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K C Rice

Publications and source records attributed to K C Rice.

At least 127 records · Page 7Linked to original sources
PubMed1994

Studies of the biogenic amine transporters. 1. Dopamine reuptake blockers inhibit [3H]mazindol binding to the dopamine transporter by a competitive mechanism: preliminary evidence for different binding domains.

The present study addressed the hypothesis that the DA transporter ligand, [3H]mazindol, labels multiple sites/states associated with the dopamine (DA) transporter in striatal membranes. Incubations with [3H]mazindol proceeded for 18-24 hr at 4 degrees C in 55.2 mM sodium phosphate buffer, pH 7.4, with a protease inhibitor cocktail. In order to obtain data suitable for quantitative curve fitting, it was necessary to repurify the [3H]mazindol by HPLC before a series of experiments. Under these conditions, we observed greater than 80% specific binding. The method of binding surface analysis was used to characterize the interaction of GBR12935, BTCP, mazindol, and CFT with binding site/sites labeled by [3H]mazindol. A one site model fit the data as well as the two site model: Bmax = 16911 fmol/mg protein, Kd of [3H]mazindol = 75 nM, Ki of GBR12935 = 8.1 nM, Ki of CFT = 50 nM and Ki of BTCP = 44 nM. The inhibitory mechanism (competitive or noncompetitive) of several drugs (GBR12935, CFT, BTCP, cocaine, cis-flupentixol, nomifensine, WIN35,065-2, bupropion, PCP, and benztropine) was determined. All drugs inhibited [3H]mazindol binding by a competitive mechanism. Although the ligand-selectivity of the [3H]mazindol binding site indicates that it is the uptake inhibitor recognition site of the classic DA transporter, the quantitative differences among the ligand-selectivities of different radioligands for the same site suggest that each radioligand labels different overlapping domains of the DA uptake inhibitor recognition site. It is likely that development of domain-selective drugs may further our understanding of the DA transporter.

Animals↗
PubMed1994

An electrophilic affinity ligand based on (+)-MK801 distinguishes PCP site 1 from PCP site 2.

The electrophilic affinity ligand, (+)-3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycl ohepten-5,10 - imine hydrochloride [(+)-MK801-NCS] was characterized for its ability to acrylate phencyclidine (PCP) and sigma binding sites in vivo. Initial studies, conducted with mouse brain membranes, characterized the binding sites labeled by [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP). The Kd values of [3H]TCP for PCP site 1 (MK801-sensitive) and PCP site 2 (MK801-insensitive) were 12 nM and 68 nM, with Bmax values of 1442 and 734 fmol/mg protein, respectively. Mice were sacrificed 18-24 hours following intracerebroventricular administration of the acylator. The administration of (+)-MK801-NCS increased [3H]TCP binding to site 2, but not to site 1. Although (+)-MK801-NCS decreased [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d; ccyclohepten-5,10-imine maleate ([3H](+)-MK801) binding to site 1, it had no effect on [3H]TCP binding to site 1. Viewed collectively with other published data, these data support the hypothesis that PCP sites 1 and 2 are distinct binding sites, and that [3H]TCP and [3H](+)-MK801 label different domains of the PCP binding site associated with the NMDA receptor.

Affinity Labels↗
PubMed1994

Kappa opioid effects on fetal behavior: central administration of U50,488.

The kappa opioid agonist U50,488 was administered to E21 rat fetuses via intracisternal (IC), intrahemispheric (IH), or intrathecal (IT) injection. The IC administration of U50,488 promoted a threefold increase in motor activity, which was predominated by movements of caudal regions of the body (rearlimbs, body trunk, and tail). The agonist effect was reversed by IC administration of the selective kappa opioid antagonist nor-binaltorphimine. The IH injection of U50,488 had little effect on fetal behavior, but IT injection resulted in pronounced increases in fetal activity, including rearlimb, trunk, and tail movements. The IT administration of nor-binaltorphimine blocked U50,488 effects, whereas IH injection of the antagonist had little influence on fetal behavior. These findings suggest that kappa opioid receptors located in the spinal cord may play a role in the regulation of fetal motor behavior.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1994

Central administration of the endopeptidase 24.15 inhibitor cFP-AAF-pAB suggests dynorphin as the endogenous ligand underlying behavioral effects of milk in the fetal rat.

