Treatment of patients with cardiovascular disease with L-4F, an apo-A1 mimetic, did not improve select biomarkers of HDL function
2010; Elsevier BV; Volume: 52; Issue: 2 Linguagem: Inglês
10.1194/jlr.m011098
ISSN1539-7262
AutoresCatherine E. Watson, Nicole Weissbach, Lise Kjems, Surya Ayalasomayajula, Yiming Zhang, Ih Chang, Mohamad Navab, Susan Hama, Greg Hough, Srinivasa T. Reddy, Daniel Soffer, Daniel J. Rader, Alan M. Fogelman, Alison D. Schecter,
Tópico(s)Lipoproteins and Cardiovascular Health
ResumoL-4F, an apolipoprotein A-I (apoA-I) mimetic peptide (also known as APL180), was administered daily by either intravenous (IV) infusion for 7 days or by subcutaneous (SC) injection for 28 days in patients with coronary heart disease in two distinct clinical studies. L-4F was well tolerated at all doses tested. Despite achieving plasma levels (mean maximal plasma concentration of 2,907 ng/ml and 395 ng/ml, following IV infusion and SC injection, respectively), that were effective in previously published animal models, treatment with L-4F, as assessed by biomarkers of HDL function such as HDL-inflammatory index (HII), and paraoxonase activity, did not improve. Paradoxically, there was a 49% increase in high-sensitivity C-reactive protein (hs-CRP) levels after seven IV infusions of 30 mg L-4F (P < 0.05; compared with placebo) and a trend for hs-CRP increase in subjects receiving 30 mg SC injection for 28 days. In a subsequent, ex vivo study, addition of L-4F at concentrations of 150, 375, or 1,000 ng/ml to plasma from subjects prior to L-4F treatment resulted in significant dose-dependent HII improvement. In conclusion, in vivo L-4F treatment, delivered by either SC injection or IV infusion, did not improve HDL functional biomarkers despite achieving plasma levels that improved identical biomarkers ex vivo and in animal models. L-4F, an apolipoprotein A-I (apoA-I) mimetic peptide (also known as APL180), was administered daily by either intravenous (IV) infusion for 7 days or by subcutaneous (SC) injection for 28 days in patients with coronary heart disease in two distinct clinical studies. L-4F was well tolerated at all doses tested. Despite achieving plasma levels (mean maximal plasma concentration of 2,907 ng/ml and 395 ng/ml, following IV infusion and SC injection, respectively), that were effective in previously published animal models, treatment with L-4F, as assessed by biomarkers of HDL function such as HDL-inflammatory index (HII), and paraoxonase activity, did not improve. Paradoxically, there was a 49% increase in high-sensitivity C-reactive protein (hs-CRP) levels after seven IV infusions of 30 mg L-4F (P < 0.05; compared with placebo) and a trend for hs-CRP increase in subjects receiving 30 mg SC injection for 28 days. In a subsequent, ex vivo study, addition of L-4F at concentrations of 150, 375, or 1,000 ng/ml to plasma from subjects prior to L-4F treatment resulted in significant dose-dependent HII improvement. In conclusion, in vivo L-4F treatment, delivered by either SC injection or IV infusion, did not improve HDL functional biomarkers despite achieving plasma levels that improved identical biomarkers ex vivo and in animal models. Epidemiologic data support that elevated levels of HDL cholesterol are associated with an improvement in cardiovascular risk (1Gordon T. Castelli W.P. Hjortland M.C. Kannel W.B. Dawber T.R. High density lipoprotein as a protective factor against coronary heart disease.Am. J. Med. 1977; 62: 707-714Abstract Full Text PDF PubMed Scopus (4082) Google Scholar). HDL particles provide atherosclerosis protection by way of their role in reverse cholesterol transport and potentially their direct anti-inflammatory, antioxidant, and antithrombotic properties. However, there is an increasing body of literature that not all HDL particles are equal in their cardioprotective effects and that certain properties of the HDL particle itself confer either pro- or anti-inflammatory effects (2Navab M. Reddy S.T. Van Lenten B.J. Anantharamaiah G.M. Fogelman A.M. The role of dysfunctional HDL in atherosclerosis.J. Lipid Res. 2009; 50: 145-149Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Thus, HDL function, rather than the absolute level of HDL-cholesterol, may be a more accurate indicator for risk of developing atherosclerosis and of manifesting its clinical sequelae (2Navab M. Reddy S.T. Van Lenten B.J. Anantharamaiah G.M. Fogelman A.M. The role of dysfunctional HDL in atherosclerosis.J. Lipid Res. 2009; 50: 145-149Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). This hypothesis has led to investigation of HDL as both a biomarker for cardiovascular risk and a therapeutic target to be functionally manipulated (3Navab M. Anantharamaiah G.M. Reddy S.T. Van Lenten B.J. Fogelman A.M. HDL as a biomarker, potential therapeutic target and therapy.Diabetes. 