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Recombinant high-density lipoproteins and their use in cardiovascular diseases
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Recombinant high-density lipoproteins and their use in cardiovascular diseases Yi-ni Cao1,2, Lu Xu1,2, Ying-chun Han1,2, Yu-nan Wang1,2, George Liu1,2 and Rong Qi1,2 1 2
Peking University Institute of Cardiovascular Sciences, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, China Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, China
The unique anti-atherosclerosis abilities and other cardioprotective properties make high-density lipoprotein (HDL) a promising solution in treating cardiovascular diseases. A number of studies showed that HDL-based therapy was well tolerated and has great potential in the future. Among all these new agents, the most studied ones including recombinant HDL, recombinant human apolipoproteins, apolipoprotein mimetic peptides and recombinant HDL used as contrast agents in cardiovascular imaging are discussed here. Recombinant HDL and apolipoproteins are promising in diagnosing and treating cardiovascular diseases.
Introduction The role of high-density lipoprotein (HDL) in cardiovascular diseases (CVD) was first demonstrated in the Framingham study [1]. Since then, the inverse correlation between HDL and CVD has been widely accepted. Although low levels of HDL-cholesterol are considered a risk factor in CVD in some studies [2], an increasing number have shown that the function of HDL is more important than HDL-cholesterol content in CVD risk evaluation and treatment [3]. Apolipoprotein A-I (apoA-I), the principal apolipoprotein of HDL, contains a sequence of 243 amino acids determined by Brewer et al. [4]. There are eight to nine a-helices formed by several 22-mer repeats, and each repeat is the duplication of a primordial sequence of 11 amino acid residues [4]. The a-helices are punctuated either by a single proline residue at the amino terminus of each 22-mer or by two unpaired 11-mers inserted between the 22mers [5]. Because the a-helices belong to amphipathic a-helices, the interaction of apoA-I and phospholipids is through the hydrophobic face, whereas the hydrophilic face of the helices interacts with the aqueous phase [6].
Corresponding author: 38 Xueyuan Road, Haidian District, Peking University Institute of Cardiovascular Sciences, Peking University Health Science Center, Beijing 100191, China. Tel: +86 10 8280 5164; Fax: +86 10 8280 5164. Qi, R. ([email protected]) 1359-6446/ß 2016 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2016.08.010
ApoA-I plays an important part in cholesterol efflux from peripheral cells, contributing to the first step of reverse cholesterol transport (RCT) [3]. Moreover, apoA-I can activate lecithin-cholesterol acyltransferase (LCAT) [7]. By transferring fatty acids from phosphatidylcholine to unesterified cholesterol, LCAT catalyzes cholesterol esterification on the surface of HDL, thus promoting cholesterol esters to enter the core of nascent HDL, forming mature HDL rich in cholesterol. Then HDL-cholesterol is taken up in liver via hepatic scavenger receptor class B type I (SR-BI) to complete the process of RCT [8]. In this review, most of the synthetic HDL and peptides are discussed, including human apoA-I, recombinant HDL and apolipoprotein mimetic peptides. Their basic characteristics and biological functions are demonstrated here.
Human apoA-I therapy One method to produce human apoA-I is to isolate it from human HDL. Reimers et al. treated atherosclerotic mice with human apoAI [9]. Mice with atherosclerosis (AS) were infused with human apoA-I twice weekly for 2 weeks. Human apoA-I reduced intraplaque hemorrhage and plaque disruption, which was in parallel with the decrease of S100A4 and matrix metalloproteinase-13 (MMP13) in the fibrous caps of the plaques and the increase of smoothmuscle-cell:macrophage ratio.
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TABLE 1
rHDL and apolipoprotein mimetics.
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Name
Protein or amino acid sequence
Other composition
Functions (Refs)
Human apoA-I
ApoA-I isolated from human HDL
MDCO-216
ApoA-I milano
POPC
Induced regression of acute plaque, had anti-inflammatory and plaque-stabilizing abilities [13] Reduced plaque volume [15]
ETC-642
PVLDLFRELLNELLEALKQKLK
Sphingomyelin, DPPC
Reduced ICAM-1 and VCAM-1 in the vascular tissue [18]
CER-001
Recombinant human apoA-I
Sphingomyelin, DPPG
Promoted RCT [20]
CSL-111
ApoA-I isolated from human HDL
Soybean phosphatidylcholine
Shortened QT interval in family dyslipidemia patients [24] Reduced atheroma volume [26]
CSL-112
ApoA-I isolated from human plasma
Phosphatidylcholine
Enhanced cholesterol efflux [27]
BL-3050
Recombinant apoA-I
Recombinant paraoxonase 1 (rPON1) variant (rePON1-G3C9), POPC
Anti-inflammatory [28]
Simvastatin-loaded rHDL
Recombinant human apoA-I
MHPC, DMPC
Suppressed inflammatory response of macrophages, markedly reduced plaque macrophage content [29]
4F
Ac-DWFKAFYDKVAEKFKEAF-NH2
Significant increase in the activity and stability of rPON1, anti-inflammation [39] Improved HII and reduced aortic lesion area and macrophage lesion area [40]
ATI-5261
EVRSKLEEWFAAFREFAEEFLARLK
Reduced atherosclerosis [48]
Ac-hE18A-NH2
LRKLRKRLLR
It was a hybrid of 18A and a sequence of apoE
Bound LDL and VLDL and facilitated their uptake by HepG2 cells [48] Lowered cholesterol levels in apoBcontaining lipoproteins [49] Increased plasma PON1 activity [50]
Gd-HDL
ApoA-I isolated from human HDL
FeO, Au or QD; Gd, phospholipid, cholesterol, triglyceride
Targeted plaque macrophages, therefore enhanced signal intensity in the aorta compared with conventional MRI contrast agents [56,57]
Enhanced the stability of plaques and reduced S100A4 and MMP-13 in the fibrous caps [9]
Abbreviations: DMPC, dimyristoyl phosphatidylcholine; DPPC, dipalmitoyl phosphatidylcholine; DPPG, dipalmitoyl phosphatidylglycerol; HDL, high-density lipoprotein; HII, HDL antiinflammatory index; ICAM, intercellular cell adhesion molecule; LDL, low-density lipoprotein; MMP, matrix metalloproteinase; MRI, magnetic resonance imaging; PON, paraoxonase; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; RCT, reverse cholesterol transport; rHDL, recombinant HDL; VCAM, vascular cell adhesion molecule; VLDL, very-low-density lipoprotein.
Another study [10] focused on the kinetics of exogenous apoA-I and its distribution in human plasma. Six men with low HDLcholesterol accepted a bolus injection and/or a 5 h infusion of apoA-I. The t1/2 of the apoA-I was longer in the groups receiving the bolus injection and the 5 h infusion. All treatments increased apoA-I content in HDL fractions, and no apoA-I was detected in very-low-density lipoprotein (VLDL) or low-density lipoprotein (LDL) fractions. The results indicated that the infused apoA-I participated in the formation of HDL, perhaps through formation of nascent HDL and interaction with lipoproteins rich in apoB to form mature HDL.
Recombinant HDL The above preliminary studies on apoA-I led to investigations on developing HDL as a therapy for CVD. When apoA-I forms complexes with phospholipids its biological activities are altered. Therefore, apoA-I–phospholipid complexes were synthesized, and their functions were evaluated in vitro, in vivo and in clinical 2
trials. Table 1 shows the various kinds of recombinant HDL (rHDL) cited in this review.
