Journal of Antimicrobial Chemotherapy Advance Access published December 23, 2014

J Antimicrob Chemother doi:10.1093/jac/dku501

Atherogenic properties of lipoproteins in HIV patients starting atazanavir/ritonavir or darunavir/ritonavir: a substudy of the ATADAR randomized study Maria Saumoy1*, Jordi Ordo´n˜ez-Llanos2,3, Esteban Martı´nez4, Elena Ferrer1, Pere Domingo5, Esteban Ribera6, Eugenia Negredo7, Jordi Curto1, Jose´ Luis Sa´nchez-Quesada2, Silvana Di Yacovo1, Ana Gonza´lez-Cordo´n4 and Daniel Podzamczer1 1

HIV Unit, Infectious Disease Service, Hospital Universitari de Bellvitge, Bellvitge Biomedical Research Institute, Hospitalet de Llobregat, Barcelona, Spain; 2Biomedical Research Institute IIB Sant Pau, Barcelona, Spain; 3Biochemistry and Molecular Biology Department, Universitat Auto`noma, Barcelona, Spain; 4Infectious Disease Service, Hospital Clı´nic, Barcelona, Spain; 5Infectious Diseases Unit, Hospital de la Santa Creu i Sant Pau, Universitat Auto`noma de Barcelona, Barcelona, Spain; 6Infectious Disease Service, Hospital Universitari Vall d’Hebron, Universitat Auto`noma de Barcelona, Barcelona, Spain; 7Fundacio´ Lluita contra la Sida, Hospital Germans Trias i Pujol, Badalona, Spain *Corresponding author. Tel: +34-93-260-7667; Fax: +34-93-260-7669; E-mail: [email protected]

Received 9 July 2014; returned 2 September 2014; revised 31 October 2014; accepted 15 November 2014 Objectives: To assess LDL subfraction phenotype and lipoprotein-associated phospholipase A2 (Lp-PLA2) in naive HIV-infected patients starting atazanavir/ritonavir or darunavir/ritonavir plus tenofovir/emtricitabine. Methods: This was a substudy of a multicentre randomized study. Standard lipid parameters, LDL subfraction phenotype (by gradient gel electrophoresis) and Lp-PLA2 activity (by 2-thio-PAF) were measured at baseline and weeks 24 and 48. Multivariate regression analysis was performed. Results are expressed as the median (IQR). Results: Eighty-six (atazanavir/ritonavir, n¼45; darunavir/ritonavir, n¼41) patients were included: age 36 (31–41) years; 89% men; CD4 319 (183–425) cells/mm3; and Framingham score 1% (0%–2%). No differences in demographics or lipid measurements were found at baseline. At week 48, a mild but significant increase in total cholesterol and HDL-cholesterol was observed in both arms, whereas LDL cholesterol increased only in the darunavir/ ritonavir arm and triglycerides only in the atazanavir/ritonavir arm. The apolipoprotein A-I/apolipoprotein B ratio increased only in the atazanavir/ritonavir arm. At week 48, the LDL subfraction phenotype improved in the darunavir/ritonavir arm (increase in LDL particle size and in large LDL particles), whereas it worsened in the atazanavir/ritonavir arm (increase in small and dense LDL particles, shift to a greater prevalence of phenotype B); the worsening was related to the greater increase in triglycerides in the atazanavir/ritonavir arm. No changes in total Lp-PLA2 activity or relative distribution in LDL or HDL particles were found at week 48 in either arm. Conclusions: In contrast with what occurred in the atazanavir/ritonavir arm, the LDL subfraction phenotype improved with darunavir/ritonavir at week 48. This difference was associated with a lower impact on plasma triglycerides with darunavir/ritonavir. Keywords: LDL size, LDL subfraction phenotype, lipoprotein-associated phospholipase A2

Introduction The risk of developing cardiovascular (CV) disease is higher in HIV-infected patients than in the general population.1 In recent years, new CV biomarkers have been evaluated, to further refine CV risk assessment. The LDL subfraction phenotype and lipoprotein-associated phospholipase A2 (Lp-PLA2) have been associated with coronary disease and stroke in the overall population.2,3

