The treatment of dyslipidemia has become critical for the early prevention and treatment of cardiovascular diseases, and the reduction of low-density lipoprotein cholesterol (LDL-C) plays a critical role in reducing the risk of cardiovascular events in both primary and secondary prevention (Cholesterol Treatment Trialists' Collaborators, et al., 2012).

Hyzetimibe (HS-25) is the second inhibitor of cholesterol absorption to be developed in China and was approved for marketing by the National Medical Products Administration of China (NMPA) in June 2021. A pharmacokinetic study of oral ¹⁴C-radiolabeled hyzetimibe revealed that a change in the hydroxyl group within the drug structure made it easier for hyzetimibe to combine with glucuronic acid, thus leading to the increased urinary excretion of hyzetimibe than ezetimibe (Liao et al., 2021). Hyzetimibe only results in a modest reduction in LDL-C and acts by inhibiting the intestinal cholesterol transporter Niemann-Pick C1-Like 1 (NPC1L1) (Ruan et al., 2014), which plays a critical role in the intestinal absorption of cholesterol (Altmann et al., 2004). Data obtained from clinical trials showed that hyzetimibe monotherapy reduced the levels of LDL-C by 15–18%. However, a significant variation was noted in the response to hyzetimibe, thus suggesting the existence of patients who show a poor response to hyzetimibe treatment or do not respond at all. As NPC1L1 is the molecular target of hyzetimibe, it follows that the expression levels of NPC1L1 reflect the efficiency of cholesterol absorption (Hui et al., 2008). Therefore, the response in variability may be partly attributed to the presence of the inherited genetic variation in NPC1L1.

Ezetimibe is another inhibitor of cholesterol absorption and was first approved for marketing by the Food and Drug Administration (FDA) in 2002 (Dujovne et al., 2002; Knopp et al., 2003). Ezetimibe monotherapy has shown to reduce the levels of LDL-C by 15–20%; an additional reduction of −13% to −20% could be attained when ezetimibe was combined with statin therapy, depending on the concentration of statin administered (Davidson et al., 2002; Ballantyne et al., 2003).

Pharmacogenomic studies were carried out in over 1,000 patients with hypercholesterolemia to investigate the impact of polymorphisms in the NPC1L1 gene on LDL-C reduction in response to ezetimibe (Simon et al., 2005). However, results were inconsistent, and even controversial; potentially due to the study design. The patients involved in this study had hypercholesterolemia and had been recruited on the ezetimibe add-on to statin for effectiveness (EASE) clinical trial and the Vytorin versus atorvastatin (VYVA) clinical trial. The EASE study was designed to evaluate the effects of ezetimibe, in combination with a stable regimen of statin therapy (any dose, any brand). In contrast, the VYVA study aimed to investigate the effects of a combination of ezetimibe and simvastatin in comparison with atorvastatin in patients with hypercholesterolemia.

These trials involved only a single dose of ezetimibe; this made it difficult to identify the specific association between the drug dose and the response exerted by the genetic variation in the NPC1L1 gene. Furthermore, neither of these studies considered ezetimibe monotherapy; this made it difficult to investigate the specific response to ezetimibe monotherapy. In addition, in the EASE cohort, ezetimibe was combined with statins (any dose and any brand); differential negative feedback mechanisms arising from different statins and doses, along with the complex background of statin therapy, made it difficult to determine the precise effects of these treatments.

Pisciotta et al. (2007) were the first to reveal the effect of the combination of ezetimibe and statins in genotype-confirmed heterozygous monotherapy in Caucasian familial hypercholesterolemia. Analysis showed that LDL-C was reduced by −18.9 ± 1.0% and −23.3 ± 2.3% (n = 65, p < 0.07) in Caucasian familial hypercholesterolemia and by −29.2 ± 1.0% and −33.7 ± 2.6% (n = 50, p < 0.06) in patients with primary hypercholesterolemia with the CC genotype and non-CC genotype of the g1679C > G SNP, respectively.

Subsequently, the Myocardial Infarction Genetics Consortium Investigators et al. (2014) found that heterozygous carriers of inactivating NPC1L1 mutations had a 53% relative risk reduction for coronary heart disease and had lower levels of LDL-C than non-carriers (p = 0.04).

Schweitzer et al. (2015) were the first to characterize the NPC1L1 gene and proteome in an exceptional responder to ezetimibe and attempted to identify genetic variations that caused functional changes. The combination of L52P-NPC1L1 and I300T-NPC1L1 amino acid changes caused a functional reduction in cholesterol uptake in the presence of ezetimibe, which was considered as a key aspect of the exceptional response.

