In Vitro Analysis of AKR1D1 Interactions with Clopidogrel: Effects on Enzyme Activity and Gene Expression

Clopidogrel, a P2Y12 receptor antagonist, is a cornerstone of dual antiplatelet therapy alongside aspirin, widely used to prevent major cardiovascular events in patients with acute coronary syndromes or undergoing percutaneous coronary interventions (1, 2). While clopidogrel has been shown to be more effective than aspirin in reducing the risks of myocardial infarction, ischemic stroke, and vascular death, there remains significant variability in treatment outcomes among patients (3, 4). This variability can range from reduced efficacy or resistance to therapy, affecting 5–44% of patients, to increased risk of bleeding due to excessive antiplatelet activity (5, 6). The phenomenon of “clopidogrel resistance” presents a major clinical challenge, particularly because the underlying mechanisms are not fully understood (7–9).

The metabolism of clopidogrel is complex. As a prodrug, it undergoes two primary processes: hydrolysis by carboxylesterase 1 (CES1), which inactivates the majority of the drug, and a two-step activation mediated by several cytochrome P450 (CYP) enzymes, which convert clopidogrel to its active form (10, 11). Among these, CYP2C19 is the most crucial, with polymorphisms in this enzyme (CYP2C19*2, *3, and *17) being major determinants of clopidogrel’s variable pharmacokinetics and treatment outcomes (12–14). However, these genetic variations explain only about 12% of the observed variability, leaving much of the interindividual differences unexplained (12, 15).

The variability in CYP enzyme activity and its effects on drug metabolism extend beyond clopidogrel, as CYP enzymes play a pivotal role in the biotransformation of many medications (16). Aldo-Keto Reductase 1D1 (AKR1D1), an enzyme critical in bile acid synthesis and steroid clearance (17–19), has been identified by Chaudhry et al. (2013) as a key trans-regulator of the CYP enzyme network, suggesting a broader regulatory mechanism. AKR1D1 regulates CYP2C19, CYP3A4, and CYP2C9 expression via its metabolic products. Specifically, the 5β-reduced steroids generated by AKR1D1 activity act as ligands for nuclear receptors such as farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), which in turn regulate CYP enzyme expression (20, 21). The AKR1D1*36 polymorphism (rs1872930), in particular, leads to increased expression of AKR1D1, resulting in the upregulation of these CYP enzymes (21).

Kapedanovska et al. (2019) explored this hypothesis in the context of clopidogrel and demonstrated that the AKR1D1*36 allele is associated with an increased risk of major adverse cardiovascular and cerebrovascular events (MACCE) in patients treated with clopidogrel, establishing AKR1D1 as an independent risk factor (22).

Building on previous findings linking AKR1D1*36 to the regulation of the cytochrome P450 enzyme network, this study seeks to elucidate the potential role of AKR1D1 in clopidogrel metabolism. Specifically, the research aims to evaluate whether clopidogrel and its inactive metabolite, 2-oxoclopidogrel, act as substrates or inhibitors of the AKR1D1 enzyme, thereby elucidating potential interactions at the protein level. Furthermore, the study investigates the influence of clopidogrel and its metabolite on AKR1D1 gene expression in HepG2 cells to explore potential mechanisms that could modulate clopidogrel metabolism. These investigations are intended to provide a deeper understanding of AKR1D1’s involvement in drug metabolism and its broader implications for pharmacological research.

The study provides valuable insights into the interaction of clopidogrel and its metabolite, 2-oxoclopidogrel, with the enzyme AKR1D1. The enzymatic assays demonstrate that neither clopidogrel nor its metabolite, 2-oxoclopidogrel, acts as a substrate or inhibitor of AKR1D1. This aligns with the known specificity of AKR1D1, which predominantly interacts with steroidal compounds and not with non-steroidal drugs like clopidogrel. The absence of inhibition is significant because it suggests that clopidogrel does not interfere with AKR1D1’s essential role in bile acid synthesis and steroid hormone clearance, thus preserving critical metabolic processes (17, 18, 20, 30). Disruption of this processes could impair digestion, absorption, and drug solubilization, which further emphasizes the physiological importance of AKR1D1’s functional integrity.

While AKR1D1’s substrate specificity appears to favor steroidal compounds, its structural homology with AKR1C enzymes - which can interact with both steroidal and non-steroidal substrates - raises the possibility that AKR1D1 could be flexible under certain conditions (19, 30, 31). Although studies have demonstrated that certain non-steroidal compounds, including indomethacin, mefenamic acid, and 4-benzoylbenzoic acid, which are potent inhibitors of AKR1C enzymes, do not inhibit AKR1D1 (31), only three non-steroidal compounds have been evaluated to date. Given the limited scope of these investigations, the possibility remains that AKR1D1 could interact with other non-steroidal substrates or inhibitors, warranting further exploration.

The potential flexibility in AKR1D1’s substrate specificity is an intriguing aspect of the enzyme’s functionality, particularly when considered in the context of genetic polymorphisms, such as the AKR1D1*36 variant that has been is associated with an increased risk of major adverse cardiovascular and cerebrovascular events (MACCE) in patients treated with clopidogrel (22). While the AKR1D1*36 polymorphism has been linked to altered expression levels and downstream effects on CYP enzyme regulation (21), its direct impact on enzymatic function and substrate specificity remains unexplored. By contrast, engineered mutations, such as the E120H substitution, have demonstrated that single amino acid changes can significantly alter AKR1D1’s enzymatic activity and substrate specificity (32). These findings highlight the potential for naturally occurring genetic variants, like AKR1D1*36, to similarly influence enzyme functionality, warranting further investigation.

Gene expression analyses further demonstrated that clopidogrel and 2-oxoclopidogrel do not modulate AKR1D1 expression at the transcriptional level. This finding implies that clopidogrel does not interfere with the production of 5β-reduced steroids, metabolic products of AKR1D1 that regulate CYP enzyme expression via nuclear receptor pathways such as FXR, CAR, and PXR (21). Consequently, the lack of AKR1D1 modulation by clopidogrel reduces the likelihood of indirect effects on CYP enzyme activity, which could otherwise alter the metabolism of co-administered drugs.

This study’s findings should be interpreted with caution regarding clinical relevance. In vivo systems involve a complex interplay of metabolic factors that may not be fully represented in the in vitro model used here. Therefore, further research is necessary to validate these observations and assess the broader implications of AKR1D1 and its genetic variations on drug metabolism as well as their potential impact on clinical outcomes particularly in the context of personalized medicine.

This study provides foundational insights into AKR1D1’s interactions with clopidogrel and its metabolite, suggesting that they do not directly influence AKR1D1 activity or expression. However, further research, particularly in vivo and genetic studies, is necessary to fully elucidate AKR1D1’s role in drug metabolism and its potential implications for clinical practice.