Benzamide, N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxy-

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2004 - 24 Feb 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2004 - 24 Feb 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 4 Apr 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium fur Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen

Radiolabelling:

yes

Remarks:

14C labelled test substance (Methoxybenzoyl-ring)

Species:

rat

Strain:

other: Wistar Hsd/Cpb: WU

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan & Winkelmann Versuchstierzucht GmbH, Borchen, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: Approx. 200 g
- Housing: Individually in Makrolon metabolism cages.
- Individual metabolism cages: Yes
- Diet: Rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, Switzerland; ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-25
- Humidity (%): 50-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the radiolabelled and non-radiolabelled test substance an adequate aliquot of the stock solution was taken and evaporated to near dryness under a gentle stream of nitrogen. The residue was suspended in 28 mL of 0.5% aqueous Tragacanth.

Duration and frequency of treatment / exposure:

single exposure

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Actual dose received: 4.94 mg/kg bw

No. of animals per sex per dose / concentration:

8 males

Control animals:

yes

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: The selected dose level was based on the dosage recommendations of the current EPA Health Effects Guidelines and was in the same range as the low dose tests in the corresponding rat metabolism studies.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, expired air
- Time and frequency of sampling: 1, 4, 8, 24, 48, 72, 96, 120, 144 and 168 h after dosing (The control animal was sacrificed after 4 h).

Preliminary studies:

no

Type:

absorption

Results:

The test substance was quickly absorbed i.e. the maximum concentration of radioactivity in almost all organs and tissues was found 1 hour after administration.

Type:

distribution

Results:

At all time-points examined, high radioactivity was mainly observed in the excretory organs kidney and liver. The residues in all other organs and tissues were fairly evenly distributed and always lower than the residues observed in blood.

Type:

excretion

Results:

The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete 48 h after administration (90% excretion). Examination of volatiles demonstrated high stability of the labelling position.

Details on absorption:

One hour after the administration of [methoxybenzoyl-ring-UL-14C]AE 0001789, the maximum concentration of radioactivity was observed in the contents of stomach and small intestine, which is an inherent result of the oral administration. High radioactivity was also observed in blood, indicating fast absorption and high bioavailability of AE 0001789.

Details on distribution in tissues:

A high concentration of radioactivity was mainly observed in the excretory organs kidney and liver. The residues in all other organs and tissues were fairly evenly distributed and always lower than the residues observed in blood.
The highest radioactivity concentration (Cmax) in all organs and tissues was observed 1 hour after the administration. The radioactivity levels observed in blood decreased quickly to an intermediate minimum observed in the animal sacrificed 8 hours after the administration (0.230 µg/g), before they increased again to a second maximum 24 hours after the administration (0.375 µg/g). This effect can be attributed to an interruption of the fast absorption in the intestine resulting from the delayed gastric emptying which was observed between 4 and 24 hours after administration.
Overall, the equivalent concentrations in most organs and tissues were relatively low compared to the actual administered amount of 4.94 mg/kg bw. Only the dose normalised concentrations (CN) at Cmax for renal medulla was significantly larger than 1, which is an indication for the efficient depletion of the compound via kidney and urine. The CN-value for blood was also slightly above 1, and the CN-values of liver and renal cortex were slightly below the equilibrium value of 1. All other CN-values were well below 1.

See Attachment 1 (background material) for tabulated distribution data.

Details on excretion:

The excretion of total radioactivity was almost complete 48 h after administration of 14C-AE 0001789. At this time more than 90% of the administered dose had been excreted via urine and faeces. The major part of the radioactivity administered was excreted by urine. The expiration of 14CO2 and other 14C-labelled volatiles was investigated with the animals sacrificed later than 24 hours. During the monitored period of 48 hours, at maximum 0.01% of the administered dose were expired as carbon dioxide or other volatiles in all cases. This demonstrates the high stability of the labelling position with regard to possible formation of volatile products.

See Attachment 2 (background material) for tabulated excretion data.

Metabolites identified:

not measured

Details on metabolites:

The metabolic profiles in urine and faeces extracts were determined by reversed phase HPLC with radio-detection. The recorded metabolic profiles are shown in Attachment 3. No isolation or structure elucidation of compounds detected in these profiles was performed in the study.

