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Plasma protein binding properties are considered as the primary determinants of the pharmacokinetic properties of drugs. Any physiological condition that causes the alteration in the albumin binding of the drugs might lead to change in the pharmacokinetic and pharmacological properties of the drugs. Drug-drug interactions thus play a vital role in the extent of plasma protein binding and consequently the therapeutic effect of the drugs.
Deferasirox mainly used as an iron chelating agent for β-thalassemic patient. Literature survey reveals that Deferasirox should not be co-administered with aluminium containing antacid, otherwise it may cause unwanted side effect like abdominal pain. So the in vitro protein binding of Deferasirox has been conducted by equilibrium dialysis method using UV spectroscopic method. In this study the free fraction of drugs and the % of protein binding of Deferasirox to Bovine serum albumin (BSA) in the presence and absence of aluminium hydroxide was calculated. This study was done to evaluate the interaction of aluminium hydroxide with Deferasirox at physiological pH (7.4) and temperature (37±0.5oc). BSA and Human serum albumin (HSA) have structural similarity.31 In this study, BSA was used instead of HSA, because of its low cost and easy availability.
Preparation of standard solutions
5 mg of Deferasirox was dissolved in 5 mL of methanol and the volume was made up with phosphate buffer of pH 7.4 to 50 mL. From this stock solution (100µg/mL) aliquots of standard solutions were prepared using phosphate buffer of pH 7.4.
Preparation of reagents
1) Buffer solution pH 7.4
50 mL of potassium dihydrogen phosphate solution was placed in 200ml volumetric flask. Add 39.1mL of 0.2N sodium hydroxide solution and make up with distilled water to produce 200mL.
2) Potassium dihydrogen phosphate solution(0.2M)
27.22 gm of potassium dihydrogen phosphate was dissolved in water and diluted with distilled water to produce 1000mL.
3) Sodium hydroxide solution (0.2N)
0.8gm of sodium hydroxide was dissolved and made up to 100mL with water.
4) Bovine serum albumin (3-10-4M)
0.99 gm of bovine serum albumin was dissolved in 25mL of buffer solution of pH 7.4 and make up the volume to 50ml.
Preparation of standard curve
For the spectrophotometric determination, standard calibration curve was plotted using different concentrations of the drug solutions which were prepared in phosphate buffer (pH 7.4) and absorbances were noted at 248 nm. Linearity range was found between 1-10 µg/mL and the correlation coefficient value was found to be 0.9997.
Equilibrium dialysis method
Equilibrium dialysis is one of the method used for the determination of protein binding and this method is used to study the complexation between BSA and the drug. If binding occurs, the drug concentration in the sac containing the protein is greater at equilibrium than the concentration of drug in the vessel outside the sac. At regular intervals samples were withdrawn and analyzed to obtain the concentration of free and complexed drug.
Study of protein binding of Deferasirox
Protein binding of Deferasirox was determined by equilibrium dialysis method. For this, 25 mL of 3-10-4M concentrations of Deferasirox was prepared in phosphate buffer pH 7.4. 25 mL of 3 x 10-4M bovine serum albumin solution (BSA) was taken in glass tube attached to semi permeable membrane (Sigma dialysis sacs, 21mm diameter, and 30cm length). The dialysis membranes were previously activated by immersing it in warm water for 30 minutes. These tubes were then immersed in beakers with 25 mL of phosphate buffer containing fixed concentration of drug solution (3-10-4M). Immediately at zero time, 1mL of the solution was pipetted out from the beaker and it was replaced with 1 mL of phosphate buffer of pH 7.4. Readings were taken at various time intervals by UV spectrophotometer at 248 nm till the absorbance values were constant.
Study of effect of Aluminium hydroxide on in vitro protein binding of Deferasirox
To study the effect aluminium hydroxide on in vitro protein binding of Deferasirox, 25 mL of 3 x 10-4M bovine serum albumin solution (BSA) was taken in each of 5 cylindrical glass tubes attached to semi permeable membrane (Sigma dialysis sacs, 21mm diameter, 30cm length). The dialysis membranes were previously activated by immersing it in warm water for 30 minutes. These tubes were then immersed in beakers with 25 mL of phosphate buffer containing fixed concentration of Deferasirox (3-10-4M)). Aluminium hydroxide was added in increasing concentrations into five beakers containing Deferasirox solution to give a final ratio (BSA: Deferasirox: Aluminium hydroxide, 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5). This system was maintained at room temperature for 8 hours. After 8 hours, 1 mL of the solution was withdrawn from the beaker, and diluted to 10 mL using potassium dihydrogen phosphate buffer and the responses of free drug was measured at a wavelength of 248 nm. The concentration of the free fraction of drug was determined from the calibration graph.
IN VITRO PROTEIN BINDING STUDY
Percentage protein binding and %free fraction of drugs
The concentration of the drug was determined from the calibration graph. The % protein binding and free fraction of Deferasirox was calculated. Percentage protein binding of Deferasirox was found to be 98.16% and the percentage free fraction of Deferasirox was found to be 1.84%.
Effect of Aluminium hydroxide on in vitro protein binding of Deferasirox
After interaction with aluminium hydroxide, the free fraction of Deferasirox increased from 1.84% to 7.27% when the ratio of aluminium hydroxide to BSA was increased from 1 to 5.
Table.1: Effect of aluminium hydroxide on in vitro protein binding of Deferasirox
% PROTEIN BINDING
% FREE DRUG CONCENTRATION