A Phase 1, Open‐Label, Fixed‐Sequence, Drug–Drug Interaction Study of Zanubrutinib with Rifabutin in Healthy Volunteers

Zanubrutinib is a second‐generation Bruton tyrosine kinase inhibitor that is primarily metabolized by CYP3A enzymes. Previous drug–drug interaction (DDI) studies have demonstrated that co‐administration of zanubrutinib with rifampin, a strong CYP3A inducer, reduces zanubrutinib plasma concentrations, potentially impacting activity. The impact of the co‐administration of zanubrutinib with less potent CYP3A inducers is unclear. This phase 1, open‐label, fixed‐sequence DDI study evaluated the pharmacokinetics, safety, and tolerability of zanubrutinib when co‐administered with steady‐state rifabutin, a known CYP3A inducer less potent than rifampin, in 13 healthy male volunteers (NCT04470908). Co‐administration of zanubrutinib with rifabutin resulted in a less than 2‐fold reduction of zanubrutinib exposures. Overall, zanubrutinib was well tolerated. The results of this study provide useful information for the evaluation of the DDI between rifabutin and zanubrutinib. In conjunction with safety and efficacy data from other clinical studies, these results will be taken into consideration to determine the appropriate dose recommendation of zanubrutinib when co‐administered with CYP3A inducers.

