Reynold Homan

Rapid separation and quantitation of combined neutral and polar lipid classes by high-performance liquid chromatography and evaporative light-scattering mass detection

Modifications are described for an innovative and widely used high-performance liquid chromatography technique that resolves a very broad spectrum of lipids for quantitation by evaporative light-scattering detection. Substitution of acetone for 2-propanol in a portion of the solvent gradient program yields consistent resolution of diacylglycerol and cholesterol without sacrificing baseline resolution of the remaining major lipid classes. Moreover, previously noted instabilities in triacylglycerol retention time are eliminated. The introduction of acetone also enables a 20% reduction in flow-rate without an increase in total run time. As a further modification of the mobile phase composition, acetic acid and ethanolamine are substituted for the serine-ethylamine combination that was originally shown to improve column performance. The combination of acetic acid and ethanolamine yields the same result but the increased volatility of these solutes over serine results in decreased baseline noise. Finally, 1,2-hexadecanediol is introduced as an internal standard that is well suited for this method. The chromatographic performance obtained with these modifications is demonstrated in compositional analyses of lipid extracts from rat liver, heart, kidney and brain.

ACAT inhibition decreases LDL cholesterol in rabbits fed a cholesterol-free diet. Marked changes in LDL cholesterol without changes in LDL receptor mRNA abundance.

Rabbits fed low-fat, cholesterol-free diets containing casein as the sole protein source develop endogenous hypercholesterolemia (EH). To test the hypothesis that lipoprotein cholesteryl esters in EH rabbits are acyl coenzyme A:cholesterol acyltransferase (ACAT) derived, we treated EH rabbits with CI-976, a potent and selective ACAT inhibitor. In addition, since cholesterol and bile acid synthesis as well as low-density lipoprotein (LDL) receptor activity are reduced in EH rabbits, we determined whether changes in gene expression for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, 7 alpha-hydroxylase, and the LDL receptor might be associated with the efficacy due to ACAT inhibition. Compared with EH controls, CI-976-treated rabbits (50 mg/kg per day for 5 weeks) had decreased plasma total cholesterol (-43%), very-low-density lipoprotein (VLDL) cholesterol (-62%), LDL cholesterol (-43%), plasma apolipoprotein B (-23%), liver cholesteryl esters (-39%), LDL size, VLDL and LDL cholesteryl ester content (percent of total lipids), cholesteryl oleate/cholesteryl linoleate ratios in VLDL and LDL (25% to 30%), and ex vivo liver ACAT activity. The triglyceride/cholesteryl ester ratio increased twofold to fourfold in these apolipoprotein B-containing lipoproteins. Endogenous cholesterol absorption appeared to be unaffected by drug treatment. CI-976 failed to alter specific hepatic mRNAs involved in cholesterol metabolism, but comparisons among dietary control groups revealed a marked reduction in 7 alpha-hydroxylase mRNA, no change in LDL receptor mRNA, and an increase in HMG-CoA reductase mRNA in EH rabbits compared with normal chow-fed controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid-lowering effects of WAY-121,898, an inhibitor of pancreatic cholesteryl ester hydrolase

WAY-121,898 is an inhibitor of pancreatic cholesteryl ester hydrolase (pCEH). After confirming its in vitro potency and relative lack of a major effect on acyl-CoA:cholesterol acyltransferase (ACAT), it was found that this compound lowers plasma cholesterol in cholesterol-fed, but not chow-fed, rats. Measures of liver cholesteryl ester content and the direct determination of cholesterol absorption (lymph-fistula model) show that inhibition of cholesterol absorption is at least one mechanism for the observed cholesterol lowering. However, WAY-121,898 was also active when administered parenterally to cholesterol-fed rats, and in cholesterol-fed hamsters cholesterol-lowering occurred with oral dosing despite no change in cholesterol absorption, suggesting other modes of action possibly relating to inhibition of liver CEH. Combination treatment in cholesterol-fed rats with the ACAT inhibitor CI-976 resulted in a greater-than-additive reduction in plasma cholesterol, imlying that both pCEH and ACAT may play a role in cholesterol absorption in this species. In rabbits, WAY-121,898 prevented the rise in plasma cholesterol due to the feeding of cholesteryl ester but not in rabbits fed (free) cholesterol. In guinea pigs, the compound induced an increase in adrenal cholesteryl ester mass. Taken together, the overall profile in these animal models suggests that WAY-121,898 inhibits more than just the intestinal (lumenal) pCEH, and that the role of this enzyme in cholesterol metabolism may be different within and across species, the former depending upon the dietary cholesterol load.

