Apabetalone

Apabetalone – BET protein inhibition in cardiovascular disease and Type 2 diabetes

Julia Brandts & KausikK Ray
1 Department of Medicine I, University Hospital RWTH Aachen, Aachen, Germany
2 Imperial Centre for Cardiovascular Disease Prevention, School of Public Health, Imperial College London, London, UK

Apabetalone is the first selective BET protein inhibitor in the field of cardiovascular diseases (CVD). BET proteins are epigenetic regulators that link upstream epigenetic modifications to downstream gene ex- pression. Inhibition of BET proteins by apabetalone has been shown to modulate reverse cholesterol trans- port, coagulation, inflammation and vascular calcification. Furthermore, apabetalone reduces circulating markers of CVD risk and plaque vulnerability. Post-hoc pooled analyses suggest a potential reduction in risk of major adverse cardiac events (MACE) in patients with Type 2 diabetes (T2D) and stable CVD. How- ever, the current cardiovascular outcomes trial BET-on-MACE failed to detect the assumed 30% reduction of MACE by apabetalone in patients with T2D after an acute coronary syndrome.

Targeting residual cardiovascular risk in patients with Type 2 diabetes by epigenetic modulation
BET protein inhibition is an evolving therapeutic concept offering the possibility to target novel pathways for pharmacological epigenetic modulation that translates into changes in gene expression and potentially into favorable clinical phenotypes. BET protein inhibitors are actively being pursued in preclinical and clinical trials for the treatment of a variety of diseases, but mainly cancer [1]. In the field of cardiovascular disease (CVD), apabetalone formerly known as RVX-208, is an orally available drug, and the first epigenetic modulator that shows beneficial effects on lipids and other contributing factors implicated in atherosclerotic diseases, that are especially prominent in patients with Type 2 diabetes (T2D). Residual cardiovascular risk is particularly high in patients with CVD when T2D is present as compared with those without T2D despite optimization of classical risk factors and recent advances in LDL-C lowering therapies [2]. For instance, it is reported, that patients with T2D who were treated with the PCSK9 inhibitor evolocumab still had a major adverse cardiac event (MACE) rate of approximately 17% at 3 years compared with 13% in patients without T2D [3]. The underlying mechanism of this excess risk is not completely understood, but factors accompanying T2D like low HDL-C, elevated triglycerides, diabetic glycemic state, chronic inflammation and hypercoagulability may be contributing factors [4,5].
Attempts to reduce the residual risk in patients with diabetes with current pharmacological interventions showed mixed results. Even though epidemiologic studies reported an inverse correlation of cardiovascular events with low HDL-C-concentrations, attempts to pharmacologically increase HDL-C do not translate into the expected cardiovascular morbidity and mortality reductions [6,7]. Low HDL-C-concentrations often occur in states of insulin resistance (IR), metabolic syndrome (MetS) and T2D; these conditions additionally impair the function of HDL-particles. Hence, increasing the amount of HDL-C alone might be insufficient and instead improving the functionality of HDL particles has been sought as an alternative approach to mitigate HDL-related risk. This is supported by findings from studies where functional HDL-particle analogues like pre-β-HDL-like particles comprising apoA-I Milano and phospholipids were infused and reduction of atheroma volume was demonstrated [8].
However other studies have not reproduced these findings [9].
Furthermore, T2D and atherosclerosis in part are both driven by chronic inflammation, supporting the hypothesis that mitigating the immune response, could be beneficial as a means to further reduce residual risk. The CANTOS trial demonstrated a relative risk reduction of 15% for MACE in patients with established CVD and elevated CRP by the administration of the monoclonal antibody targeting IL1β (canakinumab) [10,11]. The subgroup analysis showed that the MACE reduction was independent of T2D status. Further exploration would be needed to assess whether the beneficial effects of canakinumab only appear in people with established CVD or also in people with and without T2D in a primary prevention setting. However, development of canakinumab for its use in CVD prevention has been halted after rejection by the US FDA and the European committee because the CANTOS trial alone did not provide enough data on the relevance of on and pretreatment hsCRP levels and the benefit to risk ratio of canakinumab treatment [12,13]. Other attempts to repress inflammation showed mixed results [14]. For instance, in the CIRT trial, methotrexate treatment failed to reduce CV events [15], but results from the COLCOT trial, which assessed colchicine’s effect on MACE supports the inflammation hypothesis and showed a significant 23% reduction in the treatment group compared with placebo [16].
