RECOMBINANT FSH COMPOSITION FOR TREATMENT OF INFERTILITY
20230139407 · 2023-05-04
Assignee
Inventors
- JOAN CARLES ARCE SAEZ (Copenhagen, DK)
- LISBETH HELMGAARD (Copenhagen, DK)
- BJARKE MIRNER KLEIN (Copenhagen, DK)
Cpc classification
A61K38/24
HUMAN NECESSITIES
International classification
A61K38/24
HUMAN NECESSITIES
A61P5/06
HUMAN NECESSITIES
Abstract
Preparations including FSH, for example recombinant FSH, for use in the treatment of infertility.
Claims
1-19. (canceled)
20. A method of treating infertility, comprising administering a follicle stimulating hormone (FSH) composition at a dose of or equivalent to from 9 μg to 24 μg FSH to a patient identified prior to treatment as having variant Ser/Ser at position 680 of the FSH receptor and an antral follicle count (AFC)≥8 in both ovaries combined.
21. The method of claim 20, wherein the method comprises daily administration of the FSH composition.
22. The method of claim 20, wherein the method comprises administering the FSH composition at a dose of or equivalent to from greater than 12 μg FSH to 24 μg FSH.
23. The method of claim 20, wherein the FSH composition is administered starting on day one of treatment and continuing for from six to sixteen days.
24. The method of claim 20, wherein the FSH is recombinant FSH.
25. The method of claim 20, wherein the FSH is recombinant FSH that includes α2,3-sialylation and α2,6-sialylation.
26. The method of claim 20, wherein the FSH is recombinant FSH that includes α2,3-sialylation and α2,6-sialylation, wherein from 1 to 99% of the total sialylation is α2,6-sialylation and from 99% to 1% of the total sialylation is α2,3-sialylation.
27. The method of claim 20, wherein the FSH is recombinant FSH that includes α2,3-sialylation and α2,6-sialylation, wherein from 1 to 50% of the total sialylation is α2,6-sialylation and from 50% to 99% of the total sialylation is α2,3-sialylation.
28. A method of treating infertility, comprising administering a follicle stimulating hormone (FSH) composition at a dose of or equivalent to from 9 μg to 24 μg FSH to a patient identified prior to treatment as having variant Ser/Ser at position 680 of the FSH receptor, wherein the FSH is recombinant FSH that includes α2,3-sialylation and α2,6-sialylation, wherein from 1 to 50% of the total sialylation is α2,6-sialylation and from 50% to 99% of the total sialylation is α2,3-sialylation, wherein the patient is identified prior to treatment as having an antral follicle count (AFC)≥8 in both ovaries combined.
29. A method of treating infertility, comprising administering a follicle stimulating hormone (FSH) composition at a daily dose of from 9 μg to 24 μg FSH to a patient identified prior to treatment as having variant Ser/Ser at position 680 of the FSH receptor, wherein the FSH is recombinant FSH that includes α2,3-sialylation and α2,6-sialylation, wherein from 1 to 50% of the total sialylation is α2,6-sialylation and from 50% to 99% of the total sialylation is α2,3-sialylation, wherein the patient is identified prior to treatment as having an antral follicle count (AFC)≥8 in both ovaries combined.
Description
DETAILED DESCRIPTION OF THE INVENTION
[0068] The present invention will now be described in more detail with reference to the attached drawings in which:
[0069]
[0070]
[0071]
[0072]
[0073]
[0074]
[0075]
SEQUENCE SELECTION
Human FSH
[0076] The coding region of the gene for the FSH alpha polypeptide was used to according to Fiddes and Goodman. (1981). The sequence is banked as AH007338 and at the time of construction there were no other variants of this protein sequence. The sequence is referred herein as SEQ ID NO:1.
[0077] The coding region of the gene for FSH beta polypeptide was used according to Keene et al (1989). The sequence is banked as NM_000510 and at the time of construction there were no other variants of this protein sequence. The sequence is referred herein as SEQ ID NO: 2
Sialyltransferase
[0078] α2,3-Sialyltransferase—The coding region of the gene for beta-galactoside alpha-2,3-sialyltransferase 4 (α2,3-sialyltransferase, ST3GAL4) was used according to Kitagawa and Paulson (1994). The sequence is banked as L23767 and referred herein as SEQ ID NO: 3.
