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Evaluation of drug interaction potential of zanubrutinib with cocktail probes representative of CYP3A4, CYP2C9, CYP2C19, P‐gp and BCRP

Aim This study aims to assess the potential effects of zanubrutinib on the activity of cytochrome P450 (CYP) enzymes and drug transporter proteins using a cocktail probe approach. Methods Patients received single oral doses of probe drugs alone and after at least 8 days of treatment with zanubrutinib 160 mg twice daily in a single‐sequence study in 18 healthy male volunteers. Simultaneous doses of 10 mg warfarin (CYP2C9) and 2 mg midazolam (CYP3A) were administered on Day 1 and Day 14, 0.25 mg digoxin (P‐glycoprotein [P‐gp]) and 10 mg rosuvastatin (breast cancer resistance protein [BCRP]) on Day 3 and Day 16, and 20 mg omeprazole (CYP2C19) on Day 5 and Day 18. Pharmacokinetic (PK) parameters were estimated from samples obtained up to 12 h post dose for zanubrutinib; 24 h for digoxin, omeprazole and midazolam; 48 h for rosuvastatin; and 144 h for warfarin. Results The ratios (%) of geometric least squares means (90% confidence intervals) for the area under the concentration–time curve from time zero to the last quantifiable concentration in the presence/absence of zanubrutinib were 99.80% (97.41–102.2%) for S‐warfarin; 52.52% (48.49–56.88%) for midazolam; 111.3% (103.8–119.3%) for digoxin; 89.45% (78.73–101.6%) for rosuvastatin; and 63.52% (57.40–70.30%) for omeprazole. Similar effects were observed for maximum plasma concentrations. Conclusions Zanubrutinib 320 mg total daily dose had minimal or no effect on the activity of CYP2C9, BCRP and P‐gp, but decreased the systemic exposure of CYP3A and CYP2C19 substrates (mean reduction <50%).

Zanubrutinib has been granted a breakthrough therapy designation and recently received accelerated approval by the US Food and Drug Administration (FDA) for the treatment of adult patients with MCL who have received at least one prior therapy. 12 Zanubrutinib was also approved by the China National Medical Products Administration (NMPA) for CLL or SLL and MCL in adult patients who have received at least one prior therapy. 13 Zanubrutinib has been shown to be more selective than ibrutinib for the inhibition of BTK versus a panel of receptor tyrosine kinases. 13 The improved selectivity of zanubrutinib for BTK may minimize off-target inhibition of other kinases and, therefore, may result in a lower incidence and severity of off-target toxicities compared with ibrutinib. Emerging clinical data has shown that zanubrutinib is associated with high rates of objective response in patients with B-cell malignancies, including WM, MCL and CLL. [14][15][16] Clinical pharmacokinetics (PK) data has shown that zanubrutinib is rapidly absorbed after oral administration; median time to peak zanubrutinib plasma concentration was 2 h and the mean apparent terminal elimination half-life (t 1/2 ) was approximately 2-4 h in patients with B-cell malignancies. Following multiple-dose administrations of zanubrutinib, limited systemic accumulation is observed, which is consistent with the observed t 1/2 . At doses ranging from 40 mg to 320 mg, there is a dose-proportional increase in both maximum concentration (C max ) and area under the plasma concentration versus time curve (AUC). 16 Based on in vitro and clinical drug-drug interaction (DDI) studies, CYP3A is considered the primary P450 isoform responsible for zanubrutinib metabolism. 17 Results from a human absorption, metabolism, and excretion study (AME) showed that hepatic metabolism and excretion account for a substantial portion of the elimination of zanubrutinib and that renal clearance does not play an important role.
In in vitro studies, zanubrutinib showed potential for reversible inhibition of CYP2C8, CYP2C9 and CYP2C19 in human liver microsomes, with half maximal inhibition concentration (IC 50 ) values of 4.03, 5.69 and 7.80 μM, respectively. 18 In vitro, zanubrutinib also induced a ≥2-fold increase in mRNA (at 3 and 30 μM) of CYP2B6, CYP2C8, CYP2C9 and CYP3A in human hepatocytes. The average R3 values for induction of CYP3A4, CYP2B6, CYP2C8 and CYP2C9 were lower than or near the cutoff value of 0.8 according to FDA DDI guidance. Thus, zanubrutinib could theoretically have the potential to cause induction of these enzymes. In vitro studies showed that zanubrutinib was neither a significant inhibitor of P-glycoprotein This clinical drug interaction study was designed to determine the effect of multiple doses of zanubrutinib on the PK of probe substrates of CYP3A (midazolam), CYP2C9 (warfarin), CYP2C19 (omeprazole), P-gp (digoxin) and BCRP (rosuvastatin) in healthy volunteers using a cocktail approach. Simultaneous single oral doses of probe drugs, warfarin and midazolam were administered followed by a cocktail of digoxin and rosuvastatin 2 days later. Midazolam, warfarin and omeprazole have been extensively used together as probe substrates in studies using the "Modified Cooperstown Cocktail + 1" approach. 20 Omeprazole was dosed separately in this study to avoid potential pH-dependent absorption of zanubrutinib, and an exploratory objective was also included to assess whether a single dose of omeprazole had any impact on the PK of zanubrutinib. Digoxin and rosuvastatin have been safely administered together as part of a What is already known about this subject • Zanubrutinib, an irreversible BTK inhibitor, has demonstrated promising antitumour activity in patients with B-cell malignancies.
• In vitro studies show that zanubrutinib has an induction and/or inhibition potential on multiple CYPs and transporters.
• The drug interaction potential of zanubrutinib with substrates of CYP enzymes and transporters in humans has not been assessed.

