Pro-fibrotic effect of the susceptible gene PCSK5 in vascular fibrosis of Takayasu arteritis via TGF-ß and SMAD3 signaling pathway activation.

BACKGROUND: Vascular fibrosis directly causes vascular thickening in Takayasu arteritis (TAK), in which sustained transforming growth factor beta (TGF-ß) activation is critical. Understanding TGF-ß activation regulation and blocking it might yield a therapeutic effect in TAK. Proprotein convertase subtilisin/kexin type 5 (PCSK5) rs6560480 (T/C) is associated with TAK development. In this study, we assessed the association between the PCSK5 rs6560480 genotype and PCSK5 expression in TAK and explored its molecular role in TGF-ß activation and vascular fibrosis development. METHODS: In TAK patients, PCSK5 and TGF-ß expression in plasma and aortic tissue was examined by ELISA and immunohistochemical staining, and PCSK5 rs6560480 was genotyped. The correlation between PCSK5 and extracellular matrix (ECM) expression was examined by Western blotting (WB) and immunohistochemistry staining. Detection by co-immunoprecipitation was performed to detect the interaction between PCSK5 and TGF-ß in adventitial fibroblasts (AAFs). Downstream signaling pathways were detected by WB and validated with appropriate inhibitors. Potential immunosuppressive agents to inhibit the effects of PCSK5 were explored in cell culture and TAK patients. RESULTS: Patients with PCSK5 rs6560480 TT patients had significantly higher PCSK5 levels and more thickened vascular lesions than patients with PCSK5 rs6560480 CT. PCSK5 expression was significantly increased in alpha smooth muscle actin (α-SMA)-positive myofibroblasts in TAK vascular lesions. Overexpressing PCSK5 facilitated TGF-ß and downstream SMAD2/3 activation and ECM expression in AAFs and aorta in in-vitro culture. The mechanistic study supported that PCSK5 activated precursor TGF-ß (pro-TGF-ß) to the mature form by binding the pro-TGF-ß cleavage site. Leflunomide inhibited PCSK5 and pro-TGF-ß binding, decreasing TGF-ß activation and ECM expression, which was also partially validated in leflunomide-treated patients. CONCLUSION: The findings revealed a novel pro-fibrotic mechanism of PCSK5 in TAK vascular fibrosis via TGF-ß and downstream SMAD2/3 pathway activation. Leflunomide might be anti-fibrotic by disrupting PCSK5 and pro-TGF-ß binding, presenting a new TAK treatment approach.

Leflunomide alleviates obesity via activation of the TAK1-AMPK pathway and induction of lipophagy.

Lipophagy is a subset of selective autophagy that specifically degrades lipid droplets and plays an important role in obesity. Leflunomide treatment in rheumatoid arthritis (RA) patients has been associated with weight loss and decreased blood glucose levels, which cannot be attributed to its known side effects. Our prior studies showed that A77 1726, the active metabolite of leflunomide, acts as an inhibitor of S6K1 to sensitize the insulin receptor and control hyperglycemia. Whether the anti-obesity effect of leflunomide is mediated by targeting S6K1 and its underlying mechanisms remain unclear. Here, we report that A77 1726 induced LC3 lipidation and increased the formation of autophagosomes and lipoautolysosomes in 3T3-L1 adipocytes by activating TGF-ß-activated kinase 1 (TAK1), AMP-activated kinase (AMPK), and Unc-51 like autophagy-activated kinase 1 (ULK1). A77 1726 reduced the content of lipid droplets in 3T3-L1 adipocytes, which was blocked by bafilomycin or by beclin-1 knockdown. Similar observations were made in murine adipocytes differentiated from S6K1-/- embryonic fibroblasts (MEFs). Leflunomide treatment restricted bodyweight gains in ob/ob mice and reduced the visceral fat deposit and the size of adipocytes. Leflunomide treatment induced autophagy in adipose and liver tissues and reduced hepatic lipid contents. Consistently, S6K1 knockout increased the levels of LC3 lipidation in the liver, muscle, and fat of S6K-/- mice. Leflunomide treatment and S6K1 deficiency both induced TAK1, AMPK, and ULK1 phosphorylation in these tissues. These observations collectively suggest that leflunomide controls obesity in part by activating AMPK and inducing lipophagy. Our study provides insights into the mechanisms of leflunomide-mediated anti-obesity activity.