[0079] α2,6-Sialyltransferase—The coding region of the gene for beta-galactosamide alpha-2,6-sialyltransferase 1 (α2,6-sialyltransferase, ST6GAL1) was used according to Grundmann et al. (1990). The sequence is banked as NM_003032 and referred herein as SEQ ID NO: 4.
EXAMPLES
Example 1 Construction of the FSH Expression Vector
[0080] The coding sequence of FSH alpha polypeptide (AH007338, SEQ ID NO: 1) and FSH beta polypeptide (NM_003032, SEQ ID NO: 2) were amplified by PCR using the primer combinations FSHa-fw and FSHa-rev and FSHb-fw and FSHb-rec respectively.
TABLE-US-00001 FSHa-fw (SEQ ID NO: 9) 5′-CCAGGATCCGCCACCATGGATTACTACAGAAAAATATGC-3′ FSHa-rev (SEQ ID NO: 10) 5′-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3′ FSHb-fw (SEQ ID NO: 11) 5′-CCAGGCGCGCCACCATGAAGACACTCCAGTTTTTC-3′ FSHb-rev (SEQ ID NO: 12) 5′-CCGGGTTAACTTATTATTCTTTCATTTCACCAAAGG-3′
[0081] The resulting amplified FSH beta DNA was digested with the restriction enzymes Ascl and Hpal and inserted into the Ascl and Hpal sites on the CMV driven mammalian expression vector carrying a neomycin selection marker. Similarly the FSH alpha DNA was digested with BamHl and Nhel and inserted into the sites BamHl and Nhel on the expression vector already containing the FSH beta polypeptide DNA.
[0082] The vector DNA was used to transform the DH5a strain of E. coli. Colonies were picked for amplification. Colonies containing the vector containing both FSH alpha and beta were selected for sequencing and all contained the correct sequences according to SEQ ID NO: 1 and SEQ ID NO: 2. Plasmid pFSH A+B #17 was selected for transfection (
Example 2 Construction of the ST3 Expression Vector
[0083] The coding sequence of beta-galactoside alpha-2,3-sialyltransferase 4 (ST3, L23767, SEQ ID NO: 3) was amplified by PCR using the primer combination 2,3STfw and 2,3STrev.
TABLE-US-00002 2,3STfw (SEQ ID NO: 13) 5′-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3′ 2,3STrev (SEQ ID NO: 14) 5′-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3′
[0084] The resulting amplified ST3 DNA was digested with the restriction enzymes BamHl and Af/ll and inserted into the BamHl and Af/ll sites on the CMV driven mammalian expression vector carrying a hygromycin resistance marker. The vector was amplified as previously described and sequenced. Clone pST3 #1 (
Example 3 Construction of the ST6 Expression Vector
[0085] The coding sequence of beta-galactosamide alpha-2,6-sialyltransferase 1 (ST6, NM_003032, SEQ ID NO: 4) was amplified by PCR using the primer combination 2,6STfw and 2,6STrev.
TABLE-US-00003 2,6STfw (SEQ ID NO: 15) 5′-CCAGGATCCGCCACCATGATTCACACCAACCTGAAG-3′ 2,6STrev (SEQ ID NO: 16) 5′-TTTTTTTCTTAAGTTAGCAGTGAATGGTCCGG-3′
[0086] The resulting amplified ST6 DNA was digested with the restriction enzymes BamHl and Af/ll and inserted into the BamHl and Af/ll sites on the CMV driven mammalian expression vector carrying a hygromycin resistance marker. The vector was amplified as previously described and sequenced. Clone pST6 #11 (
Example 4 Stable Expression of pFSH α+β in PER.C6® Cells. Transfection Isolation and Screening of Clones
[0087] PER.C6®clones producing FSH were generated by expressing both polypeptide chains of FSH from a single plasmid (see Example 1).
[0088] To obtain stable clones a liposome based transfection agent with the pFSH α+β construct. Stable clones were selected in VPRO supplemented with 10% FCS and containing G418. Three weeks after transfection G418 resistant clones grew out. Clones were selected for isolation. The isolated clones were cultured in selection medium until 70-80% confluent. Supernatants were assayed for FSH protein content using an FSH selective ELISA and pharmacological activity at the FSH receptor in cloned cell line, using a cAMP accumulation assay. Clones expressing functional protein were progressed for culture expansion to 24 well, 6 well and T80 flasks.
