Mitochondrially targeted cytochrome P450 2D6 is involved in monomethylamine-induced neuronal damage in mouse models.

Parkinson's disease (PD) is a major human disease associated with degeneration of the central nervous system. Evidence suggests that several endogenously formed 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mimicking chemicals that are metabolic conversion products, especially ß-carbolines and isoquinolines, act as neurotoxins that induce PD or enhance progression of the disease. We have demonstrated previously that mitochondrially targeted human cytochrome P450 2D6 (CYP2D6), supported by mitochondrial adrenodoxin and adrenodoxin reductase, can efficiently catalyze the conversion of MPTP to the toxic 1-methyl-4-phenylpyridinium ion. In this study, we show that the mitochondrially targeted CYP2D6 can efficiently catalyze MPTP-mimicking compounds, i.e. 2-methyl-1,2,3,4-tetrahydroisoquinoline, 2-methyl-1,2,3,4-tetrahydro-ß-carboline, and 9-methyl-norharmon, suspected to induce PD in humans. Our results reveal that activity and respiration in mouse brain mitochondrial complex I are significantly affected by these toxins in WT mice but remain unchanged in Cyp2d6 locus knockout mice, indicating a possible role of CYP2D6 in the metabolism of these compounds both in vivo and in vitro These metabolic effects were minimized in the presence of two CYP2D6 inhibitors, quinidine and ajmalicine. Neuro-2a cells stably expressing predominantly mitochondrially targeted CYP2D6 were more sensitive to toxin-mediated respiratory dysfunction and complex I inhibition than cells expressing predominantly endoplasmic reticulum-targeted CYP2D6. Exposure to these toxins also induced the autophagic marker Parkin and the mitochondrial fission marker Dynamin-related protein 1 (Drp1) in differentiated neurons expressing mitochondrial CYP2D6. Our results show that monomethylamines are converted to their toxic cationic form by mitochondrially directed CYP2D6 and result in neuronal degradation in mice.

Prediction of clinically relevant drug-induced liver injury from structure using machine learning.

Drug-induced liver injury (DILI) is the most common cause of acute liver failure and often responsible for drug withdrawals from the market. Clinical manifestations vary, and toxicity may or may not appear dose-dependent. We present several machine-learning models (decision tree induction, k-nearest neighbor, support vector machines, artificial neural networks) for the prediction of clinically relevant DILI based solely on drug structure, with data taken from published DILI cases. Our models achieved corrected classification rates of up to 89%. We also studied the association of a drug's interaction with carriers, enzymes and transporters, and the relationship of defined daily doses with hepatotoxicity. The results presented here are useful as a screening tool both in a clinical setting in the assessment of DILI as well as in the early stages of drug development to rule out potentially hepatotoxic candidates.

The impact of CYP2D6 mediated drug-drug interaction: a systematic review on a combination of metoprolol and paroxetine/fluoxetine.

AIM: Metoprolol (a CYP2D6 substrate) is often co-prescribed with paroxetine/fluoxetine (a CYP2D6 inhibitor) because the clinical relevance of this drug-drug interaction (DDI) is still unclear. This review aimed to systematically evaluate the available evidence and quantify the clinical impact of the DDI. METHOD: Pubmed, Web of Science, Cochrane Library and Embase were searched for studies reporting on the effect of the DDI among adults published until April 2018. Data on pharmacokinetics, pharmacodynamics and clinical outcomes from experimental, observational and case report studies were retrieved. The protocol of this study was registered in PROSPERO (CRD42018093087). RESULTS: We found nine eligible articles that consisted of four experimental and two observational studies as well as three case reports. Experimental studies reported that paroxetine increased the AUC of metoprolol three to five times, and significantly decreased systolic blood pressure and heart rate of patients. Case reports concerned bradycardia and atrioventricular block due to the DDI. Results from observational studies were conflicting. A cohort study indicated that the DDI was significantly associated with the incidence of early discontinuation of metoprolol as an indicator of the emergence of metoprolol-related side effects. In a case-control study, the DDI was not significantly associated with bradycardia. CONCLUSION: Despite the contradictory conclusions from the current literature, the majority of studies suggest that the DDI can lead to adverse clinical consequences. Since alternative antidepressants and beta-blockers with comparable efficacy are available, such DDIs can be avoided. Nonetheless, if prescribing the combination is unavoidable, a dose adjustment or close monitoring of the metoprolol-related side effects is necessary.

