Rheumatology and activation markers

Treatment-emergent adverse events were comparable between treatment groups. Spebrutinib inhibited various leukocyte responses in vitro, including those of B cells and osteoclasts. In this small study in RA patients, spebrutinib was well tolerated, showed a downward trend for symptoms, significantly modulated B-cell populations, and reduced markers of chemotaxis and osteoclast activity.

The online version of this article Rheumatoid arthritis RA is a chronic, systemic autoimmune disorder initiated by dysregulation of T and B lymphocytes. Its pathology includes expansion of B cells, which leads to formation of autoantibodies [ 1 ]. BTK is required for B-cell activation by engagement of the B-cell antigen receptor BCR and is involved in B-cell differentiation, chemotaxis, and trafficking [ 2 , 3 ].

Inhibition or lack of BTK is marked by increased numbers of B cells in circulating blood and reduced B-cell chemotaxis and accumulation in secondary lymphoid organs [ 1 , 4 ]. Inhibition of BTK activity is associated with reduced lymphadenopathy in patients with chronic lymphocytic leukemia [ 5 ]. Spebrutinib CC is an oral small molecule which inhibits BTK activity by irreversible covalent binding with high affinity to the BTK adenosine triphosphate binding site in B and myeloid cells.

By providing rapid, complete, and prolonged inhibition of BTK activity, spebrutinib may be therapeutically active in RA and other B-cell-mediated autoimmune disorders. Spebrutinib has demonstrated BCR selectivity, potency, and efficacy in a collagen-induced arthritis model in mice [ 7 ].

This phase 2a multicenter, US-based clinical study was conducted to evaluate spebrutinib efficacy and safety in female patients with active RA who were receiving a stable methotrexate dose as background therapy. The study also evaluated spebrutinib pharmacodynamic effects on circulating levels of BTK, B-cell subsets, and signaling factors essential to BTK activity. B cells 0. Data analysis was performed using Milliplex Analyst Software Millipore.

Cell surface marker CDa was measured by flow cytometry. Plasmablast generation and IgG production were measured by preincubation of B cells for 1 h with spebrutinib followed by addition of anti-IgM, CD, interleukin IL and IL-2 for 5 days, as described previously [ 9 ].

Cells were then harvested and CD86 measured by flow cytometry. Whole blood was lysed, fixed, and washed. Cells were re-suspended in buffer and analyzed by flow cytometry within 2 h. Data were analyzed and calculated using FlowJo software. Raw data were normalized to positive control dimethyl sulfoxide only and half maximal inhibitory concentration IC 50 was calculated with GraphPad Prism.

Plates were stained with tartrate-resistant acid phosphatase and osteoclasts were photographed and counted using Nikon NIS software. The study was done in accordance with the International Conference on Harmonisation E6 requirements for Good Clinical Practice and in accordance with the ethical principles outlined in the Declaration of Helsinki.

The study protocol, amendments, and informed consent form were approved by the institutional review board at each investigational site or by a central review board, and all enrolled patients provided written informed consent before starting the study. Patients were not eligible for enrollment if they had any other autoimmune disease or prior inflammatory joint disease other than RA; had used any biologic agent within 8 weeks or five half-lives of randomization; were receiving treatment with a disease-modifying anti-rheumatic drug other than sulfasalazine, hydroxychloroquine or chloroquine, or methotrexate or any other contraindicated medications or medical conditions; or had laboratory abnormalities or psychiatric illness that may affect participation in the study.

Enrollment could be stopped after 48 patients were randomized for operational or administrative reasons. Patients experiencing nausea, diarrhea, vomiting, elevated liver function tests, or abnormal renal function during active treatment could have their dose of spebrutinib reduced to mg, to be taken in the morning. One spebrutinib-treated patient had a dose reduction because of abnormal renal function parameters i. At the end of the double-blind, placebo-controlled treatment period, patients continued into a 4-week, post-dose, observational follow-up period.

A post hoc analysis compared ACR20 response rates at week 4 in patients with and without prior biologics. At the time of study initiation, available toxicology study data for spebrutinib supported a 4-week proof-of-concept study and not a longer period.

Secondary endpoints assessing the safety and tolerability of spebrutinib compared with placebo included collection of treatment-emergent adverse events TEAEs , physical and ophthalmologic examinations, lead electrocardiograms, and laboratory assessments.

Subgroup analyses assessed patient demographics and disease characteristics at baseline in ACR20 responders and non-responders. Additional post hoc analyses compared the biomarker changes from baseline to week 4 in responders versus non-responders and compared baseline biomarker levels between responders and non-responders.

