Caudate-putamen dopamine and stereotypy response profiles after intravenous and subcutaneous amphetamine.

We compared the behavioral and caudate-putamen extracellular dopamine responses following intravenous (3.6 mg/kg) and subcutaneous (8 mg/kg) amphetamine administration using 2-min microdialysate sampling intervals, and doses of the drug selected to achieve comparable maximal brain concentrations. Following intravenous amphetamine, dopamine peaked within the first 2 min, then declined with a first-order decay rate of 0.018+/-0.007 min(-1). Following subcutaneous amphetamine, dopamine achieved maximum concentrations at 9 min and remained near peak levels for about 30 min before declining with a first-order decay rate of 0.019+/-0.008 min(-1). Maximal brain amphetamine levels and peak dopamine concentrations were equivalent following either route of drug administration. In contrast to the short latency to maximal extracellular dopamine, the onset of oral stereotypies was delayed until about 30 min following both routes of drug administration. Furthermore, in contrast to the behavioral response to amphetamine, apomorphine administration resulted in the rapid appearance of oral stereotypies within 5-10 min after drug administration. These results suggest that although caudate-putamen dopamine receptor activation may be a critical factor in the expression of focused oral stereotypies, other effects of amphetamine may interfere with the ability of animals to exhibit these behaviors.

Oxidation of methamphetamine and methylenedioxymethamphetamine by CYP2D6.

Methamphetamine (MeAmp) abuse has recently experienced a resurgence and approaches to the treatment of its addiction similar to those used with cocaine have been considered. As the treatment regimes are likely to use drugs whose metabolism is related to that of MeAmp, studies were initiated to establish the enzymology of the fate of MeAmp. This report describes investigations of the role of CYP2D6, the human isoform of the enzyme that catalyzes debrisoquine hydroxylation, in the 4-hydroxylation and N-demethylation of MeAmp. The results of studies with human liver microsomes including those from a genetically poor metabolizer with respect to CYP2D6, showing correlation between MeAmp and metoprolol hydroxylation and MDMA demethylenation, were consistent with a major involvement of CYP2D6 in the aromatic 4-hydroxylation of MeAmp. This was confirmed by studies with recombinant CYP2D6 expressed in yeast, which was also shown to effect the N-demethylation of MeAmp. The rate of the 4-hydroxylation reaction was substantially slower than the demethylenation of MDMA. In contrast to MeAmp, MDMA was not N-demethylated by CYP2D6. Since CYP2D6 participates in the major steps of MeAmp metabolism, pharmacokinetic interactions are likely with other drug substrates proposed for the treatment of MeAmp addiction. Furthermore, the genetic polymorphism associated with the enzyme could manifest itself in abnormal responses to MeAmp.

Arginase activity in endothelial cells: inhibition by NG-hydroxy-L-arginine during high-output NO production.

Rat aortic endothelial cells were found to contain both constitutive and lipopolysaccharide (LPS)-inducible arginase activity. Studies were performed to determine whether induction of nitric oxide synthase (NOS) by LPS and cytokines is accompanied by sufficient arginase induction to render arginine concentrations rate limiting for high-output NO production. Unactivated cells contained abundant arginase activity accompanied by continuous urea formation. LPS induced the formation of both inducible NOS (iNOS) and arginase, and this was accompanied by increased production of NO, citrulline, and urea. Immunoprecipitation experiments revealed the constitutive presence of arginase-I in both unactivated and LPS-activated cells and arginase-II induction by LPS. Arginase-I and iNOS were verified by reverse transcriptase-polymerase chain reaction. Induction of large amounts of iNOS by LPS plus several cytokines resulted in large quantities of NO, citrulline, and NG-hydroxy-L-arginine (NOHA), but urea production was markedly diminished. Decreased urea production was attributed to increased formation of NOHA, the precursor to NO and citrulline and a potent inhibitor of arginase-I activity with an inhibitory constant of 10-12 microM. Inhibition of iNOS activity by NG-methyl-L-arginine decreased NO and NOHA production and increased urea production. This study reveals for the first time that substantial arginase activity is present constitutively in rat aortic endothelial cells, a different isoform of arginase is induced by LPS, and intracellular arginase activity can be markedly inhibited during cytokine induction of iNOS because of NOHA formation. The inhibition of arginase activity that occurs by NOHA during marked iNOS induction may be a mechanism to ensure sufficient arginine availability for high-output production of NO.

