Utilization of Reconstructed Cultured Human Skin models as an Alternative Skin for Permeation Studies of Chemical Compounds

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Potts RO, Guy RH, Pharm. Res., 9, 663-669 (1992)

Abraham et al., Pestic. Sci., 55, 78-88 (1999)

�Ko/w : �� ������"���MW : ��#�R2, p2

H, Σa2H, Σb2

H, Vx :Abraham Solvation Parameter

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log P (cm/s) = 1.17Í10−7Ko/w0.751+2.73Í10−8

log P (cm/s) = –6.3+0.71Ílog Ko/w –0.0061ÍMW

Morimoto Y et al., J Pharm Pharmcol., 44, 634-639 (1992)

log P (cm/s) = –5.1+0.44R2–0.49p2H

–1.48Sa2H–3.44 Sb2

H+1.94Vx

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Agenda

• O/W���������#��in silico "&'��� ���!

• 3���)� " ������%� "&'�$�����

• "(��%� "��������!

Vertical typed diffusion cell

Silicone membraneor

Hairless rat skin

Donor compartment

Receiver compartment

Sampling port

Magnetic stirrer bar

Synchronous motor

Water jacket (32 °C) dQ/dt(Flux)

Steady-state

Non steady-state

Time, t (h)

Cum

ulat

ive

amou

nt

of d

rug

perm

eate

d

Typical skin permeation profile

Typical skin permeation profile

Skin permeation parameters• Permeation rate (flux)• Permeability coefficient• Partition coefficient• Diffusion coefficient

DLtlag 6

2

=

• Permeation profile and parameters• Drug concentration in the membrane

Cultured human skin modelor

Excised human skin

In vitro skin permeation experiments

!"#$ % = '(') =

*+,-.

Reconstructed cultured human skin models used in this experiment

Bland name(company name)

Site of skin for

the modelWell Cell type

Effective permeation area

(cm2)

Dosecondition

TESTSKINTM LSE-high(Toyobo)

Epidermis/dermis

6 Side by side 0.95 infinite

EpiDermTM 606X(MatTek/Kurabo)

Epidermis 6 Side by side 0.95 infinite

LabCyte Epi-model(J-Tec)

Epidermis 12 Franz 0.5 finite

Neoderm® -E(TegoScience)

Epidermis 6 Side by side 0.95 infinite

Vitrolife-skin(Gunze)

Epidermis/dermis

6Side by side

(Ussingchamber)

0.5 Infinite

Episkin®

(SkinEthic)Epidermis 12 Franz 0.5 finite

Culture mediumPolycarbonate

membrane

LSE-highTranswell

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

Materials

Drug Abbreviation Log Ko/w M.W.

Antipyrine ANP -1.507 188.23

Isosorbide-5-mononitrate ISMN -0.151 191.14

Caffeine CAF -0.123 194.19

Aminopyrine AMP 1.065 231.29

Isosorbidedinitrate ISDN 1.225 236.14

Benzoic acid BA 1.410 122.12

Flurbiprofen FP 2.179 224.27

Time (h)

0 2 4 6 8

Q (µ

g/cm

2 )

0

5000

10000

15000

20000

25000

30000

Human skinEpidermLabCyte LSE-high Neoderm Episkin

Vitrolife-skin

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

Antipyrine

Neoderm

Episkin

Human skinEpiderm

Vitrolife

LSE-highLabCyte

PCvL

CvKDdtdQ

Flux

=

==

Cv: Drug conc.K:Partition coefficientD:DiffusivityL:Membran thicknessP:Permeation coefficient

dQ/dt(Flux)

Steady-state

Non steady-state

Time, t (h)

Cum

ulat

ive

amou

nt

of d

rug

perm

eate

d

Calculation of permeation coefficient (P) from skin permeation profile

Typical skin permeation profile

Relationships of membrane permeability coefficients (P) of drugs

Human skin/Hairless rat skin

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

-7

-6

-5

-4

-3

LogP human = 0.98�LogP hairless rat-0.0089R2=0.98

LogP hairless rat (cm/s)