Activation of Bruton tyrosine kinase (BTK) in B cells triggers a cascade of signaling events that impact cell proliferation and survival. Although the exact mechanism underlying B-cell malignancies is unknown, aberrant BTK activation may drive the hallmarks of these malignancies and play a key role in their pathogenesis. To date, BTK inhibitors remain at the forefront of the treatment portfolio for various B-cell malignancies. 1, 2 Zanubrutinib, a second-generation covalent BTK inhibitor, was designed to maximize BTK receptor occupancy and minimize off-target kinase inhibition. 3,4 Prior pharmacokinetic (PK), safety, efficacy, and exposure-response analyses support the recommended 320 mg total daily dose of zanubrutinib (160 mg twice daily [BID] or 320 mg once daily [QD] with or without food). 5 Zanubrutinib was rapidly absorbed and eliminated after oral administration with a median time to peak plasma concentration (T max ) of 2 hours and a mean terminal elimination half-life (t 1/2 ) of 2-4 hours. 6 After multiple-dose administrations of zanubrutinib at doses ranging from 40 to 320 mg, there was a doseproportional increase in the maximum concentration (C max ) and the area under the plasma concentration-time curve (AUC) from time 0 extrapolated to infinity (AUC 0-∞ ); additionally, limited systemic accumulation was observed, which is consistent with the observed t 1/2 . 6 Hepatically, enzymatic metabolism via cytochrome P450, family 3, subfamily A (CYP3A) is considered the primary route of zanubrutinib metabolism. [6][7][8][9] Consequently, CYP3A modulators may impact zanubrutinib exposures, and dose adjustments may be necessary depending on the potency of the CYP3A inhibitors or inducers co-administered with zanubrutinib. Results from in vitro studies suggest that zanubrutinib is not a substrate of human breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP)1B1/1B3, organic cation transporter (OCT)2, or organic anion transporter (OAT)1/3 but is a potential substrate of the efflux transporter P-glycoprotein (P-gp). 10 Given the involvement of CYP3A enzymes in zanubrutinib metabolism, studies have been conducted to explore the interactions between zanubrutinib and various CYP3A modulators. A previously developed physiologically based pharmacokinetic (PBPK) model demonstrated an increase in zanubrutinib exposures when co-administered with different triazoles (voriconazole, fluconazole, and itraconazole). 11 A drug-drug interaction (DDI) study in healthy volunteers assessed the effect of co-administering zanubrutinib with a potent CYP3A inducer rifampin and CYP3A inhibitor itraconazole. 6 Therein, rifampin significantly impacted the bioavailability and apparent clearance of zanubrutinib, as demonstrated by 12.6-fold and 13.5fold decreases in zanubrutinib C max and AUC 0-∞ , respectively. Co-administration of zanubrutinib with itraconazole resulted in 2.6-fold and 3.8-fold increases in zanubrutinib C max and AUC 0-∞ , respectively. 6 In another DDI study in patients with B-cell malignancies, co-administration of zanubrutinib with the CYP3A inhibitor fluconazole resulted in 1.81-fold and 1.88fold increases in zanubrutinib C max and AUC 0-24 h , respectively, and co-administration of zanubrutinib with the CYP3A inhibitor diltiazem resulted in a 1.62fold increase for both C max and AUC 0-24 h . 12 Although the reduction of zanubrutinib exposures with the coadministration of a strong CYP3A inducer such as rifampin has been evaluated, the magnitude and extent of reduction with the co-administration of less potent CYP3A inducers is largely unknown.
The results of the clinical DDI study with rifampin indicated that co-administration of zanubrutinib with mild CYP3A inducers requires no dose reduction, whereas co-administration with strong CYP3A inducers should be avoided. 6 A dosage adjustment accounting for the magnitude of zanubrutinib exposure reduction with less potent or moderate CYP3A inducers may be warranted if co-administration cannot be avoided. Multiple treatments, including antibacterial and/or antifungal agents, may often be administered in conjunction with zanubrutinib to manage opportunistic infections in patients with B-cell malignancies. Some of these agents may be CYP3A inducers, and thus it is essential to further elucidate the PK profile of zanubrutinib when co-administered with CYP3A inducers. Rifabutin is a first-line therapeutic alternative to rifampin and is a clinically relevant anti-infective agent used in patients with B-cell malignancies. 13 Other known CYP3A inducers that are clinically relevant in their induction potential include antiviral (efavirenz and etravirine), antihypertensive (bosentan), anticancer (dabrafenib, lorlatinib, pexidartinib, and sotorasib), and antiseizure (cenobamate, phenobarbital, and primidone) medications.
Rifabutin is primarily metabolized by CYP3A enzymes, and multiple doses of rifabutin are associated with the induction of enzymes of the CYP3A subfamily. 14 Following a single oral dose of 300 mg, rifabutin was readily absorbed with a T max ranging from 2 to 4 hours and was slowly eliminated from plasma with a mean t 1/2 of 45 hours (standard deviation 17 hours; range 16-69 hours). 15 Although the systemic levels of rifabutin after multiple doses decreased by 38% due to auto-induction, its t 1/2 remained unchanged. 15 To achieve maximal induction of CYP3A, 300 mg rifabutin QD was administered for 8 days to reach a steady state. 16 Various studies have established rifabutin as a less potent CYP3A inducer relative to that of rifampin. 15,[17][18][19] One study reported the AUC 0-∞ geometric mean ratio (GMR) after co-administration of rifabutin with the index CYP3A substrate midazolam as 0.31 (≈3.2-fold reduction in exposure). 18 Prior DDI studies and PBPK models have suggested that moderately potent CYP3A inducers may decrease the AUC 0-∞ of CYP3A substrates such as zanubrutinib by 2-to 3-fold. 17,20 This open-label, fixed-sequence, phase 1 study was conducted to determine the effect of CYP3A induction by rifabutin on zanubrutinib PK when co-administered in healthy male volunteers. The results of this study in conjunction with safety and efficacy data from other clinical studies can be used to support the appropriate dose recommendation of zanubrutinib when coadministered with CYP3A inducers.

