BioDrugs (2014) 28 (Suppl 1):S5–S13 DOI 10.1007/s40259-013-0063-0

REVIEW ARTICLE

Tumour Necrosis Factor a Antagonists in the Treatment of Rheumatoid Arthritis: An Immunological Perspective Pier-Luigi Meroni • Guido Valesini

Ó Springer International Publishing Switzerland 2014

Abstract Rheumatoid arthritis (RA) is one of the most prevalent autoimmune conditions, affecting approximately 1 % of the adult population. It is associated with decreased quality of life and considerable morbidity and mortality. Various inflammatory cells, including macrophages, neutrophils, mast cells, natural killer cells, B and T cells and stromal cells play key pathophysiological roles in joint inflammation and RA progression. Several cytokines, including interleukin (IL)-1a and/or IL-1b, and tumour necrosis factor (TNF)-a, are involved at each stage of RA pathogenesis; namely, by augmenting autoimmunity, sustaining long-term inflammatory synovitis and promoting joint damage. Different cell types are involved in RA pathogenesis through upregulation of several cytokine and soluble pro-inflammatory mediators. As early as the late 1980s, TNF had been identified as a potential target in RA. Five antiTNF drugs, infliximab, adalimumab, certolizumab pegol, etanercept and golimumab, are now approved for the treatment of RA in various countries. All are bivalent monoclonal antibodies, with the exception of the monovalent certolizumab and etanercept, which is an engineered dimeric receptor. Although all react with and neutralise soluble TNF in vitro, structural differences in the molecules may contribute to differences in their therapeutic effects and the occurrence of side effects. Pegylated certolizumab permits once-monthly dosing. Other mechanisms of action proposed to be important for the efficacy of anti-TNF agents are as P.-L. Meroni (&) Divisione di Reumatologia, Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy e-mail: [email protected] G. Valesini Department of Internal Medicine and Medical Specialties, University of Rome ‘‘La Sapienza’’, Rome, Italy

follows: induction of apoptosis of both monocytes and T cells; neutralization of membrane TNF; antibody-dependent cell-mediated and complement-dependent cytotoxicity; and reverse signaling via membrane TNF.

1 Immunology of Rheumatoid Arthritis (RA) and Tumour Necrosis Factor (TNF) Blockade Rheumatoid arthritis (RA) is one of the most prevalent autoimmune conditions, affecting approximately 1 % of the adult population [1–3]. Furthermore, uncontrolled RA is associated with decreased quality of life and considerable morbidity and mortality [1, 3]. Although the precise pathogenesis of RA has not been clearly defined, significant progress has been made in the past two to three decades in the pathophysiological understanding of the disease. Various inflammatory cells, including macrophages, neutrophils, mast cells, natural killer (NK) cells, B and T cells and stromal cells (e.g. synovial fibroblasts), play key pathophysiological roles in joint inflammation and RA progression [4]. Furthermore, disease complexity is shown by the numerous inflammatory mediators and soluble factors active on maturation/ activation of different effector cells and present at raised concentrations in the joints of RA patients [4]. These molecules include interleukin (IL)-6, IL-7, IL-15, IL-17a, IL-18, IL-21, IL-23, IL-32, receptor activator of nuclear factor jB ligand (RANKL), tumour necrosis factor (TNF)a and vascular endothelial growth factor, which foster increased inflammation, angiogenesis and joint damage [1]. Of these, a major inflammatory cascade in RA involves the overexpression of the TNF gene and subsequent overproduction of TNF, leading to synovial inflammation and joint degradation [3]. This review focuses on the

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immunology of RA, with particular focus on the role of TNF in the pathogenesis of RA. 1.1 T-Cell Function in RA RA has traditionally been thought of as a condition mediated by T-helper 1 cells and therefore directed by T-cell populations producing inflammatory cytokines (e.g. TNF) [5]. More recently, it has been suggested that other T-cell populations (e.g. T-helper 17 and regulatory T cells) may also play an important pathophysiological role [6]. The activation of synovial T cells and key effector pathways is shown in Fig. 1. Synovial T cells could contribute to synovitis directly through the production of inflammatory cytokines. Dendritic cell activation within RA joints has been

