Current trends in drug metabolism and pharmacokinetics

Copyright © 2019 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Associated Data

Abstract

Pharmacokinetics (PK) is the study of the absorption, distribution, metabolism, and excretion (ADME) processes of a drug. Understanding PK properties is essential for drug development and precision medication. In this review we provided an overview of recent research on PK with focus on the following aspects: (1) an update on drug-metabolizing enzymes and transporters in the determination of PK, as well as advances in xenobiotic receptors and noncoding RNAs (ncRNAs) in the modulation of PK, providing new understanding of the transcriptional and posttranscriptional regulatory mechanisms that result in inter-individual variations in pharmacotherapy; (2) current status and trends in assessing drug–drug interactions, especially interactions between drugs and herbs, between drugs and therapeutic biologics, and microbiota-mediated interactions; (3) advances in understanding the effects of diseases on PK, particularly changes in metabolizing enzymes and transporters with disease progression; (4) trends in mathematical modeling including physiologically-based PK modeling and novel animal models such as CRISPR/Cas9-based animal models for DMPK studies; (5) emerging non-classical xenobiotic metabolic pathways and the involvement of novel metabolic enzymes, especially non-P450s. Existing challenges and perspectives on future directions are discussed, and may stimulate the development of new research models, technologies, and strategies towards the development of better drugs and improved clinical practice.

KEY WORDS: Pharmacokinetics, Drug metabolism, Drug–drug interactions, Modeling, Metabolizing enzymes, Transporters, Nuclear receptors, Noncoding RNAs

Graphical abstract

Understanding of DMPK properties is essential for drug development and precision medication. In this article, we provided an overview of recent research on DMPK with focuses on the regulatory mechanisms of pharmacokinetics, drug–drug interaction, mathematical modeling, non-classical metabolism and so on. Existing challenges and perspectives on future directions are also discussed.

1. Introduction

Pharmacokinetics (PK) is defined as the quantitative study of drug absorption, distribution, metabolism, and excretion (ADME)—i.e., the ways the body processes a drug 1 while the drug exerts its actions in the body. The scope of PK not only covers studies on healthy subjects but also includes broad research on variations under a variety of physiologic or pathologic conditions and the underlying mechanisms, potential drug–drug interactions (DDI), and possible strategies such as dose adjustment to achieve precision medication. Collectively, these aspects of PK allow customization of drug dosage regimens to enhance therapeutic outcomes 1 . Therefore, PK study is a prerequisite to establish the relations and the underlying mechanisms of a drug to its activities and clinical benefits. The information obtained is crucial for lead identification and optimization in drug discovery, as well as dosage regimen design and adjustment in clinical practice 2 . The complexity of PK has evolved, largely in relation to the rapid developments in analytical chemistry, computer science, molecular biology and biochemistry. Although much is known with regard to the PK of many drugs, and many technologies have been established for PK research, recent studies are revealing the existence of new mechanisms by which how drugs are metabolized and how PK is regulated. New experimental models and computational modeling algorithms are arising for an improved understanding of the significance of PK in a whole-body system; nonetheless, many challenges remain.

This review will provide a comprehensive overview of recent developments in the areas of PK research. First, we will provide an update of findings on drug-metabolizing enzymes and transporters in the control of PK, as well as advances in nuclear receptors and noncoding RNAs (ncRNAs) in the modulation of PK, which will provide new insights into understanding the transcriptional and posttranscriptional regulatory mechanisms behind inter-individual variations in pharmacotherapy. Second, we will review the current status and trends in assessing DDIs, especially the interactions between drugs and herbs, between drugs and therapeutic biologics, and microbiota-mediated DDIs and HDIs. Third, we will summarize recent advances in disease–drug interactions, in particular, regulation of metabolizing enzymes and transporters and alteration of PK by different diseases or physiological states. Fourth, we will summarize the trends in mathematical modeling including physiologically-based PK, which could be applied to support clinical investigations. In addition, we will discuss novel animal models such as CRISPR/Cas9-based animal models for DMPK research and overview some interesting non-classical biotransformation pathways including those utilizing novel drug-metabolizing enzymes. Existing challenges and future perspectives are also discussed. It is expected that this review will provide an update on recent advances in PK fields and may stimulate the establishment of new research models, technologies, and strategies towards the development of better drugs and improvements in clinical practice.

2. Determinants of PK

Drug-metabolizing enzymes and transporters play a very important role in the control of PK. Furthermore, transcriptional and posttranscriptional factors such as nuclear receptors and noncoding RNAs (ncRNAs) are critical in the modulation of PK and provide in-depth insight into understanding regulatory mechanisms to solve problems in PK. These mechanism-driven PK studies can improve the success of drug development related to its efficacy and safety and improve the rational use of medication in clinical practice.

2.1. Drug-metabolizing enzymes in the control of PK

Drug-metabolizing enzymes mediate the metabolism of exogenous and endogenous substances. Most drugs lose their pharmacological activities mainly through metabolic transformation, yielding metabolites with high water solubility that are readily excreted. Hence, metabolizing enzymes play an extremely important role in the control of drug PK. The biotransformation of xenobiotics by xenobiotic-metabolizing enzymes (XMEs) may be classified into Phase I and Phase II reactions. Advanced characterizations of enzymes involved in human drug metabolism are urgently needed, which help to avoid severe adverse drug reactions. Advances are being made in understanding the role of drug-metabolizing enzymes in the control of PK, including individual isoforms of many enzymes such as cytochrome P450s (CYPs) and UGTs, and their selective substrates, inducers and inhibitors. Other non-P450 oxidative enzymes and conjugative enzymes are also discussed in this section since an increasing number of drugs are metabolized via these enzymes 3 .

