Nicotine Exposure in a Phencyclidine-Induced Mice Model of Schizophrenia: Sex-Selective Medial Prefrontal Cortex Protein Markers of the Combined Insults in Adolescent Mice.

Tobacco misuse as a comorbidity of schizophrenia is frequently established during adolescence. However, comorbidity markers are still missing. Here, the method of label-free proteomics was used to identify deregulated proteins in the medial prefrontal cortex (prelimbic and infralimbic) of male and female mice modelled to schizophrenia with a history of nicotine exposure during adolescence. Phencyclidine (PCP), used to model schizophrenia (SCHZ), was combined with an established model of nicotine minipump infusions (NIC). The combined insults led to worse outcomes than each insult separately when considering the absolute number of deregulated proteins and that of exclusively deregulated ones. Partially shared Reactome pathways between sexes and between PCP, NIC and PCPNIC groups indicate functional overlaps. Distinctively, proteins differentially expressed exclusively in PCPNIC mice reveal unique effects associated with the comorbidity model. Interactome maps of these proteins identified sex-selective subnetworks, within which some proteins stood out: for females, peptidyl-prolyl cis-trans isomerase (Fkbp1a) and heat shock 70 kDa protein 1B (Hspa1b), both components of the oxidative stress subnetwork, and gamma-enolase (Eno2), a component of the energy metabolism subnetwork; and for males, amphiphysin (Amph), a component of the synaptic transmission subnetwork. These are proposed to be further investigated and validated as markers of the combined insult during adolescence.

Pharmacological Characterization of [18F]-FNM and Evaluation of NMDA Receptors Activation in a Rat Brain Injury Model.

PURPOSE: NMDA receptors (NMDARs) dysfunction plays a central role in the physiopathology of psychiatric and neurodegenerative disorders whose mechanisms are still poorly understood. The development of a PET (positron emission tomography) tracer able to selectively bind to the NMDARs intra-channel PCP site may make it possible to visualize NMDARs in an open and active state. We describe the in vitro pharmacological characterization of [18F]-fluoroethylnormemantine ([18F]-FNM) and evaluate its ability to localize activated NMDA receptors in a rat preclinical model of excitotoxicity. PROCEDURES: The affinity of the non-radioactive analog for the intra-channel PCP site was determined in a radioligand competition assay using [3H]TCP ([3H]N-(1-[thienyl]cyclohexyl)piperidine) on rat brain homogenates. Selectivity was also investigated by the displacement of specific radioligands targeting various cerebral receptors. In vivo brain lesions were performed using stereotaxic quinolinic acid (QA) injections in the left motor area (M1) of seven Sprague Dawley rats. Each rat was imaged with a microPET/CT camera, 40 min after receiving a dose of 30 MBq + / - 20 of [18F]-FNM, 24 and 72 h after injury. Nine non-injured rats were also imaged using the same protocol. RESULTS: FNM displayed IC50 value of 13.0 ± 8.9 µM in rat forebrain homogenates but also showed significant bindings on opioid receptors. In the frontal and left somatosensory areas, [18F]FNM PET detected a mean of 37% and 41% increase in [18F]FNM uptake (p < 0,0001) 24 and 72 h after QA stereotaxic injection, respectively, compared to the control group. CONCLUSIONS: In spite of FNM's poor affinity for NMDAR PCP site, this study supports the ability of this tracer to track massive activation of NMDARs in neurological diseases.

The Abuse Potential of Novel Synthetic Phencyclidine Derivative 1-(1-(4-Fluorophenyl)Cyclohexyl)Piperidine (4'-F-PCP) in Rodents.

The dissociative anesthetic phencyclidine (PCP) and PCP derivatives, including 4'-F-PCP, are illegally sold and abused worldwide for recreational and non-medical uses. The psychopharmacological properties and abuse potential of 4'-F-PCP have not been fully characterized. In this study, we evaluated the psychomotor, rewarding, and reinforcing properties of 4'-F-PCP using the open-field test, conditioned place preference (CPP), and self-administration paradigms in rodents. Using Western immunoblotting, we also investigated the expression of dopamine (DA)-related proteins and DA-receptor-mediated downstream signaling cascades in the nucleus accumbens (NAc) of 4'-F-PCP-self-administering rats. Intraperitoneal administration of 10 mg/kg 4'-F-PCP significantly increased locomotor and rearing activities and increased CPP in mice. Intravenous administration of 1.0 mg/kg/infusion of 4'-F-PCP significantly enhanced self-administration during a 2 h session under fixed ratio schedules, showed a higher breakpoint during a 6 h session under progressive ratio schedules of reinforcement, and significantly altered the expression of DA transporter and DA D1 receptor in the NAc of rats self-administering 1.0 mg/kg 4'-F-PCP. Additionally, the expression of phosphorylated (p) ERK, pCREB, c-Fos, and FosB/ΔFosB in the NAc was significantly enhanced by 1.0 mg/kg 4'-F-PCP self-administration. Taken together, these findings suggest that 4'-F-PCP has a high potential for abuse, given its robust psychomotor, rewarding, and reinforcing properties via activation of DAergic neurotransmission and the downstream signaling pathways in the NAc.

