Use of Pharmacokinetic and Pharmacodynamic Data to Develop the CDK4/6 Inhibitor Ribociclib for Patients with Advanced Breast Cancer.

Ribociclib is an orally bioavailable, selective cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor. CDK4/6 inhibition by ribociclib leads to retinoblastoma tumor suppressor protein (Rb) reactivation, thereby restoring Rb-mediated cell cycle arrest. Ribociclib is approved for the treatment of patients with hormone receptor-positive/human epidermal growth factor receptor-2-negative (HR+/HER2-) advanced breast cancer (ABC), at the dose of 600 mg once daily (QD) during cycles of 21 days on/7 days off, with optional dose reduction to 400 mg and 200 mg. Ribociclib is rapidly absorbed with a median time to reach maximum plasma concentration of 2.4 h, mean half-life of 32.0 h and oral bioavailability of 65.8% at 600 mg. It is eliminated mainly by hepatic metabolism (~ 84% of total elimination), mostly by cytochrome P450 (CYP) 3A4. Age, body weight, race, baseline Eastern Cooperative Oncology Group status, food, mild hepatic impairment, mild-to-moderate renal impairment, proton pump inhibitors, and combination partners (non-steroidal aromatase inhibitors or fulvestrant) have no clinically relevant impact on ribociclib exposure. Ribociclib inhibits CYP3A at 600 mg leading to increased exposure of CYP3A substrates. Strong CYP3A inhibitors or inducers increase or decrease, respectively, ribociclib exposure. Exposure-safety and exposure-efficacy analyses support the clinical benefit of the 600 mg QD starting dose, with potential individualized dose reductions to 400 mg and 200 mg for effective management of the adverse events neutropenia and QTcF interval prolongation, while maintaining efficacy, in patients with HR+/HER2- ABC. Overall, these clinical pharmacology data informed ribociclib dose justification and clinical development, as well as its prescribing information for clinical use in advanced breast cancer patients.

Industry Perspective on Therapeutic Peptide Drug-Drug Interaction Assessments During Drug Development: A European Federation of Pharmaceutical Industries and Associations White Paper.

Drug-drug interaction (DDI) assessments are well defined in health authority guidelines for small molecule drugs, and US Food and Drug Administration (FDA) draft guidance is now available for therapeutic proteins. However, there are currently no regulatory guidelines outlining DDI assessments for therapeutic peptides, which poses significant uncertainty and challenges during drug development for this heterogenous class of molecules. A cross-industry peptide DDI working group consisting of experts from 10 leading companies was formed under the sponsorship of the European Federation of Pharmaceutical Industries and Associations. We aimed to capture the range of DDI studies undertaken for peptide drugs by (i) anonymously surveying relevant companies involved in peptide drug development to better understand DDI study type/timing currently performed and (ii) compiling a database containing in vitro / clinical DDI data from submission packages for recently approved peptide drugs. Our analyses highlight significant gaps and uncertainty in the field. For example, the reported timing of in vitro peptide DDI studies, if performed, vary substantially across responding companies from early research to phase III. Nearly all in vitro cytochrome P450 / transporter inhibition studies reported in the survey were negative. For the few positive hits reported, no clinical follow-up studies were performed, questioning the clinical relevance of these findings. Furthermore, available submission packages reveal DDI likelihood is low for peptides >2 kDa, making it reasonable to adopt a risk-based approach during drug development for larger peptides. By benchmarking the landscape of peptide DDI activities across the industry, we set the stage for future discussions with health authorities on harmonizing peptide DDI approaches.

Novel Bruton's tyrosine kinase inhibitor remibrutinib: Assessment of drug-drug interaction potential as a perpetrator of cytochrome P450 enzymes and drug transporters and the impact of covalent binding on possible drug interactions.