At last, targeting the accompanying hypercoagulability in diabetic states, with aspirin treatment is evidence-based for secondary prevention, but in primary prevention, its beneficial effects were outweighed by the incident rate of bleeding events [17,18]. There remains an unmet need for a multifactorial approach to target the proatherogenic, prothrombotic, inflammatory status in patients with T2D. Here, we report on preclinical and clinical studies that show apabetalone’s modulatory effects and its impact on clinical outcomes.

Apabetalone – a BET protein inhibitor & upstream regulator of transcription
The epigenetic code and its modification play an important role in the regulation of gene expression and the resultant phenotype. Unlike the genetic code, the epigenetic code is dynamic, and environmental influences, as well as different diseases, are associated with changes in its pattern. Known components of the epigenetic code are for example noncoding RNAs and post-translational modifications of the DNA or histones, like acetylation, methylation or ubiquitination [19]. These modifications obtain regulatory properties on different levels. First, they may affect chromatin spatial structure and with this the accessibility of genes to the transcription complex. Second, in the case of histone acetylation, these modifications also constitute binding sites for regulatory effector proteins [20]. It is to this group of effector proteins that the BET proteins belong. BET proteins are also referred to as ‘readers’ of the epigenetic code as they exhibit two bromodomains (BD1 and BD2), which can bind to specific histone acetylation patterns and transcription factors (TF) [21,22]. Once bound to acetylated chromatin, BET proteins recruit transcription promoting enzymes like positive transcription elongation factor b and form de novo super-enhancers [22–24] to stimulate RNA polymerase II-dependent transcriptional elongation (Figure 1) [22]. By this mechanism, BET proteins couple histone modification to transcription [21].
The BET protein family consists of four members BRD2, BRD3, BRD4 and the testis-specific BRDT, where BRD4 is so far the best-described. Under physiological conditions, BET proteins commonly participate in the transcription of housekeeping genes, which involve elements of the cell cycle, cell identity and apoptosis under physiologic conditions. However, in states of chronic inflammation or other diseases, BET proteins can reallocate to other chromatin docking sites via two mechanisms. First, changes in the acetylation pattern induce movement of BET proteins to new chromatin sites. This is caused either by disease-triggered aberrant expression and/or mutagenesis that leads to deregulation of the epigenetic code-‘writing’ and ‘erasing’ enzymes, which then causes a rearrangement of the histone acetylation pattern [23]. Second, BET proteins get recruited by specific TFs. For example, in the case of inflammation, chemokine signaling will induce the translocation of NFκb to the nucleus [25]. There NFκb will form a complex that binds to promoters and enhancers of proinflammatory genes, this leads to acetylation of the RelA subunit of NFκb. Subsequently, this modification recruits BRD4 to bind the acetyled RelA subunit, with one bromodomain and potentially bind an acetylated histone in chromatin with the other. Finally, this also leads to the formation of a super-enhancer [26].
The BET protein inhibitor apabetalone mimics the acetylated lysine of histones or TFs and binds competitively through a noncovalent link to the second bromodomain (BD2) of BET proteins. This causes the dissociation of the BET protein and the associated transcription promoting complex from the chromatin and aborts the expression of downstream genes [23]. Apabetalone’s unique characteristic, among other BET protein inhibitors is its 20-fold higher selectivity for the second BD of BET proteins [27]. This leads to change in the modulated genes with different downstream effects compared with tandem BET protein inhibitors like JQ1. In HepG2 cells, only a small number of genes were affected by both JQ1 and apabetalone and inhibition by JQ1 had a stronger inhibitory effect onthese genes with almost a tenfold difference compared with apabetalone [27]. Inhibition of both BD1 and BD2 with JQ1 affects the transcription of 754 genes but in contrast 46 genes were affected by apabetalone [27]. This smaller spectrum of affected genes resulting from apabetalone’s bromodomain selectivity is probably one of the reasons why its tolerability is superior to other BET protein inhibitors. The BD2-selective inhibition of BET proteins by apabetalone has been shown to reduce some pathological effects of diabetes-induced epigenetic modulation, whereas the housekeeping genes were largely unaffected.

