Curr. Probl. Dermatol., vol. 7, pp. 58-68 (Karger, Basel 1978)
The Finite Dose Technique as a Valid in vitro Model for the Study of Percutaneous Absorption in Man Thomas J. Franz University of Washington, School of Medicine, Seattle, Wash.
Introduction The technique most frequently employed for the measurement of percutaneous absorption is the in vitro use of excised human skin mounted in diffusion chambers. Although results obtained from this model have contributed significantly to the formulation of current dogma, there is surprisingly little data available to support the belief that in vitro absorption is a totally and consistently accurate reflection of absorption in living humans. The relevance of in vitro absorption data has largely been assumed, and rests primarily on knowledge that the stratum corneum is the rate-limiting barrier of the skin (I9). It has been argued that since the stratum corneum is a 'dead', non-metabolizing structure it should function equally well in vitro as in vivo; but, aside from the one rather special situation of transepidermal water loss, this assumption has not been critically evaluated (1, 3, 4, 14, 15, 17). Recently, we have begun to examine in our laboratory the extent to which in vitro absorption can be used as a valid predictive model of absorption in living humans (10). Review of the literature revealed that many prior in vitro studies had not employed conditions generally applicable to those found in vivo in man; thus, an essential part of the work to be described has been the development of a model which attempts to approximate these conditions. Examination of the more conventional approach will help illustrate the problem.
Historically, the most frequently used in vitro method for studying absorption is the infinite dose technique (fig. 1a). The skin is mounted as a barrier between two well-stirred, fluid-filled chambers. The compound under study is
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Infinite Dose Technique
59
Percutaneous Absorption: in vitro Model Infinite dose
ro~u~t___HL=____~~;.~_____~=__~I~n
, a
2
. .,
. • r··;·
L -_ __ _ _ __ _ _ _~~~----------~
T
c
added to the solution on one side (donor) of the membrane and absorption assessed by serially sampling and assaying its concentration in the bathing solution (receptor) on the other side. Generally, the amount of solute that penetrates during the course of an experiment is small relative to the total amount available and there is no appreciable reduction in solute concentration in the donor solution. In effect, it appears to be 'infinite '. Measurements are commonly taken of the total quantity of solute that has crossed the membrane at any time (Qt), and plotted as a function of time (fig.lb). The time course of absorption can be divided into three phases: (1) a lag phase, characterized by solute movement through the membrane but during which no molecules appear in the receptor solution; (2) a nonlinear phase, during which the solute concentration in the receptor solution begins to rise , slowly at first, but at an increasing rate; and finally (3) a linear phase, during which the rate of increase in solute concentration in the receptor solution is constant. The rate of absorption or flux (1) is constant at its maximum value and steady-state conditions prevail. J can be calculated directly from the slope of the line (6Q/6t) under these steady-state conditions. Alternatively, the flux can be plotted as a function of time (fig. Ic) ; the lag phase and rise to steady-state conditions are again evident. A number of objections can be raised to the use of the infinite dose technique as a predictive model for living man. The most obvious, and certainly that of greatest significance, is the use of fully hydrated skin ; both sides of the preparation (be it isolated stratum corneum, epidermis, or full thickness skin) are bathed by aqueous solution. Among biological barriers, the skin is unique in
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Fig. 1. The infinite dose technique. (a) Schematic diagram of the skin mou nted as a barrier between two chambers. Data can be expressed as either: (b) total absorption (Q) vs. time, or (c) rate of absorption (1) vs. time.
Franz
60
that it normally functions with one surface exposed to a dry environment. It is well known that hydration drastically alters the function of the stratum corneum and induces a 5-1 O-fold increase in permeability (8, 11). Equally blatant is the fact that most compounds do not come into contact with skin in the form of aqueous solutions. Water is not a common vehicle. Yet there is ample evidence that the absorption of topically applied drugs is directly dependent upon the composition of the vehicle (13, 16). The permeability of skin to any given compound cannot be specified without knowing the vehicle in which it is applied, and the routine use of water, therefore, makes the model of limited relevance. A less obvious objection is the size of the applied dose - both vehicle and drug. Under clinical or 'use' conditions, the amounts of material applied are of the order of a few milligrams (of vehicle) per square centimeter of skin. In comparison, in vitro methods which utilize an aqueous solution (or other solvent) in contact with the skin are, in effect, applying gram per square centimeter quantities of vehicle (water or solvent, in this case). The dose of active drug in these circumstances may also be excessive due to the large (vis-a-vis infinite) amount of vehicle applied (dose = drug concentration x mass or volume of vehicle). The importance of these deviations from conditions normally associated with topical drug use will become apparent when the kinetics of absorption are discussed.
To avoid the shortcomings of the previously described approach, the following technique has been developed. Human abdominal skin obtained at autopsy is mounted in specially constructed diffusion chambers (fig. 2a). The dermis is bathed by isotonic saline, pH 7.4, and the temperature maintained at 37°C by thermostatically controlled water which is circulated through a jacket surrounding the lower chamber. Homogenous temperature distribution in the dermal bathing solution is achieved by a small teflon-covered magnetic stirring-bar, driven by an external magnet mounted on a 600 rpm timing motor. The epidermis is exposed to the ambient conditions of the laboratory environment, so that the skin is held under conditions which closely approximate the living state. The gradients of water and temperature which normally exist across the skin are both maintained in this model. Furthermore, this ar· rangement allows for the application of any type or any amount of vehicle, so that the absorption of individual compounds can be studied in the form and dose most appropriate to their actual use. Usually, 5-10 pi of a liquid vehicle containing the test compound is applied per square centimeter of skin, using a micropipette; otherwise, 2-5 mg of a non-liquid vehicle containing the test compound is applied per square centimeter of skin, using a small stirring rod. The
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Finite Dose Technique
61
Percutaneous Absorption: in vitro Model Finite dose
out
I I
a
I I
1::~:3 :I I I
I
b
T
Fig. 2. The finite dose technique. (a) Schematic diagram of the skin bathed by isotonic saline below and covered by a thin 'finite' dose on its outer surface. (b) Rate of absorption (J) vs. time.
