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The oral mucosal absorption and tissue distribution of triamcinolone acetonide in the dog studied by autoradiography
Arch\ oral Bid. Vol. 25. pp 809 lo 817 Pergamon Press Ltd 1980 Prmted m Great Brttam
THE ORAL MUCOSAL ABSORPTION AND TISSUE DISTRIBUTION OF TRIAMCINOLONE ACETONIDE IN THE DOG STUDIED BY AUTORADIOGRAPHY M. ADDS Department of Periodontology,
Dental School, Welsh National School of Medicine, Cardiff. South Wales
Summary-Triamcinolone acetonide prepared (5 mg/ml and 0.5 mg/ml) in propylene glycol and (1 mg/g) in Orabase to which [3H]-triamcinolone acetonide was added was placed on the oral mucosa in plastic wells and biopsies taken at various periods after application, together with biopsies taken after a delay of 2 or 3 h following removal of applied samples. Biopsies were processed for serial sectioning and extraction or for autoradiography. There was a trend for the tissue concentration of triamcinolone acetonide to increase with time which was significant for the 5 mg/ml samples. Both serial sectioning and autoradiographic techniques showed the absorbed steroid to he mainly within the epithelium and connective tissue immediately below the basement membrane. The total steroid in biopsies was significantly greater in the 5 mg/ml samples than in the 0.5 mg/ml or oral ointment samples. Progressive loss of steroid from the tissues following removal of the applied samples was apparent from biopsies taken 2 and 3 h after removal applied samples. - _
or
INTRODUCTION
Several of the synthetic steroids initially developed for use on the skin have been employed topically in the management of oral conditions (Cooke, 1960; Cawson, 1968; Yeoman, Greenspan and Harding, 1978). Although topical steroids have been studied quite extensively. interest has been almost entirely concerned with clinical effects. As with most topical drugs used in the mouth, little is known of the fate of the steroids once introduced into the mouth. In particular, the pattern of absorption and local tissue distribution has not been investigated. The many studies into the mucosal absorption of drugs (reviewed by Katz and Barr, 1955; Gibaldi and Kanig, 1965) including steroids (Anderson, Haymaker and Henderson, 1940; Turnoff and Rowntree, 1941; Spence, 1942) were concerned with systemic rather than local effects, or distribution. Similarly, few investigations (Scott and Katz, 1977; Kammerau, Zesch and Schaeffer, 1975; Schaeffer, Zesch and Stuttgen, 1977) of topical application to the skin have been concerned with the demonstration of local tissue concentrations, although the pertinence of such information to practical therapy has been stated (Schaeffer et al., 1977). The stratum corneum appears to be the major barrier to steroid penetration of skin (Marzulli, 1962; Scheuplein et al., 1969). Steroid absorption from the mouth may therefore not be the same as from the skin because of the lack of keratinized epithehum over much of the human mouth. Furthermore, the hydration of the epithehal lining as a result of constant bathing in saliva may accentuate absorption, as occurs with hydrated skin (Harris, Papa and Stanton, 1974) although the possible protective role of saliva cannot be discounted (Wallenius, 1966; Adams, 1974). The fate of drugs used topically in the mouth is suitable for study. My purpose was to assess the absorption and local tissue distribution of the steroid 809
triamcinolone in canine buccal mucosa employing a radiolabelled tracer. This steroid was chosen because it has been employed for the management of several acute and chronic diseases of the oral mucosa in man (Zegarelli K? al., 1960) and is normally applied in an ointment base (Rothner et al., 1949) which may be retained at certain oral sites for several hours (Kutscher et al., 1959).
MATERIALS AND METHODS
Animal model and anaesthetic technique Twelve beagle dogs aged 7-9 yr and a mean weight of 16.6 kg (range 13.2-19.5) were used. One hour prior to each experimental procedure the animals were sedated with 250 mg of diethylthiambutane hydrochloride (Themalon, Burroughs Wellcome & Co., London) injected subcutaneously into the back of the neck. This was followed immediately before commencing the experiment by slow intravenous injection of 24@300mg of pentobarbitone sodium (Sagatal, May and Baker Ltd, Dagenman, Essex) into a foreleg vein. The preparation of radiolabelled triamcinolone ucetonide in propylene glycol and Orabase Concentrations of 0.5 mg and 5 mg/ml of triamcinolone acetonide (Lederle Laboratories Cyanamid of Great Britain Ltd, Gosport, Hampshire) were prepared in propylene glycol and 1 mg/gm in a plasticised hydrocarbon gel containing hydrophilic solids (Orabase, E.R.S. Squibb & Sons Ltd, Liverpool and London). Tritium-labelled triamcinolone acetonide (The Radiochemical Centre, Amersham, Buckinghamshire) of specific activity 23 Ci/mmol (53 mCi/mg) was added to the propylene glycol steroid solutions in two concentrations of 10 and 20 &i/ml. These two initial
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M. Addy
concentrations were prepared by the addition of suitable volumes of the 1 mCi/ml labelled material. For the Orabase/steroid mixture tritium-labelled triamcinolone was added at 1&i/g. The resulting mean radioactivity in counts per minute minus background of each solution was determined by placing 0.1 ml samples into 19.9 ml of ethyl acetate. Volumes of 0.1 ml from each of these dilutions were placed into scintillant vials, evaporated to dryness and methanol (2 ml) and scintillant (IO ml) added. Scintillation counting was carried out in an Intertechnique SL.30 automatic p-counter for two IOmin periods. Background samples were similarly prepared for propylene glycol without triamcinolone acetonide. The mean radioactivity in counts per minute minus background of the Orabase mixture was determined by extracting known weights of steroid-containing Orabase with ethyl acetate. Suitable volumes for counting were then evaporated in scintillant vials and methanol and scintillant fluid added, as before. Background counts were determined from the extraction and processing of steroid-free Orabase. Drug applicution atld biopsy techtCyue The steroid preparations were applied to the buccal mucosa of the upper cheek folds in plastic wells. The wells (polystryrene) were cylindrical of 6 mm internal diameter and 5mm depth. The mucosa was dried with cotton wool before placing the wells and a watertight seal obtained using Orabase around the base of each well. The lips and cheeks were separated and the mucosa exposed in a horizontal plane using silk sutures through the skin-lip mucosal junction and tied at the side of the operating table. Mucosal biopsies of the application sites were taken at the designated time intervals for the respective experiments The well, Orabase seal and residual contents were lifted off each site with college tweezers and the mucosal surface wiped twice with cotton wool soaked in distilled water. The biopsy of the whole thickness of the mucosa was then taken to include the whole area covered by the external diameter of the well, together with a small margin of uncovered tissue. The resulting tissue specimens, approximately 10 mm in diameter, were placed epithelial surface down on a piece of card, wrapped in tin foil, frozen in liquid nitrogen and stored at - 70°C until required. Serial section and tissue extraction Samples (0.1 ml) of [3H]-labelled triamcinolone acetonide (0.5 mg/ml and 5 mg/ml with ZO&i/ml) were placed into separate wells. At intervals of 60. I20 and 150 min after application, biopsies were taken. For some I20 min application sites, following removal of the samples, biopsy was delayed for 2 or 3 h. For the Orabase mixture (I mg/g, 1 &i/g), a known weight of the material was placed in the wells and then applied directly to the mucosa. Biopsies were then obtained at 150 min. The biopsies were processed for scintillation counting using the Glick (1961) serial-sectioning technique to determine the tissue distribution of the steroid. Thus serial cryostat sections IOpm thick were cut parallel to the epithelial surface throughout the tissue. Every successive IO sections were placed into ethyl acetate (4 ml) in a scintillant vial for extraction of
contained triamcinolone acetonide. For some biopsies the section following each IO0pm series was stained with haemotoxylin and eosin and used to correlate the appearance of epithelium in the sections with radioactivity counts. After 24 h the ethyl acetate was evaporated and scintillation fluid (IO ml) added together with methanol (2 ml). The methanol removed the cloudiness in the final mixture, arising from residual moisture, observed in preliminary experiments. The radioactivity in counts/min minus background counts for the samples, was then determined, using a /I-counter. Background counts were measured using vials containing serial sections taken from three sham biopsies treated as described. From the counts previously obtained for the applied samples, the concentrations of the steroid in the sections of tissue were calculated. The extraction period of 24 h was chosen arbitrarily; however, extraction appeared to be complete because counts above background were not obtained on extracting and processing the sections on a second occasion. Autoradiography
experiments
