Mycophenolic acid

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 30th 1992 to August 4th 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The protocol follows closely the recommendations made in the Environmental Assessment Technical Assistance Handbook (Food and Drug Administration, March 1987).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: The protocol follows closely the recommendations made in the Environmental Assessment Technical Assistance Handbook (Food and Drug Administration, March 1987).

Principles of method if other than guideline:

This screening test is conducted to estimate hydrolytic potential. The test measures the loss of a test compound found to occur over a five day period at 50°C f I0C in a buffered, aqueous solution. A loss of 10% or more of the test compound is an indication of hydrolytic instability under environmen ally relevant conditions.
Test System
The test was conducted using a Lab-Line Model 3527 orbital shaker fitted with a metal shaking platform designed to hold 250 mL Erlenmeyer flasks. The unit was operated with temperature set at 50°C f I0C and shaker set at 100 rpm. The shaker was placed on the "hold" setting for a continuous, five day operation.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Mycophenolic acid

EC Number:

246-119-3

EC Name:

Mycophenolic acid

Cas Number:

24280-93-1

Molecular formula:

C17H20O6

IUPAC Name:

6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-2-benzofuran-5-yl)-4-methylhex-4-enoic acid

Details on test material:

Molecular formula: C,H,O,
CAS registry number: 24280-93-1
pK: 4.58 (COOH), 8.05 (phenolic)
Vapor pressure estimate: 3.2 x 10" Torr
Solubility, mglmL
- pH 5: 0.06
- pH 7: 3.1

Study design

Analytical monitoring:

yes

Details on sampling:

Samples from each triplicate for each pH condition were analyzed for mycophenolic acid. Each sample was analyzed in triplicate by HPLC.

Buffers:

Three 0.1 M buffered solutions were prepared from sodium acetate, monobasic sodium phosphate and sodium borate using purified water to give solutions with pH values of 5.05, 7.03 and 8.90. To prepare the pH 9 and pH 7 test solutions, 30.52 mg and 30.39 mg of test compound was added to 1.0 L of each of the pH 8.90 and pH 7.03 buffers, respectively. For the pH 5 test solution, it was necessary to predissolve 30.10 mg of mycophenolic acid in 4 mL of acetonitrile.
The solution was added to the pH 5.05 buffer, to give a total volume of 1.0 L of the pH 5 test
solution.Stock solutions of 0.05 mg/mL were prepared in each buffer. The mycophenolic acid was predissolved in 0.5 mL acetonitrile for 100 mL of the pH 5 buffer solution. The concentration of acetonitrile was one percent volume or less in the final pH 5 buffer solution. Serial dilutions were prepared from each buffer stock solution to give ten data points ranging from 0.05 to 0.005 mg/mL.
PREPARATION OF SOLUTIONS
Use purified water to prepare the following:
Buffer Solutions
pH 5: Accurately weigh 13.608 g of sodium acetate and dissolve to a total volume of 1 L. Adjust the pH with glacial acetic acid. (p0.1)
pH 7: Accurately weigh 13.800 g of monobasic sodium phosphate and dilute to a total volume of 1.0 L. Adjust the pH with 0.1 N sodium hydroxide. (~=0.1)
pH 9: Accurately weigh 20.128 g sodium borate and dilute to a total volume of 1 .O L. Adjust the pH with glacial acetic acid. (p=0.3)
Add mycophenolic acid to the buffer solutions as follows: Dissolve approximately 30 mg of mycophenolic acid in 1 L of buffer.

Details on test conditions:

TEST SYSTEM
The test was conducted using a Lab-Line Model 3527 orbital shaker fitted with a metal shaking platform designed to hold 250 mL Erlenmeyer flasks. The unit was operated with temperature set at 50°C f I0C and shaker set at 100 rpm. The shaker was placed on the "hold" setting for a continuous, five day operation.
TEST MEDIUM
Three 0.1 M buffered solutions were prepared from sodium acetate, monobasic sodium phosphate and sodium borate using purified water to give solutions with pH values of 5.05, 7.03 and 8.90. To prepare the pH 9 and pH 7 test solutions, 30.52 mg and 30.39 mg of test compound was added to 1.0 L of each of the pH 8.90 and pH 7.03 buffers, respectively. For the pH 5 test solution, it was necessary to predissolve 30.10 mg of mycophenolic acid in 4 mL of acetonitrile.

Duration of test

Duration:

5 d

Initial conc. measured:

>= 0.1 mol/L

Number of replicates:

Samples from each triplicate for each pH condition were analyzed for mycophenolic acid.

Positive controls:

no

Negative controls:

no

Statistical methods:

The raw data from each sample triplicate was tested for precision. The relative range was calculated and used to test sample triplicates.
Mycophenolic acid concentrations were then determined from the data using the slope and yintercept from linear regression analysis of data from mycophenolic acid calibration standards. The mean mycophenolic acid concentration and relative range were then calculated from the triplicate flask determinations for each pH condition. The percent hydrolysis of mycophenolic acid was determined by dividing the mean initial mycophenolic acid conc ntration less the mean final mycophenolic acid concentration by the mean initial mycophenolic acid concentration.

Results and discussion

Transformation products:

not measured

Total recovery of test substance (in %)

Total recovery of test substance (in %) 1

% Recovery:

46.15

pH:

5.05

Temp.:

50 °C

Duration:

5 d

Total recovery of test substance (in %) 2

% Recovery:

29.03

pH:

7.03

Temp.:

50 °C

Duration:

5 d

Total recovery of test substance (in %) 3

% Recovery:

36.67

pH:

8.9

Temp.:

50 °C

Duration:

5 d

Dissipation DT50 of parent compound

Dissipation DT50 of parent compound 1

pH:

5

Temp.:

50 °C

Hydrolysis rate constant:

-5 027 s-1

Dissipation DT50 of parent compound 2

pH:

7

Temp.:

50 °C

Hydrolysis rate constant:

-3 177 s-1

Dissipation DT50 of parent compound 3

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

-757 s-1

Details on results:

Values for the mean measured percent loss of mycophenolic acid at pH 5.05, 7.03 and 8.90 were 46.15%, 29.03% and 36.67%, respectively. Losses were greater than 10% of initial test concentrations and significant hydrolytic potential was established in this study.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Losses were greater than 10% of initial test concentrations and significant hydrolytic potential was established in this study.

Executive summary:

Purpose.

This study was conducted to determine the loss of mycophenolic acid under hydrolytic conditions in aqueous buffers.

Performance.

Three 0.1 M buffered solutions were prepared from sodium acetate, monobasic sodium phosphate and sodium borate to give stock solutions with pH values of 5.05, 7.03 and 8.90. The test compound, mycophenolic acid, was dissolved in the buffered solutions to yield the test solutions. The solutions were then maintained, in triplicate, in stoppered flasks at 50°C +/- 1°C for a period of five days. Initial and final samples were withdrawn from each flask for analysis by HPLC.

Results.

Values for the mean measured percent loss of mycophenolic acid at pH 5.05, 7.03 and 8.90 were 46.15%, 29.03% and 36.67%, respectively.

Losses were greater than 10% of initial test concentrations and significant hydrolytic potential was established in this study.