Pharmacokinetic interaction between itraconazole and metformin in rats: competitive inhibition of metabolism of each drug by each other via hepatic and intestinal CYP3A1/2

Abstract

BACKGROUND AND PURPOSE

Fungal infection is prevalent in patients with diabetes mellitus. Thus, we investigated whether a pharmacokinetic interaction occurs between the anti-fungal agent itraconazole and the anti-glycaemic drug metformin, as both drugs are commonly administered together to diabetic patients and are metabolized via hepatic CYP3A subfamily in rats.

EXPERIMENTAL APPROACH

Itraconazole (20 mg·kg⁻¹) and metformin (100 mg·kg⁻¹) were simultaneously administered i.v. and p.o. to rats. Concentrations (I) of each drug in the liver and intestine, maximum velocity (V_max), Michaelis–Menten constant (K_m) and intrinsic clearance (CL_int) for the disappearance of each drug, apparent inhibition constant (K_i) and [I]/K_i ratios of each drug in the liver and intestine were determined. Also the metabolism of each drug in rat and human CYPs was measured in vitro.

KEY RESULTS

After simultaneous administration of both drugs, either i.v. or p.o., the total area under the plasma concentration–time curve from time zero to infinity (AUC)s of itraconazole and metformin were significantly greater than that of either drug administered alone. The metabolism of itraconazole and metformin was significantly inhibited by each other via CYP3A1 and 3A2 in rat and 3A4 in human microsomes.

CONCLUSIONS AND IMPLICATIONS

The significantly greater AUCs of itraconazole and metformin after i.v. administration of both drugs are probably due to competitive inhibition of the metabolism of each drug by each other via hepatic CYP3A1/2. Whereas after oral administration of both drugs, the significantly greater AUCs of each drug administered together than that of either drug alone is mainly due to competitive inhibition of intestinal metabolism of each drug by each other via intestinal CYP3A1/2.

Introduction

In clinical situations drug interactions, pharmacological effects and side effects are usually evaluated by measuring changes in plasma levels of the drug. Hence, laboratory animals, such as rats, are commonly used to perform experiments that are not possible in humans, to predict drug–drug interactions based on the inhibition of cytochrome P450 (CYP)-mediated drug metabolism.

In patients with diabetes mellitus, fungal infection is prevalent (Tan and Joseph, 2004) and itraconazole, a triazole anti-fungal agent, is one of the most common and safe agents for the treatment of dermatophyte onychomycosis in these patients (Gupta et al., 2006). In humans, it has been suggested that hepatic CYP3A4 could be involved in the metabolism of itraconazole to form several metabolites, including 7-hydroxyitraconazole, the major metabolite (Penzak et al., 1999), which is an inhibitor of the CYP3A subfamily (Sagir et al., 2003). Recently, both the metabolism of itraconazole and formation of 7-hydroxyitraconazole were shown to be mediated by CYP3A1/2 in rats (Lee et al., 2009). Human CYP3A4 and rat CYP3A1 proteins have 73% homology (Lewis, 1996). After i.v., p.o., intraportal, intragastric or intraduodenal administration of itraconazole at a dose of 10 mg·kg⁻¹ to male Sprague–Dawley rats, the extent of absolute oral bioavailability (F) value was approximately 35%, the hepatic first-pass effect after absorption into the portal vein was almost negligible, and the intestinal first-pass effect was approximately 70% of the oral dose (Shin et al., 2004).

Metformin, a biguanide anti-hyperglycaemic agent, has been widely used in the management of type 2 diabetes mellitus; it lowers blood glucose concentration without causing hypoglycaemia (Scheen, 1996). In humans, approximately 20% of an i.v. dose is thought to be metabolized (Scheen, 1996). After i.v., p.o., intraportal, intragastric or intraduodenal administration of metformin at a dose of 100 mg·kg⁻¹ to male Sprague–Dawley rats, the F was approximately 29.9%, the hepatic first-pass effect after absorption into the portal vein was 26.0% and the intestinal first-pass effect was 53.8% of the p.o. dose (Choi et al., 2006). Metformin is metabolized via hepatic CYP2C11, 2D1 and 3A1/2 (but not via CYP1A2, 2B1/2 or 2E1) in male Sprague–Dawley rats (Choi and Lee, 2006). Therefore, as itraconazole and metformin are often administered together to diabetic patients and have been shown to be metabolized via the same hepatic CYP3A subfamily in rats (Choi and Lee, 2006; Lee et al., 2009), it is important to assess any possible interactions between these two drugs.

We used a rat model to study the pharmacokinetic interaction between itraconazole and metformin, after simultaneous i.v. or p.o. administration of the drugs.

Methods

Animals

Protocols for the animal studies were approved by the Animal Care and Use Committee of the College of Pharmacy, Seoul National University, Seoul, South Korea. Male Sprague–Dawley rats (5–8 weeks old, weighing 190–300 g) were purchased from Taconic Farms Inc. (Samtako Bio Korea, O-San, South Korea). The procedures used for housing and handling of the rats were similar to those reported previously (Choi and Lee, 2006; Choi et al., 2006; 2008;).

In vitro studies

Competitive inhibition for metabolism of itraconazole and metformin by each other in rat hepatic microsomes

The procedures used for the preparation of hepatic microsomes and to investigate the mode of inhibition for metabolism of metformin by itraconazole were similar to those reported previously (Choi et al., 2008). The following components were added to test tubes: hepatic microsomes (equivalent to 0.5 mg protein); 5 µL of 0.9% NaCl solution containing metformin base (as a substrate) at final concentrations of either 2.5, 5, 10, 20, 50 or 100 µM; 5 µL of dimethyl sulphoxide (DMSO) containing final itraconazole (as an inhibitor) concentrations of 0, 2, 10, 20, 50 or 100 µM; and 50 µL of 0.1 M phosphate buffer (pH 7.4) containing 1 mM β-nicotinamide adenine dinucleotide phosphate (NADPH). The volume was adjusted to 0.5 mL by adding 0.1 M phosphate buffer (pH 7.4), and the components were incubated at 37°C using a thermomixer (Thermomixer 5436; Eppendorf, Hamburg, Germany) at 500 oscillations per minute (opm). Incubation times were 5, 10 and 15 min. All of the incubation times were within the linear range of the reaction rate. At the end of the incubation time, the reaction was terminated by addition of 1 mL of acetonitrile. The procedures used to investigate the mode of inhibition of itraconazole by metformin were similar to those for mentioned above for metformin. Five microlitres of DMSO containing final itraconazole (as a substrate) concentrations of 2.5, 5, 10, 20 or 50 µM and 5 µL of 0.9% NaCl solution containing final metformin base (as an inhibitor) concentrations of 0, 2, 10, 20, 100 or 200 µM were used. Incubation times were 5, 15, 30, 45 and 50 min. At the end of the incubation time, the reaction was terminated by addition of 1 mL of methyl t-butyl ether. All of the incubation times were also within the linear range of the reaction rate.

