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Absorption of Hyaluronan Applied to the Surface of Intact Skin

1999; Elsevier BV; Volume: 113; Issue: 5 Linguagem: Inglês

10.1046/j.1523-1747.1999.00745.x

1523-1747

Tracey Brown, Daine Alcorn, J. R. E. Fraser,

Skin and Cellular Biology Research

Hyaluronan has recently been introduced as a vehicle for topical application of drugs to the skin. We sought to determine whether hyaluronan acts solely as a hydrophilic reservoir on the surface of intact skin or might partly penetrate it. Drug-free hyaluronan gels were applied to the intact skin of hairless mice and human forearm in situ, with and without [3H] hyaluronan. [3H]hyaluronan was shown by autoradiography to disseminate through all layers of intact skin in mouse and human, reaching the dermis within 30 min of application in mice. Cellular uptake of [3H]hyaluronan was observed in the deeper layers of epidermis, dermis, and in lymphatic endothelium. Absorption through skin was confirmed in mice by chromatographic analysis of blood, urine, and extracts from skin and liver, which identified 3H as intact hyaluronan and its metabolites, free acetate and water. Hyaluronan absorption was similarly demonstrated without polyethylene glycol, which is usually included in the topical formulation. [3H]hyaluronan absorption was not restricted to its smaller polymers as demonstrated by the recovery of polymers of (360–400 kDa) from both blood and skin. This finding suggests that its passage through epidermis does not rely on passive diffusion but may be facilitated by active transport. This study establishes that hyaluronan is absorbed from the surface of the skin and passes rapidly through epidermis, which may allow associated drugs to be carried in relatively high concentration at least as far as the deeper layers of the dermis. Hyaluronan has recently been introduced as a vehicle for topical application of drugs to the skin. We sought to determine whether hyaluronan acts solely as a hydrophilic reservoir on the surface of intact skin or might partly penetrate it. Drug-free hyaluronan gels were applied to the intact skin of hairless mice and human forearm in situ, with and without [3H] hyaluronan. [3H]hyaluronan was shown by autoradiography to disseminate through all layers of intact skin in mouse and human, reaching the dermis within 30 min of application in mice. Cellular uptake of [3H]hyaluronan was observed in the deeper layers of epidermis, dermis, and in lymphatic endothelium. Absorption through skin was confirmed in mice by chromatographic analysis of blood, urine, and extracts from skin and liver, which identified 3H as intact hyaluronan and its metabolites, free acetate and water. Hyaluronan absorption was similarly demonstrated without polyethylene glycol, which is usually included in the topical formulation. [3H]hyaluronan absorption was not restricted to its smaller polymers as demonstrated by the recovery of polymers of (360–400 kDa) from both blood and skin. This finding suggests that its passage through epidermis does not rely on passive diffusion but may be facilitated by active transport. This study establishes that hyaluronan is absorbed from the surface of the skin and passes rapidly through epidermis, which may allow associated drugs to be carried in relatively high concentration at least as far as the deeper layers of the dermis. cetylpyridinium chloride hyaluronan The fate of substances applied to the surface of the skin depends upon their ability to penetrate the stratum corneum, which consists of hydrophilic cornified cells embedded in a dense hydrophobic matrix. Both experimental evidence and practical experience show that passage through this layer is facilitated if the substance is lipophilic or amphiphilic (that is, both hydrophilic and lipophilic); it will be further influenced by molecular weight, the character of any solvent, and the relative solubility in the solvent and the stratum corneum (Gozzo et al., 1996Gozzo C.A. Lazarus G.S. Werth V.P. Dermatological pharmacology.in: Hardman J.G. Limberd L.E. Molinoff P.B. Ruddon R.W. Goodman and Gilman's the Pharmacological Basis of Therapeutics. 