A staged screening of registered drugs highlights remyelinating drug candidates for clinical trials

There is no treatment for the myelin loss in multiple sclerosis, ultimately resulting in the axonal degeneration that leads to the progressive phase of the disease. We established a multi-tiered platform for the sequential screening of drugs that could be repurposed as remyelinating agents. We screened a library of 2,000 compounds (mainly Food and Drug Administration (FDA)-approved compounds and natural products) for cellular metabolic activity on mouse oligodendrocyte precursors (OPC), identifying 42 molecules with significant stimulating effects. We then characterized the effects of these compounds on OPC proliferation and differentiation in mouse glial cultures, and on myelination and remyelination in organotypic cultures. Three molecules, edaravone, 5-methyl-7-methoxyisoflavone and lovastatin, gave positive results in all screening tiers. We validated the results by retesting independent stocks of the compounds, analyzing their purity, and performing dose-response curves. To identify the chemical features that may be modified to enhance the compounds’ activity, we tested chemical analogs and identified, for edaravone, the functional groups that may be essential for its activity. Among the selected remyelinating candidates, edaravone appears to be of strong interest, also considering that this drug has been approved as a neuroprotective agent for acute ischemic stroke and amyotrophic lateral sclerosis in Japan.


Purified OPC cultures
OPC were obtained from neonatal mouse primary mixed glial cultures. In brief, forebrains of newborn CD1 Swiss mice were freed of meninges, chopped into 0.2-mm sections and dissociated by mild trypsinization procedure and gentle mechanical disruption with a Pasteur pipette. The cells were seeded into poly-L-lysine (10 μg/ml, Sigma-Aldrich, St. Louis, MO) coated 60 mm diameter culture dishes at the density of 1.2×10 5 cells/cm2 and grown at 37°C in a 91.5% air-8.5% CO 2 humidified atmosphere in Dulbecco's modified eagle medium (DMEM) containing 10% Foetal Bovine Serum (FBS), 2 mM glutamine and penicillin (50 μg /ml) and streptomycin (50 μg/ml), replacing fresh medium after 1 DIV and every 2-3 days (media, sera and reagents by GIBCO, Lifetechnologies, Grand Island, NY). After 8-10 days, OPC were detached from astroglia layer by mechanical dissociation; to minimize contamination by microglial cells, the detached cell suspension was incubated for 1 hour at 37°C in a 175 cm2 culture flask. The non-adering cells were seeded in the same medium as above at the density of 1 × 105 cells/cm2 into poly-L-lysine-coated dishes. Two hours after plating, culture medium was replaced with defined serum-free DMEM without thyroid hormones (56). Macrophage/microglia contamination accounted for less than 1% of total cells, as assessed by immunostaining with the monoclonal antibody (mAb) cd11b (AbD Serotec, Oxford, UK); glial fibrillary acid protein-positive astrocytes were virtually absent and the majority of cells (>99%) belonged to the OL lineage, as assessed by specific mAbs, which bind to differentiation-regulated surface antigens of these cells.

[3H] Thymidine incorporation assay
Purified OPC subcultured in 96-well microtiter plates were exposed after 1 day to the compounds

Immunohistochemistry
Cerebellar slices were incubated in the absence or presence of compounds, fixed in 4% paraformaldehyde in PBS for 10 min and then stained using double indirect immunofluorescence techniques. After washing with PBS and a blockade with 10% normal donkey serum (Sigma-Aldrich) in PBS for 1 h, sections were incubated overnight at 4°C with anti-neurofilament heavy chain (NFH) ( pAb (1:500, AbD Serotec) and anti-MBP mAb (clone smi99, 1:1000, Covance, Denver, PA) diluted in PBS containing 1% BSA and 0.25% Triton. Bindings were visualized using a mixture of CY3-conjugated donkey anti-rabbit IgG and fluorescein-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in PBS+1% normal serum.
The incubation with the secondary Abs was carried out for 1 h at room temperature. Sections were then mounted in Vectashield (Vector Laboratories, Burlingame, CA) and sealed. Negative controls were performed by replacing primary antibodies with preimmune serum or IgG isotype control.

Terminology and data analysis of calcium mobilization experiments
The pharmacological terminology adopted in calcium mobilization experiments is consistent with IUPHAR recommendations. For potency values 95% confidence limits were indicated. Maximum change in fluorescence, expressed as percent over the baseline fluorescence, was used to determine agonist response. Agonist potencies are given as pEC50 i.e. the negative logarithm to base 10 of the molar concentration of an agonist that produces 50% of the maximal effect of that agonist.
Concentration-response curves to agonists were fitted to the classical four-parameter logistic nonlinear regression model: EC50 is the concentration of agonist producing a 50% maximal response and n is the Hill coefficient of the concentration-response curve to the agonist. Curve fitting was performed using Calcium mobilization assay was performed in HEK293mNPSR cells to test the dose response activity of NPS, edaravone, 5-methyl 7-metoxyisoflavone, lovastatin, biochanin A, benztropine and losartan. The compounds did not stimulate calcium mobilization. Data are expressed as mean ± SEM of 3 separate experiments run in duplicate.