Association Of NPC1L1 Gene Polymorphisms With The Response To Hyzetimibe

Hyzetimibe(HS-25)is the second cholesterol absorption inhibitor with a completely new chemical structure.Preclinical studies had shown that the lipid-lowering efficacy of hyzetimibe was comparable to that of ezetimibe,the first cholesterol inhibitor,and had a higher safety profile.The sterol carrier Niemann-Pick C1-like 1(NPC1L1)protein expressed at the apical membrane of enterocytes is the molecular target of cholesterol absorption inhibitor,and its gene polymorphism may exert a potential impact on the response of these drugs.Therefore,the experiment in cells about the inhibition of NPC1L1 by hyzetimibe was carried out firstly,and isotope labeling technology was adopted to investigate the main existence form,level and maintaining time of the drug in the small intestine,the receptor target organ in healthy volunteers.On the basis of these observation,a multicenter clinical trial was conducted to evaluate the response to drug treatment with hyzetimibe,then the association between lipid-lowering efficacy and NPC1L1 gene polymorphism was analyzed for identifying right SNPs affecting drug efficacy and providing the proof for better personalized lipid-lowering therapy.Objective:1)To explore the inhibitory activity of NPC1L1 by cholesterol uptake assay study.2)To evaluate the lipid-lowering effect of hyzetimibe through clinical trial.3)To analyze the relationship between the lipid-lowering effects of hyzetimibe and NPC1L1 gene polymorphism.Methods:Inhibitory activity of NPC1L1 in vitro:Using cholesterol uptake test,rat Mc Ardle RH7777 hepatoma cells which stably express human fusion protein NPC1L1-EGFP were used as the model.The labeled cholesterol uptake was measured by liquid scintillation technique.Clinical pharmacokinetic study:The main existence form,level and maintaining time of the drug in the small intestine was investigated by isotope labeling technique in healthy volunteers.In addition,the study of clinical drug-drug interaction between hyzetimibe and atorvastatin was developed with 3×3 cross-test design was used to lay the foundation for further clinical trials.Clinical study:A multicenter,randomized,placebo-controlled,multiple doses factorial design clinical study was conducted to explore the efficacy and safety of hyzetimibe.720 patients with primary hypercholesterolemia planned to be enrolled were randomized to patients were randomly assigned to 1 of 6 treatments for 12 weeks:placebo,hyzetimibe 10 mg,hyzetimibe 20 mg,atorvastatin 10 mg,atorvastatin(10 mg)plus hyzetimibe 10 mg and atorvastatin(10 mg)plus hyzetimibe20 mg with daily single dose oral.Blood samples were collected regularly for lipid test,drug trough concentration determination.The correlation analysis between efficacy and gene polymorphism:A hybridization capture chip was designed for the target gene NPC1L1.The blood samples were extracted with an automatic nucleic acid extractor and were sequenced by PE150 mode with a high-throughput sequencer.150 bp were measured at both ends of each DNA fragment.The sequencing region covered exon,exon-intron junction,5’-UTR,3’-UTR and promoter of the gene.The correlation between the clinical efficacy and the target protein gene was analyzed combined with drug trough concentration.Results:1)Inhibitory activity of NPC1L1 in vitro:In rat hepatoma cell line Mc Ardle rh7777 stably expressing human fusion protein NPC1L1-EGFP,After methyl-β-cyclodextrin treatment,with the increase of drug concentration(from 0 to 30μM),the radioactive DPM value per milligram cell ranged from 31.35×10~4PDM/mg to 17.47×10~4PDM/mg,hyzetimibe inhibited NPC1L1 dependent cholesterol absorption in a dose-dependent manner,and its activity was equivalent to that of the positive drug ezetimibe.2)Clinical pharmacokinetic study:Just hyzetimibe had abundant distribution and maintain time in the small intestine with rich NPC1L1protein.Hyzetimibewasrapidlymetabolizedinto hyzetimibe-glucuronide(M1),which was was the main component in in plasma and urine.Significant characteristics of enterohepatic circulation was observed and there did not exist significant individual difference in the distribution and absorption of hyzetimibe in the small intestine.In addition,the results of drug-drug interaction study showed that there was no clinically significant interaction between hyzetimibe and atorvastatin.3)Clinical trial of hyzetimibe:A total of 727 subjects were enrolled.The results showed that hyzetimibe 10～20 mg/day could effectively reduce the levels of LDL-C,TC,non-HDL-C and Apo B in patients with primary hypercholesterolemia and combination therapy could further reduce the blood lipid level.Adverse drug reactions were mild,and the safety and tolerability were good.4)DNA extraction and sequencing of 613 samples were completed,and 150 various ectopic sites were found.Four of them were high-frequency sites(MAF>10%),and the frequency distribution of three genotypes of g1679C>G(rs2072183)showed significant population and ethnic differences.As monotherapy,CC carriers experienced significantly higher reductions in mean LDL-C(-23.99%)than either the GG(-16.45%,p<0.01)or GC(-13.02%,p<0.01)carriers in the hyzetimibe(20 mg)group.In contrast,when co-administrated with atorvastatin,GC carriers experienced greater LDL-C reduction than non-GC carriers(-52.23%vs-45.03%)in the hyzetimibe(20 mg)plus atorvastatin group.Furthermore,the proportions of individuals experiencing a reduction in LDL-C by>50%increased as the dose of hyzetimibe increased from 16.1%to65.4%.Conclusions:1)Hyzetimibe inhibited NPC1L1 in a dose-dependent manner to limit cholesterol uptake with a comparable activity to ezetimibe.2)Hyzetimibe alone can effectively reduce the levels of LDL-C,TC,non-HDL-C and Apo B in patients with primary hypercholesterolemia,and combined with atorvastatin can further reduce the levels of various blood lipids.3)The g1679C>G SNP in the NPC1L1gene is critical and exerts differential impact on the response to hyzetimibe treatment.Patients with CC genotype were the target population for hyzetimibe alone and GC genotype for combination therapy.The findings provide a basis for the individualized treatment of reducing cholesterol in patients with primary hypercholesterolemia.