Category: Formulation and Quality
Purpose: Lisofylline (LSF) is an anti-inflammatory agent and active metabolite of pentoxifylline (PTX) with proven anti-diabetic activity. Inspite of being a potent molecule, LSF poses certain major disadvantages, including high solubility (~60 mg/mL) and rapid metabolism (Rapid interconversion between LSF and PTX), resulting in poor bioavailability (~5.9 % in human) and short half-life (~0.75 h) which limits its clinical development. In the present work, our purpose was to overcome the physicochemical and pharmacokinetic limitations associated with LSF by LSF-fatty acid prodrug approach. We focused on designing an omega-3 fatty acid based conjugate of LSF and delivering the same as a novel nanomiceller formulation. The rationale of preparing a fatty acid conjugate of LSF includes, a) imparting hydrophobicity to the drug for efficient encapsulation into the delivery system and b) protecting the hydroxyl group to reduce its metabolism which prolongs the half-life and enhances the efficacy of the drug in diabetes.
Methods: We designed a LSF-fatty acid conjugate which could self-assemble into micelles of nano size range and characterized it by various spectroscopic (1H and 13C NMR, HR-MS, FT-IR) and analytical techniques (HPLC, DSC). LSF-LA conjugate was assessed for its self-assembling ability, particle size, size distribution, zeta potential, critical micellar concentration (CMC), micellar aggregation number, protein interaction and stability in rat plasma. Further, we assessed in-vitro and in-vivo efficacy of the self-assembled micelles of LSF-LA conjugate in type 1 diabetes (T1D). For in-vitro efficacy study, LSF-LA conjugate (~20 µM to LSF) was incubated with insulin secreting murine cells, MIN-6 and evaluated for cytotoxicity of the conjugate. Further the MIN-6 cells were exposed to inflammatory challenge with cytokines (IFN-γ, TNF-α and IL-1β) and protective effect of LSF-LA conjugate on cell viability and insulin secretion was studied. It was also tested for its ability to suppress activated peripheral blood mononuclear cells (PBMC) proliferation, inflammatory cytokine production and cellular uptake (quantitative analysis by HPLC). Pharmacokinetic (PK) studies of LSF-LA conjugate and free LSF were performed in wistar rats at dose of 25 mg/kg by i.v. route to determine the improvement in PK parameters. Further, in-vivo efficacy was evaluated in Streptozotocin (STZ) induced T1D wistar rat model. For the efficacy testing, LSF-LA conjugate was given at reduced dose of 15 mg/kg once daily in comparison to free LSF (25 mg/kg twice daily and 15 mg/kg once daily) for 1 week. After 1 week of treatment, pancreatic tissues were isolated and analyzed by Hematoxylin and eosin (H&E) staining and immunohistochemistry for insulin.
Results: Synthesized LSF-LA conjugate self-assembled micelles (156.9 nm; PDI 0.187; CMC 1 μg/mL; aggregation number 54) without any surfactant had shown enhanced cellular uptake. It also protected MIN6 insulinoma cells from cytokine induced cell death and enhanced insulin production under inflammatory condition. It also suppressed the proliferation of PBMC and production of inflammatory cytokines, IFN-γ and TNF-α (Figure 1). LSF-LA micelles exhibited reduced protein binding, significantly higher half-life (5.7-fold) and higher apparent volume of distribution (5.3-fold) than free LSF (Figure 2). In T1D animals, reduced blood glucose level and increased plasma insulin level were observed at a reduced dose (~15 mg/kg, once daily of LSF-LA micelles vs. 25 mg/kg, twice daily of free LSF) which was further confirmed by immunohistochemical analysis (Figure 3).
Conclusion: Our results have demonstrated that as compared to free LSF, LSF-LA micelles consisting of hydrophilic LSF and hydrophobic LA linked through an ester linkage exhibited a much better therapeutic effect in insulin secreting MIN-6 cells as well as in STZ induced T1D model. Apart from the conjugation approach, the improvement in the therapeutic effect of the LSF-LA conjugate can also be attributed to its ability to self-assemble into micelles. Additionally, it also reduced the LSF-PTX interconversion (major concern resulting in low bioavailability of LSF) thus showing efficacy at a reduced dose and dosing frequency, which would eliminate the high-dose requirement of LSF for effective treatment of T1D. Overall, we believe that this LSF prodrug strategy based on self-assembly of amphiphilic drug fatty acid conjugate may open up new avenues in treatment of T1D and could be further explored in treatment of other autoimmune diseases where LSF has demonstrated significant therapeutic efficacy. References: Italiya, Kishan S., et al. "Self-assembling lisofylline-fatty acid conjugate for effective treatment of diabetes mellitus." Nanomedicine: Nanotechnology, Biology and Medicine 15.1 (2019): 175-187.
Samrat Mazumdar– Pilani, Rajasthan, India
Saurabh Sharma– Pilani, Rajasthan, India
Deepak Chitkara– Pilani, Rajasthan, India
Ram Mahato– Professor and Chair, University of Nebraska Medical Center, Omaha, Nebraska
Anupama Mittal– Pilani, Rajasthan, India