In vivo myocardial protection from ischemia/reperfusion injury by the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone.

BACKGROUND: Diabetes is associated with increased risk of mortality as a consequence of acute myocardial infarction. This study determined whether rosiglitazone (ROSI) could reduce myocardial infarction after ischemia/reperfusion injury. METHODS AND RESULTS: Male Lewis rats were anesthetized, and the left anterior descending coronary artery was ligated for 30 minutes. After reperfusion for 24 hours, the ischemic and infarct sizes were determined. ROSI at 1 and 3 mg/kg IV reduced infarct size by 30% and 37%, respectively (P<0.01 versus vehicle). Pretreatment with ROSI (3 mg. kg(-1). d(-1) PO) for 7 days also reduced infarct size by 24% (P<0.01). ROSI also improved ischemia/reperfusion-induced myocardial contractile dysfunction. Left ventricular systolic pressure and positive and negative maximal values of the first derivative of left ventricular pressure (dP/dt) were significantly improved in ROSI-treated rats. ROSI reduced the accumulation of neutrophils and macrophages in the ischemic heart by 40% and 43%, respectively (P<0.01). Ischemia/reperfusion induced upregulation of CD11b/CD18 and downregulation of L-selectin on neutrophils and monocytes; these effects were significantly attenuated in ROSI-treated animals. Likewise, intercellular adhesion molecule-1 expression in ischemic hearts was markedly diminished by ROSI, as was the ischemia/reperfusion-stimulated upregulation of monocyte chemoattractant protein-1. CONCLUSIONS: ROSI reduced myocardial infarction and improved contractile dysfunction caused by ischemia/reperfusion injury. The cardioprotective effect of ROSI was most likely due to inhibition of the inflammatory response.

Novel AMPA receptor potentiators LY392098 and LY404187: effects on recombinant human AMPA receptors in vitro.

The present study describes the activity of two novel potent and selective AMPA receptor potentiator molecules LY392098 and LY404187. LY392098 and LY404187 enhance glutamate (100 microM) stimulated ion influx through recombinant homomeric human AMPA receptor ion channels, GluR1-4, with estimated EC(50) values of 1.77 microM (GluR1(i)), 0.22 microM (GluR2(i)), 0.56 microM (GluR2(o)), 1.89 microM (GluR3(i)) and 0.20 microM (GluR4(i)) for LY392098 and EC(50) values of 5.65 microM (GluR1(i)), 0.15 microM (GluR2(i)), 1.44 microM (GluR2(o)), 1.66 microM (GluR3(i)) and 0.21 microM (GluR4(i)) for LY404187. Neither compound affected ion influx in untransfected HEK293 cells or GluR transfected cells in the absence of glutamate. Both compounds were selective for activity at AMPA receptors, with no activity at human recombinant kainate receptors. Electrophysiological recordings demonstrated that glutamate (1 mM)-evoked inward currents in human GluR4 transfected HEK293 cells were potentiated by LY392098 and LY404187 at low concentrations (3-10 nM). In addition, both compounds removed glutamate-dependent desensitization of recombinant GluR4 AMPA receptors. These studies demonstrate that LY392098 and LY404187 allosterically potentiate responses mediated by human AMPA receptor ion channels expressed in HEK 293 cells in vitro.

Characterization of the neurotoxic potential of m-methoxy-MPTP and the use of its N-ethyl analogue as a means of avoiding exposure to a possible Parkinsonism-causing agent.

1-Methyl-4-(3-methoxyphenyl)-1,2,3,6-tetrahydropyridine (2) produced persistent depletion of striatal dopamine in mice after four daily injections, although it was less potent than 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP has been implicated as a cause of Parkinsonism in drug abusers who inadvertently self-administered it and in industrial chemists who were exposed to it. Our results suggest that the m-methoxy compound has the same neurotoxic potential to cause destruction of nigrostriatal dopamine neurons that would lead to Parkinsonian symptoms in humans. In contrast, 1-ethyl-4-(3-methoxyphenyl)-1,2,3,6-tetrahydropyridine (11) had no effects on striatal dopamine in mice, even at doses 8 times those of MPTP. A method of preparing 11 and using it as an intermediate in the synthesis of potential analgesic drugs, thus avoiding a potentially neurotoxic intermediate, is described.