Samples (0.1 ml) of triamcinolone acetonide in propylene glycol (0.5, 5 mg/ml with IO&i/ml) were placed in wells. After 1 and 3 h, biopsies were taken from the centre of the site of well application, snapfrozen and stored as before. Some 5 mg/ml samples were maintained on the mucosa for 2 h and, after removal, biopsy was delayed for 2 and 3 h. Serial cryostat sections 7pm thick were cut to pass through the depth of the tissue and across the width of the specimen For each biopsy, a total of I2 sections, approximately one section per 30 cut, were collected on glass slides. Sections were allowed to dry in air and then dipped in autoradiographic emulsion (Ilford K5 gel form, Ilford Ltd, Ilford, Essex) with equal parts distilled water using a 3 s dip and drain procedure. After drying, the sections were placed in a light-proof box and stored in a refrigerator. After 3 weeks. a time previously determined by removing sections at 7 day intervals until the most acceptable contrast for light microscopy was attained, the sections were removed from the box and developed in Kodak D.l9B for 4min and fixed in Kodafix (Kodak). Finally, the sections were lightly counterstained with haematoxylin. Sections from sham biopsies were similarly processed. All biopsies taken at one time for one concentration of applied steroid were sectioned and processed together. The slides were then coded and viewed blind at x 400 magnification with a 10 x 10mm graticule, having I mm square divisions, in one of the eye pieces of a binocular visible-light microscope. The number of autoradiographic grains contained in each 1mm square division of the graticule was counted, counts being made perpendicular to the basement membrane through the epithelium and through the connective tissue. The counts were made at several sites of each section; selection was arbitrary except to avoid dermal papillae. A minimum of 30 counts were recorded from the sections for each biopsy. Background counts per graticule-square were taken from each slide at sites 3-5 graticule-squares above the epithelial surface. The mean of 10 counts for each slide was subtracted from the counts per graticule-square obtained
Mucosal absorption of triamcinolone acetonide
811
Table 1. Mean triamcinoline acetonide levels in 1OOpm serial sections of biopsies from the buccal mucosa applied in propylene glycol and Orabase (results expressed in ng, SD in parentheses) Propylene glycol concentration 5 mg/ml Time after application Number of biopsies
Orabase concentration 0.1%
0.5 mg/ml
60 min
120 min
150 min
60 min
120 min
10
4
7
4
5
150 min
501 (369) 537 (249) 116 (76) 89 (54) 59 (32) 49 (29) 40 (30) 31 (26) 21 (15) 18 (12) 15 (11) 13 (10) 9 (6) 7 (5) 6 (3) 5 (5) 5 (3) 4 (2) 4 (4) 4 (3) 2 (2) 2 (1) 1376 (562)
1563 (839) 706 (803) 457 (525) 236 (273) 125 (107) 82 (50) 66 (40) 47 (41) 43 (18) 41 (19) 37 (18) 31 (15) 26 (13) 24 (11) 22 (9) 20 (9) 18 (7) 18 (6) 16 (5) 11 (9) 10 (8) 7 (8) 3633 (2664)
1750 (1435) 941 (1028) 286 (217) 346 (384) 333 (488) 113 (47) 88 (32) 83 (33) 73 (28) 66 (26) 59 (26) 53 (32) 47 (22) 48 (36) 39 (20) 38 (17) 27 (8) 24 (8) 22 (7) 18 (6) 13 (7) 15 (10) 4527 (1971)
79 (59) 38 (44) 11 (11) 3 (4) 5 (4) 4 (3) 4 (3) 3 (2) 4 (3) 4 (3) 4 (3) 4 (4) 5 (5) 3 (4) 3 (4) 4 (5) 3 (4) 3 (3) 3 (5) 2 (3) 1 (2) 1 (1) 206.(96)
4
2 -
Epithelial surface 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Mean
150 min
179 (149) 38 (38) 24 (24) 10 (6) 10 (7) 8 (6) 8 (8) 8 (8) 8 (6) 7 (7) 7 (6) 6 (5) 5 (3) 3 (2) 3 (3) 3 (1) 2 (1) 2 (1) 1 (1) 1 (1) 1 (1) 0 (0) 250 (87)
-
47 (29) 75 (24) 38 (10) 28 (14) 31 (1) 27 (3) 23 (5) 22 (10) 18 (11) 16 (11) 12 (9) 11 (10) 2 (2) 7 (6) g (7) 5 (4) 4 (2) 3 (2) 3 (1) 2 (1) 2 (1) 2 (1) 394 (33)
-.-
105 (117) 66 (53) 46 (33) 44 (27) 45 (25) 26 (7) 25 (8) 22 (16) 17 (3) 18 (3) 18 (4) 16 (2) 16 (3) 18 (8) 16 (2) 15 (2) 17 (2) 17 (3) 25 (16) 23 (11) 25 (17) 13 (9) 694 (253)
Mean weight Orabase (mg) = 373.7 (7.8).
from the tissue sections for that slide. Grain counts minus background were similarly obtained for sections from the sham biopsies. RESULTS
Sections of dog buccal mucosa showed a stratified squamous epithelium overlying loose connective tissue. Rete pegs and corresponding dermal papillae were apparent. Orthokeratinization of the epithelium was not observed; however, in most sections, a narrow zone of parakeratinization was present. The mean thickness of the epithelium measured by light graticule was microscopy using a millimetre 182 + 54pm. The absorption and tissue distribution of triamcinolone acetonide in the buccal mucosa from serial-section measurements
The stained sections taken from the 100~ interfaces revealed epithelium making up the bulk of the section areas at the 10&300 pm levels. The means and standard deviations of the levels of triamcinolone acetonide through the depth of the specimens for the different application times and modes of application are shown in Table 1. For the Orabase samples, the
amount of label and the mean weight applied was also recorded. For all biopsy times and concentrations of the applied steroid, there was a progressive fall, from the epithelial surface down, in levels of triamcinolone acetonide per 100 pm serial sections. The mean fall was most marked within the first 300~; a slow rate of fall was then apparent. From the histological measurements, the highest levels of steroid were present in the epithelium and immediate sub-epithelial layers. For the 5 mg/ml samples, a one-way analysis of variance showed a significant increase in total tissue steroid with time cf= 8.32, df = 20). This arose from a significant increase at 150 and 120 min compared with 60min (p < 0.01). No significant difference was apparent between 150 and 120 min (p = >0.05). A similar mean trend for an increase in total tissue steroid with time was observed for the 0.5mg/ml samples; however, the differences did not reach significance (f = 3.23, df = 10). Comparison of the effects of concentration on steroid levels obtained was determined using the Student’s r-test. At comparable application times, the mean total steroid content of the 5 mg/ml biopsies was highly significantly greater than of both the 0.5 mg/ml and 0.1 per cent Orabase biopsies (p < 0.001). No significant difference in total
M. Addy
812
Table 2. Mean triamcinolone acetonide levels in 100 pm serial sections taken 2 and 3 h after removal of the applied samples (results expressed in ng, SD in parentheses) Biopsy time after removal Number of biopsies Epithelial surface
Mean
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
2h 6
3h 4
175 (87) 147 (134) 27 (18) 14 (10) 8 (6) 6 (2) 4 (2) 4 (2) 2 (2) 2 (2) 2 (2) 2 (2) 2 (2) 2 (1) 2 (2) 1 (1) 3 (4) 1 (1) 2 (2) 1 (1) 3 (4) 2 (2) 423 (106)
284 (125) 45 (40) 47 (51) 40 (45) 20 (25) 22 (30) 23 (28) 18 (26) 23 (33) 22 (33) 35 (49) 9 (13) 4 (4) 3 (4) 2 (4) 2 (2) 2 (2) 1 (1) 0 (0) 0 (0) 0 (0) 0 (0) 532 (371)
steroid levels was observed between the 0.5 mg/ml and the 0.1 per cent Orabase biopsies (p > 0.05). The mean concentration of triamcinolone acetonide in 100 pm serial sections of tissue from biopsies taken 2 and 3 h after removal of the applied 5 mg/ml samples are shown in Table 2. The mean total steroid levels in the biopsies are similarly recorded. Compared with the mean total level in the biopsies at 2 h (Table 1), there was a loss of greater than 85 per cent of the contained steroid by 2 and 3 h after sample removal; this loss was highly significant (p < 0.001). The steroid remaining in the tissues at these times showed the same distribution pattern as biopsies taken immediately after sample removal. The mean total steroid level at 2 and 3 h delay-periods was not significantly different (p < 0.05). Distribution of triamcinolone acetonide in the mucosal epithelium and connective tissue from autoradiographic sections
Heavy labelling was apparent in the epithelial and sub-epithelial layers (Fig. l), particularly in the connective tissue immediately below the basement membrane and especially so in the dermal papillae. A progressive and rapid fall-off in labelling was present at increasing depths through the connective tissue. Sections of normal tissue processed for autoradiography in an identical manner showed grain counts equal to, or below, the background counts. The mean and standard deviation of the grain counts minus background, per graticule square through the epithe-
lium into the connective tissue for the two concentrations and times are shown in Table 3. For both the 5 mg/ml and 0.5 mg/ml biopsies, a one-way analysis of variance showed that the mean grain counts per graticule were significantly greater at 3 h compared with 1 h (f= 11.84 and 37.6 respectively, df = 52). The distribution of grain counts through the tissue were similar at both concentration and application times. Mean counts increased through the epithelium and reached a maximum, which varied between 2-8 graticule-squares above the basement membrane. A fall in mean counts in the deeper epithelium then occurred, reaching a level below that of the mean count in the connective tissue immediately adjacent to the basement membrane. Within the connective tissue, a progressive fall in counts occurred at increasing depth. Mean grain counts from sections of biopsies obtained after the 2 and 3 h delay-periods are shown in Table 4. After 2 h, some 1abelJing was apparent in the epithelium and the mean counts increased towards the basement membrane. Very low counts were present in the connective tissue. After 3 h, virtually no labelling was observed in the epithelium or connective tissue.
