Ice: a new dosage form of an old drug.

Ice, which has been described as the drug of the 1990s, is a pure form of (+)methamphetamine hydrochloride; it is more dangerous because of its purity and because it can be inhaled. Taken by this route, the drug causes an effect similar to that from an intravenous dose, and much more intense than that from ingestion. The detailed mechanism of action differs from that of cocaine, but the overall stimulant effect of methamphetamine is similar. Methamphetamine effects, however, persist for hours, whereas cocaine effects are over in minutes. Ice is, therefore, just another agent for abuse by those seeking psychostimulation and, as with cocaine, compulsive abusers of amphetamines consume the drug repeatedly and continuously. Unlike cocaine, methamphetamine is a synthetic compound and is manufactured in illicit laboratories within the United States.

Folate and pterin metabolism by cancer cells in culture.

Malignant cells grown in culture excrete into their growth medium a folate catabolite that can be seen as a blue-fluorescent region on paper chromatograms of such media. This folate catabolite has now been identified by paper chromatography, thin-layer chromatography, and combined gas chromatography-mass spectrometry as 6- hydroxymethylpterin and not as pterin-6-carboxaldehyde as previously reported. Moreover, when pterin-6-carboxaldehyde was added to the growth medium of logarithmically growing malignant cells, it was primarily reduced to 6-hydroxymethylpterin. In contrast pterin-6-carboxylate was the principal product formed from added pterin-6-carboxaldehyde by normal established cell lines in culture. These results have been interpreted as indicative of a possible mechanism of folate catabolism in malignant cells. Folic acid or another folate derivative is oxidatively cleaved at the C-9-N-10 bond to yield pterin-6-carboxaldehyde as one of the products. This derivative is subsequently reduced to 6-hydroxymethylpterin, which is excreted into the growth medium.

Test dose response in schizophrenia: chlorpromazine blood and saliva levels.

A gas chromatographic/mass spectrometric method for measuring chlorpromazine using 2H6--labeled chlorpromazine as an internal standard has the potential for considerable precision and specificity. Results are reported of chlorpromazine levels in plasma and saliva after administration of a single test dose to 13 schizophrenic patients in a tightly designed experiment. There was a clear and substantive relationship between blood and saliva levels of chlorpromazine, both following a typical decay curve. Saliva and plasma levels were strongly associated for a particular patient, and there was even a strong consistency in saliva-plasma ratios between patients, with an overall statistically significant correlation between plasma and saliva levels for all measurement on all 13 patients. In general, it seems that saliva sampling has great potential as a simple noninvasive technique for investigation of chlorpromazine and other antipsychotic drugs in psychotic patients. Nevertheless, for the moment, it should be regarded as strictly experimental and not suitable for immediate clinical application.

Plasma and saliva levels of chlorpromazine and subjective response.

Newly admitted schizophrenic patients (N=48) were given a test does of 2.2 mg/kg of chlorpromazine hydrochloride by mouth. Chlorpromazine was measured in plasma and saliva at 1, 2, 3 (saliva only), 4, 6, 8, 12 (saliva only), and 24 hours by gas chromatography-mass spectography. Subjective response at 4 and 24 hours after test dose was not significantly related to either plasma or saliva level even for dysphoric responders. Dyschoric responses to chlorpromazine appear to be idiosyncratic--rooted perhaps in altered recepter sensitivity or in a patient's very personal interpretation of drug effect.

Enantiomeric differences in the effects of 3,4-methylenedioxymethamphetamine on extracellular monoamines and metabolites in the striatum of freely-moving rats: an in vivo microdialysis study.

The effects of (+) and (-) 3,4-methylenedioxymethamphetamine (MDMA) and racemic p-chloroamphetamine (PCA) on extracellular dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as the metabolite of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), were determined in dialysates of the striatum conscious rats by using intracerebral dialysis and high performance liquid chromatography with electrochemical detection (HPLC-EC). The (+) and (-)MDMA isomers (5, 10 mg/kg, s.c.) and PCA (2.5, 5 mg/kg, s.c.) caused a rapid increase of extracellular levels of dopamine and decreased extracellular levels of DOPAC and HVA immediately after administration in dialysates of striatum. The order of potency for this effect was PCA greater than (+)MDMA greater than (-)MDMA. The levels of 5-HIAA also decreased after the administration of drugs, but the effect had a slower time course than DOPAC and HVA and did not exhibit an enantiomeric difference. The data indicate that, although these drugs are thought to affect the 5-HT neuronal system preferentially, they also affect dopamine systems and by a mechanism in which the (+) isomer was more potent than the (-).

Inhibition of uptake of catecholamines by benzylamine derivatives.

