Papers - Nakazawa, Yosuke

TRPV1 attenuates epithelial-mesenchymal transition via calpain-protein tyrosine phosphatase pathway in lens epithelial cells

Nakazawa Y., Nishizawa F., Kawata S., Sugiyama Y., Nagai N., Yamamoto N., Funakoshi-Tago M.

Experimental Eye Research 258   110435 2025.09

ISSN  00144835

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TRPV1, which is widely expressed throughout the body, is a non-selective cation channel activated by capsaicin. We previously reported that TRPV1 activation suppressed TGFβ2-induced epithelial-mesenchymal transition (EMT) by inhibiting Epidermal Growth Factor Receptor (EGFR) phosphorylation in lens epithelial cells (Sugiyama et al., 2021). However, the detailed molecular mechanism remains unclear. In this study, we focused on the calpain–protein tyrosine phosphatase (PTP) pathway to elucidate the detailed mechanism underlying TRPV1-induced EMT suppression. Calpain and PTP inhibitors mitigated the suppressive effect of capsaicin on TGFβ2-induced EMT in vitro and ex vivo. Finally, we shown that CalpainS1 and PTPN9 overexpression abrogated the effect of capsaicin on EMT in lens epithelial cells. Our findings indicate that calpain and PTP proteins are good candidates for preventing EMT after cataract surgery.

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De novo <i>CDKN1C</i> variant in Beckwith-Wiedermann spectrum with atypical complications

Moriura, Y; Nishio, Y; Ichimura, S; Noda, H; Tanahashi, Y; Yamamoto, H; Nakazawa, Y; Oso, T; Sato, Y; Takenouchi, T; Saitoh, S; Muramatsu, Y; Ogi, T

HUMAN GENOME VARIATION 12 ( 1 ) 9 2025.05

ISSN  2054-345X

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Antagonistic effects of PU.1 on Gfi-1B-induced erythroid colony formation in human cord blood cells

Manabe N., Sakurai T., Kihara-Negishi F., Nakazawa Y., Yamada T., Iwama A., Oikawa T.

Cellular and molecular biology (Noisy-le-Grand, France) 71 ( 3 ) 48 - 56 2025.04

ISSN  0145-5680

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Gfi-1B is a hematopoietic transcription factor essential for growth and differentiation of the erythroid/megakaryocytic lineages, and PU.1 is a master regulator for myeloid development. Herein, we demonstrate that PU.1 interacted with Gfi-1B in vivo by immunoprecipitation assay. GST pull-down assays showed that the binding sites were located in the Ets domain of PU.1 and the zinc finger domain of Gfi-1B. Luciferase reporter assays revealed that PU.1 and Gfi-1B antagonized each other's transcriptional activity in a dose-dependent manner. The transduction of Gfi-1B alone in human cord blood progenitor cells strongly enhanced erythroid colony formation. However, the transduction of PU.1 along with Gfi-1B in the progenitors significantly inhibited erythroid colony formation. Co-expression of Gfi-1B with a mutant PU.1, which bound to Gfi-1B but not to GATA1, another erythroid master regulator, also inhibited Gfi-1B-induced erythroid colony formation. Our results suggest that the function of Gfi-1B in the growth and differentiation of erythroid cells is antagonized by the expression of PU.1.

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Pharmacological Behavior of Propylene Glycol/Polyvinyl Alcohol Hydrogel Incorporating Indomethacin Nanocrystals in the Skin

Otake H., Ogata F., Nakazawa Y., Misra M., Tsubaki M., Kawasaki N., Nagai N.

Gels 11 ( 4 )  2025.04

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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.

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Changes in Protein Expression in Warmed Human Lens Epithelium Cells Using Shotgun Proteomics

Otake H., Yamamoto T., Yamamoto N., Nakazawa Y., Miyata Y., Taga A., Sasaki H., Nagai N.

Medicina (Lithuania) 61 ( 2 )  2025.02

ISSN  1010660X

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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.

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Semiquantitative analysis of protein expression in heated rat lens using shotgun proteomics.

Otake H, Masuda S, Yamamoto T, Miyata Y, Nakazawa Y, Yamamoto N, Taga A, Sasaki H, Nagai N

Molecular medicine reports 31 ( 1 )  2025.01

ISSN  1791-2997

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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.

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Dermal formulation based on carbopol and Gum Arabic improves skin retention of indomethacin.

Otake H, Fumihiko O, Nakazawa Y, Misra M, Tsubaki M, Kawasaki N, Nagai N

PloS one 20 ( 6 ) e0326051 2025

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ニルバジピンナノサスペンション点眼による眼組織への 薬物送達：経口投与時における薬物挙動との比較

門脇玲太, 明和亮伍, 岩井誓治, 永田万由美, 松島博之, 中澤洋介, 山本直樹, 佐々木　洋, 長井紀章

日本白内障学会誌 (日本白内障学会)  36   95 - 99 2024.06

Research paper (scientific journal), Joint Work

亜セレン酸誘発白内障モデルの混濁過程における 水晶体弾性力の変化

門脇玲太, 浅井拓己, 出口粧央里, 大竹裕子, 岡本紀夫, 中澤洋介, 佐々木　洋, 長井紀章

日本白内障学会誌 (日本白内障学会)  36   104 - 109 2024.06

Research paper (scientific journal), Joint Work, Accepted

Identification of Ascorbate Free Radical Reductase in the Lens

Mikako Oka, Masayasu Bando, Yosuke Nakazawa, Hiroko Ushikubo, Takashi Yoshikado, Kenji Okada, Koji Chiba, Makoto Takehana

The Journal of the Japanese Society for Cataract Research (Japanese Society for Cataract Research)  36   113 - 122 2024.06

Research paper (scientific journal), Joint Work, Accepted

Design of an Oral Tablet Containing Furosemide Nanoparticles with Elevated Bioavailability.

Nagai N, Ogata F, Kadowaki R, Deguchi S, Otake H, Nakazawa Y, Misra M, Kawasaki N

Journal of oleo science (Journal of oleo science)  73 ( 4 ) 563 - 571 2024

ISSN  1345-8957

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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.

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Therapeutic Effects of Rebamipide Nanocrystals as Carbopol Gel Formulation Containing Gum Arabic in a Hamster Model of Oral Mucositis

Otake H., Masuda S., Kadowaki R., Ogata F., Nakazawa Y., Yamamoto N., Kawasaki N., Nagai N.

Journal of Oleo Science 73 ( 12 ) 1479 - 1491 2024

ISSN  13458957

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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.

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High ambient temperature may induce presbyopia via TRPV1 activation

Nakazawa Y., Kuno Y., Shimada H., Nagai N., Hiramatsu N., Takeda S., Yamamoto N., Funakoshi-Tago M., Sasaki H.

Medical Molecular Morphology 57 ( 4 ) 268 - 276 2024

ISSN  18601480

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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.

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Suppression of Neuroinflammation by Coffee Component Pyrocatechol via Inhibition of NF-κB in Microglia.

