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Rituximab (RTX) is an extensively used off-label drug for multiple sclerosis (MS).
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Repeated low-dose RTX therapy is effective and safe for RRMS.
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RTX at lower dose may be a promising option for MS with an ideal risk/benefit ratio.
Abstract
Background
Rituximab (RTX) is an extensively used off-label drug for multiple sclerosis (MS), whereas the induction and maintenance regimens vary widely among studies. Few data are available on efficacy and safety of repeated low-dose RTX therapy in MS patients.
Objective
This study aimed to evaluate the efficacy and safety of repeated low-dose RTX therapy for relapsing-remitting MS (RRMS), the most common form of MS affecting approximately 85% of patients.
Methods
Nine RRMS patients were enrolled and the medical records were retrospectively reviewed. RTX at 100 mg per week for three consecutive weeks was used as induction therapy. Maintenance therapy was reinfusions of RTX at 100 mg every 6 months during the first year, followed by 100 mg every 6 to 12 months. Main outcome measures included annualized relapse rate (ARR), expanded disability status scale (EDSS) score, and T2 lesion burden on MRI for evaluating the efficacy of low-dose RTX regimen. Meanwhile, adverse events (AEs) were recorded to assess the safety of repeated RTX infusions.
Results
All patients were females with an average onset age of 25.4 ± 6.7 years. The median disease duration before the first RTX infusion was 56 (range, 3–108) months and the median follow-up period was 30 (range, 15–40) months. No relapses were recorded in all patients after RTX therapy. Repeated low-dose RTX therapy resulted in a dramatic reduction of median ARR (pre-RTX vs post-RTX, 1.1 vs 0, p = 0.012), median EDSS score (2.0 vs 0, p = 0.007), and the number of T2 lesions on MRI (35.6 ± 18.0 vs 29.4 ± 18.1, p = 0.001). A total of 35 episodes of AEs occurred during repeated low-dose RTX therapy, and all of them were mild and transient.
Conclusion
Repeated low-dose RTX therapy is cost-effective for RRMS patients and shows a good safety profile. It may be a promising option for those having no access or poor response to first-line disease-modified drugs (DMDs), particularly in low- or middle-income countries.
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) that often results in significant neurologic deficits over time. According to the clinical course of MS, it is classified into four major phenotypes, including relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS) (
). Among them, approximately 85% initially present with RRMS, characterized by cyclic clinical deterioration followed by complete or incomplete remission. Over the past three decades, the MS treatment landscape has rapidly evolved with the development of disease-modifying drugs (DMDs), and the majority of them have been approved for the treatment of RRMS. Early DMDs use in RRMS patients can effectively reduce annualized relapse rate (ARR) and delay progression of disability (
Practice guideline recommendations summary: disease-modifying therapies for adults with multiple sclerosis: report of the Guideline Development, Dissemination, and Implementation Subcommittee of the American Academy of Neurology.
MS has been considered a T cell-dominated disease for a long time, but current evidence suggests that B cells and humoral immunity also play crucial roles in this disease. Oligoclonal bands in cerebrospinal fluid (CSF) of MS patients provide evidence of intrathecal antibody production (
). Most notably, the excellent therapeutic effects of anti-CD20 monoclonal antibodies in MS strengthen the crucial role of B cells in the pathogenesis of this disease (
). FDA has approved two CD20-depleting monoclonal antibodies, Ocrelizumab and Ofatumumab, for treatment of MS based on the convincing findings that both significantly reduced disease activity and controlled the progression of disability and demyelinating lesions (
). As another anti-CD20 monoclonal antibody originally designed for treating non-Hodgkin's lymphoma, rituximab (RTX) also showed beneficial therapeutic effects in RRMS initially in a double-blind, placebo-controlled study (
). In another prior randomized controlled study, RTX therapy dramatically delayed the time to confirmed disease progression in PPMS patients aged < 51 years, particularly those with gadolinium-enhancing lesions as compared with placebo (
Safety and clinical outcomes of rituximab treatment in patients with multiple sclerosis and neuromyelitis optica: experience from a national online registry (GRAID).
