Pregnancy Increases CYP3A Enzymes Activity as Measured by the 4ß-Hydroxycholesterol/Cholesterol Ratio.

Changes in cortisol and other hormones during pregnancy may alter CYP3A enzymes activity, but data from sub-Saharan Africa are sparse. We investigated the effect of pregnancy and CYP3A5 genotypes on CYP3A enzymes activity using the plasma 4ß-hydroxycholesterol (4ß-OHC)/cholesterol (Chol) ratio, a known endogenous biomarker. Tanzanian pregnant women (n = 110) and non-pregnant women (n = 59) controls were enrolled. Plasma 4ß-OHC and Chol were determined in the second and third trimesters for pregnant women and once for non-pregnant women using gas chromatography−mass spectrometry. Genotyping for CYP3A5 (*3, *6, *7) was performed. Wilcoxon Signed-Rank Test and Mann−Whitney U test were used to compare the median 4ß-OHC/Chol ratio between trimesters in pregnant women and between pregnant and non-pregnant women. Repeated-measure ANOVA was used to evaluate the effect of the CYP3A5 genotypes on the 4ß-OHC/Chol ratio in pregnant women. No significant effect of the pregnancy status or the CYP3A5 genotype on the cholesterol level was observed. The plasma 4ß-OHC/Chol ratio significantly increased by 7.3% from the second trimester to the third trimester (p = 0.02). Pregnant women had a significantly higher mean 4ß-OHC/Chol ratio than non-pregnant women, (p < 0.001). In non-pregnant women, the mean 4ß-OHC/Chol ratio was significantly lower in carriers of defective CYP3A5 alleles (*3, *6 or *7) as compared to women with the CYP3A5*1/*1 genotypes (p = 0.002). Pregnancy increases CYP3A enzymes activity in a gestational-stage manner. The CYP3A5 genotype predicts CYP3A enzymes activity in the black Tanzanian population, but not during pregnancy-mediated CYP3A enzyme induction.

CYP3A Specifically Catalyzes 1ß-Hydroxylation of Deoxycholic Acid: Characterization and Enzymatic Synthesis of a Potential Novel Urinary Biomarker for CYP3A Activity.

The endogenous bile acid metabolite 1ß-hydroxy-deoxycholic acid (1ß-OH-DCA) excreted in human urine may be used as a sensitive CYP3A biomarker in drug development reflecting in vivo CYP3A activity. An efficient and stereospecific enzymatic synthesis of 1ß-OH-DCA was developed using a Bacillus megaterium (BM3) cytochrome P450 (P450) mutant, and its structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A [(2)H4]-labeled analog of 1ß-OH-DCA was also prepared. The major hydroxylated metabolite of deoxycholic acid (DCA) in human liver microsomal incubations was identified as 1ß-OH-DCA by comparison with the synthesized reference analyzed by UPLC-HRMS. Its formation was strongly inhibited by CYP3A inhibitor ketoconazole. Screening of 21 recombinant human cytochrome P450 (P450) enzymes showed that, with the exception of extrahepatic CYP46A1, the most abundant liver P450 subfamily CYP3A, including CYP3A4, 3A5, and 3A7, specifically catalyzed 1ß-OH-DCA formation. This indicated that 1ß-hydroxylation of DCA may be a useful marker reaction for CYP3A activity in vitro. The metabolic pathways of DCA and 1ß-OH-DCA in human hepatocytes were predominantly via glycine and, to a lesser extent, via taurine and sulfate conjugation. The potential utility of 1ß-hydroxylation of DCA as a urinary CYP3A biomarker was illustrated by comparing the ratio of 1ß-OH-DCA:DCA in a pooled spot urine sample from six healthy control subjects to a sample from one patient treated with carbamazepine, a potent CYP3A inducer; 1ß-OH-DCA:DCA was considerably higher in the patient versus controls (ratio 2.8 vs. 0.4). Our results highlight the potential of 1ß-OH-DCA as a urinary biomarker in clinical CYP3A DDI studies.

