Identification and Characterization of ACP-5862, the Major Circulating Active Metabolite of Acalabrutinib: Both Are Potent and Selective Covalent Bruton Tyrosine Kinase Inhibitors .

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 Km (Michaelis constant) and Vmax were 2.78 µM and 4.13 pmol/pmol CYP3A/min, respectively. ACP-5862 intrinsic clearance was 23.6 µL/min per mg. Acalabrutinib weakly inhibited CYP2C8, CYP2C9, and CYP3A4, and ACP-5862 weakly inhibited CYP2C9 and CYP2C19; other cytochrome P450s, UGTs (uridine 5'-diphospho-glucuronosyltransferases), and aldehyde oxidase were not inhibited. Neither parent nor ACP-5862 strongly induced CYP1A2, CYP2B6, or CYP3A4 mRNA. Acalabrutinib and ACP-5862 were substrates of multidrug resistance protein 1 and breast cancer resistance protein but not OATP1B1 or OATP1B3. Our work indicates that ACP-5862 may contribute to clinical efficacy in acalabrutinib-treated patients and illustrates how proactive metabolite characterization allows timely assessment of drug-drug interactions and potential contributions of metabolites to pharmacological activity. SIGNIFICANCE STATEMENT: This work characterized the major metabolite of acalabrutinib, ACP-5862. Its contribution to the pharmacological activity of acalabrutinib was assessed based on covalent Bruton tyrosine kinase binding kinetics, kinase selectivity, and potency in cellular assays. The metabolic clearance and in vitro drug-drug interaction potential were also evaluated for both acalabrutinib and ACP-5862. The current data suggest that ACP-5862 may contribute to the clinical efficacy observed in acalabrutinib-treated patients and demonstrates the value of proactive metabolite identification and pharmacological characterization.

A population pharmacokinetic-pharmacodynamic model of navtemadlin, its glucuronide metabolite (M1) and serum macrophage inhibitory cykokine-1 (MIC-1).

Navtemadlin is a potent, selective, orally available inhibitor of murine double minute 2 that restores p53 activity to induce apoptosis in TP53 wild-type malignancies. Using richly sampled pharmacokinetic (PK) and pharmacodynamic (PD) data from healthy volunteers, a population PK/PD model was developed.A population PK (PPK) model described the PK characteristics of navtemadlin and its major metabolite acyl glucuronide (M1) and quantified enterohepatic recirculation (EHR). Post hoc individual PK parameters from this model were coupled with PD data for serum macrophage inhibitory cytokine-1 (MIC-1, GDF15), a cytokine biomarker of p53 activation, to construct a population PK/PD model that described plasma concentration-driven MIC-1 excursions and enabled simulation of the extent and duration of navtemadlin PD effects.The median apparent clearance (CL/F) and apparent central volume (V2/F) of navtemadlin were 36.4 L/hr and 159 L. The typical maximum stimulatory effect (Smax) was close to the median maximum MIC-1 ratio to baseline of 7.29 in observed data.Simulation revealed a dose-dependent increase of MIC-1 with steady state attained in approximately 7 days, in a 7-day-on/21-day-off dose regimen. Elevated MIC-1 concentrations persist through 17-19 days, leaving about 9-11 PD-free days in a 28-day cycle.

Pharmacokinetics and Macrophage Inhibitory Cytokine-1 Pharmacodynamics of the Murine Double Minute 2 Inhibitor, Navtemadlin (KRT-232) in Fed and Fasted Healthy Subjects.

