Pleiotropic Effects of Pure Statin May Provide More Cardiac Benets than Ezetimibe-Statin in a Comparable Intensity for Type 2 Diabetes Mellitus Patients with Extremely Atherosclerotic Cardiovascular Disease Risks

Background: Atorvastatin 40mg (ATOR 40) and ezetimibe 10mg/simvastatin 20mg (EZ-SIM 20) have comparable reductions of low-density lipoprotein cholesterol (LDL-C) but cardiovascular (CV) outcomes between these two therapies are unclear. Our real-world cohort study is to test the hypothesis of pleiotropic effects of purely higher dose statin on CV outcomes beyond comparable reductions of LDL-C, especially for extremely CV risk patients. Methods: Between January 1, 2007 and December 31, 2013, a total of 3372 patients with type 2 diabetes mellitus (T2DM) admitted due to acute coronary syndrome (ACS) or acute ischemic stroke (AIS) were selected as the study cohort from the Taiwan National Health Insurance Research Database. Clinical outcomes were evaluated by ATOR 40 group (n=1686) matched with EZ-SIM 20 group (n=1686). Primary composite outcome includes CV death, non-fatal myocardial infarction, and non-fatal stroke. Secondary composite outcome includes hospitalization for unstable angina (HUA), percutaneous coronary intervention (PCI), and coronary artery bypass grafting (CABG). Results: With a mean follow-up of 2.4 years, no signicant difference of primary composite outcome was observed between ATOR 40 and EZ-SIM 20 groups (subdistribution hazard ratio [SHR], 1.09; 95% condence interval [CI], 0.95–1.25). Nevertheless, ATOR 40 group had lower risks of HUA (SHR, 0.50; 95% CI, 0.35–0.72), PCI (SHR, 0.82; 95% CI, 0.69–0.97) and CABG (SHR, 0.62; 95% CI, 0.40–0.97) than EZ-SIM 20 group. Conclusions: For T2DM patients after ACS or AIS, ATOR 40 may have lower risks of HUA, PCI and CABG than EZ-SIM 20.


Introduction
Type 2 diabetes mellitus (T2DM) is considered as a cardiovascular (CV) disease equivalent and is at risks of CV events and mortality which are two to four times than those in the general population [1,2]. In patients with T2DM, those with myocardial infarction or ischemic stroke are vulnerable for the further CV events [3,4] and even classi ed as extremely atherosclerotic cardiovascular disease (ASCVD) risk patients by a recent de nition [5]. For these patients, high-intensity statins should be administered to achieve low-density lipoprotein cholesterol (LDL-C) ≥ 50% reduction or to meet the individual goal of LDL-C according to the different guidelines [5][6][7][8]. For either primary or secondary prevention of CV diseases, the concept of "the lower, the better" is veri ed from the results of numerous trials 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) and landmark studies of non-statin LDL-C lowering agents, such as ezetimibe [9,10] or proprotein convertase subtilsin-kexin type 9 (PCSK9) inhibitors [11,12]. However, one clinical question could be interesting and that is "Is there a different CV outcome between pure statin and ezetimibe-statin therapies when having a comparable reduction of LDL-C or goal ?" Atorvastatin 40 mg (ATOR 40) and ezetimibe 10 mg/simvastatin 20 mg (EZ-SIM 20) theoretically have a similar and comparable LDL-C lowering effect [13,14] and could be categorized as a high-intensity LDL-C lowering agent [15,16]. Nevertheless, ATOR 40 may signi cantly have more reduction of high sensitivity C-reactive protein (hs-CRP) compared to EZ-SIM 20 (reduction of hs-CRP: 28.6% versus 21.4%) [14].
Besides, the pleiotropic effects of statins, such as decreasing oxidative stress, improving endothelial dysfunction, lowering endothelial cell apoptosis, alleviating in ammation and bene cial to the immune system which are independent of LDL-C reduction and regardless of the level of LDL-C have been supported by the literatures [17][18][19]. Some researches which were conducted with randomizing healthy volunteers or patients with coronary artery disease by comparing higher dose statins with a combination of lower dose statins and ezetimibe revealed more improvements in endothelial function and vascular in ammation by the higher dose statins in a comparable reduction of LDL-C [20][21][22][23] but some other studies did not observe the same ndings [24][25][26]. Therefore, even in a comparable reduction of LDL-C, whether purely higher dose statins are better than a combination of ezetimibe and lower dose statins is still inconclusive. Moreover, these studies are not designed speci cally to examine the major and solid CV outcomes, such as CV death, non-fatal myocardial infarction (MI), non-fatal stroke, hospitalization for unstable angina (HUA), percutaneous coronary intervention (PCI), and coronary artery bypass grafting (CABG). Therefore, although there is a similar and comparable LDL-C lowering effect from ATOR 40 and EZ-SIM 20, the CV outcomes between the purely higher dose statin and ezetimibe-statin therapies are still unclear, especially for extremely CV risk patients who tend to be more vulnerable to suffer from further major CV diseases within a relatively short period and could signi cantly get more CV bene ts with adequate treatments because the effects of statins are better in secondary prevention than those in primary prevention. Given the uncertain CV effects between ATOR 40 and EZ-SIM 20 on patients with extremely CV risks, we used data from Taiwan's National Health Insurance Research Database (NHIRD) to conduct a nationwide and population-based cohort study to test the hypothesis of pleiotropic effects of the purely higher dose statins playing a role in CV outcomes beyond comparable reductions of LDL-C by analyzing T2DM patients after acute coronary syndrome (ACS) or acute ischemic stroke (AIS) who were treated ATOR 40 and EZ-SIM 20, respectively.

