MXPA96002845A - New composition of glucoprotein isoforms that have a stimulating activity of folicu - Google Patents

Abstract

The invention relates to a mixture of isoforms of a glycoprotein with follicle-stimulating activity, to a pharmaceutical composition comprising these isoforms, and to a method for inducing follicular growth and maturation. Better results are obtained if a pharmaceutical composition with a relatively high proportion of basic isoforms is administered in a controlled hyperstimulation. More oocytes could be recovered, and a lower dosage was sufficient as well as a longer treatment period.

Description

NEW COMPOSITION OF GLUCOPROTEIN ISOFORMS? THAT HAS A STIMULATING ACTIVITY OF FOLLICLES The present invention relates to a pharmaceutical composition comprising a mixture of isoforms of a glycoprotein with a follicle stimulating activity, as well as an improvement in a method for inducing follicular growth and maturation. Follicle stimulating hormone (FSH), produced by the anterior pituitary, plays an important role in female and male reproduction by stimulating gonadal differentiation and maturation through its regulatory action on the Sertoli cell in the testes, and on the granulosa cell in the ovary. Follicle-stimulating hormone is produced and secreted by the pituitary in different molecular forms (isohormones or isoforms), which vary in the overall load, receptor binding affinity, biological activity, and plasma residence time. . This microhertogoneidad r.o dobr to the differences in the amount and / or composition of the carbohydrate residues, in particular sialic acid. Multiple forms of gonadotropins have been isolated and characterized from the anterior pituitary glands, serum, and urine of several non-mammalian and mammalian species, including man. The isoforms of stimulating hormone of relatively acidic follicles, which are more heavily laclar i. , exhibit lower in vitro ideological receptor affinity activities than the most basic isoforms. However, due to their residence time in longer plasma, these more acidic forms have higher biological activities in vivo (Ulloa-Aguirre et al., 1988, Hum. Reprod., 3, 491-501). The follicle-stimulating hormone is used for the induction of ovulation and for the controlled stimulation of the ovaries in in vitro fertilization (IVF). The objective of controlled superovulation is to increase the number of retrievable mature oocytes for in vitro fertilization and the subsequent embryo transfer (ET). In general, up to three embryos are replaced by transfer. Since more than one treatment is usually necessary, in most infertility clinics loose fertilized embryos or oocytes are frozen and transferred in subsequent cycles. Assuming a normal fertilization, the more oocytes recover, the higher the number of possible transfers, and therefore, the higher the chance that a woman will become pregnant after a cycle of treatment. In the case of male infertility, the chances of establishing fertilization, and consequently, pregnancy, also increase with the number of recovered oocytes.