There are few data on these biomarkers in naive HIV-infected patients. The effect of initiating combined ART (c-ART) on plasma levels of these biomarkers is not completely understood.4 – 7 Before c-ART, an increase in the prevalence of LDL phenotype B, characterized by small proatherogenic LDL particles related to hypertriglyceridemia, was described in patients with AIDS.4 Lipid disturbances, leading to a proatherogenic pattern of increased triglycerides and decreased HDL-cholesterol concentrations, are

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common in antiretroviral-naive HIV-infected patients, probably related to chronic inflammation. Initiation of c-ART is usually associated with an improvement in lipid parameters, attributable to a return to prior metabolic health.8 We hypothesized that the decreased inflammation achieved by c-ART could improve the atherogenicity of LDL particles. Nevertheless, the impact of c-ART-related lipid disturbances on these biomarkers is unknown. The aim of this study was to analyse the LDL subfraction phenotype and Lp-PLA2 distribution in naive HIV-infected patients, the relationships between these biomarkers and lipid and virological parameters and the impact on these biomarkers of initiating c-ART with atazanavir/ritonavir or darunavir/ritonavir, two PIs with a minor impact on lipid profile, combined with tenofovir/emtricitabine.9,10

Methods Study design and patient population This is a substudy of the ATADAR study (ClinicalTrials.gov number NCT01274780),11 a multicentre, randomized, open-label clinical trial carried out in 16 Spanish hospitals that compared the effects of darunavir/ritonavir (800/100 mg/day) or atazanavir/ritonavir (300/100 mg/day) plus tenofovir/ emtricitabine (245/200 mg/day) on lipid profile in c-ART-naive HIV-infected patients. Briefly, the inclusion criteria were clinically stable patients older than 18 years, who had never received any antiretroviral drug and had plasma HIV RNA ≥1000 copies/mL. The exclusion criteria were alanine or aspartate amino transferase ≥200 IU/L, plasma creatinine ≥2.6 mg/dL, diabetes mellitus, BMI ≥30 kg/m2, use of drugs known to affect lipid or glucose metabolism 1 month prior to inclusion, any AIDS-defining event requiring parenteral therapy, hypersensitivity to or contraindication to the study drugs, and pregnancy or lactation at inclusion or expectancy to become pregnant during follow-up. The protocol was approved by the central and local ethics committees, and written informed consent was obtained from all participants prior to their inclusion in the substudy. Patients enrolled in the main study from five hospitals located in the metropolitan area of Barcelona were invited to participate. The primary endpoint was the change in the LDL subfraction phenotype and Lp-PLA2 activity at week 48 of treatment. Secondary endpoints were the changes in the LDL subfraction phenotype and Lp-PLA2 activity at week 24, and changes in the classic CV risk factors, standard lipid measures and CV risk estimated by the Framingham score at weeks 24 and 48.

Clinical assessment Clinical assessment and laboratory evaluations were performed at baseline and weeks 24 and 48. At each visit, we recorded the use of treatments other than ART, including lipid-lowering, antidiabetic and antihypertensive drugs. CV risk factors according to the National Cholesterol Education Program guidelines (NECP III) were evaluated at each visit.12 The estimated overall CV risk over the next 10 years was calculated with the Framingham score.13 Blood pressure and anthropometric measurements including height, weight and waist and hip circumferences were evaluated at each visit. The BMI and the waist-to-hip ratio were calculated.

Laboratory methods After an overnight fast, venous blood samples were drawn into glass tubes containing gelose with no additives (BD Vacutainer SST, Franklin Lakes, NJ, USA). Tubes were immediately centrifuged and sent on dry ice to a central laboratory in the Barcelona metropolitan area. Lipid measurements were performed no later than 72 h after sample collection for all parameters with the exception of Lp-PLA2, which was determined from aliquots stored at 2808C for no longer than 3 months after drawing. Total cholesterol (TC),

