International Immunopharmacology 23 (2014) 546–549
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Original article
Atorvastatin calcium in combination with methylprednisolone for the treatment of multiple sclerosis relapse Xiao-ling Li a, Zhen-chang Zhang a, Bo Zhang b, Hua Jiang a, Chun-mei Yu a, Wen-jing Zhang a, Xiang Yan a, Man-xia Wang a,⁎ a b
Department of Neurology, Second Hospital of Lanzhou University, Lanzhou 730030, China Department of Cardiology, First Hospital of Lanzhou University, Lanzhou 730030, China
a r t i c l e
i n f o
Article history: Received 12 August 2014 Received in revised form 27 September 2014 Accepted 2 October 2014 Available online 16 October 2014 Keywords: Multiple sclerosis Atorvastatin calcium Glucocorticosteroid Cytokines Expanded Disability Status Scale
a b s t r a c t This study aimed to investigate the efficacy of combined atorvastatin calcium and methylprednisolone for the treatment of multiple sclerosis relapse. Patients with multiple sclerosis (MS) at the relapse phase were randomized to receive either combined treatment of atorvastatin calcium and methylprednisolone (n = 19) or methylprednisolone alone (n = 19). Expanded Disability Status Scale (EDSS) was administered at baseline, 1 week, 2 weeks, 4 weeks, 3 months, and 6 months after treatment initiation. The number and volume of brain lesions were evaluated using magnetic resonance imaging at baseline and 6 months. The levels of IL-13, IL-35, IFN-γ, and IL-10 in the cerebrospinal fluid were examined using the enzyme-linked immunosorbent assay method. There was no significant difference in EDSS scores at 1, 2, and 4 weeks. At 3 and 6 months, the combined treatment group showed significantly lower EDSS scores than the monotherapy group (P b 0.05). The number and volume of brain lesions in the combined treatment group were significantly lower than the monotherapy group at 6 months (P b 0.001). The mean time to relapse was significantly extended in the combined treatment group than the monotherapy group (P b 0.001). At 2 and 4 weeks, the combined treatment group had significantly higher levels of IL-13, IL-35, and IL-10 in the cerebrospinal fluid than the monotherapy group (P b 0.05), but significantly lower level of IFN-γ (P b 0.001). The levels of IL-13 and IL-10 in the combined treatment group were positively correlated with EDSS scores (r = 0.632, P = 0.001; r = 0.731, P = 0.002). Combined treatment with atorvastatin calcium and methylprednisolone can improve the outcomes of MS relapse compared with glucocorticosteroid alone. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is an autoimmune disease involving the central nervous system (CNS). Focal inflammation and demyelination are the reasons for neurologic deficits in MS. Acute inflammatory lesions are initiated by activated peripheral leukocytes that enter the CNS through a breached blood–brain barrier. The specific mechanisms of MS are still not clear but are believed to be genetically associated with environmental factors. It has been shown that T cells play an important role in MS and Th1/Th2 imbalance is seen in MS relapse [1]. Glucocorticosteroid is the standard treatment for MS relapse. In recent years, statins have shown preliminary efficacy in the treatment of experimental autoimmune encephalomyelitis [2]. We used
⁎ Corresponding author at: Department of Neurology, Second Hospital of Lanzhou University, No. 82, Cuiyingmen, Chengguan District, Lanzhou 730030, China. Tel./fax: +86 931 8943704. E-mail address: [email protected] (M. Wang).
http://dx.doi.org/10.1016/j.intimp.2014.10.004 1567-5769/© 2014 Elsevier B.V. All rights reserved.
atorvastatin calcium and methylprednisolone to treat MS relapse and examined the levels of Th2 and Treg cells-associated cytokine in the cerebrospinal fluid.
2. Materials and methods 2.1. Patients Forty-five MS patients were hospitalized for disease relapse in the Department of Neurology of our hospital during March 2009 and June 2013. MS was diagnosed according to the McDonald criteria [3]. The patients were not on glucocorticosteroids and immunosuppressants in the last 3 months before hospitalization. Patients with the following conditions were excluded: other autoimmune diseases, severe infection, hypertension, diabetes mellitus, gastrointestinal ulcer, and chronic liver diseases. Informed consent was obtained from all participants. This study was approved by the Ethics Committee of The Second Hospital of Lanzhou University.
