Identification of Metabolism and Excretion Differences of Procymidone

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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Identification of Metabolism and Excretion Differences of Procymidone between Rats and Humans Using Chimeric Mice: Implications for Differential Developmental Toxicity Jun Abe,* Yoshitaka Tomigahara, Hirokazu Tarui, Rie Omori, and Satoshi Kawamura Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd, 1-98, Kasugade-Naka 3-Chome, Konohana-Ku, Osaka 554-8558, Japan S Supporting Information *

ABSTRACT: A metabolite of procymidone, hydroxylated-PCM, causes rat-specific developmental toxicity due to higher exposure to it in rats than in rabbits or monkeys. When procymidone was administered to chimeric mice with rat or human hepatocytes, the plasma level of hydroxylated-PCM was higher than that of procymidone in rat chimeric mice, and the metabolic profile of procymidone in intact rats was well reproduced in rat chimeric mice. In human chimeric mice, the plasma level of hydroxylatedPCM was less, resulting in a much lower exposure. The main excretion route of hydroxylated-PCM-glucuronide was bile (the point that hydroxylated-PCM enters the enterohepatic circulation) in rat chimeric mice, and urine in human chimeric mice. These data suggest that humans, in contrast to rats, extensively form the glucuronide and excrete it in urine, as do rabbits and monkeys. Overall, procymidone’s potential for causing teratogenicity in humans must be low compared to that in rats. KEYWORDS: species differences, developmental toxicity, pharmacokinetics, metabolism, chimeric mouse, human, rat, procymidone

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of mechanism studies were conducted.9,11−15 In vitro receptor binding assay demonstrated that procymidone has antiandrogenic activity while PCM-CH2OH also has weak inhibitory activity.12,13 In an in vivo experiment,9 metabolism of procymidone in rats, rabbits, and monkeys was examined in detail. In the research, it was identified in rabbits and monkeys that hydroxylated-PCM is conjugated in liver, transferred to blood, and then rapidly excreted in urine, leaving the presence of only a low residual concentration of hydroxylated-PCM (Figure 2A). On the other hand, the kinetics of hydroxylated-PCM was identified to be different in rats, and the glucuronide is excreted in bile and deconjugated to hydroxylated-PCM in the gastrointestinal tract and reabsorbed, resulting in recycling through the enterohepatic circulation and increased exposure in rats (Figure 2B). In another in vivo experiment,14 PCM and hydroxylated-PCM were transferred to the fetus in rats, rabbits, and monkeys, but the amounts were no less than 6 times higher in rats than in rabbits and monkeys. Further, rat fetuses tended to accumulate hydroxylated-PCM upon repeated oral administration of procymidone. From these results, it was suggested that the species difference in the developmental toxicity of procymidone is mainly due to variation in the level of exposure to the causal substance, hydroxylated-PCM, and that this variation stems from interspecies differences in the biliary excretion route of the glucuronide. The risk of procymidone was assessed in humans in an in vitro metabolism study with human hepatocytes and an in vivo metabolism study with human chimeric mice.15 The in vitro study found that procymidone was largely converted to

INTRODUCTION Procymidone (Sumilex) is a commercially available fungicide with both protective and curative properties. It is used to control plant diseases, such as fruit rot (i.e., gray mold on fruits, vines, and vegetables) and Sclerotinia rot of kidney beans and vegetable crops.1−3 The metabolic pathways of procymidone in mammals are known (Figure 1).4−6 Procymidone is first hydrox ylated at the methyl group of the imide ring to form PCMCH2OH. This hydroxylated metabolite is then metabolized in one of two ways: the hydroxymethyl group is conjugated to glucuronide or further oxidized to the carboxylated metabolite. Both the glucuronide of hydroxylated procymidone and the carboxylated procymidone are more hydrophilic and thus more readily excreted than the hydroxylated procymidone.4−6 Because the imide (cyclic) compounds (i.e., procymidone, PCM-CH2OH, and PCM-COOH) and their imide linkage-cleaved metabolites (i.e., PCM-NH-COOH, PA-CH2OH, and PA-COOH, respectively) are in equilibrium and are convertible in mammalian body7 (see Figure 1), the sum of the amounts of both the cyclic and linkage-cleaved compounds is considered to be an appropriate index to evaluate the amounts of metabolites. Therefore, in this article, the following abbreviations are used for procymidone and its metabolites: PCM (represents the sum of procymidone and PCM-NH-COOH), hydroxylated-PCM (sum of PCMCH2OH and PA-CH2OH), carboxylated-PCM (sum of PCMCOOH and PA-COOH), and hydroxylated-PCM-glucuronide (sum of PCM-CH2OH-glucuronide and PA-CH2OH-glucuronide). Since procymidone is used worldwide, many toxicity studies have been conducted to evaluate its safety. External genital abnormality has been observed as a major toxicity of procymidone in male rats but not in rabbits and monkeys.8−11 To explain the species differences in procymidone developmental toxicity, a series © XXXX American Chemical Society

