Chronopharmacology of simvastatin on hyperlipidaemia in high‐fat diet‐fed obese mice

Abstract The chronopharmacology refers to the utilization of physiological circadian rhythms to optimize the administration time of drugs, thus increasing their efficacy and safety, or reducing adverse effects. Simvastatin is one of the most widely prescribed drugs for the treatment of hypercholesterolaemia, hyperlipidemia and coronary artery disease. There are conflicting statements regarding the timing of simvastatin administration, and convincing experimental evidence remains unavailable. Thus, we aimed to examine whether different administration times would influence the efficacy of simvastatin. High‐fat diet‐fed mice were treated with simvastatin at zeitgeber time 1 (ZT1) or ZT13, respectively, for nine weeks. Simvastatin showed robust anti‐hypercholesterolaemia and anti‐hyperlipidemia effects on these obese mice, regardless of administration time. However, simvastatin administrated at ZT13, compared to ZT1, was more functional for decreasing serum levels of total cholesterol, triglycerides, non‐esterified free fatty acids and LDL cholesterol, as well as improving liver pathological characteristics. In terms of possible mechanisms, we found that simvastatin did not alter the expression of hepatic circadian clock gene in vivo, although it failed to change the period, phase and amplitude of oscillation patterns in Per2::Luc U2OS and Bmal1::Luc U2OS cells in vitro. In contrast, simvastatin regulated the expression of Hmgcr, Mdr1 and Slco2b1 in a circadian manner, which potentially contributed to the chronopharmacological function of the drug. Taken together, we provide solid evidence to suggest that different administration times affect the lipid‐lowering effects of simvastatin.


| INTRODUC TI ON
In response to the autorotation of the Earth, the majority of organisms have evolved an approximate 24-hour endogenous timing mechanism, known as the circadian clock, which controls almost all mammalian physiological and metabolic processes. 1 It is therefore not surprising that xenobiotic metabolism, which is key for the drug efficacy, also demonstrates a robust daily oscillation. 1,2 Additionally, the target receptors, transporters and enzymes of certain drugs are rhythmically expressed in various tissues. 3 These fluctuations contribute to circadian time-dependent drug behaviours such as absorption, distribution, metabolism and excretion. 4 Such rhythmicity establishes the major elements of chronopharmacology and chronopharmacodynamics. 2,5 In this sense, optimizing the administration time of the drugs during a day may increase the actions or reduce the adverse effects of drugs, benefiting patients and improving their quality of life. 5 As one of the most widely used lipid-lowering drugs, simvastatin (SV) inhibits enzymatic activity of hydroxymethylglutaryl coenzyme A reductase (Hmgcr), 6 which is rhythmically expressed in the mouse liver. 7 Conflicting statements exist in clinical research about the proper timing of SV administration. The authors of several randomized controlled clinical trials have reported that evening administration of SV is more efficient than morning administration. 8,9 However, Yoon and Kim recommended that morning administration is preferable. 10,11 Unfortunately, none of these studies provides a clear mechanism for explaining the chronopharmacology of SV.
To address these concerns, our study aimed to verify the chronopharmacology of SV for treating hypercholesterolaemia and hyperlipidemia in high-fat diet (HFD)-fed mice. We also sought to define the potential mechanisms mediating these phenomena.

| Experimental design
Thirty male mice were randomly divided into two groups. One group 79902-63-9, Aladdin) or an equal volume of 0.5% sodium carboxymethylcellulose (0.5% CMC; CAS: 9004-32-4; Aladdin) at ZT1 for nine weeks. For those three ZT13 subgroups, mice received SV or 0.5% CMC administration at ZT13 for 9 weeks. Bodyweight, food intake and water drinking were recorded weekly. When the procedure was completed, mice were killed. The mice of ZT1-ND, ZT1-HFD and ZT1-HFD + SV groups were killed at ZT1. The mice of ZT13-ND, ZT13-HFD and ZT13-HFD + SV groups were killed at ZT13. The detailed methods is reported in Appendix S1.

| Statistical analysis
All values are expressed as means ± standard deviation (SD). Oneway ANOVA was used for the data more than two groups. When the variance was homogeneous, Fisher's LSD post hoc test was conducted; otherwise, Gamos-Howell was used. Statistical significance was set at P < .05. In order to compare the efficacy of SV at ZT1 and ZT13, we used the following formulas ZT1 HFD −ZT1 HFD SV ZT1 HFD * 100% and ZT13 HFD −ZT13 HFD SV ZT13 HFD * 100% for the normalization and calculate the percentage of metabolic parameters changes of ZT1-HFD + SV and ZT13-HFD + SV. Then, a two-tailed Student's t test was used to compare the two groups.

