(T1530-09-60) Lipid-Based Formulations: How to Predict Rat In Vivo PK Profile with In Vitro + In Silico Approach

Purpose: Self-emulsifying drug delivery systems (SEDDS) are one of the primary subgroups of lipid-based formulations, providing a cost-effective approach to enhance the solubility and absorption of challenging, low-soluble BCS II drugs. However, the evaluation of SEDDS using conventional dissolution tests often fails to capture their complex in vivo behavior; therefore, advanced in vitro and in silico methods are needed to simulate gastrointestinal conditions better and characterize solubility–permeability interplay. In vivo, rat studies are often applied for the characterisation, but supra-therapeutic doses hinder the understanding of solubility, dissolution, and permeability across dose ranges. This project is a continuation of a collaboration where MacroFlux experiments were successfully used to rank-order lipid-based formulations. In this work, we evaluated 5 of these SEDDS formulations of danazol using high-dose in vitro dissolution-permeation (microFlux) assays combined with in silico mechanistic modeling to predict in vivo rat PK profile parameters. This provides an example of how advanced tools can help lipid-based formulation development and advances the efforts to reduce animal use in drug development.

Methods: Preparation of SEDDS: The formulations were prepared by weighing the excipients (in the order of oil, surfactant, co-surfactant, water/co-solvent) and mixing them through magnetic stirring at room temperature (for composition, see Figure 1). For the production of drug-loaded SNEDDS, danazol was directly weighed into glass vials, followed by the addition of SNEDDS and overnight stirring at ambient temperature. The final drug load was adjusted to correspond to 80% of the equilibrium solubility (S_eq) of danazol in the SNEDDS. In vivo rat study: Briefly, Sprague-Dawley rats were orally administered with the Danazol-SEDDS formulations (n=6 per formulation) at the dose of 28 mg/kg, and blood plasma levels were monitored over a 24-hour period in the fasted state (12 hours of fasting prior to administration). LC-MS was used for periodic sampling and analysis. In vitro dissolution-permeation (Flux) experiment: Flux assays were performed using the µFLUX™ system (Pion Inc., Billerica, MA, USA), as previously described [1,2]. The system comprises donor (FaSSIF V2 with pH and bile salt concentration adjusted to rat intestinal fluid) and receiver (Acceptor Sink Buffer) compartments separated by a vertically oriented PVDF membrane (1.54 cm², 0.45 μm pore size, 120 μm thickness) impregnated with a lipid solution ( GIT-0 lipid). Danazol-loaded SEDDs were added to the donor compartment at doses equivalent to the in vivo rat study. Danazol quantification was performed using a Rainbow R6 FO spectrometer with fiber-optic UV probes. In silico mechanistic modeling: Flux curves were used as primary input for the Predictor software (Rev. 1.0.35, Pion Inc.) to estimate in vivo absorption rates in rats. The mechanistic model assumed a GI transit time of 60 or 120 minutes and a GI volume of 3.4 mL, besides other standard rat physiological parameters. Absorption rates were converted to PK profiles predictions considering published distribution and elimination parameters of danazol (Vss = 4.55 +/- 1.77 L/kg; Kel = 4.74 +/- 1.98 L/h/kg [3]), and then AUC, C_max, and T_max were determined.

Results: Danazol is a hydrophobic, non-ionizable, and poorly soluble BCS Class II drug with a LogP of 3.56 and aqueous solubility of 0.7 μg/mL [4]. Its solubility was significantly enhanced using the SEDDS formulations, achieving 24.4, 14.6, 9.5, 15.5, and 9.0 mg/mL in formulations F1, F2, F3, F9, and F10, respectively, due to the optimized combination of oil, surfactant, and co-surfactant. In vivo, formulations F1, F2, and F3 demonstrated the highest drug exposure based on AUC₀_–8 values, whereas F9 and F10 showed comparatively lower AUC₀_–8 within the group (Figures 2 and 3). In the previous study, formulations were run at human-relevant doses, and good rank order correlation was found with in vivo results. In this study, we performed experiments at a rat-relevant dose, which is about 30x higher. F1, F2, F3, and F9 exhibited high flux values that correlated with elevated AUCs, while F10 displayed substantially lower flux. The PK profile predictions assumed 60 or 120 minute intestinal transit time, considering the variation in in vivo references. Good agreement with in vivo C_MAX (Figure 2) and AUC₀_–8 (Figure 3) for F1, F2, F3, and F10, and acceptable agreement for F9 was found, showing the potential of the in vitro + in silico approach in predicting in vivo behavior. The difference in digestibility of SEDDS formulations in vivo can potentially influence absorption and will be addressed by incorporating a lipolysis step in the in vitro assays planned for this study, hopefully further improving the prediction potential.

Conclusion: As it was shown previously, the studied SEDDS formulations improved in vivo absorption of danazol compared to the unformulated drug. By combining flux measurements with in silico modeling, in vivo performance could have been predicted with C_MAX and AUC values consistent with observed data and supporting an IVIVC approach.

References: [1] Ranking Itraconazole Formulations Based on the Flux through Artificial Lipophilic Membrane K.Tsinman et al; Pharm Res, . 2018 Jun 20;35(8):161.
[2] Food effect risk assessment in preformulation stage using material sparing μFLUX methodology C. Jankovsky et al; ADMET & DMPK 10(4) (2022) 299-314
[3] Evaluation of a Nanoemulsion Formulation Strategy for Oral Bioavailability Enhancement of Danazol in Rats and Dogs Harikrishna Devalapally et al; J Pharm Sci. 2013 October ; 102(10): 3808–3815.
[4] Exploring the Impact of Intestinal Fluid Components on the Solubility and Supersaturation of Danazol Cecilie Maria Madsen et al; Journal of Pharmaceutical Sciences Volume 110, Issue 6, June 2021, Pages 2479-2488

Acknowledgements: The authors are employees of companies whose materials, methods, and tools have been applied during this work.

Figure 1. Compositions of F1, F2, F3, F9 and F10 SEDDS.

Figure 2. Comparison of CMAX obtained for danazol-loaded SEDD formulations in vivo and predicted with in vitro + in silico. (in silico results calculated using 120 and 60 min transit times are shown in blue and green, respectively).

Figure 3. Comparison of AUC0-8h obtained for danazol-loaded SEDD formulations in vivo and predicted with in vitro + in silico.