# Drug-Drug Interaction between Pravastatin and Gliclazide in Animal Models

Authors: S. Satyanarayana; M. S. Chandrasekhar; O. Palakshi Gouda; K. Eswar Kumar
Journal: Scholarly Research Exchange
 (2008)

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## Abstract

The present study is planned to evaluate the safety of gliclazide (antidiabetic) therapy in the presence of pravastatin (antihyperlipidemic) in rats and rabbits. Studies in normal and alloxan-induced diabetic rats were conducted with oral doses of gliclazide, pravastatin, and their combination. Similarly, studies in normal rabbits were conducted with oral doses of gliclazide, pravastatin, and their combination with adequate washout periods in between the treatments. Blood samples were collected from rats and rabbits at different time intervals and were analyzed for blood serum gliclazide levels. Gliclazide produced hypoglycaemic/antihyperglycaemic activity in normal and diabetic rats with peak activity after 2 hours and 8 hours and hypoglycaemic activity in normal rabbits with peak activity after 3 hours. Pravastatin alone produced minor reduction in blood glucose levels in normal rats/diabetic rats/normal rabbits. Pravastatin increased the hypoglycaemic effect of gliclazide in normal rats/diabetic rats/normal rabbits when administered together. The serum insulin levels were increased with pravastatin treatment in rabbits. The serum gliclazide levels and pharmacokinetic parameters of gliclazide were altered significantly in presence of pravastatin in rabbits. The interaction observed appears to be pharmacokinetic interaction at metabolic and excretion levels.


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## Body

## 1. Introduction

In the present days,  the number of patients suffering from disorders like diabetes
and its associated comorbidities, that is, atherosclerosis, dyslipidemia, and
other cardiac  disorders is increasing worldwide.  Diabetes mellitusis a chronic metabolic disorder
characterized by rise in blood glucose level known as “hyperglycaemia.” It is of two types, type I accounting for 5%
prevalence and type II for 95% prevalence among diabetics. According
to WHO 40–60% type-2
diabetics are obese patients. Incidence rates of diabetes were 10 fold higher
in obese individuals, 1.5 fold higher in individual with dyslipidemia. Hence,
with oral hypoglycaemic drugs, the addition of lipid lowering drug is necessary
for the control of dyslipidemia. In such a situation, there may be chances for
drug-drug interaction between antidiabetic and antihyperlipidemic drugs.Sulfonylureas are the drugs of choice in the treatment of
type II diabetes. Currently gliclazide, a second generation sulfonylurea, was
preferred in therapy because of its selective inhibitory activity toward
pancreaticK+ATP channels [1], low incidence of producing severe hypoglycaemia [2], and
other haemobiological effects [3]. It is well established that sulfonylureas
produce insulin secretion and improve tissue utilization of glucose at cellular
level which was responsible for lowering of blood glucose level. The
sulfonylureas and related drugs used in type II diabetes stimulate insulin by
closing K+ATP channels in pancreatic β cells.Antihyperlipidemic
drugs like statins and fibratesare widely used for
prophylactic treatment in dyslipidemia and atherosclerosis. Among statins, HMG
CoA reductase inhibitor, pravastatin is widely used because it is indicated for
primary hypercholesterolemia, mixed dyslipidemia, atherosclerosis,
hypertriglyceridemia, and dysbetalipoproteinemia [4]. It has also gained
approval for primary prevention of coronary events and secondary prevention of
cardiovascular events [5]. Pravastatin is metabolized by P 450 CYP 3A4 & CYP
3A5 isozymes in the liver [6, 7], and it also has moderate inhibition on
metabolizing enzymes P 450 CYP 2C9, CYP 2D6, and CYP 3A4 [8]. Hence, there is
more possibility of pravastatin for inhibition of metabolism of gliclazide,
which is also metabolized by both CYP 2C9 and CYP 3A4.The concomitant administration of gliclazide with
pravastatin in diabetes associated with atherosclerosis may result in drug-drug
interaction with enhanced/decreased gliclazide activity, which is unwanted. The
study is planned to establish the safety of the drug combination in animal
models with respect to blood glucose level and find out the mechanisms
responsible for the interaction if any.


## 2. Materials and Methods

### 2.1. Drugs and Chemicals

Gliclazide and Pravastatin are the gift samples from Micro labs (Bangalore, India) and Biocon Ltd. (Bangalore, India), respectively. Alloxan monohydrate was purchased from Sigma Aldrich (Bommasandra, Jigani, Bangalore 560100, India), Bangalore. Glucose kits of  Span diagnostics were procured from local suppliers. The HPLC grade methanol and acetonitrile of Qualigens fine chemicals, Mumbai were procured from local chemical suppliers. All other chemicals used were of analytical grade.


