Showing papers in "Journal of Pharmacology and Experimental Therapeutics in 1996"

Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function.

Abstract: SB 203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4- pyridyl)imidazole], a selective cytokine suppressive binding protein/p38 kinase inhibitor, was evaluated in several models of cytokine inhibition and inflammatory disease. It was demonstrated clearly to be a potent inhibitor of inflammatory cytokine production in vivo in both mice and rats with IC50 values of 15 to 25 mg/kg. SB 203580 possessed therapeutic activity in collagen-induced arthritis in DBA/LACJ mice with a dose of 50 mg/kg resulting in significant inhibition of paw inflammation and serum amyloid protein levels. Antiarthritic activity was also observed in adjuvant-induced arthritis in the Lewis rat when SB 203580 was administered p.o. at 30 and 60 mg/kg. Evidence for disease-modifying activity in this model was indicated by an improvement in bone mineral density and by histological evaluation. Additional evidence for beneficial effects on bone resorption was provided in the fetal rat long bone assay in which SB 203580 inhibited 45Ca release with an IC50 of 0.6 microM. In keeping with the inhibitory effects on lipopolysaccharide-induced tumor necrosis factor-alpha in mice, SB 203580 was found to reduce mortality in a murine model of endotoxin-induced shock. In immune function studies in mice treated with SB 203580 (60 mg/kg/day for 2 weeks), there was some suppression of an antibody response to ovalbumin, whereas cellular immune functions measured ex vivo were unaffected. This novel profile of activity strongly suggests that cytokine inhibitors could provide significant benefit in the therapy of chronic inflammatory disease.

Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): identification of cannabinoid receptor subtype selective ligands.

Abstract: Two cannabinoid receptors have been identified to date; one is located predominantly in the central nervous system (CB1), whereas the other is located exclusively in the periphery (CB2). The purposes of this study were to explore further the binding requirements of the CB2 receptor and to search for compounds displaying distinct affinities for either cannabinoid receptor. The binding affinities of a series of cannabinoids tested previously at the CB1 receptor were determined at cloned human CB1 and CB2 receptors using a filtration assay. In addition, possible allosteric regulation of the CB2 receptor was examined. Sodium and a GTP analog elicited a concentration-dependent decrease in specific binding to the CB2 receptor. The affinity of cannabinol for CB2 receptors (Ki = 96.3 +/- 14 nM) was confirmed to be in approximately the same range as that of delta 9-THC (Ki = 36.4 +/- 10 nM). Affinities at cloned CB1 and CB2 receptors were compared with affinities determined in the brain. Although most of the chosen compounds did not discriminate between CB1 and CB2, several ligands were identified that showed selectivity. Affinity ratios demonstrated that two 2'-fluoro analogs of anandamide were over 23-fold selective for the CB1 receptor and confirmed the CB1 selectivity of SR141716A {N- (piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamidehydrochloride}. In addition, WIN-55, 212-2 {(R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl) methyl] pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl) methanone} and a closely related propyl indole analog were shown to be 6.75- and 27.5- fold selective, respectively, for the CB2 receptor. These ligands can now serve as a basis for the design of compounds with even greater selectivity.

Arbutin: mechanism of its depigmenting action in human melanocyte culture.

Abstract: Arbutin, a naturally occurring beta-D-glucopyranoside of hydroquinone, is effective in the topical treatment of various cutaneous hyperpigmentations characterized by hyperactive melanocyte function. We examined the mechanism of its depigmenting action in human melanocyte cultures. Arbutin inhibited the tyrosinase activity of cultured human melanocytes at noncytotoxic concentrations. It did not affect the expression of tyrosinase mRNA. Melanin production was inhibited significantly by arbutin, as determined by measuring eumelanin radicals with an electron spin resonance spectrometer. The study of the kinetics and mechanism for inhibition of tyrosinase confirms the reversibility of arbutin as a competitive inhibitor of this enzyme. The utilization of L-tyrosine or L-dopa as the substrate suggests a mechanism involving competition with arbutin for the L-tyrosine binding site at the active site of tyrosinase. These results suggest that the depigmenting mechanism of arbutin in humans involves inhibition of melanosomal tyrosinase activity, rather than suppression of the expression and synthesis of tyrosinase.

Pharmacokinetic properties of several novel oligonucleotide analogs in mice.

Abstract: Biophysical and pharmacokinetic properties of five analogs of ISIS 3082, a 20-mer phosphorothioate oligodeoxynucleotide that inhibits the expression of mouse intercellular adhesion molecule 1, were evaluated. Compared to the parent compound, ISIS 3082, the 2'-propoxy modified phosphodiester, ISIS 9044 and the 2'-propoxy phosphorothioate, ISIS 9045, had greater affinity for complementary RNA and were more lipophilic. A chimeric oligonucleotide comprised of 2'-propoxy diester wings and a phosphorothioate deoxy center (ISIS 9046) had equal affinity. It was also more lipophilic than ISIS 3082, but less so than the other 2'-propoxy modified analogs. The two analogs with 5'-lipophilic conjugates, ISIS 9047 (5'-octadecylamine) and ISIS 8005 (5'-(2'-O-hexylamino-carbonyl-oxycholesterol) were more lipophilic than ISIS 3082 (3- and 7-fold, respectively) but had similar affinity for complementary RNA. Binding of ISIS 3082 to bovine serum albumin was salt-dependent and, at physiological concentration (320 mOsmol), the dissociation constant (Kd) was 140 microM. Similarly, the 2'-propoxy phosphodiester, ISIS 9044, displayed salt-dependent bovine serum albumin binding, but not binding was measurable at physiological salt conditions. In contrast, the more lipophilic phosphorothioate analogs displayed much higher affinity to bovine serum albumin at 320 mOsmol than ISIS 3082. After bolus injection to mice, the initial volumes of distribution of the more lipophilic phosphorothioate analogs, ISIS 9045, ISIS 9047 and ISIS 8005, were less and the initial clearance from plasma was slower than ISIS 3082. The pharmacokinetics of the other analogs was similar to ISIS 3082. Distribution of ISIS 3082 into peripheral tissues was similar to that reported for other phosphorothioates with liver and kidney accumulating the highest fraction of the dose. The only modification to markedly influence distribution was the very lipophilic cholesterol conjugate (ISIS 8005), which increased substantially the fraction of the dose accumulated by the liver. Little intact drug was found in urine or feces for any analog, and the patterns of metabolites suggested that for all analogs the principal metabolic pathway was due to 3'-exonuclease activity. The metabolism of ISIS 3082 was similar to that reported for other phosphorothioates. After 2 hr, most of the radioactivity in plasma represented metabolites but, in tissues, intact ISIS 3082 was present for much longer periods of time and metabolites accumulated more slowly. The 24-hr exposure to ISIS 3082 of liver and kidney was 20.7 and 67.9 microM/hr, respectively. The rates of metabolism in plasma, liver and kidney of the two 5'-conjugates, ISIS 9047 and ISIS 8005, were similar to ISIS 3082, as was the pattern of metabolism. The rate of metabolism of ISIS 9044 (2'-propoxy phosphodiester oligonucleotide) was much more rapid in liver and plasma, but surprisingly much slower in the kidney. ISIS 9045 (full 2-propoxy phosphorothioate) was much more stable than ISIS in all tissues, the enhanced stability of ISIS 9045 resulted in increased exposure of liver and kidney to the drug, whereas the exposure of the liver to the two more lipophilic analogs, ISIS 9047 and ISIS 8005, was greater because a higher fraction of the dose was distributed to the liver. The exposure of the kidney to ISIS 9044 was also greater than that to ISIS 3082 due to the surprising stability of the drug in the kidney.

