AU2005202003B2 - Gonadotrophins - Google Patents

Description

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant: APPLIED RESEARCH SYSTEMS ARS HOLDING N.V.

Invention Title:

GONADOTROPHINS

The following statement is a full description of this invention, including the best method of performing it known to us: Gonadotrophins The entire disclosure in the complete specification of our Australian Patent Application No.

45928/00 is by this cross-reference incorporated into the present specification.

The present invention relates to the use of gonadotrophins in the treatment of anovulatory women. In particular, it relates to the use of luteinising hormone (LH) for promoting follicular development, and especially paucifollicular and monofollicular development, when inducing ovulation in anovulatory women.

Gonadotrophins are widely used in clinical practice to treat women with WHO group II and WHO group I anovulation (World Health Organisation Technical Report 514, (1973)).

Conventionally, folliculogenesis is induced by administering hMG (human menopausal gonadotrophin) or u-hFSH (urinary human follicle stimulating hormone) at a dose of 75-150 IU/day. This dose is increased after a few days (usually five) by steps of 75 IU. It is rare to exceed 450 IU/day. When there is at least one follicle having a mean diameter of at least 18 mm and no more than two follicles having a mean diameter of at least 16 mm, a high dose (of 5000 IU for example) of hCG (human chorionic gonadotrophin) is administered to induce ovulation. This "conventional protocol" has been used successfully for more than 20 years. It carries some risks however, mainly in patients with polycystic ovarian disease (PCOD). These risks include the occurrence of ovarian hyperstimulation syndrome (OHSS), and a relatively high incidence of multiple pregnancies (Schenker et al, Fertil. Steril. 35: 105-123 (1981)).

Although the majority of multiple pregnancies are twins, induction of ovulation contributes to one third of the high rank multiple births in the UK (Levene et al, Br. J. Obstet. Gynacol. 99: 607-613 (1992)).

Careful monitoring during treatment by ultrasound (US) and assessment of serum oestradiol

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2 have reduced these risks but have not been able to prevent them in all patients. These problems are directly related to the difficulty of obtaining the growth of a single dominant follicle leading to non-physiological multifollicular development.

During the last 10 years, a new protocol has been designed (the "chronic low dose H:\rochb\Keep\P56862.doc 10/05/05 protocol") and tested in order to reduce further the incidence of the complications of gonadotrophin therapy (Seibel et al, Int. J Fertil., 29:338-339 (1984); Buvat et al, Fertil. Steril., 52:553-559 (1989); Hamilton-Fairley et al, Human Reprod. 6:1095- 1099 (1991); Sagle et al, Fertil Steril., 55:56-60 (1991); Shoham et al, Fertil. Steril., 55:1051-1056 (1991); Meldrum, Fertil Steril., 55:1039-1040 (1991)). This protocol starts with a low dose of FSH or hMG (75 IU/day) and no dose adjustment before seven or preferably 14 days of treatment. If a dose adjustment is required, this is made by incremental steps of only 37.5 IU. In addition, each subsequent increase may only be effected after seven days of treatment at a given dose. The concept of this chronic low dose protocol is to find the threshold amount of FSH necessary to promote unifolliculogenesis. Encouraging results have been published so far, showing that this approach reduces the mean number ofpreovulatory follicles, the average preovulatory

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2 level and the size of the ovary at mid-luteal phase.

However, despite the use of the chronic low dose protocol, some treatment cycles still have to be cancelled due to an over-response where there are more than 3 follicles with a mean diameter of 16 mm or more). In addition, the multiple pregnancy rate, although clearly improved when compared to the conventional protocol, is still higher than in spontaneous conception cycles i.e. 5 10 in induced ovulation as opposed to 1.5 in spontaneous cycles. This is due to the fact that development of a single preovulatory follicle is obtained in only about two thirds to three quarters of the induced cycles and follicles having a mean diameter of 15 mm or less are usually not considered when assessing the number ofpre-ovulatory follicles on the day ofhCG administration (Buvat et al, Fertil. Steril., 52:553-559 (1989); Hamilton-Fairley et al, Human Reprod. 6:1095-1099 (1991)). It is however not clear whether follicles with a mean diameter of 14 to 15 mm, or even less, on the day ofhCG administration, will ovulate and lead to the release of a healthy fertilisable oocyte. Thus, it would be desirable to have improvements in FSH-induced follicular development treatment in which the rates of multiple pregnancy and cycle cancellation are reduced.

I SAntral follicle growth is induced by FSH. Continuously throughout life and up to the menopause, some follicles enter a growth phase which is interrupted by regression and Satresia before reaching the full maturity stage of preovulatory status (Hillier, Hum.

Reprod., 9:181-191 (1994)). During the growth phase, any follicle could be rescued from atresia, provided that it is exposed to a sufficient concentration of FSH. The clevel of FSH required to prevent atresia and promote further growth of a follicle is

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0called the "FSH threshold" level (Brown, Aus. NZJ. Obstet. Gynecol., 18: 47-55 S(1987). The FSH threshold level varies with time and, at a given time-point, the t follicles which are currently in a growth phase have different FSH threshold levels, 0 10 This is the rationale on which the "chronic low dose" protocol is based. A progressive and cautious increase in the dose of FSH is used for finding the threshold level of a minimal number of follicles, and hopefully achieving mono-ovulation.

It is known that luteinising hormone (LH) also contributes to the phenomenon of follicle dominance and mono-ovulation. Indeed, although some LH is essential for oestrogen synthesis during folliculogenesis, there is evidence that excessive exposure to LH will trigger follicular atresia and suppress granulosa proliferation. Developing follicles appear thus to have finite requirements for stimulation by LH, beyond which normal follicular development ceases. This is the "LH ceiling" concept (Hillier, Hum.

