Major Drug-metabolizing Cytochrome P450 Research Articles

Synthesis of 6β-hydroxy androgens from a 3,5-diene steroid precursor to test for cytochrome P450 3A4-catalyzed hydroxylation of androstenedione

6β-Hydroxytestosterone is a biomarker for the activity of human cytochrome P450 3A4 (P450 3A4), the major drug metabolizing cytochrome P450 enzyme. Despite its significance, efficient routes for the chemical synthesis of 6β-hydroxytestosterone are rare. In this study, 6β-hydroxytestosterone was synthesized through the oxidation of a 3,5-diene precursor under the Uemura-Doyle reaction conditions using a dirhodium catalyst in the presence of tert-butylhydroperoxide. Mechanistic studies showed that some oxygen is incorporated from molecular oxygen and CH abstraction is partially rate-limiting. This reaction was used to synthesize 6β-hydroxyandrostenedione, which was used as a standard to test the hypothesis of whether P450 3A4 catalyzes the hydroxylation of androstenedione. Upon incubation of P450 3A4 with androstenedione, a hydroxylated product was formed, which matched the retention time of synthetic 6β-hydroxyandrostenedione. This reaction can be exploited to study other biochemical processes involving compounds with a 6 β -hydroxy-3-keto-Δ4 steroid backbone.

The genetic landscape of major drug metabolizing cytochrome P450 genes—an updated analysis of population-scale sequencing data

Genes encoding cytochrome P450 enzymes (CYPs) are extremely polymorphic and multiple CYP variants constitute clinically relevant biomarkers for the guidance of drug selection and dosing. We previously reported the distribution of the most relevant CYP alleles using population-scale sequencing data. Here, we update these findings by making use of the increasing wealth of data, incorporating whole exome and whole genome sequencing data from 141,614 unrelated individuals across 12 human populations. We furthermore extend our previous studies by systematically considering also uncharacterized rare alleles and reveal that they contribute between 1.5% and 17.5% to the overall genetically encoded functional variability. By using established guidelines, we aggregate and translate the available sequencing data into population-specific patterns of metabolizer phenotypes. Combined, the presented data refine the worldwide landscape of ethnogeographic variability in CYP genes and aspire to provide a relevant resource for the optimization of population-specific genotyping strategies and precision public health.

Nonclinical Pharmacokinetic Evaluation of Desidustat: a Novel Prolyl Hydroxylase Inhibitor for the Treatment of Anemia.

Desidustat is a novel prolyl hydroxylase domain(PHD) inhibitor for the treatment of anemia. The objective of this study was to investigate the pharmacokinetics and drug-drug interaction properties of desidustat using invitro and invivo nonclinical models. Invitro, Caco2 cell permeability, plasma protein binding, metabolism, cytochrome P450 (CYP) inhibition, and CYP induction were examined. In vivo, pharmacokinetic studies of oral bioavailability in mice, rats, dogs and monkeys, dose linearity, tissue distribution, and excretion in rats were conducted. In Caco-2 cells, the apparent permeability of desidustat was high at low pH and low at neutral pH. The oral bioavailability (%F) of desidustat was 43-100% with a mediantime to reach peak concentration (Tmax)of about 0.25-1.3h across species. Desidustat displayed a low mean plasma clearance (CL) of 1.3-4.1mL/min/kg (approximately 1.8-7.4% of hepatic blood flow), and the mean steady-state volume of distribution (Vss) was 0.2-0.4L/kg (approximately 30-61% of the total body water). Desidustat showed a dose-dependent increase in exposures over the 15-100mg/kg dose range. It was rapidly distributed in various tissues, with the highest tissue-to-blood ratio in the liver (1.8) and kidney (1.7). Desidustat showed high plasma protein binding and was metabolically stable in human liver microsomes, hepatocytes, and recombinant CYPs. It did not show significant inhibition of major drug-metabolizing CYP enzymes (IC50>300µM) or the potential to induce CYP1A2 and CYP3A4/5 (up to 100µM) in HepG2 cells. It may have minimal potential of clinical drug-drug interaction when used in combination with iron supplements or phosphate binders. Desidustat was primarily excreted unchanged in urine (25% of the oral dose) and bile (25% of the oral dose) in rats. The mean elimination half-life of desidustat ranged from 1.0 to 5.3h and 1.3 to 5.7h across species after intravenous and oral administration, respectively. Taken together, desidustat is well absorbed orally. It showed a dose-dependent increase in exposure, did not accumulate in tissue, and was eliminated via dual routes. It is metabolically stable, has minimal potential to cause clinical drug-drug interactions (DDIs), and demonstrates discriminable pharmacokinetic properties for the treatment of anemia.

