Toxicity of the amphetamine metabolites 4-hydroxyamphetamine and 4-hydroxynorephedrine in human dopaminergic differentiated SH-SY5Y cells.

Amphetamine (AMPH) is a psychostimulant used worldwide by millions of patients in the clinical treatment of attention deficit hyperactivity disorder, narcolepsy or even obesity, and is also a drug of abuse. 4-Hydroxynorephedrine (4-OHNE) and 4-hydroxyamphetamine (4-OHAMPH) are two major metabolites known to persist in the brain longer than AMPH. The contribution of AMPH metabolites for its neurotoxicity is undetermined. We evaluated the toxicity of AMPH and its metabolites 4-OHNE and 4-OHAMPH, obtained by chemical synthesis, in human dopaminergic differentiated SH-SY5Y neurons. Cells were exposed to AMPH (concentration range 0-5mM) or 4-OHAMPH or 4-OHNE (concentration range 0-10mM) for 24 or 48h, and the viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) leakage assays. Results showed that for both AMPH and the metabolites a concentration-dependent toxicity was observed. The toxic concentration 50% (TC50) for AMPH and 4-OHNE following 24h exposure was circa 3.5mM and 8mM, respectively. For 4-OHAMPH the TC50 was not reached in the tested concentration range. N-acetyl cysteine, cycloheximide, l-carnitine, and methylphenidate were able to reduce cell death induced by AMPH TC50. Acridine orange/ethidium bromide staining showed evident signs of late apoptotic cells and necrotic cells following 24h exposure to AMPH 3.50mM. The 4-OHAMPH metabolite at 8.00mM originated few late apoptotic cells, whereas 4-OHNE at 8.00mM resulted in late apoptotic cells and necrotic cells, in a scenario similar to AMPH. In conclusion, the AMPH metabolite 4-OHNE is more toxic than 4-OHAMPH, nonetheless both are less toxic than the parent compound in vitro. The most toxic metabolite 4-OHNE has longer permanence in the brain, rendering likely its contribution for AMPH neurotoxicity.

High-performance liquid chromatography of methamphetamine and its related compounds in human urine following derivatization with fluorescein isothiocyanate.

A high-performance liquid chromatographic method with fluorescence detection for the determination of methamphetamine and its related compounds is reported. Methamphetamine, amphetamine, norephedrine, p-hydroxymethamphetamine and 1-phenylethylamine as an internal standard were extracted from human urine, derivatized with fluorescein-4-isothiocyanate, and then separated on a reversed-phase column within 36 min. The fluorescence intensity of the effluent was monitored at excitation and emission wavelengths of 496 and 518 nm, respectively. Calibration curves were confirmed to be linear up to at least 100 pmol on the column with a correlation coefficient (r) of 0.994-0.999 for the target compounds. The detection limits (S/N=3) were 55-105 fmol per 20-microl injection. The method was successfully applied to urine samples taken from methamphetamine addicts.

Lack of involvement of 4-hydroxynorephedrine in phenylpropanolamine-induced anorexia in rats.

The anorexic effects of phenylpropanolamine (PPA) appear to be qualitatively different in humans and rats. One factor that may account for these differences is that PPA is excreted essentially unchanged in humans, while nearly 30% is metabolized into 4-hydroxynorephedrine (4-OHN) in rats. To investigate the contribution of 4-OHN to the anorexic properties of PPA, this experiment compared the effects of equal doses (0.0-20.0 mg/kg, IP) of both drugs on eating and drinking during restricted feeding trials in the same group of food-deprived, female rats. Both 15.0 and 20.0 mg/kg of PPA significantly decreased eating when compared to saline vehicle, while 5.0-20.0 mg/kg of the drug reduced prandial drinking. In comparison, only the highest dose of 4-OHN (20.0 mg/kg) significantly suppressed food and water intake. When the percentage of reduction produced by corresponding doses of the two drugs was compared, PPA proved to be more than twice as potent as 4-OHN. It is concluded that, at the doses used, 4-OHN is unlikely to significantly contribute to reductions in deprivation-induced eating produced by the acute administration of PPA.