2.1.1. CYPs critical for PK

CYPs can oxidize foreign substances, enhance the water solubility and make drugs easier to be eliminated from the body. Most drugs are metabolized by CYPs, which mainly are located in the inner membrane of mitochondria or the endoplasmic reticulum of cells 4 . There are a total of 57 human CYP genes in 18 families. The members of CYP1 to CYP4 families oxidize thousands of exogenous and endogenous substrates ( Table 1 ); whereas all members of CYP5 family and higher principally metabolize endogenous substrates in a highly substrate-specific manner 5 .

Table 1

Endogenous and exogenous substrates of CYPs and ligands of transcription factors.

Most known chemical carcinogens, including aromatic amines and polycyclic aromatic hydrocarbons (PAHs), are substrates of CYP1 family, and their metabolism often results in the formation of active carcinogenic metabolites. In 2018, CYP1B1 was found in the mitochondria of cancer cells, where it reportedly metabolizes melatonin to form the metabolite N-acetylserotonin (NAS), which has antitumor effects 6 . CYP2D6, another important metabolic enzyme, is involved in the metabolism of many anti-cancer drugs, such as cyclophosphamide, tamoxifen, and gefitinib 7 . Recent research has found that in brain, CYP2D6 can metabolize both m-tyramine and p-tyramine into dopamine 8 . The CYP4 family has gained increasing attention for its potential to generate interesting metabolites and dispose of endogenous substrates in recent years. CYP4F11, together with CYP4F2, plays an important role in the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid, and participates in the metabolism of vitamin K 9 . Cyp2a5, the mouse correlate of human CYP2A6, encodes an enzyme that exhibited circadian regulation 10 . The other CYP1 to CYP4 subfamilies are involved in metabolism of different endogenous and exogenous substrates, as listed in Table 1 .

Understanding variation in mechanism-based enzyme activity is crucial for improving the clinical use of drugs. Highly selective inducers and inhibitors of CYPs have been cited in Guidance for Industry by FDA (https://www.fda.gov/drugs/drug-interactions-labeling/drug-development-and-drug-interactions-table-substrates-inhibitors-and-inducers). Recent studies have revealed new chemicals and herb products as inducers or inhibitors of CYPs. For example, CYP7A1 is upregulated by an intestinal HIF-2α inhibitor called PT2385 17 . The ketene intermediate of erlotinib can inactivate CYP3A4 and CYP3A5, which can result in liver injury 18 . Due to the complexity of components in the extract of herbs it is common that herb products exhibit different effects on the regulation of multiple enzymes. Sophora flavescens can inhibit CYP2B6, CYP2C8, CYP2C9, and CYP3A activities, while catalpol can inhibit the activity of CYP3A4, CYP2E1 and CYP2C9 19 , 20 . Other regulatory factors can also alter the expression of CYPs. For example, tumor suppressor p53 can regulate Cyp2b10 directly and thereby attenuate APAP-induced hepatotoxicity 21 .

Herbs may be used singly or in combination in the treatment of diseases 22 . It is very important to understand how drug exposure alters molecular mechanisms underlying many complex drug interactions. For example, data show that ellagic acid from pomegranate peel guava leaf extract can significantly increase the AUC of warfarin with concomitant use. A significant reduction in CYP2C8, 2C9, and 3A4 activity was the main reason for this interaction 23 .

Based on recently available data, new information on the relative content of individual isoforms of P450 has been generated. Total CYP concentrations are significantly different between Chinese and Caucasian populations and the metabolic capabilities of CYPs in Chinese liver microsomes was significantly lower (<50%) in the CL_int for substrates of CYP1A2, CYP2C9, CYP2C19 and CYP2E1 than those of Caucasian populations 24 . Large variations in protein content, mRNA levels, and intrinsic activities of ten P450s (CYP3A4, 1A2, etc) have been revealed and some single nucleotide polymorphisms had significant impact on P450 expression; for example, CYP2C19 activity varied more than 600-fold 25 . A recent human PK study further evaluated CYP1A2 content in Chinese compared with Caucasian populations, enhancing the confidence in pharmacokinetic prediction of CYP1A2 content using two substrates (caffeine and theophylline) 26 .

Other organs like kidney and intestine also have significant metabolic capacity. There is definitive evidence for CYP2B6 and 3A5 expression in human kidney, while multiple CYPs are expressed in intestine 27 , 28 . The role of renal and intestinal enzymes in herbal product metabolism has been uncovered. Aminoglycoside antibiotics are leading causes for nephrotoxicity; combination with herbs or dietary supplements at reduced dosage is possible to reduce the risk of drug-mediated renal toxicity. A recent study revealed that moringa oleifera seed oil could limit gentamicin-induced oxidative nephrotoxicity 29 . Additional herbs have been identified as having effects on intestinal metabolism, such as the extracts of Yin-Chen-Hao Tang (YCHT), a very popular hepatoprotective three-herb formula in China and Japan 30 . These findings contribute to the understanding of the metabolic characteristics of renal and intestinal metabolism.

2.1.2. Non-P450 oxidative enzymes

The contribution of non-P450 enzymes to drug metabolism can be significant and affect the overall development of drugs. Non-CYP enzymes can be divided into four general categories: namely oxidative, reductive, conjugative, and hydrolytic. Non-CYP oxidative enzymes include flavin-containing monooxygenases (FMOs), monoamine oxidases (MAOs), peroxidases, xanthine oxidases (XO), aldehyde oxidase (AO), alcohol dehydrogenase (ADHs) and aldehyde dehydrogenase (ALDHs) 31 .

