Jonathan Hadgraft&

Majella Lane

The School of Pharmacy, University of London

Spectrum on skin

λ 3km 3m 30cm 3mm 0.03mm 300nm 3nm 3pm

radiofrequency mic

row

ave

far i

nfra

red

uv x rays γ rayslog ν / Hz 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

λ 3km 3m 30cm 3mm 0.03mm 300nm 3nm 3pm

radiofrequency mic

row

ave

far i

nfra

red

uv x rays γ rayslog ν / Hz 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

NMR ESR IR neutrons

λ 3km 3m 30cm 3mm 0.03mm 300nm 3nm 3pm

radiofrequency mic

row

ave

far i

nfra

red

uv x rays γ rayslog ν / Hz 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Skin structure

transcellular intercellular

follicular eccrine

Major barrier is in stratum corneum (15 μm thick)

Role of lipids

Corneodesmosomes

Composition & structure of intercellular channels

ceramides (50%) cholesteryl sulphate (5%)cholesterol (25%) free fatty acids (15%)

Michaels et al. 1975

Albery & Hadgraft

Elias

Boddé

Potts & Francoeur

Talreja et al.

Boddé H. et al Int. J. Pharm. 53 (1989) 13-24

Lars Norlen

Cryo-sectioning and cryo-TEM

Freezing 20 ms

-180C

50 – 100 nm thick vitreous section

No cryoprotectants

Hydrogen bonding with ceramides

NO

NH

O

HO

HO

O

HO

HO

HO

NH

HO

NH

O

HOHO

HO

O

HO

HO

HO

ON

O

O

NH -0.41

-0.41-0.42

Hadgraft, J. et al. Int. J. Pharm. (1997) 141, 17-25Gay, C.L. et al. (1989) Int J. Pharm. 49, 39-45

esr

neutron scattering& monolayers

Biophysical techniques

• Infra red• Diffusion studies• Imaging• Diagnosis

• NMR• OTTER• TEWL• MS

Fourier Transform Infra Red

from

ir

sour

ce

to detector

Zn Se crystalmembrane

saturated solution

Diffusion studies

40030020010000

1

2

3

4

5

controlAzone

time (mins)

peak

are

a

120080040000

10

20

30Transcutol: water (50:50)control

time (mins)

peak

are

a

Harrison, J. et al. Pharm. Res. (1996) 13, 542-546

ATR FTIR benzoic acid – octanol:silicone membrane

0

4

8

12

0 1 2 3 4 5 6 7Time (hours)

C=O

pea

k ar

ea pre-treatmentno pre-treatment

Dias, Hadgraft, Raghavan & Tetteh. J. Pharm. Sci. 93 (2004) 186-196

Something for nothing:

InSight

chemometrics

Dias, Hadgraft, Raghavan & Tetteh. J. Pharm. Sci. 93 (2004) 186-196

Russeau

Octanol

Water vapour

Silicone membrane

Benzoic acid

0

4

8

12

0 1 2 3 4 5 6 7Time (hours)

C=O

pea

k ar

ea pre-treatmentno pre-treatment

Software enables separation of methyl and ethyl paraben

-0.5

1.5

3.5

5.5

7.5

9.5

0 100 200 300 400 500 600

Time (min)

CN

Abs

orba

nce

CNP in water

CNP in 5050 Pgwater

CNP in wet PGL

McAuley, Lane

0

0.5

1

1.5

2

0 5 10 15 20 25 30Time (mins)

CN p

eak

abso

rban

ce (A

U) Propylene glycolPEG 400IPMIPIS

Silicone membrane: pools?

Kazarian, McAuley, Lad, Lane

ipm

ipm21 h

pg

Profiles analysed using Scientist and solutions to Fick’s Laws, modelling

Infra red imaging: chemometrics

Kazarian

50 x 50 µm

Sampling the skin: tape stripping

Determination of drug concentration in the stratum corneum (SC) by sequential removal of thin layers of SC at the same site with adhesive tape.

Guy et al.

Industrial – University collaboration

Distribution profile of active across the stratum corneum (SC)

Cx

x/H

AUC

Measure drug concentration profile as a function of position in the SCRequired: (i) amount on each strip, (ii) penetration depth into SC

Evaluation of the rate and extent of active penetration into the stratum corneum - the rate-determining barrier to skin permeation.

How much?

How fast?

Howfar?

Guy et al.

