The in vivo pharmacodynamics of the novel opioid receptor antagonist, TD-1211, in models of opioid-induced gastrointestinal and CNS activity

The in vivo preclinical pharmacodynamic profile of TD-1211, a selective opioid receptor antagonist currently under development for the treatment of opioid-induced constipation, was compared to that of the clinically studied opioid antagonists, naltrexone, alvimopan, and ADL 08-0011 (the primary active metabolite of alvimopan). The oral activity of TD-1211 was evaluated in models of gastrointestinal (GI) and central nervous system (CNS) function in the rat and dog. Oral administration of TD-1211, naltrexone, and ADL 08-0011 reversed loperamide-induced inhibition of gastric emptying and castor oil-induced diarrhea in rats and nonproductive GI circular smooth muscle contractility in dogs. Alvimopan was only efficacious in the castor oil model. Oral administration of naltrexone and ADL 08-0011, but not TD-1211 or alvimopan, was associated with a CNS withdrawal response in morphine-dependent mice, inhibition of morphine-induced anti-nociception in rat and dog hot plate tests, and hypothermia and sedation in dogs. It is concluded that TD-1211 has potent in vivo GI activity, consistent with opioid receptor antagonism, but has no significant CNS activity. The data from these studies support the clinical development of TD-1211 as a novel treatment for opioid-induced GI dysfunction.     

The in vitro pharmacological profile of TD-1211, a neutral opioid receptor antagonist

The clinical efficacy of opioid receptor antagonists for the treatment of opioid-induced constipation (OIC) is established. Peripherally selective antagonists are intended to provide OIC symptom relief without compromising the analgesic effects of centrally penetrant opioid agonists. We describe the in vitro profile of a novel opioid receptor antagonist, TD-1211, at recombinant (human μ and δ, and guinea pig κ) and rodent native opioid receptors. TD-1211 bound with high affinity to human recombinant μ and δ, and guinea pig κ receptors expressed in CHO-K1 cells (pK _d?=?9.7, 8.6, and 9.9, respectively). The in vitro receptor selectivity of TD-1211 (μ?≈?κ?>?δ) is similar to that for the peripherally-selective opioid receptor antagonist methylnaltrexone, but contrasts with the μ selectivity of alvimopan. Functionally, TD-1211 behaved as an antagonist at all three receptor types in both recombinant expression systems (pK _b?=?9.6, 8.8 and 9.5, at μ, δ, and κ, respectively) and rodent native tissue preparations (μ and κ pA₂s?=?10.1 and 8.8, respectively (guinea pig ileum), and δ pK _b?=?8.4 (hamster vas deferens)). TD-1211 displayed a high degree of selectivity for opioid receptors over a broad panel of cellular targets. These in vitro data justified investigation of the preclinical in vivo activity of TD-1211 (Armstrong et al., Naunyn-Schmiedeberg’s Arch Pharm, 2013).     

(3)H]Alvimopan binding to the micro opioid receptor: comparative binding kinetics of opioid antagonists

Alvimopan is a novel peripheral μ opioid antagonist in clinical development for the management of post-operative ileus and opioid-induced bowel dysfunction. We hypothesized that the long duration of action of alvimopan might be related to a slower dissociation rate from the μ opioid receptor compared to other shorter acting antagonists. The dissociation rate of alvimopan from the μ opioid receptor (t_1/2 = 30–44 min) was comparable to that of the long acting partial agonist buprenorphine (t_1/2 = 44 min), but was slower than those of the antagonists naloxone (t_1/2 = 0.82 min) and N-methylnaltrexone (t_1/2 = 0.46 min). Also, increases in the apparent affinities and potencies of buprenorphine and alvimopan, but not of naloxone and methylnaltrexone, were observed upon preincubation with the μ opioid receptor. Consistent with its long duration of action, alvimopan has a slow dissociation rate from the μ opioid receptor compared to other shorter acting antagonists and may be more potent if administered prior to dosing with exogenous opioids.     

