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Reita Kadowaki,1 Fumihiko Ogata,1 Miku Nishida,1 Miri Komatsu,1 Hiroko Otake,1 Yosuke Nakazawa,2 Naoki Yamamoto,3 Naohito Kawasaki,1 Noriaki Nagai1 1Faculty of Pharmacy, Kindai University, Higashi-Osaka, Osaka, Japan; 2Faculty of Pharmacy, Keio University, Minato-ku, Tokyo, Japan; 3Support Office for Bioresource Research, Research Promotion Headquarters, Fujita Health University, Toyoake, Aichi, JapanCorrespondence: Noriaki Nagai, Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka, 577-8502, Japan, Tel +81 6 4307 3638, Fax +81 6 6730 1394, Email nagai_n@phar.kindai.ac.jpPurpose: Medical therapies, such as the use of anti-inflammatory agents, are commonly used for the treatment of oral mucositis (OM). However, these treatments have limited efficacy in treating severe cases of OM. In this study, we aimed to develop a carbopol gel incorporating troxipide (TRO) nanoparticles and methylcellulose (TRO-NP gel) and demonstrate its efficacy in accelerating wound healing in a hamster model of OM (OM model) induced by acetic acid injection.Methods: TRO nanoparticles were prepared using bead milling. The crystalline form was determined by powder X-ray diffraction, and the particle size was measured using a NanoSight LM10 instrument. The drug release was determined using a Franz diffusion cell, and the hamsters injected with acetic acid were selected to evaluate the therapeutic effect of OM.Results: After preparing TRO nanoparticles, we observed a mixture of crystals and amorphous TRO, and the particle size of TRO in the TRO-NP gel ranged from 50 to 280 nm. The TRO-NP gel exhibited a more uniform TRO distribution and viscosity compared to the Carbopol gel containing TRO microparticles (TRO-MP gel). However, the solubility of TRO was comparable in both TRO-MP and TRO-NP gels. The TRO-NP gel released a higher amount of TRO than that from the TRO-MP gel, with detectable release of TRO nanoparticles. TRO levels in the cheek pouches of hamsters treated with TRO-NP gel were higher than those treated with TRO-MP gel. The increased TRO levels in the cheek pouches of hamsters treated with TRO-NP gel were attenuated by treatment with 40 μM dynasore, an inhibitor of clathrin-dependent endocytosis (CME). Moreover, the therapeutic effect of the TRO-NP gel was superior to that of the TRO-MP gel in the hamster model of OM.Conclusion: We have designed a TRO-NP gel, and this gel showed excellent TRO delivery into the cheek pouch tissue through the CME pathway. Moreover, the TRO-NP gel treatment enhanced wound healing after acetic acid injection. Keywords: troxipide, nanoparticle, oral mucositis, hydrogel, endocytosis

Shuya Masuda,1,* Saori Deguchi,1,* Fumihiko Ogata,1 Joji Yoshitomi,1 Hiroko Otake,1 Kazutaka Kanai,2 Naohito Kawasaki,1 Noriaki Nagai1 1Faculty of Pharmacy, Kindai University, Osaka, Japan; 2Department of Small Animal Internal Medicine, School of Veterinary Medicine, University of Kitasato, Aomori, Japan*These authors contributed equally to this workCorrespondence: Noriaki Nagai, Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka, 577-8502, Japan, Tel +81 6 4307 3638, Fax +81 6 6730 1394, Email nagai_n@phar.kindai.ac.jpPurpose: We designed a 0.05% mometasone furoate (MF) nanocrystal dispersion and investigated whether the application of MF nanocrystals in nasal formulations enhanced local absorption compared to traditional nasal MF formulations (CA-MF).Methods: MF nanocrystal dispersions (MF-NPs) were prepared by bead milling MF microcrystal dispersions (MF-MPs) consisting of MF, 2-hydroxypropyl-β-cyclodextrin, methylcellulose, and purified water. Pluronic F-127 combined with methylcellulose, Pluronic F-68, or carbopol was used as a base for in situ gelation (thickener). MF concentrations were measured using high-performance liquid chromatography, and nasal absorption of MF was evaluated in 6 week-old male Institute of Cancer Research (ICR) mice.Results: The particle size range of MF prepared with the bead mill treatment was 80– 200 nm, and the nanoparticles