[0089] Studies to determine productivity and quality of the material from seven clones were initiated in T80 flasks to generate sufficient material. Cells were cultured in supplemented media as previously described for 7 days and the supernatant harvested. Productivity was determined using the FSH selective ELISA. The isoelectric profile of the material was determined by Isoelectric focusing (IEF), by methods known in the art. Clones with sufficient productivity and quality were selected for sialyltransferase engineering.
Example 5 Level of Sialylation is Increased in Cells that Over Express α2,3-Sialyltransferase. Stable Expression of Pst3 in FSH Expressing Per.C6® Cells; Transfection Isolation and Screening of Clones
[0090] PER.C6® clones producing highly sialylated FSH were generated by expressing α2,3 sialyltransferase from separate plasmids (Example 2) in PER.C6® cells already expressing both polypeptide chains of FSH (from Example 4). Clones produced from PER.C6® cells as set out in Example 4 were selected for their characteristics including productivity, good growth profile, production of functional protein, and produced FSH which included some sialylation. Stable clones were generated as previously described in Example 4. Clones were isolated, expanded and assayed. The α2,3-sialyltransferase clones were adapted to serum free media and suspension conditions.
[0091] As before, clones were assayed using a FSH selective ELISA, functional response in an FSH receptor cell line, IEF, metabolic clearance rate and Steelman Pohley analysis. Results were compared to a commercially available recombinant FSH (Gonal-f, Serono) and the parental FSH PER.C6®cell lines. FSH produced by most of the clones has significantly improved sialylation (i.e. on average more FSH isoforms with high numbers of sialic acids) compared to FSH expressed without α2,3-sialyltransferase. In conclusion expression of FSH together with sialyltransferase in PER.C6®cells resulted in increased levels of sialylated FSH compared to cells expressing FSH only.
Example 6 Production and Purification Overview
[0092] A procedure was developed to produce FSH in PER.C6®cells that were cultured in suspension in serum free medium. The procedure is described below and was applied to several FSH-producing PER.C6®cell lines.
[0093] FSH from α2,3-clone (Example 5) was prepared using a using a modification of the method described by Lowry et al. (1976).
[0094] For the production of PER.C6®-FSH, the cell lines were adapted to a serum-free medium, i.e., Excel 525 (JRH Biosciences). The cells were first cultured to form a 70%-90% confluent monolayer in a T80 culture flask. On passage the cells were re-suspended in the serum free medium, Excel 525+4 mM L-Glutamine, to a cell density of 0.3×10.sup.6 cells/ml. A 25 ml cell suspension was put in a 250 ml shaker flask and shaken at 100 rpm at 37° C. at 5% CO.sub.2. After reaching a cell density of >1×10.sup.6 cells/ml, the cells were sub-cultured to a cell density of 0.2 or 0.3×10.sup.6 cells/ml and further cultured in shaker flasks at 37° C., 5% CO.sub.2 and 100 rpm.
[0095] For the production of FSH, the cells were transferred to a serum-free production medium, i.e., VPRO (JRH Biosciences), which supports the growth of PER.C6®cells to very high cell densities (usually >10.sup.7 cells/ml in a batch culture). The cells were first cultured to >1×10.sup.6 cells/ml in Excel, 525, then spun down for 5 min at 1000 rpm and subsequently suspended in VPRO medium+6 mM L-glutamine to a density of 1×10.sup.6 cells/ml. The cells were then cultured in a shaker flask for 7-10 days at 37° C., 5% CO.sub.2 and 100 rpm. During this period, the cells grew to a density of >10.sup.7 cells/ml. The culture medium was harvested after the cell viability started to decline. The cells were spun down for 5 min at 1000 rpm and the supernatant was used for the quantification and purification of FSH. The concentration of FSH was determined using ELISA (DRG EIA 1288).
[0096] Thereafter, purification of FSH was carried out using a modification of the method described by Lowry et al. (1976). Purification using charge selective chromatography was carried out to enrich the highly sialylated forms by methods well known in the art.
