Phenylethylidenehydrazine (PEH), a proposed metabolite of phenelzine (PLZ), was synthesized in our laboratories and administered to rats in a series of time‐ and dose–response studies. Phenelzine is a monoamine oxidase (MAO) inhibiting antidepressant/antipanic drug that also causes marked elevations of brain levels of the inhibitory neurotransmitter (γ‐aminobutyric acid (GABA). Like PLZ, PEH inhibited GABA transaminase (GABA‐T) and caused marked, long‐lasting increases in rat brain levels of GABA. The addition of the double bond in PEH caused a marked decrease in its ability to inhibit MAO compared to PLZ. Given the ability of PEH to elevate GABA levels and its lack of significant effect on MAO, it may be a very effective and safe tool with which to study the role of GABA in anxiety disorders and may be a potentially useful compound to test against seizures, ischemia, mania, and stroke. Drug Dev. Res. 54:35–39, 2001. © 2001 Wiley‐Liss, Inc.

Alcoholic extracts of the roots and leaves of three Echinacea species (E. purpurea, E. angustifolia and E. pallida) were analysed for the presence of characteristic chemicals by HPLC directly coupled to ultraviolet absorbance and electrospray mass spectrometric detectors. The method permitted rapid characterization and tentative identification of a large number of caffeoyl conjugates and alkamides in all the samples investigated. The roots of the three species differed markedly in their contents of characteristic compounds. Cichoric acid and verbascoside predominated in extracts of E. purpurea root whereas cynarine and dodeca-2E,4E,8Z,10Z/E-tetraenoic acid isobutylamide were the major chemicals characteristic of E. angustifolia root extracts. Echinacoside and 6-O-caffeoylechinacoside predominated in extracts of E. pallida roots. Characteristic alkamides were also examined by electrospray tandem mass spectrometry (MS/MS) and these compounds provided characteristic fragmentation patterns. Extracts of the roots and leaves of all three species were found to have antioxidant properties in a free radical scavenging assay and in a lipid peroxidation assay.

A series of N1- and N2-propargylphenelzine derivatives and analogues (1–7) was synthesized. In addition to their activity as monoamine oxidase inhibitors, two of the compounds, N1- and N2-propargylphenelzines (3 and 6), were found to be potent at preventing DSP-4-induced noradrenaline (NA) depletion in mouse hippocampus, suggesting that they have neuroprotective properties.

An electron-capture gas chromatographic procedure was developed for detection and quantification of p-hydroxymephenytoin (OHMEP), a metabolite of S-mephenytoin, in human liver microsomal preparations. OHMEP was derivatized with pentafluorobenzoyl chloride (PFBC) under basic aqueous conditions prior to analysis on a gas chromatograph equipped with a capillary column and an electron-capture detector. Dextrorophan was carried through the procedure as internal standard. The structure of the PFB derivative was confirmed using combined gas chromatography-mass spectrometry (GC-MS). The procedure is rapid and reproducible and produces a stable derivative that has excellent chromatographic properties. The limit of detection was less than 5 ng/ml, and the method was applied to extracts of human liver microsomes, which had been incubated with S-mephenytoin [a probe substrate for cytochrome P450 (CYP) 2C19].

Peripheral branches of the trigeminal nerve may be damaged during maxillofacial injury or surgical procedures and trigeminal trauma may induce severe pain that is very challenging to treat. Chronic constriction injury to the infraorbital nerve (ION-CCI) by loose ligatures has proven a useful model for some types of trigeminal neuropathic pain disorder. Using ION-CCI rats, we examined the antiallodynic effects of intrathecally administered agents which are selective for 5-HT2C receptors. Allodynia was evaluated by applying von Frey filaments to skin innervated by the injured ION. Dose-dependent antiallodynic effects followed administration of three 5-HT2C receptor agonists, 6-chloro-2-(1-piperazinyl)-pyrazine (MK212: 10, 30, and 100 μg); (S)-2-(chloro-5-fluoro-indol-l-yl)-1-methyamine fumarate (RO 60-0175: 10, 30, and 100 μg); (AaR)-8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one (WAY-161503: 10, 30, and 100 μg). ED50 values for antiallodynic effects of MK212, RO 60-0175, and WAY-161503 were 39.62, 46.67, and 51.22 μg, respectively. Intrathecal administration of the 5-HT2C receptor antagonist, 8-[5-2,4-dimethoxy-5-(4-trifluoromethylphenylsulphonamido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4,5]decane-2,4-dione (RS-102221: 30 μg) did not alter the mechanical threshold. Intrathecal pretreatment with RS-102221 (10 and 30 μg) reduced the antiallodynic effects of the highest dose of 5-HT2C agonists. These results indicated that, in this rat model, the 5-HT2C receptor plays a role in spinal inhibition of trigeminal neuropathic pain.