### 2.2. Animals

Albino rats of either sex, weighing
between 160–280 g procured
from Drugs Testing Lab (Bangalore, India), were
used in the study. They were maintained under standard laboratory conditions at
ambient temp of25±2°C with 12-hour light/12-hour dark cycle. They were fed
with standard pellet diet (Venkateshwar enterprises Pvt. Ltd, Bangalore, India)
and water ad libitum. Animals
were fasted for 18 hours before experiment and during the experiment they were
withdrawn from food and water. Normal albino rabbits of either sex, weighing
between 1.35–1.72 kg were
procured from Drugs Testing Lab, Bangalore, India, were used in the study. They were maintained under standard laboratory conditions at
ambient temp of 25±2°C with 12-hour light/12-hour dark cycle. They were fed
with standard pellet diet (Venkateshwar enterprises Pvt. Ltd, Bangalore, India)
and water ad libitum. Animals
were fasted for 18 hours before experiment and during the experiment they were
withdrawn from food and water. The prior approval for conducting the
experiments in rats and rabbits was obtained from our Institutional Animal
Ethics Committee and our lab is approved by CPCSEA, Government of India (Regd.
No. GCP/CPCSEA/04/2005-06).


## 2.1. Drugs and Chemicals

Gliclazide and Pravastatin are the gift samples from Micro labs (Bangalore, India) and Biocon Ltd. (Bangalore, India), respectively. Alloxan monohydrate was purchased from Sigma Aldrich (Bommasandra, Jigani, Bangalore 560100, India), Bangalore. Glucose kits of  Span diagnostics were procured from local suppliers. The HPLC grade methanol and acetonitrile of Qualigens fine chemicals, Mumbai were procured from local chemical suppliers. All other chemicals used were of analytical grade.


## 2.2. Animals

Albino rats of either sex, weighing
between 160–280 g procured
from Drugs Testing Lab (Bangalore, India), were
used in the study. They were maintained under standard laboratory conditions at
ambient temp of25±2°C with 12-hour light/12-hour dark cycle. They were fed
with standard pellet diet (Venkateshwar enterprises Pvt. Ltd, Bangalore, India)
and water ad libitum. Animals
were fasted for 18 hours before experiment and during the experiment they were
withdrawn from food and water. Normal albino rabbits of either sex, weighing
between 1.35–1.72 kg were
procured from Drugs Testing Lab, Bangalore, India, were used in the study. They were maintained under standard laboratory conditions at
ambient temp of 25±2°C with 12-hour light/12-hour dark cycle. They were fed
with standard pellet diet (Venkateshwar enterprises Pvt. Ltd, Bangalore, India)
and water ad libitum. Animals
were fasted for 18 hours before experiment and during the experiment they were
withdrawn from food and water. The prior approval for conducting the
experiments in rats and rabbits was obtained from our Institutional Animal
Ethics Committee and our lab is approved by CPCSEA, Government of India (Regd.
No. GCP/CPCSEA/04/2005-06).


## 3. Methods

### 3.1. Pharmacodynamic Study in Normal/Diabetic Rats

A group of six rats were administered with 0.72 mg/200 g of bd wt of gliclazide, orally. The same group was administered with
pravastatin 0.72 mg/200 g bd wt orally and combination of pravastatin and
gliclazide. One-week washout period was maintained between treatments. The same
treatment was repeated in a group of six alloxan-induced diabetic rats. Blood
samples were withdrawn by retro orbital puncture [9] at 0, 1, 2, 3, 4, 6, 8, 10,
and 12 hours and were analyzed for blood glucose by GOD/POD method [10] using
commercial glucose kits (Span diagnostics).


#### 3.1.1. Induction of Diabetes

Diabetes was induced in rats by the
administration of alloxan monohydrate in two doses, that is, 100 mg and 50 mg/kg
bd wt intraperitoneally for two consecutive days [11].


### 3.2. Pharmacokinetic and Pharmacodynamic Study in Rabbits

A group of five rabbits were administered with
2.8 mg/1.5 kg bd wt of gliclazide, orally. The same group was administered with
2.8 mg/1.5 kg bd wt of pravastatin, orally after a washout period of one week.
After a further washout period the same group was administered with the
combination of pravastatin and gliclazide. After interaction study the same
group was continued with the daily treatment of interacting drug (pravastatin)
for the next eight days with regular feeding. Later after 18-hour fasting, they
were again given the combined treatment on the ninth day. Blood samples were
withdrawn from the marginal ear vein of each rabbit at 0, 1, 2, 3, 4, 6, 8, 12,
18, and 24 hours. They were analyzed for glucose by GOD/POD [10] and for
gliclazide by HPLC [12].


