Safety, pharmacokinetics, and pharmacodynamics of oral apoA-I mimetic peptide D-4F in high-risk cardiovascular patients

Patients with coronary heart disease or equivalent risk received a single dose of 30, 100, 300, or 500 mg of unformulated D-4F (n = 8, each dose) or placebo (n = 8) under fasting conditions. An additional 10 patients received 500 mg (n = 8) or placebo (n = 2) with a low-fat meal. There were no significant trends in any safety parameter. D-4F was detectable in plasma at all doses with a Tmax of 30 min, 1 h, and 2 h for 30, 100, and ⩾ 300 mg, respectively. The area under the curve(0−t) was 27.81 ng/hr/ml and 54.71 ng/hr/ml for the 300 mg and 500 mg dose groups, respectively, and 17.96 ng/hr/ml for the 500mg dose given with food. HDL from each time point for each subject was tested for its ability to inhibit LDL-induced monocyte chemotactic activity in cultures of human aortic endothelial cells. The values obtained were normalized to 1.0 for LDL alone to obtain the HDL inflammatory index. This index significantly improved at 4 h at the 300 mg dose and at 2 h at the 500 mg dose compared with placebo (P < 0.05). There were no changes in plasma lipid or lipoprotein levels. We conclude that unformulated D-4F has low bioavailability that is improved under fasting conditions, and that a single dose of D-4F is safe and well tolerated and may improve the HDL anti-inflammatory index. Patients with coronary heart disease or equivalent risk received a single dose of 30, 100, 300, or 500 mg of unformulated D-4F (n = 8, each dose) or placebo (n = 8) under fasting conditions. An additional 10 patients received 500 mg (n = 8) or placebo (n = 2) with a low-fat meal. There were no significant trends in any safety parameter. D-4F was detectable in plasma at all doses with a Tmax of 30 min, 1 h, and 2 h for 30, 100, and ⩾ 300 mg, respectively. The area under the curve(0−t) was 27.81 ng/hr/ml and 54.71 ng/hr/ml for the 300 mg and 500 mg dose groups, respectively, and 17.96 ng/hr/ml for the 500mg dose given with food. HDL from each time point for each subject was tested for its ability to inhibit LDL-induced monocyte chemotactic activity in cultures of human aortic endothelial cells. The values obtained were normalized to 1.0 for LDL alone to obtain the HDL inflammatory index. This index significantly improved at 4 h at the 300 mg dose and at 2 h at the 500 mg dose compared with placebo (P < 0.05). There were no changes in plasma lipid or lipoprotein levels. We conclude that unformulated D-4F has low bioavailability that is improved under fasting conditions, and that a single dose of D-4F is safe and well tolerated and may improve the HDL anti-inflammatory index. adverse event apolipoprotein A-I area under the curve coronary heart disease electrocardiogram type 2 diabetes mellitus total cholesterol Epidemiologically, levels of HDL cholesterol and its major protein, apolipoprotein A-I (apoA-I), are inversely related to cardiovascular risk (1.Kwiterovich Jr, P.O. The antiatherogenic role of high-density lipoprotein cholesterol.Am. J. Cardiol. 1998; 82: 13Q-21QAbstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). ApoA-I is widely recognized to be involved in the initial steps of macrophage reverse cholesterol transport (2.Rader D.J. Molecular regulation of HDL metabolism and function: implications for novel therapies.J. Clin. Invest. 