PURPOSE: Pharmacokinetic drug-drug interactions (DDIs) are investigated to ensure safety for patients receiving concomitant medications. Here, we present a strategy to characterise the DDI potential of remibrutinib, as an inhibitor of drug-metabolising enzymes and drug transporters, and as an inducer. Initial in vitro studies were performed, followed by a biomarker-based assessment of induction in a first in human study, concluded by a clinical study to verify initial results. Remibrutinib is a covalent inhibitor of Bruton's Tyrosine kinase (BTKi) carrying a reactive acrylamide moiety (warhead), thus the potential contribution of covalent binding (off-target) to observed interactions was investigated as this could lead to prolonged and more potent drug interactions. METHODS: DDI assessment was focused on the putative inhibition of key metabolic enzymes (Cytochrome P450, CYP), drug transporters and a potential effect on oral contraceptives (OC) by induction of enzymes that are involved in their clearance (CYP3A4). The impact of covalent binding was assessed by synthesising an identical reference molecule but with an inactivated warhead. RESULTS: An interaction potential of limited clinical relevance was revealed for remibrutinib for CYP enzymes and drug transporters. The reactive warhead of remibrutinib had no impact on CYP enzyme and transporter inhibition, including time-dependent inhibition of CYP3A4, but may increase the induction potential of remibrutinib. CONCLUSIONS: Observed inhibition of metabolic enzymes indicated that remibrutinib is a weak inhibitor of CYP3A4 and CYP2C9 and is not a clinically relevant inhibitor of uptake and efflux transporters, except for intestinal P-glycoprotein and breast cancer resistance protein inhibition. OC may be safely administered and are effective when given with pharmacologically relevant doses of remibrutinib.

Species-dependent hepatic and intestinal metabolism of selective oestrogen receptor degrader LSZ102 by sulphation and glucuronidation.

LSZ102 is an orally bioavailable selective oestrogen receptor degrader in clinical development for the treatment of breast cancer. Preclinical studies showed efficacy in xenograft models on oral dosing. However, oral bioavailability was relatively low in several preclinical species (7-33%), and was associated with first-pass metabolism, particularly intestinal first-pass.To investigate metabolism and first-pass effects, metabolites were analysed in human plasma samples after oral dosing of LSZ102 to patients, rat plasma samples after oral dosing of [14C]LSZ102, and in vitro incubations of [14C]LSZ102 with human and rat hepatocytes and intestinal S9 fractions. The kinetics of human sulfotransferase (SULT) enzymes potentially involved in metabolism of LSZ102 was characterised.Sulphate metabolites were found to be the major components in human plasma, as well as in human hepatocytes and intestinal S9 fractions. Contrastingly, glucuronidation was predominant in rat plasma, hepatocytes and intestinal S9. LSZ102 was found to be metabolised by several human SULTs expressed in liver and intestine. The combined metabolism data in rat and human provide supporting evidence for an extensive intestinal first-pass metabolism effect via sulphation in human but glucuronidation in rat.As LSZ102 is metabolised by a number of different SULTs, drug-drug interactions resulting from the inhibition of one SULT are unlikely.Despite the observed species difference in metabolism, the major human metabolites of LSZ102, sulphate M5, glucuronide M4, and secondary glucuronide/sulphate metabolite M12, have no or weak pharmacological activity and are not considered a toxicity risk as they are phase II conjugative metabolites.

Ribociclib Drug-Drug Interactions: Clinical Evaluations and Physiologically-Based Pharmacokinetic Modeling to Guide Drug Labeling.

Ribociclib is approved in combination with endocrine therapy as initial endocrine-based therapy for HR-positive and HER2-negative advanced breast cancer. Ribociclib is primarily metabolized by CYP3A4 and, in vitro, is an inhibitor of CYP3A and CYP1A2. Ritonavir (a strong CYP3A inhibitor) increased ribociclib 400 mg single-dose area under the plasma concentration-time curve (AUC) by 3.2-fold, whereas rifampin (a strong CYP3A inducer) decreased ribociclib AUC by 89% in healthy volunteers (HVs). Multiple 400 mg ribociclib doses increased midazolam (CYP3A substrate) AUC by 3.8-fold and caffeine (CYP1A2 substrate) AUC by 1.2-fold vs. each agent alone. A physiologically-based pharmacokinetic (PBPK) model was developed integrating in vitro, preclinical, and clinical data of HVs and patients with cancer. Data predictions indicated that multiple 600 mg ribociclib doses increased midazolam AUC by 5.85-fold and ritonavir increased ribociclib 600 mg multiple dose AUC by 1.31-fold in cancer patients. Based on pharmacokinetics, safety, and efficacy data, and PBPK modeling, dosing modifications for ribociclib recommend avoiding concurrent use of strong CYP3A inhibitors/inducers, and caution regarding using CYP3A substrates with narrow therapeutic indices.