Alterations of acoustic features of 50 kHz vocalizations by nicotine and phencyclidine in rats.

Ultrasonic vocalizations are widely used to examine affective states in rats, yet relatively few studies explore the acoustic features of vocalizations, especially in relation to drug exposure, and no studies have explored alterations in acoustic features over time. The goal of this study was to examine nicotine- and phencyclidine-induced alterations of bandwidth, duration, and frequency of 50 kHz vocalizations. The minimum and maximum frequency, bandwidth, and duration of calls were examined after 7 days of daily subcutaneous administration of phencyclidine (2.0 mg/kg) and nicotine (0.2 and 0.4 mg/kg) in male Sprague-Dawley rats. Bandwidth was significantly decreased in rats treated with both nicotine (0.2 and 0.4 mg/kg) and phencyclidine. Maximum frequency was lowest on the first day of exposure compared with all other days and was not altered by drug exposure. Call duration was not affected by time or drug exposure. These findings suggest the importance of studying alterations in acoustic features in time, especially those induced by drug exposure.

Development of MS Binding Assays targeting the binding site of MB327 at the nicotinic acetylcholine receptor.

The bispyridinium compound MB327 has been shown previously to have a positive pharmacological effect against poisoning with organophosphorous compounds (OPCs). The mechanism by which it exerts its therapeutic effect seems to be directly mediated by the nicotinic acetylcholine receptor (nAChR). In the present study, the development of mass spectrometry based binding assays (MS Binding Assays) for characterization of the binding site of MB327 at the nAChR from Torpedo californica is described. MS Binding Assays follow the principle of radioligand binding assays, but do not, in contrast to the latter, require a radiolabeled reporter ligand, as the readout is in this case based on mass spectrometric detection. For [2H6]MB327, a deuterated MB327 analogue employed as reporter ligand in the MS Binding Assays, an LC-ESI-MS/MS method was established allowing for its fast and reliable quantification in samples resulting from binding experiments. Using centrifugation for separation of non-bound [2H6]MB327 from target-bound [2H6]MB327 in saturation and autocompetition experiments (employing native MB327 as competitor) enabled reliable determination of specific binding. In this way, the affinities for [2H6]MB327 (Kd=15.5±0.9µmolL-1) and for MB327 (Ki=18.3±2.6µmolL-1) towards the nAChR could be determined for the first time. The almost exactly matching affinities for MB327 and [2H6]MB327 obtained in the MS Binding Assays are in agreement with potencies previously found in functional studies. In summary, our results demonstrate that the established MS Binding Assays represent a promising tool for affinity determination of test compounds towards the binding site of MB327 at the nAChR.

Phencyclidine-induced dysregulation of primary cilia in the rodent brain.

Significant roles of the primary cilia in the central nervous system have been reported in neural generation and cognitive functions. However, little is known about the possible pathological changes in brain primary cilia in neuropsychiatric disorders. To obtain an insight into the relationship between cilial dysregulation and schizophrenia, we presently investigated the effects of psychotomimetics, phencyclidine, MK-801 (dizocilpine), and methamphetamine, on morphological and molecular indices in the rodent brain. Using an immunohistochemical technique, we found that a subcutaneous injection of phencyclidine, an NMDA type glutamate receptor (NMDAR) antagonist, caused a reduction in the long axis length of a primary cilium in the CA1 region of the hippocampus without affecting that in the dentate gyrus and medial prefrontal cortex of rats and mice. The region-selective modulation of primary cilia was mimicked by another NMDAR antagonist, MK-801, but not by the indirect dopamine agonist methamphetamine. Furthermore, systemic administration of phencyclidine, but not methamphetamine, down-regulated mRNA expression of primary cilium morphology-related genes, including kif3a, 5-HTR6, RPGRIP1L, and TMEM67, and of genes composing the cilial Wnt/ß-catenin signaling pathway, ß-catenin, syn2 and Bcl-2, in the hippocampus, but not in the cerebral cortex of rats. These findings suggest that NMDAR hypofunction-induced dysregulation of CA1 primary cilia could be involved in the pathophysiology of dopamine transmission-independent symptoms of schizophrenia.

