Compositions for regulated ovarian stimulation

Abstract

This invention provides products for use in infertility treatment, and methods for treating infertility. [Solution] A product containing FSH for use in the treatment of infertility in patients (for example, patients with a serum AMH level of 0.05 pmol / L or higher) is provided, the product containing a dose or equivalent of 2 to 24 μg, for example, 2 to 15 μg of human recombinant follicle-stimulating hormone (FSH).

Description

[Technical Field]

[0001] This invention relates to compositions and pharmaceuticals for the treatment of infertility. [Background technology]

[0002] Assisted reproductive technology (ART) techniques such as in vitro fertilization (IVF) are well known. ART techniques generally require a controlled ovarian stimulation (COS) step, and the follicular cohort is Stimulated until fully mature. Standard COS regimens typically prevent early LH surges. To stimulate follicular development, administer a GnRH analog before and / or during stimulation. To achieve this, follicle-stimulating hormone (FSH) alone, or in combination with progesterone (LH) activity. This includes the administration of gonadotrophins in combination. Pharmaceutical compositions commonly used in COS include Recombinant follicle-stimulating hormone (rFSH), urinary FSH, recombinant FSH + LH preparation, urine Derived menotrophins [human menopausal gonadotrophins (hMG)] and highly purified human menopausal Contains gonadotrophins (HP-hMG). IVF can be life-threatening in severe cases. There may be a risk of developing ovarian hyperstimulation syndrome (OHSS).

[0003] The ability to predict a woman's responsiveness to controlled ovarian stimulation (COS) is individualized COS. This enables the development of protocols, for example, those that exhibit excessive responses to stimuli. In women being measured, reducing the risk of OHSS and / or classifying them as having a poor response. It improves pregnancy outcomes in women. Serum levels of anti-Müllerian hormone (AMH) are It is now established as a reliable marker of ovarian reserve. A decrease in AMH levels indicates This is associated with a decreased ovarian response to gonadotrophins during COS. Furthermore, high levels of A MH is a good predictor of excessive ovarian response and an indicator of the risk of OHSS.

[0004] A preliminary study of women under 35 receiving ART showed that the CONSORT administration algorithm The risk of developing OHSS (incorporating basal FSH, BMI, age, and AFC) is determined by the following factors. It was used to predict the optimal FSH initiation dose for COS in women. Olivennes et al., 2009). Individualization of administration ensures sufficient oocyte yield and good results. This resulted in a pregnancy rate. However, in the low-dose group (75 IU FSH), the response was insufficient. There was a high rate of discontinuation, and a significant proportion of patients developed OHSS (Ovarian Hyperstimulation Syndrome).

[0005] Therefore, it provides a sufficient response to stimulation and / or a reduced risk of OHSS. A composition is needed for use in individualized COS protocols.

[0006] As mentioned above, the standard COS protocol may include FSH administration. FSH is naturally In nature, it is secreted from the anterior pituitary gland and functions to maintain follicular development and ovulation. FS H is a 92-amino acid alpha subgroup common to other glycoprotein hormones, LH and CG. The unit, and the 111 amino acids specific to FSH that confer the biological specificity of the hormone. Includes the beta subunit (Pierce and Parsons, 1981). Each subunit The nit is post-translationally modified by the addition of complex carbohydrate residues. Both subunits are AL amino acids 52 and 78 of the pha subunit and amino acid residues of the beta subunit Possessing two N-linked glycan attachments at positions 7 and 24 (Rathnam and Sax ena, 1975, Saxena and Rathnam, 1976). Thus, FSH is glycosylated by approximately 30% by mass (Dias and Van Roey, 200 1. Fox et al., 2001).

[0007] FSH purified from postmenopausal human urine has been used for many years in infertility treatment: both to promote ovulation in natural reproduction and to provide oocytes in assisted reproduction medicine . Currently approved recombinant FSH (rFSH) products for ovarian stimulation, such as follitropin alfa (GONAL-F, Merck Serono / EMD Serono) and follitropin beta (PUREGON / FOLLISTIM, MSD / Scher ing-Plough), are derived from Chinese hamster ovary (CHO) cell lines . Currently, there are no rFSH products from commercially available human cell lines . There is considerable heterogeneity associated with FSH preparations regarding differences in the abundance of various isoforms . Individual FSH isoforms exhibit the same amino acid sequence but differ in the degree of post-translational modification: specific isoforms are characterized by differences in carbohydrate branching structure and the amount of sialic acid (terminal sugar) incorporation, both of which are thought to affect the biological activity of a particular isoform

[0008] . . The glycosylation of native FSH is highly complex. The glycans in native pituitary FSH include combinations of mono-, bi-, tri- and tetra-antennary glycans when present .

[0009] . . may have a wide range of structures (Pierce and Parsons, 19 81. Ryan et al., 1987. Baenziger and Green, 1988). Gly cans can also carry further modifications: core fucosylation (core fucosylatio n), bisecting glucosamine, chain elongation with N-acetyl-lactosamine, partial or complete sialylation, sialylation at α2,3 and α2,6 linkages, and substitution of galactose with sulfated galactosamine (D alpathado et al., 2006). Furthermore, there are differences in the distribution of glycan structures at individual glycosylation sites. The same level of glycan complexity has been found in FSH from the serum of individuals and FSH from the urine of postmenopausal women (Wide et al., 2007).

[0010] The glycosylation of recombinant FSH products reflects the range of glycosyltransferases present in the host cell line. Commercially available rFSH products are derived from engineered Chinese hamster ovary cells (CHO cells). The range of glycan modifications in CHO cell-derived rFSH is more limited than that found in natural products. Examples of the reduced glycan heterogeneity found in CHO cell-derived rFSH include the deletion of bisecting glucosamine and reduced levels of core fucosylation and N-acetyl-lactosamine elongation (Hard et al., 1990). Additionally, CHO cells can only add sialic acid using the α2,3 linkage (Kagawa et al., 1988, Takeuchi et al. 1988, Svensson et al., 1990); CHO cell-derived rFSH contains only α2,3- linked sialic acid and no α2,6-linked sialic acid. ning glucosamine, as well as reduced levels of core fucosylation and N-acetyl-lactosamine elongation (Hard et al., 1990). Additionally, CHO cells can only add sialic acid using the α2,3 linkage (Kagawa et al., 1988, Takeuchi et al. 1988, Svensson et al., 1990); CHO cell-derived rFSH contains only α2,3- linked sialic acid and no α2,6-linked sialic acid. linked sialic acid and no α2,6-linked sialic acid. linked sialic acid and no α2,6-linked sialic acid.

[0011] Therefore, CHO cell-derived FSH is a mixture of α2,3 and α2,6-linked sialic acids. Naturally produced FSH (e.g., human pituitary gland / ) contains glycans in which the former is predominant. This is different from serum / urinary FSH.

[0012] Furthermore, commercially available recombinant FSH preparations contain FSH from the pituitary gland, serum, or postmenopausal urine. Compared to SH, F has an isoelectric point (pI) of less than 4 (considered to be an acidic isoform). It has also been demonstrated that there are differences in the amount of SH. (Ulloa-Aguirre et al., 19 95). The amount of acidic isoforms in the urine preparation is determined by CHO cell-derived recombinant products, Gon al-f (Merck Serono) and Puregon (Schering Pl It was more than (ough) (Andersen et al., 2004). This is because sulfuric acid Because recombinant FSH has a low content of modified, negatively charged glycans, recombinant This should reflect the low molar content of sialic acid in FSH compared to natural FSH. The low sialic acid content is a characteristic of both commercially available recombinant FSH products. This may reflect limitations in the manufacturing process.

[0013] The cyclical life of FSH has been recorded for materials from various sources. Some of them are characterized by their pI (more acids have higher negative charges and It is fractionated based on the total charge of the molecules (equal to one). As already mentioned, the total charge of the molecules The main contributing factor is the total sialic acid content of each FSH molecule. For example, rFSH(Org anon has a sialic acid content of approximately 8 mol / mol, while urinary FSH is more It has a high sialic acid content (de Leeuw et al., 1996). Corresponding blood in rats The plasma clearance rates are 0.34 and 0.14 ml / min (Ulloa-Aguir). re et al., 2003). Another example in which recombinant FSH samples were separated into high-pI and low-pI fractions. Therefore, the in vivo efficacy of the high pI (low sialic acid content) fraction is reduced, and the shorter It had a plasma half-life (D'Antonio et al., 1999). It was more basic in the later stages of the ovulation cycle. The circulation of FSH is caused by an increase in estradiol levels in the anterior pituitary gland. It has also been reported that this is for the downregulation of α2,3 sialyltransferase in [the relevant region]. (Damian-Matsumara et al., 1999; Ulloa-Aguirre et al.) (2001). Results regarding α2,6 sialyltransferase have not been reported. stomach.

[0014] Therefore, as described above, recombinant proteins expressed using the CHO system have terminal They differ from their natural counterparts in the type of sialic acid bond. Carbohydrate components are molecular pharmacological This may contribute to the characteristics of the product, and therefore is relevant in the production of biologics for pharmaceutical use. This is an important matter to consider.

[0015] The applicants hereby filed international patent application PCT / GB2009 / 000978, WO2009 We developed a human-derived recombinant FSH that is intended for public release as / 127826A. Recombinant FSH containing a mixture of both α2,6-linked sialic acid and human cell lines rFS The system is engineered to express both H and α2,3 sialyltransferase. It was produced by [method]. The expressed product is highly acidic and contains α2,3- and α2 It possesses a mixture of both ,6-linked sialic acid and the latter is endogenous sialyltransferase The activity is provided by the type of sialic acid bond, α2,3- or α2,6-, which is FSH. It has been found that this can have a dramatic effect on the biological clearance of α2,3. Recombinant FSH containing a mixture of both α2,6-linked sialic acid and conventional CHO cells It has two advantages over the rFSH expressed in: one is that it has two types of sialiltra The combination of enzyme activity allows the material to be more highly sialylated; 2 The key point is that the material is similar to natural FSH. It produces only α2,3 linked sialic acid. It may be more biologically appropriate compared to recombinant products derived from CHO cells (Ka gawa et al., 1988; Takeuchi et al., 1988; Svensson et al., 1990 ), has a reduced sialic acid content (Ulloa-Aguirre et al., 1995, An Dersen et al., 2004).

[0016] The rFSH product disclosed in international patent application PCT / GB2009 / 000978 is branched It contains glycan components. FSH contains glycans (attached to FSH glycoproteins). These glycans may contain a wide variety of structures, which are well known in the art. As shown, (glycan) branching is when a glycan has 1, 2, 3, or 4 or more terminal sugar residues or " This may result in having "tena," and 1, 2, 3, or 4 terminal sugar residues or The glycans that have an "antenna" are mono-antenna, di-antenna, and tri-antenna, respectively. It is called an antenna or tetra-antenna structure. Glycan is a mono-antenna and / or This is a di-antenna and / or tri-antenna and / or tetra-antenna structure. It may have allylation. International patent application PCT / GB2009 / 000978 Examples of rFSH shown include mono-sialylation, di-sialylation, tri-sialylation, and tetra-sialylation. - The relative amounts of sialylated glycan structures are as follows: 9-15% mono-sialylated; 27-30% Di-sialylation; 30-36% tri-sialylation and 25-29% tetra-sialylation It contains. As is well known, the mono-sialylated glycan structure has one sialic acid residue. Di-sialylated glycan structure has two sialic acid residues; tri-sialylated glycan structure has two sialic acid residues; The can structure has three sialic acid residues; the tetra-sialylated glycan structure has sialic acid residues It possesses four groups. In this specification, the terms "X% mono-sialylation" and "X% di-sialylation" are used. "Lilylation," "X% tri-sialylation," or "X% tetra-sialylation," etc. (each (re) refers to the number of glycan structures on mono-, di-, tri-, or tetrasiallylated FSH. The total number of glycan structures on FSH that are sialylated (possessing sialic acid) by any means It is expressed as a percentage (X%) of a number. Therefore, the phrase "30-36% tri-sialylated The "lycan structure" is a structure on FSH that possesses a sialic acid residue (i.e., sialylated). Of the total number of glycan structures, 30-36% of these glycan structures are trisiallylated. This means that it contains three sialic acid residues. The applicants have found that a certain amount of tetra - Siallylated glycan structure (disclosed in PCT / GB2009 / 000978 as described above) Recombinant FSH currently on the market has FSH (different from the rFSH product example) We were surprised to discover that the product was significantly more effective than the H product. The amino acid sequence is a natural sequence, and it is used for natural human FSH and existing CHO-derived rFSH production. It is identical to the substance. However, the applicants have found that both α2,3 and α2,6-bonded sialic acids A mixture and / or a human-derived compound having a specific amount of tetra-siallylated glycan structure. Replacement FSH products (i.e., those produced or expressed in human cell lines, e.g., human cell lines) Recombinant FSHs (created by engineering cell lines) are (for example, individualized) (t) We found that it can be particularly effective when used in the COS protocol. did. [Overview of the project]

[0017] According to the present invention in the first aspect, 1 to 24 μg, for example 2 to 24 μg, for example 2 A patient receiving a dose or equivalent of 15 μg of human recombinant follicle-stimulating hormone (FSH). (For example, serum AMH level of 0.05 pmol / L or higher, e.g., serum AMH level of 0.5 pmol / L or higher) Products containing FSH for use in the treatment of infertility in patients with (e.g., pharmaceuticals) A composition is provided. Preferably, the product is 4.5 to 12.5 μg, for example, 5 to 1 2.5 μg, for example, 6 to 12.5 μg, for example, 6.3 to 10.5 μg of human recombinant This includes a dose or equivalent of FSH.

[0018] According to the present invention, 9-14 μg, for example 11-13 μg, for example 12 μg of human-derived compound A dose or equivalent of (e.g., daily) of follicle-stimulating hormone (FSH) <15pm Serum AMH levels in ol / L (e.g., from 0.05 pmol / L to 14.9 pmol / L) Products containing FSH for use in the treatment of infertility in patients with the condition (e.g., pharmaceutical compounds) A product is provided. Preferably, the FSH is recombinant FSH ("rFSH" or "re cFSH) is preferred. Preferably, the FSH is recombinant FSH derived from a human cell line. Dosage This provides an effective response while minimizing the risk of OHSS. Preferably, it is used in infertility treatment. This includes the steps of determining the patient's serum AMH level (e.g., measuring it), and Serum A <15 pmol / L (e.g., 0.05 pmol / L to 14.9 pmol / L) The procedure includes the step of administering a dose to a patient with MH levels.

