Phase I metabolites of mephedrone display biological activity as substrates at monoamine transporters.

BACKGROUND AND PURPOSE: 4-Methyl-N-methylcathinone (mephedrone) is a synthetic stimulant that acts as a substrate-type releaser at transporters for dopamine (DAT), noradrenaline (NET) and 5-HT (SERT). Upon systemic administration, mephedrone is metabolized to several phase I compounds: the N-demethylated metabolite, 4-methylcathinone (nor-mephedrone); the ring-hydroxylated metabolite, 4-hydroxytolylmephedrone (4-OH-mephedrone); and the reduced keto-metabolite, dihydromephedrone. EXPERIMENTAL APPROACH: We used in vitro assays to compare the effects of mephedrone and synthetically prepared metabolites on transporter-mediated uptake and release in HEK293 cells expressing human monoamine transporters and in rat brain synaptosomes. In vivo microdialysis was employed to examine the effects of i.v. metabolite injection (1 and 3 mg·kg(-1) ) on extracellular dopamine and 5-HT levels in rat nucleus accumbens. KEY RESULTS: In cells expressing transporters, mephedrone and its metabolites inhibited uptake, although dihydromephedrone was weak overall. In cells and synaptosomes, nor-mephedrone and 4-OH-mephedrone served as transportable substrates, inducing release via monoamine transporters. When administered to rats, mephedrone and nor-mephedrone produced elevations in extracellular dopamine and 5-HT, whereas 4-OH-mephedrone did not. Mephedrone and nor-mephedrone, but not 4-OH-mephedrone, induced locomotor activity. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that phase I metabolites of mephedrone are transporter substrates (i.e. releasers) at DAT, NET and SERT, but dihydromephedrone is weak in this regard. When administered in vivo, nor-mephedrone increases extracellular dopamine and 5-HT in the brain whereas 4-OH-mephedrone does not, suggesting the latter metabolite does not penetrate the blood-brain barrier. Future studies should examine the pharmacokinetics of nor-mephedrone to determine its possible contribution to the in vivo effects produced by mephedrone.

Structural Modification of the Designer Stimulant α-Pyrrolidinovalerophenone (α-PVP) Influences Potency at Dopamine Transporters.

α-Pyrrolidinovalerophenone (α-PVP, 7) is an illegal synthetic stimulant that is being sold on the clandestine market as "flakka" and "gravel". The potent pharmacological effects of α-PVP are presumably mediated by inhibition of dopamine uptake at the dopamine transporter (DAT). However, little is known about how structural modification of α-PVP influences activity at DAT. Eleven analogs of α-PVP were synthesized and examined for their ability to inhibit uptake of [(3)H]dopamine and [(3)H]serotonin in rat brain synaptosomes. None of the analogs significantly inhibited [(3)H]serotonin uptake when tested at 10 µM at the serotonin transporter (SERT). All of the analogs behaved as DAT reuptake inhibitors, but potencies varied over a >1500-fold range. Potency was primarily associated with the nature of the α-substituent, with the more bulky substituents imparting the highest potency. Expansion of the pyrrolidine ring to a piperidine reduced potency up to 10-fold, whereas conformational constraint in the form of an aminotetralone resulted in the least potent compound. Our study provides the first systematic and comparative structure-activity investigation on the ability of α-PVP analogs to act as inhibitors of DAT.

Stereoselective Actions of Methylenedioxypyrovalerone (MDPV) To Inhibit Dopamine and Norepinephrine Transporters and Facilitate Intracranial Self-Stimulation in Rats.

The designer stimulant methylenedioxypyrovalerone (MDPV) is a potent reuptake inhibitor at transporters for dopamine (DAT) and norepinephrine (NET) that produces a constellation of abuse-related behavioral effects. MDPV possesses a chiral center, and the abused formulation of the drug is a racemic mixture, but no data are available on the pharmacology of its isomers. Here, the individual optical isomers of MDPV were prepared and examined with respect to their neurochemical actions on neurotransmitter reuptake and behavioral effects in an assay of intracranial self-stimulation (ICSS) in rats. In assays of DAT uptake inhibition, S(+)MDPV (EC50 = 2.13 nM) was more potent than either (±)MDPV (EC50 = 4.85 nM) or R(-)MDPV (EC50 = 382.80 nM); the three drugs were less potent at NET uptake inhibition, with the same rank order of potency. Neither racemic MDPV nor its optical isomers inhibited the reuptake of serotonin at concentrations up to 10 µM. S(+)MDPV produced an abuse-related and dose-dependent facilitation of ICSS, and the potency of S(+)MDPV (significant facilitation at doses ≥ 0.1 mg/kg) was greater than that of the racemate (significant facilitation at doses ≥ 0.32 mg/kg). R(-)MDPV failed to alter ICSS at doses up to 100 times greater than the lowest effective dose of S(+)MDPV. The results indicate that abuse-related neurochemical and behavioral effects of racemic MDPV reside primarily with its S(+) isomer.