Synthesis and analgesic properties of N-substituted trans-4a-aryldecahydroisoquinolines.

A representative series of N-substituted derivatives of the morphine-based trans-4a-aryldecahydroisoquinoline were synthesized and evaluated for opioid analgesic activities. Compounds with potent analgesic activity and high affinities for the mu and kappa opioid receptors were discovered. The effect of varying the N-substituent in the trans-4a-aryldecahydroisoquinoline paralleled, to a certain extent, previous findings with other morphine part structures. Replacement of the N-methyl with a phenethyl group significantly increased analgesic potency. The N-cyclopropylmethyl analogue was found in rodents to have mixed agonist-antagonist properties; however, its antagonist activity was far weaker than those reported for the N-(cyclopropylmethyl)morphinan and -benzomorphan derivatives. Resolution of the stereoisomers and determination of their absolute configuration by X-ray crystallography showed that the opioid receptor effects were predominantly found with the 4aR,8aR isomer, the same relative absolute configuration of morphine. Unexpectedly, the 4aR,8aR N-cyclopropylmethyl analogue (compound 30), which in rodents had mixed agonist-antagonist properties similar to those of pentazocine, was found in rhesus monkeys to behave as a full morphine-like agonist.

Discovery of a potent, peripherally selective trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonist for the treatment of gastrointestinal motility disorders.

Structure-activity relationship studies were pursued within N-substituted-trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines in an effort to discover a peripherally selective opioid antagonist with high activity following systemic administration. Altering the size and the polarity of the N-substituent led to the discovery of 3 (LY246736). Compound 3 has high affinity for opioid receptors (Ki = 0.77, 40, and 4.4 nM for mu, kappa, and delta receptors, respectively). It is a potent mu receptor antagonist following parenteral and oral administration and distributes selectively (> 200-fold selectivity) to peripheral receptors. Thus, 3 has properties suitable for the clinical investigation of mu opioid receptor involvement in GI motility disorders.

Structure-activity relationships of trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine antagonists for mu- and kappa-opioid receptors.

A series of racemic N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines were evaluated for opioid agonist and antagonist activity at mu and kappa receptors. Several highly potent mu and kappa antagonists were discovered; however, no compounds with high selectivity for either the mu or kappa receptor were identified. Importantly, no derivative was found to have significant opioid agonist activity. Two derivatives were resolved, and the activities of the enantiomers were investigated. Only a limited stereochemical effect on opioid receptor selectivities was observed. The structure-activity relationships described establish the existence of an important lipophilic binding site distal to the nitrogen for both mu and kappa receptors and confirm the pure opioid antagonist pharmacophore nature of the trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine structure.

6-substituted decahydroisoquinoline-3-carboxylic acids as potent and selective conformationally constrained NMDA receptor antagonists.

We have prepared a series of 6-substituted decahydroisoquinoline-3-carboxylic acids, and structurally similar analogs, as potential N-methyl-D-aspartate receptor antagonists. There is a large body of evidence to support the use of such compounds as cerebroprotective agents in a variety of acute and chronic neurodegenerative disorders, where some component of glutamate-mediated excitotoxicity may exist. The compounds prepared were evaluated in vitro in both receptor binding assays ([3H]CGS19755, [3H]AMPA, and [3H]kainic acid) and in a cortical wedge preparation (versus NMDA, AMPA, and kainic acid) to determine affinity, potency, and selectivity. The new amino acids were also evaluated in vivo for their ability to block NMDA-induced lethality in mice. We synthesized many of the possible diastereomers of the decahydroisoquinoline nucleus in order to examine the spatial and steric requirements for affinity at the NMDA receptor and activity as NMDA antagonists. From our structure-activity relationship we identified two potent and selective NMDA receptor antagonists, the phosphonate- and tetrazole-substituted amino acids 31a and 32a, respectively, that show good activity in animals following systemic administration. For example, 31a and 32a selectively displaced [3H]CGS19755 binding with IC50S of 55 +/- 14 and 856 +/- 136 nM, respectively, and selectively antagonized responses due to NMDA in a cortical wedge preparation with IC50S of 0.15 +/- 0.01 and 1.39 +/- 0.29 microM, respectively. And compounds 31a and 32a blocked NMDA-induced lethality in mice with minimum effective doses of 1.25 and 2.5 mg/kg (intraperitoneal), respectively. These novel amino acids are among some of the most potent NMDA antagonists described thus far, and are excellent candidates for development as neuroprotective agents for a number of CNS disorders.

