DTG Binding Research Articles

N-Substituted-3-alkoxy-derivatives of dextromethorphan are functional NMDA receptor antagonists in vivo: Evidence from an NMDA-induced seizure model in rats

Interest in developing NMDA receptor antagonists with reduced side-effects for neurological and psychiatric disorders has been re-energized by the recent introduction of esketamine into clinical practice for treatment-resistant depression. Structural analogs of dextromethorphan bind with low affinity to the NMDA receptor ion channel, have functional effects in vivo, and generally display a lower propensity for side-effects than that of ketamine and other higher affinity antagonists. As such, the aim of the present study was to determine whether a series of N-substituted-3-alkoxy-substituted dextromethorphan analogs produce their anticonvulsant effects through NMDA receptor blockade. Compounds were studied against NMDA-induced seizures in rats. Compounds were administered intracerebroventricularly in order to mitigate confounds of drug metabolism that arise from systemic administration. Comparison of the anticonvulsant potencies to their affinities for NMDA, σ1, and σ2 binding sites were made in order to evaluate the contribution of these receptors to anticonvulsant efficacy. The potencies to block convulsions were positively associated with their affinities to bind to the NMDA receptor ion channel ([3H]-TCP binding) (r = 0.71, p < 0.05) but not to σ1 receptors ([3H]-SKF 10047 binding) (r = −0.31, p = 0.46) or to σ2 receptors ([3H]-DTG binding) (p = −0.38, p = 0.36). This is the first report demonstrating that these dextromethorphan analogs are functional NMDA receptor antagonists in vivo. Given their potential therapeutic utility and favorable side-effect profiles, such low affinity NMDA receptor antagonists could be considered for further development in neurological (e.g., anticonvulsant) and psychiatric (e.g., antidepressant) disorders.

The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes

The sigma-2 receptor (S2R) is a potential therapeutic target for cancer and neuronal diseases. However, the identity of the S2R has remained a matter of debate. Historically, the S2R has been defined as (1) a binding site with high affinity to 1,3-di-o-tolylguanidine (DTG) and haloperidol but not to the selective sigma-1 receptor ligand (+)-pentazocine, and (2) a protein of 18–21kDa, as shown by specific photolabeling with [3H]-Azido-DTG and [125I]-iodoazido-fenpropimorph ([125I]-IAF). Recently, the progesterone receptor membrane component 1 (PGRMC1), a 25kDa protein, was reported to be the S2R (Nature Communications, 2011, 2:380). To confirm this identification, we created PGRMC1 knockout NSC34 cell lines using the CRISPR/Cas9 technology. We found that in NSC34 cells devoid of or overexpressing PGRMC1, the maximum [3H]-DTG binding to the S2R (Bmax) as well as the DTG-protectable [125I]-IAF photolabeling of the S2R were similar to those of wild-type control cells. Furthermore, the affinities of DTG and haloperidol for PGRMC1 (KI=472μM and 350μM, respectively), as determined in competition with [3H]-progesterone, were more than 3 orders of magnitude lower than those reported for the S2R (20–80nM). These results clarify that PGRMC1 and the S2R are distinct binding sites expressed by different genes.

