INVOLVEMENT OF PEROXISOME PROLIFERATOR ACTIVATOR RECEPTORS (PPARs) IN THE PHOTOPROTECTIVE ACTIVITY OF BIO201

IntroductionAccording to Herzlich et al. (PPAR Research, 2008, article ID389507), PPARsplay a key role in the protection of RPE cells against AMD.

There is increasing evidence for the involvement of PPARs in the protectionagainst the development of Age-Related Macular Degeneration (AMD). PPARreceptors comprise three sub-types of nuclear receptors (PPARα, PPARβ/δ andPPARγ) involved in the control of lipid and carbohydrate metabolism, as well asinflammatory processes. They are characterized by a large ligand-binding pocketand therefore accommodate diverse types of endogenous or exogenous ligands.BIO201 is a pharmaceutical grade preparation of the di-apo-carotenoid norbixin.It has been formulated for improving its stability and per os bioavailability asMacuneos. Biophytis will start a phase 1 clinical trial in 2017 with Macuneos asan oral treatment for dry AMD. The mechanism by which BIO201 exerts in

vitro and in vivo protective effects on RPE was studied.

Materials and MethodsThe photoprotective effect of BIO201 was tested in an in vitro model ofphototoxicity of porcine primary RPE cells challenged with A2E then blue-lightilluminated. Cell survival was measured 24h after illumination. BIO201 protectiveactivity was then characterized by a pharmacological approach employingselective agonists and antagonists of the different PPAR subtypes.In order to study the antioxidant and anti-apoptotic properties of BIO201, Arpe-19cells were treated with antimycin A and PENAO, two pro-oxidant compounds, andROS and PARP cleavage were measured by flow cytometry (DCFDA) andWestern Blot, respectively, in the presence or the absence of BIO201 (30 µM).To determine whether BIO201 was able to activate the 3 PPAR isoforms, Cv-1cells were transfected with luciferase reporter plasmid (pG5-TK-pGL3) in thepresence of pGal4hPPARx (the vector expressed chimeric proteins containing theGal4 DNA-binding domain fused to a human PPAR ligand-binding domain codingsequence) expression vector, and luciferase was measured after 16h contact withvarious concentrations of BIO201 (or other substances).The expression level of the PPARs was assessed by Western Blot.Statistical analyses were performed by one-way ANOVA followed by Dunnett'stest.

Fontaine V.1, Monteiro E1., Fournie M1., Bonnard B1., On S.2, Serova M.2, Balducci C.2, Guibout L.2, Sahel J.-A.1, Veillet S.2, Dilda P.2 and Lafont R.2

1 Sorbonne Universités, UPMC Univ Paris 06, INSERM U968, CNRS UMR 7210 - Institut de la Vision, 75012 Paris, France2 Biophytis, Université Pierre et Marie Curie, 4 place Jussieu, 75005 Paris, France

Conclusions

Contacts: valerie.fontaine@inserm.fr pierre.dilda@biophytis.com

Results

Financial disclosure: Financial support came from Biophytis

# F070

BIO201, PPARa and PPARb/d agonists

protect RPE cells against A2E-mediated

phototoxicity

BIO201 protection is partially reversed

by PPARb/d and PPARg antagonists

DMSO - A2E

DMSO + A2E

BIO201 + A

2E

BIO201 + A

2E + MK

BIO201 + A

2E + GSK

BIO201 + A

2E + T0

50

100

RP

E c

ell s

urv

ival (%

of

DM

SO

-A2E

)

******

DMSO - A2E

DMSO + A2E

BIO201 + A

2E

SUL + A2E

GW + A

2E

RGZ + A2E

TGZ + A2E

0

50

100

RP

E c

ell s

urv

ival (%

of

DM

SO

-A2E

)

****

******** ****

**

seeding

+ BIO201 or agonists

- 72h

- 48h

- 19h

+ A2E

survival

24h 0h

seeding

+ BIO201

- 72h

- 48h

- 19h

+ A2E

+ antagonists

- 1h

survival

24h 0h

**p<0,01 ****p<0,0001 compared to DMSO + A2E**p<0,01 ****p<0,0001 compared to BIO201 + A2E

Molecule Abbreviation Function Conc.

(µM)

sulindac SUL PPARa agonist 200GW0742 GW PPARb/d agonist 30rosiglitazone RGZ PPARg agonist 20troglitazone TGZ PPARa, g dual agonist 20MK886 MK PPARa antagonist 5GSK3787 GSK PPARb/d antagonist 1T0070907 T PPARg antagonist 10

BIO201, as well as sulindac, GW0742and troglitazone significantly protect RPEcells against A2E-indiced phototoxicity.Rosiglitazone, a specific PPARg agonist,shows no photo-protective activity.BIO201 protective activity is partiallyreversed by PPARa and PPARb/d

antagonists.

