Measurement of CYP1A2 and CYP3A4 activity by a simplified Geneva cocktail approach in a cohort of free-living individuals: a pilot study

Introduction: The cytochrome P450 enzyme subfamilies, including CYP3A4 and CYP1A2, have a major role in metabolism of a range of drugs including several anti-cancer treatments. Many factors including environmental exposures, diet, diseaserelated systemic inflammation and certain genetic polymorphisms can impact the activity level of these enzymes. As a result, the net activity of each enzyme subfamily can vary widely between individuals and in the same individual over time. This variability has potential major implications for treatment efficacy and risk of drug toxicity, but currently no assays are available for routine use to guide clinical decision-making. Methods: To address this, a mass spectrometry-based method to measure activities of CYP3A4, CYP1A2 was adapted and tested in free-living participants. The assay results were compared with the predicted activity of each enzyme, based on a self-report tool capturing diet, medication, chronic disease state, and tobacco usage. In addition, a feasibility test was performed using a low-volume dried blood spots (DBS) on two different filter-paper supports, to determine if the same assay could be deployed without the need for repeated standard blood tests. Results: The results confirmed the methodology is safe and feasible to perform in free-living participants using midazolam and caffeine as test substrates for CYP3A4 and CYP1A2 respectively. Furthermore, though similar methods were previously shown to be compatible with the DBS format, the assay can also be performed successfully while incorporating glucuronidase treatment into the DBS approach. The measured CYP3A4 activity score varied 2.6-fold across participants and correlated with predicted activity score obtained with the self-report tool. The measured CYP1A2 activity varied 3.5-fold between participants but no correlation with predicted activity from the self-report tool was found. Discussion: The results confirm the wide variation in CYP activity between individuals and the important role of diet and other exposures in determining CYP3A4 activity. This methodology shows great potential and future cross-sectional and longitudinal studies using DBS are warranted to determine how best to use the assay results to guide drug treatments.


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Categorical Assignment of Inhibitors / Inducers Table 1.Categorical Assignment of Inhibitors / Inducers from Dietary Exposures Minor(*) inhibitors or inducers were assigned a strength score of 1.For all others, strength = 2. Minor inhibitors or inducers were assigned a strength score of 1.For all others, strength = 2.

LC-MS/MS Assay Details
The same LC-MS method was applied for both method validation and sample analysis, with details as shown below:
Carryover was below 20% of the LLOQ following injection of high calibrators, no interferences were detected in matrix double blanks.Precision for all calibrators and QC samples were within 15% and 20% CV at the LLOQ.
OH-midazolam is known to be metabolized to OH-midazolam-glucuronide, but the extent to which this would occur in our study cohort was unknown.To address this, we split each patient sample into a β-glucuronidase-treated and control group.The mean concentration of free vs glucuronide conjugated OH-midazolam was found to be 0.7 and 4.8 ng/mL respectively representing a 7-fold ratio difference.
The performance characteristics of the LCMS assay from serum are summarized in the table above and confirm that the method works well and can be used for clinical studies.The assay linear range that was sufficient for the expected range of analyte concentrations.The coefficient of variation (CV) for repeated testing was checked at lower limit of detection and was <15% for all analytes which meets the FDA standard (LLOQ <20%).The coefficient of variation at the lower limit of quantification (LLOQ) determines the assay's precision.The quality control points (low/medium/high) all fell within the criteria (known concentration ± 15%).These quality control points are samples with known concentrations that are independent of the standard curve.The use of charcoal stripped serum allowed a blank sample containing no caffeine and paraxanthine (which are commonly seen in blood samples, even criteria.Each standard curve included at least 6 points within the linear range and demonstrated high linearity (R2>0.985).The signal for each analyte in the blank charcoal-stripped serum was less than 20% of the signal at the lower limit of quantification.Also, the response for both analytes and internal standards in the double blank solvent was lower than 20% of the signal at the lower limit of quantification, showing that no carryover occurred during analysis.
The transition ratios were consistent between labeled and unlabeled standards, showing that the samples were consistent with calibrators and quality controls and did not contain unexpected interferences.
Accuracy.With respect to the accuracy of concentration measurements, the measured concentration of calibration standards based on the line of best fit, was accurate within +/-20% for each standard within the linear range for each analyte.Quality control samples (high/medium/low), consisting of charcoal-stripped serum spiked with known quantities of each analyte, were included with each batch and the measured concentration in these samples was also accurate within +/-20% of the known value.
Recovery.The recovery for each analyte was assessed by comparing the quantitation of standards in buffer, spiked into previously-extracted charcoal-stripped serum, and spiked into charcoal-stripped serum before extraction.The recovery of standards spiked prior to extraction was measured in 3 replicates at 3 concentration levels per analyte, as shown below.Reproducibility.The reproducibility of the assay was further demonstrated since each of the 3 sets of technical replicates was analyzed during the assay validation process.For each analyte, known concentrations of the standard were added into the serum at 3 levels (low, medium, high).For each level, three identical samples were extracted and analyzed in parallel.The resulting reproducibility testing at different concentrations within the linear range of the assay showed CV was <7%.These performance indicators ensure the validity of the measured concentrations for this study.
Stability.Due to the necessity of sample storage, the analytes' stability over time needed to be assessed.Samples analyzed over 90 days of storage at -80ºC showed agreement in the values generated from analysis (CVs <6%) confirming 3-month stability.incubated 16 hours at 37°C without β-glucuronidase ("control"), (ii) incubated with 500 units of βglucuronidase for 16 hours at 37°C ("on-spot"), or (iii) extracted from the DBS aqueous extraction followed by deconjugation ("aqueous extraction").Bottom panel: Measurement of OH-midazolam from pooled serum controls vs. dried blood spot samples.Serum samples (50 µL) were either (i) incubated 16 hours at 37°C without β-glucuronidase, or (ii) incubated with 500 units of βglucuronidase for 16h at 37°C, vs. (iii) DBS generated from pooled whole blood that were extracted and treated with β-glucuronidase using the "on-spot" method.

Table 2 .
Categorical Assignment of Inhibitors / Inducers from Patient-Report Medications & Supplements