Current Drug Metabolism - Volume 8, Issue 7, 2007

The ability to successfully predict pharmacokinetic properties plays a crucial role in the selection of candidate drugs and significantly reduces the number of potential failures in drug development. Current drug candidates typically show a very high affinity for the target receptors; however, the drawback is that many new lead compounds represent large, lipophilic molecules with low solubility, dissolution and/or permeability and consequently show poor absorption properties. In vitro-in vivo prediction and application of in silico methods for clearance and drug-drug interaction prediction from hepatic cytochrome P450 data have been widely accepted by both pharmaceutical companies and academia, and meet certain regulatory requirements [1]. However, the application of these approaches to extrahepatic tissues, including the intestine, has proved challenging and less definitive. Estimates of intestinal clearance are not routinely incorporated into in vitro-in vivo strategies and this may partially explain the clearance under-prediction trend often observed [2]. The aim of this special issue of Current Drug Metabolism is to provide a broad perspective, both from academia and industry, on intestinal absorption and the impact of intestinal first-pass metabolism on both clearance and drug-drug interaction prediction. The issue also considers our in silico abilities to bridge the gap between the increasing amount of intestinal in vitro data and the importance of intestinal first-pass metabolism in vivo. Overviews of the biopharmaceutical and pharmacokinetic variables and their impact on the prediction of the drug absorption from either chemical structure and/or in vitro data is presented, focusing in particular on the estimation and application of intestinal permeability [3,4]. The role of uptake and efflux transporters and their interplay with the metabolic enzymes is becoming an increasingly important issue for drug candidates. Recently, drug elimination mechanisms (e.g., via metabolism, or coupled with uptake/efflux transporters or renal clearance) have been suggested as criteria to extend the Biopharmaceutical Classification System beyond the use of drug permeability/solubility characteristics to the prediction of drug disposition [5]. The limitations of the cellular systems and potential overestimation of the significance of efflux transporters on the intestinal absorption are also discussed [3]. ROLE OF THE INTESTINE IN THE CLEARANCE PREDICTION Over recent years a number of studies have assessed the catalytic activity of intestinal metabolic enzymes in comparison to the liver, focusing predominantly on CYP3A4/CYP3A5 as well as a range of glucuronidating (UGT) enzymes. Although intuitively one would expect the activity of enzymes in both organs to be comparable, a number of in vitro studies have reported differential importance of the intestine relative to the liver. In addition, inter-individual variability in P450/UGT expression in both liver and intestine and their relative abundance in the corresponding organs have generally not being taken into account. A recent study by Galetin and Houston [6] has shown comparable intestinal and hepatic catalytic activity (when expressed per pmol of P450 enzyme) of the major P450 enzymes. Therefore, it would be reasonable to expect that once normalized for the relative abundance of the enzyme investigated, hepatic or recombinant data would be equally useful for the prediction of intestinal clearance once an appropriate mechanistic model is applied. Although this approach is applicable for P450 enzymes [4], certain specific issues need to be addressed in case of phase II enzymes. For example, the relative UGT expression levels in vivo are not clearly defined. A recent study by Cao et al. [7] indicated a 3-fold higher expression of UGTs relative to CYP3A4 in human duodenum. However, as the expression of intestinal metabolic enzymes, and transporters, follows certain gradient patterns along the intestine and within the villi, this will not necessarily reflect the UGT:P450 abundance ratio along the whole length of the gut. In addition, the identification of appropriate extrahepatic scaling factors represents a major challenge. Only recently, has a consensus on such numbers been reached for the scaling of hepatic data [8], and there is no such comprehensive data available for the analysis of the intestinal microsomal recovery......