Intraoral infusion of milk to the rat fetus promotes opioid activity that results in reduced responsiveness in a behavioral bioassay involving perioral cutaneous stimulation. Intracisternal administration of cFP-AAF-pAB, an inhibitor of endopeptidase 24.15, prolonged the opioid activity induced by milk infusion. Treatment with the selective kappa opioid antagonist nor-binaltorphimine blocked the effect of cFP-AAF-pAB on milk-induced opioid activity, but treatment with the mu antagonist CTOP or the delta antagonist naltrindole did not. These findings imply that milk may exert its effect on fetal behavior by increasing levels of dynorphin in the fetal central nervous system.

Amino Acid Sequence↗
PubMed1994

Low affinity of FMRFamide and four FaRPs (FMRFamide-related peptides), including the mammalian-derived FaRPs F-8-Famide (NPFF) and A-18-Famide, for opioid mu, delta, kappa 1, kappa 2a, or kappa 2b receptors.

The binding affinities at opioid receptor subtypes in rat or guinea pig brain membranes were determined for the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2), the two mammalian-derived FMRFamide-related peptides F-8-Famide (NPFF; Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe- NH2) and A-18-Famide (Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe -NH2), and the two other FMRFamide-related peptides Tyr-Phe-Met-Arg-Phe-NH2 (Tyr-FMRFamide) and Pro-Gln-Arg-Phe-NH2 (Pro-Gln-RFamide). The mu and delta sites were labeled in rat brain membranes using tritiated [D-Ala2, N-MePhe4,Gly-ol5] enkephalin ([3H]DAMGO) and [D-Ala2,D-Leu5]enkephalin ([3H]DADLE), respectively. The kappa sites were labeled in guinea pig brain using [3H]U-69,593 after treatment with BIT and FIT for kappa 1 and [3H]bremazocine after pretreatment with BIT and FIT for kappa 1 and [3H]bremazocine after pretreatment with BIT and FIT for kappa 2. The kappa 2a binding sites were assayed using [Leu5]enkephalin to block kappa 2b sites and the kappa 2b sites were assayed using (-)-(1S,2S)-U50,488 to block kappa 2a sites. Neither FMRFamide nor any of the FMRFamide-related peptides (up to 61.0 microM) displayed significant affinity at any of the subtypes of opioid receptor. Hence, the known ability of FMRFamide and FaRPs to interact with the opioid system does not appear to be related to direct binding to these opioid receptors.

Amino Acid Sequence↗
PubMed1994

Studies of the biogenic amine transporters. III. Demonstration of two binding sites for [3H]GBR12935 and [3H]BTCP in rat caudate membranes.

The present study addressed the hypothesis that there exist multiple sites/states associated with the dopamine (DA) transporter ligands. The authors used [3H](1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) (GBR12935) and [3H]-N-(1-(2-benzo[b]thiophenyl)cyclohexyl)piperidine ([3H]BTCP) to label binding sites present in striatal membranes and conducted experiments under nearly identical assay conditions, i.e., 18- to 24-hr incubations at 4 degrees C in 55.2 mM sodium phosphate buffer, pH 7.4, with a protease inhibitor and antioxidant cocktail. To obtain data suitable for quantitative curve fitting, it was necessary to repurify the [3H]ligands periodically by high-performance liquid chromatography. Under these conditions, greater than 90% specific binding was observed. The method of binding surface analysis was used to characterize the interaction of GBR12935, BTCP, mazindol and 2 beta-carbomethoxy-3 beta-(4-fluorophenyl) tropane with binding sites labeled by the [3H]ligands. Nonlinear least-squares curve fitting of the data to one- and two-site binding models demonstrated that, for both [3H]ligands, the two-site model fit the data far better than did the one-site model. The results indicated that [3H]GBR12935 labeled two binding sites, with higher (GBR site 1) and lower affinity (GBR site 2) for BTCP (Ki values of 5.84 nM and 1394 nM). [3H]BTCP labeled two sites with high affinity (Kd values of 9.3 nM and 6.3 nM) at which GBR12935 also had high affinity (Ki values of 8.9 nM and 0.98 nM). Intrastriatal 6-hydroxy-DA lesions decreased the density of both [3H]GBR12935 binding sites but affected site 1 much more than site 2, which indicated that a greater portion of GBR site 1 is localized on striatal nerve terminals. By contrast, the 6-hydroxy-DA lesions decreased BTCP site 2 without significantly decreasing BTCP site 1, which indicated that BTCP site 1 is not located on DA nerve terminals. The i.c.v. administration of 5,7,-dihydroxytryptamine did not decrease GBR site 1 or 2, which indicated that neither is located on serotonin striatal nerve terminals. Viewed collectively with other reports, these data support the hypothesis that DA transporter ligands label multiple binding sites in caudate membranes. The identification of selective agents for these sites may be valuable tools for identifying drugs that might modulate the effects of cocaine.

