Application of Pickering Emulsion with Cyclodextrin as an Emulsifier to a Transdermal Drug Delivery Vehicle

Abstract

The emulsion prepared with β-cyclodextrin as an emulsifier (βCDE) is considered to be a Pickering emulsion. We examined the characteristics of βCDEs using captopril (CP) as a model drug, and studied the in vitro skin permeation of CP from βCDEs through hairless mouse skin. The stability of βCDE was increased with increasing βCD concentration and conversely decreased with increasing CP concentration. The yield stress value from the rheological measurement results was suggested to be one of the factors determining the stability of the βCDE, and βCDEs with higher yield stress values were more stable. We found that the skin permeability of CP could be improved by using βCDE with isopropyl myristate as the oil phase and that the flux of CP depended on the free CP concentration in the water phase of βCDE.

INTRODUCTION

An emulsion is a thermodynamically unstable dispersion system consisting of at least two immiscible liquid phases. Normally, mixing water and oil forms an emulsion, but the obtained emulsion is usually demulsified and eventually separates into the original oil and water.¹⁾ Therefore, the addition of an emulsifier is indispensable for stabilizing an emulsion. A surfactant is an amphiphilic substance having both hydrophilic and hydrophobic moieties in the molecule and is used for many purposes, e.g., pharmaceuticals,²⁾ household and industrial detergents,³⁾ and oil drilling.⁴⁾ However, some surfactants cause skin irritation,^5,6) have hemolytic properties,⁷⁾ or poor biodegradability in nature,⁸⁾ or adversely affect humans and the environment.⁹⁾ As a means to overcome such problems, studies on surfactant-free emulsions have been conducted.¹⁰⁾ Pickering emulsions, which are stabilized by the adsorption of solid particles such as silica fine particles,^11,12) polystyrene latex,^13,14) and clay mineral¹⁵⁾ instead of a surfactant at the oil/water interface, are known as surfactant-free emulsions. Binks and Lumsdon studied the physicochemical properties of Pickering emulsions and clarified that controlling the wettability of solid particles to water and oil was important for adsorbing particles at the interface.¹⁶⁾

Cyclodextrins (CDs) are cyclic oligosaccharides consisting of 1,4-linked glucopyranose units with a bucket-like structure, a hydrophobic internal cavity and a hydrophilic exterior. αCD, βCD, and γCD, which consist of 6, 7 and 8 glucopyranose units, respectively, are known in nature. CDs can form complexes with various lipophilic guest molecules in the cavity and are therefore used for stabilization, solubilization, and sustained release of drugs, etc., especially in the medical field.¹⁷⁾ Furthermore, CDs can be used as emulsifiers, and we clarified that the emulsion prepared with CDs (CDE) was a Pickering emulsion stabilized by adsorption of the insoluble CD complex as solid particles at the oil/water interface.^18–21) However, there have only been a few studies on the application of CDEs in the medical field. For example, Leclercq and Nardello-Rataj studied CDE containing econazole nitrate salt as an antifungal azole derivative and carried out antimicrobial and antifungal tests, which found biocidal activity against Staphylococcus aureus and Candida albicans on culture media.²²⁾ Mathapa and Paunov studied cyclodextrinosomes, with the core oil of CDE removed.²³⁾

In an aging society, the use of patient-friendly medicine is important not only for improving the QOL of patients, but also for those of families and caregivers. Therefore, studies on drug delivery systems (DDSs) have been actively performed. The transdermal dosage form is one of the most important DDS. Emulsions, ointments, suspensions, lotions, tapes, patches, etc., have all been used for transdermal dosage. The main barrier to skin permeation of a drug from the transdermal dosage form is the stratum corneum. Adding a transdermal penetration enhancer to the formulation is the usual method for enhancing permeability of a drug through the skin. Isopropyl myristate (IPM) has been known to act as an enhancer²⁴⁾ and is used in many formulations.

In this study, we examined the characteristics of CDEs using CDs, IPM, and captopril (CP) which is an angiotensin converting enzyme inhibitor as a model drug. We also evaluated the in vitro skin permeation of the drug from the CDEs through hairless mouse skin, and assessed the usefulness of CDE as a transdermal dosage form.