Data source
The National Health Insurance (NHI) program in Taiwan is since 1995 and covers the medical needs of 99.8% of 23 million people in the country. All standardized information and data of in this healthcare services are prospectively recorded by the NHIRD and contains inpatient and outpatient data, including date of birth, sex, diagnosis codes (International Classification of Diseases, Ninth Revision, Clinical Modification [ICD-9-CM] codes), drug prescriptions, surgical procedures, admission dates, hospitalizations, discharge dates, and expenditure which has been validated by the previous researches [27][28][29][30][31]. The ethics approval and protocol of this study were approved by the Ethics Institutional Review Board of Linkou Chang Gung Memorial Hospital.

Identi cation of study cohort
The current study was a nationwide population-based, observational and open cohort using retrospectivecollected data from the NHIRD between January 1, 2007 and December 31, 2013. We identified patients with diagnoses of T2DM (excluding type 1 diabetes mellitus) between January 1, 2007 and December 31, 2013. Only 121,760 T2DM patients admitted with a principal diagnosis of ACS or AIS were included for analysis. After relevant exclusion, 6,959 patients with T2DM aged ≥40 years who were admitted due to ACS or AIS were eligible for analyses and after propensity score matching (PSM) in a 1:1 ratio, a total of 3,372 subjects (1,686 subjects in the ATOR 40 group and 1,686 subjects in the EZ-SIM 20 group) were included into the nal analysis ( Figure 1). The de nition of the index date was the date of discharge. The follow-up period was based on the index date to the date of death or December 31, 2013.

Exposure of study statin
During the study period, patients received these two kinds of statin-containing drugs according to the lipid-lowering guidelines in Taiwan. T2DM patients with elevated level of LDL-C ≥130 mg/dL received NHI-paid LDL-C lowering agents to reach the therapeutic goal of LDL-C. The treatment goal of T2DM patients with established CV diseases by NHI in Taiwan was less than 100 mg/dL during the study period. Eligible patients who received study drugs within 30 days of the index date were divided into two groups according to the prescribed LDL-C lowering agents, the ATOR 40 group and the EZ-SIM 20 group, respectively. This de nition of exposure of study drugs was reported in our previous publications [32][33][34].

Ascertainment of T2DM, ACS and AIS
The diagnosis of T2DM is validated according to ICD-9-CM codes where at least four visits of outpatient corresponded to an accuracy of 95.7% and with prescriptions of oral anti-diabetic agents corresponded to an accuracy of 99% [35]. We identi ed patients with T2DM based on diagnosis code and anti-diabetic agents simultaneously. The inclusions of ACS and AIS were requiring a principal diagnosis of admission. The diagnosis codes of ACS and AIS have been validated in previous NHIRD studies that have obtained high positive predictive values with ≥95% [27,29,30].

