Food Effect on Rosuvastatin Disposition and Low-Density Lipoprotein Cholesterol

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1 Food Effect on Rosuvastatin Disposition and Low-Density Lipoprotein Cholesterol Cheynne C. McLean 1,2, Wendy A. Teft 1, Bridget L. Morse 1, Steven E. Gryn 1, Robert A. Hegele 3 and Richard B. Kim 1,2 ARTICLE Rosuvastatin is commonly prescribed for the treatment of hypercholesterolemia and hepatic transporter-mediated accumulation in the liver enhances its efficacy. Current guidelines indicate no preference for fed or fasted rosuvastatin administration. We investigated the association between food intake and rosuvastatin disposition in healthy subjects and lowdensity lipoprotein cholesterol (LDL-C)-lowering effects among patients taking rosuvastatin. We demonstrate that administration with food resulted in a near 40% reduction of rosuvastatin exposure in healthy Asian (n 5 12) and Caucasian (n 5 11) subjects. Higher rosuvastatin concentrations in Asian subjects also correlated with higher allele frequency of ABCG2 c.421c>a. In mice, a greater rosuvastatin liver:plasma ratio was noted when administered with food. Among rosuvastatin patients (n 5 156), there was no difference in dose needed to reach target LDL-C, measured LDL-C, or lathosterol concentrations, when administered in a fed or fasting state. Therefore, taking rosuvastatin with food could reduce systemic concentrations, and subsequent myopathy risk, without compromising LDL-C-lowering benefit. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? þ The current guidelines for rosuvastatin, commonly prescribed to treat hypercholesterolemia, indicate no preference for fed or fasted administration. Statin-induced myopathy has been correlated with increased statin plasma concentration. WHAT QUESTION DID THIS STUDY ADDRESS? þ This study investigated the effect of concomitant food intake on rosuvastatin plasma exposure in Caucasian and Asian Canadian healthy volunteers and in mice. WHAT THIS STUDY ADDS TO OUR KNOWLEDGE þ We demonstrated that rosuvastatin exposure is significantly reduced in Caucasian and Asian subjects when administered with food compared to fasted administration. Similar results were observed in mice, with no change to rosuvastatin concentration in the liver. LDL-C and lathosterol concentrations were not different among dyslipdemic patients who took rosuvastatin with or without food. HOW THIS MIGHT CHANGE CLINICAL PHARMA- COLOGY OR TRANSLATIONAL SCIENCE þ Our data suggest that taking rosuvastatin with food could reduce systemic concentrations, and subsequent myopathy risk, without compromising the LDL-C-lowering benefit. Cardiovascular disease (CVD) is a leading cause of morbidity and mortality worldwide. 1 High plasma cholesterol is a major risk factor for CVD events, 2 and reducing circulating cholesterol, particularly low-density lipoprotein cholesterol (LDL-C), is an important goal of pharmacotherapy. Statin medications are firstline therapy for CVD prevention, and an estimated 25.2 million Americans are currently receiving statin therapy. 3 However, new guidelines from the American College of Cardiology and the American Heart Association recommend broadening the use of statin therapy to include an additional 30.8 million Americans in addition to those already receiving statins. 3,4 Statins target the liver and competitively inhibit the ratelimiting enzyme, HMG-CoA reductase, in the cholesterol biosynthesis pathway. Although generally well tolerated, a notable barrier to statin therapy is skeletal muscle toxicity, with up to 10% of statin patients experiencing some degree of muscle pain or weakness. 5 Although the exact mechanism is unknown, 6 statin-induced muscle toxicity is associated with increased systemic statin exposure. 7 Emerging evidence suggests observed interindividual variation in statin exposure in patients is associated, in part, with polymorphisms within hepatic uptake and efflux transporters such as OATP1B1 (organic aniontransporting polypeptide 1B1; SLCO1B1) and efflux transporter BCRP (breast cancer resistance protein; ABCG2). However, given the 45-fold or higher variability in plasma statin concentrations between patients on the same daily dose, 8 it is clear that known genetic polymorphisms in statin disposition pathways do not fully explain the extent of variation in attained statin concentration, suggesting that additional pathways or mechanisms may be involved. Administration of a medication with food can alter 1 Division of Clinical Pharmacology, Western University, London, ON, Canada; 2 Department of Physiology & Pharmacology, Western University, London, ON, Canada; 3 Endocrinology, Department of Medicine, Western University, London, ON, Canada. Correspondence: Richard B. Kim (Richard.Kim@lhsc.on.ca) Received 14 September 2017; accepted 30 November 2017; advance online publication 8 January doi: /cpt.973 CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 104 NUMBER 3 September