Investigation of the N-substituent conformation governing potency and mu receptor subtype-selectivity in (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonists.

A study of the binding site requirements associated with the N-substituent of (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) derivatives was undertaken using a set of rigid vs flexible N-substituents. The study showed that compounds 7-9 bearing the trans-cinnamyl N-substituent most closely reproduced the potency at the opioid receptor of the flexible N-propylphenyl or N-propylcyclohexyl analogues previously reported. Neither the N-substituted cis-cinnamyl nor the cis-phenylcyclopropylmethyl compounds 10 and 11, respectively, showed high affinity for the opioid receptor. However, the N-trans-phenylcyclopropylmethyl compound 12 closely approximated the affinity of compounds 7-9. Additionally, we found that free rotation of the phenyl ring is necessary for high affinity binding and mu receptor subtype selectivity as the planar N-substituted thianaphthylmethyl and benzofuranylmethyl compounds 13 and 14 had significantly lower binding affinities. Altogether, these findings suggest that the high binding affinity, selectivity, and antagonist potency of N-propylphenyl or N-propylcyclohexyl analogues of (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) are achieved via a conformation wherein the connecting chain of the N-substituents is extended away from piperidine nitrogen with the appended ring system rotated out-of-plane relative to the connecting chain atoms. This conformation is quite similar to that observed in the solid state for 5, as determined by single crystal X-ray analysis. Additionally, it was found that, unlike naltrexone, N-substituents bearing secondary carbons attached directly to the piperidine nitrogen of 4 suffer dramatic losses of potency vs analogues not substituted in this manner. Using a functional assay which measured stimulation or inhibition of [35S]GTP-gamma-S binding, we show that the trans-cinnamyl analogues of (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) retain opioid pure antagonist activity and possess picomolar antagonist potency at the mu receptor.

N-Substituted 9beta-methyl-5-(3-hydroxyphenyl)morphans are opioid receptor pure antagonists.

The inhibition of radioligand binding and [35S]GTPgammaS functional assay data for N-methyl- and N-phenethyl-9beta-methyl-5-(3-hydroxyphenyl)morphans (5b and 5c) show that these compounds are pure antagonists at the micro, delta, and kappa opioid receptors. Since 5b and 5c have the 5-(3-hydroxyphenyl) group locked in a conformation comparable to an equatorial group of a piperidine chair conformation, this information provides very strong evidence that opioid antagonists can interact with opioid receptors in this conformation. In addition, it suggests that the trans-3, 4-dimethyl-4-(3-hydroxyphenyl)piperidine class of antagonist operates via a phenyl equatorial piperidine chair conformation. Importantly, the close relationship between the 4-(3-hydroxyphenyl)piperidines and 5-(3-hydroxyphenyl)morphan antagonists shows that the latter class of compound provides a rigid platform on which to build a novel series of opioid antagonists.

Synthesis of a series of aryl kainic acid analogs and evaluation in cells stably expressing the kainate receptor humGluR6.

