WO2005096811A2

WO2005096811A2 - In vivo bioassay for gonadotropins

Info

Publication number: WO2005096811A2
Application number: PCT/US2005/010359
Authority: WO
Inventors: Bruce D. Weintraub; Valerie Fremont; Mariusz W Szkudlinski
Original assignee: Trophogen, Inc.
Priority date: 2004-03-31
Filing date: 2005-03-29
Publication date: 2005-10-20
Also published as: WO2005096811A3

Abstract

The present invention provides methods of determining gonadotropin activity comprising administering to a mouse at least one gonadotropin and detecting at least one endpoint, wherein said mouse has an altered response to gonadotropins. In certain embodiments, the methods provide, inter alia, for an in vivo bioassay for the quantification and standardization of gonadotropins. In certain embodiments, the invention provides, inter alia, for a murine bioassay wherein at least one endpoint is detected. In certain embodiments, the endpoint is a functional or morphometric endpoint.

Description

IN VIVO BIOASSAY FOR GONADOTROPINS

1. FIELD OF THE INVENTION

[001] The present invention provides an in vivo assay for the evaluation of gonadotropins. The invention provides a mouse bioassay for the evaluation of gonadotropins such as follicle stimulating hormone, luteinizing hormone and chorionic gonadotropin.

2. BACKGROUND

[002] The gonadotropins, follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG) are produced by the anterior pituitary (FSH and LH) and placenta (CG). This family of glycoproteins exits as heterodimers with two covalently linked subunits α- and β-. (Pierce and Parsons, Ann. Rev. Biochem., 1981, 50: 465-495). However, the gonadotropins are not single molecular structures but multiple charged isoforms that result from variations in the composition ofthe carbohydrate chains, particularly the sialic acid content. (Chappel, Hum. Reprod., 1995, 1:479-487). Sialic acids comprising a family of 9-carbon acidicmonosaccharides, are commonly found in mammals and other animals, but are absent in plants. (See, Chou, et al. PNAS USA, 2002, 99: 11736-11741).

[003] FSH and LH stimulate and promote spermatogenesis in men and regulate follicle growth in women and have been used clinically for the treatment of infertility, especially controlled ovarian stimulation during assisted reproduction. In women, FSH has also been used for the treatment of various ovulatory disorders and in men is used for the treatment of hypogonadotrophic hypogonadism.

[004] Prior to recombinant DNA technology, all gonadotropins were obtained from urine. Crude protein purification methods provided gonadotropin products with varying degrees of protein contamination. Also contributing to significant batch-to-batch inconsistencies was the highly variable isoform composition of urinary gonadotropins. Given the enormous variability in biopotency, activity and purity, the designation of protein content in any particular sample by weight or mass was, and still is, unhelpful. Hence bioassays were developed to allow samples of unknown biopotency to be estimated. Test results on bulk preparations allows filling of FSH vials or ampoules according to the desired FSH bioactivity measured in international units (IU). This bioactivity is then confirmed by a final bioassay on the finished product just prior to release. (Driebergen and Baer, Curr. Med. Res. Opin. 2003, 19(l):41-46). [005] The determination of gonadotopin activity can be made using a number of assays, including the radioligand receptor assay (RRA), the in vitro Sertoli cell bioassay, the in vitro granulosa cell bioassay, and the inhibin immunoassay. The in vitro assays require cell-culture facilities and techniques that are technically demanding. In addition, these assays are expensive and cumbersome. See, Wang, Endocr. Rev. 1988, 9(3):374-7 and Christin-Maitre, et al, Methods 2000, 21(l):51-7 for descriptions of these assays. [006] The traditional standard for the evaluation of follicle stimulating hormone is the Steelman-Pohley bioassay. (Steelman and Pohley, Endocrinology, 1953, 53:604- 616). This assay is the mainstay of pharmacopeial monographs for the statutory determination of FSH potency of therapeutic preparations. This assay however, has limited precision, requires large numbers of laboratory animals and involves cumbersome procedures for data generation and interpretation. For example, the bioassay has limited precision, with a coefficient of variation in a single determination to be about 10-20%. In order to reduce the variability to about 10%, a large sample size must be used, requiring large numbers of mice (about 100 to 150) to be sacrificed. See, Christin-Maitre, et al, Methods 2000, 21(l):51-7. Such large numbers of sacrificial animals is not only expensive but there is increasing ethical and political pressure to reduce the use of laboratory animals. 3. SUMMARY