In vivo characterization of a specific cannabinoid receptor antagonist (SR141716A): inhibition of delta 9-tetrahydrocannabinol-induced responses and apparent agonist activity.

Abstract: SR141716A has been described as a cannabinoid receptor antagonist. This study was conducted to determine whether SR141716A was capable of antagonizing the pharmacological effects of the prototypical cannabinoid agonist delta 9-THC. The AD50 (+/- 95% confidence limits) obtained after a 10 min i.v. pretreatment with SR141716A in measures of hypoactivity, hypothermia, and antinociception were: 0.12 (0.02-0.66), 0.087 (0.037-0.201), and 0.16 (0.03-1.01) mg/kg, respectively. The effect of SR141716A lasted up to 1 hr (antinociception, 10 mg/kg), 4 hr (locomotion, 1 and 3 mg/kg), or 24 hr (hypothermia, 3 mg/kg). Further evaluation revealed an AD50 value of 2.7 mg/kg (1.7-4.4) in the PPQ-stretch procedure. Additionally, the ED50 (+/- S.E.) of morphine in the tail-flick antinociception procedure was increased by SR141716A (30 mg/kg, i.v.) from 3.2 (+/- 0.3) to 5.3 (+/- 0.6) mg/kg. Finally, SR141716A produced direct effects on locomotor activity at doses greater than 3 mg/kg. Locomotion was stimulated to more than 200% of control (20 mg/kg), with an ED50 value of 4.7 (+/- 1.5) mg/kg. The ED50 value represents stimulation to levels approximately 150% of control. It is not clear whether this pharmacological activity represents an uncharacterized action of SR141716A, or an index of tonic activity of an endogenous cannabinergic system. SR141716A will be useful in establishing the biochemical events responsible for the in vivo effects of exogenous cannabinoids, as well as in establishing the existence of a putative endogenous cannabinergic system.

Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes.

Abstract: Cytochrome P450 chemical inhibitors are widely used to define the role of individual cytochrome P450 isozyme(s) in a metabolism process. In this study, cytochrome P450 isoform-dependent reactions were investigated on our human liver microsomes bank (n = 34) and characterized for both KM and VMAX values (n > or = 3). These metabolic reactions were: 7-ethoxyresorufin O-deethylation (CYP1A1), phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), tolbutamide 4-methylhydroxylation (CYP2C9), dextromethorphan O-demethylation (CYP2D6), aniline 4-hydroxylation (CYP2E1) and nifedipine dehydrogenation (CYP3A4). Literature data-based specific inhibitors were selected and characterized for both their inhibitory constant (Ki) and the inhibition-type toward their specific substrate. Results were as follows: alpha-naphthoflavone (CYP1A1; mixed-type interaction with a Ki = 0.01 microM), furafylline (CYP1A2; competitive-type interaction with a Ki = 3 microM when microsomes were incubated with both furafylline and phenacetin; noncompetitive-type interaction with a Ki = 0.6 microM when microsomes were preincubated with furafylline and NADPH), pilocarpine (CYP2A6; competitive-type interaction with a Ki = 4 microM), sulfaphenazole (CYP2C9; competitive-type interaction with a Ki = 0.3 microM), quinidine (CYP2D6; competitive-type interaction with a Ki = 0.4 microM, diallyldisulfide (CYP2E1; noncompetitive-type interaction with a Ki = 150 microM on an aniline concentration range of 10-60 microM; competitive-type interaction with a Ki = 100 microM on an aniline concentration range of 80-2000 microM) and ketoconazole (CYP3A4; mixed-type interaction with a Ki = 0.015 microM). Once the inhibitors' potency was determined, the selective effects of these inhibitors were evaluated after incubation of human hepatic microsomes with isoform-selective substrates in the presence of the different chemical inhibitors. Up to 10 times the Ki value toward the isoform-selective probe, pilocarpine, sulfaphenazole, quinidine and ketoconazole exhibited potent inhibitory and specific effects. alpha-Naphthoflavone and furafylline both inhibited phenacetin and 7-ethoxyresorufin O-deethylation processes, a consequence of the absence of CYP1A1 in noninduced human liver. Diallyldisulfide exhibited broad and nonspecific inhibitory effects. When used in their "window of selectivity," ie., up to 10-fold the Ki value, most chemical inhibitors powerfully and specifically inhibited cytochrome P450 isoform-specific reactions when analyzed at their KM values.

Frequent distribution of ultrarapid metabolizers of debrisoquine in an ethiopian population carrying duplicated and multiduplicated functional CYP2D6 alleles.

Abstract: The debrisoquine hydroxylase (CYP2D6) catalyzes the oxidative metabolism of more than 40 different clinically important drugs. The CYP2D6 gene is highly polymorphic. Defect alleles, causing the poor metabolizer phenotype, and also alleles with duplicated or multiduplicated active genes, causing ultrarapid metabolism, have been described. In the current investigation, we have evaluated the CYP2D6 phenotype (n = 115) and genotype (n = 122) among healthy Ethiopians. Only two subjects (1.8%) exhibited metabolic reaction (MR) for debrisoquine > 12.6 and were classified as poor metabolizers. A mutation in exon 1 causing a 34Pro --> Ser amino acid exchange, typical of the Chinese CYP2D6*10B (Ch1) allele and yielding an unstable enzyme, was present among 16% of the population and the carriers exhibited a high MR (0.9-5.0). Increased MR was also found among 18% of the subjects with a 107Thr --> Ile mutation associated to the CYP2D6*17(Z) allele causing diminished activity of CYP2D6 in vivo. Interestingly, 29% of the population investigated carried alleles with duplicated or multiduplicated CYP2D6 genes, indicative of ultrarapid metabolism. Xbal and EcoRI RFLP analyses identified individuals having new alleles with four or five CYP2D6*2(L) genes. Subjects with duplicated or multiduplicated CYP2D6*2 genes exhibited the lowest MR. These results suggest that the Ethiopian population, in comparison to Caucasian, Oriental and other Black populations, is genetically different with respect to the constitution of the CYP2D locus. The results also show that subjects carrying duplicated or multiduplicated active CYP2D6 genes are very common in certain ethnic groups, implicating this issue of potential global importance.

Cytochrome P450-mediated metabolism of the HIV-1 protease inhibitor ritonavir (ABT-538) in human liver microsomes.

