Menotropins
»Menotropins is an extract of human post-menopausal urine containing both follicle-stimulating hormone and luteinizing hormone,having the property in females of stimulating growth and maturation of ovarian follicles and the properties in males of maintaining and stimulating testicular interstitial cells (Leydig tissue)related to testosterone production and of being responsible for the full development and maturation of spermatozoa in the seminiferous tubules.It has a potency of not less than 40USP Follicle-Stimulating Hormone Units and not less than 40USP Luteinizing Hormone Units per mg,and it contains not less than 80percent and not more than 125percent of each of the hormone potencies stated on the label.The ratio of Units of Follicle-Stimulating Hormone to Units of Luteinizing Hormone is approximately 1.When necessary,Chorionic Gonadotropin obtained from the urine of pregnant women may be added to achieve this ratio.Not more than 30percent of the luteinizing hormone activity is contributed by Chorionic Gonadotropin,as determined by a validated method.
Packaging and storage—
Preserve in tight containers,preferably of Type Iglass,and store in a refrigerator.
USP Reference standards á11ñ—
USP Endotoxin RS.USP Human Chorionic Gonadotropin RS.USP Menotropins RS.
Bacterial endotoxins á85ñ—
It contains not more than 2.5Endotoxin Units per USP Follicle-Stimulating Hormone Unit.
Safety—
Prepare a test solution of it in Sodium Chloride Injectioncontaining 75USP Follicle-Stimulating Hormone Units per mL.Select five healthy mice,each weighing between 18g and 22g.Inject intravenously one dose of 1.0mLof the test solution into each of the mice.Observe the animals over the 48hours following the injection.If,at the end of 48hours,not more than 1of the animals shows outward symptoms of a toxic reaction,the requirements of the test are met.If 1or 2of the animals die,repeat the test on 10additional,similar animals;if all of the animals survive for 48hours and show no symptoms of a toxic reaction,the requirements of the test are met.
Water,Method Iá921ñ:
not more than 5.0%.
Assay for luteinizing hormone—
Diluent—
Dissolve 10.75g of dibasic sodium phosphate,7.6g of sodium chloride,and 1.0g of bovine serum albumin in 1Lof freshly distilled water.Adjust with 1Nsodium hydroxide or dilute with 20%phosphoric acid to a pHof 7.2±0.2.
Standard preparations—
Dissolve an accurately weighed quantity of USP Menotropins RSin the Diluentto obtain solutions having known concentrations of about 8.75,17.5,and 35.0USP Luteinizing Hormone Units per mL.
Assay preparations—
Following the procedure given under the Standard preparations,use Menotropins in place of the USP Reference Standard to obtain similar solutions.
Control solution—
Use the Diluentas the control solution.Store all solutions at 5±3
for the duration of the assay and properly dispose of any unused portions.
for the duration of the assay and properly dispose of any unused portions.
Test animals—
Select 20-to 21-day old male rats with weights within a 10g range of each other.House the animals under uniform conditions of temperature,light,food,and water.Mark the animals for identification,and divide them at random into 7groups of the same number,having not less than 6animals per group.Assign one group to each Standard preparation,one group to each Assay preparation,and one group to the Control solution.
Dose determination trial—
Use the method described for Procedureto determine a 3-dose range in which the lowest dose produces a definite response in some of the the rats in the low-dose group (as compared with the control group)and the highest dose produces a submaximal to maximal response in the high-dose group.Doses must be established in a geometric progression.The normal dose response range will occur between 3.5and 28USP Luteinizing Hormone Units total dose per rat.Useful dose ranges will vary with the sensitivity of the rat strain selected.
Procedure—
Inject each rat of each group subcutaneously in the dorsal area with 0.2mLof the solution to which it was assigned.For the Dose determination trialonly,similarly inject each rat in the control group with 0.2mLof the Control solution.Repeat these injections at approximately the same time of day after 24,48,and 72hours.Twenty-four hours after the last injection,weigh each rat,sacrifice the animals,and carefully dissect out the seminal vessicle of each rat,removing any fat and fibrous tissue.Thoroughly dry the vessicles by pressing against absorbent paper,avoiding damage to the vessicles,and immediately weigh them to the nearest 0.2mg,using a suitable balance.
Calculation—
Tabulate the observed seminal vessicle weights(y)for each dosage group of frats.For apparently outlying seminal vessicle weights,an attempt may be made to correct the organ mass relative to the mass of the rat from which it was taken.For the y-value in question,calculate for each of the frats in the appropriate group the ratio of seminal vessicle weight to total body weight.Reject the y-value if its corresponding ratio differs from the rest of the group by more than 1.5standard deviations.
If the data from one or more rats are missing,adjust to groups of equal size by suitable means (see Replacement of Missing Values á111ñ).Total the values of yin each group,and designate each total as T,using subscripts 1to 3for the three successive dosage levels and subscripts Sand Ufor the Standard and the material under test,respectively.Test both the agreement in slope of the dosage–response lines for the Standard and for the material under test,and the lack of curvature as directed for a 3-dose balanced assay (see Tests of Assay Validity under Design and Analysis of Biological Assay á111ñ).If the combined discrepancy as measured by F3exceeds its tabular value in Table 9,repeat the assay.