ApoA-I milano and MDCO-216 ApoA-I milano is a mutant form of human apoA-I, characterized by an Arg!Cys substitution in the 173 position. The mutation does not alter a-helix content of apoA-I in lipid-free or lipid-bound forms, but helps to destabilize the protein, perhaps because of the disruption of the intrahelical salt bridge formed by normal Arg 173 and Glu 169, therefore leading to enhanced interaction rates of ahelices in the N-terminal domain with lipids. Also, although plasma HDL-cholesterol and apoA-I values are lower than normal, carriers of the mutation seldom develop atherosclerotic diseases [11]. Ameli et al. [12] injected apoA-I milano/dipalmitoyl phosphatidylcholine (DPPC) complex into hypercholesterolemia rabbits with balloon injury. The complex produced a substantial reduction in intimal thickness and macrophages of carotid arteries.
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ETC-642 ETC-642 is a synthetic complex containing 22 L-amino acid residues, sphingomyelin and DPPC [16,17]. The peptide is an amphipathic apoA-I mimetic. ETC-642 is currently in a Phase I clinical trial and there are no clinical data available. Nevertheless, the complex has shown potential in treating atherosclerosis. In one study [18], after receiving ETC-642 treatment, expression of intercellular cell adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in the thoracic aorta of the rabbits induced by cholesterol was significantly reduced. Compared with HDL isolated from the control group, HDL derived from the rabbits treated by ETC-642 was more effective in inhibiting the increase of ICAM-1 and VCAM-1 induced by tumor necrosis factor (TNF)-a. It was suggested that ETC-642 could inhibit the early stage of AS, which is marked by endothelial expression of ICAM-1 and VCAM-1. Currently there are limited data available on ETC-642, and most studies have focused on its anti-inflammatory properties, probably
because the peptide in ETC-642 can only mimic the anti-inflammation effects of apoA-I. Therefore, much work is required to explore effects of ETC-642 on CVD (i.e., whether it can reduce atheroma plaques).
CER-001 CER-001 is a synthetic HDL comprising recombinant human apoA-I and phospholipids, aiming to mimic the biological benefit of pre-b HDL [19]. The apoA-I is expressed in Chinese hamster ovary cells and then purified. In a clinical study [20], 32 healthy male and female volunteers received a single intravenous dose of CER-001 ranging from 0.25 to 45 mg/kg, in a double-blinded, randomized, cross-over method. Administration of CER-001 caused elevation of plasma cholesterol and HDL-cholesterol, suggesting increased RCT. Mobilization of free cholesterol in the HDL fraction was observed with a CER-001 dose as low as 2 mg/kg, supporting the potential use of a wide dose range for patients. The CHI-SQUARE (Can HDL Infusions Significantly Quicken Atherosclerosis Regression) study was a prospective, doubleblinded, randomized Phase II clinical trial [21]. The sample size was much larger than the MDCO-216 study discussed above (507 vs 47 patients). Patients randomly received six weekly infusions of 3, 6 or 12 mg/kg of CER-001; 417 and 461 patients had paired intravascular ultrasound (IVUS) and quantitative coronary angiography (QCA) measurements, respectively. IVUS and QCA were performed to assess coronary atherosclerosis. CER-001 infusions did not reduce coronary atherosclerosis significantly compared with placebo. Furthermore, the dose-dependent increase in cholesterol mobilization did not translate into greater effects on IVUS, which doubted the predictive value of this biomarker. The disappointing results might be the result of certain limitations. For instance, differences in clinical outcomes among groups were not detected. Also, the investigators did not assess potential changes in plaque quality with virtual histology, which might have concealed its potential effects in the long term. Therefore, whether CER-001 administrated in higher doses or to other populations could affect atherosclerosis must await further study, and proper regimens should be provided with more clinical data.
CSL-111 and CSL-112 CSL-111 is another kind of recombinant HDL composed of apoA-I from human plasma and soybean phosphatidylcholine [22,23]. In nine family dyslipidemia patients [24], CSL-111 infused at 40 mg/ kg over a 4 h period shortened QT intervals in all patients. These results provided evidence for a novel function of HDL infusion that could reduce sudden cardiac death. Another study was aimed at determining mechanisms of cholesterol efflux in humans after infusion of CSL-111 [25]. It was found that enhanced cholesterol efflux from tissues required the presence of apoB-containing lipoproteins and might increase cholesterol efflux through multiple steps of the RCT pathway, not only by changing a specific HDL subfraction.In the ERASE (Effect of rHDL on Atherosclerosis Safety and Efficacy) study [26], patients with a narrowed coronary artery were recruited to receive four weekly infusions of 40 or 80 mg/kg of CSL-111. The higher dose was abolished early because of abnormal liver function issues. Although results showed no significant reduction in percentage change in atheroma volume compared with placebo, the CSL-111 treatment significantly reduced atheroma
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Another study by Ibanez et al. [13] implied that, although a similar effect was found on the regression of acute plaques after either HDL milano or wild-type HDL infusion, the HDL milano exerted superior anti-inflammatory and plaque-stabilizing effects to the wild-type HDL in a short-term treatment in rabbits, which could be attributed to the intrinsic properties of the mutated apoA-I. The results of these two studies indicate that, although apoA-I milano has increased efficiency for uptake of tissue lipids, it could exert atheroprotective effects via ways other than RCT, such as anti-inflammation. MDCO-216 (previously called ETC-216) is a complex composed of apoA-I milano and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Parolini et al. [14] evaluated its effects on atherosclerosis in rabbits with lipid-rich plaques in carotid arteries. They found that the minimal dose of MDCO-216 with five administrations to reduce plaque volume was between 20 and 40 mg/kg, the latter corresponding to the doses administrated to patients (15 mg/kg) in the clinical studies discussed below [15]. More importantly, the results showed that the highest dose given to the rabbits (150 mg/kg) could reduce plaque volume only after two intravenous injections, suggesting the possibility of reducing intravenous administration times. In one clinical trial, Nissen et al. [15] treated 47 acute coronary syndrome (ACS) patients with five weekly infusions of MDCO-216 at 15 or 45 mg/kg. The endpoints of the trial included percent of atheroma volume, total atheroma volume, mean maximum atheroma thickness, among others. Both of the doses resulted in significant changes of these endpoints compared with placebo. More importantly, compared to traditional lipid lowering drugs such as statins and niacin, MDCO-216 could reduce the formation of atheroma plaques to some extent in a short term of 5 weeks. Therefore, the authors also proposed a combined treatment for ACS patients (i.e., in the first few weeks or months after an acute event MDCO-216 was infused to promote RCT, followed by conventional lipid-modulating therapies such as simvastatin and niacin). A pilot clinical study as it was, this work is the first clinical trial of rHDL and it shows promising results.Partly as a result of the shift of the rights of recombinant apoA-I milano, investigations on MDCO-216 were halted, but there is a new clinical trial recruiting participants to evaluate its efficacy on ACS (NCT: 02678923). Whether or not MDCO-216 can be a new commercialized drug remains to be seen.
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volume compared with the condition before treatment. Plaque characterization index and coronary score on QCA were significantly different from the placebo-treated group. The study provided rationale for larger and longer clinical trials of CSL-111. Additionally, compared with the MDCO-216 study, the sample size was larger (183 vs 47 patients) and there were four rather than five infusions. Also, coronary score (including grading on fibrotic and calcific degrees, etc.) was introduced to evaluate plaque characterizations.Owing to its hepatotoxicity, CSL-111 has now been replaced by a new formulation: CSL-112, which contains less phosphocholine (PC) in the rHDL particle and shows an enhanced safety profile over its predecessor (NCT: 01129661). Although one study suggested the ability of CSL-112 to promote cholesterol efflux [27], whether it can reduce plaque or have anti-inflammation effects in AS is still unknown.