Samples for pharmacodynamic and biomarker assessments were collected before the morning dose. The primary pharmacodynamic assessment was the percent BTK target site occupancy by spebrutinib, as previously described [ 7 ]. Additional assessments included the ratio of free:total BTK and changes in the concentration of serum carboxy-terminal collagen cross-linking telopeptide CTX-I , a collagen type I fragment released by bone-resorbing osteoclasts.

Circulating B-cell subsets examined by flow cytometry included total B cells, mature naive B cells, transitional B cells, and circulating class-switched and activated memory B cells. The target sample size of 80 patients i. Study enrollment was prematurely halted due to slow recruitment.

Efficacy analyses were performed on the full analysis set, which included all randomized patients. A post hoc analysis was also performed for ACR20 at week 4 comparing prior biologics use yes vs.

The analyses of secondary efficacy endpoints of ACR50 and ACR70 response rates at week 4 were performed in a manner similar to the analysis of the primary endpoint. Secondary safety endpoints were evaluated with descriptive statistics in the safety population i. Considering the low recruitment in this study, we were interested in exploring how many patients would have been required to power the study to detect a statistically significant between-group difference based on observed ACR20 response rates for each group.

A post hoc analysis showed 80 patients would have achieved a P value of 0. For pharmacodynamic analyses, the pharmacodynamic population included all patients with baseline data and at least one post-baseline sample collected for any biomarker.

Descriptive statistics were performed using GraphPad Prism version 7. P values two-tailed were calculated based on comparisons of the active treatment versus placebo groups using a Wilcoxon signed-rank test on the median change from baseline values. To explore spebrutinib pharmacology on immune responses, various primary human cellular models were tested using lymphoid and myeloid cells in vitro.

Spebrutinib inhibited B-cell proliferation with an IC 50 of 0. B-cell differentiation to plasmablasts was inhibited, as was their ability to secrete IgG. T-cell proliferation was inhibited with an IC 50 of 4. This pharmacologic effects pattern, including suppression of adaptive and innate immune responses, combined with inhibition of osteoclastogenesis and good in vivo efficacy in the collagen-induced arthritis model [ 7 ], led to testing spebrutinib in a phase 2 proof-of-concept clinical trial of patients with RA.

Baseline patient demographics and disease characteristics were well balanced between treatment groups Table 2. Mean age for patients was Mean body mass index of participants was The primary endpoint, ACR20 response at week 4, was achieved by Some spebrutinib-treated patients achieved an ACR20 response as early as week 1, and responses continued through week 2 and week 4. For secondary efficacy endpoints, week 4 ACR50 response rates were Percent differences between spebrutinib and placebo regarding ACR50 response 8.

In post hoc analyses comparing week 4 ACR20 response rates in patients with and without prior biologic therapy, the effect observed in spebrutinib-treated patients versus placebo patients was consistent with the primary efficacy results.

Subgroup analyses of responder versus non-responder patients demonstrated that responders in both the placebo and spebrutinib treatment arms had a shorter duration of disease compared with non-responders. The mean duration of RA disease was 3. There was no difference in disease severity between responders and non-responders, as evidenced by the baseline joint count Disease Activity Score, swollen and tender joint counts, or HAQ-DI at baseline Table 3.

Examining effects on levels of circulating class switched and activated memory B cells, a trend toward reduced levels of these cell types was observed data not shown.

Changes in CTX-I expression and inflammation-associated proteins over time after administration of spebrutinib. Post hoc analyses aimed at comparing pharmacodynamic changes between baseline and week 4 among spebrutinib clinical responders and non-responders revealed the following associations.

Peroxidase activity was analyzed as previously described. All of the analyzed markers were compared between gout patients and healthy controls. Normally distributed variables were analyzed with linear regression analysis. The 90th percentile of the healthy controls was used to define positive samples. Results for the gout cohort were adjusted for possible confounding by age and gender. Patient characteristics are presented in Table 1.

The number of self-reported flares since diagnosis differed extensively between patients with a median of 10 flares IQR 4—22 flares. During the last year, patients experienced between 0 and 10 flares mean 3.

The median VAS disease activity was Mean serum urate level at baseline was 0. Neutrophil activation in patients with polyarticular gout. Levels were compared to healthy controls HC. However, markers of neutrophil activation peroxidase and calprotectin were associated with several indices of gout disease activity. Neutrophil activation markers are associated with disease activity. All statistical analyses were done with linear regression adjusted for age and gender. Our main finding is that neutrophil activation markers correlate well with different markers of disease activity, especially with characteristics of active disease and in polyarticular gout.

