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Biomedical subjects

R B Rothman

Publications and source records attributed to R B Rothman.

At least 181 records · Page 10Linked to original sources
PubMed1990

Specificity of phencyclidine-like drugs and benzomorphan opiates for two high affinity phencyclidine binding sites in guinea pig brain.

Recently, the presence of two high affinity binding sites for phencyclidine were described in guinea pig brain, with one site coupled to the glutamate excitatory amino acid receptor, specifically activated by N-methyl-D-aspartate (NMDA) (site 1) and the other site associated with the dopamine (DA) reuptake carrier (site 2). Phencyclidine and its analogs, as well as the benzomorphan opiates, are known to interact with binding sites for phencyclidine. In this study, the equilibrium dissociation constants (Kd) of these compounds for the two binding sites for phencyclidine were determined. Phencyclidine and 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP), an analog of PCP, were essentially non-selective between the two sites and also were the two drugs of the group observed to have the highest affinity for site 2. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine [(+)MK801] was the most selective agent for site 1, while none of the drugs tested showed selectivity for site 2. In humans, phencyclidine produces psychotomimetic effects, while (+)MK801 has been reported to produce minimal, if any, psychotomimetic effects, at doses sufficient to reduce seizures. These clinical observations, in conjunction with the present biochemical binding data, suggest that (+)MK801 may serve as a "marker" for site 1 and that the psychotomimetic effects of phencyclidine might be mediated by site 2.

Analgesics↗
PubMed1990

Probes for narcotic receptor mediated phenomena. 17. Synthesis and evaluation of a series of trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacet amide (U50,488) related isothiocyanate derivatives as opioid receptor affinity ligands.

A series of U50,488 related isothiocyanates was synthesized from enantiomerically pure (S,S)-(+)-trans-2-pyrrolidinyl-N-methylcyclohexylamine [(+)-7] and (R,R)-(-)-trans-2-pyrrolidinyl-N-methylcyclohexylamine [(-)-7]. DCC coupling of (+)- and (-)-7 with nitrophenylacetic acids followed by catalytic hydrogenation and treatment with thiophosgene afforded a series of six isomeric aryl isothiocyanate analogues of U50,488. Similarly, DCC coupling of (+)- and (-)-7 with (+)- and (-)-N-t-Boc-protected phenylglycines afforded four isomeric alkyl isothiocyanates. Evaluation of the isothiocyanates for their capacity to produce wash-resistant inhibition of mu, delta, and kappa sites in vitro was performed using rat and guinea pig brain membranes. None of the compounds was able to irreversibly inhibit binding of [3H]bremazocine to guinea pig and rat brain membranes (depleted of functional mu and delta receptors by pretreatment with acylating agents BIT and FIT). However, (1S,2S)-trans-2-isothiocyanato-N-methyl-N-[2- (1-pyrrolidinyl)cyclohexyl]benzeneacetamide [(-)-1] was able to specifically and irreversibly inhibit kappa receptors labeled by [3H]-U69,593: Incubation of rat brain membranes for 60 min at 25 degrees C with 1 microM of (-)-1 resulted in a wash-resistant reduction of the binding to 11.2 +/- 2.5% of the control. Binding analysis revealed the wash-resistant reduction in [3H]-U69,593 binding by (-)-1 to be through an increase in the Kd without effect on the Bmax. (-)-1 failed to effect mu or delta binding in rat or guinea pig brain under the same conditions. The enantiomer of (-)-1, (1R,2R)-trans-2-isothiocyanato-N-methyl-N-[2- (1-pyrrolidinyl)cyclohexyl]benzeneacetamide [(+)-1], failed to affect kappa receptors labeled by [3H]-U69,593 under the same conditions as for (-)-1. (1S,2S)-trans-3-Isothiocyanato-N-methyl-N-[2- (1-pyrrolidinyl)cyclohexyl]benzeneacetamide [(-)-2] inhibited to 49.6 +/- 5.1% of the control, in a wash-resistant manner, kappa receptors labeled by [3H]-U69,593. However, (-)-2 was not as selective as (-)-1 since it also reduced [3H]DADLE (delta) binding to 82.4 +/- 8.0% of the control value. (1S,2S)-trans-4-Isothiocyanato-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide [(-)-3] exhibited selective wash-resistant inhibition of delta receptors labeled by [3H]DADLE resulting in a reduction in binding to 42.9 +/- 4.2% of control.(ABSTRACT TRUNCATED AT 400 WORDS)

