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A series of imidazolylmethylthiophenes has been prepared and evaluated as ligands for the α2 adrenoceptor. These compounds were tested in two animal models that are predictive of analgesic activity in humans. The 3-thienyl compounds were generally the most potent, particularly those with substitution in the 4-position. A subset of the most active compounds was further evaluated for adverse cardiovascular effects in the anesthetized rat model. In addition to excellent binding at the α2D adrenoceptor, the 4-bromo analogues 20e and 21e were very active in the rat abdominal irritant test (RAIT) with ED50 doses of 0.38 and 0.31 mg/kg, respectively. We constructed a pharmacophore model based on the biological activity of the present series, dexmedetomidine (1), and conformationally restrained analogues 3 and 4.

A series of (imidazolylmethyl)oxazoles and -thiazoles were prepared and evaluated as α2 adrenoceptor agonists. These compounds were also tested in in vivo paradigms that are predictive of analgesic activity. Variations in both the imidazole and thiazole portions of the molecule were investigated. Some of the more potent compounds such as 22, 26, 45, and 53 displayed α2 receptor binding in the 10−20 nM range and also had significant antinociceptive activity in the mouse abdominal irritant test (MAIT).

Although it is well established that the analgesic effects of morphine are mediated by opioid receptors, previous studies have shown that some opioids additionally inhibit the uptake of serotonin and norepinephrine. The present investigation of a diverse group of opioids revealed that structurally identifiable subgroups inhibited the neuronal reuptake of these monoamines. Phenanthrene opioids with an oxygen bridge between C4 and C5, such as morphine and naloxone (group I), did not block norepinephrine or serotonin uptake, whereas phenanthrene opioids without the oxygen bridge and the C6-OH moiety, such as levorphanol and levomethorphan (group II), did inhibit uptake, as did nonphenanthrene opioids, such as d-propoxyphene and methadone (group III). Affinity at the mu opioid receptor correlated with antinociceptive potency (r = 0.87, P < .05). Although the antinociceptive activity of the "active enantiomers" of group II and III compounds also correlated with their affinity at the mu opioid receptor (r = 0.85, P = .007), additional consideration of serotonin uptake inhibiting activity (but not of norepinephrine uptake inhibiting activity) significantly improved the correlation between antinociceptive potency and the in vitro activity of these compounds (r = 0.915, P = .0017). Additionally, for group II and III (but not group I) compounds, smaller differences between enantiomers in antinociceptive potency than in mu receptor affinity were noted, presumably because of the contribution of uptake inhibition to the antinociceptive activity of group II and III compounds. Evidence also is provided suggesting a broader role for the combination of mu opioid affinity and 5-hydroxytryptamine uptake inhibition in the activity of other antinociceptive agents.

N-Oxides of centrally acting analgesics generally have minimal analgesic activity. However, the N-oxide of tramadol produced dose-related, long-lasting antinociception in the mouse abdominal irritant, 48 degrees C hot-plate, 55 degrees C hot-plate, and tail-flick tests (ED50 = 15.5, 84.7, 316.4 and 138.2 mg/kg, p.o., respectively). Tramadol N-oxide (T-N-O) (RWJ 38705) was also antinociceptive in the 51 degrees C hot-plate test in male (ED50 = 63.2 mg/kg, i.p.) and female (ED50 = 39.9 mg/kg, i.p.) rats. A characteristic feature of T-N-O was an extended duration of action in these tests (4-5 h). T-N-O had negligible affinity for opioid mu (Ki = 38.5 microM) delta. or kappa receptors (Ki > 100 microM) and, in contrast to tramadol, was essentially devoid of norepinephrine or serotonin neuronal reuptake inhibitory activity (Ki > 100 microM). However, T-N-O displayed tramadol-like characteristics in vivo. There were also significant amounts of tramadol in plasma after T-N-O administration, and the levels resulting from equal oral doses of T-N-O and tramadol were the same, suggesting that the conversion of T-N-O to tramadol was rapid and essentially quantitative. T-N-O was not readily metabolized to tramadol in rat hepatic S9 fraction (< 2%), implying that the conversion might occur in the gastrointestinal tract. Taken together, the results suggest that T-N-O acts as a prodrug for tramadol. T-N-O could offer the clinical benefits of an extended duration of action and a "blunted" plasma concentration spike, possibly leading to an enhanced side-effect profile.

