r&d - seac modelling the skin sensitisation adverse for...
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R&D - SEAC Safety & Environmental Assurance Centre
Gellatly N., Clapp C., Cubberley R., Dhadra S., Glavin, S., Hadfield S., Jacquoilleot S., Jarman A., Jowsey I., Lovell S., Maxwell G., Mayne J., Moore C., Pendlington R., Pickles J., Reynolds, J., Saib O., Sheffield D., Simpson W., Stark R., Summerfield V., Tang D., Windebank S. and MacKay C. SEAC Unilever, Colworth Science Park, Sharnbrook, Bedford, MK44 1LQ, UK
Acknowledgements
Adapted from: MacKay et al. 2013. ALTEX 30 473-486
APPLICATION OF MODEL: 2,4-DINITROCHOLROBENZENE (DNCB) CASE STUDY
1
Estimation of bioavailability in skin - based on OECD TG 428 • DNCB & ex vivo human skin - Pendlington et al 2008 Cutan. Ocul. Toxicol. 27. 283-94.
• Penetration system modelled and partitioning and diffusion rates determined by fitting - Davies et al 2011 Toxicol. Sci. 119, 308-18
Estimation of reaction kinetics in skin • DNCB in solution & ex vivo human skin (tape stripped)
• Reaction-diffusion system modelled and reaction rate determined by fitting to experimental data
Assume ‘Direct Acting’ hypothesis (unaltered proteasomal processing) and determine properties of resulting peptides
Estimate average haptenated-pMHC surface density from considerations of:
1. the fraction of nucleophiles we expect to be haptenated
2. probability that a pMHC contains a haptenated nucleophile
Prediction tools
Proteasomal cleavage
(e.g. NetChop)
MHC I binding (e.g.
NetMHCpan)
average number of
pMHC generated
per protein
average
number of
nucleophiles
per pMHC
Modelling MHC class I processing & presentation 3 Is the nature (TCR affinity) of the antigen limiting?
• what kon/koff do TCRs have for cognate hapten pMHC?
Explore effect of pMHC surface density and kon/koff on probability of T-cell triggering with the available models (Zarnitsyna & Zhu, 2012). Simulations generated using ‘confinement time’ model of Dushek, et al, 2009. Figures from: Huppa & Davis, 2013; Aleksic et al., 2010
Predicting the threshold for T cell activation 4
Davies et al. 2011. Toxicol Sci. 119 308-18
Loss from skin
Loss from
formulation
Partitioning Diffusion
Modelling skin bioavailability and reactivity 2
Reactivity
Kin
etics
Skin
Bio
availa
bity
ex vivo human skin
ex vivo
human skin
Chemical and biological events driving induction of human skin sensitisation have been investigated for many years and are now relatively well understood at a qualitative level. Informed by previous efforts, we are developing a mathematical model of human CD8+ T cell responses following topical exposure to a sensitising chemical.
These studies are increasing our fundamental understanding of human skin responses to sensitisers, and with this approach we aim to move towards an ability to quantify the relationship between the dose per unit area of a sensitising chemical applied to the skin and the likelihood that an adverse outcome that will arise as a result.
There are three main components to this model – bioavailability, antigen processing/ presentation and CD8+ T cell response. The model is underpinned by quantitative information from scientific literature, with laboratory investigations and clinical research activities being undertaken where information is not currently available.
From an Adverse Outcome Pathway (AOP) to a mathematical model
1. Skin
Penetration
3-4. Haptenation:
covalent modification
of epidermal proteins
5-6. Activation of
epidermal
keratinocytes &
Dendritic cells
7. Presentation of
haptenated protein by
Dendritic cell resulting in
activation & proliferation
of specific T cells
8-11. Allergic Contact
Dermatitis: Epidermal
inflammation following re-
exposure to substance due
to T cell-mediated cell
death
2.Electrophilic
substance:
directly or via
auto-oxidation
or metabolism
Epidermis Epidermis
Lymph
Node
Induction Elicitation
Skin Allergy Adverse Outcome Pathway
Viable Skin
Stratum Corneum
Receptor Fluid
Vehicle
Part
itio
nin
g
Dif
fusio
n Dendritic cell
Lymphatic Vessel Proliferating
CD8+ T cell
Dendritic
cell
?
Lymph Node
Naïve
CD8+ T cell
Model Output
Dose of chemical applied to skin
Pro
b. O
f hapte
n-s
pecific
T c
ell
activation
chemical X
chemical
For all Unilever presentations see:
www.TT21C.org
Figure from Yewdell et al. 2003. Nat. Rev. Immunol. 3 952-61
Characterising human T lymphocyte responses to chemical allergen p-phenylenediamine (PPD)
6
CD4
CD8
Ki-
67
0µg/ml PPD 0.01 0.1
Allergen driven proliferation of total lymphocytes and individual T cell subsets measured by intracellular Ki-67 expression.
Amy Popple, Jason Williams,
Rebecca Dearman and
Ian Kimber
Adverse
Non-Adverse
allergic immune response
time
No. C
D8
+ T
ce
lls
dose Y
dose X
Use model human immune response prediction to inform risk assessment decision
Model output for DNCB human clinical exposures
Clinical benchmark data:
Friedmann 2007 Br. J. Dermatol. 157 (6) 1093-102 • Single exposure to DNCB applied over
volar forearm in acetone vehicle
• Sensitivity assessed by challenge with DNCB 4 weeks after application
• Data shows the proportion of the cohort sensitised for a given dose
5 Model evaluation and next steps
Next steps:
• Currently performing uncertainty analysis – collaboration with J. P. Gosling (Univ. Leeds, UK; funding from NC3Rs)
• Development of model-based risk assessment approach
Insights from these analyses will guide future model development (e.g. explicit modelling of T cell subtypes, formulation effects) and may provide human biomarkers suitable for verifying model prediction
Current model scope: antigen-specific CD8+ T cell response including
• naïve (N)
CD45RO-CD62L+or CD45RA+CD27+
• central memory (CM)
CD45RO+CD62L+ or CD45RA-CD27+
• effector memory (EM)
CD45RO+CD62L- or CD45RA-CD27-
• effector (E)
CD45RO-CD62L- or CD45RA+CD27-
Human, sensitiser-specific T cell data are largely unavailable:
• Make use of literature data e.g. response to infection
• Generate sensitiser-specific, human-relevant data
Further development: Modelling CD8+ T cell response
Sheeja Krishnan,
Grant Lythe and
Carmen Molina-París
Modelling the Skin Sensitisation Adverse Outcome Pathway (AOP) for Risk Assessment
SAFETY SCIENCE IN THE 21ST CENTURY For more information visit www.tt21c.org
DNCB human clinical sensitisation data from: Friedmann 2007 Br. J. Dermatol. 157 (6) 1093-102
• Single exposure to DNCB applied over volar forearm in acetone vehicle
• Sensitivity to DNCB assessed by challenge with DNCB 4 weeks after application
Apply exposure, protein reactivity and other biological information as model inputs
Simulation of exposure scenarios cited in Friedmann 2007
• Model output = predicted synapse formation rate
• Percentages marked correspond to reported human sensitisation rates