Therapeutic efficacy of generic artemether-lumefantrine in the treatment of uncomplicated malaria in Ghana: assessing anti-malarial efficacy amidst pharmacogenetic variations.

BACKGROUND: Despite efforts made to reduce morbidity and mortality associated with malaria, especially in sub-Saharan Africa, malaria continues to be a public health concern that requires innovative efforts to reach the WHO-set zero malaria agenda. Among the innovations is the use of artemisinin-based combination therapy (ACT) that is effective against Plasmodium falciparum. Generic artemether-lumefantrine (AL) is used to treat uncomplicated malaria after appropriate diagnosis. AL is metabolized by the cytochrome P450 family of enzymes, such as CYP2B6, CYP3A4 and CYP3A5, which can be under pharmacogenetic influence. Pharmacogenetics affecting AL metabolism, significantly influence the overall anti-malarial activity leading to variable therapeutic efficacy. This study focused on generic AL drugs used in malarial treatment as prescribed at health facilities and evaluated pharmacogenomic influences on their efficacy. METHODS: Patients who have been diagnosed with malaria and confirmed through RDT and microscopy were recruited in this study. Blood samples were taken on days 1, 2, 3 and 7 for parasite count and blood levels of lumefantrine, artemisinin, desbutyl-lumefantrine (DBL), and dihydroartemisinin (DHA), the active metabolites of lumefantrine and artemether, respectively, were analysed using established methods. Pharmacogene variation analysis was undertaken using iPLEX microarray and PCR-RFLP. RESULTS: A total of 52 patients completed the study. Median parasite density from day 1 to 7 ranged from 0-2666/µL of blood, with days 3 and 7 recording 0 parasite density. Highest median plasma concentration for lumefantrine and desbutyl lumefantrine, which are the long-acting components of artemisinin-based combinations, was 4123.75 ng/mL and 35.87 ng/mL, respectively. Day 7 plasma lumefantrine concentration across all generic ACT brands was ≥ 200 ng/mL which potentially accounted for the parasitaemia profile observed. Monomorphism was observed for CYP3A4 variants, while there were observed variations in CYP2B6 and CYP3A5 alleles. Among the CYP3A5 genotypes, significant differences in genotypes and plasma concentration for DBL were seen on day 3 between 1/*1 versus *1/*6 (p = 0.002), *1/*3 versus *1/*6 (p = 0.006) and *1/*7 versus *1/*6 (p = 0.008). Day 7 plasma DBL concentrations showed a significant difference between *1/*6 and *1/*3 (p = 0.026) expressors. CONCLUSIONS: The study findings show that CYP2B6 and CYP3A5 pharmacogenetic variations may lead to higher plasma exposure of AL metabolites.

CYP2B6 and ABCB1 genotypes predict methadone plasma exposure among patients on maintenance therapy against opioid addictions in Tanzania.

AIMS: Methadone maintenance therapy (MMT) exhibits significant variability in pharmacokinetics and clinical response, partly due to genetic variations. However, data from sub-Saharan African populations are lacking. We examined plasma methadone variability and pharmacogenetic influences among opioid-addicted Tanzanian patients. METHODS: Patients attending MMT clinics (n = 119) in Tanzania were genotyped for common functional variants of the CYP3A4, CYP3A5, CYP2A6, CYP2B6, CYP2C19, CYP2D6, ABCB1, UGT2B7 and SLCO1B1 genotypes. Trough plasma concentrations of total methadone, S-methadone (S-MTD) and R-methadone (R-MTD), with their respective metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The methadone-to-EDDP metabolic ratio (MMR) was used to categorize the phenotype. RESULTS: The proportions of MMR-predicted ultrarapid, extensive, intermediate and slow methadone metabolizer phenotypes were 2.5%, 58.2%, 23.7% and 15.6%, respectively. CYP2B6 genotype significantly correlated with S-methadone (P = .006), total methadone (P = .03), and dose-normalized methadone plasma concentrations (P = .001). Metabolic ratios of R-methadone (R-MTD/R-EDDP), S-methadone (S-MTD/S-EDDP), and total methadone (MMR) were significantly higher among patients homozygous for defective variants (*6 or *18) than heterozygous or CYP2B6*1/*1 genotypes (P < .001). The metabolic ratio for S-MTD and total methadone was significantly higher among ABCB1c.3435T/T than in the C/C genotype. No significant effect of CYP2D6, CYP2C19, CYP3A4, CYP3A5, CYP2A6, UGT2B7 and SLCO1B1 genotypes on S-methadone, R-methadone, or total methadone was observed. CONCLUSIONS: Approximately one in six opioid-addicted Tanzanian patients are methadone slow metabolizers, influenced by genetic factors. Both the CYP2B6 and ABCB1 genotypes are strong predictors of methadone metabolic capacity and plasma exposure. Further investigation is needed to determine their predictive value for methadone treatment outcomes and to develop genotype-based dosing algorithms for safe and effective therapy.

