Edition: BP 2025 (Ph. Eur. 11.6 update)
Action and use
Follicle-stimulating hormone.
Preparation
Urofollitropin Injection
DEFINITION
Dried preparation containing menopausal gonadotrophin obtained from the urine of post-menopausal women. It has follicle-stimulating activity and no or virtually no luteinising activity.
Potency
Minimum 90 IU of follicle-stimulating hormone (hFSH) per milligram.
Ratio of the number of units of luteinising hormone (interstitial-cell-stimulating hormone) [hLH(ICSH)] to the number of units of follicle-stimulating hormone Maximum 1/60.
PRODUCTION
It is prepared by suitable collection and extraction procedures followed by purification steps.
The method of preparation includes steps that have been shown to remove and/or inactivate extraneous agents. In addition, the process is designed to minimise microbial contamination.
CHARACTERS
Appearance
Almost white or slightly yellowish powder.
Solubility
Soluble in water.
IDENTIFICATION
When administered to immature female rats as prescribed in the assay, it causes enlargement of the ovaries.
TESTS
Residual luteinising activity
The International Units of FSH and LH are the activities contained in stated amounts of the International Standard of human urinary follicle-stimulating hormone and luteinising hormone (interstitial-cell-stimulating hormone) which consists of a mixture of a freeze-dried extract of urine of post-menopausal women with lactose. The equivalence in International Units of the International Standard is stated by the World Health Organization.
Use immature female rats approximately 21 days old and having masses such that the difference between the heaviest and the lightest rat is not more than 10 g. Assign the rats at random to 4 equal groups of at least 6 animals. If sets of 4 litter mates are available, assign one litter mate at random from each set to each group and mark according to litter.
Inject subcutaneously into each rat 50 IU of serum gonadotrophin R on the first day and 25 IU of chorionic gonadotrophin R on the fourth day, each in 0.5 mL of phosphate-albumin buffered saline pH 7.2 R.
Choose 3 doses of the reference preparation such that the smallest dose produces a depletion of the ovarian ascorbic acid content in all the rats and the largest dose does not produce a maximal depletion in all the rats. Use doses in geometric progression; as an initial approximation, total doses of 0.5 IU, 1.0 IU and 2.0 IU may be tried although the dose to be used will depend on the sensitivity of the animals.
Choose a dose of the preparation to be examined expected to contain 60 X IU of follicle-stimulating hormone (hFSH), in which X = the number of International Units of hLH in the middle dose of the reference preparation.
Dissolve separately the total quantities of the preparation to be examined and of the reference preparation in 1.0 mL of phosphate-albumin buffered saline pH 7.2 R. Inject into a tail vein to each separate group of rats 6 days after the injection of chorionic gonadotrophin. Exactly 4 h after the injection, euthanise the rats and remove the ovaries from each animal. Remove any extraneous fluid and tissue from the ovaries and weigh the ovaries immediately.
Treat the combined ovaries from each rat separately, as follows. Crush and homogenise within 2 min in a freshly prepared 25 g/L solution of metaphosphoric acid R at a temperature of 4 °C and dilute to 7 mL with the same solution. Allow the homogenate to stand for 30 min at 4 °C and centrifuge at 4 °C at approximately 2500 g. Filter the supernatant, if necessary, through a 0.22 µm filter.
Prepare a fresh solution consisting of a mixture of 2 mL of a 45.3 g/L solution of sodium acetate R adjusted to pH 7 with
acetic acid R, 3 mL of water R and 2 mL of dichlorophenolindophenol standard solution R. Mix 2 mL of this solution with
2 mL of the clear supernatant. 30 s after mixing, measure the absorbance (2.2.25) of the solution at the maximum at about 520 nm. Use as reference a solution with a known content of ascorbic acid CRS in a 25 g/L solution of metaphosphoric acid R, treated by the same process.
Calculate the amount of ascorbic acid from the ascorbic acid standard curve obtained and express in milligrams per 0.1 g of ovary to obtain the ascorbic acid content of the ovaries. Calculate the mean and its variance of the ascorbic acid content of the ovaries of the rats treated with the preparation to be examined.