LogP

hum

an s

kin

(cm

/s)

Watanabe, T., Sugibayashi, K. et al., Altern. Animal Test. Experiment., 8, 1-14 (2001)

ANP

ISMNCAF

AMP

ISDN

BA FP

LogP LSE-high (cm/s)

LogP

hum

an sk

in (c

m/s

)

LogP

hum

an sk

in (c

m/s

)LogP LabCyte Epi-model (cm/s)

LogP

hum

an sk

in (c

m/s

)

LogP

hum

an sk

in (c

m/s

)

LogP Neoderm (cm/s)LogP Vitrolife-skin (cm/s)

LogP

hum

an sk

in (c

m/s

)

LogP Epiderm (cm/s)

a) LSE-high b) Epiderm c) LabCyte-epimodel

d) Vitrolife e) Neoderm

LogP

hum

an sk

in (c

m/s

)LogP Episkin (cm/s)

f) Episkin

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

-7

-6

-5

-4

-3

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

-7

-6

-5

-4

-3 LogP human= 1.0�LogP Epiderm+0.06

R2=0.95

LogP human= 0.91�LogP LSE-high-1.75

R2=0.84

LogP human= 0.69�LogP Episkin-2.18

R2=0.13

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

-7

-6

-5

-4

-3 LogP human= 0.72�LogP LabCyte-2.2

R2=0.13

LogP human= 1.6�LogP Neoderm+2.1

R2=0.66

LogP human=3.0�LogP Vitrolife+8.0

R2=0.63

BA

ANPANP

BA

ANPISMN

CAFAMP

ISDN

BAFP

ANPISMN

CAF

AMP ISDN

BAFP

ANP

ISMNCAF

AMP

ISDN

FP

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

-7

-6

-5

-4

-3

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

-7

-6

-5

-4

-3

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

-7

-6

-5

-4

-3

ISMNCAF

AMPISDN

FP FP

CAF

ISMN

AMP

ISDN

ANP

ISMNCAF

AMP

ISDN

FPBA

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

Local effect in skin

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Skin conc.Skin permeation

Physicochemical properties of compounds

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

Local effect in skinSkin conc.Skin

permeation

Physicochemical properties of compounds

Well-known*1 Unknown Partially-known*2

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���6��

Local effect in skinSkin conc.Skin

permeation

Physicochemical properties of compounds

Partially-known*2Well-known*1 Unknown

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

{ }vedtotvedvedvedtotscsctot

vss PPLKPPLK

LC

C /)/1(2

�����

++=

X=Lsc

Donor Stratum corneum (sc)

Viable epidermis and dermis (ved)

Cv

Ksc�Cv

X=0 X=Ltot

KscCvPtot/Pved

KscCvPtot/PvedKved/KscD : Diffusion coefficientK : Partition coefficientCv : Donor concentrationLs : Thickness of membrane C : Average membrane concentrationP : Permeability coefficient

2/••+•

=,vedtotvscvsc

scss

PPCKCKC

2/••

=,vedtotvved

vedss

PPCKC

SC concentration VED concentration

Full-thickness skin concentration

Concentration-distance profile at steady state in two-layered skin model

Sugibayashi K, Todo H, Oshizaka T, Owada Y., Pharm Res 27,134-142 (2010).

Steady state drug concentration-depth profile for full-thickness skin

0

2000

4000

6000

8000

0 0.2 0.4 0.6 0.8 1

Lido

cain

e co

ncen

tratio

n (m

g/m

L)

0

2.0

4.0

6.0

8.0

0 0.2 0.4 0.6 0.8 1.0

Normalized full-thickness skin depth

�Calculated value

�Observed lidocaine concentration instratum corneum

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�Observed lidocaine concentration inviable epidermis/dermis

�����������

Sugibayashi K, Todo H, Oshizaka T, Owada Y., Pharm Res 27,134-142 (2010).

-4

-3

-2

-1

0

1

-4 -3 -2 -1 0 1-5

-4

-3

-2

-1

0

1

-5 -4 -3 -2 -1 0 1

Full-thickness skin

Log

(obs

erve

d C

ss, t

ot/d

onor

con

c.)