Previously, it has been shown that the majority of circulating follicle stimulating hormone isoforms during the follicular and luteal phase of the normal menstrual cycle have p < 4.8, while during the middle of the 5 cycle, or after the estrogen treatment, there were considerably more alkaline follicle-stimulating hormone isoforms present (Padmanabhan et al., 1988, J. Clin. Endocrinol. Metab., 67, 465-47 ). This indicates that the stimulating hormone isoforms of More acid follicles have a role in the growth and recruitment of ovarian follicles during the folate phase. Up to now, for clinical purposes, follicle-stimulating hormone preparations have been used that have been isolated from natural sources. They have 15 reported distribution profiles of isohormones from commercially available preparations (eg, Harlin et al., 1986, Fert. Ster., 46, 1055-1061). It can be seen that these follicle-stimulating hormone compositions consist of fractions of isohormones rol ti vamito 20 acids. Surprisingly, it has now been discovered that a mixture of glycoprotein isoforms with isoelectric points therebetween and extending from the range of 4.8 to 4.2, which have a stimulating activity of 5 follicles, which consists of more than 15 percent of isoforms with isoelectric points greater than 4.8, and less dr >l 30 percent of isoforms with electrical points less than 4.2, when used in the same clinical settings, exert a better effect than known glycoprotein compositions. Clinical studies have been carried out with a glycoprotein isohormone composition according to the invention and obtained by recombinant DNA methods. A separation of isohormones from follicle-stimulating hormone according to its isoelectric point (pl) revealed that the preparation according to the invention has relatively basic isoforms. The isoelectric points as described herein were determined by means of chromatographic focusing (see example 1). The commonly known glycoprotein isohormone compositions (of which Metrodin used in this clinical study is an example) have a more acidic profile. The fact that a more basic isohormone profile gives better results in superovulation is surprising, because it has been thought in the literature that bioavailability in vivo is the most important factor for the stimulating effect of glycoprotein follicles (application European Patent Number EP 388,223). The most basic isohormones have a shorter half-life than the more acidic isoforms, which would make the basic isoimotms less suitable for use in in vitro fertilization schemes, and therefore, are discarded from the present invention. It is preferred that the glycoprotein isohormone mixture according to the invention contains a relatively high proportion of basic rolati-vamento isoforms. This can be established by making the compositions of the isoform of qlucoprotein have more than 15 percent of the isoforms with isoelectric points greater than 4.8, and less than 30 percent with isoelectric points less than 4.2; also, preferably, mixtures of isoforms may be used where more than 15 percent of the isoforms have isoelectric points greater than 4.8, and less than 25 percent have isoelectric points less than 4.2; also preferably, more than 25 percent of the isoforms have isoelectric points greater than .8, and less than? percent have isoelectric points smaller than 4.2; also preferably more than 25 percent of the isoelectric isoforms with isoelectric points greater than 4.8, and less than 20 percent have isoelectric points less than 4.2. The percentage of the isoforms as used herein is defined as the relative amount of immunoreactive isoforms recovered after the chromatographic approach. Alternatively, the protein content can also be determined by colorimetric assays. In a clinical study, as described herein, it has been found that, in controlled superovulation, the number of recovered oocytes is different in the two follicle-stimulating hormone preparations used, i.e., the preparation according to the invention , and Metrodin®, with respect to the number of oocytes recovered. More oocytes could be recovered from the treated group by a preparation according to the invention. In addition, a significantly lower dosage of follicle stimulating hormone and a shorter treatment period is sufficient. The glycoprotein mixture according to the present invention can be derived from a urinary origin. For natural follicle-stimulating hormone preparations, the number and relative amount of each species of isoform depends on the source (pituitary, serum, or urine), age, and endocrine status of the donor, and the isolation procedure applied for its purification (Wide, 1981, J. Clin Endocrinol, Metab., 55, 682-688, ide and Hobson, 1983, J. Clin Endocrinol, Metab., 56, 371-375). Alternatively, the glycoprotein could also be a recombinant glycoprotein. For the recombinant follicle stimulating hormone (recFSH) the heterogeneity of each so determines by the host cell line selected for its production, as well as the cell culture conditions. For glycoproteins, such as follicle-stimulating hormone, 5 an Ovarian Chinese Hamster (CHO) cell line is an obvious choice, since it is known that these cells produce glycoproteins with oligosaccharides identical or closely related to those found in man (Sasaki et al., J. Biol. Chem., 1987, 262, 12095-12076). However, other host cell lines can also be used. A recombinant follicle stimulating hormone preparation according to the present invention can be produced by a Chinese Hamster Ovary cell line stably transfected with a plasmid containing 15 the two subunit genes that encode the human follicle-stimulating hormone (hFSH). This Chinese Hamster Ovary cell line produces human glycosylated follicle stimulating hormone that is secreted into the culture medium, which is the source for another purification. Batches like this can be prepared, as described (van ezenbeek et al., 1990, in: From Clone to Clinic, Kluwer Academic Publichers, 245-251). Previous preclinical studies demonstrated that the binding affinity to the receptor and the in vivo and in vivo efficacy of the recombinant follicle stimulating hormone are compatible with those of the follicle stimulating hormone isolated from natural sources (Mannaerts et al. , 1991, Endocrinology, 129, 2623-2630). The follicle-stimulating hormone preparations according to the invention can be selected based on their chromatographic focusing profile. In addition, the profile can be influenced by a selection of the particular host cell line, or by adaptation of the culture conditions. As an alternative, they can also be isolated from glycoprotein isoforms according to the invention, from cell lines whose expression of the glycoprotein gene has been activated by the site-specific direction of a regulatory sequence for the glycoprotein gene. (Application of TCP Number WO 92/19255). In addition to the wild type cell lines, basic isohormones can be obtained by the expression of recombinant follicle stimulating hormone in cell lines that deteriorate in glycosylation. Said cell line could be, for example, a cell line deficient in the glucose N-acetyl transferase enzyme or in the transport of sialic acid towards the Golgi apparatus (Galway et al., 1990, Endocrinology, 127, 93-100). . Other embodiments of the present invention are mixtures of basic glycoprotein isoforms obtained by enzymatic or chemical modification. With this treatment, parts of the carbohydrate chains can be removed without affecting the amino acid sequence. Lots of glycoprotein, for example, can be treated with HF 5 (Chen et al., 1982, J. Biol. Chem. 257, 14446-14452). Partial desialylation can be performed by enzymatic hydrolysis with neura inidase (Vaitukaitis and Ross, 1971, J. Clin Endocrinol.-Metab.33, 308-311). The objects of the present invention are also \? mixtures of basic glycoprotein hormones having a biological follicle stimulating activity, i.e., which are prepared by removing one or more oligosaccharide chains linked with N, of any of the subunits. These chains can be removed by site-directed mutagenesis 15 site of the gene encoding the glycoprotein. In this way, a change of a single amino acid can be obtained, such that the binding site with the carbohydrate is no longer present. Consequently, one or more carbohydrate chains can not be bound on the a or β subunit 20 (Matzuk and Boi e, 198, J. Cell, Biol. 106, 1049-1059). The mixture of relatively basic isohoron forms can be used according to the invention for the manufacture of a medicament to be used, for example, in the treatment of ovulation induction or controlled ovarian stimulation.

