Efficacy and safety of remibrutinib, a selective potent oral BTK inhibitor, in Sjögren's syndrome: results from a randomised, double-blind, placebo-controlled phase 2 trial.

OBJECTIVES: To evaluate the safety and efficacy of remibrutinib in patients with moderate-to-severe Sjögren's syndrome (SjS) in a phase 2 randomised, double-blind trial (NCT04035668; LOUiSSE (LOU064 in Sjögren's Syndrome) study). METHODS: Eligible patients fulfilling 2016 American College of Rheumatology/European League Against Rheumatism (EULAR) criteria for SjS, positive for anti-Ro/Sjögren's syndrome-related antigen A antibodies, with moderate-to-severe disease activity (EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) (based on weighted score) ≥ 5, EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) ≥ 5) received remibrutinib (100 mg) either one or two times a day, or placebo for the 24-week study treatment period. The primary endpoint was change from baseline in ESSDAI at week 24. Key secondary endpoints included change from baseline in ESSDAI over time, change from baseline in ESSPRI over time and safety of remibrutinib in SjS. Key exploratory endpoints included changes to the salivary flow rate, soluble biomarkers, blood transcriptomic and serum proteomic profiles. RESULTS: Remibrutinib significantly improved ESSDAI score in patients with SjS over 24 weeks compared with placebo (ΔESSDAI -2.86, p=0.003). No treatment effect was observed in ESSPRI score (ΔESSPRI 0.17, p=0.663). There was a trend towards improvement of unstimulated salivary flow with remibrutinib compared with placebo over 24 weeks. Remibrutinib had a favourable safety profile in patients with SjS over 24 weeks. Remibrutinib induced significant changes in gene expression in blood, and serum protein abundance compared with placebo. CONCLUSIONS: These data show preliminary efficacy and favourable safety of remibrutinib in a phase 2 trial for SjS.

First-in-Human Study of the Safety, Pharmacokinetics, and Pharmacodynamics of MHV370, a Dual Inhibitor of Toll-Like Receptors 7 and 8, in Healthy Adults.

BACKGROUND AND OBJECTIVE: MHV370, a dual antagonist of human Toll-like receptors (TLR) 7 and 8, suppresses cytokines and interferon-stimulated genes in vitro and in vivo, and  has demonstrated efficacy in murine models of lupus. This first-in-human study aimed to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of single and multiple doses of MHV370 in healthy adults, as well as the effects of food consumption on a single dose of MHV370. METHODS: This was a phase 1, randomised, placebo-controlled study conducted in three parts. In part A, participants received (3:1) a single ascending dose (SAD) of 1, 3, 10, 20, 40, 80, 160, 320, 640 and 1000 mg MHV370 or placebo. In part B, participants received (3:1) multiple ascending doses (MAD) of 25, 50, 100, 200 and 400 mg MHV370 twice daily (b.i.d) or placebo for 14 days. In part C, participants received an open-label single dose of 200 mg MHV370 under fasted or fed conditions. Safety, pharmacokinetic and pharmacodynamic parameters were evaluated. RESULTS: MHV370 was well tolerated, and no safety signal was observed in the study. No dose-limiting adverse events occurred across the dose range evaluated. Plasma concentrations of MHV370 increased with dose (mean [SD] maximum plasma concentrations ranged from 0.97 [0.48] to 1670 [861.0] ng/mL for SAD of 3-1000 mg, 29.5 [7.98] to 759 [325.0] ng/mL for MAD of 25-400 mg b.i.d. on day 1). The intake of food did not have a relevant impact on the pharmacokinetics of MHV370. Pharmacodynamic data indicated time- and dose-dependent inhibition of TLR7-mediated CD69 expression on B cells (100% inhibition at 24 h post-dose starting from SAD 160 mg and MAD 50 mg b.i.d.) and TLR8-mediated TNF release after ex vivo stimulation (>90% inhibition at 24 h post-dose starting from SAD 320 mg and MAD 100 mg b.i.d.). CONCLUSION: The safety, pharmacokinetic and pharmacodynamic data support the further development of MHV370 in systemic autoimmune diseases driven by the overactivation of TLR7 and TLR8.

