Utilization of Reconstructed Cultured Human Skin models as
an Alternative Skin for Permeation Studies of ChemicalCompounds()

x



= n()log P (cm/s) = 1.17Í10−7Ko/w0.751+2.73Í10−8
Morimoto Y et al., J Pharm Pharmcol., 44, 634-639 (1992)

log P (cm/s) = –6.3+0.71Ílog Ko/w –0.0061ÍMW
Potts RO, Guy RH, Pharm. Res., 9, 663-669 (1992)

log P (cm/s) = –5.1+0.44R2–0.49p2H–1.48Sa2H–3.44 Sb2H+1.94Vx
Abraham et al., Pestic. Sci., 55, 78-88 (1999)

n &!&!$ &! $  

Ko/w :  "MW : #
R2, p2H, Σa2H, Σb2H, Vx :Abraham Solvation Parameter
%

&!$3h2O/Wn ]_UiOFF>
2JM[1$-*^f1#!.
gF=QG

W/O

MN:F(&3h2O/W").%,/
dN:F(&W/O").%,/#!.
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 R]_UiOF\D dN:F
Mh1?

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O/W#in silico"&'$"&'!

In vitro skin permeation experiments
Typical skin permeation profile
Donor compartment
Culturedskin model
Silicone humanmembraneor orExcised
Hairlessrat humanskin skin

Water jacket (32 °C)
Magnetic stirrer bar

Receivercompartment

Cumulative amountof drug permeatedSampling port

Synchronous motor

Vertical typed diffusion cell

• Permeation profile and parameters• Drug concentration in the membrane

Steady-state!"#$ % ='( *+ ,=').

dQ/dt(Flux)

Non steady-state
L2tlag =6DTime, t (h)Typical skin permeation profile

Skin permeation parameters• Permeation rate (flux)• Permeability coefficient• Partition coefficient• Diffusion coefficientReconstructed cultured human skin models used in this experiment
Bland name(company name)

Site ofskin forthe model

Cell type

Effectivepermeation area(cm2)

Well

Dosecondition

TESTSKINTM LSE-high(Toyobo)

Epidermis/
dermis6Side by side

0.95

infinite

EpiDermTM 606X
(MatTek/Kurabo)

Epidermis

6

Side by side

0.95

infinite

LabCyte Epi-model(J-Tec)

Epidermis

12Franz

0.5

finite

Neoderm® -E(TegoScience)

Epidermis

6

Side by side

0.95

infinite

Vitrolife-skin(Gunze)

Epidermis/
dermis6Side by side
(Ussingchamber)

0.5

Infinite

Episkin®
(SkinEthic)

Epidermis

12Franz

0.5

finiteSet-up of 3-dimensional cultured skin model for in vitro
permeation experiment (in case of LSE-high)Transwell

LSE-high

Polycarbonate
membrane

Culture medium

Materials
Drug

Abbreviation

Log Ko/w

M.W.

Antipyrine

ANP-1.507

188.23Isosorbide-5mononitrate

ISMN-0.151

191.14

Caffeine

CAF-0.123

194.19Aminopyrine

AMP

1.065

231.29Isosorbide
dinitrate

ISDN

1.225

236.14

Benzoic acid

BA

1.410

122.12

Flurbiprofen

FP

2.179

224.27

Comparison of skin permeation through 3dimensional cultured human skin models
Antipyrine

30000

Neoderm

25000

Human skinEpiderm
LabCyte
LSE-highNeodermEpiskin
Vitrolife-skin

2Q (µg/cm )

20000
15000
10000

Vitrolife

5000

Episkin

LabCyte

LSE-high

Epiderm

Human skin0024Time (h)68Calculation of permeation coefficient (P) from skinpermeation profile

Cumulative amountof drug permeatedTypical skin permeation profiledQ CvKD
Flux ==dt
L= PCv

Steady-statedQ/dt(Flux)

Non steady-state
Time, t (h)Cv: Drug conc.
K:Partition coefficient
D:Diffusivity
L:Membran thickness
P:Permeation coefficientRelationships of membrane permeability coefficients(P) of drugs
Human skin/Hairless rat skin