Very little is known about the regulation of content and activity of non-P450 oxidative enzymes. Recently, some selective substrates and inhibitors of non-P450 enzymes have been identified in natural products and other sources. FMOs are involved in the metabolism of a wide array of xenobiotics. Well-known inhibitors of FMOs include indole-3-carbinol and methimazole, and 2-mercaptobenzimidazole 32 . Classified into two different isoforms (MAO-A, MAO-B), MAOs are enzymes involved in the catabolism of monoamines. Benextramine and its derivatives were identified as novel human monoamine oxidases inhibitors, which could be considered as candidate drugs for the treatment of neurodegenerative diseases 33 . In addition, 3-(3-(dimethylamino)propanoyl)-7-hydroxy-5-methyl-2H-chromen-2-one hydrochloride has been found to function as a novel selective hMAO-B inhibitor, which is expected to be a promising multifunctional Parkinson's disease treatment agent 34 . XO and AO are involved in the oxidation of aldehydes and heterocycles, and carbazeran was used as a selective probe substrate of AO in hepatocytes 35 . Allopurinol and S-allyl cysteine (SAC) are XO inhibitors used in the treatment of gout and hyperuricemia 36 . A single-nucleotide polymorphism of human cytochrome P450 oxidoreductase (POR) in the Chinese population can regulate the content of POR and P450 isoforms 37 . Identifying specific inhibitor compounds will greatly facilitate investigation of enzyme-mediated drug disposition and drug interactions.

2.1.3. Importance of UDP-glucuronyltransferases (UGTs) in PK

UDP-glucuronyltransferases (UGTs) are a family of endoplasmic reticulum-bound enzymes which are responsible for the process of glucuronidation, a major part of phase II metabolism 38 . Human UGTs include 22 different functional enzymes and are classified into four gene families, UGT1, UGT2, UGT3 and UGT8 39 . The UGT1 and UGT2 families are primarily enzymes involved in drug glucuronidation, while the contribution of the UGT3 and UGT8 families to drug metabolism is minimal 40 .

Recently, UGT1A3 was found to be involved in the glucuronidation of alpinetin 41 . UGT1A4 is involved in the glucuronidation of metizolam 42 . Other UGT isoforms involved endogenous and exogenous substrates are listed in Table 2 23 , 43, 44, 45, 46.

Table 2

Endogenous and exogenous substrates of UGTs and ligands of transcription factors.

Highly selective substrates and selective inhibitors of UGTs have been found in natural products and from other sources. Resveratrol can activate UGT1A8 expression, and is used for breast cancer treatment 47 . Different doses of emodin can inhibit the activity of UGT2B7 48 .

In some cases, herbal products are metabolized by multiple UGTs. Linoleic acid and glutaric acid can inhibit the glucuronidation of berberrubine, a lipid-lowing metabolite of berberine, as well as the activities of UGT isoforms, such as UGT1A7, 1A8, 1A9 49 . Glucuronidation of catalposide, an active component of Veronica species, was catalyzed by gastro-intestine-specific UGTs 1A8 and 1A10 50 .

Gene polymorphisms are a key factor in the regulation of the content and activity of UGTs. UGT1A and UGT2B genetic variation can alter nicotine and nitrosamine glucuronidation in European and African American smokers 51 . In addition, the UGT1A4*3 genetic polymorphism is associated with low posaconazole plasma concentrations in patients with hematological malignancies 52 . UGT1A1*6 polymorphisms are correlated with irinotecan-induced neutropenia in cancer patients 53 .

2.1.4. Other conjugative enzymes important for PK studies

In addition to UGTs, sulfonyl transferases (SULTs) and glutathione S-transferases (GSTs) are also important conjugative enzymes mediating phase II reaction.

SULTs catalyze the transfer of the water-soluble sulfonate group from 3′-phosphoadenosine 5′-phosphosulfate to drugs or endogenous molecules that contain hydroxy or amine group(s) 54 . At present, four families of human SULTs have been discovered, namely SULT1, SULT2, SULT4 and SULT6. SULT1E1 plays an important role in the metabolism and detoxification of estrogens and flavonoids 55 . SULT2 enzymes, mainly SULT2A and SULT2B, are primarily responsible for catalyzing the sulfation of hydroxysteroids 56 . A recent study found that tumor suppressor p53 could regulate the expression of SULTs 57 .

GSTs are a group of phase II drug-metabolizing enzymes that catalyze the binding of glutathione to various electrophilic compounds. In humans, cytosolic GST isoenzymes of the alpha, zeta, theta, mu, pi, sigma and omega classes have been found. GSTA1 plays a significant role in the metabolism of acetaminophen 58 . GSTA4 metabolizes electrophilic and carcinogenic substances such as endogenous carcinogen 4-hydroxy-2-nonenal 59 . The detailed substrates of SULTs and GSTs are listed in Table 3 .

Table 3

Endogenous and xenobiotic substrates for GSTs and SULTs that are also ligands of particular transcription factors.

2.1.5. Updates on the nuclear receptor-mediated regulation of xenobiotic-metabolizing enzymes

The human nuclear receptors comprise a family of 48 ligand-regulated transcription factors that in turn regulate target genes involved in metabolism and other physiological functions. Some of these receptors (e.g., peroxisome proliferators-activated receptor (PPAR), liver X receptor (LXR), hepatocyte nuclear factor (HNF)) are of particular interest in regard to drug metabolism and disposition as they have been found to regulate many XMEs in recent years.

