(T1030-02-08) Rethinking In Vitro Testing: Using Biorelevant Media for Dispersion/Dissolution Testing for Lipid-Based Drug Delivery

Purpose: The aim of this study was to evaluate the suitability of currently employed in vitro methods for characterizing lipid-based formulations (LBFs) designed to enhance oral bioavailability. In particular, the utility of biorelevant media was assessed as a potential alternative to traditionally used dissolution and dispersion media. Data from dissolution testing in purified water (pH 1.2 and 6.8) and biorelevant media, in vitro lipolysis, and in vivo testing were compared to assess in vitro in vivo correlation (IVIVC) and to further optimize the testing methodology. Nine prototype lipid-based formulations were developed for two BCS Class II compounds—Drug X and fenofibrate—both of which exhibit poor aqueous solubility. Drug X is a novel chemical entity under investigation for cancer treatment (patent pending).

Methods: Ultra High-Performance Liquid Chromatography (UHPLC) method was developed and validated for accuracy, precision and specificity. Dissolution testing was conducted in purified water (at pH 1.2 and 6.8) and biorelevant media (Fasted state simulated gastric fluid (FaSSGF), Fasted state simulated intestinal fluid (FaSSIF), Fed state simulated intestinal fluid (FeSSIF)). Approximately 2g formulation was added to 400 mL of media at 37°C and stirred at 100 RPM in a USP type II dissolution apparatus. Aliquots were taken at 30, 60, and 120 minutes followed by UHPLC analysis. For in vitro lipolysis testing, formulations were dispersed in fasted state intestinal media at 37°C. For first 10 minutes, the formulation was allowed to disperse followed by 60 minutes of digestion using porcine pancreatic lipase enzyme. Aliquots were taken at select time points and centrifuged (21000xg and 37 °C) for 30 minutes. The centrifuged samples were analyzed to determine the amount of solubilized active using UHPLC. In vivo pharmacokinetic (PK) study was conducted using rat models for Drug X and fenofibrate. PK parameters  were calculated using GraphPad Prism based on individual plasma concentration profiles.

Results: Drug X: Formulations tested include: Labrasol^® ALF: Labrafil^® M 1944 CS: Plurol^® Oleique CC 497 (6.75: 2.25: 1). Labrasol^® ALF: Labrafil^® M 1944 CS: Transcutol^® HP (4: 4: 2). Labrasol^® ALF: Labrafil^® M 1944 CS: Capryol^® 90 (3: 3: 4). Labrasol^® ALF. Gelucire^® 50/13. Upon dissolution testing in 0.1N HCl and purified water, all formulations exhibited the same rank order, indicating that drug X’s solubility is not pH-dependent. Notably, the formulation containing Labrasol^® ALF: Labrafil^® M 1944 CS: Capryol^® 90 (3:3:4) was not dispersible in either 0.1N HCl or purified water, but was readily dispersible in all three biorelevant media, resulting in enhanced solubility of the active. Overall, most formulations showed greater active solubilization in biorelevant media compared to aqueous media (see Figures 1 and 2). Among them, the formulations composed of Labrasol^® ALF: Labrafil^® M 1944 CS: Capryol^® 90 (3:3:4) and Labrasol^® ALF: Labrafil^® M 1944 CS: Plurol^® Oleique CC (6.75:2.25:1) demonstrated higher active solubilization during in vitro lipolysis. These lipid-based formulations also achieved higher plasma concentrations in animal studies when compared to other amorphous solid dispersions, confirming improved bioavailability (see Figure 3). Fenofibrate: Formulations tested include; Labrasol^® ALF: Labrafil^® M 1944 CS: Plurol^® Oleique CC 497 (6.75: 2.25: 1), Labrasol^® ALF: Labrafil^® M 1944 CS: Transcutol^® HP (4: 4: 2), Labrasol^® ALF: Labrafil^® M 1944 CS: Transcutol^® HP (6.75: 2.25: 1), Gelucire^® 44/14: Labrafil^® M 1944 CS: Plurol^® Oleique CC 497 (6.75: 2.25: 1). During dissolution testing in 0.1N HCl and purified water, all formulations exhibited the same rank order, indicating that fenofibrate solubility is not pH dependent. Most formulations showed higher solubilization in biorelevant media compared to aqueous media. Notably, the formulation containing Labrasol^® ALF: Labrafil^® M 1944 CS: Transcutol^® HP (4:4:2) displayed phase separation in both 0.1N HCl and purified water but was fully dispersible in all biorelevant media. During in vivo studies, all four lipid-based formulations outperformed the aqueous suspension, demonstrating enhanced bioavailability. Note: Due to limitation in number of images that can be uploaded, graphs for fenofibrate are not included in abstract submission. They will presented in the poster upon abstract acceptance.

Conclusion: Biorelevant media should be considered essential for dissolution testing of lipid-based formulations. These formulations are not readily dispersible in water but demonstrate improved dispersion in biorelevant media due to its unique physicochemical properties including osmolality, ionic strength, and surface tension which closely mimic gastrointestinal conditions. When using only water, there is a risk of prematurely eliminating formulations that may, in fact, perform well in vivo - an issue that can be mitigated by incorporating biorelevant media into the evaluation process. This consideration is particularly important when working with lipid excipients with lower HLB values such as propylene glycol esters (e.g., Capryol^® 90 ) and polyoxylglycerides (e.g., Labrafil^® M 1944 CS). Additionally, in vitro lipolysis testing serves as a valuable tool for rank-ordering formulations based on their performance, thereby supporting informed selection for subsequent in vivo studies.

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Dissolution profile for Drug X in 0.1N HCl solution (pH~1.2) and purified water (pH~6.8)


Dissolution profile for Drug X in Biorelevant media (FaSSGF, FaSSIF and FeSSIF)


In vitro lipolysis and in vivo plasma concentration profile for Drug X