Cardiovascular disease (CVD) accounts for one-third of deaths globally, with 17.8 million deaths reported as a direct consequence in 2017.1 Preventative cardiology has been of great interest, reflected in an increase in publication volume, indicating a greater recognition of the importance of medical interventions at earlier points to reduce the incidence of CVD in patients.2
The use of agents in both primary and secondary prevention has formed a large aspect of preventive cardiology. There is an emerging acknowledgement that inflammatory markers on a molecular level correlate with cardiovascular outcomes. Previous attention on lowering lipid levels in the form of reduction in LDL levels still left patients with significant risk of cardiovascular adverse effects with type 2 diabetes being the biggest contributor to that risk level. Emphasis on screening of patients with type 2 diabetes and hypercholesterolaemia has highlighted that they had a higher level of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α).
It is believed that high cholesterol redirects myeloid progenitor into a state resembling higher activity. The pathology behind this relates to the accumulation of macrophages within the arterial wall, which seems to be driven by monocyte migration under the influence of elevated cytokine levels in the microenvironment. After monocytes have entered the intima, the differentiation into macrophages commences. It is this process that catalyses the progression of the atherosclerotic plaque.3 This immunological prompt seems to be driven by epigenetic changes to the chromatin and so dysfunction of this is associated with persistent immune cell activation and thus a higher incidence of atherosclerosis in blood vessels and subsequent cardiovascular death obtained from mouse models.
It is recognised that bromodomain and extra-terminal (BET) proteins play a prominent role in the transcription of cytokine-responsive genes involved in inflammation lipid metabolism and vascular function.4 Epigenetics refers to heritable changes in gene expression patterns not caused by an altered nucleotide sequence. This includes non-coding RNAs and covalent modifications of DNA and histones.
Research has focused on identifying compounds which were able to subvert the action of BET proteins to modulate activity downstream. The compound RVX-208, also called apabetalone, is a novel, synthetic compound in the family of bromodomain inhibitors, which specifically target BET proteins, genetic readers that modulate gene expression involved in vascular inflammation and calcification. This is demonstrated in vitro by preventing TNF-α, lipopolysaccharide or IL-1β-driven endothelial activation, monocyte recruitment, adhesion and plaque stabilisation.5
Apabetalone is a selective BET inhibitor for bromodomains 2 of mainly bromodomain-containing protein (BRD) 2/3, with low affinity for BRD4. Hence, it primarily acts by increasing apolipoprotein A-I (apoA-I) and HDL cholesterol.6 Apabetalone was able to show significant inhibition of transcription of BET-dependent genes and thus reduce expression of genes involved in pro-inflammatory gene expression in monocytes, endothelial vascular smooth muscle and atherosclerosis in mouse models. Apabetalone downregulates human renal mesangial cell (HRMC) activation by suppressing transforming growth factor β1 (TGF-β1) induced α-smooth muscle actin relocation to stress fibres. This is associated with reduction of cellular contraction as well as extracellular matrix overproduction and fibrosis, which contribute to the progression of chronic kidney disease.6
Apabetalone is known to have a good oral availability and is concentrated in tissues that express apoA-I, such as the small intestine and liver.7
Preclinical and clinical trials assessed the efficacy of apabetalone on conventional and non-conventional risk factors of CVD, such as atherosclerosis, plasma lipoproteins, cholesterol profile and inflammatory markers. Recent phase IIa (ASSERT), IIb (SUSTAIN and ASSURE) and III (BETonMACE) trials have also revealed potential use of apabetalone in the prevention of major adverse cardiovascular events (MACE), particularly among patients with conditions associated with increased BET activity, such as type 2 diabetes, elevated high sensitivity C-reactive protein (hsCRP) and low HDL cholesterol levels.8
Current Studies and Evidence Related to Apabetalone
