Human Cytochrome P450 2D6 Research Articles

Rational Molecular Design of a Fluorescent Probe for Selectively Sensing Human Cytochrome P450 2D6.

Cytochrome P450 2D6 (CYP2D6) is a key enzyme that mediates the metabolism of various drugs and endogenous substances in humans. However, its biological role in drug-drug interactions especially mechanism-based inactivation (MBI), and various diseases remains poorly understood, owing to the lack of molecular tools suitable for selectively monitoring CYP2D6 in complex biological systems. Herein, using a tailored molecular strategy, we developed a fluorescent probe BDPM for CYP2D6. BDPM exhibits excellent specificity and imaging capability for CYP2D6, making it suitable for the real-time monitoring of endogenous CYP2D6 activity in living bio-samples. Therefore, our tailored strategy proved useful for constructing the highly selective and enzyme-activated fluorescent probes. BDPM as a molecular tool to explore the critical roles of CYP2D6 in the pathogenesis of diseases, high-throughput screening of inhibitors and intensive investigation of CYP2D6-induced MBI in natural systems.

L-lysine supplementation attenuates experimental autoimmune hepatitis in a chronic murine model.

The incidence of autoimmune hepatitis (AIH) has increased significantly worldwide. The present study aims to explore the protective effect of L-lysine supplementation against AIH and to investigate its potential underlying mechanisms. A chronic experimental AIH mouse model was established by repeated tail vein injection of human cytochrome P450 2D6 (CYP2D6) plasmid. Starting from day 14 of the modeling, mice in the CYP2D6-AIH +L-lysine group were given 200 µl of purified water containing 10 mg/kg L-lysine by gavage until day27, once a day, and mice in the healthy control group and model group were given an equal volume of purified water by gavage. Our results showed that L-lysine supplementation partially reversed the liver injury mediated by CYP2D6 overexpression. These effects were consistent with the restraining impacts of L-lysine supplementation on decreasing pro-inflammatory cytokines expression level and CD4+ and CD8+ T lymphocytes infiltration, as well as curbing hepatic oxidative stress. Furthermore, L-lysine supplement relieved liver fibrosis in the context of AIH. In conclusion, L-lysine supplementation attenuates CYP2D6-induced immune liver injury in mice, which may serve as a novel nutrition support approach for AIH.

Investigation of the Differences in the Pharmacokinetics of CYP2D6 Substrates, Desipramine, and Dextromethorphan in Healthy African Subjects Carrying the Allelic Variants CYP2D6*17 and CYP2D6*29, When Compared with Normal Metabolizers.

This study investigated the differences in the pharmacokinetics (PK) of dextromethorphan and desipramine in healthy African volunteers to understand the effect of allelic variants of the human cytochrome P450 2D6 (CYP2D6) enzyme, namely the diplotypes of CYP2D6*1/*2 (*1*1, *1*2, *2*2) and the genotypes of CYP2D6*17*17 and CYP2D6*29*29. Overall, 28 adults were included and split into 3 cohorts after genotype screening: CYP2D6*1/*2 (n=12), CYP2D6*17*17 (n=12), and CYP2D6*29*29 (n=4). Each subject received a single oral dose of dextromethorphan 30mg syrup on day1 and desipramine 50mg tablet on day8. The PK parameters of area under the plasma concentration-time curve from time of dosing to time of last quantifiable concentration (AUClast), and extrapolated to infinity (AUCinf), and the maximum plasma concentration (Cmax) were determined. For both dextromethorphan and desipramine, AUCinf and Cmax were higher in subjects of the CYP2D6*29*29 and CYP2D6*17*17 cohorts, as compared with subjects in the CYP2D6*1/*2 diplotype cohort and with normal metabolizers from the literature. All PK parameters, including AUCinf, Cmax, and the elimination half-life, followed a similar trend: CYP2D6*17*17>CYP2D6*29*29>CYP2D6*1/*2. The plasma and urinary drug/metabolite exposure ratios of both drugs were higher in subjects of the CYP2D6*17*17 and CYP2D6*29*29 cohorts, when compared with subjects in the CYP2D6*1/*2 diplotype cohort. All adverse events were mild, except in 1 subject with CYP2D6*17*17 who had moderately severe headache with desipramine. These results indicate that subjects with CYP2D6*17*17 and CYP2D6*29*29 genotypes were 5-10 times slower metabolizers than those with CYP2D6*1/*2 diplotypes. These findings suggest that dose optimization may be required when administering CYP2D6 substrate drugs in African patients. Larger studies can further validate these findings.