[0019] According to further embodiments of the present invention, 5 to 12.5 μg, for example, 6 to 10.5 μg Contains a dose or equivalent (e.g., daily) of g of human recombinant follicle-stimulating hormone (FSH) Regarding its use in infertility treatment in patients with serum AMH levels of ≥15 pmol / L Products containing FSH (e.g., pharmaceutical compositions) are provided. Preferably, FSH is , recombinant FSH ("rFSH" or "recFSH"). Preferably, FSH is It is recombinant FSH derived from human cell lines. The dosage is effective while minimizing the risk of OHSS. Provides a response. Preferably, infertility treatment involves a step to determine the patient's serum AMH level. (For example, the step of measuring) and having a serum AMH level of ≥15 pmol / L The procedure includes the step of administering a dose to the patient. In one embodiment, the product is 15 to 24.9 It is intended for use in infertility treatment in patients with pmol / L serum AMH levels. The product contains 5 to 12 μg, for example, 7 to 12 μg, for example, 8.7 to 10 μg of human-derived Recombinant FSH (preferably 9 to 10 μg of human-derived recombinant FSH) (for example, one day ) For administration in dose or equivalent amounts. In this embodiment, infertility treatment is performed on the patient's serum AMH Steps to determine the level (e.g., a measurement step), and 15-24.9 pmo The step may include administering a dose to a patient with a serum AMH level of 1 / L. Another implementation Morphologically, the product is used in patients with serum AMH levels of 25 to 34.9 pmol / L. For use in infertility treatment, the product is 5 to 12 μg, for example, 6 to 9 μg. For example, 7 to 8 μg of human recombinant FSH (preferably 7.3 to 8 μg of human recombinant FSH) For administration of recombinant FSH (for example, daily) doses or equivalents. In this embodiment, Infertility treatment involves a step of determining the patient's serum AMH level (for example, a step of measuring it). ), and administer the dose to patients with serum AMH levels of 25 to 34.9 pmol / L. The step may include the following: In another embodiment, the product is serum AM ≥35 pmol / L It is intended for use in infertility treatment in patients with H levels, and the product is 5 to 11 μg. g, for example, 6.3 to 7 μg of human recombinant FSH (preferably 6 to 7 μg of human recombinant FSH) This is for administration of recombinant FSH (for example, a daily) dose or equivalent. In this embodiment, Infertility treatment involves a step of determining the patient's serum AMH level (for example, a step of measuring... (P) and administer doses to patients with serum AMH levels of ≥35 pmol / L. It may include the top.

[0020] The above dosages are for the treatment of infertility in the patient's (target) initial stimulation protocol. For further stimulation cycles, the dosage will be adjusted according to the actual ovarian response in the first cycle. It is understood that it can be organized.

[0021] The applicants have developed a method to enable the selection of two high-quality oocytes for transplantation, using nine eggs. We found that collecting the parent cell region is generally necessary.

[0022] The applicants have found that subjects with low AMH levels (AMH < 15 pmol / L per liter) Therefore, a moderately high dose of recombinant FSH is needed to achieve this (for example, 12 μg). g) We found that at this dose, 8 to 14 oocytes in subjects with low AMH It is taken from 60%. This is for those with low AMH who are treated with 150 IU Gonal-f. In the treatment of the target group (8 to 14 oocytes are collected from only 33% of the target group) This is an unexpected and significant improvement. The applicants believe it is necessary to adjust this dosage according to the patient's weight. I discovered that it was missing.

[0023] However, 60% of the population (and 80% of women under 30 who are being treated for infertility) have high AM It has H (i.e., AMH ≥ 15 pmol / L). For these subjects, on average 9 Collecting 11 oocytes is generally quite easy; accompanying the stimulation protocol The problem is the risk of OHSS. The applicants are administered low doses of human recombinant FSH. We found a correlation between the oocytes collected and the subject's body weight in patients. This is a risk associated with treatment with a certain dose of FSH (which is common in the art). This implies a certain possibility. The applicants believe that this is acceptable compared to well-known treatment protocols. An improved safety profile (OHSS) with or improved oocyte collection. Establish a relationship between FSH dosage (which reduces risk), AMH levels, and the subject's body weight. (See Example 10.)

[0024] According to a further embodiment of the present invention, 0.09 to 0.19 per kg of the patient's body weight μg (e.g., 0.09 to 0.17 μg) of human recombinant FSH (e.g., one day) For administration in doses or equivalent amounts, for patients with serum AMH levels of ≥15 pmol / L. Products containing follicle-stimulating hormone (FSH) for use in infertility treatment (e.g.) A pharmaceutical composition is provided. Preferably, infertility treatment involves determining the patient's serum AMH level. The steps to determine (e.g., the step to measure) and serum AMH ≥ 15 pmol / L The step includes administering a dose to a patient having a certain level. In one embodiment, the product is 15 Use in infertility treatment in patients with serum AMH levels of 24.9 pmol / L or higher. Therefore, the product contains 0.14 to 0.19 μg of human-derived substances per kg of patient body weight. Recombinant FSH (preferably 0.15 to 0.16 μg of human-derived recombinant FSH) (for example) For example, it is for administration in a daily dose or an equal dose. In this embodiment, infertility treatment is performed using the patient's blood Steps to determine the clear AMH level (e.g., step of measurement), and steps 15 to 24 This may include a step of administering a dose to a patient with a serum AMH level of 0.9 pmol / L. In another embodiment, the product has serum AMH levels of 25 to 34.9 pmol / L. It is intended for use in the treatment of infertility in patients, and the product is per kg of the patient's body weight. 0.11 to 0.14 μg of human recombinant FSH (preferably 0.12 to 0.13 μg) This is for administration of (e.g., daily) doses or equivalent amounts of human recombinant FSH (g). In terms of treatment methods, infertility treatment involves a step of determining the patient's serum AMH level (e.g., measurement (steps to be performed), and patients with serum AMH levels of 25 to 34.9 pmol / L The step may include administering a dose to the product. In further embodiments, the product is ≥35 pmo It is intended for use in infertility treatment in patients with a serum AMH level of 1 / L, and is generated The substance is 0.10-0.11 μg of human recombinant FSH per kg of the patient's body weight (for example) It is intended for administration in doses of , (daily) or equal doses. In this embodiment, infertility treatment is administered using the patient's serum. The steps include determining the AMH level (e.g., measuring it) and ≥35 pmol. The procedure may include the step of administering a dose to a patient having a serum AMH level of / L. Preferably The FSH is recombinant FSH ("rFSH" or "recFSH"). Preferably The FSH is recombinant FSH derived from human cell lines. The dosage is designed to minimize the risk of OHSS. It provides an effective response while still offering a useful response.

[0025] The above dosage is for the treatment of infertility in patients (target) using the initial stimulation protocol. Regarding further stimulation cycles, the dosage will be adjusted according to the actual ovarian response in the first cycle. It is understood that it can be organized.

[0026] According to a further embodiment of the present invention, 0.15 to 0.21 per kg of the patient's body weight μg (e.g., 0.19 to 0.21 μg) of recombinant human follicle-stimulating hormone (FSH) For administration in doses (e.g., daily) or equivalent amounts, for serum AM <15 pmol / L Products containing FSH for use in infertility treatment in patients with H levels (e.g.) A pharmaceutical composition is provided. Preferably, infertility treatment involves determining the patient's serum AMH level. The steps to determine (e.g., the step to measure) and serum AMH <15 pmol / L The step includes administering a dose to a patient with a blood level of <15 pmol / L. Patients with clear AMH levels do not need to be administered based on body weight. These doses are appropriate. Using a well-known conversion in the technical field, patients are treated with a dose appropriate to their BMI. It is understood that it can be easily converted.

[0027] The product (e.g., pharmaceutical composition) has a serum AMH level of 5.0 to 14.9 pmol / L. It may be for use in the treatment of infertility in patients, and the product may be 6 to 18 μg, for example A dose of human recombinant FSH of 8 to 11 μg, for example, 8.5 to 10.2 μg or equivalent. The amount is included. The product is found in patients with serum AMH levels of 15.0-29.9 pmol / L. It may be used for the treatment of infertility, and the product is 4.8 to 15 μg, for example, 6 to It contains a dose or equivalent of 9 μg, for example, 6.8 to 8.5 μg of human recombinant FSH. The product was used in the treatment of infertility in patients with serum AMH levels of 30-44.9 pmol / L. This may be for use, and the product may be 3.6 to 12 μg, for example 4 to 7 μg, for example Contains a dose or equivalent of 5.1 to 6.8 μg of human recombinant FSH. The product is 45 For use in infertility treatment in patients with serum AMH levels of pmol / L or higher. Often, the product is 2 to 9 μg, for example, 2.4 to 9 μg (for example, 3.4 to 5.1 μg). ) or a dose or equivalent of 2 to 5 μg of human recombinant FSH. The product is 5p Follicle stimulation for use in infertility treatment in patients with serum AMH levels below mol / L It can contain the hormone (FSH), and the product is 7.2 to 24 μg, for example, 10 to 1 Contains a dose or equivalent of 5 μg, for example, 10.2 to 13.6 μg of human recombinant FSH. The product may be for use in the treatment of infertility in patients, and the product is 4.8 18 μg, for example, 6 to 11 μg, for example, 6.8 to 10.2 μg of human recombinant F Contains a dose or equivalent of SH. Preferably, the FSH is recombinant FSH ("rFSH") or (This is "recFSH"). Preferably, the FSH is recombinant FSH derived from a human cell line. .

[0028] Preferably, rFSH (e.g., recombinant FSH derived from human cell lines) is α2,3- and α Includes 2,6-sialylation. FSH (rFSH) for use according to the present invention is total sialyl 1% to 99% of the sialylation may be α2,3-sialylation. FSH(rF) In SH), 1% to 99% of the total sialylation may be α2,6-sialylation. 50 to 70% of the total sialylation, for example 60 to 69%, or about 65%, is α2. This is 3-sialylation. Preferably, 25 to 50% of the total sialylation, for example, 30 to 5 0%, for example 31 to 38%, for example about 35% are α2,6-sialylation.

[0029] Preferably, rFSH (e.g., recombinant FSH derived from human cell lines) is mono-, di-, or t It contains Li- and tetra-siallylated glycan structures, and 15-2 of the siallylated glycan structure 4%, for example 17-23%, is a tetrasiallylated glycan structure (see example below). As shown by the wax analysis of the charged glycans described above). FSH is (FSH glycotan Contains glycans (attached to the protein). The glycans in FSH contain a wide variety of structures. It is well known that there are combinations. These are mono, bi, tri, and tetra-antenna combinations. This may include combinations of glycans. In this specification, the term "X of sialylated glycan structure" is used. "The % is a tetrasiallylated glycan structure," etc., means tetrasiallylated, i.e. This refers to the number of glycan structures on FSH that possess four sialic acid residues, and can be expressed in any way as sialic acid Percentage (X%) of the total number of glycan structures on FSH that are glycated (containing sialic acid) It is expressed as follows: Therefore, the phrase "15-24% of the sialylated glycan structure is tetrasial" "It is a sialylated glycan structure" means that it possesses a sialic acid residue (i.e., it is sialylated) Of the total number of glycan structures on FSH, 15 to 24% of these glycan structures are tetraglycerides. This means that it is trans-sialylated (having four sialic acid residues).

[0030] rFSH exists as a single isoform or as a mixture of isoforms. There are cases where this occurs.

[0031] The applicants believe that a specific dose of recombinant FSH having certain characteristics is equivalent to those specific AMs. Used to treat patients based on H levels, thereby (for example, low response possible) In patients with sexual characteristics, the likelihood of a sufficient response to stimulation increases, and / or (e.g., For example, in patients classified as having a high or excessive response rate, the risk of OHSS is low. We devised a "personalized" COS protocol to implement.

[0032] Serum levels of AMH can be determined by any method known in the art. For example, it can be measured. Preferably, the serum AMH level is AMH Gen-II e nzyme linked immunosorbent assay, a kit(B Measurements were taken using a Coulter, Inc., Webster, Texas (Eckman). This assay has a lower limit of quantification of 1.1 pmol / L and is effective at 0.57 pmol / L. This assay can detect AMH concentrations exceeding a certain level. Other assays may also be used.

[0033] In this specification, serum AMH levels are generally listed in units of pmol / L. This can be converted to ng / mL using the conversion formula 1 ng / ml AMH = 7.1 pmol / L AMH. It can be converted.

[0034] In this specification, the terms "patient" and "subject" are used interchangeably.

[0035] The products (e.g., pharmaceutical compositions) are described above, in this specification and in the claims. Preferably contains a daily dose or daily equivalent of the defined amount of human-derived rFSH. (daily) The dosage may be the initial dose (i.e., it may be reduced, increased, or maintained during treatment).

[0036] The product (e.g., pharmaceutical composition) is administered starting on day 1 of treatment and for 7 to 13 days, for example. FSH lasting for 9 to 13 days, for example 10 to 13 days, for example 10 to 11 days It may be administered (daily). The product (e.g., pharmaceutical composition) is a GnRH agonist ( For example, after administration of Synarel, Lupron, Decapeptyl (for example, 12 to 16 after the start of administration (for example, after the start of daily administration), for example, 13 to 15, for example 1 It may be administered over a 4-day period. The product (e.g., a pharmaceutical composition) contains a GnRH agonist. It may be administered in conjunction with other substances. The product (e.g., pharmaceutical composition) is a GnRH antagonist ( For example, for administration before administering ganirelix, cetrorelix, etc. It may be administered 5 or 6 days before the administration of an H antagonist. The product (e.g., pharmaceutical) The composition may be for administration with a GnRH antagonist. Preferably, the product ( For example, a pharmaceutical composition contains a high (ovulatory) dose of hCG to induce final follicular maturation (e.g., For example, 4,000 to 11,000 IU hCG, for instance 5,000 IU hCG, 10 ,000 IU hCG, etc.; or 150 to 350 micrograms recombinant hCG, for example It is intended for administration before administering 250 micrograms of recombinant hCG.

[0037] The product may be intended for administration at a frequency more than once a day (or less), in which case It is understood that the relevant dose is equal to the (daily) dose specified herein.

[0038] In this specification, the term "infertility treatment" refers to treatment by controlled ovarian stimulation (COS) or controlled eggs. Infertility treatment by methods including steps or stages of focal stimulation (COS), such as intrauterine fertilization. This includes artificial insemination (IUI), in vitro fertilization (IVF), or intracytoplasmic sperm injection (ICSI). The term "infertility treatment" refers to treatment involving ovulation induction (OI) or steps of ovulation induction (OI). This includes infertility treatment by methods including or involving stages. The term "infertility treatment" refers to treatment of the fallopian tubes or root canal. This includes infertility treatment for subjects with infertility of unknown cause, including endometriosis, e.g., stage I Or subjects with stage II endometriosis and / or anovulatory infertility, for example WHO subjects with type II anovulatory infertility and / or male infertility The treatment of infertility in subjects with endometriosis. The product (or composition) is used to treat endometriosis. The American Society of Endometriosis, which deals with the various stages of endometriosis, is a subject of interest. iety for Reproductive Medicine (ASRM) classification system [ American Society for Reproductive Medicine ne.Revised American Society for Reproduc tive medicine classification of endometr iosis:1996.Fertil Steril 1997;67,817 821 .] (Stage IV most severe; Stage I mildest) as defined, for example Stage I Or infertility treatment in subjects with stage II endometriosis (and / or controlled eggs) It may be for use in (necrotic stimulation).

[0039] The product (composition) is 1 to 16 IU / L, for example, 1 to 12 IU, during the early follicular phase. Infertility treatment (and / or ovarian regulation) in subjects with normal serum FSH levels of / L It may be for use (in the context of) stimulation.