OpenSAFELY: The impact of COVID-19 on azathioprine, leflunomide and methotrexate monitoring, and factors associated with change in monitoring rate.

AIMS: The COVID-19 pandemic created unprecedented pressure on healthcare services. This study investigates whether disease-modifying antirheumatic drug (DMARD) safety monitoring was affected during the COVID-19 pandemic. METHODS: A population-based cohort study was conducted using the OpenSAFELY platform to access electronic health record data from 24.2 million patients registered at general practices using TPP's SystmOne software. Patients were included for further analysis if prescribed azathioprine, leflunomide or methotrexate between November 2019 and July 2022. Outcomes were assessed as monthly trends and variation between various sociodemographic and clinical groups for adherence with standard safety monitoring recommendations. RESULTS: An acute increase in the rate of missed monitoring occurred across the study population (+12.4 percentage points) when lockdown measures were implemented in March 2020. This increase was more pronounced for some patient groups (70-79 year-olds: +13.7 percentage points; females: +12.8 percentage points), regions (North West: +17.0 percentage points), medications (leflunomide: +20.7 percentage points) and monitoring tests (blood pressure: +24.5 percentage points). Missed monitoring rates decreased substantially for all groups by July 2022. Consistent differences were observed in overall missed monitoring rates between several groups throughout the study. CONCLUSION: DMARD monitoring rates temporarily deteriorated during the COVID-19 pandemic. Deterioration coincided with the onset of lockdown measures, with monitoring rates recovering rapidly as lockdown measures were eased. Differences observed in monitoring rates between medications, tests, regions and patient groups highlight opportunities to tackle potential inequalities in the provision or uptake of monitoring services. Further research should evaluate the causes of the differences identified between groups.

Efficacy and safety of low-dose corticosteroids combined with leflunomide for progressive IgA nephropathy: a systematic review and meta-analysis.

BACKGROUND AND OBJECTIVE: The effectiveness of immunosuppressive and corticosteroid treatments for Immunoglobulin A (IgA) nephropathy (IgAN) remains thoroughly evaluated. We undertook a meta-analysis to investigate the efficacy and safety of low-dose corticosteroids plus leflunomide for progressive IgA nephropathy. METHODS: Eligible studies were obtained from PubMed, Embase, and Cochrane Library databases. We also searched the references of the included studies. Our protocol followed the preferred reporting items for systematic reviews and meta-analyses (PRISMA) checklist. Eligibility criteria were defined using a PICOS framework. RESULTS: Our study included three articles presenting 342 patient cases. Findings revealed that low-dose corticosteroids combined with the leflunomide group were effective in relieving urine protein excretion (UPE) [mean difference (MD) = -0.35, 95% confidence interval (CI): -0.41 to -0.30, P < 0.00001] compared with the full-dose corticosteroids group. Regarding serum creatinine (SCr), estimated glomerular filtration rate (eGFR), complete remission rate, and overall response rate, there was no difference between the groups (p > 0.05). Regarding safety, low-dose corticosteroids combined with leflunomide significantly reduced the risk of serious adverse events [odds ratio (OR): 0.11, 95% CI: 0.01 to 0.91, P = 0.04]. Besides, no significant differences were observed between the two groups in the incidence of respiratory infection, abnormal liver function, diarrhea, herpes zoster, alopecia, pruritus, insomnia, pneumonia, diabetes, and urinary tract infection (P > 0.05). CONCLUSIONS: Low-dose corticosteroids combined with leflunomide are a safe and effective treatment for progressive IgA nephropathy. TRIAL REGISTRATION: The PROSPERO registration number is CRD42022361883.

Leflunomide-induced cardiac injury in adult male mice and bioinformatic approach identifying Nrf2/NF-κb signaling interplay.