2 Table 1 Demographic characteristics of healthy Caucasian Canadian and Asian Canadian subjects Caucasian Asian Male (n 5 6) Female (n 5 5) Male (n 5 6) Female (n 5 6) Age (y) median (range) 22 (21-59) 22 (21-25) 21 (20-22) 21 (20-23) Weight (kg), mean (SD) 84.2 (11.4) 74.0 (12.9) 74.0 (11.3) 50.4 (7.5) Height (m), mean (SD) 1.77 (0.04) 1.72 (0.06) 1.70 (0.07) 1.58 (0.07) BMI (kg/m 2 ), mean (SD) 26.7 (2.3) 24.9 (4.0) 25.6 (2.6) 20.2 (3.3) BMI, body mass index. its physiochemical properties and/or change the pharmacokinetic or pharmacodynamic profiles of the drug, thus leading to variation in plasma exposure. 9 Rosuvastatin is one of the most potent statins and has demonstrated superior cholesterol-lowering abilities when compared to other statins on the market. 10,11 Current dosing guidelines for rosuvastatin indicate no preference for fed or fasted administration (Crestor product monograph), but recent pharmacokinetic data in canines and in a Chinese population of healthy volunteers suggest that rosuvastatin systemic exposure is significantly reduced when administered with food. 12,13 While these studies suggested reduced exposure was likely due to decreased intestinal absorption, increased hepatic clearance may also alter circulating concentrations. Additionally, rosuvastatin clearance among Asians is known to differ from Caucasians. 14 Accordingly, we investigated the effect of concomitant food administered on rosuvastatin pharmacokinetics among healthy Canadian subjects of Asian and European ancestry and in mice. We further examined the effect of food administration on the lipid profiles of dyslipidemic patients treated with rosuvastatin. RESULTS Concomitant food administration lowers rosuvastatin plasma concentration Subject demographics are presented in Table 1. Pharmacokinetic parameters for each study condition are presented in Table 2 and were analyzed separately based on ethnicity. Large interindividual variation in rosuvastatin area under the plasma concentration vs. time curve (AUC 0-10 ) was observed for both Caucasian and Asian subjects (Table 2, Figure 1a,c). Among Caucasian subjects, maximum plasma rosuvastatin concentration (C max ), and AUC were significantly higher when rosuvastatin was administered without food compared to administration with either a low-fat or high-fat meal (Table 2, Figure 1b). No significant differences in plasma rosuvastatin concentrations were observed between low-fat and high-fat states (Table 2, Figure 1a,b). The geometric mean ratios (GMR) for AUC 0-10 between low-fat or high-fat and fasted conditions were 0.5 (90% confidence interval (CI), ) and 0.62 (90% CI ), respectively, indicating nonbioequivalence (Supplementary Table S1). Moreover, the observed rosuvastatin clearance (Cl/F) was significantly Table 2 Pharmacokinetic parameters in healthy Caucasian Canadian and Asian Canadian subjects administered rosuvastatin (10 mg) in a fasted state or with a low-fat or high-fat meal Parameter Fasting vs. Low-fat P Fasting vs. High-fat P Low-fat vs. High-fat P Caucasian subjects (n 5 11) T max (h) 4.4 (1.8) 4.3 (1.5) (1.8) 4.4 (1.2) > (1.5) 4.4 (1.2) C max (ng/ml) 2.9 (0.6) 1.6 (0.7) < (0.6) 2.0 (0.9) < (0.7) 2.0 (0.9) AUC 0-10 (ng/ml*h) 18.3 (3.4) 9.4 (3.5) < (3.4) 11.5 (3.6) < (3.5) 11.5 (3.6) V/F (mg/(ng/ml) 2.0 (0.6) 4.4 (1.8) < (0.6) 3.4 (1.3) (1.8) 3.4 (1.3) Cl/F (mg/(mg/ml)/h 0.5 (0.1) 0.9 (0.3) < (0.1) 0.7 (0.2) < (0.3) 0.7 (0.2) Ka (1/h) 0.4 (0.3) 0.3 (0.1) > (0.3) 0.4 (0.2) > (0.1) 0.4 (0.2) >0.99 Asian subjects (n 5 12) T max (h) 3.6 (2.0) 5.3 (0.9) < (2.0) 5.1 (1.2) < (0.9) 5.1 (1.2) C max (ng/ml) 8.2 (6.1) 4.4 (3.5) (6.1) 5.7 (3.4) (3.5) 5.7 (3.4) AUC 0-10 (ng/ml*h) 50.6 (35.5) 27.4 (23.8) (35.5) 33.8 (21.5) (23.8) 33.8 (21.5) V/F (mg/(ng/ml) 1.2 (1.0) 2.1 (1.5) (1.0) 1.4 (0.9) (1.5) 1.4 (0.9) Cl/F (mg/(mg/ml)/h 0.3 (0.3) 0.5 (0.3) (0.3) 0.4 (0.2) (0.3) 0.4 (0.2) Ka (1/h) 0.6 (0.5) 0.2 (0.1) < (0.5) 0.2 (0.1) < (0.1) 0.2 (0.1) >0.999 T, time; C, concentration; AUC, area under the curve; V, volume of distribution; F, bioavailability; Cl, clearance; Ka, absorption rate constant. 526 VOLUME 104 NUMBER 3 September