Quantitative structure-activity relationship analysis of the pharmacology of para-substituted methcathinone analogues.

BACKGROUND AND PURPOSE: Methcathinone (MCAT) is a potent monoamine releaser and parent compound to emerging drugs of abuse including mephedrone (4-CH3 MCAT), the para-methyl analogue of MCAT. This study examined quantitative structure-activity relationships (QSAR) for MCAT and six para-substituted MCAT analogues on (a) in vitro potency to promote monoamine release via dopamine and serotonin transporters (DAT and SERT, respectively), and (b) in vivo modulation of intracranial self-stimulation (ICSS), a behavioural procedure used to evaluate abuse potential. Neurochemical and behavioural effects were correlated with steric (Es ), electronic (σp ) and lipophilic (πp ) parameters of the para substituents. EXPERIMENTAL APPROACH: For neurochemical studies, drug effects on monoamine release through DAT and SERT were evaluated in rat brain synaptosomes. For behavioural studies, drug effects were tested in male Sprague-Dawley rats implanted with electrodes targeting the medial forebrain bundle and trained to lever-press for electrical brain stimulation. KEY RESULTS: MCAT and all six para-substituted analogues increased monoamine release via DAT and SERT and dose- and time-dependently modulated ICSS. In vitro selectivity for DAT versus SERT correlated with in vivo efficacy to produce abuse-related ICSS facilitation. In addition, the Es values of the para substituents correlated with both selectivity for DAT versus SERT and magnitude of ICSS facilitation. CONCLUSIONS AND IMPLICATIONS: Selectivity for DAT versus SERT in vitro is a key determinant of abuse-related ICSS facilitation by these MCAT analogues, and steric aspects of the para substituent of the MCAT scaffold (indicated by Es ) are key determinants of this selectivity.

Steric parameters, molecular modeling and hydropathic interaction analysis of the pharmacology of para-substituted methcathinone analogues.

BACKGROUND AND PURPOSE: There is growing concern over the abuse of certain psychostimulant methcathinone (MCAT) analogues. This study extends an initial quantitative structure-activity relationship (QSAR) investigation that demonstrated important steric considerations of seven 4- (or para-)substituted analogues of MCAT. Specifically, the steric character (Taft's steric ES ) of the 4-position substituent affected in vitro potency to induce monoamine release via dopamine and 5-HT transporters (DAT and SERT) and in vivo modulation of intracranial self-stimulation (ICSS). Here, we have assessed the effects of other steric properties of the 4-position substituents. EXPERIMENTAL APPROACH: Definitive steric parameters that more explicitly focus on the volume, width and length of the MCAT 4-position substituents were assessed. In addition, homology models of human DAT and human SERT based upon the crystallized Drosophila DAT were constructed and docking studies were performed, followed by hydropathic interaction (HINT) analysis of the docking results. KEY RESULTS: The potency of seven MCAT analogues at DAT was negatively correlated with the volume and maximal width of their 4-position substituents, whereas potency at SERT increased as substituent volume and length increased. SERT/DAT selectivity, as well as abuse-related drug effects in the ICSS procedure, also correlated with the same parameters. Docking solutions offered a means of visualizing these findings. CONCLUSIONS AND IMPLICATIONS: These results suggest that steric aspects of the 4-position substituents of MCAT analogues are key determinants of their action and selectivity, and that the hydrophobic nature of these substituents is involved in their potency at SERT.

Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin.

