1-butylpyrrolidin-2-one

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-19 to 2016-11-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Objective of study:

distribution

excretion

metabolism

toxicokinetics

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

deviations from the protocol did not impact the quality or integrity of the study.

GLP compliance:

yes

Remarks:

in compliance with the United States FDA GLP Regulations (21 CFR Part 58), 05 Oct 1987.

Test material

Test material information

Constituent 1

Reference substance name:

1-butylpyrrolidin-2-one

EC Number:

222-437-8

EC Name:

1-butylpyrrolidin-2-one

Cas Number:

3470-98-2

Molecular formula:

C8H15NO

IUPAC Name:

1-butylpyrrolidin-2-one

Test material form:

liquid

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 98.7%
- Specific activity: 1 x2.0 mCi
- Locations of the label: The test article was [14C]-labeled in the 2'-position of the benzene ring (C=O) (please see picture below).

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

In general, this species and breed of animal is recognized to be appropriate for this type of metabolism study. The rat was utilized because it is a widely used species for which significant historical control data are available, and because it has been determined to be a pharmacologically responsive species. Additionally, this number of animals is consistent with regulatory agency expectations.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD(SD)
- Source: Charles River Raleigh, NC.
- Age at study initiation: approximately 8 weeks of age.
- Weight at study initiation: the mean: 183 g at receipt and 241 g at randomization; between 226 g and 278 g at the time of dosing.
- Housing: All animals were housed in a study-dedicated, environmentally controlled room, individually in clean, suspended wire-mesh cages. Wire-mesh caging was utilized to acclimate the animals to the wire-mesh flooring utilized in the Nalgene-type metabolism cages utilized during excreta collection. In addition, solid bottom and group housing is not appropriate for radiolabeled studies where the study objectives (quantitative analysis of test article exposure by individual animal) would be compromised by test article exposure from other animals. Cages were elevated above cage-board, which was changed at least 3 times each week. Individual cage cards were affixed to each cage displaying the animal number, group number, and study number. After dosing, animals in the Excretion Mass Balance phases were placed into glass metabolism cages for up to 168 hours. Enrichment devices were provided to each animal for environmental enrichment and to aid in maintaining the animals’ oral health. Devices were not used in the 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).
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (block) was offered ad libitum during acclimation and the biological phase of the study.
- Water (e.g. ad libitum): Reverse osmosis-treated water was available ad libitum. Water was provided using an automatic water system. Water bottles were used for animals placed into metabolism cages.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 73°F ± 5°F (22°C ± 3°C). The actual daily mean temperature ranged from to 71.9°F to 72.9°F (22.2°C to 22.7°C).
- Humidity (%): 50% ± 20%. The actual mean daily humidity ranged from 43.0% to 59.5%.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: receipt: 07 and 14 of June 2016 To: scheduled euthanasia: 7 days after the dosing

Administration / exposure

Route of administration:

other: a single oral (gavage) or intravenous (IV, bolus) dose

Vehicle:

physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Prior to dose preparation, the vial containing [14C]-test article (MET-1015A) was placed under a gentle stream of nitrogen to evaporate the ethanol almost to dryness. Using a syringe, the [14C]-test article was re-suspended with 2 mL of sterile 0.9% saline. The vial was capped, the contents inverted and vortex-mixed to ensure complete dissolution of [14C]-test article.
The solution was assigned reference no. 387082MA1-2-1.
For the Group 1/Group 3 dose formulation, a balance was used to weigh 1.1384 g of test article (TMS No. 150355) into a glass container previously calibrated to contain 38 mL with a stir bar. Using a syringe, 10 mL of sterile 0.9% saline was added to the same glass container. Using a pipette, 0.7 mL of the [14C]-test article stock solution (reference no. 387082MA1-2-1) was added to the same glass container and the contents stirred using a magnetic stir bar and stir plate. While the contents were stirring, the container was filled to the calibration mark using sterile 0.9% saline. The formulation was assigned reference no. 387082MA1-3-1 and continued to mix throughout use. The formulation was stored frozen at approximately -20°C when not in use.
For the Group 2 dose formulation, a balance was used to weigh 0.1033 g of test article (TMS No. 150355) into a glass container previously calibrated to contain 7.5 mL with a stir bar. Using a syringe, 2.5 mL of sterile 0.9% saline was added to the same glass container. Using a pipette, 0.7 mL of the [14C]-test article stock solution (reference no. 387082MA1-2-1) was added to the same glass container and the contents stirred using a magnetic stir bar and stir plate. While the contents were stirring, the container was filled to the calibration mark using sterile 0.9% saline. The formulation was sterile-filtered into a sterile septum-sealable container while in a Laminar flow hood. The formulation was assigned reference no. 387082MA1-4-2 and stored frozen at approximately -20°C when not in use.
For the Group 4/Group 5 dose formulation, a balance was used to weigh 0.9580 g of test article (TMS No. 150355) into a glass container previously calibrated to contain 32 mL with a stir bar. Using a syringe, 10 mL of sterile 0.9% saline was added to the same glass container. Sterile 0.9% saline was used to transfer the remaining 0.6 mL of the [14C]-test article stock solution (reference no. 387082MA1-2-1) into the same glass container and the contents stirred using a magnetic stir bar and stir plate. While the contents were stirring, the container was filled to the calibration mark using sterile 0.9% saline. The formulation was assigned reference no. 387082MA1-7-1 and continued to mix throughout use. The formulation was stored frozen at approximately -20°C when not in use.
The radiochemical concentration and purity of the Group 1/Group 3 [14C]-test article dose formulation (reference no. 387082MA1-3-1) was assessed prior to and following dose administration.

Duration and frequency of treatment / exposure:

single administration.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Pharmacokinetic phase (in case of iv route of exposure)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

For all phases of the study (in case of oral route of exposure)

No. of animals per sex per dose / concentration:

12/12 for pharmacokinetic phase(oral / iv routes), 2 for the Preliminary Excretion Balance (Pilot) Phase, 4 for the Excretion Mass Balance Phase, and 7 for the Tissue Distribution (QWBA) Phase

Control animals:

not specified

Positive control reference chemical:

No

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): Animals were assigned to study at random using a computer program. One animal not dosed with test material was used for matrix.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood
- Time and frequency of sampling: At approximately 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, and 72 hours postdose, blood samples of approximately 0.5 mL were collected from 4 animals/time point

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, cage washes, plasma and expired air.
- Time and frequency of sampling: Following dosing, Group 3 animals were placed into individual glass metabolism cages equipped for separate collection of expired air, urine, and feces. CO2 trapping solutions were collected at approximately 0 to 6, 6 to 12, 12 to 24, 24 to 36, and 36 to 48 hours postdose. At the end of each CO2 collection interval, the interior surface of the collection tube was rinsed with MeOH and the rinse added to the CO2 trapping solution collection.The activated carbon trap was collected at 24 and 48 hours postdose. Urine was collected on wet ice at approximately 0 to 6, 6 to 12, 12 to 24, and 24 to 48 hours postdose. At the end of each urine collection interval, the interior surface of the cage walls was rinsed with DI water and the rinse retained as a separate sample. Feces were collected on wet ice at approximately 0 to 12, 12 to 24, and 24 to 48 hours postdose (Group 3: Preliminary Excretion Balance (Pilot) Phase).

Following dosing, Group 4 animals were placed into individual glass metabolism cages equipped for separate collection of expired air, urine, and feces. CO2 trapping solutions were collected at approximately 0 to 6, 6 to 12, 12 to 24, 24 to 36, and 36 to 48 hours postdose. At the end of each CO2 collection interval, the interior surface of the collection tube was rinsed with MeOH and the rinse added to the CO2 trapping solution collection. The activated carbon trap was collected at 24 and 48 hours postdose. Urine was collected on wet ice at approximately 0 to 6, 6 to 12, and 12 to 24 hours postdose, and then every approximately 24 hours through 168 hours postdose. At the end of each urine collection, the interior surface of the cage walls was rinsed with DI water and the rinse retained as a separate sample. Feces were collected on ice at approximately 0 to 12 and 12 to 24 hours postdose, and then every approximately 24 hours through 168 hours postdose. After the final urine and feces collection, cages were washed with DI water and the washes retained as a separate sample. Following the final 168 hour collection, animals were euthanized by CO2 inhalation and carcasses weighed and retained in freezer storage (approximately -20°C) for possible analysis (Group 4: Excretion Mass Balance Phase).

