Abstract
Aims
CYP2A6 is a genetically polymorphic enzyme resulting in differential substrate metabolism and health behaviours. Current phenotyping probes for CYP2A6 exhibit limitations related to procurement (deuterated cotinine), toxicity (coumarin), specificity (caffeine) and age‐appropriate administration (nicotine, NIC). In vitro, CYP2A6 selectively forms 2‐hydroxymetronidazole (2HM) from metronidazole (MTZ). The purpose of this study was to evaluate MTZ as a CYP2A6 phenotyping probe drug in healthy adults against the well‐established method of measuring trans‐3‐hydroxycotinine (3HC)/cotinine (COT).
Methods
A randomized, cross‐over, pharmacokinetic study was completed in 16 healthy, nonsmoking adults. Separated by a washout period of at least 2 weeks, MTZ 500 mg and NIC gum 2 mg were administered and plasma was sampled over 48 hours and 8 hours, respectively. Correlations of plasma metabolite/parent ratios (2HM/MTZ; 3HC/COT) were assessed by Pearson coefficient. CYP2A6 genotyping was conducted and incorporated as a variable of plasma ratio response.
Results
Correlations between the plasma ratio 2HM/MTZ and 3HC/COT were ≥ 0.9 at multiple time points (P < 0.001), demonstrating a wide window during which 2HM/MTZ can be queried post‐MTZ dose. CYP2A6 genotype had significant impacts on both MTZ and NIC phenotyping ratios with decreased activity predicted phenotypes demonstrating 2HM/MTZ ratios ≤58% and 3HC/COT ratios ≤56% compared with extensive activity predicted phenotypes at all time points examined in the study (P < 0.05). No adverse events were reported in the MTZ arm while 38% (n = 6) of participants reported mild adverse events in the NIC arm.
Conclusions
Metronidazole via 2HM/MTZ performed well as a novel, safe phenotyping probe for CYP2A6 in healthy adults.
Keywords: antibiotics, cytochrome P450 (pharmacokinetics), cytochrome P450 enzymes, (genetics/pharmacogenetics), drug metabolism
1.
What is already known about this subject
CYP2A6 is a genetically polymorphic enzyme with variations in metabolism associated with differential drug metabolism, smoking behaviour and lung cancer risk.
Current phenotyping probes include nicotine, deuterated cotinine, caffeine and coumarin each with limitations including age‐appropriate administration, toxicity, specificity and/or accessibility.
CYP2A6 selectively forms 2‐hydroxymetronidazole from metronidazole in vitro and trans‐3‐hydroxycotinine from cotinine in vitro and in vivo.
What this study adds
A new use for a safe and widely used drug was demonstrated in this study and corroborates in vitro findings regarding the specificity of 2‐hydroxymetronidazole formation to CYP2A6 activity.
Metronidazole serves as a valid tool for phenotyping CYP2A6 activity in healthy adults and illustrates genotype‐predicted phenotype clustering.
Because metronidazole is well‐tolerated and widely available, this novel tool has the potential for utility across the lifespan, and among nonsmokers, when insight into variable CYP2A6 activity is warranted.
2. INTRODUCTION
CYP2A6 is a genetically polymorphic enzyme responsible for the biotransformation of nicotine (NIC) and several therapeutic drugs (e.g. artemisinin, efavirenz, letrozole, tegafur). Interindividual variability in excess of 100‐fold has been reported for CYP2A6 activity in vitro and in vivo. This finding is driven, in part, by genetic variations in CYP2A6 conferring intermediate (75%) or reduced (<50%) activity in 25–70% of the population depending on ethnicity,1 and exposure to endogenous (e.g. oestrogen) and exogenous compounds (e.g. clinical inhibitors isoniazid and methoxsalen) that can alter expression and/or activity.2, 3
CYP2A6 metabolically inactivates NIC and metabolically activates tobacco‐specific carcinogens thus CYP2A6 variability has typically been explored in the context of smoking behaviour (e.g. cigarette consumption, topography‐puff volume and frequency, and cessation success) and lung cancer risk. Individuals with reduced activity alleles require less frequent self‐dosing of NIC, which leads to reduced exposure to, and activation of, procarcinogens found in tobacco, thus yielding a lower risk of lung cancer.4, 5, 6, 7 In addition, CYP2A6 decreased activity variants, relative to wild type, are associated with significantly increased exposure to several medications, namely artemisinin, efavirenz and letrozole, used globally to treat deadly diseases such as malaria, human immunodeficiency virus and cancer.4 Despite these documented effects on substrate exposure, the impact of CYP2A6 variation on treatment outcomes for these medications is yet to be well described.
Phenotyping probes are a tool that can provide insight into enzyme activity allowing for potential causal associations between variation in pharmacokinetics, drug exposure and subsequent response. Current in vivo phenotyping probes for CYP2A6 include NIC, cotinine (COT), coumarin and caffeine. Each of these carries significant limitations, either due to safety concerns, feasibility for use in nonsmokers or enzyme specificity.