[0097] During all chromatographic procedures, enrichment of the sialylated forms of FSH as claimed herein was confirmed by RIA (DRG EIA 1288) and/or IEF.
Example 7 Quantification of Relative Amounts of α2,3 and α2,6 Sialic Acid
[0098] The relative percentage amounts of α2,3 and α2,6 sialic acid on purified rFSH (Example 6) were measured using known techniques.
[0099] N-Glycans were released from the samples using PNGase F under denaturative conditions and then labelled with 2-aminobenzamide. Released glycan forms were then separated and analysed by Weak Anion Exchange (WAX) column for determination of charge distribution. Labelled glycans treated with 2,3,6,8 sialidase for determination of total sialic acid and 2,3 sialidase for determination of 2,3 sialic acid, were further analyzed by wax column.
[0100] The relative percentages of the charged glycans were calculated from structures present in the undigested and digested glycan pools and are shown in
Example 8—a Multiple Dose Study Investigating FE 999049 in Comparison to GONAL-F
[0101] The following describes a randomised, controlled, assessor-blind, parallel groups, multinational, multicentre trial assessing the dose-response relationship of FE 999049 in patients undergoing controlled ovarian stimulation for in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI). The patient population was 265 IVF patients aged between 18 to 37 years, with BMI 18.5 to 32.0 kg/m.sup.2.
[0102] The trial was designed as a dose-response trial with number of oocytes retrieved as the primary endpoint. Secondary endpoints will explore the qualitative and quantitative impact of different doses of FE 999049 with regard to endocrine profile, follicular development, oocyte fertilisation, embryo quality and treatment efficiency (i.e. total gonadotropin consumption and duration of stimulation). The trial is designed to evaluate the efficacy of FE 999049 to establish pregnancy when used in controlled ovarian stimulation for IVF/ICSI cycles.
[0103] Subjects were assessed within 3 months prior to randomisation for compliance with the inclusion and exclusion criteria, including an anti-Müllerian hormone (AMH) assessment to increase homogeneity of the trial population in relation to ovarian response and minimise the number of potential poor and hyper-responders to the FE 999049 doses and GONAL-F dose used in the trial. The AMH assessment was measured using the AMH Gen-II enzyme linked immunosorbent assay kit (Beckman Coulter, Inc., Webster, Tex.). This assay can detect AMH concentrations greater than 0.57 pmol/L with a minimum limit of quantitation of 1.1 pmol/L.
[0104] On day 2-3 of their menstrual cycle, subjects were randomised in a 1:1:1:1:1:1 fashion to treatment with either 90 IU, 120 IU, 150 IU, 180 IU or 210 IU FE 999049 or 150 IU GONAL-F, and ovarian stimulation initiated. Randomisation was stratified according to AMH level at screening [5.0-14.9 pmol/L (low AMH) and 15.0 to 44.9 pmol/L (high AMH)).
[0105] Gonal-F is filled by mass (FbM) at FDA request; referring to μg dose is therefore appropriate. The Gonal-F label indicates 600 IU/44 μg, which indicates that 150 IU is 11 μg. However, there is some variation and the batch certificate for this trial indicated that 11.3 μg Gonal-F was equivalent to 150 IU. The FE999049 doses are presented by protein content (μg) rather than biological activity. Thus the doses of FE999049 were 5.2 μg (90 IU), 6.9 μg (120 IU), 8.6 μg (150 IU), 10.3 μg (180 IU) or 12.1 μg (210 IU).
[0106] The subject and dose distribution is set out as follows (data are number of subjects):
TABLE-US-00004 TABLE 1 FE 999049 GONAL-F 5.2 μg 6.9 μg 8.6 μg 10.3 μg 12.1 μg 11.3 (11) μg Total Screened 334 Randomised 42 45 44 45 46 43 265 and exposed High AMH strata 23 26 24 24 26 25 148 (15.0-44.9 pmol/L) (56%) Low AMH strata 19 19 20 20 21 18 117 (5.0-14.9 pmol/L) (44%) Per-protocol 40 42 42 44 44 43 255
[0107] The daily dose level of FE 999049 or GONAL-F is fixed throughout the entire stimulation period. During stimulation, subjects are monitored on stimulation day 1, 4 and 6 and hereafter at least every second day. When 3 follicles of ≥15 mm are observed, visits are performed daily. Subjects are treated with FE 999049 or GONAL-F for a maximum of 16 days.
