Pharmacokinetics, excretion balance, and tissue distribution of [14C]-labeled glycolipids and long chain fatty acids from Dacryopinax spathularia in rats

Food and Chemical Toxicology 109 (2017) 552e568

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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Pharmacokinetics, excretion balance, and tissue distribution of [14C]labeled glycolipids and long chain fatty acids from Dacryopinax spathularia in rats Jens Bitzer a, Thomas Henkel a, Andrey I. Nikiforov b, *, Marisa O. Rihner b, Jennifer A. Thomas c a b c

IMD Natural Solutions GmbH, Dortmund, Germany Toxicology Regulatory Services, Charlottesville, VA, USA Charles River Laboratories Ashland, LLC, Ashland, OH, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 April 2017 Received in revised form 21 August 2017 Accepted 23 August 2017 Available online 26 August 2017

The pharmacokinetics, excretion balance, and tissue distribution of [14C]-labeled glycolipids from Dacryopinax spathularia (herein referred to as “AM-1”) and [14C]-LCFA equivalents following single or repeated administration to Sprague Dawley rats were evaluated to support the safety assessment of these naturally derived jelly mushroom glycolipids for use as a food ingredient. Rats received equimolar doses of either [14C]-AM-1 or [14C]-LCFA via oral or intravenous administration followed by collection of biological samples at specified intervals. Approximately 88%e101% of the administered dose was recovered in expired air, urine, feces, and carcass following single or repeated oral administration of [14C]-AM-1 at 100 mg/kg or equimolar doses of [14C]-LCFA at 46 mg/kg. Cmax and AUClast for [14C]-AM-1and [14C]-LCFA-equivalents-derived radioactivity detected by quantitative whole body autoradiography was highest in the tissues of the GI tract, as expected following oral administration. The remaining tissues had low concentrations of test article equivalents relative to the administered dose and no target tissues for residence or accumulation were identified. AM-1 and LCFA are poorly absorbed by the oral route and are primarily eliminated in the feces without absorption. Oral bioavailability of both AM-1 and LCFA including their metabolites is low at approximately 11%. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Jelly mushroom glycolipids Natural preservative Bioavailability Long chain fatty acids

1. Introduction AM-1 represents glycolipids from Dacryopinax spathularia (Schwein.) (Martin, 1948) belonging to the phylum Basidiomycota. This edible jelly mushroom, also referred to as Cantharellus spathularius (Schwein.) (Schweinitz, 1832), is commonly known as “sweet osmanthus ear” mushroom in China. It is found and used in

Abbreviations: AUC, area under the concentration versus time curve; BLQ, below the quantitation limit; CMC, carboxymethylcellulose; Cmax, peak concentration; DN, dose normalized; iv, intravenous; LCFA, long chain fatty acids; LCMS, high performance liquid chromatography coupled with mass spectrometry; LLOQ, lower limit of quantitation; LSC, liquid scintillation counting; MW, molecular weight; QWBA, quantitative whole body autoradiography; SCFA, short chain fatty acids; Tmax, time of Cmax occurrence; T1/2, elimination half-life. * Corresponding author. Toxicology Regulatory Services, 154 Hansen Rd., Suite 201, Charlottesville, VA 22911, USA. E-mail address: [email protected] (A.I. Nikiforov). http://dx.doi.org/10.1016/j.fct.2017.08.038 0278-6915/© 2017 Elsevier Ltd. All rights reserved.

culinary applications in many tropical and subtropical regions of the world. The glycolipids are obtained as a mixture (hereinafter referred to as “AM-1”) via natural fermentation of the mushroom. The components of AM-1 are structurally closely related glycolipid congeners; the three major components are depicted in Fig. 1A, and the remaining material is comprised of congeneric compounds of the parent components. AM-1 is proposed for use as a food ingredient with antimicrobial/preservative properties preventing spoilage and extending shelf-life of foods and beverages intended for human consumption. Thus, a series of studies and scientific assessments have been conducted to evaluate the safety of AM-1 after oral ingestion (Bitzer et al., 2017a,b). The present study was performed to determine the pharmacokinetics, routes of elimination, mass balance, and tissue distribution of [14C]-labeled residues of AM-1 and its major hydrolysis product LCFA. Information about metabolism and pharmacokinetics are significant endpoints in assessing the safety of direct food additives, providing insight into possible mechanisms of

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Fig. 1. Representative structure diagrams for main components of jelly mushroom glycolipids (AM-1) (A) and LCFA (B) mixtures.

toxicity and aiding in the design and evaluation of results from other toxicity studies (FDA, 1993). A demonstration of low systemic exposure after repeated oral ingestion is a desirable attribute for a material such as AM-1, with potential application as an antimicrobial/preservative ingredient in foods and beverages. Based on the results of previous in vitro experiments in simulated gastric fluid (IMD Natural Solutions, 2013; unpublished data on file) and in vivo pharmacokinetics studies in rats with [13C]-AM-1 (Husen et al., 2013, 2014), it was hypothesized that following ingestion, AM-1 passes mostly unchanged to the lower GI tract where predominantly its ester linkages and, partially, its glycosidic linkages are prone to hydrolysis by microflora in the lower intestine to its components glucose, xylose, acetic acid, isovaleric acid, and long chain fatty acid (LCFA) molecules (Fig. 2). Since it was anticipated that its minor components, glucose, xylose, acetic acid, and isovaleric acid would be further metabolized rapidly and incorporated for normal physiological functions, the major hydrolysis component of AM-1, i.e. LCFA (Fig. 1B), was studied in parallel to AM-1. This approach allowed the pharmacokinetics, excretion

balance, and tissue distribution experiments to elucidate the ultimate fate of the primary molecule of interest, AM-1, by also studying its major hydrolysis product, LCFA. The majority of the glycolipids and their hydrolysis products were expected to be excreted within 24 h via the feces. This hypothesis was supported by the finding that in simulated gastric fluid at pH 2.0 or pH 3.2 no degradation of AM-1 was found after 1 h. After 8 h incubation time, a minor loss of ca. 5% of the acetyl and isovaleryl esters was found by LCMS analysis, while no deglycosylation was observed (IMD Natural Solutions, 2013; unpublished data on file). The in vivo study using [13C]-AM-1 in Sprague-Dawley rats revealed that bioavailability of the glycolipids is very low (0.01% based on analysis of the three main components shown in Fig. 1A) (Husen et al., 2013). The majority of the glycolipids (>90%) were excreted via the feces within 24 h, where LCMS analysis found both intact glycolipids and, to a similar extent, the hydrolysis product LCFA or corresponding metabolites obtained by oxidation, dehydrogenation or loss of acyl groups. Minor amounts of components were found in the GI tract (ca. 9%) and the carcass (<1%) (Husen et al., 2014).

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Fig. 2. Proposed degradation of jelly mushroom glycolipids (AM-1) to LCFA. First (steps a and b), sequential hydrolysis of ester groups occurs. Deglycosylation (step c) towards the free fatty acids is only observed when heated or by microbial degradation.

2. Materials and methods 2.1. General study details and design The present study was conducted at Charles River Laboratories (Ashland, Ohio) during November 2015 to April 2016 (Study No. 294501). The study was performed in compliance with the U.S. Food and Drug Administration (FDA) Good Laboratory Practice Regulations (21 CFR Part 58) (FDA, 1987) and the study protocol was designed in general accordance with U.S. FDA Redbook II Guidelines, Toxicological Principles for the Safety Assessment of Food Ingredients, Chapter V B, Metabolism and Pharmacokinetic Studies (FDA, 1993). All recommendations concerning the design and analysis of metabolism and pharmacokinetic studies as outlined in this guidance were considered in the design of the study, including details of the test compound, test species, route of administration, dosing regimen, sampling, and analysis of data. 2.2. Test article, vehicle, and dose formulations Non-radiolabeled test articles, AM-1 and LCFA were supplied as white or off-white, crystalline solids for preparation of both radiolabeled and non-radiolabeled dosing. The radiolabeled test article, [14C]-AM-1 was prepared via fermentation, using [U-14C]-glucose as feedstock, that resulted in a final glycolipid product that was a mixture of 3 main components (see representative structure diagrams in Fig. 1A) but potentially more than 30 closely related molecules, with 14C positioned uniformly throughout the molecule. Briefly, fermentation of the fungal producer strain Dacryopinax spathularia MUCL53181 was performed in 250 ml shake flasks using a nutrient medium consisting of glucose and yeast extract. Fermentation and downstream processes followed the protocol described by Stadler et al. (2012). Starting from 80 mCi of [14C]-glucose, 18.8 mCi [14C]-AM-1 was obtained with specific activity of 5.94 mCi/g, corresponding to a yield of 23.5% based on radioactivity. [14C]-LCFA was prepared from the [14C]-glycolipid product by acid hydrolysis in vitro and purification. For this, 11.6 mCi [14C]-AM-1 (ca. 2.16 g with specific activity

5.36 mCi/g) were dissolved in 108 ml of a solution of acetonitrile and 2 M hydrochloric acid (7:3; v/v) and stirred under heating (reflux) for 2 h. The mixture was allowed to cool down and the organic solvent was removed under reduced pressure in a rotary evaporator at 50
C. The aqueous residue was diluted with demineralized water to a total volume of 108 ml and stored at 6
C for 12 h. The crude LCFA was recovered as precipitate by centrifugation at 4
C and re-suspended twice in 108 ml demineralized water by thorough mixing, each followed by storage at 6
C for 4 h and recovery by centrifugation. Drying by lyophilization yielded 2.6 mCi of LCFA (ca. 0.395 g with a specific activity of 6.6 mCi/g). Representative structure diagrams for LCFA are given in Fig. 1B. For radiolabeled oral dosing formulations, non-radiolabeled AM-1 or LCFA was weighed and transferred into a mortar with an appropriate volume of 0.5% methylcellulose (MC) in physiological saline (vehicle). [14C]-AM-1 or [14C]-LCFA was weighed and transferred into the same mortar using additional vehicle. Using a pestle, a fine suspension was created in the mortar. The suspension was quantitatively transferred into a vessel containing vehicle while stirring on a magnetic stir plate. The container was filled to the calibrated volume using vehicle and the formulation sonicated for 15 min intervals at room temperature and vortex-mixed following each sonication as appropriate until all solids were visibly dissolved. The formulation was allowed to stir overnight at refrigerated conditions (approximately 2
C to 8
C). On the day of dosing, the formulation was removed from refrigerated storage and placed onto a magnetic stir plate to stir while acclimating to room temperature for at least 30 min. Non-radiolabeled formulations of AM-1 and LCFA for use in the repeated dosing phases were prepared in a similar manner as described above, with the omission of the [14C]-labeled test article. Dose formulations were divided into individual aliquots for each day of dosing and, with the exception of the day 0 aliquots, were placed on a magnetic stir plate in refrigerator storage (approximately 2
C to 8
C) until use. Each formulation was removed from refrigerator (2
C to 8
C) storage and allowed to stir on a magnetic stir plate at room temperature for approximately 30 min prior to use.

