HUMAN-DERIVED RECOMBINANT FSH FOR CONTROLLED OVARIAN STIMULATION
20200345814 · 2020-11-05
Assignee
Inventors
Cpc classification
A61P43/00
HUMAN NECESSITIES
A61K38/24
HUMAN NECESSITIES
A61P15/08
HUMAN NECESSITIES
International classification
Abstract
Preparations including FSH, for example recombinant FSH, for use in the treatment of infertility.
Claims
1. A method of treating infertility, comprising administering follicle stimulating hormone (FSH) to a patient in need of such treatment, wherein the FSH is administered at a daily dose of, or a dose equivalent to a daily dose of, 11 to 13 g recombinant FSH to the patient, wherein the patient has a serum anti-mullerian (AMH) hormone level of <15 pmol/L, and wherein the FSH is administered together with a gonadotropin releasing hormone (GnRH) antagonist.
2. The method of claim 1, wherein the method comprises a step of determining the serum AMH level of the patient, and a step of administering the dose of FSH to the patient having the serum AMH level of <15 pmol/L.
3. A method of treating infertility, comprising administering a follicle stimulating hormone (FSH) to a patient in need of such treatment, wherein the FSH is administered at a daily dose of, or a daily dose equivalent to daily dose of, 0.09 to 0.19 g recombinant FSH per kilogram body weight of the patient, wherein the has having a serum anti-mullerian hormone level of 15 pmol/L, and wherein the FSH is administered together with a gonadotropin releasing hormone (GnRH) antagonist.
4. The method of claim 3, wherein the method comprises a step of determining the serum AMH level of the patient, and a step of administering the dose of FSH to the patient having the serum AMH level of 15 pmol/L.
5. A method of treating infertility, comprising administering follicle stimulating hormone (FSH) to a patient in need of such treatment, wherein the FSH is administered at a daily dose of, or a dose equivalent to a daily dose of, 11 to 13 g recombinant FSH to the patient, wherein the patient has a serum anti-mullerian (AMH) hormone level of <15 pmol/L, and wherein the FSH is administered together with human chorionic gonadotropin (hCG).
6. The method of claim 5, wherein the method comprises a step of determining the serum AMH level of the patient, and a step of administering the dose of FSH to the patient having the serum AMH level of <15 pmol/L.
7. A method of treating infertility, comprising administering a follicle stimulating hormone (FSH) to a patient in need of such treatment, wherein the FSH is administered at a daily dose of, or a daily dose equivalent to daily dose of, 0.09 to 0.19 g recombinant FSH per kilogram body weight of the patient, wherein the patient has a serum anti-mullerian hormone (AMH) level of 15 pmol/L, and wherein the FSH is administered together with human chorionic gonadotropin (hCG).
8. The method of claim 7, wherein the method comprises a step of determining the serum AMH level of the patient, and a step of administering the dose of FSH to the patient having the serum AMH level of 15 pmol/L.
Description
DETAILED DESCRIPTION OF THE INVENTION
[0079] The present invention will now be described in more detail with reference to the attached drawings in which:
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SEQUENCE SELECTION
Human FSH
[0088] 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.
[0089] 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
[0090] 2,3-SialyltransferaseThe coding region of the gene for beta-galactoside alpha-2,3-sialyltransferase 4 (2,3-sialyltransferase, ST3GAL4) was used according to Kitagawa and Paulson (1994). The sequence is banked as L23767 and referred herein as SEQ ID NO: 3.
[0091] 2,6-SialyltransferaseThe coding region of the gene for beta-galactosamide alpha-2,6-sialyltransferase 1 (2,6-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
[0092] 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 (SEQIDNO:9) 5-CCAGGATCCGCCACCATGGATTACTACAGAAAAATATGC-3 FSHa-rev (SEQIDNO:10) 5-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3 FSHb-fw (SEQIDNO:11) 5-CCAGGCGCGCCACCATGAAGACACTCCAGTTTTTC-3 FSHb-rev (SEQIDNO:12) 5-CCGGGTTAACTTATTATTCTTTCATTTCACCAAAGG-3
[0093] 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.