Apabetalone has been shown to have both lipid-lowering and anti-inflammatory properties in animal studies. A 12-week study in mice showed an elevated circulating HDL cholesterol and decreased LDL cholesterol levels. This was associated with a significant reduction in plasma pro-inflammatory cytokine level and an associated reduction in aortic lesion formation.9 These findings were supported by the phase I clinical trial, where a 1-week trial of apabetalone in 18 healthy participants demonstrated an increase in apoA-I and HDL cholesterol, as well as α and pre-β HDL fractions, which are important substrates in the reverse cholesterol transport pathway.10,11
The ASSERT Phase IIa clinical trial was carried out on 299 patients with stable coronary artery disease (CAD). Participants were given apabetalone or a placebo for a 12-week period, on top of standard statin therapy. Apabetalone was trailed at three dosages: 50 mg, 100 mg and 150 mg, twice daily. There was a significant increase in HDL cholesterol (p=0.003) in the treatment group, more prominent at the higher doses. There was also a dose-dependent increase in apoA-I levels observed with all treatment groups (p=0.035). No difference in the levels of circulating LDL cholesterol particles was identified between the treatment and control groups. The above changes rapidly increased between 8 and 12 weeks of treatment, which suggests that longer courses could potentially result in more profound effects.12
These longer courses were reviewed in phase IIb trials SUSTAIN and ASSURE. SUSTAIN was conducted in 172 patients with atherosclerotic CVD and low levels of HDL cholesterol. Participants were randomised to receive apabetalone 100 mg twice daily or placebo for 24 weeks. An increase in HDL cholesterol (p=0.001) was demonstrated in the treatment group, which was the primary endpoint for this trial. Serum apoA-I (p=0.002) and large HDL particles (p=0.02) were increased as part of the secondary endpoints.7
The percentage change in atheroma volume was the secondary endpoint for ASSURE. In this double-blind trial, 323 participants with angiographic CAD and low HDL cholesterol were randomised to receive apabetalone 100 mg twice daily or a placebo for 26 weeks. Although apabetalone patients had a statistically significant increase in apoA-I and HDL cholesterol and a decrease in LDL cholesterol when compared to baseline, these did not reach significance when compared to placebo.12 Similarly, the total atheroma volume decreased when compared to baseline, with no significant difference between the two treatment groups. The latter was determined by intravascular ultrasound images of matched coronary artery segments at baseline and at 26 weeks.7,8
Further analysis of the 31 patients with coronary atherosclerotic plaques in this study with apabetalone revealed a negative correlation between atherosclerotic plaque (AP) index and HDL concentration (r=−0.52, p=0.006). As AP index decreased, the concentration of HDL particles increased; however, the same relationship was not observed between AP index and HDL cholesterol levels (r=−0.11, p=0.60) or apolipoprotein A-1 (r=−0.16, p=0.43), a reduction of AP length by 1 mm (p=0.003) with a correlated reduction in HDL cholesterol in those individuals.13
Pooled analysis of the 798 patients with CAD from the ASSERT, ASSURE and SUSTAIN trials revealed that a 3–6-month treatment course with apabetalone increased the levels of apoA-I, HCL cholesterol and large HDL particles and decreased hsCRP, with no change on atherogenic lipoproteins compared with placebo.12
BETonMACE is a phase III, double-blind, randomised controlled trial (RCT) conducted with 2,425 patients from 13 countries. Participants had a background of type 2 diabetes, low HDL cholesterol and a recent acute coronary syndrome (ACS) event in the preceding 7–90 days. Apabetalone 100 mg twice daily or placebo was given to patients on top of high-intensity statin therapy, and the patients were followed up for a mean duration of 26.5 months (IQR 20–32 months). More than 90% of patients were treated with inhibitors of the renin–angiotensin pathway, β-blockers and antiplatelet agents. This trial did not demonstrate an altered level of LDL cholesterol, HbA1c or fasting glucose. At 24 weeks, mean HDL cholesterol increased from 33.3 mg/dl at baseline, to 38.0 and 36.6 mg/dl in the apabetalone and placebo groups, respectively.14 From the phase II trials, patients randomised to apabetalone experienced fewer MACE than those receiving placebo (5.9% versus 10.4%, p=0.02). This effect was more prominent in patients with diabetes (5.4% versus 12.7%, p=0.02), with baseline HDL cholesterol <39 mg/dl (5.5% versus 12.8%, p=0.01) or with elevated hsCRP levels (5.4% versus 14.2%, p=0.02).12
