[Show abstract][Hide abstract] ABSTRACT: To further the development of a model for simultaneously assessing intestinal absorption and first-pass metabolism in vitro, Caco-2, LS180, T84 and SIEC cells were cultured on permeable inserts and the integrity of cell monolayers, CYP3A4 activity and the inducibility of enzymes and transporters involved intestinal drug disposition were measured. Caco-2, T84 and SIEC cells all formed tight junctions, as assessed by immunofluorescence microscopy for zonula occludens-1 (ZO-1), which was well organized into circumscribing strands in SIEC, T84 and Caco-2 cells, but was diffuse in LS180 cells. The TEER value for LS180 monolayers was lower than that for Caco-2, T84 and SIEC. In addition, the apical-to-basolateral permeability of the paracellular marker, lucifer yellow, across LS180 monolayers was higher than in SIEC, T84 and Caco-2 monolayers. The transcellular marker, propranolol exhibited similar permeability across all cells. With regard to metabolic capacity, T84 cells and LS180 cells showed comparable basal midazolam hydroxylation activity and it was inducible by rifampin and 1α,25(OH)2D3 in LS180 cells, but only marginally so in T84 cells. The basal CYP3A4 activity of SIEC and Caco-2 cells was much lower and not inducible. Interestingly, some of the drug transporters expressed in LS180 cells and Caco-2 cells were induced by either 1α,25(OH)2D3 or rifampin or both, but there were only limited effects in the other two cell lines. These results suggest that none of the cell lines tested fully replicated the drug disposition properties of the small intestine and that the search for an ideal screening tool must continue.
Preview · Article · Dec 2015 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: The cytochrome P450 (P450) enzymes are the predominant enzyme system involved in human drug metabolism. Alterations in the expression and/or activity of these enzymes result in changes in pharmacokinetics (and consequently the pharmacodynamics) of drugs that are metabolized by this set of enzymes. Apart from changes in activity due to drug-drug interactions (by P450 induction or inhibition), the P450 enzymes can exhibit substantial interindividual variation in basal expression and/or activity, leading to differences in rates of drug elimination and response. This interindividual variation can result from a myriad of factors including genetic variation in the coding region, genetic variation in the promoter region, variation in transcriptional regulators, alterations in micro-RNA that impact P450 expression, and ontogenic changes due to exposure to xenobiotics during the developmental and early post-natal periods. Other than administering a probe drug or cocktail of drugs to obtain the phenotype or conducting a genetic analysis to determine genotype, methods to determine inter-individual variation have been limited. Phenotyping via a probe drug requires exposure to a xenobiotic, and genotyping is not always well correlated with phenotype, making both methodologies less than ideal. This manuscript describes recent work evaluating the impact of some of these factors on interindividual variation in human P450-mediated metabolism and the potential utility of endogenous probe compounds to assess rates of drug metabolism among individuals.
Preview · Article · Dec 2015 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: Background:
Low blood vitamin D concentration is a concern for people living in circumpolar regions, where sunlight is insufficient for vitamin D synthesis in winter months and the consumption of traditional dietary sources of vitamin D is decreasing.
The objective was to characterize the effects of diet, genetic variation, and season on serum 25-hydroxycholecalciferol [25(OH)D3] concentrations in Yup'ik Alaska Native people living in rural southwest Alaska.
This study was a cross-sectional design that assessed the associations of traditional diet (via a biomarker, the RBC δ(15)N value), age, gender, body mass index (BMI), community location, and genotype of select single nucleotide polymorphisms (SNPs) in cytochrome P450 family 2, subfamily R, peptide 1 (CYP2R1), 7-dehydrocholesterol reductase (DHCR7), and vitamin D binding protein (GC) with serum 25(OH)D3 concentrations in 743 Yup'ik male and female participants, aged 14-93 y, recruited between September 2009 and December 2013.