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For intravenous (iv) dosing formulations, [14C]-AM-1 or [14C]LCFA was weighed into a small septum-sealable glass vial. DMSO (10% of the final volume) was added to the same glass vial and the contents stirred until dissolved. While stirring, PEG400 was added to the vial to bring the suspension to the desired volume. All formulations were stirred throughout the dosing period. Unused formulations were stored frozen at approximately 20
C, covered with aluminum foil, when not in use. The concentration of radiolabeled AM-1 or LCFA dosing formulations was confirmed by liquid scintillation counting (LSC) prior to and post-dosing. The pre- and post-dosing analyses established homogeneity of the solutions as demonstrated by the low %CV across triplicate analyses at each interval for each formulation (data not shown). Radio-HPLC analyses of the dosing solutions performed prior to and after completion of dosing demonstrated that there were no changes to the characterization of the test article over the time course of dosing; as determined by the visual similarity of the chromatograms prior to and after dosing. Radio-HPLC analyses were performed using an Agilent 1200 series HPLC system (Agilent Technologies, Inc.; Santa Clara, CA). Radioactivity in the effluent was monitored using an IN/US b-Ram Model 4B Radio-HPLC detector (IN/US Systems, Inc.; Tampa, FL). Prior to entering the radioactive flow detector, the column effluent was mixed with FlowLogic™ U scintillation cocktail (IN/US Systems, Inc.). Radiochromatograms were processed using the Laura Lite™ software package. Aliquots of the non-radiolabeled formulations were analyzed for confirmation of the AM-1 and LCFA concentration using validated LCMS methods (INS; unpublished data on file). Mean actual oral doses of [14C]-AM-1 or [14C]-LCFA (% of target) for the mass balance groups are presented in Table 1. 2.3. Animal receipt, acclimation and husbandry Sprague Dawley (Crl:CD(SD)) rats were received from Charles River Laboratories Inc. (Raleigh, NC) and used as the test system on this study. The rat is one of the preferred species chosen to assess food safety in the applicable regulatory guidelines (FDA, 1993). Each rat was inspected by a qualified technician upon receipt, judged to be in good health, and immediately placed in acclimation for approximately 1 week. Animals were weighed and uniquely

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identified by a metal ear tag displaying a permanent animal number. During the acclimation period and the biological phases of the study, each rat was observed twice daily for changes in general appearance or behavior. All animals were housed in a study-dedicated, environmentally controlled room, individually in clean, suspended, wire-mesh cages elevated above cage-board, which was changed at least 3 times each week. Enrichment devices were provided to each animal for environmental enrichment and to aid in maintaining the animals' oral health. Enrichment devices were not used in glass metabolism cages because the debris from the use of the device interferes with the analysis of the feces. The facilities at Charles River are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (block) and reverse osmosis-treated water was offered ad libitum during acclimation and the biological phase of the study. Controls were set to maintain an average daily temperature of 71
F ± 5
F (22
C ± 3
C) and an average daily relative humidity of 50% ± 20%. Timers were set to provide a daily photoperiod of 12 h fluorescent light followed by 12 h of darkness. The ventilation rate was set at a minimum of 10 air changes per hour, 100% fresh air. Prior to dose administration, animals were weighed and assigned to study groups at random using a computer program. On overview of the study design and group assignment is provided in Table 1. 2.4. Administration of test articles and sample collection For all study phases, each group of animals received equimolar doses of either [14C]-AM-1 or [14C]-LCFA via oral or iv administration followed by collection of biological samples at specified intervals. Because the test articles were uniformly radiolabeled with [14C] and based on their respective molecular weights (MW), equimolar doses of AM-1 (average MW ¼ 985) and LCFA (average MW ¼ 455) were calculated for all Groups (Table 1). For all phases, animals were weighed on the day of radiolabeled dosing. Animals in Group 9, Group 10, Group 11, and Group 12 (repeated dose phases) were also weighed on Day 0 and Day 7 for calculation of non-radiolabeled doses. Based on these weights, the dose to be administered was calculated on a mg/kg body weight

Table 1 Experimental design and group assignment during a pharmacokinetics, excretion balance, and tissue distribution study of [14C]-AM-1 and [14C]-LCFA in Sprague Dawley rats. Group Route Test Substance

MW (g/ mol)

Dose Concentration (mg/mL)

Pharmacokinetics 1 Oral AM-1 985 10 2 Oral LCFA 455 4.6 3 IV AM-1 985 5 4 IV LCFA 455 2.3 Single Dose e Excretion Balance 5 Oral AM-1 985 10 6 Oral LCFA 455 4.6 Single Dose e QWBA 7 Oral AM-1 985 10 8 Oral LCFA 455 4.6 Repeated Dose e Excretion Balance 9 Oral AM-1 985 10 10 Oral LCFA 455 4.6 Repeated Dose - QWBA 11 Oral AM-1 985 10 12 Oral LCFA 455 4.6

Target Dosage Level (mg/kg bw)a

Target Dosage Level (mmol/kg bw)

Dosage Volume (mL/kg bw)

Approximate Radioactivity (mCi/kg)b

Number of Animals

Sample Collection

100 46 10 4.6

0.10 0.10 0.010 0.010

10 10 2 2

100 100 60 24

9M/9F 9M/9F 9M/9F 9M/9F

Blood

100 46

0.10 0.10

10 10

100 100

3M/3F 3M/3F

Urine, Feces, Expired Air

100 46

0.10 0.10

10 10

100 100

7M/7F 7M/7F

Blood, Carcass for QWBA

100 46

0.10 0.10

10 10

100 100

3M/3F 3M/3F

Urine, Feces, Expired Air

100 46

0.10 0.10

10 10

100 100

7M/7F 7M/7F

Blood, Carcass for QWBA

QWBA ¼ quantitative whole body autoradiography. a Actual doses administered in mass balance phase (Groups 5, 6, 9, 10) ranged from 99% to 101% of target mg/kg dosage level. b Actual doses administered in mass balance phase (Groups 5, 6, 9, 10) ranged from 76% to 97% of target mCi/kg radioactivity.

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basis using a dosage volume of 10 ml/kg for the oral route and 2 ml/ kg for the iv route. Animals were approximately 8e10 weeks of age and weighed between 218 g and 360 g (males) and between 209 g and 237 g (females) at the time of radiolabeled dosing. For Groups 5, 6, 9, and 10, quantitation of the radiolabeled doses administered was determined by weighing each dosing syringe before and after delivery of the dose. Following dosing, Group 5, 6, 9, and 10 animals were placed into glass metabolism cages for separate collection of expired air, urine, and feces. All other animals were returned to their home cages following dosing. The pharmacokinetic phase consisted of 4 dose groups of 9 male and 9 female rats each. Group 1 and Group 2 received a single oral dose of either [14C]-AM-1 at 100 mg/kg or [14C]-LCFA at 46 mg/kg and a target radioactivity of 100 mCi/kg for both groups. Group 3 and Group 4 received a single iv dose of either [14C]-AM-1 at 10 mg/ kg or [14C]-LCFA at 4.6 mg/kg and a target radioactivity of 60 mCi/kg and 24 mCi/kg, respectively. Following dosing, blood samples were collected from 3 animals/sex rotating among 3 sub-groups at select time points through 72 h post-dosing. Blood samples were collected from the jugular vein into tubes containing K3EDTA as the anticoagulant and processed to plasma for analysis of total radioactivity by LSC. Following the final blood collection, animals were euthanized by CO2 inhalation and discarded. For the single dose - excretion balance phase, 2 dose groups consisting of 3 male and 3 female rats received a single oral dose of either [14C]-AM-1 at 100 mg/kg or [14C]-LCFA at 46 mg/kg (Group 5 and Group 6, respectively) and a target radioactivity of 100 mCi/kg for both groups. For the repeated dose - excretion balance phase, 2 dose groups consisting of 3 male and 3 female rats received a single daily oral dose of either AM-1 at 100 mg/kg or LCFA at 46 mg/kg for 14 consecutive days followed by a single oral dose of [14C]-AM-1 at 100 mg/kg or [14C]-LCFA at 46 mg/kg (Group 9 and Group 10, respectively) and a target radioactivity of 100 mCi/kg for both groups. Following dosing, animals were placed into individual glass metabolism cages for separate collection of expired air through 78 h post-dosing and urine and feces through 168 h post-dosing. The expired air from each metabolism unit was drawn through a trap containing approximately 120 ml of Carbo-Sorb E (PerkinElmer Life and Analytical Sciences) and ethylene glycol (2:1, v/v) to trap CO2 and through an activated carbon trap for volatile organic compounds. Urine and feces were collected (on ice) periodically throughout 168 h post-dosing. Animals were euthanized by CO2 inhalation following the final excreta collection and carcasses retained and stored frozen at approximately 20
C for possible analysis. Expired air, activated carbon, urine, feces, cage rinse, cage wash, and select carcass samples were analyzed for total radioactivity by LSC (details discussed below). For the single dose - quantitative whole body autoradiography (QWBA) phase, 2 dose groups consisting of 7 male and 7 female rats received a single oral dose of either [14C]-AM-1 at 100 mg/kg or [14C]-LCFA at 46 mg/kg (Group 7 and Group 8, respectively) and a target radioactivity of 100 mCi/kg for both groups. For the repeated dose - QWBA phase, 2 dose groups consisting of 7 male and 7 female rats received a single daily oral dose either AM-1 at 100 mg/kg or LCFA at 46 mg/kg for 14 consecutive days followed by a single oral dose of [14C]-AM-1 at 100 mg/kg or [14C]-LCFA at 46 mg/kg (Group 11 and Group 12, respectively) and a target radioactivity of 100 mCi/kg for both groups. At approximately 1, 2, 4, 8, 24, 48, and 168 h post-dosing, 1 animal/sex/QWBA group were anesthetized with isoflurane and a whole blood sample of approximately 4 ml was collected via cardiac puncture into tubes containing K3EDTA as the anticoagulant. Following blood collection, animals were euthanized by CO2 inhalation and carcasses frozen in a dry ice/ hexane bath for processing by QWBA.