material can be thoroughly rubbed in using the smooth, rounded tip of the stirring rod and the exact amount applied determined by weighing the rod before and after use. Thus, the small finite doses typically associated with the clinical use of dermatologic preparations are easily duplicated. Use of a finite dose alters the rate of absorption curve from that observed in the previously described model. No steady-state is achieved (compare figs. 2b and Ic). Following a lag phase, the flux rises to a peak, then falls. The rising portion of the curve is similar to that seen with an infinite dose and represents the approach to a steady state. However, as absorption proceeds, the concentration of drug on the surface of the skin falls, eventually to a point where the rate of absorption can no longer be sustained. At this point the flux also begins to fall and will continue to do so until the source is exhausted. This difference illustrates the objection to the use of the infinite dose technique alluded to earlier. On theoretical grounds alone, it seems clear that failure to approximate the amount of drug used in vivo will yield results which bear little resemblance to those found in living man. It is a serious error because it represents a failure to properly characterize the time-course of absorption.
The test of any in vitro model is its ability to simulate the in vivo system. To test the validity of the technique proposed here, use has been made of a study of percutaneous absorption in humans by Feldmann and Maibach (7), who measured the absorption of a series of organic compounds in volunteers, using radioisotopes. Their technique consisted of applying I pCi of a 14C-Iabeled compound dissolved in acetone at a dose of 4pg/cm 2 to the skin of the forearm, and thereafter assaying total urinary output over 5 days for radioactive content.
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Assessing the Validity of the Finite Dose Technique
Franz
62
Table I. Total absorption of various compounds by skin in vivo and in vitro (expressed as perc en t of applied dose)
Compound 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Hippuric Acid Nicotinic Acid Thiourea Chloramphenicol Phenol Urea Nicotinamide Acetylsalicylic Acid Salicylic Acid Benzoic Acid Caffeine Dinitrochlorobenzene
Absorption in vivol 0.2 ± 0.1 0.3 ± 0.1 0.9 ± 0.2 2.0 ± 2.5 4.4 ± 2.4 6.0 ± 1.9 11.1 ± 6.2 21.8± 3.1 22.8±13.2 42.6 ± 16.5 47.6 ± 21.0 53.1 ± 12.4
(7) (3) (3)
(6) (3)
(4) (7) (3) (17) (6) (12) (4)
Absorption in vitro' 1.2 [0.8, 2.7] 3.3 [0.7, 8.3] 3.4 [2.4,5.5] 2.9 [1.0,5.7] 10.9 [7.7, 26] 11.1 [5.2, 29] 28.8 [16, 65] 40.5[17, 49] 12.0 [2.3, 23] 44.9 [29, 53] 9.0 [5.5, 20] 27.5 [19, 33]
(15) (19) (52) (12) (7)
(22) (21 ) (14) (10) (18) (17) (18)
I Mean ± SD. , Median with 95% confidence interval given in square brackets. The values in brackets represent the number of subjects studied.
Since only urinary excretion was measured, it was necessary to correct for labeled compound which was absorbed but not excreted by the kidneys by measuring the fraction of an intravenously administered dose of the same compound which was excreted in the urine. Twelve of the compounds studied by Feldmann and Maibach were selected by us for study in vitro (see table I) so that a critical evaluation of the relevance of the finite dose technique to absorption in living man could be made (10). The same vehicles and topical doses as used in vivo were used in vitro. Comparison of the data obtained from the two studies was made on the basis of two parameters: total absorption (% of dose) and the rate of absorption (% of dose/h).
In figure 3, the time-course of absorption as determined both in vitro and in vivo is shown for three representative compounds. With eight of the twelve compounds studied (acetylsalicylic acid, benzoic acid, caffeine, dinitrochlorobenzene, hippuric acid, phenol, salicylic acid and nicotinamide) the rate of absorption was characterized by the appearance of a distinct maximum (Jmax),
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Rate of Absorption
63
Percutaneous Absorption : in vitro Model
.
ASA
10
30
24
48
72
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60
70
80
90
100 110
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as was expected from theoretical considerations. Comparison of the curves obtained in the in vitro experiments with those obtained in living man yielded excellent overall agreement, including the time at which J max occurred. Representative data for acetylsalicylic acid and dinitrochlorobenzene are presented in figure 3. Comparison of the rate of absorption profiles from the two sets of experiments is necessarily limited, in that insufficient data points were collected in vivo, particularly in the initial period of absorption. The remaining four compounds (chloramphenicol, nicotinic acid, thiourea, urea) showed an absorption pattern which displayed a tendency toward steadystate conditions and a low absorption rate. The in vitro and in vivo absorption curves had a similar overall pattern, but significant differences in the magnitude of the absorption rate were observed with all four compounds. For example, see nicotinic acid, fig. 3. The apparent steady-state in this case in fact only reflects the very low rates of absorption obtained with these compounds. Even with a finite dose, absorption proceeds so slowly that little reduction in the concentration of tracer on the skin surface occurs during the course of the experiment. When the duration of the experiment is extended to 1- 2 weeks this pseudo steady-state is revealed to be a very broad low peak, spanning several days.
