The Effect of Perfusion Rate on In Vitro Percutaneous Penetration*

Vol. 53, No. 4

ma JOURNAL OF INVESTIGATIVE DERMATOLOGY

Copyright

Printed in U.S.A.

1969 by The Williams & Wilkins Co.

THE EFFECT OF PERFUSION RATE ON IN VITRO PERCUTANEOUS PENETRATION* WILLIAM CRUTCHER, A.B. AND HOWARD I. MAIBACH, M.D.

ABSTRACT

This study presents data on the effect of Varying dermal perfusion rates in an in. vitro chamber on percutaneous penetration. Increasing the perfusion flow rates significantly increases the penetration rate with the compoimds studied. This suggests that the data obtained in in vitro studies will be more meaningful when ideal flow rates are determined for such chambers and validated with in vivo data.

Dermal perfusion rate is rarely commented upon as a significant variable in in vitro experiments on percutaneous penetration. Investigators varied chamber designs; but few attempted to approximate in vivo dermal perfusion rates or establish what effect varying

MATERIALS AND METHODS

Mature guinea pigs were sacrificed by intraperitoneal injection of 300 mg of pentobarbital and skinned. The pelt was pinned by the edges on

a tray of wax, shaved with an Oster® animal clipper, using head 10, and carefully reshaved with head 4O. The resulting skin was turned

this rate had on penetration. Attempts have not epidermal side down and repinned on a dry bed of been made to standardize epidermal surface wax. Kenwoodfli kerchiefs moistened with saline conditions, dermal perfusion rates, types of were laid over the subcutaneous muscle and exposed dermis to prevent desiccation while a sharp dissection was performed between the subcutaneous muscle and the dermis. Dissection was easiest over the belly and most difficult over the rump. A thin wall brass ring 2.4 cm in diameter with

perfusate, continuity of dermal perfusion flow, or stirring of perfusate. Tregear states that 10 jd/min/cm' is the rate beyond which increasing

perfusion fails to alter pentration (1). In that

smoothed edges was placed on the dermal side

paper no perfusion data were presented to and a metal hose clamp was brought up and validate this figure; reference was made to around it from beneati and tightened with a

Feldberg and Paton's data on perfused rabbit screw-driver to form a structure looking like an tambourine with the dermal side forming thigh skin in 1951 (2). Review of Feldberg inverted the inside bottom of a 1.5 cm tall "cup" (Fig. 1). and Paton's experiments on perfused cat skin did not include a study of percutaneous penetration. Data presented in this paper indicate a sig-

Scissors were used to trim away excess skin from

the edges. The cup was then placed in a broom clamp attached to a rotating wheel slanted at 30° such that when the cup was filled with 2 ml of saline a portion of the dermal surface was left exposed to the air but would be washed 6 times a minute as the wheel rotated and the fluid covered and then uncovered all portions of the dermis. A Pasteur pipette was used to carefully remove all

nificant increase in penetration of the substances

tested when the dermal perfusion rate is increased in the range of 2—16 d/min/cm°.

An appendix is included to help those who the fluid and 2 ml of fresh saline was immediately are interested in previous work done in this replaced in the cup when samples were taken.

This method allowed a simple comparison of the effect of the freciuency of changing the perfusate Received November 27, 1968; accepted for (and thus varying volumes of dermal perfusion).

field.

In another method, referred to as the "drip

publication April 24, 1969.

This study was supported in part by TJSPHS method," a bottle of saline was hung with intraGraduate Training Grant No. 2-Tot-AM 5372 venous tubing leading to a 30 gauge hypodermic from the National Institute of Arthritis and Meta- needle. Saline was allowed to drip on a cup simibolic Diseases, in part by Food and Drug Admin- lar to the one previously described with the existration Contract No. 68-51, and in part by a ception that the skin sides were reversed. The ring special appropriation from the State of California. Mr. Bruce D. Payne provided expert technical was placed on the epidermal side so that the cup, assistance. when inverted, would expose the outside bottom * From the Division of Dermatology, [Jniver- or dermal side to saline dripped from 5 to 10 cm sity of California School of Medicine, San Fran- above it. Perfusate was allowed to run off the cisco, California 94122. (Reprint requests to Doctor Maibach.)

sides of this apparatus from which it fell to a collection funnel conducting it to a calibrated test

264

PERCUTANEOUS PENETRATION

265

Brass Ring

Ep Id e rrn a I

Hose Clamp

Dermal Side

tpidermal Side FIG. 1. Absorption cell employed in this study (see text for description)

tube for collection. Saline rate of flow was de. termined by raising and lowering the bottle. In this method a rate of 7—8 ml per 2 hours was used.

