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

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Degenerative nerve disease Characterized by defective immune system response resulting in nerve damage Cause is unknown Difficult diagnosis

Multiple Sclerosis (MS)

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

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RR often progresses into SP over time

D

ISCOVERY

Initial Antifungal Agents

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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

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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

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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

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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

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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

Next Steps

• • 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

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

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Marketed as Gilenya First orally active treatment

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Tested on patients with RR-MS

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60% decreased in relapse rate When compared to Injection treatments growth of new lesions and plaques was significantly reduced

Questions?