At approximately 0.25, 1, 4, 8, 24, 72, and 168 hours postdose, 1 animal/time point was anesthetized with isoflurane and blood samples of approximately 4 mL were collected via cardiac puncture into tubes containing K2EDTA as the anticoagulant. Following blood collection, each anesthetized animal was euthanized by CO2 inhalation and submerged in a dry ice/hexane bath. Frozen carcasses were stored at approximately -20°C until processed for QWBA (Group 5: Tissue Distribution Phase).

- From how many animals: from all animals (samples have been pooled)
- Method type(s) for identification: Liquid scintillation counting (Groups: 1-4); Quantitative Whole Body Autoradiography (QWBA) (Group 5); HPLC-MS (Study Phase: Metabolite Identification)
- Limits of detection and quantification: For each sample analyzed, aliquots of samples collected from undosed animals were fortified with a dilution of the [14C]-test article dosing formulation. Recovery was 95% and 100% for plasma (low and high, respectively) and 97% and 101% for whole blood (low and high, respectively). Excreta recovery ranged from 81% to 112%. The sensitivity of the LSC procedures was estimated using the average background of each sample type assayed. Approximate mean aliquot size and dose were used in the calculations. e quantitation limit in terms of test article equivalents: see table 4 below.
- Other:

Statistics:

Pharmacokinetic Analysis
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). Non-compartmental analysis was employed with linear trapezoidal summation for the AUC calculations. The methods and equations for calculation of the pharmacokinetic parameters are presented in the table 5.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Test article is fully absorbed into systemic circulation following oral dosing

Main ADME results 2

Type:

distribution

Results:

Test article is widely distributed predominantly to the adrenal glands, spleen, stomach, pancreas and kidneys.

Main ADME results 3

Type:

metabolism

Results:

The test article was extensively metabolized by the rat following oral administration. Two major metabolites (hydroxylated derivatives) were formed following oral administration. Further metabolites are: N-(OH-butyl)-succinimide and glucuronide conjugate.

Main ADME results 4

Type:

excretion

Results:

The test substance is excreted predominantly via the urine. Approximately 94.1% of the recovered radioactivity was in the urine (including cage rinse), and approximately 4.28% of the dose was eliminated in feces.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Bioavailability and excretion data confirm that [14C]-test article is fully absorbed to systemic circulation following oral dosing in Crl:CD(SD) rats. After a single oral dose of [14C]-test article to male rats in Group 1 at 300 mg/kg, Cmax was 188 μg-equiv/g at 4 hours postdose; AUClast, was 3010 h•μg-equiv./g, and the half-life was 7.96 hours.
Overall, [14C]-test article-derived radioactivity was widely dispersed, slightly favoring the well-perfused organs and organs associated with oral absorption, and rapidly excreted.

Details on distribution in tissues:

The concentrations of test article equivalents in tissues of male rats are presented in Table 6 (please see attached). [14C]-test article-derived radioactivity was detected in all the tissues examined through 24 hours postdose, except fat, which was quantifiable through 8 hours postdose.
The Cmax for [14C]-test article-derived radioactivity was highest in the adrenal gland (1210 μg-equiv/g) followed by the spleen (632 μg-equiv/g), stomach (484 μg-equiv/g), pancreas (465 μg-equiv/g) and kidney (353 to 374 μg-equiv/g). The remaining tissues were all similar in concentration, ranging from 70 μg-equiv/g (fat) to small intestine (240 μg-equiv/g). There was little notable difference in the concentration of [14C]-test article-derived radioactivity in the tissues. The Tmax was generally 4 hours postdose except for bone and liver (1 hour postdose). Limited data points between Cmax and BLQ precluded calculation of the half-life except in the bone (1.1 h) and liver (5.5 h).
Overall, [14C]-test article-derived radioactivity was widely dispersed, slightly favoring the well-perfused organs and organs associated with oral absorption, and rapidly excreted.
Exposure, as measured by AUClast, generally reflected the order noted for Cmax. Exposure was lowest in fat (<450 h•μg-equiv./g) and highest for the adrenal gland, kidney (all sections), and spleen. Tissue exposure relative to the plasma (tissue:plasma AUClast ratios) were < 1 for most tissues; the highest was 2.26 for the adrenal gland.
Whole blood and plasma data collected from QWBA animals was consistent with the plasma values determined in the PK phase. The whole blood:plasma ratio for AUClast was 0.912, indicating that [14C]-test article-derived radioactivity was evenly distributed between the plasma and blood cells.

