Eric Joseph Niesor

Cholesteryl ester transfer protein (CETP), required for transferring triglyceride from very-low-density lipoprotein in exchange for cholesteryl ester from high-density lipoprotein (HDL), is also involved in lipophilic antioxidant exchange. We aimed to determine the effects of the next generation potent CETP inhibitor, obicetrapib, on concentrations and plasma distributions of lipophilic antioxidants, apolipoproteins (apo), and sphingosine-1-phosphate (S1P) in an 8-week, Phase 2 trial of placebo and 2.5, 5, and 10 mg/d obicetrapib in Japanese participants (n = 12 per group). Obicetrapib robustly increased all antioxidant levels in the HDL fraction and decreased plasma levels of oxidized low-density lipoprotein. In specific analyses of the low-dose group, obicetrapib also raised HDL2 cholesterol, apoA1, S1P, apoM, and apoE. Increased levels of monomeric-apoA1 were found in the HDL2 and HDL3 fractions and these correlated with apoM and S1P. Furthermore, we analyzed the relationship between plasma and cerebrospinal fluid (CSF) lipophilic antioxidants in a proof-of-concept study of obicetrapib 10 mg/d in early Alzheimer’s disease patients (n = 13) and demonstrated that HDL2 and CSF carotenoid and tocopherol levels were increased by obicetrapib. These results offer a first insight into the mechanism of action for obicetrapib in terms of HDL metabolism and function, with implications for both atherogenesis and neurodegeneration.

Low activity or inhibition of cholesteryl ester transfer protein (CETP), the protein required for transferring triglyceride from very-low-density lipoprotein in exchange for cholesteryl ester from high-density lipoprotein (HDL)—also involved in lipophilic antioxidant exchange—has been shown to influence several distinct disorders. We aimed to determine the effects of the next generation potent CETP inhibitor, obicetrapib, on lipoprotein disposition of plasma and cerebrospinal fluid (CSF) antioxidant markers. Therefore, we measured apolipoprotein (apo)A1, monomeric apoA1 (m-apoA1), apoE, apoM, sphingosine-1-phosphate (S1P), lipoprotein lipophilic antioxidants, and oxidized LDL (oxLDL) in plasma from a Phase 2 trial of 2.5, 5, and 10 mg/d obicetrapib or placebo taken for 8 weeks by Japanese participants (n=12); and plasma and CSF lipoprotein antioxidants from a proof-of-concept study of obicetrapib 10 mg/d taken for 24 weeks by apoE4-positive early Alzheimer’s disease patients (n=13). In the Japanese dose-range study, obicetrapib robustly increased all antioxidant levels in the HDL fraction and decreased plasma levels of oxLDL at all doses. HDL subfractions tested only in the 2.5 mg group, indicated obicetrapib also raised HDL2 cholesterol, apoA1, S1P, apoM, and apoE. In particular, m-apoA1 was found in the HDL2 and HDL3 fractions and was markedly increased and correlated with apoM and S1P in the HDL2 fraction. In the Alzheimer’s disease study, HDL2 and CSF carotenoid and tocopherol levels were increased upon obicetrapib therapy. These results are key to unraveling the mechanisms of obicetrapib’s effects on HDL metabolism and function, which has implications for atherogenesis and neurodegeneration. One Sentence Summary: Obicetrapib increased levels of plasma, HDL, and CSF lipophilic antioxidants, decreased plasma oxidized LDL, and altered HDL apolipoprotein and S1P distribution.

Voxelotor exerts its regulatory effect on the affinity of hemoglobin for oxygen through an allosteric mechanism involving binding of the drug to the α-chain of hemoglobin. Cystein 93 of the β-chain of hemoglobin is an additional allosteric regulatory site of Hb affinity for oxygen that has been investigated for several decades as a potential target for the treatment of sickle cell disease. This review covers research, in vitro and in preclinical models, on several thiol-reactive molecules such as iodoacetamide, N-ethylmaleimide, 1-bromoacetyl-3,3-dinitroazetidine, isothiocyanate derivatives, thimerosal, glutathione, triazole disulfide, and allyl disulfide, which bind to β-Cysteine 93. Although these compounds were effectively employed to demonstrate that β-Cysteine 93 is an allosteric regulator of hemoglobin function, none of them have yet provided the safety and preclinical data to allow the initiation of clinical trials for the treatment of SCD. Such an opportunity may be provided by repositioning dalcetrapib, an orally available and safe thiol derivative currently in Phase 3 clinical trials for the prevention of cardiovascular events: dalcetrapib has been shown to reduce the incidence of cardiovascular events in patients with the AA genotype of the Adenylyl Cyclase 9, rs1967309 variant, which is prevalent in people of African descent. Recently, dalcetrapib was also observed to bind to Cysteine 93 of the β-chain of hemoglobin, leading to a reduction in hemoglobin S polymerization and alterations in hemoglobin interaction with oxygen. Dalcetrapib could therefore be a therapeutic that limits red blood cell sickling in SCD patients, thus mitigating the associated complications. Nonetheless, clinical trials on dalcetrapib in this population are currently lacking.