Reprod., 9:181-191 (1994)). It is believed that, at a given time-point, the follicles which are currently in a growth phase have different LH ceiling levels. It is suggested that the more mature follicles are more resistant to the atretic action of LH than less mature follicles.

Two cases of WHO group I anovulation treated by either FSH alone or hMG using a step-up protocol have been reported (Glasier et al, Journal ofEndocrinology, 119 A- 159 (1988)). The "FSH alone" cycle had a much larger number of mature follicles than the hMG cycle, possibly supporting a role of LH in the atresia of secondary follicles. Afterwards two comparative studies were published. In a first cross-over study in 10 hypogonadotrophic hypogonadal women, a striking difference was recorded in terms ofpreovulatory E 2 levels, but follicular count was not reported S(Couzinet et al, J. Clin. Endocrinol. Metab. 66:552-556 (1988)). A second cross-over study in 9 hypogonadotrophic hypogonadal women reported a mean number of follicles having a mean diameter of more than 16 mm on the day of hCG administration of 2.0 (0.7 in hMG-treated cycles and of 1.2 in FSH-treated cycles (Shoham et al, Fertil. Steril., 55:1051-1056 (1991)). No information is available on the number of smaller follicles.

More recently, the results of administering 150 IU hFSH (human FSH) and 75 IU rhLH (recombinant human LH) to a single patient with unmeasurably low serum FSH, LH and oestradiol concentrations have been published (Hall et al, The Lancet, 344(8918):334-335 (1994)). Administration of r-hLH and hFSH caused E 2 levels to be raised, and the total number of follicles of 10 mm or more in diameter to be reduced, as compared to administration of hFSH alone. However, the number of large follicles remained sufficiently high to suggest an unacceptably high multiple pregnancy rate.

A further study compared the effect of administering r-hLH (at a dose of either 300 IU/day or 750 IU/day) and r-hFSH to normal ovulatory women after treatment with FSH for stimulating multiple follicular development prior to intrauterine implantation (Sullivan et al, Journal of Clinical Endocrinology and Metabolism, 84, 228-232, 1999)). The results indicate that serum E 2 levels were raised in those women who received LH, although no measurements of the number and size of follicles were made and a multiple pregnancy occurred in the group receiving 750 IU/day of LH.

According to a first aspect of the present invention, there is provided the use of LH and/or a biologically-active analogue thereof in the production of a medicament for inducing folliculogenesis in anovulatory women at a daily dose in the range of from 100 to 1500 IU.

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As used herein, an "IU ratio" is the ratio of the number of IU of one component to the number of IU of another component. It is noteworthy that gonadotrophins may now be expressed in (mass/gg) instead of biological IU. In this case, a conversion factor has to be used to translate the new value into IU. For convenience, references hereinafter to LH, FSH and hCG are intended to include biologically-active analogues thereof.

The inventors have found that the administration of LH at a dose of 100 to 1500 IU/day can promote paucifollicular development, that is to say, it can reduce the number of preovulatory follicles per treatment cycle in patients undergoing follicular induction, as compared to cycles where LH is not administered at a dose of 100 to 1500 IU/day. LH administered in accordance with the invention can induce unifolliculogenesis, i.e. the development of a single preovulatory follicle. Doses in the range of from 200 to 800 IU/day, and more preferably 225 to 450 IU/day, have been found to be particularly effective. The reduction in multifollicular development can reduce the number of cycles cancelled owing to excessive follicle development, i.e. it can rescue those cycles when there are an excessive number of follicles, making the process of ovulation induction more efficient. In addition, the incidence of multiple pregnancy and of OHSS can be reduced.

The required daily dose may be administered as a single dose each day. Thus, the medicament may be packaged so as to provide only the daily dose of LH, e.g. in a unit-dose container such as a vial. However, it is possible that LH may be administered on two or more occasions during the day provided of course that total LH administered during the day equals the daily dose and the medicament packaged accordingly, i.e. in a multi-dose container. It is also possible that LH could be administered on alternate days or at even longer intervals. Such decisions will be taken by the physician administering the medicament and will depend on parameters such as the patient's body mass index (BMI), medical history, stage of follicular development when receiving LH, metabolism, response to the treatment, the half-life of the medicament and so on.

Folliculogenesis will generally be induced in anovulatory women by the administration of FSH using the conventional protocol or the chronic low dose protocol described above or an alternative protocol. LH should be administered at an appropriate stage of follicular development, e.g. the mid- to late-follicular phase. This stage may be decided by the physician administering the medicament and may depend on the regime by which ovulation is induced. By way of example, the appropriate stage of follicular development may be judged to have been reached when at least a single follicle reaches a mean diameter of 8 mm, or when at least one follicle has a mean diameter in the range 10-15 mm (preferably 11-14 mm), or when there are more than 3 follicles with a mean diameter in the range of from 8 to 13 mm and no larger follicles.

The administration of LH will generally cease when ovulation is induced by the administration of the high dose ofhCG. Again, the timing ofhCG administration to induce ovulation may be decided by the physician. For example, it may be when there is at least one follicle having a diameter of 18 mm or more and no more than 3, preferably 2, follicles having a diameter of 11 mm or more.

LH can be administered only when the required stage of follicular development has been reached. In this case, the administration of FSH can be discontinued altogether or can be continued at the same dose as before, or at a lower or higher dose. It is preferred if the administration of FSH is continued but at a lower dose than previously, the dose being lower than that of LH.

Alternatively, LH can be administered concomitantly with the conventional or chronic low dose protocols, i.e. prior to follicular development reaching an appropriate stage.