Details on excretion:

Based on the excretion data collected during the pilot phase (Group 3), a small fraction (approximately 1%) of the administered dose was captured in the expired air. To ensure mass balance, expired air was also collected in the definitive excretion balance phase (Group 4). Approximately 99.5% of the dose administered to animals in Group 4 was recovered in expired air, urine, and feces from male rats following a single oral administration of [14C]-test article at 300 mg/kg. In agreement with the pilot phase, approximately 1.17% of the recovered radioactivity was in expired air. No volatile components were retained in the activated carbon trap. Approximately 94.1% of the recovered radioactivity was in the urine (including cage rinse), and approximately 4.28% of the dose was eliminated in feces. Because mass balance was achieved, carcasses were not analyzed.

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 188 μg-equiv/g at 4 hours postdose

Remarks:

after a single oral dose of [14C]-test article to male SD rats in Group 1 at 300 mg/kg

Toxicokinetic parameters 2

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: The plasma exposure, AUClast, was 3010 h•μg-equiv./g.

Remarks:

Group 1at 300 mg/kg

Toxicokinetic parameters 3

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: The terminal elimination phase was well defined (R2 = 0.997); the half-life was 7.96 hours.

Remarks:

Using the terminal elimination phase rate constant, AUCinf was extrapolated and, at 3010 h•μg-equiv./g, was virtually the same as AUClast.

Toxicokinetic parameters 4

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: After a single IV dose of [14C]-test article to male Crl:CD(SD) rats in Group 2 at 30 mg/kg, the extrapolated concentration at the time of administration (C0) was 40 μg-equiv/g. The plasma exposure, AUClast, was 212 h•μg-equiv./g.

Toxicokinetic parameters 5

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: The terminal elimination phase was well defined (R2 = 0.933); the half-life was 9.89 hours. Using the terminal elimination phase rate constant, AUCinf was extrapolated and found to be 213 μg•h/g.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The test article was extensively metabolized by the rat following oral administration; by 8 hours postdose (oral or IV administration) the test article was responsible for < 3% of the radioactivity circulating in plasma. Up to 13 individual metabolites were identified in plasma, plus 1 unknown metabolite and unchanged test article; 8 total metabolites were present at > 5% ROI in at least 1 sample of plasma collected following either route of administration. Two major metabolites were formed. Following oral administration, N-butyl-5-OH-pyrrolidone (M41) increased in relative concentration over the time course and comprised up to 70% ROI in plasma collected at 8 hours postdose (up to 120 μg/g). One other hydroxy metabolite (M35) accounted for up to 11.3% ROI at 4 hours postdose.
Following oral administration, rat urine contained up to 38 metabolites. Ten metabolites were present at > 5% ROI in at least 1 sample. Across all collections, 3 metabolites were present at > 5% of the administered dose: N-(OH-butyl)-succinimide (M22, 6%), N-butyl-5-OH-pyrrolidone (M41, 11%), and a glucuronide conjugate of OH-N-butylpyrrolidone (M44, 11%). Parent test article was present at 0.3% of the administered dose in all urine samples combined.
Twelve metabolites were identified in rat feces extractions. Eight metabolites were present at > 5% ROI in at least 1 sample. The summed % of Dose from all of the feces collections analyzed were also calculated; no summed metabolites were present at > 5% of the administered dose. Summed parent test article was present at 0.3% of the dose (please find attached a table with metabolites and their structural formula).

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

Based on AUClast (212 h•μg-equiv./g.; Group 2 - iv administration) the absolute oral bioavailability (F) of radioactivity was 142%. Calculated bioavailability > 100% is likely due to an artifact caused by a large difference in the doses administered and does not appear to be based on saturation of metabolic pathways; evaluation of the plasma curves indicates that there is no difference in clearance following administration by either route.

Any other information on results incl. tables

Dose Formulation Analyses and Actual Dose Administered (Table 1, please see attached).