Clearly, there is a lack of specific data relating to the potential impact of gene variations in NPC1L1 on the effect of cholesterol absorption inhibitors; existing data are inconsistent and unclear. Consequently, there is a critical need to carry out a high-quality clinical trial to investigate the characteristics of gene variation with exceptional responders or poor responders to drug treatment. Thus, we carried out a multicenter, randomized, double-blind, balanced-parallel, and placebo-controlled trial in 727 Chinese patients with primary hypercholesterolemia. For each patient, we characterized variability in the DNA sequence of the NPC1L1 gene and subsequently investigated the association between NPC1L1 and LDL-C reduction induced by hyzetimibe treatment.

Overall, 2,608 individuals were enrolled initially; of these, 727 were randomly assigned, and 444 were finally recruited for the analysis of genetic variation in NPC1L1; patients were excluded due to incomplete data relating to C trough and LDL-C, non-target substances in the plasma, or the absence of genotyping data (Figure 1). The treatment groups were well balanced with regards to demographics and baseline characteristics (Supplementary Table S1).

Table 1 shows the genotype frequency of the minimum allele frequency (MAF) of the four most common SNPs in the NPC1L1 gene (rs374773929, rs4720470, rs2301935, and rs2072183); the MAFs for these SNPs all exceeded 10% in Chinese patients with primary hypercholesterolemia. However, only those with the g1679C > G (rs2072183) genotype were associated with the lipid-lowering effect of hyzetimibe. The frequency of the g1679C > G genotype was similar to that in a previous Japanese population (p > 0.05) but was statistically different from the frequency recorded in a healthy population of Chinese and Japanese patients (p < 0.001), as well as in Canadian patients with primary hypercholesterolemia (p < 0.001). These findings indicated that there was a significant difference in the frequency of the g1679C > G variant when compared between healthy Chinese subjects and those with dyslipidemia; there was a clear genetic variation between Chinese and Canadian patients with dyslipidemia (Table 2) (Simon et al., 2005; Kashiwabara et al., 2014; Lou et al., 2015).

Treatment with hyzetimibe (10 and 20 mg) resulted in a mean percentage reduction from the baseline to end point in the plasma concentration of LDL cholesterol (12.82% and 16.29%, respectively) as compared to a decrease of only 3.12% in the placebo group (p < 0.01). The reduction of LDL cholesterol by hyzetimibe occurred early (2 weeks) and was maintained throughout the 12-week treatment period. The coadministration of hyzetimibe and atorvastatin resulted in a significantly greater mean reduction of LDL-C than either atorvastatin alone or hyzetimibe alone (Figure 2; Supplementary Table S2).

This is the first randomized clinical trial (based on a factorial design with six arms) to demonstrate an association between the NPC1L1 gene and the reduction of LDL-C levels in response to drug treatment with an inhibitor of cholesterol absorption. Our investigation revealed that genetic variations in NPC1L1 exert notable impacts on the extent of LDL-C reduction in response to hyzetimibe treatment in patients with primary hypercholesterolemia.

Among the common NPC1L1 haplotypes, only the g1679C > G SNP was found to exhibit a significant correlation with serum LDL-C levels. The g1679C > G SNP is located in exon 2 of the NPC1L1 gene and is embedded within the coding region. This SNP was in linkage disequilibrium with known functional variants, such as NPC1L1 -762T > C, an important transcription factor binding site, and −18C > A, in close proximity to a sterol regulatory element, thus suggesting that this SNP is related to the transcriptional level of NPC1L1; these changes lead to changes in the functionality of the NPC1L1 protein, thus leading to changes in the absorption of cholesterol (Tremblay et al., 2011).

When treated by monotherapy, patients with the CC genotype of the g1679C > G SNP tended to show significantly greater reductions in mean LDL-C (−23.99%) than either the GC genotype (−13.02% for pooled treatment groups; p < 0.01 for differences between pools) or the GG genotype (−16.45%, p < 0.01 for differences between pools) in patients who were treated with hyzetimibe (20 mg/day). When the effects of the placebo were removed, the reduction in LDL-C levels exhibited a rank order of CC (−20.49%) < GG (14.67%) < GC (−8.46%). The absolute reduction was 0.52 mmol/L for the GC genotype; this was compared to 0.65 mmol/L for the GG genotype and 0.96 mmol/L for the CC genotype.

Meta-analysis demonstrated that an absolute reduction of 1 mmol/L by statin treatment is associated with a 22% relative risk reduction in the 5-year incidence of major coronary events, ischemic stroke, and revascularization (Law et al., 2003). Most minimum doses for statins can result in an absolute reduction by more than 1 mmol/L and 0.73 mmol/L for ezetimibe. The EWTOPIA 75 (Ezetimibe Lipid-Lowering Trial on Prevention of Atherosclerotic Cardiovascular Disease in 75 or Older) was the first study to describe the clinical benefits of ezetimibe monotherapy in individuals aged ≥75 years with elevated levels of low-density lipoprotein cholesterol. In this trial, ezetimibe monotherapy was found to reduce the risks of the primary outcome by 34% (Ouchi et al., 2019).