Murata T, Tago K, Miyata K, Moriwaki Y, Misawa H, Kobata K, Nakazawa Y, Tamura H, Funakoshi-Tago M

International journal of molecular sciences (International Journal of Molecular Sciences)  25 ( 1 )  2023.12

ISSN  16616596

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According to numerous studies, it has been epidemiologically suggested that habitual coffee intake seems to prevent the onset of neurodegenerative diseases. In this study, we hypothesized that coffee consumption suppresses neuroinflammation, which is closely related to the development of neurodegenerative diseases. Using microglial BV-2 cells, we first found that the inflammatory responses induced by lipopolysaccharide (LPS) stimulation was diminished by both coffee and decaffeinated coffee through the inhibition of an inflammation-related transcription factor, nuclear factor-κB (NF-κB). Pyrocatechol, a component of roasted coffee produced by the thermal decomposition of chlorogenic acid, also exhibited anti-inflammatory activity by inhibiting the LPS-induced activation of NF-κB. Finally, in an inflammation model using mice injected with LPS into the cerebrum, we observed that intake of pyrocatechol as well as coffee decoctions drastically suppressed the accumulation of microglia and the expression of interleukin-6 (IL-6), tumor necrosis factor α (TNFα), CCL2, and CXCL1 in the inflammatory brain. These observations strongly encourage us to hypothesize that the anti-inflammatory activity of pyrocatechol as well as coffee decoction would be useful for the suppression of neurodegeneration and the prevention of the onsets of Alzheimer’s (AD) and Perkinson’s diseases (PD).

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Recovery from indomethacin-induced gastrointestinal bleeding by treatment with teprenone

Deguchi, S; Iwakami, A; Tujigiwa, M; Otake, H; Mano, Y; Yamamoto, N; Nakazawa, Y; Misra, M; Nagai, N

JOURNAL OF PHARMACEUTICAL HEALTH CARE AND SCIENCES (Journal of Pharmaceutical Health Care and Sciences)  9 ( 1 ) 44 2023.11

Accepted,  ISSN  2055-0294

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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.

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Piezo1 channel causes lens sclerosis via transglutaminase 2 activation.

Doki Y, Nakazawa Y, Sukegawa M, Petrova RS, Ishida Y, Endo S, Nagai N, Yamamoto N, Funakoshi-Tago M, Donaldson PJ

Experimental eye research (Experimental Eye Research)  237   109719 2023.11

Lead author, Corresponding author, Accepted,  ISSN  0014-4835

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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.

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The citrus flavonoid, nobiletin inhibits neuronal inflammation by preventing the activation of NF-κB.

Murata T, Ishiwa S, Lin X, Nakazawa Y, Tago K, Funakoshi-Tago M

Neurochemistry international (Neurochemistry International)  171   105613 2023.09

Accepted,  ISSN  0197-0186

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Nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone) is one of the flavonoids found in shikuwasa, a popular citrus fruit in Okinawa, Japan. It exerts various pharmacological effects, such as anti-tumor, antioxidant, and anti-inflammatory activities. We herein investigated whether nobiletin attenuated lipopolysaccharide (LPS)-induced inflammatory responses in the murine microglial cell line BV-2 and neuroinflammation in mice induced by an intracerebral injection of LPS. In BV-2 cells, nobiletin significantly inhibited the LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) by preventing the mRNA expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. Nobiletin also inhibited the LPS-induced mRNA expression of CCL2, CXCL1, IL-6, and TNFα. Nobiletin markedly attenuated the transcriptional activity of the NF-κB p65 subunit without affecting the degradation of IκBα or the nuclear localization of the NF-κB p65 subunit. Nobiletin also inhibited the LPS-induced activation of JNK, but not ERK or p38, in BV-2 cells. Furthermore, the administration of nobiletin significantly suppressed the accumulation of microglia and induction of the mRNA expression of CCL2, CXCL1, IL-6, and TNFα in the murine brain induced by injecting LPS into the striatum. Collectively, these results suggest the potential of nobiletin as a candidate anti-inflammatory drug for the prevention of neuroinflammation.

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Hesperetin treatment attenuates glycation of lens proteins and advanced‑glycation end products generation.

Doki Y, Nakazawa Y, Morishita N, Endo S, Nagai N, Yamamoto N, Tamura H, Funakoshi-Tago M

Molecular medicine reports (Molecular Medicine Reports)  27 ( 5 )  2023.05

Lead author, Corresponding author, Accepted,  ISSN  1791-2997

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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.

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Orally disintegrating tablets containing famotidine nanoparticles provide high intestinal absorbability via the energy-dependent endocytosis pathway

Nagai N., Ogata F., Kadowaki R., Deguchi S., Otake H., Nakazawa Y., Nagata M., Sasaki H., Kawasaki N.

Frontiers in Bioengineering and Biotechnology (Frontiers in Bioengineering and Biotechnology)  11   1167291 2023

Accepted,  ISSN  2296-4185

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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.

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Aquaporins contribute to vacuoles formation in Nile grass type II diabetic rats

Aihara K., Nakazawa Y., Takeda S., Hatsusaka N., Onouchi T., Hiramatsu N., Nagata M., Nagai N., Funakoshi-Tago M., Yamamoto N., Sasaki H.

Medical Molecular Morphology (Medical Molecular Morphology)  56 ( 4 ) 274 - 287 2023

Lead author, Corresponding author, Accepted,  ISSN  18601480

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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.

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Therapeutic Effects of Hydrogel Formulations Incorporating Troxipide Nanoparticles on Oral Mucositis in Hamsters

Kadowaki R., Ogata F., Nishida M., Komatsu M., Otake H., Nakazawa Y., Yamamoto N., Kawasaki N., Nagai N.

Drug Design, Development and Therapy (Drug Design, Development and Therapy)  17   3349 - 3361 2023

ISSN  1177-8881

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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.

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Role of mucoadhesive agent in ocular delivery of ganciclovir microemulsion: cytotoxicity evaluation in vitro and ex vivo

Manisha Choudhari, Kritika Nayak, Noriaki Nagai, Yosuke Nakazawa, Dignesh Khunt, Manju Misra

International Ophthalmology (Springer)  43 ( 4 ) 1153 - 1167 2022.09

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0165-5701

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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.

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Combination of Lanosterol and Nilvadipine Nanosuspensions Rescues Lens Opacification in Selenite-Induced Cataractic Rats

Deguchi Saori, Kadowaki Reita, Otake Hiroko, Taga Atsushi, Nakazawa Yosuke, Misra Manju, Yamamoto Naoki, Sasaki Hiroshi, Nagai Noriaki

PHARMACEUTICS (Pharmaceutics)  14 ( 7 )  2022.07

Accepted,  ISSN  1999-4923

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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.

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Oral Formulation Based on Irbesartan Nanocrystals Improve Drug Solubility, Absorbability, and Efficacy.

Nagai N, Ogata F, Ike A, Shimomae Y, Osako H, Nakazawa Y, Yamamoto N, Kawasaki N

Pharmaceutics (Pharmaceutics)  14 ( 2 )  2022.02

Research paper (scientific journal), Accepted,  ISSN  1999-4923

　View Summary

We previously reported that the bioavailability (BA) of irbesartan (IRB), a BSC class II drug, was improved by preparing nanocrystalline suspensions. However, nanocrystalline suspensions have chemical and physical instabilities and must be converted into tablets through drying approaches in order to overcome such instabilities. In this study, we attempted to design a molded tablet based on nanocrystalline IRB suspensions (IRB-NP tablet) and investigated the effects of this IRB-NP tablet on blood pressure (BP) in a stroke-prone spontaneously hypertensive (SHR-SP) rat. The IRB-NP tablet (with a hardness of 42.6 N) was developed by combining various additives (methylcellulose, 2-hydroxypropyl-β-cyclodextrin HPβCD, D-mannitol, polyvinylpyrrolidone, and gum arabic) followed by bead-milling and freeze-drying treatments. The mean particle size in the redispersions of the IRB-NP tablet was approximately 118 nm. The solubility and intestinal absorption of IRB in the IRB-NP tablet were significantly enhanced in comparison with the microcrystalline IRB tablet (IRB-MP tablet), and both solubility and clathrin-dependent endocytosis helped improve the low BA of the IRB. In addition, the BP-reducing effect of the IRB-NP tablet was significantly higher than that of the IRB-MP tablet. These results provide useful information for the preservation of nanocrystalline suspensions of BCS class II drugs, such as IRB.

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Novel Mechanism by a Bis-Pyridinium Fullerene Derivative to Induce Apoptosis by Enhancing the MEK-ERK Pathway in a Reactive Oxygen Species-Independent Manner in BCR-ABL-Positive Chronic Myeloid Leukemia-Derived K562 Cells

Sumi K., Tago K., Nakazawa Y., Takahashi K., Ohe T., Mashino T., Funakoshi-Tago M.

International Journal of Molecular Sciences (International Journal of Molecular Sciences)  23 ( 2 )  2022.01

Research paper (scientific journal), Accepted,  ISSN  16616596

　View Summary

In the treatment of breakpoint cluster region-Abelson (BCR-ABL)-positive chronic myeloid leukemia (CML) using BCR-ABL inhibitors, the appearance of a gatekeeper mutation (T315I) in BCR-ABL is a serious issue. Therefore, the development of novel drugs that overcome acquired resistance to BCR-ABL inhibitors by CML cells is required. We previously demonstrated that a bis-pyridinium fullerene derivative (BPF) induced apoptosis in human chronic myeloid leukemia (CML)-derived K562 cells partially through the generation of reactive oxygen species (ROS). We herein show that BPF enhanced the activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase (MEK-ERK) pathway in a ROS-independent manner. BPF-induced apoptosis was attenuated by trametinib, suggesting the functional involvement of the MEK-ERK pathway in apoptosis in K562 cells. In addition, the constitutive activation of the MEK-ERK pathway by the enforced expression of the BRAFV600E mutant significantly increased the sensitivity of K562 cells to BPF. These results confirmed for the first time that BPF induces apoptosis in K562 cells through dual pathways—ROS production and the activation of the MEK-ERK pathway. Furthermore, BPF induced cell death in transformed Ba/F3 cells expressing not only BCR-ABL but also T315I mutant through the activation of the MEK-ERK pathway. These results indicate that BPF is as an effective CML drug that overcomes resistance to BCR-ABL inhibitors.

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A bis-pyridinium fullerene derivative induces apoptosis through the generation of ROS in BCR-ABL-positive leukemia cells.

Sumi K, Tago K, Nakazawa Y, Takahashi K, Ohe T, Mashino T, Funakoshi-Tago M

European journal of pharmacology (European Journal of Pharmacology)  916   174714 2021.12

Research paper (scientific journal), Accepted,  ISSN  0014-2999

　View Summary

A fusion protein, Breakpoint cluster region-Abelson (BCR-ABL) is responsible for the development of chronic myeloid leukemia (CML) and acute lymphocytic leukemia (ALL). Inhibitors against BCR-ABL are effective for the treatment of leukemia; however, a gatekeeper mutation (T315I) in BCR-ABL results in resistance to these inhibitors, which markedly impedes their efficacy. We herein demonstrated that a bis-pyridinium fullerene derivative (BPF) significantly induced apoptosis in human CML-derived K562 cells and ALL-derived SUP-B15 cells via the generation of reactive oxygen species (ROS). BPF reduced the expression of Bcr-Abl mRNA by inhibiting expression of c-Myc through ROS production. BPF also accelerated protein degradation of BCR-ABL through ROS production. Furthermore, BPF down-regulated the expression of not only BCR-ABL but also T315I-mutated BCR-ABL in ROS-dependent manner. As a result, BPF effectively induced apoptosis in transformed Ba/F3 cells expressing both BCR-ABL and T315I-mutated BCR-ABL. Collectively, these results indicate the potential of BPF as an effective leukemia drug that overcomes resistance to BCR-ABL inhibitors.

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Capsaicin attenuates TGFβ2-induced epithelial-mesenchymal-transition in lens epithelial cells in vivo and in vitro

Sugiyama Y., Nakazawa Y., Sakagami T., Kawata S., Nagai N., Yamamoto N., Funakoshi-Tago M., Tamura H.

Experimental Eye Research (Experimental Eye Research)  213   108840 2021.12

Research paper (scientific journal), Lead author, Corresponding author, Accepted,  ISSN  00144835

　View Summary

Posterior capsule opacification (PCO), the most common complication of cataract surgery occurring in 20–50% of patients after 2–5 years of cataract surgery, is a major problem in the aging society. The epithelial-mesenchymal transition (EMT) of lens epithelial cells after cataract surgery has been proposed as a major cause of PCO. Capsaicin, widely used as a food additive and analgesic agent, is a major pungent ingredient in red pepper. Although the effect of capsaicin on EMT has been reported in cancer cells, the biological reaction of capsaicin was unique in each cell type, and there have been no reports describing its effects on EMT earlier. In this study, we demonstrated that treatment with capsaicin inhibited TGFβ2-induced EMT in vitro lens epithelial cells and ex vivo explant lens epithelial cells. Furthermore, eye drops of capsaicin inhibited the PCO model mice in vivo. Finally, we showed that capsaicin inhibited non-canonically induced Smad2/3 activation via suppression of EGFR activation and ERK phosphorylation. Our findings indicate that capsaicin and its derivatives are good candidate compounds for preventing PCO after cataract surgery.

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Regulation of the membrane trafficking of the mechanosensitive ion channels trpv1 and trpv4 by zonular tension, osmotic stress and activators in the mouse lens

Nakazawa Y., Petrova R.S., Sugiyama Y., Nagai N., Tamura H., Donaldson P.J.

International Journal of Molecular Sciences (International Journal of Molecular Sciences)  22 ( 23 )  2021.12

Lead author, Corresponding author, Accepted,  ISSN  16616596

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Lens water transport generates a hydrostatic pressure gradient that is regulated by a dual-feedback system that utilizes the mechanosensitive transient receptor potential vanilloid (TRPV) channels, TRPV1 and TRPV4, to sense changes in mechanical tension and extracellular osmolarity. Here, we investigate whether the modulation of TRPV1 or TRPV4 activity dynamically affects their membrane trafficking. Mouse lenses were incubated in either pilocarpine or tropicamide to alter zonular tension, exposed to osmotic stress, or the TRPV1 and TRPV4 activators capsaicin andGSK1016790A (GSK101), and the effect on the TRPV1 and TRPV4 membrane trafficking in peripheral fiber cells visualized using confocal microscopy. Decreases in zonular tension caused the removal of TRPV4 from the membrane of peripheral fiber cells. Hypotonic challenge had no effect on TRPV1, but increased the membrane localization of TRPV4. Hypertonic challenge caused the insertion of TRPV1 and the removal of TRPV4 from the membranes of peripheral fiber cells. Capsaicin caused an increase in TRPV4 membrane localization, but had no effect on TRPV1; while GSK101 decreased the membrane localization of TRPV4 and increased the membrane localization of TRPV1. These reciprocal changes in TRPV1/4 membrane localization are consistent with the channels acting as mechanosensitive transducers of a dual‐feedback pathway that regulates lens water transport.

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Instillation of ophthalmic formulation containing nilvadipine nanocrystals attenuates lens opacification in shumiya cataract rats

Goto R., Yamada S., Otake H., Nakazawa Y., Oka M., Yamamoto N., Sasaki H., Nagai N.

Pharmaceutics (Pharmaceutics)  13 ( 12 )  2021.12

Research paper (scientific journal), Accepted,  ISSN  1999-4923

　View Summary

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.

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Balance of Drug Residence and Diffusion in Lacrimal Fluid Determine Ocular Bioavailability in In Situ Gels Incorporating Tranilast Nanoparticles

Minami M, Otake H, Nakazawa Y, Okamoto N, Yamamoto N, Sasaki H, Nagai N.

Pharmaceutics (Pharmaceutics)  13 ( 9 ) 1425 - 1425 2021.09

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1999-4923

　View Summary

We previously designed ophthalmic formulations (nTRA) containing tranilast nanoparti-cles (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-in-corporated 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.

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Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s

Vorontsova Irene, Hall James E., Schilling Thomas F., Nagai Noriaki, Nakazawa Yosuke

CELLS (Cells)  10 ( 8 )  2021.08

Research paper (scientific journal), Accepted

　View Summary

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

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Prevention of postprandial hyperglycemia by ophthalmic nanoparticles based on protamine zinc insulin in the rabbit

Deguchi S., Ogata F., Isaka T., Otake H., Nakazawa Y., Kawasaki N., Nagai N.

Pharmaceutics (Pharmaceutics)  13 ( 3 )  2021.03

Accepted,  ISSN  1999-4923

　View Summary

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 nanopar-ticles (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.

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Effect of Alpha-Glucosyl-Hesperidin Consumption on Lens Sclerosis and Presbyopia

Yosuke Nakazawa, Yuri Doki, Yuki Sugiyama, Ryota Kobayashi, Noriaki Nagai, Naoki Morishita, Shin Endo, Megumi Funakoshi-Tago, Hiroomi Tamura

Cells (Cells)  10 ( 2 ) 382 - 382 2021.02

Research paper (scientific journal), Joint Work, Accepted

　View Summary

Presbyopia is characterized by a decline in the ability to accommodate the lens. The most commonly accepted theory for the onset of presbyopia is an age-related increase in the stiffness of the lens. However, the cause of lens sclerosis remains unclear. With age, water microcirculation in the lens could change because of an increase in intracellular pressure. In the lens, the intracellular pressure is controlled by the Transient Receptor Potential Vanilloid (TRPV) 1 and TRPV4 feedback pathways. In this study, we tried to elucidate that administration of α-glucosyl-hesperidin (G-Hsd), previously reported to prevent nuclear cataract formation, affects lens elasticity and the distribution of TRPV channels and Aquaporin (AQP) channels to meet the requirement of intracellular pressure. As a result, the mouse control lens was significantly toughened compared to both the 1% and 2% G-Hsd mouse lens treatments. The anti-oxidant levels in the lens and plasma decreased with age; however, this decrease could be nullified with either 1% or 2% G-Hsd treatment in a concentration- and exposure time-dependent manner. Moreover, G-Hsd treatment affected the TRPV4 distribution, but not TRPV1, AQP0, and AQP5, in the peripheral area and could maintain intracellular pressure. These findings suggest that G-Hsd has great potential as a compound to prevent presbyopia and/or cataract formation.

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[Look Back on the Eye with Basic Research, Clinical Science and Drug Repositioning].

Kosano H, Nakazawa Y, Hara H

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 141 ( 1 ) 33 - 34 2021

Accepted,  ISSN  0031-6903

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[Eye Diseases with Aging -for Keeping High QOV (Quality of Vision)].

Nakazawa Y, Nagai N

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 141 ( 12 ) 1305 - 1306 2021

Accepted,  ISSN  0031-6903

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Oral consumption of α-glucosyl-hesperidin could prevent lens hardening, which causes presbyopia

Yosuke Nakazawa, Miki Aoki, Yuri Doki, Naoki Morishita, Shin Endo, Noriaki Nagai, Megumi Funakoshi-Tago, Hiroomi Tamura

Biochemistry and Biophysics Reports (Elsevier)  25   100885 2020.12

Research paper (scientific journal), Joint Work, Accepted,  ISSN  2405-5808

　View Summary

© 2020 The Authors Presbyopia is one of the most well-known diseases of the eye, predominantly affecting the adult population after 50 years’. Due to hardening of the lens and failure of accommodative change, patients lose the ability to focus on near objects. This eye symptom is reported to be an early symptom of age-related nuclear cataract, and we have previously reported that hesperetin treatment could delay the onset of nuclear cataractogenesis induced by sodium selenite. In this study, we examined whether oral intake of α-glucosyl-hesperidin (G-Hsd), which has greater water solubility than hesperetin, could delay the onset of presbyopia. G-Hsd treatment protected lens elasticity, upregulated the mRNA expression of anti-oxidative enzymes like glutathione reductase and thioredoxin reductase 1 in the plasma and lens, and prevented premature cataract symptoms in selenite-induced cataract rat lens. Thus, the anti-presbyopic effects of G-Hsd were attributed, at least in part, to its antioxidant effects. G-Hsd represents the first oral treatment agent with anti-presbyopia and/or anti-cataract properties.

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Effect of a Lens Protein in Low-Temperature Culture of Novel Immortalized Human Lens Epithelial Cells (iHLEC-NY2)

Yamamoto, N., Takeda, S., Hatsusaka, N., Hiramatsu, N., Nagai, N., Deguchi, S., Nakazawa, Y., Takata, T., Kodera, S., Hirata, A., Kubo, E. and Sasaki, H.

Cells 9 ( 12 )  2020.12

Research paper (scientific journal), Accepted,  ISSN  2073-4409

　View Summary

The prevalence of nuclear cataracts was observed to be significantly higher among residents of tropical and subtropical regions compared to those of temperate and subarctic regions. We hypothesized that elevated environmental temperatures may pose a risk of nuclear cataract development. The results of our in silico simulation revealed that in temperate and tropical regions, the human lens temperature ranges from 35.0 °C to 37.5 °C depending on the environmental temperature. The medium temperature changes during the replacement regularly in the cell culture experiment were carefully monitored using a sensor connected to a thermometer and showed a decrease of 1.9 °C, 3.0 °C, 1.7 °C, and 0.1 °C, after 5 min when setting the temperature of the heat plate device at 35.0 °C, 37.5 °C, 40.0 °C, and 42.5 °C, respectively. In the newly created immortalized human lens epithelial cell line clone NY2 (iHLEC-NY2), the amounts of RNA synthesis of αA crystallin, protein expression, and amyloid β (Aβ)1-40 secreted into the medium were increased at the culture temperature of 37.5 °C compared to 35.0 °C. In short-term culture experiments, the secretion of Aβ1-40 observed in cataracts was increased at 37.5 °C compared to 35.0 °C, suggesting that the long-term exposure to a high-temperature environment may increase the risk of cataracts.

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In situ gelling systems using pluronic f127 enhance corneal permeability of indomethacin nanocrystals

Nagai N., Isaka T., Deguchi S., Minami M., Yamaguchi M., Otake H., Okamoto N., Nakazawa Y.

International Journal of Molecular Sciences (International Journal of Molecular Sciences)  21 ( 19 ) 1 - 14 2020.10

Research paper (scientific journal), Accepted,  ISSN  16616596

　View Summary

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. 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.

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Study of the Mechanisms of Maintaining the Transparency of the Lens and Treatment of Its Related Diseases for Making Anti-cataract and/or Anti-presbyopia Drugs

Yosuke Nakazawa

Yakugaku Zasshi (Yakugaku Zasshi)  140 ( 9 ) 1095 - 1099 2020.09

Research paper (scientific journal), Single Work, Accepted,  ISSN  00316903

　View Summary

© 2020 The Pharmaceutical Society of Japan The lens of the eye is an avascular and anuclear tissue that serves to focus objects on the retina. Cataract is opacity within the clear lens that changes the transparency and refractive index of the lens causing significant visual impairments. These impairments can severely restrict the ability to carry out daily activities. Cataracts is common among elderly person occurring in more than 80％ of patients aged 80 or older. Notably, we have recently identified key compounds that are eŠective against cataract formation. Presbyopia is also an ocular disease that typically develops in people over the age of 45 while aŠecting almost 100％ of people over the age of 65. Recent research suggests that age-related changes in hydrostatic pressure of the lens controlled by Na/K ATPase contribute to the development of presbyopia. In the lens, Na/K ATPase has been shown to be regulated by transient receptor potential cation channels, vanilloid 1 (TRPV1) and 4, thus suggesting the potential role of TRPV1 and TRPV4 in the development of presbyopia. This review article summarizes data obtained from our laboratory with my colleagues highlighting the critical role of aquaporin 0 (AQP0) in maintaining a healthy lens redox environment, key molecules that delay the onset of cataract in vivo, as well as potential mechanisms of lens hydrostatic pressure control that may be associated with presbyopia.

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Development of sustained-release ophthalmic formulation based on tranilast solid nanoparticles

Minami M., Seiriki R., Otake H., Nakazawa Y., Kanai K., Tanino T., Nagai N.

Materials (Materials)  13 ( 7 )  2020.04

Research paper (scientific journal), Accepted

　View Summary

© 2020 by the authors. 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.

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Aquaporin 0A is Required for Water Homeostasis in the Zebrafish Lens In Vivo

Irene Vorontsova, Alexander Vallmitjana, Yosuke Nakazawa, Belén Torrado, Thomas Schilling, James E. Hall, Enrico Gratton, Leonel S.Malacrida

Biophysical Journal (Cell Press)  118 ( 3 ) 167a 2020.02

Research paper (scientific journal), Accepted,  ISSN  0006-3495

Novel sustained‐release drug delivery system for dry eye therapy by rebamipide nanoparticles

Nagai N., Ishii M., Seiriki R., Ogata F., Otake H., Nakazawa Y., Okamoto N., Kanai K., Kawasaki N.

Pharmaceutics (Pharmaceutics)  12 ( 2 )  2020.02

Research paper (scientific journal), Accepted

　View Summary

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. 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.

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Oral intake of α-glucosyl-hesperidin ameliorates selenite-induced cataract formation

Nakazawa Y., Aoki M., Ishiwa S., Morishita N., Endo S., Nagai N., Yamamoto N., Funakoshi-Tago M., Tamura H.

Molecular Medicine Reports (Molecular Medicine Reports)  21 ( 3 ) 1258 - 1266 2020

Research paper (scientific journal), Accepted,  ISSN  17912997

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© 2020 Spandidos Publications. All rights reserved. Hesperetin is a natural flavonoid with robust antioxidant properties. Our previous study reported that hesperetin can prevent cataract formation. However, an important consideration regarding hesperetin consumption is the limited bioavailability due to its poor solubility. The present study investigated the anti-cataract effects of α-glucosyl hesperidin in vivo and in vitro using a selenite-induced cataract model. SD rats (age, 13 days) were orally administered PBS (0.2 ml) or α-glucosyl hesperidin (200 mg/kg) on days 0, 1 and 2. Sodium selenite was subcutaneously administered to the rats 4 h after the first oral administration on day 0. Antioxidant levels in the lens and blood were measured on day 6. In vitro, human lens epithelial cells were treated with sodium selenite (10 μM) and/or hesperetin (50 or 100 mM) for 24 h and analyzed for apoptosis markers using sub-G1 population and Annexin V-FITC/propidium iodide staining and DNA ladder formation. α-glucosyl hesperidin treatment significantly reduced the severity of selenite-induced cataract. The level of antioxidants was significantly reduced in the selenite-treated rats compared with in the controls; however, they were normalized with α-glucosyl hesperidin treatment. In vitro, hesperetin could significantly reduce the number of cells undergoing apoptosis induced by sodium selenite in human lens epithelial cell lines. Overall, oral consumption of α-glucosyl hesperidin could delay the onset of selenite-induced cataract, at least in part by modulating the selenite-induced cell death in lens epithelial cells.

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Degradation of connexin 50 protein causes waterclefts in human lens

Nakazawa, Y., Shibata, T., Nagai, N., Kubo, E., Tamura, H. and Sasaki, H.

Open Med (Wars) 15 ( 1 ) 1163 - 1171 2020

Research paper (scientific journal), Accepted,  ISSN  2391-5463

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Cataracts are mainly classified into three types: cortical cataracts, nuclear cataracts, and posterior subcapsular cataracts. In addition, retrodots and waterclefts are cataract subtypes that cause decreased visual function. To maintain an orderly and tightly packed arrangement to minimize light scattering, adhesion molecules such as connexins and aquaporin 0 (AQP0) are highly expressed in the lens. We hypothesized that some main and/or subcataract type(s) are correlated with adhesion molecule degradation. Lens samples were collected from cataract patients during cataract surgery, and mRNA and protein expression levels were measured by real-time RT-PCR and western blotting, respectively. The mRNA levels of adhesion molecules were not significantly different among any cataract types. Moreover, AQP0 and connexin 46 protein expressions were unchanged among patients. However, connexin 50 protein level was significantly decreased in the lens of patients with WC cataract subtype. P62 and LC3B proteins were detected in the WC patients' lenses, but not in other patients' lenses. These results suggest that more research is needed on the subtypes of cataracts besides the three major types of cataract for tailor-made cataract therapy.

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Combination with l-Menthol Enhances Transdermal Penetration of Indomethacin Solid Nanoparticles

Nagai N, Ogata F, Yamaguchi M, Fukuoka Y, Otake H, Nakazawa Y, Kawasaki N.

International Journal of Molecular Sciences (International journal of molecular sciences)  20 ( 15 ) 3644 - 3644 2019.07

Research paper (scientific journal), Accepted,  ISSN  16616596

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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).

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Verification and spatial mapping of TRPV1 and TRPV4 expression in the embryonic and adult mouse lens

Yosuke Nakazawa, Paul Donaldson, Rosica Petrova

Experimental Eye Research (Elsevier)  186   107707 - 107707 2019.06

Research paper (scientific journal), Accepted,  ISSN  00144835

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© 2019 The transient receptor protein vanilloid channels, TRPV1 and TRPV4, have recently been shown to be mechanosensors in the ocular lens that act to transduce physical changes in lens volume and internal hydrostatic pressure into the activation of signalling pathways in lens epithelial cells. These pathways in turn regulate ion and water transport to ensure that the optical properties of the lens remain constant. Despite the functional evidence that implicate the roles of TRPV1 and TRPV4 in the lens, their respective cellular expression patterns in the different regions of the lens has to date not been fully characterised. Using Western blotting we have confirmed that TRPV1 and TRPV4 are expressed throughout all regions (epithelium, outer cortex, inner cortex/core) of the adult mouse lens. Subsequent immunolabeling of lens cryosections confirmed that TRPV1 and TRPV4 are expressed throughout all regions of the lens, but revealed differentiation-dependent differences in the subcellular expression of the two channels in the different regions. In the epithelium and outer cortex, intense TRPV1 and TRPV4 labeling was predominately associated with the cytoplasm. In a discrete zone in the inner cortex, labeling for both proteins was greatly diminished, but could be enhanced by incubating sections with the detergent Triton X-100 to reveal TRPV1 and TRPV4 labelling that was associated with the membrane. This suggests that in this region of the lens there is a potential interacting protein that masks the binding of the TRPV1 and TRPV4 antibodies to their respective epitopes in the lens inner cortex. In the core of the lens, which contains the embryonic nucleus, TRPV1 and TRPV4 labelling was associated exclusively with fibre cell membranes. This labelling in the lens core of the adult mouse lens appeared to originate in early development as a similar membrane labelling was observed at embryonic day 10 (E10) of the cells in the lens vesicle that subsequently forms the embryonic nucleus in the adult lens. During subsequent stages of embryonic development TRPV1 and TRPV4 remained membranous in the inner cortex and core, while showing labelling that was associated with the cytoplasm in the superficial outer cortical region. The extent of cytoplasmic labelling for TRPV4, but not TRPV1, in this cortical region could however be dynamically regulated by cutting the zonules that normally attach the lens to the ciliary body. We have shown an early onset and continuous expression of TRPV1 and TRPV4 across all lens regions, and that TRPV4 can be dynamically trafficked into the membranes of differentiating fibre cells, results that suggests that these mechanosensitive channels may also be functionally active in lens fibre cells.

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Stability of 2-Amino-3-hydroxyacetophenone-O-beta-D-glucoside Against Near-UV Irradiation

Mikako Oka, Makoto Takehana, Yosuke Nakazawa, Hiroko Ushikubo, Kenshi Satoh

Journal of the Japanese Society for Cataract Research 31 ( 1 ) 58 - 62 2019.06

Research paper (scientific journal), Joint Work, Accepted

Energy-dependent endocytosis is responsible for drug transcorneal penetration following the instillation of ophthalmic formulations containing indomethacin nanoparticles

Noriaki Nagai, Fumiko Ogata Hiroko Otake, Yosuke Nakazawa, Naohiro Kawasaki

International Journal of nanomedicine (International Journal of Nanomedicine)  14   1213 - 1227 2019.02

Research paper (scientific journal),  ISSN  11769114

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© 2019 Nagai et al. Purpose: We previously found that ophthalmic formulations containing nanoparticles prepared by a bead mill method lead to an increase in bioavailability in comparison with traditional formulations (solution type). However, the transcorneal penetration pathway for ophthalmic formulations has not been explained yet. In this study, we investigated the mechanism of transcorneal penetration in the application of ophthalmic formulations containing indomethacin nanoparticles (IMC-NPs). Materials and methods: IMC-NPs was prepared by the bead mill method. For the analysis of energy-dependent endocytosis, corneal epithelial (HCE-T) cell monolayers and removed rabbit cornea were thermoregulated at 4°C, where energy-dependent endocytosis is inhibited. In addition, for the analysis of different endocytosis pathways using pharmacological inhibitors, inhibitors of caveolae-mediated endocytosis (54 µM nystatin), clathrin-mediated endocytosis (40 µM dynasore), macropinocytosis (2 µM rottlerin) or phagocytosis (10 µM cytochalasin D) were used. Results: The ophthalmic formulations containing 35–200 nm sized indomethacin nanoparticles were prepared by treatment with a bead mill, and no aggregation or degradation of indomethacin was observed in IMC-NPs. The transcorneal penetration of indomethacin was significantly decreased by the combination of nystatin, dynasore and rottlerin, and the decreased penetration levels were similar to those at 4°C in HCE-T cell monolayers and rabbit cornea. In the in vivo experiments using rabbits, dynasore and rottlerin tended to decrease the transcorneal penetration of indomethacin (area under the drug concentration – time curve in the aqueous humor [AUC AH ]), and the AUC AH in the nystatin-treated rabbit was significantly lower than that in non-treatment group. In addition, the AUC AH in rabbit corneas undergoing multi-treatment was obviously lower than that in rabbit corneas treated with each individual endocytosis inhibitor. Conclusion: We found that three energy-dependent endocytosis pathways (clathrin-dependent endocytosis, caveolae-dependent endocytosis and macropinocytosis) are related to the transcorneal penetration of indomethacin nanoparticles. In particular, the caveolae-dependent endocytosis is strongly involved.

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Coffee brew intake can prevent the reduction of lens glutathione and ascorbic acid levels in HFD‑fed animals

Yosuke Nakazawa, Nana Ishimori, Jun Oguchi, Noriaki Nagai, Masaki Kimura, Megumi Funakoshi‑Tago and Hiroomi Tamura

Experimental And Therapeutic Medicine (Experimental and Therapeutic Medicine)  17 ( 2 ) 1420 - 1425 2019.01

Research paper (scientific journal), Joint Work, Lead author, Corresponding author, Accepted,  ISSN  1792-0981

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© 2018, Spandidos Publications. All rights reserved. The lens has high concentrations of glutathione (GSH) and ascorbic acid (AsA) to maintain redox activity and prevent cataract formation, which is the leading cause of visual impairment worldwide. Metabolic syndrome is reported to be linked with a higher risk of age-associated cataract. As it was demonstrated previously that coffee consumption improved high-fat diet (HFD) -induced metabolic symptoms, it was hypothesized that coffee intake could delay the onset of obesity related-cataract; however, the effect of coffee consumption on this type of cataract remains unknown. Four-week-old male C57BL/6JJms SLC mice were divided into two groups and were provided ad libitum access to either a control diet (control groups) or a HFD (HFD groups). The control groups and HFD groups were further divided into three or four subgroups for each experiment. Coffee intake markedly reduced the increase in body weight in a roasting-time and concentration-dependent manner. Coffee consumption also prevented the HFD-induced decrease in the concentration of GSH and AsA, and treatment with pyrocatechol or caffeine also restored the reduction of antioxidant compounds. Plasma cholesterol and triglycerides were significantly higher in HFD groups; however, coffee brew or coffee constituent treatment in the HFD-fed mice group prevented elevation of these levels. Caffeine is a major coffee component and pyrocatechol is generated thought the roasting process. These results revealed that caffeine and pyrocatechol in coffee brew may be the key constituents responsible for preventing the reduction of lens GSH and AsA in HFD-fed animals.

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Design of a transdermal formulation containing raloxifene nanoparticles for osteoporosis treatment

Noriaki Nagai, Fumihiko Ogata, Hiroko Otake, Yosuke Nakazawa, Naohito Kawasaki

International Journal of Nanomedicine (Dovepress)  13   5215 - 5229 2018.09

Research paper (scientific journal), Accepted,  ISSN  11769114

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© 2018 Nagai et al. Purpose: In the clinical setting, raloxifene, a second-generation selective estrogen receptor modulator, is administered orally; however, the bioavailability (BA) is only 2% because of its poor solubility in aqueous fluids and its extensive first-pass metabolism. Therefore, it is expected that the development of a transdermally delivered formulation may reduce the necessary dose without compromising its therapeutic efficacy. In this study, we designed transdermal formulations containing raloxifene nanoparticles and evaluated their usefulness for osteoporosis therapy. Methods: Raloxifene was crushed with methylcellulose by the bead mill method, and the milled raloxifene was gelled with or without menthol (a permeation enhancer) by Carbopol® 934 (without menthol, Ral-NPs; with menthol, mRal-NPs). The drug release and transdermal penetration were measured using a Franz diffusion cell, and the therapeutic evaluation of osteoporosis was determined in an ovariectomized rat model. Results: The mean particle size of raloxifene in the transdermal formulation (Ral-NPs) was 173.7 nm. Although the raloxifene released from Ral-NPs remained in the nanoparticle state, the skin penetration of raloxifene nanoparticles was prevented by the stratum corneum in rat. On the other hand, inclusion of menthol in the formulation attenuated the barrier function of the stratum corneum and permitted the penetration of raloxifene nanoparticles through the skin. Moreover, macropinocytosis relates to the skin penetration of the formulation including menthol (mRal-NPs), since penetration was inhibited by treatment with 2 µM rottlerin, a macropinocytosis inhibitor. In addition, the application of 0.3% mRal-NPs (once a day) attenuated the decreases in calcium level and stiffness of the bones of ovariectomized rat. Conclusion: We prepared raloxifene solid nanoparticles by a bead mill method and designed a novel transdermal formulation containing nanoparticles and permeation enhancers. These transdermal formulations overcome the barrier properties of the skin and show high drug penetration through the transdermal route (BA 8.5%). In addition, we found that raloxifene transdermal formulations are useful for the treatment of osteoporosis in ovariectomized rat.

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Roasted Coffee Reduces β-Amyloid Production byIncreasing Proteasomal β-Secretase Degradation in HumanNeuroblastoma SH-SY5Y Cells

Kazuya Fukuyama, Shota Kakio, Yosuke Nakazawa, Kenji Kobata,Megumi Funakoshi-Tago, Toshiharu Suzuki, and Hiroomi Tamura

Molecular nutrition and food research (Molecular Nutrition and Food Research)  62 ( 18 ) 1800238 2018.08

Research paper (scientific journal), Joint Work, Accepted,  ISSN  16134125

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© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Scope: Epidemiological studies have shown that coffee consumption may be associated with a lower risk of developing several neurological disorders, including Alzheimer's disease (AD). Caffeine is a prominent candidate component underlying the preventive effects of coffee; however, the contribution of other constituents is unclear. To clarify this issue, the effect of roasting coffee beans on β-secretase (BACE1) expression in human neuroblastoma SH-SY5Y cells is investigated. Methods and results: Coffee (2%) reduces Aβ accumulation in culture medium to 80% of control levels after 24 h. Accordingly, BACE1 expression is decreased to 70% of control levels at 12 h. Experiments using cycloheximide and MG132, a proteasome inhibitor, reveal that coffee enhanced BACE1 degradation through activation of proteasomal activity. Furthermore, coffee activates cAMP-dependent protein kinase, and consequently, phosphorylation of a serine residue of proteasome 26S subunit, non-ATPase 11 (PSMD11). Pyrocatechol, a strong antioxidant known as catechol or 1,2-dihydroxybenzene, produced from chlorogenic acid during roasting, also reduces BACE1 expression by activation of proteasomal activity. Furthermore, pyrocatechol reduces Aβ production in SH-SY5Y cells. Conclusion: The data suggest that the roasting process may be crucial for the protective effects of coffee consumption in AD.

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Involvement of Endocytosis in the Transdermal Penetration Mechanism of Ketoprofen Nanoparticles

Noriaki Nagai , Fumihiko Ogata, Miyu Ishii, Yuya Fukuoka, Hiroko Otake, Yosuke Nakazawa and Naohito Kawasaki

International Journal of Molecular Sciences (International Journal of Molecular Sciences)  19 ( 7 ) 2138 2018.07

Research paper (scientific journal), Joint Work, Accepted,  ISSN  16616596

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© 2018 by the authors. Licensee MDPI, Basel, Switzerland. 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.

Therapeutic Effect of Ophthalmic Formulation containing Nilvadipine Nanoparticles on Retinal Dysfunction in Rats Injected with Streptozotocin

Nagai Noriaki, Deguchi Saori, Ishii Miyu, Fukuoka Yuya, Otake Hiroko, Nakazawa Yosuke

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE 59 ( 9 )  2018.07

Research paper (scientific journal), Accepted,  ISSN  0146-0404

Effect of hesperetin derivatives on the development of selenite‑induced cataracts in rats

Yosuke Nakazawa, Martin Pauze, Kazuya Fukuyama, Noriaki Nagai, Megumi Funakoshi-Tago, Takeshi Sugai, Hiroomi Tamura.

Molecular Medicine Reports (Molecular Medicine Reports)  18 ( 6 ) 1043 - 1050 2018.05

Research paper (scientific journal), Joint Work, Lead author, Corresponding author, Accepted,  ISSN  17912997

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Copyright © Spandidos Publications 2017. All rights reserved. Cataracts are a major cause of blindness worldwide. As anti-cataract pharmaceutical therapies require long-term treatment, identifying anti-cataract compounds that are ubiquitous in the human diet, have no adverse effects and are affordable, is of paramount importance. The present study focused on hesperetin and its derived compounds, hesperetin stearic acid ester (Hes-S) and hesperetin oleic acid ester (Hes-O), in order to investigate their therapeutic potential to treat cataracts in a selenite animal model. Thirteen-day-old Sprague Dawley rats were divided into 12 groups. Animals in groups 1 and 7 were subcutaneously injected with vehicle, those in groups 2 and 8 were administered hesperetin, those in groups 3 and 9 received stearic acid, those in groups 4 and 10 were injected with oleic acid, those in groups 5 and 11 were administered Hes-S, and those in groups 6 and 12 received Hes-O (10 nmol/kg body weight on days 0, 1 and 2). Animals in groups 7 to 12 were treated with sodium selenite (20 µmol/kg body weight given 4 h following the test compound treatment on day 0) to induce cataract. On day 6, rats had less severe central opacities and lower stage cataracts than rats in the selenite treatment-only control groups. The levels of glutathione (GSH) and ascorbic acid (AsA) in lenses with selenite-induced cataracts declined to one-third of that of controls, and the reduction in GSH and AsA levels was rescued following hesperetin, Hes-S or Hes-O treatment, with concentrations remaining to 70-80% of that of controls. However, there were no changes in the plasma levels of GSH and AsA following treatments. Administration of either hesperetin or hesperetin-derived compounds prevented the reduction of chaperone activity in the lens, and rats treated with Hes-S or Hes-O treatment had significantly greater chaperone activity than hesperetin-treated rats. Collectively, these results suggested that hesperetin and hesperetin-derived compounds may be novel drug compounds that have the potential to prevent or delay the onset of cataracts.

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水溶性薬物の角膜透過性向上を目指して：チモロールマレイン酸・マグネシウムヒドロキシドナノ粒子配合剤の開発

NAKAZAWA Yosuke

日本眼科学会雑誌 122 ( 1 ) 61 - 62 2018.01

Research paper (conference, symposium, etc.), Joint Work

Ophthalmic Formulation Containing Nilvadipine Nanoparticles Prevents Retinal Dysfunction in Rats Injected with Streptozotocin

Saori Deguchi, Hiroko Otake, Yosuke Nakazawa, Noriko Hiramatsu, Naoki Yamamoto, Noriaki Nagai

International Journal of Molecular Scinences 18   2720 - 2730 2017.12

Research paper (scientific journal), Joint Work, Accepted

Co-instillation of nano-solid magnesium hydroxide enhances corneal permeability of dissolved timolol.

Noriaki Nagai, Fumihiko Ogata, Hiroko Otake, Naohito Kawasaki, Yosuke Nakazawa, Kazutaka Kanai, Norio Okamoto, Yoshikazu Shimomura

Experimental Eye Research 165   118 - 124 2017.10

Research paper (scientific journal), Joint Work, Accepted

Administration of antioxidant compounds affects the lens chaperone activity and prevents the onset of cataracts.

Nakazawa Yosuke, Nagai Noriaki, Ishimori Nana, Oguchi Jun, Tamura Hiroomi

Biomedicine and pharmacotherapy 95   137 - 143 2017.09

Research paper (scientific journal), Accepted

Alpha-tocopherol attenuates the anti-tumor activity of crizotinib against cells transformed by NPM-ALK.

Yuki Uchihara, Fumihito Ueda, Kenji Tago, Yosuke Nakazawa, Tomoyuki Ohe, Tadahiko Mashino, Shigenobu Yokota, Tadashi Kasahara, Hiroomi Tamura, Megumi Funakoshi-Tago

PLoS One 12 ( 8 ) e0183003 2017.08

Research paper (scientific journal), Joint Work, Accepted

Model for Studying Anti- allergic Drugs for Allergic Conjunctivitis animals

Yosuke Nakazawa, Mikako Oka, Makoto Takehana

Open Medicine 12 ( 1 ) 231 - 238 2017.08

Research paper (scientific journal), Joint Work, Accepted

A Nanoparticle-based Ophthalmic Formulation of Dexamethasone Enhances Corneal Permeability of the Drug and Prolongs its Corneal Residence Time

Noriaki NAGAI, Yosuke NAKAZAWA, Yoshimasa ITO, Kazutaka KANAI, Norio OKAMOTO, Yoshikazu SHIMOMURA

Biological and Pharmaceutical Bulletin 40 ( 7 ) 1055 - 1062 2017.07

Research paper (scientific journal), Joint Work, Accepted

Roasting Enhances the Anti-Cataract Effect of Coffee Beans: Ameliorating Selenite-Induced Cataracts in Rats.

Ishimori Nana, Oguchi Jun, Nakazawa Yosuke, Kobata Kenji, Funakoshi-Tago Megumi, Tamura Hiroomi

Current Eye research 42 ( 6 ) 864 - 870 2017.06

Research paper (scientific journal), Joint Work, Accepted

細胞間接着分子による白内障発症機序

NAKAZAWA Yosuke

日本白内障学会誌 29 ( 1 ) 34 - 36 2017.06

Research paper (scientific journal), Single Work, Accepted

improvement of Dissolution rate and stability in a pirenoxine ophthalmic suspension by the beads mill methods

上野 祥奈, 山岡 咲絵, 伊藤 吉將, 小竹　武, 中澤 洋介, 長井 紀章

Yakugaku-Zasshi (日本薬学会)  137 ( 5 ) 635 - 641 2017.05

Research paper (scientific journal), Accepted

The extracellular C-loop domain plays an important role in the cell adhesion function of aquaporin 0

Yosuke Nakazawa, Mikako Oka, Megumi Funakoshi-Tago, Hiroomi Tamura, Makoto Takehana.

Current Eye Research (Taylor and Francis)  42 ( 4 ) 617 - 624 2017.04

Research paper (scientific journal), Joint Work, Accepted

Effect of hesperetin on chaperone activity in selenite-induced cataract

Yosuke Nakazawa, Mikako Oka, Hiroomi Tamura, Makoto Takehana

Open Medicine (De Gruyter)  11   183 - 189 2016.06

Research paper (scientific journal), Accepted

Evaluation of the Inhibitory Effects of Rokumigan and Hachimijiogan on Cataract Formation in a Rat Model ofStreptozotocin-induced Type 1 Diabetes -Effect of Hachimijiogan on Diabetic Cataracts-

Mikako Oka, Yosuke Nakazawa, Noriyasu Hada, Fumiyuki Kiuchi, Yukari Matsushima, Koji Chiba, Makoto Takehana.

Journal of the Japanese Society for Cataract Research 28 ( 1 ) 97 - 105 2016.06

Research paper (scientific journal), Joint Work, Accepted

アクアポリン0（AQP0）の細胞間接着に関与するアミノ酸の特定

NAKAZAWA Yosuke

日本眼科学会誌 (日本眼科学会)  120 ( 1 ) 50 - 50 2016.01

Accepted

Coffee Modulates the Function of Brain-Derived Neurotrophic Factor (BDNF) in Human Neuroblastoma SH-SY5Y Cells

Kakio Shota, Nakazawa Yosuke, Funakoshi-Tago Megumi, Tamura Hiroomi.

Neuroscience & Medicine 6 ( 4 ) 165 - 174 2015.12

Research paper (scientific journal), Accepted

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Establishment of a recessive mutant small-eye rat with lens involution and retinal detachment associated with partial deletion and rearrangement of the Cryba1 gene

Toshiyuki Yamada, Naoki Nanashima, Takeshi Shimizu, Yosuke Nakazawa, Mitsuru Nakazawa and Shigeki Tsuchida

Biochemical Journal (Portland Press)  471   293 - 305 2015.10

Research paper (scientific journal), Accepted

Hesperetin prevents selenite-induced cataract in rats

NAKAZAWA Yosuke, Mikako Oka, Masayasu Bando, Makoto Takehana.

Molecular Vision 21   804 - 810 2015.07

Research paper (scientific journal), Joint Work, Accepted

Lifespan of mRNA in the Lens.

Mikako Oka, Kazuhiro Umezawa, Yoko Nakajima, Yosuke Nakazawa, Makoto Takehana.

Journal of the Japanese Society for Cataract Research 27   50 - 62 2015.06

Research paper (scientific journal), Joint Work, Accepted

Study of Rules and Functions with Aquaporin 0

NAKAZAWA Yosuke

Journal of the Japanese Society for Cataract Research 27   9 - 13 2015.06

Research paper (scientific journal), Accepted

The effect of the interaction between aquaporin 0 (AQP0) and the filensin tail region on AQP0 water permeability.

Nakazawa Y, Oka M, Furuki K, Mitsuishi A, Nakashima E, Takehana M*.

Molecular Vision 17   3191-3199 2011.12

Research paper (scientific journal), Joint Work, Accepted

Quantitative analysis of ascorbic acid permeability of aquaporin 0 in the lens.

Nakazawa Y, Oka M, Mitsuishi A, Bando M, Takehana M*.

Biochemical and Biophysical Research Communications 415   125-130 2011.11

Research paper (scientific journal), Joint Work, Accepted

The role of ascorbic acid transporter in the lens of streptozotocin-induced diabetic rat.

Nakazawa Y, Oka M, Bando M, Inoue T, Takehana M.

Biomedicine and pahrmacotherapy 1   43 - 48 2011.05

Research paper (scientific journal), Joint Work, Accepted

Suppression of Redox Activities of Rabbit Lens Lambda-Crystallin by Glutathionylation.

Bando M, Oka M, Nakazawa Y, kawai K, Obazawa H, Takehana M*.

The Journal of the Japanese Society for Cataract Research 22   43-49 2010.06

Research paper (scientific journal), Joint Work, Accepted

Characterization and localization of side population cells inthe lens.

Oka M, Toyoda C, Kaneko Y, Nakazawa Y, Aizu-Yokota E, Takehana M*.

Molecular Vision 16   945-953 2010.06

Research paper (scientific journal), Joint Work, Accepted

The Aggregation Mechanism of Lens Crystallins.

Nakazawa Y, Takehana M*, Sano Y.

KURRI KR Report 148   16-20 2009.06

Research paper (scientific journal), Joint Work, Accepted

UV-B irradiation-induced electron transfer between 3-hydroxykynurenine and tryptophan.

Nakazawa Y, Takehana M*, Oka M, Shibuya F, Katakawa J, Sano Y.

Journal of Biological Macromolecules 26   13-22 2009.04

Research paper (scientific journal), Joint Work, Accepted

Spectroscopic Structure change of Pig alpha-crystallin coupled with 3-Hydroxykynurenine by UV-irradiation Relative to Cataract

Nakazawa Yosuke, Takehana Makoto, Katakawa Junichi, Sano Yoh.

European Spectroscopy of Biological Molecules 12 ( 1 ) 217 - 218 2007.09

Research paper (scientific journal), Joint Work, Accepted

Cooperative interaction between ETS1 and GFI1 transcription factors in the repression of Bax gene expression.

Nakazawa Y, Suzuki M, Manabe N, Yamada T, Kihara-Negishi F, Sakurai T, Tenen DG, Iwama A, Mochizuki M, Oikawa T.

Oncogene (nature publising group)  26 ( 24 ) 3541 - 3550 2007.05

Research paper (scientific journal), Joint Work, Accepted

Aggregates structural study of novel and potent immunosuppressant FTY720 in solution.

NAKAZAWA Yosuke

Photon Factory Activity Report.  2006.06

Research paper (scientific journal), Joint Work, Accepted