[0108] To prevent a premature LH surge, a GnRH antagonist (ganirelix acetate, ORGALUTRAN, MSD/Schering-Plough) may be initiated on stimulation day 6 at a daily dose of 0.25 mg and continued throughout the stimulation period. Triggering of final follicular maturation is done on the day when ≥3 follicles with a diameter ≥17 mm are observed. If there are <25 follicles with a diameter ≥12 mm, 250 μg recombinant hCG (choriogonadotropin alfa, OVITRELLE, Merck Serono/EMD Serono) is administered. If there are 25-35 follicles with a diameter ≥12 mm, 0.2 mg GnRH agonist (triptorelin acetate, DECAPEPTYL/GONAPEPTYL, Ferring Pharmaceuticals) is administered. In case of excessive ovarian response, defined as >35 follicles with a diameter ≥12 mm, the treatment is cancelled. In case of poor ovarian response, defined as <3 follicles with a diameter 0 mm observed on stimulation day 10, the cycle could be cancelled.
[0109] Oocyte retrieval takes place 36 h (±2 h) after triggering of final follicular maturation and the oocytes inseminated by IVF and/or ICSI. Fertilisation and embryo development are assessed from oocyte retrieval to the day of transfer. For subjects who underwent triggering of final follicular maturation with hCG, one blastocyst of the best quality available is transferred on day 5 after oocyte retrieval while remaining blastocysts are frozen. For subjects who undergo triggering of final follicular maturation with GnRH agonist, no embryo transfer takes place in the fresh cycle and blastocysts are instead frozen on day 5. Vaginal progesterone tablets (LUTINUS, Ferring Pharmaceuticals) 100 mg 3 times daily are provided for luteal phase support from the day after oocyte retrieval until the day of the clinical pregnancy visit. A βhCG test is performed 13-15 days after embryo transfer and clinical pregnancy will be confirmed by transvaginal ultrasound (TVU) 5-6 weeks after embryo transfer.
Results
[0110] The number of oocytes retrieved (primary endpoint) is shown in the following Table.
TABLE-US-00005 TABLE 2 Oocytes FE 999049 GONAL-F retrieved 5.2 μg 6.9 μg 8.6 μg 10.3 μg 12.1 μg 11.3 (11)μg All 5.2 (3.3) 7.9 (5.9) 9.2 (4.6) 10.6 (7.0) 12.2 (5.9) 10.4 (5.2) High AMH 5.9 (3.9) 9.1 (6.4) 10.6 (4.8) 13.6 (7.8) 14.4 (5.8) 12.4 (5.4) Low AMH 4.5 (2.2) 6.3 (4.9) 7.4 (3.8) 6.9 (3.6) 9.4 (4.9) 7.8 (3.4) Data are mean (SD)
The primary objective was met: a significant dose-response relationship was established for FE 999049 with respect to number of oocytes retrieved. This finding was observed not only for the overall trial population, but also for each of the two AMH strata used at randomisation.
A significant dose-response for FE 999049 was demonstrated for all key objective pharmacodynamic parameters, e.g. estradiol, inhibin B and inhibin A. At a similar microgram dose level, the pharmacodynamic responses with FE 999049 were larger than with GONAL-F (these results not shown).
The serum FSH concentrations after exposure to FE 999049 were significantly higher than for GONAL-F. The results confirm that the PK profile of FE 999049 differs from that of GONAL-F. Fertilisation rates, blastocyst development and pregnancy rates in IVF/ICSI patients treated with FE 999049 were within expectations.
There were no safety concerns with the use of FE 999049. A good local tolerability was documented.
Further Analysis
[0111] The applicants have further analysed the data to identify the FE 999049 dose(s) that fulfil the following criteria with respect to number of oocytes retrieved: [0112] Oocytes retrieved in the range 8-14 [0113] Minimise proportion of patients with <8 oocytes [0114] Minimise proportion of patients with <4 or ≥20 oocytes
Low AMH strata
As seen in Table 2, the dose of FE999049 which fulfilled the first criterion (Oocytes retrieved in the range 8-14) was 12.1 μg (mean 9.4 oocytes retrieved). The distribution of oocytes is shown in Table 3 below.