Animals↗
PubMed1994

Cannabinoid receptor binding and agonist activity of amides and esters of arachidonic acid.

The cannabinoid receptor in brain (CB1) specifically binds delta 9-tetrahydrocannabinol, the predominant central nervous system-active component of marijuana. An eicosanoid found in brain, N-(2-hydroxyethyl)arachidonylamide (anandamide), binds to CB1 with similar affinity. This report considers structure-activity requirements for a series of novel amides and rigid hairpin conformations typified by N-(2-hydroxyethyl)prostaglandin amides, assayed with phenylmethylsulfonyl fluoride inactivation of esterases/amidases. Arachidonyl esters were 30-fold less potent than N-(2-hydroxyethyl)arachidonylamide, showing a rank order of potency of methyl = ethyl > propyl = isopropyl. Within the N-(hydroxyalkyl)arachidonylamide series, a one-carbon increase in chain length increased the potency 2-fold, but continued extension decreased affinity. Substituting the amide for the N-(2-hydroxyethyl)amide function produced a 4-fold loss of affinity. The N-(propyl)-, N-(butyl)-, and N-(benzyl)arachidonylamide derivatives exhibited a 3-fold increase, no change, and a 5-fold decrease, respectively, in affinity, compared with N-(2-hydroxyethyl)arachidonylamide. Both the methoxy ether and the formamide derivatives suffered > 20-fold loss of potency, compared with N-(2-hydroxyethyl)arachidonylamide. N-(2-Aminoethyl)arachidonylamide interacted poorly with CB1. At 100 microM, N-(2-hydroxyethyl)amide analogs of prostaglandin E2, A2, B2, and B1 failed to alter [3H]CP55940 binding to CB1. N-(2-Hydroxyethyl)arachidonylamide inhibited adenylate cyclase with lesser potency but with similar efficacy, compared with desacetyllevonantradol. Extending the length of the hydroxyalkyl moiety by one carbon increased the apparent potency by 1 order of magnitude. The N-(propyl) derivative exhibited a 5-fold greater potency than did the N-(2-hydroxyethyl) analog. It appears that the bulk and length of the moiety appended to arachidonic acid are more important determinants of affinity for CB1 than is hydrogen-bonding capability.

Adenylyl Cyclase Inhibitors↗
PubMed1994

GBR12909 attenuates cocaine-induced activation of mesolimbic dopamine neurons in the rat.

Previous studies have shown that the dopamine (DA) reuptake inhibitor 1-(2-[bis(4-fluorophenyl)-[methoxy]ethyl)-4-(3-phenylpropyl) piperazine (GBR12909) antagonizes the increase in extracellular DA evoked by local perfusion of cocaine into the striatum. In the present work, in vivo microdialysis methods were used to examine the effects of i.v. cocaine, GBR12909 and combinations of the two drugs on DA overflow in the nucleus accumbens of awake rats. Both cocaine and GBR12909 (0.3, 1.0 and 3.0 mg/kg) caused dose-related elevations in extracellular DA when given alone. However, the temporal profile of DA overflow was different with each drug. Cocaine caused a rapid and short-lived increase in DA, whereas GBR12909 caused a slow and sustained elevation of transmitter. In drug combination studies, the rise in extracellular DA after a modest dose of cocaine (1.0 mg/kg) was significantly reduced from 250% to 175% of baseline by pretreatment with a subthreshold dose of GBR12909 (0.3 mg/kg). A high dose of cocaine (3.0 mg/kg) increased dialysate DA by 600%; this rise in DA was decreased to 450% and 325% of baseline by pretreatment with 0.3 and 1.0 mg/kg of GBR12909, respectively. The neurochemical effect of the combination of GBR12909 plus cocaine was clearly not additive. GBR12909 also blocked the DA-releasing action of amphetamine (1.0 mg/kg). Our findings show that GBR12909 antagonizes the rise in extracellular DA produced by systemic cocaine and these results provide further evidence that DA reuptake inhibitors may be useful pharmacological adjuncts in the treatment of cocaine addiction and withdrawal in human patients.