Polymorphic cytochromes P450 in non-human primates.

Cynomolgus macaques (Macaca fascicularis, an Old World monkey) are widely used in drug development because of their genetic and physiological similarities to humans, and this trend has continued with the use of common marmosets (Callithrix jacchus, a New World monkey). Information on the major drug-metabolizing cytochrome P450 (CYP, P450) enzymes of these primate species indicates that multiple forms of their P450 enzymes have generally similar substrate selectivities to those of human P450 enzymes; however, some differences in isoform, activity, and substrate specificity account for limited species differences in drug oxidative metabolism. This review provides information on the P450 enzymes of cynomolgus macaques and marmosets, including cDNA, tissue expression, substrate specificity, and genetic variants, along with age differences and induction. Typical examples of important P450s to be considered in drug metabolism studies include cynomolgus CYP2C19, which is expressed abundantly in liver and metabolizes numerous drugs. Moreover, genetic variants of cynomolgus CYP2C19 affect the individual pharmacokinetic data of drugs such as R-warfarin. These findings provide a foundation for understanding each P450 enzyme and the individual pharmacokinetic and toxicological results in cynomolgus macaques and marmosets as preclinical models. In addition, the effects of induction on some drug clearances mediated by P450 enzymes are also described. In summary, this review describes genetic and acquired individual differences in cynomolgus and marmoset P450 enzymes involved in drug oxidation that may be associated with pharmacological and/or toxicological effects.

Modulation of CYP3A4 and CYP2C9 activity by Bulbine natalensis and its constituents: An assessment of HDI risk of B. natalensis containing supplements

BackgroundBulbine natalensis is an African-folk medicinal plant used as a dietary supplement for enhancing sexual function and muscle strength in males by presumably boosting testosterone levels, but no scientific information is available about the possible herb-drug interaction (HDI) risk when bulbine-containing supplements are concomitantly taken with prescription drugs. PurposeThis study was aimed to investigate the HDI potential of B. natalensis in terms of the pregnane X receptor (PXR)-mediated induction of major drug-metabolizing cytochrome P450 enzyme isoforms (i.e., CYP3A4 and CYP2C9) as well as inhibition of their catalytic activity. ResultsWe found that a methanolic extract of B. natalensis activated PXR (EC50 6.2 ± 0.6 µg/ml) in HepG2 cells resulting in increased mRNA expression of CYP3A4 (2.40 ± 0.01 fold) and CYP2C9 (3.37 ± 0.3 fold) at 30 µg/ml which was reflected in increased activites of the two enzymes. Among the constituents of B. natalensis, knipholone was the most potent PXR activator (EC50 0.3 ± 0.1 µM) followed by bulbine-knipholone (EC50 2.0 ± 0.5 µM), and 6′-methylknipholone (EC50 4.0 ± 0.5 µM). Knipholone was also the most effective in increasing the expression of CYP3A4 (8.47 ± 2.5 fold) and CYP2C9 (2.64 ± 0.3 fold) at 10 µM. Docking studies further confirmed the unique structural features associated with knipholones for their superior inductive potentials in the activation of PXR compared to other anthraquinones. In a CYP inhibition assay, the methanolic extract as well as the anthraquinones strongly inhibited the catalytic activity of CYP2C9 while, inhibition of CYP3A4 was weak. ConclusionsThese results suggest that consumption of B. natalensis may pose a potential risk for HDI if taken with conventional medications that are substrates of CYP3A4 and CYP2C9 and may contribute to unanticipated adverse reactions or therapeutic failures. Further studies are warranted to validate these findings and establish their clinical relevancy.

