International Immunopharmacology 9 (2009) 10–25 Contents lists available at ScienceDirect International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p Review A review of the current use of rituximab in autoimmune diseases☆ Hakan M. Gürcan a,1, Derin B. Keskin b,1, Joel N.H. Stern b,1, Matthew A. Nitzberg a, Haris Shekhani a, A. Razzaque Ahmed a,⁎ a b Center for Blistering Diseases, New England Baptist Hospital, Boston, MA, USA Cancer Immunology and AIDS, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA a r t i c l e i n f o Article history: Received 26 August 2008 Received in revised form 13 October 2008 Accepted 13 October 2008 Keywords: Rituximab B cell depletion Autoimmune diseases a b s t r a c t Rituximab is a human/murine chimeric monoclonal antibody primarily used for treating non-Hodgkin's Bcell lymphoma. Recently it has also been used in the treatment of several autoimmune diseases. A literature review was conducted to determine the efficacy of rituximab in the treatment of some of these autoimmune diseases. Multiple mechanisms proposed for the rituximab mediated B cell depletion are also discussed. The efficacy of rituximab is well-established and it is FDA approved for treatment of Rheumatoid arthritis. In this review, data on the use of rituximab is presented from 92 studies involving 1197 patients with the following diseases: systemic lupus erythematosus, idiopathic thrombocytopenic purpura, anti-neutrophil cytoplasmic antibody associated vasculitis, Grave's disease, autoimmune hemolytic anemia, pemphigus vulgaris, hemophilia A, cold agglutinin disease, Sjogren's syndrome, graft vs. host disease, thrombotic thrombocytopenic purpura, cryoglobulinemia, IgM mediated neuropathy, multiple sclerosis, neuromyelitis optica, idiopathic membranous nephropathy, dermatomyositis, and opsoclonus myoclonus. The efficacy varies among different autoimmune diseases. The cumulative data would suggest that in the vast majority of studies in this review, RTX has a beneficial role in their treatment. While rituximab is very effective in the depletion of B cells, current research suggests it may also influence other cells of the immune system by reestablishing immune homeostasis and tolerance. The safety profile of RTX reveals that most reactions are infusion related. In patients with autoimmune diseases the incidence of serious and severe side effects is low. Systemic infection still remains a major concern and may result in death. © 2008 Published by Elsevier B.V. Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. B cells in autoimmunity . . . . . . . . . . . . . . . . 1.2. Mechanism of action of rituximab. . . . . . . . . . . . 1.3. Rituximab in cancer . . . . . . . . . . . . . . . . . . Rituximab in autoimmune diseases . . . . . . . . . . . . . . 2.1. FDA approved uses . . . . . . . . . . . . . . . . . . . 2.1.1. Rheumatoid arthritis . . . . . . . . . . . . . . 2.2. Off label uses . . . . . . . . . . . . . . . . . . . . . 2.2.1. Systemic lupus erythematosus . . . . . . . . . 2.2.2. Idiopathic thrombocytopenic purpura . . . . . 2.2.3. Multiple sclerosis . . . . . . . . . . . . . . . 2.2.4. Cold agglutinin disease . . . . . . . . . . . . 2.2.5. Autoimmune hemolytic anemia . . . . . . . . 2.2.6. Antineutrophil cytoplasmic antibody—associated 2.2.7. Graft versus host disease. . . . . . . . . . . . 2.2.8. Cryoglobulinemia . . . . . . . . . . . . . . . 2.2.9. Thrombotic thrombocytopenic purpura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vasculitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 11 11 11 12 12 12 13 13 13 14 14 14 14 14 14 15 ☆ The authors have no conflict of interest or competing interests to disclose. This manuscript has not previously been presented. ⁎ Corresponding author. Center for Blistering Diseases, New England Baptist Hospital, 70 Parker Hill Avenue, Suite 208 Boston, MA 02120, USA. Tel.: +1 617 738 1040; fax: +1 617 754 6434. E-mail address: aramanuscript@msn.com (A.R. Ahmed). 1 These authors contributed equally. 1567-5769/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.intimp.2008.10.004 11 H.M. Gürcan et al. / International Immunopharmacology 9 (2009) 10–25 2.2.10. Sjögren's syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.11. IgM mediated neuropathy . . . . . . . . . . . . . . . . . . . . . . . 2.2.12. Pemphigus vulgaris . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.13. Grave's disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.14. Hemophilia A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.15. Opsoclonus myoclonus . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.16. Dermatomyositis. . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.17. Neuromyelitis optica . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.18. Idiopathic membranous nephropathy . . . . . . . . . . . . . . . . . . 3. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Rituximab and intravenous immunoglobulin (IVIg) for the treatment of autoimmune 3.3. Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction Rituximab (RTX) is a human/murine chimeric monoclonal antibody (mAb) that specifically targets the transmembrane protein CD20 of B cells [1]. The Fab region of RTX recognizes a four amino acid sequence on a large extra-cellular loop of the CD20 molecule [2]. The binding of RTX to CD20 leads to significant depletion of peripheral B cells [3,4]. RTX is FDA approved for the treatment of low-grade non-Hodgkin's B cell lymphomas (NHL) [5]. Recently it is being increasingly used in the treatment of several autoimmune diseases [6,7]. This review presents the current theories on the mechanism of action of RTX-induced B cell depletion. It provides a review of the use of RTX in selected autoimmune diseases. The intent of the authors is to provide the reader with a panoramic view of the studies reported to date. This will facilitate their obtaining information derived from multiple sources. Consequently they can decide on the benefit, or lack there of, when confronted with a patient in whom RTX therapy is being considered. 1.1. B cells in autoimmunity B cells derived from hematopoietic stem-cell precursors in the bone marrow sequentially progress into pro B cells, pre B cells, immature B cells, and mature B cells. Each mature B cell leaves the bone marrow with unique antigen-specific B cell receptors (BCR) on its surface and migrates through the periphery towards follicular lymphoid tissue (lymph nodes, spleen, and mucosa associated lymphoid tissue) [8,9]. If the BCR on a mature B cell encounters its cognate antigen, the B cell proliferates and differentiates into antibody producing plasma cells or long-lived memory B cells [9]. In a healthy immune system, B cells are tolerant of self antigens and will only bind to non-self antigens. B cells acquire this immune tolerance to self-antigens during their development in the bone marrow where immature B cells that recognize self antigens undergo apoptosis or change their antigen specificity. Additionally, B cells that recognize soluble self-antigens in the periphery loose their ability to respond and are prevented from migrating to follicular lymphoid tissues where they would produce a normal immune response [9]. Autoimmune diseases result when such mechanisms fail or are bypassed resulting in the generation of pathogenic, self-reactive B cells. In 1980, Stashenko et al. described a cell surface antigen specific to B cells now known as CD20 [10]. With the exception of plasma cells, the CD20 molecule is present on all B cells after the pro B cell state [10,11]; these CD20 positive B cells are the therapeutic target of RTX. 1.2. Mechanism of action of rituximab Multiple mechanisms are proposed for RTX mediated B cell depletion. These effector mechanisms may act individually or . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 15 15 15 19 19 19 19 19 19 19 20 20 21 21 22 collectively to deplete B cells depending on the disease pathology. RTX may induce B cell killing by natural killer (NK) cells through antibody-dependent cellular cytotoxicity (ADCC) [12]. FcγRIIIA-158 polymorphisms are currently shown to enhance RTX mediated ADCC and improve clinical response to RTX [16,17] (Fig. 1-A). Direct cross linking of CD20 on B cell tumor cell lines are demonstrated to be sufficient for the induction of apoptosis [12] (Fig. 1-B). RTX induced apoptosis is reported to act through MAP kinase activation and p38 [13]. These apoptotic B cell tumors may also be taken up by dendritic cells and theoretically induce CD8 immunity against the tumors by cross-priming [14]. RTX may also induce complement dependent cytotoxicity on target B cells [12,13,18]. Differential susceptibility of malignant and pathological B cells to RTX may depend on the expression of complement regulatory proteins (CRP) such as CD46, CD55 and CD59 on target cells [17,18] (Fig. 1-C). Several studies have demonstrated that over expression of complement regulatory proteins interferes with the function of RTX and may contribute to the resistance against RTX therapy. Opsonization of B cells by RTX may also induce the clearance of B cells in circulation through phagocytosis by the reticulo-endothelial system [15]. Reticulo-endothelial system may also aid in the clearance of apoptotic B cells after RTX treatment (Fig. 1-D). All these possible mechanisms may contribute to RTX mediated B cell depletion. However, most of the resistance developed against RTX by malignant cells is reported to be through CRP's, which may suggest that complement mediated B cell depletion is the main mechanism involved in the RTX effect on malignant B cells [19–21]. Removal or killing of RTX opsonized B cells by NK cells or macrophages may also take longer since these cells have to be recruited and must interact with opsonized B cells for their function. Conversely complement mediated effects are fast acting. Apoptosis induction by RTX cross linking of CD20 on B cells may also be an effective mechanism for B cell depletion; however, most malignant tumor cells tend to rapidly develop resistance against apoptosis induction. 