Abstract
The absorption, metabolism, and excretion of ibrutinib were investigated in healthy men after administration of a single oral dose of 140 mg of 14C-labeled ibrutinib. The mean (S.D.) cumulative excretion of radioactivity of the dose was 7.8% (1.4%) in urine and 80.6% (3.1%) in feces with <1% excreted as parent ibrutinib. Only oxidative metabolites and very limited parent compound were detected in feces, and this indicated that ibrutinib was completely absorbed from the gastrointestinal tract. Metabolism occurred via three major pathways (hydroxylation of the phenyl (M35), opening of the piperidine (M25 and M34), and epoxidation of the ethylene on the acryloyl moiety with further hydrolysis to dihydrodiol (PCI-45227, and M37). Additional metabolites were formed by combinations of the primary metabolic pathways or by further metabolism. In blood and plasma, a rapid initial decline in radioactivity was observed along with long terminal elimination half-life for total radioactivity. The maximum concentration (Cmax) and area under the concentration-time curve (AUC) for total radioactivity were higher in plasma compared with blood. The main circulating entities in blood and plasma were M21 (sulfate conjugate of a monooxidized metabolite on phenoxyphenyl), M25, M34, M37 (PCI-45227), and ibrutinib. At Cmax of radioactivity, 12% of total radioactivity was accounted for by covalent binding in human plasma. More than 50% of total plasma radioactivity was attributed to covalently bound material from 8 hours onward; as a result, covalent binding accounted for 38% and 51% of total radioactivity AUC0–24 h and AUC0–72 h, respectively. No effect of CYP2D6 genotype was observed on ibrutinib metabolism. Ibrutinib was well-tolerated by healthy participants.
Introduction
Imbruvica (ibrutinib) (Reference ID: 3395788, prescribing information; http://www.accessdata.fda.gov/drugsatfda_docs/label/2013/205552lbl.pdf) is a first-in-class, potent, orally administered, covalently binding inhibitor of Bruton’s tyrosine kinase in B-cell malignancies, currently under development (Herman et al., 2011; Winer et al., 2012; Brown, 2014). The U.S. Food and Drug Administration declared ibrutinib as a breakthrough therapy in February 2013 for the treatment of relapsed/refractory mantle cell lymphoma and gave approval for this indication in November 2013 (Wang et al., 2013). Recently, in February 2014 the Food and Drug Administration provided accelerated approval for ibrutinib for chronic lymphocytic leukemia in patients who had received at least one previous therapy (Byrd et al., 2013; O’Brien et al., 2014). Ibrutinib could change the paradigm for treatment of patients with mantle cell lymphoma and chronic lymphocytic leukemia wherein intensive combination therapies are used.
Pharmacokinetic studies in rodents demonstrated that ibrutinib is rapidly absorbed following oral administration and first-pass metabolism may be a reason for low oral bioavailability (Honigberg et al., 2010). A mass balance study with a racemic mixture of 14C-ibrutinib (R-enantiomer) and 14C-PCI-32769 (S-enantiomer) in rats indicated hepatic metabolism as a primary route of elimination. Also, in vitro studies have shown that ibrutinib is extensively metabolized primarily by CYP3A4/5 and to a lesser extent by CYP2D6 enzymes (see the Imbruvica prescribing information). Pharmacokinetic findings of studies in humans also showed rapid absorption and elimination of ibrutinib (Advani et al., 2013). Also, ibrutinib does not have toxic effects on normal T cells, which distinguishes it from most regimens used for chronic lymphocytic leukemia (Byrd et al., 2013). The objective of this study was to investigate the absorption, metabolic pathways, and excretion routes of ibrutinib in healthy men after administration of a single oral dose of 140 mg (5 mg/ml solution) of unlabeled ibrutinib admixed with 14C-ibrutinib. Additionally, safety was also evaluated.
