Enhancement of Statin Effects on Lipid Lowering and Reduction of Cardiovascular Risk Score by (−)‐Epicatechin in Proof‐of‐Concept Pilot Study

ABSTRACT

Statins play an instrumental role in reducing and managing atherosclerotic cardiovascular disease (ASCVD) but can be difficult to tolerate due to muscle‐associated side effects. There remains an unmet need for strategies that improve statin tolerance and synergize their effect on atherogenic lipids. (−)‐Epicatechin (Epi) is a natural flavonoid that can improve lipid biomarkers and mitochondrial function. This study explored the capacity of Epi to augment statin's beneficial effects on lipid profile and ASCVD risk parameters. In total, 19 patients completed a randomized, double‐blind placebo‐controlled trial. The study consisted of two cohorts. Cohort 1 consisted of healthy patients with elevated low‐density lipoprotein (LDL) > 100 mg/dL and was used to determine appropriate Epi dosing. Cohort 2 consisted of patients with metabolic syndrome. Patients were randomized into statin‐only (n = 8; 5 in Cohort 2) or statin + Epi (n = 11; 8 in Cohort 2) for 3 months. VO₂ max and lipid biomarkers were assessed at baseline and at the end of 3 months. Final analysis included Cohort 2 only. The statin + Epi group saw significant beneficial changes in total cholesterol (p = 0.002) and non‐HDL cholesterol (p = 0.007). There was a significantly larger increase in HDL (p = 0.037) and significantly greater decrease in LDL particle number (p = 0.0003) and small LDL particle number (p = 0.003) among the statin + Epi group compared to statin‐only. Ten‐year ASCVD risk was significantly lower at end‐of‐study for the statin + Epi arm compared to statin‐only (p < 0.05). No VO₂ max differences were found. This is the first proof‐of‐concept study to show combination therapy of a statin with Epi is safe and effective in augmenting statin‐associated improvements in lipid biomarkers.

Trial Registration: ClinicalTrials.gov: NCT02490527

1. Introduction

Statins reduce all‐cause and cardiovascular mortality and major adverse cardiovascular events (i.e., myocardial infarction, stroke) and are the cornerstone of managing patients with atherosclerotic cardiovascular disease (ASCVD) [1, 2, 3]. Guidelines from the 2022 US Preventive Services Task Force recommend statins be used as primary prevention of ASCVD for individuals 40–75 years old with one or more ASCVD risk factors and ≥ 10% 10‐year risk of ASCVD [3, 4].

Recent findings indicate that the development of atherosclerosis and associated cardiovascular risk is not only dependent on triglyceride, total cholesterol, high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL) levels but also on LDL particle size and LDL particle number [5]. Indeed, small, dense LDL particles are now recognized as integral to the development of atherosclerosis [5]. While statins remain the gold standard of lipid‐lowering therapy, some patients with uncontrolled cholesterol have either complete or partial statin intolerance and may require more costly, less accessible alternatives such as LDL apheresis and proprotein convertase subtilisin/kexin type 9 inhibitors [6, 7]. Statin intolerance occurs in 5%–30% of patients, and adverse side effects are frequent reasons for statin discontinuation [8]. It is postulated that mitochondria may be a therapeutic target to block statin‐associated toxicity [9]. As such, there is an unmet clinical need for cost‐effective, innovative strategies that synergize with statins to decrease atherogenic lipids without the limitations of statin intolerance that may occur with increasing dosages of statins [10].

(−)‐Epicatechin (Epi), a natural flavonoid that appears as the primary mediator in dark chocolate's beneficial health effects, has shown promise in reducing ASCVD incidence and improving blood pressure, lipid biomarkers, cardiovascular, and all‐cause mortality [11, 12, 13]. Epi has beneficial effects on mitochondrial structure, function, and biogenesis, and such actions correlate with positive effects on multiple cardiometabolic endpoints [14, 15, 16]. We previously reported that Epi significantly improved lipid biomarkers and skeletal muscle mitochondrial content and function in a trial of sedentary adults [14]. Given its lipid‐lowering effects we wished to explore the capacity of Epi to augment statin's beneficial effects on lipid profile and ASCVD risk parameters.