X. Li et al. / International Immunopharmacology 23 (2014) 546–549
2.2. Treatment regimens The included patients were randomized using a random number table to receive either combined treatment of atorvastatin calcium and methylprednisolone (n = 19) or methylprednisolone alone (n = 19) (Fig. 1). Seven patients were excluded according to the exclusion criteria. Methylprednisolone was intravenously administered at a dose of 1000 mg/d in both groups. The dose of methylprednisolone was half tapered every three days until 30 mg/d. Then the drug was changed to oral prednisone 35 mg/d, and 5 mg was tapered per week until the end of the treatment. Atorvastatin calcium 40 mg (Lipitor, Pfizer, US) was orally administered every night for 6 months in the combined treatment group. 2.3. Outcome measurements Expanded Disability Status Scale (EDSS) was administered at baseline, 1 week, 2 weeks, 4 weeks, 3 months, and 6 months after treatment initiation to evaluate the neurologic deficits. Brain and spinal cord lesions were examined by T2-weighted magnetic resonance imaging (MRI) to measure the lesion counts and volume. Time to relapse was recorded. Cerebrospinal fluid was obtained through lumbar puncture at baseline, 2 weeks, and 4 weeks. The levels of IL-13, IL-35, IFN-γ, and IL-10 in the cerebrospinal fluid were examined using commercial enzyme-linked immunosorbent assay kits (Boster, Wuhan, China). Blood biochemistry, liver and kidney functions, and myocardial enzymes were monitored to record possible adverse effects. 2.4. Statistical analysis Continuous data were expressed as mean ± standard deviation (SD) and fitted the normal distribution. Statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, US). Comparisons between the two groups were made using independent samples t-test. The relationship between cytokine levels and EDSS scores was examined using Spearman analysis. Categorical data were compared using χ2 test. 3. Results
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Table 1 No significant difference in the basic information was noticed between the combined treatment group and the monotherapy group (P N 0.05).
Male Age (year) Limb weakness Paresthesia Decreased visual acuity Ataxia Lesion location White matter of the hemisphere Brain stem Spinal cord Cerebellum Evoked potentiala EDSS score
Combined treatment group (n = 19)
Monotherapy group (n = 19)
10 28.16 ± 8.69 8 5 2 1
9 26.21 ± 7.63 7 5 1 1
12 5 7 1 17 6.42 ± 1.61
10 3 7 2 15 6.81 ± 1.72
a Positive of visual evoked potential, brainstem auditory evoked potential, or somatosensory evoked potential.
3.2. EDSS scores Both groups showed a trend of decreasing EDSS scores after treatment initiation. The two groups did not differ significantly in EDSS scores at 1 week, 2 weeks, and 4 weeks. However, at 3 and 6 months, the combined treatment group showed significantly lower EDSS scores than the monotherapy group (P = 0.04 and P = 0.007, respectively) (Fig. 2). 3.3. Lesion counts and volume On T2-weighted MRI images, the combined treatment group showed significantly lower lesion counts (2.67 ± 0.75 vs 3.56 ± 0.62, P b 0.001) and volume (mm3, 3.01 ± 0.63 vs 4.63 ± 0.73, P b 0.001) compared with the monotherapy group at 6 months after treatment initiation (Fig. 3). The combined treatment group also showed significantly extended time to relapse compared with the monotherapy group (month, 4.53 ± 3.19 vs 3.13 ± 1.25, P b 0.001). 3.4. Cytokine levels
3.1. Baseline information The basic information of the patients in the two groups are listed in Table 1. No significant difference was noticed in sex, age, EDSS scores, and symptoms between the two groups (P N 0.05).
Levels of IL-13, IL-35, and IL-10 in the cerebrospinal fluid showed a trend of increase in both groups. At 2 and 4 weeks, the combined treatment group had significantly higher levels of IL-13, IL-35, and IL-10 than the monotherapy group (P b 0.05). However, there was a trend of
Fig. 1. Flow chart of the inclusion process.
Fig. 2. At 3 and 6 months after treatment initiation, the combined treatment group showed significantly lower EDSS scores than the monotherapy group. * vs the monotherapy group, P b 0.05.
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Fig. 3. Brain and spinal cord lesions on T2-weighted MRI images were significantly decreased in counts and volume in the combined treatment group compared with the monotherapy group. *, P b 0.001.