The synthesis and pharmacological characterization of a novel series of 4-aryl-substituted kainic acid analogs are described. Receptor affinities were determined on recombinantly expressed humGluR6 kainate receptors and on [3H]kainate binding to rat forebrain kainate receptors. Functional agonist potencies were assessed using whole cell voltage clamp recordings in cells expressing humGluR6 receptors. Substitution of phenyl for the methyl at the C-4 position of kainic acid produced 11 which has high affinity and agonist potency at the GluR6 receptor. Substitution on phenyl led to a series of compounds with varying affinity for this kainate receptor. Agonist potency correlated with receptor affinity and with no derivative could antagonist activity be identified. Affinities for the humGluR6 kainate receptor were approximately 10-50 less than the observed affinities at rat forebrain kainate receptors. Furthermore, within the series of 4-aryl-substituted kainic acid analogs, there was a high degree of correlation between binding affinities for humGluR6 receptors and competition with kainate binding to rat forebrain kainate receptors.

Identification of the first trans-(3R,4R)- dimethyl-4-(3-hydroxyphenyl)piperidine derivative to possess highly potent and selective opioid kappa receptor antagonist activity.

A structurally novel opioid kappa receptor selective ligand has been identified. This compound, (3R)-7-hydroxy-N-((1S)-1-[[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic, 10) demonstrated high affinity for the kappa receptor in the binding assay (kappa K(i) = 0.3 nM) and highly potent and selective kappa antagonism in the [(35)S]GTP-gamma-S assay using cloned opioid receptors (kappa K(i) = 0.006 nM, mu/kappa ratio = 570, delta/kappa ratio > 16600).

Identification of an opioid kappa receptor subtype-selective N-substituent for (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine.

A three-component library of compounds was prepared in parallel using multiple simultaneous solution-phase synthetic methodology. The compounds were biased toward opioid receptor antagonist activity by incorporating (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (a potent, nonselective opioid pure antagonist) as one of the monomers. The other two monomers, which included N-substituted or unsubstituted Boc-protected amino acids and a range of substituted aryl carboxylic acids, were selected to add chemical diversity. Screening of these compounds in competitive binding experiments with the kappa opioid receptor selective ligand [3H]U69,593 led to the discovery of a novel kappa opioid receptor selective ligand, N-¿(2'S)-[3-(4-hydroxyphenyl)propanamido]-3'-methylbutyl¿-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (8, RTI-5989-29). Additional structure-activity relationship studies suggested that 8 possesses lipophilic and hydrogen-bonding sites that are important to its opioid receptor potency and selectivity. These sites appear to exist predominantly within the kappa receptor since the selectivity arises from a 530-fold loss of affinity of 8 for the mu receptor and an 18-fold increase in affinity for the kappa receptor relative to the mu-selective ligand, (+)-N-[trans-4-phenyl-2-butenyl]-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (5a). The degree of selectivity observed in the radioligand binding experiments was not observed in the functional assay. According to its ability to inhibit agonist stimulated binding of [35S]GTPgammaS at all three opioid receptors, compound 8 behaves as a mu/kappa opioid receptor pure antagonist with negligible affinity for the delta receptor.

Synthesis and evaluation of a series of novel 2-[(4-chlorophenoxy)methyl]benzimidazoles as selective neuropeptide Y Y1 receptor antagonists.

A series of novel benzimidazoles (BI) derived from the indole 2 was synthesized and evaluated as selective neuropeptide Y (NPY) Y1 receptor antagonists with the aim of developing antiobesity drugs. In our SAR approach, the (4-chlorophenoxy)methyl group at C-2 was kept constant and a series of BIs substituted with various piperidinylalkyl groups at N-1 was synthesized to identify the optimal spacing and orientation of the piperidine ring nitrogen relative to the benzimidazole. The 3-(3-piperidinyl)propyl in 33 was found to maximize affinity for the Y1 receptor. Because of the critical importance of Arg33 and Arg35 of NPY binding to the Y1 receptor, the incorporation of an additional aminoalkyl functionality to the structure of 33 was explored. Methyl substitution was used to probe where substitution on the aromatic ring was best tolerated. In this fashion, the C-4 was chosen for the substitution of the second aminoalkyl functionality. Synthesis of such compounds with a phenoxy tether using the 4-hydroxybenzimidazole 11 was pursued because of their relative ease of synthesis. Functionalization of the hydroxy group of 45 with a series of piperidinylalkyl groups provided the dibasic benzimidazoles 55-62. Among them, BI 56 demonstrated a Ki of 0.0017 microM, which was 400-fold more potent than 33. To evaluate if there was a stereoselective effect on affinity for these BIs, the four constituent stereoisomers (69-72) of the BI 60 were prepared using the S- and R-isomers of bromide 17. Antagonist activity of these BIs was confirmed by measuring the ability of selected compounds to reverse NPY-induced forskolin-stimulated cyclic AMP. The high selectivity of several BI antagonists for the Y1 versus Y2, Y4, and Y5 receptors was also shown.