[0094] The vector DNA was used to transform the DH5 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
[0095] 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 (SEQIDNO:13) 5-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3 2,3STrev (SEQIDNO:14) 5-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3
[0096] The resulting amplified ST3 DNA was digested with the restriction enzymes BamHl and AflII and inserted into the BamHl and AflII 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
[0097] 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 (SEQIDNO:15) 5-CCAGGATCCGCCACCATGATTCACACCAACCTGAAG-3 2,6STrev (SEQIDNO:16) 5-TTTTTTTCTTAAGTTAGCAGTGAATGGTCCGG-3
[0098] The resulting amplified ST6 DNA was digested with the restriction enzymes BamHl and AflII and inserted into the BamHl and AflII 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 + in PER.C6 Cells. Transfection Isolation and Screening of Clones
[0099] PER.C6clones producing FSH were generated by expressing both polypeptide chains of FSH from a single plasmid (see Example 1).
[0100] To obtain stable clones a liposome based transfection agent with the pFSH + 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.
[0101] 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 2,3-sialyltransferase. Stable Expression of pST3 in FSH Expressing PER.C6 Cells; Transfection Isolation and Screening of Clones
[0102] PER.C6 clones producing highly sialylated FSH were generated by expressing 2,3 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 2,3-sialyltransferase clones were adapted to serum free media and suspension conditions.
[0103] 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.C6cell 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 2,3-sialyltransferase. In conclusion expression of FSH together with sialyltransferase in PER.C6cells resulted in increased levels of sialylated FSH compared to cells expressing FSH only.
Example 6 Production and Purification Overview
[0104] A procedure was developed to produce FSH in PER.C6cells that were cultured in suspension in serum free medium. The procedure is described below and was applied to several FSH-producing PER.C6cell lines.
[0105] FSH from 2,3-clone (Example 5) was prepared using a using a modification of the method described by Lowry et al. (1976).
[0106] For the production of PER.C6-FSH, the cell lines were adapted to a serum-free medium, i.e., Excell 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 0.310.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 >II0.sup.6 cells/ml, the cells were sub-cultured to a cell density of 0.2 or 0.310.sup.6 cells/ml and further cultured in shaker flasks at 37 C., 5% CO.sub.2 and 100 rpm.
[0107] 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.C6cells to very high cell densities (usually >10.sup.7 cells/ml in a batch culture). The cells were first cultured to >110.sup.6 cells/ml in Excell 525, then spun down for 5 min at 1000 rpm and subsequently suspended in VPRO medium+6 mM L-glutamine to a density of 110.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).
[0108] 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.
[0109] 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 2,3 and 2,6 Sialic Acid
[0110] The relative percentage amounts of 2,3 and 2,6 sialic acid on purified rFSH (Example 6) were measured using known techniques.
[0111] 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.
[0112] 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 Quantification of Relative Amounts Mono, Di, Tri and Tetra Sialylated Glycan Structures
[0113] The relative percentage amounts of mono, di, tri and tetra sialylated structures on glycans extracted from purified rFSH (Example 6) were measured using known techniques.
[0114] N Glycans were released from the samples using PNGase F under denaturative conditions and then were labeled with 2-aminobenzamide. Glycans were released from the samples using PNGase F under denaturative conditions and then labeled with 2-aminobenzamide. Released glycan forms were then separated and analysed by Weak Anion Exchange (WAX) column for determination of sialylation distribution. The relative amounts of neutral, mono-sialylated, di-sialylated, tri-sialylated and tetra-sialylated structures are shown in
[0115] The rFSH includes neutral , mono-sialylated, di-sialylated, tri-sialylated and tetra-sialylated glycan structures with relative amounts as follows: neutral 5-6%; 15-17% mono-sialylated; 26-30% di-sialylated; 30-32% tri-sialylated and 17-23% tetra-sialylated.
Example 8a
[0116] The relative percentage amounts of 2,6 sialic acid on purified rFSH extracted from nine samples of purified rFSH (produced by the methods of Example 6) were measured using known techniques.