decreasing IFN-γ levels in both groups. At 2 and 4 weeks, the combined treatment group had significantly lower levels of IFN-γ than the monotherapy group (P b 0.05) (Fig. 4). Spearman analysis showed that EDSS scores were positively associated with levels of IL-13 (r = 0.632, P = 0.001) and IL-10 (r = 0.731, P = 0.002) in the combined treatment group. 3.5. Adverse effects All patients showed good tolerability and compliance to the treatment. Three patients in the combined treatment group showed elevated serum levels of aminotransferases and resolved without special intervention. Treatment was not stopped in these three patients. 4. Discussion In this study, 38 patients were randomized to receive combined treatment of atorvastatin calcium and methylprednisolone or methylprednisolone alone for MS relapse. The combined treatment group showed better efficacy than the monotherapy group in terms of CNS lesion counts and volume and time to relapse. MS is an autoimmune disease induced by T cells. Naive CD4+ T cells differentiate into Th1, Th2, and Treg cells, which produce various cytokines such as IFN-γ, IL-35, IL-10, and IL-13 [4]. Specifically, Th1 cells can induce demyelination by secreting IFN-γ, TNF-α, and IL-12, while Th2 cells may impede the disease progression by producing IL-4, IL-5,
and IL-10. Therefore, Th1/Th2 cell imbalance plays an important role in the pathogenesis and prognosis of MS [5]. In addition, dysregulation of Treg cells is also involved in the development of MS [6]. Statins are a class of drugs that can lower cholesterol levels by inhibiting the enzyme HMG-CoA reductase [7]. In recent years, it was found that statins also have neurologic protective effects in MS by inhibiting inflammatory mediators and regulating immune functions [8–12]. We speculate that combining immunomodulatory drugs with different mechanisms of action can achieve additive anti-inflammatory effects. In our study, atorvastatin calcium and methylprednisolone were used in combination for MS relapse. Various inflammatory cytokines were modulated in this study and their levels were found to be associated with EDSS scores, suggesting the potential mechanisms of combined treatment of atorvastatin calcium and methylprednisolone. IL-13 and IL-10 are anti-inflammatory cytokines produced by activated Th2 cells [13]. It has been shown that IL-13 and IL-10 can inhibit the proliferation of Th1 cells, thus decreasing the cytokines produced by Th1 cells and alleviating demyelination [14,15]. IL-35 is a novel cytokine produced by Treg cells and can inhibit immune overreaction [16]. In our study, the levels of these three cytokines in cerebrospinal fluid were significantly lower in the combined treatment group than the monotherapy group at 2 and 4 weeks after treatment initiation. This might be the action mechanism for the therapeutic effects of the additional atorvastatin calcium for MS relapse. IFN-γ is a major cytokine produced by Th1 cells and can induce the release of lymphotoxin and tumor necrosis factor, leading to demyelination [17]. The levels of IFN-γ in
Fig. 4. Levels of IL-13, IL-35, IL-10, and IFN-γ in the cerebrospinal fluid of the combined treatment group and the monotherapy group at baseline, 2 weeks, and 4 weeks after treatment initiation. *, P b 0.05.
X. Li et al. / International Immunopharmacology 23 (2014) 546–549
cerebrospinal fluid were significantly lowered by the combined treatment of atorvastatin calcium and methylprednisolone in comparison of methylprednisolone alone. This suggests that atorvastatin calcium may also reduce the inflammation in MS relapse by inhibiting the production of IFN-γ. A recent study showed that atorvastatin can prevent dendrite formation during the generation of immature dendritic cells, leading to T cells with suppressive capacities and an attenuated immune response [18]. In addition, EDSS scores were positively associated with levels of IL-13 and IL-10 in the combined treatment group, suggesting that these two cytokines might server as prognostic markers for MS. In both groups, either the combination of atorvastatin calcium and methylprednisolone or methylprednisolone alone was well tolerated. No unexpected or severe adverse events occurred. In three patients in the combined treatment group, liver enzymes were elevated without clinical symptoms. The liver enzymes returned to normal levels spontaneously and the treatment was not stopped. We cannot find out whether the specific reasons for the elevated liver enzymes are associated with the combined treatment or the monotherapy. No other adverse event was noticed in both groups. Combined use of methylprednisolone as an add-on with other drugs has been found to improve the therapeutic effects than monotherapy in autoimmune encephalomyelitis [19–21]. In addition, combined treatment with atorvastatin and minocycline also showed better outcome of experimental autoimmune encephalomyelitis than minocycline alone [22]. Methylprednisolone and atorvastatin both showed their ability in improving the efficacy of other drugs in the treatment of CNS autoimmune diseases. The combination of these two drugs might provide another option for the management of CNS autoimmune diseases, including MS. Our study has limitations. Though the patients were randomized to receive atorvastatin calcium and methylprednisolone or methylprednisolone alone, they were not blind to the study design. This may affect the reliability of EDSS scores. In addition, the sample size was small and the study was performed at a single center. Finally, the follow-up time was short and we were not able to find out the long-term outcomes of the treatments. 5. Conclusion In this study, we preliminarily showed that the combined therapy of atorvastatin calcium and methylprednisolone can improve the outcomes of MS relapse compared with methylprednisolone alone. The additional treatment efficacy of atorvastatin calcium may be associated with the regulation of cerebrospinal fluid levels of IL-13, IL-35, IL-10, and IFN-γ. References [1] Zozulya AL, Clarkson BD, Ortler S, Fabry Z, Wiendl H. The role of dendritic cells in CNS autoimmunity. J Mol Med 2010;88(6):535–44.