TABLE-US-00006 TABLE 3 Oocytes FE 999049 GONAL-F retrieved 5.2 μg 6.9 μg 8.6 μg 10.3 μg 12.1 μg 11.3 (11)μg <4 32% 24% 15% 10% 10% 6% 4-7 63% 42% 45% 60% 20% 56% 8-14 5% 24% 35% 30% 60% 33% 15-19 0% 5% 5% 0% 5% 6% ≥20 0% 5% 0% 0% 5% 0% Data are % of subjects
[0115] As shown by the arrow, a dose of 12.1 μg FE999049 provides retrieval of the most desirable number of oocytes in 60% of subjects in the low AMH group. This is a marked improvement on Gonal-F (most desirable number of oocytes in only 33% of subjects). There were no indications of early OHSS of a moderate or severe nature and there were no incidences of preventative action being required; there are no concerns associated with the dose of 12.1 μg FE999049 in a patient having low AMH.
[0116] Thus the applicants have found that a dose of, or dose equivalent to, 6 to 24 μg, for example 9 to 14 μg, for example 12 μg, human derived recombinant FSH is suitable for use in the treatment of infertility in a patient having serum AMH <15 pmol/L, for example 0.05-14.9 pmol/L for example 5.0-14.9 pmol/L. The dose provides an effective response while minimising risk of OHSS.
Exploratory Evaluation
[0117] As an exploratory evaluation, the present inventors investigated the contribution of FSH receptor polymorphism on ovarian response and treatment efficiency following stimulation with FE999049.
[0118] Genomic DNA from all patients in the trial was analysed for single nucleotide polymorphism (SNP) at positions 29, 307 and 680 of the FSH-R at the University of Modena and Reggio Emilia, Italy.
[0119] The results of the clinical trial were further analysed to assess whether SNP had any effect on the duration of treatment and total dose required. This was done for the low AMH group and the high AMH group.
[0120] FSHR polymorphism was examined by PCR (Polymerase Chain Reaction) and RFLP (Restriction fragment length polymorphism) by methods known in the art. Women were classified as Asn/Asn, Asn/Ser, and Ser/Ser genotypes. The genetic analysis was described in the following overview protocol and the patients signed a special informed consent. The samples were taken as part of the other blood samples on stimulation day 1. They were measured at the University of Modena and Reggio Emilia.
Overview of Procedures Used for SNP-Analysis at the FSH Receptor Gene
[0121] General procedures: [0122] 1. Genomic DNA extraction (from blood using Nucleon Genomic DNA extraction kit, GE HEALTHCARE) [0123] 2. Operating procedures using nanodrop.
HRM procedures:
SNPs genotyping by high resolution melting (HRM) methodology (using SsoFast EvaGreen Supermix cod enzyme. 172-5201, Bio-Rad; HSP-96 plates, cat. HSP9645, Bio-Rad; and CFX96 real-time thermal cycler Bio-Rad.)
[0124] Sequencing Procedures:
In case of doubt about HRM results, (after two independent HRM on the same samples), the following sequencing procedures are utilised: [0125] 1. PCR reaction and amplification. [0126] 2. PCR product purification. [0127] 3. Quantification of purified PCR. [0128] 4. Sequence reaction protocol. [0129] 5. Sequence product purification. [0130] 6. Capillary electrophoresis run by ABI PRISM 3130 instrument. [0131] 7. Assessment and validation of the results obtained by capillary electrophoresis sequencing with ABI PRISM 3100.
Results
[0132]
[0133]
[0134] As indicated above, success (in terms of pregnancy and/or live birth) is more likely if the patient has an adequate response (expected ovarian multifollicular development, rise in circulating 17-β-estradiol) occurring within an ideal treatment window. Success is further enhanced if the response is within the centre of this treatment window; that is, not too early in the window and not too late. Reduction of the duration of treatment in patients having low AMH and variant Ser/Ser at position 680 of the FSH receptor (by increasing the dose above 12 μg) may bring the response towards the centre of the treatment window, with enhanced likelihood of success.
