Pharmacokinetic Drug Interaction between Nifedipine and Gliclazide

Abstract : Gliclazide and nifedipine have been clinically prescribed for the prevention or treatment of cardiovascular diseases with diabetes. However, these drugs have potential interaction. The pur- pose of this study was to investigate the possible effects of gliclazide on the pharmacokinetics of nifedipine and its main metabolite, dehydronifedipine, in rats.

We evaluated the effect of gliclazide on the activity of P-glycoprotein (P-gp) and cytochrome P450 (CYP)3A4. We determined the pharmacokinetic parameters of nifedipine and dehydronifedipine after oral and intravenous administration of nifedipine to rats in the presence or absence of gliclazide (1.0 and 4.0 mg/kg). Gliclazide inhibited CYP3A4 enzyme activity in a concentration-dependent manner

Nifedipine과 Gliclazide와의 약동학적 상호작용

임태환

, 김양우

, 최인

조선대학교병원 약제부

, 한국보건복지인력개발원

Pharmacokinetic Drug Interaction between Nifedipine and Gliclazide

Tae-Hwan Lim

, Yang-Woo Kim

, In Choe

Department of Pharmacy, Chosun University Hospital, 365 Pilmundaero, Dong-gu, Gwangju 501-759, Korea

, Korea Human Resource Development Institute for Health & Welfare, Cheongju-si, Chungbuk, Korea

투고일자 2016.2.2; 심사완료일자 2016.2.17; 게재확정일자 2016.2.24

�교신저자 최인 Tel:062-220-3291 E-mail:[email protected]

Original Article

INTRODUCTION

Nifedipine is a calcium channel-blocking agent that is widely used for the treatment of essential hypertension, coronary artery spasm and angina pectoris. Nifedipine inhibits the influx of extra- cellular calcium through myocardial and vascu- lar membrane pores by physically plugging the channel, which results in decreased intracellular calcium levels, inhibition of the contractile processes of smooth muscle cells, dilation of the coronary and systemic arteries, increased oxy-

gen delivery to the myocardial tissue, and decreased total peripheral resistance, systemic blood pressure and after load.

In humans, nifedipine is predominantly metabolized by CYP3A4 to its primary pyridine metabolite, dehydronifedipine.

CYP enzymes are responsible for the oxidative metabolism of many xenobiotics and play a major role in the phase I metabolism of many drugs.

CYP3A4 is the most abundant CYP enzyme (30-40%) in adult liver and metabolizes more than 50% of the clinically used drugs including nifedipine, with a 50% inhibitory concentration (IC 50 ) of 12.5 μ M. The areas under the plasma concentration-time curve (AUC 0-∞ ) and the peak concentration (C max ) of nifedipine were significantly (4.0 mg/kg, P<0.05) increased by 39.0% and 34.8%, respectively, in the presence of gliclazide, as compare to those of con- trol. In addition, the total body clearance (CL/F) was significantly (4.0 mg/kg, P<0.05) decreased by the treatment with gliclazide (47.9%). Consequently, the absolute bioavailability (AB) of nifedipine in the presence of gliclazide (4.0 mg/kg) was significantly (P<0.05) higher (39.2%) than that of the con- trol group. The metabolite-parent AUC ratio (MR) of nifedipine was significantly decreased by treat- ment with gliclazide (19.8%). The AUC 0-∞ of intravenous nifedipine was significantly (4.0 mg/kg, P<0.05) higher than that of the control group (18.1%), which suggested that gliclazide inhibited the metabolism of nifedipine.

The increased bioavailability of nifedipine in the presence of gliclazide may be due to an inhibition of the CYP3A4-mediated metabolism in the small intestine and/or in the liver and gliclazide-mediat- ed reduction of CL/F of nifedipine.

[Key words] Nifedipine, Dehydronifedipine, Gliclazide, Pharmacokinetics, Bioavailability

cyclosporine, midazolam and erythromycin.

Some studies indicate that nifedipine is a sub- strate of CYP3A4 in human.

P-gp is an adeno- sine-50-triphosphate (ATP) dependent efflux drug transporter that is constitutively expressed in normal tissues that includes gastrointestinal epithelium, canalicular membrane of the liver and capillary endothelial cells in the central nervous system.