NIC (administered orally, buccally, intravenously or self‐administered by tobacco use) and deuterium‐labelled COT have been studied in a variety of ways to determine CYP2A6 phenotype in humans. The ratios that have been explored include COT/NIC8 and 3‐hydroxycotinine (3HC)/COT.9 Notably, CYP2A6 accounts for approximately 80% of the C‐oxidation of NIC to form COT with the remaining 20% constituted by other enzymes such CYP2B6.4 By contrast, CYP2A6 accounts for 100% of the conversion of COT to 3HC,10, 11 making 3HC/COT a more specific reflection of CYP2A6 than COT/NIC. 3HC/COT has proved convenient and reliable in smokers even with variable tobacco use and can be measured in plasma, saliva or urine at any time after COT has reached steady state.12, 13 It is important to note that the absolute values of 3HC/COT measured in smokers, which have steady state levels of COT, cannot be directly compared to 3HC/COT measured after single dose NIC or deuterium‐labelled COT administration to nonsmokers and former smokers.12, 13 Although deuterium‐labelled COT has been administered to children14 and adults for measurement of 3HC/COT,15 it is not a readily available.
Alternative substrates are less desirable as phenotyping probes. Coumarin is a toxic anticancer agent no longer approved for use in the USA due to safety concerns. Caffeine has also been proposed as a probe for CYP2A6 activity, via the 8‐hydroxylation of paraxanthine; however, cross‐selectivity with CYP1A2, N‐acetyltransferase 2 and xanthine oxidase activities may compromise specificity for CYP2A6.16
Recent data17 have revealed that CYP2A6 is responsible for the biotransformation of metronidazole (MTZ), a widely used antimicrobial agent.18 MTZ is the drug of choice for treatment of T. vaginalis, the most common sexually transmitted disease globally among other infections thus, it is widely available for clinical use.19 It has been used therapeutically for over 50 years with significant evidence of favourable safety and tolerability across the lifespan from neonates to elderly.18 CYP2A6 is responsible for >96% of 2‐hydroxymetronidazole (2HM) formation in vitro 17 at pharmacologically relevant substrate concentrations. This suggests that the conversion of MTZ to 2HM might also be a useful probe of CYP2A6 activity in vivo. Hence, this prospective crossover study was designed to assess the validity of MTZ as a novel, safe CYP2A6 phenotyping probe (via measurement of 2HM/MTZ) against the commonly used 3HC/COT.
3. METHODS
3.1. Subjects
Healthy, nonpregnant adults (n = 16) between the ages of 18–65 years were eligible for enrolment. Participants were considered healthy if they had: body mass index (BMI) between 18.5 and 29.9 kg/m2; no clinically significant abnormality on screening laboratory evaluation (complete blood count, basic metabolic profile, hepatic function test and urine pregnancy test‐if applicable) or physical examination; no known pathological condition that could affect absorption, distribution, metabolism or excretion of study drug (e.g. liver or kidney disease); and no previous gastrointestinal surgery that could influence function of the gastrointestinal tract (e.g. bariatric procedure). Participants were required to be tobacco free (at least 30 days of abstinence), using no over‐the‐counter medications for 48 hours prior to or during the study, and taking no herbal supplements or prescription medication for at least 14 days prior to or during the study. Participants were also asked to refrain from consumption of foods or beverages containing alcohol, caffeine or grapefruit juice within 48 hours prior to or during the study. Participants were recruited through blast email notifications to hospital employees, postings on social media, bulletins at nearby universities and notification on an active study registry maintained by the CTSA at the University of Kansas Medical Center. The study was approved by the Institutional Review Boards at Children's Mercy and the University of Missouri–Kansas City.
3.2. Clinical study design
We conducted an open‐label, randomized, 2‐period cross‐over study to evaluate the pharmacokinetic profiles of MTZ and correlate the metabolite/parent ratio with that of NIC and COT to assess validity of MTZ as a CYP2A6 phenotyping probe. This study was conducted with a minimum 2‐week washout interval separating each treatment period to minimize the degree of interindividual variability that would be observed with an independent cohort design. Randomization was utilized to determine administration sequence of study drugs. Participants were genotyped after enrollment as a descriptive variable.
Before dosing, participants underwent full physical examination, vital signs and laboratory evaluation (as above) interpreted by a licensed health care provider. Participants arrived at the clinical research unit in the morning on the day of study after an overnight fast. During the NIC study arm, participants remained in the clinical research unit 8 hours postdose. NIC 2 mg gum (GlaxoSmithKline; lot# 15F30N) was chewed for 30 minutes, with instructions to chew for 20 out of every 30 seconds which was observed by study staff. Plasma samples for the quantitation of NIC, COT, and 3HC were obtained predose and 0.5, 1, 1.5, 2, 4, 6 and 8 hours after NIC administration. During the MTZ study arm, participants spent the first 24 hours in the clinical research unit and then returned 48 hours post dose for final sample collection. Metronidazole 500 mg (two 250 mg tablets, Watson Pharmaceuticals, Inc; lot#1059056 M) were administered by mouth. Participants were allowed water ad libitum throughout the study with the exception of the immediate 2 hours after NIC gum administration. Standard hospital meals were provided starting 2 hours after drug administration while in the clinical research unit. Meals did not contain caffeine or grapefruit juice. Plasma samples for the quantitation of MTZ and 2HM were obtained predose and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 16, 20, 24 and 48 hours after MTZ administration. Participants were restricted from strenuous physical activity during the blood sampling period.