[007] The present invention provides methods of determining gonadotropin activity comprising administering to a mouse at least one gonadotropin and detecting at least two endpoints, wherein said mouse has an altered response to gonadotropins. In certain embodiments, the methods provide, inter alia, for an in vivo bioassay for the quantification and standardization of gonadotropins. In certain embodiments, the invention provides, inter alia, for a murine bioassay wherein the mouse has an increased response to gonadotropins. In certain embodiments, the invention provides, inter alia, for a murine bioassay wherein the mouse has a decreased response to gonadotropins.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[008] Figure 1 provides a schematic comparing the conventional approach to screening gonadotropins with a novel approach High Throughput Screening;

[009] Figure 2 provides the experimental procedure, requirements and results of the High Throughput Screening by Transient Transfection;

[010] Figure 3 provides a comparison of ovulation induction in three mouse strains;

[011] Figure 4 provides a comparison of plasma hFSH levels in three mouse strains;

[012] Figure 5 provides ovarian weight, plasma hFSH levels and number of oocytes retrieved (oocytes ovulated) from immature hybrid B6D2F1 mice following various treatments with FOLLISTIM®, TR 4402 and a negative control;

[013] Figure 6 provides ovarian weight and hFSH plasma levels of immature hybrid B6D2F1 mice following various treatments with FOLLISTIM®, TR 4402, TR

4301, hCG and a negative control;

[014] Figure 7 provides a comparison of ovulation induction as measured by ovarian weight, plasma hFSH and the number of oocytes ovulated in immature hybrid

B6D2F1 mice following 3 treatments of FOLLISTIM®, TR 4402 or TR 4405, each followed with a single dose of hCG;

[015] Figure 8 provides the p values for the comparison provided in Figure 7;

[016] Figure 9 provides the combined ovarian weight following treatments with

FOLLISTIM®, TR 4405, TR 4503, FSH wild type, hCG and a positive control;

[017] Figure 10 provides the number of oocytes retrieved (oocytes ovulated) following treatments with FOLLISTIM®, TR 4405, TR 4503, FSH wild type, hCG and a positive control; and

[018] Figure 11 provides the plasma hFSH levels following treatment with

FOLLISTIM®, TR 4405, TR 4503, FSH wild type, hCG and a positive control. 5. DETAILED DESCRIPTION OF THE INVENTION

[019] Applicants have discovered a robust murine bioassay that is more sensitive than the standard Steelman Pohley bioassay. The methods ofthe invention provide for, inter alia, characterization and evaluation of gonadotropins. The new murine bioassay is advantageous in that small amounts of gonadotropin material are needed to stimulate a biological response in the target mouse and fewer mice are needed to obtain a valid sample size of acceptable variability.

[020] The methods ofthe present invention provide an improvement over the

Steelman Pohley bioassay in that, inter alia, the endpoints used in the methods ofthe present invention are a more relevant and reliable endpoint than that ofthe Steelman Pohley bioassay. The Steelman Pohley assay is based on a linear relationship between administered FSH and ovarian weight. However, ovarian weight does not indicate whether mature follicles have developed. When exogenous gonadotropins are administered for assisted reproduction, the endpoint desired is the ovulation of mature oocytes. Thus the methods ofthe present invention provide a more relevant endpoint and the methods are more reliable in terms of predicting bioactivity of gonadotropins upon administration to a subject.

[021] The methods ofthe invention are also advantageous in that by identifying a

'super-responder' mouse strain and reliable endpoints, a smaller amount of gonadotropin can be administered to the mouse. In addition, Applicants have identified a mouse strain that can be used in the methods ofthe invention which is advantageous in that it is generally accepted by those of skill in the art that mice introduce less biological variability into an in vivo experiment than do rats.

[022] The present invention provides methods of determining gonadotropin activity comprising administering to a mouse at least one gonadotropin and detecting at least two endpoints, wherein said mouse has an altered response to gonadotropins. In certain embodiments, the methods provide, ter alia, for an in vivo bioassay for the characterization, quantification and standardization of gonadotropins. In certain embodiments, the invention provides, inter alia, that the mouse has an increased response to gonadotropins. In certain embodiments, the invention provides, inter alia, that the mouse has a decreased response to gonadotropins. In certain embodiments, the endpoints are functional endpoints.

