Development and preclinical assessment of nanoemulgel loaded with phytoconstituents for the management of rheumatoid arthritis.

In recent years, natural ingredients have gained importance for therapeutic treatment due to their minimal toxicity. However, the delivery of these phytoconstituents poses a challenge to provide better efficacy. Current research reports the development of nanoemulgel (NEG) loaded with ginger oleoresin (GOR) and lipid guggul extract (LGE) for the management of rheumatoid arthritis (RA). The nanoemulsion (NE) was developed using the spontaneous emulsification technique by the pseudo-ternary method. The optimized nanoemulsion exhibited globule size of 16.08 ± 2.55 nm, PDI of 0.187 ± 0.06, and zeta potential of - 22.4 ± 0.31 mV. The cumulative release from in vitro diffusion studies at pH 7.4 was about 99.72 ± 3.47%, 57.98 ± 2.11%, and 86.42 ± 5.13% of 6-gingerol, E-guggulsterone, and Z-guggulsterone respectively at the end of 24 h. The ex vivo studies on porcine ear skin showed sustained release with 92.8 ± 3.21% for 6-gingerol, 55.61 ± 0.91% for E-guggulsterone, and 84.2 ± 4.22% for Z-guggulsterone released at the end of 24 h. The cell culture studies on RAW 264.7 cells indicated a robust inhibition of LPS-induced IL-6 and TNF-α production indicating its efficacy in the management of RA. The preclinical studies on male Wistar rats suggest that the developed NEG exhibited a comparable decrease in paw edema inflammation as compared to the marketed diclofenac sodium gel. These encouraging results demonstrate the potential of the developed nanoemulgel containing combination of GOR and LGE for the management of RA.

Relevance of circulating markers of endothelial activation for cardiovascular risk assessment in rheumatoid arthritis: a narrative review.

Rheumatoid arthritis (RA) is associated with excessive cardiovascular mortality secondary to premature atherosclerosis, in which endothelial activation (EA) plays a central role. EA is characterized by loss of vascular integrity, expression of leucocyte adhesion molecules, transition from antithrombotic to prothrombotic phenotype, cytokines production, shedding of membrane microparticles and recruitment of endothelial progenitor cells. As EA is an early event in atherogenesis, circulating markers of EA are putative markers of vascular pathology and cardiovascular (CV) risk. After a presentation of biology of EA, the present review analyzed the available data regarding changes in EA markers in RA in link with the vascular pathology and CV events, discussed their relevance as biomarkers of CV risk and proposed future directions.

Assessment of the anti-rheumatoid arthritis activity of Gastrodia elata (tian-ma) and Radix aconitic lateralis preparata (fu-zi) via network pharmacology and untargeted metabolomics analyses.

AIM: Gastrodia elata and Radix aconiti lateralis preparrata are respectively named as Tian-Ma and Fu-Zi (TF) in Chinese. We explored the active components against rheumatoid arthritis (RA) from an extensively used couplet of Chinese herbs, Gastrodia elata and Radix aconiti lateralis preparata (TF) via untargeted metabolomics and network pharmacological approaches. METHODS: Water extracts of TF were mixed at ratios 1:1, 3:2 and 2:3 (w/w). Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was then utilized as metabolomics screening. Human Metabolome (http://www.hmdb.ca/) and Lipidmaps (http://www.lipidmaps.org/) databases were used to annotate detected compounds. Further identification of vital genes and important pathways associated with the anti-RA properties of the TF preparations was done via network pharmacology, and verified by real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Four key compounds involved in unsaturated fatty acid biosynthesis and isoflavonoid biosynthesis were identified through metabolomics analyses. Three key components of TF associated with anti-RA activity were linoleic acid, daidzein, and daidzin. Results of RT-qPCR revealed that all 3 tested TF couplets (1:1, 3:2, and 2:3) markedly suppressed the transcription of PTGS2. These results were consistent with our network pharmacological predictions. CONCLUSIONS: The anti-RA properties of Tian-Ma and Fu-Zi are associated with the inhibition of arachidonic acid metabolism pathway.

A rapid response near-infrared ratiometric fluorescent probe for the real-time tracking of peroxynitrite for pathological diagnosis and therapeutic assessment in a rheumatoid arthritis model.