2006; 116: 3090-3100Crossref PubMed Scopus (472) Google Scholar). It may also be critical to the anti-inflammatory properties of HDL (3.Barter P.J. Nicholls S. Rye K.A. Anantharamaiah G.M. Navab M. Fogelman A.M. Antiinflammatory properties of HDL.Circ. Res. 2004; 95: 764-772Crossref PubMed Scopus (1056) Google Scholar). Deficiency of apoA-I in atherosclerosis prone mice results in significantly more atherosclerosis despite preserved HDL cholesterol levels (4.Moore R.E. Kawashiri M.A. Kitajima K. Secreto A. Millar J.S. Pratico D. Rader D.J. Apolipoprotein A-I deficiency results in markedly increased atherosclerosis in mice lacking the LDL receptor.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1914-1920Crossref PubMed Scopus (81) Google Scholar). Conversely, transgenic overexpression, somatic gene transfer, or intravenous infusion of apoA-I in mice and rabbits has demonstrated reduced progression and even regression of atherosclerosis (5.Badimon J.J. Badimon L. Fuster V. Regression of atherosclerotic lesions by high density lipoprotein plasma fraction in the cholesterol-fed rabbit.J. Clin. Invest. 1990; 85: 1234-1243Crossref PubMed Scopus (674) Google Scholar, 6.Benoit P. Emmanuel F. Caillaud J.M. Bassinet L. Castro G. Gallix P. Fruchart J.C. Branellec D. Denefle P. Duverger N. Somatic gene transfer of human apoA-I inhibits atherosclerosis progression in mouse models.Circulation. 1999; 99: 105-110Crossref PubMed Scopus (133) Google Scholar, 7.Plump A. Scott C. Breslow J. Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse.Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 9607-9611Crossref PubMed Scopus (546) Google Scholar). In humans, a small-scale clinical trial with intravenous recombinant apoA-I Milano complexed with phospholipids demonstrated a significant reduction in coronary atheroma volume as measured by intravascular ultrasound after 5 weeks of therapy in patients with acute coronary syndrome (8.Nissen S.E. Tsunoda T. Tuzcu E.M. Schoenhagen P. Cooper C.J. Yasin M. Eaton G.M. Lauer M.A. Sheldon W.S. Grines C.L. et al.Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial.JAMA. 2003; 290: 2292-2300Crossref PubMed Scopus (1564) Google Scholar). Given such results, it is believed that directly targeting apoA-I in humans may positively influence atherosclerosis. One approach to targeting apoA-I is to develop structurally related peptides that "mimic" apoA-I function in vivo (9.Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Yu N. Ansell B.J. Datta G. Garber D.W. et al.Apolipoprotein A-I mimetic peptides.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1325-1331Crossref PubMed Scopus (227) Google Scholar). D-4F is an apoA-I mimetic peptide composed of 18 D-amino acids, thereby preventing degradation by gut peptidases and enhancing oral absorption (9.Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Yu N. Ansell B.J. Datta G. Garber D.W. et al.Apolipoprotein A-I mimetic peptides.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1325-1331Crossref PubMed Scopus (227) Google Scholar). Like apoA-I, D-4F binds nonoxidized lipids (9.Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Yu N. Ansell B.J. Datta G. Garber D.W. et al.Apolipoprotein A-I mimetic peptides.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1325-1331Crossref PubMed Scopus (227) Google Scholar) but D-4F has a structure that enhances its ability to bind and sequester fatty acid hydroperoxides and proinflammatory oxidized phospholipids (10.Anantharamaiah G.M. Mishra V.K. Garber D.W. Datta G. Handattu S.P. Palgunachari M.N. Chaddha M. Navab M. Reddy S.T. Segrest J.P. et al.Structural requirements for antioxidative and anti-inflammatory properties of apolipoprotein A-I mimetic peptides.J. Lipid Res. 2007; 48: 1915-1923Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Oral administration of D-4F has been shown to improve HDL