Nuclear Receptor PXR in Drug-Induced Hypercholesterolemia

Atherosclerosis is a major global health concern. The central modifiable risk factors and causative agents of the disease are high total and low-density lipoprotein (LDL) cholesterol. To reduce morbidity and mortality, a thorough understanding of the factors that influence an individual’s cholesterol status during the decades when the arteria-narrowing arteriosclerotic plaques are forming is critical. Several drugs are known to increase cholesterol levels; however, the mechanisms are poorly understood. Activation of pregnane X receptor (PXR), the major regulator of drug metabolism and molecular mediator of clinically significant drug–drug interactions, has been shown to induce hypercholesterolemia. As a major sensor of the chemical environment, PXR may in part mediate hypercholesterolemic effects of drug treatment. This review compiles the current knowledge of PXR in cholesterol homeostasis and discusses the role of PXR in drug-induced hypercholesterolemia.


Introduction
Hypercholesterolemia, e.g., elevated LDL cholesterol (LDL-C) and imbalance of LDL-C and high-density lipoprotein cholesterol (HDL-C), is a central causative risk factor of atherosclerosis [1,2]. In fact, the retention of LDL-C and other cholesterol-rich apolipoprotein (Apo) B-containing lipoproteins within the arterial wall represents the key initiating event in atherogenesis [3]. Complications of atherosclerosis, ischemic heart disease and stroke, are among the leading causes of death [4]. In addition to atherosclerosis, excess cholesterol may also be involved in the pathogenesis of other diseases, such as non-alcoholic fatty liver disease and diabetes [5].
Multiple genetic and modifiable factors induce hypercholesterolemia. The most common LDL-C-elevating lifestyle factor is high saturated fat intake, but certain other nutritional factors and obesity also promote hypercholesterolemia [2,3]. Hypercholesterolemia can also be caused by secondary causes, like hypothyroidism [2]. Furthermore, multiple drugs may increase cholesterol. Some drug classes are well-known for their adverse effect on cholesterol, such as antipsychotics and immunosuppressants, but individual drugs from many different classes of drugs have been reported to affect cholesterol levels [2,6]. The mechanisms by which drugs increase cholesterol concentration are still largely unknown.
Nuclear receptor pregnane X receptor (PXR; NR1I2) is a ligand-activated transcription factor that regulates many phase I and II drug-metabolizing enzymes and drug transporters; induction of cytochrome P450 (CYP) 3A4 enzyme is a prime example [7]. Indeed, PXR is an important mediator of the induction type drug-drug interactions [8]. Unlike most other nuclear receptors, PXR accepts a wide array of structurally diverse chemicals as ligands. Many modern synthetic chemicals among environmental contaminants, industrial chemicals, and drugs are well-characterized ligands for PXR. In addition to the classical role in drug metabolism, PXR has been shown to play a role in other biological processes, such as inflammation, cellular proliferation, and glucose and lipid metabolism [9,10]. Due marker of CYP3A4 activity [31] known to be elevated by PXR activation [11], correlated with these changes. The elevations of HDL-C (4%) and total ApoB (5%) by rifampicin were non-significant after correction for multiple testing, while the largest very low-density lipoprotein (VLDL) fractions tended to decrease [30]. Serum concentration of ApoB48, the intestinal form of ApoB, was decreased 14% by rifampicin dosing. During glucose challenge (2 h oral glucose tolerance test), PXR activation by rifampicin resulted in elevated concentrations of very small VLDL particles, remnant cholesterol, and total ApoB [30]. As in a previous study [24], rifampicin elevated serum lathosterol-to-cholesterol ratio [30], and, on the other hand, decreased serum concentrations of cholesterol synthesis precursors citrate and acetate, both suggesting increased cholesterol synthesis.
Another interesting PXR agonist with a strong hypercholesterolemic effect and some human mechanistic evidence on the induction of cholesterol synthesis is mitotane, a steroidogenesis inhibitor used in Cushing's syndrome and a cytostatic treatment for adrenocortical carcinoma. Although its PXR-activating properties and high risk of drug-drug interactions were revealed only recently [32,33], mitotane has long been recognized as having a remarkable cholesterol-elevating effect. An early study on patients with Cushing's syndrome demonstrated a 59% increase in serum TC and an 81% increase in LDL-C, with an accompanying 61% increase in ApoB concentration [34]. No significant effects on HDL-C were detected in this small study (n = between 6 and 21). In a recent study in patients with adrenocortical carcinoma (n = 39), mitotane treatment led to 43% increase in TC and 69% increase in LDL-C after 6-month dosing [35]. Additionally, HDL-C was elevated by 14%. Already in 1961, mitotane was suspected to induce cholesterol synthesis as indicated by an increased rate of incorporation of l4 C-acetate into plasma cholesterol of patients [36]. Mitotane also elevates plasma levels of mevalonate, a marker of cholesterol synthesis [34].
These findings indicate that (1) drugs with PXR agonism elevate TC and LDL-C and that (2) increased cholesterol synthesis may play a role in drug-induced and PXRmediated hypercholesterolemia. Genetic studies provide additional evidence, as PXR gene polymorphisms associate with plasma LDL-C levels [37].