Accordingly, the invention relates to a pharmaceutical composition comprising these glycoprotein isoforms mixed with pharmaceutically acceptable auxiliaries. Methods for making the preparations and mixtures are described in Re ington's Pharmaceutical Sciences, pages 1463-1497 (16th edition, 1980, Mack Publ.Co. of Easton, Pa, USA). For example, ampoules containing the pharmaceutical composition according to the invention may contain from 1 to 1000 micrograms of the mixture of i .. β glycoprotein (for example, 75 international units are considered a therapeutic amount). The invention also relates to an improved method for inducing follicular growth and maturation, wherein the pharmaceutical composition containing the 15 mixture of basic glycoprotein isoforms as described above. In accordance with the present invention, there is no need to influence the administration of more mixtures of acid isoforms in the treatment program. Another method improvement consists of a repeated administration of pharmaceutical compositions containing a mixture of isoforms of a glycoprotein with follicle stimulating activity, each composition having an identical isoform profile, ie, the same isoform profile. Therefore, during a treatment, the administration of only relatively basic isoforms is sufficient. These mixtures can also be prepared by isolation of only basic isoforms, for example, by chromatographic preparation approach. Preferably, these forms have an isoelectric point greater than 4.2. It will be clear that fractionation of mixtures of isoforms can be performed on glycoprotein batches obtained from different origins, such as le, preparations isolated from urine, or recombinant-DNA cell lines that may or may not be chemically or enzymatically modified. . The pharmaceutical compositions according to the present invention can be used in treatments 15 clinical in combination with, for example, GnRH antagonists or agonists and / or LH activity, eg, HCG or LH to induce superovulation.

