the use of gÖttingen minipig skin for tts development
TRANSCRIPT
THE USE OF GÖTTINGEN MINIPIG SKIN FOR TTS DEVELOPMENT
2
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
4
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
6
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
7
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
8
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
9
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
10
PERMEATION PROFILES
Göttingen Minipig vs. Human skin – 3day application
Performance:
Similar to human skin
Or with a „stable“
correlation factor
Good differentiation
between formulations
11
PERMEATION PROFILES
Göttingen Minipig vs. Human skin – 7day application
Stable barrier function
(7 days)
Consistency of release
Similar to human skin
performance
12
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]
13
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
14
INFLUENCE OF FULL SKIN THICKNESS
Skin thickness has only
minor influence
15
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
16
USE FOR A CASE STUDY
In vitro flux data (minipig skin)
Formulation screening to
reach target flux
Step 1: In vitro permeation results
17
USE FOR A CASE STUDY
Step 2: Preclinical tolerability and in vivo permeation studies
In vivo plasma levels (minipig)
18
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
19
Contact information:
Dr. Julia Lodder-Gadaczek
Head of PhA Skin Lab, Analytical Development, R&D
LTS Lohmann Therapie-Systeme AG, Germany
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
2
• 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
3
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
5
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
6
• Preparation – 1 form – 2 phone calls
• First organ isolation – participation and mutual clarification
• Five following isolations - pickup
Region Hovedstaden
MethodsArtery tissue dissection at Rigshospitalet
7
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
10
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
11
Results
Region Hovedstaden
Results- Basic constriction and artery size
12
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
13
-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
14
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
15
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
16
-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
17
• 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