Electrophysiological effects of EGIS-7229, a new antiarrhythmic agent, in isolated mammalian and human cardiac tissues

The cellular electrophysiological effects of EGIS-7229 (5-chlor-4-[N-(3,4-dimethoxy-phenyl-ethyl)-amino-propylamino]-3(2H)-pyridazinone fumarate), a novel antiarrhythmic agent, was studied using conventional microelectrode techniques in canine cardiac Purkinje fibers and papillary muscle preparations obtained from man, rabbits and guinea pigs. Low concentration of EGIS-7229 (3 μmol/l) selectively lengthened action potential duration (both APD₅₀ and APD₉₀) in all preparations. The effect of higher concentrations (30–100 μmol/l) of EGIS-7229 on action potential duration was variable depending on the preparation studied: in rabbit and human papillary muscles both APD₅₀ and APD₉₀ were lengthened, in canine Purkinje fibers APD₉₀ was lengthened but APD₅₀ was shortened, while in guinea pig papillary muscles both APD₅₀ and APD₉₀ were shortened by high concentrations of the drug. At these higher concentrations EGIS-7229 also decreased the maximum velocity of action potential upstroke (V _max) and depressed the plateau of action potentials without affecting the resting membrane potential or action potential amplitude. Both reduction of V _max and lengthening of APD were frequency dependent. The former effect was more prominent at higher pacing frequencies, while the latter was more pronounced at lower driving rates. In guinea pig papillary muscle, the time constant of recovery from V _max-block was 719 ± 33 ms (n = 18) and the rate of onset of the block was 1.81 ± 0.06 AP^–1 (n = 16) in the presence of 100 μmol/l EGIS-7229. EGIS-7229 had a complex action on refractoriness in guinea pig papillary muscles: ERP was lengthened at low concentrations (3 to 10 μmol/l), unchanged at 30 μmol/l and shortened at 100 μmol/l. The ratio of ERP/APD₉₀, however, was significantly increased at concentrations higher than 3 μmol/l. In canine Purkinje fiber, when the delayed rectifier K current (I_K) was blocked by d-sotalol (60 μmol/l) and APD was shortened back to its control value by additional application of nicorandil (15 μmol/l), APD was not affected by 3 μmol/l but was shortened by 30 μmol/l of EGIS-7229. 100 μmol/l EGIS-7229 shortened APD in guinea pig papillary muscle. This effect of EGIS-7229 was effectively prevented by nifedipine pretreatment (10 μmol/l). In this preparation, EGIS-7229 also decreased the V _max of the slow action potential, evoked in the presence of 20 mmol/l external K⁺ plus 0.5 mmol/l Ba²⁺. It is likely that EGIS-7229 at low concentrations blocks I_K in human, canine, rabbit and guinea pig cardiac preparations, but at higher concentrations also inhibits Ca and Na currents. Therefore, EGIS-7229 appears to carry mixed class III, IV and IB antiarrhythmic properties. Received: 8 July 1996 / Accepted: 23 October 1996     

Synthesis of chroman-2-carboxylic acid N-(substituted)phenylamides and their inhibitory effect on nuclear factor-κB (NF-κB) activation

A series of chroman-2-carboxylic acid N-(substituted)phenylamides (2a-s, 3a-j) were synthesized. Their ability to inhibit nuclear factor-κB (NF-κB) activity was evaluated in lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells and their antioxidant activity was examined. NF-κB inhibition by chroman compounds was not related to their antioxidant activity. Compounds with -H, -NO₂ monosubstituents and -OCH₃, -CF₃ disubstituents on the phenyl ring were poor inhibitors of NF-kB activity. Compounds with -CH₃, -CF₃, -Cl monosubstituents or -Cl, -CH₃ disubstituents exhibited moderate to good NF-κB activity inhibition (IC₅₀: 18.2–95.8 μM). The most active NF-κB inhibitor, 2s, contained a 4-Cl (IC₅₀: 18.2 μM) substituent on the phenyl ring and was slightly more potent than the compound KL-1156 (1) (IC₅₀: 43.9 μM).     

