Involvement of Drug Transporters in Organ Toxicity - ACS Publications

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Involvement of drug transporters in organ toxicity: the fundamental basis to drug discovery and development Yaofeng Cheng, Ayman F El-Kattan, Yan Zhang, Adrian S. Ray, and Yurong Lai Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00511 • Publication Date (Web): 18 Feb 2016 Downloaded from http://pubs.acs.org on February 20, 2016

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Chemical Research in Toxicology

Involvement of drug transporters in organ toxicity: the fundamental basis of drug discovery and development Yaofeng Cheng, Ayman El-Kattan, Yan Zhang, Adrian S. Ray, Yurong Lai

Yaofeng Cheng ([email protected]); Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, 3551 Lawrenceville Road, Princeton, NJ 08540 Ayman El-Kattan ([email protected]) Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc. 610 Main St, Cambridge, MA 02139 Yan Zhang ([email protected]); Drug Metabolism and Biopharmaceutics, Incyte Corporation, 1801 Augustine Cutoff, Wilmington, DE 19803 Adrian S. Ray ([email protected]); Department of Drug Metabolism, Gilead Sciences, Inc. 333 Lakeside Drive, Foster City, CA 94404 Yurong Lai ([email protected]); Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, 3551 Lawrenceville Road, Princeton, NJ 08540

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Abstract

Membrane transporters play a pivotal role in many organs to maintain their normal physiological functions and contribute significantly to drug absorption, distribution and elimination. Knowledges gained from gene modified animal models or human genetic disorders has demonstrated that interruption of the transporter activity can lead to debilitating diseases or organ toxicities. Herein we describe transporter associated diseases and organ toxicities resulting from transporter gene deficiency or functional inhibition in the liver, kidney, gastrointestinal tract (GIT) and central nervous system (CNS). While proposing additional transporters as targets for drug-induced organ toxicity, strategies and future perspectives are discussed for transporter risk assessment in drug discovery and development.

Key words: Membrane transporters; drug-induced toxicity; drug-drug interaction; drug-nutrient interaction; asymmetric organ distribution;



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1. Introduction Biological membranes are composed of membrane bound proteins, lipids and carbohydrates in a fluid state that defines the boundary of cell space. The outer membrane forms the shape of the cell structure, whereas intracellular membrane forms various intracellular organelles that serve distinct functions. Transporting solutes across biological membranes is one of the most critical processes for living cells. This process distributes the essential nutrients, physiological chemicals and ions to the cells, and eliminates metabolic waste and environmental toxins (Figure 1). Overall, these activities are essential in maintaining the homeostasis of the living cells. Traditionally, it was thought that passive diffusion across biological membranes is the dominant route for solute transport. This concept has been challenged in the last two decades, since hundreds of membrane associated proteins have been discovered and are found to be essential as cellular uptake and efflux carriers. Indeed, there are more than 400 membrane transporters that are categorized into two distinct superfamilies, the solute carrier (SLC) and ATP-binding cassette (ABC) families. Those proteins transport solutes either through facilitative diffusion, or energy-dependent pathway by utilizing energy generated either from hydrolysis of adenosine triphosphate (ATP) or that stored in an electrochemical ion gradient. Therapeutic drugs and/or their metabolites can “hitchhike” on these transport pathways making them important determinants of drug absorption, distribution and excretion. Therefore, alteration of transporter function or expression may significantly change the exposure in plasma and/or organs, as well as could disrupt the transport of important physiological molecules. Overall, these interactions with transport proteins may represent underappreciated determinants of the tolerability and safety profiles of therapeutic agents.

To date, there is only a limited number of membrane bound transporters that are known to recognize therapeutic agents (aka drug transporters). These transporters are primarily expressed in the intestine, liver, kidney and blood-brain barrier (BBB), sites where drug transporters influence drug absorption, distribution, metabolism and elimination (ADME) of drugs.1,

2


For example, drug transporters in

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elimination organs such as the liver and kidney are functioned to facilitate the secretion of drugs and their metabolites from the body. Drug uptake transporters such as organic anion transporting polypeptide 1B1 and 1B3 (OATP1B1 and OATP1B3) that are expressed on the sinusoidal membrane of hepatocytes typically control the rate of entry of acidic molecules with large molecular weight (Mwt > 400 Dalton).3 Drugs that inhibit transporter activities or regulate gene expressions can significantly alter systemic pharmacokinetics (PK) and/or tissue exposure of other drugs that are substrates of those transporters. Consequently, the International Transporter Consortium (ITC), comprised of transporter experts from the industry, academia and regulatory agencies, recommended strategies in drug discovery and development to investigate the role of several major drug transporters, which include OATP1B1/1B3 (SLCO1B1/SLCO1B3), P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP, ABCG1), organic anion transporter (OAT1/3, SLC22A6/22A8), organic cation transporter (OCT1/2, SLC22A1/SLC22A2), multidrug and toxin extrusion proteins (MATE1/2k, SLC47A1/SLC47A2), multidrug resistance protein 2 (MRP2, ABCC2) and bile salt export pump (BSEP, ABCB11).1, 2 From drug development standpoint, these transporters are well-investigated and have displayed significant roles in drug biology and disposition as demonstrated in reported DDIs, drug responses and drug related toxicities. Consequently, both the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) have updated regulatory guidance on drug interaction studies,4,

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to include

sections on the evaluation of drug interactions with transporters.

While drug transporters have gained more recognition in the pharmaceutical industry due to their clinical relevance in DDIs and their impact on drug disposition and pharmacokinetics, many other membrane associated transporters that play pivotal roles in maintaining the body homeostasis are poorly understood, especially their association with organ specific toxicity. In this review, examples of transporter associated organ toxicity reported in the clinical studies are provided and known correlations between transporter functional defects and pathophysiological events/organ toxicity are discussed. 5 ACS Paragon Plus Environment


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Perspective directions that may lead industrial scientists to recognize the potential of transporter associated drug-induced organ toxicity are proposed.

2. Transporter related disease and transporter function in disease states

In the last two decades, an increasing number of transporter gene defects have been identified and found to be associated with severe human diseases (Table 1).

Hence, knowledge from the

association could provide new insights on mechanisms of drug-induced organ toxicity. This is of particular interest to the pharmaceutical industry in the efforts to identify drugs with improved safety profiles. For example, gene mutations of ABCB11 that encodes BSEP protein have been found to cause type 2 progressive intrahepatic cholestasis (PFIC2).

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The gene mutants disable the biogenesis or

trafficking of BSEP into the canalicular membrane of hepatocytes, which results in the decreased bile flow and increased accumulation of bile salts in hepatocytes, leading to cholestatic liver injury. In addition to gene mutations, altered transporter function or reduced expression levels due to disease state or gene polymorphisms is found to be a susceptible factor for drug-induced liver injury (DILI).7,

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Besides PFIC2, mutations of ABCB4 gene that encodes MDR3 protein located on the canalicular membrane of hepatocytes are associated with PFIC3 and several other liver diseases including adult biliary cirrhosis, transient neonatal cholestasis, drug-induced cholestasis, intrahepatic cholestasis of pregnancy, and cholesterol gallstone disease. 8-15 In an earlier review, a comprehensive list was provided to describe human diseases that are associated with defects of transporter genes. 16, 17 The links between transporter gene defects and genetic diseases has become an important reference when considering druginduced organ toxicity. For instance, inhibition of BSEP function by drug treatment may result in acquired liver disorders such as drug-induced cholestasis. Many drugs with potent inhibition of BSEP activity (IC50 < 10 µM) are reported to cause cholestatic, mixed cholestatic and hepatocellular injuries, such as cyclosporine, ritonavir, rosiglitazone, saquinavir, troglitazone, ketoconazole, pioglitazone, 6 ACS Paragon Plus Environment

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lovastatin, haloperidol, atorvastatin, cerivastatin and chlorpromazine. In that regard, BSEP is included in the EMA guidance due to its important role in bile salt homeostasis.4

Transporter expression may also be modulated by disease states. For example, in patients with inflammatory disease who are more susceptible to DILI, BSEP expression was found to be downregulated. 18, 19 Nonalcoholic fatty liver disease (NAFLD) affecting 30 to 50% of the adult population 20 appears to cause general decreases in hepatic uptake transporter expression, and increases in the expression of efflux transporters.21-23 Increased susceptibility to methotrexate-induced hepatic and renal toxicity has been reported in more advanced nonalcoholic steatohepatitis (NASH) patients.

