Multiple cytokines play a pivotal role in the pathogenesis of rheumatoid arthritis (RA). The appropriate intracellular signalling pathways must be activated via cytokine receptors on the cell surface, and the tyrosine kinases transduce the first ‘outside to in’ signals to be phosphorylated after receptor binding to its ligand. Among them, members of the Janus kinase (JAK) family are essential for the signalling pathways of various cytokines and are implicated in the pathogenesis of RA. The in vitro, ex vivo and in vivo effects of a JAK inhibitor CP-690,550 (tofacitinib) for the treatment of RA are reported. In vitro experiments indicated that the effects of tofacitinib were mediated through suppression of interleukin 17 (IL-17) and interferon γ production and proliferation of CD4 T cells, presumably Th1 and Th17. A treatment study was conducted in the severe combined immunodeficiency (SCID)-HuRAg mice, an RA animal model using SCID mice implanted with synovium and cartilage from patients. Tofacitinib reduced serum levels of human IL-6 and IL-8 in the mice and also reduced synovial inflammation and invasion into the implanted cartilage. A phase 2 double-blind study using tofacitinib was carried out in Japanese patients with active RA and inadequate response to methotrexate (MTX). A total of 140 patients were randomised to tofacitinib 1, 3, 5, 10 mg or placebo twice daily and the American College of Rheumatology 20% improvement criteria (ACR20) response rate at week 12, a primary end point, was significant for all tofacitinib treatment groups. Thus, an orally available tofacitinib in combination with MTX was efficacious and had a manageable safety profile. Tofacitinib at 5 and 10 mg twice a day appears suitable for further evaluation to optimise the treatment of RA.
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Rheumatoid arthritis (RA) is a representative autoimmune disease characterised by chronic and destructive inflammatory synovitis that causes severe disability and mortality. A new concept of ‘treat-to-target’ is emerging in treatments of RA, whereby patients are treated according to prespecified goals, such as remission. Conventional disease-modifying antirheumatic drugs (DMARDs), most commonly methotrexate (MTX), remain the cornerstone of RA treatment. Patients for whom MTX produces an inadequate response are treated with biological agents targeting tumour necrosis factor (TNF) and interleukin 6 (IL-6). The combined use of a TNF inhibitor and MTX has produced previously unseen significant improvements in clinical, structural and functional outcomes and has revolutionised the treatment goal of RA to clinical remission. However, since only about 30% of patients treated in this way attained clinical remission, next-generation treatments are a prerequisite for patients with refractory RA.1,–,3
The importance of inflammatory cytokines in the pathogenesis of RA has become apparent from the clinical efficacy of biological agents. For such cytokines to exert their biological activities, the appropriate signalling pathways must be activated via their specific receptors on the cell surface. Tyrosine kinases are the first intracellular signalling molecules to be activated after receptor binding in a cytokine response and play a part in inflammation. Among them, a Janus kinase (JAK) family has received particular attention since JAKs are essential to the signalling pathways of various cytokines and are implicated in the pathogenesis of RA. JAK3 expression is mainly limited to lymphocytes and dendritic cells and constitutively binds to the common γ (γc) chain, which is a common receptor subunit for many cytokines involved in RA (figure 1).
In view of this background, an orally available JAK inhibitor CP-690,550, which is now designated tofacitinib, was developed with the expectation that it would be a new immunosuppressant agent with few side effects.4 5 Tofacitinib improved end points of both murine collagen-induced arthritis and rat adjuvant-induced arthritis. Tofacitinib at low concentration was also reported to greatly suppress JAK3 with few side effects in a graft-versus-host disease experiment.4,–,6 Tofacitinib is currently being used in clinical trials for RA, with satisfactory effects and acceptable safety. However, the mode of action of tofacitinib in patients with RA remains unclear. We here document the in vitro, ex vivo and in vivo effects of a JAK inhibitor for the treatment of RA.
JAK-STAT pathway in inflammation
RA is characterised by systemic, chronic and destructive inflammatory synovitis. Various intercellular signalling pathways have a pivotal role during its pathological process, some of which are mediated by soluble ligands, such as cytokines and growth factors, and others are affected by cognate interaction through costimulatory molecules and adhesion molecules. For such intercellular signals to exert their biological activities, the appropriate intracellular signalling pathways must be activated by engagement of their specific receptors on the cell surface, that is ‘outside to in’ signalling. The intracellular signals are represented by the following pathway: (1) phosphorylation of a protein kinase such as serine/threonine kinase, tyrosine kinase and mitogen-activated protein kinase; (2) guanosine triphosphate-binding proteins, including small G-protein such as Rho and Ras and heterotrimeric G-protein consisting of Gα, Gβ, Gγ; (3) second messengers such as cyclic adenosine cyclic monophosphate and guanosine cyclic monophosphate; (4) protease-activating, apoptotic-related proteins such as caspase; (5) ubiquitination. The transduction of these intracellular signals leads to various cellular functions through directly, sequentially activating or regulating one another.
More than 99% of kinase proteins are serine/threonine kinases under physiological conditions. In contrast, although tyrosine kinase accounts for <1% of them under ordinary conditions, it is the first intracellular signalling molecule to be phosphorylated after receptor binding in a cytokine response and is involved in fundamental functions, such as cell proliferation, differentiation and adhesion in various pathological processes, including inflammation and cancer. Therefore, many investigators have examined tyrosine kinases as a target for the treatment of various diseases. More than 90 genes encoding tyrosine kinases have been identified from human genome-wide studies and 14 tyrosine kinases are known to be involved in RA.7
Of the tyrosine kinases, a JAK family, consisting of JAK1, JAK2, JAK3 and Tyk2, has received particular attention since JAKs are essential for the signalling pathways of various cytokines. JAKs are phosphorylated just after cytokines bind to their receptors and consecutively activate transcription factor signal transducers and activators of transcription (STAT) (figure 1).8,–,13 After the engagement of homodimeric or heterodimeric receptors for cytokines and growth factors, which are constitutively bound to JAKs, JAKs are activated by a conformational change in the receptor that allows trans- and/or autophosphorylation of the two bound JAKs. These in turn phosphorylate the cytokine receptors. STAT proteins bind the phosphorylated receptor chains, which allows the JAKs to phosphorylate the STATs. Phosphorylated STATs form dimers and translocate into the nucleus, where they regulate gene trancription and expression.
Thus, the JAK-STAT pathway regulates multiple immune functions. For instance, different STATs are involved in differential cytokine production from CD4 T cell subsets: STAT1 and STAT4 mainly induce interferon γ (IFNγ) from Th1; STAT6 induces IL-4 from Th2; STAT5 induces transforming growth factor β from regulatory T cells and STAT3 induces IL-17 from Th17. JAK3 expression is essentially limited to lymphocytes and dendritic cells and constitutively binds to the γc chain, which is a common receptor subunit for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. The deficiency or dysfunction of JAK3 leads to severe combined immunodeficiency (SCID) in both humans and mice. Thus, a number of tyrosine kinase inhibitors have recently been evaluated in clinical trials. Selective inhibition of JAK3 was considered as a potential target in the treatment of RA without affecting other organ systems.
In vitro effects of a JAK inhibitor in RA
JAKs are essential for the signalling pathways of various cytokines and are implicated in the pathogenesis of RA. An orally available JAK inhibitor CP-690,550 (tofacitinib) is undergoing clinical trials for RA, with satisfactory effects and acceptable safety. However, the mode of action of tofacitinib in patients with RA remains unclear. Walker et al reported that JAK3, STAT1, STAT4 and STAT6 were highly expressed in the synovium of patients with RA, whereas their expression was rare in the synovium of normal volunteers and patients with osteoarthritis and spondyloarthritis.14
In view of the important role of JAK3 in lymphocyte development, differentiation and proliferation, we assessed the effects of tofacitinib on CD4 T cells at local inflammatory sites in patients with RA. We have previously assessed the effects of tofacitinib on immune cells prepared from the peripheral blood and synovium tissue of patients with RA.15 The proliferation of CD4 synovial T cells in patients with RA stimulated with anti-CD3 and anti-CD28 antibodies was inhibited by tofacitinib in a dose-dependent manner. Treatment of synovial CD4 T cells with tofacitinib inhibited production of IL-17 and IFNγ, but had no effect on IL-6 and IL-8 production. However, CD14 monocytes and synovial fibroblasts isolated from the synovium in patients with RA were not affected by tofacitinib. Our in vitro results suggested that the effects of tofacitinib in RA are mediated through the suppression of IL-17 and IFNγ production and proliferation of CD4 T cells without affecting synovial fibroblasts and monocytes. Since IFNγ and IL-17 are produced by Th1 and Th17 cells, respectively, and are important drivers of destructive arthritis in mice and humans, JAK3 in CD4 T cells, presumably Th1 and Th17 cells, plays a crucial role in rheumatoid synovitis.
Ex vivo effects of a JAK inhibitor in RA
We next conducted a treatment study in SCID-HuRAg mice, an RA animal model using SCID mice implanted with synovium and cartilage from patients with RA, in which tofacitinib was administered via an implanted osmotic mini-pump.15 Male SCID mice (C.B-17/lcr), 6–8 weeks old, were housed in specific pathogen-free conditions at our university animal centre. Synovial tissue and articular cartilage and bone obtained from two patients with RA at the time of joint replacement surgery were used. The synovium was cut into sections, 5–10 mm in diameter, cartilage was cut into 2 mm3 pieces, and then synovium and cartilage were transplanted onto the back of nine SCID mice. A week after the implantation, the nine mice were randomly divided into three groups, and tofacitinib dissolved in polyethylene glycol 300 was administered continuously at 0 (n=3), 1.5 (n=3) or 15 (n=3) mg/kg/day via Alzet osmotic mini-pumps implanted subcutaneously on the back. Blood samples were collected and the serum samples were stored at −80°C until measurement of IL-6 and IL-8. Treatment of SCID-HuRAg mice with tofacitinib reduced their serum levels of human IL-6 and IL-8. However, we have previously shown that tofacitinib did not affect IL-6 and IL-8 production from CD4 T cells, synovial fibroblasts and CD14 monocytes in vitro. On the other hand, IL-17 and IFNγ production is known to induce cytokine production from monocytes and fibroblast, and IL-6 has been reported to be mainly derived from macrophages and fibroblasts of the synovium.13 14 These findings led us to speculate that tofacitinib specifically inhibited IL-17 and IFNγ production by CD4 T cells (presumably Th1 and Th17 cells), which in turn regulated synovitis by indirectly suppressing IL-6 and IL-8 from synovial fibroblasts and CD14 monocytes.
Next, implanted tissues were removed from the SCID-HuRAg mice 5 weeks after implantation, paraffin embedded and stained with haematoxylin and eosin. Histological evaluation was carried out and showed that in mice treated with vehicle alone, prominent invasion of the synovial tissue into the implanted cartilage had occurred. However, treatment with tofacitinib markedly inhibited this invasion, indicating that tofacitinib has the potential to inhibit the progression of structural damage of joints in patients with RA (figure 2).
In vivo effects of a JAK inhibitor in RA
In view of these results, an orally available JAK inhibitor, tofacitinib, is currently undergoing clinical trials for RA. Kremer et al reported a phase II dose-ranging trial which was carried out to investigate the efficacy, safety and tolerability of oral tofacitinib in 264 patients with active RA in whom MTX, etanercept, infliximab or adalimumab caused an inadequate or toxic response.16 17 Patients were randomised to placebo, 5, 15 or 30 mg tofacitinib twice daily for 6 weeks, and were followed up for an additional 6 weeks after treatment. The American College of Rheumatology 20% improvement criteria (ACR20) response rate was 26.9%, 70.5%, 81.2% and 76.8% in the placebo, 5, 15 and 30 mg twice daily groups, respectively, at 6 weeks. Thus, patients treated with tofacitinib in all treatment groups achieved the primary efficacy end point—ACR20 response rate at 6 weeks. Rapid improvements in disease activity were seen in patients treated with tofacitinib, and ACR50 and ACR70 response rates significantly improved in all treatment groups by week 4. The most common adverse events (AEs) reported were headache and nausea. The infection rate in the 15 mg twice daily group and the 30 mg twice daily group was 30.4% (26.2% in placebo) and opportunistic infections or deaths were not seen.
A phase II, double-blind study was also carried out to investigate the efficacy and safety of orally available tofacitinib in Japanese patients with active RA in whom MTX had produced an inadequate response.18 A total of 140 patients were randomised to tofacitinib 1, 3, 5, 10 mg, or placebo twice daily in this 12-week trial and continued to receive background MTX. The ACR20 response rates at week 12, a primary end point, were significant for all tofacitinib treatment groups—14.3%, 64.3%, 77.8%, 96.3%, 80.8% in the placebo, 1, 3, 5 and 10 mg twice daily groups, respectively, at 12 weeks (figure 3). Significant improvements in ACR50, ACR70 and the Health Assessment Questionnaire-Disability Index were also obtained by the use of 5 or 10 mg tofacitinib. Furthermore, in patients with high disease activity at baseline (28-joint Disease Activity Score (DAS28) >5.1), the greatest percentage of patients achieving DAS remission at week 12 was seen in the group receiving tofacitinib 10 mg twice daily (45.5%). In patients with low to moderate disease activity at baseline (DAS28 ≤5.1), the group receiving tofacitinib 5 mg twice daily contained the greatest percentage of patients achieving DAS remission at week 12 (80.0%).
The most commonly reported AEs were nasopharyngitis (n=13), and increased alanine aminotransferase (n=12) and aspartate aminotransferase (n=9). These AEs were mild or moderate in severity. Serious AEs were reported by five patients, but no deaths occurred. It is noteworthy that dose-dependent decreases in mean neutrophil counts were seen but did not result in any patients discontinuing treatment. At week 12, the mean decrease in neutrophil counts from baseline was significantly different from placebo for all tofacitinib treatment groups. Also, small changes in mean haemoglobin levels were observed across all of the tofacitinib treatment groups, although no potentially life-threatening anaemia was seen for any treatment group. Furthermore, dose-dependent increases in low-density lipoprotein, high-density lipoprotein and total cholesterol were seen, and appeared to plateau between weeks 4 and 12, although no patients stopped treatment owing to increases in serum lipids.
Neutrophil changes and anaemia may result from treatment with tofacitinib, which can inhibit signalling by haematopoietic cytokines, such as erythropoietin and granulocyte–macrophage colony-stimulating factor, through JAK2. It is interesting that some of the AEs seen here for tofacitinib, including decreases in neutrophils and increases in cholesterol and liver transaminases, were similar to those previously reported for tocilizumab, a humanised anti–IL-6 receptor antibody that blocks IL-6 signalling.19 Results from animal models of arthritis, showing decreased IL-6 levels in tofacitinib-treated animals, and the dose-dependent increase in blood lipids levels seen in clinical trials, suggest that tofacitinib may have an inhibitory effect on IL-6. Therefore, although tofacitinib has been reported to be highly specific to JAK3, it is now known that it functions as a pan-JAK inhibitor because the inhibition of JAK1 and JAK2 should also be taken into account.
In Japanese patients with active RA and in whom MTX had produced an inadequate response, an orally available JAK inhibitor tofacitinib, in combination with MTX over 12 weeks was efficacious and had a manageable safety profile. Accordingly, longer dose-ranging studies of this new JAK inhibitor tofacitinib in the treatment of patients with RA who are MTX-naïve, or who have an inadequate response to MTX/DMARDs or TNF inhibitors are continuing. Multiple global clinical examinations and efficacy studies examining the regulation of progress in structural damage and functional disability are continuing.
Biological DMARDs such as TNF inhibitors have changed the treatment strategy of RA. Clinical remission is, however, obtained in only one-third of patients treated with biological agents, and antibodies to these agents are found. Accordingly, orally available low molecular weight products such as tofacitinib, targeting intracellular signalling molecules, would provide enormous power and flexibility in the treatment of RA. It has become clear that JAK inhibition with tofacitinib in patients with RA results in a rapid and remarkable clinical effect equivalent to that of TNF inhibitors. However, the mechanism of action of tofacitinib had not been determined. Our in vitro and in vivo studies have shown that tofacitinib mainly acts on CD4 T cells, subsequently suppressing cell proliferation and production of inflammatory cytokines such as IL-17 and IFNγ and reducing synovial inflammation (figure 4). Since it is possible to design low molecular weight products recognising a particular conformation of target molecules in the signalling cascade, the success of tofacitinib will accelerate new development of multiple products for RA and for many inflammatory diseases.
The authors thank all medical staff in all institutions for providing the data.
Funding The clinical examination regarding tofacitinib (CP-690,550) was sponsored by Pfizer Inc and the compound CP-690,550 for in vitro studies was provided by Pfizer Inc. The series of studies were also supported in part by a research grant-in-aid for scientific research by the Ministry of Health, Labor and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan and the University of Occupational and Environmental Health, Japan.
Competing interests YT has received consulting fees, speaking fees, and/or honoraria from Mitsubishi-Tanabe Pharma, Chugai Pharma, Eisai Pharma, Pfizer, Abbott Immunology Pharma, Daiichi-Sankyo, Janssen Pharma, Astra-Zeneca, Takeda Industrial Pharma, Astellas Pharma, Asahi-kasei Pharma and GlaxoSmithKline and has received research grant support from Mitsubishi-Tanabe Pharma, Bristol-Myers Squibb, Takeda Industrial Pharma, MSD, Astellas Pharma, Eisai Pharma, Chugai Pharma, Pfizer and Daiichi-Sankyo.
Provenance and peer review Commissioned; externally peer reviewed.