Assessment of cytochrome P450 induction in canine intestinal organoid models.

Understanding cytochrome P450 (CYP) enzymes in the canine intestine is vital for predicting drug metabolism and developing safer oral medications. This study evaluates canine colonoids as a model to assess the expression and induction of essential intestinal CYP enzymes.Canine colonoids were cultured in expansion medium (EM) with Wnt-3A and in differentiation medium (DM) without Wnt-3A. We assessed the mRNA expression of CYP2B11, CYP2C21, CYP3A12, and CYP3A98 using qPCR and examined the effects of rifampicin and phenobarbital as inducers.Our findings show that DM significantly increased the mRNA expression of CYP3A98 and CYP2B11, but not CYP3A12, compared to EM. CYP2C21, not typically expressed in the intestine, remained unexpressed in colonoids. Rifampicin induced CYP3A98, aligning with pregnane x receptor (PXR) regulation, while phenobarbital did not, suggesting no constitutive androstane receptor (CAR) involvement. CYP2B11 did not respond to either inducer, suggesting alternative regulatory pathways in canine colonoids.This study is a pioneering effort to establish conditions for studying P450 expression in canine colonoids, confirming significant CYP3A98 expression in the canine intestine. It demonstrated colonoids can induce CYP activity post drug treatments. Further research is needed to enhance species-specific drug metabolism understanding and validate this model for broader applications.

Mechanistic Insights of Neuroprotective Efficacy of Verapamil-Loaded Carbon Quantum Dots against LPS-Induced Neurotoxicity in Rats.

Alzheimer's disease (AD) is a neurodegenerative disease that badly impacts patients and their caregivers. AD is characterized by deposition of amyloid beta (Aß) and phosphorylated tau protein (pTau) in the brain with underlying neuroinflammation. We aimed to develop a neuroprotective paradigm by loading verapamil (VRH) into hyaluronic acid-modified carbon quantum dots (CQDs) and comparing its effectiveness with the free form in an AD-like model in rats induced by lipopolysaccharide (LPS). The experimental rats were divided into seven groups: control, LPS, CQDs, early free VRH (FVRH), late FVRH, early verapamil carbon quantum dots (VCQDs), and late VCQDs. Characterizations of VCQDs, the behavioral performance of the rats, histopathological and immunohistochemical changes, some AD hallmarks, oxidative stress biomarkers, neuro-affecting genes, and DNA fragmentation were determined. VRH was successfully loaded into CQDs, which was confirmed by the measured parameters. VRH showed enhancement in cognitive functions, disruption to the architecture of the brain, decreased Aß and pTau, increased antioxidant capacity, modifiable expression of genes, and a decline in DNA fragmentation. The loaded therapy was superior to the free drug. Moreover, the early intervention was better than the late, confirming the implication of the detected molecular targets in the development of AD. VRH showed multifaceted mechanisms in combating LPS-induced neurotoxicity through its anti-inflammatory and antioxidant properties, thereby mitigating the hallmarks of AD. Additionally, the synthesized nanosystem approach exhibited superior neuroprotection owing to the advantages offered by CQDs. However, finding new actionable biomarkers and molecular targets is of decisive importance to improve the outcomes for patients with AD.

Association Between CYP2B6 Polymorphisms and Efficacy of Clopidogrel in Minor Stroke or Transient Ischemic Attack.