Materials and Methods
Study Participants
Healthy, nonsmoking men, 30–55 years old (inclusive) with a body mass index between 18 and 30 kg/m2 (inclusive) and body weight ≥50 kg were enrolled in the study. To assess the relative contribution of CYP3A4 versus CYP2D6 metabolism, 2 CYP2D6-poor metabolizers (evident from genotype analysis at screening) were enrolled. Various 2D6 genotypes were tested for each participant. Participants 1 and 2 expressed *5/*5 and *4/*4, respectively (Supplemental Material). Based on the Covance Affymetrix software (Affymetrix, Santa Clara, CA), the phenotypes were predicted to be poor metabolizers. This was done prospectively as part of the screening process. Samples were analyzed similarly for 3A4 and 3A5. All subjects had predicted 3A4 phenotypes as extensive metabolizers and all subjects had predicted 3A5 phenotypes as poor metabolizers. Participants with irregular bowel movements or clinically significant abnormal values for hematology, coagulation, and platelet function were excluded from the study. The Independent Ethics Committee approved the protocol and the study was conducted in accordance with the ethical principles, which have their origin in the Declaration of Helsinki and are consistent with good clinical practices and applicable regulatory requirements. All patients provided a written informed consent before enrollment.
Study Design
This study was a phase 1, open-label, single-center, single-dose study conducted from August 2012 to September 2012. The study consisted of screening (day 28 to day 2), baseline (day 2 to day 1; admission to study unit on day 2), open-label (15 days), end-of-study (EOS) (up to day 15), and follow up phases (30 days after EOS). Whole blood, plasma, urine, and feces were collected for a minimum of 7 days after dosing (i.e., until day 8), and possibly for up to seven more days (i.e., up to day 15) if radioactivity in the excreta (i.e., 24 hour urine and feces) on either day 6 or 7 accounted for ≥2% of the total administered dose or if <7 fecal samples were obtained by day 8. The occurrence of adverse events (AEs) was monitored throughout the study. A telephonic follow up was conducted 30 days after discharge from the study unit to collect information on any AEs.
Study Medication
An oral solution of 14C-ibrutinib targeting 5 mg base/Eq/ml, a radioactivity concentration of 52.9 kBq/ml, and a specific radioactivity of 10.6 kBq/mg base/Eq was prepared by dilution of high specific activity 14C-ibrutinib with a 30% hydroxypropyl-β-cyclodextrin formulation of unlabeled ibrutinib. The 14C label is located on the carbonyl moiety.
On day 1, following an overnight fast, each of the six participants received a single oral solution dose of 140 mg ibrutinib containing 1480 kBq (40 μCi) of 14C-ibrutinib. The radiation burden received by a human subject after an orally administered radioactivity of 1480 kBq (or 40 µCi) associated with 14C-ibrutinib has been calculated, using formulas and data recommended by the International Commission of Radiation Units and Measurements ((International Commission on Radiologic Protection, 2007). Evaluations of the absorbed radiation in single organs were based on data from a quantitative whole-body autoradiography study measuring the tissue distribution of the total radioactivity in male Lister hooded rats after administration of a single oral gavage dose of a solution of 14C-ibrutinib at 10 mg/kg. The calculations of the radiation dose to the gastrointestinal tract and the bladders (stomach, gall bladder, small intestine, upper and lower large intestine, and urinary bladder) were based on the excretion profile in rats, and have been made assuming that their exposure results entirely from radioactive material in their contents, and that the mean residence times and throughput of those contents have standard values. The weighted dose equivalents of the International Commission on Radiologic Protection recommended organs have been summed to obtain the committed effective dose to the whole body of man, after oral administration of radiolabeled ibrutinib. Oral administration of 1480 kBq (or 40 µCi) 14C-ibrutinib was calculated to result in a total radiation burden of 916 µSv. An effective dose (total body) between 100 and 1 000 µSv is categorized as a category IIa project (a minor level of risk, covering doses to the public from controlled sources) (International Commission on Radiologic Protection, 1991). The vial used for drug administration was rinsed 3× with plain noncarbonated water and the participants consumed the rinsing fluids along with additional plain noncarbonated water (240 ml).
Pharmacokinetic Evaluations
Sample Collection.