[0117] 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 (see Example 8). The analysis allows calculation of 2,6 sialic acid.
[0118] The relative percentages of the charged glycans were calculated from structures present in the undigested and digested glycan pools and are shown in the following Table. These were found to be in the ranges 25 to 50%, generally 30 to 35% for 2,6 sialylation.
[0119] The relative percentage amounts of bisecting GlcNac, GalNac and 1-Fucose Lewis on glycans extracted from the nine samples of purified rFSH (produced by the methods of Example 6) were measured using known techniques. N-Glycans were released from the glycoprotrein using PNGase F and labeled with 2-aminobenzamide (2AB). The analysis was done by two dimensional (2D) HPLC analysis in combination with enzymatic degradation of the glycans. For verification, the glycans were analyzed by MALDI-MS The relative amounts of alpha 2,6-sialic acid and the terminal residues are shown in the following table, together with those for Gonal F (CHO cell derived recombinant FSH) and Bravelle (human urinary FSH).
TABLE-US-00004 Ref. O Ref. N I-1 I-2 I-3 II II abundance abundance abundance abundance abundance abundance abundance Sample % % % % % % % 2,6 27.7 34.9 26.2 30.1 31.1 28.3 30.4 sialic acid 1GalNAc 51 44.6 50.7 44.7 49 47.6 45.3 Bisecting 10 12.4 10.2 8.9 8.7 11.8 11.4 GlcNAc 1 Fucose 21.1 16.7 23.3 16.1 20.3 18.1 17.9 Lewis 2 Fucose 4 4.1 4.3 1.9 3.1 4.2 3.8 Lewis III-1 III-2 Average Gonal F Bravelle abundance abundance abundance abundance abundance Sample % % % % % 2,6 35 33 30.7 0 55.4 sialic acid 1GalNAc 46.4 44.9 47.1 0 11.3 Bisecting 10.6 13.9 10.9 55 14 GlcNAc 1 Fucose 18.7 19.0 19.0 3.1.sup.1 2.2 Lewis 2 Fucose 3.9 4.4 3.7 n.d..sup.2 Lewis .sup.1Value of 3.1 is total Fucose Lewis. .sup.2Not determined.
[0120] It can be seen that the amount of GalNac in the FSH of the invention varies between about 44.9 and 51%, averaging about 47.1%.
[0121] It can be seen that the amount of bisecting GlcNac in the FSH of the invention varies between 8.7 and 13.9%, averaging approximately at 10.9%.
[0122] It can be seen that the amount of 1 Fucose Lewis in the FSH of the invention varies between 16.1 and 23.3%, averaging approximately at 19%.
[0123] It can be seen that the amount of 2 Fucose Lewis in the FSH of the invention varies between 1.9 and 4.4%, averaging approximately at 3.7%.
Example 9A Multiple Dose Study Investigating the Safety, Tolerability, Pharmacokinetics, Pharmacodynamics, and Immunogenicity of FE 999049 in Comparison to GONAL-F
Study Population
[0124] A total of 48 (24 on each drug) healthy women received daily doses of 14.6 g of FE 999049 (a composition according to the invention, produced according to Example 6) or 16.5 g of Gonal-F for seven days.
Safety Results
[0125] Multiple dose administration of FE 999049 and GONAL-F was safe and generally well tolerated as assessed by Adverse Events (AEs), vital signs, ECG, clinical laboratory measurements, and physical examination. No serious adverse event or death occurred during the study.
Pharmacokinetic Results
[0126] Following the administration of FE 999049 and GONAL-F over 7 days, the FSH concentration values as assessed immediately prior to the next injection increased and seemed to reach a steady state level after 6-7 days. However the exposure (AUC and Cmax) of FE 999049 was 60% higher in comparison to Gonal-F.
Pharmacodynamic Results
[0127] The concentrations of inhibin-B (see
[0128] Example 9 demonstrates that FSH having a specific amount (17-23%) of tetra-sialylated glycan structures and e.g. specific amounts of 2,3 sialylation and 2,6 sialylation is markedly more potent then recombinant FSH products which are currently on the market.
