Simultaneous Diffusion and Metabolism of Betamethasone 17-Valerate in the

Living Skin Equivalent

JOHNADEMOLA,AND HOWARD
KIYOSHIKUBOTA~, I. MAIBACH~
Received November 30, 1994, from the Department of Dermatology, University of California, San Francisco, School of Medicine,
Box 0989, Surge 110, San Francisco, CA 941434989, Accepted for publication September 27, 1995@. §Present address: Drug
Safety Research Unit, Bursledon Hall, Southampton SO31 1AA, England.

Abstract 0 Simultaneous diffusion and metabolism of betamethasone

Theoretical Section
17-valeratewas studied using betamethasone 17-valerate,betarnethasone Fraction of the Influx Metabolized in Percutaneous
21-valerate, and betamethasone as permeants. These corticosteroids Permeation-When metabolism occurs inside the skin during
were suspended in silicone adhesive and applied to an artificial living percutaneous permeation of a compound, the kinetic behavior
skin equivalent (LSE) for 72 h. When betamethasone was applied, no is described as follows
metabolites were detected in the receptor medium. Conversely, with
betarnethasone 21walerate application, only betamethasone but no
betamethasone21walerate was detected in the receptor medium indicating (1)
the metabolism of the latter by skin esterases. When tested with the
theory for simultaneous diffusion and metabolism, the result is consistent where h is the thickness of the skin layer in which the
with the enzyme rate constant in the LSE homogenate measured in a compound is metabolized (e.g., viable epidermis), C is the
previous study. When betamethasone 17-valerate was applied to the concentration, D is the diffusion coefficient, k is the first-order
LSE, more than half of the total amount of corticosteroids detected in enzyme rate constant, and x = 0 and x = h designate the
the receptor medium was unchanged, consistent with the previously donor- and receptor-side boundaries of the layer, respectively.
reported chemical (as opposed to enzymatic) degradation half-life of about At steady state, where aCBt = 0, eq 1 reduces to D 3zCf;tx2=
8 h. This result also indicated that vety little metabolism of betamethasone kC, giving the following general solution10-12
17-valerate occurred in the skin.
C(x)= C, cosh[axl + C , sinh[ajcl (2)

where u2 = k f D or a = (kfD)lIz. The constants C1 and C2 are

specified according to the boundary conditions. Flux per unit
Introduction area, J , a t steady-state is given as
Percutaneous absorption comprises ten or more pr0cesses.l
A permeant diffuses through stratum corneum, viable epi-
ac
J(x)= -D -= -Da(C, sinh[axl + C, cosh[axl) (3)
dermis, and dermis and may be metabolized in the several ibc
skin layers. Viable epidermis has been thought to be a major If no metabolism occurs, the flux a t steady-state J(x) is
site of m e t a b o l i ~ m . ~Several
-~ models have been developed constant throughout the layer. Conversely, the difference of
to study skin metabolism such as the “dynamic” culture the flux at the donor-side boundary, J(O), and that a t the
~ y s t e m ,skin-flap
~ technique,6 and living skin equivalent receptor-side boundary, J(h),accounts for metabolism inside
(LSE).7ss The LSE, constructed from cultured human epider- the layer. The fraction of the influx metabolized or “forward
mal cells and fibroblasts, consists of equivalents for stratum c o n s ~ m p t i o n ”f m, ~, ~
is given as
corneum, viable epidermis, and dermis. The LSE has both
advantages (e.g., reproducibility of experiments) and disad-
vantages (e.g., lack of skin appendages). In this study, c,
J ( o ) - J ( h )= 1 - cosh[ah] - - sinh[ah] (4)
simultaneous diffusion and metabolism of betamethasone 17- fm = J(0) c
2
valerate was studied using betamethasone 17-valerate, be-
tamethasone 21-valerate, and betamethasone as permeants. From eq 2, C1 and C2 may be specified by the concentrations
Corticosteroids were applied to the LSE, and the receptor fluid a t two boundaries, C(0) and C ( h )such that
(culture medium) is examined for 72 h to estimate degradation
and metabolism inside the skin. A silicone adhesive was used
as a vehicle. Betamethasone 17-valerate is converted to
betamethasone 21-valerate and then betamethasone. Two
first-order degradation rate constants of this serial conversion Equations 4 and 5 give
of betamethasone 17-valerate to betamethasone have been
estimated using the LSE homogenate in a previous study.g C(0) sinh2[uhl
(6)
The results of the current study are compatible with the rate
constants examined previo~sly.~ Particular attention is paid
f m = 1- C O S ~ [ U ~+
C ]( 0 )cosh[ahI - C(h)
to the finding that no betamethasone 21-valerate but only Provided that C(0) is fixed, the value of f m is the smallest
betmethasone is found in the receptor fluid when betametha- when C(h)= 0. The smallest value of f m , fm0, is given as
sone 21-valerate is applied to the LSE. This observation is
tested with a mathematical model for simultaneous diffusion
and metabolisrnl0-l2described in the theoretical section of the
paper.
~~ If the flux from the skin to the receptor fluid is constant
@ Abstract published in Advance ACS Abstracts, November 1, 1995. and chemical degradation occurs in the receptor fluid, the