[0040] The product (composition) is used to treat infertility in subjects aged 18 to 42 years, for example, 25 to 37 years. May be used for the treatment of (and / or for controlled ovarian stimulation). This includes BMI > 1 and BMI < 35 kg / m². 2 Subjects with a BMI of 18 and BMI < 25 kg / m² 2 Subjects with BMI > 20 and BMI < 25 kg / m², for example. 2 Infertility treatment (and / or controlled ovarian stimulation) in subjects having (use in It may be used for ) purposes.

[0041] rFSH prefers a trisialylated glycan structure with 27-33%, for example, 30-32%. It may contain rFSH, which is 24-33%, for example, 26-30% of the diciallylated glycan structure. It may preferably contain rFSH at 12-21%, for example, 15-17% monosiallylated rFSH. It may preferably contain a lycan structure. rFSH may be monosiallylated, diciallylated, or tricyanallylated. Lilylated and tetrasiallylated glycan structures in the following relative amounts: 15 to 17% monosially Disialylation; 26-30% Disialylation; 27-33% (e.g., 29-32%, e.g., 30-) 32% (e.g., 30-31%) tri-sialylation and 17-23% tetrasialylation Preferably includes (for example, by WAX analysis of the charged glycan described in the examples) (As shown). rFSH ranges from 0 to 7%, for example from 0.1 to 7%, for example from 3%. It may contain 6%, for example, 5 to 6%, of neutral sialylated structures. FSH is (FSH glycoprotein Contains glycans (adhering to the material). In this specification, the term "X% mono-sialylated" "X% di-sialylation", "X% tri-sialylation", or "X% tetra-sialylation" These are (each) mono-, di-, tri-, or tetra-sialylated glycosides on FSH. This refers to the number of n structures on FSH that have been sialylated (possessing sialic acid) in any way. It is expressed as a percentage (X%) of the total number of glycan structures. Therefore, the phrase "27-33% The "ly-sialylated glycan structure" has a sialic acid residue (i.e., it is sialylated) (t) Of the total number of glycan structures on FSH, 27-33% of these glycan structures are tri This means that it is sialylated (having three sialic acid residues).

[0042] rFSH is expressed as the ratio of moles of sialic acid to moles of protein. 6 mol / mol or more, for example, between 6 mol / mol and 15 mol / mol, for example, 8 Between mol / mol and 14 mol / mol, for example, from 10 mol / mol to 14 mol Between / mol, for example, between 11 mol / mol and 14 mol / mol, for example, 12 mo Between l / mol and 14 mol / mol, for example, between 12 mol / mol and 13 mol / m It may have a sialic acid content between ol. rFSH is produced in human cell lines. or it may be expressed.

[0043] FSH (rFSH) for use according to the present invention has 1% to 99% of total sialylation being α 2,3-Sialylation is acceptable. rFSH has α2,3- sialylation accounting for more than 10% of total sialylation. It can be sialylation. For example, total sialylation of 20, 30, 40, 50, 60, 70, 80 or 90% or more may be α2,3-sialylated. rFSH is total sialylated. 50 to 70% of total sialylation, for example, 60 to 69% of total sialylation, for example, total sialylation Preferably contains α2,3-sialylation in an amount of 63 to 67%, for example, about 65%. See. FSH (rFSH) for use according to the present invention is 1% to 99% of total sialylation. % may be α2,6-sialylated. The rFSH (or rFSH preparation) of the present invention is More than 5% of the total sialylation, for example, 5% to 99%, may be α2,6-sialylation. . In rFSH, less than 50% of the total sialylation may be α2,6-sialylation. H accounts for 25 to 50% of the total sialylation, for example, 30 to 50% of the total sialylation. For example, 31 to 38%, or approximately 35% of total sialylation, in terms of α2,6-sialylation. It may preferably contain the following. Siarylation is the process of modifying the sialic acid residue present on the FSH carbohydrate structure. It means quantity. α2,3-Sialylation is sialylation at the 2,3 position (in this technical field) As is well known, and α2,6 sialylation means that at the 2,6 position (similarly, this technique (This is well known in the field of technology.) Therefore, "the percentage of total sialylation is α2,3 sialylation." "It may be" refers to the total number of sialic acid residues present in FSH that are sialylated at positions 2 and 3. It means a percentage of a number. The term "percentage of total sialylation is α2,6-sialylation" means 2,6 This represents the percentage of the total number of sialic acid residues present in FSH that is sialylated at the 11th position.

[0044] rFSH is 6% or more by mass (for example, between 6% and 15%, or between 7% and 13%). For example, between 8% and 12%, for example between 11% and 15%, for example between 12% and 14% (Based on the mass of protein rather than the combined mass of protein and carbohydrates) It may have an alic acid content (amount of sialylation per FSH molecule).

[0045] rFSH is bisecting glycans if less than 16% (e.g., 0.1 to 16%) of the glycans. N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc) It may be rFSH or an rFSH preparation containing (for example, possessing) r. Preferably, r FSH (or rFSH preparations) contain 8 to 14.5% bisecting N of glycans. - Acetylglucosamine (bisecting GlcNAc or bisGlcNAc) It contains (for example, possesses) rFSH or an rFSH preparation.

[0046] It is understood that FSH contains glycans attached to the FSH glycoprotein. 100% of the glycans refers to or means all glycans attached to the FSH glycoprotein. It is understood that this is the case. Therefore, in this specification, the term "glycans 8 to 14.5" is used. "Contains (or holds) bisecting N-acetylglucosamine" means FSH glycotan 8 to 14.5% of the total number of glycans attached to the protein are bisecting N-acetyl This means that it contains / possesses luglucosamine; "less than 16% of glycans is bisected." "Containing (possessing) N-acetylglucosamine" is attached to FSH glycoprotein. Less than 16% of the total number of glycans present contains bisecting N-acetylglucosamine. It means to possess, etc.

[0047] The applicants have found that glycans in FSH glycoproteins make up less than 16% (e.g., 8-14%) of the total glycans contained in FSH glycoproteins. 0.5%) recombinant FSH (rFSH preparations) containing bisecting GlcNac; We found that FSH compositions may have advantageous pharmacokinetic properties. The amount of glycans containing bisecting GlcNac is the amount of human urine-derived product Brav Other recombinations, such as those disclosed in elle (WO2012 / 017058, etc.) This is thought to be possible because it is similar to (somewhat less than) that of FSH preparations. ru.

[0048] rFSH (or rFSH preparations) contains glycans in which more than 20% are N-acetylgalactose. For example, 20% or more of glycans containing (e.g., galnac) are terminal It may be rFSH or an rFSH preparation containing (e.g., possessing) GalNAc. In other words, rFSH (or rFSH preparations) make up 40 to 55% of the glycan, for example, 4 FSH or FSH preparations containing 2% to 52% GalNAc (e.g., retaining GalNAc) Preferably, rFSH (or rFSH preparation) is 40 to 55% of the glycan, e.g. For example, 42% to 52% contain terminal galNAc (e.g., retain FSH or FSH) It is a prepared product.

[0049] It is understood that FSH contains glycans attached to the FSH glycoprotein. 100% of the glycans refers to or means all glycans attached to the FSH glycoprotein. It is understood that this is the case. Therefore, in this specification, the term "20% or more of the glycan is G "Contains (possesses) alNAc" refers to the total number of glycans attached to the FSH glycoprotein. This means that more than 20% of it contains / possesses N-acetylglucosamine (GalNAc). shi; "40 to 55% of glycans, for example 42 to 52%, contain terminal GalNAc ( For example, "possessing" is the total number of glycans attached to the FSH glycoprotein from 40 to 5. 5% means, for example, 42% to 52% contain / possess terminal galNAc.

[0050] The ability to utilize α2,6-links is different from that of CHO cell-derived products that only have the ability to utilize α2,3-links. Compared to this, it is thought that the number of tetrasialized structures will increase. The applicants believe that this r FSH is not approved for other purposes due to its sugar composition (containing or potentially containing a specific amount of GalNAc). It was also found that the product could be distinguished from the product that was produced. This is because the 2,6-sialylation is with GalNAc. Because of this relationship, it may be related to tetrasialization and potency. In other words, The applicants have developed a material with specific features (2,6-linker moiety, GalNac) and high sial properties. It is believed that rFSH with a certain degree of fermentation will be provided, resulting in improved efficacy in vivo. We developed an rFSH product that can be obtained.

[0051] rFSH (or rFSH preparations) contains 16 to 24% of the glycan (e.g., terminally) 1 fucose-lewis, for example, from glycan 16.5 8% may contain (e.g., terminal) 1-fucose-Lewis. rFSH (or FSH) The preparation contains 1.5 to 4.5% of glycan, for example 2 to 4%, for example 3.7% ( For example, it may contain (terminal) 2-fucose Lewis. The amount of fucose Lewis affects the potency. It may have an impact.

[0052] rFSH is used in human cell lines, such as Per.C6 cell line, HEK293 cell line, and HT10. It can be generated or expressed in cell lines such as 80. This is to maintain sialylation. For example, the manipulation and regulation of cell growth media may be less critical than known processes. Therefore, the generation method can be simplified (and made more efficient). This method is Compared to the formation of well-known rFSH products, a slightly basic rFSH is produced. It is also more efficient; more acidic rFSH is produced, and the separation / removal of basic FSH is problematic. It is unlikely to become a topic. rFSH is the PER.C6(registered trademark) cell line, PER.C6(registered trademark Produced or expressed in cell lines derived from the specified () or modified PER.C6® cell line. Human cell lines (e.g., PER.C6(registered trademark) cell line, HEK293 cell line, H rFSH, which is produced and expressed in cell lines such as T1080, is endogenous [of the cell line]. Several α2,6-linked sialic acids provided by sialyltransferase activity (Contains α2,6 sialylation), provided by endogenous sialyltransferase activity. It contains several α2,3-linked sialic acids (α2,3 sialylation). The cell line is α2 It may be modified using ,3-sialyltransferase. The cell line is α2,6 - May also be modified using sialyltransferase. Alternatively or additionally rFSH is provided by the endogenous sialyltransferase activity of the cell line. May contain α2,6-linked sialic acid (α2,6 sialization). In this specification, the term "H" "Recombinant FSH derived from human cell lines (for example, engineered human cell lines)" This refers to recombinant FSH that is produced or expressed in recombinant FSH (which is manufactured).

[0053] rFSH is transmitted using α2,3- and / or α2,6-sialyltransferase. It can be produced by using α2,3-sialyltransferase. For example, rFSH can be produced using α2,3-sialyltransferase. It is produced by endogenous sialyltransferase activity. rFSH is provided by endogenous sialyltransferase activity. It may contain α2,6-linked sialic acid (α2,6 sialylation).

[0054] The product may be a pharmaceutical composition. The pharmaceutical composition is for the treatment of infertility. This may include assisted reproductive technology (ART), ovulation induction, or intrauterine insemination (IUI). The drug composition can be used, for example, in medical indications where well-known FSH preparations are used.

[0055] The product or composition can be administered via any route of drug administration, e.g., orally, rectally, parenterally, or transdermally. For example, patch technology, intravenous, intramuscular, subcutaneous, intracisional, For vaginal, intraperitoneal, topical (powder, ointment, or drops), buccal, or nasal spray use. It can be formulated in well-known compositions for this purpose. Typical compositions include, among others, Reming ton's Pharmaceutical Sciences 15th Edition (Matt Publishing Company, 1975), pp. 1405 to 1412 and 1 Pages 461-467, and the National Formulary XIV 14th edition (American Pharmaceutical Association, Aqueous solutions, salts, and preservatives, including non-toxic excipients and buffers, as described in 1975. Contains a pharmaceutically acceptable carrier.

[0056] Examples of suitable aqueous and non-aqueous pharmaceutical carriers, diluents, solvents, or vehicles include water, ethanol, and Polyols (glycerol, propylene glycol and polyethylene glycol) (such as olive oil), carboxymethylcellulose and suitable mixtures thereof, vegetable oil (olive oil, etc.) The present invention includes oils and other substances, as well as injectable organic esters such as ethyl oleate. The substances are not limited to these, but include preservatives, humectants, emulsifiers, surfactants, and dispersants. Additives may also be included. Antimicrobial and antifungal agents are included to prevent the growth of microorganisms. Common examples include m-cresol, benzyl alcohol, parabens, chlorobutanol, and fe Contains ingredients such as sorbic acid and other preservatives. If preservatives are included, benzyl alcohol and phenol are also present. While sulfur and / or m-cresol are preferred, preservatives are by no means limited to these examples. Furthermore, it may be desirable to include isotonic agents such as sugar and sodium chloride. Product or composition, Na + -or K + - Select from a group consisting of salts or combinations thereof. The salt may further contain a pharmaceutically acceptable alkali metal cation. Preferably , salt is Na+ - A salt, such as NaCl or Na2SO4.

[0057] Preferably, the product or composition is recombinant FSH and polysorbate 20, L- One of the buffers methionine, phenol, disodium sulfate, and sodium phosphate or Includes multiple items.

[0058] In some cases, to produce a sustained effect, F is administered via subcutaneous or intramuscular injection. It is desirable to slow down the absorption of SH (and other active ingredients, if present). This can be achieved by using a liquid suspension of poorly water-soluble crystalline or amorphous materials. The absorption rate of sea urchin FSH depends on the dissolution rate, and similarly on the crystal size and morphology. This may occur. Alternatively, delayed absorption of parenterally administered FSH combination forms may be beneficial for FSH combination This is achieved by dissolving or suspending the substance in an oil vehicle. The Paw form is a microencapsulation matrix of FSH (and other drugs, if present). This is produced by forming it in a biodegradable polymer such as polylactic acid-polyglycolide. This is possible, depending on the ratio of FSH to polymer and the properties of the specific polymer used. This allows for the regulation of the FSH release rate. Other examples of biodegradable polymers include polyvinyl chloride. Contains lupyrolidone, poly(orthoester), poly(acid anhydride), etc. Manufactured for depot injection. The agent encapsulates FSH in liposomes or microemulsions that are compatible with body tissues. It can also be prepared by doing so.

[0059] Injectable preparations are sterilized, for example, by filtration through a bacterial-retaining filter, or immediately before use. A sterile agent that can be dissolved or dispersed in water or other sterile injectable media can be sterilized in the form of a sterile solid composition. The injectable preparation can be provided in any suitable container, for example, a vial, prefilled syringe, injection cartridge, etc.

[0060] The product or composition can be formulated for single use or multiple uses (multiple doses). If the product or composition is formulated for multiple uses, it is preferably contains a preservative. When a preservative is included, benzyl alcohol, phenol and / or m -cresol are preferred; however, the preservative is not limited to these examples. The product or composition formulated for single use or multiple uses can further contain a salt containing a pharmaceutically acceptable alkali metal cation selected from the group consisting of Na + - or K + - salts or combinations thereof. Preferably, the salt is a Na - salt, such as NaCl or Na2SO4. +

[0061] The product or composition can be included in a container such as a vial, prefilled cartridge (e.g., for single administration or multiple uses) or an injection device such as a "pen" (e.g., for multiple-dose administration).

