Intravenous immunoglobulin does not reduce left ventricular remodeling in patients with myocardial dysfunction during hospitalization after acute myocardial infarction

International Journal of Cardiology 168 (2013) 212–218

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Intravenous immunoglobulin does not reduce left ventricular remodeling in patients with myocardial dysfunction during hospitalization after acute myocardial infarction Lars Gullestad a, e, f,⁎, Stein Ørn g, Kenneth Dickstein g, h, Christian Eek a, Thor Edvardsen a, e, Svend Aakhus a, Erik T. Askevold a, b, Annika Michelsen b, Bjørn Bendz a, Rita Skårdal a, Hans-Jørgen Smith d, e, Arne Yndestad b, Thor Ueland b, Pål Aukrust b, c, i a

Department of Cardiology, Oslo University Hospital, Rikshospitalet, Oslo, Norway Research Institute of Internal Medicine, Oslo University Hospital, Rikshospitalet, Oslo, Norway c Section of Clinical Immunology and Infectious Diseases, Oslo University Hospital, Rikshospitalet, Oslo, Norway d Department of Radiology, Oslo University Hospital, Rikshospitalet, Oslo, Norway e Faculty of Medicine and K.G. Jebsen Cardiac Research Centre, University of Oslo, Norway f Center for Heart Failure Research, Faculty of Medicine, University of Oslo, Norway g Department of Cardiology, Stavanger University Hospital, Stavanger, Norway h Institute of Internal Medicine, University of Bergen, Norway i Faculty of Medicine, University of Oslo, Oslo, Norway b

a r t i c l e

i n f o

Article history: Received 3 January 2012 Received in revised form 7 May 2012 Accepted 15 September 2012 Available online 6 October 2012 Keywords: Myocardial infarction Heart failure Inflammation

a b s t r a c t Background: Left ventricular (LV) remodeling takes place after acute myocardial infarction (MI), potentially leading to overt heart failure (HF). Enhanced inflammation may contribute to LV remodeling. Our hypothesis was that the immunomodulating effects of intravenous immunoglobulin (IVIg) would be beneficial in patients with impaired myocardial function after MI by reducing myocardial remodeling and improving myocardial function. Methods: Sixty-two patients with acute MI treated by percutaneous coronary intervention, with depressed LV ejection fraction (LVEF) were randomized in a double-blinded fashion to IVIg as induction therapy and thereafter as monthly infusions or placebo for 26 weeks. The primary end point was changes in LVEF from baseline to 6 months as assessed by MRI. Results: Our main findings were: (i) LVEF increased significantly from 38±10 (mean±SD) to 45± 13% after IVIg and from 42±9 to 49±12% after placebo with no difference between the groups. (ii) The scar area decreased significantly by 3% and 5% in the IVIg and placebo group, respectively, with no difference between the groups. (iii) During the induction therapy (baseline to day 5), IVIg induced both inflammatory (e.g., increase in tumor necrosis factor α and monocyte chemoattractant protein-1) and anti-inflammatory (e.g., increase in interleukin-10 and decrease in leukocyte counts) variables, but during maintenance therapy there were no differences in changes of inflammatory mediators between IVIg and placebo. Conclusions: IVIg therapy after ST elevation MI managed by primary PCI does not affect LV remodeling or function. This illustrates the challenges of therapeutic intervention directed against the cytokine network, to prevent post-MI remodeling. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Left ventricular (LV) remodeling takes place after acute myocardial infarction (MI), and 25–30% of the patients subsequently develop changes in the LV function, which potentially leads to overt heart failure (HF) [1]. At present it is unpredictable which patients will progress into a phase of post-MI remodeling. However, indices of remodeling during the acute phase, such as LV end systolic volume (LVESV), are powerful predictors of prognosis [2]. In addition, clinical variables ⁎ Corresponding author at: Department of Cardiology, Oslo University Hospital, Rikshospitalet, N-0027 Oslo, Norway. Tel.: + 47 23070000; fax: + 47 23073917. E-mail address: [email protected] (L. Gullestad). 0167-5273/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2012.09.092

could give important prognostic information. For example, patients with MI complicated by HF during hospitalization experience increased morbidity and mortality the first year after the event [3]. Several strategies have been used to counteract maladaptive remodeling following MI. Pharmacological agents such as angiotensinconverting enzyme (ACE) inhibitors and ß-blockers have been shown to have beneficial effects on LV after MI [4,5]. Despite modern treatment, morbidity and mortality remain high [6], and new treatment modalities to prevent or regress post-MI remodeling are needed. In addition, identifying the target population that could benefit from such intervention therapy is challenging. Acute MI is followed by an inflammatory response characterized by complement activation, generation of reactive oxygen species and

