Naringenin (NRG, >98.0%) and mefenamic acid were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Polyvinylpyrrolidone (PVP) Kollidon 25 was provided by BASF Japan Ltd. (Tokyo, Japan). Polyglycerol fatty acid ester O-50D (PG) was obtained from Mitsubishi Chemical Foods Co., Ltd. (Tokyo, Japan). Dissolution test solutions 1 and 2 were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). β-Glucuronidase from Helix pomatia Type HP-2 (No. G7017; β-glucuronidase activity ≥ 100,000 U/mL; sulfatase activity = 7,500 U/mL) and daidzein were purchased from Sigma-Aldrich Corp. (St. Louis, MO, United States). FUJI DRI-CHEM slides for clinical biochemistry analysis were purchased from FUJIFILM Corporation (Tokyo, Japan).

All animal study protocols were approved by the Animal Care and Use Committee of the Graduate School of Pharmaceutical Sciences Osaka University, Osaka, Japan. All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (25). Extra care was taken to minimize animal suffering and the number of animals used. Male Wistar rats and male and female ICR mice were obtained from Japan SLC Inc. (Shizuoka, Japan) and acclimatized under controlled environmental conditions (22 ± 1°C; 50% ± 10 relative humidity; 12-h light-dark cycle; lights on at 8:00) for at least several days before the start of the experiment. All animals were fed a certified MF diet (Oriental Yeast Co., Ltd., Tokyo, Japan) and had ad libitum access to water.

NRG, PVP, and PG were mixed in a 1:8.5:0.5 (w/w/w) ratio. To prepare the NRG SD, the physical mixtures (PMs) were heat-kneaded with a twin-screw extruder (Technovel Co., Ltd., Osaka, Japan; NARA Machinery Co., Ltd., Tokyo, Japan) (17). The extrusion temperature range was 180–200°C and the screw speed was 100 rpm. The product was cooled in the dark at 20–25°C, pulverized in an agate mortar, and passed through a 150-μm sieve. The SD was then placed in an amber bottle and stored in a desiccator at 20–25°C.

The prepared NRG SD (500 mg; equivalent to 50 mg NRG) was added to 2.5 mL dissolution test solution 2. The mixture was shaken vigorously at 37°C for 30 min and centrifuged (15, 17). The supernatant was then passed through a 0.2-μm filter and subjected to high-performance liquid chromatography (HPLC) analysis. The NRG concentration was determined using an HPLC system fitted with the LC-20AD pump and the SPD-M20 detector (Shimadzu Corp., Kyoto, Japan) (15). A reverse-phase column (CAPCELL PAK C18 MGII; 4.6 mm × 100 mm; 5 μm; Shiseido Co., Ltd., Tokyo, Japan) was operated at 40°C. The mobile phase consisted of a methanol-water (45:55, v/v) mixture with 0.05% (v/v) phosphoric acid, and it was ultrasonically degassed before use. The flow rate was set at 1 mL/min, and the detection wavelength was set at 289 nm. The sample injection volume was 10 μL. The HPLC was calibrated using standard methanol-water (50:50, v/v) NRG solutions in the concentration range of 0.125–40 μg/mL.

The dissolution test was conducted using the paddle method described in the Japanese Pharmacopoeia (26). First, 900 mL dissolution test solution 1 or 2 was placed in the dissolution tester vessel and then 30 mg of crystalline NRG, 300 mg of PM, and 300 mg of SD were added to it. The temperature (37°C) and stirring rate (100 rpm) were maintained during the experiment. Samples were collected 2, 5, 10, 20, 30, 45, 60, 90, and 120 min after sample addition. The solutions were passed through 0.2-μm filters, and the filtrates were subjected to HPLC analysis.

Sample thermograms were obtained using a differential scanning calorimeter (ThermoPlusEVOII DSC8230; Rigaku Corp., Tokyo, Japan). Samples were weighed to 5 mg, placed in covered aluminum pans, and heated from 25 to 275°C at 20°C/min in an atmosphere containing nitrogen.

The crystalline state of the sample was analyzed via automated multipurpose X-ray diffractometry (SmartLab; Rigaku Corp.) fitted with a D/teX Ultrahigh-Speed 1D X-ray Detector (Rigaku Corp.). The X-ray source was CuKα radiation fitted with a CuKβ filter. The X-ray output was 45 kV and 200 mA. Samples were analyzed over a 5–50° diffraction angle range (2θ) at 10°/min.