When the required stage of follicular development has been reached, the

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administration of FSH can be discontinued or continued as before, or at a lower or higher dose, provided that LH is administered at the appropriate dose. In a further Salternative, the medicament may formulated such that it can be used in a procedure Swhich replaces the conventional or chronic low dose protocols.

Thus, FSH and/or a biologically-active analogue thereof may be used in the Sproduction of the medicament. In this embodiment, the IU ratio of LH to FSH is N preferably in the range of from 1.5:1 to 20:1. More preferably, the ratio is in the range Nn of from 1.5:1 to 10:1.

When the medicament is for administration after the appropriate stage of follicular development has been reached, the IU ratio of LH:FSH may be about 10:1. A particularly preferred daily dose for such a medicament is 375 IU of r-hLH and 37.5 IU ofr-hFSH.

According to a second aspect of the invention, there is provided the use of LH and FSH and/or biologically-active analogues thereof in the production of a medicament for inducing folliculogenesis in women at an IU ratio of LH to FSH in the range of from 1.5:1 to 20:1.

The uses of the first and second aspects of the invention may be modified in that LH is replaced by an equivalent dose ofhCG and/or a biologically-active analogue thereof.

As used herein, an "equivalent dose" of human chorionic gonadotrophin (hCG) is calculated on the basis that 1 IU ofhCG is equivalent to 5-7 IU of LH in the pharmacopaeia Van Hell bioassay (Van Hell, H, et al, Effects of human menopausal gonadotrophin preparations in different bioassay methods, Acta Endocrin., 47: 409- 418, 1964). For convenience, references herein to luteinising hormone (LH) are intended to include hCG, with doses of LH being intended to include the equivalent dose of hCG.

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According to a third aspect of the invention, there is provided a product containing LH (or an equivalent dose of hCG) and FSH and/or biologically-active analogues thereof as a combined preparation for simultaneous, sequential or separate use in inducing folliculogenesis in women, the preparation comprising LH (or an equivalent dose of hCG) and FSH and/or biologically-active analogues thereof at an IU ratio of LH (hCG) to FSH in the range of from 1.5:1 to 20:1.

In accordance with the second and third aspects of the invention, LH or hCG and FSH may be administered to anovulatory women, preferably throughout the cycle up until the induction of ovulation by the administration of the high dose of hCG.

Alternatively, they may be administered after follicular development has reached an appropriate stage.

The invention also provides a method for the induction of folliculogenesis in anovulatory women, including the administration of luteinising hormone and/or a biologically-active analogue thereof at a dose in the range of from 100 to 1500 IU/day or an equivalent dose of human chorionic gonadotrophin and/or a biologically-active analogue thereof.

LH, FSH and hCG may be obtained from natural sources, e.g. isolated from urine, pituitary or placenta, or may be obtained using recombinant DNA technology (see W085/01959 and Loumaye et al, Human Reprod, 11: 95-107, 1996). Biologicallyactive analogues thereof include peptidic analogues, non-peptidic analogues and chimeras. It is preferred if human LH and FSH are used in the present invention.

Compounds useful in the invention may be formulated for administration by any convenient route, often in association with a pharmaceutically and/or veterinarily acceptable carrier. It is preferred that the compounds are formulated for parenteral administration.

It is preferred that the LH and FSH (when present) be administered subcutaneously, preferably into the anterior abdominal wall.

Formulations for parenteral administration will usually be sterile. Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents are also within the scope of the invention. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. The formulations can be administered through a prefilled syringe, an auto-injector or a multidose auto-injector.

Oral and other enteral formulations need not be sterile and may be presented in unit- or multi-dose form. Oral formulations may be in the form of solids, such as powders, granules, tablets, capsules (for example hard or soft gelatin capsules) or lozenges, or liquids, such as syrups or elixirs. Fillers and/or carriers may be present as appropriate, and those skilled in the art of pharmaceutical formulation will be able to provide such additional or alternative excipients as may be necessary or desirable; flavouring agents are one example. Any formulation intended for oral administration may be formulated for enteric resistance, so as to assist delivery to the small intestine by avoiding or mitigating any digestion of the compound(s) as may occur in the stomach or the proximal part of the small intestine. Tablets or capsules may be enteric coated, for example by conventional procedures. Liquid formulations may be effectively rendered enteric resistant by including or being co-administered with a suitable agent such as mediumchain triglycerides.

1^ 0 Enteral compositions other than oral compositions include rectal compositions, which may be in the form of a suppository. Suppositories will generally include a suppository base, Ssuch as cocoa butter. Again, particular formulations containing the active ingredient(s) may routinely be prepared by those skilled in the art of pharmaceutical formulation.

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Preferred features of each aspect of the invention are as for each other aspect, mutatis n mutandis.

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0It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

-l In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The invention will now be described further in the following non-limiting examples.

Example I The effect of LH when administered after FSH stimulation was examined on WHO Group II anovulatory women during a clinical study conducted according to ICH GCP (International Conference on Harmonisation Good Clinical Practice) guidelines. The patients had the following characteristics: Premenopausal; aged between 18 and 39; infertile due to ovulatory dysfunction; have had spontaneous menses, menses induced by clomiphene citrate therapy or a positive progestin-induced withdrawal bleed within the previous year; a body mass index of 35 or less (calculated as body weight in kg divided by (height x weight) in m euthyroid; no medical condition which may interfere with the absorption, distribution, metabolism or excretion of LH; no clinically systemic disease; no known allergy to gonadotrophin preparations; no persistent ovarian cyst of I I mm 3 5 or greater or ovarian N I II\IIN)--l 1 S- 1 Al. L1\Njk ,Ml 11I S \l I S2 Wl I IMI7.S-7dl 7/11111) j endometrioma (as determined by ultrasound); no previous or current hormone dependent tumour; no clinically relevant reproductive tract disease; and no active substance abuse.