Central and Peripheral Inflammation in Mild Cognitive Impairment in the Context of Alzheimer's Disease.

Mild cognitive impairment (MCI) is characterized by an abnormal decline in mental and cognitive function compared with normal cognitive aging. It is an underlying condition of Alzheimer's disease (AD), an irreversible neurodegenerative disease. In recent years, neuroinflammation has been investigated as a new leading target that contributes to MCI progression into AD. Understanding the mechanism underlying inflammatory processes involved in the early onset of the disease could help find a safe and effective way to diagnose and treat patients. In this article, we assessed over twenty different blood and cerebrospinal fluid (CSF) inflammatory biomarker concentrations with immunoassay methods in patients with MCI (mild cognitive impairment), non-impaired control (NIC), and serum healthy control (HC). We performed group comparisons and analyzed in-group correlations between the biomarkers. We included 107 participants (mean age: 64.7 ± 7.8, women: 58.9%). CSF osteopontin and YKL-40 were significantly increased in the MCI group, whereas serum C-reactive protein and interleukin-6 were significantly higher (p < 0.001) in the NIC group compared with the MCI and HC groups. Stronger correlations between interleukin-1ß and inflammasome markers were observed in the serum of the MCI group. We confirmed specific inflammatory activation in the central nervous system and interleukin-1ß pathway upregulation in the serum of the MCI cohort.

Disease Association of Anti‒Carboxyethyl Lysine Autoantibodies in Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by recurring suppurating lesions of the intertriginous areas, resulting in a substantial impact on patients' QOL. HS pathogenesis remains poorly understood. An autoimmune component has been proposed, but disease-specific autoantibodies, autoantigens, or autoreactive T cells have yet to be described. In this study, we identify a high prevalence of IgM, IgG, and IgA antibodies directed against Nε-carboxyethyl lysine (CEL), a methylglyoxal-induced advanced glycation end-product, in the sera of patients with HS. Titers of anti-CEL IgG and IgA antibodies were highly elevated in HS compared with those in healthy controls and individuals with other inflammatory skin diseases. Strikingly, the majority of anti-CEL IgG was of the IgG2 subclass and correlated independently with both disease severity and duration. Both CEL and anti-CEL‒producing plasmablasts could be isolated directly from HS skin lesions, further confirming the disease relevance of this autoimmune response. Our data point to an aberration of the methylglyoxal pathway in HS and support an autoimmune axis in the pathogenesis of this debilitating disease.

Lipidomics Profiling of Hidradenitis Suppurativa Skin Lesions Reveals Lipoxygenase Pathway Dysregulation and Accumulation of Proinflammatory Leukotriene B4.

Hidradenitis suppurativa (HS) is a chronic, recurring inflammatory dermatosis characterized by abscesses, deep-seated nodules, sinus tracts, and fibrosis in skin lesions around hair follicles of the axillary, inguinal, and anogenital regions. Whereas the exact pathogenesis remains poorly defined, clear evidence suggests that HS is a multifactorial inflammatory disease characterized by innate and adaptive immune components. Bioactive lipids are important regulators of cutaneous homeostasis, inflammation, and resolution of inflammation. Alterations in the lipid mediator profile can lead to malfunction and cutaneous inflammation. We used targeted lipidomics to analyze selected omega-3 and omega-6 polyunsaturated fatty acids in skin of patients with HS and of healthy volunteers. Lesional HS skin displayed enrichment of 5-lipoxygenase (LO)‒derived metabolites, especially leukotriene B4. In addition, 15-LO‒derived metabolites were underrepresented in HS lesions. Changes in the lipid mediator profile were accompanied by transcriptomic dysregulation of the 5-LO and 15-LO pathways. Hyperactivation of the 5-LO pathway in lesional macrophages identified these cells as potential sources of leukotriene B4, which may cause neutrophil influx and activation. Furthermore, leukotriene B4-induced mediators and pathways were elevated in HS lesions, suggesting a contribution of this proinflammatory lipid meditator to the pathophysiology of HS.