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increased myocardial and systemic expression of inflammatory cytokines such as tumor necrosis factor (TNF)α and interleukin (IL)-1β [7–9]. This inflammatory process is a prerequisite for wound repair, scar formation and compensatory hypertrophy. However, while a moderate cytokine response could be protective, an inappropriate and persistent inflammatory reaction could lead to maladaptive responses [7]. In line with this, some studies in animal models have shown that immunomodulating therapy could attenuate post-MI remodeling [7]. Therapy with intravenous immunoglobulin (IVIg) has shown beneficial effects in a wide range of immune-mediated disorders, possibly through mechanisms such as neutralization of microbial antigens and superantigens, blocking the function of Fcγ-receptors on phagocytes, impairment of leukocyte adhesion to endothelial cells, anti-apoptotic effects, neutralization of auto-antibodies, inhibition of complement activation, inhibitory effects on matrix degradation and effects on the cytokine network involving up-regulation of anti-inflammatory mediators such as IL-1 receptor anatgonist (IL-1Ra), soluble TNF receptors (sTNFRs) and IL-10 [10–12]. Some previous studies have also been performed in patients with myocardial impairment [13–16], and we have previously reported beneficial effects of IVIg in patients with chronic HF possibly involving anti-inflammatory net effects on the cytokine network [17,18]. In the present study we hypothesized that IVIg also could have beneficial effects in patients with HF following acute MI. The basis for this hypothesis was as follows. (i) Although there is an improvement in the management of these patients, there are also indications of inadequate effect of current state-of-art cardiovascular treatment [19]. (ii) Several experimental and clinical studies have shown enhanced inflammation following MI, both systemically and within the myocardium potentially related to the degree of myocardial damage [8,9]. (iii) Experimental models have shown that when the infarct size is large, the myocardial cytokine expression remains significantly elevated, correlating with impaired myocardial function [8]. (iv). In a rat model, we have demonstrated that IVIg and anti-TNF therapy may attenuate post-MI remodeling [20]. However, although inflammation during MI could have harmful effects, this response seems also to be of major importance for tissue repair. Based on these issues we: (i) avoided very early intervention immediately after symptom debut and before PCI to minimize potential impairment of the repair process after MI. (ii) Selected IVIg as the intervention drug as IVIg has a balanced effect on the cytokine network, potentially providing beneficial immunomodulatory effects without harmful consequences on tissue repair seen with more aggressive immunosuppression in these patients [21]. 2. Materials and methods 2.1. Patients Sixty two patients with acute MI followed by myocardial dysfunction during hospitalization were included in the study (Table 1). The patients were included if they: (i) had a ST elevation MI (STEMI), (ii) had LV ejection fraction (LVEF) b40%, or b45% and at least 3 adjacent dysfunctional segments as assessed by echocardiography during hospitalization, (iii) were between 18 and 80 years of age and (iv) were on optimal medical treatment and considered unsuitable for surgical intervention. Patients were not included if they had: (i) evidence of unstable disease, concomitant ischemia or unstable angina, (ii) significant concomitant diseases such as infections, pulmonary disease or connective tissue disease, (iii) diseases that required surgery, or (iv) were known to be hypersensitive to IVIg. The study was approved by the Regional Ethical Committee and the Norwegian Medicines Agency. Signed informed consent was obtained from each patient. 2.2. IVIG preparation Octagam (Octapharma, Vienna, Austria), produced from fresh frozen plasma collected from Norwegian blood banks, was dispensed in sterile water containing 10% maltose (final IgG concentration 5 g/L). The IVIg preparation contains b0.2 g/L of IgA and its IgG sub-class level was equal to that in human plasma, Half-life of IgG in Octagam is 26–34 days. As placebo, an equal amount of 5% glucose was given with

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Table 1 Baseline characteristics in the total population and the two treatment groups.

Age (yrs) Male (%) BMI (kg/m2) Current smokers (%) Previous MI (%) Known AP (%) Earlier PCI (%) Vessels diseased 1/2/3(%) DM (%) Hypertension % Stroke/TIA (%) Biochemistry Hemoglobin (g/dl) Leucocytes (109/L) Platelets (s/L) Creatinine (μmol/L) ASAT max (U/L) CKMB max (µg/L) TnT max (µg/L) NT-proBNP (pmol/L) CRP (mg/L) Medication (%) ACE inhibitors ARB β − blocker Statins Warfarin Aspirin

Total population

IVIg (n = 31)

Placebo (n = 31)

p-Value

56.8 ± 1.1 84 27.1 ± 0.7 47 10 15 3 58/35/6 13 21 5

55.2 ± 1.7 81 25.8 ± 0.8 52 7 13 0 61/29/10 7 16 7

58.4 ± 1.5 87 28.2 ± 0.6 42 13 16 7 55/42/3 19 26 3

0.15 0.49 0.015 0.69 0.39 0.72 0.15 0.39 0.13 0.35 0.55

14.2 ± 0.2 10.2 ± 0.3 269 ± 10 75 ± 2 295 ± 28 312 ± 21 7.7 ± 0.6 272 ± 31 68 ± 25

