Identification of an Epoxide Metabolite of Amitriptyline In Vitro and In Vivo.

Amitriptyline (ATL), a tricyclic antidepressant, has been reported to cause various adverse effects, particularly hepatotoxicity. The mechanisms of ATL-induced hepatotoxicity remain unknown. The study was performed to identify the olefin epoxidation metabolite of ATL and determine the possible toxicity mechanism. Two glutathione (GSH) conjugates (M1 and M2) and two N-acetylcysteine (NAC) conjugates (M3 and M4) were detected in rat liver microsomal incubations supplemented with GSH and NAC, respectively. Moreover, M1/M2 and M3/M4 were respectively found in ATL-treated rat primary hepatocytes and in bile and urine of rats given ATL. Recombinant P450 enzyme incubations demonstrated that CYP3A4 was the primary enzyme involved in the olefin epoxidation of ATL. Treatment of hepatocytes with ATL resulted in significant cell death. Inhibition of CYP3A attenuated the susceptibility to the observed cytotoxicity of ATL. The metabolic activation of ATL most likely participates in the cytotoxicity of ATL.

Dietary and genetic disruption of hepatic methionine metabolism induce acid sphingomyelinase to promote steatohepatitis.

Alcoholic (ASH) and nonalcoholic. (NASH).steatohepatitis are advanced.stages.of.fatty.liver.disease.Methionine adenosyltransferase 1A (MAT1A) plays a key role in hepatic methionine metabolism and germline Mat1a deletion in mice promotes NASH. Acid sphingomyelinase (ASMase) triggers hepatocellular apoptosis and liver fibrosis and has been shown to downregulate MAT1A expression in the context of fulminant liver failure. Given the role of ASMase in steatohepatitis development, we investigated the status of ASMase in Mat1a-/- mice and the regulation of ASMase by SAM/SAH. Consistent with its role in NASH, Mat1a-/- mice fed a choline-deficient (CD) diet exhibited macrosteatosis, inflammation, fibrosis and liver injury as well as reduced total and mitochondrial GSH levels. Our data uncovered an increased basal expression and activity of ASMase but not neutral SMase in Mat1a-/- mice, which further increased upon CD feeding. Interestingly, adenovirus-mediated shRNA expression targeting ASMase reduced ASMase activity and protected Mat1a-/- mice against CD diet-induced NASH. Similar results were observed in CD fed Mat1a-/- mice by pharmacological inhibition of ASMase with amitriptyline. Moreover, Mat1a/ASMase double knockout mice were resistant to CD-induced NASH. ASMase knockdown protected wild type mice against NASH induced by feeding a diet deficient in methionine and choline. Furthermore, Mat1a-/- mice developed acute-on-chronic ASH and this outcome was ameliorated by amitriptyline treatment. In vitro data in primary mouse hepatocytes revealed that decreased SAM/SAH ratio increased ASMase mRNA level and activity. MAT1A and ASMase mRNA levels exhibited an inverse correlation in liver samples from patients with ASH and NASH. Thus, disruption of methionine metabolism sensitizes to steatohepatitis by ASMase activation via decreased SAM/SAH. These findings imply that MAT1A deletion and ASMase activation engage in a self-sustained loop of relevance for steatohepatitis.

In-vitro and in situ assessment of the efflux of five antidepressants by breast cancer resistance protein.

OBJECTIVES: Antidepressants need to penetrate the blood-brain barrier (BBB) to exert their functions in the central nervous system. Breast cancer resistance protein (BCRP), an efflux transporter abundantly expressed in the BBB, prevents the accumulation of many drugs in the brain. This study aimed to identify whether five commonly used antidepressants (sertraline, duloxetine, fluoxetine, amitriptyline and mirtazapine) are BCRP substrates. METHODS: A combination of bidirectional transport and intracellular accumulation experiments was conducted on BCRP-overexpressing MDCKII and wild-type (WT) cells, and in situ brain perfusion was conducted in rats. KEY FINDINGS: The bidirectional transport study revealed that the net efflux ratio (NER) of sertraline reached 2.08 but decreased to 1.06 when co-incubated with Ko143, a selective BCRP inhibitor. Conversely, the other four antidepressants did not appear to be BCRP substrates, due to their low NER values (<1.5). The accumulation of sertraline in MDCKII-BCRP cells was significantly lower than that in MDCKII-WT cells. The presence of Ko143 significantly increased the sertraline accumulation in MDCKII-BCRP cells but not in MDCKII-WT cells. Brain perfusion showed that the permeability of 1 and 5 µm sertraline was significantly higher in the presence of Ko143. CONCLUSIONS: Taken together, BCRP is involved in sertraline efflux.