First-in-human clinical trial to assess pharmacokinetics, pharmacodynamics, safety, and tolerability of iscalimab, an anti-CD40 monoclonal antibody.

Iscalimab is a fully human, CD40 pathway blocking, nondepleting monoclonal antibody being developed as an immunosuppressive agent. We describe a first-in-human, randomized, double-blind, placebo-controlled study investigating the safety, tolerability, pharmacokinetics, and pharmacodynamics of iscalimab in healthy subjects and rheumatoid arthritis patients. Healthy subjects (n = 56) received single doses of intravenous iscalimab (0.03, 0.1, 0.3, 1, or 3 mg/kg), or subcutaneous iscalimab (3 mg/kg), or placebo. Rheumatoid arthritis patients (n = 20) received single doses of intravenous iscalimab (10 or 30 mg/kg) or placebo. Iscalimab exhibited target-mediated drug disposition resulting in dose-dependent and nonlinear pharmacokinetics. Complete (≥90%) CD40 receptor occupancy on whole blood B cells was observed at plasma concentrations >0.3-0.4 µg/mL. In subjects receiving 3 mg/kg iscalimab, antibody responses to keyhole limpet hemocyanin were transiently suppressed. CD40 occupancy by iscalimab prevented ex vivo human rCD154-induced expression of CD69 on B cells in whole blood. All doses were generally safe and well tolerated, with no clinically relevant changes in any safety parameters, including no evidence of thromboembolic events. Iscalimab appears to be a promising blocker of the CD40-CD154 costimulatory pathway with potential use in transplantation and other autoimmune diseases.

Assessment of the anti-CD40 antibody iscalimab in patients with primary Sjögren's syndrome: a multicentre, randomised, double-blind, placebo-controlled, proof-of-concept study.

BACKGROUND: Primary Sjögren's syndrome is an autoimmune disease that presents as dryness of the mouth and eyes due to impairment of the exocrine glands. To our knowledge, no systemic therapies for primary Sjögren's syndrome have shown efficacy. CD40-CD154-mediated T cell-B cell interactions in primary Sjögren's syndrome contribute to aberrant lymphocyte activation in inflamed tissue, leading to sialadenitis and other tissue injury. Therefore, we investigated the safety and preliminary efficacy of iscalimab (CFZ533), a novel anti-CD40 monoclonal antibody, in patients with primary Sjögren's syndrome. METHODS: This multicentre, randomised, double-blind, placebo-controlled, proof-of-concept study took place at ten investigational sites across Europe (UK, n=4; Germany, Switzerland, and Hungary, n=1 each) and the USA (n=3). Eligible patients were aged 18-75 years and fulfilled the 2002 American European consensus group diagnostic classification criteria for primary Sjögren's syndrome. In the double-blind phase of the trial, patients were randomly assigned (2:1) via computer-generated unique randomisation numbers to receive subcutaneous iscalimab (3 mg/kg) or placebo at weeks 0, 2, 4, and 8 (cohort 1) or intravenous iscalimab (10 mg/kg) or placebo at weeks 0, 2, 4, and 8 (cohort 2). Randomisation was stratified according to baseline intake of oral corticosteroids. At week 12, patients in both cohorts received open-label iscalimab (same dose and route) for 12 weeks. The primary objectives of the study were to assess the safety, tolerability, and efficacy of multiple doses of iscalimab in the two sequential dose cohorts. Safety and tolerability were assessed by adverse events and efficacy of iscalimab versus placebo was assessed by clinical disease activity, as measured by the change in European League Against Rheumatism Sjögren's syndrome disease activity index (ESSDAI) score after 12 weeks of treatment. Analyses were done on a per-protocol basis. The trial was registered with ClinicalTrials.gov, NCT02291029. FINDINGS: Between Oct 22, 2014, and June 28, 2016, we assessed 82 patients for eligibility (25 for cohort 1 and 57 for cohort 2). 38 patients were excluded because of ineligibility. In cohort 1, 12 patients were randomly assigned to receive either 3 mg/kg doses of iscalimab (n=8) or placebo (n=4), and in cohort 2, 32 patients were randomly assigned to receive either intravenous 10 mg/kg doses of iscalimab (n=21) or placebo (n=11). Adverse events were similar between iscalimab treatment groups and placebo groups, with adverse events occurring in all patients in cohort 1, and in 52% and 64% of the iscalimab and placebo groups, respectively, in cohort 2. Two serious adverse events were reported (one case of bacterial conjunctivitis in cohort 1 and one case of atrial fibrillation in cohort 2), which were unrelated to treatment with iscalimab. Intravenous treatment with iscalimab resulted in a mean reduction of 5·21 points (95% CI 0·96-9·46; one-sided p=0·0090) in ESSDAI score compared with placebo. There was no signficiant difference in ESSDAI score between subcutaneous iscalimab and placebo. INTERPRETATION: To our knowledge, this is the first randomised, placebo-controlled proof-of-concept study of a new investigational drug for primary Sjögren's syndrome that indicates preliminary efficacy. Our data suggest a role of CD40-CD154 interactions in primary Sjögren's syndrome pathology and the therapeutic potential for CD40 blockade in this disease should be investigated further. FUNDING: Novartis Pharma.