13.6 ± 0.3 10.1 ± 0.4 284 ± 17 72 ± 3 313 ± 46 343 ± 28 7.9 ± 0.8 334 ± 43 66 ± 36

14.8 ± 0.3 10.3 ± 0.5 252 ± 13 78 ± 4 278 ± 39 279 ± 30 7.5 ± 0.9 207 ± 44 70 ± 36

0.003 0.81 0.11 0.12 0.55 0.126 0.75 0.009 0.84

14 19 45 49 19 47

15 17 44 48 14 54

13 22 50 50 30 37

0.69 0.39 0.48 0.85 0.026 0.050

Values given as percentage, mean ± SEM. BMI, Body Mass Index; DM, Diabetes Mellitus; ASAT, aspartate aminotransaminase; CK, creatine kinase; TnT, troponin T; ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker.

the same infusion rate as the IVIg preparation. Using enzyme immunoassays (EIAs), we could not detect IL-1β, IL-1Ra, TNFα, sTNFRs, monocyte chemoattractant protein 1 (MCP-1) or IL-10 in the IVIg product. The endotoxin levels in the IVIg and placebo preparation were b10 pg/mL. 2.3. Study design After baseline measurements the patients were randomized to IVIg or placebo in a double-blind fashion, and stratified according to study site (i.e., Oslo University Hospital, Rikshospitalet or Stavanger University Hospital). IVIg or an equal volume of placebo was given at a rate according to the manufacturer's instruction as induction therapy (one daily infusion [0.4 mg/kg] for 5 days), and thereafter as monthly infusions (0.4 mg/kg) for a total of 5 months. Baseline measurements were repeated at the end of the study (26 weeks, 4 weeks after last IVIg or placebo infusion). One person not participating in any of the analytical procedures performed all IVIg and placebo administrations. The primary endpoint of the study was change in LVEF as assessed by magnetic resonance imaging (MRI). In addition we investigated if IVIg had any effects on (i) cardiac volumes, (ii) size of myocardial scar tissue, (iii) inflammatory and anti-inflammatory mediators and (iv) neurohormonal assessment (i.e. N terminal pro-B-type natriuretic peptide [NT-proBNP]). At baseline and at the end of the study the following tests were performed: (i) LV function and volumes as assessed by MRI; (ii) analyses of immunologic variables; (iii) clinical evaluation was assessed by NYHA classification performed by a single cardiologist each time; and (iv) measurement of plasma NT-proBNP concentration. MRI and lab test were also repeated at 12 months after inclusion, i.e. 7 months after last infusion. 2.4. Measurement of LV remodeling by MRI MR imaging was performed using 1.5 Tesla (Siemens Magnetom Sonata, Siemens, Erlangen, Germany or 1.5 Tesla Intera R10.3 Philips Medical Systems, Best, The Netherlands). Long axis and multiple short axis cine images of LV were acquired with breath-hold segmented balanced gradient echo sequences. The calculation of LV end-diastolic volumes (LVEDV) and end-systolic volumes (LVESV) was based on the summation of short axis slices. Late enhancement images were obtained 10–20 min after intravenous injection of 0.2 mmol/kg gadopentetate dimeglumine (Magnevist, Schering, Berlin, Germany) in long axis and multiple short axis projections covering the LV using a breath-hold inversion recovery turbo gradient echo sequence. The inversion time was chosen to null the signal of the normal myocardium. Scar size was assessed manually using planimetry on each short-axis slice to generate

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a volume. Non-scarred LV mass index was calculated by subtracting scarred myocardial mass index from LV mass index. A core laboratory (Oslo University Hospital, Rikshospitalet) read all the images. Postprocessing was performed using QMASS, Medis, Netherlands for LV volumes and mass and Segment version 1.8 R1145 (http://segment.heiberg.se) for scar characteristics. The protocol was not designed for the assessment of area-at-risk. The MRI measurements were assessed by a single investigator who was blinded for the study. 2.5. Echocardiography Parasternal and apical views were obtained using a GE Vivid 7 ultrasonic digital scanner (GE, Horten, Norway). We obtained standard two-dimensional images, M-mode and color Doppler as well as pulsed wave Doppler recordings of blood flow velocities in the LV outflow tract. LVEF was assessed by the biplane Simpson method. Doppler echocardiographic calculations of stroke volume, cardiac output and maximum and mean flow rates were performed on the basis of the cross-sectional area of flow and flow velocity data.

variables after treatment are expressed as mean with associated 95% confidence interval (95% CI). P values are two-sided and taken as significant when b0.05. The changes between groups in response to treatment were adjusted by baseline characteristics that were statistically imbalanced between the treatment groups using linear regression (body mass index [BMI], NTproBNP, hemoglobin and time to percutaneous coronary intervention [PCI]). The primary endpoint was change in LVEF as assessed by MRI. A previous study from our institution (the ASTAMI study [22]) showed a SD of 7.4% for the change in LVEF occurring the first 6 months after a MI measured by MRI. However, the method for measuring LVEF has been improved and we estimate a SD of 6%. We would assume a difference in LVEF of 5% to be of clinical significance. In order to observe such a difference at the end of the study with an α of 5% and power of 80%, we would need approximately 24 patients in each group. To compensate for possible drop-outs, and increase the chance of showing statistical differences in secondary end points, we included 62 patients altogether. The authors had full access to and take responsibility for the integrity of the data. All authors have read and agreed to the manuscript as written.

