Positron Emission Tomography-Based Pharmacokinetic Analysis To Assess Renal Transporter-Mediated Drug-Drug Interactions of Antimicrobial Drugs

ABSTRACT

Transporter-mediated drug-drug interactions (DDIs) are of concern in antimicrobial drug development, as they can have serious safety consequences. We used positron emission tomography (PET) imaging-based pharmacokinetic (PK) analysis to assess the effect of different drugs, which may cause transporter-mediated DDIs, on the tissue distribution and excretion of [¹⁸F]ciprofloxacin as a radiolabeled model antimicrobial drug. Mice underwent PET scans after intravenous injection of [¹⁸F]ciprofloxacin, without and with pretreatment with either probenecid (150 mg/kg), cimetidine (50 mg/kg), or pyrimethamine (5 mg/kg). A 3-compartment kidney PK model was used to assess the involvement of renal transporters in the examined DDIs. Pretreatment with probenecid and cimetidine significantly decreased the renal clearance (CL_renal) of [¹⁸F]ciprofloxacin. The effect of cimetidine (−86%) was greater than that of probenecid (−63%), which contrasted with previously published clinical data. The kidney PK model revealed that the decrease in CL_renal was caused by inhibition of basal uptake transporters and apical efflux transporters in kidney proximal tubule cells. Changes in the urinary excretion of [¹⁸F]ciprofloxacin after pretreatment with probenecid and cimetidine resulted in increased blood and organ exposure to [¹⁸F]ciprofloxacin. Our results suggest that multiple membrane transporters mediate the tubular secretion of ciprofloxacin, with possible species differences between mice and humans. Concomitant medication inhibiting renal transporters may precipitate DDIs, leading to decreased urinary excretion and increased blood and organ exposure to ciprofloxacin, potentially exacerbating adverse effects. Our study highlights the strength of PET imaging-based PK analysis to assess transporter-mediated DDIs at a whole-body level.

INTRODUCTION

Approximately 40% of the antibiotics approved between 1980 and 2009 in the United States were withdrawn from the market for safety concerns, lack of effectiveness compared to existing drugs, and weak sales (1). The rate of market withdrawal was three times higher for antibiotics than for other drugs. One important factor contributing to safety concerns are drug-drug interactions (DDIs), which may either be enzyme or transporter mediated and can lead to serious adverse effects such as hepatotoxicity or nephrotoxicity (2–4). Patients receiving antimicrobial treatment are particularly vulnerable to DDIs due to their often complex therapeutic management with polytherapy.

A few membrane transporters belonging to the solute carrier (SLC) and ATP-binding cassette (ABC) families, which are abundantly expressed in absorption organs (i.e., intestine) and clearance organs (i.e., liver, kidneys), can be critical determinants of the absorption, distribution, and excretion of certain drugs, in particular those with low passive membrane permeability (5). In transporter-mediated DDIs, the concomitant administration of multiple drugs interacting with the same drug transporter(s) can result in altered pharmacokinetics (PK) compared to when each drug is administered alone (6). Regulatory authorities require the evaluation of interactions of drug candidates with clinically relevant transporters to predict the likelihood of DDIs (7, 8). For drugs for which a DDI risk cannot be excluded based on in vitro data, conducting in vivo DDI studies is necessary (9). In vivo DDI assessment most commonly focuses on measuring plasma PK, but some transporter-mediated DDIs may cause changes in the tissue distribution without affecting plasma concentrations (10). Positron emission tomography (PET) imaging allows noninvasive measurement of the tissue PK of radiolabeled drugs and can be used to assess transporter-mediated DDIs at a tissue level (11). Analysis of PET data with PK models can provide quantitative parameters for a detailed mechanistic assessment of transporter-mediated DDIs (12–15).

The fluoroquinolone antibiotic ciprofloxacin, which has been associated with serious adverse effects (16, 17), is vulnerable to transporter-mediated DDIs since it is a substrate of several membrane transporters, has low passive membrane permeability, and is mainly excreted in unmetabolized form into the urine (18, 19). Ciprofloxacin can be labeled with the positron-emitting radionuclide fluorine-18 (¹⁸F; half-life, 109.8 min) without structural alteration (20). PET imaging with [¹⁸F]ciprofloxacin has been used to study ciprofloxacin disposition in humans (21, 22) and mice (13, 23).

To explore the potential of PET as a tool in the assessment of transporter-mediated DDIs in antimicrobial therapy, we used, in the present study, [¹⁸F]ciprofloxacin as a radiolabeled model antimicrobial drug. [¹⁸F]ciprofloxacin was intravenously (i.v.) administered to mice without or with concomitant administration of drugs, which may be potentially involved in transporter-mediated DDIs with ciprofloxacin (i.e., probenecid, cimetidine, or pyrimethamine), followed by dynamic whole-body PET imaging, which allowed assessing not only the renal excretion but also the whole-body tissue distribution of ciprofloxacin. PK modeling was employed to assess the involvement of transporters in the renal excretion of ciprofloxacin.