DISCUSSION
The distribution of triamcinolone acetonide in the tissues after topical application to the buccal mucosa of the dog, as measured by serial sectioning, was similar to that reported for healthy human skin (Schaeffer et al., 1977). Thus, the greatest concentration of the absorbed drug was in upper 30@400 pm of the mucosa. This zone of tissue would, with the measurement of epithelial thickness and the appearance of epithelium on interface sections, have included the epithelium and the connective tissue immediately below the basement membrane. The initially high levels fell rapidly within the upper 3OOpm of tissue and then progressively through the depth of the specimens. The autoradiographs supported the distribution determined from serial sections. However, the grain counts localized the high levels of labelling more precisely to either side of the basement membrane. The peak grain counts in the epithelium close to the basement membrane were interesting and were observed for both concentrations of the applied steroid at the 1 and 3 h periods. Such a distribution may have been derived from slower removal of the steroid from the corium than penetration through the epithelium. This would be consistent with the low water-, high lipidsolubility of triamcinolone acetonide which would tend to reduce removal via the tissue fluids (Tregear 1966). Certainly, removal of triamcinolone acetonide from the corium was slow compared with the findings of Malkinson and Kirschenbaum (1963) in respect of hydrocortisone. These workers postulated that the resultant depot effect in the skin may in part account for the enhanced topical effect of triamcinolone acetonide. My high grain counts in the corium close to the basement membrane may be similarly explained. Furthermore, the density of labelling in dermal papillae would be expected because of the relatively larger
Mucosal absorption of triamcinolone acetonide
813
Table 3. Mean autoradiographic counts in the buccal mucosa after application of tritium labelled triamcinolorle acetonide (10 &i/ml labelling) (results expressed as counts per graticule-square, SD in parentheses) Concentration applied Time after application
Epithelium
Basement membrane
Connective tissue
5 mgs/ml
0.5 mgs/ml
60 min
180 min
60 min
180 min
34.8 (10.0) 29.0 (12.8) 32.9 (8.9) 32.8 (10.4) 32.4 (19.0) 29.8 (22.7) 28.8 (22.4) 31.6 (29.8) 34.7 (39.0) 39.2 (44.0) 38.8 (45.9) 43.0 (50.7) 42.4 (50.4) 38.9 (47.9) 36.7 (44.5) 35.0 (43.7) 30.2 (38.5) 37.8 (41.7) 37.4 (44.2) 32.8 (41.2) 29.9 (39.2) 25.8 (35.5) 25.3 (36.0) 24.8 (30.3) 21.1 (26.2) 21.3 (27.9) 20.0 (26.3)
18.8 (19.7) 18.3 (25.5) 23.1 (31.8) 36.1 (47.4) 32.8 (40.2) 34.2 (38.3) 30.8 (33.1) 37.1 (38.2) 37.1 (39.4) 41.5 (40.8) 45.9 (42.6) 47.2 (43.5) 55.8 (49.8) 57.1 (52.4) 55.5 (52.0) 50.2 (49.5) 49.8 (52.3) 69.0 (52.4) 67.7 (52.8) 56.8 (50.1) 49.2 (44.5) 44.1 (43.8) 39.0 (43.5) 36.2 (40.6) 35.5 (39.9) 30.9 (29.5) 30.3 (34.3)
11.8 (3.5) 10.0 (3.7) 10.0 (4.1) 10.1 (5.6) 11.9 (8.3) 13.6 (9.4) 16.3 (11.9) 17.8 (19.0) 16.6 (12.3) 23.7 (22.3) 23.7 (23.3) 26.2 (29.0) 26.9 (29.9) 26.0 (29.5) 24.6 (30.2) 22.2 (32.1) 19.0 (21.5) 25.4 (24.5) 24.0 (24.7) 17.0 (16.7) 14.9 (13.2) 13.7 (13.2) 11.4 (11.2) 10.5 (8.5) 10.6 (10.0) 9.6 (7.5) 8.8 (8.0)
18.0 (10.1) 34.5 (26.8) 27.0 (33.9) 41.5 (33.9) 55.1 (48.1) 59.1 (47.2) 63.4 (49.5) 66.4 (48.5) 72.0 (49.2) 74.8 (53.9) 71.2 (51.7) 71.0 (51.3) 65.8 (47.1) 62.3 (46.4) 58.9 (46.0) 46.6 (41.3) 37.0 (35.6) 40.7 (42.3) 39.5 (41.3) 36.1 (39.3) 27.2 (32.1) 22.4 (20.6) 18.2 (16.7) 16.4 (17.2) 14.4 (13.3) 12.5 (10.2) 11.2 (9.6)
surface area of epithelium enclosing the dermal papillae and through which the steroid would penetrate. The apparent ease of penetration of triamcinolone acetonide through canine buccal mucosa, which lacks a stratum corneum, was not surprising because this layer is considered to be the major barrier to steroid penetration (Marzulli, 1962; Scheuplein et al., 1969). Moreover, considerably greater percentage absorption of applied dose has been reported for rectal and vaginal mucosa (Liddle, 1956) than for skin (Felman and Maibach, 1965; Malkinson and Ferguson, 1955; Malkinson, Ferguson and Wang, 1957). The intercellular pathway barrier which limits the penetration of some water-soluble tracers and which is present in skin (Wolff and Schreiner, 1969) and in both keratinized and non-keratinized oral epithelium (Squier, 1973; Squier and Rooney, 1974) did not appear to affect the absorption of triamcinolone acetonide. Increased labelling was not observed in the outer cell layers of the epithelium, a site which would have coincided with the intercellular barrier (Squier, 1973). Because the inter-cellular barrier is probably an aqueous channel (Squier and Johnson, 1975) along which electrolytes and lipid-insoluble substances pass (Tregear, 1966; Middleton, 1969), the virtually waterinsoluble steroid triamcinolone acetonide would be unlikely to follow such an absorption route. Absorption by lipid cell-membranes (Scheuplein, 1967) appears to be a more suitable route for non-electrolytes
with high lipid-water partition co-efficients, including of steroids. The total amounts of steroid in the tissues were approximately proportional to he applied concentration. Thus, the IO-fold difference between the 0.5 mg/ml and the 5mg/ml preparations applied in propylene glycol produced a 7-14-fold difference in total steroid content. This is consistent with a passive diffusion process being the prime mechanism of absorption across skin and mucosa (Tregear, 1966; Seigel, Hall and Stambaugh, 1971; Scheuplein, 1972; 1976), particularly for water and low molecular weight non-electrolytes (Scheuplein and Blank, 1973), including steroids (Scheuplein et al., 1969). The concentration of steroid within the tissues increased with time; this is consistent with more rapid penetration through the epithelium than removal from the corium. However, in proportion to the applied dose the total tissue levels were very small. Thus, even after I50 min application, the steroid content of the biopsies was still less than 1 per cent of the applied dose for both concentrations in propylene glycol. However, the amount of steroid in the biopsies at each time period would not indicate the actual percentage absorbed from the well because of the continuous absorption through the epithelium and loss into the circulation. The loss of steroid from the mucosa with time was apparent from the levels observed in the biopsies taken 2 and 3 h after removal of the topically
M. Addy
814
Table 4. Mean autoradiographic counts in the buccal mucosa 2 and 3 hours after the removal of applied tritium labelled triamcinolone acetonide (5 mg/ml, 10 $i/ml) (results expressed as counts per graticulesquare, SD in parentheses) Time after removal 3h 2h
Basement membrane
Connective tissue
15.5 (6.4) 5.0 (1.4) 6.3 (5.7) 12.7 (16.0) 9.4 (15.9) 7.7 (14.7) 10.4 (10.1) 14.7 (23.2) 14.1 (15.3) 17.3 (14.9) 25.1 (22.0) 31.9 (26.7) 39.0 (27.7) 36.2 (27.7) 40.8 (30.5) 46.8 (37.8) 34.7 (31.4) 5.6 (7.5) 2.9 (2.8) 3.7 (3.1) 3.2 (3.2) 3.4 (3.9) 5.0 (4.9) 4.3 (8.9) 3.3 (3.5) 3.4 (2.9) 3.8 (3.5)
5.3 (6.6) 6.6 (4.2) 5.5 (7.6) 7.2 (10.3) 5.1 (5.3) 6.5 (9.2) 8.9 (8.6) 6.8 (8.3) 6.6 (9.1) 6.3 (6.3) 7.2 (9.8) 9.4 (10.6) 9.8 (11.8) 1.5 (9.5) 7.8 (8.9) 7.1 (6.9) 7.1 (6.6) 4.1 (4.9) 4.0 (5.5) 3.3 (5.1) 3.4 (4.2) 3.3 (4.9) 3.0 (3.0) 3.3 (4.9) 2.1 (3.3) 1.6 (3.2) 1.6 (2.4)
applied samples. These levels were only I5 per cent of that measured immediately after removal of the samples. The autoradiographic counts similarly demonstrated loss of label from the tissues. The steroid content of the tissues following topical application in Orabase was 6.5 times less than that after application of 5 mg/ml samples in propylene glycol. This difference did not reflect a concentration effect because the dose in Orabase was 74 per cent of that applied in propylene glycol. The difference probably arose, under the conditions of this experiment, due to a reduced availability of steroid at the mucosa/ Orabase interface. My findings indicate that the retention of steroid within the tissues was dependent upon the maintenance of the preparation on the mucosal surface. Once removed from the mucosal surface, within a few hours, steroid levels in the tissues had fallen markedly. The oral environment in general tends to limit protracted delivery of drugs to many mucosal sites and, as with the skin (Feldman and Maibach, 1965; Malkinson, 1958), a large proportion of topically applied steroids is presumably lost and not absorbed. Even for oral ointments such as Orabase, retention at some oral sites may only be for a few minutes (Kutscher et al., 1959). The effectiveness of the delivery method may therefore be of fundamental impor-
tance to clinical results. The need for clinical methods of delivery permitting prolonged retention of preparations on the oral mucosa needs to be investigated. My study provides information relevant only to normal mucosa; the effects on absorption of pathological change require study. Such changes may increase or decrease absorption and thus increase systemic side effects or decrease pharmacological activity. REFERENCES
Adams D. 1974. The effect of saliva on the penetration of fluorescent dyes into the oral mucosa of the rat and rabbit. Archs oral Biol. 19, 503-S 10. Anderson E., Haymaker W. and Henderson E. 1940. Successful sublingual therapy in Addison’s Disease. J. Am. med. Ass. 115, 216772168. Cawson R. A. 1968. Treatment of oral lichen planus with betamethasone. Br. med. J. I, 86-89. Cooke B. E. D. 1960. Recurrent Mikulicz’s aphthae treated with topical hydrocortisone hemisuccinate sodium. Br. med. J. 1, 764766.