[Interaction of opioid analgesics at the level of biotransformation]. / Interaktionen der Opioidanalgetika auf Ebene der Biotransformation.

Opioids are an important component of the drug treatment of patients with acute and chronic pain. They differ in effectiveness, side effect profile and the risk of interactions. In this article the pharmacokinetic mechanisms of drug-drug interactions at the level of biotransformation are described and the clinical consequences which can arise are discussed. The relation of the active components to the two isoenzymes CYP2D6 and CYP3A4 is of major importance for assessing the potential drug-drug interactions of opioid analgesics at the level of the cytochrome P450 enzyme.

Effect of 24 cytochrome P450 2D6 variants found in the Chinese population on the N-demethylation of amitriptyline in vitro.

CONTEXT: Amitriptyline (AT), one of the tricyclic antidepressants, is still widely used for the treatment of the depression and control of anxiety states and panic disorders in the developing countries. OBJECTIVE: This study evaluates the catalytic activities of CYP2D6*1, CYP2D6*2, CYP2D6*10 and 22 novel alleles in Han Chinese population and their effects on the N-demethylation of AT in vitro. MATERIALS AND METHODS: CYP2D6*1 and 24 CYP2D6 allelic variants were highly expressed in insect cells, and all variants were characterized using AT as a substrate. Reactions were performed at 37 °C with 10-1000 µM substrate for 30 min. We established a HPLC method to quantify the levels of nortriptyline (NT). The kinetic parameters Km, Vmax and intrinsic clearance (Vmax/Km) of NT were calculated. RESULTS: Among the 24 CYP2D6 variants, all variants exhibited decreased intrinsic clearance values compared with wild-type CYP2D6.1. Kinetic parameters of two CYP2D6 variants (CYP2D6*92, *96) could not be determined because of absent enzyme activities. CONCLUSIONS: The comprehensive in vitro assessment of CYP2D6 variants provides significant insight into allele-specific activity towards AT in vivo.

Pharmacokinetics in neonatal prescribing: evidence base, paradigms and the future.

Paediatric patients, particularly preterm neonates, present many pharmacological challenges. Due to the difficulty in conducting clinical trials in these populations dosing information is often extrapolated from adult populations. As the processes of absorption, distribution, metabolism and excretion of drugs change throughout growth and development extrapolation presents risk of over or underestimating the doses required. Information about the development these processes, particularly drug metabolism pathways, is still limited with weight based dose adjustment presenting the best method of estimating pharmacokinetic changes due to growth and development. New innovations in pharmacokinetic research, such as population pharmacokinetic modelling, present unique opportunities to conduct clinical trials in these populations improving the safety and effectiveness of the drugs used. More research is required into this area to ensure the best outcomes for our most vulnerable patients.

Role and pharmacologic significance of cytochrome P-450 2D6 in oxidative metabolism of toremifene and tamoxifen.