Abstract: The HIV-1 protease inhibitor ritonavir (ABT-538) undergoes cytochrome P450-mediated biotransformation in human liver microsomes to three major metabolites, Ml, M2 and M11, with wide interindividual variation in the rates of metabolite formation. The structures of these metabolites were determined with the use of electrospray ionization mass spectrometry. Chemical inhibition, metabolic correlation, immunoinhibition and metabolism by microsomes derived from specific CYP cDNA-transfected B-lymphoblastoid cell lines indicated that the CYP3A subfamily of enzymes was the major contributor to the formation of M1 and M11, whereas both CYP3A and CYP2D6 contributed to the formation of M2. None of the typical CYP3A substrates/inhibitors (e.g., ketoconazole, troleandomycin) were able to completely inhibit ritonavir metabolism, even at high concentrations. Ritonavir was found to be a potent inhibitor of CYP3A-mediated biotransformations (nifedipine oxidation, IC50) = 0.07 microM; 17alpha-ethynylestradiol 2-hydroxylation, IC50 = 2 microM; terfenadine hydroxylation, IC50 = 0.14 microM). Ritonavir was also found to be an inhibitor of the reactions mediated by CYP2D6 (IC50 = 2.5 microM) and CYP2C9/10 (IC50 = 8.0 microM). The results of this study indicate the potential for in vivo inhibition of the metabolism by ritonavir of drugs that are CYP3A, CYP2D6 and, to a lesser extent, CYP2C9/10 substrates.

Preclinical characterization of the potential of the putative atypical antipsychotic MDL 100,907 as a potent 5-HT2A antagonist with a favorable CNS safety profile.

Abstract: In preclinical studies, [R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4- piperidinemethanol] [formula: see text] (MDL 100,907), a putative atypical antipsychotic, was characterized in vitro as a potent and selective ligand for the serotonin2A (5-HT2A) receptor and was evaluated in vitro and in vivo as a potent 5-HT2A receptor antagonist. Furthermore, MDL 100,9079s potential CNS safety profile and selectivity as a potential antipsychotic agent were evaluated and compared with benchmark compounds. MDL 100,907 demonstrated low nanomolar or subnanomolar binding in vitro at the 5-HT2A receptor and showed a > 100-fold separation from all other receptors measured. MDL 100,907 had subnanomolar potency as a 5-HT2A antagonist in vitro in reversing 5-HT-stimulated inositol phosphate accumulation in NIH 3T3 cells transfected with the rat 5-HT2A receptor. In vivo, MDL 100,907 potently inhibited 5-methoxy-N, N-dimethyltryptamine-induced head twitches in mice or 5-hydroxytryptophan-induced head twitches in rats. In vivo functional tests in mice revealed a > 500-fold separation between doses that produced 5-HT2A antagonism and doses that produced alpha 1-adrenergic or striatal D2 antagonism. Using inhibition of D-amphetamine-stimulated locomotion in mice as a measure of potential antipsychotic efficacy, MDL 100,907 showed a superior CNS safety index relative to the reference compounds, haloperidol, clozapine, risperidone, ritanserin, and amperozide, in each of five tests for side effect potential, including measures of ataxia, general depressant effects, alpha 1-adrenergic antagonism, striatal D2 receptor antagonism, and muscle relaxation. MDL 100,907 did not antagonize apomorphine-induced stereotypes in rats, suggesting that it potentially lacks extrapyramidal side effect liability. MDL 100,907 showed selectivity as a potential antipsychotic in that it lacked consistent activity in selected rodent models of anticonvulsant, antidepressant, analgesic, or anxiolytic activity. In summary, these preclinical data indicate that MDL 100,907 is a potent and selective ligand at the 5-HT2A receptor. MDL 100,9079s potent 5-HT2A antagonist activity might account for its activity in preclinical models of antipsychotic potential. Ongoing clinical evaluation with MDL 100,907 will test the hypothesis that 5-HT2A receptor antagonism is sufficient for antipsychotic activity in humans.

Characterization of CYP2A6 involved in 3'-hydroxylation of cotinine in human liver microsomes.

Abstract: Nicotine is primarily metabolized to cotinine, and cotinine is further metabolized to trans-3'-hydroxycotinine in human liver, which is a major metabolite of nicotine in humans. We studied the formation of trans-3'-hydroxycotinine from cotinine in human liver microsomes. trans-3'-Hydroxycotinine formation demonstrated single enzyme Michaelis-Menten kinetics (Km, 234.5 +/- 26.8 MicroM; Vmax, 37.2 +/- 2.4 pmol/min/mg protein). Significant correlation (r = .967, P < .001) between cotinine 3'-hydroxylase activities at low (50 microM) and high (1 microM) cotinine concentrations in 20 human liver microsomes suggested the contribution of a single enzyme to cotinine 3'-hydroxylation. The cotinine 3'-hydroxylase activity correlated significantly with immunoreactive cytochrome P450 (CYP)2A6 contents (r = .756, P < .01) and coumarin 7-hydroxylase activity (r = .887, P < .001). The cotinine 3'-hydroxylase activity was inhibited by coumarin, alpha-naphthoflavone, chlorzoxazone and anti-rat CYP2A1 antibodies. Microsomes of B-lymphoblastoid cells expressing human CYP2A6 exhibited cotinine 3'-hydroxylase activity. The Km value of the expressed CYP2A6 (264.7 microM) was almost identical to that of human liver microsomes. In conclusion, cotinine 3'-hydroxylation appears to be catalyzed solely by CYP2A6 in humans. Cotinine is a candidate for a new substrate for CYP2A6 in humans.

Selective N-type neuronal voltage-sensitive calcium channel blocker, SNX-111, produces spinal antinociception in rat models of acute, persistent and neuropathic pain.

Abstract: Male Sprague-Dawley rats were used to evaluate the antinociceptive properties of the selective N-type voltage-sensitive calcium channel (VSCC) blocker, SNX-111, when the compound is administered spinally by either bolus injection or continuous, constant-rate infusion into the subarachnoid space. SNX-111 produced significant, dose-dependent antinociceptive effects by suppressing both the acute (phase 1: ED50, 14 ng/hr) and tonic (phase 2: ED50, 0.82 ng/hr) phases of the formalin test when it was infused for 72 hr immediately before testing. Phase 2 nociceptive responses were suppressed by bolus injections of 100 ng SNX-111. SNX-111 was approximately 1000-fold more potent than morphine in blocking phase 2 responses when the compounds were administered by intrathecal bolus injection. In rats with an experimentally induced painful peripheral neuropathy, intrathecal bolus injections of 30 to 300 ng SNX-111 blocked mechanical allodynia in a dose-dependent manner. Subacute administration of SNX-111 (1, 10 and 100 ng/hr) by continuous intrathecal infusion produced a reversible blockade of mechanical allodynia without apparent development of tolerance. These results show that: 1) selective N-type VSCC blockers are potent and efficacious antinociceptive agents when they are administered by the spinal route; 2) selective N-type VSCC blockers are effective in rat models of acute, persistent and neuropathic pain; and 3) N-type VSCCs play a significant role in the spinal processing of noxious somatosensory input.

