Background: We previously reported that carbopol hydrogels incorporating indomethacin nanoparticles (IMC NPs) improved the low permeability and bioavailability of skin formulations in transdermal drug delivery systems. However, the combination of NPs with other types of hydrogels has not been sufficiently explored to date. Therefore, this study investigated propylene glycol (PG)/polyvinyl alcohol (PVA) hydrogel as an alternative base to carbopol hydrogel for incorporating IMC NPs. Methods: IMC NPs were prepared using bead milling treatment, and these NPs were incorporated into PG/PVA hydrogel (IMC-NP@PG/PVA hydrogel). The IMC concentration was measured using the HPLC method, and seven-week-old Wistar rats were used to evaluate skin absorption. Results: Bead milling reduced the IMC particle size in the PG/PVA hydrogels to the nanoscale (30–200 nm) without altering its crystalline form. The IMC-NP@PG/PVA hydrogel exhibited enhanced uniformity, solubility, and drug release compared to the IMC microparticle-loaded PG/PVA hydrogel (IMC-MP@PG/PVA hydrogel), with a 1.44-fold greater area under the concentration–time curve. Transdermal permeability studies revealed that IMC-NP@PG/PVA had 2.36-fold higher absorption than the IMC-MP@PG/PVA hydrogel, with dissolved IMC permeating the skin. Pharmacokinetics in the rats showed significantly increased plasma levels, absorption rates, and bioavailability for IMC-NP@PG/PVA, demonstrating its superior delivery efficiency. Moreover, the skin absorption of IMC-NP@PG/PVA was higher than that of carbopol hydrogel. Conclusions: These findings highlight the potential of PG/PVA hydrogels as an effective base for transdermal drug delivery systems based on NPs.

Background and Objectives: In previous studies, we reported that the assessment of the cumulative thermal dose in the crystalline lens, conducted through computational modeling utilizing a supercomputer and the biothermal transport equation, exhibited a significant association with the incidence of nuclear cataracts. In this study, we have investigated the types of proteins that expressed underlying 35.0 °C (normal-temp) and 37.5 °C (warming-temp) by using the shotgun liquid chromatography (LC) with tandem mass spectrometry (MS/MS)-based global proteomic approach. Materials and Methods: We have discussed the changes in protein expression in warmed iHLEC-NY2 cells using Gene Ontology analysis and a label-free semiquantitative method based on spectral counting. Results: In iHLEC-NY2, 615 proteins were detected, including 307 (49.9%) present in both lenses cultured at normal-temp and warming-temp, 130 (21.1%) unique to the lens cultured at normal-temp, and 178 (29.0%) unique to the lens cultured at warming-temp. Furthermore, LC-MS/MS analysis showed that warming decreased the expression of actin, alpha cardiac muscle 1, actin-related protein 2, putative tubulin-like protein alpha-4B, ubiquitin carboxyl-terminal hydrolase 17-like protein 1, ubiquitin-ribosomal protein eL40 fusion protein, ribosome biogenesis protein BMS1 homolog, histone H2B type 1-M, and histone H2A.J. in iHLEC-NY2. Conclusions: The decreases in the specific protein levels of actin, tubulin, ubiquitin, ribosomes, and histones may be related to cataract development under warming conditions. This investigation could provide a critical framework for understanding the correlation between temperature dynamics and the development of nuclear cataracts.

In the present study, magnetic-calcined bamboo composite adsorbents (MCBC200, MCBC400, MCBC600, MCBC800, and MCBC1000) were prepared, and their physicochemical characteristics (scanning electron microscope images, differential thermogravimetric analysis, Fourier transform-IR, specific surface area, surface functional groups, and point of zero charge [pHpzc]) were evaluated. Furthermore, the adsorption capacity of methylene blue (MB, cationic dye) using the prepared adsorbents was assessed. The value of pHpzc and the specific surface area of MCBC400 were 7.8 and 50.6 m2/g, respectively. The amounts of acidic or basic functional groups of MCBC400 were relatively greater than those of the other adsorbents. The amount of MB adsorbed onto MCBC400 (31.9 mg/g) was higher than that onto other adsorbents. The adsorption of MB using MCBC400 was evaluated in relation to various parameters, including coexistence, solution pH, adsorption temperature, and contact time. The results followed the Langmuir isotherm model and a pseudo-second-order model with correlation coefficients of 0.980-1.000 and 0.996, respectively. MB was selectively adsorbed by MCBC400 in a binary solution system containing anionic dyes. Finally, one of the adsorption mechanisms was determined by analyzing the elemental distribution and the binding energy before and after the adsorption of MB. The current findings provide important information for removing MB with MCBC400 from the aqueous phase.

Previous studies have reported that a strong correlation between the estimated cumulative thermal exposure in the crystalline lens and the incidence of nuclear cataracts; however, the precise relationship between temperature and cataracts remains to be fully elucidated. In the present study, the shotgun liquid chromatography/mass spectroscopy‑based global proteomic approach was applied to investigate cataract‑inducing factors in lens cultured at normal (35.0˚C) and slightly warmer (37.5˚C) conditions. In the rat lens, 190 proteins (total) were identified. Of these, 48 proteins (25.3%) were found in lenses cultured at both 35.0˚C and 37.5˚C. Moreover, 85 proteins (44.7%) were unique to lenses cultured at 35.0˚C, while 57 proteins (30.0%) were unique to lenses cultured at 37.5˚C. Protein expression changes in rat lenses cultured at 37.5˚C were examined using a label‑free semiquantitative approach that uses spectral counting and Gene Ontology analysis. Filensin and vimentin protein expression, key factors in maintaining lens structure, were decreased. These findings may serve as a valuable indicator for elucidating the relationship between temperature and the onset of nuclear cataracts.

Investigation of acetaminophen (APAP)-induced liver damage recently indicated the significance of phagocytic NADPH oxidase (NOX)-derived reactive oxygen species (ROS) and ferroptosis in the liver. Here, we focused on phagocytosis by iron-containing erythrocyte-devouring splenic macrophages and explored upstream factors of known APAP hepatotoxic mechanisms in vivo. Splenectomy did not alter hepatic cytochrome P450 (CYP) 2E1 activity or hepatic glutathione (GSH) content. APAP injection into splenectomized mice almost completely suppressed increases in plasma alanine aminotransferase levels and centrilobular hepatic necrosis showing the spleen to be a critical tissue in APAP-induced liver damage. Hepatic GSH was recovered to approximately 50 % content at 8 h. In non-splenectomized mice, liver damage was dramatically suppressed by a sensitive redox probe (DCFH-DA), macrophage-depleting clodronate (CL), and a NOX2 inhibitor. APAP treatment resulted in markedly stronger fluorescence intensity from DCFH-DA due to excessive ROS around splenic macrophages, which was lost upon co-treatment with a CYP inhibitor and CL. Deformed erythrocytes disappeared in mice co-treated with DCFH-DA, CL, the NOX2 inhibitor, and the CYP inhibitor. Simultaneously, these four compounds significantly improved APAP-depleted GSH levels. The CYP inhibitor also prevented the formation of APAP-cell adducts in the blood and spleen. In the spleen, CL co-treatment markedly reduced the number of adducts. Splenic ferrous iron levels were significantly elevated by APAP. Therefore, we demonstrated that splenic macrophages devoured APAP metabolite-erythrocyte adducts and subsequently splenic macrophage-related ROS caused sustained hepatic GSH depletion and excessive erythrocyte deformation around 7 h. Our data indicate in vivo upstream factors of known APAP hepatotoxic mechanisms.

Neokestose is considered to have a prebiotic function. However, the physiological activity of neokestose remains unknown. Neokestose has a blastose, a sucrose analog, in its structure. We previously demonstrated that oral administration of blastose to diabetic rats suppressed the increase in plasma glucose (PG) concentration after sucrose administration. Therefore, neokestose might have a similar effect. In this study, we investigated the effects of neokestose on PG concentrations and the mechanism of its action. We first administered neokestose orally to streptozotocin-induced diabetic rats and observed that the expected consequent increase in PG concentration was significantly suppressed. Next, we examined the inhibitory effect of neokestose on glycosidase activity, but observed only a slight inhibitory effect. Therefore, we hypothesized that neokestose might be hydrolyzed by gastric acid to produce blastose. We performed an acid hydrolysis of neokestose using artificial gastric juice. After acid hydrolysis, peaks corresponding to neokestose and its decomposition products including blastose were observed. Therefore, we suggest that neokestose and blastose, a decomposition product, synergistically inhibit glycosidase activity. These findings support the potential use of neokestose as a useful functional oligosaccharide that can help manage plasma glucose concentrations in patients with diabetes mellitus.

The prevalence of presbyopia and nuclear cataracts (NUC) is reported to be higher in tropical areas than that in other regions, suggesting a potential influence of high temperatures on lens health. Transient receptor potential vanilloid (TRPV) channels play a crucial role in detecting ambient temperatures across various species, with TRPV1 and TRPV4 expressed in lens epithelial cells. In this study, we investigated whether ambient temperatures affect TRPV1 and TRPV4 activity in the lens, potentially contributing to the development of presbyopia and NUC. We conducted experiments using cultured human lens epithelial cell lines under different temperature conditions. Our results revealed that the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and p38 pathways, downstream molecules of TRPV1, were activated, while Src family kinase, a downstream molecule of TRPV4, was inhibited at 37.5 °C culture compared to 35.0 °C. Confocal microscope images demonstrated higher expression of TRPV1 in 3D-structured cells under high-temperature culture conditions. Additionally, in organ culture lenses, higher elasticity was observed at elevated temperatures compared to that at lower temperatures. These results suggest that high ambient temperatures may induce lens sclerosis via TRPV1 activation, potentially contributing to the development of presbyopia and NUC.

BACKGROUND: The improvement in flowability and adhesion of starch powder (SP) is essential for using starch as an excipient for lactose intolerant patients. In this study, we attempted to evaluate the usefulness of hydroxypropylcellulose with molecular weight 80,000 (HPC-80) in the preparation of the starch granules (SG) as a substitute for excipient lactose. METHODS: Hydroxypropylcellulose with molecular weight 30,000 (HPC-30) and HPC-80 were used as binders to prepare the SG, and defined as HPC-30-SG and HPC-80-SG, respectively. Mean particle size (D50) was measured according to the Method, Optical Microscopy of Particle Size Determination in Japanese Pharmacopoeia, Eighteenth Edition, and storage stability were evaluated by measuring of the physical properties after vortexing the granules for 180 s (physical impact). The product loss rate was calculated from the weight change of the various excipients before and after the one dose packaging (ODP). RESULTS: The D50 of SP (30 µm) was smaller than that of the lactose powder (115 µm). The granulation with 0.75-3% HPC-30 and HPC-80 increased the particle size of SP, and the D50 in 1.5% HPC-30-SG (255 µm) and HPC-80-SG (220 µm) were higher than that of lactose. The excipient was removed from the heat seal of the ODP, and upon visual inspection, a large amount of starchy material was observed to be adhering to the paper in the SP. On the other hand, the low recovery rate in SP was attenuated by the granulation with HPC-30 and HPC-80. In the both HPC-30 and HPC-80, the improvement in recovery rate reached a plateau at 1.5%, and the levels of recovery rate was similar to that of lactose. The recovery rate in the 0.75-3% HPC-30-SG and 0.75% HPC-80-SG were decreased by the physical impact, however, the recovery rate and amount of 1.5% and 3% HPC-80-SG were not affected by the physical impact, and these levels were similar to that of lactose. CONCLUSIONS: The use of HPC-80 as a binder of SG was found to produce a higher quality granule product than conventional HPC-based SG. This finding is useful in streamlining the preparation of starch-based powdered medicine in clinical applications.

Severe oral mucositis is a major cause of a low quality of life in patients; however, the therapeutic effect of traditional treatments is insufficient. Therefore, we designed a carbopol gel based on rebamipide nanocrystals (REB NCs) and gum arabic (GA-REB@NP), and investigated its efficacy in accelerating wound healing in a hamster model of oral mucositis. REB NCs were prepared by bead milling, and GA- REB@NP were prepared by incorporating REB NCs into a carbopol gel. The REB sizes were measured using a SALD-7100 and NanoSight LM10, and both powder X-ray diffraction and differential thermal analysis were used to analyze the crystalline form. Drug release from the gel formulations and therapeutic effects were evaluated using hamsters. The particles of milled-REB without GA were microsized, whereas the particle size of milled-REB with GA was in the range of 30-180 nm, and the crystalline form was similar to that of REB with or without bead milling. Next, we evaluated the characteristics of GA-REB@NP. The particle size of REB in GA-REB@NP was in the range of 45-200 nm, and drug release from GA-REB@NP was higher than that from the gel incorporating REB microcrystals (GA-REB@MP). In addition, REB nanoparticles were released from GA-REB@NP. Moreover, inhibitors of both clathrin- (dynasore) and caveolae-dependent endocytosis (nystatin) attenuated the enhanced REB levels in the cheek pouches of hamsters treated with GA-REB@NP. GA-REB@NP also enhanced the healing of the wound area compared with GA-REB@MP in hamsters injected with acetic acid. We prepared GA-REB@NP, which provided high REB delivery into the cheek pouch tissue via endocytosis. Additionally, we demonstrated that wound healing in acetic acid-injected hamsters was promoted by the application of GA-REB@NP.