Leflunomide (LFND) is an immunosuppressive and immunomodulatory disease-modifying antirheumatic drug (DMARD) that was approved for treating rheumatoid arthritis. LFND-induced cardiotoxicity was not fully investigated since its approval. We investigated the cardiac injury in male mice and identified the role of nuclear factor erythroid 2-related factor 2/nuclear factor-κ B (Nrf2/NF-κB) signaling. Male albino mice were assigned into five groups as control, vehicle, and LFND (2.5, 5, and 10 mg/kg). We investigated cardiac enzymes, histopathology, and the mRNA expression of Nrf2, NF-κB, BAX, and tumor necrosis factor-α (TNF-α). The bioinformatic study identified the interaction between LFND and Nrf2/NF-κB signaling; this was confirmed by amelioration in mRNA expression (0.5- to 0.34-fold decrease in Nrf2 and 2.6- to 4.61-fold increases in NF-κB genes) and increased (1.76- and 2.625-fold) serum creatine kinase (CK) and 1.38- and 2.33-fold increases in creatine kinase-MB (CK-MB). Histopathological results confirmed the dose-dependent effects of LFND on cardiac muscle structure in the form of cytoplasmic, nuclear, and vascular changes in addition to increased collagen deposits and apoptosis which were increased compared to controls especially with LFND 10 mg/kg. The current study elicits the dose-dependent cardiac injury induced by LFND administration and highlights, for the first time, dysregulation in Nrf2/NF-κB signaling.

Subacute cutaneous lupus erythematosus as a rare complication of disease-modifying therapy administration in multiple sclerosis: case report.

BACKGROUND: Teriflunomide, the active metabolite of leflunomide, is a disease-modifying therapy drug used for the treatment of multiple sclerosis (MS), yet the complications associated with this drug remain not fully understood. Here we present the rare case of a 28-year-old female MS patient who developed subacute cutaneous lupus erythematosus (SCLE) following teriflunomide treatment. Though SCLE has been reported to be associated with leflunomide, the current report represents the first documented evidence demonstrating SCLE as a potential teriflunomide treatment-related complication. Additionally, a literature review on the leflunomide-induced SCLE was conducted to emphasize the association of SCLE with teriflunomide, specifically amongst the female demographic with a preexisting autoimmune diathesis. CASE PRESENTATION: A 28-year-old female first presented with MS symptoms in the left upper limb along with blurred vision in the left eye. Medical and family histories were unremarkable. The patient exhibited positive serum biomarkers including ANA, Ro/SSA, La/SSB, and Ro-52 antibodies. Relapsing-remitting MS was diagnosed according to the 2017 McDonald's diagnostic criteria, and remission was achieved upon intravenous administration of methylprednisolone followed by teriflunomide sequential therapy. Three months post-teriflunomide treatment, the patient developed multiple facial cutaneous lesions. SCLE was subsequently diagnosed and was attributed to treatment-related complication. Interventions include oral administration of hydroxychloroquine and tofacitinib citrate effectively resolved cutaneous lesions. Discontinuation of hydroxychloroquine and tofacitinib citrate treatment led to recurring SCLE symptoms under continuous teriflunomide treatment. Full remission of facial annular plaques was achieved after re-treatment with hydroxychloroquine and tofacitinib citrate. The patient's clinical condition remained stable in long-term outpatient follow-ups. CONCLUSIONS: As teriflunomide has become a standard disease-modifying therapy for MS, the current case report highlights the importance of monitoring treatment-related complications, specifically in relation to SCLE symptoms.

Retrospective evaluation of prognosis and survival with various immunosuppressants in 82 dogs diagnosed with meningoencephalitis of unknown etiology (2010-2021).

BACKGROUND: Meningoencephalomyelitis of unknown etiology (MUE) is a comprehensive term for non-infectious inflammatory brain diseases of the central nervous system (CNS) caused by abnormal autoimmune responses. This study aims to compare the differences in survival and clinical response of MUE according to the adjuvant immunosuppressant use. Medical records of 82 dogs diagnosed with MUE were reviewed retrospectively. RESULTS: The overall survival time was 769 days (range 14-2687 days). The median survival time for each adjunctive was: leflunomide 1035 days (range 126-2163 days), mycophenolate mofetil 865 days (range 39-2191 days), cyclosporin 441 days (range 11-2176 days), cytosine arabinoside 754 days (range 6-1898 days) and a combination of mycophenolate mofetil and cytosine arabinoside 132 days (range 23-1227 days). There was no significant difference in the incidence rate of adverse events according to the immunosuppressants, but moderate to severe anemia was confirmed in 3 patients (18.7%) in the leflunomide group. CONCLUSIONS: The survival time and response rate of MUE dogs differed depending on which adjunctive immunosuppressants were used. Leflunomide showed a long survival time and a relatively good response rate in dogs with MUE. However, a large-scale further study with standardized doses of immunosuppressants and supportive treatment and constant monitoring interval is needed.