Peroxynitrite (ONOO-) is a potent bio-oxidant involved in many physiological and pathological processes; however, most of the pathological effects associated with ONOO-in vivo are still ambiguous. Herein, we designed and synthesized two near-infrared ratiometric fluorescent probes, Ratio-A and Ratio-B, for the detection and biological evaluation of ONOO-. The recognition unit diene in the probes could be specifically cleaved by ONOO- with a 94-fold enhancement in the ratiometric fluorescence signal. By imaging ONOO- in immune stimulated cells and acute inflammation mice model using Ratio-A, we investigated the fluctuations of ONOO- levels in a rheumatoid arthritis (RA) model of mice. Ratio-A could be applied for the effective imaging of RA and could rapidly evaluate the response of the RA treatment with methotrexate (MTX). Thus, Ratio-A can be considered as a promising tool for pathological diagnosis and the therapeutic assessment of a wide range of diseases including RA.

Quantitative Assessment of Arthritis Activity in Rheumatoid Arthritis Patients Using [11C]DPA-713 Positron Emission Tomography.

Treatment for rheumatoid arthritis (RA) should be started as early as possible to prevent destruction of bone and cartilage in affected joints. A new diagnostic tool for both early diagnosis and therapy monitoring would be valuable to reduce permanent joint damage. Positron emission tomography (PET) imaging of macrophages is a previously demonstrated non-invasive means to visualize (sub)clinical arthritis in RA patients. We developed a kinetic model to quantify uptake of the macrophage tracer [11C]DPA-713 (N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo [1,5-a]pyrimidin-3-yl]acetamide) in arthritic joints of RA patients and to assess the performance of several simplified methods. Dynamic [11C]DPA-713 scans of 60 min with both arterial and venous blood sampling were performed in five patients with clinically active disease. [11C]DPA-713 showed enhanced uptake in affected joints of RA patients, with tracer uptake levels corresponding to clinical presence and severity of arthritis. The optimal quantitative model for assessment of [11C]DPA-713 uptake was the irreversible two tissue compartment model (2T3k). Both Ki and standardized uptake value (SUV) correlated with the presence of arthritis in RA patients. Using SUV as an outcome measure allows for a simplified static imaging protocol that can be used in larger cohorts.

Low Vitamin D Levels in Rheumatoid Arthritis Patient is Associated with Poor Disability Index and Increased Patient Global Disease Assessment Score.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory connective tissue disorder with wide spectrum of presentation from polyarthritis to multisystem involvement. Apart from bones, muscles and other soft tissues, Vitamin D receptors have been found on many immune cells and tissues. The most vital function of Vitamin D is calcium and phosphorus absorption but it can also act as an immune-modulator hormone, which can affects both innate and adaptive immune responses leading to autoimmune diseases. OBJECTIVES: To study the relationship of vitamin D insufficiency with disease activity and functional disability in patients of Rheumatoid Arthritis. MATERIAL AND METHODS: The present study was an observational, cross sectional study done in a tertiary care hospital in New Delhi, India. The inclusion criteria comprised of patients attending the inpatient (IPD) and outpatient department (OPD), age above 18 years and fulfilling 1987 American college of Rheumatology (ACR) criteria for RA. The exclusion criteria was patients suffering from any other connective tissue disorder (CTD) and patients who were taking vitamin D supplements for past 6 months. Thirty patients were enrolled in the study after satisfying inclusion and exclusion criteria and appropriate clinical data and blood sample were collected after informed consent. Joint examination were performed and swollen joint count (SJC), tender joint count (TJC), patient global assessment (PGA) and evaluator global assessment (EGA) scores were recorded. Disease activity using DAS28ESR, DAS28CRP and CDAI were calculated and disability index was assessed using Short Fries Health Assessment Questionnaire. RESULTS: In our study mean vitamin D level was 18.93 ng/ml (S.D. 6.64 ng/ml). Mean DAS28 ESR was 4.57±1.48. Mean Disability Index was 0.52±0.89. All the study population had low Vitamin D level (100%), while 50% patients had vitamin D level in deficiency range (<20ng/ml). On analysis by student t-test, statistically higher PGA (p value 0.024) and Disability Index (p value < 0.001) in vitamin D deficient patients, compared to vitamin D insufficient patient group was observed, however there was no significant difference in disease activity between the groups. CONCLUSION: Low Vitamin D levels are common in Indian rheumatoid arthritis patients. Mean PGA significantly increased, and disability index significantly increased in Vitamin D deficient group compared to insufficient group suggesting vitamin D deficient patients poor wellbeing and more disability.