Involvement of hydroxylated metabolites in amphetamine-induced hypothermia in mice.

1. The hydroxylated metabolites of amphetamine, p-hydroxyamphetamine (p-OHA) and p-hydroxynorephedrine (p-OHN), were administered intracerebroventricularly in mice in order to evaluate their ability to elicit hypothermia. 2. Intracerebroventricular (i.c.v.) administration of p-OHA and p-OHN (1, 3 and 9 micrograms/mouse) induced maximal hypothermia 30 min after injection. p-OHA and p-OHN (9 micrograms, i.c.v.) produced maximal decreases in rectal temperature of -6.48 +/- 0.44 degrees C and -3.82 +/- 0.42 degrees C, respectively. Both metabolites are more effective than amphetamine (at 9 micrograms, i.c.v., -3.32 +/- 0.75 degrees C). 3. Pretreatment with haloperidol (5 micrograms, i.c.v.) suppressed the fall in temperature produced by p-OHA (3 micrograms, i.c.v.) and reduced that produced by p-OHN (3 micrograms, i.c.v.), respectively. The selective dopaminergic D1 receptor antagonist, SCH 23390, and the D2 receptor antagonists, sultopride and metoclopramide, were without effect on the hypothermia induced by either metabolite. Similarly, amphetamine-induced hypothermia was only inhibited by haloperidol. Apomorphine (0.1 mg kg-1, i.p.) did not potentiate the hypothermia induced by either metabolite, whereas the selective dopaminergic D2 agonist, quinpirole (0.2 mg kg-1, i.p.) did. Amphetamine-induced hypothermia was potentiated by apomorphine and quinpirole. 4. Neither the 5-hydroxytryptamine (5-HT) receptor blocker, cyproheptadine, nor the 5-HT receptor agonist, quipazine, modified metabolite-induced hypothermia. In contrast, amphetamine-induced hypothermia was affected by these 5-HT drugs. 5. The neuropeptide CCK-8 (0.04 mg kg-1, i.p.) and gamma-butyrolactone (40 mg kg-1, i.p.) potentiated the hypothermia produced by amphetamine and its metabolites. Conversely, desipramine (20 mg kg-1, i.p.) antagonized it.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of brain type A monoamine oxidase and 5-hydroxytryptamine uptake by two amphetamine metabolites, p-hydroxyamphetamine and p-hydroxynorephedrine.

Two amphetamine metabolites, p-hydroxyamphetamine (p-OHA) and p-hydroxynorephedrine (p-OHN), selectively inhibited the A form of monoamine oxidase (MAO) in rat and mouse forebrain homogenates. Of these two metabolites, p-OHA inhibited MAO-A more strongly than p-OHN. This MAO-A-selective inhibition by p-OHA or p-OHN was found to be competitive with respect to deamination of its substrate, 5-hydroxytryptamine (5-HT). The degree of MAO-A inhibition was not changed by 90 min of preincubation of the enzyme preparations with either metabolite, and the activity inhibited by p-OHA after the preincubation recovered completely to the control level after repeated washing. Uptake of 5-HT or dopamine into mouse forebrain synaptosomes was highly reduced by both p-OHA and p-OHN. Both metabolites were more potent in reducing dopamine uptake than in reducing 5-HT uptake. In reduction of 5-HT and of dopamine uptake, p-OHA was more potent than p-OHN. These results indicate that p-OHA is a more selective inhibitor of brain MAO-A activity and 5-HT uptake than its subsequent metabolite, p-OHN. These two actions of p-OHA might, together with possible 5-HT efflux into the synaptic cleft, greatly contribute to head twitch, a brain 5-HT-mediated animal behavior induced by p-OHA.

Neurochemical effects of amphetamine metabolites on central dopaminergic and serotonergic systems.