Mechanisms of PXR-Induced Hypercholesterolemia
PXR is primarily expressed in the liver and intestine, which are the central tissues for drug metabolism but also those for cholesterol homeostasis. Several intestinal and hepatic mechanisms have been identified that may confer the hypercholesterolemic effect of PXR activation. However, the mechanistic evidence has been mainly gathered utilizing murine models, whose translational value is often limited by substantial differences in murine and human lipoprotein homeostasis. Mice lack cholesterol ester transfer protein (CETP), which leads to high HDL-C and very low LDL-C levels. Nevertheless, the fundamental mechanisms controlling cholesterol synthesis and several other steps in cholesterol homeostasis in humans and mice are similar, and the current evidence indicates important similarities in the mechanisms controlling PXR-mediated elevation of cholesterol in humans and mice. It should be kept in mind that the PXR ligand preference is species-specific and therefore the compound-specific results cannot be directly transferred from mouse or rat experiments to humans.

PXR in Cholesterol Synthesis
In mammals, almost all cells can synthesize cholesterol, but plasma cholesterol is only affected by cholesterol synthesis in the liver due to its central position in lipoprotein metabolism [38,39]. In the liver, as well as in other organs, the regulation of cholesterol synthesis is predominantly determined by the activity of the transcription factor sterolregulatory element-binding protein 2 (SREBP2) (Figure 1) [40]. tabolism [38,39]. In the liver, as well as in other organs, the regulation of cholesterol synthesis is predominantly determined by the activity of the transcription factor sterol-regulatory element-binding protein 2 (SREBP2) (Figure 1) [40].

