Fingolimod (Gilenya)

Presented by Matthew Brickey, Tim Robinson, Tom McGinnis, and Katie

Youmans

Outline

• Disease: Multiple Schlerosis• Discovery of Fingolimod-Gilenya• Synthesis and lead modification• Mechanism of action• Pharmacology • Questions

Multiple Sclerosis

• Degenerative nerve disease• Characterized by defective immune

system response resulting in nerve damage

• Cause is unknown• Difficult diagnosis

Multiple Sclerosis (MS)

4 types of MS• Relapsing-remitting (RR)• Primary Progressive (PP)• Secondary progressive (SP)• Progressing relapsing (PR)

• RR often progresses into SP over time

DISCOVERY

Initial Antifungal Agents

• Cyclosporin A-reported in 1976 (fungus origin)• FK506-isolated in 1987 (bacterium origin)• These set up a foundation for the screening of

new fungi and other microbes in the pursuit of new immunosuppressants.

Studying Isaria sinclairii

• Tetsuro Fujita began focusing on Isaria sinclairii in the late 1980s and early 1990s.

• This fungus is native to Asia, mainly China, Korea, and Japan.

• Classified as an entomopathogenic fungus.

Isaria sinclairii fungal development

• Spreads by infecting insect larvae with its fungal spores

• Acts as a parasite, growing in and ultimately killing the insect

• Afterwards, colonizes the insect cadaver and develops white fruiting bodies (6cm tall)

• Fruits in spring and summer

In Vitro Assay

• To screen for immunosuppressive activity• Fujita used a mouse allogeneic mixed

lymphocyte reaction (MLR) assay• Spleen cells from two different strains of mice

were cocultured and alloantigen was added to stimulate T-cell proliferation

• Samples were evaluated for inhibition of proliferation of T-cells (reported as IC50 value)

In Vivo Assay

• Performed by transplanting the dorsal skin of one rat (strain LEW) to the lateral thorax of the second rat (strain F344)

• Daily intraperitoneal administration until the skin grafts were rejected (90% necrosis)

• Compounds were scored on their ability to prolong rat skin graft survival.

Importance of Assays

• Lead to the eventual development of fingolimod

• The evaluation process guided the isolation of a compound with immunosuppressant activity– They called it ISP-I (below)

ISP-I

• Identical to previously isolated antifungal agents– Myriocin and thermozymocidine

• Pros:– Found to be 5-10 fold more potent than cyclosporin A

in the MLR assay– Also prolonged rat skin graft survival better.

• Cons: – Found to be toxic at a smaller dose than Cyclosporin A– Poorly soluble

Improving ISP-I

• Goal:– To simplify the structure and improve the physical

characteristics (e.g. solubility) and biological function

• Between 1995 and 1998, around 50 different analogues were reported, with published results from both in vitro and in vivo assays

ISP-I-28

• First analogue of interest• Less toxic, prolonged survival, but less potent

in MLR assay• Reduced carboxylic acid and 14-ketone to

alcohols

• Removed three hydroxy groups, ISP-I-36• Improved activity and survival

• Shortened chain to 14 carbons to create ISP-I-55• More potent immunosuppressant in both in vivo and

in vitro, double survival time in skin graft assay

Next Steps

Final Modification

• Introduced an aromatic moiety-believed to improve activity by restricting conformation

• Placement could increase or decrease activity of drug-one position off 10-fold less potent

• Led to fingolimod, with improved activity, less toxicity, and better solubility

Synthesis

• 13 methods for Fingolimod, Fingolimod-P• First in 1995• Yields remained at or below 25 % until 2005• Convenient method an improvement on 1995

synthesis, a three step reaction of precursor to Fingolimod (2008)

Petasis Reaction (Sugiyama, 2005)

• Five step synthesis• Couples boronic acids, amines, carbonyls• Form amino alcohols• Overall Yield of 28%

Kim 2006• Start with tris-(hydroxymethyl)aminomethane

(TRIS)• Convert to aldehyde, then alkyne• Couple to aryl iodide via Sonogashira reaction• Hydrogenate, treat with acid, purify• Cheap, practical, 64% overall yield Fingolimod

Mechanism of Action

v

G Protein Coupled Receptors

The Nuts and Bolts of it

Pharmacology

• Marketed as Gilenya• First orally active treatment• Tested on patients with RR-MS–60% decreased in relapse rate

• When compared to Injection treatments growth of new lesions and plaques was significantly reduced

Questions?