Covariates measurements
Comorbidities and histories of event at baseline were identified based on ICD-9-CM diagnosis codes (Supplemental Table 1). Comorbidities were de ned as at least two outpatient visits or anyone inpatient diagnosis in the previous year of the index date. Histories of event was detected using anyone inpatient diagnosis before the index date which can be tracked to year 1997. The baseline medication was defined as the medications prescribed in the outpatient visits or the re ll in the pharmacy within 30 days of the index date.
2.6. Ascertainment of primary, secondary and safety outcomes The primary composite outcome was an endpoint of CV death, non-fatal MI, and non-fatal stroke. The occurrences of ACS and AIS were de ned as a principal inpatient diagnosis. The de nition of CV death is the criteria of the Standardized De nitions for Cardiovascular and Stroke Endpoint Events in ClinicalTrials by the FDA in the United States.
The secondary composite CV outcome was de ned as hospitalization for unstable angina (HUA), percutaneous coronary intervention (PCI), and coronary artery bypass grafting (CABG). The occurrence of HUA was de ned as a principal inpatient diagnosis. Information of PCI and CABG was extracted using the Taiwan NHI reimbursement codes of inpatient claims. Other secondary outcomes included hospitalization for heart failure (HHF), and all-cause mortality [36].
The safety outcomes were de ned as hemorrhagic stroke, acute hepatitis, rhabdomyolysis, newly diagnosed dementia and newly diagnosed cancer during the period of follow-up.

Statistical analysis
To decrease bias due to confounding when comparing treatment effects between the ATOR 40 and EZ-SIM 20 groups, we conducted a propensity score matching analysis. The propensity score was the predicted probability of being the in one group given the values of covariates in the logistic regression. The selected covariates to calculate propensity score were listed in Table 1 where the follow up year was replaced with the index date. We adopted a greedy nearest neighbor algorithm with a caliper of 0.2 times the standard deviation of the logit of propensity score, with random matching order and without replacement. Matching quality was assessed using the absolute value of the standardized difference (STD) between the groups after matching, where a value lower than 0.1 represented negligible difference between the groups.
We compared the risks of all-cause mortality between the groups by using the Cox proportional hazards model. The risks of other time-to-event outcomes in the two groups were compared using the Fine and Gray subdistribution hazard model that considered death a competing risk. The within-pair clustering of outcomes after propensity score matching was accounted for by using a robust standard error (the marginal model) [37]. The unadjusted cumulative incidence function of the outcomes was calculated and plotted under a subdistribution hazard model. The study group (ATOR 40 versus EZ-SIM 20) was the only explanatory variable in survival analyses.
A post-hoc subgroup analysis was performed to determine whether the subdistribution hazard ratios of secondary composite CV outcomes for the ATOR 40 and EZ-SIM 20 groups were similar in the subgroups. A two-sided P value of less than 0.05 was considered statistically signi cant and no adjustment for multiple testing (multiplicity) was made in this study. All statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA), including the procedures of 'psmatch' for PSM, 'phreg' for survival analyses, and the macro of '%cif' for the cumulative incidence function.

Demographics and clinical characteristics
In total, 6,959 patients with T2DM who were admitted for ACS or AIS between January 1, 2007 and December 31, 2013, were eligible for this study. Of those, 5,249 (74.4%) patients were prescribed with ATOR 40 and 1,710 (24.6%) ones were prescribed with EZ-SIM 20. After application of PSM with 1:1 ratio, 1,686 patients in either groups ( Figure 2). After PSM, all values of absolute STD were lower than 0.1, which stands for negligible differences in demographics, comorbidities, and medications at baseline between these two groups (right panel of Table 1).
The mean follow-up period was 2.4 years (standard deviation [SD], 1.6 years) and the maximum followup duration was 6.9 years. The mean age of the patients at baseline was 66.2 years (SD, 10.6 years). The most common co-morbidity was hypertension (86.7% versus 87.8%), followed by dyslipidemia (73.6% versus 74.4%) and chronic kidney disease (33.2% versus 33.7%) in the ATOR 40 group and the EZ-SIM 20 group, respectively. In addition, patients with old MI and old ischemic stroke in the ATOR 40 group were 7.9 and 19.5%, respectively; in the EZ-SIM 20 group, those with old MI and old ischemic stroke were 8.1 and 19.5%, respectively (Table 1).