Study Design and Volunteers
This was a single-center, phase 1, open-label, fixedsequence clinical DDI study to investigate the effect of CYP3A induction by steady-state rifabutin on a single-dose PK of zanubrutinib in healthy volunteers (NCT04470908 of Helsinki, and local regulatory requirements. All volunteers provided written, informed consent before study entry. The Midlands Independent Review Board (MLIRB [now WCG IRB], Overland Park, KS, USA) reviewed and approved the protocol at the respective study center. Volunteers were screened for eligibility during the screening period (from day −28 through day −2). Those who met all the inclusion criteria and none of the exclusion criteria were admitted into the clinical research unit (CRU) on day −1 and confined to the CRU until discharge on day 13. All volunteers received study drugs in a fixed sequence as shown in Figure 1. On day 1, volunteers received a single oral dose of 320 mg zanubrutinib after an overnight fast of 8-10 hours. On days 3-10, volunteers were administered an oral dose of 300 mg rifabutin QD with food, and on day 11, volunteers were administered a single oral dose of 320 mg zanubrutinib and 300 mg rifabutin QD after an overnight fast of 8-10 hours. Volunteers were discharged on day 13 after satisfactory completion of study-related procedures.
Key inclusion criteria included healthy men of any race, between ages 18 and 65 years, with a body mass index (BMI) between 18.0 and 32.0 kg/m 2 , and good health determined by the investigator's assessment of medical history, physical examination, vital signs, electrocardiograms (ECGs), and laboratory tests at screening. Key exclusion criteria included considerable history or clinical manifestation of any metabolic, allergic, dermatological, hepatic, renal, hematological, pulmonary, cardiovascular, gastrointestinal, neurological, respiratory, endocrine, or psychiatric disorder; evidence of any infections (e.g., bacterial, viral, fungal, and parasitic) within 4 weeks before the first dose of study drug; history of significant hypersensitivity, intolerance, or allergy to any drug compound, food, or other substance; history of stomach or intestinal surgery or resection that would potentially alter absorption and/or excretion of orally administered drugs; and use or intended use of any medications/products known to alter drug absorption, metabolism, or elimination processes, including St. John's Wort, within 30 days before check-in.

Treatments
Volunteers received a single oral dose of zanubrutinib (320 mg) as 4 × 80 mg capsules in the fasted state on days 1 and 11. A single dose of rifabutin (300 mg) was given orally as 2 × 150 mg capsules with food on days 3-10 and in the fasted state on day 11.
On PK sampling days (days 1 and 11), zanubrutinib and rifabutin were administered after an overnight fast of 8-10 hours. Each dose of zanubrutinib and rifabutin was administered orally with approximately 240 mL of room temperature water. When zanubrutinib and rifabutin were administered concurrently (on day 11), an additional amount (up to 240 mL) of room temperature water was allowed to be administered.

Bioanalytical Methods
Zanubrutinib plasma concentrations were measured using a validated high-performance liquid chromatography (HPLC) with tandem mass spectrometry (LC-MS/MS; XenoBiotic Laboratories, Inc., Plainsboro, NJ, USA) assay with a lower limit of quantification (LLOQ) of 1.00 ng/mL. In this method, the analyte (zanubrutinib) and the internal standard (IS; BGB-4257) were extracted from human plasma samples by protein precipitation. The supernatants were chromatographed using reversed-phase HPLC with a Supelco Ascentis Express C18 analytical column (Sigma-Aldrich/Supelco, Bellefonte, PA, USA) with 0.1% formic acid in water and methanol:acetonitrile (1:1) as the mobile phase. The compound was detected with mass spectrometric detection, which was performed on a triple quadrupole tandem mass spectrometer API 4000 (Applied Biosystems, MDS SCIEX, Singapore) equipped with an ESI source operated in the positive ionization mode. The source parameters were collision energy 52 eV, collision gas 6 psi, gas 1 and gas 2 50 psi, and curtain gas 20 psi. Quantification was obtained by using multiple reaction monitoring modes of the transitions of mass to charge ratio [m/z] 472.2 →290.2 for BGB-3111 and m/z 460.3 → 290.2 for BGB-4257.
This method was developed and validated to determine zanubrutinib concentrations in human plasma with dipotassium ethylenediaminetetraacetic acid (K 2 EDTA) as the anticoagulant. The calibration range was 1.00-1000 ng/mL. The intra-and inter-run precision (coefficient of variation, % CV) at each quality control level (LLOQ QC, low QC, mid QC, and high QC) was ≤5.18%. The mean accuracy (% bias) at each QC level was between −3.87% and 6.67% from its respective nominal concentration. Zanubrutinib has been demonstrated to be stable in human plasma with K 2 EDTA as the anticoagulant for five cycles of freeze (−20°C) and thaw (room temperature) and 8.5 hours at room temperature under normal laboratory lighting.