We investigated the in vitro metabolism and estrogenic and antiestrogenic activity of toremifene (TOR), tamoxifen (TAM) and their metabolites to better understand the potential effects of cytochrome P-450 2D6 (CYP2D6) status on the activity of these drugs in women with breast cancer. The plasma concentrations of TOR and its N-desmethyl (NDM) and 4-hydroxy (4-OH) metabolites during steady-state dosing with TOR were also determined. Unlike TOR, TAM and its NDM metabolite were extensively oxidized to 4-OH TAM and 4-OH-NDM TAM by CYP2D6, and the rate of metabolism was affected by CYP2D6 status. 4-OH-NDM TOR concentrations were not measurable at steady state in plasma of subjects taking 80 mg of TOR. Molecular modeling provided insight into the lack of 4-hydroxylation of TOR by CYP2D6. The 4-OH and 4-OH-NDM metabolites of TOR and TAM bound to estrogen receptor (ER) subtypes with fourfold to 30-fold greater affinity were 35- to 187-fold more efficient at antagonizing ER transactivation and had antiestrogenic potency that was up to 360-fold greater than their parent drugs. Our findings suggest that variations in CYP2D6 metabolic capacity may cause significant differences in plasma concentrations of active TAM metabolites (i.e., 4-OH TAM and 4-OH-NDM TAM) and contribute to variable pharmacologic activity. Unlike TAM, the clinical benefits in subjects taking TOR to treat metastatic breast cancer would not likely be subject to allelic variation in CYP2D6 status or affected by coadministration of CYP2D6-inhibiting medications.

Is there a role for therapeutic drug monitoring with codeine?

Codeine is an old and commonly used analgesic agent for mild to moderate pain. It is the prototypical "prodrug" in that its analgesic effect is almost wholly dependent on its biotransformation to morphine, a process that is mediated by the polymorphic cytochrome P450 2D6 enzyme. As such, interindividual variability in codeine metabolism and response is a clinical reality, and there has been much progress in characterizing the genetic causes of this variability in diverse populations. Yet despite the potential for both life-threatening adverse reactions and lack of therapeutic effect, codeine is not commonly indicated for therapeutic drug monitoring. This review will discuss the relative role of pharmacogenetics and therapeutic drug monitoring in predicting and/or maintaining adequate and safe analgesia with codeine. The review will end on a discussion of how the marriage of these 2 fields may provide new insights into the mechanisms of codeine-induced toxicity and analgesia.

Serotonin toxicity: a short review of the literature and two case reports involving citalopram.

The serotonin toxicity (ST) is a potentially life-threatening adverse drug reaction results from therapeutic drug use, intentional self-poisoning, or inadvertent interactions between drugs. ST can be caused by a single or a combination of drugs with serotonergic activity due to excessive serotonergic agonism on central nervous system and peripheral serotonergic receptors (monoamine oxidase inhibitors, tricyclic antidepressants, SSRIs, opiate analgesics, over-the-counter cough medicines, antibiotics, weight-reduction agents, antiemetics, antimigraine agents, drugs of abuse, H2-antagonist and herbal products). The serotonin toxicity is often described as a clinical triad of mental-status changes (agitation and excitement with confusion), autonomic hyperactivity (diaphoresis, fever, tachycardia, and tachypnea), neuromuscular abnormalities (tremor, clonus, myoclonus, and hyperreflexia) and, in the advanced stage, spasticity; not all of these findings are consistently present. In this article, we describe two cases of ST due to interaction between Citalopram and two CYP2D6 inhibitors: Cimetidine and Topiramate and their clinical resolution after treatment discontinuation.

The role of CYP2D in rat brain in methamphetamine-induced striatal dopamine and serotonin release and behavioral sensitization.

