Effects of glucocorticoids and tumor necrosis factor-alpha inhibitors on both clinical and molecular parameters in patients with Takayasu arteritis.

OBJECTIVE: To explore the effect of sequential treatment with glucocorticoid and tumor necrosis factor-alpha inhibitors in patients with Takayasu arteritis (TA).
MATERIALS AND METHODS: In five patients with TA, the effects of the sequential treatment with prednisone for 5-7 months and then with adalimumab (ADA) + methotrexate (MTX) or infliximab + MTX, or with ADA only, for 12 months on both clinical and laboratory findings were evaluated.
RESULTS: All treatments improved both symptoms and laboratory parameters without the development of side-effects.
CONCLUSIONS: It was hypothesized that MMP-9 and neutrophil gelatinase-associated lipocalin could be markers of the response to the treatments.

INTRODUCTION

Takayasu arteritis (TA) is an idiopathic chronic inflammatory granulomatous disease affecting aorta and its main branches.[12] The pathogenesis of TA includes vessel injury due to inflammatory mediators, e.g., Tumor necrosis factor-α (TNF-α), released by T cells, natural killers, γ/δ T cells and macrophages;[345] However, an imbalance between matrix metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs) may also be involved.[6] In particular, it has been reported that MMPs 1, 3 and 9 were significantly higher in subjects with active disease.[78]

Previously, we documented an association between MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL), a protein belonging to the lipocalin family. NGAL was able to modulate the activity of MMP-9 through the activation of the NGAL/MMP-9 complex.[910] NGAL plasma concentration has been associated with vascular diseases.[11]

The clinical manifestation of TA is related to both sites of affected vessel and severity of inflammation and the acute stage is represented by fever, weight loss, and elevated C-reactive protein (CRP) levels.[12] Unfortunately, due to the delay in diagnosing, patients experience claudication, absence of pulses, hypertension, myocardial infarction and cerebrovascular diseases.[13]

The treatment include anti-inflammatory (glucocorticoids) and immunosuppressive drugs (cyclophosphamide, methotrexate [MTX] and azathioprine);[1415] in unresponsive patients, or in patients experiencing adverse drug reactions (ADRs), a treatment with TNF-α inhibitors + immunosuppressive may be used.[161718]

In this study, we reported in 5 patients with acute TA the effects of a sequential treatment with prednisone for 5 or 7 months and then with TNF-α inhibitors for 12 months on both clinical and laboratory parameters.

MATERIALS AND METHODS

In an open label, parallel groups, double clinical centers study, conducted during the period between January 2009 and December 2012 we enrolled patients with clinical, laboratory (erythrocyte sedimentation rate [ESR] >30 mm/h and CRP; >5 mg/L) and radiological findings of acute TA. Angiography and echo-color Doppler were used to evaluate the presence and the site of vessel lesions. The disease activity was determined using the Birmingham Vascular Activity Score.[19]

This study was approved by the Institutional Review Board - Independent Ethics Committee of Interuniversity Center of Phlebolymphology - International Research and Educational Program in Clinical and Experimental Biotechnology - Headquarters at University Magna Graecia of Catanzaro and before the beginning of the study, all participants were informed about the aim, procedures, risks and benefits of the study and the they provided a written informed consent. At the time of admission and during the follow-up, a blood sample was taken from each patient in order to evaluate the plasma levels of ESR, CRP, MMP-9 and NGAL. The levels of MMPs and NGAL were evaluated through Elisa test in agreement with our previous papers.[92021]

The enrolled patients received a sequential treatment with prednisone for 5 or 7 months and then with adalimumab (ADA) + MTX or infliximab (IFX) + MTX for 12 months. Follow-up was performed every month and the development of ADRs were evaluated in agreement with our previous study.[22232425262728293031]

This study may be considered to be exploratory; therefore we did not determine a power calculation.

RESULTS

During the study period, 5 new patients, of whom 4 were females and 1 male (median age 36), with acute TA were enrolled [Table 1] and signed the informed consent. Clinical evaluation documented that patients lamented fatigue, myalgia and arthralgia with a pain score of 8, measured through the visual analog scale.