Inhibition Effect of Okanin Toward Human Cytochrome P450 3A4 and 2D6 with Multi-spectroscopic Studies and Molecular Docking.

Okanin, a major flavonoid of a popular herb tea, Coreopsis tinctoria Nutt., showed strong inhibition on CYP3A4 and CYP2D6. The strong interaction between okanin and CYPs were determined by enzyme kinetics, multispectral technique and molecular docking. The inhibition type of two enzymes, CYP3A4 and CYP2D6, by okanin are mixed and non-competitive inhibition type, respectively. The IC50 values and the binding constant of okanin to CYP3A4 can be deduced that the interaction was stronger than that of CYP2D6. The Conformations of CYP3A4 and CYP2D6 were changed by okanin. The evidence from fluorescence measurement along with molecular docking verified that these two CYPs were bound with okanin by hydrogen bonds and hydrophobic forces. Our investigation suggested that okanin may lead to interactions between herb and drug by inhibiting CYP3A4 and CYP2D6 activities, thus its consumption should be taken with caution.

Effect of Human Cytochrome P450 2D6 Polymorphism on Progesterone Hydroxylation.

Herein, hydroxylation activities at the 6β-position and 21-position of progesterone mediated by human cytochrome P450 (CYP) 2D6 and its variants and the effects of psychotropic drugs on these hydroxylation activities were compared to clarify whether CYP2D6 polymorphisms and psychotropic drugs impact neurosteroid levels in the brain. Progesterone was incubated with CYP2D6.1, CYP2D6.2 (Arg296Cys, Ser486Thr), CYP2D6.10 (Pro34Ser, Ser486Thr), and CYP2D6.39 (Ser486Thr) in the absence or presence of typical psychotropic drugs (fluvoxamine, fluoxetine, paroxetine, fluphenazine, and milnacipran) and endogenous steroids (testosterone and cortisol). Then, 6β- and 21-hydroxyprogesterone levels were determined by high-performance liquid chromatography. Although the Michaelis-Menten constants (Km) for progesterone 6β- and 21-hydroxylation reactions mediated by the different CYP2D6 variants were similar, the maximal velocity (Vmax) values of the reactions mediated by CYP2D6.1 and CYP2D6.2 were the highest, followed by those mediated by CYP2D6.39 and CYP2D6.10. Thus, the of progesterone 6β- and/or 21-hydroxylation reactions mediated by CYP2D6.1 and CYP2D6.2 showed the highest Vmax/Km values, followed by the reactions mediated by CYP2D6.39. All investigated compounds inhibited progesterone 21-hydroxylation mediated by CYP2D6 variants at high concentrations. Interestingly, at low concentrations, fluoxetine increased progesterone 21-hydroxylation mediated by CYP2D6.1, but not that mediated by CYP2D6.2 or CYP2D6.10. In addition, the Km value for CYP2D6.2 was elevated in the presence of fluoxetine, whereas the value for CYP2D6.1 was unaltered; however, Vmax values of both CYP2D6.1 and CYP2D6.2 were increased. Paroxetine competitively inhibited CYP2D6.1- and CYP2D6.2-mediated progesterone 21-hydroxylation. These results suggest that CYP2D6 polymorphism can affect the stimulation/inhibition of progesterone 21-hydroxylation.

In vitro characterization of the furin inhibitor MI-1851: Albumin binding, interaction with cytochrome P450 enzymes and cytotoxicity

The substrate-analog furin inhibitor MI-1851 can suppress the cleavage of SARS-CoV-2 spike protein and consequently produces significant antiviral effect on infected human airway epithelial cells. In this study, the interaction of inhibitor MI-1851 was examined with human serum albumin using fluorescence spectroscopy and ultrafiltration techniques. Furthermore, the impacts of MI-1851 on human microsomal hepatic cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6 and 3A4 activities were assessed based on fluorometric assays. The inhibitory action was also examined on human recombinant CYP3A4 enzyme and on hepatocytes. In addition, microsomal stability (60 min) and cytotoxicity were tested as well. MI-1851 showed no relevant interaction with human serum albumin and was significantly depleted by human microsomes. Furthermore, it did not inhibit CYP1A2, 2C9, 2C19 and 2D6 enzymes. In human hepatocytes, CYP3A4 was significantly suppressed by MI-1851 and weak inhibition was noticed in regard to human microsomes and human recombinant CYP3A4. Finally, MI-1851 did not impair the viability and the oxidative status of primary human hepatocytes (up to 100 μM concentration). Based on these observations, furin inhibitor MI-1851 appears to be potential drug candidates in the treatment of COVID-19, due to the involvement of furin in S protein priming and thus activation of the pandemic SARS-CoV-2.