The BACE-1 inhibitor CNP520 for prevention trials in Alzheimer's disease.

The beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) initiates the generation of amyloid-ß (Aß), and the amyloid cascade leading to amyloid plaque deposition, neurodegeneration, and dementia in Alzheimer's disease (AD). Clinical failures of anti-Aß therapies in dementia stages suggest that treatment has to start in the early, asymptomatic disease states. The BACE-1 inhibitor CNP520 has a selectivity, pharmacodynamics, and distribution profile suitable for AD prevention studies. CNP520 reduced brain and cerebrospinal fluid (CSF) Aß in rats and dogs, and Aß plaque deposition in APP-transgenic mice. Animal toxicology studies of CNP520 demonstrated sufficient safety margins, with no signs of hair depigmentation, retina degeneration, liver toxicity, or cardiovascular effects. In healthy adults ≥ 60 years old, treatment with CNP520 was safe and well tolerated and resulted in robust and dose-dependent Aß reduction in the cerebrospinal fluid. Thus, long-term, pivotal studies with CNP520 have been initiated in the Generation Program.

Development and validation of an ELISA at acidic pH for the quantitative determination of IL-13 in human plasma and serum.

A novel sandwich ELISA for the quantitative and sensitive determination of IL-13 in human serum and plasma was established. The assay employs an incubation step at acidic pH, which was shown to decrease nonspecific binding and interference from IL-13 binding proteins. The assay was validated and was shown to be accurate and precise over the entire quantification range (0.59 to 68.4 pg/mL in human EDTA plasma). The validated assay was successfully applied to samples from healthy volunteers and patients with atopic seasonal rhinitis. The assay is suitable for use in clinical trials to monitor efficacy or pharmacodynamic effects of drug candidates.

Validation of immunoassay for protein biomarkers: bioanalytical study plan implementation to support pre-clinical and clinical studies.