Blood samples were collected for determination of ibrutinib and PCI-45227 concentrations in blood and plasma, and total radioactivity predose and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24, 48, and 72 hours postdose. Plasma protein binding was evaluated using predose samples. Covalent binding of total radioactivity to plasma proteins was evaluated in pooled human samples collected at 1, 2, 4, 8, 24 and 72 hours postdose. The plasma pellets, generated during the preparation of plasma (for metabolite identification) were used to further investigate possible covalent binding of ibrutinib and/or metabolites to proteins with different extraction steps. Plasma pellets were redissolved in 1% sodium dodecyl sulfate; proteins were homogenized in a mixture of acetonitrile/isopropyl alcohol/0.02% formic acid (6/3/1 v/v/v) and precipitated following centrifugation. Resuspension of the resulting pellet in the same organic solvent mixture and centrifugation was repeated twice. Radioactivity in the different supernatant fractions and in the final pellet (redissolved in 4 M urea + 1% sodium dodecyl sulfate) was determined by liquid scintillation counting. Urine and feces (analyzed per stool) were collected predose and at 0–2, 2–4, 4–8, 8–24, 24–48, 48–72, 72–96, 96–120, 120–144, and 144–168 hours postdose. However, for metabolic profiling, blood and plasma were collected predose and at 1, 2, 4, 8, 24, and 72 hours postdose. For metabolic profiling of urine and feces, a selection of samples was made. Baseline glomerular filtration rate was calculated based on 24 hour urine collection and serum creatinine measurement on day 1.
Bioanalytical Procedures.
Plasma samples were analyzed to determine concentrations of ibrutinib and PCI-45227 using a validated liquid chromatography tandem mass spectrometry (MS)/MS method. Blood and urine samples were analyzed to determine concentrations of ibrutinib and PCI-45227 using qualified assays (unpublished data). Total radioactivity (14C) was measured in blood, plasma, urine, and feces using liquid scintillation counting in a Packard Tri-Carb 2900 TR liquid scintillation spectrometer (PerkinElmer, Waltham, MA). Blood and feces residues were combusted in a Packard Sample Oxidizer 307 (PerkinElmer) prior to liquid scintillation counting. Concentrations of 14C-ibrutinib and 14C-metabolites were measured in blood, plasma, urine, and feces. Metabolic profiling was performed on selected blood, plasma, urine, and feces samples using reversed phase ultra-performance liquid chromatography (UPLC) with on-line [scintillation solvent: Ultima-Flo-M (14C-label) (PerkinElmer), 3.8 ml/min using switching valve] or off-line radioactivity detection (TopCount/Deepwell Lumaplate combination, Perkin Elmer) and on-line MS (or MS/MS) data acquisition for metabolite identification (Supplemental Material). The UPLC system was Acquity Binary Solvent Manager (Waters Corporation, Milford, MA) and a Waters 2777 CTC_Pal (Waters Corporation) injector equipped with a UV detector (Acquity PDA); the UPLC column was an Interchim Uptisphere Strategy 100 Å C18-2, 2.2 µm, 2 × (150 mm × 3.0 mm) (i.d.) (Interchim, Montluçon Cedex, France) (kept at 60°C, mobile phase flow rate: 0.8 ml/min). The mass spectrometer (QTOF Ultima, Waters Corporation) was equipped with a dual electrospray ionization probe and calibrated with a sodium formate solution delivered through the sample spray. The QTOF Ultima data (MS and MS/MS) were acquired in the centroid mode with a variable scan time (0.5–1.0 seconds). All data were processed using the Masslynx MS software (Waters Corporation).
Sample Preparation
Feces.
Pooled feces homogenates were extracted using a three-step extraction procedure with acetonitrile/isopropyl alcohol/0.02% formic acid (6/3/1) as the extraction solvent. Dried down fecal extracts were reconstituted in dimethylsulfoxide (1000 μl) prior to injection for UPLC analysis (750 μl) with solid phase extraction (SPE) [Interchim Uptisphere Strategy 100Å C18-2, 2.2 μm, 2 × (150 mm × 3.0 mm) i.d.] and online counting.
Urine.
Urine was thawed at room temperature and centrifuged. After centrifugation, 28.0 ml or 36.0 ml of the supernatant was injected for UPLC analysis using online SPE [Interchim Uptisphere Strategy 100Å C18-2, 2.2 μm, 2 × (150 mm × 3.0 mm) i.d.] and on-line detection of total radioactivity.
Blood.
Blood samples were thawed at room temperature. Blood (24 ml pools at 1, 2, 4, 8, 24, and 72 hours) was precipitated with acetonitrile (1/1, v/v). After vortexing and centrifugation, approximately 28.8–30.6 ml of the supernatant was injected for UPLC analysis using online SPE [Interchim Uptisphere Strategy 100 Å C18-2, 2.2 μm, 2 × (150 mm × 3.0 mm) i.d.] and off-line counting of total radioactivity using a TopCount/Deepwell Lumaplate combination (Perkin Elmer).
Plasma.
