Exploring interleukin-1b modulation in coronary and ischemic disease






Jean-Claude TARDIF, MD
Montreal Heart Institute
Montreal, CANADA

Exploring interleukin-1b modulation in coronary and ischemic disease

by J. C. Tardif, France

Cardiovascular diseases are the leading cause of death worldwide, and atherosclerosis is the main underlying etiology. Atherosclerosis is an inflammatory, dynamic, and complex disease involving multiple cell types, and many anti-inflammatory strategies have recently emerged as potential therapeutic approaches for atherosclerotic diseases. In this review, we focus on the role of the proinflammatory cytokine interleukin 1 (IL-1) in atherosclerosis and the potential benefits of IL-1 inhibition. A number of ongoing clinical studies of IL-1 inhibition will be introduced, with an in-depth look at gevokizumab. Gevokizumab is an antibody directed against IL-1 that is being tested in clinical trials of patients with coronary artery disease. The first study is evaluating the effects of gevokizumab on arterial inflammation measured by positron emission tomography.

Medicographia. 2014;36:362-370 (see French abstract on page 370)



Cardiovascular diseases remain the leading cause of mortality in the world, and atherosclerosis is the main underlying etiology. The long-standing view supporting that development of the atherosclerotic lesion solely depends on lipid deposition has been replaced by the current concept that activation of immune and inflammatory responses has a central role in plaque initiation and progression.1 Subsequently, different anti-inflammatory strategies have emerged as potential treatments of atherosclerotic disease, in addition to the existing lipid-lowering therapies.

Atherosclerosis is an inflammatory disease that consists of the formation of an atherothrombotic plaque in the arterial wall, causing stenotic and thrombotic complications. The atherosclerotic plaque formation begins early in childhood by the accumulation of lipids and inflammatory cells in the arterial wall (known as the fatty streak). Further lipid deposition and oxidation triggers the phagocytosis of the accumulated lipids by macrophages, stimulating the inflammatory reaction that contributes to the plaque’s growth. Recruitment of inflammatory cells and increased secretion of proinflammatory cytokines, such as interleukin (IL) 1, enhance the inflammatory reaction. Atherosclerosis is therefore a complex process involving diverse cell types (including monocytes and lymphocytes) and molecules (including selectins, other adhesion molecules, chemokines, and growth factors). Numerous studies dissecting the interplay between lipid deposition and oxidation, as well as inflammatory cell recruitment and cytokine secretion, have revealed multiple potential targets that could interfere with and potentially inhibit the development and progression of the atherosclerotic plaque.

Patients with chronic extracardiac inflammatory diseases such as rheumatoid arthritis or extensive psoriasis have a higher risk of cardiovascular diseases and related mortality compared with the general population.2,3 Systemic inflammation appears to be independently involved in this increased cardiovascular risk4 and leads to coronary artery disease, myocardial infarction,5 cerebrovascular disease,6 and heart failure.3,7 Indeed, the level of inflammation seems to be an independent risk marker and possibly a risk factor for cardiovascular diseases in patients with extracardiac inflammatory diseases, especially in patients with rheumatoid arthritis.8 Taken together, these studies indicate that treatment of the underlying inflammatory process could contribute to improved cardiovascular outcomes.9-11


Figure 1
Figure 1. Cholesterol crystals can promote inflammation by activating the NLRP3 inflammasome.

NLRP3 inflammasomes can activate the inflammatory cascade by binding to and cleaving pro-caspase-1, forming caspase-1, important for the cleavage and secretion of various interleukins, including IL-1β. (A,B) In a caspase-1–dependent manner, IL-1β (detected by ELISA and immunoblot analysis) is released into the supernatants of PBMC cultures treated with cholesterol crystals. Resting or LPS-primed human PBMCs were treated with cholesterol crystals as indicated, MSU crystals (250 g/mL), or ATP in the presence or absence of the caspase-1 inhibitor zYVAD-fmk (10mM). Results are represented as means and SEM of 4 donors. (C) Cleaved caspase-1 was assayed by immunoblot in the supernatants and cell lysates and (D) IL-β was measured by ELISA in supernatants from LPS-primed wild-type, NLRP3- or ASC-deficient macrophages stimulated with cholesterol crystals, dAdT, nigericin, or ATP. One out of 3 independent experiments are shown.
Abbreviations: ASC, an adaptor protein involved in inflammasome binding to pro-caspase-1; ATP, adenosine triphosphate; dAdT, transfected double-stranded DNA; ELISA, enzyme-linked immunosorbent assay; IB, immunoblot; IL, interleukin; KO, knockout; LPS, lipopolysaccharide; MSU, monosodium urate; NLRP3, nucleotidebinding domain, leucine-rich-repeat–containing family, pyrin domain–containing 3; PBMC, peripheral blood mononuclear cell; SEM, standard error of the mean; zYVAD-fmk, a caspase-1 inhibitor.
After reference 22: Duewell et al. Nature. 2010;464:1357-1361. © 2010, Nature Publishing Group.



