Dennis Yasuda

Metabolic studies of tolbutamide in the rat

Abstract Hydroxymethyltolbutamide was found to be the major oxidation product in the urine of rat and rabbit given tolbutamide. The metabolite was identified by its infrared spectrum and by paper and thin-layer chromatography. The enzyme system responsible for the oxidation of tolbutamide is a typical microsomal drug-metabolizing system subject to induction by 3,4-benzpyrene, phenobarbital, and tolbutamide itself. The only product observed after incubating tolbutamide with either the fortified microsomes or with liver homogenate was hydroxymethyltolbutamide. Only a small isotope effect was seen in the oxidation of trideuterated tolbutamide. Hydroxymethyltolbutamide was approximately half as active as tolbutamide in its hypoglycemic activity in mice.

Effects of various chemical agents on drug metabolism and cholesterol biosynthesis

Abstract The effects of representative inhibitors of drug metabolism, hypocholesterolemic agents and insecticide synergists on microsome-catalyzed drug metabolism and cholesterol biosynthesis were examined in the rat. In the experiments in vitro , both biochemical reactions were inhibited to varying degrees by all of the test compounds. Cholesterol biosynthesis appeared to be more sensitive to these compounds than was the drug metabolism system. However, in an experiment in vivo , only microsome inhibitors prolonged the sleeping time of mice induced by hexobarbital. The administration of AY-9944 and Compound A at doses sufficient to lower the plasma cholesterol level had no effect on the microsomal enzyme activity. Two effects on cholesterol metabolism were observed with microsome inducers. Administration of 3,4-benzpyrene resulted in an elevated plasma cholesterol level in the rat. However, the incorporation of 14 C-labeled acetate or mevalonic acid into cholesterol by the liver of benzpyrene-treated rats was not higher than that by the liver of the control rats. Phenobarbital did not elevate the plasma cholesterol level, but the liver from phenobarbital-treated rats showed increased 14 C-acetate incorporation into cholesterol.

AT‐1001: a high‐affinity α3β4 nAChR ligand with novel nicotine‐suppressive pharmacology

The α3β4 subtype of nicotinic acetylcholine receptors (nAChRs) has been implicated in mediating nicotine reinforcement processes. AT‐1001 has been recently described as a high‐affinity and selective α3β4 nAChR antagonist that blocks nicotine self‐administration in rats. The aim of this study was to investigate the mechanism of action underlying the nicotine‐suppressive effects of AT‐1001.

Functional Characterization of AT-1001, an α3β4 Nicotinic Acetylcholine Receptor Ligand, at Human α3β4 and α4β2 nAChR

INTRODUCTION Genome-wide association studies linking the α3, β4, and α5 nicotinic acetylcholine receptor (nAChR) subunits to nicotine dependence suggest that α3β4* nAChR may be targets for smoking cessation pharmacotherapies. We previously reported that AT-1001, a selective α3β4* nAChR ligand binds with high affinity to rat α3β4 and human α3β4α5 nAChR, antagonizes epibatidine-induced activation of rat α3β4 nAChR in HEK cells and potently inhibits nicotine self-administration in rats. METHODS Two-electrode voltage clamp was used for functional characterization of AT-1001 at recombinant human α3β4 and α4β2 nAChR expressed in Xenopus oocytes. RESULTS Concentration-response curves show that AT-1001 is a partial agonist at human α3β4 nAChR, evoking up to 35% of the maximal acetylcholine (ACh) response (50% effective concentration [EC50] = 0.37 μM). AT-1001 showed very little agonist activity at the α4β2 nAChR, evoking only 6% of the ACh response (EC50 = 1.5 μM). Pre- and co-application of various concentrations of AT-1001 with 50 μM ACh revealed a complex pattern of activation-inhibition by AT-1001 at α3β4 nAChR, which was best fitted by a 2-site equation. At α4β2 nAChR, co-exposure of AT-1001 with ACh only showed inhibition of ACh current with a shallower curve. CONCLUSIONS AT-1001 is a partial agonist at the human α3β4 nAChR and causes desensitization at concentrations at which it evokes an inward current, resulting in an overall functional antagonism of α3β4 nAChR. AT-1001 does not significantly activate or desensitize α4β2 nAChR at the same concentrations as at the α3β4 nAChR, but does inhibit ACh responses at α4β2 nAChR at higher concentrations. A combination of these mechanisms may underlie the inhibition of nicotine self-administration by AT-1001, suggesting that AT-1001 and compounds from this class may have clinical potential for smoking cessation pharmacotherapy.

