ACP-501 is recombinant human LCAT (rhLCAT). What is LCAT?
- A liver-derived enzyme
- transfers a fatty acid from lecithin to cholesterol to form cholesteryl esters
- major activator is apoA-I
- 416 amino acids, 20-25% glycosylated (65-68 kDa)
- Circulates primarily in association with high-density lipoprotein (HDL) in the bloodstream
- Normal blood concentration = 4 – 6 mg/L (apoA-I = 1200 mg/L)
- Half-life = 4 – 5 days, similar to high-density lipoproteins (HDL) on which it resides
What is the Function of LCAT?
Cholesterol must be removed from peripheral (non-hepatic) tissues or it will accumulate and cause disease. Cholesterol is removed from tissues and delivered to the liver for excretion from the body by a multistep process known as “Reverse Cholesterol Transport” (RCT, see Figure). In the first step of RCT, small high-density lipoprotein particles (HDL3) in the bloodstream acquire cholesterol from tissues by a process known as “cholesterol efflux”. Efflux can occur either by passive diffusion or active transport of cholesterol from membranes to these “acceptor” HDL3 molecules, which can be either alpha-migrating or prebeta-migrating upon agarose electrophoresis. In the second step, the enzyme lecithin: cholesterol acyltransferase (LCAT) converts the effluxed cholesterol to cholesteryl ester. The elimination of cholesterol from the HDL surface by LCAT-catalyzed esterification maintains a cholesterol concentration gradient that drives the continued movement of cholesterol from cells to HDL. This newly formed cholesteryl ester moves from the HDL surface into the core of the HDL particle, and allows the maturation of HDL from small HDL3 to large HDL2. In the third step of RCT the LCAT-derived cholesteryl esters in HDL2 are either transferred to LDL in exchange for triglyceride, or removed directly by the liver via scavenger receptor-B1 (SR-B1 mediated selected lipid uptake without whole particle uptake). LDLs are removed from the circulation by the classical LDL receptor pathway (whole particle uptake via receptor-mediated endocytosis). Once in the liver the cholesteryl esters from both the LDL and HDL are hydrolyzed back to cholesterol, which can then be converted to bile acids or excreted as cholesterol into bile.
The ability of LCAT to drive cholesterol removal has been demonstrated in several animal species, most notably in rabbits, a species that shares many common steps of RCT with humans. In rabbits the over-expression of LCAT through genetic manipulation or the injection of recombinant LCAT, results in HDL cholesterol elevation and, more importantly, increases RCT and reduces cholesterol accumulation in arteries. This suggests that LCAT could be rate-limiting with respect to RCT.
LCAT Deficiency Syndromes
The central role that LCAT plays in cholesterol and lipoprotein metabolism is evident by the characterization of patients with familial LCAT deficiency. To date close to 90 mutations in the human LCAT gene have been reported, and homozygosity or compound heterozygosity for mutations in LCAT underlies FLD. Patients with FLD have very low levels of HDL-cholesterol (HDL-C), corneal opacities, anemia and renal disease due to the accumulation of LCAT substrates (unesterified cholesterol and phospholipid) in the corneas, red blood cells and kidneys, respectively. An abnormal lipoprotein called Lp-X is also found in the blood of FLD patients. Lp-X are large vesicular structures the size of VLDL or larger but yet have the same density as low density lipoproteins (1.019-1.063 g/L. They are composed primarily of cholesterol (30%) and phospholipid (60%) which ) but unlike LDL do not contain apolipoprotein B (apoB) and trace amounts of C apolipoproteins and albumin. Several studies, preclinical and clinical, have implicated Lp-X as being responsible for the lipid deposition and glomerulosclerosis in FLD patients (see Figure below). Kidney disease is the major cause of morbidity and mortality in patients with familial LCAT deficiency. The goal of AlphaCore Pharma is to develop ACP-501 as an enzyme replacement therapy (ERT) for FLD. Orphan designations for this indication have been granted in the U.S. and Europe.