24

In

addition, evidence for decreased renal expression of OCT2 has been found in diabetes 25, 26 and bilateral ureteral obstruction results in the increased OAT1 expression in rats.

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Collectively, understanding the

impact of human diseases on transporter functions and/or gene expression is critical when considering precise medication for special populations.

3. Transporters-involved organ toxicity Transporters are key regulators of many fundamental physiological pathways, as showed in Table 1.16 Inhibition of these critical pathways may lead to drug-induced organ toxicity.16, 28 On the other hand, active uptake of drugs may also result in accumulation in a target organ to an unsafe level leading to organ-specific toxicity. Unexpected modulation of transporters by drugs may cause serious druginduced toxicities including liver cholestasis, cardiac myopathy, renal failure and brain excitotoxicity. Major organ toxicities with compelling evidence for an ideology related to transporters are highlighted below. 3.1

Transporter involved liver toxicity



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Liver transporters determine the systemic availability and biliary clearance of nutrients, drugs, and toxins absorbed from the GIT, as well as hepatic exposure of exogenous chemicals and endogenous molecules. Disruption of the function of these transporters may lead not only to hepatotoxicity, but also other organ toxicities due to increased systemic exposure. Direct evidences are genetic disorders caused by transporter gene deficiency, such as progressive familial intrahepatic cholestasis, Dubin-Johnson Syndrome, and Rotor Syndrome, as demonstrated in Table 1.16

Many mutations may not cause

syndromes directly, but may cause the carriers to be more susceptible to conditions. For example, single nucleotide polymorphism (SNP) (V444A) of ABCB11 gene was frequently observed in subjects with drug-induced cholestasis and two non-synonymous mutations of ABCB11 (2026G>T and 2563G>A) were reported to be specific for drug-induced cholestasis.29 DILI has become a major cause for drug attrition. Over 1000 approved drugs were found to be associated with DILI and several drugs, e.g. troglitazone, bromfenac and trovafloxacin, have been withdrawn from the US market due to debilitating liver injury.30 DILI can result from dose dependent adverse reactions (intrinsic) or non-predictive toxicities (idiosyncratic), of which liver transporters could be involved in either. For example, one of the major contributors to DILI is the interference with transporter-mediated biliary excretion of bile acids.1 Many drugs which inhibit BSEP are associated with hepatic cholestasis, such as troglitazone, bosentan, rifampicin, erythromycin estolate, and glibenclamide.31 Drugs inhibiting MPR2 may interrupt bilirubin hepatic excretion and lead to hyperbilirubinemia.1 In addition to direct drug-induced toxicity, DDIs involving liver transporters may also affect the disposition and biliary clearance of victim drugs or metabolites, leading to hepatotoxicity or other organ toxicity. For example, co-administration of cyclosporine and verapamil leads to an increase in the systemic exposure of cyclosporine, as well as liver enzymes (amino alanine transferase and alkaline phosphatase).32 The adverse effect (muscle pain or myopathies) of many statin drugs are an outcome of 8 ACS Paragon Plus Environment

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increased systemic exposure due to either OATP1B1/1B3 genetic variations or co-administration with OATP inhibitors (e.g. cyclosporine or rifampin). Complex DDI that involves the inhibition of both metabolizing enzymes and multiple uptake/efflux transporters located in liver and intestine can lead to a more significant interaction resembled in substantial increase in both organ and systemic exposures. For example, repaglinide is an OATP 1B1/1B3 substrate and is metabolized by CYP2C8 and to a lesser extent by CYP3A4. When repaglinide is co-administered with ketoconazole, a potent CYP3A4 inhibitor, an increase in systemic exposure of ~1.12 fold was observed. With cyclosporine, repaglinide systemic exposure was increased ~2.4 fold. Interestingly, a complex DDI is reported when repaglinide is coadministered with gemfibrozil and itraconazole with ~19 fold increase in exposure. Gemfibrozil is an inhibitor of both OATPs and CYP2C8, while itroconazole is an inhibitor of CYP3A4. This is an important example on the call for the implementation of advanced physiology based pharmacokinetics model (PBPK) to enable a prospective prediction of these complex DDI liabilities and their debilitating potential systemic and organ toxicities.33 Liver transporters are tightly regulated through transcription factors and nuclear receptors. The expression level can be modified in response to drug exposures, viral infections or disease conditions.34 Following acetaminophen overdose, the basolateral efflux transporters (MRP4 and MPR5) and canalicular efflux transporters (BCRP and P-glycoprotein) were upregulated to protect liver from further injury.35 In cholestatic patients, uptake transporters (e.g sodium taurocholate carrier polypeptide (NTCP, SLC10A1) and OAPTs) are down regulated and basolateral efflux transporters (MRP3 and MPR4) are induced, which could limit the excessive bile salts exposure in hepatocytes. However, these regulations may change the systemic exposure of drug/metabolites and lead to a greater risk of toxicities. For example, the systemic exposure of simvastatin hydroxyl acid is increased in NASH rodent model due to the down regulation of hepatic uptake transporters.36 More severe outcome may occur in subjects bearing both genetic deficiency and liver disease. 9 ACS Paragon Plus Environment


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Transporter related kidney toxicity The kidney is a key mediator of osmoregulation, electrolyte and pH balance, signaling molecule and

nutrient homeostasis, and metabolite and toxin excretion. Transporters expressed in different regions of the nephron play a central role in maintaining homeostasis in the kidney at large. Indeed, the interaction of transporters expressed in the proximal tubule with drugs has become an area of focus with the basolaterally expressed OAT1, OAT3 and OCT2, and apically expressed MATE1 and MATE2-K highlighted in various regulatory guidance. While current efforts are largely limited to predicting DDIs arising from inhibition of elimination through active tubular secretion and, thus, elevating circulating drug levels, it is becoming increasingly evident that effects on distribution to the kidney and changes in biologically active non-drug small molecules are possible and should be further considered during candidate selection and clinical development. A number of more comprehensive reviews on the interactions of drugs with renal transporters are available.37-42 Therefore, the intent of this section is not to be comprehensive but to illustrate concepts with select examples.

Given its role in the handling of drugs and their metabolites, it is not surprising that the kidney is a target organ of drug toxicity across therapeutic classes. Nephrotoxic agents that accumulate in the proximal tubule and cause direct cellular toxicity include antivirals (e.g., acyclic nucleoside phosphonates), antibiotics (e.g., β-lactams), and chemotherapeutic agents (e.g., methotrexate and cisplatin).37, 38, 41 A number of drugs subject to renal elimination can cause renal toxicity through crystal formation in urine due, in part, to transport and poor solubility (e.g. atazanavir, acyclovir, ciprofloxacin, methotrexate, sulfonamide antibiotics). The basolateral uptake transporters OAT1, OAT3 and OCT2 have been found to play a critical role in the intracellular accumulation of nephrotoxic drugs. For example, OAT1 and, to a lesser extent, OAT3 take up the acyclic nucleoside phosphonates adefovir, cidofovir and tenofovir into cells and overexpression of these transporters increases their cytotoxicity in vitro.43, 44 The understanding of the relationship of OAT-mediated uptake and cidofovir nephrotoxicity 10 ACS Paragon Plus Environment

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has led to the clinical use of the OAT inhibitor probenecid to reduce proximal tubule uptake, and therefore, greatly reduced the incidence of renal adverse events by shifting elimination to passive glomerular filtration and decreasing kidney accumulation. 45

While having the potential to increase accumulation in the proximal tubule and the resulting observation of renal adverse events of nephrotoxic agents undergoing active tubular secretion, mechanisms of efflux transport have garnered less attention than uptake transporters. The nephrotoxicity of cisplatin, an OCT2 substrate,46,

