Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 01, 2004 to April 06, 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Method According to Eric Le Ferrec et al. In vitro Models of the Intestinal Barrier, in the Report and Recommendations of ECVAM Workshop 46,

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Objective of study:
absorption
Test guideline
Qualifier:
according to
Guideline:
other: Method according to Eric Le Ferrec et al. In vitro Models of the Intestinal Barrier, in the Report and Recommendations of ECVAM Workshop 46,
Principles of method if other than guideline:
The study was performed to estimate the test substance bioavailability across the intestinal barrier in the A-B (apical to basolateral) Permeability assay using human intestinal epithelial cell line (TC-7). The uptake of the test substance from apical to basolateral cavity was analyzed by HPLC-MS/MS. A relative percent absorption of the test substance was indicated by the comparison with reference compounds. The study was performed according to ECVAM recommendations.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 2-amino-4-hydroxyethylaminoanisole sulfate
- TSIN: WR23081
- Substance type: Pure active substance
- Physical state: Pale gray powder
- Stability under test conditions: The substance on storage in dryness and darkness considered to be stable until July 2005.
- Stability in solution: The results of stability test conducted over a period of 7 days in water (5%) and DMSO (5%) confirms low degradation. The results of stability test conducted over a period of 7 days in water (0.01%), DMSO (10%) and acetone/water (0.5%) confirms a very good stability. The test solution was stored at room temperature in the absence of light during the test.
- Storage: Not reported
- Solubility: 10 g/L in water pH 2.8 (>5 weight% pH 8)
1 weight% in acetone/water 1:1 (pH 2.1)
9-10 weight% in DMSO
0.2 weight% in ethanol
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch 57
- Expiration date of the lot/batch: July 2005
- Purity test date: 22.01.2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:pale grey powder on storage in dryness and darkness until July 2005
- Solubility and stability of the test substance in the solvent/vehicle:
Stability in solution:
The stability over a total period of seven days was tested by HPLC. The test stock solutions (approx. 5 weight%) were stored at room temperature and in the absence of light.
Water solution: the results (t = 0h: 100.0%; 6h: 93.8%; 2d: 95.1%; 7d: 79.7%) confirm a low degradation (G2000/003)
DMSO solution: the results (t = 0h: 100.0%; 6h: 98.8%; 2d: 92.0%; 7d: 80.5%) confirm a low degradation (G2000/003)

Solubility: 10g/l in water pH 2.8 (>5 weight% pH 8) 1 weight% in acetone/water 1:1 (pH 2.1) 9-10 weight% in DMSO
0.2 weight% in ethanol


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The working solution for each test compound was prepared at 50µM in HBSS-MEC (5mM) at pH 6.5 from a 10 mM DMSO stock solution. 14C mannitol (approximately was also included in the working solution. The working solution was added to the apical side with a finalDMSO concentration of 1%. HBSS-HEPES (5mM), pH 7.4 was added to the basolateral side.

- Final dilution of a dissolved solid, stock liquid or gel: 50 µM


FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:
Radiolabelling:
no

Test animals

Species:
other: In Vitro TC-7 human intestinal epithelial cell line.

Administration / exposure

Vehicle:
other: HBSS buffer containing 1% DMSO
Details on exposure:
Once incubation of 60 minutes
Doses / concentrations
Dose / conc.:
50 other: µM
No. of animals per sex per dose:
duplicate
Positive control:
- Following reference compounds were tested concurrently with test substance in order to assess the assay suitability:
Propranolol (which has 90 % absorption in humans used as high permeability reference)
Ranitidine (which has 50% absorption in humans used as the low permeability reference)
- Comparison of the test substance to this reference compound can indicate a relative percent absorption of the test substance.
Details on study design:
TEST SYSTEM:
- Type of test system: The human intestinal epithelial cell line, TC-7 (13-25 days in culture and 15 passages in culture). TC7 is a sub-clone of the CacO-2 cell line.
- Medium/buffer used: 5 mM HBSS (Hank's balanced salt solution)-MES (2-(N-Morpholino)-ethanesulfonic acid) was added in apical side. 5 mM HBSS-HEPES (N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid)) was added in basolateral side.
PREPARATION OF WORKING SOLUTION: The working solution of the test substance was prepared at 50 µM in HBSS-MES (5 mM; pH: 6.5) from 10 mM DMSO stock solution of test substance.
CELL MONOLAYER INTEGRITY CHECK: A 14C-mannitol (approximately 4 µM) was included in the working solution for assessing the cell monolayer integrity. Only monolayers revealing a permeability of < 2.5x 10 (-6) cm/s were used.
Details on dosing and sampling:
TEST PROCEDURE: The working test solution was applied to apical side of the cell monolayer and HBSS-HEPES medium was added to the basolateral side. The permeability from the apical (A, pH 6.5) to the basolateral (B, pH 7.4) side of cell monolayer was investigated at 37°C in 96-well transwell plates with shaking for a 60 min contact time.
SAMPLING: The samples were collected to determine the concentration of test substance at following time intervals:
Donor (apical) samples: at 0 and 60 min
Receiver (basolateral) samples: at 60 min
REPLICATES: 2
ANALYSIS: The concentration of test substance was determined by HPLC-MS/MS.