Pharmacokinetics
Apabetalone is a small molecule, and stable in acidic milieu. It can be administered in tablet form with twice daily dosing. The compound belongs to the group of quinazolone templates and is a derivative of the plant polyphenol resveratrol (3,4r,5-trihydroxy-transstilbene) [27]. Pharmacokinetic studies in different species show that apabetalone has a bioavailability of 44% after oral administration in cynomolgus monkeys [28]. The major route of excretion is hepatic elimination by oxidation and glucuronidation [29]. Only minor amounts are removed with urine, but plasma levels were unaffected in patients with progressed chronic kidney diseases (CKD) [30].
Beyond ApoA1 induction – apabetalone an epigenetic regulator of CVD underlying pathways Apabetalone was initially found to enhance apo-A1 transcription and translation in human hepatocytes. Accordingly, patient serum levels of new and mature apo-A1 were elevated, with modest increases in HDL-C and HDL particle numbers [31]. ApoA1 is the main component of HDL-particles and promotes the cholesterol efflux from macrophages to HDL-particles. Thus, by elevating the amount of ApoA1, apabetalone may enhance the first step in reverse cholesterol transport [32]. Accordingly, a change from small-to-medium size HDL-particles and changes in the HDL-lipid composition also occurred [33]. The expression of other reverse cholesterol transport-related proteins, like peroxisome proliferator-activated receptor, liver X receptor or cholesteryl ester transfer protein remained unaltered (Johansson J, unpublished data, September 2010) [34], and the clinical routine lipid profile exhibited no changes in other lipoprotein classes [35].
Besides the amount of HDL-C (low), HDL function and composition are also impaired in individuals with T2D [31]. This is mediated by the acute phase response (APR), which alters the composition of HDL to dysfunctionalproinflammatory HDL-particles and decreases HDL-particle number [31]. Apabetalone inhibits the APR pathway and thus restores not only HDL-particle function but also diminishes the APR effect on the complement system, leading to a decrease of complement C3 in patient’s plasma after apabetalone treatment [31]. Furthermore, activation of the complement system as part of the APR is known to lead to activation of platelets, fibrin formation and inhibition of fibrinolysis [36,37], though post-hoc analysis of apabetalone-treated patients did not show changes in coagulation factors despite reduced complement protein level and activation [36]. Moreover, the activated complement system is associated with plaque development and instability [38]. Hence, modulation of the APR and complement system by apabetalone also influences mechanisms that are playing an important role in the onset of an adverse cardiovascular event like plaque vulnerability and thrombus formation.
Apart from reverse cholesterol transport and coagulation, results from microarray analysis indicate that there is modulation of additional CVD-contributing pathways like systemic and vascular inflammation. Apabetalone’sreported interaction with NFκB influences a key pathway of cellular inflammatory signaling. As expected in this context, apabetalone has been shown to reduce proinflammatory cytokines like IL-1β, IL-6 and CRP in human primary hepatocytes [39]. Accordingly, in patients treated with apabetalone, there is a 21.1% loweringin levels of hsCRP [35]. Similarly, in hyperlipidemic ApoE-knockout mice treated with apabetalone circulating inflammatory cytokines, such as IP-10, macrophage inflammatory proteins and macrophage-derived chemokine, were reduced [40]. Moreover, in this same mouse-model, apabetalone-treatment significantly reduced aortic plaque lesions up to 39% [40]. Besides reducing systemic inflammation, the chemokine ligand 2 in aortic cells of these mice were also modulated by apabetalone [40]. Chemokine ligand 2 is a chemoattractant that is involved in the migration of endothelial cells and monocyte recruitment to inflammatory sites and the vessel wall and it might accelerate the progression of atherosclerosis [41]. Further studies have evaluated apabetalone’s effect on vascular inflammation. In human endothelial cells, apabetalone reduces the vascular inflammatory processes and interferes with the expression of CD44, SELE and VCAM-1, which mediate monocytes adhesion to endothelial cells and their migration [42,43]. In macrophage cell lines, IL-6 was reduced, which again diminishes invasion of the arterial wall by macrophages that turn into foam cells by incorporating cholesterol, eventually leading to plaque formation [40].