New Psychoactive Substances 3-Methoxyphencyclidine (3-MeO-PCP) and 3-Methoxyrolicyclidine (3-MeO-PCPy): Metabolic Fate Elucidated with Rat Urine and Human Liver Preparations and their Detectability in Urine by GC-MS, "LC-(High Resolution)-MSn" and "LC-(High Resolution)-MS/MS".

BACKGROUND: 3-Methoxyphencyclidine (3-MeO-PCP) and 3-methoxyrolicyclidine (3-MeOPCPy) are two new psychoactive substances (NPS). The aims of the present study were the elucidation of their metabolic fate in rat and pooled human liver microsomes (pHLM), the identification of the cytochrome P450 (CYP) isoenzymes involved, and the detectability using standard urine screening approaches (SUSA) after intake of common users' doses using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-multi-stage mass spectrometry (LC-MSn), and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS). METHODS: For metabolism studies, rat urine samples were treated by solid phase extraction or simple precipitation with or without previous enzymatic conjugate cleavage. After analyses via LC-HR-MSn, the phase I and II metabolites were identified. RESULTS: Both drugs showed multiple aliphatic hydroxylations at the cyclohexyl ring and the heterocyclic ring, single aromatic hydroxylation, carboxylation after ring opening, O-demethylation, and glucuronidation. The transferability from rat to human was investigated by pHLM incubations, where Odemethylation and hydroxylation were observed. The involvement of the individual CYP enzymes in the initial metabolic steps was investigated after single CYP incubations. For 3-MeO-PCP, CYP 2B6 was responsible for aliphatic hydroxylations and CYP 2C19 and CYP 2D6 for O-demethylation. For 3-MeO-PCPy, aliphatic hydroxylation was again catalyzed by CYP 2B6 and O-demethylation by CYP 2C9 and CYP 2D6 Conclusions: As only polymorphically expressed enzymes were involved, pharmacogenomic variations might occur, but clinical data are needed to confirm the relevance. The detectability studies showed that the authors' SUSAs were suitable for monitoring the intake of both drugs using the identified metabolites.

N'-3-(Trifluoromethyl)phenyl Derivatives of N-Aryl-N'-methylguanidines as Prospective PET Radioligands for the Open Channel of the N-Methyl-d-aspartate (NMDA) Receptor: Synthesis and Structure-Affinity Relationships.

N-Methyl-d-aspartate (NMDA) receptor dysfunction has been linked to several neuropsychiatric disorders, including Alzheimer's disease, epilepsy, drug addiction, and schizophrenia. A radioligand that could be used with PET to image and quantify human brain NMDA receptors in the activated "open channel" state would be useful for research on such disorders and for the development of novel therapies. To date, no radioligands have shown well-validated efficacy for imaging NMDA receptors in human subjects. In order to discover improved radioligands for PET imaging, we explored structure-affinity relationships in N'-3-(trifluoromethyl)phenyl derivatives of N-aryl-N'-methylguanidines, seeking high affinity and moderate lipophilicity, plus necessary amenability for labeling with a positron-emitter, either carbon-11 or fluorine-18. Among a diverse set of 80 prepared N'-3-(trifluoromethyl)phenyl derivatives, four of these compounds (13, 19, 20, and 36) displayed desirable low nanomolar affinity for inhibition of [(3)H](+)-MK801 at the PCP binding site and are of interest for candidate PET radioligand development.

Novel Potent N-Methyl-d-aspartate (NMDA) Receptor Antagonists or σ1 Receptor Ligands Based on Properly Substituted 1,4-Dioxane Ring.

Two series of 1,4-dioxanes (4-11 and 12-19) were rationally designed and prepared to interact either with the phencyclidine (PCP) binding site of the N-methyl-d-aspartate (NMDA) receptor or with σ1 receptors, respectively. The biological profiles of the novel compounds were assessed using radioligand binding assays, and the compounds with the highest affinities were investigated for their functional activity. The results were in line with the available pharmacophore models and highlighted that the 1,4-dioxane scaffold is compatible with potent antagonist activity at NMDA receptor or high affinity for σ1 receptors. The primary amines 6b and 7 bearing a cyclohexyl and a phenyl ring or two phenyl rings in position 6, respectively, were the most potent noncompetitive antagonists at the NMDA receptor with IC50 values similar to those of the dissociative anesthetic (S)-(+)-ketamine. The 5,5-diphenyl substitution associated with a benzylaminomethyl moiety in position 2, as in 18, favored the interaction with σ1 receptors.