9th edn. McGraw-Hill, New York1996: 1593-1595Google Scholar). Unless sustained moistening hydrates the skin, hydrophilic substances will penetrate very slowly, but they can be retained on the surface as a suitably adhesive gel for protection of the skin or slow release of an amphiphilic drug. More recently hyaluronan (HA), a ubiquitous component of the extracellular matrix, has been successfully used for the application of certain drugs to the skin where it is thought to act as a sustained release and localized delivery vehicle (Moore and Willoughby, 1995Moore A.R. Willoughby D.A. Hyaluronan as a drug delivery system for diclofenac: a hypothesis for mode of action.Inter J Tiss Reac. 1995; 17: 153-156PubMed Google Scholar). HA is a linear polymer of N-acetyl glucosamine and glucuronic acid in alternating sequence and its carboxyl groups are largely ionized at the pH prevailing on the skin surface. It is therefore strongly hydrophilic, and in agreement with observations in the Franz chamber (Brown et al., 1995Brown M.B. Marriott C. Martin G.P. A study of the transdermal drug delivery properties of hyaluronan.in: Willoughby D.A. International Workshop on Hyaluronan in Drug Delivery. Royal Society of Medicine Press Limited, London1995: 53-73Google Scholar), seems unlikely to penetrate the epidermis. It adopts a stiffened random-coil molecular configuration that occupies a large volume in proportion to its polymer size (Laurent et al., 1960Laurent T.C. Ryan M. Pietruszkiewicz A. Fractionation of hyaluronic acid. The polydispersity of hyaluronic acid from the bovine vitreous body.Biochim Biophys Acta. 1960; 42: 476-485Crossref PubMed Scopus (206) Google Scholar) and overlaps at a relatively low concentration (Laurent, 1970Laurent T.C. Structure of hyaluronic acid.in: Balazs E.A. Chemistry and Molecular Biology of the Intercellular Matrix. Vol. 2. Academic Press, New York1970: 703-732Google Scholar). These properties should further hinder its passage, although they promote its ready formation of viscoelastic gels potentially suitable for surface retention, acting as a surface reservoir for therapeutic agents. Recent work, however, has revealed several kinds of intramolecular hydrogen bonds that form along the saccharide chain of HA in the hydrated state. These impart a twist that creates alternating sequences of eight exposed -CH groups and confers an amphiphilic character (Scott, 1989Scott J.E. Secondary structures in hyaluronan solutions: chemical and biological implications.in: Evered D. Whelan J. The Biology of Hyaluronan, Ciba Foundation Symposium 143. J Wiley & Sons, Chichester1989: 6-15Google Scholar). Contrary to the principles cited above, other apparently hydrophilic polymers such as insulin (Ogiso et al., 1996Ogiso T. Nishioka S. Iwaki M. Dissociation of insulin oligomers and enhancement of percutaneous absorption of insulin.Biol Pharm Bull. 1996; 19: 1049-1054Crossref PubMed Scopus (16) Google Scholar), dextrans (Ogiso et al., 1995Ogiso T. Paku T. Iwaki M. Tanino T. Percutaneous penetration of fluorescein-isothyocyanate dextrans and the mechanism for enhancement effect of enhancers on the intercellular penetration.Biol Pharm Bull. 1995; 18: 1566-1571Crossref PubMed Scopus (30) Google Scholar), interferon gamma (Short et al., 1995Short S.M. Rubas W. Paasch B.D. Mrsny R.J. Transport of biologically active interferon-gamma across human skin in vitro.Pharm Res. 1995; 12: 1140-1145Crossref PubMed Scopus (23) Google Scholar), and other glycosaminoglycans (Stuttgen et al., 1990Stuttgen G. Panse P. Bauer E. Permeation of the human skin by heparin and mucopolysaccharide polysulfuric acid ester.Arzneimittel-Forsch. 1990; 40: 484-489PubMed Google Scholar) have also been shown to penetrate skin. In the light of Scott's findings, we were therefore prompted to determine whether HA would do likewise despite its apparent impediments. Such an outcome would be pertinent not only to its therapeutic usage but also to its movements and turnover deeper in the skin, and to the use of other polymers that might be deleterious if similarly absorbed. SKh/1 hairless mice were obtained from Animal Resource Center (Perth, WA, Australia). Mice ranged in age from 3 to 6 mo and were of both sexes. The person was a healthy male without skin disease. Two batches of HA labeled with 3H in its acetyl groups were prepared by biosynthesis, purified, and reduced