A phase Ib/II randomized, open-label drug repurposing trial of glutamate signaling inhibitors in combination with chemoradiotherapy in patients with newly diagnosed glioblastoma: the GLUGLIO trial protocol.

BACKGROUND: Glioblastoma is the most common and most aggressive malignant primary brain tumor in adults. Glioblastoma cells synthesize and secrete large quantities of the excitatory neurotransmitter glutamate, driving epilepsy, neuronal death, tumor growth and invasion. Moreover, neuronal networks interconnect with glioblastoma cell networks through glutamatergic neuroglial synapses, activation of which induces oncogenic calcium oscillations that are propagated via gap junctions between tumor cells. The primary objective of this study is to explore the efficacy of brain-penetrating anti-glutamatergic drugs to standard chemoradiotherapy in patients with glioblastoma. METHODS/DESIGN: GLUGLIO is a 1:1 randomized phase Ib/II, parallel-group, open-label, multicenter trial of gabapentin, sulfasalazine, memantine and chemoradiotherapy (Arm A) versus chemoradiotherapy alone (Arm B) in patients with newly diagnosed glioblastoma. Planned accrual is 120 patients. The primary endpoint is progression-free survival at 6 months. Secondary endpoints include overall and seizure-free survival, quality of life of patients and caregivers, symptom burden and cognitive functioning. Glutamate levels will be assessed longitudinally by magnetic resonance spectroscopy. Other outcomes of interest include imaging response rate, neuronal hyperexcitability determined by longitudinal electroencephalography, Karnofsky performance status as a global measure of overall performance, anticonvulsant drug use and steroid use. Tumor tissue and blood will be collected for translational research. Subgroup survival analyses by baseline parameters include segregation by age, extent of resection, Karnofsky performance status, O6-methylguanine DNA methyltransferase (MGMT) promotor methylation status, steroid intake, presence or absence of seizures, tumor volume and glutamate levels determined by MR spectroscopy. The trial is currently recruiting in seven centers in Switzerland. TRIAL REGISTRATION: NCT05664464. Registered 23 December 2022.

Induced Chirality in Sulfasalazine by Complexation With Albumins: Theoretical and Experimental Study.

Through molecular recognition, drugs can interact and complex with macromolecules circulating in the body. The serum albumin transport protein, found in several mammals, has several interaction sites where these molecules can be located. The drug sulfasalazine (SSZ) is known in the literature to complex at drug site 1 (DS1) in human serum (HSA) and bovine serum (BSA) proteins. This complexation can be studied using various spectroscopic techniques. With the techniques used in this work, absorption in the ultraviolet and visible regions (UV-Vis) and electronic circular dichroism (ECD), a significant difference was observed in the results involving HSA and BSA. The application of theoretical methodologies, such as TD-DFT and molecular docking, suggests that the conformation that SSZ assumes in DS1 of the two proteins is different, which exposes it to different amino acid residues and different hydrophobicities. This difference in conformation may be related to the location of DS1 where the drug interacts or to the possibility of SSZ moving in the BSA site, due to its larger size, and moving less freely in HSA.

Sulfasalazine promotes ferroptosis through AKT-ERK1/2 and P53-SLC7A11 in rheumatoid arthritis.

OBJECTIVE: Ferroptosis has been reported to play a role in rheumatoid arthritis (RA). Sulfasalazine, a common clinical treatment for ankylosing spondylitis, also exerts pathological influence on the progression of rheumatoid arthritis including the induced ferroptosis of fibroblast-like synoviocytes (FLSs), which result in the perturbated downstream signaling and the development of RA. The aim of this study was to investigate the underlying mechanism so as to provide novel insight for the treatment of RA. METHODS: CCK-8 and Western blotting were used to assess the effect of sulfasalazine on FLSs. A collagen-induced arthritis mouse model was constructed by the injection of collagen and Freund's adjuvant, and then, mice were treated with sulfasalazine from day 21 after modeling. The synovium was extracted and ferroptosis was assessed by Western blotting and immunofluorescence staining. RESULTS: The results revealed that sulfasalazine promotes ferroptosis. Compared with the control group, the expression levels of ferroptosis-related proteins such as glutathione peroxidase 4, ferritin heavy chain 1, and solute carrier family 7, member 11 (SLC7A11) were lower in the experimental group. Furthermore, deferoxamine inhibited ferroptosis induced by sulfasalazine. Sulfasalazine-promoted ferroptosis was related to a decrease in ERK1/2 and the increase of P53. CONCLUSIONS: Sulfasalazine promoted ferroptosis of FLSs in rheumatoid arthritis, and the PI3K-AKT-ERK1/2 pathway and P53-SLC7A11 pathway play an important role in this process.

