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

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-ß-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.

Recovery from indomethacin-induced gastrointestinal bleeding by treatment with teprenone.

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.

Nasal Absorption Enhancement of Mometasone Furoate Nanocrystal Dispersions.

Purpose: We designed a 0.05% mometasone furoate (MF) nanocrystal dispersion and investigated whether the application of MF nanocrystals in nasal formulations enhanced local absorption compared to traditional nasal MF formulations (CA-MF). Methods: MF nanocrystal dispersions (MF-NPs) were prepared by bead milling MF microcrystal dispersions (MF-MPs) consisting of MF, 2-hydroxypropyl-ß-cyclodextrin, methylcellulose, and purified water. Pluronic F-127 combined with methylcellulose, Pluronic F-68, or carbopol was used as a base for in situ gelation (thickener). MF concentrations were measured using high-performance liquid chromatography, and nasal absorption of MF was evaluated in 6 week-old male Institute of Cancer Research (ICR) mice. Results: The particle size range of MF prepared with the bead mill treatment was 80-200 nm, and the nanoparticles increased the local absorption of MF, which was higher than that of CA-MF and MF-MPs. In addition, unlike the results obtained in the small intestine and corneal tissue, the high absorption of nanocrystalline MF in the nasal mucosa was related to a pathway that was not derived from energy-dependent endocytosis. Moreover, the application of the in situ gelling system attenuated the local absorption of MF-NPs, owing to a decrease in drug diffusion in the dispersions. Conclusion: We found that nanoparticulation of MF enhances local intranasal absorption, and nasal bioavailability is higher than that of CA-MF. In addition, we demonstrate that viscosity regulation is an important factor in the design of nasal formulations based on MF nanocrystals. These findings provide insights for the design of novel nanomedicines with enhanced nasal bioavailability.

Skin absorption of felbinac solid nanoparticles in gel formulation containing l-menthol and carboxypolymethylene.

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

Effect of Ointment Base on the Skin Wound-Healing Deficits in Streptozotocin-Induced Diabetic Rat.

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

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

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.

Function of mitochondrial cytochrome c oxidase is enhanced in human lens epithelial cells at high temperatures.

Enhancement of density via human lens epithelium (HLE) cell proliferation is the underlying cause of nuclear cataracts. Moreover, our previous epidemiological study demonstrated that the risk of nuclear cataract development is significantly higher under elevated environmental temperatures compared with under lower temperatures. The present study investigated the relationship between temperature and cell proliferation in terms of mitochondrial function, which is a nuclear cataract­inducing risk factor, using two different HLE cell lines, SRA01/04 and immortalized human lens epithelial cells NY2 (iHLEC­NY2). Cell proliferation was significantly enhanced under the high­temperature condition (37.5˚C) in both cell lines. The cell growth levels of SRA01/04 and iHLEC­NY2 cells cultured at 37.5˚C were 1.20­ and 1.16­fold those in the low­temperature cultures (35.0˚C), respectively. Moreover, the levels of cytochrome c oxidase mRNA (mitochondrial genome, cytochrome c oxidase­1­3) and its activity in SRA01/04 and iHLEC­NY2 cells cultured at 37.5˚C were higher compared with those in cells cultured at 35.0˚C. In addition, adenosine­5'­triphosphate (ATP) levels in SRA01/04 and iHLEC­NY2 cells were also significantly higher at 37.5˚C compared with those at 35.0˚C. By contrast, no significant differences in Na+/K+­ATPase or Ca2+­ATPase activities were observed between HLE cells cultured at 35.0 and 37.5˚C. These results suggested that expression of the mitochondrial genome was enhanced in high­temperature culture, resulting in a sufficient ATP content and cell proliferation for lens opacity. Therefore, elevated environmental temperatures may increase the risk of nuclear cataracts caused by HLE cell proliferation via mitochondrial activation.

Effect of storage temperature on the dispersibility of commercially available 0.1% fluorometholone ophthalmic suspension.

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

Design of a Transdermal Sustained Release Formulation Based on Water-Soluble Ointment Incorporating Tulobuterol Nanoparticles.

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

[Effects of Skin Environmental Changes by Steam Towel, Ethanol, l-Menthol and Carpronium on the Drug Behavior in the Minoxidil Nanoparticles-applied Mice].

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

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

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.

Nanocrystalline Suspensions of Irbesartan Enhance Oral Bioavailability by Improving Drug Solubility and Leading Endocytosis Uptake into the Intestine.

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

Energy-Dependent Endocytosis Is Responsible for Skin Penetration of Formulations Based on a Combination of Indomethacin Nanoparticles and l-Menthol in Rat and Göttingen Minipig.

We previously designed a Carbopol gel formulation (N-IND/MEN) based on a combination of indomethacin solid nanoparticles (IND-NPs) and l-menthol, and we reported that the N-IND/MEN showed high transdermal penetration. However, the detailed mechanism for transdermal penetration of IND-NPs was not clearly defined. In this study, we investigated whether endocytosis in the skin tissue of rat and Göttingen minipig is related to the transdermal penetration of IND-NPs using pharmacological inhibitors of endocytosis. The pharmacological inhibitors used in this study are as follows: 54 µM nystatin, a caveolae-mediated endocytosis (CavME) inhibitor; 40 µM dynasore, a clathrin-mediated endocytosis (CME) inhibitor; and 2 µM rottlerin, a micropinocytosis (MP) inhibitor. The N-IND/MEN was prepared by a bead mill method, and the particle size of solid indomethacin was 79-216 nm. In both rat and Göttingen minipig skin, skin penetration of approximately 80% IND-NPs was limited by the stratum corneum (SC), although the penetration of SC was improved by the combination of l-menthol. On the other hand, the treatment of nystatin and dynasore decreased the transdermal penetration of indomethacin in rats and Göttingen minipigs treated with N-IND/MEN. Moreover, in addition to nystatin and dynasore, rottlerin attenuated the transdermal penetration of IND-NPs in the Göttingen minipigs' skin. In conclusion, we found that l-menthol enhanced the SC penetration of IND-NPs. In addition, this study suggests that the SC-passed IND-NPs are absorbed into the skin tissue by energy-dependent endocytosis (CavME, CME, and/or MP pathways) on the epidermis under the SC, resulting in an enhancement in transdermal penetration of IND-NPs. These findings provide significant information for the design of nanomedicines in transdermal formulations.

[Effect of Shelf Life on the Physical Stability of Suspended Ophthalmic Solutions].

Quality changes associated with physical changes in suspended eye drops are difficult to predict. In this study, we attempted to evaluate the aggregation and redispersability in commercially available suspended eye drops (fluorometholone ophthalmic solutions). The 0.1% fluorometholone ophthalmic solutions (the original product and 4 generic products) were gently mixed by hand after short-term (4 months) or long-term (40 months) storage, and the drug concentration in the first drop and physical stability (redispersability and particle size) were measured. All eye drops produced a cloudy precipitate on the bottom surface of the container, and the amount of precipitate decreased with mixing time. The drug concentration per drop in the original product was approximately 70% of the labeled value after mixing 10 times, and the drug particle size was approximately 4 µm. After mixing the generic products stored short-term 10 times, the concentration ranged from less than 50% to almost 100%. In addition, some generic products after long-term storage had a reduced redispersion ability and labeled concentration. These results suggested that at least 10 mixing were required before the using of fluorometholone original product. In addition, some generic products may not provide sufficient drug exposure even when mixed in the same manner as the original products.

Prevention of Postprandial Hyperglycemia by Ophthalmic Nanoparticles Based on Protamine Zinc Insulin in the Rabbit.

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

Transdermal System Based on Solid Cilostazol Nanoparticles Attenuates Ischemia/Reperfusion-Induced Brain Injury in Mice.

Cilostazol (CIL) exerted a protective effect by promoting blood-brain barrier integrity as well as improving the status of neurological dysfunctions following cerebral ischemia/reperfusion (I/R) injury. We attempted to design a 0.5% CIL carbopol gel using solid nanoparticles (CIL-Ngel), and then investigated the relationships between energy-dependent endocytosis and the skin penetration of CIL-Ngel in this study. In addition, we evaluated whether the CIL-Ngel attenuated I/R-induced brain injury in a middle cerebral artery occlusion (MCAO)/reperfusion model mouse. The particle size of CIL was decreased using a bead mill, and the CIL particles (14.9 × 1014 particles/0.3 g) in the CIL-Ngel were approximately 50-180 nm. The release of CIL in the CIL-Ngel was higher than that in gel containing CIL powder (CIL-Mgel), and the CIL particles were released from the CIL-Ngel as nanoparticles. In addition, the percutaneous absorption of CIL from the CIL-Ngel was higher in comparison with that from CIL-Mgel, and clathrin-dependent endocytosis and caveolae-dependent endocytosis were related to the enhanced skin penetration of CIL-NPs. In the traditional (oral administration of CIL powder, 3 mg/kg) and transdermal administration (CIL-Ngel, 0.3 g) for 3 days (once a day), the area under the plasma CIL concentration-time curves (AUC) was similar, although the CIL supplied to the blood by the CIL-Ngel was more sustained than that via oral administration of CIL powder. Furthermore, the CIL-Ngel attenuated the ischemic stroke. In conclusion, we designed a gel using solid CIL-NPs, and we showed that the sustained release of CIL by CIL-Ngel provided an effective treatment for ischemic stroke in MCAO/reperfusion model mice. These findings induce the possibilities of developing novel applications of CIL solid nanoparticles.

MPC Polymer Promotes Recovery from Dry Eye via Stabilization of the Ocular Surface.

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

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

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 ß (Aß)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 Aß1-40 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.

In Situ Gelling Systems Using Pluronic F127 Enhance Corneal Permeability of Indomethacin Nanocrystals.

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.

In Situ Gel Incorporating Disulfiram Nanoparticles Rescues the Retinal Dysfunction via ATP Collapse in Otsuka Long-Evans Tokushima Fatty Rats.

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