[0135] Accordingly, tailoring the dose to patients identified as having AMH level <15 pmol/L and variant Ser/Ser prior to treatment may be possible. Identification of patients having Ser/Ser prior to treatment may allow the starting dose to be increased in these patients, compared with those having Ser/Asn and Asn/Asn.
[0136] As set out above, a dose of 12.1 μg FE999049 provides retrieval of the most desirable number of oocytes in 60% of subjects in the low AMH group (Table 3). The low AMH group shown in Table 3 included patients having variant Ser/Ser, as well as those having Ser/Asn and Asn/Asn. Administration of a higher starting dose (for example 9 to 24 μg, for example >12 to 24 μg, e.g. 12.33 μg or 13 μg human derived recombinant) of FSH to patients having low AMH [AMH level <15 pmol/L, (e.g. 0.05 pmol/L to 14.9 pmol/L, e.g. 5.0 pmol/L to 14.9 pmol/L)], as well as having variant Ser/Ser at position 680 of the FSH receptor, may be advantageous because it may provide increased probability of success (in terms of pregnancy and/or live birth) and better predictability of success.
[0137]
[0138]
[0139] The results did not show this effect related to SNP in the high AMH population.
[0140] This allows tailoring of the dose of FSH in specific patients identified as having specific AMH level, and specific polymorphism at the FSHR.
Example 9—Individualised COS Protocol (Low AMH)
[0141] The selected patients are about to undergo COS for in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) by methods known in the art. The pre-treatment protocol includes assessment/screening of the patient's serum AMH using the AMH Gen-II enzyme linked immunosorbent assay kit (Beckman Coulter, Inc., Webster, Tex.). This assay can detect AMH concentrations greater than 0.57 pmol/L with a minimum limit of quantitation of 1.1 pmol/L. AMH may be measured using other Assay kits (e.g. available from Roche). The pre-treatment protocol includes identification of the allelic variant at position 680 of the FSH receptor following extraction of genomic DNA by methods well known in the art (e.g. by means of a kit for extraction of genomic DNA from blood, and subsequent DNA sequencing, as described in e.g. Gromoll et al, Methods, 21, 83-97 (2000), Simoni et al, Journal of Clinical Endocrinology and Metabolism, Vol 84, No. 2, 751-755 (1999), Falconer et al, Acta Obstet Gynecol Scand 2005: 84: 806-811 (2005), and references therein, or by a PCR and RFLP method such as that set out in Loutradis et al, Journal of Assisted Reproduction and Genetics, Vol. 23, No. 4, April 2006).
[0142] The COS protocol proceeds in the usual manner apart from administration of the initial dose of FE 999049 according to AMH level at screening. A patient with an AMH level of <15 pmol/L and variant Ser/Asn or Asn/Asn would be administered an initial daily dose of approximately 12 μg FE 999049, a human derived recombinant FSH product manufactured according to the method of Example 6, or <12 μg FE 999049, e.g. 10 to 12 μg, e.g. 11.33 μg or 11.67 μg, of the human derived recombinant FSH. A patient with an AMH level of <15 pmol/L and variant Ser/Ser would receive a higher initial daily dose greater than 12 μg (e.g. 12.33 to 24 μg, or 13-24 μg of the human derived recombinant FSH.
REFERENCES
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TABLE-US-00007 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 TCTGCCGGGA 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 TCAGATTGAA 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 GLLAITLALH 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 GAAGGAATCC TAATTGTATG GGACCCATCT 781 GTATACCACT CAGATATCCC AAAGTGGTAC CAGAATCCGG ATTATAATTT CTTTAACAAC 841 TACAAGACTT ATCGTAAGCT GCACCCCAAT CAGCCCTTTT ACATCCTCAA GCCCCAGATG 901 CCTTGGGAGC TATGGGACAT TCTTCAAGAA ATCTCCCCAG AAGAGATTCA GCCAAACCCC 961 CCATCCTCTG GGATGCTTGG TATCATCATC ATGATGACGC TGTGTGACCA GGTGGATATT 1021 TATGAGTTCC TCCCATCCAA GCGCAAGACT GACGTGTGCT ACTACTACCA GAAGTTCTTC 1081 GATAGTGCCT GCACGATGGG TGCCTACCAC CCGCTGCTCT ATGAGAAGAA 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