Influence of Multidrug Resistance-Associated Proteins on the Excretion of the ABCC1 Imaging Probe 6-Bromo-7-[¹¹C]Methylpurine in Mice

Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (Schnelldorf, Germany) or Merck (Darmstadt, Germany). The MRP inhibitor MK571 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Before each administration, MK571 was dissolved in phosphate-buffered saline (PBS) at a concentration of 20.0 mg/ml and injected intraperitoneally (i.p.) into mice at a volume of 20 ml/kg body weight. For i.p. administration, dipyridamole was dissolved in PBS at a concentration of 3.3 mg/ml and injected at a volume of 12 ml/kg body weight. [¹¹C]Methane was produced in a PETtrace cyclotron equipped with a methane target system (GE Healthcare, Uppsala, Sweden). The unlabeled reference standards 6-bromo-7-methylpurine and S-(6-(7-methylpurinyl))glutathione were obtained as gifts from Dr. Toshimitsu Okamura (National Institute of Radiological Sciences, Chiba, Japan).

The synthesis of 6-bromo-7-[¹¹C]methylpurine was slightly modified compared to the published method [13] and is described in detail in the Electronic Supplementary Material. 6-Bromo-7-[¹¹C]methylpurine was formulated in 0.9 % (w/v) aqueous saline for i.v. injection into animals. Radiochemical purity of 6-bromo-7-[¹¹C]methylpurine was greater than 98 % and molar activity at the end of synthesis was 444 ± 203 GBq/μmol.

Female wild-type, homozygous Abcc1^(−/−) and heterozygous Abcc1^(+/−) mice with a C57BL/6J genetic background were obtained from the University of Oslo (Oslo, Norway). Female Abcc4^(−/−) mice with a C57BL/6 genetic background were obtained from the St. Jude Children’s Research Hospital (Memphis, TN, USA). At the time of experiment, animals weighed 25 ± 4 g. Animals were used in the experiments after being kept for at least one week in our animal facility. Approval was obtained from the competent authority (Amt der Niederösterreichischen Landesregierung) for conducting this study.

An overview of animal groups examined in this study is given in Table 1. Groups of wild-type, Abcc4^(−/−), Abcc1^(−/−), and Abcc1^(+/−) mice underwent a baseline PET scan with 6-bromo-7-[¹¹C]methylpurine at 30 min after i.p. injection of vehicle solution (PBS). Further groups of wild-type and Abcc4^(−/−) mice were scanned with 6-bromo-7-[¹¹C]methylpurine at 30 min after i.p. injection of the MRP inhibitor MK571 (300 mg/kg). The dosage of MK571 was selected based on previous work by Okamura et al. [12]. One group of wild-type mice underwent a 6-bromo-7-[¹¹C]methylpurine PET scan at 30 min after i.p. injection of the nucleoside transporter inhibitor dipyridamole (40 mg/kg). The dosage of dipyridamole was selected based on maximum solubility. In addition, groups of wild-type and Abcc1^(−/−) mice were used to determine the percentage of unchanged 6-bromo-7-[¹¹C]methylpurine in different tissues and fluids.

During all experimental procedures, animals were kept under isoflurane anesthesia. PET imaging was conducted on a microPET Focus220 scanner (Siemens Medical Solutions, Knoxville, TN, USA). Mice were i.p. injected under anesthesia at 30 min before start of the PET scan either with vehicle solution (PBS), or with MK571 (300 mg/kg) or with dipyridamole (40 mg/kg). Subsequently, 6-bromo-7-[¹¹C]methylpurine (29 ± 9 MBq in a volume of 0.1 ml, 0.17 ± 0.11 nmol) was administered as an i.v. bolus via the lateral tail vein, and a 90-min dynamic PET scan (energy window: 250–750 keV; timing window: 6 ns) was initiated at the start of radiotracer injection. At the end of the PET scan, a blood sample was collected from the retro-bulbar plexus and animals were euthanized. Blood was centrifuged to obtain plasma and aliquots of blood and plasma were counted for radioactivity in a gamma counter (Wizard 1470; Perkin-Elmer, Waltham, MA, USA).

