NOVEL LCAT-BASED THERAPEUTIC APPROACHES

Abstract

Purpose of review:

To review recent LCAT-based therapeutic approaches for atherosclerosis, acute coronary syndrome, and LCAT deficiency disorders.

Recent findings:

A wide variety of approaches to using LCAT as a novel therapeutic target have been proposed. Enzyme replacement therapy with recombinant human LCAT (rhLCAT) is the most clinically advanced therapy for atherosclerosis and familial LCAT deficiency (FLD), with Phase I and Phase 2A clinical trials recently completed. Liver-directed LCAT gene therapy and engineered cell therapies are also another promising approach. Peptide and small molecule activators have shown efficacy in early stage preclinical studies. Finally, lifestyle modifications, such as fat-restricted diets, cessation of cigarette smoking, and a diet rich in antioxidants may potentially suppress lipoprotein abnormalities in FLD patients and help preserve LCAT activity and renal function but have not been adequately tested.

Summary:

Preclinical and early-stage clinical trials demonstrate the promise of novel LCAT therapies as HDL-raising agents that may be used to treat not only FLD but potentially also atherosclerosis and other disorders with low or dysfunctional HDL.

INTRODUCTION

Low levels of high-density lipoprotein-cholesterol (HDL-C) in plasma are strongly inversely related to cardiovascular disease (CVD) risk. However, most therapies targeted at raising plasma HDL-C have not been successful to date in reducing CVD. Recent efforts have focused on not just raising HDL-C but on improving HDL function, in particular reverse cholesterol transport (RCT) (1) but also other functions as well (2). One such novel approach for improving HDL function involves agents that increase the activity of Lecithin:Cholesterol Acyltransferase (LCAT).

LCAT is a plasma enzyme produced by the liver that plays a key role in HDL metabolism (3). It mediates transfer of a fatty acid from the sn-2 position of phospholipids (PL) to free cholesterol (FC) to form cholesteryl ester (CE) and lysolecithin (3) (4) (5). This reaction takes place mainly on HDL and is an essential step in HDL maturation from small discoidal particles to mature spherical HDL particles. Importantly, this is thought to be a key step in RCT (3). Because of its ability to increase HDL-C and promote RCT, LCAT has long been considered as an attractive target for the development of potential therapeutics against atherosclerosis.

LCAT is defective in Familial LCAT Deficiency (FLD), an autosomal recessive disorder characterized by extremely low levels of plasma HDL-C, low LDL-C and plasma cholesteryl esters, corneal opacities, anemia, and proteinuria. The proteinuria typically progresses to nephrotic syndrome and end-stage renal failure usually by the fourth or fifth decade of life and is the main cause of morbidity and mortality in these patients. Due to mutations in specific locations on LCAT (6), FLD patients cannot esterify cholesterol on either HDL or apoB-containing lipoproteins (apoB-Lps). This results in the accumulation in plasma of unusual vesicle-like structures, termed LP-X, that are highly enriched in FC and PL. A less severe form of FLD is Fish-Eye Disease (FED). FED patients do not suffer from kidney disease but are at increased risk of CVD compared to FLD patients (7–9). They have mutations in LCAT that retain activity towards beta lipoproteins and, consequently, have low HDL-C but near-normal levels of LDL-C, likely contributing to their increased CVD risk. They also have very low or undetectable plasma LP-X levels, which may explain why they typically do not develop proteinuria and kidney disease (10, 11).

Early studies indicated that LP-X may be nephrotoxic (10) (12), providing a plausible mechanism for the increased lipid deposition observed in mesangial cells in kidneys of FLD patients (3). Recent work from our laboratory has directly demonstrated that LCAT reduces plasma and tissue accumulation of LP-X, protecting against kidney disease in mouse models of FLD nephropathy (13, 14). LP-X levels have also been shown to associate with renal disease in humans (11, 15).

These observations suggest that therapies that increase LCAT or LCAT activity will be effective not only for the treatment of kidney disease in FLD patients but also for the treatment of CVD in FED patients and possibly in patients with increased CVD risk for secondary prevention.