LogP human skin (cm/s)-3-4BAFP-5

ISDN-6AMP-7

ISMN
CAF
ANP-8-8-7-6-5-4-3LogP hairless rat (cm/s)
LogP human= 0.98LogP hairless rat-0.0089
R2=0.98
Watanabe, T., Sugibayashi, K. et al., Altern. Animal Test. Experiment., 8, 1-14 (2001)

a) LSE-high

FP
BA-5

ISDN-6CAF
ISMN-7AMP

ANP-8-8-7-6-5-4-3

LogP human= 1.0LogP Epiderm+0.06
R2=0.95-3-4FP

CAF
ISMN
ANP-7-8BA-5FP
ISDN

AMP-6CAF-7ISMN

ANP-8-8-7-6-5-4-7-6-5-4-3LogP Vitrolife-skin (cm/s)-4FPBA-5ISDN-6AMP-7ANP

CAF
ISMN-8-8-7-6-5-4FP-5ISDN-6CAF
ISMN-7-3AMPANP-8-8LogP human= 1.6LogP Neoderm+2.1
R2=0.66-3LogP human= 0.72LogP LabCyte-2.2
R2=0.13-4-3LogP Epiderm (cm/s)e) Neoderm
LogP human skin (cm/s)

LogP human skin (cm/s)-4

ISDNAMP-6-8LogP human=3.0LogP Vitrolife+8.0
R2=0.63

BA-5LogP LSE-high (cm/s)d) Vitrolife-3-3

LogP human skin (cm/s)-4c) LabCyte-epimodel-7-6-5-4-3LogP LabCyte Epi-model (cm/s)f) Episkin
LogP human skin (cm/s)

LogP human= 0.91LogP LSE-high-1.75
R2=0.84

LogP human skin (cm/s)

LogP human skin (cm/s)-3b) Epiderm

LogP Neoderm (cm/s)

LogP human= 0.69LogP Episkin-2.18
R2=0.13-3-4BAFP-5ISDN-6CAF

ISMNAMP-7ANP-8-8-7-6-5-4-3LogP Episkin (cm/s)

Kano S., Todo,H., et al., AATEX, 15, 61-70 (2010).



Physicochemical
properties ofcompounds

Skin
permeation

Skin conc.

Local effectin skin

Physicochemical
properties ofcompounds

Skinpermeation

Well-known*1

Skin conc.

Unknown

Local effectin skinPartially-known*2*1 Potts R.O., Guy R.H., Pharm. Res., 9, 663-669 (1992).
*2 Kano S., Sugibayashi K., Pharm. Res., 23, 329-335 (2006).

6
Physicochemical
properties ofcompounds

Skinpermeation

Well-known*1

Skin conc.

Unknown

Local effectin skinPartially-known*2*1 Potts R.O., Guy R.H., Pharm. Res., 9, 663-669 (1992).
*2 Kano S., Sugibayashi K., Pharm. Res., 23, 329-335 (2006).
!%&-9!.
3,;7*$6
?A@C>-9 B=#1:04.3/:+
15<2*"'6(68 )6.
45:+

Concentration-distance profile at steady state intwo-layered skin model
Donor

Stratum
corneum (sc)Viable epidermis and dermis (ved)KscCv
KscCvPtot/Pved
D : Diffusion coefficientCv
K : Partition coefficient
Cv : Donor concentration
Ls : Thickness of membrane
C : Average membrane concentration
P : Permeability coefficient

KscCvPtot/PvedKved/Ksc
X=0 X=Lsc

SC concentration

X=LtotVED concentration

K sc • Cv + K sc • Cv • Ptot / Pved
K ved • Cv • Ptot / Pved
Css , sc =Css,ved =2
2
Full-thickness skin concentration

Cv{KscLsc(1 + Ptot / Pved ) + KvedLvedPtot / Pved }Css =Sugibayashi K, Todo H, Oshizaka T, Owada Y., Pharm Res 27,134-142 (2010).
2LtotSteady state drug concentration-depth profile for fullthickness skin

Lidocaine concentration (mg/mL)

8000
8.0

6000
6.0

Calculated valueObserved lidocaine concentration instratum corneum

4000
4.0

 Observed lidocaine concentration in
viable epidermis/dermis

2000
2.00 0

00

0.2

0.2

0.4

0.4

0.6

0.6

0.8

0.8

1

1.0

Normalized full-thickness skin depth
Sugibayashi K, Todo H, Oshizaka T, Owada Y., Pharm Res 27,134-142 (2010).

