV and far-UV spectra in aqueous or organic solvents. In the far UV CD spectra, weak (20%) alpha-helicity (maximum at 193 nm and minima at 208 and 222 nm) for beta-CM-5 was obtained in trifluoroethanol (TFE); however, none of the [Trp3]-beta-CM-5 analogs showed such CD bands. Of potential relevance to gamma-turn or C7 secondary structure was the observation of a strong negative band at 245 nm for compounds II and III which was not solvent-dependent in H2O or TFE, whereas compound I showed this CD band exclusively in TFE. In the near-UV CD at 275 nm (Trp electronic transition), the relative order of intensities of this band were determined for the [Trp3]-beta-CM-5 compounds to be II greater than I greater than III, which was identical to their relative biological potencies in both the GPI and MVD assays. Fluorescence energy transfer (FET) experiments of compounds I-III provided the intramolecular distances (r) between their Tyr (donor) to Trp (acceptor) side-chains, by the Förster method, and were as follows: [Trp3]-beta-CM-5, r = 10.6 A; [Trp3, D-Pro4]-beta-CM-5, r = 9.6 A; and [D-Pro2,4,Trp3]-beta-CM-5, r = 11.0 A.(ABSTRACT TRUNCATED AT 400 WORDS)

Deltorphin analogs restricted via a urea bridge: structure and opioid activity

Eight cyclic heptapeptides related to the full sequence of deltorphin have been synthesized. The synthesis of linear peptides containing diamino acid residues in positions 2 and 4 was carried out on a 4-methylbenzhydrylamine resin. Depending on protection procedures, the N-protected peptide-resins or N-protected peptide amides with free amino groups in the side chains were obtained, which were subsequently treated with bis-(4-nitrophenyl)carbonate to form a urea unit. Opioid activities of the peptides were determined in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays. Several compounds showed high delta opioid agonist potency and high selectivity for delta receptors. The results were compared with those obtained earlier for respective 1-4 deltorphin analogs. The conformations of these peptides have been studied using 2D-NMR in H2O/D2O and molecular dynamics. We observed that the backbone rings had well defined conformations, while the Tyr and Phe side chains and the C-terminal tail had significant conformational freedom. The bioassay data and conformational parameters of these peptides were compared with those of previously described, corresponding 1-4 deltorphin analogs. This comparison permitted an assessment of the role of the C-terminal peptide segment in defining the conformation and receptor interaction of the N-terminal portion and provided insight into the relationship between the putative bioactive conformations and bioactivity.

Soymorphins, Novel μ Opioid Peptides Derived from Soy β-Conglycinin β-Subunit, Have Anxiolytic Activities

Based on the amino acid sequence YPFV found in the soy beta-conglycinin beta-subunit, which is common to an opioid peptide human beta-casomorphin-4, peptides YPFVV, YPFVVN, and YPFVVNA were synthesized according to their primary structure. On guinea pig ileum (GPI) assay, they showed opioid activity (IC50 = 6.0, 9.2 and 13 microM respectively) more potent than human beta-casomorphins, and were named soymorphins-5, -6, and -7, respectively. Their opioid activities on mouse vas deferens (MVD) assay were less potent than on GPI assay, suggesting that they are selective for the mu opioid receptor. Human beta-casomorphin-4 and soymorphin-5 were released from the soy 7S fraction (beta-conglycinin) by the action of gastrointestinal proteases. Soymorphins-5, -6, and -7 had anxiolytic activities after oral administration at doses of 10-30 mg/kg in the elevated plus-maze test in mice.

Ureido group containing cyclic dermorphin(1–7) analogues: synthesis, biology and conformation

Six cyclic peptides related to dermorphin(1-7) have been synthesized. The synthesis of linear peptides containing diamino acid residues in positions 2 and 4 was carried out on a 4-methylbenzhydrylamine resin, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate to form a urea unit. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse opioid agonist activities were observed, depending on the size of the ring. The results were compared with those obtained earlier for 1-4 dermorphin analogues. The conformations of all six dermorphin analogues were studied. The conformational space of the peptides was examined using the electrostatically driven Monte Carlo method. On the basis of NMR data, an ensemble of conformations was obtained for each peptide. The opioid activity profiles of the compounds are discussed in the light of the structural data.