This single 60-mg dose, 4-period crossover study assessed the effect of food and formulation change on navtemadlin (KRT-232) pharmacokinetics (PK) and macrophage inhibitory cytokine-1 (MIC-1) pharmacodynamics. Healthy subjects (N = 30) were randomized to 3 treatment sequences, A: new tablet, fasted (reference, dosed twice); B: new tablet, 30 minutes after a high-fat meal (test 1); C: old tablet, fasted (test 2). PK/pharmacodynamic parameters were measured over 0 to 96 hours. Adverse events were mild without any discontinuations. No serious adverse events or deaths occurred. In treatment A, navtemadlin mean (coefficient of variation) maximum concentration (Cmax ) was 525 (66) ng/mL, at median time to maximum concentration (tmax ) of 2 hours. Mean (coefficient of variation) area under the plasma concentration-time curve from time 0 to time t (AUC0-t ) was 3392 (63.3) ng • h/mL, and arithmetic mean terminal half-life was 18.6 hours. Acyl glucuronide metabolite (M1)/navtemadlin AUC0-t ratio was 0.2, and urine excretion of navtemadlin was negligible. After a meal (B vs A), navtemadlin tmax was delayed by 1 hour. Geometric least squares means ratios (90%CI) for navtemadlin Cmax and AUC0-t were 102.7% (87.4-120.6) and 81.4% (76.2-86.9), respectively. Old vs new tablet fasted formulations (C vs A) had geometric least squares means ratios (90%CI) of 78.4% (72.0-85.3) for Cmax and 85.9% (80.5-91.7) for AUC0-t . MIC-1 Cmax and AUC were comparable across groups; tmax was delayed relative to navtemadlin tmax by ≈8 hours. Navtemadlin AUC0-t and MIC-1 AUC0-t correlated significantly. In conclusion, navtemadlin can be administered safely with or without food; the new formulation does not affect navtemadlin PK. The 60-mg navtemadlin dose elicited a reproducible and robust MIC-1 response that correlated well with navtemadlin exposure, indicating that murine double minute 2 target engagement leads to p53 activation.

Evaluation of the Pharmacokinetics and Safety of a Single Dose of Acalabrutinib in Subjects With Hepatic Impairment.

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.

Bridging in vitro dissolution and in vivo exposure for acalabrutinib. Part II. A mechanistic PBPK model for IR formulation comparison, proton pump inhibitor drug interactions, and administration with acidic juices.

Acalabrutinib (Calquence®) 100 mg (bid) has received accelerated approval by FDA for the treatment of adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy. Acalabrutinib is a substrate of PgP and CYP3A4, with a significant fraction of drug metabolized by first pass gut extraction and 25% absolute bioavailability. The absorption of acalabrutinib is affected by stomach pH, with lower pharmacokinetic exposure observed following co-administration with proton pump inhibitors. During dissolution at pH values below its highest basic pKa, the two basic moieties of acalabrutinib react with protons from the aqueous solution, leading to a higher pH at the drug surface than in the bulk solution. A batch-specific product particle size distribution (P-PSD), was derived from dissolution data using a mechanistic model that was based on the understanding of surface pH and the contribution of micelles to the dissolution rate. P-PSD values obtained for various batches of acalabrutinib products in simple buffers, or in complex fluids such as fruit juices, were successfully integrated into a physiologically based pharmacokinetic (PBPK) model developed using GastroPlus v9.0™. The integrated model allowed the prediction of clinical pharmacokinetics under normal physiological stomach pH conditions as well as following treatment with proton pump inhibitors. The model also accounted for lower pharmacokinetic exposure that was observed when acalabrutinib was co-administered with the acidic beverages, grapefruit juice, (which contains CYP3A inhibitors), and orange drink (which does not contain CYP3A inhibitors), relative to administration with water. The integration of dissolution data in the PBPK model enables mechanistic understanding and the establishment of more robust in vitro-in vivo correlations (IVIVC) under a variety of conditions. The model can then distinguish the interplay between dissolution and first pass extraction and how in vivo stomach pH, saturation of gut PgP, and saturation or inhibition of gut CYP3A4, will impact the pharmacokinetics of acalabrutinib.

Evaluation of the Drug-Drug Interaction Potential of Acalabrutinib and Its Active Metabolite, ACP-5862, Using a Physiologically-Based Pharmacokinetic Modeling Approach.

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.

Bioavailability, Biotransformation, and Excretion of the Covalent Bruton Tyrosine Kinase Inhibitor Acalabrutinib in Rats, Dogs, and Humans.