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1990

Synthesis and evaluation of N-substituted cis-N-methyl-2-(1-pyrrolidinyl)cyclohexylamines as high affinity sigma receptor ligands. Identification of a new class of highly potent and selective sigma receptor probes.

Certain benzeneacetamides [(-)- and (+)-cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]benzeneacetamide] were recently reported to be potent sigma receptor ligands. In order to determine whether efficacy for the sigma receptor could be improved, a series of compounds related to the benzeneacetamides, N-substituted cis-2-(1-pyrrolidinyl)-N-methylcyclohexylamines, were synthesized and their structure-activity requirements were determined. The compounds were synthesized by starting with the previously reported (+/-)-, 1S,2R-(+)-, and 1R,2S-(-)-cis-2-(1-pyrrolidinyl)-N-methylcyclohexylamines. Analysis of sigma ([3H](+)-3-PPP), kappa ([3H]bremazocine and [3H]U69,593), dopamine-d2 ([3H](-)-sulpiride), and phencyclidine (PCP) ([3H]TCP) receptor binding in guinea pig brain revealed a number of highly potent and selective sigma receptor ligands. Notably, 1S,2R-cis-(-)-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-(2-naphthyl) acetamide [(-)-29] (Ki = 8.66 +/- 0.35 nM), (+/-)-cis-2-amino-4,5-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide [(+/-)-17] (Ki = 11 +/- 3 nM), 1S,2R-(-)-cis-N-methyl-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl ) cyclohexylamine [(-)-44] (Ki = 1.3 +/- 0.3 nM), and 1R,2S-(+)-cis-N-methyl-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl ) cyclohexylamine. [(+)-44] (Ki = 6 +/- 3 nM) exhibited very high affinity at sigma receptors, by displacement of [3H]-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [( 3H]-(+)-3-PPP). These compounds showed insignificant affinity for kappa, dopamine, or PCP receptors, making them valuable tools for the study of sigma receptors. Furthermore, these compounds also exhibited enantioselectivity ranging from 5-fold for (+)- and (-)-44 to 160-fold for (+)- and (-)-29. Several other compounds showed equivalent selectivity but displayed lower sigma receptor affinity.

Animals↗
PubMed1990

Opioid agonist and antagonist antinociceptive properties of [D-Ala2,Leu5,Cys6]enkephalin: selective actions at the deltanoncomplexed site.

The present study used the irreversibly binding enkephalin analog, [D-Ala2,Leu5,Cys6]enkephalin (DALCE) in an effort to determine whether selective agonist and antagonist properties could be demonstrated at hypothesized types of opioid delta receptors previously termed the deltanoncomplexed and the deltacomplexed sites. These putative subtypes of delta receptors have been functionally distinguished on the basis of involvement (i.e., deltacomplexed) in the modulation of mu-mediated effects such as antinociception. Intracerebroventricular administration of DALCE or the reference delta and mu agonists, [D-Pen2,D-Pen5]enkephalin (DPDPE) and morphine, to mice all produced antinociception in the warm-water tail-flick test in a dose- and time-related manner. Maximal effects with DALCE were seen at +10 min and significant antinociception could be detected for approximately 1 hr; DALCE was 3- and 90-fold more potent than i.c.v. morphine and DPDPE, respectively. The antinociceptive effects of i.c.v. DALCE and DPDPE, but not those of morphine, were antagonized by the selective delta antagonist, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH, suggesting that the antinociception associated with the peptides was mediated through a delta receptor. DALCE pretreatment up to 24 hr before testing, a time at which this compound did not produce antinociception, significantly blocked the i.c.v. DPDPE antinociceptive effect as well as that of DALCE itself, but not that of morphine, suggesting long-lasting DALCE antagonism at a delta receptor. Modulation of morphine antinociception was demonstrated with subeffective doses of i.c.v. DPDPE or [Met5]enkephalin, but not with subeffective doses of i.c.v. DALCE.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesics↗
PubMed1989

Tight binding dopamine reuptake inhibitors as cocaine antagonists. A strategy for drug development.