Hence, patients with the heterozygous genotype (the GC genotype) should not be treated by hyzetimibe monotherapy due to poor efficacy. Patients with a homozygous genotype (the CC and GG genotypes) exhibited reductions of LDL-C [0.77 mmol/L (−19.34%)] similar to those of ezetimibe; in particular, the CC genotype exhibited significantly greater reductions in LDL-C than ezetimibe, thus indicating improved clinical benefits.

In contrast, and to our surprise, when we combined hyzetimibe and atorvastatin, we found that homozygous carriers (CC or GG) did not show a favorable dose response with regards to a further reduction of LDL-C (a 7.94% increase for CC and a 6.33% increase for GG); this was compared to a 15.29% increase for GC. Patients carrying the GC genotype showed a greater median reduction in LDL-C than those with the non-GC genotype (−52.23% versus −45.03%) in the hyzetimibe (20 mg) plus atorvastatin group (Figure 6). Furthermore, the proportion of patients exhibiting a reduction in the LDL-C level by >50% increased with increasing doses of hyzetimibe in the following rank order: 16.1% for atorvastatin alone, 46.2% for atorvastatin plus hyzetimibe (10 mg), and 65.4% for atorvastatin plus hyzetimibe (20 mg). These data showed that there was no feedback regulation induced by atorvastatin on the absorption of cholesterol in patients with the GC genotype.

Although there was a significant difference in the genotype frequency when compared between Caucasian, Chinese, and Japanese patients with hyperlipidemia, the frequency of the GC genotype in patients with the g1679C > G SNP ranges from 40 to 50%, thus indicating an abundance of individuals who would not respond to ezetimibe or hyzetimibe monotherapy. Therefore, it would be very important to remove such patients from such therapies due to their weak response to cholesterol absorption inhibitors and their poor effect on LDL-C reduction. It is important to consider that patients with a high risk of arteriosclerotic cardiovascular disease (ASCVD) need to control their cholesterol levels efficiently if they are to reduce the risk of cardiovascular events, thus demanding an appropriate lipid-lowering regimen from the beginning of treatment but not to modify according to the efficacy obtained after at least one month.

With regards to combination therapy, a previous drug–drug interaction study of atorvastatin and hyzetimibe showed there was no interaction between these drugs; this was also the case for the GC genotype. Thus, when the dose of atorvastatin or hyzetimibe increases, we should see a greater reduction in LDL-C.

By deploying an appropriate study design, our study found that variations in the NPC1L1 gene had differential impacts on the efficacy of cholesterol absorption inhibitors; researchers have been attempting to address unravel these effects for the past 15 years. This is the first study to identify common SNPs and genotypes of the NPC1L1 gene with respect to LDL-C reduction by more than 6%. The inconsistencies of previous studies relating to genotype analysis can be explained by differences in genotype frequency distribution between Eastern and Western ethnic groups and differences in the extent of genotype feedback regulation when different drugs are combined. These results are expected to provide significant guidance for the development of new inhibitors of cholesterol absorption with better efficacy, the design of corresponding clinical protocols, and the optimization of clinical hyperlipidemia treatments.

This study has several important limitations that need to be considered. First, the g1679C > G SNP in the NPC1L1 gene exerts significant impact on LDL-C reduction during hyzetimibe treatment. This is because the chemical structure of hyzetimibe is different from that of ezetimibe. Furthermore, the affinities of these drugs to the NPC1L1 protein also differ; the precise impact of the NPC1L1 gene on the action of ezetimibe is yet to be investigated. Second, subjects with the CC genotype achieved levels of LDL-C that were two-fold lower in response to hyzetimibe monotherapy treatment than subjects with the GC genotype (−23.99% versus −13.02%). The extent of LDL-C reduction showed a linear relationship with the dose of hyzetimibe. However, these data were obtained from a small number of patients; future research should involve a larger sample size if we are to fully validate our findings. Third, the frequency of the GG, GC, and CC genotypes was 41%, 46%, and 13% in Chinese patients with hypercholesterolemia; this compares to 5%, 40%, and 55% in Canadian patients with hypercholesterolemia (p < 0.001), respectively. The prevalence of the G allele variants indicates that the g1679C > G SNP exhibits ethnic specificity. However, the effects of hyzetimibe treatment on Caucasian patients with hypercholesterolemia are yet to be fully elucidated. Finally, we identified the notable impact of the 1735 C > G SNP on serum LDL-C levels in response to hyzetimibe treatment; however, we still need to identify the effects of a greater dose of hyzetimibe monotherapy in patients with the CC genotype. In addition, it would be very interesting to investigate whether higher doses of hyzetimibe or atorvastatin (in combination with each other) could achieve a better cholesterol-lowering target.

