Novel fatty acyl apoE mimetic peptides have increased potency to reduce plasma cholesterol in mice and macaques

Abstract

Ac-hE18A-NH₂ is a dual-domain apoE mimetic peptide that possesses the putative receptor binding domain from apoE (LRKLRKRLLR, denoted hE; residues 141-150) covalently attached to lipid-associating peptide 18A. Like apoE, Ac-hE18A-NH₂ reduces plasma cholesterol in animal models and exhibits anti-inflammatory properties independent of its cholesterol-reducing effect. Ac-hE18A-NH₂ has already undergone phase I clinical trials as a lipid-lowering agent. To explore the therapeutic potential more, we designed and synthesized new analogues by linking ɑ-aminohexanoic acid, octanoic acid, or myristic acid to LRRLRRRLLR-18A-NH₂ ([R]hE18A-NH₂) and examined the cholesterol-lowering potency in animals. The modified peptides effectively reduced plasma cholesterol in apoE-null mice fed standard chow or a Western diet; the myristyl analogue was the most effective. A single administration of the myristyl analogue reduced plasma total and LDL cholesterol in a dose-dependent manner in hypercholesterolemic cynomolgus macaques for up to 1 week despite the continuation of a cholesterol-supplemented diet. The myristyl peptide (7.4 mg/kg) reduced total and LDL cholesterol at 24 h by 64% and 74%, respectively; plasma HDL levels were modestly reduced and returned to baseline by day 7. These new analogues should exhibit enhanced potency at lower doses than Ac-hE18A-NH₂, which may make them attractive therapeutic candidates for clinical trials.

Keywords: apolipoprotein E; dyslipidemia; lipoproteins; low density lipoprotein; peptides.

Fig. 1.

Chromatographic profiles for apoE mimetic peptides. HPLC profiles for Ac-hE18A-NH₂, Ac-[R]hE18A-NH₂, Ac-Aha-[R]hE18A-NH₂, Oct-[R]hE18A-NH₂, Myr-hE18A-NH₂, and Myr-[R]hE18A-NH₂. Peptides were injected on a C-18 reverse-phase column and eluted using an acetonitrile-water gradient (35% to 70%). UV absorbance was monitored at 220 nm (A). B: Plot of acyl chain length versus log retention time for Ac-[R]hE18A-NH₂, Ac-Aha-[R]hE18A-NH₂, Oct-[R]hE18A-NH₂, and Myr-[R]hE18A-NH₂.

Fig. 2.

ApoE mimetic peptides alter the electrophoretic mobility of LDL. Samples of human-derived LDL (5 µg) and LDL peptide complexes were diluted with loading buffer containing 10% glycerol, loaded on an agarose gel, and subjected to electrophoresis.

Fig. 3.

ApoE mimetic peptides reduce plasma cholesterol in apoE-null mice. A: A predose blood sample was collected from mice fed a standard chow diet, followed by retro-orbital injection of 5 mg/kg Ac-hE18A-NH₂ (n = 4), Ac-[R]hE18A-NH₂ (n = 4), or Ac-Aha-[R]hE18A-NH₂ (n = 4). B: Cholesterol reduction in response to 2.5 mg/kg Myr-[R]hE18A-NH₂ (n = 5), Oct-[R]hE18A-NH₂ (n = 5), and Ac-Aha[R]hE18A-NH₂ (n = 4). Data are means ± SEMs.

Fig. 4.

Effects of myristylated peptides on plasma cholesterol in apoE-null mice. Myr-hE18A-NH₂ (n = 3) or Myr-[R]hE-18A-NH₂ (n = 3) was injected in mice at a dose of 2.5 mg/kg. Both myristylated analogues induced a similar reduction in plasma cholesterol. Control mice (n = 3) received 0.2% Tween 20 as vehicle treatment. Data are means ± SEMs.

Fig. 5.

Efficacy of apoE mimetic peptides in dyslipidemic apoE-null mice. A: ApoE mimetic peptides (7.5 mg/kg) were injected in apoE-null mice fed a Western diet. Cholesterol reduction was monitored in response to vehicle control (n = 4), Ac-hE18A-NH₂ (n = 5), Ac-Aha-[R]hE18A-NH₂ (n = 5), and Myr-[R]hE18A-NH₂ (n = 5). B: Changes in plasma cholesterol over the 24 h study period were quantitated by measuring the AUCs by the least-squares method for each animal from panel A. Data are means ± SEMs. *P < 0.02 versus Ac-hE18A-NH₂; **P < 0.01 versus control; and ^†P < 0.001 versus control. AUC, area under the curve.

Fig. 6.

l-Arg decoy does not influence peptide-mediated cholesterol reduction in apoE-null mice. Myr-[R]hE18A-NH₂ was solubilized in Tween 20 in the presence of l-Arg to prevent the binding of the peptide at the injection site. The peptide was also prepared in the absence of l-Arg. Each peptide solution was injected at a dose of 2.5 mg/kg in apoE-null mice, and the effects on plasma cholesterol were monitored. Data are means ± SEMs (n = 5 per treatment). *P < 0.001 versus saline control.

Fig. 7.

Myr-[R]hE18A-NH₂ reduces LDL in cynomolgus monkeys. Plasma LDL concentration was measured at baseline and at time periods up to 7 d after the intravenous administration of increasing doses of Myr-[R]hE18A-NH₂. Changes in LDL at each time postdose were measured relative to predose LDL concentrations. Data are means ± SEMs (n = 4 per treatment). Microsoft Excel was used to first perform a single-factor ANOVA comparing the mean 24 h LDL minimums following administration at each dose level. That analysis rejected the hypothesis that all of those minimums were equal to one another. A follow-up Newman-Keuls multiple-range test was then performed to test all possible pairwise comparisons for any significant differences between minimum mean LDL levels following each dose. Based on four monkeys per group to calculate the mean minimum LDL levels, the Newman-Keuls analysis determined that all minimums were significantly different from one another at the P < 0.025 level.

Fig. 8.

Effect of Myr-[R]hE18A-NH₂ on the cholesterol profiles at different times postadministration in a cynomolgus monkey. Lipoprotein profiles for a monkey receiving Myr-[R]hE18A-NH₂ at a dose of 7.4 mg/kg at time points up to 1 day (A) and 7 days (B) after peptide administration. Plasma samples (100 uL) were injected onto a Superose column. PBS (0.4 ml/min) was used to elute the samples. Cholesterol reagent (0.2 ml/min) was then mixed with the eluent, followed by heating to 57°C and measurement of UV absorbance at 540 nm.