anti-inflammatory properties in mice and monkeys and dramatically reduce lesions in mouse models of atherosclerosis despite no change in plasma levels of HDL cholesterol (11.Navab M. Anantharamaiah G.M. Reddy S.T. Fogelman A.M. Apolipoprotein A-I mimetic peptides and their role in atherosclerosis prevention.Nat. Clin. Pract. Cardiovasc. Med. 2006; 3: 540-547Crossref PubMed Scopus (115) Google Scholar). We present data from the first trial of D-4F in humans. We evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of a single oral dose of D-4F in patients with stable coronary heart disease (CHD) or a CHD equivalent state. The study included 36 men and 14 postmenopausal women (age range, 21–75 years) with stable CHD or a CHD equivalent as defined by the National Cholesterol Education Program Adult Treatment Panel III criteria (12.Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in AdultsExecutive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III).JAMA. 2001; 285: 2486-2497Crossref PubMed Scopus (24244) Google Scholar). Eligible subjects were required to have been on a stable (>4 weeks) regimen of statin therapy. Major exclusion criteria included unstable CHD, congestive heart failure, New York Heart Association class III or IV angina pectoris, niacin >500 mg or fibrate use, uncontrolled hypertension, hemoglobin A1c >10%, serum creatine kinase above the upper limit of normal, and serum albumin <2.5 mg/dl. The protocols were approved by the General Clinical Research Center and the Institutional Review Board at the University of Pennsylvania. All subjects provided written informed consent to participate in the study. In this double-blinded study, eligible subjects were randomized to a single oral dose of D-4F at 30, 100, 300, or 500 mg or matching placebo after a 12 h fast. The first 3 dose groups included 10 subjects (8 were randomized to D-4F, 2 received placebo). The 500 mg dose group included 20 subjects (16 received D-4F, 4 received placebo). Thus, there were a total of 10 placebo subjects in these groups that were available for comparison with the subjects receiving D-4F. The first 3 dose groups and the first 10 subjects in the 500 mg dose group (500 mg, fasted) received the single dose of test article after an overnight fast and at least 2 h prior to a standardized low-fat meal and any concomitant medications. The last 10 subjects who received 500 mg or placebo had a standardized low-fat meal provided within 10 min after dosing (500 mg, fed). Blood was obtained at baseline, 15 min, 30 min, 1 h, 2 h, 6 h, 12 h, and 24 h after study drug administration for plasma D-4F levels. Subjects returned for follow-up visits 2, 7, 14, and 30 days after study drug administration to evaluate safety laboratory measures (chemistry, hematology, coagulation, creatine kinase, and cytokine profiles), adverse events (AEs), and clinical findings. AEs were judged by a study investigator as mild, moderate or severe. Relationship of AEs to study drug was assigned by the investigator as probable, possible, unlikely or none. A single study investigator reviewed all AEs at the end of the study. The peptide D-4F with the primary amino acid sequence Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 was synthesized by the solid phase method using an automated solid phase synthesizer (PS3 Protein Technologies, Woburn, MA) following Good Manufacturing Practice guidelines. D-amino acids were coupled to a Rink AM resin (Matrix Innovation) and were acetylated with acetic anhydride at the N-terminus. The peptides were cleaved from the solid support using 70% trifloroacetic acid in dichloromethane and were