SPEC disc solid-phase extraction for rapid broad-spectrum drug screening in urine.
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Broad-spectrum drug screening requires that all relevant substances be isolated, detected, and identified, regardless of their structure and/or polarity. To this end, systematic solid-phase extraction (SPE) approaches for drug isolation from biological fluids are required. Because speed and cost effectiveness are key issues in analytical toxicology, we have evaluated a disc-format extraction device for this purpose and compared the latter with an existing packed-bed column-format method. The discs were SPEC.PLUS.C18AR/MP3 cartridges with 10-mL solvent reservoirs, providing hydrophobic and cation exchange interactions. Blank human urine was spiked at 2 microg/mL with a selection of acidic, neutral, and basic drugs representing a variety of relevant drug classes. Urine specimens (2 mL) were diluted with 2 mL 0.1 M phosphate buffer (pH 5.0) and then applied to the preconditioned disc. Washing was done with 1 mL water. Acidic and neutral drugs were eluted with 1 mL ethyl acetate/acetone (1:1), and basic drugs were eluted with 1 mL ammoniated ethyl acetate. The eluates were collected separately, evaporated down to about 0.1 mL, and analyzed by gas chromatography-flame-ionization detection to check cleanliness, recoveries, and reproducibilities. The discs showed good extraction properties for all drugs and were easy to handle. Recoveries were 75-100% with coefficients of variation of around 5%. The resulting eluates showed only a few matrix interferences. As compared to our standard SPE method with packed-bed columns, the disc procedure allowed reductions in elution volumes and total processing time of approximately 60-65%. (+info)
Neuroleptics ameliorate phencyclidine-induced impairments of short-term memory.
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1. Phencyclidine (PCP), a non-competitive NMDA-receptor antagonist, is able to induce schizophrenia-like symptoms in animals and in humans. It is known that schizophrenic patients have deficits in memory processes. 2. Therefore, it was investigated whether subchronic pulsatile or continuous application of 5.0 mg kg(-1) PCP over 5 days induce short-term memory deficits in holeboard learning and the action of two different neuroleptics on this behavioural test. 3. First, an impairment in the holeboard task was described when the animals were tested 24 h after the last application but not after 15 min or 1 h after the last injection. Secondly, the influence of haloperidol and risperidone on the PCP-induced short-term memory changes was tested. 4. The combined application of PCP and risperidone led to a complete antagonism of the short-term deficits, but the combined treatment with haloperidol was accompanied by a partial abolishment of the PCP-induced deficits. 5. PCP led to an upregulation of the glutamate binding sites in striatum and nucleus accumbens whereas the D(2) binding sites were reduced in striatum. The D(1) binding sites seem to be unchanged. The receptor protein expression of glutamate receptors mGluR1, GluR2, GluR5/7 and NMDAR1 were not modified in response to PCP treatment. 6. The determination of a subpopulation of GABAergic interneurons shows a decrease of the cells within the CA3 of the hippocampal formation. 7. These findings indicate that PCP induced impairments in short term memory can be detected by holeboard learning and may provide an interesting tool for the search of new neuroleptics. (+info)
Antagonism of a PCP drug discrimination by hallucinogens and related drugs.
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Drugs such as PCP and MK-801 can cause psychotic reactions in humans by antagonizing NMDA receptors. This action is ultimately toxic to certain cortical neurons and may be one mechanism underlying neurodegenerative diseases, including schizophrenia. It has been reported that hallucinogens such as LSD, DOM, and DOI can block the neurotoxic effects of NMDA antagonists, possibly by activating inhibitory 5-HT2A receptors on GABAergic interneurons that normally inhibit glutamatergic projections to the retrosplenial and cingulate cortexes. The purpose of this experiment was to determine the extent to which similar drugs might also alter the behavioral effects of one NMDA antagonist, PCP. Rats were trained to discriminate this compound (2.5 mg/kg) from saline and were then given a series of antagonist tests. It was found that LSD (0.32 mg/kg) and DOM (4.0 mg/kg) blocked the PCP cue completely; DMT (8.0 mg/kg) and a structural congener of LSD, lisuride (LHM; 0.4 mg/kg), blocked the effects of PCP partially. The 5-HT/DA antagonists spiperone and ritanserin had no effect on the PCP cue. These data suggest that LSD, DOM, and, less effectively, DMT and LHM can block the behavioral as well as the neurotoxic effects of NMDA antagonists most likely through agonist actions at 5-HT2 receptors. (+info)
The effect of rate of drug administration on the extent and time course of phencyclidine distribution in rat brain, testis, and serum.