Stereoselective synthesis and pharmacological evaluation of [4.3.3]propellan-8-amines as analogs of adamantanamines.

Amantadine (1) exerts its anti-Parkinson effects by inhibition of the NMDA associated cation channel and its antiviral activity by inhibition of the M2 protein channel of influenza A viruses. Herein the synthesis, NMDA receptor affinity and anti-influenza activity of analogous propellanamines 3 are reported. The key steps in the synthesis of the diastereomeric propellanamines syn-3 and anti-3 are diastereoselective reduction of the ketone 7 with L-Selectride to give anti-11, Mitsunobu inversion of the alcohol anti-13 into syn-13, and SN2 substitution of diastereomeric mesylates syn-14 and anti-14 with NaN3. The affinity of the propellanamines syn-3 and anti-3 to the PCP binding site of the NMDA receptor is similar to that of amantadine (Ki=11 µM). However, both propellanamines syn-3 and anti-3 do not exhibit activity against influenza A viruses. Compared to amantadine (1), the structurally related propellanamines syn-3 and anti-3 retain the NMDA antagonistic activity but loose the antiviral activity.

Ketamine and phencyclidine: the good, the bad and the unexpected.

The history of ketamine and phencyclidine from their development as potential clinical anaesthetics through drugs of abuse and animal models of schizophrenia to potential rapidly acting antidepressants is reviewed. The discovery in 1983 of the NMDA receptor antagonist property of ketamine and phencyclidine was a key step to understanding their pharmacology, including their psychotomimetic effects in man. This review describes the historical context and the course of that discovery and its expansion into other hallucinatory drugs. The relevance of these findings to modern hypotheses of schizophrenia and the implications for drug discovery are reviewed. The findings of the rapidly acting antidepressant effects of ketamine in man are discussed in relation to other glutamatergic mechanisms.

Radiolabeling of [18F]-fluoroethylnormemantine and initial in vivo evaluation of this innovative PET tracer for imaging the PCP sites of NMDA receptors.

INTRODUCTION: The N-methyl-D-aspartate receptor (NMDAr) is an ionotropic receptor that mediates excitatory transmission. NMDAr overexcitation is thought to be involved in neurological and neuropsychiatric disorders such as Alzheimer disease and schizophrenia. We synthesized [(18)F]-fluoroethylnormemantine ([(18)F]-FNM), a memantine derivative that binds to phencyclidine (PCP) sites within the NMDA channel pore. These sites are primarily accessible when the channel is in the active and open state. METHODS: Radiosynthesis was carried out using the Raytest® SynChrom R&D fluorination module. Affinity of this new compound was determined by competition assay. We ran a kinetic study in rats and computed a time-activity curve based on a volume-of-interest analysis, using CARIMAS® software. We performed an ex vivo autoradiography, exposing frozen rat brain sections to a phosphorscreen. Adjacent sections were used to detect NMDAr by immunohistochemistry with an anti-NR1 antibody. As a control of the specificity of our compound for NMDAr, we used a rat anesthetized with ketamine. Correlation analysis was performed with ImageJ software between signal of autoradiography and immunostaining. RESULTS: Fluorination yield was 10.5% (end of synthesis), with a mean activity of 3145 MBq and a specific activity above 355 GBq/µmol. Affinity assessment allowed us to determine [(19)F]-FNM IC50 at 6.1 10(-6)M. [(18)F]-FMN concentration gradually increased in the brain, stabilizing at 40 minutes post injection. The brain-to-blood ratio was 6, and 0.4% of the injected dose was found in the brain. Combined ex vivo autoradiography and immunohistochemical staining demonstrated colocalization of NMDAr and [(18)F]-FNM (r=0.622, p<0.0001). The highest intensity was found in the cortex and cerebellum, and the lowest in white matter. A low and homogeneous signal corresponding to unspecific binding was observed when PCP sites were blocked with ketamine. CONCLUSIONS: [(18)F]-FNM appears to be a promising tracer for imaging NMDAr activity for undertaking preclinical studies in perspective of clinical detection of neurological or neuropsychological disorders.

Syntheses and N-methyl-D-aspartate receptor antagonist pharmacology of fluorinated arylcycloheptylamines.