3 Figure 1 Rosuvastatin pharmacokinetic time curve followed by area under the curve (AUC 0-10 ) for (a,b) healthy Caucasian (n 5 11), and (c,d) healthy Asian (n 5 12) subjects. Plasma was collected from blood samples taken at 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, and 10 h after a 10-mg rosuvastatin dose. Insets show PK profiles in linear scale. Data are presented as mean 6 SD. Significance of the mean difference between administration states are depicted as ***P < and ****P < longer in the fed states compared to a fasted state administration, and apparent volume of distribution (V/F) was greater when rosuvastatin was administered with a low-fat meal compared to administration without food (Table 2). As expected, significantly higher plasma rosuvastatin concentrations were measured among Asian subjects. Within this group, average time to maximum concentration (T max ) and absorption rate (Ka) were significantly longer when rosuvastatin was administered with food compared to administration without food (Table 2). A trend of lower plasma rosuvastatin concentrations (C max and AUC) was observed when rosuvastatin was administered with food but was not significantly different from concentrations attained under fasted conditions (Table 2, Figure 1c,d). ABCG2 421C>A is associated with rosuvastatin exposure independent of food administration Plasma rosuvastatin concentrations have been shown to be higher in SLCO1B1 c.521t>c and ABCG2 c.421c>a variant carriers. 8 Additionally, SLCO1B1 c.388a>g and ABCG2 c.34g>a have been associated with altered statin pharmacokinetics Here, we genotyped subjects for these polymorphisms (allele frequencies are reported in Table S2) and determined their effect on rosuvastatin exposure when rosuvastatin was administered in the presence or absence of food. Within the fasted state, ABCG2 c.421a carriers had 3.5-fold higher mean C max and AUC 0-10 when compared to noncarriers (Figure 2ai,bi). ABCG2 c.421a carriers also had significantly higher C max and AUC 0-10 compared to noncarriers when rosuvastatin was administered with a high-fat or low-fat meal (Figure 2ii,iii). To confirm that the association between ABCG2 c.421c>a and plasma rosuvastatin concentrations was not biased by ethnicity differences, ABCG2 c.421c>a was separately assessed in Asian subjects, as there were no Caucasian ABCG2 c.421a variant carriers. Rosuvastatin C max and AUC 0-10 were significantly higher in ABCG2 c.421a carriers compared to wildtype subjects when administered a high-fat or low fat-meal and a similar nonsignificant trend was noted under fasted conditions (Figure S1). No significant differences were observed for mean C max or AUC 0-10 when rosuvastatin was administered with or without food between wildtype and variant carriers of SLCO1B1 c.521t>c, SLCO1B1 c.388a>g, or ABCG2 c.34g>a (Figure S2). Food alters plasma rosuvastatin concentration in mice To investigate hepatic transport of rosuvastatin in response to food intake, we measured rosuvastatin plasma and tissue concentrations from fed and fasted wildtype C57BL/6 mice administered 10 mg/kg of rosuvastatin via oral gavage. Similar to our observations in healthy volunteers, average plasma rosuvastatin concentration was significantly greater in fasted mice (n 5 5) when compared to fed mice (n 5 4) (Figure 3a). Liver concentrations of rosuvastatin were not significantly different when the CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 104 NUMBER 3 September

