Article pubs.acs.org/JAFC
Metabolism of (Z)‑(1R,3R)‑Profluthrin in Rats Jun Abe,* Hirohisa Nagahori, Rie Omori, Kazuki Mikata, Motohiro Kurosawa, Yoshitaka Tomigahara, and Naohiko Isobe Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 1-98, 3-Chome, Kasugade-Naka, Konohana-Ku, Osaka 554-8558, Japan ABSTRACT: When [benzyl-α-14C]-labeled (Z)-(1R,3R)-profluthrin (2,3,5,6-tetrafluoro-4-methylbenzyl (Z)-(1R,3R)-2,2dimethyl-3-(prop-1-enyl) cyclopropanecarboxylate, a newly developed pyrethroid) was administered orally to rats at 1 mg/kg, around 70% was absorbed, metabolized, and mainly excreted into urine within 48 h. Radioactivity in plasma reached Cmax at 6− 8 h, and decreased (half-life; 37−52 h). A similar tendency was observed also in tissues. Absorption rate was slightly lower at high dose, while kinetics and distribution did not change. Eight metabolites were detected in urine and one in feces. Most of the 14C in feces was unabsorbed (Z)-(1R,3R)-profluthrin. The main metabolic reactions were ester cleavage, hydroxylation of the methyl group on the C4-position of the benzene ring, and its glucuronidation or oxidation to carboxylic acid. Oxidation of the geminal dimethyl on the cyclopropane-C2 to carboxylic acid, oxidation followed by hydration of the propenyl double bond, and ωoxidation to carboxylic acid and mercapturic acid conjugation of the benzyl alcohol were observed as minor reactions. KEYWORDS: metabolism, absorption, excretion, distribution, Fairytale, profluthrin, rats
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INTRODUCTION Synthetic pyrethroids are now widely used for pest control.1,2 Profluthrin, otherwise known as Fairytale, is a synthetic pyrethroid developed by Sumitomo Chemical Co., Ltd., and has high insecticidal efficacy against fabric pests (i.e., Tinea translucens, Tineola bisselliella, and Attagenus unicolor), sanitary pests (i.e., houseflies, mosquitoes, and cockroaches), and nuisance pests (i.e., moth flies and fruit flies). Since profluthrin possesses excellent insecticidal and repellent activity at ambient temperature, it has been commonly used for controlling fabric pests3 since around 2004. Profluthrin has 8 stereoisomers due to two chiral carbons and one double bond with E/Z isomerism in the structure. Among the 8 isomers, (Z)-(1R,3R)-profluthrin (2,3,5,6-tetrafluoro-4-methylbenzyl (Z)-(1R,3R)-2,2-dimethyl3-(prop-1-enyl) cyclopropanecarboxylate) (Figure 1), which
anticipated. Conventional synthetic pyrethroids such as tetramethrin, phenothrin, cyphenothrin, empenthrin, cypermethrin, and allethrin are easily cleaved at the ester linkage followed by oxidation and conjugation with glucuronic acid.1,4,5 The rate of ester cleavage is lower for the type II pyrethroids such as cyphenothrin and cypermethrin, which consist of secondary alcohol with cyano group on its α-position, than for the type I pyrethroids such as tetramethrin and phenothrin, which consist of primary alcohol,1,4,6 probably due to the difference of accessibility of esterase to the target site. It has also been revealed that the stereochemical conformations of 1and 3-positions of the cyclopropane ring can affect the efficiency of esterase, and the trans isomers generally cleave more easily than the cis isomers.7,8 Profluthrin does not have a cyano group on its α-position of the alcohol moiety and thus can be regarded as a type I pyrethroid. Since it has a unique alcohol moiety, it evokes our interest to clarify the metabolic features. Here we elucidate the metabolic fate (14C-excretion into urine, feces, expired air, and bile; 14C concentrations in tissues; isolation and identification of metabolites by spectroanalyses; and amounts of metabolites in excreta and tissues) of the most abundant isomer of profluthrin in the final product, (Z)(1R,3R)-profluthrin, in rats.