BACKGROUND: CYP2B6 (cytochrome P450 subfamily IIB polypeptide 6), encoded by the CYP2B6 gene, is a critical enzyme involved in clopidogrel metabolism. However, the association between CYP2B6 polymorphisms and the efficacy of clopidogrel in minor stroke or transient ischemic attack for secondary stroke prevention remains unclear. METHODS: Based on CHANCE (Clopidogrel in High-Risk Patients With Acute Nondisabling Cerebrovascular Events) randomized clinical trial of aspirin plus clopidogrel versus aspirin alone, we investigated the role of CYP2B6 polymorphisms and the efficacy of clopidogrel in patients with minor stroke or transient ischemic attack in China from October 2009 to July 2012. A total of 2853 patients were successfully genotyped for CYP2B6-516G>T, rs3745274 and CYP2B6-1456 T>C, rs2054675. The primary efficacy and safety outcomes were new stroke and any bleeding within 90 days. RESULTS: Among the 2853 patients, 32.8% were identified as the carriers of the CYP2B6-516 GT/TT or -1456 TC/CC genotype. The incidences of 90-day new stroke in aspirin plus clopidogrel and aspirin alone groups were 7.1% versus 11.3% among noncarriers, respectively; and 9.7% versus 12.2% among carriers, respectively. The efficacy of aspirin plus clopidogrel versus aspirin alone was not significantly different (P interaction=0.29) in noncarriers (adjusted hazard ratio, 0.61 [95% CI, 0.45-0.83]) compared to carriers (adjusted hazard ratio, 0.80 [95% CI, 0.54-1.18]). The incidence (n=51) of 90-day any bleeding in aspirin plus clopidogrel and aspirin alone groups were 2.2% (21 bleeds) versus 1.9% (18 bleeds) among noncarriers (adjusted hazard ratio, 1.11 [95% CI, 0.59-2.09]) and 1.9% (9 bleeds) versus 0.7% (3 bleeds) among carriers (adjusted hazard ratio, 3.23 [95% CI, 0.86-12.12]). Similar findings were observed during the 1-year follow-up. CONCLUSIONS: In this post hoc analysis of the CHANCE trial, we did not observe a significant difference in the efficacy of aspirin plus clopidogrel compared with aspirin in carriers versus noncarriers of CYP2B6-516 GT/TT or -1456 TC/CC genotype. Our results suggest that both carriers and noncarriers suffering from a minor stroke are likely to benefit from aspirin plus clopidogrel treatment over aspirin monotherapy for secondary prevention. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT00979589.

An mRNA Profiling Study of Vaginal Swabs from Pre- and Postmenopausal Women.

Body fluid identification by means of mRNA profiling provides valuable supplementary information in forensic investigations. In particular, the detection of vaginal mucosa mRNA markers is highly relevant in sexual assault cases. Although the vagina undergoes characteristic age-related physiological changes over a lifetime, few studies have evaluated the efficacy of vaginal mRNA markers in women of different ages. In this multicentric study, a 19-plex mRNA profiling assay including vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of 6-20-month-old vaginal swabs obtained from pre- (n = 84) and postmenopausal (n = 55) female volunteer donors. Overall, participating laboratories were able to correctly identify ~85% of samples as vaginal mucosa by mRNA profiling. The assay's success rate did not differ between the two age groups and was not affected by the time interval between swab collection and RNA analysis. MYOZ1 resulted a less sensitive vaginal marker compared to MUC4 and CYP2B7P1. A significant relative increase in the contribution to the total amplification signal was observed for MUC4, compared to CYP2B7P1 and MYOZ1, in postmenopausal women. Observation of other body fluids and tissues different from vaginal mucosa was also evaluated in connection to information on previous sexual activity and menstrual cycle phase at the time of sampling.

Genetic polymorphisms of CYP2B6 is a risk of metabolic associated fatty liver disease in Chinese population.

BACKGROUND: The expression and activity of cytochrome P450 2B6 (CYP2B6) may be related to the metabolic associated fat liver disease (MAFLD). Since constitutive androstane receptor (CAR) is a classic transcriptional regulator of CYP2B6, and the single nucleotide polymorphisms (SNPs) of CYP2B6 and CAR are both associated with adverse reactions of efavirenz, we hypothesized that genetic polymorphisms of CAR might also result in additional interindividual variability in CYP2B6. This study was devoted to explore the association between CYP2B6 and CAR SNPs and susceptibility to MAFLD. MATERIALS AND METHODS: A total of 590 objects of study (118 with MAFLD and 472 healthy control) between December 2014 and April 2018 were retrospectively enrolled. Twenty-two selected SNPs in CYP2B6 and CAR were genotyped with a custom-designed 48-plex SNP Scan TM® Kit. The frequencies of the alleles, genotypes and genetic models of the variants were compared between the two groups. The odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) were calculated. RESULTS: The T allele of rs3745274 in CYP2B6 was associated with a decreased risk for MAFLD (OR 0.610; 95% CI: 0.451-0.825, p = 0.001) which was still statistically significant after adjusting with Bonferroni method(p = 0.014) The allele, genotype and genetic model frequencies were similar in the two groups for the other twenty-one SNPs (all P > 0.05). There were no multiplicative or additive interactions between the SNPs. CONCLUSION: Our study revealed that rs3745274 variants in CYP2B6 is associated with susceptibility to MAFLD in the Han Chinese population.