Wavenumbers (cm-1)

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Abs

orba

nce

1000 2000 3000 4000

Amide II

Amide I

C=O

C-H stretching symmetric

C-H stretching asymmetric

O-H bending

Lipid extraction – lipid disorderPeak height (area) Peak shift

Dias, Guy, Hadgraft. Lane

Lipid extraction (peak area)

• Both D-octanol and D-hexanol extract the lipids after 55 min of exposure

• D-decanol does not extract the SC lipids

55 min

TS

0

0.2

0.4

0.6

0.8

1

1.2

-2 0 2 4 6 8

norm

alis

ed C

-H p

eak

area

d-decanolcontrold-octanold-hexanol

initial

30 min Stratum corneum depth (μm)

Dias, Guy, Hadgraft. Lane

Lipid disorder (peak shift)

All vehicles produce a blue shift after 30 min of exposureD-decanol disordering effect is maintained for 55 min and in ~2 μm of the SC.

initial

30 min55 min

TS

Stratum corneum depth (μm)

-8

-6

-4

-2

0

2

4

6

8

10

-2 0 2 4 6 8

C-H

pea

k fre

quen

cy s

hift

(cm

-1) control

d-hexanold-decanold-octanol

Dias, Guy, Hadgraft. Lane

-8-6-4-202468

10

0 0.2 0.4 0.6 0.8 1 1.2solvent uptake

C-H

2 asy

mm

etric

str

etch

ing

freq

uenc

y sh

ift (c

m-1

)

volunteer 1

volunteer 2

volunteer 3

volunteer 4

volunteer 5

volunteer 6

volunteer 7

volunteer 8

Solvent uptake (peak area) and lipid disorder (peak shift)

Relationship between the solvent uptake (normalised C-D peak area) and the frequency shift for each volunteer, after exposure to D-decanol for 30 min, 55 min and tape stripping.

Dias, Guy, Hadgraft. Lane

Diagnostics?Psoriasis UVA treatment

Pre-treatmentPeak areas

• C-H asymmetric = 0.07• C-H symmetric = 0.03

Post-treatmentPeak areas

• C-H asymmetric = 0.18• C-H symmetric = 0.07

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32

0.34

0.36

0.38

0.40

Abs

orba

nce

1000 1500 2000 2500 3000 3500 Wavenumbers (cm-1)

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32

0.34

Abs

orba

nce

1000 1500 2000 2500 3000 3500 Wavenumbers (cm-1)

Watkinson & Burgess 2002.

Clinical dosing: 2 mg/cm2

dynamic

Skin lipids 20% volume: limited solubility capacity

20 μm

In vitro experiments difficult to mimic dose

*

What does solvent deposited solid chemical look like on skin surface?

10 μ g/cm2 4-cyanophenol in acetone

200 μm

*

Environmental Scanning ElectronMicrographs (ESEM)

No deposited chemicalBunge

contact no contact

no contact

Bunge

Particle size and contact with intercellular channels

Permeation of propylene glycol and drug: effect of the amount of gel applied

0

200

400

600

800

1000

1200

1400

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (hours)

Flux

(ng/

cm2/

hr)

0

50

100

150

200

250

300

350

400

Flux

PG

(µg/

cm2/

hr)

Drug: 10mg/cm2 Drug: 40mg/cm2 PG: 10mg/cm2 PG: 40mg/cm2

Trottet et al. Int. J. Pharm. 274 (2004) 213-219

Mass spectroscopy

Royal Society fellow Dr Simona FranceseMalcolm Clench SHU

GARField - a magnet for planar samples

P. M. Glover, P. S. Aptaker, J. R. Bowler, E. Ciampi, P. J. McDonald J. Magn. Reson. (1996) 139, 90.

MagnetPole

Skin

RF Sensor CoilGlass slide

B0

Gy B1

Marker tape

Bo : Magnetic field (0.7 T)

Gy : Field Gradient (17 T/m)

B1 : Radio Frequency field

Fourier Transform gives 1D

projection

0

100000

200000

300000

400000

500000

600000

350 400 450 500

Position (μm)

Am

plitu

de

17 min51 min136 min238 min340 min493 min

sensorGlass Slide

Profile directionSkin

Skin dehydration – in vitro

M Dias, J Hadgraft, P M Glover and P J McDonald, J. Phys. D: Appl. Phys. (2003) 36 364

Comparison with imaging studies

Chan & Kazarian

Time course of kinetics similar

In vivo –lower arm & hand

OTTER

Imhof, London South Bank University

Air

Sample

Pulsed laser excitation

Infra red emission signal

Opto Thermal Transient Emitted Radiation

Glycerol Concentration

0

2

4

6

8

10

12

14

16

18

20

0 2 4 6 8 10 12 14

Depth [um]

Con

cent

ratio

n [a

.u.]

Control5min8min10min

Permeation into nails in vivo

Nail hydration profile

0

5

10

15

20

25

0 5 10 15

Depth [um]C

once

ntra

tion

[a.u

.]

Left ThumbNailRight ThumbNail

Hirata, Imhof, Xiao, Zheng, McAuley, Lane

Hydration profiles in vivo laser confocal Raman

Cork, Matts et al.

Skin structure

transcellular intercellular

follicular eccrine

Major barrier is in stratum corneum (15 μm thick)

Role of lipids

Corneodesmosomes