THE 18 kDa SIGMA‐2 Receptor and PGRMC1 are Derived From Separate Genes

The sigma‐2 receptor (S2R) is a unique binding site expressed in many mammalian tissues. Due to high expression in tumors, the S2R is a promising target for cancer diagnostic imaging. However, the sequence of the S2R has remained a matter of debate. Historically the identity of S2R has been determined by these criteria: (1) the S2R binds with high affinity to 1,3‐di‐o‐tolylguanidine (DTG) and haloperidol but not the selective sigma‐1 receptor ligand, (+)‐pentazocine; (2) specific photolabeling with [3H]Azido‐DTG, and [125I]‐iodoazido‐fenpropimorph ([125I]IAF) has shown the apparent molecular size of S2R to be 18‐21kDa. The progesterone receptor membrane component 1 (PGRMC1), a 25 kDa protein, has been recently reported to be the S2R. To establish the identity of the S2R, we created a PGRMC1 knockout NSC34 cell line using CRISPR/Cas9 technology. We found that in NSC34 cells devoid of PGRMC1, the maximum binding (Bmax) of haloperidol protectable [3H]‐DTG binding was similar to that of wild‐type control cells. Moreover, in both control and PGRMC1‐KO cells, [125I]IAF selectively photolabeled a DTG protectable S2R‐18kDa protein. A series of previously reported highly selective S2R compounds showed protection of [125I]‐IAF photolabeling of the S2R‐18kDa in PC12 cell membranes that contain both PGRMC1 and S2R‐18kDa. These results support the conclusion that PGRMC1 and S2R‐18kDa are separate proteins derived from two different genes. Cloning and characterization of the S2R‐18kDa will provide further clarification.

ChemInform Abstract: 3,4-Dihydro-2(1H)-quinolinone as a Novel Antidepressant Drug: Synthesis and Pharmacology of 1-[3-[4-(3-Chlorophenyl)-1-piperazinyl]propyl] -3,4-dihydro-5-methoxy-2(1H)-quinolinone and Its Derivatives.

To develop a novel antidepressant drug with central nervous system-stimulating activity, we prepared a series of 1-[omega-(4-substituted phenyl-1-piperazinyl)alkyl]-3, 4-dihydro-2(1H)-quinolinone derivatives and examined their activities by their effects at 30 and 100 mg/kg po on the sleeping time of mice anesthetized with halothane and on the time required for recovery from coma induced in mice by cerebral concussion. We examined their binding affinities for sigma receptors by evaluating their ability to inhibit [(3)H]-1,3-di(o-tolyl)guanidine ([(3)H]DTG) binding to the rat whole brain membrane in comparison with three putative sigma receptor agonists: 1,3-di(o-tolyl)guanidine (DTG, 66), (+)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2, 6-methano-3-benzazecin-8-ol (SKF10,047, 67), and (+)-1,2,3,4,5, 6-hexahydro-6,11-dimethyl-3-(3-methyl-2-butenyl)-2, 6-methano-3-benzazecin-8-ol (pentazocine, 68). Among the series of derivatives, 1-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-3, 4-dihydro-5-methoxy-2(1H)-quinolinone hydrochloride (34b) and its mesylate (34c), at a dose of 30 mg/kg po, reduced the sleeping time and the time for recovery from coma and they inhibited [(3)H]DTG binding for sigma receptors. The putative sigma receptor agonists reduced the sleeping time and the time for recovery from coma whereas two sigma receptor antagonists, alpha-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazinebutanol hydrochloride (BMY14802, 69) and cis-9-[3-(3, 5-dimethyl-1-piperazinyl)propyl]carbazole dihydrochloride (rimcazole, 70), were inactive in the two tests. Preadministration of the putative sigma receptor antagonists 69 (3 mg/kg po) and 70 (30 mg/kg po) completely antagonized the actions of 34b and the sigma receptor agonists in the test for recovery from coma. These results suggested that 34b and 34c are sigma receptor agonists. Furthermore, a single administration of 1 and 10 mg/kg po 34b and 34c showed antidepressant-like activity by reducing the immobility time in the forced-swimming test with mice, while a tricyclic antidepressant, 10, 11-dihydro-N,N-dimethyl-5H-dibenz[b,f]azepine-5-propanamine hydrochloride (imipramine, 1) (10 and 30 mg/kg po), did not reduce the time after a single administration. 1 reduced the time after repeated administration of 30 mg/kg po once a day for 4 days. The structure-activity relationship of the series of compounds is also discussed.