Despite treatment with the most powerful statins, patients remain exposed to a high risk of complications (including death and reinfarction) after an acute coronary syndrome or a stroke. Based on the model that inflammation is a central process in atherosclerotic disease development and subsequent complications and on the hypothesis that targeting specific inflammatory proteins or pathways can be effective in reducing the risk of cardiovascular events, many anti-inflammatory drugs have been developed and some have shown promising results when administered on a background of statin therapy. In this article, we focus on the potential role of IL-1β inhibition in coronary and ischemic diseases.

Involvement of IL-1β in cardiovascular diseases

IL-1β is a potent proinflammatory cytokine secreted by different cell types, including macrophages and endothelial cells,12 and is involved in the differentiation of lymphoid cells.13 IL-1β seems to be a key regulator of several inflammatory and autoimmune diseases.14 The important role of IL-1 in atherosclerosis has been highlighted by experimental studies showing reduced atherosclerosis in IL-1 knockout15 or IL-1–type I receptor knockout mice.16

The role of IL-1 is largely documented in the pathophysiology of atherosclerosis,17,18 where it specifically targets endothelial cells19 and promotes vascular smooth muscle cell proliferation20 leading to intima thickening in mice.21 IL-1β has been shown to upregulate adhesion molecules in endothelial cells, thereby increasing recruitment of inflammatory cells, such as macrophages, in response to atherogenic stimuli such as cholesterol deposits (Figure 1, page 363).22 IL-1β also promotes inflammatory-cell transmigration to the atherosclerotic site.23 In addition, IL-1β has been shown to increase the levels of matrix metalloproteinases,24 thereby promoting extracellular matrix degradation. Also, IL-1β has been shown to enhance the reactivity of endothelial cells by inducing the expression of inducible nitric oxide synthase and vascular endothelial growth factor.





In atherothrombotic coronary disease, IL-1 has been shown to promote atheromatous lesions, modulate cholesterol metabolism, enhance vascular inflammation, and contribute to plaque rupture (Figure 2).25 Indeed, IL-1 showed proadhesive activity by increasing the expression of adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1) in mice.25 The increased expression of VCAM-1 and monocyte chemotactic protein-1 (MCP-1) results in increased accumulation of monocytes in the arterial intima which will differentiate into macrophages and foam cells, leading to atherogenesis. In humans, IL-1 has been shown to result in upregulation of matrix metalloproteinases,26 which induce collagen breakdown in atheromatous plaques. Increased expression of matrix metalloproteinases by IL-1 might therefore induce atheromatous plaque rupture and lead to superimposed thrombosis.17 IL-1 may also modulate cholesterol plasma levels by serum amyloid A induction18 and stimulate angiogenesis and vessel wall inflammation via vascular endothelial growth factor27 and other inflammatory pathways.28 Of note, IL-1 levels were found to be elevated in atherosclerotic human coronary arteries.29


Figure 2
Figure 2. Microscopic and macroscopic views of atherosclerotic
lesions in apoE-deficient mice having either the IL-1β+/+ or IL-1β–/–
genotype.

(A) Microscopic appearance around the aortic valves of 12- and 24-week-old
male apoE-deficient mice (an animal model of atherosclerosis with hypercholesterolemia
when fed on a normal diet) having either the IL-1β+/+or IL-1β–/– genotype.
At both 12 and 24 weeks of age, the atherosclerotic lesion sizes of IL-1β–/–
mice were smaller than those of IL-1β+/+ mice. For each mouse, 3 sections at
100-μm intervals around the aortic valve were observed. The section where all
valves were visible was regarded as the 0-μm section. The left (+100 μm) and
right (–100 μm) photomicrographs are sections 100 μm above and below the
0-μm section, respectively. All sections were stained with Oil red O (magnification,
x40. Bar in lower right panel equals 200 μm. (B,C) Macroscopic findings of
total aortas of apoE–/–/IL-1β+/+ (B, n=14) and apoE–/–/IL-1bη–/– male mice (C, n=13)
at 24 weeks of age. These aortas were stained with Oil red O. The percentage
of the atherosclerotic lesion to total aorta area was significantly lower in apoE–/–/
IL-1β–/– mice than in apoE–/–/IL-1β+/+ mice.
After reference 25: Kirii H et al. Arterioscler Thromb Vasc Biol. 2003;23:656-660.
© 2003, American Heart Association, Inc.