). Unfortunately, a standard protocol of RTX infusions and follow-up regimens for MS is still lacking to date. In most prior studies, patients received intravenous induction infusion of 0.5 g to 1 g RTX twice 2 weeks apart or 375 mg/m2 once weekly for 4 weeks, and maintenance infusion was given every 6–9 months based on clinical functional status and patient's preference. Besides the expensive costs of long-term RTX therapy, prescriptions should be given with caution by clinicians due to the potentially serious adverse effects including death. Therefore, low-dose strategies might be more cost-effective in the context of ensuring efficacy and safety, particularly in low- or middle-income countries. This view is reinforced by several recent studies from other neuroimmune diseases such as neuromyelitis optica spectrum disorders (NMOSD) (
), in which reduced-dose RTX showed good efficacy and safety profile. In this study, we retrospectively reviewed case series receiving repeated low-dose RTX therapy in our MS center to evaluate the efficacy and safety of this modified treatment regimen in RRMS patients.
2. Methods
2.1 Subjects and study design
The MS database of Tangdu Hospital were screened and 9 eligible RRMS patients admitted between October 2016 and August 2020 were included in this retrospective observational study. A definite MS diagnosis was determined according to the revised 2010 or 2017 McDonald criteria (
). The medical records were reviewed to collect demographic and clinical data including onset age, sex, disease duration, ARR and Expanded Disability Status Scale (EDSS) score, lesion burden on T2-weighted magnetic resonance imaging (MRI), and previous treatment were collected. Low-dose RTX therapy was initiated during the remission period or after high-dose methylprednisolone administration as rescue therapy for acute attacks. Repeated brain and spinal cord MRI scans were carried out every 6 to 12 months to monitor the lesion burden on T2-weighted and fluid-attenuated inversion recovery (FLAIR) sequences, and Gd-enhanced T1-weighted imaging if necessary. The last follow-up was performed by face-to-face or telephone interview in December 2021. Main outcome measures included ARR, EDSS score, and T2 lesion burden on MRI to evaluate the efficacy of low-dose RTX therapy. Meanwhile, adverse events (AEs) were recorded to assess the safety of repeated low-dose RTX infusions. This study was approved by the Ethics Committee of Tangdu Hospital (approval number K202112–19), and all patients gave informed consent for the use of relevant data only for research purposes.
2.2 Low-dose RTX administration and circulating B cells monitoring
RTX was administered at a dose of 100 mg per week for three consecutive weeks as induction therapy. Maintenance treatment regimens include repeated infusions of RTX at 100 mg once at fixed time intervals every 6 months during the first year, and then every 6 to 12 months mainly based on the results of circulating B-cell monitoring and patient's choice. The percentage of circulating B cell subsets in total peripheral blood mononuclear cells (PBMCs) was determined before RTX induction therapy and then every 3 or 6 months. Briefly, peripheral blood samples were drawn from the cubital vein and stained with anti-CD19-PE and anti-CD27-FITC antibodies (Beckman Coulter, USA) at room temperature for 20 min, while samples stained with IgG isotypes served as controls. After erythrocytes were removed by lysis buffer (BD Pharmingen, USA), samples were analyzed by flow cytometer (Beckman Coulter, USA) for the percentages and counts of both CD19+ B cells and CD19+CD27+ memory B cells. B-cell depletion was defined as the percentage of CD19+ B cells in total PBMCs lower than 1% and memory B cells lower than 0.05%. Whenever B cell repopulation (percentage of CD19+ B cells > 1% of PBMCs and/or memory B cells > 0.05%) occurred, another infusion of RTX would be recommended.