In this work, we prepared a series of magnetic carbonaceous materials (CB200F, CB400F, CB600F, CB800F, and CB1000) from waste bamboo (RB) for the removal of cesium ions from water.

水晶体硬化は加齢性核白内障の初期症状であり，水晶体弾性の把握は白内障研究において重要である．本研究では亜セレン酸誘発白内障モデルを用い，キネティクス解析法の適用が従来の押圧時の移動度を固定した解析法（押圧法）の改善に繋がるのかを検討した．3.5 mg/kg亜セレン酸ナトリウムの皮下注射により水晶体核部の混濁が認められた．一方，0.5～2.5 mg/kg亜セレン酸ナトリウム投与群では，水晶体は透明であったが，水晶体弾性の低下がみられた．次に，これら亜セレン酸ナトリウム投与群に対し1次式を基にしたキネティクス解析を適用した．その結果，キネティクス解析を用いない従来の解析法と比較し個体間のばらつきが低値であった．本解析法は水晶体硬度変化の評価精度の向上に有用である．

Transdermal drug delivery is a formulation in which the drug is absorbed through the skin for systemic action. Its advantages include avoidance of first-pass effects, sustained drug supply, and ease of administration and discontinuation. Drugs administered transdermally transfer into the blood circulation through the stratum corneum, epidermis, and dermis. The stratum corneum on the skin surface plays a barrier function in skin absorption. Therefore, developing of transdermal drug delivery systems requires innovations that overcome the barrier function of the stratum corneum and improve skin permeation. This review examines the usefulness of transdermal formulations based on solid nanoparticles using raloxifene. Milled raloxifene was gelled with (mRal-NPs) or without menthol (Ral-NPs) using Carbopol. 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. 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. The inclusion of menthol in the formulation attenuated the barrier function of the stratum corneum and permitted raloxifene nanoparticles to penetrate through the skin. Moreover, macropinocytosis relates to the formulation's skin penetration, including menthol (mRal-NPs). Applying mRal-NPs attenuated the decreases in calcium level and stiffness of bones of ovariectomized rats. This information can support future studies aimed at designing novel transdermal formulations.

The solubility and permeability of the Biopharmaceutics Classification System (BCS) class IV drugs, such as furosemide (FUR), are low. Thus, the oral bioavailability of these drugs needs to be augmented. Here, we aimed to design orally disintegrating tablets containing FUR nanoparticles to improve bioavailability after oral administration. The FUR nanoparticles were generated by bead-milling in water containing 0.5% methylcellulose and 0.5% 2-hydroxypropyl-β-cyclodextrin (w/w%). Particle size was approximately 47-350 nm (mean particle size, 188 nm). An orally disintegrating tablet (FUR-NP tablet) comprising FUR nanoparticles (1%) was successfully produced by employing suspensions outlined above that incorporated additives (4% D-mannitol, 0.4% polyvinylpyrrolidone, and 16% gum Arabic, w/w%), followed by freeze-drying. The FUR-NP tablet disaggregated after only 5 s in water, liberating nano-sized FUR particles (172 nm). Experiments using rats showed the absorption of the FUR-NP tablet was significantly improved by comparison with a FUR tablet containing microparticles. In summary, the orally disintegrating tablet containing FUR nanoparticles markedly enhanced the bioavailability of FUR. We anticipate this formulation will also improve the bioavailability of other BCS class IV drugs.

Presbyopia is caused by age-related lenticular hardening, resulting in near vision loss, and it occurs in almost every individual aged ≥50 years. The lens experiences mechanical pressure during for focal adjustment to change its thickness. As lenticular stiffening results in incomplete thickness changes, near vision is reduced, which is known as presbyopia. Piezo1 is a mechanosensitive channel that constantly senses pressure changes during the regulation of visual acuity, and changes in Piezo1 channel activity may contribute to presbyopia. However, no studies have reported on Piezo1 activation or the onset of presbyopia. To elucidate the relevance of Piezo1 activation and cross-linking in the development of presbyopia, we analysed the function of Piezo1 in the lens. The addition of Yoda1, a Piezo1 activator, induced an increase in transglutaminase 2 (TGM2) mRNA expression and activity through the extra-cellular signal-regulated kinase (ERK) 1/2 and c-Jun-NH2-terminal kinase1/2 pathways. In ex vivo lenses, Yoda1 treatment induced γ-crystallin cross-linking via TMG2 activation. Furthermore, Yoda1 eye-drops in mice led to lenticular hardening via TGM2 induction and activation in vivo, suggesting that Yoda1-treated animals could serve as a model for presbyopia. Our findings indicate that this presbyopia-animal model could be useful for screening drugs for lens-stiffening inhibition.

BACKGROUND: Gastrointestinal injuries caused by nonsteroidal anti-inflammatory drugs (NSAIDs) is a serious side effect in patients with rheumatoid arthritis (RA). However, effective therapeutic strategies have yet to be established. In this study, we investigated the therapeutic effects of teprenone (TEP), a gastric mucosal protective drug, on NSAID-induced gastrointestinal injuries in rats with RA (AA rats). METHODS: Gastrointestinal injury was induced by oral administration of indomethacin (IMC), a typical NSAID. TEP was orally administered after IMC-induced gastrointestinal bleeding, and the stomach, jejunum, and ileum were excised. RESULTS: On day 14 of IMC administration, lesion areas in the stomach, jejunum, and ileum were significantly larger in AA rats than in normal rats. When TEP was orally administered to AA rats, the lesion areas in the stomach, jejunum, and ileum significantly decreased compared with those in control rats (IMC-induced AA rats). Therefore, we measured NOS2 mRNA and NO levels, which were significantly decreased in rats with IMC-induced AA after treatment with TEP. CONCLUSIONS: These results suggest that the oral administration of TEP may be useful for the treatment of NSAID-induced gastrointestinal injuries in patients with RA.

The metabolism of 5-aminolevulinic acid (ALA) is more efficient when combined with sodium ferrous citrate (SFC). Our previous study revealed that oral administration of ALA, which has anti-inflammatory properties, and SFC (ALA/SFC) immediately before lipopolysaccharide (LPS) inoculation suppressed endotoxin-induced uveitis (EIU) in rats. However, the therapeutic effect of ALA/SFC post-administration remains unexplored. Hence, this study aimed to evaluate the therapeutic efficacy of ALA/SFC on EIU in rats, which were administered with a gastric gavage of ALA/SFC (100/157 mg/kg) or prednisolone (Pred, 10 mg/kg) after 4 h of LPS inoculation. The treatment groups showed ameliorated clinical scores, inflammatory cells, protein levels in the aqueous humor (AqH), and histopathologic evaluation 24 h after LPS inoculation. Furthermore, the treatment groups had reduced tumor necrosis factor-α, nitric oxide, prostaglandin E2, and interleukin-6 levels in the AqH. ALA/SFC demonstrated an anti-inflammatory effect equivalent to that demonstrated by Pred. These findings indicate that ALA/SFC exerts a therapeutic effect on EIU in rats, indicating its clinical usefulness in uveitis treatment.

Regulation of ion and water microcirculation within the lens is tightly controlled through aquaporin channels and connexin junctions. However, cataracts can occur when the lens becomes cloudy. Various factors can induce cataracts, including diabetes which is a well-known cause. The most common phenotype of diabetic cataracts is a cortical and/or posterior subcapsular opacity. In addition to the three main types and two subtypes of cataracts, a vacuole formation is frequently observed; however, their origin remains unclear. In this study, we focused on the aquaporins and connexins involved in diabetes-induced cataracts and vacuoles in Nile grass type II diabetes. The results showed that the expression of aquaporin 0 and aquaporin 5 increased, and that of connexin 43 decreased in diabetic rat lenses. Additionally, aquaporin 0 and 5 were strongly localized in peripheral of vacuoles, suggesting that aquaporins are involved in vacuoles formation. Transillumination photography revealed large vacuoles at the tip of the Y-suture in the anterior capsule of the diabetic lens, and several small vacuoles were observed in the posterior capsule. Within the vacuoles, cytoplasmic degradation and aggregation of fibrous material were observed. Our findings suggest that aquaporins are potential candidate proteins for preventing vacuole formation.

BACKGROUND: It is important to design an effective formulation to enhance the skin penetration, and nanotechnologies have been used in dermal and transdermal drug delivery. In this study, we prepared formulations (gels) containing l-menthol and felbinac (FEL) solid nanoparticles (FEL-NP gel) for topical application, and investigated the local and systemic absorption of the prepared FEL-NP gel. METHODS: FEL solid nanoparticles were obtained by bead milling of FEL powder (microparticles), and a topical formulation (FEL-NP gel) consisting of 1.5% FEL solid nanoparticles), 2% carboxypolymethylene, 2% l-menthol, 0.5% methylcellulose, and 5% 2-hydroxypropyl-β-cyclodextrin (w/w %) were prepared. RESULTS: The particle size of FEL nanoparticles was 20-200 nm. The released FEL concentration from FEL-NP gel was significantly higher than that from FEL gel without bead mill treatment (carboxypolymethylene gel in which FEL microparticles (MPs) instead of FEL nanoparticles were incorporated, FEL-MP gel), and FEL was released as nanoparticles from the gel. Moreover, both transdermal penetration and percutaneous absorption of FEL-NP gel were significantly increased compared with those of FEL-MP gel, and the area under the FEL concentration-time curve (AUC) of FEL-NP gels was 1.52- and 1.38-fold of commercially available FEL ointment and FEL-MP gel, respectively. In addition, after 24 h of treatment, the FEL content in rat skin treated with FEL-NP gels was 1.38- and 2.54-fold higher than that when treated with commercially available FEL ointment and FEL-MP gel, respectively. Moreover, the enhanced skin penetration of FEL-NP gels was significantly attenuated by inhibition of energy-dependent endocytosis, such as clathrin-mediated endocytosis. CONCLUSIONS: We successfully prepared a topically applied carboxypolymethylene gel containing FEL nanoparticles. In addition, we observed that the endocytosis pathway was mainly related to the high skin penetration of FEL nanoparticles, and FEL-NP gel application resulted in high local tissue concentration and systemic absorption of FEL. These findings provide useful information for the design of topically applied nanoformulations against inflammation by providing local and systemic effects.

Sodium ferrous citrate (SFC) is involved in the metabolism of 5-aminolevulinic acid (5-ALA) and enhances its anti-inflammatory effects. The effects of 5-ALA/SFC on inflammation in rats with endotoxin-induced uveitis (EIU) have yet to be elucidated. In this study, during lipopolysaccharide injection, 5-ALA/SFC (10 mg/kg 5-ALA plus 15.7 mg/kg SFC) or 5-ALA (10 or 100 mg/kg) was administered via gastric gavage, wherein we saw that 5-ALA/SFC ameliorated ocular inflammation in EIU rats by suppressing clinical scores; by infiltrating cell counts, aqueous humor protein, and inflammatory cytokine levels; and by improving histopathological scores to the same extent as 100 mg/kg 5-ALA. Immunohistochemistry showed that 5-ALA/SFC suppressed iNOS and COX-2 expression, NF-κB activation, IκB-α degradation, and p-IKKα/β expression, and activated HO-1 and Nrf2 expression. Therefore, this study has investigated how 5-ALA/SFC reduces inflammation and revealed the pathways involved in EIU rats. 5-ALA/SFC is shown to inhibit ocular inflammation in EIU rats by inhibiting NF-κB and activating the HO-1/Nrf2 pathways.

Advanced glycation end products (AGEs) in lens proteins increase with aging, thus inducing cataracts and/or presbyopia. Hesperetin (Hst), which is an abundant plant flavanone largely derived from citrus species, and its derivatives attenuate cataracts and presbyopia in vivo and in vitro; however, no reports have described its effects on AGE formation in lens proteins. The present study demonstrated that AGEs in lens proteins increase with age in mice. Additionally, it showed that Hst can prevent AGEs and N(ε)‑carboxymethyl‑lysine generation and modification of lens proteins using in vitro in human lens epithelial cell lines and ex vivo in mouse lens organ cultures. Furthermore, treatment with Hst prevented lens hardening and decreased chaperone activity in lens proteins. These results suggested that Hst and its derivatives are good candidates for the prevention of presbyopia and cataracts.