Automated double labeling of proliferation and apoptosis in glutathione S-transferase-positive hepatocytes in rats.

Immunohistochemical markers for proliferation (bromodeoxyuridine, BrdU) and apoptosis (in situ terminal deoxynucleotide transferase dUTP nick end-labeling, TUNEL) were localized within glutathione S-transferase (GSTP)-positive hepatic foci in rats. Using the TechMate Automated Staining System (BioTek Solutions: Santa Barbara, CA), formalin-fixed, paraffin-embedded sections were run through a double-label avidin-blotin-immunoperoxidase protocol in less than 10 hr. Steam heat-induced epitope retrieval and/or proteolytic digestion preceded each labeling procedure. Color development was achieved using diaminobenzidine (DAB) with nickel enhancement for BrdU and TUNEL and VIP for GSTP. Results illustrate clear staining, brown-black BrdU-positive nuclei or TUNEL-positive apoptotic bodies within purple GSTP-positive hepatocytes. This automated procedure provides a method to easily identify and quantitate proliferating or apoptotic cells within foci of altered hepatocytes in rat liver and may have general applications for studies of cell or tissue kinetics during development, differentiation, and various pathological conditions in animals and humans.

Phencyclidine receptors in rat brain cortex.

The binding of [3H]phencyclidine (PCP) to receptors in rat brain cortex has been studied. Two receptors have been detected, a high affinity receptor site with a KD of 23.5 +/- 7.4 nM and a low affinity site with a KD of 7.6 +/- 1.8 microM. The binding of [3H]PCP to its receptors was pH and temperature dependent and was destroyed by heat-denaturation. The binding of [3H]PCP was inhibited by compounds which produce PCP-like behavioral effects including dexoxadrol, etoxadrol and ketamine as well as a novel series of benz(f)isoquinolines. The low affinity site was blocked by PCP, etoxadrol and (+)-SKF-10,047 but not morphine or leu-enkephalin, suggesting that it also represents a specific PCP site. Stereoselective displacement of PCP at the high affinity receptor was observed with the isomers of cyclazocine, cyclorphan, SKF-10,047 and dioxadrol (dexoxadrol and levoxadrol). Naloxone, 4,5,6,7-tetrahydroisoxazolo(S,4-C)pyridin-3-ol (THIP) hydrate and haloperidol inhibited binding poorly (Ki greater than 1 microM), suggesting that these compounds do not interact significantly with the high affinity PCP receptor in vivo. The affinity of ligands for the phencyclidine receptor was highly correlated (r = 0.714, P less than 0.01) with their potency to produce catalepsy in pigeons.

Phenylpiperidine opioid antagonists that promote weight loss in rats have high affinity for the kappa 2B (enkephalin-sensitive) binding site.

Certain opioid antagonists of the phenylpiperidine series (PPAs), such as LY255582, seem uniquely efficacious at producing weight loss in lean and meal-fed obese Zucker rats. Comparison of the pharmacological and receptor binding profile of PPAs that promote marked weight loss with those that do not has failed to find any obvious differences between these two groups of narcotic antagonists, which might explain the differences in their biological activities. The potent stimulatory effect of dynorphin, and other kappa agonists, on feeding behavior suggests that the antagonists that promote weight loss might have high affinity for kappa receptors. The recent demonstration by several laboratories of kappa receptor heterogeneity prompted us to test the hypothesis that the antagonists that promote weight loss might have high affinity for a subtype of kappa binding sites. In the present study, therefore, we determined the Ki values of five PPAs, naloxone, and naltrexone at mu, delta, kappa 1, kappa 2a, and kappa 2b binding sites. The data indicate that antagonists have subnanomolar Ki values and high selectivity for the kappa 2b binding site (relative to the kappa 2a binding site) are efficacious at promoting weight loss.