Yup'ik participants, on average, had adequate concentrations of serum 25(OH)D3 (31.1 ± 1.0 ng/mL). Variations in diet, BMI, age, gender, season of sample collection, and inland or coastal community geography were all significantly associated with serum 25(OH)D3 concentration. In models not adjusting for other covariates, age, diet, and seasonal effects explained 33.7%, 20.7%, and 9.8%, respectively, of variability in serum 25(OH)D3 concentrations. Of the 8 SNPs interrogated in CYP2R1 and DHCR7, only rs11023374 in CYP2R1 was significantly associated with serum 25(OH)D3, explaining 1.5% of variability. The GC haplotype explained an additional 2.8% of variability. Together, age, diet, gender, season of sample collection, BMI, geography of the community, and genotype at rs11023374 explained 52.5% of the variability in serum 25(OH)D3 concentrations.
Lower consumption of the traditional diet was associated with lower serum concentrations of 25(OH)D3. Younger adults and youth in this community may be at increased risk of adverse outcomes associated with vitamin D insufficiency compared with older members of the community, especially during seasons of low sunlight exposure, because of lower consumption of dietary sources of vitamin D.
No preview · Article · Dec 2015 · Journal of Nutrition
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to evaluate the steady-state pharmacokinetics of metoprolol during pregnancy and lactation. Serial plasma, urine and breast milk concentrations of metoprolol and its metabolite, α-hydroxymetoprolol, were measured over one dosing interval in women treated with metoprolol (25-750 mg/day) during early- (n = 4), mid- (n = 14) and late-pregnancy (n = 15), as well as postpartum (n = 9) with (n = 4) and without (n = 5) lactation. Subjects were genotyped for CYP2D6 loss-of-function allelic variants. Utilizing paired analysis, mean metoprolol apparent oral clearances were significantly higher in mid-pregnancy (361 ± 223 L/hr, n = 5, p < 0.05) and late-pregnancy (568 ± 273 L/hr, n = 8, p < 0.05) compared to ≥ 3 months postpartum (200 ± 131 L/hr and 192 ± 98 L/hr, respectively). When the comparison was limited to Extensive Metabolizers (EMs), metoprolol apparent oral clearance was significantly higher during both mid- and late-pregnancy (p < 0.05). Relative infant exposure to metoprolol through breast milk was < 1.0% of maternal weight-adjusted dose (n = 3). Due to the large, pregnancy-induced changes in metoprolol pharmacokinetics, if inadequate clinical responses are encountered, clinicians who prescribe metoprolol during pregnancy should be prepared to make aggressive changes in dosage (dose and frequency) or consider using an alternate β-blocker. This article is protected by copyright. All rights reserved
No preview · Article · Sep 2015 · The Journal of Clinical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Large interindividual variability has been observed in the metabolism of CYP2C19 substrates in vivo. The study aimed to evaluate sources of this variability in CYP2C19 activity, focusing on CYP2C19 diplotypes and the cytochrome P450 oxidoreductase (POR). CYP2C19 gene analysis was carried out on 347 human liver samples. CYP2C19 activity assayed using human liver microsomes confirmed a significant a priori predicted rank order for (S)-mephenytoin hydroxylase activity of CYP2C19*17/*17 > *1B/*17 > *1B/*1B > *2A/*17 > *1B/*2A > *2A/*2A diplotypes. In a multivariate analysis, the CYP2C19*2A allele and POR protein content were associated with CYP2C19 activity. Further analysis indicated a strong effect of the CYP2C19*2A, but not the *17, allele on both metabolic steps in the conversion of clopidogrel to its active metabolite. The present study demonstrates that interindividual variability in CYP2C19 activity is due to differences in both CYP2C19 protein content associated with gene diplotypes and the POR concentration.The Pharmacogenomics Journal advance online publication, 1 September 2015; doi:10.1038/tpj.2015.58.
Full-text · Article · Sep 2015 · The Pharmacogenomics Journal
[Show abstract][Hide abstract] ABSTRACT: Background and aims:
In previous analyses, we identified three dietary patterns from food frequency questionnaire data among a sample of Yup'ik Alaska Native people living in Southwest Alaska: a "subsistence foods" dietary pattern and two market-based dietary patterns "processed foods" and "fruits and vegetables". In this analysis, we aimed to characterize the association between the dietary patterns and cardiometabolic (CM) risk factors (lipids, blood pressure, glucose, adiposity).