4 Figure 2 Effect of ABCG2 c.421c>a on rosuvastatin (a) maximum concentration (C max ) and (b) area under the plasma concentration vs. time curve (AUC 0-10 ) in healthy Caucasian and Asian (n 5 23) subjects administered 10 mg oral rosuvastatin (i) in a fasted state, (ii) with a low-fat meal, or (iii) with a high-fat meal. Top and bottom of the boxes represent the 25 th and 75 th percentile, respectively; with midline representing the median. The whiskers depict the 5 th and 95 th percentile. ****P < drug was administered with or without food (Figure 3b). Mean liver-to-plasma ratio of rosuvastatin was 2.1-fold greater, in fed mice but was not significantly different when compared to fasted mice (Figure 3c). LDL-C concentration or rosuvastatin dose among patients on therapy do not differ whether rosuvastatin is consumed in a fed or fasted state To investigate the effect of concomitant food administration with rosuvastatin dose on the lipid profiles of statin patients, we analyzed data from 156 previously recruited dyslipidemic patients. 8 Patients were stratified into groups presumed to take rosuvastatin with (n 5 75) or without food (n 5 82). Patient characteristics are presented in Table S3. The average dose of rosuvastatin was not significantly different between individuals presumed to take rosuvastatin with or without food (Figure 4a), suggesting that food administration does not alter LDL-Clowering, as rosuvastatin dose was titrated to achieve therapeutic benefit. Taking rosuvastatin with or without food did not significantly affect average LDL-C or lathosterol concentrations for individuals taking daily doses between 5 and 40 mg (Figure 4b,c). Together, these results suggest that food administration does not alter the ability of rosuvastatin to inhibit endogenous cholesterol production. DISCUSSION Statin-induced muscle toxicities most commonly present as muscle pain or weakness known as myalgia, occurring in up to 10% of statin patients. 5 In rare cases, a life-threatening form of muscle toxicity, rhabdomyolysis, may occur as a result of statin therapy. Switching statins to avoid statin-induced muscle toxicities may be efficacious. Remarkably, during the clinical trials that led to the approval of statins for lowering LDL-C, there were pharmacokinetic data showing plasma concentrations of commonly prescribed statins, such as pravastatin and atorvastatin, were lower when administered with food compared to a fasted administration. 18,19 However, such findings did not have any impact on the overall LDL-C-lowering effect. Therefore, no specific guidance was provided in terms of whether statins should be administered with or without food. Indeed, the current product monograph for statins do not indicate any preference with regard to taking the medication in a fed or fasted state. However, we hypothesized that lower circulating statin concentration, without compromising its efficacy, would be a desirable and clinically meaningful strategy for reducing the risk for statin myopathy. Therefore, we investigated the effect of concomitantly administered meals (both high-fat and low-fat meals) on plasma rosuvastatin concentrations in healthy volunteer subjects. We demonstrate for the first time that concomitant administration with food substantially reduced mean plasma rosuvastatin C max and AUC in healthy Caucasians and have validated the results previously observed in Asian subjects. 13 We found that, in healthy Caucasian individuals administered 10 mg oral rosuvastatin with a low-fat or high-fat meal, resulted in an average reduction in mean plasma rosuvastatin concentrations (average of C max and AUC 0-10 )of 46.7% and 34.1%, respectively when compared to values observed under fasting conditions. We found similar reductions of 46.1% and 31.8% for the low-fat and high-fat states, respectively, among Asian subjects. 528 VOLUME 104 NUMBER 3 September

5 1,046 kcal and 43 g of fat compared to 712 kcal and 37 g for the high-fat meal and 661 kcal and 15 g for the low-fat meal used in our study. It is possible that had our test meals been more calorically dense with a higher fat content that a larger food effect could have been demonstrated. However, in designing the test meals for this work we chose items that are not contraindicated in hyperlipidemic patients. Therefore, we suspect that the magnitude of the rosuvastatin-food effect found in our healthy Caucasian and Asian cohorts more closely resembles that which would be found in statin patients. Li et al. concluded that rosuvastatin systemic exposure was likely lower when administered with food due to impaired intestinal absorption. In support of this, we observed a longer time to maximum concentration (T max ) and reduced absorption rate when rosuvastatin was administered with food compared to a fasted administration in our Asian cohort. Figure 3 Rosuvastatin concentrations in (a) plasma, (b) liver, and (c)the liver-to-plasma concentration ratio in fed (n 5 4) and fasted (n 5 5) C57BL/6 mice 2 h after a 10 mg/kg oral rosuvastatin dose. Plasma and liver were collected 2 h postdose. Rosuvastatin concentrations in the plasma and liver homogenate samples were measured by LC-MS/MS. Data presented as mean with SD. *P < We note that Li et al. found that administering 10 mg oral rosuvastatin to healthy Chinese subjects directly following a large meal resulted in a >90% decrease in mean AUC and C max values when compared to fasting values. 