by sonication (Fraser et al., 1988Fraser J.R.E. Kimpton W.G. Laurent T.C. Cahill R.N.P. Vakakis N. Uptake and degradation of hyaluronan in lymphatic tissue.Biochem J. 1988; 256: 153-158Crossref PubMed Scopus (153) Google Scholar) to a modal Mr of 2.5 × 105 and 4.0 × 105, respectively. These were lyophilized, redissolved in pyrogen-free distilled water, and incorporated in the therapeutic gels by repeated vortex mixing and centrifugation at 500 × gav to ensure thorough mixing. Radiochemical purity was > 99.5% as determined by digestion with Streptomyces hyaluronidase (refer to Methods of Analysis). Two series of mouse and one series of human experiments were conducted (refer to Table 1 for components of experimental gels).Table 1Formulations of gels used in the mouse and human experimentsExperimentComponents per gram of gelPolyethylene glycol (mg)Benzyl alcohol (%vol/vol)Non-radioactive HAaMr 4 × 105 Daltons. (mg)[3H] HA(mg)MrSpecific activity (dpm/mg total HA)Mouse studySeries 1 (repeated applications) n = 161430.717.90.52.5 × 10 56.4 × 106Series 2 (single application)n = 101080.513.43.34.0 × 10 531.6 × 106n = 10–0.513.43.34.0 × 10 531.6 × 106Series 2 (single application) n = 251080.512.514.0 × 10 523.3 × 106Human study–0.513.43.34.0 × 10 531.6 × 106a Mr 4 × 105 Daltons. Open table in a new tab Liver Each was excised, weighed, and frozen immediately after death. For measurement of total radioactivity per g wet wt, 100–300 mg was solubilized in 3 ml of Wallac Optisolve (LKB, Melbourne, Victoria, Australia) for 24 h at 22°C. The remainder was dispersed in phosphate-saline buffer, 1:1 wt/vol with sodium azide, 0.02% wt/vol, for 1 min in a Polytron PCU homogenizer (Kinematica, Steinhofhalde, Switzerland), held at 4°C for 3 d, then frozen and thawed three times. The supernatant was recovered after centrifugation at 113,000 × gav for 2 h at 4°C. The washed or swabbed tissue (mouse, 219–293 mg, human, 3.4 mg; see later) was solubilized in 2 ml of Wallac Optisolve (LKB) for 15 h at 22°C. For identification of [3H] molecules in the skin, a section of swabbed skin was digested by incubating with 400 U of collagenase type IV (Worthington Biochemicals, NJ) in 50 mM phosphate/NaCl buffer containing 1 mM calcium chloride, pH 7.25, for 12 h at 37°C. The digest was centrifuged at 113,000 × gav for 30 min; analysis was performed on the supernatant. Serum was prepared from blood collected immediately after death; 50 μl aliquots were decolorized with 100 μl of H2O2 30% vol/vol, for measurement of 3H content. This was centrifuged at 14,000 × gav for 10 min. Chromatography of serum, urine, and tissue extracts or digests was performed as follows: (i) in Superose 12 (Pharmacia, Uppsala, Sweden), column 1 cm × 30 cm, sample volume 0.5 ml, fractions 0.5 ml, elution rate 24 ml per h; (ii) in Sephacryl S-300 (Pharmacia), column 1.6 cm × 70 cm, sample volume 2 ml, fractions 2 ml, elution rate 14 ml per h; (iii) in Sephacryl S-1000 (Pharmacia), column 1.6 cm × 63 cm, sample volume 2 ml, fraction size 2 ml, elution rate 18 ml per h. A 0.15 M NaCl-phosphate buffer, pH 7.25 (Fraser et al., 1981Fraser J.R.E. Laurent T.C. Pertoft H. Baxter E. Plasma clearance, tissue distribution and metabolism of hyaluronic acid injected intravenously in the rabbit.Biochem J. 1981; 200: 415-424Crossref PubMed Scopus (282) Google Scholar) was used for tissue extraction or sample dilution with the addition of 0.2% Triton X-100 (New England Nuclear, MA) for chromatography. HA was identified in macromolecular fractions by repeating chromatography in Superose 12 to determine the proportion reduced to oligosaccharide after digestion with Streptomyces hyaluronidase, 10 turbidity reducing units (TRU) per ml, for 6 h at 37°C. HA was identified in skin by pretreating de-waxed paraffin sections with Streptomyces or testicular hyaluronidases (Calbiochem, CA), 50 TRU per ml dissolved in 0.1 M phosphate buffer, pH 6.8, containing protease inhibitors ovomucoid 250 μg, soybean trypsin inhibitor and pepstatin each 2 μg, iodoacetic acid 1 mM, and ethylenediamine tetraacetic acid 1.5 mM. A Wallac 1410 counter (Pharmacia) was used. Column fractions were monitored for [3H] activity by adding 3 ml of Wallac Hisafe II scintillant (LKB) to 0.5 ml of the column fractions. Activity in tissue