2.5. Sample processing and analytical procedures 2.5.1. Pharmacokinetic analysis Pharmacokinetic analysis was conducted using Phoenix WinNonlin v 6.3 software. The individual and/or composite concentration data were used to calculate standard pharmacokinetic parameters such as area under the concentration versus time curve (AUClast, AUCinf), peak concentration (Cmax) and the time of its occurrence (Tmax), and elimination half-life (T1/2). Values above the upper limit of quantitation were set at the upper quantitation limit for pharmacokinetic analysis; values below the lower limit of quantitation were set to zero. Non-compartmental analysis was employed with linear trapezoidal summation for the AUC calculations. 2.5.2. Sample combustion and liquid scintillation counting Radioactivity was determined using a Beckman Model LS 6500 liquid scintillation counter (Beckman Coulter Instruments, Inc.; Fullerton, CA). Plasma samples from PK and QWBA animals were weighed into scintillation vials and mixed with an appropriate scintillation cocktail for LSC analysis. Expired air collections were weighed into scintillation vials and mixed with an appropriate scintillation cocktail and Triton X-100 for LSC analysis. Whole blood samples were weighed for combustion and LSC analysis. Fecal samples were homogenized by addition of deionized water (DI water) at an approximate ratio of 1 part DI water to 2 parts feces (w/ w) and use of a stainless steel spatula, then weighed for combustion and LSC analysis. Urine, cage rinse, and cage wash samples were weighed into scintillation vials and mixed with an appropriate scintillation cocktail for LSC analysis. Each activated carbon layer of the trap was divided into approximately equal aliquots for combustion and LSC analysis. Carcass samples were chopped using an autopsy knife, ground using an LEM™ meat grinder, and homogenates were weighed for combustion and LSC analysis. Aliquots of whole blood, fecal homogenates, carcass, and activated carbon layer of the trap were combusted prior to LSC analysis in a Harvey Biological Materials Oxidizer Model OX-500 or OX-501 (R. J. Harvey Instrument Corporation; Hillsdale, NJ). The released 14 CO2 was trapped in 15 ml of Permafluor and Eþ:Carbosorb E (2:1, v/v) liquid scintillation cocktail and CO2 absorber (PerkinElmer Life Analytical Sciences; Boston, MA). 2.5.3. Quantitative whole body autoradiography Frozen carcasses were individually set in a mold, submerged in 5% (w/v) low viscosity carboxymethylcellulose (CMC), and embedded by placing the stage (mold) in a dry ice/hexane bath. Before sectioning, one-fourth inch holes were drilled into the sample blocks and filled with QC standards. Calibration and QC standards for QWBA were prepared in CMC using [14C]-glucose. Sagittal, 30-mm thick sections of the CMC-embedded rat carcasses, including QC standards, and of the CMC-embedded calibration standards were sectioned using a Vibratome 9800 Cryomicrotome (Vibratome; St. Louis, MO). Whole body sections were placed in a cassette with a section containing CMC calibration standards and in direct contact with phosphor imaging plates (IP), BAS-SR 2025 (FujiFilm; Tokyo, Japan). The IPs were exposed for approximately 4 days while stored at room temperature in a leadshielded box to minimize background radiation. Digitized phosphor images (autoradioluminograms) were generated using a GE Healthcare Typhoon 7000 Phosphor Imager (Raytest GmbH, Berlin, Germany) and analyzed using AIDA software (Raytest GmbH, Berlin, Germany). Radioactivity is indicated by darkened areas in the autoradioluminogram. The radioactivity concentration in selected tissues was determined by digital analysis of the photostimulated light/unit area (PSL/mm2) on the autoradioluminogram. Using the

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AIDA software, the PSL/mm2 response (y-axis) and the radioactive concentration (x-axis) of the calibration standards were fit with least-squares regression analysis to the linear function with 1/x2 weighting. The results of the regression analysis were used to calculate concentrations of radioactivity in QC standards and selected tissues, as follows: adrenal gland; bone (femur); bone marrow (femur); brain; cecum; eye; fat; heart; kidney; kidney (cortex); kidney (medulla); large intestine; liver; lung; muscle (femoral); ovaries (females); pancreas; pituitary gland; prostate (males); skin; small intestine; spleen; stomach; thymus; thyroid gland; testes (males); urinary bladder; uterus (females).

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points in this region would not have guaranteed that all curves could be fit. However, based on all of the available PK data in Tables 2, 7 and 8, and examination of Fig. 3, allows one to conclude that the time courses are comparable for all groups receiving an oral dose of AM-1, and T1/2 for Group 1 females would be expected to be similar compared to males receiving the same dose level (see

3. Results 3.1. Concentration and kinetics in plasma following oral administration (groups 1 and 2) After a single oral dose of [14C]-AM-1 administered at 100 mg/ kg, Cmax was approximately 2.5 mg/g and 3.1 mg/g at 8 (males) and 24 (females) h post-dosing, respectively (Table 2). Exposure to AM1 equivalents, as measured by AUClast, was 119 (males) and 140 (females) mgh/g. The terminal elimination phase half-life (T1/2) was approximately 27 h for males, but could not be calculated with reasonable accuracy for females (insufficient data points after Tmax). In Group 1 females the 24 h time point demonstrated an unusually high standard deviation between the three samples. This inter-animal variability and differences between groups in a sparsesampling design led to a slightly higher value at 24 h post-dosing versus the 8 h Tmax observed in Group 1 males; therefore, there was insufficient data to accurately calculate the termination elimination phase as defined by the acceptance criteria of the study protocol. Inter-animal and analytical variability are not unexpected at low plasma concentrations, and changing the data collection

Fig. 3. Mean concentration of test substance equivalents in rat plasma (mg/g) following oral administration of [14C]-AM-1 or [14C]-LCFA.

Table 2 Mean Plasma Concentration and Pharmacokinetics of Test Article Equivalents in Group 1 and Group 2 Rats following an Oral Dose of [14C]-AM-1 at 100 mg/kg or [14C]-LCFA at 46 mg/kg. Time Post-Dose

Group 1 (AM-1)

Group 2 (LCFA)

Males

Females

Mean

SD

(h)

(mg/g)

0.25 0.5 1 2 4 8 24 48 72

0.0427 0.0878 0.186 0.282 0.637 2.46 2.46 1.40 0.719

PK Parameter (Units) AUClast (h*mg equiv./g) AUCinf (h*mg equiv./g) DN AUClast AUCinf Extrap (%) Cmax (mg equiv./g) Tmax (h) T1/2 (h) Rsq F (%)

119 147 1.19 19.1 2.46 8 27 0.998 10.7

Males SD

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

0.0180 0.0268 0.0555 0.0633 0.0873 0.642 0.387 0.385 0.128

0.0610 0.185 0.238 0.285 0.869 2.56 3.06 1.49 1.21

0.00704 0.0453 0.0550 0.055 0.155 0.259 1.49 0.353 0.167

0.625 1.32 2.17 2.94 3.39 2.74 1.84 0.982 0.646

0.0479 0.223 0.160 0.209 0.0943 0.380 0.0471 0.0727 0.0450

0.974 1.72 1.99 2.88 2.65 2.49 1.55 0.932 0.648

0.143 0.0838 0.365 0.262 0.367 0.492 0.136 0.126 0.119

140 NC 1.40 NA 3.06 24 NA NA 12.7

Mean

Females

Mean

112 141 2.44 20.1 3.39 4 30.3 0.992 12.6

SD

Mean

SD

101 131 2.19 23.2 2.88 2 32.6 0.986 8.98

Note: Results are discussed in text Section 3.1, Concentration and Kinetics in Plasma Following Oral Administration (Groups 1 and 2). Underlined values are below the approximate quantitation limit (0.167 mg/g Group 1, 0.056 mg/g Group 2). AUClast ¼ area under the analyte concentration versus time curve from the time of dosing to the time of the last concentration > LLOQ; AUCinf ¼ estimate of the area under the concentration versus time curve from the time of dosing to infinity; DN ¼ Dose normalized; AUCinf Extrap ¼ percentage of AUCinf due to extrapolation from Tlast infinity; Cmax ¼ maximum measured concentration of the analyte in matrix; Tmax ¼ sampling time at which Cmax was reached; T1/2 ¼ half-life for the analyte in matrix; Rsq ¼ R-squared (coefficient of determination); F ¼ Bioavailability.

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Section 3.4.2. Repeated Administration of AM-1). In addition, T1/2 values for oral administration were similar to the IV phase, indicating that the AM-1 elimination phase was fully characterized for both sexes using the available data. Using the terminal elimination phase rate constant, AUCinf was extrapolated for male rats and found to be 147 mgh/g; only 19% of the total value was extrapolated. After a single oral dose of [14C]-LCFA administered at 46 mg/kg, Cmax was approximately 3.4 mg/g and 2.9 mg/g at 4 (males) and 2 h (females) post-dosing, respectively (Table 2). The initial phase in the PK profile, whose primary character is determined by absorption, differed between AM-1 and LCFA equivalents (Fig. 3), and Tmax was much earlier for LCFA equivalents. Exposure to LCFA equivalents, as measured by AUClast, was 112 and 101 mgh/g, and AUCinf was 141 and 131 mgh/g for males and females, respectively. AUClast was comparable for AM-1 and LCFA equivalents, but dose normalized (DN) AUClast for LCFA equivalents was approximately 2times the DN AUClast for AM-1 equivalents. The terminal elimination phase half-life (T1/2) was approximately 31 h, which was very similar to the half-life reported for AM-1 equivalents (i.e. 27 h for males). Thus, the elimination kinetics were similar for AM-1 and LCFA equivalents. Oral bioavailability (F) of AM-1 equivalents was determined using DN AUClast values for oral group and DN AUCinf values for the iv group (because oral AUCinf values were not calculable for Group 1 females because of only two data points after Tmax as previously discussed). Oral bioavailability of AM-1 equivalents (including its hydrolysis products, glucose, xylose, acetate, isovalerate, and LCFA) was approximately 10.7% for males and 12.7% for females (Table 2). Oral bioavailability (F) of LCFA equivalents was determined using dose-normalized AUClast values for the oral group and dosenormalized AUCinf values for the iv group (for consistency of

calculations among AM-1 and LCFA groups and because oral AUCinf values were >20% extrapolated). Oral bioavailability of LCFA equivalents was approximately 12.6% for males and 8.98% for females (Table 2). 3.2. Concentration and kinetics in plasma following iv administration (groups 3 and 4) After a single iv (slow push) dose of [14C]-AM-1 administered at 10 mg/kg, C0 was approximately 89 mg/g and 109 mg/g for males and females, respectively (Table 3; Fig. 4A,B). The highest measured concentration at the first time point collected was approximately 44.7 mg/g and 38.7 mg/g for males and females, respectively. This corresponds to approximately 13% of the dose circulating in the plasma at Cmax (the first time point collected) and 31% of the dose circulating at C0. Exposure to AM-1 equivalents, as measured by AUClast, was 88 mgh/g regardless of sex. The terminal elimination phase was well defined and the half-life of AM-1 equivalents was approximately 32 h. After a single iv dose of [14C]-LCFA administered at 4.6 mg/kg, C0 was approximately 8.26 mg/g and 8.48 mg/g for males and females, respectively (Table 3; Fig. 5A,B). The highest measured concentration at the first time point collected was approximately 5.36 mg/g and 5.74 mg/g for males and females, respectively. These highest measured concentrations correspond to approximately 3.8% of the dose circulating in the plasma at Cmax (the first time point collected) and 5.7% of the dose circulating at C0. Exposure to LCFA equivalents, as measured by AUClast, was 68.4 (males) to 76.5 (females) mgh/g. The terminal elimination phase was well-defined and the half-life was approximately 35 h for males and 48 h for females. There was no apparent difference in the terminal elimination phase behavior between AM-1 and LCFA equivalents and the