The efficacy of immunosuppressive drugs induction therapy for lupus nephritis: a systematic review and network meta-analysis.

OBJECTIVE: This study was to assess the safety and effectiveness of immunosuppressive agents, specifically Voclosporin, when used in conjunction with mycophenolate mofetil (MMF) induction therapy for the management of lupus nephritis (LN). METHODS: A systematic review and network meta-analysis (NMA) was conducted on randomized controlled trials investigating the efficacy of immunosuppressant-induced therapy for LN. The random effects model was used in the analysis. I2 was used to evaluate the heterogeneity of the model. Odds ratios (OR) and 95% credible intervals (CrI) were computed to assess and compare the relative effectiveness and safety of various treatment protocols. RESULTS: The study included a total of 16 randomized controlled trials (RCTs) involving 2444 patients with LN. The analysis results indicated that there was no significant difference in terms of partial remission (PR) between the drugs. However, when considering complete remission (CR), the combination of Voclosporin with MMF showed the highest remission rate, followed by Tacrolimus (TAC). Unfortunately, Voclosporin in combination with MMF had the highest risk of infection and serious infection, indicating a lower safety profile. CONCLUSIONS: Voclosporin in combination with MMF demonstrated the highest efficacy as an induction therapy for LN. However, it should be noted that the risk of infection and serious infection was found to be high with this regimen. On the other hand, TAC not only showed efficacy but also had a lower risk of infection and serious infection, making it a favorable option in terms of safety. This study did' not include results on other adverse events.

Development and validation of HPLC method for simultaneous determination of Leflunomide and folic acid in the nanoparticulate system by reversed-phase HPLC.

OBJECTIVE: The main objective of this study was to develop a highly sensitive, accurate, and reproducible analytical method for the simultaneous detection of LEF and FA in polymeric nanocarriers. SIGNIFICANCE: Leflunomide (LEF), is widely employed in the treatment of rheumatoid arthritis (RA). However, long-term delivery of the drug is associated with systemic side effects. Therefore, folate (FA) conjugated LEF nanocarriers were fabricated for targeting the nanocarriers toward activated macrophages. HPLC is considered one of the most sensitive and precise analytical techniques for the simultaneous detection and estimation of different components in a particular sample. METHODS: Analysis was performed on HPLC (Shimadzu 10 A), having a reversed-phase C-18 column (Beckmen, 250 X 4.6 mm, 5 µm) equipped with a photodiode detector set at a wavelength of 260 nm (LEF) and 285 nm (Folic acid). The isocratic mobile phase was composed of acetonitrile, water, and trimethylamine in a ratio of 65:35:0.5 at pH 4. Rapid analysis of both agents was performed, with a total run time of 10 min (FA = 2.1 ± 0.1 min, LEF = 5.9 ± 1 min) at a 1 mL/min flow rate. RESULTS: The assay demonstrated good linearity of 0.9989 of 0.9997 for LEF and FA respectively with a recovery in the range of 95-100%. The method also depicted good specificity, and intra and inter-day precision based on relative standard deviation (RSD) values. CONCLUSIONS: The study concludes, that the developed method was helpful in the detection and quantitation of lower values of both agents from polymeric nanocarriers.

HighlightsOptimization and validation of the RP-HPLC method were performed for the simultaneous detection of LEF and FA.Validation was performed on the basis of linearity, accuracy, precision, LOD, LOQ, and robustness in accordance with ICH criteria.Validated analytical procedure was employed for the simultaneous detection of LEF and FA from polymeric nanocarriers.The proposed analytical method is reliable, fast, robust, and can be successfully applied for quantification of % EE, and % DL in polymeric nanocarriers.

Glucocorticoids in the treatment of IgG4-related disease-Prospects for new international treatment guidelines.