The behavioral response elicited in mice by an i.p. injection of endothelin-1 (ET-1) (0.1 mg/kg) was differentiated from that elicited by standard agents such as acetylcholine (ACh) (5.5 mg/kg) or phenyl-p-quinone (PpQ) (1.25 mg/kg). First, there was lack of two-way "cross-tolerance' between test paradigms. That is, at equieffective doses, a 60-min prior i.p. injection of ET-1 blocked the behavioral response to a subsequent i.p. injection of ET-1 or PpQ, but not of ACh, whereas a 60-min prior injection of ACh or of PpQ had no effect on a subsequent i.p. injection of ACh, PpQ or ET-1. Second, differential antagonism of ET-1-, ACh- or PpQ-induced responses was observed in an examination of 36 test compounds. For example, cyclo-oxygenase inhibitors such as indomethacin and ibuprofen did not block the ET-1-induced response at > 10 times the doses that blocked ACh- or PpQ-induced responses, whereas other compounds (such as certain benzodiazepines) inhibited ET-1-induced, but not ACh- or PpQ-induced, responses. These findings suggest that ET-1 produces a novel nociceptive stimulus, mechanistically distinct from ACh and PpQ. Hence, the ET-1-induced behavioral response in mice serves as a rapid and convenient measure of in vivo endothelin activity. In addition, this test might be a model for clinical pains not adequately treated by present analgesic agents or adequately tested by preclinical antinociceptive screens using ACh or PpQ. As such, it is a potentially valuable model for the identification of novel analgesic and other agents.

The role of mu and delta opioid receptors in the spinal and supraspinal analgesic actions of morphine and [D-Pen2, L-Pen5] enkephalin were examined in the tail-flick test utilizing the mu opioid receptor deficient CXBK mouse and BALB/cBy and C57BL/6By, the progenitor strains of CXBK. The analgesic effects of i.c.v. administered morphine were equivalent in the CRS-CD1 (Swiss) standard laboratory mice and the progenitor strains of CXBK. Morphine did not, however, produce analgesia in the CXBK mice at doses greater than 10 times the ED50 dose in the progenitor strains. Similarly, the analgesic effect of i.c.v. [D-Ala2, NMePhe4, Gly-ol]enkephalin, a highly selective mu receptor peptide agonist, also was reduced greatly in the CXBK mice. These data are consistent with the deficiency in mu opioid receptors observed autoradiographically in this strain. In contrast, the highly selective delta opioid receptor peptide agonist [D-Pen2, L-Pen5]enkephalin was equipotent i.c.v. in the CXBK mice and in the progenitor strains of CXBK. In contrast to the effects produced by i.c.v. administration, the analgesic effects of intrathecally administered morphine were similar between CRS-CD1 and CXBX strains of mice. These results suggest that 1) both mu and delta opioid receptors can mediate supraspinal analgesia and 2) that the receptor(s) involved in spinally mediated analgesia is (are) quite distinct from those involved supraspinally.

The present study examined the effects of three heavy metals on the acetylcholine (ACh) contracture and Ca++ kinetics of the anterior byssus retractor muscle of Mytilus edulis. An isolated tissue bioassay using anterior byssus retractor muscle was prepared according to standard procedures and the isometric tension produced in response to ACh was measured. Ten millimolar Ni++, Co++ or Cd++ reduced the maximum contracture response to ACh in zero-Ca medium in a time-dependent manner. The inhibition was reversed upon restoration of medium containing 10 mM Ca++. The loss (and re-establishment) of contracture response to ACh corresponded to the influx (and efflux) of the heavy metal ions opposite to the direction of Ca++ flow. These results are consistent with the concept that the loss of the ACh contracture response is attributable to the displacement of tissue Ca++ from release sites by heavy metals.