RESULTS

Groups of mice underwent PET scans after intravenous (i.v.) administration of [¹⁸F]ciprofloxacin either without or with pretreatment with the renal anion and cation transporter inhibitors probenecid (150 mg/kg), cimetidine (50 mg/kg), or pyrimethamine (5 mg/kg). Since PET measures total radioactivity and cannot distinguish radiolabeled parent drug from radiolabeled metabolites, a prerequisite for PET-based PK analysis is good metabolic stability of the radiolabeled drug. We analyzed tissue and fluid samples collected at the end of the PET scan with radio-thin-layer chromatography (radio-TLC), which revealed that the majority of radioactivity was composed of unmetabolized [¹⁸F]ciprofloxacin (Table 1). This supported that the measured PET signal mainly represented unmetabolized [¹⁸F]ciprofloxacin.

TABLE 1.

Metabolism of [¹⁸F]ciprofloxacin

Group	No. of subjects	% unchanged [18F]ciprofloxacin in tissue or fluida
		Plasma	Liver	Bile	Kidney	Urine
Baseline	6	99 ± 2	73 ± 3	78 ± 4	86 ± 2	91 ± 3
Probenecid (150 mg/kg)	6	>99	>99b	93 ± 5b	90 ± 13	94 ± 4
Cimetidine (50 mg/kg)	5	99 ± 1	94 ± 4	94 ± 3b	80 ± 14	95 ± 4
Pyrimethamine (5 mg/kg)	6	>99	>99b	92 ± 7b	92 ± 8	97 ± 1b

^aValues are given as mean ± SD percentage of unchanged [¹⁸F]ciprofloxacin in different tissues and fluids determined with radio-TLC at the end of the 90-min PET scan.

^bKruskal-Wallis test followed by a Dunn’s multiple-comparison test against the baseline group; P ≤ 0.05.

Representative coronal PET summation images obtained after injection of [¹⁸F]ciprofloxacin are shown in Fig. 1, revealing high [¹⁸F]ciprofloxacin concentrations in the kidneys and urinary bladder of untreated animals. In probenecid- and cimetidine-treated animals, [¹⁸F]ciprofloxacin content in the urinary bladder was markedly decreased, and kidney concentrations were increased (Fig. 1).

FIG 1.

Representative coronal PET summation images (0 to 40 min) of one untreated mouse (baseline) and one mouse pretreated with either probenecid (150 mg/kg), cimetidine (50 mg/kg), or pyrimethamine (5 mg/kg). Radioactivity concentration is expressed as percentage of injected dose per milliliter (%ID/mL). Anatomical regions are labeled with arrows (L, liver; LK, left kidney; RK, right kidney; UB, urinary bladder).

Due to difficulties in performing repeated blood sampling in mice, blood concentration-time curves of [¹⁸F]ciprofloxacin were derived from the PET data by placing a region of interest (ROI) into the left ventricle of the heart. Image-derived blood concentration measurements were validated by correlating them with gamma countermeasured blood concentrations from samples collected at the end of the PET scan (see Fig. S1 in the supplemental material). PET measurements overestimated the gamma counter measurements by a factor of ~2, but there was an excellent correlation (Spearman rank correlation coefficient [r_s] = 0.914) between both measurements, supporting the utility of the image-derived blood curve for a relative comparison of PK parameters among groups.

Mean concentration-time curves (expressed in units of percentage of injected dose per milliliter [%ID/mL]) in blood, brain, and lung are shown in Fig. 2, and those in excretory organs (kidney, liver, urinary bladder, and intestine) are given in Fig. 3. Among all investigated organs, [¹⁸F]ciprofloxacin concentrations were lowest in the brain. In probenecid- and cimetidine-treated animals, blood and organ exposures to [¹⁸F]ciprofloxacin measured as the area under the concentration-time curve (AUC [%ID/mL × min]) were increased compared with untreated animals, while pretreatment with pyrimethamine caused only minor changes (Table 2). Organ-to-blood AUC ratios were not significantly different among groups, with the exception of AUC_liver/AUC_blood, which was significantly reduced in probenecid-treated animals, suggesting inhibition of an uptake transporter in hepatocytes (Table 2). The amount of [¹⁸F]ciprofloxacin excreted into the urinary bladder at the end of the PET scan was markedly decreased after pretreatment with probenecid (26.0 ± 18.1%ID) and cimetidine (13.6 ± 11.0%ID) compared with untreated mice (58.1 ± 6.7%ID), but only a minor reduction was observed after pyrimethamine pretreatment (46.0 ± 8.0%ID) (Fig. 3C).

FIG 2.

Mean concentration-time curves (%ID/mL ± SD) of [¹⁸F]ciprofloxacin in blood (image-derived blood curve from the left ventricle of the heart) (A), brain (B), and right lung (C) of ciprofloxacin mice without (baseline) and with pretreatment with either probenecid (150 mg/kg), cimetidine (50 mg/kg), or pyrimethamine (5 mg/kg).

FIG 3.