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November 22, 2017 January 5, 2018 January 9, 2018 January 9, 2018 DOI: 10.1021/acs.jafc.7b05463 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Proposed main metabolic pathways of procymidone in mammals. This figure was reproduced from a previous article.9 Copyright Pesticide Science Society of Japan. All rights reserved.

Figure 2. Flow diagrams of procymidone and its metabolites (A) in rats and (B) in rabbits and monkeys. This figure was reproduced from a previous article.9 Copyright Pesticide Science Society of Japan. All rights reserved. B

DOI: 10.1021/acs.jafc.7b05463 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article


Animal Treatment. Pharmacokinetics, Metabolism, and Excretion Studies (Groups 1−4). After the quarantine period of over 2 days, 14 C-procymidone in corn oil was administered as the single oral dose at 37.5 mg/kg/10 mL (Groups 1 and 2, low dose) or 62.5 mg/kg/10 mL (Groups 3 and 4, high dose). The low dose (37.5 mg/kg) was the lowest toxicologically effective dose to present the developmental toxicity as confirmed in our laboratory and the high dose (62.5 mg/kg) was the toxicological effect level showing decreased spontaneous activity.8 After administration, animals in Groups 1 and 3 were housed individually in polycarbonate cages (175 × 245 × 125 mm3) and animals in Groups 2 and 4 were housed individually in metabolic cages (MM-1STZ, Sugiyamagen Iriki, Tokyo, Japan) to allow separate collection of urine and feces. Animals had free access to the diet (CRF-1 containing vitamin C, sterilized by γ-ray irradiation, Oriental Yeast Co., Ltd., Tokyo, Japan) and distilled water throughout the study. Blood (approximately 75 μL) was collected using heparin-treated glass capillary from the orbital vein plexus at 1, 4, 8, and 12 h after administration from the animals in Groups 1 and 3, and at 2, 24, and 48 h after administration from the animals in Groups 2 and 4. Approximately 250 μL was collected in the same manner from the orbital vein plexus under anesthesia at 72 h after administration from all animals. Plasma was obtained by blood sample centrifugation. Urine and feces from each animal in Groups 2 and 4 were collected at 24, 48, and 72 h after administration, and the cages were washed with water to recover 14C (cage washing). The carcasses of the animals in Groups 2 and 4 were collected after the final blood samples of ∼250 μL were drawn. Biliary Excretion and Metabolism Studies (Group 5). During the period of quarantine and 2 day acclimation before administration, their general health was monitored and their body weights were measured at least once a day. The mice were anesthetized (by intraperitoneal administration of a mixture of xylazine hydrochloride and ketamine hydrochloride) and gall bladders were cannulated with polyethylene tubes. After recovery from anesthesia, 14C-procymidone was orally administered at 37.5 mg/kg to each mouse. Bile, urine, and feces were collected separately at 24 and 48 h after administration. Upon completion of the above, the gastrointestinal contents, bladder urine contents, and residual carcasses were collected. Sample Processing. Pharmacokinetics, Metabolism, and Excretion Studies (Groups 1−4). An aliquot (10 μL) of plasma was diluted with distilled water (40−90 μL) and an aliquot (20 μL) of each diluted sample was measured for radioactivity. The radioactivity in 10−50 mg of urine and 1000 mg of cage-wash was measured in duplicate to calculate the recovery. Radioactivity in the cage-wash was included in the urinary excretion radioactivity. The feces samples were weighed and extracted three times with 3-fold volume of acetonitrile. The radioactivity in the extracts (100 mg) and the unextractable residue (200 mg) was measured in duplicate. The carcasses were dissolved in about 100 mL of 3 mol/L potassium hydroxide, and the radioactivity in approximately 1 mL of this solution was measured in duplicate. Biliary Excretion and Metabolism Studies (Group 5). The radioactivity in aliquots (10−50 mg) of the excreted urine, bile, and urine in the bladder was measured in duplicate. The bladder urine radioactivity was included in the urinary excretion radioactivity. Feces and carcasses samples were processed in a similar manner as described above. The intestinal contents were weighed and homogenized in a 3-fold volume of distilled water. The radioactivity was measured in 200 mg of each homogenate. Radioanalysis. Radioactivity in the liquefied samples including organosoluble fractions, dosing formulation, urine, bile, fecal extract, and residual carcass was quantified by scintillation counting with a TriCarb 2500TR Liquid Scintillation Analyzer (PerkinElmer Inc., Waltham , MA) after mixing the samples with scintillation cocktail, Emulsifier Scin tillator Plus (PerkinElmer Inc.). The unextractable residues of the feces after extraction and the homogenates of the intestinal contents were taken separately, dried, and treated in a sample combustion apparatus (sample oxidizer: System 307, PerkinElmer Inc.), and their radioactivity measured by using a Tri-Carb 2500TR Liquid Scintillation Analyzer. Metabolites in Plasma, Bile, Urine, and Feces. Precoated silica gel 60 F254 chromatoplates (20 × 20 cm2, 0.25 or 0.5 mm layer