Bremazocine reduces unrestricted free-choice ethanol self-administration in rats without affecting sucrose preference

It has been postulated that opioid systems in the brain may play a role in ethanol reinforcement. In this respect, μ- and δ-opioid receptors may mediate the rewarding effects whereas κ receptors are thought to mediate the aversive effects of opioids. Accordingly, long-acting benzomorphans such as bremazocine, that simultaneously act as μ and δ receptor antagonists and κ receptor agonists may be particularly effective in reducing ethanol self-administration. Therefore, we studied the effect of bremazocine on oral ethanol self-administration in rats using a paradigm [unrestricted free-choice drinking of 10% (v/v) ethanol], previously shown to cause long-term neuroadaptations in the nucleus accumbens and caudate putamen. Bremazocine (0.1 mg/kg, once daily for five consecutive days) reduced ethanol drinking by about 50% during the active period of the animals, whereas the intake of sucrose (3–10% w/v) was affected neither in naive nor in ethanol-experienced rats. This effect of bremazocine appeared not to be secondary to its acute sedative effect or the slight increase in total fluid consumption. Unlike bremazocine, the selective κ-opioid receptor agonist U50,488H (10 mg/kg, once daily) inhibited ethanol drinking only during the first of 5 treatment days and the opioid receptor antagonist naltrexone (0.3–10 mg/kg, once daily) only caused a modest (about 20%) suppression of ethanol drinking during the first hours after drug injection. Thus, bremazocine appears to be far more potent than the clinically applied drug naltrexone in this respect. Our data further support the role of opioid receptors in ethanol reinforcement and indicate that long-acting mixed-action opioids such as bremazocine may be useful as adjuvants for the clinical management of ethanol addiction. Received: 1 July 1998/Final version: 3 September 1998     

The µ opioid irreversible antagonist beta-funaltrexamine differentiates the discriminative stimulus effects of opioids with high and low efficacy at the μ opioid receptor

 The purpose of the present study was to determine the relative intrinsic efficacy of various opioids using the irreversible μ opioid antagonist beta-funaltrexamine (βFNA). To this end, pigeons were trained to discriminate 3.0 (n=6) or 1.8 (n=1) mg/kg morphine from distilled water in a two-key, food-reinforced, drug discrimination procedure. The μ opioids fentanyl, l-methadone, buprenorphine, butorphanol, nalorphine, nalbuphine and levallorphan, as well as the δ opioid BW373U86, substituted completely for the morphine stimulus. The stimulus effects of morphine were antagonized (i.e., produced a significant increase in the ED₅₀ value) by a 10 mg/kg but not a 5 mg/kg dose of βFNA. Antagonist effects of βFNA were observed following a 2-h pretreatment, but not following 26-, 50-, 74-, 98- or 146-h pretreatments. The stimulus effects produced by fentanyl, l-methadone and buprenorphine were not antagonized by doses of βFNA as high as 20, 10 and 10 mg/kg, respectively. The lowest dose of βFNA required to antagonize the stimulus effects of butorphanol was 10 mg/kg, whereas the effects of nalorphine, nalbuphine and levallorphan were antagonized by a dose of βFNA as low as 5 mg/kg. The δ opioid BW373U86 substituted for the morphine stimulus, and this effect was not antagonized by 10 mg/kg βFNA. The pkB values for naloxone (1.0 mg/kg) against the stimulus effects of fentanyl (6.70) and morphine (6.52) were considerably higher than that for BW373U86 (4.60), indicating further that the morphine-like stimulus effects produced by BW373U86 were not mediated by activity at the μ opioid receptor. These findings indicate that the strategy of irreversible antagonism can be used effectively to differentiate opioids with varying degrees of intrinsic efficacy at the μ opioid receptor in a pigeon drug discrimination procedure. In particular, the ranking of these drugs by relative intrinsic efficacy at the μ opioid receptor is: l-methadone=fentanyl≥buprenorphine≥morphine≥ butorphanol>nalorphine=nalbuphine=levallorphan. Additionally, the short-acting effect of βFNA in the pigeon suggests that the recovery of μ opioid receptor function varies across species. Received: 19 August 1997 / Final version: 28 March 1998     