An integrated assessment of the ADME properties of the CDK4/6 Inhibitor ribociclib utilizing preclinical in vitro, in vivo, and human ADME data.

Ribociclib (LEE011, Kisqali ®) is a highly selective small molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), which has been approved for the treatment of advanced or metastatic breast cancer. A human ADME study was conducted in healthy male volunteers following a single oral dose of 600 mg [14 C]-ribociclib. Mass balance, blood and plasma radioactivity, and plasma ribociclib concentrations were measured. Metabolite profiling and identification was conducted in plasma, urine, and feces. An assessment integrating the human ADME results with relevant in vitro and in vivo non-clinical data was conducted to provide an estimate of the relative contributions of various clearance pathways of the compound. Ribociclib is moderately to highly absorbed across species (approx. 59% in human), and is extensively metabolized in vivo, predominantly by oxidative pathways mediated by CYP3A4 (ultimately forming N-demethylated metabolite M4) and, to a lesser extent, by FMO3 (N-hydroxylated metabolite M13). It is extensively distributed in rats, based on QWBA data, and is eliminated rapidly from most tissues with the exception of melanin-containing structures. Ribociclib passed the placental barrier in rats and rabbits and into milk of lactating rats. In human, 69.1% and 22.6% of the radiolabeled dose were excreted in feces and urine, respectively, with 17.3% and 6.75% of the 14 C dose attributable to ribociclib, respectively. The remainder was attributed to numerous metabolites. Taking into account all available data, ribociclib is estimated to be eliminated by hepatic metabolism (approx. 84% of total), renal excretion (7%), intestinal excretion (8%), and biliary elimination (1%).

Fevipiprant has a low risk of influencing co-medication pharmacokinetics: Impact on simvastatin and rosuvastatin in different SLCO1B1 genotypes.

Fevipiprant, a prostaglandin D2 receptor 2 antagonist, is in clinical development as a treatment for asthma. The goal of this study was to assess the potential of fevipiprant to cause drug-drug interactions (DDI) as a perpetrator, that is, by altering the pharmacokinetics (PK) of co-medications. In vitro drug interaction studies of clinically relevant drug metabolizing enzymes and transporters were conducted for fevipiprant and its acyl glucuronide (AG) metabolite. Comparison of Ki values with unbound systemic or portal vein steady-state plasma exposure of fevipiprant and its AG metabolite revealed the potential for inhibition of organic anion transporting polypeptide 1B1 (OATP1B1) transporters (R-value of 5.99), while other targets including cytochrome P450 enzymes were not, or only marginally, inhibited. Consequently, an open-label, two-part, two-period, single-sequence clinical study assessed the effect of fevipiprant 450 mg QD on the pharmacokinetics of simvastatin 20 mg and rosuvastatin 20 mg, two statins with different dependency in OATP1B1-mediated hepatic uptake, in healthy adult volunteers. The study also assessed the pharmacogenetics of the SLCO1B1 gene, which encodes OATP1B1. Clinically, fevipiprant 450 mg QD showed a low potential for interaction and increased the peak concentrations of simvastatin acid and rosuvastatin by 2.23- and 1.87-fold, respectively, with little or no impact on total exposure. Genotype analysis confirmed that SLCO1B1 genotype influences statin pharmacokinetics to a similar extent either with or without fevipiprant co-administration. In summary, fevipiprant at 450 mg QD has only minor liabilities as a perpetrator for DDI.