Dr. Martin Bdtker Mortensen and Brge Grnne Nordestgaard recently reported the outcomes of the Copenhagen General Population Study (CGPS) in The Lancet (2020) (Mortensen and Nordestgaard, 2020). Individuals aged 80–100 years with an LDL cholesterol level of 5.0 mmol/L or higher had the highest absolute risk of myocardial infarction and atherosclerotic cardiovascular disease than those aged 70–79 years [hazard ratio (HR): 2.99 versus 1.82], thus suggesting that active preventive strategies aimed at reducing the burden of myocardial infarction and atherosclerotic cardiovascular disease in the growing population aged 70–100 years should be adopted and may need more appropriate drugs to reduce the increased levels of LDL-C. Interestingly, at the same time and in the same journal, Gencer et al. also reported outcomes related to the efficacy and safety of lowering LDL cholesterol in older patients aged ≥75 years. This analysis involved 21,492 individuals from 29 trials; 54.7% were treated with statins, 28.9% were treated with ezetimibe, and 16.4% were treated with PCSK9 inhibitors. The risk of major vascular events in older patients was reduced by 26% per 1 mmol/L reduction in LDL-C; there was no significant difference in this respect when compared to patients aged <75 years. Furthermore, there were no significant differences between statin and non-statin treatments (Gencer et al., 2020). The clinical benefits of non-statin treatment (ezetimibe monotherapy) in individuals aged ≥75 years were also reported in the EWTOPIA 75 study (Ouchi et al., 2019). However, the HPS2-THRIVE (heart protection study 2-treatment of HDL to reduce the incidence of vascular events) study reported a rate of myopathy that was 10 times higher in Chinese patients receiving treatment with statins than that in European patients (HPS2-THRIVE Collaborative Group, 2013). In addition, in 2017, Wu et al. revealed that just 2.9% of high-intensity statins were used in China, thus resulting in an extremely low proportion of patients (<30%) of patients with CAD achieving a target LDL-C level (<1.8 mol/L) (Wu et al., 2017).

Therefore, ezetimibe or hyzetimibe monotherapy may be potentially useful for the treatment of older patients with dyslipidemia, especially those with statin-related adverse events (e.g., rhabdomyolysis) and in Chinese patients with hypercholesterolemia who are intolerant to high doses of statins.

The 2018 AHA/ACC treatment guidelines for LDL-C reduction recommended individualized treatment approaches for LDL-C reduction based on an individual patient’s CV risk category (Grundy et al., 2019). Cholesterol absorption inhibitors and PCSK9 inhibitors are the only drugs that are currently recommended as second-line treatment options as add-on therapies for patients with a high risk of CV who require additional LDL-C reduction. Patient access to PCSK9 inhibitors has been challenging due to expense and the refusal of payers to provide an appropriate cover. Inhibitors of cholesterol absorption remain as an optional choice with cheap expense; however, LDL-C reduction and the relative risk reduction of cholesterol absorption inhibitors are relatively small when compared to statins or PCSK9 inhibitors. Therefore, it appears to be important and of clinical significance to identify poor responders and non-responders and get rid of them for more effective interventions.

Elevated cholesterol is an established risk factor for cardiovascular (CV) disease. Hyzetimibe represents a new inhibitor of cholesterol absorption and provides an additional therapeutic option for high-risk patients who cannot meet clinical goals with standard forms of therapy (maximally tolerated statin). The IMPROVE-IT trial first demonstrated that the coadministration of an inhibitor of cholesterol and a statin was associated with a lower incidence of cardiovascular events than statin monotherapy (Cannon et al., 2015; Giugliano et al., 2018). The EWTOPIA 75 trial further demonstrated the clinical benefits of monotherapy with cholesterol absorption inhibitors in individuals aged ≥75 years with elevated levels of low-density lipoprotein cholesterol (Ouchi et al., 2019). However, the modest treatment effects of hyzetimibe on LDL-C levels and the significant variability in response to this drug demands a comprehensive and targeted study of gene variation and the effects of such variation on drug response. The g1679C > G SNP in the NPC1L1 gene is critical and can exert notably different impacts on the response to hyzetimibe treatment. Patients who are heterozygous (the GC genotype) will exhibit poor efficacy with hyzetimibe monotherapy. However, when co-administered with atorvastatin, patients with heterozygous genotypes achieved much better improvements in LDL-C levels. The results of our trial indicate that the precise treatment of hypercholesterolemia with hyzetimibe would be beneficial for patients and highlights the potential necessity for identifying the most appropriate subjects for treatment with ezetimibe.