Effects of Renal Impairment on the Pharmacokinetics of Morinidazole: Uptake Transporter-Mediated Renal Clearance of the Conjugated Metabolites

Abstract

Morinidazole is a novel 5-nitroimidazole antimicrobial drug that undergoes extensive metabolism in humans via N⁺-glucuronidation (N⁺-glucuronide of S-morinidazole [M8-1] and N⁺-glucuronide of R-morinidazole [M8-2]) and sulfation (sulfate conjugate of morinidazole [M7]). Our objectives were to assess the effects of renal impairment on the pharmacokinetics (PK) of morinidazole and to elucidate the potential mechanisms. In this parallel-group study, healthy subjects and patients with severe renal impairment received an intravenous infusion of 500 mg of morinidazole. Plasma and urine samples were collected and analyzed. The areas under the plasma concentration-time curves (AUC) for M7, M8-1, and M8-2 were 15.1, 20.4, and 17.4 times higher, respectively, in patients with severe renal impairment than in healthy subjects, while the AUC for morinidazole was 1.5 times higher. The urinary recovery of the major metabolites was not significantly different between the two groups over 0 to 48 h, but the renal clearances of M7, M8-1, and M8-2 in patients were 85.3%, 92.5%, and 92.2% lower, respectively. In vitro transporter studies revealed that M7 is a substrate for organic anion transporter 1 (OAT1) and OAT3 (K_m = 28.6 and 54.0 μM, respectively). Only OAT3 transported M8-1 and M8-2. Morinidazole was not a substrate for the transporter-transfected cells examined. These results revealed that the function or activity of renal uptake transporters might be impaired in patients with severe renal impairment, which accounted for dramatically increased plasma exposure and reduced renal clearance of the conjugated metabolites of morinidazole, the substrates of renal transporters in patients. It will help clinicians to adjust the dose in patients with severe renal impairment and to predict possible transporter-based drug-drug interactions.

INTRODUCTION

Morinidazole [R,S-1-(2-methyl-5-nitro-1H-imidazol-1-yl)-3-morpholinopropan-2-ol] is a novel 5-nitroimidazole antimicrobial agent indicated for the treatment of infections caused by amoeba, Trichomonas vaginalis, and anaerobic bacteria. 5-Nitroimidazole antimicrobial drugs act via nitroreduction of the molecules in the pathogens (1). As an analog of metronidazole, morinidazole appears to show greater antiparasitic potency against T. vaginalis and amoebic protozoa, but it exhibited relatively weaker toxicity in vitro and in preclinical animal studies compared with metronidazole (2).

Following intravenous administration, morinidazole was reported to undergo extensive metabolism in healthy humans, principally via N⁺-glucuronidation (yielding the N⁺-glucuronide of S-morinidazole [M8-1] and N⁺-glucuronide of R-morinidazole [M8-2]) and O-sulfation (yielding the sulfate conjugate of morinidazole [M7]) (Fig. 1) (3). Renal elimination of morinidazole and its main metabolites accounted for about 71% of a 500-mg dose over 0 to 36 h (3). The primary components in urine were M8-2 (28.4% of the dose), followed by morinidazole (21.2%), M8-1 (6.6%), and M7 (13.0%) (unpublished data). Because of the significant role of the kidney in elimination of morinidazole and its metabolites, it is necessary to evaluate the potential effects of renal impairment on the pharmacokinetics (PK) and elimination of morinidazole.

FIG 1.

Major metabolic pathways of morinidazole in humans.

In our preliminary clinical pharmacokinetic study, we observed an increase in the area under the concentration-time curve (AUC) and delayed elimination of morinidazole and its major metabolites in patients with severe renal impairment compared with healthy subjects. Significant differences in the pharmacokinetics of other drugs have been found between patients with normal renal function and healthy subjects. For example, the exposure of dabigatran was 6.3 times higher in patients with severe renal impairment (4). Meanwhile, after a single oral dose of 10 mg saxagliptin, systemic exposure in terms of the AUC for saxagliptin and the 5-hydroxy metabolite were 2.1 and 4.5 times higher, respectively, in patients with severe renal impairment than in healthy subjects (5).

Some explanations have been proposed to explain the reasons for the significant differences in pharmacokinetic characteristics of drugs in healthy individuals and patients with renal impairment. It is generally acknowledged that chronic renal failure could reduce the renal clearance of drugs through a decrease in the glomerular filtration rate (GFR) (6) and may ultimately affect plasma exposure. The GFR is often considered to be the best clinical marker of renal function (7). Impaired renal function can also adversely affect the hepatic and renal metabolism of various drugs by downregulating members of the cytochrome P450 (CYP450) family and suppressing phase II metabolic enzymes (8–12). Furthermore, several studies have demonstrated that changes in renal transporters may affect renal clearance (6, 8). Additionally, abnormal plasma protein binding was reported to affect drug disposition in patients with renal impairment (13).

