(M1530-03-15) Evaluating Physicochemical Properties of Cyclic Peptides and Their Interactions with Bile Acids Using Micellar Electrokinetic Chromatography

Purpose: Oral delivery of peptide therapeutics is gaining increased attention, especially with the growing success of approved peptide drugs on the market¹. However, oral absorption creates a significant challenge for most peptide drugs due to the limited enzymatic stability and poor membrane permeability. Additionally, recent evidence has demonstrated the critical role of the intestinal micellar system, composed of bile salt micelles and bile salt-phospholipid mixed micelles, in influencing the behavior of macrocyclic peptides^2,3. These micelles have been found to interact with peptide therapeutics and may negatively impact drug absorption, both in vitro and in vivo^2–4. Therefore, it is extremely valuable to understand and evaluate the interactions between macrocyclic peptide drugs and the intestinal micellar system to develop efficient oral peptide formulations. In this work, we aimed to deconvolute the effect of specific amino acid substitution and the overall physicochemical properties of the peptide on the interaction with the intestinal micelles using a newly developed micellar electrokinetic chromatography (MEKC) method. The hypothesis is that the peptide with a higher propensity to interact with the micelles would have a longer migration time. Furthermore, MEKC would enable the assessment of the overall net forces that drive the peptide-bile interactions (i.e., hydrophobic and/or electrostatic forces).

Methods: MEKC analysis was done using a capillary electrophoresis system with a UV detector; different bile salt solutions (di- and trihydroxy bile salts) were used as the background electrolyte (BGE). Octreotide was used as a model cyclic peptide drug, and several mutated analogs were generated by employing the same principles used in the alanine scanning technique, as described in the previous work by Dening et al³. Briefly, single amino acids in octreotide sequence were substituted at key residues (position 4 and 5); Trp4 was substituted with Ala4, Phe4, and Leu4, while Lys5 was substituted with Ala5 and Asp5. These specific amino acid modifications were used to deconvolute the role of specific amino acids with differing hydrophobicity and charge on the molecular interactions between peptides and bile salts. Additionally, distribution coefficients (logD) were determined using the n-octanol/buffer system, and each phase was analyzed using liquid chromatography-mass spectrometry (LC-MS/MS), as reported previously⁵.

Results: As seen in Table 1, the analogs have a similar migration time when no micelles are present in the BGE due to similar charge/mass ratios and, therefore, similar electrophoretic mobility (μep). The only exceptions are [Ala5] and [Asp5] analogs, which migrate later due to a lower positive charge (lower μep). However, when di- or trihydroxy bile salts are used as the BGE, the peptide analogs would have different migration times depending on the relative partitioning with the micelles. Overall, all the analogs seem to interact more with the dihydroxy bile salts compared to trihydroxy, as evident by their longer migration times (Fig. 1 and Fig. 2). Our data shows that there is a correlation between the overall peptide hydrophobicity and the interaction with the micelles; the more hydrophobic peptide interacts more with the micelle and has a slower migration time (Fig. 1A and B). Additionally, there is an electrostatic contribution to the overall interaction, which plays a major role in the case of dihydroxy bile salt (Fig. 2A and B). These results are consistent with the previous data, which compared the interaction between octreotide and mutated analogs with the di- and trihydroxy bile salts using diffusive flux experiments and nuclear magnetic resonance (NMR) spectroscopy^2,3.

Conclusion: The proposed MEKC method was efficiently used as a screening tool to assess the interactions between oral peptide analogs and the endogenous bile salt micelles. These interactions depend on the nature of the bile salt (i.e., dihydroxy or trihydroxy bile salt) and the physicochemical properties of the peptide. Specific amino acids in the peptide sequence can alter the interactions with the intestinal micelles and influence the driving force in the peptide-bile interaction (i.e., hydrophobic and electrostatic forces). Current and future work investigates the peptide interactions with different types of bile salts and uses a more realistic biorelevant model containing bile acids and phospholipids. Additionally, the feasibility of using this method to assess the interaction between the intestinal micellar system and different small-molecule drugs is under investigation.

References: (1) Pereira, A. J.; de Campos, L. J.; Xing, H.; Conda-Sheridan, M. Peptide-Based Therapeutics: Challenges and Solutions. Med. Chem. Res. 2024, 33 (8), 1275–1280. https://doi.org/10.1007/s00044-024-03269-1.
(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.
(3) Dening, T. J.; Napolitano, J. G.; Ochoa, J. L.; Douglas, J. T.; Hageman, M. J. Characterization of Macrocyclic Peptide Drug Interactions with Bile Salts and Biorelevant Colloids via Single Amino Acid Mutations and 1H Nuclear Magnetic Resonance (NMR) Spectroscopy. J. Pharm. Sci. 2025, 114 (1), 434–448. https://doi.org/10.1016/j.xphs.2024.10.021.
(4) Fricker, G.; Drewe, J.; Vonderscher, J.; Kissel, T.; Beglinger, C. Enteral Absorption of Octreotide. Br. J. Pharmacol. 1992, 105 (4), 783–786.
(5) Lou, H.; Feng, M.; Al-Tamimi, Z.; Kuczera, K.; Hageman, M. J. Predicting Distribution Coefficients (LogD) of Cyclic Peptides Using Molecular Dynamics Simulations. Pharm. Res. 2025. https://doi.org/10.1007/s11095-025-03850-2.

Acknowledgements: This work is supported by the Valentino J. Stella Endowment funds.


Table 1. Migration times of the peptide analogs using capillary zone electrophoresis, where no micelles are present in the background electrolyte (BGE). BGE: Phosphate buffer, pH = 6.5. The concentration of each analog was 0.1mg/mL.


Fig. 1. MEKC-UV Electropherograms for peptides with single amino acid substitution at residue 4 with di- and trihydroxy bile salts as the background electrolyte (BGE); (A) BGE: NaTDC* (15mM). (B) BGE: NaTC* (15mM). The concentration of each analog was 0.1mg/mL. Detection wavelength = 254nm.
*NaTDC: Sodium taurodeoxycholate. NaTC: Sodium taurocholate


Fig. 2. MEKC-UV Electropherograms for peptides with single amino acid substitution at residue 5 with di- and trihydroxy bile salts as the background electrolyte (BGE); (A) BGE: NaTDC* (15mM). (B) BGE: NaTC* (15mM). The concentration of each analog was 0.1mg/mL. Detection wavelength = 280nm.
*NaTDC: Sodium taurodeoxycholate. NaTC: Sodium taurocholate