Mean concentration-time curves (%ID/mL or %ID ± SD) of [¹⁸F]ciprofloxacin in excretory organs of mice without (baseline) and with pretreatment with either probenecid (150 mg/kg), cimetidine (50 mg/kg), or pyrimethamine (5 mg/kg). (A) Left kidney (corticomedullary region); (B) liver; (C) urinary bladder (assumed to represent excreted urine); (D) intestine.

TABLE 2.

Pharmacokinetic parameters from noncompartmental pharmacokinetic analysis describing the organ distribution of [¹⁸F]ciprofloxacin^b

Parameter	Baseline	Probenecid (150 mg/kg)	Cimetidine (50 mg/kg)a	Pyrimethamine (5 mg/kg)
AUCblood (%ID/mL × min)	286 ± 49	373 ± 64	506 ± 94c	299 ± 64
AUCbrain (%ID/mL × min)	45 ± 3	65 ± 12c	78 ± 17c	58 ± 15
AUClung (%ID/mL × min)	113 ± 21	138 ± 29	210 ± 38c	129 ± 46
AUCkidney (%ID/mL × min)	510 ± 134	1,030 ± 635	1,329 ± 494c	560 ± 170
AUCliver (%ID/mL × min)	432 ± 105	401 ± 60	663 ± 199	430 ± 80
AUCbrain/AUCblood	0.16 ± 0.03	0.19 ± 0.05	0.16 ± 0.04	0.20 ± 0.03
AUClung/AUCblood	0.40 ± 0.04	0.37 ± 0.03	0.42 ± 0.04	0.42 ± 0.08
AUCkidney/AUCblood	1.79 ± 0.30	2.61 ± 1.22	2.61 ± 0.72	1.86 ± 0.27
AUCliver/AUCblood	1.51 ± 0.22	1.08 ± 0.04c	1.28 ± 0.18	1.46 ± 0.26

^aOne animal of the cimetidine-treated group received a 100 mg/kg dose of cimetidine.

^bData are given as mean ± SD. AUC is the area under the concentration-time curve (corrected for the amount of [¹⁸F]ciprofloxacin in the organ blood fraction).

^cKruskal-Wallis test followed by a Dunn’s multiple-comparison test against the baseline group; P ≤ 0.05.

The total clearance of [¹⁸F]ciprofloxacin from the blood (CL_T) was significantly reduced after pretreatment with cimetidine (−44%), and it was moderately but not significantly reduced after pretreatment with probenecid (−25%) (Fig. 4A; Table 3). CL_T remained unchanged after pretreatment with pyrimethamine (Fig. 4A; Table 3). The decrease in CL_T after probenecid and cimetidine treatment was due to a significant decrease in the renal clearance (CL_renal) of [¹⁸F]ciprofloxacin (Fig. 4B; Table 3), while the intestinal clearance (CL_intestinal) was not significantly different among all investigated groups (Fig. 4C; Table 3).

FIG 4.

Pharmacokinetic parameters (mean ± SD) obtained with noncompartmental pharmacokinetic analysis in untreated mice (baseline) and mice pretreated with either probenecid (150 mg/kg), cimetidine (50 mg/kg), or pyrimethamine (5 mg/kg). One animal of the cimetidine-treated group received a 100 mg/kg dose of cimetidine (open triangle). (A) CL_T is the total blood clearance. (B) CL_renal is the renal clearance with respect to the blood concentration. (C) CL_intestinal is the intestinal clearance with respect to the blood concentration. Kruskal-Wallis test followed by a Dunn’s multiple-comparison test against the baseline group; *, P ≤ 0.05; **, P ≤ 0.01.

TABLE 3.

Pharmacokinetic parameters from noncompartmental and compartmental pharmacokinetic analysis describing the excretion of [¹⁸F]ciprofloxacin^b

Parameter	Baseline	Probenecid (150 mg/kg)	Cimetidine (50 mg/kg)a	Pyrimethamine (5 mg/kg)
CLT (mL/min)	0.36 ± 0.07	0.27 ± 0.05	0.20 ± 0.05c	0.35 ± 0.07
CLrenal (mL/min)	0.210 ± 0.050	0.078 ± 0.060c	0.030 ± 0.026c	0.1610 ± 0.049
CLintestinal (mL/min)	0.077 ± 0.009	0.059 ± 0.014	0.057 ± 0.009	0.075 ± 0.022
CL1 (mL/min)	0.631 ± 0.363 (6.5–19.0)	0.132 ± 0.218c (3.2–19.3)	0.055 ± 0.055c (6.0–15.6)	0.792 ± 0.424 (11.5–28.9)
k2 (min−1)	1.141 ± 1.011 (16.6–60.8)	0.307 ± 0.700 (24.5–123.0)	0.198 ± 0.285 (7.3–19.2)	1.775 ± 1.026 (16.4–44.1)
k3 (min−1)	1.012 ± 0.393 (3.2–6.4)	0.164 ± 0.256c (4.7–72.6)	0.002 ± 0.001c (45.2–84.2)	0.672 ± 0.361 (3.6–6.8)

^aOne animal of the cimetidine-treated group received a 100 mg/kg dose of cimetidine.