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is potentiated in mice in the presence of the inhibitor

pyrimethamine or genetic knockout of the efflux transporter MATE1.48 MATE1 is inhibited by a large number of drugs that bind with a relatively high affinity,49 perhaps of the greatest clinical significance for cisplatin are the often co-administered antiemetic ondansetron and kinase inhibitor vandetanib. Both drugs are potent MATE1 inhibitors and have been shown to affect cisplatin toxicity either in mice or transfected cell lines.50, 51 While renal transporters have been therapeutic targets for gout (inhibitors of urate transporter 1/URAT1/SLC22A12, probenecid and benzbromarone), hyperglycemia (inhibitors of the sodium glucose co-transporter-2/SGLT-2/SLC5A2 from the gliflozin class) and hypertension (inhibitors of the sodium chloride cotransporters/NCCT/SLC12A family from the loop diuretic and thiazide classes), the interactions of drugs with transporters mediating reabsorption, apical uptake or basolateral efflux have been underexplored. Interestingly, a few studies have observed DDIs resulting in seemingly paradoxical increases in the renal elimination of drugs that may best be explained by inhibition of reabsorption.52-54

Creatinine is a functional indicator of renal glomerular filtration. Creatinine is also actively secreted by renal proximal tubule and the inhibition of creatinine tubular secretion by medications can increase serum creatinine. While not known to be intrinsically toxic itself, the number of drugs observed to affect serum creatinine levels illustrates the common interaction of drugs with the transport of 11 ACS Paragon Plus Environment


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endogenous compounds. The active tubular secretion of creatinine is inhibited by over a dozen drugs from across therapeutic areas, primarily caused by inhibition of MATE1.55 Further, a recent report showed that the MATE1 and MATE2-K inhibitor pyrimethamine markedly (>2-fold) reduced the excretion of a number of endogenous compounds including thiamine, 2’-deoxycitidine, Nmethylnicotinamide and carnitine in healthy human volunteers.56 Metabolomics studies conducted in knockout mice have also shown that loss of OAT1 or OAT3 causes changes in endogenous metabolites, nutrients, uremic toxins and signaling molecules.57, 58 Previously, inhibition of organic cation/carnitine transporter (OCTN2; SLC22A5) mediated carnitine reabsorption at the apical membrane of the proximal tubule has been proposed as the mechanism for cephaloridine-induced carnitine deficiency.59 Renal transporters are also involved in the handling of environmental toxins including ethidium, aristolochic acid and mercury suggesting that drug-toxin interactions could affect the impact of exposure to these agents.37, 60, 61 3.3

Transporter associated toxicity and malabsorption in gastrointestinal tract Although transporter-mediated GIT toxicity has not been reported or studied as extensively as those

in liver and kidney, many potential cases involving GIT toxicity to drugs may be associated with altered nutrient and/or ion transporter function. These transporters include, but are not limited to, sodiumglucose cotransporter (SGLT1, SLC5A1), creatine transporter (CRT, SLC6A8), apical sodium-dependent bile salt transporter (ASBT, SLC10A2), sodium-dependent serotonin transporter (SERT, SLC6A4), as well as other transporters that are co-transporting sodium and/or chloride with their substrates. Many of these transporters belong to the SLC family and are important in cell homeostasis and metabolism.

SGLT1 is encoded by gene SLC5A1 and is responsible for the sodium-dependent transport of sugars, anions, and other nutrients such as amino acids as well as vitamins.62 The SGLT1 cotransports sodium ion and sugar across the brush-border of the intestine. D-Glucose and D-galactose are the natural 12 ACS Paragon Plus Environment

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substrates for SGLT1. Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder caused by defects in SGLT1 originally discovered in newborns and characterized by disrupted dietary glucose and galactose absorption in the intestine. It was reported that mutations in the SLC5A1 gene are the cause of GGM.62 Newborns with congenital defects in SGLT1 present with severe diarrhea and dehydration and without treatment, the condition can become fatal. To date, more than 20 missense mutations of SGLT1 have been reported by Wright and colleagues. Their data have suggested that the impairment of sugar transport is mainly due to that the mutant SGLT1 being either truncated or improper trafficking to the cell membrane, resulting in a reduction or elimination of transport function.62, 63

Creatine is a nonessential dietary compound that is both endogenously synthesized, primarily in the liver, and naturally ingested through the diet. Oral supplementation of creatine is routinely used by many athletes to improve exercise performance and muscle mass. The initial absorption of ingested creatine in the gut is mediated by CRT that is localized on the brush-border of the small intestine.64 The CRT, encoded by gene SLC6A8, is a member of the superfamily of Na+- and Cl--dependent transporters that are responsible for the uptake of creatine, certain neurotransmitters and amino acids.64 The most common side effect of creatine supplements use is gastrointestinal upset, including stomach discomfort, diarrhea, and/or nausea. These side effects are primarily due to the presence of large quantities of undissolved creatine particles residing within the intestinal compartment especially after a large loading dose.65 Since the absorption process of creatine mediated by CRT involves the cotransport of Na+ and Cl-, it has the propensity to draw water into the body compartment where the transport process is occurring. If the body compartment is the intestine, then excessive water absorption may lead to diarrhea and intestinal cramps. These side effects can often be largely circumvented through use of more soluble salt forms of creatine, such as creatine hydrochloride, creatine citrate or creatine pyruvate.66



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Bile acids are physiological detergents synthesized from cholesterol in the liver, stored in the gall bladder, and released into the small intestine to aid in the absorption of fats and fat-soluble nutrients.67, 68 Through enterohepatic recycling, most of the secreted bile acids are reabsorbed in the ileum, which is primarily achieved by carrier-mediated mechanisms involving multiple transporters. One such carrier is the ASBT, which transports bile acid from the lumen of the intestine. The ASBT is identified at the gene level as SLC10A2.68 It was reported that inherited loss-of-functions in the SLC10A2 gene can cause primary bile acid malabsorption (PBAM).69 PBAM is an idiopathic intestinal disorder associated with chronic watery diarrhea, excess fecal bile acids, steatorrhea and interruption of the enterohepatic circulation of bile acids. ASBT inhibitors have been used in clinic for treating hypercholesterolemia and can be divided into two classes: bile acid-derivatives, including bile acid dimers, and non-bile acid compounds, including benzothiazepine and benzothiepine analog.70 In addition to these classical inhibitors, many FDA-approved drugs have been discovered to function as ASBT inhibitors, including the dihydropyridine calcium channel blockers and HMG-CoA reductase inhibitors.71 Inhibition of ASBT may result in greater passage of bile acids into the colon, and consequently may explain the potential side effects experienced with these drugs, such as bile acid-induced diarrhea.71 The finding that mutations in SLC10A2 gene as well as inhibition of ASBT cause significant bile acid malabsorption in the absence of ileal diseases indicates the importance of ASBT, the ileal Na+/bile acid cotransporter, in maintaining the enterohepatic circulation of bile acids and cholesterol homeostasis.69, 72

GIT toxicity to drugs may be caused either through a direct effect on the intestinal epithelium by the ingested drugs, nutrients and/or their metabolites, or through an indirect effect involving interference with normal transporter processes in the intestinal cells. Diarrhea, which was proposed by Phillips73 as a hallmark of intestinal failure, may be the simplest measure of intestinal toxic effect, because it reflects an impairment of the ability of the intestine to conserve water and electrolytes, which is as important as conservation of nutrients. Given the important roles of many transporters, including those responsible 14 ACS Paragon Plus Environment

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for transporting nutrients, dietary supplements, endogenous ions and molecules, in maintaining physiological homeostasis, the importance of these transporters in gastrointestinal toxicity has been underexplored. The above mentioned transporters all share a common fact, which is their transport functions are dependent on the extracellular Na+ and/or Cl− gradient and are cotransporters of Na+ and Cl−. Therefore, when the function of these transporters was disrupted by either a gene mutation or direct effects from a substrate, water absorption would be reduced and resulted in an increase of water retention in the intestinal lumen. Other transporters which could potentially be involved in gastrointestinal toxicity may include amino acid transporter bo,+ (SCL6A15) and thiamine transporter 2 (THTR2, SLC19A3). GIT side effects have also been reported with arginine based dietary supplement ingestion which shares the amino acid transporter bo,+ with other dibasic amino acids for intestinal absorption.74 Metformin and fluoxetine inhibition of SERT has been implicated in associated GIT adverse effects, 17, 75 a hypothesis supported by changes in colon motility in SERT knockout animals. 76 And most recently, studies from Zhang and colleagues

