Human Pharmacokinetics of LYS006, an Oral Leukotriene A4 Hydrolase Inhibitor Displaying Target-Mediated Drug Disposition.

LYS006 is a potent leukotriene A4 hydrolase inhibitor currently in clinical development for long-term treatment of various neutrophil-driven inflammatory conditions. Here, we present pharmacokinetics from the first-in-human study with complementary metabolism and transporter profiling data. The randomized first-in-human study included nine cohorts receiving 5-2*100 mg of LYS006 or placebo, a crossover food-effect part, and a multiple-dose part consisting of two fasted (5 mg and 15 mg once daily) and three fed cohorts (20-80 mg twice a day) of LYS006 or placebo. LYS006 and metabolites were assessed in plasma and urine, and transporters involved in LYS006 disposition were analyzed in vitro. Systemic plasma exposure increased with dose; steady-state exposure was dose proportional up to 40 mg twice a day. Steady state was achieved after ∼3 days, with mean accumulation of 2.1-fold for 5 mg once daily and ≤1.4-fold for all higher doses. Despite limited accumulation, a long terminal half-life (T1/2) was observed. The long T1/2 and saturable binding to blood cells, which causes a highly nonlinear blood-to-plasma distribution, reflect a strong impact of target binding on drug distribution at lower concentrations. Skin biopsy and blister fluid concentration data indicated saturable binding in the former but not the latter, suggesting saturable binding in tissues beyond blood. Major excretion of LYS006 (∼90% of dose) through urine at steady state triggered renal transporter investigations that identified LYS006 as a substrate of organic anion transporter (OAT)3, OAT4, breast cancer resistance protein, and multidrug resistance-associated protein 4. Seven metabolites were identified in human plasma and urine, comprising only 4% of the dose recovered in urine at steady state. SIGNIFICANCE STATEMENT: Pharmacokinetic data from a first-in-human study combined with in vitro work support dose and regimen selection for patient studies with LYS006 and provide guidance on drug interaction investigations and other clinical pharmacology work needed for further development. Mass balance information at steady state without the use of a radiolabel, skin concentrations, and identification of the major clearance pathway, as well as the transporters driving elimination, make this a particularly conclusive early study despite nonlinear pharmacokinetics impacted by target binding.

Double Trap Interface: A novel gas interface for high throughput analysis of biomedical samples by AMS.

Although Accelerator Mass Spectrometry (AMS) offers unparalleled sensitivity by investigating the fate of 14C-labeled compounds within the organism, its widespread use in ADME (absorption, distribution, metabolism, excretion) studies is limited. Conventional approaches based on Liquid Scintillation Counting (LSC) are still preferred, in particular because of complexity and costs associated with AMS measurements. Progress made over the last decade towards more compact AMS systems increased the interest in a combustion-based AMS approach allowing the analysis of samples in gaseous form. Thus, a novel gas Double Trap Interface (DTI) was designed, providing high sample throughput for the analysis of biomedical samples. DTI allows the coupling of an Elemental Analyzer (EA) for sample combustion to the hybrid ion source of a MICADAS (MIni CArbon DAting System) AMS system. The performance was evaluated in two studies through the analysis of more than 1000 samples from 14C-labeled biomatrices and fractions collected after liquid chromatography (LC). The covered activity ranged from 1 to 1000 mBq/g for labeled biomatrices and from 1 to 10000 mBq/g(C) for LC fractions. The implemented routine allows automated measurements requiring less than 5 min per sample (12-13 analyses per hour) without the need for sample conversion to graphite.

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%).

Comparison of 19F NMR and 14C Measurements for the Assessment of ADME of BYL719 (Alpelisib) in Humans.

The human mass balance study is the definitive study for the assessment of absorption, distribution, metabolism, and excretion (ADME) properties of a new chemical entity in humans. Traditionally this has been carried out by the administration of radiolabeled drug substances, typically 14C or occasionally 3H, as detection methods for these isotopes allow the absolute quantification of drug-related material (DRM) in blood, plasma, and excreta. Coupled with the use of analytical techniques such as liquid chromatography-mass spectrometry, a picture of the metabolic fate of a compound can be elucidated. In this study, we demonstrate the capabilities of 19F nuclear magnetic resonance (NMR) spectroscopy, applied as an alternative to radiolabeling, for the determination of mass balance and for metabolite profiling of an orally administered fluorinated drug. To demonstrate the capabilities of NMR, the study was conducted on remaining samples from a 14C human mass balance study conducted on Alpelisib (BYL719), a compound in late stage development at Novartis for the treatment of solid tumors. Quantitative 14C data were used to cross-validate the data obtained by NMR. The data show that, using 19F NMR, comparable data can be obtained for key human ADME endpoints including mass balance, total DRM determination in plasma and metabolite profiling and identification in plasma and excreta. Potential scenarios where NMR could be employed as an alternative to radiolabeling for the conduct of an early human ADME study are discussed.

Microbial production of phase I and phase II metabolites of midazolam.

Midazolam, a potent benzodiazepine derivative and a typical substrate of CYP3A4/3A5, is essentially metabolized in human into 1'-hydroxymidazolam, then eliminated as the corresponding phase II metabolite, the 1'-O-ß-D-glucuronide derivative. A high yield alternative to the current multistep synthesis of 1'-hydroxymidazolam is described, using a biotransformation of midazolam by a fungal microorganism, Beauveria bassiana. The corresponding phase II metabolite, 1'-hydroxymidazolam-1'-O-ß-D-glucuronide, has been then prepared by chemical synthesis (3 steps, 20% yield), or by microbial glucuronidation (one step, 20% yield) using a Streptomyces sp. strain. The use of the same Streptomyces strain allows a direct and expeditive synthesis of the same glucuronide conjugate from midazolam itself in an advantageous 17% yield.

Microbial production of phase I and phase II metabolites of propranolol.

1. The production in multimilligram amounts of 4- and 5-hydroxylated metabolites of (R)- or (S)-propranolol by biotransformation with two fungal strains, an Absidia sp. M50002 and a Cunninghamella sp. M50036, was carried out, starting from either the racemic drug or pure enantiomers. 2. While both enantiomers of propranolol were hydroxylated in the 5-position by incubation with strain M50002, the strain M50036 operated a chiral discrimination, resulting in the exclusive formation of the 4-hydroxy-(R)-enantiomer. 3. In addition, a Streptomyces sp. strain M52104, isolated from a soil sample, was selected for the high-yield regioselective ß-glucuronidation of propranolol and its 4- and 5-hydroxylated derivatives. 4. NMR and mass spectroscopic data have been extensively used for the unambiguous characterization of 4- and 5-hydroxylated and glucuronidated derivatives, all of them corresponding to the major animal and human metabolites of propranolol, a typical substrate of CYP2D6.