High affinity α3β4 nicotinic acetylcholine receptor ligands AT-1001 and AT-1012 attenuate cocaine-induced conditioned place preference and behavioral sensitization in mice

Cholinergic signaling via the nicotinic acetylcholine receptors (nAChRs) in the mesolimbic circuitry is involved in the rewarding effects of abused drugs such as cocaine and opioids. In mouse studies, nonselective nAChR antagonist mecamylamine blocks cocaine-induced conditioned place preference (CPP) and behavioral sensitization. Among subtype-selective nAChR antagonists, the β2-selective antagonist dihydrobetaerythroidine and α7 antagonist methyllycaconitine (MLA), but not MLA alone prevent behavioral sensitization to cocaine. Since the role of the α3β4 nAChR subtype in the rewarding and behavioral effects of cocaine is unknown, the present study investigated the effect of two potent and selective α3β4 nAChR ligands, AT-1001 and AT-1012, on the acquisition of cocaine-induced CPP and behavioral sensitization in mice. At 5-30mg/kg, cocaine produced robust CPP, whereas behavioral sensitization of locomotor activity was only observed at the higher doses (20-30mg/kg). Pretreatment with AT-1001 (1-10mg/kg) or AT-1012 (3-10mg/kg) blocked CPP induced by 5mg/kg cocaine, but not by 30mg/kg cocaine. Lower doses of AT-1001 (0.3-1mg/kg) and AT-1012 (1-3mg/kg) did not affect the increase in locomotor activity induced by 5 or 30mg/kg cocaine. But AT-1001, at these doses, blocked locomotor sensitization induced by 30mg/kg cocaine. These results indicate that the α3β4 nAChR play a role in the rewarding and behavioral effects of cocaine, and that selective α3β4 nAChR ligands can attenuate cocaine-induced behavioral phenomena. Since the selective α3β4 nAChR functional antagonist AT-1001 has also been shown to block nicotine self-administration in rats, the present results suggest that α3β4 nAChRs may be a target for the treatment of cocaine addiction as well as for cocaine-nicotine comorbid addiction.

In vitro functional characterization of novel nociceptin/orphanin FQ receptor agonists in recombinant and native preparations.

Nociceptin/Orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP). In this study novel nonpeptide NOP ligands were characterized in vitro in receptor binding and [35S]GTPγS stimulated binding in membranes of cells expressing human NOP and classical opioid receptors, calcium mobilization assay in cells coexpressing the receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, the electrically stimulated mouse vas deferens and the mouse colon bioassays. The action of the AT compounds were compared with standard NOP agonists (N/OFQ and Ro 65-6570) and the NOP selective antagonist SB-612111. AT compounds displayed high NOP affinity and behaved as NOP agonists in all the functional assays consistently showing the following rank order of potency AT-127≥AT-090≥AT-035>AT-004= AT-001. AT compounds behaved as NOP full agonists in the calcium mobilization and mouse colon assays and as partial agonists in the [35S]GTPγS and BRET assays. Interestingly AT-090 and AT-127, contrary to standard nonpeptide agonists that display G protein biased agonism, behaved as an unbiased agonists. AT-090 and AT-127 displayed higher NOP selectivity than Ro 65-6570 at native mouse receptors. AT-090 and AT-127 might be useful pharmacological tools for investigating the therapeutic potential of NOP partial agonists.

Synthesis of Δ11-9α-fluoro-steroids: X-ray crystal and molecular structure of 17α,21-dipropionyloxy-9α-fluoro-16β-methylpregna-1,4,11-triene-3,20-dione

Reaction of 9α-fluoro-corticosteroids with NN-dialkylaminosulphur trifluorides gives Δ11-9α-fluoro-steroids (2a,b); a single-crystal X-ray analysis of (2a) is reported.

Buprenorphine requires concomitant activation of NOP and MOP receptors to reduce cocaine consumption