Log (calculated Css , tot /donor conc.)

Data range: 122.12<M.W.<234.30, -5.54<ClogP<3.57 Mean � S.E. (n = 4)

Log

(obs

erve

d C

ss, v

ed/d

onor

con

c.)

Log (calculated Css , ved /donor conc.)

Viable epidermis and dermis

Relationship between calculated skin conc. and observed skin conc.

MP

EP

PPBP

M-PABA

E-PABA

P-PABA

B-PABA

Dopa

CAFEpi

ANP

AMP

BA

LCMP

EPPP BP

M-PABAE-PABA

P-PABA B-PABA

Dopa CAFEpi ANP

AMP

BA

LC

ISMN

ISDN

ISMN

ISDN

1:1 correlation1:1 correlation

In vitro'(,-����*�!"#$ % = '(

') =*+,-.

dQ/dt(Flux)

Steady-state

Non steady-state

Time, t (h)

Cum

ulat

ive

amou

nt

of d

rug

perm

eate

d Typical skin permeation profile

DLtlag 6

2

=

= /+-01.

/+ = 0+×*+

Log P (cm/h) = -2.7 + 0.71×log Ko/w-0.0061×MWPotts RO, Guy RH., Pharm Res. 9, 663-669 (1992)

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r2=0.67 (93 compounds)

, = 30+ 01

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MaterialCompound Chemical structure log Ko/w M.W. Solubility in water

(Swater, mg/mL)

Flurbiprofen(FP) 2.2 224.3 0.048

Saturated solubility of FP in the ester oils

Oil name (Abbre.) Saturated solubility (Soil, mg/mL)

2-Ethylhexyl isononanoate (EHIN) 38.62-Ethylhexyl palmitate (EHP) 23.9Diisopropyl adipate (DIPA) 135Di-2-ethylhexyl sebacate (DEHS) 58.5Propylene glycol isostearate (PGIS) 77.7Diethyl sebacate (DES) 1612-Octyldodecyl ricinoleate (ODR) 65.02-Hexyldecyl isostearate (HDIS) 11.3Isononyl isononanoate (ININ) 31.9Neopentyl glycol di(2-ethylhexanoate) (NPGDEH) 52.3

Isotridecyl isononanoate (ITDIN) 23.3Dioctyl carbonate (DOC) 37.4

LABOLUTIONÒ

(PRIMIX Corp.)

Stored at 32℃

Emulsion

OilFP solution (10 mg/g) was prepared by completely dissolving FP with ester oil

at 80ºC followed by stirring at 32ºC for 48 h.

Preparation of applied formulations

Composition of o/w emulsionsEster oil conc.

Component 5% 10% 20% 30%FP 0.2Ester oil 1 2 4 6Purified water 17.13 16.13 14.13 12.132.0% acrylates/C10-30 alkyl acrylate crosspolymer gel

1.6

20% KOH 0.05Phenoxy ethanol 0.02Total 20

Total : 20 g 4,500 rpm, 3 min, 80℃

4,500 rpm, 3 min, 40℃

4,500 rpm, 5 min, 70℃

mix20� KOH aq

mixPhenoxyethanol

Excised skin

Desiccator

Oil solvent-uptake experiment

� �SC�����

SC���������Desiccator Temperature: 20�5 °C

Humidity: 75�5%Hydration: 24 h

SC sheet in dish

Stirrer bar

Saturated sodium chloride solution

SC�������������mg/mg������SC�������mg�����SC������mg�

Measured

Measured

Measured

Soaked in oil or emulsion at 32°C for 1 h

Put at 3°C for 16 hPut at 32°C for 5 h

Peeled

Dried

Hydration

Silica gel

0.1% Trypsin solution

stratum corneum sheet(SC sheet)

SC

�

EHIN-20

EHP-20

DIPA-20

DEHS-20

PGIS-20

DES-20

ODR-20

HDIS-20

NPGDEH-20ITDIN-20

DOC-20

0

0.2

0.4

0.6

0.8

1

1.2

1.4

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1

!"# $%&'()*% = 1.04×!"# $23( + 0.52 (r2=0.61)