Application of test material to the epidermis was carried out with a 100 lambda pipette to gently and evenly spread the acetone solution containing '4C-testosterone or testosterone propionate.

(x)

counts obtained from the standard

X gm of sample in the standard X 100 Y

y=

Standards were made with the same pipette. The

percent of applied sample recovered RESULTS

standard consisted of 100 l of testosterone or

testosterone propionate added to 10 ml. of methRepresentative data are included in Figures anol. One-hundred l of this solution was added to 2 and 3. The perfusate was changed at inter2 ml of saline in the Packard low potassium vals of from ½ to 4 hours as noted. The notacounting vials, and 17 ml of counting solution was tion as to the "drip method" refers to a conadded. To determine the radioactivity of the perfusate, stant flow rate of 7—8 ml/2 hrs. These reprea 2 ml aliquot of the sample was added to 17 ml sentative curves show the data until just prior

of the counting solution containing: to reaching the steady state. In most experi6% PPO (2,5-diphenyloxazole) 2: 1 Baker reagent grade toluene: Filtered Rohm ments sample collections continued for 4 to 8 hours after reaching a steady state. and Hass Triton X-100

The filtration was carried out by gravity using In Figure 2, utilizing testosterone, the rate 1 filter paper. Radioassay was performed in a of penetration approximately doubled each time Beckman liquid scintillation system using a win- the dermal perfusate doubled (i.e. changing at dow of 110—300, having a counting efficiency of 56 per cent and figure of merit of 191. The figure of 1, 2, and 4 hours). It is emphasized that this merit (efficiency/background) reached a maxi- was not a continuous flow but the data may be mum at 110—300 with a 14C standard. considered roughly equivalent to 2, 4, 8, and From the counts obtained and the volumes of 16 cl/min/cm2. When a continuous flow of 2 recovered perfusate, percentage of sample recovery per hour was calculated by the following ml/hr was employed, the penetration more than formula and graph:

doubled. The data from testosterone pene-

tration studies show slightly increasing penetration rates for 2 ml/hr, 1 ml/hr, and ½ ml/hr samples at 24 hours whieh are near their evenX counts obtained from the aliquot of perfusate tual steady states based on previous experi-

volume of perfusate recovered volume of the aliquot of perfusate

ments.

266

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY PENETRATION RATES WITH VARYING PERFUSATE

TESTOSTERONE, 1 MICROCURIL IN 8 MICROGRAMS AREA OF APPLICATION 4.45 sq. c.m.

dr.p rrrhod (with snnstonr Hoo(

PER CENT OF THE

4a1/hr) (tO 89% racn.r.d)

APPLIED SAMPLE RECOVERED PER HOUR

perfucat. changed all haur Internal.

perfosat. changed at 2 hour ,nr.snafc

parfuiera chang.d at 4 hour nror.of. C/crrrljhr) (757. racao.rad)

6

8

10

(2

14

16

18

20

24

22

HOURS AFTER APPLICATION

FIG. PER CENT OF THE

APPLIED SAMPLE RECOVERED PER HOUR -

.50%

2

PENETRATION RATES WITH VARYING PERFUSATE

TESTOSTERONE PROPRIONATE (2 MICROCURIES IN 23 MICROGRAMS) AREA OF APPLICATION 4.45 sq. cm.

perfocate changed at T/, hour internal;

internals (.63% recovered)

rift 01

0

16

HOURS AFTER APPLICATION

Fia. 3

Figure 3 demonstrates similar findings with ing steady state at 22 hours. The 1 and '/2 hour testosterone propionate. Doubling the flow rate sample changes were discontinued in their doubles the penetration rate, Testosterone pro- steady states at 16 hours as graphed. pionate samples taken at 4 hour intervals During steady state, the perfusion rate conreached a steady state at 16 hours and the sam- tinued to directly affect the recovery rate. The ples changed at 2 hour intervals showed an in- experiment was not carried beyond 24 hours as significant rise in penetration rate before achiev- it was felt that the cutaneous barrier could not

267

PERCUTANEOUS PENETRATION

be assumed to remain intact beyond this time.