samples was determined after adding 10 or 15 ml of Wallac Hisafe II scintillant. Definitive activity was measured for 20 min after decay of photo- and chemiluminescence was verified by stabilization of repeated preliminary counting. Fixed skin was dehydrated stepwise to 100% ethanol and embedded in paraffin. Sections of 2–4 μm were placed in pairs on slides, dewaxed, and rehydrated. One of each pair was immersed in Streptomyces or testicular hyaluronidase in 0.1 M phosphate buffer, pH 6.8, with protease inhibitors as previously described, and the other in the same solution without enzyme. The slides were held in a humidified chamber for 4 h at 37°C, washed, drained, dipped in diluted Ilford K5 autoradiographic emulsion (Ilford Australia, Melbourne, Australia) at 40°C, and drained again. After 2 or 3 wk in airtight chambers at 22°C, the emulsions were developed in Kodak D19 and fixed in Hypam Rapid Fixer (Ilford). The sections were then washed and lightly stained with hematoxylin. Mice To estimate amounts applied, the gel was weighed on a tared smooth latex fingerstall. In mice, about 50 mg (range 44.7–63.1 mg) was gently rubbed on a marked area of 5–6 cm2 on the dorsum of the trunk. Fingerstalls were then thoroughly soaked for 48 h in 2 ml of distilled water to measure the residue as 3H. Mice were allotted to two groups of four. In each, four were treated with radioactive gel and four with nonradioactive gel. At 12 hourly intervals, one group received three applications of gel and the other 12. The animals were killed for removal of tissues 12–16 h after the last application. Mice were treated with a single application of [3H]HA gel (range: 51.3–70.3 mg), held individually, with free access to food and water, in soft nonwettable plastic enclosures and constantly observed to ensure that they had not licked or rubbed the treated skin. Mice were killed 0.5, 1, 2, 4, 8, 16, and 24 h after application of the gel. Two mice were included in each series for histologic comparison of untreated skin. The treated skin of the mice was excised, weighed, and pinned on a flat base to its original size. Half was removed for solubilization and further analysis. The rest remained pinned while fixed for 16 h with 4% formaldehyde (i) in 0.174 M phosphate buffer, pH 7.5, and cetylpyridinium chloride (CPC), 0.5% wt/vol (series 1), or (ii), in 0.06 M phosphate, pH 7.5, and CPC 1.0% wt/vol (series 2). The fixative was recovered for chromatographic analysis of its3H content. Before solubilization, the unfixed skin in series 1 was gently agitated for 30 min three times in 3 ml of saline (0.9%NaCl solution); in series 2 the surface of the whole specimen was first swabbed with cotton moistened with saline. Initial trials in mice showed that massage of the gel on the skin for 7 min generated small cohesive fragments and left only some 20% of the 3H content on the skin. Thereafter application was ceased at the first sign of stickiness, which appeared within 2–5 min. In the human study two applications of 56.3 and 56.4 mg were made to an area of 1.8 cm2, 6 cm below the antecubital crease and 3 cm lateral to the medial border, and again 12 h later, with a nonradioactive control applied to the other forearm. Seven hours after the second application, the human skin was swabbed as previously described. Samples were then removed from both the treated and control areas with a diagnostic trephine of 3 mm diameter and adherent subcutaneous fat removed before fixation. 3H activity removed before fixation was measured in each case. The fraction of the gel application transferred to the skin ranged from 52 to 99% (mean ± SD: 71.4% ± 11.7, n = 44) in series 1, and from 70 to 91% (mean ± SD: 81.4% ± 4.6, n = 38), in the other mice. In the human study 85% of the second application was transferred to the skin. Both the stratum corneum and deeper layers of the epidermis were much thinner in the mouse than the human skin. Nevertheless comparison in mice with untreated skin showed no epidermal breach, erythrocyte extravasation or inflammatory cell infiltration in the dermis. Optimum autoradiographic development occurred after 3 wk. Thereafter, some fading of latent image was noted. Background development was very slight in the matched specimens treated with nonradioactive