Example 10A Multiple Dose Study Investigating FE 999049 in Comparison to GONAL-F
[0129] 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.
[0130] 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.
[0131] Subjects were assessed within 3 months prior to randomisation for compliance with the inclusion and exclusion criteria, including an anti-Mllerian 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.
[0132] 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)).
[0133] Gonal-F is filled by mass (FbM) at FDA request; referring to g dose is therefore appropriate. The Gonal-F label indicates 600 IU/44 g, which indicates that 150 IU is 11 g. However, there is some variation and the batch certificate for this trial indicated that 11.3 g Gonal-F was equivalent to 150 IU. The FE999049 doses are presented by protein content (g) rather than biological activity. Thus the doses of FE999049 were 5.2 g (90 IU), 6.9 g (120 IU), 8.6 g (150 IU), 10.3 g (180 IU) or 12.1 g (210 IU).
[0134] The subject and dose distribution is set out as follows (data are number of subjects):
TABLE-US-00005 TABLE 1 GONAL-F FE 999049 11.3 (11) 5.2 g 6.9 g 8.6 g 10.3 g 12.1 g g Total Screened 334 Randomised 42 45 44 45 46 43 265 and exposed High AMH 23 26 24 24 26 25 148 strata (56%) (15.0-44.9 pmol/L) Low AMH 19 19 20 20 21 18 117 strata (44%) (5.0-14.9 pmol/L) Per-protocol 40 42 42 44 44 43 255
[0135] 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.
[0136] 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 g 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 mm, the treatment is cancelled. In case of poor ovarian response, defined as <3 follicles with a diameter 10 mm observed on stimulation day 10, the cycle could be cancelled.
[0137] 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 hCG 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
[0138] The number of oocytes retrieved (primary endpoint) is shown in the following Table.
TABLE-US-00006 TABLE 2 GONAL- F FE 999049 11.3 (11) 5.2 g 6.9 g 8.6 g 10.3 g 12.1 g g Oocytes retrieved 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 5.9 (3.9) 9.1 (6.4) 10.6 (4.8) 13.6 (7.8) 14.4 (5.8) 12.4 (5.4) AMH Low 4.5 (2.2) 6.3 (4.9) 7.4 (3.8) 6.9 (3.6) 9.4 (4.9) 7.8 (3.4) AMH Data are mean (SD)
[0139] 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.
[0140] 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).
[0141] 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.
[0142] There were no safety concerns with the use of FE 999049. A good local tolerability was documented.
Further Analysis
[0143] 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: [0144] Oocytes retrieved in the range 8-14 [0145] Minimise proportion of patients with <8 oocytes [0146] Minimise proportion of patients with 20 oocytes
[0147] The applicants also investigated the impact of body weight. If relevant, the dose is converted into g/kg for an average subject. This value of g/kg and 0.01 g/kg are evaluated in a model with respect to distribution of oocytes retrieved as well as safety profile, and the optimal dose is identified.
Low AMH Strata
[0148] As seen in Table 2, the dose of FE999049 which fulfilled the first criterion (Oocytes retrieved in the range 8-14) was 12.1 g (mean 9.4 oocytes retrieved). The distribution of oocytes is shown in Table 3 below.
[0149] As shown by the box and arrow, a dose of 12.1 g 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).
[0150] Table 4 below shows the analysis of signs of excessive response in the low AMH strata (data are number of subjects). It can be seen that 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 g FE999049 in a patient having low AMH.
TABLE-US-00007 TABLE 4 GONAL-F FE 999049 11.3 (11) 5.2 g 6.9 g 8.6 g 10.3 g 12.1 g g All subjects 19 19 20 20 21 18 Early OHSS, 0 0 0 0 0 0 mod/sev GnRH agonist 0 0 0 0 0 0 triggering Preventive action* 0 0 0 0 0 0 15 oocytes 0 2 1 0 2 1 Any of the above 0 2 1 0 2 1
[0151]
[0152] Thus the applicants have found that a dose of, or dose equivalent to, 6 to 18 g, for example 9 to 14 g, for example 12 g, 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.