Methods and results:
We used multilevel linear regression to estimate the mean of each CM risk factor, comparing participants in the 4th to the 1st quartile of each dietary pattern (n = 637). Models were adjusted for age, sex, past smoking, current smoking, and physical activity. Mean log triglyceride levels were significantly higher among participants in the 4th compared to the 1st quartile of the processed foods dietary pattern (β = 0.11). Mean HbA1c percent was significantly lower (β = -0.08) and mean diastolic blood pressure (DBP) mm Hg was significantly higher (β = 2.87) among participants in the 4th compared to the 1st quartile of the fruits and vegetables dietary pattern. Finally, mean log triglyceride levels and mean DBP mm Hg were significantly lower among participants in the 4th compared to the 1st quartile of the subsistence foods dietary pattern (β = -0.10 and β = -3.99 respectively).
We found increased CM risk, as reflected by increased triglycerides, associated with eating a greater frequency of processed foods, and reduced CM risk, as reflected by lower triglycerides and DBP, associated with eating a greater frequency of subsistence foods.
No preview · Article · Aug 2015 · Nutrition, metabolism, and cardiovascular diseases: NMCD
[Show abstract][Hide abstract] ABSTRACT: CYP2C19 rs12769205 alters an intron 2 branch point adenine leading to an alternative mRNA in human liver with complete inclusion of intron 2 (exon 2B). rs12769205 changes the mRNA reading frame, introduces 87 amino acids, and leads to a premature stop codon. The 1000 genomes project indicated rs12769205 is in LD with rs4244285 on CYP2C19*2, but found alone on CYP2C19*35 in Blacks. Minigenes containing rs12769205 transfected into HepG2 cells demonstrated this SNP alone leads to exon 2B and decreases CYP2C19 canonical mRNA. A residual amount of CYP2C19 protein was detectable by quantitative proteomics with tandem mass spectrometry in CYP2C19*2/*2 and *1/*35 liver microsomes with an exon 2 probe. However, an exon 4 probe, downstream of rs12769205, but upstream of rs4244285, failed to detect CYP2C19 protein in livers homozygous for rs12769205 demonstrating rs12769205 alone can lead to complete loss of CYP2C19 protein. CYP2C19 genotypes and mephenytoin phenotype were compared in 104 Ethiopians. Poor metabolism of mephenytoin was seen in persons homozygous for both rs12769205 + rs4244285 (CYP2C19*2/*2), but with little effect on mephenytoin disposition of CYP2C19*1/*2, CYP2C19*1/*3 or CYP2C19*1/*35 heterozygous alleles. Extended haplotype homozygosity tests of the Hapmap Yorubans (YRI) showed both haplotypes carrying rs12769205 (CYP2C19*35 and CYP2C19*2) are under significant natural selection, with CYP2C19*35 having a higher REHH score. The phylogenetic tree of the YRI CYP2C19 haplotypes revealed rs12769205 arose first on CYP2C19*35 and that rs4244285 was added later creating CYP2C19*2. In conclusion, rs12769205 is the ancestral polymorphism leading to aberrant splicing of CYP2C19*35 and CYP2C19*2 alleles in liver.
The American Society for Pharmacology and Experimental Therapeutics.
Full-text · Article · May 2015 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: Members of the CYP3A family of drug metabolizing enzymes exhibit developmental changes in expression in human liver characterized by a transition between CYP3A7 and CYP3A4 over the first few years of life. In contrast, the developmental expression of CYP3A5 is less well-understood due to polymorphic expression of the enzyme in human tissues as a result of the prevalence of the CYP3A5*3 allele that leads to alternative splicing. The objective of this paper was to further explore the expression of CYP3A5 and the impact of alternative splicing on the variability of CYP3A5 functional activity in a large bank of human prenatal liver samples (7 to 32 weeks of age post-conception). Expression of normally spliced CYP3A5 mRNA in all human fetal liver samples varied 235-fold, while CYP3A5 SV1 mRNA was only detected in fetal liver samples with at least one CYP3A5*3 allele. Formation of 1'-OH midazolam (MDZ) varied 79-fold, while the ratio of 1'-OH MDZ to 4-OH MDZ varied 8-fold and was dependent on the presence or absence of the CYP3A5*3 allele. Formation of 4-OH MDZ was significantly associated with 1'-OH MDZ (r(2)=0.76, p<0.0001) but varied (36-fold) independently of CYP3A5 genotype or expression. In conclusion, substantial interindividual variability that remains even after stratification for CYP3A5 genotype suggests that factors, such as environmental exposure and epigenetic factors, act in addition to genetic variation to contribute to variability of CYP3A5 expression in human prenatal liver.