increased the local absorption of MF, which was higher than that of CA-MF and MF-MPs. In addition, unlike the results obtained in the small intestine and corneal tissue, the high absorption of nanocrystalline MF in the nasal mucosa was related to a pathway that was not derived from energy-dependent endocytosis. Moreover, the application of the in situ gelling system attenuated the local absorption of MF-NPs, owing to a decrease in drug diffusion in the dispersions.Conclusion: We found that nanoparticulation of MF enhances local intranasal absorption, and nasal bioavailability is higher than that of CA-MF. In addition, we demonstrate that viscosity regulation is an important factor in the design of nasal formulations based on MF nanocrystals. These findings provide insights for the design of novel nanomedicines with enhanced nasal bioavailability.Plain Language Summary: Mometasone furoate (MF) is administered nasally to treat nasal inflammatory diseases. In this study, we designed MF nanocrystal dispersions (MF-NPs) and investigated whether the application of MF nanocrystals in nasal MF formulations enhanced local absorption compared to traditional nasal MF formulations. MF-NPs were obtained via bead-milling of dispersions, consisting of MF, 2-hydroxypropyl-β-cyclodextrin, methylcellulose, and purified water, and the MF-NPs with in situ gelation were prepared by the addition of a gel base consisting of Pluronic F-127 combined with methylcellulose, Pluronic F-68, or carbopol. The nasal absorption of MF was evaluated in 6 week-old ICR mice. The bead-milled MF particles in the dispersions were crystalline with sizes of 80– 200 nm. Moreover, MF-NPs exhibited lower viscosity and higher local absorption than commercially available nasal formulations and dispersions containing microcrystalline MF. However, the application of in situ gelling systems attenuated the local absorption of the MF-NPs. This is the first study to investigate the nasal absorption of dispersions containing nanocrystalline MF and the basis of in situ gelation, providing important information for the design of nanomedicines with enhanced nasal bioavailability.Graphical Abstract: Keywords: mometasone furoate, nanocrystal, nasal absorption, drug delivery, in situ gel

Hiroko Otake,1,* Ryoka Goto,1,* Fumihiko Ogata,1 Takumi Isaka,1 Naohito Kawasaki,1 Shinichiro Kobayakawa,2 Toru Matsunaga,3 Noriaki Nagai1 1Faculty of Pharmacy, Kindai University, Higashi-Osaka, Osaka, 577-8502, Japan; 2Department of Ophthalmology, Nippon Medical School, Musashi-Kosugi Hospital, Kawasaki, Kanagawa, 211-8533, Japan; 3Design and Development, SEED Co., Ltd., Kounosu-shi, Saitama, 369-0131, Japan*These authors contributed equally to this workCorrespondence: Noriaki NagaiFaculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka, 577-8502, JapanTel +81 6 4307 3640Fax +81 6 6730 1394Email nagai_n@phar.kindai.ac.jpPurpose: The multi-instillation of three commercially available (CA) eye drops [fluorometholone (FL)-, bromfenac (BF)- and levofloxacin (LV)-eye drops] has been used to manage pain and inflammation post-intraocular surgery. However, the multi-instillation of these three eye drops causes corneal damage, and the FL drops have the disadvantage of low ocular bioavailability. To overcome these problems, we prepared fixed-combination eye drops based on FL nanoparticles (FL-NPs) and BF/LV solution (nFBL-FC), and evaluated the corneal toxicity and transcorneal penetration of the nFBL-FC eye drops.Methods: FL powder was mixed in 2-hydroxypropyl-β-cyclodextrin solution containing benzalkonium chloride, mannitol and methylcellulose, and milled with a Bead Smash 12 (5500 rpm for 30 s× 30 times). The BF/LV solution was then added to the milled-dispersions to be used as nFBL-FC. The FL, BF and LV concentrations were measured by HPLC methods, and transcorneal penetration was evaluated in rabbits.Results: The FL particle size in nFBL-FC was 40– 150 nm, with only 0.0018% in liquid form. No aggregation of FL particles in the nFBL-FC was observed for 1 month. The viability