Pharmacokinetic Analyses
Noncompartmental analysis was conducted using Phoenix ® WinNonlin TM version 8.1 (Certara USA, Inc., Princeton, NJ, USA). PK parameters were derived for zanubrutinib alone and in combination with rifabutin, including AUC, C max , T max , t 1/2 , apparent total oral clearance (CL/F), and apparent volume of distribution during the terminal elimination phase.

Safety Assessments
Adverse events (AEs; classified based on the Medical Dictionary for Regulatory Activities [MedDRA] Version 24.0), serious adverse events (SAEs), clinical laboratory tests, physical examinations, and vital signs were monitored and recorded. Safety was measured by the incidence, timing, and severity of treatmentemergent adverse events (TEAEs), according to the National Cancer Institute Common Terminology Criteria for Adverse Events Version 5.0 (NCI CTCAE v5.0). An AE that started during or after the first dose or started before the first dose and increased in severity after the first dose was defined as a TEAE.

Statistical Analyses
The study sample size was based on precedent set by other PK studies of a similar nature and was not based on power calculations. Target enrollment was 15 volunteers to ensure that at least 12 volunteers completed the study.
All patients who received ≥1 dose of zanubrutinib were included in the safety analysis. Incidence of AEs, severity of AEs, change from baseline values, and abnormal values for all relevant parameters such as AEs, laboratory parameters, vital signs, and physical examination were assessed for safety and tolerability.
The GMRs of the PK parameters of zanubrutinib with and without co-administration of rifabutin and the associated 90% confidence intervals (CIs) were constructed based on the least squares mean (LSM) and intra-subject CV from a mixed-effects model of log-transformed PK parameters. The geometric least squares mean (GLSM) and their ratios were obtained by taking the exponential of the corresponding estimates of LSM and their differences on the natural logarithm scale, where ratio = test/reference. Within-subject CV was calculated as 100 × square root[exp(mean square error from the analysis model) -1]. Estimates of GMRs and the corresponding 90% CIs were derived for the comparisons of the area under the concentrationtime curve from time zero to the time of the last quantifiable concentration (AUC 0-t ), AUC 0-∞ , and C max for zanubrutinib co-administration with rifabutin (test) versus zanubrutinib alone (reference).

Demographics and Baseline Characteristics
A total of 13 volunteers were enrolled and 12 completed the study. One volunteer was unable to return for the follow-up visit and was therefore noted as lost to followup. All 13 volunteers completed the study through day 13. Baseline demographics are shown in Table S1.    Volunteers were men aged between 28 and 63 years with BMI values ranging from 23.5 to 31.3 kg/m 2 . Twelve of the volunteers were White and one volunteer was an American Indian or Alaska Native.

Pharmacokinetics
After administration of zanubrutinib alone (day 1) and co-administration with 300 mg rifabutin (day 11), zanubrutinib was rapidly absorbed with a median T max of 1.5 and 2.0 hours postdose, respectively (Figure 2). The zanubrutinib arithmetic mean t 1/2 was similar when zanubrutinib was administered alone (7.2 hours) compared to when zanubrutinib was co-administered with rifabutin (7.0 hours; Table 1).
The arithmetic mean plasma concentration-time profiles and PK parameters of zanubrutinib in the absence and presence of rifabutin are presented in Figure 2 and Table 2, respectively. The results of statistical analyses of AUCs and C max are summarized in Table 2. Plasma concentrations of zanubrutinib were significantly lower after co-administration of 320 mg zanubrutinib with 300 mg rifabutin compared with the administration of 320 mg zanubrutinib alone. The GLSM for AUC 0-t , AUC 0-∞ , and C max values were lower when zanubrutinib was co-administered with rifabutin than when zanubrutinib was administered alone, with GLSM ratios of 0.57, 0.56, and 0.52 for AUC 0-t , AUC 0-∞ , and C max , respectively (Table 2).

Safety
Zanubrutinib monotherapy and in combination with rifabutin demonstrated a favorable safety and tolerability profile in all volunteers.
Overall, 6 of 13 volunteers (46.2%) experienced 7 TEAEs, all of which were grade 1 in severity; 1 TEAE was related to zanubrutinib on day 1 (Table S2). All TEAEs resolved by the end of the study, no volunteers discontinued study treatment because of a TEAE, and no deaths or SAEs were reported.