The explanation for the co-existence of opioid and nonopioid components of tramadol-induced antinociception appears to be related to the different, but complementary and interactive, pharmacologies of its enantiomers. The (+) enantiomer had Ki values of only 1.33, 62.4 and 54.0 microM at mu, delta and kappa receptors, respectively. The (-) enantiomer had even lower affinity at the mu and delta sites (Ki = 24.8, 213 and 53.5 microM, respectively. The (+) enantiomer was the most potent inhibitor of serotonin uptake (Ki = 0.53 microM) and the (-) enantiomer was the most potent inhibitor of norepinephrine uptake (Ki = 0.43 microM). Basal serotonin release was preferentially enhanced by the (+) enantiomer and stimulation-evoked norepinephrine release was preferentially enhanced by the (-) enantiomer. The (+) and (-) enantiomers each independently produced centrally mediated antinociception in the acetylcholine-induced abdominal constriction test (ED50 = 14.1 and 35.0 micrograms i.t., respectively). Racemic tramadol was significantly more potent (P < .05) than the theoretical additive effect of the enantiomers (antinociceptive synergy). Synergy was also demonstrated (P < .1) in the mouse 55 degrees C hot-plate test (i.p. route) and (P < .05) the rat Randall-Selitto yeast-induced inflammatory nociception model (i.v. and i.p. routes). Critically, the enantiomers interacted less than synergistically in two side-effects of inhibition of colonic propulsive motility and impairment of rotarod performance. The racemate and the (+) enantiomer were active in a chronic (arthritic) inflammatory pain model. Taken together, these findings provide a rational explanation for the coexistence of dual components to tramadol-induced antinociception and might form the basis for understanding its clinical profile.

The temperature dependence of the dissociation constant for the interaction of an opioid delta selective ligand and its receptor was evaluated in three tissues. The change in free energy of this interaction was similar in mouse brain, mouse spinal cord, and NG 108-15 mouse neuroblastoma-rat glioma hybrid cells (delta G(o)' = -13.44, -13.34, and -13.66 kcal.mol-1, respectively). However, the reaction was endothermic and occurred with an increase in entropy in mouse brain and NG 108-15 cells, but it was exothermic and occurred with a negligible change in entropy in mouse spinal cord. These data are consistent with the existence of multiple subtypes of opioid delta receptor, and they further suggest that the opioid delta receptor recently cloned from the NG 108-15 cell line is of the brain subtype. Subtypes of opioid delta receptors may mediate analgesia, but not side-effects, of opiates and thus could be targets for future drug design.

The thermodynamic quantities of change in free energy (delta G degree'), change in enthalpy (delta H degree') and change in entropy (delta S degree') were determined for the interaction of norepinephrine with the alpha-1 adrenoceptor of vascular smooth muscle. Specifically, a standard isolated rabbit thoracic-aorta preparation was used to examine the effect of temperature on norepinephrine-induced isometric tension development. Dissociation constants (KA) for norepinephrine were determined at several temperatures over the range 25-40 degrees C from equiactive concentrations obtained before (A) and after (A') partial irreversible receptor blockade by phenoxybenzamine, plotted as 1/A against 1/A' (KA = (slope-1)/intercept). The values of KA increased with temperature over the range 25-40 degrees C, indicating that the affinity of norepinephrine for the alpha-1 adrenoceptor is an inverse function of temperature over this range. From these results, the thermodynamic quantities delta H degree' and delta S degree' were determined from a van't Hoff plot of In (KA) against 1/T. The relative magnitudes of the change in enthalpy (delta H degree' = -25.58 kcal mol-1) and the change in entropy (delta S degree' = -0.052 kcal mol-1 deg-1) suggest that the reaction between norepinephrine and the alpha-1 adrenoceptor (delta G degree' = -9.15 kcal mol-1) is enthalpy driven, which is consistent with radioligand binding studies of other adrenoceptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelins (ET-1, ET-2 or ET-3) or endothelin precursors (big-ET-1[1-38], big-ET-2[1-37] or big-ET-3[1-41]) injected i.p. in mice have previously been shown to elicit a characteristic nociceptive behavioral response. In this study, we investigated the endothelin receptor type (ETA or ETB) that mediates this behavioral response. Mice were injected i.p. with ET-1, ET-2, ET-3, big-ET-1[1-38], big-ET-2[1-37], big-ET-3[1-41], sarafotoxin S6a, sarafotoxin S6b, sarafotoxin S6c, ET-1 with Ala substitutions for Cys3 and Cys11 or His-Leu-Asp-Ile-Ile-Trp, and quantal dose-response curves were obtained for each of the compounds (except the latter). Co-administration of enzyme inhibitors with the big-endothelins was used to establish the requisite conversion to endothelins and big-ET-1[22-38], big-ET-2[22-37] and ET-3[22-41] amide, and the ETA-selective antagonist cyclo[-D-Asp-Pro-D-Val-Leu-D-Trp-] was used to determine receptor specificity. The ED50 values were 2.9, 3.3 and 23.9 micrograms/kg i.p. for ET-1, ET-2 and ET-3, respectively, 0.6, 0.6 and 13.1 micrograms/kg i.p. for sarafotoxin S6a, sarafotoxin S6b and sarafotoxin S6c, respectively, and 5.3 micrograms/kg i.p. for ET-1 with Ala substitutions for Cys3 and Cys11. Big-ET-1[22-38], big-ET-2[22-37], big-ET-3[22-41] amide and ET-C produced less than 25% effect up to 2000 micrograms/kg. The big-ET-1-induced effects were blocked by the enzyme inhibitors phosphoramidon and thiorphan (ID50 = 0.9 mg/kg) but not by ubenimex (bestatin), captopril or perindopril. Cyclo[-D-Asp-Pro-D-Val-Leu-D-Trp-] blocked ET-1- and ET-2-induced effects but not ET-3-, ACh- or phenyl-p-quinone-induced effects. These results suggest that endothelin-induced nociceptive behavioral response in mice can be mediated via both ET receptor types, ETA and ETB. Further, the ET-1 carboxy-terminal hexapeptide is insufficient to produce the effect, and the Cys3-Cys11 disulfide bridge of ET-1 is not required.