Selective mechanism-based inactivation of rat CYP2D by 4-allyloxymethamphetamine.

The high selectivity of amphetamine and its derivatives for CYP2D-mediated oxidations suggested the use of the phenylisopropylamine skeleton as a template for a selective inhibitor of this important enzyme. Accordingly, 4-allyloxymethamphetamine-amine (ALLMA) was synthesized and its ability to selectively inactivate CYP2D was investigated both in in vitro and in vivo experiments. Incubation studies with rat liver microsomes demonstrated that this compound suppressed the CYP2D-mediated methylenedioxymethamphetamine (MDMA) demethylation in time- and dose-dependent manner and that the inhibition required the presence of NADPH. The development of irreversible inhibition was associated with oxidation at position 4 of the aromatic ring, the common site of CYP2D-mediated oxidation of this group of compounds. In in vivo studies doses of ALLMA (1-10 mg/kg) were administered to adult male Sprague-Dawley rats and liver microsomes were obtained 3 hr later. Methamphetamine p-hydroxylation and low Km MDMA demethylation activities, both mediated by CYP2D, were reduced by more than 80% after a dose of 10 mg/kg. Cytochrome P-450 reactions attributed to P-450s other than CYP2D, such as aniline p-hydroxylation, the high Km system of MDMA demethylation and the N-demethylation of methamphetamine, benzphetamine, aminopyrine and erythromycin, all appeared to be minimally affected. The importance of aromatic ring oxidation in the metabolism is such that inhibition of CYP2D would be expected to cause a significant change in the pharmacokinetics of these compounds. The kinetics of MDMA metabolic activity in microsomes from ALLMA-pretreated rats were comparable to those from female Dark-Agouti rats, an animal model for CYP2D1 deficiency.

Pharmacokinetic and pharmacodynamic analysis of the actions of D-amphetamine and D-methamphetamine on the dopamine terminal.

To establish whether the actions of D-amphetamine (Amp) and D-methamphetamine (MeAmp) on the striatal dopamine system were equipotent, pharmacokinetic profiles of each drug were applied to an analysis of their respective induced dopamine efflux profiles. Amp or MeAmp (1 and 5 mg/kg i.v.) was administered to chloral hydrate-anesthetized rats; plasma and brain kinetics were then assessed from 5 to 60 min. Dose-dependent increases in Amp and MeAmp plasma levels resulted in proportional increases in striatum levels that were equivalent for both drugs; elimination rates also were similar and were characterized by a first-order decay process. After MeAmp administration, low levels of brain MeAmp metabolites were detected throughout the 1-hr time period; relative to MeAmp, Amp and p-hydroxy-MeAmp levels were less than 10 and 1%, respectively. The drug-induced dopamine efflux profiles in the striatum were characterized by microdialysis; Amp and MeAmp (1, 2.5 and 5 mg/kg i.v.) effected equivalent, dose-dependent increases in extracellular dopamine levels. For both drugs at 5- and 10-min postinjection, increases in drug striatum levels preceded increases in dopamine efflux. In contrast, from the time of the peak dopamine responses observed at 10 to 20 min until the end of the study at 90 min, changes in striatal drug levels were correlated with extracellular dopamine levels; this correlation was similar for both drugs. These results indicate that Amp and MeAmp pharmacokinetics and their subsequent dopamine responses in the striatum are equivalent. The pharmacokinetic analysis can be extended to the interpretation of other comparative studies that assess effects of Amp and MeAmp.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of constitutive hepatic cytochromes P450 by phencyclidine in the rat.