The American Society for Pharmacology and Experimental Therapeutics.
No preview · Article · May 2015 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: Pharmacogenetic testing is projected to improve health outcomes and reduce the cost of care by increasing therapeutic efficacy and minimizing drug toxicity. American Indian and Alaska Native (AI/AN) people historically have been excluded from pharmacogenetic research and its potential benefits, a deficiency we sought to address. The vitamin K antagonist warfarin is prescribed for prevention of thromboembolic events, although its narrow therapeutic index and wide interindividual variability necessitate close monitoring of drug response. Therefore, we were interested in variation in CYP2C9, VKORC1, CYP4F2, CYP4F11, and GGCX, which encode enzymes important for the activity of warfarin and synthesis of vitamin K-dependent blood clotting factors.
We resequenced these genes in 188 AI/AN people in partnership with Southcentral Foundation in Anchorage, Alaska and 94 Yup'ik people living in the Yukon-Kuskokwim Delta of southwest Alaska to identify known or novel function-disrupting variation. We conducted genotyping for specific single nucleotide polymorphisms in larger cohorts of each study population (380 and 350, respectively).
We identified high frequencies of the lower-warfarin dose VKORC1 haplotype (-1639G>A and 1173C>T) and the higher-warfarin dose CYP4F2*3 variant. We also identified two relatively common, novel, and potentially function-disrupting variants in CYP2C9 (M1L and N218I), which, along with CYP2C9*3, CYP2C9*2, and CYP2C9*29, predict that a significant proportion of AI/AN people will have decreased CYP2C9 activity.
Overall, we predict a lower average warfarin dose requirement in AI/AN populations in Alaska than that seen in non-AI/AN populations of the USA, a finding consistent with clinical experience in Alaska.
No preview · Article · May 2015 · Pharmacogenetics and Genomics
[Show abstract][Hide abstract] ABSTRACT: Purpose Telaprevir inhibits CYP3A resulting in drug-drug interactions (DDI) of unprecedented magnitude. We investigated the mechanisms by which telaprevir inhibits the oxidation of midazolam and tacrolimus in human liver microsomes (HLM). Methods We performed a static mechanistic DDI prediction to evaluate whether previously reported competitive inhibition of CYP3A by telaprevir and its diastereomeric metabolite - VRT-127394 is sufficient to explain the remarkable reduction in oral clearance observed with oral midazolam and tacrolimus. To further explore the inhibitory mechanisms of telaprevir, we assessed whether telaprevir-mediated inhibition of the oxidation of midazolam and tacrolimus is time-dependent in human liver microsomes, and whether any observed time-dependency was irreversible or reversible in nature. Results The competitive inhibition model failed to account for the magnitude of telaprevir interactions in human subjects. In comparing HLM incubations with and without a prior 30-min exposure to telaprevir, a respective 4- and 11-fold reduction in IC50 was observed with midazolam and tacrolimus as substrates. This time-dependent inhibition was shown to be NADPH-dependent. Upon dilution of microsomes following pre-incubation with telaprevir, time-dependent inhibition of midazolam metabolism was completely reversed, whereas partial reversal occurred with tacrolimus. Conclusions The interaction between telaprevir and midazolam or tacrolimus involves both competitive and time-dependent inhibition. The time-dependent component is not explained by irreversible inactivation of CYP3A. Formation of potent inhibitory metabolites may contribute to the remarkable in vivo inhibitory potency of telaprevir. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
No preview · Article · Mar 2015 · Journal of Pharmacy and Pharmaceutical Sciences