2.6. Blood sampling protocol Blood samples were collected into pyrogen-free EDTA tubes from an antecubital vein. The tubes were immediately immersed in melting ice, centrifuged within 15 min at 2000 g for 20 min and platelet-poor plasma was stored at −80 °C until analysis. Samples were thawed only once. 2.7. Laboratory analyses TNFα and IL-10 were measured by a fluorokine multiplex TNF assay from R&D Systems (Minneapolis, MN). Soluble TNFR type 1 (sTNFR1) and MCP-1 were analyzed by EIAs obtained from R&D Systems. IL-1Ra was analyzed by EIA from Biosource (Camarillo, CA). NT-proBNP and C-reactive protein (CRP) were assayed on a MODULAR platform (Roche Diagnostics, Basel, Switzerland). The coefficients of variation were b10% for all assays. 2.8. Statistical analyses The primary objective of statistical analysis was to compare the effect of IVIg therapy with placebo on LVEF between enrolment and 6 months. The primary statistical analyses were undertaken on the ‘intention-to-treat’ population, whereas secondary, exploratory analyses were conducted on the ‘per protocol’ population. Differences between groups were compared with Student t test or Mann–Whitney U Rank-Sum test for unpaired data as appropriate. In the paired situation, multiple analyses of variance (MANOVA) were performed a priori, and if significant, Wilcoxon's Rank-Sum test for paired data was performed. Relations between variables were tested using Spearman's Rank-Correlation test. The results are given as mean ± SEM, while the changes in

3. Results Sixty-two out of 485 consecutively screened patients were included. The major reasons for exclusion were small infarctions (51%), need of reintervention (13%), age (12%) and administrative reasons (12%) (Fig. 1). Of the 62 patients randomized, 31 were assigned to IVIg and 31 to placebo. There were 3 drop outs during IVIg treatment (1 developed a pancreatic cancer and 2 developed exanthema), while there was one drop out during placebo treatment (unwilling to continue because of worsening of diabetes mellitus). The baseline demographic and hemodynamic parameters are shown in Table 1. There were some differences in the baseline characteristics between the study groups. BMI and hemoglobin levels were lower and NT-proBNP levels were higher in the IVIg group (significant), and the proportion of patients with diabetes and hypertension was higher in the placebo group (non-significant). For infarct related characteristics, time to PCI from symptom debut was longer in the IVIg group, while time to treatment (IVIg or placebo), the localization of the infarct and infarct size as assessed by biochemical values (aspartate aminotransaminase [ASAT] max, creatine kinase [CK] MB max, and troponin T [TnT] max) were similar (Tables 1 and 2).

Fig. 1. Flow chart of the study.

L. Gullestad et al. / International Journal of Cardiology 168 (2013) 212–218 Table 2 Infarct related values.

Time symptoms PCI (h) Time symptoms treatment (h) Anterior MI (%) LAD prox (%) LAD mid (%) LAD distal (%) Circumflex (%) RCA VT/VF during hosp (%) Atrial fibrillation (%)

Total population

IVIg (n = 31)

Placebo (n = 31)

p-Value

5.6 ± 0.7 46.3 ± 3.5 94 60 45 3 15 13 16 2

7.0 ± 1.2 48.0 ± 4.6 90 68 58 3 10 19 19 1

4.2 ± 0.7 44.5 ± 5.4 97 52 52 3 20 7 13 1

0.045 0.55 0.31 0.208 0.31 1.00 0.28 0.13 0.49 1.0

Values given as percentage, mean±SEM. MI, Myocardial infarction; LAD, left anterior descending artery; RCA, right coronary artery; VT/VF, ventricular tachycardia/fibrillation.

3.1. LV function and infarct size LV volumes, LVEF and infarct size as assessed by MRI are shown in Table 3. During follow-up, there was a significant increase in LVEF in both the IVIg (7 units) and the placebo (8 units) group with no difference between the treatment groups. While there were no changes in LVEDV during follow-up, LVESV decreased during both placebo and IVIg therapy with no differences in changes between the treatment groups. Infarct size, measured as gram of tissue, remained unchanged and scar size decreased in both treatment groups with no differences between IVIg and placebo. Finally, LV volumes, LVEF, and scar size remained unchanged in both treatment groups from 6 to 12 months (i.e., after cessation of therapy [IVIg or placebo], data not shown). When changes in volumes, LVEF and infarct size were adjusted by baseline LVEF, no further treatment effects were observed, indicating that the lower LVEF at baseline in the IVIg group did not influence the effect of treatment (Table 3). Similar findings were also seen after adjustment for other baseline characteristics that were imbalanced between the treatment groups (BMI, NTproBNP, hemoglobin and time to PCI) (Table 3). Data from echocardiography showed similar results as from MRI (data not shown). 3.2. Inflammatory variables during induction therapy During the induction therapy from baseline to day 5, there was an increase of TNFα, sTNFR1, IL-10, TNFα/IL-10 ratio, IL-Ra and MCP-1 during IVIg, while only TNFα and IL-Ra increased and MCP-1 decreased during placebo, resulting in significant difference in changes for TNFα, IL-10 and MCP-1 between the two treatment groups (adjusted values, see the Materials and methods section) (Fig. 2). In addition, the number of lymphocytes and neutrophils decreased significantly during IVIg therapy, with only a modest decrease in neutrophils during placebo, resulting in a significant difference in the