In this review we discuss the various approaches for LCAT therapies, including protein replacement biologics, gene and cell therapies, and peptide and small molecule LCAT activators (summarized in Table I). In particular, protein replacement therapy has recently entered clinical evaluation (16–18) and the data are likely to be relevant for not only LCAT therapeutics but the broader area of HDL therapies.

Table 1:

Indications for LCAT activators

Disorder	Mutation Type Or Defect	rhLCAT, WT	rhLCAT, modified	Activating Antibody	Gene Therapy	Peptides	Small Molecules
FLD	Active Site	+	+	−	+	−	−
FLD	Major Truncation	+	+	−	+	−	−
FLD	Small Truncation	+	+	−/+	+	−/+	−/+
FLD	Secretion defects	+	+	−	+	−	−
FLD	Stability/ Structure	+	+	−/+	+	−/+	−/+
FLD	Partial loss of activity	+	+	−/+	+	−/+	−/+
Cholestasis	All types	+	+	+	+	+	+
Atherosclerosis	Low HDL	+	+	+	+	+	+
ACS	Endothelial NO production	+	+	+	+	+	+
Oxidative stress disorders	Oxidative inactivation of endogenous LCAT	+	+	+	+	+	+

LCAT enzyme replacement therapy and gene therapy would not require the presence of functional LCAT. Activating antibodies, peptides and small molecule activators of LCAT require expression of LCAT protein, either wild-type or with a mutation permissive for enzyme activation by the activating agent.

BIOLOGICS

Several LCAT-raising biologic approaches have been developed or are currently under development as potential therapeutics for atherosclerosis, acute coronary syndrome (ACS), FLD, FED, and cholestasis.

rhLCAT as a therapeutic for atherosclerosis and ACS

Recombinant human LCAT (rhLCAT) for enzyme replacement therapy was first developed for clinical testing by AlphaCore Pharmaceuticals. In a first-in-human randomized, blinded, placebo-controlled, dose-escalation Phase I trial in subjects with stable atherosclerosis, this early formulation of rhLCAT, ACP501, raised HDL-C by as much as 50%, improved cholesterol efflux, and was found to be safe and well-tolerated (19). MedImmune developed a newer and more potent formulation of rhLCAT, MEDI6012, which was also found to raise HDL-C in stable atherosclerosis patients for at least a week after a single infusion. A Phase I and Phase 2a randomized, blinded, placebo-controlled, dose-escalation study of intravenous (IV) trials with MEDI6012 in stable atherosclerosis patients showed that MEDI6012 was safe, well-tolerated and also increased HDL-C, HDL-CE, and, interestingly, decreased apoB and small LDL particles in human subjects (16–18).

Preclinical work using wild-type (WT) and LCAT-knockout (KO) mice showed that treatment with MEDI6012 dramatically increased in-vivo macrophage cholesterol efflux in LCAT-KO mice (20). More recent findings suggest that in addition to improving RCT, LCAT treatment may also improve HDL functionality. Specifically, HDL from myocardial infarction (MI) patients is impaired in its ability to stimulate NO production from endothelial cells and this impairment is reversed in HDL isolated from MI patient plasma incubated with LCAT ex-vivo (21). The ability of rhLCAT to restore HDL-mediated NO production suggests that rhLCAT may be a potential therapeutic target for restoring HDL function in not only ACS but in other cardiovascular diseases as well.

