MATERIALS AND METHODS

The present investigation was carried out from April 2010 to March 2011 at Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka, Bangladesh.

Drugs and reagents: Experimental drugs Amlodipine besylate and Atorvastatin Calcium were purchased from Eskayef Bangladesh Ltd. Warfarin Sodium and Diazepam, used as probe, were purchased from Incepta Pharmaceuticals Ltd. Bangladesh. Vehicle Methanol, Ethanol and Acetone used in this experiment all were of analytical grade and purchased from BDH, UK. Buffer chemicals Disodium hydrogen phosphate (Na₂HPO₄) and Potassium dihydrogen phosphate (KH₂PO₄) were purchased from GSK, UK. Cellulose Membrane (molecular weight cut off at 3500 Daltons) used in the experiment was purchased from Medical International Limited in England. Bovine serum albumin (fatty acid free, fraction V, 96-98%) was purchased from the Sigma Chemical CO. USA. The molecular weight of the protein was approximately 66,210 Da with 581 amino acids and 4.7 of isoelectric point (Bogusz, 2000).

Instruments: The following instruments were used: pH Meter (HANNA Microprocessor pH Meter, Portugal), SP8-400 UV/VIS Spectrophotometer (Thermospectronic, England), Metabolic Shaking Incubator (Nical electro Ltd., England), Micro syringe (Hamilton, Switzerland).

Preparation of membrane: The supplied membrane was cut into small pieces (9 cm in length) and taken in 500 mL beaker containing de-ionized water. The membranes were immersed beneath the de-ionized water and heated for more than 8 h in order to remove sulfur as sulfur may interfere in the overall binding process. The temperature was controlled between 65-70°C and hot water was replaced by fresh de-ionized water at every one hour interval for efficient removal of sulfur. The prepared pieces of membrane were kept in a beaker containing fresh de-ionized water and preserved in a refrigerator (chilling chamber) until use.

Preparation of Bovine Serum Albumin (BSA) Solution: In this study, Bovine Serum Albumin (BSA) was used in stead of Human serum albumin (HAS) due to structural similarity, good stability in various media, reproducibility, availability and cost effectiveness (Brown, 1976; Simion et al., 2009). To prepare 100 mL of 2x10^-5 M solution of Bovine serum albumin (BSA) 0.133 of protein was accurately measured and dissolved in a 100 mL volumetric flask with the previously prepared phosphate buffer solution of 6.4. This was done carefully and gently so that it did not form foam. The protein solution was kept in a refrigerator until use.

Same procedure was followed for the preparation of 100 mL of 2x10^-5 M protein solutions in phosphate buffer of pH 7.4.

Preparing standard curve
Amlodipine besylate: In each of seven test tubes Phosphate buffer solution of pH 6.4 was taken. Amlodipine Besylate solution at pH 6.4 and conc. of 1x10^-3 M was added to make the concentration 0.5x10^-5 M, 0.8x10^-5 M, 1x10^-5 M, 2x10^-5 M, 3x10^-5 M, 4x10^-5 M and 5x10^-5 M, respectively. The same procedure is followed for preparing standard curve of Amlodipine Besylate at pH 7.4.

The absorbance values of the solutions were determined by a UV spectrophotometer at λ_max 361 nm. As a control or reference sample, phosphate buffer solution of pH 6.4 and pH 7.4 were used, respectively. The standard curve was obtained by plotting the absorbance values against the corresponding concentration.

Atorvastatin calcium: Phosphate buffer solution of pH 6.4 was taken in each of seven test tubes. Atorvastatin Calcium solution at pH 6.4 and conc. of 1x10^-3M was added in different volume to seven test tubes to have the following concentration: 0.1x10^-5 M, 0.3x10^-5 M, 0.5x10^-5 M, 0.6x10^-5 M, 0.8x10^-5 M, 1x10^-5 M and 2x10^-5 M. The same procedure is followed for preparing standard curve of Atorvastatin Calcium at pH 7.4.

The absorbance values of the solutions were determined by a UV spectrophotometer at λ_max 247 nm. As a control or reference sample, phosphate buffer solution of pH 6.4 and pH 7.4 were used, respectively. The standard curve was obtained by plotting the absorbance values against the corresponding concentration.

Warfarin sodium and diazepam: Phosphate buffer solution of pH 7.4 was taken in each of seven test tubes. Warfarin Sodium stock solution at pH 7.4 and concentration of 1x10^-3 M was added in different concentrations to the seven test tubes, to have the following concentrations: 0.5x10^-5 M, 0.8x10^-5 M, 1x10^-5 M, 2x10^-5 M, 3x10^-5 M, 4x10^-5 M and 5x10^-5 M.