Comparative performance of oral midazolam clearance and plasma 4ß-hydroxycholesterol to explain interindividual variability in tacrolimus clearance.

AIMS: We compared the CYP3A4 metrics weight-corrected midazolam apparent oral clearance (MDZ Cl/F/W) and plasma 4ß-hydroxycholesterol/cholesterol (4ß-OHC/C) as they relate to tacrolimus (TAC) Cl/F/W in renal transplant recipients. METHODS: For a cohort of 147 patients, 8 h area under the curve (AUC) values for TAC and oral MDZ were calculated besides measurement of 4ß-OHC/C. A subgroup of 70 patients additionally underwent intravenous erythromycin breath test (EBT) and were administered the intravenous MDZ probe. All patients were genotyped for common polymorphisms in CYP3A4, CYP3A5 and P450 oxidoreductase, among others. RESULTS: MDZ Cl/F/W, 4ß-OHC/C/W, EBT and TAC Cl/F/W were all moderately correlated (r = 0.262-0.505). Neither MDZ Cl/F/W nor 4ß-OHC/C/W explained variability in TAC Cl/F/W in CYP3A5 expressors (n = 29). For CYP3A5 non-expressors (n = 118), factors explaining variability in TAC Cl/F/W in a MDZ-based model were MDZ Cl/F/W (R2  = 0.201), haematocrit (R2  = 0.139), TAC formulation (R2  = 0.107) and age (R2  = 0.032; total R2  = 0.479). In the 4ß-OHC/C/W-based model, predictors were 4ß-OHC/C/W (R2  = 0.196), haematocrit (R2  = 0.059) and age (R2  = 0.057; total R2  = 0.312). When genotype information was ignored, predictors of TAC Cl/F/W in the whole cohort were 4ß-OHC/C/W (R2  = 0.167), MDZ Cl/F/W (R2  = 0.045); Tac QD formulation (R2  = 0.036), and haematocrit (R2  = 0.032; total R2  = 0.315). 4ß-OHC/C/W, but not MDZ Cl/F/W, was higher in CYP3A5 expressors because it was higher in CYP3A4*1b carriers, which were almost all CYP3A5 expressors. CONCLUSIONS: A MDZ-based model explained more variability in TAC clearance in CYP3A5 non-expressors. However, 4ß-OHC/C/W was superior in a model in which no genotype information was available, likely because 4ß-OHC/C/W was influenced by the CYP3A4*1b polymorphism.

On the formation of 7-ketocholesterol from 7-dehydrocholesterol in patients with CTX and SLO.

A new mechanism for formation of 7-ketocholesterol was recently described involving cytochrome P-450 (CYP)7A1-catalyzed conversion of 7-dehydrocholesterol into 7-ketocholesterol with cholesterol-7,8-epoxide as a side product. Some patients with cerebrotendinous xanthomatosis (CTX) and all patients with Smith-Lemli-Opitz syndrome (SLO) have markedly increased levels of 7-dehydrocholesterol in plasma and tissues. In addition, the former patients have markedly upregulated CYP7A1. We hypothesized that these patients may produce 7-ketocholesterol from 7-dehydrocholesterol with formation of cholesterol-7,8-epoxide as a side product. In accord with this hypothesis, two patients with CTX were found to have increased levels of 7-ketocholesterol and 7-dehydrocholesterol, as well as a significant level of cholesterol-7,8-epoxide. The latter steroid was not detectable in plasma from healthy volunteers. Downregulation of CYP7A1 activity by treatment with chenodeoxycholic acid reduced the levels of 7-ketocholesterol in parallel with decreased levels of 7-dehydrocholesterol and cholesterol-7,8-epoxide. Three patients with SLO were found to have markedly elevated levels of 7-ketocholesterol as well as high levels of cholesterol-7,8-epoxide. The results support the hypothesis that 7-dehydrocholesterol is a precursor to 7-ketocholesterol in SLO and some patients with CTX.

Immediate effect of passive smoking on microcirculatory flow.

