Tomohiro Hikima

Effect of Ultrasound Application on Skin Metabolism of Prednisolone 21-Acetate

AbstractPurpose. The effect of ultrasound on skin penetration and metabolism of prednisolone (PN) and prednisolone 21-acetate (PNA) was investigated in the hairless mouse skin in vitro. Methods. The abdominal skin excised freshly was pretreated under different ultrasound intensities (4.32, 2.88, and 1.50 W/cm2) for 10, 30, and 60 min. The penetration/metabolism rate of PNA and its metabolite (PN) was then measured using a side-by-side diffusion cell. Results. The skin penetration of PN was enhanced by the ultrasound pretreatment. This enhancement was attributed to the decrease in the stratum corneum barrier capacity by ultrasound energy. The steady-state appearance rate of PN following the skin bioconversion of PNA decreased appreciably with increasing the product of the duration of pretreatment (Dp, min) and the intensity of ultrasound applied (Iu W/cm2). When the product value was less than 40 W/cm2 ⋅ min, the steady-state appearance rate of the PN hardly increased in spite of the penetration enhancement of PNA. Conclusions. These findings indicated a possible deactivation of the skin enzymes by ultrasound energy.

Diffusion and Metabolism of Prednisolone Farnesylate in Viable Skin of the Hairless Mouse

The diffusion and metabolism of prednisolone 21-farnesylate were investigated in viable skin of the hairless mouse in vitro. The pro-drug ester was extensively metabolized in viable skin, while it was stable in the donor and receptor solutions. The rate of appearance of the prodrug and its metabolite prednisolone was markedly influenced by the direction of the skin placed between the in vitro diffusion half-cells. The rate of bioconversion of the prodrug was determined as a function of the distance from the surface of the skin. The prodrug was increasingly metabolized with the distance from the surface of the skin, indicating that the responsible enzymes are enriched in the lower layers of the viable skin. A model with linearly increasing enzyme activity in the viable skin accounts for the in vitro profiles of the diffusion/metabolism of the prodrug in the viable skin of hairless mouse.

Comparison of skin distribution of hydrolytic activity for bioconversion of β-estradiol 17-acetate between man and several animals in vitro

We have investigated the distribution of hydrolytic enzymes which metabolize beta-estradiol 17-acetate (EA) to beta-estradiol (E) in man and animal skins in vitro. The distribution of hydrolytic enzymes in human cadaver, hairless dog, rat and hairless mouse skin, was investigated by a skin-slicing technique. We performed histological studies with hematoxylin and eosin stain. The highest amount of metabolite (E) appeared in the layers of 80-120 microm from the skin surface, the basement layer in human skin, while the amount of metabolite was distributed evenly in the hairless dog skin from 0 to 180 microm. In the rat and hairless mouse skin, on the other hand, peak levels of metabolite were observed in the basement layer of dermis, the surrounding area of the cutaneous plexus. The total metabolic activities in the area of epidermis in human, hairless dog and hairless mouse skin were 2.59, 8.03 and 0.33 x 10(-4) microg/ml/microm/h, respectively. The values in whole skin layers in the hairless dog and hairless mouse skin were 3.35 and 1.85 x 10(-4)microg/ml/microm/h, respectively. EA transported across the human and hairless dog skin can be effectively metabolized before entering the capillary. Among animal models investigated, hairless dog skin might be the most facile model in simulating drug metabolism for human skin under the clinical (in vivo) conditions. Hairless mouse skin, on the other hand, was also an excellent model in excised human skin under in vitro conditions.