purified via reverse-phase HPLC column. The purity of the peptides was verified via analytical reverse-phase HPLC and mass spectral analysis. Because there is no difference other than oral bioavailability between the peptide 4F when synthesized from all D-amino acids (D-4F) compared with the same peptide synthesized from all L-amino acids (L-4F), the latter was used for some in vitro experiments. D-4F is highly water-soluble, and each dose was diluted in 30 ml of 25% sucrose-water solution, yielding a test article concentration ranging from 1 mg/ml (lowest dose) to 16.67 mg/ml (highest dose). The vehicle control (25% sucrose solution) served as the placebo. The chilled diluted study agent was administered immediately after dilution and filter sterilization. Samples from each dose group were validated for purity, content uniformity, and concentration of D-4F by an independent laboratory (Alta Analytical Laboratory, Inc., El Dorado Hills, CA). Plasma levels of D-4F were determined by Alta Analytical Laboratory, Inc., using HPLC and liquid chromatography atmospheric pressure ionization tandem mass spectrometry (LC-API-MS-MS) with 13C, 15N-4F as the internal standard. The validated lower limit of quantification was 1.0 ng/ml. Intra-assay accuracy and precision values were within ±15%. The calibration curves were linear over a range of 1–250 ng/ml. Pharmacokinetic parameters including the area under the curve (AUC) from time zero to the time of the last measurable concentration [AUC(0−t)], the AUC from time zero to infinity [AUC(o−inf)], the terminal elimination rate (Kel), the apparent elimination half-life (T1/2), maximum drug concentration (Cmax), and time of the maximum drug concentration following dosing (Tmax) were computed by MDS Pharma (Lincoln, NE) from the plasma drug concentration time data for D-4F by noncompartmental analysis, using WinNonlin version 4.0 and SAS version 8.2 (SAS Institute). Lipid parameters were analyzed from EDTA plasma collected after a 12 h fast in a Centers for Disease Control and Prevention standardized lipid laboratory. Plasma total cholesterol (TC), HDL cholesterol, and triglycerides (TG) were measured enzymatically on a Cobas Fara II autoanalyzer (Roche Diagnostic Systems Inc.) using Sigma reagents (Sigma Chemical Co.). LDL cholesterol and VLDL cholesterol levels were determined after ultracentrifugation at a density of 1.006 g/ml. ApoA-I was measured using Wako reagents (Wako Chemicals USA, Inc.). ApoA-I and HDL cholesterol were measured over 24 h at the same time points as the HDL inflammatory index. Samples for analysis of the HDL inflammatory index were coded and shipped on dry ice from the University of Pennsylvania by overnight courier to the University of California-Los Angeles where the determinations were made blinded to treatments. The samples for the HDL inflammatory index were sucrose cryopreserved and the HDL inflammatory index was determined as previously described (13.Ansell B.J. Navab M. Hama S. Kamranpour N. Fonarow G. Hough G. Rahmani S. Mottahedeh R. Dave R. Reddy S.T. et al.Inflammatory/antiinflammatory properties of high-density lipoprotein distinguish patients from control subjects better than high-density lipoprotein cholesterol levels and are favorably affected by simvastatin treatment.Circulation. 