[023] In certain embodiments, the methods provide for the determination of gonadotropin activity. The pharmacologic and pharmacokinetic characteristics of gonadotropins can be determined by the methods ofthe invention. For example, the isoforms of FSH vary in molecular weight, biological potency and elimination half-life. (Stanton, et al, Endocrinology, 1992, 130(5): 2820-32). Isoforms with increased sialic acid content are more acidic and have longer biological half-lives. (Stanton, et al, Endocrinology, 1992, 130(5): 2820-32 and Robertson, et al, Endocrinology, 1982, 111:385-391). In certain embodiments ofthe invention, the methods provide for the determination of pharmacologic or pharmacokinetic parameters of gonadotropin activity, such as potency, onset of activity, biological half-life and ED₅o (Effective Dose 50, the amount of material required to produce 50% of a specified effect). The determination or calculation of such parameters are known to those of skill in the art and have been described. See, Basic and Clinical Pharmacology 2000, Katzung, ed., McGraw Hill/Appleton and Lange, Textbook of Receptor Pharmacology, 2nd edition, 2002, Foreman and Johanson, eds., CRC Press, Goodman and Gilman's The Pharmacologic Basis of Therapeutics, 10th edition, Hardman et al, eds., McGraw Hill and Applied Biopharmaceutics and Pharmacokinetics, 4th edition, 1999, Shargel and Yu eds., McGraw Hill/Appleton and Lange the contents of each of which is incorporated herein by reference in their entireties. In certain embodiments, the methods provide that the gonadotropin activity provides indirect assessment of biological half-life and stability. [024] In certain embodiments the methods provide, inter alia, for the determination of gonadotropin activity. The in vivo bioassay, can be used for pharmacopoeial monographs for the determination of gonadotropin potency of therapeutic preparations. In the traditional Steelman Pohley assay, a linear relationship between ovarian weight and gonadotropin activity has been established. To standardize a gonadotropin of unknown activity, a parallel group of rats is given the gonadotropin of unknown activity and the ovarian weight determined at necropsy. A comparison ofthe gonadotropin activity/ovarian weight dose-response curve provided in the group with known gonadotropin activity with the ovarian weight in the parallel group provides the gonadotropin activity for the parallel group.

[025] The methods ofthe present invention can be used to standardize gonadotropin activity by establishing a dose-response curve using more relevant functional endpoints. In certain embodiments, the methods provide for the standardization of gonadotropin activity using a dose-response curve wherein the response is the number of oocytes ovulated in combination with ovarian weight anόVor in vitro fertilization.

[026] In certain embodiments, the methods provide for the determination ofthe potency of a gonadotropin. In certain embodiments, the determination of potency can be used to determine the acceptance range or the fiducial limits of a gonadotropin composition. See, Doody, "Follicle stimulating hormone: isoforms, consistency and fill- by-mass. Biochemical and clinical considerations" (abstract). 57th Meeting ofthe American Society of Reproductive Medicine, 20-25 October 2001, Orlando, Florida, USA.

[027] The Steelman-Pohley bioassay is widely acknowledged to have only limited precision - its coefficient of variation (CN) in a single determination is 10-20%. See, Driebergen and Baer, Curr. Med. Res. Opin. 2003, 19(1): 41-46. In certain embodiments, the methods provide a murine bioassay with a coefficient of variation of less than about 20%. In certain embodiments, the methods provide a murine bioassay with a coefficient of variation of less than about 18%, less than about 15%, less than about 12%, less than about 10%, less than about 8% or less than about 5%. In a preferred embodiment, the methods provide a murine bioassay with a coefficient of variation of about 6 to about 12%.

[028] In certain embodiments, the methods provide for the quantification and standardization of gonadotropins. In preferred embodiments, the methods provide for the quantification and standardization of follicle stimulating hormone (FSH) as expressed in International Units (IU).

[029] In certain embodiments, the methods provide for the determination ofthe specific activity of a gondotropin. The specific activity is a ratio ofthe bioactivity determined by bioassay and the protein content expressed in units of IU/mg protein. The protein content of a sample or batch of gonadotropin can be determined by size exclusion high performance liquid chromatography (SE-HPLC). Such methods are known in the art, for example, as reported in Driebergen and Baer, Curr. Med. Res. Opin. 2003, 19(1): 41-6, which is herein incorporated by reference in its entirety.