Figure 1. Activation of PXR stimulates cholesterol synthesis and induces
Pcsk9 in the liver. The processes and molecules upregulated and downregulated by PXR activation have been indicated with blue and red coloring in the figure, respectively. PXR activation leads to nuclear accumulation of SREBP2, which consequently induces cholesterogenic genes, including the rate-limiting enzyme HMGCR, and thus induces cholesterol synthesis. Furthermore, PXR activation directly induces Sqle to increase the rate of squalene epoxidation. Induction of DHCR24 enzyme targets cholesterol synthesis flux to the Kandutsch-Russell pathway instead of the Bloch pathway. PXR activation appears to bypass the usual negative feedback mechanism controlling cholesterol synthesis, including inhibition of SREBP2-SCAP complex translocation from the ER to the Golgi apparatus by high cellular cholesterol levels and desmosterol, as well as the inhibitory effect of cholesterol on SQLE activity. Accumulation of plasma LDL-C level is potentiated by induction of the hepatic Pcsk9 gene and a consequent increase in circulating PCSK9 levels.
Inactive SREBP2 resides in the endoplasmic reticulum (ER) in a complex with SREPBcleavage activating protein (SCAP) and insulin-induced gene 1 (INSIG1), in which the former functions as a sterol sensor and the latter as a negative regulator of SREBP2 activation [40]. Sterol depletion in the ER incites conformational changes to SCAP, which are required for the translocation of the SREBP2-SCAP complex to the Golgi apparatus. There, SREBP2 is proteolytically cleaved to yield active SREBP2 monomers, which homodimerize, translocate to the nucleus and induce gene expression ( Figure 1). In the nucleus, SREBP2 induces genes for cholesterol synthesis, including the rate-controlling enzyme HMGCR. Repletion of sterols in the ER represses SCAP, thus forming a feedback loop to control cholesterol synthesis. In addition to fluctuating ER sterol levels, SREBP2 The processes and molecules upregulated and downregulated by PXR activation have been indicated with blue and red coloring in the figure, respectively. PXR activation leads to nuclear accumulation of SREBP2, which consequently induces cholesterogenic genes, including the rate-limiting enzyme HMGCR, and thus induces cholesterol synthesis. Furthermore, PXR activation directly induces Sqle to increase the rate of squalene epoxidation. Induction of DHCR24 enzyme targets cholesterol synthesis flux to the Kandutsch-Russell pathway instead of the Bloch pathway. PXR activation appears to bypass the usual negative feedback mechanism controlling cholesterol synthesis, including inhibition of SREBP2-SCAP complex translocation from the ER to the Golgi apparatus by high cellular cholesterol levels and desmosterol, as well as the inhibitory effect of cholesterol on SQLE activity. Accumulation of plasma LDL-C level is potentiated by induction of the hepatic Pcsk9 gene and a consequent increase in circulating PCSK9 levels.
Inactive SREBP2 resides in the endoplasmic reticulum (ER) in a complex with SREPBcleavage activating protein (SCAP) and insulin-induced gene 1 (INSIG1), in which the former functions as a sterol sensor and the latter as a negative regulator of SREBP2 activation [40]. Sterol depletion in the ER incites conformational changes to SCAP, which are required for the translocation of the SREBP2-SCAP complex to the Golgi apparatus. There, SREBP2 is proteolytically cleaved to yield active SREBP2 monomers, which homodimerize, translocate to the nucleus and induce gene expression ( Figure 1). In the nucleus, SREBP2 induces genes for cholesterol synthesis, including the rate-controlling enzyme HMGCR. Repletion of sterols in the ER represses SCAP, thus forming a feedback loop to control cholesterol synthesis. In addition to fluctuating ER sterol levels, SREBP2 activity is regulated by several modulators of cellular energy metabolism [41]. For instance, factors affecting INSIG1 expression may affect SREBP2 activation, and SREBP2 is regulated by several post-translational modifications conveyed by hormonal signals and signaling cascades, such as insulin and mammalian target of rapamycin (mTOR). Thus, the need to adjust cholesterol synthesis rates may rise from multiple sources, and cholesterol synthesis presents a highly adaptable biological phenomenon.
Our recently published results indicate that rifampicin treatment increases human plasma cholesterol by activating PXR and hepatic cholesterol synthesis [30]. The same cholesterol synthesis-inducing effect could be repeated also in high-fat diet-fed mice, which were treated with selective murine PXR agonist, pregnenolone-16α-carbonitrile (PCN). This enabled us to study the molecular mechanisms in more detail. In the liver, PXR activation led to nuclear accumulation of active SREBP2 protein, widespread induction of its target genes, and increases in cholesterol and markers of cholesterol synthesis [30].
INSIG1 inhibits SREBP2 activation by retaining the SREBP2-SCAP complex in the ER [42]. PXR has been shown to transcriptionally induce Insig1 mRNA [43]. With this in mind, the stimulation of SREBP2 activity by PXR was an unexpected finding [30]. We also detected increased Insig1 mRNA expression in response to PXR activation but were unable to detect increased INSIG1 protein expression [30]. In their study, Roth and colleagues [43] did not report the INSIG1 protein level; furthermore, the study did not evaluate the effect of INSIG1 on the activity of SREBP2 but SREBP1, an SREBP isoform mainly controlling triglyceride synthesis [40]. SREBP1 inhibition by PXR activation is in line with studies describing how PXR induces hepatic steatosis independent of SREBP1 [44,45]. Altogether, these findings suggest that PXR may affect INSIG1 translation or protein degradation and selectively affect SREBP1 and SREBP2 activities. In addition to INSIG1, PXR has been shown to suppress fibroblast growth factor 21 (FGF21), a negative regulator of SREBP2 expression [46,47], which may play an additional role in PXR-stimulated SREBP2 activity, although Srebp2 mRNA expression remained unaffected in our experiments.
Cholesterol is synthesized from acetyl-CoA, which undergoes multiple enzymatic reactions to form squalene, subsequently converted to (S)-2,3-epoxysqualene by squalene epoxidase (SQLE) and further to lanosterol, which is converted to cholesterol either in the Bloch or in the Kandutsch-Russell pathway [48,49]. Recently, an antiretroviral efavirenz was shown to induce cholesterol synthesis in mice by activating PXR and inducing hepatic Sqle, a novel PXR target gene [50]. Importantly, the effect of efavirenz on cholesterol was abolished in mice lacking hepatic PXR. In our study, PCN treatment induced hepatic Sqle among other genes of cholesterol synthesis [30]. Furthermore, all PCN-treated mice had lower liver squalene levels than controls, which possibly indicated faster squalene metabolism and increased SQLE activity. Interestingly, besides HMGCR, SQLE is another rate-limiting enzyme of cholesterol synthesis and cholesterol constitutes its inhibitory feedback signal, in addition to downregulation of proteolytic processing of SREBP2, also by directly inhibiting SQLE activity [51,52]. Besides the direct PXR mediated regulation [50], distortion of normal inhibitory regulation of SREBP2 by PXR activation may in part account for the induction of Sqle gene expression [30].
We showed that increased cholesterol synthesis due to PXR activation was caused by the induction of the Kandutsch-Russell pathway, as evidenced by increased plasma and hepatic markers of the pathway, lathosterol and zymostenol, and induced DHCR24, an enzyme that directs cholesterol synthesis to the Kandutsch-Russell pathway [30]. PXR did not affect desmosterol, a marker of the Bloch pathway and a negative regulator of SREBP2 [53], meaning that by inducing the Kandutsch-Russell pathway instead of the Bloch pathway PXR may evade normal SREBP2 downregulation by desmosterol ( Figure 1). Overall, these results are in line with the previous reports regarding PXR and cholesterol synthesis and, most importantly, with the human findings, and suggest that PXR activation stimulates hepatic SREBP2 activity with harmful effects on circulating atherogenic lipids.