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[2] Weber MS, Youssef S, Dunn SE, Prod'homme T, Neuhaus O, Stuve O, et al. Statins in the treatment of central nervous system autoimmune disease. J Neuroimmunol 2006;178(1–2):140–8. [3] Polman CH, Reingold SC, Banwell B, Clanet M, Cohen JA, Filippi M, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 2011;69(2):292–302. [4] Frohman EM, Racke MK, Raine CS. Multiple sclerosis—the plaque and its pathogenesis. N Engl J Med 2006;354(9):942–55. [5] Boppana S, Huang H, Ito K, Dhib-Jalbut S. Immunologic aspects of multiple sclerosis. Mt Sinai J Med 2011;78(2):207–20. [6] Venken K, Hellings N, Hensen K, Rummens JL, Medaer R, D'hooghe MB. Secondary progressive in contrast to relapsing-remitting multiple sclerosis patients show a normal CD4+CD25+ regulatory T-cell function and FOXP3 expression. J Neurosci Res 2006;83(8):1432–46. [7] Prospective Studies Collaboration, Lewington S, Whitlock G, Clarke R, Sherliker P, Emberson J, et al. Blood cholesterol and vascular mortality by age, sex, and blood pressure: a meta-analysis of individual data from 61 prospective studies with 55,000 vascular deaths. Lancet 2007;370(9602):1829–39. [8] Kamm CP, El-Koussy M, Humpert S, Findling O, von Bredow F, Burren Y, et al. Atorvastatin added to interferon beta for relapsing multiple sclerosis: a randomized controlled trial. J Neurol 2012;259(11):2401–13. [9] Ciurleo R, Bramanti P, Marino S. Role of statins in the treatment of multiple sclerosis. Pharmacol Res 2014;87:133–43. [10] Kamm CP, El-Koussy M, Humpert S, Findling O, Burren Y, Schwegler G, et al. Atorvastatin added to interferon beta for relapsing multiple sclerosis: 12-month treatment extension of the randomized multicenter SWABIMS trial. PLoS One 2014;9(1): e86663. [11] Abtahi Froushani SM, Delirezh N, Hobbenaghi R, Mosayebi G. Synergistic effects of atorvastatin and all-trans retinoic acid in ameliorating animal model of multiple sclerosis. Immunol Invest 2014;43(1):54–68. [12] Feng X, Han D, Kilaru BK, Franek BS, Niewold TB, Reder AT. Inhibition of interferonbeta responses in multiple sclerosis immune cells associated with high-dose statins. Arch Neurol 2012;69(10):1303–9. [13] de Waal Malefyt R1, Figdor CG, Huijbens R, Mohan-Peterson S, Bennett B, Culpepper J, et al. Effects of IL-13 on phenotype, cytokine production, and cytotoxic function of human monocytes. Comparison with IL-4 and modulation by IFN-gamma or IL-10. J Immunol 1993;151(11):6370–81. [14] Yang J, Jiang Z, Fitzgerald DC, Ma C, Yu S, Li H, et al. Adult neural stem cells expressing IL-10 confer potent immunomodulation and remyelination in experimental autoimmune encephalitis. J Clin Invest 2009;119(12):3678–91. [15] Oreja-Guevara C, Ramos-Cejudo J, Aroeira LS, Chamorro B, Diez-Tejedor E. TH1/TH2 cytokine profile in relapsing-remitting multiple sclerosis patients treated with Glatiramer acetate or Natalizumab. BMC Neurol 2012;12:95. [16] Collison LW, Workman CJ, Kuo TT, Boyd K, Wang Y, Vignali KM, et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 2007;450(7169): 566–9. [17] Lees JR, Golumbek PT, Sim J, Dorsey D, Russell JH. Regional CNS responses to IFNgamma determine lesion localization patterns during EAE pathogenesis. J Exp Med 2008;205(11):2633–42. [18] Leuenberger T, Pfueller CF, Luessi F, Bendix I, Paterka M, Prozorovski T, et al. Modulation of dendritic cell immunobiology via inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. PLoS One 2014;9(7):e100871. [19] Chen X, Pi R, Liu M, Ma X, Jiang Y, Liu Y, et al. Combination of methylprednisolone and minocycline synergistically improves experimental autoimmune encephalomyelitis in C57 BL/6 mice. J Neuroimmunol 2010;226(1–2):104–9. [20] Shu YQ, Yang Y, Wang YG, Dai YQ, Xiao L, Qiu W, et al. Combined therapy with methylprednisolone and ulinastatin in experimental autoimmune encephalomyelitis. Chin Med J (Engl) 2013;126(18):3439–45. [21] Chen X, Hu X, Zou Y, Pi R, Liu M, Wang T, et al. Combined treatment with minocycline and prednisone attenuates experimental autoimmune encephalomyelitis in C57 BL/6 mice. J Neuroimmunol 2009;210(1–2):22–9. [22] Luccarini I, Ballerini C, Biagioli T, Biamonte F, Bellucci A, Rosi MC, et al. Combined treatment with atorvastatin and minocycline suppresses severity of EAE. Exp Neurol 2008;211(1):214–26.