OBJECTIVE: Exposure to SHS, as by passive smoking, seems to increase the incidence of cardiovascular events. It has been shown that active smoking of a single cigarette causes an immediate and significant decrease in microcirculatory blood flow velocity, whereas the acute effects of exposure to SHS on microcirculatory flow have as yet not been demonstrated. METHODS: Healthy nonsmoking volunteers of both genders were studied during acute exposure to SHS of two cigarettes burning up to 10 minutes. Microvessels were examined by in vivo vital capillaroscopy (Capiflow(®)), allowing continuous assessment of CBV. RESULTS: CBV decreased from 514 mm/sec (CI 383-646) at baseline to 306 mm/sec (CI 191-420) at end of SHS exposure with a further decrease to a nadir of 240 mm/sec (CI 155-325) four minutes after the end of this exposure (p < 0.0001; ANOVA). CONCLUSIONS: The result of this study shows that passive inhalation of secondhand cigarette smoke induces an immediate and prolonged marked reduction in CBV in nonsmoking healthy volunteers.

Reduction of nevirapine-driven HIV mutations by carbamazepine is modulated by CYP3A activity.

OBJECTIVES: The reduction in mother-to-child transmission of HIV-1 by single-dose nevirapine given at birth onset is achieved at the expense of de novo HIV-1 resistance mutations. In the VITA1 study, single-dose carbamazepine accelerated nevirapine elimination, but the accompanying trend towards fewer de novo HIV-1 mutations was statistically non-significant. METHODS: We investigated if the effect of carbamazepine was confounded by the individual variability in nevirapine metabolism and transport. RESULTS: Nine of 34 (26%) single-dose nevirapine-treated women had one or more nevirapine-associated resistance mutations, compared with 3 of 34 (9%) in the single-dose nevirapine/carbamazepine arm. The genetic polymorphisms in CYP2B6 and MRP7 affected neither nevirapine kinetics nor the development of HIV-1 resistance. In contrast, the reduction in HIV-1 mutations by single-dose carbamazepine reached statistical significance at P = 0.04 with an OR of 0.1 (95% CI 0.01-0.90) upon consideration of CYP3A activity, defined as the ratio of 4ß-hydroxycholesterol to cholesterol, and it was more likely in women with higher CYP3A activity. These findings were in agreement with CYP3A induction in carbamazepine-treated patients. Likewise, carbamazepine induced CYP3A4, but not CYP2B6, in vitro when combined with nevirapine. CONCLUSIONS: The induction of nevirapine elimination reduces HIV-1 resistance mutations, but this effect is modulated by individual CYP3A activity. The study suggests that CYP3A4 activity could be monitored using an endogenous marker and, if needed, boosted to improve clinical endpoints.

Amidoxime reductase system containing cytochrome b5 type B (CYB5B) and MOSC2 is of importance for lipid synthesis in adipocyte mitochondria.

Reduction of hydroxylamines and amidoximes is important for drug activation and detoxification of aromatic and heterocyclic amines. Such a reductase system was previously found to be of high activity in adipose tissue and liver, and furthermore, in vitro studies using recombinant truncated and purified enzymes suggested the participation of cytochrome b(5) reductase (CYB5R), cytochrome b(5) (CYB5), and molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and -2). Here, we show that purified rat liver outer mitochondrial membrane contains high amidoxime reductase activity and that MOSC2 is exclusively localized to these membranes. Moreover, using the same membrane fraction, we could show direct binding of a radiolabeled benzamidoxime substrate to MOSC2. Following differentiation of murine 3T3-L1 cells into mature adipocytes, the MOSC2 levels as well as the amidoxime reductase activity were increased, indicating that the enzyme is highly regulated under lipogenic conditions. siRNA-mediated down-regulation of MOSC2 and the mitochondrial form of cytochrome b(5) type B (CYB5B) significantly inhibited the reductase activity in the differentiated adipocytes, whereas down-regulation of MOSC1, cytochrome b(5) type A (CYB5A), CYB5R1, CYB5R2, or CYB5R3 had no effect. Down-regulation of MOSC2 caused impaired lipid synthesis. These results demonstrate for the first time the direct involvement of MOSC2 and CYB5B in the amidoxime reductase activity in an intact cell system. We postulate the presence of a novel reductive enzyme system of importance for lipid synthesis that is exclusively localized to the outer mitochondrial membrane and is composed of CYB5B, MOSC2, and a third unknown component (a CYB5B reductase).

