Nuclear Magnetic Resonance (NMR) Spectroscopy on the Effect of 1 PCSK9 Inhibitor on Lipoprotein Particles in Patients With Acute 2 Coronary Syndromes(ACS)

Objective : To assess the effects of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor 2 (evolocumab) on blood lipid level, lipoprotein particles, and their subfractions with Nuclear Magnetic 3 Resonance (NMR) spectroscopy in patients with acute coronary syndromes(ACS). 4 Methods : A total of 99 consecutive patients with ACS and poor lipid control were enrolled and assigned to 5 either the experimental group (n = 54) or the control group (n = 45). The combination therapy of PCSK9 6 inhibitor (Repatha ® , 140mg, q2w) and moderate statin (rosuvastatin, 10 mg, qn) was administered in the 7 experimental group, with moderate statin therapy (rosuvastatin, 10 mg, qn) alone in the control group. The 8 therapeutic effects on blood lipid levels and lipoprotein particle subfractions were assessed with NMR 9 spectroscopy after eight weeks of treatment, and the achievement of LDL-C treatment target in both 10 groups was analyzed . 11 Results : In the experimental group, after eight weeks of evolocumab and moderate statin combination 12 therapy, the level of blood lipids (TC, LDL-C and its subfractions [LDL-1 to 6], VLDL-C and its 13 subfractions [VLDL-1 to 5], IDL-C, and HDL-C), lipoprotein particles, and their subfractions (VLDL-P, 14 IDL-P, LDL-P, and its subfractions [LDL-P1 to 6], apoB, and LP(a)) demonstrated therapeutic benefits with 15 statistical significance (P < 0.05). Lowered level of LDL-P was attributed to the significant decrease of small 16 LDL-P (LDL-P5+6), which was significantly more prominent than the decrease in medium LDL-P (LDL- 17 P3+4) and large LDL-P (LDL-P1+2) (P < 0.001). According to lipid control target recommended by the 18 latest China Cholesterol Education Program (CCEP) Expert Consensus in 2019, heterogeneous and to lipoproteins blood ,

Objective: To assess the effects of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor 2 (evolocumab) on blood lipid level, lipoprotein particles, and their subfractions with Nuclear Magnetic 3 Resonance (NMR) spectroscopy in patients with acute coronary syndromes(ACS). 4 Methods: A total of 99 consecutive patients with ACS and poor lipid control were enrolled and assigned to 5 either the experimental group (n = 54) or the control group (n = 45). The combination therapy of PCSK9 6 inhibitor (Repatha ® , 140mg, q2w) and moderate statin (rosuvastatin, 10 mg, qn) was administered in the 7 experimental group, with moderate statin therapy (rosuvastatin, 10 mg, qn) alone in the control group. The 8 therapeutic effects on blood lipid levels and lipoprotein particle subfractions were assessed with NMR 9 spectroscopy after eight weeks of treatment, and the achievement of LDL-C treatment target in both 10 groups was analyzed.

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Results: In the experimental group, after eight weeks of evolocumab and moderate statin combination 12 therapy, the level of blood lipids (TC, LDL-C and its subfractions [LDL-1 to 6], VLDL-C and its 13 subfractions [VLDL-1 to 5], IDL-C, and HDL-C), lipoprotein particles, and their subfractions (VLDL-P, 14 IDL-P, LDL-P, and its subfractions [LDL-P1 to 6], apoB, and LP(a)) demonstrated therapeutic benefits with 15 statistical significance (P < 0.05). Lowered level of LDL-P was attributed to the significant decrease of small 16 LDL-P (LDL-P5+6), which was significantly more prominent than the decrease in medium LDL-P (LDL-17 P3+4) and large LDL-P (LDL-P1+2) (P < 0.001). According to lipid control target recommended by the 18 latest China Cholesterol Education Program (CCEP) Expert Consensus in 2019, the percentage of patients 19 reaching the treatment target differed significantly between the experimental group and the control group 20 (96.3% and 13.3%, respectively, P < 0.001).  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  , making it the primary subject in the prevention and treatment of cardiovascular diseases to 7 control blood lipids. In the Chinese Guidelines on Prevention and Treatment of Dyslipidemia in 8 Adults issued in 2016, LDL-C was regarded as the most important indicator for early warning, 9 medication modification, and lipid monitoring in ASCVD patients. However, in clinical practice, 10 many individuals with normal or even low concentration of LDL-C (< 70 mg/dL) experience ASCVD-related 11 events or progression of atherosclerosis [3][4] . This residual risk indicates that a focus solely on the measurement of 12 LDL-C is not an optimal strategy for all patients [5,6] . LDL is a heterogeneous lipoprotein fraction comprising 13 different LDL subclasses that vary in size, density, and composition due to continuous remodeling of lipoproteins 14 in the blood [7] , whose chemical components and physiologic functions differ a lot from each other. 15 LDL particles of different sizes might not play the same role in the pathogenesis of ASCVD [8] , 16 indicating that the size of LDL particles closely correlates with their functions, which is of grander 17 clinical significance [9][10][11] . 18 Statins are the first choice for lipid-lowering drugs in clinical applications, which could also effectively 19 decrease the risk of cardiovascular diseases. Nonetheless, studies revealed that even with high-dose statins, 20 cardiovascular events were still of elevated incidence in high-risk patients. Furthermore, some patients show 21 poor tolerance for high-dose statin therapy. In recent years, novel lipid-lowering medications, such as 22 PCSK9 inhibitors, are receiving more attention, and impressive progresses were made in relevant studies. 23 With the conclusion of a series of randomized clinical trials, the novel lipid-lowering medication, PCSK9 24 inhibitors, are gradually proven to be effective in lowering blood lipid levels and preventing cardiovascular 25 diseases [12] . 26 This study aims to evaluate the effect of statins , the traditional lipid-lowering drug, on lipoprotein particles 27 subfractions. In addition, the effect of PCSK9 inhibitor (Repatha®), a novel lipid-lowering drug, on 28 lipoprotein particles subfractions will also be explored. It has been demonstrated in a large number of studies 29 that statins in combination with PCSK9 inhibitor can further decrease LDL-C level by 50% -70% [ 54 At baseline and after 8 weeks of drug therapy, the participants in the two groups were collected peripheral 55 venous blood at the fasting and resting state in the morning for examination .