CHOLESTERYL ESTER TRANSFER PROTEIN INHIBITION BY PD 140195

The presence of plasma cholesteryl ester transfer protein (CETP) activity may be atherogenic, and, therefore, strategies to inhibit its activity or production may result in a beneficial effect on lipoprotein profiles and the disease process. The current report describes 4-phenyl-5-tridecyl-4H-1,2,4-triazole-3-thiol (PD 140195), a novel CETP inhibitor. The concentration-dependent inhibition of CETP by PD 140195 and the inhibitory monoclonal antibody (Mab) TP2 is demonstrated in a variety ofin vitro assay systems. Molecular models of PD140195 suggest a spatial mimicry of the cholesteryl ester structure. Despite the structural similarity, kinetic studies with a fluorescent cholesteryl ester analog suggest that the inhibition of transfer is not competitive. PD 140195 also selectively inhibited cholesteryl ester but not triglyceride transfer, while the Mab TP2 blocked CETP transfer of both. Studies were carried out to determine whether CETP inhibition observedin vitro could also be demonstratedin vivo. When PD 140195 was intravenously infused to anesthetized rabbits (up to 20 mg/kg), only transient CETP inhibition was observed.In vitro reconstitution studies in the presence of bovine serum albumin resulted in marked reduction of PD 140195 inhibitory activity. Thus, the low activity of PD 140195 in whole plasma probably results from binding to other plasma proteins.

Lack of correlation between ACAT mRNA expression and cholesterol esterification in primary liver cells.

A partial rabbit cDNA clone (14b) for ACAT has been characterized and used to demonstrate that hepatic and aortic ACAT mRNA14b abundance increased 2-3-fold in rabbits receiving a high fat/high cholesterol-diet compared to chow fed animals (Pape et al. (1995) J. Lipid Res. 36, 823-838). Because of those data we hypothesized that increased hepatic cholesteryl ester mass and synthesis rates in rabbit liver cells are associated with an increase in ACAT mRNA14b levels. To test this hypothesis we altered cellular cholesteryl ester mass and synthesis rates in primary parenchymal and nonparenchymal cells using various extracellular agents and measured the accumulated mass of ACAT mRNA14b. Parenchymal cells incubated with rabbit beta VLDL or mevalonolactone displayed a 6-10-fold increase in cellular cholesteryl ester mass over a three day treatment with no significant changes in cellular free cholesterol, triacylglycerols, or ACAT mRNA14b levels; HMG CoA reductase and LDL receptor mRNA mass decreased initially as a result of cholesteryl ester loading. Treatment of parenchymal cells with CI-976, an ACAT inhibitor, showed a marked reduction in cholesteryl ester synthetic rate compared to beta VLDL controls but displayed no change in ACAT mRNA14b levels. A mixed population of rabbit hepatic nonparenchymal cells was incubated with beta VLDL for 24 h in culture which resulted in a 6-fold increase in cellular cholesteryl ester mass; there was no change in ACAT mRNA14b levels. In an in vivo study, rabbits consuming a high fat/high cholesterol-diet for three weeks showed a 10-fold increase in hepatic cholesteryl ester with no significant changes in ACAT mRNA14b levels. Together these data indicate that rabbit liver cellular cholesteryl ester mass increases of up to 10-fold are not correlated with ACAT mRNA14b changes. Thus, hepatic ACAT mRNA14b expression and cellular cholesterol esterification do not appear to be coordinately regulated at this level of cholesteryl ester loading.

Safety and Tolerability of ACP-501, a Recombinant Human Lecithin:Cholesterol Acyltransferase, in a Phase 1 Single-Dose Escalation Study

RATIONALE Low high-density lipoprotein-cholesterol (HDL-C) in patients with coronary heart disease (CHD) may be caused by rate-limiting amounts of lecithin:cholesterol acyltransferase (LCAT). Raising LCAT may be beneficial for CHD, as well as for familial LCAT deficiency, a rare disorder of low HDL-C. OBJECTIVE To determine safety and tolerability of recombinant human LCAT infusion in subjects with stable CHD and low HDL-C and its effect on plasma lipoproteins. METHODS AND RESULTS A phase 1b, open-label, single-dose escalation study was conducted to evaluate safety, tolerability, pharmacokinetics, and pharmacodynamics of recombinant human LCAT (ACP-501). Four cohorts with stable CHD and low HDL-C were dosed (0.9, 3.0, 9.0, and 13.5 mg/kg, single 1-hour infusions) and followed up for 28 days. ACP-501 was well tolerated, and there were no serious adverse events. Plasma LCAT concentrations were dose-proportional, increased rapidly, and declined with an apparent terminal half-life of 42 hours. The 0.9-mg/kg dose did not significantly change HDL-C; however, 6 hours after doses of 3.0, 9.0, and 13.5 mg/kg, HDL-C was elevated by 6%, 36%, and 42%, respectively, and remained above baseline ≤4 days. Plasma cholesteryl esters followed a similar time course as HDL-C. ACP-501 infusion rapidly decreased small- and intermediate-sized HDL, whereas large HDL increased. Pre-β-HDL also rapidly decreased and was undetectable ≤12 hours post ACP-501 infusion. CONCLUSIONS ACP-501 has an acceptable safety profile after a single intravenous infusion. Lipid and lipoprotein changes indicate that recombinant human LCAT favorably alters HDL metabolism and support recombinant human LCAT use in future clinical trials in CHD and familial LCAT deficiency patients. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01554800.