2009; 58: 2711-2717Crossref PubMed Scopus (91) Google Scholar). Increasing the plasma concentrations of the main apolipoprotein in HDL, apolipoprotein A-I (apoA-I), through intravenous (IV) administration has been considered an attractive approach. However, treatment with recombinant HDL or even apoA-I is difficult because the commercial production of apoA-I (a 243-amino acid protein) is not trivial and requires complexing with lipid prior to IV administration. Therefore, smaller peptides that "mimic" or augment apoA-I activity have been explored as novel therapeutics (4Navab M. Shechter I. Anantharamaiah G.M. Reddy S.T. Van Lenten B.J. Fogelman A.M. Structure and function of HDL mimetics.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 164-168Crossref PubMed Scopus (92) Google Scholar). The apoA-I mimetic peptides D-4F and L-4F (APL180) have shown promise in a number of animal models (4Navab M. Shechter I. Anantharamaiah G.M. Reddy S.T. Van Lenten B.J. Fogelman A.M. Structure and function of HDL mimetics.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 164-168Crossref PubMed Scopus (92) Google Scholar) and in early human trials with D-4F (5Bloedon L.T. Dunbar R. Duffy D. Pinell-Salles P. Norris R. DeGroot B.J. Movva R. Navab M. Fogelman A.M. Rader D.J. Safety, pharmacokinetics and pharmacodynamics of oral apoA-I mimetic peptide D-4F in high-risk cardiovascular patients.J. Lipid Res. 2008; 49: 1344-1352Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). The mechanism of action of these peptides has been reported to be due to their extraordinary ability to bind oxidized and oxidizable lipids with four to six orders of magnitude higher affinity, as compared with native apoA-I (6Van Lenten B.J. Wagner A.C. Jung C.L. Ruchala P. Waring A.J. Lehrer R.I. Watson A.D. Hama S. Navab M. Anantharamaiah G.M. Anti-inflammatory apoA-I mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I.J. Lipid Res. 2008; 49: 2302-2311Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). After oral administration in mice, these mimetic peptides have been reported to improve the ability of HDL to inhibit LDL-induced monocyte chemoattractant protein-1 (MCP-1) production by human aortic endothelial cells as measured by monocyte migration in vitro and quantified as the HDL-inflammatory index (HII) (6Van Lenten B.J. Wagner A.C. Jung C.L. Ruchala P. Waring A.J. Lehrer R.I. Watson A.D. Hama S. Navab M. Anantharamaiah G.M. Anti-inflammatory apoA-I mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I.J. Lipid Res. 2008; 49: 2302-2311Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 7Navab M. Anantharamaiah G.M. Hama S. Garber D.W. Chaddha M. Hough G. Lallone R. Fogelman A.M. Oral administration of an apo A-I mimetic peptide synthesized from D-amino acids dramatically reduces atherosclerosis in mice independent of plasma cholesterol.Circulation. 2002; 105: 290-292Crossref PubMed Scopus (367) Google Scholar, 8Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Wagner A.C. Frank J.S. Datta G. Garber D. 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 (311) Google Scholar, 9Navab M. Ruchala P. Waring A.J. Lehrer R.I. Hama S. Hough G. Palgunachari M.N. Anantharamaiah G.M. Fogelman A.M. A novel method for oral delivery of apolipoprotein mimetic peptides synthesized from all L-amino acids.J. Lipid Res. 2009; 50: 1538-1547Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Ansell B.J. Navab M. Hama S. Kamranpour N. Fonarow G. Hough G. Rahmani S. Mottahedeh R. Dave R. Reddy S.T. Inflammatory/anti-inflammatory 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 (521) Google Scholar, 11Navab M. Anantharamaiah G.M. Hama S. Hough G. Reddy S.T. Frank J.S. Garber D.W. Handattu S. Fogelman A.M. D-4F and statins synergize to render HDL anti-inflammatory in mice and monkeys and cause lesion regression in old apolipoprotein E-null mice.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1426-1432Crossref PubMed Scopus (143) Google Scholar, 12Van 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). The maximal plasma concentration (Cmax) of 4F (L or D) in the mouse studies were on the order of 100 to 300 ng/ml of peptide after oral administration (8Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Wagner A.C. Frank J.S. Datta G. Garber D. 