A large body of evidence supports the hypothesis that mesolimbic dopamine (DA) mediates, in animal models, the reinforcing effects of central nervous system stimulants such as cocaine and amphetamine. The role DA plays in mediating amphetamine-type subjective effects of stimulants in humans remains to be established. Both amphetamine and cocaine increase norepinephrine (NE) via stimulation of release and inhibition of reuptake, respectively. If increases in NE mediate amphetamine-type subjective effects of stimulants in humans, then one would predict that stimulant medications that produce amphetamine-type subjective effects in humans should share the ability to increase NE. To test this hypothesis, we determined, using in vitro methods, the neurochemical mechanism of action of amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), (+)-methamphetamine, ephedrine, phentermine, and aminorex. As expected, their rank order of potency for DA release was similar to their rank order of potency in published self-administration studies. Interestingly, the results demonstrated that the most potent effect of these stimulants is to release NE. Importantly, the oral dose of these stimulants, which produce amphetamine-type subjective effects in humans, correlated with the their potency in releasing NE, not DA, and did not decrease plasma prolactin, an effect mediated by DA release. These results suggest that NE may contribute to the amphetamine-type subjective effects of stimulants in humans.

Uptake and release effects of diethylpropion and its metabolites with biogenic amine transporters.

Three metabolites of diethylpropion (1), (+/-)-2-ethylamino-1-phenyl-propan-1-one (2), (1R,2S)-(-)-N,N-diethylnorephedrine (3a) and (1S,2R)-(-)-N,N-diethylnorephedrine (3b) were synthesized. Their uptake and release effects with biogenic amine transporters were evaluated. A major finding of this study is that the in vivo activity of diethylpropion on biogenic amine transporters is most likely due to metabolite 2 as diethylpropion (1) and the metabolites 3a and 3b showed little or no effect in the assays studied. These studies also revealed that 2 acted as a substrate at the norepinephrine (IC50 = 99 nM) and serotonin transporters (IC50 = 2118 nM) and an uptake inhibitor at the dopamine transporter (IC50 = 1014 nM). The potent action of 2 at the NE transporter supports the hypothesis that amphetamine-type subjective effects may be mediated in part by brain norepinephrine.

Methamphetamine dependence: medication development efforts based on the dual deficit model of stimulant addiction.

Converging lines of evidence indicate that withdrawal from prolonged exposure to stimulants and alcohol results in synaptic deficits of both dopamine (DA) and serotonin (5-HT). According to the dual deficit model proposed by the authors, DA dysfunction during cocaine or alcohol withdrawal underlies anhedonia and psychomotor retardation, whereas 5-HT dysfunction gives rise to depressed mood, obsessional thoughts, and lack of impulse control. This model predicts that pharmacotherapies which correct only one of the two neurochemical deficits will not be effective. On the other hand, pharmacotherapies which "correct" both of the proposed DA and 5-HT abnormalities should be effective in treating stimulant and alcohol dependence. This paper reviews two approaches, based on the dual deficit model, taken by our laboratory to develop medications to treat stimulant abuse.

Serotonin transporters, serotonin release, and the mechanism of fenfluramine neurotoxicity.

Administration of d,l-fenfluramine (FEN), or the more active isomer d-fenfluramine (dFEN), causes long-term depletion of forebrain serotonin (5-HT) in animals. The mechanism underlying FEN-induced 5-HT depletion is not known, but appears to involve 5-HT transporters (SERTs) in the brain. Some investigators have postulated that 5-HT release evoked by FEN is responsible for the deleterious effects of the drug. In the present work, we sought to examine the relationship between drug-induced 5-HT release and long-term 5-HT depletion. The acute 5-HT-releasing effects of dFEN and the non-amphetamine 5-HT agonist 1-(m-chlorophenyl)piperazine (mCPP) were evaluated using in vivo microdialysis in rat nucleus accumbens. The ability of dFEN and mCPP to interact with SERTs was assessed using in vitro assays for [3H]-transmitter uptake and release in rat forebrain synaptosomes. Drugs were subsequently tested for potential long-lasting effects on brain tissue 5-HT after repeated dosing (2.7 or 8.1 mg/kg, ip x 4). dFEN and mCPP were essentially equipotent in their ability to stimulate acute 5-HT release in vivo and in vitro. Both drugs produced very selective effects on 5-HT with minimal effects on dopamine. Interestingly, when dFEN or mCPP was administered repeatedly, only dFEN caused long-term 5-HT depletion in the forebrain at 2 weeks later. These data suggest that acute 5-HT release per se does not mediate the long-term 5-HT depletion associated with dFEN. We hypothesize that dFEN and other amphetamine-type releasers gain entrance into 5-HT neurons via interaction with SERTs. Once internalized in nerve terminals, drugs accumulate to high concentrations, causing damage to cells. The relevance of this hypothesis for explaining clinical side effects of FEN and dFEN, such as cardiac valvulopathy and primary pulmonary hypertension, warrants further study.

