EFFECT OF GLUCOSE ON PROTEIN BINDING OF LOSARTAN POTASSIUM WITH BOVINE SERUM ALBUMIN
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1 EFFECT OF GLUCOSE ON PROTEIN BINDING OF LOSARTAN POTASSIUM WITH BOVINE SERUM ALBUMIN Dr. M. Eswar Gupta, N. krishnasri *, and I. Sudheer Babu Department of Pharmacology, SIR C. R. Reddy College of Pharmaceutical Sciences, Eluru * Corresponding author N. krishnasri Department of Pharmacology, SIR C. R. Reddy College of Pharmaceutical Sciences, Eluru, Andhra Pradesh, India krishnasri532@gmail.com Research Article Abstract: Protein binding studies of losartan potassium with bovine serum albumin was carried out by equilibrium dialysis method using HIMEDIA LA 387 dialysis membrane (molecular weight cut off ,000 daltons).the Diffusion of losartan potassium through this membrane followed first order kinetics. In the presence of high glucose concentration (corresponding to elevated blood glucose levels in diabetics), protein binding of losartan potassium to bovine serum albumin was reduced significantly. Statistical student t-test was applied to the results to know weather there was significant difference or not. It was concluded that, elevated high glucose concentrations reduces the protein binding of losartan potassium with Bovine serum albumin. However in vivo studies have to be done to confirm this. KEYWORDS: Losartan potassium, bovine serum albumin, equilibrium dialysis method, Glucose, Protein binding, Diabetics. INTRODUCTION: The phenomenon of complex formation of drugs with proteins of blood is called protein binding. A number of drugs are extensively bound to plasma proteins.only the unbound form of drug is available for distribution, metabolism, and elimination. The intensity of pharmacological action and toxic effects of a drug if any depends on the free drug concentration in plasma. Higher the free drug concentration, higher the intensity of pharmacological action and toxic effects if any.due to concurrent administration of drugs or changes in diet, the extent of drug bound to plasma proteins may get altered. As a result the free drug concentration in the blood changes and the intensity of pharmacological action is altered [1]. Losartan potassium is a widely used antihypertensive drug. It is bound to the plasma protein albumin to an extent of 99 % [2]..As a result only around 0.3 % of the administered dose is available for drug distribution and elimination from the body. Any factor which influences the protein binding of a drug will alter the free drug concentration in blood, thus altering the intensity of therapeutic action, For example: if 0.3 % of losarton potassium is displaced from the protein due to any reason, there will be a 100 % increase in free drug concentration in blood leading to increase in intensity of drug action and toxic effects if any. Khaja Nazim Uddin Ahmed and et al [3] worked on protein binding of losartan potassium and valsartan, two anti-hypertensive drugs to bovine serum albumin (BSA) by equilibrium dialysis method. Losartan potassium showed slightly higher affinity towards BSA than Valsartan. During concurrent administration of losartan potassium and valsartan, both drugs have been found to increase the respective free concentration of one another causing reduced binding to BSA. Ferdosi Kabir A and et al [4] worked on binding of losartan potassium with bovine serum albumin by equilibrium dialysis method in presence and absence of palmitic acid. They reported that palmitic acid reduces protein binding of losarton potassium with BSA. An extensive survey of the literature indicated that no one studied the influence of glucose concentration on Krishnasri et al IJAPS (1) 6-18 Page 6
2 the extent of binding of Losartan potassium with bovine serum albumin. Most of the diabetic patients also suffer with hypertension and may be prescribed losartan potassium drug also. Increased blood glucose levels may alter the extent of losarton potassium binding with albumin. Hence an attempt was made to study this influence by an in vitro method.out of the many methods available, in vitro equilibrium dialysis method was used to study the effect of glucose on binding of Losartan potassium with bovine serum albumin [5]. MATERIALS AND METHODS: Dialysis membrane (molecular cut off ,000 daltons) and bovine serum albumin used in the experiment were purchased from Himedia Ltd., and SDFCL (Sd fine-chem. Limited) respectively. Losartan potassium was a kind gift sample from Hetero pharmaceutical company