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)
Feb 24, 2016
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