77

suggested that Janus kinase (JAK2) inhibitor

fedratinib was a potent inhibitor of THTR2, which is responsible for the intestinal absorption of thiamine. While the clinical development of fedratinib being terminated due to Wernicke’s encephalopathy, a thiamine deficiency neurological disorder, nausea, diarrhea, and vomiting were major adverse events noted.78

The discontinuation of the clinical development of fedratinib has drawn

attention on drug-nutrient interactions.77 3.4

Transporter related CNS dysfunction

Blood brain barrier (BBB) is a dynamic barrier that separates the circulatory blood system from the brain, and plays a key role in protecting the brain from harmful endotoxins and chemicals.79, 80 BBB maintains the brain homeostasis by regulating the transport of nutrients, the volume and composition of the interstitial fluid bathing the brain parenchyma. The barrier function of the BBB is in part attributed 15 ACS Paragon Plus Environment


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to the expression of membrane bound efflux transporters that determines the chemicals transcellularly crossing the barrier.80 Examples on these transporters are P-gp, MRPs and BCRP. Alterations in the expression of these transporters in the BBB are known to affect the biological activities of agents affecting the CNS. For example, epilepsy is neurological disorder where the nerve cell activity is disrupted leading to epileptic seizures that vary in intensity. It is interesting to note that around one third of the epileptic patients are pharmaco-resistant also known as intractable epilepsy.81 This has a debilitating effect on resistant patients. It reduces their life span and leads to bodily injury and social disability. These patients typically don’t respond to several antiepileptic agents with different mechanism of actions.81 It should be emphasized that the systemic exposure of these patients is comparable to those that are normal. In 1995, Tishler et al attributed the intractable epilepsy to the overexpression of efflux drug transporters in the brain capillary endothelium for these patients with this devastating ailment.81 It is also generally accepted that drug resistant epilepsy is also associated with upregulation of P-gp, which is driven by the seizure activity rather than the epileptic drug itself. 82 Similar up-regulation findings were also reported with BCRP, MRP1, and MRP2 in intractable epilepsy patients.83-85 Alzheimer is a devastating progressive neurodegenerative disease that is associated with the loss of social skills and behavioral dysfunction. It is associated with neurofibrillary tangles and the plaques of amyloid beta protein. These are protein aggregates that are associated with microglia and astrocyte inflammation and the secretion of neurotoxins that lead to nerve cells injury and death. A set of research reports indicated amyloid protein is a P-gp substrate.86,

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Interestingly, increasing vascular P-gp

expression is associated with a reduction clearance of amyloid protein from the brain, which could potentially be an effective approach to either prevent or treat Alzheimer disease.88, 89 As discussed in previous section, fedratinib is a potent inhibitor of THTR2, which is responsible for the intestinal absorption of thiamine. Preclinical studies suggested that the distribution of fedratinib into 16 ACS Paragon Plus Environment

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the brain was high in rats.77 Therefore, in addition to inhibition of thiamine uptake at the intestine, fedratinib may also inhibit thiamine transport into the brain through the inhibition of THTR2.

4. Strategies to assess transporter involved drug toxicity As aforementioned, primary transporter assessments in drug discovery and development are mainly focusing on how membrane transporters affect drug absorption and elimination through GIT, liver and kidney. Regulatory guidance recommends evaluating systemic PK changes for both interacting and interacted drugs, and requires including these information in the drug labels. As such, evaluation of drug transporter interactions are routinely assessed using in vitro systems in the early stage of drug discovery for a number of transporters which have demonstrated clinical significance on drug ADMEPK. These transporters include OATP1B1/1B3, P-gp, BCRP, OAT1/3, OCT1/2, MATE1/2k and BSEP. Additional transporters including MRP2, the peptide transporters (PEPTs, SLC15 family) and nucleotide transporters (NTs, SLC28 and SLC29 family) are identified to have an emerging importance in drug disposition, DDIs and drug-interactions with endogenous compounds in humans.1

Early efforts for conducting substrate assays and understanding transporter roles in elimination pathways are important steps when considering the drug tissue accumulation or active liver/renal secretion in animals or clinical studies (Figure 2). These assessments provide important parameters to help predict drug disposition, and understand potential effect of DDI on systemic exposure or tissue partitioning, therefore to generate a more accurate estimation on the safety margins towards target organs. It is becoming increasingly important trend to understand transporter related asymmetric tissue distribution. Transporter interactions associated with increased exposure in peripheral tissues to an unsafe level need to be carefully investigated when evaluating drug-induced organ toxicities (Figure 2). Often, the interactions on tissue exposure are not associated with systemic PK changes. For example, OCT2 and MATE1/2k work in concert to eliminate cisplatin from the proximal tubule cells in the 17 ACS Paragon Plus Environment


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kidney. Co-administration of antiemetic agent ondansetron with cisplatin can result in a decrease in systemic exposure of cisplatin; 90 however, nephrotoxicity is significantly enhanced due to accumulation of cisplatin in the kidney, as ondansetron is a more potent inhibitor of human MATEs than that of human OCT2.50

Drug-induced organ toxicities are usually not well predicted if only based on in vitro results. This is largely due to our insufficient knowledge of membrane proteins in each organ. Most of the times, an investigation is not initiated until in vivo toxicity is observed. The concentrations of drug and its metabolites are usually first evaluated in systemic and in injured organs to determine if it is a dose dependent adverse reaction (Figure 2). In preclinical species, this can be easily completed by analyzing isolated tissues or using quantitative whole body autoradiography. In human, the tissue concentration is usually predicted based on results from in vitro assays and animal models. Noninvasive approaches are emerging areas to investigate the functional activity of transporters in vivo. Magnetic resonance imaging (MRI) uses contrast agents, tracers or fluorescent dyes to determine the OATPs-MRP2 transport pathway to better understand the transporter functions under liver diseases.

91

Positron emission

tomography (PET) is also used to measure the tissue distribution of drug related components in human. However, this technology may not differentiate the parent drug from its metabolites. Biopsy may provide an alternative solution but only for limited cases. A higher tissue to plasma ratio indicates the possibility of drug/metabolites cellular accumulation in the organ, which could result from active uptake or impaired efflux, therefore, in vitro substrate assays should be conducted to identify the transporter(s). This will provide valuable information for the development of backup molecules of the drug candidates. In addition, pharmacogenomics of the patients should be assessed to unravel if such toxicity is resulted from genetic variance of transporters. Membrane proteins transport various endogenous molecules including nutrients, hormones or biosynthesis intermediates. It is very challenging to directly study transporters in vivo, therefore 18 ACS Paragon Plus Environment

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biomarker has become a very useful tool to monitor transporter activity and diagnose transporter involved DDI and toxicity. However, it is very difficult to find a specific biomarker for each transporter because of extensive substrate overlap. So far, only a few endogenous molecules have been identified which could reflect the function of one transporter or a group of transporters. For example, bile salt excretion is strongly correlated with BSEP activity.92 A high plasma level of bilirubin conjugates indicates the probability of MRP2 malfunction.93 Untargeted metabolomics on the plasma and urine from Oat1 gene knockout mice identifies three metabolites, indoxyl sulfate, kynurenine, and xanthurenic acid, being elevated in the plasma.58 Recently, 6β-hydroxycortisol is proposed as a novel endogenous biomarker for detection of OAT3 related DDIs94 and N-Methylnicotinamide is thought to be an endogenous probe for DDIs involving MATE1 and MATE2-K.95 As more and more biomarkers are being identified, they can greatly facilitate our understanding of transporter functions in vivo and to reduce DDI risk and toxicity. The first step before initiating a transporter experiment is to select the most appropriate assay system. Based on the transporter characteristics, various in vitro methods to support transporter evaluation are discussed by ITC in 2013.96 Since ABC efflux transporters commonly mediate the inside-out uphill transport by utilizing the energy of ATP hydrolysis, bidirectional transport in polarized cell monolayers such as Caco-2 cells or inside-out oriented membrane vesicles are commonly used. Cell lines such as human embryonic kidney (HEK293) or Madin–Darby canine kidney (MDCK) overexpressing transporter proteins are used for characterizing uptake transporters. In addition, primarily isolated hepatocytes are also widely accepted as a holistic system to study the interactions with hepatic transporters. The compound of interest could be tested in these systems for determination of substrate or the inhibitory effects on the transporter. Transporter evaluation strategy may also involve multiple steps using different testing systems and the information obtained from various models are then integrated, in either a stepwise or parallel fashion. Figure 2 recommends a paradigm to determine transporter 19 ACS Paragon Plus Environment