Selective uncompetitive antagonists of the phencyclidine (PCP) binding site of the N-methyl-D-aspartate receptor (NMDAR) are known to have therapeutic potential as anticonvulsants and neuroprotective agents. Several fluorinated molecules with each containing a cycloheptane ring were designed to probe the PCP pharmacophore and test the influence of fluorine substitution on NMDAR binding and in vivo efficacy. Syntheses and analyses of six novel compounds, 1-(4- fluorophenyl)cycloheptanamine (3), 1-(1-(4-fluorophenyl)cycloheptyl)piperidine (4), 1-(1-(4-fluorophenyl)cycloheptyl) pyrrolidine (5), 1-(3-fluorophenyl)cycloheptanamine (6), 1-(1-(3-fluorophenyl)cycloheptyl)piperidine (7), 1-(1-(3-fluorophenyl) cycloheptyl)pyrrolidine (8) and several related reference arylcyloalkylamines are described. Receptor binding was performed at the PCP site of NMDAR for each compound using [(3)H]-(+)-MK-801 displacement. Unexpectedly, the 3- fluoro- primary amine 6 had the greatest affinity of the series and these binding results support a different structure activity relationship (SAR) profile for arylcycloheptylamines when compared to arylcyclohexylamines like PCP. Five of the novel compounds have affinity (Ki) in the hundred nM (10(-7)) range. In addition, compounds 3, 5, 6, 7 and 8 were evaluated and found to exhibit neuroprotective effects from NMDA induced toxicity in vitro and compounds 6, 7 and 8 exhibited anticonvulsant activities in rats. An ED50 of 13.84 mg/kg was found for compound 6 in rat maximal electroshock (MES) test with a protective index (PI) of 3.66 against ataxia. These results support further investigation of the arylcycloheptylamine class.

The ketamine analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor.

In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as 'designer drugs' and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS)-2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone) and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenyl)cyclohexanamine) and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenyl)cyclohexyl]piperidine and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine), were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.

Enantiomerically pure 1,3-dioxanes as highly selective NMDA and σ1 receptor ligands.

We synthesized and investigated the NMDA and σ1 receptor affinity of enantiomerically pure 2-(2-phenyl-1,3-dioxan-4-yl)ethanamines 17-26. The primary amines (R,R)-18-20 with an axially oriented phenyl moiety in position 2 interacted with high enantioselectivity (eudismic ratios 70-130) and high affinity (K(i)((R,R)-19) = 13 nM) with the PCP binding site of the NMDA receptor. Introduction of an N-benzyl moiety led to potent σ1 ligands including compound (S,R)-22 (K(i) = 6 nM) with an equatorially oriented phenyl moiety in position 2.

Chemistry, pharmacology, and metabolism of emerging drugs of abuse.

In recent years, besides the classic designer drugs of the amphetamine type, a series of new drug classes appeared on the illicit drugs market. The chemistry, pharmacology, toxicology, metabolism, and toxicokinetics is discussed of 2,5-dimethoxy amphetamines, 2,5-dimethoxy phenethylamines, beta-keto-amphetamines, phencyclidine derivatives as well as of herbal drugs, ie, Kratom. They have gained popularity and notoriety as rave drugs. The metabolic pathways, the involvement of cytochrome P450 isoenzymes in the main pathways, and their roles in hepatic clearance are also summarized.

1,2-ethane bis-1-amino-4-benzamidine is active against several brain insult and seizure challenges through anti-NMDA mechanisms targeting the 3H-TCP binding site and antioxidant action.

Five bis-benzamidines were screened towards murine magnesium deficiency-dependent audiogenic seizures, unravelling two compounds with efficacious doses 50 (ED(50)) less than 10mg/kg. They were also screened against maximal electroshock and subcutaneous pentylenetetrazole-induced seizures, and explored for superoxide -scavenging activity. 1,2-Ethane bis-1-amino-4-benzamidine (EBAB) was selected and evaluated in 6 Hz seizure test (ED(50)=49 mg/kg) and at 4 microg/kg in focal cerebral ibotenate poisoning in pups (sizes of both white and grey matter wounds were halved). EBAB was further tested on NMDA-induced seizures in mice (ED(50)=6 mg/kg) and on (3)H-TC -binding to a rodent cerebral preparation (IC(50)=1.4 microM). Taken as a whole, present data emphasise the suitability of bis-benzamidines as templates for designing brain protective compounds.

Early candidacy for differentiation into heterocysts in the filamentous cyanobacterium Anabaena sp. PCC 7120.