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triglycerides—corrected for free glycerol—and glucose were measured by standard enzymatic methods; apolipoprotein B (Apo B) and apolipoprotein A-I (Apo A-I) were measured by immunoturbidimetry; and HDL-cholesterol concentrations were determined by a homogeneous direct method. All assays were from Roche Diagnostics (Basel, Switzerland). TC/HDL and Apo A-I/Apo B ratios were calculated. LDL-cholesterol was calculated by the Friedewald formula when triglyceride concentrations were ,300 mg/dL or measured after separation of lipoproteins by ultracentrifugation at 105000 g for 18 h at 48C when triglycerides were ≥300 mg/dL. All measurements were controlled by national and international external quality assurance programmes and fulfilled the international recommendations for inaccuracy and total imprecision. LDL size and LDL subfraction phenotype were determined by gel electrophoresis in 3% polyacrylamide tubes using a commercial system (Lipoprint, Quantimetrix Co., Redondo Beach, CA, USA). This method classifies individuals into three phenotypes: A (LDL size .268 A˚ with predominance of large buoyant LDL subfractions), intermediate (LDL size 260 – 268 A˚) and B [LDL size ,260 A˚ with a predominance of small and dense LDL (sd-LDL) subfractions]. Results are expressed as LDL size (A˚) and as cholesterol content (mg/dL) in LDL particles. Lp-PLA2 activity was measured in serum (total Lp-PLA2) and in lipoproteins using 2-thio-PAF (Cayman Chemical Company, Ann Arbor, MI, USA) as the substrate, as previously described;14 activity was expressed as mmol/min.mL. To determine Lp-PLA2 in lipoprotein subfractions, we precipitated Apo B-containing lipoproteins [very LDL, intermediate-density lipoprotein, LDL and lipoprotein(a)] from serum using dextran sulphate; Lp-PLA2 activity measured in the supernatant corresponded to the activity associated with HDL (HDL-Lp-PLA2).15 Lp-PLA2 activity in Apo B-containing lipoproteins was obtained by subtracting HDL-Lp-PLA2 from total Lp-PLA2. Since most of this activity is due to PLA2 associated with LDL, it is referred to hereafter as LDL-Lp-PLA2. Insulin and C-peptide were measured by immunochemiluminometric assays (Roche Diagnostics, Basel, Switzerland). Insulin sensitivity was estimated with the homeostasis model assessment insulin resistance (HOMA-IR) index as the product of the concentrations of fasting glucose (mmol/L) and the fasting insulin expressed as mIU/L, divided by 22.5.

Statistical analysis Sample size could not be estimated in advance since the biological variability of the biochemical measures established as the primary endpoint is unknown. Therefore, a significance level of 0.05 was considered biologically relevant. Prior to the statistical analyses, the normality of distribution and homogeneity of variance were tested. The Student’s t-test or Mann– Whitney U-test were used to compare continuous variables between arms. Qualitative variables were compared using the x2 or Fisher exact test. Comparisons between baseline and follow-up measures in each arm were carried out with the paired t-test or Wilcoxon signed rank test. Continuous variables are expressed as the median and IQR. The Pearson correlation coefficient was calculated to estimate the strength of association between continuous clinical and laboratory variables. Univariate and multivariate analyses were done by regression analysis. Variables with P ≤0.1 in the univariate analysis were selected for multivariate study. Multivariate analysis was adjusted by sex and age. The final model was selected using the stepwise method, with P¼0.1 to enter the model and P, 0.05 to remain in the model. Analyses were performed using PASW Statistics 18 (Chicago, IL, USA).

Results Baseline characteristics The ATADAR study included 178 patients, of whom 86 (atazanavir/ ritonavir, n ¼ 45; darunavir/ritonavir, n ¼ 41) participated in this

JAC

LDL phenotype and PIs

Randomized n = 86

Allocated to atazanavir/ritonavir n = 45

Allocated to darunavir/ritonavir n = 41

Kaposi’s sarcoma 1 Lost to follow-up 1 Consent withdrawal 1

Lost to follow-up 1 Consent withdrawal 1

Month 6 n = 39

Month 6 n = 42

Lost to follow-up 1 Consent withdrawal 1

Consent withdrawal 2

Month 12 continued on study medication n = 40

Month 12 continued on study medication n = 37

Figure 1. CONSORT flow diagram.

metabolic substudy and were included between 23 May 2011 and 22 September 2011 (see Figure 1). There were no differences in baseline characteristics between the participants of the present substudy and participants of the main ATADAR study (data not shown). Participants were mainly men (89%), with a median age of 36 (31 – 41) years and a low CV risk measured by the Framingham score [1% (0% – 2%)]. No differences in demographic, anthropometric, CV risk factor and lipid variables were found between arms at baseline (Table 1). Nine patients discontinued the study medication (five in the atazanavir/ritonavir arm and four in the darunavir/ritonavir arm), five owing to withdrawal of consent, three lost to follow-up and one with Kaposi’s sarcoma. Only one participant started lipid-lowering therapy during the study.