Identification of rhythmic human CYPs and their circadian regulators using synchronized hepatoma cells

Cytochromes P450 (CYPs) catalyze a great number of metabolic reactions that have profound effects on the biological activities of xenobiotics and endobiotics. In this study, we aimed to characterize rhythmic expressions of drug-metabolizing CYPs using synchronized hepatoma cells, and to investigate the potential roles of cis-elements of circadian clock system (E-box, D-box and RevRE or RORE) in generating the rhythms.Serum was used to synchronize circadian cycles and to induce circadian gene expression in cultured hepatoma cells (HepRG and HepG2 cells). Regulation of CYP genes by circadian clock components was investigated by performing luciferase reporter, overexpression and knockdown experiments. mRNA and protein expression were determined by qPCR and Western blotting assays, respectively.Of ten major drug-metabolizing CYP genes, six are rhythmically expressed (CYP1A2, 2B6, 2C8, 2D6, 2E1 and 3A4), whereas other four are non-rhythmic (CYP1B1, 2A6, 2C9 and 2C19).The E-box binding protein BMAL1 directly controls the rhythmic expression of CYP1A2. Rhythmic expressions of CYP2E1 and CYP3A4 are generated via both E-box and D-box elements. The RevRE binding protein REV-ERBα contributes to rhythmic oscillations in CYP2B6 and CYP2C8.In conclusion, rhythmic expressions of five human CYPs (CYP1A2, 2B6, 2C8, 2E1 and 3A4) are generated and regulated by E-box-, D-box-, and/or RevRE-acting clock components. Our findings may have implications for understanding chronopharmacokinetic events in humans.

Isolation, synthesis, and drug interaction potential of secondary metabolites derived from the leaves of miracle tree (Moringa oleifera) against CYP3A4 and CYP2D6 isozymes

BackgroundMoringa oleifera Lam. is known as a drumstick tree that is widely cultivated in various subtropical and tropical provinces. Previous studies indicated that both aqueous and methanolic extracts of M. oleifera leaves have potent inhibitory effects on two major drug metabolizing Cytochrome P450 enzymes, namely, CYP3A4 and CYP2D6. PurposeThe current study was aimed to isolate the secondary metabolites from M. oleifera and investigate their cytotoxicity and inhibitory effects on CYP3A4 and CYP2D6 to assess their herb-drug interaction (HDI) potential. MethodsChemical structure elucidation was achieved by interpreting the spectroscopic data (UV, IR, 1D, and 2D NMR experiments), confirming by HR-ESI-MS, and comparing with the previously reported data in the literature. All the isolates were evaluated for their cytotoxicity against a panel of cell lines (SK-MEL, KB, BT-549, SK-OV-3, VERO, LLC-PK1, and HepG2) and inhibition of two principal CYP isozymes (CYP3A4 and CYP2D6). ResultsPhytochemical investigation of M. oleifera leaves resulted in the isolation and characterization of one new compound, namely omoringone (1), along with twelve known secondary metabolites (2–13) belonging to several chemical classes including flavonoids, terpenoids, lignans, and phenylalkanoids. A plausible biosynthetic pathway for compound 1 was provided. Because of the low isolation yield and limited supply, omoringone (1) and niazirin (12) were successively synthesized. No cytotoxicity was observed on any of the tested cell lines up to 50 µM. The extract exhibited an inhibitory effect on CYP3A4 isoform (IC50 = 52.5 ± 2.5 µg/ml). Among the isolates, 1–4 and 7–9 inhibited CYP3A4 with the IC50 values ranging from 41.5 to 100 µM with no remarkable effect on CYP2D6 isozyme. ConclusionThis work aided in ascertaining components of M. oleifera contributing to CYP3A4 inhibition exhibited by the extract using an in vitro assay. Nonetheless, further studies are warranted to determine the bioavailability of the phytochemicals and extrapolate these findings in more physiologically relevant conditions to further establish the clinical relevance of in vitro observations.