1478 / Journal of Pharmaceutical Sciences 0022-3549/95/3184-1478$09.00/0 0 1995, American Chemical Society and
Vol. 84, No. 12, December 1995 American Pharmaceutical Association
fraction of the compound degradated in the receptor fluid a t
steady state, fdg, may be estimated as

In eq 8, kdg is the first-order degradation rate constant and

t is time when the receptor fluid is collected in the static cell
after it is replenished.
When both metabolism within the skin and chemical
degradation in the receptor fluid occur subsequently, the
fraction of the influx metabolized or degradated, f m t d g , may
be approximated as

0 24 48 72
Time (h)
where f, and fdg are given in eqs 6 and 8, respectively. The
minimum value of fmtdg, fm+d@, may be estimated as Figure 1-The average (n = 6 each) cumulative amount of corticosteroids
eliminated to the receptor medium till 72 h after the application of betarnethasone
17-valerate (0),betamethasone 21-valerate (O), and betamethasone (A) to the
(10) LSE. Total amounts of betamethasoneequivalents of three corticosteroids detected
in the receptor medium are plotted.
where fmo is given in eq 7. Note that the fraction of the
compound found to have been metabolized or degradated in 0.075% (w/v) for betamethasone 21-valerate. Three milliliters of each
the receptor fluid sample should be larger than the possible solution was applied on Scotchpak (Type 1009 backing membrane,
smallest value fm+dgO because fm (eq 6) is larger than f , ~
(eq 3M, St. Paul, MN) and then developed into a n approximately 6 x 20
7) when C(h) > 0 and metabolism or degradation may also cm area by a casting device (2.5 in. in width and 0.010 in. in
occur in other layers of skin (e.g., stratum corneum and thickness, designed and made by Stanford Research International
dermis). (Menlo Park, CA)). All of the adhesive containing the three corti-
Estimation of the First-Order Enzyme Rate Con- costeroids turned cloudy after the solvent evaporated overnight, which
stant-When a piece of viable skin is homogenized and suggests a suspension was obtained. Low adhesion polyester film,
suspended in the receptor medium, the first-order enzyme rate Scotchpak (Type 1022 release liner, 3M, St. Paul, MN), was then
placed on BIOPSA, and the patches were stored at room temperature.
constant in the receptor medium, k,,, may be estimated as The drug load was 38 ,ug/cm2 for betamethasone, 38 ,ug/cm2 for
in eq 11below, if the chemical degradation and metabolism betamethasone 17-valerate (31 ,ug/cm2as betamethasone equivalent),
by the skin enzyme are assumed to occur linearly in the and 19 ,ug/cm2 for betamethasone 21-valerate (16 ,ug/cm2 as be-
receptor medium tamethasone equivalent) since the mixture was developed at a
thickness of 0.010 in. (0.0254 cm).
(11) SimultaneousDiffusion and Metabolism of Betamethasone
17-Valerate, Betamethasone 21-Valerate, and Betamethasone-A
where kcmf and kcm- are the first-order degradation rate patch containing one of three corticosteroids was cut into round pieces
(0.5 in. in diameter with a n area of 1.27 cm2) and applied centrally
constants in the receptor medium with and without skin onto the LSE ( n = 6 each). Skin models with the patches were
homogenate, respectively. Presuming that the enzyme activ- incubated in a tissue incubator at 37 "C for 72 h. The receptor
ity in the viable skin is the same as that in the receptor medium (2 mL) provided by Organogenesis Inc. (Canton, MA) was
medium and that the enzyme rate constant is proportional to composed of 6.6 g L Dulbecco's Modified Eagle's medium, 5.3 g/L
the amount of the enzyme molecules per unit volume, the first- Ham's F-12 powder, 0.35 g/L sodium bicarbonate, 2.6 g/L HEPES
order enzyme rate constant in the skin layer, k, may be (sodium salt), and 1.0 ,ug/mL gentamicin sulfate (pH = 7.4). The
estimated as receptor medium was removed and replenished by a fresh medium
a t 10, 24, 34, 48, 58, and 72 h.
Measurement-Corticosteroids were extracted immediately, and
(12) the receptor medium was collected and then measured within 24 h
as described p r e v i o ~ s l y .At
~ 72 h, the patch used in the experiment
was removed from the LSE and the drug in the patch was analyzed
where Vcmis the volume of the receptor medium in which skin as previously described.14
homogenate is suspended and V,l is the volume of the skin
layer where the compound is metabolized.