Measurement of maximum velocity (V_max), Michaelis–Menten constant (K_m), intrinsic clearance (CL_int) and inhibition constant (K_i) for the disappearance of itraconazole and metformin in the presence and absence of each drug in rat hepatic and intestinal microsomes

The procedures used for the preparation of hepatic and intestinal microsomes and for measurement of V_max and K_m (the concentration at which the rate is one half of the V_max) for the disappearance of itraconazole in the absence and presence of metformin in hepatic and intestinal microsomes were similar to those reported previously (Choi et al., 2008). The following components were added to test tubes: microsomes (equivalent to 0.5 and 1.0 mg proteins for hepatic and intestinal microsomes respectively); 5 µL of DMSO containing final itraconazole concentrations of 2.5, 5, 10, 20 or 50 µM; 5 µL of 0.9% NaCl solution with and without a final metformin base concentration of 20 µM; and 50 µL of 0.1 M phosphate buffer (pH 7.4) containing 1 mM NADPH. The volume was adjusted to 0.5 mL by adding 0.1 M phosphate buffer (pH 7.4), and the components were incubated at 37°C using a thermomixer (at 500 opm). Incubation times were 5, 15, 30, 50 and 60 min for both hepatic and intestinal microsomes. The reaction was terminated by addition of 1 mL of methyl t-butyl ether at the end of the incubation for both hepatic and intestinal microsomes.

The methods used to determine the V_max and K_m for the disappearance of metformin in the absence and presence of itraconazole in hepatic and intestinal microsomes were similar to those mentioned above for itraconazole. Five microlitres of 0.9% NaCl solution containing final metformin base concentrations of 2.5, 5, 10, 20, 50 or 100 µM and 5 µL of DMSO with and without final itraconazole concentration of 20 µM were used. Incubation times were 5, 10 and 15 min for hepatic microsomes and 15, 30, 45 and 60 min for intestinal microsomes. At the end of the incubation period for both hepatic and intestinal microsomes, the reaction was terminated by addition of 1 mL of acetonitrile. All of the above incubation times were also within the linear range of the reaction rate.

The kinetic constants (K_m and V_max) for the disappearance of itraconazole and metformin in the absence and presence of each other were calculated using a non-linear regression method (Duggleby, 1995). The CL_int was calculated by dividing the V_max by the K_m.

The apparent K_i of itraconazole and metformin for the disappearance of metformin and itraconazole, respectively, in hepatic and intestinal microsomes was also measured. The K_i of an inhibitor (metformin or itraconazole) was calculated using the following equation for competitive inhibition (Segel, 1975):

where [I] is the concentration of an inhibitor and K_m′ is the apparent Michaelis–Menten constant of the substrate with an inhibitor. For competitive inhibition, the K_i is equivalent to the concentration of an inhibitor that doubles the slope of the inverse of velocity for the disappearance plotted against the inverse of the substrate concentration.

Bechmann and Lewis (2005) suggest that if, for a putative inhibitory drug–drug interaction, the [I]/K_i ratio is >2, the drug–drug interaction is due to CYP inhibition, but if the ratio is <0.01, the mechanism is not attributable to CYP inhibition.

Measurement of disappearance rate of itraconazole and metformin in rat microsomes from Baculovirus-infected insect cells expressing CYP3A1 and 3A2 and human Baculovirus-infected insect cells expressing CYP3A4

To find which CYP isozymes are responsible for the inhibition of the metabolism of itraconazole and metformin by each other, rat microsomes from Baculovirus-infected insect cells expressing CYP3A1 and 3A2 and human Baculovirus-infected insect cells expressing CYP3A4 were incubated with itraconazole and/or metformin. The procedures used were similar to those described previously (Yang et al., 2008). The disappearance rates of itraconazole in the presence and absence of metformin were determined after incubating the rat Baculovirus-infected insect cells (n = 4; equivalent to 30 pmol; each) or human Baculovirus-infected insect cells (n = 4; equivalent to 30 pmol; each) in 5 µL of 0.9% NaCl solution containing 20 µM itraconazole in the absence and presence of 20 µM of metformin base, and 50 µL of 0.1 M phosphate buffer of pH 7.4 containing 1 mM NADPH. The volume was adjusted to 0.5 mL by adding 0.1 M phosphate buffer of pH 7.4, and the incubations were carried out at 37°C using a thermomixer (at 500 opm), for 20 min; the reaction was terminated by addition of 1 mL methyl t-butyl ether.

The method used to measure the disappearance rates of metformin in the presence and absence of itraconazole was similar to those mentioned above for itraconazole. Five microlitres of 0.9% NaCl solution containing 20 µM metformin base were incubated with the cells in the absence and presence of 20 µM itraconazole. After the 20 min incubation, the reaction was terminated by addition of 1 mL of acetonitrile.

Rat plasma protein binding of itraconazole and metformin with and without each other using equilibrium dialysis

Protein binding values for itraconazole and metformin with and without each other to fresh plasma from control rats (n = 5; each) were measured using equilibrium dialysis (Choi et al., 2006). One millilitre of plasma was dialysed against 1 mL of isotonic Søresnsen phosphate buffer (pH 7.4) containing 3% (w/v) dextran (‘the buffer’) in a 1 mL dialysis cell (Spectrum Medical Industries, Los Angeles, CA, USA) using a Spectra/Por 4 membrane (mol. wt. cut-off 12–14 kDa; Spectrum Medical Industries). After 24 h incubation, two 50 µL samples were collected from each compartment and stored at −70°C (Revco ULT 1490 D-N-S; Western Mednics, Ashville, NC, USA) until used for the HPLC analysis of itraconazole and metformin (Hale et al., 2002; Shin et al., 2004; Choi and Lee, 2006).

In vivo studies

Single i.v. and p.o. administration of itraconazole, metformin and both drugs together to rats

The rats were anaesthetized with ether (in the early in the morning) before cannulation of the jugular vein (for drug administration in the i.v. study) and the carotid artery (for blood sampling), using methods similar to those described previously (Choi and Lee, 2006; Choi et al., 2006; 2008;). The rats were not restrained in the present study.

