EVIDENCE FOR THE INVOLVEMENT OF PPARs IN THE PHOTOPROTECTIVE - - PowerPoint PPT Presentation

EVIDENCE FOR THE INVOLVEMENT OF PPARs IN THE PHOTOPROTECTIVE ACTIVITY OF BIO201 (NORBIXIN) ON RETINAL PIGMENT EPITHELIUM.

Fontaine V.1, Monteiro E.1, Fournie M.1, On S.2, Serova M.2, Dilda P.2 and Lafont R.2

1 Sorbonne Universités, UPMC Univ. Paris 06, INSERM, CNRS, Institut de la Vision, 17 Rue

Moreau, 75012 Paris, France

2 Biophytis, Université Pierre et Marie Curie, 4 place Jussieu, 75005 Paris, France

Financial disclosure: Financial support came from Biophytis.

According to Herzlich (PPAR Res. 2008 Article ID 389507 ), PPARs play a key role in the protection of RPE cells against Age-Related Macular Degeneration (AMD)

PPARs comprise three subtypes that can accommodate various types of ligands. The aim of the present work was to investigate the respective roles of PPARa, PPARb/d and PPARg in response to BIO201 (norbixin) on the protection against the deleterious effects of A2E.

INTRODUCTION

A2E sensitizes RPE cells to blue light damage and reduces PPARa protein

• levels. Both effects are blunted by BIO201, a preparation of the diapo-

carotenoid norbixin developed by Biophytis1. No significant effect is observed on other PPARs levels. PPARa PPARb/d PPARg

The effect on PPARa is post-translational and is also observed with inverted protocols (where treatment with A2E precedes that with norbixin)

DMSO NBX A2E NBX + A2E 0.0 0.5 1.0 1.5

relative expression compared to DMSO **** **** ****

NBX: norbixin 20 µM A2E: 30 µM

DMSO NBX A2E NBX + A2E 0.0 0.5 1.0 1.5

relative expression compared to DMSO

DMSO NBX A2E NBX + A2E 0.0 0.5 1.0 1.5

relative expression compared to DMSO

1(Fontaine et al., PLoS ONE 2016, DOI:10.1371/journal.pone.0167793).

BIO201(norbixin) activates hPPARa and even more efficiently hPPARb/d

Cell plating into 96- well dark plate (morning) Luciferase activity measurement

Day 1 Day 2 Day 3 Day 4

Norbixin treatment (afternoon) CV1 cell plating into flask Cell transfection with pG5TkpGI3 (reporter vector) & pGal4-hPPAR

Norbixin

0,00E+00 3,00E-08 1,00E-07 3,00E-07 1,00E-06 6,00E-06 1,00E-05 3,00E-05 1,00E-04 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025

109% 121% 158% * 231% *** 285% *** 321% *** 358% *** 212% *** 100%

Norbixin (M)

Luciferase assay (Arbitrary units)

hPPARa

Norbixin

0,00E+00 3,00E-08 1,00E-07 3,00E-07 1,00E-06 6,00E-06 1,00E-05 3,00E-05 1,00E-04 0.0000 0.0005 0.0010 0.0015

125% 16% 50% 52% 61% 116% 207% 202% 100%

Norbixin (M)

Luciferase assay (Arbitrary units)

hPPARg

BIO201

0,00E+00 1,00E-08 1,00E-07 3,00E-07 1,00E-06 3,00E-06 1,00E-05 3,00E-05 1,00E-04 0.0 0.2 0.4 0.6 0.8

86%113% 123% 135% 281% 1727% 5219% *** 8851% *** 100%

BIO201 (M)

Luciferase assay (Arbitrary units)

hPPARb/d

Norbixin (M)

Such experiments do not tell us whether these effects are direct or indirect

DIRECT BINDING TESTS (competition with radioactive ligands):

BIO201 (norbixin) binds to PPARa with a Ki of 16.5 µM, and to PPARg with a Ki of 1.15 µM

FRET ACTIVITY TESTS (induction of PPAR coactivator or corepressor recruitment):

BIO201 (norbixin) shows no agonist effect on any of the three PPARs, but has an antagonist effect on PPARb/d (Ka ca. 3.2 µM)

Compound Effect PPARa agonist Sulindac does not protect RPE cells and does not maintain PPARa levels PPARa antagonist MK886 protects RPE cells and maintains PPARa levels PPARb/d agonist GW0742 protects RPE cells and maintains PPARa levels PPARb/d antagonist GSK3787 reduces protection by norbixin PPARg agonist Troglitazone protects RPE cells and maintains PPARa levels PPARg antagonist T0070907 reduces protection by norbixin and troglitazone

Effect of various pharmacological agents

APOPTOSIS: BIO201 (norbixin) cytoprotective activity correlates with a significant reduction of both ROS levels (p<0.01) and apoptosis induction upon treatment with antimycin A.

CONCLUSIONS

• These experiments have revealed the difficulty of interpreting experimental data,

arising from the limited specificity of current PPAR agonists and antagonists, a potential source of multiple cross-reactions (e.g. troglitazone).

• There is some discrepancy between activation studies and binding experiments, which

suggests that PPAR activation is mostly indirect.

• A2E induces PPARa degradation, and all the tested compounds showing a protection
• f RPE cells against blue light damage in the presence of A2E are able to sustain PPARa
• levels. Thus the presence of PPARa appears required for photoprotection.
• Agonists of PPARg and PPARb/d protect RPE cells, whereas their antagonists reduce the

protective effect of BIO201 (norbixin). Thus, both PPARs seem involved in BIO201 action.

• Additional strategies (siRNA, shRNA, …) are under way in order to relieve remaining

ambiguities and assess definitively the role of the three PPARs in BIO201 (norbixin) protective action.

• Norbixin is the active principle of BIO201, a 97% pure 9’-cis isomer prepared from the

seeds of Bixa orellana. BIO201 has been formulated for improving its stability and per os bioavailability as Macuneos. Biophytis will start a phase 1 clinical trial in 2017 with Macuneos as an oral treatment for dry AMD.

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