Gender-related differences in the antinociceptive properties of morphine.

Abstract: As part of an effort to examine gender-related differences in the abuse liability of morphine, the present parametric study was undertaken to systematically establish whether there are gender-related differences in the antinociceptive activity of morphine in rats. Our results showed that male rats were uniformly more sensitive to the antinociceptive properties of morphine than were females in three different assays, i.e., the hot-plate, tail-flick and abdominal-constriction tests. This enhanced sensitivity to morphine was reflected in the peak antinociceptive effect, the magnitude of antinociception (i.e., area under the time-action curve), the duration of the antinociceptive response and the 50% effective dose. These differences appear to reflect markedly enhanced central nervous system sensitivity to morphine in males, compared with females, because we observed no gender-linked differences in serum levels of morphine after its injection, at the time when peak antinociceptive effects were observed. Furthermore, these gender-related differences appear to be reflected in antinociception thought to be mediated by both spinal and supraspinal mechanisms. Finally, our results suggest that the acute effects of steroids play little role in the gender-related differences observed, because short-term castration did not alter the gender-related differences we observed. Rather, it appears more probable that the organizational effects of steroids during critical periods in development, which determine gender-related distinctions, may be significant in the male-female differences we have observed. In view of a great deal of largely anecdotal data for humans that suggest that there may be gender-related differences in the abuse liability of psychoactive substances, the model described in this paper may provide a means to examine this important issue.

Investigation of brain sites mediating cannabinoid-induced antinociception in rats: evidence supporting periaqueductal gray involvement.

Abstract: The purpose of this study was to elucidate brain areas that mediate cannabinoid-induced antinociception as assessed in the tail-flick test. Intracerebroventricular administration of the prototypical cannabinoid, delta 9-tetrahydrocannabinol, and the potent bicyclic analog, CP-55,940, produced antinociception at ED50 values of 373 and 64 nmol/rat, respectively. Hypothermic and cataleptic effects also were observed after i.c.v. administration of CP-55,940, but not delta 9-tetrahydrocannabinol. In contrast, the endogenous cannabinoid, anandamide, failed to elicit any apparent pharmacological effects. Administration of CP-55,940 into the caudate putamen produced catalepsy, but failed to produce either antinociception or hypothermia. Micro-injection of CP-55,940 into the ventrolateral aspect of the periaqueductal gray (PAG), in the region of the dorsal raphe, produced antinociception (ED50 dose = 28 nmol/rat), catalepsy and hypothermia. CP-56,667, the inactive stereoisomer of CP-55,940, failed to produce any effects when injected into the same site. Additional studies demonstrated that pertussis toxin completely prevented the pharmacological effects of CP-55,940 when both agents were administered into the posterior ventrolateral PAG. In contrast, dibutyryl-cAMP failed to attenuate cannabinoid-induced antinociception. Finally, CP-55,940 administered into either the posterior dorsolateral or the anterior ventrolateral areas of the PAG was without effect. These results indicate that the antinociceptive and cataleptic effects of cannabinoids in the PAG are dose-related, exhibit regional specificity and are enantioselective. Moreover, the complete prevention of these pharmacological effects by pertussis toxin pretreatment in the PAG is consistent with the involvement of G proteins. These findings suggest that the posterior ventrolateral PAG may be an important brain area for the antinociceptive and cataleptic effects of the cannabinoids.

Pharmacological evaluation of novel Alzheimer's disease therapeutics: acetylcholinesterase inhibitors related to galanthamine.

Abstract: Acetylcholinesterase (AChE) inhibitors from several chemical classes have been tested for the symptomatic treatment of Alzheimer's disease; however, the therapeutic success of these compounds has been limited. Recently, another AChE inhibitor, galanthamine hydrobromide (GAL), has shown increased clinical efficacy and safety. Using biochemical, behavioral and pharmacokinetic analyses, this report compares GAL with two of its analogs, 6-O-acetyl-6-O-demethylgalanthamine hydrochloride (P11012) and 6-O-demethyl-6-O[(adamantan-1-yl)-carbonyl]galanthamine hydrochloride (P11149), for their therapeutic potential. P11012 and P11149 were found to be potent, competitive and selective inhibitors of AChE, demonstrating central cholinergic activity, behavioral efficacy and safety. P11012 and P11149, though pharmacokinetic analyses, were shown to act as pro-drugs, yielding significant levels of 6-O-demethylgalanthamine. In vitro, 6-O-demethylgalanthamine was 10- to 20-fold more potent than GAL as an inhibitor of AChE, and it demonstrated greater selectivity for inhibition of AChE vs. butyrylcholinesterase. Like GAL, both P11012 and P11149 showed central cholinergic activity biochemically, by significantly inhibiting rat brain AChE; physiologically, by causing hypothermia; and behaviorally, by attenuating scopolamine-induced deficits in passive avoidance. In addition, GAL, P11012 and P11149 enhanced step-down passive avoidance, another measure of behavioral efficacy. By comparing efficacious doses with primary overt effects, P11012 and P11149 had better oral therapeutic indices than GAL. Oral pharmacokinetic analyses of GAL, P11012 and P11149 revealed differences. Although P11012 and P11149 exhibited similar area under the curve values, 191149 had slower, lower and more sustained concentration maximum levels. P11012 and GAL rapidly reached their concentration maximums, but GAL, in brain had the highest area under the curve and concentration maximum. Because of its composite profile, including duration of action, oral therapeutic index and pharmacokinetics, P11149 is considered the better therapeutic candidate for the treatment of Alzheimer's disease.

Triazolam biotransformation by human liver microsomes in vitro: effects of metabolic inhibitors and clinical confirmation of a predicted interaction with ketoconazole.

Abstract: Biotransformation of the triazolobenzodiazepine triazolam to its hydroxylated metabolites, alpha-hydroxy (OH)- and 4-OH-triazolam, was studied in vitro using microsomal preparations of human liver. Mean values of Vmax (10.3 nM/min/mg of protein) and Km (304 microM) for the 4-OH pathway exceeded values for the alpha-OH pathway (2.4 and 74, respectively). However the mean Vmax/Km ratios for the two pathways were nearly identical, indicating that both contribute approximately equally to intrinsic clearance. Ketoconazole was a powerful inhibitor of triazolam biotransformation, having mean competitive Ki values of 0.006 and 0.023 microM for the alpha-OH and 4-OH pathways. This is consistent with the role of P450-3A isoforms in mediating triazolam biotransformation. The serotonin2 antagonist antidepressant nefazodone inhibited the alpha-OH and 4-OH pathways (Ki = 0.6 and 1.7 microM, respectively), but with considerably less activity than ketoconazole. Among six selective serotonin reuptake-inhibitor antidepressants, norfluoxetine was the most potent inhibitor (Ki = 2.7 and 8.0 microM) and fluoxetine itself was the weakest (Ki = 7.0 and 44.3 microM). In a double-blind clinical pharmacokinetic-pharmacodynamic study, administration of triazolam (0.125 mg) preceded by ketoconazole, compared to triazolam preceded by placebo, produced a nearly 9-fold reduction in apparent oral clearance of triazolam (41 vs. 337 ml/min) and a 4-fold prolongation of half-life (13.5 vs. 3.4 hr). Pharmacodynamic testing indicated enhancement of electroencephalographic beta activity, and enhanced decrements in digit-symbol substitution test performance, attributable to coadministration of ketoconazole. Plasma ketoconazole concentrations measured in the clinical study ranged from 0.02 microgram/ml (projected minimum) to 4.95 micrograms/ml (maximum). An in vitro-in vivo scaling model, using these plasma ketoconazole concentrations together with liver partition ratios and the in vitro Ki values, predicted a decrement of triazolam clearance due to ketoconazole coadministration that was consistent with the 88% decrement in clearance actually observed in vivo.

