Current Drug Metabolism - Volume 11, Issue 9, 2010

Oral bioavailability is a function of intestinal absorption and first-pass metabolism. Future chemistry space is predicted to be relatively hydrophilic; where the development issues associated with toxicity are expected to be low [1]. This may result in limited passive membrane permeability and increased reliance on membrane transporters for intestinal absorption. A growing list of membrane transporters has been recognized in almost all tissues, although a few of them are well-characterized and appreciated to be determinants of drug disposition. Enterocytes express several transporters, belonging to the adenosine triphosphate binding cassette (ABC) and the solute carrier (SLC) superfamilies, on the apical and basolateral membranes for the influx or efflux of endogenous and drug substances.Several successful chemistries have been reported to target some of the vital intestinal uptake transporters or avoid efflux pumps to enhance the absorption. In the light of this, the contribution by Varma et al. reviews the molecular and functional characteristics along with the structure-activity relationships of the vital intestinal transporters. This chapter also provides several case examples on targeting uptake transporters and circumventing efflux pumps via chemistry and prodrug approaches that would be helpful to the discovery teams working in this direction. Although the small intestine is regarded as an absorptive organ and may act as a rate-limiting barrier, it also has ability to metabolize drugs by several pathways involving both phase I and phase II reactions and may lead to limited systemic exposure. CYP3A4, the most abundant P450 present in human hepatocytes and intestinal enterocytes is implicated in the metabolic elimination of many drugs [2, 3]. The next common metabolic elimination pathways are due to glucuronidation and ester hydrolysis. It has also been proposed that drug interactions involving CYP3A inhibition and induction may be largely occurring at the level of the intestine [4, 5]. In a recent analysis of 309 drugs with intravenous and oral clinical pharmacokinetic data, we noted that roughly 30% of the drugs in the data set show more than 20% intestinal extraction, underscoring the importance of considering intestinal metabolism in predicting bioavailability and dose projections in drug discovery and development settings [6]. Although, the average human intestinal content of CYP3A has been estimated to be only about 1% of the average hepatic content [2], the data set indicated that intestinal metabolism may contribute to first-pass extraction more than the hepatic metabolism for certain drugs. This could be a result of better access to the enzymes in the enterocytes; a function of transcellular flux and the large absorptive area, and/or due to reduced access to hepatic enzymes because of potential plasma protein binding [7]. The intestinal first-pass metabolism in humans is indirectly estimated under certain assumption, by comparing the plasma AUCs following intravenous and oral dosing. Early studies in liver transplant patients during the anhepatic phase indicated the relative importance of the gut extraction to the first-pass metabolism for drugs such as midazolam and cyclosporine [8]. Further clinical evidences were obtained in the grape-fruit juice interaction studies, where coadministration of grape-fruit juice result in the inhibition of gut CYP3A4 without significantly affecting the hepatic metabolism of drugs like felodipine [9]. However, assessment of the quantitative contribution of intestinal and hepatic extraction in first-pass metabolism is limited by ethical and technical challenges. There exist gaps in predicting the gut extraction before the clinical development stage due to shortcomings in the in vitro-in vivo extrapolation (Eg. utilizing human intestinal microsomal stability). Also species differences exist where rat and monkey typically under-predicts the fraction escaping gut extraction (Fg) in human [10, 11]. Recently, transgenic mice model with constitutive expression of human CYP3A4 in liver or intestine that provides quantitative estimation of the contribution of hepatic and gut extraction to the first-pass metabolism has been generated [5]. Overall, due to limited access to the sophisticated models and complexities with in vitroin vivo extrapolation and species differences, intestinal metabolic disposition is far from consistently predictable. Recent studies demonstrated that efflux transporters present on the apical membrane of enterocytes, in particular P-glycoprotein, can affect the intestinal metabolism by prolonging the enterocytic transit time and consequent exposure to CYP3A enzymes [12]. A significant overlap has also been identified between substrates and inhibitors of CYP3A4 and Pglycoprotein, suggesting that these two proteins may act complementarily in further limiting Fg of CYP3A substrates. Due to the complexity in these biochemical processes and the lack of availability of extensive experimental models, application of physiologically-based pharmacokinetic (PBPK) models and systems biology seem to provide quantitative prediction of first-pass metabolism. The chapters by Darwich et al. and Fan et al. describe the mechanistic models and explore new PBPK models to achieve improved predictions. These emerging tools aim towards appropriate reconstruction of the physicochemical, anatomical and biochemical complexities in mathematical terms. Utilizing experimental data majorly derived from in vitro tools, Darwich et al. recognized the combination of parameter (passive permeability, P-glycoprotein efflux kinetic and enzyme kinetic) values where the gut extraction would be expected to be high. Fan et al. evaluated the intestinal and liver PBPK models to predict the contributions of enzymes and transporters on intestinal and hepatic availability, and studied the impact of the model variables on oral bioavailability. With the involvement of saturable processes in the transport and metabolism, the prediction of non-linear pharmacokinetics and drug-drug interactions (DDIs) is likely to play a large role in preclinical and clinical development. The contribution from Tachibana et al. reviews the semi-quantitative and quantitative methods for predicting intestinal DDIs caused by inhibition of CYP3A4 and P-glycoprotein. They point to the importance of accuracy in the in vitro enzyme and transporter kinetics parameters to achieve reliable predictions. While the discussion in the above three articles majorly focus on the CYP3A and P-glycoprotein, these models can be conceptually applied to the rest of the transporters and enzymes. Won et al. discuss the effect of dietary substances on the drug disposition, which is often overlooked. While reviewing the advances in the understanding of mechanisms involved in the drug-dietary substances interactions, authors argued on the need to further characterize the specific dietary components that alter drug disposition, in the process of predicting such interactions. It is also apparent that systemic availability of ester drugs and prodrugs may be hindered by the hydrolytic enzymes present in the enterocytes. Understanding the esterase activity is therefore valuable with drug industry diverting significant resources to prodrugs discovery and development. Imai and Ohura discussed the characteristics of human intestinal carboxylesterase and its role in the absorption of prodrugs and drug candidates with ester functionalities. Authors also discussed the pros and cons of available tools for predicting the gut extraction of prodrugs. Collectively, the articles of this Current Drug metabolism issue provide comprehensive and updated information on several vital areas of intestinal drug disposition and points to the current challenges and scientific gaps. As guest editor, I owe great thanks to each one of the authors for the excellent contributions. I also owe a special thanks to the reviewers for providing valuable inputs.