2. Methods

This clinical study was approved by the University of California, San Diego (UCSD) Institutional Review Board (IRB #150171, approved June 2015), and conducted in accordance with the Helsinki Declaration of 1975 (as revised in 1983). All patients provided informed written consent.

2.1. Study Design

This was a pilot proof‐of‐concept double‐blind, placebo‐controlled trial in which eligible patients were randomized into either a statin‐only or statin + Epi group for 3 months. This study involved two cohorts. The first cohort included six patients total (three statin‐only, three statin + Epi) and was enrolled from 2016 to 2018. The second cohort was enrolled from 2018 to 2021 and comprised 13 patients (five statin‐only, eight statin + Epi). Both the research team and patients were blinded to the treatment. Patients were unblinded upon study completion or study withdrawal.

2.2. Patient Selection

Patients were screened and recruited from UCSD Health internal medicine, endocrinology, and cardiology clinics from 2015 to 2021. Patients were randomized to either statin alone or Epi plus statin groups (Figure 1). In Cohort 1, patients aged 30–75 years old with an LDL > 100 mg/dL or on statin therapy and VO₂ max > 25 mL/kg/min, and without ASCVD were enrolled. For Cohort 2, eligibility criteria were changed to individuals aged 30–75 years old with LDL > 100 mg/dL or on statin therapy, VO₂ max > 18 mL/kg/min, and metabolic syndrome defined as triglycerides > 150 mg/dL (or on drug treatment for elevated triglycerides) with at least 2 of the following: waist circumference ≥ 88 cm (female) or ≥ 102 cm (male), fasting glucose ≥ 100 mg/dL (or on drug treatment of elevated blood glucose), reduced HDL‐C < 40 mg/dL (men) or < 50 mg/dL (women) (or on drug treatment for reduced HDL‐C), and systolic blood pressure ≥ 130 mmHg and/or diastolic blood pressure ≥ 85 mmHg (or on drug treatment for hypertension). Patients with a history of ASCVD were excluded. Eligibility criteria were changed after an interim analysis that showed no differences between the statin‐only and statin + Epi groups in Cohort 1, which consisted of healthy subjects. In addition, the dose of Epi was increased from the pilot study (from 50 mg daily to 50 mg twice daily) as it was felt the dosing may be too low.

FIGURE 1.

Flowchart overview of patients from screening to study completion. A total of 50 patients were screened, 19 were randomized (5 in Cohort 1, 13 in Cohort 2), and 19 completed the study.

For both cohorts, patients with a history of liver or kidney disease, acute or chronic infectious disease, autoimmune/inflammatory disease, cancer, chronic obstructive pulmonary disease, anemia, insulin‐dependent diabetes, smoking, illicit drug use, and/or psychiatric diagnosis were excluded. Patients with a history of previous knee surgery, active history of knee pain, neuromuscular disease, or musculoskeletal conditions that prevented exercise on a stationary bike were excluded. Patients taking medications that interfere with statin metabolism (e.g., calcium channel blockers, colchicine) were excluded. Patients on statin medication before study enrollment were required to undergo a washout period of 30 days before their screening visit.

2.3. Cardiopulmonary Exercise Protocol (CPX)

Patients completed CPX during their screening visit to determine study eligibility. CPX with exhaled gas analysis was performed after an overnight fast [17]. Caffeine and alcohol were prohibited for 18 h before the test. Breath‐by‐breath gas analysis was obtained during 30 min of rest and during ramped exercise on a stationary bicycle. Before each gas collection, the Orca cardiopulmonary exercise testing system with a CardioPulmonary Technologies pulsed non‐dispersive infrared spectroscopy CO₂ sensor and Oxigraf laser diode absorption spectroscopy oxygen sensor was calibrated with a known gas mixture. We measured VO₂ max during their screening visit to determine study eligibility and establish a baseline VO₂ max, as well as after study intervention.