gels. Series 1. Twelve to 16 h after the last application positive development was found mainly in the dermis, from the outermost layer down to the lymphatic and blood vessels just above the platysma. Where counterstaining was sufficient to stain the collagen bundles, the silver grains were clearly confined to the ground substance between them Figure 1a). Grains were also aggregated at points in the keratinous layers of the epidermis but very few in its cellular layers or hair follicles. These findings indicated failure to penetrate these areas, more rapid transit through them, or degradation within these layers. Radioactive content was not significantly higher after 12 than after three applications (Table 1). In an attempt to capture activity in these sites further observations were therefore made after a single application of gels prepared with a much higher specific activity. Series 2. Positive development was found in the same distribution within dermis as early as 2 h after a single application and later in platysma (Figure 1b). Grains were also concentrated within cell boundaries at three levels: in the basal epidermis, in the dermal matrix and in the lining cells of the lymphatic sinuses (Figure 1c). We also found grains developed in the keratinized layer of the skin and in the rudimentary hair follicles. These were not completely removed by Streptomyces or testicular hyaluronidase Figure 2a). As there were no grains at these sites in the matched controls (Figure 2b), this observation could not be attributed to artifact. Although the density of grains was less intense overall, there was similar aggregation of grains in the keratinized layer Figure 3a), epidermis (Figure 3b), and clear penetration of activity to the deeper dermis with concentration notably at that level and just beneath the epidermis (Figure 3c). With the exception of that noted in the keratinized layers, positive autoradiographic development was reduced to background levels in all specimens by digestion with Streptomyces hyaluronidase. The fractions of the applied HA recovered from the surface of the skin at the end of each study were small from 8 h onward. In series 2, the quantity of applied HA recovered from within the skin was high 30 min after application, but at 1–8 h after application there was very little difference in the amount recovered from within the skin, suggesting that HA turnover took approximately 1 h to equilibrate. Mice receiving one or several applications of HA did demonstrate a lower proportion of [3H] radioactivity in the skin 12 and 16 h after application. To confirm that [3H] in the skin was HA or its metabolites the skin was digested with collagenase. After collagenase digestion the 3H molecules in the skin were identified as HA, identical in molecular weight to the applied HA Figure 4), and as its metabolites of acetate and water. No oligosaccharides were found which confirmed that the collagenase was hyaluronidase-free.Table 2Table 2Distribution of radioactivity retained in and on the skin after application of [3H]HA formulation to the surface of the skin aFigures represent mean ± SD, where n = 4 or 5.No. of applicationsTime after last application (h)[3H] HA appliedfRetained on skin immediately after last application. (μ g HA per g skin)[H3 H] HA remaining on surface of skingImmediately before removal of skin specimen. (μ g HA per g skin)3H recovered in skinbPercentage was determined by separation in a Superose 12 size exclusion gel.(i) as polymeric HA(ii) as HA metabolitesμg HA per g skin% of 3HcUndegraded HA polymer eluted at Kav 0 (Vo).[3H]AcetatedAcetate eluted at Kav 0.9.[3H]WatereWater eluted at Kav 1 (Vt).Series 13121352.3 ± 72.817.4 ± 1.70.04 ± 0.021.0 ± 1.03.0 ± 2.196.0 ± 4.812161081.3 ± 42.131.4 ± 1.2002.0 ± 0.598.0 ± 1.1Series 210.51361.4 ± 89.0121.9 ± 13.773.9 ± 10.9100 ± 000111295.1 ± 61.086.0 ± 12.749.6 ± 12.699.6 ± 0.40.4 ± 0.20121403.3 ± 71.043.4 ± 9.832.9 ± 8.895.2 ± 0.82.1 ± 0.42.7 ± 0.6141213.1 ± 68.485.2 ± 18.042.5 ± 12.789.0 ± 2.94.0 ± 0.57.0 ± 0.9181751.0 ± 112.445.3 ± 6.024.5 ± 4.977.2 ± 1.75.0 ± 1.117.8 ± 2.21161242.0 ± 66.72.5 ± 1.5002.0 ± 0.498.0 ± 0.41242558.8 ± 81.92.0 ± 1.3006.1 ± 2.193.9 ± 0.3Human skin2729,606 ± 05462.1 ± 029.6 ± 0hExpressed as μg HA/g skin, but not discriminated as