For each dose-group of the reference preparation, plot the mean ascorbic acid content of the ovaries as a function of the logarithm of the dose and analyse the regression of the ascorbic acid content on the logarithm of the dose injected, using standard methods of analysis (the method of least squares).
The test is not valid unless:
— the slope constant b is significant at the 0.05 significance level;
— for the groups treated with the reference preparation, the sum of squares due to linear regression is equal to at least 95 per cent of the total sum of squares of the ascorbic acid content;
— the within-group variance of the ascorbic acid content of the group receiving the preparation to be examined is not significantly different at the 0.05 significance level from the within-group variance of the ascorbic acid content of the groups receiving the reference preparation.
The mean ascorbic acid content of the ovaries of the rats treated with the preparation to be examined is not significantly lower than that of the rats treated with the middle dose of the reference preparation (calculated from the regression equation) at the 0.05 significance level.
Water (2.5.32)
Maximum 5.0 per cent.
Bacterial endotoxins (2.6.14, Method C)
Less than 0.40 IU per International Unit of urofollitropin, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins.
ASSAY
The follicle-stimulating activity of urofollitropin is estimated by comparing under given conditions its effect in enlarging the ovaries of immature rats treated with chorionic gonadotrophin with the same effect of the International Standard preparation of human urinary follicle-stimulating hormone and luteinising hormone or of a reference preparation calibrated in International Units. The International Units of FSH and LH are the activities contained in stated amounts of the International Standard of human urinary follicle-stimulating hormone and luteinising hormone (interstitial-cell-stimulating hormone) which consists of a mixture of a freeze-dried extract of urine of post-menopausal women with lactose. The equivalence in International Units of the International Standard is stated by the World Health Organization.
Use immature female rats of the same strain, 19-28 days old, differing in age by not more than 3 days and having masses such that the difference between the heaviest and the lightest rat is not more than 10 g. Assign the rats at random to 6 equal groups of at least 5 animals. If sets of 6 litter mates are available, assign one litter mate from each set to each group and mark according to litter.
Choose 3 doses of the reference preparation and 3 doses of the preparation to be examined such that the smallest dose produces a positive response in some of the rats and the largest dose does not produce a maximal response in all the rats. Use doses in geometric progression and as an initial approximation total doses of 1.5 IU, 3.0 IU and 6.0 IU may be tried although the dose will depend on the sensitivity of the animals used, which may vary widely.
Dissolve separately the total quantities of the preparation to be examined and of the reference preparation corresponding to the daily doses to be used in sufficient phosphate-albumin buffered saline pH 7.2 R such that the daily dose is administered in a volume of about 0.5 mL. The buffer solution shall contain in the daily dose not less than 14 IU of chorionic gonadotrophin to ensure complete luteinisation. Add a suitable antimicrobial preservative such as 4 g/L of phenol or 0.02 g/L of thiomersal. Store the solutions at 5 ± 3 °C.
Inject subcutaneously into each rat the daily dose allocated to its group. Repeat the injection of each dose 24 h and 48 h after the first injection. About 24 h after the last injection, euthanise the rats and remove the ovaries from each animal.
Remove any extraneous fluid and tissue from the ovaries and weigh the 2 combined ovaries of each animal immediately. Calculate the results by the usual statistical methods (for example, 5.3) using the mass of the 2 combined ovaries as the response (a suitable correction of the organ mass with reference to the mass of the animal from which it was taken may be applied; an analysis of covariance may be used).
The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated potency. The confidence limits (P = 0.95) of the estimated potency are not less than 64 per cent and not more than 156 per cent of the stated potency.
STORAGE
In an airtight container, protected from light, at a temperature of 2 °C to 8 °C. If the substance is sterile, store in a sterile, airtight, tamper-evident container.
LABELLING
The label states:
— the number of International Units of follicle-stimulating hormone per container;
— the potency, in International Units of follicle-stimulating hormone per milligram.