PPARα induces the expression of CYP4A in response to a heterogeneous group of peroxisome proliferators. PPARγ also regulates the expression of CYP4V2, a fatty acid metabolizing enzyme, in human tetrahydropyranyl 1 (THP1) macrophages 60 . LXR controls the transcription of Cyp7a1 and Cyp27a1, Cyp3a11 and Cyp2e161, 62, 63.

Traditional transcriptional factors can bind directly to specific DNA sequences and thus control the gene expression. However, epigenetic regulation like histone modification and DNA methylation modulates transcription of UGTs or CYPs mainly by changing chromatin architecture. For example, the UGT1A gene can be repressed by the recruitment of histones in females 64 . Several studies determined that microRNAs (miRNAs), could down-regulate the expression of metabolizing enzymes, which will be further reviewed in Section 2.3.

In summary, the expression and activity of metabolizing enzymes can be regulated by multiple factors, including drugs, nuclear receptors, gene polymorphisms, and even ethnic categories. Non-P450 enzymes and other conjugative metabolizing enzymes have gained attention in drug metabolism in recent years. It is desirable to illustrate the key factors responsible for variable expression and activity of drug metabolizing enzymes, as it may be beneficial in the prediction of potential therapeutics, drug–drug interactions, and in modifying the PK of drugs.

2.2. Transporters in the control of PK

2.2.1. Introduction of transporters

Transporters are membrane-bound proteins expressed on the cell membrane in most tissues with varying abundance. They can transport a variety of endogenous or exogenous substrates (such as drugs and their metabolites) in and out of cells. For drugs, transporters are the gatekeepers for cells and control the uptake and efflux of drugs. Transporters are involved in the ADME process of drugs. Therefore, transporters play critical roles in the pharmacokinetics, efficacy and toxicity of drugs. Alteration of transporter function or expression may significantly change the blood and/or tissue exposure of drugs, leading to significant changes in pharmacokinetics. Furthermore, the induction or inhibition of transporters by co-administered drugs can change PK and pharmacodynamics of therapeutic drugs and produce DDI.

There are more than 400 membrane transporters belonging to two major superfamilies: adenosine triphosphate (ATP)-binding cassette (ABC) and solute carrier (SLC) transporters. They utilize the energy that is released by ATP hydrolysis or an electrochemical ion gradient to translocate drugs across the membrane.

2.2.1.1. The ABC family of drug transporters

ABC transporters mainly act as exporters and pump drug molecules out of cells by utilizing the energy released by the hydrolysis of ATP. According to the organization and sequence of ATP-binding domains, 49 ABC transporters are classified into seven subfamilies: ABC1/ABCA, multidrug resistance (MDR)/TAP/ABCB, MRP/ABCC, ALD/ABCD, OABP/ABCE, GCN20/ABCF and White/ABCG 65 . Among them, P-glycoprotein (P-gp, MDR1, ABCB1), MRPs/ABCCs, breast cancer resistance protein (BCRP/ABCG2) and bile salt export pump (BSEP/ABCB11) are recognized for their importance in drug disposition 66 . P-gp, which is expressed at a high level in the intestine, liver, kidney, brain and placenta, is the most studied ABC transporter. Many substrates of P-gp including antibiotics, statins, immunosuppressants, anticancer drugs and a broad spectrum of drugs overlap with the substrates of CYPs. The expression of P-gp is regulated by several transcription factors including PXR, CAR, vitamin D receptor (VDR) and CCAAT/enhancer binding protein (C/EBP) and some microRNA such as miR-451, miR-27a and miR-145 67 , 68 . Furthermore, P-gp is usually overexpressed in cancer cells and plays a critical role in MDR. For example, during chemotherapy, P-gp may be an obstacle for drug exposure if the therapeutic drugs are P-gp substrates 69 . Besides its role in MDR induction, P-gp plays a critical role in pharmacokinetics, pharmacology and toxicology. Through pumping multiple drugs out of cells, P-gp decreases the bioavailability of oral drugs and increases drug efflux into urine or bile. Furthermore, P-gp also plays a vital role in the maintenance of the blood–brain barrier by pumping drugs or toxins out of the CNS 70 . Another important ABC transporter group is the MRP family that consists of 9 MRP proteins (MRP1–MRP9). Among them, MRP2 is important in drug pharmacokinetics. MRP2, once known as the canalicular multispecific organic anion transporter, is highly expressed in liver, intestine and kidney. Chemotherapeutics such as methotrexate, melphalan, and statins are the classical substrates of MRP2. Since co-expressed in the liver, many liver-enriched transcription factors such as LXR, farnesoid X receptor (FXR), HNF and C/EBP regulate the transcription of MRP2. Another efflux transporter BCRP is a half transporter and is expressed at a high level in a wide variety of tissues such as intestine, kidney, liver, testis and brain. BCRP is modulated by the progesterone receptor B (PRB) and estrogen receptor (ER). Another ABC family drug transporter, BSEP, is primarily expressed in the liver and pumps bile acids and non-bile acid drugs such as pravastatin into bile.