Anticonvulsant effects of phencyclidine-like drugs: relation to N-methyl-D-aspartic acid antagonism.

Various compounds that have been identified in the literature as binding to the [3H]phencyclidine receptor site and as producing behavioral effects similar to phencyclidine (phencyclidine-like) protected mice from maximal electric shock-induced tonic-extensor seizures. These anticonvulsant effects appear to be due to blockade of the N-methyl-D-aspartic acid receptor, as recently reported for phencyclidine-like compounds. Phencyclidine-like compounds produced their anticonvulsant effects at doses that were also neurologically impairing.

Central administration of the opioid antagonist, LY255582, decreases short- and long-term food intake in rats.

A variety of opioid antagonists have been reported to decrease short-term food intake, but few appear to reduce long-term intake. In the present study we evaluated the effect of a relatively new class of opioid antagonists, 3,4-dimethyl-4-phenylpiperidines, on short-term and long-term food intake after central administration. We also evaluated their affinities for the mu and kappa opioid receptor sites in synaptosomal membranes derived from rat whole brain tissue (minus cerebellum) and guinea-pig cortex, respectively. The affinities for the mu receptor sites were LY255582 greater than LY217273 greater than LY256897 greater than naloxone greater than LY227444. The affinities for the kappa receptor sites were LY255582 greater than LY256897 = LY217273 greater than LY227444. LY255582 reduced food intake for up to 24 h after a single intraventricular injection. Doses as low as 1 microgram of LY255582 decreased food intake for up to 4 h. All other drugs were much less powerful. Naloxone and LY256897 only decreased food intake after injection of the 100 microgram dose. LY227444 and LY217273 failed to decrease intake at all doses tested. LY255582 (100 micrograms) decreased food intake over a 7 day period when injected intraventricularly once per day. The body weight of the rats also decreased during the 7 day period. Upon cessation of drug administration body weights and food intake approached control levels. Thus, LY255582 appears to be a very potent and long-acting anorectic agent which may be useful in the treatment of obesity. The mu and kappa binding profile of the phenylpiperidines does not seem to clearly correlate with their anorectic activity.

N-methyl-D-aspartic acid-induced lethality in mice: selective antagonism by phencyclidine-like drugs.

N-Methyl-D-aspartic acid (NMDA) produced a dose-related increase in lethality in mice, with 200 mg/kg (i.p.) effecting 100% lethality. Upon daily dosing, acutely sublethal doses of NMDA produced deaths. This NMDA-induced lethality was stereoselective; N-methyl-L-aspartic acid had no effects at doses as high as 400 mg/kg. Moderate doses of phencyclidine (PCP) and drugs having PCP-like behavioral effects blocked the NMDA-induced lethality. Other classes of psychoactive drugs, including opioids, anticonvulsants and antipsychotics, were ineffective in preventing NMDA-induced lethality. The potency of PCP-like drugs to block the NMDA-induced lethality correlates highly with the dose necessary to produce PCP-like catalepsy and PCP-like discrimination in pigeons. These data support the hypothesis that PCP-like drugs produce many of their effects by impairing the normal functioning of the NMDA-defined excitatory neurotransmitter receptor in the central nervous system.

Novel phenylpiperidine opioid antagonists and partial agonists: effects on fluid consumption.

The effects of five opioid antagonists, a racemate partial agonist and its agonist and antagonist optical isomers were studied on deprivation-induced drinking. All compounds had a phenylpiperidine nucleus. The antagonists produced dose-related decreases in drinking, and the potencies for decreasing drinking correlated with morphine-antagonist doses. The racemic partial agonist and its agonist isomer decreased drinking at doses higher than those which produced marked analgesia. Within the class of phenylpiperidine drugs studied, some had less specificity than naloxone for the mu-receptors as compared to the delta-receptor, but the suppression of drinking was not related to changes in mu-to-delta ratios.