The efficacy of Mycophenolate mofetil for the treatment of Chinese Takayasu’s arteritis

To investigate the therapeutic effect of mycophenolate mofetil(MMF) on Chinese Takayasu’s arteritis(TAK) patients. Thirty consecutive TAK outpatients were prospectively enrolled during 2013 to 2015. MMF combined with glucocorticoid was the primary treatment regimen. If clinical stable disease could not be reached, another traditional immunosuppressive agent could be added. All patients were evaluated and followed up every 3 months and vascular image studies by Doppler ultrasonography were repeated every 6 months. The effectiveness of MMF was defined as:(1) ESR < 20 mm/hr;(2) CRP < 10 mg/L or hs-CRP<3 mg/L;(3) stable or improved in vascular image studies;(4) clinical assessment is stable, improved or in remission;(5) the dosage of glucocorticoid could be tapered to less than 15 mg/day. ESR < 40 mm/hr, CRP < 20 mg/L or hs-CRP < 6 mg/L, but meet the other three criteria is defined as partial effectiveness. MMF alone combined with corticosteroid was effective in 12(40.0%) patients. When MMF combined with methotrexate less than 15 mg/week, the effective rate was 30.0%(9/30), including partial effective in 3 patients. When MMF combined with azathioprine 100–150 mg/day, the effective rate was 10.0%(3/30), including partial effective in 1 patient. Four patients withdrew due to side effects. Two patients failed to show response. The overall effective rate of therapy including MMF in treating TAK is 80%.


Results
Demographic data, baseline data, and previous medication summary. Thirty TAK patients were treated with MMF in this study. The demographic and baseline data were summarized in Table 1. 90.0% (27/30) patients were female, and the median age at onset was 24.5 (19.8, 32.0) years old. The median disease duration at diagnosis was 11.5 (3.8, 27.0) months, and the median follow-up time was 17.0 (11.0, 28.0) months. Prior to commencing MMF usage, the median disease duration was 12.0 (7.5, 36.0) months (Table 1).
Malaise (63.3%) and headache (53.3%) were the common complaints. Vascular bruit (83.3%) was the most common findings in physical examination in this group of patients, followed by asymmetric blood pressure (80.0%), pulsation (66.7%) and hypertension (56.7%). Elevated C-reaction protein (CRP) and/or high sensitive CRP (hs-CRP) (86.7%) were the most common abnormal laboratory tests, and elevated erythrocyte sediment rate (ESR) was found in 76.7% of patients. Mild anemia was found in 30.0% patients (10/30), in whom the level of hemoglobin was less than 110 g/L ( Table 1).
The angiographic classification 13 at the baseline was shown in Table 1. Twelve patients (40.0%) were classified as type I, seven patients (23.3%) were type IIa, six patients (20.0%) were type V, three patients (10.0%) were type IIb, and two patients (6.7%) were type IV.
Seventeen patients (17/30, 56.7%) received MMF as the first immunosuppressive agent, while the other thirteen patients received MMF as an alternative or additive immunosuppressive drug (ISD) due to uncontrolled disease activity. All patients received glucocorticoids (GCs) at the same time, and the dosage of steroids were tapered during the study period ( Table 1).
The mean dosage of GCs when MMF was initiated was 40.0 (14.4, 40.0) mg in 24 patients who showed effectiveness during follow-up, and the mean dosage of GCs on last follow-up in these patients was 10.0 (7.5, 10.0) mg. The duration for dosage of GCs less than 15 mg was 12.0 (9.0, 21.8) months, and the duration of GCs less than 10 mg was 9.0 (3.0, 16.5) months. Three patients among them (3/24, 12.5%) stopped GCs at the last follow-up (Table 1).
Response towards the treatment of MMF. In this study, 4 patients withdrew MMF due to adverse events. One patient developed skin rash 2 weeks after MMF administration, 2 patients complained of severe gastrointestinal discomfort, and HBV flare occurred in 1 patient. Fourteen patients received MMF and steroid combined with other immunosuppressive agent when their diseases was still active. The follow-up time after initiation of MMF in MMF combined with GCs group (median 24.5 months) was longer than that in patients with MMF combined with another immunosuppressive agent treatment group (median 11.0 months) ( Table 1). Excluding 4 patients withdrawal due to side effect of MMF, 26 patients were followed up for more than 6 months. 40.0% (12/30) patients in the MMF combined with GCs group could reach and maintain stable disease. With adding MTX less than 15 mg/week or AZA 100-150 mg/day, the dosage of steroid could be tapered to less than 15 mg/day in 12 patients (40.0%). Two patients failed to reach stable disease after being treated with combination of several ISDs including MMF (Table 2). Thus the effective rate of MMF alone, or combined with methotrexate or azathioprine, with low-dosage GCs in most patients, was effective in the treatment of TAK, and the overall effective rate was 80.0%.