[030] In the methods ofthe present invention, the mouse has an altered response to gonadotropins. In certain embodiments, the mouse has an increased response to gonadotropins as compared with other strains of immature female mice. In certain embodiments, the mouse is about 90% more responsive to gonadotropins than the provided by the Steelman Pohley assay. In certain embodiments, the mouse is about 80%, about 70%, about 60% or about 50% more responsive than the Steelman Pohley assay. The methods ofthe invention therefore provide an assay that is about 50%, about 60%, about 70%, about 80% or about 90% more sensitive than the Steelman Pohley assay.

[031] In certain embodiments, the methods provide for a bioassay that utilizes less gonadotropin than the Steelman Pohley assay. In certain embodiments, the methods provide that about 50% less gonadotropin can be used in the present methods than in the Steelman Pohley assay to obtain a given endpoint. In certain embodiments, the methods provide that about 60% less gonadotropin can be used than in the Steelman Pohley assay. Applicants have found that using the methods described herein about 2 fold less to about 1.6 fold less gonadotropin can stimulate the same functional response in the immature female B6D2F1 mice.

[032] The methods ofthe present invention provide for an in vivo bioassay that is more sensitive than the previous gold standard Steelman-Pohley bioassay and hence less animals are needed to obtain a valid sample size of acceptable variability. The methods provide, inter alia, for the detection of at least two endpoints in a super-responder mouse. In certain embodiments, the super-responder mouse is an immature female B6D2F1 mouse.

[033] The methods ofthe present invention also provide for an in vivo bioassay that can be used, for example, as a model for anovulatory or infertile women. The methods provide, inter alia, for the detection of unexplained infertility using a low- responder mouse. In certain embodiments, the low-responder mouse strain is a C3H mouse.

[034] In certain embodiments, the endpoint is a functional endpoint. For example, in certain embodiments, the methods provide for ovulation induction in the mouse. In certain embodiments, the endpoint can be ovarian weight, the number of oocytes ovulated, serum level of FSH, LH or CG in the mouse, the number of blastocytes (embryos) developed from in vitro fertilization, the number of pregnancies, the presence of DNA markers or the number of successful in vitro fertilizations that be performed on the ovulated oocytes. In preferred embodiments, the function endpoints can be any two of: ovarian weight, the number of oocytes ovulated or serum FSH level. In certain embodiments, results using all endpoints can be verified or evaluated relative to the serum level of FSH, LH and CG remaining at the end of each experiment. [035] In certain embodiments, the methods ofthe invention provide, inter alia, for the determination of at least two endpoints. In certain embodiments, the methods provide for the number of oocytes ovulated as an endpoint.

[036] In certain embodiments, the endpoint is a nonfunctional, morphometric evaluation ofthe oocyte wherein the quality ofthe oocyte produced is evaluated. In certain embodiments, the oocytes produced can be visually evaluated for relative size, color and symmetry. In addition, the oocytes can be evaluated for the presence and morphology of at least one polar body and/or signs of fragmentation. [037] The present invention provides methods for an in vivo bioassay for the quantification and standardization of gonadotropins. In one aspect the invention provides for a murine bioassay for the functional characterization of gonadotropins. In certain embodiments, the methods use an immature hybrid female mouse. Using an immature mouse ensures that endogenous gonadotropins will not be present which would interfere with the assay. Most laboratory mice have contributions from both Mus musculus musculus and Mus musculus domesticus. However, laboratory mice should not be referred to by species name, but rather by use of a specific strain or stock name. Laboratory rat strains derive from the Rattus norvegicus species. [038] In certain embodiments, the methods use a B6D2F1 female mouse. Mouse and rat strain names can be found and registered through the Mouse Genome Database (MGD) or Rat Genome Database (RGD), respectively. (Blake, et al, Nucleic Acids Res. 2003, 31: 193-195, Eppig, et al, Genesis 2002, 32:63-65, Steen, et al, 1999. Research Genetics, Rat Genome Database, ftp://rgd.mcw.edU/pub/publications/l 999/steen__genome_research/.) The International Committee on Standardized Genetic Nomenclature for Mice and the Rat Genome and Nomenclature Committee have agreed to a joint set of rules for strain nomenclature. For hybrid mice or rats that are the progeny of two inbred strains, crossed in the same direction, the designation consists of upper case abbreviations ofthe two parents (maternal strain listed first), followed by FI. In the case ofthe B6D2F1 mouse, the mouse is an offspring of a C57BL6/J mother and a DBA/2 father. Reciprocal FI hybrids are not genetically identical, and their designations are, therefore, different. For example, a D2B6F1 mouse is the offspring of a DBA/2 mother and C57BL6/J father. Committee on Standardized Genetic Nomenclature for Mice, Chair: Eppig, Rat Genome and Nomenclature Committee, Chair: Guenther, 2004, http://www.informatics.jax.org/mgihome/nomen/strains.shtml. See also, Committee on Rat Nomenclature. 1992, Definition, nomenclature, and conservation of rat strains. ILAR News 34: S1-S26, Committee on Standardized Genetic Nomenclature for Mice. 1952, Standardized nomenclature for inbred strains of mice. Cancer Res. 12:602-613, Committee on Standardized Genetic Nomenclature for Mice, 1960, Standardized nomenclature for inbred strains of mice, second listing. Cancer Res. 20:145-169, Committee on Standardized Genetic Nomenclature for Mice, 1976, Nomenclature for inbred strains of mice preserved by freezing. Mouse News Lett. 54:2-3, Committee on Standardized Genetic Nomenclature for Mice, Chair: Lyon, M.F. Rules for nomenclature of inbred strains, pp. 368-372, In: Genetic Variants and Strains ofthe Laboratory Mouse, Green, M.C. (ed.), First Edition, Gustav Fischer Nerlag, Stuttgart, 1981, Committee on Standardized Genetic Nomenclature for Mice, Chair: Lyon, M.F. Rules for nomenclature of inbred strains, pp. 632-635. In: Genetic Variants and Strains ofthe Laboratory Mouse, Lyon, M.F., A.G. Searle (eds.), Second Edition, Oxford University Press, Oxford, 1989 and Committee on Standardized Genetic Nomenclature for Mice, Chair: Davisson, M.T.