The filamentous cyanobacterium Anabaena sp. PCC 7120 fixes dinitrogen facultatively. Upon depletion of combined nitrogen, about 10% of vegetative cells within the filaments differentiate terminally into nitrogen-fixing cells. The heterocyst has been studied as a model system of prokaryotic cell differentiation, with major focus on signal transduction and pattern formation. The fate of heterocyst differentiation is determined at about the eighth hour of induction (point of no return), well before conspicuous morphological or metabolic changes occur. However, little is known about how the initial heterocysts are selected after the induction by nitrogen deprivation. To address this question, we followed the fate of every cells on agar plates after nitrogen deprivation with an interval of 4 h. About 10% of heterocysts were formed without prior division after the start of nitrogen deprivation. The intensity of fluorescence of GFP in the transformants of hetR-gfp increased markedly in the future heterocysts at the fourth hour with respect to other cells. We also noted that the growing filaments consisted of clusters of four consecutive cells that we call quartets. About 75% of initial heterocysts originated from either of the two outer cells of quartets at the start of nitrogen deprivation. These results suggest that the future heterocysts are loosely selected at early times after the start of nitrogen deprivation, before the commitment. Such early candidacy could be explained by different properties of the outer and inner cells of a quartet, but the molecular nature of candidacy remains to be uncovered.

Subchronic effects of phencyclidine on dopamine and serotonin receptors: implications for schizophrenia.

Changes in representative dopamine (D(1), D(2), and D(4)) and serotonin (5-HT(1A) and 5-HT(2A)) receptors that have been implicated in the pathophysiology and treatment of schizophrenia were autoradiographically quantified after subchronic phencyclidine (PCP) treatment (2 mg/kg for 7 days, bi-daily followed by 7 days drug free). This treatment has consistently induced robust and long-lasting cognitive deficits in adult rats, although the molecular mechanisms contributing to PCP-induced cognitive deficits remain undefined. Repeated PCP treatment significantly decreased labeling of D(1) receptors in the medial and lateral caudate-putamen (22% and 23%, respectively) and increased 5HT(1A) receptor binding in the medial-prefrontal (26%) and dorsolateral-frontal cortex (30%). No changes in D(1) or 5HT(1A) receptors were detected in other brain regions. These findings suggest that downregulation of striatal D(1) receptors and upregulation of cortical 5HT(1A) receptors may contribute to PCP-induced impairment of cognitive functions in rats. Subchronic PCP treatment did not alter levels of D(2), D(4), and 5HT(2A) receptors in all brain regions examined, which suggests a minimal role for these receptors in mediating subchronic actions of PCP in adult rats.

Use of fission yeast heterologously expressing human cytochrome P450 2B6 in biotechnological synthesis of the designer drug metabolite N-(1-phenylcyclohexyl)-2-hydroxyethanamine.

Standards of drug metabolites are required for drug metabolism studies as a basis for toxicological risk assessment with respect to drug interactions and pharmacogenetic polymorphisms. They are further needed as reference compounds in analytical toxicology. However, metabolite standards are often not commercially available, particularly in the case of new designer drugs. As an alternative to often cumbersome chemical synthesis, human cytochrome P450 (CYP) isoenzymes heterologously expressed in the fission yeast Schizosaccharomyces pombe can be used for the biotechnological synthesis of drug metabolites. In the present study this concept was applied to the synthesis of N-(1-phenylcyclohexyl)-2-hydroxyethanamine (PCHEA), the common O-dealkyl metabolite of the designer drugs N-(1-phenylcyclohexyl)-2-methoxyethanamine (PCMEA) and N-(1-phenylcyclohexyl)-2-ethoxyethanamine (PCEEA). After adding 250 micromol PCEEA x HCl (62 mg), a 1 l culture of CAD65 (S. pombe strain co-expressing human CYP reductase and CYP2B6) was fermented over 65 h (pH 8, 30 degrees C) and centrifuged. PCHEA and remaining parent drug were isolated from the supernatant by solid-phase extraction (SPE). The eluate was evaporated to dryness and reconstituted in HPLC solvent. Aliquots were separated by semi-preparative HPLC. From the respective fraction, PCHEA was extracted by liquid-liquid extraction and precipitated as hydrochloric salt. Approximately 80% of PCEEA was converted to PCHEA. The final yield of PCHEA x HCl was 9 mg (35 micromol). Its identity was confirmed by GC-MS, (1)H NMR and (13)C NMR. The product purity, as determined by HPLC-UV, was 95%.