Dissociation constants (KB) for naloxone inhibition of the actions of DPDPE in the mouse isolated vas deferens preparation (inhibition of electrically induced twitch) were determined at five temperatures ranging between 25 and 40 degrees C. The values of KB tended to increase with temperature over the range examined, indicating that the affinity of naloxone for the opioid delta receptor is an inverse function of temperature. Using these data, the thermodynamic quantities delta G zero' (change in free energy), delta H zero' (change in enthalpy) and delta S zero' (change in entropy) were calculated from a van't Hoff plot of in (KB) against 1/T. The thermodynamic quantities determined in this study in vivo (delta G zero' = -10.59 kcal mol-1, delta H zero' = -15.73 kcal mol-1 and delta S zero' = -0.0168 kcal mol-1 zero K-1) are consistent with data reported from radioligand binding studies in vitro and suggest that the interaction between naloxone and the opioid delta receptor in the mouse isolated vas deferens is enthalpy driven. These data represent the first evaluation of the thermodynamics of opioid antagonist/receptor interaction in a physiological assay.

Tramadol hydrochloride produced dose-related antinociception in mouse abdominal constriction [ED50 = 1.9 (1.2-2.6) mg/kg i.p.], hot-plate [48 degrees C, ED50 = 21.4 (18.4-25.3) mg/kg s.c.; 55 degrees C, ED50 = 33.1 (28.2-39.1) mg/kg s.c.] and tail-flick [ED50 = 22.8 (19.2-30.1) mg/kg s.c.] tests. Tramadol also displayed antinociceptive activity in the rat air-induced abdominal constriction [ED50 = 1.7 (0.7-3.2) mg/kg p.o.] and hot-plate [51 degrees C, ED50 = 19.5 (10.3-27.5) mg/kg i.p.] tests. The antinociceptive activity of tramadol in the mouse tail-flick test was completely antagonized by naloxone, suggesting an opioid mechanism of action. Consistent with this, tramadol bound with modest affinity to opioid mu receptors and with weak affinity to delta and kappa receptors, with Ki values of 2.1, 57.6 and 42.7 microM, respectively. The pA2 value for naloxone obtained with tramadol in the mouse tail-flick test was 7.76 and was not statistically different from that obtained with morphine (7.94). In CXBK mice, tramadol, like morphine, was devoid of antinociceptive activity after intracerebroventricular administration, suggesting that the opioid component of tramadol-induced antinociception is mediated by the mu-opioid receptor. In contrast to the mouse tail-flick test and unlike morphine or codeine, tramadol-induced antinociception in the mouse abdominal constriction, mouse hot-plate (48 degrees or 55 degrees C) or rat hot-plate tests was only partially antagonized by naloxone, implicating a nonopioid component. Further examination of the neurochemical profile of tramadol revealed that, unlike morphine, it also inhibited the uptake of norepinephrine (Ki = 0.79 microM) and serotonin (0.99 microM). The possibility that this additional activity contributes to the antinociceptive activity of tramadol was supported by the finding that systemically administered yohimbine or ritanserin blocked the antinociception produced by intrathecal administration of tramadol, but not morphine, in the rat tail-flick test. These results suggest that tramadol-induced antinociception is mediated by opioid (mu) and nonopioid (inhibition of monoamine uptake) mechanisms. This hypothesis is consistent with the clinical experience of a wide separation between analgesia and typical opioid side effects.

