Frédéric AMARAL

L’Oréal Research and Innovation , Aulnay, France

In vitro assessment of shampoos eye stinging potential using Transient Receptor Potential

Vanilloid Type 1 (TRPV1)

ESTIV 2012, 16-19 October 2012

Presentation Outline

Role of TRPV1 in nociception

Objectives of the study/experimental design

Results

Conclusions and next steps

ESTIV 2012, 16-19 October 2012

Role of TRPV1 in nociception (1)

Receptor type: calcium permeable ion channel belonging

to the TRP (Transient Receptor Potential) family

Location: C-fibers, CNS but also in skin, cornea and

mucosal tissue

Activation: heat, acidic conditions, Capsaicin (CP)

calcium flux from extracellular sources

Release of neurotransmiters, neuropeptides

induction of pain, burning, pruritus

ESTIV 2012, 16-19 October 2012

Role of TRPV1 in nociception (2)

Steinhoff and Biro, 2009 (J ID 129)

TRPV1

fluorophore Quantification

intracellular Ca2+

Target for 1st

generation assay

Need for an in vitro assay for identifying the stinging potential of personal care products (ingredients and formulae)

ESTIV 2012, 16-19 October 2012

Objectives of the Study/experimental design

To demonstrate the PoC with the « Nociocular » in vitro

assay (based on SH-SY5Y overexpressing TRPV1)

Collaboration with the University of Stockholm (A Forsby)

Read out of the assay: calcium influx (fura2-AM)

Test articles used:TRPV1 agonist: capsaicine (CP) for sensitivityTRPV1 antagonist: capsazepine (CPZ) for specificityA selection of 10 coded shampoos (adults and children/baby)

ESTIV 2012, 16-19 October 2012

Results: in vitro assay data

3 categories of profiles have been identified

Product A (n=2)

-4 -3 -2 -1 00

25

50

75

100

125

150No capsazepine+capsazepine

Concentration (log %)

Ca2+

influ

x(%

of 1

0 nM

cap

saic

in)

Case A

No induction of Ca2+ flux

Product H (n=2)

-4 -3 -2 -1 00

255075

100125150175200 No capsazepine

+capsazepine

Concentration (log %)

Ca2+

influ

x(%

of 1

0 nM

cap

saic

in)

Case B

induction of Ca2+ fluxDecreased response with CPZTRPV1-mediated effect

Product B (n=3)

-6 -5 -4 -3 -2 -1 00

25

50

75

100

125

150 No capsazepine+capsazepine

Concentration (log %)

Ca2+

influ

x(%

of 1

0 nM

cap

saic

in)

Case C

induction of Ca2+ fluxNo impact of CPZTRPV1-mediated effect?

ESTIV 2012, 16-19 October 2012

Results: in vitro/in vivo correlation

Good correlation, especially for extreme classesGood trend for the moderate class except for formula G

Effect at 0,1% concentration

Effect at 0,1% concentration + CPZ

Shampoo code % Calcium influx of CP % Calcium influx of CP In vitro discomfort class Clinical discomfort class

A 8 4 No Good

E 7 4 No Good

B 50 50 Mild Moderate

F* 17 4 Mild Moderate

G 103 71 Mild Good

C 96 73 Mild Moderate

D 109 92 Mild Moderate

I 130 118 Mild ModerateH 137 120 High Mild: Important stingingJ 152 148 High Mild: Important stinging

* Based on concentrations up to 0,1% due to massive cytotoxicity at higher concentrations

v

v

ESTIV 2012, 16-19 October 2012

Conclusions

We have demontrated that: The « Nociocular » assay could be used to evaluate eye stinging

personal care productsThe assay was applicable to complex water-soluble formulaFor some formulae, the response was TRPV1-mediatedThe in vitro responses correlated well with clinical data

Next steps:

To evaluate a more diverse set of formulae and ingredientsTo confirm the robustness of the assayTo refine the discomfort classes proposed

To develop a model for non water-soluble formulaeTo sick for additional assays to get a better understanding of the

responses observed

ESTIV 2012, 16-19 October 2012

Acknowledgments

L’OREALLinda Bourouf Reine Note

Thomas DelanneGladys Ouédraogo

Sophie Loisel-JoubertJosé Cotovio

Jean-Roch Meunier

University of Stockholm Hanegraaf Maaike

Anna Forsby