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Fig. 3. Comparison of the rate of absorption (1) determined both in vitro and in vivo for acetylsalicylic acid (ASA), dinitrochlorobenzene (DNCB) , and nicotinic acid.
64
Franz Caffeine
• 24hr wash '" 48hrwash o no wash
08 06
10
20
30
40
50
60
70
80
Hours
Fig. 4. The effect of a brief wash ('V 1 min) of the outer surface of the skin at 24 or 48 h on the rate of absorption of caffeine. Three rinses consisting of acetone, then water, followed by acetone were utilized.
In addition to the absorption rate, total absorption can be taken as a parameter for comparing the in vitro model with in vivo conditions. An interesting question arises - how long should one follow absorption in an in vitro model? At least one crucial function of great importance to the process of absorption that cannot be duplicated in the model is desquamation of the epidermis. In living man, this process will result in a continuing reduction in the amount of material remaining on the skin following a single dose, even in the absence of significant permeation, whereas in the model, material will remain on the skin sample and continue to serve as a source for absorption. This is illustrated in figure 4; a very brief wash of the skin surface at 24 or 48 h results in significant reduction in the subsequent rate of absorption. Theoretically, one might expect to observe 100% absorption in vitro for most compounds over a sufficiently long time period. From data on the turnover and thickness of the stratum corneum, it can be calculated that there is an average loss of one cell layer per day (2, 18), making this factor potentially important in experiments lasting longer than 1 day. As a first approximation it seemed reasonable to select 2 days as the time limit for the calculation of total absorption in vitro, for three reasons. First, it is compatible with the simple model that if the entire surface layer is lost at the end of day 1, at least another day must be allowed for the absorption of material in transit through the deeper regions of the stratum corneum and thus not affected by events at the surface. This is illustrated in figure 4. Second, for the majority of the compounds studied (those displaying a distinct J max ) most absorption takes place in the first 2 days and that which takes place subsequently can be essentially neglected in computing totai absorption. It should be noted, however, that
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Total Absorption
Percutaneous Absorption: in vitro Model
65
this latter argument does not hold for the four compounds displaying pseudo steady-state kinetics. Third, it makes sense to use a shorter time for the calculation of total absorption in vitro than in vivo, because of the possibility of delayed excretion in vivo , due to drug binding or slow metabolism in the body. Data for total in vitro (2 day) and in vivo (5 day) absorption are presented in table I. There is reasonable agreement between the two sets of data with regard to compounds that are well absorbed (i.e. greater than 10% absorption) , but not for compounds that are poorly absorbed. For hippuric acid, nicotinic acid and thiourea, the in vitro values are 4-1O-fold higher than those obtained in vivo. There is, however, a trend toward positive correlation. Compounds with high absorption rates can be distinguished by either set of data from those with low absorption rates and the rank order of rates found in the in vitro experiments is similar to that found in vivo. Statistical evaluation of the data using Spearman's test for rank order correlation (5) showed high significance (p = 0.01); the correlation coefficient was 0.734.
This first attempt to evaluate the relevance of the finite dose model was sufficiently promising to prompt further investigation (Franz and Barker: to be published). One new factor introduced was the use of abdominal skin in vitro and forearm skin in vivo , since differences in the permeability of skin from different regions of the body have previously been reported (6, 9). Also, the use by Feldmann and Malbach (7) of only a 5-day urine collection period might be questioned, especially since in only two of the twelve cases studied was there no radioactivity in the urine on day 5. Indeed, for nicotinic acid, more radioactivity was present in the urine on day 5 than in all the first four days taken together. It is conceivable that, for some compounds, excretion from the body may be rate-limiting because of slow metabolism , binding, or distribution to a 'deep compartment' (12). Thus, a 5-day collection period might be inadequate and lead to significant underestimation of the actual amount absorbed . In view of these potential sources of error in the investigations just described, new data have been obtained on four compounds (hippuric acid, nicotinic acid, thiourea, and caffeine). All four are compounds for which there was previously poor agreement between the in vitro and in vivo findings (table I). In both the in vitro and in vivo tests , abdominal skin with acetone as vehicle was used. Urine was collected in the in vivo experiments until background levels of radioactivity were approached. Early urine samples were taken so that the rate curve data would be as complete as possible. To minimize the differences which might be caused by desquamation in vivo , the surface of the skin was washed 24 h after application of the test compounds in both sets of experiments. The
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Further Studies
Franz
66
Table II. Total absorption of various compounds by skin in vivo and in vitro (modified tests) (expressed as percent of applied dose) Compound 1. 2. 3. 4.
Nicotinic Acid Hippuric Acid Thiourea Caffeine
Absorption in vivo'
T2
Absorption in vitro'
0.32 1.0 3.7 22.1
21 3 21 7
2.3 1.25 4.6 24.1
± ± ± ±
0.10 0.4 1.3 15.8
(3)
(6) (4) (4 )
± ± ±
±
0.9 0.5 2.3 7.8
(4) (4) (5) (4)
, Mean ± SD. The values in brackets represent the number of subjects studied. 2 Number of days urine was collected.
