Category: Formulation and Quality
Purpose: Drug permeability and solubility in gastrointestinal track (GIT) are key parameters influencing oral absorption. Solubility-enabling formulations are often employed for improving drug solubility but the interplay between apparent solubility enhancement and permeability reduction was recently reported, which was attributed to the lowering free drug fraction in solubilized formulations for permeation. For cyclodextrin-based formulation, Sun et al.  also demonstrated that bile-salts play a role on modulating apparent permeability due to the competition with cyclodextrin on solubilizing low solubility drugs and mutual solubilization between bile-salts and cyclodextrin. The purpose of this study was to develop an in-vitro absorption model to understand the interplay effect among drug, HPßCD and bile-salts on the drug permeability and to explain the in-vivo results obtained from a rat study.
Methods: A BCS class I drug, BI-XYZ and Hydroxypropyl betacyclodextrin (HPßCD) with MW 1,460 was used in this study. Solubility measurements were performed by adding excess amount of drug in aqueous solutions of HPßCD. The binding constant of the drug to HPßCD was determined based on Schonbeck et al. 
Eigth formulations were evaluated with µFlux apparatus (Pion Inc.) using rat physiology and also tested in rats. Flux measurements were performed with FaSSIF media in the donor compartment and Acceptor Sink Buffer (ASB pH 7.4) in the acceptor, separated by a lipophilic membrane (GIT Lipid, Pion Inc.). Drug concentrations in both compartments were monitored using in-situ fiber optic probes connected to a Rainbow UV spectrometer (Pion Inc.). Formulations at the fixed drug concentration of 1.6 x 10-3 mM containing different HPßCD concentrations (0, 2.8, 5.6 and 34.2 mM) and varied amount of sodium taurocholate (NaTC, 0, 1.5, 6.5, 11.5 and 20 mM) was placed in donor compartment. Flux was monitored up to 4 hours. Second derivative analysis and lower path length (1 mm) were used to further improve donor compartment concentration analysis. Statistical analysis were done using SASTM System (SAS Institute Inc., Cary, North Carolina) version 9.4.
Results: BI-XYZ apparent solubility increases linearly with increasing HPbCD concentration suggesting a 1:1 complexation between drug and HPßCD. K1:1 extrapolated from solubility data was found to be 353 M-1. BI-XYZ flux in water increases linearly (1.8 and 2.35 μg min-1 cm-2 ) with increasing drug concentration. In the absence of bile-salts, BI-XYZ flux decreased (1.804-0.185 μg min-1 cm-2) with increasing HPßCD concentration. Flux was the highest in the absence of HPßCD suggesting that with increasing HPßCD concentrations in the donor, in spite of the increase in apparent solubility, more drug molecules form inclusion complex and the free drug fraction in solution decreases. In the absence of HPßCD, BI-XYZ flux initially stays constant and decreases with increasing NaTC (1.804 to 0.780 μg min-1 cm-2). This may be due to the fact that below critical micelle concentration (CMC) flux is not affected by the presence of bile-salts while above CMC, bile-salts form mixed-micelle including BI-XYZ resulting in a decrease in unbound drug available to permeate across the membrane. With 20 mM NaTC/5 mM lecithin, BI-XYZ flux stays constant initially and decreases (0.780-0.2820 μg min-1 cm-2) with increasing HPßCD. We hypothesize that during initial state, when flux was constant even after addition of HPBCD, drug molecules forming inclusion complexes with HPßCD are displaced by NaTC molecules. For stochiometric ratio drug: HPßCD of 1:21, flux was the lowest (0.282 μg min-1 cm-2) and comparable to flux without NaTC (0.185 μg min-1 cm-2) possibly due to drug and NaTC molecules mostly bind to cyclodextrin. Flux measurement trends of tested formulations correlate well with in-vivo rat data. Although Cmax in animal group dosed at low level of HPßCD was lower than expected, and was not consistent with in-vitro flux measurement this could be explained by the content of bile salts in-vivo. Statistical analysis indicated that both NaTC and HPßCD have statistically important contributions to the flux. However, a larger sample size is needed to evaluate the level of correlation between each variables.
Conclusion: This work shows that for a BCI class I drug, when using solubility-enabling formulations with HPßCD for high dose studies, fraction of dose absorbed is governed by a trade-off between apparent solubility increase and permeability decrease. Interplay between drug, HPßCD and bile-salts are strongly depend on physiological conditions, content and concentration of bile-salts in the intestinal fluid, and difficult to predict. This work also shows µFlux apparatus is an easy in-vitro methodology that can assess free drug concentration in solution to help us study interplay between components and evaluate the performance of solubility enabling formulations using HPßCD.  L. Sun et al, J.Pharm. Sci. 2018, 107, 488-494.  Schönbeck et al., International Journal of Pharmaceutics, 2017, 531, 504-511