Other kinds of rHDL The component of certain rHDL varies from the complexes described above. These rHDL are mainly used to test the structure and/or the function of native HDL. An rHDL containing recombinant paraoxonase (PON)1 variant rPON1-G3C9, POPC and apoA-I, dubbed BL-3050, was synthesized [28]. Owing to the special antiinflammatory property of rPON1, this rHDL could have potential advantages in treating chronic CVD. A simvastatin-loaded rHDL suppressed the inflammatory response of macrophages, markedly reduced plaque macrophage content but did not exhibit myotoxic or hepatotoxic effects [29]. This study indicated that, with its unique properties, rHDL can be used as a tool to carry specific agents to detect and/or cure diseases.
ApoA-I mimetic peptides The rHDL therapies listed above seem promising. However, whether wild type or mutant, the nature of apoA-I being a protein means that the manufacturing is never easy or inexpensive. Besides, the best, maybe the only, way to deliver the complexes is by injection, which will require patient incompliance. As a result, smaller peptides possessing some of the functions of apoA-I are created [30]. The structure and function of these peptides have been studied for years [31]. One of the most important factors is that they have class A amphipathic helices with positively charged amino acids at the polar–nonpolar interface and negatively charged amino acids at the center of the polar face. This structure is essential for its lipid binding and cholesterol efflux promoting functions. The first designed peptide was 18A, because it has 18 amino acids and forms a class A amphipathic helix [32–34]. Its sequence has no homology to apoA-I [35] but it could mimic lipid-binding properties of apoA-I. When the amino and carboxyl termini are blocked by an acetyl group and amide group, respectively, stability and lipid-binding properties are improved and the peptide is called 2F because of the two phenylalanine residues on the hydrophobic face. 2F also had lipid-binding properties but failed to reduce lesions in a mouse model of atherosclerosis [36]. The disappointing effect of 2F on atherosclerosis lesions led to the creation of 4F, the most studied peptide [37,38]. 4Fs synthesized from L and D amino acids are called L-4F and D-4F, respectively. As expected, if taken orally, L-4F will be digested by 4
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proteases in the gastrointestinal tract, whereasD-4F will not be recognized by proteases, making it possible to be taken orally. One study proposed that L-4F removed oxidized lipids from plasma that bound to L-4F with much higher affinity compared with apoA-I but would not remove lipids that bound equally well to apoA-I and L-4F [38]. In the study carried out by Mishra et al. [39], interaction of rPON1 with 4F–POPC complex was studied. Significant increase in the activity and stability of rPON1 in the presence of the 4F–POPC complex was found and a structural model of the 4F–POPC–PON1 ternary complex was formed. The study demonstrated that the ability of 4F to bind proinflammatory oxidized lipids probably accounted for its remarkable anti-inflammatory properties. In the study of safety, pharmacokinetics and pharmacodynamics of D-4F [40], unformulated D-4F had low bioavailability but this could be improved under fasting conditions. A single dose of D-4F was safe and well tolerated and might improve HDL anti-inflammatory index (HII). However, a study in patients indicated that L4F delivered by either subcutaneous injection or intravenous infusion did not improve HDL functions [41]. Explanations of these contradictions have not been reported yet. A novel method was to deliver L-4F orally [42]. L-4F and niclosamide were co-incubated to form L-4F–niclosamide complex, which was resistant to trypsin digestion and could significantly improve HII and reduce aortic lesion area and macrophage lesion area. Another example, mFc-2X4, a peptibody built by fusing two tandem repeats of 4F to the C terminus of a murine IgG Fc fragment, was synthesized [43]. The peptibody was constructed to examine whether multiple helices were superior in cholesterol efflux compared with a monomeric helix. The new complex dosedependently promoted cholesterol efflux in vitro, and its efflux potency was superior to monomeric 4F peptide and apoA-I. It was also capable of forming nascent HDL particles in vitro and plasma from mFc-2X4F-treated mice had higher large a-1 HDL but lower a-3 HDL. These results show the prospect of 4F and its derivatives as cardiovascular protective agents. 6F is another 2F analog [44]. In one study, transgenic tomatoes expressing 6F were planted and added to the Western diet given to mice [45]. Although HDL-cholesterol and PON were increased in the transgenic-tomato-treated group, transgenic tomatoes and empty vector tomatoes reduced plasma 5-hydroxy-eicosatetraenoicacid (5-HETE) and 15-HETE levels, suggesting that the wildtype tomatoes had antioxidative activity as well. Thus, more studies and evidence are needed to support further use of this transgenic tomato. Also, the detailed physical and biological properties of 6F are yet to be explored.
ApoE mimetic peptides ATI-5261 The C-terminal domain of apoE is a potent mediator of ABCA1dependent cholesterol efflux [46,47]. Therefore, a peptide based on an apoE segment, ATI-5261, was synthesized. ATI-5261 is a 26-mer peptide having a single class A amphipathic a-helix [48]. The peptide has a favorable solubility in physiological buffers. It reduced atherosclerosis to roughly the same extent as MDCO-216 in an apoE null mouse, and might also serve as a useful tool to study the structure–function relationship and anti-atherosclerosis mechanism of apolipoprotein mimetic peptides.
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Ac-hE18A-NH2 Ac-hE18A-NH2 is a hybrid of 18A and an apoE sequence [49]. The unique sequence in Ac-hE18A-NH2, which is the receptor-binding domain of apoE, allowed it to bind LDL and VLDL and facilitated their uptake by HepG2 cells via a heparin sulfate proteoglycan mediated pathway, which could lead to reduced plasma cholesterol level [48]. The peptide could lower cholesterol levels in apoBcontaining lipoproteins [49], promote plasma PON1 activity [50], increase phospholipid affinity [51] and thus form phospholipidrich particles, which are efficient mediators of cellular cholesterol efflux [52].
mR18L Based on the lytic class L peptide 18L [53], aromatic residues are clustered in the nonpolar face similar to 4F, resulting in modified 18L (m18L) [54]. The Lys residues of 18L are replaced with Arg to reduce lytic properties, forming the peptide mR18L (modified R18L) [55]. Compared with Ac-hE18A-NH2, antioxidant abilities of mR18L were weaker, and it reduced lesion and macrophage area in the aortic sinus to a lesser extent [53]. Owing to the simple structure, these synthetic peptides are superior to natural apolipoproteins in manufacturing and drug delivery. However, a specific peptide can only mimic part of the functions of apoA-I, a protein containing various segments of peptides. Investigations on these peptides are limited to preclinical studies. More clinical trials are needed, of course, to prove feasibility of developing these peptides as drugs. Also, researchers can focus on combinative therapies of several peptides to exploit their advantages.