1. Five N,N-dialkylated amphetamines, N-methyl-N-propargylamphetamine (deprenyl; DEP), N-benzyl-N-methylamphetamine (benzphetamine; BPA), N-allyl-N-methylamphetamine (AMA), N,N-diallylamphetamine (DAA) and N-methyl-N-propylamphetamine (MPA), were metabolized in vitro with a microsomal preparation from cells expressing human CYP2D6 to determine what influence the N,N-dialkyl substituents had on the extent of N-dealkylation and/or aromatic ring oxidation. 2. The results obtained from experiments with the first two substrates, DEP and BPA, were surprisingly different. Whereas DEP was N-demethylated and N-depropargylated by the CYP2D6 enzyme system, no metabolites were formed from BPA. Subsequently, it was determined that AMA, DAA and MPA also underwent CYP2D6-catalysed N-dealkylation. Both N-methyl- and N-allylamphetamine were identified as products of AMA metabolism; similarly, metabolism of MPA produced both N-methyl- and N-propargylamphetamine, and N-allylamphetamine was the sole metabolite of DAA. 3. No N,N-didealkylated product (i.e. amphetamine) was isolated from incubates of any of the five substrates, and none of the N,N-dialkylated substrates was metabolized to a ring-hydroxylated product. 4. Rates of these CYP2D6-catalysed reactions were dependent on the nature and degree of unsaturation of the N-substituents.

The effects of Ginkgo biloba leaf extract on rat brain or livermonoamine oxidase (MAO)-A and -B activity, biogenic amine concentration in nervous tissue, N-methyl-D-aspartate (NMDA)- and N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)-induced neurotoxicity and antioxidant activity was investigated to determine the effects of the extract on monoamine catabolism and neuroprotection. Ginkgo biloba leaf extract was shown to produce in-vitro inhibition of rat brain MAO-A and -B. The Ginkgo biloba extract was chromatographed on a reverse-phase HPLC system and two of the components isolated were shown to be MAO inhibitors (MAOIs). These MAOIs were identified by high-resolution mass spectrometry as kaempferol and isorhamnetin. Pure kaempferol and a number of related flavonoids were examined as MAOIs in-vitro. Kaempferol, apigenin and chrysin proved to be potent MAOIs, but produced more pronounced inhibition of MAO-A than MAO-B. IC50 (50% inhibition concentration) values for the ability of these three flavones to inhibit MAO-A were 7 x 10(-7), 1 x 10(-6) and 2 x 10(-6) M, respectively. Ginkgo biloba leaf extract and kaempferol were found to have no effect ex-vivo on rat or mouse brain MAO or on concentrations of dopamine, noradrenaline, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid. Kaempferol was shown to protect against NMDA-induced neuronal toxicity in-vitro in rat cortical cultures, but did not prevent DSP-4-induced noradrenergic neurotoxicity in an in-vivo model. Both Ginkgo biloba extract and kaempferol were demonstrated to be antioxidants in a lipid-peroxidation assay. This data indicates that the MAO-inhibiting activity of Ginkgo biloba extract is primarily due to the presence of kaempferol. Ginkgo biloba extract has properties indicative of potential neuroprotective ability.