Carbon monoxide releasing molecule 401 (CORM-401) modulates phase I metabolism of xenobiotics.

Next to its well-studied toxicity, carbon monoxide (CO) is recognized as a signalling molecule in various cellular processes. Thus, CO-releasing molecules (CORMs) are of considerable interest for basic research and drug development. Aim of the present study was to investigate if CO, released from CORMs, inhibits cytochrome P450-dependent monooxygenase (CYP) activity and modulates xenobiotic metabolism. CORM-401 was used as a model CO delivering compound; inactive CORM-401 (iCORM-401), unable to release CO, served as control compound. CO release from CORM-401, but not from iCORM-401, was validated using the cell free myoglobin assay. CO-dependent inhibition of CYP activity was shown by 7-ethoxyresorufin-O-deethylation (EROD) with recombinant CYP and HepG2 cells. Upon CORM-401 exposure EROD activity of recombinant CYP decreased concentration dependently, while iCORM-401 had no effect. Treatment with CORM-401 decreased EROD activity in HepG2 cells at concentrations higher than 50 µM CORM-401, while iCORM-401 showed no effect. At the given concentrations cell viability was not affected. Amitriptyline was selected as a model xenobiotic and formation of its metabolite nortriptyline by recombinant CYP was determined by HPLC. CORM-401 treatment inhibited the formation of nortriptyline whereas iCORM-401 treatment did not. Overall, we demonstrate CO-mediated inhibitory effects on CYP activity when applying CORMs. Since CORMs are currently under drug development, the findings emphasize the importance to take into account that this class of compounds may interfere with xenobiotic metabolism.

Metabolism and bioactivation of the tricyclic antidepressant amitriptyline in human liver microsomes and human urine.

AIM: Amitriptyline is a widely used tricyclic antidepressant, but the metabolic studies were conducted almost 20 years ago using high-performance liquid chromatography coupled with ultraviolet detector or radiolabeled methods. RESULTS: First, multiple ion monitoring (MIM)- enhanced product ion (EPI) scan was used to obtain the diagnostic ions or neutral losses in human liver microsome incubations with amitriptyline. Subsequently, predicted multiple reaction monitoring (MRM)-EPI scan was used to identify the metabolites in human urine with the diagnostic ions or neutral losses. Finally, product ion filtering and neutral loss filtering were used as the data mining tools to screen metabolites. Consequently, a total of 28 metabolites were identified in human urine after an oral administration using LC-MS/MS. CONCLUSION: An integrated workflow using LC-MS/MS was developed to comprehensively profile the metabolites of amitriptyline in human urine, in which five N-acetyl-l-cysteine conjugates were characterized as tentative biomarkers for idiosyncratic toxicity.

Design and nuclear magnetic resonance (NMR) structure determination of the second extracellular immunoglobulin tyrosine kinase A (TrkAIg2) domain construct for binding site elucidation in drug discovery.

The tyrosine kinase A (TrkA) receptor is a validated therapeutic intervention point for a wide range of conditions. TrkA activation by nerve growth factor (NGF) binding the second extracellular immunoglobulin (TrkAIg2) domain triggers intracellular signaling cascades. In the periphery, this promotes the pain phenotype and, in the brain, cell survival or differentiation. Reproducible structural information and detailed validation of protein-ligand interactions aid drug discovery. However, the isolated TrkAIg2 domain crystallizes as a ß-strand-swapped dimer in the absence of NGF, occluding the binding surface. Here we report the design and structural validation by nuclear magnetic resonance spectroscopy of the first stable, biologically active construct of the TrkAIg2 domain for binding site confirmation. Our structure closely mimics the wild-type fold of TrkAIg2 in complex with NGF ( 1WWW .pdb), and the (1)H-(15)N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution.