Feldman R. J. and Maibach H. I. 1965. Penetration of Cl4 hydrocortisone through normal skin. The effect of stripping and occlusion. Archs Derm. 91, 661-666. Gibaldi M. and Kanig J. L. 1965. Absorption of drugs through the oral mucosa. J. oral Thu. Pharmuc. 1. 440-150.
Glick D. 1961, Preparation titntiue
Chemical
of biological
Techniques
c$Histo-
sample.
In:
Quan-
and Cytochemistry.
Vol. 1, pp. l-33. Interscience Wiley, New York. Harris D. R., Papa C. M. and Stanton R. 1974. Percutaneous absorption and the surface area of occluded skin. A scanning electron microscope study. Br. J. Derm. 91. 27-32. Kammerau B., Zesch A. and Schaeffer H. 1975. Absolute concentrations of Dithranol and Triacetyl-dithranol in the skin layers after local treatment. I)7 uiuo investigations with four different types of pharmaceutical vehicle. J. invest. Derm. 64, 145-149. Katz M. and Barr M. 1955. A study of sublingual absorption-1. Several factors influencing the rate of absorption. J. Am. pharm. Ass. 44. 419423. Ku&her A. H., Zegarelli E. V., Beube F. E., Chilton N. W., Berman C., Mercandante J. L., Stern I. B. and Rolan J. N. 1959. A new vehicle (Orabase) for the application of drugs to the oral mucous membranes. Oral Surg. 12. 108GlO90.
Liddle G. N. 1956. A9a-Fluorohydrocortisone: investigative tool in adrenal physiology. J. c/in. Metab.
A new Endocr.
16, 557-559.
Malkinson F. D. 1958. Studies on the percutaneous absorption of C I4 labelled steroids by the use of a gas flow cell. J. invest. Derm. 31, 19-28. Malkinson F. D. and Ferguson E. M., 1955. Preliminary and short report. Percutaneous absorption of hydrocortisone 4-C14 in two human subjects. J. invest. Derm. 25, 281-283.
Malkinson F. D. and Kirschenbaum M. B. 1963. Percutaneous absorption of C I4 labelled triamcinolone acetonide. Archs Derm. 88, 427439. Malkinson F. D., Ferguson E. M. and Wang M. C. 1957. Percutaneous absorption of cortisone-4-C’4 in two human subjects. J. invest. Derm. 28, 21 l-216. Marzulli F. J. 1962. Barriers to skin penetration. 1. imust. Derm. 38. 251-253. Middleton J. D. 1969. Pathways of penetration of electrolytes through stratum corneum. Br. J. Derm. 81, Suppl. 4, 5662. Rothner J. T., Herbert M. C., Rosenthal S. L. and Bailin J. 1949. An adhesive penicillin ointment for topical application. J. dent. Res. 28, 544-548.
Mucosal
absorption
of triamcinolone
Schaeffer H., Zesch A. and Stuttgen G. 1977. Penetration, permeation and absorption of triamcinolone acetonide in normal and psoriatic skin. Archs derm. Res. 258, 241-249.
Scheuplein R. J. 1967. Mechanism of percutaneous absorption-11. Transient diffusion and the relative importance of various routes of skin penetration. J. inuesr. Derm. 48, 79- 89.
Scheuplein R. J. 1972. Properties of the skin as a membrane. In: Pharmacology and the Skin (Edited by Montagna W.. Van Scott E. J. and Stoughton R. B.) Chap. 10, pp. 125-I 52. Scheuplein R. J. 1976. Permeability of the skin: A review of major concepts and some new developments. J. invest. Derm. 67. 672-676. Scheuplein R. J. and Blank I. H. 1973. Mechanism of percutaneous absorption. IV. Penetration of non-electrolytes (alcohols) from aqueous solutions and from pure liquids. J. inoest. Derm. 60, 286-296. Scheuplein R. J., Blank I. H., Braunder G. J. and MacFarlane D. J. 1969. Percutaneous absorption of steroids. J. incest. Derm. 52, 63-70. Scott A. and Katz F. 1956. The penetration and distribution of CL4 hydrocortisone in human skin after topical applicatron. J. invest. Derm. 26, 149-158. Siegel 1. A., Hall S. H. and Stambaugh L. 1971. Permeabilitv of the oral mucosa. In: Current Conceots of the Histolog> of the Oral Mucosu (Edited by Squier c’. A. and Meyer J.) Chap. 17, pp. 274-286. Thomas, Springfield, Ill. Spence A. A. 1942. Sublingual administration of methyl testerone. Br. med. J. 1, 668.
acetonide
815
Squier C. A. 1973. The permeability of keratinised and non-keratinised oral epithelium to horse radish peroxidase. J. Ultrastruct. Res. 43, 16&l 77. Squier C. A. and Johnson N. W. 1975. Permeability of the oral mucosa. In: Research in Dentistry, Br. med. Bull. 31, 169-175. Squier C. A. and Rooney L. 1974. The in vitro permeability of keratinised and non-keratinised oral epithelium to lanthanum. J. dent. Res. 53. 1071 Abstract. Tregear R. J. 1966. Physical Funcrions of Skin. Academic Press, London. Turnoff D. and Rowntree L. G. 1941. Successful subhngual therapy in Addison’s Disease: Confirmative report. J. Am. med. Ass. 116, 2016. Wallenius K. 1966. Influence of salivation on oral tumours induction in white rats. Acta. path. microhiol. stand. Suppl. 180, l-91. Wolff K. and Schreiner E. 1969. Aufnahme intracellularer Transport und abban exogenen Proteins in Koratinocyten. elektranenmikroskopisch-cytochemische Eine Studie mit Peroxidase als Markierungs-substanz. Arch. klin. exp. Derm. 235. 203-220.
Yeoman c. M., Greenspan J. S. and Harding S. M. 1978. Recurrent oral ulceration. A double blind comparison of treatment with betamethasone valerate aerosol and placebo. Br. dent. J. 144, 114-l 16. Zetrarelli E. V.. Kutscher A. H.. Silvers H. F.. Beube F. E.. Stern I. B., Berman C. L. and Herlands R. E. 1960. Triamcinolone acetonide in the treatment of acute and chronic lesions of the oral mucous membranes. Preliminary report. Oral Surg. 13, 17@175.
Plate 1 overleaf.