its glucuronide via hydroxylated-PCM and the relevant pathways of this conversion were similar in humans, rabbits, and monkeys. The in vivo study found that the main metabolite in the chimeric mice was hydroxylated-PCM-glucuronide, suggesting that the metabolic profile of procymidone is similar in humans, rabbits, and monkeys, but not in rats. Collectively, the evidence indicated that hydroxylated-PCM is the active developmental toxicant in rats, and the difference in the teratogenicity of procymidone between rats and other tested species resulted from a difference in the level of exposure to hydroxylated-PCM, which is excreted more slowly in rats owing to the enterohepatic recycling of hydroxylated-PCM in rats. An in vitro study and an in vivo study with human hepatocytes supported the conclusion that the metabolic profiles of procymidone in humans are similar to those in rabbits and monkeys but differ from those in rats, suggesting that procymidone is unlikely to be teratogenic in humans. To further assess the teratogenic risk associated with procymidone, the excretion route and rate of hydroxylated-PCM-glucuronide, which are the key factor of the mechanism of the toxicity to elucidate the extent of exposure to hydroxylated-PCM, must be clarified. In order to accomplish the objective to compare the metabolic profiles of procymidone in humans and rats, we planned to conduct in vivo experiments using the chimeric mice. We first tried to reveal that the metabolic profiles of procymidone between intact rats and rat chimeric mice are reflective, in order to evaluate whether the chimeric mice with rat hepatocytes are reflective of rats and they can be a useful model to investigate the metabolism and excretion of procymidone. Then, in order to confirm that the metabolic and excretion profiles of procymidone are similar among humans, rabbits, and monkeys but differ between rats and these other species, we compared the metabolic and pharmacokinetic profiles of procymidone, its metabolism in liver, and its biliary excretion between rat chimeric mice and human chimeric mice.

