Bicarbonate Buffer Dissolution Test Using the Floating Lid Method: Inter-Laboratory Reproducibility of pH Maintenance

Abstract

Bicarbonate buffer (BCB) has been difficult to use in conventional dissolution tests because its pH rapidly increases as CO₂ escapes from the air–water interface. Recently, the floating lid method was introduced as a convenient method for using BCB in dissolution tests. This study aimed to confirm the inter-laboratory reproducibility of pH maintenance of BCB using the floating lid method for both paddle and flow-through cell (FTC) methods. Three pharmaceutical companies and 1 academic research institute participated in this study. A BCB solution (pH 6.5, 15 mM) was employed as the test solution. In the paddle method, the pH values of BCB rapidly increased without the floating lid. The pH change (ΔpH) at 6 h ranged from +1.66 to +1.82 (50 rpm) and +1.96 to +2.02 (100 rpm). The floating lid effectively maintained the pH values in all laboratories, with ΔpH ranging from +0.13 to +0.17 (50 rpm) and +0.21 to +0.25 (100 rpm). The standard deviation of ΔpH was within 0.05 at both 50 and 100 rpm. Similarly, in the FTC method, without the floating lid, ΔpH ranged from +1.71 to +1.77 (reservoir), +1.59 to +1.72 (FTC), and +1.73 to +1.76 (sampling tube). With the floating lid, ΔpH ranged from +0.05 to +0.10 (reservoir), +0.05 to +0.09 (FTC), and +0.26 to +0.39 (sampling tube). The standard deviation of ΔpH was within 0.05. In conclusion, the inter-laboratory reproducibility of pH maintenance of BCB using the floating lid method was confirmed for both the paddle and FTC methods.

Introduction

Dissolution tests are widely used in drug development and quality control of orally administered pharmaceuticals.¹⁾ For successful drug development, dissolution tests must accurately predict the oral absorption of a drug in humans. To improve this prediction, the conditions of dissolution tests, such as pH, buffer concentration, and buffer species, should closely resemble those of gastrointestinal (GI) fluids.^2–6) Physiologically, bicarbonate buffer (BCB) maintains the pH in the small intestine.^7–10) However, most dissolution tests employ phosphate buffer (PPB) solutions, because the pH of BCB rapidly increases as CO₂ escapes from the air–water interface in conventional dissolution tests.¹¹⁾

BCB undergoes a unique pH neutralization reaction, in which the hydration reaction from CO₂ to H₂CO₃ is much slower than the dehydration reaction.¹²⁾

  

$${\mathrm{C}\mathrm{O}}_2+{\mathrm{H}}_2\mathrm{O}\rightleftarrows {\mathrm{H}}_2\mathrm{C}{\mathrm{O}}_3\rightleftarrows \mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}+{\mathrm{H}}^{+}$$

There is a 700-fold difference in the average reaction time between the hydration and dehydration reactions (hydration reaction: 9.6 s; dehydration reaction: 0.013 s).¹²⁾ In addition, the buffer efficacies of BCB and PPB are different even when their buffer capacities at neutral pH are similar because the acid dissociation constants (pK_a) of these buffers are different (BCB: pK_a = 6.05 and 9.79; PPB: pK_a = 1.94, 6.69, and 11.61 at 37°C; ionic strength = 0.15 M).^13,14) These features cause a marked difference between BCB and PPB in pH neutralization at the surface of dissolving drug particles. Many drug products show different dissolution profiles between BCB and PPB.^15–17) In the case of enteric drugs, their dissolution profiles in BCB provide a disintegration time that correlates with clinical plasma concentration–time profiles and improves discrimination between different formulations.^18–23) In the case of immediate-release formulations of dissociable drugs, the dissolution profiles in BCB are significantly different from those in PPB.^24–26) According to bioequivalence studies, the in vitro dissolution profiles in BCB show better correlation with clinical pharmacokinetic parameters than those in PPB.^21,25–28) In the case of solid dispersion formulations using ionic polymers, the dissolution profiles in BCB differ from those in PPB.^29,30) Furthermore, the precipitation profiles of dissociable drugs in BCB are markedly different from those in PPB.^31,32)