Gender Differences of Enzymatic Activity and Distribution of 17β-Hydroxysteroid Dehydrogenase in Human Skin in vitro

The interconversion of estrone (E1) and 17β-estradiol (E2) is catalyzed by 17β-hydroxysteroid dehydrogenase (17β-HSD) in peripheral steroidogenic organs such as the skin. To investigate gender differences of activity and skin distribution of 17β-HSD in human skin, enzymatic activity was measured in skin homogenates and skin horizontally sliced by 10 µm thickness in vitro. Reductive 17β-HSD (E2 formation from E1) in female skin has a lower substrate affinity than in male skin; Km (Michaelis-Menten constant) of female and male skin is 11.8 ± 6.5 and 2.0 ± 2.0 µM, respectively. Female skin had a tendency to activate estrogen; Vmax (maximum rate) for E2 formation, 5.8 ± 4.0 pmol/min/mg protein, is 1.7 times larger than E1 formation, 3.5 ± 1.5 pmol/min/mg protein, and, on the other hand, male skin tends to deactivate estrogen; Vmax for E1 and E2 is 10.5 ± 6.1 and 4.2 ± 3.7 pmol/min/mg protein, respectively. The concentration of metabolite had a peak value at 80–120 µm from the skin surface. Therefore, these in vitro results suggest that the enzymatic activities of 17β-HSDs have a gender difference in estrogen formation/metabolism and are distributed around the basement layer of the epidermis irrespective of sex. 17β-HSDs distributed around the basement epidermis may be effectively supplied with circulating estrogen from the papillary plexus to maintain the estrogen level in skin. This distribution pattern having a peak surrounding 100 µm from the skin surface indicates the importance for defense from noxae (e.g. detoxication) and maintenance of the internal environment (e.g. biosynthesis of hormones). Future studies should increase sample size and confirm these results by stricter statistical analysis.

Skin metabolism in transdermal therapeutic systems.

Skin has at least two barriers with protective functions: the stratum corneum physical barrier and a biochemical barrier in the epidermis and dermis. Numerous chemical and physical enhancers exist for transdermal therapeutic systems; some cause irritation, and possibly influence enzyme deactivation. Knowledge of enzymatic skin reactions is important for developing safe and efficacious transdermal systems for treatment not only of skin diseases but also for systemic application. This paper overviews the effects of (a) chemical enhancers and additives, (b) drug structure, and (c) physical enhancement on skin metabolism.

Binding of Prednisolone and Its Ester Prodrugs in the Skin

AbstractPurpose. Skin binding of prednisolone and its esters was investigated in the hairless mouse skin in vitro.Methods. The distribution of the amount of drugs bound in the skin was determined by a skin slicing technique. The model drugs used were prednisolone (PN, M.W. 360) and its esters, senesyonate (PN-C5, M.W. 442), geranate (PN-C10, M.W. 510), farnesylate (PN-C15, M.W. 578), and geranylgeranate (PN-C20, M.W. 646). Results. The distribution of bound drug was nonhomogeneous in the skin; the concentration of PN-C10 and PN-C15 in the skin increased gradually with the distance from the skin surface. The parent drug, PN, however, was hardly bound in the viable skin. Conclusions. These findings suggest that the prodrugs of prednisolone may prolong the dermal retention of the parent drug and minimize to delivery into the systemic circulation of the prodrug and metabolite.

Skin accumulation and penetration of a hydrophilic compound by a novel gemini surfactant, sodium dilauramidoglutamide lysine.

We investigated a novel peptide-based gemini amphiphilic compound, sodium dilauramidoglutamide lysine (DLGL), as a chemical enhancer for the skin penetration of l-ascorbic acid 2-glucoside (AAG). A three-dimensional cultured human skin product, TESTSKIN™ LSE-high (LSE-high), was used as a skin model. The penetration flux of AAG with DLGL and that obtained with sodium lauramidoglutamide (LG) as a conventional surfactant across LSE-high were increased by 12.56 and 69.29 times compared to the control, respectively. The ratio of AAG amount with DLGL in the skin (21.78% total dose) was significantly increased (p<0.05) compared to the control (7.23%) and to the AAG amount with LG (8.13%). The AAG amounts in receptor were 1.06% (control), 3.19% (+DLGL) and 21.00% (+LG). Thus, DLGL preserved AAG in skin, resulting in enhanced AAG penetration flux. However, LG might create the pathways through the skin. We conclude that DLGL is a gemini surfactant that accumulates a hydrophilic compound in skin and enhances the penetration flux. DLGL may therefore be a novel addition agent for skin local therapy.