of human corneal epithelial cells treated with nFBL-FC was remarkably higher than that of cells subjected to the multi-instillation of the corresponding three CA-eye drops. In addition, the corneal penetrations (AUC) of the FL, BF and LV in nFBL-FC were 4.9-, 1.8-, and 7.1-fold those of the corresponding CA-eye drops, respectively. Moreover, the caveolae-dependent endocytosis (CavME) inhibitor (nystatin) significantly prevented the transcorneal penetration of these drugs.Conclusion: We prepared fixed-combination eye drops based on FL-NPs and BF/LV solution (nFBL-FC), and show that high levels of FL-NPs and dissolved BF/LV (liquid drugs) can be delivered into the aqueous humor by the instillation of nFBL-FC. Further, we show that CavME is mainly related to the enhancement of transcorneal penetration of both the solid (NPs) and liquid drugs.Keywords: fluorometholone, bromfenac, levofloxacin, fixed-combination eye drops, corneal permeability, endocytosis

Noriaki Nagai,1 Fumihiko Ogata,1 Hiroko Otake,1 Yosuke Nakazawa,2 Naohito Kawasaki1 1Faculty of Pharmacy, Kindai University, Higashi-Osaka 577-8502, Japan; 2Faculty of Pharmacy, Keio University, Minato-ku, Tokyo 105-8512, Japan Purpose: We previously found that ophthalmic formulations containing nanoparticles prepared by a bead mill method lead to an increase in bioavailability in comparison with traditional formulations (solution type). However, the transcorneal penetration pathway for ophthalmic formulations has not been explained yet. In this study, we investigated the mechanism of transcorneal penetration in the application of ophthalmic formulations containing indomethacin nanoparticles (IMC-NPs).Materials and methods: IMC-NPs was prepared by the bead mill method. For the analysis of energy-dependent endocytosis, corneal epithelial (HCE-T) cell monolayers and removed rabbit cornea were thermoregulated at 4°C, where energy-dependent endocytosis is inhibited. In addition, for the analysis of different endocytosis pathways using pharmacological inhibitors, inhibitors of caveolae-mediated endocytosis (54 µM nystatin), clathrin-mediated endocytosis (40 µM dynasore), macropinocytosis (2 µM rottlerin) or phagocytosis (10 µM cytochalasin D) were used.Results: The ophthalmic formulations containing 35–200 nm sized indomethacin nanoparticles were prepared by treatment with a bead mill, and no aggregation or degradation of indomethacin was observed in IMC-NPs. The transcorneal penetration of indomethacin was significantly decreased by the combination of nystatin, dynasore and rottlerin, and the decreased penetration levels were similar to those at 4°C in HCE-T cell monolayers and rabbit cornea. In the in vivo experiments using rabbits, dynasore and rottlerin tended to decrease the transcorneal penetration of indomethacin (area under the drug concentration – time curve in the aqueous humor [AUCAH]), and the AUCAH in the nystatin-treated rabbit was significantly lower than that in non-treatment group. In addition, the AUCAH in rabbit corneas undergoing multi-treatment was obviously lower than that in rabbit corneas treated with each individual endocytosis inhibitor.Conclusion: We found that three energy-dependent endocytosis pathways (clathrin-dependent endocytosis, caveolae-dependent endocytosis and macropinocytosis) are related to the transcorneal penetration of indomethacin nanoparticles. In particular, the caveolae-dependent endocytosis is strongly involved. Keywords: drug delivery system, bead mill, caveolae-dependent endocytosis, clathrin-dependent endocytosis, macropinocytosis