Experimental Section Permeation of Corticosteroid through LSE from the

Living Skin Equivalent-Living skin equivalent (LSE), TEST- Patches Containing Betamethasone 17-Valerate, Be-
SKIN, with a standard six-well plate, was a generous gift from tamethasone 21-Valerate, and Betamethasone-Figure 1
Organogenseis Inc. (Canton, MA). Methods for preparing the LSE shows the time courses of the average (n = 6) cumulative
and some relevant information have been published amount of corticosteroids that permeated through the LSE
Corticosteroid Patch as a D o n o r Device-Betamethaosne 17- from BIOPSA containing betamethasone 17-valerate (circles),
valerate and betamethasone 21-valerate were generous gifts from betamethasone 21-valerate (squares), and betamethasone
Schering Plough (Bloomfield, NJ). Betamethasone was purchased (triangles). The amounts of the three corticosteroids detected
from Sigma (St. Louis, MO). Medical grade pressure sensitive silicone
adhesive, BIOPSA, in trichlorotrifluoroethane as solvent carrier, was in the receptor fluid sample (culture medium) in the static
purchased from Dow Corning (Midland, MI). Corticosteroids were cell (well on the plate) are expressed as betamethasone
dissolved in the mixture of dichloromethane-BIOPSA in solvent equivalents. When more than two corticosteroids are detected
carrier [64:36, (w/w)l. The drug concentrations in the mixtures were in the receptor medium, the total amount is shown in Figure
0.15% (w/v) for betamethasone 17-valerate and betamethasone and 1.

Journal of Pharmaceutical Sciences / 1479

Vol. 84, No. 12, December 7995
 mL of the medium). The mean wet weight and diameter of
the epidermis equivalent of the LSE measured at 72 h were
approximately 60 mg and 1.8 cm, respectively. According to
loo 1 the manufacturer, the thickness of viable epidermis equivalent
is approximately 70% of the total epidermis thickness. Thus,

L h (eq 1)was estimated as h 0.017 cm assuming the same

density one for wet stratum corneum equivalent and viable

-
X
51 60 epidermis equivalent of the LSE. The volume of viable
ii
epidermis equivalent in one piece of LSE was therefore