Rules for nomenclature of inbred strains, pp. 1532-1536, In: Genetic Nariants and Strains ofthe Laboratory Mouse, Lyon MF, Rastan S, Brown SDM (eds.), Third Edition, Oxford

University Press, Oxford, 1996, each of which is incorporated herein by reference in its entirety.

[039] Immature female mice, about 21 to about 22 days old, when pretreated with human chorionic gonadotropin (hCG), are sensitive to exogenous FSH. FSH is administered parenterally, preferably subcutaneously, once daily for 3 days with a necropsy performed after 20 to 22 hours after the last injection.

[040] In certain embodiments, the methods provide for the first administration of a gonadotropin when the mouse is about 21 to about 22 days of age. Immature mice are used to eliminate the confounding effects of endogenous gonadotropins. The mice useful in the methods ofthe present invention, both the super-responders and the low responders, are those that are less than about 22 days old. In certain embodiments, the mice are about 19 to about 22 days old.

[041] In certain embodiments, the mouse weights about 8 to about 13 grams at the time of first dosing. In certain embodiments, the mouse weights about 9 to about 12 grams at the time of first dosing.

[042] In certain embodiments, the methods provide for the administration of a gonadotropin to a mouse about three times. In certain embodiments, the gonadotropin is administered about every 24 hours. In certain embodiments, the gonadotropin is administered about every 12 hours. In preferred embodiments, an ovulatory dose of hCG is administered with the final dose of gonadotropin.

[043] The modified glycoprotein hormones or compositions thereof can be administered to the murine model by any suitable route that ensures bioavailability in the circulation. This can best be achieved by parenteral routes of administration, including subcutaneous (SC), intravenous (IN), intramuscular (IM), intradermal and intraperitoneal

(IP) injections. However, other routes of administration can be used.

[044] In certain embodiments the gonadotropin can be a modified gonadotropin, such as a modified FSH, modified LH or modified CG. Such modified glycoprotein hormones have been described in U.S. Patent No. 6,361,992, U.S. Application Nos. 10/057113 (filed January 25, 2002), 09/813398 (filed March 20, 2001) and U.S.

Provisional Application No. (Attorney Docket No. 56815-5001 PR)

(filed March 19, 2004) and PCT Publications 00/17360, 97/42322 and 96/06483 the contents of which are hereby incorporated by reference in their entireties. 6. EXAMPLES

A. Oocyte Retrieval

[045] The following example describes a protocol for the collection of ovulated oocytes from rodents.

[046] The identity ofthe rodent scheduled for euthanasia is verified and the animal is euthanized in accordance with standard laboratory procedures in a vented workstation in a necropsy room.