BACKGROUND:There have been conflicting reports in the literature about whether or not tranylcypromine is metabolized to amphetamine. In the current report, we investigated this possible route of metabolism in both rats and humans. Body fluid samples from patients and rats and brain, liver and heart samples from rats were analyzed for levels of amphetamine and 1-amino-3-phenylpropane, another potential product of cleavage of the cyclopropyl ring of tranylcypromine after administration of tranylcypromine. Extracted samples were reacted with pentofluorobenzenesulfonyl chloride and analyzed using electron-capture gas chromatography.RESULTS:Amphetamine or 1-amino-3-phenylpropane were not found in any of the samples, indicating that opening of the cyclopropyl ring of tranylcypromine is not a significant route of metabolism for this drug at usual doses.LIMITATIONS:The assay procedure did not permit analysis of 1-amino-2-phenylpropane (another possible product of cleavage of the cyclopropyl ring of tranylcypromine) or of N-methylamphetamine.CONCLUSIONS:These studies support the growing body of evidence indicating that opening of the cyclopropyl ring of tranylcypromine to form amphetamine, a drug of abuse, is not significant at usual doses of tranylcypromine.

AIM: To determine the concentrations of chemical characteristic to extracts of leaves and flowers of Hypericum perforatum (St John's wort) in a number of selected samples and, following chemical characterization, to investigate the effects of these extracts on several pharmacological properties including effects of the extracts on inhibition of 5-hydroxytryptamine (5-HT) uptake and on antioxidant properties. METHODS: The samples were analyzed for the presence of characteristic chemicals by high performance liquid chromatography (HPLC) directly coupled to ultraviolet wavelength absorbance and positive or negative mode electrospray mass spectrometric detection. The effects of extracts on 5-HT uptake were determined by quantifying H-3-5-HT incorporation into rat hippocampal prisms. Estimates of effects of extracts on free radical scavenging capacity were made using a dynamic assay based on the ability of compounds to prevent the initiation of a colored reaction produced by the horseradish peroxidase catalyzed formation of hydroxyl free radicals from hydrogen peroxide using 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) as the color indicator. RESULTS: The chemical profile of a number of extracts were determined and found to differ substantially from each other. Inhibition of 5-HT uptake was found to correlate with hyperforin content and free radical scavenging capacity was found to correlate with the content of several flavonoids including quercetin and hyperoside. CONCLUSION: Standardized extracts of H perforatum varied substantially in the concentration of several characteristic chemicals. The correlation between pharmacological activity and certain characteristic chemicals found in these extracts indicates that the medicinal benefit derived from selected extracts will vary considerably depending on their chemical composition.

1. This review summarizes the major known aspects of the metabolism of second-generation (iprindole, viloxazine, bupropion, mianserin, maprotiline, and trazodone) and fourth-generation (nefazodone and venlafaxine) antidepressants.

1. Amphetamine (AM) and five amphetamine derivatives, N-ethylamphetamine (NEA), N-butylamphetamine (NBA), 4-methoxyamphetamine (M-AM), 4-methoxy-Nethylamphetamine (M-NEA) and 4-methoxy-N-butylamphetamine (M-NBA) were incubated with microsomal preparations from cells expressing human CYP2D6 to determine whether the enzyme was capable of catalyzing the direct ring oxidation of all substrates; the N-dealkylation of NEA, NBA, M-NEA and M-NBA; and the O-demethylation of M-AM, M-NEA and M-NBA. 2. None of the six compounds examined was N-dealkylated to any extent. 3. The only metabolites produced from AM, NEA and NBA were the corresponding ring 4-hydroxylated compounds, and the rates of formation were low. 4. All ring 4-methoxylated substrates were efficiently O-demethylated by CYP2D6 to their corresponding phenols. The size of the N-alkyl group influenced the rates of formation of these phenolamines. In contrast to reported findings with 2- and 3-methoxyamphetamines, none of the 4-methoxyamphetamines was ring-oxidized in the CYP2D6 enzyme system to 2- or 3-hydroxy-4-methoxyamphetamines or to dihydroxyamphetamines.

Dextromethorphan, a constituent of many over-the-counter cough syrups, is used as a probe drug for phenotyping subjects for their cytochrome P450 2D6 (CYP2D6) enzyme activity and for measuring CYP2D6 activity of preparations such as microsomes. In such studies, formation of the metabolite dextrorphan is used as indicator of the activity of this CYP enzyme. The present report describes an electron-capture gas chromatographic procedure developed for detection and quantification of dextrorphan in human liver microsomal preparations in vitro. After basification of the incubation mixture, dextrorphan was derivatized with pentafluorobenzoyl chloride under aqueous conditions prior to analysis on a gas chromatograph equipped with a capillary column, an electron capture detector, and a printer-integrator. Para-hydroxymephenytoin was carried through the procedure as internal standard. The procedure, which involves the derivatization of dextrorphan under aqueous conditions, is rapid and involves the use of the relatively economical procedure of electron-capture gas chromatography. The derivative is stable and possesses excellent chromatographic properties.