Conclusions:

The toxicokinetic behaviour of the test compound was investigated in a GLP-compliant study on rats according to OECD 417. The study is therefore considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat. The 14C labelled test substance (Methoxybenzoyl-ring) was absorbed quickly from the gastrointestinal tract. The maximum concentration of radioactivity in almost all organs and tissues was found 1 hour after the administration. The absorption process was apparently interrupted, probably due to delayed gastric emptying occurring between 4 and 24 hours after dosing. The high radioactivity observed in kidney already 1 hour after dosing indicated that renal excretion commenced immediately after absorption.
The absorbed radioactivity was not uniformly distributed in the body. At all time-points examined, high radioactivity was mainly observed in the excretory organs kidney and liver. The residues in all other organs and tissues were fairly evenly distributed and always lower than the residues observed in blood.
Residues in all organs and tissues decreased rapidly between 1 and 48 hours. In all organs and tissues residues were < LOD or < LOQ at later time-points between 72 and 168 hours after dosing. There was no sign for retention of radioactivity in specific organs or tissues. Residues from glandular organs or tissues responsible for hormonal regulation (such as adrenal, testis, or thyroid gland) were rapidly depleted in parallel with the depletion from the other organs and tissues.
The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete at the end of the study period. No significant expiration of 14C-labelled volatiles was observed.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Jan 2005 - 12 Jun 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 4 Apr 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium fur Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen

Radiolabelling:

yes

Remarks:

14C labelled test substance (Methoxybenzoyl-ring)

Species:

rat

Strain:

other: Wistar Hsd/Cpb: WU

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan & Winkelmann Versuchstierzucht GmbH, Borchen, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: Approx. 200 g
- Housing: Individually in Makrolon metabolism cages.
- Individual metabolism cages: Yes
- Diet: Rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, Switzerland; 16 g/day
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 27-40
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the radiolabelled and non-radiolabelled test substance an adequate aliquot of the stock solution was taken and evaporated to near dryness under a gentle stream of nitrogen. The residue was suspended in 16 mL of 0.5% aqueous Tragacanth.

Duration and frequency of treatment / exposure:

single exposure

Dose / conc.:

2 mg/kg bw/day (actual dose received)

Remarks:

2.05 mg/kg bw (actual dose received)

No. of animals per sex per dose / concentration:

4 males

Control animals:

no

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: The dose level of 2 mg/kg bw was selected based on the toxicological properties of the test substance. This dose level is in the range of the NOEL, but high enough to allow for metabolite identification in excreta.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, plasma
- Time and frequency of sampling: 4, 8, 12, 24, 48, 72 and 96 h (urine); 24, 48, 72 and 96 h (faeces); 0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 24, 32, 48, 72 and 96 h (plasma)

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine, faeces, tissues
- Time and frequency of sampling: 4, 8, 12, 24, 48, 72 and 96 h (urine); 24 h (faeces); at sacrifice (tissues)
- From how many animals: Samples pooled
- Method types for identification: HPLC-UV, TLC-UV, LC-MS

ANALYTICAL METHOD
HPLC/HPTLC-UV combined with a radioactivity detector and LC-MS were used for measuring radioactivity, quantitation of parent compound and/or metabolites in composite urine samples and faeces extracts, and quantitation of the test substance in the administered dose formulations.

Measurement of radioactivity
- Liquid samples: Liquid scintillation counting (LSC). Small organs or tissues (adrenal glands, thyroid, ovaries, perirenal fat,and uterus ) were solubilised by means of a tissue solubiliser (e.g.BTS-450).
- Solid samples: All samples with the exception of those dissolved in BTS were weighed and combusted in an oxygen atmosphere. The released 14C02 was trapped in an alkaline absorber and radio-assayed by LSC using a scintillator.
- The limit of detection (LOD) was established at 20 dpm measured per aliquot after correction for the background radioactivity. The limit of quantification (LOQ) for each individual measurement was established as 2 to 3 times of the background radioactivity (dpm) of each instrument/method.

Statistics:

The values were checked for outliers in accordance with the outlier test by Nalimov, if appropriate. Values identified as outliers were marked and not used for the calculations of arithmetic means and coefficients of variations (in %). In addition, the concentrations or amounts of radioactivity were checked for values below the limit of detection (LOD). Values below the LOD were not considered in calculations of arithmetic means.

Preliminary studies:

no

Type:

absorption

Results:

The test substance was quickly absorbed i.e. the maximum concentration of radioactivity in almost all organs and tissues was found approx. 40 min after administration.

Type:

distribution

Results:

No significant residues remained in the bodies of the rats at sacrifice, 96 h after administration (approx. 0.07% of the administered dose). No radioactive residues were accumulated in any of the tissues, organs or glands.