Serum levels of 25-hydroxyvitamin D and the CYP3A biomarker 4ß-hydroxycholesterol in a high-dose vitamin D supplementation study.

The primary aim was to study the relationship between individual serum levels of 25-hydroxyvitamin D and 4ß-hydroxycholesterol, which is an endogenous biomarker of the drug-metabolizing CYP3A enzymes. In addition, the relationship between this biomarker and inflammation, measured as C-reactive protein (CRP), was investigated. Serum samples were used from a recently performed clinical trial in patients with antibody deficiency or increased susceptibility to respiratory tract infections that were randomized to either placebo or high-dose (4000 IU/day) vitamin D for 12 months. One hundred sixteen patients were included in the final analyses, and serum samples collected 6 months after study start were analyzed. At this time point, 25-hydroxyvitamin D levels were found to range between 10 and 284 nM. Individual levels of 25-hydroxyvitamin D as well as CRP were compared with 4ß-hydroxycholesterol levels. In addition, all participants were genotyped for two polymorphisms (Taq1 and Foq1) in the vitamin D receptor gene. There was no significant correlation between individual serum levels of 25-hydroxyvitamin D and 4ß-hydroxycholesterol. However, a moderate, but statistically significant, negative correlation between CRP and 4ß-hydroxycholesterol levels was observed. This study in patients with highly variable serum levels of 25-hydroxyvitamin D could not reveal any relationship between vitamin D and 4ß-hydroxycholesterol, an endogenous biomarker of CYP3A activity. However, the negative correlation between CRP and 4ß-hydroxycholesterol supports earlier experimental results that inflammation may suppress hepatic CYP3A activity, a finding of potentially high clinical relevance that warrants further exploration.

Comparison of endogenous 4ß-hydroxycholesterol with midazolam as markers for CYP3A4 induction by rifampicin.

CYP3A4, considered the most important enzyme in drug metabolism, is often involved in drug-drug interactions. When developing new drugs, appropriate markers for detecting CYP3A4 induction are needed. Our study compared endogenously formed 4ß-hydroxycholesterol with the midazolam clearance in plasma and the 6ß-hydroxycortisol/cortisol ratio in urine as markers for CYP3A4 induction. To this end, we performed a clinical trial in which 24 healthy subjects were randomized to 10, 20, or 100 mg daily doses of rifampicin for 14 days (n = 8 in each group) to achieve a low and moderate CYP3A4 induction. The CYP3A4 induction could be detected even at the lowest dose of rifampicin (10 mg) via the estimated midazolam clearance, the 4ß-hydroxycholesterol ratio (both P < 0.01), and the 6ß-hydroxycortisol ratio (P < 0.05). For the three dosing groups (10, 20, and 100 mg), the median fold induction from baseline was 2.0, 2.6, and 4.0 for the estimated midazolam clearance; 1.3, 1.6, and 2.5 for the 4ß-hydroxycholesterol/cholesterol ratio; and 1.7, 2.9, and 3.1 for the 6ß-hydroxycortisol/cortisol ratio. In conclusion, the 4ß-hydroxycholesterol ratio is comparable to midazolam clearance as a marker of CYP3A4 induction, and each may be used to evaluate CYP3A4 induction in clinical trials evaluating drug-drug interactions for new drugs.

A comparison of 4ß-hydroxycholesterol : cholesterol and 6ß-hydroxycortisol : cortisol as markers of CYP3A4 induction.