Caffeine
16
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procedure consisted of two acetone washes, then two water washes, followed by a further two acetone washes. Each wash lasted only 20-30 seconds. Data are presented in table II. With the exception of nicotinic acid, quantitative agreement between the two sets of experiments was excellent, with regard to both total absorption and rates of absorption. As can be seen with caffeine (fig. 5), agreement over the whole time course is quite good. The lack of agreement between the in vivo and in vitro data for nicotinic acid is similar to that observed in the previous study. This discrepancy was resolved by studies on its rate of excretion (fig. 6) which showed that less than 15% of the radioactivity from an intravenously administered dose of 14C-nicotinic acid was excreted in the urine, and at a very slow rate (up to 8 weeks before background levels were reached). Thus, the value of 0.32% absorption observed in vivo in humans, when corrected for incomplete urinary excretion, becomes 2.1 %, in better agreement with the value observed in the model. This also explains discrepancies in the original study of Feldmann and Maibach. Although they corrected for incomplete urinary excretion, not all correc-
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Fig. 5. Comparison of the rate or absorption (1) determined both in vitro and in vivo for caffeine. The surface of the skin was washed at 24 h in both cases.
Percutaneous Absorption: in vitro Model
67
10
.
I
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Fig. 6. Semi-logarithmic plot of the rate of excretion of nico tinic acid and caffeine following intravenous injection in rhesus monkeys.
tions were based on direct measurements of excretion of the compound in question after its i.v. administration. The extent of urinary excretion for some compounds, including nicotinic acid, was assessed on the basis of the chemical structure of the molecule. Nicotinic acid was assumed to behave like salicylic acid , a compound in which 90% of the radioactivity is excreted in the urine in less than 2 days. In light of our later study it can be concluded that the finite dose technique is a relevant in vitro model for the study of percutaneous absorption. Based on data obtained from the compounds studied to date, it appears that the model accurately portrays the phenomenon of absorption as it occurs in living man.
An in vitro model of percutaneous absorption has been developed which permits close simulation of conditions commonly associated with topical drug use in living man. Quantitative comparison of the absorption of selected compounds in th e model and in living man
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Summary
Franz
68
was made to test the validity of the model. Excellent agreement has been found between the two sets of data, both with respect to the total amount absorbed and the kinetics of absorption.
2
2 3 4
5
6 7 8 9 10
11
12
13 14
15 16
17 18
19
Baker, H and Kligman, A.M.: Measurement of transepidermalloss by electrical hygrometry. Archs Derm. 96: 441-452 (1967). Baker, H and Kligman, A.M: Technique for estimating turnover time of human stratum corneum. Archs Derm. 95: 408- 411 (1967). Bettley, F.R. and Grice, KA .: A method for measuring the transepidermal water loss and a means of inactivating sweat glands. Br. J. Derm. 77: 627 - 638 (1965). Blank, I.H: Factors which influence the water content of the stratum corneum. J. invest. Derm. 18: 443 - 440 (1952). Bradley, J. V: Distribution-free statistical tests. pp. 91 - 96 (Pren tice-Hall, New York 1968). Cronin, E. and Stoughton, R.B.: Percutaneous absorption. Regional variations and the effect of hydration and ep1dermal stripping. Br. J. Derm. 74: 265 - 272 (1962). Feldmann, RJ. and Maibach, HI.: Absorption of some organic compounds through the skin in man. 1. invest. Derm. 54: 399-404 (1970). Feldmann , RJ. and Maibach, HI.: Penetration of 14C-hydrocortisone through normal skin. Archs Derm. 91: 661 - 666 (1965). Feldmann, RJ. and Maibach, HI.: Regional variation in percutaneous penetration 14C cortisol in man. J. invest. Derm. 48: 181 - 183 (1967). Franz, T.J.: Percutaneous absorption on the relevance of in vitro data. J. invest. Derm. 64: 190-195 (1975). Fritsch, WC and Stoughton , R.B.: The effect of temperature and humidity on the penetration of C 14 acetylsalicylic acid in excised human skin. J. invest. Derm. 41: 307-311 (1963). Garrett, E.R.: Classical pharmacokinetics to the frontier. J. Pharmacokin. Biopharm. 1: 341 - 361 (1973). Maibach, HI.: In vivo percutaneous penetration of corticoids in man and unresolved problems in their efficacy. Dermatologica 152: (suppl.) 11 - 25 (1976). Maili, J. WH: The transport of water through the human epidermis. 1. invest. Derm. 27: 451-469 (1956). Onken, HD. and Moyer, CA .: The water barrier of human epidermis. Archs Derm. 87: 584 - 590 (1963). Ostrenga, J.; Steinmetz, C, and Poulsen, B.: Significance of vehicle composition I: Relationship between topical vehicle composition, skin penetrability, and clinical efficacy. J. pharm. Sci. 60: 1175-1179 (1971). Rosenberg, E. W.; Blank, H, and Resnik, S.: Sweating and water loss through the skin. J. Am. med. Ass. 179: 809-811 (1962). Rothberg, 8.; Crounse, R.C, and Lee, J.L.: Glycine-C 14 incorpora tion into the proteins of normal stratum corneum and the abnormal stratum corneum of psoriasis. 1. invest. Derm. 37: 497-506 (1961). Scheuplein, RJ. and Blank, I.H: Permeability of the skin. Physiol. Rev. 51: 702-747 (1971).
Dr. T.J. Franz, University of Washington , School of Medicine, Seattle, WA 98105 (USA)
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References
The Finite Dose Technique as a Valid in vitro Model for the Study of Percutaneous Absorption in Man Thomas J. Franz University of Washington, School of Medicine, Seattle, Wash.
