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Original Article

Effect of Ca (II) ion on the in vitro availability andprotein binding of Amlodipine besylate

Marzina Ajrin*, Newton Sen, Irfan Newaz Khan, Maria Islam Khan

University of Science and Technology Chittagong (USTC), Foy’s Lake, Pahartali, Chittagong 4202, Bangladesh

a r t i c l e i n f o

Article history:

Received 17 July 2013

Accepted 14 August 2013

Available online 8 September 2013

Keywords:

Amlodipine besylate

Ca (II) ion

Drugemetal interaction

Protein binding

* Corresponding author. Tel.: þ88 659070 1x1E-mail address: marzina-ajrin@hotmail.c

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.08.017

a b s t r a c t

Background: The present study explicates the effect of metals ion (Ca2þ) on the in vitro

availability of Amlodipine besylate owing to drugemetal interaction.

Methods: Spectral studies were performed in an aqueous system at a fixed temperature

(37 � 0.5)�C and under different pH by UV spectrophotometric method at various con-

centrations of drug and metal. A Job plot was used to determine the stoichiometry of a

binding event and The Ardon’s method confirmed the complexation. An in vitro study of

protein binding of Amlodipine besylate and their 1:1 mixture with Ca2þ ion had been

conducted by equilibrium dialysis method at (37 � 0.5)�C and at pH 7.4 by using Bovine

Serum Albumin (BSA).

Results: Spectral studies detected the initial complexation. By Job’s plot it was found that

the interaction of Amlodipine besylate with metal ion (Ca2þ) form one complex with metal

at composition of 1:1. The Ardon’s spectrophotometric method confirmed the 1:1

complexation and the value of stability constant was higher at pH 7.4 (0.11). The percentage

of protein binding of Amlodipine besylate with BSA was found to be 86% and 42% at high

and low concentration range respectively. In presence of Ca2þ the percentage of protein

binding of drug increased 46% at lower concentration range and 94% at higher concen-

tration zone. The results were statistically significant ( p < 0.05).

The Scatchard plots showed that in class I binding sites, the value of affinity constant

and number of binding sites of 1:1 complexes with Ca2þ was 1.04 and 20.8 respectively.

Conclusion: Drugemetal complex might, therefore, decrease the free drug in plasma and

tissue systems. This may change the pharmacokinetic properties of the drug and may

affect the pharmacological effects. It is thus inferred that care and monitoring must be

taken during combination therapy of Amlodipine besylate and Ca2þ.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction drug andmetal may inadvertently reduce or increase the drug

Study of interaction between drugs and metals is an active

research area in bioinorganic chemistry. Interactions between

24 (office), þ88 017192340om (M. Ajrin).2013, JPR Solutions; Publi

effect.1 Amlodipine besylate is a widely used anti-

hypertensive drug. It selectively inhibits calcium influx

across cell membranes in cardiac and vascular smooth

22 (mobile).

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 6 7 1e6 7 6672

muscle. It is a peripheral arteriolar vasodilator; thus it reduces

after load.2 It is useful for the treatment of angina, essential

hypertension, congestive heart failure and Reynaud’s dis-

ease.3 Calcium is the 5th most abundant element in the body

and the major fraction is in the bony structure. Calcium plays

important physiological roles in the maintenance of the

functional integrity of the nervous, muscular and skeletal

systems, cell membrane and capillary permeability.

Protein binding generally refers to the binding of a drug to

plasma proteins. The amount of drug bound to protein de-

termines how effective the drug is in the body. Serum albu-

min, the most abundant protein in the blood, plays a very

important role in the binding phenomenon and serves as a

depot and transport protein for numerous endogenous com-

pounds.4 Among the plasma protein, albumin is mostly

bounds to ligands or drug. Since number of protein binding

sites is limited, competition will exist between two drugs and

the drug with higher affinity will displace the other, causing

increased free drug concentration, which leads to higher

toxicity or short duration of action of the related drug.5 The

ability of one drug to inhibit the other is a function of their

relative concentration, binding affinities and specifically of

binding.6 BSA and HSA have structural similarity.7 In this

study BSA is in lieu of HAS, because of low cost and easy

availability.

This study was aimed to evaluate interaction of Amlodi-

pine besylate with (Ca2þ) present in multi-vitamins and foods

as well as influence of (Ca2þ) on protein binding of drug.