Our published data in an animal model of FLD (LCAT knockout mice) provide proof-of-concept for enzyme replacement therapy. The administration of ACP-501 normalized the lipoprotein profile (increased HDL-C and reduced particles the size of Lp-X) and enhanced the cholesterol efflux potential of plasma (Rousset, et al, 2010a). Four daily injections of ACP-501 redued the level of cholesterol in kidneys, spleen and RBCs in this mouse model (Rousset, et al, 2010b). We also demonstrated that ACP-501 can decrease the amount of Lp-X when incubated in vitro with the plasma from an FLD patient (Rousset, et al, 2010a).
The “hallmark” or “root cause” of atherosclerosis is thought to be the accumulation of cholesterol in arteries. Cholesterol is delivered to arteries by low density lipoproteins (LDL). The retention of LDL is thought to initiate a cascade of events including inflammation, decreases in endothelium-dependent vasorelaxation, promotion of plaque instability, and in later stages, narrowing of the vessel lumen. Therefore, the removal of cholesterol from arteries is an important clinical goal, especially since plaque instability can cause plaque rupture, the major cause of most heart attacks.
Reduction of plasma LDL, and thus the delivery of cholesterol to arteries, is the foundation for treatment guidelines designed to lower the risk of coronary heart disease (CHD). Despite the lowering of LDL with drugs (e.g. statins), however, significant residual risk for CHD still remains. The magnitude of the problem is illustrated by the fact that 1-2 million patients per year are hospitalized for either chest pain (unstable angina) or heart attack (myocardial infarction or MI), collectively referred to as the acute coronary syndromes (ACS). Clinical studies suggest that 12% to 15% of ACS patients will have a subsequent event within 6 months. Therefore, complementary approaches that increase the removal of cholesterol from the arterial wall are being explored as therapeutic approaches for the acute treatment of atherosclerosis.
Proof-of-concept imaging studies in humans have demonstrated that rapid reductions (within weeks) in atherosclerotic plaque burden can be achieved in ACS patients by infusing synthetic HDL particles (apoA-I or apoA-IMilano complexed to phospholipids) to stimulate reverse cholesterol transport. However, we believe that stimulating the endogenous production of HDL by driving cholesterol esterification may potentially provide a more “natural” way of enhancing the flux of cholesterol through the RCT pathway (see below). ACP-501 need not be complexed to other lipids, has the potential to be administered subcutaneously, and unlike synthetic HDLs and other apoA-I mimetics does not elevate plasma triglycerides. The goal of AlphaCore Pharma is to develop ACP-501 as a new pharmaceutical agent that has the potential to rapidly remove cholesterol from arteries, by stimulating RCT, thus reducing event rates in patients who have suffered an MI.
Preclinical Evidence that LCAT Enhances RCT and/or Decreases Atherosclerosis in Relevant Animal Models (i.e. CETP-Competent Species)
- LCAT transgenic rabbits have elevated HDL and less atherosclerosis when fed a cholesterol diet (Hoeg, et al, 1996)
- Hamsters overexpressing human LCAT have elevated HDL and increased biliary sterol excretion (Zhang, et al, 2004)
- Overexpression of LCAT in rabbits causes regression of established atherosclerotic lesions (Van Craeyveld, et al, 2009)
- Injection of LCAT (subcutaneous) enhances RCT, specifically, cholesterol efflux from peripheral tissues and fecal sterol excretion. It also reduced atherosclerosis (Zhou, et al, 2009).
Clinical Evidence Supporting the Role of LCAT in RCT and Atherosclerosis
- The severity of coronary heart disease (number of diseased vessels) is correlated with LCAT activity (Solajic-Bozicevic, et al, 1994)
- Carriers of LCAT mutations have been shown by some investigators to have increased atherosclerosis (Ayyobi, et al, 2004; Hovingh, et al, 2005; Duivenvoorden, et al, 2010)
- Pre-β-HDL levels (LCAT substrate) are elevated in patients with coronary heart disease (Miida, et al, 1996; Asztalos, et al, 2000; Lamon-Fava, et al, 2008; Tashiro, et al, 2009; Guey, et al, 2011)
- There is an inverse correlation between pre-β-HDL levels and LCAT activity in patients with heart disease (Sethi, et al, 2010)
- Infusion of ACP-501 increases HDL-cholesterol in patients with stable atherosclerosis (see October 9, 2012 Press Release)