Phase II metabolism in human skin: skin explants show full coverage for glucuronidation, sulfation, N-acetylation, catechol methylation, and glutathione conjugation.

Although skin is the largest organ of the human body, cutaneous drug metabolism is often overlooked, and existing experimental models are insufficiently validated. This proof-of-concept study investigated phase II biotransformation of 11 test substrates in fresh full-thickness human skin explants, a model containing all skin cell types. Results show that skin explants have significant capacity for glucuronidation, sulfation, N-acetylation, catechol methylation, and glutathione conjugation. Novel skin metabolites were identified, including acyl glucuronides of indomethacin and diclofenac, glucuronides of 17ß-estradiol, N-acetylprocainamide, and methoxy derivatives of 4-nitrocatechol and 2,3-dihydroxynaphthalene. Measured activities for 10 µM substrate incubations spanned a 1000-fold: from the highest 4.758 pmol·mg skin(-1)·h(-1) for p-toluidine N-acetylation to the lowest 0.006 pmol·mg skin(-1)·h(-1) for 17ß-estradiol 17-glucuronidation. Interindividual variability was 1.4- to 13.0-fold, the highest being 4-methylumbelliferone and diclofenac glucuronidation. Reaction rates were generally linear up to 4 hours, although 24-hour incubations enabled detection of metabolites in trace amounts. All reactions were unaffected by the inclusion of cosubstrates, and freezing of the fresh skin led to loss of glucuronidation activity. The predicted whole-skin intrinsic metabolic clearances were significantly lower compared with corresponding whole-liver intrinsic clearances, suggesting a relatively limited contribution of the skin to the body's total systemic phase II enzyme-mediated metabolic clearance. Nevertheless, the fresh full-thickness skin explants represent a suitable model to study cutaneous phase II metabolism not only in drug elimination but also in toxicity, as formation of acyl glucuronides and sulfate conjugates could play a role in skin adverse reactions.

The potential of Hypoxis hemerocallidea for herb-drug interaction.

CONTEXT: Aqueous decoction of Hypoxis hemerocallidea Fisch. & C.A. Mey. (Hypoxidaceae) (Hypoxis) is widely consumed in Southern Africa by people living with HIV/AIDS, some of whom are on ARV and other medications. OBJECTIVE: The aim of this study was to investigate the potential of the crude aqueous extracts of Hypoxis to inhibit major forms of CYP450 and transport proteins. MATERIALS AND METHODS: Corms of Hypoxis were water-extracted and incubated (in graded concentrations: 1-100 µg/mL) with human liver microsomes (20 min) to monitor the effects on phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, midazolam 1'-hydroxylation and testosterone 6ß-hydroxylation as markers for the metabolic activities of CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4/5, respectively. The generation of metabolites were monitored and quantified with the aid of LC-MS/MS. The potential of the extracts to inhibit human ATP-binding cassette transporter activity was assessed using recombinant MDCKII and LLC-PK1 cells over-expressing human breast cancer resistant protein and human P-glycoprotein , respectively (with Ko143 and cyclosporin A as positive controls). Similar assessment was performed with human organic anion transporting polypeptide (OATP1B1 and OATP1B3) using recombinant HEK293 cells over-expressing OATP1B1 and OATP1B3, respectively (with rifamycin and 10 µM atorvastatin as positive controls). RESULTS: Extracts of Hypoxis inhibited the production of the metabolites of the substrates of the following enzymes (as compared to controls) with the indicated IC50 values (µg/mL): CYP1A2 (120.6), CYP2A6 (210.8), CYP2B6 (98.5), CYP2C8 (195.2), CYP2C9 (156) and CYP3A4/5 (185.4). The inhibition of the uptake activity of OATP1B1 and OATP1B3 were also observed with IC50 values of 93.4 and 244.8 µg/mL, respectively. DISCUSSION: Extract concentrations higher than the estimated IC50 values are achievable in the gastrointestinal tract when traditional doses of Hypoxis are considered. This may have profound effects on presystemic metabolism of the drug substrates. If absorbed, systemic inhibition of metabolic enzymes/transporters by Hypoxis may be expected. CONCLUSION: The result suggests that there is the potential for HDI between Hypoxis and the substrates of the affected enzymes/transporters, if sufficient in vivo concentration of Hypoxis extracts is attained.