^bParameter estimates are given as mean ± SD. Values in parentheses represent the range in percent coefficient of variation (%CV; parameter precision) of the parameters obtained with the 3-compartment kidney PK model. CL_T, CL_renal, and CL_intestinal are the total, renal, and intestinal clearances of [¹⁸F]ciprofloxacin calculated with noncompartmental PK analysis. CL₁, k₂, and k₃ are the parameters obtained with the 3-compartment kidney PK model and represent the renal uptake clearance and glomerular filtration of [¹⁸F]ciprofloxacin (CL₁) and the rate constants defining the transfer of [¹⁸F]ciprofloxacin from the corticomedullary region to the sink compartment (blood) (k₂) and from the corticomedullary region into excreted urine (k₃).

^cKruskal-Wallis test followed by a Dunn’s multiple-comparison test against the baseline group; P ≤ 0.05.

To assess the role of renal transporters as a possible cause for the reduction in CL_renal in the probenecid- and cimetidine-treated groups, data were further analyzed with a previously developed 3-compartment kidney PK model (Fig. S2) (13). Since the inhibitory effect of probenecid and cimetidine appeared to be reversible 40 min after radiotracer administration (Fig. 3A), the model was applied only to the first 40 min of the PET data. The PK model provided good fits for both kidney and excreted urine curves (Fig. S3 to S6), and parameter precision was generally acceptable (Table 3). The model parameter representing the renal uptake clearance and glomerular filtration of [¹⁸F]ciprofloxacin (CL₁) and the parameter describing the transfer of [¹⁸F]ciprofloxacin from the renal corticomedullary region into excreted urine (k₃) were significantly reduced in probenecid- (−79% and −84%, respectively) and cimetidine-treated (−91% and −99.9%, respectively) animals (Fig. 5A and C) with respect to the untreated group. The rate constant describing the transfer of [¹⁸F]ciprofloxacin from the renal corticomedullary region to the sink compartment (blood) (k₂) showed a trend toward decreases without reaching statistical significance (Fig. 5B). There was a trend toward a k₃ reduction (−34%) in pyrimethamine-treated animals, but statistical significance was not reached (Fig. 5C). CL_renal calculated from the model parameters was 0.30 ± 0.08 mL/min for the untreated group, which agreed well with CL_renal calculated by noncompartmental analysis using only the first 40 min of the PET data (CL_renal = 0.25 ± 0.08 mL/min).

FIG 5.

Pharmacokinetic parameters (mean ± SD) obtained with the 3-compartment kidney pharmacokinetic model in untreated mice (baseline) and mice pretreated with either probenecid (150 mg/kg), cimetidine (50 mg/kg), or pyrimethamine (5 mg/kg). One animal of the cimetidine-treated group received a 100 mg/kg dose of cimetidine (open triangle). (A) CL₁ represents both the renal uptake clearance and the glomerular filtration rate of [¹⁸F]ciprofloxacin. (B) k₂ is the rate constant describing the transfer of [¹⁸F]ciprofloxacin from the kidney into the sink compartment (blood). (C) k₃ is the rate constant describing the transfer of [¹⁸F]ciprofloxacin from the kidney into the excreted urine. Kruskal-Wallis test followed by a Dunn’s multiple-comparison test against the baseline group; *, P ≤ 0.05; **, P ≤ 0.01.

DISCUSSION

In this study, we demonstrated the utility of dynamic PET imaging in combination with PK modeling for a detailed mechanistic assessment of renal transporter-mediated DDIs involving the fluoroquinolone ciprofloxacin as a victim drug. Fluoroquinolones represent a drug class which has been associated with serious adverse effects, including musculoskeletal and central nervous system disorders, hepatotoxicity, and photosensitivity (16, 17). From 1980 to 2009, nine fluoroquinolones were withdrawn from the market, mainly for safety reasons (1), and the use of fluoroquinolones has been restricted by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Many fluoroquinolones possess low passive membrane permeability, and their tissue distribution and excretion are, to a large extent, controlled by membrane transporters (19). Transporter-mediated DDIs may thus contribute to the adverse effects of these drugs.

Renal clearance accounts for approximately two-thirds of the total clearance of ciprofloxacin (18). The unbound renal clearance of ciprofloxacin exceeds the normal glomerular filtration rate, indicating that ciprofloxacin undergoes tubular secretion (18, 19). This process involves an interplay between SLC transporters in the basal membrane of kidney proximal tubule cells, which mediate the uptake of drugs from blood into the cells, and apical ABC or SLC transporters, which mediate the excretion into the urine (19). Ciprofloxacin is a low-permeability compound (octanol-water partition coefficient logP, −1.08) which predominantly (82%) exists at physiological pH as a zwitterion (19, 24, 25). Therefore, both renal anion and cation transporters may accept the drug as their substrates (19). However, the transporters involved in the tubular secretion of ciprofloxacin have not yet been identified. We have previously invesigated the role of apical ABC transporters, i.e., breast cancer resistance protein (BCRP/ABCG2), multidrug resistance-associated proteins 4 and 2 (MRP4/ABCC4 and MRP2/ABCC2), and P-glycoprotein (P-gp/ABCB1), in the tubular secretion of [¹⁸F]ciprofloxacin in mice, employing the same kidney PK model as in this study (13). Although ciprofloxacin is a known substrate of mouse and human BCRP and MRP4 (26, 27), single and combined knockout of the Abcg2 and Abcc4 genes did not reduce renal excretion of [¹⁸F]ciprofloxacin in mice, which suggested that Bcrp and Mrp4 were not involved in the tubular secretion of [¹⁸F]ciprofloxacin.