Itraconazole (diluted in 0.9% NaCl solution) at a dose of 20 mg·kg⁻¹ (n = 8), metformin base (metformin hydrochloride dissolved in 0.9% NaCl solution) at a dose of 100 mg·kg⁻¹ (n = 9) and both drugs together (n = 8) were manually infused i.v. (total infusion volume of 2 mL·kg⁻¹) for 1 min via the jugular vein. Blood samples (approximately 0.12 mL for each drug alone or 0.22 mL for both drugs together) were collected via the carotid artery at 0 (control), 1 (end of the infusion), 5, 15, 30, 60, 120, 180, 240, 360, 480, 600, 720, 1440, 1800, 2160 or 2880 min after the start of the i.v. infusion of the drug(s). Blood samples were immediately centrifuged and a 50 µL plasma sample (two 50 µL samples for both drugs) was collected in a 1.5 mL polyethylene tube, and stored at −70°C until used for the HPLC analysis of itraconazole, 7-hydroxyitraconazole and metformin (Hale et al., 2002; Shin et al., 2004; Choi and Lee, 2006). The procedures used for preparation and handling of a 48 h urine sample (Ae_0–48h) and the gastrointestinal tract (including its contents and faeces) sample at 48 h (GI_48h) were similar to methods reported previously (Choi and Lee, 2006; Choi et al., 2006; 2008;).

Itraconazole (the same solution used in the i.v. study) at a dose of 20 mg·kg⁻¹ (n = 6), metformin base (the same solution used in the i.v. study) at a dose of 100 mg·kg⁻¹ (n = 7) and both drugs together (n = 9) were administered p.o. (total oral volume of 6 mL·kg⁻¹) using a gastric gavage tube. Blood samples (approximately 0.12 mL for each drug alone or 0.22 mL for both drugs) were collected via the carotid artery at 0, 15, 30, 60, 90, 120, 180, 240, 360, 480, 600, 720, 960, 1200, 1400, 2160 or 2880 min after the p.o. administration of the drug(s). Other procedures were similar to those for the i.v. study.

Measurement of hepatic (after both i.v. and p.o. administration) and intestinal (after p.o. administration) concentrations of itraconazole and metformin after i.v. and p.o. administration of both drugs together

The procedures used were similar to those reported previously (Choi et al., 2006). The same doses of itraconazole and metformin were administered i.v. and p.o. At 5, 15, 30, 60, 120, 240, 480, 720 or 1880 min (for both i.v. or p.o. administration), as much blood as possible was collected via the carotid artery, and each rat was killed by cervical dislocation. Approximately 1 g of the liver (for both i.v. and p.o. studies) and intestine (for p.o. study) was excised and blotted on paper tissue (n = 3 at each time point for each route of administration).

HPLC analysis of itraconazole and metformin

Concentrations of itraconazole and 7-hydroxyitraconazole in the samples were determined by HPLC (Shin et al., 2004). Concentrations of metformin in the samples were determined by a slight modification of the method used by Choi et al. (2006) and Hale et al. (2002); ipriflavone instead of hydrocodeine was used as an internal standard.

Pharmacokinetic analysis

Standard methods (Gibaldi and Perrier, 1982) were used to calculate the following pharmacokinetic parameters using a non-compartmental analysis (WinNonlin; Pharsight Corporation, Mountain View, CA, USA): the total area under the plasma concentrations–time curve from time zero to infinity (AUC) (Chiou, 1978), the time-averaged total body, renal and non-renal clearances (CL, CL_R and CL_NR respectively), the terminal half-life, the first moment of AUC (AUMC), the mean residence time (MRT), the apparent volume of distribution at a steady state (V_ss) and the F. The peak plasma concentration (C_max) and time to reach C_max (T_max) were obtained directly from the experimental data.

Statistical analysis

A P-value <0.05 was deemed to be statistically significant; Student's t-test was used to determine differences between the two means for the unpaired data. All results are expressed as means ± SD except for T_max, which was expressed as a median (range).

Chemicals

Sporanox® i.v. solution [10 mg·mL⁻¹ as itraconazole as a solution in hydroxylpropyl-β-cyclodextrin (HP-β-CD)], itraconazole, 7-hydroxyitraconazole and R51012 (internal standard for the HPLC analysis of itraconazole and 7-hydroxyitraconazole) were supplied by Janssen Korea (Seoul, South Korea). Metformin hydrochloride was donated by Daelim Pharmaceutical Company (Seoul, South Korea). The reduced form of NADPH (as a tetrasodium salt), tris(hydroxymethyl)aminomethane (Tris)-buffer, ethylenediamine tetraacetic acid (EDTA; as a disodium salt) were purchased from Sigma–Aldrich Corporation (St. Louis, MO, USA). Human CYP enzymes (CYP3A cDNA-expressed P450 enzymes from rat), Baculovirus-infected insect cells expressing CYP3A1 and 3A2 (Supersomes) and human Baculovirus-infected insect cells expressing CYP3A4 were obtained from Gentest Corp. (Woburn, MA, USA). Other chemicals were of reagent or HPLC grade.

Results

In vitro studies

Competitive inhibition of metabolism of itraconazole and metformin by each other in rat hepatic microsomes

To investigate the kinetics of the inhibitory effects of metabolism of itraconazole and metformin by each other, the rates of disappearance of itraconazole and metformin in the hepatic microsomes were examined in the absence and presence of each other. The Lineweaver–Burk (Lineweaver and Burk, 1934) plots for the disappearance of itraconazole and metformin in the presence and absence of each other in hepatic microsomes are shown in Figure 1. A linear relationship between the inverse of substrate (itraconazole or metformin) concentrations and the inverse of velocities (primarily metabolism of itraconazole or metformin) of substrate was observed. Also on increasing the inhibitor (metformin or itraconazole) concentration, the slope between the inverse of the substrate concentration and the inverse of velocities of substrate increased and the y-intercept moved one point, indicating that inhibition occurred in a competitive manner.

Figure 1.

Lineweaver–Burk plots showing inhibition of the disappearance of itraconazole (A) and metformin (B) by each other in rat hepatic microsomes. The ‘s’ represents itraconazole (A) and metformin (B) concentration. The ‘v’ represents the velocity for the disappearance of itraconazole (A) and metformin (B). The symbols represent the concentrations of inhibitors, metformin (A) and itraconazole (B). Inset shows up to 0.10 of 1/s for the disappearance of itraconazole and metformin.

