Evaluation of CYP1A2 activity: Relationship between the endogenous urinary 6-hydroxymelatonin to melatonin ratio and paraxanthine to caffeine ratio in dried blood spots.

The suitability of the endogenous 6-hydroxymelatonin/melatonin urinary metabolic ratio as a surrogate for the paraxanthine/caffeine ratio to predict cytochrome P450 1A2 (CYP1A2) activity was assessed in this study. Twelve healthy volunteers completed four study sessions spread over 1 month (including overnight urine collection with first morning voids collected separately). Except for the third session, volunteers were asked to abstain from methylxanthine-containing beverages and foods at least 24 h before urine collection. At the end of urine collection, subjects were given a caffeinated beverage and capillary blood samples were collected 2 h after the drink administration. A significant linear relationship between the 6-hydroxymelatonin/melatonin ratios from 12-h urine samples and first morning voids was observed (R2  = 0.876, p < 0.0001). In contrast to the paraxanthine/caffeine ratio, consumption of methylxanthine-containing beverages during session three did not significantly influence the 6-hydroxymelatonin/melatonin ratios compared with the other sessions requiring abstinence from caffeine. A larger intra- and interindividual variability in the 6-hydroxymelatonin/melatonin ratios compared with the paraxanthine/caffeine ratio was also observed. A very weak correlation was observed between the paraxanthine/caffeine ratio and both of the endogenous 6-hydroxymelatonin/melatonin ratios (Pearson r < 0.35, p < 0.05). All these results question whether this endogenous metric could adequately reflect CYP1A2 activity or substitute for the probe caffeine. Additional studies with larger study samples are needed to examine this endogenous metric in more details.

Metabolomics reveals biomarkers in human urine and plasma to predict cytochrome P450 2D6 (CYP2D6) activity.

BACKGROUND AND PURPOSE: Individualized assessment of cytochrome P450 2D6 (CYP2D6) activity is usually performed through phenotyping following administration of a probe drug to measure the enzyme's activity. To avoid any iatrogenic harm (allergic drug reaction, dosing error) related to the probe drug, the development of non-burdensome tools for real-time phenotyping of CYP2D6 could significantly contribute to precision medicine. This study focuses on the identification of markers of the CYP2D6 enzyme in human biofluids using an LC-high-resolution mass spectrometry-based metabolomic approach. EXPERIMENTAL APPROACH: Plasma and urine samples from healthy volunteers were analysed before and after intake of a daily dose of paroxetine 20 mg over 7 days. CYP2D6 genotyping and phenotyping, using single oral dose of dextromethorphan 5 mg, were also performed in all participants. KEY RESULTS: We report four metabolites of solanidine and two unknown compounds as possible novel CYP2D6 markers. Mean relative intensities of these features were significantly reduced during the inhibition session compared with the control session (n = 37). Semi-quantitative analysis showed that the largest decrease (-85%) was observed for the ion m/z 432.3108 normalized to solanidine (m/z 398.3417). Mean relative intensities of these ions were significantly higher in the CYP2D6 normal-ultrarapid metabolizer group (n = 37) compared with the poor metabolizer group (n = 6). Solanidine intensity was more than 15 times higher in CYP2D6-deficient individuals compared with other volunteers. CONCLUSION AND IMPLICATIONS: The applied untargeted metabolomic strategy identified potential novel markers capable of semi-quantitatively predicting CYP2D6 activity, a promising discovery for personalized medicine.

The 1ß-Hydroxy-Deoxycholic Acid to Deoxycholic Acid Urinary Metabolic Ratio: Toward a Phenotyping of CYP3A Using an Endogenous Marker?

In this study, we assessed the potential use of the 1ß-hydroxy-deoxycholic acid (1ß-OH-DCA) to deoxycholic acid (DCA) urinary metabolic ratio (UMR) as a CYP3A metric in ten male healthy volunteers. Midazolam (MDZ) 1 mg was administered orally at three sessions: alone (control session), after pre-treatment with fluvoxamine 50 mg (12 h and 2 h prior to MDZ administration), and voriconazole 400 mg (2 h before MDZ administration) (inhibition session), and after a 7-day pre-treatment with the inducer rifampicin 600 mg (induction session). The 1ß-OH-DCA/DCA UMR was measured at each session, and correlations with MDZ metrics were established. At baseline, the 1ß-OH-DCA/DCA UMR correlated significantly with oral MDZ clearance (r = 0.652, p = 0.041) and Cmax (r = -0.652, p = 0.041). In addition, the modulation of CYP3A was reflected in the 1ß-OH-DCA/DCA UMR after the intake of rifampicin (induction ratio = 11.4, p < 0.01). During the inhibition session, a non-significant 22% decrease in 1ß-OH-DCA/DCA was observed (p = 0.275). This result could be explained by the short duration of CYP3A inhibitors intake fixed in our clinical trial. Additional studies, particularly involving CYP3A inhibition for a longer period and larger sample sizes, are needed to confirm the 1ß-OH-DCA/DCA metric as a suitable CYP3A biomarker.