The experiments reported in this study tested the hypothesis that tight binding dopamine (DA) reuptake inhibitors might act as cocaine antagonists. Binding studies demonstrated that the high affinity dopamine reuptake inhibitor, 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine (GBR12909) produced a wash-resistant inhibition of the DA transporter in rat striatal membranes as labeled by [3H]cocaine or [3H]1-[2-(diphenyl- methoxy)ethyl]-4-(3-phenylpropyl)piperazine [( 3H]GBR12935), indicative of tight binding. In vivo microdialysis experiments showed that administration of 25 mg/kg GBR12909 to rats produced a modest, but not statistically significant, increase in the extracellular levels of striatal DA, while this same dose of GBR12909 inhibited the ability of cocaine to elevate extracellular DA levels by 64%. These data suggest that tight binding DA reuptake blockers may provide a fruitful approach for developing a cocaine antagonist.

Animals↗
PubMed1989

Modulation of mu-mediated antinociception by delta agonists: characterization with antagonists.

The functional interactions between supraspinal mu and delta receptors were characterized in the mouse using mu receptor-selective antagonists. The effects of pretreatment with the mu opioid antagonists, beta-funaltrexamine (beta-FNA) and naloxonazine on the modulation of morphine antinociception by the delta agonists [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2,Met5]enkephalinamide (DAMA) were studied. When co-administered in the same i.c.v. injection, a sub-antinociceptive dose of DPDPE consistently and significantly increased the antinociceptive potency of morphine in control animals, while a sub-effective dose of DAMA decreased morphine antinociception; both the respective increase and the decrease of morphine potency by DPDPE and DAMA had been previously shown to be blocked by ICI 174,864, a delta antagonist. Pretreatment of mice with the non-equilibrium mu antagonist beta-FNA 4 h prior to testing, a pretreatment which had no effect on i.c.v. DPDPE or DAMA antinociception, prevented the modulation of morphine antinociception by both DPDPE and DAMA. Pretreatment with the long acting mu 1 antagonist naloxonazine, 24 h prior to testing, failed to affect the modulation of morphine antinociception by either DPDPE or DAMA; such a pretreatment had no effect on the antinociceptive effects of DPDPE or DAMA when given alone. These results provide further support for the concept of a functionally coupled mu-delta receptor complex which is sensitive to antagonism by beta-FNA, but not naloxonazine, and support the notion that subtypes of opioid mu and delta (i.e. complexed and non-complexed) receptors may exist.

Analgesics↗
PubMed1989

Pharmacological activities of optically pure enantiomers of the kappa opioid agonist, U50,488, and its cis diastereomer: evidence for three kappa receptor subtypes.

