(W0930-07-41) Analysis of the API Sustained-Release Mechanism of Hyaluronic Acid Nanogel (HA nanogel) Formulation

Purpose: We have been developing cholesterol-substituted hyaluronic acid (CHHA) as a new pharmaceutical excipient and manufacturing it. Previously, it has been reported that CHHA self-assembles in water to form nanosized particles (HA nanogel), encapsulates various types of APIs simply by mixing, and functions as a sustained-release material through salting out in physiological saline environments¹⁾. The drug release mechanism of similar materials such as Cholesterol bearing pullulan (CHP) nanogel has been reported to primarily be based on the exchange of drugs with Bovine Serum Albumin (BSA)²⁾. We believed that HA nanogel would work in the same way. However, it had not been confirmed whether HA nanogel formulations exchanged drugs with proteins such as BSA. Therefore, to advance the development of HA nanogel as a sustained-release material, we analyzed the drug release mechanism using several fluorescently labeled APIs.

Methods: Preparation of Fluorescently Labeled Compounds: Alexa Fluor™ 488 NHS Ester was used to fluorescently label semaglutide dissolved in carbonate buffer (Alexa488-semaglutide). The labeled compound was purified using HPLC. Similarly, Alexa Fluor™ 647 NHS Ester was used to fluorescently label BSA dissolved in carbonate buffer (Alexa647-BSA), and the labeled compound was purified using a MWCO 3KDa ultrafiltration membrane. Both solutions were lyophilized to obtain Alexa488-semaglutide and Alexa647-BSA. Preparation of CHHA-Semaglutide Formulation: CHHA was dissolved in water and mixed with Alexa488-semaglutide powder. The mixture was stirred overnight at room temperature to prepare the CHHA-semaglutide formulation (CHHA concentration: 25 mg/mL, Alexa488-semaglutide concentration: 4 mg/mL). In vitro release test: 100 µL of the CHHA-semaglutide formulation was added to a 2 mL microtube, followed by gently adding 500 µL of either 1x phosphate buffer saline (PBS) or 0.8% BSA/PBS containing Alexa647-BSA. The mixture was incubated at 37°C. At predetermined time points, 60 µL of the supernatant was collected, and 60 µL of the release medium was replenished. Analysis of Drug Release: The sampling solution was diluted five-fold with water and analyzed using size exclusion chromatography (SEC). 10mM phosphate buffer was used as the mobile phase, and TSKgel G3000SWXL(Tosho) was used for the SEC column. A fluorescence detector was used to measure the release amount of Alexa488-semaglutide and to determine the fraction in which Alexa488-semaglutide was present (Figure 1).

Results: After adding the release medium to the CHHA-semaglutide formulation and incubating at 37°C, the formation of pellets was confirmed. Analysis of the sampling solution over time showed a gradual release of alexa488-semaglutide, and it was confirmed that alexa488-semaglutide was released even in 1xPBS as the release medium. Furthermore, using 0.8% BSA/PBS as the release medium resulted in a higher release of alexa488-semaglutide. Additionally, when the concentration of alexa488-semaglutide was calculated from the Alexa-488 derived peak in the fractions where CHHA, BSA, and alexa488-semaglutide eluted, it was found that alexa488-semaglutide was mostly not present within the HA nanogels. Instead, the majority of alexa488-semaglutide existed either alone or bound to BSA.

Conclusion: In vitro release tests were conducted using nanogel formulations encapsulating fluorescently labeled drugs, and the drug release mechanism was verified by analyzing the fractions where the fluorescent peaks appeared in the supernatant. For the formulation using alexa488-semaglutide, it was suggested that the drug release mechanism differs from that of similar nanogels, as alexa488-semaglutide was released even when PBS was used as the release medium. Additionally, the concentration of alexa488-semaglutide in the supernatant increased with the replacement of the release medium over time, and further analysis indicated that alexa488-semaglutide existed independently. These results suggested that the drug release mechanism would involve drug dissociation from the nanogels by tilting the dissociation equilibrium towards the dissociation side due to concentration gradients. As a future work, we will focus on identifying the interaction sites between nanogels and drugs to deepen the understanding of the drug release mechanism.

References: 1) T. Nakai, et al. Macromolecular Bioscience, 2012, 475-483
2) K. Akiyoshi, et al. Journal of Controlled Release, 1998, 313-320

Acknowledgements: This research was funded by Asahi Kasei Corporation.


Figure 1. Representative SEC chromatograms of the HA nanogel, the Alexa488- Semaglutide, the Alexa647-BSA, and the sampling solution at 336h.


Figure 2. The cumulative release of semaglutide using 1xPBS(left) or 0.8% BSA/PBS (right) as the release media (mean±S.D, n=3).