rhLCAT as a therapeutic for FLD

Preclinical studies in mice demonstrated that ACP501 injection restored a normal lipid phenotype in LCAT-KO mice and also increased cholesterol efflux (22). FLD patients have very low HDL, and it is conceivable that reduced HDL may drive kidney diseases in these patients. However, other conditions with very low HDL levels, such as FED, Tangier disease and apoA-I deficiency, also have very low HDL, yet patients do not develop kidney disease. These findings indirectly suggest that LP-X is the causative factor in FLD nephropathy. Consistent with this, we recently demonstrated that infusing LP-X into LCAT-deficient mice causes renal disease; importantly, the presence of LCAT in WT mice prevents kidney damage from injected LP-X (13). Moreover, in SREBP-Tg x LCAT-KO mice, which produce abundant quantities of LP-X on a protein-rich/carbohydrate-low (PRCL) diet, rhLCAT treatment decreases LP-X and protects against renal disease (14). Finally, a very recent publication documented that LP-X plasma levels, as determined by a new LP-X quantification assay (11), is in fact a major discriminator of the presence or absence of renal disease among two patients carrying identical LCAT mutations (15).

In a single FLD patient tested, ACP-501 treatment rapidly increased plasma HDL-C (23), with rapid disappearance of the small discoidal preβ-HDL and α-4 particles and subsequent appearance of large spherical α-HDL particles. LDL-C also increased but more slowly than HDL-C, likely due to transfer of newly formed CE to LDL by CETP. Despite the patient’s advanced renal disease, anemia improved significantly and renal function parameters remained stable or slightly improved (23). Like the Phase I study, ACP501 was safe and well-tolerated in the one FLD patient given the therapy, who was treated once or twice a week for approximately 7 months.

The data so far indicating LP-X as a causative factor in FLD nephropathy and the availability of a recently published standardized LP-X quantification assay (11) should expedite clinical development of such protein replacement therapies for FLD and possibly other disorders with highly elevated plasma LP-X.

Cholestasis

As mentioned above, patients with primary biliary cholestasis (PBC) also have LP-X in their plasma. In fact, the most common cause of increased LP-X is liver disease and in particular the type of liver disease that leads to cholestasis. Even though the LP-X in cholestatic patients appears to be of hepatic origin, in contrast to FLD where LP-X is formed in the circulation (24), recent data in our laboratory show that LCAT overexpression or MEDI6012 treatment dramatically suppresses plasma LP-X levels in a model of chemically induced cholestasis in mice (25). Although LP-X does not seem to be causative for liver damage in PBC, it was thought to be responsible for xanthoma formation in these patients (26). Whether LP-X has other negative consequences like renal disease in cholestatic patients is not known, but it can reach such high levels in these patients to cause hyperviscosity syndrome requiring plasmapheresis (27). Therefore, LCAT protein therapy may also be useful for cholestatic liver disease.

Modified rhLCAT therapeutics

As noted below, LCAT Cys31Tyr substitution, like other Cys31 modifications with large bulky hydrophobic groups, increases LCAT activity by 10-fold (28). LCAT Cys31Tyr is a natural rare mutation in humans (29). Moreover, injecting LCAT Cys31Tyr into rabbits resulted in large HDL particles, increased cholesterol efflux from peripheral tissues into plasma, increased neutral sterol excretion into feces, and decreased progression of atherosclerosis (30). Other modifications of LCAT may increase LCAT stability and plasma half-life (31). This modified, more active recombinant LCAT holds promise to deliver LCAT as a more potent enzyme replacement therapy and has been patented by Amgen (31–33).

Finally, single administration of 27C3, an antibody against LCAT Cys31Tyr, into cynomolgus monkeys rapidly increased plasma LCAT enzymatic activity and led to a 35% increase in HDL-C that persisted up to 32 days. This class of antibodies that appears to stabilize LCAT represents another novel biological approach for the treatment of dyslipidemia and cardiovascular disease (28).

GENE AND CELL THERAPY

Although gene therapy for the treatment of human genetic diseases was first conceptualized in the 1970s, it is only recently that gene therapies have started to emerge as real therapies in humans. Following the early failure of gene therapy trials with retroviral vectors and highly immunogenic adenovirus vectors in the late 1990s and early 2000s, huge gaps have been closed concerning virus biology, vector dynamics, immune interaction, and vector safety with the development of a class of viral vectors called adeno-associated viruses (AAVs) as a leading platform for new gene therapies (34).

