Adapting ADME and Pharmacokinetic Analysis to the Next Generation of Therapeutic Modalities.

The development of multiple drug modalities over the past 20 years has dramatically expanded the therapeutic space for intervention in disease processes. Rather than being alternative therapeutic approaches, these modalities tend to be complimentary both in the scope of target space and the biological mechanisms harnessed for disease control. Realization of these therapeutic opportunities requires an understanding of the physiological, biochemical and biological barriers that control exposure to the drug target and resulting biological response.

siRNA-mediated knockdown of P450 oxidoreductase in rats: a tool to reduce metabolism by CYPs and increase exposure of high clearance compounds.

Purpose: To develop a tool based on siRNA-mediated knockdown of hepatic P450 oxidoreductase (POR) to decrease the CYP-mediated metabolism of small molecule drugs that suffer from rapid metabolism in vivo, with the aim of improving plasma exposure of these drugs.

Methods: siRNA against the POR gene was delivered using lipid nanoparticles (LNPs) into rats. The time course of POR mRNA knockdown, POR protein knockdown, and loss of POR enzyme activity was monitored.

RNA-induced silencing complex-bound small interfering RNA is a determinant of RNA interference-mediated gene silencing in mice.

Deeper knowledge of pharmacokinetic and pharmacodynamic (PK/PD) concepts for RNA therapeutics is important to streamline the drug development process and for rigorous selection of best performing drug candidates. Here we characterized the PK/PD relationship for small interfering RNAs (siRNAs) targeting luciferase by examining siRNA concentration in plasma and liver, the temporal RNA-induced silencing complex binding profiles, mRNA reduction, and protein inhibition measured by noninvasive bioluminescent imaging. A dose-dependent and time-related decrease in bioluminescence was detected over 25 days after a single treatment of a lipid nanoparticle-formulated siRNA targeting luciferase messenger RNA.

Effect of P-glycoprotein-mediated efflux on cerebrospinal fluid concentrations in rhesus monkeys.

Brain penetration of drugs which are subject to P-glycoprotein (Pgp)-mediated efflux is attenuated, as manifested by the fact that the cerebrospinal fluid concentration (C(CSF)), a good surrogate of the unbound brain concentration (C(ub)), is lower than the unbound plasma concentration (C(up)) for Pgp substrates. In rodents, the attenuation magnitude of brain penetration by Pgp-mediated efflux has been estimated by correlating the ratio of CSF to plasma exposures (C(CSF)/C(p)) with the unbound fraction in plasma (f(u)) upon the incorporation of the in vivo or in vitro Pgp-mediated efflux ratios (ERs). In the present work, we investigated the impact of Pgp-mediated efflux on C(CSF) in monkeys.

First demonstration of cerebrospinal fluid and plasma A beta lowering with oral administration of a beta-site amyloid precursor protein-cleaving enzyme 1 inhibitor in nonhuman primates.

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated.

Strategies toward improving the brain penetration of macrocyclic tertiary carbinamine BACE-1 inhibitors.

This letter describes replacements for the P3 amide moiety present in previously reported tertiary carbinamine macrolactones. Although P-gp efflux issues associated with these amide-macrolactones were solved and full brain penetration was measured in one case, potency was compromised in the process.

Design, synthesis, and SAR of macrocyclic tertiary carbinamine BACE-1 inhibitors.

This Letter describes the design and synthesis of tertiary carbinamine macrocyclic inhibitors of the beta-secretase (BACE-1) enzyme. These macrocyclic inhibitors, some of which incorporate novel P2 substituents, display a 2- to 100-fold increase in potency relative to the previously described acyclic analogs while affording greater stability.

Discovery and SAR of isonicotinamide BACE-1 inhibitors that bind beta-secretase in a N-terminal 10s-loop down conformation.

A series of low-molecular weight 2,6-diamino-isonicotinamide BACE-1 inhibitors containing an amine transition-state isostere were synthesized and shown to be highly potent in both enzymatic and cell-based assays. These inhibitors contain a trans-S,S-methyl cyclopropane P(3) which bind BACE-1 in a 10s-loop down conformation giving rise to highly potent compounds with favorable molecular weight and moderate to high susceptibility to P-glycoprotein (P-gp) efflux.

Identification of novel, orally bioavailable spirohydantoin CGRP receptor antagonists.

A rapid analogue approach to identification of spirohydantoin-based CGRP antagonists provided novel, low molecular weight leads. Modification of these leads afforded a series of nanomolar benzimidazolinone-based CGRP receptor antagonists. The oral bioavailability of these antagonists was inversely correlated with polar surface area, suggesting that membrane permeability was a key limitation to absorption.

Development and characterization of LLC-PK1 cells containing Sprague-Dawley rat Abcb1a (Mdr1a): comparison of rat P-glycoprotein transport to human and mouse.

Introduction: P-glycoprotein is localized in numerous tissues throughout the body and plays an important role in the disposition of many xenobiotics. The contribution of P-glycoprotein-mediated drug transport is being evaluated in early drug discovery stages, particularly for compounds targeted to the central nervous system, using in vitro tools including cell lines expressing P-glycoprotein. Previous work in our laboratory suggests there are species differences in P-glycoprotein transport activity between humans and animals.

Interactions of human P-glycoprotein with simvastatin, simvastatin acid, and atorvastatin.

Purpose: In this study, P-glycoprotein (P-gp) mediated efflux of simvastatin (SV), simvastatin acid (SVA), and atorvastatin (AVA) and inhibition of P-gp by SV, SVA, and AVA were evaluated to assess the role of P-gp in drug interactions.

Methods: P-gp mediated efflux of SV, SVA, and AVA was determined by directional transport across monolayers of LLC-PK1 cells and LLC-PK1 cells transfected with human MDR1. Inhibition of P-gp was evaluated by studying the vinblastine efflux in Caco-2 cells and in P-gp overexpressing KBV1 cells at concentrations of SV, SVA, and AVA up to 50 microM.

Acyl glucuronidation and glucosidation of a new and selective endothelin ET(A) receptor antagonist in human liver microsomes.

Compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl) cyclopenteno [1,2-b] pyridine 6-carboxylic acid] is a new and selective endothelin ET(A) receptor antagonist. It underwent significant acyl glucuronidation and acyl glucosidation in human liver microsomes supplemented with UDP-glucuronic acid (UDPGA) and UDP-glucose (UDPG). These two conjugations were observed in a panel of human liver microsomal samples (n = 16) that gave rise to varying activities but with no significant correlation with each other in the native and activator-treated microsomal preparations (r(2) View Article and Find Full Text PDF

Evaluation of drug interactions with P-glycoprotein in drug discovery: in vitro assessment of the potential for drug-drug interactions with P-glycoprotein.

The pharmacological effects of a drug are highly dependent on the absorption, metabolism, elimination, and distribution of the drug. In the past few years it has become apparent that transport proteins play a major role in regulating the distribution, elimination and metabolism of some drugs. As a consequence of our new understanding of the influence of transport proteins on the pharmacokinetic and pharmacodynamic behavior of drugs, increasing attention has been focused on the potential for drug-drug interactions arising from interactions with drug transport proteins.