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 <1 hour and elimination half-life values of <2 hours. Low concentrations of radioactivity persisted longer in the blood cell fraction and a peripheral blood mononuclear cell subfraction (enriched in target BTK) relative to plasma. [14C]Acalabrutinib was metabolized to more than three dozen metabolites detectable by liquid chromatography-tandem mass spectrometry, with primary metabolism by CYP3A-mediated oxidation of the pyrrolidine ring, thiol conjugation of the butynamide warhead, and amide hydrolysis. A major active, circulating, pyrrolidine ring-opened metabolite, ACP-5862 (4-[8-amino-3-[4-(but-2-ynoylamino)butanoyl]imidazo[1,5-a]pyrazin-1-yl]-N-(2-pyridyl)benzamide), was produced by CYP3A oxidation.Novel enol thioethers from the 2-butynamide warhead arose from glutathione and/or cysteine Michael additions and were subject to hydrolysis to a ß-ketoamide. Total radioactivity recovery was 95.7% ± 4.6% (n = 6), with 12.0% of dose in urine and 83.5% in feces. Excretion and metabolism characteristics were generally similar in rats and dogs. Acalabrutinib's highly selective, covalent mechanism of action, coupled with rapid absorption and elimination, enables high and sustained BTK target occupancy after twice-daily administration.

Population Pharmacokinetics of the BTK Inhibitor Acalabrutinib and its Active Metabolite in Healthy Volunteers and Patients with B-Cell Malignancies.

INTRODUCTION: Bruton tyrosine kinase (BTK) is a key component of B-cell receptor signalling, critical for cell proliferation. Acalabrutinib, a selective, covalent BTK inhibitor, recently received an accelerated approval in relapsed/refractory mantle cell lymphoma. This analysis characterized the population pharmacokinetics (PK) of acalabrutinib and its metabolite ACP-5862. METHODS: Data were obtained from six phase I/II trials in adult patients with B-cell malignancy and seven phase I trials in healthy volunteers. Pooled concentration-time data, at dose levels ranging from 15 to 400 mg, were analysed using non-linear mixed-effects modelling. Base model parameters were scaled with body weight and normalized to 70 kg (fixed exponents: 0.75 and 1 for clearance and volumes, respectively). A full covariate approach was used to evaluate any relevant effects of dose, health group/disease status, hepatic and renal impairment, use of acid-reducing agents, race and sex. RESULTS: A total of 11,196 acalabrutinib and 1068 ACP-5862 concentration-time samples were available. The PK of both analytes were well described using two-compartment disposition models. Acalabrutinib absorption was characterized using sequential zero- and first-order constants and a lag time. Apparent clearance (CL/F) of acalabrutinib was 169 L/h (95% CI 159-175). Relative to the 100 mg dose group, the 15 and 400 mg dose groups showed a 1.44-fold higher and 0.77-fold lower CL/F, respectively. The clearance for ACP-5862 was 21.9 L/h (95% CI 19.5-24.0). The fraction metabolized was fixed to 0.4. The central and peripheral volumes of distribution were 33.1 L (95% CI 24.4-41.0) and 226 L (95% CI 149-305) for acalabrutinib, and 38.5 L (95% CI 31.6-49.2) and 38.4 L (95% CI 32.3-47.9) for ACP-5862. None of the investigated covariates led to clinically meaningful changes in exposure. CONCLUSION: The PK of acalabrutinib and its metabolite ACP-5862 were adequately characterized. Acalabrutinib CL/F decreased with increasing dose, but the trend was small over the 75-250 mg range. No dose adjustment was necessary for intrinsic or extrinsic covariates.

Acalabrutinib in relapsed or refractory mantle cell lymphoma (ACE-LY-004): a single-arm, multicentre, phase 2 trial.