Table 3 Mean Plasma Concentration and Pharmacokinetics of Test Article Equivalents in Group 3 and Group 4 Rats following an iv Dose of [14C]-AM-1 at 10 mg/kg or [14C]-LCFA at 4.6 mg/kg. Time Post-Dose

Group 3 (AM-1)

Group 4 (LCFA)

Males Mean

Females SD

Mean

Males SD

Mean

Females SD

Mean

SD

(h)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

0.083 0.25 0.5 1 2 4 8 24 48 72

44.7 11.2 2.45 1.51 1.55 1.58 1.82 1.31 0.693 0.489

27.3 0.817 0.264 0.452 0.105 0.256 0.527 0.0309 0.0460 0.0623

38.7 4.80 2.04 1.69 1.75 1.64 1.93 1.35 0.682 0.485

22.2 0.476 0.532 0.453 0.0746 0.295 0.309 0.125 0.0400 0.118

5.36 2.25 2.31 2.06 2.05 2.05 1.42 1.02 0.666 0.396

1.37 0.256 0.0303 0.0971 0.332 0.227 0.0571 0.0635 0.0560 0.0194

5.74 2.62 2.35 2.61 2.24 2.13 1.90 1.04 0.681 0.518

1.58 0.112 0.291 0.271 0.136 0.152 0.114 0.0813 0.0575 0.0122

PK Parameter (Units) AUClast (h*mg equiv./g) AUCinf (h*mg equiv./g) AUCinf Extrap (%) DN AUCinf C0 (mg equiv./g) Cl (g/min/kg) Vss (g/kg) T1/2 (h) Rsq

87.6 111 20.9 11.1 89.1 1.51 3900 32.8 0.986

88.4 110 19.8 11.0 109 1.51 3770 31.1 0.982

68.4 88.6 22.7 19.3 8.26 0.866 2470 35.3 0.998

76.5 112 31.9 24.4 8.48 0.682 2540 47.9 0.985

Note: Results are discussed in text Section 3.2, Concentration and Kinetics in Plasma Following iv Administration (Groups 3 and 4). Approximate quantitation limit (0.023 mg/g Group 3, 0.022 mg/g Group 4). AUClast ¼ area under the analyte concentration versus time curve from the time of dosing to the time of the last concentration > LLOQ; AUCinf ¼ estimate of the area under the concentration versus time curve from the time of dosing to infinity; AUCinf Extrap ¼ percentage of AUCinf due to extrapolation from Tlast infinity; DN ¼ Dose normalized; C0 ¼ estimated analyte concentration in matrix immediately following intravenous administration; Cl ¼ apparent systemic clearance for the analyte in matrix; Vss ¼ estimate of the volume of distribution for the analyte at steady state; T1/2 ¼ half-life for the analyte in matrix; Rsq ¼ R-squared (coefficient of determination).

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Fig. 4. A. Mean Concentration of Test Substance Equivalents in Rat Plasma (mg/g) following iv Administration of [14C]-AM-1 (0e72 h post-dosing), B. Mean Concentration of Test Substance Equivalents in Rat Plasma (mg/g) following iv Administration of [14C]-AM-1 (0e4 h post-dosing).

Fig. 5. A. Mean Concentration of Test Substance Equivalents in Rat Plasma (mg/g) following iv Administration of [14C]-LCFA (0e72 h post-dosing), B. Mean Concentration of Test Substance Equivalents in Rat Plasma (mg/g) following iv Administration of [14C]-LCFA (0e4 h post-dosing).

initial absorption/distribution phase is a negligible contributor to overall AUC. Dose-normalized AUC for AM-1 vs LCFA was calculated to elucidate the relationship between the equimolar administered dose and the plasma, whole blood, and tissue concentrations, which are all reported in standard mg/g (e.g. mass not molar) units. Because of the labeling of the test article in many locations, the conversion between the equimolar dose and the mass could be misinterpreted. There was approximately a 2-fold difference in the dose-normalized AUCinf between AM-1 (approximately 11) and LCFA (approximately 22) equivalents.

male and female rats following a single oral administration of [14C]AM-1 at 100 mg/kg. Approximately 16%e20% of the recovered radioactivity was captured in the CO2 trap for expired air, but no volatile components were retained in the activated carbon trap. Approximately 1% of the recovered radioactivity was in the urine, and approximately 74%e83% of the dose was eliminated in feces. A maximum of approximately 2% of the recovered radioactivity was found in the individual carcass of analyzed animals while mean values of 0.73% and 0.53% were calculated for males and females, respectively (Table 4). Following repeated administration of [14C]-AM-1, approximately 92% (overall average, males and females combined) of the administered dose was recovered in expired air, urine, feces, and carcass from male and female rats. Approximately 19%e21% of the recovered radioactivity was captured in the CO2 trap for expired air, but no volatile components were retained in the activated carbon

3.3. Excretion balance 3.3.1. AM-1 excretion balance Based on the excretion data (Table 4; Fig. 6), approximately 98% (overall average, males and females combined) of the administered dose was recovered in expired air, urine, feces, and carcass from

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Table 4 Recovery of AM-1 and LCFA equivalents (% of dose administered) from rats following oral dosing. Test Article:

Group 5 (AM-1)

Group 6 (LCFA)

Group 9 (AM-1)

Group 10 (LCFA)

Dosing Regimen:

Single Dosea

Single Doseb

Repeated Dosec

Repeated Dosed

M

F

M

F

M

F

M

F

Mean

(SD)

Mean

(SD)

Mean

(SD)

Mean

(SD)

Mean

(SD)

Mean

(SD)

Mean

(SD)

Mean

(SD)

Expired Air Activated Carbon Urine Feces Cage Rinse

16.3 0.02 1.11 82.6 0.28

(15.70) (0.01) (0.65) (17.13) (0.15)

20.2 0.03 0.96 73.7 0.38

(11.48) (0.02) (0.16) (9.91) (0.19)

14.7 0.02 2.75 70.6 0.95

(2.56) (0.01) (0.59) (5.06) (0.20)

16.4 0.02 2.94 72.1 0.98

(2.49) (0.01) (0.59) (5.75) (0.25)

20.5 0.05 1.55 62.8 0.88

(1.61) (0.01) (0.49) (10.76) (0.59)

18.6 0.03 1.02 75.6 0.48

(2.75) (0.00) (0.47) (6.93) (0.15)

16.1 0.03 3.05 67.6 0.92

(5.53) (0.01) (0.91) (3.55) (0.24)

17.5 0.02 3.19 71.7 0.80

(1.39) (0.00) (0.18) (5.57) (0.07)

Cage Wash DI Water MeOH Carcass TOTAL

0.02 0.00 0.73 101.0

(0.01) (0.00) NA (16.24)

0.01 0.00 0.53 95.8

(0.01) (0.00) NA (10.01)

0.07 0.00 0.82 90.0

(0.08) (0.00) NA (3.85)

0.01 0.00 0.96 93.4

(0.01) (0.00) NA (7.52)

0.10 0.01 1.81 87.7

(0.11) (0.01) NA (9.62)

0.01 0.00 NA 95.7

(0.01) (0.00) NA (3.63)

0.01 0.00 1.41 89.2

(0.01) (0.00) NA (3.37)

0.01 0.00 0.43 93.7

(0.01) (0.00) NA (4.88)

NA e Not Applicable (only  1 of 3 animals analyzed based on measured radioactivity [>90%] in excreta samples for remaining animals; mean values calculated using all 3 animals; i.e. with negligible amounts of radioactivity [value of 0] assumed for carcasses not analyzed). a Group 5 received a single oral dose of [14C]-AM-1 at 100 mg/kg and target radioactivity of 100 mCi/kg. b Group 6 received a single oral dose of [14C]-LCFA at 46 mg/kg and target radioactivity of 100 mCi/kg. c Group 9 received a single daily oral dose of AM-1 at 100 mg/kg for 14 consecutive days followed by a single oral dose of [14C]-AM-1 at 100 mg/kg and target radioactivity of 100 mCi/kg. d Group 10 received a single daily oral dose of LCFA at 46 mg/kg for 14 consecutive days followed by a single oral dose of [14C]-LCFA at 46 mg/kg and target radioactivity of 100 mCi/kg.

trap. Approximately 1% of the recovered radioactivity was in the urine; approximately 63%e76% of the dose was eliminated in feces. Approximately 2%e3% (range of maximum values) of the recovered radioactivity was found in the individual carcass of analyzed animals while a mean value of 1.81% was calculated for males (Table 4).

3.3.2. LCFA excretion balance Based on the excretion data (Table 4; Fig. 7), approximately 92% (overall average, males and females combined) of the administered dose was recovered in expired air, urine, feces, and carcass from male and female rats following a single oral administration of [14C]LCFA at 46 mg/kg. Approximately 15%e16% of the recovered radioactivity was captured in the CO2 trap for expired air. Approximately 3% of the recovered radioactivity was in the urine, and approximately 71%e72% of the dose was eliminated in feces. Approximately 1%e2.5% (range of maximum values) of the recovered radioactivity was found in the individual carcass of analyzed animals while mean values of 0.82% and 0.96% were calculated for males and females, respectively (Table 4). Following repeated administration of [14C]-LCFA, approximately 91% (overall average, males and females combined) of the administered dose was recovered in expired air, urine, feces, and carcass from male and female rats. Approximately 16%e18% of the recovered radioactivity was captured in the CO2 trap for expired air. Approximately 3% of the recovered radioactivity was in the urine; approximately 68%e72% of the dose was eliminated in feces. Approximately 1%e2.5% (range of maximum values) of the recovered radioactivity was found in the individual carcass of analyzed animals while mean values of 1.41% and 0.43% were calculated for males and females, respectively (Table 4). There appeared to be no difference in the excretion of AM-1 versus LCFA equivalents, with the exception that there may have been a slightly greater amount of LCFA equivalents in the urine versus AM-1 equivalents (3% versus 1%). In all cases, the majority of the elimination occurred in the first 48 h post-dosing; however, some radioactivity was still detected in both urine and feces collected at 168 h post-dosing. Overall, mass balance was achieved for all test articles, sexes, and dosing regimens.