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Figure 1. Chemical structure and proton assignments of (Z)-(1R,3R)profluthrin for determination of the chemical structure by NMR spectrometry.
Chemicals. [Benzyl-α- 14 C]-labeled (Z)-(1R,3R)-profluthrin (shown in Figure 1) was chemically synthesized with the previously reported schemes9−11 in our laboratory, with a specific activity of 2.15 GBq/mmol. Chemical names, high-performance liquid chromatography (HPLC) retention times, and thin layer chromatography (TLC)
has R chirality for the 1-position of cyclopropane, trans- for 1and 3-substituents of cyclopropane (i.e., 3R), and the Zconformation for the propenyl group, is the main active ingredient of the final product. The structure of the alcohol moiety of profluthrin is quite different from that of most other pyrethroid compounds. Tefluthrin has a similar alcohol moiety, but its metabolic pathways have not been elucidated, thus the metabolic fate and the behavior of profluthrin in mammals cannot be clearly © XXXX American Chemical Society
MATERIALS AND METHODS
Received: July 6, 2015 Revised: September 9, 2015 Accepted: September 11, 2015
A
DOI: 10.1021/acs.jafc.5b03321 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Table 1. Chemical Names, HPLC Retention Times, and TLC Rf Values for (Z)-(1R,3R)-Profluthrin and Its Metabolites HPLC compound (Z)-(1R,3R)profluthrin M1 M2 M3 M4 M5 M6 M7 M8 a
tR [min]
chem name 2,3,5,6-tetrafluoro-4-methylbenzyl (Z)-(1R,3R)-2,2-dimethyl-3-(prop-1-enyl) cyclopropanecarboxylate 2,3,5,6-tetrafluoro-4-(hydroxymethyl)benzoic acid 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene N-acetyl-S-[2,3,5,6-tetrafluoro-4-(hydroxymethyl)benzyl]cysteine 2,3,5,6-tetrafluoro-4-methylbenzoic acid 2,3,5,6-tetrafluoro-4-methylbenzyl alcohol 2,3,5,6-tetrafluoro-4-(hydroxymethyl)benzyl (1R,3R)-2-(1,2-dihydroxypropyl)-3,3dimethylcyclopropanecarboxylate 2,3,5,6-tetrafluoro-4-(hydroxymethyl)benzyl (Z)-(1R,3R)-2-carboxy-2-methyl-3-(prop-1-enyl) cyclopropanecarboxylate 2,3,5,6-tetrafluoro-4-methylbenzyl (Z)-(1R,3R)-2-(2-carboxyvinyl)-3,3dimethylcyclopropanecarboxylate
TLC Rf value syst A a
syst B
50.8
ND
11.0 16.0 21.0 34.1 36.6 30.7, 31.9, 33.9, 35.3b 41.1
ND 0.82 ND 0.76 0.89 0.71
ND 0.43 ND 0.41 0.63 0.15
0.86
0.45
45.2
0.91
0.66
ND
ND: Not determined. bThis metabolite contains 4 isomers and can be separated under the HPLC conditions. Spectroscopic and Spectrometric Analysis. Chemical structures of purified metabolites were determined using NMR spectrometry and mass spectrometry together with high performance liquid chromatography (LC/MS). 1H NMR spectra were obtained with a Unity 400 plus spectrometer (Varian, Palo Alto, CA, USA) operating at 400 MHz with methanol-d4, DMSO-d6, or chloroform-d as the solvent. LC/MS spectra were obtained with an LTQ XL (Thermo Fisher Scientific, Waltham, MA, USA). Treatment of Animals. Male and female Crl:CD(SD) untreated rats at the age of 6 weeks and bile duct cannulated rats at the age of 8 weeks were purchased from Charles River Japan Inc. (Kanagawa, Japan) and maintained under constant environmental conditions: room temperature of 21−25 °C; relative humidity of 55 ± 15%; ventilation of 10 air exchanges per hour; and artificial lighting from 8:00 am to 8:00 pm. Animals had free access to pelleted diet and water throughout the study. All animal experiments were conducted in accordance with the Guidance for the Care and Use of Laboratory Animals