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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.

Tranilast (TL), an antiallergic agent, has been clinically used in the treatment of bronchial asthma, although its clinical use has been limited by its poor solubility in water, photodegradation and systemic side effects. In this study, we prepared a gel ointment containing TL nanoparticles (TL nano gel ointment), and investigated its usefulness. In addition, we demonstrated the preventive effects of the TL nano gel ointment on inflammation in adjuvant-induced arthritis (AA) rats. The TL nano gel ointment was prepared using Bead Smash 12 (a bead mill) and additives including sodium docusate, 2-hydroxypropyl-β-cyclodextrin, methylcellulose and Carbopol 934; the mean particle diameter of the TL nanoparticles was 71.0 25.4 nm. In in vitro skin penetration experiments, the amount of penetrated TL, the penetration rate (J c ) and the penetration coefficient through the skin (K p ) of the TL nano gel ointment were significantly higher than those of a gel ointment containing TL microparticles (TL micro gel ointment; particle diameter 50.5 26.3 µm). The TL concentrations in the skin tissue and plasma of rats receiving the TL nano gel ointment were also higher than in rats receiving the TL micro gel ointment. In addition, the application of the TL nano gel ointment attenuated the increase in paw edema of the hind feet of AA rats in comparison with AA rats treated with the TL micro gel ointment. These results suggest that TL nanoparticles can be applied to the formulation of a transdermal system, and that a transdermal formulation using TL nanoparticles might be a delivery option for the clinical treatment of RA.

PurposeIn 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.MethodsRaloxifene 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.ResultsThe 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.ConclusionWe prepared raloxifene solid nanoparticles by a bead mill method and designed a novel transdermal formulation containing nanoparticles and permeation enhancers. These trans-dermal 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.

We previously reported that dermal application using nanoparticles improves skin penetration. In this study, we prepared novel topical formulations containing ketoprofen (KET) solid nanoparticles (KET nano gel ointment) and investigated the antiinflammatory effect of the KET nanoparticle formulations on rheumatoid arthritis using adjuvant-induced arthritis (AA) rats. The KET nano gel ointment was prepared using a bead mill method and additives including methylcellulose and Carbopol 934; the mean particle size of the KET nanoparticles was 83 nm. In the in vitro skin penetration experiment, the penetration rate (J c ) and penetration coefficient through the skin (K p ) values of the KET nano gel ointment were significantly higher than those of gel ointment containing KET microparticles (KET micro gel ointment; mean particle size 7.7 µm). On the other hand, in the in vivo percutaneous absorption experiment, the apparent absorption rate constant (k a ) and the areas under the KET concentration-time curve values in the skin of rats receiving the KET nano gel ointment were significantly higher than those of rats receiving the KET micro gel ointment, and the amounts of KET in the skin tissues of rats receiving the KET nano gel ointment were also significantly higher than those of rats receiving the KET micro gel ointment. In addition, the application of the KET nano gel ointment attenuated the enhancement of paw edema of the hind feet of AA rats more than the application of the KET micro gel ointment. Our findings suggest that a topical drug delivery system using nanoparticles could lead to expansion in the therapeutic use of KET.Key words nanoparticle; ketoprofen; gel ointment; drug delivery; adjuvant-induced arthritis Ketoprofen (KET) is a reversible inhibitor of cyclooxygenases 1 and 2 whose action leads to a reduction in the formation of prostaglandin precursors. 1) Thus, KET acts as an antiinflammatory agent, and is used in the treatment of rheumatoid arthritis (RA) and osteoarthritis. However, its usefulness is limited due to its low water solubility and the fact that oral administration of KET tends to cause gastrointestinal lesions in 10-30% of patients. These gastrointestinal lesions lead to an interruption of drug therapy in about 5-15% patients. 2,3)Topical and transdermal delivery routes of drug administration reduce the incidence of gastrointestinal lesions and improve patient compliance. Furthermore, the transdermal route of application avoids hepatic first pass metabolism, meaning that therapeutic serum drug concentrations can be applied with a reduced risk of nephrotoxicity and drug-drug interactions.2) However, the transdermal route of application is limited by the barrier properties of the skin. Therefore, it is necessary to develop enhancement techniques to assist the skin penetration of KET, and extensive research has been done to find both topical and transdermal KET formulations.