Permeability--in vitro assays for assessing drug transporter activity

The accumulating evidence has revealed that drug transporters have essential roles in the delivery and excretory processes of drugs and their metabolites. Inhibition or induction of drug transporters can affect pharmacokinetic properties and therapeutic efficacy of a drug. Thus, the characterization of drug-transporter interactions becomes important for the selection of compounds to avoid transporter associated absorption, distribution, metabolism, excretion and toxicity (ADME/Tox) issues. Additionally, the potential use of absorptive transporters for drug delivery has been recognized for drug design. In vitro and in vivo approaches have been developed for studying the transporter activities. In vitro assays can rapidly provide the information for identifying interaction of a compound and a particular transporter and have proved to be amenable to high throughput approaches. Therefore, the studies are conducted in early drug discovery. In this article, in vitro methods are reviewed, including cell free and cell-based assays. Their applications, limitations and impact on drug discovery are discussed.     

Transporters as a determinant of drug clearance and tissue distribution

Transporters play an important role in the processes of drug absorption, distribution and excretion. In this review, we have focused on the involvement of transporters in drug excretion in the liver and kidney. The rate of transporter-mediated uptake and efflux determines the rate of renal and hepatobiliary elimination. Transporters are thus important as a determinant of the clearance in the body. Even when drugs ultimately undergo metabolism, their elimination rate is sometimes determined by the uptake rate mediated by transporters. Transporters regulate the pharmacological and/or toxicological effect of drugs because they limit their distribution to tissues responsible for their effect and/or toxicity. For example, the liver-specific distribution of some statins via organic anion transporters helps them to produce their high pharmacological effect. On the other hand, as in the case of metformin taken up by organic cation transporter 1, drug distribution to the tissue(s) may enhance its toxicity. As transporter-mediated uptake is a determinant of the drug elimination rate, drug–drug interactions involving the process of transporter-mediated uptake can occur. In this review, we have introduced some examples and described their mechanisms.

More recently, some methods to analyze such transporter-mediated transport have been reported. The estimation of the contributions of transporters to the net clearance of a drug makes it possible to predict the net clearance from data involving drug transport in transporter-expressing cells. Double transfected cells, where both uptake and efflux transporters are expressed on the same polarized cells, are also helpful for the analysis of the rate of transporter-mediated transcellular transport.     

Oral drug delivery utilizing intestinal OATP transporters

Transporters play important roles in tissue distribution and urinary- and biliary-excretion of drugs and transporter molecules involved in those processes have been elucidated well. Furthermore, an involvement of efflux transporters such as P-glycoproteins, multidrug resistance associated protein 2, and breast cancer resistance protein as the intestinal absorption barrier and/or intestinal luminal secretion mechanisms has been demonstrated. However, although there are many suggestions for the contribution of uptake/influx transporters in intestinal absorption of drugs, information on the transporter molecules responsible for the intestinal absorptive process is limited. Among them, most studied absorptive drug transporter is peptide transporter PEPT1. However, utilization of PEPT1 for oral delivery of drugs may not be high due to the chemical structural requirement of PEPT1 limited to peptide-mimetics. Recently, organic anion transporting polypeptide (OATP) family such as OATP1A2 and OATP2B1 has been suggested to mediate intestinal absorption of several drugs. Since OATPs exhibit species difference in expressed tissues and functional properties between human and animals, human studies are essential to clarify the intestinal absorption mechanisms of drugs via OATPs. Recent pharmacogenomic studies demonstrated that OATP2B1 is involved in the drug absorption in human. In addition, information of drug-juice interaction in the intestine also uncovered the contribution of OATP1A2 and OATP2B1 in drug absorption. Since OATP1A2 and OATP2B1 exhibit broader substrate selectivity compared with PEPT1, their potential to be applied for oral delivery should be high. In this review, current understanding of characteristics and contribution as the absorptive transporters of OATPs in small intestine in human is described. Now, it is getting clearer that OATPs have significant roles in intestinal absorption of drugs, therefore, there are higher possibility to utilize OATPs as the tools for oral delivery.     

Transporters and drug discovery: why, when, and how

Drug transporters are now increasingly recognized as important determinants of variable drug disposition and response. In addition, transporter associated problems appear to be occurring with greater frequency during the drug discovery and development process. What has not been clear is whether drug transporter related issues are a truly new problem, or whether such issues had existed all along, but were previously unrecognized or ignored. In this review, a brief overview of key drug transporters will be outlined. In addition, a commentary on specific issues of relevance to pharmaceutical sciences in terms of the role and relevance of drug transporters to the drug discovery and development process is provided.     