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MATERIALS AND METHODS

Chemicals. Phenyl-14C-labeled procymidone (specific radioactivity: 4.48 GBq/mmol, radiochemical purity > 99%, chemical purity > 98%) was synthesized in our laboratory as described previously.14,15 Unlabeled procymidone (chemical purity 99.2%) and its metabolites including PCM-NH-COOH (chemical purity 98.6%), PCM-CH2OH (chemical purity 99.9%), PCM-COOH (chemical purity 94.7%), PACH2OH (chemical purity 98.8%), and PA-COOH (chemical purity 96.9%) were also synthesized14,15 in our laboratory and used for metabolite identification. The structures of procymidone and metabolites are shown in Figure 1. The 14C-procymidone was diluted with unlabeled procymidone to achieve the appropriate specific radioactivity, and was dissolved in corn oil to prepare the desired dose of 37.5 or 62.5 mg/150 MBq/kg/10 mL. Other chemicals were of reagent grade. Animal Husbandry. Procedures involving the use and care of animals were approved by the IACUC of our laboratory and in accord with guidelines compliant with current Japanese law. Fourteen 10−14week-old female chimeric mice with rat hepatocytes and 15 10−14week-old female chimeric mice with human hepatocytes were supplied by PhoenixBio Co., Ltd. (Hiroshima, Japan). Animals were maintained in an air-conditioned room at 20−26 °C with an alternating 12 h light and 12 h dark cycle for at least 2 days prior to use for the study. Both rat chimeric mice and human chimeric mice were separated into five groups; Groups 1, 2, and 5 received the low dose of procymidone, and Groups 3 and 4 received the high dose. Groups 1−4 were used in the pharmacokinetics study, Groups 2 and 4 were used for the excretion study, and Group 5 was used for the biliary excretion study. All groups contained three rat chimeric mice or three human chimeric mice, except for Group 5 which had only two rat chimeric mice. C


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Table 2. Pharmacokinetics Parameters of 14C in Plasma after Single Oral Administration of 14C-Procymidone to Chimeric Mice with Rat or Human Hepatocytesa

thickness, Merck, Darmstadt, Germany) were used for thin layer chromatography (TLC). The solvent systems were toluene/ethyl formate/ formic acid (5/7/1, by volume). Samples were developed with the authentic standards of procymidone, PCM-NH-COOH, PCM-CH2OH, PCM-COOH, PA-CH2OH, and PA-COOH. Unlabeled standards and radioactivity on TLC plates were detected as described previously.16 The Rf values of the compounds and representative chromatograms are presented in our previous articles.4,7 Calculations. The amount of 14C was calculated from the weight of solution administered to each mouse. The maximal radioactivity and time to reach maximal radioactivity were used as values for the maximal concentration in plasma (Cmax) and the time to reach Cmax (Tmax). Area under the plasma concentration time curve (AUC) from 0−72 or 0−48 h (AUC0−72h or AUC0−48h) was calculated using the trapezoidal approximation method. The AUC after the last sampling point (AUC72h−∞) was calculated by monoexponential fitting to the data in the elimination phase curve, and the AUC from time 0 to infinity (AUC0h−∞) was the sum of AUC0−72h and AUC72h−∞.

(A) 37.5 mg/kg parameter Cmax [μg equiv/mL] Tmax [h] AUC0−72h [μg equiv·h/mL] AUC0h−∞ [μg equiv·h/mL]

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RESULTS Radioactivity Concentrations in Plasma. Low Dose (Groups 1 and 2). The results are shown in Table 1A, and the

a

(A) 37.5 mg/kg concentration [μg equiv of procymidone/mL plasma]

1 2 4 8 12 24 48 72

chimeric mice with rat hepatocytes 7.0 7.1 7.0 8.9 8.9 0.5 0.1 0.1

± ± ± ± ± ± ± ±

chimeric mice with human hepatocytes

1.70 1.24 0.87 0.91 0.93 0.08 0.02 0.03

4.9 14.3 7.9 7.4 4.6 2.9 0.6 0.2

± ± ± ± ± ± ± ±

2.04 5.30 2.49 2.29 2.16 0.84 0.29 0.07

(B) 62.5 mg/kg concentration [μg equiv of procymidone/mL plasma] time [h] 1 2 4 8 12 24 48 72 a

chimeric mice with rat hepatocytes 8.6 9.3 9.9 13.7 14.8 1.5 0.2 0.1

± ± ± ± ± ± ± ±

2.52 1.04 1.48 4.38 6.03 0.41 0.05 0.04

Chimeric mice with human hepatocytes 10.4 11.4 9.7 13.3 9.5 3.8 1.3 0.5

± ± ± ± ± ± ± ±

8.9 12 158 164 (B) 62.5 mg/kg

chimeric mice with human hepatocytes 14.3 2 184 185

parameter

chimeric mice with rat hepatocytes


Cmax [μg equiv/mL] Tmax [h] AUC0−72h [μg equiv·h/mL] AUC0h−∞ [μg equiv·h/mL]

