Category: Formulation and Quality
Purpose: Our Idea: To develop a liposomal formulation of carboplatin for the treatment of lung cancer. Problem: Patients suffering from cancer are treated with anticancer agents. Toxic effects associated with anticancer agents are bone marrow suppression, peripheral neuropathy, nephrotoxicity, ototoxicity, nausea, vomiting. These toxic effects are related with dose of cytotoxic agents.
The Problem That Our Idea May Solve: Liposomal formulation of anticancer agent is developed by us with an aim to improve quality of life and lifespan of cancer patients. We aim to achieve it by improving drug utilization as well as by reducing drug-dose related toxicity. To achieve this objective, formulation develop by us should be administered to lung cancer patients via pulmonary route.
Methods: HPLC Method for Carboplatin Estimation: HPLC system was utilized to estimate carboplatin. The system was coupled with PDA detector. Chromatographic detection was implemented using the Analytical column and guard column. Mobile phase comprised a blend (40:60) of acetonitrile and Phosphate buffer(0.02mM pH6.8).
Preparation of Liposomal Dispersions: Lecithin and cholesterol were dissolved in ethanol at 60 ±2. Carboplatin was dissolved in aqueous phase (Phosphate buffer, pH6.8) and temperature of aqueous phase was maintained at 60 ±2. Organic phase was taken in 1ml syringe fitted with 25gauge needle. Organic phase was added to aqueous phase, drop by drop, within time limit of 2min ±20sec and continuous stirring using magnetic stirrer.Contents were continued to be stirred at 60 ±2 and 300-320 rpm for a period of 30 min after completion of addition of organic phase.The liposomal dispersion so obtained was filled in 10ml vial fitted with rubber plug and sealed with aluminum cap.
Plackett-Burman Screening Design: PB screening design is used to identify the most important factors early in the experimentation phase when complete knowledge about the system is usually unavailable Each factor was set at a HighLevel, and LowLevel. The actual values and coded values of independent variables & dependent variables are given in Table-1 and Table-2 respectively. Design Expert® software was employed for data analysis.
Lyophilization of Liposomal Dispersion: Liquid liposomal dispersion has disadvantage in terms of drug leakage through liposomal membrane during storage.
Determination of Entrapment Efficiency and Drug Leakage: Separation between the free drug and the encapsulated drug could also be achieved by the use of a dialysis membrane with an appropriate cut-off. The liposome sample was dialyzed against a buffer solution for total 48hours using Tube-O-Dialyzer. After 24hours,a sample of dialysis fluid was withdrawn and analyzed for carboplatin content by HPLC method.
Determination of Particle Size and PDI: The vesicle size and PDI of carboplatin loaded liposomes was determined by dynamic light scattering technique using Malvern nanoS90,Ver.6.01(Figure 2).
Transmission Electron Microscopy (TEM):TEM was performed on a Tecnai F20 operating at 200kV. The Liposomal dispersion was pipetted and stain on Carbon-filmed copper mesh grids with Urinal acetate dye.
Stability Study of Liquid Liposomal Dispersion and Lyophilized Liposomes: Vials where filled with 1ml of liquid liposomal dispersion and lyophilized liposomes. Vials were then stored in a refrigerator at a temperature of 2-8℃.Samples were withdrawn at an interval of 1-week, 2-week and 3-week time period. At each time, samples were analyzed for following parameters. 1.Vesicle Size 2.Vesicle Size Distribution 3.Drug leakage (%).
Results: HPLC Method for Estimation of Carboplatin: Table-3 indicates independent variables employed for Carboplatin estimation. Figure-1 shows the HPLC Chromatogram. The Method employed is simple and provides reproducible results.
Effect of Independent Variables on Dependent Variables: Table-4 indicates actual value of independent & dependent variables for 12 batches of liposomes prepared as per PB Design. Table-5 describes whether independent variables have significant/nonsignificant effect on dependent variables it also indicates whether proposed model is good fit or not. Desirability function was employed to formulate and identify the best batch. Composition and characteristics of the optimized batch are indicate in Table-6. The optimized batch was further evaluated.
Stability of Liposomes: Drug leakage occurs during 1st week of storage then after, drug leakage is negligible. As per reported data liposomes did not show significant changes in particle size or PDI during 14-weeks, Stability study also indicate that there was no aggregation and non-significant drug leakage during storage at 2- 4°C.Table-6 and 7 indicates parameters determined for stability studies of liposomal liquid dispersion and lyophilized liposomes respectively.
Transmission Electron Microscopy Study: From the images (1,2) of two different liposomal dispersions, it can be concluded that lyophilized liposomes have greater uniformity in size and shape in comparison of liquid liposomal dispersion (before and after dialysis). Reported data indicate that the Lyophilization process mainly by preventing liposome fusion/aggregation.
Conclusion: Liposome encapsulated carboplatin product was developed with vesicle size 70.16 nm. The product characteristics are acceptable from pulmonary delivery via nebulization. However, this product need to be further evaluated for its efficacy and safety at Preclinical and Clinical level. It is also require to be evaluated with respect to size of droplet after nebulization.