BACKGROUND: Bruton tyrosine kinase is a clinically validated target in mantle cell lymphoma. Acalabrutinib (ACP-196) is a highly selective, potent Bruton tyrosine kinase inhibitor developed to minimise off-target activity. METHODS: In this open-label, phase 2 study, oral acalabrutinib (100 mg twice per day) was given to patients with relapsed or refractory mantle cell lymphoma, until disease progression or unacceptable toxicity. The primary endpoint was overall response assessed according to the Lugano classification, and safety analyses were done in all participants. This trial is registered with ClinicalTrials.gov, number NCT02213926. FINDINGS: From March 12, 2015, to Jan 5, 2016, 124 patients with relapsed or refractory mantle cell lymphoma were enrolled and all patients received treatment; median age 68 years. Patients received a median of two (IQR 1-2) previous therapies. At a median follow-up of 15·2 months, 100 (81%) patients achieved an overall response and 49 (40%) patients achieved a complete response. The Kaplan-Meier estimated medians for duration of response, progression-free survival, and overall survival were not reached; the 12-month rates were 72% (95% CI 62-80), 67% (58-75), and 87% (79-92%), respectively. The most common adverse events were primarily grade 1 or 2 and were headache (47 [38%]), diarrhoea (38 [31%]), fatigue (34 [27%]), and myalgia (26 [21%]). The most common grade 3 or worse adverse events were neutropenia (13 [10%]), anaemia (11 [9%]), and pneumonia (six [5%]). There were no cases of atrial fibrillation and one case of grade 3 or worse haemorrhage. The median duration of treatment was 13·8 months. Treatment was discontinued in 54 (44%) patients, primarily due to progressive disease (39 [31%]) and adverse events (seven [6%]). INTERPRETATION: Acalabrutinib treatment provided a high rate of durable responses and a favourable safety profile in patients with relapsed or refractory mantle cell lymphoma. These findings suggest an important role for acalabrutinib in the treatment of this disease population. FUNDING: Acerta Pharma, a member of the AstraZeneca Group.

Pharmacokinetics, Biotransformation, and Excretion of [14C]Etelcalcetide (AMG 416) Following a Single Microtracer Intravenous Dose in Patients with Chronic Kidney Disease on Hemodialysis.

Etelcalcetide (AMG 416) is a novel synthetic peptide calcium-sensing receptor activator in clinical development as an intravenous calcimimetic for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease (CKD) on hemodialysis. Etelcalcetide is composed of seven D-aminoacids with an L-cysteine linked to a D-cysteine by a disulfide bond. A single intravenous dose of [14C]etelcalcetide (10 mg; 26.3 kBq; 710 nCi) was administered to patients with CKD on hemodialysis to elucidate the pharmacokinetics, biotransformation, and excretion of etelcalcetide in this setting. Blood, dialysate, urine, and feces were collected to characterize the pharmacokinetics, biotransformation product profiles, mass balance, and formation of anti-etelcalcetide antibodies. Accelerator mass spectrometry was necessary to measure the microtracer quantities of C-14 excreted in the large volumes of dialysate and other biomatrices. An estimated 67 % of the [14C]etelcalcetide dose was recovered in dialysate, urine, and feces 176 days after dose administration. Etelcalcetide was primarily cleared by hemodialysis, with approximately 60 % of the administered dose eliminated in dialysate. Minor excretion was observed in urine and feces. Biotransformation resulted from disulfide exchange with endogenous thiols, and preserved the etelcalcetide D-amino acid backbone. Drug-related radioactivity circulated primarily as serum albumin peptide conjugate (SAPC). Following removal of plasma etelcalcetide by hemodialysis, re-equilibration occurred between SAPC and L-cysteine present in blood to partially restore the etelcalcetide plasma concentrations between dialysis sessions. No unanticipated safety signals or anti-etelcalcetide or anti-SAPC antibodies were detected.

Evaluation of Evolocumab (AMG 145), a Fully Human Anti-PCSK9 IgG2 Monoclonal Antibody, in Subjects With Hepatic Impairment.