Axillary lymph-node metabolic activity assessment on 18F-FDG-PET/CT in rheumatoid arthritis patients treated with biologic therapies.

Objective: Recent studies have provided new insights into the role of lymph nodes (LNs) in rheumatoid arthritis (RA). The aim of this study was to evaluate the metabolic activity of the axillary LNs in relation to that of the upper limb joints and the clinical assessment of disease activity in RA patients treated with biologic therapies.Method: 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (18F-FDG-PET/CT) scans were acquired for 64 patients with RA at baseline and after 6 months of biologic therapy, and the patients' clinical status was evaluated. The maximum standardized uptake value (SUVmax), metabolic active volume, and total lesion glycolysis (TLG) were used to assess glucose metabolism in the LNs and 12 joints. Clinical evaluations included serum markers and the Disease Activity Score based on 28-joint count-erythrocyte sedimentation rate (DAS28-ESR).Results: Changes in the SUVmax and TLG for the axillary LNs correlated significantly with those of the ipsilateral wrist joints. There was a positive correlation between the changes in the three metabolic parameters of the axillary LNs and the changes in disease activity after treatment. After 6 months of biologic therapy, all metabolic parameters for the axillary LNs in patients with a DAS28-ESR < 3.2 were significantly lower than those of patients with a DAS28-ESR ≥ 3.2.Conclusion: A relationship between the glucose metabolism of the axillary LNs and the ipsilateral wrist joints was demonstrated by the 18F-FDG-PET/CT parameters. The metabolic activity and active volume of axillary LNs may reflect the therapeutic response to the biologic treatment of RA.

Atomic force microscopy technique used for assessment of the anti-arthritic effect of licochalcone A via suppressing NF-κB activation.

Atomic force microscopy (AFM) is appropriately applied to the examination of hard surfaces and soft samples with extremely high resolution and ultrasensitive force, which cannot be obtained by other imaging techniques, including optical and electron microscopy. In the current study, AFM was employed to evaluate the anti-arthritic effect of licochalcone A (LCA), a flavonoid isolated from the root of Chinese medicinal herb Glycyrrhiza inflate, on rheumatoid arthritis synovial fibroblasts (RASFs) at the nanoscale for the first time. The morphology, ultrastructure and stiffness of RASFs was modified by LCA as determined by AFM, suggesting that LCA most likely exerts an anti-arthritic effect based on the key role of RASFs in the progression of RA. Further studies showed that the inhibitory effect of LCA on IκBα phosphorylation and degradation as well as on p65 nuclear translocation and phosphorylation contributed to altering the morphology, ultrastructure and stiffness of the RASF membrane. Interestingly, IKKß phosphorylation was not detectable in RASFs, indicating that LCA altered the morphology, ultrastructure and stiffness of the RASF membrane by inhibiting NF-κB activation independent of IKKß phosphorylation. Antigen-induced arthritis (AIA) was established in Sprague Dawley (SD) rats to validate the anti-arthritic effect of LCA, and LCA significantly decreased both the arthritis scores and paw swelling in the AIA rats, suggesting that LCA inhibits the progression and development of arthritis in vivo. Collectively, AFM provides evidence at the nanoscale to predict the anti-arthritic effect of drugs on RASFs, and LCA should be further investigated as a candidate agent for the treatment of arthritis.

The potential impact of monitoring disease activity biomarkers on rheumatoid arthritis outcomes and costs.

Rheumatoid arthritis (RA) management requires monitoring of disease activity to determine course of treatment. Global assessments are used in clinical practice to determine RA disease activity. Monitoring disease activity via biomarkers may also help providers optimize biologic and nonbiologic drug use while decreasing overall drug spend by delaying use of expensive biologic therapies. By testing multiple biologic domains at the same time, a multibiomarker disease activity test may have utility in RA patient management, through improved intra- and inter-rater reliability. This report provides a comprehensive review of studies of objective measures, single biomarkers and multibiomarker disease activity tests as disease activity measures to decrease uncertainty in treatment decisions, and of biomarkers' potential impact on economic and clinical outcomes of treatment choices.

