Preformulation and stability in biological fluids of the retrocyclin RC-101, a potential anti-HIV topical microbicide

Abstract

Background: RC-101, a cationic peptide retrocyclin analog, has in vitro activity against HIV-1. Peptide drugs are commonly prone to conformational changes, oxidation and hydrolysis when exposed to excipients in a formulation or biological fluids in the body, this can affect product efficacy. We aimed to investigate RC-101 stability under several conditions including the presence of human vaginal fluids (HVF), enabling the efficient design of a safe and effective microbicide product. Stability studies (temperature, pH, and oxidation) were performed by HPLC, Circular Dichroism, and Mass Spectrometry (LC-MS/MS). Additionally, the effect of HVF on formulated RC-101 was evaluated with fluids collected from healthy volunteers, or from subjects with bacterial vaginosis (BV). RC-101 was monitored by LC-MS/MS for up to 72 h.

Results: RC-101 was stable at pH 3, 4, and 7, at 25 and 37°C. High concentrations of hydrogen peroxide resulted in less than 10% RC-101 reduction over 24 h. RC-101 was detected 48 h after incubation with normal HVF; however, not following incubation with HVF from BV subjects.

Conclusions: Our results emphasize the importance of preformulation evaluations and highlight the impact of HVF on microbicide product stability and efficacy. RC-101 was stable in normal HVF for at least 48 h, indicating that it is a promising candidate for microbicide product development. However, RC-101 stability appears compromised in individuals with BV, requiring more advanced formulation strategies for stabilization in this environment.

Figure 1

Effects of temperature on RC-101 (500 μg/mL) solutions. A) HPLC analysis for RC-101 stored at (solid circle) 25 ± C, (open square) 37 ± C, and (solid triangle) 65 ± C. B)MALDI-TOF MS spectrum of RC-101 in water, exposed for 10 days at room temperature, 100% intensity = 38291 counts.

Figure 2

Effect of pH on RC-101. A) RC-101 under different pH conditions analyzed over time by HPLC. (open square) pH 3, (solid circle) pH 4, (open triangle) pH 7, and (solid square) pH 12. B) CD spectra of RC-101 solution (500 μg/mL) under different pH conditions.

Figure 3

Effect of hydrogen peroxide on RC-101. A) RC-101 under different concentrations of hydrogen peroxide over time analyzed by HPLC. B) RC-101 exposed to hydrogen peroxide 0.002% without EDTA (solid circle), and in the presence of EDTA (open square), over time, analyzed by HPLC.

Figure 4

Representative LC-MS/MS chromatograms for A) Sample A supernatant (blank HVF) at time 0, B) Sample B supernatant (RC-101 solution + HVF) at 72 h, C) Sample C supernatant (RC-101 solution control), and D) Sample D (RC-101 film + HVF) supernatant at 48 h.