Bioelectrochemistry of Human Liver Microsomes Attached to Magnetic Nanoparticles

Human liver microsomes (HLM) containing major drug metabolizing cytochrome P450 (CYP) enzymes and its reductase was electrostatically immobilized onto positively charged amine-functionalized magnetic nanoparticles (MNPamine, 100 nm hydrodynamic diameter). The MNP-adsorbed HLM biomaterial (MNPamine/HLM) was then coated onto a solid graphite disk electrode via a polycation glue to accomplish electrocatalysis and prodrug biosensing. Amperometric biosensing of CYP-specific drug candidates and electrocatalyzing drug conversion into metabolites were accomplished. The MNPamine/HLM bioelectrodes exhibited a formal potential of -0.46 V vs Ag/AgCl that was in agreement with the FAD/FMN cofactor containing CYP-reductase as the electron receiver from the electrode. A heterogeneous electron transfer rate constant of 19±5 s- 1 was determined from the MNPamine/HLM film voltammetry. Amperometric i-t curves with diclofenac drug concentration yielded the apparent Michaelis-Menten affinity constant (KM app ) of 302±33 µM for the MNPamine/HLM film, and the KM app was 365±36 µM in the case of only HLM film with no MNPamine. This is the first electrocatalytic study of bioelectrodes featuring HLM-bound MNPamine for applications in prodrug activity assays, enhanced metabolite production, and sensitive biosensing of CYP-specific prodrugs and harmful environmental pollutants.

Survey of Drug Oxidation Activities in Hepatic and Intestinal Microsomes of Individual Common Marmosets, a New Nonhuman Primate Animal Model.

Background: Common marmosets (Callithrix jacchus) are potentially useful non-human primate models for preclinical studies. Information for major drug-metabolizing cyto-chrome P450 (P450) enzymes is now available that supports the use of this primate species as an animal model for drug development. Here, we collect and provide an overview of infor-mation on the activities of common marmoset hepatic and intestinal microsomes with respect to 28 typical human P450 probe oxidations.Results: Marmoset P450 2D6/8-dependent R-metoprolol O-demethylation activities in hepatic microsomes were significantly correlated with those of midazolam 1′- and 4-hydroxylations, testosterone 6β-hydroxylation, and progesterone 6β-hydroxylation, which are probe reactions for marmoset P450 3A4/5/90. In marmosets, the oxidation activities of hepatic microsomes and intestinal microsomes were roughly comparable for midazolam and terfenadine. Overall, multiple forms of marmoset P450 enzymes in livers and intestines had generally similar sub-strate recognition functionalities to those of human and/or cynomolgus monkey P450 enzymes.Conclusion: The marmoset could be a model animal for humans with respect to the first-pass extraction of terfenadine and related substrates. These findings provide a foundation for un-derstanding individual pharmacokinetic and toxicological results in nonhuman primates as pre-clinical models and will help to further support understanding of the molecular mechanisms of human P450 function.

Effects of Khat (Catha edulis) use on catalytic activities of major drug-metabolizing cytochrome P450 enzymes and implication of pharmacogenetic variations

In a one-way cross-over study, we investigated the effect of Khat, a natural amphetamine-like psychostimulant plant, on catalytic activities of five major drug-metabolizing cytochrome P450 (CYP) enzymes. After a one-week Khat abstinence, 63 Ethiopian male volunteers were phenotyped using cocktail probe drugs (caffeine, losartan, dextromethorphan, omeprazole). Phenotyping was repeated after a one-week daily use of 400 g fresh Khat leaves. Genotyping for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A5 were done. Urinary cathinone and phenylpropanolamine, and plasma probe drugs and metabolites concentrations were quantified using LC-MS/MS. Effect of Khat on enzyme activities was evaluated by comparing caffeine/paraxanthine (CYP1A2), losartan/losartan carboxylic acid (CYP2C9), omeprazole/5-hydroxyomeprazole (CYP2C19), dextromethorphan/dextrorphan (CYP2D6) and dextromethorphan/3-methoxymorphinan (CYP3A4) metabolic ratios (MR) before and after Khat use. Wilcoxon-matched-pair-test indicated a significant increase in median CYP2D6 MR (41%, p < 0.0001), and a marginal increase in CYP3A4 and CYP2C19 MR by Khat. Repeated measure ANOVA indicated the impact of CYP1A2 and CYP2C19 genotype on Khat-CYP enzyme interactions. The median MR increased by 35% in CYP1A2*1/*1 (p = 0.07) and by 40% in carriers of defective CYP2C19 alleles (p = 0.03). Urinary log cathinone/phenylpropanolamine ratios significantly correlated with CYP2D6 genotype (p = 0.004) and CYP2D6 MR (P = 0.025). Khat significantly inhibits CYP2D6, marginally inhibits CYP3A4, and genotype-dependently inhibit CYP2C19 and CYP1A2 enzyme activities.