Evaluation of biotransformation capacity of transplastomic plants and hairy roots of Nicotiana tabacum expressing human cytochrome P450 2D6

Cytochrome P450 monooxygenases (CYPs) are important tools for regio- and stereoselective oxidation of target molecules or engineering of metabolic pathways. Functional heterologous expression of eukaryotic CYPs is often problematic due to their dependency on the specific redox partner and the necessity of correct association with the membranes for displaying enzymatic activity. Plant hosts offer advantages of accessibility of reducing partners and a choice of membranes to insert heterologous CYPs. For the evaluation of plant systems for efficient CYP expression, we established transplastomic plants and hairy root cultures of Nicotiana tabacum carrying the gene encoding human CYP2D6 with broad substrate specificity. The levels of CYP2D6 transcript accumulation and enzymatic activity were estimated and compared with the data of CYP2D6 transient expression in N. benthamiana. The relative level of CYP2D6 transcripts in transplastomic plants was 2–3 orders of magnitude higher of that observed after constitutive or transient expression from the nucleus. CYP2D6 expressed in chloroplasts converted exogenous synthetic substrate loratadine without the need for co-expression of the cognate CYP reductase. The loratadine conversion rate in transplastomic plants was comparable to that in N. benthamiana plants transiently expressing a chloroplast targeted CYP2D6 from the nucleus, but was lower than the value reported for transiently expressed CYP2D6 with the native endoplasmic reticulum signal-anchor sequence. Hairy roots showed the lowest substrate conversion rate, but demonstrated the ability to release the product into the culture medium. The obtained results illustrate the potential of plant-based expression systems for exploiting the enzymatic activities of eukaryotic CYPs with broad substrate specificities.

Development of a New Murine Model of Type 2 Autoimmune Hepatitis Using a Human Liver Protein

Autoimmune hepatitis (AIH) is a chronic inflammatory condition of the liver characterized by parenchymal destruction, hypergammaglobulinemia, specific autoantibody production, and hepatic fibrosis and necrosis. Murine models of AIH have been described; however, little is known about the immunologic mechanisms of tissue destruction. In this study, a new murine model of type 2 AIH was developed using recombinant human cytochrome P450 (CYP) 2D6 emulsified with complete Freund's adjuvant (CFA). BALB/c mice were immunized with 2 μg/mL i.p. of CYP2D6 in CFA. The control group received CFA or phosphate-buffered saline alone. Alanine aminotransferase activity, autoantibody production, IgG concentrations, histologic damage, and specific T-cell response were evaluated. Persistent AIH, characterized by cellular infiltration, hepatic fibrosis, elevated alanine aminotransferase, and the production of anti-liver kidney microsomal antibody type 1 developed in CFA/CYP2D6-immunized mice. These mice presented high levels of IgG and its subclasses IgG1, IgG2a, and IgG2b against liver self-proteins. Interestingly, IL-2+ and interferon γ-positive Cyp2d6-specific T cells were present in greater concentrations in mice immunized with CFA/CYP2D6 compared with control. Immunization with CFA, in combination with a natural human autoantigen like CYP2D6, was demonstrated to break tolerance, resulting in a chronic form of autoimmune-related liver damage. This murine model of type 2 AIH is expected to be instrumental in understanding the immunologic mechanisms of the pathogenesis of this autoimmune liver disease.