2.3 Statistical analysis
Variables with normal distribution are expressed as mean ± standard deviation (SD) and those with skewed distribution as median with range. As previously described (
), ARR was calculated only when disease duration before the first RTX infusion lasted for at least 6 months to avoid the potential overestimation. Paired Student's t-test was used to compare the number of T2 lesions between pre- and post-RTX therapy, and Wilcoxon matched-pairs signed-rank test was used to compare ARR and EDSS scores. All statistical analyses were performed by the SPSS 26.0 software, and differences were considered statistically significant at a p value lower than 0.05.
3. Results
3.1 Patient characteristics
All the 9 patients with RRMS were females with an average onset age of 25.4 ± 6.7 years. The median disease duration before the first RTX infusion was 56 (range, 3–108) months. Of them, 7 (77.8%) received low-dose RTX following rescue therapy for acute attacks, whereas other 2 (22.2%) initiated low-dose RTX therapy at the remission stage. Additionally, 3 patients had ever received other immunosuppressants including azathioprine (AZA), mycophenolate mofetil (MMF), or interferon beta-1b (IFN β−1b) before RTX induction therapy. The median follow-up period was 30 (range, 15–40) months from the first RTX infusion to the last follow-up visit. The detailed demographic and clinical characteristics of each patient are summarized in Table 1.
Table 1Detailed demographic and clinical characteristics of each RRMS patient and responses to repeated low-dose RTX therapy.
Patient No.
Sex
Onset age, y
Disease duration before RTX, m
Previous treatment
Interval of RTX infusion, m
Cycles of RTX
Follow-up from 1st RTX, m
Number of T2 lesions before RTX
Number of T2 lesions after RTX
ARR before RTX
ARR after RTX
EDSS score before RTX
EDSS score after RTX
1
F
32
58
Cort, AZA
6
5
30
52
43
1.0
0
2.0
0
2
F
21
20
Cort
6, 12
6
39
26
22
2.4
0
3.5
1.5
3
F
30
89
Cort
6
4
24
52
47
0.5
0
3.0
3.0
4
F
35
102
Cort, AZA, MMF
6
5
28
35
35
0.4
0
2.0
0
5
F
21
18
Cort
6
3
19
17
11
1.3
0
2.0
0
6
F
25
8
Cort
6
6
36
15
12
3.0
0
2.0
0
7
F
30
56
Cort
6, 12
4
40
45
37
0.9
0
2.0
0
8
F
20
108
Cort, AZA, IFN β−1b
6
6
35
62
55
1.1
0
4.5
2.5
9
F
15
3
Cort
6
3
15
16
3
NA
0
1.5
1
Abbreviations: No., number; F, female; y, year; m, month; RTX, rituximab; Cort, corticosteroids; AZA, azathioprine; MMF, mycophenolate mofetil; IFN β−1b, interferon beta-1b; ARR, annualized relapse rate; EDSS, Kurtzke Expanded Disability Status Scale; NA, not applicable.
3.2 Effect of repeated low-dose RTX therapy on circulating B cells
All patients were treated with repeated low-dose RTX over a median of 5 (range, 3–6) cycles. Detailed dynamics in percentage and count of circulating B cell subsets of each patient during induction therapy and follow-up period were presented in Fig. 1 and Supplemental Figure 1, respectively. As shown in Fig. 2, the median percentage of CD19+ B cells in total PBMCs was 13.15% (range, 9.37%–18.85%) before induction therapy and reduced dramatically to 0.02% (0%–1.45%) at 3 months after the first RTX infusion, 2.40% (0.57%–5.59%) at 6 months, 2.82% (0.31%–9.02%) at 12 months, 2.82% (0.03%–5.51%) at 18 months, 3.46% (1.05%–7.52%) at 24 months, 4.82% (0.05%–7.10%) at 30 months, and 3.15% (1.18%–5.12%) at 36 months. The percentages of CD19+CD27+memory B cells at the corresponding time points were 3.26% (range, 1.39%–8.56%), 0.02% (0%–1%), 0.25% (0.08%–0.69%), 0.38% (0.08%–0.72%), 0.19% (0.03%–0.89%), 0.34% (0.18%–0.82%), 0.16% (0%–0.38%), and 0.38% (0.30%–0.45%), respectively. Similar tendencies were observed in the median counts of CD19+ and memory B cell subsets across the same time points, as illustrated in Supplemental Figure 2.