3.3. Bioanalytical method
Concentrations of all analytes in plasma were determined by ultraperformance liquid chromatography–tandem mass spectrometer (UPLC‐MS/MS) (Waters Acquity TQD, Milford, MA, USA). The analytical method for determination of MTZ and 2HM was developed and validated according to the Food and Drug Administration guidelines for a bioanalytical assay.20 Briefly, samples were prepared for injection onto UPLC‐MS/MS using solid phase extraction in a 96‐well plate (Oasis Hydrophilic–Lipophilic Balance microelution plate; Waters Corp.). Analytes were separated on a Waters Acquity BEH reversed phase column (BEH 1.7 um, 2.1 × 50 mm) using gradient elution and mobile phases consisting of 0.1% formic acid‐methanol (A) and 0.1% formic acid‐water (B) with total run time of 8 minutes. Deuterium‐labelled compounds served as internal standards. Lower limits of quantification (LLOQs) for MTZ and 2HM were both 0.1 μM.
The analytical method for determination of NIC, COT, and 3HC in plasma was adapted from a previously published method.21 The modified method used a lower plasma volume (100 vs 500 μL) with proportional reduction in wash volumes from 1000 to 200 μL. After elution, samples were evaporated to dryness and then reconstituted in 100 μL 100% acetonitrile. Deuterium‐labelled analytes served as internal standards and were added prior to solid phase extraction. Interday variability was within acceptable limits according to the Food and Drug Administration guidelines for a bioanalytical assay (coefficients of variation [CV] < 15% and accuracy 85–115%; LLOQ CV < 20% and accuracy 80–120%). LLOQs for NIC, COT and 3HC were 1, 5 and 1 nM, respectively.
3.4. Pharmacokinetic analysis
Pharmacokinetic analyses were conducted using Kinetica version 5.0 (Thermo Scientific, Philadelphia, PA, USA). Pharmacokinetic parameters for each participant were estimated based on noncompartmental analyses. The area under the plasma concentration–time curve during the sampling period (AUC0‐t) was calculated using the mixed log‐linear rule and extrapolated (Clast/λ z) to infinity (AUC0‐inf). Weight‐adjusted apparent oral clearance was calculated by dividing the apparent oral clearance (CL/F) by weight (kg). Rate constants for 2HM were determined by compartmental analysis to fully characterize formation and elimination of this metabolite. Half‐lives were calculated using the following equations: t1/2 elim = 0.693/λ z and t1/2 form = 0.693/ka. The apparent terminal elimination rate constant (λ z) and formation rate constant (ka) were determined from weighted linear least squares regression of log‐linear plasma concentration–time curves. Each curve was assessed for goodness of fit based on mathematical criteria (objective function, Akaike, Schwartz). CV < 30% was used to determine final parameter estimates from the model‐dependent analyses.
3.5. CYP2A6 genotyping
Blood was also obtained for CYP2A6 genotyping to use as a covariate in the statistical analysis. Genomic DNA was extracted from whole blood (3 mL) using Qiagen All‐Prep DNA/RNA mini kit (Valencia, CA, USA) according to manufacturer's recommendations. Isolated genomic DNA samples were frozen at −80°C and shipped to the University of Toronto for CYP2A6 genomic inquiry of known allelic variants (CYP2A6*2, *4, *7, *9, *12, *17, *20, *23‐*28, *31, *35) using TaqMan assays. Because no participants were found to carry a *7 allelic variant, samples were not tested for *8 or *10.22, 23
3.6. Safety
Occurrence of any adverse events, subsequent actions taken and outcomes observed were assessed throughout all study days. Determination of relatedness to the study drug was done by the principal investigator.
3.7. CYP2A6 expression and activity in vitro
In preparation for the in vivo study to assess the potential of MTZ as a phenotyping probe drug, studies were carried out to examine the effects of MTZ on the inhibition and induction of CYP2A6 expression and activity in vitro. Further detail regarding the methods is provided in the supplementary materials.
3.8. Statistical analysis
A priori power analysis revealed that 13 subjects would be required to detect a difference in correlation coefficient (r) of 0.9 compared with the null value of 0.5 at 80% power. Therefore, we enrolled 16 subjects to exceed minimum power analysis calculations while allowing for attrition.
Pearson correlation coefficient, ANOVA and linear regression were performed using SPSS version 23 (IBM Corp., Armonk, NY, USA) to describe the relationships between variables of interest. Student t (independent, 2‐tailed) tests were utilized to evaluate differences between groups using Excel 2013 (Microsoft, Redmond, WA, USA). The level of significance accepted for all statistical analyses was α = 0.05.
3.9. Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18.24
4. RESULTS
4.1. Study population
Characteristics of the study participants are listed in Table 1. Sixteen healthy adult volunteers completed all study procedures. One participant was noted to have high levels of baseline COT in plasma (10× threshold for distinguishing smokers from nonsmokers25) and, after confirmation of tobacco use, was excluded from COT and 3HC data analyses. All other participants had baseline (predose) COT levels below the accepted threshold for nonsmokers25 indicating a negligible impact of baseline COT on interpretation of this study data.
