Comparison of Various Cell Lines and Three-Dimensional Mucociliary Tissue Model Systems to Estimate Drug Permeability Using an In Vitro Transport Study to Predict Nasal Drug Absorption in Rats.

Recently, various types of cultured cells have been used to research the mechanisms of transport and metabolism of drugs. Although many studies using cultured cell systems have been published, a comparison of different cultured cell systems has never been reported. In this study, Caco-2, Calu-3, Madin-Darby canine kidney (MDCK), EpiAirway and MucilAir were used as popular in vitro cell culture systems, and the permeability of model compounds across these cell systems was evaluated to compare barrier characteristics and to clarify their usefulness as an estimation system for nasal drug absorption in rats. MDCK unexpectedly showed the best correlation (r = 0.949) with the fractional absorption (Fn) in rats. Secondly, a high correlation was observed in Calu-3 (r = 0.898). Also, Caco-2 (r = 0.787) and MucilAir (r = 0.750) showed a relatively good correlation with Fn. The correlation between Fn and permeability to EpiAirway was the poorest (r = 0.550). Because EpiAirway forms leakier tight junctions than other cell culture systems, the paracellular permeability was likely overestimated with this system. On the other hand, because MDCK formed such tight cellular junctions that compounds of paracellular model were less likely permeated, the paracellular permeability could be underestimated. Calu-3, Caco-2 and MucilAir form suitable cellular junctions and barriers, indicating that those cell systems enable the precise estimation of nasal drug absorption.

Improving the Solid-State Photostability of Furosemide by Its Cocrystal Formation.

The photostability of three types of furosemide (FUR) cocrystal (FUR-caffeine, FUR-urea, and FUR-nicotinamide cocrystals) was studied under irradiation with a D65 fluorescent lamp. The coloration of the FUR-urea pellets was significantly faster than that of the intact FUR, whereas the coloration of FUR-nicotinamide was suppressed compared with that of intact FUR and the other cocrystals. In the case of FUR-urea, the chemical degradation of FUR increased by approximately 6.6% after irradiation for 90 d. On the other hand, FUR-nicotinamide showed better chemical stability, with only 1.3% of FUR degraded, which was significantly lower than the other cocrystals. The FUR-urea pellets showed a UV-Visible absorption spectrum similar to that of intact FUR, while the absorption range of FUR-nicotinamide shifted to a shorter wavelength. The light sensitivity of FUR-nicotinamide was improved because of the much lower emission of the D65 fluorescent lamp in the absorption range of the cocrystal.

Novel Strategy for the Systemic Delivery of Furosemide Based on a New Drug Transport Mechanism.

We reported a novel transport mechanism of curcumin, independent of improved solubility, which involved direct contact of amorphous solid particles with the cell membrane. This mechanism has potential as a novel systemic delivery system of poorly water-soluble drugs. In this study, the transport mechanism of furosemide (FUR), which is transported by the same novel mechanism, was examined. In vitro cell permeation studies under air-interface conditions (AICs) revealed that the permeation from powders sprayed on cell monolayers was significantly higher than that under liquid-covered conditions (LCCs) from their solutions. The permeation from amorphous solid particles was faster than that from crystals. Similar results were derived from in vitro studies using an artificial membrane, with which the permeation of FUR could be examined without water. These findings clearly indicated that the transport mechanism of FUR is the same as that of curcumin. For the application of this new transport mechanism, the in vivo absorption of FUR was examined after pulmonary insufflation, which allows the solid particles to make direct contact with the epithelial cells. Pulmonary absorption of FUR from the amorphous powder was almost complete and was faster than that after intragastric administration of the solution, suggesting that FUR was absorbed from the lung by the same mechanism as the in vitro study. This new transport mechanism, which is independent of water dissolution, could be exploited to develop a novel delivery system for poorly water-soluble drugs, using pulmonary powder inhalation.

Quantitative Estimation of the Effect of Nasal Mucociliary Function on in Vivo Absorption of Norfloxacin after Intranasal Administration to Rats.