The use of recombinant viruses expressing LCAT for gene therapy for “treating or preventing dyslipoproteinaemia-related diseases” was first proposed in 2001 (35). Santamarina, Hoeg and Brewer subsequently patented methods to increase LCAT activity in plasma by either cell transfection with vectors containing LCAT, by upregulating LCAT gene expression pharmaceutically, and/or by administering LCAT itself, to prevent or treat atherosclerosis or to treat FLD or FED (36). An early study using adenovirus encoding hLCAT in non-human primates showed a sustained elevation of HDL-C after a single treatment (37).

The development of AAV vectors with tissue tropism, for example AAV8, which preferentially infects hepatocytes (38), has reinvigorated the idea of developing AAV LCAT gene delivery therapeutics for the treatment of FLD. Early optimization of such vectors regarding the use of naturally occurring LCAT variants with increased activity, codon optimization, and effective dose determination in rodent FLD models have been recently described (38, 39).

Despite these promising developments for an effective LCAT gene therapy, there are issues that need to be addressed before such therapy becomes broadly available. These issues include transfection efficiency for adequate LCAT production to sustain physiologic LCAT plasma levels, pre-existing anti-viral vector neutralizing antibodies, and immunogenicity, especially with high viral infection loads. Nevertheless, it is encouraging that clinical investigators have taken up these challenges proposing to optimize such therapies in humans (see for example: http://grantome.com/grant/NIH/P01-HL059407-16A1-8339).

Given the potential limitations with direct gene therapy, indirect gene delivery has also been considered. The basic concept is to isolate adipose tissue cells from patients, transfect them with an LCAT expressing vector, and then after in vitro expansion, return (implant) these LCAT-producing cells to the same patient (40). Although this is conceptually attractive, particularly since it is autologous cell transplantation which avoids immunogenic issues, it also has limitations. First, re-integration of the cells in the tissue and long-term survival may not be optimal, as implied by efforts to develop biomaterials (Fibrin glue) to support survival of these cells and optimal LCAT production after cell implantation (41). Second, the efficiency of LCAT transport from the implanted tissue to the circulation and the number of implanted cells will determine whether physiological LCAT plasma levels can be achieved. Finally, this is an autologous cell transplantation and, therefore, it represents a procedure rather than a drug that can be used to treat all patients. Despite these limitations, the concept of genetically engineered cell therapy for FLD is a valid approach, especially if one can develop an efficient genetically engineered hematopoietic stem cell that can engraft and proliferate leading to sustained production of LCAT (see for example reference (42)).

PEPTIDE ACTIVATORS

LCAT has very low intrinsic activity and requires apolipoproteins for full activity. The major activator of LCAT on HDL is apoA-I (43). ApoA-IV, apoC-I, and apoE, but not apoA-II, apoC-II or apoC-III, can also significantly activate LCAT (43). LCAT does have some activity on apoB-containing lipoprotein particles (LCAT beta activity) and apoE is the major physiological activator of LCAT on these lipoproteins (44). In the brain, nascent apoE-containing lipoproteins are secreted from glial cells and apoE activates LCAT on these lipoprotein particles (45).

Based on the above findings, synthetic peptides that activate LCAT were developed (Fig. 1). All of these peptides contain an amphipathic helix and are thus like apoA-I mimetic peptides, which are also being developed into a form of HDL therapy (46). One of the first such synthetic “peptides” to activate LCAT was full-length, mature apoC-I (47). Truncated versions of apoC-I showed 50–100% activation compared to full-length apoC-I (48) (Fig. 1A). Figure 1B shows other mimetic peptides, based mostly on apoA-I, that can activate LCAT (49) (50) (51, 52) (53) (54) (Table 2). The peptide farthest along in clinical development is ESP24218 (aka P-642), a 22-aa apoA-I mimetic peptide designed by Esperion Therapeutics (55) (56). P-642 complexed with phospholipids is 1.25X more efficient in activating LCAT than proteoliposomes made with human apoA-I (57). Phase I studies were completed but development of the peptide was discontinued (56). Finally, an apoA-I polypeptide or mimetic peptide that activates LCAT fused to an antibody Fc region to enhance plasma half-life has been described (58).