Beyond its ability to modulate the inflammatory components of the atherosclerotic plaque, potentially influencing plaque formation and/or stability, apabetalone also influences pathways that contribute to calcification. In a model of vascular calcification, apabetalone showed inhibitory effects on extracellular calcium deposition and transdifferentiation of vascular smooth muscle cells of human coronary arteries [44]. ALP, a regulator of tissue mineralization and also a robust, independent predictor of all-cause mortality in the general population and in patients with CKD is reduced by apabetalone [44–46]. Furthermore, ALP is a marker of calcification, plaque vulnerability and CVD and its modulation is thought to be a potential treatment target for reducing CVD, in patients with CKD and T2D [45].

Clinical efficiency & safety of apabetalone
Apabetalone was initially found in a screen for inducers of apoA1-mRNA in hepatocyte cell cultures, and showed an increasing effect on HDL-C concentrations in vitro and in vivo. A total of ten clinical studies with apabetalone have been conducted comprising tolerability, effects on lipoproteins, glucose, kidney function, coronary plaque and major cardiovascular events. Apabetalone is the first BET-inhibitor that was found to be safe and effective enough to enter a Phase III clinical trial in the field of CVD.
In Phase I and II clinical trials, the in vitro findings of apabetalone’s effect on HDL-C were replicated in healthy humans, patients with established coronary artery diseases (CAD) and patients with diabetes [28]. The ASSERT (ApoA-I Synthesis Stimulation Evaluation in Patients Requiring Treatment for Coronary Artery Disease) study was a placebo controlled dose-finding trial that investigated the effect of apabetalone 50–150 mg twice daily lipid and lipoprotein parameters in 299 statin-treated patients with stable CAD [34]. The SUSTAIN (Study of Quantitative Serial Trends in Lipids with Apolipoprotein A-I Stimulation) study also evaluated the longer term effect of 100 mg apabetalone on lipid and lipoprotein parameters over 24 weeks [47]. The ASSURE (ApoA-I Synthesis Stimulation and Intravascular Ultrasound for Coronary Atheroma Regression Evaluation) trial investigated apabetalone’s effect on coronary plaque progression in 323 patients with prevalent CAD and low HDL-C levels [48].
The pooled analysis of those three Phase II trials (ASSERT, ASSURE and SUSTAIN; Table 1), which all included patients with prevalent CAD and low HDL-C-levels, showed that there was a dose-dependent effect of apabetalone on apolipoprotein A-I (apoA-I) and HDL-C. It significantly increased apolipoprotein A-I (apoA-I) concentrations up to 6.7% and accordingly HDL-C concentrations up to 6.5%. Additionally, alteration of HDL-particle composition was observed with an increased occurrence of larger HDL-particles by 23.3% [35]. This shift in the composition of HDL particles enhances their function and improves the reverse cholesterol transport, albeit the effect on HDL-C levels was modest. Beyond these changes, the standard clinical lipid-profile and other atherogenic lipoproteins remained unaltered [35].
Even though the primary end point of the ASSURE trial, a significant reduction in plaque burden, was not met, additional investigations revealed beneficial alterations on plaque composition. In the relatively short study duration of 26 weeks in the ASSURE-trial, the intravascular ultrasound examinations showed a trend (p = 0.08) for plaque regression from baseline in percent atheroma volume in the treatment group compared with the placebo group [48]. Furthermore, a post-hoc analysis of the ASSURE trial aimed to evaluate apabetalone’s effect on signs of plaque instability. For this, the attenuated plaque index (AP index) was calculated to separately investigate the small group of patients which showed signs of plaque instability in the IVUS. In this group the AP index decreased significantly compared with baseline after treatment with apabetalone (57.8 mm◦ [42.4, 79.8] vs 41.3 mm◦ [0, 75.7]; p = 0,003). Moreover, its change correlated with the on-treatment concentration of HDL particles, but not HDL-C or ApoA1 levels [50]. These results indicated a beneficial impact of apabetalone on plaque stability and highlighted the importance of HDL-particle concentration in reverse cholesterol transport compared with HDL-C concentration. These findings were supported by the observation of reduced circulating ALP levels, as a serum marker for plaque vulnerability, after apabetalone treatment [46].