7
c
0
$
estimated as x 0.04 mL. Using Vcm= 20 mL, Vsl = 0.04 mL,
and k,, = 0.619 h-l, k in eq 12 was estimated as k x 300 h-l.
40:
20 The diffusion coefficient of betamethasone 21-valerate in
viable epidermis equivalent was presumed to be of the same
magnitude as that of betamethasone 17-valerate estimated
07
in human dermis or 0.74 x cm2/s.15 Using this value for
D, a in eq 2 was estimated as a x 340 cm-l and fmo in eq 7 as
fmo x 0.994.
The fraction of the compound degradated in the receptor
fluid, f d g , in eq 8 was estimated by assuming that the
1001' - - - - a 1001. - - - - '
degradation rate constant k d g is equal to kc,- or 0.178 h-l.
The value of f d g was estimated as 0.53 (t = 10 h for the samples
collected 10 h after the receptor medium was replenished) to
0.63 (t = 14 h). The minimum fraction of the influx metabo-
lized or degradated, f m + d g o , (eq lo), is estimated as 0.997 (t =
B 4 0 1 - . ~ ~, - . 10 h) to 0.998 (t = 14 h). This indicates that when a patch
20 containing betamethasone 21-valerate is applied to the LSE,
a t least 99.7-99.8% of corticosteroids in the receptor fluid
0 24 48 72 '0 24 48 72 collected every 10 to 14 h should be found as betamethasone.
Time (h) Time (h)
Figure 2-The average (n = 6 each) percentage of flux of betamethasone 17-
valerate (O),betamethasone 21-valerate (m), and betamethasone (A)plotted on Discussion
the midpoint in each sampling period. Plots in parts a, b, and c show those after
the application of betamethasone 17-valerate, betamethasone 21walerate, and The smallest fraction of the influx metabolized or forward
betamethasone to the LSE, respectively. consumption,13fmo in eq 7, and the fraction of the compound
degradated in the receptor fluid, f d g in eq 8, were estimated
Figure 2a shows the average (n = 6) percentage of flux of assuming the steady state while the drug flux tended to
the three corticosteroids recovered in each sampling period decrease during the experiment in the current study (Figure
following the application of the patch containing betametha- 1). However, it is unlikely that the average values at
sone 17-valerate. Betamethasone 21-valerate (squares) in the nonsteady state are significantly different from those a t steady
receptor fluid was always less than 10% of the total corti- state. When the patch containing betamethasone 21-valerate
costeroids measured. The percentage of flux of betamethasone was applied to the LSE, no betamethasone 21-valerate but
17-valerate (circles) decreased while betamethasone (tri- only betamethasone was detected in the receptor medium
angles) increased with time. Figure 2b shows the average ( n (Figure 2b). This finding was compatible with the theoretical
= 6) percentage of flux of betamethasone 21-valerate and value of f m + d p o (eq 10) estimated t o be more than 0.997.
betamethasone when the patch containing betamethasone 21- Approximately half of betamethasone 21-valerate, which
valerate was applied. Only betamethasone but no betametha- might have escaped from the metabolism by the skin enzyme
sone 21-valerate was detected in the receptor fluid. Figure and reached the receptor fluid, was predicted to be degradated
2c shows the average (n = 6) percentage of flux of betametha- into betamethasone by the chemical degradation in the
sone when the patch containing betamethasone was applied. receptor fluid because f d g in eq 8 was estimated as 0.53 to 0.63.
No compound which was potentially a metabolite of be- Nevertheless, the observation that only betamethasone but
tamethasone was detected even when the chromatograph was no betamethasone 21-valerate is detected in the receptor fluid
carefully traced until 150 min. indicates that almost complete metabolism of the compound
When the corticosteroid remaining in the patch which had has occurred already inside the LSE. The LSE contains
been applied to the LSE for 72 h was analyzed, only the sufficient esterase to eventually metabolize almost all the
corticosteroid initially loaded (i.e., betamethasone 17-valerate, betamethasone 21-valerate molecules that permeated from the
betamethasone 21-valerate, or betamethasone) but no other vehicle.
compound was recovered in BIOPSA. On the other hand, when betamethasone 17-valerate was
Estimation of Parameters f o r the Metabolism of applied to the LSE, more than 50% of corticosteroids were
Betamethasone 21-Valerate-The smallest fraction of the detected as the unchanged compound in the receptor medium
influx metabolized or forward consumption,13fmo, in eq 7 was (Figure 2a). Since the receptor medium was replaced only
estimated as follows for the patches containing betamethasone every 10-14 h, the fraction of betamethasone 17-valerate of
21-valerate (Figure 2b). It is assumed that esterase is the flux from the LSE should be higher than the values
uniformly distributed in the viable epidermis equivalent of obtained in the experiment (Figure 2a). In the previous
LSE but not in other layers. In a previous paper using the studies, the degradation half-life from betamethasone 17-
homogenate of LSE suspended in the culture medium, kcm+ valerate to betamethasone 21-valerate was x8 h with or
and kc,- in eq 8 were estimated as 0.797 and 0.178 h-l, without skin homogenates or esterase.l6 Therefore, the
respe~tively.~ Therefore, k,, (eq 11)was estimated as 0.619 degradation of betamethasone 17-valerate should have oc-
h-l. In a previous experiment, four pieces of the LSE were curred in the receptor medium after penetrating the LSE as
suspended in 80 mL of the culture medium (one piece per 20 well as within the LSE. The small amount of betamethasone