Fig. 1 Activation of synovial T cells in rheumatoid arthritis and key effector pathways and interactions among dendritic cells, T cells and B cells. ADAMTS a disintegrin and metalloprotease with thrombospondin-1-like domains, DAMP damage-associated molecular pattern, Dkk-1 dickkopf-1, FcR Fc receptor, FceRI high-affinity IgE receptor, FGF fibroblast growth factor, GM-CSF granulocyte macrophage colony-stimulating factor, HA hyaluronan, HSP heat-shock protein, IFN-a/b interferon-a/b, MMP matrix metalloproteinase, NLR

P.-L. Meroni, G. Valesini

attributed to TNF and IL-17, and synergistic activity has even been reported between low concentrations of IL-17, IL-1b and TNF, leading to increased intrasynovial fibroblast activation and further cytokine production [7]. T cells contribute to synovial inflammation via direct interaction with neighbouring macrophages and synovial fibroblasts that promote their activation. In vitro, synovial T cells have also been reported to promote TNF and IL-1b production after cell contact with syngeneic macrophages [8]. 1.2 Role of Cytokines in the Pathogenesis of RA Several cytokines are implicated in the pathogenesis of RA, which has been extensively reviewed by McInnes and Schett [4]. Cytokines are involved at each stage of RA

nucleotide-binding oligomerization domain-like receptor, PAMP pathogen-associated molecular pattern, PAR2 protease-activated receptor 2, PDGF platelet-derived growth factor, RANKL receptor activator of nuclear factor jB ligand, TGFb transforming growth factor b, Th0 type 0 helper T cell, Th1 type 1 helper T cell, Th17 type 17 helper T cell, TLR Toll-like receptor, TNFa tumour necrosis factor a, VEGF vascular endothelial growth factor. Reproduced with permission from McInnes and Schett [4]

TNFa Antagonists in RA: Immunology

pathogenesis, and their pattern may change over time depending on the stage of the disease [4]. They play a role in augmenting autoimmunity, sustaining long-term inflammatory synovitis and promoting joint damage [4]. IL-1a and/or IL-1b and TNF are the main players in RA pathogenesis. TNF is expressed in monocytes, B cells and synovial fibroblasts, but is also expressed in T cells, NK cells, polymorphonuclear leukocytes, mast cells, endothelial cells and osteoblasts. Its potential function in the pathogenesis of RA is to increase monocyte activation, cytokine and prostaglandin release; increase polymorphonuclear leukocyte priming, apoptosis and oxidative burst; increase endothelial cell activation; increase matrix metalloproteinase (MMP) and cytokine release; increase adipocyte free fatty acid release; facilitate T-cell apoptosis, clonal regulation and T-cell receptor dysfunction; decrease synovial fibroblast proliferation and collagen synthesis; and other endocrine effects [4]. 1.2.1 Osteoclasts and Bone Erosion Together with IL-1, IL-6 and IL-17, TNF is a major promoter of osteoclast formation, and may act additively in this respect with RANKL [9]. RANKL, a member of the TNF superfamily, induces the final differentiation of osteoclasts and their bone-resorption activity, and its expression is regulated by inflammatory cytokines such as TNF and IL-1b [10, 11]. 1.2.2 Articular Destruction and Degradation of Cartilage Synovial fibroblasts are pivotal in integrating the inflammatory and destructive phases of inflammatory arthritis that are regulated by cytokines. TNF and IL-1b are important cytokines involved in synovial fibroblast activation, and facilitate matrix enzyme release (e.g. MMP) [4]. The release of IL-1b and TNF also encourages a switch from anabolism to catabolism of cartilage [4].