The [14C]-test article dosing formulations administered to rats via oral (gavage) were formulated to provide each animal a target dosage of 300 mg/kg and 200 μCi/kg of radioactivity. The [14C]-test article dosing formulation administered to rats via IV (bolus) injection was formulated to provide each animal a target dosage of 30 mg/kg and 200 μCi/kg of radioactivity. The pre and postdose analyses established homogeneity of the solutions as demonstrated by the low % CV, < 3.6% across triplicate analyses at each interval (Table 3-2, Table 3-3, Table 3-4; Appendix 3). The mean radioconcentration of the formulation for Group 1 and Group 3 on the day of administration was 17.8 μCi/g, or 96.1% of the target. The mean radioconcentration of the formulation for Group 2 on the day of administration was 89.1 μCi/g, or 94.1% of the target. The mean radioconcentration of the formulation for Group 4 and Group 5 on the day of administration was 21.1 μCi/g, or 112.5% of the target.

Radio-HPLC analyses of the dosing solution were performed prior to (all formulations) and after completion of dosing (Group 1 and Group 3, and Group 4 and Group 5 formulations). As indicated by similar pre and postdose radiopurity results, the radiolabeled test article was stable over the dosing period (Table 3-5, Figure 3-1, Figure 3-2, Figure 3-3, Appendix 3). There was no adverse impact resulting from the lack of postdose radiopurity analysis for the Group 2 formulation; the test article stability was adequately established for the other formulations prepared in the same vehicle. The mean radiopurity for each formulation ranged from 98.4% to 98.9%.

The amount of test article to be administered was based on the body weights of the animals on the day of dosing (Table 4-1; Appendix 4). For Group 1, Group 2, and Group 5, the nominal amount delivered was based on the volume administered to each animal. For the purpose of determining mass balance, the actual amount of dosing formulation administered to each animal in Group 3 and Group 4 was quantified by weighing the dosing syringe before and after dosing. Calculations of actual administered doses for individual animals are presented in Table 5-1 (Appendix 5) and mean actual administered dosages of test article are presented in Table 1. Rats received 98% to 102% of the target mass (mg/kg) and 90% to 103% of the target radioactivity (μCi/kg).

Applicant's summary and conclusion

Conclusions:

Bioavailability and excretion data confirm that [14C]-test article is fully absorbed to systemic circulation following oral dosing in Crl:CD(SD) rats. After a single oral dose of [14C]-test article to male rats in Group 1 at 300 mg/kg, Cmax was 188 μg-equiv/g at 4 hours postdose; AUClast, was 3010 h•μg-equiv./g, and the half-life was 7.96 hours. After a single IV dose of [14C]-test article to male SD rats at 30 mg/kg, the extrapolated concentration at the time of administration (C0) was 40 μg-equiv/g, AUClast was 212 h•μg-equiv./g, and the half-life was 9.89 hours. The test article is widely distributed to all tissues, and tissue concentrations were correlated to the degree of perfusion by circulating blood. Test article equivalents are rapidly and completely excreted in the urine (94.1%); < 6% of the administered dose was recovered in the feces and expired air.
The test article was extensively metabolized by the rat following oral administration; by 8 hours postdose (oral or IV administration) the test article was responsible for < 3% of the radioactivity circulating in plasma. Two major metabolites were formed: N-butyl-5-OH-pyrrolidone (M41) and 1 other hydroxy metabolite (M35). Rat urine contained up to 38 metabolites; 3 metabolites were present at > 5% of the administered dose: N-(OH-butyl)-succinimide (M22, 6%), N-butyl-5-OH-pyrrolidone (M41, 11%), and a glucuronide conjugate of OH-N-butylpyrrolidone (M44, 11%). Parent test article was a minor component of urine, present at 0.3% of the administered dose in all urine samples combined. Twelve metabolites were identified in rat feces extractions and 8 were present at > 5% ROI in at least 1 sample, but no metabolites were present at > 5% of the administered dose. As with urine, parent test article was a minor component (0.3% of the dose).

Executive summary:

The objectives of this study were to determine the plasma pharmacokinetics of [14C]-N-butyl pyrrolidone ([14C]-test article)-derived radioactivity in male Crl:CD(SD) rats following a single oral (gavage) or intravenous (IV, bolus) dose. The routes of elimination and excretion mass balance of [14C]-test article-derived radioactivity, and the tissue distribution and tissue pharmacokinetics of [14C]-test article-derived radioactivity using QWBA methods, were determined in male Crl:CD(SD) rats following a single oral (gavage) dose. Plasma, urine, and fecal homogenate samples were also used for metabolite profiling of [14C]-test article-derived radioactivity.