The ATP-binding cassette transporters and their implication in drug disposition: a special look at the heart

The passage of drugs across cell membranes dictates their absorption, distribution, metabolism, and excretion. This process is determined by several factors including the molecular weight of the compounds, their shape, degree of ionization, and binding to proteins. Accumulation of xenobiotics into tissues does not depend only on their ability to enter cells, but also on their ability to leave them. For instance, the role of efflux transporters such as ATP-binding cassette (ABC) proteins in the disposition of drugs is now well recognized. Actually, ABC transporters act in synergy with drug-metabolizing enzymes to protect the organism from toxic compounds. The most studied transporter from the ABC transporter superfamily, P-glycoprotein, was found to be overexpressed in tumor cells and associated with an acquired resistance to several anticancer drugs. P-glycoprotein, thought at first to be confined to tumor cells, was subsequently recognized to be expressed in normal tissues such as the liver, kidney, intestine, and heart. Even though information remains rather limited on the functional role of ABC transporters in the myocardium, it is hypothesized that they may modulate efficacy and toxicity of cardioactive agents. This review addresses recent progress on knowledge about the ABC transporters in drug disposition and more precisely their role in drug distribution to the heart.     

应用药物转运体的药代动力学评价

药物转运体在药代动力学方面起到非常重要的作用,它与药物的吸收、分布、排泄、药效发挥、以及药物毒性作用等密切相关。基于转运体的药物间相互作用,还能影响到临床合并治疗药物之间的药代动力学关系。本研究室已经建立了一整套的实用于药代动力学研究和药物间相互作用评价,高效表达人药物转运体蛋白的体外筛选系统,并已将其应用于新药的筛选研究。此方法的特点在于,以表达人药物转运体蛋白的宿主培养细胞为研究对象,在临床前期提供与临床试验相似的研究结果,今后必将成为药物研发领域有用的评价方法。     

Computational approaches to modeling drug transporters

Computational modeling has advanced our understanding of drug absorption, tissue distribution, excretion and toxicity profiles by providing both direct and indirect knowledge of drug–transporter interactions that would otherwise be unavailable using experimental methods. Currently, two complementary approaches are available in modeling transporters: substrate-based and transporter-based methods. The transporter-based approach directly predicts the transporter's three-dimensional structure to assist in understanding the drug transport process, whereas substrate-based models infer such information by studying a group of substrates or inhibitors with measured activities. In this review, the available strategies in both transporter-based and substrate-based approaches are explained and illustrated with applications and case studies. With increasing computational power and continuously improving modeling algorithms, computational techniques can assist in further understanding transporter–substrate interactions as well as, the optimization of transporter-directed drug design.     

The genetic polymorphism of drug transporters: functional analysis approaches

Evidence is accumulating to strongly suggest that drug transporters are one of the determining factors governing the pharmacokinetic profile of drugs. To date, a variety of drug transporters have been cloned and classified as solute carriers and ATP-binding cassette transporters. Such drug transporters are expressed in various tissues such as the intestine, brain, liver, and kidney, and play critical roles in the absorption, distribution and excretion of drugs. However, at the present time, information is limited regarding the genetic polymorphism of drug transporters and its impact on their function. In this context, we have undertaken the functional analyses of the polymorphisms identified in drug transporter genes. This article aims to provide an overview on the functional aspects of the non-synonymous polymorphisms of drug transporters and to present standard methods for the evaluation of the effect of polymorphisms on their function.     

Herb-drug interactions involving drug metabolizing enzymes and transporters

Herbal medicines and their active ingredients are widely used worldwide, and they have become an important part of clinical medicine. The combined use of herbs and drugs has increased the possibility of pharmacokinetic and pharmacodynamic interactions. Clinical studies have demonstrated that the combined use of herbs and drugs can enhance or attenuate the drug efficacy and toxicity. The herb-drug combinations may reduce a drug efficacy and lead to treatment failure when long-term administration. Case reports detailing serious clinical adverse reactions have promoted studies on the interactions between herbs and drugs. This review highlights recent knowledge to discuss herb-drug interactions involving metabolizing enzymes and drug transporters. Drug transporters are widely present in body and play an important role in the absorption, distribution, excretion and metabolism, efficacy, and toxicity of drugs. Investigation of transporters has developed rapidly since 1990s, the effects of many transporters on the pharmacokinetics of drugs and herb-drug interactions have been reported. Some concepts on drug transporters issued experimentally and clinically drug-drug and herb-drug interactions have applied in many studies. Methodology studies are very important for understanding the mechanism, considerations and evaluation of experiments and clinical studies on drug metabolizing enzymes and transporters in drug-drug interactions.     

