(M1530-06-36) Development of Polymeric Dual-Drug Nanoformulation: Enhancing Bioavailability and Preclinical Efficacy in Diabetic Nephropathy Treatment

Purpose: Diabetes Mallitus (DM) is steadily increasing worldwide and is expected to rise by 51% from 2019 to 2045, reaching 0.7 billion cases, with developing countries most affected. It can lead to complications like diabetic nephropathy (DN), characterized by reduced glomerular filtration rate and persistent albuminuria. Current treatments focus on managing blood pressure, glucose, and triglycerides. However, the underlying molecular mechanisms of DN-particularly fibrosis, oxidative stress, and inflammation, are often overlooked despite their critical role in disease progression. In this study, Lisofylline (LSF) and C-peptide (CPep) are the drugs of choice due to their significant clinical potential in treating both T1DM and T2DM individually. They modulate immune cell function to reduce autoimmunity and oxidative stress while preserving β-cell insulin secretion under inflammatory conditions. Although these molecules have shown pharmacological effects in trials, achieving therapeutic efficacy remains challenging due to their short half-life (5–30 minutes), even with continuous intravenous infusion. The purpose of preparing this polymeric nanoformulation provided a synergistic efficacy, which helped in restoring the diabetic aortic dysfunction and improving management of diabetic nephropathy (Figure 1).

Methods: In this study, we combined the therapeutic benefits of the LSF prodrug and C-peptide (CPep) through co-delivery using an in-house synthesized high molecular weight cationic polymer. The approach involved encapsulating the LSF prodrug within polymeric nanoparticles, followed by lyophilization and electrostatic complexation with CPep. The resulting combination formulation was evaluated for its antioxidant, anti-apoptotic, and proliferative effects on metabolically stressed kidney and insulinoma cells. After confirming hemocompatibility, we generated pharmacokinetic profiles and compared them with free LSF, LSF prodrug, and CPep nano-complexes^1,2. The efficacy of the co-delivery system was further assessed in a streptozotocin-induced diabetic nephropathy model in Wistar rats, focusing on its impact on pancreatic islets and diabetic kidney complications.

Results: Prodrugs and a cationic polymer were synthesized to develop a nanoformulation for combination therapy using LSF and CPep, aimed at treatment of T1D induced DN animal model. A co-loaded nanoformulation (mPLM-LSF-OA-CPep) was developed by encapsulating LSF in cationic nanoparticles (mPLM-LSF-OA) and complexing with CPep. mPLM-LSF-OA showed antioxidative and proliferative effects in stressed MIN-6 and NRK-52E cells, increasing GSH levels 1.62- and 1.27-fold, while mPLM-LSF-OA-CPep further enhanced them to 2.06- and 1.8-fold, respectively. The non-compartmental analysis showed improved pharmacokinetics for LSF and CPep when delivered as mPLM-LSF-OA and mPLM-LSF-OA-CPep. mPLM-LSF-OA increased LSF’s half-life by 15.42- and 2.60-fold compared to free LSF and its prodrug, respectively. mPLM-LSF-OA-CPep extended CPep’s half-life 6.45-fold and reduced clearance by 12.09-fold versus free CPep. In vivo, mPLM-LSF-OA-CPep treatment reduced IL-6 and TNF-α levels by ~2.69- and ~2.05-fold compared to STZ-induced DN controls. The formulation also improved insulin levels ~6.8-fold and increased Ki-67+ cells in pancreatic islets, indicating enhanced cell proliferation. H&E and PAS staining showed reduced inflammation, mesangial expansion, matrix deposition, and near-normal glomerular structure, supporting the therapeutic potential of the LSF-OA and CPep combination in treating DN.

Conclusion: This study investigated a co-delivery approach combining LSF and CPep for the treatment of DN. Notably, this combination has not been previously explored for its therapeutic efficacy or formulation. The findings emphasize the potential synergistic effects and enhanced effectiveness of this combined therapy in managing DN.

References: 1. Arihant Kumar Singh, Kishan Italiya, Saibhargav Narisepalli, Deepak Chitkara, Anupama Mittal, Role of chain length and degree of unsaturation of fatty acids on physicochemical and pharmacological behaviour of drug-fatty acid conjugates in diabetes, Journal of Medicinal Chemistry (2021) 64, 14217-29.
2. Arihant Kumar Singh, Shubham A. Salunkhe, Deepak Chitkara, Anupama Mittal, Potent anti-inflammatory and anti-apoptotic activities of electrostatically complexed C-peptide nanospheres for ameliorating diabetic nephropathy, Biomaterial Advances (2024) 163, 213935.


Rationale and Purpose of Nanoformulation Design