[0062] The product or composition can be a preparation (e.g., an injectable preparation) containing FSH (optionally together with hCG, LH, LH activity, etc.). The LH activity, if present, can be provided by LH or human chorionic gonadotropin, hCG. If there is more than one active ingredient (i.e., FSH and, for example, hCG or LH), these can be administered separately or together. It may be suitable for [this purpose]. If administered separately, the administration may be sequential. The product is optional. It may be supplied in appropriate packaging. For example, the product may be either FSH or hCG. or a number of containers containing a combination of both FSH and hCG (e.g., prefill syringes) It may contain (di or vial). hCG may be recombinant hCG or urinary hCG. The product contains FSH, for example, multiple containers containing recombinant FSH (e.g., prefill silins). If a vial is included, each container may contain the same amount of FSH. The containers may contain different amounts of FSH. Syringes or vials may be blister-packaged. Alternatively, it may be packaged with other means to maintain sterility. Any product may be FSH(and If present, it may include instructions for using, for example, hCG preparations. The pH and precise concentration of various components of a pharmaceutical composition are routinely determined in the field. It will be adjusted according to convention. GOODMAN and GILMAN's THE PHA RMACOLOGICAL BASIS FOR THERAPEUTICES, 7th edition Please refer to the following. In a preferred embodiment, the composition of the present invention is a composition for parenteral administration and It is supplied as such. General methods for preparing parenteral formulations are well known in the art. REMINGTON; THE SCIENCE AND PRACTICE OF PHARMACY is described on pages 780-820 above. Parenteral compositions are liquid. It may be supplied as a preparation or as a solid to be mixed with a sterile injection medium immediately before administration. Particularly preferred In a more accurate embodiment, the parenteral composition is administered in units for ease of administration and dose uniformity. It is supplied in various forms.

[0063] According to a further embodiment of the present invention, (a) a stool for measuring the serum AMH level of a subject (b) 1-24 μg, e.g., 2-24 μg, e.g., 2-15 μg of human extract A treatment method for infertility that includes the step of administering a dose or equivalent of recombinant FSH Provided. Preferably, the dose is 4.5 to 12.5 μg, for example, 5 to 12.5 μg. For example, is it 6 to 12.5 μg, or for example, 6.3 to 12 μg of human recombinant FSH? , or equal to.

[0064] According to a further embodiment of the present invention, (a) a step for determining the serum AMH level of the subject (b) the step (e.g., measuring step); and (b) < 15 pmol / L (e.g., 0.05 pmol / L). Subjects with serum AMH levels (from ol / L to 14.9 pmol / L) from 9 to 14 μg, for example 11 to 13 μg, for example 12 μg of recombinant human follicle-stimulating hormone Treatment of infertility comprising the step of administering a dose or equivalent of (FSH) (for example, daily), A method is provided. Preferably, the FSH is recombinant FSH ("rFSH" or "rec") The FSH is preferably recombinant FSH derived from a human cell line. The dosage is It provides an effective response while minimizing the risk of OHSS.

[0065] According to a further embodiment of the present invention, (a) a step for determining the serum AMH level of the subject (b) step (for example, step to measure); and (b) serum AMH level ≥ 15 pmol / L The subject having (the disease) is given 5 to 12.5 μg of human recombinant follicle-stimulating hormone (FS) A method of treating infertility comprising the step of administering a (for example, daily) dose or equivalent of H) Provided. (For example, daily) dose is 6 to 10 μg of human recombinant follicle-stimulating hormone. (FSH) may be or may be equal to (FSH). Preferably, FSH is recombinant FSH( It is "rFSH" or "recFSH". Preferably, the FSH is derived from a human cell line. This is recombinant FSH. The dosage provides an effective response while minimizing the risk of OHSS. .

[0066] In one embodiment, the method involves having a serum AMH level of 15 to 24.9 pmol / L (The) target is given 5 to 12 μg, for example 7 to 12 μg, for example 8.7 to 10 μg, Recombinant FSH derived from (preferably 9 to 10 μg of human-derived recombinant FSH) (for example, The method includes the step of administering a daily dose or an equivalent amount. In another embodiment, the method is 25 Subjects with a serum AMH level of 34.9 pmol / L received 5 to 12 μg of human Recombinant FSH derived from (e.g., 7 to 12 μg, e.g., 6 to 9 μg, e.g., 7 to 8 μg) For example, a daily dose (e.g., 7.3 μg to 8 μg) of human recombinant FSH or equivalent. The method includes the step of administering a dose. In another embodiment, the method involves administering serum A ≥35 pmol / L. 5 to 11 μg of human recombinant FSH (e.g., 6 μg) is administered to the subject with MH levels. The daily dose (for example, 6.3 to 7 μg) of human recombinant FSH is 7 μg or more. The procedure includes the step of administering an equal amount.

[0067] According to a further embodiment of the present invention, a) a step to determine the target serum AMH level (e.g., step of measurement); and (b) 0.09 to 0 per kg of the subject's body weight 0.19 μg (e.g., 0.09 to 0.17 μg) of human recombinant FSH (e.g., one The procedure includes administering a dose or equivalent amount, and the subject has a serum AMH level of ≥15 pmol / L. A method for treating infertility is provided, having a certain level of effectiveness. Preferably, the FSH is recombinant FSH. ("rFSH" or "recFSH"). Preferably, the FSH is derived from human cell lines. This is recombinant FSH. The dosage provides an effective response while minimizing the risk of OHSS. ru.

[0068] In one embodiment, the method involves administering 0.14 to 0.19 μg of human glycerin per kg of body weight of the subject. Recombinant FSH (preferably 0.15 to 0.16 μg of human-derived recombinant FSH) For example, the step may include administering a daily dose or an equivalent amount, and the target population is between 15 and 24.9 pm. The subject has a serum AMH level of ol / L. In another embodiment, the method involves the subject's body weight of 1 kg 0.11 to 0.14 μg of human recombinant FSH (preferably 0.12 to 0. Steps involving administering a dose (e.g., daily) or equivalent of 13 μg of human recombinant FSH. The subjects include those with serum AMH levels ranging from 25 to 34.9 pmol / L. Another implementation In terms of morphology, the method involves administering 0.10 to 0.11 μg of human recombinant DNA per kg of body weight of the subject. The step includes administering a dose or equivalent of FSH (e.g., daily), and the subject is ≥35 p.m. The serum AMH level is ol / L. Preferably, the FSH is recombinant FSH ("rFS"). It is either "H" or "recFSH". Preferably, the FSH is recombinant F derived from a human cell line. These are SH doses that provide an effective response while minimizing the risk of OHSS. .

[0069] According to a further embodiment of the present invention, (a) a step for determining the serum AMH level of the subject (b) a step (for example, a step to measure); and (b) 0.15 per kg of the subject's body weight 0.21 μg (e.g., 0.19 to 0.21 μg) of human recombinant FSH (e.g., The step includes administering a daily dose or equivalent, and the subject has serum AM <15 pmol / L. A treatment method for infertility with H levels is provided.

[0070] The administration is a daily dose or one dose of the amount of FSH as defined above and in the claims. It preferably contains the same amount per day. The (daily) dose may be the initial dose (which may be reduced or increased during treatment). (or it may be maintained).

[0071] The method is a treatment method for infertility in the patient's (target's) initial stimulation protocol. For further stimulation cycles, the dosage is adjusted according to the actual ovarian response in the first cycle. It is understood that it can be organized.

[0072] According to a further embodiment of the present invention, (a) a step for determining the serum AMH level of the subject Step (for example, a step to measure); and (b) the subject is <15 pmol / L (for example, from 0.05 pmol / L to 14.9 p) If the subject has a serum AMH level of (mol / L), administer 10 to 14 μg, for example, 11 or For example, a dose of 13 μg, or 12 μg, of human recombinant follicle-stimulating hormone (FSH) or The step is to administer an equal amount; or If the subject has a serum AMH level of 15 to 24.9 pmol / L, the subject's body weight per kilogram 0.14 to 0.19 μg of human recombinant FSH per g (preferably 0.15 to 0 A step of administering a dose or equivalent of 0.16 μg of human recombinant FSH; Taha If the subject has a serum AMH level of 25 to 34.9 pmol / L, 0.11 to 0.14 μg of human recombinant FSH (preferably 0) per kg of elephant body weight The target dose is 0.12 to 0.13 μg of human recombinant FSH, or an equivalent dose. Step; or If the subject has a serum AMH level of ≥35 pmol / L, the subject's body weight 1 The target dose or equivalent of 0.10 to 0.11 μg of human recombinant FSH per kg. A method for treating infertility is provided, which includes the step of administering a drug to the patient.

[0073] For patients (subjects) with serum AMH levels of 5.0 to 14.9 pmol / L, 6 to 1 8 μg, e.g., 8 to 11 μg, e.g., 8.5 to 10.2 μg of human recombinant FSH The dose or equivalent may be administered. The patient has a serum AMH level of 15.0-29.9 pmol / L. For patients (subjects), 4.8 to 15 μg, for example 6 to 9 μg, for example 6.8 to 8 A dose of 0.5 μg of human recombinant FSH, or an equivalent dose, may be administered. 30-44.9 pm For patients (subjects) with serum AMH of ol / L, the dose should be 3.6 to 12 μg, for example, 4 to A dose or equivalent of 7 μg, for example, 5.1 to 6.8 μg of human recombinant FSH, is administered. For patients (subjects) with serum AMH of 45 pmol / L or higher, 2 to 9 μg. For example, 2.4 to 9 μg (e.g., 3.4 to 5.1 μg) or 2 to 5 μg of human free A dose or equivalent of recombinant FSH may be administered. Serum AMH levels of 5 pmol / L or less are present. For the patient (subject), the dose is 7.2 to 24 μg, for example, 10 to 15 μg, for example, 10. A dose or equivalent of 2 to 13.6 μg of human recombinant FSH may be administered. In this example, 4.8 to 18 μg, for example 6 to 11 μg, for example 6.8 to 10.2 μg A dose or equivalent of human recombinant FSH is administered. Preferably, the FSH is recombinant FSH ("rFSH" or "recFSH") is preferred. It is recombinant FSH derived from the cell line. The administration is F as defined above and in the claims. The daily dose or equivalent of SH is preferably included. The (daily) dose is the initial dose. Good (may be reduced, increased, or maintained during treatment).

[0074] The present invention is described in more detail herein with reference to the accompanying drawings. [Brief explanation of the drawing]

[0075] [Figure 1] This figure shows the plasmid map of the pFSH alpha / beta expression vector. [Figure 2] This figure shows the α2,3-sialyltransferase (ST3GAL4) expression vector. [Figure 3] This figure shows an α2,6-sialyltransferase (ST6GAL1) expression vector. [Figure 4] This figure shows the percentage abundance of sialic acid distribution in an example of recombinant FSH produced by PER.C6® cells that stably express FSH after engineering manipulation with α2,3-sialyltransferase. [Figure 5] This figure shows the percentage abundance of glycan charge distribution in an example of recombinant FSH produced by PER.C6® cells that stably express FSH after engineering manipulation with α2,3-sialyltransferase. [Figure 6] This figure shows a comparison of inhibin-B concentrations after administration of 225 IU of Gonal f (underlined, dotted line) and 225 IU of the present invention example (overlined, solid line). [Figure 7] This figure shows the effect of body weight on oocytes collected in the low AMH treatment group (Examples 10, 10A). [Figure 8]This figure shows the effect of body weight on oocytes collected in the high-AMH treatment group. [Modes for carrying out the invention]

[0076] Array selection Human FSH According to Fiddes and Goodman (1981), FSH alpha polypeptide The coding region of the gene for [the gene] was used. The sequence was deposited as AH007338. At the time of construction, no other variants of this protein sequence existed. The sequence is referred to herein by sequence number. It is referred to as No. 1.

[0077] According to Keene et al. (1989), the gene for FSH beta polypeptide A data region was used. The sequence was deposited as NM_000510, and this was used during construction. No other variants of the protein sequence were found. The sequence is referred to herein as Sequence ID 2.

[0078] sialyltransferase α2,3-Sialyltransferase-Kitagawa and Paulson(1 Beta-galactoside alpha-2,3-sialyltransferase according to 994) The coding of the gene for 4 (α2,3-sialyltransferase, ST3GAL4) The do region was used. The sequence was deposited as L23767, and is referred to herein as sequence number 3. It is called that.

[0079] α2,6-Sialyltransferase - According to Grundmann et al. (1990) Beta-galactosamide alpha-2,6-sialyltransferase 1(α2,6 -The coding region of the gene for sialyltransferase (ST6GAL1) is used. It was deposited under the accession number NM_003032 and is referred to herein as SEQ ID NO:4 and so on.

Example

[0080] [Example 1] Construction of FSH expression vector The coding sequences of the FSH alpha polypeptide (AH007338, SEQ ID NO:1) and the FSH beta polypeptide (NM_003032, SEQ ID NO:2) were amplified by PCR using primer pairs FSHa-fw and FSHa-rev and FSHb-fw and FSHb-rec, respectively. The amplified FSH beta DNA obtained was digested with the restriction enzymes AscI and HpaI and inserted into the AscI and HpaI sites of a CMV promoter mammalian expression vector carrying a neomycin selection marker. Similarly, the FSH alpha DNA was digested with BamHI and NheI and inserted into the BamHI and NheI sites on an expression vector already containing the FSH beta polypeptide DNA. FSHa-fw 5'-CCAGGATCCGCCACCATGGATTACTACAGAAAAATATGC-3' (SEQ ID NO:9) FSHa-rev 5'-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3' (SEQ ID NO:10) FSHb-fw 5'-CCAGGCGCGCCACCATGAAGACACTCCAGTTTTTC-3' (SEQ ID NO:11) FSHb-rev 5'-CCGGGTTAACTTATTATTCTTTCATTTCACCAAAGG-3' (SEQ ID NO:12)

[0081] The amplified FSH beta DNA obtained was digested with the restriction enzymes AscI and HpaI and inserted into the AscI and HpaI sites of a CMV promoter mammalian expression vector carrying a neomycin selection marker. Similarly, the FSH alpha DNA was digested with BamHI and NheI and inserted into the BamHI and NheI sites on an expression vector already containing the FSH beta polypeptide DNA.

[0082] The vector DNA was used to transform the DHα strain of Escherichia coli (E. coli). ​​​​Colonies were taken for amplification. Beta containing both FSH alpha and beta Colonies containing the tar were selected for sequencing, and all of them were sequence number 1 and sequence number 2. It contained the exact sequence according to No. 2. Plasmid pFSH A+B#17 was transfused. This was chosen for the following reason (Figure 1).