[047] The rodent is placed ventral side up. The abdominal cavity is opened with scissors and forceps. Each ovary with oviduct is removed and placed in a petri dish or equivalent container, with about 2 ml of 0.9% saline. The petri dish is labeled according to animal number. The animal carcass is discarded in accordance with standard laboratory techniques.

[048] The ovary and oviduct are examined visually with a dissection microscope.

If ovulation has occurred, a swollen, translucent portion ofthe oviduct near the infundibulum will be visible, called the ampulla. The ampulla contains clusters of ovulated oocytes surrounded and adhered to by cumulus cells. Grasp the adjacent oviduct tissue with a pair of forceps and gently tear open the ampula with another set of forceps. The oocytes should flow out ofthe ampulla. Allow the oocytes to flow out of the ampulla into a second petri dish or equivalent with fresh 0.9% saline solution. Oocyte retrieval may be done without use of a vented workstation in the teratology lab.

[049] Using a microcapillary pipette, transfer the oocytes into fresh 0.9% saline solution containing hyaluronidase (500 IU/ml). Alternatively, the oocytes may be released directly into clean 0.9% saline solution containing hyaluronidase. Gently pipette the cluster of oocytes up and down using the microcapillary pipette aspirator assembly and transfer the pipette in the hyaluronidase solution until most ofthe cumulus cells have disassociated from the oocytes. This step should be done as quickly as possible to avoid damage to the oocytes themselves by the hyaluronidase.

[050] Transfer the oocytes through several washes of fresh 0.9% saline solution to remove any remaining hyaluronidase and cellular debris. If the oocytes are being photographed, transfer to a clean drop of 0.9% saline solution on a 10-well slide labeled with the animal number.

[051] Oocytes are counted at least twice to verify the count. If preservation is not required, discard tissue according to current version of SOP.

B. High Throughput Screening & Bioassay

[052] This example demonstrates a High Throughput Screening Method used to screen FSH analogs employing the methods described, supra. The results demonstrate the longer plasma half life and superior in vivo performance of FSH analog TR 4405 to that of a prior FSH analog lead candidate (TR 4402) and to recombinant FSH (FOLLISTIM®, Organon). These data were obtained in a highly sensitive, robust and novel mouse bioassay system.

[053] A schematic providing a comparison ofthe conventional approach to screening and a novel approach High Throughput Screening are provided in Figure 1. As can be seen, a conventional approach requires about five steps; in vitro selection, establishment of lead candidates; production; purification and bioassay. In the novel approach High Throughput Screening (HTS), only 3 steps are needed; transient transfection; purification and bioassay. Bioassay is comprised of an in vivo and in vitro component. In the conventional approach, the time to test one analog is about 15 months as compared with about 4 months required in the novel approach high throughput screening. The bioassay in mice in a conventional screening uses the Steelman Pohley assay and observes morphometric and ovarian weight changes. The novel approach High Throughput Screening uses a mouse bioassay with endpoints of ovarian weight, gonadotropin plasma levels oocytes ovulated, oocytes retrieved and the morphometric evaluation of the quality ofthe eggs. The in vitro follicle bioassay uses endpoints of folliculogenesis, oogenesis and steroidogenesis. [054] Transfection and Purification Methods

[055] DNA was made by MaxiPrep DNA Production from 500 ml of bacterial culture to yield 0.8 to 1.2 mg of DNA. Transient Transfection was performed with

Lipofectamine on 20 x 150mm dishes per analog to provide 15 μg of DNA/dish at a ratio of α:β of 3 : 1. The recombinant protein was expressed in serum free medium for 48 hours. The protein was purified by immunoaffinity and quantified by FSH IRMA radioimmunoassay. In vitro follicule bioassay and bioassay in immature hybrid female mice were performed. Other transfection methods can be used.

[056] Transfection Results

[057] The recombinant protein expression production averaged 100-200 rnlU/ml with an amount of analog produced per transfection of about 100 IU. The results ofthe in vitro follicule bioassay shows that the amount of analog required to perform a biopotency study is about 200 to 300 mlU. The results ofthe bioassay in immature hybrid female mice show that the amount of analog required to test a high and low dose is about 20-25 IU.

[058] Bioassay Methods

[059] For the comparative mouse assay, immature female C57BL/6, C3H and

B6D2F1 mice between the ages of 19 and 21 days were used. For methods comparing ovulatory responses to various gonadotropin doses, immature hybrid female B6D2F1 mice between 19 and 21 days of age were used. The mice weighed between about 8 to about 13 grams at the time of first dosing. The various gonadotropin regimens were administered subcutaneously once every 24 hours for 3 doses. For certain protocols an ovulatory dose of hCG was administered as the last dose in the regiment.