1.3. Rituximab in cancer The FDA approved the use of RTX in certain classes of low-grade NHL in 1997. Since then, RTX has been used in over 500,000 NHL patients [6]. The current indications of RTX use in NHL are: (i) refractory CD20+ B-cell low grade NHL; (ii) retreatment of relapsed NHL patients after first RTX therapy; (iii) first-line treatment for follicular NHL combined with cyclophosphamide (CYP); (iv) maintenance therapy for CYP induced NHL stability; (v) first line treatment for diffuse large B-cell lymphoma combined with a CYP–doxorubicin– vincristine–prednisone regimen; (vi) maintenance therapy for refractory indolent NHL [22]. The overall efficacy and safety of RTX therapy 12 H.M. Gürcan et al. / International Immunopharmacology 9 (2009) 10–25 Fig. 1. A. RTX induce B cell killing by natural killer (NK) cells through antibody-dependent cellular cytotoxicity (ADCC), which results in the apoptosis of the target B cell. B. Direct cross linking of CD20 on B cell tumor cell lines are also demonstrated to be sufficient for the induction of apoptosis. C. When RTX bind to surface CD20 on target B cells it may induce complement dependent cytotoxicity on target B cells and cell death through membrane attack complex. D. Opsonization of B cells by RTX may also induce the clearance of B cells in circulation through phagocytosis by the reticulo-endothelial system cells such as monocytes and macrophages. with B-Cell cancer stimulated the use of RTX in the treatment of B-Cell related autoimmune diseases. 2. Rituximab in autoimmune diseases 2.1. FDA approved uses 2.1.1. Rheumatoid arthritis Rheumatoid arthritis (RA) is an autoimmune inflammatory disorder commonly affecting the synovial membrane of joints. Manifestations include pain, tenderness and symmetrical swelling of the hand, wrist, foot and knee joints bilaterally eventually leading to loss of function [23]. RA affects approximately 1% of the population, is more common in women and increases in prevalence with age [24]. Macrophages, fibroblasts, endothelial cells, T cells, and multiple cytokines may all be involved in the pathogenesis. Several hypothesized mechanisms include B cell dependent activation of synovial T cells, B cell secretion of proinflammatory cytokines and B cell production of the rheumatic factor autoantibody found in aggressive cases of RA. The efficacy of RTX implies that B cells play an integral role [25]. There are three randomized controlled trials that demonstrate the efficacy of RTX on RA [26–28] (Table 2). In the first study, 161 patients were given either RTX, methotrexate (MTX), RTX with MTX, or RTX with cyclophosphamide (CYP). The American College of Rheumatology 20% response scores (ACR20) at 24 weeks were 38, 65, 76, and 73% respectively [26]. In the second study, 465 patients were given either placebo, 500 mg infusions of RTX, or 1000 mg infusions of RTX while simultaneously receiving weekly MTX and varying doses of corticosteroids (CS) (Table 2). The ACR20 responses were 28, 55, and 54% respectively at 24 weeks; pretreatment with IV GC helped reduce acute infusion reactions [27]. In the third study, 520 patients received either RTX with MTX or MTX alone; the ACR20 responses were 51 and 18% respectively at 24 weeks [28]. All three studies demonstrated that RTX showed efficacy in RA that is refractory to other treatments. It is of interest to note that in 12, 15, and 21% of patients, there were worsening of the clinical disease in the 3 studies. The quantification of the worsening of the RA in these patients was not provided. A major limitation of the published data on RA is that since there is only a six months follow-up, it is not possible to know if RTX professed a longterm safe remission. Moreover in a two to five year period how many patients will continue to require additional therapy? Adverse events occurred in both RTX and control groups, which may be a combined effect of both medications. Infection rate was 35–41%, however serious infections occurred in 1.2–3.7% of patients amongst these three studies (Table 2). Depletion of peripheral blood B cells persisted through the 24 week follow-up in all three studies [26–28]. In two studies some recovery of B-cells was observed between 16–20 weeks [27,28]. Immunoglobulin levels were either stable or never below normal levels [26–28]. In a different study of 24 patients with RA, patients were treated with RTX and repopulation of peripheral B cells were studied. More than 80% of residual B cells showed a memory or plasma cell precursor phenotype. B cell repopulation occurred at a mean of 8 months. In patients who experienced a relapse of RA, the phenotype of the B cells upon repopulation showed a higher number of memory B cells [29]. H.M. Gürcan et al. / International Immunopharmacology 9 (2009) 10–25 In 2006 the FDA approved RTX for the treatment of patients with RA non-responsive to tumor necrosis factor (TNF)-blocking agents. The current recommended protocol is: two 1000 mg IV infusions separated by two weeks; each infusion is preceded by 100 mg IV methylprednisolone (or equivalent); weekly MTX is given as an adjuvant for increased efficacy. A second cycle of RTX therapy should be considered after 24 weeks if there is still residual disease activity or if a relapse of symptoms occurs [30]. 2.2. Off label uses A PubMed literature search of MEDLINE was conducted using the terms “rituximab” and each autoimmune disease in Table 1. Diseases were included in this review if the corresponding literature met the following criteria: 1) English language; 2) minimum of 5 patients treated; 3) the intent of study was to determine the efficacy of RTX. 2.2.1. Systemic lupus erythematosus Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by the deposition of immune complexes throughout the body and the presence of anti-DNA autoantibodies [31]. Clinical manifestations are extensive and often include a photosensitive rash, arthralgia, or renal dysfunction [32]. The prevalence of SLE in the United States is 52 per 100,000. The disease occurs at all ages, in both genders and in all ethnic groups although the prevalence is significantly higher in women—particularly women of African or Hispanic descent [33]. Compared to the normal, the distribution of subsets of B cells during their development process is altered in patients with SLE. There are increased level of pre-plasma cell, memory cell, and pre-germinal center cells and depleted levels of naive B cells [11]. In the 17 studies presented in this paper, RTX was used because the use of antimalarials, immunosuppressive agents (ISA), disease modifying antirheumatic drugs (DMARD), and corticosteroids (CS) was not effective as first line treatments [34–50]. RTX led to clinical response in 159 of 208 (79%) treated SLE patients with improvement in mean lupus assessment scores (BILAG, SLEDAI, SLAM), renal function, and decreased steroid dosage. There was a particularly dramatic improvement in patients with neuropsychiatric symptoms [42]. The antibodies to dsDNA and serum C3 levels are significantly improved following B cell depletion matching the clinical improvement in one study [47]. However in a different study clinical im- Table 1 Autoimmune diseases with reported use of Rituximab therapy FDA approved Rheumatoid arthritis Diseases meeting inclusion criteria of this review Systemic lupus erythematosus Idiopathic thrombocytopenic purpura ANCA associated vasculitis Autoimmune hemolytic anemia Cold agglutinin disease Graft vs host disease Pemphigus vulgaris Hemophilia A Graves disease Primary Sjögren's Syndrome IgM mediated neuropathy Thrombotic thrombocytopenic purpura Cryoglobulinemia Neuromyelitis optica Dermatomyositis Idiopathic membranous nephritis Multiple sclerosis Opsoclonus myoclonus Diseases not meeting inclusion criteria Autoimmune neutropenia Multifocal motor neuropathy Myasthenia gravis Evan's syndrome Pure red cell aplasia Ankylosing spondylitis Bullous pemphigoid Antiphospholipid syndrome Chronic inflammatory demyelinating polyneuropathy Chronic focal encephalitis Acquired angioedema Chronic urticaria 13 provement did not match the fall in the levels of ds-DNA and C3 [34]. The likelihood and timing of relapse was predicted by baseline antiENA and C3 levels [46]. The treatment protocol