McN-5195 [(+/-)-trans-3-(2-bromophenyl)-octahydroindolizine] inhibited at nontoxic doses the nociceptive response in tail-pinch, tail-flick and 48 degrees C hot-plate tests of mice, with ED50 values of 38.2, 33.9 and 30.9 mg/kg i.p., respectively, and of rats, with ED50 values (i.p.) of 33.2 mg/kg (tail-flick) and 33.3 mg/kg (hot-plate). The compound was p.o. active in the acetylcholine-induced irritant test (ED50 = 20.1 mg/kg) in mice and the air-induced irritant test (ED50 = 33.2 mg/kg) in rats. McN-5195 blocked thalamic activity (multiunit recordings from the ventral posterolateral nucleus) evoked by noxious stimulation of the contralateral hindlimb of anesthetized rats, but did not alter thalamic activity during non-noxious stimulation. The antinociceptive action of McN-5195 was not blocked by naloxone and was not diminished in morphine-tolerant animals. McN-5195 did not affect arachidonate metabolism and was not active against carrageenan-induced paw edema or in an adjuvant arthritis test in rats. McN-5195 did not bind to opiate, serotonin S1 or S2, dopamine D2, alpha-1, alpha-2, beta adrenergic or gamma-aminobutyric acid-A receptors and did not inhibit the synaptic uptake of norepinephrine, serotonin, dopamine or gamma-aminobutyric acid. McN-5195-induced antinociception was not affected by reserpine or phentolamine pretreatment and was not reduced in clonidine-tolerant animals. Ketanserin and yohimbine inhibited McN-5195-induced antinociception by an indirect mechanism. Tolerance did not develop to chronic administration of McN-5195 (120 mg/kg 3 times per day for 10 days). We conclude that McN-5195 is a structurally novel (indolizine) antinociceptive agent that produces its analgesic action via a nonopioid mechanism, not involving products of arachidonate metabolism.

The effect of several i.c.v.-administered FMRFamide (Phe-Met-Arg-Phe-NH2)-like peptides (FaRPs) on mouse colonic propulsive motility was examined. Dose-related inhibition of propulsive motility (measured as an increase in the time of colonic bead expulsion) was produced by analogs with either the sequence (-)F[X]RFamide or with the FMRFamide sequence containing D-amino acid substitutions. The C-terminal dipeptide sequence Arg-Phe-NH2 was sufficient to produce this effect. D-amino acid substitution in the second position, i.e., [D-Met2]-FMRFamide (DMFa), conferred significantly enhanced activity (nearly maximal obtainable response under the test conditions) in this preparation (ED25 = 2.3 micrograms = 3.8 nmol). DMFa did not block the action of morphine but, like morphine, was blocked by the opioid antagonist naloxone and was attenuated by 24-hr pretreatment with the selective mu-1 opioid antagonist naloxonazine (35 mg/kg s.c.). It is concluded that a variety of FaRPs, particularly those with a nonpolar residue in the first position and with Arg in the third position, behave as opioid-like agonists, not antagonists, on mouse colonic propulsive motility. DMFa is identified as the most active FaRP studied to date on this endpoint.