Transporter-mediated DDIs in the kidneys (3, 4) may lead to PK variability and may potentially influence the therapeutic effects as well as the adverse effects of ciprofloxacin. Cimetidine and pyrimethamine are recommended prototypical inhibitors of renal cation transporters, i.e., organic cation transporter 2 (OCT2/SLC22A2) and multidrug and toxic extrusion 1 and 2-K (MATE1/SLC47A1 and MATE2-K/SLC47A2) proteins for clinical DDI studies (9). Probenecid is a recommended prototypical inhibitor of renal anion transporters, i.e., organic anion transporter 1 and 3 (OAT1/SLC22A6 and OAT3/SLC22A8) (9). In a clinical DDI study, probenecid was shown to reduce renal clearance and increase plasma exposure to ciprofloxacin (28, 29). Cimetidine reduced the renal clearance of several fluoroquinolones (i.e., temafloxacin, enoxacin, and gemifloxacin) (30–32), but not that of ciprofloxacin (33). In previous clinical DDI studies, drug concentrations were only assessed in plasma and urine and not in the kidneys, which did not allow for distinguishing the individual roles of basal uptake and efflux transporters and apical efflux transporters in the tubular secretion of ciprofloxacin. In the present study, we applied our previously developed PET imaging-based kidney PK model (13) for a mechanistic assessment of the DDIs between [¹⁸F]ciprofloxacin and probenecid, cimetidine, and pyrimethamine in mice. The perpetrators were administered at doses similar to their clinically used doses. [¹⁸F]ciprofloxacin was administered as a microdose (~8 μg), which is several times lower than the clinically used dose. The low administered ciprofloxacin dose largely excludes the possibility of saturation of renal transporters handling ciprofloxacin. Ciprofloxacin PK has been found to be linear over a large dose range (34), which suggests that microdose PK data can be extrapolated to therapeutic dose data. A potential advantage of using microdoses of the victim drug in clinical DDI studies is improved safety (35).

Noncompartmental PK analysis revealed a significant decrease in the renal clearance (CL_renal) of [¹⁸F]ciprofloxacin (−63%) (Fig. 4B) and an increase in the blood exposure (AUC_blood) to [¹⁸F]ciprofloxacin (Table 2) after pretreatment with probenecid. In humans, the probenecid-induced reduction in renal clearance of ciprofloxacin was of similar magnitude (−64%) and has been attributed to OAT3 inhibition (29). Indeed, VanWert et al. reported that ciprofloxacin is transported by mouse Oat3 and that Slc22a8^−/− mice had a decreased plasma clearance of ciprofloxacin (−36% in female mice), which was of similar magnitude as the effect of probenecid observed in our study (CL_T reduced by −25%) (Fig. 4A) (36). In addition, other reported DDIs with probenecid, in which renal clearance of several drugs was decreased, have also been attributed to the involvement of OATs (3, 4, 6). Analysis with the kidney PK model (13) revealed a significant decrease in the renal uptake clearance (CL₁) of [¹⁸F]ciprofloxacin after probenecid administration (Fig. 5A), which supported the involvement of mouse Oat3 in the interaction between both drugs. However, the decrease in urinary excretion (k₃) (Fig. 5C), together with the accumulation of [¹⁸F]ciprofloxacin in the renal corticomedullary region (Fig. 3A), cannot be explained by the inhibition of a basal uptake transporter alone. This suggested that the probenecid-sensitive transporter(s) in the apical membrane of kidney proximal tubule cells also contributed to the probenecid-ciprofloxacin DDI.