High AMH Strata
[0153] As seen in Table 2, three doses of FE999049 fulfilled the first criterion (oocytes retrieved in the range 8-14): 6.9 g (mean 9.1 oocytes retrieved), 8.6 g (mean 10.6 oocytes retrieved), and 10.3 g (mean 13.6 oocytes retrieved).
[0154]
[0155] Table 5a below shows a further breakdown of oocytes retrieved (from Table 2) by AMH. This shows the doses which fulfilled the first criterion (oocytes retrieved in the range 8-14) for each sub strata of AMH (boxes).
[0156] Table 5 b below shows the analysis of patients where treatment was cancelled due to either excessive response or agonist triggering, for these subgroups. For example, one patient in the 25-34 pmol/L AMH strata cancelled due to excessive response following the dose of 10.3 g and one patient in the 25-34 pmol/L AMH strata cancelled due to excessive response following the dose of 12.1 g; one patient in the 35-45 pmol/L AMH strata cancelled following agonist triggering following dose of 10.3 g; and one patient in the 35-45 pmol/L AMH strata cancelled following agonist triggering following dose of 6.9 g.
TABLE-US-00008 TABLE 5b FE 999049 5.2 g 6.9 g 8.6 g 10.3 g 12.1 g OHSS***, cancellation due to excessive response or agonist triggering**** 15-24 pmol/L 0 0 0 0 0 25-34 pmol/L 0 0 0 1*** 1*** 35-45 pmol/L 0 1**** 0 1**** 0
[0157] It can be seen therefore that tailoring of dose by bodyweight (
[0158] The applicants have found that the following doses provide an effective response while minimising risk of OHSS (kg is kg body weight of patient).
TABLE-US-00009 Serum AMH dose (Max dose) <15 pmol/L 12 g (12 g) 15-24 pmol/L 0.14-0.19 g/kg, for example 0.15-0.16 (12 g) g/kg, preferably 0.15 g/kg 25-34 pmol/L 0.11-0.14 g/kg; for example 0.12-0.13 (12 g) g/kg, preferably 0.13 g/kg 35 pmol/L 0.10-0.11 g/kg, preferably 0.11 g/kg (12 g)
[0159] The following are appropriate if dosing by bodyweight is not desired.
TABLE-US-00010 Serum AMH dose (Max dose) <15 pmol/L 12 g 12 g 15-24 pmol/L 9.3-10 g (12 g) 25-34 pmol/L 7.3-8 gg (12 g) 35 pmol/L 6.3-7 (12 g)
[0160] The following are appropriate if fewer categories of AMH are required.
TABLE-US-00011 4 AMH 3 AMH 2 AMH categories categories categories One dose AMH Dose AMH Dose AMH Dose AMH Dose <15 12 g <15 12 g <15 12 g 0.16 g/kg 15-24 0.15-0.16 15-24 0.15-0.16 15 0.14 g/kg g/kg g/kg 25-34 0.12-0.13 25 0.12 g/kg g/kg 35 0.10-0.11 g/kg
[0161] The following are appropriate if dosing by bodyweight is not desired.
TABLE-US-00012 4 AMH categories 3 AMH categories 2 AMH categories One dose AMH Dose AMH Dose AMH Dose AMH Dose <15 12 g <15 12 g <15 12 g 9.3 g or 10 g 15-24 9.3-10 g 15-24 9.3-10 g 15 8.7 g 25-34 7.3-8 g 25 7.3 g 35 6.3-7 g
[0162] Thus the applicants have found that a dose of, or dose equivalent to, 9 to 14 g, for example 12 g, 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.
[0163] The applicants have found that a dose of, or dose equivalent to, 5 to 12.5 g, for example 6 to 10.5 g, human derived recombinant FSH is suitable for use in the treatment of infertility in a patient having serum AMH pmol/L. The dose provides an effective response while minimising risk of OHSS.