Because of such tissue local- ized and its broad substrate specificity, P-gp appears to play a key role in absorption, distri- bution and elimination of many drugs.

It is generally known that the substrate and/or inhibitors of CYP3A4 and P-gp overlap with each other. Dorababu et al.

reported that nifedipine belonged to a group of P-gp sub- strate. Since P-gp is co-localized with CYP3A4 in the small intestine, P-gp and CYP3A4 may act synergistically to promote presystemic drug metabolism, which may result in the limited absorption of drugs.

Gliclazide, 3-(7-azabicyclo [3.3.0] oct-7-yl)-1- (4-methylphenyl) sulfonylurea is a second-gen- eration, widely used for the treatment of non- insulin-dependent diabetes mellitus.

Oral hypoglycaemic agents remain the cornerstone of the treatment of Type 2 diabetes in patients not responsive to diet, exercise and weight reduc- tion. Of the various oral hypoglycaemic agents available, sulphonylureas and biguanides are considered the first-line treatment. Amongst the sulphonylureas, gliclazide is widely used.

Considerable interindividual variability in meta- bolic clearance is a feature of the sulphonylureas

and differences in elimination are believed to contribute to therapeutic out- come and the occurrence of adverse effects.

Despite the widespread use of gliclazide, factors that contribute to pharmacokinetic variability have received less attention than for other

sulphonylureas.

In vitro, the effects of gli- clazide on the CYP3A4-inhibition and P-gp- inhibition activities have not been reported.

Thus, we attempted to evaluate the CYP3A4 activity of gliclazide, and furthermore, to evalu- ate the P-gp activity using rhodamine-123 retention assay in P-gp-overexpressed MCF- 7/ADR cells.

Although a combination of gliclazide and nifedipine have been clinically prescribed for the prevention or treatment of cardiovascular dis- eases with diabetes, the pharmacokinetic inter- action between gliclazide and nifedipine has not been reported in vivo thus far. Therefore, in this study we aimed to investigate the possible effects of gliclazide on the activities of CYP3A4 and P-gp and bioavailability & the pharmacoki- netics of nifedipine and its active metabolite, dehydronifedipine, after oral and intravenous administration of nifedipine with gliclazide in rats.

METHODS Materials

Nifedipine, dehydronifedipine, gliclazide and amlodipine [internal standard for the high-per- formance liquid chromatographic (HPLC) analy- sis of nifedipine] were purchased from the Sigma-Aldrich Co. (St. Louis, MO, USA).

Methanol, isooctane, methyl-tert-butyl ether (MTBE), analytical grade acetic acid and triethy- lamine (TEA) were products from Merck Co.

(Darmstadt, Germany). Rhodamine was from Calbiochem (USA), the CYP inhibition assay kit was from GENTEST (Woburn, MA, US). Other chemicals were of reagent or HPLC grade.

Apparatus used in this study included an HPLC

system equipped with a Waters 1515 isocratic

HPLC Pump, a Waters 717 plus auto sampler and a Waters

2487 scanning UV detector (Waters Co., Milford, MA, USA), an HPLC col- umn temperature controller (Phenomenex Inc., CA, USA), a Bransonic

Ultrasonic Cleaner (Branson Ultrasonic Co., Danbury, CT, USA), a vortex-mixer (Scientific Industries Co., NY, USA), and a high-speed microcentrifuge (Hitachi Co., Tokyo, Japan).

Animal studies

All animal study protocols were approved by the Animal Care Committee of Chosun University (Gwangju, Republic of Korea). Male Sprague-Dawley rats (270-300 g) were pur- chased from Dae Han Laboratory Animal Research Co. (Eumsung, Republic of Korea), and they were given free access to a normal stan- dard chow diet (No. 322-7-1; Superfeed Co., Wonju, Republic of Korea) and tap water.

Throughout the experiments, the animals were housed, four or five per cage, in laminar flow cages maintained at 22 ± 2℃, 50-60% relative humidity, under a 12 h light-dark cycle. The rats were acclimated under these conditions for at least 1 week. Each rat was fasted for at least 24 h before the experiment. The left femoral artery (for blood sampling) and left femoral vein (for iv administration of the drug) were cannu- lated using a polyethylene tube (SP45; i.d., 0.58 mm, o.d., 0.96 mm; Natsume Seisakusho Company, Tokyo, Japan) while each rat was under light ether anesthesia.