Selective Time- and NADPH-Dependent Inhibition of Human CYP2E1 by Clomethiazole.

The sedative clomethiazole (CMZ) has been used in Europe since the mid-1960s to treat insomnia and alcoholism. It has been previously demonstrated in clinical studies to reversibly inhibit human CYP2E1 in vitro and decrease CYP2E1-mediated elimination of chlorzoxazone. We have investigated the selectivity of CMZ inhibition of CYP2E1 in pooled human liver microsomes (HLMs). In a reversible inhibition assay of the major drug-metabolizing cytochrome P450 (P450) isoforms, CYP2A6 and CYP2E1 exhibited IC50 values of 24 µM and 42 µM, respectively with all other isoforms exhibiting values >300 µM. When CMZ was preincubated with NADPH and liver microsomal protein for 30 minutes before being combined with probe substrates, however, more potent inhibition was observed for CYP2E1 and CYP2B6 but not CYP2A6 or other P450 isoforms. The substantial increase in potency of CYP2E1 inhibition upon preincubation enables the use of CMZ to investigate the role of human CYP2E1 in xenobiotic metabolism and provides advantages over other chemical inhibitors of CYP2E1. The KI and kinact values obtained with HLM-catalyzed 6-hydroxylation of chlorzoxazone were 40 µM and 0.35 minute(-1), respectively, and similar to values obtained with recombinant CYP2E1 (41 µM, 0.32 minute(-1)). The KI and kinact values, along with other parameters, were used in a mechanistic static model to explain earlier observations of a profound decrease in the rate of chlorzoxazone elimination in volunteers despite the absence of detectable CMZ in blood.

PP09.5 – 2350: Pharmacokinetic evaluation of eteplirsen, SRP-4045, and SRP-4053; Three phosphorodiamidate morpholino oligomers (PMOs) for the treatment of patients with Duchenne muscular dystrophy (DMD)

Background DMD is a rare, X-linked recessive, degenerative neuromuscular disorder caused by mutations in the dystrophin gene. The most prevalent mutations result in a reading frame shift and premature translation termination, resulting in a lack of dystrophin, a protein that plays a key structural role in muscle fiber function. Exon-skipping PMOs direct alternative splicing of the dystrophin pre-mRNA to restore the mRNA reading frame and enable translation of an internally truncated yet functional dystrophin protein. Eteplirsen, an investigational exon-skipping PMO, has been dosed at up to 50 mg/kg/wk for over 3 years in a clinical study with no reported clinically significant treatment-related adverse events. Preclinical analysis enables comparison of the pharmacokinetic properties of eteplirsen, SRP-4045, and SRP-4053, three PMOs with specific sequences developed to treat patients with mutations amenable to skipping exon 51, 45 and 53, respectively. Methods Studies conducted to evaluate the in vitro pharmacokinetic properties of eteplirsen, SRP-4045 and SRP-4053 included analysis of plasma protein binding and metabolic stability in hepatic microsomes of mice, rats, monkeys, and humans. Induction and inhibition of cytochrome P450 isoenzymes were also assessed. Results Pharmacokinetic profiles for the three PMOs were similar across species. All three compounds showed comparable Cmax, AUC, and clearance, and displayed similar dose proportionality across the three doses examined (5, 40, and 320 mg/kg). Each exhibited low protein binding in all species, no evidence of in vitro metabolism by hepatic microsomes, no extensive inhibition of the major drug metabolizing cytochrome P450 isoenzymes CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, or CYP3A4/5, and no induction of CYP1A2, CYP2B6, or CYP3A4 at biologically relevant concentrations. Conclusions The consistent preclinical profiles and lack of any significant, sequence-specific toxicities for eteplirsen, SRP-4045, and SRP-4053 demonstrate PMOs to be a well-tolerated therapeutic class, with potential applications in a wide variety of disease indications.