AIM: To compare plasma 4ß-hydroxycholesterol : cholesterol with urinary 6ß-hydroxycortisol : cortisol as markers of cytochrome P4503A4 activity before and after treatment with rifampicin for 2 weeks. METHOD: 6ß-hydroxycortisol and cortisol were determined by liquid chromatography tandem mass spectrometry and 4ß-hydroxycholesterol was determined by gas chromatography-mass spectrometry in three groups of healthy volunteers. RESULTS: Induction ratios for 6ß-hydroxycortisol : cortisol were 1.8, 3.9 and 4.5 for 20 mg day(-1) , 100 mg day(-1) or 500 mg day(-1) of rifampicin, respectively. The corresponding ratios for 4ß-hydroxycholesterol : cholesterol were 1.5, 2.4 and 3.8. CONCLUSIONS: Plasma 4ß-hydroxycholesterol : cholesterol gave similar induction ratios to urinary 6ß-hydroxycortisol : cortisol.

Microvascular reactivity in response to smoking and oral antioxidants in humans.

OBJECTIVE: To investigate whether a daily intake of a moderate dose of antioxidants modifies the microcirculatory response to smoking, assuming a major influence of oxidative stress on microcirculation. METHODS: The microvascular response to smoking was assessed in individual capillaries by capillaroscopy before and after two weeks of treatment with oral antioxidants. RESULTS: Smoking prolonged time to peak (TtP) capillary blood flow velocity in all subjects. When the subjects were pre-treated with ascorbate, TtP was comparable to baseline values of untreated subjects. No significant effect of vitamin E was observed either before or after smoking. Capillary blood flow velocity increased after treatment with ascorbate as well as after vitamin E. However, significant reductions in velocity were still observed in response to smoking even after subjects consumed ascorbate and vitamin E (p<0.0004 and p<0.000008 respectively). CONCLUSIONS: This study focused on individual capillaries, and confirms that smoking has a very pronounced, direct and reproducible microvascular effect possible to demonstrate in vivo in human capillaries. Moderate intake of the antioxidant ascorbate clearly mitigated the effects induced by smoking. TtP after smoking in subjects treated with ascorbate was similar to that observed in untreated subjects before smoking a cigarette. Thus, oxidative stress could be assumed to play a role in the effects of smoking on microcirculation.

FTY720 stimulates 27-hydroxycholesterol production and confers atheroprotective effects in human primary macrophages.

RATIONALE: The synthetic sphingosine analog FTY720 is undergoing clinical trials as an immunomodulatory compound, acting primarily via sphingosine 1-phosphate receptor activation. Sphingolipid and cholesterol homeostasis are closely connected but whether FTY720 affects atherogenesis in humans is not known. OBJECTIVE: We examined the effects of FTY720 on the processing of scavenged lipoprotein cholesterol in human primary monocyte-derived macrophages. METHODS AND RESULTS: FTY720 did not affect cholesterol uptake but inhibited its delivery to the endoplasmic reticulum, reducing cellular free cholesterol cytotoxicity. This was accompanied by increased levels of Niemann-Pick C1 protein (NPC1) and ATP-binding cassette transporter (ABC)A1 proteins and increased efflux of endosomal cholesterol to apolipoprotein A-I. These effects were not dependent on sphingosine 1-phosphate receptor activation. Instead, FTY720 stimulated the production of 27-hydroxycholesterol, an endogenous ligand of the liver X receptor, leading to liver X receptor-induced upregulation of ABCA1. Fluorescently labeled FTY720 was targeted to late endosomes, and the FTY720-induced upregulation of ABCA1 was NPC1-dependent, but the endosomal exit of FTY720 itself was not. CONCLUSIONS: We conclude that FTY720 decreases cholesterol toxicity in primary human macrophages by reducing the delivery of scavenged lipoprotein cholesterol to the endoplasmic reticulum and facilitating its release to physiological extracellular acceptors. Furthermore, FTY720 stimulates 27-hydroxycholesterol production, providing an explanation for the atheroprotective effects and identifying a novel mechanism by which FTY720 modulates signaling.

Metabolism of alprazolam (a marker of CYP3A4) in hemodialysis patients with persistent inflammation.