14.8 12 259 271

13.3 8 290 292

Each value is the mean of three animals.

for radioactivity in plasma was 14.8 μg equiv/mL at 12 h after administration and the AUC0−72h and AUC0h−∞ of radioactivity were 259 and 271 μg equiv·h/mL, respectively. In the chimeric mice with human hepatocytes, they were 13.3 μg equiv/mL at 8 h after administration and 290 and 292 μg equiv·h/mL, respectively. The AUCs showed dose-related increase between low and high doses in both rat chimeric mice and human chimeric mice, although there was no clear difference in Cmax. Radioactivity Excretions in Urine and Feces. Low Dose (Group 2). In the chimeric mice with rat hepatocytes, the radioactivity excreted in urine and feces within 72 h after administration was 77.2% and 18.7% of the dose (total 95.9%), respectively, as shown in Table 3A. The radioactivity in the carcass was 0.5% of the dose and the total recovery ratio within 72 h after administration was thus 96.3%. In the chimeric mice with human hepatocytes, the radioactivity excreted in urine and feces within 72 h after administration was 86.4% and 10.7% of the dose (total 97.1%), respectively. The radioactivity in the carcass was 0.7% of the dose and the total recovery ratio for radioactivity within 72 h after administration was 97.7%. 14 C-procymidone was excreted rapidly and almost completely within 72 h after administration mostly in urine in both species. High Dose (Group 4). In the chimeric mice with rat hepatocytes, the radioactivity excreted in urine and feces within 72 h after administration was 84.7% and 11.3% of the dose (total 96.0%), respectively, as shown in Table 3B. The radioactivity in the carcass was 0.4% of the dose and the total recovery ratio for radioactivity within 72 h after administration was thus 96.5%. In the chimeric mice with human hepatocytes, the radioactivity excreted in urine and feces within 72 h after administration was 83.5% and 14.4% of the dose (total 97.9%), respectively. The radioactivity in the carcass was 0.6% of the dose and the total recovery ratio for radioactivity within 72 h after administration was 98.5%. Also at the high dose, 14C was excreted rapidly and completely, and there was no difference between the doses. Metabolites in Plasma. Low Dose (Groups 1 and 2). The concentrations of metabolites in plasma and the pharmacokinetics parameters of the metabolites are presented in Figure 3 and Table 4, respectively, for both low and high dose groups. Details of the metabolites’ quantification are shown in the Supporting Information. In chimeric mice with rat hepatocytes, the main radioactive components in plasma were PCM, hydroxylated-PCM, and carboxylated-PCM, with hydroxylated-PCM-glucuronide

Table 1. 14C Concentrations in Plasma after Single Oral Administration of 14C-Procymidone to Chimeric Mice with Rat or Human Hepatocytesa

time [h]


6.13 6.53 3.78 2.89 2.77 1.24 0.43 0.06

Data are presented as the mean value ± SD of three animals.

pharmacokinetics parameters are presented in Table 2A. In the chimeric mice with rat hepatocytes, the Cmax for radioactivity in plasma was 8.9 μg equiv/mL at 12 h after administration, and the AUC0−72h and AUC0h−∞ of radioactivity were 158 and 164 μg equiv·h/mL, respectively. In the chimeric mice with human hepatocytes, they were 14.3 μg equiv/mL at 2 h after administration, and 184 and 185 μg equiv·h/mL, respectively. There were no notable differences between species except the delayed Tmax observed in rat chimeric mice. High Dose (Groups 3 and 4). The results are shown in Table 1B, and the pharmacokinetics parameters are presented in Table 2B. In the chimeric mice with rat hepatocytes, the Cmax D