Distribution of hydrolytic activity catalyzes the biotransformation of prednisolone 21-acetate in human skin.

We investigated the distribution of hydrolytic enzymes which metabolize prednisolone 21-acetate (PNA) to prednisolone (PN) in human skin. Km (Michaelis-Menten constant) and Vmax (maximum rate) of hydrolytic enzyme in human skin was 25.1 µM and 0.46 nmol/min/mg protein, respectively. Specific activities of hydrolysis in dermis and epidermis were similar and, in consideration to their thickness, hydrolytic activity in epidermis was 12.1 times higher than in dermis. Moreover, the highest amount of metabolite (PN) was found at 80–120 µm from the skin surface by skin slicing. Therefore, hydrolytic activity which metabolized PNA was distributed in epidermis, especially in the basement membrane area; epidermis borders dermis in this area and the papillary plexus is reached just beneath the dermal papillae. These results suggest that the distribution of hydrolytic activity in human skin may prevent certain substances from entering the systemic circulation in their unhydrolyzed form.

Metabolism of Prednisolone 21-Acetate in Hairless Mouse Skin

We investigated the hydrolytic activity of prednisolone 21-acetate (PNA) to prednisolone (PN) in an enzyme solution composed of esterase and skin homogenates from hairless mice. The values of the Michaelis-Menten constant obtained from hairless mouse skin and esterase solution were 14.2 and 10.2 µM, respectively; conversely, the value of the maximum rate from hairless mouse and esterase solution were 0.67 and 1,886 nmol/min/mg protein, respectively. To examine the effect of enzymatic inhibitors on hydrolytic activity, five enzymatic inhibitors, 3,4-dichloroisocoumarine (DCIC), N-tosyl-L-phenylalanine chloromethyl ketone, iodoacetamide, p-hydroxymercuribenzoic acid (HMBA) and sodium dodecylsulfate, were added to the enzyme solution. Sixty-eight percent of hydrolytic activity in skin homogenates were not deactivated by DCIC which completely inhibited the enzymatic activity in esterase solution. We also studied the localization of hydrolytic enzyme with a subcellular faction: 66 and 11% of specific activity existed in microsome (Ms) and cytosol (Cp) fractions, indicating that the hydrolytic activity of PNA was included mainly in the Ms fraction. Hydrolytic activity in Ms and Cp fractions was different from sensitivity to enzymatic inhibitor; DCIC inhibited activity in the Ms fraction and, on the other hand, HMBA inhibited it in the Cp fraction. Therefore, Ms and Cp fractions in skin homogenates include a different esterase isoform and the metabolism of PNA to PN in hairless mouse skin is mediated by these isoforms.

Combined Use of Iontophoresis and Other Physical Methods

The progress of transdermal therapeutic systems is based on the development of chemical and physical penetration enhancers. Especially, physical enhancers transiently disrupt the stratum corneum’s barrier function using electrical methods (iontophoresis and electroporation), other energy sources such as ultrasound (sonophoresis), and mechanical force (microneedles). Iontophoresis (a direct current ≤ 0.5 mA/cm2) can enhance and control the skin penetration flux of low molecular weight drugs (≤300 Da), while the flux of high molecular weight drugs (≥ 7 kDa) is difficult to increase. The other enhancers are able to improve the flux of macromolecules (i.e., proteins and nucleic acid) by creating and expanding pores in the stratum corneum by high-voltage pulses (electroporation), changing the stratum corneum structure by cavitation (sonophoresis), and creating microchannels through the stratum corneum (microneedles). This chapter provides a review about the combined use of iontophoresis and other physical enhancement methods, and the advantages and disadvantages of the combination will be discussed.