Introduction The technique most frequently employed for the measurement of percutaneous absorption is the in vitro use of excised human skin mounted in diffusion chambers. Although results obtained from this model have contributed significantly to the formulation of current dogma, there is surprisingly little data available to support the belief that in vitro absorption is a totally and consistently accurate reflection of absorption in living humans. The relevance of in vitro absorption data has largely been assumed, and rests primarily on knowledge that the stratum corneum is the rate-limiting barrier of the skin (I9). It has been argued that since the stratum corneum is a 'dead', non-metabolizing structure it should function equally well in vitro as in vivo; but, aside from the one rather special situation of transepidermal water loss, this assumption has not been critically evaluated (1, 3, 4, 14, 15, 17). Recently, we have begun to examine in our laboratory the extent to which in vitro absorption can be used as a valid predictive model of absorption in living humans (10). Review of the literature revealed that many prior in vitro studies had not employed conditions generally applicable to those found in vivo in man; thus, an essential part of the work to be described has been the development of a model which attempts to approximate these conditions. Examination of the more conventional approach will help illustrate the problem.
Historically, the most frequently used in vitro method for studying absorption is the infinite dose technique (fig. 1a). The skin is mounted as a barrier between two well-stirred, fluid-filled chambers. The compound under study is
Downloaded by: Weizmann Inst. of Science 198.143.58.1 - 4/11/2016 4:54:43 PM
Infinite Dose Technique
59
Percutaneous Absorption: in vitro Model Infinite dose
ro~u~t___HL=____~~;.~_____~=__~I~n
, a
2
. .,
. • r··;·
L -_ __ _ _ __ _ _ _~~~----------~
T
c
added to the solution on one side (donor) of the membrane and absorption assessed by serially sampling and assaying its concentration in the bathing solution (receptor) on the other side. Generally, the amount of solute that penetrates during the course of an experiment is small relative to the total amount available and there is no appreciable reduction in solute concentration in the donor solution. In effect, it appears to be 'infinite '. Measurements are commonly taken of the total quantity of solute that has crossed the membrane at any time (Qt), and plotted as a function of time (fig.lb). The time course of absorption can be divided into three phases: (1) a lag phase, characterized by solute movement through the membrane but during which no molecules appear in the receptor solution; (2) a nonlinear phase, during which the solute concentration in the receptor solution begins to rise , slowly at first, but at an increasing rate; and finally (3) a linear phase, during which the rate of increase in solute concentration in the receptor solution is constant. The rate of absorption or flux (1) is constant at its maximum value and steady-state conditions prevail. J can be calculated directly from the slope of the line (6Q/6t) under these steady-state conditions. Alternatively, the flux can be plotted as a function of time (fig. Ic) ; the lag phase and rise to steady-state conditions are again evident. A number of objections can be raised to the use of the infinite dose technique as a predictive model for living man. The most obvious, and certainly that of greatest significance, is the use of fully hydrated skin ; both sides of the preparation (be it isolated stratum corneum, epidermis, or full thickness skin) are bathed by aqueous solution. Among biological barriers, the skin is unique in
Downloaded by: Weizmann Inst. of Science 198.143.58.1 - 4/11/2016 4:54:43 PM
Fig. 1. The infinite dose technique. (a) Schematic diagram of the skin mou nted as a barrier between two chambers. Data can be expressed as either: (b) total absorption (Q) vs. time, or (c) rate of absorption (1) vs. time.
Franz
60
that it normally functions with one surface exposed to a dry environment. It is well known that hydration drastically alters the function of the stratum corneum and induces a 5-1 O-fold increase in permeability (8, 11). Equally blatant is the fact that most compounds do not come into contact with skin in the form of aqueous solutions. Water is not a common vehicle. Yet there is ample evidence that the absorption of topically applied drugs is directly dependent upon the composition of the vehicle (13, 16). The permeability of skin to any given compound cannot be specified without knowing the vehicle in which it is applied, and the routine use of water, therefore, makes the model of limited relevance. A less obvious objection is the size of the applied dose - both vehicle and drug. Under clinical or 'use' conditions, the amounts of material applied are of the order of a few milligrams (of vehicle) per square centimeter of skin. In comparison, in vitro methods which utilize an aqueous solution (or other solvent) in contact with the skin are, in effect, applying gram per square centimeter quantities of vehicle (water or solvent, in this case). The dose of active drug in these circumstances may also be excessive due to the large (vis-a-vis infinite) amount of vehicle applied (dose = drug concentration x mass or volume of vehicle). The importance of these deviations from conditions normally associated with topical drug use will become apparent when the kinetics of absorption are discussed.
To avoid the shortcomings of the previously described approach, the following technique has been developed. Human abdominal skin obtained at autopsy is mounted in specially constructed diffusion chambers (fig. 2a). The dermis is bathed by isotonic saline, pH 7.4, and the temperature maintained at 37°C by thermostatically controlled water which is circulated through a jacket surrounding the lower chamber. Homogenous temperature distribution in the dermal bathing solution is achieved by a small teflon-covered magnetic stirring-bar, driven by an external magnet mounted on a 600 rpm timing motor. The epidermis is exposed to the ambient conditions of the laboratory environment, so that the skin is held under conditions which closely approximate the living state. The gradients of water and temperature which normally exist across the skin are both maintained in this model. Furthermore, this ar· rangement allows for the application of any type or any amount of vehicle, so that the absorption of individual compounds can be studied in the form and dose most appropriate to their actual use. Usually, 5-10 pi of a liquid vehicle containing the test compound is applied per square centimeter of skin, using a micropipette; otherwise, 2-5 mg of a non-liquid vehicle containing the test compound is applied per square centimeter of skin, using a small stirring rod. The
Downloaded by: Weizmann Inst. of Science 198.143.58.1 - 4/11/2016 4:54:43 PM
Finite Dose Technique
61
Percutaneous Absorption: in vitro Model Finite dose
out
I I
a
I I
1::~:3 :I I I
I
b
T
Fig. 2. The finite dose technique. (a) Schematic diagram of the skin bathed by isotonic saline below and covered by a thin 'finite' dose on its outer surface. (b) Rate of absorption (J) vs. time.