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swelling and improve clinical scores in murine models of arthritis [17]. In another animal study, transgenic mice in which TNF gene expression was deregulated developed chronic inflammatory arthritis; treatment of these mice with anti-TNF antibodies before the development of arthritis resulted in the animals showing no signs of the disease [18]. Those studies demonstrate the widespread regulatory effects of anti-TNF antibodies, that TNF is at the top of the pro-inflammatory cascade, and that it is an essential component of the processes leading to the development of arthritis. This combination of in vitro and in vivo studies provided the rationale for evaluating anti-TNF therapy in RA in humans. However, although clinical benefit was shown with the first generation of monoclonal antibodies, initial studies were hampered by the formation of human antimouse neutralizing antibodies [19]. This led to the development of ‘chimeric’ antibodies developed by re-engineering mouse antibodies with functionally equivalent human amino acids, initially replacing the mouse immunoglobulin (Ig) constant domains with human equivalents in order to reduce the overall immunogenicity of the molecule [20, 21]. One of these, infliximab, was developed by converting a mouse monoclonal antibody specific for TNF into a partly human IgG1/j by grafting the human constant region onto the mouse variable region [22]. Initial trials in human subjects with RA showed that treatment with infliximab decreased serum levels of cytokines such as IL-6 and IL1b, decreased the inflammatory marker C-reactive protein, and improved the clinical symptoms of RA [23, 24]. In the late 1990s, infliximab became the first monoclonal antibody approved for RA treatment in patients who were inadequate responders to methotrexate after showing superior response rates compared with methotrexate alone, and significantly slowing disease progression in large-scale clinical trials [25].

2 Anti-TNF Drugs: Similarities and Differences 1.3 TNF as a Therapeutic Target for RA As early as the late 1980s, TNF had been identified as a potential target in RA [12–14]. In vitro studies revealed that the production of IL-1, considered important in inflammatory arthritides, was dependent on TNF [12]. TNF was also shown to induce the pro-inflammatory granulocyte macrophage colony-stimulating factor (GM-CSF), in in vivo studies of cytokine expression in synovium [15, 16], and blockade of TNF expression is known to prevent GM-CSF expression [16]. Subsequently, TNF blockade was shown to reduce IL-1 production in human primary synovial membrane cultures, and also to reduce paw

Five anti-TNF drugs, infliximab, adalimumab, certolizumab pegol, etanercept and golimumab, are now approved for the treatment of RA in various countries (Table 1). All drugs are administered subcutaneously, except for infliximab, which is given by intravenous infusion. However, the duration of dosing varies between weekly and every 8 weeks, and is largely dependent on the structure of the molecule. Furthermore, key differences in the structures of these anti-TNF drugs (Fig. 2) [32] may contribute to differences in their properties and are discussed in the following sections.

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Table 1 Currently approved anti-tumour necrosis factor therapies for the treatment of patients with rheumatoid arthritis [26–31] Drug name

Antibody format

Route/frequency of administration

Incidence of anti-drug antibodies (%)

US FDA black box warning Risk of infection

Hepatosplenic T-cell lymphoma

Adalimumab

Human IgG1

SC 40 mg eow

\1–87

x

x

Certolizumab pegol

Pegylated human Fab0 2

SC 400 mg qw 0, 2 and 4 followed by 200 mg eow

3–25

x

x

Etanercept

Soluble TNF receptor fusion protein

SC 50 mg weekly

0–18

x

x

Golimumab

Human IgG1

SC 50 mg monthly

0–7

x

x

Infliximab

Chimeric IgG1

IV infusion 3 mg/kg q8w

6–61

x

x

eow every other week, Ig immunoglobulin, IV intravenous, SC subcutaneous, TNF tumour necrosis factor; qxw every x weeks, qw every week, US FDA US Food and Drug Administration

Fig. 2 The structures of currently available anti-tumour necrosis factor agents. Reproduced with permission from Horiuchi et al. [32]

2.1 Antibody Structure Despite attempts to reduce the immunogenicity of monoclonal antibodies using humanization strategies, the long-term use of chimeric agents can result in antibody formation [1]. More recently, the use of recombinant technology, such as phage display of human antibody components or transgenic methods, has been used to produce fully human monoclonal antibodies (e.g. adalimumab [phage display] or golimumab [transgenic mice]) [1]. Complementarity determining region loop grafting to produce humanized monoclonal antibodies was developed to reduce some of the immunogenic issues associated with chimeric antibodies [1].