Single-cell RNA-seq of rheumatoid arthritis synovial tissue using low-cost microfluidic instrumentation.

Droplet-based single-cell RNA-seq has emerged as a powerful technique for massively parallel cellular profiling. While this approach offers the exciting promise to deconvolute cellular heterogeneity in diseased tissues, the lack of cost-effective and user-friendly instrumentation has hindered widespread adoption of droplet microfluidic techniques. To address this, we developed a 3D-printed, low-cost droplet microfluidic control instrument and deploy it in a clinical environment to perform single-cell transcriptome profiling of disaggregated synovial tissue from five rheumatoid arthritis patients. We sequence 20,387 single cells revealing 13 transcriptomically distinct clusters. These encompass an unsupervised draft atlas of the autoimmune infiltrate that contribute to disease biology. Additionally, we identify previously uncharacterized fibroblast subpopulations and discern their spatial location within the synovium. We envision that this instrument will have broad utility in both research and clinical settings, enabling low-cost and routine application of microfluidic techniques.

Assessment of local and systemic 25-hydroxy-vitamin D, RANKL, OPG, and TNF levels in patients with rheumatoid arthritis and periodontitis.

The present study aimed to evaluate proinflammatory cytokine and vitamin D levels in rheumatoid arthritis (RA) and chronic periodontitis (CP) patients and healthy individuals before and after initial periodontal treatment. Overall, 17 CP patients with RA (RA + CP), 18 systemically healthy CP patients (CP), and 18 healthy controls (C) were included. Clinical periodontal measurements were recorded and gingival crevicular fluid (GCF) and blood samples were recorded. RA + CP and CP patients received nonsurgical periodontal treatment. Vitamin D, tumor necrosis factor (TNF)-α, receptor activator of nuclear factor-KB ligand (RANKL), and OPG levels were determined in GCF and serum. Baseline clinical parameters were similar in all periodontitis groups (P > 0.05) but were higher than that in controls (P < 0.05). Periodontal treatment improved clinical parameters in all periodontitis groups (P < 0.05). GCF vitamin D levels were higher in RA + CP and CP groups than in healthy controls, but these levels decreased in the RA + CP group after periodontal treatment (P < 0.05). Serum RANKL and GCF TNF-α levels in RA patients decreased after periodontal treatment (P < 0.05). Within the limitations of this study, the results suggested that GCF vitamin D levels are increased in RA patients and decrease after periodontal treatment; therefore, local vitamin D levels might be an important indicator of periodontal bone loss.

Exposure-Response Modeling and Power Analysis of Components of ACR Response Criteria in Rheumatoid Arthritis (Part 2: Continuous Model).

Population pharmacokinetic/pharmacodynamic (PK/PD) models were developed to quantitate the exposure-response relationships using continuous longitudinal data on American College of Rheumatology (ACR) subcomponents, that is, tender-joint count (TJC), swollen-joint count (SJC), C-reactive protein, patient's assessment of pain, patient's global assessment of disease activity, physician's global assessment of disease activity, and patient's assessment of physical function for 5 biologics approved for use in rheumatoid arthritis. The models were then used to simulate the time courses of clinical outcomes following different treatment regimens. The relative sensitivity of the 7 subcomponents was assessed using Monte Carlo simulation-based power analysis. The developed population PK/PD models adequately described the relationship between serum concentrations and changes in ACR subcomponents. The trial simulation and subsequent power analysis showed that SJC and TJC appeared to be more sensitive than the other 5 ACR subcomponents to detect treatment effect over placebo/methotrexate. These 7 ACR subcomponents had similar power in detecting the treatment difference between different doses. In addition, the continuous measures of ACR subcomponents did not appear to be more sensitive than binary measures.

Exposure-Response Modeling and Power Analysis of Components of ACR Response Criteria in Rheumatoid Arthritis (Part 1: Binary Model).

