Effect of protein and calorie malnutrition on drug metabolism in rat - in vitro.

PURPOSE: To study the effect of protein and calorie malnutrition on in vitro drug metabolism of protein and calorie malnourished juvenile and adult rats. METHOD: Microsomal incubation was used as a means of monitoring drug metabolism changes, HPLC was employed to quantify metabolites and enzyme immunoassay (EIA) was used for rat growth hormone (rGH) monitoring. RESULTS: Protein and calorie malnutrition significantly decreased levels of microsomal protein and total P450. Microsome of protein and calorie malnourished rats showed impaired testosterone 16alpha- and 2alpha- hydroxylation (CYP2C11), testosterone 6beta-hydroxylation (CYP3A), and testosterone 7alpha-hydroxylation (CYP2A1). Testosterone 16beta-hydroxylation (CYP2B1) did not show any significant change, neither in capacity nor affinity. The quantity and the secretion pattern of rGH were not altered in protein and calorie malnourished rats compared to those in healthy animals. CONCLUSIONS: Serum albumin is not a good indicator of malnutrition. The capacity and affinity of CYP2C11, CYP3A and CYP2A1 were compromised by protein and calorie malnutrition. The impairment of drug metabolism in protein and calorie malnourished rats was not caused by the alteration of rGH.

Synthesis of N-propargylphenelzine and analogues as neuroprotective agents.

A series of N(1)- and N(2)-propargylphenelzine derivatives and analogues (1-7) was synthesized. In addition to their activity as monoamine oxidase inhibitors, two of the compounds, N(1)- and N(2)-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.

Comparison of chemical components and antioxidants capacity of different Echinacea species.

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.

Failure to detect amphetamine or 1-amino-3-phenylpropane in humans or rats receiving the MAO inhibitor tranylcypromine.

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.

Neurochemical and metabolic aspects of antidepressants: an overview.

Antidepressants, in addition to being effective therapeutic agents for depression, have also proved to be multifaceted drugs useful for treating a number of other psychiatric and neurologic disorders. Despite the widespread use of these drugs, much remains to be understood about their mechanisms of action and other important aspects, such as their metabolism and potential interactions with other drugs. This article reviews research conducted in the authors' laboratories on various aspects of antidepressants, including trace amines and antidepressants, gamma-aminobutyric acid and antidepressants, drug metabolism, development and application of rapid, sensitive assay procedures for antidepressants and their metabolites; and drug development based on analogues of the antidepressants phenelzine and tranylcypromine. The significance of this work to future drug development is also discussed.

Identification of kaempferol as a monoamine oxidase inhibitor and potential Neuroprotectant in extracts of Ginkgo biloba leaves.

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.

Metabolism of N,N-dialkylated amphetamines, including deprenyl, by CYP2D6 expressed in a human cell line.

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.

Determination of paroxetine levels in human plasma using gas chromatography with electron-capture detection.

A simple, rapid and sensitive procedure using gas chromatography with electron-capture detection to measure paroxetine levels in human plasma has been developed. The analyte was extracted from plasma with ethyl acetate after basification of the plasma and then derivatized with heptafluorobutyric anhydride before gas chromatographic separation. The calibration curves were linear, with typical r2 values >0.99. The assay was highly reproducible and gave peaks with excellent chromatographic properties.

Chemical and pharmacological evaluation of Hypericum perforatum extracts.

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 3H-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.

A rapid electron-capture gas chromatographic procedure for the analysis of p-hydroxymephenytoin.

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].

Involvement of CYP2D6 in the in vitro metabolism of amphetamine, two N-alkylamphetamines and their 4-methoxylated derivatives.

1. Amphetamine (AM) and five amphetamine derivatives, N-ethylamphetamine (NEA), N-butylamphetamine (NBA), 4-methoxyamphetamine (M-AM), 4-methoxy-N-ethylamphetamine (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.

Metabolism of some "second"- and "fourth"-generation antidepressants: iprindole, viloxazine, bupropion, mianserin, maprotiline, trazodone, nefazodone, and venlafaxine.

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. 2. Discussions about specific enzymes involved and about possible pharmacokinetic drug-drug interactions, particularly as they relate to cytochrome P450 enzymes, are provided.

Polymorphic cytochromes P450 and drugs used in psychiatry.

1. The cytochrome P450 monooxygenases, CYP2D6, CYP2C19, and CYP2C9, display polymorphism. CYP2D6 and CYP2C19 have been studied extensively, and despite their low abundance in the liver, they catalyze the metabolism of many drugs. 2. CYP2D6 has numerous allelic variants, whereas CYP2C19 has only two. Most variants are translated into inactive, truncated protein or fail to express protein. 3. CYP2C9 is expressed as the wild-type enzyme and has two variants, in each of which one amino acid residue has been replaced. 4. The nucleotide base sequences of the cDNAs of the three polymorphic genes and their variants have been determined, and the proteins derived from these genes have been characterized. 5. An absence of CYP2D6 and/or CYP2C19 in an individual produces a poor metabolizer (PM) of drugs that are substrates of these enzymes. 6. When two drugs that are substrates for a polymorphic CYP enzyme are administered concomitantly, each will compete for that enzyme and competitively inhibit the metabolism of the other substrate. This can result in toxicity. 7. Patients can be readily phenotyped or genotyped to determine their CYP2D6 or CYP2C19 enzymatic status. Poor metabolizers (PMs), extensive metabolizers (EMs), and ultrarapid metabolizers (URMs) can be identified. 8. Numerous substrates and inhibitors of CYP2D6, CYP2C19, and CYP2C9 are identified. 9. An individual's diet and age can influence CYP enzyme activity. 10. CYP2D6 polymorphism has been associated with the risk of onset of various illnesses, including cancer, schizophrenia, Parkinson's disease, Alzheimer's disease, and epilepsy.