IL-1 antagonism

Pharmacological inhibition of IL-1 or IL-1 receptor has been shown to decrease experimental atherosclerotic plaque formation. The selective loss of IL-1 signaling in the vessel wall by bone marrow transplantation reduced plaque burden in a mouse atherosclerosis model.30 This reduction was associated with restored endothelium-dependent vasodilation and decreased levels of arterial oxidative stress. These findings were further confirmed by studies targeting the IL-1 receptor.31,32 Subsequently, therapies including soluble truncated IL-1 receptor, IL-1–receptor antagonist, IL-1 trap fusion protein, or anti–IL-1 antibodies have been developed and tested in animal models and in clinical settings.

The soluble IL-1–receptor antagonist molecule is an IL-1 antagonist, which binds to the IL-1 receptor, but does not activate the IL-1–receptor signaling pathway. IL-1–receptor antagonist has a key role in the development of atherogenesis; indeed, mice lacking the IL-1Ra gene develop lethal arterial inflammation.28 IL-1–receptor antagonist has also been shown to inhibit neointima formation after coronary artery injury.21 This impact on vascular inflammation and atherosclerosis has been proposed to be dependent on the ratio of IL-1 to IL-1– receptor antagonist.33 Finally, genetic associations between IL-1Ra and coronary artery disease as well as the development of restenosis after stenting have been reported.34,35

Ongoing clinical trials evaluating the effects of anti–IL-1 therapies

There are a number of clinical trials studying the effects of anti-IL–1 therapy currently under way. Briefly introduced here are studies evaluating the effects of IL-1 inhibition with anakinra, and more selectively, IL-1inhibition with canakinumab. We will then take a more in-depth look at the IL-1inhibitor gevokizumab and the ongoing initial clinical assessment of its effects on plaque inflammation.

The IL-1 inhibitor anakinra
Anakinra is a recombinant form of human IL-1–receptor antagonist that competitively inhibits IL-1 by binding the IL-1 type I receptor, acting as an antagonist to the IL-1 receptor. A clinical trial (NCT01566201) is ongoing to evaluate the effects of anakinra on vascular processes (ie, coronary flow reserve, aortic deformation) and ventricular function (ie, systolic and diastolic function on echocardiography) as well as apoptotic and inflammatory biomarkers in 80 patients with both rheumatoid arthritis and coronary artery disease.

The IL-1β inhibitor canakinumab
A human monoclonal antibody against IL-1β is currently indicated for the treatment of autoinflammatory diseases such as cryopyrin-associated periodic syndromes (CAPS). Canakinumab selectively neutralizes IL-1β, resulting in a rapid and sustained inhibition of the inflammatory acute phase response. CANTOS (Canakinumab ANti-inflammatory Thrombosis Outcomes Study) is a large randomized, double-blinded, placebocontrolled, event-driven trial (NCT01327846) of 17 200 stable post–myocardial infarction patients with persistent elevation of high-sensitivity C-reactive protein (hs-CRP) that is evaluating the effects of canakinumab, at doses of 50, 150, or 300 mg administered subcutaneously every 3 months, as an add-on to optimized treatment. All participants are followed up over an estimated period of up to4years for the trial’s primary endpoint (ie, cardiovascular death, nonfatal myocardial infarction, and nonfatal stroke) as well as for other end points including vascular events, total mortality, adverse events, and specific end points associated with inflammation, including new-onset diabetes, venous thrombosis, and atrial fibrillation. CANTOS is the first large study evaluating the effect of anti–IL-1 therapies on atherosclerosis-related end points. If such therapy has beneficial effects, it would open new avenues for direct targeting of inflammation toreduce atherosclerosis andits complications.

Focus on the IL-1β inhibitor gevokizumab
Gevokizumab is a potent anti–IL-1β neutralizing antibody,36 which acts as a regulator of the IL-1 pathway (Figure 3, page 366).37 It has been shown to reduce pathologically high IL-1β activity, while allowing homeostatic signaling in biologically important processes. Gevokizumab is a recombinant Human EngineeredTM monoclonal antibody that binds human IL-1β with 0.3pM affinity and regulates the activation of IL-1 receptors.36,37 The antibody was humanized using proprietary technology with the goal of reducing the probability of eliciting antidrug immunogenic responses. Gevokizumab is produced in Chinese hamster ovary (CHO) cells. It is an immunoglobulin G subclass 2 (IgG2) isotype, thereby reducing the probability of antibody-dependent cell-mediated cytotoxicity should binding to the cell surface occur. The terminal half-life of the antibody is estimated to be between 22 to 28 days. The high affinity and potency of gevokizumab is expected to allow for lower doses and more convenient dosing regimens than currently available with other IL-1β inhibitors.38

