Plasma protein binding is one of the important and useful pharmacokinetics
parameters of a drug. There are multiple binding sites on a protein molecule.
The pharmacokinetics and pharmacodynamic behavior of a drug is governed by the
strength of a complex formed by the drug molecules with the protein (Singlas,
1987). Drugs generally form reversible complexes with the plasma proteins that
act as a reservoir releasing the free drug to the circulation.
Structures of ibuprofen, gliclazide and glipizide
The free form of the drug shows the pharmacologic response, is metabolized
and excreted (Salam and Hossain, 2001). The bound drug is gradually released
to maintain an equilibrium and thus the pharmacologic response is maintained.
The type and nature of protein binding depend on the physicochemical properties
of the drug molecule, its concentration, pH of the medium and also on the concentration
and number of available binding sites of the plasma proteins (Mohiuddin et
al., 2009a). Protein binding of a drug is a limiting factor for the drug
effect (Hossain and Amran, 1999). Simultaneous administration of two or more
drugs into the systemic circulation can modify the affinity of the drug to bind
with plasma protein and thus percentage of protein binding. Due to this influence,
a combined therapy can change the volume of distribution, renal and hepatic
clearance and hence drug effects (Begum et al., 1988).
The concentration of drug in blood is easily accessible. The pharmacological response is influenced by the plasma concentration of drug. It is observed that there is an optimum or desired therapeutic or pharmacological concentration range over which the drug produces its characteristic effect (Mohiuddin et al., 2009b; Rahat et al., 1999). Drug-drug interaction result when one drug alters the known therapeutic response of another drug that has been administered concurrently. The result may be enhanced or diminished effects of one or both of the drugs. It is a common practice for the medical practitioners to prescribe more than one drugs in a single prescription and patients receive those drugs simultaneously. The effect of this simultaneous intake sometimes be neither safe nor effective but may be deleterious (Cadwallader, 1985).
The aim of the present study is to evaluate the effect of glipizide and gliclazide (Fig. 1) on the protein binding and plasma concentration of ibuprofen (Fig. 1) and thus to infer about the consequences of combined drug therapy for these drugs.
MATERIALS AND METHODS
Protein Binding Study
Protein binding was determined by the equilibrium dialysis method. For this
purpose the membrane pieces (12 cm long and 3 mL capacity) were activated by
digestion with 1.0 M sodium bicarbonate at 70°C for 4 h and washing thoroughly
with deionized water thereby and immersing them in 0.067 M phosphate buffer
of pH 7.4. Activated membrane bags were filled with solutions of serum bovine
albumin with different concentration of the drugs and their mixtures. The membrane
bags were immersed in a fixed amount (50 mL) of phosphate buffer and the system
was shaken gently for 6 h in a metabolic shaking incubator at 37±1°C.
After this period the absorbance of the buffer samples (outside the membrane
bags) were measured at 240 nm and the concentrations of the bound and unbound
drugs were calculated with the help of the relationship shown in the next page
using a standard curve (Singlas, 1987; Mohiuddin et al., 2009a).
Preparation of Standard Curve
A standard curve for ibuprofen was prepared using solutions of different
concentrations of the drug in the buffer using a fixed concentration of either
gliclazide or glipizide and measuring their absorbance at 223 nm which was found
to be the ëmax of ibuprofen at lower concentrations (5-100 μM).
The absorbance were plotted against the concentration of ibuprofen keeping the
concentration of glipizide and gliclazide constant.
Calculation of Percentage of Protein Binding
The percentage of protein binding (F) is given by
||Molar concentration of free drug in buffer compartment after
Molar concentration of total drug in protein compartment before dialysis
Calculation of Affinity Constants and Number of Binding Sites
The Scatchard method (1949) was used for this purpose and a curve was thus
produced by plotting r/[D] versus r using the equation:
where, r is the ratio between the molar concentration of the bound drug and
the molar concentration of protein. The curve so obtained when extrapolated
gave an intercept on the Y axis representing nK, the intersection on the X-axis
representing n and the slope of the line being K, where, n is the number of
binding sites on the protein available to bind drug molecules or their complexes
and K is affinity constant for the binding of a drug molecule or its complex.
Study of Plasma Concentration
Test Animals and Administration of Drugs
Plasma samples were taken at various time intervals after a drug was orally
administrated. Sixteen adult rats of 250±25 g body weight were used.
They were kept rest for 7 days with normal diet. These rats were divided into
4 groups each having 4, marked as 1, 2 and 3 and one group was used as control.
Here, caffeine alone and its 1:1 mixture with gliclazide and metformin HCl were
administrated by orogastric tube individually in each group. They were over
night fasted before drug administration. Blood samples (1 mL) were collected
from cutting the tip of the tail into centrifuge tubes before drug administration
and at 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 h after drug administration. All blood
samples were protected from light, immediately centrifuged at 3000 rev min-1
f or 10 min and the plasma samples were separated into vials and kept into deep
freeze until taking the absorbance. The absorbance was measured at 273 nm and
the plasma concentration of caffeine was determined by using calibration curve
(Ara et al., 2008; Mohiuddin et al., 2009b; Rahat et al.,
Study of Blood Sugar, Cholesterol and Creatinine Level
A total of 42 rats were brought into diabetic type-2 status under the application
of streptozocine, which is a potentially effective hyperglycemic agent and the
required does was 90 μg kg-1. For each combination of drugs
such as ibuprofen-glibenclamide against glibenclamide alone, ibuprofen-glipizide
against glipizide alone and ibuprofen-gliclazide against gliclazide alone. 12
rats (two groups, 6+6), aged about three months, each weighing 150-200 g were
used for each drug combination. The six other rats were used as control. Blood
samples (1 mL) were collected from cutting the tip of the tail into centrifuge
tubes at fasting condition. All blood samples were protected from light, immediately
centrifuged at 3000 rpm for 10 min and the plasma samples separated into vials
and kept into freezer until taking the absorbance (Baki and Hossain, 1999).