Nuclear receptors and the regulation of drug-metabolizing enzymes and drug transporters: implications for interindividual variability in response to drugs

Erratic or unpredictable response to drugs remains a challenge of modern drug therapy. An important determinant of such interindividual differences in drug response is variability in the expression of drug-metabolizing enzymes and/or transporters at sites of absorption and/or tissue distribution. Variable drug-metabolizing enzyme and transporter expression can result in unpredictable exposure and tissue distribution of drugs and may manifest as adverse effects or therapeutic failure. In the past decade, important new insights have been made relating to the regulatory mechanisms governing the expression of drug-metabolizing enzymes and transporters by ligand-activated nuclear receptors. Specifically, there is compelling evidence to demonstrate that PXR, CAR, FXR, LXR, VDR, HNF4alpha, and AhR form a battery of nuclear receptors that regulate the expression of many important drug-metabolizing enzyme and transporters. In this review, the authors focus on clinically important drug-metabolizing enzymes such as CYP3A4, CYP2B6, CYP2C9, CYP2C19, UGT1A1, SULT2A1, and glutathione S-transferases and their regulation by nuclear receptors. They also review the nuclear receptor-mediated regulation of drug transporters such as MDR1, MRP2, MRP4, BSEP, BCRP, NTCP, OATP1B3, and OATP1A2. Finally, they outline how the drug development process has been affected by the current understanding of the involvement of nuclear receptors in the regulation of drug disposition genes.     

Scientific perspectives on drug transporters and their role in drug interactions

Recently, increased interest in drug transporters and research in this area has revealed that drug transporters play an important role in modulating drug absorption, distribution, and elimination. Acting alone or in concert with drug metabolizing enzymes they can affect the pharmacokinetics and pharmacodynamics of a drug. This commentary will focus on the potential role that drug transporters may play in drug-drug interactions and what information may be needed during drug development and new drug application (NDA) submissions to address potential drug interactions mediated by transporters.     

Label-free kinetic binding data as a decisive element in drug discovery

The emerging possibilities to obtain label-free, kinetic-based binding data for drug–target and drug absorption, distribution, metabolism and excretion (ADME)–marker interactions have proven useful in many drug discovery related issues. Multiple reports have demonstrated that the common use of affinity as an early measure of drug potency may be directly misleading. This review summarises findings in the literature related to compound selection in the drug discovery process. It is important to understand the different properties of association and dissociation rates, the former being related to both structure and dosage and the latter depending solely on molecular structure. By performing parallel optimisations of association and dissociation rates, compounds with desirable kinetic profiles for target binding may be generated. In addition, compound selection may also consider the kinetic properties of the drug–ADME–marker binding profiles, further refining the quality of compounds early in the drug discovery process. The promising results found in the literature indicate that kinetic data on drug–protein interactions may soon become a crucial decisive element in modern drug discovery.     

Transporter targeted drug delivery

The pharmacological behavior of various drugs is severely affected by biological barriers such as epithelial tight junctions, efflux proteins and metabolizing enzymes. Apart from the biological barriers, physicochemical properties of drug molecules such as molecular weight, lipophilicity, surface charge and solubility also play an important role in absorption characteristics of drug candidates. Pharmacological properties affected by efflux pumps such as P-gp and MRPs include bioavailability, hepatobiliary and urinary excretion of drugs as well as drug metabolites. This leads to sub-therapeutic concentrations of various potential drugs at the target site. One of the strategies to overcome these biological barriers is transporter targeted prodrug design. Prodrug derivatization targeting membrane transporters and receptors improves drug absorption. Various prodrugs which have been synthesized so far demonstrated enhanced bioavailability and tissue specificity. This review mainly focuses on the efflux pumps which play an important role in drug absorption and a few strategies to overcome these efflux pumps.     