The tyrosine kinase inhibitor nilotinib inhibits SARS-CoV-2 in vitro.

Since the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019, no vaccine has been approved to counter this infection and the available treatments are mainly directed against the immune pathology caused by the infection. The coronavirus disease 2019 (COVID-19) is currently causing a worldwide pandemic, pointing the urgent need for effective treatment. In such emergency, drug repurposing presents the best option for a rapid antiviral response. We assess here the in vitro activity of nilotinib, imatinib and dasatinib, three Abl tyrosine kinase inhibitors, against SARS-CoV-2. Although the last two compounds do not show antiviral efficacy, we observe inhibition with nilotinib in Vero-E6 cells and Calu-3 cells with EC50s of 1.44 µM and 3.06 µM, respectively. These values are close to the mean peak concentration of nilotinib observed at steady state in serum, making this compound a potential candidate for treatment of COVID-19 in vivo.

Toward precision medicine in pediatric population using cytochrome P450 phenotyping approaches and physiologically based pharmacokinetic modeling.

The activity of drug-metabolizing enzymes (DME) shows high inter- and intra-individual variability. Genetic polymorphisms, exposure to drugs, and environmental toxins are known to significantly alter DME expression. In addition, the activity of these enzymes is highly age-dependent due to maturation processes that occur during development. Currently, there is a vast choice of phenotyping methods in adults using exogenous probes to characterize the activity of these enzymes. However, this can hardly be applied to children since it requires the intake of non-therapeutic xenobiotics. In addition, sampling may be challenging in the pediatric population for a variety of reasons: limited volume (e.g., blood), inappropriate sampling methods for age (e.g., urine), and metric requiring invasive or multiple blood samples. This review covers the main existing methods that can be used in the pediatric population to determine DME activity, with a particular focus on cytochrome P450 enzymes. Less invasive tools are described, including phenotyping using endogenous probes. Finally, the potential of pediatric model-informed precision dosing using physiologically based pharmacokinetic modeling is discussed.

Interaction between Fexofenadine and CYP Phenotyping Probe Drugs in Geneva Cocktail.

Drug metabolic enzymes and transporters are responsible for an important variability in drug disposition. The cocktail approach is a sound strategy for the simultaneous evaluation of several enzyme and transporter activities for a personalized dosage of medications. Recently, we have demonstrated the reliability of the Geneva cocktail, combining the use of dried blood spots (DBS) and reduced dose of phenotyping drugs for the evaluation of the activity of six cytochromes and P-glycoprotein (P-gp). As part of a study evaluating potential drug-drug interactions between probe drugs of the Geneva cocktail, the present paper focuses on the impact of cytochromes (CYP) probe drugs on the disposition of fexofenadine, a P-gp test drug. In a randomized four-way Latin-square crossover study, 30 healthy volunteers (15 men and 15 women) received caffeine 50 mg, bupropion 20 mg, flurbiprofen 10 mg, omeprazole 10 mg, dextromethorphan 10 mg, midazolam 1 mg, and fexofenadine 25 mg alone (or as part of a previously validated combination) and all together (Geneva cocktail). The determination of drug concentrations was performed in DBS samples and pharmacokinetic parameters were calculated. Fexofenadine AUC0-8 h and Cmax decreased by 43% (geometric mean ratio: 0.57; CI 90: 0.50-0.65; p < 0.001) and 49% (geometric mean ratio: 0.51; CI 90: 0.44-0.59; p < 0.001), respectively, when fexofenadine was administered as part of the Geneva cocktail in comparison to fexofenadine alone. Consequently, the apparent oral clearance (Cl/F) increased 1.7-fold (CI 90: 1.49-1.93; p < 0.001). There was no interaction between the remaining probes. In conclusion, an unexpected interaction occurred between fexofenadine and one or several of the following substances: caffeine, bupropion, flurbiprofen, omeprazole, dextromethorphan, and midazolam. Further studies are necessary to elucidate the mechanism of this interaction.

Phenotyping of Human CYP450 Enzymes by Endobiotics: Current Knowledge and Methodological Approaches.

Drug response is subject to an important within- and between-individual variability owing, mainly, to pharmacokinetic and pharmacodynamic factors. Pharmacokinetics includes metabolism by cytochrome P450 (CYP450), major enzymes of phase I reactions that are responsible for the biotransformation of around 60% of the currently approved drugs. CYP450 activity and/or expression are influenced by multiple intrinsic and extrinsic factors, such as drug-drug interactions or genetic polymorphisms. Present phenotyping strategies with xenobiotics used to assess CYP450 activity could be replaced by less invasive procedures using endogenous CYP450 biomarkers. In this work, we review existing knowledge on endobiotics and their ability to characterise variability of the CYP1A2, CYP2C19, CYP2D6 and CYP3A enzymes in humans. To date, it appears that there is a lack of clinical data for the majority of the endogenous compounds described in the literature or some important limitations to allow their use in clinical practice. Additional studies are needed to fill the gap or to identify new candidates, in particular through the use of metabolomics. The use of multivariate models is also a very promising approach to enhance prediction by combining several endogenous phenotyping metrics and other covariates.