V_max, K_m and CL_int for the disappearance of itraconazole and metformin in the absence and presence of each other, and K_i of itraconazole and metformin for inhibition of metabolism by each other in rat hepatic and intestinal microsomes

The V_max, K_m and CL_int for the disappearance of itraconazole in the absence and presence of metformin in rat hepatic and intestinal microsomes are listed in Table 1. The V_max values for itraconazole with and without metformin were comparable in both hepatic and intestinal microsomes, suggesting that the V_max for the disappearance (primarily metabolism) of itraconazole was not affected by metformin. However, the K_m values of itraconazole in the presence of metformin in the hepatic and intestinal microsomes were significantly higher (by 87.9% and 238% respectively) than those in the absence of metformin, suggesting that the affinity of the enzyme(s) for itraconazole was decreased by metformin. As a result, the CL_ints for itraconazole in the presence of metformin were significantly slower both in the hepatic and intestinal microsomes (by 33.1% and 58.1% respectively), suggesting that metabolism of itraconazole was inhibited by metformin. The apparent K_i values of metformin for inhibition of metabolism of itraconazole in rat hepatic and intestinal microsomes were from 6.12 to 8.98 and from 3.42 to 15.4 µM respectively.

Table 1.

K_m, V_max, CL_int and K_i for the disappearance of itraconazole and metformin with and without each other in hepatic and intestinal microsomes

	Itraconazole			Metformin
Parameter	Without metformin	With metformin	Parameter	Without itraconazole	With itraconazole
Hepatic	(n = 4)	(n = 5)	Hepatic	(n = 5)	(n = 5)
Km (µM)	11.6 ± 3.12	21.8 ± 7.20*	Km (µM)	38.0 ± 6.32	65.4 ± 17.6*
Vmax (nmol·min−1·mg−1 protein)	0.132 ± 0.0368	0.165 ± 0.0461	Vmax (nmol·min−1·mg−1 protein)	0.986 ± 0.250	0.777 ± 0.419
CLint (µL·min−1·mg−1 protein)	11.5 ± 1.87	7.69 ± 1.17**	CLint (µL·min−1·mg−1 protein)	25.6 ± 3.41	13.2 ± 8.49*
Ki (µM) for the inhibition of metabolism of metformin	6.12 to 8.98		Ki (µM) for the inhibition of metabolism of itraconazole	5.76 to 13.4
Intestinal	(n = 5)	(n = 4)	Intestinal	(n = 4)	(n = 4)
Km (µM)	8.55 ± 2.87	28.9 ± 11.2**	Km (µM)	26.9 ± 7.59	38.7 ± 1.85*
Vmax (nmol·min−1·mg−1 protein)	0.0807 ± 0.0140	0.116 ± 0.0615	Vmax (nmol·min−1·mg−1 protein)	0.212 ± 0.0729	0.197 ± 0.0698
CLint (µL·min−1·mg−1 protein)	9.79 ± 1.39	4.10 ± 1.61***	CLint (µL·min−1·mg−1 protein)	7.72 ± 0.833	5.04 ± 1.57*
Ki (µM) for the inhibition of metabolism of metformin	3.42 to 15.4		Ki (µM) for the inhibition of metabolism of itraconazole	4.89 to 16.4

Data are expressed as means ± SD.

Significantly different from without metformin or without itraconazole

^*P < 0.05

^**P < 0.01

^***P < 0.001.

CL_int, intrinsic clearance; K_i, inhibition constant; K_m, apparent Michaelis–Menten constant; V_max, maximum velocity.

The values for V_max, K_m and CL_int for the disappearance of metformin in the absence and presence of itraconazole in rat hepatic and intestinal microsomes are listed in Table 1. The V_max values of metformin in both hepatic and intestinal microsomes were comparable in the absence and presence of itraconazole, suggesting that the V_max for the disappearance (primarily metabolism) of metformin was not altered by itraconazole. However, the K_m values of metformin with itraconazole in the hepatic and intestinal microsomes were significantly higher (by 72.1% and 43.9% respectively) than those obtained in the absence of metformin, suggesting that the affinity of the enzyme(s) for metformin was decreased by itraconazole. As a result, the CL_ints of metformin in the presence of itraconazole were significantly slower in both hepatic and intestinal microsomes (by 48.4% and 34.7% respectively). The apparent K_i values of itraconazole for the inhibition of metabolism of metformin in rat hepatic and intestinal microsomes were from 5.76 to 13.4 and 4.89 to 16.4 µM respectively.

The above data suggest that the inhibition of the metabolism of itraconazole and metformin by each other in both the liver and intestine occurs in a competitive manner.

In vitro disappearance of itraconazole and metformin in rat Baculovirus-infected insect cells expressing CYP3A1 and 3A2 and human Baculovirus-infected insect cells expressing CYP3A4

The amounts of itraconazole (Figure 2A) and metformin (Figure 2B) remaining after the addition of either itraconazole, metformin or both drugs together to rat microsomes from Baculovirus-infected insect cells expressing CYP3A1 and 3A2 and human Baculovirus-infected insect cells expressing CYP3A4 are shown in Figure 2, expressed as a percentage of the amount added to the cells. In the presence of metformin, the amounts of itraconazole (% of amount added) remaining via CYP3A1 and 3A2 were significantly greater (by 34.8% and 41.6% respectively) than those in the absence of metformin. Similarly, in the presence of itraconazole, the amounts of metformin remaining via CYP3A1 and 3A2 were also significantly greater (by 15.4% and 20.4% respectively) than those in the absence of itraconazole. In addition, in the presence of metformin, the amount of itraconazole remaining via CYP3A4 was significantly greater (by 21.6%) than that in the absence of metformin. Similarly, in the presence of itraconazole, the amount of metformin remaining via CYP3A4 was significantly greater (by 19.7%) than that in the absence of itraconazole. This suggests that metabolism of itraconazole and metformin was significantly inhibited by each other via CYP3A1 and 3A2 in rats and 3A4 in humans.

Figure 2.

Mean quantities (expressed as a percentage of the amounts of drug added to the cells) of itraconazole (A) and metformin (B) remaining after a 20 min incubation in rat Baculovirus-infected insect cells expressing CYP3A1 and 3A2 and human Baculovirus-infected insect cells expressing CYP3A4 in the absence (solid columns) and presence (shaded columns) of 20 µM metformin (A), or itraconazole (B). Columns show means and bars represent SD. *Significantly different (P < 0.05) from the quantity remaining in the absence of metformin (A) or itraconazole (B).