However, BCB has been challenging to use in the dissolution tests. The CO₂ sparging method has been introduced in dissolution tests to maintain the pH.¹⁶⁾ Although this method is sophisticated, it requires special equipment and is both costly and time-consuming. In addition, CO₂ sparging causes foaming of the surfactant⁷⁾ and induces mechanical stress that may affect the dissolution and precipitation profiles.^7,31)

The floating lid method was recently developed for the convenient use of BCB in dissolution tests. It does not require expensive equipment or complex operations. This method has already been applied to the compendial paddle apparatus,^{24–27,31,33,34)} mini-scale paddle apparatus,²⁹⁾ flow-through cell (FTC) apparatus,^27,28) Wood’s apparatus,³⁰⁾ and μFlux apparatus.³⁵⁾ It can also be combined with pH shift techniques to mimic the pH changes that occur during passage through the GI tract.³⁶⁾ However, good inter-laboratory reproducibility must be confirmed to encourage the use of this method in the pharmaceutical industry and regulatory settings.

The purpose of this study was to confirm the inter-laboratory reproducibility of pH maintenance of BCB using the floating lid method for both paddle and FTC methods. Three pharmaceutical companies and 1 academic research institute participated in this study. A BCB solution (pH 6.5, 15 mM) was employed as the test solution for both the paddle and FTC methods.

Results and Discussion

Effect of BCB Concentration on pH Maintenance

Previously, Sakamoto et al. investigated pH maintenance in the 2–50 mM BCB range using an in-house polystyrene foam floating lid with the paddle dissolution test apparatus.³³⁾ Here, we first confirmed pH maintenance using a newly designed acrylic floating lid with the paddle apparatus (Fig. 1A) at 2 BCB concentrations (5 and 20 mM) before conducting an inter-laboratory comparison. In the paddle method, the pH change (ΔpH) at 6 h was +0.13 and +0.14 for 5 and 20 mM BCB, respectively (Fig. 2A). The pH gradually increased up to 24 h with both BCB concentrations. No significant difference in ΔpH was observed between the concentrations. Sakamoto et al. reported that the ΔpH after 22 h was maintained at less than +0.7.³³⁾ This result is consistent with our findings, although the BCB concentrations were different. The pH maintenance at the same BCB concentrations was also investigated using a floating lid for the reservoir tank in the FTC method (Fig. 1B). ΔpH in the reservoir at 6 h was +0.07 and +0.09 for 5 and 20 mM BCB, respectively (Fig. 2B). No significant difference in ΔpH was observed between the concentrations. The floating lid effectively maintained the pH in both the vessel and the FTC reservoir tank at 5–20 mM.

Fig. 1. Floating Lids for (A) Compendial Paddle Apparatus (Made of Acrylic, 5 mm Thick), (B) CE7 Smart FTC Apparatus, and (C) DF-7 FTC Apparatus (Made of Polypropylene, 3 mm Thick)

Fig. 2. Changes in pH (ΔpH) over Time in 5 and 20 mM BCB Observed Using the (A) Paddle Apparatus and (B) FTC Apparatus (in the Reservoir Tank) (Mean ± Standard Deviation [S.D.], N = 3)

The solubility of CO₂ in water is approximately 25 mM (1 atm, 37°C).³⁷⁾ Therefore, CO₂ was sufficiently dissolved in water within the investigated concentration range. Based on these results, 15 mM BCB (pH 6.5) was employed for the inter-laboratory reproducibility studies.

Inter-laboratory Reproducibility (Paddle Method)

For the paddle method, the inter-laboratory comparison was conducted at 50 and 100 rpm (Fig. 3). The pH value was measured up to 24 h, considering its use for sustained-release formulations. Without the floating lid, the mean ΔpH at 6 h ranged from +1.66 to +1.82 (50 rpm) and from +1.96 to +2.02 (100 rpm) in all the laboratories. With the floating lid, the mean ΔpH at 6 h ranged from +0.13 to +0.17 (50 rpm) and from +0.21 to +0.25 (100 rpm). ΔpH gradually increased over 24 h, ranging from +0.48 to +0.54 at 50 rpm and from +0.83 to +1.01 at 100 rpm. The standard deviation of ΔpH at 6 h was within 0.05 for both 50 and 100 rpm in all the laboratories.

Fig. 3. ΔpH–Time Profiles in the Paddle Apparatus (Mean ± S.D., N = 3) at (A) 50 rpm and (B) 100 rpm

The data without the floating lid show the mean ± S.D. of 4 laboratories (N = 3 at each laboratory).

Although the manufacturer and model number of the paddle apparatus differed between laboratories (Table 1), the pH maintenance of BCB tested with the floating lid was reproducible between laboratories. As the inner diameters of the compendial vessels were standardized, the surface areas of the solutions were identical. The same manufacturer provided the floating lid to all laboratories. The reduced inter-laboratory variation in ΔpH may be attributed to these factors.

Table 1. Dissolution Test Apparatuses

Laboratory	Paddle dissolution apparatus	FTC dissolution apparatus
A	708-DS (Agilent Technologies, Inc., Santa Clara, CA, U.S.A.)	CE7 Smart (SOTAX AG, Aesch, Switzerland)
B	NTR-6200ACT (Toyama Sangyo Co., Ltd., Osaka, Japan)	DF-7 (Dainippon Seiki Co., Ltd., Kyoto, Japan)
C	NTR-6200A (Toyama Sangyo Co., Ltd.)	CE7 Smart (SOTAX AG)
D	NTR-8600AS (Toyama Sangyo Co., Ltd.)	DF-7 (Dainippon Seiki Co., Ltd.)

As the floating lid did not completely seal the surface, the pH increased slightly over time. However, this gradual increase in pH is also observed along the GI tract.³⁸⁾ Therefore, the floating lid method may be useful for testing the dissolution of sustained-release formulations. ΔpH increased when the paddle rotation speed was increased. Increasing the paddle rotation speed may facilitate the generation of microbubbles, thereby enlarging the gas–liquid interfacial area and enhancing CO₂ exchange with the atmosphere, eventually resulting in a greater pH increase of the BCB. However, it has been suggested that the agitation strength in the human GI tract corresponds to 50 rpm or less in the paddle apparatus.³⁹⁾

Inter-laboratory Reproducibility (FTC Method)

In the FTC method, the pH in the reservoir tank, FTC, and sampling tube of the FTC apparatus was measured (Fig. 4). Without the floating lid, ΔpH at 6 h ranged from +1.71 to +1.77 (reservoir tank), +1.59 to +1.72 (FTC), and +1.73 to +1.76 (sampling tube) in laboratories A, B, and D (N = 1 at each laboratory). With the floating lid, ΔpH at 6 h ranged from +0.05 to +0.10 (reservoir tank), +0.05 to +0.09 (FTC), and +0.26 to +0.39 (sampling tube) in all 4 laboratories (N = 3 at each laboratory). Although a slightly higher ΔpH was observed in the sampling tube, the increase in pH was suppressed in all 3 positions. The standard deviation of ΔpH at 6 h was within 0.05 in all 3 positions.

Fig. 4. ΔpH–Time Profiles in the FTC Apparatus, Specifically in the (A) Reservoir Tank, (B) FTC, and (C) Sampling Tube (Mean ± S.D., N = 3 at Each Laboratory)

The data without the floating lid show the mean ± S.D. of laboratories A, B, and D (N = 1 at each laboratory).

The increase in pH was suppressed more effectively than in the paddle apparatus because the test solution was not continuously stirred in the reservoir tank after the FTC experiments were initiated. The pH was slightly higher in the sampling tube than in the reservoir tank and FTC. To investigate the reason for this difference, ΔpH was measured before and after adding a BCB solution to the sampling tube using a peristaltic pump. It was confirmed that the ΔpH increased when the BCB was dropped from the delivery tube into the sampling tube (data not shown). The increase in pH within the sampling tube is potentially attributable to the increased surface area resulting from droplet formation during sample recovery from the delivery tube. Despite this increase in pH, the pH of the supplied BCB was maintained, indicating that the BCB within the sampling tube showed a constant ΔpH over time.

Effect of Bile Micelles on pH Maintenance in the Paddle Apparatus

The pH of 15 mM bicarbonate-based fasted state simulated intestinal fluid (BCB-FaSSIF) was measured for 24 h using the compendial paddle apparatus (Fig. 3A). The ΔpH at 6 h was +0.15 or less, showing no difference compared to 15 mM BCB without bile micelles. No foaming was observed with BCB-FaSSIF.

The solubility and dissolution rate of poorly soluble drugs are enhanced by bile micelles. Galia et al. proposed the use of FaSSIF, which mimics the intestinal solution in the fasting state, to investigate the dissolution profile of poorly soluble drugs.²⁾ Previously, PPB or maleate buffer had been used for FaSSIF. The CO₂ sparging method is difficult to apply to FaSSIF because it induces foaming.⁷⁾ Recently, Sakamoto et al. used BCB with FaSSIF in the mini-paddle apparatus with an in-house floating lid.³⁴⁾ In the present study, foaming was not observed when using the compendial paddle apparatus with a commercial floating lid, suggesting that it enables the use of BCB with FaSSIF.

Practical Use of the Floating Lid Method in the Drug Industry

For routine use in the drug industry, a test should be inexpensive, simple, robust, and highly efficient. The floating lid method does not require any special equipment or reagents. This test can be performed by placing a floating lid on the test solution in a vessel or reservoir tank. This is convenient in practice, requiring minimal additional operation compared to conventional dissolution test solutions. The manufacturers and grades of the reagents used in the preparation of BCB were not specified in this study. Similarly, the type of water used (ultrapure, pure, or distilled) was not specified. Heat and vacuum/sonic degassing was performed before adding NaHCO₃ and HCl. These features render floating lids suitable for routine use in the drug industry.

The PPB concentration may possibly be adjusted to match the dissolution profiles of each drug formulation in PPB and BCB.^15,40,41) However, the optimal PPB concentration varies for each formulation. Therefore, finding a PPB concentration that is universally applicable for formulation development is difficult. Because the floating lid method is simple, easy, and robust, BCB can be considered the 1st choice as a dissolution test medium for more efficient formulation development.

Limitations

In this study, we focused only on the change in pH incurred by BCB because the reproducibility of the dissolution profile is affected by other factors, such as the selection of a standard formulation, sample pretreatment methods, and quantification conditions. The inter-laboratory reproducibility of dissolution profiles should be investigated in future studies.

Conclusion

In conclusion, the pH maintenance of BCB using the floating lid method was reproducible among the laboratories for both the paddle and FTC methods. Owing to the difficulties in using BCB, the dissolution behavior of many drug substances and products in BCB is yet to be determined. The results of this study will facilitate the successful use of BCB in both industrial and academic research.

Experimental

Materials

Laboratories A, B, and D purchased NaHCO₃ and NaCl from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), while laboratory C purchased NaHCO₃ from Nacalai Tesque, Inc. (Kyoto, Japan) and NaCl from Kanto Chemical Co., Inc. (Tokyo, Japan).

HCl (6 N) was purchased from FUJIFILM Wako Pure Chemical Corporation (laboratories A and B), Kanto Chemical Co., Inc. (laboratory C), and Nacalai Tesque, Inc. (laboratory D).

Ultrapure water, pure water, or distilled water was prepared at each laboratory (Table 2). Sodium taurocholate and egg yolk lecithin were purchased from Nacalai Tesque, Inc. A 5-mm-thick acrylic floating lid for the paddle apparatus (Fig. 1A) was purchased from PHYSIO MCKINA Co., Ltd. (Tokyo, Japan). The 3-mm-thick polypropylene floating lid for the FTC apparatus (Figs. 1B, 1C) was provided by PHYSIO MCKINA Co., Ltd. The adapter for the pH meter used with the FTC dissolution test apparatus (DF-7) was provided by Dainippon Seiki Co., Ltd. (Tokyo, Japan). The reservoir tank (20 L, SUS304) was purchased from MONOVATE Co., Ltd. (Tokyo, Japan). The paddle and FTC dissolution test apparatuses are listed in Table 1. Portable pH meters (laboratory A: S-20, Mettler Toledo, Greifensee, Switzerland; laboratories B and C: LAQUA F-72, HORIBA, Ltd., Kyoto, Japan; laboratory D: LAQUA D-74, HORIBA, Ltd.) were used.

Table 2. Degassing Methods

Laboratory	Water	Degassing method
A	Ultrapure water	Vacuum with sonication (room temperature, 15 min)
B	Distilled water	Heating (45°C, 2 h)
C	Distilled water	Heating (45°C, 2 h)
D	Pure water	Heating (45°C, 2 h)

Methods

Effect of Bicarbonate Concentration on pH Maintenance

In this experiment, 5 and 20 mM BCB solutions were prepared as previously reported for the paddle apparatus.³³⁾ Briefly, a NaHCO₃ solution containing NaCl (490 mL) was added to a 1 L vessel. The surface of the solution was covered with a floating lid in the paddle method (Fig. 1A). The solution was then heated to 37°C. HCl solution (0.085 or 0.33 N, 10 mL) was added to obtain the BCB solution (pH 6.5, 5 or 20 mM, ionic strength = 0.14 M, adjusted by NaCl). The paddle speed was set at 50 rpm. The pH was measured at 0, 1, 2, 4, 6, and 24 h.

When performing the FTC method, BCB was prepared by adding NaHCO₃ and HCl solutions containing NaCl to the reservoir, as described in section Inter-laboratory Reproducibility Study (FTC Method). For the FTC method, the surface of the solution in the reservoir was covered with a floating lid (Fig. 1B). The flow rate was set at 4 mL/min, and the mixture was maintained at 37°C. The test solution in the reservoir was mixed with a stirrer to ensure that the initial pH was 6.5 ± 0.1. The pH was measured at 0, 0.5, 1, 2, 4, and 6 h.

Inter-Laboratory Reproducibility Study (Paddle Method)

The following procedure was used for inter-laboratory comparisons of BCB pH maintenance using the paddle method. The temperature of the dissolution apparatus was set to 37°C. A 46.8 mM HCl solution was prepared and degassed. A 16.7 mM NaHCO₃ solution was prepared by dissolving NaHCO₃ in a degassed 138.9 mM NaCl solution. The prepared solutions were then stored in airtight containers until further use. The NaHCO₃ solution (450 mL) and HCl solution (50 mL) were added to the vessel. A floating lid was immediately placed on the surface of the solution. The paddle was rotated at 50 or 100 rpm. The hole in the floating lid was sealed except when the pH was measured, at 0, 1, 2, 4, 6, and 24 h.

ΔpH was defined as the change in pH from the initial pH (0 h). The pH values without the floating lid were measured for comparison. These experiments were performed in triplicate.

Inter-Laboratory Reproducibility Study (FTC Method)

The following procedure was used for inter-laboratory comparisons of BCB pH maintenance using the FTC method. A 46.8 mM HCl solution (1.4 L) and water (11.34 L) were degassed and added to the reservoir tank. Then, a 167 mM NaHCO₃ solution was prepared by dissolving NaHCO₃ in a degassed 1.39 M NaCl solution, and 1.26 L of this solution was added to the reservoir tank. The floating lid (Figs. 1B, 1C) was immediately placed on the surface of the solution, with the inlet nozzle passed through the hole in the lid. The solution in the reservoir tank was stirred using a magnetic stirrer before the FTC experiments. The pH of the solution in the reservoir tank was confirmed to be within 6.5 ± 0.1. Ruby beads with diameters of 5 mm were placed at the bottom of the FTC. The flow rate was set to 4 mL/min, and the test solutions were maintained at 37°C. The flow path was equilibrated with the test solution. The pH of the solutions in the reservoir tank, FTC, and sampling tube was measured at 0, 0.5, 1, 2, 4, and 6 h.

In the FTC method, ΔpH was defined as the pH change from the initial pH in the reservoir tank. The pH change without the floating lid was measured for comparison (N = 1 in each laboratory). The experiments were performed in triplicate.

Effect of Bile Micelles on BCB pH Maintenance in the Paddle Apparatus

BCB-FaSSIF was prepared by adding sodium taurocholic acid and lecithin (final concentrations: taurocholic acid = 3.0 mM, egg lecithin = 0.75 mM) to BCB prepared as described in section Inter-laboratory Reproducibility Study (Paddle Method). The pH change was measured at 0, 1, 2, 4, 6, and 24 h using the paddle apparatus at 50 rpm. The experiments were performed in triplicate.

Acknowledgments

We thank PHYSIO MCKINA Co., Ltd. for their generous support in providing the floating lid for the FTC used in this research.

Conflict of Interest

Kiyohiko Sugano is a consultant for Towa Pharmaceutical Co., Ltd. and Nipro Corporation.

References

• 1)  Dressman J. B., Amidon G. L., Reppas C., Shah V. P., Pharm. Res., 15, 11–22 (1998).
• 2)  Galia E., Nicolaides E., Hörter D., Löbenberg R., Reppas C., Dressman J. B., Pharm. Res., 15, 698–705 (1998).
• 3)  Jantratid E., Janssen N., Reppas C., Dressman J. B., Pharm. Res., 25, 1663–1676 (2008).
• 4)  Cristofoletti R., Dressman J. B., Eur. J. Pharm. Biopharm., 105, 134–140 (2016).
• 5)  Fuchs A., Leigh M., Kloefer B., Dressman J. B., Eur. J. Pharm. Biopharm., 94, 229–240 (2015).
• 6)  Ramtoola Z., Corrigan O. I., Drug Dev. Ind. Pharm., 15, 2359–2374 (1989).
• 7)  Boni J. E., Brickl R. S., Dressman J., J. Pharm. Pharmacol., 59, 1375–1382 (2007).
• 8)  Litou C., Psachoulias D., Vertzoni M., Dressman J., Reppas C., Pharm. Res., 37, 42 (2020).
• 9)  Mudie D. M., Amidon G. L., Amidon G. E., Mol. Pharm., 7, 1388–1405 (2010).
• 10)  Amaral Silva D., Al-Gousous J., Davies N. M., Bou Chacra N., Webster G. K., Lipka E., Amidon G., Löbenberg R., Eur. J. Pharm. Biopharm., 142, 8–19 (2019).
• 11)  Mudie D. M., Samiei N., Marshall D. J., Amidon G. E., Bergström C. A. S., AAPS J., 22, 34 (2020).
• 12)  Al-Gousous J., Salehi N., Amidon G. E., Ziff R. M., Langguth P., Amidon G. L., Mol. Pharm., 16, 2626–2635 (2019).
• 13)  Tarumi Y., Sugano K., J. Pharm. Sci., 114, 477–485 (2025).
• 14)  Avdeef A., “Absorption and drug development: Solubility, permeability, and charge state,” John Wiley & Sons, Hoboken, NJ, 2012.
• 15)  Krieg B. J., Taghavi S. M., Amidon G. L., Amidon G. E., J. Pharm. Sci., 104, 2894–2904 (2015).
• 16)  McNamara D. P., Whitney K. M., Goss S. L., Pharm. Res., 20, 1641–1646 (2003).
• 17)  Sheng J. J., McNamara D. P., Amidon G. L., Mol. Pharm., 6, 29–39 (2009).
• 18)  Liu F., Merchant H. A., Kulkarni R. P., Alkademi M., Basit A. W., Eur. J. Pharm. Biopharm., 78, 151–157 (2011).
• 19)  Shibata H., Yoshida H., Izutsu K.-I., Goda Y., J. Pharm. Pharmacol., 68, 467–474 (2016).
• 20)  Al-Gousous J., Ruan H., Blechar J. A., Sun K. X., Salehi N., Langguth P., Job N. M., Lipka E., Loebenberg R., Bermejo M., Amidon G. E., Amidon G. L., Eur. J. Pharm. Biopharm., 139, 47–58 (2019).
• 21)  Amaral Silva D., Gomes Davanço M., Davies N. M., Krämer J., de Oliveira Carvalho P., Löbenberg R., Int. J. Pharm., 605, 120857 (2021).
• 22)  Amaral Silva D., Davies N. M., Doschak M. R., Al-Gousous J., Bou-Chacra N., Löbenberg R., J. Control. Release., 325, 323–334 (2020).
• 23)  Fadda H. M., Basit A. W., J. Drug Deliv. Sci. Technol., 15, 273–279 (2005).
• 24)  Okamoto N., Higashino M., Yamamoto H., Sugano K., Pharm. Res., 41, 959–966 (2024).
• 25)  Higashino M., Sugano K., Chem. Pharm. Bull., 72, 298–302 (2024).
• 26)  Higashino M., Sugano K., Chem. Pharm. Bull., 72, 989–995 (2024).
• 27)  Ikuta S., Nakagawa H., Kai T., Sugano K., Eur. J. Pharm. Sci., 180, 106326 (2023).
• 28)  Ikuta S., Nakagawa H., Kai T., Sugano K., Eur. J. Pharm. Sci., 192, 106622 (2024).
• 29)  Sakamoto A., Sugano K., Pharm. Res., 38, 2119–2127 (2021).
• 30)  Bapat P., Schwabe R., Paul S., Tseng Y.-C., Bergman C., Taylor L. S., J. Pharm. Sci., 114, 185–198 (2025).
• 31)  Yamamoto H., Sugano K., Mol. Pharm., 21, 2854–2864 (2024).
• 32)  Jede C., Wagner C., Kubas H., Weigandt M., Weber C., Lecomte M., Badolo L., Koziolek M., Weitschies W., Mol. Pharm., 16, 3938–3947 (2019).
• 33)  Sakamoto A., Izutsu K.-I., Yoshida H., Abe Y., Inoue D., Sugano K., J. Drug Deliv. Sci. Technol., 63, 102447 (2021).
• 34)  Sakamoto A., Sugano K., Pharm. Res., 40, 989–998 (2023).
• 35)  Ishida S., Lee S., Sinko B., Box K., Sugano K., ADMET DMPK, 13, 2603 (2025).
• 36)  Matsui F., Sakamoto A., Sugano K., J. Drug Deliv. Sci. Technol., 83, 104438 (2023).
• 37)  Krieg B. J., Taghavi S. M., Amidon G. L., Amidon G. E., J. Pharm. Sci., 103, 3473–3490 (2014).
• 38)  Aburub A., Fischer M., Camilleri M., Semler J. R., Fadda H. M., Int. J. Pharm., 544, 158–164 (2018).
• 39)  Katori N., Aoyagi N., Terao T., Pharm. Res., 12, 237–243 (1995).
• 40)  Krollik K., Lehmann A., Wagner C., Kaidas J., Kubas H., Weitschies W., Eur. J. Pharm. Biopharm., 171, 90–101 (2022).
• 41)  Matsui K., Nakamichi K., Nakatani M., Yoshida H., Yamashita S., Yokota S., Int. J. Pharm., 631, 122531 (2023).