[060] Bioassay Results

[061] A comparison of mouse strains, C57BL/6, C3H and B6D2F1, given 6 treatments and using an endpoint of oocytes retrieved is provided in Figure 3. The mice were given one of 6 different treatment regimens: a) three doses of 20 IU of FSH and 1 IU of hCG, b) three doses of 2 IU of FSH and 1 IU of hCG, c) three doses of 20

IU of FSH, d) three doses of 2 IU FSH, e) one dose of 15 IU of PMSG (pregnant mare serum gonadotropin used as a positive control for FSH and LH activity) or three doses of BSA/PBS (bovine serum albumin/phosphate buffered saline used as a negative control). All three mice strains had increased numbers of oocytes ovulated (e.g. oocytes retrieved) with the treatment of three doses of 20 IU FSH and 1 IU hCG or the treatment of a single dose of 15 IU of PMSG. The B6D2F1 mouse had a significant increase in the number of oocytes ovulated over the C57BL/6 and C3H mice. Thus, the B6D2F1 was identified as a super-responder mouse. The C3H mouse was identified the low-responder. [062] A comparison of mouse strains, C57BL/6, C3H and B6D2F1 given 6 treatments and using an endpoint of hFSH plasma levels at terminal kill is provided in Figure 4. The mice were given one of 6 different treatment regimens as described above. All three mice strains had an increased number of oocytes ovulated with the treatment of three doses of 20 IU FSH and 1 IU hCG or the treatment of three doses of 20 IU FSH. The C57BL/6 mice had the highest plasma levels of hFSH following three doses of 20 IU FSH and 1 IU hCG. The C3H mice had the highest plasma levels of hFSH following the three doses of20 IU FSH.

[063] Figure 5 provides a comparison of FSH gondotropins, FOLLISTIM®, a recombinant FSH (Organon), with TR 4402, a candidate FSH analog (Trophogen) in B6D2F1 mice. The mice were treated with one ofthe following treatment regimens: a) three doses of BSA/PBS, b) three doses of 10 IU of an FSH gonadotropin with 1 IU of hCG, c) three doses of 5 IU of an FSH gonadotropin with 1 IU of hCG, d) three doses of 2.5 IU of an FSH gonadotropin with 1 IU of hCG, or e) three doses of 1IU of an FSH gonadotropin with 1 IU of hCG. The mice treated with TR 4402 provided an increased number of oocytes retrieved (ovulated) and increased ovarian weights over mice treated with FOLLISTIM®, for all treatment regimens. In addition, plasma hFSH levels were higher in mice treated with FOLLISTIM® than with mice treated with FSH 4402, indicating that the mice responded with greater robustness and lower circulating gonadotropin levels to the TR 4402 than to the FOLLISTIM®. [064] Figure 6 provides the ovarian weight and FSH plasma levels after ovulatory induction in B6D2F1 mice following various treatments with various FSH gonadotropins. The FSH gonadotropins used were: FOLLISTIM®, a recombinant FSH (Organon), TR 4402, a candidate FSH analog (Trophogen), TR 4301, a candidate FSH analog (Trophogen), wild type FSH, BSA PBS or hCG. The treatment regimens were: a) three doses of HU hCG, b) three doses of BSA/PBS, c) three doses of 10 IU of an FSH gonadotropin with 1 IU of hCG, d) three doses of 5 IU of an FSH gonadotropin with 1 IU of hCG, e) three doses of 1 IU of an FSH gonadotropin with 1 IU of hCG. [065] Figure 7 provides a comparison of ovulation induction in B6D2F1 mice following three doses of an FSH gonadotropin and one ovulatory dose of 15 IU hCG. The FSH gonadotropins were FOLLISTIM, TR 4402, TR 4405 or hCG. The terms immunopurified, stable and transient refer to the method of making or purifying the gonadotropin.