13 Li et al. used generic rosuvastatin provided by Dyne Pharma, a manufacturer in Shandong, China. Formulation differences between these tablets and the Crestor tablets used in our study may account for the difference in the magnitude of the response to food. Moreover, the test meal used by Li et al. was significantly larger than either of the treatment breakfasts used in our study and consisted of Figure 4 Average (a) dose of rosuvastatin, (b) low-density lipoprotein cholesterol (n 5 146) concentrations, and (c) lathosterol (n 5 154) concentrations in dyslipidemic patients presumed to take their dose with (n 5 75) or without (n 5 82) food. Data are stratified by dose of daily rosuvastatin and are presented as mean 6 SD. Lipid parameter values were compared at each dose. CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 104 NUMBER 3 September

6 Interestingly, we did not observe these effects in our Caucasian cohort, and instead observed an increased rosuvastatin clearance in the fed states and greater apparent volume of distribution when rosuvastatin was administered with a low-fat meal compared to a fasted administration, suggesting ethnic differences in the response to food, although the overall average percent reduction in rosuvastatin systemic exposure was similar between Caucasian and Asian subjects. Our findings in mice also suggest that lower systemic exposure could also potentially be explained by increased hepatic uptake of rosuvastatin in the presence of food. This is supported by our findings of significantly higher plasma rosuvastatin concentrations in fasted mice when compared to fed mice and a mean liver-to-plasma concentration ratio that was 2.1-fold higher in fed mice when compared to fasted mice administered oral rosuvastatin. However, we did not observe a difference in liver rosuvastatin concentration, likely due to the large intermouse variability. Further investigations are indicated to explore hepatic uptake of rosuvastatin under fed and fasted administration. Although the OATP family of transporters has been viewed as the principal mediators for the hepatic uptake of statins, our group had demonstrated that the liver-specific bile acid transporter, NTCP, may account for nearly one-third of rosuvastatin uptake. 20 Polymorphisms in the gene encoding for NTCP, SLC10A1, are rare and we previously identified four nonsynonymous variants in 370 subjects. 21 Of these variants, SLC10A1 c.800c>t was found exclusively in 7% Chinese American subjects and demonstrated a near complete loss of activity for bile acid uptake, but demonstrated normal transport of estrone sulfate. Interestingly, SLC10A1 c.800c>t, has demonstrated greater transport of atorvastatin and rosuvastatin compared to wildtype, and ethnic differences in allele frequency have been noted to be 5% in Asian populations. 22,23 Moreover, SLC10A1 c.190g>a has been identified in Korean subjects (frequency of 1.0%) and has demonstrated decreased uptake of taurocholate and rosuvastatin compared to wildtype NTCP. 23 The clinical relevance of these variants remains to be evaluated. Increased expression or activity of NTCP associated with food intake may aid in clearance of rosuvastatin from portal circulation, and thereby lower circulating statin concentrations. NTCP is a major transporter involved the enterohepatic recirculation of bile acids, and its expression and activity are highly regulated to allow for efficient management of a high bile acid load associated with food ingestion. 