#### 3.2.1. Data and Statistical Analysis

Data was expressed as mean ± standard error of mean (SEM). The significance was determined by applying Student’s pairedt-test.


## 3.1. Pharmacodynamic Study in Normal/Diabetic Rats

A group of six rats were administered with 0.72 mg/200 g of bd wt of gliclazide, orally. The same group was administered with
pravastatin 0.72 mg/200 g bd wt orally and combination of pravastatin and
gliclazide. One-week washout period was maintained between treatments. The same
treatment was repeated in a group of six alloxan-induced diabetic rats. Blood
samples were withdrawn by retro orbital puncture [9] at 0, 1, 2, 3, 4, 6, 8, 10,
and 12 hours and were analyzed for blood glucose by GOD/POD method [10] using
commercial glucose kits (Span diagnostics).


### 3.1.1. Induction of Diabetes

Diabetes was induced in rats by the
administration of alloxan monohydrate in two doses, that is, 100 mg and 50 mg/kg
bd wt intraperitoneally for two consecutive days [11].


## 3.1.1. Induction of Diabetes

Diabetes was induced in rats by the
administration of alloxan monohydrate in two doses, that is, 100 mg and 50 mg/kg
bd wt intraperitoneally for two consecutive days [11].


## 3.2. Pharmacokinetic and Pharmacodynamic Study in Rabbits

A group of five rabbits were administered with
2.8 mg/1.5 kg bd wt of gliclazide, orally. The same group was administered with
2.8 mg/1.5 kg bd wt of pravastatin, orally after a washout period of one week.
After a further washout period the same group was administered with the
combination of pravastatin and gliclazide. After interaction study the same
group was continued with the daily treatment of interacting drug (pravastatin)
for the next eight days with regular feeding. Later after 18-hour fasting, they
were again given the combined treatment on the ninth day. Blood samples were
withdrawn from the marginal ear vein of each rabbit at 0, 1, 2, 3, 4, 6, 8, 12,
18, and 24 hours. They were analyzed for glucose by GOD/POD [10] and for
gliclazide by HPLC [12].


### 3.2.1. Data and Statistical Analysis

Data was expressed as mean ± standard error of mean (SEM). The significance was determined by applying Student’s pairedt-test.


## 3.2.1. Data and Statistical Analysis

Data was expressed as mean ± standard error of mean (SEM). The significance was determined by applying Student’s pairedt-test.