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The goal of these studies was to examine the relationship between the rate of phencyclidine (PCP) administration and PCP tissue distribution. The time course of PCP distribution in serum, brain, and testis after rapid (i.v.) and slow (s.c.) administration was studied. Brain and serum PCP concentrations after an i.v. bolus dose (1 mg/kg at 900 microg/min) were highest at 30 s and decreased biphasically, with serum concentrations decreasing 30 times faster than brain concentrations during the early phase. Consequently, the brain-to-serum PCP concentration ratio increased from 8:1 at 30 s to 14:1 at 20 min before equilibrating at a ratio of 3:1 that remained constant from 1 to 8 h. In contrast, the testis-to-serum ratio increased slowly from 1:1 to 12:1 over 4 h, and then remained constant. In a separate group of animals, an s.c. infusion of PCP (18 mg/kg/day or 3.6 microg/min) produced a brain-to-serum ratio (6:1) that remained constant throughout the 96-h infusion. Testis-to-serum ratios increased from 4:1 at 1 h to 12:1 at 8 h and then remained constant for 96 h. Steady-state infusion of a pharmacologically inactive dose (2.5 mg/kg/day) produced a brain-to-serum ratio (3:1) that was significantly lower than the ratio (6:1) after infusion of the three pharmacologically active doses (10-25 mg/kg/day). The temporary high brain PCP concentrations and the dynamic disequilibrium between brain and serum concentrations after rapid i.v. administration could provide a better understanding of the preference of the human drug abuser for rapid rates (e.g., i.v. or smoking) of drug administration. (+info)
Mechanisms of N-methyl-D-aspartate-induced apoptosis in phencyclidine-treated cultured forebrain neurons.
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Chronic administration of phencyclidine (PCP) to rats has been demonstrated to produce a sensitized locomotor response to PCP challenge that is associated with apoptotic cell death and an up-regulation of the N-methyl-D-aspartate (NMDA) receptor. To determine the underlying mechanisms, dissociated forebrain cultures were treated for 2 days with 3 microM PCP. After washout of PCP, NMDA was added (in the presence of Mg(2+)) for 20 h. The uptake of a vital dye and the release of lactate dehydrogenase measured cell viability. Apoptosis was assessed by an enzyme-linked immunosorbent assay that was specific for fragmented (histone-associated) DNA and an in situ assay for nicked DNA, terminal dUTP nick-end labeling. These assays showed that the effect of a nontoxic concentration of NMDA (30 microM) became lethal to approximately one-third of the neurons after chronic (48-h) PCP treatment. This treatment also resulted in a 47% increase in NR1 subunit mRNA, suggesting that NMDA-induced neuronal cell death after chronic PCP is due to NMDA receptor up-regulation. Furthermore, exposure of PCP-treated cultures to NMDA led to increased expression of Bax and decreased expression of Bcl-X(L). The Bcl-X(L)/Bax ratio was markedly decreased by 30 microM NMDA in the PCP-treated, but not control, cultures. Addition of superoxide dismutase and catalase prevented the decrease in Bcl-X(L)/Bax. This study suggests that NMDA-induced changes in Bax and/or Bcl-X(L) involve the formation of reactive oxygen species. By extrapolation, these data suggest that PCP-induced apoptosis in vivo may involve similar mechanisms and that cultured neurons may be a suitable model for the mechanistic study PCP toxicity in vivo. (+info)
Blockade of phencyclidine-induced effects by a nitric oxide donor.