The patients underwent routine ovulation induction with FSH until there were 4 or more follicles in the range of from 8-13 mm in diameter, no larger follicles and an endometrium of 8 mm or more thickness. They were then randomised into 3 blinded groups, one to receive a placebo, one to receive 225 IU/day of r-hLH and one to receive 450 IU/day of r-hLH.

0 Table 1 below summarises the respective groups of patients: Table 1 Mean ±SD Placebo r-hLH 225 IU/day r-hLH 450 IU/day No of patients 5 4 8 Age (yrs) 29.2 5.7 26.8 6.2 30.9 3.9 (min-max) (23-35) (20.35) (25-38) Weight (kg) 62.8 15.9 60.0 1.6 66.8 15.4 (min-max) (47-86) (58-62) (48-97) BMI 24.6 4.7 22.8 1.9 24.7 4.9 (min-max) (20-31) (21-25) (18-34) r-hLH (LHadi Serono) was used in vials containing 75 IU r-hLH and 47.75 mg of sucrose, phosphate buffer and Tween 20 in a lyophilised form. LHadi is produced in genetically engineered Chinese hamster Ovary (CHO) cells in which the genes encoding the alpha and beta chains of human LH have been introduced through recombinant technology. The specific activity of LHadi is approximately 15000 IU LH/mg.

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For a dose of 225 IU, 3 vials were used. One vial was reconstituted in 1 ml of water and gently agitated, taking care to avoid contact with the rubber stopper. The totality of the resulting solution was aspirated and used for reconstitution of the second vial.

After gentle agitation, the totality of the resulting solution was aspirated and used for reconstitution of the third vial. After further gentle agitation, the totality of the resulting solution was aspirated and immediately injected subcutaneously in the anterior abdominal wall using a new needle. For a dose of 450 IU, two injections of 225 IU were made.

The placebo was in vials matching the r-hLH vials but containing only sucrose, phosphate buffer and Tween The r-hLH/placebo treatment was continued for 7 days unless at least one follicle reached a mean diameter of at least 18 mm and there were 3 or fewer follicles having a mean diameter of 11 mm or greater. In this case, a single dose of 5000 IU of u-hCG (Profasi Serono) was given subcutaneously.

Prior to and during the r-hLH/placebo treatment, ultrasound (US) was used at intervals of 1-2 days to measure the mean diameter of the follicles (determined as the mean of the two longest perpendicular diameters) and the endometrial thickness (assessed as the distance from the hyperechogenic interface of the endometrium and the myometrium to the opposite interface including the stronger midline echo (endometrial interface)). All follicles with a mean diameter of 11 mm or greater were recorded.

Prior to and each time an ultrasound scan was carried out during the r-hLH/placebo treatment, a blood sample was taken and the resulting serum was analysed for E 2 (oestradiol), P 4 (progesterone), LH, FSH and androstenedione.

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2 and P 4 were analysed using DPC Coat-a-count, RIA solid phase coated tube Cseparation, LH (serum and urinary) and FSH were analysed using MAIACLONE SIRMA, and androstenedione was analysed using Diagnostic System Laboratories Smethod, RIA.

The results are summarised in Tables 2-4 and in Figure 1 of the accompanying on drawings which is a graph showing the size and number of follicles on the day of hCG 0administration (or the last day of treatment of no hCG was administered) for each of 0the patients.

0 10 It can be seen that the administration of LH at 225 or 450 IU/day subsequent to FSH treatment resulted in a more marked follicular regression than in the administration of placebo, as suggested by patients with complete follicular regression, a smaller number of follicles on the day of hCG administration and a reduction in follicle median size from 15 mm in the placebo group to 14 mm in the 225 IU r-hLH group and 13 mm in the 450 IU r-hLH group.

The efficacy of r-hLH in promoting mono-ovulation is illustrated by the emergence of a dominant follicle (as evidenced by the median size), the absence of follicular phase luteinisation and a comparatively lower P 4 level at the mid-luteal phase.

Example 2 The effect of LH and FSH administered during the late follicular phase was examined on WHO Group I anovulatory women during a clinical trial conducted according to ICH GCP guidelines. The patients had the following characteristics: premenopausal; aged between 18 and 39; a clinical history of hypogonadotrophic hypogonadism; have stopped treatment (if any) with pulsatile GnRH, gonadotrophins or oestrogen progesterone treatment therapy at least one month before the screening procedure; have had a negative progesterone challenge test performed curing the screening period; had the following hormonal values in a fasting blood sample (between 7 and 9.30 AM) drawn within 6 months before the treatment period: FSH: 5 mIU/ml LH: 1.2 mlU/ml Thyroid stimulating hormone (TSH): 6.5 U/ml Free T 4 >11 and 24 pmol/1 Testosterone: 3.5 nmol/1 Prolactin (PRL): 520 mIU/l; no clinically significant abnormal finding, within 6 months prior to study start, in pre-treatment haematology, in clinical chemistry and urinalysis parameters or results of no pathological significance of outside normal limits; have, on file, if clinically indicated, a CT scan or MRI of the hypothalamic pituitary region to document current putative tumoral status of the region; a body mass index of between 18.4 (percentile 10 for 18 years) and 31.4 (percentile 90 for 38 years); no medical condition which may interfere with the absorption, distribution, metabolism or excretion of LH or FSH; no clinically systemic disease; no known allergy to gonadotrophin preparations; no persistent ovarian cyst of 11 mm or greater or ovarian endometrioma (as determined by ultrasound); no previous or current hormone dependent tumour; no clinically relevant reproductive tract disease; and no active substance abuse.