Nasal drug delivery has attracted significant attention as an alternative route to deliver drugs having poor bioavailability. Large-molecule drugs, such as peptides and central nervous system drugs, would benefit from intranasal delivery. Drug absorption after intranasal application depends on the nasal retention of the drug, which is determined by the nasal mucociliary clearance. Mucociliary clearance (MC) is an important determinant of the rate and extent of nasal drug absorption. The aim of the present study was to clarify the effect of the changes in MC on in vivo drug absorption after nasal application, and to justify the pharmacokinetic model to which the MC parameter was introduced, to enable prediction of bioavailability after intranasal administration. The pharmacokinetics of norfloxacin (NFX) after intranasal administration were evaluated following the modification of nasal MC by pretreatment with the MC inhibitors propranolol and atropine and the MC enhancers terbutaline and acetylcholine chloride. From the relationship between nasal MC and bioavailability after nasal application, prediction of drug absorption was attempted on the basis of our pharmacokinetic model. Propranolol and atropine enhanced the bioavailability of NFX by 90 and 40%, respectively, while the bioavailability decreased by 30% following terbutaline and 40% following acetylcholine chloride. As a result of changes in the MC function, nasal drug absorption was changed depending on the nasal residence time of the drug. On the basis of our pharmacokinetic model, the nasal drug absorption can be precisely predicted, even when the MC is changed. This prediction system allows the quantitative evaluation of changes in drug absorption due to changes in nasal MC and is expected to contribute greatly to the development of nasal formulations.

The relationship between in vivo nasal drug clearance and in vitro nasal mucociliary clearance: Application to the prediction of nasal drug absorption.

Drug absorption after nasal application is dependent on drug clearance from the nasal cavity, which is determined by nasal mucociliary clearance (MC). We previously developed an in vitro method to evaluate MC via the translocation velocity of fluorescent microspheres (VFMS) applied to excised rat nasal mucosa. In the present study, the relationship between in vivo nasal MC and in vitro VFMS was examined to optimize our PK model for the prediction of nasal drug absorption. Appropriate inhibitors (propranolol and atropine) and enhancers (terbutaline and acetylcholine chloride) of MC were utilized to modify MC. In vivo clearance of drug from the nasal cavity was determined from the disappearance of fluorescent microspheres (FMS) from the nasal cavity following nasal application to rats. The first order elimination rate constant, kmc, was determined from the disappearance profiles of FMS. kmc was decreased to 35.8% by propranolol and 52.6% by atropine, but increased to 117% by terbutaline and 168% by acetylcholine chloride. A significant linear correlation was observed between kmc and VFMS (r2 = 0.9745, p < 0.001). These results indicate that in vivo kmc can be estimated from the in vitro parameter, VFMS. By introducing linear correlation into our PK model, nasal drug absorption may be precisely estimated, even with changes in MC.

Delivery of Oxytocin to the Brain for the Treatment of Autism Spectrum Disorder by Nasal Application.

Oxytocin (OXT) is a cyclic nonapeptide, two amino acids of which are cysteine, forming an intramolecular disulfide bond. OXT is produced in the hypothalamus and is secreted into the bloodstream from the posterior pituitary. As recent studies have suggested that OXT is a neurotransmitter exhibiting central effects important for social deficits, it has drawn much attention as a drug candidate for the treatment of autism. Although human-stage clinical trials of the nasal spray of OXT for the treatment of autism have already begun, few studies have examined the pharmacokinetics and brain distribution of OXT after nasal application. The aim of this study is to evaluate the disposition, nasal absorption, and therapeutic potential of OXT after nasal administration. The pharmacokinetics of OXT after intravenous bolus injection to rats followed a two-compartment model, with a rapid initial half-life of 3 min. The nasal bioavailability of OXT was approximately 2%. The brain concentration of OXT after nasal application was much higher than that after intravenous application, despite much lower concentrations in the plasma. More than 95% of OXT in the brain was directly transported from the nasal cavity. The in vivo stress-relief effect by OXT was observed only after intranasal administration. These results indicate that pharmacologically active OXT was effectively delivered to the brain after intranasal administration. In conclusion, the nasal cavity is a promising route for the efficient delivery of OXT to the brain.

Novel strategy for improving the bioavailability of curcumin based on a new membrane transport mechanism that directly involves solid particles.

Amorphization has been widely recognized as a useful solubilization technique for poorly water-soluble drugs, such as curcumin. We have recently reported the novel finding that the membrane transport of curcumin was markedly enhanced when amorphous solid particles of curcumin came into direct contact with the lipid membrane surface, but this was not true for crystalline solid particles. The increase in the permeation of curcumin was found to be independent of the improvements in aqueous solubility brought about by amorphization. Thus, we have identified a novel membrane transport mechanism that directly involves solid particles. In addition, it might represent a novel strategy for improving the bioavailability of curcumin that does not focus on the aqueous solubility of the drug. In this study, the direct effects of the administration of amorphous nanoparticles of curcumin (ANC) on the in vivo intestinal absorption of curcumin were investigated. After the intraduodenal administration of a curcumin suspension, the area under the curve of the plasma concentration of curcumin increased in a manner that was dependent on the curcumin concentration of the suspension, while no significant absorption was observed from a saturated solution. This finding is consistent with the results from our in vitro transepithelial transport study. In the latter experiment, the bioavailability of curcumin was found to be 1-2%. The intrapulmonary insufflation of ANC powder resulted in a significant increase in the bioavailability of curcumin (it was two orders of magnitude higher than that seen after the application of a crystalline suspension). This was due to the ANC particles coming into contact with epithelial cells in a more efficient manner after the pulmonary application of the ANC powder than after the intestinal application of the ANC suspension. Therefore, the pulmonary insufflation of amorphous powder is a novel approach to improving the bioavailability of curcumin and might be a useful way of increasing the bioavailability of poorly water-soluble drugs, such as curcumin.

Differences in the rheological properties and mixing compatibility with heparinoid cream of brand name and generic steroidal ointments: The effects of their surfactants.

Most steroidal ointments contain propylene glycol (PG) and surfactants, which improve the solubility of corticosteroids in white petrolatum. Surfactants aid the uniform dispersal of PG within white petrolatum. Since the surfactants used in generic ointments are usually different from those used in brand name ointments, we investigated the effects of surfactants on the rheological properties of three brand name ointments and six equivalent generic ointments. We detected marked differences in hardness, adhesiveness, and spreadability among the ointments. Further examinations of model ointments consisting of white petrolatum, PG, and surfactants revealed that the abovementioned properties, especially hardness and adhesiveness, were markedly affected by the surfactants. Since steroidal ointments are often admixed with moisturizing creams prior to use, we investigated the mixing compatibility of the ointments with heparinoid cream and how this was affected by their surfactants. We found that the ointments containing glyceryl monostearate demonstrated good mixing compatibility, whereas those containing non-ionic surfactants with polyoxyethylene chains exhibited phase separation. These results were also consistent with the findings for the model ointments, which indicates that the mixing compatibility of steroidal ointments with heparinoid cream is determined by the emulsifying capacity of the surfactants in their oily bases.

Prominent efficiency in skin delivery of resveratrol by novel sucrose oleate microemulsion.

OBJECTIVES: To achieve an efficient skin delivery of resveratrol using sucrose fatty acid ester microemulsions and to clarify the mechanism of enhanced penetration. METHODS: Skin delivery of resveratrol using different sucrose fatty acid ester microemulsions was examined in vitro. Vehicle-skin interaction was assessed by applying blank microemulsions to skin. Skin incorporation of microemulsion components was also assessed. KEY FINDINGS: The microemulsion consisting of sucrose oleate (SO), ethanol, isopropyl myristate (IPM) and water (MESO-E) showed a prominent increase in the amount of skin incorporation of resveratrol, which was more than 5-fold higher than those of all microemulsions we previously examined. Using MESO-E, resveratrol was rapidly incorporated into skin and mainly located in the dermis. When applied in the concentration range of 5-55 mm, the amount of skin incorporation of resveratrol increased with the applied concentration up to 30 mm, whereas skin incorporation efficiency was inversely proportional to the concentration. The microemulsion-skin interaction seemed to be involved in the enhanced skin delivery process of resveratrol by MESO-E. Stratum corneum modification due to the penetration of IPM, ethanol and SO is also involved in this interaction. CONCLUSIONS: MESO-E would be a promising vehicle for the efficient skin delivery of resveratrol, especially when applied at a low concentration.

Microemulsion Using Polyoxyethylene Sorbitan Trioleate and its Usage for Skin Delivery of Resveratrol to Protect Skin against UV-Induced Damage.

We examined the phase behavior of various polyoxyethylene sorbitan fatty acid ester (polysorbates)/ethanol/isopropyl myristate (IPM)/150 mM NaCl solution (NaClaq) systems in order to prepare a microemulsion containing a low ratio of ethanol, which is more suitable for in vivo application. Using polyoxyethylene sorbitan trioleate (Tween 85), which has a large lipophilic moiety, as a surfactant component, single-phase domain of the phase diagram was the largest of all the polysorbates examined, and in particular a large oil-rich single-phase domain was obtained. When the ratio of Tween 85 to ethanol was changed from 1 : 1 to 3 : 1, the oil-rich single-phase domain further expanded, which led to a reduced ethanol concentration in the preparation. Thus, we determined the composition of the microemulsion to be Tween 85 : ethanol : IPM : NaClaq=30 : 10 : 53 : 7, and used it for skin delivery of resveratrol. Microemulsion gel was also prepared by adding 6.5% Aerosil) 200 into the microemulsion for ease of topical application. When applied with each vehicle, delivery of resveratrol into guinea pig skin in vitro was significantly enhanced compared with that by IPM, and resveratrol incorporated into the skin by microemulsion gel decreased lipid peroxidation to 29.5% compared with that of the control. Pretreatment of guinea pig dorsal skin with the microemulsion gel containing resveratrol almost completely prevented UV-B-induced erythema formation in vivo. These findings demonstrate that the microemulsion using Tween 85 containing a minimal concentration of ethanol enhanced the skin delivery of resveratrol and the incorporated resveratrol exhibited a protective effect against UV-induced oxidative damage.

Effects of mixing procedure itself on the structure, viscosity, and spreadability of white petrolatum and salicylic acid ointment and the skin permeation of salicylic acid.

White petrolatum is a mixture of solid and liquid hydrocarbons and its structure can be affected by shear stress. Thus, it might also induce changes in its rheological properties. In this study, we used polarization microscopy to investigate how different mixing methods affect the structure of white petrolatum. We used two different mixing methods, mixing using a rotation/revolution mixer and mixing using an ointment slab and an ointment spatula. The extent of the fragmentation and dispersal of the solid portion of white petrolatum depended on the mixing conditions. Next, we examined the changes in the structure of a salicylic acid ointment, in which white petrolatum was used as a base, induced by mixing and found that the salicylic acid solids within the ointment were also dispersed. In addition to these structural changes, the viscosity and thixotropic behavior of both test substances also decreased in a mixing condition-dependent manner. The reductions in these parameters were most marked after mixing with a rotation/revolution mixer, and similar results were obtained for spreadability. We also investigated the effects of mixing procedure on the skin accumulation and permeation of salicylic acid. They were increased by approximately three-fold after mixing. Little difference in skin accumulation or permeation was detected between the two mixing methods. These findings indicate that mixing procedures themselves affect the utility and physiological effects of white petrolatum-based ointments. Therefore, these effects should be considered when mixing is required for the clinical use of petrolatum-based ointments.

Efficient delivery and distribution in skin of chlorogenic acid and resveratrol induced by microemulsion using sucrose laurate.

To achieve efficient skin delivery of polyphenols, we prepared a novel oil-in-water (o/w)-type microemulsion (MESL) using sucrose laurate as a surfactant and ethanol, isopropyl myristate and water as other components. We examined its usefulness by in vitro studies on skin delivery of chlorogenic acid and resveratrol as hydrophilic and hydrophobic polyphenols using Yucatan micropig skin, and also examined the difference in the distribution of these polyphenols in skin. MESL significantly improved skin incorporation of these polyphenols at all time points examined (6, 20, 40 h) in the epidermis and at 20 and 40 h in the dermis, compared with the microemulsion using Tween 80 as a surfactant component (MEK), although the solubilization capacity of MESL was lower than that of MEK. Using MESL, the incorporation amount in the dermis of each polyphenol increased with time, while the amount in the epidermis was almost constant during the time examined. Incorporation efficiencies into skin of chlorogenic acid and resveratrol induced by MESL at 40 h after application were about 6-fold and 19-fold higher in the epidermis and 3.5-fold and 15-fold higher in the dermis, respectively, than those by MEK. The increase was more prominent for resveratrol. Hydrophilic chlorogenic acid was distributed slightly more in the epidermis, while hydrophobic and smaller-molecular-weight resveratrol was mainly distributed in the dermis. These findings suggest that MESL could be a promising vehicle for the efficient skin delivery of chlorogenic acid and resveratrol, especially for resveratrol to the dermis.

Distribution of polyphenols and a surfactant component in skin during Aerosol OT microemulsion-enhanced intradermal delivery.

As for most other polyphenols, intradermal delivery of curcumin and resveratrol is limited; however, it was significantly improved by a microemulsion using Aerosol OT (Aerosol OT microemulsion) and Tween 80 (Tween 80 microemulsion) as surfactants. Aerosol OT microemulsion was more effective and the incorporation ratio of these polyphenols into skin by Aerosol OT microemulsion was five-fold or ten-fold that by Tween 80 microemulsion. To clarify the mechanism of the enhancement we examined the distribution of these polyphenols and the surfactant component, Aerosol OT, using excised guinea pig skin and Yucatan micropig (YMP) skin. During permeation, polyphenols distributed deep in the skin. In particular, a small molecule, resveratrol, was mainly present in the dermis in YMP skin. Aerosol OT also distributed deep in the skin. These findings suggest the possible involvement of the interaction of surfactant molecules with skin components in the enhanced delivery process of polyphenols. The distribution ratio between the dermis and epidermis of the polyphenols, including quercetin, in the presence of Aerosol OT microemulsion decreased with the increase of molecular weight in YMP skin, suggesting the possibility that distribution to the dermis is regulated by the molecular size.