Evolocumab binds PCSK9, increasing low-density lipoprotein cholesterol (LDL-C) receptors and lowering LDL-C. Target-mediated evolocumab elimination is attributable to PCSK9 binding. As circulating PCSK9 and LDL-C levels are primarily regulated by the liver, we compared evolocumab pharmacokinetics, pharmacodynamics, and safety in individuals with and without hepatic impairment. An open-label, parallel-group study evaluated the pharmacokinetics of evolocumab in hepatic-impaired (Child-Pugh Class A or B) or healthy adults. Participants were classified as having no, mild, or moderate hepatic impairment (n = 8/group) and received a single 140-mg evolocumab dose. Assessments of unbound evolocumab and PCSK9 were made predose and postdose. Adverse events were monitored throughout the study. No significant association was observed between baseline PCSK9 and increasing level of hepatic impairment. No difference in extent and time course of PCSK9 or LDL-C reduction was observed despite an apparent decrease in mean unbound evolocumab exposure with increasing hepatic impairment (Jonckheere-Terpstra trend test; maximum serum concentration P = .18; area under the curve P = .09). Maximum reductions were observed in moderately impaired subjects vs healthy individuals: mean maximum serum concentration -34%; mean area under the concentration-time curve (AUC) -47%. On average, unbound PCSK9 serum concentrations fell by >80% at 4 hours after a single evolocumab dose. Mean (95% confidence interval) maximum LDL-C reductions in the healthy, mild, and moderate groups were -57% (-64% to -48%), -70% (-75% to -63%), and -53% (-61% to -43%), respectively. No safety risks were identified. These results support evolocumab use without dose adjustment in patients with active liver disease and mild or moderate hepatic impairment.

An open-label study in healthy men to evaluate the risk of seminal fluid transmission of denosumab to pregnant partners.

AIMS: Denosumab is a fully human monoclonal immunoglobulin G2 antibody that inhibits bone resorption and increases bone mass and strength. The present clinical study assessed serum and seminal fluid pharmacokinetics following a single denosumab dose in healthy men, and evaluated whether denosumab in seminal fluid poses any risk to a fetus in the event of unprotected sexual intercourse with a pregnant partner. METHODS: An open-label, single-dose study in 12 healthy men was conducted over a 106-day period. Subjects received a single subcutaneous dose of 60-mg denosumab on day 1. Serum and seminal fluid samples were collected at specified time points to assess denosumab pharmacokinetics. Adverse events were recorded. RESULTS: Denosumab was measurable at low concentrations in seminal fluid (~2% of serum concentrations). The mean [standard deviation (SD)] maximum observed drug concentration (Cmax ) was 6170 (2070) ng ml(-1) (serum) and 100 (81.9) ng ml(-1) (seminal fluid). The median time to Cmax (tmax ) was 8 days (serum) and 21 days (seminal fluid). The mean (SD) area under the plasma concentration-time curve (AUC) from time zero to the time of the last quantifiable concentration (AUClast ) was 333 000 (122 000) day•ng ml(-1) (serum) and 5220 (4880) day•ng ml(-1) (seminal fluid). The mean (SD) Cmax and AUC ratios between seminal fluid and serum were 0.0217 (0.0154) and 0.0170 (0.0148), respectively. Using conservative assumptions for ejaculate volume (6 ml), vaginal absorption (100%) and placental transfer (100%), the measured mean denosumab seminal fluid Cmax would result in fetal exposure that was more than 110 times below the preclinically derived 'no effect level' for denosumab. CONCLUSIONS: These results indicate a negligible risk to a fetus exposed to denosumab via seminal fluid transfer to a pregnant partner.

Therapeutic protein drug-drug interactions: navigating the knowledge gaps-highlights from the 2012 AAPS NBC Roundtable and IQ Consortium/FDA workshop.

The investigation of therapeutic protein drug-drug interactions has proven to be challenging. In May 2012, a roundtable was held at the American Association of Pharmaceutical Scientists National Biotechnology Conference to discuss the challenges of preclinical assessment and in vitro to in vivo extrapolation of these interactions. Several weeks later, a 2-day workshop co-sponsored by the U.S. Food and Drug Administration and the International Consortium for Innovation and Quality in Pharmaceutical Development was held to facilitate better understanding of the current science, investigative approaches and knowledge gaps in this field. Both meetings focused primarily on drug interactions involving therapeutic proteins that are pro-inflammatory cytokines or cytokine modulators. In this meeting synopsis, we provide highlights from both meetings and summarize observations and recommendations that were developed to reflect the current state of the art thinking, including a four-step risk assessment that could be used to determine the need (or not) for a dedicated clinical pharmacokinetic interaction study.