Study design

For the pharmacokinetic phase, 2 dose groups, each consisting of 12 male rats, received either a single oral dose of [14C]-test article at 300 mg/kg and a target radioactivity of 200 μCi/kg or a single IV dose of [14C]-test article at 30 mg/kg and a target radioactivity of 200 μCi/kg. Following dosing, blood samples were collected from 4 animals/group/time point at approximately 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, and 72 hours. Blood samples were processed to plasma for analysis by LSC. For the preliminary excretion balance (pilot) phase, 1 dose group consisting of 2 male rats received a single oral dose of [14C]-test article at 300 mg/kg and a target radioactivity of 200 μCi/kg. Following dosing, animals were placed into glass metabolism cages for separate collection of urine, feces, and expired air through 48 hours. For the excretion balance phase, 1 dose group consisting of 4 male rats received a single oral dose of [14C]-test article at 300 mg/kg and a target radioactivity of 200 μCi/kg. Following dosing, animals were placed into glass metabolism cages for separate collection of expired air through 48 hours and urine and feces through 168 hours. Urine, cage rinse, feces, cage wash, expired air, and carbon trap samples from both excretion balance phases were analyzed by LSC. For the tissue distribution (QWBA) phase, 1 dose group consisting of 7 male rats received a single oral dose of [14C]-test article at 300 mg/kg and a target radioactivity of 200 μCi/kg. At approximately 0.25, 1, 4, 8, 24, 72, and 168 hours postdose, a whole blood sample was collected from 1 animal/time point. Following blood collection, animals were euthanized by CO2 inhalation and carcasses frozen in a dry ice/hexane bath for analysis by QWBA. Whole blood and plasma samples were analyzed by LSC. Select plasma, urine, and feces samples were selected for radioprofiling and metabolite identification by HPLC-MS/MS with in-line radiodetection.

Results and Key findings

Bioavailability and excretion data confirm that [14C]-test article is fully absorbed to systemic circulation following oral dosing in male rats. After a single oral dose of [14C]-test article to male rats in Group 1 at 300 mg/kg, Cmax was 188 μg-equiv/g at 4 hours postdose; AUClast, was 3010 h•μg-equiv./g, and the half-life was 7.96 hours. After a single IV dose of [14C]-test article to male rats at 30 mg/kg, the extrapolated concentration at the time of administration (C0) was 40 μg-equiv/g, AUClast was 212 h•μg-equiv./g, and the half-life was 9.89 hours. [14C]-test article is widely distributed to all tissues, and tissue concentrations were correlated to the degree of perfusion by circulating blood. At Cmax, most tissue concentrations were within an approximate 5-fold range. Tissue exposure, as measured by AUClast, was lowest in the fat (<450 h•μg-equiv./g) and highest for the adrenal gland, kidney (all sections), and spleen (> 3750 h•μg-equiv./g). Tissue exposure relative to the plasma (tissue:plasma AUClast ratios) were < 1 for most tissues; the highest was 2.26 for the adrenal gland.

Test article equivalents are rapidly and completely excreted in the urine (94.1%); < 6% of the administered dose was recovered in the feces and expired air.

The test article was extensively metabolized by the rat following oral administration; by 8 hours postdose, the test article was responsible for < 3% of the radioactivity circulating in plasma. Two major plasma metabolites were identified: N-butyl-5'-OH-pyrrolidone (M41) and 1 other hydroxy metabolite (M35). Rat urine contained up to 38 metabolites; 3 metabolites were present at > 5% of the administered dose: N-(OH-butyl)-succinimide (M22, 6%), N-butyl-5'-OH-pyrrolidone (M41, 11%), and a glucuronide conjugate of OH-N-butylpyrrolidone (M44, 11%). Twelve metabolites were identified in rat feces extractions and 8 were present at > 5% ROI in at least 1 sample, but no metabolites were present at > 5% of the administered dose. Parent test article was a minor component of urine and feces, present at a total of 0.3% of the administered dose in all samples for each matrix.