The primary outcome for the BETonMACE was a composite of the time to the first occurrence of cardiovascular death, non-fatal MI or stroke, defined as MACE. During this trial, 274 primary endpoints occurred, most commonly acute MI. This was evenly distributed between ST-elevation MI (STEMI) and non-ST-elevation MI (NSTEMI) subtypes. Although the trends appeared encouraging, this trial did not demonstrate a significant change in MACE between apabetalone and placebo.14,15
Effects of Apabetalone on High-sensitivity C-reactive Protein
Chronic systemic inflammation contributes to CVD and correlates with the abundance of acute-phase response (APR) proteins in the liver and plasma. Apabetalone has been shown to reduce basal expression of APR genes and cytokine-induced gene expression and represses CRP gene expression in response to cytokines in vitro and mice studies. Apabetalone was shown to regulate circulating cytokine targets in patients with stable CAD on standard-of-care therapy (n=55) when compared with placebo.16 However, the ASSERT, SUSTAIN and ASSURE trials did not show any significant associations between on treatment hsCRP or absolute change of hsCRP level from baseline to weeks 12–14 and risk of MACE.17 BETonMACE also did not demonstrate a change in the hsCRP for participants on apabetalone when compared to placebo.15
Effects of Apabetalone on Alkaline Phosphatase
The reduction in MACE associated with lower alkaline phosphatase (ALP) is independent of age, sex, race, established cardiovascular risk factors, chronic kidney disease (CKD), baseline ALP, hsCRP, calcium and liver function. This was true at 12–14 weeks and maintained at 24–26 weeks in these trials. Among all patients, each decrease in ALP by 1 SD from baseline to weeks 12–14 was associated with a lower risk of MACE. There was also a similar association with the absolute change in ALP.17,18
Elevated serum ALP has a known link to inflammation and oxidative stress. It independently predicts MACE by contributing to vascular calcification and endothelial dysfunction arising in CKD and CVD. Clinical and non-clinical trials suggest that apabetalone reduced tissue non-specific ALP (TNALP) expression, which accounts for approximately half of the circulating ALP.18 The post hoc analyses of patients recruited in the ASSERT, SUSTAIN and ASSURE trials demonstrated a decrease in ALP in all three studies. From ASSERT, ALP levels were decreased under apabetalone treatment in a dose-dependent manner, with the decrease in ALP observed by week 4 and sustained throughout the study.17 In SUSTAIN, the mean correction of ALP with apabetalone compared with placebo at week 12 was 13.1% (p<0.001) and maintained at 24 weeks. From ASSURE, the mean placebo-corrected reduction of ALP at 14 weeks was 9.9% (p=0.003), which was again maintained at 26 weeks. The pooled analysis showed a mean placebo-corrected decrease in ALP from baseline of 9.2% (p<0.001) after 12–14 weeks and 7.7% (p<0.001) after 24–26 weeks of apabetalone treatment. These changes were not accompanied by changes in other liver enzymes. The placebo groups did not show a change in ALP levels, consistent with phase II trials. This effect was also noted in the BETonMACE trial, which also demonstrated a significant reduction in ALP by 6.8 U/l with apabetalone at 24 weeks.15
Apabetalone and Major Adverse Cardiovascular Events
A meta-analysis of RCTs (n=3,223) demonstrated that apabetalone significantly reduced MACE and hospitalisation for heart failure compared to placebo. No significant differences were observed in death or coronary revascularisation.15,16 In the recently completed phase III cardiovascular outcomes trial (BETonMACE), 2,425 patients with type 2 diabetes and recent ACS were treated with apabetalone or placebo and followed for 26 months. The primary endpoint of the study involved time to MACE defined as cardiovascular death, non-fatal MI or stroke. The methodology adopted in many of these trials had similar approaches in the form of large cohort groups with randomisation.