Full-thickness skin1

1:1 correlation

P-PABA B-PABABA PP BPEPISDN
E-PABAMPAMP LC M-PABA
ANP
CAF

0-1-2

EpiDopa

ISMN-3-4-5-5-4-3-2-101Log (calculated Css , tot /donor conc.)

Log (observed Css , ved /donor conc.)

Log (observed Css , tot /donor conc.)

Relationship between calculated skin conc. and observed skin conc.

Viable epidermis and dermis
1:1 correlation

10-1
Dopa-2
EpiP-PABA
BA EP
ISDN
E-PABAMP
B-PABA
M-PABA
LC
AMP
BP
PP
ANP
CAF
ISMN-3-4-4-3-2-101Log (calculated Css , ved /donor conc.)

Data range: 122.12<M.W.<234.30, -5.54<ClogP<3.57

Mean  S.E. (n = 4)

In vitro'(,-*

Cumulative amountof drug permeatedTypical skin permeation profile'( *+ ,!"#$ % ==').
/+ =01 .

Steady-statedQ/dt(Flux)

Non steady-state

L2tlag =6D/+ = 0+ ×*+0, = +301

Time, t (h))%
/ . 
Log P (cm/h) = -2.7 + 0.71×log Ko/w-0.0061× MW
Potts RO, Guy RH., Pharm Res. 9, 663-669 (1992)

r2=0.67 (93 compounds))%'( .  (K))%" # (*+ ))%'(   (D)'(0 1 (L))%

$&  (/+ ))%

/  (0+ ))%'(/  (01 ) /!+)%
/ . 








/%"# !"# 12()*+’ -’ ==/0
3
$"# 2
=&' 3

 / !  (K) (!"# )   (D)" ($"# )  "(%"# )()*+’’ -’’ =/%4567 !4# 14# 2
=/0
3
$4# 2
=&' 3

 / !  (Kep) (Cep)   (D)" (Aep) "  (&' )MaterialCompound

Chemical structure

Flurbiprofen(FP)log Ko/w

M.W.

Solubility in water(Swater, mg/mL)

2.2

224.3

0.048

Saturated solubility of FP in the ester oils
Oil name (Abbre.)
2-Ethylhexyl isononanoate (EHIN)
2-Ethylhexyl palmitate (EHP)
Diisopropyl adipate (DIPA)
Di-2-ethylhexyl sebacate (DEHS)
Propylene glycol isostearate (PGIS)
Diethyl sebacate (DES)
2-Octyldodecyl ricinoleate (ODR)
2-Hexyldecyl isostearate (HDIS)
Isononyl isononanoate (ININ)

Saturated solubility(Soil, mg/mL)
38.6
23.9
135
58.5
77.7
161
65.0
11.3
31.9

Neopentyl glycol di(2-ethylhexanoate) (NPGDEH)

52.3

Isotridecyl isononanoate (ITDIN)
Dioctyl carbonate (DOC)

23.3
37.4

Preparation of applied formulations
Oil
FP solution (10 mg/g) was prepared by completely dissolving FP with ester oil
at 80ºC followed by stirring at 32ºC for 48 h.

Emulsion

LABOLUTIONÒ(PRIMIX Corp.)

Total : 20 g
Composition of o/w emulsions
ComponentFP
Ester oil
Purified water
2.0% acrylates/C1030 alkyl acrylatecrosspolymer gel
20% KOH
Phenoxy ethanol
Total

5%
117.13

1.6
0.05
0.02
20mix

mix

4,500 rpm,
3 min, 80℃

Ester oil conc.
10%20%0.2
2416.13
14.13

Phenoxyethanol

20 KOH aq

4,500 rpm,5 min, 70℃

4,500 rpm,
3 min, 40℃

Stored at 32℃
30%612.13

Oil solvent-uptake experimentSC

Desiccator

0.1% Trypsin solution

PeeledExcised skin

Put at 3°C for 16 h
Put at 32°C for 5 hSCDesiccator

stratum corneum sheet(SC sheet)

SCDriedTemperature: 20 5 °C
Humidity: 755%Hydration: 24 h

Silica gel

MeasuredSC sheet in dishSoaked in oil or emulsion
at 32°C for 1 h

MeasuredStirrer bar
Saturated sodium chloride solution

MeasuredHydration
SC mg/mgSC mg

SC mg

Prediction of skin permeation of FP from o/w emulsions!"# $%&'()*% = 1.04×!"# $23( + 0.52 (r2=0.61)