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associations in DDI or drug induced organ toxicity. Transporter interactions can be assessed from ether a “bottom-up” (discovery) or “top-down” (development) approach. The “bottom-up” approach is to proactively assess the potential liabilities of “known” transporters. The transporter data obtained prior to clinical development are extrapolated or modeled, to gain insight into the clinical relevance of the interaction. Often the factors such as the therapeutic indications, potential co-medications, targeted populations, dose regimens and routes of administration should be also taken into account. Information obtained could be used to develop early screening plans to support lead candidate optimization to avoid the organ toxicity. However, potential drawback of the “bottom-up” approach is that data generated from preclinical studies may not be relevant and sometimes difficult to interpret. In addition, there is only limited number of transporters that are well-studied and could be tested at discovery phase. On the other hand, the “top-down” investigation is commonly triggered first followed by the observation of clinical data. The approach is designed to identify transporter mechanisms explaining clinical findings and subsequently pathing forward a study plan for further clinical development. As “top-down” approach may divulge critical issues of transporter-related organ toxicity found in late stage of drug development, the issues may no longer be mitigated if activity is eased to develop backup molecules. Nevertheless, as various tools are available for testing transporter-related hypotheses, it is important to outline thought-processes as below to integrate in vitro and in vivo data and determine the role of transporters on organ toxicity.

A) Asymmetric tissue distribution is a common indication of the involvement of uptake transporters such as hepatoselective OATPs. Substrate determination could better understand the consequence of the DDIs that may sometimes be deceptive if human DDI studies only monitor the changes of systemic exposure, as the systemic exposure does not always reflect the tissue concentration for compounds that are substrates of transporters.


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B) When selecting in vitro and in vivo testing methods, special attention should be given to the pros and cons of each assay.

While in vitro cell assays are useful in shedding light on the potential

contribution of a particular transporter, it is pivotal to confirm the outcomes in an in vivo model. Gene knockout models are currently considered the “gold” standard for the functional assessment of transporter roles in the disposition of new molecular entities (NMEs). However, transporter compensatory effects followed by the gene knockout, and species differences in transporter expression, structure and activities need to be investigated if animal testing data are to be extrapolated to human predictions.

C) In vitro kinetic parameters could be used to predict potential relevance of transporter interactions in clinical studies, as compared to the projected human exposure of the drug or predicted concentration in target organs. The use of physiology based pharmacokinetic models to project the human plasma and tissue exposures of NMEs that are transporter substrates is important and should be considered to put the potential organ toxicity of the NME in perspective. Also of importance is the fact that empirical scaling factors are commonly used to translate in vitro transporter activity to in vivo. The scaling factors could be obtained from preclinical species and used for the prediction of human interactions.97 In addition, the scaling factors could be transporter and/or compound specific.97, 98

D) It is of the growing interest in developing biomarkers for studying transporter related toxicity and physiological regulations. The use of “omics” approaches, such as transcriptomics, proteomics or metabolomics may be promising approaches to discover specific biomarkers for early detection of transporter modulations or transporter-related organ toxicity, despite very few biomarkers related transporter function are available and data are controversial so far.

E) The redundancy of drug transporter expressions with overlapping substrate specificity remains a mystery. This further complicates the distribution of substrate drugs in target organs and affects the 21 ACS Paragon Plus Environment


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ability of prediction from in vitro to in vivo. The use of gene knockout mice or functional knock down animals becomes a routine approach to study the in vivo role of drug transporters in drug disposition and organ toxicity. Transporters that are associated with severe human diseases or causing lethality followed by the gene deletion are considered high risk targets for drug-induced organ toxicity. Drug candidate being a potent inhibitor for these transporters should be avoided.

F) Genetic polymorphisms or disease-state in humans may cause reduced expression or function of specific transporters, which may vary the susceptibility of organ toxicity in human. The results of experiments in preclinical species could also not explain the potential outcomes of genetic polymorphisms in human. Since reliable in vitro models for transporter studies to address transporter polymorphisms are still lacking, studies on quantitative transporter expression in human subjects with genetic polymorphisms or at disease-state have been instrumental to predict individual susceptibility to adverse effects.

Drug interactions with the transport of endogenous molecules such as serotonin, bile salts, bilirubin, carnitine, and thiamine may lead to organ toxicities. “Bottom-up” investigation should also include the studies on the inhibition of transporters which are known to transport endogenous molecules. Positive inhibition results lead to further investigations using more complicated in vitro systems (i.e. human primary cells), animal models and simulation software. Low toxicity risk is expected from transporter interactions if no interaction (substrate and inhibition) is observed in in vitro screening assays. Since many membrane bound transporters are critical for normal cell functions, in addition to the transporters that are discussed in regulatory guidance or ITC white papers,1

a dozen of disease associated

transporters are proposed in Table 2. These transporters should be considered for further investigation depend upon the observations of preclinical or clinical findings (Figure 2).

5. Summary and future perspectives 22 ACS Paragon Plus Environment

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While the field of membrane transporter research is rapidly advancing, the majority of the membrane proteins (>90%) are poorly understood and further studies are warranted to elucidate their substrate specificity and associations with organ functions. Based on regulatory guidance, the earlier studies with transporters are conducted primarily to assess changes of systemic exposure for both interacting drugs and interacted drugs. In our view, transporter mediated asymmetric tissue distribution, changes in organ exposure due to DDIs, or drug interactions with nutrients, hormone and neurotransmitters, which likely do not affect pharmacokinetics, should be considered equally important and evaluated in early drug discovery.

A variety of in vitro assays are currently available to investigate drug transporter interactions, including membrane vesicles, transporter gene expressing cell lines and hepatocyte models. 96 However, significant disconnect often occurs when attempting to translate results from these in vitro systems into clinical observations. These disconnects are likely due to simplified in vitro assays inability to appropriately represent the physiological microenvironment including transporter interplay and establishment of appropriate electrochemical gradients. Therefore, more comprehensive and physiological relevant in vitro models are in increasing demand. Lately, efforts have been invested to develop new methodologies which can better simulate in vivo tissue functionality, such as 3D culture, primary cell co-cultures and microfluidic cell culture (organ-on-a-chip).99-101 These new technologies may greatly facilitate our understanding of the in vivo characteristics and mechanisms of transporters and their significance in organ physiology and disease etiology. They are also great in vitro tools for drug candidate optimization and toxicity evaluation in early discovery. Gene modulation technologies have become one of the most important tools, which are applied in many steps in preclinical toxicology and safety studies. The applications of transgenic or gene knockout animal models provide new mechanistic perspectives, particularly to gain better understanding of not only overlapping substrate specificities of different transporters and the possible effects of inhibitors on 23 ACS Paragon Plus Environment