Type:

metabolism

Results:

AE 0001789 was metabolised to a low extent: Parent compound (76%), AE 0001789-descyclopropylamino (5%), AE 0001789-desmethyl (0.6%), AE 0001789-anisic acid (0.3%).

Type:

excretion

Results:

The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete 24 h after administration (98% excretion of the recovered dose).

Details on absorption:

The test substance was very rapidly absorbed and absorption commenced immediately after oral dosing. The maximum plasma concentration was reached approximately 40 min after the administration (tmax). Absorption was nearly complete; approximately 82% of the dose recovered was renally excreted or remained in the body at sacrifice (without gastrointestinal tract).

Details on distribution in tissues:

The maximum plasma concentration (Cmax = 4.36 µg/g) was reached approx. 40 minutes after dosage (tmax = 0.7 h) reflecting rapid absorption. Afterwards, the plasma concentrations declined to less than 1% of the maximum concentration within 72 hours post administration, indicating continuous distribution and elimination and that no retention of the compound related residues in the body of the animals took place.

At sacrifice, only approx. 0.07% of the administered dose was found in the bodies of the rats. The major fraction was detected in organs and tissues (0.04%) and a minor proportion in the gastrointestinal tract (0.01%). Among the organs and tissues, the highest residues were found in the carcass (0.03%), followed by the residues in the skin (0.02%) and liver (0.01%). All other analysed organs and tissues contained far less than 0.01% of the administered radioactivity.

Only approx. 85% of the radioactivity administered was recovered. This was probably due to losses of small amounts of urine for two animals, which contained a high radioactivity concentration.

See Attachment 4 for tabulated on radioactive residues in organs and tissues.
See Attachment 5 for tabulated data of the time course of 14C-concentration in the plasma.
See Attachment 6 for tabulated data of the pharmacokinetic parameters.

Details on excretion:

Elimination of the test substance was almost complete 24 h after administration. At this time more than 83% of the administered dose (corresponding to >98% of the recovered dose) had been excreted with urine and faeces. At the time of sacrifice, 96 h after administration, more than 85% of the administered and more than 99% of the recovered dose had been excreted. Approx. 82% of the recovered radioactivity was excreted by urine and approx. 18% was excreted by the faeces.

See Attachment 2 for tabulated data of excretion profiles - Urine and Faeces.
See Attachment 3 for a balance of radioactivity in excreta and tissues, and percentage total radioactivity recovered.

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 4.36 µg/g

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: 40 min (0.7 h)

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 13.43 mg/L*h

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 22.9 h

Key result

Test no.:

#1

Toxicokinetic parameters:

other: Total clearance: 2.48 mL/min

Key result

Test no.:

#1

Toxicokinetic parameters:

other: Mean residence time: 4.85 h

Metabolites identified:

yes

Details on metabolites:

AE 0001789 was metabolised to a low extent. Parent compound was the major component and represented in total approx. 76% of the administered dose. Three metabolites were detected in urine: AE 0001789-descycloprpylamino, representing approx. 5% of the administered dose and the minor metabolites AE 0001789-desmethyl and AE 0001789-anisic acid. Minor amounts of AE 0001789-descyclopropylamino were also identified in faeces, as well as minor amounts of AE 0001789-desmethyl.

The main metabolic reactions of [methoxy-benzoyl-ring-UL-14C]AE 0001789 observed in the rat are:
- Elimination of the cyclopropylamine moiety by hydrolysis of the carboxamide bond in the sulfonylbenzamide moiety to give AE 0001789-descyclopropylamino,
- Desmethylation of the methoxybenzoyl moiety results in AE 0001789-desmethyl,
- Hydrolytic cleavage of the carboxamide bond in the methoxybenzoyl moiety to form AE 0001789-anisic acid.

See Attachment 1 for the proposed metabolic pathway in rat.
See Attachment 7 for the balance of test substance and metabolites excreted with urine and faeces.