The absorbance values of the solutions were determined by a UV spectrophotometer at λ_max 306 nm. As a control or reference sample, phosphate buffer solution of pH 7.4 was used. Same procedure is followed for Diazepam. The absorbance values of the solutions were determined by a UV spectrophotometer at λ_max 235 nm. The standard curve of Warfarin Sodium and Diazepam were obtained by plotting the absorbance values against the corresponding concentrations.

Experimental design
Estimation of association constant: The value of association constant (ka) and the number of corresponding binding sites (n) of Amlodipine Besylate and Atorvastatin Calcium bound to BSA were determined by Scatchard plot utilizing equilibrium dialysis technique (Singlas, 1987a, b). In this method, a curve was obtained by plotting r/D_f versus r values where D_f stands for the molar concentration of free drug and r describes the ratio of the molar concentration of bound drug to the molar concentration of protein, i.e.,:

The extrapolation of the plot gives an intercept on Y-axis representing nKa values and the intercept thus obtained on X-axis represents n; the slope of the line being Ka.

Estimating association constant of amlodipine besylate bound to BSA at pH 6.4 and pH 7.4 at temperature 28°C: 5 mL of 2x10^-5M BSA solution at pH 6.4 was taken in each of 9 test tubes and Amlodipine Besylate was added to make the concentrations 1.5x10^-5 M, 1.75x10^-5 M, 2x10^-5 M, 4x10^-5 M, 6x10^-5 M, 10x10^-5 M, 14x10^-5 M, 16x10^-5 M and 20x10^-5 M, respectively. Another test tube containing only BSA solution at pH 6.4 was marked as control. The solutions were then properly mixed and allowed to stand for 30 min in order to ensure maximum binding of Amlodipine Besylate to BSA.

From each of the test tube, 3.5 mL of solution was pipetted out and poured into 10 tubes containing semipermeable membrane. Both ends of the membrane tubes were clipped so that there was no leakage. The membrane tubes were then immersed in ten separate 50 mL conical flasks containing 20 mL of phosphate buffer solution of pH 6.4. The mouths of the conical flasks were covered by foil paper. The conical flasks were then placed in a metabolic shaker for dialysis for 12 h at 28°C and 20 rpm. Same procedure is followed for pH 7.4.

After completion of dialysis samples were collected from each flask and free concentrations of Amlodipine Besylate were measured by a UV spectrophotometer at a wavelength of 361 nm.

Estimating association constant of atorvastatin calcium bound to bsa at ph 6.4 and pH 7.4 at temperature 28°C: 5 mL of 2x10^-5 M BSA solution at pH 6.4 was taken in each of 9 test tubes and Atorvastatin Calcium was added to make the concentrations 0.1x10^-5 M, 0.2x10^-5 M, 0.3x10^-5 M, 0.5x10^-5 M, 0.8x10^-5 M, 1x10^-5 M, 1.5x10^-5 M, 1.8x10^-5 M and 2x10^-5 M, respectively. The rest of the procedure is same for estimating association constant of Amlodipine Besylate.

After completion of dialysis samples were collected from each flask and free concentrations of Atorvastatin Calcium were measured by a UV spectrophotometer at a wavelength of 247 nm.

Identification and characterization of binding site of amlodipine besylate and atorvastatin calcium by site specific probe method: Site-specific probes were used here to enhance our understanding of the drug-BSA interaction and thereby characterization of binding sites of the drugs used in the study on the BSA molecule.

The present investigation was carried out by utilizing Equilibrium Dialysis. Warfarin Sodium (Site-I specific) and Diazepam (Site-II specific) were used as probe to determine the binding site of Amlodipine Besylate and Atorvastatin Calcium on Bovine Serum Albumin (BSA).

In the presence of the mixture of the probe (Warfarin Sodium and Diazepam) and BSA at a constant ratio (1:1, 2x10^-5 M: 2x10^-5 M), different concentrations of drugs (Amlodipine Besylate and Atorvastatin Calcium) were added. Free concentrations of the probe were then determined by the equilibrium dialysis method to see whether there was any change in the free concentrations of the probe by the addition of the drugs.

Drug-drug displacement study between amlodipine besylate and atorvastatin calcium in the presence and absence of diazepam: Here in drug-drug displacement study was carried out in the presence and absence of diazepam (a site-II specific probe). The reason for using diazepam is because site-II binding site is more specific than site-I (Kabir et al., 2010).