Lipid changes Changes in lipids at weeks 24 and 48 are shown in Figure 2. TC increased significantly in both arms at week 48 (P¼0.026 in atazanavir/ritonavir and P¼0.0038 in darunavir/ritonavir). LDL-cholesterol increased significantly at week 48 only in the darunavir/ritonavir arm (P¼0.031). HDL-cholesterol increase was observed in both arms at week 48 (P¼0.0006 in atazanavir/ritonavir and P¼0.017 in darunavir/ritonavir). Apo A-I increased only in the atazanavir/ritonavir arm at week 48 (P¼0.019). There were no changes in Apo B or TC/HDLcholesterol ratio in either arm during the study. Nevertheless, the Apo A-I/Apo B ratio increased at week 48 only in the atazanavir/ ritonavir arm (P¼0.018). Finally, triglycerides significantly increased only in the atazanavir/ritonavir arm at week 48 (P¼0.007). No differences between arms were found in any variable at week 48 except in triglycerides, which showed a greater increase in the atazanavir/ritonavir arm (P ¼0.036). It is of note, as can be seen in Figure 2, that changes in most of the lipid variables occurred at week 24 and stabilized over the next 24 weeks.

Regarding LDL subfraction phenotype, at baseline there was a predominance of the more favourable phenotype A in both arms. As can be seen in Figure 3, there was an increase in LDL size (P ¼ 0.017) and cholesterol content in large and buoyant LDL particles (P ¼ 0.008), but not in sd-LDL particles in the darunavir/ ritonavir arm at week 48. In contrast, in the atazanavir/ritonavir arm, an increase in cholesterol content in sd-LDL particles (P ¼ 0.015) was observed at week 48 (Figure 3), as well as a shift to a more atherogenic phenotype (phenotype B) (Figure 3). Table 2 shows the correlation analysis between LDL size and metabolic variables at baseline. Factors associated with a change in LDL size at week 48 in the univariate and multivariate analyses are summarized in Table S1 (available as Supplementary data at JAC Online). Increases in plasma triglyceride concentrations and in cholesterol content in large and buoyant LDL particles (LDL 1, 2, 3) at week 48 were negatively and positively, respectively, associated with the change in LDL size on multivariate analysis. HIV viral load and CD4 cell count were not associated with LDL size.

Lp-PLA2 At baseline, Lp-PLA2 activity distribution was 69.1% (60.4%–73.1%) in LDL-Lp-PLA2 and 30.9% (26.9% – 39.6%) in HDL-Lp-PLA2 particles with no differences between arms. At week 48 there was no change in total Lp-PLA2 activity and no shift in its relative distribution in LDL or HDL particles (Figure S1). Table 2 shows the correlation analysis between total Lp-PLA2 activity and metabolic variables at baseline. Neither HIV viral load nor CD4 cell count was associated with Lp-PLA2 activity.

Changes in CV risk assessment and anthropometric and glucose parameters Baseline CV risk factors are shown in Table 1. Around half of the participants were active smokers. At week 48, tobacco consumption

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Table 1. Baseline characteristics of participants Atazanavir/ritonavir, n ¼45

Variable

Darunavir/ritonavir, n¼41

P

Age (years), median (IQR)

35 (31 –41)

36 (31 –41)

0.942

Male, n (%)

40 (88.9)

37 (90.2)

1

23 (51.1) 17 (37.8) 3 (6.7) 3 (6.7)

22 (53.7) 16 (39) 5 (12.2) 1 (2.4)

0.813 0.905 0.470 0.618

4 (9.3)

1 (2.6)

