The Role of Hepatic Transporters in Drug-induced Liver Injury (DILI) Research and Preclinical Evaluation Recommendations

Drug-induced liver injury (DILI) refers to liver injury induced by various types of prescription or over-the-counter (OTC) chemical drugs, biological agents, traditional Chinese medicine (TCM), natural medicine (NM), health products (HP), dietary supplements (DS), and their metabolites and even excipients^[1]. HepaTox in China and LiverTox in the United States have documented information on liver injury in more than 1,100 common drugs. In recent years, there has been a rise in the incidence of DILI, making it the fourth most prevalent liver disease. Liver diseases caused by drugs account for 20% to 50% of non-viral liver diseases and 15% to 30% of fulminant liver failure. Therefore, DILI has become a severe public health issue that urgently needs attention. In addition, hepatotoxicity is also one of the critical reasons for drug withdrawal, restricted use, and failure of marketing. Table 1 lists drug withdrawals due to hepatotoxicity.

Table 1. Partial Withdrawals for Hepatotoxicity

DILI has become the most common reason for safety-related drug withdrawals from the market over the past 60 years, resulting in considerable losses to the pharmaceutical industry. This article will elaborate on the role of hepatic transporters from aspects such as the pathogenesis and classification of DILI and the expression and function of hepatic transporters.

Pathogenesis of drug-induced liver injury (DILI)

The pathogenesis of DILI is complex, involving multiple factors, and shows diverse clinical manifestations. In 2019, Weaver, R.J. et al. summarized six Pathogenesis of DILI: #1 Mitochondrial impairment; #2 Inhibition of biliary efflux; #3 Lysosomal impairment; #4 Reactive metabolites (chemical stress, oxidative stress, protein modification); #5 Endoplasmic reticulum stress; and #6 Immune system (innate, adaptive and inflammation) (Figure 1) ^[14]. It could be found that the direct damage of hepatocytes by drugs and the inhibition of biliary efflux are considered important reasons for DILI, and the occurrence of these processes is closely related to the changes in the function of hepatic transporters.

Figure 1. Pathogenesis and clinical presentations of DILI. Source: adapted from Weaver, R. J 2020 (figure 1) ^[14]

Classification of drug-induced liver injury (DILI)

According to the pathogenesis, DILI can be divided into three types: direct hepatotoxicity, idiosyncratic hepatotoxicity, and indirect hepatotoxicity ^[15]. The main characteristics are shown in Table 2.

Table 2. Classification and characteristics of DILI by pathogenesis

Direct hepatotoxicity is more common and refers to the direct damage to the liver caused by drugs and/or their metabolites ingested in the body, which is often dose-dependent and usually predictable, also known as intrinsic DILI.

Idiosyncratic hepatotoxicity is rare, and its pathogenesis has become a research hotspot in recent years. The occurrence of idiosyncratic hepatotoxicity does not correlate with drug dosage, and is unpredictable. It cannot be reconstructed in animal models, with variable latency periods ranging from a few days to several years.

Indirect hepatotoxicity is caused by the action of drugs rather than by the inherent hepatotoxicity or immunogenicity of drugs, and it is a common response to an entire class of drugs rather than a specific drug. Indirect DILI is usually not correlated with drug dosage and is partially predictable. It can be reconstructed in animal models.

According to the course of disease, DILI can be divided into acute DILI and chronic DILI. In most cases of acute DILI, liver function can return to normal after discontinuation of the drug. Still, in a small number of patients, liver function cannot recover even after long-term treatment for more than six months, and acute DILI gradually develops into chronic DILI.

Based on the clinical features, DILI can be divided into hepatocellular injury, cholestasis, mixed type, and hepatic vascular injury. Changes in serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), and total bilirubin (Tbil) are currently the leading laboratory indicators for determining liver injury and diagnosing DILI. The criteria for the first three types of DILI established by the Council for International Organizations of Medical Sciences (CIOMS) are shown in Table 3 ^[1]. When hepatocytes are damaged, the permeability of the cell membrane changes and ALT in the cells can be released into the blood, causing an increase in ALT activity in the blood. ALP and GGT are excreted with bile, and when bile excretion is blocked, ALP and GGT will increase significantly in the hepatocytes. When Tbil is not entirely excreted, and bile flow is obstructed, it easily accumulates in the blood, resulting in hyperbilirubinemia. Hepatic transporters play a crucial role in all these processes.