Combinatorial intervention with dental pulp stem cells and sulfasalazine in a rat model of ulcerative colitis.

BACKGROUND: Ulcerative colitis is an inflammatory bowel disease (IBD) that involves inflammation of the colon lining and rectum. Although a definitive cure for IBD has not been identified, various therapeutic approaches have been proposed to mitigate the symptomatic presentation of this disease, primarily focusing on reducing inflammation. The aim of the present study was to evaluate the therapeutic potential of combining dental pulp stem cells (DPSCs) with sulfasalazine in an acetic acid-induced ulcerative colitis rat model and to assess the impact of this combination on the suppression of inflammatory cytokines and the regulation of oxidative stress in vivo. METHODS: Ulcerative colitis was induced in rats through transrectal administration of 3% acetic acid. The therapeutic effect of combining DPSCs and sulfasalazine on UC was evaluated by measuring the colonic weight/length ratio and edema markers; performing histopathological investigations of colon tissue; performing immunohistochemical staining for NF-κB-P65 and IL-1ß; and evaluating oxidative stress and antioxidant indices via ELISA. Moreover, the proinflammatory markers NF-κB-P65, TNF-α and TLR-4 were assessed in colon tissue via ELISA. Furthermore, qRT‒PCR was used to estimate the expression levels of the TLR-4, NF-κB-P65, and MYD88 genes in colon tissue. RESULTS: The investigated macroscopic and microscopic signs of inflammation were markedly improved after the combined administration of sulfasalazine and DPSCs, where a noticeable improvement in histological structure, with an intact mucosal epithelium and mild inflammatory infiltration in the mucosa and submucosa, with slight hemorrhage. The administration of either DPSCs or sulfasalazine, either individually or in combination, significantly reduced ROS levels and significantly increased XOD activity. The immunohistochemical results demonstrated that the combined administration of DPSCs and sulfasalazine attenuated NFκB-p65 and IL-1ß expression. Finally, the combined administration of DPSCs and sulfasalazine significantly downregulated MyD88, NF-κB and TLR4 gene expression. CONCLUSIONS: Cotreatment with DPSCs and sulfasalazine had synergistic effects on ulcerative colitis, and these effects were relieved.

The Dual Benefit of Sulfasalazine on Pneumocystis Pneumonia-Related Immunopathogenesis and Antifungal Host Defense Does Not Require IL-4Rα-Dependent Macrophage Polarization.

Pneumocystis is a respiratory fungal pathogen that is among the most frequent causes of life-threatening pneumonia (PcP) in immunocompromised hosts. Alveolar macrophages play an important role in host defense against Pneumocystis, and several studies have suggested that M2 polarized macrophages have anti-Pneumocystis effector activity. Our prior work found that the immunomodulatory drug sulfasalazine (SSZ) provides a dual benefit during PcP-related immune reconstitution inflammatory syndrome (IRIS) by concurrently suppressing immunopathogenesis while also accelerating macrophage-mediated fungal clearance. The benefits of SSZ were associated with heightened Th2 cytokine production and M2 macrophage polarization. Therefore, to determine whether SSZ improves the outcome of PcP through a mechanism that requires Th2-dependent M2 polarization, RAG2-/- mice lacking interleukin 4 receptor alpha chain (IL-4Rα) on macrophage lineage cells were generated. As expected, SSZ treatment dramatically reduced the severity of PcP-related immunopathogenesis and accelerated fungal clearance in immune-reconstituted RAG2-/- mice. Similarly, SSZ treatment was also highly effective in immune-reconstituted RAG2/IL-4Rα-/- and RAG2/gamma interferon receptor (IFN-γR)-/- mice, demonstrating that neither IL-4Rα-dependent M2 nor IFN-γR-dependent M1 macrophage polarization programs were required for the beneficial effects of SSZ. Despite the fact that macrophages from RAG2/IL-4Rα-/- mice could not respond to the Th2 cytokines IL-4 and IL-13, M2-biased alveolar macrophages were identified in the lungs following SSZ treatment. These data demonstrate that not only does SSZ enhance phagocytosis and fungal clearance in the absence of macrophage IL-4Rα signaling, but also that SSZ promotes M2 macrophage polarization in an IL-4Rα-independent manner. These findings could have implications for the treatment of PcP and other diseases in which M2 polarization is beneficial.