Protein precipitation for plasma sample preparation was performed using acetonitrile (1/1, v/v). Individual 8 ml samples were analyzed at 1, 2, and 4 hours, and three 24 ml pools across participants were made for the 8, 24, and 72 hour plasma samples. After vortexing and centrifugation, total radioactivity was determined in a subsample using liquid scintillation counting to determine the recovery of total radioactivity. The supernatant was injected for UPLC analysis using online SPE [Interchim Uptisphere Strategy 100Å C18-2, 2.2 μm, 2 × (150 mm × 3.0 mm) i.d.] and off-line counting of total radioactivity using a TopCount/Deepwell Lumaplate combination (Perkin Elmer).
Pharmacokinetic Parameters.
The following pharmacokinetic parameters were estimated from total radioactivity or unchanged drug and metabolite concentrations in whole blood, plasma, urine, and feces samples: maximum concentration (Cmax); time to reach Cmax; area under the concentration-time curve (AUC) from time 0 to 24 hours (AUC24); AUC from time 0 to time of the last quantifiable concentration (AUClast); AUC from time 0 to infinite time (AUC∞), calculated as the sum of AUClast and Clast/λz, in which Clast is the last observed quantifiable concentration; elimination half-life time to last quantifiable concentration; apparent volume of distribution based on the terminal phase, calculated as D/(λz*AUC∞); renal clearance of drug, calculated as Ae∞/AUC∞; and total clearance of drug after extravascular administration, calculated as D/AUC∞.
Pharmacogenomic Evaluations.
Blood samples (10 ml) of participants were collected for pharmacogenomics evaluation at screening to determine their CYP2D6 metabolizer status by genotyping. The DNA samples from enrolled participants were also analyzed for CYP3A4 and CYP3A5 status.
Safety Evaluations.
Safety and tolerability were evaluated throughout the study and consisted of assessment of AEs from the time of obtaining informed consent to EOS or early withdrawal assessment. Scheduled safety assessments of 12-lead ECG, physical examination, vital signs, and clinical laboratory investigations (hematology, serum chemistry, and urinalysis) occurred before the administration of the study drug and at the EOS assessment (after collection of the final fecal sample). The AE severity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events grading system, version 4.03 (http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf).
Statistical Methods
Sample Size.
The sample size of six patients with a minimum of four to complete the study was considered adequate to generate meaningful descriptive measures of the pharmacokinetics (absorption, metabolism, and excretion) of ibrutinib.
Analysis Sets.
All enrolled participants completed the study and were included in the pharmacokinetic and safety analysis population.
Analysis.
Individual and mean blood and plasma ibrutinib and PCI-45227 concentration-time profiles were plotted. Blood and plasma concentration data at each time point were summarized with mean, median, geometric mean, minimum, maximum, S.D., and CV%. All estimated pharmacokinetic parameters of ibrutinib and PCI-45227 were summarized with mean, median, geometric mean, minimum value, maximum value, S.D., and CV%. Individual genotype status for CYP3A4/5 and CYP2D6 was listed.
Results
Study Participants
All participants (n = 6) were white healthy men with median (range) age of 51 years (35–55 years); baseline weight of 82.5 (61.7–87.3) kg; height of 176 (163–179) cm; and baseline body mass index 25.8 (22.7–28.9) kg/m2.
Pharmacokinetic Results
Drug Concentration Measurements and Total Radioactivity in Whole Blood and Plasma.
After dosing, mean concentration-time profiles in blood and plasma were similar for ibrutinib and PCI-45227 (Fig. 1). Mean ibrutinib and PCI-45227 concentrations were lower than the total radioactivity in both blood and plasma (Fig. 2). All participants had measurable blood concentrations of total radioactivity for 4 hours after dosing. In total, five out of six participants had measurable radioactivity for 8 hours and only one participant had measurable radioactivity at 24 hours. Concentrations of total radioactivity in plasma were measurable for all participants for 72 hours after dosing. The fact the blood concentrations could not be measured at a later time point was due to a higher limit of quantification for blood compared with plasma. In blood and plasma, a rapid initial decline in radioactivity was observed along with long terminal half-life for total radioactivity. Most of the total radioactivity was associated with plasma rather than the cellular components of whole blood (Fig. 2). The mean blood to plasma concentration ratio of total radioactivity was approximately 0.7.
Mean (S.D.) logarithmic-linear (A) ibrutinib and (B) PCI-45227 concentration-time profiles in plasma and whole blood.