Cimetidine is a prototypical inhibitor of the renal cation transport system and is known to be involved in multiple DDIs, which have been mainly attributed to the inhibition of MATE1 and MATE2-K in the apical membrane of kidney proximal tubule cells (37). Of note, we observed some toxicity of cimetidine in mice that has not been described in previous studies, and the mechanism of which is unknown. PK modeling revealed a significant reduction in CL₁ in cimetidine-treated mice, pointing to inhibition of renal uptake transporter(s) (Fig. 5A). Since cimetidine is not only an OCT inhibitor but also inhibits mouse, rat, and human OAT3 (38, 39), the observed CL₁ reduction can most likely be attributed to the inhibition of mouse Oat3 at the basal membrane of proximal tubule cells. Despite the fact that cimetidine is a less potent inhibitor of human OAT3 than probenecid (38), the reduction in CL₁ caused by cimetidine (−91%) was greater than that of probenecid (−79%) in mice. A possible explanation for this could be species differences in inhibitory potencies and/or the involvement of additional basal uptake transporters, which are inhibited by cimetidine, such as mouse Oct1/2. Next to the CL₁ decrease, PK modeling revealed a significant and pronounced decrease in k₃ in cimetidine-treated animals (Fig. 5C), which suggests inhibition of apical efflux transporters. Since ciprofloxacin was shown to be transported by rat Mate1 (40) and this transport was inhibited by cimetidine, the interaction between cimetidine and ciprofloxacin at the apical membrane of kidney proximal tubule cells may be attributable to the inhibition of mouse Mate1-mediated [¹⁸F]ciprofloxacin excretion. The pronounced DDI between cimetidine and ciprofloxacin in mice contrasts with data in humans, in whom no DDI was observed between the two drugs (33), pointing to species differences in the transporters involved in the tubular secretion of ciprofloxacin. While ciprofloxacin was found to be a substrate of rat Mate1 (40), there is no evidence of its transport by human MATE1 or MATE2-K (41).

Unexpectedly, no significant reduction in CL_renal was observed after pretreatment with pyrimethamine (Fig. 4B), although a moderate (but not significant) decrease in k₃ was observed (Fig. 5C). Pyrimethamine is a potent inhibitor of mouse and human MATEs (42), and MATE-mediated interactions with pyrimethamine have been reported to affect the urinary excretion of various drugs, such as metformin and sulpiride (43–46). The chosen pyrimethamine dose inhibited Mate1-mediated tubular secretion of [¹¹C]metformin in mice (44). A possible explanation for the only moderate effect of pyrimethamine on the urinary excretion of [¹⁸F]ciprofloxacin may be that the Mate1-inhibitory effect of pyrimethamine is substrate dependent and that the employed pyrimethamine dose was too low to achieve sufficiently high pyrimethamine plasma and kidney concentrations for Mate1 inhibition. A limitation of our study is that plasma and organ concentrations of the administered inhibitors were not measured, which would have enabled a better comparison with available clinical data. PET experiments in mice genetically lacking Oat3 or Mate1 may help to confirm these transporters’ role in the tubular secretion of ciprofloxacin.

A prerequisite for PET-based PK analysis is good metabolic stability of the radiolabeled drug over the time course of the PET experiment. This is supported by the results of radio-TLC analysis, which showed that in all mouse groups, the majority of radioactivity (≥80%) in plasma, kidneys, and urine, i.e., the organs/fluids analyzed with the PK model, was composed of unmetabolized [¹⁸F]ciprofloxacin (Table 1). All treatments decreased the percentage of radiolabeled metabolites of [¹⁸F]ciprofloxacin in the liver, which may be caused by inhibition of hepatic metabolism. However, given the low extent of metabolism, this effect appears negligible compared to the observed transporter effects.

Since ciprofloxacin is also partly eliminated via direct secretion from the blood into the intestine (47), we assessed the intestinal clearance (CL_intestinal) of [¹⁸F]ciprofloxacin, which corresponded to about 20% of the total clearance in untreated wild-type mice (Table 3). No changes in CL_intestinal were observed after pretreatment with any of the drugs (Fig. 4C), which indicated the absence of a DDI at the level of intestinal epithelial cells. We furthermore assessed the distribution of [¹⁸F]ciprofloxacin to an organ which may be involved in adverse effects (i.e., brain) and an organ of therapeutic interest (i.e., lungs). The reduction in urinary excretion after pretreatment with probenecid or cimetidine led to increased blood exposure to [¹⁸F]ciprofloxacin, which, in turn, led to increased brain (AUC_brain) and lung (AUC_lung) exposure (Table 2). However, brain-to-blood and lung-to-blood AUC ratios were unaffected (Table 2), indicating the lack of involvement of transporters in brain and lung capillary endothelial cells in the observed DDIs. These data suggest that renal transporter-mediated DDIs could exacerbate ciprofloxacin side effects in organs such as the brain, while they may simultaneously enhance the efficacy for the treatment of respiratory infections.

With the upcoming availability of clinical PET systems with long axial fields of view (FOVs) (up to 194 cm) (48, 49), the methodology employed in this work bears great potential for translation to humans. These long axial FOV PET systems allow for dynamic imaging of the whole body so that concentration-time curves of organs that are distant from each other can be simultaneously measured (e.g., brain and kidneys). This opens up the possibility for a detailed assessment of transporter-mediated DDIs at a whole-body level, including both pharmacological target organs and organs relevant to drug excretion and toxicity. Extended axial FOV PET imaging, in combination with PK models such as the one employed in this work, may thus prove helpful in the clinical assessment of transporter-mediated DDIs of novel antimicrobial drugs.

Conclusion.