Example 1 Comparison of the immunoreactive isohormone profile of recombinant human follicle stimulating hormone (Org) 32489) and urinary follicle stimulating hormone (Metrodin®). In the present study, the isohormone profile of several batches of Org 32489 (recombinant human follicle stimulating hormone, NV Organon), derived from Chinese Hamster Ovary cells transfected with genes encoding hormone-stimulating hormone, was examined. human follicles, and compared with that of commercially available batches of urinary human follicle stimulating hormone, ie, Metrodin®. Preparations were obtained from the final and bulk product of purified Org 32489 (> 99 percent) in Diosynth and Organon (The Netherlands), respectively. The stimulating hormone i of human urinary follicles, that is, Metrodin® (Serono, Rome Italy; 75 international units / ampoule declared a bioactivity live in relation to IS 70/45), was used as a reference. In total, twelve bulk lots of Org 32489, five clinical preparations (CP) of Org 32489 were used, 15 and twelve batches of Metrodin® for comparison of isohormones. The lots of Metrodin® indicated with an enclosed CP code have been used in a trial with batches of clinical preparations of Org 32489 to compare the clinical efficacy (see Example 2). By using the chromatographic approach, the different human follicle stimulating hormone isohormones were separated based on their isoelectric point (pl). The distribution of human follicle stimulating hormone was determined after quantification by an enzyme immunoassay using antibodies that showed comparable capabilities towards all isoforms. The chromatographic approach was carried out in the pH range of 6 to 3 on a column of fast protein liquid chromatography (FPLC) HR 5/20 (Pharmacia, Oerden, Holland) 5 packed with poly-buffer pH 94 exchanger ( PBE-94; Pharmacia), and equilibrated with 0.034 moles / liter of L-histidine (Aldrich Chemie, Steinheim, FRG), adjusted to a pH of 6.2 with HCl. Before use, each column was eluted with 53 milliliters of poly-buffer from Ph 74 (Pharmacia) diluted in l. 1:11 (volume / volume) with distilled water, and adjusted to a pH of 3.0 with HCl (elution buffer) until a pH curve was obtained with a constant decline from pH 6 to 3. Before each test experimental, the column was eluted with elution buffer and 2 milliliters of 0.03415 moles / liter of L-histidine, containing 100 micrograms / milliliter of human serum albumin (USA; Behring, Marburg, FRG). Of each hormone preparation, approximately 225 international units of hormone were dissolved 20 follicle stimulant in terms of bioactivity in vivo, in 3 milliliters of equilibrium regulator (material solution) and 2 milliliters of this solution (150 international units) were applied to the column. Subsequently, the column was eluted with the elution buffer. They were collected 25 fractions of 1 milliliter at a flow rate of 1 milliliter / minute. After 53 fractions, 2 milliliters of a 2 mol / liter NaCl solution was applied to the column, and 7 additional fractions were collected. For the quantification of follicle-stimulating hormone, fractions 1, 2, and 3, and fractions 4, 5, and 6 had to be pooled together. All other fractions were adjusted to 2.5 milliliters with a mixture of 1: 1 HAM F12 and DMEM (Gibco, Grands Islands, NY, USA) supplemented with 1 gram / liter of bovine serum albumin (BSA; Sigma, St. l * and Louis, MO, USA) (medium +). In addition, 0.3 milliliters of the material solution of follicle stimulating hormone was adjusted to 2.5 milliliters with 2.2 milliliters of medium +. All fractions of follicle-stimulating hormone were desalted by applying each fraction to a 15 column PD-10 (Pharmacia), equilibrated with 12 milliliters of medium +. After elution with 3.5 milliliters of medium +, the collected samples were stored at -20 ° C until the determination of the follicle-stimulating hormone in unreactivity. The follicle-stimulating hormone immunoreactivity was measured in a two-site sandwich immunoassay, using a capture antibody directed to β (monoclonal antibody 4B) and a detection antibody labeled with HRP targeting aa (monoclonal antibody 116B). as described above (Mannaerts et al., 1991, Endocrinology, 129, 2623-2630). This assay recognizes only intact dimers, and was found to detect all isoforms of follicle-stimulating hormone equally well. The sensitivity of the assay in terms of IS 70/45 was 0.4 international units / liter, and the coefficients of intra- and inter-assay variations were 7 percent and 8 percent, respectively. The cross-reactivity with hLH and hCG was < 0.01 percent and < 0.01 percent, respectively. In order to quantify the isohormone composition of Org 32489 and Metrodin®, the distribution of follicle-stimulating hormone immunoreactivity after the chromatographic approach was divided into the following six pH scales: > 5.3, 5.30-4.81 4.80-4.20 4.19-3.60 3.59-3.00 < 3.0 (fraction of salt).