Table 1.
Demographics of study population
| Healthy participants (n = 16) | ||
|---|---|---|
| Variable | Value | Range |
| Sex (male/female) | 9/7 | ‐ |
| Age (y) | 30.6 ± 13.9 | 18–56 |
| BMI | 24.9 ± 2.4 | 19.7–28.4 |
| Weight (kg) | 76.5 ± 12.2 | 50.1–93.3 |
| CYP2A6 *1/*1, n (%) | 10 (63%) | ‐ |
| CYP2A6 *1/*2, n (%) | 1 (6%) | ‐ |
| CYP2A6 *1/*9, n (%) | 3 (19%) | ‐ |
| CYP2A6 *1/*17, n (%) | 2 (13%) | ‐ |
| Ethnicity, n (%) | ‐ | |
| White, non‐Hispanic | 14 (88%) | |
| Black | 1 (6%) | |
| Middle‐Eastern | 1 (6%) | |
BMI, body mass index
4.2. Pharmacokinetic parameters of MTZ, NIC and metabolites
Pharmacokinetic parameters of parent compounds and metabolites are listed in Table 2. The plasma concentration–time curves for NIC, COT and 3HC shown as a function of CYP2A6 genotype are illustrated in Figure 1. Predicted genotype–phenotype relationships are present for MTZ and 2HM (see Figure 1). For reduced metabolizers (RM), 2HM AUC0‐inf was significant different compared with wild type (WT; AUC0‐inf 262.6 ± 44.9 μM h for WT vs 178.9 ± 76.2 μM h for RM, P < 0.05). Additionally, significant differences were noted in 2HM apparent Cmax (9.4 ± 2.0 μM for WT vs 5.4 ± 2.5 μM for RM, P < 0.05) between these two genotype groups. Given that 2HM formation is the most proximal phenotype for CYP2A6 when using MTZ as a substrate, compartmental analyses were conducted to determine the rate‐limiting step, formation or elimination, of 2HM (see Supplementary Text for further detail).
Table 2.
Mean ± SD (range) of pharmacokinetic parameters of parent compounds and metabolites in the study population
| MTZ n = 16 | 2HM n = 16 | NIC n = 16 | COT n = 15 | |
|---|---|---|---|---|
| Wt‐adj dose (mg/kg) | 6.7 ± 1.2 (5.4–10.0) | ‐ | 0.027 ± 0.005 (0.021–0.040) | ‐ |
| Cl/F (L/h) | 4.4 ± 1.0 (2.7–6.2) | ‐ | 178.3 ± 70.6 (34.5–296.6) | ‐ |
| Cl/F/W (L/h/kg) | 0.057 ± 0.010 (0.039–0.072) | 2.4 ± 0.9 (0.5–4.2) | ‐ | |
| AUC0‐inf | 701.6 ± 172.5 (473.5–1091) μM h | 231.2 ± 70.0 (108.1–340.8) μM h | 90.9 ± 75.6 (41.6–357.7) nM h | 546.8 ± 191.3* (131.6–820.0) nM h |
| Tmax (hour) | 1.2 ± 0.8 (0.3–3.0) | 9.9 ± 3.7 (6.0–20.0) | 0.8 ± 0.3 (0.5–1.0) | 4.1 ± 1.8 (2.0–8.0) |
| Cmax | 65.7 ± 17.2 (42.0–91.6) μM | 7.9 ± 2.9 (3.3–13.9) μM | 25.9 ± 14.1 (11.8–70.6) nM | 86.0 ± 30.9 (19.8–132.4) nM |
| T1/2 (hour) | 8.5 ± 1.8 (5.3–12.7) | 12.6 ± 2.6 (9.1–20.4) | 2.3 ± 0.7 (1.4–4.4) | 15.2 ± 7.2 (7.5–31.8) |
Pharmacokinetic parameters above determined by noncompartmental analysis. AUC0‐inf, area under the curve; COT, cotinine; Cl/F, apparent oral clearance; MTZ, metronidazole; NIC, nicotine; T1/2, half‐life; Wt‐adj, weight‐adjusted; 2HM, 2‐hydroxymetronidazole.
COT AUC0–8, area under the curve.
Figure 1.
Concentration–time curves in plasma (mean) in healthy volunteers with CYP2A6 WT (*1/*1, n = 10) and CYP2A6 variants (*1/*2, n = 1; *1/*9, n = 3; *1/*17, n = 2). C, Nicotine (total n = 16). D, Cotinine. E, 3‐hydroxycotinine (total n = 15, one individual with *1/*9 omitted due to confirmed tobacco use as described in the text). To provide unobstructed visualization of data by genotype groups, errors bars were not included
4.3. Correlation of 2‐HM/MTZ ratio with NIC probe measures
NIC probe measures, COT/NIC and 3HC/COT, have both been used to evaluate CYP2A6 activity. When NIC gum is administered to nonsmokers, COT/NIC ratio in plasma has been measured 2 hours post dose.8, 26 In our cohort, COT/NIC measured at 2 hours postdose correlated well with 2HM/MTZ between 2–20 hours (r = 0.68–0.82; P < 0.05). 3HC/COT is more widely used than COT/NIC as a proxy for CYP2A6 activity. Dempsey et al. suggest measuring 3HC/COT in the plasma of nonsmokers 2–8 hours after oral administration of labelled NIC.9 In our study, the 2HM/MTZ ratio correlated well with 3HC/COT, measured after administration of NIC gum, across a wide continuum of postexposure time points (Table 3).