ALOX5 inhibition protects against dopaminergic neurons undergoing ferroptosis.

Oxidative disruption of dopaminergic neurons is regarded as a crucial pathogenesis in Parkinson's disease (PD), eventually causing neurodegenerative progression. (-)-Clausenamide (Clau) is an alkaloid isolated from plant Clausena lansium (Lour.), which is well-known as a scavenger of lipid peroxide products and exhibiting neuroprotective activities both in vivo and in vitro, yet with the in-depth molecular mechanism unrevealed. In this study, we evaluated the protective effects and mechanisms of Clau on dopaminergic neuron. Our results showed that Clau directly interacted with the Ser663 of ALOX5, the PKCα-phosphorylation site, and thus prevented the nuclear translocation of ALOX5, which was essential for catalyzing the production of toxic lipids 5-HETE. LC-MS/MS-based phospholipidomics analysis demonstrated that the oxidized membrane lipids were involved in triggering ferroptotic death in dopaminergic neurons. Furthermore, the inhibition of ALOX5 was found to significantly improving behavioral defects in PD mouse model, which was confirmed associated with the effects of attenuating the accumulation of lipid peroxides and neuronal damages. Collectively, our findings provide an attractive strategy for PD therapy by targeting ALOX5 and preventing ferroptosis in dopaminergic neurons.

Regulation of BCRP expression and sulfasalazine pharmacokinetics by the nuclear receptor REV-ERBα.

BCRP (breast cancer resistance protein) is a crucial efflux transporter involved in the regulation of the pharmacokinetics and pharmacodynamics of a wide range of drugs. Herein, we aimed to investigate a potential role for the nuclear receptor REV-ERBα in the regulation of BCRP expression and sulfasalazine (a BCRP probe substrate) pharmacokinetics.Regulation of BCRP expression by REV-ERBα was assessed using Rev-erbα-/- mice and AML12 and CT26 cells. Pharmacokinetic analysis was performed with Rev-erbα-/- and wild-type mice after sulfasalazine administration.We found that the expression levels of BCRP mRNA and protein were downregulated in the liver and small intestine of Rev-erbα-dificient mice. In line with this, Rev-erbα ablation increased the systemic exposures of oral sulfasalazine.Positive regulation of BCRP expression and function by REV-ERBα was furtherly confirmed in AML12 and CT26 cells. Moreover, indirect regulation of Bcrp expression by REV-ERBα was potentially mediated by a negative transcription factor DEC2, which is a downstream target of REV-ERBα.In conclusion, REV-ERBα positively regulates BCRP expression in mice, thereby affecting sulfasalazine pharmacokinetics.

3,6-Epidioxy-1,10-bisaboladiene and sulfasalazine synergistically induce ferroptosis-like cell death in human breast cancer cell lines.

3,6-Epidioxy-1,10-bisaboladiene (EDBD) is an endoperoxide compound isolated from edible wild plants that induces iron-dependent ferroptosis-like cell death in HL-60 cells by decreasing the expression of GPX4 and glutathione. In contrast, sulfasalazine (SSZ), a clinically used anti-inflammatory drug, induces ferroptosis through the system xc-. In this study, we investigated the synergistic effects of these 2 compounds on 3 human breast cancer cell lines (HBC-5, MCF-7, and MDA-MB-231). EDBD-induced cell death was relieved by the lipid peroxidation inhibitor ferrostatin-1 and the iron chelator deferoxamine mesylate (DFOM), indicating that EDBD induced ferroptosis-like cell death. Moreover, cotreatment with EDBD and SSZ synergistically induced cell death in all 3 cell lines. Because the cytotoxicity of the cotreatment was inhibited by DFOM and ferrostatin-1, the combination of EDBD and SSZ synergistically induced ferroptosis. Collectively, EDBD enhanced the effects of SSZ as a clinical anti-inflammatory and anticancer drug candidate.