varied widely among studies (Table 3). A total of 13 serious infections were reported and one patient infected with histoplasmosis died [39]. SLE can be more serious in children and often requires aggressive treatment [37]. There is a correlation between Fc receptor type and age of onset of SLE [51] suggesting SLE children may have a unique set of Fc receptors. As previously mentioned, RTX efficacy is dependent upon the genotype of the FcγRIIIa receptor [16]. It is therefore possible that children may have a substantially different outcome in the treatment of SLE than found in adults [37]. Two studies examined the efficacy of RTX in childhood SLE. Both studies reported high efficacy (84% response in 18 patients) although they differed in the safety outcomes. Willems et al. [37] reported a 45% frequency of severe adverse events; much higher than seen in adults, while Marks et al. [36] reported no severe adverse events. Studies in which RTX was used to treat SLE patients highlighted the observation that pathogenesis of SLE is a consequence of abnormal interaction between B and T cells. The cumulative evidence suggests that RTX has multiple mechanisms of action in SLE patients, which may act alone, simultaneously, or synchronously. The observations of these studies have demonstrated that some of the mechanisms by which RTX influences the outcome in SLE patients are as follows: the depletion of B cells is dependent on FcγRIIIa allele of the effector cell [16]. B cell depletion normalizes the abnormalities in peripheral B cell lymphocyte homeostasis that are characteristic of active disease [11]. RTX therapy decreases the expression of CD40L, CD69, and ICOS on T cells, and HLA-DR suggesting that not only B cells, but also T cells are affected. It also results in rapid falls in the percentages of CD40 and CD80 expressing CD19 cells, suggesting possible disturbance of T cell activation through these costimulatory molecules [38,42,43]. RTX therapy enhances the number and function of regulatory T cells (Tregs) [39]. RTX efficacy for refractory SLE appears promising and RTX appears to be well tolerated in most patients. Specifics with regards to indications for its use and treatment protocols and frequency of side effects need to be determined. Two randomized controlled trials are currently underway to address these and other issues [52]. 2.2.2. Idiopathic thrombocytopenic purpura Idiopathic Thrombocytopenia Purpura is an autoimmune hematological disorder characterized by low platelet counts (b150 × 103); it is caused by the production of autoantibodies against platelets surface glycoproteins [53]. Intracranial hemorrhage is the most serious complication in ITP [54]. It occurs in 0.2–1% of patients with ITP when platelet counts are less than 20 × 10 ^ 3 and can result in death [55]. CS, IVIg, and anti-D therapy can increase platelet counts by 60% to 80%, but these effects are often transient [56]. Splenectomy is effective in 70% to 80% of patients [57]. Immunosuppressive drugs also have been effective, but are toxic and do not always alleviate symptoms [58]. RTX was given to patients who are refractory to these treatments [58–67]. RTX was effective in elevating the platelet counts in 114 out of 198 patients with majority of patients achieving normal platelet counts (N150 × 109 cells/L). The mean response rate was 57% (25–85) among the studies. Most patients received four 375 mg/m2 RTX infusions weekly for four consecutive weeks. Adverse events were generally mild, though one patient died of pneumonia [67]. Splenectomy did not influence the outcome of RTX therapy [66]. The response of childhood ITP to RTX was similar to the response observed in adults [65]. The authors of a systematic review of RTX on ITP included published abstracts that have not been included in this review. Of the 313 patients reported, the overall response rate was 62.5% with a median time response of 5.5 weeks and clinical improvement was maintained for a median10.5 months [68]. 14 H.M. Gürcan et al. / International Immunopharmacology 9 (2009) 10–25 2.2.3. Multiple sclerosis Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease affecting the brain and spinal cord. It usually is progressive and often results in permanent disability [69]. It's typically seen in patients between 20 and 45 years [70]. The course of the disease is relapsing remitting (RRMS) in 85% of patients. Glucocorticosteroids are effective in shortening acute relapses, while beta-interferons, glatiramer acetate, azathioprine and IVIg are used long-term to reduce the number of relapses [71]. A total of 111 patients were treated with rituximab in 3 studies [72–74]. In one study, 16 patients received 4 weekly infusions of RTX (375 mg/m2). Cerebral spinal fluid B cell levels dropped in 24 weeks, but there were no significant changes in clinical status [72]. In a double-blind trial, 69 patients were given RTX and compared to 39 patients receiving placebo. A single course of RTX reduced inflammatory brain lesions and clinical relapses for 48 weeks [73]. In another recent study, 26 patients received 4 infusions of RTX at weeks 0, 2, 24, and 26. Fewer new gadolinium-enhancing lesions were seen starting from week 4 and through week 72. A significant reduction in relapses was also reported in the same study [74]. No significant serious adverse events were reported. 2.2.4. Cold agglutinin disease Cold agglutinin disease (CAD) is a type of Autoimmune hemolytic anemia (AIHA) in which IgM-type autoantibodies bind to the I antigen on RBC in colder temperatures leading to agglutination and hemolysis [75,76]. CS, alkylating agents, splenectomy, interferon-α (IFN-α) monotherapy, and purine analogs, have been used with variable success. Consequently several patients are non-responsive to them [76]. In a cohort of 105 patients in four studies, 62% (45–71) of patients had a positive clinical response to RTX [75–78]. A clinical response was defined as improvements in hemoglobin levels, and serum IgM levels. RTX was used according to the lymphoma protocol. No serious adverse events were reported. 2.2.5. Autoimmune hemolytic anemia Autoimmune hemolytic anemia (AIHA) is characterized by the presence of autoantibodies against red blood cells (RBC). The autoantibodies are classified as either warm or cold-activated, and their presence is often associated with other underlying diseases such as SLE, chronic lymphocytic leukemia and others. The incidence of AIHA is 1–3 cases per 100,000 [64]. CS, splenectomy, and immunosuppression are considered first-line therapies. Not all patients respond to them. In eight studies the use of RTX produced a clinical response in 62 of 76 patients. The mean response rate was 87% (range 40–100) [64,79– 85]. Responses were assessed based on the following: a reduction in reticulocyte count; absence of hemolysis; decreased need of transfusion; normalization of hemoglobin, neutrophil, and platelet counts. Patients typically received three to four infusions at a dose of 375 mg/ m2 at weekly intervals, often in combination with other therapies (Table 3). One particularly effective protocol combined dexamethazone and CYP with RTX therapy leading to a response in 8 of 8 patients [84]. The association of AIHA with CLL did not affect the response rate to RTX [83,84]. All studies concluded that RTX is a safe and promising mode of therapy for CS refractory AIHA, but recommended that further studies are warranted to establish its precise role in the management of AIHA. 2.2.6. Antineutrophil cytoplasmic antibody—associated vasculitis Antineutrophil cytoplasmic antibody (ANCA)—associated vasculitis (AAV) is a multisystem autoimmune disorder affecting the smaller vasculature of the body [86]. The disorder manifests itself in two main forms, Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA); both are characterized by necrotizing vasculitis of the small to medium vessels with the former distinguished by granulomatous inflammation of the respiratory tract [87]. Though AAV is frequently linked to the presence of ANCA, there are ANCA negative symptomatic patients and vice versa [88]. Patients with WG generally have proteinase 3 specific ANCA while patients with MPA have myeloperoxidase specific ANCA [88]. If left untreated, the mortality rate of AAV has been reported as high as 90% within 2 years of diagnosis with a mean survival time of 5 months [89]. The current standard therapy is an aggressive CS regimen combined with cyclophosphamide (CYP) [90]. The clinical outcome of the therapy shows that 10% of patients do not experience remission [91], 50% of patients relapse [92], and patients over 60 years old have a 1 year mortality rate of 30% [41]. RTX was used in eight studies [41,90–96] for AAV patients who were refractory to the standard therapy and subjected to the severe dose-limiting side effects of CYP. RTX infusion significantly improved AAV in 65 of 73 patients with 58 patients experiencing complete remission defined by a Birmingham Vasculitis Activity Score of zero. The mean response rate was 89% (38–100). Prednisone dosage, renal function, gangrene, neuropathy, arthritis, musculoskeletal pain, and pulmonary symptoms improved in responders. Granulomatous lesions especially orbital pseudotumors were slow to respond [92,94]. ANCA levels became negative in the majority of patients with successful outcomes. In 14 patients, ANCA levels remained negative after B cell reconstitution, suggesting that RTX therapy may reintroduce immune tolerance in AAV patients. The majority of patients received four RTX infusions of 375 mg/m2 over four consecutive weeks. Adverse events were rarely severe and readily controlled. The exact pathogenic origin of AAV and the impact of RTX therapy remains unclear as illustrated by some of the following observations: clinical remission often preceded the slower and occasionally incomplete disappearance of ANCA [41,93]; ANCA levels do not always correlate with disease activity [92]; it appears that AAV is more responsive to RTX than several other diseases in which it is used [40]; patients with refractory granulomatous diseases were not responsive to RTX [92,94]. Nonetheless the current view of many investigators is that RTX therapy for nongranulomatous aspects of AAV appears safe and effective in treating patients refractory to other conventional therapies. RCTs are currently underway to confirm these earlier observations. 