Conclusions:

The toxicokinetic behaviour of the test compound was investigated in a GLP-compliant study on rats according to OECD 417. The study is therefore considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat.
A group of male rats was orally administered by gavage with a single dose of 14C labelled test substance (Methoxybenzoyl-ring) in a 0.5% aqueous Tragacanth suspension at a nominal dose level of 2 mg/kg bw. The animals were sacrificed 96 hours after administration.
The test substance was absorbed very quickly; the maximum plasma concentration was reached approx. 40 min after administration. Absorption was nearly complete in the test; approximately 82% of the dose recovered was renally excreted or remained in the body at sacrifice (without GIT).
The compound related radioactivity was quickly and completely excreted, approximately 85% of the administered dose (>99% of the recovered dose) had been excreted via urine and faeces at the time of sacrifice, 96 hours after administration. The predominant route of excretion was via urine, accounting for 70% of the dose administered, 15% was excreted with the faeces.
The test substance was efficiently eliminated from the body as illustrated by the terminal half-life of 23 h, and by the total clearance of 2.5 mL/min. More than 83% of the administered dose (98% of the recovered dose) was excreted within 24 h after administration.
Total radioactive residues in the organs and tissues were very low (<0.0073 µg/g), in all cases near or below the limit of detection.
The test substance was metabolised to a low extent. The main compound detected in urine and faeces was parent compound. AE 0001789-descyclopropylamino was formed as major metabolite by hydrolytic cleavage of the carboxamide bond in the sulfonylbenzamide moiety and the loss of the descyclopropylamine moiety. Hydrolysis of the carboxamide bond in the methoxybenzoyl moiety led to the minor label-specific metabolite AE 0001789-anisic acid. Minor metabolite AE 0001789-desmethyl resulted from desmethylation of the parent compound.

Cross-reference 3

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Sep 2004 - 29 Jun 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 4 Apr 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium fur Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen

Radiolabelling:

yes

Remarks:

14C labelled test substance (Sulfonylbenzamide-ring)

Species:

rat

Strain:

other: Wistar Hsd/Cpb: WU

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan & Winkelmann Versuchstierzucht GmbH, Borchen, Germany
- Age at study initiation: 7 weeks (males); 10 weeks (females)
- Weight at study initiation: approx. 200 g
- Housing: Individually in Makrolon metabolism cages.
- Individual metabolism cages: Yes
- Diet: Rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, Switzerland; ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 44-67
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the radiolabelled and non-radiolabelled test substance an adequate aliquot of the stock solution was taken and evaporated to near dryness under a gentle stream of nitrogen. The residue was suspended in 16 mL of 0.5% aqueous Tragacanth.

Duration and frequency of treatment / exposure:

single exposure

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

Actual dose received (m/f): 2.17/2.20 mg/kg bw

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Actual dose received (m/f): 190.23/193.46 mg/kg bw

No. of animals per sex per dose / concentration:

4

Control animals:

no

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: The dose levels were selected based on the toxicological properties of the test substance. A dose level of 200 mg/kg was selected for the high dose experiments, which is sufficiently below the LD50. The low dose level was fixed at 2 mg/kg, and thus in the range of the NOEL.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, plasma
- Time and frequency of sampling: 4, 8, 24, 48, 72 and 96 h (urine; high-dosed males were additionally sampled at 12 h); 24, 48, 72 and 96 h (faeces); 0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 24, 32, 48, 72 and 96 h (plasma)

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine, faeces, tissues
- Time and frequency of sampling: 4, 8, 12, 24, 48, 72 and 96 h (urine); 24 h (faeces), at sacrifice (tissues)
- From how many animals: Samples pooled
- Method types for identification: HPLC-UV, TLC-UV, LC-MS

ANALYTICAL METHOD
HPLC/HPTLC-UV combined with a radioactivity detector and LC-MS were used for measuring radioactivity, quantitation of parent compound and/or metabolites in composite urine samples and faeces extracts, and quantitation of the test substance in the administered dose formulations.

Measurement of radioactivity
- Liquid samples: Liquid scintillation counting (LSC). Small organs or tissues (adrenal glands, thyroid, ovaries, perirenal fat,and uterus ) were solubilised by means of a tissue solubiliser (e.g.BTS-450).
- Solid samples: All samples with the exception of those dissolved in BTS were weighed and combusted in an oxygen atmosphere. The released 14C02 was trapped in an alkaline absorber and radio-assayed by LSC using a scintillator.
- The limit of detection (LOD) was established at 20 dpm measured per aliquot after correction for the background radioactivity. The limit of quantification (LOQ) for each individual measurement was established as 2 to 3 times of the background radioactivity (dpm) of each instrument/method.

Statistics:

The values were checked for outliers in accordance with the outlier test by Nalimov, if appropriate. Values identified as outliers were marked and not used for the calculations of arithmetic means and coefficients of variations (in %). In addition, the concentrations or amounts of radioactivity were checked for values below the limit of detection (LOD). Values below the LOD were not considered in calculations of arithmetic means.