2003; 108: 2751-2756Crossref PubMed Scopus (526) Google Scholar). Briefly, a normal control human LDL was added to human aortic endothelial cells in culture at 100 μg LDL cholesterol per ml without added HDL or together with the test HDL prepared by fast protein liquid chromatography and added at a concentration of 50 μg HDL cholesterol per ml. After 8 h the supernatants were collected and assayed in triplicate for monocyte chemotactic activity as previously described (13.Ansell B.J. Navab M. Hama S. Kamranpour N. Fonarow G. Hough G. Rahmani S. Mottahedeh R. Dave R. Reddy S.T. et al.Inflammatory/antiinflammatory properties of high-density lipoprotein distinguish patients from control subjects better than high-density lipoprotein cholesterol levels and are favorably affected by simvastatin treatment.Circulation. 2003; 108: 2751-2756Crossref PubMed Scopus (526) Google Scholar). The monocyte chemotactic activity produced in this assay is virtually all due to the production of monocyte chemotactic protein-1 by the endothelial cells (14.Navab M. Imes S.S. Hama S.Y. Hough G.P. Ross L.A. Bork R.W. Valente A.J. Berliner J.A. Drinkwater D.C. Laks H. et al.Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein.J. Clin. Invest. 1991; 88: 2039-2046Crossref PubMed Scopus (645) Google Scholar). The values in the absence of HDL were normalized to 1.0. Values >1.0 after the addition of HDL indicated proinflammatory HDL; values <1.0 indicated anti-inflammatory HDL. The HDL inflammatory index has been shown to correlate with atherosclerotic lesion area and with serum amyloid A levels in cholesterol-fed rabbits (15.Van Lenten B.J. Wagner A.C. Navab M. Anantharamaiah G.M. Hama S. Reddy S.T. Fogelman A.M. Lipoprotein inflammatory properties and serum amyloid A levels but not cholesterol levels predict lesion area in cholesterol-fed rabbits.J. Lipid Res. 2007; 48: 2344-2353Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Determination of LDL lipid hydroperoxide levels and paraoxonase activity were performed as previously described (16.Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Wagner A.C. Frank J.S. Datta G. Garber D. et al.Oral D-4F causes formation of pre-beta high-density lipoprotein and improves high-density lipoprotein-mediated cholesterol efflux and reverse cholesterol transport from macrophages in apolipoprotein E-null mice.Circulation. 2004; 109: 3215-3220Crossref PubMed Scopus (312) Google Scholar). The data from the University of California-Los Angeles and from Alta Analytical Lab (El Dorado Hills, CA) were sent to the Data Management Center at MDS Pharma (Lincoln, NE), where the samples were decoded and statistical analyses were performed. D-4F or scrambled D-4F (Ac-DWFAKDYFKKAFVEEFAK-NH2), a peptide that does not promote α-helix formation, was added at concentrations up to 300 ng/ml to human plasma from subjects with CHD or a CHD equivalent (prior to administration of the test article). The plasma was incubated under argon for 1 h and fractionated by fast protein liquid chromatography and the HDL inflammatory index, lipoprotein lipid hydroperoxide contents and HDL-paraoxonase activity were determined as previously described (13.Ansell B.J. Navab M. Hama S. Kamranpour N. Fonarow G. Hough G. Rahmani S. Mottahedeh R. Dave R. Reddy S.T. et al.Inflammatory/antiinflammatory properties of high-density lipoprotein distinguish patients from control subjects better than high-density lipoprotein cholesterol levels and are favorably affected by simvastatin treatment.Circulation. 2003; 108: 2751-2756Crossref PubMed Scopus (526) Google Scholar, 16.Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Wagner A.C. Frank J.S. Datta G. Garber D. et al.Oral D-4F causes formation of pre-beta high-density lipoprotein and improves high-density lipoprotein-mediated cholesterol efflux and reverse cholesterol transport from macrophages in apolipoprotein E-null mice.Circulation. 