MATERIALS AND METHODS

Materials

Cyclodextrins (α, β, and γCD) were purchased from Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan) and used after drying under vacuum. Captopril (CP), isopropyl myristate (IPM), liquid paraffin (LP), soybean oil (Soy), and Dulbecco’s phosphate saline (PBS) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Polyoxyethylene (60) hydrogenated castor oil (HCO60) was obtained from Nikko Chemical Co., Ltd. (Tokyo, Japan). Water was purchased from Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan). Acetonitrile was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Other reagents were of the purest grade supplied by Wako Pure Chemical Industries, Ltd.

Preparation of Emulsion

The required amounts of emulsifier (CD or HCO60), CP and water were mixed in a stainless steel container, followed by addition of oil. The mixture was mixed at 10000 rpm for 5 min using a homogenizer (Excel Auto Homogenizer, Nihonseiki Kaisha Ltd., Tokyo, Japan) at 25°C. The type of emulsion was determined by electric conductivity measurements and dilution testing.

To compare the characteristics of the CDEs, various vehicles were prepared in the same manner as described above except for one or more ingredients of the emulsion, as shown in Table 1.

Table 1. Composition of Vehicles

Vehicles	Emulsifier (wt%)		Water (wt%)	Oil (wt%)
	CDs	HCO60		IPM	LP	Soy
15%βCDE (IPM)	15		46	39
20%βCDE (IPM)	20		43	37
25%βCDE (IPM)	25		40	35
20%βCDE (LP)	20		43		37
20%βCDE (Soy)	20		42			38
20%αCDE (IPM)	20		43	37
20%γCDE (IPM)	20		43	37
Aq sol.			100
20%βCD/Aq susp.	20		80
IPM susp.				100
20%βCD/IPM susp.	20			80
HCO60E		20	43	37

The volume ratio of oil to water in the emulsion is 1 : 1. The standard formulation is 20%βCDE (IPM) with 5 wt%CP.

Stability of Emulsion

The phase state of the emulsions was observed visually and microscopically. The stability of the emulsions was evaluated by measuring the time–course of the volume fractions of the emulsion phase, the separated oil phase, and the separated water phase in a screw-top vial at 25°C.

Rheological Measurement

Rheograms of the emulsions were obtained using a stress-controlled rheometer (RheoStress 600, HAAKE GmbH, Karlsruhe, Germany) equipped with parallel-plate geometry (diameter, 35 mm; gap distance, 1.0 mm) at 25°C.

Skin Permeation Studies

The excised skin of hairless mice (Laboskin^®, Hos: HR-1 Male, 7 weeks old, Hoshino Laboratory Animals Inc., Ibaraki, Japan) was used in the permeation studies. The permeation apparatus was a Franz-type diffusion cell (Hanson Research Co., Chatsworth, CA, U.S.A.) with a receptor volume ranging from 7.0 to 7.3 mL and a diffusion area of 1.74 cm².

The receptor cell was kept at 32°C by circulating water through an external jacket. PBS as a receptor phase was added and the mixture was stirred using a magnetic stirrer with wire at 550 rpm. Skin was placed on the cell, and 0.5 g of vehicles with CP was applied on the skin in the donor compartment. A 0.5 mL sample of the receptor phase was removed from the cells at predetermined times and replaced with fresh PBS. The concentration of CP in the sample was determined using HPLC.

The flux and lag time were determined from the slope of the linear portion and the X-axis intercept of the skin permeation profile of CP, respectively.

HPLC Analysis of CP

The quantitative determination of CP in the samples was performed on a HPLC system under the following conditions: pump: PU-2080 Plus (JASCO, Tokyo, Japan); UV detector: UV-2075 Plus (JASCO) at 220 nm; injection volume of sample: 5 µL with autosampler AS-2051 Plus (JASCO); column: L-column2 ODS (150 × 4.6 mm i.d., Chemical Evaluation and Research Institute, Tokyo, Japan) at 40°C; mobile phase: acetonitrile/water/phosphoric acid (20/80/0.05, v/v/v); flow rate: 1.0 mL/min; chromatographic data processing: ChromNAV (JASCO).

The CP concentration of each phase in the CDE was measured by HPLC after separating the liquid (water and oil phase) and precipitation by centrifugal filtration (3000 rpm) using a Merck Millipore Ultrafree^®-CL centrifugal filter device at 25°C.

Data Analysis

All skin permeation data are expressed as the mean ± standard deviation (S.D.) of at least three experiments. The significance of the differences was determined by ANOVA followed by using freely available “EZR” as the statistical software.²⁵⁾ A p value of <0.05 after Bonferroni correction was considered indicative of statistical significance in all cases.

RESULTS AND DISCUSSION

Preparation and Properties of CDEs

Emulsions prepared using CD as an emulsifier (CDE) are considered to be Pickering emulsions, which are stabilized by adsorption of the oil-CD inclusion complex precipitate at the oil-water interface. An O/W emulsion is prepared when the oil/water contact angle (θ_ow) of the precipitate is θ_ow < 90°, and a W/O emulsion is prepared when θ_ow > 90°. When IPM is used as an oil and natural CDs are used as an emulsifier, an O/W emulsion is formed.^20,22) The static stability of CDEs prepared using alkanols, hydrocarbons, and common oils (liquid paraffin, soybean oil, and squalane) has been studied,²¹⁾ but the stability is not known in the case of IPM. Figure 1 shows the static stabilities of CDEs (IPM) with 5% CP. In the case of 15%βCDE (IPM), separation of the water and oil phases occurred by coalescence and creaming after several days, and the volume fraction of the O/W emulsion phase decreased over time (Fig. 1a). In the case of 20%βCDE (IPM), only a slight separation of the oil phase occurred (Fig. 1b). In the case of 25%βCDE (IPM), phase separation did not occur at all and the emulsion was stable for 30 d (Fig. 1c). When the natural CDs concentration was fixed at 20%, the stability was on the order of βCDE (IPM) > αCDE (IPM) >> γCDE (IPM), as shown in Figs. 1b, d and e.

Fig. 1. Static Stability of CDEs with 5 wt% CP

(a) 15%βCDE (IPM), (b) 20%βCDE (IPM), (c) 25%βCDE (IPM), (d) 20%αCDE (IPM), (e) 20%γCDE (IPM). The hatched region shows the separated oil phase. The dashed region shows the separated water phase. The white region shows the O/W emulsion phase.

Figure 2 shows photographs of CDEs (IPM) with 5% CP. We found that the precipitate among emulsion particles increased with an increase in βCD concentration, as shown in Figs. 2a–c. In the case of 20%αCDE (IPM) (Fig. 2d), the state of the precipitate showed an appearance similar to that of 20% βCDE (IPM). On the other hand, in the case of γCDE (IPM) with low stability, precipitates were observed (Fig. 2e), but not much adsorbed at the emulsion particle surface. In addition, the size of the emulsion particles was non-uniform and large compared to other natural CDs.

Fig. 2. Photographs of CDEs with 5 wt% CP

(a) 15%βCDE (IPM), (b) 20%βCDE (IPM), (c) 25%βCDE (IPM), (d) 20%αCDE (IPM), (e) 20%γCDE (IPM).

The rheological properties of an emulsion serve as predictors of deformation and stability under static conditions and shear stress and as an index of feeling when using cosmetics and daily necessities. To investigate the stability of CDEs in detail, rheology measurements were carried out. Figure 3 shows the rheograms of βCDE (IPM) with 5% CP as a function of βCD concentration. All the βCDE (IPM) samples showed Bingham flow with a yield stress value. This is considered to be due to the formation of a strong three-dimensional network structure by the dispersed phase and the complex precipitate that formed at higher CD concentrations in CDE. The CDEs could not flow by the yield stress value, but above this value the structure collapses and flows at once. The yield stress values were in the order of 15%βCDE (IPM) < 20%βCDE (IPM) < 25%βCDE (IPM) and we found that the more stable emulsions had higher values. Therefore, it was suggested that the yield value was one of the factors determining the stability of the CDE. The results of the yield stress values of CDEs are summarized in Table 2.

Fig. 3. Rheograms of βCDEs with 5 wt% CP

Table 2. Yield Stress Value of βCDEs

Vehicles	CP added (wt%)	Yield stress value (Pa)
15%βCDE (IPM)	5	45
20%βCDE (IPM)	5	92
25%βCDE (IPM)	5	621
20%βCDE (IPM)	1	1172
20%βCDE (IPM)	10	73
20%βCDE (Soy)	5	92
20%βCDE (LP)	5	489

We also examined the effect of CP on the stability of 20%βCDE (IPM). Twenty percent βCDE (IPM) prepared with 1% CP and 5% CP were stable over 30 d and 14 d, respectively, while that with 10% CP was only stable for 1 d after preparation (Fig. 4). Both IPM and CP are known to form a complex with βCD.^22,26) Therefore, we believe that the formation of the IPM-CD complex might be competitively inhibited. It is known that Pickering emulsion is more stable when θ_ow having the maximum adsorption energy at the interface is close to 90°.¹⁶⁾ Not all of the precipitated βCD complexes are involved in the formation of βCDE but adsorb at IPM/water interface or precipitate in the water phase depending on the wettability. It is known that the θ_ow of IPM-βCD complex is 64°.¹⁹⁾ The θ_ow of CP-βCD complex including CP with higher hydrophilicity than IPM is expected to be smaller than that of IPM-βCD complex. Since as the addition amount of CP increases, the amount and the θ_ow of complexes involved in the formation of βCDE decrease relatively, we consider that the obtained emulsion becomes unstable.

Fig. 4. Effect of CP on the Static Stability of 20%βCDE (IPM)

The data of 5 wt% CP in this figure is the same as the volume ratio of the emulsion phase in Fig. 1b.

From the above results, we suggested that a formulation consisting of a stable O/W Pickering emulsion containing a drug can be prepared using an appropriate amount of βCD as an emulsifier.

Skin Permeation of CP from CDEs

To investigate the usefulness of CDE as a transdermal dosage form, we carried out in vitro skin permeation studies of CP from CDEs through hairless mouse skin. Here, the standard formulation was 20%βCDE (IPM) with 5% CP and the skin permeability of the formulation was evaluated as compared with various vehicles as shown in Table 1. Figure 5 shows the permeation profiles of 20%βCDE (IPM) and various formulations with 5% CP. In each vehicle, the CP permeated the skin in a steady-state fashion. The cumulative amount of CP from 20%βCDE (IPM) by 24 h after application was ca. 6.7 mg/cm² (ca. 46%: the cumulative percentage to CP in the formulation) and about 15-fold that of Aq. sol.

Fig. 5. Permeation Profiles of CP from Various Vehicles with 5 wt% CP

Each point represents the mean ± S.D. of at least three experiments.

Figures 6a and b show the flux of CP through the skin and the lag time, respectively, from various vehicles. In the case of Aq. sol., the solubility of CP in water is ca. 10%, thus 5% CP was completely dissolved in Aq. sol. However, CP is hydrophilic, so it had difficulty penetrating the skin barrier and hence the flux value was low. In the case of 20%βCD/Aq. susp., which was prepared by mixing Aq. sol. and CD, the flux was lower than that without CD. The free drug concentration decreases when the CD complex is formed by adding CD to the drug solution. Moreover, a drug with a molecular weight of 500 or less can permeate through the skin, but since the CD complex is larger than 500, it has difficulty penetrating the skin barrier. Therefore, we consider that the flux decreases due to a decrease in the free CP concentration involved in skin permeation with the addition of CD. On the other hand, the flux from vehicles with IPM as an enhancer such as IPM susp., 20%CD/IPM susp., and HCO60E was higher than that of Aq. sol. In addition, we examined the effects of oil on the flux. 20%βCDE (IPM) was found to have significantly higher skin permeability than 20%βCDE (LP) and 20%βCDE (Soy), thus IPM was confirmed to act as an enhancer.

Fig. 6. Flux and Lag Time of CP from Various Vehicles with 5 wt% CP

(a) Flux, (b) Lag time. Each point represents the mean ± S.D. of at least three experiments a) Significant difference relative to Aq sol. (p < 0.05). b) Significant difference relative to 20%βCDE (IPM) (p < 0.05).

The flux of 20%βCDE (IPM) was significantly larger compared with other formulations containing IPM, e.g., IPM susp. and HCO60E. HCO60E was emulsion containing the same amount of water and IPM, but it differ in the type of emulsifier. To improve the skin permeation of CP, we found that not only the application of an emulsion containing water and IPM, but also CD as an emulsifier, should coexist in the emulsion.

We investigated the effect of CP concentration on skin permeation of CP from 20%βCDE (IPM). Figure 7 shows the flux of 20%βCDE (IPM) systems. The flux increased dose-dependently to 7.5%, and became constant above 7.5%. On the other hand, the lag time showed no difference with CP content (data not shown). It is well known that the flux of a drug from the same vehicle correlates with drug concentration if the drug is dissolved homogenously. In our case, the flux increased with CP concentration but it was not linear. To clarify the reason for these results, we determined the concentration of CP in the water and oil phases of the CDEs. Figure 8 shows the relationship between the addition amounts of CP to 20%βCDE (IPM) and the CP concentration in the water and oil phases. The CP concentration in the water phase increased with additional amounts of CP, but that in the oil phase was consistently low. The volume ratio of the water and oil phases was 1 : 1 (Table 1). Almost all CP existed in water under 5% in CDEs, but CP precipitated over 5% in CDEs because the CP solubility in water is ca.125 mg/mL.²⁷⁾

Fig. 7. Effect of Amount of CP on Flux from 20%CDE (IPM) Each Point Represents the Mean ± S.D. of at Least Three Experiments

Fig. 8. Relationship between the Addition Amount of CP to 20%βCD (IPM) and the CP Concentration in the Water and Oil Phases

βCD make inclusion complex with CP and the stability constant (K) for the CP-βCD complex in a molar ratio of 1 : 1 is obtained from Eq. 1,

  

(1)

where [CP-βCD], [CP] and [βCD] are molarity of the CP-βCD complex, free CP and free βCD, respectively. By using total CP concentration ([CP]_tot) and total βCD concentration ([βCD]_tot), Eq. 2 is obtained from Eq. 1.

  

(2)

When we make provisional assumptions that CP forms a complex with βCD in a molar ratio of 1 : 1 in the water phase of CDEs, the presence of IPM in CDE is ignored and the density of the water phase is 1, the apparent [CP] in the water phase can be calculated from the [CP]_tot ([CP] + [CP-βCD]) in the water phase (Fig. 8), the addition amount of βCD, and the K for a 1 : 1 CP-βCD complex, which is known to be 58 ± 8 M⁻¹ at 25°C.²⁶⁾ In the case of 20%βCDE (IPM), the apparent [βCD]_tot is 0.410 M. [CP-βCD] is obtained by substituting K (58 M⁻¹), [CP] _tot (Fig. 8) and [βCD]_tot (0.410 M) into Eq. 2 and solving the quadratic equation (note, that [CD] _tot > [CP-βCD]). Finally, the apparent [CP] is obtained by subtracting [CP-βCD] from [CP]_tot. Figure 9 shows the relationship between the flux of CP from βCDE (IPM) and the apparent [CP] in the water phase. The approximate curve shows a highly linear relationship with a determination coefficient square value (R²) of 0.9775. Consequently, it became clear that the flux of CP was proportional to the free CP concentration in the water phase of CDE. This agrees with the result of CP skin permeability through excised rabbit abdominal skin by Wu et al.²⁸⁾

Fig. 9. Relationship between the Flux of CP from βCDE (IPM) and the Free CP Concentration in the Water Phase

The approximate straight line in this figure was obtained using the least squares method.

CONCLUSION

To evaluate the usefulness of emulsions prepared with CD (CDE) as a drug delivery vehicle, the stability of CDEs and in vitro skin permeability were examined using CP as a model drug. Stable βCDEs could be prepared with increasing βCD concentrations and conversely the stability decreased with increasing addition amounts of CP. Furthermore, it was found that the skin permeability of CP could be improved by using CDE containing water, IPM and βCD when compared with vehicles not containing one or more of them. Our results indicate that CDE can serve as a transdermal dosage form. Currently, we are examining further usefulness of CDE using other drugs.

Acknowledgments

Thanks are due to Ms. Eri Ishikawa and Ms. Maika Ikemura for their technical assistance in the experimental work.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) McClements DJ. Advances in fabrication of emulsions with enhanced functionality using structural design principles. Curr. Opin. Colloid Interface Sci., 17, 235–245 (2012).
• 2) Strickley RG. Solubilizing excipients in oral and injectable formulations. Pharm. Res., 21, 201–230 (2004).
• 3) Kawase T, Fujii M, Kikukawa M, Sakai Y, Watanabe S, Shimizu T. Interfaces and Surfactants. Japan Oil Chemists’ Society, 2 Ed., Tokyo, Chapter 11, pp. 265–300 (2009).
• 4) Nabipour M, Ayatollahi S, Keshavarz P. Application of different novel and newly designed commercial ionic liquids and surfactants for more oil recovery from an Iranian oil field. J. Mol. Liq., 230, 579–588 (2017).
• 5) Hall-Manning TJ, Holland GH, Rennie G, Revell P, Hines J, Barratt MD, Basketter DA. Skin irritation potential of mixed surfactant systems. Food Chem. Toxicol., 36, 233–238 (1998).
• 6) Attwood D, Florence AT. Surfactant Systems: their chemistry, pharmacy, and biology, Chapman and Hall, pp. 614–631 (1983).
• 7) Kondo T. Mechanisms of hemolysis by surface active agent. Adv. Colloid Interface Sci., 6, 139–172 (1976).
• 8) Miura K, Masuda M. Biodegradation of surfactants. J. Jpn. Oil Chem. Soc., 43, 332–339 (1994).
• 9) Kikuchi M. Aquatic toxicology and chemistry of some surfactants. J. Jpn Oil Chem. Soc., 45, 1189–1199 (1996).
• 10) Sakai T. Surfactant-free emulsions: Growth processes and stabilization of oil droplets dispersed in water. Oleoscience, 8, 539–547 (2008).
• 11) Binks BP, Lumsdon SO. Catastrophic phase inversion of water-in-oil emulsions stabilized by hydrophobic silica. Langmuir, 16, 2539–2547 (2000).
• 12) Binks BP, Lumsdon SO. Transitional phase inversion of solid-stabilized emulsions using particle mixtures. Langmuir, 16, 3748–3756 (2000).
• 13) Binks BP, Lumsdon SO. Pickering emulsions stabilized by monodisperse latex particles: Effects of particle size. Langmuir, 17, 4540–4547 (2001).
• 14) Strohm H, Löbmann P. Porous TiO₂ hollow spheres by liquid phase deposition on polystyrene latex-stabilised Pickering emulsions. J. Mater. Chem., 14, 2667–2673 (2004).
• 15) Nonomura Y, Kobayashi N. Phase inversion of the Pickering emulsions stabilized by plate-shaped clay particles. J. Colloid Interface Sci., 330, 463–466 (2009).
• 16) Binks BP, Lumsdon SO. Influence of particle wettability on the type and stability of surfactant-free emulsions. Langmuir, 16, 8622–8631 (2000).
• 17) Uekama K. Novel approach of cyclodextrin-based pharmaceutical formulation. Yakugaku Zasshi, 132, 85–105 (2012).
• 18) Hashizaki K, Kageyama T, Inoue M, Taguchi H, Ueda U, Saito Y. Study on Preparation and Formation mechanism of n-alkanol/water emulsion using α-cyclodextrin. Chem. Pharm. Bull., 55, 1620–1625 (2007).
• 19) Inoue M, Hashizaki K, Taguchi H, Saito Y. Formation and characterization of emulsions using β-cyclodextrin as an emulsifier. Chem. Pharm. Bull., 56, 668–671 (2008).
• 20) Inoue M, Hashizaki K, Taguchi H, Saito Y. Emulsion preparation using β-cyclodextrin and its derivatives acting as an emulsifier. Chem. Pharm. Bull., 56, 1335–1337 (2008).
• 21) Inoue M, Hashizaki K, Taguchi H, Saito Y. Emulsifying ability of β-cyclodextrins for common oils. J. Disp. Sci. Tech., 31, 1648–1651 (2010).
• 22) Leclercq L, Nardello-Rataj V. Pickering emulsions based on cyclodextrins: A smart solution for antifungal azole derivatives topical delivery. Eur. J. Pharm. Sci., 82, 126–137 (2016).
• 23) Mathapa BG, Paunov VN. Cyclodextrin stabilised emulsions and cyclodextrinosomes. Phys. Chem. Chem. Phys., 15, 17903–17914 (2013).
• 24) Sato K, Sugibayashi K, Morimoto Y. Effect and mode of action of aliphatic esters on the in vitro skin permeation of nicorandil. Int. J. Pharm., 43, 31–40 (1988).
• 25) Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant., 48, 452–458 (2013).
• 26) Ikeda Y, Motoune S, Matsuoka T, Arima H, Hirayama F, Uekama K. Inclusion complex formation of captopril with α- and β-cyclodextrins in aqueous solution: NMR spectroscopic and molecular dynamic studies. J. Pharm. Sci., 91, 2390–2398 (2002).
• 27) Wu P-C, Huang Y-B, Lin H-H, Tsai Y-H. In vitro percutaneous absorption of captopril through excised rabbit skin. Int. J. Phram., 143, 119–123 (1996).
• 28) Wu P-C, Huang Y-B, Fang J-Y, Tsai Y-H. In vitro percutaneous absorption of captopril. Int. J. Phram., 148, 41–46 (1997).