OBJECTIVE: To investigate the impact of persistent inflammation in hemodialysis (HD) patients on the pharmacokinetics of alprazolam, a cytochrome P450 (CYP) 3A4 substrate, and its metabolites and the role of HD in the impact of persistent inflammation in this clinical context. METHODS: The study population comprised 26 HD patients (mean age 64 years, range 27-79 years; 19 men, 7 women) who were given 1 mg of alprazolam orally in the evening before the day of HD. Unconjugated and conjugated alprazolam and its 4-hydroxy and α-hydroxy metabolites were measured by liquid chromatography-mass spectrometry at 10, 34 (start of HD) and 38 (end of HD) h after intake. C-reactive protein (CRP) was measured weekly beginning 2 months before study initiation, and alpha 1-acid glycoprotein and 4ß-hydroxycholesterol were measured at baseline. CYP3A4 activity was estimated as the ratio of unconjugated alprazolam to 4-hydroxyalprazolam between 10 and 34 h following alprazolam intake. RESULTS: After a single dose of alprazolam, plasma concentrations of unconjugated alprazolam and its metabolites decreased gradually, and unconjugated 4-hydroxyalprazolam was eliminated more rapidly than unconjugated alprazolam by HD. In contrast, the plasma concentrations of conjugated alprazolam and its conjugated metabolites increased during the 34 h following drug intake and the subsequent HD decreased their levels by almost 80%. The ratio of unconjugated alprazolam to 4-hydroxyalprazolam was correlated with CRP levels (r(s) = 0.49, P = 0.01). There was no significant correlation between CYP3A4 activity measured by alprazolam (4-hydroxylation) and alpha 1-acid glycoprotein or 4ß-hydroxycholesterol. Conjugated alprazolam was also found in the plasma. CONCLUSIONS: The correlation between CYP3A4 activity (assessed by alprazolam 4-hydroxylation) and CRP level suggests that inflammation may downregulate CYP3A4 activity. If confirmed, this could have major implications for drug dosing in persistently inflamed patients.

4ß-Hydroxycholesterol, an endogenous marker of CYP3A4/5 activity in humans.

We have proposed that 4ß-hydroxycholesterol (4ß-OHC) may be used as an endogenous marker of CYP3A activity. The cholesterol metabolite 4ß-OHC is formed by CYP3A4. Treatment of patients with strong inducers of CYP3A enzymes, e.g. anti-epileptic drugs, resulted in 10-fold increased concentrations of plasma 4ß-OHC, while treatment with CYP3A inhibitors such as ritonavir or itraconazole resulted in decreased plasma concentrations. There was a relationship between the 4ß-OHC concentration and the number of active CYP3A5*1 alleles showing that 4ß-OHC was not only formed by CYP3A4, but also by CYP3A5. The concentration of 4ß-OHC was higher in women than in men, confirming previous studies indicating a gender difference in CYP3A4/5-activity. The rate of elimination of 4ß-OHC is slow (half-life 17 days) which results in stable plasma concentrations within individuals, but limits its use to study rapid changes in CYP3A activity. In short-term studies exogenous markers such as midazolam or quinine may be superior, but in long-term studies 4ß-OHC is a sensitive marker of CYP3A activity, especially to assess induction but also inhibition. Under conditions where the cholesterol concentration is changing, the ratio of 4ß-OHC:cholesterol may be used as an alternative to 4ß-OHC itself. The use of an endogenous CYP3A marker has obvious advantages and may be of value both during drug development and for monitoring CYP3A activity in patients.

Cytochrome P450 3A activity in mothers and their neonates as determined by plasma 4ß-hydroxycholesterol.