Comparisons of clinical manifestations, laboratory findings, and angiographic presentations.
We compared the clinical features and angiographic types between patients who responded to MMF combined with GCs (n = 12), and MMF combined with other ISD and GCs (n = 12) ( Table 1). There was no significant difference in clinical manifestations between these groups. But the disease duration at diagnosis (5.0 vs 18.5 months, p = 0.014) and prior to initiation of MMF (10.0 vs 18.5 months, p = 0.024) were shorter in MMF combined with GCs group (n = 12) than in MMF combined with other ISD and GCs group (n = 12). And the follow-up time after initiating MMF was longer in MMF combined with GCs group (27.5 vs 11.0 months, p = 0.005).
There was no significant difference in the dosage of GCs used when MMF treatment was initiated and on last follow-up between MMF combined with GCs group (n = 12) and MMF combined with other ISD and GCs group (n = 12). But the duration for the dosage of GCs tapered to less than 15 mg (21.5 vs 10.0 months, p = 0.024) and 10 mg (14.0 vs 6.0 months, p = 0.033) was longer in the former group.

Comparisons of involved vessels distribution.
For the vessels involved in these 30 TAK patients, carotid artery and subclavian artery were involved in 22 patients (73.3%). The distribution of vessels involved between the MMF combined with GCs group (n = 12) and the combined treatment including MMF group (n = 12) was not significantly different (Table 3).

Discussion
TAK is a chronic progressive large vessel vasculitis 3 . Autopsy in Japanese TAK patients disclosed various lesions, including acute exudative inflammation, chronic non-specific productive inflammation, and granulomatous inflammation in the media and adventitia through the casa vasorum. The new active lesions were often observed near the old fibrotic ones, which suggested that TAK might be a progressive disease 3 .The disease activity assessment and treatment are still challenging. Due to its relatively low prevalence, there has been no controlled trial in TAK. Diverse immunosuppressive drugs had been examined in small sample-size case series or case reports, including CTX, MTX, AZA, cyclosporine and leflunomide 4,5,[7][8][9] . MMF has been shown to be effective as an alternative steroid-sparing agent in three case series, including three, ten and twenty-one TAK patients, respectively [10][11][12] .
As an inosine monophosphate dehydrogenase inhibitor, MMF could interfere DNA synthesis by inhibiting guanine nucleotide synthesis. Since lymphocytes depend primarily on de novo purine synthesis while neutrophils utilize salvage pathway, MMF inhibits proliferation of lymphocytes specifically 14     Continued demonstrated the role of MMF in preventing acute transplant rejection 15 . And in experimental models, MMF prevents arterial smooth muscle-cell proliferation and proliferative arteriopathy, which were common mechanisms involved in chronic transplant rejection 16,17 . Based on these mechanisms, MMF has been suggested to inhibit the vasculitis mediated by lymphocytes, such as TAK. In fact, MMF had been demonstrated to be effective in various systemic vasculitides, including ANCA-associated vasculitis 18 and lupus nephritis 19 .
In this study, we have demonstrated that MMF may be effective in controlling disease activity and vessel damage progression, either combined with low-dosage GCs or combined with MTX or AZA, with the total effective rate up to 80.0%. But in the combination of immunosuppressive agents (the combination regimen) and MMF group, the follow-up time after administration of MMF (median 11.0 months) is shorter than in MMF plus GC group (median 27.5 months) (p = 0.005). And four patients in the combination regimen group is partial effective, demonstrating no progression of vascular lesions but with elevated level of ESR/CRP, which was lower than two times of the upper normal limit.
When the age at disease onset, history of medication usage, clinical symptoms, physical examination findings, laboratory tests, angiographic classification, and the distribution of involved vessels were compared between patients based on the efficacy and regimen used, no significant difference could be found. It suggests that MMF could be indicated in any TAK patients with active disease. In this study, in patients of the MMF plus GCs group (n = 12), the disease duration at diagnosis (median 5.0 months) and prior to commencing of MMF (median 10.0 months) were significantly shorter than in the combined regimen group (n = 12).The immunosuppressive effect of MMF in autoimmune disease was speculated by inhibiting lymphocyte-mediated immune process. Therefore, we may speculated that early administration of MMF in TAK patients may be beneficial in slowing down or preventing the disease progression.
For the side effects of MMF, Shinjo et al. 11 had reported headache as the only adverse events, and Goel et al. 10 had reported skin rash and sepsis. In this study, two patients experienced unbearable gastrointestinal discomfort, one had pruritic rashes, and one patient suffered a flare of previous HBV infection 6 weeks after administration of MMF. Discontinuation of MMF and addition of entecavir suppressed the replication of HBV, and later addition of MTX and AZA did not induce replication of HBV within 5 months follow-up. No cytopenia, liver enzyme elevation, or renal dysfunction was observed in our patients.
Doppler ultrasonic imaging was repeated to measure and compare the thickness of the vessel walls and lumen diameter of vessels by one technician specialized in vascular ultrasonic examinations. As there is no widely accepted image modality to evaluate the disease activity of angiographic lesions, various image modalities have been used in different clinical trials. Magnetic resonance angiography (MRA) 20 and positron emission tomography (PET) 21,22 are reported to be sensitive in evaluating vessel inflammatory changes, but their role in daily   practice and disease activity follow-up has not been well established. The high cost of MRA and PER-CT limits its usage in Chinese TAK patients.
The major limitation of this study is the relatively small sample size, though this is the largest cohort reported in the literature according to our knowledge. Another limitation is the relatively short follow-up time for a chronic disease as TAK. In this study, the median follow-up time was 17.0 months. As TAK is a chronic disease, so 17 months follow-up duration might not be long enough to provide solid evidence to assess disease progression. Perhaps further follow-up of these patients is needed to confirm the effectiveness of these regimens in delaying or halted the progress of the disease. Therefore, large, long-term follow up studies are needed to verify the effectiveness of MMF in TAK treatment.
In summary, this study has demonstrated that, MMF, and MMF combined with MTX or AZA, when combined with daily low dose GCs, are tolerable and effective in 80.0% of TAK patients. Neither angiographic progression shown on Doppler ultrasonography nor toxicity of vital organ/system has been observed during follow-up. Therefore, we may conclude that MMF may be an effective alternative for disease control and tapering of steroid in the treatment of TAK. It offers a good choice for active TAK patients.