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multiple transport systems, but also the endogenous roles of the transporters, in morphogenesis and organ development, remote communication, and responses to environmental changes. As summarized in Table 1, the rapid progress in generating gene knockout mice and characterizing transporter functions in these animals has greatly increased our knowledge of the roles of transporters in ADME-PK of xenobiotics, and their association with disease progression. It should be noted that species differences have been well documented regarding the involvement of transporters in organ toxicity. For example, individuals with PFIC-2 syndrome caused by ABCB11 gene polymorphisms typically will develop progressive cholestasis and then lead to failure to thrive, enlarged liver or hepatic failure. However, mice with Bsep gene knockout were viable and fertile, and developed a relatively mild phenotype and nonprogressive intrahepatic cholestasis.102 Additionally, one of the reasons that preclinical animals fail to predict organ toxicity, especially asymmetric toxicity, in human is the gene homogeneity of those models. The expression of transporters are greatly influenced by the genetic background, disease condition, diet variance, and living habit in human, which are not well represented in the in-breed animal models. In this case, out-breed mice have demonstrated the potential to better identify toxicity mechanisms and may provide key information to predict asymmetric toxicity risks in the future.103, 104 Although it is still in its infancy, application of humanized animal model has shed some light on evaluating and predicting drug metabolism and toxicity in human.103-105 Before starting to dose in humans, it is expected that drug candidates undergo extensive preclinical toxicology assessment to provide critical drug safety information for establishing safe starting dose in human subjects. Drug candidates are given to animals e.g. a rodent and upper preclinical model such as dogs or monkeys where short and long-term toxic damages to organs are monitored.106 A combination of in vitro model using human primary cells, animal safety data and modeling software may improve the toxicity risk prediction in human. The pathophysiological events or organ toxicity found in in preclinical models suggest the follow up studies so that potential mechanisms can be identified. 24 ACS Paragon Plus Environment

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In summary, drug-induced toxicities in major organs are highlighted and additional dozen transporters that are known to be associated with serious diseases in clinic are listed for consideration of investigation. Strategies and future perspective are discussed that early eliminating drug candidates that are associated with toxic findings and advancing safe molecules that will most likely succeed is achieved by the introduction of well-defined testing strategy that ensures the implementation of the appropriate in vitro/in vivo test models.



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Abbreviation list ABC

ATP-binding cassette family

ADME

Absorption, distribution, metabolism and elimination

ASBT

apical sodium-dependent bile salt transporter

BBB

Blood-brain barrier

BCRP

breast cancer resistance protein

BSEP

bile salt export pump

CNS

Central nervous system

CRT

creatine transporter

DDI

Drug-drug interaction

DILI

drug-induced liver injury

GGM

Glucose-galactose malabsorption

GIT

Gastrointestinal tract

MATE

Multidrug and toxin extrusion proteins

MRP

multidrug resistance protein

NASH

nonalcoholic steatohepatitis

NMEs

new molecular entities

NT

nucleotide transporters

NTCP

sodium taurocholate carrier polypeptide

OCT

organic cation transporter

OCTN

organic cation/carnitine transporter

OAT

organic anion transporter

OATP

organic anion transporting polypeptide

PBAM

primary bile acid malabsorption

PEPT

peptide transporters

PFIC

progressive intrahepatic cholestasis 26 ACS Paragon Plus Environment

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P-gp

P-glycoprotein

PK

pharmacokinetics

SERT

sodium-dependent serotonin transporter

SGLT

sodium-glucose cotransporter

SLC

Solute carrier family

THTR

thiamine transporter



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AUTHOR BIOGRAPHIES

Yaofeng Cheng, PhD Dr. Yaofeng Cheng obtained his PhD degree in Medical Pharmacology from University of Arizona, and then completed two years of post-doctoral training at Novartis Institute of Biomedical Research. After that, Yaofeng joined Bristol-Myers Squibb and leading projects focusing on drug metabolism, disposition and drug-drug interactions. He has 7 years of experience in drug metabolism and transporter research, and is the leading inventor for a patent and authors of 9 peer reviewed publications. His current research interests are transporters involved liver bile acids homeostasis and transporter biomarkers in preclinical animals and clinical subjects.

Ayman El-Kattan, B. Pharm., PhD Dr. Ayman El-Kattan is Associate Research Fellow at the PDM Department, Pfizer Inc. He earned his B.Sc. in pharmacy from University of Jordan and a Ph.D. at University of South Carolina. His research interests are focused on understanding the role of transporters in influencing drug ADME. He is also an Adjunct Professor at College of Pharmacy, University of Rhode Island where he lectures in the graduate-level pharmacokinetic courses and serves as external advisor on dissertation committees. He serves at Drug Transporter focus group of AAPS. He has published over 100 papers in Journals, book chapters and proceedings.


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Yan Zhang, PhD Dr. Yan Zhang is a Senior Principal Investigator in the Department of Drug Metabolism & Biopharmaceutics (DMB) at Incyte Corporation in Wilmington, Delaware. She received her M.Sc. degree from the Pharmacology Department at South Dakota State University and her PhD from the Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center. After completing her post-doctoral training in the PDM Department at Pfizer in Ann Arbor, Michigan, she joined the DMB Department at Incyte Corporation. She has over ten years’ experiences as an ADME/PK/PD scientist in oncology drug discovery and over fifteen years’ experience in the field of drug transporters. Dr. Zhang is the author of over 30 peer-reviewed publications, book chapters, and abstracts. She is currently leading the drug absorption and transporter strategies to support Incyte discovery and development programs. Research interests include the pharmacokinetics and pharmacodynamics, transporter mediated drug-drug interactions and in vitro/in vivo transporter correlation.

Adrian S. Ray, PhD Dr. Adrian S. Ray is Senior Director Drug Metabolism at Gilead Sciences, Inc. where he has been a scientist for over 13 years. He received his Ph.D. from the departments of Pharmacology and Molecular, Cellular and Developmental Biology at Yale University. His accomplishments include over 70 peerreviewed publications, serving as a primary responder at 3 FDA advisory committee meetings, and being the recipient of the 2014 William H. Prusoff award presented by the International Society for Antiviral Research. Related to this review, transporter mediated drug interactions with endobiotics, nutrients and toxins has been an area of focused research within his team.



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Yurong Lai, PhD Dr. Lai received his M.D from Fujian Medical University in China and his Ph.D. (Toxicology) from Sapporo Medical University in Japan in 1998. He was a research fellow in Hokkaido University and research associate in University of Washington. In 2004, he Joined PDM Pfizer Inc. In 2013, he become a Sr. Principal Scientist in BMS and leads the transporter labs for the implementation of drug transporter strategies and in vitro/in vivo transporter investigations for regulatory filings. Dr Lai holds an Adjunct faculty position in University of Rhode Island and is a patent inventor and the author of a book, book chapters and over 120 original publications in peer-reviewed journals.


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Table 1. Major pathophysiological events found in human subject with genetic polymorphism or transporter gene knockout mice

Organs

Generic name

Primary endogenous Substrate

Symptoms in humans with genetic deficiency

Mouse gene

ABCB4

Multidrug resistance protein 3 (MDR3)

phospholipids

Cholestasis progressive familial intrahepatic 3

Abcb4 107, 108

ABCB11

Bile Salt Export Pump (BSEP)

Bile acids

Cholestasis progressive familial (severe) intrahepatic 2

Abcb11, Abcb1a/1b 109

Severe cholestasis, liver injury, and liver cancer

Bilirubin

Dubin-Johnson syndrome

glutathione and its adducts

Cystic fibrosis; congenital bilateral absence of the vas deferens;

Transporter gene

ABCC2

ABCC7 Liver

Multidrug resistanceassociated protein 2 (MRP2) Cystic fibrosis transmembrane conductance regulator (CFTR)

Symptoms in gene knockout mice Lack of biliary phospholipid excretion and development of progressive liver disease

ATP7b

coppertransporting P-type ATPase

copper

Wilson disease

Atp7b 110

Marked hepatic accumulation of copper and develop liver cirrhosis and neurological problems

ATP8B1

Probable phospholipidtransporting ATPase IC

Phosphatidylserine, phosphatidylethanolamine

Progressive familial intrahepatic cholestasis type 1

Atp8b1 111

Type-1 progressive familial intrahepatic cholestasis (PFIC1)

Slc30a10 112

Multiple abnormalities, including osteopenia, characterized by the loss of most trabecular bone at the metaphyses; dystonia

SLC30A10

Zinc transporter 8

Hepatic Cirrhosis, Dystonia, Polycythemia, and Hypermanganesemia

Zinc



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and adult-onset parkinsonism

SLC37A4

Glucose-6phosphate transporter

glucose-6-phosphate

Glycogen storage disease, type 1B/1C

SLCO1B1/1B3

Organic aniontransporting polypeptide 1B1/1B3 (OATP1B1/1B3)