in our laboratory, which corresponds to the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan). All tested animals except bile duct cannulated rats showed normal weight gain and no abnormal clinical symptoms during 7 days of quarantine and acclimation, and their body weights were 250−306 g for males and 177−215 g for females when dosed. Bile duct cannulated rats were dosed on the day of arrival. All animals were euthanized by bleeding from the abdominal artery under anesthesia. Preparation of Dosing Solutions. [Benzyl-α-14C]-labeled (Z)(1R,3R)-profluthrin was radiochemically diluted with unlabeled (Z)(1R,3R)-profluthrin with a specific activity of 6.51 MBq/mg for low dose and 108 kBq/mg for high dose. The test compound was dissolved in corn oil to give 1 and 60 mg/5 mL in the dosing solutions. The radiochemical purities of the 14C-labeled compound in the dosing formulations were analyzed by HPLC, and were confirmed to be 100% for all the dose groups. 14 C-Excretion Study. Male and female rats at 7 weeks old were given a single oral dose of [benzyl-α-14C]-labeled (Z)-(1R,3R)profluthrin at 1 and 60 mg/kg/5 mL of corn oil at 4 animals per group, and the animals were housed separately in glass metabolism cages (Metabolica CO2, Sugiyamagen Iriki, Tokyo, Japan) to collect urine, feces, and expired air. Urine and feces from each rat were collected at 6 and 12 h (urine only), and 1, 2, 3, 5, and 7 days after administration. Expired air was passed through an alkaline trap containing 10% NaOH solution for 1 day after administration to collect expired CO2 gas. Following the collection of feces and urine, each metabolism cage was washed with water to recover the remaining 14C (cage-wash). Radioactivity in the cage-wash was included in the urinary excretion. Biliary Excretion Study. Bile duct cannulated rats at 8 weeks old were given a single oral dose of [benzyl-α-14C]-labeled (Z)-(1R,3R)profluthrin at 1 mg/kg/5 mL of corn oil at 4 animals per group, and the animals were housed separately in glass metabolism cages. In addition to collecting urine and feces, bile was collected with a free
Rf values for authentic metabolite standards are shown in Table 1, and their structures are shown in Figure 5. Unlabeled (Z)-(1R,3R)profluthrin (purity: 99.3%) and 6 authentic standards of M1, M2, M4, M5, M6, and M8 were synthesized, and their structures were confirmed using nuclear magnetic resonance (NMR) and mass spectrometry (MS). The spectroscopic information on these 6 metabolites is as follows. M1: 1H NMR (DMSO-d6, δ in ppm) 4.597 (t, 2H, J = 2 Hz, PhCH2OH); APCI-MS (negative), m/z = 223 ([M − H]−). M2: 1H NMR (CD3OD, δ in ppm) 4.716 (t, 4H, J = 1 Hz, PhCH2OH); ESI-MS (positive), m/z = 218 ([M + Na + H]+). M4: 1H NMR (CD3OD, δ in ppm) 2.346 (t, 3H, J = 3 Hz, PhCH3); ESI-MS (negative), m/z = 207 ([M − 1]−). M5: 1H NMR (CD3OD, δ in ppm) 2.274 (t, 3H, J = 2 Hz, PhCH3), 4.687 (t, 2H, J = 2 Hz, PhCH2OH); ESI-MS (positive), m/z = 234 ([M + Na + H]+). M6: 1H NMR (CD3OD, δ in ppm) 1.111 (d, 3H, J = 6 Hz, CH(OH)CH3), 1.175 (m, 6H, C(CH3)2), 1.505 (m, 1H, CHCH(OH)), 1.602 (d, 1H, J = 6 Hz, CHCOO), 3.054 (m, 1H, CHCH(OH)CH(OH)), 3.630 (m, 1H, CH(OH)CH(OH)CH3), 4.718 (t, 2H, J = 1 Hz, PhCH2OH), 5.251 (q, 2H, J = 2, 5 Hz, PhCH2OCO); ESI-MS (negative), m/z = 379 ([M − H]−). M8: 1H NMR (CDCl3, δ in ppm) 1.219 (s 3H, CCH3), 1.342 (s, 3H, CCH3), 1.727 (d, 1H, J = 7 Hz, CHCOO), 3.313 (m, 1H, CHCHCH), 5.227 (s, 2H, PhCH2OCO), 5.918 (m, 1H, CHCHCH), 5.918 (m, 1H, CHCHCOOH); ESI-MS (negative), m/z = 359 ([M − H]−). The two other authentic standards, those of M3 and M7, were isolated and purified in our laboratory. Their spectroscopic information is described in Identification of Isolated Metabolites in Results. Other chemicals were reagent grade and were purchased from Nacalai Tesque (Japan) or Wako Pure Chemicals (Japan). Chromatographic Procedures. HPLC was conducted with a system consisting of an HPLC pump (L-2130, Hitachi, Tokyo, Japan), a UV detector (L-2400, Hitachi), and an online radioactivity monitor (Radiomatic 610TR, PerkinElmer, Waltham, MA, USA) fitted with an ODS column (Atlantis dC18, 5 μm, 4.6 mm i.d. × 150 mm, Waters, MA, USA). The mobile phases were A, 0.05% trifluoroacetic acid, and B, acetonitrile, and gradient conditions were % B = 5% (0 min), 5% (5 min), 25% (15 min), 25% (30 min), 100% (50 min), 100% (55 min) for metabolite analyses and % B = 10% (0 min), 60% (30 min) for purification and for purity analysis of metabolites, at a flow rate of 1 mL/min. Precoated silica gel 60 F254 chromatoplate (20 × 20 cm, 0.25 or 0.5 mm layer thickness, Merck, Darmstadt, Germany) was used for TLC. The solvent systems were A, ethyl acetate/water/formic acid (35:2:2, v/v/v), and B, toluene/ethyl acetate/formic acid (50:50:1, v/v/v). Unlabeled standards on TLC plates were detected by viewing under UV light (around 254 nm). Radioactivity on TLC plates was detected by scraping−LSC (liquid scintillation counter) method or autoradiography using an imaging plate processed with a fluoro-image analyzer (FLA-5000, Fujifilm, Tokyo, Japan). B
DOI: 10.1021/acs.jafc.5b03321 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 2. Cumulative 14C-excretion after single oral administration of [benzyl-α-14C]-labeled (Z)-(1R,3R)-profluthrin to rats. Points and bars are mean values and standard deviations for data from four rats. moving system adapted to the cages at 6 and 12 h (urine and bile), and 1, 2, and 3 days after administration. Plasma 14C Concentration Study. Male and female rats at 7 weeks old were given a single oral dose of [benzyl-α-14C]-labeled (Z)(1R,3R)-profluthrin at 1 and 60 mg/kg/5 mL of corn oil at 3 animals per group. Blood samples were collected from tail vein at 15 and 30 min, and 1, 2, 4, 6, 8, 12, 24, 48, 72, 120, and 168 h after the administration, and aliquoted into vials. The rest of the blood was centrifuged at 3000g for 10 min, and separated blood cells and plasma were transferred to vials. Maximum plasma concentration (Cmax, μg equiv of (Z)-(1R,3R)-profluthrin/g) and time to peak concentration (Tmax, h) were obtained from the actual measured values. Area under the concentration−time curve to 168 h (AUC0−168h, μg equiv of (Z)(1R,3R)-profluthrin·h/g) was determined by trapezoid method for the time interval of actual measurement of concentration, assuming the value of “0 μg/mL” as the concentration at time 0. Half-life of radioactivity was obtained by calculating ln 2/α of the slope (α) of a regression line fitted to the β phase of plasma concentration curve (48 to 168 h without including values less than the quantification limit), with the least-squares method. 