These pH scales were selected to obtain a symmetric division of the bell-shaped distribution of follicle-stimulating hormone immunoreactivity after the Org 32489 chromatographic approach. The amount of follicle-stimulating hormone immunoreactivity that was eluted within each scale pH was expressed as a percentage of total eluted follicle stimulating hormone immunoreactivity. In addition, recovery was determined, i.e. total eluted follicle stimulating hormone immunoreactivity expressed as a percentage of the follicle stimulating hormone immunoreactivity applied to the column, the pH scale, and the upper pH. Recoveries below 75 percent and greater than 125 percent were considered unacceptable, resulting in rejection of the chromatographic focus test. Recovery, pH scale, upper pH, and distribution of follicle-stimulating hormone immunoreactivity after the chromatographic focus of batches of Org 32489 in bulk and Metrodin® are shown in Table 1. Focus profiles Representative chromatographic analyzes of Org 32489 are shown in Figure 1. The recovery of follicle-stimulating hormone immunoreactivity after the chromatographic focus of Org 32489 and Metrodin® was comparable, being 93.7 (standard deviation: 8.9) percent and 89.1 (standard deviation: 6.1) percent, respectively. Org 32489 exhibited a bell-shaped distribution of follicle-stimulating hormone immunoreactivity on a pH scale of 5.68 (standard deviation: 0.11) -3.18 (standard deviation 0.09) with an upper one at a pH of 4.53 (standard deviation: 0.08). (n = 12). The immunoreactive profile of follicle stimulating hormone of Metrodin® was 5.58 (standard deviation: 0.18) to 3.07 (standard deviation: 0.05) with an upper one at a pH of 4.28 (standard deviation: 0.13) (n = 12). The distribution of follicle-stimulating hormone immunoreactivity on the six pH scales showed that, compared to Metrodin®, Org 32489 contained an approximately two-fold higher percentage of relative isoforms relative to an isoelectric point >; 4.8 (20.9 percent vs. 12.7 percent) and an approximately twice lower percentage of relatively acidic isoforms with an isoelectric point < 4.2 (21.1 percent against 41.7 percent) (Tables 1 and 2). In addition, all batches of Metrodin® contained more follicle-stimulating hormone immunoreactivity in the salt fraction (3.3 percent vs. 0.8 percent for Org 32489), representing isoforms of follicle-stimulating hormone with an isoelectric point less than 3.0 . In addition to the bulk batches of Org 32489 described above, the chromatographic focusing profile of five clinical preparations was investigated. The average distribution of follicle-stimulating hormone from these clinical preparations was very comparable with that found for bulk lots (Table 1). As the twelve batches of Org 32489 and Metrodin used for comparison of isohormones, the clinical preparations of Org 32489 and Metrodin used to compare the clinical efficacy (see Example 2) showed the same difference in the relative contribution of basic and acid isoforms, that is, the clinical preparations of Org 32489 contained more basic isoforms than the clinical preparations of Metrodin (17,120.2 percent vs. 12.7 percent if the isoelectric point > 4.8). This comparative study, comprising chromatographic focusing profiles of Org 32489 and urinary human follicle stimulating hormone, ie Metrodin®, revealed that Org 32489 contains more basic isoforms with an isoelectric point > 4.8 and fewer acid isoforms with an isoelectric point < 4.2 what Metrodin.

Example 2 Comparative study of efficacy of a preparation of Org 32489 in patients undergoing in vitro fertilization.

Study Design The study was designed as a multiple-center, randomized, blinded advisor, group comparison study, where the safety and efficacy of Org 32489 and Metrodin® were compared in subjects infertile with the suppressed pituitary who underwent fertilization in vitro (IVF) and embryo transfer (ET). Approximately 1000 subjects were included in this study with a relationship between subjects treated with Org 32489 and with Metrodin® of 3: 2. The study period covered no more than 3 treatment cycles. The efficacy analysis included the first treatment cycles only. The inclusion of the subjects was based on the following criteria to enter: Criteria for inclusion: at least 18 and at the most 39 years of age at the time of classification; cause of infertility of the potential subject being resolvable by in vitro fertilization; maximum of three previous attempts of in vitro fertilization, intrafallopian transfer of gametes (GIFT), or intrafallopian transfer of zygote (ZIFT), where oocytes were collected at least once; - normal ovulatory cycles with an average duration of between 24 and 35 days, and an intra-individual variation of plus or minus 3 days (but never outside the range of 24-35 days); Good physical and mental health; - body weight between 80 and 130 percent of ideal body weight? and desire to give written informed consent. 5 Criteria for Exclusion: infertility caused by endocrine abnormalities such as hyperprolactinemia, polycystic ovary syndrome, and lack of ovarian function; male infertility defined according to AC / the following criteria: < 10x106 sperm per milliliter and / or 40 percent normal morphology, and / or 40 percent normal mobility; contraindications for the use of GnRH analogues, follicle stimulating hormone, hMG, and / or hCG; 15 - any ovarian and / or abdominal abnormality that would interfere with adequate investigation with ultrasound; hypertension (diastolic blood pressure> 90 mm Hg and / or systolic blood pressure> 150 mm Hg); 20 - cardiovascular, hepatic, renal, or chronic pulmonary disease; history (within 12 months) or current abuse of alcohol or drugs; and administration of research drugs 5 within three months before classification.