Rat plasma protein binding of itraconazole and metformin using equilibrium dialysis

Protein binding values of itraconazole to fresh rat plasma were 95.1 ± 11.3% and 94.8 ± 9.57% in the presence and absence of metformin, respectively; they were not significantly different. The values of metformin were 13.5 ± 6.01% and 14.9 ± 5.05% in the presence and absence of itraconazole, respectively; they were also not significantly different.

In a preliminary study, plasma protein binding values of itraconazole and metformin were constant; the values were 94.9% and 10.3% for itraconazole (from 1 to 50 µg·mL⁻¹) and metformin (from 0.1 to 200 µg·mL⁻¹) concentrations respectively. Thus, a concentration of 5 µg·mL⁻¹ for each drug was used in the plasma protein binding study.

In vivo studies

Pharmacokinetics of itraconazole and 7-hydroxyitraconazole after i.v. administration of itraconazole with and without metformin

After i.v. administration of itraconazole with and without metformin, the mean arterial plasma concentration–time profiles of itraconazole and 7-hydroxyitraconazole are shown in Figure 3A and B respectively. The relevant pharmacokinetic parameters are listed in Table 2. Changes in the pharmacokinetic parameters of itraconazole in the presence of metformin compared with in its absence were as follows: the AUC was significantly greater (by 20.9%); terminal half-life and MRT were significantly longer (by 38.1% and 27.7% respectively); CL and CL_NR were significantly slower (by 24.6% and 24.7% respectively); and GI_48h was significantly greater (by 38.1%). The CL_R, Ae_0–48h and GI_48h of itraconazole were almost negligible. Thus, the changes in the above-mentioned parameters did not have a significant effect on the other pharmacokinetic parameters of itraconazole.

Figure 3.

Mean arterial plasma concentration–time profiles of itraconazole (A) and 7-hydroxyitraconazole (B) after single i.v. administration of itraconazole 20 mg·kg⁻¹ in the absence and presence of metformin 100 mg·kg⁻¹, and of metformin (C) after single i.v. administration of metformin 100 mg·kg⁻¹ in the absence and presence of itraconazole 20 mg·kg⁻¹ to rats. Symbols show means and bars represent SD.

Table 2.

Pharmacokinetic parameters of itraconazole, 7-hydroxyitraconazole and metformin after single i.v. administration of itraconazole 20 mg·kg⁻¹, metformin 100 mg·kg⁻¹ or both drugs together to rats

	Itraconazole			Metformin
Parameter	Without metformin (n = 8)	With metformin (n = 8)	Parameter	Without itraconazole (n = 9)	With itraconazole (n = 8)
Itraconazole			Metformin
AUC (µg·min−1·mL−1)	2390 ± 461	2890 ± 354*	AUC (µg·min−1·mL−1)	6840 ± 553	9510 ± 1370**
Terminal half-life (min)	753 ± 160	1040 ± 178*	Terminal half-life (min)	144 ± 37.5	268 ± 92.1***
MRT (min)	716 ± 148	914 ± 166*	MRT (min)	26.3 ± 5.48	38.2 ± 12.6*
Vss (mL·kg−1)	4390 ± 1340	4060 ± 609	Vss (mL·kg−1)	383 ± 69.7	403 ± 123
CL (mL·min−1·kg−1)	6.55 ± 1.35	4.94 ± 0.579**	CL (mL·min−1·kg−1)	14.7 ± 1.18	10.7 ± 1.58***
CLR (mL·min−1·kg−1)	0.0730 ± 0.0605	0.0618 ± 0.0623	CLR (mL·min−1·kg−1)	11.6 ± 1.64	9.10 ± 1.23**
CLNR (mL·min−1·kg−1)	6.48 ± 1.36	4.88 ± 0.598**	CLNR (mL·min−1·kg−1)	3.11 ± 1.47	1.61 ± 0.823*
Ae0–48h (% of itraconazole dose)	1.54 ± 1.07	1.30 ± 1.46	Ae0–48h (% of metformin dose)	78.9 ± 9.65	85.3 ± 6.93
GI48h (% of itraconazole dose)	0.226 ± 0.0870	0.312 ± 0.374*	GI48h (% of metformin dose)	1.06 ± 0.750	1.61 ± 0.823
7-Hydroxyitraconazole
AUC (µg·min−1·mL−1)	1930 ± 463	1490 ± 236*
Terminal half-life (min)	1270 ± 427	757 ± 127
Cmax (µg·mL−1)	0.907 ± 0.227	0.936 ± 0.206
Tmax (min)	360 (120 to 720)	480 (180 to 720)
Ae0–48h (% of itraconazole dose)	0.220 ± 0.206	0.332 ± 0.377
GI48h (% of itraconazole dose)	0.170 ± 0.0874	0.216 ± 0.362
AUC7-hydroxyitraconazole/AUCitraconazole	81.6 ± 16.8	52.2 ± 9.84**

Data are expressed as means ± SD.

Significantly different from without metformin or without itraconazole

^*P < 0.05

^**P < 0.01

^***P < 0.001.

Ae_0–48h, percentage of the dose excreted in the 48 h urine; AUC, total area under the plasma concentration–time curve from time zero to infinity; CL, time-averaged total body clearance; CL_NR, time-averaged nonrenal clearance; CL_R, time-averaged renal clearance; C_max, peak plasma concentration; GI_48h, percentage of the dose recovered from the entire gastrointestinal tract (including its contents and feces) at 48 h; MRT, mean residence time; T_max, time to reach C_max, V_ss, apparent volume of distribution at steady state.

After i.v. administration of itraconazole with or without metformin, formation of 7-hydroxyitraconazole was rapid; 7-hydroxyitraconazole was detected in plasma at the second blood sampling time point (15 min) in both the absence and presence of metformin (Figure 3B). After i.v. administration of both drugs together, the AUC of 7-hydroxyitraconazole and AUC_{7-hydroxyitraconazole}/AUC_itraconazole ratio were significantly smaller (by 22.8% and 36.0% respectively) than those of itraconazole alone.

Pharmacokinetics of metformin after its i.v. administration with and without itraconazole

The mean arterial plasma concentration–time profiles of metformin after its i.v. administration with and without itraconazole are shown in Figure 3C. The relevant pharmacokinetic parameters are also listed in Table 2. Changes in the pharmacokinetic parameters of metformin in the presence of itraconazole compared with in its absence were as follows: the AUC was significantly greater (by 39.0%); terminal half-life and MRT were significantly longer (by 86.1% and 45.2% respectively); and CL, CL_R and CL_NR were significantly slower (by 27.2%, 21.6% and 48.2% respectively).