σ1 and σ2 receptor binding affinity and selectivity of SA4503 and fluoroethyl SA4503

SA4503, a potent sigma(1) receptor agonist, is reported as having 103-fold higher affinity for sigma(1) (IC(50) = 17.4 nM) than sigma(2) (IC(50) = 1,784 nM) sites in guinea pig brain membranes. Modest structural changes appear to have major effects on sigma(1)/sigma(2) selectivity. The fluoroethyl analog, FE-SA4503, is described as having high primary affinity for sigma(2) sites (IC(50) = 2.11 nM) and a weaker interaction with sigma(1) sites (IC(50) = 6.48 nM). SA4503 and FE-SA4503 have been radiolabeled for PET studies, and both bind selectively to sigma(1) receptors in animal and human brain in vivo. We prepared SA4503 and FE-SA4503 as reference compounds for radioligand development efforts. In our hands, SA4503 is 14-fold selective for sigma(1) (K(i) = 4.6 nM) over sigma(2) (K(i) = 63.1 nM) sites in guinea pig brain homogenates. Further, FE-SA4503 exhibits the same 14-fold selectivity for sigma(1) (K(i) = 8.0 nM) over sigma(2) (K(i) = 113.2 nM) receptors. The main differences from previously reported values stem from sigma(2) affinity determinations. This protocol, displacement of [(3)H]DTG binding to sigma(2) sites using (+)-pentazocine (200 nM) to mask sigma(1) sites, was validated by the proper rank order of sigma(2) inhibitory potencies shown by a panel of additional ligands: ifenprodil > haloperidol > DTG >> (+)-pentazocine. Robust Pearson correlation (r = 1.0, P = 0.002; slope = 0.97) was observed for our pK(i) values against those from a prior study by others. The findings have bearing on structure-activity relationships for this active series, and on conclusions that might be drawn from experiments relying upon defined sigma(1)/sigma(2) binding selectivity.

Synthesis of (+)-cis-N-(4-isothiocyanatobenzyl)-N-normetazocine, an isothiocyanate derivative of N-benzylnormetazocine as acylant agent for the sigma(1) receptor.

(+)-cis-N-(4-Isothiocyanatobenzyl)-N-normetazocine (BNIT) (+)-(4) was designed and synthesized as a derivative of the potent and selective sigma(1) receptor ligand (+)-cis-N-benzyl-N-normetazocine for irreversibly blocking sigma(1) binding sites. Pretreatment of guinea pig brain membranes with BNIT (0.1, 1, and 5 microM) caused a concentration-dependent loss of binding of the selective sigma(1) ligand [(3)H]-(+)-pentazocine. Binding experiments with [(3)H]-1,3-di(2-tolyl)guanidine ([(3)H]-DTG), a ligand of sigma(1) and sigma(2) receptors, showed that pretreatment with BNIT blocked only the sigma(1) component of [(3)H]-DTG binding.

Repeated haloperidol treatment decreases σ 1 receptor binding but does not affect its mRNA levels in the guinea pig or rat brain

The effects of chronic treatment with haloperidol on sigma (σ) receptors were investigated across brain regions and species. The regional distribution of [ 3H](+)-pentazocine binding to σ 1 receptor was similar between the guinea pig and rat brains. The highest level of binding was detected in the brain stem and lowest in the striatum and hippocampus. The regional distribution of [ 3H]1,3-di (2-tolyl) guanidine ([ 3H]DTG) binding in the presence of 100 nM (+)-pentazocine to σ 2 receptor was similar to that of the [ 3H](+)-pentazocine binding in the guinea pig brain, while in the rat brain high levels of [ 3H]DTG binding were detected in the cortex, frontal cortex and cerebellum. The intraperitoneal administration of 2 mg/kg of haloperidol to guinea pig and rats once a day for 21 days produced inhibition of [ 3H](+)-pentazocine binding but did not affect [ 3H]DTG binding to σ 2 receptors in any brain region examined. The effects of haloperidol on [ 3H](+)-penazocine binding in the rat were much weaker than those in the guinea pig. The regional distribution of the level of σ 1 receptor mRNA determined by the ribonuclease protection assay was similar to that of the [ 3H](+)-pentazocine binding activity, except in the cortex and cerebellum where the levels of σ 1 receptor mRNA were low in guinea pig and rat. Treatment with haloperidol did not affect the levels of σ 1 receptor mRNA in any brain region in either species. These findings suggested that the σ receptors differentially distributed in brain regions are down regulated by treatment with haloperidol across σ receptor subtypes and animal species without changing the transcriptional activity of the σ 1 receptor. The mechanisms by which σ receptors could be differently regulated in vivo by chronic treatment with haloperidol in different species may contribute to the therapeutic efficacy of haloperidol.