Bioavailability of orally administered drugs can be influenced by a number of factors including release from the formulation, dissolution, stability in the gastrointestinal (GI) environment, permeability through the gut wall and first-pass gut wall and hepatic metabolism. Although there are various enzymes in the gut wall which may contribute to gut first pass metabolism, Cytochrome P450 (CYP) 3A has been shown to play a major role. The efflux transporter P-glycoprotein (P-gp; MDR1/ABCB1) is the most extensively studied drug efflux transporter in the gut and might have a significant role in the regulation of GI absorption. Although not every CYP3A substrate will have a high extent of gut wall first-pass extraction, being a substrate for the enzyme increases the likelihood of a higher first-pass extraction. Similarly, being a P-gp substrate does not necessarily pose a problem with the gut wall absorption however it may reduce bioavailability in some cases (e.g. when drug has low passive permeability). An on-going debate has focused on the issue of the interplay between CYP3A and P-gp such that high affinity to P-gp increases the exposure of drug to CYP3A through repeated cycling via passive diffusion and active efflux, decreasing the fraction of drug that escapes first pass gut metabolism (FG). The presence of P-gp in the gut wall and the high affinity of some CYP3A substrates to this transporter are postulated to reduce the potential for saturating the enzymes, thus increasing gut wall first-pass metabolism for compounds which otherwise would have saturated CYP3A. Such inferences are based on assumptions in the modelling of oral drug absorption. These models should be as mechanistic as possible and tractable using available in vitro and in vivo information. We review, through simulation, this subject and examine the interplay between gut wall metabolism and efflux transporters by studying the fraction of dose absorbed into enterocytes (Fa) and FG via systematic variation of drug characteristics, in accordance with the Biopharmaceutics Classification System (BCS) within one of the most physiological models of oral drug absorption currently available, respectively ADAM. Variables studied included the intrinsic clearance (CLint) and the Michaelis- Menten Constant (Km) for CYP3A4 and P-gp (CLint-CYP3A4 and Km-CYP3A4, CLint-P-gp and Km-P-gp). The impact of CYP3A4 and P-gp intracellular topography were not investigated since a well-stirred enterocyte is assumed within ADAM. An increased CLint-CYP3A4 resulted in a reduced FG whereas an increase in CLint-P-gp resulted in a reduced Fa, but interestingly decreased FG too. The reduction in FG was limited to certain conditions and was modest. Non-linear relationships between various parameters determining the permeability (e.g. Papp, CLint-P-gp, and Km-P-gp) and gut wall metabolism (e.g. CLint-CYP3A4, Km-CYP3A4) resulted in disproportionate changes in FG compared to the magnitude of singular effects. The results suggest that P-gp efflux decreases enterocytic drug concentration for drugs given at reasonably high dose which possess adequate passive apparent permeability (high Papp), by de-saturating CYP3A4 in the gut resulting in a lower FG. However, these findings were observed only in a very limited area of the parameters space matching very few therapeutic drugs (a group with very high metabolism, high turn-over by efflux transporters and low Fa). The systematic approach in this study enabled us to recognise the combination of parameter values where the potential interplay between metabolising enzymes and efflux transporters is expected to be highest, using a realistic range of parameter values taken from an intensive literature search.

