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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From: 2010-03-01 To: 2012-03-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Objective of study:
distribution
excretion
metabolism
other: mass balance
Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MAFF (12-Nousan-No.8147, 2-3-1)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Cas Number:
915972-17-7
Molecular formula:
C33H39NO9
IUPAC Name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Test material form:
solid
Details on test material:
- Analytical purity: >94 %
Specific details on test material used for the study:
NON RADIOLABELED TEST SUBSTANCE

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Meiji Seika Pharrna Co., Ltd., TA10224601
- Purity: 98.50 %

RADIOLABELED TEST SUBSTANCE

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Institute of lsotopes Co., Ltd. (Hungary), XVII/53

RADIOLABELLING INFORMATION
- Radiochemical purity: 95.87 % by radio-HPLC
- Specific activity: 3.29 MBq/mg (1955 MBq/mmol)
- Locations of the label: nicotinic acid 9-14CShort

STORAGE CONDITIONS OF TEST MATERIAL
- in a refrigerator at ca. 5 °C ( defined range: 1 to 10 °C)

Radiolabelling:
yes

Test animals

Species:
rat
Strain:
other: F344/DuCrlCrlj, SPF/VAF
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Japan, Inc. (Kanagawa, Japan)
- Age at study initiation: 9 weeks
- Housing: animals of the same sex were house together
- Diet: MF pellet (lot. 100108A2, Oriental Yeast Co., Ltd., Tokyo, Japan) ad libitum
- Water: tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23±2 °C
- Humidity: 55±15 %
- Air changes: > 20 times/hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2010-04-05 To: 2010-05-19

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 3 % hydroxylpropyl cellulose solution (3% HPC, w/w). The vehicle was prepared by dissolving hydroxylpropyl cellulose (Wako pure chemical industries, Ltd., Osaka, Japan) in Otsuka distilled water (Otsuka pharmaceutical factory, Inc., Tokushima, Japan).
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Appropriate aliquot of the stock solution of radiolabeled test substance was mixed with appropriate amount of the non-radiolabeled test substance (as the stock solution for the low dose or the intact crystal for the high dose). The organic solvent was completely removed under gentle stream of nitrogen, and the dried test substance was resuspended in appropriate volume of 3 % HPC solution. The suspension was kept at ambient temperature under the dark condition with continuous stirring until use. The low and high dose formulations were used within 7 days after preparation.
Duration and frequency of treatment / exposure:
single gavage
Doses / concentrationsopen allclose all
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose / concentration:
1 per sex per dose
Control animals:
no
Positive control reference chemical:
no
Details on study design:
The test substance was delivered to the stomach of each rat via a gavage tube with a glass syringe. The amount of dose to be administered to each rat was determined based on the body weight measured immediately prior to the dose administration. The oral route was selected for dose administration to be consistent with the relevant toxicological studies, and also as this is expected to be an important route for human exposure.
Details on dosing and sampling:
Samples collected
Expired air: The exhaust from each metabolic cage was successively passed through two gas-trapping towers, one contained monoethanolamine/methylcellosorb mixture (1/1, v/v) and the other contained methylcellosorb, to collect radioactive components in the expired air. The solutions in two traps were replaced to fresh ones at sampling intervals of 6, 24 and 48 hr post dose. The solutions from two traps were combined as an expired air sample, and subjected to radioassay. The sample collection was terminated at 48 hr post dose, since no significant radioactivity accounting > 1 %AR was detected in 24 hr after dose.

Urine, feces, and cage wash: The urine samples were collected at 6, 12, 24, 48, 72 and 96 hr post dose and feces were collected at 24, 48, 72 and 96 hr post dose. During the sample collection, the containers were kept under a chilled condition. After the last sampling, the inner wall of the glass metabolic cage was rinsed with water and methanol, and the rinsate was pooled as the cage wash sample.

Tissues: At termination (96 hr post dose), rats were anesthetized with ethyl ether to collect blood sample from the inferior vena cava, then sacrificed by cervical vertebra dislocation. The organs/tissues - (brain, pituitary gland, thyroid, thymus, lungs, heart muscle, liver, kidneys, adrenal glands, spleen, pancreas, urinary bladder, adipose tissue (around kidneys), skin with hair (back), skeletal muscle (a portion of the gastrocnemius muscle), bone marrow (femur and tibia), bone (humerus), prostate gland, testes, epididymides, ovaries, uterus (including oviduct), and gastro-intestinal tract (with contents) - and the residual carcass were excised from the animals.