Patients with sickle cell disease (SCD) exhibit high levels of reactive oxygen species and low plasma levels of lipophilic antioxidants, which may contribute to end‐organ damage and disease sequelae. Apolipoprotein A1, the major apolipoprotein of high‐density lipoprotein (HDL), is mainly secreted by the intestine and liver in the form of monomeric ApoA1 (mApoA1) present in plasma. Cholesterol and α‐tocopherol are delivered to ApoA1 via the ATP‐binding cassette transporter, subfamily A, member 1 (ABCA1). We measured cholesterol, mApoA1, ApoA1, and lipophilic antioxidants in the plasma of 17 patients with SCD and 40 healthy volunteers. Mean HDL cholesterol (‐C) levels in SCD patients and healthy subjects were 59.3 and 48.1 mg/dL, respectively, and plasma lutein, zeaxanthin, and α‐tocopherol were 64.0%, 68.7%, and 9.1% lower, respectively. To compare SCD to healthy subjects with similar HDL‐C, we also performed subgroup analyses of healthy subjects with HDL‐C above or below the mean. In SCD, the mApoA1 level was 30.4 μg/mL; 80% lower than 141 μg/mL measured in healthy volunteers with similar HDL‐C (56.7 mg/dL). The mApoA1 level was also 38.4% greater in the higher versus lower HDL‐C subgroups (p = .002). In the higher HDL‐C subgroup, lutein and zeaxanthin transported by HDL were 48.9% (p = .01) and 41.9% (p = .02) higher, respectively, whereas α‐tocopherol was 31.7% higher (p = .003), compared to the lower HDL‐C subgroup. Plasma mApoA1 may be a marker of the capacity of HDL to capture and deliver liposoluble antioxidants, and treatments which raise HDL may benefit patients with high oxidative stress as exemplified by SCD.

Cell membrane lipid composition, especially cholesterol, affects many functions of embedded enzymes, transporters and receptors in red blood cells (RBC). High membrane cholesterol content affects the RBCs’ main vital function, O₂ and CO₂ transport and delivery, with consequences on peripheral tissue physiology and pathology. A high degree of deformability of RBCs is required to accommodate the size of micro-vessels with diameters significantly lower than RBCs. The potential therapeutic role of high-density lipoproteins (HDL) in the removal of cholesterol and its activity regarding maintenance of an optimal concentration of RBC membrane cholesterol have not been well investigated. On the contrary, the focus for HDL research has mainly been on the clearance of cholesterol accumulated in atherosclerotic macrophages and plaques. Since all interventions aiming at decreasing cardiovascular diseases by increasing the plasma level of HDL cholesterol have failed so far in large outcome studies, we reviewed the potential role of HDL to remove excess membrane cholesterol from RBC, especially in sickle cell disease (SCD). Indeed, abundant literature supports a consistent decrease in cholesterol transported by all plasma lipoproteins in SCD, in addition to HDL, low- (LDL) and very low-density lipoproteins (VLDL). Unexpectedly, these decreases in plasma were associated with an increase in RBC membrane cholesterol. The concentration and activity of the main enzyme involved in the removal of cholesterol and generation of large HDL particles—lecithin cholesterol ester transferase (LCAT)—are also significantly decreased in SCD. These observations might partially explain the decrease in RBC deformability, diminished gas exchange and tendency of RBCs to aggregate in SCD. We showed that incubation of RBC from SCD patients with human HDL or the HDL-mimetic peptide Fx5A improves the impaired RBC deformability and decreases intracellular reactive oxygen species levels. We propose that the main physiological role of HDL is to regulate the cholesterol/phospholipid ratio (C/PL), which is fundamental to the transport of oxygen and its delivery to peripheral tissues.