2. Methods

Amlodipine besylate were given by Square Pharmaceuticals

Ltd., Bangladesh, Bovine serum albumin (Fatty acid free,

fraction V, 96e98%, Sigma) and Dialysis Membrane (Medicell,

England) and Calcium chloride and all other reagents were

purchased from Merck, India. Following methods were used

for the commencement of the experiment:

2.1. Spectral studies

Initial detection of complexation of Amlodipine besylate and

Ca2þ had done from the nature of spectra of pure drug as well

as their 1:1mixtures in buffer solutions of pH 1.2, 2.2, 6.4, 7.4 at

a fixed concentration (0.1� 10�4) Mwere comparedwith those

of each interacting species.8 The various concentrations of the

samples were kept at very dilute levels in each case and

recorded between 300 and 400 nm using a UVeVIS automatic

recording instrument with a constant temperature cell

compartment and automatic recording unit.

2.2. Job’s spectrophotometric method of continuousvariation

This study was done by method of Vogel.9 A Job plot was used

to determine the stoichiometry of a binding event. In this

method, absorbance was measured at pH 1.2, 2.2, 6.4 and 7.4

at various concentrations (1 � 10�5 to 8 � 10�5 M) of Amlo-

dipine besylate with Ca2þ (2 � 10�5 to 9 � 10�5) at 365 nm. The

observed absorbance of the mixtures at various mole

fractions was subtracted from the sum of the values for free

drug and free metal. The absorbance differences (D) were

then plotted against the mole fractions of drugs in the

mixtures.

2.3. The Ardon’s spectrophotometric methods

This method was conducted according to Ardon.10 In this

method, concentrations of drugwere variedwhile keeping the

concentration of the metal fixed (2 � 10�5 M). All the experi-

ments were performed in buffer at pH 1.2, 2.2, 6.4 and pH 7.4.

The absorbance was measured at 365 nm by using

UVeVIS spectrophotometer. From Ardon’s plot, the value of

stability constants of the drugemetal complexwas calculated.

For calculation, the Ardon’s equation was used. This

equation is given below:

1ðD� ˛ACÞ ¼

1

KCð˛com � ˛AÞ½B�nþ 1Cð˛com � ˛AÞ

Here D ¼ Absorbance of the mixture; B ¼ Molar concentration

of the drug; C¼Molar concentration of themetal;˛com¼Molar

extinction co-efficient of the complex and ˛A ¼ Molar

extinction co-efficient of the drug.

2.4. Equilibrium dialysis

Equilibrium dialysis is one of the methods used for the

determination of the protein binding of any compound

developed by Singlass.11 Before conducting this method the

dialysis membrane are activated.12 The membrane pieces

were filled with BSA solution with different concentrations of

drug and their (1:1) drugemetal mixture, keeping the total

volume 4 ml. The membrane bags were immersed in 60 ml of

solution having pH 7.4 and were shaken gently at (37 � 0.5)�Cfor about 6 h in metabolic shaker. The absorbance of buffer

(outside the membrane bags) was measured at 365 nm using

the UVeVIS spectrophotometer and the concentrations of the

bound and unbound drugs were calculated using a standard

curve. The percentage of protein binding (F) was determined

by the formula:

F ¼ ½B� � ½A�½Total drug� � 100

where, A and B was the Molar concentration of free drug in

buffer compartment and Molar concentration of total drug in

protein compartment respectively.

2.5. Protein binding sites and the affinity constantscalculations

The Scatchard method13,14 was used for this purpose and a

curve was produced by plotting ‘r/[A]’ versus ‘r’ using the

equation:

r ¼ ½B� � ½A�½Protein�

where, r ¼ the ratio between the molar concentration of the

bound drug and the molar concentration of protein.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

300 310 320 330 340 350 360 365 370 380 390 400

Abs

orba

nce

Wavelength (nm)

D

D + M

Fig. 1 e Comparison between the spectral plot of

Amlodipine besylate alone and with Ca2D at pH 1.2 & 2.2

respectively (The data are shown as mean ± SME).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

300 310 320 330 340 350 360 365 370 380 390 400

Abs

orba

nce

Wavelength (nm)

D

D+M

Fig. 3 e Comparison between the spectral plot of

Amlodipine besylate alone and with Ca2D at pH 6.4 & 7.4

respectively (The data are shown as mean ± SME).

0.35

0.4D

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 6 7 1e6 7 6 673

2.6. Statistical analysis

The results were expressed as Mean � SEM values for each

experiment. Differences in mean values between experi-

mental groups were analyzed by unpaired t-test. A probability

values less than 0.05 ( p < 0.05) was defined to be significant.15

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

300 310 320 330 340 350 360 365 370 380 390 400

Abs

orba

nce

Wavelength (nm)

D+M

Fig. 4 e Comparison between the spectral plot of

Amlodipine besylate alone and with Ca2D at pH 6.4 & 7.4

respectively (The data are shown as mean ± SME).