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Original article
Atorvastatin calcium in combination with methylprednisolone for the treatment of multiple sclerosis relapse Xiao-ling Li a, Zhen-chang Zhang a, Bo Zhang b, Hua Jiang a, Chun-mei Yu a, Wen-jing Zhang a, Xiang Yan a, Man-xia Wang a,⁎ a b
Department of Neurology, Second Hospital of Lanzhou University, Lanzhou 730030, China Department of Cardiology, First Hospital of Lanzhou University, Lanzhou 730030, China
a r t i c l e
i n f o
Article history: Received 12 August 2014 Received in revised form 27 September 2014 Accepted 2 October 2014 Available online 16 October 2014 Keywords: Multiple sclerosis Atorvastatin calcium Glucocorticosteroid Cytokines Expanded Disability Status Scale
a b s t r a c t This study aimed to investigate the efficacy of combined atorvastatin calcium and methylprednisolone for the treatment of multiple sclerosis relapse. Patients with multiple sclerosis (MS) at the relapse phase were randomized to receive either combined treatment of atorvastatin calcium and methylprednisolone (n = 19) or methylprednisolone alone (n = 19). Expanded Disability Status Scale (EDSS) was administered at baseline, 1 week, 2 weeks, 4 weeks, 3 months, and 6 months after treatment initiation. The number and volume of brain lesions were evaluated using magnetic resonance imaging at baseline and 6 months. The levels of IL-13, IL-35, IFN-γ, and IL-10 in the cerebrospinal fluid were examined using the enzyme-linked immunosorbent assay method. There was no significant difference in EDSS scores at 1, 2, and 4 weeks. At 3 and 6 months, the combined treatment group showed significantly lower EDSS scores than the monotherapy group (P b 0.05). The number and volume of brain lesions in the combined treatment group were significantly lower than the monotherapy group at 6 months (P b 0.001). The mean time to relapse was significantly extended in the combined treatment group than the monotherapy group (P b 0.001). At 2 and 4 weeks, the combined treatment group had significantly higher levels of IL-13, IL-35, and IL-10 in the cerebrospinal fluid than the monotherapy group (P b 0.05), but significantly lower level of IFN-γ (P b 0.001). The levels of IL-13 and IL-10 in the combined treatment group were positively correlated with EDSS scores (r = 0.632, P = 0.001; r = 0.731, P = 0.002). Combined treatment with atorvastatin calcium and methylprednisolone can improve the outcomes of MS relapse compared with glucocorticosteroid alone. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is an autoimmune disease involving the central nervous system (CNS). Focal inflammation and demyelination are the reasons for neurologic deficits in MS. Acute inflammatory lesions are initiated by activated peripheral leukocytes that enter the CNS through a breached blood–brain barrier. The specific mechanisms of MS are still not clear but are believed to be genetically associated with environmental factors. It has been shown that T cells play an important role in MS and Th1/Th2 imbalance is seen in MS relapse [1]. Glucocorticosteroid is the standard treatment for MS relapse. In recent years, statins have shown preliminary efficacy in the treatment of experimental autoimmune encephalomyelitis [2]. We used
⁎ Corresponding author at: Department of Neurology, Second Hospital of Lanzhou University, No. 82, Cuiyingmen, Chengguan District, Lanzhou 730030, China. Tel./fax: +86 931 8943704. E-mail address: [email protected] (M. Wang).
http://dx.doi.org/10.1016/j.intimp.2014.10.004 1567-5769/© 2014 Elsevier B.V. All rights reserved.
atorvastatin calcium and methylprednisolone to treat MS relapse and examined the levels of Th2 and Treg cells-associated cytokine in the cerebrospinal fluid.
2. Materials and methods 2.1. Patients Forty-five MS patients were hospitalized for disease relapse in the Department of Neurology of our hospital during March 2009 and June 2013. MS was diagnosed according to the McDonald criteria [3]. The patients were not on glucocorticosteroids and immunosuppressants in the last 3 months before hospitalization. Patients with the following conditions were excluded: other autoimmune diseases, severe infection, hypertension, diabetes mellitus, gastrointestinal ulcer, and chronic liver diseases. Informed consent was obtained from all participants. This study was approved by the Ethics Committee of The Second Hospital of Lanzhou University.