Table 3.
Correlation coefficients (r) of metabolite/parent ratios (probe measure) in plasma at selected time points post‐administration of probe drugs
| 3HC/COT ratio in plasma (n = 15) | |||||
|---|---|---|---|---|---|
| Hours | 2 | 4 | 6 | 8 | |
| 2HM/MTZ ratio in plasma (n = 15) | 1 | 0.87 | 0.76 | 0.70 | 0.71 |
| 1.5 | 0.88 | 0.78 | 0.74 | 0.75 | |
| 2 | 0.92 | 0.85 | 0.80 | 0.81 | |
| 2.5 | 0.91 | 0.89 | 0.86 | 0.86 | |
| 3 | 0.92 | 0.86 | 0.81 | 0.80 | |
| 4 | 0.91 | 0.86 | 0.83 | 0.83 | |
| 6 | 0.93 | 0.92 | 0.90 | 0.90 | |
| 8 | 0.89 | 0.93 | 0.94 | 0.94 | |
| 10 | 0.81 | 0.90 | 0.93 | 0.94 | |
| 12 | 0.77 | 0.87 | 0.93 | 0.94 | |
| 16 | 0.71 | 0.80 | 0.87 | 0.89 | |
| 20 | <0.70 | 0.74 | 0.81 | 0.82 | |
| 24 | <0.70 | <0.70 | 0.75 | 0.76 | |
| 48 | <0.70 | <0.70 | <0.70 | 0.73 | |
3HC, trans‐3‐hydroxycotinine; COT, cotinine; MTZ, metronidazole; 2HM, 2‐hydroxymetronidazole
2HM/MTZ at 8, 10 and 12 hours was found to correlate best with 3HC/COT at 6 and 8 hours, yielding the highest correlation coefficient of r = 0.94 (P < 0.0001). These 3HC/COT time points are within range of those suggested after oral NIC dosing by Dempsey et al.9 Of note, numerous time points widely bracketing the Tmax of 2HM demonstrated association (i.e. r ≥ 0.9) in the matrix as demonstrated by grey shading.
When considering clinical convenience, the MTZ and NIC probe measures 2 hours post‐dose correlated well (r = 0.92), indicating a wide window of usage for 2HM/MTZ. Figure 2 further illustrates the correlation at this early time point by plotting individual ratios labelled with genotype‐predicted phenotype.
Figure 2.
Correlation between metabolite/parent ratios (probe measures) of nicotine and metronidazole in healthy volunteers (n = 15). Correlation coefficient (r) reflects comparison between 3‐hydroxycotinine (3HC)/cotinine (COT) and 2‐hydroxymetronidazole (2HM)/metronidazole (MTZ) at 2 hours postdose (i.e. earliest 2HM/MTZ time point demonstrating r ≥ 0.9 within 3HC/COT gold standard range). Solid circles represent CYP2A6 WT homozygotes. Grey circles represent CYP2A6 genotype predicted intermediate metabolizers. Open circles represent CYP2A6 genotype predicted slow metabolizers
4.4. Performance of genotype‐predicted phenotype
Four CYP2A6 diplotypes were identified on descriptive genetic analyses, with predicted phenotypes classified as WT (*1/*1, n = 10), intermediate (*1/*9, n = 3) and slow (*1/*2, n = 1; *1/*17, n = 2).3, 27, 28, 29, 30, 31 Due to small numbers, the genotype‐predicted intermediate and slow metabolizers were grouped together and designated as RM for analysis as commonly performed.3 The NIC probe measures, 3HC/COT and COT/NIC, have demonstrated genotype predicted segregation.8, 25, 30 2HM/MTZ mirrors this in the current study. Compared with WT (n = 10), RM (n = 5) demonstrated 2HM/MTZ ratios ≤58% and 3HC/COT ratios ≤56% at all time points examined in the study, with greatest separation observed in the 3HC/COT plasma ratio at 4 hours (43.6%, P = 0.025) and in the 2HM/MTZ at 16 hours (50.7%, P = 0.002). Table S1 lists the ratios for 3HC/COT and 2HM/MTZ by individual genotype groups along with the percent remaining activity compared with WT for all time points. Figure 3 illustrates the genotype‐predicted phenotype at 2 hours for both NIC and MTZ probe measures. Importantly, the two genotype‐predicted activity groups (i.e. WT and RM) had significantly different 2HM/MTZ ratios measured at 2 hours, thus providing evidence for the clinical utility of this CYP2A6 probe measure early after MTZ administration.
Figure 3.