Immunoglobulin G4-related disease (IgG4-RD) is a chronic fibro-inflammatory disease that may cause dysfunction in various organs. Worldwide multidisciplinary experts attending the Fourth International Symposium on IgG4-Related Disease in Japan in 2021 discussed treatments for IgG4-RD, especially glucocorticoid (GC) therapy. This review describes the efficacy, safety, and cost of treatments for IgG4-RD based on findings presented at the international symposium. A medium dose of GC was considered appropriate for the initial treatment of IgG4-RD. A randomized controlled trial and an open-label prospective study have shown that long-term maintenance GC therapy (prednisolone ≥ 5 mg/day) could prevent disease relapse. In addition, two open-label randomized controlled trials reported the effects of combinational use of GC and synthetic immunosuppressive agents, mycophenolate mofetil and leflunomide, on relapse prevention. Moreover, an open-label single-arm study showed an excellent rate of clinical response to rituximab. Many observational studies have shown the efficacy of an appropriate GC regimen in patients with IgG4-RD. Synthetic immunosuppressive agents and a molecular-targeted agent can be potent alternatives to GCs, but additional studies are required comparing their efficacy, risk of infection, and costs.

Comparison of the efficacy and safety of methotrexate alone or in combination with leflunomide in the treatment of juvenile idiopathic arthritis: a double-blind, placebo-controlled, randomized trial.

Introduction: Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disorder in children. Although methotrexate (MTX) is the first line disease-modifying antirheumatic drug for JIA, many patients do not respond well or cannot tolerate MTX. The aim of this study was to compare the effect of combination therapy of MTX and leflunomide (LFN) with MTX in patients who do not respond to MTX. Material and methods: Eighteen patients (2-20 years old) with polyarticular, oligoarticular or extended oligoarticular subtypes of JIA who did not respond to conventional JIA therapy participated in this double-blind, placebo-controlled, randomized trial. The intervention group received LFN and MTX for 3 months while the control group received oral placebo and MTX at a similar dose to the intervention group. Response to treatment was assessed every 4 weeks using the American College of Rheumatology Pediatric criteria (ACRPed) scale. Results: Clinical criteria, including number of active joints and restricted joints, physician and patient global assessment, Childhood Health Assessment Questionnaire (CHAQ38) score, and serum erythrocyte sedimentation ratelevel, did not differ significantly between groups at baseline and at the end of the 4th and 8th weeks of treatment. Only the CHAQ38 score was significantly higher in the intervention group at the end of the 12th week of treatment. Analysis of the effect of treatment on study parameters revealed that only the global patient assessment score differed significantly between groups (p = 0.003). Conclusions: The results of this study showed that combining LFN with MTX does not improve clinical outcomes of JIA and may increase side effects in patients who do not respond to MTX.

Development and validation of a prognostic model for leflunomide discontinuation with abnormal blood tests during long-term treatment: cohort study using data from the Clinical Practice Research Datalink Gold and Aurum.

OBJECTIVE: To develop and validate a prognostic model for LEF discontinuation with abnormal blood test results. METHODS: Data from the Clinical Practice Research Datalink Gold and Aurum were used for model development and external validation, respectively. Participants prescribed LEF between 1 January 2007 and 31 December 2019 were followed up from 6 months after the first general practitioner prescription to the earliest of date of outcome, death, 5 year follow-up or 31 December 2019. Candidate prognostic factors were ascertained using theory and data-driven approaches. Penalized Cox regression was performed to develop the risk equation, followed by internal validation using 500 bootstraps to correct for optimism. Multiple imputation was applied to handle missing data. Model performance was assessed in terms of calibration and discrimination. RESULTS: Data for 1487 and 2329 participants contributing 3140 and 5246 person-years follow-up were included in the development and validation cohorts, respectively. Thirteen candidate predictors were included in the model. Epilepsy and either cytopenia or elevated liver enzymes during the first 6 months of shared-care LEF prescription were strong predictors of drug discontinuation with a hazard ratio of 4.39 (95% CI 1.74, 11.06) and 3.06 (2.15, 4.35), respectively. The unadjusted and optimism-adjusted calibration slope in development data was 1.00 (95% CI 0.75, 1.25) and 0.72 (95% CI 0.47, 0.97), respectively. The calibration slope in validation data was 0.91 (95% CI 0.74, 1.07). The model showed prognostic separation with an optimism-adjusted Royston D statistic of 0.73 (95% CI 0.44, 1.02). CONCLUSION: We have developed and externally validated an easy-to-use prognostic model that may be used to risk stratify monitoring for LEF toxicity and to make informed choices about risks when choosing treatments.

