1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d] pyrimidin-5-yl)phenylsulphonyl]-4-methylpiperazine

Partition coefficient

Administrative data

Endpoint:

partition coefficient

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-10-13 to 2010-12-1

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

UK 92480 is a intermediate in the

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 107 (Partition Coefficient (n-octanol / water), Shake Flask Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 830.7550 (Partition Coefficient, n-octanol / H2O, Shake Flask Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of method:

flask method

Partition coefficient type:

octanol-water

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

not available

Test material form:

solid - liquid: aqueous solution

Remarks:

10:1 (v/v) ethanol/water

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot No.of test material: Sponsor and 00703777-020-WH0001
- Expiration date of the lot/batch: Not available
-Purity Test Date: 2010-07-29

RADIOLABELLING INFORMATION
- Radiochemical purity: >97%
- Specific activity: 30.6 µCi/mg

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was stored under frozen conditions in darkness, initially at approximately -18°C followed by a transfer to approximately -28°C storage and finally placed in an ultra-cold freezer (at approximately -80°C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
A primary stock solution of the 14C-Sildenafil test substance was prepared in methanol by using a calibrated positive displacement pipette to transfer a 0.500 mL aliquot of the test substance solution into a 5.00-mL volumetric flask. The aliquot was evaporated to dryness at room temperature under nitrogen. The test substance was re-dissolved in methanol and the flask was sonicated for approximately 5 minutes before the solution was brought to final volume in methanol. The activity of the resulting primary stock solution was 2.93 µCi/mL as determined by LSC analysis. Based on a specific activity of 30.6 µCi/mg for the radiolabeled test substance, the calculated 14 C-Sildenafil concentration in this primary stock solution was 95.9 µg/mL. The primary stock solution was used to fortify the partition samples, the n-octanol matrix fortification quality control (QC) samples and the pH 4 aqueous matrix fortification QC sample.
An aliquot of the primary stock solution was diluted 10-fold with methanol in a glass vial to create a secondary stock solution. The activity of the resulting secondary stock solution was determined by LSC analysis to be 0.308 µCi/mL which corresponds to a 14C-Sildenafil calculated concentration of 10.1 µg/mL (based on a specific activity of 30.6 µCi/mg). This secondary stock solution was used to fortify the pH 7 and 9 aqueous matrix fortification QC samples.

FORM AS APPLIED IN THE TEST
Partition Sample Preparation for Feasibility Trial
For each aqueous phase pH test condition, an initial feasibility trial was conducted with equal volumes of saturated aqueous buffer and n-octanol solutions (n-octanol:aqueous buffer solution volume ratio of 1:1). For each pH test condition, duplicate samples were prepared by fortifying each of two empty glass vials with 0.100 mL of the primary stock solution and evaporating the solvent under a gentle stream of nitrogen. To each sample, 10.0 milliliters of the appropriate aqueous buffer solution (saturated with n-octanol) was added, followed by vortexing, approximately five minutes of sonication, and 10.0 milliliters of n-octanol (saturated with the appropriate buffer solution). For all three pH test conditions, the nominal fortified mass of
14 C-Sildenafil was 9.59 micrograms in each partition sample.

Partition Sample Preparation for Definitive Test
Based on the findings of the feasibility trial, partition samples in the definitive tests were prepared at n-octanol/water volume ratios of 1:1, 1:2 and 1:4 for all three pH test conditions. The definitive tests for each pH test system were performed on separate sample preparation days. For each pH test condition, the partition samples were prepared in duplicate at each volume ratio in 40-mL glass vials. Positive displacement pipettes were used to add an appropriate volume of the 95.9 µg/mL primary stock solution to each empty sample vial and the solvent was evaporated under a gentle stream of nitrogen. For pH 4, the nominal fortified mass of 14 C-Sildenafil was 6.71 µg. For pH 7 the nominal fortified mass of 14 C-Sildenafil was 9.59 µg. For pH 9 the nominal fortified mass of 14 C-Sildenafil was 7.67 µg.

Study design

Analytical method:

liquid chromatography

Results and discussion

Partition coefficient

Partition coefficient 1

Key result

Type:

log Pow

Partition coefficient:

ca. 0.424

Temp.:

25 °C

pH:

ca. 4

Partition coefficient 2

Key result

Type:

log Pow

Partition coefficient:

ca. 1.78

Temp.:

25 °C

pH:

ca. 7

Partition coefficient 3

Key result

Type:

log Pow

Partition coefficient:

ca. 1.56

Temp.:

25 °C

pH:

ca. 9

Details on results:

Partition coefficients (Pow) were calculated from the measured concentrations of 14C-Sildenafil in each phase for each of six samples for each pH test condition. Overall mean n-octanol/water partition coefficients and overall mean of the logarithm (base 10) of the n-octanol/water partition coefficients were calculated for each of the six samples for each pH test condition.

In the pH 4 partition test system, the mean Pow values for 14C-Sildenafil at n-octanol/water solvent ratios of 1:1, 1:2, and 1:4 were determined to be 2.71, 2.83 and 2.45, respectively (Table 6). The corresponding mean log Pow values were 0.432, 0.452 and 0.389, respectively. The overall mean log Pow was 0.424 ± 0.034 (n = 6, CV = 8.0%).

In the pH 7 partition test system, the mean Pow values for 14C-Sildenafil at n-octanol/water solvent ratios of 1:1, 1:2, and 1:4 were determined to be 45.2, 60.6 and 79.5, respectively (Table 7). The corresponding mean log Pow values were 1.66, 1.78 and 1.90, respectively. The overall mean log Pow was 1.78 ± 0.11 (n = 6, CV = 6.2%).

In the pH 9 partition test system, the mean Pow values for 14C-Sildenafil at n-octanol/water solvent ratios of 1:1, 1:2, and 1:4 were determined to be 28.5, 51.4 and 33.5, respectively (Table 8). The corresponding log Pow values were 1.46, 1.71 and 1.52, respectively. The overall mean log Pow was 1.56 ± 0.13 (n = 6, CV = 8.4%).

Applicant's summary and conclusion

Conclusions:

The n-octanol/water partition coefficients (Pow) of 14C-Sildenafil at 25ºC as a function of water pH were effectively determined by this study.