The objectives of this study were to evaluate the effects of severe renal impairment on the pharmacokinetics and elimination of morinidazole and to elucidate the potential mechanisms.

MATERIALS AND METHODS

Chemicals.

A reference standard of racemic morinidazole (99.9% purity) was kindly provided by Jiangsu Hansoh Pharmaceutical Co. Ltd. (Lianyungang, China). The sulfate conjugate of morinidazole (M7), the N⁺-glucuronide of S-morinidazole (M8-1), and the N⁺-glucuronide of R-morinidazole (M8-2) were isolated and purified from human urine as previously described (3). Metronidazole (internal standard), cimetidine, probenecid, para-aminohippurate (PAH), estrone-3-sulfate (E3S), 1-methyl-4-phenylpyridinium (MPP⁺), Hanks' balanced salt solution (HBSS), bicinchoninic acid (BCA) protein assay kits, and hygromycin B were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum, Dulbecco's modified Eagle's medium, 0.05% trypsin-EDTA, penicillin G, and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Deionized water was generated using a Millipore Milli-Q gradient water purification system (Molsheim, France).

Study design, dosing, and sample collection.

This single-center, parallel-group, open-label study was conducted in patients with severe renal impairment and a matched group of healthy subjects with normal renal function. The study protocol and informed consent documents were approved by the Ethics Committee of the Third Xiangya Hospital (Changsha, China). The study was conducted in accordance with the principles of the Declaration of Helsinki and good clinical practice. Written informed consent was obtained from all of the subjects before enrollment.

Severe renal impairment was defined as a creatinine clearance rate (CL_CR) of <30 ml · min⁻¹ estimated by the Cockcroft-Gault formula. Healthy subjects were required to have a CL_CR of >90 ml · min⁻¹ and were matched to patients with severe renal impairment for sex, age (within 5 years), and body mass index (within 15%). A sample size of 10 subjects per group was chosen for studies of renal impairment. Male and female participants aged 18 to 65 years were eligible for the study if their body weight was ≥50 kg for males or ≥45 kg for females. Subjects with renal impairment were enrolled if they met the CL_CR inclusion criterion and did not require hemodialysis. Patients with renal impairment were excluded if they had recent hepatitis B infection, were HIV antibody positive, had hepatic impairment, or preexisting conditions (other than renal impairment) that might interfere with the disposition, metabolism, or excretion of the study drug. Healthy subjects were in good physical health as evaluated by medical examinations and vital signs, no history of drug or alcohol abuse, were nonsmokers, and were not using other medications at the time of the study. All subjects were required to abstain from wine, orange, or grapefruit products from 3 days before administration of the study drug until the collection of the final pharmacokinetic sample. However, the principal investigator allowed patients with renal impairment to use medically necessary concomitant drugs provided they were deemed unlikely to affect morinidazole exposure.

Subjects were given an intravenous infusion of 500 mg of racemic morinidazole over 0.75 h. Blood samples (5 ml) were collected for pharmacokinetic analyses in heparinized tubes at 0 (predose), 0.375, 0.75, 1.25, 1.5, 1.75, 2.75, 4.75, 6.75, 8.75, 12.75, 24.75, 36.75, and 48.75 h after starting the infusion. Samples were centrifuged at 3,000 × g for 10 min to separate the plasma fractions and were stored at −20°C until analysis. Urine samples were collected 2 h before infusion and for 0 to 4, 4 to 8, 8 to 12, 12 to 24, and 24 to 48 h after starting infusion. Urine samples were stored at −20°C until analysis.

Binding of morinidazole and its major metabolites to human plasma protein.

The binding of morinidazole, M7, M8-1, and M8-2 to human plasma proteins was measured by ultrafiltration with Amicon Ultra 0.5-ml centrifugal filter units (molecular size > 10 kDa; Millipore Canada, Ltd., Etobicoke, ON, Canada). Morinidazole, M7, M8-1, and M8-2 were spiked with blank human plasma to obtain three concentration levels (200/10.0/10.0/50.0, 2,000/50.0/50.0/250, and 10,000/250/250/1,600 ng · ml⁻¹, respectively) in triplicate at each level. Plasma samples with known concentrations were first equilibrated for 2 h at 37°C. Then, 300 μl of each sample was transferred to the ultrafiltration devices and equilibrated for another 30 min at 37°C. After equilibration, the samples were centrifuged at 14,000 × g for 25 min, and the filtrates were collected. The relative amount of unbound drug or metabolite (percent unbound) was calculated using the following equation: % unbound = (C_filtered/C_total) × 100 where C_filtered is the concentration of the drug or metabolite in the filtrate and C_total is the total concentration spiked in plasma.