1.4

(2%¯

HDIS-20%

1.2
ITDIN-20µ )NPGDEH-20
ODR-20/1(/DOC-200.8

1 /0/ (2%PGIS-200.6

DEHS-200.4

EHIN-20

DIPA-200.2DES-20

EHP-200-0.4-0.2

00.20.4/ (//µ )

0.6
%0.81

In silico modeling approach
Multi-regression analysis: JMPâ Pro (ver. 13.1.0, SAS Institute, Cary, NC, USA).
Stepwise regression analysis

Cross-validation: 75% training and 25% validation
Measuredparameters

Molecular descriptors of oils and measured parameters used in this experiment
Abbre.
Descriptors and measured parameters
Qoil
The cumulative amount of FP permeated from the oil base
SCoil
Amount of oil uptake into SC sheet
Swater and Soil
Solubility of FP in purified water and ester oilST
Surface tension
Abrahamdescriptor

Molecular descriptors!"#VxR2∑%"#∑&"#
Kgas/hex
M.W.
dMref
HAtPSAFRBIRPa

Dipolarity/polarizability
McGowan characteristic volume
Excess molar refractivity
Hydrogen bond donor acidity
Hydrogen bond acceptor basicit
Partitioning coefficient between gas phase and hexadecane
Molecular weight of oil
Molecular density
Excessive molar refraction
Number of hydrogen bond acceptors
Topological polar surface area
Freely rotatable bonds
Index of refraction
Hetero ratio
C ratio
N ratio
NO ratio
Parachor, Pa='()/+ , -. /./0Multi-regression analysis and cross-validation!"#$ = −10.3 ±2.7 ./0 + 81.8 ±20.9 4 + 22.9 ±2.5 67 − 0.07 ±0.02 6 − 63.5(±17.0)"#$./0dSCoil
Soil

"#$

Dipolarity/polarizability; = 69, = / = 0.68, >? (4,64) = 30.1, (A < 0.001)
Molecular density
Amount of oil uptake into SC sheet
Solubility of FP in ester oil

Trial #4

Cross-validation result/=CD=0.66  0.05
(the mean  S.D.)()µ75% training and 25% validation/=CD: R2 for each cross-validation result(µFP permeation from o/w emulsions
Col 1 vs 5
Col 1 vs 10

Col 1 vs 5

Col 1 vs 5

Col 1 vs 10
Col 1 vs 20a) PGIS

1430

1240308¡; 5%¨; 10%¯; 20%20)µµ)

20

Col 1 vs 30¡; 5%¨; 10%¯; 20%10)µb) DES3525

Col 1 vs 30c) ODR

Col 1 vs 20

Col 1 vs 30¡; 5%¨; 10%¯; 20%r; 30%

Col 1 vs 20

Col 1 vs 10

15610452000

12345(PGIS: Propylene glycol isostearate
DES: Diethyl sebacate
ODR: 2-Octyldodecyl ricinoleate100123(4500123(45Prediction of skin permeation of FP from o/w emulsions containing 5%, 10% and 20% ester oil
1.6
ODR-5)/1.4

PGIS-5¡5

PGIS-10

1.2/%¯5

HDIS-20

ININ-20

ODR-10ITDIN-20

NPGDEH-20
ODR-201µ¨5)/)/ %)00

DES-52 01

DOC-205-20 %

0.8

PGIS-20(0 ) /Least squared method
log Qemul5%=0.97×&'( )*+, + 1.25
(r2=0.80)

0.6
DEHS-20

DES-100.4

EHIN-20

DIPA-200.2
PGIS-30

DES-20

EHP-200-0.4-0.2log Qemul10%=1.11×&'( )*+, + 0.97
(r2=0.91)&'( )123,45% = 1.04×&'( )*+, + 0.52
(r2=0.61)

0Todo H., et al, Prediction of skin

0.2(0 )00.4

0µ0.6/%

0.8

1 permeation of flurbiprofen from neat esteroils and their o/w emulsions, Chem. Pharm.
Bull, accepted, 2018

Conclusion
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QTH\ -YS.?SB92P.3U=QTW_7,CAR/^\0N874J@