Results and discussion

Main ADME results
Type:
absorption
Results:
The mean apparent permeability coefficient (Papp) of test substance was 73.3 x 10 (-6) cm/s and test substance is classified to be of high permeability.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
The apparent permeability coefficient (Papp) of test substance was 69.78 x 10 (-6) and 76.75 x 10 (-6) for first and second experiment respectively. The mean Papp value of test substance was 73.3 x 10 (-6) cm/s and therefore test substance is classified to be of high permeability.

Any other information on results incl. tables

Table 1: Product-ion spectra of test substance-in HPLC-MS/MS screening (study # 84747)

Molecular Weight (g)

Formula Weight (g)

Selected ESI (+) precursor Ion (m/z)

 

Product Ion (m/z)

 

Collision Offset (V)

 

Ionization classification

182.22

280.30

183.2

138.0

-20

2.0

Ionization Classification: The classification is as follows:

1 = Highly ionizable compound

2 = Intermediately ionizable compound

3 = Poorly ionizable compound

 

Result of recovery (%):The mean recovery of the test substance was 33%.

Result of assay validation:The assay was validated as suitability criteria were met for reference compounds. The permeability values for the test substance and reference compounds are as follows:

Table 2: Results of A-B Permeability assay (study # 84747)

Treatment

Concentration (µM)

Papp (10(-6) cm/s)

Recovery (%)

1st

2nd

Mean

1st

2nd

Mean

Test substance

50

69.78

76.75

73.30

34

32

33

Reference compound

Propranolol

50

23.85

27.93

25.90

86

59

72

Ranitidine

50

0.20

0.20

0.20

93

89

91

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: High permeability
In an A-B (apical to basolateral) permeability assay performed using the TC-7 cell line, 2-amino-hydroxyethylaminoanisole sulfate showed a Papp of 73.3 x 10 (-6) cm/sec. Therefore test substance was classified to be of high permeability, indicating a nearly 100% absorption from the gastrointestinal tract.
Executive summary:

The in-vitro A-B (apical to basolateral) permeability study of 2-amino-4-hydroxyethylaminoanisole sulfate  was conducted to determine bioavailability of the test substance across the intestinal barrier using the TC-7 human intestinal epithelial cell line.

The permeability from the apical (A, pH 6.5) to the basolateral (B, pH 7.4) side of a cell monolayer was investigated at 37°C in 96-well transwell plates with shaking for a 60 min contact time. The test solution (50 µM test substance in HBSS) was applied to the apical side and HBSS-HEPES buffer was added to the basolateral side. Analysis of the donor (apical) and receiver (basolateral) samples was performed by means of HLPC-MS/MS. For analysis, donor samples were collected at 0 and 60 minutes and receiver samples were collected at 60 min. The apparent permeability coefficient (Papp) was calculated for two independent experiments. 14C-mannitol was used to demonstrate the integrity of the cell monolayer. Only monolayers revealing a permeability of < 2.5 x 10 (-6) cm/sec were used.

The reference compounds Propranolol and Ranitidine were analysed concurrently to demonstrate the validity of the test system.

The mean apparent permeability coefficient (Papp) of test substance was 73.3 x 10 (-6) cm/s.The mean recovery of the test substance was 33%.

The Papp value for reference compounds Propanolol (25.90x 10 (-6)) and ranitidine (0.20x 10 (-6)) were within the acceptable range and demonstrated the validity of the assay.

In an A-B (apical to basolateral) permeability assay performed using the TC-7 cell line, 2-amino-hydroxyethylamino-anisole sulfate showed a Papp of 73.3 x 10 (-6) cm/sec. Therefore test substance was classified to be of high permeability, indicating a nearly 100% absorption from the gastrointestinal tract.