Fig. 6. Cumulative recovery of AM-1 equivalents from group 5 male rats following single oral administration of [14C]-AM-1.

3.4. Tissue distribution 3.4.1. Single administration of AM-1 Following a single oral administration of [14C]-AM-1 at 100 mg/ kg to male and female rats, [14C]-AM-1 equivalents and/or metabolites were detected by quantitative whole body autoradiography in all tissues measured, except the eye, testes (males only), and brain (Tables 5 and 6). Tissue Tmax was generally 8 h post-dosing in

J. Bitzer et al. / Food and Chemical Toxicology 109 (2017) 552e568

Fig. 7. Cumulative recovery of LCFA equivalents from group 6 male rats following single oral administration of [14C]-LCFA.

561

males (see representative autoradioluminogram in Fig. 8), and 824 h post-dosing in females, consistent with the plasma Tmax. Cmax for [14C]-AM-1- equivalents-derived radioactivity was highest in the tissues associated with the GI tract and excretion following oral administration: primarily the stomach (561,000 ng/g) followed by the cecum, pancreas, large and small intestine (74,400 ng/g to 17,000 ng/g), and urinary bladder (16,500 ng/g, consistent with urinary excretion), in males and the cecum (105,000 ng/g) followed by the large and small intestine, urinary bladder, pancreas, liver, adrenal gland, and stomach (64,800 ng/g to 11,600 ng/g) in females. The remaining tissues had concentrations of approximately 3000 ng/g to 10,000 ng/g in both sexes. Most tissue concentrations were near or below the lower limit of quantitation (LLOQ) by 168 h post-dosing. For the majority of the tissues, T1/2 estimations calculated by the software were not reported because they did not meet the laboratory's rigorous acceptance criteria for reporting robust pharmacokinetic data. Due to limited absorption and rapid disposition, tissue concentrations were typically < LLOQ by 168 h. Based on the low concentrations and disappearance of AM-1 from plasma between 48 and 168 h post-dose, it can be assumed that the 120 h period represents at least 3e5 half-lives during the terminal elimination phase for AM-1 and a reasonable estimate of T1/2 for AM-1 in tissues is approximately 24e40 h, which is consistent with the plasma elimination phase half-life of AM-1. Exposure, as measured by AUClast, was highest in cecum, stomach, adrenal gland (2,050,000 hng/g to 897,000 hng/g) of males, and highest in large intestine, cecum, adrenal gland, and liver (2,080,000 hng/g to 1,080,000 hng/g) of females. The low concentration and exposure in the remaining tissues, relative to the magnitude of the dose, is consistent with low circulating concentrations in the whole blood and plasma, and with the majority of the radioactivity in the tissues being due to whole blood perfusion. Tissue:plasma ratios (calculated using AUClast), were generally

Table 5 Pharmacokinetic Parameters of AM-1 Equivalents in Plasma, Whole Blood, and Tissues of Group 7 Male Rats following a Single Oral Dose of [14C]-AM-1 at 100 mg/kg. Tissue

Cmax (ng equiv./g)

Tmax (h)

T½ (h)

AUClast (ng equiv$h/g)

AUCinf (ng equiv$h/g)

%AUCext (%)

Rsq

Tissue: Plasmaa

Plasma Whole blood Adrenal gland Bone (Femur) Bone marrow (Femur) Brain Cecum Eye Fat Heart Kidney Kidney (Cortex) Kidney (Medulla) Large intestine Liver Lung Muscle (Femoral) Pancreas Pituitary gland Prostate Skin Small intestine Spleen Stomach Testes Thymus Thyroid gland Urinary bladder

2380 2090 7330 6200 7520 0 74,400 0 4620 4800 5250 5740 4560 17,200 9730 4050 3640 30,800 4200 6040 3030 17,000 9500 561,000 0 3570 5500 16,500

8 24 24 8 24 NC 8 NC 48 1 8 8 8 8 1 8 8 8 8 8 24 2 8 2 NC 8 8 4

42.5 NC NC NC NC NC NC NC NC NC NC NC NC NC 47.5 NC NC NC NC NC NC 32.8 NC 30.9 NC NC NC NC

169,000 211,000 897,000 215,00 271,000 0 2,050,000 0 129,000 70,500 214,000 232,000 180,000 417,000 370,000 165,000 67,000 445,000 148,000 296,000 24,200 284,000 297,000 1,030,000 0 145,000 201,000 155,000

181,000 NC NC NC NC NC NC NC NC NC NC NC NC NC 744,000 NC NC NC NC NC NC 436,000 NC 1,150,000 NC NC NC NC

6.5 NC NC NC NC NC NC NC NC NC NC NC NC NC 50 NC NC NC NC NC NC 35 NC 10 NC NC NC NC

0.997 NC NC NC NC NC NC NC NC NC NC NC NC NC 0.895 NC NC NC NC NC NC 0.958 NC 0.968 NC NC NC NC

1.00 1.25 5.31 0.13 1.60 0.00 12.13 0.00 0.76 0.42 1.27 1.37 1.07 2.47 2.19 0.98 0.40 2.63 0.88 1.75 0.14 1.68 1.76 6.09 0.00 0.86 1.19 0.92

NC ¼ Not calculated due to insufficient data. Bolded values are approximations. a Tissue:plasma ratio based on AUClast.

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Table 6 Pharmacokinetic Parameters of AM-1 Equivalents in Plasma, Whole Blood, and Tissues of Group 7 Female Rats following a Single Oral Dose of [14C]-AM-1 at 100 mg/kg. Tissue

Cmax (ng equiv./g)

Tmax (h)

T½ (h)

AUClast (ng equiv$h/g)

AUCinf (ng equiv$h/g)

%AUCext (%)

Rsq

Tissue: Plasmaa

Plasma Whole blood Adrenal gland Bone (Femur) Bone marrow (Femur) Brain Cecum Eye Fat Heart Kidney Kidney (Cortex) Kidney (Medulla) Large intestine Liver Lung Muscle (Femoral) Ovary Pancreas Pituitary gland Skin Small intestine Spleen Stomach Thymus Thyroid gland Urinary bladder Uterus

3390 2570 12,000 5440 6170 0 105,000 0 4690 3940 4990 5920 4350 64,800 13,300 4840 2650 6090 14,100 5040 2980 16,600 6020 11,600 4320 6600 28,200 5840

24 24 24 8 8 NC 8 NC 168 1 8 24 8 8 24 24 8 48 8 24 168 8 24 8 24 24 2 4

NC NC NC NC NC NC 139 NC NC 136 163 NC NC 58.7 NC NC NC NC NC NC NC NC NC NC NC NC 219 1140

228,000 209,000 1,310,000 105,000 242,000 0 1,970,000 0 530,000 149,000 622,000 667,000 163,000 2,080,000 1,080,000 191,000 5290 801,000 288,000 204,000 369,000 503,000 238,000 287,000 175,000 740,000 598,000 508,000

NC NC NC NC NC NC 2,550,000 NC NC 758,000 1,240,000 NC NC 2,380,000 NC NC NC NC NC NC NC NC NC NC NC NC 1,530,000 5,020,000

NC NC NC NC NC NC 23 NC NC 80 50 NC NC 13 NC NC NC NC NC NC NC NC NC NC NC NC 61 90

NC NC NC NC NC NC 0.478 NC NC 0.616 0.999 NC NC 0.655 NC NC NC NC NC NC NC NC NC NC NC NC 0.795 0.696

1.00 0.92 5.75 0.46 1.06 0.00 8.64 0.00 2.32 0.65 2.73 2.93 0.71 9.12 4.74 0.84 0.02 3.51 1.26 0.89 1.62 2.21 1.04 1.26 0.77 3.25 2.62 2.23

NC ¼ Not calculated due to insufficient data. Bolded values are approximations. a Tissue:plasma ratio based on AUClast.

consistent with this hypothesis, and were less than 0.7 for poorly perfused tissues e bone, skin (males only), muscle, heart, fat (males only). For the peripheral tissues, the tissue:plasma ratios generally ranged from 1 to <4 and indicated that the majority of the radioactivity in the tissues was due to whole blood perfusion. For tissues associated with the GI tract, the tissue:plasma ratio was as high as 12 (cecum, males). 3.4.2. Repeated administration of AM-1 Following repeated oral dosing of non-radiolabeled AM-1 at 100 mg/kg to male and female rats for 14 days with a final single administration of [14C]-AM-1, AM-1 equivalents and/or metabolites were similarly broadly distributed in the tissues as when only a single dose of [14C]-AM-1 was administered (Tables 7 and 8). AM-1 equivalents were below the quantitation limit (BLQ) in the brain and eye; a low concentration of AM-1 equivalents were measured in testes (males) at 24 h post-dosing only. Cmax for [14C]-AM-1equivalents-derived radioactivity was highest in the tissues of the

GI tract following oral administration. For other tissues, the concentration was of the same order of magnitude as measured in animals receiving a single administration of AM-1. Tmax was generally 24 h post-dosing in males, and 8e24 h post-dosing in females. Most tissue concentrations were near or below LLOQ by 168 h post-dosing. For both the single and repeated dose groups, the concentration of AM-1 equivalents was above LLOQ at 168 h post-dosing for a greater number of tissues in female animals versus male animals, but the difference was a minimal contributor to overall exposure. Exposure, as measured by AUClast, was highest in stomach (above the upper limit of quantitation, i.e. 2,660,000 hng/g), followed by the cecum, large and small intestine, and adrenal gland (2,000,000 hng/g to 867,000 hng/g) of males, and highest in large and small intestine, adrenal gland, and liver (2,190,000 hng/g to 1,180,000 h ng/g) of females. For the majority of the tissues, T1/2 estimations were not robust enough to be reported or were reported as approximations. As occurred in the single dose administration phase, most tissues were BLQ by 168 h

Fig. 8. Whole Body Autoradioluminogram Showing Tissue Distribution of Radioactivity at 8 Hours following a Single Oral Dose of [14C]-AM-1 to a Group 7 Male Rat at 100 mg/kg.

