Jerome H. Hochman

Mechanisms of absorption enhancement and tight junction regulation

Abstract In this article, current knowledge on the regulation of tight junction permeability is used as a platform for an analysis of the mechanisms of some common absorption enhancers. Epithelial barriers are considered with an emphasis on the intestinal epithelium. The analysis reveal that different absorption enhancers may share some previously unrecognised characteristics but also display unique mechanisms of action.

Cultured Rat Hepatocytes

The use of in vitro and in vivo systems to evaluate hepatic drug uptake and metabolism, cytochrome P450 induction, drug interactions affecting hepatic metabolism, hepatotoxicity, and cholestasis is an essential part of the drug development process (Tavoloni and Boyer, 1980; Powis et al., 1989; Bertrams and Ziegler, 1991; Jurima-Romet and Huang, 1992; Komai et al., 1992; Jurima-Romet and Huang, 1993). Primary hepatocyte culture is one of many techniques used to address these issues. Although primary hepatocytes maintained under conventional culture conditions have been broadly used to study drug metabolism and hepatotoxicity, the rapid loss of liver-specific functions and the failure of cultured hepatocytes to reestablish normal bile canaliculi (cell polarity, cell architecture) have limited their application.

Drug-drug interaction studies: regulatory guidance and an industry perspective.

Recently, the US Food and Drug Administration and European Medicines Agency have issued new guidance for industry on drug interaction studies, which outline comprehensive recommendations on a broad range of in vitro and in vivo studies to evaluate drug–drug interaction (DDI) potential. This paper aims to provide an overview of these new recommendations and an in-depth scientifically based perspective on issues surrounding some of the recommended approaches in emerging areas, particularly, transporters and complex DDIs. We present a number of theoretical considerations and several case examples to demonstrate complexities in applying (1) the proposed transporter decision trees and associated criteria for studying a broad spectrum of transporters to derive actionable information and (2) the recommended model-based approaches at an early stage of drug development to prospectively predict DDIs involving time-dependent inhibition and mixed inhibition/induction of drug metabolizing enzymes. We hope to convey the need for conducting DDI studies on a case-by-case basis using a holistic scientifically based interrogative approach and to communicate the need for additional research to fill in knowledge gaps in these areas where the science is rapidly evolving to better ensure the safety and efficacy of new therapeutic agents.

First Demonstration of Cerebrospinal Fluid and Plasma Aβ Lowering with Oral Administration of a β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitor in Nonhuman Primates

β-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 Aβ42 peptides in Alzheimers disease. Although previous mouse studies have shown brain Aβ 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 Aβ in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC50 ∼ 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain Aβ levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma Aβ levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPPβ, Aβ40, Aβ42, and plasma Aβ40 levels. CSF Aβ42 lowering showed an EC50 of ∼20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF Aβ lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

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. The efflux transporter P-glycoprotein (P-gp) has received the most attention with regard to its role in restricting drug absorption and distribution and as a potential source for variability in drug pharmacokinetics and pharmacodynamics. This review will focus on the evaluation of drug candidates to assess the potential for drug interactions at the level of P-gp. We will discuss the role of P-gp in drug disposition, the biochemistry of P-gp efflux as it relates to model systems to study drug interactions with P-gp, and the implementation of P-gp assay models within the drug discovery process.

Simultaneous in vitro measurement of intestinal tissue permeability and transepithelial electrical resistance (TEER) using Sweetana-Grass diffusion cells.

A simple modification of the commercially available Sweetana–Grass (S-G) side-by-side diffusion cells, allowing the simultaneous measurement of tissue permeability and transepithelial electrical resistance (TEER), has been described and validated for rat excised, muscle-free intestinal tissue. The TEER-lowering effects of a series of acylcarnitines were shown to be correlated with previously reported in vitro (i.e., membrane perturbation) and in vivo (i.e., absorption enhancement) activity. The TEER-lowering effect of palmitoyl carnitine chloride (PCC) was also shown to be reversible. The effects of PCC on TEER and the permeability of poorly absorbed compounds (cefoxitin and lucifer yellow) were simultaneously determined. Compared to controls (mannitol-treated), PCC immediately produced a rapid drop in colon TEER. By 5 min post-PCC addition, colon TEER was 50% of control; by 10 min post-PCC addition, colon TEER was 17% of control. After a lag of about 5–10 min post-PCC addition, the cefoxitin or lucifer yellow permeability coefficient increased more than 20-fold. The modified S-G cells provide a simple and reproducible method whereby flux and TEER can be simultaneously determined, providing a valuable link between the effect of absorption enhancers on TEER measurements and the increased permeability of poorly absorbed compounds.