Patients and Methods
Patients. Thirty consecutive TAK patients were treated with oral MMF, and were followed-up at the outpatient Vasculitis Clinic, Department of Rheumatology of Peking Union Medical College Hospital (PUMCH), a Chinese nationwide referral center during January 2013 to September 2015. All patients fulfilled the classification criteria of modified 1990 American College of Rheumatology (ACR) criteria 23 . Patients who had one of these criteria were excluded: (1) intolerant or failed to response to MMF in the past history of treatment; (2) thiopurine methyl transferase (TPMT) gene test showed mutations; (3) un-controlled diabetes or hypertension before entry; (4) active digestive tract bleeding 3 months before entry; (5) severe liver and kidney dysfunction, and damage defined as the alanine aminotransferase (ALT) or aspartate aminotransferase (AST) level were 3 times above the upper limits of the normal range and the serum creatinine level was 1.5 times above the upper limits; (6) patients with active infection, including bacteria, virus, fungi and mycobacteria. All patients were in active disease based on the National Institutes of Health (NIH) criteria 2 , defined as new-onset or worsening in at least   two of the following: (1) systemic features, such as fever, musculoskeletal involvement (excluded other causes); (2) manifestations of ischemia or inflammation in vessels, such as claudication, pulse deficit, bruits, vascular pain (carotidynia), asymmetric blood pressure in upper and/or lower limbs; (3) elevated ESR and/or CRP protein levels, excluding the existence of active infection; (4) typical angiographic features. These patients received oral MMF, 1.5 to 2 gram per day, and were followed up for a minimum period of 6 months. All patients were evaluated and followed up every 3 months and vascular image studies were repeated every 6 months. Clinical manifestations of these patients were reviewed, including symptoms, physical findings, laboratory findings, and vascular involvement. Their medication records and response to MMF were categorially summarized. The effectiveness of MMF was defined as: (1) ESR < 20 mm/hr after treatment; (2) CRP < 10 mg/L or hs-CRP < 3 mg/L after treatment; (3) stable or improved in vascular image studies; (4) clinical assessment of disease activity is stable, improved or in remission after treatment; (5) the dosage of corticosteroid could be tapered to less than 15 mg/day (equivalent dosage of prednisone). ESR < 40 mm/hr, CRP < 20 mg/L or hs-CRP < 6 mg/L, but fulfilled for the other three criteria was defined as partial effective. The primary end-point of this study is the percentage of patients with full and partial effectiveness.
Methods. These patients were prospectively enrolled and followed-up every 3 months. The demographic data, disease duration, clinical symptoms, physical findings, disease activity status, laboratory findings, angiographic presentations, and treatment modalities, including medication regimens and adverse drug reactions, were collected at baseline and at each follow-up visit. During follow up, we repeated the examination of involved vessels by vascular Doppler ultrasonic imaging, measuring and comparing the thickness of the vessel walls and lumen diameter of vessels.
The study protocol was approved by Institutional Review Board of PUMCH, and all methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained for each participant. This study was registered in Chinese Clinical Trial Registry (http://www.chictr.org.cn) on Jul 25, 2016. The registration number is ChiCTR-OPC-16008907.

Statistical analysis.
Due to the small sample size of this study, the variables were not distributed in a normal pattern. We described the numerical variables as median (quartiles), and the categorical variables as number (percentage). Comparisons between groups were made using Mann-Whitney U test for numerical data, and Chi-square tests for categorical data. Fisher's exact tests were performed when the expected frequencies were less than 5. A two-sided P-value less than 0.05 was considered to be statistically significant. Analysis was performed with the SPSS software (version 19.0).