(M1130-07-41) Development and Verification of Novel Agarose Gel Based Dialysis Cassette System as an In Vitro Release Model for Long-Acting Injectable Formulations

Purpose: This research aims to develop a robust, physiologically relevant in vitro model for gel based long-acting injectable (LAI) formulations by utilizing a novel agarose gel-based dialysis cassette system and correlate the in vitro release to the in vivo performance. The study also aims to establish In vitro-in vivo correlation (IVIVC) through in-silico modeling and provide biorelevant insights into the varied drug release mechanism across different types of LAI gel formulations.

Methods: 2.1 Experimental-LAI gel formulations were prepared using 20 wt% drug and 20 wt% polymer, with Resomer RH-502 for Gel-1 and Resomer RH-504 for Gel-2. Both formulations formed clear solutions upon the addition of 60 wt% N-methyl pyrrolidone (NMP) to the drug-polymer mixtures. 2.2.1 In vitro release studies- For in vitro release testing, G3 Slide-A-Lyzer dialysis cassettes (1kDa) were utilized. Agarose gel solution was loaded and allowed to set inside the dialysis cassettes, creating a controlled environment for drug diffusion. The LAI formulations were injected into the gel cassettes and placed inside beakers containing PBS buffer, which acted as the sink media. Sample quantification was performed using a Waters Alliance e2695 HPLC with a UV/VIS detector (210 nm). The mobile phase consisted of 10 mM phosphate buffer (pH 6.6) in water at a 1 mL/min flow rate, with a 2 μL injection volume. Reverse-phase chromatography was conducted using a Zorbax® C18 column (150 × 4.6 mm, 5 μm; Agilent Technologies) and data analysis was performed using Empower software (Waters Corporation, Milford, MA) 2.2.2 In vivo animal studies- The in vivo studies were performed using rat models with plasma samples collected at regular timepoints over a period of two weeks. 2.2.3 In silico modeling- The in vitro release data obtained from the gel-based assay is modeled along with the in vivo data from the rat model using Gastroplus 9.9 software.

Results: 3.1 Results 3.1.1 In vivo release- In vivo, both gel formulations showed an initial burst release within the first two hours of post-injection, followed by a sustained release over two weeks. 3.1.2 In vitro release- In vitro release studies demonstrated a continuous increase in drug concentration, with Gel-1 exhibiting faster release kinetics compared to Gel-2. 3.2 Discussions: Upon injection, the clear gel solutions transformed into a white opaque gel-like suspension inside the body of the animals, as observed in MRI images at the injection site. A similar white gel formation was noted in vitro within the agarose gel matrix, indicating a physiological response replicated effectively in the in vitro system. The LAI gel formulations followed a biphasic release profile: an initial burst phase due to rapid drug diffusion from the loosely formed gel structure, followed by a sustained release phase over several days as the gel matrix degraded, allowing deeper-embedded drug molecules ^[1]. to diffuse. 3.3 In silico modeling- The in-silico modeling of in vitro data for both LAI gel formulations demonstrated a strong fit to the double Weibull model, as shown in Figures 1(a) and 2(a).  overlay of the predicted and observed in vivo profile as shown in Fig. 1(b) and Fig 2(b). closely mirrors the sustained release pattern for LAI gel formulations. Deconvoluted in vivo release profile and its overlay with the in vitro release data, illustrated in Fig. 3(a), shows an increasing trend in the fraction of drug released over time with in vitro release being faster than in vivo and gel-1 exhibiting faster kinetics compared to gel-2. Further, convolution analysis of the in vitro data to predict in vivo performance showed strong alignment, with gel-1 having slightly higher exposure than gel-2, as depicted in Fig. 3(b). This aligns with in vitro findings and in vivo exposure data, where gel-1 showed greater systemic exposure than gel-2 The final IVIVC graph in Fig. 3(c) demonstrated an exceptionally strong correlation ( >99%), reinforcing the robustness of the in vitro assay in predicting in vivo performance.

Conclusion: By bridging the gap between in vitro release testing and in vivo pharmacokinetics, this innovative gel-based dialysis cassette system offers a reproducible, physiologically relevant, and scalable approach for LAI formulation screening. This advancement facilitates a more efficient pathway for LAI development, by reducing extensive in vivo studies and expediting regulatory approval processes.

References: 1. Journal of Pharmaceutics: In Vitro Evaluation of Poly(lactide-co-glycolide) In Situ Forming Gels for Bedaquiline Fumarate Salt and Pharmacokinetics Following Subcutaneous Injection in Rats. DOI: 10.3390/pharmaceutics13081231

Acknowledgements: The authors would like to acknowledge Pham Cheim from AbbVie Irvine and Nathaniel Catron from AbbVie Lake County for helping with the formulation preparation