material can be thoroughly rubbed in using the smooth, rounded tip of the stirring rod and the exact amount applied determined by weighing the rod before and after use. Thus, the small finite doses typically associated with the clinical use of dermatologic preparations are easily duplicated. Use of a finite dose alters the rate of absorption curve from that observed in the previously described model. No steady-state is achieved (compare figs. 2b and Ic). Following a lag phase, the flux rises to a peak, then falls. The rising portion of the curve is similar to that seen with an infinite dose and represents the approach to a steady state. However, as absorption proceeds, the concentration of drug on the surface of the skin falls, eventually to a point where the rate of absorption can no longer be sustained. At this point the flux also begins to fall and will continue to do so until the source is exhausted. This difference illustrates the objection to the use of the infinite dose technique alluded to earlier. On theoretical grounds alone, it seems clear that failure to approximate the amount of drug used in vivo will yield results which bear little resemblance to those found in living man. It is a serious error because it represents a failure to properly characterize the time-course of absorption.
The test of any in vitro model is its ability to simulate the in vivo system. To test the validity of the technique proposed here, use has been made of a study of percutaneous absorption in humans by Feldmann and Maibach (7), who measured the absorption of a series of organic compounds in volunteers, using radioisotopes. Their technique consisted of applying I pCi of a 14C-Iabeled compound dissolved in acetone at a dose of 4pg/cm 2 to the skin of the forearm, and thereafter assaying total urinary output over 5 days for radioactive content.
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Assessing the Validity of the Finite Dose Technique
Franz
62
Table I. Total absorption of various compounds by skin in vivo and in vitro (expressed as perc en t of applied dose)
Compound 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Hippuric Acid Nicotinic Acid Thiourea Chloramphenicol Phenol Urea Nicotinamide Acetylsalicylic Acid Salicylic Acid Benzoic Acid Caffeine Dinitrochlorobenzene
Absorption in vivol 0.2 ± 0.1 0.3 ± 0.1 0.9 ± 0.2 2.0 ± 2.5 4.4 ± 2.4 6.0 ± 1.9 11.1 ± 6.2 21.8± 3.1 22.8±13.2 42.6 ± 16.5 47.6 ± 21.0 53.1 ± 12.4
(7) (3) (3)
(6) (3)
(4) (7) (3) (17) (6) (12) (4)
Absorption in vitro' 1.2 [0.8, 2.7] 3.3 [0.7, 8.3] 3.4 [2.4,5.5] 2.9 [1.0,5.7] 10.9 [7.7, 26] 11.1 [5.2, 29] 28.8 [16, 65] 40.5[17, 49] 12.0 [2.3, 23] 44.9 [29, 53] 9.0 [5.5, 20] 27.5 [19, 33]
(15) (19) (52) (12) (7)
(22) (21 ) (14) (10) (18) (17) (18)
I Mean ± SD. , Median with 95% confidence interval given in square brackets. The values in brackets represent the number of subjects studied.
Since only urinary excretion was measured, it was necessary to correct for labeled compound which was absorbed but not excreted by the kidneys by measuring the fraction of an intravenously administered dose of the same compound which was excreted in the urine. Twelve of the compounds studied by Feldmann and Maibach were selected by us for study in vitro (see table I) so that a critical evaluation of the relevance of the finite dose technique to absorption in living man could be made (10). The same vehicles and topical doses as used in vivo were used in vitro. Comparison of the data obtained from the two studies was made on the basis of two parameters: total absorption (% of dose) and the rate of absorption (% of dose/h).
In figure 3, the time-course of absorption as determined both in vitro and in vivo is shown for three representative compounds. With eight of the twelve compounds studied (acetylsalicylic acid, benzoic acid, caffeine, dinitrochlorobenzene, hippuric acid, phenol, salicylic acid and nicotinamide) the rate of absorption was characterized by the appearance of a distinct maximum (Jmax),
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Rate of Absorption
63
Percutaneous Absorption : in vitro Model
.
ASA
10
30
24
48
72
ONeB • In vItro
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, In VIVO
72
96 Hours
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004 J
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20
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40
50
60
70
80
90
100 110
Hours
as was expected from theoretical considerations. Comparison of the curves obtained in the in vitro experiments with those obtained in living man yielded excellent overall agreement, including the time at which J max occurred. Representative data for acetylsalicylic acid and dinitrochlorobenzene are presented in figure 3. Comparison of the rate of absorption profiles from the two sets of experiments is necessarily limited, in that insufficient data points were collected in vivo, particularly in the initial period of absorption. The remaining four compounds (chloramphenicol, nicotinic acid, thiourea, urea) showed an absorption pattern which displayed a tendency toward steadystate conditions and a low absorption rate. The in vitro and in vivo absorption curves had a similar overall pattern, but significant differences in the magnitude of the absorption rate were observed with all four compounds. For example, see nicotinic acid, fig. 3. The apparent steady-state in this case in fact only reflects the very low rates of absorption obtained with these compounds. Even with a finite dose, absorption proceeds so slowly that little reduction in the concentration of tracer on the skin surface occurs during the course of the experiment. When the duration of the experiment is extended to 1- 2 weeks this pseudo steady-state is revealed to be a very broad low peak, spanning several days.