Metabolic ratios and SNPs implicated in tramadol-related deaths.

Tramadol (TR) metabolism is performed by polymorphic enzymes that are influenced by genetic polymorphisms. Within this scope, the study presented here aimed to describe 41 genetic variants within CYP2D6, CYP2B6, and CYP3A4 genes in 48 cases of TR-related death that may be involved in the response to TR and to assess whether there is a correlation between these genetic variants and metabolic ratios (MRs). Blood samples from 48 victims of a TR-related death were analyzed to determine the concentrations of TR and its metabolites [O-desmethyltramadol (M1) & N-desmethyltramadol (M2)] using a LC-MS/MS method. All the samples were also genotyped for 41 common CYP2D6, CYP2B6, and CYP3A4 single nucleotide polymorphisms (SNPs) using the HaloPlex Target Enrichment system. Cases with the T/- genotype (rs35742686 in CYP2D6) had significantly higher M2/M1 ratio than cases with T/T genotype and cases with the G/A genotype (rs35599367 in CYP3A4) had significantly higher MR2 (TR/M2) ratio than cases with G/G genotype. The frequency of tested SNPs which belong to CYP2D6, CYP2B6, and CYP3A4 revealed the over-presentation of 2 SNPs (rs1058172 in CYP2D6 and rs4803419 in CYP2B6) in TR overdose group, which could have toxicological implications. These results indicate these polymorphisms in CYP2D6, CYP2B6, and CYP3A4 might influence the function and could increase the risk of toxicity. However, these findings should be supported in future studies with larger groups of cases.

Relating CYP2B6 genotype and efavirenz resistance among post-partum women living with HIV with high viremia in Uganda: a nested cross-sectional study.

BACKGROUND: We investigated the association between CYP2B6 polymorphisms and efavirenz drug resistance among women living with HIV who started on antiretroviral therapy during pregnancy and with high viremia during post-partum. METHODS: This was a cross-sectional study of women with viral loads greater than 1000 copies/ml who were at least 6 weeks postpartum. Sanger sequencing was used to detect resistant mutations, as well as host genotyping, and efavirenz resistance was compared among the metabolizer genotypes. RESULTS: Over the course of one year (July 2017-July 2018), 322 women were screened, with 110 (34.2%) having viral loads of 1000 copies/ml and 62 having whole blood available for genotyping. Fifty-nine of these women had both viral resistance and human host genotypic results. Efavirenz resistance according to metabolizer genotype was; 47% in slow, 34% in extensive and 28% in intermediate metabolizers, but the difference was not statistically significant due to the small sample size. CONCLUSIONS: There was no statistically significant difference in EFV resistance between EFV metabolizer genotypes in women who started antiretroviral therapy during pregnancy and had high viremia in the postpartum period. However, a numerical trend was discovered, which calls for confirmation in a large, well-designed, statistically powered study.

Association of CYP3A5*3, CYP3A4*18 & CYP2B6*6 polymorphisms with imatinib treatment outcome in Azerbaijani chronic myeloid leukaemia patients.

Background & objectives: Imatinib mesylate (IM) is a reliable first line treatment for chronic myeloid leukaemia (CML). Nevertheless, despite promising results, a considerable proportion of patients develop resistance to the drug. Cytochrome P450 (CYP) enzymes play a crucial role in IM metabolism. Thus, point mutations in CYP genes may modify IM enzyme activity resulting in insufficient treatment response. This investigation was aimed to identify the functional impact of CYP3A5*3, CYP3A4*18 and CYP2B6*6 polymorphisms on the IM response in patients with CML in Azerbaijan. Methods: Genotyping of CYP3A5*3, CYP3A4*18 and CYP2B6*6 was performed in 153 patients (102 IM non-responders and 51 IM responders) with CML by the PCR-restriction fragment length polymorphism (RFLP) assays. The odds ratios (ORs) with 95 per cent confidence intervals (CIs) were applied to assess the association between allelic variants and IM therapy outcome. The results were validated by sequencing. Results: The frequency of the CYP3A4*18 allele was considerably lower in the responder's group (97.1 vs. 100%; P=0.036). For CYP3A5*3, the allelic frequency was slightly higher among the IM responders (100 vs. 99.02%) with no significant difference. Although patients heterozygous (TC) for CYP2B6*6 demonstrated a higher risk of acquiring resistance (OR 1.04; 95% CI: 0.492-2.218), differences were not significant (P=0.909). In addition, the homozygous genotype (TT) demonstrated a lower risk of unresponsiveness (OR 0.72; 95% CI: 0.283-1.836), but associations were not significant (P=0.491). Interpretation & conclusions: Our results demonstrated that CYP3A4*18 was significantly associated with IM treatment response in patients with CML in Azerbaijan, whereas rather common CYP3A5*3 was identified to have no such association.