[0083] [Example 2] Construction of ST3 expression vector Beta-galactoside alpha-2,3-sialyltransferase 4 (ST3, L The code sequence of 23767 (sequence number 3) is a pair of 2,3STfw and 2,3STrev The material was amplified by PCR using an imer combination. 2,3STfw 5'-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3'(Sequence ID 13) 2,3STrev 5'-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3'(Sequence ID 14)

[0084] The resulting amplified ST3DNA was digested with restriction enzymes BamHI and AflII, and hygroscopic DNA was obtained. BamHI on CMV-promoting mammalian expression vectors possessing mycin resistance markers It was inserted into the AflII region. The vector was amplified and sequenced as previously described. Loan pST3#1 (Figure 2) contains the exact sequence according to Sequence ID No. 3, and is a translocation. I chose it for that reason.

[0085] [Example 3] Construction of ST6 expression vector Beta-galactosamide alpha-2,6-sialyltransferase 1 (ST6, The code sequence for NM_003032 (sequence number 4) is 2,6STfw and 2,6STre The sample was amplified by PCR using primer combination v. 2,6STfw 5'-CCAGGATCCGCCACCATGATTCACACCAACCTGAAG-3'(Sequence ID 15) 2,6STrev 5'-TTTTTTTCTTAAGTTAGCAGTGAATGGTCCGG-3'(Sequence ID 16)

[0086] The resulting amplified ST6 DNA was digested with restriction enzymes BamHI and AflII, and hygroscopic DNA was obtained. BamHI on CMV-promoting mammalian expression vectors possessing mycin resistance markers It was inserted into the AflII region. The vector was amplified and sequenced as previously described. Lone pST6#11 (Figure 3) contains the exact sequence according to Sequence ID No. 4, and translocation I chose it for that reason.

[0087] [Example 4] Stable expression of pFSHα+β in PER.C6(registered trademark) cells. Transfusion isolation and Clone screening. A PER.C6(registered trademark) clone that generates FSH was created from a single plasmid. This was produced by expressing both polypeptide chains of SH (see Example 1). .

[0088] Liposome-based translocation agent containing pFSHα+β construct for obtaining stable clones Stable clones were selected from VPRO containing G418 and supplemented with 10% FCS. Three weeks after transduction, G418-resistant clones were developed. Clones were selected for isolation. The isolated clones were cultured in selective medium until 70-80% confluence. The supernatant was then used. The FSH protein content was determined using FSH-selective ELISA, and the clones were also analyzed. The pharmacological activity of the FSH receptor in invasive cell lines was assessed using a cAMP accumulation assay. We selected. Clones expressing the functional protein were transferred to 24-well, 6 well and T80 flasks for culture expansion.

[0089] Research to determine the productivity and quality of materials from 7 clones was initiated in T80 flasks to produce sufficient materials. The cells were cultured in the previously described supplemented medium for 7 days, and the supernatant was collected. Productivity was determined using FSH-selective ELISA. The isoelectric point profile of the materials was determined by isoelectric focusing electrophoresis (IEF) by a method well-known in the art. Clones with sufficient productivity and quality were selected for engineering the sialyltransferase. The cells were cultured in the previously described supplemented medium for 7 days, and the supernatant was collected. Productivity was determined using FSH-selective ELISA. The isoelectric point profile of the materials was determined by isoelectric focusing electrophoresis (IEF) by a method well-known in the art. Clones with sufficient productivity and quality were selected for engineering the sialyltransferase. The cells were cultured in the previously described supplemented medium for 7 days, and the supernatant was collected. Productivity was determined using FSH-selective ELISA. The isoelectric point profile of the materials was determined by isoelectric focusing electrophoresis (IEF) by a method well-known in the art. Clones with sufficient productivity and quality were selected for engineering the sialyltransferase. The isoelectric point profile of the materials was determined by isoelectric focusing electrophoresis (IEF) by a method well-known in the art. Clones with sufficient productivity and quality were selected for engineering the sialyltransferase.

[0090] [Example 5] The level of sialylation is increased in cells overexpressing α2,3-sialyltransferase. Stable expression of pST3 in FSH-expressing PER.C6® cells; transfection isolation and clone screening. PER.C6® clones producing highly sialylated FSH were generated by expressing α2,3-sialyltransferase from another plasmid (Example 2) in PER.C6® cells (from Example 4) that already express both polypeptide chains of FSH. Clones generated from PER.C6® cells as described in Example 4 were selected for their productivity, good growth profile, production of functional protein, and their characteristics including generated FSH with some sialylation. Stable clones were prepared as already described in Example 4. The clones were isolated and grown. PER.C6® clones producing highly sialylated FSH were generated by expressing α2,3-sialyltransferase from another plasmid (Example 2) in PER.C6® cells (from Example 4) that already express both polypeptide chains of FSH. PER.C6® clones producing highly sialylated FSH were generated by expressing α2,3-sialyltransferase from another plasmid (Example 2) in PER.C6® cells (from Example 4) that already express both polypeptide chains of FSH. [[ID=三十二]]PER.C6® clones producing highly sialylated FSH were generated by expressing α2,3-sialyltransferase from another plasmid (Example 2) in PER.C6® cells (from Example 4) that already express both polypeptide chains of FSH. as described in Example 4. Clones generated from PER.C6® cells as described in Example 4 were selected for their productivity, good growth profile, production of functional protein, and their characteristics including generated FSH with some sialylation. Stable clones were prepared as already described in Example 4. The clones were isolated and grown. as described in Example 4. Clones generated from PER.C6® cells as described in Example 4 were selected for their productivity, good growth profile, production of functional protein, and their characteristics including generated FSH with some sialylation. Stable clones were prepared as already described in Example 4. The clones were isolated and grown. as described in Example 4. Clones generated from PER.C6® cells as described in Example 4 were selected for their productivity, good growth profile, production of functional protein, and their characteristics including generated FSH with some sialylation. Stable clones were prepared as already described in Example 4. The clones were isolated and grown. Stable clones were prepared as already described in Example 4. The clones were isolated and grown. ​​The assay was performed. α2,3-sialyltransferase clones were tested in serum-free medium and suspended. It was adapted to turbidity conditions.

[0091] As mentioned above, the clones were subjected to FSH-selective ELISA and functional evaluation in FSH receptor cell lines. Answer: Using IEF, metabolic clearance rate and Steelman-Pohley analysis, The results were obtained using commercially available recombinant FSH (Gonal-f, Serono). And compared with the parental FSH PER.C6® cell line. Most clones The FSH produced is expressed without α2,3-sialyltransferase. It had significantly improved sialylation compared to SH (i.e., generally, a large number of sialylations) (More FSH isoforms that contain acid). In conclusion, PER.C6(registered trademark) FSH expression accompanied by sialyltransferase in cells is equivalent to the expression of FSH alone. This results in increased sialylated FSH levels compared to cells.

[0092] [Example 6] Overview of Production and Purification FSH is produced in PER.C6(registered trademark) cells cultured in suspension in serum-free medium. We developed a procedure for this purpose. The procedure is described below, and several FSH-generating PER.C6( (Registered trademark) Applicable to cell lines.

[0093] FSH was described by Lowry et al. (1976) from α2,3-clones (Example 5). The preparation was made using a modified version of the method described.

[0094] For the production of PER.C6(registered trademark)-FSH, the cell line is placed in serum-free medium, i.e., E The cells were initially adapted to xcell 525 (JRH Biosciences). The cultures were grown in 80°C culture flasks to form a 70%–90% confluent monolayer. Subculturing was performed. Then, the cells were placed in serum-free medium, Excell 525 + 4 mM L-glutamine, and the cell density was measured. cell 0.3×10 6 The cells were resuspended at a concentration of cells / ml. 25 ml of cell suspension was placed in a 250 ml shaking flask. The cells were placed in a container and shaken at 100 rpm, 37°C, and 5% CO2. Cell density > 1x10 cells 6 pieces / m After reaching l, the cell density is reduced to 0.2 or 0.3 × 10⁻¹⁶ cells. 6 Subculture at a rate of cells / ml The samples were further cultured in a shaking flask at 37°C, 5% CO2, and 100 rpm.

[0095] For FSH production, serum-free culture medium, i.e., PER.C6(registered trademark) cells Proliferate to a very high cell density (typically >10 cells in batch culture). 7 (pieces / ml) Supporting V Cells were transplanted into PRO (JRH Biosciences). The cells were processed using Excel 5. In 25 cells, the first cell > 1x10 6 Culture until cells / ml, then at 1000 rpm for 5 minutes. Centrifugation, then density of cells 1x10 in VPRO medium + 6 mM L-glutamine. 6 pieces / ml The cells were then suspended in a shaking flask for 7-10 days at 37°C, 5% CO2, and 10 The cells were cultured at 0 rpm. During this period, the cell density was increased to 10. 7 The cells were grown to a concentration of cells / ml. The culture medium was collected after the cell viability began to decline. The cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was used for FSH quantification and purification. The FSH concentration was measured using ELISA (D The determination was made using RG EIA 1288).

[0096] Therefore, the purification of FSH is performed using the method described by Lowry et al. (1976). The method was modified and used in the implementation. Purification using charge-selective chromatography is used in this technical field. This was carried out to concentrate highly sialized forms using well-known methods.

[0097] During all chromatography procedures, the sial of FSH claimed herein The enrichment of the ionized form is performed by RIA (DRG EIA 1288) and / or IEF. I confirmed it.

[0098] [Example 7] Quantification of the relative amounts of α2,3 and α2,6 sialic acids The relative percentage amounts of α2,3 and α2,6 sialic acid on purified rFSH (Example 6) The measurement was performed using intelligence technology.

[0099] PNGase F was used under denaturing conditions to release N-glycans from the sample, and then 2- It was labeled with aminobenzamide. Then the free glycan form was separated and the charge distribution was measured. For analysis, a weak anion exchange (WAX) column was used. For the measurement of total sialic acid, 2 For the measurement of 2,3-sialic acid, treatment with 3,6,8-sialidase was performed. The labeled glycans were further analyzed using a wax column.

[0100] The relative percentage of charged glycans is calculated from the structure present in the undigested and digested glycan pools. And as shown in Figure 4 (for 8 samples). These showed 50% or more α2,3 sialylation. For 70% (e.g., about 60% or 65%) and α2,6 sialylation, see 28. It is generally seen to be in the range of 50%, or generally between 30 and 35% (for example, about 31% or 35%). I took it out.

[0101] [Example 8] Quantification of the relative structural amounts of mono, di, tri, and tetrasiallylated glycans Mono, di, tri, and tetracyan glycans extracted from purified rFSH (Example 6) The relative percentage of the lylized structure was measured using well-known techniques.

[0102] PNGase F was used under denaturing conditions to release N-glycan from the sample, and then 2-A The glycans were labeled with minobenzamide. Under denaturation conditions, the glycans were processed using PNGase F. The free glycan was then released and labeled with 2-aminobenzamide. The free glycan form was then separated. Furthermore, the sialylation distribution was analyzed using a weak anion exchange (WAX) column. Neutral, mono-sialylation, di-sialylation, tri-sialylation, and tetra-sialylation The relative quantities of the structures are shown in Figure 5 (Figure 4 shows the quantities for the eight samples).

[0103] rFSH is neutral, mono-siallylated, di-siallylated, tri-siallylated, and tetra The sialylated glycan structure is divided into the following relative amounts: neutral 5-6%; mono-sialyl 15-17%. Compounding; 26-30% di-sialylation; 30-32% tri-sialylation and 17-23% tetralysis It is included by trans-sialylation.

[0104] [Example 8a] Purified rFSH (produced by the method in Example 6) was extracted from 9 samples. The relative percentage of α2,6-sialic acid on FSH was measured using a well-known technique.

[0105] PNGase F was used under denaturing conditions to release N-glycans from the sample, and then 2- It was labeled with aminobenzamide. The free glycan form was then separated and the charge distribution was measured. For analysis, a weak anion exchange (WAX) column was used. For the measurement of total sialic acid, 2 3,6,8-sialidase is used to measure 2,3-sialic acid, which is labeled with 2,3-sialidase. The lycan was processed and further analyzed by a wax column (see Example 8). Analysis enables the calculation of α2,6-sialic acid.

[0106] The relative percentage of charged glycans is calculated from the structure present in the undigested and digested glycan pools. These are shown in the following table. For α2,6 sialylation, these range from 25 to 50%, and generally 3. It was found that the range was between 0 and 35%.

[0107] Glycan extracted from 9 purified rFSH samples (produced by the method of Example 6) The relative percentages of bisecting GlcNac, GalNAc, and 1-Fucose Lewis. The percentage was measured using a well-known technique. PNGase F was used to convert N-glycans into glycoproteins. The substance was released from the chlorine and labeled with 2-aminobenzamide (2AB). Analysis was performed using two-dimensional (2) spectroscopy. D) HPLC analysis was performed in combination with enzymatic glycan degradation. For verification, glycan was used. Analysis was performed by MALDI-MS. The relative amount of alpha-2,6-sialic acid and terminal residues were analyzed. The following table shows Gonal F (CHO cell-derived recombinant FSH) and Bravelle ( This is shown along with information on human urinary FSH.

[0108] JPEG2026113577000002.jpg110155

[0109] The amount of GalNAc in FSH according to the present invention varies between approximately 44.9% and 51%. It can be observed that the average is approximately 47.1%.

[0110] The amount of bisecting GlcNac in FSH according to the present invention is 8.7 to 13.9% It can be observed that it varies between these ranges, with an average of approximately 10.9%.

[0111] The amount of 1 fucose Lewis in the FSH of this invention varies between 16.1% and 23.3%. And it can be acknowledged that the average is approximately 19%.

[0112] The amount of 2-fucose Lewis in the FSH of the present invention varies between 1.9 and 4.4%. It can be observed that the average is approximately 3.7%.

[0113] [Example 9] The safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity of FE999049 were investigated using GONAL- Multiple dose studies to investigate in comparison with F Target population for the survey FE999049 (composition according to the present invention, produced by Example 6) with a daily dose of 14.6 μg. A total of 48 people (for each drug) received either (or 16.5 μg of Gonal-F for 7 days) 24 healthy women.

[0114] safety results Multiple doses of FE999049 and GONAL-F are safe and do not cause adverse events (AEs). s) Generally good, as assessed by vital signs, ECG, clinical laboratory measurements, and physical examination. It was well tolerated. No serious adverse events or deaths occurred during the study.

[0115] Pharmacokinetic results After 7 days of administration of FE999049 and GONAL-F, evaluate immediately before the next injection. The FSH concentration levels increased, and it was thought that they reached a steady state level after 6-7 days. However, the exposure (AUC and Cmax) of FE999049 is 6 compared to Gonal-F. It was 0% higher.

[0116] Pharmacodynamic results Inhibin-B (see Figure 6), estradiol and progesterone concentrations The degree increased in all cases after administration of FE999049 and GONAL-F, but GONAL- The degree was greater after administration of FE999049 compared to F. (Number and size of follicles) Both distributions showed a greater response for FE999049 compared to GONAL-F. .

[0117] Example 9 involves a specific amount (17-23%) of the tetra-sialylated glycan structure, as well as For example, FSH having specific amounts of α2,3 sialylation and α2,6 sialylation is currently This demonstrates that it is significantly more effective than commercially available recombinant FSH products.