FIGURE 1: Synthetic peptide activators of LCAT.

(A) ApoC-I-based LCAT activators. The full-length, mature apoC-I amino acid sequence (amino acids 1–57) is shown on the top line, followed by truncated versions of apoC-I. The activity of full-length apoC-I is defined as 100%. Note that peptide 32–57 encompasses one of the major phospholipid-binding domains of apoC-I. (B) ApoA-I mimetic peptide/amphipathic helix-based LCAT activators. A helical grid representation of the GALA peptide demonstrates stacking of glutamic acid residues (previously published, from Reference (52)). Activities relative to full-length apoA-I are shown in Table 2.

TABLE 2.

Percent activation of synthetic peptide LCAT activators relative to apoA-I

	Percent activity (compared to full-length apoA-I)
Peptide	Egg PC (Vesicular)	Egg PC (Discoidal)	DMPC
ApoA-I	100	100	100*
LAP-20			50
AP	18
GALA			85
18A	18	30
37pA	Up to 100**	30
[Glu1,8Leu11,17]18A	34		460
ESP24218	93 – 125

^*35% as active as apo A-l in egg PC vesicular assay system

^**Biphasic; low affinity compared to apoA-I (ref. 53).

Adapted from Reference (43)

SMALL MOLECULES

A small heterocyclic amine called Compound A ([3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile]) (Fig. 2A) (59) covalently binds Cys31 in LCAT (Fig. 2B, C) (60)) and increases plasma CE and HDL-C levels in mice and hamsters (59) (61). Interestingly, in vitro, Compound A was able to rescue some naturally occurring LCAT mutations that cause FLD. These mutations are not in the active site but rather promote either the binding or orientation of its lipid to enhance catalysis (60). Site-directed mutagenesis of Cys31 demonstrated that charged residues (Glu, Arg, and Lys) at amino acid #31 decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) at this position increased activity (60). Consistently, the wild-type chicken LCAT sequence contains a Phe in place of Cys at position 31 (62) and chickens have very high HDL (63). Modeling studies suggested that Cys31 alterations that activate LCAT all show increased movement of the membrane-binding domain. Movements of the lid and substrate binding region were also altered and correlated with changes in LCAT activity. Large hydrophobic adducts to Cys31 appear to activate LCAT by promoting either the binding of its hydrophobic lipid substrates and/or a more favorable orientation of the lipid substrate for catalysis (60). Our studies also revealed the importance of specific functional groups of Compound A for LCAT activation (Figure 2B, C) (60).

FIGURE 2.

(A) Compound A. (B,C) Compound A plus LCAT – 3D model (B) and functional groups of Compound A important for LCAT binding (C). The α,β-unsaturated system (cyanopyrazine ring) and the presence of Lewis bases (e.g., nitrogen atoms in the cyanopyrazine ring) in Compound A were important for its ability to activate LCAT. The S-ethyl group tail of compound A also appears to be necessary for appropriately situating the molecule via hydrogen-bonding toward the Cys31 for nucleophilic attack. From Reference (60), previously published. (D) Piperidinylpyrazolopyridine and related activators. Structure of compounds 1 (patent example 95, Ref. (65)), 2 (patent example 46, Ref. (65)), and 3 (patent example 3, Reference (68)) (From Figure 1a of Reference (69)). Altering functional groups on these compounds highlighted the importance of a hydroxyl group and chirality at C4 and the pyrazine ring structure and led to a novel derivative, Compound 8, with even greater ability to activate LCAT and rescue Arg244 mutations (8: Compound 8 from Figure 7 of Reference (69) [7-(trifluoromethyl)−1-(1-(5-(trifluoromethyl)pyrazin-2-yl)piperidin-4-yl)−1H-imidazo[4,5-b]pyridin-5(4H)-one]). (E) Structure of Daiichi compound 1 bound to LCAT (From Figure 2 of Reference (69), previously published).

