Simvastatin (SIM)-induced myopathy is a dose-dependent adverse drug reaction (ADR) that has been reported to occur in 18.2% of patients receiving a 40- to 80-mg dose. The pharmacokinetics of SIM hydroxy acid (SIMA), the bioactive form of SIM, and the occurrence of SIM-induced myopathy are linked to the function of the organic anion transporting polypeptide (Oatp) hepatic uptake transporters. Genetic polymorphisms in SLCO1B1, the gene for human hepatic OATP1B1, cause decreased elimination of SIMA and increased risk of developing myopathy. Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease, and is known to alter drug transporter expression and drug disposition. The purpose of this study was to assess the metabolism and disposition of SIM in a diet-induced rodent model of NASH. Rats were fed a methionine- and choline-deficient diet for 8 weeks to induce NASH and SIM was administered intravenously. Diet-induced NASH caused increased plasma retention and decreased biliary excretion of SIMA due to decreased protein expression of multiple hepatic Oatps. SIM exhibited increased volume of distribution in NASH as evidenced by increased muscle, decreased plasma, and no change in biliary concentrations. Although Cyp3a and Cyp2c11 proteins were decreased in NASH, no alterations in SIM metabolism were observed. These data, in conjunction with our previous data showing that human NASH causes a coordinated downregulation of hepatic uptake transporters, suggest that NASH-mediated transporter regulation may play a role in altered SIMA disposition and the occurrence of myopathy.
Statins are a widely prescribed and relatively safe class of drugs used to treat hyperlipidemia and reduce the risk of heart disease and stroke (Bruckert et al., 2005; Fernandez et al., 2011). The most common adverse drug reactions (ADRs) related to statin therapy are the musculoskeletal toxicities, broadly termed myopathy, which range from myalgia to potentially fatal rhabdomyolysis (Thompson et al., 2006). Although myopathy is usually not life-threatening, the cost to the healthcare system is concerning because of the resources required for management of this adverse event (Fernandez et al., 2011). In addition, the cost to a patient’s health is potentially great because it has been estimated that up to 60% of elderly patients prescribed a statin will discontinue therapy within 2 years, at least partially due to myopathy, thus placing them at increased risk of heart disease and stroke (Fernandez et al., 2011). Simvastatin (SIM) is one statin for which multiple risk factors for myopathy have been identified, including dose, genetic variation in hepatic uptake transporter organic anion transporting polypeptide-1B1 (OATP1B1), and hepatic impairment (Thompson et al., 2006; Link et al., 2008; Fernandez et al., 2011; Watson and McLeod, 2011).
Pharmacogenetic analyses have identified a single nucleotide polymorphism (SNP) (rs4149056) in SLCO1B1, the gene for OATP1B1, that increases systemic exposure of simvastatin hydroxy acid (SIMA), the bioactive metabolite of SIM, and increases the risk of SIM-induced myopathy (Wilke et al., 2012). It was shown that the presence of two rs4149056 alleles caused a 221% increase in SIMA area under the curve (AUC) and a 200% increase in SIMA maximum plasma concentration (Cmax) (Pasanen et al., 2006). In patients taking 80 mg of simvastatin daily, the odds ratio for myopathy per copy of rs4149056 was 4.5, with greater than 60% of the myopathy cases attributable to the SNP (Link et al., 2008). These rs4149056-associated changes in SIMA pharmacokinetics and increased risk of myopathy led to publication of dose adjustment guidelines in the context of this SNP by the Clinical Pharmacogenomics Implementation Consortium (Wilke et al., 2012).