This study aimed to investigate the anti-inflammatory effect of 5-aminolevulinic acid (5-ALA) on endotoxin-induced uveitis (EIU) in rats. EIU was induced in male Sprague Dawley rats by the subcutaneous injection of lipopolysaccharide (LPS). During LPS injection, 5-ALA diluted with saline was administered via gastric gavage. After 24 h, clinical scores were assessed after which aqueous humor (AqH) samples were obtained. The number of infiltrating cells, protein concentration, and levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nitric oxide (NO), and prostaglandin E2 (PGE2) in AqH were measured. For histological examination, both eyes of some rats were enucleated. In vitro, a mouse macrophage cell line (RAW264.7 cells) was stimulated by LPS with or without 5-ALA. Western blot was used to analyze the expression of inducible NO synthase (iNOS) and cyclooxygenase-2. 5-ALA suppressed the EIU clinical scores, infiltrating cell number, and protein concentration while improving the histopathologic scores. In particular, 100 mg/kg 5-ALA reduced the concentrations of NO, PGE2, TNF-α, and IL-6 in AqH, similar to 1 mg/kg prednisolone. In addition, 5-ALA suppressed iNOS upregulation in LPS-stimulated RAW264.7 cells. Therefore, 5-ALA has an anti-inflammatory effect on EIU through the inhibition of the upregulation of inflammatory mediators.

In this study, waste biomass adsorbents produced from mangosteen shells (MGS) were prepared (denoted as MGS500 and MGS1000). The physical and chemical characteristics, such as scanning electron microscopy, thermogravimetric-differential thermal analysis, specific surface area, pore volumes, surface functional groups, and point of zero charge of the prepared MGS samples were determined, and the adsorption capacity of cadmium ions from aqueous media was assessed. The effects of pH, adsorption time, temperature, and coexistence on adsorption were carefully assessed using an inductively coupled plasma optical emission spectrometer under several experimental conditions. The adsorption capacity decreased in the order, MGS < MGS500 < MGS1000. The optimal pH for cadmium ion removal was 5.0. The amount of cadmium ions adsorbed gradually increased with time, and adsorption equilibrium was achieved within 24 h after adsorption. Additionally, the amount of adsorbed cadmium ions increased with increasing adsorption temperature. To elucidate the adsorption mechanism in detail, the elemental distribution and X-ray photoelectron spectra of the prepared adsorbents were analyzed. Finally, desorption solutions such as HNO3, H2O, and NaOH were used to desorb the absorbed cadmium ions from MGS1000. Under our experimental conditions, the desorption percentage of cadmium ions was approximately 98.8% using HNO3. In conclusion, MGS1000 exhibited a good adsorption capacity of 12.0 mg/g for adsorbing cadmium ions from aqueous media and desorption capacity with HNO3 at 1000 mmol/L.

N,N-diethyl-meta-toluamide (DEET) is a widely used insect repellent, with minimal skin permeation and sustained repellent activity in the superficial layers of the skin. In this study, we prepared a 10% DEET formulation consisting of 40% ethanol with or without 2% poly(oxyethylene)/poly(oxypropylene) butyl ether (POE-POP), an amphiphilic random copolymer. Further, we demonstrated the effects of POE-POP on tensile stress (stickiness), hydrophobicity, skin retention, permeation, and repellent activity of DEET. Stickiness was measured in male ICR mice (7-week old), and skin retention and permeation were evaluated in male Wistar rats (7-week old). In addition, female Aedes albopictus were used to measure the repellent action of DEET. The addition of POE-POP did not affect stickiness, volatility, and degradability but decreased logP and increased viscosity of DEET. Next, we demonstrated the behavior of DEET formulations in the rat skin. POE-POP prolonged the retention of DEET in the superficial layers of the rat skin (skin surface and stratum corneum) and decreased the penetration of DEET into rat skin tissues (epithelium and dermis). The repellent effect of DEET was also enhanced by the addition of POE-POP. However, severe skin damage was not observed after repetitive treatment with DEET formulations containing POE-POP for one month (twice a day). In conclusion, we demonstrated that a 10% DEET formulation consisting of 40% ethanol and 2% POE-POP attenuated the skin penetration and prolonged the repellent action of DEET without causing stickiness and skin damage. We conclude that the combination of ethanol and POE-POP is useful as a safe and effective delivery system for the development of insect repellent formulations containing DEET.

A colloidal silicate granulated nickel-aluminum-zirconium (CSG-NAZ) was prepared, and the chromium(VI) (Cr(VI)) ions recovery capacity was evaluated using a sodium sulfate solution in a column experiment. The amount adsorbed and breakthrough time were enhanced by decreasing the flow rate (flow rate is in the order of 3.0 > 2.0 > 0.5 mL). The breakthrough curves and model parameters were estimated using the Thomas and Yoon-Nelson models. The obtained data confirmed to fit both the Yoon-Nelson model (0.858-0.906) and the Thomas model (0.813-0.906). Additionally, Cr(VI) ions that adsorbed onto CSG-NAZ could be desorbed using a sodium sulfate solution in a column experiment. The total recovery percentage of Cr(VI) ions was 80.9% after six repetitions of adsorption/desorption. Finally, the obtained results revealed that CSG-NAZ was a candidate adsorbent for the recovery of Cr(VI) ions owing to its applicability toward a continuous system.

PURPOSE: 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.

PURPOSE: 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.

This study investigated the preventive effect of 5-aminolevulinic acid combined with sodium ferrous citrate (5-ALA/SFC) on blood-aqueous barrier (BAB) breakdown induced after anterior chamber paracentesis (ACP) in beagles. 5-ALA/SFC (1/0.64 mg/kg or 3/1.92 mg/kg) or carprofen (4.0 mg/kg) was orally administered daily for 7 days prior to ACP. Then, a sample of the aqueous humor (AH) was collected from one eye via ACP (first sample) and again 60 min later (second sample). The protein and prostaglandin E2 (PGE2) concentrations in both samples were measured. Compared with the control group, high-dose 5-ALA/SFC and carprofen significantly reduced the AH protein and PGE2 concentrations in the second sample. Our findings suggest that 5-ALA/SFC suppresses BAB breakdown in dogs.

The permeability of the Biopharmaceutics Classification System (BCS) class III drugs are low, and their oral bioavailability needs to be improved. In this study, we attempted to design oral formulations containing famotidine (FAM) nanoparticles to overcome the limitations of BCS class III drugs. Dispersions containing FAM nanoparticles with a particle size of approximately 50-220 nm were produced by the bead-milling treatment. Moreover, we succeeded in preparing an orally disintegrating tablet containing FAM nanoparticles using the dispersions described above, additives (D-mannitol, polyvinylpyrrolidone, and gum arabic), and freeze-dry treatment (FAM-NP tablet). The FAM-NP tablet was disaggregated 3.5 s after addition to purified water, and the FAM particles in the redispersion of the FAM-NP tablet stored for 3 months were nano-sized (141 ± 6.6 nm). The ex-vivo intestinal penetration and in vivo absorption of FAM in rats applied with the FAM-NP tablet were significantly higher than those in rats applied with the FAM tablet containing microparticles. In addition, enhanced intestinal penetration of the FAM-NP tablet was attenuated by an inhibitor of clathrin-mediated endocytosis. In conclusion, the orally disintegrating tablet containing FAM nanoparticles improved low mucosal permeability and low oral bioavailability and overcame these issues of BCS class III drugs as oral formulations.

Wound-healing deficits of the skin, one of the most common complications in patients with diabetes, delay wound healing, significantly reducing the patient's QOL. Therefore, the topical treatment of wound areas with drug-containing ointments and dressings is important. In this study, we investigated the effect of various ointment bases on skin wound healing in normal and streptozotocin-induced diabetic rats (STZ rats). Three ointment bases were used: white ointment (oil-based), absorbent cream (emulsion-based, w/o), and macrogol ointment (water-based). Skin wound healing in STZ rats was delayed compared with that in normal rats. Each of the three ointment bases was applied to the skin wound area in normal rats, and there was no difference in the therapeutic effect. The therapeutic effect of both white ointment and absorbent cream was higher in the STZ rats group than that in the non-treated group, and delayed wound healing was observed in STZ rats treated with macrogol ointment. In conclusion, skin wound healing in STZ rats is affected by the properties of the ointment base, and it is important to use an ointment base that controls the drying of the wound area in STZ rats. These findings provide information for the selection of ointment bases useful for application to skin wounds in patients with diabetes.

Enhancement of density via human lens epithelium (HLE) cell proliferation is the underlying cause of nuclear cataracts. Moreover, our previous epidemiological study demonstrated that the risk of nuclear cataract development is significantly higher under elevated environmental temperatures compared with under lower temperatures. The present study investigated the relationship between temperature and cell proliferation in terms of mitochondrial function, which is a nuclear cataract‑inducing risk factor, using two different HLE cell lines, SRA01/04 and immortalized human lens epithelial cells NY2 (iHLEC‑NY2). Cell proliferation was significantly enhanced under the high‑temperature condition (37.5˚C) in both cell lines. The cell growth levels of SRA01/04 and iHLEC‑NY2 cells cultured at 37.5˚C were 1.20‑ and 1.16‑fold those in the low‑temperature cultures (35.0˚C), respectively. Moreover, the levels of cytochrome c oxidase mRNA (mitochondrial genome, cytochrome c oxidase‑1‑3) and its activity in SRA01/04 and iHLEC‑NY2 cells cultured at 37.5˚C were higher compared with those in cells cultured at 35.0˚C. In addition, adenosine‑5'‑triphosphate (ATP) levels in SRA01/04 and iHLEC‑NY2 cells were also significantly higher at 37.5˚C compared with those at 35.0˚C. By contrast, no significant differences in Na+/K+‑ATPase or Ca2+‑ATPase activities were observed between HLE cells cultured at 35.0 and 37.5˚C. These results suggested that expression of the mitochondrial genome was enhanced in high‑temperature culture, resulting in a sufficient ATP content and cell proliferation for lens opacity. Therefore, elevated environmental temperatures may increase the risk of nuclear cataracts caused by HLE cell proliferation via mitochondrial activation.

Dry eye disease (DED) is a frequently observed eye complaint, which has recently attracted considerable research interest. Conventional therapy for DED involves the use of artificial tear products, cyclosporin, corticosteroids, mucin secretagogues, antibiotics and nonsteroidal anti-inflammatory drugs. In addition, ocular drug delivery systems based on nanotechnology are currently the focus of significant research effort and several nanotherapeutics, such as nanoemulsions, nanosuspensions, microemulsions, liposomes and nanomicelles, are in clinical trials and some have FDA approval as novel treatments for DED. Thus, there has been remarkable progress in the design of nanotechnology-based approaches to overcome the limitations of ophthalmic formulations for the management of anterior eye diseases. This review presents research results on diagnostic methods for DED, current treatment options, and promising pharmaceuticals as future therapeutics, as well as new ocular drug delivery systems.

When regenerated tissue is generated from induced pluripotent stem cells (iPSCs), it is necessary to track and identify the transplanted cells. Fluorescently-labeled iPSCs synthesize a fluorescent substance that is easily tracked. However, the expressed protein should not affect the original genome sequence or pluripotency. To solve this problem, we created a cell tool for basic research on iPSCs. Iris tissue-derived cells from GFP fluorescence-expressing mice (GFP-DBA/2 mice) were reprogrammed to generate GFP mouse iris-derived iPSCs (M-iris GFP iPSCs). M-iris GFP iPSCs expressed cell markers characteristic of iPSCs and showed pluripotency in differentiating into the three germ layers. In addition, when expressing GFP, the cells differentiated into functional recoverin- and calbindin-positive cells. Thus, this cell line will facilitate future studies on iPSCs.

We aimed to investigate which base was suitable for preparing transdermal formulations incorporating tulobuterol (TUL) nanoparticles (30-180 nm) in this study. Three bases (water-soluble, absorptive, and aqueous ionic cream) were selected to prepare the transdermal formulations, and TUL nanoparticles were prepared with a bead-milling treatment. In the drug release study, the TUL release from the water-soluble ointment was higher than that from the other two ointments. Moreover, the addition of l-menthol enhanced TUL nanoparticle release from the ointment, and the rat skin penetration of the TUL water-soluble ointment was also significantly higher than that of the other two ointments. In addition, the drug penetration of the TUL water-soluble ointment with l-menthol sustained zero-order release over 24 h, and the skin permeability of TUL increased with TUL content in the ointment. On the other hand, this penetration was significantly inhibited by treatment with a caveolae-mediated endocytosis inhibitor (nystatin). In conclusion, we found that the water-soluble base incorporating TUL nanoparticles and l-menthol was the best among those assessed in this study. Furthermore, the pathway using caveolae-mediated endocytosis was related to the skin penetration of TUL nanoparticles in the TUL water-soluble ointment with l-menthol. These findings are useful for the design of a transdermal sustained-release formulation based on TUL nanoparticles.