2.2.1.2. The SLC family of drug transporters

The SLC family consists of 55 gene subfamilies and more than 360 family members. SLC transporters mainly utilize the energy stored in the ion gradients across membranes, but do not depend directly on ATP hydrolysis 71 . Several SLC family transporters play important roles in drug disposition including organic anion-transporting proteins (OATPs/SLC21/SLCO), organic anion and cation transporters (OATs and OCTs/SLC22), peptide transporters (PEPTs/SLC15) and sodium-dependent bile acid transporters (NTCP/SLC10A1). The OATP family consists of 11 members. Among them, four transporters including OATP1A2 (SLCO1A2), OATP1B1 (SLCO1B1), OATP1B3 (SLCO1B3) and OATP2B1 (SLCO2B1) are involved in drug transport 72 . OATP1A2 is expressed in the intestinal epithelium, renal epithelium and brain capillary endothelial cells, while OATP1B1, OATP1B3 and OATP2B1 are expressed predominantly in hepatocytes. Statins and anti-cancer drugs like paclitaxel, sorafenib and methotrexate are known as the substrates of OATPs. The SLC22 family consists of 23 members, including OCTs, zwitterion/cation transporters (OCTNs) and OATs. Among OCTs, OCT1 (SLC22A1) is mainly expressed in the liver, OCT2 (SLC22A2) is located at a high level in proximal tubular cells, and OCT3 (SLC22A3) has a broader expression range. Several drugs have been identified as OCT substrates including anesthetic drugs, the anti-diabetic drug metformin, antidepressants, β-blockers and anti-cancer chemotherapeutics. Among OATs, OAT1 (SLC22A6) and OAT3 (SLC22A8) have a broader expression range with the highest expression in kidney, while OAT2 (SLC22A7) is primarily expressed in the liver. OAT1 substrates include antiviral drugs, antibiotics, diuretics and angiotensin-converting enzyme (ACE) inhibitors. For the SLC15 subfamily, PEPT1 and PEPT2 are the most studied transporters. Both mediate oligopeptide uptake. PEPT1 is highly expressed in the intestine and mediates drug absorption, while PEPT2 is mainly expressed in kidney and affects renal reabsorption.

2.2.2. Transporters are critical for PK

The ADME process determines the blood and tissue concentration of drugs, as well as subsequent pharmacological or toxicological effects. The intestine and liver, both of which tightly regulate the entry of drugs into the blood circulation, are important organs in determining the bioavailability of oral drugs. Elimination of drugs or their active metabolites occurs either by metabolism to inactive metabolites that are excreted, or by direct excretion of drugs or active metabolites in the kidney. The transporters expressed in intestine, liver and kidney are involved in the absorption, distribution and excretion processes of drugs, and are the major determinant in blood and tissue concentration of drugs.

2.2.2.1. Transporter-mediated oral drug absorption

Oral drug absorption primarily occurs in the intestine, which is the major determinant of drug bioavailability, together with the first-pass extraction in the liver. Drug molecules pass through the membranes in the intestine through two pathways: passive diffusion and transporter-mediated absorption.

The process of transporter-mediated oral drug absorption consists of two parallel transport processes including transporter-mediated uptake and transporter-mediated efflux 73 , 74 ( Fig. 1 A). In general, net drug absorption depends on multiple uptake and efflux transporters in the intestine. Uptake transporters such as OATP2B1, PEPT1 and sodium-dependent bile acid transporter (ASBT/SLC10A2) are involved in the intestinal uptake of drugs across the brush border membrane 75 . For example, PEPT1 transports di/tripeptides-like anticancer drugs such as bestatin and β-lactam antibiotics into enterocytes76, 77, 78. Efflux transporters expressed on the brush border membrane of the intestine, are considered as the barriers for intestinal drug absorption. P-gp, MRP2 and BCRP are three major efflux transporters in the intestine. P-gp, the most studied efflux transporter, has broad substrate specificity and significantly limits the bioavailability of many oral drugs 79 . For example, co-treatment with verapamil, a P-gp inhibitor, increases the intestinal absorption of afatinib or bestatin due to P-gp inhibition in the intestine 80 , 81 . On the contrary, rifampin, a P-gp inducer, decreases the oral absorption of cyclosporine and tacrolimus through the induction of P-gp in the intestine 82 . BCRP is another efflux transporter expressed in the intestine and suppresses the intestinal absorption of drugs 83 . Due to only one ATP binding site and six putative transmembrane helices, BCRP is considered a “half-transporter”. The substrates of BCRP include statins (pitavastatin, rosuvastatin), antiviral drugs (lamivudine, zidovudine, abacavir), anticancer drugs (methotrexate, SN-38, irinotecan, gefitinib, imatinib, erlotinib) and antibiotics (nitrofurantoin, ciprofloxacin) 84 . The efflux transporter MRP2 is also expressed on the brush border membrane of the intestine and transports a variety of substrates conjugated with sulfate, glutathione and glucuronide, as well as various unmodified drugs. Previous studies showed that resveratrol inhibited MRP2 and thereby increased the intestinal absorption of methotrexate 85 .

Drug transporter expression in tissues. Drug transporter expression in the intestine (A), liver (B) and kidney (C). The arrows indicate the general directions in which the substrates are transported.