Neurochemical neutralization of methamphetamine with high-affinity nonselective inhibitors of biogenic amine transporters: a pharmacological strategy for treating stimulant abuse.

The abuse of methamphetamine (METH) and other amphetamine-like stimulants is a growing problem in the United States. METH is a substrate for the 12-transmembrane proteins which function as transporters for the biogenic amines dopamine (DA), serotonin (5-HT), and norepinephrine (NE). Increased release of CNS DA is thought to mediate the addictive effects of METH, whereas increased release of NE in both the peripheral and CNS is thought to mediate its cardiovascular effects. The neurotoxic effects of METH on both dopaminergic and serotonergic nerves requires the transport of METH into the nerve terminals. Thus, transport of METH into nerve terminals is the crucial first step in the production of METH-associated pharmacological and toxicological effects. A single molecular entity which would block the transport of METH at all three biogenic amine transporters might function to neurochemically neutralize METH. This agent would ideally be a high-affinity slowly dissociating agent at all three transporters, and also be amenable to formulation as a long-acting depot medication, such as has been accomplished with an analog of GBR12909. As a first step towards developing such an agent, we established an in vitro assay which selectively detects transporter substrates and used this assay to profile the ability of a lead compound, indatraline, to block the releasing effects of METH and MDMA at the DA, 5-HT, and NE transporters. The major finding reported here is that indatraline blocks the ability of METH and MDMA to release these neurotransmitters. Synapse 35:222-227, 2000. Published 2000 Wiley-Liss, Inc.

Neurochemical neutralization of amphetamine-type stimulants in rat brain by the indatraline analog (-)-HY038.

Amphetamine-type stimulants are substrates for the proteins that serve as transporters for the biogenic amines dopamine (DA), serotonin (5HT), and norepinephrine (NE) and release these neurotransmitters from neurons located in the peripheral and central nervous system. Using indatraline as a lead compound, we sought to develop a long-acting depot medication that would neutralize the deleterious effects of amphetamine-type stimulants. Our first efforts produced (+/-)-HY038, and its two stereoisomers, which are hydroxy-substituted analog of indatraline. The K(i) values for [(3)H]DA reuptake inhibition by (-)-HY038 and (+)-HY038 were 3.2 +/- 0.1 and 32 +/- 1 nM. Similar results were obtained for [(3)H]5HT reuptake inhibition. (-)-HY038 and (+)-HY038 were slightly less potent at inhibiting [(3)H]NE reuptake (K(i) values of 20 +/- 2 and 159 +/- 12 nM). Low doses of (-)-HY038 blunted the ability of AMPH to release [(3)H]DA by shifting the AMPH dose-response curve to the right in a dose-dependent manner. (-)-HY038 also inhibited the ability of (+)-methamphetamine and (+/-)-3,4-methylenedioxymethamphetamine ((+/-)-MDMA) to release [(3)H]DA. Low doses of (-)-HY038 blunted the ability of these stimulants to release [(3)H]NE and [(3)H]5HT by shifting their dose-response curves to the right in a manner similar to that seen for inhibition of [(3)H]DA release. These data indicate that (-)-HY038 inhibits the ability of AMPH, (+)-methamphetamine and (+/-)-MDMA to release DA, NE, and 5HT and therefore might have the potential to neutralize the neurotoxic and cardiovascular side-effects of substrate-type stimulants.

Opioid peptide receptor studies. 12. Buprenorphine is a potent and selective mu/kappa antagonist in the [35S]-GTP-gamma-S functional binding assay.