Brain efflux index as a novel method of analyzing efflux transport at the blood-brain barrier.

Abstract: A brain efflux index method has been developed to characterize an efflux transport system for substrates from the cerebrum to the circulating blood across the blood-brain barrier. The brain efflux index value is defined as the relative amount of test drug effluxed from cerebrum compared with that of a reference compound, [14C]carboxylinulin, which has limited blood-brain barrier permeability. Microinjection of 0.2, 0.5 or 1.0 microliter into the parietal cortex area 2 region was found to be an appropriate procedure for obtaining a high recovery and for restricting the test drug and reference compound to the ipsilateral cerebrum. No significant increase in influx clearance of [14C]carboxylinulin into the brain was observed for the ipsilateral cerebrum after the sham microinjection compared with the contralateral cerebrum, which demonstrated that only limited physical damage is caused. Microinjection of [3H]H2O into the cerebrum devoid of cerebral blood flow yielded no elimination from the brain. Analysis based on a pharmacokinetic model demonstrated that the elimination of a highly permeable compound [3H]H2O from the brain was governed by cerebral blood flow. The apparent elimination rate constant (Kel) of [3H]3-O-methyl-D-glucose from the brain was determined as 0.129 +/- 0.014 (min-1) and was reduced significantly by a preinjection of excess cold 3-O-methyl-D-glucose and phloridzin, whereas no significant elimination was observed for L[3H]glucose. The efflux clearance of 3-O-methyl-D-glucose was calculated from the Kel value and the compound's distribution volume, the value being close to that of the influx clearance. These results demonstrate that the brain efflux index method is a useful technique for analyzing an efflux process from the brain across the blood-brain barrier involving a carrier-mediated transport system.

Pharmacological characterization of A-127722: an orally active and highly potent ETA-selective receptor antagonist.

Abstract: Endothelins (ET) are potent vasoactive peptides implicated in the pathogenesis of a number of vascular diseases. The effects of ET on mammalian organs and cells are initiated by binding to ETA or ETB receptors. In this report, we document the pharmacology of A-127722, a novel ETA-selective receptor antagonist. A-127722 inhibits [125I]ET-1 binding to cloned human ETA and ETB receptors competitively with Ki values of 69 pM and 139 nM, respectively. A-127722 exhibits a dose-dependent inhibition of ET-1-induced arachidonic acid release in human pericardium smooth muscle cells with a pA2 value of 10.5 and inhibits ET-1-induced vasoconstriction in isolated rat aorta with a pA2 value of 9.2. In vivo, A-127722 dose-dependently blocks the pressor response to ET-1 (0.3 nmol/kg i.v.) in conscious rats. Statistically significant (P < .05) antagonism is seen at doses greater than 0.1 mg/kg p.o. Maximal inhibition, at 10 mg/kg, remains constant for at least 8 hr after dosing. No effect is seen on the ETB-mediated transient vasodepressor effect of exogenous ET-1. In conclusion, A-127722 is ETA-selective, orally bioavailable and efficacious for inhibiting the effects of ET in the rat, and A-127722 is the most potent ET receptor antagonist yet reported.

Identification of the human cytochromes P450 responsible for the in vitro formation of the major oxidative metabolites of the antipsychotic agent olanzapine.

Abstract: The formation kinetics of 2-hydroxymethyl olanzapine (2-OH olanzapine), 4'-N-oxide olanzapine (N-O olanzapine) and 4'-N-desmethyl olanzapine (NdM olanzapine) were analyzed in vitro. Biphasic kinetics were observed for formation of 2-OH and NdM olanzapine. The high-affinity enzyme responsible for 2-OH olanzapine formation by two human liver samples exhibited an intrinsic clearance (CLint) of 0.2 microliter/min/mg. NdM olanzapine formation by two human liver samples exhibited a CLint of 1.0 microliter/min/mg for the high affinity enzyme. The formation of N-O olanzapine was linear up to 300 microM olanzapine, yielding a CLint of 0.32 to 1.70 microliters/min/mg. The formation of 7-hydroxy olanzapine (7-OH olanzapine) exhibited an apparent Km of 24.2 microM. The rates of 2-OH olanzapine formation correlated with CYP2D6 levels and activity, and it was formed to the greatest extent by cDNA-expressed CYP2D6. N-O olanzapine formation correlated with human liver flavin-containing monooxygenase (FMO3) levels and activity. NdM olanzapine and 7-OH olanzapine formation correlated with CYP1A2 catalytic activities and they were formed to the greatest extent by expressed CYP1A2. These results suggest that CYP1A2 catalyzes NdM olanzapine and 7-OH olanzapine formation, CYP2D6 catalyzes 2-OH olanzapine formation and FMO3 catalyzes N-O olanzapine formation.

Electrically evoked acetylcholine release from hippocampal slices is inhibited by the cannabinoid receptor agonist, WIN 55212-2, and is potentiated by the cannabinoid antagonist, SR 141716A.

Abstract: This study examined the effect of the cannabinoid receptor agonist, WIN 55212-2, on the electrically evoked release of [14C]acetylcholine (ACh) from superfused brain slices from the hippocampus, a region with a high density of cannabinoid receptors. A comparison was also made with [14C]ACh release from the nucleus accumbens, which has relatively fewer cannabinoid receptors. In the hippocampal slices, WIN 55212-2 produced a dose-dependent inhibition of [14C]ACh release, with an EC50 of 0.03 microM and a maximal inhibition of 81% at 1 microM. In the nucleus accumbens slices, WIN 55212-2 produced a weak inhibition of [14C]ACh release, which did not quite reach statistical significance. The inhibition of electrically evoked hippocampal [14C]ACh release by WIN 55212-2 could be prevented by the cannabinoid receptor antagonist, SR 141716A (EC50, 0.3-1.0 microM). In addition to antagonizing the effects of WIN 55212-2, SR 141716A alone produced a 2-fold potentiation of the electrically stimulated [14C]ACh release in this region (EC50, 0.1-0.3 microM). By contrast, in nucleus accumbens slices, no potentiation of the stimulated release of [14C]ACh release by SR 141716A was observed. Basal [14C]ACh release was unaffected by WIN 55212-2 or SR 141716A in either area. These results suggest that cannabinoid receptor activation can produce a strong inhibition of ACh release in the hippocampus. Furthermore, the potentiation of ACh release in the hippocampus by SR 141716A alone suggests either that this compound is an inverse agonist at cannabinoid receptors or it is antagonizing the actions of an endogenous ligand acting on these receptors.