RATIONALE: Cytochrome P450 2D (CYP2D) enzymes metabolize many addictive drugs, including methamphetamine. Variable CYP2D metabolism in the brain may alter CNS drug/metabolite concentrations, consequently affecting addiction liability and neuropsychiatric outcomes; components of these can be modeled by behavioral sensitization in rats. METHODS: To investigate the role of CYP2D in the brain in methamphetamine-induced behavioral sensitization, rats were pretreated centrally with a CYP2D irreversible inhibitor (or vehicle) 20 h prior to each of 7 daily methamphetamine (0.5 mg/kg subcutaneous) injections. In vivo brain microdialysis was used to assess brain drug and metabolite concentrations, and neurotransmitter release. RESULTS: CYP2D inhibitor (versus vehicle) pretreatment enhanced methamphetamine-induced stereotypy response sensitization. CYP2D inhibitor pretreatment increased brain methamphetamine concentrations and decreased the brain p-hydroxylation metabolic ratio. With microdialysis conducted on days 1 and 7, CYP2D inhibitor pretreatment exacerbated stereotypy sensitization and enhanced dopamine and serotonin release in the dorsal striatum. Day 1 brain methamphetamine and amphetamine concentrations correlated with dopamine and serotonin release, which in turn correlated with the stereotypy response slope across sessions (i.e., day 1 through day 7), used as a measure of sensitization. CONCLUSIONS: CYP2D-mediated methamphetamine metabolism in the brain is sufficient to alter behavioral sensitization, brain drug concentrations, and striatal dopamine and serotonin release. Moreover, day 1 methamphetamine-induced neurotransmitter release may be an important predictor of subsequent behavioral sensitization. This suggests the novel contribution of CYP2D in the brain to methamphetamine-induced behavioral sensitization and suggests that the wide variation in human brain CYP2D6 may contribute to differential methamphetamine responses and chronic effects.

Cytochrome P450 2D6 and outcomes of adjuvant tamoxifen therapy: results of a meta-analysis.

Pharmacological evidence shows that cytochrome P450 2D6 (CYP2D6) function is important in the conversion of tamoxifen to its active metabolites. Many retrospective analyses have assessed the role of both CYP2D6 genotype and concurrent administration of drug inhibitors of CYP2D6 on outcome of tamoxifen therapy. These studies have frequently been of small size and their data highly variable. A published data meta-analysis of trials evaluating outcomes of tamoxifen therapy in early breast cancer was undertaken. Hazard ratios (HRs) were extracted for disease-free survival (DFS) and overall survival (OS). Pooled estimates were computed using inverse-variance and random-effect modeling. Data from 10 studies assessing CYP2D6 genotype were included in a meta-analysis. There was significant heterogeneity in the definition of comparison groups between studies. When compared to reduced CYP2D6 function, normal function was associated with a trend toward improved DFS (HR 2.07, 95% CI 0.96-4.49, P = 0.06) but not OS (HR 1.36, 95% CI 0.73-2.52, P = 0.34). Pooling of data from two studies evaluating CYP2D6 drug inhibitors showed that concomitant administration of these with tamoxifen was associated with a non-significant association with DFS (HR 1.37, 95% CI 0.69-2.73, P = 0.37). Analysis of the effect of CYP2D6 drug inhibitors on OS was not possible. The effect of CYP2D6 genotype on breast cancer seems to be relatively small and may not warrant testing of CYP2D6 genotype in all women with hormone receptor positive breast cancer. The effect of CYP2D6 genotype on outcome in low-risk patients may not be clinically relevant, while the upfront use of aromatase inhibitors is a reasonable alternative to tamoxifen in high-risk post-menopausal women, irrespective of CYP2D6 genotype. There are limited data supporting the association of potent inhibitors of CYP2D6 and detrimental outcome, but avoidance of such drugs seems reasonable.

Metabolism of sesamin by cytochrome P450 in human liver microsomes.

Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest k(cat)/K(m) value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9.

Variations in critical morphine biosynthesis genes and their potential to influence human health.

Endogenous morphine has been detected in human tissues from the vascular, immune and nervous systems. The genes/enzymes (CYP2D6, COMT and PNMT) that are involved in the biosynthesis of morphine have variations that affect their functionality. Some of these variations are the result of single nucleotide polymorphisms of DNA sequences. This review highlights some of the functional differences in the critical enzymes required for the biosynthesis of morphine that may affect human health. These variations have been shown to change the way animals react to stressors, perceive pain and behave. The presence of morphine signaling in almost all organ systems suggests that it is most likely playing a role in maintaining the health and promoting the normal functioning of these physiological systems.

[Genomic based approach to individualized cancer therapy].