0.360

a

Risk group, n (%) homosexual men heterosexual intravenous drug user other/unknown AIDS–C stage, n (%)b Viral load (log copies/mL), median (IQR) CD4 count (cells/mm3), median (IQR) Weight (kg), median (IQR) 2

BMI (kg/m ), median (IQR) Waist (cm), median (IQR)

4.6 (4.2 –5.4)

4.5 (3.9– 4.9)

0.427

285 (144 –420)

362 (247– 429)

0.118

67.5 (63.5–70)

0.133

22.7 (21.6–24.5)

0.287

81 (76.5–85.5)

0.383

70 (64.7– 77.9) 23.2 (22.1– 25.3) 83 (78 –87)

Waist-to-hip ratio, median (IQR)

0.89 (0.86– 0.93)

0.87 (0.84–0.92)

0.394

Systolic blood pressure (mmHg), median (IQR)

119 (110 –129)

114 (109– 123)

0.188

Diastolic blood pressure (mmHg), median (IQR)

72 (66 –79)

70 (65 –75)

0.178

CV risk factors, n (%) smoking hypertension diabetes personal history of CHD family history of premature CHD other atherosclerotic diseases

21 (46.7) 4 (8.9) 0 0 3 (6.7) 0

23 (56.1) 0 0 0 7 (17.1) 0

0.382 0.118 — — 0.183 —

CV risk estimation, Framingham score (%)

0 (0– 3)

1 (0–2)

0.769

Lipid-lowering therapy, n (%)

0

0

0.424

Lipid measurements, median (IQR) TC (mg/dL) LDL-cholesterol (mg/dL) HDL-cholesterol (mg/dL) triglycerides (mg/dL) TC/HDL-cholesterol Apo A-I (g/L) Apo B (g/L) Apo A-I/Apo B

146.3 (129.3–163.3) 85.2 (71.8– 107.8) 35.6 (29.4– 41.8) 101.8 (72.6– 133.6) 4.2 (3.3 –5.1) 1.2 (1.0 –1.4) 0.8 (0.6 –0.9) 1.6 (1.2 –1.9)

145.5 (133.1– 169.5) 91.2 (74.6–110.3) 36.0 (31.7–41.8) 85.8 (65.5–120.4) 4.0 (3.5– 5.2) 1.2 (1.1– 1.3) 0.8 (0.7– 0.9) 1.5 (1.2– 1.7)

0.817 0.638 0.663 0.369 0.625 0.813 0.877 0.976

LDL subfraction phenotype, median (IQR) LDL size (A˚) cholesterol content in LDL particles 4 –6 (sd-LDL) (mg/dL)

271.6 (268.4–273.4) 20.2 (11.2– 34.6)

271 (268.9– 272.1) 24.9 (18.1–34.4)

0.405 0.343

LDL particle phenotype, n (%) A intermediate B unavailable data Lp-PLA2 activity, median (IQR) total (mmol/min.mL) LDL-Lp-PLA2 (%)

0.341 33 (73.3) 10 (22.2) 0 (0.0) 2 (4.4) 21.4 (19 –26.2) 67.6 (60.2– 72.5)

33 (80.5) 7 (17.1) 1 (2.4) 0 (0.0) 22.2 (17.1–27.2) 69.5 (60.4–73.5)

0.729 0.679 Continued

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LDL phenotype and PIs

Table 1. Continued Variable

Atazanavir/ritonavir, n ¼45

Darunavir/ritonavir, n¼41

P

32.4 (27.5– 39.7)

30.5 (26.4–39.6)

0.679

4.7 (4.4 –5.2) 7.1 (5.4 –13.5) 1.6 (1.1 –2.6)

4.6 (4.3– 5.0) 6.5 (4.6– 12.3) 1.3 (0.9– 2.8)

0.531 0.379 0.394

HDL-Lp-PLA2 (%) Glycaemia measures, median (IQR) glucose (mmol/L) insulin (pmol/L) HOMA-IR (mIU/L×mmol/L) CHD, coronary heart disease. a Patients can belong to more than one risk group. b Two patients with unknown data in both arms.