The non-xanthine heterocyclic compound SCH 58261 is a new potent and selective A2a adenosine receptor antagonist.

Abstract: We have characterized the in vitro pharmacological profile of the new potent and selective A2a adenosine receptor antagonist SCH 58261 [7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine]. In binding studies on rat and bovine brain tissues, SCH 58261 showed affinity in the low nanomolar range at A2a adenosine striatal receptors and good A2a adenosine vs. A1 adenosine selectivity (about 50- to 100-fold in rat and bovine brain, respectively). SCH 58261 did not show affinity for either the A3 adenosine receptor or other receptors at concentrations up to 1 microM. Saturation experiments on rat A1 and A2a adenosine receptors indicated the competitive nature of the antagonism. SCH 58261 antagonized competitively the effects induced by the A2a adenosine-selective agonist CGS 21680 (2-[4-(2-carboxyethyl)-phenethyl-amino]-5'-N- ethylcarboxamidoadenosine) in two functional assays, such as inhibition of rabbit platelet aggregation and porcine coronary artery relaxation. Specifically, the compound showed pA2 values of 7.9 and 9.5, respectively. SCH 58261 (300 nM) failed to antagonize 5'-N-ethylcarboxamidoadenosine-induced vasorelaxation in the isolated guinea pig aorta, a response mediated by A2b adenosine receptors. Likewise, at the same concentration, the compound weakly inhibited the A1 adenosine-mediated negative chronotropic effect induced by 2-chloro-N6-cyclopentyladenosine in the isolated rat atria. These data show that SCH 58261 is a potent and selective non-xanthine A2a adenosine antagonist which has competitive properties in biological responses mediated by this receptor subtype. The compound is of interest for investigating the biological role of A2a adenosine receptors and deserves further attention to clarify the therapeutic potential of A2a antagonists.

Characterization of opioid agonist efficacy in a C6 glioma cell line expressing the mu opioid receptor.

Abstract: In C6 glioma cells stably expressing a homogeneous population of the cloned rat mu opioid receptor, the binding affinities of opioid agonists and subsequent activation of G protein were examined. Opioid receptor number in membranes of these cells was high (10-30 pmol/mg protein [3H]diprenorphine binding sites). Opioids were found to bind to the receptor with high affinity [Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO) 0.23 nM; sufentanil 0.034 nM; morphine 0.16 nM]. Activation of G protein by opioid agonists was examined by measuring the stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding. Sufentanil increased [35S]GTP gamma S binding by 326% with an EC50 value of 2.39 nM. Agonist stimulation of [35S]GTP gamma S binding was stereoselective, naltrexone-reversible, and pertussis toxin-sensitive. The "intrinsic activity" of opioids at the mu receptor was reflected by the magnitude of agonist-mediated activation of G protein. The rank order of the stimulation of [35S]GTP gamma S binding was etonitazene = sufentanil = DAMGO = PLO17 = fentanyl > morphine > profadol > meperidine > butorphanol = nalbuphine = pentazocine > cyclazocine = nalorphine > levallorphan > naltrexone. High affinity binding of ligands to the mu opioid receptor was reduced by the addition of sodium and guanosine diphosphate at concentrations used in the [35S]GTP gamma S binding assay. Ligand affinity was reduced in a manner correlating with "intrinsic activity". DAMGO, 1229-fold, nalbuphine 35-fold, naltrexone, 3-fold. The results presented show that the stable expression of the rat mu opioid receptor in C6 cells provides an effective tool to examine opioid receptor signal transduction mechanisms and evaluate the activity of novel opioids at the mu receptor.

Metabolism of cocaine and heroin is catalyzed by the same human liver carboxylesterases.

Abstract: Concomitant i.v. use of cocaine and heroin ("speedballing") is prevalent among drug-abusing populations. Heroin is rapidly metabolized by sequential deacetylation of two separate ester bonds to yield 6-monoacetylmorphine and morphine. Hydrolysis of heroin to 6-monoacetylmorphine is catalyzed by pseudocholinesterase. The pathway for hydrolysis of 6-monoacetylmorphine to morphine in vivo has yet to be established. Pseudocholinesterase and two human liver carboxylesterases [human liver carboxylesterase form 1 (hCE-1) and human liver carboxylesterase form 2 (hCE-2)] catalyze the rapid hydrolysis of ester linkages in cocaine. This investigation examined the relative catalytic efficiencies of hCE-1, hCE-2 and pseudocholinesterase for heroin metabolism and compared them with cocaine hydrolysis. Enzymatic formation of 6-monoacetylmorphine and morphine was determined by reverse-phase high-performance liquid chromatography. All three enzymes rapidly catalyzed hydrolysis of heroin to 6-monoacetylmorphine (hCE-1 kcat = 439 min-1, hCE-2 kcat = 2186 min-1 and pseudocholinesterase kcat = 13 min-1). The catalytic efficiency, under first-order conditions, for hCE-2-catalyzed formation of 6-monoacetylmorphine (314 min-1 mM-1) was much greater than that for either hCE-1 or pseudocholinesterase (69 and 4 min-1 mM-1, respectively). Similarly, the catalytic efficiency for hydrolysis of 6-monoacetylmorphine to morphine by hCE-2 (22 min-1 mM-1) was substantially greater than that for hCE-1 (0.024 min-1 mM-1). Cocaine competitively inhibited hCE-1-, hCE-2- and pseudocholinesterase-catalyzed hydrolysis of heroin to 6-monoacetylmorphine (Ki = 530, 460 and 130 microM, respectively) and 6-monoacetylmorphine hydrolysis to morphine (Ki = 710, 220 and 830 microM, respectively). These data demonstrate that metabolism of cocaine and heroin in humans is mediated by common metabolic pathways. The role of hepatic hCE-2 is particularly important for the hydrolysis of heroin to 6-monoacetylmorphine and of 6-monoacetylmorphine to morphine.

Stable expression, pharmacologic properties and regulation of the human neuronal nicotinic acetylcholine alpha 4 beta 2 receptor.