[0164] The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.09 to 0.19 g human derived recombinant FSH per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of 15 pmol/L. The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.14 to 0.19 g human derived recombinant FSH (preferably 0.15 to 0.16 g human derived recombinant FSH) per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of 15 to 24.9 pmol/L. The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.11 to 0.14 g human derived recombinant FSH (preferably 0.12 to 0.13 g human derived recombinant FSH) per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of 25 to 34.9 pmol/L. The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.10 to 0.11 g human derived recombinant FSH per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of 35 pmol/L. These doses provide an effective response while minimising risk of OHSS.
[0165] The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.15 to 0.21 g (e.g. 0.16 g) human derived recombinant FSH per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of <15 pmol/L, for example for the first stimulation cycle with human derived recombinant FSH. However, it is not required that patients are dosed by body weight at this level of AMH.
Example 10AIndividualised COS Protocol (Low AMH)
[0166] 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).
[0167] 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 <14.9 pmol/L would be administered an initial daily dose of approximately 12 g FE 999049, a human derived recombinant FSH product manufactured according to the method of Example 6. A patient with an AMH level of 15 to 24.9 pmol/L would receive an initial daily dose of 0.15 to 0.19 g of the human derived recombinant FSH per kg bodyweight of the patient. A patient with an AMH level of 25 to 34.9 pmol/L would receive an initial daily dose of 0.11 to 0.13 g of the human derived recombinant FSH per kg bodyweight of the patient. A patient with an AMH level of 35 pmol/L would receive an initial daily dose of 0.10 to 0.11 g of the human derived recombinant FSH per kg bodyweight of the patient.
Example 11Individualised COS Protocols
[0168] The doses in this protocol are less preferred that Example 10A.
[0169] 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.
[0170] The COS protocol proceeds in the usual manner apart from administration of the initial dose of FE 999049 according to AMH level at screening in line with the following table. Thus a patient with an AMH level of 5-14.8 pmol/L would be administered 180 IU FSH in the form of approximately 8-11 g FE 999049, a human derived recombinant FSH product manufactured according to the method of Example 6. A patient with an AMH level of 30-44.9 pmol/L would be administered 120 IU FSH in the form of approximately 4-7 g FE 999049, a human derived recombinant FSH product manufactured according to the method of Example 6. If the AMH level is not available, the patient recombinant would be administered 120-180 IU FSH in the form of approximately 6-11 g FE 999049, a human derived recombinant FSH product manufactured according to the method of Example 6.