PCSK9 Induction by PXR Activation
Identification of proprotein convertase subtilisin kexin-type 9 (PCSK9) and development of PCSK9 inhibitors for drug therapy has remarkably improved the understanding of plasma cholesterol regulation [54,55]. PCSK9 is secreted from the liver to the circulation, where it functions to induce the degradation of hepatic LDL receptors. This results in decreased LDL clearance and increased circulating LDL. Inhibition of plasma PCSK9 by antibodies has proven to be an efficient therapeutic strategy to lower plasma LDL [56].
In the liver, the expression of Pcsk9 is regulated by SREBP2 [57]. As PXR activation was shown to stimulate SREBP2 activity, this raised the hypothesis that PXR could also induce Pcsk9 expression. Indeed, this was found to be the case in both mice and humans [30]. LDL receptor is another SREBP2 target gene and could counteract the negative effect of PCSK9. Expectedly, the LDL receptor mRNA was also induced by PXR activation in mouse liver; however, the response was very minor compared with the Pcsk9 response [30].
Statins stimulate SREBP2 activity and Pcsk9 expression as a consequence of cholesterol synthesis inhibition and lower sterol content in the hepatocyte ER. Interestingly, lipophilic statins (atorvastatin, simvastatin) have been shown to increase PCSK9 more efficiently than hydrophilic statins (rosuvastatin, pravastatin) [58]. Atorvastatin and simvastatin are also PXR ligands, and it could be speculated that, in addition to the decreased cellular sterol content, the PXR agonism plays an additional role in the PCSK9 increase by these statins.
The current list of drugs that induce PCSK9 include statins, fibrates, mTOR inhibitors, nilotinib, and rifampicin [30,[59][60][61][62]. The mechanisms are probably diverse but given the recently discovered role of PXR in Pcsk9 regulation, further research on the effects of PXR agonists on Pcsk9 expression is warranted. Of the currently known PCSK9-inducing drugs, several (atorvastatin, simvastatin, rifampicin, and nilotinib) are PXR ligands, although they involve also other cellular mechanisms.