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Fig. 3. Comparison of the rate of absorption (1) determined both in vitro and in vivo for acetylsalicylic acid (ASA), dinitrochlorobenzene (DNCB) , and nicotinic acid.
64
Franz Caffeine
• 24hr wash '" 48hrwash o no wash
08 06
10
20
30
40
50
60
70
80
Hours
Fig. 4. The effect of a brief wash ('V 1 min) of the outer surface of the skin at 24 or 48 h on the rate of absorption of caffeine. Three rinses consisting of acetone, then water, followed by acetone were utilized.
In addition to the absorption rate, total absorption can be taken as a parameter for comparing the in vitro model with in vivo conditions. An interesting question arises - how long should one follow absorption in an in vitro model? At least one crucial function of great importance to the process of absorption that cannot be duplicated in the model is desquamation of the epidermis. In living man, this process will result in a continuing reduction in the amount of material remaining on the skin following a single dose, even in the absence of significant permeation, whereas in the model, material will remain on the skin sample and continue to serve as a source for absorption. This is illustrated in figure 4; a very brief wash of the skin surface at 24 or 48 h results in significant reduction in the subsequent rate of absorption. Theoretically, one might expect to observe 100% absorption in vitro for most compounds over a sufficiently long time period. From data on the turnover and thickness of the stratum corneum, it can be calculated that there is an average loss of one cell layer per day (2, 18), making this factor potentially important in experiments lasting longer than 1 day. As a first approximation it seemed reasonable to select 2 days as the time limit for the calculation of total absorption in vitro, for three reasons. First, it is compatible with the simple model that if the entire surface layer is lost at the end of day 1, at least another day must be allowed for the absorption of material in transit through the deeper regions of the stratum corneum and thus not affected by events at the surface. This is illustrated in figure 4. Second, for the majority of the compounds studied (those displaying a distinct J max ) most absorption takes place in the first 2 days and that which takes place subsequently can be essentially neglected in computing totai absorption. It should be noted, however, that
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Total Absorption
Percutaneous Absorption: in vitro Model
65
this latter argument does not hold for the four compounds displaying pseudo steady-state kinetics. Third, it makes sense to use a shorter time for the calculation of total absorption in vitro than in vivo, because of the possibility of delayed excretion in vivo , due to drug binding or slow metabolism in the body. Data for total in vitro (2 day) and in vivo (5 day) absorption are presented in table I. There is reasonable agreement between the two sets of data with regard to compounds that are well absorbed (i.e. greater than 10% absorption) , but not for compounds that are poorly absorbed. For hippuric acid, nicotinic acid and thiourea, the in vitro values are 4-1O-fold higher than those obtained in vivo. There is, however, a trend toward positive correlation. Compounds with high absorption rates can be distinguished by either set of data from those with low absorption rates and the rank order of rates found in the in vitro experiments is similar to that found in vivo. Statistical evaluation of the data using Spearman's test for rank order correlation (5) showed high significance (p = 0.01); the correlation coefficient was 0.734.
This first attempt to evaluate the relevance of the finite dose model was sufficiently promising to prompt further investigation (Franz and Barker: to be published). One new factor introduced was the use of abdominal skin in vitro and forearm skin in vivo , since differences in the permeability of skin from different regions of the body have previously been reported (6, 9). Also, the use by Feldmann and Malbach (7) of only a 5-day urine collection period might be questioned, especially since in only two of the twelve cases studied was there no radioactivity in the urine on day 5. Indeed, for nicotinic acid, more radioactivity was present in the urine on day 5 than in all the first four days taken together. It is conceivable that, for some compounds, excretion from the body may be rate-limiting because of slow metabolism , binding, or distribution to a 'deep compartment' (12). Thus, a 5-day collection period might be inadequate and lead to significant underestimation of the actual amount absorbed . In view of these potential sources of error in the investigations just described, new data have been obtained on four compounds (hippuric acid, nicotinic acid, thiourea, and caffeine). All four are compounds for which there was previously poor agreement between the in vitro and in vivo findings (table I). In both the in vitro and in vivo tests , abdominal skin with acetone as vehicle was used. Urine was collected in the in vivo experiments until background levels of radioactivity were approached. Early urine samples were taken so that the rate curve data would be as complete as possible. To minimize the differences which might be caused by desquamation in vivo , the surface of the skin was washed 24 h after application of the test compounds in both sets of experiments. The
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Further Studies
Franz
66
Table II. Total absorption of various compounds by skin in vivo and in vitro (modified tests) (expressed as percent of applied dose) Compound 1. 2. 3. 4.
Nicotinic Acid Hippuric Acid Thiourea Caffeine
Absorption in vivo'
T2
Absorption in vitro'
0.32 1.0 3.7 22.1
21 3 21 7
2.3 1.25 4.6 24.1
± ± ± ±
0.10 0.4 1.3 15.8
(3)
(6) (4) (4 )
± ± ±
±
0.9 0.5 2.3 7.8
(4) (4) (5) (4)
, Mean ± SD. The values in brackets represent the number of subjects studied. 2 Number of days urine was collected.