2004; 109: 3215-3220Crossref PubMed Scopus (312) Google Scholar). In other in vitro studies L-4F was added to the endothelial cells at concentrations ranging from 1–1,000 ng/ml with LDL in the absence of HDL. All individual and group mean data for pharmacokinetics pattern of D-4F were tabulated along with descriptive statistics [N, N missing, mean, SD, CV (%), SEM, median, minimum, and maximum]. Changes in pharmacodynamic measures were assessed by mean change from predose to postdose time points by dose group, dose group versus placebo, treatment versus placebo, and time. All descriptive and lipid statistical analyses were performed by MDS Pharma (Lincoln, NE) using SAS statistical software version 8.2 (SAS Institute, Cary, NC). The statistical analysis of the lipid concentration and HDL inflammatory index was performed for change from baseline values within each treatment group and compared with placebo. The analysis was conducted using ANOVA using a linear mixed model of repeated measurements to test the differences between D-4F dose groups and placebo at each time point. Pairwise comparisons of the LS means between D-4F dose groups and placebo at each time points of collection were examined. Analysis of data from the in vitro studies after addition of D-4F or scrambled D-4F was performed by ANOVA using GraphPad InStat version 3.05, 32 bit for Windows 95/NT (GraphPad Software, San Diego, CA). For the in vitro studies, the appropriate statistical analysis of change from baseline for HDL inflammatory index for each individual time point was based on the normality of the data within each time point and treatment. For the comparison between DF-4 and placebo at each time point, the Mann-Whitney test (nonparametric test) was used if the data were not normal. For the comparison of each postdose time point to baseline for each treatment, the Wilcoxon's signed rank test (nonparametric test) was used if the data were not normal. If the data were normal for the pairwise comparisons, ANOVA with Satterhwaite's adjustment was performed. For all statistical analyses, the α level was defined at the 0.05 statistical significance level. Because the primary objective was assessment of safety and tolerability, no formal sample size or power calculations were performed. The baseline demographics of the subjects are presented in Table 1 . The majority of subjects were male, Caucasian (subjects were given the option to report more than one race), and obese. Thirty-two percent of the subjects had CHD; 70% had type 2 diabetes mellitus (T2DM).TABLE 1.Baseline demographics by population and dose groupTrait30 mg100 mg300 mg500 mg fasted500 mg fedAll PlaceboOverallMale sex, n67645836Female sex, n21243214Race American Indian/Alaska Native0001203 Asian/Pacific Islander0000101 Black34210313 Caucasian54665733 Hispanic0210014Age (mean ± SD), y63.5 ± 5.652.6 ± 9.458.5 ± 6.658.4 ± 6.764.5 ± 6.361.6 ± 8.859.9 ± 8.1Weight (mean ± SD), kg91.5 ± 20.091.3 ± 13.695.0 ± 18.182.9 ± 17.389.3 ± 14.395.9 ± 15.591.2 ± 16.3Body mass index (mean ± SD), kg/m231.7 ± 5.830.0 ± 2.231.4 ± 4.729.7 ± 3.931.7 ± 4.132.0 ± 3.531.3 ± 4.0CHD, n30433316Receiving an angiotensin receptor blocker or angiotensin-converting enzyme inhibitor37515526T2DM, n47556835Other forms of cardiovascular disease, n0031116>20% risk if no CHD or T2DM1100002Systolic blood pressure (mean ± SD), mm Hg134 ± 12.6138 ± 31.7136 ± 16.7116 ± 11.3142 ± 23.0136 ± 22.4—–Diastolic blood pressure (mean ± SD), mm Hg76 ± 14.581 ± 19.079 ± 10.572 ± 6.776 ± 10.080 ± 12.6—–HDL cholesterol (mean ± SD), mg/dl41 ± 11.6946 ± 14.2446 ± 13.8445 ± 11.7548 ± 14.9754 ± 21.03—– Open table in a new tab Fig. 1 shows the mean plasma