polymer or metabolites and NA refers to "not analyzed".NANANAa Figures represent mean ± SD, where n = 4 or 5.b Percentage was determined by separation in a Superose 12 size exclusion gel.c Undegraded HA polymer eluted at Kav 0 (Vo).d Acetate eluted at Kav 0.9.e Water eluted at Kav 1 (Vt).f Retained on skin immediately after last application.g Immediately before removal of skin specimen.h Expressed as μg HA/g skin, but not discriminated as polymer or metabolites and NA refers to "not analyzed". Open table in a new tab Chromatography in Superose 12 showed a significant macromolecular content as early as 30 min after gel application, which was identified as HA by its complete degradation with Streptomyces hyaluronidase (results not shown). The metabolic end-products of acetate and water first appeared in the serum at 2 h after application, indicating that 1–2 h was required to generate detectable catabolism. The molecular weight profile of HA recovered in serum was only slightly lower (modal Mr of 3.6 × 105 Da, Figure 4b) than that of HA applied to skin, demonstrating that passage of HA through the skin was not restricted to its smaller polymers. Furthermore, in contrast to sheep (Fraser et al., 1988Fraser J.R.E. Kimpton W.G. Laurent T.C. Cahill R.N.P. Vakakis N. Uptake and degradation of hyaluronan in lymphatic tissue.Biochem J. 1988; 256: 153-158Crossref PubMed Scopus (153) Google Scholar), there had been no degradation or selective absorption of the larger polymers in their passage through the lymphatic system or in the bloodstream.Table 3Table 3Time sequence in appearance of hyaluronan and its metabolites in the mouse bloodstreamHA metabolitesbPercentage of HA metabolites was determined by separation in a Superose 12 size exclusion gelTime after last application (h)% of the total applied [3H] recovered in serumaThe d.p.m. appearing in the mouse serum was converted to the percentage of the applied [3H]HA dose by: mouseweight(g)×0.075(blood volume)×0.59(serumvolume of mouse)×[H]d.p.m./ml serum × 100[H]d.p.m. transferred to skinUndegraded polymeric [3H]HAcUndegraded HA polymers eluted at Kav0 (Vo) (%)AcetatedAcetate eluted at Kav 0.9 (%)WatereWater eluted at Kav1 (Vt) (%)0.50.36 ± 0.021000010.02 ± 0.041000020.40 ± 0.06Range: 20–99; Mean: 69Range: 1–6; Mean: 4Range: 0–74; Mean: 2740.96 ± 0.37Range: 4–95; Mean: 51Range: 0–7; Mean: 2Range: 0–95; Mean: 4784.46 ± 1.09Range: 0–9; Mean: 3Range: 0–4; Mean: 1Range: 90–100; Mean: 96164.82 ± 0.590Range: 0–5; Mean: 3Range: 95–100; Mean: 97243.24 ± 0.4200100Figures represent mean ± SD. n = 4 or 5.a The d.p.m. appearing in the mouse serum was converted to the percentage of the applied [3H]HA dose by:mouseweight(g)×0.075(blood volume)×0.59(serumvolume of mouse)×[H]d.p.m./ml serum × 100[H]d.p.m. transferred to skinb Percentage of HA metabolites was determined by separation in a Superose 12 size exclusion gelc Undegraded HA polymers eluted at Kav0 (Vo)d Acetate eluted at Kav 0.9e Water eluted at Kav1 (Vt) Open table in a new tab Figures represent mean ± SD. n = 4 or 5. There was negligible difference between activity in solubilized whole liver and aqueous extracts (extraction efficiency, mean 97.2%) as in other species (Fraser et al., 1981Fraser J.R.E. Laurent T.C. Pertoft H. Baxter E. Plasma clearance, tissue distribution and metabolism of hyaluronic acid injected intravenously in the rabbit.Biochem J. 1981; 200: 415-424Crossref PubMed Scopus (282) Google Scholar). Recovery from the extracts is shown in Table IV. From 4 to 8 h after application, increased amounts of activity were found, but mainly as the metabolites of the labeled acetyl group of HA. As in serum, relatively little was found as intact HA after 4 h, but there was clear evidence that HA had been absorbed and its metabolic degradation had begun within 1–2 h after application to the skin. Figures represent mean ± SD. n = 4 or 5. Seventeen specimens were obtained 0.73–8 h after application of HA. The percentage of [3H] in the urine ranged from 0.0008% to 6.065 (mean ± SD: 0.49% ± 2.31) of the applied dose. In Superose 12 chromatography, labeled HA and its metabolites, acetate and water, were identified in varying amounts as early as 50 min after application. Analysis in Sephacryl S-300 showed HA as small polymers and oligosaccharides, with