In vitro binding studies show that gevokizumab binds with similar affinity to human, rat, rabbit, and cynomolgus and rhesus monkey IL-1β and with over 1000-fold less affinity tomouse IL-1β.36 Gevokizumab shows selective binding to recombinant human IL-1β, but not to recombinant human IL-1β (another inactive subtype of IL-1).39 In vitro cell culture studies show the ability of gevokizumab to inhibit IL-1β–mediated IL-6 expression: the inhibition induced by gevokizumab was greater than that induced by the recombinant IL-1–receptor antagonist anakinra (data on file). Evaluation of Toll-like–receptor agonist stimulation in human whole blood cultures also indicate that 0.1pM gevokizumab inhibited 50% of the production and release of IL-1β, IL-1α, and interferon gamma, and to a lesser degree tumor necrosis factor α and IL-6, but not IL-1–receptor antagonist and IL-8. These results demonstrate that gevokizumab was able to reduce, but not fully suppress, the cytokine induction under physiological conditions of this assay.


Figure 3
Figure 3. Effect of gevokizumab on activity of interleukin 1β in cell-based activity assays.

(A) Gevokizumab neutralizes interleukin (IL)-1β stimulation of IL-6 release by MRC-5 human lung fibroblast cells with an IC50 of approximately 2pM at the EC50 for this assay (100 pg/mL IL-1β). This activity is comparable with that of control blocking Ab 6, with an IC50 of around 6pM. (B) Gevokizumab attenuates the dose response of MRC-5 cells to IL-1β ~50-fold (EC50 values of 12pM and 815pM with an anti-KLH antibody and gevokizumab, respectively), whereas blocking Ab 6 almost completely inhibits IL-1β activity. (C) Gevokizumab neutralizes IL-1β stimulation of NF-κB activation in HeLa cells stably expressing an NF-κB–luciferase reporter construct with an IC50 of approximately 1pM at the EC50 for this assay (25 pg/mL IL-1β). Under these conditions, the potency of the control blocking antibody is 36-fold lower, with an IC50 of 36pM. (D) Gevokizumab attenuates the dose response of HeLa cells stably expressing an NF-κB–luciferase reporter construct to IL-1β stimulation around 20-fold, from an EC50 of 1.7pM in the presence of a nonbinding anti-KLH antibody to 36pM with gevokizumab. For A,C, and D, error bars show standard deviation of 3 replicates for test samples and duplicates for the anti-KLH antibody control. For B, error bars show standard deviation between duplicate samples. All experiments were run at an n=2.
Abbreviations: Ab, antibody; EC50, half maximal effective concentration; IC50, half maximal inhibitory concentration; IL, interleukin; KLH, keyhole limpet hemocyanin; NF-κB, nuclear factor κB; RLU, relative light units.
After reference 37: Roell et al. J Biol Chem. 2010;285:20607-20614. © 2010, The American Society for Biochemistry and Molecular Biology, Inc.



In vivo studies confirmed in mice the ability of gevokizumab to inhibit, in a dose-dependent manner, recombinant human IL-1β induction of IL-6. Approximately 70% inhibition of activity was obtained with the highest dose tested (3 μg/mouse, data on file). The in vivo pharmacological evaluation of gevokizumab has included studies that show that it is safe in a rat model of myocardial infarction and has beneficial effects in a mouse model of atherosclerosis. When administered at the time of reperfusion following 30 minutes of complete occlusion of a coronary artery, gevokizumab (10 mg/kg) did not exert a deleterious effect on left ventricular scar formation or on scar size (J. C. Tardif et al, unpublished observations, 2012). Gevokizumab was recently shown to have antiatherosclerotic effects in apolipoprotein E (apoE)-knockout mice: indeed, lesion area in the aorta was reduced by 37%, 22%, and 29% at doses of 0.1, 1, and 10 mg/kg administered twice weekly for 16 weeks, respectively, with no significant differences between doses (Figure 4, page 367).40


Figure 4
Figure 4. Effect of a chimeric murine version of gevokizumab (XMA052 MG1K) on atherosclerotic lesions in the aortas and brachiocephalic
arteries of apoE-deficient mice.