Nanocrystal core rHDL HDL can be seen as a vehicle carrying its ‘cargo’ (i.e., apolipoproteins, lipids and enzymes – all of which render HDL with cardiovascular protection activities). Therefore, replacing some of the natural compounds in HDL with inorganic imaging particles is an exciting and promising strategy. In the work of Cormode et al. [56], gold, iron oxide or quantum dot (QD) nanocrystals were introduced into rHDL, replacing the lipid content to become the core of the rHDL. In addition, fluorescent and paramagnetic lipids were included in the phospholipid corona to make the particle magnetic resonance imaging (MRI)or fluorescence-active. The diameters of the synthetic HDL were all within the normal size range of natural HDL and the particles remained separated in plasma. The Au-HDL was superior to the iodine-based contrast agent Omnipaque1 (Nycomed Imaging, Oslo, Norway) in computed tomography (CT) imaging. In MRI imaging, cells incubated with Au-HDL were much brighter than the cells incubated with Au-PEG. FeO-HDL in MRI imaging and
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QD-HDL in fluorescence imaging demonstrated better profiles than their PEG-coated counterparts. More importantly, the particles were mainly taken up into the aorta wall. These results, together with the observation that the particles specifically bound to macrophages and were subsequently taken up by phagocytosis, support their further clinical use. Skajaa et al. [57] synthesized a FeO-HDL. The particle was a potential contrast agent for optical imaging, MRI and transmission electron microscopy. The particles can be excreted in a similar way to cholesterol, because they appeared in the bile and the feces. The close resemblance to natural HDL not only accounts for the superb ability of these particles as contrast agents but also makes it possible to investigate diseases where HDL plays a key part. Moreover, it is possible to incorporate certain drugs into these rHDL to make them theranostic agents.
Concluding remarks Owing to the complicated functions of either native HDL or their recombinant counterparts, the precise structure–function relationship of them remains to be determined. Despite the unknown properties of HDL, a considerable number of studies have provided evidence for the cardiovascular protective properties of rHDL and apolipoprotein mimetic peptides. MDCO-216, CER-001 and CSL-112, which have undergone clinical trials, are promising rHDL to be developed as commercialized drugs for CVD if major problems and questions are solved, including high cost of manufacturing recombinant apoA-I and verification of their safety and cardiovascular protective effects in further clinical trials. In addition, rHDL can be carriers for drugs and imaging substances, which is a very attractive research field. Apolipoprotein mimetic peptides, by contrast, are a more flexible choice in that they are smaller in size and easier to produce. However, little information is available from clinical studies. In addition, the endeavor to produce oral peptides is also hindered by the problem of poor absorption. Much work needs to be done to develop suitable peptide therapies. Compared with conventional CVD drugs, rHDL with rapid plaque reduction and cardiovascular protective effects can serve as efficient therapy for patients with ACS. There is no doubt that rHDL will be a rising star in CVD treatment in the future.
Conflicts of interest The authors have no conflicts of interest to declare.
Acknowledgments The study was supported by grants from National Natural Science Foundation of China (no. 81270368, 81360054) and the National Basic Research Program of China (2015CB932100).
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4 Brewer, H.B., Jr et al. (1978) The amino acid sequence of human APOA-I, an apolipoprotein isolated from high density lipoproteins. Biochem. Biophys. Res. Commun. 80, 623–630 5 Anantharamaiah, G.M. et al. (1990) Use of synthetic peptide analogues to localize lecithin:cholesterol acyltransferase activating domain in apolipoprotein A-I. Arteriosclerosis 10, 95–105 6 Segrest, J.P. et al. (1994) The amphipathic alpha helix: a multifunctional structural motif in plasma apolipoproteins. Adv. Protein Chem. 45, 303–369
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7 Fielding, C.J. et al. (1972) A protein cofactor of lecithin:cholesterol acyltransferase. Biochem. Biophys. Res. Commun. 46, 1493–1498 8 Brown, W.V. et al. (2010) HDL as a treatment target. J. Clin. Lipidol. 4, 5–16 9 Reimers, G.J. et al. (2011) Inhibition of rupture of established atherosclerotic plaques by treatment with apolipoprotein A-I. Cardiovasc. Res. 91, 37–44 10 Nanjee, M.N. et al. (1996) Effects of intravenous infusion of lipid-free apo A-I in humans. Arterioscler. Thromb. Vasc. Biol. 16, 1203–1214 11 Weisgraber, K.H. et al. (1980) A-Imilano apoprotein. Isolation and characterization of a cysteine-containing variant of the A-I apoprotein from human high density lipoproteins. J. Clin. Invest. 66, 901–907 12 Ameli, S. et al. (1994) Recombinant apolipoprotein A-I Milano reduces intimal thickening after balloon injury in hypercholesterolemic rabbits. Circulation 90, 1935–1941 13 Ibanez, B. et al. (2012) Recombinant HDL(Milano) exerts greater anti-inflammatory and plaque stabilizing properties than HDL(wild-type). Atherosclerosis 220, 72–77 14 Parolini, C. et al. (2008) Dose-related effects of repeated ETC-216 (recombinant apolipoprotein A-I Milano/1-palmitoyl-2-oleoyl phosphatidylcholine complexes) administrations on rabbit lipid-rich soft plaques: in vivo assessment by intravascular ultrasound and magnetic resonance imaging. J. Am. Coll. Cardiol. 51, 1098–1103 15 Nissen, S.E. et al. (2003) Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial. JAMA 290, 2292–2300 16 Iwata, A. et al. (2011) Antiatherogenic effects of newly developed apolipoprotein A-I mimetic peptide/phospholipid complexes against aortic plaque burden in Watanabe-heritable hyperlipidemic rabbits. Atherosclerosis 218, 300–307 17 Busseuil, D. et al. (2008) Regression of aortic valve stenosis by ApoA-I mimetic peptide infusions in rabbits. Br. J. Pharmacol. 154, 765–773 18 Di Bartolo, B.A. et al. (2011) The apolipoprotein A-I mimetic peptide, ETC-642, reduces chronic vascular inflammation in the rabbit. Lipids Health Dis. 10, 224 19 Tardy, C. et al. (2014) CER-001, a HDL-mimetic, stimulates the reverse lipid transport and atherosclerosis regression in high cholesterol diet-fed LDL-receptor deficient mice. Atherosclerosis 232, 110–118 20 Keyserling, C.H. et al. (2011) CER-001, a synthetic HDL-mimetic, safely mobilizes cholesterol in healthy dyslipidemic volunteers. Circulation 124, A15525 21 Tardif, J.C. et al. (2014) Effects of the high-density lipoprotein mimetic agent CER001 on coronary atherosclerosis in patients with acute coronary syndromes: a randomized trial. Eur. Heart J. 35, 3277–3286 22 Patel, S. et al. (2009) Reconstituted high-density lipoprotein increases plasma highdensity lipoprotein anti-inflammatory properties and cholesterol efflux capacity in patients with type 2 diabetes. J. Am. Coll. Cardiol. 53, 962–971 23 Shaw, J.A. et al. (2008) Infusion of reconstituted high-density lipoprotein leads to acute changes in human atherosclerotic plaque. Circ. Res. 103, 1084–1091 24 Den Ruijter, H.M. et al. (2011) Reconstituted high-density lipoprotein shortens cardiac repolarization. J. Am. Coll. Cardiol. 