Presented at EADV 2012- These slides are reproduced with the permission of Dr.

Norsgaard

New evidence on skin atrophy and immune regulation by the two-compound fixedcombination gel

Hanne Norsgaard LEO Pharma Department of Molecular Biomedicine LEO Satellite Symposium EADV Prague 28 September 2012

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12 October, 2012 | Slide 1

Introduction

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

The calcipotriol and betamethasone dipropionate twocompound, fixed-combination product is widely used for topical treatment of psoriasis vulgaris The mode-of-action of this combination in relation to both cutaneous side effects (skin atrophy) and immune modulation (effect on key T-cell subsets) has not been fully characterised and directly compared with corticosteroid monotherapy

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12 October, 2012 | Slide 2

Objectives

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To compare the effects of the calcipotriol/betamethasone dipropionate combination with the monotherapies on biomarkers for skin atrophy using in vitro and in vivo models, and on differentiation and activity of human Th17/Th1 cells in vitro

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12 October, 2012 | Slide 3

Treatments

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Gel formulations for topical application in vivo and on a reconstructed skin model

DMSO solutions added to in vitro cultures with concentrations based on skin exposure data:

Calcipotriol 100 nM : betamethasone dipropionate 1 M (ratio 1:10) Calcipotriol 100 nM

Betamethasone dipropionate 1 M
Clobetasol 1 M (positive control for skin atrophy biomarkers)

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12 October, 2012 | Slide 4

Outline

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

Part I: Skin atrophy Background on biomarkers for skin atrophy Experimental results Part II: Immune regulation of Th17/Th1 cells Background Experimental results Conclusion

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12 October, 2012 | Slide 5

Corticosteroid-induced skin atrophy

Epidermal changes Reduction in keratinocyte proliferation Change in tissue modelling and structure Reduction in hyaluronic acid (HA) Decrease in matrix metalloproteinases (MMPs) Decrease in lipid synthesis Decrease in antimicrobial peptides Dermal changes
Reduced numbers of epidermal layers and reduced cellular volume lead to epidermal thinning

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

Impaired skin barrier and water loss

Reduction in collagen synthesis and turnover Collagen I/III mRNA and propeptides MMPs
Reduction in GAG synthesis HA content Thinning and fragmentation of elastin fibres in the upper layers

Reduced matrix network and dermal thinning

Reduced water-binding capacity Reduced skin flexibility/elasticity

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12 October, 2012 | Slide 6

Calcipotriol prevents steroid-induced decrease in collagen I synthesis

EpiDermFT

EpiDerm full-thickness human reconstructed skin model

Clobetasol

Calcipotriol

Combination

Betamethasone

48 hours of treatment with topical application of gel formulations (n=3)

P946 Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

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12 October, 2012 | Slide 7

Calcipotriol and betamethasone in combination counteracts the suppression of MMPs by steroids

MMP3 secretion --fibroblasts MMP1 secretion fibroblasts MMP1 secretion keratinocytes MMP3 secretion - -keratinocytes
*** *** *** * *** *** **** *** ** * *

48 hours of treatment (n=4)

Clobetasol Clobetasol Clobetasol Clobetasol

Calcipotriol Combination Betamethasone Betamethasone Calcipotriol Combination Calcipotriol Combination Calcipotriol Combination Betamethasone Betamethasone

48hours hoursof oftreatment treatment(n=3) (n=4) (n=3) 48

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12 October, 2012 | Slide 8

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In contrast to steroids, calcipotriol does not suppress levels of hyaluronic acid secreted from fibroblasts and keratinocytes

*** ***

* *** ***

*** ***

**

Clobetasol Clobetasol

Calcipotriol Calcipotriol

Combination Combination

Betamethasone Betamethasone

48 hours of treatment (n=3)

P946

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12 October, 2012 | Slide 9

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

Calcipotriol in combination with betamethasone in a gel formulation prevents steroid-induced epidermal thinning in minipigs
Gttingen minipigs were dosed topically with gel formulations on the flanks once daily, 5 days a week for 4 consecutive weeks

P946

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12 October, 2012 | Slide 10

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

Calcipotriol may counteract the atrophic effects of steroids

Epidermal changes Prevent epidermal thinning Prevent changes in tissue modelling and structure Increased hyaluronic acid (HA) production Increased MMP secretion Increase lipid synthesis Increase production of antimicrobial peptides Dermal changes