2.2.2.2. Transporter-mediated drug distribution

Transporters also affect the tissue distribution and contribute to the selective distribution of drugs to specific tissues. For example, OATP1B1 and OATP1B3 are the major uptake transporters in the liver for cilostazol, and MRP2, BCRP, P-gp pump cilostazol out of the liver into bile 86 . These transporters assist the liver-specific distribution of cilostazol. Another example is pravastatin, which enters into the liver through OATP1B1 and OATP1B3. After being excreted into the bile, pravastatin is reabsorbed in the intestine to the portal vein and taken up by the liver, and effectively undergoes enterohepatic circulation 87 . Therefore, the liver concentration should be higher than that in the circulating blood, leading to a high pharmacological effect at a relatively low plasma concentration. Transporters are also expressed on the blood–brain barrier and play critical roles in restricting the distribution of drugs into the brain. Increasing evidence has demonstrated that P-gp on the blood–brain barrier can suppress the distribution of drugs into the CNS 88 , 89 . Also, BCRP is recognized as an efflux transporter on the blood–brain barrier suppressing drug entry into the brain. Except for efflux transporters, uptake transporters are also expressed on the blood–brain barrier and play key roles in the uptake of neuroactive drugs. OAT3 is highly expressed on the basolateral membrane of brain capillaries 90 , and OCT2 is expressed in neurons and the choroid plexus. OCT2 is involved in the reabsorption of many drugs such as serotonin, norepinephrine, dopamine, choline and histamine from the cerebrospinal fluid.

2.2.2.3. Transporter-mediated drug excretion

Drug elimination primarily occurs in the liver and kidney. Hepatobiliary elimination processes can be summarized as follows: (1) the uptake of drugs into hepatocytes via uptake transporters or passive diffusion; (2) drug metabolism in hepatocytes including CYP metabolism (phase I metabolism) and conjugation (phase II metabolism); (3) excretion from hepatocytes into bile or portal blood via efflux transporters. Hepatobiliary transport of drugs is attributable to transporters located on the basolateral (sinusoidal) or canalicular (apical) membrane of hepatocytes ( Fig. 1 B). SLC superfamily transporters are responsible for drug uptake from the portal blood into hepatocytes. Among them, OAT2, OCT1, OATPs and NTCP are major uptake transporters. Efflux transporters such as P-gp, BCRP, MRP2 and BSEP are responsible for the hepatobiliary excretion of drugs and their metabolites. In addition, the efflux transporter MRP3, 4 and 6 expressed on the basolateral membrane are responsible for the basolateral efflux of drugs from the liver into the blood circulation. The hepatic transporters OAT2, OATP1B1/1B3 and OCT1 are highly expressed in the liver and are considered to be of particular importance for hepatic drug elimination, PK and efficacy. Much like the interplay of transport and metabolic enzymes at the intestinal barrier, these transporters also have a “gatekeeper” function in the drug movement from the blood into hepatocytes; they regulate both the number of drugs available for metabolism by liver enzymes and the subsequent biliary excretion. Efflux transporters including P-gp, BCRP, MRP2 and BSEP are responsible for the biliary excretion of endogenous and exogenous molecules. Many studies have shown that P-gp transports amphiphilic cationic drugs such as doxorubicin, digoxin and vinblastine into bile 91 . BCRP is involved in the biliary excretion of sulfated conjugates of steroids and drugs such as doxorubicin, mitoxantrone and daunorubicin, while BSEP transports drugs including vinblastine and taxol, et al. Due to their important roles in hepatobiliary efflux, the inhibition of BSEP, BCRP and MRP2 may lead to cholestasis. Therefore, the effects of chemicals on transporter-mediated hepatobiliary excretion must be determined in drug discovery 92 .

The kidney is the major organ of drug excretion. Renal clearance of drugs consists of glomerular filtration, tubular secretion and reabsorption. The proximal tubule region is responsible for the active secretion and reabsorption of drugs. Many transporters are located at the renal tubular epithelial cells and are involved in the proximal tubular secretion and reabsorption ( Fig. 1 C). These transporters include OCTs, OATs, multidrug and toxin extrusion proteins (MATE1 and MATE2-K), sodium-phosphate transporter (NPT/SLC17A1), OATPs and PEPTs, as well as equilibrium and concentration nucleoside transporters (ENTs and CNTs/SLC28A). Among them, OCTs, OATs and MATEs play critical roles in the active secretion of renal proximal tubule. These transporters work in concert with efflux transporters to transfer drugs into urine. OATs mainly transport anionic drugs such as beta-lactam antibiotics and anti-inflammatory drugs. The competitive inhibition of OATs may lead to a decrease in renal tubular secretion and an increase in the systemic concentration of drugs. For example, co-administration of probenecid, one OAT inhibitor, decreases renal secretion, leading to an increase in the plasma concentration of bestatin 93 . JBP485, a dipeptide with potential protective activity against kidney, liver and intestinal injury, has been demonstrated to be a substrate of OATs. Co-administration of JBP485 and cephalexin decreased the accumulative renal excretion and renal clearance of both compounds 77 . When JBP485 and lisinopril were co-administered, the competitive inhibition of OAT1 and OAT3 were also observed in OAT1/3-HEK293 cells 94 . In addition, acyclovir, an antiviral drug, was also a substrate of OAT1/3 and JBP485 can inhibit its renal excretion 95 . Furthermore, the DDIs between JBP485 and entecavir through the competitive inhibition of OAT1 and OAT3 significantly decreased the renal excretion of both compounds 96 . On the other hand, OATs are involved in drug-related nephrotoxicity. Probenecid, by inhibiting OAT1 and OAT3, reduced the accumulation of cephaloridine and subsequently nephrotoxicity 97 , 98 .