Rats aged 9 weeks were fasted for 16 h and intragastrically administered 100 mg/kg body weight (BW) crystalline NRG, PM equivalent to 100 mg/kg BW NRG, or SD equivalent to 100 mg/kg BW NRG. Each solution was suspended in ion-exchange water immediately prior to administration. Blood was collected from the tail vein 0.125, 0.25, 0.5, 1, 2, 4, and 8 h after administration. Blood samples were also collected from the heart under isoflurane anesthesia 24 h after administration. The blood was stored in heparinized hematocrit tubes, and the plasma was isolated by centrifugation.

NRG SD equivalent to 200 mg/kg BW NRG was intragastrically administered to 9-week-old rats that were fasted for 16 h. The same amount of NRG SD was intragastrically administered 5 min later. Blood was collected from the heart under isoflurane anesthesia 35 min after the first administration and stored at 4°C for several hours. Serum was then isolated by centrifugation and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The serum contained 34.8 μg/mL unchanged NRG and 134.5 μg/mL NRG metabolites.

Serum containing the unchanged NRG and NRG metabolites was administered via the tail vein at a dose of 500 μL/rat. Blood was collected from the tail vein 2.5, 5, 7.5, 10, 12.5, 20, 40, and 80 min after administration and stored in heparinized hematocrit tubes. The plasma was isolated by centrifugation.

To extract the unchanged NRG, 5 μL of plasma derived from rats that were orally or intravenously administered NRG SD was mixed with 20 μL of normal rat plasma, 5 μL of 90% (v/v) acetonitrile, and 50 μL of acetonitrile containing 2 μM daidzein and 0.1 μM mefenamic acid. The mixture was centrifuged at 4°C, and 25 μL supernatant was collected and mixed with 75 μL of 30% (v/v) acetonitrile.

To prepare NRG metabolites, 6 μL of plasma derived from rats that were orally or intravenously administered NRG SD was mixed with 3 μL of 0.2 M sodium acetate buffer (pH 5.0) containing 0.2% (w/v) ascorbic acid, 1.2% (w/v) EDTA, and 10 U/μL β-glucuronidase (Type HP-2 from Helix pomatia). The samples were incubated at 37°C overnight and extracted using the same method that was employed for those containing the unchanged NRG.

LC-MS/MS analysis was performed using an ACQUITY UPLC system (Waters Corp., Milford, MA, United States) fitted with a cooling autosampler and a column oven with temperature control. Mass spectrometry was performed using a Waters Xevo TQ-S tandem quadrupole mass spectrometer (QQ-MS; Waters Corp.). Chromatographic separation was performed on an ACQUITY UPLC BEH C18 1.7 μm column (50 mm × 2.1 mm; Waters Corp.). The mobile phase A was 0.1% formic acid in water (v/v), and acetonitrile containing 0.1% formic acid was used as mobile phase B. The flow rate was set at 0.5 mL/min. The sample injection volume was 5 μL. The injected sample was eluted from the column with the following gradient program: 1.8-min linear gradient in solvent B from 2 to 98%, 0.7-min linear gradient in solvent B from 98 to 2%, and 2.5-min hold at 2% B. The sample injection volume was 5 μL. Positive electrospray ionization was used to generate the parental ions. Multiple reaction monitoring, using precursor → product ion transitions of m/z 273.04 → 152.97 and m/z 255.10 → 198.99, was used to quantify NRG and daidzein (internal standard), respectively.

Pharmacokinetic analyses after oral or intravenous NRG administration were performed using the free Napp software, which was provided by the University of Tokyo Hospital (27).

The acetic acid-induced writhing test was performed according to the method of Pinho-Ribeiro et al. (8) with modifications. Rats aged 5–7 weeks were fasted for 16 h and divided into four groups of eight animals per group. The first group was intragastrically administered 10 mL/kg saline as a control. The second group was intragastrically administered crystalline NRG at 100 mg/kg BW. The third and fourth groups were intragastrically administered NRG SD equivalent to 30 mg/kg and 100 mg/kg BW NRG, respectively. After 5 min, each rat was intraperitoneally injected with 10 mL/kg of 0.8% (v/v) acetic acid in saline solution. Visceral pain intensity was quantified by enumerating the abdominal writhing within 0–25 min after the stimulus. The writhing response was characterized by the contraction of abdominal muscles and stretching of the hind limbs.

Mice aged 6 weeks were randomly divided into two groups of 10 mice (five males and five females) per group. The experimental group was prepared by mixing NRG SD with powdered food to a concentration of 1% (w/w) NRG SD. The control group was prepared by mixing the base material (PVP:PG = 8.5:1 weight ratio) with powdered food to a concentration of 1% (w/w). After the mixed diet was administered, body weight, food, and water consumption were measured for each mouse group for 28 days. On day 28, all animals were anesthetized with chloral hydrate, and blood samples were collected from the heart and stored in heparinized tubes. Portions of these samples were utilized to evaluate hematological parameters using an XT-2000i hematology analyzer (Sysmex Corp., Hyogo, Japan). The remaining blood samples were centrifuged to obtain the plasma for which comprehensive clinical chemistry test parameters were evaluated using a DRI-CHEM 4000V veterinary chemistry analyzer (FUJIFILM Corp., Tokyo, Japan). The livers, spleens, hearts, kidneys, lungs, and brains were excised and weighed. The organ weights were divided by body weight measured on day 28.

All data are presented as mean ± standard error. Statistical analysis was performed with the Tukey-Kramer method or an unpaired t-test method using Statcel v.4.0 (OMS Publishing Inc., Saitama, Japan).

Preliminary experiments were conducted on the base materials and their mixtures with various ratios via previously reported handmade hot-melt technology (15). The preparation of SD by heat-kneading the materials at a 1:8.5:0.5 (w/w/w) NRG:PVP:PG mixing ratio substantially increased the solubility of NRG in water (data not shown). We prepared NRG SD using a twin-screw extruder by feeding a 1:8.5:0.5 (w/w/w) NRG:PVP:PG mixture into the extruder and mixing it at high temperature (17). We then compared the water solubility of NRG SD, crystalline NRG, and PM. The latter was the product formed by mixing NRG, PVP, and PG at 1:8.5:0.5 (w/w/w). The water solubility values were 70 μg/mL, 4 mg/mL, and 15 mg/mL for crystalline NRG, PM, and SD, respectively (Figure 1A). Therefore, using the twin-screw extrusion method, the synthesized NRG SD had excellent solubility.

The dissolution characteristics of the highly water-soluble NRG SD were evaluated using Japanese Pharmacopoeia-based dissolution tests (26). Dissolution test solutions 1 (pH 1.2) and 2 (pH 6.8) were used. Only 40 and 60% of the crystalline NRG was dissolved in test solutions 1 and 2, respectively, after 120 min (Figures 1B,C). Only 40 and 70% of crystalline NRG and PM were dissolved in test solution 1, respectively, after 120 min. By contrast, 100% of NRG SD was dissolved in test solution 1 after 30 min (Figure 1B). Approximately, 70% of crystalline NRG was dissolved in test solution 2 after 120 min, whereas 100% of the PM and SD were dissolved in test solution 2 after 90 min and 20 min, respectively (Figure 1C). Hence, NRG SD dissolved faster in test solutions 1 and 2 than either crystalline NRG or PM and hence, it is expected to be rapidly absorbed in the body.

The water solubility of the prepared NRG SD was superior to those of crystalline NRG and PM. However, it was unknown whether the SD was, in fact, amorphous. We investigated the crystalline state of NRG SD via differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD). The DSC thermogram of crystalline NRG showed a sharp endothermic peak near 250°C corresponding to the NRG melting point (Figure 2A). However, this peak was absent in the PM and SD thermograms. The PXRD analysis revealed strong diffraction peaks between 10° and 30° for the NRG crystals, thereby confirming their crystalline form (Figure 2B). For PM, the characteristic crystalline NRG peaks remained intact and their intensity only slightly changed. In contrast, for SD, the crystalline NRG exhibited a diffuse broad peak and lacked the intensity of the peaks observed for PM. For these reasons, NRG underwent a phase transition from the crystalline to the amorphous form during preparation of SD.

To investigate gastrointestinal NRG SD absorption, we measured the plasma levels of crystalline NRG, PM, and SD that were orally administered to rats. The area under the plasma concentration-time curve (AUC_0–∞) was 0.74 ± 0.24 μg⋅h/mL, the maximum plasma concentration (C_max) was 0.13 ± 0.01 μg/mL, and the time required to reach maximum plasma concentration (t_max) was 4.00 ± 1.26 h in the rats administered 100 mg/kg crystalline NRG (Figure 3A and Table 1). The results for the rats that were administered with SD at a dose equivalent to 100 mg/kg NRG were AUC_0–∞ = 1.99 ± 0.46 μg⋅h/mL, C_max = 1.67 ± 0.33 μg/mL, and t_max = 0.29 ± 0.04 h. The rats administered SD showed a 2.7-fold increase in AUC_0–∞, a 12.8-fold increase in C_max, and a 3.7 h decrease in t_max compared to those administered crystalline NRG. A 1.5-fold increase in AUC, a 1.8-fold increase in C_max, and a 2.4 h decrease in t_max were observed in rats administered PM at a dose equivalent to 100 mg/kg NRG. In terms of AUC_{0–1 h}, there was a 3.7-fold increase in PM and a 26.4-fold increase in SD relative to crystalline NRG. The amorphization of unchanged NRG enabled it to be rapidly absorbed in the gut. No significant differences in AUC_{0–1 h} were observed among crystalline NRG, PM, and SD in terms of plasma NRG glucuronide and sulfate conjugates. However, SD showed a 3.9-fold increase in C_max and a 1.9 h decrease in t_max compared to crystalline NRG (Figure 3B and Table 1). NRG SD demonstrated substantially improved gastrointestinal absorption, especially at a very high plasma NRG concentration immediately after oral administration.

We evaluated the pharmacokinetic parameters of NRG to characterize NRG SD. We prepared sera containing unchanged NRG and NRG metabolites, intravenously administered them to the rats, and measured their unchanged NRG and NRG metabolite levels in the plasma. The transitions in the concentrations of unchanged NRG and NRG metabolites in the plasma were biphasic (Figure 4). Hence, their pharmacokinetic parameters were evaluated using a two-compartment model (Table 2). The absolute bioavailability values (F) of crystalline NRG, PM, and SD were 0.52, 0.75, and 1.43%, respectively. Therefore, the amorphization of NRG via hot-melt extrusion technology increased its F by ∼2.7-fold. These results suggest that NRG SD may strongly exhibit the physiological functions of NRG.

We conducted an in vivo functional evaluation of NRG SD to confirm its efficacy and assessed its analgesic effect because this property is a characteristic of NRG. To this end, we applied the acetic acid writhing test (8) by intraperitoneally administering acetic acid to the rats 5 min after oral administration of crystalline NRG and NRG SD and then enumerating the writhings after 25 min of administering acetic acid. Both the crystalline NRG and the SD treatments significantly decreased the number of writhings compared to the control (Figure 5). Rats administered SD equivalent to 30 mg/kg NRG showed the same degree of writhing suppression as those administered crystalline NRG but at one-third dose. This result suggests that SD demonstrated improved functionality than crystalline NRG.

To investigate the safety of NRG SD, we examined its in vivo effects when administered to mice for 28 days. Male and female mice were fed with a powdered diet including 1% (w/w) base materials (control) or 1% (w/w) NRG SD equivalent to 0.1% (w/w) NRG. There were no significant differences between the control and NRG SD groups or males and females in terms of body weight or water consumption (Supplementary Figure 1). Food intake increased significantly by day 9 for females administered SD and decreased by day 14 for males administered SD. Nevertheless, these changes were still within the normal range (28). The weights of the livers of the female mice administered NRG SD significantly increased relative to those of the control. However, there were no significant differences between different treatment groups or sexes in terms of the weights of any other organs (Table 3). We evaluated the hematological parameters of mice using blood samples collected after 28 days of feeding. There were no significant differences between the control and NRG SD groups in terms of these factors (Table 4). Biochemical and chemical serum parameters were then measured in mouse plasma to understand the effects of SD administration for 28 days on blood chemistry (Table 5). Relative to the control, the levels of alanine aminotransferase (ALT) were reduced significantly in male mice administered NRG SD. Nevertheless, the change was still within the normal range (29). There were no significant differences in any other clinical chemistry parameters associated with the liver, kidney, heart, or other organs between different treatment groups or sexes. The results suggest that NRG SD can be considered safe, and oral ingestion of this material should cause no adverse effects.