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Table 3. Cumulative 14C-Excretions in Urine and Feces after Single Oral Administration of 14C-Procymidone to Chimeric Mice with Rat or Human Hepatocytesa (A) 37.5 mg/kg excretion [% of dose] chimeric mice with rat hepatocytes time [h]

urine

feces

0−24 0−48 0−72 carcass total

72.1 ± 3.22 76.0 ± 3.68 77.2 ± 3.75

15.4 ± 4.16 18.1 ± 3.52 18.7 ± 3.81

77.2 ± 3.75

18.7 ± 3.82

chimeric mice with human hepatocytes total 87.5 ± 1.92 94.1 ± 1.41 95.9 ± 1.07 0.5 ± 0.06 96.3 ± 1.13 (B) 62.5 mg/kg

urine

feces

69.1 ± 4.85 83.4 ± 0.92 86.4 ± 0.45

8.0 ± 0.43 10.3 ± 0.41 10.7 ± 0.27

86.4 ± 0.45

10.7 ± 0.27

total 77.1 93.7 97.1 0.7 97.7

± ± ± ± ±

5.28 1.28 0.52 0.25 0.70

excretion [% of dose] chimeric mice with rat hepatocytes

a

time [h]

urine

feces

0−24 0−48 0−72 carcass total

76.6 ± 4.07 81.8 ± 3.92 84.7 ± 2.92

8.9 ± 1.43 11.1 ± 2.51 11.3 ± 2.50

84.7 ± 2.92

11.3 ± 2.50

chimeric mice with human hepatocytes total

urine

feces

± ± ± ± ±

61.2 ± 8.20 80.7 ± 9.15 83.5 ± 8.58

11.2 ± 9.08 14.1 ± 9.31 14.4 ± 9.40

83.5 ± 8.58

14.4 ± 9.40

85.5 92.9 96.0 0.4 96.5

3.53 2.34 1.00 0.19 0.91

total 72.4 94.8 97.9 0.6 98.5

± ± ± ± ±

7.49 0.22 0.83 0.02 0.82

Data are presented as the mean value ± standard deviation of three animals.

Cmax and AUC of PCM were comparable between rat chimeric mice and human chimeric mice, while AUC was larger and Tmax delayed for hydroxylated-PCM in rat chimeric mice when compared to those in human chimeric mice. High Dose (Groups 3 and 4). In both rat chimeric mice and human chimeric mice, the proportion of metabolites was similar between high dose and low dose groups. In the chimeric mice with rat hepatocytes, the Cmax and AUC0−72h were 4.80 μg equiv/mL at 1 h after administration and 48.3 μg equiv·h/mL, respectively, for PCM and 9.57 μg equiv/mL at 12 h after administration and 120.8 μg equiv·h/mL, respectively, for hydroxylatedPCM. In the chimeric mice with human hepatocytes, they were 3.01 μg equiv/mL at 2 h after administration and 44.3 μg equiv·h/mL, respectively, for PCM and 1.77 μg equiv/mL at 1 h after administration and 36.1 μg equiv·h/mL, respectively, for hydroxylated-PCM. Similar to the low dose results, the high dose pharmacokinetics parameters of PCM were comparable between the two chimeric mice, while the AUC and Cmax of hydroxylated-PCM were 3 and 5 times larger, respectively, in rat chimeric mice than in human chimeric mice. Also, the Tmax of hydroxylated-PCM was delayed compared to that of PCM in rat chimeric mice. When the results of low and high doses were compared, the Cmax of PCM and metabolites was almost similar between the doses in human chimeric mice despite the dose difference (Figure 3B and D). However, the AUC was almost doubled at high dose (Table 4), which is consistent to the difference of the dosed amount, 62.5 vs 37.5 mg/kg. These data can suggest that the rate of the intestinal absorption was plateaued at the high dose. The elimination of PCM and metabolites from 2 h toward 24 h was slower at high dose as shown in the graphs, which was considered to be another evidence of the continued absorption at the latter time points. Metabolites in Urine and Feces. Low Dose (Groups 1 and 2). The metabolite composition in excreta is presented in Table 5 for both the low and high dose groups. Details of the metabolite analysis are shown in the Supporting Information. In the chimeric mice with rat hepatocytes, the main metabolites were carboxylated-PCM (58.3% of the dose) followed by

Figure 3. Concentration of procymidone and its metabolites in plasma after single oral administration of 14C-procymidone to chimeric mice with rat or human hepatocytes. X axis: Time after administration [h]. Y axis: concentration [μg equiv of procymidone/mL]. Points are mean values from three animals.