PXR in Intestinal Cholesterol Absorption
Intestinal absorption of biliary and dietary cholesterol is an important contributor to hypercholesterolemia, and ezetimibe or food additives (plant stanols or sterols) that limit intestinal cholesterol absorption decrease circulating cholesterol [63,64]. Interestingly, Niemann-Pick C1-like 1 (NPC1L1), an intestinal cholesterol transporter protein and the molecular target of ezetimibe, has been shown to be directly regulated by PXR [65]. Tributyl citrate, a common plasticizer, and quetiapine, an atypical antipsychotic with known adverse effects on lipid metabolism, have been shown to induce hypercholesterolemia in mice by activating intestinal PXR, which is associated with increased Npc1l1 expression [50,65]. It is noteworthy that in mice NPC1L1 is expressed only in the intestine, whereas in humans NPC1L1 is also strongly expressed in the liver, which may affect the translational value of these findings [66]. Rifaximin is a gut-specific human PXR activator, which does not increase serum cholesterol in PXR-humanized mice, although it induces intestinal triglyceride absorption [67,68]. However, rifaximin induced hepatic cytochrome P450 7A1 (CYP7A1) enzyme, the gatekeeper of bile acid synthesis, which suggests that increased bile acid synthesis may have protected the mice from hypercholesterolemia.

PXR in Bile Acid Synthesis and Cholesterol Metabolism
Synthesis of bile acids from cholesterol and biliary secretion of bile acids and cholesterol present a major route of excess cholesterol disposal. Increasing bile acid disposal by inhibiting bile acid absorption from the intestine to the circulation by bile acid sequestrants, such as cholestyramine and colesevelam, lowers plasma cholesterol, highlighting the importance of hepatic cholesterol disposal as a regulatory mechanism of circulating cholesterol.
PXR activation has been shown to have a complex role in endogenous bile acid homeostasis. Activation of PXR by some bile acids is critical to accelerate the detoxification of otherwise hepatotoxic bile acids [69][70][71]. Bile acids are detoxified mainly by known PXR target genes; CYP3A enzymes, bile acid conjugation enzymes SULT2A1 and UGTs, and bile acid transporter MRP2 [72,73].
Several studies have reported that PXR activation represses CYP7A1, the rate-limiting enzyme of bile acid synthesis, thus forming a negative feedback loop to regulate bile acid homeostasis [72,[74][75][76]. Although PXR activation represses CYP7A1 in several animal and cell models, rifampicin dosing does not seem to affect CYP7A1 expression in humans [77] and even increases bile acid synthesis, as evidenced by increased serum levels of 7αhydroxy-4-cholestene-3-one, a marker of bile acid synthesis [24,77].

PXR in HDL Homeostasis
HDL is a central factor in reverse cholesterol transport, a mechanism by which tissues and cells get rid of excess cholesterol; HDL functions to transport cholesterol from extrahepatic tissues to the liver and intestine [78]. Although PXR activation appears to have mainly harmful effect on cholesterol homeostasis, there is some evidence that PXR activation could increase HDL-C. For instance, some drugs with PXR-activating properties, such as carbamazepine and phenytoin, appear to increase HDL-C [14,[79][80][81]. Furthermore, we have reported that rifampicin induces CYP3A4-mediated formation of 4β-hydroxycholesterol (4βHC) and, as a liver X receptor (LXR) agonist, 4βHC in turn stimulates cholesterol efflux transporters in macrophages, possibly promoting HDL-C-mediated reverse cholesterol transport [82]. Thus, elevation of circulating 4βHC could partly explain the HDL-C increasing effect of PXR-activating drugs (PXR-4βHC-LXR circuit). For more information on this topic, please see our recent review [11].