Table 1

Demographic characteristic of each patient enrolled in this study

Patients were treated with Prednisone for 5 or 7 months and then with ADA + MTX for 1 year or with IFX + MTX for 1 year [Table 2].

Table 2

Protocol treatment used in this study

In these patients, laboratory findings revealed an improvement of inflammatory index (ESR and CRP) after 2 months from the beginning of prednisone treatment. Moreover, a complete normalization of inflammatory index was recorded 4 months after the beginning of prednisone and these values maintained steady during treatment with ADA or IFX without difference between groups [Table 3].

Table 3

Laboratory findings of ESR (mm/h) and CRP (mg/L) in enrolled patients

A patient (patient 5) with relapse, enrolled in one of our previously published study [Table 1],[14] who developed ADRs to corticosteroid and intolerance to MTX received a treatment with ADA only, with a good control of laboratory index [Table 3]. All patients also showed an improvement in their clinical conditions (VAS from 8 to 1 at the end of the study). No relapses and no ADRs occurred during the study and during the follow-up of 34 months.

In all patients (Case 1-5) ELISA findings revealed higher levels of both plasma MMP-9 (mean 140 ± 4 ng/mL) and NGAL (mean 176 ± 5 ng/mL) with respect to control patients (5 healthy volunteers: 4 F, 1 M, median age 34; MMP-9: 65 ng/mL; NGAL: 20 ng/mL). In enrolled patients (Cases 1-4) the prednisone treatment induced a time-related decrease in both plasma MMP-9 and NGAL levels [Table 4], that have been maintained in normal values during the treatment with monoclonal antibodies [Table 4].

Table 4

Elisa values of MMP-9 (ng/mL) and NGAL (ng/mL) in enrolled patients

DISCUSSION

In this study, we evaluated the effects of a different drug treatment of TA in patients with acute TA symptoms.

Till date the etiology of TA disease is enigmatic since several mechanisms such as post-infective, autoimmune, ethnic susceptibility and genetic predisposition have been postulated.[32] Oxidative stress (i.e., reactive oxygen species) and reactive nitrosative species represent a cardinal feature of inflammatory process and are involved in vascular abnormalities influencing extracellular matrix remodeling through the activation of MMPs.[8] MMPs regulate extracellular structural proteins and consequent tissue remodeling and seem to be involved in various vascular disease and related complications.[1133] NGAL is a protein belonging to the lipocalin family, is expressed by activated neutrophils and is able to modulate the activity of MMP-9 through the activation of the NGAL/MMP-9 complex. In vessels MMPs influence the migration, proliferation and apoptosis of vascular smooth muscle, endothelial cells and inflammatory cells. NGAL plasma concentrations have been associated with vascular diseases.[34353637] In agreement with these data, in our study we enrolled 5 patients with fever myalgia, claudication intermittens; angiography documented the presence of stenosis in upper diaphragmatic vessels. Previously it has been showed that MMP-1, 3 and 9 were significantly higher in subjects with active disease when compared to those study subjects who were in remission.[333435]

The limitation of this study is related to the very low number of patients enrolled that is related with the very low frequency of this disease.

CONCLUSION

Elisa test revealed higher levels of both plasma MMP-9 and NGAL in Takayasu patients respect to control patients. Prednisone administered for 5 months showed the same effects on both clinical and laboratory findings of a longer treatment (7 months). Moreover, we showed that both ADA and IFX administered on-label with MTX improved the inflammatory indexes inducing a significant decrease in plasma levels of CRP, ESR, MMP-9 and NGAL. The patient treated on-label with ADA in monotherapy documented a good control of symptoms and of laboratory findings. No side effects appeared during the treatments. The limitation of this study is very low number of patients due to very low frequency of this disease. In conclusion, monoclonal antibodies are able to improve the symptoms of Takayasu without the development of side effects and we suggest that treatment with prednisone for 5 months leads to a good clinical outcome, although further studies are needed to validate these data. Finally, the evaluation of MMP-9 and NGAL plasma levels could be useful during the follow-up of this disease.