Preliminary studies:

no

Type:

absorption

Results:

The test substance was quickly absorbed i.e. the maximum concentration of radioactivity in almost all organs and tissues was found approx. 10-60 min after administration.

Type:

distribution

Results:

No significant residues remained in the bodies of the rats at sacrifice, 96 h after administration (<0.06% of the administered dose). No radioactive residues were accumulated in any of the tissues, organs or glands.

Type:

metabolism

Results:

AE 0001789 was metabolised to a low extent: Parent compound (80-95%), AE 0001789-descyclopropylamino (3-8%), AE 0001789-desmethyl (0.17-0.4%), AE 0001789-cyclopropyl-sulfamoylbenzamide (0-0.64%).

Type:

excretion

Results:

The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete 72 h after administration (98% excretion of the recovered dose).

Details on absorption:

After administration of 2 mg/kg bw AE 0001789 to male and female rats, between 90 and 91% of the recovered total radioactivity were excreted with the urine. Only between 9 and 10% of the dose were detected in the faeces and the GIT of rats.
After administration of 200 mg/kg bw AE 0001789, renal excretion was slightly lower with values between 69 and 74% of the dose recovered, although still indicating a high level of absorption at this dose level. The absorption rate (renal excretion plus residues in body without GIT), based on the low dose tests, therefore amounted to more than 90% of the dose recovered.
The absorption of AE 0001789 commenced immediately after administration as shown by the concentration of radioactivity in the plasma. See Attachment 5 for tabulated data of the time course of 14C-concentration in the plasma.

Details on distribution in tissues:

After a single oral administration of 2 mg/kg bw AE 0001789 to male and to female rats the maximum of the plasma concentration of radioactivity was reached approximately 10 to 40 minutes after dosage (tmax). The equivalent concentrations Cmax were in a similar range for males and females with approximately 5.6 µg/g.
In the high dose tests with 200 mg/kg bw AE 0001789, the maximum of the plasma concentration was observed 40 to 60 minutes after dosing. The maximum dose normalised concentrations were considerably lower than for the low dose tests for both sexes. This indicates that a saturation of the absorption process has occurred at the high dose level.
The curves obtained in the high-dose tests feature a double-peaked curve for both, male and female rats. A second plasma maximum of the mean plasma concentrations is apparent 32 hours after the administration for female rats and 48 hours after dosing for male rats. These double peak phenomena in the plasma concentration-time profile are well in line with the excretion profiles, which also show that a large proportion of compound-related radioactivity is renally excreted at later time points, after 24 hours post dose.
Double peak phenomena in plasma profiles have been described in the pharmacokinetic literature as effects caused by discontinuous absorption and/or delayed gastric emptying of a portion of the dose. In the case of AE 0001789 it is assumed, that delayed gastric emptying causes the double peak phenomena since a large proportion of compound-related radioactivity (up to 47% of the administered dose) was excreted later than 24 hours after dosing.
Apart from these observations, no significant differences were apparent with regard to the levels of the dose normalised concentrations in the high dose and in the low dose experiment. The dose normalised concentrations in the distribution/elimination phase and the decrease of the plasma levels were similar in all tests.
The mean residence time (MRT) of AE 0001789-related radioactivity was short for all tests included in the study, ranging from 5 to 26 hours.
The area under the plasma curve (AUC) was similar for males and females in the low dose tests, with 17 and 13 mg/L*h. In the corresponding high dose tests it was significantly higher, with 545 and 567 mg/L*h. The AUC-ratios of high dose versus low dose were 31 for males and 44 for females, i.e. under-proportional to the increase in dose, indicating a certain saturation of the absorption process in the high dose.
The terminal half-lives (t1/2) were calculated using the last data points, only, and were in the range of 13 to 21 hours. The half-lives were in the same order of magnitude for all tests and thus apparently unaffected by the dose levels. The total clearance (CL) was in the range of 2 to 6 mL/min. These values demonstrate the efficient elimination of the AE 0001789-related radioactivity from the rats.
At sacrifice, only between 0.02% and 0.06% of the administered dose was found in the bodies of the rats. The major fraction, in total between 0.01% and 0.04%, was detected in organs and tissues and a minor proportion in the gastro-intestinal tract. Very low dose normalised concentrations were detected in all organs and tissues in all tests. The individual values, ranged from
See Attachment 4 for tabulated on radioactive residues in organs and tissues.
See Attachment 5 for tabulated data of the time course of 14C-concentration in the plasma.
See Attachment 6 for tabulated data of the pharmacokinetic parameters.