PURPOSE: The purpose of this study was to determine the 4ß-hydroxycholesterol to cholesterol ratio in mothers and neonates at the time of birth and 4 months post-partum. METHOD: 21 mothers and 22 neonates were recruited at the delivery ward at Karolinska University Hospital, Huddinge, Sweden. Blood samples taken from mothers and neonates at birth and 4 months post-partum were analysed for 4ß-hydroxycholesterol and cholesterol. RESULTS: The median plasma concentration of 4ß-hydroxycholesterol was higher in mothers at delivery (50 ng/mL) compared to healthy non-pregnant women (29 ng/mL). The pregnant women had a higher median cholesterol concentration (6.2 mmol/L) compared to healthy non-pregnant women (4.6 mmol/L) but this could only partly explain the increased 4ß-hydroxycholesterol. The major cause is an increased CYP3A activity during pregnancy. The median 4ß-hydroxycholesterol/cholesterol ratio·10(4) was elevated in mothers at time of birth compared to non-pregnant women (0.19 and 0.15, respectively) but decreased to 0.15 4 months post-partum. Neonates had a median 4ß-hydroxycholesterol/cholesterol ratio·10(4) (0.19) comparable to adults already at birth, but lower 4ß-hydroxycholesterol (12 ng/mL) and cholesterol (1.8 mmol/L) concentrations. CONCLUSION: Pregnancy leads to increased CYP3A enzyme activity as determined by the 4ß-hydroxycholesterol/cholesterol ratio. Neonates have low 4ß-hydroxycholesterol and cholesterol concentrations but similar total CYP3A activity as adults already at birth.

Evaluation of 1ß-Hydroxylation of Deoxycholic Acid as a Non-Invasive Urinary Biomarker of CYP3A Activity in the Assessment of Inhibition-Based Drug-Drug Interaction in Healthy Volunteers.

In this study, we aimed to evaluate the utility of endogenous 1ß-hydroxy-deoxycholic acid/total deoxycholic acid ratio (1ß-OH-DCA/ToDCA) in spot urine as a surrogate marker of cytochrome P450 3A (CYP3A) activity in the assessment inhibition-based drug-drug interactions in healthy volunteers. This was accomplished through an open-label, three-treatment parallel-arm study in healthy male volunteers from Zimbabwe. Each group received itraconazole (ITZ; 100 mg once daily; n = 10), fluconazole (FKZ; 50 mg once daily; n = 9), or alprazolam (APZ; 1 mg once daily; n = 8) orally. Midazolam (MDZ), dosed orally and intravenously, was used as a comparator to validate the exploratory measures of CYP3A activity and the effects of known inhibitors. Urinary metabolic ratios of 1ß-OH-DCA/ToDCA before and after CYP3A inhibitor treatment showed a similar magnitude of inhibitory effects of the three treatments as that measured by oral MDZ clearance. The maximum inhibition effect of a 75% reduction in the 1ß-OH-DCA/ToDCA ratio compared to the baseline was achieved in the ITZ group following six once-daily doses of 100 mg. The correlations of the two markers for CYP3A inhibitor treatment were significant (rs = 0.53, p < 0.01). The half-life of urinary endogenous 1ß-OH-DCA/ToDCA was estimated as four days. These results suggested that 1ß-OH-DCA/ToDCA in spot urine is a promising convenient, non-invasive, sensitive, and relatively quickly responsive endogenous biomarker that can be used for CYP3A inhibition-based drug-drug interaction in clinical studies.

Studies on CYP3A activity during the menstrual cycle as measured by urinary 6ß-hydroxycortisol/cortisol.

The 6ß-OH-cortisol/cortisol ratio (6ß-OHC/C) in urine is an endogenous marker of drug-metabolizing enzyme cytochrome P450 3A (CYP3A). The primary aim of this single center, prospective, non-interventional cohort study, was to investigate the variability of 6ß-OHC/C during the menstrual cycle. In addition, possible associations between the CYP3A activity and sex hormones, gut microbiota metabolite trimethylamine-N-Oxide (TMAO) and microRNA-27b, respectively, were investigated. Serum and urinary samples from healthy, regularly menstruating women followed for two menstrual cycles were analyzed. Twenty-six complete menstrual cycles including follicular, ovulatory, and luteal phase were defined based on hormone analyses in serum. 6ß-OHC/C were analyzed in urine and sex hormones, TMAO and miRNA-27b were analyzed in serum at the same time points. 6ß-OHC/C did not vary between the follicular, ovulatory, or luteal phases. There was a difference in the relative miRNA-27b expression between the follicular and ovulatory phase (p = .03). A significant association was found between 6ß-OHC/C and progesterone during the follicular (p = .005) and ovulatory (p = .01) phases (n = 26 for each phase). In addition, a significant association was found between the ratio and TMAO during the ovulatory (p = .02) and luteal (p = .002) phases. 6ß-OHC/C and gut microbiota TMAO were significantly associated (p = .003) when evaluating all values, for all phases (n = 78). Interestingly, the finding of an association between 6ß-OHC/C in urine and levels of TMAO in serum suggest that gut microbiota may affect CYP3A activity.