β-Methyl Substitution of Cyclohexylalanine in Dmt-Tic-Cha-Phe Peptides Results in Highly Potent δ Opioid Antagonists

The opioid peptide TIPP (H-Tyr-Tic-Phe-Phe-OH, Tic:1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was substituted with Dmt (2',6'-dimethyltyrosine) and a new unnatural amino acid, beta-MeCha (beta-methyl-cyclohexylalanine). This double substitution led to a new series of opioid peptides displaying subnanomolar delta antagonist activity and mu agonist or antagonist properties depending on the configuration of the beta-MeCha residue. The most promising analog, H-Dmt-Tic-(2S,3S)-beta-MeCha-Phe-OH was a very selective delta antagonist both in the mouse vas deferens (MVD) assay (Ke = 0.241 +/- 0.05 nM) and in radioligand binding assay (K i delta = 0.48 +/- 0.05 nM, K i mu/K i delta = 2800). The epimeric peptide H-Dmt-Tic-(2S,3R)-beta-MeCha-Phe-OH and the corresponding peptide amide turned out to be mixed partial mu agonist/delta antagonists in the guinea pig ileum and MVD assays. Our results constitute further examples of the influence of Dmt and beta-methyl substitution as well as C-terminal amidation on the potency, selectivity, and signal transduction properties of TIPP related peptides. Some of these compounds represent valuable pharmacological tools for opioid research.

Synthesis and biological activity of homoarginine‐containing opioid peptides

Two tris-alkoxycarbonyl homoarginine derivatives, Boc-Har{omega,omega'-[Z(2Br)]2}-OH and Boc-Har{omega,omega'-[Z(2Cl)]2}-OH, were prepared by guanidinylation of Boc-Lys-OH, and used for the synthesis of neo-endorphins and dynorphins. The results were compared with that obtained in the synthesis in which Boc-Lys(Fmoc)-OH was incorporated into the peptide chain, and after removing Fmoc protection, the resulting peptide-resin was guanidinylated with N,N'-[Z(2Br)]2- or N,N'-[Z(2Cl)]2-S-methylisourea. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. The results indicated that replacement of Arg by Har may be a good avenue for the design of biologically active peptides with increased resistance to degradation by trypsin-like enzymes.

In vitro and in vivo pharmacological characterization of the nociceptin/orphanin FQ receptor ligand Ac-RYYRIK-ol

It was recently reported that the hexapeptide Ac-RYYRIK-ol binds with high affinity nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors and competitively antagonizes N/OFQ actions in the mouse vas deferens assay. Here we further describe the in vitro and in vivo pharmacological features of this NOP receptor ligand. In mouse brain homogenate the degradation half life of Ac-RYYRIK-ol (2.48 min) was significantly higher than that of the parent compound Ac-RYYRIK-NH 2 (1.20 min). In the electrically stimulated mouse vas deferens, Ac-RYYRIK-ol (10–1000 nM) competitively antagonized the inhibitory effect of N/OFQ (pA 2 = 8.46), while in the isolated mouse colon the hexapeptide mimicked N/OFQ contractile effects thus behaving as a NOP receptor agonist (pEC 50 = 9.09). This latter effect was no longer evident in colon tissues taken from mice knock out for the NOP receptor gene (NOP −/−). In vivo in mice, similarly to N/OFQ, Ac-RYYRIK-ol (dose range 0.001–1 nmol) produced: i) pronociceptive effects after intracerebroventricular (i.c.v.) administration and antinociceptive actions when given intrathecally (i.t.) in the tail withdrawal assay; ii) inhibition of locomotor activity and iii) stimulation of food intake after supraspinal administration. Finally, in the forced swimming test, Ac-RYYRIK-ol was inactive per se, but reversed the antidepressant-like effects elicited by the NOP receptor selective antagonist UFP-101 ([Nphe 1,Arg 14,Lys 15]N/OFQ-NH 2). Thus, in all these in vivo assays Ac-RYYRIK-ol mimicked the actions of N/OFQ showing however higher potency. In conclusion, Ac-RYYRIK-ol displayed a complex pharmacological profile which is likely due to the low efficacy agonist nature of this novel ligand of the NOP receptor. The high potency, selectivity of action, and in vivo effectiveness make Ac-RYYRIK-ol a useful pharmacological tool for future studies in the field of N/OFQ and its NOP receptor.

Cyclic dermorphin tetrapeptide analogues obtained via ring-closing metathesis.

The dermorphin-derived cyclic tetrapeptide analogues H-Tyr-c[D-Cys-Phe-Cys]NH(2) and H-Tyr-c[D-Cys-Phe-D-Cys]NH(2) are opioid agonists at the mu and delta receptor. To enhance the metabolic stability of these peptides, we replaced the disulfide bridge with a bis-methylene moiety. This was achieved by solid-phase synthesis of the linear precursor peptide containing allylglycine residues in place of the Cys residues, followed by ring-closing metathesis. In the case of the peptide with L-configuration in the 4-position both the cis and the trans isomer of the resulting olefinic peptides were formed, whereas the cis isomer only was obtained with the peptide having the D-configuration in position 4. Catalytic hydrogenation yielded the saturated -CH(2)-CH(2)- bridged peptides. In comparison with the cystine-containing parent peptides, all olefinic peptides showed significantly reduced mu and delta agonist potencies in the guinea pig ileum and mouse vas deferens assays. The -CH(2)-CH(2)-bridged peptide with l-configuration in the 4-position was equipotent with its cystine-containing parent in both assays, whereas the bis-methylene analogue with D-configuration in position 4 was 10-27-fold less potent compared to its parent. The effect of the disulfide replacements with the -CH=CH- and -CH(2)-CH(2)- moieties on the conformational behavior of these peptides was examined by theoretical conformational analysis which provided plausible explanations in terms of structural parameters for the observed changes in opioid activity.