The study was divided into an open phase of a maximum of 28 days and a blinded phase of a maximum of 7 days.

In the open phase, all patients received 225 IU/day of r-hLH and 112.5 IU/day of rhFSH. If there was no rise in E 2 levels or sign of follicular growth after 7 days, the dose of r-hFSH was raised to 150 IU/day. After a further 7 days, the dose of r-hFSH was raised to 187.5 IU/day if there was no rise in E 2 levels or sign of follicular growth and after a further 7 days, the dose of r-hFSH was raised to 262.5 IU/day if there was no rise in E 2 levels or sign of follicular growth. The dose of r-hLH remained constant throughout the open phase.

When a patient had at least one follicle with a mean diameter in the range of from 13 mm, she entered the blinded phase. In this phase, the patients were randomised into 3 blinded groups, one to receive a LH placebo and continue the dose of r-hFSH received on the last day of the open phase, one to receive 225 IU/day of r-hLH and continue the dose ofr-hFSH received on the last day of the open phase, and one to receive 225 IU/day of r-hLH and a FSH placebo.

Table 5 below summarises the respective groups of patients.

Table Mean±SD FSH/Placebo r-hLH/placebo FSH/r-hLH No of patients 6 6 8 Age (yrs) 31.9 6.2 31.0 3.0 30.8 4.6 (min-max) (21-39) (27-34) (25-37) Weight (kg) 70.3 10.0 51.7 ±4.4 66.9 15.9 (min-max) (60-88) (46-59) (50-89) BMI 25.2 2.3 19.8 1.1 24.6 4.3 (min-max) (21-28) (19-21) (20-30) r-hFSH (Gonal-F Serono) was used in ampoules containing 75 IU r-hFSH and mg sucrose and phosphate buffer in a lyophilised form, up to 3 of which were dissolved in 1 ml of water for injection. Matching ampoules containing only sucrose and phosphate buffer were provided for the FSH placebo.

r-hLH (LHadi Serono) was provided and administered as in Example 1. The LH placebo was in vials matching the r-hLH vials but containing only sucrose, phosphate buffer and Tween All injections were made subcutaneously into the anterior abdominal wall.

The blinded phase was continued for 7 days unless at least one follicle reached a mean diameter of at least 18 mm and there were 2 or fewer follicles having a mean diameter of 11 mm or greater. In this case, a single dose of 10000 IU of u-hCG (Profasi Serono) was given subcutaneously.

On the first, fifth and eight days of the open phase, and at regular intervals 1 to 2 days) during the blinded phase, ultrasound was used to measure the mean diameter of the follicles and the endometrial thickness. All follicles with a mean diameter of 11 mm or greater were recorded.

On the first day of the open phase, and at regular intervals 1 to 2 days) during the blinded phase, a blood sample was taken and the resulting serum was analysed for E 2 P4, LH, FSH and androstenedione as in Example 1.

The results are summarised in Tables 6-9 and in Figure 2 of the accompanying drawings which is a graph showing the size and number of follicles on the day of hCG administration (or the last day of treatment of no hCG was administered) for each of the patients.

It can be seen that stopping FSH and administering r-hLH at 225 IU/day resulted in a marked and excessive follicular regression.

The efficacy of r-hLH in promoting mono-ovulation in the presence of FSH is 17 illustrated by a reduction in the mean number of follicles having a diameter of 14 mm or greater, an increase in the proportion of patients with only 1 or 2 follicles having a diameter of 14 mm or greater, the emergence of a dominant follicle (as evidenced by a median follicle size of 12 mm as compared to 15 mm for the FSH/placebo group), and the absence of follicular phase luteinisation.

2005202003 11I May 2005 Table 2 Summary Data on Number and Size of Follicles and hCG Cancellation Treatment Group Patient Id Number of Number of Number of Follicles =8 mm Follicles =I mmnu Follicles 14 mm at BaselineLatULstS Placebo 20002 854 30003 -22 1 0 40001 2032 '40005 12 3 1__ P40008 8 5 14.00 ±6.635.04738±.6 r-hLH 225 lU/day 20001. 510 30001 120 40003 1852 40007 4 N=4 9.75±6.55 2.25±2.22 -0 4391 1.25±1.50 -0.2342 r-hH 450 lU/~day 10001' 6 0 0 20003 10 13 4 30002 9 5 3 40002 17 3 1 40004 7 3 1 40009 4 3 2 50001 9 0 0 70001 9 3 1 N 81 8.88±3.83 3.75±4.10 -0.8684 1.50± 1.41 v 0.2731 p-values from comparison with Placebo group (ANCOVA adjusted for number offollicles at baseline) P: pregnant patient hCG Received Reason Comment No Risk of OHSS Yes Yes Yes Yes._ 4 Yes/ I No No Follicles regressed No Failure of treatment Yes Yes 2 Yes/2 No No all follicles became atretic No Risk of QHSS No Failure of treament Yes Yes Yes No Failure of treatment Yes__ 4 Y7/4N 2005202003 11 May 2005 Table 3 Number of Patients with 0, 1, 2, 3 or >3 Follicles on the Day of hCG or on the Last Day of Treatment if No hCG was Administered Treatment Randomised i-.alnr t 4 riaceoo r-flLkl L~ Contrast One-sided Two-sided laceoO r-ILH 223 IU/dav r-lLH 45U IU/dav Contrast* One-sided Two-sided Variable Number of Follicles N N N Asymptotic Exact Asymptotic Exact Follicles 0 foil.> =Ilmm 0 0.0% 1 25.0% 2 25.0% Placebo vs. r-hLH 225 IU 0.0562 0.1429 0.1124 0.1746 11mm 1 fol.>=11 mm 0 0.0% 1 25.0% 0 0.0% Placebo vs. r-hLH 450 IU 0.1108 0.2339 0.2217 0.3590 2 foil. 11 mm 0 0.0% 0 0.0% 0 0.0% r-hLH 225 IU vs. r-hLH 450 IU 0.2987 0.3879 0.5973 0.7192 3 foil. =11 mmn 3 60.0% 1 25.0% 4 50.0% Overall comparison 0.2064 0.2222 0.4128 0.4378 >3 foil.> =II nun 2 40.0% 1 25.0% 2 25.0% 5 100.0% 4 100.0% 8 100.0% Follicles Ofoll.> =14mm 0 0.0% 2 50.0% 2 25.0% Placebo vs. r-hLH 225 IU 0.0774 0.1429 0.1547 0.2857 14mm I foll.> =14 mm I 20.0% 0 0.0% 3 37.5% Placebo vs. r-hLH 450 IU 0.0817 0.1298 0.1635 0.2416 2fol. =14mm 2 40.0% 1 25.0% 1 12.5% r-hLH 225 IU vs. r-hLH 450 IU 0.3786 0.4788 0.7572 0.8323 3foil.> =14mm 0 0.0% 1 25.0% 1 12.5% Overall comparison 0.1259 0.1354 0.2519 0.2675 >3foil.>= 14mm 2 40.0% 0 0.0% 1 12.5% 5 100.0% 4 100.0% 8 100.0% Contrast. Overall Comparison: Jockheere-Terpstra test Pairwise Comparison: Cochran-Armitage test for trend.