Mean (S.D.) ibrutinib and PCI-45227 concentration-time and total radioactivity-time profiles in (A) blood and (B) plasma.
Ibrutinib and PCI-45227 in Urine.
In total, five of the six participants had measurable but very low levels of ibrutinib in urine from 0 to 2 hours after dosing and one participant also had measurable but very low levels between 2 and 4 hours. The mean total amount excreted was 0.000176% of the dose. PCI-45227 was measurable in urine in all participants through 72 hours after dosing and in 2/6 participants through 96 hours. The mean total amount (ibrutinib and PCI-45227) excreted was 0.12% of the dose.
Total Radioactivity in Urine and Feces.
The mean (S.D.) cumulative excretion of radioactivity in urine was 7.8% (1.4%) of the dose and the majority of the radioactivity was excreted within 24 hours after dosing. In feces, mean (S.D.) cumulative excretion of radioactivity accounted for 80.6% (3.1%) of the dose and the majority of the radioactivity was excreted within 48 hours after dosing (Fig. 3, A–D). Total excretion in the 0–168 hour period postdose amounted to 88.5% (4.3%).
(A) Mean (S.D.) cumulative excretion of total radioactivity in urine and feces. (B) Representative radio-chromatograms for human feces after a single dose of 140 mg 14C-ibrutinib; pools of the stools collected at 6 and 10 hours postdose (B1) and pools of the stools collected at 23, 36, and 53 hours postdose (B2) of a study participant. (C) Representative radio-chromatogram for human urine after a single dose of 140 mg 14C-ibrutinib. (D) Radio-chromatogram plasma—off-line counting—representative sample at 1 hour postdose.
Pharmacokinetic Parameters.
The pharmacokinetic parameters of ibrutinib and PCI-45227 were similar for blood and plasma (Table 1). Total radioactivity Cmax and AUC in both plasma and blood were several-fold higher than that for ibrutinib and PCI-45227. Mean (S.D.) unbound ibrutinib was 2.3% (0.3%) or 97.7% bound. Creatinine clearance ranged from 112 to 159 ml/min and confirmed that participants had normal renal function.
Mean (S.D.) pharmacokinetic parameters and total radioactivity of ibrutinib and PCI-45227
Metabolite Identification
Three main metabolic pathways for ibrutinib were distinguished based on the nature of the identified metabolites: hydroxylation of the phenyl (M35), opening of the piperidine with further reduction to a primary alcohol (M34) or oxidation to a carboxylic acid (M25), and epoxidation of the ethylene on the acryloyl moiety followed by hydrolysis to a dihydrodiol (M37, also referred to as PCI-45227). Most remaining metabolites were formed by combinations or by further secondary metabolism of these main metabolites (Fig. 4).
Proposed metabolic scheme.
In feces, 12 metabolites and unchanged drug were identified as the main entities (40% of the administered dose) and ibrutinib was present in minor quantity (0.77% of the administered dose). The main metabolites identified in feces were M20 (oxidation product of M25), M25 and M34, each between 5% and 10% of the dose. Of note, M34 appeared to coelute with other metabolites in urine and feces, but was the main entity under the radioactive peak using MS detection.
The observed metabolites in blood and feces were very similar (Fig. 5). In urine, the majority of the observed metabolites were assigned to the piperidine ring-opening metabolic pathway (3.84% of the administered dose). Table 2 provides an overview of the identified metabolites.
Mass balance bar graph showing UD (unchanged drug) and metabolite abundance in feces and urine expressed as percentage of the dose.
Identification of in vivo metabolites of ibrutinib
Because the metabolites were generally below the quantification limit of the radio-detector, metabolite profiles in blood and plasma could only be qualitatively compared, and were found to be similar. The main circulating entities in blood and plasma were M21 (sulfate conjugate of a monooxidized metabolite on the phenoxyphenyl), M25, M34, M37, and unchanged drug. These metabolites could be observed predominantly at 1 hour post oral administration and rapidly eliminated through 4 hours postdose, except for M37, which was still detectable at 24 hours post oral administration at 140 mg. Small amounts of M39 and M40 (both addition of one oxygen on the piperidine ring) were also detected in blood and plasma. Minor unlabeled metabolites were observed as well. M23 (unlabeled metabolite resulting from amide hydrolysis) and downstream metabolites M30 (+O–2H), M1 (sulfate of monooxidized M23), and M4 (glucuronide of monooxidized M23) were detected with MS. In plasma M23, M30, M1, and M4 were observed; M1 was not observed in blood. The abundance of these unlabeled metabolites in blood (based on MS response) was comparable to the abundance in plasma.