Of the currently available anti-TNF agents, infliximab is a chimeric TNF-specific monoclonal antibody with mouse hypervariable domains and a human antibody backbone; adalimumab is a recombinant human TNF-specific monoclonal antibody; etanercept is a fully human construct comprising the p75 TNF receptor and crystallizable fragment (Fc) antibody portion only; and golimumab is a fully human TNF-specific monoclonal antibody (Fig. 2) [26–28, 32]. In contrast, certolizumab pegol is a Fab0 fragment of a humanized monoclonal antibody conjugated with a 40 kDa polyethylene glycol (PEG) moiety that, unlike other antiTNF agents, does not contain the Fc domain (Fig. 2) [32– 34]. Pegylation reduces immunogenicity by shielding the protein from recognition by the immune system, and

TNFa Antagonists in RA: Immunology

increases the half-life of the conjugated molecule in the blood [35]. The use of biological agents in the first-line treatment of RA has also allowed the evaluation and approval of other antibody or fusion protein-based therapies, including rituximab (B-cell depletion therapy), abatacept (inhibition of co-stimulation), and tocilizumab (anti-IL-6 receptor therapy) [1]. 2.2 Development of Immunogenicity All anti-TNF drugs are biologically active molecules that can elicit immune reactions in humans (Table 1) [36]. Moreover, approximately one-third of patients treated with anti-TNF agents do not respond adequately to treatment or lose efficacy over time, and many patients stop treatment because of adverse events or tachyphylaxis resulting, in part, from immunogenicity [1, 37]. One cause may be the development of anti-drug or neutralizing antibodies by the patient (Table 1). With infliximab, for example, the lack of response in some patients has been attributed to an antidrug immune response and formation of an immune complex (of the therapeutic drug antibody with the anti-drug antibody). This results in greater drug clearance, reduced drug efficacy and an increased risk of infusion reactions in these patients [38]. Anti-drug antibody formation has also been reported with etanercept, adalimumab and certolizumab pegol (Table 1). The production of anti-drug antibodies is not surprising, as therapy with biological TNF inhibitors resembles common vaccination procedures, in which repeated, and in most cases subcutaneous, administration of non-self proteins are used to elicit an immune response. Anti-drug antibodies may be directed against non-human molecular structures, as is the case for infliximab in which the main immunogenic component is represented by the murine part of the Fab0 fragment. However, antibodies against these drugs can also be found for drugs that are ‘humanized’ through genetic engineering (i.e. certolizumab pegol), or following the use of so-called fully human antibodies (i.e. adalimumab, golimumab), or even for human antibody components fused with the extracellular part of human TNF receptor 2 (etanercept). Anti-drug antibodies can be directed against non-self Ig allotypes and/or idiotypes. However, additional neoepitopes originating at molecular reconstruction sites, or generated by drug aggregation or from non-human glycosylation, may be able to trigger an immune response [39]. Anti-drug antibodies are less frequently detected when humanized molecules are used [40]. The frequency of anti-drug antibody formation varies widely in the literature; a recent review put the prevalence in patients with RA at 0–87 %, depending on the drug examined (Table 1) [29]. While some of this variability is

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due to differing immunogenicity based on the structure of the drug (e.g. the presence of murine-derived antibody fragments in a biological molecule can increase immunogenicity when used in humans) [22] and individual patient factors (genetics and possible demographic characteristics currently not well characterized), evidence suggests that accurate detection of anti-drug antibodies can depend heavily on the assay used [29]. Standard enzyme-linked immunosorbent assays (ELISA) may have a high rate of false-positive results and a large amount of non-specific binding, but two-site ELISA and the radioimmunoassay antigen binding test have both high specificity and high sensitivity. However, two-site ELISA may preferentially detect IgM antibodies over monovalent IgG4 antibodies, and the antigen binding test, while capable of detecting IgG antibodies, uses radioactivity, which is a disadvantage [29, 36, 41]. Anti-drug antibody detection is also affected by the presence of rheumatoid factor, IgM antibodies that complex with the Fc portion of IgG, which can mask the epitopes bound in the assays [29]. Assays are also confounded by the presence of drugs, which in a sample containing anti-drug antibodies can lead to the formation of immune complexes that hinder detection in vitro [42]. Assays designed to overcome the limitations of currently available methods are under development, but to date none have become routinely used in clinical practice. 2.3 Potential Risk of Infection and Malignancy All anti-TNF agents have been associated with a potentially increased risk of bacterial infections (e.g. sepsis, cellulitis and abscesses), fungal infections (e.g. candidiasis) and viral infections (e.g. herpes zoster), and appropriate screening (chest radiography, skin testing, or whole-blood testing for Mycobacterium tuberculosis) is advocated to reduce the risk of latent tuberculosis reactivation [1, 3, 43, 44]. However, debate continues about whether anti-TNF agents are definitively associated with an increased risk of malignancy, particularly lymphoproliferative malignancies [1, 44, 45]. One meta-analysis of nine randomized, placebo-controlled trials of infliximab and adalimumab showed a dose-dependent increase in the risk of malignancies with anti-TNF treatment [44], while another showed no significant increase in solid malignancies when comparing a large cohort of etanercept, infliximab or adalimumab-treated patients with the general population [46]. A third meta-analysis that included 63 randomized, controlled trials investigating the use of adalimumab, certolizumab, etanercept, golimumab and infliximab in 29,423 patients with RA also showed no increased risk of malignancy when patients were treated with anti-TNF agents compared with other disease-modifying anti-rheumatic