Amphetamine↗
PubMed1994

Studies of the biogenic amine transporters. IV. Demonstration of a multiplicity of binding sites in rat caudate membranes for the cocaine analog [125I]RTI-55.

The drug 3 beta-[4'-iodophenyl]tropan-2 beta-carboxylic acid methyl ester (RTI-55) is a cocaine congener with high affinity for the dopamine transporter (Kd < 1 nM). The present study characterized [125I]RTI-55 binding to membranes prepared from rat, monkey and human caudates and COS cells transiently expressing the cloned rat dopamine (DA) transporter. Using the method of binding surface analysis, two binding sites were resolved in rat caudate: a high-capacity binding site (site 1, Bmax = 11,900 fmol/mg of protein) and a low-capacity site (site 2, Bmax = 846 fmol/mg of protein). The Kd (or Ki) values of selected drugs at the two sites were as follows: (Ki for high-capacity site and Ki for low-capacity site, respectively): RTI-55 (0.76 and 0.21 nM), 1-[2-diphenyl-methoxy)ethyl]-4-(3-phenylpropyl)piperazine (0.79 and 358 nM), mazindol (37.6 and 631 nM), 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (45.0 and 540 nM) and cocaine (341 and 129 nM). Nisoxetine, a selective noradrenergic uptake blocker, had low affinity for both sites. Serotonergic uptake blockers had a high degree of selectivity and high affinity for the low-capacity binding site (Ki of citalopram = 0.38 nM; Ki of paroxetine = 0.033 nM). The i.c.v. administration of 5,7-dihydroxytryptamine to rats pretreated with nomifensine (to protect dopaminergic and noradrenergic nerve terminals) selectively decreased the Bmax of site 2, strongly supporting the idea that site 2 is a binding site on the serotonin (5-HT) transporter. This serotonergic lesion also increased the affinity of [125I]RTI-55 for the DA transporter by 10-fold. The ligand selectivity of the caudate 5-HT transporter was different from the [I125]RTI-55 binding site on the 5-HT transporter present in membranes prepared from whole rat brain minus caudate. The [125I]RTI-55 binding to the DA transporter was further resolved into two components, termed sites 1a and 1b, by using human and monkey (Macaca mulatta) caudate membranes but not the membranes prepared from rat caudate or COS cells that transiently expressed the cloned cocaine-sensitive DA transporter complementary DNA. Similar experiments also resolved two components of the caudate 5-HT transporter. Viewed collectively, these data provide evidence that [125I]RTI-55 labels multiple binding sites associated with the DA and 5-HT transporters.

Animals↗
PubMed1993

Synthesis and binding properties of MK-801 isothiocyanates; (+)-3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten- 5,10-imine hydrochloride: a new, potent and selective electrophilic affinity ligand for the NMDA receptor-coupled phencyclidine binding site.