Difference between CYP2A6 wild type (*1/*1) and CYP2A6 genotype predicted reduced metabolizers (*1/*2, *1/*9, *1/*17) in metabolite/parent ratio in plasma at 2 hours. Nicotine metabolite ratio (n = 15). Metronidazole metabolite ratio (n = 16). Solid circles represent CYP2A6 WT homozygotes. Grey circles represent CYP2A6 genotype predicted intermediate metabolizers. Open circles represent CYP2A6 genotype predicted slow metabolizers
4.5. Variables associated with CYP2A6 probe measures and MTZ exposure
The relative contribution of CYP2A6 genotype and nongenetic factors (e.g. sex, BMI, age) to the observed variability in the CYP2A6 phenotypic measures, 3HC/COT and 2HM/MTZ, was assessed by multiple linear regression. Table 4 lists the contribution of variables evaluated individually by univariate analysis as well as all factors incorporated into a full model. For 3HC/COT and 2HM/MTZ, CYP2A6 genotype was the single most important factor contributing to variability in the metabolite ratios, accounting for 52% (P = 0.015) and 55% (P = 0.007) of the observed variability, respectively. None of the demographic variables contributed significantly to the full model. In contrast, CYP2A6 genotype accounted for 36% and only 8% of the observed variability in 2HM AUC0‐inf and MTZ AUC0‐inf, respectively.
Table 4.
Variables associated with CYP2A6 probe measures and MTZ exposure
| 3HC/COT at 6 h (n = 15) | |||||
|---|---|---|---|---|---|
| Predictor | R 2 | B coefficient (95% CI) | P value | Full model B coefficient (95% CI) | Full model P value |
| CYP2A6 genotype | 0.52 | 0.141 (0.060–0.223) | 0.002 | 0.135 (0.032–0.238) | 0.015 |
| Sex | 0.36 | −0.110 (−0.199‐(−)0.021) | 0.019 | −0.018 (−0.124–0.088) | 0.712 |
| BMI | 0.18 | −0.016 (−0.037–0.005) | 0.120 | −0.001 (−0.018–0.017) | 0.922 |
| Age | 0.16 | 0.003 (−0.001–0.006) | 0.136 | 0.003 (−0.001–0.006) | 0.147 |
| All factors | 0.71 | 0.009 | |||
| 2HM/MTZ at 8 h (n = 16) | |||||
|---|---|---|---|---|---|
| CYP2A6 genotype | 0.55 | 0.152 (0.074–0.230) | 0.001 | 0.148 (0.051–0.246) | 0.007 |
| Sex | 0.34 | −0.116 (−0.209‐(−)0.022) | 0.019 | −0.005 (−0.114–0.105) | 0.924 |
| BMI | 0.13 | −0.015 (−0.038–0.007) | 0.167 | 0.000 (−0.017–0.018) | 0.989 |
| Age | 0.18 | 0.003 (−0.001–0.007) | 0.104 | 0.003 (−0.001–0.007) | 0.114 |
| All factors | 0.72 | 0.005 | |||
| MTZ AUC0‐inf (n = 16) | |||||
|---|---|---|---|---|---|
| CYP2A6 genotype | 0.08 | −98.127 (−287.687–91.432) | 0.286 | −260.021 (−500.64‐(−)19.403) | 0.037 |
| Sex | 0.01 | −26.252 (−218.628–166.124) | 0.774 | −251.512 (−521.53–18.501) | 0.065 |
| BMI | 0.01 | −4.797 (−45.660–36.065) | 0.805 | −23.992 (−67.028–19.044) | 0.245 |
| Age | 0.03 | −1.981 (−9.027–5.066) | 0.556 | −7.685 (−16.704–1.333) | 0.087 |
| All factors | 0.39 | 0.202 | |||
| 2HM AUC0‐inf (n = 16) | |||||
|---|---|---|---|---|---|
| CYP2A6 genotype | 0.36 | 83.687 (19.368–148.006) | 0.014 | 5.487 (−38.124–49.099) | 0.787 |
| Sex | 0.38 | −84.444 (−145.995‐(−)22.893) | 0.011 | −66.621 (−115.56‐(−)17.682) | 0.012 |
| BMI | 0.62 | −22.749 (−33.052‐(−)12.445) | <0.001 | −20.181 (−27.981‐(−)12.381 | <0.001 |
| Age | 0.11 | 1.677 (−1.055–4.409) | 0.209 | 0.121 (−1.514–1.755) | 0.874 |
| All factors | 0.88 | <0.001 | |||
Rationale for inclusion of specific time points in analysis above: 2HM/MTZ at 8 hours was chosen as it was the earliest time point with the highest correlation (r = 0.94) with 3HC/COT (which occurred earliest at 6 hours).
2HM, 2‐hydroxymetronidazole; 3HC, trans‐3‐hydroxycotinine; BMI, body mass index; COT, cotinine; MTZ, metronidazole.
4.6. Effects of MTZ on CYP2A6 expression and activity in vitro
To determine if MTZ itself would interfere with CYP2A6 activity in a meaningful way, the ability of MTZ to induce or inhibit CYP2A6 was investigated in primary human hepatocytes and human liver microsomes, respectively. Our data show that MTZ concentrations corresponding to those seen in plasma after therapeutic doses are unlikely to cause induction and should cause no more than a slight inhibition (Ki 443.8 ± 93.0 μM) of CYP2A6 activity in vivo (see Figures S1‐S2 and Supplementary Text).