3. Results

3.1. Spectral study

It was seen that Amlodipine besylate gives a sharp peak at

365 nm. But when (Ca2þ) mixed with Amlodipine besylate in

1:1 ratio, the intensity of the peak of Amlodipine besylate

changes remarkably (absorbance decreases) i.e., absorption

characteristics are altered due to interaction but the position

of the compound do not shift (Figs. 1e4).

3.2. Study of job’s method

At experimental pH, Amlodipine besylate form strong 1:1

complexes with Ca2þ ion. Absorbance differences at pH 1.2,

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

300 310 320 330 340 350 360 365 370 380 390 400

Abs

orba

nce

Wavelength (nm)

D

D+ M

Fig. 2 e Comparison between the spectral plot of

Amlodipine besylate alone and with Ca2D at pH 1.2 & 2.2

respectively (The data are shown as mean ± SME).

Fig. 5 e Job’s Plot for Complexation of Amlodipine besylate

and Ca (II) ion at pH 1.2&2.2 (The data are shown as

mean ± SME).

Fig. 6 e Job’s Plot for Complexation of Amlodipine besylate

and Ca (II) ion at pH 1.2&2.2 (The data are shown as

mean ± SME).

Fig. 8 e Job’s Plot for Complexation of Amlodipine besylate

and Ca (II) ion at pH 6.4&7.4 (The data are shown as

mean ± SME).

Fig. 9 e Ardon’s plot for Amlodipine besylate with Ca (II)

ion at pH 1.2 & 2.2 (The data are shown as mean ± SME).

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 6 7 1e6 7 6674

2.2, 6.4 and 7.4 were (Figs. 5e8) indicated as “̂” shaped curves

and the break points were found at absorbance difference of

0.15, 0.16, 0.17 and 0.18 at pH 1.2, 2.2, 6.4 and 7.4 respectively. It

confirmed the formation of 1:1 complexes of Amlodipine

besylate with Ca (II) ion.

3.3. Study of Ardon’s method

Ardon’s plot confirmed the formation of 1:1 complex of

Amlodipine besylate with Ca (II) ion at pH 1.2, 2.2, 6.4 and 7.4,

since the method is valid for only 1:1 complexes. The Ardon’s

plots gave straight lines intercept which are presented in Figs.

9e12 indicate the formation of 1:1 complexes at experimental

pH.

3.4. Estimation of stability constant

The value of stability constant for the complexation of

Amlodipine besylate with Ca (II) ion at pH 1.2, 2.2, 6.4 and 7.4

were obtained from the spectral data using Ardon’s plot. The

values of stability constant were given as [(Intercept)/(slope)]

by using Ardon’s equation. The values of stability constants

for the drugemetal system at pH 1.2, 2.2, 6.4 and 7.4 presented

in Table 1

Fig. 7 e Job’s Plot for Complexation of Amlodipine besylate

and Ca (II) ion at pH 6.4&7.4 (The data are shown as

mean ± SME).

3.5. Study of protein binding

The in vitro determination of percentage of protein binding of

Amlodipine besylate and their 1:1 mixture with Ca (II) ion was

done by equilibrium dialysis method at physiological tem-

perature (37 � 0.5)�C and at pH 7.4. The observed values of

protein binding for drug alone and with metal are given in

Fig. 13.

4. Discussion

The spectra of drug molecules alone and (1:1) mixture of drug

and metal showed significant change in their absorption

Fig. 10 e Ardon’s plot for Amlodipine besylate with Ca (II)

ion at pH 1.2 & 2.2 (The data are shown as mean ± SME).

Fig. 11 e Ardon’s plot for Amlodipine besylate with Ca (II)

ion at pH 6.4 & 7.4 (The data are shown as mean ± SME).

Fig. 12 e Ardon’s plot for Amlodipine besylate with Ca (II)

ion at pH 6.4 & 7.4 (The data are shown as mean ± SME).

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8

% P

rotr

in b

indi

ng

Conc. ( M X10-5)

% protein binding (drug alone)% protein binding with metal

Fig. 13 e % of protein binding of drug alone and in presence

of metal (1:1 mixture) to BSA at pH 7.4 (The data are shown

as mean ± SME).