2.4. Intervention

Pure Epi powder (> 98% purity) was purchased from Sigma‐Aldrich (Sigma‐Aldrich E1753). Epi or placebo was identically encapsulated by UCSD's investigational pharmacy using good manufacturing practices. In Cohort 1, the statin‐only group took 40 mg simvastatin and placebo orally once daily for 3 months (n = 3). The statin + Epi group was started on 40 mg simvastatin and 50 mg Epi orally once daily for 3 months (n = 3). In Cohort 2, the statin‐only group took 40 mg simvastatin daily and placebo orally twice daily (n = 5). The statin + Epi group took 40 mg simvastatin daily and 50 mg Epi orally twice daily for 3 months (n = 8). The 50 mg daily dose for Epi in Cohort 1 was chosen based on a previous study that showed 50 mg daily is safe, associated with changes in mitochondrial function biomarkers, and yields steady‐state serum Epi levels of ~200 nM [18]. However, a preliminary analysis showed no significant changes in VO₂ max, ascorbyl radical, LDL, triglycerides, or cholesterol between the statin‐only and statin + Epi. Thus, we increased the daily Epi dose to 100 mg (which has been shown to improve systemic inflammation and the lipid profile in patients with hypertriglyceridemia) [19] in Cohort 2.

To assess compliance with the capsule consumption and possible adverse effects and to review any dietary or lifestyle changes, patients were contacted one to two times per month. Epi levels were checked in the statin + Epi group after 2 weeks of treatment by a non‐blinded research assistant to ensure compliance and plasma Epi levels were in the 200 nM range. The final analysis was performed including data from Cohort 2 only. Data analysis was performed by individuals blinded to the treatment groups.

All patients received blood tests to assess for cardiometabolic health (lipids with NMR and Direct LDL) at the beginning and end of statin‐only or statin + Epi treatment. Patients were required to keep the same baseline activity level and not engage in any new exercise regimens. They were also provided a list of foods to refrain from consuming, such as those with high Epi content (e.g., green tea).

2.5. Statistical Methods

We tested the effects of Epi treatment on the VO₂ max, lipid biomarkers (total cholesterol, high density lipid [HDL] cholesterol, non‐HDL cholesterol, triglycerides and LDL cholesterol), and 10‐year ASCVD risk [20]. Levels were assessed at baseline (if applicable, after the 30‐day washout period) and after the 3‐month intervention period. An unpaired t‐test was performed to assess pre‐ and post‐intervention outcomes (Table 1), and a paired and unpaired t‐test was performed to estimate p‐values for changes in each outcome in each treatment arm (Table 2). Post hoc power was calculated as 0.8.

TABLE 1.

Anthropometric and lipid panel data before and after randomization.

Variable	Statin‐only (n = 5)		p	Statin + Epi (n = 8)		p
	Before	After		Before	After
Age (years)	63.8 ± 1.96			53.38 ± 2.91
Body weight (kg)	85.70 ± 3.91	86.32 ± 3.85	0.723	87.20 ± 4.07	87.78 ± 4.29	0.495
BMI (kg/m2)	30.43 ± 2.16	30.67 ± 2.22	0.687	29.65 ± 0.63	29.83 ± 0.72	0.509
Cholesterol (mg/dL)	229.2 ± 23.82	184.4 ± 15.47	0.129	237.5 ± 9.81	170.9 ± 10.68	0.002
HDL (mg/dL)	43.6 ± 4.0	38.00 ± 3.51	0.078	46.0 ± 3.1	47.88 ± 4.17	0.341
LDL (mg/dL)	143.0 ± 21.08	94.4 ± 21.15	0.0017	156.3 ± 7.61	88.75 ± 6.1	0.0003
Non‐HDL cholesterol (mg/dL)	185.6 ± 21.75	146.4 ± 15.96	0.1776	180.6 ± 13.7	117.12 ± 5.81	0.0045
Triglycerides (mg/dL)	253.8 ± 49.79	280.6 ± 69.54	0.749	252.9 ± 34.01	201.8 ± 42.77	0.099
Triglycerides/HDL	6.496 ± 2.08	8.208 ± 2.587	0.531	5.661 ± 0.83	4.45 ± 1.04	0.096
LDL particle number	1887 ± 197.9	1431 ± 209.9	0.0067	2022 ± 76.53	1286 ± 130.5	0.0035
Small LDL particle number	919 ± 120.6	686 ± 132.4	0.092	1007 ± 60.14	610.1 ± 78.48	0.002
VO2 max (mL/kg/min)	28.69 ± 6.491	28.44 ± 6.353	0.945	27.95 ± 8.848	30.15 ± 9.598	0.5809
AHA ACC Risk Estimator of 10‐year ASCVD risk	16.60 ± 3.7	17.12 ± 4.77	0.842	7.1 ± 1.15 (n = 6)	4.95 ± 1.11 (n = 6)	0.387

Note: Bolded font indicates p < 0.05.