Quantifying rare, deleterious variation in 12 human cytochrome P450 drug-metabolism genes in a large-scale exome dataset

The study of genetic influences on drug response and efficacy ('pharmacogenetics') has existed for over 50 years. Yet, we still lack a complete picture of how genetic variation, both common and rare, affects each individual's responses to medications. Exome sequencing is a promising alternative method for pharmacogenetic discovery as it provides information on both common and rare variation in large numbers of individuals. Using exome data from 2203 AA and 4300 Caucasian individuals through the NHLBI Exome Sequencing Project, we conducted a survey of coding variation within 12 Cytochrome P450 (CYP) genes that are collectively responsible for catalyzing nearly 75% of all known Phase I drug oxidation reactions. In addition to identifying many polymorphisms with known pharmacogenetic effects, we discovered over 730 novel nonsynonymous alleles across the 12 CYP genes of interest. These alleles include many with diverse functional effects such as premature stop codons, aberrant splicesites and mutations at conserved active site residues. Our analysis considering both novel, predicted functional alleles as well as known, actionable CYP alleles reveals that rare, deleterious variation contributes markedly to the overall burden of pharmacogenetic alleles within the populations considered, and that the contribution of rare variation to this burden is over three times greater in AA individuals as compared with Caucasians. While most of these impactful alleles are individually rare, 7.6-11.7% of individuals interrogated in the study carry at least one newly described potentially deleterious alleles in a major drug-metabolizing CYP.

Influence of gold(I) complexes involving adenine derivatives on major drug–drug interaction pathway

A series of considerably anti-inflammatory active gold(I) mixed-ligand complexes, involving the benzyl-substituted derivatives of N6-benzyladenine (HLn) and triphenylphosphine (PPh3) as ligands and having the general formula [Au(Ln)(PPh3)]·xH2O (1–4; n=1–4 and x=0–1), was evaluated for the ability to influence the expression of CYP1A1/2 and CYP3A4 and transcriptional activity of glucocorticoid (GR) and aryl hydrocarbon (AhR) receptors in primary human hepatocytes and HepG2 cells. In both tests, evaluating the ability of the complexes to modulate the expression of CYP1A1, CYP1A2 and CYP3A4 in primary human hepatocytes and influence the transcriptional activity of AhR and GR in the reporter cell lines, no negative influence on the major drug-metabolizing cytochrome P450 isoenzymes and their signaling pathway (through GR and AhR receptors) was observed. These positive findings revealed another substantial evidence that could lead to utilization of the complexes as effective and relatively safe drugs for the treatment of hard-to-treat inflammation-related diseases, such as rheumatoid arthritis, comparable or even better than clinically used gold-containing drug Auranofin.

Assessment of Extracts from Red Yeast Rice for Herb-Drug Interaction by in-vitro and in-vivo assays

Red yeast rice (RYR) is made by fermenting the yeast Monascus purpureus over rice. It is a source of natural red food colorants, a food garnish and a traditional medication. Results of the current study demonstrated that polar fractions of the RYR preparations contained herbal-drug interaction activity, which if left unremoved, enhanced P-glycoprotein activity and inhibited the major drug metabolizing cytochromes P450, i,e, CYP 1A2, 2C9 and 3A4. The data from Caco-2 cell absorption and animal model studies further demonstrated that the pharmacokinetic modulation effect by RYR preparations containing the polar fractions (“untreated” preparation) was greater than that from RYR preparations with the polar fractions removed (“treated” preparation). The data indicates a potential for herb-drug interactions to be present in RYR commonly sold as nutritional supplements when the polar fractions are not removed and this should be taken into consideration when RYR is consumed with medications, including verapamil.

Altered Human CYP3A4 Activity Caused by Antley-Bixler Syndrome-Related Variants of NADPH-Cytochrome P450 Oxidoreductase Measured in a Robust In Vitro System