Chemotherapy plays an important role in cancer therapy. The major problem of chemotherapy is that only a part of patients can receive benefit of chemotherapy. The low efficacy of cancer chemotherapy is mostly due to heterogeneity in cancers. Recent advance in molecular biology has made possible to analyze expressions of a large number of genes simultaneously. To identify cancers effective in the chemotherapy, differences in signatures of genes expressions are compared between in chemotherapy effective cancer and in ineffective ones. Such attempts have been applied to molecular diagnostic tools to predict clinical outcome of the cancer. Using such tools, individualization of cancer chemotherapy is now on clinical trial. In this article, we review molecular diagnostic tools focusing on breast cancer therapy.

New insights into the structural characteristics and functional relevance of the human cytochrome P450 2D6 enzyme.

To date, the crystal structures of at least 12 human CYPs (1A2, 2A6, 2A13, 2C8, 2C9, 2D6, 2E1, 2R1, 3A4, 7A1, 8A1, and 46A1) have been determined. CYP2D6 accounts for only a small percentage of all hepatic CYPs (< 2%), but it metabolizes approximately 25% of clinically used drugs with significant polymorphisms. CYP2D6 also metabolizes procarcinogens and neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1,2,3,4-tetrahydroquinoline, and indolealkylamines. Moreover, the enzyme utilizes hydroxytryptamines and neurosteroids as endogenous substrates. Typical CYP2D6 substrates are usually lipophilic bases with an aromatic ring and a nitrogen atom, which can be protonated at physiological pH. Substrate binding is generally followed by oxidation (5-7 A) from the proposed nitrogen-Asp301 interaction. A number of homology models have been constructed to explore the structural features of CYP2D6, while antibody studies also provide useful structural information. Site-directed mutagenesis studies have demonstrated that Glu216, Asp301, Phe120, Phe481, and Phe483 play important roles in determining the binding of ligands to CYP2D6. The structure of human CYP2D6 has been recently determined and shows the characteristic CYP fold observed for other members of the CYP superfamily. The lengths and orientations of the individual secondary structural elements in the CYP2D6 structure are similar to those seen in other human CYP2 members, such as CYP2C9 and 2C8. The 2D6 structure has a well-defined active-site cavity located above the heme group with a volume of approximately 540 A(3), which is larger than equivalent cavities in CYP2A6 (260 A(3)), 1A2 (375 A(3)), and 2E1 (190 A(3)), but smaller than those in CYP3A4 (1385 A(3)) and 2C8 (1438 A(3)). Further studies are required to delineate the molecular mechanisms involved in CYP2D6 ligand interactions and their implications for drug development and clinical practice.

The CYP2D6*4 polymorphism affects breast cancer survival in tamoxifen users.

Cytochrome P450 2D6 (CYP2D6) plays an important role in the formation of endoxifen, the active metabolite of tamoxifen. In this study the association between the most prevalent CYP2D6 null-allele in Caucasians (CYP2D6*4) and breast cancer mortality was examined among all incident users of tamoxifen in a population-based cohort study. Breast cancer mortality was significantly increased in patients with the * 4/*4 genotype (HR = 4.1, CI 95% 1.1-15.9, P = 0.041) compared to wild type patients. The breast cancer mortality increased with a hazard ratio of 2.0 (CI 95% 1.1-3.4, P = 0.015) with each additional variant allele. No increased risk of all-cause mortality or all-cancer mortality was found in tamoxifen users carrying a CYP2D6*4 allele. The risk of breast cancer mortality is increased in tamoxifen users with decreased CYP2D6 activity, consistent with the model in which endoxifen formation is dependent on CYP2D6 activity.

Enantioselective analysis of citalopram and escitalopram in postmortem blood together with genotyping for CYP2D6 and CYP2C19.

