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