X. Li et al. / International Immunopharmacology 23 (2014) 546–549
2.2. Treatment regimens The included patients were randomized using a random number table to receive either combined treatment of atorvastatin calcium and methylprednisolone (n = 19) or methylprednisolone alone (n = 19) (Fig. 1). Seven patients were excluded according to the exclusion criteria. Methylprednisolone was intravenously administered at a dose of 1000 mg/d in both groups. The dose of methylprednisolone was half tapered every three days until 30 mg/d. Then the drug was changed to oral prednisone 35 mg/d, and 5 mg was tapered per week until the end of the treatment. Atorvastatin calcium 40 mg (Lipitor, Pfizer, US) was orally administered every night for 6 months in the combined treatment group. 2.3. Outcome measurements Expanded Disability Status Scale (EDSS) was administered at baseline, 1 week, 2 weeks, 4 weeks, 3 months, and 6 months after treatment initiation to evaluate the neurologic deficits. Brain and spinal cord lesions were examined by T2-weighted magnetic resonance imaging (MRI) to measure the lesion counts and volume. Time to relapse was recorded. Cerebrospinal fluid was obtained through lumbar puncture at baseline, 2 weeks, and 4 weeks. The levels of IL-13, IL-35, IFN-γ, and IL-10 in the cerebrospinal fluid were examined using commercial enzyme-linked immunosorbent assay kits (Boster, Wuhan, China). Blood biochemistry, liver and kidney functions, and myocardial enzymes were monitored to record possible adverse effects. 2.4. Statistical analysis Continuous data were expressed as mean ± standard deviation (SD) and fitted the normal distribution. Statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, US). Comparisons between the two groups were made using independent samples t-test. The relationship between cytokine levels and EDSS scores was examined using Spearman analysis. Categorical data were compared using χ2 test. 3. Results
547
Table 1 No significant difference in the basic information was noticed between the combined treatment group and the monotherapy group (P N 0.05).
Male Age (year) Limb weakness Paresthesia Decreased visual acuity Ataxia Lesion location White matter of the hemisphere Brain stem Spinal cord Cerebellum Evoked potentiala EDSS score
Combined treatment group (n = 19)
Monotherapy group (n = 19)
10 28.16 ± 8.69 8 5 2 1
9 26.21 ± 7.63 7 5 1 1
12 5 7 1 17 6.42 ± 1.61
10 3 7 2 15 6.81 ± 1.72
a Positive of visual evoked potential, brainstem auditory evoked potential, or somatosensory evoked potential.
3.2. EDSS scores Both groups showed a trend of decreasing EDSS scores after treatment initiation. The two groups did not differ significantly in EDSS scores at 1 week, 2 weeks, and 4 weeks. However, at 3 and 6 months, the combined treatment group showed significantly lower EDSS scores than the monotherapy group (P = 0.04 and P = 0.007, respectively) (Fig. 2). 3.3. Lesion counts and volume On T2-weighted MRI images, the combined treatment group showed significantly lower lesion counts (2.67 ± 0.75 vs 3.56 ± 0.62, P b 0.001) and volume (mm3, 3.01 ± 0.63 vs 4.63 ± 0.73, P b 0.001) compared with the monotherapy group at 6 months after treatment initiation (Fig. 3). The combined treatment group also showed significantly extended time to relapse compared with the monotherapy group (month, 4.53 ± 3.19 vs 3.13 ± 1.25, P b 0.001). 3.4. Cytokine levels
3.1. Baseline information The basic information of the patients in the two groups are listed in Table 1. No significant difference was noticed in sex, age, EDSS scores, and symptoms between the two groups (P N 0.05).
Levels of IL-13, IL-35, and IL-10 in the cerebrospinal fluid showed a trend of increase in both groups. At 2 and 4 weeks, the combined treatment group had significantly higher levels of IL-13, IL-35, and IL-10 than the monotherapy group (P b 0.05). However, there was a trend of
Fig. 1. Flow chart of the inclusion process.
Fig. 2. At 3 and 6 months after treatment initiation, the combined treatment group showed significantly lower EDSS scores than the monotherapy group. * vs the monotherapy group, P b 0.05.
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X. Li et al. / International Immunopharmacology 23 (2014) 546–549
Fig. 3. Brain and spinal cord lesions on T2-weighted MRI images were significantly decreased in counts and volume in the combined treatment group compared with the monotherapy group. *, P b 0.001.