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change of neutrophil and lymphocyte counts between the two treatment groups (adjusted values, see the Materials and methods section) (Fig. 2). Thus, it seems that IVIg induced both inflammatory (i.e. increase in TNFα and MCP-1) and potentially anti-inflammatory (i.e. increase in IL-10 and decrease in lymphocyte and neutrophil counts) effects during the induction therapy. 3.3. Inflammatory variables during maintenance therapy During follow-up (baseline versus 6 months), there was a decrease in MCP-1, sTNFR1 and IL-1Ra in both the IVIg and placebo group, with no differences in changes between the two treatment groups. Also, TNFα and TNFα/IL-10 ratio increased while IL-10 decreased during IVIg therapy, with no change in the placebo group, but again, there were no differences in changes between the treatment groups (Table 4). Finally, during follow-up there was a decrease in CRP, leukocyte counts and neutrophil counts, but not in lymphocyte count, in both treatment groups, with no differences in changes between IVIg and placebo (Table 4). A similar pattern was seen after adjustment for baseline characteristics that were imbalanced between the treatment groups (BMI, NTproBNP, hemoglobin and time to PCI) (Table 4). 3.4. Effect on NT-proBNP The measurement of plasma NT-proBNP, which correlates with myocardial wall stress and provides important prognostic information in HF patients, was one of the pre-defined secondary endpoints in the present study. Plasma NT-proBNP concentrations were elevated in both groups at baseline, with significantly higher levels in the IVIg group (pb 0.01). During follow-up, NT-proBNP decreased in both treatment groups, with a particularly marked decrease during IVIg therapy, but with no difference in changes between IVIg and placebo, also after adjustment for imbalanced baseline characteristics (Table 4). 3.5. Effect of treatment in subgroups We next looked at the effect of IVIg and placebo in relevant subgroups; i.e., the patients were dicotomized according to the median values of infarct size, time from symptoms to PCI, time from symptoms to intervention, ventricular function as assessed by NT-proBNP or LVEF and the degree of systemic inflammation as assessed by CRP. In essence, there were no differences in the changes for the primary endpoint, LVEF, between IVIg and placebo groups within any of these subgroups (data not shown). 3.6. Side effects and laboratory status The induction therapy was well tolerated, but during 6 month follow-up seven subjects in the IVIg group (exanthema [n = 3], chest

Table 3 Ventricular function and infarct size parameters before and after six months of therapy with IVIG or placebo as assessed by MRI. IVIG

MRI LVEDV (mL) LVESV (mL) LVEF (%) LVM (g) Scar (%)

Placebo

Baseline

6 months

Change

Baseline

6 months

Change

161 ± 10 102 ± 9 38 ± 2 128 ± 5 23 ± 2

158 ± 11 94 ± 12⁎ 45 ± 2⁎⁎

−4 (−13,5) −9 (−20,2) 7 (3,11)⁎ −8 (−18,3) −3 (−6,−0)⁎

140 ± 5 82 ± 4 42 ± 2 121 ± 5 21 ± 2

139 ± 7 70 ± 6⁎ 49 ± 2⁎⁎⁎

−3 (−12,7) −12 (−21,−4) 8 (4,11) −7 (−16,2) −5 (−8,−1)⁎

117 ± 5 19 ± 2

113 ± 5 17 ± 2

Differences in changes between groups

0.52/0.60† 0.95/0.86† 0.74/0.89† 0.54/0.71† 0.80/0.43†

Data are given as mean ± SEM at baseline and 6 months. Changes are given as mean and 95% CI in parenthesis. LVESV; left ventricular end systolic volume; LVEDV, left ventricular end diastolic volume; LVEF, left ventricular ejection fraction; LVM, left ventricular myocardial infarction; Scar (%), percent myocardial infarct. †p-Values adjusted by BMI, NTproBNP, Hemoglobin and time to PCI. ⁎ p b 0.05 versus baseline. ⁎⁎ p b 0.01 versus baseline. ⁎⁎⁎ p b 0.001 versus baseline.