| RE SULTS AND D ISCUSS I ON
The experimental design is illustrated in Figure 1A. After 2 months of HFD feeding, mice developed significant obesity when compared to those fed with ND. However, SV treatment decreased the weight gain in these HFD-fed obese mice ( Figure S1A). In addition, SV administration did not alter the amount of food intake and water drinking ( Figure S1B,C).
We next assessed the pharmacological difference of circadian SV treatment at ZT1 and ZT13, respectively. As shown in Table 1, HFD feeding significantly increased the serum lipid levels of TC, TG, NEFA, LDL-C and the liver injury biomarkers, such as ALT, AST and LDH, whereas the SV treatment reversed nearly all the above parameters at both examined time-points. Consistent with these observations, liver histological and lipid examinations demonstrated that SV treatment alleviated HFD-induced liver injury and lipid accumulation ( Figure 1B). Of note, such a hypolipidaemic and hepatoprotective action of SV was more functional at ZT13 compared to that at ZT1 (Table 1). For instance, serological TC levels were decreased by 31.09% in the HFD-fed obese mice followed by the SV treatment at ZT13, but it was only decreased by 14.30% at ZT1. For TG, the decrease was 49.11% at ZT13% vs 24.91% at ZT1.
At the molecular level, we examined the hepatic mRNA expression of key genes involved in the cholesterol and lipid metabolism regulated by SV. As shown in Figure 1C, the expression levels of Ldlr (uptake of exogenous cholesterol), Abca1/Abcg8 (cholesterol efflux), Fabp1/Fatp2 (uptake of fatty acids) and Dgat2 (triglyceride synthesis) were increased in the liver of HFD-fed mice, but were decreased significantly when treated with SV. Interestingly, the overall changes of these genes were more robust when observed at ZT13 compared to ZT1. Collectively, these findings suggest that SV exerts its lipid-lowering and hepatoprotective effects in a circadian manner.
In order to determine whether the chronopharmacology of SV is mediated by the circadian clock, we measured the hepatic expression levels of key clock genes. As shown in Figure S2, whereas the HFD feeding altered the expression pattern of these clock genes, SV treatment showed a modest effect on them when compared to the corresponding HFD group. Consistently, SV failed to modulate the circadian rhythm in both Per2::Luc and Bmal1::Luc U2OS cells, including the period, amplitude and phase ( Figure S3). Therefore, the chronopharmacology of SV does not appear to be directly mediated by the circadian clock either in vitro or in vivo.
To further explore the possible mechanism of the chronopharmacological effect of SV, we detected the expression of Hmgcr, the known target of SV. 6 We found that SV administration was partially recovered the hepatic Hmgcr expression in HFD-fed mice at both transcriptional and translational levels ( Figure 1D, Figure S4A). This finding suggests that SV effectively reduced the accumulation of cholesterol, leading to the compensatory increase of Hmgcr expression. In addition, the drug metabolism may also play a vital role in the F I G U R E 1 Time of simvastatin (SV dosing influences liver pathological characteristics and the expressions of lipid metabolic and drug transport genes. SV was administered to high-fat diet (HFD) + SV groups at ZT1 or ZT13, respectively, for nine weeks, and at the same time, 0.5% CMC was administrated to normal diet (ND) and HFD groups (n 3.32 ± 0.18 pharmacodynamics of SV. 12 Given that Mdr1, Mrp2 and Slco family members are key transporters regulating the SV efflux and uptake in the liver, [13][14][15] we next checked the hepatic mRNA expression levels of these genes. As shown in Figure 1E,F and Figure S4B,C, the expression of Mrp2, Slco1a4 and Slco2b1 was induced by HFD feeding at both ZT1 and ZT13. In contrast, Mdr1 mRNA expression was decreased by the HFD feeding. When treated with SV, the protein level of Mdr1 was further decreased ( Figure 1E, Figure S4D).
Regarding the SV uptake transporters, Slco1a4 and Slco2b1 were expressed at a higher level at ZT13 than that at ZT1. Specifically, the hepatic protein levels of Slco2b1 were induced by SV treatment to 3.48-fold (ZT13) and 2.07-fold (ZT1), respectively, when compared to their HFD-feeding controls ( Figure 1F, Figure S4E).
These findings indicated that at ZT13, the expression of SV uptake transporter was induced more robustly, whereas a decrease in the expression of SV efflux transporter was observed, leading to the higher drug accumulation of SV in the liver and the better effective drug concentration.
In summary, this study demonstrated that SV administrated at both ZT1 and ZT13 produced anti-hypercholesterolaemia and anti-hyperlipidemia effects in HFD mice, whereas evening administration of SV was more effective. The chronopharmacological function of SV was attributed to the comprehensive action of multiple aspects: the circadian rhythm of the targeted gene Hmgcr, as well as the differential expression of the drug transport genes Sloc2b1 and Mdr1. Moreover, we found that the chronopharmacology of SV was not directly mediated by the circadian clock. We expect that our results will be beneficial to the clinical utilization of SV and will strengthen future researchers' attention to its chronopharmacology.

ACK N OWLED G EM ENTS
This work was financially supported by grants from the National

CO N FLI C T O F I NTE R E S T
The authors have declared that no conflict of interest exists.