BACKGROUND:Metabolic drug-drug interactions can occur between drugs that are substrates or inhibitors of the same cytochrome P450 (CYP) isoenzymes, but can be prevented by knowing which isoenzymes are primarily responsible for a drug's metabolism. m-Chlorophenylpiperazine (mCPP) is a psychopharmacologically active metabolite of four different psychiatric drugs. The present experiments were designed to identify the CYP isoenzymes involved in the metabolism of mCPP to its main metabolite p-hydroxy-mCPP (OH-mCPP). METHODS:The rate of production of OH-mCPP from mCPP was correlated with isoform activities in a panel of human liver microsomes, was assessed using a panel of individual complementary DNA-expressed human CYP isoenzymes, and was investigated in the presence of a specific inhibitor of CYP2D6. RESULTS:OH-mCPP production correlated significantly with CYP2D6 activity in human liver microsomes. Furthermore, incubations with microsomes from cells expressing CYP2D6 resulted in OH-mCPP formation, whereas no mCPP was formed from incubations with microsomes from cells expressing other individual isoforms. Finally, when the specific CYP2D6 inhibitor quinidine was preincubated with either human liver microsomes or cells expressing human CYP2D6, there was a concentration-dependent decrease in the production of OH-mCPP. CONCLUSIONS:These results confirm that CYP2D6 is the isoform responsible for the p-hydroxylation of mCPP, and indicate that caution should be exercised in coprescribing inhibitors or substrates of CYP2D6 with drugs that have mCPP as a metabolite.

BAKER, G.B., J. FANG, S. SINHA AND R. T. COUTTS. Metabolic drug interactions with selective serotonin reuptake inhibitor (SSRI) antidepressants. NEUROSCI BIOBEHAV REV 22(2) 325–333, 1998. The selective serotonin reuptake inhibitor (SSRI) antidepressants have become an important component of the therapeutic armamentarium in psychiatry and have attracted a great deal of public attention. Another interesting aspect of the SSRIs is their interaction with various isozymes of the cytochrome P450 (CYP) system which are responsible for metabolism of numerous drugs. This effect on the CYP isozymes has drawn attention to the importance of metabolic drug–drug interactions when dealing with drugs used to treat psychiatric disorders. Such interactions are of great relevance since psychiatry patients are frequently treated with multiple drugs and often these drugs undergo extensive biotransformation to metabolites which contribute to therapeutic and/or adverse effects. The present review deals with various aspects of metabolism mediated by CYP isozymes, particularly as they relate to pharmacokinetic interactions between the SSRIs and other drugs which are coadministered with them.

Objective: To assess the frequency of adverse drug reaction in patients with fibromyalgia in relation to medications prescribed for this condition, To evaluate the potential role of the P450IID6 phenotype in the pathogenesis of these adverse drug reactions.Methods: Thirty-five patients with fibromyalgia were assessed using a structured questionnaire with demographic and clinical data and perceived adverse drug reactions, A sample of 60 patients with rheumatoid arthritis and 62 patients with localized back pain served as controls, The P450IID6 phenotype was determined for each of the fibromyalgia patients.Results: Overall, 141 patients had used NSAID and 79 (56%) of them reported adverse effects, Antidepressant drugs were used by 68 patients and 35 (51%) patients had adverse effects, Muscle relaxant drugs were used by 48 patients and 15 (31%) of them reported side effects, Analgesics were used by 122 patients and 22 (18%) had experienced adverse effects, Statistical differences in the frequency of adverse effects were found with antidepressant drugs in the fibromyalgia group, compared with rheumatoid arthritis (p=0.01) and back pain (p=0.02), Four of the 35 patients (11.4%) had a metabolic ratio (M.R.) greater than 0.30 (log M.R.=-0.52) indicative of the poor metabolizers (PM) phenotype, M.R. varied from 0.005 (log M.R.=-2.30) to 4.99 (log M.R.=0.70).Conclusions: The problem of adverse drug reactions in fibromyalgia patients does not appear to correlate with the PM phenotype of the P450IID6 oxidative enzyme, It also is unlikely that altered xenobiotic detoxification attributable to this PM phenotype would have a significant role in the development of fibromyalgia.