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Fig. 2. Change in different cytokines and leucocyte counts during induction treatment with IVIg or placebo. ⁎p b 0.05, ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 versus baseline. †p b 0.05, †† p b 0.01 and †††p b 0.001 comparing differences in changes between IVIg and placebo. The p-values for differences between treatment groups were adjusted by BMI, NTproBNP, hemoglobin and time to PCI. Data are given as mean±SEM.

pain [n = 2], cancer pancreas [n = 1], and general discomfort [n = 1]) and three in the placebo group (Non STEMI [n = 1], post-MI syndrome [n = 1], and syncope [n = 1]) experienced a clinically serious adverse event. Three of these patients (2 in the IVIg group and 1 on placebo) had to withdraw from the study. Blood was collected every month, but except for creatinine that increased significantly during IVIg, but still within normal limit, no differences between the groups were observed (data not shown). 4. Discussion This present, randomized, double-blind, placebo-controlled clinical trial demonstrated that IVIg, given as an induction therapy over

5 days followed by one infusion each month for 6 months to patients with STEMI and signs of myocardial dysfunction during the subacute phase, did not improve myocardial function or influence LV remodeling or infarct size during follow-up. After an acute MI, a maladaptive phase with LV remodeling takes place, and is associated with inflammation, wall thinning, fibrosis, apoptosis, ventricular enlargements and hypertrophy [7]. These events may contribute to depressed cardiac function with subsequent development of overt HF. Our hypothesis was that intervention with IVIg, with a potential balanced anti-inflammatory net effect [17], could be beneficial when given in the subacute phase following MI, mitigating the remodeling process and improving LV function in patients with sign of myocardial dysfunction. However, our study demonstrated that IVIg had no effect on infarct size, LV function or LV remodeling during follow-up to one year after MI. We have previously shown that IVIg given to patients with chronic HF in a stable phase improved LV function, associated with an antiinflammatory net effect on the cytokine network [17]. Why IVIg given in the subacute phase after a MI was not efficacious on LV systolic function, is at present unclear, but several non-elusive mechanisms could have contributed to the failure of IVIg in the present study. First, the selection of patients could be wrong. In fact, there was a significant improvement in the markers of myocardial failure and remodeling in the placebo group, and potentially, patients with more severe HF and/or more severe myocardial damage should have been selected. Likewise, there was a marked reduction in several of the inflammatory parameters during follow-up in the placebo group, and it is possible that a selection of patients with more sustained inflammation could have been of interest. Second, the time point for intervention is not clear. Although we tried to avoid interference with the beneficial repair process, forthcoming studies on immunomudulation in post-MI HF should more carefully address this issue. Third, while IVIg has been reported to modulate innate immunity at least partly by increasing IL-1Ra levels, this was not seen in the present study. A more rational approach could be to give IL-1Ra itself, and indeed, there are ongoing studies with the use of this medication in post-MI remodeling [23]. Finally, whereas immunomodulatory intervention in chronic disorders in a stable phase has been shown beneficial in several diseases, invention during the acute phase is more challenging (e.g., during septicemia), potentially moving from one side of the road (“inflammatory”) to the other (“immunosuppression”). Although several effects of IVIg may be of relevance in HF (e.g., neutralization of microbial antigens, superantigens and autoantibodies), the potential anti-inflammatory net effect on the cytokine network may be of particular importance [24]. However, the failure of IVIg in the present study, even to modulate inflammation,

Table 4 Plasma levels of cytokines and their endogenous modulators in 62 HF patients before and after six months of treatment with IVIG (n = 31) or placebo (n = 31). IVIG

TNFα (pg/mL) sTNFR1 (ng/mL) IL-10 (pg/mL) TNFα/IL-10 IL-1Ra (pg/mL) MCP-1 (pg/mL) NT-proBNP CRP Leucocytes Lymphocytes Neutrophils

Placebo

Baseline

6 months

Change

Baseline

6 months

Change

1.38 ± 0.42 1.27 ± 0.38 0.31 ± 0.14 5.82 ± 0.53 46 ± 8 84 ± 40 320 ± 220 30 ± 36 10.0 ± 2.3 1.9 ± 0.8 6.7 ± 2.2

1.48 ± 0.44⁎ 0.92 ± 0.22⁎⁎⁎ 0.26 ± 0.10⁎ 7.28 ± 0.68⁎⁎ 23 ± 3⁎⁎⁎ 76 ± 27⁎ 58 ± 56⁎⁎⁎ 1.8 ± 1.7⁎⁎⁎ 6.2 ± 1.3⁎⁎⁎

0.16 (0.06,0.27) −329 (−429,−230) −0.06 (−0.12,0.00) 1.86 (0.51,3.22) −24 (−39,−10) −9 (−17,−1) −262 (−367,−157) −28 (−42,−15) −3.7 (−4.9,−2.7) −0.0 (−0.3,0.3) −3.1 (−4.2,−2.0)