2005202003 11 May 2005 Table 4 Descriptive Statistics of Hormone Levels Measured at TI and on the Day of hCG or on the Last Day of Treatment if No hCG was Administered TI (first day of stimulation) I Day of hCG or last day of treatment if no hCG was Au .h~~ac v Variable Treatment n Mean SD SEM Median Range n Mean SD SEM Median Range FSH (IU/L) Placebo 5 12.20 5.60 2.50 9.50 (8-21) 5 6.54 3.84 1.72 7.90 (2-11) r-hLH 225 IU/day 4 12.53 6.75 3.37 11.45 (6-21) 4 7.35 2.98 1.49 6.05 (6-12) r-hLH 450 IU/day 7 11.10 3.71 1.40 9.80 (8-19) 8 6.94 2.05 0.73 6.25 (5-10) 16 11.80 4.84 1.21 9.65 (6-21) 17 6.92 2.70 0.66 6.20 (2-12) LH (IU/L) Placebo 5 7.84 6.30 2.82 4.80 (3-18) 5 6.12 2.74 1.23 6.90 (2-9) r-hLH 225 IU/day 4 5.25 2.69 1.34 6.20 4 6.80 4.48 2.24 6.40 (2-12) r-hLH 450 IU/day 7 4.60 4.14 1.57 3.40 (1-13) 7 6.67 4.14 1.56 4.80 (3-15) All 16 5.78 4.58 1.15 4.55 (1-18) 16 6.53 3.60 0.90 6.00 (2-15) E2 (pmol/L) Placebo 5 4031.6 3759.9 1681.5 3612.0 (598-10017) 5 4780.6 4612.7 2062.9 3540.0 (313-11040) r-hLH 225 IU/day 4 1491.8 1633.5 816.8 851.5 (384-3880) 4 2560.0 4715.7 2357.8 227.0 (153-9633) r-hLH 450 IU/day 7 1376.7 885.8 334.8 1315.0 (123-2809) 8 1966.9 2665.1 942.3 297.0 (133-7269) 16 2235.1 2486.8 621.7 1304.5 (123-10017) 17 2934.0 3763.6 912.8 378.0 (133-11040) P4 (nmol/L) Placebo 5 4.56 1.80 0.80 4.30 5 8.86 10.83 4.84 4.50 (2-28) r-hLH 225 IU/day 4 3.08 1.02 0.51 3.05 4 2.68 1.15 0.57 2.50 (2-4) r-hLH 450 IU/day 7 2.47 1.03 0.39 2.30 8 2.89 1.73 0.61 2.25 (1-6) 16 3.28 1.53 0.38 2.85 17 4.59 6.24 1.51 2.80 (1-28) Androstenedione Placebo 5 17.42 11.62 5.20 16.50 (5-35) 5 15.74 7.03 3.14 14.40 (8-27) (nmol/L) r-hLH 225 IU/day 4 8.63 0.88 0.44 8.30 (8-10) 4 11.75 1.92 0.96 12.00 (9-14) r-hLH 450 IU/day 7 10.53 7.11 2.69 8.00 (5-26) 8 12.18 9.56 3.38 8.95 (6-35) _All 16 12.21 8.38 2.09 9.10 (5-35) 17 13.12 7.49 1.82 11.40 (6-35) 2005202003 11 May 2005 Table 6 Summary Data on Stimulation Open and Blinded Phases and hCG Cancellation F Open Phase Blinded Phase Treatment Patient First Dose Last Dose Number Cumulative Cumulative LH Number Cumulative Cumulative LH hCG Group Id of FSH (IU) of FSH (IU) of Days FSH Dose Dose of Days FSH Dose Dose Received Gonal- 10002 112.5 112.5 8 900 1800 2 225 No F/Placebo 10004 112.5 150.0 13 1688 -2925 3 450 No 20001 112.5 112.5 7 788 1575 3 338 No 30002 112.5 150.0 10 1238 2250 1 150 Yes 40002 112.5 150.0 13 1688 2925 2 300 Yes 50001 112.5 112.5 7 788 1575 5 563 Yes N=6 112.5 131.3±20.5 9.7±2.8 1181.3±425.4 2175.0±631.1 2.7±1.4 337.5±150.0 3 Yes/3 No r-hLH/Placebo 10003 112.5 150.0 15 1988 3375 3 675 No 10005 112.5 112.5 2 225 450 5 1125 No 30003 112.5 112.5 7 788 1575 7 1575 No 40001 112.5 112.5 7 788 1575 5 1125 Yes 50002. 112.5 112.5 5 563 1125 7 1575 No 60002 112.5 150.0 12 1538 2700 4 900 Yes N=6 112.5 125.0±19.4 8.0±4.7 981.3±654.9 1800.0±1064.9 5.2±1.6 1162.5:360.5 2 Yes/4 No Gonal-F/r-hLH 10001 112.5 187.5 17 2363 3825 3 563 675 Yes 10006 112.5 112.5 3 338 675 2 225 450 No 20002 112.5 112.5 11 1238 2475 1 113 225 Yes 40003 112.5 112.5 7 788 1575 3 338 675 Yes 40004 112.5 112.5 7 788 1575 2 225 450 Yes 50003 112.5 112.5 6 675 1350 7 788 1575 No 50004 112.5 150.0 11 1388 2475 3 450 675 Yes 60001 112.5 112.5 6 675 1350 2 225 450 No N=8 112.5 126±27.9 8.5±4.3 1031.3±632.0 1912.5±977.1 2.9±1.8 365.6±223.0 646.9±406.7 5 Yes/3 No 2005202003 11 May 2005 Table 7 Summary Data on Number and Size of Follicles and hCG Cancellation I rr~ ILast