decreased only in the darunavir/ritonavir arm (56.1% to 44.4%, P¼0.039), and there were no changes in the remaining CV risk factors assessed or in CV risk estimation by the Framingham score, which remained very low [1% (0%–3%)]. BMI increased in the atazanavir/ritonavir arm [1.01 (20.47 – 2.21) kg/m2, P ¼ 0.004] at week 48, with differences approaching significance compared with patients receiving darunavir/ritonavir (P ¼ 0.059). Waist circumference also increased only in the atazanavir/ritonavir arm [3 (0 –9) cm; P ≤0.001], with significant differences compared with darunavir/ritonavir (P ¼ 0.027). No change in waist-to-hip ratio was observed in any arm at week 48. Glucose plasma levels had not changed in either arm at week 48. Insulin increased only in the atazanavir/ritonavir arm [3.38 (20.25 – 7.35) pmol/L; P ¼ 0.017] with no differences between arms; however, HOMA-IR showed no changes in either arm at week 48.

Discussion In this study conducted in antiretroviral-naive HIV-infected patients, initiation of c-ART based on darunavir/ritonavir plus tenofovir/emtricitabine was associated with an improvement in the LDL subfraction phenotype. There were no changes in total Lp-PLA2 activity in the darunavir/ritonavir arm or in the atazanavir/ritonavir arm at week 48. A mild increase in quantitative lipid measurements occurred in both arms at week 48, with a favourable increase in Apo A-I and Apo A-I/Apo B ratio observed in the atazanavir/ritonavir arm, whereas triglycerides showed a greater increase in this arm, in accordance with the main ATADAR study.16 LDL particles can be separated into subfractions that differ in size, charge, density, physicochemical composition and atherogenicity. A predominance of sd-LDL particles is strongly associated with coronary artery disease risk in the general population.12,17 We found an improvement in the atherogenic properties of LDL particles (increase in LDL size and large LDL particles) in patients on darunavir/ritonavir. The increase in LDL size and the decrease in Apo B concentrations that we found after 48 weeks of darunavir/ritonavir suggest that, irrespective of the increased LDL-cholesterol concentration, the number of LDL particles decreased since Apo B concentrations dropped and, consequently, that the increased LDL-cholesterol was related to an increased number of large LDL particles, i.e. the less atherogenic LDL particles. In contrast, patients starting

atazanavir/ritonavir showed a shift to the less favourable LDL phenotype B in association with an increase in cholesterol content in sd-LDL particles, related to an elevation of triglyceride levels, the major determinant of LDL size. Moreover, an increase in the BMI and waist circumference, reflecting a probable increase in abdominal adiposity, was found only in the atazanavir/ritonavir arm, in accordance with previous publications.18,19 Abdominal adiposity and insulin, which also increased only in the atazanavir/ritonavir arm, have both been related to sd-LDL synthesis in the general population.20 Several studies have analysed LDL subfraction phenotype in HIV-infected patients,4 – 6,21 – 25 but most have used laboratory techniques different from ours, i.e. NMR spectroscopy; this makes any comparison among studies difficult. c-ART initiation in the Strategies for Management of Anti-Retroviral Therapy (SMART) study was associated with an increase in large LDL particles,21 in agreement with what occurred in our patients receiving darunavir/ritonavir. Nonetheless, initiation of fosamprenavir/ ritonavir and lopinavir/ritonavir has been associated with an increase in the number of LDL particles, a decrease in LDL size and an increase in triglyceride plasma concentrations, similar to the atazanavir/ritonavir arm in our study.5,6 Taken together, control of viral replication by c-ART could improve the LDL subfraction phenotype, likely because of a decrease in HIV-associated inflammation status. Chronic infection and inflammatory diseases, including HIV infection, are related to increased triglyceride levels mediated by cytokines.26 On the other hand, the lipid disturbances caused by c-ART, mainly triglyceride increases, are associated with the deterioration observed in the current study and other studies. Lp-PLA2 is a vascular-specific inflammatory marker. In the general population, plasma Lp-PLA2 is associated with future coronary events and stroke.9 Lp-PLA2 circulates in association with lipoproteins, mainly LDL particles and, preferentially, sd-LDL particles.27 In our study, total Lp-PLA2 activity and the percentage distribution in lipoproteins showed no changes in either arm at week 48. In the general population, a correlation has been reported between Lp-PLA2 activity and several lipid measurements, in accordance with our results.27 Various studies have analysed Lp-PLA2 in HIV-infected patients in different scenarios.7,23,24,28 – 30 The effect of initiating c-ART on Lp-PLA2 activity was analysed in the ARIES study, in which a progressive decrease in Lp-PLA2 activity was found at weeks 84 and 144 of follow-up in patients on an atazanavir-based therapy. 7