PURPOSE: The aim of the present study was to investigate increase in delivery of drug upon formulation as mucoadhesive microemulsion system and further to investigate possibility of any cytotoxic effects using such formulation. MATERIAL AND METHODS: Considering hydrophilic and small molecular nature of the drug, it was attempted to be formulated as microemulsion, by using pseudo ternary phase diagram method. Thus, three types of microemulsions were prepared; oil in water, water in oil type and chitosan-coated microemulsion. These microemulsions were characterized for several physicochemical properties like size, zeta potential, Polydispersity index, and compared for in vitro cell viability and ex vivo corneal irritation study. RESULTS: All three microemulsions were quite stable, transparent and homogenous systems. They showed similar drug release pattern, but highest ex vivo goat corneal permeation was observed with Chitosan coated microemulsion when compared with ganciclovir solution. CONCLUSION: All microemulsions were found to be non-irritant in in vitro cell viability assay and ex vivo corneal irritation study, indicating the potential of using such systems for delivery of drug to eye.

Induced pluripotent stem (iPS) cells are widely used as a research tool in regenerative medicine and embryology. In studies related to lens regeneration in the eye, iPS cells have been reported to differentiate into lens epithelial cells (LECs); however, to the best of our knowledge, no study to date has described their formation of three-dimensional cell aggregates. Notably, in vivo studies in newts have revealed that iris cells in the eye can dedifferentiate into LECs and regenerate a new lens. Thus, as basic research on lens regeneration, the present study investigated the differentiation of human iris tissue-derived cells and human iris tissue-derived iPS cells into LECs and their formation of three-dimensional cell aggregates using a combination of two-dimensional culture, static suspension culture and rotational suspension culture. The results revealed that three-dimensional cell aggregates were formed and differentiated into LECs expressing αA-crystallin, a specific marker protein for LECs, suggesting that the cell-cell interaction facilitated by cell aggregation may have a critical role in enabling highly efficient differentiation of LECs. However, the present study was unable to achieve transparency in the cell aggregates; therefore, we aim to continue to investigate the degradation of organelles and other materials necessary to make the interior of the formed cell aggregates transparent. Furthermore, we aim to expand on our current work to study the regeneration of the lens and ciliary body as a whole in vitro, with the aim of being able to restore focusing function after cataract surgery.

It has recently been reported that lanosterol (LAN) plays a preventive role against lens opacification through the reversal of crystalline aggregation. However, the effect of LAN is not sufficient to restore lens transparency. In this study, we designed ophthalmic nanosuspensions (LAN-ONSs and NIL-ONSs) based on LAN and nilvadipine (NIL), which can counteract cataract-related factors (e.g., enhanced Ca2+ and calpain levels), and investigated whether the combination of LAN-ONSs and NIL-ONSs can restore the nuclear lens opacity in sodium-selenite-induced cataractic rats (cataractic rats). The mean particle sizes of the LAN-ONSs and NIL-ONSs were 108.8 nm and 89.0 nm, respectively. The instillation of the LAN-ONSs or NIL-ONSs successfully delivered the drugs (LAN or NIL) into the lenses of the rats, although the instillation of LAN-ONSs or NIL-ONSs alone did not increase lens transparency in the cataractic rats. On the other hand, the cataract-related factors (enhanced Ca2+ and calpain levels) were significantly alleviated by the combination of LAN-ONSs and NIL-ONSs; furthermore, the perinuclear refractile ring in the lens nucleus and enhanced number of swollen fibers were attenuated by the LAN-ONS and NIL-ONS combination. Moreover, the opacity levels in the cataractic rats were reduced after treatment with the combination of LAN-ONSs and NIL-ONSs. It is possible that the combination of LAN and NIL will be useful for the treatment of lens opacification in the future.

To increase the adsorption capability of Hg2+ from aqueous media, we prepared sodium-type fine zeolite grains with various particle sizes (denoted as ZE1, ZE2 and ZE3). The particle sizes of ZE1, ZE2 and ZE3 were 16.363 ± 0.365, 1.454 ± 0.357 and 0.607 ± 0.377 μm, respectively. Moreover, the CEC, specific surface area and pore volume were in the order ZE1 (42 mmol/g and 23.5 m2/g) < ZE2 (72 mmol/g and 67.1 m2/g) < ZE3 (135 mmol/g and 176.6 m2/g). Subsequently, the Hg2+ adsorption capability was investigated. The performance of tested agents on Hg2+ adsorbed was in the order ZE1 (5.0 mg/g) < ZE2 (9.4 mg/g) < ZE3 (20.2 mg/g). It was concluded that fine crystalline zeolite was important in enhancing the adsorption capability of Hg2+. In addition, the mechanism of adsorption of Hg2+ on the ZE samples was evaluated. Our results suggested that Hg2+ was exchanged with sodium ions in the interlayers of ZE samples with correlation coefficients of 0.966-0.979. Our findings revealed that these ZE samples constitute potential agents for the adsorption of Hg2+ from aqueous media.

We investigated the effect of fenofibrate nano-eyedrops (FenoNano) on impaired retinal blood flow regulation in type 2 diabetic mice. Six-week-old db/db mice were randomly divided into an untreated group (n = 6) and treated group, which received FenoNano (n = 6). The longitudinal changes in retinal neuronal function and blood flow responses to systemic hyperoxia and flicker stimulation were evaluated every 2 weeks in diabetic db/db mice treated with FenoNano (n = 6) or the vehicle (n = 6) from ages 8-14 weeks. The retinal blood flow was assessed using laser speckle flowgraphy. We also evaluated the expressions of vascular endothelial growth factor (VEGF), glial fibrillary acidic protein (GFAP), and aquaporin 4 (AQP4) and the phosphorylation of peroxisome proliferator-activated receptor alpha (PPAR-α) by immunofluorescence. In db/db mice treated with FenoNano, both responses were restored from 8 to 14 weeks of age compared with the diabetic mice treated with the vehicle. At 14 weeks of age, the impaired regulation of retinal blood flow during systemic hyperoxia and flicker stimulation improved to about half of that in the db/db mice treated with FenoNano compared with the db/m control group (n = 5). FenoNano prevented the activation of VEGF and GFAP expression and increased the AQP4 expression and the phosphorylation of PPAR-α detected by immunofluorescence compared with the diabetic mice treated with the vehicle eyedrop. Our results suggested that the fenofibrate nano-eyedrops prevent retinal glial dysfunction via the phosphorylation of PPAR-α and improves the retinal blood flow dysregulation in type 2 diabetic mice.

We previously designed the formulation containing minoxidil (MXD) nanoparticles (MXD-NPs), and found that the MXD-NPs can mainly deliver MXD into hair bulbs via hair follicles pathway, and that the therapeutic efficiency for hair growth is higher in comparison with the formulation containing dissolved MXD. In this study, we investigated whether the skin environmental changes by the treatment of steam towel, ethanol, l-menthol and commercially available (CA) carpronium affect the drug behavior in the MXD-NPs-applied mice. The steam towel, ethanol, l-menthol and CA-carpronium were pre-treated 3 min before MXD-NPs application, and the MXD content in the hair bulge, bulb, skin tissue and blood of mice were measured 4 h after MXD-NPs application. No significant difference of MXD levels in the blood was observed by the pre-treatment of steam towel, ethanol, l-menthol and CA-carpronium. On the other hand, the pre-treatment of steam towel and l-menthol enhanced the MXD levels in hair bulge and/or bulb. Although, the MXD levels in hair bulge and bulb were not changed by the pre-treatment of ethanol, the MXD levels in skin tissue was higher than that of saline-pre-treated group (control). The MXD levels in hair bulge, bulb and skin tissue of mice pre-treated with CA-carpronium were remarkably higher in comparison with control. In conclusion, we showed that the changes in skin environment by the steam towel, ethanol, l-menthol and CA-carpronium affected the absorption of MXD-NPs, and these increased MXD levels in the hair bulb and blood by the combination may enhance the therapeutic efficiency without side effects.

In this study, we focused on the storage conditions and investigated the effects of low-temperature storage (10°C) on the dispersibility of active components in three formulations of fluorometholone (FLU) suspension eye-drops (one original drug and two generic drugs, P1-P3). For all three eye-drop products, before shaking by hand, white sediment anticipated to be the principal active component was seen at the vial base. In the ordinary-temperature storage group, the FLU contents per drop after shaking by hand were 0.076% in P1, 0.023% in P2, and 0.100% in P3, and the content in P2 was significantly lower than that in P1 and P3. In contrast, almost no dispersion was observed in the low-temperature group. The results after sufficient shaking of these samples with a vortex, in contrast, were such that the FLU contents per drop were 0.063% in P1, 0.086% in P2, and 0.088% in P3; the content in P1 was significantly lower than that in P2 and P3, and there was no difference between P2 and P3. Moreover, we evaluated the dispersibility according to the evaluation "Vs / (ρg - ρf) g." In both the low- and ordinary-temperature storage groups, the value of Vs / (ρg - ρf) g, proportional to the terminal velocity, decreased in the following order: P3 > P1 ≫ P2, and each value in the ordinary-temperature was higher than that in low temperature. The zeta potential decreased in the following order: P2 > P3 ≫ P1. In conclusion, when FLU suspension eye drops are stored at low temperatures until use, such as in a refrigerator, ordinary shaking does not help achieve dispersion to the specified concentration, and even with vigorous shaking with some formulations, the specified concentration cannot be achieved.

Coal fly ash (FA) was treated by hydrothermal activation with sodium hydroxide solution at different concentrations to optimize the conversion method. Zeolite of the sodium type is prepared from coal FA by 1, 1.5, and 3 mol/L sodium hydroxide solutions (ZE1, ZE1.5, and ZE3). These adsorbents' morphology, crystal structure, scanning electron microscopy, Fourier transform (FT)-IR spectra, cation exchange capacity (CEC), specific surface area and pore volumes, and pHpzc were determined. An adsorption experiment was performed to evaluate the effects of contact time, pH, temperature, and coexistence. From the results, the values of CEC and specific surface area of prepared samples was in the order ZE3 < ZE1.5 < ZE1. The similar trends were observed in lead ions adsorption. In addition, our obtained data elucidate that the ion exchange with sodium ions in the interlayer ZE1 is one of the adsorption mechanisms of Pb2+ from water layer. Finally, lead ions adsorbed on ZE1 could be desorbed using a hydrochloric acid solution, showing that ZE1 could be reused as a water purification agent.

We developed ophthalmic formulations based on nilvadipine (NIL) nanocrystals (NIL-NP dispersions; mean particle size: 98 nm) by using bead mill treatment and investigated whether the instillation of NIL-NP dispersions delivers NIL to the lens and prevents lens opacification in hereditary cataractous Shumiya cataract rats (SCRs). Serious corneal stimulation was not detected in either human corneal epithelial cells or rats treated with NIL-NP dispersions. The NIL was directly delivered to the lens by the instillation of NIL-NP dispersions, and NIL content in the lenses of rats instilled with NIL-NP dispersions was significantly higher than that in the ophthalmic formulations based on NIL microcrystals (NIL-MP dispersions; mean particle size: 21 µm). Moreover, the supply of NIL prevented increases in Ca2+ content and calpain activity in the lenses of SCRs and delayed the onset of cataracts. In addition, the anti-cataract effect in the lens of rats instilled with NIL-NP dispersions was also significantly higher than that in NIL-MP dispersions. NIL-NPs could be used to prevent lens opacification.

This study aimed to compare the in vitro and in vivo retention, bacterial adhesion, and biofilm formation between anionic and zwitterionic bandage contact lenses (BCLs) in healthy canines. BCL retention and tolerance were evaluated in 10 healthy canines via a single-masked, crossover study for 7 days. To compare in vitro bacterial adhesion and biofilm formation, four Staphylococcus strains were incubated with the BCLs at 37 °C for 2 or 24 h, and the bacterial colony forming units (CFUs) adhering to the BCLs were counted. Next, to compare in vivo bacterial adhesion, the CFUs of bacteria adhering to the BCLs worn by canines for 24 h were counted. Anionic lenses significantly retained and reduced in vitro bacterial adhesion than in the zwitterionic lenses. However, the amount of in vitro biofilm formation was more likely to be higher on anionic lenses than on zwitterionic lenses. In vivo bacterial adhesion was not significantly different between the two types of BCLs. Nevertheless, both BCLs were well-tolerated by the canines; thus, their short-term use in dogs can be recommended as safe.