Three new site-directed irreversible (wash-resistant) ligands for the high-affinity phencyclidine (PCP) binding site associated with the N-methyl-D-aspartate (NMDA) receptor were synthesized and their binding characteristics were studied. (+)-3- And (+)-2-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycl ohepten-5,10 - imine hydrochloride ((+)-8a,b.HCl) were prepared in four steps from the corresponding nitro derivatives (+)-4a,b, which were obtained by nitration of (+)-3 (MK-801). In the same way the optical antipode (-)-8a.HCl was synthesized from (-)-3. At a concentration of 100 nM, the 3-isothiocyanate derivative (+)-8a irreversibly labeled approximately 50% of the (+)-[3H]-3 binding sites, compared to 20 microM needed for its optical antipode (-)-8a and the 2-isothiocyanate (+)-8b. The apparent Ki values for reversible inhibition of (+)-[3H]-3 binding by (+)- and (-)-8a and (+)-8b were 37,838, and 843 nM, respectively. In contrast, metaphit (1b) and etoxadrol m-isothiocyanate (2b), two previously reported irreversible ligands for the PCP binding site, label about 50% of the (+)-[3H]-3 binding sites at 100 microM and 250 nM, respectively, with apparent Ki values for reversible inhibition of 535 and 94 nM. Compound (+)-8a is also a selective affinity ligand, displaying little or no irreversible in vitro affinity at 100 microM for opioid, benzodiazepine, muscarinic, and dopamine receptors. At a 25 microM concentration, (+)-8a caused an irreversible 52% reduction of binding to sigma 1-receptors. Compound (+)-8a is the most potent known electrophilic affinity label for the PCP binding site. Its potency and selectivity should enable it to be a valuable tool for the elucidation of the structure and function of the NMDA receptor-associated PCP binding site in the mammalian central nervous system.

Animals↗
PubMed1993

Synthesis and evaluation of imidazo[1,5-a][1,4]benzodiazepine esters with high affinities and selectivities at "diazepam-insensitive" benzodiazepine receptors.

A series of imidazo[1,5-a][1,4]benzodiazepine esters have been synthesized with varying ester side chains and 8-position substituents. The affinities of these compounds were evaluated at both "diazepam-insensitive" (DI) and diazepam-sensitive (DS) subtypes of the benzodiazepine receptor (BZR). A profound steric effect of the 3-position ester side chain moiety was observed on ligand affinity at DI. In contrast, ester size had a less robust effect on ligand affinity at DS. The tert-butyl ester compound 8 displayed the highest affinity (Ki = 1.7 nM) for DI within a series of 8-chloro esters. Furthermore, halogens at the 8-position resulted in an enhancement of both ligand affinity and selectivity at DI among the series of tert-butyl esters examined. The 8-nitro derivative 23 and 8-isothiocyanato congener 25 had high affinities for both DI and DS but exhibited little subtype selectivity (10.8 and 2.7 nM at DI versus 14 and 3.7 nM at DS, respectively). The 8-azido tert-butyl ester 29 exhibited a significantly higher affinity (Ki = 0.43 nM) and selectivity (DI/DS ratio of 0.2) than the corresponding ethyl ester, the prototypic DI ligand 1 (Ro 15-4513). Among the compounds synthesized, 29 is the highest affinity ligand for DI described to date while its 8-bromo analog 18 is the most selective ligand (DI/DS ratio of 0.17) for this novel BZR subtype.

Animals↗
PubMed1993

Identification of a GBR12935 homolog, LR1111, which is over 4,000-fold selective for the dopamine transporter, relative to serotonin and norepinephrine transporters.

The di-substituted piperazines, GBR12909 (1-[2-[bis(4-fluorophenyl)-methoxy]ethyl]-4-[3- phenylpropyl]piperazine) and GBR12935 (1-[2-(diphenyl-methoxy)-ethyl]-4-(3-phenylpropyl)piperazine), are potent and selective (20-to 100-fold) inhibitors of [3H]dopamine reuptake, relative to [3H]5-HT and [3H]norepinephrine uptake. The GBR12935 analog, 1-(2-(diphenylmethoxy)ethyl)-4-(3-phenylpropyl)homopiperazine (LR1111), was synthesized as part of a systematic structure-activity study of analogs of GBR12935 and GBR12909. LR1111 differs from GBR12935 by the addition of a methylene group into the piperazine ring to yield a compound with a seven-member homopiperazine ring. The IC50 values for LR1111 at the dopamine, norepinephrine, and serotonin transporters were 7.2 nM, 34,072 nM, and greater than 20,000 nM, respectively, whereas the IC50 values of GBR12935 were 3.7 nM, 289 nM, and 1261 nM for these same transporters. This demonstrates that the addition of a single methylene group in the piperazine ring results in a compound with similar affinity but significantly higher selectivity for the dopamine transporter. LR1111 increased motoric activity in rats after intravenous administration. These indicate that LR1111 is a potent and highly selective inhibitor of the dopamine transporter.