Mutual effect of Atorvastatin Calcium and Amlodipine Besylate to displace each other
Effect of amlodipine besylate on atorvastatin calcium: To the mixture of Atorvastatin Calcium and BSA (1:1, 2x10^-5 M: 2x10^-5 M) different concentrations of Amlodipine Besylate were added both in the absence and presence of diazepam. The changes in the free concentration of Atorvastatin Calcium were measured by a UV spectrophotometer at a wavelength of 247 nm.

Here the previously described procedure for estimating of association constant was followed to observe the effect.

Effect of atorvastatin calcium on amlodipine besylate: To the mixture of Amlodipine Besylate and BSA (1:1, 2x10^-5 M: 2x10^-5 M) different concentrations of Atorvastatin Calcium were added both in the absence and presence of diazepam. The changes in the free concentration of Amlodipine Besylate were measured by a UV spectrophotometer at a wavelength of 361 nm.

Here the previously described procedure of estimating of association constant was followed to observe the effect.

RESULT AND DISCUSSION

Regression equations for standard curves are presented in Table 1. Regression equations are utilized to calculate the value of free drug concentration by putting the absorbance value.

Determining association constant and number of binding sites: Drug-protein binding are mainly of two types-strong affinity binding to a small number of sites and weak affinity binding to a large number of sites. Since binding is almost exclusively to albumin and the number of sites available is limited, the protein binding of some drugs depends on plasma albumin concentration (Rahman and Sharker, 2009).

In this present investigatin, the binding parameters of Amlodipine Besylate and Atorvastatin Calcium have been characterized by Scatchard plot. The non-liner curve of the plot suggests the presence of at least two classes of binding site of experimental drugs to BSA (Kabir et al., 2010).

By ploting data from Table 2 Scatchard plot analysis revealed that at pH 6.4 association constant and No. of binding site of Amlodipine Besylate is about 3.75x10⁵M^-1 and 1.2, respectively at high affinity binding site. On the other hand, at low affinity binding site, association constant and No. of binding site is found about 0.08410^-5 M^-1 and 13, respectively (Fig. 1). At pH 7.4, association constant and No. of binding site of Amlodipine Besylate at high affinity binding site is about 6.84x10⁵ M^-1 and 1.9, respectively. At low affinity binding site, association constant and No. of binding site is found about 0.273x10⁵ M^-1 and 12.8, respectively (Fig. 2).

By ploting data from Table 3 Scatchard plot analysis revealed that at pH 6.4 association constant and No. of binding site of Atorvastatin Calcium at high affinity binding site is about 35.7x10⁵ M^-1 and 0.14, respectively. Association constant and No. of binding site at low affinity binding site is found about 0.977x10⁵ M^-1 and 1.33, respectively (Fig. 3). At pH 7.4 association constant and No. of binding site of Atorvastatin Calcium at high affinity binding site is about 142.1x10⁵ M^-1 and 0.19, respectively. At low affinity binding site, association constant and No. of binding site is found about 5.45x10⁵ M^-1 and 1.1, respectively (Fig. 4).

So, it is seen that, association constants of Amlodipine Besylate and Atorvastatin Calcium in both high and low affinities were found to be greater in pH 7.4 than that of in pH 6.4. The difference in association constants at different pH is not fully understood. It may be due to the structural modification of protein molecule at a given pH value.

Determining binding sites: Binding site of Amlodipine Besylate and Atorvastatin Calcium are determined by calculating the free concentration of Warfarin Sodium and Diazepam. The basic principle is that-If a drug is able to displace a probe from its binding site, it is assumed that the drug also binds to that particular site (Alam et al., 2009).

From Table 4, it is seen that addition of Amlodipine Besylate displace Warfarin Sodium more (as % of initial, from 100 to 198%) than that of Diazepam (as % of initial, from 100 to 144%). As the increment of free concentration of warfarin Sodium is greater than that of diazepam it can be concluded that Amlodipine Besylate preferentially bind to Site I. Alam et al. (2008) also found that the same result.

Accordingly, as Atorvastatin Calcium displace Warfarin Sodium more (as % of initial, from 100 to 194%) than that of Diazepam (as % of initial, from 100 to 150%) it can be concluded that Atorvastatin Calcium also preferentially bind to Site I (Table 5).

Table 1:	Regression equation of standard curve of Amlodipine Besylate, Atorvastatin Calcium, Warfarin and Dizepam

However, as the displacement of diazepam is quite pronounced in both cases of Amlodipine Besylate and Atorvastatin Calcium, it can be also assumed that the investigated drugs Amlodipine Besylate and Atorvastatin Calcium preferentially bind with site-I. In addition to Site I, they also bind with Site II on the BSA molecule but to a lower extent.