present as a relatively minor metabolite. Cmax and AUC0−72h were 3.54 μg equiv/mL at 1 h after administration and 26.1 μg equiv·h/mL, respectively, for PCM and 2.95 μg equiv/mL at 8 h after administration and 47.4 μg equiv·h/mL, respectively, for hydroxylated-PCM. In the chimeric mice with human hepatocytes, the predominant metabolite in plasma was hydroxylatedPCM-glucuronide followed by carboxylated-PCM, PCM, and hydroxylated-PCM. Cmax and AUC0−72h were 3.55 μg equiv/mL at 2 h after administration and 22.2 μg equiv·h/mL, respectively, for PCM and 2.19 μg equiv/mL at 2 h after administration and 20.8 μg equiv·h/mL, respectively, for hydroxylated-PCM. E


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Table 4. Pharmacokinetics Parameters of PCM and Hydroxylated-PCM in Plasma after Single Oral Administration of 14CProcymidone to Rats and Chimeric Micea (A) 37.5 mg/kg

a

ratsb

chimeric mice with rat hepatocytes PCM

parameter

PCM

hydroxylated-PCM

Cmax [μg equiv/mL] Tmax [h] AUC0−72h [μg equiv·h/mL]

3.7 6 40.9

8.2 12 225.3

3.54 1 26.1 (B) 62.5 mg/kg


hydroxylated-PCM

PCM

hydroxylated-PCM

2.95 8 47.4

3.55 2 22.2

2.19 2 20.8

ratsb



parameter

PCM

hydroxylated-PCM

PCM

hydroxylated-PCM

PCM

hydroxylated-PCM

Cmax [μg equiv/mL] Tmax [h] AUC0−72h [μg equiv·h/mL]

6.8 4 89.9

10.4 12 332.1

4.80 1 48.3

9.57 12 120.8

3.01 2 44.3

1.77 1 36.1

Data are presented as the mean value of three animals. bReproduced from a previous article.9

(12.5% of the dose) in urine, and PCM (4.5% of the dose) in feces. In the chimeric mice with human hepatocytes, they were hydroxylated-PCM-glucuronide (37.8% of the dose) and carboxylated-PCM (38.4% of the dose) in urine and PCM (3.1% of the dose) in feces. The excretion patterns were the same at both the high and low doses. Biliary Excretion and Metabolism Studies (Group 5). Distribution and Excretion of Radioactivity. The radioactivity in urine, bile, feces, intestinal contents, and residual carcasses after single oral administration of 14C-procymidone is shown in Table 6. In the chimeric mice with rat hepatocytes, the radioactivity excreted in urine, bile, and feces was 29.4%, 24.8%, and 6.5% of the dose, respectively, and the total was 60.7% within 48 h after administration. The radioactivity in the intestinal contents and carcasses was 2.0% and 34.5% of the dose, respectively, and the total recovery within 48 h after administration was thus 97.2%. In the chimeric mice with human hepatocytes, the radioactivity excreted in urine, bile, and feces was 75.7%, 7.0%, and 2.6% of the dose, respectively, and the total was 85.4% within 48 h after administration. The radioactivity in the intestinal contents and carcass were 0.4% and 10.9% of the dose, respectively, and the total recovery within 48 h after administration was thus 96.7%. In human chimeric mice, main excretion route was urine. Metabolites in Urine, Bile, and Feces. The results of the metabolite analysis in urine, bile, and feces are presented in Table 7. In the chimeric mice with rat hepatocytes, the main metabolites were carboxylated-PCM (21.5% of the dose) and hydroxylated-PCM-glucuronide (4.3% of the dose) in urine and hydroxylated-PCM-glucuronide (19.7% of the dose) in bile. In the chimeric mice with human hepatocytes, they were hydroxylated-PCM-glucuronide (45.8% of the dose) and carboxylated-PCM (25.6% of the dose) in urine and hydroxylated-PCM-glucuronide (5.4% of the dose) in bile. The data showed a clear difference in routes of hydroxylated-PCMglucuronide excretion between rat chimeric mice and human chimeric mice (i.e., urine in human chimeric mice and bile in rat chimeric mice).