5. Cronin, E. and Stoughton, R. B.: Nicotine

Time lag in reaching steady rate of pene-

acid and ethyl nicotinate in excised human

tration varied with the perfusion flow rate. This was an inverse relationship i.e., by increasing dermal perfusion rate, time lag before reaching steady state was reduced. We can not explain

6. Elfbaum, S. G. and Laden, K.: The effect of dimethylsulfoxide on percutaneous absorp-

skin. Arch. Derm., 87:

445, 1963.

tion. J. Soc. Cosm. Chem., 19: 119, 1968.

7. Feldmann, R. J. and Maibach, H. I.: Per-

the mechanism involved, but suspect this

cutaneous penetration of steroids in man. J.

phenomenon of importance in comparing in

8. Frisch, W. C. and Stoughton, R. B.: The effect of temperature and humidity on the penetra.-

vitro data to in vivo experience where perfusion rates are high. DISCUSSION

These data demonstrate that the rate of pene-

tration in an in vitro chamber is related to

Invest. Derm., 52: 89, 1969.

tion of 14 acetylsalicylic acid in excised

human skin. J. Invest. Derm., 41: 307, 1963. 9. Garby, L. and Linderhoim, H.: The permeabil.-

ity of frog skin to heavy water and ions with special reference to the effect of some diuretics. Acta Physiol. Scand., 28: 336, 1953.

the rate of dermal perfusion. We submit that the rising concentration of penetrant in the

10. Linderholm, H.: Ion permeability and electri-

perfusate of in vitro chambers limits the rate of

11. Macey, R. I. and Meyers, S.: Dependence of

percutaneous penetration. In the physiologic

isolated frog skin. Amer. J. Physiol. 204:

situation, penetrant concentration in the dermal capillaries is insignificant, hence chamber work

should strive to duplicate this with frequent perfusate changes or preferably a continuous trickle at the same rate as the normal cutaneous perfusion. We performed similar experiments with other

cal conductivity of the isolated frog skin. Acta Physiol. Scand., 20: 185, 1950.

chloride permeability on sodium in the 1095, 1963.

12. Mali, J. W. H., Van Kooten, W. J. and Van Neer, F. C. J.: Some aspects of the behavior of chromium compounds in the skin. J. Invest. Derm., 41: 111, 1963.

13. Mali, J. W. H., Van Kooten, W. 3., Van Neer, F. C. J. and Spruit, D.: Quantitative aspects of chromium sensitization. Acta Dermato-

vener., If4: 44, 1964. compounds of varying solubilities including 14. Marzulli, F. N.: Barriers to skin penetration.

testosterone acetate, salicylic acid, benzoic acid, hydrocortisone, butter yellow, hexachiorophene, urea and thiourea; always the same dependence

15. Marzulli, F. N., Callahan, 3. F. and Brown,

are not strictly controlled hour by hour and

methylacetamide and dimethylformamide ef-

J. Invest. Derm., 39: 387, 1962.

D. W.: Chemical structure and skin penetrat-

ing capacity of a short series of organic on perfusion rate occurred. These compounds phosphates and phosphoric acid. J. Invest. span a wide range of solubility characteristics. Derm., 44: 339, 1965. Hence, any experiment in which perfusion rates 16. Munroe, D. D. and Stoughton, R. B.: Difect on percutaneous penetration. Arch.

from sample to sample may have artifact introDerm., 92: 585, 1965. duced into its results. We do not feel that the 17. Scheuplein, R. J.: Mechanism of percutaneous absorption, I. Routes of penetration and the dependence of penetration in vitro on perfusion influence of solubility. J. Invest. Derm., 45: rate precludes the validity of in vitro studies. 334, 1965. It does suggest the necessity for obtaining back- 18. Scheuplein, R. J. and Blank, I. H.: Progress report from Edgewood Arsenal, Oct. 1965. ground data on perfusion rates that may yield 19. Sweeney, T. M., Downes, A. M. and Matoltsy, results comparable to the in vivo situation. A. G.: The effect of dimethyl sulfoxide on the epidermal water barrier. J. Invest. Derm., REFERENCES 46: 300, 1966. 1. Tregear, R. T.: Epidermis as a thick mem- 20. Tregear, R. T.: Relative permeability of hair

brane. J. Physiol., 153: 54, 1960. (In

the appendix under Proceedings of the

follicles and epidermis. J. Physiol., 156: 307, 1961.

Physiologic Society). R. T.: Physical Functions of Skin. 2. Feldberg, W. and Paton, W. D. M.: Release 21. Tregear, Academic Press, New York, 1966. of histamine from skin and muscle in the cat by opium alkaloids and other histamine 22. Tregear, R. T.: The permeability of mam-

liberators. J. Physiol., 114: 490, 1951.