In the ASSET Phase IIa trial, a large group of patients had exposure to apabetalone or placebo in addition to the standard statin therapy with the treatment group having three subsets of doses, with the measurements of changes in Apo-I levels after a 4-month time interval. The study demonstrated a dose-related positive effect; however, there were no primary endpoint measures.
BETonMACE focused on a post-ACS cohort with those admitted for treatment of either unstable angina or MI within 7–90 days. This study focused on primary endpoints of death, further MI or cerebral vascular attack (CVA). Akin to previous studies, this involved exposure to apabetalone or placebo in addition to rosuvastatin or atorvastatin. There was no interim analysis during this round. This trial demonstrated significant reduction of events when reviewing MACE and hospitalisation for congestive heart failure but failed to establish significance.15 Various endpoint analyses demonstrated that apabetalone-treated patients experienced a lower rate of first hospitalisation for heart failure (2.4% versus 4.0%, HR 0.59; 95% CI [0.38–0.94]; p=0.03), total number of hospitalisations for heart failure (35 versus 70, HR 0.47; 95% CI [0.27–0.83]; p=0.01) and the combination of cardiovascular death or hospitalisation for heart failure (5.7% versus 7.8%, HR 0.72; 95% CI [0.53–0.98]; p=0.04).12
Apabetalone and Diabetes
There is evidence to suggest that patients with type 2 diabetes on insulin have increased benefit from apabetalone for MACE prevention when compared to those who are not on insulin treatment, although statistical significance was not reached.19 Apabetalone may also have protective effects on pre-diabetic patients against the development of type 2 diabetes, with delayed and reduced oral glucose absorption (p=0.003), endogenous glucose production (p=0.014) and total glucose appearance and disappearance rate (p=0.016). This was based on a small-scale study of 20 unmedicated pre-diabetic patients who were given 100 mg apabetalone twice daily versus placebo.20
Apabetalone and Pulmonary Arterial Hypertension
BRD4 inhibition caught the attention of research in pulmonary arterial hypertension (PAH) based on the understanding of its effects on a molecular level. Many of the molecular results and interplay in pathways previously explored in the context of CAD and metabolic syndrome also link to PAH.21 This was an open-label, single-arm, 16-week study evaluating apabetalone 100 mg twice daily in addition to guideline-recommended therapy in PAH. The exploratory trial APPRoAcH-p consisted of a 16-week open-label study, where patients received apabetalone (100 mg twice daily) alongside standard PAH therapy. Seven patients of European ancestry participated, with two excluded for low baseline pulmonary vascular resistance (PVR). Key findings after 16 weeks of treatment included a decrease in PVR by 140 dyn⋅s⋅cm−5 (95% CI [−200, −79]), increase in cardiac output by 0.73 l/min−1 (95% CI [−0.22, 1.68]) and increase in stroke volume by 8 ml (95% CI [−4, 20]). No significant changes were noted in the 6-minute walk distance (6MWD) and N-terminal pro b-type natriuretic peptide (NT-proBNP). This pilot study suggests potential benefits of apabetalone in PAH treatment, but further research with a larger sample size is needed for conclusive results.22
Apabetalone and Effects Within Chronic Kidney Disease
In CKD patients, the association between ALP with adverse cardiovascular outcomes has previously been reported and may be a result of increased vascular calcification and inflammation.23 A post hoc analysis of the SUSTAIN and ASSURE trials demonstrated that patients (n=48) with estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m2 who received apabetalone showed significantly lower ALP levels when compared to placebo (14% versus 6.3%, p=0.02) after 6 months.24 The mean eGFR of the apabetalone group was 54 ml/min/1.73 m2 and placebo 53 ml/min/1.73 m2. The eGFR in the apabetalone group increased by 3.4% (1.7 ml/min/1.73 m2) and decreased by 5.8% (2.9 ml/min/1.73 m2) in the control. The reduction in blood urea nitrogen (BUN) was similar between the two groups, as were the serum markers of bone metabolism and liver function.