The time course of covalently bound radioactivity in plasma was very flat with an apparent maximum at 8 hours postdose while the peak of total radioactivity in plasma (covalently and noncovalently bound) was observed at 1 hour postdose. A total 6-fold decline in total plasma radioactivity and slight decrease in covalently bound radioactivity was observed from peak to 24 hours postdose, with the apparent long half-life confirmed based on the 72 hour time point. Based on a comparison of Cmax values, at Cmax of radioactivity 12% of total radioactivity was accounted for by covalent binding in human plasma. Covalent binding accounted for 38% and 51% of total radioactivity AUC0–24 h and AUC0–72 h, respectively (Table 3).
Levels of covalently bound radioactivity in plasma
As part of an in vitro plasma protein binding estimation, radiolabeled ibrutinib was spiked to human plasma at 500 ng/ml and covalent binding was assessed at various time points. Covalent binding gradually increased from 7.3% at 1 hour to 24.9% at 8 hours. Also, an experiment assessing the covalent binding of ibrutinib to human plasma proteins was conducted at various concentrations (50, 500, and 1000 ng/ml) up to 30 minutes and did not highlight a concentration dependency of the covalent binding; at 30 minutes covalent binding averaged about 3%. A covalent binding experiment using purified human plasma proteins (α1 AGP and human serum albumin) and ibrutinib at 150 ng/ml showed that ibrutinib was mainly binding covalently to human serum albumin (10.2%) and minimally to α1 AGP (0.4%–0.6%).
Pharmacogenomics
No difference in unchanged ibrutinib exposure (Cmax and AUC) was observed between CYP2D6 poor metabolizers (n = 2) and extensive metabolizers (n = 4). PCI-45227 exposure (Cmax and AUC) for one of the CYP2D6-poor metabolizers was approximately 40% higher than the median values for all other participants.
Safety
Two out of six participants in the safety analysis set experienced at least one AE. The reported AEs were abdominal pain, diarrhea, headache, and skin irritation with grade 1 or 2 severities and were reported one time. No clinically relevant changes were observed in physical examination, clinical laboratory analyses, vital signs measurements, and ECGs.
Discussion
In this open-label phase 1 pharmacokinetic study, the absorption, metabolic pathways, and route of excretion of orally administered radiolabeled ibrutinib were studied. Minimal excretion of unchanged ibrutinib, high levels of oxidative metabolites formed due to liver and gut metabolism, and lack of reduction products in feces suggests complete absorption of ibrutinib from the gastrointestinal tract. This study also supports the nonclinical data suggesting first-pass metabolism is the reason for low bioavailability of orally administered ibrutinib rather than poor absorption (see the Imbruvica prescribing information). Also, bioavailability is in line with other studies in healthy participants who were administered ibrutinib in a 40 or 140 mg capsule formulation (de Jong et al., 2014).
The pharmacokinetic parameters of ibrutinib and its dihydrodiol metabolite PCI-45227 were similar for blood and plasma. Higher plasma Cmax and AUC for total radioactivity may be attributed to measurable concentration for a longer time period in plasma (up to 72 hours) compared with blood (up to 8 hours) due to the more sensitive plasma assay. The shorter mean half-life of ibrutinib compared with PCI-45227 was not consistent with previous clinical studies (see the Imbruvica prescribing information). However, the estimate of ibrutinib half-life may have been limited by the absence of measurable ibrutinib concentrations after 16 hours. Also, the longer radioactivity half-life in plasma compared with blood may be due to fewer time points in the elimination phase.
Ibrutinib was extensively metabolized after a single dose of 14C-ibrutinib with three main primary metabolic clearance pathways: hydroxylation of the phenyl, opening of the piperidine and epoxidation of the ethylene on the acryloyl moiety with further hydrolysis to a dihydrodiol. Most of the ibrutinib metabolites (>95%) were oxidative in nature. Nonoxidative metabolites (<5%) were derived from glutathione addition to the acryloyl C=C. Other phase II metabolites were minor and secondary to phase I [mostly sulfates of hydroxy metabolites (<5% of dose)]. Direct sulfates or glucuronides of the parent were not observed. Metabolism at the acryloyl C=C resulted in mostly dihydrodiol formation, which was consistent with in vitro studies and accounted for a relatively small proportion of the observed metabolites.