Quantification of morinidazole and its major metabolites in human plasma and urine.

Morinidazole, M7, M8-1, and M8-2 were simultaneously determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as previously described (14).

Pharmacokinetic analysis.

A noncompartmental model was used to calculate the pharmacokinetic parameters for morinidazole and its major metabolites using WinNonlin software version 5.3 (Pharsight, Mountain View, CA, USA). The maximum plasma drug concentration (C_max) and the time to the C_max (t_max) were taken directly from the data obtained. The areas under the concentration-time curves from 0 h to the last sampling time (AUC_0–t) were calculated using the trapezoidal rule. The terminal elimination rate constant (k_el) was estimated by least-squares regression of the terminal log linear phase of the plasma drug concentration-time profiles, and the apparent elimination half-life (t_1/2) was calculated as 0.693/k_el. The amounts of morinidazole and its major metabolites recovered from urine during each collection interval were calculated by multiplying the concentration of each analyte by the volume of excreted urine collected during the collection interval. All results are presented as means ± standard deviations. Differences in pharmacokinetic parameters between patients with severe renal impairment and healthy subjects were determined using Student's t test. The level of significance was set at P < 0.05. The renal clearance rate (CL_R) was estimated as the amount of each drug/metabolite excreted in urine from 0 h to the last time point (t_last) divided by AUC_0–t.

Cell culture.

Human embryonic kidney (HEK293) cells transfected with human organic anion transporter 1 (OAT1), OAT3, or organic cation transporter 2 (OCT2) and empty-vector-transfected control cells were established at HD Biosciences Co., Ltd. (Shanghai, China). The functions of these transporters in these cells have been identified and validated using typical substrates and inhibitors. The cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetal bovine serum, 100 U · ml⁻¹ of penicillin, 100 mg · ml⁻¹ of streptomycin, and 100 μg · ml⁻¹ of hygromycin B at 37°C in an atmosphere with 95% relative humidity and 5% (vol/vol) CO₂. The cells were seeded into the wells of 24-well BD Biocoat poly-d-lysine-coated plates (BD Biosciences, Bedford, MA, USA) at an initial density of 2.0 × 10⁵ cells/well with 0.5 ml per well. The cell culture medium was replaced with fresh culture medium containing 5 mM sodium butyrate 24 h before the transport studies to induce the expression of the transporters. Transport experiments were performed on day 3, when the cells had grown to confluence.

In vitro uptake experiments.

Before the in vitro uptake experiments were conducted, the cells were washed three times with 1 ml of prewarmed HBSS (pH 7.4). After preincubation with 300 μl of HBSS for 15 min at 37°C, the equilibration buffer was removed, and uptake was initiated by adding 300 μl of HBSS containing the test compound to plated cells in the absence or presence of the indicated inhibitor. The uptake experiments were terminated at the designated times by aspirating the incubation solution and washing the cells three times with ice-cold HBSS. The cells were lysed by the addition of 300 μl of deionized water by the multigelation method. To exclude the possible effects of passive uptake and nonspecific binding, the uptake studies were performed in parallel with empty-vector-transfected control HEK293 cells.

After cell lysis, 50 μl of the solubilized cell samples, 25 μl of the internal standard (50.0 ng · ml⁻¹ metronidazole), and 150 μl of acetonitrile were added to centrifuge tubes. After vortex mixing and centrifugation at 14,000 × g for 5 min, the supernatants were evaporated to dryness under a gentle stream of nitrogen at 40°C. The residues were then reconstituted in 100 μl of methanol-water (1:1, vol/vol) and 5.0 μl of the resulting solution was used for LC-MS/MS. The protein content of the solubilized cells was measured using the BCA protein assay kit.

Kinetic parameters were obtained by plotting the substrate concentration versus uptake velocity using the following equation: v = (V_max × S)/(K_m + S) where v, S, K_m, and V_max represent the uptake velocity of the substrate (in picomoles · minute⁻¹ · milligram of protein⁻¹), the substrate concentration in the medium (in micromolar), the Michaelis-Menten constant (in micromolar), and the maximum uptake rate (in pmol · min⁻¹ · mg protein⁻¹), respectively. Fitting was performed with the nonlinear least-squares method using GraphPad Prism software version 6 (GraphPad Software, La Jolla, CA, USA).

LC-MS/MS determination of morinidazole and its major metabolites in transporter experiments.