Increase collagen synthesis and turnover Collagen I/III mRNA and propeptides MMPs
Do not suppress GAG synthesis HA content

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12 October, 2012 | Slide 11

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

Outline
Part I: Skin atrophy Background on biomarkers for skin atrophy Experimental results Part II: Immune regulation of Th17/Th1 cells Background Experimental results Conclusion

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12 October, 2012 | Slide 12

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

In vitro Th17/Th1 cultures

CD4+ T cells were isolated from human peripheral blood of healthy volunteers T-cell activation was mediated by anti-CD3/CD28, and IL-2 supported survival and growth of the T-cell cultures In vitro differentiation into Th17/Th1 cells occurred in the presence of a defined differentiation cocktail (IL-1, IL-6, IL-23, TGF, and anti-IL-4 and anti-IL-13 Ab)
Removal of differentiation cocktail

Differentiation

(67 days)

Activity

(1 day)

CD4+ T cell

Th1/Th17 cells

Read-outs: Expression of Th1-, Th2-, Th17- and Treg-associated transcription factors and cytokines

Drug treatment

Drug treatment

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12 October, 2012 | Slide 13

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

Calcipotriol and betamethasone inhibit the expression of key pathogenic cytokines

Differentiation Activity Release of inflammatory cytokines

Th17-related cytokine (n=6)

IL-17A expression

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12 October, 2012 | Slide 14

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Calcipotriol and betamethasone have opposing effects on Th2 and Treg cytokines
Differentiation Activity Release of inflammatory cytokines

Th2-related cytokine (n=6)

IL-13 expression

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12 October, 2012 | Slide 15

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

Effects of calcipotriol and betamethasone on the differentiation of Th17/Th1 cells

Selective pressure for inducing and maintaining a Th17/Th1 cell lineage

Th1 T-bet

INFg

Th0
T-bet T-bet ROR RORg gt t GATA3 GATA3 FOXP3 FOXP3

Th17 RORgT

IL-17 IL-22

Calcipotriol or combination of calcipotriol and BDP

Th2 GATA3

IL-4

Treg FOXP3

IL-10

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12 October, 2012 | Slide 16

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

Effects of calcipotriol and betamethasone on differentiated Th17/Th1 cells

Calcipotriol BDP Combination of calcipotriol and BDP

Selective pressure for inducing and maintaining a Th17/Th1 cell lineage

Th1 T-bet

INFg

Th1 T-bet

INFg

Th0
T-bet T-bet RORgt RORgt RORgt GATA3 GATA3 FOXP3 FOXP3

Th17 RORgT

IL-17 IL-22

Th17 RORgT

IL-17 IL-22

Calcipotriol

Calcipotriol

Th2 GATA3

IL-4

Treg FOXP3

IL-10

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12 October, 2012 | Slide 17

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

Outline
Part I: Skin atrophy Background on biomarkers for skin atrophy Experimental results Part II: Immune regulation of Th17/Th1 cells Background Experimental results Conclusion

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12 October, 2012 | Slide 18

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

Conclusion
The presented data suggest that the calcipotriol component in the two-compound, fixed-combination gel:
counteracts the corticosteroid-induced effects on biomarkers related to skin atrophy has an immunomodulatory effect by suppressing the key Th17/Th1 pathogenic mediators and simultaneously inducing a Th2/Treg response in contrast to the broader immunosuppressive effect observed by corticosteroid monotherapy

The new data provide a probable mechanism behind the favourable safety and efficacy profile seen with calcipotriol and betamethasone fixed-combination gel for topical treatment of psoriasis

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12 October, 2012 | Slide 19

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

Acknowledgements
LEO Pharma
Paola Lovato Mads Rpke Tatiana Gonzalez Georg Duenstl Paul Kidson Markus Latta

Thank you!

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12 October, 2012 | Slide 20

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard

Meet the Expert Sessions

LEO Pharma Booth at EADV 2012

Daivobet Mode of action: effects on Th1, Th2, Th17 and Treg subsets 28 September 2012, 15:0015:30 29 September 2012, 10:0010:30 Daivobet Mode of action: effects on markers of skin

atrophy
29 September 2012, 13:3014:00

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12 October, 2012 | Slide 21

Presented at EADV 2012- These slides are reproduced with the permission of Dr. Norsgaard