3,4-dihydro-2(1H)-quinolinone as a novel antidepressant drug: synthesis and pharmacology of 1-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-3,4- dihydro-5-methoxy-2(1H)-quinolinone and its derivatives.

To develop a novel antidepressant drug with central nervous system-stimulating activity, we prepared a series of 1-[omega-(4-substituted phenyl-1-piperazinyl)alkyl]-3, 4-dihydro-2(1H)-quinolinone derivatives and examined their activities by their effects at 30 and 100 mg/kg po on the sleeping time of mice anesthetized with halothane and on the time required for recovery from coma induced in mice by cerebral concussion. We examined their binding affinities for sigma receptors by evaluating their ability to inhibit [(3)H]-1,3-di(o-tolyl)guanidine ([(3)H]DTG) binding to the rat whole brain membrane in comparison with three putative sigma receptor agonists: 1,3-di(o-tolyl)guanidine (DTG, 66), (+)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2, 6-methano-3-benzazecin-8-ol (SKF10,047, 67), and (+)-1,2,3,4,5, 6-hexahydro-6,11-dimethyl-3-(3-methyl-2-butenyl)-2, 6-methano-3-benzazecin-8-ol (pentazocine, 68). Among the series of derivatives, 1-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-3, 4-dihydro-5-methoxy-2(1H)-quinolinone hydrochloride (34b) and its mesylate (34c), at a dose of 30 mg/kg po, reduced the sleeping time and the time for recovery from coma and they inhibited [(3)H]DTG binding for sigma receptors. The putative sigma receptor agonists reduced the sleeping time and the time for recovery from coma whereas two sigma receptor antagonists, alpha-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazinebutanol hydrochloride (BMY14802, 69) and cis-9-[3-(3, 5-dimethyl-1-piperazinyl)propyl]carbazole dihydrochloride (rimcazole, 70), were inactive in the two tests. Preadministration of the putative sigma receptor antagonists 69 (3 mg/kg po) and 70 (30 mg/kg po) completely antagonized the actions of 34b and the sigma receptor agonists in the test for recovery from coma. These results suggested that 34b and 34c are sigma receptor agonists. Furthermore, a single administration of 1 and 10 mg/kg po 34b and 34c showed antidepressant-like activity by reducing the immobility time in the forced-swimming test with mice, while a tricyclic antidepressant, 10, 11-dihydro-N,N-dimethyl-5H-dibenz[b,f]azepine-5-propanamine hydrochloride (imipramine, 1) (10 and 30 mg/kg po), did not reduce the time after a single administration. 1 reduced the time after repeated administration of 30 mg/kg po once a day for 4 days. The structure-activity relationship of the series of compounds is also discussed.

Up-regulation of [formula omitted]DTG but not [formula omitted](+)-pentazocine labeled σ sites in mouse spinal cord by chronic morphine treatment