24 It should be noted that posttranscriptional regulation, especially via plasma membrane insertion/retrieval, is a key pathway that mediates the rapid up- and downregulation of NTCP expression and function in the presence of changing bile acid load. 24 Since a larger bile acid load accompanies the ingestion of a high-fat meal, 25 we hypothesized that plasma rosuvastatin concentrations would be lowest when a rosuvastatin dose was administered with a high-fat meal. Indeed, Baek et al. found that C max and AUC of rosuvastatin were significantly lower when a 10 mg oral dose was administered to dogs fed a high-fat meal when compared to those fed a low-fat meal prior to dosing. 12 Interestingly, we did not observe any significant differences in C max or AUC values when rosuvastatin was administered following a high-fat compared to administration following a lowfat meal in our healthy human cohorts. This disparity may, in part, be explained by differences in gastrointestinal physiology and biochemistry and/or variability in expression of important hepatobiliary transporters between humans and dogs. 26,27 For example, hepatic blood flow is greater in dogs than in humans, and the absolute amount of BCRP protein in liver tissue has been shown to be over four times more in dogs than in humans. 28 An important question to be addressed is whether the observed lower plasma concentration of rosuvastatin when taken with food results in reduced benefit, in terms of an LDL-C-lowering effect, from rosuvastatin therapy. Previous research had shown that LDL-C reductions were not significantly different in healthy volunteers following administration of 10 mg atorvastatin tablets with or without food for 15 days. 29 Similarly, taking pravastatin or fluvastatin with or without food did not significantly affect their LDL-C-lowering capacities. 18 However, since statins are taken for years and sometimes life-long, we sought to address the potential food effects to rosuvastatin dosing and LDL-Clowering effects among patients on rosuvastatin therapy. Here we demonstrated that mean plasma concentrations of LDL-C and lathosterol were not significantly different among patients presumed to take their daily rosuvastatin dose with or without food. Importantly, the average dose of rosuvastatin was not significantly different between patients presumed to take their rosuvastatin with or without food. A further prospective study including the use of medication and food diaries is needed to more conclusively determine the effect of food administration on rosuvastatin efficacy. In the current study, we also assessed the role of statin transporter pharmacogenetics. Particularly, we investigated the efflux transporter ABCG2 (BCRP), which is known to play an important role in the disposition of rosuvastatin. 30 ABCG2 c.421c>a has been associated with higher plasma rosuvastatin concentrations and this loss-of-function variant is known to be more common among Asians compared to Caucasians. 14,31 Here we noted that ABCG2 c.421c>a allele frequency was much higher among Asian subjects compared to Caucasians. ABCG2 c.421a carriers had higher rosuvastatin C max and AUC 0-10 values when compared to noncarriers. However, the reduced rosuvastatin AUC observed with food administration was independent of ABCG2 c.421c>a genotype, suggesting that taking rosuvastatin with a meal may be particularly important for variant carriers to reduce myopathy risk. It is now thought that for compounds reliant on transporters for absorption and distribution, such as rosuvastatin, allele frequencies of pertinent transporters can contribute significantly to ethnic variability. 32 Moreover, recent work suggests that rosuvastatin exposure does not differ between Asians and Caucasians when all subjects are wildtype carriers for SLCO1B1*1a and ABCG2 c In our study, few subjects (three Caucasian and two Asian) were wildtype for both alleles, thus we were unable to assess the role of ethnic variability in the absence of SLCO1B1 and ABCG2 polymorphisms on rosuvastatin exposure under fed and fasted administration. In summary, although the current United States and Canadian dosing guidelines indicate no preference for fed or fasted 530 VOLUME 104 NUMBER 3 September