Because of apabetalone’s ability to modulate systemic inflammation, and the potential beneficial effect of this on IR, we might expect an improvement in glucose metabolism. However, a clinically relevant effect of apabetalone on glucose metabolism and IR has yet to be demonstrated [53]. In the Phase III BET-on-MACE trial that included 2425 individuals with T2D, apabetalone did not change glucose or HbA1c levels compared with placebo [51]. Earlier studies testing apabetalone’s impact on blood glucose levels and insulin secretion during an oral glucose tolerance test in participants with prediabetes observed a delayed peak of glucose for 30 min in the apabetalone treatment group, but otherwise the glucose levels and insulin sensitivity were unaffected [33]. Additionally, by using orally administered stable isotope tracer for glucose metabolism this study detected a decreased total appearance rate of glucose caused by both lower ingestion rate and endogenous production in the treatment group compared with the placebo group. The unaltered overall glucose concentrations may be explained by the concomitant decrease of glucose uptake from the circulation. This might be either a direct effect by apabetalone or a compensatory mechanism to maintain glucose homeostasis. However, the underlaying biochemical pathway by that apabetalone causes those changes that are not yet determined.
A post-hoc pooled analysis of 798 patients in the ASSURE, ASSERT and SUSTAIN trials suggested a reduction in MACE (death, myocardial infarction, coronary revascularization and hospitalization for cardiovascular causes) by apabetalone treatment. In the apabetalone group, the frequency of MACE was 5.9% compared with 10.4% in the placebo group (p = 0.02). Hazard ratios (HR) for MACE were 0.51 (95% CI: 0.27–0.93; p = 0.03) in the pooled study sample and 0.38 (95% CI: 0.15–0.99; p = 0.04) in the subgroup-analysis for patients with diabetes after adjustment for differences in baseline risk factors and study duration [35]. Notably, this reduction was not explained by the amount of HDL-C increase induced by apabetalone. Furthermore, the subgroup analysis showed that patients with lower HDL-C levels at baseline (5.5 vs 12.8%; p = 0.01) and/or higher hsCRP concentrations (5.4 vs 14.2%; p = 0.02) had fewer cardiovascular events after treatment with apabetalone compared with placebo [35]. However, in the cardiovascular outcome trial BET-on-MACE the primary end point reduction of time-to-first occurrence of MACE (CV death, nonfatal myocardial infarction or stroke) did not achieve statistical significance (hazard ratio [HR], 0.82; 95% CI: 0.65–1.04; p = 0.11) [51]. The trial investigated 2425 participants with T2D within 3 months after an acute coronary syndrome (myocardial infarction in 75% and unstable angina in 25% of the overall study population) on high-intensity statin therapy and followed them for 26.5 months [51,52]. The median (interquartile range [IQR]) age was 62 (55–68) years and 26% of participants were female, median LDL-C and HDL-C levels at baseline were 65 (49–85) mg/dl and 33 (30–37) mg/dl and median HbA1C was 7.30 (6.40–8.70)% at baseline. The occurrence of the primary end point showed a difference in frequency with a 10.3% rate in the treatment group versus 12.4% in the placebo group. The finding that apabetalone did not reduce the time-to-first occurrence of MACE may be due to insufficient power, as the effect size was initially assumed to be about 30% and the observed event rate was slightly lower than expected. Furthermore, it should be acknowledged that the study collective differed from the study population in the Phase II trials in their state of CAD. In the Phase II studies, patients had documented, but mainly stable CAD, in the BET-on-MACE trial participants had just experienced an acute event. Indeed, former studies had pointed to higher efficacy of apabetalone in patients with elevated CRP levels, which is the case for this patient population. It is likely that the BET-on-MACE trial was underpowered to show a true treatment effect of 20%, even though a smaller treatment effect had been considered in the trial design compared to results from phase II studies, as those smaller studies often overestimate benefit. Although no effects on systemic markers of inflammation such as hsCRP were detected in the BET-on-MACE trial, this does not preclude effects at the level of the vessel wall. However, the exploratory results of the BET-on-MACE trial also intriguingly demonstrated benefits on hospitalization for heart failure with a hazard ratio of 0.9 (95% CI: 0.38–0.94) for first hospitalization and a hazard ratio of 0.47 (95% CI: 0.27–0.83) for first and reoccurring hospitalization and furthermore an overall trend of the primary end point components in favor for apabetalone. These results may suggest that further attempts should be made to investigate apabetalone’s potentially favorable effect on CVD and heart failure. Its hypothetical beneficial effect on heart failure is supported by reduced pressure overload induced cardiac hypertrophy in apabetalone-treated mice [54] and also by the pathophysiology of heartfailure where inflammation is a considerable contributor, including upregulation of proinflammatory cytokines like TNF, IL-1β and IL-6, chemokines, and the complement system [55] which have previously been shown to be modulated by apabetalone.