(/ 1 /0

( 2 % ¯

/ (/ / µ ) %

/(

2%

/ µ

)%

Prediction of skin permeation of FP from o/w emulsions

Multi-regression analysis: JMPâ Pro (ver. 13.1.0, SAS Institute, Cary, NC, USA). ��������Stepwise regression analysis

In silico modeling approach

Cross-validation: 75% training and 25% validationMolecular descriptors of oils and measured parameters used in this experiment

Abbre. Descriptors and measured parameters

Measured

parameters

Qoil The cumulative amount of FP permeated from the oil baseSCoil Amount of oil uptake into SC sheetSwater and Soil Solubility of FP in purified water and ester oilST Surface tension

Molecular descriptors

Abraham

descriptor

!"# Dipolarity/polarizabilityVx McGowan characteristic volumeR2 Excess molar refractivity∑%"# Hydrogen bond donor acidity∑&"# Hydrogen bond acceptor basicitKgas/hex Partitioning coefficient between gas phase and hexadecaneM.W. Molecular weight of oild Molecular densityMref Excessive molar refractionHA Number of hydrogen bond acceptorstPSA Topological polar surface areaFRB Freely rotatable bondsIR Index of refraction

Hetero ratioC ratioN ratioNO ratio

Pa Parachor, Pa='()/+ , -././0

!"#$ = −10.3 ±2.7 ./0 + 81.8 ±20.9 4 + 22.9 ±2.5 67"#$− 0.07 ±0.02 6

"#$− 63.5(±17.0)

; = 69, =/ = 0.68, >?(4,64) = 30.1, (A < 0.001)

Multi-regression analysis and cross-validation

./0 Dipolarity/polarizabilityd Molecular densitySCoil Amount of oil uptake into SC sheetSoil Solubility of FP in ester oil

Cross-validation result

75% training and 25% validation

Trial #4

()

µ

( µ

=CD/ =0.66 � 0.05 (the mean � S.D.)

=CD/ : R2 for each cross-validation result

0 1 2 3 4 50

5

10

15

20

25

30

35

Col 1 vs 5

Col 1 vs 10

Col 1 vs 20

Col 1 vs 30

)µ

0 1 2 3 4 50

2

4

6

8

10

12

14

Col 1 vs 5

Col 1 vs 10

Col 1 vs 20

Col 1 vs 30

)µ

0 1 2 3 4 50

10

20

30

40

Col 1 vs 5

Col 1 vs 10

Col 1 vs 20

Col 1 vs 30

)µ

( ( (

a) PGIS b) DESc) ODR

¡; 5%¨; 10%¯; 20%r; 30%

¡; 5%¨; 10%¯; 20%

¡; 5%¨; 10%¯; 20%

FP permeation from o/w emulsions

PGIS: Propylene glycol isostearateDES: Diethyl sebacateODR: 2-Octyldodecyl ricinoleate

EHIN-20

EHP-20

DIPA-20

DEHS-20

PGIS-20

DES-20

ODR-20

HDIS-20ININ-20

NPGDEH-20ITDIN-20

DOC-20

PGIS-5

PGIS-10

PGIS-30

ODR-5

ODR-10

DES-5

DES-10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1

(0)

/5-

20 % 0

µ/

%) / ¨5 ) / % ¯5

)0 2 01

(0 )0 0 µ /%

log Qemul5%=0.97×&'( )*+, + 1.25(r2=0.80)

&'( )123,45% = 1.04×&'( )*+, + 0.52(r2=0.61)

log Qemul10%=1.11×&'( )*+, + 0.97(r2=0.91)

Least squared method

) / ¡5

Prediction of skin permeation of FP from o/w emulsions containing 5%, 10% and 20% ester oil

Todo H., et al, Prediction of skin permeation of flurbiprofen from neat ester oils and their o/w emulsions, Chem. Pharm. Bull, accepted, 2018

Conclusion

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