Our study highlights the great potential of PET-based PK analysis for a mechanistic assessment of transporter-mediated DDIs in antimicrobial drug development. Our results support that tubular secretion of ciprofloxacin is mediated by multiple membrane transporters, with possible species differences between mice and humans. As a zwitterion, ciprofloxacin can interact with renal anion and cation transporters. Renal transporters may be involved in clinically relevant DDIs, which may contribute to the clinical adverse effects of ciprofloxacin and also affect its efficacy.

MATERIALS AND METHODS

Chemicals.

Chemicals were purchased from Sigma-Aldrich Chemie (Schnelldorf, Germany) unless otherwise stated. An i.v. injection solution of cimetidine (200 mg/2 mL; ratiopharm GmbH, Ulm, Germany) was obtained from a local pharmacy. Cimetidine solution was diluted 1:1 with physiological saline (0.9%, wt/vol) and i.v. injected at a volume of 2 μL per g body weight (administered dose, 50 mg/kg body weight). Probenecid (3.75 mg) was dissolved in 100 μL physiological saline (pH adjusted to 8.0 with aqueous sodium hydroxide) and injected intraperitoneally (i.p.) at a volume of 4 μL per g body weight (administered dose, 150 mg/kg). Pyrimethamine (12.5 mg) was dissolved in sterile water (5 mL) containing 50 μL of 1 M aqueous sulfuric acid (50), and the solution (pH 3.0) was i.v. injected at a volume of 2 μL per g body weight (administered dose, 5 mg/kg). Due to the short radioactive half-life of ¹⁸F (109.8 min), [¹⁸F]ciprofloxacin was newly synthesized on each study day as described previously (20) and formulated in phosphate-buffered saline (pH 5.0 to 6.0) for i.v. injection. The radiochemical purity of all synthesized batches (n = 5) was >98%, and molar activity at the end of synthesis was 2.15 ± 1.59 GBq/μmol.

Animals.

Female mice with an FVB genetic background (n = 26) were obtained from Charles River Laboratories (Sulzfeld, Germany). Female mice were used to enable comparison of our data with our previous study (13). At the time of the experiment, animals weighed 23.9 ± 1.7 g. Animals were housed in type III individually ventilated cage system (IVC) cages under controlled environmental conditions (21.8 ± 1.0°C, 40 to 70% humidity, 12-h light/dark cycle) with free access to a standard laboratory rodent diet (LASQCdiet; LASvendi, Soest, Germany) and water. An acclimatization period of at least 1 week was allowed before the animals were used in the experiments. The study was approved by the Intramural Committee for Animal Experimentation of the Medical University of Vienna and by the Austrian Federal Ministry of Education, Science and Research (approval number 2021-0.798.870). All study procedures were in accordance with the European Communities Council Directive of 22 September 2010 (2010/63/EU). The data reported in this study are in compliance with the Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines.

Experimental design.

Four groups of mice underwent 90-min dynamic whole-body PET scans after i.v. administration of [¹⁸F]ciprofloxacin. No randomization was applied. The mean injected amount of [¹⁸F]ciprofloxacin was 6.4 ± 1.0 MBq, corresponding to 8 ± 19 μg of unlabeled ciprofloxacin. One group of animals (n = 6) received no pretreatment before the start of the PET scan (baseline group), and a second group (n = 6) received an i.p. injection of probenecid (150 mg/kg) 15 min before the start of the PET scan. A third group (n = 8) received an i.v. injection of cimetidine 5 min before the start of the PET scan. Cimetidine was initially administered to two animals at a dose of 100 mg/kg. One animal died immediately after cimetidine administration, and the dose was reduced to 50 mg/kg for the remaining animals. Despite the dose reduction, two additional animals died after cimetidine (50 mg/kg) administration, so there were, in total, data from 5 animals available in this group. A fourth group (n = 6) received an i.v. injection of pyrimethamine (5 mg/kg) 30 min before the start of the PET scan. The administered probenecid, cimetidine, and pyrimethamine doses were based on previous work (44–46, 51) and corresponded to human-equivalent doses of 12.2 mg/kg for probenecid, 4.1 mg/kg for cimetidine, and 0.41 mg/kg for pyrimethamine (52), which were in comparable ranges to the respective clinically used oral doses of the drugs (probenecid, 500 mg; cimetidine, 400 mg; pyrimethamine, 50 mg).

PET imaging.

A microPET Focus 220 scanner (Siemens Medical Solutions, Knoxville, TN, USA; axial FOV, 7.6 cm) was used for PET imaging. Experiments were performed under isoflurane/air anesthesia, and animals were warmed while monitoring body temperature and respiratory rate. [¹⁸F]ciprofloxacin was administered i.v. over 1 min, and a 90-min dynamic PET scan was initiated at the start of radiotracer injection. List mode data were acquired with a timing window of 6 ns and an energy window of 250 to 750 keV. At the end of the PET scans, blood was collected from the retrobulbar plexus, and animals were sacrificed by cervical dislocation while still under deep anesthesia. The liver and kidneys were removed, and urine and bile were collected. Blood was centrifuged to obtain plasma, and all samples were assessed for radiolabeled metabolites of [¹⁸F]ciprofloxacin using radio-TLC as previously described (23). Radioactivity in a weighed blood aliquot was measured in a gamma counter.