Intravenous and oral administration of nifedipine

The rats were divided into six groups (n=6, each): oral groups [10 mg/kg of nifedipine dis- solved in distilled water (1.0 mL/kg)] without

(control) or with 1.0 and 4.0 mg/kg of gliclazide (mixed in distilled water; total oral volume of 1.0 mL/kg) and intravenous groups (2.5 mg/kg of nifedipine; the same solution used: 0.9% NaCl- injectable solution; total injection volume of 1.0 mL/kg) without (control) or with 1.0 and 4.0 mg/kg of gliclazide. A feeding tube was used to administer nifedipine and gliclazide intragastri- cally. Gliclazide was administered 30 min prior to oral administration of nifedipine. A blood sample (0.3-mL aliquot) was collected from the femoral artery into heparinized tubes at 0.017 (at the end of infusion), 0.1, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h for the intravenous study, and 0.25, 0.5, 0.75, 1, 2, 4, 8, 12 and 24 h for the oral study. Whole blood (approximately 1.2 mL) col- lected from untreated rats was infused via the femoral artery at 0.75, 4 and 8 h, respectively, to replace blood loss caused by blood sampling.

The blood samples were centrifuged (13,000 rpm, 3 min), and a 150-μ L aliquot of plasma samples was stored in the deep freezer at -40℃

until the HPLC analysis.

HPLC assay

The plasma concentrations of nifedipine were

determined using an HPLC assay with a modifi-

cation to the method reported by Grundy et al.

Briefly, 50-μ L of amlodipine (3 μ g/mL), as the

internal standard and 50-μ L of 1.0 M sodium

hydroxide were added to 0.15-mL of the plasma

sample. It was then mixed for 3 s and 1-mL

MTBE-isooctane (75:25, v/v) was added. The

resultant mixture was vortex-mixed for 1 min

and centrifuged at 3,000 rpm for 5 min. The

organic layer (0.8 mL) was transferred into a

clean test tube and evaporated under a gentle

stream of nitrogen gas (no heat applied). The

dried extract was reconstituted with 200 μ L of

the mobile phase vortex-mixed for 1 min and aliquots of 160 μ L were transferred to a clean autosampler vial. A 70-μ L aliquot of the super- natant was injected into the HPLC system. The UV detector wavelength was set to 350 nm; and the column, a Nova-pack C8 (100 mm×8 mm i.d., 4 μ m; Waters Co., Milford, MA, USA), was used at room temperature. A mixture of methanol:water (62:38, v/v, pH 4.5, adjusted with acetic acid, 320 μ L of TEA/1,000 mL mix- ture) was used as the mobile phase at a flow rate of 1.0 mL/min. The retention times were:

internal standard at 16.8 min, nifedipine at 8.2 min, and dehydronifedipine at 6.5 min. The detection limits of nifedipine and dehy- dronifedipine in rat plasma were all 5 ng/mL.

The coefficients of variation for nifedipine and dehydronifedipine were all below 5.0%.

CYP3A4 inhibition assay

The assay of inhibitory activities on the human CYP3A4 enzyme activity was performed in a multiwell plate using CYP inhibition assay kit (GENTEST, Woburn, MA) as described previous- ly.

Briefly, human CYP enzyme was obtained from baculovirus-infected insect cells. CYP substrate (7-BFC for CYP3A4) was incubated with or without test compounds in the enzyme /substrate buffer with 1 pmol of P450 enzyme and an NADPH-generating system (1.3 mM NADP, 3.54 mM glucose 6-phosphate, 0.4 U/mL glucose 6-phosphate dehydrogenase and 3.3 mM MgCl 2 ) in potassium phosphate buffer (pH 7.4). Reactions were terminated by adding stop solution after 45 min incubation. Metabolite concentrations were measured using spectroflu- orometer (Molecular Device, Sunnyvale, CA) at an excitation wavelength of 409 nm and an emis- sion wavelength of 530 nm. Positive control (1 μ

M ketoconazole for CYP3A4) was run on the same plate and showed 99% inhibition. All experiments were done in duplicate, and the results were expressed as the percent of inhibi- tion.