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ATV/r change at 24 weeks DRV/r change at 24 weeks

ATV/r change at 48 weeks DRV/r change at 48 weeks 2.5

25.0 23.01

** 20.0

2

18.58 15.04

15.0

** 10.84 **

10.0 6.58 5.0

1.5

13.27

13.16 9.66 9.87

1

8.85

** ** 6.2 5.03 4.64

**

** **

0.5

3.48 2.71

**

**

0.11

Median change TC/HDL-c

Median change (mg/dL)

**

0.06 0

0.0 0.036*

–0.04 –0.16

–1.76

–0.5

–5.0 LDL-cholesterol

TC

HDL-cholesterol

ATV/r change at 24 weeks DRV/r change at 24 weeks

TC/HDL-cholesterol

TG ATV/r change at 48 weeks DRV/r change at 48 weeks

0.3

0.3

Median change (g/L)

0.2

0.2

0.14 0.13

**

**

0.11

0.1

** 0.06 0.06

0.05

0.1

0.04

0.05

0.03

0.0 –0.01 –0.01

–0.02

–0.1

–0.1 Apo A-I

Apo B

Apo A-I/Apo B

Figure 2. Changes in lipid parameters from baseline to weeks 24 and 48. *Significant change between arms at week 48 (Mann– Whitney U-test). No other significant P values were found at weeks 24 or 48 between arms. **Within-group significant change (P,0.05) from baseline at weeks 24 and 48 (Student’s t-test for repeated measures or Wilcoxon test). ATV/r, atazanavir/ritonavir; DRV/r, darunavir/ritonavir; TG, triglycerides.

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JAC

LDL phenotype and PIs

ATV/r

DRV/r 15

1.5

10

1.0 Median change (A)

** 0.5

0.5

5

** 2.31

2.25

**

Median change (mg/dL)

8.31

0

0.0

–0.3 –0.5 P value*

–3.31 –5

0.006

0.005

0.094

LDL particle size

Cholesterol content in LDL-cholesterol particles 1,2,3

Cholesterol content in LDL-cholesterol particles 4,5,6 (sd-LDL)

100 A

LDL particle phenotype (%)

80

Intermediate

B

0.020***

0.257***

60

40

20

0

Baseline

24 weeks

48 weeks

ATV/r (n = 45)

Baseline

24 weeks

48 weeks

DRV/r (n = 41)

Figure 3. Change in LDL subfraction phenotype at week 48. *P value of the comparison of the change between arms at week 48 (Student’s t-test or Mann–Whitney U-test). **Within-group significant change (P,0.05) from baseline at week 48 (Student’s t-test for repeated measures or Wilcoxon test). ***Marginal homogeneity test to compare percentages at baseline, and weeks 24 and 48 in each arm. ATV/r, atazanavir/ritonavir; DRV/r, darunavir/ ritonavir.

Our study has some limitations. First, the number of patients included in the study is small; nevertheless we have found significant and congruent results that support the conclusions. Plasma triglyceride concentrations have large intra-individual variability, which may explain the discordance between our

results and those of the ACTG 5257 study, in which no differences were observed in triglyceride levels between patients receiving darunavir/ritonavir or atazanavir/ritonavir.31 However, the relationship found between triglycerides and LDL subfraction phenotype was consistent. Finally, the relatively short follow-up

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Table 2. Pearson correlation coefficients between LDL size, total Lp-PLA2 activity and metabolic variables at baseline

Variable TC LDL-cholesterol HDL-cholesterol Apo A-I Apo B Triglycerides TC/HDL-cholesterol Apo A-I/Apo B Cholesterol content in sd-LDL Lp-PLA2 Insulin HOMA-IR Framingham score