decreasing IFN-γ levels in both groups. At 2 and 4 weeks, the combined treatment group had significantly lower levels of IFN-γ than the monotherapy group (P b 0.05) (Fig. 4). Spearman analysis showed that EDSS scores were positively associated with levels of IL-13 (r = 0.632, P = 0.001) and IL-10 (r = 0.731, P = 0.002) in the combined treatment group. 3.5. Adverse effects All patients showed good tolerability and compliance to the treatment. Three patients in the combined treatment group showed elevated serum levels of aminotransferases and resolved without special intervention. Treatment was not stopped in these three patients. 4. Discussion In this study, 38 patients were randomized to receive combined treatment of atorvastatin calcium and methylprednisolone or methylprednisolone alone for MS relapse. The combined treatment group showed better efficacy than the monotherapy group in terms of CNS lesion counts and volume and time to relapse. MS is an autoimmune disease induced by T cells. Naive CD4+ T cells differentiate into Th1, Th2, and Treg cells, which produce various cytokines such as IFN-γ, IL-35, IL-10, and IL-13 [4]. Specifically, Th1 cells can induce demyelination by secreting IFN-γ, TNF-α, and IL-12, while Th2 cells may impede the disease progression by producing IL-4, IL-5,
and IL-10. Therefore, Th1/Th2 cell imbalance plays an important role in the pathogenesis and prognosis of MS [5]. In addition, dysregulation of Treg cells is also involved in the development of MS [6]. Statins are a class of drugs that can lower cholesterol levels by inhibiting the enzyme HMG-CoA reductase [7]. In recent years, it was found that statins also have neurologic protective effects in MS by inhibiting inflammatory mediators and regulating immune functions [8–12]. We speculate that combining immunomodulatory drugs with different mechanisms of action can achieve additive anti-inflammatory effects. In our study, atorvastatin calcium and methylprednisolone were used in combination for MS relapse. Various inflammatory cytokines were modulated in this study and their levels were found to be associated with EDSS scores, suggesting the potential mechanisms of combined treatment of atorvastatin calcium and methylprednisolone. IL-13 and IL-10 are anti-inflammatory cytokines produced by activated Th2 cells [13]. It has been shown that IL-13 and IL-10 can inhibit the proliferation of Th1 cells, thus decreasing the cytokines produced by Th1 cells and alleviating demyelination [14,15]. IL-35 is a novel cytokine produced by Treg cells and can inhibit immune overreaction [16]. In our study, the levels of these three cytokines in cerebrospinal fluid were significantly lower in the combined treatment group than the monotherapy group at 2 and 4 weeks after treatment initiation. This might be the action mechanism for the therapeutic effects of the additional atorvastatin calcium for MS relapse. IFN-γ is a major cytokine produced by Th1 cells and can induce the release of lymphotoxin and tumor necrosis factor, leading to demyelination [17]. The levels of IFN-γ in
Fig. 4. Levels of IL-13, IL-35, IL-10, and IFN-γ in the cerebrospinal fluid of the combined treatment group and the monotherapy group at baseline, 2 weeks, and 4 weeks after treatment initiation. *, P b 0.05.
X. Li et al. / International Immunopharmacology 23 (2014) 546–549
cerebrospinal fluid were significantly lowered by the combined treatment of atorvastatin calcium and methylprednisolone in comparison of methylprednisolone alone. This suggests that atorvastatin calcium may also reduce the inflammation in MS relapse by inhibiting the production of IFN-γ. A recent study showed that atorvastatin can prevent dendrite formation during the generation of immature dendritic cells, leading to T cells with suppressive capacities and an attenuated immune response [18]. In addition, EDSS scores were positively associated with levels of IL-13 and IL-10 in the combined treatment group, suggesting that these two cytokines might server as prognostic markers for MS. In both groups, either the combination of atorvastatin calcium and methylprednisolone or methylprednisolone alone was well tolerated. No unexpected or severe adverse events occurred. In three patients in the combined treatment group, liver enzymes were elevated without clinical symptoms. The liver enzymes returned to normal levels spontaneously and the treatment was not stopped. We cannot find out whether the specific reasons for the elevated liver enzymes are associated with the combined treatment or the monotherapy. No other adverse event was noticed in both groups. Combined use of methylprednisolone as an add-on with other drugs has been found to improve the therapeutic effects than monotherapy in autoimmune encephalomyelitis [19–21]. In addition, combined treatment with atorvastatin and minocycline also showed better outcome of experimental autoimmune encephalomyelitis than minocycline alone [22]. Methylprednisolone and atorvastatin both showed their ability in improving the efficacy of other drugs in the treatment of CNS autoimmune diseases. The combination of these two drugs might provide another option for the management of CNS autoimmune diseases, including MS. Our study has limitations. Though the patients were randomized to receive atorvastatin calcium and methylprednisolone or methylprednisolone alone, they were not blind to the study design. This may affect the reliability of EDSS scores. In addition, the sample size was small and the study was performed at a single center. Finally, the follow-up time was short and we were not able to find out the long-term outcomes of the treatments. 5. Conclusion In this study, we preliminarily showed that the combined therapy of atorvastatin calcium and methylprednisolone can improve the outcomes of MS relapse compared with methylprednisolone alone. The additional treatment efficacy of atorvastatin calcium may be associated with the regulation of cerebrospinal fluid levels of IL-13, IL-35, IL-10, and IFN-γ. References [1] Zozulya AL, Clarkson BD, Ortler S, Fabry Z, Wiendl H. The role of dendritic cells in CNS autoimmunity. J Mol Med 2010;88(6):535–44.