We utilized the [(35)S]-GTP-gamma-S functional binding assay to determine the selectivity of opioid receptor agonists in guinea pig caudate membranes. The study focused on two opioid agonists used for treating opioid-dependent patients: methadone and buprenorphine. Selective antagonists were used to generate agonist-selective conditions: TIPP + nor-BNI to measure mu receptors, CTAP + nor-BNI to measure gamma receptors and TIPP + CTAP to measure kappa receptors. The assay was first validated with opioid agonists of known subtype specificity (DAMGO for mu, SNC80 for delta, and U69, 593 for kappa receptors). Methadone-stimulated [(35)S]-GTP-gamma-S binding was mu-specific and less potent and efficacious than etorphine (K(d) = 1,537 nM vs. K(d) = 7.8 nM). Buprenorphine failed to stimulate [(35)S]-GTP-gamma-S binding but inhibited agonist-stimulated [(35)S]-GTP-gamma-S binding. The antagonist-K(i) values (nM) of buprenorphine at mu, delta, and kappa receptors were 0.088 nM, 1.15 nM, and 0.072 nM, respectively. The antagonist-K(i) values (nM) of naloxone at mu, delta, and kappa receptors were 1.39 nM, 25.0 nM, and 11.4 nM, respectively. Autoradiographic studies showed that buprenorphine failed to stimulate [(35)S]-GTP-gamma-S binding in caudate-level rat brain sections but blocked DAMGO-stimulated [(35)S]-GTP-gamma-S binding. In cells expressing the cloned rat mu receptor, buprenorphine was a partial agonist and potent mu antagonist. Administration of buprenorphine to rats produced a long-lasting (>24 h) decrease in mu and kappa2 receptor binding and attenuated mu-stimulated [(35)S]-GTP-gamma-S binding. Viewed collectively, these data indicate that, in this assay system, buprenorphine is a potent mu and gamma receptor antagonist. The clinical implications remain to be elucidated. Synapse 34:83-94, 1999. Published 1999 Wiley-Liss, Inc.

Region-specific up-regulation of opioid receptor binding in enkephalin knockout mice.

Mu and delta opioid receptors were labeled in enkephalin knockout mice by quantitative autoradiography. Discrete, large increases (100-300%) were found in limbic forebrain structures for mu binding and striatum and pallidum for delta binding. The up-regulation of opioid receptors may reflect a form of 'denervation supersensitivity. ' The receptor up-regulation in limbic areas is consistent with the increased emotional and aggressive behaviors observed in the enkephalin knockout mice.

Opioid peptide receptor studies, 11: involvement of Tyr148, Trp318 and His319 of the rat mu-opioid receptor in binding of mu-selective ligands.

Previous data obtained with the cloned rat mu opioid receptor demonstrated that the "super-potent" opiates, ohmefentanyl (RTI-4614-4) and its four enantiomers, differ in binding affinity, potency, efficacy, and intrinsic efficacy. Molecular modeling (Tang et al., 1996) of fentanyl derivatives binding to the mu receptor suggests that Asp147, Tyr148, Trp318, and His319 are important residues for binding. According to this model, Asp147 interacts with the positively charged opiate agonist to form potent electrostatic and hydrogen-bonding interactions. In this study, the role of weak electrostatic and hydrogen-bonding "pi-pi" interactions of the O atom of the carbonyl group and the phenyl ring structures of RTI-4614-4 and its four enantiomers with residues Tyr148, Trp318, and His319 were explored via site-directed mutagenesis. Tyr148 (in transmembrane helix 3 {TMH3}), Trp318 (TMH7), and His319 (TMH7) were individually replaced with phenylalanine or alanine. Receptors transiently expressed in COS-7 cells were labeled with [125I]IOXY according to published procedures. Mutation of Tyr148 to phenylalanine reduced the binding affinities of some mu-selective agonists (2-7 fold) but did not alter the affinities of DAMGO, naloxone, and the non-selective opiates etorphine and buprenorphine. In contrast, this mutation significantly increased the binding affinities (decreased the Kd values) of [D-Ala2,D-Leu5]enkephalin, IOXY, and dermorphin. Mutation of Trp318 decreased opioid receptor binding to almost undetectable levels. Substitution of alanine for His319 significantly reduced binding affinities for the opioid ligands tested (1.3- to 48-fold), but did not alter the affinities of naloxone and bremazocine. These results indicate the importance of Tyrl48 and His319 for the binding of fentanyl derivatives to the mu receptor. Functional studies using the mutant receptors will provide additional insight into the mechanism of action of RTI-4614-4 and its four enantiomers.