LDL size, r (P)

Total Lp-PLA2 activity, r (P)

20.215 (0.051) 20.175 (0.114) 0.453 (,0.001) 0.243 (0.027) 20.414 (,0.001) 20.630 (,0.001) 20.575 (,0.001) 0.473 (,0.001) 20.808 (,0.001) 20.252 (0.031) 20.229 (0.041) 20.227 (0.043) 20.266 (0.015)

0.345 (0.002) 0.324 (0.005) 20.248 (0.032) 20.171 (0.143) 0.426 (,0.001) 0.321 (0.005) 0.401 (,0.001) 20.419 (,0.001) 0.344 (0.003) 20.252 (0.031) 0.232 (0.046) 0.210 (0.072) 0.277 (0.016)

period of our study compared with other similar studies7 could prevent the finding of similar results in our study to those observed in lengthy follow-up periods. In conclusion, an improvement in the LDL subfraction phenotype was found in patients initiating c-ART including darunavir/ritonavir plus tenofovir/emtricitabine, whereas worsening, related to a greater increase in plasma triglyceride levels, was found in those initiating atazanavir/ritonavir. No changes in Lp-PLA2 activity were seen in this population. Despite the differences found between arms in lipid profile, the clinical significance of our findings is not clear, especially in this very low CV risk population. Assessment of these CV biomarkers in patients initiating even more ‘lipid-friendly’ regimens may improve our understanding of the differences in CV risk and metabolic parameters occurring with the use of c-ART.

Acknowledgements We thank all of the patients who participated in the study. We pay tribute to the memory of Ignacio Pe´rez, beloved friend and insightful researcher, who died shortly after the completion of the ATADAR study.

Funding This work was supported in part by research grants from BristolMyers Squibb and Janssen-Cilag, and Red Tema´ tica Cooperativa de Investigacio´n en SIDA G03/173 (RIS-EST11), Ministerio de Sanidad, Servicios Sociales e Igualdad, Spain (registration number: NCT01274780; registry name: ATADAR; EUDRACT: 2010-021002-38).

Transparency declarations The following authors have received research funding, consultancy fees or lecture sponsorships from or served on advisory boards for the following companies: M. S., Abbott, Bristol-Myers Squibb, Gilead Sciences and Tibotec; J. O.-L., Abbott Diagnostics, Alere Diagnostics, Astute Diagnostics, Critical Diagnostics, Roche Diagnostics, Siemens Medical Solutions, Stat Diagnostics and ThermoFischer; E. M., Abbott, Boehringer-Ingelheim,

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Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Merck Sharp and Dohme, Theratechnologies, Tibotec and ViiV Healthcare; P. D., Abbott, Boehringer-Ingelheim, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Merck Sharp and Dohme, Theratechnologies, Tibotec and ViiV Healthcare; E. R., Abbott, Boehringer-Ingelheim, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Merck Sharp and Dohme, Tibotec and ViiV Healthcare; E. N., Abbott, Boehringer-Ingelheim, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Merck Sharp and Dohme, Tibotec and ViiV Healthcare; S. D. Y., Boehringer- Ingelheim, Bristol-Myers Squibb, Gilead Sciences, Merck Sharp and Dohme, Tibotec and ViiV Healthcare; A. G.-C., Bristol-Myers Squibb and Tibotec; and D. P., Abbott, Boehringer-Ingelheim, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Merck Sharp and Dohme, Tibotec and ViiV Healthcare. E. F., J. C. and J. L. S.-Q.: none to declare.

Author contributions M. S., D. P. and E. M. designed the study and drafted the paper. J. O.-L. and J. L. S.-Q. designed and conducted laboratory studies. M. S., E. M., E. F., P. D., E. R., E. N., A. G.-C. and S. D. Y. included and collected data from participants. J. C. performed data analyses. All authors reviewed and approved the final version of the manuscript.

Supplementary data Table S1 and Figure S1 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).

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ritonavir: a substudy of the ATADAR randomized study.

To assess LDL subfraction phenotype and lipoprotein-associated phospholipase A2 (Lp-PLA2) in naive HIV-infected patients starting atazanavir/ritonavir...
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