Sustained Delivery Of IL-1Ra From Pluronic F127-based Thermosensitive Gel And Its Prolonged Therapeutic Effects On Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) has been considered as an auto-inflammatory syndrome which is characterized by progressive β-cell dysfunctioning and insulin resistance. These two factors are recognized as a major cause for unproductiveness of pancreatic islets leading to insufficient insulin secretion in response to high blood glucose and metabolic demand. The role of various pro-inflammatory mediators in pathogenesis of T2DM is considered to be crucial; thereby, the insight of inflammatory responses in T2DM may provide a new gateway for the better treatment of T2DM. Conventional anti-diabetic agents are becoming less prescribed to treat T2DM as most of them cause hazardous effects. Currently, many anti-inflammatory therapeutic modalities are being investigated to abate the infuriating effects of inducers of T2DM and among them, interleukin-1 receptor antagonist (IL-1Ra) is considered one of an effective anti-diabetic agent. IL-1Ra is a naturally occurring anti-inflammatory antagonist of pro-inflammatory cytokines belonging to IL-1 family. The broad spectrum anti-inflammatory effects of IL-1Ra have been investigated against various auto-immune diseases. Despite of its outstanding broad spectrum anti-inflammatory effects, IL-1Ra has short biological half-life and to cope with this problem, up till now, many strategies have been applied either to extend the half-life and/or prolong the sustained release of IL-1Ra from its target site, but none of these approaches have been utilized to investigate the anti-diabetic effects of IL-1Ra.To overcome the limitation of short half-life of IL-1Ra and to investigate its therapeutic potentials, we first performed a cross-species comparison of amino acid sequences of IL-1Ra_human and IL-1Ra_rat, and their computational docking on rat receptor by utilizing various bioinformatics tools for structure determination, sequence alignment and protein docking of concerned proteins. The similarity and identity of sequences and structures of IL-1Ra_human and IL-1Ra_rat were 85.4% and 73.6% respectively. Two of the three receptor residues i.e. LYS290 and ASP259 were common in the docking of both ligands on rat receptor which elucidates that the binding sites for both ligands are apparently similar signifying that both ligands will probably possess the same therapeutic efficacy. So, there was need to prove whether human IL-1Ra would effectively work in rat or not. Thereby, we extended our approach to investigate the therapeutic effects of EL-1Ra using diabetic GK-rats as an experimental model. We administered IL-1Ra (10 mg/kg twice day) subcutaneously into GK-rats for one month. IL-1Ra decreased the onset of hyperglycemia, but did not affect the body weight and/or food intake. Moreover, IL-1Ra also improved the glucose tolerance and insulin sensitivity in response to exogenously administered glucose. The results of this study revealed that IL-1Ra improved normoglycemia, but high doses with frequent dosing intervals were required to achieve desired therapeutic effects.To overcome the problem of short biological half-life of IL-1Ra, for the very first time, we developed a new dosage form of IL-1Ra using pluronic F127 (PF127) as thermosensitive gel and investigated the in vitro and in vivo characteristics of IL-1Ra loaded in PF127. As a result, PF127 gel showed in vitro sustained release of IL-1Ra depending upon the concentration of PF127 used. SDS-PAGE confirmed the stability of IL-1Ra during its in vitro release. When compared directly with IL-1Ra solution, IL-lRa loaded in PF127 gel exhibited prolonged in vivo release, greater efficacy to induce hypoglycemia and inhibited IL-1β-stimulated production of IL-6 in Wistar-rats. We further extended this approach to investigate the stability of IL-1Ra from PF127 gel stored at 4℃,25℃ and 40℃ for 3 months. From the results, it was found that rheological properties and in vitro release pattern of IL-1Ra from PF127 gel stored at 4℃ were almost same throughout the stability study period. No drug-polymer interaction were found throughout the stability study period whereas results of SDS-PAGE confirmed the stability of IL-1Ra loaded in PF127 gel.Subsequently, we progressed this approach to look at in vivo efficacy and pharmacokinetics of IL-1Ra using diabetic GK-rats to test the anti-diabetic efficacy of IL-1Ra loaded in PF127 gel. We administered IL-1Ra (10 mg/kg/day) loaded in 25% PF127 gel subcutaneously for one month. IL-1Ra loaded in PF127 gel exhibited a sustained and prolonged hypoglycemic effects on treated animals. IPGTT results showed that IL-1Ra loaded in PF127 gel increased the glucose tolerance in treated rats along with increased insulin sensitivity and β-cell’s secretory function. Moreover, significant reduction in pro-insulin/insulin ratio, lipid profiles and IL-6 were also observed. Immunohistochemical analysis showed slight macrophages infiltration in pancreatic islets in IL-1Ra loaded in PF127 gel treated rats as compared to that of saline treated GK-rats. Histochemical analysis revealed no PF127-induced alteration in the normal physiology of skin and kidneys of treated animals. Hence, we concluded that IL-1Ra loaded in PF127 gel has a potential to exhibit broad spectrum anti-inflammatory effects alleviating the symptoms of T2DM. The significant and prolonged therapeutic effects of IL-1Ra on T2DM using PF127 as sustained delivery systems might be helpful in future as treatment strategy for the management of T2DM in patients who are genetically deficient of IL-1Ra. Moreover, this study also highlight the safety profile of IL-1Ra loaded in PF127 having negligible side effects because of which this approach can be translated into significant clinical outcomes in future. We believe that this methodology for sustained delivery of IL-1Ra loaded in PF127 gel might be one of the best dosage form suitable and convenient for the patients to achieve desired therapeutic potentials without exceeding the dose limits-and frequent administration.