Efficacy of sulfasalazine for the prevention of Pneumocystis pneumonia in patients with rheumatoid arthritis: A multicentric self-controlled case series study.

INTRODUCTION: Pneumocystis pneumonia (PCP) is an opportunistic lung infection and has been reported among patients with rheumatoid arthritis (RA). An animal study revealed that sulfasalazine enhances Pneumocystis clearance from the lung by accelerating macrophage activity. METHODS: The self-controlled case series (SCCS) method was used to investigate the association between sulfasalazine use and PCP development in patients with RA without the effect of time-invariant, interpatient confounders. PCP episodes which developed in patients with RA at five hospitals between 2003 and 2019 were identified. PCP was defined by the following criteria: 1) detection of Pneumocystis jirovecii in respiratory specimens by polymerase chain reaction; 2) clinical symptoms (pyrexia, dry cough, dyspnea or hypoxia); 3) diffuse interstitial infiltrate on chest imaging; and 4) absence of PCP prophylaxis. The PCP incidence rate ratio (IRR) was compared between periods with and without sulfasalazine use by conditional Poisson regression. RESULTS: Fifty episodes of PCP were identified in 49 patients. Thirty patients received sulfasalazine at some point during their observation. While 49 episodes of PCP developed in 170.3 person-years without sulfasalazine use, only one episode of PCP developed in 103.7 person-years with sulfasalazine use. Sulfasalazine use was associated with a decreased PCP risk (adjusted IRR <0.01; 95% confidence interval <0.01-0.03) after adjusting for age and glucocorticoid, methotrexate, and tumor necrosis factor inhibitor administration. CONCLUSION: Our study demonstrated a preventive effect of sulfasalazine against PCP in patients with RA.

Effects of sulfasalazine in axial spondyloarthritis on COVID-19 outcomes: real-life data from a single center.

INTRODUCTION: Compared to biological agents, little is known about the impact of sulfasalazine therapy on COVID-19 outcomes in patients with Axial Spondyloarthritis (AxSpA). Therefore, we aimed to evaluate the COVID-19 severity in AxSpAs receiving sulfasalazine and biologic-agent. MATERIALS AND METHODS: A total of 219 SARS-CoV-2 positive AxSpA patients were retrospectively analyzed. COVID-19 pneumonia, hospitalization rate, and length of stay were used to determine COVID-19 severity. AxSpA patients were mainly grouped and compared as sulfasalazine and non-sulfasalazine. Afterward, we excluded no-treatment patients to reveal the drug's effects more clearly and regrouped AxSpA patients as sulfasalazine-monotherapy (34.3%), biologic-monotherapy (33.7%), and sulfasalazine + biologic (7.3%). RESULTS: Fifty-nine percent of the patients were male and the mean age was 45.0 years. Peripheral arthritis was 35% and uveitis 15%. In total, 41.5% of them have received sulfasalazine and 41.0% biologic agents, and the remaining patients with no AxSpA-specific treatment. In the first comparison, the sulfasalazine group had a higher age, more frequent COVID-19 pneumonia, hospitalization, and longer hospitalization than a non-sulfasalazine group. In the pairwise comparison of 3 treatment groups, the demographic and clinical features, the hospitalization rate and the length of hospital stay were similar but the sulfasalazine-monotherapy group had a higher frequency of COVID-19 pneumonia than the biologic-monotherapy group (23% vs. 7%, p = 0.008). CONCLUSION: Our results imply sulfasalazine may be related to more severe COVID-19 in AxSpA patients. These patients should be followed more carefully in the presence of COVID-19, regardless of reasons such as age, comorbidity, and extra-axial disease, and consideration of discontinuing sulfasalazine maybe even thought.