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drugs or placebo [47]. As the risk of lymphomas is increased in patients with severe RA [48, 49], these patients are more likely to receive treatment with anti-TNF agents. However, evidence to date suggests that it is the disease itself that increases the risk of lymphoma in patients with RA, not the treatment [50]. Nevertheless, all anti-TNF agents carry US Food and Drug Administration black box warnings describing cases of lymphoma and other malignancies in patients treated with these agents. Adalimumab and infliximab also contain warnings regarding post-marketing cases of hepatosplenic T-cell lymphoma in patients with inflammatory bowel diseases [26–28, 30]. 3 Distinctive Signatures of Certolizumab: Insights into its Potential Mechanism of Action While infliximab has shown efficacy in the management of symptoms in patients with RA and active Crohn’s disease [51], etanercept failed to show efficacy in a double-blind, placebo-controlled phase II study of Crohn’s disease [52], suggesting that neutralization of soluble TNF is not the only mechanism of action of these drugs [53]. A number of other reasons have been suggested to account for these differences, including variations in the penetration of the diseased tissue among the anti-TNF agents, differences in stability of the immune complexes with TNF, or insufficient neutralization of soluble TNF. Other mechanisms of action proposed to be important for the efficacy of antiTNF agents are induction of apoptosis of both monocytes and T cells; neutralization of membrane tumour necrosis factor (mTNF)-a; antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC); and reverse signaling via mTNF [53]. An overview of in vitro comparisons between adalimumab, etanercept, infliximab and certolizumab pegol is shown in Table 2. Structural differences of certolizumab pegol, such as the absence of an Fc domain, may translate into important Table 2 Summary of in vitro properties of anti-tumour necrosis factor drugs [54] Drug

Induction of apoptosis

Induction of CDC/ ADCC

Neutrophil death/ degranulation

NK cell reverse signaling

Certolizumab pegol

No

No

No

Yes

Etanercept Adalimumab

Yes/noa Yes

Yes/noa Yes

Yes/noa Yes

Yes Yes

Infliximab

Yes

Yes

Yes

Yes

a

clinical differences in its mode of action relative to other anti-TNF agents [33, 53]. For example, certolizumab pegol has shown a decreased efficacy compared with other antiTNF agents when used to treat inflammatory bowel diseases. In placebo-controlled trials of anti-TNF agents in patients with Crohn’s disease, a greater proportion of patients treated with certolizumab pegol were not in remission at week 4 than those treated with infliximab or adalimumab [40, 62–64], and this result was also seen in maintenance studies, in which ‘no remission’ rates were higher in certolizumab than infliximab- and adalimumabtreated patients in the longer term [40, 65–67]. These differences in efficacy between certolizumab pegol and other anti-TNF agents could be related to the structure of certolizumab pegol, when it is considered that certolizumab pegol does not display the range of biological activity shown by the fully humanized anti-TNF agents with respect to apoptosis, cytotoxicity and interaction with the Fc receptors. The effect of these differences on the mechanism of action in the treatment of RA is discussed in the following sections, focusing on insights into the potential mechanism of action of certolizumab pegol. 3.1 Drug Exposure, TNF Binding and Neutralization One important consideration in the effective treatment of inflammatory disorders such as RA is drug exposure at the site of inflammation. For instance, while certolizumab pegol, adalimumab and infliximab were distributed more effectively in inflamed than non-inflamed tissues in animal models, certolizumab pegol appears to effect a more prolonged duration of exposure of the inflamed tissues, and the accumulation of certolizumab pegol was more responsive to the severity of inflammation than either adalimumab or infliximab [68]. All biological agents have a high affinity for soluble TNF. While etanercept has the highest affinity of all the biological agents, certolizumab pegol has a higher affinity than either infliximab or adalimumab, showing that affinity of the pegylated Fab0 fragment is comparable, if not superior, to the other full antibodies studied [53]. Furthermore, with certolizumab pegol, the Fab0 fragment is pegylated at a position distant from the TNF binding site, thereby ensuring that the high antigen-binding affinity and in vitro potency of the variable region are retained [69]. Furthermore, in vitro, all four anti-TNF drugs potently neutralize soluble TNF, and also bind to mTNF and neutralize mTNF-mediated effects [53]. 3.2 Apoptosis of Monocytes and T cells