The PET data were sorted into 25 frames with a duration increasing from 5 s to 20 min. Fourier re-binning of the three-dimensional sinograms followed by a two-dimensional filtered back-projection with a ramp filter resulting in a voxel size of 0.4 × 0.4 × 0.796 mm³ was used to reconstruct the PET data. Using AMIDE software [15], brain, right lung, left kidney, urinary bladder, liver, duodenum, intestine, and gall bladder were manually outlined on the PET images to derive concentration-time curves expressed in units of standardized uptake value (SUV = (radioactivity per g/injected radioactivity) × body weight). In addition, the left ventricle of the heart was outlined to obtain an image-derived blood curve. For all organs, the area under the concentration-time curve (AUC) from 0 to 90 min after radiotracer injection was calculated using Prism 7 software (GraphPad Software, La Jolla, CA, USA) and organ-to-blood AUC ratios were calculated. From the log-transformed concentration-time curves in blood, brain, lung, kidney, and liver, the elimination rate constant (k_elimination) of radioactivity was determined by linear regression analysis from 17.5 to 80 min after radiotracer injection. A graphical analysis method (integration plot) [16] was used to estimate the rate constant for transfer of radioactivity from blood into different organs (brain, kidney, and liver, k_uptake, ml/min/g tissue) using data measured from 0.3 to 3.5 min after radiotracer injection, as described in detail before [17]. Moreover, the rate constant for tubular secretion of radioactivity (k_urine, min⁻¹) was determined from 12.5 to 65 min after radiotracer injection using integration plot analysis [17].

In separate groups of wild-type and Abcc1^(−/−) mice, the percentage of unchanged 6-bromo-7-[¹¹C]methylpurine and its glutathione conjugate S-(6-(7-[¹¹C]methylpurinyl))glutathione in different organs and fluids was analyzed by radio-thin layer chromatography (TLC) as described in the Electronic Supplementary Material.

MK571 concentrations in plasma and kidneys were determined as described in the Electronic Supplementary Material.

Differences between two groups were analyzed with a two-sided t test and between multiple groups by one-way ANOVA followed by a Tukey’s multiple comparison test using Prism 7 software. The level of statistical significance was set to a p value of less than 0.05. All values are given as mean ± standard deviation (SD) unless stated otherwise.

We used radio-TLC to determine the percentage of unchanged 6-bromo-7-[¹¹C]methylpurine and its glutathione conjugate S-(6-(7-[¹¹C]methylpurinyl))glutathione in different tissues and fluids of wild-type and Abcc1^(−/−) mice at 15 min after radiotracer injection (Table 2). In none of the investigated tissues and fluids unchanged 6-bromo-7-[¹¹C]methylpurine could be detected. In urine and bile, the majority of radioactivity was composed of the radiolabeled glutathione conjugate, while in plasma, brain, kidney, and liver approximately one-half of radioactivity was composed of the glutathione conjugate and the other half of unidentified radiolabeled species, which were not identical with 6-bromo-7-[¹¹C]methylpurine. In urine and plasma, the percentage of S-(6-(7-[¹¹C]methylpurinyl))glutathione was significantly lower in Abcc1^(−/−) mice than in wild-type mice.

To assess the influence of MRPs on tissue distribution and excretion of 6-bromo-7-[¹¹C]methylpurine-derived radioactivity, we performed PET scans in wild-type mice, which were pre-treated before the scan either with vehicle or with MK571. Whole-body PET images depicting the distribution of 6-bromo-7-[¹¹C]methylpurine-derived radioactivity over time in one vehicle-treated and one MK571-treated wild-type mouse are shown in Fig. 1. To obtain image-derived blood concentration-time curves, a region of interest was drawn around the left ventricle of the heart. A good correlation was found between the image-derived blood radioactivity concentrations and radioactivity measured in venous blood at the end of the PET scan (Pearson correlation coefficient r = 0.980, p < 0.0001, slope = 0.77 ± 0.04). Following pre-treatment with MK571, blood radioactivity concentrations were significantly increased and elimination of radioactivity from the blood was markedly delayed (Fig. 2a). In MK571-pre-treated animals, the AUC of the image-derived blood concentration-time curves was increased by 2.8-fold and k_{elimination,blood} was decreased by 4.9-fold, as compared with vehicle-treated animals. MK571 pre-treatment caused in parallel to changes in blood pharmacokinetics pronounced changes in the shape of the concentration-time curves in all investigated organs (Figs. 2 and 3). In brain, lung, kidney, and liver, k_elimination values were significantly decreased by 1.5-, 3.0-, 8.5-, and 4.3-fold, respectively (Fig. 5). In MK571-treated animals, organ-to-blood AUC ratios were significantly decreased, by 2.2-fold in the brain and by 1.5-fold in the liver, but not in the other investigated organs. We also estimated the rate constants for transfer of radioactivity from blood into the respective organs (k_uptake) with integration plot analysis (Fig. 6). K_uptake values in vehicle-treated wild-type mice were 0.749 ± 0.228 ml/min/g tissue in the kidneys and 0.211 ± 0.057 ml/min/g tissue in the liver, which corresponded to extraction ratios of 0.18 and 0.21, respectively (assuming renal and hepatic blood flow rates of 4.1 and 1.0 ml/min/g tissue) [18]. The only organ in which k_uptake was significantly changed after MK571 treatment were the kidneys, showing a decrease by 3.5-fold (Fig. 6a). Over the time course of the PET scan, radioactivity appeared to mainly undergo urinary excretion in vehicle-treated wild-type mice (Fig. 1). We estimated the rate constant for transfer of radioactivity from the kidneys into urine (k_urine) using integration plot analysis (Fig. S1). Pre-treatment with MK571 completely abolished urinary excretion of radioactivity (Fig. 3c) and decreased k_urine by 42.6-fold (Fig. 5f). We also found uptake of radioactivity in the intestine, but intestinal concentrations of radioactivity were considerably lower than in the urinary bladder. Intestinal AUCs were significantly (2.1-fold) higher in MK571-treated mice compared to vehicle-treated mice. The rate constant for transfer of radioactivity from the liver via bile into the intestine (k_bile) could not be estimated as the slopes of the linear parts of the integration plots were negative.