An electron-capture gas chromatographic procedure was developed for the analysis of p-trifluoromethylphenol, an O-dealkylated metabolite of fluoxetine, in biological samples. A basic extraction of the biological sample was employed, followed by derivatization with pentafluorobenzenesulfonyl chloride. The internal standard, 2,4-dichlorophenol, was added to all samples used in the procedure to aid in quantitation. The practical limit of detection (signal-to-noise ratio>3) for p-trifluoromethylphenol was <5 ng/ml in human plasma samples, <10 ng/g of rat brain tissue, <25 ng/g of rat liver tissue and <25 ng/ml in human and rat urine samples. In the rat, the levels of free p-trifluoromethylphenol in the liver were 10-fold higher than those in the brain, and a substantial amount was excreted in the urine. Human urine samples contained levels of free p-trifluoromethylphenol approximately 30-fold higher than those found in human plasma samples. The procedure described is useful for the detection and quantitation of free p-trifluoromethylphenol in humans and rats treated with fluoxetine.

1. Expressed human cytochrome P450 enzyme CPY2D6 was used to metabolize amitriptyline (AMI). It was established that CYP2D6 not only catalyzed ring 10-hydroxylation of AMI, but also mediated its N-demethylation to nortriptyline (NT), as well as the formation of 10-hydroxy-NT from NT. When the metabolism of AMI by CYP2D6 was repeated in the presence of quinidine, none of the metabolites, 10-hydroxy-AMI, NT and 10-hydroxy-NT, was formed. 2. Biochemical parameters of NT formation from AMI were determined, yielding Km = 47.48 +/- 1.32 microM; Vmax = 3.95 +/- 0.11 nmol/h/mg protein. The same parameters were calculated for the formation of 10-hydroxy-AMI (E + Z-isomers) from AMI, yielding Km = 10.70 +/- 0.20 microM; Vmax = 8.99 +/- 0.47 nmol/h/mg protein. 3. The formation of 10-hydroxy-NT from AMI proceeded primarily via NT and to a much lesser extent via 10-hydroxy-AMI. 4. Quantitative analyses of AMI and its metabolites were difficult to reproduce when the metabolites were analysed underivatized. Two derivatization procedures, acetylation and trifluoroacetylation, were employed to improve assay reproducibility.

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.

Antidepressant drugs are widely used in the treatment of different psychiatric disorders, as well as in conjunction with antipsychotics for the treatment of major depressive disorder. In this study, a simple and rapid ultrasound-assisted low-density solvent dispersive liquid–liquid microextraction (UA-LDS-DLLME) method was developed for the simultaneous determination of 12 new antidepressants (norfluoxetine, fluoxetine, fluvoxamine, agomelatine, mirtazapine, moclobemide, melitracen, N-desmethylmirtazapine, maprotiline, sertraline, citalopram, paroxetine) and 2 antipsychotics (clozapine and haloperidol) in human whole blood by gas chromatography–mass spectrometry (GC–MS). Different parameters affecting the UA-LDS-DLLME were optimized and the optimal conditions were as follows: 100 μL of toluene as extraction solvent, extraction pH 12 and 3 min of ultrasound stirring. Good linearity (R2 ≥ 0.991) was obtained at the concentration range of 15–1500 ng/mL for norfluoxetine, fluoxetine, fluvoxamine, melitracen, maprotiline and citalopram, and 5–500 ng/mL for agomelatine, mirtazapine, moclobemide, N-desmethylmirtazapine, sertraline, paroxetine, clozapine and haloperidol. The intra-day and inter-day precision were all less than 10%, and accuracy of intra-day and inter-day were in the range of −12.7% to 7.9% and −13.9 to 11.8%, respectively. The extraction recoveries of most analytes were more than 60%. The UA-LDS-DLLME/GC–MS method was demonstrated with acceptable precision, accuracy and good specificity for the simultaneous determination of 12 antidepressants and 2 antipsychotics, and has been successfully applied in a real case.