## 4. Results

Gliclazide produced biphasic
hypoglycaemic activity with maximum reduction of38.34±4.63% & 36.34±2.97%  after 2 hours
and 8 hours in normal rats (Table 1 and Figure 1) and antihyperglycaemic activity with maximum
reduction of 36.7±1.27% & 37.04±0.64%  after  2 hours and 8 hours in diabetic rats,
respectively (Table 2 and Figure 2). It produced peak activity of 33.1±3.23% reductions  after  3 hours in normal
rabbits (Table 3 and Figure 3). Pravastatin alone produced 9.10±1.29% & 8.33±1.54% and 18.92±1.04 & 8.71±0.67% decrease in the blood glucose in normal and diabetic
rats after 2 hours and
8 hours, respectively and 8.35±1.83% in normal rabbits after 3 hours. When gliclazide given in
combination with pravastatin produced enhanced hypoglycaemic effect with
maximum reduction of 48.44±2.50% & 47.26±6.25% and 48.99±0.72% &
48.25±0.56% in the blood glucose in normal and diabetic rats after 2 hours and 8 hours,
and 39.77±2.66% in normal rabbits after 3 hours, respectively (Tables 1, 2, 3 and Figures 1, 2, 3). The serum gliclazide levels and
pharmacokinetic parameters of gliclazide like AUC, AUMC, T1/2, clearance,
Vdss, Vdarea, Cmax, and Tmax were altered significantly with single- and multiple-dose
treatments of pravastatin in normal rabbits (Tables 4,   and Figure 4). The serum insulin levels were
increased with pravastatin treatment in normal rabbits.Table 1
Mean percent blood glucose changes after oral administration of gliclazide, Pravastatin, and their combination in normal rats (n=6).
Time (h)GliclazidePravastatatinGliclazide + pravastatatin00001−31.14±4.22−05.24±1.60−35.45±4.152−38.34±4.63−09.10±1.29−48.44±2.503−22.65±3.70−10.85±1.46−39.29±3.14†4−13.21±3.80−09.66±1.50−41.51±2.64*6−32.52±2.85−07.78±1.58−41.14±6.458−36.34±2.97−08.33±1.54−47.26±6.25†10−28.06±3.31−07.88±1.54−34.18±2.3212−19.61±2.90−06.69±2.16−28.48±1.87*Significant at P<.01: †Significant at P<.05 compared to gliclazide
control.Table 2
Mean percent blood glucose changes after oral administration of gliclazide, Pravastatin, and their combination in diabetic rats (n=6).
Time (h)GliclazidePravastatatinGliclazide + pravastatatin00001−29.25±0.60−14.68±1.10−32.23±1.71‡2−36.70±1.27−18.92±1.04−48.99±0.72*3−32.57±0.51−17.64±0.74−41.39±0.57*4−30.28±0.52−14.21±0.64−37.25±0.69‡6−34.29±0.61−11.60±0.95−39.21±0.54†8−37.04±0.64−08.71±0.67−48.25±0.56*10−31.54±0.81−04.35±0.82−31.47±1.1912−27.30±0.91−03.17±0.55−28.20±0.73*Significant at P<.001; †Significant at P<.01; ‡Significant
at P<.05 compared to gliclazide control.Table 3
Mean percent blood glucose changes after oral administration of gliclazide, pravastatin, and their combination in normal rabbits (n=5).
Time (h)GliclazidePravastatatinGliclazide + pravastatatin (acute)Gliclazide + pravastatatin (chronic)000001−22.17±2.21−03.71±1.82−27.60±3.21†−26.99±2.48‡2−23.44±3.88−06.93±1.87−32.02±1.87‡−33.25±4.83‡3−33.10±3.23−08.35±1.84−39.77±2.66†−41.41±2.52†4−26.94±3.55−08.60±1.93−33.04±2.03−37.12±3.15‡6−24.72±2.63−09.90±2.00−28.89±2.48‡−29.14±2.348−22.49±2.91−10.83±2.06−27.94±2.06†−28.83±0.78‡12−16.71±3.60−10.21±1.54−15.70±2.18−17.48±2.1018−04.38±1.62−11.33±1.70−11.01±1.71‡−13.50±3.60‡24−02.79±2.45−11.95±1.98−05.97±1.87−07.36±1.61‡*Significant at P<.001; †Significant at P<.01; ‡Significant at P<.05 compared to gliclazide control.Table 4
Mean serum gliclazide concentration (ng/ml) before and after oral administration of pravastatin in normal rabbits (n=5).
Time (h)GliclazideGliclazide + pravastatatin (acute)Gliclazide + pravastatatin (chronic)0000198.54±06.29116.4±03.43†121.9±08.60‡2246.6±10.54168.7±03.58†178.3±06.60†3343.4±11.54393.7±07.31†396.1±17.57‡4294.8±12.19294.6±06.55301.8±07.396261.2±09.03312.8±04.56*316.7±09.81*8222.3±07.33342.3±15.80*343.8±08.12*12151.1±09.34264.5±06.02*259.7±07.58*18105.1±06.59108.1±02.76112.5±06.242474.09±02.2296.43±01.74*98.46±06.27‡*Significant at P<.001; †Significant at P<.01; ‡Significant at P<.05 compared to gliclazide control.Table 5
Mean pharmacokinetic parameters of gliclazide before and after oral administration of  pravastatin in normal rabbits (n=5).
Kinetic parametersGliclazideGliclazide + pravastatin (acute)Gliclazide + pravastatin (chronic)AUC0–24 (ng/mL/h)3905±66.665034±95.01*5083±117.10*AUMC0–24 (ng/mL/h*h)47503±165357651±911.50*59091±2941.00Kel (h−1)0.07±0.010.08±0.01‡0.07±0.0063AUC0–∞ (ng/mL/h)4963±98.566182±106.60*6342±262.20‡AUMC0–∞ (ng/mL/h*h)88101±452998882±1565‡105736±10157T1/2 (h)9.86±0.398.23±0.11‡8.70±0.63Ka (h−1)1.15±0.001.15±0.001.15±0.00Clearance (mL/h)637.80±24.05513.10±21.50‡490.30±19.07‡Clearance (mL/h/kg)375.50±8.34301.40±5.44*296.00±11.81Vdss (mL)10704±328.307765±346.90†7664±479.10‡Vdss (mL/kg)6314±181.504562±110.00†4598±144.30†Vdarea (mL)9037±202.506109±273.80†6145±453.60‡Vdarea (mL/kg)5335±155.003589±90.08†3682±157.70†MRT (h)17.71±0.6415.99±0.1416.53±0.94Cmax⁡ (ng/mL)325.80±12.47393.70±7.37†396.10±17.57‡Tmax⁡ (h)3.00±0.003.00±0.003.00±0.00*Significant
at P<.0001; †Significant at P<.001; ‡Significant at P<.05
compared to gliclazide control.Figure 1
The mean percent
blood glucose change with gliclazide alone, pravastatin alone, and in
combination in normal rats (n=6).Figure 2
The mean
percent blood glucose change with gliclazide alone, pravastatin alone, and in
combination in diabetic rats (n=6).Figure 3
Effect of acute and chronic administration of
pravastatin on the percent blood glucose change with gliclazide in normal rabbits
(n=5).Figure 4
Serum gliclazide concentration versus time in
normal rabbits treated with pravastatin (n=5).