4.7. Adverse events
Adverse events considered to be related to the study drug occurred in 6 participants who complained of dizziness after NIC administration (38%). These events were mild and self‐limiting with no significant intervention required. No participants experienced any adverse events post MTZ administration.
5. DISCUSSION
An ideal in vivo phenotyping probe should provide a real‐time composite reflection of factors affecting the enzymatic pathway of interest (e.g. inhibition or induction) and the probe drug itself should not directly impact the pathway in such a way that could markedly impact the actual activity of the enzyme of interest. Additional considerations when selecting a probe drug include specificity (for the reaction measured), safety, tolerability, cost and commercial availability of the compound.32, 33 We propose that MTZ meets many of these criteria and should be considered as an alternative to NIC and COT for estimating CYP2A6 activity. Data presented indicate that the proposed measure, the plasma 2HM/MTZ ratio, correlates well with the 3HC/COT ratio across numerous time points. Even at 2 hours post‐dose, 2HM/MTZ correlated extremely well (r > 0.9) with 3HC/COT. 2HM/MTZ demonstrated a higher correlation with 3HC/COT, compared with COT/NIC, providing additional evidence for similar specificity of MTZ conversion to 2HM by CYP2A6 in vivo. This is supported by in vitro evidence that demonstrated the affinity and specificity of MTZ for CYP2A6.17
2HM/MTZ and 3HC/COT appear to have a similar dependence on CYP2A6 in the multivariate analysis which provides additional evidence for the specificity of 2HM/MTZ being comparable to 3HC/COT. Interestingly, CYP2A6 genotype alone accounted for more than half of the variability of 2HM/MTZ and 3HC/COT (see Table 4). Seventy‐one and 72% of the variability in 3HC/COT and 2HM/MTZ, respectively, could be explained when additional factors (e.g. sex, BMI and age) were added to the model. Sex, through an oestrogen‐sensitive mechanism (e.g. oestrogen receptor α), has been associated with differential CYP2A6 activity.34, 35, 36 Important to note is the similarity in the contribution of the factors to both probe measures. Clearly, additional factors leading to variability in these probe measures may be identified. Possibilities include dietary components not yet known to affect CYP2A6, endogenous variables leading to altered CYP2A6 expression and/or function, and novel CYP2A6 variant alleles not yet characterized.
A potential benefit worth further exploration is the use of 2HM/MTZ to directly compare ratios in smokers and nonsmokers, a notable limitation of current NIC‐derived CYP2A6 probe measures. Evaluation of 2HM/MTZ in this context would determine reliability of this CYP2A6 activity probe in the presence of steady state COT and variable NIC concentrations but was not within the scope of the current study.
MTZ as a probe drug also offers the benefits of safety and tolerability. MTZ at low doses, such as that used in this study, has a large body of evidence to document safety across the continuum of age, including lack of teratogenicity to developing human fetuses if taken during pregnancy.37, 38 The MTZ dose in this study was chosen based on clinical availability and ability to provide quantifiable plasma concentrations over the study time course. Future studies could evaluate the performance of low‐dose MTZ as a CYP2A6 probe. Given the well‐documented safety, the 2HM/MTZ ratio can also be used to shed light on the impact of ontogeny on CYP2A6 activity in the very young and as a potential tool to understand the risks for smoking or vaping initiation in adolescents and the effects of environmental NIC exposure (e.g. active vs passive smoke exposure) if validated in this context.
Limitations of the current work frame the application of its findings. The study design was chosen based on published work with NIC, the in vitro 3 and in vivo assessment of CYP2A6 activity,39 and clinical feasibility pertaining to the investigation of MTZ as a phenotyping probe. With this approach, full pharmacokinetic analysis of NIC metabolites was limited due to the long t1/2 of COT (16–20 h)9, 14, 31 and 3HC (6.6 h),40 but this did not affect study findings. In fact, the pharmacokinetic parameters of NIC and COT in our study are consistent with available data from healthy adults after dosing with NIC gum (see Table 2).8 In addition, pharmacokinetic parameters of MTZ and 2HM in our study are also consistent with available data from healthy adults after similar MTZ oral doses.41, 42, 43, 44 We recognize that alterations in ancillary metabolic pathways may impact 2HM/MTZ, such as glucuronidation (contributing <15% of total dose recovered) and formation of MTZ‐acetic acid metabolite (found in trace amounts in patients with normal renal function).44, 45 It is important to note that absolute values for the 3HC/COT ratio in plasma reported in our study following administration of NIC gum in nonsmokers are not directly comparable to the 3HC/COT ratio measured at COT steady state in smokers in other published studies. Yet, the 3HC/COT ratio measured between 2–8 hours in nonsmokers after oral NIC is highly specific for CYP2A6 and has been previously reported to correlate well with NIC clearance.9 Thus, it was used as the main comparator. Finally, the goal of our study was to assess the suitability of MTZ as a CYP2A6 phenotyping probe, and specifically the 2HM/MTZ ratio, using 3HC/COT as the gold standard. We were able to assess the correlation between the two metabolite ratios over a 10‐fold range of values with the random sampling approach we used for the study. We recognize that we may have been able to expand the dynamic range for the comparison with inclusion of poor metabolizers, but doing so was statistically unlikely given their expected low frequency in the recruitment population.1, 46 Alternative approaches, such as a drug–drug interaction study with a CYP2A6 inhibitor or an a priori genotype‐stratified study was beyond the scope of the present study, but could be considered in the future to further validate 2HM/MTZ as an alternative to 3HC/COT as a phenotyping measure.