The change in ALP was also noted in the BETonMACE analysis as participants with CKD showed a greater change when compared to those without CKD (mean change of 7.8 versus 1.4 U/l) resembling a decrease.23 Fewer MACE occurred in patients from the BETonMACE trial within the CKD subgroup with apabetalone (11%) when compared to placebo (21%), p=0.032. There were also fewer heart failure-related hospitalisations, with 3% of patients on apabetalone versus 9% in the control group. The aggregate CKD subgroup included 186 participants (65%) with CKD stage IIIa and 102 participants (35%) with CKD stage IIIb with a mean eGFR among those with CKD 49 ml/min/1.73 m2. The benefits of apabetalone on MACE and heart failure occurred without an effect of treatment of kidney function as measured by eGFR. Apabetalone also demonstrated a modest increase in HDL-c that was of similar magnitude in CKD and non-CKD groups.18,23
Adverse Events of Apabetalone
BETonMACE concluded that apabetalone at a dosage of 100 mg twice daily did not cause more adverse events or serious adverse events when compared to the control group. However, patients on apabetalone had an increased risk of elevated alanine transaminase (ALT) more than five times the upper limits of normal (3.3% versus 0.7%), which was shown to return to normal within 4 weeks of stopping the drug.15 This was supported by the previous trials.16 Apabetalone has also shown to be well tolerated in CKD patients with patients on apabetalone experiencing similar numbers of adverse events and fewer serious adverse events (SUSTAIN and ASSURE: 14.3% versus 15.4%; BETonMACE 29% versus 43%, p=0.02).23,24
Discontinuation for abnormal liver function tests (LFT) in the BETonMACE trial were lower than previous studies with an absolute difference of 2.9% in comparison to 2%. There were no significant adverse events noted during the period of treatment with apabetalone.19,20
Discussion
On reflection of the molecular mechanism underpinning apabetalone, there is reasonable evidence to show its utility as a conductor, influencing the epigenetic regulation of gene expression.15 The multifaceted nature of its mechanism lies in its role as a BET protein inhibitor, specifically targeting the BRD4 protein. This inhibition disrupts the reading of acetylated histones, a pivotal step in the transcription of genes involved in atherosclerotic processes, such as those regulating inflammation, lipid metabolism, and plaque stability.16
Further delving into its cellular influence, studies have elucidated that apabetalone selectively modulates the expression of vascular endothelial genes critical for maintaining endothelial integrity, which is often compromised in atherosclerosis.25 Additionally, it has been observed to exert a downregulating effect on the expression of complement component C5 and serum amyloid A, which are implicated in plaque formation and instability.12 On a molecular level, the attenuation of pathological cholesterol transport by the downregulation of the ATP-binding cassette transporters ABCA1 and ABCG1 has also been documented, further substantiating the role of apabetalone in mitigating atherogenic profiles.26
The cardioprotective efficacy of apabetalone has been underpinned by a series of rigorous clinical trials, each contributing pivotal insights into its potential to reduce MACE. The ASSURE trial, although not demonstrating a significant reduction in the primary composite endpoint of MACE, provided an initial hint at the potential of apabetalone in modifying coronary atherosclerosis in patients with ACS when used in conjunction with statin therapy.13 Bolstering these findings, the subsequent SUSTAIN and ASSERT trials suggested improvements in biomarkers associated with vascular inflammation and plaque stability.12 The culmination of this investigative journey was the BETonMACE trial, which stands as the most comprehensive study to date on the impact of apabetalone on cardiovascular outcomes. BETonMACE targeted high-risk type 2 diabetics with recent ACS and showcased a reduction in MACE, driven by a significant decrease in cardiovascular death, MI or stroke, positing apabetalone as a clinically relevant therapeutic agent for this patient demographic.16