Table 5. Amounts of Metabolites in Urine and Feces within 72 h after Single Oral Administration of 14C-Procymidone to Chimeric Micea (A) 37.5 mg/kg excretion [% of dose] chimeric mice with rat hepatocytes metabolite PCM hydroxylated-PCM carboxylated-PCM hydroxylated-PCMglucuronide othersb unextractable total


urine

feces

total

urine

feces

total

0.5 3.9 58.3 10.4

6.0 3.1 1.9 0.5

6.5 7.0 60.2 10.9

0.4 3.5 42.2 37.4

0.5 0.9 1.7 1.0

0.8 4.3 43.9 38.3

4.1 0.8 4.9 NA 6.4 6.4 77.2 18.7 95.9 (B) 62.5 mg/kg

2.9 NA 86.3

0.5 6.3 10.7

3.4 6.3 97.1

excretion [% of dose] chimeric mice with rat hepatocytes metabolite PCM hydroxylated-PCM carboxylated-PCM hydroxylated-PCMglucuronide othersb unextractable total


urine

feces

total

urine

feces

total

0.7 4.6 62.4 12.5

4.5 1.5 0.5 0.5

5.2 6.1 62.8 13.1

0.5 3.4 38.4 37.8

3.1 2.0 0.9 0.9

3.6 5.5 39.4 38.7

4.5 NA 84.7

0.6 3.8 11.3

5.1 3.8 96.0

3.4 NA 83.5

0.7 6.6 14.3

4.1 6.6 97.9

a

Values were obtained from pooled samples of three animals. NA: Not applicable. bSum of the amounts of five or six unidentified metabolites.

hydroxylated-PCM-glucuronide (10.4% of the dose) in urine and PCM (6.0% of the dose) in feces. In the chimeric mice with human hepatocytes, they were hydroxylated-PCM-glucuronide (37.4% of the dose) and carboxylated-PCM (42.2% of the dose) in urine and carboxylated-PCM (only 1.7% of the dose) in feces. In summary, carboxylated-PCM was abundant in excreta of both rat chimeric mice and human chimeric mice, but urinary excretion of the glucuronide was considerable only in human chimeric mice. High Dose (Groups 3 and 4). In the chimeric mice with rat hepatocytes, the main metabolites were carboxylated-PCM (62.4% of the dose) and hydroxylated-PCM-glucuronide

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DISCUSSION Chimeric mice with human hepatocytes (humanized liver) are obtained by transplanting human hepatocytes into urokinasetype plasminogen activator-transgenic SCID mice (uPA/SCID mice). The uPA/SCID mice are immunodeficient and undergo liver failure. The transplanted human hepatocytes can then F


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Table 6. Cumulative 14C-Excretions in Urine, Bile, and Feces after Single Oral Administration of 14C-Procymidone to Chimeric Mice at 37.5 mg/kg excretion [% of dose] chimeric mice with rat hepatocytesa

a

chimeric mice with human hepatocytesb

time [h]

urine

bile

feces

total

urine

bile

feces

0−24 0−48 GI contents carcass total

14.2 29.4

9.5 24.8

3.7 6.5

46.6 ± 1.9 75.7 ± 10.9

4.8 ± 2.1 7.0 ± 1.7

1.6 ± 1.3 2.6 ± 1.1

29.4

24.8

6.5

27.5 60.7 2.0 34.5 97.2

75.7 ± 10.9

7.0 ± 1.7

2.6 ± 1.1

total 53.0 85.4 0.4 10.9 96.7

± ± ± ± ±

2.3 9.2 0.1 8.2 1.1

Data are the mean values for two animals. bData are the mean values ± SD for three animals.

Table 7. Amounts of Metabolites in Urine, Bile, and Feces within 48 h after Single Oral Administration of 14C-Procymidone to Chimeric Mice at 37.5 mg/kga excretion [% of dose] chimeric mice with rat hepatocytes

b

chimeric mice with human hepatocytesc

metabolite

urine

bile

feces

total

urine

bile

feces

total

PCM hydroxylated-PCM carboxylated-PCM hydroxylated-PCM-glucuronide othersd unextractable total

0.4 1.4 21.5 4.3 1.8 NA 29.4

0.2 0.6 3.5 19.7 0.9 NA 24.8

4.5 0.7 0.3

Identification of Metabolism and Excretion Differences of Procymidone

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