Familial lecithin:cholesterol acyltransferase deficiency: First-in-human treatment with enzyme replacement

BACKGROUND Humans with familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) have extremely low or undetectable high-density lipoprotein cholesterol (HDL-C) levels and by early adulthood develop many manifestations of the disorder, including corneal opacities, anemia, and renal disease. OBJECTIVE To determine if infusions of recombinant human LCAT (rhLCAT) could reverse the anemia, halt progression of renal disease, and normalize HDL in FLD. METHODS rhLCAT (ACP-501) was infused intravenously over 1 hour on 3 occasions in a dose optimization phase (0.3, 3.0, and 9.0 mg/kg), then 3.0 or 9.0 mg/kg every 1 to 2 weeks for 7 months in a maintenance phase. Plasma lipoproteins, lipids, LCAT levels, and several measures of renal function and other clinical labs were monitored. RESULTS LCAT concentration peaked at the end of each infusion and decreased to near baseline over 7 days. Renal function generally stabilized or improved and the anemia improved. After infusion, HDL-C rapidly increased, peaking near normal in 8 to 12 hours; analysis of HDL particles by various methods all revealed rapid sequential disappearance of preβ-HDL and small α-4 HDL and appearance of normal α-HDL. Low-density lipoprotein cholesterol increased more slowly than HDL-C. Of note, triglyceride routinely decreased after meals after infusion, in contrast to the usual postprandial increase in the absence of rhLCAT infusion. CONCLUSIONS rhLCAT infusions were well tolerated in this first-in-human study in FLD; the anemia improved, as did most parameters related to renal function in spite of advanced disease. Plasma lipids transiently normalized, and there was rapid sequential conversion of small preβ-HDL particles to mature spherical α-HDL particles.

A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase.

We describe a simple but sensitive fluorescence method to accurately detect the esterification activity of lecithin:cholesterol acyltransferase (LCAT). The new assay protocol employs a convenient mix, incubate, and measure scheme. This is possible by using the fluorescent sterol dehydroergosterol (DHE) in place of cholesterol as the LCAT substrate. The assay method is further enhanced by incorporation of an amphiphilic peptide in place of apolipoprotein A-I as the lipid emulsifier and LCAT activator. Specific fluorescence detection of DHE ester synthesis is achieved by employing cholesterol oxidase to selectively render unesterified DHE nonfluorescent. The assay accurately detects LCAT activity in buffer and in plasma that is depleted of apolipoprotein B lipoproteins by selective precipitation. Analysis of LCAT activity in plasmas from control subjects and sickle cell disease (SCD) patients confirms previous reports of reduced LCAT activity in SCD and demonstrates a strong correlation between plasma LCAT activity and LCAT content. The fluorescent assay combines the sensitivity of radiochemical assays with the simplicity of nonradiochemical assays to obtain accurate and robust measurement of LCAT esterification activity.

Biology, Pathology, and Interfacial Enzymology of Pancreatic Phospholipase A2

The phospholipase A2 (PLA2) secreied by Ihe exocrine pancreas is one of the earliest known members of a large class of enzymes that catalyze hydrolysis of the ester-bond linking fatty acids to the sn-2 position of glycerophospholipids (Fig. 1). The phospholipase A2 enzyme class has been intensely scrutinized, in large part because the sn-2-spccific deacylation of glycerophospholipids is a critical regulatory step in a variety of metabolic pathways, ranging from digestion and defense mechanisms to cicosanoid synthesis and signal transduction.

Inhibitors of acyl-coa:cholesterol acyltransferase (ACAT). 15. sulfonylurea inhibitors with excellent hypocholesterolemic activity in vivo.

Abstract A series of sulfonylureas were prepared and tested for the ability to inhibit the enzyme acyl-CoA: cholesterol acyltransferase (ACAT) in vitro and lower plasma cholesterol in cholesterol-fed rats in vivo. Although compounds from this series were generally weak inhibitors of ACAT in vitro, several displayed excellent hypocholesterolemic activity in vivo.