We previously designed ophthalmic formulations (nTRA) containing tranilast nanoparticles (Tra-NPs) with high uptake into ocular tissues. In this study, we used in situ gel (ISG) bases comprising combinations of pluronic F127 (F127) and methylcellulose (MC/F127), pluronic F68 (F68/F127), and Carbopol (Car/F127), and we developed in situ gels incorporating Tra-NPs (Tra-NP-incorporated ISNGs) such as nTRA-F127, nTRA-MC/F127, nTRA-F68/F127, and nTRA-Car/F127. Moreover, we demonstrated the therapeutic effect on conjunctival inflammation using lipopolysaccharide-induced rats. Each Tra-NP-incorporated ISNG was prepared by the bead mill method, the particle size was 40-190 nm, and the tranilast release and diffusion from formulation were nTRA > nTRA-F127 > nTRA-F68/F127 > nTRA-Car/F127 > nTRA-MC/F127. In the Tra-NP-incorporated ISNGs, the tranilast residence time in the lacrimal fluid, cornea, and conjunctiva was prolonged, although the Cmax was attenuated in comparison with nTRA. On the other hand, no significant difference in conjunctival inflammation between non- and nTRA-F127-instilled rats was found; however, the nTRA-F68/F127, nTRA-Car/F127, and nTRA-MC/F127 (combination-ISG) attenuated the vessel leakage, nitric oxide, and tumor necrosis factor-α expression. In particular, nTRA-F68/F127 was significant in preventing the conjunctival inflammation. In conclusion, we found that the combination-ISG base prolonged the residence time of Tra-NPs; however, Tra-NP release from the formulation was attenuated, and the Tmax was delayed longer than that in nTRA. The balance of drug residence and diffusion in lacrimal fluid may be important in providing high ocular bioavailability in formulations containing solid nanoparticles.

We attempted to design irbesartan nanocrystalline (IRB-NC) suspensions by the bead mill method, and we evaluated the bioavailability (BA) in the oral administration of the nanocrystalline drug. The mean particle size of the IRB-NC suspensions was approximately 140 nm, and the crystalline structure of irbesartan in these suspensions was different using the bead mill method. The aggregation and degradation of irbesartan were not observed for one month, and the solubility increased. Moreover, the inclusion complex formation of IRB-NC suspensions with 2-hydroxypropyl-β-cyclodextrin was higher than that in traditional IRB powder (IRB-P). In addition, the intestinal absorption of IRB-NC suspensions was higher than that of IRB-P suspensions, and the reducing effect on blood pressure in spontaneously hypertensive SHR-SP rats orally administered IRB-NC suspensions was significantly higher than in those administered IRB-P suspensions. On the other hand, the intestinal penetration of IRB-NC suspensions was attenuated by the inhibitors of clathrin-dependent endocytosis (CME). In conclusion, we improved the low oral BA of irbesartan by preparing IRB-NC suspensions and showed that both the solubility and CME are related to the enhanced intestinal absorption of IRB-NC suspensions, resulting in an increase in their antihypertensive effect. These findings provide significant information for the development of oral nanomedicines.

To identify risk factors for the prognosis of prostate cancer (PC), we retrospectively analyzed the impact of lifestyle-related disorders as well as PC characteristics at initial diagnosis on the progression to castration-resistant PC (CRPC) in PC patients undergoing hormone therapy. Of 648 PC patients, 230 who underwent hormone therapy and met inclusion criteria were enrolled in this study. CRPC developed in 48 patients (20.9%). Univariate analysis using Cox proportional hazard model indicated that newly developed diabetes mellitus (DM) following hormone therapy (postDM), but not preexisting DM, as well as PC characteristics at initial diagnosis including prostate-specific antigen (PSA) ≥ 18 were significantly associated with the progression to CRPC. A similar tendency was also observed in the relationship between newly developed hypertension following hormone therapy and CRPC progression. On the other hand, neither dyslipidemia nor hyperuricemia, regardless the onset timing, exhibited any association with CRPC progression. In multivariate analysis, postDM and PSA ≥ 18 were extracted as independent risk factors for CRPC progression (adjusted hazard ratios, 3.38 and 2.34; p values, 0.016 and 0.019, respectively). Kaplan-Meier analysis and log-rank test clearly indicated earlier progression to CRPC in PC patients who developed postDM or had relatively advanced initial PC characteristics including PSA ≥ 18. Together, the development of lifestyle-related disorders, particularly DM, following hormone therapy, as well as advanced PC characteristics at initial diagnosis is considered to predict earlier progression to CRPC and poor prognosis in PC patients undergoing hormone therapy.

Timolol maleate (TM), a beta-adrenergic receptor antagonist, is widely used for canine antiglaucoma eye drops; however, its bioavailability is <5%. Our previous study revealed that magnesium hydroxide nanoparticles (nMH) have potency in improving the bioavailability of fixed-combined TM in rodent models. This study aimed to investigate whether the fixed combination with nMH improves the ocular hypotensive effect of TM and affects pupil size (PS), heart rate (HR), and mean arterial pressure (MAP) in clinically healthy dogs. Five clinically healthy dogs were administered topical saline, commercial 0.5% TM, and a 0.01% or 0.1% nMH-0.5% TM fixed combination (0.01% or 0.1% nMH-TM) twice daily in one eye for 7 days with at least a 28-day interval. The changes from baseline were calculated and were statistically analyzed for each drug. IOP was significantly reduced in both 0.01% and 0.1% nMH-TM-treated-dogs compared with saline- and TM-treated dogs. Meanwhile, 0.01% and 0.1% nMH did not exacerbate the side effects of TM. From these results, nMH improved the ocular hypotensive effect of TM without enhancing side effects. Topical nMH-TM is potentially more effective for canine ocular hypotensive eye drops than TM.

Postprandial hyperglycemia, a so-called blood glucose spike, is associated with enhanced risks of diabetes mellitus (DM) and its complications. In this study, we attempted to design nanoparticles (NPs) of protamine zinc insulin (PZI) by the bead mill method, and prepare ophthalmic formulations based on the PZI-NPs with (nPZI/P) or without polyacrylic acid (nPZI). In addition, we investigated whether the instillation of the newly developed nPZI and nPZI/P can prevent postprandial hyperglycemia in a rabbit model involving the oral glucose tolerance test (OGTT). The particle size of PZI was decreased by the bead mill to a range for both nPZI and nPZI/P of 80-550 nm with no observable aggregation for 6 d. Neither nPZI nor nPZI/P caused any noticeable corneal toxicity. The plasma INS levels in rabbits instilled with nPZI were significantly higher than in rabbits instilled with INS suspensions (commercially available formulations, CA-INS), and the plasma INS levels were further enhanced with the amount of polyacrylic acid in the nPZI/P. In addition, the rapid rise in plasma glucose levels in OGTT-treated rabbits was prevented by a single instillation of nPZI/P, which was significantly more effective at attenuating postprandial hyperglycemia (blood glucose spike) in comparison with nPZI. In conclusion, we designed nPZI/P, and show that a single instillation before OGTT attenuates the rapid enhancement of plasma glucose levels. These findings suggest a better management strategy for the postprandial blood glucose spike, which is an important target of DM therapy.

The polymer that includes 2-methacryloyloxy ethyl phosphorylcholine (MPC) is well-known as an effectively hydrating multifunction agent. In this study, we prepared an MPC polymer (MPCP) using radical polymerization with co-monomers-MPC/Stearyl Methacrylate/N,N-dimethylacrylamide-and evaluated the MPCP's usefulness for dry eye treatment using a rabbit model treated with N-acetylcysteine. The MPCP particle size was 50-250 nm, and the form was similar to that of micelles. The MPCP viscosity (approximately 0.95 mPa·s) was 1.17-fold that of purified water, and a decrease in the transepithelial electrical resistance value (corneal damage) was not observed in the immortalized human corneal epithelial cell line HCE-T cell (HCE-T cell layer). The MPCP enhanced the water maintenance on the cornea, and the instillation of MPCP increased the lacrimal fluid volume and prolonged the tear film breakup time without an increase in total mucin contents in the lacrimal fluid of the normal rabbits. The therapeutic potential of the MPCP for dry eye was evaluated using an N-acetylcysteine-treated rabbit model, and, in our investigation, we found that MPCP enhanced the volume of lacrimal fluid and promoted an improvement in the tear film breakup levels. These findings regarding the creation and characteristics of a novel MPCP will provide relevant information for designing further studies to develop a treatment for dry eyes.

The incidence rate of post-cataract surgery posterior capsule opacification (PCO) and lens turbidity is about 20% in 5 years. Soemmering's ring, which is a type of PCO also called a regenerated lens with similar tissue structure to that of a human lens, is an important proxy for elucidating the mechanism of lens regeneration and maintenance of transparency. The authors created new human immortalized crystalline lens epithelial cells (iHLEC-NY1s) with excellent differentiation potential, and as a result of culturing the cells by static and rotation-floating methods, succeeded in producing a three-dimensional cell structure model (3D-iHLEC-NY1s) which is similar to Soemmering's ring in tissue structure and expression characteristics of αA-crystalline, βB2-crystalline, vimentin proteins. 3D-iHLEC-NY1s is expected to be a proxy in vitro experimental model of Soemmering's ring to enable evaluation of drug effects on suppression of cell aggregate formation and transparency. By further improving the culture conditions, we aim to control the cell sequence and elucidate the mechanism underlying the maintenance of lens transparency.

The use of eye drops is a well-established practice in the treatment of ophthalmic diseases, although the bioavailability of traditional eye drops, which are either solutions or suspensions, is insufficient, as the corneal barrier and dilution by lacrimation prevent the transcorneal penetration of drugs. Additionally, frequent instillation may cause undesirable systemic side effects and local corneal toxicity. To overcome these problems, micro- and nanoparticles, hydrogels, and viscous solutions have been tested, and solid nanoparticles are also expected to be applied. This review examines the usefulness of ophthalmic formulations based on solid nanoparticles, by using the specific example of indomethacin (IMC). Ophthalmic formulations based on solid IMC nanoparticles (IMC-NP dispersions) have been prepared using various additives (benzalkonium chloride, mannitol, methylcellulose, and cyclodextrin) and a rotation/revolution pulverizer (NP-100), to produce particles of 50-220 nm in size. The solubility of IMC in IMC-NP dispersions was 4.18-fold higher than that in the suspensions containing IMC microparticles (IMC-MP suspensions), and IMC-NP dispersions were better tolerated than commercially available NSAIDs eye drops, such as IMC, pranoprofen, diclofenac, bromfenac, and nepafenac eyedrops, in human corneal epithelial cells. Moreover, the corneal penetration in IMC-NP dispersions was higher than that in commercially available IMC and IMC-MP suspensions, and three energy-dependent endocytosis pathways (clathrin-dependent endocytosis, caveolae-dependent endocytosis, and macropinocytosis) were related to the high ophthalmic bioavailability of IMC-NP dispersions. This information can be used to support future studies aimed at designing novel ophthalmic formulations.

We previously designed an ophthalmic dispersion containing indomethacin nanocrystals (IMC-NCs), showing that multiple energy-dependent endocytoses led to the enhanced absorption of drugs from ocular dosage forms. In this study, we attempted to prepare Pluronic F-127 (PLF-127)-based in situ gel (ISG) incorporating IMC-NCs, and we investigated whether the instillation of the newly developed ISG incorporating IMC-NCs prolonged the precorneal resident time of the drug and improved ocular bioavailability. The IMC-NC-incorporating ISG was prepared using the bead-mill method and PLF-127, which yielded a mean particle size of 50-150 nm. The viscosity of the IMC-NC-incorporating ISG was higher at 37 °C than at 10 °C, and the diffusion and release of IMC-NCs in the IMC-NC-incorporating ISG were decreased by PLF-127 at 37 °C. In experiments using rabbits, the retention time of IMC levels in the lacrimal fluid was enhanced with PLF-127 in the IMC-NC-incorporating ISG, whereby the IMC-NC-incorporating ISG with 5% and 10% PLF-127 increased the transcorneal penetration of the IMCs. In contrast to the results with optimal PLF-127 (5% and 10%), excessive PLF-127 (15%) decreased the uptake of IMC-NCs after instillation. In conclusion, we found that IMC-NC-incorporating ISG with an optimal amount of PLF-127 (5-10%) resulted in higher IMC corneal permeation after instillation than that with excessive PLF-127, probably because of the balance between higher residence time and faster diffusion of IMC-NCs on the ocular surface. These findings provide significant information for developing ophthalmic nanomedicines.