Routine blood lipid testing: The participants were fasted for 8 hours. The blood was collected in a serum tube 1 containing an inert separating gel. After the blood was fully coagulated, it was centrifuged at 3 000 rpm for 2 10 min, and the supernatant was taken for testing.The levels of plasma TC, HDL-C, LDL-C, and TG were 3 measured by enzymatic measures using Roche c701 automated clinical chemistry analyzer. Apolipoprotein 4 A1 (Apo-A1) and Apolipoprotein B (Apo-B) were measured by immunoturbidimetric methods. Lp(a) was 5 measured by latex enhanced immuno-turbidimetry method. 6 Nuclear magnetic resonance (NMR) spectroscopy testing: 4 ml of the participant's whole blood (fasting for 8 7 h, BD blood vessels containing EDTA-K2 anticoagulant) was collected, centrifuged at 1500 g for 10 min, 8 and transferred the upper plasma into the cryopreserved tube, which was stored at -80 ℃ for future testing. 9 During the test, the samples were taken out of the refrigerator, and after thawing completely, 400μl plasma 10 was taken and mixed with NMRS lipid buffer (Bruker Biospin, USA) 1:1, fully mixed, and then placed in a 5 11 mm NMR tube, and loaded into an automatic sample injector for testing [14] . 12 13 1.3 Nuclear magnetic resonance (NMR) spectroscopy methods and testing program 14 According to the standard operating procedure of AVANCE IVDr magnetic resonance spectrometer 15 system( Bruker Biospin) [15,16] . The spectra were normalized to the same quantitative scale using Bruker's In the experimental group, after a combined therapy of evolocumab and moderate statins for eight weeks, 3 benefits in LDL-C concentrations and other blood lipids measurements were revealed with statistical 4 significance (P < 0.05). TC, LDL-C and its subfractions (LDL-1 to -6), VLDL-C and its subfractions 5 (VLDL-1 to -5), and IDL-C significantly decreased compared to baseline (P < 0.001). The decreased level of 6 LDL-C was significantly attributed to the decrease in the level of small LDL particles (LDL 5+6). In contrast, a significant increase in HDL-C was observed (P < 0.05), which could be attributed to an increase in small 1 HDL particles (HDL 3+4). The levels of TC, LDL-C, VLDL-C and IDL-C were reduced by 48.4%, 65.5%, 2 26.3%, and 62.0%, respectively, while the HDL-C level was increased by 7.6%, compared to baseline. 3 4 After eight weeks of single moderate statins therapy in the control group, levels of TC, IDL-C, and LDL-C 5 significantly decreased (P < 0.05), by 16.9%, 20.7%, and 18.1%, respectively. But the concentration of 6 VLDL-C and HDL-C did not decrease significantly after treatment(P > 0.05). 7 8 After eight weeks of treatment, the absolute reduction in the levels of TC, LDL-C, VLDL-C, and IDL-C in 9 the experimental and control groups were (14.7 ± 15.4 vs. 5.2 ± 12.9), (81.6 ± 28.7 vs. 22.9 ± 25.1), (14.7 ± 10 15.4 vs. 5.2 ± 12.9), and (10.0 ± 7.9 vs. 4.3 ± 6.4), respectively, with significant differences discerned 11 between the two groups (P < 0.05).