Differential Interactions of Selected Phytocannabinoids with Human CYP2D6 Polymorphisms

Cytochrome P450 2D6 (CYP2D6) is primarily expressed in the liver and in the central nervous system. It is known to be highly polymorphic in nature. It metabolizes several endogenous substrates such as anandamide (AEA). Concomitantly, it is involved in phase 1 metabolism of several antidepressants, antipsychotics, and other drugs. Research in the field of phytocannabinoids (pCBs) has recently accelerated owing to their legalization and increasing medicinal use for pain and inflammation. The primary component of cannabis is THC, which is well-known for its psychotropic effects. Since CYP2D6 is an important brain and liver P450 and is known to be inhibited by CBD, we investigated the interactions of four important highly prevalent CYP2D6 polymorphisms with selected phytocannabinoids (CBD, THC, CBDV, THCV, CBN, CBG, CBC, β-carophyllene) that are rapidly gaining popularity. We show that there is differential binding of CYP2D6*17 to pCBs as compared to WT CYP2D6. We also perform a more detailed comparison of WT and *17 CYP2D6, which reveals the possible regulation of AEA metabolism by CBD. Furthermore, we use molecular dynamics to delineate the mechanism of this binding, inhibition, and regulation. Taken together, we have found that the interactions of CYP2D6 with pCBs vary by polymorphism and by specific pCB class.

Interaction study of engeletin toward cytochrome P450 3A4 and 2D6 by multi-spectroscopy and molecular docking

The inhibitory effects of engeletin on the activities of human cytochrome P450 3A4 and 2D6 (CYP3A4 and CYP2D6) were investigated by enzyme kinetics, multi-spectroscopy and molecular docking. Engeletin was found to strongly inhibit CYP3A4 and CYP2D6, with the IC50 of 1.32 μM and 2.87 μM, respectively. The inhibition modes of engeletin against CYP3A4 and CYP2D6 were a competitive type and a mixed type, respectively. The fluorescence of the two CYPs was quenched statically by engeletin, which was bound to CYP3A4 stronger than to CYP2D6 at the same temperature. Circular dichroism spectroscopy, three-dimensional fluorescence, ultraviolet–visible spectroscopy and synchronous fluorescence confirmed that the conformation and micro-environment of the two CYPs protein were changed after binding with engeletin. Molecular docking, ultraviolet–visible spectroscopy and the fluorescence data revealed that engeletin had strong binding affinity to the two CYPs through hydrogen and van der Waals forces. The findings here suggested that engeletin may cause the herb-drug interactions for its inhibition of CYP3A4 and CYP2D6 activities.

Interaction study of astilbin, isoastilbin and neoastilbin toward CYP2D6 by multi-spectroscopy and molecular docking.

Astilbin, isoastilbin and neoastilbin are the three flavonoid isomers prevalent in Rhizoma Smilax glabra. The interactions between human cytochrome P450 2D6 (CYP2D6) and the three isomers were investigated by multiple spectroscopic coupled with molecular docking. As a result, the fluorescence intensity of CYP2D6 was quenched statically by the three isomers. Meanwhile, astilbin had the strongest binding ability to CYP2D6, followed by isoastilbin and neoastilbin under the identical temperature. Synchronous fluorescence, three-dimensional fluorescence, ultraviolet-visible spectroscopy, circular dichroism and Fourier-transform infrared spectra confirmed that the conformation and micro-environment of CYP2D6 protein were changed after binding with the three isomers. As suggested from molecular docking, the three isomers had strong binding affinity to CYP2D6 via the bonding of hydrogen and van der Waals forces, and the results were in agreement with the fluorescence results. The findings here suggested that astilbin, isoastilbin and neoastilbin may cause the herb-drug interactions for their inhibition of CYP2D6 activity.

Interaction of silymarin components and their sulfate metabolites with human serum albumin and cytochrome P450 (2C9, 2C19, 2D6, and 3A4) enzymes

Silymarin is a mixture of flavonolignans isolated from the fruit of milk thistle (Silybum marianum (L.) Gaertner). Milk thistle extract is the active ingredient of several medications and dietary supplements to treat liver injury/diseases. After the oral administration, flavonolignans are extensively biotransformed, resulting in the formation of sulfate and/or glucuronide metabolites. Previous studies demonstrated that silymarin components form stable complexes with serum albumin and can inhibit certain cytochrome P450 (CYP) enzymes. Nevertheless, in most of these investigations, silybin was tested; while no or only limited information is available regarding other silymarin components and metabolites. In this study, the interactions of five silymarin components (silybin A, silybin B, isosilybin A, silychristin, and 2,3-dehydrosilychristin) and their sulfate metabolites were examined with human serum albumin and CYP (2C9, 2C19, 2D6, and 3A4) enzymes. Our results demonstrate that each compound tested forms stable complexes with albumin, and certain silymarin components/metabolites can inhibit CYP enzymes. Most of the sulfate conjugates were less potent inhibitors of CYP enzymes, but 2,3-dehydrosilychristin-19-O-sulfate showed the strongest inhibitory effect on CYP3A4. Based on these observations, the simultaneous administration of high dose silymarin with medications should be carefully considered, because milk thistle flavonolignans and/or their sulfate metabolites may interfere with drug therapy.