We attempted to design an ophthalmic in situ gel formulation incorporating disulfiram (DIS) nanoparticles (Dis-NPs/ISG) and demonstrated the therapeutic effect of Dis-NPs/ISG on retinal dysfunction in 15-month-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a rat model of diabetes. The DIS particles were crushed using a bead mill to prepare the nanoparticles, and the Dis-NPs/ISG was prepared using a combination of the DIS nanoparticles and an in situ gelling system based on methylcellulose (MC). The particle size of the Dis-NPs/ISG was 80-250 nm, and there was no detectable precipitation or aggregation for 1 month. Moreover, the Dis-NPs/ISG was gelled at 37 °C, and the drug was delivered into the retina by instillation. Only diethyldithiocarbamate (DDC) was detected in the retina (DIS was not detected) when the Dis-NPs/ISG was instilled in the right eye, and the DDC levels in the right retina were significantly higher than those in the left retina. In addition, the retinal residence time of the drug was prolonged by the application of the in situ gelling system, since the DDC levels in the retinas of rats instilled with Dis-NPs/ISG were higher than those in DIS nanoparticles without MC. Furthermore, repetitive instillation of the Dis-NPs/ISG attenuated the deterioration of electroretinograms (ERGs) in 15-month-old OLETF rats by preventing the collapse of ATP production via excessive nitric oxide and recovered the decrease in retinal function. These findings provide important information for the development of novel therapeutic approaches to diabetic retinopathy.

We attempted to prepare ophthalmic in situ gel formulations containing lanosterol (Lan) nanoparticles (LA-NPs/ISG) and investigated the characteristics, delivery pathway into the lens, and anti-cataract effects of LA-NPs/ISG using SCR-N (rats with slight lens structure collapse) and SCR-C (rats with a combination of remarkable lens structure collapse and opacification). LA-NPs/ISG was prepared by bead milling of the dispersions containing 0.5% Lan powder, 5% 2-hydroxypropyl-β-cyclodextrin, 0.5% methylcellulose, 0.005% benzalkonium chloride, and 0.5% mannitol. The particle size distribution of Lan was 60-250 nm. The LA-NPs/ISG was gelled at 37 °C, and the LA-NPs/ISG was taken into the cornea by energy-dependent endocytosis and then released to the intraocular side. In addition, the Lan contents in the lenses of both SCR-N and SCR-C were increased by the repetitive instillation of LA-NPs/ISG (twice per day). The space and structure collapse in the lens of SCR-N with aging was attenuated by the instillation of LA-NPs/ISG. Moreover, the repetitive instillation of LA-NPs/ISG attenuated the changes in cataract-related factors (the enhancement of nitric oxide levels, calpain activity, lipid peroxidation levels, Ca2+ contents, and the decrease of Ca2+-ATPase activity) in the lenses of SCR-C, and the repetitive instillation of LA-NPs/ISG delayed the onset of opacification in the SCR-C. It is possible that the LA-NPs/ISG is useful in maintaining lens homeostasis.

A mouthwash formulation of rebamipide (REB) is commonly used to treat oral mucositis; however, this formulation does not provide sufficient treatment or prevention in cases of serious oral mucositis. To improve treatment, we attempted to design a hydrogel incorporating REB nanocrystals (R-NPs gel). The R-NPs gel was prepared by a bead mill method using carbopol hydrogel, methylcellulose and 2-hydroxypropyl-β-cyclodextrin, and another hydrogel incorporating REB microcrystals (R-MPs gel) was prepared following the same protocol but without the bead mill treatment. The REB particle size in the R-MPs gel was 0.15-25 μm, and while the REB particle size was 50-180 nm in the R-NPs gel. Next, we investigated the therapeutic effect of REB nanocrystals on oral mucositis using a hamster model. Almost all of the REB was released as drug nanocrystals from the R-NPs gel, and the REB content in the cheek pouch of hamsters treated with R-NPs gel was significantly higher than that of hamsters treated with R-MPs gel. Further, treatment with REB hydrogels enhanced the healing of oral wounds in the hamsters. REB accumulation in the cheek pouch of hamsters treated with the R-NPs gel was prevented by an inhibitor of clathrin-dependent endocytosis (CME) (40 μM dynasore). In conclusion, we designed an R-NPs gel and found that REB nanocrystals are taken up by tissues through CME, where they provide a persistent effect resulting in an enhancement of oral wound healing.

We investigated whether the accumulation of amyloid β-protein (Aβ) is enhanced in the lenses of diabetic patients. Lens epithelium samples were collected from Japanese patients during cataract surgery, and the Aβ levels and gene expression of Aβ-producing and -degrading enzymes in the samples were measured by ELISA and real-time RT-PCR, respectively. The Aβ 1-43 levels in lenses of non-diabetic patients were low (0.11 pmol/g protein), while the levels in lenses of diabetic patients were significantly (6-fold) higher. Moreover, the Aβ1-43/total-Aβ ratio in the lenses of diabetic patients was also significantly higher than non-diabetic patients (p < 0.05). In addition, the mRNA levels for Aβ-producing enzymes were also enhanced in the lenses of diabetic patients. In contrast to the results for Aβ-producing enzymes, the mRNAs for the Aβ-degrading enzymes in the lenses of diabetic patients were significantly lower than in non-diabetic patients (p < 0.05). Furthermore, Aβ 1-43/total-Aβ ratio in lenses was found to increase with plasma glucose level. In conclusion, these results suggest that high glucose levels cause both an increase in Aβ production and a decrease in Aβ degradation, and these changes lead to the enhancement in Aβ1-43 accumulation in the lenses of diabetic patients. These findings are useful for developing therapies for diabetic cataracts and for developing anti-cataract drugs.

Eye drops containing Tranilast (TL), N-(3,4-dimethoxycinnamoyl) anthramilic acid, are used as an anti-allergic conjunctivitis drug in the ophthalmic field. Traditional eye drops are very patient compliant, although the bioavailability (BA) of most eye drops is low since eye drops cannot be instilled beyond the capacity of the conjunctival sac due to its limited volume. Thus, traditional eye drops have low BA and a short duration of the drug on the ocular surface, so solutions to these problems are highly anticipated. In this study, we designed a sustained-release drug-delivery system (DDS) for TL nanoparticles. TL nanoparticles were prepared by bead mill treatment, and the gel formulations containing TL nanoparticles (TL-NPs-Gel, particle size 50 nm-100 nm) were provided by carboxypolymethylene. The crystal structure of TL with and without bead mill treatment is the same, but the TL solubility in formulations containing nanoparticles was 5.3-fold higher compared with gel formulations containing TL microparticles (TL-MPs-Gel). The photo and thermal stabilities of TL-NPs-Gel are also higher than those of dissolved TL. Moreover, when TL-NPs-Gel is applied to the upper eyelid skin (outside), the TL is released as nanoparticles, and delivered to the lacrimal fluid through the meibomian glands. In addition, the TL release profile for TL-NPs-Gel was sustained over 180 min after the treatment. These findings can be used to develop a sustained-release DDS in the ophthalmic field.

Meloxicam (MLX) is widely applied as a therapy for rheumatoid arthritis (RA); however, it takes far too long to reach its peak plasma concentration for a quick onset effect, and gastrointestinal toxicity has been observed in RA patients taking it. To solve these problems, we designed MLX solid nanoparticles (MLX-NPs) by the bead mill method and used them to prepare new oral formulations. The particle size of the MLX-NPs was approximately 20-180 nm, and they remained in the nano-size range for 1 month. The tmax of MLX-NPs was shorter than that of traditional MLX dispersions (MLX-TDs), and the intestinal penetration of MLX-NPs was significantly higher in comparison with MLX-TDs (P < 0.05). Caveolae-dependent endocytosis (CavME), clathrin-dependent endocytosis (CME), and micropinocytosis (MP) were found to be related to the high intestinal penetration of MLX-NPs. The area under the plasma MLX concentration-time curve (AUC) for MLX-NPs was 5-fold higher than that for MLX-TDs (P < 0.05), and the AUC in rats administered 0.05 mg/kg MLX-NPs were similar to rats administered the therapeutic dose of 0.2 mg/kg MLX-TDs. In addition, the anti-inflammatory effect of the MLX-NPs was also significantly higher than that of MLX-TDs at the corresponding dose (P < 0.05), and the therapeutic effect of 0.2 mg/kg MLX-TDs and 0.05 mg/kg MLX-NPs in adjuvant-induced arthritis (AA) rats showed no difference. Furthermore, the gastrointestinal lesions in AA rats treated repetitively with 0.05 mg/kg MLX-NPs were fewer than in rats receiving 0.2 mg/kg MLX-TDs (P < 0.05). In conclusion, we demonstrate that MLX solid nanoparticles allow a quick onset of therapeutic effect and that three endocytosis pathways, CavME, CME, and MP, are related to the high absorption of solid nanoparticles. In addition, we found that MLX solid nanoparticles make it possible to reduce the amount of orally administered drugs, and treatment with low doses of MLX-NPs allows RA therapy without intestinal ulcerogenic responses to MLX. These findings are useful for designing therapies for RA patients.

The commercially available rebamipide ophthalmic suspension (CA-REB) was approved for clinical use in patients with dry eye; however, the residence time on the ocular surface for the traditional formulations is short, since the drug is removed from the ocular surface through the nasolacrimal duct. In this study, we designed a novel sustained-release drug delivery system (DDS) for dry eye therapy by rebamipide nanoparticles. The rebamipide solid nanoparticle-based ophthalmic formulation (REB-NPs) was prepared by a bead mill using additives (2-hydroxypropyl-β-cyclodextrin and methylcellulose) and a gel base (carbopol). The rebamipide particles formed are ellipsoid, with a particle size in the range of 40-200 nm. The rebamipide in the REB-NPs applied to eyelids was delivered into the lacrimal fluid through the meibomian glands, and sustained drug release was observed in comparison with CA-REB. Moreover, the REB-NPs increased the mucin levels in the lacrimal fluid and healed tear film breakup levels in an N-acetylcysteine-treated rabbit model. The information about this novel DDS route and creation of a nano-formulation can be used to design further studies aimed at therapy for dry eye.

We designed an intravitreal injection formulation containing lanosterol nanoparticles (LAN-NPs) via the bead mill method and evaluated the therapeutic effect of LAN-NPs on lens structure collapse and opacification using two rat cataract models (SCR-N, rats with slight lens structure collapse; SCR-C, rats with the combination of a remarkable lens structure collapse and opacification). The particle size of lanosterol in the LAN-NPs was around 50-400 nm. A single injection of LAN-NPs (0.5%) supplied lanosterol into the lens for 48 h, and no irritation or muddiness was observed following repeated injections of LAN-NPs for 6 weeks (once every 2 days). Moreover, LAN-NPs repaired the slight collapse of the lens structure in SCR-N. Although the remarkable changes in the lens structure of SCR-C were not repaired by LAN-NP, the onset of opacification was delayed. In addition, the increase of cataract-related factors (Ca2+ contents, nitric oxide levels, lipid peroxidation and calpain activity levels) in the lenses of SCR-C was attenuated by the repeated injection of LAN-NPs. It is possible that a deficiency of lanosterol promotes the production of oxidative stress. In conclusion, it is difficult to improve serious structural collapse with posterior movement of the lens nucleus with a supplement of lanosterol via LAN-NPs. However, the intravitreal injection of LAN-NPs was found to repair the space and structural collapse in the early stages in the lenses.

The percutaneous absorption of a fentanyl (FEN)-patch is affected by various external factors including the volume of sebum secretion, which causes changes in the skin surface environment. In this study, we prepared a lard-based sebum-like secretion (SLS), and applied it to investigate the effect of different skin surface conditions on the drug penetration of a FEN-patch. In vitro work to test drug release using the Franz diffusion cell indicated that drug release was significantly suppressed by treatment with 5% SLS, which is equivalent to the amount of daily human sebum secretion. Conversely, in ex vivo experiments using rat skin, the amount of FEN that accumulated in the skin tissue of the 5% SLS-treated rats was higher in comparison with the non-SLS treated group. Furthermore, in vivo experiments indicated that the plasma FEN concentration in rats treated with the FEN-patch was significantly increased by treatment with 5% SLS. These results suggest that the sebum affected the release, accumulation, and absorption of FEN from the FEN-patch, and the FEN concentration in the blood was reflected by the balance of the suppression of drug release and the enhancement of drug accumulation in the skin with SLS.