## 5. Discussion

Drug interactions are
usually seen in clinical practice and the mechanisms of interactions are
evaluated usually in animal models. We studied the influence of pravastatin on
the pharmcodynamics and pharmacokinetics of gliclazide in normal and diabetic
rats and in normal rabbits. The normal rat model served to quickly identify the
interaction and diabetic rat model served to validate the same response in the
actually used condition of the drug. The rabbit model is another dissimilar
species to validate the occurrence of the interaction.The gliclazide produced biphasic
response in rat model may be due to its enterohepatic circulation in rats [13, 14]
and in humans [15]. Such effect is not seen in rabbit model. Gliclazide is known to produce
hypoglycaemic activity by pancreatic [16] (stimulating insulin secretion by
blocking K+channels in the pancreatic β cells) and extra pancreatic
[17] (increasing tissue uptake of glucose) mechanisms. Pravastatin had minor effect
on blood glucose levels and enhanced hypoglycaemic effect of gliclazide when
administered in combination in rats and rabbits.There was significant rise
in serum gliclazide levels and pharmacokinetic parameters like AUC, AUMC, T1/2, clearance,
Vdss, Vdarea, Cmax, and Tmax of gliclazide with single- and multiple-dose
treatments of pravastatin. The increase in AUC, AUMC indicates improved
availability of gliclazide in presence of pravastatin. The increased
bioavailability cannot be due to improved absorption, since absorption rate and
absorption half-life of gliclazide were not altered. There might not be
interaction at absorption level since oral absorption of pravastatin is poor.
Gliclazide is highly protein bound drug (85–99%) [16], whereas pravastatin is
bound to proteins to the extent of 43–55% [4, 18]. Hence,
the possibility of displacing gliclazide from protein bound sites by
pravastatin was low. Hence, the rise of gliclazide blood levels in the presence
of pravastatin might be other than improved absorption and altered
distribution.Pravastatin is metabolized
by hepatic P450 CYP 3A4, CYP 2C9 isozymes [6, 8], and there is more possibility
of pravastatin for inhibition of metabolism of gliclazide, which is also
metabolized by both CYP 2C9 and CYP 3A4 [19]. Further gliclazide is eliminated
through renal (80%) and biliary (20%) routes [16, 20]. Pravastatin is also
eliminated by both the routes 20% being in urine and 71% in feacal matter [18].
Hence, there is possibility for interaction between pravastatin and gliclazide
at biliary excretion also. However, the drug pravastatin did not change the
pattern of biphasic response of gliclazide indicating that it did not interfere
with the reabsorption of gliclazide in its enterohepatic circulation in
rats.  Hence, the interaction at hepatic
metabolism with reduced gliclazide metabolism by pravastatin leading to raised
serum levels is possible.Alternatively, since
pravastatin is acidic drug, it might promote reabsorption of gliclazide (which
is also acidic) by tubular reabsorption process, and hence raises the blood
gliclazide levels. Hence, there is possibility for interaction at urinary
excretion. So, the rise in serum levels of gliclazide in the presence of pravastatin
may be because of the combined influence of its metabolism and urinary
excretion.


## 6. Conclusions

The
interaction observed appears to be pharmacokinetic interaction at metabolic and
excretion levels. Since the interaction was observed in two dissimilar species,
it is likely to occur in humans also. Hence, the combination of gliclazide and pravastatin
should be contraindicated or used with caution in a clinical situation.



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*Source: 620489-2008-09-03.xml*