Even though, a significant effect on CV events could not be shown in the BET-on-MACE trial, apabetalone’s potential to target multiple atherosclerosis promoting pathways (Figure 2) should not be discarded. The therapeutic concept of epigenetic modulation is new in the field of CVD and apabetalone is the first agent that opens the op-portunity to explore and understand interdependencies of epigenetic regulation in large-scale studies. Furthermore, apabetalone’s effect may not be limited to atherosclerosis and its accompanying diseases, as these pathways are also related to other conditions. Ongoing studies evaluate apabetalone’s effect on pulmonary arterial hypertension (APPRoAcH-p study, ClinicalTrials.gov Identifier: NCT03655704), CKD (NCT03160430), vascular dementia (prespecified, exploratory end point in BETonMACE) and Fabry’s disease (NCT03228940).
Apabetalone’s safety and tolerability profile differs substantially from other BET protein inhibitors. Apabetalone’s selectivity to the second BD might be an explanation for its good tolerability, compared with tandem inhibitors, which showed dose-dependent on-target side effects like thrombocytopenia, anemia, neutropenia, gastrointestinal toxicities, fatigue and bilirubin increase [56]. For apabetalone so far, in terms of adverse events, there were elevations of transaminases over three-times the upper limit of normal at around 8 weeks of treatment [35]. This occurred in approximately 8% of patients in the treatment group, compared with zero in the placebo group [35]. There was no accompanying serum total bilirubin elevation and in less severe cases where study medication was continued regression occurred spontaneously. Also, in patients who developed a liver-enzyme increase greater than eight- times the upper limit of normal, transaminase levels normalized within 2 weeks after treatment discontinuation [34]. Predisposing factors for transaminase elevations are assumed to be simvastatin treatment, high-dose statin treatment and liver enzyme elevations at baseline [34,57]. Nevertheless, most patients in the Phase II trials were on background statin treatment, and in the BET-on-MACE trial all patients received high-intensity statin treatment or maximum tolerable dose without showing major interactions with apabetalone [35]. The BET-on-MACE trial provided safety data on over 5,000 patient years of treatment with apabetalone [51]. Despite similar incidence of adverse events in the apabetalone and the placebo group, the frequency of transaminase elevations exceeding five-times the upper limit of normal was more frequent in the apabetalone group than in the placebo group (3.3 vs 0.7%) and also bilirubin levels were 11% higher in relative terms compared with placebo, albeit a rare occurance. However, there were no cases of Hy’s Law. Similar to earlier studies after apabetalone discontinuation transaminase levels returned to baseline levels within 4 weeks. The other significant difference of safety outcomes was the higher frequency of nausea in the apabetalone group with 2.1 versus 0.6%.
Despite apabetalone’s effect on coagulation pathways and inflammation, no bleeding, sepsis or similar events were reported. Additionally, apabetalone could be safely administered to patients with CKD and might even havefavorable effects here in this patient group, which is important because of the great overlap and simultaneous occurrence of CKD with CVD and T2D [30].

Conclusion
In conclusion, apabetalone is a compound with a good tolerability profile and beneficial effects on key factors implicated in the development of atherosclerosis and their acute manifestation as ischemic cardiovascular events that are especially prominent in the high-risk group of patients with established CVD accompanied by T2D. Apabetalone downregulates proinflammatory, prothrombotic and proatherogenic pathways leading beyond others to favorable changes in the lipid profile and plaque stability (Figure 2). The effect of these changes on cardiovascular morbidity and mortality is certainly, if any, smaller than expected according to Phase II results and the impact in different patient groups remains to be established. However, taking these limitations into account the results of the BET-on-MACE study suggest enough potential for further trials of this promising therapy to be warranted.

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