NADPH-cytochrome P450 oxidoreductase (CYPOR) variants have been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR variants were coexpressed with CYP3A4, resulting in relative expression levels comparable to those found in hepatic tissue. Dibenzylfluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375-384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver microsomes. CYP3A4, combined with WT CYPOR, demonstrated kinetic parameters (k(cat) and K(m)) equal to those for pooled human liver microsomes. CYPOR variants Y181D, Y459H, V492E, L565P, and R616X all demonstrated maximal loss of CYP3A4 catalytic efficiency, whereas R457H and G539R retained ∼10 and 30% activities, respectively. Conversely, variants P228L, M263V, A287P, and G413S each showed WT-like capacity (k(cat)/K(m)), with the A287P variant being formerly reported to exhibit substantially lower catalytic efficiency. In addition, Q153R exhibited 60% of WT CYPOR capacity to support the DBF O-debenzylation reaction, contradicting increased catalytic efficiency (k(cat)/K(m)) relative to that for the WT, reported previously. Our data indicate the importance of use of simulated, validated in vitro systems, employing full-length proteins with appropriate stoichiometric incorporation of protein partners, when pharmacogenetic predictions are to be made for P450-mediated biotransformation.

Clinical Pharmacokinetics of Fingolimod

Fingolimod (FTY720), a sphingosine 1-phosphate receptor modulator, is the first in a new class of therapeutic compounds and is the first oral therapy approved for the treatment of relapsing forms of multiple sclerosis (MS). Fingolimod is a structural analogue of endogenous sphingosine and undergoes phosphorylation to produce fingolimod phosphate, the active moiety. Fingolimod targets MS via effects on the immune system, and evidence from animal models indicates that it may also have actions in the central nervous system. In phase III studies in patients with relapsing-remitting MS, fingolimod has demonstrated efficacy superior to that of an approved first-line therapy, intramuscular interferon-β-1a, as well as placebo, with benefits extending across clinical and magnetic resonance imaging measures. The pharmacokinetic profiles of fingolimod and fingolimod phosphate have been extensively investigated in studies in healthy volunteers, renal transplant recipients (the indication for which fingolimod was initially under clinical development, but the development was subsequently discontinued) and MS patients. Results from these studies have demonstrated that fingolimod is efficiently absorbed, with an oral bioavailability of >90%, and its absorption is unaffected by dietary intake, therefore it can be taken without regard to meals. Fingolimod and fingolimod phosphate have a half-life of 6-9 days, and steady-state pharmacokinetics are reached after 1-2 months of daily dosing. The long half-life of fingolimod, together with its slow absorption, means that fingolimod has a flat concentration profile over time with once-daily dosing. Fingolimod and fingolimod phosphate show dose-proportional exposure in single- and multiple-dose studies over a range of 0.125-5 mg; hence, there is a predictable relationship between dose and systemic exposure. Furthermore, fingolimod and fingolimod phosphate exhibit low to moderate intersubject pharmacokinetic variability. Fingolimod is extensively metabolized, with biotransformation occurring via three main pathways: (i) reversible phosphorylation to fingolimod phosphate; (ii) hydroxylation and oxidation to yield a series of inactive carboxylic acid metabolites; and (iii) formation of non-polar ceramides. Fingolimod is largely cleared through metabolism by cytochrome P450 (CYP) 4F2. Since few drugs are metabolized by CYP4F2, fingolimod would be expected to have a relatively low potential for drug-drug interactions. This is supported by data from in vitro studies indicating that fingolimod and fingolimod phosphate have little or no capacity to inhibit and no capacity to induce other major drug-metabolizing CYP enzymes at therapeutically relevant steady-state blood concentrations. Population pharmacokinetic evaluations indicate that CYP3A inhibitors and CYP3A inducers have no effect or only a weak effect on the pharmacokinetics of fingolimod and fingolimod phosphate. However, blood concentrations of fingolimod and fingolimod phosphate are increased moderately when fingolimod is coadministered with ketoconazole, an inhibitor of CYP4F2. The pharmacokinetics of fingolimod are unaffected by renal impairment or mild-to-moderate hepatic impairment. However, exposure to fingolimod is increased in patients with severe hepatic impairment. No clinically relevant effects of age, sex or ethnicity on the pharmacokinetics of fingolimod have been observed. Fingolimod is thus a promising new therapy for eligible patients with MS, with a predictable pharmacokinetic profile that allows effective once-daily oral dosing.