The chromatographic conditions are the same as those in an earlier report (14). For MS detection, an API 6500 triple-quadrupole MS (Applied Biosystems, Concord, Ontario, Canada) with multiple reaction monitoring (m/z 271 → 144 for morinidazole, m/z 351 → [271 + 144] for M7, and m/z 447 → [320 + 144 + 100] for M8-1 and M8-2) were used in the positive electrospray ionization mode. All MS parameters were optimized to achieve the highest response. Analyst 1.6 software (Applied Biosystems) was used for data acquisition and processing. Calibration curves were constructed by plotting the peak area ratio of the analyte to the internal standard (i.e., analyte/internal standard) versus the nominal concentration using a linear-weighted (1/x²) least-squares regression method. The standard curves for morinidazole, M7, M8-1, and M8-2 in the control transporter matrix ranged from 0.100 to 300 ng · ml⁻¹.

RESULTS

Pharmacokinetic evaluation and renal elimination of morinidazole and its major metabolites in healthy subjects and patients with severe renal impairment.

The plasma drug concentration-time profiles of morinidazole and its metabolites after an intravenous infusion of 500 mg of morinidazole to patients with severe renal impairment and healthy subjects are shown in Fig. 2. Table 1 presents a summary of the pharmacokinetic characteristics of morinidazole and its metabolites.

FIG 2.

Mean plasma drug concentration-time profiles of morinidazole (A), M7 (B), M8-1 (C), and M8-2 (D) following an intravenous infusion of 500 mg of morinidazole to healthy subjects and patients with severe renal impairment.

TABLE 1.

Pharmacokinetic characteristics of morinidazole and its major metabolites^a

Group	Pharmacokinetic parameterb	Value for pharmacokinetic parameter forc:
		Morinidazole M0	Sulfate conjugate M7	Glucuronide conjugate
				M8-1	M8-2
Healthy subjects	Cmax (μg · ml−1)	10.8 ± 1.6	0.206 ± 0.049	0.312 ± 0.120	2.66 ± 0.78
	tmax (h)	0.9 ± 0.2	1.4 ± 0.3	2.3 ± 1.0	1.6 ± 0.4
	t1/2 (h)	5.5 ± 0.7	6.0 ± 0.5	5.7 ± 0.6	3.5 ± 0.4
	AUC0–t (μg · h · ml−1)	60.8 ± 12.0	1.71 ± 0.34	3.28 ± 1.32	19.5 ± 4.0
	CLR (liters · h−1)	1.28 ± 0.47	40.1 ± 11.6	17.0 ± 8.9	10.6 ± 3.1
	CL (liters · h−1)	8.51 ± 1.79
	Vss (liters)	56.6 ± 5.8
Patients with severe renal impairment	Cmax (μg · ml−1)	11.2 ± 3.6	1.15 ± 0.51	2.14 ± 0.82	11.1 ± 3.8
	tmax (h)	0.9 ± 0.3	5.8 ± 2.7	9.2 ± 2.8	9.8 ± 5.8
	t1/2 (h)	7.4 ± 1.2	10.0 ± 2.6	29.5 ± 31.6	22.1 ± 17.6
	AUC0–t (μg · h · ml−1)	89.5 ± 17.5	25.8 ± 16.0	66.9 ± 36.3	339 ± 170
	CLR (liters · h−1)	0.350 ± 0.160	5.90 ± 4.17	1.28 ± 1.02	0.824 ± 0.551
	CL (liters · h−1)	5.75 ± 1.31
	Vss (liters)	53.9 ± 11.4

^aPharmacokinetic characteristics of morinidazole and its major metabolites following an intravenous infusion of 500 mg of morinidazole to healthy subjects and patients with severe renal impairment.

^bC_max, maximum plasma concentration; t_max, time to the C_max; t_1/2, apparent elimination half-life; AUC_0–t, area under the concentration-time curve from 0 h to the last sampling time; CL_R, renal clearance; CL, total body clearance; V_ss, volume of distribution at steady state.

^cData are expressed as arithmetic means ± standard deviations.

In healthy subjects, morinidazole was the major circulating drug-related component, while the major metabolite was M8-2 with a systemic exposure of 19.4% relative to the parent drug. The systemic exposures of M7 and M8-1 were approximately 2.2% and 3.3% of the parent drug (based on molar concentrations), respectively. The concentration of morinidazole declined rapidly within 12 h after the dose. The plasma clearance of morinidazole was 8.51 liters · h⁻¹. The CL_R values for morinidazole, M7, M8-1, and M8-2 were 1.28, 40.1, 17.0, and 10.6 liters · h⁻¹, respectively.