The majority (84%) of the 50 most-sold pharmaceutical products in the US and European markets are given orally. The dominating role of this route in drug therapy is a consequence of it being safe, efficient and easily accessible with minimal discomfort to the patient in comparison with other routes of drug administration. A successful drug discovery and development of oral pharmaceutical products require an in-depth understanding of multiple biochemical and physiological processes that determine the dissolution rate, intestinal permeability, gastrointestinal transit, first-pass extraction and systemic exposure-time profiles of drugs. It is crucial to realize that these basic biopharmaceutic and pharmacokinetic properties are crucial to focus on to allow successful drug development. Identification of the rate-limiting step(s) in order to overcome these barriers and understanding of the sources of variability are important in the selection of suitable candidate molecules in drug development. Several reports based on in vitro investigations in various cell models have suggested that carrier-mediated intestinal efflux may be a major reason for incomplete absorption and variable bioavailability of drugs, as well being a site for drug-drug and specific food-drug interactions. However, many drugs which were initially suggested to undergo significant efflux in vitro were later shown to be completely absorbed in vivo. This apparent discrepancy between in vitro and in vivo results may be due to several factors that will be discussed in this review. Novel data on solubility and dissolution in human gastrointestinal derived fluids will be reviewed. The effect of food intake on solubility and dissolution rate of a range of drugs including felodipine, danazol, griseofulvin, cyclosporine, probucol and ubiquinone in simulated and real intestinal fluids is discussed. The biopharmaceutic and physicochemical data discussed here can potentially be used as a benchmark set for validation of new experimental techniques or in silico models in future. Factors such as structural diversity, commercial availability, price and a suitable analytical technique for quantification were considered in the selection of a specific drug set. Using the compiled data set lipophilicity as determined by reverse phase HPLC and permeability across Caco-2 cell monolayers were determined; means to overcome the experimental difficulties due to the diversity of the data are also discussed.

Although the liver has long been thought to play the major role in drug metabolism, also the metabolic capacity of the intestine is more and more recognized. In vivo studies eventually pointed out not only the significance of first-pass metabolism by the intestinal wall for the bioavailability of several compounds, but also the relevance of transporters in this process. Only a few methods are available to study drug metabolism in vivo or in situ and with most of these methods it remains difficult to discriminate between the contribution of liver and extrahepatic tissues. To study intestinal drug metabolism in vitro, apart from subcellular fractions, several intact cell systems are nowadays available. This review discusses the available intestinal in vitro methods to study drug metabolism. The advantages and limitations of intact cell systems (isolated intestinal perfusion, everted sac, Ussing chamber preparations, biopsies, precision-cut slices, primary cells), subcellular fractions (S9 fractions, microsomes) and intestinal cell lines (caco-2, LS180 cells amongst others) are discussed. Their applicability to different species and to study phase I and II metabolism/transport and drug-drug interactions are summarized. Furthermore, causes of variation within and between methods are discussed and metabolic rates obtained with different methods are compared. Whereas subcellular fractions and cell lines are efficient methods to study mechanistic aspects of drug metabolism at the enzyme level, the isolated intestinal perfusion, everted sac and Ussing chamber appear particularly useful for studying drug metabolism of rapidly metabolised drugs and interactions with transporters. Biopsies, precision-cut slices and primary cells seem all appropriate to study induction and metabolism of slowly metabolised drugs.

Despite a lower content of many drug metabolising enzymes in the intestinal epithelium compared to the liver (e.g. intestinal CYP3A abundance in the intestine is 1% that of the liver), intestinal metabolic extraction may be similar to or exceed hepatic extraction. Modelling of events on first-pass through the intestine requires attention to the complex interplay between passive permeability, active transport, binding, relevant blood flows and the intrinsic activity and capacity of enzyme systems. We have compared the predictive accuracy of the “well-stirred” gut model with that of the “QGut” model. The former overpredicts the fraction escaping first-pass gut metabolism; the latter improves the predictions by accounting for interplay between permeability and metabolism.

