(M0930-07-37) Enabling Oral Delivery of Therapeutic Peptide Salmon Calcitonin: Impact of Hydrophobic Ion Pairs and Self-Nanoemulsifying Drug Delivery Systems on Proteolytic Protection and Pharmacological Activity

Purpose: Despite the desire among patients and industry, developing oral peptide delivery systems remains a major challenge¹. This arises from the biological and physicochemical properties of peptides, resulting in degradation by low pH, enzymes, and low permeability across the gastrointestinal tract(GI). Lipid-based drug delivery systems, such as self-nano emulsifying drug delivery systems (SNEDDS), can enhance oral peptide absorption². SNEDDS can inherently contain permeation enhancers that can enhance peptide absorption³.Aditionally, the emulsion droplets formed during SNEDDS dispersion protect incorporated peptides from acid and enzymes during transit of the GI.  However, incorporating a hydrophilic peptide into SNEDDS requires prior lipophilization of the peptide through hydrophobic ion pairing (HIP) with an oppositely charged surfactant⁴. In this study, HIP complexes were formed between model peptide salmon calcitonin (sCT), involved in calcium regulation, and the anionic surfactants (AnS) sodium docusate (SDOCS) and sodium decanoate (C10). C10 was further chosen for its permeation-enhancing properties⁵, which could promote co-localization with sCT, thereby improving absorption. Both HIP complexes were evaluated for their protective effect and oral bioavailability, with and without SNEDDS. This comparison allowed us to assess the standalone impact of each HIP complex and to evaluate the effectiveness of SNEDDS in enhancing the protection and oral delivery of the model peptide complex.

Methods: One mL of AnS solution was added dropwise to the peptide solution with various sCT: AnS molar ratios (1:1 to 1:8) under constant stirring. The resulting precipitate and supernatant were separated and collected by centrifugation at 14.700 g for 10 mins. The amount of peptide in the supernatant was quantified and the complexation efficiency (CE %) was calculated using the following equation (Eq.1). Subsequently, the precipitates were washed and lyophilized at 0.1 mBar and -50°C (Figure.1).

CE%=((𝐶_𝑖 −𝐶_𝑚))/𝐶_𝑖 )∗100%                     (Eq.1)

C_i = initial amount of sCT          C_m = amount of sCT in the supernatant.sCT:AnS 1:4 complexes were then incorporated into SNEDDS formulation (30% w/w medium chain triglycerides: MCT, 30% w/w mono and diglycerides: MGDG, 30% w/w Kolliphor RH40, and 10% w/w propylene glycol). Free sCT, lyophilized sCT:AnS complexes alone, or complexes loaded into SNEDDS were tested in vitro to evaluate their protection effect against protease degradation. Each formulation was added to 10 ml of tris buffer (100 mM, pH 6.8) containing trypsin, and proteolysis was carried out for 120 mins at 37°C. Samples were taken at set intervals and analyzed by HPLC for %sCT remaining. The pharmacological hypocalcemic effect of free sCT, sCT:SDOCS, sCT:C10 complexes, and the complexes loaded into SNEDDS was assessed by administration to rats via oral gavage at a dose of 1 mg/kg, and compared to a subcutaneous dose (s.c) of sCT at 0.1 mg/kg.