Based on the conclusions from the BETonMACE study, the incidence of hospital admissions for heart failure was evaluated by compartmentalising events as either patients with known history of heart failure prior to study entry or those with no prior history of heart failure prior to study. Although apabetalone was shown to reduce incidence, there was no statistical significance to support this.16
Even with the approach of sequential gatekeeping to minimise false-positive incidence as an argument for limiting confidence of the proposed p-values, there are many limitations to extrapolating the primary endpoint. A large component relates to the number and power of the study specific for categories of patients who had heart failure before or after enrolment into the study. BETonMACE does well to show the subgroup analysis to justify that there is a trend within this domain and expand on this by comparing the HRs for both arms of the study in relation to categories ALP, GFR, HbA1c, HDL, hsCRP, LDL, insulin and sodium-glucose cotransporter 2 inhibitors (SGLT2I). This list is not exhaustive, but this availability of interaction p values provides further reinforcement of the molecule targets. The paucity of measurements of brain natriuretic peptide (BNP) or NT-proBNP or echocardiographic data limited the approach to differentiate patients existing as either heart failure with preserved ejection fraction (HFpEF) or reduced ejection fraction (HFrEF). This distinction is crucial to consider when analysing for the effect on primary endpoint: hospitalisation for heart failure.
A further expansion of this point resides with the number of days of admission for patients admitted with heart failure. Exploration of these aspects would provide more grounding to make conclusions related to heart failure. Exploring the use of apabetalone on patients with heart failure not confined to those without diabetes to capture a larger patient sample and focus on biochemical and other measurable parameters in relation to the length of admission would give more robust data to further interrogate the primary endpoints. Apabetalone has reasonable demonstration to show its ability to normalise adverse profiles, which are common occurrences after cardiac events.
At present, BETonMACE2 is a planned phase III cardiovascular outcomes trial of apabetalone in patients with type 2 diabetes who are established on an SGLT2I. The justification for this study was derived from the observation of a subgroup of patients from BETonMACE who encountered a higher hazard reduction in MACE and heart failure-related hospitalisations when they were taking an SGLT2I or dipeptidyl peptidase-4 (DPP-4) inhibitor. In relation to the controlled variables; established diabetes, HbA1c 7.3 g/dl, baseline LDL 70 mg/dl, HDL <33 mg/dl, the outcome study revealed a 30% relative RR with 274 events in the trial with apabetalone HR 0.82 with upper CI 1.04, absolute difference 2% between this and the placebo. This was not statistically significant.
In individuals in whom deaths of uncertain cause were removed from the post hoc analysis, total events of 251 with HR 0.79 with upper CI 1.01 (p<0.06) were observed. Despite every component showing a reduction in events failing to meet significance, the results are considered exploratory and the rationale is that certain subgroups benefit more from the therapy.
Conclusion
While clinical endpoints like reduction in MACE remain the ultimate measure of therapeutic efficacy, these molecular insights provide a compelling narrative that underpins the therapeutic potential of apabetalone. A comprehensive review of the cellular studies elucidating these pathways not only bolsters our understanding of its clinical benefits but also paves the way for precision medicine approaches that leverage epigenetic modifiers to combat cardiovascular disease. Collectively, these trials sketch a narrative of apabetalone not just as a lipid-modulating agent but as a novel therapeutic player with the potential to alter the clinical landscape of cardiovascular risk reduction. Further meta-analyses and long-term follow-up studies are warranted to fully distil the essence of these trials into actionable clinical strategies.