Lazaroid Formulations for Brain Delivery: Development of Nano-structured Lipid Carriers, Optimization, and Pharmacokinetic Evaluation

Purpose and Specific Aims: There are several limitations associated with the current gliobalstoma therapy, namely radiation induced lipid peroxidation and resistance to chemotherapy, resulting in recurrence of glioblastoma. In order to overcome these problems, alternative agents such as lazaroid U74389-G (LAZ) need to be explored. LAZ, a 21-aminosteroid, is a known lipid peroxidation inhibitor in vivo and has also demonstrated anti-proliferative activity in vitro. However, LAZ a potential substrate of P-glycoprotein (P-gp) efflux transporter, is extensively metabolized by Phase I enzymes and has shown high hepatic clearance on intravenous administration leading to poor brain penetration and restricting its potential in treating glioblastoma. One approach to overcome the limitations associated with LAZ is to design drug-loaded nano-carriers engineered to have surface properties and sub-micron size conducive for delivery across the blood brain barrier (BBB) while escaping the clearance by liver. The goal of this study was to investigate the utility of LAZ loaded nano-carriers such as nanostructured lipid carriers (NLCs), increasing the brain exposure while decreasing the liver exposure of LAZ. Three specific aims were proposed in order to achieve our goal; 1) Development, optimization and in-vitro characterization of lazaroid loaded NLCs, 2) development and validation of UPLC-MS/MS for quantification of lazaroid in bio-matrices and 3) evaluation of pharmacokinetics and bio-distribution of lazaroid formulations in Sprague-Dawley rat model. Method: A 2-factor, 5-level Central Composite Design (CCD) along with response surface plots were used to determine the effect of independent variables (amount of DSPE-PEG 2k and % liquid lipid) on dependent variables (particle size, zeta potential and encapsulation efficiency), and providing numerical optimization for LAZ-NLC composition. The optimal LAZ-NLCs were characterized for their physico-chemical properties such as particle size and morphology, surface charge, encapsulation efficiency, crystallinity, hemolytic potential and storage stability using various analytical techniques. A sensitive UPLC/MS-MS analytical method was developed and validated for the analytical quantification of LAZ in rat plasma and brain, liver and lung tissue samples. Male Spargue-Dawley rat groups were dosed intravenously with optimal LAZ-NLC (15 mg/kg) and comparative LAZ citrate (5 mg/kg) and LAZ co-solvent (15 mg/kg) solutions and the plasma pharmacokinetics and brain, liver and lung bio-distribution profiles were evaluated for up to 8 hours. Additionally, male Spargue-Dawley rats were dosed intravenously with increasing dose of the optimal LAZ-NLC from 15 to 60 mg/kg. The brain levels of LAZ 20 minutes post-dose were evaluated in each dose group and were evaluated using ANOVA and power model for assessing dose linearity. Results: The DSPE-PEG 2k had an inverse effect on particle size and zeta potential and a synergistic effect on encapsulation efficiency of LAZ-NLCs. The liquid lipid Labrasol had an inverse effect on particle size and a slight synergistic effect on zeta potential without having any effect on encapsulation efficiency of LAZ-NLC. The optimal LAZ-NLCs measured 172.3 ± 3.54 nm in diameter with surface charge of -4.54 ± 0.87 mV and encapsulation efficiency of 85.01 ± 2.60 %. The optimal LAZ-NLCs were spherical in shape as per transmission electron microscopy and in vitro hemolytic potential was within acceptable limits (<15%) for formulation to blood ratio up to 1. LAZ was solubilized in the lipid matrix of the NLC in an amorphous state as highlighted by differential scanning calorimetry and x-ray diffraction analysis. The optimal LAZ-NLC was stable on storage at 4°C for up to 3 months with slight increase (1.2 times) in particle size and slight decrease (3 times) in zeta potential and insignificant changes in encapsulation efficiency. Although the optimal LAZ-NLC had similar plasma pharmacokinetic profile compared to LAZ citrate solution and LAZ co-solvent groups, different inter-compartmental clearance between groups resulted in differential organ bio-distribution profiles. The LAZ exposure in brain was enhanced by two times with NLC and co-solvent compared to citrate group while a decrease in liver exposure by half was observed for NLC compared to citrate and co-solvent group. The optimal surface properties of optimal LAZ-NLC and presence of DSPE-PEG 2k and Polysorbate 80 were instrumental in increasing the LAZ brain permeability while decreasing its hepatic exposure. A combination of transport mechanisms such as passive diffusion, active endocytic uptake and/or inhibition of efflux transport were proposed for passage of free unbound LAZ as well as intact LAZ-NLC across the BBB. The optimal LAZ-NLC was retained twice longer than citrate group in the lungs. A dose-linearity study indicated non-proportional (11.6 times) increase in brain levels of LAZ at 20 minutes post-dose with linear (4 times) increase in dose of optimal LAZ-NLC from 15 to 60 mg/kg. Conclusion and Significance: We demonstrated the utility of NLCs designed using logical methodology with favorable properties in enhancing LAZ exposure to the brain. We established 1) a CCD matrix for determining the effect of NLC composition on particle size, zeta potential and encapsulation efficiency, 2) a selection criteria for formulation composition in enhancing brain delivery using CCD matrix with the resulting optimal LAZ-NLCs measuring < 200 nm, with a neutral surface charge and high drug payload, 3) the physico-chemical properties of optimal LAZ-NLCs viz. spherical in shape, retention of lipid crystallinity and conversion of LAZ to amorphous state, low hematotoxic potential, and stable on storage, 4) the optimal LAZ-NLC had similar plasma pharmacokinetics as LAZ citrate and co-solvent groups, but a disparity in the bio-distribution pattern was seen with two times increase in brain levels of LAZ for both NLC and co-solvent compared to citrate group with reduction in LAZ liver levels by half for LAZ-NLC compared to citrate and co-solvent group, and 5) a non-linear increase in amount of LAZ in brain on linearly increasing the dose of optimal LAZ-NLC. Our study is significant since it laid the foundation for the pre-clinical efficacy testing of LAZ-NLC in treatment of glioblastoma and its potential translation to clinical setting.