[0118] [Example 10] A multi-dose study investigating FE999049 compared to GONAL-F. Below is a description of controlled ovarian stimulation for in vitro fertilization (IVF) / intracytoplasmic sperm injection (ICSI). A randomized controlled assessment to evaluate the dose-response relationship of FE999049 in patients receiving this treatment. This document describes a blinded, parallel-group, multinational, multicenter clinical trial. The patient population consisted of 265 IVF patients, aged 18 years. From age 37, BMI 18.5 to 32.0 kg / m² 2 That was the case. The clinical trial was designed as a dose-response trial with the number of oocytes collected as the primary endpoint. Secondary endpoints included endocrine profile, follicular development, oocyte fertilization, embryo quality, and FE9 of various doses regarding therapeutic efficiency (i.e., total gonadotropin use and duration of stimulation) The qualitative and quantitative effects of 99049 will be examined. The clinical trial involves IVF / ICSI cycles. FE999049 is used in controlled ovarian stimulation to establish pregnancy. Designed to evaluate the effectiveness of [the system / method].

[0119] The target group was selected to increase the homogeneity of the clinical trial population regarding ovarian response, and the FE9 used in the clinical trial was selected. Potential low-responders and over-responders to the doses of 99049 and GONAL-F Selection criteria including anti-Müllerian hormone (AMH) assessment to minimize the number of cases and Eligibility with exclusion criteria was assessed within 3 months prior to randomization. AMH assessment was , AMH Gen-II enzyme linked immunosorbent assay kit (Beckman Coulter, Inc., Webster, T. Measurement was performed using exas. This assay has a lower limit of quantification of 1.1 pmol / L. It can detect AMH concentrations exceeding 0.57 pmol / L.

[0120] On the 2nd to 3rd day of the menstrual cycle, administer 90 IU, 120 IU, 150 IU, 180 IU, or Treatment with either 210 IU FE999049 or 150 IU GONAL-F. For this purpose, participants were randomized to a 1:1:1:1:1:1 ratio, and ovarian stimulation was initiated. Randomization was performed in a 1:1:1:1:1:1 ratio. Stratified by AMH levels during leaning [5.0~14.9 pmol / L (low A MH) and 15.0 to 44.9 pmol / L (high AMH).

[0121] Gonal-F is mass-filled (FbM) as required by the FDA; therefore, the μg dose is... It is appropriate to mention the Gonal-F label, which indicates 600 IU / 44 μg. This indicates that 150 IU is equivalent to 11 μg. However, there is some variation, and in this clinical trial, The test showed that 11.3 μg of Gonal-F was equivalent to 150 IU. FE9 The 99049 dose was expressed by protein content (μg) rather than biological activity. Therefore, the dosage of FE999049 is 5.2 μg (90 IU) and 6.9 μg (120 IU). ), 8.6 μg (150 IU), 10.3 μg (180 IU), or 12.1 μg (21 It was 0 IU.

[0122] [Table 1]

[0123] The daily dose level of FE999049 or GONAL-F should remain constant throughout the entire stimulation period. Determine. The subject will be subjected to stimulation on days 1, 4, and 6, and at least every other day thereafter. Monitor. If three follicles of ≥15mm are observed, daily visits will be conducted. The subjects are FE Treat with 999049 or GONAL-F for up to 16 days.

[0124] To prevent a premature LH surge, GnRH antagonists (ganirelix acetate, ORG) are used. ALUTRAN (MSD / Schering-Plough) was administered on day 6 of stimulation at a daily dose of 0 Treatment may begin with 0.25 mg and continue throughout the stimulation period. Induction of final follicular maturation is performed as follows: Perform the procedure on the day when 3 or more follicles with a diameter of ≥17 mm are observed. If there are <25 units, 250 μg recombinant hCG (human chorionic gonadotropin alpha, OV) Administer ITRELLE (Merck Serono / EMD Serono). ≥1 If there are 25-35 follicles measuring 2mm, 0.2mg GnRH agonist (acetate) Liptorelin, DECAPEPTYL / GONAPEPTYL, Ferring Pha Administer rmaceuticals. This is defined as having >35 follicles with a diameter of ≥12mm. In cases of excessive ovarian response, treatment should be discontinued. Eggs with a diameter of ≥10 mm on day 10 of stimulation. If the ovarian response is poor, defined as <3 follicles being observed, the cycle may be terminated. There is.

[0125] Oocyte retrieval is performed 36 hours (±2 hours) after induction of final follicular maturation, and the oocytes are used for IVF. Fertilization was performed by oocyte collection and / or ICSI. Fertilization and embryo development were carried out from oocyte collection to transplantation. Evaluation will be conducted until [date]. For subjects who underwent induction of final follicular maturation with hCG, the best available One quality blastocyst is transferred 5 days after oocyte collection, and the remaining blastocysts are frozen. GnR For subjects whose final follicular maturation was induced with an H agonist, embryo transfer occurred in the new cycle. Instead of transplanting, the blastocyst is frozen on day 5. one tablets(LUTINUS, Ferring Pharmaceutic als) 100 mg, three times a day, to support the luteal phase, starting from the day of oocyte retrieval, during a clinical pregnancy check-up. Continue giving until [date]. Perform a βhCG test 13-15 days after embryo transfer, and confirm clinical pregnancy [after embryo transfer]. The condition will be checked again in 5-6 weeks using transvaginal ultrasound (TVU).

[0126] result The number of oocytes collected (primary endpoint) is shown in the table below.

[0127] [Table 2]

[0128] The main objectives were aligned. Regarding the number of oocytes collected, the results for FE999049 A remarkable dose-response relationship was established. This finding is significant not only for the entire clinical trial population, but also for the entire population. This was also observed in each of the two AMH strata used in the randomization. FE999049 The significant dose-response relationship for all pharmacokinetic parameters is the main objective, for example This was demonstrated for estradiol, inhibin B, and inhibin A. Similar results were obtained for My At chlorogram dose levels, the pharmacodynamic response of FE999049 is greater than that of GONAL-F. (These results are not described.) Serum FSH concentrations after exposure to FE999049 were significantly higher than those after exposure to GONAL-F. It was expensive. The result was that the PK profile of FE999049 was different from that of GONAL-F. Confirm that it will happen. Fertilization rates, blastocyst development, and pregnancy rates in IVF / ICSI patients treated with FE999049 The pregnancy rate was within the predicted range. No safety concerns were raised regarding the use of FE999049. Good local tolerability was recorded. Ta.

[0129] Further analysis The applicants have found that FE999049 meets the following criteria regarding the number of oocytes to be collected. Further analysis of the data was conducted to identify the appropriate dosage: • The number of oocytes collected ranges from 8 to 14. • Minimize the proportion of patients with <8 oocytes • Minimize the proportion of patients with ≥20 oocytes.

[0130] The applicants also investigated the effect of body weight. Where relevant, the dose was μg for the average subject. This is converted to μg / kg. The μg / kg and ±0.01 μg / kg values ​​are obtained from the oocytes from which the sample was taken. We evaluate the cell distribution and safety profile in the model and identify the optimal dose. ru.

[0131] low AMH layer As shown in Table 2, the dose of FE999049 that meets the first criterion (the amount of oocytes collected) The weight of 8-14 cells was 12.1 μg (average of 9.4 oocytes collected). The distribution of parent cells is shown in Table 3 below.

[0132] [Table 3]

[0133] As indicated by the box and arrows, the dose of 12.1 μg FE999049 is for the low AMH group. This provides the most desirable number of oocytes collected from 60% of the subjects. There was a significant improvement in nal-F (the most desirable number of oocytes, but only 33% of the subjects). ru.

[0134] Table 4 below shows an analysis of signs of overresponse in the low AMH group (data represents the number of subjects). There were no indications for moderate or severe early-stage OHSS, and no occurrences requiring prophylactic treatment. It may be observed that 12.1 μg FE99904 was administered to patients with low AMH. There are no concerns related to dosage 9.

[0135] [Table 4]

[0136] Figure 7 shows the shadow of body weight on oocytes collected for various doses (with respect to the low AMH layer). The arrow indicates the range of subjects weighing between 45 kg and 90 kg treated with a dose of 12.1 μg. The number of oocytes collected is shown. As observed (text box) Weight 45kg The difference between a patient with a weight of 90 kg and a patient with a weight of 90 kg is less than approximately 0.5 oocytes; in other words Therefore, with this dosage, there is no significant change in body weight in the oocytes that are collected, When the dose of 999049 is at least 12 μg, in patients with low AMH Therefore, dosage based on body weight is not necessary.

[0137] Therefore, the applicants have found that 6 to 18 μg, for example 9 to 14 μg, for example 12 μg The dose or equivalent of human recombinant FSH should be <15 pmol / L, e.g., 0.05-14 For patients with serum AMH levels of 0.9 pmol / L, for example, 5.0-14.9 pmol / L It has been found to be suitable for use in infertility treatment. The dosage is important as it reduces the risk of OHSS. To provide an effective response while minimizing the impact.

[0138] High AMH layer As shown in Table 2, the three dosages of FE999049 are the first standard (when the collected oocytes are Meet the criteria of 8-14 cells: 6.9 μg (average 9.1 oocytes collected), 8. 6 μg (average 10.6 oocytes collected) and 10.3 μg (average oocytes collected) average 13.6 pieces).

[0139] Figure 8 shows the body weight shadows on oocytes collected for various doses (with respect to the high AMH layer). The image shows the effects. The arrows indicate the weight of a 4-gram body treated with doses of 6.9 μg, 8.6 μg, and 10.3 μg. This shows the number of oocytes collected from subjects weighing between 5 kg and 90 kg. As observed ( (Text box) The change is significant: Compared to a 90kg patient with a dose of 6.9μg Six additional oocytes are collected from a 45kg patient; 9 for a dose of 8.6μg Compared to a patient weighing 0 kg, a patient weighing 45 kg will yield 4 additional oocytes; dosage Regarding 10.1 μg, patients weighing 45 kg showed 2.5 additional units compared to patients weighing 90 kg. Oocytes are collected. In other words, administration based on body weight is used in patients with high AMH levels. When the dose of 99049 is less than 12 μg, the oocytes collected at these doses are Because it changes significantly with weight, it has an effect.

[0140] Table 5a below shows the further degradation of collected oocytes (from Table 2) by AMH. This is the first criterion for each sublayer of AMH (where 8 to 14 oocytes are retrieved). The dosage that satisfies the requirement is shown (in the box).

[0141] [Table 5a]

[0142] Table 5b below shows that for these subgroups, either an overresponse or agonist induction occurred. This section presents an analysis of patients who discontinued treatment. For example, the 25-34 pmol / L AMH layer. One patient discontinued treatment after a dose of 10.3 μg due to an excessive response, with a dose of 25-34 pmol / LA. One patient in the MH group discontinued treatment after a dose of 12.1 μg due to an excessive response; 35-45 pmol One patient in the / L AMH layer discontinued treatment after agonist induction following a dose of 10.3 μg; 35~ One patient with a 45 pmol / L AMH level discontinued treatment after agonist induction following a dose of 6.9 μg. It was done.

[0143] [Table 5b]

[0144] Therefore, dosage adjustment based on body weight (Figure 8) and AMH levels are important in the high AMH group. This may be useful in minimizing discontinuation and maximizing oocyte collection.

[0145] The applicants have demonstrated that the following doses provide an effective response while minimizing the risk of OHSS. This was found (kg represents the patient's weight in kg).

[0146] [Table 6]

[0147] The following is appropriate when weight-based dosage is not required.

[0148] [Table 7]

[0149] The following is appropriate when a smaller classification of AMH is needed.

[0150] [Table 8]

[0151] The following is appropriate when weight-based dosage is not required.

[0152] [Table 9]

[0153] Therefore, the applicants have found that 9 to 14 μg, for example 12 μg, of human recombinant FSH A dose or equivalent is <15 pmol / L, for example 0.05-14.9 pmol / L, for example Use in infertility treatment for patients with serum AMH levels of 5.0-14.9 pmol / L It was found to be suitable for this purpose. The dosage is effective while minimizing the risk of OHSS. Provide the answer.

[0154] The applicants have developed a human recombinant FS in a dose of 5 to 12.5 μg, for example, 6 to 10.5 μg. Infertility treatment in patients with serum AMH levels of ≥15 pmol / L in doses or equivalents of H It was found to be suitable for use in [specific context]. The dosage minimizes the risk of OHSS. This provides an effective response.

[0155] The applicants have found that 0.09 to 0.19 μg of human recombinant F per kg of patient body weight is sufficient. A dose or equivalent of SH (for example, daily) is given to a serum AMH level of ≥15 pmol / L We found it suitable for use in the treatment of infertility in patients. , 0.14 to 0.19 μg of human recombinant FSH per kg of patient body weight (preferably) This is the (e.g., daily) dose of 0.15 to 0.16 μg of human recombinant FSH or equivalent. Infertility treatment in patients with serum AMH levels of 15 to 24.9 pmol / L We found it suitable for use in medical treatment. The applicants found that per kg of patient body weight 0.11 to 0.14 μg of human recombinant FSH (preferably 0.12 to 0.13 μg) The (e.g., daily) dose or equivalent of g of human recombinant FSH is 25 to 34.9 pg. Suitable for use in infertility treatment in patients with serum AMH levels of mol / L. We have found that the patient's body weight is 0.10 to 0.11 μg A (e.g., daily) dose or equivalent of human recombinant FSH in blood ≥35 pmol / L It has been found to be suitable for use in the treatment of infertility in patients with clear AMH levels. These doses provide an effective response while minimizing the risk of OHSS.

[0156] The applicants have shown that, for example, regarding the initial stimulation cycle with human recombinant FSH, the patient's body 0.15 to 0.21 μg (e.g., 0.16 μg) of human-derived recombinant F per 1 kg of weight A dose or equivalent of SH (for example, daily) is used to control serum AMH levels <15 pmol / L. It was found to be suitable for use in the treatment of infertility in patients. However, this Patients do not need to be administered based on their AMH levels and body weight.

[0157] [Example 10A] Individualized COS protocol (low AMH) The selected patients will undergo in vitro fertilization (IVF) / egg cell regeneration using methods well known in this field. I am about to undergo COS (Cognitive Sperm Preparation) for intra-costal sperm infusion (ICSI). Pre-treatment protocol. This is AMH Gen-II enzyme linked immunosorbent assay kit (Beckman Coulter, Inc., Webster, This assay includes evaluation / screening of patient serum AMH using the Texas assay. AMH concentrations exceeding 0.57 pmol / L have a lower limit of quantification of 1.1 pmol / L. It can be measured using other assay kits (e.g., available from Roche). AMH can be measured using other assay kits. It can be determined.

[0158] Except for the initial dose of FE999049 administered based on AMH levels at screening, CO The S protocol proceeds in the usual manner. AMH levels <14.9 pmol / L Patients with this condition take approximately 12 μg of FE999049 (produced by the method in Example 6). The initial daily dose of recombinant FSH product derived from [unspecified source] is administered. 15 to 24.9 pmol Patients with an AMH level of / L should receive 0.15 to 0.19 μg per kg of body weight. Receive the initial daily dose of human recombinant FSH, ranging from 25 to 34.9 pmol / L. Patients with MH levels should take 0.11 to 0.13 μg of human free meth per kg of body weight. The patient will receive the initial daily dose of recombinant FSH and has an AMH level of ≥35 pmol / L. The patient received an initial dose of human recombinant FSH at a dose of 0.10 to 0.11 μg per kg of body weight. Receive the daily dose.

