[Development of Transdermal Formulation Based on Nanotechnology and Elucidation of Its Drug Delivery Pathways].

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

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.

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

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.

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.

Poly(oxyethylene)/Poly(oxypropylene) butyl ether prolongs the repellent effect of N,N-diethyl-3-toluamide on the skin.

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

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.

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.

Novel drug delivery systems for the management of dry eye.

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

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.

Instillation of Ophthalmic Formulation Containing Nilvadipine Nanocrystals Attenuates Lens Opacification in Shumiya Cataract Rats.

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.

Effect of inner physical properties on powder adhesion in inhalation capsules in case of a high resistance device.

The inhalation performance of a dry powder inhaler (DPI) depends on the inhalation patterns of patients, inhalation particle characteristics and inhalation devices. In capsule-based DPIs, the capsule plays an important role in the dispersion of inhalation particles. The present study investigated the effects of inner physical properties of capsules on drug release from capsules-based DPIs with high resistance device. Atomic force microscopy (AFM) was used to evaluate the capsule physical properties, such as the capsule inner structure and surface potential, of three capsules with different compositions (G-Cap, PEG/G-Cap, and HPMC-Cap). As a model dry powder for capsule-based DPIs, the dry powder in Spiriva® Inhalation Capsules containing tiotropium bromide was used. Inhalation performance was evaluated using a twin-stage liquid impinge and Handihaler® (flow rate 30 l/min). The results indicated that the capsule inner surface presented with numerous valleys and mountains, regardless of the capsule type. Furthermore, the valley and mountain areas on the capsule inner surface showed a significantly higher or lower surface potential. Following inhalation of capsule-based DPIs, the drug remained in the valleys on the capsule inner surface; however, no significant difference was observed in the drug release from capsule and lung drug delivery. Therefore, inhalation performance in capsule-based DPIs when a high resistance device, such as Handihaler®, is used at an appropriately flow rate is not markedly affected by the physical properties of the capsule inner surface due to capsule composition.

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.

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

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

Fixed-Combination Eye Drops Based on Fluorometholone Nanoparticles and Bromfenac/Levofloxacin Solution Improve Drug Corneal Penetration.

PURPOSE: The multi-instillation of three commercially available (CA) eye drops [fluorometholone (FL)-, bromfenac (BF)- and levofloxacin (LV)-eye drops] has been used to manage pain and inflammation post-intraocular surgery. However, the multi-instillation of these three eye drops causes corneal damage, and the FL drops have the disadvantage of low ocular bioavailability. To overcome these problems, we prepared fixed-combination eye drops based on FL nanoparticles (FL-NPs) and BF/LV solution (nFBL-FC), and evaluated the corneal toxicity and transcorneal penetration of the nFBL-FC eye drops. METHODS: FL powder was mixed in 2-hydroxypropyl-ß-cyclodextrin solution containing benzalkonium chloride, mannitol and methylcellulose, and milled with a Bead Smash 12 (5500 rpm for 30 s×30 times). The BF/LV solution was then added to the milled-dispersions to be used as nFBL-FC. The FL, BF and LV concentrations were measured by HPLC methods, and transcorneal penetration was evaluated in rabbits. RESULTS: The FL particle size in nFBL-FC was 40-150 nm, with only 0.0018% in liquid form. No aggregation of FL particles in the nFBL-FC was observed for 1 month. The viability of human corneal epithelial cells treated with nFBL-FC was remarkably higher than that of cells subjected to the multi-instillation of the corresponding three CA-eye drops. In addition, the corneal penetrations (AUC) of the FL, BF and LV in nFBL-FC were 4.9-, 1.8-, and 7.1-fold those of the corresponding CA-eye drops, respectively. Moreover, the caveolae-dependent endocytosis (CavME) inhibitor (nystatin) significantly prevented the transcorneal penetration of these drugs. CONCLUSION: We prepared fixed-combination eye drops based on FL-NPs and BF/LV solution (nFBL-FC), and show that high levels of FL-NPs and dissolved BF/LV (liquid drugs) can be delivered into the aqueous humor by the instillation of nFBL-FC. Further, we show that CavME is mainly related to the enhancement of transcorneal penetration of both the solid (NPs) and liquid drugs.

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.

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.

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.