To monitor the possible effect of morphine on σ sites, binding characteristics of [ 3 H] (+)-pentazocine and [ 3 H] 1,3-di-(2-tolyl)guanidine (DTG) to brain and spinal cord membranes of morphine-treated and control mice were compared. For morphine treatment, a single injection (100 mg/kg, s.c.) of morphine was followed 4 h later by pellet implantation (75 mg morphine free base). Animals were sacrificed 24, 72 h or 7 days later. The equilibrium dissociation value ( K d) and the density ( B max) of [ 3 H] (+)-pentazocine binding remained unaffected by morphine treatment. Also, no change was found in K d and B max values of [ 3 H] DTG labeled σ 2 subtypes after any morphine treatment schedule when measured in the presence of 100 nM (+)-pentazocine. However, the B max of [ 3 H] DTG binding in the spinal cord in the absence of 100 nM (+)-pentazocine, was significantly elevated 72 h after implantation of the morphine pellet and recovered by 7 days, a time when the antinociceptive effect produced by the morphine pellet had dissipated. These data suggest that one population of σ sites, that has a high affinity for [ 3 H] DTG, but is not equivalent with the [ 3 H] (+)-pentozocine labeled σ 1 subtype or the [ 3 H] DTG labeled σ 2 subtype, is upregulated by morphine and, therefore, may play a role in the development of tolerance to or dependence on the effects of morphine.

Uncompetitive inhibition of [ [formula omitted]]1,3-di- o-tolyl-guanidine-defined σ binding sites by desipramine, propranolol and alprenolol in rat brain

Desipramine, imipramine, clomipramine, (−)-propranolol, (−)-alprenolol, (±)-pentazocine and risperidone caused a concentration-dependent inhibition of 6 nM [ 3 H ]DTG (1,3-di- o-tolylguanidine)-defined sigma ( σ) binding with K i values of about 0.5–2.5 μM in well-washed homogenates obtained from rat cerebral cortex. The saturation studies revealed that the inhibition by desipramine (1–4 μM), (−)-propranolol (1 μM) and (−)-alprenolol (3 μM) resulted from a reduction of the B max value without alteration of the K d of [ 3 H ]DTG binding to the cortex or hippocampus. In contrast, imipramine, (±)-pentazocine, clomipramine and risperidone competitively attenuated the cortical or hippocampal [ 3 H ]DTG binding. These findings demonstrate the uncompetitive inhibition of [ 3 H ]DTG binding by neuroactive drugs, thereby providing further support for the possible multiple regulation of cerebral σ receptors.

99mTc-labeled sigma-receptor-binding complex: synthesis, characterization, and specific binding to human ductal breast carcinoma (T47D) cells.

sigma-Receptors have recently been shown to be expressed in a variety of human tumor cells. In an attempt to prepare 99mTc chelates that would bind to sigma-receptors and be useful for imaging sigma-receptor-positive tumors, we have synthesized and characterized a bisaminothiol (BAT) chelate appended with a sigma-receptor pharmacophore. The synthesis of target ligand VII was accomplished in three steps starting from bicyclic imidazolidino[1,2-d]dithiazapine. The labeling of the BAT ligand with 99mTc was carried out in high yields (> 80%) using stannous tartarate as a reducing agent, resulting in the target sigma-receptor-binding chelate [99mTc]BAT-EN6, III. Similarly, 99gTc chelate with ligand VII was prepared from ammonium pertechnetate by reduction with stannous tartarate. 99nTc-radiolabeled chelate was purified by reversed phase HPLC, and cell binding with human breast ductal carcinoma (T47D) was performed. A high degree of specific binding (90-97%) was obtained when sigma-receptor ligands such as halogenated phenylethylenediamines were used to determine nonspecific binding. A modest affinity dose-dependent inhibition of binding was found with BD1008, I, and 4-IPEMP, II (IC50 = 47 +/- 2 and 59 +/- 5 nM, respectively), known sigma-ligands. No specific binding was found with [99mTc]BAT, VIII [without appended sigma-pharmacophore (N-alkyl-substituted ethylenediamine)], showing that biological activity resulted from the pendent pharmacophore. 99gTc complex was found to be a potent inhibitor (Ki = 42.7 +/- 8.5 nM) of [3H]DTG binding in guinea pig brain membranes. Scatchard analysis of [99mTc]BAT-EN6 (spiked with [99gTc]BAT-EN6) binding in T47D breast cancer cells showed a saturable binding, with a Kd of 43.5 +/- 14.7 nM and a Bmax of 3121 +/- 130 fmol/(mg of protein). A biodistribution study of [99mTc]BAT-EN6 chelates in Sprague Dawley rats showed hepatic clearance, as expected. A blocking study at 4 h postinjection using 2 mumol of BD1008 with [99mTc]BAT-EN6 showed a significant decrease of radiopharmaceutical in liver (15.32 vs 22.31% ID/organ) and kidney (1.01 vs 2.21% ID/ organ), organs known to possess high concentrations of sigma-receptors. These results imply that [99mTc]BAT-EN6 binds with high affinity to sigma-receptors expressed in human breast tumor cells, and it may be useful for imaging breast cancer.