TABLE 2.

Average change in lipid biomarkers from baseline.

	Change with statin‐only	Change with statin + Epi	p
Cholesterol (mg/dL)	−48.80 ± 23.48 mg/dL (p = 0.129)	−66.63 ± 13.94 mg/dL (p = 0.002)	0.409
HDL (mg/dL)	−5.5 ± 2.379 mg/dL (p = 0.078)	+0.4286 ± 1.307 mg/dL (p = 0.754)	0.037
LDL (mg/dL)	−48.6 ± 6.53 mg/dL (p = 0.0017)	−67.5 ± 10.05 mg/dL (p = 0.0003)	0.2002
Non‐HDL cholesterol (mg/dL)	−39.02 ± 23.99 mg/dL (p = 0.1776)	−68.14 ± 16.97 mg/dL (p = 0.007)	0.33
Triglycerides (mg/dL)	+26.80 ± 78.22 mg/dL (p = 0.7491)	−52.29 ± 31.10 mg/dL (p = 0.1437)	0.315
Triglycerides/HDL	+1.712 ± 2.5 (p = 0.531)	−1.205 ± 0.62 (p = 0.096)	0.1892
LDL particle number	−455.6 ± 88.4 (p = 0.0067)	−736.14 ± 158.6 (p = 0.0035)	0.0003
Small LDL particle number	−233 ± 105.4 (p = 0.092)	−391 ± 96.26 (p = 0.0066)	0.003
AHA ACC Risk Estimator of 10‐year ASCVD risk	0.52% ± 2.25 (p = 0.842)	−2.12 ± 2.189 (p = 0.387)	0.44

Note: Bolded font indicates p < 0.05.

3. Results

All patients reported no adverse effects with treatment. Anthropometric and lipid panel data before and after randomization for each study arm are shown in Table 1 and Figure 2. In total, 13 patients were enrolled (8 statin + Epi, 5 statin‐only) (Figure 1). In the statin + Epi group, two were female, six were male, and the average age was 53.38 ± 2.91 years old. In the statin‐only group, one was female, four were male, and the average age was 63.8 ± 1.96 years old.

FIGURE 2.

Scatterplot of lipid panel data from baseline to end‐of‐study. Scatterplot data for (A) Cholesterol, (B) HDL cholesterol, (C) LDL cholesterol, (D) Triglycerides, (E) Non‐HDL cholesterol, (F) Triglycerides/HDL ratio, (G) LDL particle number, and (H) Small LDL particle number outcomes. *p < 0.05, **p < 0.01. EC = (‐)‐Epicatechin.

There were no observed changes in VO₂ max for either treatment arm (Table 1). In the statin‐only group, there was a reduction in LDL (p = 0.0017) and LDL particle number (p = 0.0067). The statin + Epi group saw a significant reduction in cholesterol (p = 0.002), LDL cholesterol (p = 0.0003), non‐HDL cholesterol (p = 0.0045), LDL particle number (p = 0.0035), and small LDL cholesterol (p = 0.002).

Changes in lipid biomarkers for each study arm are shown in Table 2. The statin + Epi group saw a significantly larger increase in HDL (p = 0.037) and significantly lower reductions in LDL particle number (p = 0.0003) and small LDL particle number (p = 0.003) compared to the statin‐only arm. End‐of‐study 10‐year ASCVD risk score was significantly lower (p < 0.05) in the statin + Epi group compared to the statin‐only group (Figure 3).

FIGURE 3.

Scatterplot of AHA/ACC 10‐year ASCVD risk estimation. Ten‐year ASCVD risk score was significantly lower at the end of the study for the statin + Epi group compared to the statin‐only group. *p < 0.05. EC = (‐)‐Epicatechin.

4. Discussion

In this novel proof‐of‐concept study, we found simvastatin with Epi supplementation augmented statin's lipid‐lowering effects and reduced 10‐year ASCVD risk in a small cohort of patients with metabolic syndrome. We found no difference in VO₂ max between the statin‐only and statin + Epi groups. Our findings have important clinical implications as Epi supplementation may be especially beneficial in a primary prevention cohort that has partial or complete statin intolerance or that needs further improvements in their lipid profile.