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M. Addy
Plate I. Fig. I. Autoradiographic triamcinolone acetonide
section of the buccal mucosa of dog following the application (5 mg;ml, IO $I.‘ml) for I h. Labelling is apparent throughout subepithelial layers. x 300
of [3H]-labelled the epithelial and
Mucosal absorption
of triamcinolone
Plate I.
acetonide
817
THE ORAL MUCOSAL ABSORPTION AND TISSUE DISTRIBUTION OF TRIAMCINOLONE ACETONIDE IN THE DOG STUDIED BY AUTORADIOGRAPHY M. ADDS Department of Periodontology,
Dental School, Welsh National School of Medicine, Cardiff. South Wales
Summary-Triamcinolone acetonide prepared (5 mg/ml and 0.5 mg/ml) in propylene glycol and (1 mg/g) in Orabase to which [3H]-triamcinolone acetonide was added was placed on the oral mucosa in plastic wells and biopsies taken at various periods after application, together with biopsies taken after a delay of 2 or 3 h following removal of applied samples. Biopsies were processed for serial sectioning and extraction or for autoradiography. There was a trend for the tissue concentration of triamcinolone acetonide to increase with time which was significant for the 5 mg/ml samples. Both serial sectioning and autoradiographic techniques showed the absorbed steroid to he mainly within the epithelium and connective tissue immediately below the basement membrane. The total steroid in biopsies was significantly greater in the 5 mg/ml samples than in the 0.5 mg/ml or oral ointment samples. Progressive loss of steroid from the tissues following removal of the applied samples was apparent from biopsies taken 2 and 3 h after removal applied samples. - _
or
INTRODUCTION
Several of the synthetic steroids initially developed for use on the skin have been employed topically in the management of oral conditions (Cooke, 1960; Cawson, 1968; Yeoman, Greenspan and Harding, 1978). Although topical steroids have been studied quite extensively. interest has been almost entirely concerned with clinical effects. As with most topical drugs used in the mouth, little is known of the fate of the steroids once introduced into the mouth. In particular, the pattern of absorption and local tissue distribution has not been investigated. The many studies into the mucosal absorption of drugs (reviewed by Katz and Barr, 1955; Gibaldi and Kanig, 1965) including steroids (Anderson, Haymaker and Henderson, 1940; Turnoff and Rowntree, 1941; Spence, 1942) were concerned with systemic rather than local effects, or distribution. Similarly, few investigations (Scott and Katz, 1977; Kammerau, Zesch and Schaeffer, 1975; Schaeffer, Zesch and Stuttgen, 1977) of topical application to the skin have been concerned with the demonstration of local tissue concentrations, although the pertinence of such information to practical therapy has been stated (Schaeffer et al., 1977). The stratum corneum appears to be the major barrier to steroid penetration of skin (Marzulli, 1962; Scheuplein et al., 1969). Steroid absorption from the mouth may therefore not be the same as from the skin because of the lack of keratinized epithehum over much of the human mouth. Furthermore, the hydration of the epithehal lining as a result of constant bathing in saliva may accentuate absorption, as occurs with hydrated skin (Harris, Papa and Stanton, 1974) although the possible protective role of saliva cannot be discounted (Wallenius, 1966; Adams, 1974). The fate of drugs used topically in the mouth is suitable for study. My purpose was to assess the absorption and local tissue distribution of the steroid 809
triamcinolone in canine buccal mucosa employing a radiolabelled tracer. This steroid was chosen because it has been employed for the management of several acute and chronic diseases of the oral mucosa in man (Zegarelli K? al., 1960) and is normally applied in an ointment base (Rothner et al., 1949) which may be retained at certain oral sites for several hours (Kutscher et al., 1959).
MATERIALS AND METHODS
Animal model and anaesthetic technique Twelve beagle dogs aged 7-9 yr and a mean weight of 16.6 kg (range 13.2-19.5) were used. One hour prior to each experimental procedure the animals were sedated with 250 mg of diethylthiambutane hydrochloride (Themalon, Burroughs Wellcome & Co., London) injected subcutaneously into the back of the neck. This was followed immediately before commencing the experiment by slow intravenous injection of 24@300mg of pentobarbitone sodium (Sagatal, May and Baker Ltd, Dagenman, Essex) into a foreleg vein. The preparation of radiolabelled triamcinolone ucetonide in propylene glycol and Orabase Concentrations of 0.5 mg and 5 mg/ml of triamcinolone acetonide (Lederle Laboratories Cyanamid of Great Britain Ltd, Gosport, Hampshire) were prepared in propylene glycol and 1 mg/gm in a plasticised hydrocarbon gel containing hydrophilic solids (Orabase, E.R.S. Squibb & Sons Ltd, Liverpool and London). Tritium-labelled triamcinolone acetonide (The Radiochemical Centre, Amersham, Buckinghamshire) of specific activity 23 Ci/mmol (53 mCi/mg) was added to the propylene glycol steroid solutions in two concentrations of 10 and 20 &i/ml. These two initial
810
M. Addy
concentrations were prepared by the addition of suitable volumes of the 1 mCi/ml labelled material. For the Orabase/steroid mixture tritium-labelled triamcinolone was added at 1&i/g. The resulting mean radioactivity in counts per minute minus background of each solution was determined by placing 0.1 ml samples into 19.9 ml of ethyl acetate. Volumes of 0.1 ml from each of these dilutions were placed into scintillant vials, evaporated to dryness and methanol (2 ml) and scintillant (IO ml) added. Scintillation counting was carried out in an Intertechnique SL.30 automatic p-counter for two IOmin periods. Background samples were similarly prepared for propylene glycol without triamcinolone acetonide. The mean radioactivity in counts per minute minus background of the Orabase mixture was determined by extracting known weights of steroid-containing Orabase with ethyl acetate. Suitable volumes for counting were then evaporated in scintillant vials and methanol and scintillant fluid added, as before. Background counts were determined from the extraction and processing of steroid-free Orabase. Drug applicution atld biopsy techtCyue The steroid preparations were applied to the buccal mucosa of the upper cheek folds in plastic wells. The wells (polystryrene) were cylindrical of 6 mm internal diameter and 5mm depth. The mucosa was dried with cotton wool before placing the wells and a watertight seal obtained using Orabase around the base of each well. The lips and cheeks were separated and the mucosa exposed in a horizontal plane using silk sutures through the skin-lip mucosal junction and tied at the side of the operating table. Mucosal biopsies of the application sites were taken at the designated time intervals for the respective experiments The well, Orabase seal and residual contents were lifted off each site with college tweezers and the mucosal surface wiped twice with cotton wool soaked in distilled water. The biopsy of the whole thickness of the mucosa was then taken to include the whole area covered by the external diameter of the well, together with a small margin of uncovered tissue. The resulting tissue specimens, approximately 10 mm in diameter, were placed epithelial surface down on a piece of card, wrapped in tin foil, frozen in liquid nitrogen and stored at - 70°C until required. Serial section and tissue extraction Samples (0.1 ml) of [3H]-labelled triamcinolone acetonide (0.5 mg/ml and 5 mg/ml with ZO&i/ml) were placed into separate wells. At intervals of 60. I20 and 150 min after application, biopsies were taken. For some I20 min application sites, following removal of the samples, biopsy was delayed for 2 or 3 h. For the Orabase mixture (I mg/g, 1 &i/g), a known weight of the material was placed in the wells and then applied directly to the mucosa. Biopsies were then obtained at 150 min. The biopsies were processed for scintillation counting using the Glick (1961) serial-sectioning technique to determine the tissue distribution of the steroid. Thus serial cryostat sections IOpm thick were cut parallel to the epithelial surface throughout the tissue. Every successive IO sections were placed into ethyl acetate (4 ml) in a scintillant vial for extraction of
contained triamcinolone acetonide. For some biopsies the section following each IO0pm series was stained with haemotoxylin and eosin and used to correlate the appearance of epithelium in the sections with radioactivity counts. After 24 h the ethyl acetate was evaporated and scintillation fluid (IO ml) added together with methanol (2 ml). The methanol removed the cloudiness in the final mixture, arising from residual moisture, observed in preliminary experiments. The radioactivity in counts/min minus background counts for the samples, was then determined, using a /I-counter. Background counts were measured using vials containing serial sections taken from three sham biopsies treated as described. From the counts previously obtained for the applied samples, the concentrations of the steroid in the sections of tissue were calculated. The extraction period of 24 h was chosen arbitrarily; however, extraction appeared to be complete because counts above background were not obtained on extracting and processing the sections on a second occasion. Autoradiography
experiments
