(T0930-07-47) Pharmacokinetic Study of the Effect of Buffer on Long-Acting Injectable Formulations

Purpose: Long-acting injectable formulations are highly sought after to mitigate side effects, reduce dosing frequency, and ultimately enhance patient adherence. However, obstacles such as lengthy manufacturing processes, dosage limitations, initial burst release, injection site reactions, and the necessity for large needles impede their widespread adoption. Our group has developed cholesterol-modified hyaluronic acid (CHHA), which forms nanosized hydrogels (HA nanogel) in water and exhibits versatile encapsulation properties for small molecules, peptides, and proteins ¹⁾. Previous studies have shown that HA nanogel works as a highly functional excipient for sustained-release material through salting out in physiological saline environments. However, detailed investigations into how buffers affect the sustained-release properties of these HA nanogel formulations have been lacking. This study aims to enhance formulation design by analyzing the pharmacokinetics (PK) of Cyclosporin A (CyA) in buffer formulations or without, evaluating PK parameters, and assessing residual API in the gel post-PK study.

Methods: Preparation of CHHA-Cyclosporine A Formulation without buffer: CHHA (Mw of HA: 10k, Degree of substitution of cholesterol: 30%) was dissolved in a 10% sucrose solution and mixed with Cyclosporine A powder. The mixture was stirred overnight at room temperature to prepare the CHHA-CyA formulation (CHHA concentration: 30 mg/mL, Cyclosporine A: 3 mg/mL). Preparation of CHHA-Cyclosporine A buffer-containing Formulation: CHHA was dissolved in a 10% sucrose solution. Then, Cyclosporine A (21 mg) was weighed into a clean 20 mL vial. Subsequently, 630 μL of 20 mM sodium acetate buffer was added. Following this, 4666.7 μL of 45 mg/mL HA nanogel aqueous solution was added. The mixture was stirred for 30 seconds. Next, 1563.3 μL of a 10% sucrose aqueous solution was added and stirred for 30 seconds. Then, 140 μL of 100 mM phosphate buffer was added and stirred for 30 seconds. Finally, the mixture was stirred overnight (CHHA concentration: 30 mg/mL, Cyclosporine A: 3 mg/mL, acetate buffer concentration: 2 mM, phosphate buffer concentration: 2.0 mM). After that, a pharmacokinetic study was performed. The mean plasma concentration versus time plot was obtained after subcutaneous administration of 15 mg/kg Cyclosporine A to Sprague Dawley rats. We compared two types of formulations: acetate and phosphate buffer systems and hyaluronic acid nanogel alone. The solution formulation of CyA with 10% α-Cyclodextrin was also injected subcutaneously (2.5 mg/kg) for the control. CyA concentrations at various time points were measured. Concentrations of Cyclosporin A in the plasma, in the formulation, and in the collected gel were analyzed using an LC-MS/MS method. WinNonlin (PhoenixTM, version 8.3) software was used for pharmacokinetic calculations. The following pharmacokinetic parameters were calculated, whenever possible, from the plasma concentration versus time data.

Results: The pharmacokinetic (PK) profile of CyA following subcutaneous administration was evaluated in rats. The results indicated that CHHA-CyA formulation exhibited a prolonged release profile with sustained plasma concentrations over the observed period (840 h). The area under the curve (AUC) was calculated to assess the overall exposure of CyA in the systemic circulation. The data demonstrated a significant AUC, indicating effective absorption and prolonged presence of the drug in the bloodstream (Figure 1). An analysis of pharmacokinetics showed that the area under the plasma concentration-time curve from zero to 35 days (AUClast) appeared to increase in the buffer HA nanogel formulation (Figure 2). The acetate and phosphate buffer formulation demonstrated improved release of CyA, with lower residual API in the gel. This suggests that buffer formulations are promising as sustained-release materials for HA nanogel formulation development.

Conclusion: Overall, the PK profile of CyA supports its potential for long-term therapeutic applications, providing consistent and sustained drug levels in the plasma. Pharmacokinetic analysis of CyA in acetate and phosphate buffer formulations confirmed enhanced release properties. This indicates that acetate and phosphate buffer systems are effective in the design of sustained-release formulations using HA nanogel, providing valuable insights for formulation development.

References: 1) T. Nakai, et al. Macromolecular Bioscience, 2012, 475-483.


Figure 1. Plasma concentration vs time profile for Cyclosporin A after Subcutaneous injection in Sprague Dawley Rat (mean±S.D., n=3).


Figure 2. Comparative analysis of AUC/Dose (left axis, bar graph) and drug concentration in gel matrix post-PK study (right axis, line graph).