US

I

I I I rimmentl kroup rauent Id Number of Foll Icles Number of Follicles I mm Number of Follicles >l=14mxn

I

hCG Received No 10mm on TI 1 n Uonal-R/Placebo 1I0021 1 I 5 Reason/Comment possible risk of multiple pregnancy I follicle 18 mm plus 3>11 mm; not within 1 t I* luUVA No 20001 1 4 3 Nomultiple follicles 30002 4 5 4 Yes 40002 1 1 1 4 Yes 50001 2 4 4 Yes N=6 1.83+1.17 4 174-0 5 417 1)1 r.hT UIDI...h I innn-2 I L VV/jf I J 1CSIJPNO j Y'U ~L~IIU II III1 11 n 4_ U 4 U JNo 10005 I 1,40 regression of follicles regression of follicles 30003 rrro radure of treatment 40001 2 3 2 Yes failureoftreatment 50002 2 4 0 No failure of treatment 60002 1 1 1 Yes N=6 I i2n rj A o-ovlll 11 1II~II ro r+rn nrn 7 V-rll L1^ y VA I Ubt-t n %jonal-rlr-aLLn IUUU1 V, 10006 13 1 No risk of OHSS 20002 2 2 2 Yes P40003 4 3 1 Yes P40004 3 3 1 Yes 50003 2 0 0 No failure of treatment 50004 1 4 1 Yes_ 60001 2 19 8 No risk of OHSS 9Afl4.M1 07 a -inn. n P-.WJS I &.YUT.a1 O41D4J2 I 9 YeI Nn I p values adjustedfor BMI: contrast p-value with the previous treatment group *Gonal-F/-hL- vs. Gonal-FiPlacebo 4'Gonal-FiPlacebo vs.r-hLfJ/Placebo r-hLH/Placebo vs. Gonal-Flr-hLH P: pregnant patient 2005202003 11I May 2005 Table 8 Number of Patients with 0, 1, 2, 3 or 3 Follicles on the Day of hCG or on the Last Day of Treatment if No IiCG was Administered Treatment Randomised I .t 4 uonai-r r-nLRl iJonai-z- r -Contrast** One-sided Two-sided Gonal-F r-nLn Gonal-F r- Contrast- One-sided Two-sided Variable Number of Follicles N N %h N Asymptotic Exact Asymptotic Exact Follicles 0 foil. =11 mm 0 0.0% 2 33.3% 1 12.5% Gonal-FlPlacebo vs r-hLH/placebo 0.0057 0.0141 0.0115 0.0281 -1 lnmmI foil. =I mm 0 0.0% 2 33.3% 0 0.0% Gonal-F/Placebo vs Gonal-F/r-hLH 0.0820 0.1538 0.1641 0.2887 2 foil. I1I mm 0 0.0% 0 0.0% I 12.5% r-hLI{IPlacebo vs Gonal-F/r-hLH 0.0399 0.0653 0.0799 0.1016 3 fol.>=-11 mmar 1 16.7% 1 16.7% 2 25.0% Overall comparison 0.2051 0.2184 0.4101 0.4325 3 foil. =1I mm 5 83.3% 1 116.7% 4 50.0% All 6 100.0% 6 100.0% 8 1100.0% Follicles 0 foil. 14mm 0 0.0% 4 66.7% 1 12.5% Gonal-FlPlacebo vs r-hLH/placebo 0.0046 0.0076 0.0092 0.01527 >=14mm I foil. 14 mm 1 16.7% 1 16.7% 4 50.0% Gonal-F/Placebo vs Gonal-F/r-bLH 0.0424 0.0766 0.0848 0.1485 2 foil. =14 mm 2 33.3% 1 16.7% 2 25.0% r-/iLlfPlacebo vs Gonal-Flr-hLH 0.0461 0.0776 0.0922 0.1575 3 foil. 14 mmu 1 16.7% 0 0.0% 0 0.0% Overall comparison 0.1330 0.1377 0.2660 0.2769 3foil. >=l14 nu 2 133.3% 10 10.0% 1 12.5% r_ All 6 1 10.% 16 1100.0% 1T 100.0% Contrast Overall Comparison: Jonckhieere-Terpstra test Pairwise Comparison: Cochran-A rmtage test for trend 2005202003 11 May 2005 Table 9 Descriptive Statistics of Hormone Levels Measured at Ti and on the Day of hCG or on the Last Day of Treatment if No hCG was Administered TI (first day of stimulation) Day of hCG or last day of treatment if no ICG was Variable I I Trp~ritnwn I nI I Pr 1~ I Treatm ent -i n M SDr I iF an Range I n r3n IIUIL) 13onal-F/Placebo r-hLH/Placebo Gonal-F/r-hLH 8.58 12.37 9.68 3.19 6.73 3.44 1.30 2.75 1.22 8.05 9.85 In i( (5-14) (9-26) Mean 8.52 3.33 3.44 122 1015 (415 03 LII 10.t I 4h7i ml I 07 o7( I* rr\ 12 I (4-6I LH (lU/L) Gonal-F/Placebo r-hLH/Placebo Gonal-F/r-hLH 1 1.08 1.30 1.58 0.16 0.60 0.87 0.07 0.24 1.00 1.00 i n (1-1) (1-3) 7.09 1.00 1.88 1I .r I IL- 4 .4A J 0