TH-030418: a potent long-acting opioid analgesic with low dependence liability

Numerous efforts have been made on the chemical modification of opioid compounds, with the ultimate goal of developing new opioid analgesics that is highly potent and low/non-addictive. In a search for such compounds, TH-030418 [7α-[(R)-1-hydroxy-1-methyl-3-(thien-3-yl)-propyl]-6,14-endo-ethanotetrahydrooripavine] was synthesized. Here, we evaluated the pharmacological activities of TH-030418, in comparison with morphine, the prototype opioid analgesic. In radioligand binding assays, TH-030418 bound potently and nonselectively to μ-, δ-, κ-, and ORL1 (opioid receptor-like 1) receptors stably expressed in CHO (Chinese hamster ovary) cells with K _i values of 0.56, 0.73, 0.60, and 1.55 nM, respectively. When administered subcutaneously, TH-030418 was much more potent than morphine in analgesia, with the ED₅₀ values of 1.37 μg/kg and 1.70 μg/kg in hot plate and acetic acid writhing tests, respectively. The opioid antagonist naloxone blocked the antinociceptive effect of TH-030418, indicating that the action of TH-030418 was mediated by opioid receptors. The antinociceptive effect of s.c. TH-030418 in hot plate test lasted for more than 12 h, which is much longer than those of morphine (2.5 h) and dihydroetorphine (1.5 h). In addition, naloxone did not precipitate withdrawal syndrome in the mice treated with TH-030418 previously. Most importantly, TH-030418 did not induce conditioned place preference in mice after chronic treatment. These results indicate that TH-030418 is a potent long-acting opioid analgesic with low dependence liability and may be of some value in the development of new analgesics.     

Diminution of contractile response by κ-opioid receptor agonists in isolated rat ventricular cardiomyocytes is mediated via a pertussis toxin-sensitive G protein

Opioids directly decrease the contractile response of isolated ventricular cardiomyocytes to electrical stimulation. To investigate whether these effects are mediated via GTP-binding G_i/o proteins we examined the influence of pertussis toxin on the effects of the κ-opioid receptor agonist trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide (U-50,488) methanesulphonate and on the as yet undescribed effects of the opioid peptide dynorphin A (1–8) on contraction. In isolated, electrically driven, rat ventricular cardiomyocytes both agents concentration dependently reduced cell shortening within 15 min, decreasing the contractile response by 79±4% (n=5) and 62±2% (n=6) of control values at maximal effective concentrations of 10 μM (U-50,488) and 1 μM [dynorphin A (1–8)], respectively. Pertussis toxin pre-treatment (200 ng/ml; 4.5–5 h) completely abolished the effects of U-50,488 and dynorphin A (1–8) on the contractile response, indicating that these effects are mediated via G_i/o proteins. In addition, the non-selective opioid receptor antagonist (–)-naloxone and the κ-opioid receptor antagonist nor-binaltorphimine antagonized the effects of U-50,488 and dynorphin A (1–8) on the contractile response. Furthermore, the μ- and δ-opioid receptor agonist (D-Ala², D-Leu⁵)-enkephalin (DADLE) had no effects on contraction. These results indicate that the decrease in cell shortening is due to stimulation of κ-opioid receptors. The direct effect of κ-opioid receptor agonists on the contractile response thus represents an additional mechanism for decreasing cardiac contractility, besides the M-cholinoceptor- or adenosine receptor-mediated pathway. It is conceivable that increased release of endogenous dynorphins from the heart during hypoxia may protect the heart in a similar manner to adenosine. Received: 16 September 1997 / Accepted: 15 June 1998     

Synthesis,antimicrobial evaluation,and QSAR analysis of 2-isopropyl-5-methylcyclohexanol derivatives

A series of 2-isopropyl-5-methylcyclohexanol derivatives were synthesized and evaluated for their antibacterial activity against Gram-positive Staphylococcus aureus and Bacillus subtilis and Gram-negative Escherichia coli and in vitro antifungal activity against Candida albicans and Aspergillus niger. The results of antimicrobial activity demonstrated that the compounds 10, 20, and 21 were the most active ones among the synthesized compounds. The QSAR studies revealed the importance of dipole moment (μ), total energy (Te), and topological parameters (κ₁ and κ₃) in describing the antimicrobial activity of 2-isopropyl-5-methylcyclohexanol derivatives.     