Although genetic variation clearly plays a role in the occurrence of ADRs, other factors such as liver diseases are also known to alter drug pharmacokinetics and contribute to the occurrence of ADRs (Verbeeck, 2008; Merrell and Cherrington, 2011; Gandhi et al., 2012; Naik et al., 2013). Liver diseases that impair hepatic function may require dose adjustments to maintain drug concentrations within the therapeutic window and avoid ADRs (Delcò et al., 2005; Verbeeck, 2008). Nonalcoholic steatohepatitis (NASH) is one such liver disease that is becoming increasingly prevalent (up to 17% of US adults) and has been shown to cause alterations in drug transporters and drug disposition (McCullough, 2006; Ali and Cusi, 2009; Hardwick et al., 2011, 2012; Lake et al., 2011; Merrell and Cherrington, 2011). We previously showed that altered expression of multiple hepatic Oatp transporters in rats caused increased plasma concentrations and decreased biliary excretion of sulfobromophthalein, a dye used in liver function tests (Fisher et al., 2009). We have also shown that NASH in human liver samples caused a coordinated downregulation of hepatic uptake transporters (Lake et al., 2011). These findings in NASH and the results presented here suggest that other Oatp substrates, such as the statins, may also exhibit altered disposition in NASH patients and increase the risk of statin-induced myopathy.
Materials and Methods
SIM and SIMA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Simvastatin-d6 (SIM-d6) and simvastatin-d6 hydroxy acid (SIMA-d6) were purchased from Toronto Research Chemicals (Toronto, ON, Canada). Ammonium acetate and urethane were purchased from Sigma-Aldrich (St. Louis, MO). Acetonitrile, methanol, and high-performance liquid chromatography–grade water were purchased from Thermo Fisher Scientific (Pittsburg, PA). The Oasis HLB μElution 96-well plates were purchased from Waters Corporation (Milford, MA).
Eight-week-old male Sprague-Dawley rats were purchased from Harlan Laboratories (Indianapolis, Indiana). Rats were maintained in 12-hour light and 12-hour dark cycles in a University of Arizona animal facility and were allowed water and standard laboratory diet ad libitum during the 1-week acclimation period. All animal treatments were performed in accordance with protocols approved by the University of Arizona’s Institutional Animal Care and Use Committee. After acclimation, rats were divided into two groups and fed either a methionine- and choline-deficient (MCD) diet or methionine- and choline-replete (control) diet from Dyets Inc. (Bethlehem, PA). After 8 weeks of feeding, animals were anesthetized with a 1.2 g/kg intraperitoneal dose of urethane (5 ml/kg). During anesthesia, animal body temperature was monitored and maintained at 37°C with a TCAT-2V heat pad from Physitemp Instruments Inc (Clifton, NJ). The carotid artery and jugular vein were cannulated with PE-50 polyethylene tubing and the common bile duct was cannulated with PE-10 polyethylene tubing from Braintree Scientific (Braintree, MA). SIM lactone was administered into the carotid artery at a dose of 1 mg/kg (5 ml/kg) in 10% ethanol, 45% propylene glycol, 15% glycerine, and 30% water. Blood samples were collected from the jugular vein into heparinized tubes 0, 2, 10, 20, 40, 60, 90, and 120 minutes after SIM administration, and bile was collected at 15-minute intervals over the course of the 120-minute experiment. Whole blood was centrifuged at 10,000 rpm for 1 minute to collect plasma. After the final blood and bile collections, the rats were euthanized, and liver, kidney, and soleus tissues were collected. A slice of liver was taken and fixed in formalin at 4°C overnight followed by 70% ethanol and embedded in paraffin for hematoxylin and eosin staining. Kidney, soleus, and the remaining liver tissue were flash-frozen in liquid nitrogen and stored at −80°C.