The purpose of this study was to determine whether phencyclidine (PCP) inhibits constitutive hepatic cytochrome P450 (CYP) isozymes when administered to naive adult male Sprague-Dawley rats. Animals were pretreated with PCP (25 mg/kg/day for 2 days), killed 3 and 16 hr after the last dose, and liver microsomes prepared. The washed microsomes were then assayed for benzphetamine, methamphetamine (MA), and methylenedioxymethamphetamine (MDMA) N-demethylation together with MDMA demethylenation and MA 4-hydroxylation activities. MDMA demethylenation (low substrate concentration), MA 4-hydroxylation, and metoprolol alpha-hydroxylation reactions, which are catalyzed by CYP2D isozymes, were reduced > 74% 3 hr after the last PCP dose and were only partially restored 13 hr later. Benzphetamine and (-)-MDMA N-demethylation activities were restored to control values 16 hr after the last dose. These results indicate that PCP suppresses constitutive isozymes, including CYP2C11 and members of the CYP2D subfamily. The suppression of cytochromes P450 activity by PCP in vivo is consistent with its in vitro actions found in this and other studies, and demonstrates that alteration of CYP activity is another pharmacological effect of this compound.

The demethylenation of methylenedioxymethamphetamine ("ecstasy") by debrisoquine hydroxylase (CYP2D6).

The metabolism of methylenedioxymethamphetamine (MDMA, "ecstasy") was examined in a microsomal preparation of the yeast Saccharomyces cerevisiae expressing human debrisoquine hydroxylase, CYP2D6. Only one product, dihydroxymethylamphetamine (DHMA), was detected in the incubation mixture, and this product accounted for all of the substrate consumption at low concentration (10 microM). Mean +/- SD values of apparent Km(microM) and Vmax (nmol/min per nmol P450) for the demethylenation of (+) and (-)-MDMA at low concentrations (1-100 microM) were 1.72, 0.12 and 6.45, 0.10 and 2.90, 0.10 and 7.61, 0.06, respectively. At high concentrations (> 1000 microM) substrate inhibition was noted, with Ki values of 14.2 and 28.2 mM, respectively, for the (+) and (-) enantiomers. Incubation of MDMA isomers with human liver microsomes indicated that their demethylenation is deficient in the poor metabolizer phenotype. Thus, MDMA is converted to the catecholamine DHMA by CYP2D6, and this may give rise to genetically-determined differences in toxicity.

A behavioral and pharmacokinetic study of the actions of phenylcyclohexyldiethylamine and its active metabolite, phenylcyclohexylethylamine.

Phenylcyclohexyldiethylamine (PCDE) is an analog of phencyclidine with low affinity for the N-methyl-d-aspartate receptor that is metabolized to an active monoethyl derivative, phenylcyclohexylethylamine (PCE). In a pharmacokinetic analysis of the ataxia response of rats to i.p. administered PCDE and PCE, ataxia intensity was determined together with plasma and cerebrospinal fluid concentrations of the drugs. The role of PCE as the active metabolite of PCDE was assessed quantitatively by correlating the response with both the plasma and cerebrospinal fluid drug levels. Increased PCE concentrations in the cerebrospinal fluid and plasma were associated with increased ataxia response when either PCDE or PCE was the administered drug. However, the concentration-response curves did not superimpose and the curve after PCDE was shifted to the left of that after PCE, suggesting that PCDE was contributing an effect not accountable by PCE concentration. This apparent potentiation must involve an interaction at sites other than the N-methyl-daspartate receptor. In the analysis of the behavior responses, PCDE was found to induce a greater backpedalling response which has been attributed to interaction with dopamine or serotonin systems, suggesting that other transmitter systems may contribute to the overall ataxia response.

A pharmacokinetic study of phenylcyclohexyldiethylamine. An analog of phencyclidine.

The pharmacokinetics of three phencyclidine analogs--phenylcyclohexyl-diethylamine (PCDE), phenylcyclohexylethylamine (PCE), and phenylcyclohexylamine (PCA)--were determined in rats after intravenous administration of each drug. Because PCE and PCA are major metabolites of PCDE, their plasma levels were also measured after administration of PCDE. Similarly, PCA concentrations was determined after administration of PCE. The data were combined and analyzed by nonlinear regression procedures using compartmental and noncompartmental models to determine the kinetic parameters of PCDE metabolism. The object was to estimate the kinetic constants for the metabolic sequence, PCDE to PCE to PCA. A 6-compartment model (two pools for each analyte) that included saturable components for the conversion of PCDE to PCE and PCE to PCA gave the best fit to the combined data. Despite large uncertainties for some microparameters, useful estimates were obtained for clearances, distribution volumes, and fraction of PCDE or PCE converted to PCE and PCA in vivo under nonsaturating conditions. The estimated fraction of PCDE converted to PCA and the apparent Km value for the conversion of PCDE to PCE were comparable to values obtained in vitro with microsomal preparations, suggesting that metabolic studies in vitro provide reasonable predictors of the biotransformation process in vivo for this class of compounds.