Synthesis and nuclear factor-κB inhibitory activities of 6- or 7-methylchroman-2-carboxylic acid N-(substituted) phenylamides

A series of 6- or 7-methylchroman-2-carboxylic acid N-(substituted) phenylamides (2a-s, 3a-s) were synthesized. Their abilities to inhibit nuclear factor-κB (NF-κB) activity were evaluated in lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells. Compounds with substituents such as -H, -CH₃, and -CF₃ on the phenyl ring were poor inhibitors of NF-κB. The most active NF-κB inhibitors contained 4-Cl (3s) and 4-OMe (3g) in the 7-methylchroman-2-carboxamide derivatives and 2-OH (2b) and 4-Cl (2s) in the 6-methylchroman-2-carboxamide derivatives (IC₅₀: 20.2–24.0 μM). These were slightly more potent than a reference compound, KL-1156 (1) (IC₅₀: 43.9 μM).     

Characterization of the cloned guinea pig leukotriene B4 receptor: comparison to its human orthologue

A cDNA clone coding for the guinea pig leukotriene B₄ (BLT) receptor has been isolated from a lung cDNA library. The guinea pig BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respectively. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein–Bar Virus Nuclear Antigen) cells showed that both receptors displayed high affinity for leukotriene B₄ (K_d value of 0.4 nM) and were expressed at high levels (B_max values ranging from 9 to 12 pmol/mg protein). The rank order of potency for leukotrienes and related analogs in competition for [³H]leukotriene B₄ specific binding at the recombinant guinea pig BLT receptor is leukotriene B₄>20-OH-leukotriene B₄>12(R)-HETE ((5Z,8Z,10E,12(R)14Z)-12-hydroxyeicosatetraen-1-oic acid)>12(S)-HETE ((5Z,8Z,10E,12(S)14Z)-12-Hydroxyeicosatetraen-1-oic acid)>20-COOH-leukotriene B₄>U75302 (6-(6-(3-hydroxy-1E,5Z-undecadienyl)-2-pyridinyl)-1,5-hexanediol)leukotriene C₄=leukotriene D₄=leukotriene E₄. For the human receptor the rank order of 12(S)-HETE, 20-COOH-leukotriene B₄ and U75302 was reversed. Xenopus melanophore and HEK aequorin-based reporter gene assays were used to demonstrate that the guinea pig and human BLT receptors can couple to both the cAMP inhibitory and intracellular Ca²⁺ mobilization signaling pathways. However, in the case of the aequorin-expressing HEK cells (designated AEQ17-293) transfected with either the guinea pig or human BLT receptor, expression of G_α16 was required to achieve a robust Ca²⁺ driven response. Leukotriene B₄ was a potent agonist in functional assays of both the guinea pig and human BLT receptors. U-75302 a leukotriene B₄ analogue which possesses both agonistic and antagonistic properties behaved as a full agonist of the guinea pig and human BLT receptors in AEQ17-293 cells and not as an antagonist. The recombinant guinea pig BLT receptor will permit the comparison of the intrinsic potencies of leukotriene B₄ receptor antagonists used in guinea pig in vivo models of allergic and inflammatory disorders.     