While oral exposure continues to be the major focus, the chemical space of recent drug discovery is apparently trending towards more hydrophilic libraries, due to toxicity and drug-interactions issues usually reported with lipophilic drugs. This trend may bring in challenges in optimizing the membrane permeability and thus the oral absorption of new chemical entities. It is now apparent that the influx transporters such as peptide transporter 1 (PepT1), organic-anion transporting polypeptides (OATPs), monocarboxylate transporters (MCT1) facilitate, while efflux pumps (e.g. P-glycoprotein (P-gp), breast cancer resistance protein (BCRP)) limit oral absorption of drugs. This review will focus on intestinal transporters that may be targeted to achieve optimal clinical oral plasma exposure for hydrophilic and polar drugs. The structure, mechanism, structure-activity relationships and the clinical examples on the functional role of these transporters in the drug absorption was discussed. Physicochemical properties, lipophilicity and hydrogen-bonding ability, show good correlation with transport activity for efflux pumps. Although several attempts were made to describe the structural requirements based on pharmacophore modeling, lack of crystal structure of transporters impeded identification of definite properties for transporter affinity and favorable transport activity. Furthermore, very few substrate drug datasets are currently available for the influx transporters to derive any clear relationships. Unfortunately, gaps also exist in the translation of in vitro end points to the clinical relevance of the transporter(s) involved. However, it may be qualitatively generalized that targeting intestinal transporters are relevant for drugs with high solubility and low passive permeability i.e. a class of compounds identified as Class III according to the Biopharmaceutic Classification System (BCS) and the Biopharmaceutic Drug Disposition Classification System (BDDCS). A careful considerations to oral dose based on the transporter clearance (Vmax/Km) capacity is needed in targeting a particular transporter. For example, low affinity and high capacity uptake transporters such as PEPT1 and MCT1 may be targeted for high oral dose drugs.