Expression of CYP2B6 Enzyme in Human Liver Tissue of HIV and HCV Patients.

Background and Objectives: Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections present significant public health challenges worldwide. The management of these infections is complicated by the need for antiviral and antiretroviral therapies, which are influenced by drug metabolism mediated by metabolic enzymes and transporters. This study focuses on the gene expression of CYP2B6, CYP3A4, and ABCB1 transporters in patients with HIV, HCV, and HIV/HCV co-infection, aiming to assess their potential association with the choice of therapy, patohistological and clinical parameters of liver damage such as the stage of liver fibrosis, serum levels of ALT and AST, as well as the grade of liver inflammation and other available biochemical parameters. Materials and Methods: The study included 54 patients who underwent liver biopsy, divided into HIV-infected, HCV-infected, and co-infected groups. The mRNA levels of CYP2B6, CYP3A4, and ABCB1 was quantified and compared between the groups, along with the analysis of liver fibrosis and inflammation levels. Results: The results indicated a significant increase in CYP2B6 mRNA levels in co-infected patients, a significant association with the presence of HIV infection with an increase in CYP3A4 mRNA levels. A trend towards downregulation of ABCB1 expression was observed in patients using lamivudine. Conclusions: This study provides insight into gene expression of CYP2B6 CYP3A4, and ABCB1 in HIV, HCV, and HIV/HCV co-infected patients. The absence of correlation with liver damage, inflammation, and specific treatment interventions emphasises the need for additional research to elucidate the complex interplay between gene expression, viral co-infection, liver pathology, and therapeutic responses in these particular patients population.

Amphipol-facilitated elucidation of the functional tetrameric complex of full-length cytochrome P450 CYP2B4 and NADPH-cytochrome P450 oxidoreductase.

Interactions of membrane-bound mammalian cytochromes P450 (CYPs) with NADPH-cytochrome P450 oxidoreductase (POR), which are required for metabolism of xenobiotics, are facilitated by membrane lipids. A variety of membrane mimetics, such as phospholipid liposomes and nanodiscs, have been used to simulate the membrane to form catalytically active CYP:POR complexes. However, the exact mechanism(s) of these interactions are unclear because of the absence of structural information of full-length mammalian CYP:POR complexes in membranes. Herein, we report the use of amphipols (APols) to form a fully functional, soluble, homogeneous preparation of full-length CYP:POR complexes amenable to biochemical and structural study. Incorporation of CYP2B4 and POR into APols resulted in a CYP2B4:POR complex with a stoichiometry of 1:1, which was fully functional in demethylating benzphetamine at a turnover rate of 37.7 ± 2.2 min-1, with a coupling efficiency of 40%. Interestingly, the stable complex had a molecular weight (Mw) of 338 ± 22 kDa determined by multiangle light scattering, suggestive of a tetrameric complex of 2CYP2B4:2POR embedded in one APol nanoparticle. Moreover, negative stain electron microscopy (EM) validated the homogeneity of the complex and allowed us to generate a three-dimensional EM map and model consistent with the tetramer observed in solution. This first report of the full-length mammalian CYP:POR complex by transmission EM not only reveals the architecture that facilitates electron transfer but also highlights a potential use of APols in biochemical and structural studies of functional CYP complexes with redox partners.

Through a glass, darkly? HepaRG and HepG2 cells as models of human phase I drug metabolism.

The pharmacokinetic and safety assessment of drug candidates is becoming increasingly dependent upon in vitro models of hepatic metabolism and toxicity. Predominant among these is the HepG2 cell line, although HepaRG is becoming increasingly popular because of its perceived closer resemblance to human hepatocytes. We review the functionality of these cell lines in terms of Phase I protein expression, basal cytochrome P450-dependent activity, and utility in P450 induction studies. Our analysis indicates that HepG2 cells are severely compromised: proteomic studies show that they express few key proteins in common with hepatocytes and they lack drug-metabolizing capacity. Differentiated HepaRGs are more hepatocyte-like than HepG2s, but they also have limitations, and it is difficult to assess their utility because of the enormous variability in data reported, possibly arising from the complex differentiation protocols required to obtain hepatocyte-like cells. This is exacerbated by the use of DMSO in the induction protocol, together with proprietary supplements whose composition is a commercial secret. We conclude that, while currently available data on the utility of HepaRG generates a confusing picture, this line does have potential utility in drug metabolism studies. However, to allow studies to be compared directly a standardized, reproducible differentiation protocol is essential and the cell line's functionality in terms of known mechanisms of P450 regulation must be demonstrated. We, therefore, support the development of regulatory guidelines for the use of HepaRGs in induction studies as a first step in generating a database of consistent, reliable data.