2.2.7. Graft versus host disease Chronic graft versus host disease (GVHD) is a complication of bone marrow transplantation. The condition occurs when donor lymphocytes recognize the recipient as foreign and result in multiple organ damage. GVHD occurs in 33–80% of transplantations depending on donor–recipient compatibility. Long term immunosuppression is the standard treatment of GVHD, however some patients do not respond to it [97]. Four studies using RTX for GVHD are presented in this review [98– 101]. Overall, 66% (50–82) of the 72 patients treated with RTX responded. Patient response included improvements in hair growth, cutaneous and musculoskeletal manifestations, sclerosis-associated dyspnea and dysphagia, serum bilirubin levels, and visual analog scores for pain and fatigue. Most patients were treated according to the lymphoma protocol. Adverse events were infection related (Table 3). Ten serious infections were reported and 3 patients died of infection. 2.2.8. Cryoglobulinemia Cryoglobulinemia (CG) is a chronic immune disorder characterized by the presence of cold-precipitable immunoglobulins called cryoglobulins. Precipitation factors include temperature, pH, and cryoglobulin concentration [102]. The disorder manifests as a systemic vasculitis often affecting, but not limited to the skin, joints, peripheral nerves, liver, and kidneys [103]. CG is usually secondary to multiple H.M. Gürcan et al. / International Immunopharmacology 9 (2009) 10–25 underlying disorders such as Hepatitis C virus (HCV), lymphoproliferative disorders, and Sjögren's syndrome [104]. Treatment of CG usually targets the underlying disease and includes interferon, CS, plasmapheresis, and cytotoxic agents [104]. RTX is a promising nontoxic therapy for CG as demonstrated by the six studies [104–109]. There are 61 patients in the literature that have been treated with RTX for CG of which 55 were responsive. Overall, 90% (75–100) of the patients demonstrated a clinical response. These patients were unresponsive to at least one of the following: ISA, CS, plasma exchange (PE), Interferon alpha (IFα), disease modifying anti-rheumatic drugs (DMARDS), anti-viral therapy, plasmapheresis, intravenous immunoglobulin (IVIg), mycophenolate mofetil (MMF), or cyclosporine A (CSA). 55 of the 61 patients responded to RTX therapy with response defined as improvement in cutaneous vasculitis, renal function, neuropathy, arthralgia, parasthesia, cryocrit levels, RF levels, C4 levels, comorbid conditions, or CS dosage. The majority of patients received the lymphoma protocol; other patients received the same RTX dosage, but received a different number of infusions (Table 2). Two of the studies reported significant side effects of RTX therapy in CG. In one study with renal transplant associated CG [108], 2 patients developed severe infections: one patient acquired disseminated herpes simplex and one patient died from disseminated cryptococcosis. In another study with HCV associated CG, HCV RNA levels doubled from baseline after RTX therapy [106]. 2.2.9. Thrombotic thrombocytopenic purpura Thrombotic thrombocytopenic purpura (TTP) is a rare disorder that occurs when pathogenic antibodies of the IgG or IgM type inhibit the enzyme ADAMTS13 that normally cleaves the multimers of von Willebrand factor (VWF). Inhibition of ADAMTS13 leads an increase in of VWF multimers resulting in the aggregation of platelets [110]. TTP can cause ischemia to various organs, decreased blood supply to the brain, intravascular hemolysis with erythrocyte fragmentation, and extreme elevations of serum lactate dehydrogenase (LDH). Up to 90% of TTP patients respond to plasma exchange as a first line therapy [111]. In five studies, RTX therapy was effective in 60 of 61 (98%) patients [110,112–115]. Patients receiving RTX were refractory to at least one of the following: PE, CS, vincristine, splenectomy, ISA, IVIg. Most patients were treated with RTX using lymphoma protocol and all patients received concomitant PE. The objective parameters to measure response included improvements in hemoglobin levels, platelet counts, LDH elevation, ischemic signs and need for PE. One study reported a decrease in ADAMTS13 titers after RTX therapy [110]. There were no major adverse events reported in the studies. RTX was a welltolerated addition to treatment of TTP. 2.2.10. Sjögren's syndrome Sjögren's Syndrome (SS) is an autoimmune disorder characterized by persistent dryness of the salivary and lacrimal glands with one third of patients presenting with extraglandular manifestations [116]. The disorder can be further classified as primary (PSS) or secondary depending on the presence of an associated underlying disease in the latter [116]. SS affects 0.2–3% of the population [117] and most therapies are only fleetingly successful in treating the short term sicca symptoms [118]. Recently, B cells have been suggested to play a role in the pathogenesis of PSS [119]. Four clinical studies are presented in this review with a total of 49 patients [35,118,120,121]. The subjective patient assessment of improvement on RTX therapy was high. Reported benefits for PSS included fatigue, dryness, pain, dry mouth, dry skin, and dry trachea visual analog scale (VAS) scores [118]; decreased tender point and tender joint counts [118]; decreased parotid swelling [120,121]; decreased steroid dosage [35,121]; improvement in associated cryglobulinemia [35,118,121], pulmonary involvement [118,121], polysynovitis, and mononeuritis [121]; improvement in quality of life [118,120]. 15 It is difficult to provide an objective analysis of the efficacy of RTX on PSS due to the diverse manifestations of disease presentation, the subjective nature of response assessment, and the potentially confounding role of concomitant therapies. 2.2.11. IgM mediated neuropathy The definition of IgM mediated neuropathy (MMN) is implicit within its name. The disorder occurs when autoantibodies of the IgM type bind to components of sensory and motor neurons leading to neuropathy. The studies currently presented used RTX to treat patients with MMN caused by anti-myelin associated glycoprotein (MAG) and anti-GM1 ganglioside antibodies. Current treatments include IVIg, CYP, chlorambucil, plasma exchange, and interferon alpha [122,123]. RTX therapy was effective in 35 of 46 patients determined by improvements in neurological examinations, motor nerve conduction velocity, strength, coordination, gait stability, and IgM titers [122– 126]. RTX was typically given in four 375 mg/m2 infusions once per week although one study used two cycles of RTX therapy with the second cycle at a higher dose (750 mg/m2). Two patients unresponsive to the first 375 mg/m2 cycle responded to the higher dosage in the second cycle [122,125]. One study found that patients with low titers of anti-MAG had a significantly enhanced response to RTX therapy over patients with high anti-MAG at baseline [126]. Authors of another study speculate that advanced cases of MMN are more difficult to treat with RTX than earlier stages of the disease implying RTX therapy is most beneficial as an early treatment [125]. No significant side effects were reported. 2.2.12. Pemphigus vulgaris Pemphigus vulgaris is a rare and potentially fatal autoimmune mucocutaneous blistering disease that affects the skin, oral cavity, and other mucosal surfaces. The disease is characterized by the presence of desmoglein 3 specific IgG autoantibodies which result in the loss of adhesion between keratinocytes causing acantholysis [127]. Conventional therapy consists of systemic corticosteroids and/or immunosuppressive agents. Patients who are non-responsive to or develop significant side effects to these treatments are treated with intravenous immune globulin (IVIg) [127]. RTX has been shown to be beneficial in the treatment of Pemphigus vulgaris with rapid healing of skin and mucosal lesions. In one study, patients were given combination of RTX and IVIg therapy. Each patient received 10 infusions of RTX during a six month interval. IVIg was given to prevent infection. There were no observed adverse effects and combination of RTX and IVIg therapy proved very effective in producing a sustained and a complete remission in all 11 patients [127]. In a second study, patients were given RTX once weekly for 4 weeks and 60% of patients responded. No adverse infusion reactions occurred, but two patients did experience infection [128]. In two other recent studies, a single course of four weekly infusions of 375 mg/m2 of RTX induced a complete clinical remission in 24 patients [129,130]. Two infections and 1 death from septicemia were reported in one of these studies [130]. The cumulative evidence from these studies, indicates that RTX can induce a long-term remission PV patients. 2.2.13. Grave's disease Graves' disease (GD) is a thyroid disorder where antibodies specific for the thyroid stimulating hormone (TSH) receptor stimulate the thyroid gland to overproduce thyroid hormone leading to the clinical manifestations of hyperthyroidism [131]. Grave's ophthalmopathy (GO) is a significant swelling of the extraocular muscles causing proptosis and can lead to compression of the optic nerve [132]. Two studies have examined the efficacy of RTX therapy in GD and concurrent GO involving 29 patients. One study compared the effect of RTX treatment to that of CS. Nine patients receiving RTX therapy 16 Table 2 Rituximab in the treatment of rheumatoid arthritis Patients Previous (n) Rx Indications for RTX Cohen et al. [28] 520 Anti-TNFα MTX Emery et al. [27] 465 DMARDs Study protocol Side effects of therapy Infection rate Clinical outcome – Inadequate response 1000 mg on to TNF inhibitors days 1 and 15 Group 1: Placebo +MTX Group 2: RTX + MTX All groups received IV GC and oral GC RA, Headache, URI, Nasopharyngitis, Nausea, Fatigue, Hypertension, Diarrhea, Arthralgia, Pyrexia, Dizziness, Bronchitis, Cough, Sinusitis, UTI, pruritis, urticaria, flushing, hot flush, throat irritation, tachycardia, ear pruritis, oropharyngeal swelling, hypotension, vomiting, rash – 41% in RTX group – 38% in placebo group – 7 (1.3%) serious infections – 51% of patients responded to combined RTX compared to 18% placebo; measured by the American College of Rheumatology improvement criteria at 24 weeks – ACR50 and ACR70 responses were also significant at week 24 – 21% of RTX treated patients experienced an exacerbation RA – RA for 6 months Group 1A: Placebo Group 1B: Placebo+IV GC Group 1C: Placebo + IV and oral GC Group 2A: RTX 500 Group 2B: RTX 500 + IV GC Group 2C: RTX 500 + IV and oral GC Group 3A: RTX 1000 Group 3B: RTX 1000 +IV GC Group 3C: RTX 1000 + IV and oral GC All groups received weekly MTX RA, Headache, Nausea, URI, UTI, Nasopharyngitis, Arthralgia, Diarrhea, Fatigue, Hypertension, Rigors, Dizziness, cerebral infarction, fatal cerebrovascular accident, convulsion, MI, supraventricular tachycardia, drug hypersensitivity, implant failure, lower limb fracture, interstitial lung disease, epilepsy, serotonin syndrome, chest pain, generalized edema, exacerbation of RA, satus asthmaticus, intestinal obstruction, metrorrhagia, thromboangiitis obliterations – 35% in RTX group – 28% in placebo group – 6 (1.2%) serious infections – 1000 mg RTX, 500 mg RTX, and placebo yielded 54%, 55%, and 28% patient response respectively; measured by the ACR20 improvement criteria at 24 weeks – ACR50 and ACR70 responses were also significant at week 24 – 15% of patients experienced exacerbated RA; pretreatment with IV GC ameliorated adverse infusion reactions RA, hypotension, bronchopneumonia, staphylococcal septicemia, renal impairment, hypertension, arthralgia, rash, back pain, cough, pruritus, nausea, dyspnea – 6 (3.7%) serious infections – % of infections not available – Combined RTX and MTX – 73% response; combined RTX and CYP – 76% response; RTX alone – 65% response; MTX alone – 38% response; measured by ACR20 improvement criteria at 24 weeks – ACR70 responses were also significant at week 24, ACR50 responses were only numerically higher – 12% of patients experienced exacerbation of RA – MTX for 12 weeks prior to study – Failure of 1 to 5 DMARDs Edwards et al. 161 [26] MTX – Active RA despite 10 mg MTX/week RTX dosage (infusion) 500/1000 mg on days 1 and 15 1000 mg on days Group Group 1 and 15 Group Group 1: 2: 3: 4: MTX RTX RTX + CYP RTX + MTX CYP, cyclophosphomide; IV GC, Intravenous glucocorticoid; MTX, methotrexate; RA; rheumatoid arthritis; RTX, rituximab; TNF, tumor necrosis factor alpha; URI, upper respiratory infection; UTI, urinary tract infection. H.M. Gürcan et al. / International Immunopharmacology 9 (2009) 10–25 Reference Disease Studies Patients (n) (n) Previous Rx Dose of RTX Concomitant Tx Side effects of therapy Clinical outcome Systemic lupus erythematosus 17 [34–50] 208 Steroids, CYP, AZA, MMF, IVIG, TCR, PP, HCQ, CSA, MTX, LFN, antimalarial agents, ETE, IFX, MPC, MZB, VCR, CCC – 4 × 375 mg/m2 weekly – 4 × 500 mg/m2 weekly – 2 × 1000 mg 2 weeks apart – 2 × 500 mg 2 weeks apart – 2 × 750 mg 2 weeks apart – 2 × 375 mg 2 weeks apart – 1 × 100 mg/m2 – 1 × 350 mg/m2 – 1 × 450 mg/m2 – 1 × 375 mg/m2 – 1 × 750 mg/m2 ACM, DPH, steroids, CYP, MMF, AZA, PP, HCQ, CPM, PCM, CSA, IVIg Bronchospasm, transient ischemic attack, Bell's Palsy, parotid swelling, hypersensitivity rxn, fever, rash, neutropenia, deep vein thrombosis, pulmonary embolism, massive pulmonary hemorrhage, HACA, photosensitive eruption, microscopic hematuria, thrombocytopenia, nausea, vomiting, hypogammaglobulinemia, severe hematologic toxicity, lymphopenia S. aureus abscess of the thigh, Herpes Zoster (4), septic elbow complicated by bacteremia and necrotizing fasciitis of group A streptococci, S. bovis septicemia, E. coli septicemia, invasive histoplasmosis and mucormycosis in a coronary artery, UTI (2), pneumococcal pneumonia and septicemia, pneumonia (3), enteritis, subcutaneous abscess, limited RTI/UTI – 159 of 208 (76%) patients responded to RTX – 18 serious infections reported and 1 patient died of histoplasmosis Idiopathic thrombocytopenic purpura [58–67] 198 Steroids, SPL, IVIg, CYP, CSP, Anti-D, DNL, AZP, VNC, DXM, RBM, MMF, PE, BMT, eradication of H. Pylori, Vit C – 4 × 375 mg/m2 weekly – 4 × 50–375 mg/ m2 weekly Anti-D, IVIg, CYP, CC, DXM, DZL, Steroids, VCR, VNB, ACT, BMT, H1 and H2 receptor blockers, PCM, DPH, SRA, CPM Serum sickness, pruritis, throat tightness, urticaria, headache, chest pain, low ANC, dizziness, nausea, fever, chills, asthenia, vomiting, hypotension, thrombocytosis Pneumonia, limited RTI/UTI – 114 of 198 (57%) patients responded to RTX Betaseron, avonex, copaxone, rebif – 4 × 375 mg/m2 weekly – 2 × 1000 mg 2 weeks apart –1000 mg at week 0, 2, 24, 26 β-IFN 1a, β-IFN 1b, glatiramer acetate Headache, back pain, general pain, limb pain, heat sensations, pruritus, rash, ischemic coronary-artery syndrome, malignant thyroid neoplasm Gastroenteritis, bronchitis, limited RTI/UTI – 4 × 375 mg/m2 weekly RTX + IFN-α, RTX + FLU Multiple sclerosis [72–74] Cold agglutinin disease [75–78] 9 3 4 111 105 CHL + CYP, CHL + PDN, CHL, alkylating agents, PA, CDB, SPL Fever, cough, headache, nausea, diarrhea, shiver or dizziness, hypotension, muscular pain, transient neutropenia, fever, flulike symptoms, limited RTI/UTI H.M. Gürcan et al. / International Immunopharmacology 9 (2009) 10–25 Table 3 Autoimmune diseases with at least 50 patients in the literature treated with rituximab – 1 serious infection reported and the patient died of pneumonia – no significant change in clinical status in most patients in one study – Mean relapse rates and inflammatory brain lesions were reduced in two studies at 48 and 72 week follow-up respectively – 2 serious infections reported – 65 of 105 (62%) patients responded to RTX – No serious infections reported 17 (continued on next page) 18 Table 3 (continued) Disease Studies Patients (n) (n) Previous Rx Dose of RTX Concomitant Tx Side effects of therapy Clinical outcome Autoimmune hemolytic anemia [64,79–85] 7 76 MPL, CY, VCR CHOP, CVP, Flu-CY; CHL, FN, DOF, PDN, IVIG, CYP, PE,SPL, DZL,, MTX, DNZ, AZA, SPL, IV MPL, oral PDN, IV IgG, Cs-A, PDN, MPM, MFT, DOR, BMC, VNB, DCN, IV high dose IG, – 4 × 375 mg/m2 weekly – 3 × 375 mg/m2 weekly –MPL, DZL, CYP, AMG, BMT, CSPA, steroids, AZA, DXM, APL, BP, PDN, CVP Fever, chills, upper airway edema, nausea, mild headache, itching, rash, hypotension, Grade IV neutropenia E. coli pyelonephritis, febrile bronchitis, primary varicella zoster, sepsis (2), limited RTI – 62 of 76 (87%) patients responded to RTX – 5 serious infections reported and 2 patients died of septicemia ANCA Associated Vasculitis [41,90–96] 8 73 (62 WG) CSs, cyp, TNF agonists, AZA, (10 PMA) (1 CS) MTX, IVIg, CSA, trimethoprim/ sulfamthoxazole, IFX, MTX, LFN, ETE, MMF, ETS, AMG, plasmapheresi, PE, ELT – 4 × 375 mg/m2 weekly – 2 – 4 × 500 mg weekly – 2 × 1000 mg 2 weeks apart LFN as maintenance, PDN, ACM, DPH, CYP, MTX, AHM, CSs, MF, AZA, Mild angioedema, hypertension, petechiae, dizziness, HACA, rigors, chills, postherpetic neuropathy, polyarthritis – serum sickness related, dizziness, lower extremity petechiae, thrombocytopenia, hypertension, fever, nausea, urticarial rash, limited RTI/UTI Herpes zoster (2), Influenza, septic arthritis – 65 of 73 (89%) patients responded to RTX Gastrointestinal hemorrhage, nephrolithiasis with acute renal colic, infusional reaction to RTX, renal failure, tremors, ischemic stroke Hepatitis B reactivation, septic arthritis, diarrhea (3), viral conjunctivitis, pneumonia, gram (–) sepsis – 48 of 72 (66%) patients responded to RTX Neutropenia, bradycardia, hypotension, left sided amaurosis, thrombosis of retinal artery Cryptococcosis, disseminated herpes simplex type 2 – 55 of 61 (90%) patients responded to RTX – 2 serious infections reported and 1 patient died of cryptoccocus Fever, chills, headache, hypotension, skin rash – 60 of 61 (98%) patients responded to RTX – No serious infections reported 4 72 –CSs, stem cell grafts, transplantation, PPX, TCR, MPL MPM, MFT, HCQ, TLM, ACT, ECP – 4 × 375 mg/m weekly PPX, CSs, MPL, AA, and HCN Cryoglobulinemia [104–109] 6 61 – 4 × 375 mg/m2 \ISA, CS, PE, IFNα, CS, DMARDs, CSs, CNI, MMF anti-viral therapy, plasmapheresis, weekly IVIg, MMF, or CSA. – 2 – 4 × 375 mg/m2 weekly – 4 – 8 × 375 mg/m2 weekly Thrombotic thrombocytopenic purpura [110,112–115] 5 61 PE, CS, vincristine, splenectomy, ISA, IVIg – 4 × 375 mg/m2 weekly PE, CSs – 4 serious infections reported – 10 serious infections reported and 3 patients died of infections (causative organisms not identified) AA, aerosolized albuterol; ACM, acetamenophine; ACT, acitretin; ADM, Adriamycin; AHM, antihistamine; AMG, antithymocyte globulin; AMI, amicar; APOL, allopurinol; AZA, azathioprine; BMC, Bleomycin; BMT, Bone Marrow Transplant; BP, Blood products; CCC, colchicin; CDP, Cladribine; CHL, chlorambucil; CHOP, Cytoxan, Hydroxyrubicin (Adriamycin), Oncovin (Vincristine), Prednisone; CLE, Clestamine; CPM, chlorophenamine; CSP, cyclosporine; CSPA, Cyclosporine A; CVP: Cyclophosphamide + Vincristine + Prednisone; Cy, cytoxan; CYP, Cyclophosmomide; DCN, dacarbazine; DOF, Deossicoformicin; DOR, deoxyrubicin; DPH, diphenhydramine; DXM, dexamethasone; DZL, Danazol; ECP, extracorporeal photopheresis; ELT, elemtuzumab; ETE, etenercept; ETS, etoposide; FLU, fludarabine; Flu-Cy, fludarabine, Cytoxan; HCQ, hydroxychloroquine; HCN, hydrocortisone; FN, fludarabine; IFN, Interferon; IFX, infliximab; IV IgG, intravenous immunoglobulin; IVGC, Intravenous glucocorticoid; KGT-FS, Kogenate FS; LFN, lefunomid; LTX, L-thyroxine; NVS, novoseven; MFT, mofetil; MMI, Methimazole; MMF, mycophenolate mofetil; MPC, mepacrin; MPL, methylprednisolone; MPM, mycophenolate; MTX, methotrexate; MX, mitoxantrone; MZB, mizoribine; PA, purine analogues; PCM, paracetamol; PDN, prednisolone; PE, plasma exchange; PPL, propanolol; PPX, prophylaxis; rFVIIa, recombinant human activated factor VII; RTX, rituximab; SPL, splenectomy; SRA, serotonin receptor agonist; TA, tranexamic acid; TCR, tacrolimus; TLM, thalidomide; TNF, tumor necrosis factor alpha; UTI, urinary tract infection; VCR, vincristine; VNB, Vinblastin; VZV, varicella-zoster virus. H.M. Gürcan et al. / International Immunopharmacology 9 (2009) 10–25 Graft versus host disease [98–101] 2 H.M. Gürcan et al. / International Immunopharmacology 9 (2009) 10–25 (1000 mg I.V. twice in 2 week intervals) were compared to 20 patients treated with CS. RTX had no effect on hyperthyroidism or the causative autoantibody levels, but RTX improved GO symptoms in all patients by decreasing proptosis and inflammation. The relapse rates of symptoms of GO for RTX and intravenous glucocorticoid treated patients were 0 and 10% respectively [133]. In another controlled study with 20 patients, 10 patients received RTX in combination with methimazole (MMI) and 10 patients received MMI alone. Four patients in the RTX group sustained remission in the follow up period (435–904 days) while all of the patients in the MMI group relapsed by day 393. The influence on autoantibody levels was the same in both groups [134]. No significant adverse events were reported in either study. 2.2.14. Hemophilia A Acquired Hemophilia A (AHA) is a disorder characterized by the production of autoantibodies specific for Factor VIII (FVIII) in the clotting cascade [135]. The disorder has an incidence of 0.2–1 per million per year with a mortality rate estimated between 8 and 22%. It is often associated with other autoimmune disorders, malignancies, and may be drug induced. 10% of cases occur in post partum women [135]. Therapy depends on the underlying pathology. It consists of systemic CS combined with CYP. However up to 30% of patients are refractory to such therapy [135]. RTX has been used in three studies in which a positive clinical response was observed in 20 of 21 (95%) patients [136–138]. RTX improved bleeding, compartment syndrome, epistaxis, bruising, hematoma development, and ecchymoses. RTX was typically given in four weekly infusions at a dose of 375 mg/m2. In one study, RTX was beneficial when combined with human FVIII administration. 4 of 5 patients experienced a reduction in inhibitor titers while the one patient that did not receive human FVIII did not have a reduction [136]. Two other studies reported a decline in the inhibitor titer with the use of the drug RTX without the need for long-term immunosuppressive therapy [137,138]. Two patients died of sepsis related to RTX therapy [138]. RTX therapy for AHA has an added complication in that the depletion of FVIII inhibitor levels may cause a dangerous rise in FVIII. In one study, 3 patients developed venous thrombosis when hospitalized for unrelated purposes [138]. There is a review on RTX treatment for AHA in which many of the reports do not conform to our inclusion criteria. However fifty seven of 65 patients in this review had a complete response to RTX therapy [135]. The literature would suggest that, RTX therapy seems beneficial in the treatment of AHA. 2.2.15. Opsoclonus myoclonus Opsoclonus myoclonus syndrome (OMS) is a rare neurological autoimmune disorder characterized by random, chaotic eye motion and spontaneous skeletal muscle contractions. Patients may also exhibit behavioral dysfunction, impaired reflexes, speech pathology, and sleep disturbances. OMS can be drug induced or can be caused by neural crest derived tumors and infection [139]. The disease is usually treated with CS, adrenocorticotropic hormone (ACTH) [139], and IVIG [140]. In one controlled study, 16 OMS pediatric patients were treated with a weekly 375 mg/m2 infusion of RTX for 4 weeks. RTX maybe an effective addition to concurrent IVIg and ACTH therapy for OMS [141]. 2.2.16. Dermatomyositis Dermatomyositis (DM) is a chronic autoimmune disorder characterized by a cutaneous rash and a progressive symmetrical weakening of proximal muscles. The disease often has articular, gastrointestinal, cardiac, pulmonary manifestations and a high cancer comorbidity. DM has an annual incidence of 0.6–1 per 100,000 and can occur at any age [142]. CS and ISA are the mainstay of DM therapy, though IVIg has been successful [143]. 19 RTX was used for the treatment of 15 DM patients in two studies [144,145]. Patients were unresponsive to at least one of the following prior to RTX therapy: CS, ISA, CSA, TNF-α inhibitors, MMF, AMA, or IVIg. In the earliest study, 3 patients received weekly 100 mg/m2 RTX infusions and 4 patients received weekly 375 mg/m2 infusions; all patients received a total of 4 infusions. Response to therapy was measured by an increase in strength over baseline ranging from 36– 113%, measured by manual testing and quantitative myometry. Improvements were not dose related. Response also involved improvements in creatine phosphokinase (CPK) levels, rash, body weight, hair growth, and pulmonary function. Minor adverse events included infusion-related hypertension and shortness of breath; one patient developed treatable grade III cellulitis [144]. In the other study, 8 patients received two 1000 mg infusions of RTX two weeks apart. Three of 8 patients had a 50% improvement in muscle strength deficit and thus met the study criteria for an objected response to RTX. There were no substantial improvements in skin disease, CPK levels, or patient assessed subjective improvement. The DM in the patients in this study was mild at baseline. The authors suggested that differences in DM severity, and RTX protocol could explain the difference in response rates between the two studies [145]. 2.2.17. Neuromyelitis optica Neuromyelitis optica (NMO) is a demyelinating disease of the central nervous system affecting the optic nerves and the spinal cord. In one study, 8 patients received 4 weekly infusions of RTX (375 mg/ m2). CS was given only for acute attacks of NMO during the study period. Seven of 8 patients responded to the RTX therapy with improvement in pyramidal, sensory, visual, bowel, and bladder nervous function. Expanded disability status scale scores decreased from a median of 7.5 to 5.5 [146]. 2.2.18. Idiopathic membranous nephropathy Idiopathic membranous nephropathy is an immune disorder in which IgG and complement components damage the basement membrane of the glomerular capillary wall. In one study 8 patients received 4 weekly infusions of RTX (375 mg/m2). Six patients showed improvement in proteinuria and serum albumin levels. Adverse events were minor [147]. 