Comparative pharmacokinetic profile of cimicoxib in dogs and cats after IV administration

Cimicoxib is a selective COX-2 inhibitor (coxib) registered for the treatment of pain and inflammation in dogs. Pharmacokinetics of some coxibs have been described in dogs and cats. In cats, total body clearance values are lower and terminal half-lives of the coxibs are longer than those in dogs. The aim of this work was to evaluate if this is also the case for cimicoxib. For this purpose, blood pharmacokinetics and urinary excretion after IV administration were compared between these species. The in vitro metabolism of cimicoxib was also evaluated using canine and feline microsomes.In canine and feline microsomes, the formation rate of demethyl-cimicoxib, a phase 1 metabolite, was decreased in presence of quinidine, a specific human cytochrome P450 (CYP)2D6 inhibitor. IC50 values were 1.6 μM and 0.056 μM with canine and feline microsomes, respectively. As quinidine was about 30 times more potent in feline microsomes, the affinity of cimicoxib to the enzyme was considered to be about 30 times lower than that in canine microsomes. Total body clearance (ClB) of cimicoxib, was 0.50 L/h kg in dogs and 0.14 L/h kg in cats (P < 0.01) and terminal half-life, T½λz, was 1.92 and 5.25 h, respectively (P < 0.01). Dose eliminated in urine was 12.2% in dogs and 3.12% in cats (P < 0.01). Conjugated demethyl-cimicoxib represented 93.7% of this amount in dogs and 67.5% in cats. Thus cimicoxib, like other veterinary coxibs, was eliminated more slowly in cats. Both CYP2D15 (the canine ortholog of CYP2D6) and UDP-glucuronyltransferase enzyme systems have reduced ability to produce metabolites of cimicoxib in cats.

Plasma proteomic analysis of autoimmune hepatitis in an improved AIH mouse model

BackgroundThe prevalence of autoimmune hepatitis (AIH) is increasing, and its early clinical diagnosis is difficult. The pathogenesis of AIH remains unclear, and AIH-related studies are largely limited because of lack of suitable mouse models.MethodsTo obtain a good tool for research on AIH, we first established an improved immune-mediated mouse model that can mimic the pathological process of AIH as in the human body, through repeated injections of human cytochrome P450 2D6 (CYP2D6) plasmid. Next, a proteomic analysis based on isobaric tag (IBT) technology was performed to detect the differentially expressed proteins (DEPs), and related biological functions and pathways in the plasma of AIH and normal mice. Finally, we performed enzyme-linked immunosorbent assay (ELISA) to further confirm the most abundant DEP in the plasma of patients with AIH.ResultsAutoantibodies and the characteristic pathology of AIH were observed in our mouse model. Inflammatory infiltration also increased in the livers of AIH mice over time and plateaued by day 42 post the first injection. Chronic hepatitis was most severe on day 35 with the development of fibrosis as well, and the plasma of AIH mice were collected for proteomic analysis. A total of 176 DEPs were found in this experiment, of which 148 DEPs were up-regulated and 28 DEPs were down-regulated. Thirty significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (P < 0.05) were detected. Arginine biosynthesis was found to be the most significant pathway involved in the AIH process. During the Gene Ontology (GO) analysis, most DEPs were found to be involved in the binding, cellular, and metabolic processes. Using ELISA, the most overexpressed DEP, serum amyloid A 1 (SAA1), was confirmed to be increased specifically in the plasma of patients with AIH compared to other chronic hepatitis. Different plasma levels of SAA1 were also found related to different grades of inflammation and stages of fibrosis in the liver of patients with AIH.ConclusionsOur study is the first to describe the proteomics analysis of a true sense of AIH mouse model, which is beneficial for a better understanding of AIH pathogenesis and identifying potential biomarkers for its clinical diagnosis.

Conversion of Cytochrome P450 2D6 of Human Into a FRET-Based Tool for Real-Time Monitoring of Ajmalicine in Living Cells.