I

LIII I1 I 1 f I flT( I 1iV 20] 0 E2(pmol/L) Gonal-F/Placebo r-hLH.Placebo Gonal-F/r-hLH 691.50 669.33 1416.50 737.24 483.03 IE6e6;n 300.98 197.20 ZRO fl? 474.5 630.00 r;(n An (160-2171) (129-1311) 110Y AOOC.

1.52 725.80 116.33 3452.86 administered SD SEM 3.13 1.40 2.13 0.87 2.66 0.94 3.62 0.83 0.00 0.00 1.56 0.64 0.71 0.25 1.00 0.23 989.66 442.59 102.12 41.69 3843.18 1452.59 2803.84 660.87 0.8 0.4 1.5 0.6 53.0 20.0 33.2 7.8 2.35 1.18 2.37 0.97 6.19 2.19 5.18 1.22 Median 7.00 3.00 9.55 7.00 1.00 1.25 1.35 1.00 302.00 100.00 1537.00 309.00 1.3 1.2 2.8 Range (5-12) (1-6) (4-13) (1-13) (1-1) (1-3) (163-2483) (33-316) (251-11257) (33-11257) (1-3) (2-143) (1-143) 141650 166601 58902 65000 9fl I O7AR~ I I1~7QO I IC I AIAEn r 20 97485 11 1I 4~ 3 1Y4~3 1 1831 89 26 1 I 17t !IUUoUL) Gonal-rFlacebo r-hLH/Placebo Gonal-F/r-hL 1.4 2.1 213 0.6 1.1 1.1 1.6 In (1-2) (1-4) 1.6 1.9 23 13 20 22.9 All I

I

~v L V I 1 II ~1 10 In n 20 20 11 1 5 0- 1 18 1 10.0 1 Androstenedione Gonal-F/Placebo 4 87 2 1.9 (nmo!IL) r-hLH/Placebo Gonal-F/r-hLH 5.93 7.71 2.50 3.78 1.02 1134 5.65 *1 In (3-I1) (3-9) I2IA\ 4.08 5.63 3.20 5.15 10.80 5.90 (2-8) (3-10) (2-22) 3.78 134 730 4 10.58 11 I h I 3 10 I A 71 I C ot I.- 1 633 319 071 7.48

Claims (7)

1. 2. A method for inducing paucifolliculogenesis or unifolliculogenesis in an anovulatory Swoman, the method comprising administering to the anovulatory woman in the mid-to-late- t~ 10 follicular phase, a medicament providing a daily dose of LH and/or a biologically-active O analogue thereof, in the range of from 100 to 1500 IU.
2. 3. The use as claimed in claim I or method as claimed in claim 2, wherein the LH is r-hLH.
3. 4. The use as claimed in claim I or method as claimed in claim 2, wherein the daily dose is in the range of from 200 to 800 IU. The use as claimed in claim I or method as claimed in claim 2, wherein the daily dose is in the range of from 225 to 450 IU.
4. 6. The use or method as claimed in any preceding claim, wherein FSH and/or a biologically-active analogue thereof is used in the production of the medicament.
5. 7. The use or method as claimed in claim 6, wherein the IU ratio of LH to FSH is in the range of from 1.5:1 to 20:1.
6. 8. The use or method as claimed in claim 7, wherein the ratio is in the range of from 1.5:1 to 10:1.
7. 9. The use or method as claimed in any preceding claim, modified in that LH and/or a biologically-active analogue thereof is replaced by an equivalent dose of hCG and/or a biologically-active analogue thereof. N \\Ic I I%)..4.I9J I l I \Aj I\.S 1 I 15 A: I 2L1Ni iI.)in 2IXI7-9-7., 7/I)X/117 0. The method or use as claimed in any proceeding claim, wherein the medicament is for administering when there are more than 3 follicles with a mean diameter in the range of from 8 to 13 mmn and no larger follicles. 005 11. The use wherein the medicament is for inducing pauIcifolliculogenesis or unifolliculogenesis in women. N\.kh VW I N I) AP GIIN] S A U I \Sxv.AI'J3S 15 AUbI I, W~~u 2M7X 7 d-7/08/17