Fig. 1Detailed CD19+ B cell and CD19+CD27+ memory B cell dynamics of each patient with RRMS during repeated low-dose RTX therapy. Percentage of specific B cell subgroups in total PBMCs was shown before RTX induction therapy and at each time point during follow-up period. Initiation of induction therapy was denoted as 0 at x-axis. Asterisks indicate rituximab infusion.
Fig. 2The dynamics of median percentage of CD19+ B cell and CD19+CD27+ memory B cell in total PBMCs of patients with RRMS during repeated low-dose RTX therapy. Initiation of induction therapy was denoted as 0 at x-axis.
3.3 Efficacy of repeated low-dose RTX therapy in RRMS
As revealed in Table 2, a dramatic reduction of median ARR was observed at the end of this study compared to before RTX induction therapy (1.1 vs 0, p = 0.012). Notably, all patients were relapse-free during the post-RTX follow-up period. Similarly, a significant decrease in median EDSS score was achieved (2.0 vs 0, p = 0.007; Table 2). Compared to pre-RTX therapy, 8 (88.9%) patients showed an obvious improvement of disability as demonstrated by the EDSS score, and 5 of them scored 0 eventually. In addition, only one patient (11.1%; patient 3) remained stable EDSS score and no patients experienced the increase of EDSS score (Table 1). Low-dose RTX therapy also resulted in a significant decrease in the number of T2 lesions on MRI (pre-RTX vs post-RTX, 35.6 ± 18.0 vs 29.4 ± 18.1, p = 0.001; Table 2). As revealed in Table 1, most (8/9, 88.9%) patients showed to different extent the decrease of MS lesion burden including the number of lesions and lesion volume (Fig. 3). Although the number of lesions remained unchanged after RTX therapy compared to before RTX therapy in one patient (patient 4), an obvious decrease of lesion volume was confirmed in this patient.
Table 2Comparisons of main outcome measures between pre- and post-RTX therapy.
Measures
Pre-RTX
Post-RTX
P value
ARR
1.1 (0.4–3)
0 (0–0)
0.012
EDSS score
2.0 (1.5–4.5)
0 (0–3.0)
0.007
Number of T2 lesions
35.6 ± 18.0
29.4 ± 18.1
0.001
Abbreviations: RTX, rituximab; ARR, annualized relapse rate; EDSS, Kurtzke Expanded Disability Status Scale. ARR and EDSS scores were presented as
Paired Student's t-test was used to compare the difference in the number of T2 lesions between pre- and post-RTX therapy, and Wilcoxon matched-pairs signed-rank test was used for comparisons of ARR and EDSS score between pre- and post-RTX therapy.
Fig. 3Changes of lesion burden on MRI in patients with RRMS treated with repeated low-dose RTX therapy. Brain axial T2-weighted (A,B, E, F) and FLAIR (C, D, G, H) images show the decrease of the number of MS lesions after RTX therapy (B, D, F, H) compared to before RTX therapy (A, C, E, G) in patient 6 (A–D) and patient 9 (E–H). Brain axial T2-weighted (I, J) images show the decrease of lesion volume but not the number after RTX therapy (J) compared to before RTX therapy (I) in patient 4. Spinal sagittal (K, L) and axial (M, N) T2-weighted images show the decrease of lesion volume but not the number after RTX therapy (N) compared to before RTX therapy (M) in patient 2.