1480 / Journal of Pharmaceutical Sciences

Vol. 84, No. 12, December 1995
21-valerate (less than 10% of all corticosteroids) detected in 7. Parenteau, N. L.; Nolte, C. M.; Bilbo, P.; Rosenberg, M.; Wilkins,
the receptor medium was probably formed chiefly by the L. M.; Johnson, E. W.; Watson, S.; Mason, V. S.; Bell, E. J . Cell.
Biochem. 1991,45, 245-251.
degradation of betamethasone 17-valerate that had already 8. Nolte, C. J. M.; Oleson, M. A.; Bilbo, P. R.; Parenteau, N. L.
reached the receptor fluid. Arch. Dermatol. Res. 1993,285, 466-474.
In conclusion, an artificial living skin equivalent was useful 9. Kubota, K.; Ademola, J.; Maibach, H. I. Dermatology 1994,188,
in assessing the metabolism of a topical drug. Although there 13-17.
may be large differences in diffusivity and enzyme activity 10. Ando, H. Y.; Ho, N. F. H.; Higuchi, W. I. J . Pharm. Sci. 1977,
between the LSE and human skin in vivo, the present study 66, 1525-1528.
suggests that a certain fraction of betamethasone 17-valerate 11. Fox, J . L.; Yu, C. D.; Higuchi, W. I.; Ho, N. F. H. Znt. J . Pharm.
1979,2, 41-57.
applied to the skin probably reaches viable epidermis and
12. Yu, C. D.; Fox, J. L.; Ho, N. F. H.; Higuchi, W. I. J . Pharrn. Sci.
dermis as the unchanged compound. On the other hand, the 1979, 68, 1341-1346.
fraction of betamethasone 21-valerate is probably small in the 13. Siegel, R. A. J . Phys. Chem. 1991, 95, 2556-2565.
skin and it is rapidly metabolized into betamethasone by skin 14. Kubota, K.; Twizell, E. H.; Maibach, H. I. J . Pharm. Sci. 1994,
esterase. 83, 1593-1599.
15. Kubota, K.; Maibach, H. I. J . Pharrn. Sci. 1993,82,1039-1045.
16. Cheung, Y. W.; Po, A. L. W.; Irwin, W. J. Znt. J. Pharm. 1985,
References and Notes 26,175-189.
1. Wester, R. C.; Maibach, H. I. Drug Metab. Rev. 1983,14, 169-
205.
2. Kao, J.; Carver, M. P. Drug Metab. Rev. 1990,22, 363-410. Acknowledgments
3. Martin, R. J.; Denyer, S. P.; Hadgraft, J. Znt. J . Pharm. 1987,
39, 23-32. We thank Drs. David Friend and Harold W. Nolen 111, of Stanford
4. Pannatier, A,; Jenner, P.; Testa, B.; Etter, J . C. Drug Metab. Research Institute (SRI),Menlo Park, CA, for their help in developing
Rev. 1978, 8, 319-343. the patches containing corticosteroids. We also thank Ms. Nicole
5. Kao, J.; Hall, J. J . Pharmacol. Exp. Ther. 1987,241, 482-487. Kukutsch and Mrs. E. Gogoleva for their technical assistance.
6. Carver, M. P.; Levi, P. E.; Riviere, J. E. Pestic. Biochem. Physiol.
1990,38, 245-254. JS940655W

Journal of Pharmaceutical Sciences / 1481

Vol. 84, No. 12, December 1995