SIM and SIMA quantification was performed according to a previously published protocol with slight modifications for extraction from other biologic matrices (Yang et al., 2005). For plasma and bile samples, 25 µl of each sample was mixed with 10 µl of 100 mM ammonium acetate (pH 4.5) and 2 µl of internal standards simvastatin-d6 and simvastatin-d6 hydroxy acid (6.2 µg/ml). Samples were centrifuged at 10,000 rpm for 5 minutes at 4°C. Ninety-six–well Waters Oasis HLB µElution plates were conditioned sequentially with 200 µl of acetonitrile and 200 µl of methanol followed by equilibration with 200 µl of water. Thirty microliters of the supernatant was applied to a preconditioned and equilibrated Oasis HLB µElution plate (30 μm). For tissues, an entire soleus or approximately 250 mg of liver or kidney was weighed and cut into small pieces with scissors. Tissues were homogenized in 600 µl of 100 mM ammonium acetate (pH 4.5) with a Dounce homogenizer. The homogenate was transferred to a microcentrifuge tube, vortexed, and frozen at −80°C to ensure complete lysis of the cells. Samples were then thawed and centrifuged at 10,000 rpm for 5 minutes at 4°C. Three-hundred microliters of the homogenate were removed, mixed with 2 µl of internal standards simvastatin-d6 and simvastatin-d6 hydroxy acid (6.2 µg/ml), and 250 µl was applied to a preconditioned and equilibrated HLB plate. The above-mentioned volumes of prepared biologic matrices were applied to the plate followed by two sequential washes with 400 µl of 5% methanol. Samples were eluted with 50 µl of 95/5 (v/v) acetonitrile and water followed by dilution with 50 µl of 1 mM methyl ammonium acetate (pH 4.5). Samples in the autosampler were maintained at 10°C, and 10 µl was injected onto the column. Ultra-performance liquid chromatography was performed with a Waters Acquity system connected to a Waters Acquity BEH C18 1.7 µm 2.1 × 50 mm column maintained at 40°C. An isocratic mobile phase consisting of 80% acetonitrile and 20% 1 mM methyl ammonium acetate (pH 4.5) was used to separate the compounds at a flow rate of 0.3 ml/min. The eluent from the column was delivered to a Waters Micromass Quattro Premier XE tandem mass spectrometer that operated in polarity switching mode to detect ions in both positive and negative mode. Compounds were quantified by multiple reaction monitoring of precursor-product ion transitions in positive mode at m/z 450 to 303 (SIM) and 456 to 285 (SIM-d6), and negative mode at 435 to 319 (SIMA) and 441 to 319 (SIMA-d6).
RNA isolation and mRNA quantification were performed as previously described with specific probes for Cyp2c11, Cyp3a1/23, Oatp1b2, Oatp1a1, and Oatp1a4 (Hardwick et al., 2012).
Liver whole-cell lysates were collected as previously described (Hardwick et al., 2012). Membrane proteins were isolated from a portion of the whole-cell lysate by ultracentrifugation at 100,000g for 60 minutes. The supernatant was discarded, and the pellet was resuspended in 0.1 M Tris buffer with Complete Protease Inhibitor Cocktail (Roche, Indianapolis, IN) added per the manufacturer’s instruction. Sixty micrograms of whole-cell lysate or membrane preparation was reduced and denatured in Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA) and 5% 2-mercaptoethanol at 37°C for 30 minutes. Samples were separated on 7.5% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The following antibodies were used to detect each protein: Cyp2c11 (ab3571; Abcam, Cambridge, MA), Cyp3a1 (sc-53246; Santa Cruz Biotechnology), Oatp1b2 (sc-376904; Santa Cruz Biotechnology), Oatp1a1 (OATP11-A; Alpha Diagnostics International, San Antonio, TX), Oatp1a4 (OATP21-A; Alpha Diagnosis International), extracellular signal-regulated kinase 1 (ERK1) (sc-93; Santa Cruz Biotechnology), and pan-cadherin (ab16505; Abcam). Pan-cadherin and ERK1 were used as loading control for blots from membrane and whole preparations, respectively. Images were captured using the Image Laboratory Imaging System (Bio-Rad Laboratories) and relative protein quantities were determined using ImageJ software (National Institutes of Health, Bethesda, MD).
The AUC was calculated using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). SIM clearance (Cl) was calculated using the dose, AUC, and body weight for each animal. SIM volume of distribution (Vd) was calculated by dividing the Cl value by the slope of the linear regression of the logarithm plasma concentration by time plot for each animal.
A t test within GraphPad Prism software was used to determine differences between control and MCD groups. All data represent the mean ± S.E.M.