Of the 9 RRMS patients, 5 (55.6%) experienced at least one adverse event (AE) during the follow-up period: one patient experienced a total of three AEs (influenza-like symptoms, sweating, and alopecia), two patients each experienced two AEs (influenza-like symptoms and skin rash for one; and palpitations and fatigue for the other); one patient experienced influenza-like symptoms; and one patient experienced alopecia. As shown in Table 3, a total of 35 episodes of AEs occurred during repeated low-dose RTX therapy, of which infusion-related reactions occurred most frequently (26/35; 74.3%), followed by fatigue (7/35; 20%) and alopecia (2/35; 5.7%). All the infusion-related reactions were mild and transient and could be rapidly relieved by lowering the rate of infusion or anti-allergic therapy.
Table 3Adverse events associated with repeated low-dose RTX therapy.
). In this study, we reported a single-center experience regarding the use of low-dose RTX in RRMS patients from Northwest China. During a median follow-up of 30 months, a significant reduction in ARR was obtained and no clinical relapses were recorded. The therapeutic effect on disability progression was also reflected by the decline of EDSS scores in almost all of the patients. In addition, both the number and volume of lesions decreased significantly after repeated low-dose RTX therapy. These findings appear to be astonishing when considering that the dose of RTX in this study was much lower than those in prior studies, and some patients had never achieved sustained remission when taking other immunosuppressants earlier.
The doses and infusion frequency of RTX in treating MS vary widely among studies. In initial randomized controlled trials, the induction regimen was 1000 mg twice with an interval of two weeks (
). Considering the lower lymphocyte burden in an autoimmune disease such as MS, the effective dose of RTX could be probably lower than what is administrated for lymphoma. Supporting evidence is obtained from several autoimmune diseases such as immune thrombocytopenia (
) in which low-dose RTX has shown comparable effectiveness to high-dose RTX. Till now, studies comparing the efficacy of different RTX doses in treating MS remain to be scarce. In a large retrospective study involving 822 MS patients, the ARR and the number of contrast-enhancing lesions did not differ between patients receiving maintenance doses of RTX at 500 mg every 6 months and 1000 mg every 6 months, while AEs were slightly less common in the lower-dose group (
). Subsequent studies evaluating the effect of RTX dose reduction showed that the clinical and radiologic parameters remained stable in MS patients switching from high-dose (1000–2000 mg per year) to low-dose RTX therapy (500–1000 mg per year) (
). The findings from this study strengthened the feasibility of low-dose RTX therapy in MS. However, there are several differences between this study and prior studies. First, we further reduced the dose of RTX, that is, 100 mg per week for 3 consecutive weeks as induction therapy, and 100 mg every 6 months as maintenance therapy, which is so far the lowest dose regimen to our knowledge. Second, all patients started with a low-dose regimen rather than transitioning from a high-dose regimen. Third, disease control in most patients was not ideal before receiving RTX therapy. For instance, patient 4 and patient 8 had used several immunosuppressants prior to RTX, but none of these prevented relapses and disability progression. Considering a much lower dose of RTX in this study, our regimen may be more cost-effective.
A major concern regarding the low-dose RTX regimen is whether it can effectively deplete circulating B cells and how long the effect could last. In this study, 7 of 9 patients (77.8%) maintained the level of B-cell depletion at 3 months after the first RTX infusion and repopulated at 6 months, whereas in patient 8 and patient 9 B cells had repopulated at 3 months. The time to B-cell repopulation was a little shorter than that demonstrated by Ellwardt et al. who showed a mean time to B-cell repopulation of 8.3 months after high-dose RTX therapy (
). Similar more rapid repopulation of B cells were observed with low-dose strategy of CD20-depleting antibody compared to high-dose in several other autoimmune diseases such as neuromyelitis optica spectrum disorder (
Ocrelizumab, a humanized anti-CD20 monoclonal antibody, in the treatment of patients with rheumatoid arthritis: a phase I/II randomized, blinded, placebo-controlled, dose-ranging study.