Animals↗
PubMed1993

Studies of the biogenic amine transporters. II. A brief study on the use of [3H]DA-uptake-inhibition to transporter-binding-inhibition ratios for the in vitro evaluation of putative cocaine antagonists.

The cocaine receptor on the dopamine transporter is a logical target binding site for the design and synthesis of novel agents for evaluation as possible cocaine antagonists. Although there is no widely accepted and validated assay for detecting a cocaine antagonist, one commonly accepted strategy is to compare the IC50 value of a test agent for inhibition of [3H]dopamine uptake and its IC50 value for inhibition of the binding of a transporter ligand such as [125I]RTI-55. The goal of such a comparison is to guide the synthesis of agents which have high "uptake-to-binding ratios", i.e. agents which are much more potent in the binding assay than they are in the uptake assay. In the present study we tested the hypothesis that ratios different from unity can result from the fact that the two assays are conducted under markedly different conditions. The results showed that conducting the uptake and binding assays under identical conditions reduced the GBR12935 uptake-to-binding ratio of 6.20 (under standard assay conditions) to 0.36. These data indicate that uptake-to-binding ratios must be interpreted with caution, and emphasizes the need for simpler and less expensive methods than cocaine self-administration paradigms to screen compounds as modulators of cocaine reinforcement.

Animals↗
PubMed1993

Comparison of bolus and infusion methods for receptor quantitation: application to [18F]cyclofoxy and positron emission tomography.

Positron emission tomography studies with the opiate antagonist [18F]cyclofoxy ([18F]CF) were performed in baboons. Bolus injection studies demonstrated initial uptake dependent on blood flow. The late uptake showed highest binding in caudate nuclei, amygdala, thalamus, and brainstem and the least accumulation in cerebellum. By 60 min postinjection, regional brain radioactivity cleared at the same rate as metabolite-corrected plasma, i.e., transient equilibrium was achieved. Compartmental modeling methods were applied to time-activity curves from brain and metabolite-corrected plasma. Individual rate constants were estimated with poor precision. The model estimate of the total volume of distribution (VT), representing the ratio of tissue radioactivity to metabolite-corrected plasma at equilibrium, was reliably determined. The apparent volume of distribution (Va), the concentration ratio of tissue to metabolite-corrected plasma during transient equilibrium, was compared with the fitted VT values to determine if single-scan methods could provide accurate receptor measurements. Va significantly overestimated VT and produced artificially high image contrast. These differences were predicted by compartment model theory and were caused by a plasma clearance rate that was close to the slowest tissue clearance rate. To develop a simple method to measure VT, an infusion protocol consisting of bolus plus continuous infusion (B/I) of CF was designed and applied in a separate set of studies. The Va values from the B/I studies agreed with the VT values from both B/I and bolus studies. This infusion approach can produce accurate receptor measurements and has the potential to shorten scan time and simplify the acquisition and processing of scan and blood data.

Algorithms↗
PubMed1993

Cannabinoid structure-activity relationships: correlation of receptor binding and in vivo activities.

Although a receptor exists for cannabinoid drugs, it is uncertain which pharmacological actions this receptor mediates. This structure-activity relationship investigation was initiated to determine which effects might correspond to binding affinity for the cannabinoid receptor, as well as to explore the binding requirements of this site. The ability of nearly 60 cannabinoids to displace [3H]CP-55,940 [(-)-3-[2-hydroxy-4-(1,1-dimethylheptyl) phenyl]-4-[3-hydroxy propyl] cyclohexan-1-ol] was determined before establishing correlations between receptor affinity and in vivo pharmacological potency. Analysis of [3H]CP-55,940 binding indicated a Hill coefficient of 0.97, a Bmax of 499 pM (3.3 pmol/mg of protein) and an apparent Kd of 924 pM. Closer inspection indicated the binding assay exhibited "zone B" characteristics, and use of correction equations indicated a true Kd for CP-55,940 of 675 pM. The structure-activity relationship indicated the importance of side chain structure to high-affinity binding, with the most potent analogs (K1 < 10 nM) possessing either a dimethylheptyl side-chain, a similarly complex branched side chain or a halogen substituent at the 5' position. Comparative analysis of K1 values to in vivo potency in a mouse model indicated a high degree of correlation between parameters for the depression of spontaneous locomotor activity (r = 0.91) and for the production of antinociception (r = 0.90), hypothermia (r = 0.89) and catalepsy (r = 0.85). Similarly high correlations were demonstrated between binding affinity and in vivo potency in both the rat drug discrimination model (r = 0.81) and for psychotomimetic activity in humans (r = 0.88).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Evidence for mutually exclusive binding of cocaine, BTCP, GBR 12935, and dopamine to the dopamine transporter.