Pharmacokinetics of itraconazole and 7-hydroxyitraconazole after p.o. administration of itraconazole with and without metformin

After p.o. administration of itraconazole with and without metformin, the mean arterial plasma concentration–time profiles of itraconazole and 7-hydroxyitraconazole are shown in Figure 4A and B respectively. The relevant pharmacokinetic parameters are listed in Table 3. After p.o. administration of itraconazole with or without metformin, absorption of itraconazole from the gastrointestinal tract and formation of 7-hydroxyitraconazole were rapid; itraconazole and 7-hydroxyitraconazole were detected in plasma at the second blood sampling time point (15 min) in both the absence and presence of metformin. Changes in the pharmacokinetic parameters of itraconazole and 7-hydroxyitraconazole with metformin compared with in its absence were as follows: the AUC and T_max of itraconazole were significantly greater (69.3% increase) and shorter (by 25.0%), respectively; the AUC, C_max and T_max of 7-hydroxyitraconazole were significantly smaller (by 13.9%), lower (by 35.1%) and longer (by 100%), respectively; and the AUC_{7-hydroxyitraconazole}/AUC_itraconazole ratio was significantly smaller (by 48.2%).

Figure 4.

Mean arterial plasma concentration–time profiles of itraconazole (A) and 7-hydroxyitraconazole (B) after single p.o. administration of itraconazole 20 mg·kg⁻¹ in the absence and presence of metformin 100 mg·kg⁻¹, and of metformin (C) after single p.o. administration of metformin 100 mg·kg⁻¹ in the absence and presence of itraconazole 20 mg·kg⁻¹ to rats. Symbols show means and bars represent SD.

Table 3.

Pharmacokinetic parameters of itraconazole, 7-hydroxyitraconazole and metformin after single p.o. administration of itraconazole 20 mg·kg⁻¹, metformin 100 mg·kg⁻¹ or both drugs together to rats

	Itraconazole			Metformin
Parameter	Without metformin (n = 6)	With metformin (n = 9)	Parameter	Without itraconazole (n = 7)	With itraconazole (n = 9)
Itraconazole			Metformin
AUC (µg·min−1·mL−1)	1370 ± 377	2320 ± 525*	AUC (µg·min−1·mL−1)	2930 ± 660	5340 ± 1260**
Terminal half-life (min)	969 ± 268	1140 ± 533	Terminal half-life (min)	253 ± 38.8	490 ± 177*
CLR (mL·min−1·kg−1)	0.0307 ± 0.0308	0.00643 ± 0.00795	CLR (mL·min−1·kg−1)	30.9 ± 8.27	16.9 ± 3.44**
Cmax (µg·mL−1)	1.20 ± 0.318	1.29 ± 0.403	Cmax (µg·mL−1)	13.9 ± 2.11	8.15 ± 1.74**
Tmax (min)	480 (120 to 720)	360 (120 to 720)***	Tmax (min)	60 (15 to 120)	120 (15 to 360)**
Ae0–48h (% of itraconazole dose)	0.182 ± 0.199	0.0670 ± 0.0742	Ae0–48h (% of metformin dose)	86.2 ± 11.2	87.2 ± 12.1
GI48h (% of itraconazole dose)	0.309 ± 0.153	0.342 ± 0.195	GI48h (% of metformin dose)	6.05 ± 3.70	4.76 ± 2.21
F (%)	57.3	68.4	F (%)	42.8	56.2
7-Hydroxyitraconazole
AUC (µg·min−1·mL−1)	2520 ± 316	2170 ± 274***
Terminal half-life (min)	1180 ± 426	1190 ± 381
CLR (mL·min−1·kg−1)	0.00564 ± 0.00453	0.00379 ± 0.00174
Cmax (µg·mL−1)	1.30 ± 0.453	0.844 ± 0.109***
Tmax (min)	720 (720 to 1800)	1440 (30 to 360)***
Ae0–48h (% of itraconazole dose)	0.187 ± 0.161	0.0919 ± 0.0394
GI48h (% of itraconazole dose)	0.315 ± 0.108	0.346 ± 0.152
AUC7-hydroxyitraconazole/AUCitraconazole	190 ± 34.4	98.5 ± 27.7**

Data are expressed as means ± SD.

Significantly different from without metformin or without itraconazole

^*P < 0.01

^**P < 0.001

^***P < 0.05.

Ae_0–48h, percentage of the dose excreted in the 48 h urine; AUC, total area under the plasma concentration–time curve from time zero to infinity; CL_R, time-averaged renal clearance; C_max, peak plasma concentration; F, extent of absolute oral bioavailability; GI_48h, percentage of the dose recovered from the entire gastrointestinal tract (including its contents and feces) at 48 h; T_max, time to reach C_max.

Pharmacokinetics of metformin after its p.o. administration with and without itraconazole

The mean arterial plasma concentration–time profiles of metformin after its p.o. administration with and without itraconazole are shown in Figure 4C. The relevant pharmacokinetic parameters are also listed in Table 3. Changes in the pharmacokinetic parameters of metformin in the presence of itraconazole compared with in its absence were as follows; the AUC was significantly greater (by 82.3%); terminal half-life was significantly longer (by 93.7%); CL_R was significantly slower (by 45.3%); C_max was significantly lower (by 41.4%); and T_max were significantly longer (by 100%).

Liver and intestinal concentrations of itraconazole and metformin after i.v. and p.o. administration of both drugs together

After i.v. and p.o. administration of both drugs together, the concentrations and tissue-to-plasma ratios of each drug in the liver and intestine are listed in Table 4. After i.v. administration of both drugs together, the liver concentration/K_i ratios of metformin up to 240 min and the liver concentration/K_i ratios of itraconazole up to 1800 min were greater than (or in the range of) 2.0 (Table 4). After i.v. administration of both drugs together, the AUC_0–240min/AUC ratio of itraconazole and AUC_0–1800min/AUC ratio of metformin were high; the values were 34.9% and 100% respectively. After p.o. administration of both drugs together, the liver concentration/K_i ratios of metformin up to 240 min and intestinal concentration/K_i ratios of metformin up to 720 min, liver concentration/K_i ratios of itraconazole up to 1800 min, and intestinal concentration/K_i ratios of itraconazole up to 1800 min were greater than (or in the range of) 2.0 (Table 4). After p.o. administration of both drugs together, the AUC_0–240min/AUC ratio of itraconazole, AUC_0–720min/AUC ratio of itraconazole and AUC_0–1800min/AUC ratio of metformin were high; 16.8%, 45.5% and 100% respectively. The above data suggest that the competitive inhibition of metabolism of each drug by each other in the liver and intestine was mainly due to inhibition of CYP (Bechmann and Lewis, 2005).