TABLE-US-00013 Starting Approx AMH Level Dose FSH equivalent in g <5 pmol/l 210 IU 10-15 g 5-14.9 pmol/l 180 IU 8-11 g >15-29.9 pmol/l 150 IU 6-9 g >30-44.9 pmol/l 120 IU 4-7 g >45 pmol/l 90 IU 2-5 g Not Available 120-180 IU 6-11 g
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TABLE-US-00014 Folliclestimulatinghormonealphapolypeptide AccessionnumberAH007338 NucleotidesequenceofFSHalpha SEQIDNO:1 1 ATGGATTACTACAGAAAATATGCAGCTATCTTTCTGGTCACATTGTCGGTGTTTCTGCAT 61 GTTCTCCATTCCGCTCCTGATGTGCAGGATTGCCCAGAATGCACGCTACAGGAAAACCCA 121 TTCTTCTCCCAGCCGGGTGCCCCAATACTTCAGTGCATGGGCTGCTGCTTCTCTAGAGCA 181 TATCCCACTCCACTAAGGTCCAAGAAGACGATGTTGGTCCAAAAGAACGTCACCTCAGAG 241 TCCACTTGCTGTGTAGCTAAATCATATAACAGGGTCACAGTAATGGGGGGTTTCAAAGTG 301 GAGAACCACACGGCGTGCCACTGCAGTACTTGTTATTATCACAAATCTTAA ProteinsequenceofFSHalpha (SEQIDNO:5) 1 MDYYRKYAAIFLVTLSVFLHVLHSAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRA 61 YPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS Folliclestimulatinghormonebetapolypeptide AccessionnumberNM_000510 NucleotidesequenceofFSHbeta SEQIDNO:2 1 ATGAAGACACTCCAGTTTTTCTTCCTTTTCTGTTGCTGGAAAGCAATCTGCTGCAATAGC 61 TGTGAGCTGACCAACATCACCATTGCAATAGAGAAAGAAGAATGTCGTTTCTGCATAAGC 121 ATCAACACCACTTGGTGTGCTGGCTACTGCTACACCAGGGATCTGGTGTATAAGGACCCA 181 GCCAGGCCCAAAATCCAGAAAACATGTACCTTCAAGGAACTGGTATATGAAACAGTGAGA 241 GTGCCCGGCTGTGCTCACCATGCAGATTCCTTGTATACATACCCAGTGGCCACCCAGTGT 301 CACTGTGGCAAGTGTGACAGCGACAGCACTGATTGTACTGTGCGAGGCCTGGGGCCCAGC 361 TACTGCTCCTTTGGTGAAATGAAAGAATAA ProteinsequenceofFSHbeta (SEQIDNO:6) 1 MKTLQFFFLFCCWKAICCNSCELTNITIAIEKEECRFCISINTTWCAGYCYTRDLVYKDP 61 ARPKIQKTCTFKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCDSDSTDCTVRGLGPS 121 YCSFGEMKE Beta-galactosidealpha-2,3-sialyltransferase4 AccessionNumberL23767 NucleotidesequenceofST3GAL4 SEQIDNO:3 1 ATGTGTCCTGCAGGCTGGAAGCTCCTGGCCATGTTGGCTCTGGTCCTGGTCGTCATGGTG 61 TGGTATTCCATCTCCCGGGAAGACAGGTACATCGAGCTTTTTTATTTTCCCATCCCAGAG 121 AAGAAGGAGCCGTGCCTCCAGGGTGAGGCAGAGAGCAAGGCCTCTAAGCTCTTTGGCAAC 181 TACTCCCGGGATCAGCCCATCTTCCTGCGGCTTGAGGATTATTTCTGGGTCAAGACGCCA 241 TCTGCTTACGAGCTGCCCTATGGGACCAAGGGGAGTGAGGATCTGCTCCTCCGGGTGCTA 301 GCCATCACCAGCTCCTCCATCCCCAAGAACATCCAGAGCCTCAGGTGCCGCCGCTGTGTG 361 GTCGTGGGGAACGGGCACCGGCTGCGGAACAGCTCACTGGGAGATGCCATCAACAAGTAC 421 GATGTGGTCATCAGATTGAACAATGCCCCAGTGGCTGGCTATGAGGGTGACGTGGGCTCC 481 AAGACCACCATGCGTCTCTTCTACCCTGAATCTGCCCACTTCGACCCCAAAGTAGAAAAC 541 AACCCAGACACACTCCTCGTCCTGGTAGCTTTCAAGGCAATGGACTTCCACTGGATTGAG 