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[2] Weber MS, Youssef S, Dunn SE, Prod'homme T, Neuhaus O, Stuve O, et al. Statins in the treatment of central nervous system autoimmune disease. J Neuroimmunol 2006;178(1–2):140–8. [3] Polman CH, Reingold SC, Banwell B, Clanet M, Cohen JA, Filippi M, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 2011;69(2):292–302. [4] Frohman EM, Racke MK, Raine CS. Multiple sclerosis—the plaque and its pathogenesis. N Engl J Med 2006;354(9):942–55. [5] Boppana S, Huang H, Ito K, Dhib-Jalbut S. Immunologic aspects of multiple sclerosis. Mt Sinai J Med 2011;78(2):207–20. [6] Venken K, Hellings N, Hensen K, Rummens JL, Medaer R, D'hooghe MB. Secondary progressive in contrast to relapsing-remitting multiple sclerosis patients show a normal CD4+CD25+ regulatory T-cell function and FOXP3 expression. J Neurosci Res 2006;83(8):1432–46. [7] Prospective Studies Collaboration, Lewington S, Whitlock G, Clarke R, Sherliker P, Emberson J, et al. Blood cholesterol and vascular mortality by age, sex, and blood pressure: a meta-analysis of individual data from 61 prospective studies with 55,000 vascular deaths. Lancet 2007;370(9602):1829–39. [8] Kamm CP, El-Koussy M, Humpert S, Findling O, von Bredow F, Burren Y, et al. Atorvastatin added to interferon beta for relapsing multiple sclerosis: a randomized controlled trial. J Neurol 2012;259(11):2401–13. [9] Ciurleo R, Bramanti P, Marino S. Role of statins in the treatment of multiple sclerosis. Pharmacol Res 2014;87:133–43. [10] Kamm CP, El-Koussy M, Humpert S, Findling O, Burren Y, Schwegler G, et al. Atorvastatin added to interferon beta for relapsing multiple sclerosis: 12-month treatment extension of the randomized multicenter SWABIMS trial. PLoS One 2014;9(1): e86663. [11] Abtahi Froushani SM, Delirezh N, Hobbenaghi R, Mosayebi G. Synergistic effects of atorvastatin and all-trans retinoic acid in ameliorating animal model of multiple sclerosis. Immunol Invest 2014;43(1):54–68. [12] Feng X, Han D, Kilaru BK, Franek BS, Niewold TB, Reder AT. Inhibition of interferonbeta responses in multiple sclerosis immune cells associated with high-dose statins. Arch Neurol 2012;69(10):1303–9. [13] de Waal Malefyt R1, Figdor CG, Huijbens R, Mohan-Peterson S, Bennett B, Culpepper J, et al. Effects of IL-13 on phenotype, cytokine production, and cytotoxic function of human monocytes. Comparison with IL-4 and modulation by IFN-gamma or IL-10. J Immunol 1993;151(11):6370–81. [14] Yang J, Jiang Z, Fitzgerald DC, Ma C, Yu S, Li H, et al. Adult neural stem cells expressing IL-10 confer potent immunomodulation and remyelination in experimental autoimmune encephalitis. J Clin Invest 2009;119(12):3678–91. [15] Oreja-Guevara C, Ramos-Cejudo J, Aroeira LS, Chamorro B, Diez-Tejedor E. TH1/TH2 cytokine profile in relapsing-remitting multiple sclerosis patients treated with Glatiramer acetate or Natalizumab. BMC Neurol 2012;12:95. [16] Collison LW, Workman CJ, Kuo TT, Boyd K, Wang Y, Vignali KM, et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 2007;450(7169): 566–9. [17] Lees JR, Golumbek PT, Sim J, Dorsey D, Russell JH. Regional CNS responses to IFNgamma determine lesion localization patterns during EAE pathogenesis. J Exp Med 2008;205(11):2633–42. [18] Leuenberger T, Pfueller CF, Luessi F, Bendix I, Paterka M, Prozorovski T, et al. Modulation of dendritic cell immunobiology via inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. PLoS One 2014;9(7):e100871. [19] Chen X, Pi R, Liu M, Ma X, Jiang Y, Liu Y, et al. Combination of methylprednisolone and minocycline synergistically improves experimental autoimmune encephalomyelitis in C57 BL/6 mice. J Neuroimmunol 2010;226(1–2):104–9. [20] Shu YQ, Yang Y, Wang YG, Dai YQ, Xiao L, Qiu W, et al. Combined therapy with methylprednisolone and ulinastatin in experimental autoimmune encephalomyelitis. Chin Med J (Engl) 2013;126(18):3439–45. [21] Chen X, Hu X, Zou Y, Pi R, Liu M, Wang T, et al. Combined treatment with minocycline and prednisone attenuates experimental autoimmune encephalomyelitis in C57 BL/6 mice. J Neuroimmunol 2009;210(1–2):22–9. [22] Luccarini I, Ballerini C, Biagioli T, Biamonte F, Bellucci A, Rosi MC, et al. Combined treatment with atorvastatin and minocycline suppresses severity of EAE. Exp Neurol 2008;211(1):214–26.