Intrastriatal administration of the hydroxylated metabolites of amphetamine, p-hydroxyamphetamine (p-OHA) or p-hydroxy-norephedrine (p-OHNor), decreased local concentrations of dopamine and serotonin in a dose-dependent manner. Although both compounds reduced concentrations of the metabolites of dopamine, 5-hydroxyindoleacetic acid concentrations were elevated. After systemic treatment with p-OHA, striatal dopamine was also reduced. In contrast, only hypothalamic and hippocampal serotonin stores were altered significantly in rats treated with p-OHA systemically. Neither compound decreased the activities of tryptophan hydroxylase or tyrosine hydroxylase. Because p-OHA is metabolized to p-OHNor via dopamine beta-hydroxylase present in noradrenergic neurons, the direct effects of these compounds on dopaminergic and serotonergic variables can be observed in rats which receive intrastriatal drug treatment. p-OHA and p-OHNor were equally potent in decreasing dopamine concentrations. However, p-OHNor was more potent than p-OHA in decreasing serotonin concentrations. Both compounds more readily depleted dopamine compared to serotonin stores. Complete recovery of p-OHA-induced decreases in striatal dopamine occurred within 48 hr of intrastriatal administration and concurrent treatment with the dopamine uptake blocker, amfonelic acid, significantly attenuated the p-OHA-induced effects on dopamine.

Modification of behavioral responses to methamphetamine evoked by the stimulant's metabolite p-hydroxynorephedrine in rats.

The central effect of p-hydroxynorephedrine (OH-NE), one of the p-hydroxylated metabolites of methamphetamine (MAP) and amphetamine (AMP), was investigated in rats. Locomotion and stereotypy were examined after SC injections of 0.5-5 mg/kg of MAP or 0.02-0.5 mg/kg of apomorphine (APO) in animals treated with either saline or 5-50 mg/kg of OH-NE IP 20 hr before behavioral assessment. The locomotor stimulating effect of both 0.5-2 mg/kg of MAP and 0.2 mg/kg of APO was enhanced by 5 mg/kg of OH-NE. On the other hand, 30 mg/kg of OH-NE severely suppressed the stimulating effect of MAP but had no influence on that induced by 0.2 mg/kg of APO. The stereotypy induced by 5 mg/kg of MAP or 0.5 mg/kg of APO was enhanced and prolonged in the OH-NE-treated rats. Subsequently, examinations were performed to determine whether OH-NE had any effect on the dopaminergic mechanism. Hypomotility induced by 0.02 mg/kg of APO was alleviated by 5 mg/kg of OH-NE, but was aggravated by 30 mg/kg. These results suggest that OH-NE administered prior to SC injections of MAP or APO influences their behavioral effects via the dopaminergic mechanism. The possibility that other neural mechanisms may be involved in this OH-NE-induced behavioral modification is also discussed.

P-hydroxy-norephedrine as a possible mediator causing the reduction of oral intake of D-amphetamine in rats.

In rats D-amphetamine is predominantly metabolized by hydroxylation to p-hydroxy-norephedrine (p-HNE); in guinea pigs, however, by deamination to benzoic acid. After 2-3 days on dosages of 1 mg/kg per day and more rats begin to reduce their oral intake of the stimulant whereas guinea pigs do not. In the present study we examined the hypothesis that the formation of p-HNE in the CNS is partially responsible for this aversion. To determine the elimination of D-amphetamine and the increase in p-HNE, groups of male Wistar rats were given various doses (0.5-5 mg/kg per day) of D-amphetamine in their drinking water intragastrically and intravenously. D-Amphetamine in the brain was determined by radioimmunoassay, p-HNE by high performance liquid chromatography followed by electrochemical detection. In contrast to the concentration of D-amphetamine, the p-HNE-content is independent of the route of administration; after oral treatment it showed a linear increase. The results reveal that p-HNE induces the aversion to the stimulant and that the ratio of D-amphetamine to its metabolite determines the onset of this aversion. No p-HNE was found in the brain of guinea pigs. Guinea pigs do not show any aversion to drinking D-amphetamine solutions, even in high dosages.

Determination of amphetamine, norephedrine, and their phenolic metabolites in rat brain by gas chromatography.