[0159] [Example 11] Personalized COS Protocol The dose in this protocol is preferably less than that in Example 10A.

[0160] The selected patients will undergo in vitro fertilization (IVF) / egg cell regeneration using methods well known in this field. I am about to undergo COS (Cognitive Sperm Preparation) for intra-costal sperm infusion (ICSI). Pre-treatment protocol. This is AMH Gen-II enzyme linked immunosorbent assay kit (Beckman Coulter, Inc., Webster, This assay includes evaluation / screening of patient serum AMH using the Texas assay. AMH concentrations exceeding 0.57 pmol / L have a lower limit of quantification of 1.1 pmol / L. It can be produced.

[0161] The COS protocol is based on the AMH level at screening, which matches the table below. The procedure proceeds in the usual manner, except for the initial dose of E999049. Therefore, 5-14 For patients with an AMH level of 0.8 pmol / L, approximately 8-1 doses of 180 IU FSH are recommended. 1 μg of FE999049, human-derived recombinant FSH product prepared by the method of Example 6 It is administered in the following form. For patients with AMH levels of 30-44.9 pmol / L, 12 Approximately 4-7 μg of 0 IU FSH was produced using the method of Example 6. Administer in the form of a human-derived recombinant FSH product. If AMH levels are not available, Patients were given 120-180 IU FSH, approximately 6-11 μg of FE999049, Example 6 The product is administered in the form of a human-derived recombinant FSH product manufactured by the method described above.

[0162] [Table 10]

[0163] References Andersen CY, Westergaard LG, and van Wely M. (2004). FSH isoform composition of commercial gonadotrophin preparations: a neglected aspect? Reprod Biomed Online. 9(2) , 231-236. Arey BJ, Stevis PE, Deecher DC, Shen ES, Frail DE, Negro-Vilar A, and Lopez FJ. (1997) Induction of promiscuous G protein coupling of the follicle-stimulating h ormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms. Molecule Endocrinol.11(5) , 517-526. Baenziger JU and Green ED. (1988). Pituitary glycoprotein hormone oligosaccharid es: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. Biochim Biophys Acta. 947(2), 287-306. Bassett RM, and Driebergen R. (2005). Continued improvements in the quality and consistency of follitropin alfa, recombinant human FSH. Reprod Biomed Online. 10 (2), 169-177. Damian-Matsumura P, Zaga V, Maldonado A, Sanchez-Hernandez C, Timossi C, and Ull oa-Aguirre A. (1999). Oestrogens regulate pituitary alpha2,3-sialyltransferase m essenger ribonucleic acid levels in the female rat. J Mol Endocrinol. 23(2), 153 -165. D'Antonio M., Borrelli F. , Datola A., Bucci R. , Mascia M. , Polletta P., Pisci telli D., and Papoian R. (1999) Biological characterization of recombinant human follicle stimulating hormone isoforms. Human Reproduction 14 , 1160-1167 Dalpathado DS, Irungu J, Go EP, Butnev VY, Norton K, Bousfield GR, and Desaire H . (2006). Comparative glycomics of the glycoprotein follicle stimulating hormone : glycopeptide analysis of isolates from two mammalian species. Biochemistry. 45 (28), 8665-8673. No copy Dias JA, Van Roey P. (2001). Structural biology of human follitropin and its rec eptor. Arch Med Res. 32(6), 510-519 Fiddes, J. C. and Goodman, H. M. (1979) Isolation, cloning and sequence analysis of the cDNA for the alpha-subunit of human chorionic gonadotropin. Nature, 281, 351-356. Flack, M.R., Bennet, A.P., Froehlich, J. Anasti, JN and Nisula, B. (1994). Incre ased biological activity due to basic isoforms in recombinant human follicle-sti mulating hormone produced in a human cell line. J. Clin. Endocrinol. Metab., 79 , 756-760 Fox KM, Dias JA, and Van Roey P. (2001). Three-dimensional structure of human fo llicle-stimulating hormone. Mol Endocrinol. 15(3), 378-89 Grabenhorst E, Hoffmann A, Nimtz M, Zettlmeissl G, and Conradt HS. (1995). Const ruction of stable BHK-21 cells coexpressing human secretory glycoproteins and hu man Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase alpha 2,6-linked NeuAc is preferentially attached to the Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)-branc h of diantennary oligosaccharides from secreted recombinant beta-trace protein. Eur J Biochem. 232(3) , 718-25. Green ED and Baenziger JU. (1988). Asparagine-linked oligosaccharides on lutropi n, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated ol igosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J Bi ol Chem. 263(1) , 36-44. Grundmann,U., Nerlich,C., Rein,T. and Zettlmeissl, G. (1990). Complete cDNA sequ ence encoding human beta-galactoside alpha-2,6-sialyltransferase. G Nucleic Acid s Res. 18 (3), 667 Howles, C.M. (1996). Genetic engineering of human FSH (Gonal-F). Hum Reprod. Upd ate, 2 ,172-191. Kagawa Y, Takasaki S, Utsumi J, Hosoi K, Shimizu H, Kochibe N, and Kobata A. (19 88). Comparative study of the asparagine-linked sugar chains of natural human in terferon-beta 1 and recombinant human interferon-beta 1 produced by three differ ent mammalian cells. J Biol Chem. 263(33), 17508-17515. Keene, J.L., Matzuk, M.M., Otani, T., Fauser, B,C,J,M., Galway, A.B., Hsueh, A.J .W. and Boime, I. (1989). Expression of Biologically active Human Follitropin in Chinese Hamster Ovary Cells. The Journal of Biological Chemistry, 264(9) , 4769- 4775. Kitagawa,H. and Paulson,J.C (1994) Cloning of a novel alpha 2,3-sialyltransferas e that sialylates glycoprotein and glycolipid carbohydrate groups. J. Biol. Chem . 269(2) , 1394-1401. Lee EU, Roth J, and Paulson JC (1989) Alteration of terminal glycosylation seque nces on N-linked oligosaccharides of Chinese hamster ovary cells by expression o f beta-galactoside alpha 2,6-sialyltransferase. J Biol Chem. 264(23) , 13848-1385 5. de Leeuw, R., Mulders, J., Voortman, G. Rombout, F. Damm, J. and Kloosterboer, L . (1996) Structure-function relationship of recombinant follicle stimulating hor mone (Puregon). Mol. Hum. Reprod., 2 , 361-369. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. (1951) Protein measurement with th e Folin phenol reagent. J Biol Chem. 193(1) , 265-75. Lowry, PJ, McLean, C, Jones RL and Satgunasingam N. (1976) Purification of anter ior pituitary and hypothalamic hormones Clin Pathol Suppl (Assoc Clin Pathol). 7 , 16-21. Olivennes F, Howles CM, Borini A, Germond M, Trew G, Wikland M, Zegers-Hochschil d F, Saunders H (2009) Individualizing FSH dose for assisted reproduction using a novel algorithm: the CONSORT study. Reprod Biomed Online. 2009 Feb; 18(2): 195 -204. Pierce JG, and Parsons TF (1981) Glycoprotein hormones: structure and function A nnu Rev Biochem. 50, 465-495. Pricer WE Jr, and Ashwell G. (1971). The binding of desialylated glycoproteins b and plasma membranes of rat liver. J Biol Chem. 246(15), 4825-3 Rathnam P, and Saxena BB. (1975). Primary amino acid sequence of follicle-stimulus ating hormone from the human pituitary gland. I. alpha subunit. J Biol Chem.; 250(17 ):6735-6 Regoeczi E, Debanne MT, Hatton MC, Koj A. (1978) Elimination of asialofetuin and Asialoorosomucoid by the intact rat. Quantitative aspects of the hepatic cl earance mechanism. Biochim Biophys Acta. 541(3), 372-8 [ PubMed ] [ Cross Ref ] Royle L, Radcliffe CM, Dwek RA and Rudd PM (2006). ed I Brockhausen-Schutzbach (Humana Press), 347: Glycobiology protocols, 125-144 . . . . [ PMC free article ] [ PubMed ] Ryan RJ, Keutmann HT, Charlesworth MC, McCormick DJ, Milius RP, Calvo FO and Vut Yavanich T. (1987). Structure-function relationships of gonadotropins. Recent Pr and Horm Res.; 43 ,:383-4 Saxena BB and Rathnam P. (1976) Amino acid sequence of the beta subunit of folli cle-stimulating hormone from human pituitary glands. J Biol Chem. 251(4), 993-10 05 Steelman SL, and Pohley FM. (1953) Assay of the follicle stimulating hormone bas ed on the augmentation with human chorionic gonadotropin. Endocrinology. 53(6) , 604-616. Steer CJ, and Ashwell G. (1980) Studies on a mammalian hepatic binding protein s pecific for asialoglycoproteins. Evidence for receptor recycling in isolated rat hepatocytes. J Biol Chem. 255(7), 3008-13. Svensson EC, Soreghan B, and Paulson JC. (1990) Organization of the beta-galacto side alpha 2,6-sialyltransferase gene. Evidence for the transcriptional regulati on of terminal glycosylation. J Biol Chem. 265(34):20863-20868. Takeuchi M, Takasaki S, Miyazaki H, Kato T, Hoshi S, Kochibe N, and Kobata A (19 88). Comparative study of the asparagine-linked sugar chains of human erythropoi etins purified from urine and the culture medium of recombinant Chinese hamster ovary cells. J Biol Chem. 263(8) , 3657-3663. Timossi CM, Barrios de Tomasi J, Zambrano E, Gonzalez R, Ulloa-Aguirre A. (1998) . A naturally occurring basically charged human follicle-stimulating hormone (FS H) variant inhibits FSH-induced androgen aromatization and tissue-type plasminog en activator enzyme activity in vitro. Neuroendocrinology. 67(3), 153-163. Timossi CM, Barrios-de-Tomasi J, Gonzalez-Suarez R, Arranz MC, Padmanabhan V, Co nn PM, and Ulloa-Aguirre A. (2000). Differential effects of the charge variants of human follicle-stimulating hormone. J Endocrinol. 165(2), 193-205. Ulloa-Aguirre, A., Espinoza, R., Damian-Matsumura, P. and Chappel, S.C. (1988) I mmunological and biological potencies of the different molecular species of gona dotrophins. Hum. Reprod. 3 , 491-501. Ulloa-Aguirre, A., Cravioto, A., Damian-Matsumura, P. Jimenez, M, Zambrano, E an d Diaz-Sanchez, V. (1992) Biological characterization of the naturally occurring analogues of intrapituitary human follicle stimulating hormone. Hum. Reprod. 7 , 23-30. Ulloa-Aguirre A, Midgley AR Jr, Beitins IZ, and Padmanabhan V. (1995). Follicle- stimulating isohormones: characterization and physiological relevance. Endocr Re v. 16(6), 765-787. Ulloa-Aguirre A, Maldonado A, Damian-Matsumura P, and Timossi C (2001). Endocrin e regulation of gonadotropin glycosylation. Arch Med Res. 32(6) , 520-532. Ulloa-Aguirre A, Timossi C, Barrios-de-Tomasi J, Maldonado A, and Nayudu P. (200 3). Impact of carbohydrate heterogeneity in function of follicle-stimulating hor mone: studies derived from in vitro and in vivo models. Biol Reprod. 69(2), 379- 389. Van Lenten L, and Ashwell G. (1972) The binding of desialylated glycoproteins by plasma membranes of rat liver. Development of a quantitative inhibition assay. J Biol Chem. 247(14), 4633-40. Wide, L. and Albertsson-Wikland, K. (1990) Change in electrophoretic mobility of human follicle-stimulating hormone in serum after administration of gonadotropi n-releasing hormone. J. Clin. Endocrinol. Metab. 70, 271-276. Wide, L. and Bakos, O. (1993). More basic forms of both human follicle-stimulati ng hormone and luteinizing hormone in serum at midcycle compared with the follic ular or luteal phase. J. Clin. Endocrinol. Metab., 76, 885-889. Wide L, Naessen T, Sundstrom-Poromaa I, Eriksson K. (2007) Sulfonation and sialy lation of gonadotropins in women during the menstrual cycle, after menopause, an d with polycystic ovarian syndrome and in men. J Clin Endocrinol Metab.; 92(11), 4410-4417. Zambrano E, Zarinan T, Olivares A, Barrios-de-Tomasi J, and Ulloa-Aguirre A. (19 99). Receptor binding activity and in vitro biological activity of the human FSH charge isoforms as disclosed by heterologous and homologous assay systems: impl ications for the structure-function relationship of the FSH variants. Endocrine. 10(2), 113-121. Zhang X, Lok SH, and Kon OL (1998) Stable expression of human alpha-2,6-sialyltr ansferase in Chinese hamster ovary cells: functional consequences for human eryt hropoietin expression and bioactivity. Biochim Biophys Acta. 1425(3), 441-452.