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Table 7 Pharmacokinetic Parameters of AM-1 Equivalents in Plasma, Whole Blood, and Tissues of Group 11 Male Rats following Repeated Oral Doses of AM-1 and a Single Oral Dose of [14C]-AM-1 at 100 mg/kg. Tissue

Cmax (ng equiv./g)

Tmax (h)

T½ (h)

AUClast (ng equiv$h/g)

AUCinf (ng equiv$h/g)

%AUCext (%)

Rsq

Tissue: Plasmab

Plasma Whole blood Adrenal gland Bone (Femur) Bone marrow (Femur) Brain Cecum Eye Fat Heart Kidney Kidney (Cortex) Kidney (Medulla) Large intestine Liver Lung Muscle (Femoral) Pancreas Pituitary gland Prostate Skin Small intestine Spleen Stomacha Testes Thymus Thyroid gland Urinary bladder

4470 3320 10,300 4540 13,000 0 162,000 0 5260 4070 8560 9020 6670 25,800 18,700 6530 3730 9770 7490 5200 3880 68,200 11300 2,140,000 2810 7600 11,700 21,800

24 24 24 24 24 NC 8 NC 8 24 24 24 24 24 24 24 24 8 24 24 24 2 24 1 24 24 24 8

NC NC NC NC NC NC NC NC 392 NC NC NC NC NC NC NC NC NC NC NC NC 70.2 NC 28.6 NC NC NC NC

291,000 284,000 969,000 79,300 387,000 0 2,000,000 0 742,000 135,000 314,000 334,000 253,000 1,590,000 564,000 229,000 29,800 350,000 237,000 222,000 119,000 867,000 371,000 2,660,000 22,500 247,000 341,000 414,000

NC NC NC NC NC NC NC NC 2,950,000 NC NC NC NC NC NC NC NC NC NC NC NC 1,730,000 NC 2,900,000 NC NC NC NC

NC NC NC NC NC NC NC NC 75 NC NC NC NC NC NC NC NC NC NC NC NC 50 NC 8.1 NC NC NC NC

NC NC NC NC NC NC NC NC 0.993 NC NC NC NC NC NC NC NC NC NC NC NC 0.125 NC 0.922 NC NC NC NC

1.00 0.98 3.33 0.27 1.33 0.00 6.87 0.00 2.55 0.46 1.08 1.15 0.87 5.46 1.94 0.79 0.10 1.20 0.81 0.76 0.41 2.98 1.27 9.14 0.08 0.85 1.17 1.42

NC ¼ Not calculated due to insufficient data. Bolded values are approximations. a Cmax is equivalent to the upper limit of quantitation; the actual concentration at 1 h post-dosing was above the upper limit of quantitation. b Tissue:plasma ratio based on AUClast.

post-dosing and can be reasonably expected to have tissue halflives of approximately 24e40 h. Tissue:plasma ratios were comparable to those determined during the single-dose phases. Overall, repeated dosing versus a single administration did not appear to alter the tissue distribution of AM-1 equivalents. 3.4.3. Single dose administration of LCFA Following a single oral administration of [14C]-LCFA at 46 mg/kg to male and female rats, [14C]-LCFA equivalents and/or metabolites were detected by quantitative whole body autoradiography in all tissues measured, except the eye, testes, and brain of male rats (both the eye and brain in female rats had concentrations of LCFA equivalents > LLOQ at 2 h post-dosing) (Tables 9 and 10). Cmax for [14C]-LCFA-equivalents-derived radioactivity was highest in the tissues associated with the GI tract and excretion following oral administration: primarily the cecum (347,000 ng/g), followed by the large and small intestine, urinary bladder, stomach, pancreas, and liver (111,000 ng/g to 10,400 ng/g) in males and the cecum (72,300 ng/g), followed by the urinary bladder, small intestine, liver, and pancreas (68,600 ng/g to 10,400 ng/g) in females. The remaining tissues had concentrations of approximately 1300 ng/g to 8500 ng/g in both sexes. Tmax was generally 1 h post-dosing in males and 2 h post-dosing in females. For the majority of the tissues, T1/2 estimations were not robust enough to be reported or were reported as approximations. As observed for AM-1, most tissue concentrations of LCFA were BLQ by 168 h post-dosing and can be reasonably expected to have tissue half-lives of approximately 24e40 h. Exposure, as measured by AUClast, was highest in cecum (3,690,000 hng/g in males and 1,120,000 hng/g in females). All other tissues, with the exception of those noted above, had some exposure, but most tissue concentrations were near or below LLOQ

by 168 h post-dosing for both males and females. Tissue:plasma ratios were calculated using AUClast and generally ranged from 0.3 to 3.7 for males and 0.01 to 3.6 for females. For tissues associated with the GI tract, the tissue:plasma ratio was as high as 21 (cecum, males). As with AM-1, tissue:plasma ratios indicate that most of the radioactivity detected in the tissues results from the low concentration of test article equivalents circulating in the plasma, and no target tissues were identified. 3.4.4. Repeated administration of LCFA Following repeated oral dosing of LCFA at 46 mg/kg to male and female rats for 14 days with a final single administration of [14C]LCFA, LCFA equivalents and/or metabolites were similarly broadly distributed in the tissues as when only a single dose of [14C]-LCFA was administered (Tables 11 and 12). Tmax was generally 1e8 h post-dosing in males, and ranged from 1 to 48 h post-dosing in females. Cmax for [14C]-LCFA-equivalents-derived radioactivity was highest in the tissues associated with the GI tract and excretion following oral administration: primarily the cecum, small and large intestine, liver and urinary bladder. LCFA equivalents were not detected in the brain (females only), eye, or testes (males only) at any time point, but was present at a measureable concentration in brain of males at 1 and 2 h post-dosing. The tissue concentrations were of the same order of magnitude as measured in animals receiving a single administration of [14C]-LCFA. For the majority of the tissues, T1/2 estimations were not robust enough to be reported or were reported as approximations. As observed following a single administration of LCFA, following repeated administration most tissue concentrations of LCFA were BLQ by 168 h post-dosing and can be reasonably expected to have tissue half-lives of approximately 24e40 h.

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Table 8 Pharmacokinetic Parameters of AM-1 Equivalents in Plasma, Whole Blood, and Tissues of Group 11 Female Rats following Repeated Oral Doses of AM-1 and a Single Oral Dose of [14C]-AM-1 at 100 mg/kg. Tissue

Cmax (ng equiv./g)

Tmax (h)

T½ (h)

AUClast (ng equiv$h/g)

AUCinf (ng equiv$h/g)

%AUCext (%)

Rsq

Tissue: Plasmaa

Plasma Whole blood Adrenal gland Bone (Femur) Bone marrow (Femur) Brain Cecum Eye Fat Heart Kidney Kidney (Cortex) Kidney (Medulla) Large intestine Liver Lung Muscle (Femoral) Ovary Pancreas Pituitary gland Skin Small intestine Spleen Stomach Thymus Thyroid gland Urinary bladder Uterus

4200 2670 12,700 9740 9620 0 14,700 0 4740 4720 6560 7170 5150 58,900 14,400 6000 4960 8010 13,800 5150 4240 24,300 7690 5590 7630 6720 5990 4910

8 24 48 8 24 NC 24 NC 168 8 8 8 1 8 24 24 8 8 2 24 24 8 1 2 8 24 24 8

58.1 NC NC NC NC NC NC NC NC NC NC 129 77.7 72.6 NC NC NC 116 23.8 NC NC 97.5 126 NC NC NC NC NC

320,000 286,000 1,600,000 120,000 384,000 0 469,000 0 386,000 153,000 250,000 746,000 205,000 2,190,000 1,200,000 222,000 9920 936,000 308,000 200,000 147,000 1,180,000 304,000 196,000 243,000 258,000 652,000 160,000

372,000 NC NC NC NC NC NC NC NC NC NC 1,270,000 604,000 2,560,000 NC NC NC 1,490,000 439,000 NC NC 1,710,000 1,410,000 NC NC NC NC NC

14 NC NC NC NC NC NC NC NC NC NC 41 66.0 15 NC NC NC 37 30 NC NC 31 78 NC NC NC NC NC

0.989 NC NC NC NC NC NC NC NC NC NC 0.982 0.786 0.878 NC NC NC 0.998 0.822 NC NC 0.74 0.846 NC NC NC NC NC

1.00 0.89 5.00 0.38 1.20 0.00 1.47 0.00 1.21 0.48 0.78 2.33 0.64 6.84 3.75 0.69 0.03 2.93 0.96 0.63 0.46 3.69 0.95 0.61 0.76 0.81 2.04 0.50

NC ¼ Not calculated due to insufficient data. Bolded values are approximations. a Tissue:plasma ratio based on AUClast.

Table 9 Pharmacokinetic Parameters of LCFA Equivalents in Plasma, Whole Blood, and Tissues of Group 8 Male Rats following a Single Oral Dose of [14C]-LCFA at 46 mg/kg. Tissue

Cmax (ng equiv./g)

Tmax (h)

T½ (h)

AUClast (ng equiv$h/g)

AUCinf (ng equiv$h/g)

%AUCext (%)

Rsq

Tissue: Plasmaa

Plasma Whole blood Adrenal gland Bone (Femur) Bone marrow (Femur) Brain Cecum Eye Fat Heart Kidney Kidney (Cortex) Kidney (Medulla) Large intestine Liver Lung Muscle (Femoral) Pancreas Pituitary gland Prostate Skin Small intestine Spleen Stomach Testes Thymus Thyroid gland Urinary bladder

2890 1810 6060 3890 6040 0 347,000 0 4150 3520 6540 8030 4630 111,000 10,400 2710 5330 13,800 2930 4320 1620 25,600 6430 16100 0 2410 3630 36,000

4 4 24 1 1 NC 8 NC 1 1 1 1 1 2 1 48 1 1 4 8 48 1 4 4 NC 4 8 2

44.5 69.1 NC NC 81.1 NC NC NC 1330 111 95.8 93.6 103 137 45.1 NC 82.3 22.5 560 NC NC 23.2 129 49.3 NC 1300 115 51.2

172,000 158,000 639,000 73,800 207,000 0 3,690,000 0 388,000 103,000 454,000 481,000 146,000 1,710,000 321,000 117,000 60,000 178,000 112,000 121,000 54,400 428,000 177,000 164,000 0 104,000 377,000 611,000

184,000 190,000 NC NC 608,000 NC NC NC 3,180,000 457,000 628,000 656,000 616,000 1,980,000 644,000 NC 230,000 251,000 2,100,000 NC NC 586,000 761,000 312,000 NC 4,400,000 579,000 720,000

6.4 17 NC NC 66 NC NC NC 88 77 28 27 76 14 50 NC 74 29 95 NC NC 27 77 48 NC 98 35 15

0.992 0.983 NC NC 0.924 NC NC NC 0.00677 0.331 0.938 0.947 0.334 0.614 0.932 NC 0.729 0.666 0.0454 NC NC 0.628 0.976 0.42 NC 0.0128 0.971 0.818

1.00 0.92 3.72 0.43 1.20 0.00 21.45 0.00 2.26 0.60 2.64 2.80 0.85 9.94 1.87 0.68 0.35 1.03 0.65 0.70 0.32 2.49 1.03 0.95 0.00 0.60 2.19 3.55

NC ¼ Not calculated due to insufficient data. Bolded values are approximations. a Tissue:plasma ratio based on AUClast.