58, 40–44 25 Hoang, A. et al. (2012) Mechanism of cholesterol efflux in humans after infusion of reconstituted high-density lipoprotein. Eur. Heart J. 33, 657–665 26 Tardif, J.C. et al. (2007) Effects of reconstituted high-density lipoprotein infusions on coronary atherosclerosis: a randomized controlled trial. JAMA 297, 1675–1682 27 Diditchenko, S. et al. (2013) Novel formulation of a reconstituted high-density lipoprotein (CSL112) dramatically enhances ABCA1-dependent cholesterol efflux. Arterioscler. Thromb. Vasc. Biol. 33, 2202–2211 28 Gaidukov, L. et al. (2009) In vivo administration of BL-3050: highly stable engineered PON1–HDL complexes. BMC Clin. Pharmacol. 9, 18 29 Duivenvoorden, R. et al. (2014) A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation. Nat. Commun. 5, 3065 30 Degoma, E.M. and Rader, D.J. (2011) Novel HDL-directed pharmacotherapeutic strategies. Nat. Rev. Cardiol. 8, 266–277 31 Anantharamaiah, G.M. et al. (2007) Structural requirements for antioxidative and anti-inflammatory properties of apolipoprotein A-I mimetic peptides. J. Lipid Res. 48, 1915–1923 32 Anantharamaiah, G.M. et al. (1985) Studies of synthetic peptide analogs of the amphipathic helix. Structure of complexes with dimyristoyl phosphatidylcholine. J. Biol. Chem. 260, 10248–10255
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33 Venkatachalapathi, Y.V. et al. (1993) Effect of end group blockage on the properties of a class A amphipathic helical peptide. Proteins 15, 349–359 34 Yancey, P.G. et al. (1995) Efflux of cellular cholesterol and phospholipid to lipid-free apolipoproteins and class A amphipathic peptides. Biochemistry 34, 7955–7965 35 Mendez, A.J. et al. (1994) Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol. J. Clin. Invest. 94, 1698–1705 36 Datta, G. et al. (2001) Effects of increasing hydrophobicity on the physical–chemical and biological properties of a class A amphipathic helical peptide. J. Lipid Res. 42, 1096–1104 37 Navab, M. et al. (2007) Peptide mimetics of apolipoproteins improve HDL function. J. Clin. Lipidol. 1, 142–147 38 Imaizumi, S. et al. (2010) L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL in mice. Drug Metab. Lett. 4, 139–148 39 Mishra, V.K. et al. (2013) Lipid complex of apolipoprotein A-I mimetic peptide 4F is a novel platform for paraoxonase-1 binding and enhancing its activity and stability. Biochem. Biophys. Res. Commun. 430, 975–980 40 Bloedon, L.T. et al. (2008) Safety, pharmacokinetics, and pharmacodynamics of oral apoA-I mimetic peptide D-4F in high-risk cardiovascular patients. J. Lipid Res. 49, 1344–1352 41 Watson, C.E. et al. (2011) Treatment of patients with cardiovascular disease with L4F, an apo-A1 mimetic, did not improve select biomarkers of HDL function. J. Lipid Res. 52, 361–373 42 Navab, M. et al. (2009) A novel method for oral delivery of apolipoprotein mimetic peptides synthesized from all L-amino acids. J. Lipid Res. 50, 1538–1547 43 Lu, S.C. et al. (2012) An apoA-I mimetic peptibody generates HDL-like particles and increases alpha-1 HDL subfraction in mice. J. Lipid Res. 53, 643–652 44 Navab, M. et al. (2005) Apolipoprotein A-I mimetic peptides. Arterioscler. Thromb. Vasc. Biol. 25, 1325–1331 45 Chattopadhyay, A. et al. (2013) A novel approach to oral apoA-I mimetic therapy. J. Lipid Res. 54, 995–1010 46 Bhargava, H.N. et al. (1987) Proliferation of thyrotropin releasing hormone receptors in specific brain regions during the development of hypertension in spontaneously hypertensive rats. Peptides 8, 231–235 47 Chou, C.K. et al. (1987) Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate postreceptor effects of insulin. J. Biol. Chem. 262, 1842–1847 48 Bielicki, J.K. et al. (2010) A new HDL mimetic peptide that stimulates cellular cholesterol efflux with high efficiency greatly reduces atherosclerosis in mice. J. Lipid Res. 51, 1496–1503 49 Datta, G. et al. (2010) Anti-inflammatory and recycling properties of an apolipoprotein mimetic peptide, Ac-hE18A-NH(2). Atherosclerosis 208, 134–141 50 Garber, D.W. et al. (2003) Effect of an arginine-rich amphipathic helical peptide on plasma cholesterol in dyslipidemic mice. Atherosclerosis 168, 229–237 51 Gupta, H. et al. (2005) Apolipoprotein E mimetic peptide dramatically lowers plasma cholesterol and restores endothelial function in watanabe heritable hyperlipidemic rabbits. Circulation 111, 3112–3118 52 Datta, G. et al. (2000) The receptor binding domain of apolipoprotein E, linked to a model class A amphipathic helix, enhances internalization and degradation of LDL by fibroblasts. Biochemistry 39, 213–220 53 Handattu, S.P. et al. (2013) Two apolipoprotein E mimetic peptides with similar cholesterol reducing properties exhibit differential atheroprotective effects in LDLR null mice. Atherosclerosis 227, 58–64 54 Nayyar, G. et al. (2012) Apolipoprotein E mimetic is more effective than apolipoprotein A-I mimetic in reducing lesion formation in older female apo E null mice. Atherosclerosis 224, 326–331 55 Segrest, J.P. et al. (1990) Amphipathic helix motif: classes and properties. Proteins 8, 103–117 56 Cormode, D.P. et al. (2008) Nanocrystal core high-density lipoproteins: a multimodality contrast agent platform. Nano Lett. 8, 3715–3723 57 Skajaa, T. et al. (2011) The biological properties of iron oxide core high-density lipoprotein in experimental atherosclerosis. Biomaterials 32, 206–213
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Recombinant high-density lipoproteins and their use in cardiovascular diseases Yi-ni Cao1,2, Lu Xu1,2, Ying-chun Han1,2, Yu-nan Wang1,2, George Liu1,2 and Rong Qi1,2 1 2
Peking University Institute of Cardiovascular Sciences, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, China Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, China
The unique anti-atherosclerosis abilities and other cardioprotective properties make high-density lipoprotein (HDL) a promising solution in treating cardiovascular diseases. A number of studies showed that HDL-based therapy was well tolerated and has great potential in the future. Among all these new agents, the most studied ones including recombinant HDL, recombinant human apolipoproteins, apolipoprotein mimetic peptides and recombinant HDL used as contrast agents in cardiovascular imaging are discussed here. Recombinant HDL and apolipoproteins are promising in diagnosing and treating cardiovascular diseases.
Introduction The role of high-density lipoprotein (HDL) in cardiovascular diseases (CVD) was first demonstrated in the Framingham study [1]. Since then, the inverse correlation between HDL and CVD has been widely accepted. Although low levels of HDL-cholesterol are considered a risk factor in CVD in some studies [2], an increasing number have shown that the function of HDL is more important than HDL-cholesterol content in CVD risk evaluation and treatment [3]. Apolipoprotein A-I (apoA-I), the principal apolipoprotein of HDL, contains a sequence of 243 amino acids determined by Brewer et al. [4]. There are eight to nine a-helices formed by several 22-mer repeats, and each repeat is the duplication of a primordial sequence of 11 amino acid residues [4]. The a-helices are punctuated either by a single proline residue at the amino terminus of each 22-mer or by two unpaired 11-mers inserted between the 22mers [5]. Because the a-helices belong to amphipathic a-helices, the interaction of apoA-I and phospholipids is through the hydrophobic face, whereas the hydrophilic face of the helices interacts with the aqueous phase [6].