De Costa et al. (FEBS Lett. 223, 335; 1987) recently described the synthesis of optically pure enantiomers of (+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488). In the present study we examined the in vitro opioid receptor selectivity of (-)-(1S,2S)-U50,488, (+)-(1R,2R)-U50,488 and (+/-)-cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide (the cis diasteromers of U50,488), as well as their pharmacological activities in rhesus monkeys. Using [3H]5 alpha,7 alpha,8 beta-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro (4,5)dec-8-yl]-phenyl-benzeneacetamide ([3H]U69,593) to label kappa binding sites of guinea pig membranes, the apparent dissociation constants of the enantiomers of U50,488 were 0.89 and 299 nM, for the (S,S) and (R,R) enantiomers, respectively. The (-)-cis and (+)-cis diastereomers had apparent Kds of 167 and 2715 nM, respectively. Binding surface analysis of the interaction of (-)-(1S,2S)-U50,488 with kappa binding sites labeled by [3H]bremazocine resolved two binding sites at which (-)-(1S,2S)-U50,488 had Kds of 30 and 10,485 nM, respectively. The (+/-)-cis, (-)-cis and (+)-cis diastereomers of U50,488 (1 microM) did not inhibit [3H]bremazocine binding. Rhesus monkeys were trained to discriminate ethylketocyclazocine (EKC) and saline. All compounds tested substituted completely for EKC. The order of potency was (-)-(1S,2S)-U50,488 greater than (+/-)-U50,488 greater than (+/-)-cis diastereomer of U50,488 greater than (+)-(1R,2R)-U50,488.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1989

Synthesis of an affinity ligand ('UPHIT') for in vivo acylation of the kappa-opioid receptor.

The isothiocyanate analog (1S,2S-trans-2-isothiocyanato-4,5-dichloro-N- methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide, 3a) of the highly selective kappa-opioid receptor agonist, U50,488, was prepared as a potential site-directed affinity ligand for acylation of kappa-opioid receptors in vivo. The isothiocyanate (3a) which we have designated UPHIT and its enantiomer (3b) were synthesized in 3 steps starting from optically pure (1S,2S)-(+)-trans-2-pyrrolidinyl-N-methyl-cyclohexylamine (4a) and its enantiomer (4b), respectively, thus defining their absolute stereochemistry. Binding in vitro of the 1S,2S enantiomer 3a to kappa receptors labelled by [3H]U69,593 was shown to occur with an IC50 value of 25.92 +/- 0.36 nM, whereas 827.42 +/- 5.88 and 115.10 +/- 1.23 nM were obtained for the IC50 value of the 1R,2R enantiomer (3b) and (+/-)-3 respectively. Intracerebroventricular (ICV) injection of 100 micrograms of (+/-)-3 into guinea-pig brain followed by analysis of remaining kappa-binding sites 24 h later revealed that (+/-)-3 depleted 98% of the kappa receptors that bind [3H]U69,593 and 40% of those that bind [3H]bremazocine. These preliminary data suggest exciting uses for these compounds in furthering our knowledge of the kappa-opioid receptor.

Acylation↗
PubMed1989

Cross-linking of [125I]beta-endorphin to mu-opioid receptors during development.

Radioiodinated human beta-endorphin was cross-linked to opioid receptors from rat brain membranes using the bifunctional reagents bis-[2-(succinimidooxycarbonyloxy)ethyl] sulfone (BSCOES) and disuccinimidyl suberate (DSS). Major radiolabeled bands migrated with Mr values of 65,000, 55,000 and 33,000, however the presence of the 55 kDa band was variable. The 65 kDa band was characterized as the mu-receptor: the binding of [125I]beta-endorphin to this band was displaced by mu-selective ligands and blocked by alkylation of the receptor by mu-specific, but not delta-specific alkylating agents. The cross-linked receptor underwent alterations in mol. wt. during development. Early in development, embryonic day 18 and postnatal day 1, the [125I]beta-endorphin-labeled material migrated as a single band of mol. wt. 55 kDa. By day 21 postnatally the higher mol. wt. band of 65 kDa was present, as was material of 53, 47 and 43 kDa. Although the protein labeled early in development migrated with a mol. wt. of 55 kDa similar to the delta-receptor isolated from NG108-15 neuroblastoma-glioma cells, competition studies suggest this protein is not the delta-receptor. The 65 kDa band, tentatively identified as the mu-receptor, was present in adults but not detected in neonates, despite competition binding data indicating the presence of mu-sites. The results suggest that the 55 kDa band found in the 1-day-old neonate may be an immature form of the mu-opioid receptor that undergoes posttranslational modification, perhaps glycosylation, during development.

Alkylation↗