Comparison between leflunomide and sulfasalazine based triple therapy in methotrexate refractory rheumatoid arthritis: an open-label, non-inferiority randomized controlled trial.

To compare efficacy and safety of two different combination csDMARD therapy in Methotrexate-failed Rheumatoid arthritis patients. In this 24-week open-label, parallel-group non-inferiority, single-center clinical trial, Methotrexate-failed Rheumatoid arthritis patients with disease duration < 2 years, were randomized to either of the two treatment regimens-Methotrexate + Leflunomide + Hydroxychloroquine or Methotrexate + Sulfasalazine + Hydroxychloroquine. Primary endpoint was proportion of patients achieving EULAR good response at 12 weeks. Non-inferiority of Leflunomide based therapy was confirmed if the upper limit of the 2-sided 95% confidence interval of treatment difference between the 2 groups was lower than the selected non-inferiority margin of (- 20%) in primary endpoint at 12 weeks. Secondary endpoints were improvement in DAS28, functional outcome and adverse events at 24 weeks. 136 eligible patients were randomized to either Leflunomide or Sulfasalazine group (68 in each group).63 and 59 patients in Leflunomide and 66 and 61 patients in Sulfasalazine group completed 12 and 24 weeks of trial, respectively. In Intension-to-treat analysis, EULAR good response was achieved by 58.8% and 54.4% patients (p = 0.7) at the end of 12 weeks, and 61.7% and 64.7% patients (p = 0.8) at the end of 24 weeks-in Leflunomide and Sulfasalazine group respectively. At 12 weeks, the difference in EULAR good response with 2-sided 95% confidence interval between 2 groups was 4.4% (- 12%, 20%) in intention-to-treat and 5.8% (- 11%, 23%) in perprotocol analysis.15 and 21 adverse events were recorded in Leflunomide and Sulfasalazine group respectively. Parenteral Methotrexate was required more in Sulfasalazine group due to gastrointestinal intolerance. Leflunomide based csDMARD therapy is non-inferior to Sulfasalazine based csDMARD therapy in Methotrexate-failed Rheumatoid arthritis patients with comparable safety profile. Trial registered at clinicaltrials.gov (NCT02930343) dated 10.09.2016.

Efficacy and safety of leflunomide combined with corticosteroids for the treatment of IgA nephropathy: a Meta-analysis of randomized controlled trials.

OBJECTIVE: We performed a meta-analysis of randomized controlled trials (RCTs) to evaluate the efficacy and safety of leflunomide combined with corticosteroids, compared with corticosteroids alone, for IgA nephropathy. MATERIALS AND METHODS: Studies were retrieved by searching of PubMed, Embase, Cochrane's Library, China National Knowledge Infrastructure (CNKI), and Wanfang databases on 11 October 2021. A random-effect model incorporating the heterogeneity was used to pool the results. The efficacy outcomes included the complete remission rate of proteinuria, overall response rate (the combined rates of patients with complete and partial remission of proteinuria), changes of urine protein excretion (UPE), serum creatinine (SCr), and estimated glomerular infiltrating rate (eGFR). RESULTS: Nineteen studies were included. Patients receiving the combined therapy had a higher complete remission rate (relative risk [RR]: 1.29, 95% CI: 1.08-1.55, p = 0.006; I2 = 0%) and overall response rate (RR: 1.18, 95% CI: 1.10-1.26, p < 0.001, I2 = 0%) compared to patients who received CS alone. Besides, combined therapy was associated with significantly reduced levels of UPE (mean difference [MD]: -0.30 g/24h, 95% CI: -0.43 to -0.16, p < 0.001; I2 = 34%) and SCr (MD: -7.55 mmol/L, 95% CI: -11.06 to -4.04, p < 0.001; I2 = 34%), and increased level of eGFR (MD: 6.51 mL/min/1.73 m2, 95% CI: 4.06-8.97, p < 0.001; I2 = 0%). The incidence of adverse events was not significantly different. CONCLUSIONS: Combined treatment with leflunomide and corticosteroids was more effective than corticosteroids alone for patients with IgA nephropathy.

Saliva as Blood Alternative in Therapeutic Monitoring of Teriflunomide-Development and Validation of the Novel Analytical Method.