Aromatic hydroxylation of methylenedioxybenzene (MDB) and methylenedioxymethamphetamine (MDMA) by rabbit liver microsomes.

1. Metabolites formed during incubation of methylenedioxybenzene (MDB) and methylenedioxymethamphetamine (MDMA) with rabbit liver microsomes were examined by h.p.l.c.-electrochemical detection and g.l.c.-mass spectrometry. 2. The trifluoroacetyl derivative of metabolite M-1, obtained from MDB, had a molecular ion at m/z 234 and was identified as 3,4-methylenedioxy-6-hydroxybenzene (sesamol) by comparison with authentic material. 3. The trifluoroacetyl derivative of metabolite M-2, obtained from MDMA, exhibited a molecular ion at m/z 401. Experiments with the deuterium substituted variants of MDMA indicated that the product was hydroxylated on the aromatic ring. 4. The formation of these hydroxylated metabolites required NADPH and was inhibited by carbon monoxide, indicating the possible participation of cytochrome P-450. Phenobarbital (PB) induction caused a marked enhancement of MDP hydroxylase activity whereas MDMA hydroxylation was not affected. 5. The aromatic hydroxylation of MDB and MDMA was also observed in a reconstituted system with cytochrome P-450 isozyme IIB4.

Pharmacokinetic and pharmacodynamic properties of some phencyclidine analogs in rats.

The pharmacodynamics and pharmacokinetics of three phencyclidine analogs, differing from phencyclidine (PCP) only in the nature of the amine structure, were determined after intravenous doses of equimolar amounts to rats. The purpose of the study was to assess the role of pharmacokinetics in the in vivo potency of the compounds. The compounds examined were phenylcyclohexyl-pyrrolidine (PCPY), diethylamine (PCDE), ethylamine (PCE), and phencyclohexylamine (PCA). The behavior responses monitored included ataxia and others previously shown to be characteristic of PCP. In contrast to their relative affinities for the MK 801 binding site, the behavioral potencies of PCE, PCDE and PCPY were comparable to PCP. The major discrepancy occurred with PCDE, whose affinity for the NMDA receptor was 1/20th of PCP. The pharmacokinetic studies showed that the discrepancy between in vivo and in vitro activity of PCDE could be partially accounted for by its conversion to PCE, a relatively potent PCP-like agent.

Metabolism of methylenedioxyphenyl compounds by rabbit liver preparations. Participation of different cytochrome P450 isozymes in the demethylenation reaction.

The cytochrome P450-mediated oxidative demethylenation of the benzo-1,3-dioxoles (methylenedioxyphenyl compounds, MDPs), methylenedioxybenzene (MDB), methylenedioxyamphetamine (MDA), and methylenedioxymethamphetamine (MDMA), by rabbit liver microsomes and cytochrome P450IIB4 (CYP2B4) was examined. Material balance studies indicated that demethylenation to catechol derivatives is a major metabolic pathway for MDB, MDA and MDMA. The reactions required NADPH and were inhibited by CO/O2 (4:1, v/v). Biphasic double-reciprocal plots of MDMA, MDA and MDB oxidation suggested participation of more than one isozyme of cytochrome P450 in the reaction. Phenobarbital (PB) induction was selective in that the Vmax values for MDB were increased but not those for MDA and MDMA. Exposure of liver microsomes from PB-pretreated animals to phencyclidine (PCP) markedly suppressed MDB oxidation but had little effect on MDA and MDMA demethylenation. Reconstitution experiments with CYP2B4 demonstrated that MDB is a good substrate for the isozyme; but the relative demethylenation activities for MDA and MDMA were 1 and 2% of that for MDB. These results indicate that the PB-inducible isozymes such as CYP2B4 appear to play an important role in MDB demethylenation, whereas MDA and MDMA oxidation is mediated mainly by constitutive isozymes.