Experimental strategies have long been applied for in vitro or in vivo evaluation of the effect of transporters and/or enzymes on the bioavailability. However, the lack of specific inhibitors or inducers of transporters and enzymes and the multiplicity of nuclear receptors in gene regulation and cross-talk have led to compromised assessments of these effects in vivo. These and other causes have resulted in confusion and controversy in transporter-enzyme interplay. In this review, physiologically-based pharmacokinetic (PBPK) intestinal and liver models are utilized to predict the contributions of enzymes and transporters on intestinal availability (FI) and hepatic availability (FH), with the aim to fully understand the impact of these variables on bioavailability (Fsys) in vivo. We emphasize the often overlooked impact of influx and efflux clearances, and apply the PBPK models and their solutions to examine individual organ clearances of the intestine and liver. In order to accurately predict oral bioavailability, these organ models are incorporated into the whole body PBPK model, and additional complicated scenarios such as segmental differences and zonal heterogeneity of transporters and enzymes in the intestine and liver, and segregated blood flow patterns of the intestine are further discussed. The sequential metabolism of a drug to form primary and secondary metabolites in the first-pass organs is considered in PBPK modeling, revealing that the segregated flow model (SFM) of the intestine is more appropriate than the traditional PBPK intestinal model (TM). Examples are included to highlight the potential application of these PBPK models on the quantitative prediction of bioavailability.

Recently, interest has grown in drug-drug interactions (DDIs) involving the inhibition of intestinal CYP3A4, P-glycoprotein (P-gp), and other drug efflux transporters. The criteria for intestinal DDIs are described in the draft guidances of the US Food and Drug Administration (FDA) and European Medicines Agency (EMA). Substrate drugs with small fraction absorbed (Fa) and/or low intestinal availability (Fg) as a result of intestinal efflux transport and metabolism are important as “victim” drugs because these substrates are likely to show considerable interactions. The susceptibility of a victim drug to intestinal interactions can be evaluated from its FaFg. In this review, methods for estimating the FaFg of substrate drugs are discussed. The nonlinear pharmacokinetics of substrate drugs caused by the saturation of intestinal CYP3A4/P-gp is also discussed. The methods for predicting intestinal DDIs caused by inhibitor drugs are then summarized. Because the prediction accuracy of intestinal DDIs also depends on the inhibition constant (Ki) estimated in in vitro studies, these in vitro methods of estimating Ki are also discussed. Standardized methods for predicting intestinal DDIs have not yet been established. Further studies are required to establish more accurate and standardized prediction methods.

Successful delivery of promising new chemical entities via the oral route is rife with challenges, some of which cannot be explained or foreseen during drug development. Further complicating an already multifaceted problem is the obvious, yet often overlooked, effect of dietary substances on drug disposition and response. Some dietary substances, particularly fruit juices, have been shown to inhibit biochemical processes in the intestine, leading to altered pharmacokinetic (PK), and potentially pharmacodynamic (PD), outcomes. Inhibition of intestinal CYP3A-mediated metabolism is the major mechanism by which fruit juices, including grapefruit juice, enhances systemic exposure to new and already marketed drugs. Inhibition of intestinal non-CYP3A enzymes and apically-located transport proteins represent recently identified mechanisms that can alter PK and PD. Several fruit juices have been shown to inhibit these processes in vitro, but some interactions have not translated to the clinic. The lack of in vitro-in vivo concordance is due largely to a lack of rigorous methods to elucidate causative ingredients prior to clinical testing. Identification of specific components and underlying mechanisms is challenging, as dietary substances frequently contain multiple, often unknown, bioactive ingredients that vary in composition and bioactivity. A translational research approach, combining expertise from clinical pharmacologists and natural products chemists, is needed to develop robust models describing PK/PD relationships between a given dietary substance and drug of interest. Validation of these models through well-designed clinical trials would facilitate development of common practice guidelines for managing drug-dietary substance interactions appropriately.