In vitro experimental data exist for both results [54–61]

ADCC antibody-dependent T-cell-mediated cytotoxicity, CDC complement-dependent cytotoxicity, NK natural killer

It is currently unclear what role apoptosis plays in the mechanism of action of anti-TNF treatments; however, in

TNFa Antagonists in RA: Immunology

contrast to infliximab, adalimumab and etanercept, certolizumab pegol does not mediate increased levels of apoptosis in vitro (no apoptotic effect is observed on activated lymphocytes and monocytes, and granulocyte degranulation and necrosis are not increased), suggesting that these mechanisms are not essential for the efficacy of anti-TNF agents [53]. This may have important clinical implications regarding a potentially unique mechanism of action, and possibly a lower-than-anticipated incidence of infections with certolizumab pegol. 3.3 Cell-Dependent and Antibody-Dependent CellMediated Cytotoxicity Certolizumab pegol does not mediate CDC and ADCC in vitro [53], in contrast with both infliximab and adalimumab [70, 71], as would be expected due to the absence of an Fc region in the certolizumab pegol molecule. In addition, NK cells are the only type of lymphocyte that express high levels of mTNF; therefore, it has been suggested that they may also play a potential role in the pathogenesis of RA. It has been suggested that anti-TNF drugs may increase NK cell-mediated cytotoxicity via reverse signaling through constitutively expressed mTNF by promoting the release of multiple cytotoxic effector molecules and inflammatory cytokines [54]. In vitro, certolizumab pegol, etanercept, adalimumab and infliximab stimulated NK cell degranulation, but only certolizumab pegol can activate NK cells and does not mediate ADCC. The authors concluded that this was due to the absence of an Fc region in the certolizumab pegol molecule [54]. Differences in the mode of action of certolizumab pegol, adalimumab, infliximab and etanercept may also be due to the differential effect of the anti-TNF agents on reverse signaling mediated via mTNF. Unlike the other anti-TNF agents, certolizumab pegol is a steric inhibitor of TNF receptor binding. Also, because the different anti-TNF drugs react with different epitopes expressed on the TNF molecule (including mTNF), it has been suggested that selective epitope binding may induce different downstream cell signaling. This finding may also explain some of the differences in their mode of action [72].

4 Conclusions The role of TNF in the pathogenesis of RA has been well established. Several anti-TNF drugs are now available for the treatment of RA. However, differences in their structures and mechanisms of action allow different dosing and may be responsible for differences in efficacy and tolerability. Structural differences in certolizumab pegol may

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result in advantages over other currently available antiTNF agents under certain conditions. Acknowledgments This manuscript was prepared with financial assistance from UCB Pharma SpA, Milan, Italy. The authors would like to thank Jane Caple and David Murdoch, inScience Communications, Springer Healthcare, for their assistance in the preparation of the manuscript. Assistance with post-submission requirements was provided by Sheridan Henness, PhD, inScience Communications, Springer Healthcare. This article was published in a supplement sponsored by UCB Pharma SpA, Italy. The supplement was guest edited by Daniel Aletaha and peer reviewed by Leonard H. Calabrese who both received a small honorarium from Springer Healthcare to cover out-of-pocket expenses. D.A. has received honoraria and research grants from UCB, and honoraria from Abbvie, Gru¨nenthal, Janssen, Merck, Medac, Mitsubishi Tanabe, Pfizer, AstraZeneca, Eli Lilly, Novo Nordisk, and Sanofi/Regeneron. L.H.C. has consulted for UCB, Roche, Janssen, Pfizer and BMS. Conflict of interest declare.

The authors have no conflicts of interest to

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