Table 3. Clinical criteria of DILI

Note: ULN, upper limit of normal.

R = (measured ALT/ULN ALT)/ (measured ALP/ULN ALP).

Expression and function of hepatic transporters

The liver is the major metabolic and clearance organ in the human body. In addition to the expression of abundant metabolic enzymes, many functional proteins are expressed on the membrane of hepatocytes. The proteins that can transport drugs are called drug transporters, which have a critical function in drug absorption, distribution, and elimination. In recent years, about a quarter of drugs have been withdrawn from the market due to various toxic side effects related to drug transporters. Therefore, it is essential to understand drug transporters for the investigation of the mechanism of DILI.

The distribution of hepatic transporters is shown in Figure 2. The transporters involved in transporting endogenous and exogenous substances are located on the sinusoidal and canalicular membranes of hepatocytes. Among them, transmembrane transporters of the soluble carrier (SLC) family mediate the entry of endogenous and exogenous substances into hepatocytes. In contrast, those of the ATP-binding cassette (ABC) family mediate their efflux.

The absorption of exogenous substances by hepatocytes can be mediated by organic anion-transporting polypeptides (OATPs), organic anion transporters (OATs), and organic cation transporters (OCTs). Sodium taurocholate cotransporting polypeptide (NTCP) is expressed on the sinusoidal membrane of hepatocytes and mediates sodium-dependent bile acid uptake. NTCP plays an essential role in the enterohepatic circulation of bile acids. After bile acids are excreted into the small intestine, about 95% can be reabsorbed by apical sodium ion/bile acid transporter (ABST), of which 80% could be ingested into hepatocytes by NTCP and re-secreted into bile.

The transportation of drugs and their metabolites from hepatocytes back into the blood is mainly mediated by multidrug resistance protein 3 (MRP3) and MRP4. In contrast, the elimination into the bile canaliculus is mediated by several different transporters, such as MRP2, breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), and multidrug and toxin extrusion 1 (MATE1). Since bile acids and their salts have certain toxicity to hepatocytes, they must be efficiently cleared, and BSEP mainly mediates this task.

Figure 2. Distribution of hepatic transporters. Source: adapted from Morrissey 2012 ^[16]

Transporter-mediated drug-induced liver injury (DILI)

Changes in transporter function are closely related to the occurrence of direct hepatotoxicity, and drugs can generally affect the function of transporters to cause DILI through the following two mechanisms:

1. Transporter-mediated drug interactions

The impact of drugs on the function of almost all hepatic transporters may lead to DILI. For example, a recent study found that a COVID-19 patient who was treated with Remdesivir also took chloroquine and amiodarone, resulting in severe hepatotoxicity. Since Remdesivir is a substrate of P-gp, and chloroquine and amiodarone are inhibitors of P-gp, coadministration of the two drugs with Remdesivir decreased the clearance of Remdesivir, leading to a concentration of Remdesivir in hepatocytes that exceeded the threshold of toxicity, thereby causing hepatotoxicity ^[17].

2. Impact of drugs or their metabolites on transporters related to bile formation, secretion, and excretion

Bile acids serve crucial physiological functions in the human body but also have inherent toxicity. When bile acid formation, secretion, and excretion are obstructed, high concentrations of bile acids will accumulate in hepatocytes and blood. High concentrations of bile acids in hepatocytes can damage the cell membrane and destroy the normal liver function. The obstruction of biliary excretion can also lead to an increase of bilirubin in hepatocytes and blood. Therefore, the dynamic balance of bile acid formation, secretion, and excretion is the basis for maintaining the normal physiological function of hepatocytes. A variety of hepatic transporters expressed on the canalicular surface and on the sinusoidal surface of the liver are involved in the formation, secretion, and excretion of bile acids and bilirubin (Table 4).