Assessment of extracts from mistletoe ( Viscum album) for herb–drug interaction by inhibition and induction of cytochrome P450 activities

Three commercially available extracts from mistletoe (Viscum album L.) grown on ash tree (abnobaVISCUM(®) Fraxini 20 mg), on fir (abnobaVISCUM(®) Abietis 20 mg), and on pine (abnobaVISCUM(®) Pini 20 mg) were tested in vitro for their potential to interfere with the major drug metabolizing cytochromes P450 by hepatocyte viability, by inhibition of cytochromes P450 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4, and by the induction of cytochromes P450 1A2, 2B6, 2C9, 2E1 and 3A4. As the three extracts are produced from mistletoe plants belonging to three different subspecies of Viscum album L. they have explicit differences in the content and spectrum of various active ingredients, e.g. mistletoe specific lectins. Cytotoxic effects on liver cells were observed for abnobaVISCUM(®) Fraxini with a high lectin content with an EC(50) value of 2.56 µg/mL, for abnobaVISCUM(®) Abietis with a moderate lectin content with an EC(50) value of 5.79 µg/mL and for abnobaVISCUM(®) Pini with a low lectin content with an EC(50) value of 30.86 µg/mL. The induction of cytochromes P450 was tested on human liver cells from three donors. Inhibition of cytochromes P450 was carried out on human liver microsomes. No or minor induction and inhibition was observed for all three extracts. The data indicate no or minor potential for herb-drug interactions by interference with cytochromes P450 by any of the three mistletoe extracts.

Assessment of Extracts from Mistletoe (Viscum album) for Herb–Drug Interaction by Inhibition and Induction of Cytochrome P450 Activities

Three commercially available extracts from mistletoe (Viscum album L.) grown on ash tree (abnobaVISCUM(®) Fraxini 20 mg), on fir (abnobaVISCUM(®) Abietis 20 mg), and on pine (abnobaVISCUM(®) Pini 20 mg) were tested in vitro for their potential to interfere with the major drug metabolizing cytochromes P450 by hepatocyte viability, by inhibition of cytochromes P450 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4, and by the induction of cytochromes P450 1A2, 2B6, 2C9, 2E1 and 3A4. As the three extracts are produced from mistletoe plants belonging to three different subspecies of Viscum album L. they have explicit differences in the content and spectrum of various active ingredients, e.g. mistletoe specific lectins. Cytotoxic effects on liver cells were observed for abnobaVISCUM(®) Fraxini with a high lectin content with an EC(50) value of 2.56 µg/mL, for abnobaVISCUM(®) Abietis with a moderate lectin content with an EC(50) value of 5.79 µg/mL and for abnobaVISCUM(®) Pini with a low lectin content with an EC(50) value of 30.86 µg/mL. The induction of cytochromes P450 was tested on human liver cells from three donors. Inhibition of cytochromes P450 was carried out on human liver microsomes. No or minor induction and inhibition was observed for all three extracts. The data indicate no or minor potential for herb-drug interactions by interference with cytochromes P450 by any of the three mistletoe extracts.

Scaling Down of a Clinical Three-Dimensional Perfusion Multicompartment Hollow Fiber Liver Bioreactor Developed for Extracorporeal Liver Support to an Analytical Scale Device Useful for Hepatic Pharmacological In Vitro Studies

Within the scope of developing an in vitro culture model for pharmacological research on human liver functions, a three-dimensional multicompartment hollow fiber bioreactor proven to function as a clinical extracorporeal liver support system was scaled down in two steps from 800 mL to 8 mL and 2 mL bioreactors. Primary human liver cells cultured over 14 days in 800, 8, or 2 mL bioreactors exhibited comparable time-course profiles for most of the metabolic parameters in the different bioreactor size variants. Major drug-metabolizing cytochrome P450 activities analyzed in the 2 mL bioreactor were preserved over up to 23 days. Immunohistochemical studies revealed tissue-like structures of parenchymal and nonparenchymal cells in the miniaturized bioreactor, indicating physiological reorganization of the cells. Moreover, the canalicular transporters multidrug-resistance-associated protein 2, multidrug-resistance protein 1 (P-glycoprotein), and breast cancer resistance protein showed a similar distribution pattern to that found in human liver tissue. In conclusion, the down-scaled multicompartment hollow fiber technology allows stable maintenance of primary human liver cells and provides an innovative tool for pharmacological and kinetic studies of hepatic functions with small cell numbers.