Pharmacokinetics of Amitriptyline HCl and Its Metabolites in Healthy African Grey Parrots ( Psittacus erithacus ) and Cockatoos (Cacatua Species).

Amitriptyline, a tricyclic antidepressant, is used clinically to treat feather-destructive behavior in psittacine birds at a recommended dosage of 1-5 mg/kg PO q12-24h, which has been extrapolated from human medicine and based on anecdotal reports. The purpose of this pilot study was to describe the individual and population pharmacokinetic parameters of amitriptyline after a single oral dose at 1.5 mg/kg, 4.5 mg/kg, and 9 mg/kg in healthy African grey parrots ( Psittacus erithacus , n = 3) and cockatoos (Cacatua species, n = 3). Three birds received an initial 1.5 mg/kg oral dose, and blood samples were collected for 24 hours at fixed time intervals. Serum concentrations of amitriptyline and its metabolites were determined by polarized immunofluorescence. After determining the initial parameters and a 14-day washout period, 2 African grey parrots and 1 cockatoo received a single oral dose at 4.5 mg/kg, and 3 cockatoos and 1 African grey parrot received a single oral dose at 9 mg/kg. Concentrations reached the minimum therapeutic range reported in people (60 ng/mL) in 4 of 10 birds (4.5 and 9.0 mg/kg). Concentrations were within the toxic range in 1 African grey parrot (9 mg/kg), with regurgitation, ataxia, and dullness noted. Serum concentrations were nondetectable in 3 birds (1.5 and 4.5 mg/kg) and detectable but below the human therapeutic range in 3 birds (1.5 mg/kg and 9 mg/kg). Drug concentrations were continuing to increase at the end of the study (24 hours) in 1 bird. Elimination half-life varied from 1.6 to 91.2 hours. Population pharmacokinetics indicated significantly varied absorption, and elimination constants varied between species. Although amitriptyline appeared to be tolerated in most birds, disposition varies markedly among and within species, between the 2 genera, and within individual birds. The current recommended dosage of 1-5 mg/kg q12h in psittacine birds appears insufficient to achieve serum concentrations within the human therapeutic range and does not yield predictable concentrations. Results of this study suggest doses of up to 9 mg/kg may be necessary, although that dose may produce adverse events in some birds, and elimination half-life is sufficiently variable that dosing intervals are not predictable. Therapeutic drug monitoring combined with response to therapy is indicated to determine individual therapeutic ranges.

Liquid-phase microextraction in a microfluidic-chip--high enrichment and sample clean-up from small sample volumes based on three-phase extraction.

In this work, a microfluidic-chip based system for liquid-phase microextraction (LPME-chip) was developed. Sample solutions were pumped into the LPME-chip with a micro-syringe pump at a flow rate of 3-4 µL min(-1). Inside the LPME chip, the sample was in direct contact with a supported liquid membrane (SLM) composed of 0.2 µL dodecyl acetate immobilized in the pores of a flat membrane of polypropylene (25 µm thickness). On the other side of the SLM, the acceptor phase was present. The acceptor phase was either pumped at 1 µL min(-1) during extraction or kept stagnant (stop-flow). Amitriptyline, methadone, haloperidol, loperamide, and pethidine were selected as model analytes, and they were extracted from alkaline sample solution, through the SLM, and into 10 mM HCl or 100mM HCOOH functioning as acceptor phase. Subsequently, the acceptor phase was either analyzed off-line by capillary electrophoresis for exact quantification, or on-line by UV detection or electrospray ionization mass spectrometry for time profiling of concentrations. The LPME-chip was found to be highly effective, and extraction efficiencies were in the range of 52-91%. When the flow of acceptor phase was turned off during extraction (stop-flow), analyte enrichment increased linearly with the extraction time. After 10 min as an example, amitriptyline was enriched by a factor of 42 from only 30 µL sample solution, and after 120 min amitriptyline was enriched by a factor of 500 from 320 µL sample solution. This suggested that the LPME-chip has great potentials for very efficient analyte enrichments from limited sample volumes in the future.

