(T1130-05-28) Toward IVIVC (In Vitro-In Vivo Correlation) for Glycosidic Compounds: Optimization Study of β-Glucosidase Addition Levels in Membrane Permeation Tests

Purpose: In recent years, there has been a growing interest in artificial membrane permeability assessment for predicting drug absorption in vitro in the pharmaceutical field. In these assessments, test solutions using biomimic fluids, such as FaSSIF and FeSSIF are commonly employed. FaSSIF and FeSSIF reflect the concentration and composition of the intestinal components (bile salts, lecithin, and fatty acids), enabling their evaluation in a manner that closely mimics biological systems. However, they do not contain enzymes; therefore, external enzyme addition is necessary when evaluating components that undergo metabolism by digestive enzymes or intestinal bacteria. Glycoside-type compounds, such as soy isoflavones, are examples of components affected by enzymes. Soy isoflavones consist of three aglycones (daidzein, genistein, and glycitein) and their glycosides. In soybeans, they primarily exist as glycosides, and when consumed by humans, they undergo hydrolysis by β-glucosidase secreted from intestinal enzymes or bacteria to form aglycones before being absorbed, metabolized, and subjected to various conjugation reactions in the enterohepatic circulation. Conversely, aglycone-type compounds did not require such metabolic processes. Therefore, using conventional evaluation methods, accurate permeability assessment is possible for aglycone-type compounds; however, for glycoside-type compounds, enzymes, such as β-glucosidase must be added externally. As the permeability assessment results vary significantly depending on the amount of enzyme added, optimizing enzyme addition is extremely important for enhancing the reliability of the evaluation system for glycoside-type compounds. In this study, we evaluated the optimal amount of enzyme for membrane permeability assessment of glycoside-type compounds, using soy isoflavone glycosides as model compounds. Specifically, we compared the membrane permeability of glycoside and aglycone compounds by gradually adjusting the amount of enzyme added to FaSSIF and compared these results with human clinical trial data obtained from our initial studies.

Methods: Preparation of Test Tablets: Soy isoflavone glycoside ingredients were used as glycoside-type compounds, and soy aglycone ingredients were used as aglycone-type compounds. Two types of tablets were prepared, with each tablet adjusted to contain 25 mg of aglycone. Membrane Permeability Evaluation Test: The test solution was prepared by adding two tablets of each type to 250 mL of FaSSIF, followed by a 3-h test conducted using BioFlux (dissolution-permeation test system). For the test solutions containing tablets with soy isoflavone glycoside ingredients, varying amounts of β-glucosidase were added in multiple stages. Sampling and filtration of the donor-side solution (FaSSIF) was conducted at specific time points (15 min, 30 min, 1 h, 2 h, and 3 h). The concentration of soy aglycone in the FaSSIF solution was quantified using HPLC. Additionally, at the conclusion of the 3-h test, the acceptor-side solution was sampled and the concentration of permeated soy aglycone was quantified using HPLC. Human Clinical Trials: An open-label, two-treatment, two-period crossover study was conducted in 16 healthy Japanese female participants. Each period included a 6-day washout interval. On the test days, blood samples were collected before to the consumption of the test food. After breakfast, the participants consumed the test food with 150 mL of water 5 minutes post-meal. Blood samples were collected at 1, 2, 4, 6, 8, 10, 12, and 24 h after test food intake. The concentration of isoflavones in blood was subsequently analyzed using LC-MS/MS.

Results: In the evaluation of membrane permeability tests, tablets containing glycoside-type compounds showed a tendency for improved membrane permeability as the amount of β-glucosidase increased, which enhanced the conversion rate to the aglycone form（Fig1）. However, it exceeding a certain enzyme concentration did not further enhance the dissolution rate or membrane permeability. Furthermore, regarding the dissolution profile, under all test conditions, the amount of dissolved aglycone increased with time during the first hour of testing, however, plateaued thereafter (Fig2). Additionally, in clinical trials on human subjects, the AUC0-24h for total blood isoflavone concentrations measured after a single intake of the test food were significantly higher in tablets containing aglycone-type compounds than in those containing glycoside-type compounds (Fig３). These results suggest that the addition of approximately 0.1 mg of β-glucosidase may lead to a favorable correlation between the membrane permeability test results of glycoside-type compounds and human clinical trial outcomes. This insight provides valuable knowledge for the future design of evaluation systems and for improving the reliability of the tests.

Conclusion: This study established a method for evaluating the permeability of glycoside compounds using β-glucosidase-supplemented FaSSIF in an artificial membrane system. Our findings emphasize the importance of optimizing the enzyme concentration to improve the reliability of the evaluation system, particularly for glycoside-type compounds. While this approach correlates well with human clinical data under optimized conditions, it does not account for enzyme activity derived from the gut microbiota, which plays a role in in vivo metabolism. Further refinement of this system is required to better simulate the human intestinal conditions, thereby enhancing the predictive accuracy of drug absorption studies.

References: Chie Tarumizu ,Satomi Takahashi, Aina Hirata ,Yoshimithu Takai , Shinji Fukuhara , Yuto Nagae, Yuri Ishii ,Nomoyuki Abe,Hideki Hoshino, Sachiyuki Teramoto,Study on The Absorption of Isoflavones after Ingestion of Soy-containing Supplements Composed of Different Ingredients, J.New Rem.＆Clin 2025; 74: 1: 3-16.

Acknowledgements: The clinical data presented in this poster were previously published in J New Rem Clin. The original study was approved by the Institutional Review Board of Medical Corporation Jikokai Shiroishi Internal Medicine Clinic on April 15, 2024. The trial was registered in the UMIN Clinical Trials Registry (UMIN ID: UMIN000054337).

Comparison of aglycone concentrations after membrane permeability. The first four graphs show data from test foods prepared using glycoside-type compounds, illustrating the results of gradually varying the amount of β-glucosidase added. The amount of β-glucosidase added is indicated at the bottom of the figure. The graph on the far right presents data from test foods prepared using aglycone-type compounds. The data points corresponding to the aglycone-type compounds are represented with dotted lines.

Comparison of the dissolution profile. Data related to glycoside-type compounds are shown with solid lines, while data for aglycone-type compounds are represented with dotted lines. For glycoside-type compounds, the plot symbols vary according to the amount of enzyme added as follows: × plot indicates no enzyme added, ■ plot represents the addition of 0.1 mg of β-glucosidase, ▲ plot corresponds to 10 mg of β-glucosidase added, and ● plot indicates the addition of 210 mg of β-glucosidase.

Blood total isoflavone AUC0-24h in a human clinical study. The bar graph on the left shows the results of a human clinical trial using test foods prepared with glycoside-type compounds. The bar graph on the right presents the results of a human clinical trial using test foods prepared with aglycone-type compounds. Both graphs represent the AUC₀–₂₄h of total blood isoflavone concentration measured after a single intake of the test food.