What this study adds
• The cocktail approach to assess drug-drug interaction (DDI) has been commonly used, and this work was one of few studies utilizing this combination of CYP and transporter substrates.
• At clinically relevant concentrations, zanubrutinib did not impact the activity of CYP2C9 and BCRP, but it had a weak induction effect on CYP3A and CYP2C19. recent cocktail transporter study. 21 Probe substrates for CYP2C8 and CYP2B6 were not included in this study mainly because of lack of validated probes for these enzymes in cocktail DDI approaches.
Results from this study will be used to assess clinical DDI risk for concomitant administration of zanubrutinib with substrates of these CYP enzymes or transporters.

| METHODS
This cocktail DDI study was registered on ClinicalTrials.gov (NCT03561298). The study protocol was reviewed and approved by the study centres' Independent Ethics Committee and was conducted in compliance with the protocol, and in accordance with the International Conference on Harmonization Good Clinical Practice principles and the Declaration of Helsinki. All participating subjects provided written informed consent prior to screening.

| Subjects
Healthy male subjects of any race aged between 18 and 60 years and with a body mass index (BMI) between 18.0 and 32.0 kg/m 2 were selected according to the inclusion criteria listed in the protocol.
Subjects were excluded if they had consumed or intended to use any medications/products known to alter the absorption, metabolism or elimination processes of the study drugs, within 30 days prior to the first dose administration, or if they had used any metabolic enzyme inhibitors or inducers that would affect the study drugs, within 30 days of the first dose administration. Eighteen subjects were enrolled to ensure that 12 subjects completed the study. The sample size chosen for this study was based on precedent set by other cocktail DDI studies of similar nature (assuming similar within-subject variation of substrate drugs). The fixed-sequence crossover design used in this study is typical for cocktail DDI studies 20 because it reduces intersubject variability from the comparison between treatments; thus, a relatively small number of subjects are required.

| Study design
This was a phase 1, open-label, single-centre, single-sequence study to investigate the effect of multiple doses of zanubrutinib on the PK of probe drugs in healthy male subjects. All subjects received study drugs in a fixed sequence, all of which were administered orally, first without zanubrutinib and then with zanubrutinib. A summary of the study design is shown in Figure 1. In brief, simultaneous single oral doses of probe drugs, warfarin and midazolam were administered followed by dosing of digoxin and rosuvastatin together 2 days later.
Omeprazole was dosed separately on Day 5. Subjects were in a fasted state on the day of probe drug administration and PK collection. On Days 7-19, subjects received twice-daily doses of 160 mg zanubrutinib; after a sufficient enzyme induction period, the probe drugs (using the same sequence) were administered again starting on Pharmacogenetic blood samples were collected, and in the event of aberrant PK outliers, genotype testing was conducted.