Corresponding author: 38 Xueyuan Road, Haidian District, Peking University Institute of Cardiovascular Sciences, Peking University Health Science Center, Beijing 100191, China. Tel: +86 10 8280 5164; Fax: +86 10 8280 5164. Qi, R. ([email protected]) 1359-6446/ß 2016 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2016.08.010
ApoA-I plays an important part in cholesterol efflux from peripheral cells, contributing to the first step of reverse cholesterol transport (RCT) [3]. Moreover, apoA-I can activate lecithin-cholesterol acyltransferase (LCAT) [7]. By transferring fatty acids from phosphatidylcholine to unesterified cholesterol, LCAT catalyzes cholesterol esterification on the surface of HDL, thus promoting cholesterol esters to enter the core of nascent HDL, forming mature HDL rich in cholesterol. Then HDL-cholesterol is taken up in liver via hepatic scavenger receptor class B type I (SR-BI) to complete the process of RCT [8]. In this review, most of the synthetic HDL and peptides are discussed, including human apoA-I, recombinant HDL and apolipoprotein mimetic peptides. Their basic characteristics and biological functions are demonstrated here.
Human apoA-I therapy One method to produce human apoA-I is to isolate it from human HDL. Reimers et al. treated atherosclerotic mice with human apoAI [9]. Mice with atherosclerosis (AS) were infused with human apoA-I twice weekly for 2 weeks. Human apoA-I reduced intraplaque hemorrhage and plaque disruption, which was in parallel with the decrease of S100A4 and matrix metalloproteinase-13 (MMP13) in the fibrous caps of the plaques and the increase of smoothmuscle-cell:macrophage ratio.
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TABLE 1
rHDL and apolipoprotein mimetics.
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Name
Protein or amino acid sequence
Other composition
Functions (Refs)
Human apoA-I
ApoA-I isolated from human HDL
MDCO-216
ApoA-I milano
POPC
Induced regression of acute plaque, had anti-inflammatory and plaque-stabilizing abilities [13] Reduced plaque volume [15]
ETC-642
PVLDLFRELLNELLEALKQKLK
Sphingomyelin, DPPC
Reduced ICAM-1 and VCAM-1 in the vascular tissue [18]
CER-001
Recombinant human apoA-I
Sphingomyelin, DPPG
Promoted RCT [20]
CSL-111
ApoA-I isolated from human HDL
Soybean phosphatidylcholine
Shortened QT interval in family dyslipidemia patients [24] Reduced atheroma volume [26]
CSL-112
ApoA-I isolated from human plasma
Phosphatidylcholine
Enhanced cholesterol efflux [27]
BL-3050
Recombinant apoA-I
Recombinant paraoxonase 1 (rPON1) variant (rePON1-G3C9), POPC
Anti-inflammatory [28]
Simvastatin-loaded rHDL
Recombinant human apoA-I
MHPC, DMPC
Suppressed inflammatory response of macrophages, markedly reduced plaque macrophage content [29]
4F
Ac-DWFKAFYDKVAEKFKEAF-NH2
Significant increase in the activity and stability of rPON1, anti-inflammation [39] Improved HII and reduced aortic lesion area and macrophage lesion area [40]
ATI-5261
EVRSKLEEWFAAFREFAEEFLARLK
Reduced atherosclerosis [48]
Ac-hE18A-NH2
LRKLRKRLLR
It was a hybrid of 18A and a sequence of apoE
Bound LDL and VLDL and facilitated their uptake by HepG2 cells [48] Lowered cholesterol levels in apoBcontaining lipoproteins [49] Increased plasma PON1 activity [50]
Gd-HDL
ApoA-I isolated from human HDL
FeO, Au or QD; Gd, phospholipid, cholesterol, triglyceride
Targeted plaque macrophages, therefore enhanced signal intensity in the aorta compared with conventional MRI contrast agents [56,57]
Enhanced the stability of plaques and reduced S100A4 and MMP-13 in the fibrous caps [9]
Abbreviations: DMPC, dimyristoyl phosphatidylcholine; DPPC, dipalmitoyl phosphatidylcholine; DPPG, dipalmitoyl phosphatidylglycerol; HDL, high-density lipoprotein; HII, HDL antiinflammatory index; ICAM, intercellular cell adhesion molecule; LDL, low-density lipoprotein; MMP, matrix metalloproteinase; MRI, magnetic resonance imaging; PON, paraoxonase; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; RCT, reverse cholesterol transport; rHDL, recombinant HDL; VCAM, vascular cell adhesion molecule; VLDL, very-low-density lipoprotein.
Another study [10] focused on the kinetics of exogenous apoA-I and its distribution in human plasma. Six men with low HDLcholesterol accepted a bolus injection and/or a 5 h infusion of apoA-I. The t1/2 of the apoA-I was longer in the groups receiving the bolus injection and the 5 h infusion. All treatments increased apoA-I content in HDL fractions, and no apoA-I was detected in very-low-density lipoprotein (VLDL) or low-density lipoprotein (LDL) fractions. The results indicated that the infused apoA-I participated in the formation of HDL, perhaps through formation of nascent HDL and interaction with lipoproteins rich in apoB to form mature HDL.
Recombinant HDL The above preliminary studies on apoA-I led to investigations on developing HDL as a therapy for CVD. When apoA-I forms complexes with phospholipids its biological activities are altered. Therefore, apoA-I–phospholipid complexes were synthesized, and their functions were evaluated in vitro, in vivo and in clinical 2
trials. Table 1 shows the various kinds of recombinant HDL (rHDL) cited in this review.
ApoA-I milano and MDCO-216 ApoA-I milano is a mutant form of human apoA-I, characterized by an Arg!Cys substitution in the 173 position. The mutation does not alter a-helix content of apoA-I in lipid-free or lipid-bound forms, but helps to destabilize the protein, perhaps because of the disruption of the intrahelical salt bridge formed by normal Arg 173 and Glu 169, therefore leading to enhanced interaction rates of ahelices in the N-terminal domain with lipids. Also, although plasma HDL-cholesterol and apoA-I values are lower than normal, carriers of the mutation seldom develop atherosclerotic diseases [11]. Ameli et al. [12] injected apoA-I milano/dipalmitoyl phosphatidylcholine (DPPC) complex into hypercholesterolemia rabbits with balloon injury. The complex produced a substantial reduction in intimal thickness and macrophages of carotid arteries.
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ETC-642 ETC-642 is a synthetic complex containing 22 L-amino acid residues, sphingomyelin and DPPC [16,17]. The peptide is an amphipathic apoA-I mimetic. ETC-642 is currently in a Phase I clinical trial and there are no clinical data available. Nevertheless, the complex has shown potential in treating atherosclerosis. In one study [18], after receiving ETC-642 treatment, expression of intercellular cell adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in the thoracic aorta of the rabbits induced by cholesterol was significantly reduced. Compared with HDL isolated from the control group, HDL derived from the rabbits treated by ETC-642 was more effective in inhibiting the increase of ICAM-1 and VCAM-1 induced by tumor necrosis factor (TNF)-a. It was suggested that ETC-642 could inhibit the early stage of AS, which is marked by endothelial expression of ICAM-1 and VCAM-1. Currently there are limited data available on ETC-642, and most studies have focused on its anti-inflammatory properties, probably
because the peptide in ETC-642 can only mimic the anti-inflammation effects of apoA-I. Therefore, much work is required to explore effects of ETC-642 on CVD (i.e., whether it can reduce atheroma plaques).