Novel endomorphin-2 analogs with mu-opioid receptor antagonist activity

A series of position 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) analogs containing 3-(1-naphthyl)-alanine (1-Nal) or 3-(2-naphthyl)-alanine (2-Nal) in L- or D-configuration, was synthesized. The opioid activity profiles of these peptides were determined in the mu-opioid receptor representative binding assay and in the Guinea-Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot-plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the mu-opioid receptor was reduced compared with endomorphin-2. The two most potent analogs were [D-1-Nal(4)]- and [D-2-Nal4]endomorphin-2, with IC50 values 14 +/- 1.25 and 19 +/- 2.1 nM, respectively, compared with 1.9 +/- 0.21 nM for endomorphin-2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot-plate test. Antinociception induced by endomorphin-2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [D-1-Nal4]- and [D-2-Nal4]-endomorphin-2, indicating that these analogs were mu-opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 microg per animal naloxone almost completely inhibited antinociceptive action of endomorphin-2, while [D-1-Nal4]endomorphin-2 in about 46%.

Allosteric Modulation of the Cannabinoid CB1 Receptor

We investigated the pharmacology of three novel compounds, Org 27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide), Org 27759 (3-ethyl-5-fluoro-1H-indole-2-carboxylic acid [2-94-dimethylamino-phenyl)-ethyl]-amide), and Org 29647 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid (1-benzyl-pyrrolidin-3-yl)-amide, 2-enedioic acid salt), at the cannabinoid CB1 receptor. In equilibrium binding assays, the Org compounds significantly increased the binding of the CB1 receptor agonist [3H]CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol], indicative of a positively cooperative allosteric effect. The same compounds caused a significant, but incomplete, decrease in the specific binding of the CB1 receptor inverse agonist [3H]SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride], indicative of a limited negative binding cooperativity. Analysis of the data according to an allosteric ternary complex model revealed that the estimated affinity of each Org compound was not significantly different when the radioligand was [3H]CP 55,940 or [3H]SR 141716A. However, the estimated cooperatively factor for the interaction between modulator and radioligand was greater than 1 when determined against [3H]CP 55,940 and less than 1 when determined against [3H]SR 141716A. [3H]CP 55,940 dissociation kinetic studies also validated the allosteric nature of the Org compounds, because they all significantly decreased radioligand dissociation. These data suggest that the Org compounds bind allosterically to the CB1 receptor and elicit a conformational change that increases agonist affinity for the orthosteric binding site. In contrast to the binding assays, however, the Org compounds behaved as insurmountable antagonists of receptor function; in the reporter gene assay, the guanosine 5'-O-(3-[35S]thio)triphosphate binding assay and the mouse vas deferens assay they elicited a significant reduction in the Emax value for CB1 receptor agonists. The data presented clearly demonstrate, for the first time, that the cannabinoid CB1 receptor contains an allosteric binding site that can be recognized by synthetic small molecule ligands.

Synthesis of long-chain amide analogs of the cannabinoid CB1 receptor antagonist N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide (SR141716) with unique binding selectivities and pharmacological activities

An extended series of alkyl carboxamide analogs of N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl- 1 H-pyrazole-3-carboxamide (SR141716; 5) was synthesized. Each compound was tested for its ability to displace the prototypical cannabinoid ligands ([ 3H]CP-55,940, [ 3H] 2; [ 3H]SR141716, [ 3H] 5; and [ 3H]WIN55212-2, [ 3H] 3), and selected compounds were further characterized by determining their ability to affect guanosine 5′-triphosphate (GTP)-γ-[ 35S] binding and their effects in the mouse vas deferens assay. This systematic evaluation has resulted in the discovery of novel compounds with unique binding properties at the central cannabinoid receptor (CB1) and distinctive pharmacological activities in CB1 receptor tissue preparations. Specifically, compounds with nanomolar affinity which are able to fully displace [ 3H] 5 and [ 3H] 2, but unable to displace [ 3H] 3 at similar concentrations, have been synthesized. This selectivity in ligand displacement is unprecedented, in that previously, compounds in every structural class of cannabinoid ligands had always been shown to displace each of these radioligands in a competitive fashion. Furthermore, the selectivity of these compounds appears to impart unique pharmacological properties when tested in a mouse vas deferens assay for CB1 receptor antagonism.