7 rosuvastatin administration (Crestor product monograph), the findings from this work indicate that rosuvastatin patients could take their dose with a meal as a way to lower systemic statin exposure, hence lowering the risk of statin myopathy, without compromising its LDL-C-lowering effect. This strategy may be particularly relevant for patients requiring high-dose statins or for carriers of known loss-of-function genetic variants in statin transporters. METHODS Rosuvastatin pharmacokinetic assessment in healthy subjects A prospective, randomized, crossover, pharmacokinetic study was performed in healthy Canadian volunteers of European and Asian descent. Asian subjects were self-identified as Chinese or Korean Canadians. All participants provided written, informed consent. The study was approved by the Research Ethics Board of the University of Western Ontario, London, Canada. Participants were deemed healthy by the study physician upon physical examination, a brief medical history, and analysis of routine serum chemistry. Subjects did not take other medications (with the exception of oral contraceptives) within 1 month of the study. Participants completed three separate study days, each beginning by fasting from 12 AM the morning of each study day, refraining from alcohol consumption 24 h prior to each study day, and refraining from caffeine consumption on each study day. Urine pregnancy tests were conducted for female volunteers prior to drug administration. On each study day, patients were instructed to consume the entirety of the predesigned low-fat (20.8% fat, 10.9% protein, 58.1% carbohydrates), or highfat (46.9% fat, 10.1% protein, 39.91% carbohydrates) breakfast and were administered rosuvastatin (10 mg) immediately following meal completion or were administered rosuvastatin under fasting conditions. The order of study days was randomized, with a minimum washout period of 1 week between study days. Ethylenediamine-tetraacetic acid (EDTA)- blood samples were collected prior to rosuvastatin administration and at 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, and 10 h postdose. Samples were centrifuged at 48C, 2000g for 10 min and plasma was collected and stored at 808C until further analysis. Subjects were provided with a meal of their choosing 5 h after drug administration with the same meal provided on each study day. Genotypic assessment in healthy subjects DNA was isolated from whole blood using the MagNA Pure Compact Nucleic Acid Isolation Kit I and MagNA Pure Compact Instrument (Roche, Indianapolis, IN). DNA samples were analyzed using TaqMan allelic discrimination assays for the following polymorphisms: SLCO1B1 c.388a>g (*1b), SLCO1B1 c.521t>c (*5), ABCG2 c.421c>a, and ABCG2 c.34g>a. Rosuvastatin pharmacokinetic assessment in mice Wildtype, 10-week-old male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME), were housed in a temperature-controlled environment with a 12-h light/dark cycle where they received standard murine chow and water ad libitum. Mice received rosuvastatin (10 mg/kg) in phosphate-buffered saline (total volume of 200 ll) by oral gavage under fed or fasting conditions. Food was removed from the fasting mouse group 6 h prior to drug dosing. Mice were euthanized by isoflurane 120 min after rosuvastatin administration. Blood was collected via cardiac puncture into EDTA-containing tubes and centrifuged at 48C, 2,000 rcf for 10 min for plasma isolation. Plasma was stored at 808C until further analysis. Livers were excised postmortem, rinsed in phosphate-buffered saline, blotted, and weighed; liver samples were flashfrozen in liquid nitrogen and stored at 808C. This study protocol was approved by the Animal Use Subcommittee of the University of Western Ontario, London, Canada. Determination of plasma statin concentrations Plasma rosuvastatin concentrations were determined using a modified liquid chromatography/tandem mass spectrometry (LC-MS/MS) method. 34 Rosuvastatin calcium-salt and rosuvastatin-d6 was purchased from Toronto Research Chemicals (North York, ON, Canada). Standards and quality control samples (QCs) were prepared in drug-na ıve human plasma (K2 EDTA, BioreclamationIVT, New York, NY). Standards, QCs, and plasma samples (100 ll) from each timepoint were precipitated with 300 ll acetonitrile spiked with rosuvastatin-d6 and centrifuged for 20 min, 14,000 rpm at 48C. The supernatant was transferred to glass autosampler vials and diluted 1:1 in 0.05% formic acid in water. Mobile phases included 0.05% formic acid in water and acetonitrile. Chromatographic separation was performed using an Agilent Technologies 1200 LC injector HTS (Agilent