b79daf4ce199cb49d797abea537561a8 ijms-26-11311.pdf 9ee3cb1d3543bad7f17c5d4a790db540c7906cf7 ijms-26-11311.pdf e6414d84864493f5425241791970b3e80943410008a948ac884e6161fa147901 ijms-26-11311.pdf Title: Cladribine Preserves Normal Central Nervous System Cellular Activity and Promotes Neuroprotection to Oxidative Stress Damage Subject: Multiple sclerosis (MS) is a chronic neuroinflammatory and demyelinating disease that causes disability in patients. Cladribine is an oral treatment that is used in relapsing–remitting and active secondary progressive MS. T and B lymphocytes are especially sensitive to cladribine, which are transiently depleted upon short treatment courses. However, cladribine crosses the blood–brain barrier (BBB), supporting the hypothesis that cladribine may affect central nervous system (CNS)-resident cells. In this study, we used human primary cells and human cell lines to test the effect of cladribine, at therapeutic concentrations, on cells of the CNS. In these conditions, cladribine did not affect survival, proliferation and the capacity of producing cytokines of human microglial cells (HMC3 cell line) or primary human astrocytes but enhanced the production of oxygen reactive species in both cell types. The initial differentiation of primary human neuronal progenitor cells was impaired when continuously exposed to the maximum therapeutic concentration of cladribine, but not when lower concentrations were used. However, cladribine protected differentiated SH-SY5Y human neuroblastoma cell line from oxidative stress-related cell death. In conclusion, using different in vitro cell models, we demonstrate that cladribine maintains the normal function of CNS glia and protects neuronal cells from oxidativestress damage. Keywords: cladribine; multiple sclerosis; disease modifying treatment; central nervous system; immunomodulation; neuroprotection; neurodegeneration Author: Herena Eixarch, Laura Calvo-Barreiro, Nicolás Fissolo, Ursula Boschert, Arnau Hervera, Manuel Comabella, Xavier Montalban and Carmen Espejo Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Sat Nov 22 12:32:28 2025 CET ModDate: Sat Nov 22 12:56:51 2025 CET Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 22 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3163038 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- GDOREV+URWPalladioL-Bold Type 1 Custom yes yes yes 164 0 MIKRSI+VnURWPalladioL Type 1 Custom yes yes yes 165 0 DZIWAW+URWPalladioL-Roma Type 1 Custom yes yes yes 166 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463 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 464 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 465 0 CZKGAS+CMR10 Type 1 Builtin yes yes yes 587 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-04-21 04:15:09 CEST RepresentationInformation: ijms-26-11311.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-04-20 15:28:30 CEST Size: 3163038 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 714 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Results Children: Item: Title: Effect of Cladribine on Human Microglial Cells and Primary Astrocytes Children: Item: Title: Steady-State Cladribine Concentration Does Not Affect Cell Survival or Proliferation of HMC3 Human Microglia Cell Line and Human Primary Astrocytes Item: Title: Immune Function of HMC3 Human Microglia Cell Line and Human Primary Astrocytes Exposed to Cladribine Item: Title: Effect of Cladribine on Neural and Oligodendroglial Cells Children: Item: Title: Cladribine Does Not Impair the Differentiation of Neuronal Progenitors into Neurons Item: Title: Cladribine Does Not Affect the Differentiation of MO3.13 Human Oligodendroglia Cell Line Item: Title: Effect of Cladribine at Estimated Brain Concentrations in Neural- and Oligodendroglial-like Cells Exposed to Inflammatory, Oxidative and Excitotoxic Stress Item: Title: Discussion Item: Title: Materials and Methods Children: Item: Title: Human Primary Cells and Cell Lines, Media and Differentiation Protocols Item: Title: Cladribine and Deoxycytidine Item: Title: Cytokine Stimulation Item: Title: Determination of Cell Death Item: Title: Proliferation Assay Item: Title: Cytokine Release Quantification Item: Title: Determination of Oxidative Stress Item: Title: Cytotoxicity Detection by LDH Release Item: Title: Excitotoxic Insult in Differentiated SH-SY5Y Cells Item: Title: Immunocytochemistry Item: Title: Image Acquisition and Analysis Item: Title: Statistical Analysis Item: Title: Conclusions Item: Title: References Info: Title: Cladribine Preserves Normal Central Nervous System Cellular Activity and Promotes Neuroprotection to Oxidative Stress Damage Author: Herena Eixarch, Laura Calvo-Barreiro, Nicolás Fissolo, Ursula Boschert, Arnau Hervera, Manuel Comabella, Xavier Montalban and Carmen Espejo Subject: Multiple sclerosis (MS) is a chronic neuroinflammatory and demyelinating disease that causes disability in patients. Cladribine is an oral treatment that is used in relapsing–remitting and active secondary progressive MS. T and B lymphocytes are especially sensitive to cladribine, which are transiently depleted upon short treatment courses. However, cladribine crosses the blood–brain barrier (BBB), supporting the hypothesis that cladribine may affect central nervous system (CNS)-resident cells. In this study, we used human primary cells and human cell lines to test the effect of cladribine, at therapeutic concentrations, on cells of the CNS. In these conditions, cladribine did not affect survival, proliferation and the capacity of producing cytokines of human microglial cells (HMC3 cell line) or primary human astrocytes but enhanced the production of oxygen reactive species in both cell types. The initial differentiation of primary human neuronal progenitor cells was impaired when continuously exposed to the maximum therapeutic concentration of cladribine, but not when lower concentrations were used. However, cladribine protected differentiated SH-SY5Y human neuroblastoma cell line from oxidative stress-related cell death. In conclusion, using different in vitro cell models, we demonstrate that cladribine maintains the normal function of CNS glia and protects neuronal cells from oxidativestress damage. 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