Samples (0.1 ml) of triamcinolone acetonide in propylene glycol (0.5, 5 mg/ml with IO&i/ml) were placed in wells. After 1 and 3 h, biopsies were taken from the centre of the site of well application, snapfrozen and stored as before. Some 5 mg/ml samples were maintained on the mucosa for 2 h and, after removal, biopsy was delayed for 2 and 3 h. Serial cryostat sections 7pm thick were cut to pass through the depth of the tissue and across the width of the specimen For each biopsy, a total of I2 sections, approximately one section per 30 cut, were collected on glass slides. Sections were allowed to dry in air and then dipped in autoradiographic emulsion (Ilford K5 gel form, Ilford Ltd, Ilford, Essex) with equal parts distilled water using a 3 s dip and drain procedure. After drying, the sections were placed in a light-proof box and stored in a refrigerator. After 3 weeks. a time previously determined by removing sections at 7 day intervals until the most acceptable contrast for light microscopy was attained, the sections were removed from the box and developed in Kodak D.l9B for 4min and fixed in Kodafix (Kodak). Finally, the sections were lightly counterstained with haematoxylin. Sections from sham biopsies were similarly processed. All biopsies taken at one time for one concentration of applied steroid were sectioned and processed together. The slides were then coded and viewed blind at x 400 magnification with a 10 x 10mm graticule, having I mm square divisions, in one of the eye pieces of a binocular visible-light microscope. The number of autoradiographic grains contained in each 1mm square division of the graticule was counted, counts being made perpendicular to the basement membrane through the epithelium and through the connective tissue. The counts were made at several sites of each section; selection was arbitrary except to avoid dermal papillae. A minimum of 30 counts were recorded from the sections for each biopsy. Background counts per graticule-square were taken from each slide at sites 3-5 graticule-squares above the epithelial surface. The mean of 10 counts for each slide was subtracted from the counts per graticule-square obtained
Mucosal absorption of triamcinolone acetonide
811
Table 1. Mean triamcinoline acetonide levels in 1OOpm serial sections of biopsies from the buccal mucosa applied in propylene glycol and Orabase (results expressed in ng, SD in parentheses) Propylene glycol concentration 5 mg/ml Time after application Number of biopsies
Orabase concentration 0.1%
0.5 mg/ml
60 min
120 min
150 min
60 min
120 min
10
4
7
4
5
150 min
501 (369) 537 (249) 116 (76) 89 (54) 59 (32) 49 (29) 40 (30) 31 (26) 21 (15) 18 (12) 15 (11) 13 (10) 9 (6) 7 (5) 6 (3) 5 (5) 5 (3) 4 (2) 4 (4) 4 (3) 2 (2) 2 (1) 1376 (562)
1563 (839) 706 (803) 457 (525) 236 (273) 125 (107) 82 (50) 66 (40) 47 (41) 43 (18) 41 (19) 37 (18) 31 (15) 26 (13) 24 (11) 22 (9) 20 (9) 18 (7) 18 (6) 16 (5) 11 (9) 10 (8) 7 (8) 3633 (2664)
1750 (1435) 941 (1028) 286 (217) 346 (384) 333 (488) 113 (47) 88 (32) 83 (33) 73 (28) 66 (26) 59 (26) 53 (32) 47 (22) 48 (36) 39 (20) 38 (17) 27 (8) 24 (8) 22 (7) 18 (6) 13 (7) 15 (10) 4527 (1971)
79 (59) 38 (44) 11 (11) 3 (4) 5 (4) 4 (3) 4 (3) 3 (2) 4 (3) 4 (3) 4 (3) 4 (4) 5 (5) 3 (4) 3 (4) 4 (5) 3 (4) 3 (3) 3 (5) 2 (3) 1 (2) 1 (1) 206.(96)
4
2 -
Epithelial surface 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Mean
150 min
179 (149) 38 (38) 24 (24) 10 (6) 10 (7) 8 (6) 8 (8) 8 (8) 8 (6) 7 (7) 7 (6) 6 (5) 5 (3) 3 (2) 3 (3) 3 (1) 2 (1) 2 (1) 1 (1) 1 (1) 1 (1) 0 (0) 250 (87)
-
47 (29) 75 (24) 38 (10) 28 (14) 31 (1) 27 (3) 23 (5) 22 (10) 18 (11) 16 (11) 12 (9) 11 (10) 2 (2) 7 (6) g (7) 5 (4) 4 (2) 3 (2) 3 (1) 2 (1) 2 (1) 2 (1) 394 (33)
-.-
105 (117) 66 (53) 46 (33) 44 (27) 45 (25) 26 (7) 25 (8) 22 (16) 17 (3) 18 (3) 18 (4) 16 (2) 16 (3) 18 (8) 16 (2) 15 (2) 17 (2) 17 (3) 25 (16) 23 (11) 25 (17) 13 (9) 694 (253)
Mean weight Orabase (mg) = 373.7 (7.8).
from the tissue sections for that slide. Grain counts minus background were similarly obtained for sections from the sham biopsies. RESULTS
Sections of dog buccal mucosa showed a stratified squamous epithelium overlying loose connective tissue. Rete pegs and corresponding dermal papillae were apparent. Orthokeratinization of the epithelium was not observed; however, in most sections, a narrow zone of parakeratinization was present. The mean thickness of the epithelium measured by light graticule was microscopy using a millimetre 182 + 54pm. The absorption and tissue distribution of triamcinolone acetonide in the buccal mucosa from serial-section measurements
The stained sections taken from the 100~ interfaces revealed epithelium making up the bulk of the section areas at the 10&300 pm levels. The means and standard deviations of the levels of triamcinolone acetonide through the depth of the specimens for the different application times and modes of application are shown in Table 1. For the Orabase samples, the
amount of label and the mean weight applied was also recorded. For all biopsy times and concentrations of the applied steroid, there was a progressive fall, from the epithelial surface down, in levels of triamcinolone acetonide per 100 pm serial sections. The mean fall was most marked within the first 300~; a slow rate of fall was then apparent. From the histological measurements, the highest levels of steroid were present in the epithelium and immediate sub-epithelial layers. For the 5 mg/ml samples, a one-way analysis of variance showed a significant increase in total tissue steroid with time cf= 8.32, df = 20). This arose from a significant increase at 150 and 120 min compared with 60min (p < 0.01). No significant difference was apparent between 150 and 120 min (p = >0.05). A similar mean trend for an increase in total tissue steroid with time was observed for the 0.5mg/ml samples; however, the differences did not reach significance (f = 3.23, df = 10). Comparison of the effects of concentration on steroid levels obtained was determined using the Student’s r-test. At comparable application times, the mean total steroid content of the 5 mg/ml biopsies was highly significantly greater than of both the 0.5 mg/ml and 0.1 per cent Orabase biopsies (p < 0.001). No significant difference in total
M. Addy
812
Table 2. Mean triamcinolone acetonide levels in 100 pm serial sections taken 2 and 3 h after removal of the applied samples (results expressed in ng, SD in parentheses) Biopsy time after removal Number of biopsies Epithelial surface
Mean
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
2h 6
3h 4
175 (87) 147 (134) 27 (18) 14 (10) 8 (6) 6 (2) 4 (2) 4 (2) 2 (2) 2 (2) 2 (2) 2 (2) 2 (2) 2 (1) 2 (2) 1 (1) 3 (4) 1 (1) 2 (2) 1 (1) 3 (4) 2 (2) 423 (106)
284 (125) 45 (40) 47 (51) 40 (45) 20 (25) 22 (30) 23 (28) 18 (26) 23 (33) 22 (33) 35 (49) 9 (13) 4 (4) 3 (4) 2 (4) 2 (2) 2 (2) 1 (1) 0 (0) 0 (0) 0 (0) 0 (0) 532 (371)
steroid levels was observed between the 0.5 mg/ml and the 0.1 per cent Orabase biopsies (p > 0.05). The mean concentration of triamcinolone acetonide in 100 pm serial sections of tissue from biopsies taken 2 and 3 h after removal of the applied 5 mg/ml samples are shown in Table 2. The mean total steroid levels in the biopsies are similarly recorded. Compared with the mean total level in the biopsies at 2 h (Table 1), there was a loss of greater than 85 per cent of the contained steroid by 2 and 3 h after sample removal; this loss was highly significant (p < 0.001). The steroid remaining in the tissues at these times showed the same distribution pattern as biopsies taken immediately after sample removal. The mean total steroid level at 2 and 3 h delay-periods was not significantly different (p < 0.05). Distribution of triamcinolone acetonide in the mucosal epithelium and connective tissue from autoradiographic sections
Heavy labelling was apparent in the epithelial and sub-epithelial layers (Fig. l), particularly in the connective tissue immediately below the basement membrane and especially so in the dermal papillae. A progressive and rapid fall-off in labelling was present at increasing depths through the connective tissue. Sections of normal tissue processed for autoradiography in an identical manner showed grain counts equal to, or below, the background counts. The mean and standard deviation of the grain counts minus background, per graticule square through the epithe-
lium into the connective tissue for the two concentrations and times are shown in Table 3. For both the 5 mg/ml and 0.5 mg/ml biopsies, a one-way analysis of variance showed that the mean grain counts per graticule were significantly greater at 3 h compared with 1 h (f= 11.84 and 37.6 respectively, df = 52). The distribution of grain counts through the tissue were similar at both concentration and application times. Mean counts increased through the epithelium and reached a maximum, which varied between 2-8 graticule-squares above the basement membrane. A fall in mean counts in the deeper epithelium then occurred, reaching a level below that of the mean count in the connective tissue immediately adjacent to the basement membrane. Within the connective tissue, a progressive fall in counts occurred at increasing depth. Mean grain counts from sections of biopsies obtained after the 2 and 3 h delay-periods are shown in Table 4. After 2 h, some 1abelJing was apparent in the epithelium and the mean counts increased towards the basement membrane. Very low counts were present in the connective tissue. After 3 h, virtually no labelling was observed in the epithelium or connective tissue.