On-chip electro membrane extraction with online ultraviolet and mass spectrometric detection.

Electro membrane extraction was demonstrated in a microfluidic device. The device was composed of a 25 µm thick porous polypropylene membrane bonded between two poly(methyl methacrylate) (PMMA) substrates, each having 50 µm deep channel structures facing the membrane. The supported liquid membrane (SLM) consisted of 2-nitrophenyl octyl ether (NPOE) immobilized in the pores of the membrane. The driving force for the extraction was a 15 V direct current (DC) electrical potential applied across the SLM. Samples containing the basic drugs pethidine, nortriptyline, methadone, haloperidol, loperamide, and amitriptyline were used to characterize the system. Extraction recoveries were typically in the range of 65-86% for the different analytes when the device was operated with a sample flow of 2.0 µL/min and an acceptor flow of 1.0 µL/min. With the sample flow at 9.0 µL/min and the acceptor flow at 0.0 µL/min, enrichment factors exceeding 75 were obtained during 12 min of operation from a total sample volume of only 108 µL. The on-chip electro membrane system was coupled online to electrospray ionization mass spectrometry and used to monitor online and real-time metabolism of amitriptyline by rat liver microsomes.

Lipophilic cationic drugs increase the permeability of lysosomal membranes in a cell culture system.

Lysosomes accumulate many drugs several fold higher compared to their extracellular concentration. This mechanism is believed to be responsible for many pharmacological effects. So far, uptake and release kinetics are largely unknown and interactions between concomitantly administered drugs often provoke mutual interference. In this study, we addressed these questions in a cell culture model. The molecular mechanism for lysosomal uptake kinetics was analyzed by live cell fluorescence microscopy in SY5Y cells using four drugs (amantadine, amitriptyline, cinnarizine, flavoxate) with different physicochemical properties. Drugs with higher lipophilicity accumulated more extensively within lysosomes, whereas a higher pK(a) value was associated with a more rapid uptake. The drug-induced displacement of LysoTracker was neither caused by elevation of intra-lysosomal pH, nor by increased lysosomal volume. We extended our previously developed numerical single cell model by introducing a dynamic feedback mechanism. The empirical data were in good agreement with the results obtained from the numerical model. The experimental data and results from the numerical model lead to the conclusion that intra-lysosomal accumulation of lipophilic xenobiotics enhances lysosomal membrane permeability. Manipulation of lysosomal membrane permeability might be useful to overcome, for example, multi-drug resistance by altering subcellular drug distribution.

Role of human UGT2B10 in N-glucuronidation of tricyclic antidepressants, amitriptyline, imipramine, clomipramine, and trimipramine.

The role of human UDP glucuronosyltransferase (UGT) 2B10 in the N-glucuronidation of a number of tricyclic antidepressants was investigated and compared with that of UGT1A4 in both the Sf9 expressed system and human liver microsomes. The apparent K(m) (S(50)) values for the formation of quaternary N-glucuronides of amitriptyline, imipramine, clomipramine, and trimipramine were 2.60, 16.8, 14.4, and 11.2 microM in UGT2B10 and 448, 262, 112, and 258 microM in UGT1A4, respectively. The kinetics of amitriptyline and imipramine glucuronidation in human liver microsomes exhibited a biphasic character, where the high- and low-affinity components were in good agreement with our results in expressed UGT2B10 and UGT1A4, respectively. The kinetics of clomipramine and trimipramine glucuronidation in human liver microsomes were sigmoidal in nature, and the S(50) values were similar to those found for expressed UGT1A4. The in vitro clearances (CL(int) or CL(max)) were comparable between UGT2B10 and UGT1A4 for glucuronidation of imipramine, clomipramine, and trimipramine, whereas CL(int) of amitriptyline glucuronidation by UGT2B10 was more than 10-fold higher than that by UGT1A4. Nicotine was found to selectively inhibit UGT2B10 but not UGT1A4 activity. At a low tricyclic antidepressant concentration, nicotine inhibited their glucuronidation by 33 to 50% in human liver microsomes. Our results suggest that human UGT2B10 is a high-affinity enzyme for tricyclic antidepressant glucuronidation and is likely to be a major UGT isoform responsible for the glucuronidation of these drugs at therapeutic concentrations in vivo.