Hepatic CYP2B10 is highly induced by binge ethanol and contributes to acute-on-chronic alcohol-induced liver injury.

BACKGROUND: The chronic-plus-binge model of ethanol consumption, where chronically (8-week) ethanol-fed mice are gavaged a single dose of ethanol (E8G1), is known to induce steatohepatitis in mice. However, how chronically ethanol-fed mice respond to multiple binges of ethanol remains unknown. METHODS: We extended the E8G1 model to three gavages of ethanol (E8G3) spaced 24 h apart, sacrificed each group 9 h after the final gavage, analyzed liver injury, and examined gene expression changes using microarray analyses in each group to identify mechanisms contributing to liver responses to binge ethanol. RESULTS: Surprisingly, E8G3 treatment induced lower levels of liver injury, steatosis, inflammation, and fibrosis as compared to mice after E8G1 treatment. Microarray analyses identified several pathways that may contribute to the reduced liver injury after E8G3 treatment compared to E8G1 treatment. The gene encoding cytochrome P450 2B10 (Cyp2b10) was one of the top upregulated genes in the E8G1 group and was further upregulated in the E8G3 group, but only moderately induced after chronic ethanol consumption, as confirmed by RT-qPCR and western blot analyses. Genetic disruption of Cyp2b10 worsened liver injury in E8G1 and E8G3 mice with higher blood ethanol levels compared to wild-type control mice, while in vitro experiments revealed that CYP2b10 did not directly promote ethanol metabolism. Metabolomic analyses revealed significant differences in hepatic metabolites from E8G1-treated Cyp2b10 knockout and WT mice, and these metabolic alterations may contribute to the reduced liver injury in Cyp2b10 knockout mice. CONCLUSION: Hepatic Cyp2b10 expression is highly induced after ethanol binge, and such upregulation reduces acute-on-chronic ethanol-induced liver injury via the indirect modification of ethanol metabolism.

Methadone pharmacogenetics in vitro and in vivo: Metabolism by CYP2B6 polymorphic variants and genetic variability in paediatric disposition.

AIMS: Methadone metabolism and clearance are determined principally by polymorphic cytochrome P4502B6 (CYP2B6). Some CYP2B6 allelic variants affect methadone metabolism in vitro and disposition in vivo. We assessed methadone metabolism by CYP2B6 minor variants in vitro. We also assessed the influence of CYP2B6 variants, and P450 oxidoreductase (POR) and CYP2C19 variants, on methadone clearance in surgical patients in vivo. METHODS: CYP2B6 and P450 oxidoreductase variants were coexpressed with cytochrome b5 . The metabolism of methadone racemate and enantiomers was measured at therapeutic concentrations and intrinsic clearances were determined. Adolescents receiving methadone for surgery were genotyped for CYP2B6, CYP2C19 and POR, and methadone clearance and metabolite formation clearance were determined. RESULTS: In vitro, CYP2B6.4 was more active than wild-type CYP2B6.1. CYPs 2B6.5, 2B6.6, 2B6.7, 2B6.9, 2B6.17, 2B6.19 and 2B6.26 were less active. CYPs 2B6.16 and 2B6.18 were inactive. CYP2B6.1 expressed with POR variants POR.28, POR.5 and P228L had lower rates of methadone metabolism than wild-type reductase. In vivo, methadone clinical clearance decreased linearly with the number of CYP2B6 slow metabolizer alleles, but was not different in CYP2C19 slow or rapid metabolizer phenotypes, or in carriers of the POR*28 allele. CONCLUSIONS: Several CYP2B6 and POR variants were slow metabolizers of methadone in vitro. Polymorphisms in CYP2B6, but not CYP2C19 or P450 reductase, affected methadone clearance in vivo. CYP2B6 polymorphisms 516G>T and 983T>C code for canonical loss of function variants and should be assessed when considering genetic influences on clinical methadone disposition. These complementary translational in vitro and in vivo results inform on pharmacogenetic variability affecting methadone disposition in patients.