CER-001 CER-001 is a synthetic HDL comprising recombinant human apoA-I and phospholipids, aiming to mimic the biological benefit of pre-b HDL [19]. The apoA-I is expressed in Chinese hamster ovary cells and then purified. In a clinical study [20], 32 healthy male and female volunteers received a single intravenous dose of CER-001 ranging from 0.25 to 45 mg/kg, in a double-blinded, randomized, cross-over method. Administration of CER-001 caused elevation of plasma cholesterol and HDL-cholesterol, suggesting increased RCT. Mobilization of free cholesterol in the HDL fraction was observed with a CER-001 dose as low as 2 mg/kg, supporting the potential use of a wide dose range for patients. The CHI-SQUARE (Can HDL Infusions Significantly Quicken Atherosclerosis Regression) study was a prospective, doubleblinded, randomized Phase II clinical trial [21]. The sample size was much larger than the MDCO-216 study discussed above (507 vs 47 patients). Patients randomly received six weekly infusions of 3, 6 or 12 mg/kg of CER-001; 417 and 461 patients had paired intravascular ultrasound (IVUS) and quantitative coronary angiography (QCA) measurements, respectively. IVUS and QCA were performed to assess coronary atherosclerosis. CER-001 infusions did not reduce coronary atherosclerosis significantly compared with placebo. Furthermore, the dose-dependent increase in cholesterol mobilization did not translate into greater effects on IVUS, which doubted the predictive value of this biomarker. The disappointing results might be the result of certain limitations. For instance, differences in clinical outcomes among groups were not detected. Also, the investigators did not assess potential changes in plaque quality with virtual histology, which might have concealed its potential effects in the long term. Therefore, whether CER-001 administrated in higher doses or to other populations could affect atherosclerosis must await further study, and proper regimens should be provided with more clinical data.
CSL-111 and CSL-112 CSL-111 is another kind of recombinant HDL composed of apoA-I from human plasma and soybean phosphatidylcholine [22,23]. In nine family dyslipidemia patients [24], CSL-111 infused at 40 mg/ kg over a 4 h period shortened QT intervals in all patients. These results provided evidence for a novel function of HDL infusion that could reduce sudden cardiac death. Another study was aimed at determining mechanisms of cholesterol efflux in humans after infusion of CSL-111 [25]. It was found that enhanced cholesterol efflux from tissues required the presence of apoB-containing lipoproteins and might increase cholesterol efflux through multiple steps of the RCT pathway, not only by changing a specific HDL subfraction.In the ERASE (Effect of rHDL on Atherosclerosis Safety and Efficacy) study [26], patients with a narrowed coronary artery were recruited to receive four weekly infusions of 40 or 80 mg/kg of CSL-111. The higher dose was abolished early because of abnormal liver function issues. Although results showed no significant reduction in percentage change in atheroma volume compared with placebo, the CSL-111 treatment significantly reduced atheroma
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Another study by Ibanez et al. [13] implied that, although a similar effect was found on the regression of acute plaques after either HDL milano or wild-type HDL infusion, the HDL milano exerted superior anti-inflammatory and plaque-stabilizing effects to the wild-type HDL in a short-term treatment in rabbits, which could be attributed to the intrinsic properties of the mutated apoA-I. The results of these two studies indicate that, although apoA-I milano has increased efficiency for uptake of tissue lipids, it could exert atheroprotective effects via ways other than RCT, such as anti-inflammation. MDCO-216 (previously called ETC-216) is a complex composed of apoA-I milano and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Parolini et al. [14] evaluated its effects on atherosclerosis in rabbits with lipid-rich plaques in carotid arteries. They found that the minimal dose of MDCO-216 with five administrations to reduce plaque volume was between 20 and 40 mg/kg, the latter corresponding to the doses administrated to patients (15 mg/kg) in the clinical studies discussed below [15]. More importantly, the results showed that the highest dose given to the rabbits (150 mg/kg) could reduce plaque volume only after two intravenous injections, suggesting the possibility of reducing intravenous administration times. In one clinical trial, Nissen et al. [15] treated 47 acute coronary syndrome (ACS) patients with five weekly infusions of MDCO-216 at 15 or 45 mg/kg. The endpoints of the trial included percent of atheroma volume, total atheroma volume, mean maximum atheroma thickness, among others. Both of the doses resulted in significant changes of these endpoints compared with placebo. More importantly, compared to traditional lipid lowering drugs such as statins and niacin, MDCO-216 could reduce the formation of atheroma plaques to some extent in a short term of 5 weeks. Therefore, the authors also proposed a combined treatment for ACS patients (i.e., in the first few weeks or months after an acute event MDCO-216 was infused to promote RCT, followed by conventional lipid-modulating therapies such as simvastatin and niacin). A pilot clinical study as it was, this work is the first clinical trial of rHDL and it shows promising results.Partly as a result of the shift of the rights of recombinant apoA-I milano, investigations on MDCO-216 were halted, but there is a new clinical trial recruiting participants to evaluate its efficacy on ACS (NCT: 02678923). Whether or not MDCO-216 can be a new commercialized drug remains to be seen.
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volume compared with the condition before treatment. Plaque characterization index and coronary score on QCA were significantly different from the placebo-treated group. The study provided rationale for larger and longer clinical trials of CSL-111. Additionally, compared with the MDCO-216 study, the sample size was larger (183 vs 47 patients) and there were four rather than five infusions. Also, coronary score (including grading on fibrotic and calcific degrees, etc.) was introduced to evaluate plaque characterizations.Owing to its hepatotoxicity, CSL-111 has now been replaced by a new formulation: CSL-112, which contains less phosphocholine (PC) in the rHDL particle and shows an enhanced safety profile over its predecessor (NCT: 01129661). Although one study suggested the ability of CSL-112 to promote cholesterol efflux [27], whether it can reduce plaque or have anti-inflammation effects in AS is still unknown.
Other kinds of rHDL The component of certain rHDL varies from the complexes described above. These rHDL are mainly used to test the structure and/or the function of native HDL. An rHDL containing recombinant paraoxonase (PON)1 variant rPON1-G3C9, POPC and apoA-I, dubbed BL-3050, was synthesized [28]. Owing to the special antiinflammatory property of rPON1, this rHDL could have potential advantages in treating chronic CVD. A simvastatin-loaded rHDL suppressed the inflammatory response of macrophages, markedly reduced plaque macrophage content but did not exhibit myotoxic or hepatotoxic effects [29]. This study indicated that, with its unique properties, rHDL can be used as a tool to carry specific agents to detect and/or cure diseases.