Rhodamine-123 retention assay

The procedure used for the Rho-123 retention assay was similar to that reported previously.

MCF-7/ADR cells were seeded in 24-well plates.

At 80% confluence, the cells were incubated in FBS-free DMEM for 18 h. The culture medium was changed to Hanks’balanced salt solution and the cells were incubated at 37℃ for 30 min.

After incubation of the cells with 20 μ M rho- damine-123 in the presence or absence of gli- clazide (10, 30 and 100 μ M) and verapamil (posi- tive control, 100 μ M) for 90 min, the medium was completely removed. The cells were then washed three times with ice-cold phosphate buffer (pH 7.0) and lysed in EBC lysis buffer.

Rhodamine-123 fluorescence in the cell lysates was measured using excitation and emission wavelengths of 480 and 540 nm, respectively.

Fluorescence values were normalized to the total protein content of each sample and were pre- sented as the ratio to control.

Pharmacokinetic analysis

The plasma concentration data were analyzed

by the non-compartmental method using

Thermo Kinetica Software Version 5.0 (Thermo

Fisher Scientific Inc., Miami, OK, USA). The

parameter values were obtained by fitting to the

pharmacokinetic model using the simplex algo-

rithm. The area under the plasma concentra-

tion-time curve (AUC 0-∞ ) was calculated by a

trapezoidal rule. The peak concentration (C max )

of nifedipine in plasma and time to reach peak concentration (T max ) were obtained by visual inspection of the data from the concentration- time curve. The terminal half-life (t 1/2 ) was cal- culated by 0.693/K el. Total body clearance (CL/F) was calculated using the equation dose/AUC.

The absolute bioavailability (AB) was calculated using the equation AUC oral /AUC i.v. ×dose i.v. / dose oral , and the relative bioavailability (RB) of nifedipine were calculated using the equation AUC nifedipine with gliclazide / AUC control. The metabo- lite-parent AUC ratio (MR) was calculated using the equation AUC dehy-dronifedipine /AUC nifedipine.

Statistical analysis

All the means were presented with their stan- dard deviation. The pharmacokinetic parameters were compared with a one-way ANOVA, fol- lowed by a posteriori testing with the use of the Dunnett correction. A P value < 0.05 was con- sidered statistically significant.

RESULTS

Inhibitory Effect of Gliclazide on CYP3A4 Activity

The inhibitory effect of gliclazide on CYP3A4 activity is shown in Fig. 1. Gliclazide inhibited CYP3A4 activity in a concentration-dependent manner. Gliclazide inhibited CYP3A4 activity with an IC50 value of 12.5 μ M.

Rhodamine-123 Retention Assay

Accumulation of rhodamine-123, a P-glyco- protein substrate, was decreased in MCF-7/ADR cells overexpressing P-glycoprotein compared to that in MCF-7 cells lacking P-glycoprotein, as shown in Fig. 2. The concurrent use of gliclazide did not enhance the cellular uptake of rho- damine-123. This result suggests that gliclazide did not inhibit the P-gp activity.

Effect of Gliclazide on the Pharmacokinetics of oral Nifedipine

The mean plasma concentration-time profiles of nifedipine without or with of gliclazide (1.0

Fig. 1 Inhibitory effect of ketoconazole and gliclazide on CYP3A4 activity.

The results were expressed as the percent of inhibition.

Log concentration of ketoconazole(μM) Log concentration of gliclazide(μM)

and 4.0 mg/kg) are shown in Fig. 3. The phar- macokinetic parameters of nifedipine are sum- marized in Table 1. Gliclazide (4.0 mg/kg) signif- icantly (P<0.05) increased the area under the plasma concentration-time curve from time zero to time infinity (AUC 0-∞ ) of nifedipine by 39.0%,

and peak concentration (C max ) of nifedipine by 34.8%. The total body clearance (CL/F) was sig- nificantly decreased (4.0 mg/kg, P<0.05) by gli- clazide (47.9%). Accordingly, the absolute bioavailability (AB) values of nifedipine in the presence of gliclazide (4.0 mg/kg) were signifi- Fig. 2 Effects of gliclazide on the cellular accumulation of rhodamine-123 in MCF-7 and MCF-7/ADR cells.