Hydroxyl radical mediated demethylenation of (methylenedioxy)phenyl compounds.

The oxidative demethylenation reactions of (methylendioxy)phenyl compounds (MDPs), (methylenedioxy)benzene (MDB), (methylenedioxy)amphetamine (MDA), and (methylenedioxy)methamphetamine (MDMA), were evaluated by using two hydroxyl radical generating systems, the autoxidation of ascorbate in the presence of iron-EDTA and the iron-catalyzed Haber-Weiss reaction conducted by xanthine/xanthine oxidase with iron-EDTA. Reaction products generated when MDB, MDA, and MDMA were incubated with the ascorbate or xanthine oxidase system were catechol, dihydroxyamphetamine (DHA), and dihydroxymethamphetamine (DHMA), respectively. The reaction required the presence of either ascorbic acid or xanthine oxidase. Levels of each catechol increased in proportion to ferric ion concentration and were suppressed by desferrioxamine B methanesulfonate (desferal). Catalase (CAT) inhibited the oxidation by the ascorbate system whereas superoxide dismutase (SOD) had little effect. The addition of hydrogen peroxide to the reaction mixture stimulated the oxidation, but the reaction was not initiated by hydrogen peroxide alone, suggesting that hydrogen peroxide acts as a precursor of hydroxyl radical. SOD and CAT suppressed the demethylenation reactions in the xanthine oxidase system. Hydroxyl radical scavenging agents such as ethanol, benzoate, DMSO, and thiourea effectively inhibited the oxidation by both systems. Urea, which has little effect on hydroxyl radical, was without any effect. These results indicated that hydroxyl radical can effect the cleavage of methylenedioxy group on MDPs.

A comparative in vitro metabolic study of methaphenilene and pyribenzamine.

1. In vitro metabolism of methaphenilene (MFN) and pyribenzamine (PBZ) was compared to that of methapyriline (MPH) in rat, because chronic treatment with MPH causes cancer in rats, whereas MFN and PBZ cause no cancer. 2. G.l.c. and mass spectrometry were used to identify 7 metabolites of MFN and 6 of PBZ in extracts of rat liver microsome incubations. 3. Quantification of the metabolic pathways revealed that N-oxide formation is considerably more important for both MFN and PBZ than for MPH, and only MPH forms an amide as a metabolic product. 4. Quantitative balance studies show that a lower recovery is apparent for metabolic experiments with MPH than for either MFN or PBZ under all conditions examined, indicating that significant metabolic pathways for MPH exist which are not being measured under these conditions.

The in vivo metabolism of methapyrilene, a hepatocarcinogen, in the rat.

1. The metabolism of methapyrilene (I), was examined in vivo by g.l.c. and g.l.c.-mass spectrometric analysis of rat urinary extracts. 2. Dosing the animals with tetradeuterium-labelled I helped identify 7 different metabolites of I in the urine, including (5-hydroxylpyridyl)-methapyrilene, which was identified by comparison with a synthetic reference standard. 3. After 4 weeks of treatment with I, rats also excrete detectable amounts of the 3- and (6-hydroxylpyridyl)-methapyrilene metabolites suggesting that pretreatment with I alters the metabolism of the pyridine ring. 4. Metabolic removal of the 2-thienylmethylene moiety is also facile, as large amounts of N'-(2-pyridyl)-N,N-dimethylethylenediamine and its metabolite N'-[2(5-hydroxylpyridyl)]-N,N-dimethylethylenediamine are excreted under all dosing regimens. 5. Urinary concn of both I and metabolites decline with time, despite continuous dosing, indicating a change in absorption, metabolism, and/or excretion of I on repeated dosing.

Species differences in the in vitro metabolism of methapyrilene.