The anti-atherogenic properties of high-density lipoproteins (HDL) have been explained mainly by reverse cholesterol transport (RCT) from peripheral tissues to the liver. The RCT seems to agree with most of the negative epidemiological correlations between HDL cholesterol levels and coronary artery disease. However, therapies designed to increase HDL cholesterol failed to reduce cardiovascular risk, despite their capacity to improve cholesterol efflux, the first stage of RCT. Therefore, the cardioprotective role of HDL may not be explained by RCT, and it is time for new paradigms about the physiological function of these lipoproteins. It should be considered that the main HDL apolipoprotein, apo AI, has been highly conserved throughout evolution. Consequently, these lipoproteins play an essential physiological role beyond their capacity to protect against atherosclerosis. We propose HDL as bidirectional lipid vectors carrying lipids from and to tissues according to their local context. Lipid influx mediated by HDL appears to be particularly important for tissue repair right on site where the damage occurs, including arteries during the first stages of atherosclerosis. In contrast, the HDL-lipid efflux is relevant for secretory cells where the fusion of intracellular vesicles drastically enlarges the cytoplasmic membrane with the potential consequence of impairment of cell function. In such circumstances, HDL could deliver some functional lipids and pick up not only cholesterol but an integral part of the membrane in excess, restoring the viability of the secretory cells. This hypothesis is congruent with the beneficial effects of HDL against atherosclerosis as well as with their capacity to induce insulin secretion and merits experimental exploration.

Following the neutral results of the dal-OUTCOMES trial, a genome-wide study identified the rs1967309 variant in the adenylate cyclase type 9 ( ADCY9 ) gene on chromosome 16 as being associated with the risk of future cardiovascular events only in subjects taking dalcetrapib, a CETP (cholesterol ester transfer protein) modulator. Homozygotes for the minor A allele (AA) were protected from recurrent cardiovascular events when treated with dalcetrapib, while homozygotes for the major G allele (GG) had increased risk. Here, we present the current state of knowledge regarding the impact of rs1967309 in ADCY9 on clinical observations and biomarkers in dalcetrapib trials and the effects of mouse ADCY9 gene inactivation on cardiovascular physiology. Finally, we present our current model of the interaction between dalcetrapib and ADCY9 gene variants in the arterial wall macrophage, based on the intracellular role of CETP in the transfer of complex lipids from endoplasmic reticulum membranes to lipid droplets. Briefly, the concept is that dalcetrapib would inhibit CETP-mediated transfer of cholesteryl esters, resulting in a progressive inhibition of cholesteryl ester synthesis and free cholesterol accumulation in the endoplasmic reticulum. Reduced ADCY9 activity, by paradoxically leading to higher cyclic AMP levels and in turn increased cellular cholesterol efflux, could impart cardiovascular protection in rs1967309 AA patients. The ongoing dal-GenE trial recruited 6145 patients with the protective AA genotype and will provide a definitive answer to whether dalcetrapib will be protective in this population.

Cholesterol efflux from macrophages to HDL, measured in vitro, is augmented by treatment with agents which raise HDL cholesterol. In vitro, cholesterol depletion by statins is known to trigger a positive feedback on the cholesterol synthetic pathway via sterol regulatory element-binding protein (SREBP) transcription and changes in expression of SREBP regulated genes including microRNA33 (miR33) which is co-transcribed with SREBP and down-regulates ABCA1 and ABCG1 expression. We investigated whether miR33 up-regulation, associated with SREBP increased transcription by statins, reduces macrophage ATP-binding cassette (ABC) transporter expression, thereby decreasing HDL-mediated cholesterol efflux at the tissue level. In human macrophage THP-1 cells cholesterol-loaded with acetylated LDL, incubation with 1 μM atorvastatin increased miR33 by 33 % (P < 0.05), and decreased ABCA1 messenger RNA (mRNA) and ABCG1 mRNA by 47 % (P < 0.05) and 27 % (NS), respectively. In J774A.1 mouse macrophage, labelled with 3H-cholesterol, ABCA1 mRNA and ABCA1-mediated cholesterol efflux were decreased by 1 μM statin: simvastatin > pitavastatin > atorvastatin > rosuvastatin > pravastatin. HDL incubated with rhCETP and dalcetrapib increased ABCA1-mediated cholesterol efflux. However, incremental simvastatin concentrations decreased cholesterol efflux to HDL treated with rhCETP and dalcetrapib. When HDL was incubated with rhCETP, addition of dalcetrapib augmented ABCA1-mediated cholesterol efflux from J774A.1 macrophages. However, simvastatin ≥1 μM virtually eliminated any HDL-ABCA1-mediated cholesterol efflux and any augmentation of that process by dalcetrapib. In vitro, statins increase miR33 expression, and decrease ABCA1 expression and cholesterol efflux from peripheral tissues; this may counteract the potential benefit of agents that raise HDL and apolipoprotein A-I in statin-treated patients.