The present study addressed the possibility that there are distinct but allosterically interacting populations of binding sites for dopamine/cocaine and BTCP/GBR (N-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine/1-(2-diphenylmethox y) - ethyl]-4-(3-phenylpropyl)piperazine) (selective dopamine uptake blockers) on the dopamine transporter in the rat striatum. Dopamine uptake sites were labeled in vitro with the cocaine analog [3H]CFT (2 beta-carbomethoxy-3 beta-(4-fluorophenyl)-tropane), and the inhibition of binding by CFT or cocaine was measured. A graphic method was adopted for studying shifts in inhibitory potency resulting from the addition of a second compound. Under the conditions used, the co-presence of dopamine, GBR 12935, or BTCP decreased the inhibitory potency of CFT or cocaine to the extent predicted by a model in which all compounds bind to the same site or the binding of all compounds is mutually exclusive. No evidence for negative allosteric interactions between CFT and BTCP was found in experiments comparing inhibition of [3H]CFT binding by BTCP at a low and high concentration of [3H]CFT.

Animals↗
PubMed1992

Probes for narcotic receptor mediated phenomena. 18. Epimeric 6 alpha- and 6 beta-iodo-3,14-dihydroxy-17-(cyclopropylmethyl)-4,5 alpha-epoxymorphinans as potential ligands for opioid receptor single photon emission computed tomography: synthesis, evaluation, and radiochemistry of [125I]-6 beta-iodo-3,14-dihydroxy-17-(cyclopropylmethyl)-4,5 alpha-epoxymorphinan.

The epimeric 6 beta- and 6 alpha-iodo-3,14-dihydroxy-17-(cyclopropylmethyl)-4,5 alpha-epoxymorphinans (1, ioxy) and (2, epioxy), respectively, were each synthesized in five steps starting with naltrexone. The configuration of the 6-iodo group of 1 was unequivocally determined to be beta-based on single crystal X-ray analysis of its precursor 3-acetoxy-6 beta-iodo-14-hydroxy-17-(cyclopropylmethyl)-4,5 alpha-epoxymorphinan (10). Both 1 and 2 as well as their corresponding 3-O-acetates 10 and 11 were found to readily cross the blood-brain barrier and completely reverse the analgesic effects of a 10 mg/kg intraperitoneal dose of morphine sulfate as determined by the paw withdrawal latency test. Compounds 1 and 2 were found to bind with high affinity to mu, delta, and kappa receptors in vitro. In general, 1 and 2 exhibited higher affinity for mu and kappa receptors than naltrexone while the 6 beta-iodo epimer 1 (ioxy) was more potent than its epimer 2. In a comparison of the 6 beta-halogen substituent on binding affinity across opioid receptor subtypes, it was generally found that I greater than Br greater than F. On the basis of the results of in vitro and in vivo testing, 1 was selected as a target for radioiodination and evaluation as a potential single photon emission computed tomography imaging agent for opioid receptors. Carrier-free [125I]-1 was synthesized in near quantitative yield by the sequence of reaction of excess 3-acetoxy-6 alpha-[[(trifluoromethyl)sulfonyl]oxy]-14-hydroxy-17- (cyclopropylmethyl)-4,5 alpha-epoxymorphinan (8) with anhydrous Na125I in dry acetonitrile for 90 min at 76 degrees C followed by deacetylation of the product with 1:1 aqueous ammonia/acetonitrile at 25 degrees C. The potential of [125I]-1 as an in vivo imaging agent for opioid receptors is evaluated and discussed.

Animals↗