[Show abstract][Hide abstract] ABSTRACT: FFQ data can be used to characterise dietary patterns for diet-disease association studies. In the present study, we evaluated three previously defined dietary patterns - 'subsistence foods', market-based 'processed foods' and 'fruits and vegetables' - among a sample of Yup'ik people from Southwest Alaska. We tested the reproducibility and reliability of the dietary patterns, as well as the associations of these patterns with dietary biomarkers and participant characteristics. We analysed data from adult study participants who completed at least one FFQ with the Center for Alaska Native Health Research 9/2009-5/2013. To test the reproducibility of the dietary patterns, we conducted a confirmatory factor analysis (CFA) of a hypothesised model using eighteen food items to measure the dietary patterns (n 272). To test the reliability of the dietary patterns, we used the CFA to measure composite reliability (n 272) and intra-class correlation coefficients for test-retest reliability (n 113). Finally, to test the associations, we used linear regression (n 637). All factor loadings, except one, in CFA indicated acceptable correlations between foods and dietary patterns (r>0·40), and model-fit criteria were >0·90. Composite and test-retest reliability of the dietary patterns were, respectively, 0·56 and 0·34 for 'subsistence foods', 0·73 and 0·66 for 'processed foods', and 0·72 and 0·54 for 'fruits and vegetables'. In the multi-predictor analysis, the dietary patterns were significantly associated with dietary biomarkers, community location, age, sex and self-reported lifestyle. This analysis confirmed the reproducibility and reliability of the dietary patterns in the present study population. These dietary patterns can be used for future research and development of dietary interventions in this underserved population.
No preview · Article · Feb 2015 · British Journal Of Nutrition
[Show abstract][Hide abstract] ABSTRACT: We sought to discover endogenous urinary biomarkers of human CYP2D6 activity.
Healthy pediatric subjects (n = 189) were phenotyped using dextromethorphan and randomized for candidate biomarker selection and validation. Global urinary metabolomics was performed using liquid chromatography quadrupole time-of-flight mass spectrometry. Candidate biomarkers were tested in adults receiving fluoxetine, a CYP2D6 inhibitor.
A biomarker, M1 (m/z 444.3102) was correlated with CYP2D6 activity in both the pediatric training and validation sets. Poor metabolizers had undetectable levels of M1, whereas it was present in subjects with other phenotypes. In adult subjects, a 9.56-fold decrease in M1 abundance was observed during CYP2D6 inhibition.
Identification and validation of M1 may provide a noninvasive means of CYP2D6 phenotyping.
No preview · Article · Dec 2014 · Pharmacogenomics
[Show abstract][Hide abstract] ABSTRACT: Glyburide is commonly prescribed for the treatment of gestational diabetes mellitus; however, fetal exposure to glyburide is not well understood and may have short- and long-term consequences for the health of the child. Glyburide can cross the placenta; fetal concentrations at term are nearly comparable to maternal levels. Whether or not glyburide is metabolized in the fetus and by what mechanisms has yet to be determined. In this study, we determined the kinetic parameters for glyburide depletion by CYP3A isoenzymes; characterized glyburide metabolism by human fetal liver tissues collected during the first or early second trimester of pregnancy; and identified the major enzyme responsible for glyburide metabolism in human fetal livers. CYP3A4 had the highest metabolic capacity towards glyburide, followed by CYP3A7 and CYP3A5 (Clint,u = 37.1, 13.0, and 8.7 ml/min/nmol P450, respectively). M5 was the predominant metabolite generated by CYP3A7 and human fetal liver microsomes (HFLMs) with approximately 96% relative abundance. M5 was also the dominant metabolite generated by CYP3A4, CYP3A5, and adult liver microsomes; however, M1-M4 were also present, with up to 15% relative abundance. CYP3A7 protein levels in HFLMs were highly correlated with glyburide Clint, 16α-OH DHEA formation, and 4′-OH midazolam formation. Likewise, glyburide Clint was highly correlated with 16α-OH DHEA formation. Fetal demographics as well as CYP3A5 and CYP3A7 genotype did not alter CYP3A7 protein levels or glyburide Clint. These results indicate that human fetal livers metabolize glyburide predominantly to M5 and that CYP3A7 is the major enzyme responsible for glyburide metabolism in human fetal livers.