The potential of Sutherlandia frutescens for herb-drug interaction.

In Africa, Sutherlandia frutescens is a popular medicinal herb widely consumed by people living with human immunodeficiency virus/AIDS. Concomitant use with antiretroviral drugs has generated concerns of herb-drug interaction (HDI). This study investigated the inhibitory effects of the crude extracts of S. frutescens on the major cytochrome P450 isozymes with the use of pooled human liver microsomes. Its effect on the metabolic clearance of midazolam using cryopreserved hepatocytes was also monitored. The potential of S. frutescens to inhibit human ATP-binding cassette transporters (P-gp and BCRP) and the human organic anion transporting polypeptide (OATP1B1 and OATP1B3) activity was assessed using cell lines overexpressing the transporter proteins. S. frutescens showed inhibitory potency for CYP1A2 (IC(50) = 41.0 µg/ml), CYP2A6 (IC(50) = 160 µg/ml), CYP2B6 (IC(50) = 20.0 µg/ml), CYP2C8 (IC(50) = 22.4 µg/ml), CYP2C9 (IC(50) = 23.0 µg/ml), CYP2C19 (IC(50) = 35.9 µg/ml), and CYP3A4/5 (IC(50) = 17.5 µg/ml [with midazolam1'-hydroxylation]; IC(50) = 28.3 µg/ml [with testosterone 6ß-hydroxylation]). Time-dependent (irreversible) inhibition by S. frutescens was observed for CYP3A4/5 (K(I) = 296 µg/ml, k(inact) = 0.063 min(-1)) under the conditions of this study. S. frutescens also delays the production of midazolam metabolites in the hepatocytes, decreasing its clearance by 40%. Furthermore, S. frutescens inhibited P-gp (IC(50) = 324.8 µg/ml), OATP1B1 (IC(50) = 10.4 µg/ml), and OATP1B3 (IC(50) = 6.6 µg/ml). The result indicates the potential for HDI between S. frutescens and the substrates of the affected enzymes, if sufficient in vivo concentration of the extract is attained.

Do rat cardiac myocytes release ATP on contraction?

ATP is released by numerous cell types in response to mechanical strain. It then acts as a paracrine or autocrine signaling molecule, inducing a variety of biological responses. In this work, we addressed the question whether mechanical force acting on the membranes of contracting cardiomyocytes during periodic longitudinal shortening can stimulate the release of ATP. Electrically stimulated isolated adult rat cardiomyocytes as well as spontaneously contracting mouse cardiomyocytes derived from embryonic stem (ES) cells were assayed for ATP release with the use of luciferase and a sensitive charge-coupled device camera. Sensitivity of soluble luciferase in the supernatant of cardiomyocytes was 100 nM ATP, which is approximately 10-fold below the EC(50) values for most purinergic receptors expressed in the heart (1.5-20 microM). Light intensities were not different between resting or contracting adult rat cardiomyocytes. Similar results were obtained with ES-cell-derived contracting mouse cardiomyocytes. ATP release was measurable only from obviously damaged or permeabilized cells. To increase selectivity and sensitivity of ATP detection we have targeted a recombinant luciferase to the sarcolemmal membrane using a wheat germ agglutinin-IgG linker. Contraction of labeled adult rat cardiomyocytes was not associated with measurable bioluminescence. However, when human umbilical vein endothelial cells were targeted with membrane-bound luciferase, shear stress-induced ATP release could be clearly detected, demonstrating the sensitivity of the detection method. In the present study, we did not detect ATP release from contracting cardiomyocytes on the single cell level, despite adequate sensitivity of the detection system. Thus deformation of the contracting cardiomyocyte is not a key stimulus for the release of cellular ATP.