Therapeutic drug monitoring (TDM) is extremely helpful in individualizing dosage regimen of drugs with narrow therapeutic ranges. It may also be beneficial in the case of drugs characterized by serious side effects and marked interpatient pharmacokinetic variability observed with leflunomide and its biologically active metabolite, teriflunomide. One of the most popular matrices used for TDM is blood. A more readily accessible body fluid is saliva, which can be collected in a much safer way comparing to blood. This makes it especially advantageous alternative to blood during life-threatening SARS-CoV-2 pandemic. However, drug's saliva concentration is not always a good representation of its blood concentration. The aim of this study was to verify whether saliva can be used in TDM of teriflunomide. We also developed and validated the first reliable and robust LC-MS/MS method for quantification of teriflunomide in saliva. Additionally, the effect of salivary flow and swab absorptive material from the collector device on teriflunomide concentration in saliva was evaluated. Good linear correlation was obtained between the concentration of teriflunomide in plasma and resting saliva (p < 0.000016, r = 0.88), and even better between plasma and the stimulated saliva concentrations (p < 0.000001, r = 0.95) confirming the effectiveness of this non-invasive method of teriflunomide's TDM. The analyzed validation criteria were fulfilled. No significant influence of salivary flow (p = 0.198) or type of swab in the Salivette device on saliva's teriflunomide concentration was detected. However, to reduce variability the use of stimulated saliva and synthetic swabs is advised.

The STIM1/2-Regulated Calcium Homeostasis Is Impaired in Hippocampal Neurons of the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of age-related dementia. Neuronal calcium homeostasis impairment may contribute to AD. Here we demonstrated that voltage-gated calcium (VGC) entry and store-operated calcium (SOC) entry regulated by calcium sensors of intracellular calcium stores STIM proteins are affected in hippocampal neurons of the 5xFAD transgenic mouse model. We observed excessive SOC entry in 5xFAD mouse neurons, mediated by STIM1 and STIM2 proteins with increased STIM1 contribution. There were no significant changes in cytoplasmic calcium level, endoplasmic reticulum (ER) bulk calcium levels, or expression levels of STIM1 or STIM2 proteins. The potent inhibitor BTP-2 and the FDA-approved drug leflunomide reduced SOC entry in 5xFAD neurons. In turn, excessive voltage-gated calcium entry was sensitive to the inhibitor of L-type calcium channels nifedipine but not to the T-type channels inhibitor ML218. Interestingly, the depolarization-induced calcium entry mediated by VGC channels in 5xFAD neurons was dependent on STIM2 but not STIM1 protein in cells with replete Ca2+ stores. The result gives new evidence on the VGC channel modulation by STIM2. Overall, the data demonstrate the changes in calcium signaling of hippocampal neurons of the AD mouse model, which precede amyloid plaque accumulation or other signs of pathology manifestation.

Outcome of solid and cavitary pulmonary nodules in rheumatoid arthritis patients-case series.

BACKGROUND: Rheumatoid pulmonary nodule can be detected in up to 32% of rheumatoid arthritis (RA) patients and approximately one-third of nodules may cavitate. We aimed to evaluate characteristics of patients with RA developing cavitary pulmonary nodular (CPN) lesions under disease-modifying antirheumatic drugs (DMARDs), follow-up of both cavitary and solid nodules, and their outcome with the treatment. METHODS: RA patients who presented with CPN lesions during follow-up were recruited retrospectively in this case series analysis. Total numbers and mean diameters of cavitary and solid nodules in each thorax computed tomography (CT) have been determined and followed up by two experienced pulmonary physicians. Moreover, changes in treatment after the development of the CPN lesions and characteristics of cavitary nodules were collected. RESULTS: Eleven patients with CPN lesions were reported. At the time of CPN diagnosis, more patients were taking leflunomide than methotrexate (81% vs 19%). Half of the patients were receiving biologic therapy and only 18% were taking anti-TNF drugs. After a median of 24 (3-65) months of follow-up, the regression of CPN lesions was determined in 45% (5/11) of patients. Four of these 5 (80%) patients were switched to a treatment regimen without leflunomide and three of them to nonanti-TNF biologic treatment or targeted synthetic DMARDs (tocilizumab, tofacitinib, and rituximab). DISCUSSION: CPN lesions seen in RA patients are often pulmonary manifestations of the underlying disease; however, one must rule out malignancies or infections. If lesions progress under DMARDs, it is advised to discontinue synthetic DMARDs (LEF/MTX) and switch to another biological DMARD with different modes of action.