Three OCT isoforms including OCT1, OCT2 and OCT3 have been found in the kidney. Among the three OCTs, OCT2 is the major transporter for renal secretion of a variety of drugs such as memantine, metformin and amantadine. DDIs may also occur through the competitive inhibition of OCTs. For example, through inhibiting OCT2, cimetidine decreases the renal excretion of metformin and increases its plasma concentration 99 . On the other hand, OCT2, by modulating the exposure of drugs to renal proximal tubule cells regulates the nephrotoxicity of anticancer drug cisplatin and its analogs 100 . Substrates taken up from the systemic circulation may subsequently undergo efflux across the brush border membrane of proximal tubule cells by various ABC efflux transporters such as P-gp and BCRP. For example, a probe P-gp substrate, methotrexate, is actively secreted into urine. Co-treatment with bestatin, another P-gp substrate, increases plasma concentrations and decreases the renal clearance of methotrexate 101 . MATE1 and MATE2-K are expressed on the brush border membrane of proximal tubular cells. MATE1 mediated the renal secretion of fluoroquinolones including gatifloxacin, ciprofloxacin, levofloxacin, enoxacin, pazufloxacin, norfloxacin and tosufloxacin.

In summary, the expression and activity of transporters can be regulated by drugs and competitive inhibition may occur after co-administration of more than one drug. Furthermore, species differences in transporters complicate pharmacokinetic scaling from preclinical species to humans. Additionally, the expression of transporters may also be regulated by disease progression 102 . Modulation of transporter expression by disease states can potentially modify the PK of drugs.

2.3. ncRNAs in the regulation of drug metabolism and pharmacokinetics

ncRNAs are genome-derived RNA molecules that are not translated into proteins. Indeed, the human genome is comprised of over 95% of noncoding sequences 103 that are transcribed into various forms of functional ncRNAs including miRs, transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small nucleolar RNAs (snoRNAs), and long noncoding RNAs (lncRNAs). Among them, miRNAs usually lead to translation inhibition or enhance mRNA degradation in cells through complementary base pairing with target transcripts. Many miRNAs have been shown to modulate the expression of drug-metabolizing enzymes or transporters, and consequently alter cellular drug metabolism and transport capacity, as well as drug responses (see recent reviews104, 105, 106). For instance, miR-27b reduces CYP1B1 protein expression in human carcinoma cells and thus suppresses CYP1B1 enzymatic activity, as indicated by a P450-Glo™ luminescent assay 107 . Meanwhile, miR-27b modulates CYP3A4 expression through direct targeting of CYP3A4 3ʹ-untranslated region (3ʹUTR) and “indirect” targeting of transcriptional factors such as NR1I1/VDR 108 , which may significantly alter CYP3A4-mediated drug metabolism 109 , 110 . Furthermore, miR-27a/b regulates the expression of a number of transporters such as ABCB1/P-gp111, 112, 113, and thus influences intracellular drug accumulation and chemosensitivity. In addition, a number of Phase 2 drug-metabolizing enzymes such as the UGTs are regulated by miRNAs at the posttranscriptional level114, 115, 116, 117, 118, 119. Findings on miRNA-controlled regulation of DMPK provide new insights into mechanisms behind inter-individual variations in pharmacotherapy.

Recent studies on miRNA regulation in DMPK also led to the development of novel research approaches and technologies. For example, while the luciferase reporter assay, gene mutagenesis and correlation analysis are helpful methods for the assessment of the interactions between miRNAs and target transcripts, a more direct approach has been established which is based on the change of RNA mobility after binding to miRNA, namely RNA electrophoretic mobility shift assay (EMSA) 120 , 121 . Using this RNA EMSA and other methods, a number of CYP genes (e.g., CYP2C19, CYP2E1 and CYP2D6) and regulators have been shown to be regulated post-transcriptionally by particular miRNAs121, 122, 123, 124, 125. It is also noteworthy that miRNA research is limited to the use of miRNA-expressing plasmids or viruses, or chemically-synthesized or chemo-engineered miRNA mimics126, 127, 128. To better capture the properties of biologic RNA molecules and cellular miRNA machinery, a novel RNA bioengineering technology has been established for the production of biologic miRNA agents in living cells 109 , 129, 130, 131, 132, 133. With such novel bioengineered miRNA agents produced cost effectively and on a large scale, extensive functional studies have been conducted and the results showed rather a modest change in the PK of major CYP probe drugs in mouse models 134 . Further studies have demonstrated the utility of miRNAs as therapeutics or sensitizing agents for the treatment of human diseases in various animal models 133 , 135, 136, 137, 138, 139.

There is also growing evidence that lncRNAs may regulate the expression of drug-metabolizing enzymes and transporters. For example, expression of HNF1α antisense RNA 1 (HNF1α-AS1) and HNF4α antisense RNA 1 (HNF4α-AS1) shows a significant influence on the basal and drug-induced expression of drug-metabolizing enzymes in human cells 140 . H19, an lncRNA highly expressed in liver tissues, induces the expression of efflux transporter P-gp/MDR1/ABCB1 in drug-resistant HepG2 cells 141 . The lncRNA MRUL confers the overexpression of ABCB1 in drug-resistant gastric cancer cells 142 . Furthermore, some studies have demonstrated that a lncRNA modulates drug sensitivity through its action on miRNA-transporter axis 143 , 144 . In addition, as RNA editing and posttranscriptional modifications are critical for RNA stability and biological function, very recent studies have also demonstrated the alteration of DMPK gene expression following RNA editing145, 146, 147. Future studies in these areas will undoubtedly advance our understanding of RNA-based regulation in DMPK.

In summary, research on miRNA-controlled regulation of DMPK provides new insights into understanding the posttranscriptional regulatory mechanisms behind inter-individual variations. Novel technologies and research approaches are also established during the investigation of ncRNA regulation of DMPK gene expression, which should have broad impact on biomedical research. Evidence is accumulating that some lncRNAs may be involved in the regulation of DMPK, which represents a new area of research.