This study also characterized the ex vivo plasma protein binding of ibrutinib in man, which is consistent with the results obtained from in vitro studies. The slight decrease in covalently bound radioactivity from peak to 24 hours postdose suggested a slower process of protein turnover to clear covalently bound radioactivity from the circulation. The nature of the covalently bound radioactivity (parent versus metabolite) has not been investigated at this stage. Overall, the observation of significant covalent binding to plasma proteins is not unexpected because ibrutinib is by nature a covalent binder due to the presence of the Michael acceptor acryloyl moiety, which is also preserved in most of the observed metabolites.
When comparing total plasma radioactivity and covalently bound plasma radioactivity in the current human mass balance study at 1 hour, as close as possible to Cmax (0.5 hour), covalently bound plasma radioactivity (47 ng⋅Eq/ml) represented around 8.5% of total plasma radioactivity (549 ng⋅Eq/ml). However, the liver extraction ratio of ibrutinib is known to be high, which will lead to much higher ibrutinib concentrations in the portal vein compared with the systemic circulation. Considering 92% liver extraction, the ibrutinib Cmax of 37.1 ng/ml implies a portal vein concentration of around 464 ng/ml. Assuming a 30 minute time window available for covalent binding in the portal vein based on a time to reach Cmax of around 0.5 hour in the mass balance study, 3% or about 14 ng⋅Eq/ml would be covalently bound, which is around 30% of the observed 1 hour postdose level of 47 ng⋅Eq/ml.
Covalent binding of radioactivity to the cellular fraction of blood was not assessed in this clinical study, but was assessed in rat and dog following oral dosing of 14C-labeled Ibrutinib. The in vivo radioactive studies in these animal species show a lot of similarities with the human study. In rat and dog, the time course of total radioactivity observed in blood and plasma was comparable to human (rapid initial decline followed by a long terminal half-life), with also an important contribution of circulating metabolites to the total radioactivity.
The blood to plasma ratio of total radioactivity was consistently lower than 1, similar to human [around 0.7 in dog up to 48 hours postdose; around 0.75 in rat, increasing to 1 or above at the later time points (24–48 hours)]. Similar to human plasma, the relative importance of covalent binding versus total radioactivity increased as a function of time postdose, with covalent binding accounting for the majority of circulating radioactivity (both in plasma and blood) at the later time points, in line with the long terminal half-life.
In both rat and dog, the contribution of covalent binding to total radioactivity was higher for cellular components of blood than for plasma: covalent binding accounted for 30%–40% of total plasma radioactivity (AUC0–48 h), in line with human, and for 60%–70% of total radioactivity in cellular components of blood (AUC0–48 h). In the rat, at 1 hour postdose (close to time to reach Cmax), covalent binding in blood cellular components accounted for around 20% of total radioactivity in these cellular components, with a total blood radioactivity ranging between 694 (males) and 888 (females) ng⋅Eq/ml. When spiking 440 ng⋅Eq/ml Ibrutinib to blank rat blood and following incubation at 37°C for 1 hour, covalent binding in blood cellular components accounted for maximum 7% of total radioactivity. As a result, and in line with the previous reasoning for human plasma, it is not expected that the circulating levels of parent ibrutinib post liver can generate the observed amount of covalent binding in blood cellular components; the observed covalent binding most probably results from high levels of unchanged drug in the portal vein (Ibrutinib also shows a high liver extraction ratio in rat and dog) with possible additional contribution of metabolites.
In conclusion, it is likely that the observed covalent binding in plasma can be attributed to ibrutinib for a significant part (around 30% as an estimated upper limit), with albumin as the main target protein, and ibrutinib covalent binding occurring mainly presystemically. The remainder of the covalent binding in plasma can be attributed to ibrutinib metabolites (logically, those bearing the acryloyl moiety intact), which can be formed via first-pass gut or liver metabolism. Based on the structural similarity of metabolites with parent ibrutinib (except for the fact that some metabolites bear a ring-opened piperidine moiety), albumin would appear as a logical target protein for covalent binding of ibrutinib metabolites as well.
The small percentage of radioactivity recovered and the negligible amount of unchanged drug present in urine suggested renal excretion to be a minor elimination pathway. Poor and extensive CYP2D6 metabolizers did not show apparent differences in ibrutinib pharmacokinetics and metabolism, which was consistent with the results from an in vitro study. The identification of cytochrome P450 isozymes responsible for the metabolism of ibrutinib was evaluated in vitro using recombinant CYP450 expressing different isozymes and human liver microsomes in the presence of CYP450-specific chemical inhibitors. CYP3A4/5 was identified to be the major human microsomal enzyme responsible for the metabolism of ibrutinib with no apparent involvement of CYP1A, CYP2B6, CYP2C8, CYP2C9, CYP2C19, or CYP2D6. Using the selective mechanism–based CYP3A4 inhibitor CYP3cide and comparing the metabolism of ibrutinib in selected lots of human liver microsomes with functional or low to null CYP3A5 activity, the relative contribution of CYP3A5 of the overall metabolism of ibrutinib was estimated to be low (<20%).
Excretion profiles in poor and extensive metabolizers were highly comparable; no difference in the levels of any of the metabolites was detected between poor and extensive metabolizers. In plasma multiple metabolites were observed both in poor and extensive metabolizers. Next to unchanged drug, other major metabolites are circulating. There is no indication that one of the metabolites is more predominant in extensive metabolizers in comparison with poor metabolizers. There is no indication that the poor metabolizers are different from the extensive metabolizers with regard to excretion pathways and/or metabolism. No notable safety findings were reported during the study.
In summary, this study demonstrated that a single oral dose of 140 mg ibrutinib was completely absorbed from the gastrointestinal tract followed by oxidative metabolism with three major pathways and minimal renal excretion. The Cmax and AUC for total radioactivity were higher in plasma compared with blood. Ibrutinib and PCI-45227 each constituted less than 10% of the total circulating radioactivity. Approximately 12% of total radioactivity was accounted for by covalent binding in human plasma based on Cmax; covalent binding accounted for 38% and 51% of total radioactivity AUC0–24 h and AUC0–72 h, respectively. No apparent effect of poor and extensive CYP2D6 metabolizers was observed on pharmacokinetics and metabolism of ibrutinib. Also, ibrutinib was tolerated well by healthy participants.
Acknowledgments
The authors thank Rishabh Pandey (SIRO Clinpharm Pvt., Ltd.) for providing writing assistance and Dr. Namit Ghildyal (Janssen Research & Development, LLC) for additional editorial assistance. The authors also thank Dr. H. Thierens (State University of Ghent, Belgium), for evaluation of the radiation exposure; licensed radiopharmacist Dr. F. De Vos (State University of Ghent, Belgium), and the study participants, without whom this study would not have been accomplished.
Authorship Contributions
Participated in research design: Scheers, Leclercq, de Jong, Bode, Bockx, Laenen, Cuyckens, Skee, Murphy, Sukbuntherng, Mannens
Conducted experiments: Skee, de Jong, Murphy, Cuyckens, Scheers, Laenen, Bockx
Performed data anaysis: Scheers, Leclercq, de Jong, Bode, Bockx, Laenen, Cuyckens, Skee, Murphy, Sukbuntherng, Mannens
Wrote or contributed to the writing of the manuscript: Scheers, Leclercq, de Jong, Bode, Bockx, Laenen, Cuyckens, Skee, Murphy, Sukbuntherng, Mannens
Footnotes
- Received July 22, 2014.
- Accepted December 4, 2014.
This study was supported by funding from Janssen Research & Development, LLC. This study is registered at ClinicalTrials.gov [NCT01674322].
All authors met International Committee of Medical Journal Editors (ICMJE) criteria and all those who fulfilled those criteria are listed as authors. All authors had access to the study data and made the final decision about where to present these data. G.M., L.L., N.B., F.C., E.S., A.L., M.B., J.d.J., D.S., and J.M. are employees of Janssen R&D. The Janssen companies are Johnson & Johnson companies. G.M., L.L., N.B., F.C., J.d.J., and J.M. hold stocks in Johnson & Johnson. J.S. is an employee of and holds stocks in Pharmacyclics.
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This article has supplemental material available at dmd.aspetjournals.org.
Abbreviations
- AE
- adverse event
- AUC
- area under the concentration-time curve
- Cmax
- maximum concentration
- EOS
- end of study
- MS
- mass spectrometry
- SPE
- solid phase extraction
- UPLC
- ultra-performance liquid chromatography
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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Absorption, Metabolism, and Excretion of Ibrutinib