12 13 2.4 Changes in lipoprotein particle concentrations after treatment in the two groups 14 Changes in lipoprotein particle concentrations were presented in Table 3. After eight weeks of combined 15 therapy of evolocumab and moderate statins therapy, statistically significant benefits in the concentrations of 16 LDL-P and other lipoprotein particle concentrations were observed (P < 0.05). VLDL-P, IDL-P, LDL-P and 17 its subfractions (LDL-P1 to 6), ApoB and LP(a) all decreased compared to baseline levels (P < 0.001). The 18 concentration of LDL-P before and after medication were 1573.9 ± 375.3 vs 463.6 ± 246.7 respectively, 19 showing a reduction of 71.1% (P < 0.001), which could be accounted for by a decrease in small LDL-P 20 (LDL-P5+6). In our study, LDL-P were further classified into large (LDL-P1+2), medium (LDL-P3+4) and non-high-density lipoprotein cholesterol, and increased HDL-C [20] . In addition, compared to statins, PCSK9 7 inhibitor exhibit stronger efficacy in lowering LDL-C and TC, as well as increasing HDL-C [21] . Compared to 8 statin alone, a combination therapy of PCSK9 inhibitor and statin demonstrates more advantages in lowering 9 LDL-C [22] .Although the effect of evolocumab on LDL-C levels is well characterized, little is known about 10 its effects on lipoprotein particles or particle subfractions.

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It is well-known that lipoprotein particle play an important role in atherosclerosis, including IDL-P, as well 13 as VLDL and LDL-P. More LDL particles could increase the risk of atherosclerosis. Studies found that small 14 LDL particles, which posed a higher threat than larger ones on causing atherosclerosis, were potential 15 predictors of atherosclerosis and coronary artery disease [23] , as LDL particles of smaller size and higher 16 density were more difficult to be removed. Smaller size leads to an increased particle density and three-17 dimensional conformation changes of ApoB. From a pure biophysics perspective, smaller LDL particles 18 were more likely to penetrate the vascular endothelial barrier, because they are smaller and denser, and do 19 not require too much subendothelial space during penetration.LDL particles of smaller size and higher 20 density were also more prone to oxidation, and oxidized LDL particles were recognized as the primary target 21 lipoprotein of sub-endothelial phagocytosis for macrophages. As remnant lipoproteins (small molecule 22 VLDL and IDL) are also found in the atherosclerotic plaques, reduction of remnant lipoproteins is also 23 important in addition to the reduction of LDL. representing the level of circulating numbers of atherogenic lipoproteins, decreased by 60.9%. These 3 revealed significantly different lipid profiles compared to baseline. The decreased level of LDL-C was 4 attributed to decreased small LDL particles (LDL5+6), and the reduction of VLDL-C was due to that of 5 small VLDL particles (VLDL4+5). Eight weeks later, the absolute reductions in TC, LDL-C, VLDL-C and 6 IDL-C level in both the experimental and control groups were (14.7 ± 15.4 vs. 5.2 ± 12.9), (81.6 ± 28.7 vs. 7 22.9 ± 25.1), (14.7 ± 15.4 vs. 5.2 ± 12.9), and (10.0 ± 7.9 vs. 4.3 ± 6.4), respectively, exhibiting differences 8 of statistical significance (P < 0.05). Thus, the combination therapy showed significant advantages over 9 statins alone. 10 11 In terms of lipoprotein particles subfractions, after eight weeks of treatment, the experimental group showed 12 statistically significant benefits in LDL-P concentration and other lipoprotein particles concentrations(P < 13 0.05). VLDL-P, IDL-P, LDL-P and its subfractions (LDL-P1 to 6), and LP(a), were significantly decreased 14 compared to baseline (P < 0.001). The concentration of LDL-P was reduced by 71.1% compared to baseline 15 (P < 0.001), which could be mainly attributed to the reduction in small LDL-P (LDL-P5+6). The level of 16 small LDL-P decreased by 76.8%, to a significantly larger extent compared to medium and large LDL-P (P < 17 0.001). Levels of VLDL-P, IDL-P, and LP(a) decreased by 20%, 53.3%, and 21%, respectively. After eight 18 weeks of treatment, the absolute reductions of which in the experimental group were also more prominent 19 compared with the control group, with statistical significance (P < 0.05). 20 21 It is well accepted that smaller size of lipoprotein particles leads to an increased risk for atherosclerosis [24,25] . 22 In our study, evolocumab exhibited significant advantages in lowering the levels of small lipoprotein 23 particles. After further classifying LDL and VLDL into small, medium, and large particles, we found a 24 significant larger reduction in small LDL (LDL5+6) and VLDL (VLDL4+5), compared to medium and large 25 particles. Meanwhile, the size of LDL-P before and after medication in the experimental group were 20.2 ± 26 0.4 vs 20.9 ± 0.5, respectively, suggesting an increase of 3.6% in the size of lipoproteins after treatment with 27 evolocumab. The FOURIER trial found that evolocumab could reduce the incidence of primary endpoint 28 event by 15%, which might be associated with a decrease of particles which could result in atherosclerosis., 29 in addition to lower levels of LDL-C.  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57