Differential involvement of opioidergic and serotonergic systems in the antinociceptive activity of N-arachidonoyl-phenolamine (AM404) in the rat: comparison with paracetamol

It is recognized that paracetamol undergoes a metabolic transformation to N-arachydonylphenolamine (AM404), a CB₁ receptor ligand and anandamide uptake inhibitor. Using hot-plate and paw pressure tests, we decided to establish whether AM404 may act through opioidergic and serotonergic mechanisms. Thus, we pretreated rats with opioid μ₁ (naloxonazine) and κ (MR2266) or 5-HT_1A (NAN-190), 5-HT₂ (ketanserin), and 5-HT₃ (ondansetron) receptor antagonists. We investigated the possible changes in 5-hydroxyindoleacetic acid/serotonin ratio in the frontal cortex and pons. The antinociceptive effect of AM404 (10 mg/kg, intrapertoneally) or paracetamol (400 mg/kg, intrapertoneally) in either test was abolished by naloxonazine or MR2266. Ondansetron prevented AM404 activity; NAN-190 and ketanserin were ineffective. Ketanserin antagonized paracetamol activity; NAN-190 and ondansetron were inactive. AM404 did not change serotonergic activity, while paracetamol decreased serotonin turnover. The diverse antinociceptive potency of the compounds might be explained by the different influence on the serotonergic system, despite a similar involvement of opioidergic one.     

N-nitroso-N-methylurea and N-nitroso-N-ethylurea induce upregulation of cellular NF-κ B activity through protein kinase C-dependent pathway in human malignant keratinocytes

The upregulatory mechanism of cellular NF-κB activity by carcinogens, N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) in human malignant keratinocytes was investigated. To elucidate the role of protein kinase C (PKC) in the upregulation of NF-κB by NMU and NEU, two known PKC inhibitors, staurosporine and H-7 were studied. Treatment of cells with PKC inhibitors decreased NF-κB activity in a dose responsive manner at concentrations of 20∼200 nM. Staurosporine (160 nM) and H-7 (200 nM) downregulated the cellular NF-κB activation up to 20 and 60% compared to the NF-κB activity that was upregulated by NMU (5 μM) and NEU (5 μM), respectively. These results indicated that the PKC activity was responsible for the upregulation of NF-κB activity. The level of phosphorylation of I-κBα, the predominant form of the I-κB family represented by NMU and NEU, was quantified. The relative amount of I-κBα phosphorylation (serines-32 and -36) determined using the cellular activation of signaling ELISA assay method showed that NMU (5 μM) and NEU (5 μM) increased the amount of I-κBα phosphorylation up to 17 and 10% compared to the control, respectively. The results demonstrate the upregulatory effect of NMU and NEU on cellular NF-κB activity in human keratinocytes via the protein kinase C-mediated pathway.     

Phenacetin O-deethylation by human liver microsomes in vitro: inhibition by chemical probes,SSRI antidepressants,nefazodone and venlafaxine

Biotransformation of phenacetin via O-deethylation to acetaminophen, an index reaction reflecting activity of Cytochrome P450-1A2, was studied in microsomal preparations from a series of human livers. Acetaminophen formation was consistent with a double Michaelis-Menten system, with low-K_m (mean K_m1 = 68 μM) and high-K_m (mean K_m2 = 7691 μM) components. The low-K_m enzyme accounted for an average of 96% of estimated intrinsic clearance, and was predicted to contribute more than 50% of net reaction velocity at phenacetin concentrations less than 2000 μM. Among index inhibitor probes, α-naphthoflavone was a highly potent inhibitor of the low-K_m enzyme (K_i1 = 0.013 μM); furafylline also was a moderately active inhibitor (K_i1 = 4.4 μM), but its inhibiting potency was increased by preincubation with microsomes. Ketoconazole was a relatively weak inhibitor (K_i1 = 32 μM); quinidine and cimetidine showed minimal inhibiting activity. Among six selective serotonin reuptake inhibitor (SSRI) antidepressants, fluvoxamine was a potent inhibitor of 1A2 (mean K_i1 = 0.24 μM). The other SSRIs were more than tenfold less potent. Mean K_i1 values were: fluoxetine, 4.4 μM; norfluoxetine, 15.9 μM; sertraline, 8.8 μM; desmethylsertraline, 9.5μM; paroxetine, 5.5 μM. The antidepressant nefazodone and four of its metabolites (meta-chloro-phenylpiperazine, two hydroxylated derivatives, and a triazoledione) were very weak inhibitors of P450-1A2. Venlafaxine and its O- and N-desmethyl metabolites showed minimal inhibitory activity. Received: 18 March 1996/Final version: 10 July 1996     