J. Bitzer et al. / Food and Chemical Toxicology 109 (2017) 552e568

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Table 10 Pharmacokinetic Parameters of LCFA Equivalents in Plasma, Whole Blood, and Tissues of Group 8 Female Rats following a Single Oral Dose of [14C]-LCFA at 46 mg/kg. Tissue

Cmax (ng equiv./g)

Tmax (h)

T½ (h)

AUClast (ng equiv$h/g)

AUCinf (ng equiv$h/g)

%AUCext (%)

Rsq

Tissue: Plasmaa

Plasma Whole blood Adrenal gland Bone (Femur) Bone marrow (Femur) Brain Cecum Eye Fat Heart Kidney Kidney (Cortex) Kidney (Medulla) Large intestine Liver Lung Muscle (Femoral) Ovary Pancreas Pituitary gland Skin Small intestine Spleen Stomach Thymus Thyroid gland Urinary bladder Uterus

3840 2360 5760 2930 4270 2730 72,300 1310 1490 3350 7910 8440 6610 8410 14,800 3650 2180 3930 10,400 4950 1660 24,000 4370 5580 3780 4340 68,600 3680

4 4 48 2 2 2 8 2 4 2 2 2 2 2 4 4 4 48 2 2 2 1 2 2 2 2 2 2

43.2 69.2 NC 105 67.1 NC NC NC NC 55.0 62.1 56.8 75.6 NC 25.4 141 NC NC 23.6 186 976 71 118 32.4 226 108 16.3 281

189,000 139,000 674,000 35,200 178,000 1360 1,120,000 657 32,700 95,200 152,000 165,000 130,000 145,000 325,000 113,000 20,200 174,000 149,000 132,000 51,800 244,000 141,000 112,000 121,000 165,000 292,000 121,000

200,000 166,000 NC 259,000 446,000 NC NC NC NC 236,000 413,000 415,000 427,000 NC 467,000 555,000 NC NC 211,000 818,000 2,180,000 558,000 582,000 193,000 842,000 578,000 325,000 771,000

5.6 17 NC 86 60 NC NC NC NC 60 63 60 70 NC 30 80 NC NC 29 84 98 56 76 42 86 72 10 84

1.00 1.00 NC 0.17 0.79 NC NC NC NC 0.59 0.45 0.54 0.26 NC 0.68 0.45 NC NC 0.72 1.00 0.03 0.98 0.93 0.68 0.39 0.30 0.67 0.02

1.00 0.74 3.57 0.19 0.94 0.01 5.93 0.00 0.17 0.50 0.80 0.87 0.69 0.77 1.72 0.60 0.11 0.92 0.79 0.70 0.27 1.29 0.75 0.59 0.64 0.87 1.54 0.64

NC ¼ Not calculated due to insufficient data. Bolded values are approximations. a Tissue:plasma ratio based on AUClast.

Table 11 Pharmacokinetic Parameters of LCFA Equivalents in Plasma, Whole Blood, and Tissues of Group 12 Male Rats following Repeated Oral Doses of LCFA and a Single Oral Dose of [14C]-LCFA at 46 mg/kg. Tissue

Cmax (ng equiv./g)

Tmax (h)

T½ (h)

AUClast (ng equiv$h/g)

AUCinf (ng equiv$h/g)

%AUCext (%)

Rsq

Tissue: Plasmaa

Plasma Whole blood Adrenal gland Bone (Femur) Bone marrow (Femur) Brain Cecum Eye Fat Heart Kidney Kidney (Cortex) Kidney (Medulla) Large intestine Liver Lung Muscle (Femoral) Pancreas Pituitary gland Prostate Skin Small intestine Spleen Stomach Testes Thymus Thyroid gland Urinary bladder

3320 1980 4140 2740 5470 1420 23,800 0 2820 3160 5790 6140 5220 13,500 12,800 3180 1430 9780 2730 2060 1390 15,900 4450 9260 0 2690 3810 19,500

4 4 4 8 8 2 8 NC 48 2 1 1 1 8 4 8 8 8 4 8 1 4 8 1 NC 2 24 2

63.9 115 155 NC NC NC NC NC NC NC 39.3 39.7 77.0 NC 24.1 NC NC NC 90.7 NC NC 16.7 NC 78.7 NC 81.8 NC 21.1

166,000 125,000 443,000 47,300 145,000 2110 341,000 0 317,000 17,900 114,000 122,000 96,200 257,000 251,000 94,800 8820 173,000 94,900 81,500 2050 275,000 133,000 108,000 0 102,000 139,000 133,000

194,000 192,000 838,000 NC NC NC NC NC NC NC 218,000 233,000 266,000 NC 343,000 NC NC NC 307,000 NC NC 315,000 NC 301,000 NC 307,000 NC 174,000

14 35 47 NC NC NC NC NC NC NC 48 48 64 NC 27 NC NC NC 69 NC NC 13 NC 64 NC 67 NC 24

0.999 0.996 0.999 NC NC NC NC NC NC NC 0.767 0.801 0.834 NC 0.842 NC NC NC 0.983 NC NC 0.999 NC 0.85 NC 0.913 NC 0.993

1.00 0.75 2.67 0.28 0.87 0.01 2.05 0.00 1.91 0.11 0.69 0.73 0.58 1.55 1.51 0.57 0.05 1.04 0.57 0.49 0.01 1.66 0.80 0.65 0.00 0.61 0.84 0.80

NC ¼ Not calculated due to insufficient data. Bolded values are approximations. a Tissue:plasma ratio based on AUClast.

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Table 12 Pharmacokinetic Parameters of LCFA Equivalents in Plasma, Whole Blood, and Tissues of Group 12 Female Rats following Repeated Oral Doses of LCFA and a Single Oral Dose of [14C]-LCFA at 46 mg/kg. Tissue

Cmax (ng equiv./g)

Tmax (h)

T½ (h)

AUClast (ng equiv$h/g)

AUCinf (ng equiv$h/g)

%AUCext (%)

Rsq

Tissue: Plasmaa

Plasma Whole blood Adrenal gland Bone (Femur) Bone marrow (Femur) Brain Cecum Eye Fat Heart Kidney Kidney (Cortex) Kidney (Medulla) Large intestine Liver Lung Muscle (Femoral) Ovary Pancreas Pituitary gland Skin Small intestine Spleen Stomach Thymus Thyroid gland Urinary bladder Uterus

3130 1790 7290 2620 4320 0 7350 0 3150 3230 6490 7080 6050 5540 12,800 2560 1780 3540 6090 2170 1800 30,200 2910 6710 3230 4450 16,900 2850

4 4 48 1 24 NC 4 NC 48 2 2 2 2 8 1 24 1 24 4 48 48 4 24 4 4 8 1 48

46.9 73.0 NC NC NC NC 31.1 NC NC 60.5 153 143 1560 NC 59.8 NC NC NC 40.3 NC NC 65.9 NC 56.5 NC NC 20.1 NC

183,000 132,000 775,000 5810 165,000 0 169,000 0 83,200 97,300 138,000 152,000 111,000 184,000 292,000 111,000 888 157,000 144,000 90,700 59,800 268,000 130,000 147,000 109,000 183,000 148,000 101,000

198,000 162,000 NC NC NC NC 287,000 NC NC 251,000 711,000 744,000 4,840,000 NC 672,000 NC NC NC 263,000 NC NC 534,000 NC 349,000 NC NC 204,000 NC

7.6 19 NC NC NC NC 41 NC NC 61 81 80 98 NC 57 NC NC NC 45 NC NC 50 NC 58 NC NC 28 NC

1 0.998 NC NC NC NC 0.421 NC NC 0.675 0.292 0.349 0.829 NC 1 NC NC NC 0.716 NC NC 0.922 NC 0.444 NC NC 0.608 NC

1.00 0.72 4.23 0.03 0.90 0.00 0.92 0.00 0.45 0.53 0.75 0.83 0.61 1.01 1.60 0.61 0.00 0.86 0.79 0.50 0.33 1.46 0.71 0.80 0.60 1.00 0.81 0.55

NC ¼ Not calculated due to insufficient data. Bolded values are approximations. a Tissue:plasma ratio based on AUClast.

Exposure, as measured by AUClast, was highest in adrenal gland, cecum, fat, small and large intestine, and liver (443,000 hng/g to 251,000 hng/g) of males, and highest in the adrenal gland, liver, and small intestine (775,000 hng/g to 268,000 hng/g) of females. All other tissues, with the exception of the eye, testes (male only), and brain (females only) had some exposure. Most tissue concentrations were near or below LLOQ by 168 h post-dosing. Tissue:plasma ratios were calculated using AUClast, and were less than 0.7 for poorly perfused tissues e bone, skin, muscle, heart, testes (males only), but also the uterus, thymus, pituitary, kidney (medulla) and lung in females. For remaining tissues including those associated with the GI tract, the tissue:plasma ratio was as high as 4 (adrenal gland, females). As with AM-1, repeated dosing did not appear to alter the tissue distribution of LCFA equivalents versus a single administration. Excluding the GI tract, tissue radioactivity was primarily associated with circulating whole blood and no target tissues were identified. 3.4.5. Tissue distribution of AM-1 and LCFA For both AM-1 and LCFA, the exposure of tissues to test article related equivalents was generally within 2-fold the plasma exposure as determined by tissue:plasma ratios calculated from AUClast; ratios were higher for those tissues associated with the GI tract and with excretion, and lower for poorly perfused tissues (fat was sometimes an exception). Tissue concentrations and the magnitude of the exposure were generally similar for AM-1 and LCFA equivalents and were low relative to the administered dose. No target tissues were identified. The only notable difference between AM-1 and LCFA equivalents was a tendency for Tmax to occur earlier relative to the time of dosing for LCFA than for AM-1. Although there were some high values noted for AUClast and tissue:plasma ratios, overall the concentrations measured for the

adrenal gland were comparable to the other tissues over the entire time course of the study with the exception that at 168 h concentrations were just marginally above the LLOQ. Generally concentrations for the other tissues were below the LLOQ at the terminal time point. The region of the curve from 48 to 168 h contributed a significant amount to the overall AUC for the adrenal gland, resulting in what appeared upon initial review to be aberrantly high tissue:plasma ratios relative to other tissues. 4. Discussion The objective of this study was to obtain information on the pharmacokinetics, excretion balance, and tissue distribution of [14C]-AM-1 and [14C]-LCFA equivalents following single or repeated administration to Sprague Dawley rats. Rats received equimolar doses of either [14C]-AM-1 or its major hydrolysis product, [14C]LCFA, via oral or iv administration followed by collection of biological samples at specified intervals. After a single oral dose of [14C]-AM-1 at 100 mg/kg or equimolar 14 [ C]-LCFA at 46 mg/kg, circulating radioactivity at Cmax was equal to <0.1% (AM-1 equivalents) and 0.2% (LCFA equivalents) of the administered dose, if the approximate amount of circulating plasma in a 250 g rat is assumed to be 7.8 g. The initial phase in the PK profile, whose primary character is determined by absorption, differed between AM-1 and LCFA equivalents and Tmax was much earlier for LCFA equivalents. We interpret this observation as indicating that orally administered LCFA was directly and rapidly absorbed, whereas AM-1 underwent a number of hydrolysis steps before its components were absorbed; therefore, accounting for the time delay in Tmax for AM-1. Also, based on the iv profiles, slow absorption of AM-1 followed by hydrolysis in circulation may be occurring. Exposure to the test articles as measured by AUClast, was