Determine the logarithm of luteinizing hormone potency of the Menotropins taken by the formula:
M=(4iTA/3TB)+log R,
in which TA=S(TU-TS);TB=S(T3-T1);iis the interval between successive log doses of both the Standard preparationand the Assay preparation;and R=vS/vUis the ratio of the high dose of the Standard in USP Luteinizing Hormone Units (vS)to the high dose of the Menotropins in mg (vU).Compute the log confidence interval (see Design and Analysis of Biological Assays á111ñ).
Replication—
Repeat the entire determination at least once.Test the agreement among the two or more independent determinations,and compute the weight for each (see Combination of Independent Assays á111ñ).Calculate the weighted mean log-potency Mand its confidence interval,LC(see Confidence Intervals for Individual Assays á111ñ).The potency,P*,is satisfactory if P*=antilog Mis not less than 80%and not more than 125%of the labeled potency and if the confidence interval does not exceed 0.18.
Assay for follicle-stimulating hormone—
Diluting solution—
Using the Diluentunder Assay for luteinizing hormone,dissolve an accurately weighed quantity of USP Human Chorionic Gonadotropin RSto obtain a solution having a concentration of 70USP Chorionic Gonadotropin Units per mL,readjusting the pH,if necessary,to 7.2±0.2.
Standard preparations—
Dissolve an accurately weighed quantity of USP Menotropins RSin the Diluting solutionto obtain solutions having known concentrations of about 2.5,5.0,and 10.0USP Follicle-Stimulating Hormone Units per mL.
Assay preparations—
Following the procedure given under the Standard preparations,use Menotropins in place of the USP Reference Standard to obtain similar solutions.
Control solution—
Use the Diluting solutionas the control solution.Store all solutions at 5±3for the duration of the assay,and properly dispose of any unused portions.
for the duration of the assay,and properly dispose of any unused portions.
Test animals—
Select 20-to 21-day old female rats with weights within a 10-g range of each other.Proceed as directed under Test animalsin the Assay for luteinizing hormonebeginning with “House the animals.”
Dose determination trial—
Use the method described for Procedureto determine a 3-dose range in which the lowest dose produces a definite response in some of the rats in the low-dose group (as compared with the control group)and the highest dose produces a submaximal to maximal response in the high-dose group.Doses must be established in a geometric progression.The normal dose response range will occur between 0.5and 6.0USP Follicle-Stimulating Hormone Units total dose per rat.Useful dose ranges will vary with the sensitivity of the rat strain selected.
Procedure—
Inject each rat of each group subcutaneously in the dorsal area with 0.2mLof the solution to which it was assigned.For Dose determination trialonly,similarly inject each rat in the control group with 0.2mLof the Control solution.Repeat these injections at approximately the same time of day after 24hours and 48hours.Twenty-four hours after the last injection,weigh each rat,sacrifice the animals,and carefully dissect out the ovaries of each rat,removing any fat and fibrous tissue.Thoroughly dry the ovaries by pressing against absorbent paper,avoiding damage to follicles on the ovary surface,and immediately weigh them to the nearest 0.2mg,using a suitable balance.
Calculation—
Tabulate the observed ovarian pair weight for each rat designated by the symbol y,for each dosage group of frats.For apparently outlying ovarian weight gain,an attempt may be made to correct the organ mass relative to the mass of the rat from which it was taken.For the y-value in question,calculate for each of the frats in the appropriate group the ratio of ovarian weight to the total body weight.Reject the y-value if its corresponding ratio differs from the rest of the group by more than 1.5standard deviations.
If the data from one or more rats are missing,adjust to groups of equal size by suitable means (seeReplacement of Missing Values under Design and Analysis of Biological Assays á111ñ).Total the values of yin each group,and designate each total as T,using subscripts 1to 3for the three successive dosage levels and subscripts Sand Ufor the Standard and the material under test,respectively.Test both the agreement in slope of the dosage-response lines for the Standard and for the material under test,and the lack of curvature as directed for a 3-dose balanced assay (see Tests of Assay Validity under Design and Analysis of Biological Assays á111ñ).If the combined discrepancy as measured by F3exceeds its tabular value in Table 9(see Combination of Independent Assays under Design and Analysis of Biological Assays á111ñ),regard these data as preliminary,and repeat the assay.
Determine the logarithm of follicle-stimulating hormone potency of the Menotropins taken by the formula:
M=(4iTA/3TB)+log R,
in which TA=S(TU-TS);TB=S(T3-T1);iis the interval between successive log doses of both the Standard preparationand Assay preparation;and R=vS/vUis the ratio of the high dose of the Standard in USP Units (vS)to the high dose of the Menotropins in mg (vU).Compute the log confidence interval (see Design and Analysis of Biological Assays á111ñ).
Replication—
Repeat the entire determination at least once.Test the agreement among the two or more independent determinations,and compute the weights/mean log-potency Mand its confidence interval,LC(see Confidence Intervals for Individual Assays under Design and Analysis of Biological Assays á111ñ).If this exceeds 0.18,repeat the assay until the confidence interval of the combined results is 0.18or less.
The potency P*is satisfactory if P*=antilog Mis not less than 80%and not more than 125%of the labeled potency and if the log confidence interval does not exceed 0.18.
Auxiliary Information—
Staff Liaison:Larry N.Callahan,Ph.D.,Scientist
Expert Committee:(BNT)Biotechnology and Natural Therapeutics/Diagnostics
USP28–NF23Page 1205
Pharmacopeial Forum:Volume No.29(6)Page 1923
Phone Number:1-301-816-8385