(T1130-11-72) Estimation of Drug Apparent Solubility in Gastric Contents after the Standard High-Calorie High-Fat Meal: Comparison of Data in Biorelevant Media with Data in Aspirates

Purpose: To clarify the required level of biorelevance and the composition of the medium which is comparatively the most useful for estimating the solubility of danazol (nonionizable, logP 4.2), and the solubility of ketoconazole [pkas 2.94 and 6.51 (both alkaline), clogP 4.45] in gastric contents after the standard high-calorie high-fat meal.

Methods: Composition of the tested media was based on the physicochemical characteristics of gastric aspirates after the administration of high-fat high-calorie meal [1]. Three levels of biorelevance were considered [2]. Within each level, the average pH and the average buffer capacity of gastric contents was simulated by using hydrochloric acid, acetates, and FEDGAS buffer concentrate (biorelevant.com). In Level II and Level III biorelevant media the osmolality was adjusted using sodium chloride and the presence of lipid species was simulated using various amounts of FEDGAS gel (biorelevant.com). In Level III biorelevant media, the presence of proteins was simulated using powdered skimmed milk. HGFs had been aspirated 75, 105, 135, 165 and 225 minutes, after initiation of administration of the standard high-calorie high-fat meal [3]. Thirty minutes after initiation of meal administration 240 mL of aqueous solution was administration to simulate the drug administration conditions in relevant drug bioavailability / bioequivalence studies in the fed state [3]. Aspirated samples were maintained at -70 °C until used. On the day of solubility experiment, HGF samples were brought to room temperature and were pooled by taking 1mL from each sample, to create the HGF₇₅, the HGF₁₀₅, the HGF₁₃₅, the HGF₁₆₅ and the HGF₂₂₅ pooled samples. Solubility of danazol and of ketoconazole was measured in triplicate with the shake-flask method at 37 °C. The duration of incubation for achieving equilibrium was evaluated with preliminary experiments in Level III biorelevant media and in HGFs and was found to be 4h, at most. At equilibrium, each sample was filtered through a regenerated cellulose 0.45 mm filter (Level I and Level II biorelevant media) or centrifuged at 11,400 g for 10 min at 37 °C (Level III biorelevant media and HGFs) and the pH was measured. Drug concentration was determined using HPLC-UV [4, 5]. In all cases, quantification was based on standard curves constructed usin the corresponding medium.

Results: For danazol, data in Level I biorelevant media vastly underestimated the data in HGFs. Mean solubility in Level II biorelevant media was within the range of mean HGF solubility values estimated at each aspiration time point, i.e., within the 90-200 μg/ml range, when the presence of lipid species in gastric aspirates was simulated by using 2.8 g FEDGAS gel [corresponding to 17 % of the amount that is currently recommended (biorelevant.com)]. In Level III biorelevant media, buffer species impacted substantially the solubility data; the interplay of proteins with the buffer species could be a potential reason. Variability of individual data collected in a specific Level III biorelevant medium and in HGF aspirated at a specific time was high, i.e., 7-14 %RSD and 20-60 %RSD, respectively; variability in the vesicle/micellar structures in the presence of proteins could be a potential reason. For ketoconazole, mean data in HGFs ranged from 0.77 to 11.7 mg/ml and were reflective of the pH of the medium at equilibrium. Data in Level I biorelevant media were in line with the ionization characteristics of the compound. Data in Level II biorelevant media indicated that when hydrochloric acid is used for the preparation of the biorelevant medium (pH 3), the presence of lipid species controls solubility (pH at equilibrium = 4.5 > pka+1 in both cases) as the non-ionizable form of ketoconazole seems to determine its solubility; however, when the pH at equilibrium does not exceed the pKa value more than one unit and acetates or FEDGAS buffer concentrate is used, solubility is not affected by the presence of lipid species. Data in Level III biorelevant media indicated that, when the pH at equilibrium does not exceed the pKa value more than one unit and acetates or FEDGAS buffer concentrate are used as buffer systems, proteins can increase ketoconazole solubility by 30-50%.

Conclusion: Based on solubility data collected for first time in gastric aspirates after the standard high-calorie high-fat meal, a Level II biorelevant medium, prepared by using acetates or FEDGAS buffer concentrate as buffer system and 17% of the amount of FEDGAS gel that is currently recommended (biorelevant.com) was useful for achieving comparatively the best estimate for both danazol and ketoconazole solubility in gastric contents after the standard high-calorie high-fat meal.

References: 1. Pentafragka et al. Eur. J. Pharm. Sci. 155:105535 (2020).
2. Markopoulos, Andreas et al. Eur. J. Pharm. Biopharm. 93:173 (2015).
3. Pentafragka et al. Eur. J. Pharm. Sci. 149:105351 (2020).
4. Vertzoni et al. J. Chromatogr. B. 839:62 (2006).
5. Vertzoni et al. Pharm. Res. 24:909 (2007).

Acknowledgements: The authors would like to thank biorelevant.com for providing FEDGAS buffer concentrate and gel.