2021 年 69 巻 9 号 p. 862-871
The aim of the present study was to determine whether solid dispersions (SDs) are applicable to gummi formulations. Amenamevir was selected as a model of a poorly water-soluble drug, and polyvinyl alcohols (PVAs) with various degrees of hydrolysis (PVA 66, PVA 80, PVA 88, and PVA 66/88) were used as SD carriers. Design of experiments (DOE) was used to develop a gummi formulation that was suitable for an amenamevir SD using SD with PVA 66. Dissolution studies and clinical sensory tests on 11 formulations calculated by DOE revealed that a gummi formulation comprising 10.5% gelatin and 22.8% water was suitable for SD of the drug. Gummi formulations comprising amenamevir SDs with various PVAs were prepared using the determined gummi formulation, and their ability to dissolve amenamevir, their stability, and their oral absorption in dogs were evaluated. The results suggested that PVA 66, PVA 66/88, and PVA 80 were appropriate in terms of dissolution, stability, and in vivo absorption, respectively. Considering these results comprehensively, it was concluded that PVA 80, which enabled the highest degree of absorption, was the most suitable SD carrier for gummi formulations. Thus, it was possible to apply a PVA SD of amenamevir to gummi formulations.
An increase in the number of poorly water-soluble drugs is one of trends in recent drug candidate compound.1,2) Low solubility in the gastrointestinal tract can lead to low in vivo absorption, so technologies that improve solubility often effectively enhance the in vivo absorption of drugs.3–6) Solid dispersion (SD) is a solubilizing technology that has been commonly used in the development of solid oral formulations.7) An SD is a formulation in which a drug is dispersed in a carrier such as a polymer; the drug generally exists in an amorphous state. Selecting an appropriate carrier for the drug is key to utilizing SD as a solubilizing technology.3)
We focused on polyvinyl alcohol (PVA) as the carrier for SD to improve the bioavailability of poorly water-soluble drugs. PVA is a non-ionic, pH-independent polymer. It can be expected to solubilize poorly water-soluble drugs in the whole gastrointestinal environment and achieve high bioavailability.8) There are many grades of PVA with properties that are determined by two parameters: the degree of hydrolysis (the ratio of hydroxyl to acetyl groups) and the degree of polymerization. Because both parameters affect the water solubility of this polymer, different PVAs have different properties.9,10) In a previous study, we evaluated the ability of various PVAs with different degrees of hydrolysis and polymerization to solubilize poorly water-soluble drugs, and found that the optimal PVA had a degree of hydrolysis of approximately 66% and a low degree of polymerization.11)
Many solid formulation products such as tablets and capsules containing SDs have been developed.12) By contrast, as an improvement of medication adherence is also taken into consideration in recent drug therapies,13) many patient-oriented dosage forms have been developed. These include orally disintegrating tablets,14,15) mini-tablets,16) film formulations,17) jelly formulations,18) and gummi formulation.19,20)
Among these dosage forms, gummi formulations have been attractive ones for children and older adults, who sometimes have difficulty in swallowing; such formulations are easy to ingest by chewing, without the need for water to aid swallowing. Gummi is a dried jelly prepared by cooling a mixture of gelatin and a concentrated syrup of sugar, such as starch syrup, and gummi formulations are made by adding a drug to the gummi.21) However, to the best of our knowledge, research on the application of SD to water-rich dosage forms such as gummi formulations has not yet been reported. It is generally considered difficult to employ an amorphous drug in SD to water-rich environment, owing to its physical instability. However, the development of gummi formulations would increase the number of options for designing dosage forms of poorly water-soluble drugs, which would improve medication adherence.
In the present study, we investigated the possibility of applying SD to gummi formulations using PVA as the SD carrier. When PVA forms a film, it has lower moisture permeability than other water-soluble polymers such as hydroxypropylmethylcellulose (HPMC).22) Therefore, the drug in an SD with PVA as the carrier was expected to be less susceptible to water in gummi formulations. Amenamevir (Fig. 1) was used as a model of a poorly water-soluble drug.23) A gummi formulation suitable for an amenamevir SD was first examined by dissolution studies and clinical sensory tests using formulations calculated by design of experiments (DOE). Gummi formulations comprising amenamevir SDs with various PVAs were prepared using the determined gummi formulation, and their dissolution, their stability, and their oral absorption in dogs were evaluated.
The notation “PVA X” indicates a polyvinyl alcohol with a degree of hydrolysis of X mol%. PVA 66 (POVAL JMR-10M) was kindly provided by JAPAN VAM & POVAL Co., Ltd. (Osaka, Japan). PVA 80 (GOHSENOL™ KL-05) and PVA 88 (GOHSENOL™ EG-05P) were kindly provided by Mitsubishi Chemical Corporation (Tokyo, Japan). HPMC (TC-5® E) was purchased from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Amenamevir and ASP2986 were provided by Astellas Pharma Inc. (Tokyo, Japan). Mannitol, pentagastrin, gelatin (AP-50), and hydrogenated maltose starch syrup (AMALTY® syrup) were purchased from Roquette Frères (Lestrem, France), Sigma-Aldrich (St. Louis, MO, U.S.A.), Nippi, Inc. (Tokyo, Japan), and Mitsubishi Shoji Foodtech Co., Ltd. (Tokyo, Japan), respectively. The D-sorbitol solution (75%) was of Japanese Pharmacopoeia grade. All other reagents were of analytical grade.
Preparation of the SDsThe amenamevir SDs were prepared by ball milling. Amenamevir and each of the carriers (PVA 66, PVA 80, PVA 88, PVA 66/88, and HPMC) were co-milled in a drug:carrier ratio of 1 : 3. PVA 66/88 was prepared in a PVA 66 : PVA 88 ratio of 1 : 2. Ball milling was performed for 24 h using an LA-PO planetary ball mill (Ito Seisakusho, Tokyo, Japan) with agate balls at a rotation speed of 200 rpm. The milling scale was 3 g/container.
Preparation of the Physical Mixture (PM)Amenamevir and PVA 66 were mixed in a 1 : 3 ratio using a mortar and pestle to prepare PVA 66 PM, which had the same composition as the amenamevir SD.
Development of Gummi Formulation by DOEDOEGummi formulations and experimental numbers were determined by DOE using JMP version 10 (SAS Institute Japan Ltd., Tokyo, Japan). An extreme vertices design in the JMP software was used as the mixture design of this DOE. According to previous findings,19,20) the ranges of the gummi components were as follows: 5–14% gelatin, 15–35% water, and 51–80% of a mixture comprising hydrogenated maltose starch syrup and D-sorbitol solution (in a 61 : 39 w/w ratio). The water content was at least 1.5-fold higher than the gelatin content. The calculated compositions of the gummi formulations are listed in Table 1.
| Component (%) | Formulation No. | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | |
| X1 | 5.0 | 5.0 | 5.0 | 7.5 | 9.5 | 9.6 | 10.0 | 12.0 | 14.0 | 14.0 | 14.0 |
| X2 | 15.0 | 25.0 | 35.0 | 15.0 | 35.0 | 24.2 | 15.0 | 18.0 | 21.0 | 28.0 | 35.0 |
| X3 | 80.0 | 70.0 | 60.0 | 77.5 | 55.5 | 66.2 | 75.0 | 70.0 | 65.0 | 58.0 | 51.0 |
X1 = gelatin; X2 = water; and X3 = a mixture of hydrogenated maltose starch syrup and D-sorbitol solution.
Hydrogenated maltose starch syrup and the D-sorbitol solution were mixed and heated (up to 135 °C) to concentrate the solution. Separately, gelatin was dissolved in purified water by heating at approximately 60 °C. The solutions were then mixed and kept at 70 °C. During this process, purified water was added to the mixed solution to adjust the water content of the gummi. Subsequently, 3.4 g of the mixed solution was injected into a plastic mold (Fig. 2) using a syringe, and 100 mg of amenamevir SD with PVA 66 (containing 25 mg of amenamevir) was immediately added and mixed. Finally, the gummi formulation was formed by overnight cooling at room temperature.
The dissolution profiles of the gummi formulations were evaluated using United States Pharmacopeia (USP) Apparatus 2 (paddle) in 900 mL of purified water at 37 °C. The paddle rotation speed was 50 rpm. At 10, 20, 30, 45, 60, 120, and 180 min, a sample was withdrawn and filtered through a TORAST Disc Syringe Filter (hydrophilic mixed cellulose ester, diameter: 25 mm, pore size: 0.45 µm; Shimadzu GLC Ltd., Tokyo, Japan). The filtrate was diluted 10-fold with acetonitrile and purified water (2/1, v/v). The dissolved amenamevir was quantitatively analyzed by HPLC (CMB-20 A and SPD-M20A; Shimadzu Corporation, Kyoto, Japan) using a YMC-Pack Pro C18 column (4.6 × 150 mm, 5 µm; YMC Co., Ltd., Kyoto, Japan), which was maintained at 40 °C. The mobile phase consisted of acetonitrile and 5 mM ammonium acetate (65/35, v/v). The flow rate was 0.5 mL/min, and the detection wavelength was 273 nm. The maximum dissolution values of the gummi formulations were determined during 3-h dissolution tests.
To evaluate the preliminary stability of the gummi formulations in terms of the dissolution performance of the amenamevir, the gummi formulations formed in the plastic mold were covered with a film of polyvinylidene chloride and stored in an aluminum-laminated pouch at 25 °C for 1 week. After storage, dissolution tests were conducted using the method mentioned above. The change ratios in the maximum dissolution of the drug after storage for 1 week were calculated by comparison with the maximum dissolution before storage.
Clinical Sensory TestsClinical sensory tests were carried out on 10 healthy volunteers (6 females and 4 males; mean age ± standard deviation (S.D.), 22.5 ± 1.7 years) who participated after providing written informed consent. The tests were conducted in accordance with the Declaration of Helsinki and its amendments. The study protocol was approved by the Ethics Committee of the University of Shizuoka, Japan (approved letter number 1–29). Each sensory test was conducted by evaluation using a visual analogue scale (VAS). Each VAS score was obtained by placing a mark along a 100-mm line. The volunteers were asked to provide a score based on sense of touch (0: very bad, 100: very good), stickiness (0: not sticky at all, 100: very sticky), and hardness (0: not hard at all, 100: very hard) after handling a gummi formulation.
Data AnalysisThe predicted values of the five evaluation items—i.e., the maximum value of dissolution, the change ratio of dissolution after storage for 1 week, and the VAS scores for sense of touch, stickiness, and hardness—were calculated from observed values by a least-squares method using JMP version 10 to draw the diagrams. The criteria were as follows: maximum value of dissolution, ≥85%; change ratio of dissolution after storage for 1 week, >−5%; VAS score for sense of touch, >50; and VAS score for stickiness, <60. The VAS score for hardness was set at 50–65 because gummi formulations with that score range can be ingested preferably, according to the relationship among the VAS score, the penetrated distance into the gummi by a penetrometer, and human preference.20) Finally, the area in which all the criteria were met was determined using JMP version 10.
Measurements of Pharmaceutical Properties of Gummi FormulationsPreparation of Gummi FormulationsGummi formulations with various amenamevir SDs (comprising PVA 66, PVA 80, PVA 88, PVA 66/88, and HPMC) or PVA 66 PM were prepared using the gummi formulation determined in the DOE study, as described above.
Dissolution StudiesThe dissolution profiles of the gummi formulations were evaluated using USP Apparatus 2 with a paddle revolution rate of 50 rpm. During the dissolution tests, the gummi formulations were divided into four pieces to simulate patients chewing the gummi drug and dividing it into several pieces within the mouth. The dissolution media were purified water, USP simulated gastric fluid without pepsin (SGF, pH 1.2), and phosphate buffer (pH 6.8). The maximum values of dissolution of each formulation were determined during 1-h dissolution tests. The other test conditions were the same as those described above.
AssayEach gummi sample was dissolved with 100 mL of acetonitrile and purified water (2/1, v/v) by warming at 60 °C. The dissolved solution was filtered through a TORAST Disc Syringe Filter (hydrophilic mixed cellulose ester, diameter: 25 mm, pore size: 0.45 µm). The filtrate was diluted 100-fold with acetonitrile and purified water (2/1, v/v). The dissolved amenamevir was quantitatively analyzed by HPLC using the YMC-Pack Pro C18 column. The mobile phase consisted of acetonitrile and 5 mM ammonium acetate (65/35, v/v). The flow rate was 0.5 mL/min, and the detection wavelength was 273 nm.
Stability StudiesThe gummi formulations formed in the plastic mold were covered with a film of polyvinylidene chloride and stored in an aluminum-laminated pouch at 4 °C for 1 month and 30 °C for 1 month. After storage, the dissolution test in purified water and the amenamevir assay were conducted.
Oral Absorption of Amenamevir in DogsThe present study was approved by the Institutional Animal Care and Use Committee of Astellas Pharma Inc., Yaizu Pharmaceutical Research Center, which is accredited by AAALAC International.
Five male beagle dogs aged 12–41 months and weighing 10.1–12.4 kg were enrolled in the present study. The dogs were fasted overnight and water was withheld for 0.5 h prior to administration of the gummi formulations. Pentagastrin was injected intramuscularly at a dose of 0.015 mg/kg at 0.5 h before, and at 0.5 and 1.5 h after oral administration of the formulations to ensure an acidic gastric pH similar to the stomach pH in fasted humans.24) Amenamevir (100 mg/body), which was in four gummi formulations divided into four pieces (i.e., 16 units in total), was orally administered with 50 mL of water. Suspensions of the corresponding amenamevir SDs and PVA 66 PM—wherein each dosing powder was mixed with the same amount of mannitol and suspended in 20 mL of water—were also orally administered to the dogs. The suspensions were administered via an oral catheter, and 30 mL of water was additionally administered to flush out the dosing syringe and catheter. Access to water was withheld for 2 h after administration of the formulations, after which the dogs were given free access to water. The dogs were fed 8 h after dosing. Blood samples (2.5 mL) were collected from the forelimb vein before dosing, and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, and 24 h after dosing. Immediately after blood sampling, plasma was isolated from the whole blood by centrifugation at 1811 × g at 4 °C for 15 min and stored at −20 °C until measurement of the plasma concentration of the drug.
Plasma Concentration MeasurementThe plasma concentration of amenamevir was quantitatively measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), consisting of an HPLC (LC-20AD and SIL 20AC, Shimadzu Corporation) and a mass spectrometer (TSQ Quantum Ultra, Thermo Fischer Scientific, Waltham, MA, U.S.A.). A plasma sample (50 µL) was added to 150 µL of acetonitrile containing an internal standard (IS; ASP2986). The mixture was centrifuged at 16000 × g for 5 min, and the supernatant was used for the subsequent analysis. A TSKgel ODS-100Z 5 µm analytical column (2-mm i.d. ×5 cm; Tosoh Corporation, Tokyo, Japan) was used at 40 °C. The mobile phase consisted of 20 mM ammonium formate and acetonitrile (50/50, v/v), and the flow rate was 0.2 mL/min. The product ions with mass-to-charge ratios (m/z) of 322.1 (amenamevir) and 308.1 (IS) produced from parent ions with m/z of 481.1 and 468.1, respectively, were detected in the selected reaction monitoring mode using electrospray ionization (positive). The lower limit of quantification was 10 ng/mL under these analytical conditions.
Pharmacokinetic and Statistical AnalysesThe maximum plasma concentration of amenamevir (Cmax), the time to reach Cmax (Tmax), and the area under the plasma concentration–time curve (AUC) from 0 to 24 h (AUC0–24 h) were calculated from each plasma concentration–time profile by non-compartmental analysis using Phoenix® WinNonlin® version 7.0 (Certara, L.P., Princeton, NJ, U.S.A.). These pharmacokinetic parameters were evaluated by one-way ANOVA with Tukey’s post-tests for multiple comparisons, in which a p-value <0.05 was considered statistically significant, using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, U.S.A.). The geometric mean ratio (GMR) and 90% confidence interval of Cmax and AUC0–24 h after administration of each gummi formulation were calculated by comparison with those of the suspensions.
To develop a gummi formulation suitable for amenamevir SD, 11 formulations (Table 1) calculated by DOE were evaluated. Amenamevir SD was prepared using PVA 66 by the ball milling method. The drug was in an amorphous state in the SD, as reported in the literature.11) The SD was added to each gummi and dispersed homogeneously in each gummi formulation.
The drug dissolution of each gummi formulation was evaluated by dissolution tests in water. Table 2 shows the maximum values of dissolution before and after storage at 25 °C for 1 week and the change ratios after storage. Except for formulation number 3, each gummi formulation met the maximum value of dissolution criterion (≥85%) before storage. However, only three formulations (numbers 4, 6, and 8) met the change ratio of dissolution criterion (> − 5%) after storage for 1 week.
| Formulation No. | Dissolution | VAS score | ||||
|---|---|---|---|---|---|---|
| Maximum value before storage (%) | Maximum value after storage for 1 week (%) | Change ratio after storage for 1 week (%) | Sense of touch | Stickiness | Hardness | |
| 1 | 87.9 ± 3.4 | 80.4 ± 5.1 | −8.6 | 3.4 ± 4.4 | 96.5 ± 3.2 | 2.1 ± 2.8 |
| 2 | 87.9 ± 6.1 | 81.6 ± 3.8 | −7.2 | 28.7 ± 21.6 | 83.4 ± 10.0 | 15.6 ± 14.0 |
| 3 | 83.7 ± 12.5 | 76.2 ± 7.9 | −9.0 | 38.0 ± 22.1 | 56.8 ± 25.1 | 20.2 ± 13.2 |
| 4 | 85.1 ± 9.0 | 82.6 ± 0.2 | −2.9 | 32.3 ± 17.4 | 68.7 ± 22.6 | 38.2 ± 19.7 |
| 5 | 87.1 ± 3.7 | 77.4 ± 6.9 | −11.2 | 73.0 ± 14.7 | 36.2 ± 22.3 | 52.9 ± 12.2 |
| 6 | 88.6 ± 6.5 | 85.4 ± 3.8 | −3.6 | 47.6 ± 13.8 | 63.1 ± 25.1 | 59.3 ± 14.4 |
| 7 | 89.2 ± 1.8 | 82.5 ± 1.3 | −7.5 | 37.5 ± 13.4 | 54.8 ± 27.5 | 58.7 ± 20.1 |
| 8 | 85.6 ± 2.8 | 83.7 ± 4.4 | −2.2 | 32.7 ± 13.3 | 67.2 ± 27.9 | 63.0 ± 18.7 |
| 9 | 85.0 ± 5.1 | 78.9 ± 6.0 | −7.1 | 47.7 ± 23.9 | 39.2 ± 26.9 | 74.6 ± 20.3 |
| 10 | 88.3 ± 1.7 | 72.8 ± 1.3 | −17.6 | 67.2 ± 16.1 | 36.9 ± 20.5 | 67.2 ± 12.4 |
| 11 | 88.7 ± 7.9 | 70.8 ± 6.6 | −20.2 | 68.5 ± 16.2 | 30.2 ± 20.1 | 71.1 ± 11.3 |
| Criteria | ≥85 | — | >−5 | >50 | <60 | 50–65 |
Each value represents the mean ± S.D. (dissolution: n = 3; VAS score: n = 10).
The clinical sensory tests were conducted by evaluating the VAS scores for sense of touch, stickiness, and hardness of each gummi formulation (Table 2). The following formulations met the criteria: VAS score for sense of touch (>50), numbers 5, 10, and 11; VAS score for stickiness (<60), numbers 3, 5, 7, 9, 10, and 11; and VAS score for hardness (50–65), numbers 5, 6, 7, and 8.
Predicted values of the evaluation items mentioned above were calculated from the observed results, and the resulting ternary diagrams are shown in Fig. 3. The maximum value of dissolution was predicted to be highest when the gummi formulation had a composition near the center of the ternary diagram among the formulations evaluated. The change ratio of dissolution after storage for 1 week was predicted to increase with decreasing water content. The higher the contents of gelatin and water, the higher the predicted VAS score for sense of touch. Conversely, the lower the contents of gelatin and water, the higher the expected VAS score for stickiness. An increased gelatin content corresponded to a higher VAS score for hardness. The area in which all the criteria were met was determined from these predicted diagrams. The white area in Fig. 4 shows the gelatin and water composition that met all the criteria of the gummi formulation (the content of a mixture of hydrogenated maltose starch syrup and D-sorbitol solution was determined automatically). The red dot in Fig. 4 represents the center of the white area, and the gummi formulation comprising 10.5% gelatin and 22.8% water was selected as the optimal formulation for amenamevir SD.
(a) Maximum value of dissolution (%); (b) Change ratio of dissolution after storage for 1 week (%); (c) Visual analogue scale (VAS) score for sense of touch; (d) VAS score for stickiness; and (e) VAS score for hardness. X1 = gelatin; X2 = water; and X3 = a mixture of hydrogenated maltose starch syrup and D-sorbitol solution. (Color figure can be accessed in the online version.)
Each borderline means: (a) Maximum value of dissolution; (b) Change ratio of dissolution after storage for 1 week; (c) Visual analogue scale (VAS) score for sense of touch; (d) VAS score for stickiness; and (e) VAS score for hardness. The red dot represents the formulation selected in the present study. (Color figure can be accessed in the online version.)
Gummi formulations containing amenamevir SDs with various PVAs were prepared using the determined gummi formulation to evaluate their amenamevir dissolution profiles. Four PVAs with different degrees of hydrolysis (PVA 66, PVA 80, PVA 88, and PVA 66/88) were used. Gummi formulations containing HPMC SD and PVA 66 PM were also evaluated. It was confirmed that the drug in each SD existed in an amorphous state.11)
Figures 5a–f show the results of dissolution tests on the gummi formulations and the corresponding SDs and PVA 66 PM in water. There were no significant differences in the maximum dissolution values between the gummi formulations containing SDs with PVAs and the corresponding SDs, although the gummi formulations had slower dissolution rates than the SD suspensions. In contrast, the gummi formulations comprising HPMC SD and PVA 66 PM had higher amenamevir dissolution values than the corresponding suspensions. The rank order of the maximum dissolution values among the gummi formulations was: PVA 66> PVA 66/88> PVA 80> HPMC > PVA 88> PVA 66 PM.
(a) PVA 66; (b) PVA 80; (c) PVA 88; (d) PVA 66/88; (e) HPMC; and (f) PVA 66 PM. Water was used as the dissolution medium. Each value represents the mean with a vertical bar showing the S.D. (n = 3).
The dissolution profiles of the gummi formulations were also determined in SGF (pH 1.2) and phosphate buffer (pH 6.8) (Fig. 6). The rank order of the maximum dissolution values in those media was: PVA 66> PVA 66/88> HPMC > PVA 80> PVA 88> PVA 66 PM, which was almost the same as in water.
(●) PVA 66; (■) PVA 80; (▲) PVA 88; (▼) PVA 66/88; (○) HPMC; and (□) PVA 66 PM. Each value represents the mean with a vertical bar showing the S.D. (n = 3).
Stability studies on the gummi formulations containing amenamevir SDs were carried out. Each gummi formulation was stored at 4 °C for 1 month and at 30 °C for 1 month. The results of the amenamevir assays are shown in Table 3. The amenamevir content remained constant in each gummi formulation during both storage conditions. However, the maximum values obtained from the dissolution tests in water were greatly influenced by storage (Table 4). There was a more significant decrease in dissolution at 30 °C than at 4 °C in each formulation. The PVA 66 formulation exhibited the largest decrease among the formulations tested, with change ratios of −35.3% at 4 °C and −53.9% at 30 °C. On the other hand, PVA 66/88 exhibited the smallest change in the maximum dissolution value (−5.1% at 4 °C) among all the PVAs. There was also no significant change in the HPMC formulation.
| Formulation | Assay (%) | ||
|---|---|---|---|
| Before storage | After 1 month at 4 °C | After 1 month at 30 °C | |
| PVA 66 | 100.2 ± 0.2 | 100.0 ± 0.8 | 99.4 ± 1.5 |
| PVA 80 | 98.6 ± 0.7 | 99.6 ± 1.1 | 99.4 ± 1.2 |
| PVA 88 | 98.5 ± 1.8 | 98.0 ± 2.8 | 98.0 ± 2.4 |
| PVA 66/88 | 98.0 ± 0.5 | 98.2 ± 0.8 | 98.2 ± 1.0 |
| HPMC | 100.6 ± 0.5 | 99.3 ± 1.9 | 98.0 ± 0.3 |
Each value represents the mean ± S.D. (n = 3).
| Formulation | Before storage | After 1 month at 4 °C | After 1 month at 30 °C | ||
|---|---|---|---|---|---|
| Maximum value (%) | Maximum value (%) | Change ratio (%) | Maximum value (%) | Change ratio (%) | |
| PVA 66 | 91.4 ± 2.8 | 59.2 ± 9.1 | −35.3 | 42.2 ± 2.5 | −53.9 |
| PVA 80 | 84.4 ± 1.2 | 74.6 ± 6.5 | −11.6 | 66.6 ± 0.5 | −21.1 |
| PVA 88 | 67.8 ± 2.8 | 56.0 ± 3.2 | −17.3 | 51.8 ± 3.2 | −23.5 |
| PVA 66/88 | 88.2 ± 1.4 | 83.7 ± 11.3 | −5.1 | 76.8 ± 5.4 | −12.9 |
| HPMC | 81.9 ± 9.3 | 84.7 ± 0.9 | +3.4 | 72.2 ± 4.1 | −11.9 |
Each value represents the mean ± S.D. (n = 3).
Oral absorption of the drug products in dogs was evaluated to determine in vivo absorption of the gummi formulations containing amenamevir SDs with various PVAs. Tests were carried out on gummi formulations comprising SDs with PVA 66, PVA 80, PVA 88, PVA 66/88, and HPMC, and PVA 66 PM. The amenamevir dose was 100 mg/body, and four gummi formulations—each of which was divided into four pieces (16 units in total)—were used for each administration.
The plasma concentration–time profiles of amenamevir following oral administration of the gummi formulations are presented in Fig. 7a, and the pharmacokinetic parameters estimated from the plasma concentration–time data are summarized in Table 5. The rank order of Cmax and AUC0–24 h among the gummi formulations was: PVA 80> PVA 66/88> PVA 66≈ PVA 88> PVA 66 PM ≈ HPMC. PVA 80 had significantly higher Cmax and AUC0–24 h values than the other formulations. The rank order of Tmax was: PVA 66< PVA 88 = PVA 66/88≈ PVA 80< HPMC < PVA 66 PM.
(●) PVA 66; (■) PVA 80; (▲) PVA 88; (▼) PVA 66/88; (○) HPMC; and (□) PVA 66 PM. Each value represents the mean with a vertical bar showing the S.D. (n = 5).
| Formulation | Pharmacokinetic parameters | ||
|---|---|---|---|
| Tmax (h) | Cmax (ng/mL) | AUC0–24 h (ng h/mL) | |
| PVA 66 PM | 5.60 ± 1.67 | 1294 ± 156 | 19884 ± 2841 |
| PVA 66 | 2.20 ± 0.76 | 1897 ± 194a) | 27774 ± 3172a) |
| PVA 80 | 3.10 ± 1.02 | 3346 ± 659b, d) | 51709 ± 7393b, d) |
| PVA 88 | 3.00 ± 1.37 | 1772 ± 197f) | 26354 ± 4162f) |
| PVA 66/88 | 3.00 ± 1.37 | 2414 ± 421b, e, g) | 35779 ± 5818b, c, f, g) |
| HPMC | 4.40 ± 1.67 | 1224 ± 169c, f, h) | 19636 ± 3645c, f, h) |
Each value represents the mean ± S.D. (n = 5). a) p < 0.05, b) p < 0.001 (Tukey’s multiple comparison tests, vs PVA 66 PM), c) p < 0.05, d) p < 0.001 (Tukey’s multiple comparison tests, vs PVA 66), e) p < 0.01, f) p < 0.001 (Tukey’s multiple comparison tests, vs PVA 80), g) p < 0.05 (Tukey’s multiple comparison tests, vs PVA 88), h) p < 0.001 (Tukey’s multiple comparison tests, vs PVA 66/88).
The pharmacokinetics of the corresponding suspensions of amenamevir SDs and PVA 66 PM were also evaluated in dogs (Fig. 7b and Table 6). The suspension comprising PVA 66 exhibited the highest Cmax and AUC0–24 h values, and the shortest Tmax, in accordance with the results observed in rats.11) The Cmax and AUC0–24 h values of the gummi formulations were compared with those of the corresponding suspensions, and the GMRs and 90% confidence intervals are provided in Table 7. The gummi formulations comprising PVA 66 and HPMC exhibited significantly lower oral absorption values than the suspensions, with GMRs of approximately 0.5. There was a slight decrease in the GMRs of Cmax and AUC0–24 h (approx. 0.7) in the PVA 66/88 gummi formulation. With regard to PVA 88, there was no statistically significant difference between the gummi formulation and the suspension. Surprisingly, PVA 80 and PVA 66 PM had high Cmax and AUC0–24 h GMRs (1.32 and 1.41, and 5.20 and 5.09, respectively).
| Formulation | Pharmacokinetic parameters | ||
|---|---|---|---|
| Tmax (h) | Cmax (ng/mL) | AUC0–24 h (ng h/mL) | |
| PVA 66 PM | 4.20 ± 1.10 | 248 ± 26 | 3884 ± 341 |
| PVA 66 | 1.20 ± 0.57 | 3979 ± 626a) | 54516 ± 3656a) |
| PVA 80 | 3.50 ± 2.78 | 2504 ± 228a, c) | 36565 ± 4689a, c) |
| PVA 88 | 4.00 ± 1.41 | 1942 ± 384a, c) | 28979 ± 4391a, c, d) |
| PVA 66/88 | 2.00 ± 1.06 | 3313 ± 449a, b, d, h) | 49403 ± 3597a, e, h) |
| HPMC | 2.80 ± 1.30 | 2653 ± 490a, c, f, i) | 36548 ± 2762a, c, g, j) |
Each value represents the mean ± S.D. (n = 5). a) p < 0.001 (Tukey’s multiple comparison tests, vs PVA 66 PM), b) p < 0.05, c) p < 0.001 (Tukey’s multiple comparison tests, vs PVA 66), d) p < 0.01, e) p < 0.001 (Tukey’s multiple comparison tests, vs. PVA 80), f) p < 0.05, g) p < 0.01, h) p < 0.001 (Tukey’s multiple comparison tests, vs. PVA 88), i) p < 0.05, j) p < 0.001 (Tukey’s multiple comparison tests, vs. PVA 66/88).
| Formulation | Geometric mean ratio | |
|---|---|---|
| Cmax | AUC0–24 h | |
| PVA 66 PM | 5.20 (4.57–5.91) | 5.09 (4.54–5.72) |
| PVA 66 | 0.48 (0.43–0.53) | 0.51 (0.46–0.56) |
| PVA 80 | 1.32 (1.15–1.51) | 1.41 (1.26–1.59) |
| PVA 88 | 0.93 (0.69–1.23) | 0.91 (0.72–1.15) |
| PVA 66/88 | 0.73 (0.65–0.81) | 0.72 (0.64–0.80) |
| HPMC | 0.46 (0.36–0.59) | 0.53 (0.44–0.65) |
Each geometric mean ratio between two phases is given with the corresponding 90% confidence interval.
A gummi formulation suitable for amenamevir SD was first investigated using DOE. Three attributes of the gummi (the contents of gelatin, water, and a mixture of hydrogenated maltose starch syrup and D-sorbitol solution) were set based on previous findings.19,20) It was possible to prepare all 11 gummi formulations developed using DOE with homogeneously dispersed amenamevir SD. The DOE results for five test items (maximum value of dissolution; change ratio of dissolution after storage for 1 week; VAS scores for sense of touch, stickiness, and hardness) indicated that the water content of the gummi formulation had a different impact on the change ratio of dissolution after 1-week storage and the VAS scores for sense of touch and stickiness. Such trade-offs make it very difficult to develop gummi formulations with optimal properties. Therefore, an appropriate setting of quality target product profile is essential in the development of gummi formulations comprising SDs. The current research revealed that a gummi composition comprising 10.5% gelatin and 22.8% water met all the requisite criteria.
Amenamevir SDs with various PVAs were housed in the gummi formulation optimized in the present study. PVAs with a high degree of hydrolysis such as PVA 88 are slightly soluble in cold water because they have numerous hydroxyl groups that form hydrogen bonds; they also have low moisture permeability as films.22) However, PVA 66, which has fewer hydroxyl groups, was predicted to have higher water permeability because it dissolves rapidly in water. Therefore, it was speculated that the stability of an amorphous drug in a gummi formulation comprising PVA 66 would be inferior, although the solubility of amenamevir using this polymer was likely to improve significantly. Therefore, a formulation using PVA 66/88 was prepared, which was expected to combine the high amenamevir dissolution capability of PVA 66 with the high stability of PVA 88. The rank order of the maximum dissolution values among the gummi formulations was: PVA 66> PVA 66/88> PVA 80> HPMC > PVA 88> PVA 66 PM, which was similar to that among the corresponding suspensions of SDs and PVA 66 PM. With respect to the SDs comprising PVAs, because there were no significant differences in the maximum values of dissolution between the gummi formulations and the corresponding suspensions of SDs, the gummi formulations exhibited comparable amenamevir solubilizing capability to the suspensions. The dissolution capability of PVA 66/88—which was expected to have both the high dissolution characteristics of PVA 66 and the high stability of PVA 88—was comparable to that of PVA 66. Interestingly, the gummi formulations containing HPMC SD and PVA 66 PM had better dissolution capabilities than the suspensions. HPMC is reportedly less capable of maintaining amenamevir supersaturation than PVAs.11) The supersaturation capability of HPMC may have been improved by dispersing the SD into gelatin in the gummi in the present study. The maximum dissolution value of the gummi formulation comprising PVA 66 PM was approximately two times higher than that of the suspension. This may have been because part of the amenamevir was changed to the amorphous state during the preparation of the gummi formulation such as heating and dispersing in gelatin. This possibility is corroborated by research in which the oral absorption of a different drug was enhanced by a gummi formulation.19) The pH-independent dissolution of gummi formulations containing SDs with PVAs in water, SGF (pH 1.2), and phosphate buffer (pH 6.8) was observed in the present study, and the result suggested that a constant in vivo performance of the gummi formulations can be expected throughout the entire gastrointestinal tract after oral administration.
The stabilities of the gummi formulations containing amenamevir SDs were evaluated after storage at 4 °C for 1 month and after storage at 30 °C for 1 month. Although the contents of amenamevir in all the gummi formulations remained almost unchanged, the dissolution rates were negatively affected. This was presumed to have been caused by the recrystallization of a part of the drug by water in the gummi formulations during storage. There was a more significant decrease in the dissolution capability at 30 °C than at 4 °C. This was assumed to be because storage at 4 °C is thermodynamically advantageous, and amorphous drugs are stable under such conditions. Among all the formulations, PVA 66 exhibited the most significant decrease in dissolution after storage. It was predicted that the amenamevir in the formulation comprising PVA 66 would be easily affected by water because PVA 66 itself is readily soluble in water. PVA 66/88, which was expected to resolve the issue associated with PVA 66, had improved stability, and its dissolution capability remained almost unchanged following storage for 1 month at 4 °C. PVA 80 and PVA 88 were also more stable than PVA 66; this was assumed to be owing to the low water permeability of these PVAs. Although it was not clear why PVA 66/88 was more stable than PVA 88, the presence of two types of PVAs with different degrees of hydrolysis might have resulted in the formation of a structure different from that of a single PVA. HPMC, which was expected to be inferior to PVA in water permeability and stability, was stable. Although the reason for this has not been clarified, it is possible that the HPMC stably dispersed the amorphous drug in the gummi formulation, because the dissolution was improved by the gummi formulation. A polarized light microscope was used to investigate the changes taking place during storage in terms of the crystallinity of amenamevir in the gummi formulations. There were no appreciable changes in the crystallinity of the drug during storage in any of the gummi formulations (data not shown), suggesting that the result of the polarized light microscope could not explain the decrease in dissolution after storage. Better methods of detecting and quantifying crystallinity may be required for an improved understanding of this phenomenon.
The oral absorption of amenamevir from the gummi formulations was evaluated in the dogs. The gummi formulations comprising PVA 80 and 66/88 had higher Cmax and AUC0–24 h values than PVA 66, which had the highest dissolution capability in vitro. Furthermore, when the absorption capabilities of the gummi formulations were compared with those of the corresponding suspensions, the Cmax and AUC0–24 h values of the gummi formulations comprising PVA 80, PVA 66/88, and PVA 88 could be assumed to be comparable to the suspensions, whereas those of PVA 66 were halved. The poor stability of the PVA 66 gummi formulation (i.e., the crystallization of the drug in vivo) might have contributed to the low absorption. There was a significant improvement in absorption in the gummi formulation comprising PVA 66 PM compared with the absorption in the corresponding suspension. This might have occurred because some of the amenamevir became amorphous during the preparation of the gummi formulation, as described above. On the other hand, the drug absorption of the HPMC formulation was low and halved by forming gummi as well as PVA 66, although the gummi formulation with HPMC SD was stable and showed high dissolution close to that of the PVA 80 formulation. Therefore, a phenomenon that cannot be detected in vitro might occur in vivo, and further investigations may be required to elucidate the reason for this. Although dissolution tests at a higher drug concentration were performed assuming high drug concentration in the gastrointestinal tract, similar results to those reported herein were observed (data not shown). As an alternative method, the in vitro determination of the particle size of the dissolved SDs that correlates with in vivo absorption may be useful because the presence of large amorphous particles in dissolved SDs in an aqueous solution has been reported.25) Furthermore, methods for the investigation of hydrogel formulations and amorphous solid dispersions—in which in vitro biorelevant dissolution testing is coupled with in silico modeling and simulation to predict oral absorption—may be relevant to the current gummi with SD formulations.26,27)
The findings of the current study suggest that PVA 66, PVA 66/88, and PVA 80 are suitable in terms of dissolution, stability, and in vivo absorption, respectively, for the development of gummi formulations comprising PVA SDs of amenamevir. Although the correlation between in vitro dissolution and in vivo absorption has not yet been clearly demonstrated in gummi formulations, PVA 80, which had the highest absorption capability, was the most suitable SD carrier for gummi formulations among those evaluated in the present study. Thus, it was possible to apply a PVA SD of amenamevir to gummi formulations although a further development of the formulation would be needed.
A gummi formulation suitable for amenamevir SD was first developed using DOE. A gummi composition comprising 10.5% gelatin and 22.8% water was formulated based on the results of dissolution studies and clinical sensory tests. Gummi formulations containing amenamevir SDs with various PVAs were prepared using the determined gummi formulation, and their in vitro dissolution, stability, and in vivo oral absorption characteristics were evaluated. Considering these results comprehensively, it was concluded that PVA 80, which enabled the highest degree of absorption, was the most suitable SD carrier for gummi formulations. Thus, it was possible to produce gummi formulations comprising a PVA SD of poorly-soluble amenamevir. The findings of the present study will facilitate the development of patient-friendly formulations with poorly water-soluble drugs.
We thank Frank Kitching, MSc., for editing a draft of this manuscript.
The authors declare no conflict of interest.