Altered Disposition of SIM and SIMA in Diet-Induced NASH.
After 8 weeks, animals on the MCD diet had characteristic pathologic features of NASH, including macrovesicular steatosis, fibrosis, and inflammation (Fig. 1), similar to previous reports (Lickteig et al., 2007; Hardwick et al., 2012). To determine how NASH affects hepatobiliary disposition of SIM and SIMA, concentrations of each compound were measured in plasma and bile over the course of the study. The plasma concentrations of SIM were reduced by more than 50% in animals with NASH (Fig. 2A), whereas the biliary excretion was not different between the two groups (Fig. 2B). For SIMA, NASH caused a 160% increase in plasma retention (Fig. 2C) with a concomitant 28% decrease in biliary excretion (Fig. 2D). Further investigation into the pharmacokinetic parameters of SIM indicated that the volume of distribution was increased 360% in NASH animals (Table 1), potentially due to increased muscle concentrations of SIM (Fig. 3). Within the kidney, SIMA concentrations were approximately 3-fold higher in animals who received the MCD diet (Fig. 3). The higher SIMA plasma concentrations (Fig. 2C) and lower liver concentrations (Fig. 3) caused a 91% lower SIMA liver to plasma ratio in NASH animals, whereas this ratio for SIM did not reach statistical significance (Fig. 4). Together, these data indicate that NASH impaired the uptake of SIMA into the liver.
OATP Expression in Diet-Induced NASH.
Hepatic Oatp uptake transporter mRNA and protein expression was measured by branched DNA and Western blot, respectively. Oatp1b2 and Oatp1a1 mRNA expression was significantly reduced by 52% and 46%, respectively, in NASH livers, whereas the reduction in Oatp1a4 mRNA did not reach statistical significance (Fig. 5A). At the protein level, expression of Oatp1b2, Oatp1a1, and Oatp1a4 was significantly reduced in NASH livers by 30%, 39%, and 83%, respectively (Fig. 5B). These data suggest that the decrease in hepatic uptake transporters is responsible for the NASH-induced increase in plasma concentrations, decreased biliary elimination, and decreased liver concentrations of SIMA.
SIM Metabolism Is Not Altered in NASH.
To determine whether NASH altered the metabolism of SIM, we measured the mRNA and the protein expression of Cyp2c11 and Cyp3a1/23 and used the AUC values for SIM and SIMA in bile and plasma. Diet-induced NASH caused a dramatic 92% and 87% decrease in Cyp2c11 and Cyp3a1/23 mRNA, respectively (Fig. 6A). At the protein level, diet-induced NASH caused an 87% and 84% decrease in Cyp2c11 and Cyp3a1/23 protein, respectively (Fig. 6A). In spite of these changes in metabolism enzymes, our data do not indicate that SIM metabolism was altered in NASH because the decrease in P450 enzymes in NASH would lead to higher SIM concentrations in these animals, which is opposite to what was observed in the plasma (Fig. 2A). Metabolites other than SIM and SIMA have been reported in rodents (Ishigami et al., 2001) and although we did not directly measure any other metabolites, our data indicate that NASH did not induce differential metabolism because the sums of SIM and SIMA AUCs were not different between the treatment groups in plasma or bile (Fig. 6B).
The incidence of NAFLD and NASH in the United States has increased in recent years and, as our understanding of the disease has grown, the clinical challenges facing this patient population have increased as well. For example, an increasing number of publications show that NASH alters the expression of hepatic transporters and the disposition of multiple xenobiotic substrates, potentially placing NASH into the category of liver diseases that impair hepatic function and require drug dose adjustments (Lickteig et al., 2007; Fisher et al., 2009; Lake et al., 2011; Merrell and Cherrington, 2011; Canet et al., 2012; Clarke and Cherrington, 2012; Hardwick et al., 2012). NAFLD patients are also at increased risk of developing and dying from cardiovascular disease and are thus frequently prescribed lipid-lowering drugs such as statins (Targher et al., 2005, 2006, 2007; Ekstedt et al., 2006; Fan, 2008; Ali and Cusi, 2009; Nseir et al., 2012). In light of our recent data showing decreased expression of hepatic uptake transporters in NASH (Fisher et al., 2009; Lake et al., 2011) and the fact that many of the statins rely heavily on these uptake transporters (Elsby et al., 2012), we undertook the present investigation to determine how an experimental model of NASH affects the metabolism and disposition of SIM and how this may influence the occurrence of statin-induced myopathy.