601 ACCATCCTGAGTGATAAGAAGCGGGTGCGAAAGGGTTTCTGGAAACAGCCTCCCCTCATC 661 TGGGATGTCAATCCTAAACAGATTCGGATTCTCAACCCCTTCTTCATGGAGATTGCAGCT 721 GACAAACTGCTGAGCCTGCCAATGCAACAGCCACGGAAGATTAAGCAGAAGCCCACCACG 781 GGCCTGTTGGCCATCACGCTGGCCCTCCACCTCTGTGACTTGGTGCACATTGCCGGCTTT 841 GGCTACCCAGACGCCTACAACAAGAAGCAGACCATTCACTACTATGAGCAGATCACGCTC 901 AAGTCCATGGCGGGGTCAGGCCATAATGTCTCCCAAGAGGCCCTGGCCATTAAGCGGATG 961 CTGGAGATGGGAGCTATCAAGAACCTCACGTCCTTCTGA ProteinSequenceofST3GAL4 (SEQIDNO:7) 1 MCPAGWKLLAMLALVLVVMVWYSISREDRYIELFYFPIPEKKEPCLQGEAESKASKLFGN 61 YSRDQPIFLRLEDYFWVKTPSAYELPYGTKGSEDLLLRVLAITSSSIPKNIQSLRCRRCV 121 VVGNGHRLRNSSLGDAINKYDVVIRLNNAPVAGYEGDVGSKTTMRLFYPESAHFDPKVEN 181 NPDTLLVLVAFKAMDFHWIETILSDKKRVRKGFWKQPPLIWDVNPKQIRILNPFFMEIAA 241 DKLLSLPMQQPRKIKQKPTTGLLAITLALHLCDLVHIAGFGYPDAYNKKQTIHYYEQITL 301 KSMAGSGHNVSQEALAIKRMLEMGAIKNLTSF Beta-galactosamidealpha-2,6-sialyltransferase1 AccessionnumberNM_003032 NucleotidesequenceofST6GAL1 SEQIDNO:4 1 ATGATTCACACCAACCTGAAGAAAAAGTTCAGCTGCTGCGTCCTGGTCTTTCTTCTGTTT 61 GCAGTCATCTGTGTGTGGAAGGAAAAGAAGAAAGGGAGTTACTATGATTCCTTTAAATTG 121 CAAACCAAGGAATTCCAGGTGTTAAAGAGTCTGGGGAAATTGGCCATGGGGTCTGATTCC 181 CAGTCTGTATCCTCAAGCAGCACCCAGGACCCCCACAGGGGCCGCCAGACCCTCGGCAGT 241 CTCAGAGGCCTAGCCAAGGCCAAACCAGAGGCCTCCTTCCAGGTGTGGAACAAGGACAGC 301 TCTTCCAAAAACCTTATCCCTAGGCTGCAAAAGATCTGGAAGAATTACCTAAGCATGAAC 361 AAGTACAAAGTGTCCTACAAGGGGCCAGGACCAGGCATCAAGTTCAGTGCAGAGGCCCTG 421 CGCTGCCACCTCCGGGACCATGTGAATGTATCCATGGTAGAGGTCACAGATTTTCCCTTC 481 AATACCTCTGAATGGGAGGGTTATCTGCCCAAGGAGAGCATTAGGACCAAGGCTGGGCCT 541 TGGGGCAGGTGTGCTGTTGTGTCGTCAGCGGGATCTCTGAAGTCCTCCCAACTAGGCAGA 601 GAAATCGATGATCATGACGCAGTCCTGAGGTTTAATGGGGCACCCACAGCCAACTTCCAA 661 CAAGATGTGGGCACAAAAACTACCATTCGCCTGATGAACTCTCAGTTGGTTACCACAGAG 721 AAGCGCTTCCTCAAAGACAGTTTGTACAATGAAGGAATCCTAATTGTATGGGACCCATCT 781 GTATACCACTCAGATATCCCAAAGTGGTACCAGAATCCGGATTATAATTTCTTTAACAAC 841 TACAAGACTTATCGTAAGCTGCACCCCAATCAGCCCTTTTACATCCTCAAGCCCCAGATG 901 CCTTGGGAGCTATGGGACATTCTTCAAGAAATCTCCCCAGAAGAGATTCAGCCAAACCCC 961 CCATCCTCTGGGATGCTTGGTATCATCATCATGATGACGCTGTGTGACCAGGTGGATATT 1021 TATGAGTTCCTCCCATCCAAGCGCAAGACTGACGTGTGCTACTACTACCAGAAGTTCTTC 1081 GATAGTGCCTGCACGATGGGTGCCTACCACCCGCTGCTCTATGAGAAGAATTTGGTGAAG 1141 CATCTCAACCAGGGCACAGATGAGGACATCTACCTGCTTGGAAAAGCCACACTGCCTGGC 1201 TTCCGGACCATTCACTGCTAA Op-ProteinSequenceofST6GAL1 (SEQIDNO:8) 1 MIHTNLKKKFSCCVLVFLLFAVICVWKEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDS 61 QSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMN 121 KYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGP 181 WGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTE 241 KRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQM 301 PWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFF 361 DSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC