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Acalabrutinib is a covalent Bruton tyrosine kinase (BTK) inhibitor approved for relapsed/refractory mantle cell lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma. A major metabolite of acalabrutinib (M27, ACP-5862) was observed in human plasma circulation. Subsequently, the metabolite was purified from an in vitro biosynthetic reaction and shown by nuclear magnetic resonance spectroscopy to be a pyrrolidine ring-opened ketone/amide. Synthesis confirmed its structure, and covalent inhibition of wild-type BTK was observed in a biochemical kinase assay. A twofold lower potency than acalabrutinib was observed but with similar high kinase selectivity. Like acalabrutinib, ACP-5862 was the most selective toward BTK relative to ibrutinib and zanubrutinib. Because of the potency, ACP-5862 covalent binding properties, and potential contribution to clinical efficacy of acalabrutinib, factors influencing acalabrutinib clearance and ACP-5862 formation and clearance were assessed. rCYP (recombinant cytochrome P450) reaction phenotyping indicated that CYP3A4 was responsible for ACP-5862 formation and metabolism. ACP-5862 formation K

Acalabrutinib received approval for the treatment of adult patients with mantle cell lymphoma who received at least 1 prior therapy and adult patients with chronic lymphocytic leukemia or small lymphocytic lymphoma. This study investigated the impact of hepatic impairment (HI) on acalabrutinib pharmacokinetics (PK) and safety at a single 50-mg dose in fasted subjects. This study was divided into 2 parts: study 1, an open-label, parallel-group study in Child-Pugh class A or B subjects and healthy subjects; and study 2, an open-label, parallel-group study in Child-Pugh class C subjects and healthy subjects. Baseline characteristics and safety profiles were similar across groups. Acalabrutinib exposure (area under the plasma concentration-time curve) increased slightly (1.90- and 1.48-fold) in subjects with mild (Child-Pugh class A) and moderate (Child-Pugh class B) hepatic impairment compared with healthy subjects. In severe hepatic impairment (Child-Pugh class C), acalabrutinib exposure (area under the plasma concentration-time curve and maximum plasma concentration) increased ≈5.0- and 3.6-fold, respectively. Results were consistent across total and unbound exposures. Severe hepatic impairment did not impact total/unbound metabolite (ACP-5862) exposures; the metabolite-to-parent ratio decreased to 0.6 to 0.8 (vs 3.1-3.6 in healthy subjects). In summary, single oral dose of 50-mg acalabrutinib was safe and well tolerated in subjects with mild, moderate, and severe hepatic impairment and in healthy control subjects. In subjects with severe hepatic impairment, mean acalabrutinib exposure increased by up to 5-fold and should be avoided. Acalabrutinib does not require dose adjustment in patients with mild or moderate hepatic impairment.

Acalabrutinib is a targeted, covalent inhibitor of Bruton tyrosine kinase (BTK) with a unique 2-butynamide warhead that has relatively lower reactivity than other marketed acrylamide covalent inhibitors. A human [14C] microtracer bioavailability study in healthy subjects revealed moderate intravenous clearance (39.4 l/h) and an absolute bioavailability of 25.3% ± 14.3% (n = 8). Absorption and elimination of acalabrutinib after a 100 mg [14C] microtracer acalabrutinib oral dose was rapid, with the maximum concentration reached in

Background: KRT-232 is a potent, selective, orally available, targeted inhibitor of human MDM2 homolog interactions with tumor suppressor protein 53 (p53). KRT-232 is under development for treatment of myeloproliferative neoplasms, acute myeloid leukemia and Merkel cell carcinoma. KRT-232 is a highly permeable 568.6 g/mol, monoprotic carboxylic acid (pKa 4.35); elimination involves primarily biliary-fecal excretion of an acyl glucuronide metabolite (M1) and enteric glucuronide hydrolysis with enterohepatic recirculation of parent drug. M1 is a 744.7 g/mol monoprotic acid with pKa 3.32 and 1/5th the MDM2 inhibitory potency of KRT-232. Plasma protein binding (fup) of KRT-232 and M1 were ~0.026 and ~0.006, respectively. Static model drug-drug interaction (DDI) calculations (FDA-CDER. 2020) indicated a potential minor DDI with substrates of cytochrome P450s (CYP) CYP3A4 and CYP2C8, UGT1A1, P-glycoprotein 1 (P-gp) and OATP1B1/3. Here, a 'fit-for-purpose' PBPK model for KRT-232 was developed using physicochemical, preclinical (in vitroandin vivo) and clinical pharmacokinetic (PK) data (KartosData on file; Ye et al.Xenobiotica. 2015; Gluck et al.Invest New Drugs. 2020 [Study A]). Methods:Simcyp Simulator V17 (release 1; 27/11/2017; 17.0.90.0) was used. In vitro kinetic parameters: In vitroKi for competitive inhibition of CYP2C8 was 3.8 and 0.17 µM for KRT-232 and M1, respectively.In vitroKI and kinact for mechanism-based inhibition of CYP3A4 by KRT-232 were 28 µM and 4.14 h-1, respectively. Estimatedin vitroKi values for KRT-232-mediated inhibition of UGT1A1, P-gp, OATP1B1 and OATP1B3, were 1.9, 14.8, 4.9 and 6.9 µM. For M1-mediated inhibition of UGT1A1, OATP1B1 and OATP1B3, values were 2.3, 0.393 and 0.606 µM. Model development and verification:KRT-232 absorption was described by a first-order process; fraction absorbed was assumed to be 100% and the absorption rate constant was manually optimized using observed mean plasma concentration data from Part 1 of Study A. KRT-232 clearance was based onin vitroformation clearance of M1 in human liver microsomes, scaled up 4-fold based on observed oral clearance of KRT-232 (Wood et al.Drug Metab Dispos. 2017). KRT-232 distribution volume was described by a minimal PBPK model. KRT-232 and M1 DDI parameters were based onin vitrodata. M1 clearance was manually optimized using mean observed data from Study A (Part 1, 240 mg dose), and distribution volume was predicted by the Simulator using Method 1 and a full PBPK model. PBPK simulated data were compared with patient PK data collected during Part 2 of Study A. Model application:The model predicted potential DDI after steady state (SS) 240 mg once daily KRT-232 administration with substrates of CYP3A4, CYP2C8, UGT1A1, P-gp and OATP1B1/3. DDI simulations included 100 virtual subjects assigned to 10 virtual trials (N=10 each). Population estimates of geometric mean and median Cmax and AUC0-∞ ratios were generated. A "worst case" sensitivity analysis was performed by using a 10-fold increase inin vitroCYP3A4 interaction potency (KI, EC50) relative to the measuredin vitrovalue. Results:Figure 1shows simulated SS mean and individual observed KRT-232 and M1 concentration data from Part 2 of Study A. SS KRT-232 Cmax, AUC0-24h and t1/2 were predicted within 0.80-1.25-fold of observed PK data and tmax was under-predicted (0.36-fold) by the model (Table 1). M1 Cmax and AUC0-24h were within 0.80-1.25-fold of observed PK data on Day 1 (not shown). M1 Cmax on Day 7 was within 0.80-1.25-fold of observed PK data and SS AUC0-24h, t1/2, and tmax were underpredicted (0.72, 0.79 and 0.61-fold) relative to observed PK data. In all simulated DDIs, no clinically significant interaction was predicted for SS 240 mg doses of KRT-232 since predicted Cmax and AUC0-∞ geometric mean ratio (GMR) changes were less than 1.25-fold (Table 2). The CYP3A4 DDI simulation gave a population GMR (90% CI) for Cmax and AUC0-∞ of 1.07 (1.06, 1.09) and 1.15 (1.12, 1.17), respectively. In a sensitivity check, predicted midazolam Cmax and AUC0-∞ after a 10-fold reduction ofin vitroKI and EC50 were 1.44-fold and 2.18-fold, respectively. Although unlikely, a weak CYP3A4 DDI could not be definitively excluded. Conclusions: Mean plasma exposure of KRT-232 and M1 and inter-individual variability were adequately described by the PBPK model. No clinically significant DDI were predicted based on Cmax and AUC0-∞ GMR changes of less than 1.25-fold. Disclosures Templeton: Certara UK, LTD.:Current Employment.Podoll:IV/PO, LLC:Consultancy.Krejsa:Kartos Therapeutics:Current Employment;AstraZeneca:Current equity holder in publicly-traded company;Seattle Genetics:Current equity holder in publicly-traded company;Acerta Pharma:Current equity holder in private company.Slatter:Kartos Therapeutics:Current Employment;AstraZeneca:Current equity holder in publicly-traded company;Amgen:Divested equity in a private or publicly-traded company in the past 24 months.

Acalabrutinib, a selective, covalent Bruton tyrosine kinase inhibitor, is a CYP3A substrate and weak CYP3A/CYP2C8 inhibitor. A physiologically‐based pharmacokinetic (PBPK) model was developed for acalabrutinib and its active metabolite ACP‐5862 to predict potential drug–drug interactions (DDIs). The model indicated acalabrutinib would not perpetrate a CYP2C8 or CYP3A DDI with the sensitive CYP substrates rosiglitazone or midazolam, respectively. The model reasonably predicted clinically observed acalabrutinib DDI with the CYP3A perpetrators itraconazole (4.80‐fold vs. 5.21‐fold observed) and rifampicin (0.21‐fold vs. 0.23‐fold observed). An increase of two to threefold acalabrutinib area under the curve was predicted for coadministration with moderate CYP3A inhibitors. When both the parent drug and active metabolite (total active components) were considered, the magnitude of the CYP3A DDI was much less significant. PBPK dosing recommendations for DDIs should consider the magnitude of the parent drug excursion, relative to safe parent drug exposures, along with the excursion of total active components to best enable safe and adequate pharmacodynamic coverage.

Emixustat potently inhibits the visual cycle isomerase retinal pigment epithelium protein 65 (RPE65) to reduce the accumulation of toxic bisretinoid by-products that lead to various retinopathies. Orally administered emixustat is cleared rapidly from the plasma, with little excreted unchanged. The hydroxypropylamine moiety that is critical in emixustat’s inhibition of RPE65 is oxidatively deaminated to three major carboxylic acid metabolites that appear rapidly in plasma. These metabolites greatly exceed the plasma concentrations of emixustat and demonstrate formation-rate-limited metabolite kinetics. This study investigated in vitro deamination of emixustat in human vascular membrane fractions, plasma, and recombinant human vascular adhesion protein-1 (VAP-1), demonstrating single-enzyme kinetics for the formation of a stable aldehyde intermediate (ACU-5201) in all in vitro systems. The in vitro systems used herein established sequential formation of the major metabolites with addition of assay components for aldehyde dehydrogenase and cytochrome P450. Reaction phenotyping experiments using selective chemical inhibitors and recombinant enzymes of monoamine oxidase, VAP-1, and lysyl oxidase showed that only VAP-1 deaminated emixustat. In individually derived human vascular membranes from umbilical cord and aorta, rates of emixustat deamination were highly correlated to VAP-1 marker substrate activity (benzylamine) and VAP-1 levels measured by enzyme-linked immunosorbent assay. In donor-matched plasma samples, soluble VAP-1 activity and levels were lower than in aorta membranes. A variety of potential comedications did not strongly inhibit emixustat deamination in vitro.

Acalabrutinib (Calquence®) is a potent, selective, orally administered, covalent inhibitor of Bruton tyrosine kinase (BTK) that received accelerated approval for relapsed/refractory mantle cell lymphoma from the US FDA in October 2017. Profiling of acalabrutinib metabolites in human plasma revealed a late-eluting, +16 Da metabolite circulating at concentrations higher than parent drug. Metabolite regiochemistry could not be determined by mass spectrometry. In vitro metabolism and preparative HPLC was used to generate a pure sample of the metabolite for structural characterization by NMR. Confirmatory chemical synthesis revealed a pyrrolidine ring-opened ketone. The structure of the metabolite, designated ACP-5862, and a smaller -2 Da peak, identified as dehydropyrrolidine, M25, inferred a common carbinolamide intermediate in their genesis. Both metabolites retained the butynamide electrophile responsible for the inactivation of BTK. In vitro studies on the inhibition of BTK and related Tec and Src kinases revealed that ACP-5862 was active against BTK with similar selectivity and potency to acalabrutinib (Kaptein et al, 2019) This work then investigated the in vitro metabolism and drug transport features of acalabrutinib, and the metabolite ACP-5862, to establish the potential for clinical drug-drug interactions (DDI) via CYPs, UGTs and drug transporters. CYP reaction phenotyping indicated CYP3A4 was responsible for both the formation and further metabolism of ACP-5862. Km and Vmax values for the formation of ACP-5862 using rCYP3A4 were 2.78 μM and 4.13 pmol/pmol CYP/min, respectively. The in vitro intrinsic clearance of ACP-5862 was 23.6 μL/min/mg. Acalabrutinib weakly inhibited CYP2C8, CYP2C9 and CYP3A4 in vitro, and ACP-5862 weakly inhibited CYP2C9 and CYP2C19, with no inhibition of CYP1A2, CYP2B6, or CYP2D6. Similarly, UGT1A1, UGT2B7, and aldehyde oxidase were not inhibited. Neither parent or ACP-5862 strongly induced CYP1A2, CYP2B6, or CYP3A4 mRNA. Acalabrutinib and ACP-5862 were substrates of MDR1 and BCRP in vitro, but were not substrates of OATP1B1 or OATP1B3. Acalabrutinib was not a substrate of OAT1, OAT3, and OCT2. Based on static PK model calculations, acalabrutinib may cause a modest increase in exposure to coadministered BCRP substrates by inhibition of intestinal BCRP, but with no inhibition of BCRP at the systemic level. The PK of substrates of MDR1, MATE1, MATE2-K, OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 are not likely to be altered by acalabrutinib or ACP-5862. These data were combined with clinical DDI data (Izumi et al, 2017) to simulate DDI in the presence of CYP3A inhibitors and inducers. PBPK models confirmed that acalabrutinib and ACP-5862 were not likely to perpetrate CYP2C8 or CYP3A4 mediated drug interactions (Zhou et al., 2019). Overall, acalabrutinib and major metabolite, ACP-5862 have a favorable drug interaction profile. Citation Format: Terry Podoll, Paul G. Pearson, Jerry Evarts, Tim Ingallinera, Hao Sun, Stephen Byard, Adrian J. Fretland, J. Greg Slatter. Structure elucidation, metabolism, and drug interaction potential of ACP-5862, an active, major, circulating metabolite of acalabrutinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 13.

Incubations with human liver and gut microsomes revealed that the antibiotic, clindamycin, is primarily oxidized to form clindamycin sulfoxide. In this report, evidence is presented that the S-oxidation of clindamycin is primarily mediated by CYP3A. This conclusion is based upon several lines of in vitro evidence, including the following. 1) Incubations with clindamycin in hepatic microsomes from a panel of human donors showed that clindamycin sulfoxide formation correlated with CYP3A-catalyzed testosterone 6beta-hydroxylase activity; 2) coincubation with ketaconazole, a CYP3A4-specific inhibitor, markedly inhibited clindamycin S-oxidase activity; and 3) when clindamycin was incubated across a battery of recombinant heterologously expressed human cytochrome P450 (P450) enzymes, CYP3A4 possessed the highest clindamycin S-oxidase activity. A potential role for flavin-containing monooxygenases (FMOs) in clindamycin S-oxidation in human liver was also evaluated. Formation of clindamycin sulfoxide in human liver microsomes was unaffected either by heat pretreatment or by chemical inhibition (e.g., methimazole). Furthermore, incubations with recombinant FMO isoforms revealed no detectable activity toward the formation of clindamycin sulfoxide. Beyond identifying the drug-metabolizing enzyme responsible for clindamycin S-oxidation, the ability of clindamycin to inhibit six human P450 enzymes was also evaluated. Of the P450 enzymes examined, only the activity of CYP3A4 was inhibited (approximately 26%) by coincubation with clindamycin (100 microM). Thus, it is concluded that CYP3A4 appears to account for the largest proportion of the observed P450 catalytic clindamycin S-oxidase activity in vitro, and this activity may be extrapolated to the in vivo condition.