Treatment of Coronavirus Disease 2019 Patients With Prolonged Postsymptomatic Viral Shedding With Leflunomide: A Single-center Randomized Controlled Clinical Trial.

BACKGROUND: We aimed to evaluate the efficacy and safety of leflunomide, an approved dihydroorotate dehydrogenase inhibitor, to treat coronavirus disease 2019 (COVID-19) patients with prolonged postsymptomatic viral shedding. METHODS: We conducted a prospective, randomized controlled, open-label trial involving hospitalized adult COVID-19 patients with prolonged polymerase chain reaction (PCR) positivity. Patients were randomly assigned to receive either leflunomide (50 mg every 12 hours, 3 consecutive times, orally; then 20 mg once daily for 8 days), in addition to nebulized interferon alpha 2a (IFN-α-2a, 3 million IU each time, twice daily for 10 days), or nebulized IFN-α-2a alone for 10 days. The primary endpoint was the duration of viral shedding. RESULTS: A total of 50 COVID-19 patients with prolonged PCR positivity were randomized into 2 groups: 26 were assigned to the leflunomide plus IFN-α-2a group, and 24 were assigned to the interferon-alone group. Treatment with leflunomide was not associated with a difference from the interferon-alone group in the duration of viral shedding (hazard ratio for negative reverse-transcription PCR, 0.70 [95% confidence interval, .391-1.256]; P = .186). In addition, the patients given leflunomide did not have a substantially shorter length of hospital stay than patients treated with interferon alone, with median durations of 29.0 (interquartile range [IQR], 19.3-47.3) days and 33.0 (IQR, 29.3-42.8) days, respectively (P = .170). Two leflunomide recipients were unable to complete the full 10-day course of administration due to adverse events. CONCLUSIONS: In COVID-19 patients with prolonged PCR positivity, no benefit in terms of the duration of viral shedding was observed with the combined treatment of leflunomide and IFN-α-2a beyond IFN-α-2a alone.

Inhibition of canine distemper virus replication by blocking pyrimidine nucleotide synthesis with A77 1726, the active metabolite of the anti-inflammatory drug leflunomide.

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

What is the incidence of methotrexate or leflunomide discontinuation related to cytopenia, liver enzyme elevation or kidney function decline?

OBJECTIVES: To examine incidence of treatment changes due to abnormal blood-test results and, to explore rates of treatment changes due to liver, kidney and haematological blood-test abnormalities in autoimmune rheumatic diseases (AIRD) treated with low-dose MTX or LEF. METHODS: Data for people with AIRDs prescribed MTX or LEF were extracted from the Clinical Practice Research Datalink. Participants were followed-up from first prescription of MTX or LEF in primary care. Primary outcome of interest was drug discontinuation, defined as a prescription gap of ≥90 days following an abnormal (or severely abnormal) blood-test result. Dose reduction was examined between consecutive prescriptions. Incidence rates per 1000 person-years were calculated. RESULTS: 15, 670 and 2,689 participants contributing 46, 571 and 4,558 person-years follow-up were included in MTX and LEF cohorts, respectively. The incidence of MTX and LEF discontinuation with abnormal (severely abnormal) blood-test was 42.24 (6.16) and 106.53 (9.42)/1000 person-years in year 1, and 22.44 (2.84) and 31.69 (4.40)/1000 person years, respectively, thereafter. The cumulative incidence of MTX and LEF discontinuation with abnormal (severely abnormal) blood tests was 1 in 24 (1 in 169), 1 in 9 (1 in 106) at 1 year; and 1 in 45 (1 in 352), 1 in 32 (1 in 227) per-year, respectively, thereafter. Raised liver enzymes were the commonest abnormality associated with drug discontinuation. MTX and LEF dose reduction incidence were comparable in year 1, however, thereafter MTX dose was reduced more often than LEF [16.60 (95% CI 13.05, 21.13) vs 8.10 (95% CI 4.97, 13.20)/1000 person-years]. CONCLUSION: MTX and LEF were discontinued for blood-test abnormalities after year 1 of treatment, however, discontinuations for severely abnormal results were uncommon.