| Pharmacokinetic sample analysis
To determine the PK of the probe drugs, blood samples were collected for the analysis of plasma concentrations of S-warfarin, midazolam, digoxin, rosuvastatin, omeprazole and zanubrutinib ( Figure 1 and Table 1). The analyses were performed by XenoBiotic

| Pharmacokinetic data analyses
The PK parameters included C max , time to C max (T max ), area under the plasma concentration-time curve from time zero to last measurable timepoint (AUC 0-t ), AUC from time zero extrapolated to infinity (AUC 0-∞ ), and t 1/2 , using noncompartmental methods with Phoenix WinNonlin (Certara USA, Inc.) Version 6.4. Pharmacokinetic analysis was carried out using actual postdose times. C max and T max values were obtained directly from the plasma concentration-time profiles.
Area under the concentration-time curve over a dosing interval τ (AUC 0-12 , where τ = 12 h) for zanubrutinib was also calculated when it was administered alone or co-administered with omeprazole.
Regression-based parameters (AUC 0-∞ , t 1/2 ) were calculated only if the R 2 -adjusted value of the regression line was ≥0.7. Values for AUC 0-∞ where the percentage extrapolation was greater than 30% were reported but excluded from descriptive statistics.

| Pharmacokinetic statistical analysis
The effect of zanubrutinib on the PK of each probe drug was evaluated using a confidence interval (CI) approach. In each statistical analysis, zanubrutinib given in combination with each probe drug was the test treatment, and each probe drug given alone was the reference treatment. The PK parameters AUC 0-t , AUC 0-∞ (if allowed by the available data), and C max of each probe drug were log-transformed prior to analysis and analysed using a mixed model 26 for each part of the study. The model included treatments as fixed effects and subject as a random effect. For AUC 0-t , AUC 0-∞ , and C max , the geometric least squares (LS) means were calculated for the test and reference treatments; the test-to-reference geometric LS means ratios (GMRs) and the corresponding 90% CI were also calculated. The T max was analysed nonparametrically using the Wilcoxon signed-rank test.

| Safety analyses
Clinical chemistry, haematology, urinalysis, vital signs and electrocardiogram (ECG) assessments were collected for safety evaluation.
Adverse events (AEs) that occurred following the administration of any probe drug on Day 1 until the administration of zanubrutinib on Day 7 were assigned to be associated with "probe drugs only". Adverse events that occurred following the administration of zanubrutinib on Day 7 until the administration of warfarin, vitamin K or midazolam on Day 14 were assigned to be associated with "zanubrutinib only". Any AEs that occurred at the administration of warfarin, vitamin K or midazolam on Day 14 through the follow-up visit were assigned to be associated with "probe drugs plus zanubrutinib". Treatment-emergent AEs (TEAEs) were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events v4.03.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the T A B L E 1 Investigational product administration and blood sampling times

| Study population
A total of 18 subjects were enrolled and 17 completed the study. All 18 enrolled subjects were included in the safety and PK populations.
One subject withdrew consent prior to dosing on Day 3 of the study.
Demographic data are summarized in Table 2. Subjects enrolled in the study were males between the ages of 21 and 58 years with BMI values ranging from 22.6 to 32.0 kg/m 2 .

| Effect of zanubrutinib on PK of probe drugs
Statistical comparisons for each probe drug administered alone or coadministered with zanubrutinib are presented in Table 3, and concentration-time profiles are shown in Figure 2. In all statistical comparisons of T max for each probe drug analyte, no statistically significant differences were observed when the probe drugs were coadministered with zanubrutinib compared to when administered alone. In general, within-subject variation in C max and AUC was low for S-warfarin (≤14.2%), midazolam (≤17.6%), digoxin (≤24.4%) and rosuvastatin (≤27.5%), and low to moderate for omeprazole (15% and 35% for AUC and C max , respectively).
The PK data showed that one subject had a much longer

| Plasma PK profile of zanubrutinib
The PK data of zanubrutinib are available from 17 subjects ( Table 4).
The geometric mean AUC 0-t and C max values of zanubrutinib administered alone were similar to those of zanubrutinib co-administered with omeprazole. Zanubrutinib showed a similar median T max and a similar arithmetic mean t 1/2 when administered alone or co-administered with omeprazole.