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 (311) Google Scholar, 9Navab M. Ruchala P. Waring A.J. Lehrer R.I. Hama S. Hough G. Palgunachari M.N. Anantharamaiah G.M. Fogelman A.M. A novel method for oral delivery of apolipoprotein mimetic peptides synthesized from all L-amino acids.J. Lipid Res. 2009; 50: 1538-1547Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In ex vivo studies with human plasma, addition of 250 to 1,000 ng/ml of peptide resulted in a decreased HII (13Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Yu N. Ansell B.J. Datta G. Garber D.W. Apolipoprotein A-I mimetic peptides.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1325-1331Crossref PubMed Scopus (226) Google Scholar, 14Vaziri N.D. Moradi H. Pahl M.V. Fogelman A.M. Navab M. In vitro stimulation of HDL anti-inflammatory activity and inhibition of LDL pro-inflammatory activity in the plasma of patients with end-stage renal disease by an apoA-1 mimetic peptide.Kidney Int. 2009; 76: 437-444Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In a previous clinical study with D-4F, administration of a single oral dose of 300 mg or 500 mg achieved low Cmax plasma levels (approximately 10 ng/ml) due to the poor bioavailability of the oral formulation. Despite this fact, this oral formulation of D-4F improved the HII in patients with coronary heart disease (CHD) or a CHD equivalent compared with placebo, whereas lower oral doses did not have an effect (5Bloedon L.T. Dunbar R. Duffy D. Pinell-Salles P. Norris R. DeGroot B.J. Movva R. Navab M. Fogelman A.M. Rader D.J. Safety, pharmacokinetics and pharmacodynamics of oral apoA-I mimetic peptide D-4F in high-risk cardiovascular patients.J. Lipid Res. 2008; 49: 1344-1352Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Oxidized lipids bind with similar high affinity to D-4F and L-4F (6Van Lenten B.J. Wagner A.C. Jung C.L. Ruchala P. Waring A.J. Lehrer R.I. Watson A.D. Hama S. Navab M. Anantharamaiah G.M. Anti-inflammatory apoA-I mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I.J. Lipid Res. 2008; 49: 2302-2311Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). D-4F is synthesized from d-amino acids which are poorly degraded in mammals, as discussed previously (15Garber D.W. Venkatachalapathi Y.V. Gupta K.B. Ibdah J. Phillips M.C. Hazelrig J.B. Segrest J.P. Anantharamaiah G.M. Turnover of synthetic class A amphipathic peptide analogues of exchangeable apolipoproteins in rats. Correlation with physical properties.Arterioscler. Thromb. 1992; 12: 886-894Crossref PubMed Google Scholar). This led to prolonged tissue retention times of D-4F, particularly in liver and kidney, in preliminary studies in animals (data not shown). In contrast, L-4F is synthesized from l-amino acids and was rapidly degraded in mammalian tissues (data not shown). Despite these differences, the effects of D-4F and L-4F on biomarkers and lesion area were similar when administered by subcutaneous (SC) injection in cholesterol-fed rabbits (12Van 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). Therefore, human studies with L-4F were initiated to probe the HDL anti-inflammatory effects of apoA-I mimetics. The studies described herein are the first clinical studies of L-4F administered to humans by either IV infusion (ClinicalTrials.gov number, NCT00568594) or by SC injection (ClinicalTrials.gov number, NCT00907998). For these initial short studies in CHD patients, in addition to safety and pharmacokinetics, biomarkers of HDL function and systemic inflammation were assessed. The following pharmacodynamic (PD) biomarker endpoints were measured: HII using a cell-based assay, paraoxonase (PON), high-sensitivity C-reactive protein (hs-CRP), and interleukin-6 (IL-6) levels. We report here that despite achieving plasma levels of L-4F comparable to or well above those achieved in mouse models (8Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Wagner A.C. Frank J.S. Datta G. Garber D. 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 (311) Google Scholar, 9Navab M. Ruchala P. Waring A.J. Lehrer R.I. Hama S. Hough G. Palgunachari M.N. Anantharamaiah G.M. Fogelman A.M. A novel method for oral delivery of apolipoprotein mimetic peptides synthesized from all L-amino acids.J. Lipid Res. 2009; 50: 1538-1547Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and in the early human studies with D-4F (5Bloedon L.T. Dunbar R. Duffy D. Pinell-Salles P. Norris R. DeGroot B.J. Movva R. Navab M. Fogelman A.M. Rader D.J. Safety, pharmacokinetics and pharmacodynamics of oral apoA-I mimetic peptide D-4F in high-risk cardiovascular patients.J. Lipid Res. 2008; 49: 1344-1352Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), no significant change in HII or PON were observed. Unexpectedly, there was a trend toward increases