3. Drug–drug interactions

3.1. Current status of research on drug–drug interactions

DDIs may result in favorable or toxic effects. Patients frequently use more than one medication at a time. Depending on the clinical settings and the number of drugs prescribed, the incidence of potential DDIs ranges between 15% and 80% 148 . DDIs can be classified mechanistically into 3 major types: physio-chemical incompatibility, PK interactions, and pharmacodynamic interactions 149 . Physio-chemical interactions usually occur when positively and negatively charged compounds are mixed before they are administrated or absorbed. Pharmacokinetics-based DDIs, characterized by altered concentration of unbound drugs that exert pharmacological effects, can be caused by several mechanisms, including: 1) alteration of drug metabolizing enzymes (e.g., CYPs) 150 , 2) alteration of transporters involved in the absorption, distribution and excretion of drugs (e.g., MDR1, OAT, OCT, etc) 150 , 3) influence on plasma protein binding affinity 149 , and 4) changes in the function of organs (e.g., gut motility or stomach content pH) 149 . Pharmacodynamics-based DDIs are characterized by a shift of the unbound drug concentration versus response curve 149 . New responses that are not present when either of the drugs is given alone may also be observed when drugs are used in combination.

In vitro, in vivo and clinical studies are usually conducted to identify any potential DDIs. The in vitro studies are usually simple systems that can be used for high throughput screening and provide mechanistic information for potential DDIs. In vivo animal studies are often conducted using clinically relevant dosages and pharmacodynamic endpoints to confirm the in vitro observations. If evidence obtained from in vitro and in vivo animal models suggests strong DDIs potential further clinical trials are recommended 150 , 151 . Recently, mathematical modeling, particularly physiologically-based pharmacokinetic (PBPK) modeling has also been applied to investigate potential pharmacokinetic-based DDIs. A recent review by Min et al. 152 depicted how pharmacokinetic modeling improves and simplifies the investigation on DDIs. In addition, systematic reviews and databases summarize all the experimental and predicted data on DDIs, which are useful for providing warning and proper advice to patients in clinical practice 153 .

Although DDIs between small molecule drugs have been well investigated and documented, knowledges on interactions between drugs and herbs, interactions between therapeutic biologics, and interactions mediated by the gut microbiome are currently not well understood. The cutting-edge investigations on these aspects are briefly introduced in the following sections.

3.2. Current status of research on herb–drug interactions

Herbal plants and herbal products are commonly used as remedies and dietary supplements. When herbs are concurrently administered with drugs unrecognized herb–drug interactions (HDIs) may lead to side effects and toxicity. HDIs basically share the same mechanisms as DDIs. To avoid physio-chemical interactions between herbal components and drugs, it is usually recommended that herbs should be taken at two hours before or after the drugs. Moreover, herbs may sometimes alter the PK and/or pharmacodynamics of the concurrently administered drugs. PK and pharmacodynamic interactions have been reported between herbs and drugs with narrow therapeutic indexes, especially drugs for CNS and cardiovascular diseases 154 . For example, St John's wort (Hypericum perforatum) was reported to decrease warfarin plasma concentrations via inducing the activity of CYPs, leading to the loss of anticoagulant activity 155 . A traditional Chinese herb Danshen (Salvia miltiorrhiza) was reported to interact with warfarin on both its PK profiles and pharmacodynamic effects, resulting in over-anticoagulation and increased risk of bleeding 155 .

Investigation of HDIs is often more complicated than those of DDIs, due to the complex herbal components and the batch-to-batch variation of herbal products. As demonstrated in Table 4 , compared with DDIs, research on HDIs is still insufficient. In vitro screening assays, which are efficient ways for detecting potential DDIs, may not be applicable for testing crude herbs or herb extracts, due to the fact that some of the herbal components may not be bioavailable, and adding such herbal components to the in vitro cell/microsome systems may alter results. By using LC–MS/MS, several multi-compound pharmacokinetic studies allowed the simultaneous detection of the plasma/tissue concentrations of multiple components after ingestion of the studied herb, facilitating the discovery of the bioavailable active components and subsequent in vitro and in vivo mechanistic studies on potential HDIs 156 , 157 . Most of the reported HDIs are based on in vitro and in vivo animal models, providing evidence with low clinical relevance. Moreover, many clinical studies were conducted among healthy populations, where the impact of the herbs on the pharmacodynamics effects of the concurrent drug may not be determined. On the other hand, the wide variation between different batches of herbal products also leads to poor reproducibility of the tests. Although not true in all countries, herbal products in China are generally regulated and used as medicine with standardization of the content of the major active components, and the herbal products are sometimes investigated not only as the effector but also as the affected agent of HDIs. In addition to experimental approaches based on the pre-clinical and clinical data, mathematical models have been established to predict HDIs, demonstrating the feasibility of using PBPK modeling for the prediction of HDIs 152 . For example, PBPK modeling of two major active components from Wuzhi capsule (Schisandra sphenanthera extract) predicts its interaction with tacrolimus metabolism by CYP3A4 inhibition 158 . However, the application of modeling and simulation on the investigation of HDIs is still restricted by the limited human pharmacokinetic data of herbal components 152 . More sophisticated designs of clinical studies are warranted to evaluate the safety and efficacy of the concomitant use of herbs and drugs.

Table 4

Comparison between investigations on DDIs and HDIs 151 , 153 .