Chronic exposure to antibodies directed against anti-opiate peptides alter delta-opioid receptor levels.

The development of addictive states in response to chronic opioid use may be regulated partially by the release of endogenous peptides. These anti-opiate peptides (AOP) are secreted or released into the CNS and produce diverse actions that counterbalance the effects of prolonged opiate exposure. Though the mechanism(s) by which these peptides exert their physiological properties remain largely unknown, there is some indication that AOP's modulate opioid receptor levels. In this study, we investigated the effects of chronically infused alpha-melanocyte stimulating hormone (alpha-MSH), dynorphin(1-8) (DYN(1-8)), dynorphin A (DYNA), and NPFF antibodies on delta-opioid receptor expression in rat brains. Quantitative autoradiographic experiments revealed that antibodies directed against alpha-MSH and DYNA produced significant increases in delta receptor levels in the caudate, claustrum, and cingulate cortex of the rat brain. Conversely, NPFF monoclonal antibodies caused significant decreases in the caudate, nucleus accumbens, olfactory tubercle, and cingulate cortex. These results suggest that the density of delta-opioid receptors is affected by changes in the levels of the anti-opioid peptides in the extracelluar fluid in the rat brain.

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.

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.

Opioid peptide receptor studies. 9. Identification of a novel non-mu- non-delta-like opioid peptide binding site in rat brain.

Quantitative binding studies resolved two high-affinity [3H][D-Ala2,D-Leu5]enkephalin binding sites in rat brain membranes depleted of mu binding sites by pretreatment with the irreversible agent BIT. The two binding sites had lower (delta ncx-2, Ki = 96.6 nM) and higher (delta ncx-1, Ki = 1.55 nM) affinity for DPDPE. The ligand-selectivity profile of the delta ncx-1 site was that of a classic delta binding site. The ligand-selectivity profile of the delta ncx-2 site was neither mu- or delta-like. The Ki values of selected agents for the delta ncx-2 site were: [pCl]DPDPE (3.9 nM), DPLPE (140 nM), and DAMGO (2.6 nM). Under these assay conditions, [3H][D-Ala2,D-Leu5]enkephalin binding to the cells expressing the cloned mu receptor is very low and pretreatment of cell membranes with BIT almost completely inhibits [3H]DAMGO and [3H][D-Ala2,D-Leu5]enkephalin binding. Intracerebroventricular administration of antisense DNA to the cloned delta receptor selectively decreased [3H][D-Ala2,D-Leu5]enkephalin binding to the delta ncx-1 site. Administration of buprenorphine to rats 24 h prior to preparation of membranes differentially affected mu, delta ncx-1, and delta ncx-2 binding sites. Viewed collectively, these studies have identified a novel non-mu- non-delta-like binding site in rat brain.

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.

Opioid peptide receptor studies. 8. One of the mouse brain deltaNCX binding sites is similar to the cloned mouse opioid delta receptor: further evidence for heterogeneity of delta opioid receptors.

Quantitative ligand binding studies resolved two subtypes of the delta opioid receptor, termed delta(ncx1) and delta(ncx2), in mouse brain membranes depleted of mu receptors by pretreatment with the irreversible ligand, BIT. The purpose of the present study was to compare the binding parameters, ligand-selectivity profile and pharmacological properties of the cloned mouse delta receptor (MDOR) stably expressed in a cell line to the delta(ncx) binding sites of mouse brain. [3H][D-Ala2,D-Leu5]enkephalin labeled a single binding site in membranes prepared from MDOR cells under several different assay conditions including BIT-pretreatment. The MDOR had high affinity for delta agonists and antagonists. [3H][D-Ala2,D-Leu5]enkephalin labeled two binding sites in mouse brain membranes depleted of mu receptors by pretreatment with BIT: the delta(ncx1) site (high affinity for DPDPE and deltorphin) and the delta(ncx2) site (low affinity for DPDPE and deltorphin). Some agents were moderately selective for the delta(ncx2) site: [pCl]DPDPE (10.9-fold), JP41 (5.9-fold) and JP45 (3.8-fold). The Ki values of 12 opioids at the mouse MDOR were determined. These values were highly correlated with their values at the delta(ncx1) site but not the delta(ncx2) site. These data suggest that the delta(ncx2) site may be distinct from the cloned delta opioid receptor.