in hs-CRP and IL-6 levels. The peptide L-4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2) was synthesized from l-amino acids by solid-phase synthesis as described (9Navab M. Ruchala P. Waring A.J. Lehrer R.I. Hama S. Hough G. Palgunachari M.N. Anantharamaiah G.M. Fogelman A.M. A novel method for oral delivery of apolipoprotein mimetic peptides synthesized from all L-amino acids.J. Lipid Res. 2009; 50: 1538-1547Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). For in vitro studies, a control peptide, scrambled L-4F, with the same l-amino acids as in L-4F but in a sequence that does not promote α-helical formation (Ac-D-W-F-A-K-D-Y-F-K-K-A-F-V-E-E-F-A-K-NH2) was synthesized by solid-phase synthesis as described previously (8Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Wagner A.C. Frank J.S. Datta G. Garber D. 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 (311) Google Scholar). For both the IV and SC studies, the peptides were provided to the clinical trial sites as a lyophilized powder in a sterile trehalose-phosphate buffer. Prior to use, the powder was reconstituted with sterile water for injection (SWFI) to produce vials containing 0.2 or 3 mg/ml (IV study) and 10 or 30 mg/ml (SC study). Placebo consisted of lyophilized trehalose-phospate buffer that was reconstituted with SWFI. Multiple vials (APL180 and/or placebo) were used to produce the required dose for the IV study. Results presented herein are from two separate studies, an IV infusion study (APL180A2201; Fig. 1A) and an SC injection study (APL180A2210B; Fig. 1B). The IV infusion study initially included eight subjects at each dose level treated with IV infusions daily for 7 days to establish safety/tolerability. Additional patients were enrolled in the 30 mg dose group to increase the number of subjects and allow for more-robust statistical analyses. The SC study involved a single SC injection daily for 28 days. For both the IV and SC studies, males and females (post-menopausal or surgically sterile) between the ages of 18 and 75 years with stable CHD or a CHD equivalent and on a stable dose of a statin (>8 weeks) were eligible. CHD and equivalents were defined by the National Cholesterol Education Program Adult Treatment Panel III criteria (16Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults.Executive 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). 2001; 285: 2486-2497Google Scholar), and CHD patients were required to be event- and procedure-free (e.g., no documented myocardial infarction (MI), unstable angina, coronary revascularization) for at least 6 months prior to inclusion. CHD equivalent patients included those with a history of symptomatic carotid artery disease (e.g., transient ischemic attack or stroke of carotid origin); patients with peripheral artery disease; diabetes mellitus (excluded if HbA1c ≥9%); 20% 10 year risk of CHD (Framingham point score ≥16 for men and ≥23 for women); or patients with other clinical forms of atherosclerotic disease, including >50% stenosis on angiography or ultrasound or other forms of clinical atherosclerotic disease (e.g., renal artery disease). All subjects provided informed written consent before participating in any study procedures. The study was approved by the Independent Ethics Committee and/or Institutional Review Board at each study center and local health authorities. The studies were conducted in accordance with good clinical practice and the ethical principles of the Declaration of Helsinki. Both studies were registered in accord with the International Committee of Medical Journal Editors (http://prsinfo.clinicaltrials.gov/icmje.html). As shown in Fig. 1A, the IV study was a randomized, double-blinded, placebo-controlled study with ascending multiple doses of L-4F given as daily IV infusions for seven consecutive days. Initially for this study, each ascending dose cohort consisted of eight patients randomized to receive L-4F (3, 10, 30, or 100 mg) or placebo (6:2) (APL180A2201), and then a larger cohort of 40 patients (randomized 1:1 to receive 30 mg L-4F or placebo) were added. Eligible patients reported to the site for baseline assessments 24 h prior to their dose (day −1) and remained domiciled until day 2. On day 1, following an overnight fast, patients received an IV dose of L-4F or placebo. The dose was administered as a 2 h IV infusion, where 47% of the dose was given at a constant rate for the first 15 min, then the rate of infusion was decreased and the remaining 53% was administered over the next 1 h and 45 min. This dosing regime was chosen such that the exposures obtained would approximate those expected