MK571 concentrations at approximately 120 min after i.p. administration of MK571 were 251 ± 58 μmol/L in the plasma and 544 ± 58 μmol/kg in the kidneys of wild-type mice.

To investigate which MRP subtypes were involved in the tissue distribution and urinary excretion of 6-bromo-7-[¹¹C]methylpurine-derived radioactivity, we examined Abcc1^(−/−) and Abcc4^(−/−) mice. Knockout of Abcc1 caused pronounced changes in blood, brain, and lung concentration-time curves, which were comparable to the changes seen in MK571-treated wild-type mice (Figs. 2 and Fig. 4). Blood AUCs in Abcc1^(−/−) mice were increased by 3.0-fold as compared with wild-type mice. Plasma-to-blood ratios of radioactivity, determined from the venous blood sample collected at the end of the PET scan, were decreased by 2.4-fold (wild-type: 1.46 ± 0.14, Abcc1^(−/−): 0.60 ± 0.04). Abcc1^(−/−) mice showed 3.6-, 9.4-, and 5.7-fold reduced k_elimination values in the blood, the brain, and the lungs, respectively (Fig. 5). K_elimination values were also significantly decreased in the kidneys and the liver of Abcc1^(−/−) mice relative to wild-type mice, but to a lesser degree than in the brain and the lungs. K_urine values were significantly decreased in Abcc1^(−/−) mice by 3.4-fold. In the liver of Abcc1^(−/−) mice, k_uptake values were significantly increased by 1.6-fold relative to wild-type mice (Fig. 6b).

Abcc4^(−/−) mice also showed significantly decreased k_elimination values in some of the investigated organs, i.e., brain (1.5-fold) and kidneys (1.4-fold) (Fig. 5). K_urine values of Abcc4^(−/−) mice were significantly lower (2.3-fold) than in wild-type mice. We also examined a group of Abcc4^(−/−) mice which was pre-treated with MK571. The effect of MK571 pre-treatment in different organs of Abcc4^(−/−) mice was comparable to the effect seen in wild-type mice (Figs. 5 and Fig. 6).

To assess the sensitivity of 6-bromo-7-[¹¹C]methylpurine PET to measure moderate changes in ABCC1 expression, we examined heterozygous Abcc1 knockout mice (Abcc1^(+/−)), which are expected to have a 50 % reduction in ABCC1 expression levels in different tissues. Heterozygous Abcc1^(+/−) mice showed, relative to wild-type mice, a much smaller but significant reduction in k_elimination values from the brain as compared with homozygous Abcc1^(−/−) mice (1.3-fold versus 9.4-fold reduction in k_{elimination,brain} relative to wild-type mice), but not in the other investigated organs (Fig. 5).

To determine whether nucleoside transporters play a role in the distribution of 6-bromo-7-[¹¹C]methylpurine-derived radioactivity from blood into brain, we examined wild-type mice pre-treated with the nucleoside transporter inhibitor dipyridamole. Brain concentration-time curves were similar in vehicle- and dipyridamole-treated wild-type mice (Fig. S2) and k_uptake,brain values were not significantly different between the two groups (k_uptake,brain vehicle: 0.113 ± 0.028 ml/min/g brain, dipyridamole: 0.120 ± 0.004 ml/min/g brain).