No preview · Article · Oct 2014 · Biochemical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Gestational diabetes mellitus is a major complication of human pregnancy. The oral clearance (CL) of glyburide, an oral anti-diabetic drug, increases 2-fold in pregnant women during late gestation versus non-pregnant controls. In this study, we examined gestational age-dependent changes in maternal-fetal pharmacokinetics (PK) of glyburide and metabolites in a pregnant mouse model. Non-pregnant and pregnant FVB mice were given glyburide by retro-orbital injection. Maternal plasma was collected over 240 min on gestation days (gd) 0, 7.5, 10, 15 and 19; fetuses were colleceted on gd 15 and 19. Glyburide and metabolites were quantified using HPLC-MS, and PK analyses were performed using a pooled data bootstrap approach. Maternal CL of glyburide increased ≈2-fold on gd 10, 15, and 19 compared to non-pregnant controls. CLint of glyburide in maternal liver microsomes also increased as gestation progressed. Maternal metabolite/glyburide AUC ratios were generally unchanged or slightly decreased throughout gestation. Total fetal exposure to glyburide was < 5% of maternal plasma exposure, and was doubled on gd 19 versus gd 15. Fetal metabolite concentrations were below the limit of assay detection. This is the first evidence of gestational age-dependent changes in glyburide PK. Increased maternal glyburide clearance during gestation is attributable to increased hepatic metabolism. Metabolite elimination may also increase during pregnancy. In the mouse model, fetal exposure to glyburide is gestational age-dependent and low compared to maternal plasma exposure. These results indicate that maternal glyburide therapeutic strategies may require adjustments in a gestational-age dependent manner if these same changes occur in humans.
Preview · Article · Jun 2014 · Journal of Pharmacology and Experimental Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Pharmacogenetics is a subset of personalized medicine that applies knowledge about genetic variation in gene-drug pairs to help guide optimal dosing. There is a lack of data, however, about pharmacogenetic variation in underserved populations. One strategy for increasing participation of underserved populations in pharmacogenetic research is to include communities in the research process. We have established academic-community partnerships with American Indian and Alaska Native people living in Alaska and Montana to study pharmacogenetics. Key features of the partnership include community oversight of the project, research objectives that address community health priorities, and bidirectional learning that builds capacity in both the community and the research team. Engaging the community as coresearchers can help build trust to advance pharmacogenetic research objectives.
No preview · Article · Jun 2014 · Pharmacogenomics
[Show abstract][Hide abstract] ABSTRACT: Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors, have proven efficacy in both lowering low-density-lipoprotein levels and preventing major coronary events, making them one of the most commonly prescribed drugs in the United States. Statins exhibit a class-wide side effect of muscle toxicity and weakness, which has led regulators to impose both dosage limitations and a recall. This review focuses on the best-characterized genetic factors associated with increased statin muscle concentrations, including the genes encoding cytochrome P450 enzymes (CYP2D6, CYP3A4, and CYP3A5), a mitochondrial enzyme (GATM), an influx transporter (SLCO1B1), and efflux transporters (ABCB1 and ABCG2). A systematic literature review was conducted to identify relevant research evaluating the significance of genetic variants predictive of altered statin concentrations and subsequent statin-related myopathy. Studies eligible for inclusion must have incorporated genotype information and must have associated it with some measure of myopathy, either creatine kinase levels or self-reported muscle aches and pains. After an initial review, focus was placed on seven genes that were adequately characterized to provide a substantive review: CYP2D6, CYP3A4, CYP3A5, GATM, SLCO1B1, ABCB1, and ABCG2. All statins were included in this review. Among the genetic factors evaluated, statin-related myopathy appears to be most strongly associated with variants in SLCO1B1.Genet Med advance online publication 8 May 2014Genetics in Medicine (2014); doi:10.1038/gim.2014.41.
No preview · Article · May 2014 · Genetics in medicine: official journal of the American College of Medical Genetics