Critical review of preclinical approaches to investigate cytochrome p450-mediated therapeutic protein drug-drug interactions and recommendations for best practices: a white paper.

Drug-drug interactions (DDIs) between therapeutic proteins (TPs) and small-molecule drugs have recently drawn the attention of regulatory agencies, the pharmaceutical industry, and academia. TP-DDIs are mainly caused by proinflammatory cytokine or cytokine modulator-mediated effects on the expression of cytochrome P450 enzymes. To build consensus among industry and regulatory agencies on expectations and challenges in this area, a working group was initiated to review the preclinical state of the art. This white paper represents the observations and recommendations of the working group on the value of in vitro human hepatocyte studies for the prediction of clinical TP-DDI. The white paper was developed following a "Workshop on Recent Advances in the Investigation of Therapeutic Protein Drug-Drug Interactions: Preclinical and Clinical Approaches" held at the Food and Drug Administration White Oak Conference Center on June 4 and 5, 2012. Results of a workshop poll, cross-laboratory data comparisons, and the overall recommendations of the in vitro working group are presented herein. The working group observed that evaluation of TP-DDI for anticytokine monoclonal antibodies is currently best accomplished with a clinical study in patients with inflammatory disease. Treatment-induced changes in appropriate biomarkers in phase 2 and 3 studies may indicate the potential for a clinically measurable treatment effect on cytochrome P450 enzymes. Cytokine-mediated DDIs observed with anti-inflammatory TPs cannot currently be predicted using in vitro data. Future success in predicting clinical TP-DDIs will require an understanding of disease biology, physiologically relevant in vitro systems, and more examples of well conducted clinical TP-DDI trials.

Activity-based exposure comparisons among humans and nonclinical safety testing species in an extensively metabolized drug candidate.

1. Extensive metabolism of a drug candidate can complicate the interpretation of comparative safety and efficacy data from humans and preclinical species. 2. The 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitor, AMG 221 underwent extensive oxidative metabolism to structurally similar but differentially active primary and secondary metabolites. Relative potency data from synthetic metabolites enabled a stepwise quantitative assessment of AMG 221 in vivo metabolism that compared relative exposure to metabolites in plasma across species and discerned which active metabolites to monitor in preclinical and clinical safety and efficacy studies. 3. Pooled plasma samples from AMG 221-dosed human subjects were profiled using high-resolution liquid chromatography-mass spectrometry (LC-MS) with a mass-defect-filter. The most abundant peak, M1 accounted for 47%-59% of peaks followed by AMG 221 at 27%-40%. Other metabolites were each less than 7%. Activity-normalized data indicated both M1 and AMG 221 should be monitored to assist pharmacokinetic-pharmacodynamic (PK-PD) modeling. 4. Rat and dog area under the plasma concentration time curve (AUC) exposures to M1 at preclinical no observable adverse effect level (NOAEL) doses were 2-42-fold higher than human, indicating M1 was not a disproportionate metabolite, as defined by International Committee on Harmonization (ICH) M3(R2) guidance. 5. Development decisions regarding active metabolite monitoring and potentially disproportionate metabolites in extensively metabolized drug candidates are enabled by metabolite synthesis and liquid chromatography high-resolution mass spectrometry (LC-HRMS)-based assessment of potency-normalized plasma metabolite AUCs.

Effects of interleukin 1ß (IL-1ß) and IL-1ß/interleukin 6 (IL-6) combinations on drug metabolizing enzymes in human hepatocyte culture.