ApoE-deficient mice were fed an atherogenic diet for 16 weeks and dosed as indicated. Aortic lesion area was measured by en face analysis and expressed as percent Sudan IV–positive pixels. (A) En face images from representative individuals with lesion size approximating the mean are shown. (B) Lesion area was reduced by 37%, 22%, and 29% at the 0.1, 1.0, and 10 mg/kg doses, respectively. Results are represented as mean ± SEM (n=10). *P<0.05 versus IgG and vehicle. (C) Representative images of brachiocephalic arteries for IgG and XMA052 MG1K (1.0 mg/kg).
Abbreviations: mIgG, mouse immunoglobulin G; SEM, standard error of the mean; XMA052 MG1K, chimeric murine version of gevokizumab.
After reference 40: Bhaskar et al. Atherosclerosis. 2011;216:313-320. © 2011, Elsevier Ireland Ltd.



The initial clinical cardiovascular assessment of gevokizumab will target arterial inflammation evaluated by positron emission tomography (PET) in patients at high cardiovascular risk. Symptomatic unstable arteriosclerotic plaques accumulate more 18fluorodeoxyglucose (18FDG) than asymptomatic lesions, and 18FDG uptake in the ascending aorta and the left main coronary artery is higher in patients with a recent acute coronary syndrome as compared with patients with stable angina.41,42 In addition, recent studies demonstrated that 18FDG uptake is frequently elevated in high-risk patients with type 2 diabetes, coronary artery disease, or obesity.43 In order to maximize the possibility of observing a difference between placebo and gevokizumab, the study population includes patients with active plaques: high-risk patients with a high 18FDG uptake in at least 1 main arterial region (thoracic aorta or carotid) following a recent acute coronary syndrome.

This first study is a prospective, international, multicenter, randomized, double-blinded, parallel-group trial. As a pilot exploratory study, the multiple administration of gevokizumab is compared with placebo, and the randomization is unbalanced (2:1 ratio). The duration of treatment (12 weeks between first and last monthly injection) was chosen in order to allow sufficient exposure to therapy allowing the assessment of drug activity on plaque inflammation. The assessment of plaque inflammation with PET will be performed both before study drug initiation and approximately 2 weeks after the last administration of study drug, at the theoretical peak of plasma concentration. There is a 3-month observation period following the last drug intake; its duration is considered to be sufficient given the expected gevokizumab pharmacokinetic profile (half-life of 22 to 26 days, residual concentration of gevokizumab 3 months after last injection <15%). The standardized uptake value is the unit used to quantify 18FDG uptake in body tissue and is determined as the decay- corrected tissue concentration of 18FDG (in kBq/mL) divided by the injected dose per body weight. The target-tobackground ratio (TBR) was developed in order to reduce the interpatient variability due to injected dose and body weight. It is calculated by dividing the arterial wall standardized uptake value by the venous blood value measured in the corresponding venous area (vena cava or jugular vein). A significant correlation has been demonstrated to exist between the TBR measured in the arterial wall and macrophage staining from the corresponding histological sections (r=0.85; P<0.0001).44 An elevated TBR has been shown to be a strong predictor of subsequent cardiovascular events45 and was associated with several cardiovascular risk factors.

The goal of this pilot study is to evaluate the effect of 4 successive monthly subcutaneous administrations of gevokizumab versus placebo on the reduction of arterial wall inflammation in patients with marked arterial wall inflammation following a recent acute coronary syndrome. The primary objective is to evaluate the effect of gevokizumab compared with placebo on arterial wall inflammation assessed by 18FDG PET in the most diseased region of interest of both carotid and thoracic aortic walls. The secondary objectives are to evaluate the effects of gevokizumab compared with placebo on cardiac and vascular biological blood biomarkers, including hs-CRP and IL-6, on the safety profile of gevokizumab, and on its pharmacokinetics in this specific population.

All 18FDG PET measurements will be performed in 3 regions of interest: left carotid, right carotid, and thoracic aorta. All comparisons will be performed within the most diseased region of interest (region with the highest maximum mean TBR at baseline). The study end points include the changes of the maximum TBR, mean TBR, and most diseased segment TBR assessed by 18FDG PET.

Conclusion

IL-1 appears to play a significant role in the pathophysiology of coronary artery disease. Thus, IL-1 inhibition is being investigated in patients with cardiovascular diseases. Clinical trials, such as those touched on in this review, are required to test in a definitive way the hypothesis that anti-inflammatory approaches like IL-1 inhibition will improve cardiovascular outcomes in patients with coronary disease treated with optimal standard of care including intensive statin use.





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Keywords: anti–IL-1 therapy; atherosclerosis; coronary disease; gevokizumab; inflammation; IL-1; IL-1