Table 4.

Concentrations (I; µM) and [I]/K_i of itraconazole and metformin in the liver (i.v. and p.o. studies) and intestine (p.o. study) after simultaneous single i.v. and p.o. administration of itraconazole (20 mg·kg⁻¹) and metformin (100 mg·kg⁻¹) to rats

Metformin
	i.v.			p.o.
Time (min)	Liver concentration	[I]/Ki	Time (min)	Liver concentration	[I]/Ki	Intestine concentration	[I]/Ki
Metformin
5	58.7 ± 22.1 (0.159 ± 0.0496)	6.54–9.59	30	27.1 ± 9.95 (1.21 ± 0.425)	3.02–4.43	25.7 ± 9.83 (1.15 ± 0.435)	1.67–7.51
15	123 ± 19.0 (1.05 ± 0.177)	13.7–20.1	60	49.5 ± 25.7 (2.09 ± 1.22)	5.51–8.09	34.0 ± 10.7 (1.35 ± 0.0336)	2.21–9.94
30	65.6 ± 43.6 (1.09 ± 0.528)	7.31–10.7	120	23.5 ± 2.04 (1.20 ± 0.314)	2.62–3.84	34.0 ± 2.52 (1.78 ± 0.649)	2.21–9.94
60	42.2 ± 1.23 (1.12 ± 0.135)	4.70–6.89	240	14.9 ± 2.41 (0.996 ± 0.152)	1.66–2.43	39.7 ± 3.34 (2.66 ± 0.364)	2.58–11.6
120	33.7 ± 11.4 (5.64 ± 2.03)	3.75–5.51	480	11.1 ± 3.22 (1.31 ± 0.550)	1.24–1.81	26.7 ± 2.70 (3.16 ± 1.17)	1.73–7.81
240	14.4 ± 6.58 (7.14 ± 6.57)	1.60–2.35	720	5.99 ± 1.52 (1.08 ± 0.344)	0.667–0.979	15.5 ± 4.72 (2.82 ± 1.08)	1.01–4.53
480	3.21 ± 1.00 (2.15 ± 1.22)	0.357–0.525
Itraconazole
5	27.4 ± 2.66 (2.22 ± 0.730)*	2.04–4.76	30	21.3 ± 7.59 (21.2 ± 7.96)	1.59–3.70	27.2 ± 11.4 (27.2 ± 12.0)	1.66–5.56
15	37.7 ± 4.68 (4.09 ± 1.02)	2.81–6.55	60	38.0 ± 4.48 (9.11 ± 0.815)	2.84–6.60	52.1 ± 21.8 (12.5 ± 5.17)	3.18–10.7
30	37.5 ± 10.0 (6.40 ± 1.27)	2.80–6.51	120	37.7 ± 3.60 (14.8 ± 1.42)	2.81–6.55	36.2 ± 4.40 (14.3 ± 2.64)	2.21–7.40
60	43.0 ± 13.2 (10.7 ± 0.802)	3.21–7.47	240	39.6 ± 12.4 (23.4 ± 9.10)	2.96–6.88	38.4 ± 9.73 (21.9 ± 3.73)	2.34–7.85
120	39.9 ± 1.26 (22.4 ± 6.46)	2.98–6.93	480	34.1 ± 3.87 (31.4 ± 3.78)	2.54–5.92	33.0 ± 2.70 (30.4 ± 2.61)	2.01–6.77
240	24.6 ± 9.79 (19.0 ± 10.9)	1.84–4.27	720	22.3 ± 4.70 (25.5 ± 10.9)	1.66–3.71	24.6 ± 10.2 (26.8 ± 13.7)	1.50–5.03
480	22.9 ± 7.72 (40.8 ± 8.65)	1.71–3.98	1800	15.0 ± 2.48 (41.9 ± 9.98)	1.13–2.62	17.0 ± 8.93 (48.0 ± 27.2)	1.04–3.48
720	17.6 ± 2.53 (55.1 ± 5.68)	1.31–3.06
1800	12.6 ± 2.42 (50.7 ± 24.5)	0.940–2.188

Data are expressed as means ± SD.

^*The values in parentheses represent the tissue-to-plasma (T/P) ratios.

Discussion

The doses of itraconazole, 20 mg·kg⁻¹, and of metformin, 100 mg·kg⁻¹, used in this study are within the ranges of dose-proportional AUCs of each drug based on results from previous studies done by Shin et al. (2004) and Choi et al. (2006); they determined dose-proportional AUCs for itraconazole and metformin, respectively, after both i.v. and p.o. administration to rats.

To find which hepatic CYP isozymes inhibit metabolism of itraconazole and metformin by each other, itraconazole and metformin were incubated with microsomes from rat Baculovirus-infected insect cells expressing CYP3A1 and 3A2 and with human Baculovirus-infected insect cells expressing CYP3A4 in vitro. The results suggest that metabolism of itraconazole and metformin are mainly inhibited by each other via CYP3A1/2 in rats and 3A4 in humans (Figure 2). A similar homology between rat and human CYP isozymes was reported by Lewis (1996).

It has been reported that changes in the CL_NRs of itraconazole (Shin et al., 2004) and metformin (Choi et al., 2006) were due to changes in metabolism of each drug in rats. Thus, the changes in the CL_NRs of itraconazole and metformin after administration of both drugs together, listed in Table 2, could represent changes in the metabolism of each drug. Following i.v. administration of both drugs together, the CL_NRs of each drug were significantly slower than that of each drug alone (Table 2). Both itraconazole (Shin et al., 2004) and metformin (Choi et al., 2006) are drugs with low hepatic extraction ratios in rats. Thus, their hepatic clearances depend more on the hepatic CL_ints for the disappearance of each drug than on the hepatic blood flow rate (Wilkinson and Shand, 1975). The significantly slower CL_NRs of itraconazole and metformin administered together than that of either drug alone (Table 2) is consistent with the significantly slower in vitro hepatic CL_ints for the disappearance of itraconazole and metformin induced by each other respectively (Table 1). The slower CL_ints of each drug (Table 1) could have been due to a competitive inhibition for the metabolism of either drug by each other via hepatic CYP3A1/2 (Figure 1). Rat plasma protein binding values of itraconazole and metformin were not altered by each other.