For certain CYP3A4 substrates intestinal first-pass metabolism makes a substantial contribution to low oral bioavailability and extent of drug-drug interactions (DDI). In order to include the contribution of enzyme inhibition in the gut wall in the assessment of DDI potential, the ratio of the intestinal wall availability in the presence and absence of an inhibitor (FG’ and FG, respectively) has been incorporated into a prediction equation based on hepatic enzyme interactions. This approach has been applied for both reversible and irreversible DDIs, involving 36 different inhibitors and 11 CYP3A4 substrates. The aim was to investigate the use of maximal (complete) inhibition of intestinal CYP3A4 (FG’=1) as a pragmatic measure of the intestinal enzyme interaction and to compare this approach with observed in vivo values (where available) and predicted FG ratios from an intestinal model. The latter was obtained from the decrease in the intestinal intrinsic clearance in the presence of an inhibitor, using an estimated inhibitor concentration in the intestinal wall during absorption phase (IG) and an in vitro obtained Ki. In addition, the impact of variability in the enterocytic blood flow on the estimated IG and subsequently the model predicted FG ratio was investigated. The maximal FG ratios for the 11 CYP3A4 substrates investigated ranged from 1.06-7.14 for alprazolam and tacrolimus, respectively. In 91% of the studies investigated the model predicted FG ratio was within 40% of the maximal value. Maximal FG ratio is proposed as an initial indicator of the magnitude of intestinal enzyme interaction; the implications for drug elimination involving substrates cleared either by metabolism or by a combination of metabolism and efflux transporters are discussed.

Over the past decade, our knowledge concerning the importance of intestinal metabolism in the disposition of xenobiotics has significantly improved. Compounds such as midazolam can be extensively extracted in the intestine upon first-pass metabolism after oral dosing. Conversely, the intestine plays a less important, albeit less characterized role in systemic metabolism. This manuscript is meant to review the published examples of pharmaceutical industry research on the intestinal metabolism of xenobiotics, including the various in vitro and in vivo models used. While it is clear that some examples exist of published characterization of the role of intestinal metabolism in drug disposition from the pharmaceutical industry, the majority of industry literature ignores its contribution. The opportunity exists to increase the examination of intestinal metabolism of drugs and drug candidates in industry.

RNA interference (RNAi) is a powerful technique that utilizes RNA molecules to specifically knock down the expression of targeted gene at posttranscriptional level. These small interfering RNAs (siRNAs) not only have broad application to basic biomedical research but may be developed as therapeutic agents. Drug-metabolizing enzymes (DMEs) and drug transporters (DTs) are molecular determinants of pharmacokinetic property of a drug. Transcriptional gene expression of DMEs and DTs is controlled by xenobiotic-sensing nuclear receptors (NRs). Because of complexity in studying the function of individual DMEs, DTs and NRs, siRNAs can be an excellent addition to chemical inhibitors and inhibitory antibodies in delineating their specific roles in drug metabolism and transport, gene regulation, and drug-drug interactions. RNAi may be employed to modulate DT expression to overcome multidrug resistance. Recent studies using RNAi to silence gene expression of specific DME, DT and NR, and the impact on drug metabolism and transport are discussed in this review. Concerns remain about the efficiency, specificity, and off-target effects when interpreting data obtained from RNAi studies. Furthermore, potential role for endogenous siRNAs, microRNA (miRNA) molecules, in controlling the posttranscriptional gene regulation of DMEs, DTs and NRs is discussed.