Details on excretion:

The excretion of total radioactivity was almost complete 72 h after a single oral administration of AE 0001789. At this time more than 84% of the administered dose (corresponding to >98% of the recovered dose) had been excreted via urine and faeces in all four tests. At the time of sacrifice, 96 h after administration, more than 85% of the administered and more than 99% of the recovered dose had been excreted in all cases.
Between 79 and 90% of the administered radioactivity was excreted by urine in the low dose tests, and between 69 and 72% in the high dose tests. Faecal excretion accounted for between 7 and 10% of the administered dose in the low dose tests and between 25 and 31% in the high dose tests. The higher faecal excretion in the high dose tests was probably due to an incomplete absorption of AE 0001789 from the GIT into plasma. No sex difference was apparent in the faecal excretion rate.
The profile of excretion over time shows that a large proportion of compound-related radioactivity was excreted after 24 hours post dose in the high dose tests. Up to 47% of the administered dose was excreted per day between 24 and 72 hours after dosing. This indicates most probably delayed gastric emptying.

See Attachment 2 for tabulated data of excretion profiles - Urine and Faeces.
See Attachment 3 for a balance of radioactivity in excreta and tissues, and percentage total radioactivity recovered.

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 62.6 µg/g

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: 40 min (0.7 h)

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 545.18 mg/L*h

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 13 h

Key result

Test no.:

#2

Toxicokinetic parameters:

Cmax: 81.5 µg/g

Key result

Test no.:

#2

Toxicokinetic parameters:

Tmax: 1 h

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 566.69 mg/L*h

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 15.1 h

Key result

Test no.:

#3

Toxicokinetic parameters:

Cmax: 5.57 µg/g

Key result

Test no.:

#3

Toxicokinetic parameters:

Tmax: 40 min (0.7 h)

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 17.49 mg/L*h

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 1st: 21.4 h

Key result

Test no.:

#4

Toxicokinetic parameters:

Cmax: 5.57 µg/g

Key result

Test no.:

#4

Toxicokinetic parameters:

Tmax: 10 min (0.17 h)

Key result

Test no.:

#4

Toxicokinetic parameters:

AUC: 12.84 mg/L*h

Key result

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: 19.5 h

Metabolites identified:

yes

Details on metabolites:

AE 0001789 was metabolised to a low extent. Parent compound was the major component and represented in total approx. 80-95% of the administered dose. Three metabolites were detected in urine: AE 0001789-descyclopropylamino (3-8%) was formed as major metabolite by hydrolytic cleavage of the carboxamide bond in the sulfonylbenzamide moiety and the loss of the descyclopropylamine moiety. Hydrolysis of the carboxamide bond in the methoxybenzoyl moiety led to the minor metabolite AE 0001789-cyclopropylsulfamoylbenzamide, which can also be formed from metabolite AE 0001789-desmethyl. AE 0001789-desmethyl resulted from desmethylation of the parent compound.

See Attachment 1 for the proposed metabolic pathway.
See Attachment 7 for the balance of test substance and metabolites excreted with urine and faeces.

Conclusions:

The toxicokinetic behaviour of the test compound was investigated in a GLP-compliant study on rats according to OECD 417. The study is therefore considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat.
Groups of 4 male and female rats were orally administered by gavage with a single dose of 14C labelled test substance (Sulfonylbenzamide-ring) in a 0.5% aqueous Tragacanth suspension at nominal dose levels of 2 and 200 mg/kg bw. The animals were sacrificed 96 hours after administration.
The test substance was rapidly absorbed in all tests, the maximum plasma concentrations were reached between approximately 10 and 60 minutes after administration. Good absorption was also detected at the high dose level, though renal excretion was slightly lower than in the low dose tests. Absorption was complete in the low dose tests, more than 90% of the dose recovered were renally excreted or remained in the body at sacrifice (without GIT).
The compound was quickly and efficiently eliminated from the body in all tests as illustrated by the short terminal half-lives (t1/2) in the range of 13 to 21 h, and by the total clearance in the range of 2 to 6 mL/min. Excretion of total radioactivity was almost complete 72 h after administration. More than 84% of the administered dose (corresponding to >98% of the recovered dose) was excreted 72 h after the administration.
Delayed gastric emptying was observed in the high dose tests, leading to double peak phenomena in the plasma concentration-time curves and late excretion of significant portions of the administered dose (up to 47% per day) between 24 and 72 h after administration.
The predominant route of excretion was via urine, accounting for 69 to 90% of the radioactivity administered. In the high dose tests, a certain saturation of the absorption process could be observed. The maximum dose-normalised concentrations in plasma were lower than in the low dose tests, and faecal excretion increased by a factor of 2 - 3 compared to the low dose tests.
No significant residues remained in the bodies of the rats at sacrifice, 96 h after administration (<0.06% of the administered dose). No radioactive residues were accumulated in any of the tissues, organs or glands.
AE 0001789-descyclopropylamino was formed as major metabolite by hydrolytic cleavage of the carboxamide bond in the sulfonylbenzamide moiety and the loss of the descyclopropylamine moiety. Hydrolysis of the carboxamide bond in the methoxybenzoyl moiety led to the minor metabolite AE 0001789-cyclopropylsulfamoylbenzamide, which can also be formed from metabolite AE 0001789-desmethyl. AE 0001789-desmethyl resulted from desmethylation of the parent compound.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Objective of study:

absorption

distribution

excretion

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 4 Apr 1984

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium fur Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

485-320-2

EC Name:

-

Cas Number:

221667-31-8

Molecular formula:

C18H18N205S

IUPAC Name:

N-[4-(cyclopropylcarbamoyl)benzenesulfonyl]-2-methoxybenzamide

Radiolabelling:

yes

Remarks:

14C labelled test substance (Sulfonylbenzamide-ring)

Test animals

Species:

rat

Strain:

other: Wistar Hsd/Cpb: WU

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan & Winkelmann Versuchstierzucht GmbH, Borchen, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: approx. 200 g
- Housing: Individually in Makrolon metabolism cages.
- Individual metabolism cages: Yes
- Diet: Rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, Switzerland; ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25
- Humidity (%): 50-67
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the radiolabelled and non-radiolabelled test substance an adequate aliquot of the stock solution (test substance dissolved in acetonitrile) was taken and evaporated to near dryness under a gentle stream of nitrogen. The residue was suspended in 28 mL of 0.5% aqueous Tragacanth.

Duration and frequency of treatment / exposure:

single exposure

Doses / concentrations

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Actual dose received: 4.95 mg/kg bw/day

No. of animals per sex per dose / concentration:

8 males recieved radiolabelled test substance
1 male recieved non-radiolabelled test substance (control)

Control animals:

yes

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: The selected dose level was based on the dosage recommendations of the current EPA Health Effects Guidelines and was in the same range as the low dose tests in the corresponding rat metabolism studies.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, expired air
- Time and frequency of sampling:
Urine: 1, 4, 8, 24, 48, 72, 96, 120, 144 and 168 h after dosing
Faeces: 24, 48, 72, 96, 120, 144 and 168 h after dosing
Expired air: 24 and 48 hours after dosing
QBWA: 24 and 48 hours after dosing (for 4 animals)

The control animal for autoradiography of the carcass was sacrificed after 4 h.

ANALYTICAL METHOD
- Measurement of Liquid Samples Using Liquid Scintillators: The measurement of the radioactivity in liquid samples was carried out by liquid scintillation counting (LSC).
- Measurement of Solid Samples Using Liquid Scintillators: Solid faeces samples were weighed and combusted in an oxygen atmosphere using the "Oxidizer 387" (Packard Instruments). The released 14C02 was trapped in an alkaline absorber (Carbosorb E, Packard Instruments) and radio-assayed by LSC using Permafluor E+ (Packard Instruments) as scintillator.
- Quantitative Whole Body Autoradiography (Radioluminography): The digital images of the autoradiograms obtained by the Fuji BAS 5000 system were used for the assessment of the distribution of radioactivity concentrations in different organs and tissues.
- Reversed phase HPLC (Agilent HP 1100) in combination with UV / radio-detector (Ramona star) / mass spectrometry (TSQ 7000) were used for quantification as well as for co-chromatographic investigations.

Results and discussion

Preliminary studies:

no

Main ADME results

Main ADME results 1

Type:

absorption

Results:

The radiolabelled test substance was absorbed quickly from the gastrointestinal tract. The maximum concentration of radioactivity in almost all organs and tissues was found 1 hour after administration.