Eight benzylamine analogues of bretylium were synthesized, including N-(2-chloroethyl)-N-ethyl-2-methylbenzylamine (5), and evaluated as inhibitors of accumulation of norepinephrine and dopamine in rat brain homogenates. All compounds gave an I50 value (concentration of inhibitor that causes 50% reduction in control accumulation) considerably lower against norepinephrine in cortex that against dopamine in striatum. High potency (low I50) and high specificity (preference for inhibition of norepinephrine transport compared to dopamine transport) are associated with a (2-chloroethyl) moiety, tertiary amino center, and ortho substitution of the aromatic function in the benzylamino group. 5 also inhibited the uptake of norepinephrine in rabbit aorta, indicating its effect against the uptake process in general. Cocaine protects against the effects of 5 in coincubation studies when compared to the appropriate controls, indicating that 5 acts at or close to the site of action of cocaine which is thought to be the uptake carrier site.

Determination of the mechanism of demethylenation of (methylenedioxy)phenyl compounds by cytochrome P450 using deuterium isotope effects.

The mechanism of demethylenation of (methylenedioxy)benzene (MDB), (methylenedioxy)amphetamine (MDA), and (methylenedioxy)methamphetamine (MDMA) by purified rabbit liver cytochrome P450IIB4 has been investigated by using deuterium isotope effects. A comparison of the magnitude and direction of the observed kinetic isotope effects indicates that the three compounds are demethylenated by different mechanisms. The different mechanisms of demethylenation have been proposed on the basis of comparisons of the observed biochemical isotope effects with the isotope effects from purely chemical systems.

Conversion of N-hydroxyamphetamine to phenylacetone oxime by rat liver microsomes.

These in vitro studies indicate that N-oxidation of N-hydroxyamphetamine (NOHA) by rat liver homogenates yields phenylacetone oxime (PAOx) as the major metabolite. This oxidation was NADPH and oxygen dependent but was not appreciably increased in microsomes from phenobarbital-pretreated animals. The addition to microsomal incubations of superoxide dismutase (SOD), catalase (CAT), azide or mannitol did not alter the rate of oxidation, suggesting that O2-, H2O2, or OH' are not involved in this process. The reaction was minimally inhibited by a 2:1 ratio of CO/O2, and there was no significant reduction in the formation of product by the presence of diethylaminoethyl diphenylvalerate (SKF-525A) or 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) in micromolar concentrations. Thus, although this NADPH-dependent N-oxidation pathway was catalyzed by rat hepatic microsomes, the data suggest that is was not a cytochrome P-450 mediated monooxygenase reaction.

Disposition of methylenedioxymethamphetamine and three metabolites in the brains of different rat strains and their possible roles in acute serotonin depletion.

3,4-Methylenedioxymethamphetamine (MDMA) affects both dopamine and serotonin (5-HT) systems. One of its acute actions is to cause a reversible fall in steady-state brain 5-HT concentrations. To investigate the chemical basis of this acute effect, the brain levels of the parent compound and three major metabolites, 3,4- 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymethamphetamine (DHMA) and 6-hydroxy-3,4-methylenedioxymethamphetamine (6-OHMDMA), were monitored, together with 5-HT levels, over a period of 6 hr in male Sprague-Dawley (SD) rats. The temporal relationships between drug concentrations of both stereoisomers and depletions were evaluated first. There was no correlation between the concentrations of the compounds measured and the extent of 5-HT depletion. Brain levels of MDMA and MDA were higher than plasma levels and exhibited a stereoselectivity in that (-)-MDMA and (+)-MDA levels were higher than those of enantiomers. The relationship between the dose of ((+)-MDMA and reduction in 5-HT levels was next investigated in SD male, SD female, and Dark Agouti (DA) female rats. These animals exhibit different capabilities of MDMA metabolism. There is a lower level of MDA, the N-demethylated metabolite of MDMA, in female SD rats than in males. Female DA rats are deficient in CYP2D isozymes, one of the enzymes responsible for demethylenation of MDMA to DHMA at pharmacological concentrations of substrate. there was a significant accuulation of MDMA in the brain and plasma of DA rats, but their 5-HT depletion was somewhat attenuated. The results indicated that MDMA ++ was apparently not the single, causative agent for the acute 5-HT depletion, which may also involve a metabolite formed by CYP2D.

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.

Induction of phencyclidine metabolism by phencyclidine, ketamine, ethanol, phenobarbital and isosafrole.