Previous research has noted the potential of combining statins with flavonoid supplements to enhance the reduction of cardiovascular disease risk factors, including lipid biomarkers, markers of oxidative stress and inflammation, and blood pressure [21, 22, 23]. However, we are the first to show combination therapy of a statin with Epi can further enhance lipid biomarker improvement and lower ASCVD risk. Enhanced improvements in lipid biomarkers in the statin + Epi group may be explained by Epi's antioxidant effects on LDL oxidation via free radical scavenging and inhibition of pro‐oxidative enzymes including NADPH oxidase, myeloperoxidase, and lipoxygenases [24, 25]. While Epi's antioxidation mechanism is yet to be fully understood, some suggest Epi prevents oxidized LDL formation and action by inhibiting the MPO/H₂O₂/nitrite reaction system and Cu²⁺. Interestingly, oxidized LDL has been shown to be associated with triglycerides and triglyceride/HDLratio—which saw improving trends in the statin + Epi group [26]. Epi may also embed itself in lipoprotein particle membranes, decreasing membrane peroxidation [27]. Recently published research has also suggested flavanols may increase LDL receptor expression, facilitating greater LDL clearance [28]. Taken together, these actions may contribute to the significantly greater improvements in lipid biomarkers from statin + Epi therapy observed in our study. These mechanisms may also attenuate the pathogenesis of atherosclerosis [29, 30]. While our findings provide initial evidence of Epi's beneficial effects on lipid biomarkers, further study is needed to fully elucidate its mechanism. For instance, studies should investigate changes in inflammatory, oxidative stress, and antioxidant‐related biomarkers, such as C‐reactive protein and interleukin‐6.

Importantly, Epi supplementation can serve as an important clinical tool and easier to tolerate alternative to high‐intensity statins, particularly for high‐risk primary patients, which recent studies such as the CLEAR OUTCOMES (Cholesterol Lowering via Bempedoic Acid, an ACL‐Inhibiting Regimen) trial have shown is an undertreated patient population [31]. Notably, Epi is commonly found in US diets, household pantries, and grocery stores, including dark chocolate, ginger, blackberries, apples, pears, and green and black tea, while prior research on statin‐flavonoid combination therapy has been done with flavonoids that are inaccessible for most individuals (i.e., chokeberries, buckwheat‐enriched bread) [15, 16, 21, 22]. Thus, daily supplementation of 100 mg of Epi through diet and Epi medication can be reasonably suggested as an adjunct to statin therapy.

This study has some limitations to consider. The first is the small number of participants enrolled, which may limit statistical power. However, small study samples are vulnerable to type II (false‐negative) errors, but we found several statistically significant results. Second, a longer study duration may be warranted to assess potential long‐term effects of combined statin + Epi therapy. It is also important to note the imbalance of women and men in the study, which may be emblematic of the wider issue of underrepresentation of women in clinical trials as well as lower rates of statin prescription and use among statin‐eligible women [32, 33]. Finally, in future studies it would be important to assess lipid biomarkers at different time points to verify biomarker changes over time. Despite these limitations, our study provides important initial evidence of Epi's promise in enhancing statin therapy and future research with larger sample sizes is warranted.

In conclusion, this is the first proof‐of‐concept study to our knowledge to show that combination therapy of simvastatin with Epi among patients with metabolic syndrome is safe and effective in augmenting statin‐associated improvements in lipid biomarkers. Given that this is a pilot study, further studies are needed with larger cohorts to confirm our findings.

Author Contributions

C.K.O., F.V., and P.R.T. wrote the manuscript; F.V. and P.R.T. designed the research; C.K.O., A.P., A.R., and P.R.T. performed the research; C.K.O., G.C., F.V., and P.R.T. analyzed the data.

Conflicts of Interest

Dr. Pam Taub reports a relationship with Novartis Pharmaceuticals Corporation, Esperion Therapeutics Inc., Amarin, Amgen Inc., Novo Nordisk Inc., Medtronic Inc., Edwards Inc., and Boehringer Ingelheim, Lilly that includes consulting or advisory. Drs. Taub and Villarreal (co‐founders) and Dr. Ceballos are stockholders of Epirium Bio Inc. All other authors declared no competing interests for this work.

Supporting information

Figure S1

Table S1