DMD058750 163..181

ABSTRACT During the process of drug discovery, the pharmaceutical industry is faced with numerous challenges. One challenge is the successful prediction of the major routes of human clearance of new medications. For compounds cleared by metabolism, accurate predictions help provide an early risk assessment of their potential to exhibit significant interpatient differences in pharmacokinetics via routes of metabolism catalyzed by functionally polymorphic enzymes and/or clinically significant metabolic drug-drug interactions. This review details the most recent and emerging in vitro strategies used by drug metabolism and pharmacokinetic scientists to better determine rates and routes of metabolic clearance and how to translate these parameters to estimate the amount these routes contribute to overall clearance, commonly referred to as fraction metabolized. The enzymes covered in this review include cytochrome P450s together with other enzymatic pathways whose involvement in metabolic clearance has become increasingly important as efforts to mitigate cytochrome P450 clearance are successful. Advances in the prediction of the fraction metabolized include newly developed methods to differentiate CYP3A4 from the polymorphic enzyme CYP3A5, scaling tools for UDP-glucuronosyltranferase, and estimation of fraction metabolized for substrates of aldehyde oxidase. Introduction In an era where combination drug therapy to treat several conditions simultaneously is common, drug companies emphasize the need for optimal absorption, distribution, metabolism, and excretion (ADME) properties, with the purpose of optimization of efficacy and minimization of the risk of adverse events. This includes a proper assignment and an extensive understanding of the routes of metabolism to aid in the prediction of human pharmacokinetics, and to help avoid a potential "object drug" scenario when coadministration is likely. The attributes of the coadministered drug and/or the patient has the potential to increase the probability of a drug-drug interaction (DDI), i.e., enzyme inhibitor, inducer, polymorphic genotype. Hence, the greater the percentage attributed to a single metabolic route, the greater the potential for a DDI and possible "black box warning" being issued as part of the drug package insert. Furthermore, the pharmacokinetic implications of a single polymorphic enzyme being responsible for a majority of the metabolism of a drug may have either an efficacy (extensive metabolizers) or toxicological (poor metabolizers) impact on exposure. To address these concerns, early drug discovery teams strive for balanced metabolism across multiple enzymes and clearance mechanisms (hepatic, renal, biliary). These discovery efforts center on in vitro reaction phenotyping to support enhanced chemical design. There is a general agreement among the major regulatory agencies that pharmaceutical companies should provide a characterization of the metabolic profile of a new chemical entity (NCE) and understand the enzymology of the major clearance mechanisms (reaction phenotyping) using various in vitro study tools (http://www.fda.gov/downloads/Drugs/ GuidanceComplianceRegulatoryInformation/Guidances/ucm292362.pdf). Reaction phenotyping is the semiquantitative in vitro estimation of the relative contributions of specific drug-metabolizing enzymes to the metabolism of a test compound. The relative enzyme contributions quantified The authors have no conflicts of interest to declare and have not received compensation for the preparation of this manuscript. d

Optimum design of experiments for enzyme inhibition kinetic models

We find closed-form expressions for the D-optimum designs for three- and four-parameter nonlinear models arising in kinetic models for enzyme inhibition. We calculate the efficiency of designs over a range of parameter values and make recommendations for design when the parameter values are not well known. In a three-parameter experimental example, a standard design has an efficiency of 18.2% of the D-optimum design. Experimental results from a standard design with 120 trials and a D-optimum design with 21 trials give parameter estimates that are in close agreement. The estimated standard errors of these parameter estimates confirm our theoretical results on efficiency and thus on the serious savings that can be made by the use of D-optimum designs

The flavone hispidulin, a benzodiazepine receptor ligand with positive allosteric properties, traverses the blood–brain barrier and exhibits anticonvulsive effects

1. The functional characterization of hispidulin (4′,5,7-trihydroxy-6-methoxyflavone), a potent benzodiazepine (BZD) receptor ligand, was initiated to determine its potential as a modulator of central nervous system activity. 2. After chemical synthesis, hispidulin was investigated at recombinant GABA(A)/BZD receptors expressed by Xenopus laevis oocytes. Concentrations of 50 nM and higher stimulated the GABA-induced chloride currents at tested receptor subtypes (α(1−3,5,6)β(2)γ(2)S) indicating positive allosteric properties. Maximal stimulation at α(1)β(2)γ(2)S was observed with 10 μM hispidulin. In contrast to diazepam, hispidulin modulated the α(6)β(2)γ(2)S-GABA(A) receptor subtype. 3. When fed to seizure-prone Mongolian gerbils (Meriones unguiculatus) in a model of epilepsy, hispidulin (10 mg kg(−1) body weight (BW) per day) and diazepam (2 mg kg(−1) BW per day) markedly reduced the number of animals suffering from seizures after 7 days of treatment (30 and 25% of animals in the respective treatment groups, vs 80% in the vehicle group). 4. Permeability across the blood–brain barrier for the chemically synthesized, (14)C-labelled hispidulin was confirmed by a rat in situ perfusion model. With an uptake rate (K(in)) of 1.14 ml min(−1) g(−1), measurements approached the values obtained with highly penetrating compounds such as diazepam. 5. Experiments with Caco-2 cells predict that orally administered hispidulin enters circulation in its intact form. At a concentration of 30 μM, the flavone crossed the monolayer without degradation as verified by the absence of glucuronidated metabolites