The in vitro metabolism of phencyclidine (PCP) was investigated in 9000 g supernatant fractions of both control and PCP-, ketamine-, ethanol-, phenobarbital- or isosafrole-pretreated rats. Levels of PCP, trans-4-phenyl-4-piperidinocyclohexanol (I), 1-(1-phenylcyclohexyl)-4-hydroxypiperidine (II), N-(5-hydroxypentyl)-1-phenylcyclohexylamine (IX), and 5-(1-phenylcyclohexylamino)-valeric acid (X) were monitored by gas chromatographic analysis in all cases. The inhibition of metabolism by N2, CO, SKF-525A or 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), or deletion of NADPH or protein, implied the involvement of cytochrome P-450 in the reactions. The various inducing agents affected the metabolism of PCP in different ways, implying that at least several isozymes of cytochrome P-450 were involved in the total metabolism. The majority of the consumed PCP was not accounted for by the measured metabolites so that some other metabolic pathways of major quantitative importance must be operative.

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.

Comparison of the behavioral and biochemical effects of the NMDA receptor antagonists, MK-801 and phencyclidine.

The behavioral and biochemical effects of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 [+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate) were compared with those of phencyclidine (PCP). In the dose range used in this study, MK-801 (0.125-0.5 mg/kg i.p.) produced ataxia and other behavioral responses which were similar to PCP (5-10 mg/kg i.p.). However, turning and backpedalling induced by MK-801 were not dose-dependent and less intense at the dose producing approximately the same level of ataxia as PCP. Neurochemically, MK-801 (0.5 mg/kg i.p.) increased dopamine turnover in the cortex and striatum, but had no effect on 5-HT systems. It was also 3.4 times less potent in inhibiting 5-HT uptake than PCP. These results suggest that the behavioral responses induced by MK-801 involve primarily the PCP receptor and the dopamine system, and that the differences from PCP reflect a reduced effect on the 5-HT neuronal system.

Inhibition of dopamine release by methylenedioxymethamphetamine is mediated by serotonin.

(+/-)-3,4-Methylenedioxymethamphetamine (MDMA), at doses of 0.1, 1 and 10 mg/kg, produced a long-lasting decrease in extracellular dopamine concentration in the neostriatum of anesthetized rats, as measured by in vivo voltammetry. Since MDMA has been shown to release serotonin from rat brain slices and synaptosomes, we examined the possibility that increased serotonin release might be the cause of the decrease in dopamine release. Rats were treated with d,l-p-chloroamphetamine seven days prior to acute MDMA administration. Rats pretreated with p-chloroamphetamine, which produced a marked decrease in serotonin content, showed no significant decrease in extracellular dopamine concentration when administered 10 mg/kg MDMA. These data suggest that MDMA produces a significant decrease in dopamine release when administered acutely, and that this decrease is an indirect effect mediated by an increase in serotonin release.

A pharmacokinetic analysis of 3,4-methylenedioxymethamphetamine effects on monoamine concentrations in brain dialysates.

Interpretation of the in vivo actions of 3,4-methylenedioxymethamphetamine (MDMA) is complicated by the formation of the active metabolite, 3,4-methylenedioxyamphetamine (MDA). This study evaluates the role of MDA in the dopamine releasing actions of (+)-MDMA. In the study, rats were given subcutaneous doses of (+)-MDMA and concentrations of monoamines and their metabolites in striatal dialysate were measured at 15 min intervals. In parallel experiments, plasma concentrations of (+)- and (-)-MDMA and MDA were determined by GC/MS procedures. The time course of MDMA levels was comparable for the two isomers as were their bioavailabilities. In contrast, the plasma levels of MDA were about three times higher after (+)-MDMA. (+)-MDMA caused a rapid increase in striatal dialysate levels of dopamine and decreased extracellular levels of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). There was a significant correlation between dopamine concentration in striatal dialysate and plasma MDMA concentration, but not with plasma MDA. These results indicate that MDMA itself has stereoselective actions on dopamine neurons. However, the higher plasma MDA levels after (+)-MDMA may account for part of the enantiomeric differences in the behavioral and neurotoxicological effects of MDMA.

MK-801 ameliorates delayed amnesia, but potentiates acute amnesia induced by CO.

The effects of non-competitive N-methyl-D-aspartate receptor antagonists on amnesia induced by carbon monoxide (CO) were investigated, since they have neuroprotective effects on delayed degeneration induced by ischemia. In the mice exposed to CO, acute and delayed amnesia were induced. (+)-MK-801 and (-)-MK-801 improved the delayed amnesia, but the effects of phencyclidine (PCP) were weak. (+)-MK-801 and PCP potentiated the acute amnesia. From these results, it is suggested that there is a stereoselectivity in the effects of MK-801 on CO-induced amnesia and that CO-induced delayed amnesia animals could be used as an ischemic amnesia model.