BIO201 activates hPPARa and, even more efficiently, hPPARb/d

BIO201

0,00

E+0

0

3,00

E-0

8

1,00

E-0

7

3,00

E-0

7

1,00

E-0

6

3,00

E-0

6

1,00

E-0

5

3,00

E-0

5

1,00

E-0

4

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

109%121%

158%*

231%***

285%***

321%***

358%***

212%***

100%

BIO201 (M)

Lu

cifera

se a

ssay (

Arb

itra

ry u

nit

s)

PPARa Troglitazone

0,00

E+0

0

3,00

E-0

8

1,00

E-0

7

3,00

E-0

7

1,00

E-0

6

3,00

E-0

6

1,00

E-0

5

3,00

E-0

5

1,00

E-0

4

0.000

0.002

0.004

0.006

0.008

97%109%176%

358%394%

637%***

1085%***

91%100%

Troglitazone (M)

Lu

cifera

se a

ssay (

Arb

itra

ry u

nit

s) PPARa

BIO201

0,00

E+00

1,00

E-08

1,00

E-07

3,00

E-07

1,00

E-06

3,00

E-06

1,00

E-05

3,00

E-05

1,00

E-04

0.0

0.2

0.4

0.6

0.8

86%113%123%135%281%

1727%

5219%***

8851%***

100%

BIO201 (M)

Lu

cifera

se a

ssay (

Arb

itra

ry u

nit

s)

PPARb/d BIO201

0,00

E+0

0

3,00

E-0

8

1,00

E-0

7

3,00

E-0

7

1,00

E-0

6

3,00

E-0

6

1,00

E-0

5

3,00

E-0

5

1,00

E-0

4

0.0000

0.0005

0.0010

0.0015

125%

16% 50%52% 61%

116%

207%202%

100%

BIO201 (M)

Lu

cifera

se a

ssay (

Arb

itra

ry u

nit

s)

PPARg

BIO201 is able tosignificantly trans-activatePPARa (C ≥ 0.3 µM) andPPARb/d (C ≥ 3 µM), butit does not trans-activatePPARg.

Troglitazone, which is infact a dual PPARa/gagonist indeed activatesPPARa (C ≥ 1 µM). Thisresult suggests that thephotoprotective effect oftroglitazone on RPE cellsis mediated by itsinteraction with PPARa.

These experimentshowever do not prove adirect binding of BIO201to PPARs.

*p<0,05 **p<0,001 compared to not treated

BIO201 reduces ROS production and abolishes PARP cleavage in

Arpe-19 cells

BIO201 (30 µM) significantly protectsARPE-19 cells against ROS induced byPENAO (A, B, C) or Antimycin A (D, E, F).

*p<0,05, **p<0,01 ***p<0,00, Mann-Whitney test

BIO201 (30 µM) decreases c-PARP expression induced byAntimycin A in ARPE-19 cells,suggesting the protection fromROS-induced apoptosis.

BIO201 counteracts PPARa protein degradation induced by A2E

DMSO

DMSO + A2E

BIO201 + A

2E

GW + A

2E

RGZ + A2E

TGZ + A2E

0.0

0.5

1.0

1.5

pro

tein

rela

tive e

xp

ressio

n

**** ******

**p<0,01 ****p<0,0001 compared to DMSO + A2E

seeding

+ BIO201 or agonists

- 72h

- 48h

- 19h

+ A2E

0h

PPAR analysis (western blot)

We observe here for the first time a rapidand profound reduction of PPARa proteinexpression upon A2E treatment. This isabolished when RPE cells are treated withBIO201 before A2E exposure. GW0742and troglitazone, two photoprotective PPARagonists, display similar patterns. On thecontrary, rosiglitazone, which is lackingcytoprotective activity, is unable to sustainPPARa protein expression. PPARa

expression is not modified by BIO201 orPPAR agonists when used alone.PPARb/d and g protein expression were notaffected by A2E treatment.

- Both photoprotection and transactivaction results indicate thatBIO201’s beneficial effect on RPE cells is linked with PPARα and/orPPARβ/δ activation.

- BIO201 inhibits ROS production and apoptosis.- Prevention of PPARα protein degradation appears to be a key feature

in BIO201 photoprotective activity.- Further experiments are underway with BIO201 (i) to better

understand the crosstalk between the PPARs and (ii) to elucidate thesignaling pathways involved in RPE cell survival.

NEW THERAPEUTICS FOR AGING DISEASES