Effect of chain length on binding of fatty acids to Pluronics in microemulsions.

We investigated the effect of fatty acid chain length on the binding capacity of drug and fatty acid to Pluronic F127-based microemulsions. This was accomplished by using turbidity experiments. Pluronic-based oil-in-water microemulsions of various compositions were synthesized and titrated to turbidity with concentrated Amitriptyline, an antidepressant drug. Sodium salts of C(8), C(10), or C(12) fatty acid were used in preparation of the microemulsion and the corresponding binding capacities were observed. It has been previously determined that, for microemulsions prepared with sodium caprylate (C(8) fatty acid soap), a maximum of 11 fatty acid molecules bind to the microemulsion per 1 molecule of Pluronic F127 and a maximum of 12 molecules of Amitriptyline bind per molecule of F127. We have found that with increasing the chain length of the fatty acid salt component of the microemulsion, the binding capacity of both the fatty acid and the Amitriptyline to the microemulsion decreases. For sodium salts of C(8), C(10) and C(12) fatty acids, respectively, a maximum of approximately 11, 8.4 and 8.3 molecules of fatty acid molecules bind to 1 Pluronic F127 molecule. We propose that this is due to the decreasing number of free monomers with increasing chain length. As chain length increases, the critical micelle concentration (cmc) decreases, thus leading to fewer monomers. Pluronics are symmetric tri-block copolymers consisting of propylene oxide (PO) and ethylene oxide (EO). The polypropylene oxide block, PPO is sandwiched between two polyethylene oxide (PEO) blocks. The PEO blocks are hydrophilic while PPO is hydrophobic portion in the Pluronic molecule. Due to this structure, we propose that the fatty acid molecules that are in monomeric form most effectively diffuse between the PEO "tails" and bind to the hydrophobic PPO groups.

An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics.

Many adverse drug reactions are caused by the cytochrome P450 (CYP) dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-mediated toxicity and to improve safety of drug candidates, we developed two in vitro cell-based assays by combining an activating system (human CYP3A4) with target cells (HepG2 cells): in the first method we incubated microsomes containing cDNA-expressed CYP3A4 together with HepG2 cells; in the second approach HepG2 cells were transiently transfected with CYP3A4. In both assay systems, CYP3A4 catalyzed metabolism was found to be comparable to the high levels reported in hepatocytes. Both assay systems were used to study ten CYP3A4 substrates known for their potential to form metabolites that exhibit higher toxicity than the parent compounds. Several endpoints of toxicity were evaluated, and the measurement of MTT reduction and intracellular ATP levels were selected to assess cell viability. Results demonstrated that both assay systems are capable to metabolize the test compounds leading to increased toxicity, compared to their respective control systems. The co-incubation with the CYP3A4 inhibitor ketoconazole confirmed that the formation of reactive metabolites was CYP3A4 dependent. To further validate the functionality of the two assay systems, they were also used as a "detoxification system" using selected compounds that can be metabolized by CYP3A4 to metabolites less toxic than their parent compounds. These results show that both assay systems can be used to screen for metabolic activation, or de-activation, which may be useful as a rapid and relatively inexpensive in vitro assay for the prediction of CYP3A4 metabolism-mediated toxicity.

Microsomal binding of amitriptyline: effect on estimation of enzyme kinetic parameters in vitro.