CYP3A Activity in End-of-Life Cancer Patients Measured by 4ß-Hydroxycholesterol/cholesterol Ratio, in Men and Women.

More than 50% of all drugs are metabolized by the cytochrome P450 3A enzyme (CYP3A). The aim of this study was to investigate if the CYP3A activity, measured by the endogenous marker 4ß-hydroxycholesterol/cholesterol ratio (4ß-OHC/C), is changed during the last weeks and days of life in men and women. To this end, serum samples from 137 deceased patients (median age 70 years) collected at a single time point 1-60 days before death, were analyzed and compared to 280 young (median 27 years), and 30 elderly (median age 70 years) non-cancer controls. There were no significant differences in the 4ß-OHC/C ratio between men and women in end-of-life patients (p < 0.25). The median 4ß-OHC/C was significantly higher in end-of-life male patients compared to both young (p < 0.0001) and elderly (p < 0.05) male controls. In a similar manner, 4ß-OHC/C in end-of-life female patients was significantly higher compared to young and elderly female controls, p < 0.0001 and p < 0.001, respectively. There was no significant correlation between 4ß-OHC/C and survival time. The results from this study suggest maintained CYP3A activity to the very last days of life and even a capacity of induction of the enzyme in end-of-life cancer patients.

Early or deferred initiation of efavirenz during rifampicin-based TB therapy has no significant effect on CYP3A induction in TB-HIV infected patients.

BACKGROUND AND PURPOSE: In TB-HIV co-infection, prompt initiation of TB therapy is recommended but anti-retroviral treatment (ART) is often delayed due to potential drug-drug interactions between rifampicin and efavirenz. In a longitudinal cohort study, we evaluated the effects of efavirenz/rifampicin co-treatment and time of ART initiation on CYP3A induction. EXPERIMENTAL APPROACH: Treatment-naïve TB-HIV co-infected patients (n = 102) were randomized to efavirenz-based-ART after 4 (n = 69) or 8 weeks (n = 33) of commencing rifampicin-based anti-TB therapy. HIV patients without TB (n = 94) receiving efavirenz-based-ART only were enrolled as control. Plasma 4ß-hydroxycholesterol/cholesterol (4ß-OHC/Chol) ratio, an endogenous biomarker for CYP3A activity, was determined at baseline, at 4 and 16 weeks of ART. KEY RESULTS: In patients treated with efavirenz only, median 4ß-OHC/Chol ratios increased from baseline by 269% and 275% after 4 and 16 weeks of ART, respectively. In TB-HIV patients, rifampicin only therapy for 4 and 8 weeks increased median 4ß-OHC/Chol ratios from baseline by 378% and 576% respectively. After efavirenz/rifampicin co-treatment, 4ß-OHC/Chol ratios increased by 560% of baseline (4 weeks) and 456% of baseline (16 weeks). Neither time of ART initiation, sex, genotype nor efavirenz plasma concentration were significant predictors of 4ß-OHC/Chol ratios after 4 weeks of efavirenz/rifampicin co-treatment. CONCLUSION AND IMPLICATIONS: Rifampicin induced CYP3A more potently than efavirenz, with maximum induction occurring within the first 4 weeks of rifampicin therapy. We provide pharmacological evidence that early (4 weeks) or deferred (8 weeks) ART initiation during anti-TB therapy has no significant effect on CYP3A induction. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.