Description of analytical procedures
Sample Processing for Radioassay
Radioactivity in liquid sample was determined by LSC. Generally, duplicate or triplicate aliquots of a liquid sample were dissolved in either AtomlightTM, Pico-FluorTM plus or Hionic-FluorTM scintillation cocktail (all PerkinElmer Japan Co., Ltd.) prior to LSC Analysis. Samples of blood, plasma and erythrocytes were solubilized prior to LSC. Solid samples were subjected either to solubilization (fecal homogenate) or oxidative combustion (bone, homogenate of residual carcass, residues on a filter paper, etc.).
Expired air: Duplicate aliquots (2.0 mL) of each sample were taken for radioassay.
The urine samples were filtered and duplicate aliquots (0.1 mL) were radioassayed. Residues on the filter paper were subjected to oxidative combustion prior to radioassay. Filtered urine samples were radioassayed on the day of sampling and stored frozen until use for metabolite analysis. Residues on the filter paper were stored for up to 7 days under a frozen condition before combustion. Feces was homogenized with chilled water using a Polytron® homogenizer under ice-chilled condition and triplicate aliquots (ca. 150 to 200 mg) of the homogenate were solubilized prior to radioassay. These processes were completed on the day of sampling. The remaining homogenate was stored frozen until use for metabolite analysis. Cage wash was filtered, rinsed with methanol/water (1/1, v/v), and duplicate aliquots (2 mL) of the combined sample were radioassayed. Residues on the filter paper were subjected to oxidative combustion prior to radioassay. Filtered cage wash samples were radioassayed on the day of sampling. Residues on the filter paper were stored for up to 3 days under a frozen condition before combustion.
Duplicate aliquots (ca. 150 mg) of freshly collected whole blood sample were radioassayed and the remainder was immediately centrifuged at 3500 rpm (approx. 2200 xg) for 5 minutes to separate plasma and erythrocytes. A 1.0 to 1.2-mL aliquot was taken from plasma for radioassay. Duplicate aliquots of erythrocytes (ca. 70 mg) were also radioassayed. Immediately after excise, all collected tissues/organs were weighed and radioassayed as follows: The following tissues were entirely subjected for radioassay: pituitary gland, thyroid, thymus, adrenal glands, urinary bladder, bone marrow, bone, prostate gland, epididymides, ovaries, and uterus. For the following relatively large tissues, duplicate or triplicate aliquots were excised (ca. 50 mg for adipose tissue and ca. 200 mg for others) for radioassay: brain, lungs, heart muscle, liver, kidneys, spleen, pancreas, testis, adipose tissue, skin with hair, and skeletal muscle. The gastro-intestinal tract including contents was entirely solubilized with approximately 90 mL of HCl/2-propanol mixture (1/2, v/v) at approximately 50°C for 18 hrs, and triplicate aliquots of the digest were radioassayed. The residual carcass was homogenized with 10 to 15 mL of 2-propanol/water mixture (1/1, v/v) with an appropriate amount of dry ice using a blender, and triplicate aliquots (ca. 300 mg) of the homogenate were radioassayed following oxidative combustion. All these process were completed within 3 days after sacrifice of the animals.