A specific analytical procedure for the quantitation of amphetamine (I), norephedrine (III), and their amphoteric metabolites, p-hydroxy-amphetamine (II) and p-hydroxynorephedrine (IV), in biological samples using electron-capture gas chromatography (GC-EC) is described. The procedure utilizes the ion-pairing reagent, bis(2-ethylhexyl)phosphoric acid, which frees the amines from most contaminants and permits the efficient extraction of the amphoteric compounds (as acetates) from the aqueous solution. Amines I and III and acetylated amines II and IV were perfluoroacetylated prior to GC-EC analysis. Metabolism of I, II, and III in the rat brain was studied. Results indicate that both in vivo and in vitro amines I and III are p-hydroxylated to II and IV, respectively, and II is beta-hydroxylated to give IV. Norephedrine (III) was not detected as a rat brain metabolite of amphetamine (I).

Evidence that a behavioral augmentation following repeated amphetamine administration does not involve peripheral mechanisms.

Repeated administration of amphetamine (AMPH) to rats results in an augmentation of the drug-induced locomotion and stereotypy. The studies reported below were directed at examining the potential role for some dispositional and peripheral sympathomimetic factors in mediating the enhanced stereotypy response. These included three factors associated with repeated AMPH administration: (1) the possible accumulation of AMPH in a peripheral mobilizable pool; (2) repeated sympathetic activation; and (3) AMPH metabolite-induced depletion of peripheral stores of norepinephrine. The approach utilized was to selectively reduce or mimic the peripheral actions of AMPH through the use of non-pharmacological or pharmacological manipulations which are relatively lacking in AMPH-like central stimulant effects. The results indicate that these factors cannot account for the augmentation of the behavioral response to AMPH and suggest that these behavioral alterations reflect changes in the responsiveness of brain mechanisms which mediate the behavioral effects of the drug.

Blood pressure and heart rate response evoked by p-hydroxyamphetamine and by p-hydroxynorephedrine II. A quantitative assessment of the role of amphetamine metabolites in acute responses evoked by d-amphetamine.

At postganglionic sympathetic sites, p-hydroxyamphetamine is neither a receptor agonist nor a receptor antagonist; in addition the drug does not act presynaptically to antagonize or synergize d-amphetamine. p-Hydroxyamphetamine is an indirectly acting sympathomimetic amine with a potency approximately twice that of d-amphetamine. In the rat, a large fraction (approximately 0.5) of d-amphetamine is biotransformed to p-hydroxyamphetamine. However, the rate of biotransformation to p-hydroxyamphetamine (approximately 0.0099 . min-1) is slow compared to the rate of elimination of p-hydroxyamphetamine (0.049 . min-1). As a result, plasma levels of d-amphetamine exceed those of p-hydroxyamphetamine. The kinetic data suggest that: 1) p-hydroxyamphetamine plays little role in immediate responses to single injections of d-amphetamine; 2) p-hydroxyamphetamine is not involved in tachyphylactic responses to repeated injections of d-amphetamine; and 3) p-hydroxynorephedrine plays no role in immediate or tachyphylactic responses to d-amphetamine.

Interindividual and interspecies variation in the metabolism of the hallucinogen 4-methoxyamphetamine.

1. The qualitative and quantitative aspects of the urinary elimination of orally administered 4-methoxy[14C]amphetamine have been examined in the rat and guinea-pig and in three volunteer human subjects, to determine interspecies and interindividual variations in disposition of the drug. 2. Both rat and guinea-pig excreted 70--80% of the administered dose(6 mg/kg) in the urine within 24 h, mainly as metabolites. 3. In the guinea-pig, the drug was metabolized by O-demethylation to give 4-hydroxyamphetamine, which was excreted free (4% dose) and conjugated (73%). No other metabolite was detected. 4. The rat metabolizes the drug both by O-dealkylation and by side-chain oxidation, the products being 4-hydroxyamphetamine (5% of dose free and 60% conjugated) and 1-(4'-methoxyphenyl)propan-2-one oxime (5% dose, free and conjugated). 5. In man the drug (dose 5 mg) is metabolized by O-demethylation and by side-chain oxidation. Marked intersubject variations were observed both in the array and quantitative aspects of metabolite excretion. Two subjects excreted mainly 4-hydroxyamphetamine (free and conjugated) together with smaller amounts of 1-(4'-methoxyphenyl)propan-2-one oxime and 4-hydroxynorephedrine. The third subject, however, who was previously known to exhibit a genetically determined defect in drug oxidation, was defective in O-dealkylation of 4-methoxyamphetamine, and the main excretion products were the unchanged drug together with products of side-chain oxidation, namely, 1-(4'-methoxyphenyl)propan-2-one oxime, 1-(4'-methoxyphenyl)propan-2-one and 4-methoxybenzoic acid. 6. Inter-individual differences in oxidative O-demethylation of the drug are discussed in relation to current theories on the aetiology of schizophrenia and reported fatalities arising from abuse of the drug.