). Although B cells repopulated faster in our low-dose regimen, we did not observe disease progression after B-cell repopulation in any cases. Not coincidentally, Maarouf et al. showed B-cell repopulation in half of the patients, but none of them relapsed. Similarly, an increasing probability of B-cell repopulation was confirmed after prolonging the interval of RTX reinfusion, whereas no obvious disease progression was observed (
). Therefore, we speculated that it might be reasonable to prolong the interval of RTX reinfusion as long as the frequency of B cells maintained a low level. This hypothesis is reinforced by the favorable efficacy obtained in this study.
Regarding the safety profile, repeated low-dose RTX therapy was well tolerated in this study. A total of 35 episodes AEs were recorded during the follow-up, all of which were mild to moderate and reversible. Infusion-related reactions were the most common, followed by fatigue and alopecia. No serious AEs were recorded such as leukopenia, infections, malignancies and cardiovascular events. Collectively, the low-dose RTX regimen in this study shows good safety profile.
This study has several limitations: (1) This was a single-center retrospective study in the Chinese population, thus selection bias could not be avoided as patients were included retrospectively; (2) This was a single-arm study with a small sample size; (3) The percentages of circulating B cells and memory B cells were not monitored regularly; (4) For some patients in the acute attack, the administration of low-dose RTX was subsequent to high-dose methylprednisolone as rescue therapy, so the decrease of EDSS scores and MS lesion burden may be partly attributed to the effect of glucocorticoids. Therefore, prospective randomized controlled studies with a larger sample size and standardized design are required to verify our findings in the future.
In conclusion, repeated low-dose RTX therapy may be effective for RRMS patients in preventing subsequent relapses and disability progression, and our regimen in this study may be more cost-effective than those reported high-dose regimen. Moreover, due to its good safety profile, low-dose RTX may be a promising option with an ideal risk/benefit ratio for RRMS patients who have no access or poor response to first-line DMDs, particularly in low- or middle-income countries.
Data availability statement
The data that support the findings of this study are available from the corresponding author on reasonable request.
Ethics statement and patient consent
This study was approved by the Ethics Committees of Tangdu Hospital, Air Force Medical University (approval number: K202112–19). Informed consent was obtained from all patients participating in this study.
Funding
This study was supported by the National Natural Science Foundation of China (Nos. 82171339 and 81901226), Key Research and Development Project of Shaanxi Province (No. 2022ZDLSF02–04), and the Excellent Personnel Foundation of Tangdu Hospital in 2021.
CRediT authorship contribution statement
Daidi Zhao: Conceptualization, Methodology, Investigation, Formal analysis, Writing – original draft. Cong Zhao: Methodology, Investigation, Formal analysis, Funding acquisition, Writing – original draft. Jiarui Lu: Data curation, Resources. Yu Han: Investigation. Tangna Sun: Data curation, Resources. Kaixi Ren: Data curation, Validation. Chao Ma: Data curation. Chao Zhang: Data curation, Investigation. Hongzeng Li: Conceptualization, Methodology, Validation, Writing – review & editing. Jun Guo: Conceptualization, Methodology, Funding acquisition, Project administration, Supervision, Writing – review & editing.
Declaration of Competing Interest
The authors declare that there is no conflict of interest.
Acknowledgments
The authors highly appreciate Mr. Jiafeng Ren for his professional assistance in statistical analysis.
Ocrelizumab, a humanized anti-CD20 monoclonal antibody, in the treatment of patients with rheumatoid arthritis: a phase I/II randomized, blinded, placebo-controlled, dose-ranging study.
Practice guideline recommendations summary: disease-modifying therapies for adults with multiple sclerosis: report of the Guideline Development, Dissemination, and Implementation Subcommittee of the American Academy of Neurology.
Safety and clinical outcomes of rituximab treatment in patients with multiple sclerosis and neuromyelitis optica: experience from a national online registry (GRAID).
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