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up to 140 mgh/g for AM-1 equivalents and up to 112 mgh/g for LCFA equivalents. There was approximately a 2-fold difference in the dose-normalized AUClast between AM-1 (approximately 1.3) and LCFA (approximately 2.3) equivalents, which is proportional to the higher molecular weight of AM-1. However, there was no apparent difference in the terminal elimination phase half-life between AM-1 and LCFA equivalents, which was determined to be approximately 30 h. After a single iv (slow push) dose of [14C]-AM-1 at 10 mg/kg or equimolar [14C]-LCFA at 4.6 mg/kg, C0 was up to 109 mg/g for AM-1 equivalents and up to 8.48 mg/for LCFA equivalents. Higher C0 for AM-1 versus LCFA equivalents is tentatively attributed to a transient contribution from glucose, xylose, acetate, and isovalerate, following the hydrolysis of AM-1. Owing to the short half-life of these compounds, they would be rapidly removed from the circulating blood, whereas LCFA would not, resulting in the initially higher C0 for AM-1 compared with LCFA, followed by similar plasma concentrations for both substances. Exposure to the test articles as measured by AUClast, was 88 mg h/g for AM-1 equivalents (regardless of sex) and up to 76.5 mg h/g for LCFA equivalents. There was approximately a 2-fold difference in the dose-normalized AUCinf between AM-1 (approximately 11) and LCFA (approximately 22) equivalents; which mirrors the two-fold difference in MW of these compounds. Based on the similarity of the PK analysis, and subsequent tissue distribution and excretion balance behavior, the data is interpreted to mean that an equivalent concentration of LCFA (the component hydrolysis product of AM-1) was present in systemic circulation following equimolar administration of AM-1 or LCFA, suggesting that AM-1 can be hydrolyzed to LCFA in circulation following an iv dose. Because the molecule is uniformly labeled, the calculations transforming administered radioactivity necessarily assume that test article equivalents result from AM-1 (average MW ¼ 985) following administration of AM-1, when it is likely a two-fold concentration of AM-1 derived LCFA (average MW ¼ 455) with similar total mass. Thus, as a function of moles of test article administered, the pharmacokinetic behavior of AM-1 and LCFA equivalents following iv administration were virtually indistinguishable (Figs. 4A,B and 5A,B). However, there was no apparent difference in the terminal elimination phase half-life between AM-1 and LCFA equivalents, which was determined to be approximately 37 h (overall average). The similarity between AM-1 and LCFA equivalents in the terminal elimination phase following iv administration also indicates that AM-1 is at least partially hydrolyzed in circulation to LCFA. Based on the excretion data, approximately 88%e101% of the administered dose was recovered in expired air, urine, feces, and carcass from male or female rats following single or repeated oral administration of [14C]-AM-1 at 100 mg/kg or single or repeated oral administration of equimolar [14C]-LCFA at 46 mg/kg. There was no difference in the excretion of AM-1 or LCFA based on sex (males versus females) or single versus repeated exposures. Approximately 15%e21% of the recovered radioactivity was captured in the CO2 trap for expired air, approximately 1%e3% of the recovered radioactivity was in the urine, and approximately 63%e83% of the dose was eliminated in feces. Possible sources of CO2 include intestinal metabolism of hydrolysis products by microbiota. It is likely that only a very small fraction of the AM-1 is hydrolyzed before elimination, because if a significant fraction were hydrolyzed, then a strong contribution of the metabolites (other than LCFA) on the PK and excretion balance would be seen, especially differences in the percent of dose in the urine. Approximately 2% of the recovered radioactivity was in the carcass of select animals. The majority of both test articles are eliminated in the feces without absorption. Thus, there appeared to be no difference in the excretion of AM-1 or LCFA equivalents based on sex (males versus females) or single

567

versus repeated exposures. The apparent oral bioavailability (F) of AM-1 and LCFA equivalents (approximately 11%; overall average for males and females combined) determined in the pharmacokinetic phase may reflect the absorption of degradation products of AM-1 and LCFA formed in the gut including CO2 (which must be present systemically to be eliminated in the expired air), monosaccharides (from AM-1 only) and short chain fatty acids (SCFA). Quantitative analysis of the disposition of the radioactivity into the possible metabolic products formed prior to or after absorption is not practical due to the number of radiolabeled species and the number of absorption, distribution, and elimination processes occurring concurrently, but the existing data support the conclusion that oral bioavailability of both AM-1 and LCFA equivalents is very low with 0.2% of the administered dose present in the plasma at Cmax, and less than 3.5% of the dose recovered in the urine. [14C]-AM-1 or [14C]-LCFA equivalents-derived radioactivity was detected by quantitative whole body autoradiography in all tissues measured. Cmax and AUClast for [14C]-AM-1- and [14C]-LCFA-equivalents-derived radioactivity was highest in the tissues of the GI tract, as expected following oral administration. The eye, testes (males only), and brain had only very low concentrations at a small number of time points in select animals. The remaining tissues had concentrations of test article equivalents ranging from just above the lower limit of quantitation to <30,000 ng/g (excluding GI tract), which represented a low concentration relative to the administered dose, and no target tissues were identified. Tmax was variable depending on the tissue, but generally correlated with the plasma or whole blood Tmax, and was often earlier for LCFA than for AM-1. For the majority of the tissues, T1/2 was considered not to be reportable or was an approximation; however, both AM-1 and LCFA concentrations were BLQ by 168 h following either single or repeated dosing suggesting tissue half-lives of approximately 24e40 h, which is consistent with the observed plasma T1/2. Tissue:plasma ratios (calculated using AUClast) indicated that test article equivalents in tissue were primarily associated with the perfusion of tissues by whole blood, and were <0.7 for poorly perfused tissues. In addition to the similarity in tissue distribution between AM-1 and LCFA, there was no notable impact of single versus repeated dosing on the tissue distribution of either test article. Variations in the concentrations of AM-1 equivalents in tissues of the GI tract are expected based on individual variability in intestinal motility depending on the rat and when diet was ingested. The high concentrations in the various tissues of the GI tract, low concentration of circulating radioactivity, and low concentrations in the remaining tissues are consistent with low oral bioavailability, which suggests only a small portion of the administered AM-1 was absorbed into systemic circulation, as also evident in the autoradioluminograms. In conclusion, AM-1 and LCFA and their GI tract metabolites are poorly absorbed by the oral route and are primarily eliminated in the feces without absorption. The pharmacokinetic, tissue distribution, and excretion balance data derived in this study are consistent with an interpretation that following ingestion, AM-1 is partially hydrolyzed to its components, glucose, xylose, acetate, isovalerate and LCFA. The expected small primary metabolites glucose, xylose, acetate, and isovalerate are expected to have a fast and high bioavailability but rapid clearance and thus to contribute marginally to the observed test article equivalents in blood and tissues after oral administration of AM-1. These results support an interpretation that systemic exposure to AM-1 or its metabolites would be very limited following oral ingestion. AM-1 is proposed for use as a food ingredient with antimicrobial/preservative properties without offering nutritional, taste or other technical benefits to foods or beverages. Therefore, limited absorption of AM-1 is a

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desirable property and serves to minimize exposure by the consumer to AM-1 or its major hydrolysis product, LCFA. In addition to limited exposure after a single dose there was no change to the pharmacokinetics, distribution, and elimination after repeated exposures, which is positive in regards to its application as a food ingredient where repeated ingestion is expected. Conflict of interest Authors Bitzer and Henkel are employees of IMD Natural Solutions GmbH (INS), the funding sponsor of this study and submitter of an associated patent application. Authors Rihner and Nikiforov are consultants to INS, the study sponsor. Author Thomas is employed by Charles River Laboratories Ashland which received funding to conduct the study. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2017.08.038. References Bitzer, J., Henkel, T., Nikiforov, A.I., Rihner, M.O., Leuschner, J., 2017a. A 90-day oral

toxicity study of glycolipids from Dacryopinax spathularia in CD(®) rats. Food Chem. Toxicol. http://dx.doi.org/10.1016/j.fct.2017.06.040, 2017 Jun 27. pii: S0278e6915(17)30358-7. [Epub ahead of print]. Bitzer, J., Henkel, T., Nikiforov, A.I., Rihner, M.O., Henderson, K.A., 2017b. A 90-day oral toxicity study of glycolipids from Dacryopinax spathularia in Beagle dogs. Food Chem. Toxicol. http://dx.doi.org/10.1016/j.fct.2017.07.026, 2017 Jul 13. pii: S0278e6915(17)30398-8. [Epub ahead of print]. FDA, 1987. Good Laboratory Practice Regulations. Final Rule. 21 Code of Federal Regulations (CFR) Part 58. Food and Drug Administration, US (FDA) Department of Health and Human Services, Washington, DC. FDA, 1993. Toxicological Principles for the Safety Assessment of Food Ingredients: Redbook II, Draft Guidance. Chapter V B, Metabolism and Pharmacokinetic Studies. Food and Drug Administration, US (FDA), Center for Food Safety and Applied Nutrition (CFSAN), Washington, DC. Husen, B., Hurst, L., Biemel, K., 2013. Pharmacokinetic Evaluation of IMD AM-1 after Intravenous and Oral Administration to Rats and Identification of Components and Major Metabolites in Plasma. Pharmacelsus Project No. 2012IMD001. Unpublished Report submitted to IMD Natural Solutions GmbH. Husen, B., Hurst, L., Biemel, K., 2014. Excretion and Orientating Mass Balance Analysis of IMD AM-1 after Oral Administration to Rats. Pharmacelsus Project No. 2014IMD001/2014IMD001b. Unpublished Report submitted to IMD Natural Solutions GmbH. Martin, G.W., 1948. New or noteworthy tropical fungi. IV. Lloydia 11 (2), 116. Schweinitz, L.D., 1832. Synopsis fungorum in America boreali media degentium. Trans. Am. Philosophical Soc. 4 (2), 153. € pcke, B., Reinhardt, K., Moldenhauer, J., 2012. Long Chain Stadler, M., Bitzer, J., Ko Glycolipids Useful to Avoid Perishing or Microbial Contamination of Materials. WO 2012/167920A1.