Buprenorphines clinical use is approved for the treatment of heroin addiction; however, evidence supporting its efficacy in cocaine abuse also exists. While for heroin it has been demonstrated that the effect of buprenorphine is mediated by its ability to activate μ‐opioid peptide receptor (MOP) receptors, the mechanism through which it attenuates cocaine intake remains elusive. We explored this mechanism using operant models where rodents were trained to chronically self‐administer cocaine for 2 hours daily. Buprenorphine (0.3, 1.0 and 3.0 mg/kg) given intraperitoneally 90 minutes before access to cocaine significantly and dose dependently reduced its intake. Pre‐treatment with naltrexone or with the selective nociceptin/orphanin FQ peptide (NOP) antagonist SB‐612111 did not prevent buprenorphine‐induced reduction of cocaine intake. However, when naltrexone and SB‐612111 were combined, the effect of buprenorphine on cocaine was completely prevented. To confirm that co‐activation of MOP and NOP receptors is the underlying mechanism through which buprenorphine reduces cocaine intake, three compounds, namely, AT‐034, AT‐201 and AT‐202, with a range of affinity and intrinsic activity profiles for MOP and NOP receptors, but weak ability for kappa‐opioid peptide receptor (KOP) transmission, were tested. Consistent with our hypothesis based on buprenorphines effects, results demonstrated that AT‐034 and AT‐201, which co‐activate MOP and NOP receptors, reduced cocaine self‐administration like buprenorphine. AT‐202, which selectively stimulates NOP receptors, was not effective. Together, these data demonstrate that for buprenorphine, co‐activation of MOP and NOP receptors is essential to reduce cocaine consumption. These results open new vistas on the treatment of cocaine addiction by developing compounds with mixed MOP/NOP agonist properties.

The α3β4 nAChR partial agonist AT-1001 attenuates stress-induced reinstatement of nicotine seeking in a rat model of relapse and induces minimal withdrawal in dependent rats

HighlightsAT‐1001, a high affinity, selective &agr;3&bgr;4 nAChR partial agonist attenuates reinstatement of nicotine seeking induced by pharmacological stress in a rat model of relapse.When administered to nicotine‐dependent rats, AT‐1001 induces minimum withdrawal signs.The robust efficacy of &agr;3&bgr;4 nAChR‐selective compounds such as AT‐1001 to block reinstatement of nicotine‐seeking differentiate it from current smoking cessation medications like bupropion and varenicline which do not attenuate relapse to nicotine seeking.Targeting the &agr;3&bgr;4 nAChR offers a new approach for smoking cessation treatment and improving long term abstinence rates. Abstract The strong reinforcing effects of nicotine and the negative symptoms such as anxiety experienced during a quit attempt often lead to relapse and low success rates for smoking cessation. Treatments that not only block the reinforcing effects of nicotine but also attenuate the motivation to relapse are needed to improve cessation rates. Recent genetic and preclinical studies have highlighted the involvement of the &agr;3, &bgr;4, and &agr;5 nicotinic acetylcholine receptor (nAChR) subunits and the &agr;3&bgr;4 nAChR subtype in nicotine dependence and withdrawal. However, the involvement of these nAChR in relapse is not fully understood. We previously reported that the &agr;3&bgr;4 nAChR partial agonist AT‐1001 selectively decreases nicotine self‐administration in rats without affecting food responding. In the present experiments, we examined the efficacy of AT‐1001 in attenuating reinstatement of nicotine‐seeking behavior in a model of stress‐induced relapse. Rats extinguished from nicotine self‐administration were treated with the pharmacological stressor yohimbine prior to AT‐1001 treatment and reinstatement testing. We also examined whether AT‐1001 produced any withdrawal‐related effects when administered to nicotine‐dependent rats. We found that AT‐1001 dose‐dependently reduced yohimbine stress‐induced reinstatement of nicotine seeking. When administered to nicotine‐dependent rats at the dose that significantly blocked nicotine reinstatement, AT‐1001 elicited minimal somatic withdrawal signs in comparison to the nicotinic antagonist mecamylamine, which is known to produce robust withdrawal. Our data suggest that &agr;3&bgr;4 nAChR‐targeted compounds may be a promising approach for nicotine addiction treatment because they can not only block nicotine’s reinforcing effects, but also decrease motivation to relapse without producing significant withdrawal effects.

Semisynthetic aminoglycoside antibacterials. Part II. Synthesis of gentamicin X2 and related compounds

Gentamicin X2, naturally occurring aminoglycoside antibiotic produced as a minor component together with the clinically important gentamicin C complex by Micromonospora purpurea, has been synthesized by glycosylation of suitably protected garamine derivatives. The synthesis of the α-glycoside was accomplished by means of the Lemieux–Nagabhushan reaction as well as by using a Koenigs–Knorr reaction. The latter reaction was also used to prepare 4-O-β-analogue of gentamicin X2. The syntheses of other analogues of gentamicin X2, namely 4-O-[2-amino-2-deoxy-α-D-mannopyranosyl], 4-O-[2-amino-2-deoxy-α-D-galactopyranosyl], 4-O-[2-amino-2-deoxy-α- and -β-D-glucofuranosyl], 4′-O-[2-amino-2-deoxy-α-D-glucopyranosyl], 4-O-α-D-glucopyranosyl, 4-O-α-D-talopyranosyl, and 4-O-2-deoxy-α-D-galactopyranosyl derivatives of garamine, are also described.