Introduction
Cladribine (CdA), also known as 2-chlorodeoxyadenosine, is a synthetic deoxyadenosine analogue that selectively depletes lymphocytes (1-3). CdA is a prodrug, and accumulation of the active drug CdA triphosphate depends on the ratio of deoxycytidine kinase (DCK), that catalyzes the first of three phosphorylation steps, and 5’-nucleotidases (5’-NTases), that converts phosphorylated cladribine to cladribine nucleoside. In lymphocytes due to a high DCK to 5’-NTase ratio, accumulation of CdA triphosphate results in apoptosis by several mechanisms (2, 3).
Cladribine tablets has been approved for the treatment of relapsing MS, based in part on a placebo-controlled, double-blind, multicenter phase-III trial (CLARITY). 3.5 mg/kg oral CdA reduced the annual relapse rate by around 57% (4), and the relative reduction in the risk of 3-month sustained progression of disability was 33% compared to placebo. The mean number of active T2 lesions on MRI was reduced by 73.4% (4). The extension of the phase-III trial showed a durable effect on MRI and clinical outcomes (5, 6).
The dose of 3.5 mg/kg CdA applied orally in short treatment cycles causes a short-term lymphocyte depletion followed by immune-reconstitution (7). However, CdA passes the blood-brain barrier (BBB), and gives rise to 25% of the plasma concentration in CSF resulting in a maximum CSF concentration of 0.019-0.025 µM (2, 3), and this may suggest a potential effect on CNS resident cells as well.
Microglia comprise 10-15% of all glial cells in the CNS (8). Pro-inflammatory microglia secrete pro-inflammatory cytokines, chemokines, reactive oxygen and nitrogen species, which are toxic for oligodendrocytes and neurons (9, 10). Lipopolysaccharides (LPS) induce a pro-inflammatory phenotype of microglia characterized by the expression of interleukin-1β (IL-1β), IL-6, major histocompatibility complex II (MHC II), tumor necrosis factor (TNF) and inducible nitric oxide synthase (iNOS) (11, 12). Microglia also exert neuroprotective and repairing functions, and can produce neurotrophic factors and anti-inflammatory cytokines (11, 13, 14). The anti-inflammatory cytokine IL-4 induces the anti-inflammatory phenotype characterized by arginase 1 (Arg1) and IL-10 expression among others (11, 12, 15).
Data about the effect of CdA on microglia are scarce (16). When investigating the effect of the drug in murine cells, higher CdA concentrations are required because the activity of DCK is ten times lower in mice than in humans (17, 18). Here, we examined the effect of CdA on the phenotype and function of naïve and activated mouse microglia in concentrations that putatively overlap with the levels in the human CSF.
Materials and Methods