DISCUSSION
The distribution of triamcinolone acetonide in the tissues after topical application to the buccal mucosa of the dog, as measured by serial sectioning, was similar to that reported for healthy human skin (Schaeffer et al., 1977). Thus, the greatest concentration of the absorbed drug was in upper 30@400 pm of the mucosa. This zone of tissue would, with the measurement of epithelial thickness and the appearance of epithelium on interface sections, have included the epithelium and the connective tissue immediately below the basement membrane. The initially high levels fell rapidly within the upper 3OOpm of tissue and then progressively through the depth of the specimens. The autoradiographs supported the distribution determined from serial sections. However, the grain counts localized the high levels of labelling more precisely to either side of the basement membrane. The peak grain counts in the epithelium close to the basement membrane were interesting and were observed for both concentrations of the applied steroid at the 1 and 3 h periods. Such a distribution may have been derived from slower removal of the steroid from the corium than penetration through the epithelium. This would be consistent with the low water-, high lipidsolubility of triamcinolone acetonide which would tend to reduce removal via the tissue fluids (Tregear 1966). Certainly, removal of triamcinolone acetonide from the corium was slow compared with the findings of Malkinson and Kirschenbaum (1963) in respect of hydrocortisone. These workers postulated that the resultant depot effect in the skin may in part account for the enhanced topical effect of triamcinolone acetonide. My high grain counts in the corium close to the basement membrane may be similarly explained. Furthermore, the density of labelling in dermal papillae would be expected because of the relatively larger
Mucosal absorption of triamcinolone acetonide
813
Table 3. Mean autoradiographic counts in the buccal mucosa after application of tritium labelled triamcinolorle acetonide (10 &i/ml labelling) (results expressed as counts per graticule-square, SD in parentheses) Concentration applied Time after application
Epithelium
Basement membrane
Connective tissue
5 mgs/ml
0.5 mgs/ml
60 min
180 min
60 min
180 min
34.8 (10.0) 29.0 (12.8) 32.9 (8.9) 32.8 (10.4) 32.4 (19.0) 29.8 (22.7) 28.8 (22.4) 31.6 (29.8) 34.7 (39.0) 39.2 (44.0) 38.8 (45.9) 43.0 (50.7) 42.4 (50.4) 38.9 (47.9) 36.7 (44.5) 35.0 (43.7) 30.2 (38.5) 37.8 (41.7) 37.4 (44.2) 32.8 (41.2) 29.9 (39.2) 25.8 (35.5) 25.3 (36.0) 24.8 (30.3) 21.1 (26.2) 21.3 (27.9) 20.0 (26.3)
18.8 (19.7) 18.3 (25.5) 23.1 (31.8) 36.1 (47.4) 32.8 (40.2) 34.2 (38.3) 30.8 (33.1) 37.1 (38.2) 37.1 (39.4) 41.5 (40.8) 45.9 (42.6) 47.2 (43.5) 55.8 (49.8) 57.1 (52.4) 55.5 (52.0) 50.2 (49.5) 49.8 (52.3) 69.0 (52.4) 67.7 (52.8) 56.8 (50.1) 49.2 (44.5) 44.1 (43.8) 39.0 (43.5) 36.2 (40.6) 35.5 (39.9) 30.9 (29.5) 30.3 (34.3)
11.8 (3.5) 10.0 (3.7) 10.0 (4.1) 10.1 (5.6) 11.9 (8.3) 13.6 (9.4) 16.3 (11.9) 17.8 (19.0) 16.6 (12.3) 23.7 (22.3) 23.7 (23.3) 26.2 (29.0) 26.9 (29.9) 26.0 (29.5) 24.6 (30.2) 22.2 (32.1) 19.0 (21.5) 25.4 (24.5) 24.0 (24.7) 17.0 (16.7) 14.9 (13.2) 13.7 (13.2) 11.4 (11.2) 10.5 (8.5) 10.6 (10.0) 9.6 (7.5) 8.8 (8.0)
18.0 (10.1) 34.5 (26.8) 27.0 (33.9) 41.5 (33.9) 55.1 (48.1) 59.1 (47.2) 63.4 (49.5) 66.4 (48.5) 72.0 (49.2) 74.8 (53.9) 71.2 (51.7) 71.0 (51.3) 65.8 (47.1) 62.3 (46.4) 58.9 (46.0) 46.6 (41.3) 37.0 (35.6) 40.7 (42.3) 39.5 (41.3) 36.1 (39.3) 27.2 (32.1) 22.4 (20.6) 18.2 (16.7) 16.4 (17.2) 14.4 (13.3) 12.5 (10.2) 11.2 (9.6)
surface area of epithelium enclosing the dermal papillae and through which the steroid would penetrate. The apparent ease of penetration of triamcinolone acetonide through canine buccal mucosa, which lacks a stratum corneum, was not surprising because this layer is considered to be the major barrier to steroid penetration (Marzulli, 1962; Scheuplein et al., 1969). Moreover, considerably greater percentage absorption of applied dose has been reported for rectal and vaginal mucosa (Liddle, 1956) than for skin (Felman and Maibach, 1965; Malkinson and Ferguson, 1955; Malkinson, Ferguson and Wang, 1957). The intercellular pathway barrier which limits the penetration of some water-soluble tracers and which is present in skin (Wolff and Schreiner, 1969) and in both keratinized and non-keratinized oral epithelium (Squier, 1973; Squier and Rooney, 1974) did not appear to affect the absorption of triamcinolone acetonide. Increased labelling was not observed in the outer cell layers of the epithelium, a site which would have coincided with the intercellular barrier (Squier, 1973). Because the inter-cellular barrier is probably an aqueous channel (Squier and Johnson, 1975) along which electrolytes and lipid-insoluble substances pass (Tregear, 1966; Middleton, 1969), the virtually waterinsoluble steroid triamcinolone acetonide would be unlikely to follow such an absorption route. Absorption by lipid cell-membranes (Scheuplein, 1967) appears to be a more suitable route for non-electrolytes
with high lipid-water partition co-efficients, including of steroids. The total amounts of steroid in the tissues were approximately proportional to he applied concentration. Thus, the IO-fold difference between the 0.5 mg/ml and the 5mg/ml preparations applied in propylene glycol produced a 7-14-fold difference in total steroid content. This is consistent with a passive diffusion process being the prime mechanism of absorption across skin and mucosa (Tregear, 1966; Seigel, Hall and Stambaugh, 1971; Scheuplein, 1972; 1976), particularly for water and low molecular weight non-electrolytes (Scheuplein and Blank, 1973), including steroids (Scheuplein et al., 1969). The concentration of steroid within the tissues increased with time; this is consistent with more rapid penetration through the epithelium than removal from the corium. However, in proportion to the applied dose the total tissue levels were very small. Thus, even after I50 min application, the steroid content of the biopsies was still less than 1 per cent of the applied dose for both concentrations in propylene glycol. However, the amount of steroid in the biopsies at each time period would not indicate the actual percentage absorbed from the well because of the continuous absorption through the epithelium and loss into the circulation. The loss of steroid from the mucosa with time was apparent from the levels observed in the biopsies taken 2 and 3 h after removal of the topically
M. Addy
814
Table 4. Mean autoradiographic counts in the buccal mucosa 2 and 3 hours after the removal of applied tritium labelled triamcinolone acetonide (5 mg/ml, 10 $i/ml) (results expressed as counts per graticulesquare, SD in parentheses) Time after removal 3h 2h
Basement membrane
Connective tissue
15.5 (6.4) 5.0 (1.4) 6.3 (5.7) 12.7 (16.0) 9.4 (15.9) 7.7 (14.7) 10.4 (10.1) 14.7 (23.2) 14.1 (15.3) 17.3 (14.9) 25.1 (22.0) 31.9 (26.7) 39.0 (27.7) 36.2 (27.7) 40.8 (30.5) 46.8 (37.8) 34.7 (31.4) 5.6 (7.5) 2.9 (2.8) 3.7 (3.1) 3.2 (3.2) 3.4 (3.9) 5.0 (4.9) 4.3 (8.9) 3.3 (3.5) 3.4 (2.9) 3.8 (3.5)
5.3 (6.6) 6.6 (4.2) 5.5 (7.6) 7.2 (10.3) 5.1 (5.3) 6.5 (9.2) 8.9 (8.6) 6.8 (8.3) 6.6 (9.1) 6.3 (6.3) 7.2 (9.8) 9.4 (10.6) 9.8 (11.8) 1.5 (9.5) 7.8 (8.9) 7.1 (6.9) 7.1 (6.6) 4.1 (4.9) 4.0 (5.5) 3.3 (5.1) 3.4 (4.2) 3.3 (4.9) 3.0 (3.0) 3.3 (4.9) 2.1 (3.3) 1.6 (3.2) 1.6 (2.4)
applied samples. These levels were only I5 per cent of that measured immediately after removal of the samples. The autoradiographic counts similarly demonstrated loss of label from the tissues. The steroid content of the tissues following topical application in Orabase was 6.5 times less than that after application of 5 mg/ml samples in propylene glycol. This difference did not reflect a concentration effect because the dose in Orabase was 74 per cent of that applied in propylene glycol. The difference probably arose, under the conditions of this experiment, due to a reduced availability of steroid at the mucosa/ Orabase interface. My findings indicate that the retention of steroid within the tissues was dependent upon the maintenance of the preparation on the mucosal surface. Once removed from the mucosal surface, within a few hours, steroid levels in the tissues had fallen markedly. The oral environment in general tends to limit protracted delivery of drugs to many mucosal sites and, as with the skin (Feldman and Maibach, 1965; Malkinson, 1958), a large proportion of topically applied steroids is presumably lost and not absorbed. Even for oral ointments such as Orabase, retention at some oral sites may only be for a few minutes (Kutscher et al., 1959). The effectiveness of the delivery method may therefore be of fundamental impor-
tance to clinical results. The need for clinical methods of delivery permitting prolonged retention of preparations on the oral mucosa needs to be investigated. My study provides information relevant only to normal mucosa; the effects on absorption of pathological change require study. Such changes may increase or decrease absorption and thus increase systemic side effects or decrease pharmacological activity. REFERENCES
Adams D. 1974. The effect of saliva on the penetration of fluorescent dyes into the oral mucosa of the rat and rabbit. Archs oral Biol. 19, 503-S 10. Anderson E., Haymaker W. and Henderson E. 1940. Successful sublingual therapy in Addison’s Disease. J. Am. med. Ass. 115, 216772168. Cawson R. A. 1968. Treatment of oral lichen planus with betamethasone. Br. med. J. I, 86-89. Cooke B. E. D. 1960. Recurrent Mikulicz’s aphthae treated with topical hydrocortisone hemisuccinate sodium. Br. med. J. 1, 764766.