Metabolite Analysis
Sample processing: Pooled samples were prepared for the filtered urine (0-72 hr post dose) and fecal (0-96 hr post dose) homogenate to use for metabolite analysis by taking proportional amounts (10%, by volume for the urine and by weight for the fecal homogenate) of the samples obtained at each time interval and combining them for the same test group. The pooled samples were stored frozen when not in use. An aliquot of the pooled urine was directly subjected to radio-HPLC analysis
For feces, a portion of the pooled fecal homogenate (ca. 2.5 to 5 g) was extracted with approximately 15 mL of acetonitrile using the Polytron® homogenizer under an ice-chilled condition. The homogenate was sonicated for 5 minutes and then centrifuged at 6000 rpm (approx. 4000 x g) for 5 minutes at 4 °C to separate the supernatant and the pellet. A similar extraction process was repeated for the pellet once with acetonitrile and an additional two times with acetonitrile/water (1/1, v/v). All extracts (supernatant after centrifugation) were combined to obtain an extract fraction. The final pellet was suspended in 2 to 3 mL of acetonitrile. Duplicate aliquots of the extract and triplicate aliquots of the pellet suspension were radioassayed. The samples of the pellet suspension were solubilized prior to radioassay. The extractions were performed in duplicate. Duplicate extracts were combined and concentrated by rotary-evaporation prior to radio-HPLC analysis. The extraction efficiency of the radioactive residues in the feces was >98%.

Metabolite quantitation
Pooled urine and fecal extract were analyzed by a radio-HPLC to quantitate radioactive metabolites. An aliquot of the sample was analyzed using HPLC method 2 monitored by a flow-through radioactivity detector (RAD). Non-radioactive components were detected by the UV detector. Percent distribution of radio area counts on the chromatogram was determined for individual 14C peaks, and was applied to the sample %AD to calculate %AD of individual 14C-peaks. HPLC recovery was checked for each quantitative HPLC and HPLC recoveries were found to be over 94% throughout the experiment. Major 14C-components were primarily identified/characterized by HPLC co-chromatography (co-injection) with the reference substances.

Detection limit for radio-analysis: For LSC analysis, the detection limit was fixed at 40 dpm/LSC sample which is approximately twice the background radioactivity. This means LSC output showing < 40 dpm was defined as “not detected”. The detection limit for HPLC analysis was defined as 4.66σ (σ= sqr(BKG×t) , BKG: rate of the background area counts (counts per minute), t: counting time (min)) based on HPLC background radio area counts. The background area counts were obtained from the HPLC region where radioactive peaks were not detected (e.g. HPLC area counts of 0 to 3 and 57 to 60 min for the HPLC method 2). HPLC detection limits were determined for individual runs.

Data expression and calculation: The quantity of radioactivity in a sample was expressed as percentage of the administered radioactivity (%AD) as shown below.

Percentage of the administered dose (%AD) = [(total radioactivity found in the sample, dpm) / (dosed radioactivity, dpm)] × 100

Storage stability
The urine and feces samples were stored frozen when not in use, and quantitative metabolite analyses in these samples were completed within 18 days after sample collection. The sample analyses were considered to be completed reasonably soon after sample collection, so that the storage stability of these samples was not evaluated further.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:
At 96 hr post dose, only 0.48 to 1.03 % of the applied radioactivity remained in the whole body. The highest distribution was found in the gastro-intestinal tracts (with contents, 0.05 to 0.61 %AD), followed by the liver (0.03 to 0.06 %AD). Residue level in the other tissues was less than 0.01 %AD. No significant sex difference was found.

For the low dose experiment, the highest residue levels were found in liver (0.0276 to 0.0418 mg eq./kg), adrenals (0.0293 to 0.0383 mg eq./kg), heart muscle (0.0316 to 0.0335 mg eq./kg) and kidneys (0.0252 to 0.0285 mg eq./kg) at 96 hr post dose. Residue levels for all other tissues were less than 0.02 mg eq./kg. The plasma 14C-concentration was very low as 0.0003 to 0.0004 mg eq./L. Tissue to plasma concentration ratios (TIP) greater than 50 were observed in liver (62.8 to 134.3), adrenals (66.5 to 123.0), heart muscle (76.1 to101.3), kidneys (57.2 to 91.6), skeletal muscle (60.5, in male only) and spleen (51.7, in male only). The concentrations of all tissues except for adipose, pituitary and thyroid were at least more than 8 times higher than that of plasma. The 14C-concentrations in the adipose, pituitary and thyroid tissues were below the limit of detection.