ApoA-I mimetic peptides The rHDL therapies listed above seem promising. However, whether wild type or mutant, the nature of apoA-I being a protein means that the manufacturing is never easy or inexpensive. Besides, the best, maybe the only, way to deliver the complexes is by injection, which will require patient incompliance. As a result, smaller peptides possessing some of the functions of apoA-I are created [30]. The structure and function of these peptides have been studied for years [31]. One of the most important factors is that they have class A amphipathic helices with positively charged amino acids at the polar–nonpolar interface and negatively charged amino acids at the center of the polar face. This structure is essential for its lipid binding and cholesterol efflux promoting functions. The first designed peptide was 18A, because it has 18 amino acids and forms a class A amphipathic helix [32–34]. Its sequence has no homology to apoA-I [35] but it could mimic lipid-binding properties of apoA-I. When the amino and carboxyl termini are blocked by an acetyl group and amide group, respectively, stability and lipid-binding properties are improved and the peptide is called 2F because of the two phenylalanine residues on the hydrophobic face. 2F also had lipid-binding properties but failed to reduce lesions in a mouse model of atherosclerosis [36]. The disappointing effect of 2F on atherosclerosis lesions led to the creation of 4F, the most studied peptide [37,38]. 4Fs synthesized from L and D amino acids are called L-4F and D-4F, respectively. As expected, if taken orally, L-4F will be digested by 4
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proteases in the gastrointestinal tract, whereasD-4F will not be recognized by proteases, making it possible to be taken orally. One study proposed that L-4F removed oxidized lipids from plasma that bound to L-4F with much higher affinity compared with apoA-I but would not remove lipids that bound equally well to apoA-I and L-4F [38]. In the study carried out by Mishra et al. [39], interaction of rPON1 with 4F–POPC complex was studied. Significant increase in the activity and stability of rPON1 in the presence of the 4F–POPC complex was found and a structural model of the 4F–POPC–PON1 ternary complex was formed. The study demonstrated that the ability of 4F to bind proinflammatory oxidized lipids probably accounted for its remarkable anti-inflammatory properties. In the study of safety, pharmacokinetics and pharmacodynamics of D-4F [40], unformulated D-4F had low bioavailability but this could be improved under fasting conditions. A single dose of D-4F was safe and well tolerated and might improve HDL anti-inflammatory index (HII). However, a study in patients indicated that L4F delivered by either subcutaneous injection or intravenous infusion did not improve HDL functions [41]. Explanations of these contradictions have not been reported yet. A novel method was to deliver L-4F orally [42]. L-4F and niclosamide were co-incubated to form L-4F–niclosamide complex, which was resistant to trypsin digestion and could significantly improve HII and reduce aortic lesion area and macrophage lesion area. Another example, mFc-2X4, a peptibody built by fusing two tandem repeats of 4F to the C terminus of a murine IgG Fc fragment, was synthesized [43]. The peptibody was constructed to examine whether multiple helices were superior in cholesterol efflux compared with a monomeric helix. The new complex dosedependently promoted cholesterol efflux in vitro, and its efflux potency was superior to monomeric 4F peptide and apoA-I. It was also capable of forming nascent HDL particles in vitro and plasma from mFc-2X4F-treated mice had higher large a-1 HDL but lower a-3 HDL. These results show the prospect of 4F and its derivatives as cardiovascular protective agents. 6F is another 2F analog [44]. In one study, transgenic tomatoes expressing 6F were planted and added to the Western diet given to mice [45]. Although HDL-cholesterol and PON were increased in the transgenic-tomato-treated group, transgenic tomatoes and empty vector tomatoes reduced plasma 5-hydroxy-eicosatetraenoicacid (5-HETE) and 15-HETE levels, suggesting that the wildtype tomatoes had antioxidative activity as well. Thus, more studies and evidence are needed to support further use of this transgenic tomato. Also, the detailed physical and biological properties of 6F are yet to be explored.
ApoE mimetic peptides ATI-5261 The C-terminal domain of apoE is a potent mediator of ABCA1dependent cholesterol efflux [46,47]. Therefore, a peptide based on an apoE segment, ATI-5261, was synthesized. ATI-5261 is a 26-mer peptide having a single class A amphipathic a-helix [48]. The peptide has a favorable solubility in physiological buffers. It reduced atherosclerosis to roughly the same extent as MDCO-216 in an apoE null mouse, and might also serve as a useful tool to study the structure–function relationship and anti-atherosclerosis mechanism of apolipoprotein mimetic peptides.
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Ac-hE18A-NH2 Ac-hE18A-NH2 is a hybrid of 18A and an apoE sequence [49]. The unique sequence in Ac-hE18A-NH2, which is the receptor-binding domain of apoE, allowed it to bind LDL and VLDL and facilitated their uptake by HepG2 cells via a heparin sulfate proteoglycan mediated pathway, which could lead to reduced plasma cholesterol level [48]. The peptide could lower cholesterol levels in apoBcontaining lipoproteins [49], promote plasma PON1 activity [50], increase phospholipid affinity [51] and thus form phospholipidrich particles, which are efficient mediators of cellular cholesterol efflux [52].
mR18L Based on the lytic class L peptide 18L [53], aromatic residues are clustered in the nonpolar face similar to 4F, resulting in modified 18L (m18L) [54]. The Lys residues of 18L are replaced with Arg to reduce lytic properties, forming the peptide mR18L (modified R18L) [55]. Compared with Ac-hE18A-NH2, antioxidant abilities of mR18L were weaker, and it reduced lesion and macrophage area in the aortic sinus to a lesser extent [53]. Owing to the simple structure, these synthetic peptides are superior to natural apolipoproteins in manufacturing and drug delivery. However, a specific peptide can only mimic part of the functions of apoA-I, a protein containing various segments of peptides. Investigations on these peptides are limited to preclinical studies. More clinical trials are needed, of course, to prove feasibility of developing these peptides as drugs. Also, researchers can focus on combinative therapies of several peptides to exploit their advantages.
Nanocrystal core rHDL HDL can be seen as a vehicle carrying its ‘cargo’ (i.e., apolipoproteins, lipids and enzymes – all of which render HDL with cardiovascular protection activities). Therefore, replacing some of the natural compounds in HDL with inorganic imaging particles is an exciting and promising strategy. In the work of Cormode et al. [56], gold, iron oxide or quantum dot (QD) nanocrystals were introduced into rHDL, replacing the lipid content to become the core of the rHDL. In addition, fluorescent and paramagnetic lipids were included in the phospholipid corona to make the particle magnetic resonance imaging (MRI)or fluorescence-active. The diameters of the synthetic HDL were all within the normal size range of natural HDL and the particles remained separated in plasma. The Au-HDL was superior to the iodine-based contrast agent Omnipaque1 (Nycomed Imaging, Oslo, Norway) in computed tomography (CT) imaging. In MRI imaging, cells incubated with Au-HDL were much brighter than the cells incubated with Au-PEG. FeO-HDL in MRI imaging and
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QD-HDL in fluorescence imaging demonstrated better profiles than their PEG-coated counterparts. More importantly, the particles were mainly taken up into the aorta wall. These results, together with the observation that the particles specifically bound to macrophages and were subsequently taken up by phagocytosis, support their further clinical use. Skajaa et al. [57] synthesized a FeO-HDL. The particle was a potential contrast agent for optical imaging, MRI and transmission electron microscopy. The particles can be excreted in a similar way to cholesterol, because they appeared in the bile and the feces. The close resemblance to natural HDL not only accounts for the superb ability of these particles as contrast agents but also makes it possible to investigate diseases where HDL plays a key part. Moreover, it is possible to incorporate certain drugs into these rHDL to make them theranostic agents.
Concluding remarks Owing to the complicated functions of either native HDL or their recombinant counterparts, the precise structure–function relationship of them remains to be determined. Despite the unknown properties of HDL, a considerable number of studies have provided evidence for the cardiovascular protective properties of rHDL and apolipoprotein mimetic peptides. MDCO-216, CER-001 and CSL-112, which have undergone clinical trials, are promising rHDL to be developed as commercialized drugs for CVD if major problems and questions are solved, including high cost of manufacturing recombinant apoA-I and verification of their safety and cardiovascular protective effects in further clinical trials. In addition, rHDL can be carriers for drugs and imaging substances, which is a very attractive research field. Apolipoprotein mimetic peptides, by contrast, are a more flexible choice in that they are smaller in size and easier to produce. However, little information is available from clinical studies. In addition, the endeavor to produce oral peptides is also hindered by the problem of poor absorption. Much work needs to be done to develop suitable peptide therapies. Compared with conventional CVD drugs, rHDL with rapid plaque reduction and cardiovascular protective effects can serve as efficient therapy for patients with ACS. There is no doubt that rHDL will be a rising star in CVD treatment in the future.
Conflicts of interest The authors have no conflicts of interest to declare.
Acknowledgments The study was supported by grants from National Natural Science Foundation of China (no. 81270368, 81360054) and the National Basic Research Program of China (2015CB932100).
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