[0164] SEQ ID NO: 1 Follicle stimulating hormone alpha polypeptide Accession number AH007338 Nucleotide sequence of FSH alpha 1 ATGGATTACT ACAGAAAATA TGCAGCTATC TTTCTGGTCA CATTGTCGGT GTTTCTGCAT 61 GTTCTCCATT CCGCTCCTGA TGTGCAGGAT TGCCCAGAAT GCACGCTACA GGAAAACCCA 121 TTCTTCTCCC AGCCGGGTGC CCCAATACTT CAGTGCATGG GCTGCTGCTT CTCTAGAGCA 181 TATCCCACTC CACTAAGGTC CAAGAAGACG ATGTTGGTCC AAAAGAACGT CACCTCAGAG 241 TCCACTTGCT GTGTAGCTAA ATCATATAAC AGGGTCACAG TAATGGGGGG TTTCAAAGTG 301 GAGAACCACA CGGCGTGCCA CTGCAGTACT TGTTATTATC ACAAATCTTA A Protein sequence of FSH alpha (SEQ ID NO: 5) 1 MDYYRKYAAI FLVTLSVFLH VLHSAPDVQD CPECTLQENP FFSQPGAPIL QCMGCCFSRA 61 YPTPLRSKKT MLVQKNVTSE STCCVAKSYN RVTVMGGFKV ENHTACHCST CYYHKS SEQ ID NO: 2 Follicle stimulating hormone beta polypeptide Accession number NM_000510 Nucleotide sequence of FSH beta 1 ATGAAGACAC TCCAGTTTTT CTTCCTTTTC TGTTGCTGGA AAGCAATCTG CTGCAATAGC 61 TGTGAGCTGA CCAACATCAC CATTGCAATA GAGAAAGAAG AATGTCGTTT CTGCATAAGC 121 ATCAACACCA CTTGGTGTGC TGGCTACTGC TACACCAGGG ATCTGGTGTA TAAGGACCCA 181 GCCAGGCCCA AAATCCAGAA AACATGTACC TTCAAGGAAC TGGTATATGA AACAGTGAGA 241 GTGCCCGGCT GTGCTCACCA TGCAGATTCC TTGTATACAT ACCCAGTGGC CACCCAGTGT 301 CACTGTGGCA AGTGTGACAG CGACAGCACT GATTGTACTG TGCGAGGCCT GGGGCCCAGC 361 TACTGCTCCT TTGGTGAAAT GAAAGAATAA Protein sequence of FSH beta (SEQ ID NO: 6) 1 MKTLQFFFLF CCWKAICCNS CELTNITIAI EKEECRFCIS INTTWCAGYC YTRDLVYKDP 61 ARPKIQKTCT FKELVYETVR VPGCAHHADS LYTYPVATQC HCGKCDSDST DCTVRGLGPS 121 YCSFGEMKE SEQ ID NO: 3 Beta-galactoside alpha-2,3-sialyltransferase 4 Accession Number L23767 Nucleotide sequence of ST3GAL4 1 ATGTGTCCTG CAGGCTGGAA GCTCCTGGCC ATGTTGGCTC TGGTCCTGGT CGTCATGGTG 61 TGGTATTCCA TCTCCCGGGA AGACAGGTAC ATCGAGCTTT TTTATTTTCC CATCCCAGAG 121 AAGAAGGAGC CGTGCCTCCA GGGTGAGGCA GAGAGCAAGG CCTCTAAGCT CTTTGGCAAC 181 TACTCCCGGG ATCAGCCCAT CTTCCTGCGG CTTGAGGATT ATTTCTGGGT CAAGACGCCA 241 TCTGCTTACG AGCTGCCCTA TGGGACCAAG GGGAGTGAGG ATCTGCTCCT CCGGGTGCTA 301 GCCATCACCA GCTCCTCCAT CCCCAAGAAC ATCCAGAGCC TCAGGTGCCG CCGCTGTGTG 361 GTCGTGGGGA ACGGGCACCG GCTGCGGAAC AGCTCACTGG GAGATGCCAT CAACAAGTAC 421 GATGTGGTCA TCAATTACT CAATGCCCCA GTGGCTGGCT ATGAGGGTGA CGTGGGCTCC 481 AAGACCACCA TGCGTCTCTT CTACCCTGAA TCTGCCCACT TCGACCCCAA AGTAGAAAAC 541 AACCCAGACA CACTCCTCGT CCTGGTAGCT TTCAAGGCAA TGGACTTCCA CTGGATTGAG 601 ACCATCCTGA GTGATAAGAA GCGGGTGCGA AAGGGTTTCT GGAAACAGCC TCCCCTCATC 661 TGGGATGTCA ATCCTAAACA GATTCGGATT CTCAACCCCT TCTTCATGGA GATTGCAGCT 721 GACAAACTGC TGAGCCTGCC AATGCAACAG CCACGGAAGA TTAAGCAGAA GCCCACCACG 781 GGCCTGTTGG CCATCACGCT GGCCCTCCAC CTCTGTGACT TGGTGCACAT TGCCGGCTTT 841 GGCTACCCAG ACGCCTACAA CAAGAAGCAG ACCATTCACT ACTATGAGCA GATCACGCTC 901 AAGTCCATGG CGGGGTCAGG CCATAATGTC TCCCAAGAGG CCCTGGCCAT TAAGCGGATG 961 CTGGAGATGG GAGCTATCAA GAACCTCACG TCCTTCTGA Protein Sequence of ST3GAL4 (SEQ ID NO: 7) 1 MCPAGWKLLA MLALVLVVMV WYSISREDRY IELFYFPIPE KKEPCLQGEA ESKASKLFGN 61 YSRDQPIFLR LEDYFWVKTP SAYELPYGTK GSEDLLLRVL AITSSSIPKN IQSLRCRRCV 121 VVGNGHRLRN SSLGDAINKY DVVIRLNNAP VAGYEGDVGS KTTMRLFYPE SAHFDPKVEN 181 NPDTLLVLVA FKAMDFHWIE TILSDKKRVR KGFWKQPPLI WDVNPKQIRI LNPFFMEIAA 241 DKLLSLPMQQ PRKIKQKPTT GLLAITALH LCDLVHIAGF GYPDAYNKKQ TIHYYEQITL 301 KSMAGSGHNV SQEALAIKRM LEMGAIKNLT SF SEQ ID NO: 4 Beta-galactosamide alpha-2,6-sialyltransferase 1 Accession number NM_003032 Nucleotide sequence of ST6GAL1 1 ATGATTCACA CCAACCTGAA GAAAAAGTTC AGCTGCTGCG TCCTGGTCTT TCTTCTGTTT 61 GCAGTCATCT GTGTGTGGAA GGAAAAGAAG AAAGGGAGTT ACTATGATTC CTTTAAATTG 121 CAAACCAAGG AATTCCAGGT GTTAAAGAGT CTGGGGAAAT TGGCCATGGG GTCTGATTCC 181 CAGTCTGTAT CCTCAAGCAG CACCCAGGAC CCCCACAGGG GCCGCCAGAC CCTCGGCAGT 241 CTCAGAGGCC TAGCCAAGGC CAAACCAGAG GCCTCCTTCC AGGTGTGGAA CAAGGACAGC 301 TCTTCCAAAA ACCTTATCCC TAGGCTGCAA AAGATCTGGA AGAATTACCT AAGCATGAAC 361 AAGTACAAAG TGTCCTACAA GGGGCCAGGA CCAGGCATCA AGTTCAGTGC AGAGGCCCTG 421 CGCTGCCACC TCCGGGACCA TGTGAATGTA TCCATGGTAG AGGTCACAGA TTTTCCCTTC 481 AATACCTCTG AATGGGAGGG TTATCTGCCC AAGGAGAGCA TTAGGACCAA GGCTGGGCCT 541 TGGGGCAGGT GTGCTGTTGT GTCGTCAGCG GGATCTCTGA AGTCCTCCCA ACTAGGCAGA 601 GAAATCGATG ATCATGACGC AGTCCTGAGG TTTAATGGGG CACCCACAGC CAACTTCCAA 661 CAAGATGTGG GCACAAAAAC TACCATTCGC CTGATGAACT CTCAGTTGGT TACCACAGAG 721 AAGCGCTTCC TCAAAGACAG TTTGTACAAT GAAGATCC TAATTGTATG GGACCCATCT 781 GTATACCACT SPECIALSTCCC AAAGTGGTAC SPECIALS ATTATAATTT CTTTAACAAC 841 TACAAGACTT ATCGTAAGCT GCACCCCAAT CAGCCCTTTT ACATCCTCAA GCCCCAGATG 901 CCTTGGGAGC TATGGGACAT TCTTCAAAGAA ATCTCCCCAG AAGAGATTCA GCCAAACCCC 961 CCATCCTCTG GGATGCTTGG TATCATCATC ATGATGACGC TGTGTGACCA GGTGGATATT 1021 TATGAGTTCC TCCCATCCAA GCGCAAGACT GACGTGTGCT ACTACTACCA GAAGTTCTTC 1081 GATAGTGCCT GCACGATGGG TGCCTACCAC CCGCTGCTCT ATGAGAAA TTTGGTGAAG 1141 CATCTCAACC AGGGCACAGA TGAGGACATC TACCTGCTTG GAAAAGCCAC ACTGCCTGGC 1201 TTCCGGACCA TTCACTGCTA A 0p- Protein Sequence of ST6GAL1 (SEQ ID NO: 8) 1 MIHTNLKKKF SCCVLVFLLF AVICVWKEKK KGSYYDSFKL QTKEFQVLKS LGKLAMGSDS 61 QSVSSSSTQD PHRGRQTLGS LRGLAKAKPE ASFQVWNKDS SSKNLIPRLQ KIWKNYLSMN 121 KYKVSYKGPG PGIKFSAEAL RCHLRDHVNV SMVEVTDFPF NTSEWEGYLP KESIRTKAGP 181 WGRCAVVSSA GSLKSSQLGR EIDDHDAVLR FNGAPTANFQ QDVGTKTTIR LMNSQLVTTE 241 KRFLKDSLYN EGILIVWDPS VYHSDIPKWY QNPDYNFFNN YKTYRKLHPN QPFYILKPQM 301 PWELWDILQE ISPEEIQPNP PSSGMLGIII MMTLCDQVDI YEFLPSKRKT DVCYYYQKFF 361 DSACTMGAYH PLLYEKNLVK HLNQGTDEDI YLLGKATLPG FRTIHC

[0165] Figures 1, 2, and 3: pFSH alpha / beta, pST3, and pST6 expression vectors Plasmid map. CMV = cytomegalovirus promoter, BGHp(A) = bovine Growth hormone polyadenylated sequence, fl ori = fl replication start site, SV40 = salui Russ 40 promoter, Neo = neomycin resistance marker, Hyg = hygromycin Resistance marker, SV40p(A) = monkey virus 40 polyadenylated sequence, FSH A = Follicle-stimulating hormone alpha polypeptide, FSH B = follicle-stimulating hormone beta polypeptide Tide, ST3GAL4 = α2,3-sialyltransferase, ST6GAL1 = α2 ,6-Sialyltransferase,ColEl=ColEl replication start site,Amp=Amp Picillin resistance marker.

Claims

1. Infertility in patients (e.g., patients with serum AMH levels of 0.05 pmol / L or higher) A product containing FSH for therapeutic use, comprising 2 to 24 μg, for example, 2 to 15 A product containing a dose or equivalent of μg of human recombinant follicle-stimulating hormone (FSH).

2. <For use in the treatment of infertility in patients with a serum AMH level of 15 pmol / L A product for use according to claim 1, comprising 9 to 14 μg of human-derived FSH. Products containing a dose or equivalent of recombinant follicle-stimulating hormone (FSH).

3. The use according to claim 2, comprising a dose or equivalent of 12 μg of human-derived recombinant FSH A product for the eye.

4. Infertility treatment involves the step of determining the patient's serum AMH level, and <15 pmol / Claim 2 or the following step comprises administering a dose to a patient having a serum AMH level of L Products for use as described in item 3.

5. For use in the treatment of infertility in patients with serum AMH levels of ≥15 pmol / L A product for use according to claim 1, comprising 5 to 12.5 μg of human FSH Products containing a dose or equivalent of recombinant follicle-stimulating hormone (FSH) derived from [source].

6. Infertility treatment involves the step of determining the patient's serum AMH level, and ≥15 pmol / Claim 5 includes the step of administering a dose to a patient having a serum AMH level of L. Products for use.

7. For use in the treatment of infertility in patients with serum AMH levels of ≥15 pmol / L A product for use according to claim 1, comprising FSH, wherein 0 per kg of patient body weight A dose or equivalent of 0.09 to 0.19 μg of human recombinant follicle-stimulating hormone (FSH). A product intended for administration in [location / medicine].

8. Claims relating to the treatment of a patient having a serum AMH level of 15 to 24.9 pmol / L The product for use as described in 7, containing 0.14 to 0.19 μg per kg of patient's body weight. A product for administering a dose or equivalent of g of human-derived recombinant FSH.

9. Claim 7 in the treatment of a patient having a serum AMH level of 25 to 34.9 pmol / L The product for use as described, containing 0.11 to 0.14 μg per kg of the patient's body weight. Products for administration of human-derived recombinant FSH in doses or equivalent amounts.

10. The treatment of a patient having a serum AMH level of ≥35 pmol / L, as described in claim 7. A product for use, containing 0.10 to 0.11 μg of human-derived ingredients per kg of patient body weight. Products intended for administration in doses or equivalent amounts of recombinant FSH.

11. Infertility treatment involves the step of determining the patient's serum AMH level, and the prescribed serum AMH The step of administering a dose to a patient having a certain level, as described in any one of claims 7 to 10. Products intended for use on the vehicle.

12. The follicle-stimulating hormone (FSH) is human recombinant FSH, in any of the above claims. The products listed.

13. The FSH comprises α2,3- and α2,6-sialylation, as described in any of the claims. Products of [company name].

14. 25 to 50% of the total sialylation, for example, 30 to 50%, is α2,6-sialylation. The product according to claim 13.

15. Claim 13 or claim that 50 to 70% of the total sialylations are α2,3-sialylations. Products as described in item 14.

16. FSH contains mono-, di-, tri- and tetra-sialylated glycan structures, and sialy 15-24% of the glycan structure, for example, 17-23%, is the tetrasiallylated glycan structure. The product according to any of the above claims.

17. Na + - or K + - A pharmaceutically acceptable salt or a combination thereof selected from the group. The infertility according to any of the claims, further comprising a salt containing an alkali metal cation. Products containing follicle-stimulating hormone (FSH) for use in the treatment of certain conditions.

18. (a) the step of measuring the serum AMH level of the subject; and (b) A dose or equivalent of 2 to 24 μg, for example, 2 to 15 μg of human recombinant FSH Steps to administer to the target Infertility treatment methods, including [mention specific treatment methods].

19. (a) A step of determining the serum AMH level of the subject; and (b) <15 pmol / L Subjects with serum AMH levels are given 9 to 14 μg of human recombinant follicle-stimulating hormone ( A method for treating infertility, comprising the step of administering a dose or equivalent of FSH.

20. (a) A step to determine the serum AMH level of the subject; and (b) ≥ 15 pmol / L Subjects having the serum AMH level (as described above) are given 5 to 12.5 μg of human recombinant follicle extract. A method for treating infertility, comprising the step of administering a dose or equivalent of a fasting hormone (FSH).

21. (a) A step of determining the serum AMH level of the subject; and (b) A step of determining the body weight of the subject per 1 kg Administer a dose or equivalent of 0.09 to 0.19 μg of human recombinant FSH. A treatment method for infertility that includes a suppository and involves a subject with a serum AMH level of ≥15 pmol / L. 。

22. The dose of human recombinant FSH at 0.14 to 0.19 μg per kg of body weight of the subject, or The procedure includes the step of administering an equal amount, wherein the subject has a serum AMH level of 15 to 24.9 pmol / L. The method according to claim 21, comprising a bell.

23. The dose of human recombinant FSH is 0.11 to 0.14 μg per kg of body weight of the subject. The procedure includes the step of administering an equal amount, wherein the subject has a serum AMH level of 25 to 34.9 pmol / L. The method according to claim 21, comprising a bell.

24. The dose of human recombinant FSH is 0.10 to 0.11 μg per kg of body weight of the subject. The procedure includes the step of administering an equal amount, wherein the subject has a serum AMH level of ≥35 pmol / L. The method according to claim 21.

25. (a) A step of determining the serum AMH level of the subject; and (b) A step of determining the body weight of the subject per 1 kg Administer a dose of 0.15 to 0.17 μg of human recombinant FSH or an equivalent dose. A treatment method for infertility that includes a suppository and applies to subjects with a serum AMH level of <15 pmol / L. 。

26. Infertility in patients (e.g., patients with serum AMH levels of 0.05 pmol / L or higher) A product containing FSH for use in the manufacture of pharmaceuticals used for therapeutic purposes, wherein 2 to 24 A dose of human recombinant follicle-stimulating hormone (FSH) of, for example, 2 to 15 μg or Products containing an equal amount.

Images