Interaction of xylamine with peripheral sympathetic neurons.

Xylamine (XYL) administered to intact rats caused a 70-80% reduction in norepinephrine (NE) uptake by the vas deferens but had little or no effect on NE content in that tissue. The vas deferens accumulates 3H-XYL in vitro by a desmethylimipramine (DMI)-sensitive mechanism. Vasa deferentia from 6-hydroxydopamine (6OHDA) pretreated animals exhibited a 80% reduction in both NE content and XYL uptake activity. These results indicate that XYL is taken up by sympathetic nerve terminals and can reduce NE uptake activity without depleting terminals of neurotransmitter.

Spectral and inhibitory interactions of (+/-)-3,4-methylenedioxyamphetamine (MDA) and (+/-)-3,4-methylenedioxymethamphetamine (MDMA) with rat hepatic microsomes.

Incubation of racemic methylenedioxyamphetamine (MDA) or methylenedioxymethamphetamine (MDMA) with rat hepatic microsomes, in the presence of NADPH, generated a spectrally observed inhibitory complex with cytochrome P-450. The complex inhibited product formation from MDA and MDMA as well as other P-450 dependent reactions such as benzphetamine demethylation and CO binding. In the absence of NADPH, MDMA and MDA generated type I and type IIa difference spectra, respectively, suggesting differences in their binding to the enzyme active site. The N-demethylation of MDMA was partially inhibited by methimazole suggesting involvement of the hepatic flavin-containing monooxygenase.

3H-xylamine binds to presynaptic rat striatal membranes.

3H-xylamine (3H-XYL), an irreversible catecholamine uptake inhibitor, was incubated with rat striatal synaptosomes, and the membrane fraction was examined by fluorography of a sodium dodecyl sulfate-polyacrylamide gel. A number of peptides were labeled. To determine their location, the striatal dopaminergic presynaptic nerve terminals were destroyed by unilateral electrolytic lesions through the nigrostriatal fibers prior to 3H-XYL exposure. The 3H-XYL bound to membranes from lesioned striata was about 29% of that bound to control membranes, which is consistent with the 83% reduction in dopamine (DA) uptake and the 68% reduction in DA content in the lesioned tissue. The decrease in peptide-bound 3H-XYL paralleled the decrease in DA content, with the exception of a 45% decrease in binding to a 45K peptide. These data show that 3H-XYL binding is predominantly localized in the dopaminergic presynaptic nerve terminals of the striatum.

Cytochrome P450 enzymes involved in the enhancement of propranolol N-desisopropylation after repeated administration of propranolol in rats.

Repeated oral administration of propranolol (PL, 100 mg/kg daily, for 5, 10 and 15 days) to male Wistar rats increased PL N-desisopropylase and decreased PL 4-,5- and 7-hydroxylase activities in liver microsomes. The increase was highest at the 10 day time point whereas the decrease was relatively constant over the 15 day treatment period. There were no significant changes in the total content of cytochromes P450 (P450) or cytochrome b5 or in NADPH-cytochrome c reductase activity during the PI, treatment. The enhanced N-desisopropylase activities were markedly inhibited by alpha-naphthoflavone (a P450-1A1/2 inhibitor), and moderately by triacetyloleandomycin (a P450-3A1/2 inhibitor) and diethyldithiocarbamate (a P450-2E1 inhibitor). Phenacetin O-deethylase activity, an index of P450-1A2, was significantly increased on day 5, 10 and 15 of the treatment, whereas p-nitrophenol hydroxylase activity was elevated on day 10 only. The PL N-desisopropylation showed a strong and significant correlation with phenacetin O-deethylation, and a weaker but significant correlation with p-nitrophenol hydroxylation. Immunoblot analysis revealed that a protein band corresponding to P450-1A2 was increased by PL pretreatment, and protein band corresponding to P450-3A tended to be increased slightly, but other protein band corresponding to the subfamily of P450-2B, -2C, or -2E was not changed. Pretreatment of rats with P450 inducers (beta-naphthoflavone, phenobarbital, acetone and dexamethasone) increased PL N-dealkylase activity in liver microsomes. Furthermore, antibodies raised against P450-1A and -3A enzymes suppressed PL N-desisopropylation in a concentration-dependent manner, but P450-2E antibody did not. Reconstitution studies showed that P450-1A1, -1A2, -2E1 and -3A2 exhibited catalytic activities for PL N-dealkylation. These results suggest that P450-1A2 is a major PL N-desisopropylase in the PL-treated rats, and P450-3A related enzyme(s) and P450-2E1 as a moderate or minor enzyme are also involved in PL N-dealkylation in native and PL-treated rats.