A novel, orally available LCAT activator, DS-8190, that increased LCAT activity, promoted reverse cholesterol transport (RCT) and prevented atherosclerosis progression in mice was developed by Daiichi (64), as well as 4 other patented additional small-molecule LCAT activators (65–68). Activator Compounds 1–3 (Figure 2D) activated LCAT but did not alter LCAT’s affinity for HDL (69). Structural studies of LCAT bound to Compound 1 indicated that it binds within the membrane-binding domain of LCAT and changes the conformation of the lid, likely stabilizing the membrane-binding domain and facilitating entry of the substrate into the active site (Figure 2D, E) (69). Most importantly, Compounds 1–3 were able to rescue acyltransferase activities of some naturally occurring mutations that cause FLD. Like Compound A, these would not be active site mutations but rather mutations that alter LCAT structure or stability.

LIFESTYLE MODIFICATIONS

Fat Restricted Diets

In contrast to PBC, where LP-X appears to originate from the liver, in FLD patients LP-X is formed in the circulation, most likely from surface components shed off from the lipolysis of triglyceride-rich lipoprotein metabolism (70). Therefore, reduction or limited availability of chylomicrons will be expected to reduce LP-X precursors and, therefore, LP-X plasma levels in FLD patients. Consistently, significant improvements in renal function and proteinuria following the adoption of a fat-restricted diet (70) along with administration of an angiotensin II receptor blocker were seen in a few patients on this diet (71). More recently, treatment of FLD patients with a combination of nicotinic acid and fenofibrate reduced LP-X plasma levels and urine albumin excretion (72). Based on these data, it is reasonable to suggest that newer, more effective triglyceride reducing agents may potentially also possibly be effective in suppressing LP-X plasma levels and kidney dysfunction in FLD patients but this remains to be tested in clinical trials.

Cigarettes and oxidative stress

Cigarette smoke reduces LCAT activity (i.e. acquired LCAT deficiency) by aldehyde-induced modification of its free thiols (73) (74, 75) (76). Oxidative stress also reduces LCAT activity (77). Cessation of smoking and a diet rich in antioxidants are lifestyle modifications anticipated to preserve LCAT activity and raise HDL. LCAT modifications to resist such oxidative stress-induced damage may also be of value in raising HDL in diseases with increased oxidative stress, such as hemolytic anemia in sickle cell disease (78) or psoriasis (79) (80).

CONCLUSION

LCAT-based therapeutics have come a long way in the past few years. Phase I and Phase 2A rhLCAT enzyme replacement therapy trials for atherosclerosis have been completed and have shown safety and tolerability as well as an improved lipoprotein profile. A first-in-human trial demonstrated that rhLCAT raised HDL and stabilized or improved renal function parameters in a single FLD patient. Gene therapy trials are just over the horizon and peptide and small molecule LCAT activators show promise as well. Lifestyle modifications, such as fat-restricted diets for FLD patients, has shown positive results and cessation of cigarette smoking, which is recommended in any case, has the added benefit of preserving LCAT functionality. LCAT-based therapeutics may be of general utility for raising HDL-C and improving HDL functionality in a number of disorders. There is a critical need for LCAT-based therapeutics to treat FLD patients, who have only limited and mostly palliative treatment options available.

KEY POINTS.

1. LCAT is a plasma enzyme that raises high-density lipoprotein (HDL) levels in plasma and may protect against atherosclerosis.

2. LCAT is defective in patients with Familial LCAT Deficiency (FLD), who progress to nephrotic syndrome and end-stage renal failure in the fourth or fifth decade of life.

3. Numerous LCAT-based therapies are under development and some have completed clinical trials.

4. Enzyme replacement therapy, gene and cell-based therapy, peptide and small-molecule LCAT activators, and lifestyle modifications are appropriate for atherosclerosis, ACS, and other disorders resulting in low or dysfunctional HDL.

5. Enzyme replacement therapy with rhLCAT and gene therapy are promising avenues for treatment of FLD.