of Hyderabad. Glucose was of company Merck. Table 1: Solutions used in the protein binding studies UV-VIS spectrophotomer (Shimadzu) and magnetic stirrer (Remi), burette stand with clamps, Electronic balance (Shimadzu), organ bath tubing were used in the experiment. Equilibrium Dialysis method was employed in this study. Experimental procedure:- U.V analysis of Losartan potassium:- A series of losartan potassium standard solutions 8, 12, 16, 20, 24 µg /ml were prepared in water, and their absorbance was measured at 240 nm using a UV spectrophotometer. Distilled water was used as a blank. A beer s plot was constructed and was used for the analysis of losartan potassium solutions in the research work. S.NO Solution Concentration 1 Losartan potassium solution in water 1 mg / ml (A) 2 Losartan potassium solution in water 10 mg / ml (B) 3 Bovine serum albumin solution in water 10 mg / ml (C) 4 Glucose solution 10 mg / ml (D) 5 Glucose solution 40 mg / ml (E) Fig I : Instrumental set up used to carry drug protein binding studies Krishnasri et al IJAPS (1) 6-18 Page 7
3 Procedure to carry drug diffusion studies: To know the order associated with drug diffusion through the dialysis membrane the following procedure was followed. 20 cm of dialysis membrane was cut from the roll and was tied to the organ bath tubing and was suspended in a beaker containing water as shown in the figure 2.The membrane was allowed to soak for 24 hrs in water. Approximately 18 cm of the dialysis membrane tubing was immersed in distilled water. Ten ml of solution A (1mg/ml losarton potassium drug solution) was poured in to dialysis tubing and was immersed in 200 ml distilled water at 30 0 C which was stirred by a magnetic stirrer. Five ml sample from the beaker was collected at 15 minutes interval for 2 hrs. Whenever a sample was taken, it was replaced with 5 ml distilled water. The absorbance of the samples was measured at 240 nm using UV spectrophotometer. Distilled water was used as a blank. Protein binding studies in presence of normal glucose (Normal Glucose level in blood is 100 mg/dl or 1mg/1ml): Test Solution: The drug solution (B), protein solution (C) and glucose solution (D) were mixed in different ratios as given in table2. Control Solutions: The solutions were prepared by mixing solution B and D in different ratios as shown in table 3. Protein binding studies in presence of high glucose :( High Glucose level in blood is 400 mg/dl or 4 mg/1ml): Test Solution: The drug solution B, protein solution C and glucose solution E were mixed in different ratios as given in table 4. Control Solutions: The solutions were prepared by mixing solution B and E in different ratios as shown in table 5. 2) Results of losartan potassium release from dialysis membrane are given in the table 6 and the corresponding graph is graph is fig 3. 3) The first order graphical plots obtained in dialysis experiments of protein binding studies of Losartan potassium in presence of Normal glucose concentration and High glucose concentration are given in fig 4 to 19 4) From the above graphical plots the percentage of Losartan potassium bound to bovine serum albumin was calculated by the procedure given in martin[6]. 5) The results are given in the table 6 6) Statistical t-test was applied to know weither high glucose significantly influences binding of Losartan potassium with bovine serum albumin. Results of Students T- test: Calculated t value at 0.05 level of significance at 7 degrees of freedom is Critical t-value at 0.05 level of significance at 7 degrees of freedom is 2.3. Calculated t-value is greater than the critical t-value, so protein binding of losartan potassium decreased significantly in presence of high glucose. Discussion:- 1) As log of amount of drug remaining to be released versus time (fig 4 to 19) is a straight line it indicates that diffusion of losartan potassium through the dialysis membrane followed first order kinetics. 20 cm of dialysis membrane was cut from the roll and was tied to the organ bath tubing and was suspended in a beaker containing water as shown in the figure 2.The membrane was allowed to soak for 24 hrs in water. Approximately 18 cm of the dialysis membrane tubing was immersed in distilled water. Fifteen ml of test solution / control was poured in to dialysis tubing and was immersed in 200 ml distilled water at 30 0 C which was stirred by a magnetic stirrer. Five ml sample from the beaker was collected at 15 minutes interval for 2 hrs. Whenever a sample was taken, it was replaced with 5 ml distilled water. The absorbance of the samples was measured at 240 nm using UV spectrophotometer. Distilled water was used as a blank. Experimental Results:- 1) The standard graph of Losartan potassium is give as fig 1. It was linear over the range 8 to 24 µg /ml. Krishnasri et al IJAPS (1) 6-18 Page 8