After i.v. administration of both drugs together, the significantly slower CL_NRs of each drug alone (Table 2) are also consistent with the values obtained for apparent K_i of each drug and [I]/K_i ratios in the liver (Tables 1 and 4). The [I]/K_i ratios of metformin of greater than (or in the ranges of) 2 for inhibition of metabolism of itraconazole up to 240 min and of itraconazole for inhibition of metabolism of metformin up to 1800 min (Table 4) further indicate that the metformin–itraconazole interaction is primarily due to the competitive inhibition of hepatic CYP3A1/2 in the liver. The significantly smaller AUCs of 7-hydroxyitraconazole and AUC_{7-hydroxyitraconazole}/AUC ratio (Table 2) could have been due to a result of inhibition of metabolism of itraconazole to form 7-hydroxyitraconazole by metformin.

After p.o. administration of both drugs together, the AUCs of metformin and itraconazole were also significantly greater than those of each drug alone (Table 3). However, this was unlikely to be due to the increased gastrointestinal absorption of metformin and itraconazole by each other. The mean ‘true’ fractions of the p.o. dose of metformin and itraconazole unabsorbed (‘F_unabs’) in the absence and presence of each other were estimated based on the equation reported by Lee and Chiou (1983). The ‘F_unabs’ values of metformin thus estimated were 5.64% and 3.66% in the presence and absence of itraconazole respectively. The corresponding values for itraconazole were 0.212% and 0.167% for with and without metformin. Thus, more than 94% of the p.o. dose of metformin and itraconazole were absorbed for both with and without each other.

The significantly greater AUC of itraconazole after p.o. administration of both drugs together than that obtained after itraconazole alone (Table 3) is consistent with the values obtained for CL_int, apparent K_i, concentrations of each drug, and ratios of [I]/K_i in the liver and intestine. Metformin inhibits the hepatic metabolism of itraconazole after p.o. administration of both drugs together as mentioned earlier. However, the contribution of the inhibition of hepatic metabolism of itraconazole by metformin to the AUC of itraconazole obtained after its administration p.o. was considerably smaller than that of intestinal metabolism; the intestinal first-pass effect of itraconazole was approximately 70% of the p.o. dose, but the hepatic first-pass effect is almost negligible in rats (Shin et al., 2004). Thus, the significantly greater AUC of itraconazole after p.o. administration of both drugs together (Table 3) is probably mainly due to the competitive inhibition of intestinal metabolism of itraconazole by metformin via intestinal CYP3A1/2. Therefore, the greater magnitude of the increase in AUC of itraconazole after the administration of both drugs together p.o. compared with their i.v. administration, 69.3% compared with 20.9% increase (Tables 3 and 2 respectively), is likely to be mainly due to the inhibition of intestinal metabolism of itraconazole by metformin.

The significantly greater AUC of metformin after p.o. administration of both drugs together could also have been due to a competitive inhibition of hepatic and intestinal metabolism of metformin by itraconazole (Table 3). This is supported by the values obtained for CL_int, apparent K_i, concentrations of each drug and ratios of [I]/K_i in the liver and intestine. Itraconazole inhibited the hepatic metabolism of metformin after p.o. administration of both drugs together as mentioned earlier. However, the contribution of inhibition of hepatic metabolism of metformin by itraconazole to the AUC of metformin given p.o. was considerably smaller than that of intestinal metabolism, as the gastrointestinal first-pass effect of metformin was 53.8% of the p.o. dose, but the hepatic first-pass effect was only 11.3% of oral dose in rats (Choi et al., 2006). Thus, the significantly greater AUC of metformin, after p.o. administration of both drugs together (Table 3), is likely to have been due mainly to the competitive inhibition of intestinal metabolism of metformin by itraconazole via intestinal CYP3A1/2. Therefore, the greater magnitude of the increase in AUC of metformin after p.o. administration of both drugs together, 82.3% increase (Table 3), than after i.v. administration (Table 2), 39.0% increase, is probably mainly been due to the inhibition of intestinal metabolism of metformin by itraconazole.

The significantly slower CL_R of metformin by itraconazole after simultaneous single i.v. (Table 2) or p.o. (Table 3) administration of both drugs could also contribute to the significantly greater AUCs of metformin after both routes of administration (Tables 2 and 3). Metformin is mainly excreted in the urine via organic cation transporter 2 (Kimura et al., 2005), but is not a substrate for P-glycoprotein (P-gp) (Song et al., 2006). Itraconazole is a substrate and inhibitor for P-gp (Wang et al., 2002). Similar results have also been reported for the CL_R of metformin; it was significantly slower after co-administration of udenafil (a substrate for P-gp) in rats (Choi et al., 2008). However, the exact reason for the slower CL_R of metformin in the presence of itraconazole is not clear.

In summary, after i.v. administration of itraconazole and metformin together to rats, the significantly slower CL_NRs of each drug than that of each drug alone (Table 2) could be due to a competitive inhibition of metabolism of each drug by each other via hepatic CYP3A1/2. Whereas, after p.o. administration of both drugs, the significantly greater AUCs of each drug than that of each drug alone (Table 3) could be mainly due to a competitive inhibition of intestinal metabolism of each drug by each other via intestinal CYP3A1/2 in addition to a competitive inhibition of hepatic metabolism of each drug by each other via hepatic CYP1A1/2. These results could be useful to predict and design clinical studies to investigate the interaction between itraconazole and metformin. Also if the present data obtained in rats is extrapolated to humans, the dosage regimen of itraconazole and metformin used in the clinical situation should be modified to take account of the decreased metabolism of the drugs, especially with chronic administration schedules. Although similar results have been obtained in studies involving rats and humans in vivo, further experiments in humans are required to confirm the above hypothesis.

Glossary

Abbreviations

Ae_0–24h

percentage of the dose excreted in the 24 h urine

AUC

total area under the plasma concentration–time curve from time zero to infinity

CL

time-averaged total body clearance

CL_int

intrinsic clearance

CL_NR

time-averaged non-renal clearance

CL_R

time-averaged renal clearance

C_max

peak plasma concentration

F

extent of absolute oral bioavailability

GI_24h

percentage of the dose recovered from the entire gastrointestinal tract (including its contents and faeces) at 24 h

K_i

inhibition constant

K_m

Michaelis–Menten constant

MRT

mean residence time; s, substrate

T_max

time to reach C_max

v

velocity

V_max

maximum velocity

V_ss

apparent volume of distribution at steady state

Conflict of interest

The authors state no conflict of interest.