Data represents mean ± SD (n=6).

Fig. 3 Mean arterial plasma concentration-time profiles of nifedipine after oral (10 mg/kg) administration of nifedipine with gliclazide to rats (mean ± SD, n=6).

● - Control (nifedipine alone, 10 mg/kg)

○ - with 1.0 mg/kg gliclazide

▼ - with 4.0 mg/kg gliclazide

* - P<0.05

cantly (P<0.05) higher (39.2%) than that of the control group. Gliclazide increased the relative bioavailability (RB) of nifedipine by 1.11- to 1.39-fold. There were no significant differences in the time to reach peak plasma concentration (T max ), terminal half-life (t 1/2 ) of nifedipine in the

presence of gliclazide.

Effect of Gliclazide on the Pharmacokinetics of Dehydronifedipine

The plasma concentration-time profiles of

0-∞

C

max

T

max

t

1/2

CL/F (ml∙min^-1) 1.81 ± 0.31 1.40 ± 0.28 0.94 ± 0.18*

AB (%) 15.8 ± 2.9 17.8 ± 3.5 21.8 ± 3.7*

RB (%) 100 111 139

Nifedipine + gliclazide

Parameter Control

1.0 mg∙kg

4.0 mg∙kg

Table 1. Mean (± SD, n=6) pharmacokinetic parameters of nifedipine after oral (10 mg/kg) administration of nifedipine without or with gliclazide in rats

* P<0.05, statistically significant different from the control

Abbreviations: AUC

: area under the plasma concentration-time curve from 0 h to infinity; C

: peak plasma concentration; T

: time to reach peak plasma concentration; t

: terminal half-life; CL/F: total body clearance; AB: absolute bioavailability; RB: relative bioavailability.

Fig. 4 Mean arterial plasma concentration-time profiles of dehydronifedipine after oral administration of nifedipine (10 mg/kg) with gliclazide to rats (mean ± SD, n=6).

● - Control (nifedipine alone, 10 mg/kg)

○ - with 1.0 mg/kg gliclazide

▼ - with 4.0 mg/kg gliclazide

dehydronifedipine are shown in Figure 4. The pharmacokinetic parameters of dehydronifedip- ine are summarized in Table 2. The AUC 0-∞ of dehydronifedipine was not significant greater than that of the control group by gliclazide. The MR ratio of nifedipine was significantly decreased (19.8%) by gliclazide, which suggested

that gliclazide inhibited metabolism of nifedip- ine.

Effect of Gliclazide on the Pharmacokinetics of Intravenous Nifedipine

Mean plasma concentration-time profiles of

AUC

0-∞

C

max

T

max

t

1/2

RB (%) 100 108 112

MR 36.4 ± 3.6 35.3 ± 3.4 29.2 ± 3.1*

Nifedipine + gliclazide

Parameter Control

1.0 mg∙kg

4.0 mg∙kg

Table 2. Mean (± SD, n=6) pharmacokinetic parameters of dehydronifedipine following an oral adminis- tration of nifedipine (10 mg/kg) without or with gliclazide in rats

* P<0.05, statistically significant different from the control,

Abbreviations: AUC

: area under the plasma concentration-time curve from 0 h to infinity; C

: peak plasma concentration; T

: time to reach peak plasma concentration; t

: terminal half-life; RB: relative bioavailability; MR: metabolite-parent AUC ratio.

Fig. 5 Mean arterial plasma concentration-time profiles of nifedipine after intravenous (2.5 mg/kg) administration of nifedipine with gliclazide to rats (mean ± SD, n=6).

● - Control (nifedipine alone, 2.5 mg/kg)

○ - with 1.0 mg/kg gliclazide,

▼ - with 4.0 mg/kg gliclazide, *, P<0.05.