Primary composite outcome
The primary composite outcome occurred in 422 patients (25.0%) in the ATOR 40 group and 398 patients  (Table 2).

Subgroup analysis
There was a signi cant result of the secondary composite outcome between these two groups. Therefore, a post-hoc subgroup analysis was performed to evaluate whether the SHRs of the secondary composite outcome were similar in the selected subgroups. The results demonstrated that the effect of ATOR 40 did not differ signi cantly in the subgroups of age, gender, duration of T2DM, admitted due to ACS or AIS, heart failure, chronic kidney disease, atrial brillation, hypertension, and dyslipidemia ( Figure 4). Noticeably, the values of all SHRs were less than 1 which favored the ATOR 40 group regardless of the statistical signi cance.

Discussion
In this nationwide, population-based, non-crossover and observational cohort study, we tested the hypothesis of "the pleiotropic effects of purely higher dose statin may play a role in CV bene ts than a combination of ezetimibe and lower dose statin in a comparable reduction of LDL-C". Although the primary composite outcome (CV death, non-fatal MI and non-fatal stroke) was no different between the ATOR 40 group and the EZ-SIM 20 group in T2DM patients after ACS or AIS, treatment with ATOR 40 had the lower risks of HUA, PCI and CABG than EZ-SIM 20 which is compatible with coronary plaque stabilization is independent of LDL-C reduction [38]. The use of ATOR 40 reduced the risk of HUA by 50%, PCI by 18% and CABG by 38% with a mean follow-up of 2.4 years. For decreasing risks of HUA, PCI and CABG, the numbers needed to treat were 41.7, 37 and 100, respectively. There was a consistent bene t of ATOR 40 versus EZ-SIM 20 on the secondary composite outcome across all subgroups.
As studies aforementioned in the section of introduction, whether statin's clinical CV bene ts are partially due to its pleiotropic effects or just due to the reduction of LDL-C and even in a equal intensity of LDL-C lowering, whether purely higher dose statin is better than a combination of ezetimibe and lower dose statin are still debated; positive ndings were limited to the discovery of improvements of endothelial function and vascular in ammation which could only be a surrogate marker for CV outcomes in our realworld patients. Therefore, the valuable strength of our research is that it is the rst real-world and nationwide population-based cohort study to evaluate the solid CV outcomes of the pleiotropic effects of statins at the similar reduction of LDL-C and provided the evidence of the pleiotropic effects on extremely CV risk patients who tend to be vulnerable to the further major CV diseases over a relatively short period and could signi cantly get more cardiac bene ts with the pleiotropic effects by the purely higher dose statin beyond a comparable reduction of LDL-C. In other words, our study complemented to answer the question from the IMPROVE-IT study that "We know the lower is the better but is there a different CV outcome between pure statin and ezetimibe-statin therapy when having a similar LDL-C reduction or goal?" and our answer could be yes based on reducing risks of HUA, PCI and CABG by ATOR 40 compared with EZ-SIM 20 in the present study which could imply the pleiotropic effects made these differences. Nevertheless, our results of study still supported the concept that LDL-C is a key role in the pathogenesis of ASCVD [39,40] and the capacity of medication to lower of LDL-C regardless of statin or non-statin therapies (ezetimibe and PCSK9 inhibitors) is de nitely the rst concern rather than the pleiotropic effects because the primary composite outcome (CV death, non-fatal MI, and non-fatal stroke) in our study showed no difference between the ATOR 40 group and EZ-SIM 20 group. The similar or equivalent reduction of LDL-C brings the similarly major CV outcomes.
However, according to our data, something in detail should be more addressed as follows. Although the reduction of LDL-C of ATOR 40 is similar to EZ-SIM 20, the reduction of LDL-C by ATOR 40 is still less than that by EZ-SIM 20 numerically according to the previous data; the average reductions of LDL-C by ATOR 40 is 48.3-49% [14,[41][42][43] but those by EZ-SIM 20 is 50.6-51.9% [14,44,45]. As a result, in theory, based on the concept of "the lower, the better", EZ-SIM 20 could be more effective to reduce ASCVD complications but our ndings were inconsistent. For CV death and non-fatal stroke, the SHR of ATOR 40 versus EZ-SIM 20 was worse insigni cantly; for non-fatal MI, HUA, PCI and CABG, the SHR of ATOR 40 versus EZ-SIM 20 were better (insigni cant for SHR of non-fatal MI but signi cant for SHRs of HUA, PCI and CABG. But because of the discrepancy in these ndings, they highlighted the role of pleiotropic effects which could contribute to these results. The pleiotropic effects could compensate for less reductions of LDL-C by ATOR 40 and resulted in no signi cant differences in the primary composite outcome. Moreover, the pleiotropic effects, especially the ability for anti-in ammation may have more in uence on coronary atherosclerosis than coratid atherosclerosis which is the pathogenesis of large artery ischemic stroke. The destabilization of atherosclerotic lesions due to systemic in ammatory could lead to the plaques rupture and subsequent thrombosis. The levels of hs-CRP level are associated with higher risks for coronary atherosclerosis but not for carotid atherosclerosis [46]. ATOR 40 could lower hs-CRP more than EZ-SIM 20 [14]. Besides, the plaque instability due to increased intracellular adhesion molecule-1 (ICAM-1) has been noted in coronary atherosclerosis but not in carotid atherosclerosis [47]. Higher dose of statins may decrease more ICAM-1 [48]. As a result, our study showed patients in the ATOR 40 group may get more cardiac bene ts particularly without decreasing stroke events than those in the EZ-SIM 20 group.