Exposure to cytokines can down-regulate hepatic cytochrome P450 enzymes. Accordingly, relief of inflammation by cytokinetargeted drug therapy has the potential to up-regulate cytochrome P450s and thereby increase clearance of co-administered drugs. This study examined the effects of the inflammatory cytokine, interleukin 1ß (IL-1ß), and IL-1ß/interleukin 6 (IL-6) combinations on drug metabolizing enzymes in human hepatocyte culture. Treatment of hepatocytes with IL-1ß revealed suppression of mRNA expression of several clinically important cytochrome P450 isoenzymes, with EC50 values that differed by isoenzyme. Suppression of CYP1A2 activity by IL-1ß could not be measured in 3 of 5 donors due to lack of response, and in the two remaining donors the average EC50 was 450 pg/mL. CYP3A activity had an EC50 of suppression of 416 ± 454 pg/mL. Measurable EC50s were obtained for all 5 donors for CYP2C8, 3A4, 3A5, 4A11 and IL-6R mRNA with fold differences which varied between 9.5-fold (CYP2C8) to 109-fold (CYP4A11). When hepatocytes were treated with IL-1ß and IL-6 in combination at concentrations which ranged from 1-100 pg/mL, IL-6 was the main determinant of increases in acute phase response marker mRNA and of decreases in CYP3A4 mRNA. There was no synergy between IL-1ß and IL-6 in the regulation of cytochrome P450 mRNA when dosed in combination, although the effects of the two cytokines in combination were additive in certain instances. These data indicate that IL-1ß and IL-6 both suppress cytochrome P450 mRNA and enzyme levels in vitro and that, at similar physiologically-relevant concentrations in vitro, IL-6 is more potent than IL-1ß.

Murine collagen antibody induced arthritis (CAIA) and primary mouse hepatocyte culture as models to study cytochrome P450 suppression.

Changes in cytochrome P450 expression incurred by inflammatory disease were studied in a murine collagen antibody induced arthritis (CAIA) model and compared to bacterial lipopolysaccharide-treated mice and to cytochrome P450 changes in primary mouse hepatocytes following combination treatments with cytokines IL-1ß, IL-6, or TNFα. CAIA in female mice increased serum IL-1ß, IL-6 and hepatic serum amyloid A (SAA) mRNA and significantly altered cytochrome P450 mRNA and activity levels. Most cytochrome P450 isoforms were down-regulated, although some, such as Cyp3a13, were up-regulated. Cytokine effects on cytochrome P450 levels in mouse hepatocytes were compared at in vitro cytokine concentrations similar to those measured in CAIA mouse serum in vivo. In vivo and in vitro cytochrome P450 suppression by cytokines was congruent for some cytochrome P450 isoforms (Cyp1a2, Cyp2c29, and Cyp3a11) but not for others (cytochrome P450 oxidoreductase (POR) and Cyp2e1). In mouse hepatocytes, IL-6 and IL-1ß in combination in vitro caused a synergistic increase in SAA mRNA expression, but not in cytochrome P450 suppression. IL-1ß and IL-6 were equipotent in the suppression of cytochrome P450 gene expression, while TNFα caused mild suppression only at the highest concentrations used. TNFα in combination with IL-1ß, IL-6, or both had a protective effect against IL-1ß and IL-6-mediated cytochrome P450 suppression. When IL-1ß or IL-6 was combined with low concentrations of TNFα, several P450 isoforms were induced rather than suppressed. These data highlight the complexities of performing in vitro-in vivo comparisons using disease models for cytochrome P450 regulation by cytokines.

Regiospecific and stereospecific triangulation of the structures of metabolites formed by sequential metabolism at multiple prochiral centers.

Structures of in vivo secondary metabolites of a norbornane-containing drug candidate with multiple prochiral centers were triangulated, in a regio- and stereospecific fashion, using in vitro metabolism data from synthetic primary metabolites and in vivo metabolism data from the separate administration of a radiolabeled primary metabolite, [(14)C]-(S)-2-((1R,2S,4R,5S)-5-hydroxybicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (M1). A mass balance study on the 11ß hydroxysteroid dehydrogenase type 1 enzyme inhibitor [(14)C]-(S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221) in rats was dosed at 2 mg/kg. Radioactivity was excreted mainly in urine. Metabolites of AMG 221 were quantified by high-performance liquid chromatography with radiometric detection and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS revealed at least 38 metabolites. Seven monohydroxylated metabolites mediated formation of the other 31 metabolites. Twenty-eight metabolites were identified regio- and stereo-specifically. Little parent drug was observed in urine or feces. Monohydroxy metabolite M1 was the major metabolite comprising 17 to 24% of excreted dose, and seven monohydroxy metabolites comprised 29 (male) and 37% (female) of dose. Of 11 quantifiable isobaric dihydroxy metabolites that comprised 8.3 (male) and 24% (female) of dose, 10 were identified regio- and stereospecifically by triangulation. A single trihydroxy metabolite comprised approximately 10% of dose. Complex secondary metabolism of drugs with multiple prochiral centers can be elucidated in a regio- and stereospecific fashion without NMR through synthesis and in vitro and in vivo studies on the metabolism of chiral primary oxidation products.