Discrepancies in characterization of σ sites in the mouse central nervous system

The characteristics of [ 3H](+)-pentazocine and [ 3H]1,3-di(2-tolyl)guanidine (DTG) binding to mouse whole brain, cortex, cerebellum and spinal cord membranes were investigated in radioreceptor assays. [ 3H](+)-Pentazocine bound to a single, high affinity site ( K d = 1.2 − 1.6 nM) with increasing density along the neuraxis from the cortex ( B max = 543 fmol/mg protein) to the spinal cord ( B max = 886 fmol/mg protein). Hot saturation studies resolved the presence of one binding site for [ 3H]DTG showing no tissue variations in terms of density ( B max = 1075−1264 fmol/mg protein) or affinity ( K d = 16.6−22.3 nM). Incubation with 100 nM (+)-pentazocine revealed two classes of high affinity [ 3H]DTG labeled binding sites corresponding to σ 1 and σ 2 subtypes. A preponderance of σ 2 sites was revealed in all investigated tissues. Different pharmacological profiles were demonstrated for the σ 2 sites in mouse whole brain compared to mouse spinal cord. However, competition studies indicated that the whole brain and spinal [ 3H](+)-pentazocine labeled σ 1 binding sites exhibited similar pharmacological properties. The density of [ 3H](+)-pentazocine labeled σ 1 population was found not to match that of [ 3H]DTG labeled σ 1 site throughout the mouse central nervous system. The presence of low affinity [ 3H]DTG labeled sites was demonstrated in cold saturation experiments. Equilibrium binding data for the low affinity [ 3H]DTG binding site resulted in an increasing density ( B max = 1973−11369 fmol/mg protein) with a decreasing affinity ( K d = 242−943 nM) in mouse cortex through the spinal cord.

Substance P analogs displace sigma binding differentially in the brain and spinal cord of the adult mouse

We have previously observed similarities in the behavioral effects produced by the NH2-terminus of the undecapeptide substance P (SP) and by 1,3-di(2-tolyl)-guanidine (DTG) in the adult mouse. The present series of experiments indicate differences in the rank-order of potency of sigma ligands [DTG; haloperidol (HAL)], SP analogs [SP; SP(1-7); SP(5-11); [D-Pro2, D-Phe7]-SP(1-7) (D-SP(1-7))] and miscellaneous compounds [morphine (MOR), naloxone (NAL)] at competing for [3H]-DTG binding sites in the mouse brain and spinal cord in vitro: Brain; DTG = HAL >> SP = MOR = NAL >> SP(1-7) >> D-SP(1-7) >> SP(5-11): Spinal cord; DTG = HAL >> SP(1-7) = MOR = NAL >> SP >> D-SP(1-7) = SP(5-11). The observed difference in the rank-order potencies of the displacing ligands at these same binding sites supports the notion of two distinct populations of sigma binding sites in these tissues in the adult mouse. Given the low (micromolar) potency of SP analogs at displacing [3H]-DTG binding in the present series of experiments, it is unlikely that the similar behavioral effects we have previously observed elicited by SP(1-7) and DTG in the adult mouse are a result of a direct action of SP(1-7) at the sigma binding site.

Binding properties of KB-5492, a novel anti-ulcer agent, at sigma receptors in porcine gastric fundic mucosa.