PET data analysis.

Dynamic PET data were sorted into 25 time frames with a duration increasing from 5 s to 20 min. PET images were reconstructed using Fourier rebinning of the 3-dimensional sinograms followed by a 2-dimensional filtered back projection with a ramp filter giving a voxel size of 0.4 × 0.4 × 0.796 mm³. Using the medical image data examiner software AMIDE (53), the left ventricle of the heart (image-derived blood curve), brain, liver, left kidney (corticomedullary region, excluding the renal pelvis), right lung, intestine (representing all the visible intestinal radioactivity), and urinary bladder (excreted urine) were manually outlined on the PET images as ROIs. Since biliary excretion of ciprofloxacin is very low (<1% of the administered dose) (54), intestinal radioactivity was assumed to mainly represent luminal content originating from direct secretion from blood. Concentration-time curves were extracted for each ROI and were expressed in %ID/mL for blood, brain, liver, kidney, and lung and in %ID for intestine and urinary bladder by multiplication of the image-derived radioactivity concentration with the ROI volume. Time points were set to the midpoint of each PET frame.

Pharmacokinetic analysis.

(i) Noncompartmental PK analysis. For each concentration-time curve, the AUC (%ID/mL × min) was calculated from 0 to 80 min after radiotracer injection using Prism 8.0 software (GraphPad, La Jolla, CA, USA). Due to the short measurement time, the AUC was not extrapolated to infinity. AUCs in brain, lung, kidney, and liver were corrected for the amount of [¹⁸F]ciprofloxacin in the blood fraction in the organ by subtracting AUC_blood multiplied by the organ blood fraction obtained from the literature (brain, 0.05; lung, 0.16; kidney, 0.25; and liver, 0.25) from the AUC_organ (55–58). Since the AUC is dose normalized, total clearance (equal to dose/AUC) of [¹⁸F]ciprofloxacin from blood (CL_T [mL/min]) can be calculated as (1/AUC_blood) × 100. The renal and intestinal clearances of [¹⁸F]ciprofloxacin with respect to the blood concentration (CL_renal and CL_intestinal [mL/min]) were calculated by dividing the total amount of [¹⁸F]ciprofloxacin at the last time point (i.e., 80 min) in the urinary bladder or intestine, respectively, by AUC_blood. To assess the organ distribution of [¹⁸F]ciprofloxacin, the organ-to-blood AUC ratios (AUC_organ/AUC_blood) were determined.

(ii) Compartmental PK analysis. A previously developed 3-compartment kidney PK model (see Fig. S2 in the supplemental material) (13) was used for a mechanistic assessment of the involvement of renal transporters in the investigated DDIs. The analysis was performed using custom-written MATLAB scripts (R2018a; MathWorks Inc.). The employed model is a typical PET PK model in which the input to the system is a directly measured arterial blood curve (image derived) (59). Therefore, although the blood supply is represented as a physical compartment, in PET PK modeling, blood is usually not considered a mathematical compartment. In addition to the model input function, PET PK models typically contain one or two compartments that describe the kinetics of the radiotracer in the tissue of interest. The employed model contains a kidney compartment representing the corticomedullary region and an excreted urine compartment corresponding to the urinary bladder ROI (Fig. S2). The [¹⁸F]ciprofloxacin amount in the corticomedullary region was corrected for the amount of [¹⁸F]ciprofloxacin in the blood fraction in the kidneys (~0.25). The model assumes that no metabolism of [¹⁸F]ciprofloxacin occurs during the PET scan (13). The model parameters were CL₁ (mL/min), which represented the renal uptake clearance and glomerular filtration rate of [¹⁸F]ciprofloxacin, and the rate constants describing the transfer of [¹⁸F]ciprofloxacin from the corticomedullary region to the sink compartment (blood) (k₂ [min⁻¹]) and from the corticomedullary region into excreted urine (k₃ [min⁻¹]) (Fig. S2). From the estimated model parameters, CL_renal (mL/min) can be calculated as (CL₁ × k₃)/(k₂ + k₃).

Statistical analysis.

Data are reported as mean ± standard deviation (SD). The GINGER tool of the Institute of Clinical Biometrics at the Medical University of Vienna, available at https://clinicalbiometrics.shinyapps.io/GINGER/, was used for sample size calculations. Based on published data from the study by Jaehde et al. (28) in which the renal clearance of ciprofloxacin was reduced in humans from 373 ± 79 mL/min to 134 ± 45 mL/min following probenecid intake (effect size [difference in mean value divided by standard deviation] = 3), the minimum required sample size was 4 in order to detect differences between three pretreated groups and the untreated group, with a two-sided significance level of 0.05 and a power of 80%. Statistical analysis was performed with Prism 8.0 software. Differences between pretreated groups and the untreated group were analyzed by a Kruskal-Wallis test followed by a Dunn’s multiple-comparison test. The Spearman rank correlation coefficient, r_s, was calculated to assess correlations. The level of statistical significance was set to a P value of ≤0.05.