Ophthalmic preservatives are indispensable in eye drop formulations, but may be toxic to corneal structures. Corneal damage necessitates the discontinuation of treatment with ophthalmic solutions. Therefore, the development of a new and safe preservative system without corneal toxicity is needed. The present study investigated the effects of mannitol on the antimicrobial activities and corneal toxicities of various preservatives using Escherichia coli and a human corneal epithelial cell line (HCE-T cells). The following preservatives were examined: boric acid (BA), benzalkonium chloride (BAC), methyl parahydroxybenzoate (MP), propyl parahydroxybenzoate (PP), sodium chlorite (SC), and zinc chloride (ZC). The antimicrobial activities and HCE-T-cell toxicities of 50 µg/mL BA, MP, PP, SC, and ZC were reduced by a co-treatment with mannitol (0-300 µg/mL). The suppressed antimicrobial activities of BA, MP, PP, and SC by the co-treatment with mannitol were restored by the application of a mannitol content higher than 500 µg/mL. In contrast to these 5 preservatives, the addition of mannitol did not affect the antimicrobial activity of BAC and attenuated its HCE-T cell toxicity. Therefore, the balance between the contents of mannitol and preservatives is important in co-treatments. The present results will serve as a guide for the future development of eye drop formulations without corneal toxicity.

We previously developed ophthalmic formulations containing tranilast nanopartaicles (ophthalmic TL-NPs formulations), and found them to show high uptake into ocular tissues. In this study, we aimed to design an in situ gel incorporating TL-NPs with 0.5-3% methylcellulose (MC, type SM-4) to ensure long residence time of the drug at the ocular surface. The ophthalmic TL-NPs formulations were prepared by the bead mill method, which yielded a mean particle size of ~93 nm with or without MC (0.5-3%). Although the dispersibility of TL particles in ophthalmic formulations increased with the MC content, the diffusion behavior of TL particles in the dispersion medium decreased with MC content. In an in vivo study using rats, the TL content in the lacrimal fluid was enhanced with MC content in the ophthalmic TL-NPs formulations, and the optimum amount of MC (0.5-1.5%) enhanced the TL content in the cornea and conjunctiva, and an anti-inflammatory effect of TL in rats instilled with ophthalmic TL-NPs formulations was observed. On the other hand, excessive MC (3%) prevented the corneal uptake of TL-NPs after instillation, and the anti-inflammation effect of TL was lower than that of ophthalmic TL-NPs formulations with optimum MC (0.5-1.5%). In conclusion, we found that gel formulations of TL-NPs with 0.5 and 1.5% MC provided a prolonged pre-corneal and pre-conjunctival contact time of TL, and resulted in higher TL contents in the cornea and conjunctiva following instillation in comparison with TL-NPs with or without 3% MC. This is probably due to the balance between the higher residence time and faster diffusion of TL-NPs on the ocular surface. These findings provide significant information that can be used to design further studies aimed at developing ophthalmic nanomedicines.

Indomethacin (IMC)-induced gastrointestinal (GI) injuries are more common in rheumatoid arthritis (RA) patients than in other IMC users, and the overexpression of nitric oxide (NO) via inducible NO synthase (iNOS) is related to the seriousness of IMC-induced GI injuries. However, sufficient strategies to prevent IMC-induced GI injuries have not yet been established. In this study, we designed dispersions of rebamipide (RBM) solid nanocrystals (particle size: 30-190 nm) by a bead mill method (RBM-NDs), and investigated whether the oral administration of RBM-NDs is useful to prevent IMC-induced GI injuries. The RBM nanocrystals were spherical and had a solubility 4.71-fold greater than dispersions of traditional RBM powder (RBM-TDs). In addition, the RBM-NDs were stable for 1 month after preparation. The RBM contents in the stomach, jejunum, and ileum of rats orally administered RBM-NDs were significantly higher than in rats administered RBM-TDs. Moreover, the oral administration of RBM-NDs decreased the NO levels via iNOS and area of the GI lesions in IMC-stimulated RA (adjuvant-induced arthritis rat) rats in comparison with the oral administration of RBM-TDs. Thus, we show that the oral administration of RBM-NDs provides a high drug supply to the GI mucosa, resulting in a therapeutic effect on IMC-induced GI injuries. Solid nanocrystalline RBM preparations may offer effective therapy for RA patients.

OBJECTIVE: This study evaluated the clinical value of diffusion-weighted magnetic resonance imaging (DW-MRI) in the diagnosis and staging of congenital cholesteatoma (CC). PATIENTS AND METHODS: We retrospectively reviewed 24 patients with CC. All the patients underwent computed tomography (CT) and DW-MRI preoperatively; thereafter, surgery was performed. DW-MRI examination was performed with a 3 T MRI system using three-dimensional reversed fast imaging with steady-state precession and diffusion-weighted magnetic resonance sequence. The preoperative and operative CT and DW-MRI findings were compared. RESULTS: Using DW-MRI, cholesteatoma was successfully detected in 17 (71%) of the 24 patients with CC. Among the seven patients with false-negative results, the cholesteatoma mass diameter was <5 mm in six patients and ≥5 mm in one patient. One of these patients had open type congenital cholesteatoma (OTCC). The detection rates for closed type cholesteatoma and OTCC were 85% (17/20) and 0% (0/4), respectively, using DW-MRI. Using CT and DW-MRI, the correct stage was identified in 88% (15/17) and 59% (10/17) of the patients with aeration around the CC and in 0% (0/7) and 100% (7/7) of those without aeration around the CC, respectively. CONCLUSION: CT is the primary imaging tool for evaluating suspected CC in patients with aeration around the CC. However, CT is unreliable for the detection of the extension and staging of CC when the middle ear is filled with nonspecific imaging. DW-MRI is useful for the preoperative diagnosis and staging of CC > 5 mm in diameter with or without surrounding granulation tissue. Thus, we recommend using DW- MRI at least when CT fails to localize CC as a soft tissue mass because of non-specific tissue filling the middle ear and the mastoid.

This study designed the transdermal formulations containing indomethacin (IMC)-1% IMC was crushed with 0.5% methylcellulose and 5% 2-hydroxypropyl-β-cyclodextrin by the bead mill method, and the milled IMC was gelled with or without 2% l-menthol (a permeation enhancer) by Carbopol® 934 (without menthol, N-IMC gel; with menthol, N-IMC/MT gel). In addition, the drug release, skin penetration and percutaneous absorption of the N-IMC/MT gel were investigated. The particle sizes of N-IMC gel were approximately 50-200 nm, and the combination with l-menthol did not affect the particle characterization of the transdermal formulations. In an in vitro experiment using a Franz diffusion cell, the skin penetration in N-IMC/MT gel was enhanced than the N-IMC gel, and the percutaneous absorption (AUC) from the N-IMC/MT gel was 2-fold higher than the N-IMC gel. On the other hand, the skin penetration from the N-IMC/MT gel was remarkably attenuated at a 4 °C condition, a temperature that inhibits all energy-dependent endocytosis. In conclusion, this study designed transdermal formulations containing IMC solid nanoparticles and l-menthol, and found that the combination with l-menthol enhanced the skin penetration of the IMC solid nanoparticles. In addition, the energy-dependency of the skin penetration of IMC solid nanoparticles was demonstrated. These findings suggest the utility of a transdermal drug delivery system to provide the easy application of solid nanoparticles (SNPs).

We previously found the instillation of sericin to be useful as therapy for keratopathy with or without diabetes mellitus. In this study, we investigated whether a combination of solid magnesium hydroxide nanoparticles (MHN) enhances epithelial corneal wound healing by sericin using rabbits, normal rats and type 2 diabetes mellitus rats with debrided corneal epithelium (ex vivo and in vivo studies). Ophthalmic formulations containing sericin and MHN (N-Ser) were prepared using a bead mill method. The mean particle size of the N-Ser was 110.3 nm at the time of preparation, and 148.1 nm one month later. The instillation of N-Ser had no effect on the amount of lacrimal fluid in normal rabbits (in vivo), but the MHN in N-Ser was found to expand the intercellular space in ex vivo rat corneas. In addition, the instillation of N-Ser increased the phosphorylation of Extracellular Signal-regulated Kinase (ERK)1/2, a factor involved in cell adhesion and cell proliferation in the corneal epithelium, in comparison with the instillation of sericin alone. The combination with MHN enhanced epithelial corneal wound healing by sericin in rat debrided corneal epithelium (in vivo). This study provides significant information to prepare potent drugs to cure severe keratopathy, such as diabetic keratopathy.

Purpose: We designed formulations based on minoxidil (MXD) nanoparticles (N-MXD) and examined whether N-MXD can increase drug delivery into the follicles. In addition, we investigated the effect of N-MXD on hair growth in C57BL/6 mice. Methods: N-MXD (1%) was prepared as follows: methylcellulose, p-hydroxyalkylbenzoates, mannitol, and MXD were dispersed in purified water and milled using zirconia beads under refrigeration (5500 rpm, 30 s×15 times, intermittent milling). C57BL/6 mice were used to evaluate hair-growth effects. The expression levels of mRNA and protein for vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) were determined by real-time PCR and ELISA methods, respectively. Results: The ratio of solid-MXD was approximately 60% in N-MXD, and the MXD nanoparticles (90-300 nm) were oblong in shape. For the design of nanomedicines, usability is important. Therefore, we measured the stability and toxicity after N-MXD treatment. No agglutination of MXD nanoparticles was detected for 2 weeks, and no redness or MXD powder residue was observed in the skin after repetitive applications of N-MXD. Next, we evaluated hair-growth effects by N-MXD treatment. MXD contents in the skin tissue from N-MXD were lower than for commercially available MXD formulations (CA-MXD). Conversely, MXD contents in the hair bulbs were higher for N-MXD than for CA-MXD, and the drug efficacy of N-MXD was also higher than that of CA-MXD. In addition, the mRNA and protein levels of IGF-1 and VEGF were enhanced by the repetitive application of N-MXD and CA-MXD, and the enhanced IGF-1 and VEGF levels were significantly higher for N-MXD than for CA-MXD. Conclusion: We designed a novel nanomedicine based on MXD nanoparticles and showed that N-MXD can deliver MXD into hair bulbs via hair follicles and that the therapeutic efficiency for hair growth is higher than for CA-MXD (solution type).

We previously designed a novel transdermal formulation containing ketoprofen solid nanoparticles (KET-NPs formulation), and showed that the skin penetration from the KET-NPs formulation was higher than that of a transdermal formulation containing ketoprofen microparticles (KET-MPs formulation). However, the precise mechanism for the skin penetration from the KET-NPs formulation was not clear. In this study we investigated whether energy-dependent endocytosis relates to the transdermal delivery from a 1.5% KET-NPs formulation. Transdermal formulations were prepared by a bead mill method using additives including methylcellulose and carbopol 934. The mean particle size of the ketoprofen nanoparticles was 98.3 nm. Four inhibitors of endocytosis dissolved in 0.5% DMSO (54 μM nystatin, a caveolae-mediated endocytosis inhibitor; 40 μM dynasore, a clathrin-mediated endocytosis inhibitor; 2 μM rottlerin, a macropinocytosis inhibitor; 10 μM cytochalasin D, a phagocytosis inhibitor) were used in this study. In the transdermal penetration study using a Franz diffusion cell, skin penetration through rat skin treated with cytochalasin D was similar to the control (DMSO) group. In contrast to the results for cytochalasin D, skin penetration from the KET-NPs formulation was significantly decreased by treatment with nystatin, dynasore or rottlerin with penetrated ketoprofen concentration-time curves (AUC) values 65%, 69% and 73% of control, respectively. Furthermore, multi-treatment with all three inhibitors (nystatin, dynasore and rottlerin) strongly suppressed the skin penetration from the KET-NPs formulation with an AUC value 13.4% that of the control. In conclusion, we found that caveolae-mediated endocytosis, clathrin-mediated endocytosis and macropinocytosis are all related to the skin penetration from the KET-NPs formulation. These findings provide significant information for the design of nanomedicines in transdermal formulations.

The creation of the National Health Insurance program has greatly contributed to giving Japan the world's highest level of life expectancy.  However, the cost of medical care in Japan has increased as a result of an aging society.  In response to this reality, the Japanese government initiated a campaign to promote the use of generic drugs (GEs).  In order to clarify some of the trends that contribute to different clinical medicine department usages of GEs, we carried out a survey of 400 pharmacies.  The survey data was analyzed using linear regression analysis.  Analysis of linear equations derived "utilization" that indicated ease of use of GEs, and a "saturation acceptable value (maximum allowed)" that indicated usage of GEs.  The breakdown for different clinical medicine department usages of GEs was determined as the following: psychosomatic medicine or psychiatry was 11±0.13%, internal medicine was 29±0.18%, orthopedics was 18±0.14%, ophthalmology or otolaryngology was 15±0.14%, other departments was 17±0.15%.  Furthermore, the highest utilization derived by linear regression analysis was orthopedics.  The highest acceptable saturation value was for psychosomatic medicine or psychiatry, while the lowest acceptable saturation value was orthopedics.  The results of the study confirm the importance of establishing evaluation methods for GE usage, and that linear regression analysis is a powerful tool for revealing trends in GE usage among different departments.  Additionally, the study suggests that determining GE spread measures is valuable, since they can serve as an aid to future pharmaceutical administration consideration.