Predicting oral drug absorption and hepatobiliary clearance: Human intestinal and hepatic in vitro cell models

Membrane transport proteins control the uptake and efflux of many drugs in tissues including the intestine, liver and kidneys and thus play important roles in drug absorption, distribution and excretion. With the development of high throughput screening in an industrial environment, the importance of having appropriate in vitro systems to study drug transporter function, regulation, and interactions are invaluable. Cell lines are efficient tools in screening individual transport processes. In this review, we focus on the processes involved in the absorption and hepatobiliary clearance of drugs and the potential of cell lines to model such process, paying particular attention to the use of Caco-2 and HepG2 cells.     

转运体和核受体影响药物代谢的研究进展

转运体是存在于体内几乎所有器官上的有转运功能的蛋白,对药物在体内的吸收、分布、代谢和排泄有非常重要的作用.而核受体是存在于细胞内的配体依赖性转录因子,可以激活配体影响代谢酶和转运体的表达和活性,进而影响药物在机体内代谢.本文综述了转运体和核受体家族中几个重要的成员以及它们对药物代谢影响的研究进展.     

A regulatory viewpoint on transporter-based drug interactions

1. Pharmacokinetic drug interactions can lead to serious adverse events and the evaluation of a new molecular entity's (NME) drug-drug interaction potential is an integral part of drug development and regulatory review before its market approval. Clinically relevant interactions mediated by transporters are of increasing interest in clinical development and research in this emerging area and it has been revealed that drug transporters can play an important role in modulating drug absorption, distribution, metabolism and elimination. 2. Acting alone or in concert with drug-metabolizing enzymes transporters can affect the pharmacokinetics and/or pharmacodynamics of a drug. The newly released drug interaction guidance by the US Food and Drug Administration (USFDA) includes new information addressing drug transporter interactions with a primary focus on P-glycoprotein (P-gp, ABCB1). 3. This paper provides a regulatory viewpoint on transporters and their potential role in drug-drug interactions. It first outlines information that might be needed during drug development and ultimately included in new drug application (NDA) submissions to address potential transporter-mediated drug interactions. Next, it explains criteria that may warrant conduct of in vivo P-gp-mediated drug interaction studies based on in vitro assessment. In addition, it includes a review case that describes the evaluation of data suggesting a P-gp-based induction interaction.     

Targeting intestinal transporters for optimizing oral drug absorption

While the 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/or low passive permeability i.e. a class of compounds identified as Class III and Class IV 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 (V(max)/K(m)) 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.     

Intestinal drug transporters: in vivo function and clinical importance

The oral route of drug administration remains the most popular and convenient route of administration, despite its many shortcomings and challenges. Although the advantages associated with this route far outweigh any limitations, a prominent limitation relates to the interactions of drugs with intestinal membrane transporters. The complexities of these interactions and their impact on drug absorption and absorption variability are only now becoming recognized. The rapidly growing awareness of transporter-mediated secretion, saturable absorption, and even the concerted actions of transporters in intestinal drug absorption and secretion has attracted the attention of pharmaceutical scientists in academia, the pharmaceutical industry and the regulatory agencies. This is evidenced by the recent rapid accumulation of data in the literature, the routine conducting of transport studies in the discovery and development of drugs, and finally by the recognition of the importance of transporter (e.g. P-glycoprotein and OATP) mediated secretion of drugs by regulatory authorities such as the U.S. Food and Drug Administration. In this mini-review, we focus on the handful of absorptive and secretory transporters that have been relatively well studied and illustrate the impact of these intestinal transporters on oral drug absorption using published reports from preclinical and clinical studies.     

Knowledge-based approaches in the design and selection of compound libraries for drug discovery

In the past decade, the pharmaceutical industry has realized the increasing significance of impacting the early phase hit-to-lead development in the drug discovery process. In particular, knowledge-based approaches emerged and evolved to address a multitude of issues such as absorption, distribution, metabolism and excretion (ADME), potency, toxicity and overall drugability. Each of these approaches seeks to bring together all relevant pieces of information and create a knowledge-oriented process to deploy such information in drug discovery. This review focuses on work relating to drugability, which aims at obtaining hits (or leads) that have enhanced likelihoods of leading to successful clinical candidates by medicinal chemistry efforts. The period covered in this review is from 1997 (since the publication of Lipinski's rule of 5) to March 2002.