1. Fingolimod (Gilenya)
Presented by Matthew Brickey, Tim Robinson, Tom McGinnis, and Katie Youmans

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

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

4. 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
Relapsing Remitting– 85-90%

5. Discovery

6. 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.
The same fungus was used to isolate a cyclic depsipeptide that was an active antibiotic (1980s-1990s)

7. 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.

8. 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

9. 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)

10. 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.

11. 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)

12. ISP-I Identical to previously isolated antifungal agents Pros: Cons:
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

13. 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
Tim’s going to hit the improve button as we move on through a few of the analogues.

14. 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

15. 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

16. 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
The SPT inhibition assay evaluates immunosuppressant activity based on a compound’s ability to inhibit the enzyme serine palmitoyltransferase. (worked for Cyclosporin A, FK506 and ISP-I
The necessity of the MLR assay for ISP-I-55 and fingolimod indicated a different MOA
Needed MLR assay, ISP-I-55 and fingolimod not active in normal SPT assay

17. From 2 to 3, they reduced the ketone to an alcohol
From 2 to 3, they reduced the ketone to an alcohol. ~less toxic, better survival
From 3 to 4, they removed 3 hydroxy groups. ~better activity and survival
From 4 to 5, they shortened the chain. ~more potent and longer term
From 5 to 1, they introduced the aromatic group. ~improve activity by restricting conformation, more potent in body, less toxic, better survival

18. Synthesis 13 methods for Fingolimod, Fingolimod-P First in 1995
Yields remained at or below 25 % until 2005
Convenient method an improvement on synthesis, a three step reaction of precursor to Fingolimod (2008)

19. Petasis Reaction (Sugiyama, 2005)
Five step synthesis
Couples boronic acids, amines, carbonyls
Form amino alcohols
Overall Yield of 28%
Chemical reaction of amine, aldehyde, boronic acid to form substituted amines

20. 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
-cross coupling reaction, forms carbon to carbon bonds, uses palladium catalyst to form bond between terminal alkyne and aryl or vinyl halide, may be performed in mild conditions, halide copper salt catalyst

21. Mechanism of Action

22. v

24. G Protein Coupled Receptors

25. The Nuts and Bolts of it

31. 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

32. Questions?