Noriaki Nagai,1 Fumihiko Ogata,1 Hiroko Otake,1 Yosuke Nakazawa,2 Naohito Kawasaki1 1Faculty of Pharmacy, Kindai University, Higashi-Osaka, Osaka, Japan; 2Faculty of Pharmacy, Keio University, Minato-ku, Tokyo, Japan Purpose: In the clinical setting, raloxifene, a second-generation selective estrogen receptor modulator, is administered orally; however, the bioavailability (BA) is only 2% because of its poor solubility in aqueous fluids and its extensive first-pass metabolism. Therefore, it is expected that the development of a transdermally delivered formulation may reduce the necessary dose without compromising its therapeutic efficacy. In this study, we designed transdermal formulations containing raloxifene nanoparticles and evaluated their usefulness for osteoporosis therapy. Methods: Raloxifene was crushed with methylcellulose by the bead mill method, and the milled raloxifene was gelled with or without menthol (a permeation enhancer) by Carbopol® 934 (without menthol, Ral-NPs; with menthol, mRal-NPs). The drug release and transdermal penetration were measured using a Franz diffusion cell, and the therapeutic evaluation of osteoporosis was determined in an ovariectomized rat model. Results: The mean particle size of raloxifene in the transdermal formulation (Ral-NPs) was 173.7 nm. Although the raloxifene released from Ral-NPs remained in the nanoparticle state, the skin penetration of raloxifene nanoparticles was prevented by the stratum corneum in rat. On the other hand, inclusion of menthol in the formulation attenuated the barrier function of the stratum corneum and permitted the penetration of raloxifene nanoparticles through the skin. Moreover, macropinocytosis relates to the skin penetration of the formulation including menthol (mRal-NPs), since penetration was inhibited by treatment with 2 µM rottlerin, a macropinocytosis inhibitor. In addition, the application of 0.3% mRal-NPs (once a day) attenuated the decreases in calcium level and stiffness of the bones of ovariectomized rat. Conclusion: We prepared raloxifene solid nanoparticles by a bead mill method and designed a novel transdermal formulation containing nanoparticles and permeation enhancers. These transdermal formulations overcome the barrier properties of the skin and show high drug penetration through the transdermal route (BA 8.5%). In addition, we found that raloxifene transdermal formulations are useful for the treatment of osteoporosis in ovariectomized rat. Keywords: nanomedicine, transdermal delivery system, permeation enhancer, endocytosis, skin

In order to gain insights on the conditions of aqueous alteration on asteroid Ryugu and the origin of water in the outer solar system, we developed the measurement of water content in magnetite at the micrometer scale by secondary ion mass spectrometry (NanoSIMS) and determined the H and Si content of coarse‐grained euhedral magnetite grains (polyhedral magnetite) and coarse‐grained fibrous (spherulitic) magnetite from the Ryugu polished section A0058‐C1001. The hydrogen content in magnetite ranges between ~900 and ~3300 wt ppm equivalent water and is correlated with the Si content. Polyhedral magnetite has low and homogenous silicon and water content, whereas fibrous magnetite shows correlated Si and water excesses. These excesses can be explained by the presence of hydrous Si‐rich amorphous nanoinclusions trapped during the precipitation of fibrous magnetite away from equilibrium and testify that fibrous magnetite formed from a hydrous gel with possibly more than 20 wt% water. An attempt to determine the water content in sub‐μm framboids indicates that additional calibration and contamination issues must be addressed before a safe conclusion can be drawn, but hints at elevated water content as well. The high water content in fibrous magnetite, expected to be among the first minerals to crystallize at low water–rock ratio, points to the control of water content by local conditions of magnetite precipitation rather than large‐scale alteration conditions. Systematic lithological variations associated with water‐rich and water‐poor magnetite suggest that the global context of alteration may be better understood if local water concentrations are compared with millimeter‐scale distribution of the various morphologies of magnetite. Finally, the high water content in the magnetite precursor gel indicates that the initial O isotopic composition in alteration water must not have been very different from that of the earliest magnetite crystals. [ABSTRACT FROM AUTHOR]