Drug-Induced Hypercholesterolemia
Increase in circulating cholesterol during drug treatment is not uncommon, as several antihypertensives, antihyperglycemics, antipsychotics, antiretrovirals, and immunosuppressants may have an unfavorable effect on cholesterol. Many of these drugs are used long-term, increasing the potential for cardiovascular risk. Drug-induced hypercholesterolemia may be directly caused by the drug or secondarily caused by other effects of the drug, such as weight gain. For example, some antipsychotics, antidepressants, anticonvulsants, and hormones may increase weight and negatively affect cholesterol status [83].
To obtain a systematic overview on drug-induced hypercholesterolemia, we identified clinically used drugs that increase TC or LDL-C in the commercially available Drug Laboratory Effects database (Multirec Ltd., Turku, Finland) ( Table 1). To increase the clinical significance of the table, we removed drugs that have been withdrawn from the market or were reported to have both lowering and increasing effects on cholesterol. Furthermore, we added bexarotene, mitotane, and rifampicin to the table, as they have reported effects on cholesterol but were not included in the original database [22,30,35]. Table 1 consists of 106 drugs, of which 48 drugs increase both LDL-C and TC, 54 drugs increase LDL-C, and 100 drugs increase TC. Among the drugs that increase LDL-C, the largest drug classes are immunosuppressants with 15 drugs (28%), antineoplastics with 10 drugs (19%), antipsychotics with eight drugs (15%), and antiretrovirals with six drugs (11%). Together these four classes comprise 72% of the drugs listed.
A similar pattern is seen in the 100 drugs that increase TC ( Table 1): 62% of the drugs that increase cholesterol are either antineoplastic (22 drugs; 22%), immunosuppressants (19 drugs; 19%), antiretrovirals (12 drugs; 12%), or antipsychotics (9 drugs; 9%). In addition, the list of drugs that increase TC include several commonly used glucocorticoids, serotonin reuptake inhibitors, and non-steroidal anti-inflammatory drugs. Naturally, the lists of drugs increasing TC and LDL-C are heavily overlapping. It should be noted that sometimes the original publications used as the source for the database did not report all the relevant cholesterol values. Furthermore, in many cases there exist conflicting data on the effect of drugs on plasma cholesterol levels. Finally, among the drugs that have been reported to increase TC and/or LDL-C, the magnitude of effect and the clinical significance are very variable. Some drug classes are clearly overrepresented in the list of cholesterol-elevating drugs and their effects may seem like a class effect of certain drugs. However, there are still significant differences between the drugs within the therapeutic groups. For instance, a recent meta-analysis described marked differences between atypical antipsychotics in the metabolic side effects, including TC and LDL-C, with olanzapine and clozapine displaying the worst profiles [130]. Furthermore, changing from quetiapine, risperidone, or olanzapine to aripiprazole has been reported to be beneficial for cholesterol status and 10-year cardiovascular disease risk [131]. Notably, these antipsychotics have also significant differences in their target receptor profiles that could potentially play a role in their varying effects on metabolic parameters.
The mechanistic understanding of the molecular mediators of drug-induced dyslipidemia is elusive and the data to date are limited by the lack of sophisticated human studies. The hypercholesterolemic effects of several antipsychotics have been linked to increased activation of SREBP2 due to inhibition of INSIG-2 in murine models [132]. Furthermore, INSIG-2 gene polymorphisms in schizophrenia patients are associated with weight gain and prevalence of metabolic syndrome [133].
Inflammation and drugs that modulate inflammation, non-steroidal anti-inflammatory drugs (NSAIDS), glucocorticoids, and immunosuppressants, are known regulators of cholesterol levels. Acute infections tend to decrease LDL cholesterol, and the more severe the infection, the more pronounced the effects on cholesterol [134,135]. The same seems to apply to chronic inflammatory diseases, as a decrease in HDL and LDL cholesterol is a common finding in cases of rheumatoid arthritis and the reductions in HDL and LDL correlate with disease severity in ankylosing spondylitis [136,137]. As inflammation attenuates during anti-inflammatory drug treatment, cholesterol levels return to normal, which may explain why many anti-inflammatory drugs seem to increase cholesterol [138,139]. However, some drugs used to treat inflammatory diseases may have more specific effects on lipid metabolism.
Interestingly, immunosuppressants used in organ transplant patients, cyclosporin (calcineurin inhibitor) and especially the mTOR inhibitors sirolimus, tacrolimus, and everolimus, increase cholesterol [140,141]. Immunosuppressant-mediated dyslipidemia is common and occurs in 60% of organ transplant patients [142]. The mechanisms by which immunosuppressants induce dyslipidemia are not well understood, but they seem to involve LDL metabolism and PCSK9 [143,144]. mTOR is a central regulator of lipid metabolism and SREBP, which already hints at mechanisms of mTOR inhibitor-induced dyslipidemia [145]. Antiretroviral therapies have improved the life expectancy of human immunodeficiency virus (HIV)-infected patients. This success has brought up HIV-associated risks for other comorbidities, including increased risk for cardiovascular diseases [146,147]. One significant contributing factor is the adverse effect of antiretrovirals on lipid metabolism [146]. Protease inhibitors, non-nucleoside reverse transcriptase inhibitors, and integrase inhibitors may all increase TC or LDL-C [148]. Most of the approved protease inhibitors and about a quarter of the approved reverse transcriptase inhibitors are included in the list of drugs that increase cholesterol (Table 1). Mechanistically, protease inhibitors have been shown to stimulate SREBP activity in the liver [148].