1.63 ± 0.47 1.44 ± 0.65 0.28 ± 0.11 7.32 ± 0.63 45 ± 4 73 ± 22 199 ± 253 28 ± 32 10.3 ± 2.5 2.0 ± 0.9 7.1 ± 1.7

1.68 ± 0.49 1.13 ± 0.56⁎⁎⁎ 0.31 ± 0.26 7.32 ± 0.54 29 ± 3⁎⁎⁎ 62 ± 18⁎⁎⁎ 58 ± 97⁎⁎⁎ 2.0 ± 1.6⁎⁎⁎ 6.4 ± 1.5⁎⁎⁎

0.09 −306 0.03 0.38 −16 −11 −141 −26 −4.1 −0.1 −3.5

1.9 ± 0.6 3.7 ± 1.1⁎⁎⁎

1.9 ± 0.6 3.6 ± 0.9⁎⁎⁎

(−0.06,0.24) (−414,−199) (−0.07,0.12) (−0.68,1.44) (−23,−9) (−17,−6) (−227,−57) (−39,−13) (−5.2,3.0) (−0.1,0.1) (−4.3,−2.7)

Differences in changes between groups 0.73/0.33† 0.98/0.59† 0.16/0.36† 0.05/0.33† 0.52/0.81† 0.51/0.16† 0.07/ 0.77† 0.53/0.94† 0.97/0.87† 0.33/0.43† 0.90/0.30†

Data are given as mean±SEM at baseline and 6 months. Changes are given as mean and 95% CI in parenthesis. TNFα/IL-10 ratio is defined as [TNFα(trimer)(pmol/L)/(IL-10)(pmol/L)]. † p-Values adjusted by BMI, NTproBNP, Hemoglobin and time to PCI. ⁎ p b 0.05 versus baseline. ⁎⁎ p b 0.01 versus baseline. ⁎⁎⁎ p b 0.001 versus baseline.

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suggests that other anti-inflammatory treatment modalities should also be investigated. Alternative approaches could include inhibition of triggers (e.g., danger associated molecular patterns), inhibition of sensors such as toll-like receptors or Nucleotide Oligomerization Domain (NOD) like receptor proteins, or inhibition of up-stream mediators in the inflammatory response such as IL-1 [25]. In particular, inflammasomes and IL-1-related inflammation could be of importance during myocardial ischemia–reperfusion damage as a proto-typical form of sterile inflammation [26], and as discussed above, more direct inhibition of IL-1 (e.g., Canakinumab and kineret) [23,27] could potentially be more attractive than indirect inhibition by IVIg. Several studies have shown anti-inflammatory effect of IVIg which include interaction between the Fc part of IgG in the IVIg preparation with negative Fc receptors [11]. However, while we in our previous study in chronic HF found that IVIg, but not placebo, induced a marked increase in the anti-inflammatory mediators IL-1Ra, sTNFRI and IL-10 [17], this was not seen in the present study. In fact, these markers decreased during 6 month follow-up, with no difference between the placebo and IVIg group. The reason for this finding is not clear, but could involve different degree of cell pre-action between the acute-MI study and the chronic HF study. IVIg has been shown to directly induce the production of IL-1Ra in monocytes, involving both F(ab’)2 fragments and Fcy fragments of IVIg, and interesting, endotoxin pre-activation of monocytes inhibited the IVIg induced IL-1Ra secretion in these cells [28]. This illustrates a potential link between cellular pre-activation and the effect of IVIg [24]. Moreover, although there was an increase in IL-Ra during IVIg therapy during the induction phase in the present study, a similar increase was seen in the placebo group, further underscoring a potential effect of cellular pre-activation on the effect of IVIg. However, these issues are far from clear, and should be explored in forthcoming mechanistic studies. The present study has some limitations, such as a relatively low number of patients and some differences between treatment groups at baseline (i.e., BMI, NT-proBNP, hemoglobin, and in particular the significant difference in time between symptom debut time and PCI). It is also possible that including patients with even larger infarcts and clinically more advance HF that is more likely to undergo myocardial remodeling during follow up could have the benefit of IVIg therapy. Moreover, while our previous IVIg study was performed in patients with chronic HF in a stable condition [17], the patients in the present study, that were included short time after a major event (i.e., MI), represent a more heterogeneous group of patients, which also could have resulted in an underpowered study. It can also be argued that an increase in LVEF in 5% that was used in the power calculation in the present study was somewhat arbitrary, and that an increase in 4% also could be of clinical significance. Moreover, besides IgG, there were some differences between the placebo and the IVIg preparation (5% glucose as opposed to 10% maltose and lack of protein in the placebo preparation). Finally, the present study used surrogate markers as the primary endpoint, and the lack on effect on such markers does not exclude effects on clinical end-points such as hospitalization and mortality. However, to study the effect of IVIg on clinical end-points, a much larger study population will be needed.