Estimation of Blood Glucose Level
The blood glucose level was measured by the glucose oxidase method starting
at the initial and after every three days of application of therapy in all experimental
rats as well as for the control. Blood samples were collected from cutting the
tip of the tail into centrifuge tubes and were centrifuged for 10 min. Five
microliter of the standard/sample solution (1, 2, 4, 8, 12, 16, 20 mmol L-1)
were kept in different well of the microfiltration plate. Two hundred and fifty
microliter reagent (oxidase enzyme) was added to the solution of the each well
and the plate was kept in a incubator for 18 min at 37°C. Absorbance at
490 nm was taken and concentration of glucose was determined (Baki and Hossain,
Estimation of Blood Cholesterol Level
The blood cholesterol level was measured by enzymatic method. Blood samples
were collected and prepared for experiments in an analogous method and absorbance
at 500 nm was taken and the concentration of cholesterol was determined (Baki
and Hossain, 1999).
Estimation of Blood Creatinine Level
The blood creatinine level was measured by enzymatic method. Blood samples
were collected and prepared for experiments in an analogous method and absorbance
at 510 nm was taken and the concentration of creatinine was determined (Baki
and Hossain, 1999).
RESULTS AND DISCUSSION
The percentage of protein binding of ibuprofen was 91% and in 1:1 mixtures with glipizide and gliclazide were 80 and 82%, respectively, at saturation level (Fig. 2). It is seen that the percentage of protein binding of ibuprofen is markedly decreased in presence of glipizide and gliclazide, though the extents become almost similar at high concentrations of the drugs. It is known that the more is the protein binding of a drug the less will be its therapeutic effect.
The number of binding sites for ibuprofen alone were 0.75 and 1.37 for class
I and class II binding sites and the binding constants were 16 and 4.8, respectively.
The number of binding sites for the ibuprofen-glipizide systems were found to
be 1.01 and 6.17 and the binding constant being 2.32 and 0.17, respectively.
binding of ibuprofen in presence of glipizide and gliclazide
||Scatchard plot for protein binding of ibuprofen and its 1:1
mixtures with glipizide and gliclazide
|| The values for number of binding sites and affinity constants
The binding sites for ibuprofen-gliclazide systems were found to be 3.1 and
2.11 and the binding constants were 0.37 and 0.45, respectively (Fig.
3, Table 1). Since, the value of n1k1
and n2k2 are 12 and 4.8, respectively, the lone drug (ibuprofen)
has large affinities for serum protein binding and the interacting drug molecules
(glipizide and gliclazide lower these affinities (values being 2.35, 1.15, 1.05
and 0.95, respectively)). These two drugs compete with ibuprofen sufficiently
to occupy the binding sites in all the conditions studied.
Effects of Glipizide and Gliclazide on the Plasma Concentration of Ibuprofen
The pharmacological response is influenced by the plasma concentration of
drug. The plasma concentration of ibuprofen alone was 95 ng mL-1
and in the case of ibuprofen-glipizide it has been found to be 102 ng mL-1
while, in the case of ibuprofen-gliclazide the value was 115 ng mL-1
(Fig. 4). Therefore, the enhancement of the values of ibuprofen
in presence of glipizide and gliclazide is supportive of the values of protein
binding of ibuprofen in presence of these molecules (Mohiuddin et al.,
Influence of Ibuprofen on Hypoglycemic Properties of Glipizide and Gliclazide
The affect of ibuprofen on hypoglycemic effects of glipizide and gliclazide
were measured on rats after inducing diabetes in then by administration of streptozotocine
keeping a control group only on normal diet. The values of serum glucose, cholesterol
and creatinine were measured initially, after 3 days, 6 days (data not shown
in the Table 2) and after 9 days of drug administration (Table
It is seen that ibuprofen has good effects on antiglycemic activities of gliclazide and glipizide in streptozotocine-induced diabetic rats. However, the management of cholesterol and creatinine by gliclazide and glipizide are difficult tasks and leads to complications in many cases.
concentration of Ibuprofen after oral administration of Ibuprofen alone
and its mixture with glipizide and gliclazide in rats
||Serum glucose (mmol L-1), cholesterol (mg dL-1)
and creatinine (mg dL-1) levels of the controls and streptozotocine-induced
diabetic rats at initial and after 9 days of drug administration in the
cases of antidiabetic molecules and their mixtures with ibuprofen
The comparison of this study with our previous drug-drug interaction studies
(Mohiuddin, 2009a-c; Amran et al., 2006, 2008) infer that the results
of those studies do not differ significantly as shown in the current study as
Polypharmacy is a common practice in cases of patients undergoing a major operation, hospitalized patients and also in geriatric patients. We conclude on the basis of protein binding data and serum levels of ibuprofen in presence of glipizide and gliclazide that co-administration of ibuprofen with glipizide and gliclazide might be safe and effective.