Over the last few decades, understanding of the mechanisms involved in the process of neuronal cell death has grown. Recent findings have established that DNA damage, and specifically ataxia telangiectasia mutated protein (ATM), is key to the cascade of regulation of neuronal apoptosis. Another characteristic common to all neurodegenerative diseases is oxidative stress. Likewise, a common feature in the brain of patients with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases and other neurological disorders is the expression of proteins involved in cell-cycle control. In the process of re-entry in the cell cycle, an additional component, transcription factor E2F-1, also involved in the regulation of apoptosis, is expressed. Finally, in this complex puzzle, mitochondrial activation with the release of proteins and the activation of cystein proteases, specifically caspase-3, is prominent in the last step of neuronal apoptosis. This review focuses on the role of ATM activation and its re-entry into the cell cycle in neurons as a potential target for the prevention of neuronal apoptosis. We suggest the mechanisms by which ATM and E2F-1 orchestrate the apoptotic process. Among them, p53 could be a common point on this apoptotic route. Finally, we put forward drugs that are now being studied experimentally, such as p53 inhibitors, ATM inhibitors and cyclin-dependent kinase (CDKs) inhibitors, for the treatment of neurodegenerative diseases.

It has long been suggested that some of the neuropharmacological, neurochemical and behavioural effects of ethanol are mediated by its first metabolite, acetaldehyde. In spite of the well documented psychoactivity of acetaldehyde, the precise role of this compound in alcohol abuse remains a matter of intense debate among scientists devoted to the study of alcoholism. Very frequently, the main drawback has been related to the presence of adequate levels of acetaldehyde or its derivatives inside the brain after ethanol ingestion. Since penetration into the central nervous system from blood of peripherically derived acetaldehyde is very low due to the high aldehyde dehydrogenase activity at the blood-brain barrier, several authors called into question the acetaldehyde implication in the toxicity and neurobehavioral effects of ethanol. The confirmation in several laboratories of the existence of enzymatic mechanisms of ethanol oxidation in the brain has revitalized the old theories supporting the acetaldehyde contribution to alcohol abuse and alcoholism. In this paper, we review current data on the brain metabolism of ethanol. We focused on the description of the enzymatic mechanisms involved in this metabolic process, reviewing the constitutive expression, catalytic activity and inhibition and inducibility of the enzymes involved in brain ethanol metabolism. We also analyze old and recent data on their regional distribution and cellular localization in the central nervous system, with special reference to the mesocorticolimbic system, a dopaminergic brain pathway that plays an important role in drug and ethanol reinforcement.

Cytochromes P450 (CYPs) comprise a number of enzyme subfamilies responsible for the oxidative metabolism of a wide range of therapeutic agents, environmental toxicants, mutagens, and carcinogens. In particular, cytochrome P450 2E1 (CYP2E1) is implicated in the oxidative bioactivation of a variety of small hydrophobic chemicals including a number of epoxide-forming drugs and environmentally important toxicants including urethane, acrylamide, acrylonitrile, benzene, vinyl chloride, styrene, 1-bromopropane, trichloroethylene, dichloroethylene, acetaminophen, and butadiene. Until recently, chemical modulators (inducers and inhibitors) were used in order to characterize the enzymatic basis of xenobiotic metabolism and the relationships between CYP-mediated bioactivation and chemical-induced toxicity/carcinogenicity. With the advent of genetically engineered knockout mice, the ability to evaluate the roles of specific CYPs in the metabolism of xenobiotics has become more attainable. The main focus of the current review is to present studies that characterized the enzymatic, metabolic, and molecular mechanisms of toxicity, genotoxicity, and carcinogenicity of various xenobiotics using Cyp2e1-/- mice. Data presented in this review demonstrated that the most comprehensive studies using Cyp2e1-/- mice, encompassing the entire paradigm of metabolism to toxicity, genotoxicity, and carcinogenicity were possible when a substrate was primarily metabolized via CYP2E1 (e.g. urethane and acrylamide). In contrast, when multiple CYP enzymes were prevalent in the oxidation of a particular substrate (e.g.: trichloroethylene, methacrylonitrile, crotononitrile), investigating the relationships between oxidative metabolism and biological activity became more complicated and required the use of chemical modulators. In conclusion, the current review showed that Cyp2e1-/- mice are a valuable animal model for the investigation of the metabolic and molecular basis of toxicity, genotoxicity, and carcinogenicity of xenobiotics.