Effects of interleukin-6 (IL-6) and an anti-IL-6 monoclonal antibody on drug-metabolizing enzymes in human hepatocyte culture.

The cytokine-mediated suppression of hepatic drug-metabolizing enzymes by inflammatory disease and the relief of this suppression by successful disease treatment have recently become an issue in the development of drug interaction labels for new biological products. This study examined the effects of the inflammatory cytokine interleukin-6 (IL-6) on drug-metabolizing enzymes in human hepatocyte culture and the abrogation of these effects by a monoclonal antibody directed against IL-6. Treatment of human hepatocytes with IL-6 (n = 9 donors) revealed pan-suppression of mRNA of 10 major cytochrome P450 isoenzymes, but with EC(50) values that differed by isoenzyme. Some EC(50) values were above the range of clinically relevant serum concentrations of IL-6. Marker activities for CYP1A2 and CYP3A4 enzyme were similarly suppressed by IL-6 in both freshly isolated and cryopreserved hepatocytes. IL-6 suppressed induction of CYP1A2 enzyme activity by omeprazole and CYP3A4 enzyme activity by rifampicin but only at supraphysiological concentrations of IL-6. Glycosylated and nonglycosylated IL-6 did not significantly differ in their ability to suppress CYP1A2 and CYP3A4 enzyme activity. A monoclonal antibody directed against IL-6 abolished or partially blocked IL-6-mediated suppression of CYP1A2 and CYP3A4 enzyme activity, respectively. These data indicate that experimentation with IL-6 and anti-IL-6 monoclonal antibodies in human hepatocyte primary culture can quantitatively measure cytochrome P450 suppression and desuppression and determine EC(50) values for IL-6 against individual cytochrome P450 isoenzymes. However, the complex biology of inflammatory disease may not allow for quantitative in vitro-in vivo extrapolation of these simple in vitro data.

Systematic genetic and genomic analysis of cytochrome P450 enzyme activities in human liver.

Liver cytochrome P450s (P450s) play critical roles in drug metabolism, toxicology, and metabolic processes. Despite rapid progress in the understanding of these enzymes, a systematic investigation of the full spectrum of functionality of individual P450s, the interrelationship or networks connecting them, and the genetic control of each gene/enzyme is lacking. To this end, we genotyped, expression-profiled, and measured P450 activities of 466 human liver samples and applied a systems biology approach via the integration of genetics, gene expression, and enzyme activity measurements. We found that most P450s were positively correlated among themselves and were highly correlated with known regulators as well as thousands of other genes enriched for pathways relevant to the metabolism of drugs, fatty acids, amino acids, and steroids. Genome-wide association analyses between genetic polymorphisms and P450 expression or enzyme activities revealed sets of SNPs associated with P450 traits, and suggested the existence of both cis-regulation of P450 expression (especially for CYP2D6) and more complex trans-regulation of P450 activity. Several novel SNPs associated with CYP2D6 expression and enzyme activity were validated in an independent human cohort. By constructing a weighted coexpression network and a Bayesian regulatory network, we defined the human liver transcriptional network structure, uncovered subnetworks representative of the P450 regulatory system, and identified novel candidate regulatory genes, namely, EHHADH, SLC10A1, and AKR1D1. The P450 subnetworks were then validated using gene signatures responsive to ligands of known P450 regulators in mouse and rat. This systematic survey provides a comprehensive view of the functionality, genetic control, and interactions of P450s.