THE USE OF GÖTTINGEN MINIPIG SKIN FOR TTS DEVELOPMENT

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THE USE OF GÖTTINGEN MINIPIG SKIN FOR TTS DEVELOPMENT

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Presented by:

Dr. Julia Lodder-Gadaczek

Head of PhA Skin Lab, Analytical Development, R&D

LTS Lohmann Therapie-Systeme AG

TTS – A BRIEF INTRODUCTION TO FUNCTION

3

TTS (Stratum Corneum)

Epidermis

Dermis

A known principle applies:

A – Adhesion

O – Occlusion

L – Liberation

A – Absorption

D – Distribution

M – Metabolism

E – Elimination

~ 10 – 50 μm

~ 50 – 250 μm

~ 70 – 100 μm

Backing Layer

Drug Containing Adhesive

Matrix

Release Liner

DESIGN OF TRANSDERMAL SYSTEMS

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Monolithic Transdermal Matrix System – DIA – Drug in adhesive

Most common TDS design

Can be expanded to multiple layers, if required

Adhesive with good solubility properties, like acrylates

SKIN PENETRATION PATHWAYS

5

Penetration routes through the

stratum corneum

intercellular route

(for most transdermal drugs):

diffusion along alkyl-chains of

lipophilic substance

follicular route

transcellular transport

Marie-Alexandrine Bolzinger et al, Curr. Opin. Colloid & Interface Sci. Journal, 2012, 17(3), 156-165

SKIN PENETRATION PATHWAYS

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Donor-compartment:

liberation of the drug

Penetration in the skin

Transport into acceptor

compartment = transport into the

organism (passive diffusion)

Acceptor: aqueous medium =

systemic resorption (blood vessels)

Perfect sink conditions

Diffusion cells

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IN VITRO PERMEATION FOR TTS

Göttingen Minipig: Preparation of the skin

1 Unprepared skin (flank) 2 Prepared and „trimmed“ 3 Dermatomized (500–800 µm)

4 Sample preparation 5 Sample

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SKIN PROPERTIES RELATED TO

IN VITRO PERMEATION

Stratum corneum thickness (abdomen)

Human skin(n=8)

Minipig skinage 3-4 month (n=6)

Minipig skinage 6-8-month (n=6)

14.7 13.6 21.5

Cholesterol

Cer (NS)

Cer (NP)

Cer (C24-AS)

Cer (C16-AS)

Minipig human skin

Epidermal

lipids

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PERMEATION PROFILES – EXAMPLE

Kinetic profile of API transport – Formulations with

different area coating weights

Permeated amounts as

time-dependend function

Information:

Cmax

Steady state concentration

Consistency of release

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PERMEATION PROFILES

Göttingen Minipig vs. Human skin – 3day application

Performance:

Similar to human skin

Or with a „stable“

correlation factor

Good differentiation

between formulations

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PERMEATION PROFILES

Göttingen Minipig vs. Human skin – 7day application

Stable barrier function

(7 days)

Consistency of release

Similar to human skin

performance

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MINIPIG MODEL – LIMITS

In case of big difference in

human skin vs. Minipig skin

> the flux profile might be

different

Example: human skin >

„drop“ in the profile caused

by a low residue in TTS,

Minipig skin > no effectΔFl

ux

dat

a[µ

g/cm

²h]

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MINIPIG SKIN – DIFFERENCES

Test of skin integrity

Influence of age

SC thickness

Hair follicle density

Surface lipid concentration

Siblings > same

performance

Optimal age: 3–4 month

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INFLUENCE OF FULL SKIN THICKNESS

Skin thickness has only

minor influence

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INFLUENCE OF STRATUM CORNEUM

THICKNESS AND SURFACE LIPID DENSITY

High permeability

depends on SC

thickness

In case of high conc.

of surface lipids the

permeabilty is low

CAVE: stress reaction

(high conc. of sebum

lipids)

0

20

40

60

80

100

120

0 8 16 24 32 40 48 56

Cu

mu

late

d d

ata

[µg/

cm²]

Time point [h]

skin, 800 µm (ref)skin, 800 µm, stripped (5x)skin, 800 µm, stripped (1x)