Pharmacological properties of cardiovascular histamine H1 receptor binding sites: characterisation with 2-phenylhistamines

We determined and compared the molecular properties of histamine H₁ receptor binding sites in bovine thoracic aorta smooth muscle and guinea pig myocardial membranes from ventricles with saturation and inhibition binding assay, using ³H-mepyramine to label the receptor and specific and selective H₁ receptor agonists of the 2-phenylhistamine group as displacers of specific ³H-mepyramine binding. ³H-mepyramine binds in a saturable manner to a homogenous population of binding sites with a K _D of 5.6 nM and a B _max of 57 fmol/mg of protein in bovine aorta vascular smooth muscle membranes, whereas in the guinea pig myocardium high and low affinity ³H-mepyramine binding sites exist having the following molecular characteristics: a K _D of 1.6 nM and a B _max of 99 fmol/mg of protein (high affinity site) and a K _D 15.0 nM and a B _max of 466 fmol/mg of protein (low affinity site). Halogenated 2-phenylhistamines: 2-(3-fluoro-) (2-FPH), 2-(3-trifluoromethyl-) (2-triFMPH), 2-(3-chloro-) (2-CPH), 2-(3-bromo-) (2-BPH) and 2-(3-iodophenyl)histamine (2-IPH), which showed high selectivity and potency for H₁ receptors in the functional pharmacological studies, were potent inhibitors of specific radioligand binding in comparison with histamine and parent nonhalogenated 2-phenylhistamine (2-PH). Their rank order of potencies and affinities differ significantly for the vascular and cardiac H₁ receptor binding sites: Specific ³H-mepyramine binding to H₁ receptors in bovine vascular smooth muscle membranes was displaced in a biphasic manner by 2-(3-fluoro-), 2-(3-trifluoromethyl-), 2-(3-chloro-), 2-(3-bromo-), 2-(3-iodophenyl)histamine and histamine. In guinea pig ventricular myocardium the rank order was 2-(3-iodo-), 2-(3-bromo-), histamine, 2-(3-chloro-), and 2-(3-fluorophenyl)histamine showing better correlation with the lipophilicity of the derivatives than in vascular tissue (order of lipophilicity: 2-triFMPH >2-IPH >2-BPH >2-CPH >2-FPH >>2-PH). Displacement of the radioligand binding to myocardial H₁ receptor by the above drugs is (except for 2-(3-fluorophenyl)histamine), better fitted to a two-site model. 2-phenylhistamine, which acted as a moderate agonist in functional studies, displaced the radioligand in a monophasic manner and was the weakest displacer of specific radioligand binding in both model systems (pK _i = 5.76 – vascular and 5.57 – cardiac tissue). The agonistic nature of the halogenated 2-phenylhistamine derivatives was confirmed on the molecular level, since their interaction with the H₁ receptor is regulated by guanine nucleotides. GTP (0.1 mM) significantly lowered the affinities of all tested halogenated 2-phenylhistamines and histamine for H₁ receptor binding site converting biphasic displacement curves, to monophasic ones, whereas GTP had no effect on the affinity of 2-PH. The results of this study support the conclusions that bovine vascular and guinea pig myocardial histamine H₁ receptors differ in their molecular properties. Selective and potent H₁ receptor agonists of 2-phenylhistamine class can discriminate between vascular and cardiac receptor. Received: 3 July 1996 / Accepted: 16 December 1996     

Acute exposure to saccharin reduces morphine analgesia in the rat: evidence for involvement of N-methyl-d-aspartate and peripheral opioid receptors