In conclusion, MTZ is a well‐tolerated probe drug, and the 2HM/MTZ ratio is a specific biomarker of CYP2A6 activity in healthy adults. The benefits of using this probe to further describe CYP2A6 variability include ease of use (e.g. 2 hours postdose), drug availability, excellent safety profile and potential for use in all ages regardless of smoking status.
CONTRIBUTORS
S.L.S., R.E.P., R.F.T., G.L.K., C.A.V., S.A.R. and J.S.L. wrote the manuscript. S.L.S., R.E.P., G.L.K., C.A.V., S.A.R. and J.S.L. designed the research. S.L.S. and R.F.T. performed the research. S.L.S., S.A.R., R.E.P., C.A.V., J.S.L. and R.F.T. analysed the data.
COMPETING INTERESTS
R.F.T. has consulted for Apotex, for Quinn Emmanuel, for Ethismos, and is a member of several scientific advisory boards (e.g. Canadian Centre for Substance Abuse, Health Canada (Vaping), and Brain Canada).
Supporting information
Table S1. Probe measures at various time points by individual genotype
Figure S1. Dixon plot of the effects of various concentrations of metronidazole (0, 5, 10, 30, 50, 100, 200 or 300 μM) on the conversion of nicotine to cotinine. Data are presented as the mean of 3 separate experiments. Interday coefficients of variation were ≤ 15.5% (range 1.0–15.5%).
Figure S2. Effects of metronidazole treatment on CYP2A6 expression and activity in primary human hepatocytes (PHHs). PHHs from 4 female donors (n = 3 replicates/treatment group) were treated with vehicle (DMSO 0.1%), positive control (CITCO 500 nM), or metronidazole (300 μM) for 72 hours. Panel A shows expression levels of CYP2A6 mRNA as determined via qRT‐PCR and normalized to GAPDH. Panel B shows the effects of test compound treatment on the conversion of metronidazole to 2‐hydroxymetronidazole, a marker reaction for CYP2A6 activity. Data for the 8‐hour time points are shown, reflecting maximum conversion of metronidazole to its metabolite. In both data sets, significance was determined by comparing values from treatment groups with vehicle treated control PHHs (*P < .05; **P < .01). Error bars indicate standard deviation from the mean.
ACKNOWLEDGEMENTS
The authors would like to warmly thank the study volunteers for their participation and meaningful contribution to this work. The authors would also like to thank Jaylene Weigel and Krista LaDue Wright for superb coordination of the clinical study and Ewa Hoffmann for assistance with genotyping analyses. This research was supported by the Katharine B. Richardson Award and the Young Investigator Award, both administered by Children's Mercy Kansas City. We also acknowledge these sources of funding: Canada Research Chair in Pharmacogenomics (R.F.T.) and the Canadian Institutes of Health Research (CIHR) grant FDN‐154294 (R.F.T.); the Campbell Family Mental Health Research Institute of the Centre for Addiction and Mental Health (CAMH).
Stancil SL, Pearce RE, Tyndale RF, et al. Evaluating metronidazole as a novel, safe CYP2A6 phenotyping probe in healthy adults. Br J Clin Pharmacol. 2019;85:960–969. 10.1111/bcp.13884
The authors confirm that the PI for this paper is Stephani Stancil and that she had direct clinical responsibility for patients.
The 3rd affiliation is the formal affiliation of Stephani L. Stancil while completing this work as a student.
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Supplementary Materials
Table S1. Probe measures at various time points by individual genotype
Figure S1. Dixon plot of the effects of various concentrations of metronidazole (0, 5, 10, 30, 50, 100, 200 or 300 μM) on the conversion of nicotine to cotinine. Data are presented as the mean of 3 separate experiments. Interday coefficients of variation were ≤ 15.5% (range 1.0–15.5%).
Figure S2. Effects of metronidazole treatment on CYP2A6 expression and activity in primary human hepatocytes (PHHs). PHHs from 4 female donors (n = 3 replicates/treatment group) were treated with vehicle (DMSO 0.1%), positive control (CITCO 500 nM), or metronidazole (300 μM) for 72 hours. Panel A shows expression levels of CYP2A6 mRNA as determined via qRT‐PCR and normalized to GAPDH. Panel B shows the effects of test compound treatment on the conversion of metronidazole to 2‐hydroxymetronidazole, a marker reaction for CYP2A6 activity. Data for the 8‐hour time points are shown, reflecting maximum conversion of metronidazole to its metabolite. In both data sets, significance was determined by comparing values from treatment groups with vehicle treated control PHHs (*P < .05; **P < .01). Error bars indicate standard deviation from the mean.