There were no significant differences in the C_max or t_max for morinidazole between patients with severe renal impairment and healthy subjects, as the mean C_max and t_max were about 11 μg · ml⁻¹ and 0.9 h, respectively, in both groups. The AUC_0–t for morinidazole was about 1.5 times higher, and the t_1/2 was about 1.3 times longer in patients with severe renal impairment than in healthy subjects. The volume of distribution and total body clearance of morinidazole were similar in both groups. In contrast, compared with healthy subjects, the plasma concentration-time profiles of the major metabolites were altered dramatically in patients with severe renal impairment. The semilog plot (Fig. 2) shows that the concentrations of the major metabolites were markedly higher in patients with severe renal impairment at all times. The C_max values for M7, M8-1, and M8-2 were 5.6, 6.9, and 4.2 times higher, respectively, in patients with severe renal impairment than in healthy subjects. Meanwhile, the AUC_0–t values for M7, M8-1, and M8-2 were 15.1, 20.4, and 17.4 times higher, respectively, in patients with severe renal impairment, which means that the AUC_0–t for M8-2 was 3.8 times higher than that of the parent drug. The metabolites M7, M8-1, and M8-2 took longer to reach the C_max, as the t_max values were increased from 1.4 to 2.3 h in healthy subjects to 5.8 to 9.8 h in patients with severe renal impairment. Similarly, the t_1/2 values for M7, M8-1, and M8-2 were 1.7, 5.2, and 6.3 times longer, respectively.

The total cumulative recovery of major metabolites in urine samples collected for 0 to 48 h after the dose was nearly the same between healthy subjects and patients with severe renal impairment. However, the CL_R values for M7, M8-1, and M8-2 were 85.3%, 92.5%, and 92.2% lower, respectively, in patients with severe renal impairment.

Binding of morinidazole and its major metabolites to human plasma proteins.

The binding of morinidazole, M7, M8-1, and M8-2 to human plasma proteins at clinically relevant concentrations are listed in Table 2. The percentages of unbound morinidazole and its metabolites were consistently >70%, which indicates that the free drug fractions (F_u) of these compounds are high.

TABLE 2.

Binding of morinidazole, M7, M8-1, and M8-2 to human plasma proteins at clinically relevant concentrations

Analyte	Concn (ng · ml−1)	Fua (%)
Morinidazole	200	72.8
	2,000	77.9
	10,000	76.9
Sulfate conjugate M7	10.0	82.1
	50.0	75.4
	250	83.0
Glucuronide conjugates
M8-1	10.0	86.5
	50.0	108
	250	104
M8-2	50.0	99.6
	250	105
	1,600	98.9

^aF_u, free drug fraction.

Uptake of morinidazole and its major metabolites by transfected HEK293 cells.

To elucidate the roles of renal uptake transporters in the elimination of morinidazole and its major metabolites, we examined their uptake by three transporters, namely, OAT1, OAT3, and OCT2. As shown in Fig. 3, the rates of M7 uptake (5 μM) by OAT1 and OAT3 were 180 and 133 times higher, respectively, in uptake transporter-transfected cells than in empty-vector-transfected cells. OAT3-transfected cells also displayed greater uptake of M8-1 and M8-2 (5 μM) than control cells (by 274 and 180 times, respectively). The accumulation of M7, M8-1, and M8-2 in OCT2-transfected cells was increased by <2 times in these cells compared with empty-vector-transfected cells. The uptake of morinidazole by the transporters examined in this study was comparable to that in empty-vector-transfected cells, suggesting that the parent drug was not a substrate for OAT1, OAT3, or OCT2.

FIG 3.

Uptake of standard substrates (A to C) and test compounds (D to G) in the absence or presence of specific transporter inhibitors in transporter-transfected and empty-vector-transfected HEK293 cells. (A) PAH; (B) E3S; (C) MPP⁺; (D) morinidazole; (E) M7; (F) M8-1; (G) M8-2.

Inhibitory effects of probenecid and cimetidine on the uptake of M7, M8-1, and M8-2 by OAT1-, OAT3- and OCT2-transfected HEK293 cells.

As shown in Fig. 3, OAT1-mediated uptake of the positive-control substrate PAH, OAT3-mediated uptake of E3S, and OCT2-mediated uptake of MPP⁺ were suppressed significantly in the presence of 200 μM probenecid, an OAT inhibitor, or 500 μM cimetidine, an OCT2 inhibitor. Probenecid also reduced the uptake of M7, M8-1, and M8-2 by their respective transporters by >90%, which suggests that OAT1 and OAT3 are involved in the renal secretion of the major metabolites of morinidazole. However, the uptake of morinidazole was not significantly affected by these inhibitors, further confirming that morinidazole is not a substrate for OAT1, OAT3, or OCT2.