Caffeine
16
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11
12 08 04
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~
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10
20
30
40
50
60
70
80
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procedure consisted of two acetone washes, then two water washes, followed by a further two acetone washes. Each wash lasted only 20-30 seconds. Data are presented in table II. With the exception of nicotinic acid, quantitative agreement between the two sets of experiments was excellent, with regard to both total absorption and rates of absorption. As can be seen with caffeine (fig. 5), agreement over the whole time course is quite good. The lack of agreement between the in vivo and in vitro data for nicotinic acid is similar to that observed in the previous study. This discrepancy was resolved by studies on its rate of excretion (fig. 6) which showed that less than 15% of the radioactivity from an intravenously administered dose of 14C-nicotinic acid was excreted in the urine, and at a very slow rate (up to 8 weeks before background levels were reached). Thus, the value of 0.32% absorption observed in vivo in humans, when corrected for incomplete urinary excretion, becomes 2.1 %, in better agreement with the value observed in the model. This also explains discrepancies in the original study of Feldmann and Maibach. Although they corrected for incomplete urinary excretion, not all correc-
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Fig. 5. Comparison of the rate or absorption (1) determined both in vitro and in vivo for caffeine. The surface of the skin was washed at 24 h in both cases.
Percutaneous Absorption: in vitro Model
67
10
.
I
\
\
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... Nicoti niC a Cid
•I
,
• Caffeine
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Fig. 6. Semi-logarithmic plot of the rate of excretion of nico tinic acid and caffeine following intravenous injection in rhesus monkeys.
tions were based on direct measurements of excretion of the compound in question after its i.v. administration. The extent of urinary excretion for some compounds, including nicotinic acid, was assessed on the basis of the chemical structure of the molecule. Nicotinic acid was assumed to behave like salicylic acid , a compound in which 90% of the radioactivity is excreted in the urine in less than 2 days. In light of our later study it can be concluded that the finite dose technique is a relevant in vitro model for the study of percutaneous absorption. Based on data obtained from the compounds studied to date, it appears that the model accurately portrays the phenomenon of absorption as it occurs in living man.
An in vitro model of percutaneous absorption has been developed which permits close simulation of conditions commonly associated with topical drug use in living man. Quantitative comparison of the absorption of selected compounds in th e model and in living man
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Summary
Franz
68
was made to test the validity of the model. Excellent agreement has been found between the two sets of data, both with respect to the total amount absorbed and the kinetics of absorption.
2
2 3 4
5
6 7 8 9 10
11
12
13 14
15 16
17 18
19
Baker, H and Kligman, A.M.: Measurement of transepidermalloss by electrical hygrometry. Archs Derm. 96: 441-452 (1967). Baker, H and Kligman, A.M: Technique for estimating turnover time of human stratum corneum. Archs Derm. 95: 408- 411 (1967). Bettley, F.R. and Grice, KA .: A method for measuring the transepidermal water loss and a means of inactivating sweat glands. Br. J. Derm. 77: 627 - 638 (1965). Blank, I.H: Factors which influence the water content of the stratum corneum. J. invest. Derm. 18: 443 - 440 (1952). Bradley, J. V: Distribution-free statistical tests. pp. 91 - 96 (Pren tice-Hall, New York 1968). Cronin, E. and Stoughton, R.B.: Percutaneous absorption. Regional variations and the effect of hydration and ep1dermal stripping. Br. J. Derm. 74: 265 - 272 (1962). Feldmann, RJ. and Maibach, HI.: Absorption of some organic compounds through the skin in man. 1. invest. Derm. 54: 399-404 (1970). Feldmann , RJ. and Maibach, HI.: Penetration of 14C-hydrocortisone through normal skin. Archs Derm. 91: 661 - 666 (1965). Feldmann, RJ. and Maibach, HI.: Regional variation in percutaneous penetration 14C cortisol in man. J. invest. Derm. 48: 181 - 183 (1967). Franz, T.J.: Percutaneous absorption on the relevance of in vitro data. J. invest. Derm. 64: 190-195 (1975). Fritsch, WC and Stoughton , R.B.: The effect of temperature and humidity on the penetration of C 14 acetylsalicylic acid in excised human skin. J. invest. Derm. 41: 307-311 (1963). Garrett, E.R.: Classical pharmacokinetics to the frontier. J. Pharmacokin. Biopharm. 1: 341 - 361 (1973). Maibach, HI.: In vivo percutaneous penetration of corticoids in man and unresolved problems in their efficacy. Dermatologica 152: (suppl.) 11 - 25 (1976). Maili, J. WH: The transport of water through the human epidermis. 1. invest. Derm. 27: 451-469 (1956). Onken, HD. and Moyer, CA .: The water barrier of human epidermis. Archs Derm. 87: 584 - 590 (1963). Ostrenga, J.; Steinmetz, C, and Poulsen, B.: Significance of vehicle composition I: Relationship between topical vehicle composition, skin penetrability, and clinical efficacy. J. pharm. Sci. 60: 1175-1179 (1971). Rosenberg, E. W.; Blank, H, and Resnik, S.: Sweating and water loss through the skin. J. Am. med. Ass. 179: 809-811 (1962). Rothberg, 8.; Crounse, R.C, and Lee, J.L.: Glycine-C 14 incorpora tion into the proteins of normal stratum corneum and the abnormal stratum corneum of psoriasis. 1. invest. Derm. 37: 497-506 (1961). Scheuplein, RJ. and Blank, I.H: Permeability of the skin. Physiol. Rev. 51: 702-747 (1971).
Dr. T.J. Franz, University of Washington , School of Medicine, Seattle, WA 98105 (USA)
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References