14 C-Tissue Distribution Study. Male and female rats at 7 weeks old were given a single oral dose of [benzyl-α-14C]-labeled (Z)(1R,3R)-profluthrin at 1 and 60 mg/kg/5 mL of corn oil at 4 time points per dose and sex, and 3 or 4 animals each per group. The animals were euthanized after the defined time points, and the tissues were dissected for the radioanalysis. Duplicate aliquots of blood were collected, and the remaining blood was separated into blood cells and plasma by centrifugation at 2000g for 10 min. The residual carcasses were solubilized with 3 M KOH alkaline solution. The percentage of the 14C-distribution to tissue was calculated on the basis of the weight of each tissue dissected, except blood, bone, fat, muscle, and skin, for which the actual weight cannot be measured. Measurement of Radioactivity. Radioactivity in urine, cage wash, bile, 14CO2-trapped alkaline solution, solubilized carcass (decolorized by hydrogen peroxide), and organosoluble fraction was directly quantified with a Tri-Carb 2700TR liquid scintillation (LS)
analyzer (PerkinElmer) after mixing with scintillation cocktail, Emulsifier Scintillator Plus or Hionic Fluor (PerkinElmer). Feces (homogenized with 2 times weight of water), blood, plasma, blood cell, tissues, and unextractable fecal residues were combusted with a 308 sample oxidizer (PerkinElmer) prior to LS analysis. As the 14CO2 absorbent and the scintillator, 8 mL of Carbo Sorb and 8 mL of Permafluor E+ (PerkinElmer) were used, respectively. Isolation and Purification of Metabolites. Two metabolites (M3 and M7 in Table 1) were isolated from rat excreta by HPLC and TLC. Three female rats were given a single oral dose of [benzylα-14C]-labeled (Z)-(1R,3R)-profluthrin at 50 mg/kg/5 mL of corn oil and housed in metabolism cages. Urine sample was collected 1 day after administration, and a small part of the sample was applied to HPLC analysis. Fractions eluted at 22 and 42 min were collected, and these fractions were used as crude standards for the further purification procedures of M3 and M7, respectively. The rest of the urine sample was applied to preparative TLC analysis (solvent system A) after extraction with ethyl acetate or HCl-acidified ethyl acetate. Separated organosoluble fractions were collected, concentrated, and then applied to preparative HPLC for purification. The purified metabolites were identified by spectroanalyses, and then used as metabolite standards of M3 and M7. Metabolite Analysis. Urine, bile, and homogenate of feces in the excretion study and plasma and homogenate of liver and kidney in the tissue distribution study were analyzed with HPLC. Homogenates of feces, liver, and kidney and plasma samples were applied to HPLC after extraction with 3-fold volume of methanol and concentrated. Major unknown metabolites were isolated by HPLC and applied to enzyme hydrolysis under the following conditions: metabolites were incubated with β-glucuronidase/aryl sulfatase (from Helix pomatia, Merck) in 0.2 M acetic acid buffer (pH 5.0) with or without βglucuronidase inhibitor, D-saccharic acid 1,4-lactone, at 37 °C for 4 h. Assay was terminated by adding 2 times of acetonitrile, and the supernatant was applied to 2D TLC analysis (first D, solvent system A; second D, solvent system B). Consistency of metabolites was confirmed against authentic standards by 2 of the following 3 C
DOI: 10.1021/acs.jafc.5b03321 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 3. Cumulative 14C-excretion after single oral administration of [benzyl-α-14C]-labeled (Z)-(1R,3R)-profluthrin at the dose of 1 mg/kg to bile duct cannulated rats. Points and bars are mean values and standard deviations for data from four rats.