Agonist effects of beta-phenethylamines on the noradrenergic cyclic adenosine 3',5'-monophosphate generating system in rat limbic forebrain. Stereoisomers of p-hydroxynorephedrine.

Significant agonist activity for the specific noradrenergic cyclic adenosine 3',5'-monophosphate (cAMP) generating system in rat limbic forebrain requires a beta-3,4-dihydroxyphenethylamine with a beta-hydroxyl group in the R configuration. Thus, neither of the enantiomers of p-hydroxynorephedrine nor of p-hydroxynorpseudoephedrine mimics the agonist activity of (R)-norepinephrine. It has yet to be established whether or not one of the p-hydroxynorephedrines exhibits antagonist activity in this same system.

Radioimmunoassay of amphetamines in rat parotid saliva.

Amphetamines, a commonly abused class of drugs, have been detected in various biological specimens, in particular, urine and blood. However, little information is available concerning the detection of these drugs in saliva. This investigation, utilizing the rat salivary secretions, has been attempted to establish the ability of amphetamines to be secreted in saliva and to determine the feasibility of using radioimmunoassay (RIA) for drug detection in saliva. The results of this investigation showed that (1) d-amphetamine and methamphetamine decreased salivary flow, (2) after d-amphetamine RIA tests were demonstrated in both saliva and plasma for a period of fifty minutes, and (3) positive RIA reactions were obtained by the following metamphetamine metabolites: amphetamine, 4-hydroxynorephedrine and 4-hydroxyamphetamine. Methamphetamine and 4-hydroxy-N-methylamphetamine were found to be non-reactive in the radioimmunoassay procedure. The results indicate that saliva could be radioimmunoassayed for the detection of amphetamine or amphetamine derivatives after the administration of either d-amphetamine and methamphetamine.

Mechanism of D-amphetamine inhibition of protein synthesis.

At 1 h after intraperitoneal administration of D-amphetamine sulphate (15 mg/kg), rat brain polyribosomes show disaggregation accompanied by reduced capacity for in vitro peptide chain elongation. The direct action of amphetamine on cell-fine protein-synthesizing systems was therefore explored. When brain or liver polyribosomes from untreated rats were incubated with pH 5 enzyme, peptide chain elongation was not inhibited by the addition 4 mM amphetamine to the medium. On the other hand, an initiation-dependent system consisting of rat liver of brain mRNA and wheat germ S-30 fraction showed inhibition of [3H]leucine incorporation by 50% when 4 mM amphetamine were added. The metabolites of amphetamine, p-hydroxyamphetamine and p-hydroxynorephedrine, had no inhibitory action in either system, but the potent neurotoxin p-chloroamphetamine was a more powerful inhibitor of initiation than amphetamine. By using [3H]amphetamine, it was shown that amphetamine binds to the 80-S ribosomes of the wheat germ system. This binding depended on the presence in the system of natural liver or brain mRNA or several synthetic mRNAs, but was not promoted by polyuridylic acid as the messenger. Significantly, polyuridylic acid-dependent polyphenylalanine synthesis by the wheat germ system was not inhibited by amphetamine or p-chloroamphetamine. Therefore, it was concluded that amphetamine inhibits protein synthesis by interfering with initiation through a step related to formation of the mRNA ribosome complex.