Main ADME results 2

Type:

distribution

Results:

At all time-points examined, high radioactivity was mainly observed in the excretory organs kidney and liver. The residues in all other organs and tissues were fairly evenly distributed and always lower than the residues observed in blood.

Main ADME results 3

Type:

excretion

Results:

The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete 48 h after administration (90% excretion).

Toxicokinetic / pharmacokinetic studies

Details on absorption:

One hour after the administration of [Sulfonylbenzamide-ring-UL-14C]AE 0001789, the maximum concentration of radioactivity was observed in the contents of stomach and small intestine, which is an inherent result of the oral administration. High radioactivity was also observed in blood, indicating fast absorption and high bioavailability of AE 0001789.

Details on distribution in tissues:

A high concentration of radioactivity was mainly observed in the excretory organs liver and kidney, indicating commencing clearance of the compound immediately after absorption mainly by the renal route.
The highest radioactivity concentration (Cmax) in all organs and tissues was observed 1 hour after the administration. The radioactivity levels observed in blood decreased quickly to an intermediate minimum observed in the animal sacrificed 24 hours after the administration (0.189 µg/g), before they increased again to a second maximum 48 hours after the administration (0.234 µg/g). This effect can be attributed to an interruption of the fast absorption in the intestine resulting from the delayed gastric emptying which was observed between 4 and 48 hours after administration.
Overall, the equivalent concentrations in most organs and tissues were relatively low compared to the actual administered amount of 4.95 mg/kg bw. Only the dose normalised concentrations (CN) at Cmax for renal medulla was close to the equilibrium value of 1, which is an indication for the efficient depletion of the compound via kidney and urine. The CN-values for blood, liver and renal cortex were between 0.5 and 0.7. All other CN-values were well below 0.5.

See Attachment 1 (background material) for tabulated distribution data.

Details on excretion:

The excretion of total radioactivity was almost complete 48 h after administration of 14C-AE 0001789. At this time more than 90% of the administered dose had been excreted via urine and faeces. The major part of the radioactivity administered was excreted by urine. The expiration of 14CO2 and other 14C-labelled volatiles could not be determined quantitatively due to a malfunction of the collection equipment. However, the volatile radioactivity was determined in the course of an autoradiography test with AE 0001789 labelled in the methoxybenzoyl moiety. This test demonstrated the high stability of the labelling position with regard to possible formation of volatile products (key, 2006, M-274337-01-1, rat, oral, metabolism, RL1). As no molecule cleavage was observed in the metabolism studies performed in rat (key, 2006, M-274975-01-2, rat, oral, metabolism, RL1; key, 2006, M-276053-01-2, rat, oral, metabolism, RL1) with both radiolabels, a repetition of the test for volatile radioactivity with the [sulfonylbenzamide-ring-UL-C]-label was not considered necessary.

See Attachment 2 (background material) for tabulated excretion data.

Metabolite characterisation studies

Metabolites identified:

not measured

Details on metabolites:

The metabolic profiles in urine and faeces extracts were determined by reversed phase HPLC with radio-detection. The recorded metabolic profiles are shown in Attachment 3. No isolation or structure elucidation of compounds detected in these profiles was performed in the study.

Applicant's summary and conclusion

Conclusions:

The toxicokinetic behaviour of the test compound was investigated in a GLP-compliant study on rats according to OECD 417. The study is therefore considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat. The 14C labelled test substance (Sulfonylbenzamide-ring) was absorbed quickly from the gastrointestinal tract. The maximum concentration of radioactivity in almost all organs and tissues was found 1 hour after the administration. The absorption process was apparently interrupted, probably due to delayed gastric emptying occurring between 4 and 48 hours after dosing. The high radioactivity observed in kidney already 1 hour after dosing indicated that renal excretion commenced immediately after absorption.
The absorbed radioactivity was not uniformly distributed in the body. At all time-points examined, high radioactivity was mainly observed in the excretory organs kidney and liver. The residues in all other organs and tissues were fairly evenly distributed and always lower than the residues observed in blood.
Residues in all organs and tissues decreased rapidly between 1 and 72 hours. In all organs and tissues residues were < LOD or < LOQ at later time-points between 120 and 168 hours after dosing.There was no sign for retention of radioactivity in specific organs or tissues. Residues from glandular organs or tissues responsible for hormonal regulation (such as adrenal, testis, or thyroid gland) were rapidly depleted in parallel with depletion from the other organs and tissues.
The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete at the end of the study period.