Treatment Program Treatment with Buserelin began on the first day of the menstrual cycle. The first day of the menstrual cycle was defined as the first day the person woke up with menstrual bleeding. Treatment with Org 32489 (75 international units / ampule) or Metrodin® (75 international units / ampule) was started 14 to 18 days after the establishment of Buserelin administration when this treatment had resulted in a hypogonadotropic state (ie , serum E2 <50 pg / mL). In the event that this condition had not been reached, treatment with Org 32489 or Metrodin was postponed, and the dose of Buserelin was increased to 4 x 300 micrograms / day. This increased dose of Buserelin was sustained throughout the additional treatment period until the follicle-stimulating hormone treatment was stopped. The previous treatment with Buserelin was not allowed to exceed a period of five weeks. If after a complete regulation on the first day of treatment with follicle-stimulating hormone a follicular cyst of > 20 millimeters, this cyst was perforated before the first injection of follicle-stimulating hormone. In each treatment cycle, the dosage was made according to the following program: HCG (10,000 international units) was administered when there were at least three follicles > 17 millimeters present. No more than three embryos were replaced, and the frozen embryos were replaced in natural cycles or in stimulated cycles. A cycle was considered canceled if an embryo transfer had not taken place. The luteal phase was monitored by progesterone evaluations. Luteal phase support was given (for example, three injections of 1,500 international units of hCG or at least 50 milligrams of intramuscular progesterone per day, b 400 milligrams of progesterone intravaginally per day (for at least two weeks after the injection of 2,000 units international hCG.

Statistical Evaluation The total number of oocytes recovered in the first treatment cycle was a variable of primary efficacy. Other variables analyzed in the first treatment cycle were the number of follicle-stimulating hormone vials administered for ovarian stimulation, and the duration of treatment with follicle-stimulating hormone.

The result of these parameters was analyzed by means of the Cochran method of combining the results of the individual centers. In addition, the Wilcoxon rank sum test and a variation analysis (ANOVA) were applied to consolidate the result of the Cochran method.

Results of the First Treatment Cycles of the Study In total, 981 subjects started treatment with follicle-stimulating hormone in 18 study centers. In total, 907 subjects had a sting, 546 from the Org 32489 group and 361 from the Metrodin group. With this number of chopped subjects, it was possible to detect with a probability of 0.8 (the so-called study power) at least a difference of 1.2 oocytes between the two treatment groups. The number of oocytes recovered in each treatment group per center was. presented in Table 3. In all. l: -. centers, the Org 32489 group had a higher average number of retrieved oocytes than the Metrodin group. The overall average number of oocytes (heavy on the centers) was 10.84 oocytes in the Org 32489 group, and 8.95 oocytes in the Metrodin group, resulting in a treatment difference of 1.89 oocytes in favor of Org 32489. The difference of 1.89 oocytes is more than five times its standard error and is highly significant (P <0.00 () l). The resulting 95 percent confidence interval indicated that, on average, subjects treated with Org 3? .4 C > They finished with at least 1.2 and at most 2.6 oocytes more than those treated with Metrodin®. The exclusion of immature oocytes from the total number of recovered oocytes had no influence on the treatment effect (see Table 4). The total number of ampoules administered to chopped subjects is presented per center in Table 5. The total dosage of Org 32489 was, in 14 of the 18 centers, lower than the total metrodin dosage *. The average global follicle stimulating hormone dosage (measured over the centers) was 28.5 ampoules in the Org 32489 group, and 31.8 ampoules in the Metrodin group, resulting in a treatment difference of 3.3 ampoules, which is highly statistically significant (P < 0.0001). The resulting 95 percent confidence interval indicated that, on average, subjects treated with Org 32489 receive minimally 2.1 and maximally 4. 5 ampules less than those treated c * on Metrodi «n (S). The duration of follicle-stimulating hormone treatment of chopped subjects is presented by center in Table 6. The duration of treatment was, in 13 of 18 centers, shorter in the Org 32489 group than in the Metrodin group. The overall average treatment duration (measured over the centers) was 10.7 days in the Org 32489 group, and 11.3 days in the Metrodin group, resulting in a 0.6 day treatment difference that is highly statistically significant (P < 0.0001). The resulting 95 percent confidence interval indicated that, on average, for subjects treated with Org 32489, the treatment period is minimally 0.3 days and maximally 0.9 days shorter than for those treated with Metrodin *. The result of the Wilcoxon rank sum test and the variation analysis confirmed all the previous discoveries. Therefore, it can be concluded that in this study, controlled superovulation by means of Org 32489 treatment demonstrated a superiority over Metrodin® with respect to the number of oocytes recovered with a significantly lower follicle stimulating hormone dosage and a treatment period. shorter.