Bilirubin, bile salts

Rotor syndrome

SLC1A1

Excitatory aminoacid transporter 3 (EAAT3)

L-Glu, D/L-Asp

Dicarboxylic amino aciduria

glucose, galactose, mannose, glucosamine

Fanconi-Bickel syndrome

Uric acid (glucose, fructose)

Renal hypouricemia, gout, Alzheimer’s disease

neutral/dibasic amino acids

Cystinuria-lysinuria type I, hypotonia-cystinuria syndrome

HCO3- (and/or CO32-)

Renal tubular acidosis, proximal, with ocular abnormalities

glucose

glycosuria renal

neutral amino acids

Iminoglycinuria and hyperglycinuria

sodium, chloride

Gitelman syndrome 1

SLC2A2

SLC2A9

SLC3A1/SLC7A9 Kidney SLC4A4

SLC5A2

SLC6A19

SLC12A3

Glucose transporter 2 (GLUT2) Glucose transporter 9 (GLUT9) Neutral and basic amino acid transport protein Electrogenic sodium bicarbonate cotransporter 1 sodium/glucose cotransporter 2 (SGLT2) system B(0) neutral amino acid transporter sodium-chloride symporter


Slco1b2 113

Completely resistant to phalloidin-induced hepatotoxicity

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SLC16A12

SLC17A1

Sodium-dependent phosphate transport protein 1

monocarboxylic acid

Juvenile cataract, microcornea and renal glycosuria

organic acids, phosphate, chloride

Gout development

SLC17A2

Sodium-dependent phosphate transport protein 3

phosphate

SLC22A5

Organic Cation/Carnitine Transporter 2 (OCTN2)

Carnitine

SLC22A12

Urate transporter 1 (URAT1)

urate

Renal hypo-uricemia, (gout)

SLC36A2

Proton/Amino Acid Symporter 2

glycine, alanine, and proline

Iminoglycinuria, hyperglycinuria, type 1

Slc17a2 114

Primary carnitine deficiency

Slc22a5 115

Slc26a7 112, 116,

SLC26a7

Anion exchange transporter

SLC5A1

Sodium/glucose cotransporter 1 (SGLT1)

glucose, galactose

SLC9A3

Sodium–hydrogen exchanger 3 (NHE3)

Na+, H+, Li+, NH4+

117

Glucose-galactose malabsorption

GI Tract Congenital secretory diarrhea; hypertension


Urinary elimination of phosphate, hypophosphatemia, and elevation in the blood levels of 1,25dihydroxyvitamin D and calcium Excessive urinary loss of carnitine, systemic carnitine deficiency, and disorder of fatty acid oxidation

Distal renal tubular acidosis, reduced gastric acid secretion and distal renal tubular acidosis


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

SLC10A2

Apical Sodium Dependent Bile Acid Transporter (ASBT)

bile acids

SLC26A3

Chloride anion exchanger

Chloride

Bartter syndrome familial chloride diarrhea

SLC46A1

Proton-coupled folate transporter

folates and antifolates

Congenital folate malabsorption

Nervous system

Primary bile acid malabsorption

plant sterols and cholesterols

ABCG5/8

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Sitosterolemia

Abcg5/Abcg8 118

SLC5A7

High-affinity choline transporter (ChT)

Choline

SLC6A1

GABA transporter 1 (GAT1)

gamma-aminobutyric acid

Orthostatic intolerance, Attention deficit hyperactivity disorder

Slc6a2 120, 121

Slc6a3 122

Slc5a7 119

SLC6A2

Norepinephrine transporter (NET)

norepinephrine, dopamine

Orthostatic intolerance, Attention deficit hyperactivity disorder

SLC6A3

Dopamine active transporter, (DAT)

dopamine

Major affective disorder, ADHD


Increased blood concentration of phytosterols, and accumulation of phytosterols in brain Limited capacity of movement, dropping forelimbs, and death within 1 h of birth from respiratory distress

Excessive Tachycardia and Elevated Blood Pressure With Wakefulness and Activity Elevated spontaneous locomoter activity. Took longer to habituate to the open field test and more active

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


SLC6A4

Serotonin transporter

Serotonin

SLC6A9

Sodium- and chloride-dependent glycine transporter 1

Glycine

SLC17A6

Vesicular glutamate transporter 2

Anxiety-related personality traits (obsessive-compulsive disorder) (schizophrenia)

Slc6a4 123

Slc6a9 124

glutamate

Slc17A6 125

Die immediately after birth due to dysfunctional respiratory central pattern generator.

glutamate

Slc17a7 126

Lethality in the third postnatal week

(VGLUT2)

SLC17A7

Vesicular glutamate transporter

Increased anxiety-like behaviors, reduced aggression, and exaggerated stress responses severe motor and respiratory deficits, died within 12 h of birth

1 (VGULT1)

SLC18A2

Vesicular monoamine transporter 2 (VAMT2)

monoamine

SLC19A2

Thiamine transporter 1 (THTR1)

Thiamine

Slc18a2 127, 128

Thiamine responsive megaloblastic anemia


Slc19a2 129

Null mice result in neonatal death; heterozygotes display prolonged QT intervals and reduced amphetamineconditioned reward, enhanced amphetamine locomotion, and enhanced MPTP toxicity. On a thiamine-free diet, null mice develop diabetes mellitus with reduced insulin secretion and an enhanced response to insulin


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SLC19A3

Thiamine transporter 1 (THTR2)

Thiamine

SLC23A1

Sodium-dependent vitamin C transporters 2 (SVCT2)

vitamin C

SLC26A4

Pendrin

I-, Cl-, HCO3-

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Biotin-responsive basal ganglia disease

Slc23a1 131 Neurosensory deafness Pendred syndrome

SLC30A3

Zinc transporter 3

zinc

SLC25A3

Phosphate carrier protein, mitochondrial

phosphate

Cardiomyopathy hypertrophic with lactic acidosis

SLC25A4

ADP/ATP translocase 1

ADP, ATP

Congenital cataract and progressive external ophthalmoplegia 2

SLC2A3

Glucose transporter 3 (GLUT3)

Glucose

Lethal

cardiovascular system

Slc19a3 130

Slc26a4 116

Deaf without hypothyroidism

Slc30a3 132

Marked decreased plaque load and less insoluble beta amyloid in the brain; contribute to the accumulation of amyloid plaques in Alzheimer’s disease

Slc2a3 133

Embryonic lethality

Slc19a1 134

Embryonic lethal and severe abnormalities. Rescued RFC null neonates appear to be markedly growth retarded with multiple

Reproductive systems SLC19A1

Reduced folate carrier 1 (RFC1)

Reduced folate


Premature death within a year of age, appear to be lethargy, cachexia, injured liver parenchyma, hepatic necrosis and, liver and kidney inflammation Undetectable ascorbic acid levels in the brain at birth and die perinatally

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compromised organ systems

Others organs

ABCA1

Cholesterol efflux regulatory protein (Cerp)

ABCC6

Multidrug resistanceassociated protein 6 (MRP6)

cholesterol

Familial hypoalphalipoproteinemia and Tangier disease

Abca1135

Decreased blood levels of total and high-density lipoproteins (HDL) cholesterol

Angioid streaks; pseudoxanthoma elasticum

Abcc6 136

Mineralization of connective tissues

ABCG2

Breast cancer resistance protein (BCRP)

Abcg2 137

SLC6A6

Sodium- and chloride-dependent taurine transporter

SLC6a6 138

SLC16A1

Monocarboxylate transporter 1 (MCT1)

Monocarboxylate

SLC16A2

Monocarboxylate transporter 1 (MCT8/OATP1c1)

Monocarboxylate

Hyperinsulinemic hypoglycemia, familial 7

Allan–Herndon–Dudley syndrome


Slc16a1 139

Slc16a2/Slco1c1 140

Protoporphyria and diet-dependent phototoxicity; severe, sometimes lethal phototoxic lesions on light-exposed skin Severe and progressive retinal degeneration, a small brain, and shrunken kidneys Null mutation was embryonically lethal; MCT1+/− mice displayed resistance to development of dietinduced obesity, as well as less insulin resistance and no hepatic steatosis Pronounced locomotor abnormalities. lower T3 in brain but higher T3 in liver, resulting in a decrease in serum cholesterol and an increase in alkaline