For the high dose experiment, the highest residue levels were found in liver (2.15 to 3.46 mg eq./kg), heart muscle (2.84 to 2.89 mg eq./kg), adrenals (2.23 to 2.53 mg eq./kg), and kidneys (2.10 to 2.42 mg eq./kg) at 96 hr post dose. Residue levels for all other tissues were less than 2 mg eq./kg. The plasma 14C-concentration was low as 0.03 to 0.04 mg eq./L. Tissue to plasma concentration ratios greater than 50 were observed in liver (68.4 to 95.7), heart muscle (80.0 to 90.0), adrenals (61.7 to 80.2), kidneys (66.6 to 67.0), and skin with hair (54.7, in male only). The concentrations of all tissues except for adipose, pituitary and thyroid were at least more than 10 times higher than that of plasma. The 14C-concentrations in the adipose, pituitary and thyroid tissues were below the limit of detection.
Details on excretion:
The material balance (sum of the excreted radioactivity in the expired air, urine, feces and present in the body and cage wash) was approximately 95 % of administered dose (%AD). The radioactivity in the expired air was less than 0.1 %AD in any test groups indicating excretion through expired air is not a significant route for elimination of the test substance. The excretion into the urine and feces amounted to 5.73 to 6.75 %AD and 87.37 to 87.86 %AD, respectively, for the low dose, and 13.60 to 15.65 %AD and 78.71 to 81.14 %AD, respectively, for the high dose. The excreted radioactivity totaled approximately 93 to 95 %AD at 96 hour post dose. The results indicate fecal excretion is the predominant route for elimination of the test substance, irrespective of the dose level and sex. The radioactivity remaining in the gastro-intestinal tracts (with contents) and carcass are 0.05 to 0.61 %AD and 0.41 to 0.52 %AD respectively at termination.

Metabolite characterisation studies

Metabolites identified:
yes
Remarks:
major metabolites (> 5%AD) = ME5343-Tl, ME5343-T2, ME5343-T8, "HPLC Reg.#25"
Details on metabolites:
In any test groups, less than 2 % AD remained in the post extraction solids. Radioactivity excreted in the feces accounted for 78.71 to 87.86 %AD, irrespective of the dose level. For the low dose experiment, 14C-components/metabolites found greater than 5 %AD were unchanged test substance (27.90 to 38.52 %AD), followed by ME5343-Tl (19.88 to 23.80 %AD), ME5343-T2 (7.62 to 8.27 %AD), an unknown metabolite "HPLC Reg.#25" (3.16 to 5.54 %AD) and ME5343-T8 (3.87 to 6.01 %AD). Minor metabolites are also found at a range of 2.3 to 4.3 %AD. For the high dose experiment, 14C-components/metabolites found greater than 5 %AD were unchanged test substance (33.48 to 37.42 %AD), followed by ME5343-Tl (13.54 to 13.98 %AD), ME5343-T2 (8.99 to 10.49 %AD), ME5343-T8 (7.95 to 9.61 %AD), and an unknown metabolite "HPLC Reg.#25" (3.65 to 5.54 %AD). Minor metabolites are also found at a range of 1.5 to 3.6 %AD. No significant sex related differences were found for the metabolite profiles in the feces.
Radioactivity excreted in the urine amounted for 5.73 to 6.75%AD and 13.60 to 15.65%AD for the low and high dose experiments, respectively (Table 3). For the low dose experiment, none of the 14C-metabolites were found to be greater than 5%AD). The minor metabolite found at the highest level was ME5343-Tl (3.08 to 3.34 %AD), followed by an unknown metabolite "HPLC Reg.#39" (1.04 to 2.12%AD) and ME5343-T2 (0.58 to 1.52%AD). No other metabolites formed over 1 %AD were detected. For the high dose experiment, 14C-metabolites formed greater than 5%AD were only ME5343-Tl (7.06 to 7.14%AD). The other minor metabolites found were ME5343-T2 (1.45 to 3.36 %AD), an unknown metabolite “HPLC Reg.#25" (1.55 to l.85%AD) and ME5343-T8 (0.83 to l.09%AD). No other metabolites formed over 1 %AD were detected. Unchanged [NCA-14C]ME5343 was not detected in the urine of the low and high dose experiments. Minimal dose related difference was noted for the formation of the unknown "HPLC Reg.#39". No significant sex related differences were found for the metabolite profiles in the urine.

See also attached document - proposed metabolic pathway.

Applicant's summary and conclusion