(M1230-12-78) Disruption of Octreotide Interactions with the Bile Acid-Phospholipid Mixed Micelles Using Bile Acid Sequestrants

Purpose: Peptide drugs have become one of the most valuable therapeutic modalities on the market. Despite the significant advancement in peptide drug development, there is still a challenge in formulating and delivering peptide drugs orally. Current oral peptide drugs have very low bioavailability ( < 0.5%), which could be attributed, in part, to enzymatic instability, poor membrane permeability/flux and the sequestration by intestinal colloids composed of bile acid and bile acid-phospholipid mixed micelles (BAMM). Previous studies have found a 6-fold increase in the bioavailability of octreotide in bile duct-cannulated rats, indicating that bile acid had reduced peptide drug absorption¹. Furthermore, it has been shown that the interaction between peptide drugs and bile salts can reduce peptide membrane flux (negative impact) and protect from enzymatic degradation (positive impact)². In this work, we investigated how perturbing the components of the colloidal system (bile acid-phospholipid) with bile acid sequestrants (BAS) would affect the membrane flux of octreotide, which could ultimately correlate to peptide drug absorption and bioavailability.

Methods: Fasted state and fed state simulated intestinal Fluid (FaSSIF and FeSSIF, respectively) were used as a source of in vitro bile salt-phospholipid mixed micelles with octreotide as a model peptide drug. In vitro octreotide flux rates were tested using side-by-side diffusion cells separated by a regenerated cellulose dialysis membrane with a molecular weight cutoff (MWCO) of 3.5kDa in different donor conditions, i.e., Acetate buffer, pH=5 (as a control), FeSSIF, and bile acid sequestrants (i.e., commercially marketed drugs cholestyramine, colestipol, or colesevelam) added to FeSSIF. Sample analyses were conducted using ultra-performance liquid chromatography (UPLC). Additionally, The effect of micellar perturbation by BAS on octreotide stability toward pancreatic enzymes was tested using a method adapted from Dening et al.². Briefly, octreotide was incubated for 3 hours with 10 mg/mL pancreatin and FeSSIF (pH=6.5) in the presence of BAS, and the percent remaining peptide was determined by UPLC.

Results: The flux of octreotide decreased by 4.5- fold in the presence of FeSSIF. Adding BAS with FeSSIF in the donor chamber led to an increase in octreotide flux that depended on the polymer used (Fig. 1). Cholestyramine, colestipol, and colesevelam increased the flux rate by 1.7-, 2.7-, and 3.1-fold, respectively, compared to FeSSIF alone in the donor chamber. The effect of BAS on octreotide stability toward pancreatic enzymes is summarized in Table 1. The data shows that the micelles present in FeSSIF would protect the peptide from enzymatic degradation when compared with the buffer alone by around 2.9-fold (which is similar to the previous findings²). Moreover, although adding BAS would disrupt the protective micellar structure, the peptide drug was still more stable to degradation by 3.4- and 2.1-fold in the presence of colestipol and cholestyramine, respectively. This protection could be attributed to encapsulation inside the remaining bile salt micelles or because of the remaining phospholipids in the solution; however, more stability studies are needed to explain these findings. Additionally, BAS adsorption to octreotide was tested over a 24 hours period. As presented in Fig.2, colestipol tends to adsorb almost none of the peptide drug from the solution.

Conclusion: The results suggest that bile acid sequestration by BAS decreases the bile acid levels and disrupts the micellar structure, leading to a higher concentration of the free peptide to diffuse across the membrane. Therefore, in vitro bile acid sequestration enhanced the overall peptide flux rate without compromising enzymatic stability. These results warrant the future investigation of oral peptide delivery in the presence and absence of BAS.

References: (1) Fricker, G.; Drewe, J.; Vonderscher, J.; Kissel, T.; Beglinger, C. Enteral Absorption of Octreotide; Br. J. Pharmacol; 1992; Vol. 105, pp 783–786.
(2) Dening, T. J.; Douglas, J. T.; Hageman, M. J. Do Macrocyclic Peptide Drugs Interact with Bile Salts under Simulated Gastrointestinal Conditions? Mol. Pharm. 2021, 18 (8), 3086–3098. https://doi.org/10.1021/acs.molpharmaceut.1c00309.

Figure 1. Cumulative permeated amount (Qt) versus time plot of octreotide in the receiver chamber when different donor conditions are used in the side-by-side diffusion cell. Flux can be calculated using the following equation: J= ΔQt / (A*Δt). Data are reported as mean ± standard deviation, n=3, *n=2. (R2> 0.998 in all cases)

Figure 2. Octreotide adsorption to different bile acid sequestrants as represented by octreotide concentration versus time plot. Data are reported as mean ± standard deviation, n=3.

Table 1. Percentage of octreotide remaining after incubation for 3 hours with pancreatin in different media. Values are represented as mean ± standard deviation, n=3.