Ajmalicine is naturally present in the root bark of Catharanthus roseus L. and Rauvolfia serpentina (L.) Benth ex.Kurz. It has been extensively utilized in the treatment of hypertension across the world. The increased demand, overconsumption, and low content of the alkaloid in the plants have raised the issue of the depletion of natural sources. The metabolic engineering approach has not been successful in improving the content of the ajmalicine because the metabolic regulation of this metabolite is not known. The regulation of a metabolite in the metabolic pathway requires a tool that can carry out real-time measurement of the flux of the metabolite in living system. Given this, the present study was conducted to develop a genetically encoded FRET-based nanosensor by engineering human Cytochrome P450-2D6, an ajmalicine binding protein. The Cytochrome P450-2D6 was sandwiched between two FRET fluorophores. The design of the nanosensor brings two fluorescent proteins in conjunction with the ajmalicine binding protein, such that it undergoes FRET (Fluorescence Resonance Energy Transfer) upon binding of the ligand. The nanosensor, named as FLIP-Ajn (Fluorescence Indicator Protein for Ajmalicine), was pH stable and ajmalicine specific. The affinity of the FLIP-Ajn was 582 μM. The FLIP-Ajn successfully performed real-time measurement of ajmalicine in prokaryotic (bacteria) and eukaryotic systems (yeast, animal cell line, and plant suspension culture), thereby, establishing its biocompatibility in monitoring of ajmalicine in living cells. Besides, several affinity mutants of the nanosensor were generated through mutations in the ajmalicine binding protein to increase the detection range of the nanosensor at varying physiological scales. The non-invasiveness and high spatial and temporal resolution of the tool holds a great significance in the bio-imaging of a highly compartmentalized metabolic pathway. The flux study of ajmalicine will help in identifying the regulatory steps involved in the synthesis of the alkaloids and, hence, will improve the production rate of ajmalicine from its natural sources.

Effects of adenovirus‐induced hepatocyte damage on chronic bile duct inflammation in a sclerosing cholangitis mouse model

Four major autoimmune diseases target the liver. They develop because of bile duct destruction, leading to chronic cholestasis or result from hepatocyte damage like autoimmune hepatitis (AIH). Interestingly, some patients simultaneously show features of both cholangitis and AIH. Our goal was to mimic such concurrent characteristics in a mouse model that would help deciphering mechanisms possibly involved in an inflammatory crosstalk between cholestatic disease and hepatitis. Mdr2-/- mice, which spontaneously develop sclerosing cholangitis because of accumulation of toxic bile salts, were infected with adenovirus (Ad) encoding human Cytochrome P4502D6 (hCYP2D6), the major target autoantigen in type-2 AIH, to trigger hepatocyte injury. Wild type FVB mice were controls. Resulting Ad-Mdr2-/- mice presented with cholangitis, fibrosis and cellular infiltrations that were higher than in Mdr2-/- or Ad-FVB mice. Increased levels of anti-neutrophil cytoplasmic antibodies but similar anti-hCYP2D6 antibody titres were detected in Ad-Mdr2-/- compared to Mdr2-/- and Ad-FVB mice respectively. IFNγ-expressing hCYP2D6-specific CD4 T cells declined, whereas hCYP2D6-specific CD8 T cells increased in Ad-Mdr2-/- compared to Ad-FVB mice. The overall T cell balance in Ad-Mdr2-/- mice was a combination of a type 17T cell response typically found in Mdr2-/- mice with a type 1 dominated T cell response characteristic for Ad-FVB mice. Simultaneously, the type 2T cell compartment was markedly reduced. Experimental hepatitis induction in a mouse with sclerosing cholangitis results in a disorder which represents not simply the sum of the individual characteristics but depicts a more complex entity which urges on further analysis.

Effects of catalpol on the activity of human liver cytochrome P450 enzymes

1. Catalpol possesses numerous pharmacological activities, and however, little data available for the effects of catalpol on the activity of human liver cytochrome P450 (CYP) enzymes.2. This study investigates the inhibitory effects of catalpol on the main human liver CYP isoforms. In this study, the inhibitory effects of catalpol on the eight human liver CYP isoforms 1A2, 2A6, 2E1, 2D6, 2C9, 2C19, 2C8 and 3A4 were investigated in human liver microsomes.3. The results indicated that catalpol could inhibit the activity of CYP3A4, CYP2E1 and CYP2C9, with IC50 values of 14.27, 22.4 and 14.69 μM, respectively, but those other CYP isoforms were not affected. Enzyme kinetic studies showed that catalpol was not only a noncompetitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP2E1 and CYP2C9, with Ki values of 7.40, 10.75 and 7.37 μM, respectively. In addition, catalpol is a time-dependent inhibitor for CYP3A4, with maximum inactivation (kinact) and 50% maximum inactivation (KI) values of 0.02 min−1 and 1.86 μM, respectively.4. The in vitro studies of catalpol with CYP isoforms suggest that catalpol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4, CYP2E1 and CYP2C9. Further in vivo studies are needed in order to evaluate the significance of this interaction.