Time courses and concentration dependence of M7, M8-1, and M8-2 uptake by OAT1/3-transfected HEK293 cells.

We further evaluated the roles of OAT1 and OAT3 in the transport of M7, M8-1, and M8-2 by measuring the time- and concentration-dependent uptake of these metabolites in HEK293 cells transfected with OAT1 or OAT3. Figure 4 shows that the apparent uptake of M7 by OAT1- and OAT3-transfected cells was significantly greater than that in empty-vector-transfected cells at all times and increased in a linear time-dependent manner after incubation for 4 min. The rates of M8-1 and M8-2 uptake by OAT3-transfected HEK293 cells were linear for 15 min. Thus, in the present study, the uptake of M7 by OAT1- and OAT3-transfected HEK293 cells was evaluated at 1 min, and the uptake of M8-1 and M8-2 by OAT3-transfected HEK2933 was evaluated at 3 min.

FIG 4.

Time course of M7, M8-1, and M8-2 uptake by OAT1- and OAT3-transfected HEK293 cells (◆) and vector-transfected HEK293 cells (○). Data are expressed as means plus standard deviations (error bars) (n = 3). (A) Uptake of M7 by OAT1; (B) uptake of M7 by OAT3; (C) uptake of M8-1 by OAT3; (D) uptake of M8-2 by OAT3.

Figure 5 indicates that OAT1- and OAT3-dependent uptake of M7 was saturable, and the Eadie-Hofstee plot displayed a straight line for both cell types. The kinetic parameters are summarized in Table 3. There were differences in the affinities or uptake capacities of M7 between OAT1- and OAT3-transfected HEK293 cells, as the estimated K_m value for the transport of M7 was 2 times higher in OAT3-transfected cells (54.0 ± 3.4 μM) than in OAT1-transfected cells (28.6 ± 4.4 μM). The V_max values of M7 uptake in OAT1- and OAT3-transfected cells were 485 ± 29 and 864 ± 27 pmol · min⁻¹ · mg protein⁻¹, respectively. However, the uptake clearance of M7, expressed as V_max/K_m, was almost identical in OAT1- and OAT3-transfected cells, with values of 17.0 and 16.0 μl · min⁻¹ · mg protein⁻¹, respectively.

FIG 5.

Concentration dependence of M7, M8-1, and M8-2 uptake by OAT1- and OAT3-transfected HEK293 cells. OAT1- or OAT3-mediated transport was determined by subtracting the transport velocity in empty-vector-transfected cells from that in OAT1- and OAT3-transfected HEK293 cells. Data are expressed as means ± standard deviations (error bars) (n = 3). (A) Uptake of M7 by OAT1; (B) uptake of M7 by OAT3; (C) uptake of M8-1 by OAT3; (D) uptake of M8-2 by OAT3. M7 and M8-2 were assessed at concentrations of 0.5, 1, 2, 5, 10, 20, 50, and 100 μM. M8-1 was assessed at concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 500, and 1,000 μM.

TABLE 3.

Uptake kinetic parameters for OAT1- and OAT3-mediated uptake of M7, M8-1, and M8-2

Metabolite	Transporter	Km (μM)a	Vmax (pmol · min−1 · mg protein−1)a	Vmax/Km (μl · min−1 · mg protein−1)a
M7	OAT1	28.6 ± 4.4	485 ± 29	17.0
	OAT3	54.0 ± 3.4	864 ± 27	16.0
M8-1	OAT3	209 ± 9	218 ± 3	1.0
M8-2	OAT3	26.2 ± 3.9	44.5 ± 2.6	1.7

^aK_m, Michaelis-Menten constant; V_max, maximum uptake rate; OAT1, organic anion transporter 1. Data are expressed as means ± standard deviations.

In contrast, we observed marked differences for M8-1 and M8-2 in OAT3-transfected cells, with estimated K_m values of 209 ± 9 and 26.2 ± 3.9 μM, respectively, and V_max values of 218 ± 3 and 44.5 ± 2.6 pmol · min⁻¹ · mg protein⁻¹, respectively. Although the K_m and V_max values for M8-1 and M8-2 differed, the uptake clearances of these metabolites were comparable in OAT3-transfected cells (1.0 and 1.7 μl · min⁻¹ · mg protein⁻¹, respectively).

Taken together, these results indicate that OAT1 is involved in M7 uptake, while OAT3 is involved in the uptake of M7, M8-1, and M8-2. The relative affinities for OAT3 were M8-2 > M7 > M8-1, but the relative uptake clearances were M7 > M8-2 > M8-1.

DISCUSSION

In the present study, we evaluated the effects of renal impairment on the pharmacokinetics of morinidazole. We also examined the mechanism involved in the renal excretion of morinidazole and its metabolites using in vitro transporter systems.