3. Ainsworth, M.: Methods for measuring percutaneous absorption. J. Soc. Cosm. Chem.,

malian skin to ions. 3. Invest. Derm., 46: 16, 1966.

23. Treherne, J. E.: Permeability of skin to some

11: 69,1960.

non-electrolytes. J. Physiol., 133: 171, 1956.

Invest. Derm., 43: 414, 1964.

matologica, 132: 141, 1966.

24. Van Kooten, W. J. and Mali, J. W. H.: Significance of sweat-ducts in permeation experiments on isolated cadaverous skin. Dercentration of alcohol and type of vehicle, J.

4. Blank, I. H.: Penetration of low molecular weight alcohols into skin, I. Effect of con-

THE JOTJBNAL OF INVESTIGATIVE DERMATOLOGY

268

APPENDIX I Summary of In Vitro Penetration Studies Author

Ainsworth, M.

Skin

Rabbit

Area cm'

3.5 at max.

Perfusate

Ringer's

Aint. of perfusate

5 ml.!

hr. at

Con-

stant flow

Stirred

yes yes,

teflon

Epidermal surface

dry with one drop

no

no

yes

wet with pene-

no

no

dry with one

Blank, I. H. (A.)

Human

3.0

.85% NaG! 2—3 ml.! yes hr.

Blank, I. H. (B.)

Human

3.0

.85%NaC1 2—3 ml.

Cronin, E. and Stoughton, R. B.

Human 23.8

.9% NaCl 7 ml.

trant reservoir trant reservoir drop

Elfbaum, S. G. and Guinea Laden, K. Pig Fritsch, W. C. and Human

3.0

pene-

trant wet with pene-

bar

max.

pene-

15 ml.

no

no

trant wet with pene-

.9% NaCI 10 ml.

no

no

dry with one

Buffer

trant reservoir

.78

Stoughton, H. B.

and .9%

drop

trant

benzyl alcohol

pene-

Garby, L. and Lin- Frog

7.1

Ringer's

8 ml.

yes

no

wet with pene-

derholm, H. Linderhoim, H.

Frog

7.1

Ringer's

8 ml.

yes

no

wet with pene-

Frog

2.5

Ringer's

5 ml.

no

no

wet with pene-

Macey, R. I. and Meyers, S.

trant reservoir trant reservoir

trant reservoir

with SO4

& Man-

nitol

Mali, J. W. H., Van Human

7.1

HOH

Mali, J. W. H., Van Human Kooten, W. J., Van Neer, F. C. J. and Spruit, D. Marzulli, F. N. Human Marzulli, F. N., Callahan, J. F.

Human

8

1% NaC1

no

no

50 ml./2 no

no

.2 .2

.9% NaCl 10-20 yes ml./hr.

no

.9% NaCI 10—20

no

yes

.78

.9% NaCl 10 ml.

trant

no

no

drop

yes,

teflon

Scheuplein, R. J. and Human not given not given Blank, I, H.

pene-

dry with one trant

not given no

pene-

dry with one drop

and .9%

Human not given not given

dry with one drop

ml./hr. Human

wet with pene-

trant

benzyl alcohol

Scheuplein, R. J.

wet with pene-

trant reservoir

days

and Brown, D. W.

Munro, D. D. and Stoughton, R. B.

400 ml.

trant reservoir

Kooten, W. J. and Van Neer, F. C. J.

pene-

wet with penetrant reservoir

bar not given no

yes, teflon

wet with penetrant reservoir

bar Sweeney, T. M., Downes, A. M. and Matoitsy,

A. G.

Hairless mice

.71

.9% NaCI not given yes

no

wet with penetrant reservoir

269

PERCUTANEOUS PENETRATION

APPENDIX I—Continued Author

Tregear, R. T.

.

Skin

Rabbit

Area cm2

Aint f

Perfusate

not given Ringer's

perfusate

Con-

stant

at least yes

Stirred

not given

lOisl/

Treherne, J. E.

Rabbit 3.5(?) .9% NaC1 & human Rabbit not given Ringer's

Van Kooten, W. J.

Human

dry with one drop

trant

cm2!

Tregear, R. T.

Epidermal surface

pene-

mm. not given yes

no

8 ml

no

yes

wet with pene-

no

no

wet with pene-

and Mali, J. W. H.

This summarizes the pertinent experimental variables.

wet with penetrant reservoir trartt reservoir

trant reservoir

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

94

linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.

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