from a SC injection. Following dosing on day 1, patients continued to fast for an additional 4 h. Safety, pharmacokinetic (PK), and PD assessments were conducted predose and continued throughout the first 24 h. Patients received their second daily IV dose at the clinic, were closely monitored for at least 1 h post dose, and then were released from the clinic. Patients returned to the site each morning for their daily IV infusions. Patients were readmitted to the site on day 6 prior to supper and stayed domiciled until day 8. Following an overnight fast, the procedures on day 1 were repeated on day 7. The patients were released from the site 24 h after their seventh dose, then returned 7, 14, and 28 days later (study days 14, 21, and 35) to undergo immunogenicity and safety assessments. As shown in Fig. 1B, the SC study (APL180A2210B) was a parallel, randomized, double-blind, placebo-controlled, multiple-dose (daily SC injections for 28 days) design. A total of 104 patients were randomized to three distinct dose arms (10 or 30 mg L-4F, or placebo) in a 1:1:1 ratio. Eligible patients reported to the site for baseline assessments on day −1 and remained domiciled until day 2. On day 1, following an overnight fast, the first injection was administered. The doses were given in the abdomen, and the injection sites were rotated daily between the four quadrants (upper and lower right, upper and lower left). Safety, PK, and PD assessments were conducted predose and continued throughout the first 24 h. Patients received their second dose at the clinic on the morning of day 2, were closely monitored for at least 4 h, and then were released from the clinic. The daily SC doses were administered by a trained nurse on an out-patient basis until day 27, at which time the nurse assessed local tolerance of SC injections, other adverse events (AEs) and changes to comedication. On days 8, 15, and 22, additional safety, PK, and PD assessments were made. On day 28, patients reported to the site after an overnight fast and continued to fast for 4 h postdose. PK/PD samples were collected predose and for 24 h postdose. The patients were released from the site 24 h after their 28th dose, then returned 7 and 14 days later (days 35 and 42) to undergo immunogenicity and safety assessments. In both studies, the choice of a placebo control over a scrambled peptide control was chosen due to the time and resources required to qualify the scrambled peptide for use in humans. All AEs and serious adverse events (SAEs) were recorded during the study period. The treating physician assessed all AEs for severity and determined the likelihood of a possible relationship to the study drug. In addition, blood hematologic and chemistry profiles and urinalysis, vital signs, body weight, physical condition, and 12-lead electrocardiogram (ECG) were monitored regularly throughout the studies. In the SC study, injection site reactions (ISRs) were monitored daily for severity of pain, redness, induration, swelling, hemorrhage, and itching, and it was left to the discretion of the investigator whether to report the ISR as an AE. Blood samples for pharmacokinetic assessment were obtained at regular intervals up to 24 h postdose on day 1 and days 7/28 (IV/SC, respectively). In the SC study, weekly samples (days 8, 15, and 22) were also obtained to determine trough levels. In brief, blood samples (2 ml) were collected in sodium-heparin tubes, plasma was extracted, and samples were frozen at –70°C until analysis. Plasma levels of intact L-4F were determined using a validated LC-MS/MS methodology with a lower limit of quantitation of 2.5 ng/ml. For analysis, protein was precipitated from the plasma sample using acetonitrile and centrifugation in a 96-well format on a TomTec Quadra 96 System. Solid-phase extraction was performed on the supernatant using a Waters Oasis HLB µElution plate and using an elution mixture of methanol-water-acetic acid. Samples were then injected onto an Ace C8 5 µm column, and eluted with a gradient of acetonitrle in formic acid. MS was carried out on an API3000 Applied Biosystem using TurboIonspray® with positive-ion mode. The steady-state pharmacokinetic parameters, including peak plasma concentrations (Cmax), area under the plasma concentration-time curve during dosing interval of 24 h (AUCtau), time to reach peak concentrations (Tmax), total clearance following IV dosing (Cl), terminal half-life (T1/2), and average daily concentration (Cavg) were estimated using noncompartmental methods (WinNonlin, version 5.2). Blood samples for biomarker assessment were obtained predose and at 0.25 h, 2 h (end of