AUC

0-∞

CL

t

t

1/2

RB (%) 100 107 118

Nifedipine + gliclazide

Parameter Control

1.0 mg∙kg

4.0 mg∙kg

Table 3. Mean (± SD, n=6) pharmacokinetic parameters of nifedipine after intravenous (2.5 mg/kg) administration of nifedipine without or with gliclazide in rats

* P<0.05, statistically significant different from the control,

Abbreviations: AUC

: area under the plasma concentration-time curve from 0 h to infinity; CL

: total body clearance; t

: terminal half-life; RB: relative bioavailability

nifedipine following an intravenous administra- tion of nifedipine (2.5 mg/kg) to rats without or with of gliclazide (1.0 and 4.0 mg/kg) are shown in Fig. 5, while the corresponding pharmacoki- netic parameters are shown in Table 3. The AUC 0-∞ of intravenous nifedipine was signifi- cantly higher than that of the control group (18.1%), which suggested that gliclazide inhibited metabolism of nifedipine.

Accordingly, the increased of nifedipine in the presence of gliclazide may be attributed to inhi- bition of the CYP3A4-mediated metabolism of nifedipine in the small intestine and/or in the liver by gliclazide.

DISCUSSION

The study of mechanisms of drug interactions is of much value in selecting the drug combina- tions to provide rational therapy.

The drug interaction studies assume much importance especially for drugs that have narrow margin of safety and where the drugs are used for pro- longed period of time. Diabetes mellitus is one such metabolic disorder that needs treatment for prolonged periods. Chronic diabetes leads to

several complications, which includes cardiovas- cular, renal, retinal and fungal infections etc., out of which cardiovascular complications are more prevalent.

Angina pectoris one such complication which is commonly seen in many chronic diabetics.

According to EUROPA study, the incidence of angina is more in people with type II diabetes and 12% of people from a normal population with angina or heart disease also had diabetes.

Nifedipine is widely used for the treatment of essential hypertension, coro- nary artery spasm, and angina pectoris.

Gliclazide is a second-generation sulfonylurea, widely used for the treatment of non-insulin- dependent diabetes mellitus.

In clinic, gliclazide and nifedipine could be prescribed for the pre- vention or treatment of cardiovascular diseases as complications of diabetes.

In Fig. 1, the inhibitory effect of gliclazide on

CYP3A4 activity is shown and gliclazide inhibit-

ed CYP3A4 activity with an IC50 value of 12.5 μ

M. In Figure 2, The concurrent use of gliclazide

did not enhance the cellular uptake of rho-

damine-123. This result suggested that gli-

clazide did not inhibit P-gp activity. Some in-

vitro and in-vivo studies have indicated that

nifedipine is metabolized to dehydronifedipine mainly by CYP3A4 enzymes.

As CYP3A4 expressed in rat is corresponding to the fuction of CYP3A4 in human.

This study evaluated the influence of gliclazide on the pharmacokinetics of nifedipine in rats in order to assess the potential drug interactions between gliclazide and nifedipine. Considering that nifedipine is a substrate of both CYP3A4 enzymes, gliclazide may significantly impact the pharmacokinetics and bioavailability of nifedipine.

As shown in Table 1, the coadministration of gliclazide (4.0 mg/kg) significantly altered the pharmacokinetic profiles of nifedipine compared with the administration of nifedipine alone.

Consequently, the relative bioavailability of nifedipine increased by 1.11- to 1.39-fold than those of the control group. Our results were consistent with those reported previously, which showed that oral diallyl trisulfide (major organosulfur compounds derived from garlic) significantly increased the bioavailability of nifedipine by inhibition of CYP3A4 in rats.

Our results are also consistent with those reported that showed that ketoconazole, Coenzyme Q10 and licochalcone A significantly increased the AUC 0-∞ and Cmax of nifedipine.

After intra- venous administration of nifedipine with gli- clazide, the AUC 0-∞ of nifedipine was signifi- cantly increased by gliclazide (Table 3). The MR ratios of nifedipine was significantly decreased (19.8%) by gliclazide, which suggested that gli- clazide inhibited metabolism of nifedipine (Table 2). Our results are also consistent with those reported that showed that licochalcone A signif- icantly decreased the MR ratios of nifedipine.

In conclusion, the increased bioavailability of nifedipine in the presence of gliclazide could be attributed to the inhibition of CYP3A4-mediated metabolism in the small intestine and/or in the

liver and/or to the reduction of CL/F of nifedip- ine by gliclazide. Concomitant use of nifedipine with gliclazide may require close monitoring for potential drug interactions. Further studies should be performed to determine drug interac- tions associated with in human.

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