Fig. 14 e Scatchard plot for protein binding of Amlodipine

besylate to BSA (The data are shown as mean ± SME).

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 6 7 1e6 7 6 675

intensities. This may be due to interaction of Ca2þ with drug

that may alter the absorption intensities but the position of

the compound does not shift.

Job’s plots showed, for a constant total concentration of

drug andmetal, the complex was at its greatest concentration

at a pointwhere the species of drug andmetal are combined in

the ratio in which they occur in complex. The straight lines

which cross each other showed a break at nearly 5 mol frac-

tions indicating the 1:1 complexes for all the systems. At

experimental pH, Amlodipine besylate forms strong 1:1 com-

plexes with Ca2þ indicated as ‘̂’ shaped curves. These curves

may indicate strong kinetics of complexation between

Amlodipine besylate with Ca2þ.

The stability constants obtained from the Ardon’s plot for

AmlodipineeCa2þ system was remain quite close at all pH

systems except at pH 7.4. At pH 7.4 the stability constant was

0.11, higher than all other systems. So, we can conclude that a

stable complex was formed at pH 7.4 i.e. in blood.

In protein binding studies it was found that at a low drug

concentration the percentage of protein binding attains a

steady state plateau condition (84%). This indicated the

Table 1 e Values of stability constant.

System Stability constant K

Amlodipine besylate & Ca2þ ion pH 1.2 0.054

pH 2.2 0.053

pH 6.4 0.06

pH 7.4 0.11

saturation of the sites of protein by the drugs or its complexes

as observed by other investigators.16 In our experiment, the

percentage of binding of Amlodipine besylate with BSA was

found to be 86% and 42% at high and low concentration range

respectively. In presence of Ca (II) ion the percentage of pro-

tein binding of drug increased (42e46) % at lower concentra-

tion range and (82e91) % at higher concentration zone. In

brief, Ca2þ caused an increase in protein binding of Amlodi-

pine besylate leading to the formation of stable 1:1 Amlodi-

pine besylateeCa 2þ complex. This means that the increase in

percentage of protein binding may be due to capture of bind-

ing sites in the protein by Ca2þ or Amlodipine besylate &

Amlodipine besylateeCa2þ complex. Thus possibility of

Fig. 15 e Scatchard plot for protein binding of Amlodipine

besylate to BSA presence of Ca2D (The data are shown as

mean ± SME).

Table 2 e Binding parameters of Amlodipine besylate.

Systems Class I Class II

n1 k1 n1$k1 n2 k2 n2$k2

Amlodipine besylate alone 80.3 1.02 0.819 0.172 0.226 0.0388

Amlodipine besylateeCa

(II) ion

20.8 1.04 0.216 0.476 0.172 0.0818

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 6 7 1e6 7 6676

adverse effect of Amlodipine besylatemay become prominent

in presence of Ca or similar drugs in the body system.

The subsequent non-linear shape of the Scatchard plots

(Figs. 14 and 15) describes both high and low affinity binding

sites of the drug on proteinmolecules. There were at least two

classes (Class 1 and Class II) of binding sites in BSA for

Amlodipine besylate and its (1:1) complex with Ca (II) ion

(Table 2). We saw that in class I binding sites, the value of

affinity constant for Amlodipine besylate alone 1.02 was lower

than its 1:1 complexeswith Ca (II) ion 1.04 (Table 2), that is, the

presence of Ca 2þ with Amlodipine besylate at physiological

temperature and pH conditions, cause an increase in values of

affinity constant. In class-I, the number of binding site

decrease in presence of Ca (II) ion 2.08 than that of alone

Amlodipine besylate i.e. 8.03.

Since it is almost exclusively limited to albumin and the

number of available binding sites is limited, the binding

properties of drugs depend on plasma albumin concentration.

So, due to increase in affinity of the Amlodipine besylate to

plasma protein in class I binding site in presence of Ca (II) ion,

the volume of distribution (Vd) as well as bioavailability of the

drug (Amlodipine besylate)may decrease.17,18 So the proposed

drugemetal interactions could interfere substantiallywith the

intestinal absorption of Amlodipine besylate owing to the

lower solubility of the chelates in intestinal tract.19 So

concomitant administration of Amlodipine besylate with food

products containing Calcium, nutritional supplements and

multivitamins containing Ca (II) ion could impair the clinical

efficacy of the drug and reduce its bioavailability. More

detailed research may reveal the mechanism of increase

binding of drug to the protein in presence of calcium.

Conflicts of interest

All authors have none to declare.

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