concentrations of D-4F over time. Absorption of D-4F was rapid, less than 1%, and dose-dependent in the fasted groups. The mean maximum plasma concentrations for 30 mg, 100 mg, 300 mg, 500 mg fasted, and 500 mg fed were: 1.62 ± 1.92 ng/ml, 7.75 ± 6.43 ng/ml, 8.13 ± 5.66 ng/ml, 15.9 ± 6.53 ng/ml, and 4.49 ± 5.47 ng/ml, respectively. Five of the eight subjects (62.5%) randomized to 30 mg D-4F had at least one time point with measurable plasma levels of D-4F compared with 7 of 8 (87.5%) of the subjects in the 100 mg and 300 mg dose groups; 8 (100%) in the 500 mg (fasted) dose group and 6 (75%) in the 500 mg (fed) group. The AUC(0−t) values for each dose group were determined by averaging the detectable values for each dose group and were 2.17 ± 2.51 ng/h/ml (30 mg), 23.11 ± 26.19 ng/h/ml (100 mg), 27.81 ± 17.04 ng/h/ml (300 mg), 54.71 ± 22.10 ng/h/ml (500 mg fasted), and 17.96 ± 17.96 ng/h/ml (500 mg fed). The Tmax by dose group was 0.5 ± 0 (30 mg), 0.938 ± 0.8 (100 mg), 2.45 ± 1.6 (300 mg), 2.0 ± 0 (500 mg fasted), and 1.98 ± 2.1 h (500 mg fed). No formal sample size calculations were performed for pharmacodynamic readouts, because these were not the primary objective of this study. The 10 subjects in the 500 mg fed group were not analyzed for any pharmacodynamic parameter, becuase they were only included in the pharmacokinetic and safety studies. There was no change in HDL cholesterol or apoA-I levels over 24 h at any dose over time compared with placebo (Fig. 2) or when comparing all D-4F dose groups to placebo (data not shown). The change from baseline was significant for VLDL cholesterol and triglycerides at 24 h within several treatment groups (including placebo for triglycerides), but there were no differences in the change from baseline for any other plasma lipids and lipoproteins at any dose over time compared with change from baseline in placebo (Table 2) or when combining data from all D-4F-treated subjects to placebo (data not shown). Nor was there a significant change in LDL lipid hydroperoxide levels or HDL paraoxonase activity (data not shown). The mean ± SD for the baseline HDL inflammatory index values for all fasting subjects was 1.18 ± 0.32 (range, 0.59–1.97). Of the 40 subjects studied in the fasted groups, 30 subjects (75%) had an HDL inflammatory index >1.0 at baseline despite statin treatment. The number of subjects with an HDL inflammatory index >1.0 at baseline in the 30 mg, 100 mg, 300 mg, and 500 mg fasted groups was: 4 (40%), 8 (80%), 9 (90%), and 9 (90%), respectively. The change in HDL inflammatory index within each treatment group compared with placebo is presented in Table 3 . There was a significant improvement in the HDL inflammatory index at 4 h at 300 mg and 2 h at 500 mg compared with placebo. In all treatment groups, including the placebo group, the mean HDL inflammatory index was <1.0 at 8 h post dose which returned to >1.0 at 24 h. Compared with baseline within each treatment group, the change at 8 h was significant in the 100, 300, and 500 mg dose groups, but not placebo.Fig. 2.There was no significant change in plasma apoA-I concentrations (A) or HDL cholesterol levels (B) after administration of D-4F.