Subject Number :S-17-MS-2012
Support Type :Common use (including technical support necessary for the training),
Proposal Title (English) : Probing the Penetration of Tofacitinib and Tofacitinib Citrate in Inflamed Murine Skin by Soft X-Ray Spectromicroscopy
Username (English) : K. Yamamoto1, A. Klossek1, J. Berkemeyer1, T. Ohigashi2, F. Rancan3, R. Flesch1, A. Vogt3, U. Blume-Peytavi3, M. Radbruch4, H. Pischon4, A.D. Gruber4, L. Mundhenk4, N. Kosugi2, and E. Rühl1
Affiliation (English) : 1Institute for Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany
2UVSOR Synchrotron Facility, Institute for Molecular Science, Okazaki 444-8585, Japan
3Charité Universitätsmedizin, 10117 Berlin, Germany
4Veterinary Pathology, Freie Universität Berlin, 14163 Berlin, Germany

The penetration of hydrophilic and hydrophobic drugs into human skin is systematically investigated by label-free detection employing soft X-ray microscopy. The FDA-approved anti-inflammatory drug tofacitinib citrate (C16H20N6O • C6H8O7, molecular weight 504.49 g/mol), marketed under the name Xeljanz, is chosen as a hydrophilic drug, which acts as a JAK inhibitor by interfering with the JAKSTAT signaling pathway. It is a low molecular weight drug that is commonly used for oral drug delivery for treating e.g. rheumatoid arthritis. Its use for treating plaque psoriasis is currently under investigation [1]. We also included the free base of tofacitinib (C16H20N6O, molecular weight 312.37 g/mol) as a hydrophobic complement to tofacitinib citrate. This enables us to investigate the influence of hydrophilicity on dermal drug penetration.
The experiments made use of excised murine skin, which was inflamed and exposed to tofacitinib citrate and the free base obtained from LC Labs for penetration times between 10 and 1000 min, similar to earlier work using dexamethasone as a drug [2-3].
Spectromicroscopy studies were performed at the BL4U beamline at UVSOR III using a scanning X-ray microscope. The chemical selectivity for probing the drugs in EPON-fixed skin was achieved by exciting the samples selectively at the O 1s-edge in the pre-edge regime, i.e. at 528 eV (pre-edge) and 531.8 eV, respectively, corresponding to the O 1s * transition of the drugs. Fig. 1 shows the O 1s regime of fixed murine skin, tofacitinib, and tofacitinib citrate.

Fig. 1. X-ray absorption of tofacitinib citrate, the free base, and fixed murine skin near the O 1s-edge.
Fig. 1. This provides chemical selectivity for label-free probing of tofacitinib. Differential absorption maps corresponding to the drug distribution as a function of depth were derived similar to our previous work [2-3]. Fig. 2 shows the drug distribution in the top skin layers, consisting of stratum corneum (SC) and viable epidermis (VE). There are distinct differences between the drug distributions after 100 min penetration time. The hydrophilic drug tofacitinib citrate is primarily found in the top layers of the stratum corneum and no drug is found in deeper skin layers (see Fig. 2(a)). In contrast, the free base is found in deeper layers of the stratum corneum and in the viable epidermis (see Fig. 2(b)). There, the drug is preferably found in the thin membranes of the keratinocytes surrounding the cell nuclei. This corresponds to the site of action of this drug. The present results also indicate that topically applied hydrophobic drugs can more efficiently penetrate the top layers of skin than the hydrophilic analogue.

Fig. 2. Differential absorption of (a) tofacitinib citrate and (b) the free base of tofacitinib penetrating the top skin layers of murine skin for 100 min, respectively. High drug concentration is indicated by yellow color.

[1] V. Di Lernia et al., Drug Design Develop. Therap. 10 (2015) 533.
[2] K. Yamamoto et al., Anal. Chem. 87 (2015) 6173.
[3] K. Yamamoto et al., Eur. J. Pharm. Biopharm. 118 (2017) 30.