Limitation
Our present study has some limitations. First, the record of smoking was lacking in the NHIRD database and smoking is an important confounding factor of CV outcomes. Therefore, chronic obstructive pulmonary disease (COPD) was used as a proxy variable instead of smoking for matching at baseline to mitigate this important confounding factor because smoking is strongly associated with the prevalence of COPD [49]. Second, because the lab data in the NHIRD database were not available, the actual effects of lipid-lowering agents were unknown and the patients could only be assumed to have theoretically therapeutic effects based on previous literatures. Finally, unfortunately in this claim database study, we had di culty in clarifying the subtypes of the upcoming ischemic stroke. Because the strong association between cholesterol and carotid atherosclerosis supporting that cholesterol is a main pathogenesis of the large artery ischemic stroke, the large artery ischemic stroke is one subtype of ischemic strokes that could be proven to get bene ts from statins treatment. This may limit the further discussion of the pleiotropic effects on stroke events because the bene cial effects of statins therapy on the other subtypes of ischemic stroke are less certain.
In spite of these limitations, our real-world and nationwide population-based cohort study is still valuable to answer the uncertain question and to ll the gap of evidence of pleiotropic effects by purely higher dose statin on CV outcomes beyond a comparable reduction of LDL-C.

Conclusions
In summary, the pleiotropic effects could play a role on cardiac bene ts with lowering risks of HUA, PCI and CABG beyond a similar reduction of LDL-C. For more cardiac bene ts of T2DM patients after a recent ACS or AIS, ATOR 40 could be considered prior to EZ-SIM 20 to achieve the reduction of LDL-C ≥ 50% or the goal of LDL-C and this kind of clinical practice is in line with the concept of current clinical practice guidelines which suggest ezetimibe should be added to statins when patients do not achieve the reduction of LDL-C ≥ 50% or the goal of LDL-C after a maximal and tolerable intensi cation of statins therapy.

Declarations
Ethics approval and consent to participate The protocol of this study and informed consent of all participants were approved by the Ethics Institutional Review Board of Chang Gung Memorial Hospital.

Consent for publication
Authors give full consents for publication of this present article.

Availability of data and materials
This datasets used and analyzed in this study are available from the corresponding author on reasonable requests.

Competing interests
The authors report no nancial relationships or con icts of interest regarding the content herein.  Data were presented as frequency and percentage or mean ± standard deviation.   The cumulative incidence of the secondary composite outcome (A), and the individual outcomes of hospitalization for unstable angina (B), percutaneous coronary intervention (C) and coronary artery bypass grafting (D) in the propensity score matched cohorts