Technologies, Mississuaga, Ontario, Canada) and a Kintex 5 lm EVO C18 100Å column fitted with guard cartridge, (Phenomenex, Torrance, CA) with an injection volume of 50 ll and a gradient over 6.5 min starting at a ratio of 70:30 with a gradient to a ratio of 10:90 with a flow rate of 0.5 ml/min. The mass spectrometer (Thermo TSQ Vantage, Thermo Scientific, Pittsburgh, PA) with heated electrospray ionization was operated in positive mode, with mass transitions: rosuvastatin (482!258) and rosuvastatin-d6 (488!64). The lower limit of quantification for rosuvastatin was 0.1 ng/ml. Assay bias and precision (coefficient of variation) were 1.1% and 8.0%, respectively. For analysis of plasma and liver rosuvastatin concentrations from mice, standards were prepared in drug-na ıve mouse plasma (K2 EDTA, BioreclamationIVT) and homogenized, wildtype, untreated, C57BL/6 liver. Liver samples and standards were homogenized 1:1 (weight to volume) in 0.05% formic acid in water. Plasma samples and liver homogenate samples (5 ll of each) were precipitated using 20 ll acetonitrile spiked with internal standard (rosuvastatin d6) and centrifuged for 20 min, 14,000 rpm at 48C. Supernatants were diluted 1:2 in 0.05% formic acid in water and analysis was carried out as described above with an injection volume of 30 ll. Dyslipidemic study population The study population included 156 previously recruited individuals from the Lipid Clinic at London Health Sciences Centre (London, ON, Canada) from August 2009 to May All patients provided informed written consent. Each patient was on daily rosuvastatin therapy at the time of enrolment and provided a blood sample as previously described. 8 Data collected upon enrolment included a daily dose of rosuvastatin, time of sample collection, and time since last dose. Plasma concentrations of lathosterol were measured from the provided blood samples as previously described. 8 Clinically measured LDL-C concentrations were used where possible; if LDL-C values were not recorded, these values were obtained using the Friedewald equation and clinically recorded HDL-C, total cholesterol, and triglyceride values. Patients were stratified as follows: patients who reported taking their last rosuvastatin dose between 5 9 AM (inclusive) or 5 7 PM (inclusive) were presumed to have taken the medication with food (n 5 75), while patients who reported last dose at or after 8 PM were presumed to have taken rosuvastatin without food (n 5 82). Patients who reported dosing at alternative times were excluded from analysis. Time of reported dosing was presumed to be the patient s typical dosing routine for rosuvastatin. Pharmacokinetics and statistical analysis Rosuvastatin pharmacokinetic parameters in healthy volunteers and mice were calculated using PKSolver. 35 Of note, as blood samples were only collected up to 10 h postdose for the healthy volunteer study, the elimination phase of rosuvastatin and corresponding pharmacokinetic parameters (T 1/2 and AUC 0-8 ) could not be accurately reported. Statistical analysis was performed using GraphPad Prism 6 (La Jolla, CA). Values for the three study days for each subject were compared by one-way analysis of variance (ANOVA) and the Tukey multiple comparison test. AUC 0-10 and C max values were compared between variant-allele carriers and wildtype individuals for each polymorphism using Student s t-test or CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 104 NUMBER 3 September

8 Mann Whitney U-test, as appropriate. Pharmacokinetic data from the fed and fasted mouse groups were compared by Mann Whitney U-test. Liver-to-plasma ratios were calculated by dividing the tissue rosuvastatin concentration by the plasma concentration at the terminal timepoint (120 min). Dyslipidemic stratified patients were categorized based on daily rosuvastatin dose: 5 mg, 10 mg, 20 mg, and 40 mg. For each dose, plasma LDL-C (n 5 146) and lathosterol (n 5 154) concentrations were compared between stratified patients using Student s t-test or Mann Whitney U-test, as appropriate. Additional Supporting Information may be found in the online version of this article. ACKNOWLEDGMENTS We thank Cameron Ross, Sara E. Mansell, and Yinyin Liao for technical assistance. CONFLICT OF INTEREST The authors declare no competing interests for this work. 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