infusion), 4, 8, 12, and 24 h postdose on day 1 and day 7 for the IV study. During the SC study, samples were collected predose on days 1, 2, 8, 15, and 28, as well as at 2, 4, and 8 h postdose on day 28. Biomarker measurements included the HII, which was determined using previously described methods (10Ansell B.J. Navab M. Hama S. Kamranpour N. Fonarow G. Hough G. Rahmani S. Mottahedeh R. Dave R. Reddy S.T. Inflammatory/anti-inflammatory 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 (521) Google Scholar, 12Van 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). To assess the reproducibility of the cell-based assay used for determining the HII, a single plasma sample from one subject was divided into four aliquots, and each was processed on a different fast-protein liquid chromatography (FPLC) system to obtain the HDL-containing fractions; these fractions were then tested on four different plates containing the same human aortic endothelial cells exposed to the same standard control LDL on the same day. MCP-1 was then assayed by bioassay using the same normal human peripheral blood monocytes from a single normal donor but using different chambers on the same day. The results are shown in Table 1 and indicate a coefficient of variation of 21%.TABLE 1Determining the reproducibility of the HIIAssayHII ValueIntra-assay variability#11.04#21.15#31.13#41.61Mean ± SD (CV)1.23 ± 0.26 (21%)Inter-day variationDay 11.34 ± 0.36Day 21.36 ± 0.44CV, coefficient of variation; HII, HDL-inflammatory index. To determine the intra-assay reproducibility of the HII, plasma from one subject was divided into four aliquots and each was processed on a different fast-protein liquid chromatography (FPLC) system to obtain the HDL-containing fractions. These fractions were tested on four different plates containing the human aortic endothelial cells from the same preparation and exposed to the same standard control LDL on the same day. Monocyte chemoattractant protein-1 was then assayed by monitoring membrane migration of the same normal human monocytes but using different chambers on the same day. To determine the inter-day reproducibility of the HII, four subjects from the multi-dose IV study were randomly selected, and their plasma samples were divided into two aliquots that were processed on different days and assayed using different human aortic endothelial cells and different monocytes. Open table in a new tab CV, coefficient of variation; HII, HDL-inflammatory index. To determine the intra-assay reproducibility of the HII, plasma from one subject was divided into four aliquots and each was processed on a different fast-protein liquid chromatography (FPLC) system to obtain the HDL-containing fractions. These fractions were tested on four different plates containing the human aortic endothelial cells from the same preparation and exposed to the same standard control LDL on the same day. Monocyte chemoattractant protein-1 was then assayed by monitoring membrane migration of the same normal human monocytes but using different chambers on the same day. To determine the inter-day reproducibility of the HII, four subjects from the multi-dose IV study were randomly selected, and their plasma samples were divided into two aliquots that were processed on different days and assayed using different human aortic endothelial cells and different monocytes. To further support the reproducibility of the cell-based assay used for determining the HII, four subjects from the IV study were randomly selected, and their plasma samples were divided into two aliquots that were then processed on different days and assayed using different human aortic endothelial cells and different monocytes. The mean ± SD of the HII values obtained on the two different assay days are shown in Table 1 and indicate a mean difference of 1.5%. A third approach to characterize the reproducibility of the HII was as follows: the HII values obtained at each time point on the first and seventh days following infusion (time points assayed after infusion were 15 min, and 2, 4, 8, 12, and 24 h) for the four subjects described in Table 1 were divided by the predose HII value on the first and seventh day, respectively, and plotted against each other. The results are shown in Fig. 2 and confirmed the reproducibility of the method using samples obtained on different days. Other biomarkers assessed in these studies were PON activity, hs-CRP, and IL-6. PON activity was assessed, in sodium-heparin plasma using paraoxon as a substrate, as previously described (7Navab M. Anantharamaiah G.M. Hama S.
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