Active hepatic uptake of SIMA has been estimated to contribute at least 75% of the hepatic extraction ratio (Elsby et al., 2012). There is a high degree of overlap in substrate specificity among the OATP transporters; within the human liver, OATP1B1 and OATP1B3 are the main OATP isoforms responsible for drug uptake (Kalliokoski and Niemi, 2009). Many of the statins are substrates for the different OATP isoforms (Kalliokoski and Niemi, 2009) but data showing that SIMA is a substrate for the OATPs have been challenging, potentially because of its physical properties and methodological difficulties (Elsby et al., 2012). Despite this, there is clear evidence that the SLCO1B1 SNP rs4149056 increases SIMA exposure and the risk of SIM-induced myopathy (Voora et al., 2009; Niemi, 2010; Wilke et al., 2012). The overlap in substrate specificity creates a complex situation in which other OATPs that are fully functional can compensate for the genetic polymorphisms that affect the function of only one transporter. We have shown that human NASH potentially reduces this compensatory transport mechanism through a coordinated decrease in hepatic uptake transporter expression (Lake et al., 2011). The results from the current study show that in the context of diet-induced NASH, there is reduced expression of multiple rodent hepatic Oatp isoforms leading to an increase in SIMA plasma retention, as indicated by increased plasma concentrations, decreased liver concentrations, and decreased biliary excretion. Other data from the literature support this mechanism of compensatory transport of statins by other uptake transporters. In Oatp1b2 knockout mice, the function of Oatp1a transporters in rodents has been suspected to compensate for the loss of Oatp1b2 (Degorter et al., 2012), which may explain why a previous report showed that SIMA did not have an altered liver to plasma ratio in Oatp1b2 knockout mice (Chen et al., 2008). As further evidence for this mechanism, it has been shown that knockout of the entire Oatp1a/1b cluster potentiated the increase in pravastatin plasma exposure compared with Oatp1b2 knockout alone (Zaher et al., 2008; Iusuf et al., 2012). These data indicate that NASH-specific alterations in uptake transporters may have a compounding effect on drug disposition and the occurrence of ADRs because of changes in multiple compensatory transporters.
Our current data also show that SIMA concentrations were higher in the kidneys of rats fed an MCD diet. The mechanism behind this change in disposition is currently unclear but will be the focus of future investigations for drugs that are highly dependent on renal elimination. For the current study, because statins are primarily dependent on hepatobiliary elimination (Shitara and Sugiyama, 2006), it is not anticipated that this change in kidney statin concentrations will influence the metabolism and disposition of SIM.
SIM is administered as the prodrug lactone that must be metabolized into SIMA to elicit its cholesterol-lowering properties. SIM can be metabolized into SIMA and other metabolites through both enzymatic and nonenzymatic pathways (Prueksaritanont et al., 1997). The enzymatic metabolism of SIM into SIMA and several other metabolites is reported to be carried out by the Cyp2c11 isoform in male rats and the Cyp3a isoform in female rats (Ishigami et al., 2001). Nonenzymatic hydrolysis from SIM lactone into SIMA has also been reported to be carried out by esterases and paraoxonases (Prueksaritanont et al., 1997; Neuvonen et al., 2006). In this study, we administered the lactone form of SIM and observed conversion into SIMA in both control and MCD diet-induced NASH animals, in which we observed a sharp decrease in Cyp2c11 and Cyp3a1/23 expression. If these changes in P450 enzyme levels affected the metabolism of SIM, then it would be expected that NASH animals would have much higher SIM levels and lower SIMA levels in plasma. Our data show exactly the opposite result, suggesting that P450-mediated metabolism does not account for the differences in SIM and SIMA concentrations observed in our study. In addition, our data indicate that SIM was not metabolized differentially in control and MCD animals into alternate metabolites since the sums of the AUCs for SIM and SIMA in plasma or bile were not different between the treatment groups. Changes in P450 expression and activity are common in liver diseases, and we have previously shown that the expression and activity of multiple human P450 isoforms are altered in human NASH. The mechanism behind these changes in P450s and impact of these changes in human populations requires further investigation. These data indicate that MCD diet-induced NASH did not alter the metabolism of SIM and, therefore, the differences observed in SIMA concentrations are a result of transporter-mediated processes.