The effect of binding of amitriptyline to human liver microsomes and to microsomes from human B-lymphoblastoid cells on the estimation of enzyme kinetic parameters describing N-demethylation to nortriptyline was investigated using a combination of microsomal binding and in vitro enzyme kinetic studies. Quantitative binding in both matrices increased with higher microsomal protein concentrations (free fractions 0.88-0.32 at 100-500 microg protein/ml in human liver microsomes and 0.82-0.26 at 250-1000 microg protein/ml in microsomes from B-lymphoblastoid cells) and was independent of amitriptyline concentration over a concentration range of 0.2 to 200 microM. Addition of heat-inactivated microsomal protein (50-450 microg/ml) to native human liver microsomes (50 microg/ml) reduced the amitriptyline N-demethylation rate in a protein concentration dependent manner. This effect was greater at lower substrate concentrations and was overcome by saturating concentrations of substrate, thereby decreasing the apparent affinities of the high- and low-affinity components of the N-demethylation process, with minimal effect on the net V(max). Addition of metabolically inactive microsomes from untransfected human lymphoblastoid cells (750 microg/ml) to CYP2C19 (250 microg/ml protein) increased the apparent K(m) value for amitriptyline N-demethylation by 3.5-fold and increased the uncompetitive substrate inhibition constant (K(s)) by 2.2-fold, making substrate inhibition essentially undetectable. A similar effect was seen with CYP3A4, with a 1.8-fold increase in the S(50) (substrate concentration at which half-maximal velocity of a Hill enzyme is achieved). Microsomal binding did not alter the V(max) of either CYP isoform to any appreciable extent. These findings emphasize the importance of incorporating microsomal binding in the estimation of enzyme kinetic parameters in vitro and making appropriate corrections for unbound drug concentrations.

Five distinct human cytochromes mediate amitriptyline N-demethylation in vitro: dominance of CYP 2C19 and 3A4.

The human cytochromes P450 (CYPs) mediating amitriptyline N-demethylation have been identified using a combination of enzyme kinetic and chemical inhibition studies. Amitriptyline was N-demethylated to nortriptyline by microsomes from cDNA transfected human lymphoblastoid cells expressing human CYPs 1A2, 2C9, 2C19, 2D6, and 3A4. CYP 2E1 showed no detectable activity. While CYP 2C19 and CYP 2D6 showed high affinity, CYP 3A4 showed low affinity; CYP 2C9 and 1A2 showed intermediate affinities. Based on these kinetic parameters and estimated relative abundance of the different CYPs in human liver, CYP 2C19 was identified as the major amitriptyline N-demethylase at low (therapeutically relevant) amitriptyline concentrations, whereas CYP 3A4 may be more important at higher amitriptyline concentrations. Chemical inhibition studies with ketoconazole and omeprazole indicate that CYP 3A4 is the major amitriptyline N-demethylase at 100 mumol/L amitriptyline, while CYP 2C19 is equally important at a substrate concentration of 5 mumol/L. The CYP 1A2 inhibitor alpha-naphthoflavone and the CYP 2C9 inhibitor sulfaphenazole produced much less inhibition of amitriptyline N-demethylation at both substrate concentrations. Quinidine produced no detectable inhibition. The kinetics of amitriptyline N-demethylation by human liver microsomes were consistent with a two enzyme model, with the high affinity component exhibiting Michaelis Menten kinetics and the low affinity component exhibiting Hill enzyme kinetics. No difference was apparent in the kinetics of amitriptyline N-demethylation in two liver samples with low levels of CYP 2C19 activity compared with two other samples with relatively normal 2C19 activity. This may reflect the importance of higher substrate concentration values in estimation of kinetic parameters in vitro.

Cooperativity in oxidations catalyzed by cytochrome P450 3A4.

