In studies described earlier IRL was

In studies described earlier, IRL-1620 was administered intravenously; however, because of its short half-life (Rubin and Levin, 1994) (about 7–8min), the dosing had to be repeated three times. Also, a dose-dependent hypotension was observed with repeated doses of IRL-1620 in rats (Leonard and Gulati, 2009). To overcome these limitations concerning delivery of IRL-1620, we planned to develop a stable liposomal formulation of IRL-1620 with a long circulation time. It is anticipated that the “PEG” coating (Fig. 2) will help the liposomes evade the reticuloendothelial system (RES) delaying their clearance from the body, thereby increasing the half-life of IRL-1620. We believe that an increase in plasma circulation time of IRL-1620 and greater concentration of IRL-1620 in the 69 8 australia will significantly enhance its efficacy as an agent that can reduce the damage and produce neurovascular plasticity of the damaged brain.
Experimental procedures
Results
Discussion This research work was carried out to determine whether the liposomal encapsulated IRL-1620 has greater efficacy in terms of neuroprotection. One of the principal aims of this project is to enhance the permeability of IRL-1620 across the blood–brain barrier using the PEGylated liposomal formulation. Liposomes are small unilameller or mutilamellar phospholipid bilayers surrounding central aqueous compartment. They are made up of natural biological lipids, structurally similar to the cell membrane, and therefore are considered to be biologically compatible with low toxicity. Liposomes have been extensively studied nanocarriers because of their low toxicity and ability to deliver both hydrophilic as well as lipophilic drugs. Some liposomal formulations are also used clinically. Liposomal nanocarriers have been shown to effectively deliver a range of drugs including antifungals, chemotherapeutic drugs antiretrovirals etc. across the BBB because of their composition, size and surface properties (Kakinuma et al., 1996, Zhang et al., 2003, Soni et al., 2005, Garg et al., 2006, Sharma et al., 2006). Ligand targeted liposomal nanocarriers have also been shown to be effective in treatment of brain tumors (Qin et al., 2011, Liu et al., 2014) and AD (Oesterling et al., 2014) Because of the smaller size and the PEG coating on the liposomes allows them to circulate in the body for a longer duration of time. Hydrophilicity due to PEG coating can prevent them from being cleared by the RES. PEG can also inhibit P-glycoprotein (P-gp) efflux pump (Beduneau et al., 2008). We anticipate that all these factors will contribute toward an increased residence time of liposomal nanocarriers in the brain. In addition to this hypothesis other supporting experiments were also carried out that dealt with mechanism of action of free IRL-1620 as a neuroprotectant. In another experiment to probe the mechanism of action of IRL-1620, neuronal growth and differentiation, which is characterized by measurement of length of the extension of neuronal processes termed neurites, was studied. NGF was administered to adhered, undifferentiated PC-12 cells. Once differentiated, the PC-12 cells depend on repeated NGF administration in order to survive, while in the absence of NGF the cells undergo apoptosis. In previous studies in our lab, IRL-1620 was found to neurogenic in vivo (Leonard and Gulati, 2013). In this assay the ability of IRL-1620 to sustain the differentiated PC-12 cells in the absence of NGF was studied. The neurite outgrowth due to treatment with IRL-1620 was determined, using NGF as a positive control. Two different doses of IRL-1620 were used, 1nM and 10nM. The average neurite length, average soma diameter, and average neurite length to soma diameter ratios were measured and quantified with an EVOS FL cell imaging system in phase contrast mode. Statistical analysis using an unpaired T-test indicated that there were no significant differences between the control group and the two treatment groups (p>0.05 defining statistical significance). Because there are no significant differences in the neurite lengths or cell diameters between the three groups, we can conclude that IRL-1620 is able to promote the retention of neurites, at a level comparable to NGF providing evidence that IRL-1620 has similar capabilities in terms of neuroprotection. It can be concluded that ETB receptor stimulation by IRL-1620 will not cause undesirable retraction of characteristic neuronal structures, but rather maintain them. To further elucidate the mechanism of action of IRL-1620 via apoptotic protein pathways, western blots were run on IRL-1620 treated serum-deprived NGF-treated PC-12 cells. The results of these western blots again were found to be in agreement with earlier experiments that showed increased viability of serum-deprived NGF-differentiated PC-12 cells in the presence of IRL-1620. BCL-2 regulates cell death by inhibiting apoptosis. Therefore, higher BCL-2 gene expression leads to reduced apoptotic behavior of the cells. Higher BCL-2 protein expression in the 1nM liposomal IRL-1620 treatment group suggests that the liposomal IRL-1620 not only promotes cell proliferation to prevent apoptosis, but also increases the production of BCL-2 to reverse the apoptotic behavior of differentiated PC-12 cells. The lower BCL-2 gene expression in the positive control group (Fig. 10), might be due to NGF acting via a pathway other than BCL-2 to keep the cells alive. This is supported by a study by Maroto et al. that showed that long term NGF treatment in serum-deprived PC-12 cells did not induce any significant changes in BCL-2 gene expression level when compared to serum-deprived PC-12 cells without NGF treatment (Maroto and Perez-Polo, 1997). With this background and a minimal difference in BCL-2 expression in positive and negative control groups the assessment of higher BCL-2 expression in 1nM liposomal IRL-1620 has its own limitations. Cells treated with liposomal IRL-1620 were found to have a significantly higher BCL-2 protein expression when compared to those treated with free IRL-1620 (p<0.05). This, again, can be explained by the enhanced delivery system using nanoparticles that allow the differentiated PC-12 cells to utilize the IRL-1620 more effectively. Previous studies conducted in our lab also showed a higher cell viability in serum-deprived differentiated PC-12 cells treated with liposomal IRL-1620 when compared to free IRL-1620. Other studies using an immunosuppressant drug FK506 showed a neuroprotective effect on experimental stroke models, and a neuroactive drug Galantamine used for symptomatic treatment of AD, also showed that liposomal encapsulation increases the therapeutic efficacy of those drugs when using PC-12 cells as a model (Ishii et al., 2012, Ishii et al., 2013).