The bioavailability of therapeutic agents can be improved by using prodrugs which have better passive diffusion than the active agents. Intestinal hydrolysis is an important reaction in the bioconversion of prodrugs, and may be the rate-limiting factor in their absorption. Carboxylesterase (CES) is ubiquitous in most organs and is located in the endoplasmic reticulum. Single-pass perfusion experiments in rat intestine have shown that CES is the main enzyme involved in intestinal first-pass hydrolysis. In man, intestinal CESs belong to the CES2 gene family and their activity is nearly constant along the jejunum and ileum. The predominant human intestinal CES, hCE2, preferentially hydrolyzes prodrugs in which the alcohol group of a pharmacologically active molecule has been modified by the addition of a small acyl group. In preclinical animal models, CES2 isozymes are also the major intestinal enzymes although they have different substrate specificities to human CES2, while CES1 isozymes and other unidentified enzymes are also present. It is therefore difficult to predict human intestinal absorption from animal experiments. Caco-2 cells mainly express the human CES1 isozyme, hCE1, which shows quite different substrate specificity from hCE2, making Caco-2 cells unsuitable for prediction of human intestinal absorption of prodrugs. However, we have developed a novel experimental method for predicting the human intestinal absorption of prodrugs using Caco-2 cells in which CES-mediated hydrolysis has been inhibited. The expression of hCE2 shows inter-individual variation and is regulated by several mechanisms, such as gene polymorphism and epigenetic processes. There are no reports suggesting that severe toxicity is associated with prodrugs due to genetic polymorphism of the CES2 gene.

The screening of new drug candidates for nuclear receptor activation can identify agents with the potential to produce drugdrug interactions or elicit adverse drug effects. The nuclear receptors of interest are those that control the expression of drug metabolizing enzymes and drug transporters, and include the constitutive androstane receptor (CAR, NR1I3), the pregnane X receptor (PXR, NR1I2) and the aryl hydrocarbon receptor (AhR). This review will focus on the methods currently used to assess activation of these receptors. Assessment of nuclear receptor activation can be accomplished using direct or indirect approaches. Indirect methods quantify specific gene products that result from nuclear receptor activation while direct approaches measure either the binding of ligands to the receptors or the transcriptional events produced by ligand binding. Assays that directly quantify nuclear receptor activation are growing in popularity and, importantly, are amenable to high throughput screening (HTS). Several ligand binding assays are currently being utilized, including radioligand competition binding, where compounds compete with radiolabelled ligand for binding to PXR or CAR, such as the scintillation proximity binding assay that measures the reaction of ligands with receptor-coated beads. A fluorescence resonance energy transfer assay has also been developed, where the fluorescent signal is generated via the ligand-dependent interaction between the fluorescently- labeled ligand binding domain of a nuclear receptor and co-activator proteins. Other in vitro activation assays include transient- and stably-transfected cell lines incorporating an expression vector for PXR, CAR or AhR plus a reporter gene vector containing response elements. The methods focused on in this review will be limited to the more direct in vitro approaches that are amenable to high throughput screening.

A review with 132 references. Several kinds of anxiolytic and hypnotic drugs are currently available on the market. Although BZDs are surely the most frequently prescribed among them, several chemically unrelated compounds have been commercialised, which can provide similar or even higher efficacy and tolerability. These drugs can prove useful for patients who are non-responder or intolerant to benzodiazepine treatment, thus giving broader therapeutic options to the clinician. The most important studies on the metabolic characteristics of several non-benzodiazepine anxiolytics and hypnotics are reported and briefly discussed in this review; moreover, the analytical methods related to these studies are also described and commented upon and their characteristics are highlighted. Finally, an update is included on recent (2007-2010) metabolism and pharmacokinetic studies on benzodiazepines. A monograph is included for each of the following drugs: zolpidem, zaleplon, zopiclone, ramelteon, buspirone and tandospirone; updates are included for the following benzodiazepines: alprazolam, bromazepam, diazepam, flunitrazepam, lorazepam, midazolam, oxazepam and triazolam.