Pharmacogenetic predictors of variability in efavirenz pharmacokinetics in an admixed Brazilian HIV cohort.

AIMS: To investigate the influence of pharmacogenetic polymorphisms on efavirenz (EFV) exposure and metabolism in HIV-infected Brazilians under treatment with EFV-containing antiretroviral (ART) regimens. METHODS: HIV-positive adults (n = 82) on stable ART regimens containing 600 mg EFV once daily for at least 6 months were recruited at 2 university hospitals. Blood samples collected at mid-dose interval were used to quantify the plasma concentrations of EFV (denoted [EFV]), its major metabolite 8-OH-EFV ([8-OH-EFV]) and [8-OH-EFV]/[EFV] metabolic ratio, and to genotype single nucleotide polymorphisms in CYP2B6 (rs3745274, c.516G > T; rs28399499, c.983 T > C) and ABCB1 (rs3842, c.4036G > A). CYP2B6 metabolic phenotypes were inferred from the CYP2B6 diplotypes. Linear regression modelling was applied to identify sociodemographic, clinical and pharmacogenetic predictors of [EFV] and [8-OH-EFV]/[EFV] metabolic ratio. RESULTS: Wide (50-fold) interindividual variation in [EFV], [8-OH-EFV] and [8-OH-EFV]/[EFV] was observed; 69.5% of participants had [EFV] within the nominal therapeutic range (1000-4000 ng/mL), while 19.5 and 11.0% had [EFV] below and above this range, respectively. Multiple regression modelling retained only CYP2B6 metabolic phenotypes or the combined rs3745274 and rs28399499 genotypes, as significant predictors of [EFV] and [8-OH-EFV]/[EFV]. CONCLUSION: EFV exposure and disposition varied widely among HIV-infected Brazilians under stable treatment with EFV-containing ART regimens. About 1/10 of the participants had [EFV] exceeding nominal supratherapeutic concentration (4000 ng/mL), but reported tolerance to the ARV regimens, while 1/5 of participants had nominal subtherapeutic [EFV] (<1000 ng/mL) but adequate virological response. Genotype for the 2 CYP2B6 single nucleotide polymorphisms studied explained 48% of variation in [EFV] and 35% of variation in [8-OH-EFV]/[EFV].

Selective deuteration of bupropion slows epimerization and reduces metabolism.

Strategically replacing hydrogen with deuterium at sites of metabolism in small molecule drugs can significantly alter clearance and potentially enhance clinical safety. Bupropion is an antidepressant and smoking cessation medication with the potential to cause seizures. We hypothesized that incorporating deuterium at specific sites in bupropion may greatly reduce epimerization, potentially slow metabolism, and reduce the formation of toxic metabolites, namely hydroxybupropion which has been associated with bupropion's toxicity. Four deuterated analogues were synthesized incorporating deuterium at sites of metabolism and epimerization with the aim of altering the metabolic profile of bupropion. Spectroscopic binding and metabolism studies with bupropion and R-or S-d4 and R-or S-d10 analogs were performed with recombinant CYP2B6, human liver microsomes, and human hepatocytes. Results demonstrate that deuterated bupropion analogues exhibited 20-25% decrease in racemization and displayed a significant decrease in the formation of CYP2B6-mediated R,R - or S,S-hydroxybupropion with recombinant protein and human liver microsomes. In primary human hepatocytes, metabolism of deuterated analogs to R,R - and S,S-hydroxybupropion and threo- and erythro-hydrobupropion was significantly less than R/S-d0 bupropion. Selective deuterium substitution at metabolic soft spots in bupropion has the potential to provide a drug with a simplified pharmacokinetic profile, reduced toxicity and improved tolerability in patients.

Lack of association of CYP2B6 pharmacogenetics with cyclophosphamide toxicity in patients with cancer.