4 Table 2:Test solutions preparation in presence of Normal glucose S.NO Drug: protein ratio Drug solution (B) in ml(10mg/ml) Protein solution in (C) ml(10 mg/ml) Glucose solution in (D) ml (10mg/ml) Distilled water in ml Total volume Taken in dialysis tubing in ml Amount of drug in 15 ml system mg Amount of protein in 15ml system mg Amount of Glucose in 15ml system mg : : : : : : : : Krishnasri et al IJAPS (1) 6-18 Page 9
5 Table 3:Control solutions preparation in presence of Normal glucose S.NO Drug: protein ratio Drug solution (B) in ml (10mg/ml) Glucose solution in (D) ml (10mg/ml) Distilled water in ml Total volume Taken in dialysis tubing in ml Amount of drug in 15 ml system mg Amount of Glucose in 15 ml system mg 1 1: : : : : : : : Krishnasri et al IJAPS (1) 6-18 Page 10
6 Table 4 :Test solutions preparation in presence of High glucose S.NO Drug: protein ratio Drug solution (B), ml Protein solution (C), ml Glucose solution in (E), ml Distilled water in ml Total volume Taken in dialysis tubing in ml Amount of drug in 15 ml system mg Amount of protein in 15ml system mg Amount of Glucose in 15 ml system mg 1: : : : : : : : Krishnasri et al IJAPS (1) 6-18 Page 11
7 able 5: Control solutions preparation in presence of High glucose S.NO Drug:protein ratio Drug solution (B) ml Glucose solution in (E), ml Distilled water in ml Total volume Taken in dialysis tubing in ml Amount of drug in 15 ml system mg Amount of Glucose in 15 ml system mg 1 1: : : : : : : : Krishnasri et al IJAPS (1) 6-18 Page 12
8 Table 7: % PROTEIN BINDING RESULTS S.NO Drug: Protein ratio Presence of normal glucose Presence of high glucose 1 1: : : : : : : : Average Krishnasri et al IJAPS (1) 6-18 Page 13
9 Table 6: Data of losartan potassium released through dialysis membrane S.NO Time in hrs Amount released Amount remaining to be released (ARR) Log amount remaining Krishnasri et al IJAPS (1) 6-18 Page 14
10 Krishnasri et al IJAPS (1) 6-18 Page 15
11 Krishnasri et al IJAPS (1) 6-18 Page 16
12 1) From the figures (fig 4 to 19) it can be seen that the slope of the line for test solution is less than that of control solution, indicating that the diffusion rate of drug in presence of bovine serum albumin is less. This indicates that losartan potassium is being bound to bovine serum albumin. 3) bound to bovine serum albumin, where as in presence of high glucose (corresponding to high glucose blood levels in diabetic patients), on an average 21.9 % of losarton potassium was bound to bovine serum From the Students t-test results, it can be concluded that elevated blood glucose levels may reduce 2) By mathematical treatment of the lines in figures 4 to 19 as per the procedure given in reference six, % of losarton potassium bound to bovine serum was calculated and the data is presented in table 7. In presence of normal glucose (corresponding to normal blood glucose levels in diabetic patients), on an average, 28.3 % of losartan potassium was albumin. Statistical T test of the data in table VII indicates this decrease as stastically significant. CONCLUSION the protein binding of losartan potassium with albumin. The protein binding of losartan potassium decreased by 7% in Krishnasri et al IJAPS (1) 6-18 Page 17
13 presence of high glucose. Hence the dose of losartan potassium should be decreased by 7% in presence of high glucose. So dosage adjustment of losarton potassium in case of diabetic patients is necessary. However in vivo studies have to be done to confirm this. References:- 1. MiloGibaldi, Bio pharmaceutics & Clinical Pharmacokinetics, Fourth edition. 2. Losartan drug information Available from drugs/db00678 Google search. 3. KhajaNazimuddinAhmed,Shahanasharmin,IshtiagAhmed, Protein Binding of Angiotensin Receptor II Antagonist, Journal of pharmacy and pharmacology FerdosiKabir.A,Nizimuddin.K,Interaction of palmiticacid with Losarton potassium at the binding site of Bovine serum Albumin,ASR Pharmacetica,Vol.51;1;28-36; dialysis.php 6. Patrick J.Sinko, Martin s Physical pharmacy & pharmaceutical sciences Fifth edition, Lippincot Williams & Willikins, USA, Krishnasri et al IJAPS (1) 6-18 Page 7
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