Results: For both sCT complexes, the CE% increased with increasing AnS molar ratio, reaching ≥ 88% at 1:4 sCT:AnS. Increasing the AnS molar ratio further caused a decrease or a plateau in the CE% (Figure. 2A). The optimal molar ratio depends on the ability of the AnS to neutralize the sCT. sCT complexes loaded into SNEDDS exhibited significant protection against trypsin when compared to free sCT and the complexes alone, with SDOCS complex exhibiting over 55% protection as complex alone and retaining 70% of sCT remaining intact after 120 mins of proteolysis when loaded into SNEDDS. In contrast, sCT:C10 complex showed higher protection only when loaded into SNEDDS with > 55% sCT remaining after 120 mins, compared with C10 standalone complex which was almost fully degraded after 30 mins (Figure. 2B). sCT:SDOCS complex alone reduced calcium level to 72% of the initial, while complex-loaded SNEDDS significantly reduced calcium level to reach 49% exhibiting a significant pharmacological activity (PA) of 8.4%, as a measure of hypocalcemic effect relative to (s.c) dose.  In comparison, sCT:C10 complex did not significantly differ from free sCT in reducing calcium levels 85%, however when loaded into SNEDDS it reduced calcium level to 65% achieving a 5% PA. These findings highlight the superior performance of SNEDDS loaded complexes over their standalone counterparts, with the docusate complex consistently demonstrating greater efficacy despite the permeation properties of C10 (Figure. 2C, Table 1). This shows that the type of HIP is a key parameter for an effective peptide complex, alongside the advantages of SNEDDS as a drug delivery system.

Conclusion: Our results demonstrated that high complexation efficiency was achieved through the ionic interactions between the positively charged sCT and AnS. Loading these complexes into SNEDDS significantly enhanced protection against proteolysis compared to free sCT or standalone complexes. Oral administration of the complex-loaded SNEDDS resulted in significantly greater pharmacological activity than both free sCT and the complexes alone. The lipophilic docusate complex outperformed the C10 complex, indicating that the permeation-enhancing properties of C10 did not significantly aid absorption compared to other counterions. These findings highlight that the type of HIP is a crucial factor in the effectiveness of SNEDDS for oral sCT delivery.

References: 1 Ibrahim, Y. H. Y., Regdon, G., Jr., Hamedelniel, E. I. & Sovany, T. Review of recently used techniques and materials to improve the efficiency of orally administered proteins/peptides. Daru 28, (2020).
2 Haddadzadegan, S., Dorkoosh, F. & Bernkop-Schnurch, A. Oral delivery of therapeutic peptides and proteins: Technology landscape of lipid-based nanocarriers. Adv Drug Deliv Rev 182, (2022).
3 Li, P., Nielsen, H. M. & Mullertz, A. Oral delivery of peptides and proteins using lipid-based drug delivery systems. Expert Opin Drug Deliv 9, 1289-1304, (2012).
4 Ristroph, K. D. & Prud'homme, R. K. Hydrophobic ion pairing: encapsulating small molecules, peptides, and proteins into nanocarriers. Nanoscale Adv 1, 4207-4237,(2019).
5 Twarog, C. et al. Intestinal Permeation Enhancers for Oral Delivery of Macromolecules: A Comparison between Salcaprozate Sodium (SNAC) and Sodium Caprate (C(10)). Pharmaceutics 11, (2019).

Acknowledgements: Passant M. Al-Maghrabi would like to thank the Egyptian government scholarship (Egyptian Ministry of Higher Education & Scientific Research) for the financial support.

Figure 1: Schematic representation showing the sCT:AnS complex formation applying Hydrophobic Ion Pairing (HIP) method.

Figure 2: [A] Complexation efficiency (CE%) of sCT:AnS complexes at different molar ratios of sCT to AnS; [B] % Salmon calcitonin remaining during 120 min proteolysis of sCT:AnS complexes and complexes loaded into SNEDDS (Data are shown as mean±SD, n=3); [C] Plasma calcium level % after per oral (p.o) dose of free sCT(green line), sCT:C10 complex (purple dotted line),sCT:SDOCS complex (blue dotted line), sCT:C10 complex loaded into SNEDDS formulation (purple line), sCT:SDOCS complex loaded into SNEDDS formulation (blue line), at a dose of (1mg/kg), and a subcutaneous (s.c) dose of sCT (at 0.1mg/kg, grey line). All in vivo data are shown as (mean±SD, n=5-6).

Table 1. Pharmacodynamic parameters after dosing of sCT formulations.