Table 4. Role of hepatic transporters in the transport of bile acids and bilirubin

Taking Troglitazone as an example, the US FDA approved Troglitazone for the treatment of type 2 diabetes in January 1997, but it was demanded for withdrawal by the UK Medicines and Healthcare products Regulatory Agency (MHRA) due to hepatotoxicity in the same year ^[18]. In March 2000, after 90 cases of liver failure and 63 cases of deaths were reported, the FDA notified the withdrawal of this drug, and subsequently, it was withdrawn from the Japanese market. The main cause of DILI is the inhibitory effect of Troglitazone and its metabolites on the transporters related to bile formation, secretion, and excretion. Troglitazone and its primary metabolites are BSEP inhibitors, inhibiting bile acid excretion. Meanwhile, Troglitazone is also an inhibitor of NTCP, which can reduce the reabsorption of bile acids back into the liver. This simultaneously increases the content of bile acids in the liver and blood, leading to cholestasis (Figure 3).

Figure 3. Mechanism of Troglitazone-induced DILI

BSEP and MRP2 primarily mediate the excretion of bile acids and bilirubin into the bile canaliculus, and the abnormal function of these two transporters is the main reason for the occurrence of cholestasis and hyperbilirubinemia.

BSEP is a transmembrane protein expressed on the canalicular membrane of hepatocytes. BSEP not only plays an important role in maintaining normal liver function but also participates in the excretion of bile acid and the transport of bile salts, which are the main driving forces of bile enterohepatic circulation. If the function of BSEP is inhibited, it will lead to cholestatic liver injury. The further development of cholestasis can cause liver fibrosis and cirrhosis, eventually resulting in liver failure and death. Studies have shown that BSEP is significantly related to DILI ^[19]. In addition, BSEP is also involved in the transport of bilirubin, and the inhibition of BSEP can lead to hyperbilirubinemia. Therefore, drug-related BSEP inhibition is considered to be the main cause of cholestasis, mixed liver injury, as well as hyperbilirubinemia.

MRP2 is primarily distributed in the liver, kidneys, and small intestine; in the liver, like BSEP, MRP2 is also expressed on the canalicular of hepatocytes and mediates the excretion of metabolites and exogenous compounds into the bile. It is a very important transmembrane transporter to balance bile acid metabolism. The substrates of MRP2 are mainly lipophilic organic anions. Therefore, MRP2 can regulate liver injury in obstructive jaundice by regulating the secretion of acid-base substances, as well as increasing the lipid solubility of bile salts to promote bile secretion. MRP2 is involved not only in the efflux of bile acids, but also in the transport process of conjugated bilirubin. The abnormal expression of MRP2 can lead to the disorder of bilirubin excretion, resulting in bilirubin accumulation, clinically significant hyperbilirubinemia, and jaundice symptoms.

Regulatory agency recommendations for transporter evaluation

Table 5 summarizes the transporters recommended for evaluation by the three major regulatory agencies: the National Medical Products Administration (NMPA, 2021), the Food and Drug Administration (FDA, 2020), and the European Medicines Agency (EMA, 2013) ^[20-22]. NMPA and FDA recommendations consistently focus on gastrointestinal absorption, hepatic uptake, and renal clearance. EMA added BSEP and OCT1 in addition to the FDA recommendations.

Table 5. Regulatory agency recommendations for transporter evaluation

Summary

Whether a drug candidate can be successfully marketed depends on the balance between efficacy and safety. One important reason for the failure of most drug candidates is their lack of good safety ^[23]. Hence, early identification of safety risks in drug research and development can significantly save time and costs. DILI is one of the most common reasons for the withdrawal of drugs from the market due to safety concerns. Evaluating the interactions between drug candidates and hepatic transporters associated with DILI at the early stages of drug development is crucial for advancing the process of new drug development and promoting rational drug usage.

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Authors: Qing Liu, Tao Xiong, Genfu Chen

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