Discussion
This was a single-center, phase 1, open-label, fixedsequence clinical DDI study to investigate the effect of CYP3A induction by steady-state rifabutin on the single-dose PK of zanubrutinib in healthy volunteers and to evaluate dose-adjustment recommendations. Overall, co-administration of zanubrutinib with rifabutin resulted in a decrease in zanubrutinib exposure by approximately 44% for AUC 0-t and AUC 0-∞ , and 48% for C max , respectively, compared with administration of zanubrutinib alone. Thus, in the current study, rifabutin reduced zanubrutinib exposure to 56% of control (based on the ratios of AUC 0-∞ ), compared to rifampin reducing exposures to 7.4% of control in a previous DDI study. 6 A 1.8-to 1.9-fold decrease in exposure (AUC and C max ) when zanubrutinib was coadministered with rifabutin is significantly lower than the 13-fold decrease in exposures observed for rifampin in a previous DDI study. 6 Patients with B-cell malignancies are at an increased risk of infection due to immune defects associated with the disease. 21,22 The current therapies to treat opportunistic infections, such as mycobacterial infections, include rifampin and rifabutin. Since many anticancer agents are metabolized by CYP3A, rifabutin may be considered a clinically relevant anti-infective agent in patients with B-cell malignancies. In zanubrutinib clinical trials, rifabutin has been prescribed for the treatment of mycobacterial infections and was selected in the current study as it is more clinically relevant as a CYP3A inducer in the target patient population of zanubrutinib. Hence, this study was conducted to elucidate the DDI potential of zanubrutinib when co-administered with rifabutin, specifically. Additionally, these study results could aid in the development and validation of a PBPK model for rifabutin, which would be a useful development tool to predict the clinical DDI potential of rifabutin when used with other anticancer agents that are CYP3A substrates.
In a previous clinical DDI study in healthy volunteers, the reported mean t 1/2 was 6.8 hours, where PK samples were collected up to 48 hours post dose. 6 In the current study, the t 1/2 of zanubrutinib was 7 hours, where PK samples were collected up to 36 hours post dose; this was longer than the reported t 1/2 of 2-4 hours for a single oral dose of 160 or 320 mg zanubrutinib in patients with B-cell malignancies (where samples were collected up to 8 hours post dose). 4,6 All TEAEs resolved by the end of the study, no volunteers discontinued because of a TEAE, and no deaths or SAEs were reported during the study. Clinical laboratory evaluations, vital signs, 12-lead ECGs, and physical examinations were unremarkable and showed no apparent association with plasma drug concentrations of zanubrutinib.
The results of this study provide useful information for the evaluation of clinical DDI between rifabutin and zanubrutinib, and management of clinical DDIs for zanubrutinib when a moderately potent CYP3A inducer was co-administered. In conjunction with safety and efficacy data from other clinical studies, the results from this study further contribute to the totality of data to help determine the appropriate dose recommendation of zanubrutinib when co-administered with CYP3A inducers. In addition, our study is one of the few reported clinical studies that could be used to aid in the development and validation of a PBPK model for rifabutin, which would be a useful tool to predict the impact of CYP3A inducers similarly potent to rifabutin on CYP3A substrates, especially in patients with B-cell malignancies.

Conclusions
The results of the current study show that the coadministration of zanubrutinib with a CYP3A inducer, rifabutin, resulted in an approximately 2-fold reduction in exposures of zanubrutinib. No new safety signals were identified. Single doses of 320 mg zanubrutinib administered alone or co-administered with 300 mg rifabutin were well tolerated in healthy volunteers in this study.

Funding Information
This study was sponsored by BeiGene.

Conflicts of Interest
Bilal Tariq and Stephanie Conto are employed by BeiGene and hold stock with BeiGene. Srikumar Sahasranaman, Ying C. Ou, and Aileen Cohen are employed by BeiGene, are in a leadership position at BeiGene, hold stock with BeiGene, and have received funding for travel and/or research funding from BeiGene.