We examined the effects of KB-5492, 4-methoxyphenyl 4-(3,4,5-trimethoxybenzyl)-1-piperazine acetate monofumarate monohydrate, a novel anti-ulcer agent and a selective sigma receptor ligand, on specific [3H](+)-N-allyl-normetazocine (SKF 10,047) and [3H]1,3-di(2-tolyl)guanidine (DTG) binding in porcine gastric fundic mucosa. KB-5492 inhibited specific [3H](+)-SKF 10,047 binding in a competitive manner and specific [3H]DTG binding in a non-competitive manner. The Ki value of KB-5492 on specific [3H]DTG binding (Ki = 4.6 microM) was 8.4-fold higher than that on specific [3H](+)-SKF 10,047 binding (Ki = 0.55 microM). Computer-assisted analysis of the displacement curve of KB-5492 for specific [3H]DTG binding indicated the best fit for a two-site model rather than a one-site model, but not for specific [3H](+)-SKF 10,047 binding. Anti-ulcer agents such as omeprazole, cetraxate, cimetidine, sofalcone, sucralfate, teprenone and troxipide had weak or little effect on specific [3H](+)-SKF 10,047 and [3H]DTG binding at a concentration of 100 microM, except that omeprazole exhibited a low affinity for specific [3H](+)-SKF 10,047 binding. These findings suggest that KB-5492 is a unique anti-ulcer agent which binds to sigma receptors in porcine gastric fundic mucosa.

Receptor binding profiles of KB-5492, a novel anti-ulcer agent, at σ receptors in guinea-pig brain

We studied the receptor binding profile of 4-methoxyphenyl 4-(3,4,5-trimethoxybenzyl)-1-piperazine acetate monofumarate monohydrate (KB-5492), a novel anti-ulcer agent, for the σ receptor in guinea-pig brain membranes. KB-5492 selectively inhibited specific [ 3H]1,3-di(2-tolyl)guanidine (DTG) binding to the σ receptor ( IC 50 = 3.15 μ M ) with a pseudo-Hill coefficient of 0.3. Computer-assisted analysis revealed that KB-5492 bound to high- and low-affinity sites. Although KB-5492 had weak affinity for α 2- adrenoceptors at 10 μM, it was almost inactive at a concentration of 10 μM in 33 other binding assays for receptors, second messenger system and ion channels. σ Receptor ligands such as haloperidol, DTG, (+)-3-(3-hydroxyphenol)- N-(1-propyl)piperidine (3-PPP), rimcazole and (−)-3-PPP inhibited specific [ 3H]DTG binding and their IC 50 values were 0.003, 0.044, 0.33, 0.67 and 1.03 μM, respectively. On the other hand, various anti-ulcer agents such as cetraxate, cimetidine, omeprazole, sofalcone, sucralfate, teprenone and troxipide could hardly displace specific [ 3H]DTG binding at 100 μM. Scatchard-Rosenthal analysis indicated that [ 3H]DTG bound to a single site, and K D and B max values for [ 3H]DTG were 87 .3 nM and 679.3 fmol/mg protein, respectively. KB-5492 significantly decreased the B max value, but did not affect the K D value . In contrast, haloperidol and DTG significantly increased the K D values, but did not affect the B max values. These findings indicate that KB-5492 selectively bound to the [ 3H]DTG-labeled σ receptor and that other anti-ulcer agents had little affinity for the σ receptor. Furthermore, KB-5492 inhibited specific [ 3H]DTG binding in a non-competitive manner.