Feldman R. J. and Maibach H. I. 1965. Penetration of Cl4 hydrocortisone through normal skin. The effect of stripping and occlusion. Archs Derm. 91, 661-666. Gibaldi M. and Kanig J. L. 1965. Absorption of drugs through the oral mucosa. J. oral Thu. Pharmuc. 1. 440-150.
Glick D. 1961, Preparation titntiue
Chemical
of biological
Techniques
c$Histo-
sample.
In:
Quan-
and Cytochemistry.
Vol. 1, pp. l-33. Interscience Wiley, New York. Harris D. R., Papa C. M. and Stanton R. 1974. Percutaneous absorption and the surface area of occluded skin. A scanning electron microscope study. Br. J. Derm. 91. 27-32. Kammerau B., Zesch A. and Schaeffer H. 1975. Absolute concentrations of Dithranol and Triacetyl-dithranol in the skin layers after local treatment. I)7 uiuo investigations with four different types of pharmaceutical vehicle. J. invest. Derm. 64, 145-149. Katz M. and Barr M. 1955. A study of sublingual absorption-1. Several factors influencing the rate of absorption. J. Am. pharm. Ass. 44. 419423. Ku&her A. H., Zegarelli E. V., Beube F. E., Chilton N. W., Berman C., Mercandante J. L., Stern I. B. and Rolan J. N. 1959. A new vehicle (Orabase) for the application of drugs to the oral mucous membranes. Oral Surg. 12. 108GlO90.
Liddle G. N. 1956. A9a-Fluorohydrocortisone: investigative tool in adrenal physiology. J. c/in. Metab.
A new Endocr.
16, 557-559.
Malkinson F. D. 1958. Studies on the percutaneous absorption of C I4 labelled steroids by the use of a gas flow cell. J. invest. Derm. 31, 19-28. Malkinson F. D. and Ferguson E. M., 1955. Preliminary and short report. Percutaneous absorption of hydrocortisone 4-C14 in two human subjects. J. invest. Derm. 25, 281-283.
Malkinson F. D. and Kirschenbaum M. B. 1963. Percutaneous absorption of C I4 labelled triamcinolone acetonide. Archs Derm. 88, 427439. Malkinson F. D., Ferguson E. M. and Wang M. C. 1957. Percutaneous absorption of cortisone-4-C’4 in two human subjects. J. invest. Derm. 28, 21 l-216. Marzulli F. J. 1962. Barriers to skin penetration. 1. imust. Derm. 38. 251-253. Middleton J. D. 1969. Pathways of penetration of electrolytes through stratum corneum. Br. J. Derm. 81, Suppl. 4, 5662. Rothner J. T., Herbert M. C., Rosenthal S. L. and Bailin J. 1949. An adhesive penicillin ointment for topical application. J. dent. Res. 28, 544-548.
Mucosal
absorption
of triamcinolone
Schaeffer H., Zesch A. and Stuttgen G. 1977. Penetration, permeation and absorption of triamcinolone acetonide in normal and psoriatic skin. Archs derm. Res. 258, 241-249.
Scheuplein R. J. 1967. Mechanism of percutaneous absorption-11. Transient diffusion and the relative importance of various routes of skin penetration. J. inuesr. Derm. 48, 79- 89.
Scheuplein R. J. 1972. Properties of the skin as a membrane. In: Pharmacology and the Skin (Edited by Montagna W.. Van Scott E. J. and Stoughton R. B.) Chap. 10, pp. 125-I 52. Scheuplein R. J. 1976. Permeability of the skin: A review of major concepts and some new developments. J. invest. Derm. 67. 672-676. Scheuplein R. J. and Blank I. H. 1973. Mechanism of percutaneous absorption. IV. Penetration of non-electrolytes (alcohols) from aqueous solutions and from pure liquids. J. inoest. Derm. 60, 286-296. Scheuplein R. J., Blank I. H., Braunder G. J. and MacFarlane D. J. 1969. Percutaneous absorption of steroids. J. incest. Derm. 52, 63-70. Scott A. and Katz F. 1956. The penetration and distribution of CL4 hydrocortisone in human skin after topical applicatron. J. invest. Derm. 26, 149-158. Siegel 1. A., Hall S. H. and Stambaugh L. 1971. Permeabilitv of the oral mucosa. In: Current Conceots of the Histolog> of the Oral Mucosu (Edited by Squier c’. A. and Meyer J.) Chap. 17, pp. 274-286. Thomas, Springfield, Ill. Spence A. A. 1942. Sublingual administration of methyl testerone. Br. med. J. 1, 668.
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815
Squier C. A. 1973. The permeability of keratinised and non-keratinised oral epithelium to horse radish peroxidase. J. Ultrastruct. Res. 43, 16&l 77. Squier C. A. and Johnson N. W. 1975. Permeability of the oral mucosa. In: Research in Dentistry, Br. med. Bull. 31, 169-175. Squier C. A. and Rooney L. 1974. The in vitro permeability of keratinised and non-keratinised oral epithelium to lanthanum. J. dent. Res. 53. 1071 Abstract. Tregear R. J. 1966. Physical Funcrions of Skin. Academic Press, London. Turnoff D. and Rowntree L. G. 1941. Successful subhngual therapy in Addison’s Disease: Confirmative report. J. Am. med. Ass. 116, 2016. Wallenius K. 1966. Influence of salivation on oral tumours induction in white rats. Acta. path. microhiol. stand. Suppl. 180, l-91. Wolff K. and Schreiner E. 1969. Aufnahme intracellularer Transport und abban exogenen Proteins in Koratinocyten. elektranenmikroskopisch-cytochemische Eine Studie mit Peroxidase als Markierungs-substanz. Arch. klin. exp. Derm. 235. 203-220.
Yeoman c. M., Greenspan J. S. and Harding S. M. 1978. Recurrent oral ulceration. A double blind comparison of treatment with betamethasone valerate aerosol and placebo. Br. dent. J. 144, 114-l 16. Zetrarelli E. V.. Kutscher A. H.. Silvers H. F.. Beube F. E.. Stern I. B., Berman C. L. and Herlands R. E. 1960. Triamcinolone acetonide in the treatment of acute and chronic lesions of the oral mucous membranes. Preliminary report. Oral Surg. 13, 17@175.
Plate 1 overleaf.
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M. Addy
Plate I. Fig. I. Autoradiographic triamcinolone acetonide
section of the buccal mucosa of dog following the application (5 mg;ml, IO $I.‘ml) for I h. Labelling is apparent throughout subepithelial layers. x 300
of [3H]-labelled the epithelial and
Mucosal absorption
of triamcinolone
Plate I.
acetonide
817