Analysis of dextrorphan, a metabolite of dextromethorphan, using gas chromatography with electron-capture detection.

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.

Human CYP2D6 and metabolism of m-chlorophenylpiperazine.

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.

Elucidation of individual cytochrome P450 enzymes involved in the metabolism of clozapine.

The atypical antipsychotic clozapine has been reported to be metabolised mainly to its N-oxide and N-demethylated products. In the present study, individual recombinant cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) enzymes were used to elucidate which enzymes are responsible for these metabolic conversions. In vitro metabolism of clozapine was investigated using human CYP1A1, CYP1A2, CYP2C8, CYP2E1, CYP2C9-arg144, CYP2C9-cys144, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and FMO3 supplemented with an NADPH generating system. Clozapine and its N-oxidation and N-demethylation metabolites were determined by an HPLC method with a Hypersil CN column and a UV detector. Of the enzymes investigated, CYP1A2, CYP3A4, CYP2D6, CYP2C8, CYP2C19 and, to a lesser extent, CYP2C9-cys, CYP2C9-arg and CYP3A5 were apparently involved in N-demethylation, while CYP1A2, CYP3A4, FMO3 and, to a lesser extent, CYP2C8, CYP2C19 and CYP3A5 were found to catalyse the formation of clozapine N-oxide. A bank of 16 human liver microsome preparations was investigated for ability to catalyze the production of clozapine N-oxide and N-desmethylclozapine. Attempts were made to correlate the rates of formation of these metabolites of clozapine to previously determined catalytic activities of CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. At a clozapine concentration of 20 microM, the rate of formation of clozapine N-oxide showed significant correlations with activities of CYP3A4 (P<0.01) and CYP1A2 (P<0.05). The formation of N-desmethylclozapine exhibited significant correlations with CYP1A2 (P<0.01) and CYP3A4 (P<0.01). Similar correlations were observed when the clozapine concentration was increased to 300 microM except that the formation of clozapine N-oxide no longer correlated with CYP1A2 activity. It can be seen from these results that although some recombinant enzymes individually are capable of metabolising clozapine, the activities of several of these enzymes did not correlate with clozapine metabolism when mixtures of the enzymes are used. By combining the results of the current study and those reported in the literature, it is proposed that CYP3A4 and FMO3 are primarily responsible for the production of clozapine N-oxide, and CYP3A4 and CYP1A2 are primarily responsible for the formation of N-desmethylclozapine. The present study demonstrates the importance of the use of multiple techniques for the elucidation of the enzymes involved in the metabolism of certain drugs.

Metabolic drug interactions with selective serotonin reuptake inhibitor (SSRI) antidepressants.

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.

Determination of p-trifluoromethylphenol, a metabolite of fluoxetine, in tissues and body fluids using an electron-capture gas chromatographic procedure.

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.

A rapid and sensitive electron-capture gas chromatographic procedure for analysis of metoprolol in rat brain and heart.

A procedure for analysis of metoprolol-utilizing extraction followed by derivatization with pentafluoropropionic anhydride and analysis on a gas chromatograph equipped with a fused silica capillary column, an electron-capture detector and a printer/integrator is described. Propranolol was carried through the procedure as internal standard. The pentafluoropropionyl derivative of metoprolol yields a sharp peak on the gas chromatograph, and the structure of the derivative was confirmed using combined gas chromatography-mass spectrometry. The analytical method is linear, sensitive and reproducible and has been applied to analysis of metoprolol in brain and heart from rats treated with metoprolol intraperitoneally. Pretreatment of the rats with the antidepressant desipramine prior to metoprolol administration resulted in a marked increase in levels of metoprolol in both brain and heart, indicating a pharmacokinetic drug-drug interaction between desipramine and metoprolol.

Effects of intravenous infusion of lidocaine on its pharmacokinetics in conscious instrumented dogs.

In this study, potential alterations in hepatic blood flow, plasma protein binding, hepatic tissue binding, and enzyme activities induced by LD iv infusion of lidocaine (LD) were evaluated using a chronically instrumented dog model. Four conscious female mongrel dogs (19.0-23.5 kg) were each given, on days 1 and 10, a 5-min infusion of a mixture of unlabeled LD at approximately 2 mg/kg and 14C-labeled LD at approximately 25 microCi and, on day 8, a 12-h constant rate iv infusion of LD (approximately 76 microg/kg/min). During LD infusion, there was a 11-79% increase in total hepatic blood flow, mainly due to a 1.6-9.2-fold increase in hepatic arterial flow. Despite similar blood clearance (27.5 +/- 6.0 mL/min/kg vs 27.5 +/- 3.5 mL/min/kg), volume of distribution at steady state (1.38 +/- 0.08 L/kg vs 1.36 +/- 0.17 L/kg), and free fraction values of LD between days 1 and 10 (p > 0.05), intrinsic clearance values were consistently reduced (1224 +/- 859 mL/ min/kg vs 285 +/- 104 mL/min/kg; p = 0.034). Furthermore, hepatic tissue uptake of LD and/or its metabolites was less on day 10 than on day 1 (39.7 +/- 14.5 micromol vs 30.1 +/- 15.1 micromol; p = 0.072). The extent of N-dealkylation of LD to MEGX was unaltered, whereas sequential biotransformation of MEGX was impaired. Hence, these findings suggested that LD infusion led to a reduction of hepatic intrinsic clearance, although the change was not significant enough to alter its conventional kinetic parameters.