| Safety
During the administration of the probe drugs alone, one subject dis-

| DISCUSSION
The results of this cocktail DDI study indicate that at clinically relevant concentrations, zanubrutinib had minimal or no effect on the activity of CYP2C9, BCRP and P-gp, but decreased the systemic exposure of CYP3A-and CYP2C19-sensitive substrates by less than 50%.
While the cocktail approach to assess DDI has been commonly used, to our knowledge, the current study was one of the first studies to use this particular combination of probe substrates to assess DDI liability of these CYP enzymes and transporters together in one study.
The cocktail approach used in this study is built upon reports of previous cocktail studies, including the "Modified Cooperstown Cocktail + 1" approach 20 (midazolam, warfarin and omeprazole probes) and the Geneva phenotyping cocktail approach (six CYP isoforms and P-gp probes). 28 Digoxin (P-gp) was safely used in evaluating clinical DDIs with metabolic enzymes. 29 In a recent cocktail transporter study, the utility of using digoxin (P-gp) and rosuvastatin (BCRP) probe drugs together has been validated. 21 Building upon these previous studies, T A B L E 3 Summary of the pharmacokinetic parameters of probe drugs administered alone and with zanubrutinib 160 mg b.i.d.    is an important metabolizer for apixaban and rivaroxaban but not the other DOACs. It has been shown that the fractions of rivaroxaban and apixaban metabolized by CYP3A4/5 was 18% and 15%, respectively. 31 Co-administration of rivaroxaban with the strong CYP3A4 inducer rifampicin led to an approximate 50% decrease in mean rivaroxaban AUC, with parallel decreases in its pharmacodynamic effects. Product labelling includes a recommendation that the concomitant use of rivaroxaban (or apixaban) with strong CYP3A4 inducers (e.g., rifampicin, phenytoin, carbamazepine, phenobarbital and St. John's Wort) should be avoided. 32 Given that the extent of CYP3A induction by zanubrutinib is far less than that of rifampicin, it can be concluded that zanubrutinib can be safely used with DOACs due to its low DDI potential.
There are limitations in the current study design. Due to the number of PK samples planned for each probe substrate and zanubrutinib (six together) and the limited blood volume that can be drawn per subject, the current PK sampling schedule may not be ideal to capture the C max of substrate drugs. This was reflected in the wide confidence interval in the mean estimate of C max for omeprazole and digoxin.
More intensive PK sampling would have been ideal to characterize the effects of zanubrutinib on omeprazole, which was absorbed with a wide T max range with the delayed-release capsules used in the study.
Another limitation of this study is that substrates of CYP2B6 and CYP2C8 were not included in this study due to the lack of sensitive and validated probes for these enzymes to be used in a cocktail DDI Since zanubrutinib exhibits pH-dependent solubility, the potential impact of omeprazole on the exposure of zanubrutinib was assessed.
The AUC and C max values of zanubrutinib administered alone were similar to those when co-administered with omeprazole 20 mg.
Although the interaction was assessed after only a single dose of omeprazole, studies have shown its effect on gastric pH occurs within 1 h, with the maximum effect (increasing the gastric pH to approximately 4.5) occurring within 2 h. The lack of impact for omeprazole on the PK of zanubrutinib is consistent with results from a population PK analysis, showing that co-administration with proton-pump inhibitors and acid-reducing agents does not appear to significantly impact the PK of zanubrutinib.
In conclusion, at clinically relevant concentrations, zanubrutinib does not affect the PK of drugs metabolized by CYP2C9 or transported by BCRP. This result supports the recommendation that no dosage adjustments are necessary for drugs that are substrates of CYP2C9 and BCRP when given concomitantly with zanubrutinib.
Zanubrutinib decreased the systemic exposure of CYP3A-and CYP2C19-sensitive substrates with a mean reduction less than 50%.
The results from this study provide important information on the potential clinical impact of zanubrutinib on these CYPs and transporter activities.
the study data and was responsible for the decision to submit the manuscript for publication.

DATA AVAILABILITY STATEMENT
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