Cytochrome P450 (P450) 3A4 is the most abundant human P450 and oxidizes a diversity of substrates, including various drugs, steroids, carcinogens, and macrolide natural products. In some reactions, positive cooperativity has been reported in microsomal studies. Flavonoids, e.g., 7,8-benzoflavone (alpha-naphthoflavone, alpha NF), have been shown to stimulate some reactions but not others. In systems containing purified recombinant bacterial P450 3A4, positive cooperativity was seen in oxidations of several substrates, including testosterone, 17 beta-estradiol, amitriptyline, and most notably aflatoxin (AF) B1. With these and other reactions, alpha NF typically reduced cooperativity (i.e., the n value in a Hill plot) while either stimulating or inhibiting reactions. With the substrate AFB1, alpha NF both stimulated 8,9-epoxidation and inhibited 3 alpha-hydroxylation. The same patterns were seen with AFB1 in a fused P450 3A4-NADPH-P450 reductase protein. alpha NF did not alter patterns of activity plotted as a function of NADPH-P450 reductase concentration in systems containing the individual proteins. The patterns of AFB1 oxidation to the two products were modified considerably in systems in which NADPH-P450 reductase was replaced with a flavodoxin or ferredoxin system, iodosylbenzene, or cumene hydroperoxide. AFB2, which differs from AFB1 only in the presence of a saturated 8,9-bond, was not oxidized by P450 3A4 but could inhibit AFB1 oxidation. These and other results are considered in the context of several possible models. The results support a model in which an allosteric site is involved, although the proximity of this putative site to the catalytic site cannot be ascertained as of yet. In order to explain the differential effects of alpha NF and reduction systems on the two oxidations of AFB1, a model is presented in which binding of substrate in a particular conformation can facilitate oxygen activation to enhance catalysis.

Identification of human liver cytochrome P450 isoforms involved in the in vitro metabolism of cyclobenzaprine.

Cyclobenzaprine (Flexeril) is a muscle relaxant, possessing a tricyclic structure. Numerous therapeutic agents containing this structure are known to be metabolized by polymorphic cytochrome P4502D6. The aim of this study was to determine if cytochrome P4502D6 and other isoforms are involved in the metabolism of cyclobenzaprine in human liver microsomes. Selective cytochrome P450 inhibitors for CYP1A1/2 (furafylline and 7,8-benzoflavone) and CYP3A4 (troleandomycin, gestodene, and ketoconazole) inhibited the formation of desmethylcyclobenzaprine, a major metabolite of cyclobenzaprine, in human liver microsomes. Antibodies directed against CYP1A1/2 and CYP3A4 inhibited the demethylation reaction whereas anti-human CYP2C9/10, CYP2C19, and CYP2E1 antibodies did not show any inhibitory effects. When a panel of microsomes prepared from human B-lymphoblastoid cells that expressed specific human cytochrome P450 isoforms were used, only microsomes containing cytochromes P4501A2, 2D6, and 3A4 catalyzed N-demethylation. In addition, demethylation catalyzed by these recombinant cytochromes P450 can be completely inhibited with selective inhibitors at concentrations as low as 1 to 20 microM. Interestingly, cyclobenzaprine N-demethylation was significantly correlated with caffeine 3-demethylation (1A2) and testosterone 6 beta-hydroxylation (3A4) but not with dextromethorphan O-demethylation (2D6) in human liver microsomes. To further determine the involvement of cytochrome P4502D6 in cyclobenzaprine metabolism, liver microsomes from a human that lacked CYP2D6 enzyme activities was included in this study. The data showed that cyclobenzaprine N-demethylation still occurred in the incubation with this microsome. These results suggested that cytochrome P4502D6 plays only a minor role in cyclobenzaprine N-demethylation whereas 3A4 and 1A2 are primarily responsible for cyclobenzaprine metabolism in human liver microsomes. Due to the minimum involvement of CYP2D6 in the vitro metabolism of cyclobenzaprine, the polymorphism of cytochrome P4502D6 in man should not be of muci concern in the clinical use of cyclobenzaprine.

Muscarinic receptor subtypes and sexual behavior in female rats.

