No abstract

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate in clinical development for the treatment of human epidermal growth factor receptor 2 (HER2)-positive cancers. Herein, we describe a series of studies to assess T-DM1 absorption, distribution, metabolism, and excretion (ADME) in rats as well as to assess human exposure to T-DM1 catabolites. Following administration of unlabeled and radiolabeled T-DM1 in female Sprague Dawley rats as a single dose, plasma, urine, bile and feces were assessed for mass balance, profiling and identification of catabolites. In rats, the major circulating species in plasma was T-DM1, while DM1 concentrations were low (1.08 to 15.6 ng/mL). The major catabolites found circulating in rat plasma were DM1, [N-maleimidomethyl] cyclohexane-1- carboxylate-DM1 (MCC-DM1), and Lysine-MCC-DM1. These catabolites identified in rats were also detected in plasma samples from patients with HER2-positive metastatic breast cancer who received single-agent T-DM1 (3.6 mg/kg every 3 weeks) in a phase 2 clinical study. There was no evidence of tissue accumulation in rats or catabolite accumulation in human plasma following multiple dosing. In rats, T-DM1 was distributed nonspecifically to the organs without accumulation. The major pathway of DM1-containing catabolite elimination in rats was the fecal/biliary route, with up to 80% of radioactivity recovered in the feces and 50% in the bile. The rat T-DM1 ADME profile is likely similar to the human profile, although there may be differences since trastuzumab does not bind the rat HER2- like receptor. Further research is necessary to more fully understand the T-DM1 ADME profile in humans.

The majority of marketed small-molecule drugs undergo metabolism by hepatic Cytochrome P450 (CYP) enzymes (Rendic 2002). Since these enzymes metabolize a structurally diverse number of drugs, metabolism-based drug-drug interactions (DDIs) can potentially occur when multiple drugs are coadministered to patients. Thus, a careful in vitro assessment of the contribution of various CYP isoforms to the total metabolism is important for predicting whether such DDIs might take place. One method of CYP phenotyping involves the use of potent and selective chemical inhibitors in human liver microsomal incubations in the presence of a test compound. The selectivity of such inhibitors plays a critical role in deciphering the involvement of specific CYP isoforms. Here, we review published data on the potency and selectivity of chemical inhibitors of the major human hepatic CYP isoforms. The most selective inhibitors available are furafylline (in co-incubation and pre-incubation conditions) for CYP1A2, 2-phenyl-2-(1-piperidinyl)propane (PPP) for CYP2B6, montelukast for CYP2C8, sulfaphenazole for CYP2C9, (-)-N-3-benzyl-phenobarbital for CYP2C19 and quinidine for CYP2D6. As for CYP2A6, tranylcypromine is the most widely used inhibitor, but on the basis of initial studies, either 3-(pyridin-3-yl)-1H-pyrazol-5-yl)methanamine (PPM) and 3-(2-methyl-1H-imidazol-1-yl)pyridine (MIP) can replace tranylcypromine as the most selective CYP2A6 inhibitor. For CYP3A4, ketoconazole is widely used in phenotyping studies, although azamulin is a far more selective CYP3A inhibitor. Most of the phenotyping studies do not include CYP2E1, mostly because of the limited number of new drug candidates that are metabolized by this enzyme. Among the inhibitors for this enzyme, 4-methylpyrazole appears to be selective.

The well accepted "free drug hypothesis" for small-molecule drugs assumes that only the free (unbound) drug concentration at the therapeutic target can elicit a pharmacologic effect. Unbound (free) drug concentrations in plasma are readily measurable and are often used as surrogates for the drug concentrations at the site of pharmacologic action in pharmacokinetic-pharmacodynamic analysis and clinical dose projection in drug discovery. Furthermore, for permeable compounds at pharmacokinetic steady state, the free drug concentration in tissue is likely a close approximation of that in plasma; however, several factors can create and maintain disequilibrium between the free drug concentration in plasma and tissue, leading to free drug concentration asymmetry. These factors include drug uptake and extrusion mechanisms involving the uptake and efflux drug transporters, intracellular biotransformation of prodrugs, membrane receptor-mediated uptake of antibody-drug conjugates, pH gradients, unique distribution properties (covalent binders, nanoparticles), and local drug delivery (e.g., inhalation). The impact of these factors on the free drug concentrations in tissues can be represented by K p,uu , the ratio of free drug concentration between tissue and plasma at steady state. This review focuses on situations in which free drug concentrations in tissues may differ from those in plasma (e.g., K p,uu > or <1) and discusses the limitations of the surrogate approach of using plasmafree drug concentration to predict free drug concentrations in tissue. This is an important consideration for novel therapeutic modalities since systemic exposure as a driver of pharmacologic effects may provide limited value in guiding compound optimization, selection, and advancement. Ultimately, a deeper understanding of the relationship between free drug concentrations in plasma and tissues is needed.

ABSTRACT:(R)-N-(3-(6-(4-(1,4-Dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide (GDC-0834) is a potent and selective inhibitor of Bruton's tyrosine kinase (BTK), investigated as a potential treatment for rheumatoid arthritis. In vitro metabolite identification studies in hepatocytes revealed predominant formation of an inactive metabolite (M1) via amide hydrolysis in human. The formation of M1 appeared to be NADPHindependent in human liver microsomes. M1 was found in only minor to moderate quantities in plasma from preclinical species dosed with GDC-0834. Human clearance predictions using various methodologies resulted in estimates ranging from low to high. In addition, GDC-0834 exhibited low clearance in PXB chimeric mice with humanized liver. Uncertainty in human pharmacokinetic prediction and high interest in a BTK inhibitor for clinical evaluation prompted an investigational new drug strategy, in which GDC-0834 was rapidly advanced to a single-dose human clinical trial. GDC-0834 plasma concentrations in humans were below the limit of quantitation (<1 ng/ml) in most samples from the cohorts dosed orally at 35 and 105 mg. In contrast, substantial plasma concentrations of M1 were observed. In human plasma and urine, only M1 and its sequential metabolites were identified. The formation kinetics of M1 was evaluated in rat, dog, monkey, and human liver microsomes in the absence of NADPH. The maximum rate of M1 formation (V max ) was substantially higher in human compared with that in other species. In contrast, the Michaelis-Menten constant (K m ) was comparable among species. Intrinsic clearance (V max /K m ) of GDC-0834 from M1 formation in human was 23-to 169-fold higher than observed in rat, dog, and monkey.