Patients with severe renal impairment exhibited quantitatively distinct pharmacokinetic profiles, with AUC values 15.1, 20.4, and 17.4 times higher and CL_R values 85.3%, 92.5%, and 92.2% lower for sulfate conjugate M7, glucuronide conjugate M8-1, and M8-2, respectively, compared to those in healthy subjects. The total urinary excretion of the metabolites over 0 to 24 h in patients with severe renal impairment was less than that in healthy subjects, which confirmed that the urinary elimination rates of the metabolites were lower in patients with severe renal impairment. However, the total cumulative urinary recovery of the major metabolites was nearly the same in these two groups over 0 to 48 h, suggesting that the metabolism of morinidazole was not significantly affected in patients with severe renal impairment. Somewhat differently, it was found that the cumulative urinary recovery of morinidazole in patients with severe renal impairment (∼7.8%) was less than that in the healthy subjects (∼15.9%). This may be attributed to other elimination pathways of morinidazole, such as biliary excretion, or other metabolism pathways. These findings imply that renal elimination, rather than the overall metabolism of morinidazole, was significantly affected by renal impairment. Considering the differences in AUC_0–t between these two groups, renal impairment had greater effects on the pharmacokinetics of the conjugated metabolites than those of the parent drug. It is also noteworthy that the pharmacokinetics of M7, M8-1, and M8-2 also showed greater individual variation than morinidazole in patients with severe renal impairment, because the coefficient of variation for AUC_0–t was 62.0%, 54.3%, and 50.1%, for M7, M8-1, and M8-2, respectively, versus 19.6% for morinidazole. In addition, the AUCs of the conjugated metabolites, not the parent drug, had a good correlation with renal function in patients with severe renal impairment. Given all that, a decrease in glomerular filtration function may not be the main cause of changes in the pharmacokinetics of morinidazole metabolites.

Impaired renal function was reported to affect the extent of plasma protein binding, which might affect the distribution and elimination of drugs (13, 15). However, according to the U.S. Food and Drug Administration guidance (16), plasma protein binding of drugs with a relatively low extent of plasma protein binding (e.g., F_u > 20%) is not markedly affected by impaired renal function. Thus, regarding to the morinidazole and its metabolites (F_u > 70%), plasma protein binding has little contribution to the significantly greater AUC and reduced renal elimination of morinidazole and its metabolites in patients with severe renal impairment.

Considering that the sulfate conjugate M7 and glucuronide conjugates M8-1 and M8-2 are hydrophilic compounds with clogD (calculated logarithm of a compound's partition coefficient between n-octanol and water) values (at pH 7.4) of −6.15, −8.92, and −8.92, respectively, which were calculated using ACD software version 12.01 (ACD Laboratories, Advanced Chemistry Development Inc., Toronto, ON, Canada), they do not readily cross the plasma membrane via passive diffusion. Therefore, it was speculated that some transporters are involved in the uptake and disposition of morinidazole conjugated metabolites. Meanwhile, in healthy subjects, the CL_R values of M7 (40.1 liters · h⁻¹), M8-1 (17.0 liters · h⁻¹), and M8-2 (10.6 liters · h⁻¹), but not morinidazole (1.28 liters · h⁻¹), exceeded the products of the glomerular filtration rate (7.5 liters · h⁻¹) and their F_u values (17), which suggests that these metabolites may be secreted by active transporters. On the basis of these findings, we hypothesized that renal transporter systems may account for the different pharmacokinetic properties of morinidazole conjugated metabolites between healthy subjects and patients with severe renal impairment.

The OAT family of transporters, especially OAT1 and OAT3, is predominantly expressed on the basolateral membrane of the proximal tubules in kidneys and is involved in the initial transport of organic anions (18, 19). Members of the OCT family are involved in the transport of xenobiotics and endogenous organic cations. Three human OCTs have been identified, namely, OCT1 (SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3) (20), of which OCT2 is the most abundantly expressed OCT in the human kidney (21). Therefore, in the present study, we examined the transport of morinidazole and its conjugated metabolites by the three major renal uptake transporters OAT1, OAT3, and OCT2. The uptake of morinidazole was increased by <2-fold by OAT1, OAT3, and OCT2, indicating that morinidazole is not a substrate of these transporters. Pilot studies showed that the uptake of M7 was significantly higher in OAT1- and OAT3-transfected cells than in empty-vector-transfected cells. Inhibition of OATs using probenecid further confirmed that M7 was a substrate of these transporters. The lower K_m value for OAT1-mediated M7 uptake indicates that OAT1 has a higher affinity for M7 than OAT3 does. However, both OAT1 and OAT3 transporters have similar contributions to the renal uptake of M7 for comparable transport efficiencies. Considering that M7 is transported by both OAT1 and OAT3, it is not surprising that M7 had the greatest CL_R among the three major metabolites in healthy subjects. Although the substrate spectrum for OAT1 overlapped with that for OAT3 (22), there was a significant accumulation of the glucuronide conjugates M8-1 and M8-2 in OAT3-transfected cells but not in OAT1-transfected cells. Furthermore, it seems that the uptake of M8-1 by OAT3 was characterized by low-affinity, high-capacity transport, while the uptake of M8-2 by OAT3 was characterized by high-affinity, low-capacity transport. However, the transport efficiencies of OAT3 for M8-1 and M8-2 were similar. Therefore, it can be speculated that inhibition or reduced expression of renal uptake transporters could decrease the renal clearance of the major metabolites of morinidazole.