SIM is a lipophilic compound that is not dependent on active transport to cross membrane barriers (Pasanen et al., 2006). The lipophilic nature of SIM has also been reported to contribute to its larger volume of distribution compared with SIMA (Prueksaritanont et al., 2005). In this study, we observed that MCD diet-induced NASH caused an increase in the volume of distribution for SIM. This change in volume of distribution was caused by SIM more readily partitioning into the muscle of NASH animals, thus decreasing the plasma concentrations without affecting the amount of SIM excreted into the bile. These results are particularly striking since SIM-induced myopathy is a dose-dependent ADR that occurs in the muscle, suggesting that the altered disposition of SIMA in NASH may increase the risk of SIM-induced myopathy. Although further investigation into the cause of this increased volume of distribution is needed, these data clearly indicate that NASH alters the disposition of SIM and may contribute to the occurrence of SIM-induced myopathy.
The work presented here further highlights the need for more directed investigation into how liver disease affects SIMA disposition and the occurrence of SIM-induced myopathy, especially since SIM and other statins have been proposed as treatment options for NAFLD and NASH (Nseir et al., 2012). To date, the small preliminary investigations for SIM have concluded that it is safe in patients with NAFLD or NASH. Unfortunately, these studies only considered SIM-induced hepatotoxicity but did not evaluate, nor did they have the statistical power to detect, an increased risk of myopathy in NASH (Abel et al., 2009; Nelson et al., 2009). Furthermore, one of these studies looked at NAFLD broadly defined as elevated liver enzyme levels in the absence of other kinds of liver diseases rather than NASH specifically. Multiple previous reports suggest altered drug disposition and dose-dependent ADRs may occur only following the development of NASH, highlighting the need for well designed studies that will examine the safety of SIM specifically in patients diagnosed with NASH (Fisher et al., 2009; Lake et al., 2011). Conducting these studies will be challenging due to the difficulty in obtaining a liver biopsy, which continues to be the only effective method of NASH diagnosis (Mofrad et al., 2003; Verbeeck, 2008). Collectively, the data shown here provide further impetus for the design of specific clinical trials that will evaluate the disposition of SIMA and the occurrence of SIM-induced myopathy in NASH patients.
Participated in research design: Clarke, Hardwick, Cherrington.
Conducted experiments: Clarke, Hardwick, Lake, Canet.
Contributed new reagents or analytic tools: Hardwick.
Performed data analysis: Clarke.
Wrote or contributed to the writing of the manuscript: Clarke, Hardwick, Lake, Canet, Cherrington.
- Received November 14, 2013.
- Accepted January 6, 2014.
This work was supported by the National Institutes of Health Eunice Kennedy Shriver National Institute of Child Health and Human Development [Grant HD062489]; the National Institutes of Health National Institute of Allergy and Infectious Diseases [Grant AI083927]; and the National Institutes of Health National Institute of Environmental Health Sciences [Toxicology Training Grant T32-ES007091].
- adverse drug reaction
- area under the curve
- extracellular signal-regulated kinase 1
- methionine and choline deficient
- nonalcoholic fatty liver disease
- nonalcoholic steatohepatitis
- organic anion transporting polypeptide
- simvastatin hydroxy acid
- simvastatin-d6 hydroxy acid
- single nucleotide polymorphism
- volume of distribution
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics