Background: ATI-450, is an investigational small molecule inhibitor of the MAPK-activated protein kinase 2 (MK2) signaling pathway. This pathway drives the expression of multiple cytokines including TNFα, IL-1α and β, and IL-6. Objectives: We evaluated the safety and tolerability of ATI-450 in healthy volunteers as well as pharmacokinetics (PK) and pharmacodynamics (PD). Here we present data from single and multiple ascending dose cohorts. The aim was to select a dose for evaluation in phase 2 in patients with rheumatoid arthritis. Methods: Safety, PK and PD were assessed in a randomized, observer-blind, placebo-controlled, phase 1 study in male and female healthy subjects aged 18-55 (n=77). Part A: Single Ascending Dose (SAD) (n=32, 8 subjects per dose cohort - 2 placebo, 6 active). A single dose of 10mg, 30mg, 50mg and 100mg was tested. Part B: Multiple Ascending Dose (MAD) (n=30, 10 subjects per dose cohort - 2 placebo, 8 active). 10mg BID, 30mg BID and 50mg BID doses were tested over 7 days of administration. Safety and tolerability of ATI-450 was evaluated based on adverse events, clinical laboratory, vital signs, 12-lead ECG, Holter monitoring, and physical examination. Blood was drawn for PK analysis at 0.5, 1, 2, 4, 6, 8, 12 hours, 24, 36, and 48 hours post dose in the SAD cohort and on day 7 of the MAD cohort. PD of ATI-450 were explored by investigating the inhibition of a target biomarker, phospho-HSP27 (pHSP27) and proinflammatory cytokines, TNFα, IL1β, IL6 and IL8 in ex-vivo LPS-stimulated blood samples collected 4 and 12 hours post dose on day 7 from subjects in the MAD cohorts. Results: ATI-450 was generally well tolerated. No serious adverse events or severe adverse events were reported, and no adverse events led to discontinuation of the study medication. The most common adverse events (reported by 2 or more subjects who received ATI-450) observed during the trial were dizziness, headache, upper respiratory tract infection, constipation, nausea, and abdominal pain. All adverse events were mild. A trend of a decrease in absolute neutrophil count (ANC) was observed without correlated clinical sequelae. ATI-450 had dose proportional PK with a terminal half-life (t½) of 9-12 hours in the MAD cohort on day 7. A dose and concentration dependent inhibition of ex vivo stimulated cytokines and target biomarker was observed. On day 7, patients in the 50mg BID dose (the dose with the highest degree of inhibition) recorded mean trough drug levels (12 hours post dose) that were 1.4, 2.5, 2.5 and 2.4 times greater than the IC 80 for TNFα, IL1β, IL8 and pHSP27 respectively. Mean Cmax drug levels (4 hours post dose) were 3.6, 6.4, 6.2 and 6.0 times greater than the IC 80 for TNFα, IL1β, IL8 and pHSP27 respectively. IL6 levels were inhibited by more than 50% for part of the dosing interval. Conclusion: Oral ATI-450 was generally well tolerated at all doses with dose proportional PK. The t½ suggests that once or twice daily oral dosing may be possible. At the 50mg BID dose, marked inhibition of TNFα, IL1β and IL8, IL6 and pHSP27 was observed. ATI-450 has the potential to be an oral, small molecule drug which can target multiple cytokines. Exploration of its benefit to risk profile in patients with rheumatoid arthritis is warranted. Disclosure of Interests: Judy Schnyder Shareholder of: aclaris therapeutics, Employee of: aclaris therapeutics, Joe Monahan Shareholder of: aclaris therapeutics, Employee of: aclaris therapeutics, Walter Smith Shareholder of: aclaris therapeutics, Employee of: aclaris therapeutics, Heidi Hope Shareholder of: aclaris therapeutics, Employee of: aclaris therapeutics, Deborah Kelly Shareholder of: aclaris therapeutics, Employee of: aclaris therapeutics, David Burt Shareholder of: aclaris therapeutics, Employee of: aclaris therapeutics, E Huff Shareholder of: aclaris therapeutics, Employee of: aclaris therapeutics, A Kaul Shareholder of: aclaris therapeutics, Employee of: aclaris therapeutics, A Hildebrand Shareholder of: aclaris therapeutics, Employee of: aclaris therapeutics, B Burnette Shareholder of: aclaris therapeutics, Employee of: aclaris therapeutics, N Klug Shareholder of: aclaris therapeutics, Employee of: aclaris therapeutics, M Bangs Shareholder of: Aclaris Therapeutics, Employee of: Aclaris Therapeutics, David Gordon Shareholder of: aclaris therapeutics, Employee of: aclaris therapeutics

The acute-phase reactant rabbit serum amyloid A 3 (SAA3) was identified as the major difference product in Ag-induced arthritis in the rabbit, a model resembling in many aspects the clinical characteristics of rheumatoid arthritis (RA) in humans. In Ag-induced arthritis, up-regulated SAA3 transcription in vivo was detected in cells infiltrating into the inflamed joint, in the area where pannus formation starts and, most notably, also in chondrocytes. The proinflammatory cytokine IL-1 beta induced SAA3 transcription in primary rabbit chondrocytes in vitro. Furthermore, rSAA3 protein induced transcription of matrix metalloproteinases in rabbit chondrocytes in vitro. In the human experimental system, IL-1 beta induced transcription of acute-phase SAA (A-SSA; encoded by SAA1/SAA2) in primary chondrocytes. Similar to the rabbit system, recombinant human A-SAA protein was able to induce matrix metalloproteinases' transcription in chondrocytes. Further, immunohistochemistry demonstrated that A-SAA was highly expressed in human RA synovium, A new finding of our study is that A-SSA expression was also detected in cartilage in osteoarthritis. Our data, together with previous findings of SAA expression in RA synovium, suggest that A-SAA may play a role in cartilage destruction in arthritis.

Plasminogen activator and urokinase are often used as biological markers of cell activation. However, the methods currently used are cumbersome, make no discrimination between tissue-type plasminogen activator and urokinase, and do not allow expression of the results of the overall reaction in International Units. The one-step method described in this paper lacks these drawbacks. Moreover, we propose use of H-D-Val-Phe-Lys-4-nitroanilide as substrate which has a lower Km than the standard H-D-Val-Leu-Lys-4-nitroanilide which is commercially available. Low concentrations of sodium dodecyl sulfate in the reaction mixture dramatically and preferentially accelerate the reaction catalyzed by tissue-type plasminogen activators. Identical results are obtained under kinetic or fixed-time assay conditions using either a photometer or 96-well plate reader. The corresponding formulae are provided.

Compound IX 207-887 is a novel antiarthritic agent which inhibits the release of interleukin-1 (IL-1) from human monocytes and mouse peritoneal macrophagesin vitro at concentrations which are achieved therapeutically in human rheumatoid arthritis and in animal models of arthritis.

Cytokines such as Interleukin-1 (IL-1) are important modulators of the cell-mediated immune response and play a paramount role in inflammatory autoimmune disease. We report on preliminary clinical experiences with a new, tricyclic substance [( 10-Methoxy-4H-benzo[4,5]cyclo-hepta-[1,2-b]thiophene-4- ylidene]acetic acid, MW 284), which inhibits the release of interleukin-1 alpha and -beta from cultured murine macrophages or human mononuclear cells. The study included 12 patients (rheumatoid arthritis, n = 9; hemochromatotic arthropathy, n = 1; psoriatic arthropathy, n = 1; seronegative spondylarthropathy, n = 1). Eight patients were treated for a total of 8 weeks, receiving a median dose of 800 mg/d of the substance. Due to significant clinical benefits, two patients continued for a total of six months. Administration of the drug was discontinued in two patients because of severe urticaria and lack of compliance, respectively. Four out of 10 patients showed clinical improvement according to Ritchie-Index, pain score, ESR and CRP. Side effects were diffuse gastrointestinal symptoms (4/12), temporary impairment of liver function (4/12) and allergic skin reactions (3/12).

The sterol-like fungal metabolite wortmannin and a number of natural and chemically-derived analogues were found to block the induction of the respiratory burst during phagocytosis. 17-Hydroxy wortmannin, the most active compound tested, showed a 50% inhibition of the burst in neutrophils and mononuclear phagocytes at concentrations ranging between 0.8 and 17 nM, while wortmannin itself was about half as potent. Chemical derivation showed that a furane structure between ring A and B with adjacent carbonyl functions is essential for activity. At concentrations that entirely prevented superoxide or hydrogen peroxide production, the wortmannins were not cytotoxic and did not inhibit phagocytosis. At even higher concentrations (10 μM), 17-hydroxy wortmannin had no effect on the NADPH oxidase, once activated. This suggests that the wortmannins interfere with the signal transduction sequence initiated by the particulate stimulus and leading to the activation of the respiratory burst oxidase.

Macrophage products induce production of proteases that contribute to cartilage degradation in various joint diseases. In these studies we stimulated rabbit chondrocytes with various cytokines in vitro in order to determine which were responsible for changes in the release of prostaglandin, plasminogen activator, and a metalloproteinase. The metalloproteinase assayed in these studies is a latent enzyme whose activity can rapidly be measured with fluorogenic casein. Conditioned media from stimulated human peripheral blood mononuclear cells; purified human monocyte IL 1, pI 7,6, and 5; and recombinant human IL 1, beta or alpha forms, all changed the secretory pattern of rabbit articular chondrocytes in a similar manner: production and secretion of a latent metalloproteinase(s) and prostaglandin E were stimulated in a concentration-dependent fashion, whereas the activity of plasminogen activator was strongly reduced. Antibodies against human monocyte IL 1 blocked the active principle in various mononuclear cell-conditioned media, suggesting that uncharacterized factors present in these supernatants do not affect the metalloproteinase response. When added to confluent chondrocytes, phorbol myristate acetate, concanavalin A, IL 2, lipopolysaccharide, indomethacin, and prostaglandin E2, which interfere with lymphocyte proliferation assays for IL 1, failed to influence chondrocyte metalloproteinase secretion. Recombinant human IFN-alpha or IFN-gamma in the presence or absence of IL 1 had no effect on rabbit chondrocytes, whereas recombinant human tumor necrosis factor decreased plasminogen activator but had no effect on prostaglandin or metalloproteinase production. These results support the concept that IL 1 specifically induces chondrocytes to produce metalloproteinases, and hence may play an important role in destructive joint diseases.

Synthetic calcium pyrophosphate dihydrate crystals and, to a lesser extent, synthetic hydroxyapatite crystals increased the amount of interleukin-1/mononuclear cell factor released by human blood monocytes, as measured by collagenase and prostaglandin E2 production by rabbit chondrocytes, human dermal fibroblasts, and adherent rheumatoid synovial cells. The same crystals also directly induced collagenase and prostaglandin E2 secretion by rabbit chondrocytes, and potentiated the action of interleukin-1/mononuclear cell factor on chondrocytes. These mechanisms may be important in the pathogenesis of the destructive arthropathies associated with these crystals.

Stimulation of macrophages by zymosan phagocytosis triggers the respiratory burst and induces an early release of lysosomal hydrolases and E-type prostaglandins (PGE). We have studied whether antigen presentation by macrophages to helper T-cells elicits a comparable sequence of events. Cloned T-helper cells specific for hen egg albumin (EA) were added to histocompatible or histoincompatible resident mouse peritoneal macrophages in the presence of EA or an unrelated antigen, and the changes in biochemical parameters were monitored. The interaction between macrophages. T-helper cells and EA induced the production of PGE, but no release of lysosomal hydrolases or activation of the respiratory burst. In addition T-cell proliferation was observed. By contrast, no proliferation and no biochemical changes were observed when histoincompatible macrophages or unrelated antigen were used. When the experiments were done in the presence of indomethacin to inhibit PGE release, T-cell proliferation was enhanced. These results suggest that the PGE released may exert a feed-back control of the T-cell response.

Journal Article Effect of Interleukin-1 and 2 on Enzyme Secretion and Prostaglandin Fromation by Chondrocytes Get access J. Schnyder, J. Schnyder Search for other works by this author on: Oxford Academic PubMed Google Scholar T. Payne T. Payne Search for other works by this author on: Oxford Academic PubMed Google Scholar Rheumatology, Volume XXIV, Issue suppl_1, January 1985, Pages 128–132, https://doi.org/10.1093/rheumatology/XXIV.suppl_1.128 Published: 01 January 1985

We describe the effects of products of mononuclear phagocytes on the secretory activity of chondrocytes. The primary confluent cultures of rabbit articular chondrocytes were exposed to standard medium alone or enriched with conditioned medium obtained from cultures of rabbit peritoneal macrophages, the mouse macrophage cell line P388D1 or human blood mononuclear cells. Four markers of release were assessed, the neutral proteinases plasminogen activator and collagenase, the acid hydrolase beta-glucuronidase and prostaglandin E2, and the kinetics of their changes were monitored. Chondrocytes that were cultured in standard medium secreted large amounts of plasminogen activator, some beta-glucuronidase, but no collagenase, and released only minor amounts of prostaglandin E2. The addition of conditioned medium from rabbit macrophages induced a rapid release of large quantities of prostaglandin E2 and an abundant secretion of collagenase, while abolishing or strongly decreasing plasminogen activator secretion. In addition, beta-glucuronidase secretion was markedly enhanced. The decrease in secretion of plasminogen activator appeared to reflect a diminished production, since no evidence was found for the generation of inhibitors or for an accelerated extracellular breakdown of the enzyme. Conditioned media of the mouse and human mononuclear cells influenced the secretory activities of rabbit articular chondrocytes in a similar way, suggesting that the factor (or factors) acting on chondrocytes is produced by a variety of macrophages, and that its action is not species-restricted. The time course and concentration-dependence of the effects observed indicate that the secretion of plasminogen activator and collagenase are influenced in a strictly reciprocal fashion by the macrophage products. The release of prostaglandin E2 paralleled that of collagenase.

Mouse peritoneal macrophages infected with Leishmania parasites were exposed in vitro to the electron carriers methylene blue (MB), toluidine blue 0(TB), phenazine methosulfate (PMS) and crystal violet (CV). This led to parasite destruction without harm to the macrophages. The kinetics of intracellular killing depended on both the drug concentration and the duration of exposure; over 80% of the microorganisms were inactivated within 2.5 min of incubation of the parasitized cells with 10−4 MMB. On a molar basis, the drugs were considerably more active against intracellular compared to free parasites, suggesting that the macrophages themselves play a role in the observed anti-parasite toxicity. Intracellular killing by macrophages exposed to MB, TB and PMS correlated with the stimulation of oxygen uptake and hexose monophosphate shunt activity in the cells. Cytochrome c markedly inhibited MB-induced intracellular parasite destruction as well as completely blocking parasite killing in macrophages activated by lymphokines, pointing to O−2, H2O2 or products derived therefrom as possible mediators of macrophage toxic activity in both instances. Cytochrome c did not protect free parasites from the direct toxicity of the drug, however. Lipopolysaccharide promoted parasite destruction by lymphokine-activated macrophages, but failed to do so for electron carrier-stimulated cells. These observations suggest that intracellular killing induced by electron carriers results from a direct interaction of the drugs with cellular redox systems, leading to the generation of oxygen metabolites toxic for the parasites.