Multiple [ 3H]DTG binding sites in guinea pig cerebellum: evidence for the presence of non-specific binding

The characteristics of the low affinity component of 1,3-di(2- 3H]tolyl)guanidine binding to the guinea pig cerebellum were investigated. Saturation binding assays where σ 1 receptors were masked with dextrallorphan indicated that 1,3-di(2-[5- 3H]tolyl)guanidine bound to cerebellar membranes in a fashion best described by a 1 site + non-specific binding model with a low density of specific binding sites ( B max ≈ 200 fmol/mg protein). Boiling the cerebellar membranes before addition to the saturation assay had no effect on the density of 1,3-di(2-[5- 3H]tolyl)guanidine binding. In contrast, both the K d and B max for 1,3-di(2-[5- 3H]tolyl)guanidine binding to liver membranes was significantly reduced by boiling, as was the density of [ 3H](+)-pentazocine binding to cerebellum and liver. Thus, a substantial component of 1,3-di(2-[5- 3H]tolyl)guanidine binding in the guinea pig cerebellum is to non-specific, proteinaceous binding sites with some of the pharmacological characteristics of the σ 2 binding site.

NE-100, a novel potent σ ligand, preferentially binds to σ 1 binding sites in guinea pig brain

The selectivity of N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ehthylamine monohydrochloride (NE-100) for σ 1 and σ 2 binding sites was studied by means of binding of [ 3H](+)-pentazocine and [ 3 H]1,3- di-o- tolylguanidine ([ 3H]HTG) in guinea pig brain. NE-100 inhibited [ 3H](+)-pentazocine binding to σ 1 binding sites potently with an IC 50 value of 1.54±0.26 nM while it had a weak effect on [ 3H]DTG binding to σ 2 binding sites. The inhibitory effect of NE-100 on [ 3H](+)-pentazocine was 55 times more potent than that on [ 3H]DTG binding. These results suggest that NE-100 is a potent and selective ligand for σ 1 binding sites.

An absence of changes in σ receptor subtypes in the brains of genetically dystonic ( dt) rats

Binding sites for the σ ligand [ 3 H] di-o- tollylguanidine [ 3H]DTG) have been reported to be altered in the brains of genetically dystonic rats. In the present study, selective σ 1 and σ 2 assay conditions were utilized in an effort to define which subpopulation of [ 3H]DTG binding sites is altered in the dystonic strain ( dt). Both this approach and a re-examination using conditions similar to the previous report failed to confirm a difference between the brains of dystonic and normal rats in terms of σ binding. Although not directly negating the possible involvement of σ receptors in dystonia, these results indicate that σ 1 and σ 2 receptors appear unchanged in dystonic rats.

Discrimination of (+)-3-PPP sites from DTG sites by FH-510, a novel potent σ ligand, in rat brain

The effect of 5,8-dimethyl-4-(2-di- n-propylaminoethyl)carbazol monohydrochloride (FH-510) on the binding of σ ligands such as [ 3 H](+)-3-(3- hydroxyphenyl)-N-(1- propyl)piperidine([ 3 H](+)-3- PPP) and [ 3 H]1,3- di-o- tolylguanidine ([ 3H]DTG) to rat brain membranes was studied. The inhibitory effect of FH-510 on [ 3H](+)-3-PPP binding to membranes of rat brain was 260 times more potent than that on [ 3H]DTG binding. Scatchard plot analysis showed that FH-510 inhibited [ 3H](+)-3-PPP binding in a competitive manner, while the inhibitory effect of FH-510 on [ 3H]DTG binding was noncompetitive. These results suggest that (+)-3-PPP sites could be discriminated from DTG sites, and that FH-510 binds preferentially to (+)-3-PPP recognition sites in rat brain.

Loss of sigma binding sites in the CA1 area of the anterior hippocampus in Alzheimer's disease correlates with CA1 pyramidal cell loss

The densities of [ 3H]1, 3-di- o-tolyguanidine ([ 3H]DTG) binding to sigma sites in the CA1 stratum pyramidale region in 7 hippocampi affected by Alzheimer's disease, were compared with densities in 7 normal hippocampi. There was an average reduction of 26% in [ 3H]DTG binding in this area, which was correlated with an average 29% pyramidal cell loss in the same region. These results are consistent with experiments in animals indicating that sigma binding sites are preferentially associated with the somata of large cells.