Regioselective hydroxylation of an antiarrhythmic drug, propafenone, mediated by rat liver cytochrome P450 2D2 differs from that catalyzed by human P450 2D6

1. Propafenone, an antiarrhythmic drug, is a typical human cytochrome P450 (P450) 2D6 substrate used in preclinical studies. Here, propafenone oxidation by mammalian liver microsomes was investigated in vitro.2. Liver microsomes from humans and marmosets preferentially mediated propafenone 5-hydroxylation, minipig, rat and mouse livers primarily mediated 4′-hydroxylation, but cynomolgus monkey and dog liver microsomes differently mediated N-despropylation.3. Quinine, ketoconazole or anti-P450 2D antibodies suppressed propafenone 4′/5-hydroxylation in human and rat liver microsomes. Pretreatments with β-naphthoflavone or dexamethasone increased N-despropylation in rat livers.4. Recombinant rat P450 2D2 efficiently catalysed propafenone 4′-hydroxylation in a substrate inhibition manner, comparable to rat liver microsomes, while human P450 2D6 displayed propafenone 5-hydroxylation. Human and rat P450 1A, 2C and 3A enzymes mediated propafenone N-despropylation with high capacities.5. Carbon-4′ of propafenone docked favourably into the active site of P450 2D2 based on an in silico model; in contrast, carbon-5 of propafenone docked into human P450 2D6.6. These results suggest that the major roles of individual P450 2D enzymes in regioselective hydroxylations of propafenone differ between human and rat livers, while the minor roles of P450 1A, 2C and 3A enzymes for propafenone N-despropylation are similar in livers of both species.

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 β-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-β-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.

Cytochrome P450 2D6 profiles and their relationship with outcomes of primaquine anti-relapse therapy in Australian Defence Force personnel deployed to Papua New Guinea and East Timor

BackgroundPrimaquine, an 8-aminoquinoline with anti-hypnozoite activity against Plasmodium vivax, is metabolized by human cytochrome P450 2D6 (CYP2D6) to its active metabolite. Human CYP2D6 activities may influence the metabolism of primaquine and the risk of experiencing Plasmodium relapses following primaquine anti-relapse therapies (PART). In this study, the CYP2D6 profile and its relationship with outcomes of PART in Australian Defence Force (ADF) personnel is retrospectively investigated.MethodsGenomic DNA was isolated from stored and de-identified serum or blood samples from ADF personnel deployed on peacekeeping duties to Papua New Guinea (PNG) (1999) and East Timor (1999–2000) who received PART before returning to Australia and after experiencing relapses. CYP2D6 allelic type was determined by PCR and Sanger sequencing. CYP2D6 allele frequency, predicted phenotypes and activity scores were compared among personnel who did not experience P. vivax (ADF-NR, n = 48) and those who experience at least one (ADF-R, n = 109) relapse, as well as between those who experienced 1 (n = 79), 2 (n = 21) and 3–5 (n = 9) relapses within the ADF-R group.Results16 CYP2D6 alleles were observed in 157 ADF personnel. Alleles *1, *4, *2 and *41 were major alleles (> 5%). The CYP2D6 allele frequency profile in the ADF-NR group matched that of a European population. There was an increased proportion of non-functional CYP2D6 alleles in the ADF-R group compared to the European population and ADF-NR group. However, frequencies of predicted CYP2D6 phenotype and activity score were not different between the ADF-R and ADF-NR groups, nor among sub-groups experiencing multiple relapses within the ADF-R group.ConclusionsCYP2D6 phenotype or activity score based on the allele classification was not a major contributor to P. vivax relapse in this ADF cohort. Other factors such as adherence and/or parasite tolerance to primaquine are likely contributing factors to P. vivax relapses in this cohort.