Citalopram is marketed as a racemate (50:50) mixture of the S(+)-enantiomer and R(-)-enantiomer and the active S(+)-enantiomer (escitalopram) that possess inhibitory effects. Citalopram was introduced in Sweden in 1992 and is the most frequently used antidepressant to date in Sweden. In 2002, escitalopram was introduced onto the Swedish market for treatment of depression and anxiety disorders. The main objective of this study was to investigate S(+)-citalopram [i.e., the racemic drug (citalopram) or the enantiomer (escitalopram)] present in forensic autopsy cases positive for the presence of citalopram in routine screening using a non-enantioselective bioanalytical method. Fifty out of the 270 samples found positive by gas chromatography-nitrogen-phosphorus detection were further analyzed using enantioselective high-performance liquid chromatography. The 50 cases were genotyped for CYP2D6 and CYP2C19, as these isoenzymes are implicated in the metabolism of citalopram and escitalopram. In samples positive for racemic citalopram using the screening method for forensic autopsy cases, up to 20% would have been misinterpreted in the absence of an enantioselective method. An enantioselective method is thus necessary for correct interpretation of autopsy cases, after the enantiomer has been introduced onto the market. The percentage of poor metabolizers was 6% for CYP2D6 and 8% for CYP2C19.

The endocannabinoid anandamide is a substrate for the human polymorphic cytochrome P450 2D6.

Members of the cytochrome P450 (P450) family of drug-metabolizing enzymes are present in the human brain, and they may have important roles in the oxidation of endogenous substrates. The polymorphic CYP2D6 is one of the major brain P450 isoforms and has been implicated in neurodegeneration, psychosis, schizophrenia, and personality traits. The objective of this study was to determine whether the endocannabinoid arachidonoylethanolamide (anandamide) is a substrate for CYP2D6. Anandamide is the endogenous ligand to the cannabinoid receptor CB1, which is also activated by the main psychoactive component in marijuana. Signaling via the CB1 receptor alters sensory and motor function, cognition, and emotion. Recombinant CYP2D6 converted anandamide to 20-hydroxyeicosatetraenoic acid ethanolamide and 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid ethanolamides (EET-EAs) with low micromolar K(m) values. CYP2D6 further metabolized the epoxides of anandamide to form novel dioxygenated derivatives. Human brain microsomal and mitochondrial preparations metabolized anandamide to form hydroxylated and epoxygenated products, respectively. An inhibitory antibody against CYP2D6 significantly decreased the mitochondrial formation of the EET-EAs. To our knowledge, anandamide and its epoxides are the first eicosanoid-like molecules to be identified as CYP2D6 substrates. Our study suggests that anandamide may be a physiological substrate for brain mitochondrial CYP2D6, implicating this polymorphic enzyme as a potential component of the endocannabinoid system in the brain. This study also offers support to the hypothesis that neuropsychiatric phenotype differences among individuals with genetic variations in CYP2D6 could be ascribable to interactions of this enzyme with endogenous substrates.

Humanized mouse lines and their application for prediction of human drug metabolism and toxicological risk assessment.

Cytochrome P450s (P450s) are important enzymes involved in the metabolism of xenobiotics, particularly clinically used drugs, and are also responsible for metabolic activation of chemical carcinogens and toxins. Many xenobiotics can activate nuclear receptors that in turn induce the expression of genes encoding xenobiotic metabolizing enzymes and drug transporters. Marked species differences in the expression and regulation of cytochromes P450 and xenobiotic nuclear receptors exist. Thus, obtaining reliable rodent models to accurately reflect human drug and carcinogen metabolism is severely limited. Humanized transgenic mice were developed in an effort to create more reliable in vivo systems to study and predict human responses to xenobiotics. Human P450s or human xenobiotic-activated nuclear receptors were introduced directly or replaced the corresponding mouse gene, thus creating "humanized" transgenic mice. Mice expressing human CYP1A1/CYP1A2, CYP2E1, CYP2D6, CYP3A4, CY3A7, pregnane X receptor, and peroxisome proliferator-activated receptor alpha were generated and characterized. These humanized mouse models offer a broad utility in the evaluation and prediction of toxicological risk that may aid in the development of safer drugs.