Figure 4. Plasma concentration after single oral administration of [benzyl-α-14C]-labeled (Z)-(1R,3R)-profluthrin to rats. methods: (1) comparing their retention time in radio-HPLC analysis against coeluted authentic standards, (2) cochromatography of TLC analysis, and (3) comparing MS and MS/MS spectra in LC/MS analysis against those of authentic standards.
thrin at a dose of 1 mg/kg (low dose) to bile duct cannulated rats are shown in Figure 3. 14C-Excretion was rapid in both males and females with average rates for 3 animals of 93.6% in males (41.0% in feces, 15.3% in urine, and 37.3% in bile), and 92.5% in females (52.4% in feces, 15.7% in urine, and 24.4% in bile) within 3 days after administration. Plasma 14C Concentration Study. The plasma−concentration curves after a single oral administration of [benzylα-14C]-labeled (Z)-(1R,3R)-profluthrin at low and high doses are shown in Figure 4, and the kinetic parameters are shown in Table 2. At both low and high dose, plasma 14C concentration was rapidly increased and reached Cmax at 6 to 8 h after the
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RESULTS C-Elimination into Urine, Feces, and Bile. 14CExcretion profiles into urine, feces, and expired air during 7 days after a single oral administration of [benzyl-α-14C]-labeled (Z)-(1R,3R)-profluthrin to normal rats are shown in Figure 2. 14 C was rapidly and almost completely excreted into urine and feces when dosed at 1 and 60 mg/kg. 14C-Excretion into expired air was not more than 1% at 1 day after the administration at low dose, thus it was not examined further nor at high dose since it could be regarded as negligible. 14CExcretion within 7 days after administration was 99.0% (feces, 32.8%; urine, 65.6%; and expired air, 0.5%) in male rats and 99.7% (feces, 27.3%; urine, 71.8%; and expired air, 0.6%) in female rats at 1 mg/kg groups. At high dose, 14C-excretion was 98.1% (feces, 37.8%; and urine, 60.2%) in male rats and 97.6% (feces, 38.2%; and urine, 59.4%) in female rats, which revealed that the ratio excreted through urine was approximately 10% lower at high dose. No marked sex-related differences were observed in the rate of 14C-elimination. The 14C-excretion rates within 2 days after a single oral administration of [benzyl-α-14C]-labeled (Z)-(1R,3R)-proflu14
Table 2. Pharmacokinetic Parameters of 14C in Plasma after Single Oral Administration of [Benzyl-α-14C]-Labeled (Z)(1R,3R)-Profluthrin to Male and Female Rats at 1 and 60 mg/kg 1 mg/kg
D
60 mg/kg
parameters
male
female
male
female
Tmax [h] Cmax [μg equiv of profluthrin/g of plasma] T1/2 [h] AUC [μg equiv of profluthrin·h/g of plasma]
6 0.148
8 0.091
6 3.54
6 3.87
52.2 2.82
37.3 1.97
46.2 88.9
51.8 104.8
DOI: 10.1021/acs.jafc.5b03321 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Table 3. Concentrations of Radioactivity in Tissues after Single Oral Administration of [Benzyl-α-14C]-Labeled (Z)-(1R,3R)Profluthrin to Male and Female Rats at 1 mg/kg μg equiv of (Z)-(1R,3R)-profluthrin/g of tissue (ppm) male
female
tissue
6h
12 h
48 h
168 h
8h
12 h
48 h
168 h
adrenal blood blood cell plasma bone bone marrow brain eye fat hair and skin heart kidney liver lung mandibular gland muscle ovary pancreas pituitary gland sciatic nerve spleen spinal cord testis thymus thyroid uterus cecum large intestine small intestine stomach cecum contents large intestine contents small intestine contents stomach contents
0.0572 0.0781 0.0409 0.115 0.0163 0.0351 0.0147 0.0212 0.0344 0.0950 0.0372 0.381 0.686 0.0525 0.0472 0.0234
0.0347 0.0347 0.0190 0.0495 0.0081 0.0192 0.0053 0.0139 0.0267 0.0466 0.0165 0.160 0.492 0.0261 0.0244 0.0124
0.0079 0.0056 0.0046 0.0064 0.0015 0.0039 0.0012 0.0022 0.0067 0.0083 0.0025 0.0179 0.155 0.0056 0.0030 0.0028
0.0024 0.0016 0.0025 0.0010 0.0007 0.0007