Cholinergic muscarinic systems are involved in the regulation of female sexual behavior in rats and hamsters. This series of experiments was designed to determine whether sexual behavior in female rats is controlled preferentially by one of the traditional muscarinic receptor subtypes. Intraventricular infusion of the muscarinic antagonist scopolamine (10 micrograms bilaterally) which binds with high affinity to both M1 and M2 subtypes inhibited sexual behavior, as indicated by the incidence of lordosis, in ovariectomized rats treated with estrogen and progesterone. In contrast, the M1-selective antagonist pirenzepine failed to reduce the incidence of lordosis following intraventricular infusion (10 to 80 micrograms bilaterally). Biochemical analyses revealed that intraventricular infusion of scopolamine (10 micrograms bilaterally) inhibited both M1 and M2 binding in brain tissues while intraventricular infusion of pirenzepine (10 micrograms bilaterally) completely inhibited M1 binding without affecting M2 binding. Intraventricular infusions of the acetylcholinesterase inhibitor physostigmine (10 micrograms bilaterally), the cholinergic agonist carbachol (1 microgram bilaterally), and the muscarinic agonist oxotremorine-M (0.1 micrograms bilaterally) activated lordosis in ovariectomized females primed with low doses of estrogen. In contrast, the putative M1 agonist McN-A-343 failed to significantly increase lordosis following intraventricular infusions (1, 10, 20 micrograms bilaterally). According to biochemical results, the ability of these agents to activate lordosis in female rats was related to their affinities for M2 binding sites not M1 binding sites. In a final experiment, estrogen treatment of ovariectomized rats did not alter muscarinic subtype binding in several brain areas as measured by the M1-selective ligand [3H] pirenzepine and the M2-selective ligand [3H] oxotremorine-M. The results of these experiments confirm that muscarinic systems contribute to the regulation of lordosis in female rats and indicate that M2 binding sites rather than M1 binding sites may be a critical component of this regulation.

Decreased drug absorption in a patient with Behçet's syndrome.

Observing a lack of response to orally administered drugs in a patient with Behçet's disease, we studied the absorption of amitriptyline, diazepam, carbamazepine, phenytoin, and acetaminophen in this patient after single and (or) multiple dose administrations. The relative oral/intramuscular bioavailability of amitriptyline was only 13%, and the steady-state concentrations of this drug on four consecutive days were acutely subtherapeutic (i.e., 3.6, 3.7, 3.9, and 3.7 micrograms/L). The concentrations of diazepam, phenytoin, and acetaminophen in plasma were nonmeasurable. Examination of the gastrointestinal tract by endoscopy and by light and electronic microscopy of a biopsy section revealed inflammatory and vascular changes in the duodenum. In the absence of clinical evidence for malabsorption syndrome, we believe that the decreased drug absorption observed in this patient was caused by inflammatory changes associated with Behçet's syndrome.

Slow hydroxylation of tricyclic antidepressants--relationship to polymorphic drug oxidation.

There are marked interindividual differences in Css of tricyclic antidepressants. These are due mainly to corresponding differences in the rate of oxidative metabolism of these drugs. Twin, family, and cross-over studies with nortriptyline (NT) and desmethylimipramine (DMI) show that their kinetics and hydroxylation (Css, Kel, and Vd) are controlled mainly by genetic factors (in drug-free individuals). Slow hydroxylators are at risk of developing excessive plasma concentrations of NT and DMI when given per se or when formed from the tertiary amines amitriptyline and imipramine. Classic antidepressants have fairly well established concentration-effect curves in endogenous depression. Severe toxicity usually occurs at supratherapeutic plasma levels and might be prevented by tailoring the dosage according to the individual's drug hydroxylating capacity. Monitoring drug plasma levels is particularly relevant in slow hydroxylators (Sjöqvist et al, 1980). There is a strong association between an individual's ability to hydroxylate NT and DMI and his debrisoquine (D) hydroxylation phenotype. The D hydroxylation index will predict the patient's capacity to hydroxylate NT and DMI and hence Css during therapy. Possibly, similar hydroxylases are involved in the 4-hydroxylation of debrisoquine, in the stereospecific E-10-hydroxylation of NT, and in the 2-hydroxylation of DMI. By contrast, demethylation of AT (and probably other tertiary tricyclics) does not significantly correlate to debrisoquine hydroxylation except in non-smoking individuals. The increasing knowledge about the clinical pharmacokinetics of tricyclic antidepressants is a distinct advantage over that of the new generation of antidepressants, where little is known about concentration-effect relationships and factors governing their rate of metabolism. Possible interethnic differences in the metabolism of these essential drugs should be explored in epidemiologically sound investigations.