Abstract: (-)-Nicotine, the prototypical agonist for neuronal nicotinic acetylcholine receptors (nAChR) has been shown to bind with high affinity to the rodent and avian alpha 4 beta 2 nAChR subtype. This subtype may represent a primary molecular target for some of the beneficial central nervous system effects i.e., cognitive enhancement, anxiolysis, analgesia, neuroprotection, of (-)-nicotine and related ligands. However, a detailed study of the human alpha 4 beta 2 subunit combination has not yet been reported. In this study, we stably coexpressed the human neuronal alpha 4 and beta 2 nAChR subunits in human embryonic kidney (HEK) 293 cells and studied its pharmacological and regulatory properties. [3H]Cytisine bound to stably transfected cells with high affinity (KD value, 0.2 +/- 0.04 nM) and with a Bmax value of 1359 +/- 91 fmol/mg protein. A good correlation (r = 0.98) was observed between binding affinities in transfected cells and in native neuronal preparations for a series of nAChR ligands. 86Rb+ efflux studies showed that stably transfected cells express functional ion channels that are sensitive to blockade by dihydro-beta-erythroidine. (+/-)-Epibatidine, (-)-nicotine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418), acetylcholine and (-)-cytisine stimulated 86Rb+ efflux with EC50 values of 0.02, 3.9, 2.5, 10, 44 and 38 microM, respectively. Treatment of transfected cells with (-)-nicotine for 7 days led to a significant increase in the density of [3H](-)-cytisine binding sites (EC50 = 0.56 microM) and a significant enhancement in the sensitivity of ACh. Specific binding or (-)-nicotine-evoked cation efflux was not detected in untransfected cells. Analysis of total cellular RNA from transfected, but not untransfected cells, showed the expected fragment sizes corresponding to the human alpha 4 and beta 2 subunit mRNA. These results demonstrate that stable expression of the human alpha 4 beta 2 nAChR subunit combination can give rise to functional ion channels that bind [3H](-)-cytisine with high affinity, exhibit homologous regulation and evoke agonist-induced cation flux with pharmacological properties consistent with native neuronal alpha 4 beta 2 nAChR.

Block of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by polyamines and polyamine toxins.

Abstract: A variety of polyamine spider and wasp toxins are known to block N-methyl-D-aspartate receptor channels and recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the edited glutamate receptor (GluR)2 subunit. Recently, inward rectification of GluR2-lacking AMPA receptors was shown to be caused by voltage-dependent block by intracellular spermine. Here we demonstrate that, when applied extracellularly, the endogenous polyamines spermine and spermidine, as well as monoacylated spermine analogs and the polyamine toxins ageltoxin-489 and philanthotoxin-433, exerted a use-dependent and weakly voltage-dependent block of AMPA receptors that lack the edited GluR2 subunit, when the recombinant receptors were expressed in Xenopus oocytes. External spermine and polyamine toxins were also effective blockers of AMPA receptor mutants that did not not show inwardly rectifying kainate responses but had high calcium permeability. The polyamines and polyamine toxins also markedly reduced inwardly rectifying currents of native AMPA receptors expressed by a class of hippocampal interneurons in rat CA3 stratum radiatum that appear not to express the GluR2 subunit. In contrast, polyamines had little or no effect on the linear or outwardly rectifying kainate responses of other interneurons or CA3 pyramidal cells in which GluR2 mRNA was routinely detected. Together with previous reports, these data suggest that endogenous polyamines may bind to GluR2-lacking AMPA receptors at two or more distinct sites, one near the cytoplasmic side of the pore and the other nearer the outer side of the pore.

Protection of acetaminophen-induced hepatocellular apoptosis and necrosis by cholesteryl hemisuccinate pretreatment.

Abstract: This study of acetaminophen (AAP) hepatotoxicity examined whether some aspects of the highly integrated process of drug-induced toxicity involves apoptosis, in addition to necrosis in vivo; and if so, whether cholesteryl hemisuccinate (CS) pretreatment would selectively interfere with apoptotic or necrotic liver cell death. We have previously demonstrated that CS preexposure in vivo, protects hepatocellular necrosis and necrosis-related events induced by carbon tetrachloride (CCl4) administration. Our study demonstrates that administration of hepatotoxic doses of AAP (350-500 mg/kg, i.p.) to ICR mice (CD-1) results in severe liver injury leading to cell death both by necrosis and apoptosis. AAP-induced cell death was preceded by massive elevation in serum alanine aminotransferase coupled with rapid loss of large genomic DNA (2-24 hr), fragmentation of DNA in the form of a ladder (2-24 hr), apoptotic nuclear condensation at early hours (2-6 hr) followed by massive fragmentation and margination of heterochromatin at later (6-24) hours and a near total loss of glycogen in pericentral areas. Although CS (100 mg/kg, i.p.) alone had no noticeable biochemical or morphological effects, its administration before AAP (350-500 mg/kg, i.p.) abrogated histological and biochemical diagnostics of both apoptosis and necrosis. These include near total absence of loss of large genomic DNA and glycogen, and dramatic protection from escalating levels of liver injury. CS pretreatment also arrested AAP-induced ultrastructural changes typical of both apoptosis and necrosis. Histopathological examination of periodic acid-Schiff stained liver sections mirrored the biochemical and ultrastructural findings. In conclusion, this study for the first time establishes that apoptosis, in addition to necrosis, significantly contributes to AAP hepatotoxicity in vivo, and preexposure of mice to CS prevents AAP-induced hepatic apoptosis and necrosis.

Sensitization of gamma-aminobutyric acidA receptors to neuroactive steroids in rats during ethanol withdrawal.

Abstract: The anxiolytic and anticonvulsant effects of benzodiazepines, barbiturates, ethanol and neuroactive steroids are mediated by selective interactions with gamma-aminobutyric acidA (GABA(A)) receptors. Chronic ethanol exposure decreases the sensitivity of GABA(A) receptors to benzodiazepines, barbiturates and ethanol. Ethanol withdrawing rats are cross-tolerant to the anticonvulsant effects of benzodiazepines as shown by a 16% decrease in the anticonvulsant efficacy of diazepam compared to controls. In contrast, ethanol withdrawing rats are sensitized to the anticonvulsant effects of the neuroactive steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-THP), exhibiting a 46% increase in the anticonvulsant effect against bicuculline-induced seizures compared to control rats. This effect may involve a change in the sensitivity of GABA(A) receptors to 3 alpha,5 alpha-THP because potentiation of GABA(A) receptor mediated chloride uptake into cerebral cortical synaptoneurosomes is enhanced by 3 alpha,5 alpha-THP up to 50% in ethanol withdrawing rats compared to controls. 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (THDOC) potentiation of GABA(A) receptor-mediated chloride uptake is also enhanced during ethanol withdrawal. Moreover, the plasma levels of 3 alpha,5 alpha-THP and progesterone did not differ in ethanol withdrawing rats compared to controls. These alterations in neurosteroid sensitivity were also accompanied by selective alterations in cortical GABA(A) receptor subunit mRNA levels. Levels for the alpha 1 and alpha 4 subunit showed only slight alteration during withdrawal whereas we had previously observed a significant decrease in alpha 1 and a significant increase in alpha 4 mRNA levels in ethanol dependent (not withdrawing) animals. beta 2, beta and gamma 1 mRNA levels significantly increased during ethanol withdrawal. Taken together, these results suggest that ethanol withdrawal produces alterations in GABA(A) receptors that sensitize rats to the pharmacological effects of neuroactive steroids. Because ethanol-dependent or withdrawing rats are tolerant to the intoxicating, anxiolytic and anticonvulsant effects of ethanol and cross-tolerant to many effects of benzodiazepines and barbiturates, sensitization to the effects of neuroactive steroids could have significant therapeutic potential.

Investigation of the effects of opiate antagonists infused into the nucleus accumbens on feeding and sucrose drinking in rats.