Mini Review on Molecular Modeling of P-Glycoprotein (Pgp)

Membrane bound P-glycoprotein (Pgp) acts as an active transport pump. It plays a major role as a cause of multidrug resistance (MDR) and acts as a component of the blood-brain barrier. Pgp transports a wide variety of structurally unrelated compound from the cell interior into the extracellular space. Recent molecular modeling efforts, mostly in homology modeling and QSAR studies, have brought some understanding to the interactions between the protein and the drugs at the atomic level. We review the recent developments from the point of view of methodology.

Quantitation of Physiological and Biochemical Barriers to siRNA Liver Delivery via Lipid Nanoparticle Platform

Effective delivery of small interfering RNA (siRNA) requires efficient cellular uptake and release into cytosol where it forms an active complex with RNAi induced silencing complex (RISC). Despite rapid developments in RNAi therapeutics, improvements in delivery efficiency of siRNA are needed to realize the full potential of this modality in broad therapeutic applications. We evaluated potential physiological and biochemical barrier(s) to the effective liver delivery of siRNA formulated in lipid nanoparticle (LNP) delivery vehicles. The comparative siRNA delivery performance of three LNPs was investigated in rats. They were assembled with either C14- or C18-anchored PEG-lipid(s), cationic lipid(s), and various helper lipid(s) and contained the same siRNA duplex. These LNPs demonstrated differentiated potency with ED50s ranging from 0.02 to 0.25 mg/kg. The two C14-PEG-LNPs had comparable siRNA exposure in plasma and liver, while the C18-PEG-LNP demonstrated a higher plasma siRNA exposure and a slower but sustained liver uptake. RISC bound siRNA within the liver, a more proximal measure of the pharmacologically active siRNA species, displayed loading kinetics that paralleled the target mRNA knockdown profile, with greater RISC loading associated with more potent LNPs. Liver perfusion and hepatocyte isolation experiments were performed following treatment of rats with LNPs containing VivoTag-fluorescently labeled siRNA. One hour after dosing a majority of the siRNA within the liver was associated with hepatocytes and was internalized (within small subcellular vesicles) with no significant cell surface association, indicating good liver tissue penetration, hepatocellular distribution, and internalization. Comparison of siRNA amounts in hepatocytes and subcellular fractions of the three LNPs suggests that endosomal escape is a significant barrier to siRNA delivery where cationic lipid seems to have a great impact. Quantitation of Ago-2 associated siRNA revealed that after endosomal escape further loss of siRNA occurs prior to RISC loading. This quantitative assessment of LNP-mediated siRNA delivery has highlighted potential barriers with respect to endosomal escape and incomplete RISC loading for delivery optimization efforts.

Drug–Drug Interactions Related to Altered Absorption and Plasma Protein Binding: Theoretical and Regulatory Considerations, and an Industry Perspective

Drug-drug interactions (DDIs) related to altered drug absorption and plasma protein binding have received much less attention from regulatory agencies relative to DDIs mediated via drug metabolizing enzymes and transporters. In this review, a number of theoretical bases and regulatory framework are presented for these DDI aspects. Also presented is an industry perspective on how to approach these issues in support of drug development. Overall, with the exception of highly permeable and highly soluble (BCS 1) drugs, DDIs related to drug-induced changes in gastrointestinal (GI) physiology can be substantial, thus warranting more attentions. For a better understanding of absorption-associated DDI potential in a clinical setting, mechanistic studies should be conducted based on holistic integration of the pharmaceutical profiles (e.g., pH-dependent solubility) and pharmacological properties (e.g., GI physiology and therapeutic margin) of drug candidates. Although majority of DDI events related to altered plasma protein binding are not expected to be of clinical significance, exceptions exist for a subset of compounds with certain pharmacokinetic and pharmacological properties. Knowledge of the identity of binding proteins and the binding extent in various clinical setting (including disease states) can be valuable in aiding clinical DDI data interpretations, and ensuring safe and effective use of new drugs.

Role of Mechanistic Transport Studies in Lead Optimization

During the drug discovery process an average of five to ten thousand compounds are evaluated to identify the small subset of structures with appropriate properties to become a drug. A potential drug is distinguished from a potent agonist /antagonist based on multiple factors affecting safety, exposure and marketability including target selectivity, chemical stability, physical chemical properties, and drug metabolism properties. From the drug metabolism standpoint unfavorable pharmacokinetics is one of the primary barriers to overcome in drug discovery. In the case of most CNS drugs, this is further complicated by the requirement for the compound to traverse the blood-brain barrier in order for it to be efficacious. Thus, for CNS drugs, a compound must balance chemical properties conferring good CNS penetration, favorable metabolic characteristics, and good oral absorption in addition to high potency against the target activity.