Very few studies have documented the changes in renal uptake transporters in patients with renal impairment. Sakurai et al. reported that of the four members of the OAT family (i.e., OAT1 to -4), only the mRNA expression level of OAT1 was significantly lower and that of OAT3 was slightly lower in patients with renal diseases than in individuals with normal renal function (23). Most of the evidence for changes in OAT expression is derived from in vitro and in vivo preclinical animal studies. Animal studies revealed that the protein and mRNA expression of many transporters, including OAT1, OAT2, OAT3, Oatp1, and Oatp4c1, were significantly reduced in 5/6 nephrectomized chronic renal failure rats (6, 8, 11, 24). Furthermore, in patients with renal impairment, endogenous organic anionic compounds, such as indoxyl sulfate, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, indoleacetate, and hippuric acid, accumulated to high levels in uremic serum. Since these uremic toxins are also substrates of OAT1 and OAT3 (25), the accumulation of these toxins could inhibit the renal excretion of morinidazole conjugated metabolites by competitively inhibiting transporters. Thus, we cannot exclude the possibility that inhibition of renal transporters by endogenous uremic toxins was responsible for the increased plasma exposure of M7, M8-1, and M8-2 in the patients with severe renal impairment in our study.

The substrates of OAT1 include antiviral drugs (e.g., adefovir, cidofovir, and tenofovir) and endogenous compounds (e.g., cyclic nucleotides, dicarboxylates, prostaglandins, and uremic toxins) (18, 19, 22). OAT3 exhibits a broad-substrate spectrum, like OAT1, and its substrates include β-lactam antibiotics (benzylpenicillin and cephalosporins), 3-hydroxy-3-methyl-glutaryl reductase inhibitors (pravastatin and rosuvastatin), H₂ receptor antagonists (cimetidine and ranitidine), and endogenous compounds, such as uremic toxins and conjugated steroids (26). Except for endogenous conjugates (such as dehydroepiandrosterone sulfate, estradiol-17β-glucuronide, and estrone-3-sulfate [E3S]) (19), there is scant information on the interactions between OATs and sulfate- and glucuronide-conjugated metabolites. Mizuno et al. reported that the sulfate conjugate of edaravone was a substrate of OAT3 (27). In addition, Han et al. reported that the renal uptake of a 3-O-sulfate conjugate of 17α-ethinylestradiol was mediated by OAT3 and OAT4 (28). In terms of uptake clearance, the sulfate conjugate M7 was a better substrate for OAT3 than the glucuronide conjugates were. In particular, the clearance of M7 by OAT1 (V_max/K_m = 17.0 μl · min⁻¹ · mg protein⁻¹) was comparable with that of its typical substrate, para-aminohippurate (PAH) (V_max/K_m = 13.1 μl · min⁻¹ · mg protein⁻¹) (data not shown), indicating that M7 is a good substrate for OAT1.

In summary, our study confirmed that the sulfate conjugate morinidazole is a substrate for OAT1 and OAT3, while the glucuronide conjugates are substrates for OAT3. In contrast, morinidazole is not a substrate for the kidney uptake transporters OAT1, OAT3, and OCT2. The significantly greater AUC and reduced renal clearance of these conjugated metabolites compared with the parent drug suggest that patients with severe renal impairment may have reduced expression and/or increased inhibition of renal uptake transporters compared with healthy subjects. In contrast, the slightly increased exposure of the parent drug in patients with severe renal impairment is likely to be due to the reduction in the glomerular filtration rate (GFR). Understanding the mechanisms involved in the renal excretion of morinidazole will help clinicians to adjust its dose in patients with severe renal impairment and to predict possible transporter-based drug-drug interactions. Our results also highlight the importance of the changes in the function and activity of renal transporters that may interfere with the pharmacokinetic properties of drugs in patients with severe renal impairment.