0

10

20

30

40

50

60

70

0 8 16 24 32 40 48 56

Cu

mu

late

d d

ata

[µg/

cm²]

Time point [h]

skin, 800 µm, lipid surfaceskin, 800 µm (ref)

OilRed stain

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USE FOR A CASE STUDY

In vitro flux data (minipig skin)

Formulation screening to

reach target flux

Step 1: In vitro permeation results

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USE FOR A CASE STUDY

Step 2: Preclinical tolerability and in vivo permeation studies

In vivo plasma levels (minipig)

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SUMMARY

Göttingen Minipig skin has a high similarity to human skin,

both morphologically and in drug permeability

Good differentiation between different prototype formulations in in

vitro permeation studies

The age of the animals, the SC thickness, the hair follicle density

and the concentration of surface lipids has an influence on the

permeabilty > the skin must be qualified in a skin integrity test

Usefull model for early development stage and for the selection of

prototype formulations for preclinical studies

EXPLORE WITH US

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Contact information:

Dr. Julia Lodder-Gadaczek

Head of PhA Skin Lab, Analytical Development, R&D

LTS Lohmann Therapie-Systeme AG, Germany

Julia.Lodder-Gadaczek@ltslohmann.de

Region Hovedstaden

Application of Göttingen Minipig biological material in In Vitro studies

Neuronal and endothelial regulation of vasculature in the Göttingen Minipigs

Anette Sams, Kristian Haanes,

Majid Sheykhzade and Lars Edvinsson

Clinical Experimental Research,

Forskerparken, Copenhagen University Hospital, Glostrup

1

Region Hovedstaden

Cardioprotection with a neurovascular rescue mechanism

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• Anette Sams

• Pharmacist and PhD at University of Copenhagen (1996-2000)

• Neurovascular and metabolic pharmacology in vitro

• 13 years with Novo Nordisk R&D (2001-2015)

• Diabetes - metabolism – inflammation – vascular biology

• 5 years at Copenhagen University Hospital (2016-2021)

• Chasing a new cardioprotective principle

Region Hovedstaden

Aim of current In Vitro study

• Comparison of sympathetic, parasympathetic, sensory and endothelial regulation of

• coronary, cerebral and mesenteric artery segments

• form Göttingen Minipigs

• by functional myography

• Investigation of the robustness of the vascular response after 24h storage of vascular tissue

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Region Hovedstaden

Why

• Very limited number of vascular studies of minipigs in vitro

• Potential closure of translational gap (and scientific mis-interpretation) within

• Cardiovascular research and development (e.g. heart failure)

• Neurovascular research and development (e.g. migraine)

• Pharmacology and safety pharmacology

• Improved characterization of current best cardiovascular model

4

Region Hovedstaden

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Methods

6 male Göttingen Minipigs over 6 weeks

Age: 3.8 months ± 0.4

Body weight: 8.3 kg ± 0.3

(mean ± SEM)

Region Hovedstaden

MethodsOrgan isolation at Ellegaard Göttingen Minipigs A/S

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• Preparation – 1 form – 2 phone calls

• First organ isolation – participation and mutual clarification

• Five following isolations - pickup

Region Hovedstaden

MethodsArtery tissue dissection at Rigshospitalet

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Coronary LAD

(CA)Middle cerebral

(MCA)Mesenteric

(MA)

Region Hovedstaden

MethodsArtery tissue dissection at Rigshospitalet

Region Hovedstaden

MethodsMyography - mounting

Göttingen Minipig

similar to human heart

1st branch of LAD

Region Hovedstaden

MethodsMyography - experimentation

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1

2

X 243

4

Sheykhzade at al., Vascular and molecular pharmacology of the metabolically stable CGRP analogue, SAX. Eur J Pharmacol., 2018

Region Hovedstaden

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Results

Region Hovedstaden

Results- Basic constriction and artery size

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Coronary (CA) Cerebral (MCA) Mesenteric (MA)