PURPOSE: Cyclophosphamide is a commonly used cancer agent that is metabolically activated by polymorphic enzymes. This study aims to investigate the association between predicted activity of candidate pharmacogenes with severe toxicity during cyclophosphamide treatment. METHODS: Genome-wide genetic data was collected from an institutional genetic data repository for CYP2B6, CYP3A4, CYP2C9, CYP2C19, GSTA1, GSTP1, ALDH1A1, ALDH3A1, ABCC1, ABCB1, and ERCC1. Treatment and toxicity data were retrospectively collected from the patient's medical record. The a priori selected primary hypothesis was that patients who have CYP2B6 reduced metabolizer activity (poor or intermediate (PM/IM) vs. normal (NM) metabolizer) have lower risk of severe toxicity or cyclophosphamide treatment modification due to toxicity. RESULTS: In the primary analysis of 510 cyclophosphamide-treated patients with available genetic data, there was no difference in the odds of severe toxicity or treatment modification due to toxicity in CYP2B6 PM/IM vs. NM (odds ratio = 0.97, 95% Confidence Interval: 0.62-1.50, p = 0.88). In an exploratory, statistically uncorrected secondary analysis, carriers of the ALDH1A1 rs8187996 variant had a lower risk of the primary toxicity endpoint compared with wild-type homozygous patients (odds ratio = 0.31, 95% Confidence Interval: 0.09-0.78, p = 0.028). None of the other tested phenotypes or genotypes was associated with the primary or secondary endpoints in unadjusted analysis (all p > 0.05). CONCLUSION: The finding that patients who carry ALDH1A1 rs8187996 may have a lower risk of cyclophosphamide toxicity than wild-type patients contradicts a prior finding for this variant and should be viewed with skepticism. We found weak evidence that any of these candidate pharmacogenetic predictors of cyclophosphamide toxicity may be useful to personalize cyclophosphamide dosing to optimize therapeutic outcomes in patients with cancer.

So many roads traveled: A career in science and administration.

I have traveled many roads during my career. After spending my first 19 years in Los Angeles, I became somewhat of an academic nomad, studying and/or working in six universities in the United States and three in Sweden. In chronological order, I have a B.A. in Scandinavian languages and literature from UCLA, a Ph.D. in biochemistry from Uppsala University, and an M.S. in toxicology from the Karolinska Institute. I have been in schools of natural science, pharmacy, and medicine and have worked in multiple basic science departments and one clinical department. I have served as a research-track and tenured faculty member, department chair, associate dean, and dean. My research has spanned toxinology, biochemistry, toxicology, and pharmacology. Through all the moves, I have gained much and lost some. For the past 40 years, my interest has been cytochrome P450 structure-function and structure-activity relationships. My lab has focused on CYP2B enzymes using X-ray crystallography, site-directed mutagenesis, deuterium-exchange MS, isothermal titration calorimetry, and computational methods in conjunction with a variety of functional assays. This comprehensive approach has enabled detailed understanding of the structural basis of the remarkable substrate promiscuity of CYP2B enzymes. We also have investigated the mechanisms of CYP3A4 allostery using biophysical and advanced spectroscopic techniques, and discovered a pivotal role of P450-P450 interactions and of multiple-ligand binding. A major goal of this article is to provide lessons that may be useful to scientists in the early and middle stages of their careers and those more senior scientists contemplating an administrative move.

CYP2B6 Genotype and Weight Gain Differences Between Dolutegravir and Efavirenz.

BACKGROUND: Dolutegravir is associated with more weight gain than efavirenz. Loss-of-function polymorphisms in CYP2B6 result in higher efavirenz concentrations, which we hypothesized would impair weight gain among people living with human immunodeficiency virus (HIV; PLWH) starting efavirenz-based antiretroviral therapy (ART). METHODS: We studied ART-naive participants from the ADVANCE study randomized to the efavirenz /emtricitabine/tenofovir disoproxil fumarate (TDF) and dolutegravir/emtricitabine/TDF arms. We compared changes in weight and regional fat on DXA from baseline to week 48 between CYP2B6 metabolizer genotypes in the efavirenz arm, and with the dolutegravir arm. RESULTS: There were 342 participants in the dolutegravir arm and 168 in the efavirenz arm who consented to genotyping. Baseline characteristics were similar. Weight gain was greater in women than men. In the efavirenz arm CYP2B6 metaboliser genotype was associated with weight gain (P = .009), with extensive metabolizers gaining the most weight, and with changes in regional fat in women, but not in men. Weight gain was similar in CYP2B6 extensive metabolizers in the efavirenz arm and in the dolutegravir arm (P = .836). The following variables were independently associated with weight gain in all participants: baseline CD4 count, baseline human immunodeficiency virus type 1 (HIV-1) RNA, and CYP2B6 metaboliser genotype. CONCLUSIONS: CYP2B6 metaboliser genotype was associated with weight gain in PLWH starting efavirenz-based ART. Weight gain was similar between CYP2B6 extensive metabolizers in the efavirenz arm and in the dolutegravir arm, suggesting that impaired weight gain among CYP2B6 slow or intermediate metabolizers could explain the increased weight gain on dolutegravir compared with efavirenz observed in ADVANCE and other studies.