Identification of PXR Ligands among the Drugs Inducing Hypercholesterolemia
To gain a systematic overview of the potential role of PXR in drug-induced hypercholesterolemia, we identified in the literature the PXR-activating drugs among the drugs that increase TC or LDL-C (Table 1).
Only studies that provided direct evidence of PXR binding were considered. For instance, induction of classical PXR target genes, CYP3A and CYP2C, was not considered a proof of PXR agonism, as the expression of these genes is also regulated by other nuclear receptors. PXR activation is often evaluated based on CYP induction and consequent drug-drug interactions. However, this may not always reveal actual PXR activation status, as some inducers are also CYP inhibitors [149]. Indeed, several drugs that are PXR agonists also inhibit CYP3A4 activity. In these cases, the other effects of PXR activation could still be significant. On the other hand, the results of reporter gene assays or other in vitro methods may overestimate the receptor activation, especially if performed only with high ligand concentrations.
Among the 106 drugs that increase either LDL-C or TC, 23 (21.7%) were identified as PXR activators, and three, brigatinib, apalutamide, and ruxolitinib, as likely activators. Thus, altogether, of the 106 drugs that increase cholesterol levels, 26 (24.5%) are most likely to have PXR-activating property. Among the drugs that increase LDL cholesterol but have not been identified as PXR agonists, clozapine, and nevirapine have been shown to have some inducing effect on CYP3A4, but only nevirapine is a clinically significant CYP3A4 inducer. Among the drugs that increase TC, 10 drugs have not been identified as PXR agonists but induce CYP3A4.
PXR and its sister receptor constitutive androstane receptor (CAR) both regulate drug metabolism but also glucose and lipid metabolism and also share some ligands. However, while PXR activation is detrimental to metabolic performance, CAR activation has been shown to be beneficial. Of importance, CAR activation has been shown to lower cholesterol by increasing fecal cholesterol disposal and by suppressing cholesterol synthesis [21,150].

Conclusions
A large number of drugs have been reported to display adverse effects on plasma cholesterol level. For many, the effect size is rather small, but there is still a significant number of drugs for which the effect is considered clinically significant. In long-term treatment, the unfavorable effect of drug therapy on cholesterol may predispose to atherosclerosis, especially if combined with other risk factors.
Recent studies have indicated that activation of nuclear receptor PXR increases plasma cholesterol and several potential mechanisms have been reported, including increased hepatic cholesterol synthesis, induction of PCSK9, increased intestinal absorption, and decrease of bile acid synthesis. In humans, there is currently evidence for the first two of these mechanisms. So far, rifampicin, efavirenz, and quetiapine have been reported to induce hypercholesterolemia through PXR-mediated mechanisms, either in humans or mice [30,50,96]. Among the known cholesterol-increasing drugs, about a quarter are certain or possible PXR ligands. Thus, PXR activation is likely to at least partly mediate their hypercholesterolemic effects but verifying the role of PXR requires further studies in each case in the future.

Data Availability Statement:
The data presented in this study are available within this article.