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phosphatase

SLC25A1

Mitochondrial tricarboxylate transport protein

Tricarboxylate

SLC27A4

Long-chain fatty acid transport protein 4 (FATP4)

Long-chain fatty acid

SLC30A5

Zinc transporter 5

SLC39A13

ZIP13

Inborn errors of metabolism, Combined D-2- and L-2Hydroxyglutaric Aciduria

Congenital ichthyosis 2

Slc25a1

112

Slc27a4 141

Slc30a5 142

Spondylocheirodysplasia, Ehlers-Danlos syndromelike


Slc39A13 112

A generalized hypoplasia that was most severe in liver, bone and bone marrow Hyperproliferative hyperkeratosis with a disturbed epidermal barrier, a flat dermal– epidermal junction, a reduced number of pilo-sebaceous structures, and a compact dermis. Poor growth and a decrease in bone density due to impairment of osteoblast maturation to osteocyte. 60% null mice died suddenly because of the bradyarrhythmias Growth retardation, a generalized low bone mass phenotype and dysplasia of the epiphyseal growth plate cartilage.

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Table 2. Proposed transporters that should be considered for further evaluation in drug discovery and development and testing rationales

Related Organ

Transporter

Transporter Gene

Liver

Multi-drug resistant protein 3 (MDR3/Mdr2 (rodent)).

ABCB4

Central nervous system

Excitatory amino acid transporter 1 (EAAT1)

SLC1A3

Excitatory amino acid transporter 2 (EAAT2)

SLC1A2

Sodium- and chloridedependent GABA transporter 1 (GAT1).

SLC6A1

Norepinephrine transporter (NET)

SLC6A2

Rationales MDR3 is expressed on the apical membrane of hepatocytes. The function of MDR3 is to excrete biliary phospholipids that are needed to maintain solubility of cholesterol and neutralize toxic detergent such as bile salts. Functional inhibition of MDR3 is associated with itraconazole-induced cholestasis. 143 Inhibition or functional defects of MDR3, along with MRP2 and BSEP, are potential liabilities of the formation of cholesterol calculus 144 and found to be associated with drug-induced cholestasis, intrahepatic cholestasis of pregnancy, and cholesterol gallstone disease. 8, 15 EAAT1 transports glutamate and aspartic acid from the synaptic cleft against concentration gradient by coupling with sodium, potassium and proton. Inhibition of EAAT1 may lead to episodic ataxia, and other disorders in the CNS including epilepsy, schizophrenia and excitotoxicity. 145 EAAT2 maintains extracellular glutamate concentrations by reuptaking the synaptically released glutamate. Functional defects of EAAT2 result in pathogenesis of neurodegenerative diseases of the motor neuron system including amyotrophic lateral sclerosis. 146 GAT1 is expressed on neurons and glia in the brain. It is a voltagedependent gamma-aminobutyric acid (GABA) transporter that regulates neurotransmitter levels in the synaptic cleft. Mutations in SLC6A1 gene are associated with epilepsy in humans. 147 GABA is an inhibitory neurotransmitter to counterbalance neuronal excitation in the brain. Inhibition of GAT1 activity reduces the re-uptake of GABA from the synapse, leading to seizures. 148 NET belongs to the family of the Na+/Cl- dependent GABA/NET transporter. Its substrate, norepinephrine, synthesized from dopamine by dopamine beta-hydroxylase is stored in vesicles under normal condition, and released by neuronal activation into the synapse. NET mediates the reuptake of extracellular norepinephrine, which is an essential process to terminate neuron actions by regulating



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Gastrointestinal track

Dopamine transporter (DAT)

SLC6A3

Serotonin transporter (SERT)

SLC6A4

High-affinity thiamine transporter 2 (THTR-2)

SLC19A3

The proton-coupled folate transporter (PCFT)

SLC46A1

The apical sodiumdependent bile salt transporter (ASBT)

SLC10A2

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concentrations of monoamine neurotransmitters in the synaptic cleft. Inhibition of NET result in the high norepinephrine levels in plasma and is found to be associated with orthostatic intolerance. 149 DAT is expressed in dopaminergic neurons and functions to reuptake extraneuronal dopamine. It is a principal regulator of the duration and amplitude of dopaminergic action through regulating the released dopamine back up into presynaptic terminals. Inhibition of DAT leads to the defects of dopamine reuptake, and causes parkinsonismdystonia, Tourette syndrome and alcohol dependence. 150, 151 SERT is expressed in brain and many peripheral tissues and is responsible for the active transport of serotonin (5-HT) that is used by neurons to communicate with each other. The concentration of synaptic serotonin is controlled directly by its uptake transporter, SERT, to uptake the serotonin into the pre-synaptic terminal. Inhibition of the transporter function may affect the rate of serotonin uptake, which takes part in numerous serotonin related clinic conditions including autism, depression, neuroticism, and aggressive behavior in Alzheimer disease patients. 152-155 THTR2 is expressed in various tissues on the apical membrane of polarized cells and is the major absorptive transporter for thiamine and other substrates in the intestine. Inhibition of THTR2 activity results in significantly reduced intestinal thiamine absorption, as well as its uptake into various tissues including CNS. Thiamine deficiency is associated with the development of Wernicke's encephalopathy and biotin-responsive basal ganglia disease. 156 PCFT is expressed on apical membrane of the intestine and the bloodchoroid plexus cerebrospinal barriers, where the transporter absorbs folates from the intestine and delivers it to the CNS. Similar to thiamine, folates are co-factors for many biosynthetic enzymes. Inhibition of PCFT may lead to folate deficiency in systemic circulation and in the CNS. 157 ASBT is expressed on the brush boarder membrane of enterocytes, and works in concert with the basolateral heterodimer of organic solute transporter (OSTalpha-OSTbeta) for enterohepatic circulation of bile acids. As discussed above, inhibition of ASBT could result in 50

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Heart

Sodium dependent carnitine transporter 2 (OCTN2)

SLC22A5

Circulation system

High-affinity thiamine transporter 1 (THTR-1)

SLC19A2

bile acid malabsorption, leading to congenital diarrhea, steatorrhea and disorders of cholesterol homeostasis. OCTN2 is widely expressed in many organs including the kidneys, pancreas, prostate, skeletal muscle and heart and appears to be a cation/zwitterion sodium dependent high affinity carnitine transporter. Its substrate, carnitine, is a natural substance involved in transporting activated fatty acids into mitochondria for energy production. In the kidney, OCTN2 reabsorbs the filtered carnitine to prevent its excretion from urine. Inhibition of OCTN2 is associated with the condition of systemic primary carnitine deficiency. Some organs including the liver and kidney can biosynthesize carnitine, while many other tissues such as skeletal and heart muscle are highly dependent on OCTN2 mediated carnitine uptake from blood. Functional deficiency of OCTN2 often leads to severe cardiomyopathy rhabdomyolysis and heart failure. 158, 159 THTR-1 is widely expressed in many organs including muscle, heart, liver and kidney and transports thiamine that is an important co-factor for enzymes including pyruvate dehydrogenase and alphaketaglutarate dehydrogenase for the krebs cycles, a part of cellular respiration. Inhibition of THTR-1 results in low concentration of intracellular thiamine in erythrocytes and low activities of thiamindependent enzymes, leading to thiamine responsive megaloblastic anemia. 160



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Figure 1. Membrane bound transporters distribute the essential nutrients, physiological chemicals and ions into the cells, and eliminate metabolic waste and environmental toxins.

Drugs Hormones Vitamin Neurotransmitters Amino acids Peptides Folate

Fatty acids Amino acid Dicarboxylates Nucleotides Glucose, etc.

Citrates, Phosphonates ATP, H+, Ions, etc.

Drugs Bile acid Bilirubin Intermediate metabolites Environmental toxins


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Figure 2 General procedures to evaluate transporter related DDI and organ toxicity



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Involvement of Drug Transporters in Organ Toxicity - ACS Publications

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