3. Discussion 3.1. Protocols There are two FDA approved uses for RTX. Each uses a different protocol. Treatment of lymphoma patients involved four infusions of RTX at a dose of 375 mg/m2, given on four consecutive weeks [22]. The protocol for RA was two infusions at a dose of 1000 mg of RTX, given two weeks apart [30]. The majority of the patients in this review received the lymphoma protocol. Some studies had minor variations. Many used concomitant therapies usually involving corticosteroids and immunosuppressive agents (Table 3). In the majority of studies investigators have utilized a treatment protocol often used for patients with lymphomas. This therapeutic approach requires careful scrutiny and reconsideration. The biology of B cells in lymphomas and in autoimmunity are more than likely to be different. It is fair to presume that B cells in autoimmune diseases are abnormal, compared to normal B cells, but are not malignant. Moreover the primary purpose of using RTX in autoimmunity has a different goal compared to lymphoma. Therefore investigators considering the use of RTX in autoimmune diseases, should design and implement protocols, which provide evidence based decision making choices for a clinician intending to use RTX. Furthermore, each autoimmune disease has different underlying bases for its pathobiology. Recognizing these differences will facilitate developing treatment strategies that are more appropriate and effective for each disease. 20 H.M. Gürcan et al. / International Immunopharmacology 9 (2009) 10–25 Like RTX, IVIg has been successfully used to treat many autoimmune diseases [148]. In several studies in this review, IVIg was used in combination with RTX therapy [37,81,82]. The combination produced an excellent clinical response with minimal adverse events. This observation raises an important issue. Would the combination of RTX and IVIg work in harmony and symbiotically to produce long-term remission in autoimmune diseases? 3.2. Rituximab and intravenous immunoglobulin (IVIg) for the treatment of autoimmune diseases A recent study demonstrated the beneficial effects of inducing long-term remission using a combination therapy of IVIg and rituximab in treatment resistant patients with pemphigus vulgaris [127]. Since plasma cells lack CD20 expression they are not affected by rituximab treatment. Our review of the literature suggests that in many patients, rituximab therapy did not completely decrease the autoantibody titers. One of the suggested mechanisms of IVIg in autoimmune disease is its ability to bind idiotipic determinants of auto-antibodies. It is proposed that by this mechanism IVIg modulates B cells via the antigen receptor. IVIg can also reduce the activity of various auto-antibodies and/or block the binding of auto-antibodies to their relevant auto-antigens in vitro. In some autoimmune diseases monotherapy with IVIg has been demonstrated to decrease autoantibody titers [149,150]. Hence there is emerging evidence that the combination of IVIg and rituximab might be a better choice for the treatment of autoimmune disease where the main pathway for the pathogenesis is autoantibody mediated [127]. IVIg may also aid in the re-establishment of immune tolerance by inhibiting pathogenic cytokine production. IVIg selectively up regulates the expression of an IL-1 receptor agonist on macrophages, thus in turn effectively downregulates the production of inflammatory cytokines such as IL-1α, IL-1β, IL-6, and TNF-α [151–155]. IVIg preparations also contain anti-inflammatory cytokines such as TGFβ, which may further help in the re-establishment of immune tolerance [156]. IVIg is also demonstrated to inhibit maturation of dendritic cells (DC) and IL-12 production, and increase IL-10 production. Resulting tolerogenic DC may also be beneficial in the re-establishment of immune-tolerance [157]. Auto-antigens may be retained in secondary lymphoid tissues in the manner of immune complexes on follicular dendritic cells (FDC). These immune complexes retained on FDC are hypothesized to maintain long-term humoral immune responses by continuous providing antigen presentation to B cells and T cells. Since rituximab does not uniformly decrease auto-antibody titers it is possible that these immune-complexes retained on FDC might facilitate the production of autoantibody. IVIg may hypothetically remove these immune complexes by binding to FcγRIII-A receptors on FDC. This process have the potential to then reestablish immune tolerance [158–160]. IVIg may also modulate immune responses through inhibitory Fcγ receptors on cells of the reticulo-endothelial system (RES). Occupation of Fc receptors on the cells of the RES might also increase rituximab titers in the serum and increase its half life. These supplementary functions of IVIg might be beneficial during the combined treatment using IVIg with RTX. 3.3. Safety A 2005 report on the safety of RTX in patients with cancer and RA concluded that serious adverse reactions occur only in a small minority of patients but overall RTX therapy is safe [161]. The adverse event data included in this RTX review are compared to information in the 2005 report. The 2005 report found 84% of patients experience infusion related reactions including nausea, headache, fatigue, rash, and flu-like symptoms after RTX administration. 97% of these reactions are grade 1–2 intensity on the National Cancer Institute Toxicity Scale. The incidence of these symptoms is highest after the first infusion and decreases with each subsequent infusion [161]. These observations correlate well with the RTX use in autoimmune disease. Most studies reported a high incidence of minor infusion related reactions with a minority of patients requiring cessation of RTX (Table 3). The use of paracetamol, antihistamines, and CS are recommended as pretreatment to help control infusion related reactions [161]. RTX associated infections are a reasonable concern due to the depletion of the B cell component of the immune system. The 2005 report stated that 30% of RTX treated patients acquire some type of infection, however, only 1–2% of patients acquire severe infections. The report suggested that the low incidence of infection is due to steady serum immunoglobulin levels and T-cells activity, neither of which appears to be directly affected by RTX [161]. These observations again correlate well with RTX use in this review. Minor, treatable infections were common in B cell depleted patients whereas lethal severe infection occurred in a small minority (Table 3). One study supports the hypothesis that T cells are crucial to fight off infection during B cell depletion consequently patients treated with RTX who simultaneously receive concomitant T cell immunosuppressive agents can experience serious or even fatal systemic infection [108]. In the analysis of the data in this review we observed that in 25 studies, involving 389 patients, serious infections were reported. The incidence varied from 2.8% to 45% (mean 12.5%) [34,37,39,41,43– 46,49,50,67,73,80–83,90,96,100,101,108,128,130,138,144]. The authors have used the criteria established by individual investigator to consider an infection “serious”. Several of these infections were grade 3 or 4 infections based on the National Cancer Institute's criteria. The validity of the frequency of infection in patients with autoimmune diseases treated with RTX is a multi-factorial phenomenon and becomes difficult to analyze because there is no unifying factor in these different diseases. Moreover patients are frequently on concomitant immunosuppressive therapies which also enhance or contribute to susceptibility to infection. Also patients are at various points during the clinical evolution of their disease which can influence susceptibility to infection. Furthermore the age of the patient, the presence of other medical problems and unrelated yet consequential drug therapies could play a significant role. The use of RTX has been directly attributed as a cause of death in 8 studies [39,67,80,83,101,108,130,138]. In these studies which represent 147 patients, the death rate varied from 2.8% to 33% (mean 7.4%). The accurate and complete interpretation of this data is difficult, because the patients who died as a consequence of RTX had a wide spectrum of diseases. Furthermore the time at which RTX was used in the course of the disease, the severity of the disease, and the concomitant therapies that contributed to profound immune suppression were different in each study. However it does appear that RTX in a certain group of patients in several diseases in which it has been used, may predispose patients to developing severe infections which can infrequently lead to death. The incidence of human anti-chimeric antibodies (HACA) against RTX was very low in the 2005 report and HACA did not influence the efficacy or toxicity of RTX therapy [161]. This does not correlate with the presence of HACA in patients with autoimmune disease. Studies have reported the appearance of significantly high titers of HACA