Mean SEM n Mean SEM n Mean SEM n

K60, mN/mm 8,8 0,7 12 7,4 0,8 12 4,6*** 0,4 12

U46619, mN/mm 4,0 0,8 12 8,1* 0,9 12 6,6 1,1 12

K30, mN/mm 10,4 0,8 12 7,2* 1,0 12 5,0*** 1,1 12

IC1, µm 2442 76 12 2023* 114 12 664*** 24 12

U46/K60 0,5 1,2** 1,4**

K30/K60 1,2 1,1 1,0

Paired t-test (to CA). *: p < 0.05; ***: p < 0.001

Region Hovedstaden

Results– sympathic, parasympathic, sensory

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-10 -9 -8 -7 -6 -5 -4

0

100

200

300

400

Log [Norepinephrine], M

Rem

ain

ing

co

nstr

icti

on

(% o

f U

46

61

9 c

on

tra

cti

on

)

CA

MCA

MA

-10 -9 -8 -7 -6 -5 -4

-200

0

200

400

600

800

Log [Carbachol], M

Rem

ain

ing

co

nstr

icti

on

(% o

f U

46

61

9 c

on

tra

cti

on

)

CA

MCA

MA

Data are given as mean ± SEM (n = 6)

-11 -10 -9 -8 -7 -6 -5

0

25

50

75

100

Manus 1 - Nonlin fit of CGRP

Log [CGRP], M

Rem

ain

ing

co

nstr

icti

on

(% o

f 3

0 m

M K

PS

S c

on

tra

cti

on

)

CA

MCA

MA

Norepinephrine

Sympathic agonist

Carbachol

Parasympathic agonist

CGRP

Sensory agonist

Region Hovedstaden

Results– day to day storage

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Data are given as mean ± SEM (n = 6)

Endothelin

Endothelial autoregulator

-12 -11 -10 -9 -8 -7 -6

0

50

100

150

200

250

300

350

400

ET-1 day to day

Log [ET-1], M

Co

nstr

icti

on

(% o

f 6

0 m

M K

PS

S c

on

tra

cti

on

)

CA

MCA

MA

MA 1

MCA 1

CA 1

-11 -10 -9 -8 -7 -6 -5

-50

0

50

100

150

CGRP day to day

Log [CGRP], M

Rem

ain

ing

co

nstr

icti

on

(% o

f 3

0 m

M K

PS

S c

on

tra

cti

on

)

CA

MCA

MA

MA 1

MCA 1

CA 1

CGRP

Sensory neuropeptide

Region Hovedstaden

Results– endothelial contribution

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Data are given as mean ± SEM (n = 6)

Endothelin

Endothelial regulator

CGRP

Sensory neuropeptide

-12 -11 -10 -9 -8 -7 -6

0

50

100

150

200

250

300

ET1

Log [ET1], M

Co

nstr

icti

on

(% o

f 6

0 m

M K

PS

S c

on

tra

cti

on

)

CA

CA - endothel

MCA

MCA - endothel

MA

MA - endothel

-11 -10 -9 -8 -7 -6 -5

0

50

100

150

CGRP

Log [CGRP], M

Rem

ain

ing

co

nstr

icti

on

(% o

f 3

0 m

M K

PS

S c

on

tra

cti

on

)

CA

CA - endothel

MCA

MCA - endothel

MA

MA - endothel

Region Hovedstaden

Results- functional endothelial removal

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-11 -10 -9 -8 -7 -6 -5

0

50

100

150

200

BK med og uden EC

Log [BK], M

Rem

ain

ing

co

nstr

icti

on

(% o

f U

46

61

9 c

on

tra

cti

on

)

CA

CA-EC

MCA

MCA-EC

MA

MA-EC

Data are given as mean ± SEM (n = 6)

Region Hovedstaden

Conclusions

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• Standardisation, comparison, statistics and conclusions of high quality

• Evaluation of robustness is feasible (not possible in rodents)

• High degree of human translation of Göttingen Minipig’s vascular

regulation

Region Hovedstaden

Kristian HaanesMSc, PhD Senior Scientist, Clinical Experimental

Research, Rigshospitalet.

Lars EdvinssonDr Med Professor, Lund University and Clinical

Experimental Research, Rigshospitalet.

Majid SheykhzadePhD, Cand. Pharm.Associate Professor, Molecular and Cellular

Pharmacology, University of Copenhagen.

Ellegaard Göttingen Minipigs Research Foundation

Thank you