5. Conclusions We have shown that IVIg therapy in the subacute phase after STEMI managed by primary PCI does not affect LV remodeling or function. This does not exclude that IVIg could have potential beneficial effects in other cardiovascular disorders such as myocarditis, chronic HF and atherosclerosis, in particular in a more stable phase [29]. Our study illustrates the challenges in the quest for an efficient modulation of the inflammatory response following reperfused MI; there still is a need to identify the correct timing, and the most appropriate immunomodulation therapy in this patient cohort.

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Funding This work was supported by The Norwegian Research Council, the South-Eastern Norway Regional Health Authority, a gift from an anonymous subject, and an unrestricted grant from Sparebanken1 SR bank. Conflict of interest None of the authors have any potential conflicts of interest to report. There is no relationship with the industry. Clinical Trial Registration Information — URL: http://www. clinicaltrials.gov. Unique identifier: NCT00430885. Acknowledgment The authors wish to thank Torbjørn Aarsland, Jorunn Nielsen, Fredrikke Wick, Eva Staal and Ole Jacob Greve for their important contributions during the study. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. References [1] Cohn JN, Ferrari R, Sharpe N. On behalf on an international forum on cardiac remodeling. Cardiac remodeling-concepts and clinical implications: a consensus paper from an international forum on cardiac remodeling. J Am Coll Cardiol 2000;35:569-82. [2] White HD, Norris RM, Smith H. Left ventricular end-systolic volume as the major determinant of survival after recovery from myocardial infarction. Circulation 1987;76:44-51. [3] Møller JE, Brendorp B, Ottesn M, et al. Congestive heart failure with preserved left ventricular systolic function after acute myocardial infarction: clinical and prognostic implications. Eur J Heart Fail 2003;5:811-9. [4] Sharpe N, Murphy J, Smith H. Treatment of patients with symptomless left ventricular dysfunction after myocardial infarction. Lancet 1988:255-9. [5] Pfeffer M, Lamas GA, Vaughan D. Effect of captopril on progressive ventricular dilation after myocardial infarction. N Engl J Med 1988;319:80-6. [6] Doughty RN, Whalley GA, Walsh HA, Gamble GD, Lopez-Sendon J, Sharpe N. Effects of carvedilol on left ventricular remodeling after acute myocardial infarction. The CAPRICORN echo substudy. Circulation 2004;109:201-6. [7] Frangogiannis NG, Smith CW, Entman ML. The inflammatory response in myocardial infarction. [Review] [181 refs]. Cardiovasc Res 2002;53:31-47. [8] Ono K, Matsumori A, Shioi T, Furukawa Y, Sasayama S. Cytokine gene expression after myocardial infarction in rat hearts. Circulation 1998;98:149-56. [9] Orn S, Manhenke C, Ueland T, et al. C-reactive protein, infarct size, microvascular obstruction, and left-ventricular remodelling following acute myocardial infarction. Eur Heart J 2009;30:1180-6. [10] Ballow M. Mechanism of action of intravenous immune serum globulin in autoimmune and inflammatory diseases. J Allergy Clin Immunol 1997;100:151-7. [11] Mobini N, Sarela A, Ahmed AR. Intravenous immunoglobulins in the therapy of autoimmune and systemic inflammatory disorders. Ann Allergy Asthma Immunol 1995;74:119-28. [12] Aukrust P, Damås JK, Gullestad L. Immunomodulating therapy-new treatment modality in congestive heart failure. Congest Heart Failure 2003;9:64-9. [13] Takada H, Kishimoto C, Hiraoka Y. Therapy with immunoglobulin suppresses myocarditis in a murine coxsackievirus B3 model. Circulation 1995;92:1604-11. [14] McNamara DM, Rosenblum WD, Janosko KM, et al. Intravenous immune globulin in the therapy of myocarditis and acute cardiomyopathy. Circulation 1997;95: 2476-8. [15] McNamara DM, Holubkov R, Starling RC, et al. Controlled trial of intravenous immune globulin in recent-onset dilated cardiomyopathy. Circulation 2001;103: 2254-9. [16] Kishimoto C, Shioji K, Kinoshita M, et al. Treatment of acute inflammatory cardiomyopathy with intravenous immunoglobulin ameliorates left ventricular function associated with suppression of inflammatory cytokines and decreased oxidative stress. Int J Cardiol 2003;91:173-8. [17] Gullestad L, Aass H, Fjeld JG, et al. Effect of immunomodulating therapy with intravenous immunoglobulin in chronic congestive heart failure. Circulation 2001;103: 220-5. [18] Damås JK, Gullestad L, Aass H, et al. Enhanced gene expression of chemockines and their corresponding receptors in mononuclear blood cells in chronic heart failure-modulatory effects of intravenous immunoglobulin. J Am Coll Cardiol 2001;38:187-93. [19] Pitt B, Remme WJ, Zannad F, et al. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med 2003;348:1309-21. [20] Gurantz D, Yndestad A, Halvorsen B, et al. Etanercept or intravenous immunoglobulin attenuates expression of genes involved in post-myocardial infarction remodeling. Cardiovasc Res 2005;67:106-15.

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