Chronic pain is a major clinical problem that is difficult to treat and requires novel therapies. Although most pain therapies primarily target neurons, neuroinflammatory processes characterized by spinal cord and dorsal root ganglion production of proinflammatory cytokines play an important role in persistent pain states and represent potential therapeutic targets. Anti-inflammatory cytokines are attractive candidates to regulate aberrant neuroinflammatory processes, but the therapeutic potential of these cytokines as stand-alone drugs is limited. Their optimal function requires concerted actions with other regulatory cytokines, and their relatively small size causes rapid clearance. To overcome these limitations, we developed a fusion protein of the anti-inflammatory cytokines interleukin 4 (IL4) and IL10. The IL4-10 fusion protein is a 70 kDa glycosylated dimeric protein that retains the functional activity of both cytokine moieties. Intrathecal administration of IL4-10 dose-dependently inhibited persistent inflammatory pain in mice: three IL4-10 injections induced full resolution of inflammatory pain in two different mouse models of persistent inflammatory pain. Both cytokine moieties were required for optimal effects. The IL4-10 fusion protein was more effective than the individual cytokines or IL4 plus IL10 combination therapy and also inhibited allodynia in a mouse model of neuropathic pain. Mechanistically, IL4-10 inhibited the activity of glial cells and reduced spinal cord and dorsal root ganglion cytokine levels without affecting paw inflammation. In conclusion, we developed a novel fusion protein with improved efficacy to treat pain, compared with wild-type anti-inflammatory cytokines. The IL4-10 fusion protein has potential as a treatment for persistent inflammatory pain.
The results of current preliminary study suggest that IL4-10 FP has DMOAD potentials since it shows chondroprotective and anti-inflammatory effects in vitro, as well as potentially analgesic effect in a canine in vivo model of osteoarthritis.
SummaryThe objective of this study was to test the capacity of a newly developed fusion protein of interleukin 4 (IL‐4) and IL‐10 [IL4‐10 fusion protein (FP)] to shift multiple pro‐inflammatory pathways towards immune regulation, and to inhibit pro‐inflammatory activity in arthritis models. The effects of IL4‐10 FP in comparison with IL‐4, IL‐10 and IL‐4 plus IL‐10 on pro‐ and anti‐inflammatory mediators, T cells and immunoglobulin (Ig) receptors in favour of immunoregulatory activity were studied. In addition, the capacity of IL4‐10 FP to inhibit pro‐inflammatory activity in ex‐vivo and in‐vivo arthritis models was investigated. IL4‐10 FP robustly inhibited pro‐inflammatory cytokine [IL‐1β, tumour necrosis factor (TNF)‐α, IL‐6 and IL‐8] production in whole blood cultures, mediated by both the IL‐10 and the IL‐4 moiety. IL4‐10 fusion protein induced IL‐1 receptor antagonist (IL‐1RA) production and preserved soluble TNF receptor (sTNFR) levels, strongly increasing IL‐1RA/IL‐1β and sTNFR/TNF‐α ratios. In addition, IL4‐10 FP strongly inhibited T helper (Th) type 1 and 17 cytokine secretion, while maintaining FoxP3 expression and up‐regulating Th2 activity. In addition, while largely leaving expression of activating Fc gamma receptor (FcγR)I, III and Fc epsilon receptor (FcεR) unaffected, it significantly shifted the FcγRIIa/FcγRIIb ratio in favour of the inhibitory FcγRIIb. Moreover, IL4–10 FP robustly inhibited secretion of pro‐inflammatory cytokines by rheumatoid arthritis synovial tissue and suppressed experimental arthritis in mice, without inducing B cell hyperactivity. IL4‐10 fusion protein is a novel drug, signalling cells to induce immunoregulatory activity that overcomes limitations of IL‐4 and IL‐10 stand‐alone therapy, and therefore has therapeutic potential for inflammatory diseases such as rheumatoid arthritis.
New therapeutic approaches to resolve persistent pain are highly needed. We tested the hypothesis that manipulation of cytokine receptors on sensory neurons by clustering regulatory cytokine receptor pairs with a fusion protein of interleukin (IL)-4 and IL-10 (IL4–10 FP) would redirect signaling pathways to optimally boost pain-resolution pathways. We demonstrate that a population of mouse sensory neurons express both receptors for the regulatory cytokines IL-4 and IL-10. This population increases during persistent inflammatory pain. Triggering these receptors with IL4–10 FP has unheralded biological effects, because it resolves inflammatory pain in both male and female mice. Knockdown of both IL4 and IL10 receptors in sensory neurons in vivo ablated the IL4–10 FP-mediated inhibition of inflammatory pain. Knockdown of either one of the receptors prevented the analgesic gain-of-function of IL4–10 FP. In vitro, IL4–10 FP inhibited inflammatory mediator-induced neuronal sensitization more effectively than the combination of cytokines, confirming its superior activity. The IL4–10 FP, contrary to the combination of IL-4 and IL-10, promoted clustering of IL-4 and IL-10 receptors in sensory neurons, leading to unique signaling, that is exemplified by activation of shifts in the cellular kinome and transcriptome. Interrogation of the potentially involved signal pathways led us to identify JAK1 as a key downstream signaling element that mediates the superior analgesic effects of IL4–10 FP. Thus, IL4–10 FP constitutes an immune-biologic that clusters regulatory cytokine receptors in sensory neurons to transduce unique signaling pathways required for full resolution of persistent inflammatory pain.
Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.
Purpose Modulating sialylation of therapeutic glycoproteins may be used to influence their clearance and systemic exposure. We studied the effect of low and high sialylated IL4-10 fusion protein (IL4-10 FP) on in vitro and in vivo bioactivity and evaluated the effect of differential sialylation on pharmacokinetic parameters. Methods CHO cell lines producing low (IL4-10 FP lowSA) and high sialylated (IL4-10 FP highSA) fusion protein were generated. Bioactivity of the proteins was evaluated in an LPS-stimulated whole blood assay. Pharmacokinetics were studied in rats, analyzing plasma levels of IL4-10 FP upon intravenous injection. In vivo activity was assessed in an inflammatory pain mice model upon intrathecal injection. Results IL4-10 FP lowSA and IL4-10 FP highSA had similar potency in vitro. The pharmacokinetics study showed a 4-fold higher initial systemic clearance of IL4-10 FP lowSA, whereas the calculated half-life of both IL4-10 FP lowSA and IL4-10 FP highSA was 20.7 min. Finally, both IL4-10 FP glycoforms inhibited persistent inflammatory pain in mice to the same extent. Conclusions Differential sialylation of IL4-10 fusion protein does not affect the in vitro and in vivo activity, but clearly results in a difference in systemic exposure. The rapid systemic clearance of low sialylated IL4-10 FP could be a favorable characteristic to minimize systemic exposure after administration in a local compartment.
p ¼< 0.000 and p ¼< 0.000 and in total WOMAC score, p ¼< 0.000 and p ¼< 0.000) over TA group. Primary efficacy end point outcome (improvement ! 40%) of total WOMAC scores at 12 weeks, in Hylan G-F 20 and TA groups respectively was 30 (66.7%) and 5 (13.2%) in target knee joints (p ¼< 0.00) and at 26 weeks 35 (77.8%) and 10 (26.3%) in target knee joints (p ¼< 0.001). Within the group, VAS for pain reduction reported by patients and the investigator were significant statistically both at 12 and 26 weeks and in between groups, at weeks 12 (patient, p ¼ 0.002; investigator, p ¼ 0.001) and at weeks 26 (patient, p ¼ 0.000; investigator, p ¼ 0.000) respectively. Joint space improvement was also significant both at12 weeks (p ¼ 0.044) and 26 weeks (p ¼ 0.003) for Hylan G-F 20 than TA. Conclusions: A significant pain reduction, improvement in joint rheology and quality of life was observed with use of high molecular weight visco-supplementation (Hylan G-F 20) in patients suffering from primary knee OA refractory to standard care. Besides comparable efficacy both agents were found safe. Large scale project with longer follow up might answer long term outcomes.
BackgroundCartilage damage and inflammation are clear characteristics of joint degeneration in OA. In human and animal in vivo and in vitro experimental models, the immunoregulatory cytokines IL-4 and IL-10, both individually or combined, were shown to be effective in joint degeneration [1]. The additive effects of the cytokines are of importance to achieve this effect. We recently developed IL4-10 synerkine, a fusion protein composed of IL-4 and IL-10, with preserved activities of the individual cytokines.ObjectivesThis study evaluates the cartilage protective and anti-inflammatroy effects of IL4-10 synerkine ex vivo.MethodsOA cartilage (n=7, age 60 years) was cultured for 4 days in presence or absence of IL-4 (10ng/ml), IL-10 (10ng/ml), a combination of IL-4 (10ng/ml) and IL-10 (10ng/ml), or IL4-10 synerkine (20ng/ml). Changes in proteoglycan (PG) synthesis and PG release were studied. OA synovial tissue (n=8, age 66 years) was cultured for 3 days under similar circumstances. Cytokine levels in culture medium supernatants of both cartilage and synovium cultures were measured by ELISA for IL-6, IL-8 and TNFα. In addition, conditioned medium of synovial tissue cultures (n=3, age 61 years) cultured in presence or absence of IL4-10 was tested on healthy cartilage (changes in PG synthesis and release) to demonstrate indirect synovial effects on cartilage.ResultsCulturing OA cartilage in presence of IL4-10 synerkine increased proteoglycan (PG) synthesis with 47.6% (p=0.018), +26.6% for IL-4 alone, +31.2% for IL-10 alone, or +6.9% for IL-4 and IL-10 combined. In addition, a reduction of -8.7% (p=0.018) in PG release was found for IL4-10 synerkine (-4.9% for IL-4 alone, -8.6% for IL-10 alone, or -6.7% for IL-4 and IL-10 combined). The overall improved proteoglycan turnover was in cocurrence with a significant inhibition of Il-6, IL-8 and TNF-α cytokine production by the cartilage in presence of the IL4-10 synerkine (IL-6 -81.9%, IL-8 -65.9% and -2.0% TNF-α, resp.). Moreover, IL4-10 synerkine also reduced the production of IL-6, IL-8 and TNFα in osteoarthritic synovium (-83.5%, -85.6% and -25.9% resp., all p<0.01). When conditioned culture medium of synovial tissue was tested on healthy cartilage, a changed PG turnover was seen (-77% PG synthese, +21%, PG release). This PG turnover was normalized to healthy levels by IL4-10 treated synovium. There was no direct effect of IL4-10 synerkine on healthy cartilage.ConclusionsThese data show that IL4-10 synerkine induces both structural repair and reduce inflammation in OA. Ex vivo, IL4-10 synerkine has a direct effect on OA cartilage by affecting its proteoglycan turnover and cytokine production. Inhibiting the synovial cytokine production, adds indirectly to this structural repair of the cartilage. Together with the pain inhibiting effects found in animal models of OA, these data suggest disease modifying characteristics of the IL4-10 synerkine.ReferencesVan Roon JA, Lafeber FP, Bijlsma JW. (2001). Synergistic activity of interleukin-4 and interleukin-10 in suppression of inf...
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