Legends Figure 1 Distribution of follicle stimulating hormone immunoreactivity after chromatographic focus of a specific lot of Org 32489. The profile of a batch is shown as an example. The upper and lower panels represent separate tests. (sf = fraction of salt). Total recovery of 91.9 and 91.3 percent, respectively.

Table 1 Average recovery, pH scale, upper pH, and distribution of follicle-stimulating hormone immunoreactivity on pH scales after the chromatographic approach of 12 bulk batches of Org 32489, five final products of Org 32489, ie, clinical preparations. • cas (CP) and twelve lots of Metrodm • (JO.

Table 2 Percentage of basic isoforms, defined as follicle-stimulating hormone immunoreactivity at an isoelectric point > 4.8, and percentage of acid isoforms, defined as follicle-stimulating hormone immunoreactivity at an isoelectric point < 4.2 of the clinical preparations of Org 32489 and Metrodin® used to compare the clinical efficacy (see Example 2).

Table 3 Statistical analysis (Cochran approach) of the total number of retrieved oocytes. Including all the chopped subjects.

Table 4 Statistical analysis (Cochran approach) of the number of mature oocytes recovered. Including all the chopped subjects.

Table 5 Statistical analysis (Cochran approach) of the total follicle stimulating hormone dose administered. Including all the chopped subjects.

Table 6 Statistical analysis (Cochran's approach) of the duration of treatment with follicle-stimulating hormone. Including all the chopped subjects.

Claims (9)

1. A pharmaceutical composition comprising a mixture of isoforms of a glycoprotein hormone with isoelectric points between and extending from the scale of 4.8 to 4.2, having follicle-stimulating activity, mixed with pharmaceutically acceptable auxiliaries, characterized in that more than 15 percent of isoforms have isoelectric points greater than 4.8, and less than 30 percent have isoelectric points smaller than 4.

2. 2. The composition according to the claim 1, characterized because more than 15 percent of the isoforms have isoelectric points greater than 4.8, and less than 25 percent 15 percent have isoelectric points smaller than 4.2.

3. The composition according to the claim 2, characterized because more than 25 percent of the isoforms have isoelectric points greater than 4.8, and less than 25 percent have isoelectric points smaller than 4.2.

4. The composition according to the claim 3, characterized in that more than 25 percent of the isoforms have isoelectric points greater than 4.8, and less than 20 percent have isoelectric points smaller than 4.2.

5. The composition according to claims 1 to 4, characterized in that the glycoprotein is recombinant follicle stimulating hormone.

6. The composition according to claims 1 to 5, characterized in that the glycoprotein is modified by enzymatic or chemical treatment.

7. The composition according to the claim 5, characterized in that the recombinant follicle stimulating hormone is modified by the elimination of; at least one of the oligosaccharide chains linked to N.

8. A method for inducing follicular growth and maturation in females, by administering a pharmaceutical composition according to claims 1 to 7.

9. A method for inducing growth and maturation follicular by administering, during the ovulatory cycle, identical pharmaceutical compositions of a mixture of isoforms of a glycoprotein with follicle stimulating activity, characterized in that the isoforms have an isoelectric point greater than 4.2.