Biomarkers have emerged as an important tool to optimize the benefit/risk ratio of therapeutics. The scientific impact of biomarker studies is directly related to the quality of the underlying data. It is therefore important that guidance be established for validation of assays used to support drug development. This paper specifically focuses on validation of immunoassay for protein biomarker to support pre-clinical and clinical studies. Therapeutics (small- and macro-molecules) and their respective target/ligand are out of scope. This paper describes the implementation of a bioanalytical study plan for the validation of immunoassays to support decision-making biomarkers and biomarker selection during preclinical and clinical studies. It establishes the complete operating procedure as well as the parameters and their respective acceptance criteria and defines milestones and decision points to be followed during the assay validation that should result in high quality bioanalytical data in a limited timeframe and with reduced costs. The bioanalytical study plan can be applied to the validation of a wild range of immunoassay technology such as monoplex ELISA, automated analyzer, multiplex assays or cutting edge technology. Before any validation, a feasibility study is performed to assess the performance of the immunoassay using biological samples which should mimic the clinical population. The feasibility study addresses the likelihood that an assay will be able to achieve its intended purpose with parallelism being the most critical element (milestone 1). At the end of the feasibility study, a decision is taken to either continue with the validation or change the assay (milestone 2). The milestone 3 consists of the establishment of the nominal value of quality control to be used during the validation. The quality controls used to validate an assay should preferentially be prepared using neat (non-spiked) biological matrix (ideally derived from the specific trial population). The last milestone (milestone 4), the formal validation, includes demonstration of the assay performance meeting accuracy and precision acceptance criteria within (intra-run) and between (inter-run) validation runs for each QC sample. Validation also includes the assessment of stability of the protein biomarker in the biological matrix. It is recognized that the extent of the validation should be correlated to the intended use of the data and the assay acceptance criteria should take into consideration the study objective(s), nature of the methodology and the biological variability of the biomarker.

A novel method for quantitative measurement of a therapeutic monoclonal antibody in the presence of its target protein using enzymatic digestion.

Over the past decades, the use of therapeutic monoclonal antibodies (mAbs) has become an important strategy in the treatment of various diseases. To enable pharmacokinetic (PK) assessment, specific immunoassays need to be developed to quantify mAbs in blood. In these assays, the presence of bound target protein can lead to severe underestimation of mAb concentration. Here we describe a novel approach for the quantification of total (free plus bound) human mAb concentration, in human and non-human primate serum, in the presence of a high level of target protein. The method is based on sample digestion with pepsin under optimized conditions to fully digest the target while keeping the mAb in the form of immunoreactive fragments. The quantification of mAb is then performed by ELISA without interference from the target. This method allows accurate quantification of as low as 50ng/ml mAb in the presence of up to 100-fold target molar excess. Intra- and inter-run precision is better than 10%, and intra- and inter-run accuracy in the range of 89.3-106.7%. In conclusion, this general and simple approach allows the accurate and sensitive measurement of preclinical and clinical samples avoiding target interference.

Reduction of matrix interferences by the combination of chaotropic salt and DMSO in a broadly applicable target-based ELISA for pharmacokinetic studies of therapeutic monoclonal antibodies.

Use of a synergistic effect of DMSO together with a chaotropic salt (NaSCN or MgCl2) allowed to drastically reduce matrix interferences in an ELISA for therapeutic monoclonal antibodies. Optimum combinations were found to be 0.4 M NaSCN together with 10.0% DMSO, and 1.0 M MgCl2 with 15.0% DMSO. At this optimum combination, quality controls spiked with mAb at 50.0 ng/ml in eighteen individual human sera and plasmas were quantified with an overall accuracy of 102.0%. All of these QCs fulfilled the acceptance criteria of 80.0-120.0% accuracy and precision below 20.0%. The assay was also successfully applied to the quantification of two other mAbs in human serum. Furthermore, the use of the assay was extended to pre-clinical species (cynomolgus monkey and rat serum). Here, the performed validation experiments confirmed the utility of the assay and demonstrated that the assay allowed quantification of mAb from 50.0 ng/ml to 100.0 microg/ml in cynomolgus monkey serum. The method has then been applied to a pharmacokinetic study in cynomolgus monkeys. In summary, this work demonstrates the efficacy of the combination of a chaotropic salt with DMSO to minimize matrix interferences in an ELISA. The robustness thus obtained allowed the successful establishment of a cost effective, target-based ELISA format for use in pharmacokinetic studies, that is easily applicable for the quantification of mAbs in various matrices such as human, cynomolgus monkey or rat serum and plasma.