In our opinion, this is an interesting and clinically relevant finding that could well be indicative of chronic systemic inflammation in gout. Furthermore, this study, for the first time, demonstrates the presence of NETs in peripheral blood of gout patients. Earlier studies have described neutrophil activation and NET formation locally in tophi and in the precursor form of urate micro aggregates UMA but not widespread in the circulation [ 8 ].

An explanation to the presence of NETs and neutrophil activation markers in the peripheral blood of gout patients could be the presence of small circulating MSU crystals which activate neutrophils in the circulation leading to phagocytosis and afterwards NET formation [ 8 ]. Another explanation could be that local inflammation triggers a systemic inflammatory environment, priming neutrophils for activation and NET formation even in absence of the direct stimulus of a crystal.

Recent work from our group demonstrates that levels of NETs are associated with disease activity and inflammation in two rheumatic diseases: juvenile dermatomyositis and rheumatoid arthritis [ 13 , 14 ]. Although elevated, the levels of NETs did not correspond with disease activity in our gout cohort. This finding is similar to earlier findings in SLE patients where levels of NETs were elevated though not associated with ongoing disease activity [ 6 ]. It is an unexpected and interesting finding, suggesting that what we define as NETs may be a broad definition of more or less pathogenic DNA complexes.

Indeed, we, and others, have demonstrated that the cargo of NETs varies depending on mechanism of induction, with certain NETs containing more granular enzymes [ 15 ] and other NETs containing more oxidized mitochondrial DNA, the latter being highly inflammatory [ 12 ]. Furthermore, pioneering work from the group of Herrmann et al.

Further studies are needed to investigate the composition of the gout-derived NETs and determine why they are less inflammatory, other than the potential presence of proteases [ 2 ]. In clinical practice, specific biomarkers for disease activity in gout are lacking. The level of serum urate is associated with the risk of acute arthritis, and lowering serum urate levels leads to a reduction of tissue deposits and risk of flares [ 16 ]. On the other hand, serum urate has been shown to be only weakly associated with patient subjective outcomes such as disability and health-related quality of life HRQOL [ 17 ].

Our findings indicate that neutrophil activation markers are promising novel biomarkers for both clinical and patient reported outcomes of disease activity in gout. These observations however should be validated in other large gout cohorts. In this cohort, there were differences in baseline characteristics between the two groups age and gender.

Earlier cohort studies however have showed no association between age, gender, and the levels of neutrophil activation markers or NET formation [ 6 ]. Similarly, also in the current study, we found no association between levels of neutrophil activation and cell death markers on age and gender.

Currently, we are investigating the effect of therapy, especially urate lowering therapy on disease activity as well as neutrophil activation markers. These future results will further clarify the value of these biomarkers in monitoring of disease activity. Neutrophil activation markers are associated with characteristics of active polyarticular gout. Furthermore, NETs are present in the peripheral blood of gout patients: however, they do not associate with markers of disease activity or inflammation.

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Sci Transl Med. Article Google Scholar. Some limitations of this study have to be discussed. First, the sample size is rather small; however, APS is a rare disease and the investigations had to be performed with freshly collected blood samples. Due to the relatively low number of participants, we were not able to evaluate the influence of comorbidities. Second, only one mechanism of NET formation including the citrullination of histone H3 was investigated.

Apart from histone citrullination, additional mechanisms of NET formation exist [ 39 ]; thus, it would be interesting to analyse other NETosis pathways in this context. This is, however, hampered by the lack of parameters specific for NET formation and distinguishing to cell death programs.

Currently, measurement of H3Cit is one of the most widely used and accepted methods to measure NET formation. Furthermore, detection of NET formation could be performed by other methods where neutrophils and NETs can adhere to the surface and may not be washed away, which is a frequent problem with staining procedures.

In conclusion, these experiments are the first to systematically investigate the potential of activation and H3Cit production of HDNs and LDNs in different stimulation settings in healthy controls and patients with APS. Our findings strengthen the hypothesis that neutrophils play a role in APS and the increased thrombotic risk in this patient cohort.

These results could help to further unravel underlying mechanisms of this rare, but in some cases serious, immunologic disease and to potentially introduce novel treatment options for patients with APS. Supplementary data are available at Rheumatology online. Antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of 1, patients.

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