American College of Rheumatology (ACR) response criteria is used to assess improvement in tender and swollen joint counts and in 3 of the 5 core measures (acute-phase reactant, physician global assessment, patient global assessment, pain, and physical function). From the clinical trial data on 5 approved biological products for the treatment of rheumatoid arthritis, population pharmacokinetic/pharmacodynamic models were developed to quantitatively describe the relationship between exposure and response rates of 3 individual components of ACR response criteria. The models were then used to simulate the clinical outcomes at various time points following different treatment regimens. The relative sensitivity of these criteria components was assessed using power analysis. As compared to the composite endpoints (ACR20/ACR50/ACR70), the individual ACR criteria components had adequate power and higher sensitivity in distinguishing treatment effects over placebo/methotrexate control. The 3 individual ACR criteria components appeared to have similar powers at different dose levels after long-term treatment. This research provides a unique approach to assess the relative sensitivity of the 3 binary components of ACR response criteria which would be useful to support future dose selection and trial design in the treatment of rheumatoid arthritis.

Evaluating osteoporotic fracture risk with the Fracture Risk Assessment Tool in Chinese patients with rheumatoid arthritis.

This study aims to evaluate the discriminative and predictive capacity of the Fracture Risk Assessment Tool (FRAX) to determine the 10-year risk of osteoporotic fracture in Chinese rheumatoid arthritis (RA) patients.This study included 168 RA patients and 168 healthy individuals as controls. The Chinese mainland FRAX model was applied to calculate the 10-year risk of osteoporotic fractures, defined as fracture of the spine, forearm, hip, or shoulder.The incidence of osteoporosis was significantly increased in RA patients compared to controls (P < .05). Bone mineral density (BMD), lumbar vertebra T-score, and femoral neck T-score were significantly lower in RA patients compared to controls (P < .05). BMD, disease duration, DAS28, and glucocorticoid use were important risk factors for osteoporotic fractures in Chinese RA patients. Ten-year osteoporotic fracture risk in Chinese RA patients was higher when BMD was incorporated in FRAX.There was a higher incidence of osteoporosis and reduced BMD in RA patients compared to controls. The FRAX model should integrate femoral neck BMD with other risk factors to evaluate osteoporotic fracture risk in RA patients, making it a valuable screening tool.

Identification of significant pathway cross-talk in rheumatoid arthritis by the Monte Carlo cross-validation method.

We attempted to identify significant pathway cross-talk in rheumatoid arthritis (RA) by the Monte Carlo cross-validation (MCCV) method. We therefore obtained and preprocessed the gene expression profile of RA. MCCV involves identifying differentially expressed genes (DEGs), identifying differential pathways (DPs), calculating the discriminating score (DS) of the pathway cross-talk, and random forest (RF) classification. We carried out 50 bootstrap iterations of MCCV to identify the key instances of pathway cross-talk involved in RA. We identified a total of 17 significant DEGs and 15 significant DPs by comparing RA samples and normal controls. We found the most significant difference between RA and the normal controls in the eIF4 and p70S6K signaling regulation pathway. Furthermore, we identified 10 instances of pathway cross-talk with the best classification performance for RA and normal controls, using the RF classification model. All of the top 10 pathway pairs involved cross-talk with eIF4 and p70S6K signaling regulation, and the other 10 pathways were immune-related. By MCCV, we identified one critical DP and 10 significant instances of pathway cross-talk in RA. We propose that the eIF4 and p70S6K signaling regulation pathway and the other significant instances of pathway cross-talk play key roles in the occurrence and development of RA, and are potential predictive and prognostic markers for RA.

Use of Hydroxychloroquine Is Associated With Improved Lipid Profile in Rheumatoid Arthritis Patients.