and that the presence of HACA was associated with failure to deplete B cells [26,34,47,120,121]. Patients with autoimmune diseases may be more susceptible to the development of HACA after RTX therapy compared to lymphoma patients due to polyclonal B cell activity [45]. Humanized RTX has been used to avoid HACA associated complications [162]. It would appear that these RTX resistant B cells now have one or more different biological features from the original or naïve CD20+ B-cells that they were before rituximab. This drug resistance has the potential to create additional features of this phenomenon. Exactly what the consequences of these changes will be remains to be studied or observed, especially the specific function of CD20 is still unknown. H.M. Gürcan et al. / International Immunopharmacology 9 (2009) 10–25 Patients with B cell cancers can experience cytokine release and tumor lysis syndrome after RTX therapy. Since these events do not occur in RA, the 2005 report found the overall incidence and severity of adverse reactions to be less in RA patients compared with cancer patients [161]. By inference, this finding should also apply to other autoimmune diseases beyond RA. The similarities between the findings of 2005 safety report and the adverse event observations in this review suggest that RTX is a well tolerated therapy for autoimmune disease. The 2005 report, however, was based on the fact that RTX had been used in over 540,000 cancer patients while there are far fewer patients treated with autoimmune diseases. Safety studies for RTX in autoimmunity with larger sample sizes are needed to provide a definitive answer regarding RTX tolerability in autoimmune diseases. 4. Conclusion This review of the current literature demonstrates that RTX induced B cell depletion resulted in a significant improvement of morbidity in many autoimmune diseases (Table 3). It appears that in diseases where the pathogenesis is antibody related, all of the treatment protocols depleted B cells, however a decrease in the pathologic antibody titers among the different diseases varied. Post-RTX treatment, B cell counts returned to normal levels, however autoimmune disease did not relapse in a significant portion of patients. These observations suggest that RTX mediated B cell depletion may have a positive influence on other cells of the immune system and may aid in the re-establishment of immune tolerance and homeostasis. Current studies suggest that RTX mediated depletion of B cells results in generation of FoxP3+ T regulatory cells along with down regulation of CD40 expression [163]. Regulatory T cells are effective suppressors of autoimmune responses in many animal models of autoimmune diseases [164–169]. Down regulation of CD40–CD40 ligand interactions may also aid in the re-establishment of immune tolerance by down regulating T cell help to the B cells during the re-population of B cell compartment after post-RTX therapy. It is known that B cells are capable of processing the antigen and presenting it to T cells. Antigen presentation by immature B cell can result in T cell tolerance. On the contrary, activated mature B cells are frequently inducers of T cell immunity. Activated B cells are implicated in a variety of autoimmune diseases as primary antigen presenting cells (APC) responsible for the pathogenesis [170–173]. Depletion of B cells could possibly alter the balance between autoimmune and tolerogenic mechanisms and may provide the opportunity for other APC such as tolerogenic dendritic cells to re-establish immune tolerance [174–180]. During the re-population of B cell compartments, post-RTX treatment, it is possible that most of the regenerated B cells are immature and they may further tolerize autoimmune T cells [181,182]. Recognition of MHC-Peptide complexes by naïve T cells are not sufficient enough to induce autoimmune responses. Signaling through co-stimulatory molecules such as CD28/CD80–CD86 is necessary. B cells also require T cell help to produce antibodies through CD40–CD40L interactions [183,184]. RTX treatment of SLE patients demonstrated prolonged inhibition of CD40 and CD80 on re-populated B cells [185]. This may further inhibit autoimmune T cells and reestablish immuno-tolerance. It is also possible that pathogenic B cells may influence the function of other immune cells by their production of inflammatory cytokines and chemokines in the pathogenesis of autoimmune disease. Removal of the B cells may result in the down regulation of these pro-inflammatory cytokines and consequently down-regulate the pathogenesis of the autoimmune responses. IL-10 activates T helper 2 (Th-2) cells which are inducers of tolerogenic dendritic cells [186–188]. RTX mediated B cell depletion may activate these tolerogenic dendritic cells which may also benefit the re-establishment of the immune tolerance. There is also current research that suggests RTX treatment down regulates pathogenic 21 macrophage responses [189]. Blocking of Fc inhibitory receptors due to RTX binding on effector phagocytes such as macrophages, neutrophils and monocytes may also down regulate inflammatory reactions to immune complexes involved in the pathogenesis of some diseases such as SLE, RA, and ITP. This review contains information on several studies so that a reader can benefit from the knowledge of the experience of the use of RTX. However there are several limitations to the analysis. The lack of uniform criteria for indication of use of RTX limits the ability to determine the ideal time to use it. The lack of objective criteria to evaluate clinical response also makes it difficult to determine what to expect after using it. In a significant number of studies the concomitant use of CSs and ISAs create an environment in which it is difficult to isolate the beneficial effect of RTX. In some studies the lack of information on timing of repopulation of B cells and its correlation with clinical disease creates the dilemma of establishing a clear-cut relationship between depletion of B cells and clinical recovery. The authors recognize that the use of RTX in autoimmune diseases is still at an early and possibly investigational stage. As new studies are planned it would be vital if the investigators defined their objective clearly. For example, is the purpose of using RTX a method to produce the dose of systemic CSs or to decrease the use of concomitant immunosuppressive agents, or an attempt to produce a lasting and sustained clinical remission. The effect of RTX on the clinical course of the disease can only be determined if a significant long-term followup is provided. Hence a minimum of two years follow-up should be a yard stick for such an assessment. Many studies did not evaluate B cell levels after initiating RTX treatment. Since each disease has a separate pathobiology documenting the time at which complete depletion of B cells occurs is important. Similarly it is critical to know the time at which the B cells repopulate to a normal level. Some patients may not achieve a B cell recovery for 18–24 months and during this period are susceptible to infection. Consequently such late side effects of RTX maybe missed if careful follow-up is not provided. A very major area concern for any clinician using RTX is its long term effects and potential long-term hazards. While the majority of current studies indicate that the use of RTX does not affect the serum immunoglobulin levels, there is no concrete evidence to indicate that repeated long-term use could not result in hypogammaglobulinemia. It is neither clear nor apparent in any study thus far that, in patients with autoimmune diseases who have had malignant disease in the past and are free of tumor at the time of the administration of RTX, there may be enhanced predisposition to having recurrence of the tumor secondary to the profound immunosupression caused by RTX. While the loss of B-cells is limited in time in most patients with autoimmune disease, the recovery to normal levels of B-cells can be delayed in certain diseases and often in elderly patients. Prolonged immunosupression has been associated with increased incidence of cancer. Hence, it seems appropriate and justified that clinicians using RTX should be aware of the unknown but possible development of malignancies in patients who have received RTX. The use of RTX in autoimmune diseases is rapidly increasing [6]. Its efficacy and safety varies among different autoimmune diseases. The vast majority of studies in this review suggest that RTX may be beneficial in their treatment. Further investigation with RCTs will provide more insight into the specifics of the role of RTX in the overall management of each disease. The cumulative information provided in this review would be of significant benefit in designing these protocols, identifying definitive objectives and outcome measures, and deciding when, how and in what therapeutic regimen will RTX most benefit a disease or a patient. Acknowledgements The authors are grateful to Olga Lyczmanenko, BA, MA, MLIS, library manager and Christopher Vaillancourt, BA, MA, MLIS of the 22 H.M. 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