As both of the drugs compete for site-I preferentially concurrent administration of these two drugs will alter the therapeutic efficacy of each other. This finding will be helpful to understand the poor clinical result of these two drugs incase of some individuals when used simultaneously.

Table 2:	Data for association constant of Amlodipine Besylate bound to BSA at pH 6.4 and pH 7.4 at temperature 28°C

Fig. 1:

Scatchard plot for the binding of Amlodipine Besylate to BSA at pH 6.4

Fig. 2:

Scatchard plot for the binding of Amlodipine Besylate to BSA at pH 7.4

Mutual effect of amlodipine besylate and atorvastatin calcium on the binding to bsa in the absence and presence of diazepam: The interactions at binding sites on BSA were measured between Amlodipine Besylate and Atorvastatin Calcium in the absence or in presence of site specific probes diazepam.

Table 3:	Data for association constant of Atorvastatin Calcium bound to BSA at pH 6.4 and pH 7.4 at temperature 28°C

Fig. 3:

Scatchard plot for the binding of Atorvastatin Calcium to BSA at pH 6.4

Fig. 4:

Scatchard plot for the binding of Atorvastatin Calcium to BSA at pH 7.4

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In absence of diazepam, the free fraction of Atorvastatin Calcium increased from 20 to 40% with the addition of Amlodipine Besylate from 0x10^-5 M to 10x10^-5 M. Whereas, in presence of diazepam, Amlodipine Besylate at the same concentration, increased the free fraction of Atorvastatin Calcium little more (from 25 to 53%) than that in the absence of diazepam (Table 6).

From Table 7, it can be assumed that, the ability of Atorvastatin Calcium to displace Amlodipine Besylate is more than the ability of Amlodipine Besylate to displace Atorvastatin Calcium from the binding site on BSA.

Table 4:	Determination of binding site of Amlodipine Besylate at pH 7.4 and 28°C using Warfarin Sodium as site-I specific probe
BSA: Warfarin (1:1) (2x10-5 M: 2x10-5M) when using Warfarin Sodium as site-I specific probe, BSA: Diazepam (1:1) (2x10-5M: 2x10-5 M) when using Diazepam as site-II specific probe

Table 5:	Determination of binding site of atorvastatin calcium at pH 7.4 and 28°C using warfarin sodium as site-I specific probe
BSA: Warfarin (1:1) (2x10-5M: 2x10-5M) when using warfarin sodium as site-I specific probe, BSA: Diazepam (1:1) (2x10-5M: 2x10-5 M) when using diazepam as site-II specific probe

Table 6:	Effect of Amlodipine Besylate on Atorvastatin Calcium binding to BSA in the absence and presence of Diazepam
In the absence of Diazepam, concentration of Atorvastatin:BSA (1:1) (2x10-5M: 2x10-5M), In the presence of Diazepam, concentration of Atorvastatin:BSA:Diazepam (1:1: 2) (2x10-5M: 2x10-5M: 4x10-5M)

Table 7:	Effect of Atorvastatin Calcium on Amlodipine Besylate binding to BSA in the absence and presence of Diazepam
In absence of Diazepam, concentration of Amlodipine:BSA (1:1) (2x10-5M: 2x10-5 M), In presence of Diazepam, concentration of Amlodipine:BSA:Diazepam (1:1: 2) (2x10-5M: 2x10-5M: 4x10-5M)

In absence diazepam, the free fraction of Amlodipine Besylate increased from 30 to 63% with the addition of Atorvastatin Calcium from 0x10^-5 M to 2.5x10^-5 M. Whereas, in presence of diazepam, Atorvastatin Calcium at the same concentration, increased the free fraction of Amlodipine Besylate significantly more (from 34 to 82%) than that in the absence of diazepam.

CONCLUSION

During concurrent administration of Amlodipine and Atorvastatine, both the drugs compete for the same binding site on the albumin molecule. This results in notable increase in the free concentration of one drug by another on the basis of direct competitive displacement. So, care should be exercised if it is necessary to administer both of these drugs.

ACKNOWLEDGMENT

The authors gratefully acknowledge the contribution of Department of Clinical Pharmacy and Pharmacology and Department of Pharmaceutical Chemistry, University of Dhaka, Bangladesh to give necessary support along with different instruments and platform to continue the research work. The authors also grateful to Prof. Nazmul Qais (Chairman, Department of Clinical Pharmacy and Pharmacology) and Prof. ABM Faroque (Dean, Faculty of Pharmacy) for giving their consent to conduct the research work.