Rationale: Pairings of a sweet taste and injection of morphine result in a learned avoidance of that taste and learned analgesic tolerance. This avoidance is mediated by the drug’s peripheral effect, while learned tolerance involves activation of N-methyl-d-aspartate (NMDA) receptors. Exposure to a sweet taste also reduces morphine analgesia. We studied whether this taste-mediated reduction was reversed by an NMDA or peripheral opioid receptor antagonist. Objectives: To determine whether an intraoral infusion of saccharin would modulate morphine analgesia in rats, and to study the contribution of NMDA as well as peripheral opioid receptors to this modulation. Methods: Six experiments used the rat’s tail-flick response to study the effect of an intraoral infusion of a sodium saccharin solution on morphine analgesia, and the effects of the quaternary opioid receptor antagonist methylnaltrexone as well as the non-competitive NMDA receptor antagonist MK-801 on this modulation of analgesia. Results: An intraoral infusion of saccharin reduced the analgesic effects of an intraperitoneal (i.p.) injection of morphine across a range of doses (experiment 1a), which was not attributable to an influence on tail-skin temperature (experiment 1b). This reduction was mediated by opioid receptors in the periphery and activation of NMDA receptors because morphine analgesia was reinstated by an i.p. injection of either methylnaltrexone (experiment 2a) or MK-801 (experiment 3a), which was not due to the effect of methylnaltrexone (experiment 2b) or MK-801 (experiment 3b) on morphine analgesia in the absence of saccharin. Conclusions: These results document evidence for an antagonism of morphine analgesia by actions of the drug at peripheral opioid receptors and excitatory amino-acid activity at NMDA receptors. They are discussed with reference to the aversive motivational effects of peripheral opioid receptors and pain facilitatory circuits. Received: 12 June 1999 / Final version: 1 November 1999     

Relaxant and antispasmodic effects of extracts of the orchid <Emphasis Type="Italic">Encyclia michuacana</Emphasis> on isolated guinea pig ileum

The antispasmodic effects of hexane-, chloroform-, methanol- and water-extracts of the orchid Encyclia michuacana were studied in vitro on guinea pig ileum against three spasmogens: acetylcholine (Ach), histamine and barium chloride. The chloroform extract exerted a significant antispasmodic effect on ileum contractions induced by Ach, histamine and barium chloride (IC₅₀ = 90.64, 73.12 and 115.2 μg/mL, respectively). Furthermore, the chloroform extract of E. michuacana provoked a concentration-dependent inhibition of spontaneous contractions of guinea pig ileum with potencies comparable to those of papaverine. The antagonism against the spasmogens used suggests that the action of the chloroform extract of E. michuacana could be due mainly to the presence of gigantol. Hexane-, methanol- and water-extracts did not elicit any significant spasmolytic activity.     

Conditioned place preference: no tolerance to the rewarding properties of morphine

The effect of repeated morphine administration on conditioned place preference (CPP) using a novel treatment schedule, i.e., drug treatment was always contingent with the conditioned environmental stimuli, was investigated. We also examined whether changes in the μ- and κ-opioid receptor binding occurred in the brain of morphine-treated animals. Intraperitoneal (i.p.) administration of morphine (2 and 10 mg/kg) induced a place preference after 8 daily conditioning trials (4 morphine injections on alternate trials), the level of preference being the same with the two doses of the opiate. No change in place preference was observed in the morphine-treated rats at 2 mg/kg, when animals were further trained up to a total of 32 conditioning trials (16 morphine injections). Conversely, after 20 conditioning trials (10 morphine injections), a stronger CPP response developed in the morphine-treated rats at 10 mg/kg. Signs of morphine withdrawal were never detected in morphine-treated rats during the experiment. Loss of body weight (index of opiate dependence) was not observed either 24 h or 48 h after the last morphine administration. μ- and κ-opioid receptor density and affinity were not affected by repeated morphine administrations at either dose. The results demonstrate that no tolerance develops to the rewarding properties of morphine. Indeed, a sensitisation effect may occur at increasing doses of the opiate. Furthermore, changes in the rewarding effect of morphine are not dependent upon alterations in opioid receptors involved in the reinforcing mechanisms. Received: 31 October 1996 / Accepted: 7 February 1997