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1. Phencyclidine (PCP) is widely used as an animal model of schizophrenia. The aim of this study was to better understand the role of nitric oxide (NO) in the mechanism of action of PCP and to determine whether positive NO modulators may provide a new approach to the treatment of schizophrenia. 2. The effects of the NO donor, sodium nitroprusside (SNP), were studied in PCP-treated rats. Following drug administration, behavioural changes and the expression of c-fos, a metabolic marker of functional pathways in the brain, were simultaneously monitored. 3. Acute PCP (5 mg kg(-1), i. p.) treatment induced a complex behavioural syndrome, consisting of hyperlocomotion, stereotyped behaviours and ataxia. Treatment with SNP (2 - 6 mg kg(-1), i.p.) by itself produced no effect on any behaviour studied but completely abolished PCP-induced behaviour in a dose- and time-dependent manner. 4. PCP had differential regional effects on c-fos expression in rat brain, suggesting regionally different patterns of neuronal activity. The most prominent immunostaining was observed in the cortical regions. Pre-treatment with SNP blocked PCP-induced c-fos expression at doses similar to those that suppress PCP-induced behavioural effects. 5. These results implicate the NO system in the mechanism of action of PCP. The fact that SNP abolished effects of PCP suggests that drugs targeting the glutamate-NO system may represent a novel approach to the treatment of PCP-induced psychosis and schizophrenia. (+info)
Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by 2-phenyl-2-(1-piperidinyl)propane.
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2-Phenyl-2-(1-piperidinyl)propane (PPP), an analog of phencyclidine, was tested for its ability to inactivate cytochrome P450s (P450s) 2B1 and 2B6. PPP inactivated the 7-(benzyloxy)resorufin O-dealkylation activity of liver microsomes obtained from phenobarbital-induced rats with a K(I) of 11 microM. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified rat liver P450 2B1 and expressed human P450 2B6 was inactivated by PPP in a reconstituted system containing NADPH-cytochrome P450 reductase and lipid. In the presence of NADPH, the loss of activity was time- and concentration-dependent, and followed pseudo first order kinetics. The rate of inactivation for P450 2B1 was 0.3 min(-1), and the concentration of PPP required to achieve half-maximal inactivation was 12 microM. The time for 50% of the P450 2B1 to become inactivated at saturating concentrations of PPP was 2.5 min. P450 2B6 was inactivated with a k(inact) of 0.07 min(-1), a K(I) of 1.2 microM, and a t(1/2) of 9.5 min. The inactivated P450s 2B1 and 2B6 lost about 25 and 15%, respectively, of their ability to form a CO-reduced complex, suggesting that the loss of activity was caused by a PPP modification of the apoprotein rather than the heme. The estimated partition ratio for P450s 2B1 and 2B6 with PPP was 31 and 15, respectively. The inactivation was not reversible and reductase activity was not affected. Coincubation of P450 2B1 and 2B6 with PPP and NADPH in the presence of an alternate substrate protected both enzymes from inactivation. The exogenous nucleophile GSH did not affect the rate of inactivation. PPP-inactivated P450s 2B1 and 2B6 were recognized on Western blots by an antibody generated to phencyclidine that had been conjugated to BSA. Stoichiometries of 1.4:1 and 0.7:1 were determined for the binding of a [3H]PPP metabolite to P450 2B1 and 2B6, respectively. (+info)
Role of catecholaminergic and cyclic AMP systems in psychological dependence on phencyclidine: a study in mutant mice.
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Catecholaminergic and/or cyclic AMP (cAMP) systems have been demonstrated to be involved in the development of drug dependence. We investigated the involvement of both systems in psychological dependence on phencyclidine (PCP) by using tyrosine hydroxylase (TH) heterozygous (TH+/-) and cAMP response element binding protein (CREB) binding protein (CBP) heterozygous (CBP+/-) mice. PCP (8 mg/kg) induced place preference in wild-type mice pretreated with PCP (10 mg/kg once a day for 28 days). In these mice, the level of cAMP in the striatum, but not in the thalamus, was increased one day after the last injection of PCP (10 mg/kg). In TH+/- and CBP+/- mice pretreated with PCP (10 mg/kg per day for 28 days), however, no PCP (8 mg/kg)-induced place preference was observed. The level of cAMP in the striatum was increased in CBP+/- mice, but not TH+/- mice. Furthermore, we have demonstrated that the place preference induced by PCP is attenuated by 6-hydroxydopamine, a dopaminergic neurotoxin, and (+) SCH-23390, a dopamine-D1 receptor antagonist, but not by DSP-4, a noradrenergic neurotoxin, and (-) sulpiride, a dopamine-D2 receptor antagonist. These findings suggest that catecholamines and CBP are involved in the development of psychological dependence on PCP and that changes in dopaminergic and/or cAMP systems induced by repeated PCP treatment play an important role in the addiction to PCP. (+info)