白内障予防および治療において，その発症機構を把握し，エビデンスに基づいた治療薬を開発することは非常に重要である．本研究では，3 種の遺伝性白内障モデルラット(UPLR，SCR，ICR)を用い，いずれの水晶体混濁機構においても一酸化窒素(NO)がかかわることを見出すとともに，NOを標的とした点眼製剤の開発とその抗白内障効果を評価した．その混濁機構として，UPLR では過剰なNO がミトコンドリアゲノム傷害とATP 産生減少を招き，その結果としてCa2+-ATPase の機能不全に伴う細胞内Ca2+増加や水晶体混濁がみられた．一方，SCR およびICR では過剰なNO が脂質過酸化を引き起こし，これによりCa2+-ATPase 活性が低下，結果として細胞内Ca2+が増加し水晶体混濁につながることを明らかにした．さらに，2- ヒドロキシプロピル-β-シクロデキストリンを用い，ラジカルスカベンジャーでありNO 産生阻害作用を有するジスルフィラム点眼製剤の調製法を確立し，本点眼製剤がNO を標的とした白内障薬物療法に有用であることを明らかにした．

Objective: Recently, the cost of medical care in Japan has increased as a result of an aging society.  In response to this reality, the Japanese government initiated a campaign to promote the use of generic drugs.  In spite of this campaign, Japanese consumers have doubts about the safety and reliability of generic drugs, resulting in lower usage of these drugs compared to usage in Europe and the US.Methods: In order to clarify some of the factors that contribute to low rates of generic drug use, we carried out a survey of 400 pharmacies.  The survey data was analyzed using factor analysis and cluster analysis, which is a technique known as multivariate analysis.Results: The results from factor analysis derived four factors: 1) generic drug usage related to generic drug prescription class, 2) the amount of generic drug prescriptions related to patient preferences, 3) patient willingness to use generic drug prescriptions, and 4) pharmacy willingness.  Cluster analysis was used to classify pharmacies participating in the survey.  The results of cluster analysis revealed three main pharmacy groups: a) low usage of generic drugs, b) moderate usage of generic drugs, and c) high usage of generic drugs.Conclusion: The results of multivariate analysis showed that pharmacists are more willing to issue generic drugs unless doctors instruct them to use a brand-name drug.

Kalimate^® is often administered to patients via a simple suspension method by feeding tube. However, this method has a low recovery ratio and tube obstruction can occur. In this study, we investigated whether adding dextrin to decrease the adhesiveness and dispersibility improves the low recovery ratio and reduce tube obstruction. The addition of 0.6% dextrin significantly increased the dispersibility and fluidity of kalimate^® compared to the dispersibility and fluidity of kalimate in purified water, but the addition of 0.1% and 0.2% dextrin did not. On the other hand, the 0.1 - 0.6% dextrin improved the low recovery ratio of kalimate^® in the simple suspension method by feeding tube. These results show that the addition of dextrin improves the low recovery ratio and prevents tube obstruction in the simple suspension method of kalimate^®. In addition, it was suggested that the decrease in recovery ratio of kalimate^® is related to the adhesiveness, and the increase of dispersibility suppressed the tube obstruction in the simple suspension method by feeding tube. These findings provide significant information that can be used in improving the low recovery ratio and tube obstruction in the simple suspension method by feeding tube.

眼房水の調節には誘導型一酸化窒素合成酵素（iNOS）が産生するNOが働いていることが明らかとされている。ジスルフィラムの活性体であるジエチルジチオカルバミン酸はiNOSの阻害剤であり、緑内障で高まった房水産生量を低下させる可能性がある。本研究では、種々ジスルフィラム点眼薬製剤をウサギに点眼し実験的高眼圧を抑制することができた。

網膜疾患は中途失明の主たる原因であるが、現在の点眼薬では眼後部に位置する網膜に十分な薬物量を送達することが難しいのが現状である。本研究ではこれら課題を克服すべく、薬物ナノ結晶を装填したin situゲル化ナノ点眼液の処方設計を検討し、従来点眼液以上の眼内への薬物送達が可能であることを見出した。また、眼疾患モデルを用い、これら点眼製剤の有用性について評価した。
１）In situゲル化ナノ点眼液の処方設計とその物性評価：各種添加物を用いたビーズミル法（乾式・水中破砕）にて、トラニラスト、イルベサルタン、ブリンゾラミド、ニルバジピン等数種のナノ結晶製剤の調製法を確立した。また、メチルセルロースやPluronic F-127及び68を始めとした温度応答性in situゲル基剤をナノ結晶製剤に適用した際の粘性変化や現表面での薬物滞留性を測定することで、ナノ結晶がゲルから持続的に放出できる最適な組成・濃度の設定を行った。 ２）In situゲル化ナノ点眼液の眼疾患治療への応用性：In situゲル化ナノ点眼液は従来の溶液型製剤と比較し、薬物眼表面滞留性、角膜透過性及び眼内組織（水晶体や網膜）への薬物移行性が高まることを明らかとした。また、今回調製した製剤の1つであるブリンゾラミドナノ点眼製剤点眼が糖尿病モデル動物の視機能改善に有効であることを明らかとした。
以上、数種のin situゲル化ナノ点眼液を作成し、眼疾患に対する有用性について検討を行った。来年度は、今回作成したin situゲル化ナノ点眼液の薬効について病態モデルを用いて評価する予定である。

発生学的観点からin vivoにおいて多能性幹細胞から多様な細胞へ分化する過程では，細胞間の因子やシグナルが適切かつ必要な部位に作用する必要があるが，その機序は明らかではない。本研究では，ヒト虹彩由来iPS細胞から生理的機能を有する網膜神経細胞への分化において二次元培養の細胞間相互作用と三次元オルガノイド培養を中心とした網膜神経細胞の再生，およびそれらの細胞の移植時における拒絶反応に対する免疫抑制剤などの薬物送達と移植効率を検討する。 さらに，既に臨床で使用されている薬物の安全性や薬効評価について，iPS細胞から分化誘導後に不死化させた網膜前駆細胞および網膜神経細胞を用いた薬効評価を行う。 2020年度では，我々が初めて樹立することに成功したヒト虹彩由来iPS細胞の多分化能に関する検証実験を行い，さらにヒト虹彩由来iPS細胞を二次元培養系において，Recoverin陽性の網膜神経細胞，およびNeurofilament-MとBrn-3b陽性の網膜神経節細胞に分化誘導を行った。分化誘導した細胞について，電気生理学的機能の有無についてパッチクランプ法による検証を行い，Naチャネル（内向き電流）が検出された。TTXを添加することにより、Naチャネルは消失し、Wash outすることで再びNaチャネルが再検出されたことから、電気生理学的に機能する網膜神経細胞であることが確認された。 以上の結果より，ヒト虹彩由来iPS細胞を二次元培養系による分化誘導を行ったところ，電気生理学的な機能を有する網膜神経系細胞に分化できることがわかった。これらの結果をまとめて論文として報告した。

点眼により非侵襲的に薬物を眼後部へ到達させ、安全で効果的な新規の網膜症治療を可能とする「ナノ点眼製剤の開発」を目指し、令和1年度は血管拡張作用を有するシロスタゾールやニルバジピンをはじめ、レバミピド、インドメタシンといった様々な薬物ナノ結晶の調製法確立や組織に対する安全性と移行性について検討した。 １）ナノ結晶点眼製剤の角膜に対する傷害性について検討する： 再結晶化を基盤とした結晶形制御技術により結晶多型を同定・作成し、よりナノ結晶化しやすい結晶構造を解析した。また、本検討から得られた結晶に対し、セルロースやシクロデキストリンを添加剤としたビーズミル法（乾式・水中破砕）を適用することで、シロスタゾール、ニルバジピン、レバミピド、インドメタシン等数種のナノ結晶製剤の調製法を確立した。さらにこれら薬物ナノ結晶分散液を、ヒト角膜上皮細胞や家兎へ適用した際の細胞傷害性を検討し、本ナノ製剤が従来製剤と比べ細胞傷害性が少ないことを明らかとした。 ２）ナノ結晶点眼製剤適用後の眼内薬物分布及び動態を確認する： 昨年の検討で示唆された点眼後におけるエンドサイトーシスによる取込みと薬物粒子サイズの関係性をより詳細に検討し、クラスリン介在性エンドサイトーシス、カベオラ介在性エンドサイトーシス、マクロピノサイトーシス等がナノ結晶の組織移行に関与していることを明らかとした。また、本性質を応用することで、眼瞼適用による眼表面への持続的な薬物供給システムや眼後部に位置する水晶体への薬物供給に伴う白内障治療への応用化が可能であることを示した。 以上、結晶形制御技術とビーズミル法の融合により得られたナノ結晶点眼製剤は高い眼内移行能を有することを見出した。来年度は、これら高い眼内移行性に徐放能を付加することでより高い治療効果が期待できる製剤開発を目指すとともに、網膜疾患モデルを用いてその効果を評価する予定である。

ナノ化技術を用いた貼付型眼瞼透過性製剤“貼る目薬”を作成した。本“貼る目薬”は、刺激性が少なく、涙液中に安定した薬物放出を示した。また、ナノ結晶含有ゲルパッチの眼内移行ルートは、眼瞼表面から侵入した薬物が、マイボーム腺に移行し、マイバムとして眼表面に排出されることで、涙液に移行していることを見出した。本研究結果は、抗菌や抗炎症をはじめとする眼領域疾患に対し、より効果的な治療を実現する上で重要な知見であると思われる。

臨床において、非ステロイド性抗炎症薬（NSAIDs）による消化管障害は重篤な問題である。我々は、このNSAIDs起因性消化管障害の制御を目指し、湿式ビーズミル法を用い、メロキシカム（MLX）ナノ結晶経口製剤を作製した（粒子径48±138 nm, 平均値±標準偏差）。本製剤は、従来の製剤と比較し、4.3倍バイオアベイラビリティが高まり、薬物投与量の減量を可能とした。さらに、これら製剤化に伴うNSAIDs投与量の減少が、薬剤の消化管粘膜直接刺激の低下を介し、障害誘発を軽減することを示した。本成果が安全なNSAIDs療法の確立に繋がることを期待する。

緑内障による失明患者の減少を目指すべく、平成25年度はシロスタゾール（CLZ）ナノ粒子含有点眼液の処方設計及び点眼後の眼内動態眼の測定（眼後部薬物到達量の測定）について検討した。１）高い安定性及び保存性を有するCLZナノ粒子含有点眼液の調製：界面活性剤（ソディウムドキュセート）、ベンザルコニウム塩化物、セルロース及びシクロデキストリンを添加剤として用い、ビーズミル法により乾式及び水中破砕を行うことで、安定な分散性及び粒子状態を有するCLZナノ結晶点眼液を調製した。さらにこれらは日本薬局方に従った保存性試験（大腸菌使用）にて高い保存効果を有していることが確認できた。２）CLZナノ粒子含有点眼液点眼後の体内薬物分布及び動態を確認：上記にて調製したCLZナノ粒子含有点眼製剤は、点眼後速やかに角膜を透過し、眼内へ移行することが確認できた。また、結膜や眼周辺部に対しても高い透過性を示し、眼周辺部を介した網膜への薬物到達が可能であることを明らかとした。３）CLZナノ粒子含有点眼液の眼内降下作用：ブドウ糖負荷による高眼圧モデルに点眼することで、市販点眼薬（プロスタグランジン系）と同程度の高い眼圧降下作用を示した。以上、CLZナノ粒子含有点眼製剤の調製法を確立した。さらに、これら点眼製剤は点眼による網膜への薬物送達が可能であり、眼圧降下作用も有しているという知見が得られた。

L-ピログルタミン酸は一酸化窒素を抑制することで、眼房水産生及び眼圧低下作用を有すること、また、これらL-ピログルタミン酸は、視神経賦活効果も有していることを明らかとした。さらに、点眼製剤より高濃度の薬物供給が可能である眼瞼適用ゲルパッチを開発した。以上、L-ピログルタミン酸と眼瞼適用ゲルパッチを用いることで、眼圧降下と視神経保護を同時標的とした新しい緑内障治療薬の開発に繋がる可能性を示した。

急速かつ強いレベルの酸化的刺激は、ヒト水晶体上皮においてミトコンドリア傷害を引き起こすこと、また、この傷害には、刺激に対するミトコンドリアの感受性に加え、細胞膜の反応性・抵抗性が密接に関わることを明らかとした。さらに、これらミトコンドリア機能低下は、早期において可逆的であるが、機能回復には時間を有することを見出した。以上、一過性のミトコンドリア傷害の繰り返しは、水障体白濁に繋がる可能性を示した。