Abstract: Previous work has shown that opiate agonist infusion into the nucleus accumbens, a region implicated in reinforcement, stimulates food intake. In the present study, the effects of opiate antagonist infusion into this region were examined in two behavioral paradigms. In the feeding test, food-deprived animals were tested for intake of laboratory chow. In the sucrose intake test, sated animals familiar with a 20% sucrose solution were tested. Before these tests, the following drugs were bilaterally infused into the accumbens: naloxone (0, 1, 10 and 30 micrograms; equivalent to 2.8, 28 and 83 nmol, respectively), naltrexone (0, 0.2, 2 and 20 micrograms; 0.55, 5.5 and 55 nmol, respectively), beta-funaltrexamine (0 and 15 micrograms; 31 nmol), naloxonazine (0 and 10 micrograms; 15 nmol), naltrindole (trial 1: 0, 1, 10 and 20 micrograms; 2.2, 22 and 44 nmol, respectively; trial 2: 0, 0.1 and 0.5 micrograms; 0.22 and 1.1 nmol, respectively) and nor-binaltorphimine (0, 0.1, 1 and 10 micrograms; 0.14, 1.4 and 14 nmol, respectively). Naloxone and naltrexone both significantly reduced sucrose drinking and did not affect feeding. Naloxone infused into the dorsolateral striatum, as a control, had no effect on sucrose drinking. Accumbens infusion of the mu antagonist beta-funaltrexamine reduced both sucrose drinking and feeding. The mu 1 antagonist naloxonazine did not influence intake behaviors, with the exception of a decrease in duration of chow feeding. In contrast, the delta antagonist naltrindole markedly potentiated both sucrose drinking and duration of chow feeding. In a replication of this effect, systemic naltrexone given concurrently blocked the enhancement. The kappa antagonist nor-binaltorphimine did not influence any parameters of ingestive behavior. Although some treatments also decreased motor activities, the overall profile of behavior suggested specific effects on ingestive behavior. The putative contributions of mu and delta receptors within the nucleus accumbens to modulation of food reward are discussed.

Role of extracellular dopamine in the initiation and long-term expression of behavioral sensitization to cocaine.

Abstract: Repeated intermittent administration of cocaine has been shown to sensitize animals to the locomotor-activating effects of this agent. The neurobiochemical basis of this phenomenon, however, remains only partially understood. The present study sought to characterize basal dialysate dopamine (DA) concentrations within the nucleus accumbens (NAc), 2, 12 or 22 days after the cessation of either repeated cocaine (20 mg/kg/day x 5 days) or saline (1.0 ml/kg/day x 5 days) treatment. Locomotor activity and dialysate DA levels in response to a subsequent cocaine administration (20 mg/kg i.p.) were assessed at the same time intervals. Cocaine-pretreated animals exhibited an enhanced motor response to a cocaine injection 2 days after cessation of cocaine treatment. The magnitude of this effect increased progressively over time. Basal DA overflow was elevated 2 days after termination of cocaine treatment; at this time, however, a blunted response of DA neurons to the cocaine administration was observed. As the duration of withdrawal increased, basal dialysate DA concentrations gradually declined, whereas the response of DA neurons to cocaine progressively increased. By day 22 of withdrawal, a significant enhancement of cocaine-induced DA overflow was seen. These findings demonstrate that increased DA overflow in response to cocaine cannot account for the short-term expression of behavioral sensitization to cocaine. Rather, an enhanced DA response develops during later stages of the sensitization process and, therefore, may be one of the mechanisms responsible for the long-term expression of cocaine sensitization.

RJR-2403: a nicotinic agonist with CNS selectivity II. In vivo characterization.

Abstract: We have evaluated the physiological and behavioral effects of the CNS-selective nicotinic agonist (E)-N-methyl-4-(3-pyridinyl) -3-butene-1-amine (RJR-2403) using a number of different methods, including 1) reversal of pharmacologically induced amnesia in a step-through passive avoidance paradigm, 2) radial arm maze performance in rats with chemically induced brain lesions, 3) changes in HR and blood pressure in rats and 4) changes in body temperature, Y-maze activity, acoustic startle response and respiration in mice. Our results indicate that RJR-2403 is equal to or better than nicotine on measures of CNS function and cognitive enhancement. Specifically, RJR-2403 significantly improved passive avoidance retention after scopolamine-induced amnesia and enhanced both working and reference memory in rats with ibotenic acid lesions of the forebrain cholinergic projection system in an 8-arm radial maze paradigm. By comparison, RJR-2403 was 15 to 30-fold less potent than nicotine in decreasing body temperature, respiration, Y-maze rears and crosses and acoustic startle response. RJR-2403 also demonstrated greatly reduced cardiovascular effects. RJR-2403 was approximately 10-fold less potent than nicotine in increasing HR and 20-fold less potent in increasing blood pressure. These results are consistent with in vitro data indicating this compound's high selectivity for CNS nicotinic ACh receptor subtypes relative to peripheral ganglionic and muscle-type nicotinic ACh receptors. Therefore, RJR-2403 may be a valuable tool for understanding the central and peripheral pharmacology of nicotinic cholinergic systems as well as a potential lead compound for the development of nicotinic therapeutics to treat neurological diseases where cholinergic neurotransmission has been compromised.

Pharmacogenetic determination of the effects of codeine and prediction of drug interactions.

Abstract: To define the differences in codeine pharmacodynamics in extensive (EMs) and poor (PMs) metabolizers of debrisoquin and to determine whether the inhibition of codeine's metabolism by quinidine produces phenotypically dependent pharmacodynamic changes, we studied 16 healthy nonsmoking males, 10 EMs and 6 PMs of debrisoquin. The subjects received in random double-blind fashion 120 mg of codeine plus placebo, 120 mg of codeine plus 100 mg of quinidine and 100 mg of quinidine plus placebo. Blood was obtained over 24 hr and urine was collected for 48 hr. Respiratory, psychomotor and pupillary effects of codeine were greater in the EMs than in the PMs (P < .01). Morphine and morphine metabolites were detectable only in plasma from EMs. Codeine metabolic clearance by O-demethylation was almost 200-fold greater in the EMs than in the PMs. After coadministration of quinidine, morphine and morphine metabolites were not detectable in the plasma of either phenotype and mean (+/- S.E.M.) O-demethylation clearance was reduced in the EMs from 162.7 +/- 36.6 to 17.0 +/- 5.0 ml/min (P < .003), but not in the PMs. The diminished production of morphine in the EMs was associated with significantly reduced respiratory, psychomotor and pupillary effects (P < .01). Thus, CYP2D6 mediated O-demethylation of codeine to morphine is central to its pharmacodynamic effects. Patients who lack CYP2D6 or whose CYP2D6 is inhibited would not be expected to benefit from codeine. Thus, phenotyping for CYP2D6 and the avoidance of CYP2D6 inhibitors is justified in patients with chronic path before initiating long-term therapy with analgesics whose in vivo activation is dependent on CYP2D6 activity (i.e., codeine, hydrocodone and oxycodone.