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2.Lipids over 48 hours by dose group compared with placebo30 mg (n = 8)100 mg (n = 8)300 mg (n = 8)500 mg (fasted) (n = 8)All Placebo (n = 10)Variable (mg/dl)0 h24 h48 h0 h24 h48 h0 h24 h48 h0 h24 h48 h0 h24 h48 hTotal cholesterol160 (13)166 (14)168 (14)174 (13)174 (14)174 (14)151 (13)151 (14)156 (14)153 (13)155 (14)156 (14)145 (12)149 (13)154 (13)Δ = 6Δ = 8Δ = 0Δ = 0Δ = 0Δ = 5Δ = 2Δ = 3Δ = 3.50Δ = 9.20P = 0.06P = 0.21P = 0.94P = 0.92P = 0.84P = 0.36P = 0.52P = 0.61P = 0.22P = 0.09δ = 18δ = 13δ = 25.4δ = 20.1δ = 2.5δ = 1.7δ = 6.2δ = 2.0P = 0.35P = 0.48P = 0.18P = 0.29P = 0.89P = 0.93P = 0.74P = 0.92VLDL cholesterol21 (5)24 (5)26 (5)33 (5)39 (5)33 (5)15 (5)21 (5)18 (5)27 (5)35 (5)34 (5)15 (5)19 (5)19 (5)Δ = 3Δ = 5Δ = 6Δ = 0Δ = 6Δ = 3Δ = 8Δ = 7Δ = 4.4Δ = 3.8P = 0.30P = 0.09P = 0.04P = 0.80P = 0.04P = 0.26P = 0.01P = 0.01P = 0.09P = 0.14δ = −1.4δ = 1.2δ = 1.6δ = −3.1δ = 1.6δ = −0.6δ = 3.7δ = 3.5P = 0.69P = 0.78P = 0.65P = 0.47P = 0.65P = 0.90P = 0.29P = 0.42LDL cholesterol102 (10)100 (10)99 (10)96 (10)93 (10)94 (10)89 (10)86 (10)91 (10)82 (10)77 (10)77 (10)76 (9)79 (9)80 (9)Δ = −2Δ = −3Δ = −3Δ = −2Δ = −3Δ = 2Δ = −5Δ = −5Δ = 1.4Δ = 2.7P = 0.64P = 0.59P = 0.25P = 0.70P = 0.25P = 0.81P = 0.07P = 0.42P = 0.56P = 0.59δ = −2.7δ = −5.7δ = −4.5δ = −4.8δ = −4.5δ = −1.3δ = −6.4δ = −7.2P = 0.47P = 0.38P = 0.22P = 0.46P = 0.22P = 0.84P = 0.08P = 0.27Triglycerides129 (19)141 (19)121 (19)149 (19)188 (19)150 (19)99 (19)126 (19)94 (19)143 (19)171 (19)146 (19)100 (17)128 (17)95 (17)Δ = 12Δ = −8Δ = 39Δ = 1Δ = 27Δ = −5Δ = 28Δ = 3Δ = 28.65Δ = −4.55P = 0.34P = 0.53P < 0.01P = 0.92P = 0.03P = 0.69P = 0.020.76P = 0.01P = 0.68δ = −16.8δ = −3.4δ = 9.8δ = 5.7δ = −1.7δ = −0.5δ = 0.1δ = 8.4P = 0.30P = 0.83P = 0.57P = 0.72P = 0.92P = 0.97P = 1.0P = 0.60Results are reported as LS means (SE). Δ = Change compared with baseline within treatment group. δ = Net change from baseline in each dose group compared with change from baseline in placebo. Statistical comparison is to placebo. Open table in a new tab TABLE 3.Change in HDL inflammatory index by time point and treatment group compared with placeboTime Point30 mg (n = 8)100 mg (n = 8)300 mg (n = 8)500 mg (fasted) (n = 8)All Placebo (n = 10)Baseline0.99 (0.11)1.15 (0.11)1.38 (0.11)1.30 (0.11)1.10 (0.11)2 hours post dose1.01 (0.08)1.01 (0.08)1.21 (0.08)1.07 (0.08)1.09 (0.08)Δ = 0.02Δ = −0.14Δ = −0.17Δ = −0.23Δ = −0.01P = 0.87P = 0.11P = 0.07P = 0.01P = 0.90δ = 0.03δ = −0.13δ = −0.15δ = −0.22P = 0.80P = 0.22P = 0.14P = 0.034 hours post dose1.02 (0.10)0.94 (0.10)1.02 (0.10)1.05 (0.12)1.05 (0.10)Δ = 0.03Δ = −0.21Δ = −0.36Δ = −0.25Δ = −0.05P = 0.79P = 0.02P < 0.001P = 0.01P = 0.62δ = 0.07δ = −0.16δ = −0.31δ = −0.20P = 0.55P = 0.17P = 0.009P = 0.098 hours post dose0.80 (0.12)0.89 (0.12)0.92 (1.12)0.90 (0.12)0.92 (0.12)Δ = −0.19Δ = −0.26Δ = −0.46Δ = −0.40Δ = −0.17P = 0.06P = 0.01P < 0.0001P < 0.001P = 0.09δ = −0.02δ = −0.08δ = −0.28δ = −0.23P = 0.92P = 0.60P = 0.08P = 0.1624 hours post dose1.11 (0.12)1.12 (0.12)1.19 (0.12)1.17 (0.12)1.16 (0.12)Δ = 0.12Δ = −0.03Δ = −0.19Δ = −0.13Δ = 0.06P = 0.23P = 0.82P = 0.07P = 0.19P = 0.57δ = 0.06δ = −0.08δ = −0.25δ = −0.19P = 0.71P = 0.63P = 0.15P = 0.26Results are reported as LS means (SE). Δ = Change compared with baseline within treatment group. δ = Net change from baseline in each dose group compared with change from baseline in placebo. Open table in a new tab Results are reported as LS means (SE). Δ = Change compared with baseline within treatment group. δ = Net change from baseline in each dose group compared with change from baseline in placebo. Statistical comparison is to placebo. Results are reported as LS means (SE). Δ = Change compared with baseline within treatment group. δ = Net change from baseline in each dose group compared with change from baseline in placebo. Adding D-4F to human plasma in vitro at 250 ng/ml was previously shown to improve HDL inflammatory index. Given the low plasma concentrations achieved in this study, concentrations of D-4F starting at 25 ng/ml up to 300 ng/ml were added to plasma from 3 p