fractions of 16–20 kDa, 2 kDa, and 0.7 kDa. No significant activity was detected in any of the human urine samples. In series 1 after tissues were fixed with formalin in 0.174 M phosphate with 0.5% CPC, the fixative contained a 4 kDa fraction identified by enzymic digestion as HA, in addition to [3H]acetate and 3H2O. When formalin was buffered in 0.06 M phosphate and its CPC content raised to 1% for the rest of the study, only [3H]acetate and 3H2O were detected in the fixative. Penetration of the topically applied HA from gels with and without polyethylene glycol (10 animals per group, Table 2), showed no difference in the kinetics, quantity or metabolism of the absorbed HA. The human study showed penetration throughout the dermis without polyethylene glycol (Figure 3b,c). Despite the strongly hydrophilic properties inherent in the primary structure of HA, this work clearly shows, by autoradiography and radiochemical analysis, that HA applied to the surface of the skin penetrates normal epidermis to accumulate at least briefly in the dermis before its disposal and degradation via known metabolic pathways. The rate-limiting steps in metabolic degradation of HA in vivo occur mainly at the level of monosaccharides and free acetate (Fraser et al., 1988Fraser J.R.E. Kimpton W.G. Laurent T.C. Cahill R.N.P. Vakakis N. Uptake and degradation of hyaluronan in lymphatic tissue.Biochem J. 1988; 256: 153-158Crossref PubMed Scopus (153) Google Scholar,Fraser et al., 1989Fraser J.R.E. Dahl L.B. Kimpton W.G. Cahill R.N.P. Brown T.J. Vakakis N. Elimination and subsequent metabolism of circulating hyaluronic acid in the fetus.J Devel Physiol. 1989; 11: 235-242PubMed Google Scholar), which should be removed in the aqueous and ethanolic phases of tissue preparation leaving only labeled HA. This was confirmed by the reduction of autoradiographic development to background levels after treatment with Streptomyces hyaluronidase, which degrades no other glycosaminoglycan. As noted in the Results section, the development of grains within the stratum corneum did not appear to be an artifact. As it resisted both Streptomyces and testicular enzymes, which are, respectively, exoglycosidases and endoglycosidases, this observation suggests an unexpectedly close association of HA with some component of this layer which is sufficient to hinder access by the enzymes' active site, temporarily concentrating the HA at this level. Passage of HA through skin in mice was confirmed by the recovery of labeled HA and the products of its metabolic degradation in skin, blood, liver, and urine. The failure to find metabolites in human urine is explicable by the much greater volume of body water relative to the applied amount of isotope, and the consequent dilution of any H2O generated. The Mr of the first batch of [3H]HA was lower than that of the unlabeled HA content, due to an unexpected response to sonication (see Materials and Methods). Nevertheless, the difference between 250 kDa and 400 kDa makes little difference to the Stokes radius of HA (seeFraser et al., 1996Fraser J.R.E. Cahill R.N.P. Kimpton W.G. Laurent T.C. The lymphatic system.in: Comper W.D. Extracellular Matrix. Vol. 1. Harwood Academic, London1996: 109-130Google Scholar). In the outcome, the larger labeled material, corresponding with that in the pharmaceutical preparation, still penetrated all layers of the skin with unexpected speed. With the exception of the stratum corneum, the positive autoradiographic development in all levels of the skin unquestionably represented intact HA, for the reasons just given. In series 1, it was consistently less intense in the deeper layers of the epidermis than in the dermis. This difference was still apparent with the higher specific activity of the gels and shorter intervals in the subsequent studies. The transit of HA through these layers is therefore remarkably rapid. As there is no inward movement of extracellular fluid at this point, its passage must be mediated by extracellular diffusion, active transport through the cells, or combinations thereof. The detection of grains within the basal epidermal cells supports a role for active transport. An analogy is provided by the facilitated vesicular transport of nutrients through intestinal mucosa, whi