BACKGROUND/PURPOSE: We examined the association between hydroxychloroquine (HCQ) and plasma lipid and glucose levels in rheumatoid arthritis (RA) cohort. METHODS: This is a retrospective cohort analysis of 1261 RA patients comparing fasting lipid profiles and plasma glucose between patients who were and were not taking HCQ. We divided patients into 3 groups based on HCQ exposure during follow-up: those who had never taken HCQ, those who took it intermittently, and those who took it continuously. We used multivariable models and propensity scoring to compensate for the effect of nonrandom treatment assignment. RESULTS: We followed 1261 RA patients for a total of 4605 observations between 1996 and 2014. After adjusting for age, sex, ethnicity, other disease-modifying antirheumatic drugs (DMARDs), lipid-lowering medications, body mass index (BMI), and smoking, patients taking HCQ at baseline had significantly lower total cholesterol (TC) (P ≤ 0.001), low-density lipoprotein (LDL) (P ≤ 0.001), triglycerides (P = 0.013), and lipid profile ratios TC/high-density lipoprotein (HDL) (P ≤ 0.001) and LDL/HDL (P ≤ 0.001), as well as higher HDL (P ≤ 0.001).In longitudinal analyses, after adjusting for confounders, patients who continuously took HCQ showed significantly lower TC, LDL, TC/HDL, and LDL/HDL and higher HDL (P ≤ 0.01). Fasting plasma glucose levels were not significantly associated with HCQ exposure. CONCLUSIONS: Hydroxychloroquine use was associated with lower lipid levels but not with the plasma glucose in this RA cohort. These findings support the need for a randomized trial to establish the role of HCQ in cardiovascular disease prevention in RA patients.

Longitudinal assessment of monocyte chemoattractant protein-1 in lupus nephritis as a biomarker of disease activity.

Urinary MCP-1 (uMCP-1) levels reflect lupus nephritis (LN) disease activity. However, long-term prospective studies evaluating it as a biomarker are lacking. SLE patients with active nephritis (AN), active disease without nephritis (ANR), and inactive disease (ID) were enrolled. AN patients were followed up every 3 months for 1 year. Urine and serum samples were collected at baseline from all and at follow-up visits in AN group. Urine samples from healthy subjects (HC), rheumatoid arthritis (RA), and diabetic nephropathy (DM) patients (20 each) served as controls. Serum (sMCP-1) and uMCP-1 was measured using ELISA. Urinary values were normalized for creatinine excretion. Nonparametric tests were used. A total of 121 SLE patients were enrolled. Baseline uMCP-1 was significantly higher in AN as compared to ANR, ID, HC, and RA (p < 0.001), but it was not different from DM and showed good correlation with rSLEDAI and SLEDAI (r = 0.52 and 0.47, p < 0.001) but not with sMCP-1. On ROC analysis to differentiate between AN and ANR, uMCP-1 performed better than sMCP-1, anti-dsDNA antibodies, C3 and C4. uMCP-1 and not sMCP-1 decreased significantly at all follow-up visits (p < 0.001). uMCP-1 remained persistently elevated in a patient who developed CKD and rose before conventional markers in two patients with relapse of LN. uMCP-1 correlates well with LN disease activity and helps differentiate between AN and ANR patients. Its levels fall with treatment and may have a potential to predict poor response and relapse of LN. uMCP-1 is most likely generated locally in the kidney.

Characterization and non-clinical assessment of the proposed etanercept biosimilar GP2015 with originator etanercept (Enbrel(®)).

BACKGROUND AND OBJECTIVE: Biosimilars are approved biologics that are comparable to an originator product with respect to quality, safety and efficacy. Herein, the authors describe the functional and non-clinical studies designed to determine the biosimilarity of GP2015 and originator etanercept (Enbrel®). METHODS: The development of an Enbrel biosimilar (GP2015) involved extensive characterization of the originator. A step-wise target-directed and iterative technical development program involving state-of-the-art functional characterization studies and non-clinical evaluations (pharmacokinetics, pharmacodynamics and safety/toxicology) was applied with the aim of confirming that GP2015 is comparable to originator (Enbrel) at the non-clinical level. RESULTS: In in vitro tests, GP2015 and Enbrel had comparable binding affinities to TNF-α, C1q complement and a complete panel of Fc-Receptors. Comprehensive functional characterization testing confirmed the comparability of GP2015 with Enbrel in terms of its ability to bind to and neutralize TNF-α, which reflects the primary mechanism of action of etanercept. Non-clinical data confirmed that the proposed biosimilar to Enbrel, GP2015, is comparable with regards to its pharmacokinetic properties and pharmacodynamic activity, and efficacy as well as safety/toxicity. CONCLUSION: The proposed Enbrel biosimilar, GP2015, was shown to be comparable to its originator product in studies designed to confirm biosimilarity.