1. The metabolism of N,N-dimethyl-N'-(2-pyridyl)-N'-(2-thienylmethyl)-1,2- ethanediamine(methapyrilene, I) by liver microsomes from rat, guinea pig, and rabbit has been examined. 2. Methapyrilene-N-oxide, (III), normethapyrilene, (II), 2-thiophene methanol, (VI), 2-thiophene carboxylic acid, (VII), N-(2-pyridyl)-N',N'-dimethylethylenediamine, (IX), and methapyrilene amide, (XIV) were found in all species. 3. N-(2-Thienylmethyl)-2-amino pyridine, (VIII), 2-aminopyridine, (X), and (5-hydroxypridyl)-methapyrilene, (XII), were detected in rat and rabbit only. 4. N-Hydroxynormethapyrilene, (XXI), was tentatively identified by mass spectral fragmentation patterns only in rabbit liver microsomes incubations; however, it was found in 9000 g supernatant fraction incubations of rabbit, rat and guinea pig. 5. The formation of IX and XII was quantitatively more important in the rat than in either rabbit or guinea pig.

Lidocaine metabolism by rabbit-liver homogenate and detection of a new metabolite.

Metabolism of lidocaine in rabbit liver 9000 g supernatant fraction was examined. A capillary g.l.c. assay was developed to separate seven known metabolites of lidocaine, and all seven metabolites were identified in extracts of incubations of lidocaine with rabbit-liver fractions. These metabolites were monoethylglycinexylidide(I), glycinexylidide(II), 3-hydroxymonoethylglycinexylidide(III), 3-hydroxylidocaine(IV), 4-hydroxylidocaine(V), xylidine(VI) and 4-hydroxyxylidine(VII). A new metabolite, 2-amino-3-methylbenzoic acid(VIII), was identified in extracts of incubations of lidocaine with rabbit-liver fractions, by comparison of the mass-spectral fragmentation patterns and g.l.c. retention time with those of the authentic compound. The formation of VIII is dependent on protein, NADPH, time, O2, and the presence of soluble enzymes. Quantitative analysis of metabolites I-VIII after a two hour incubation accounts for 89% of the metabolized lidocaine.

Metabolism of methapyrilene by rat-liver homogenate.

The metabolism of methapyrilene(I) in rat-liver 9000 g supernatant fraction produced four new metabolites positively identified by comparison of g.l.c. retention times and mass-spectral fragmentation patterns with those of authentic materials. These compounds are 2-thiophene-methanol(VI), 2-thiophenecarboxylic acid(VII), N-2-pyridyl-N'-dimethylethylenediamine(IX) and 2-aminopyridine(X). In addition, the previously known metabolite 2-[(2-thienylmethyl)amino]-pyridine(VIII) was also positively identified. Six other metabolites were tentatively identified by analysis of the mass-spectral fragmentation patterns of both the trimethylsilyl and the tertiary butyldimethylsilyl derivatives of each compound. These compounds are tentatively identified as: normethapyrilene(II), (hydroxypyridyl)-methapyrilene(XII), methapyrilenamide(XIV), (hydroxypyridyl)-normethapyrilene(XVI), (hydroxypyridyl)-desmethylmethapyrilenamide(XVII), and (hydroxypyridyl) methapyrilenamide(XVIII). Quantification of II, VI-X, XII and XIV account for approx. 65% of the metabolized methapyrilene.

Species differences in the in vitro metabolism of phencyclidine.

The metabolism of 1-(1-phenylcyclohexyl)-piperidine (phencyclidine or PCP) by liver preparations from cat, monkey, rabbit and rat has been studied. 4-Phenyl-4-piperidinocyclohexanol (I), 1-1-phenylcyclohexyl-4-hydroxy-piperidine (II), N-(5-hydroxypentyl)-1-phenylcyclohexylamine (IX) and 5-(1-phenylcyclohexylamino)-valeric acid (X) were found in all species, but liver preparations of rat and rabbit were much more active than those of cat or monkey in metabolizing PCP. Only rabbit produced 4-(4'-hydroxypiperidino)-4-phenylcyclohexanol (III) in amounts detectable by g.l.c. Mass balance calculations of PCP, I, II, III, IX and X in the cat, monkey and rat indicate that other metabolic pathways not measured in this study are operative.