[066] The results ofthe comparison provided in Figure 7 is given in Figure 8. A comparison of TR 4405 with FOLLISTIM® for an endpoint of ovarian weight at a dose of 2.5 IU provided a p value of p=0.067. For the same endpoint and dose, TR 4405 vs. TR 4402 provided a p value of ρ=0.23. At a lower dose of 1 IU, FSH 4405 vs. FOLLISTIM® provided a p value of p=0.032 while TR 4405 vs. TR 4402 provided a p value of p=0.041. Other p values for other endpoints are provided. [067] Figure 9 provides the combined ovarian weight following one of 6 treatments with one of 5 FSH gonadotropins or two controls. The treatments were: a) a single dose of 15 IU of PMG, b) three doses of 0.1 IU of an FSH gonadotropin and 1 IU of hCG, c) three doses of 0.25 IU of an FSH gonadotropin and 1 IU of hCG, d) three doses of 1 IU of an FSH gonadotropin and lhCG, e) three doses of 2IU of an FSH gonadotropin and 1 IU of hCG, f) three doses of HU of hCG. The FSH gonadotropins used were FOLLISTIM, TR 4405, TR 4503, a candidate FSH analog (Trophogen), stable FSH wild-type or PMSG.

[068] Figure 10 provides the number of oocytes retrieved following one of 6 treatments, described above, with one of 5 FSH gonadotropins or two controls, described above.

[069] Figure 11 provides the plasma hFSH levels following one of 6 treatments, described above, with one of 5 FSH gonadotropins or two controls, described above. [070] The disclosures of all publications referenced throughout this application are hereby incorporated by referenced in their entireties. The invention is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects ofthe invention, and functionally equivalent methods and components are within the scope ofthe invention. Indeed various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope ofthe appended claims.

Claims

We claim:

1. A method of determining gonadotropin activity comprising administering to an immature female mouse at least one gonadotropin and detecting at least two endpoints, wherein said mouse has an altered response to gonadotropins.

2. The method of Claim 1 wherein the mouse has an increased response to gonadotropins.

3. The method of Claim 1 wherein the gonadotropin activity is potency.

4. The method of Claim 1 wherein the gonadotropin activity is onset of activity.

5. The method of Claim 1 wherein the gonadotropin activity provides indirect assessment of biological half-life and stability.

6. The method of Claim 1 wherein the gonadotropin activity is the specific activity.

7. The method of Claim 1 wherein the gonadotropin activity is ED50.

8. The method of Claim 1 wherein the mouse is a hybrid mouse.

9. The method of Claim 8 wherein the hybrid mouse is a B6D2F1 mouse.

10. The method of Claim 9 wherein the B6D2F1 female mouse is about age 19 days to about age 22 days at the time of first dosing.

11. The method of Claim 9 wherein the B6D2F1 female mouse weighs about 8 to about 13 grams at the time of first dosing.

12. The method of Claim 9 wherein the B6D2F1 female mouse is dosed three times.

13. The method of Claim 1 wherein the administration is parenteral.

14. The method of Claim 13 wherein the administration is subcutaneous.

15. The method of Claim 1 wherein one endpoint is ovarian weight.

16. The method of Claim 1 wherein one endpoint is number of oocytes ovulated.

17. The method of Claim 1 wherein one endpoint is serum FSH or LH.

18. The method of Claim 1 wherein one endpoint is ovulation induction.

19. The method of Claim 1 wherein one endpoint is the development of a blastocyte from an in vitro fertilized, ovulated oocyte.

20. The method of Claim 19 wherein one endpoint is the number of pregnancies conceived through in vitro fertilization.

21. The method of Claim 1 wherein one endpoint is the detection of a DNA marker in the induced oocyte.

22. The method of Claim 1 wherein one endpoint is a visual evaluation ofthe induced oocyte for relative size, color, symmetry and the presence and morphology of at least one polar body or signs of fragmentation.

23. The method of Claim 1 wherein one gonadotropin administered is a modified gonadotropin.

24. The method of Claim 23 wherein the modified gonadotropin is a modified FSH, modified LH or modified CG.

25. A method of determining the potency of an FSH molecule, said method comprising administering subcutaneously to an immature female B6D2F1 mouse a modified FSH once daily for three days and determining the number of oocytes ovulated and ovarian weight, wherein said mouse has an increased response to gonadotropins and wherein said determining ofthe number of oocytes ovulated and ovarian weight provides the potency of said modified FSH.

26. The method of Claim 25 wherein the FSH is a modified FSH having increased activity as compared to wild type FSH.

27. The method of Claim 1 wherein the mouse has a decreased response to gonadotropins.

28. The method of Claim 27 wherein the mouse is a C3H mouse.

29. The method of Claim 27 wherein the methods provide a model for anovulatory or infertile human females.