WO1995019991A1 - New composition of glycoprotein isoforms having follicle stimulating activity - Google Patents

New composition of glycoprotein isoforms having follicle stimulating activity Download PDF

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Publication number
WO1995019991A1
WO1995019991A1 PCT/EP1995/000212 EP9500212W WO9519991A1 WO 1995019991 A1 WO1995019991 A1 WO 1995019991A1 EP 9500212 W EP9500212 W EP 9500212W WO 9519991 A1 WO9519991 A1 WO 9519991A1
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Prior art keywords
isoforms
mean
fsh
org
glycoprotein
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Application number
PCT/EP1995/000212
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English (en)
French (fr)
Inventor
Renato De Leeuw
Ferdinand Rombout
Original Assignee
Akzo Nobel N.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Akzo Nobel N.V. filed Critical Akzo Nobel N.V.
Priority to EP95907574A priority Critical patent/EP0739352A1/en
Priority to BR9506557A priority patent/BR9506557A/pt
Priority to JP7519345A priority patent/JPH09508114A/ja
Priority to AU15751/95A priority patent/AU1575195A/en
Publication of WO1995019991A1 publication Critical patent/WO1995019991A1/en
Priority to FI962923A priority patent/FI962923A/fi

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is concerned with a pharmaceutical composition
  • a pharmaceutical composition comprising a mixture of isoforms of a glycoprotein with follicle stimulating activity as well as an improvement in a method to induce follicular growth and maturation.
  • Follicle stimulating hormone (FSH) produced by the anterior pituitary plays a major role in female and male reproduction by stimulating gonadal differentiation and maturation via its regulatory action on the Sertoli cell in the testes and granulosa cell in the ovary.
  • FSH is produced and secreted by the pituitary in different molecular forms (isohormones or isoforms), which vary in overall charge, receptor binding affinity, biological activity, and plasma residence time. This micro-heterogeneity is due to differences in the amount and/or composition of the carbohydrate residues, in particular sialic acid. Multiple forms of gonadotropins have been isolated and characterised from anterior pituitary glands, serum and urine of several non-mammalian and mammalian species, including man. Relatively acidic FSH isoforms, which are more heavily sialylated, exhibit lower receptor affinity and in vitro biological activities than more basic isoformes. However, due to their longer plasma residence time these more acidic forms have greater in vivo biological activities (UUoa- Aguirre et al., 1988, Hum.Reprod., 3, 491-501).
  • FSH is used for ovulation induction and controlled ovarian stimulation in in vitro fertization (INF).
  • the aim of controlled superovulation is to increase the number of retrievable mature oocytes for INF and subsequent embryo transfer (ET). Generally, up to three embryos are replaced per transfer. As usually more than one treatment is necessary, in most infertility clinics spare embryos or fertilized oocytes are frozen and transferred in subsequent cycles. Assuming normal fertilization, the more oocytes retrieved the higher the number of possible transfers and thus the higher the chance of a woman to become pregnant after one treatment cycle. In case of male infertility, the chances of establishing fertilization, and thus pregnancy, also increases with the number of oocytes recovered.
  • FSH preparations have been used which have been isolated from natural sources. Isohormone distribution profiles of commercially available preparations have been reported (e.g. Harlin et al, 1986, Fert. Ster., 46, 1055-1061). It appears that these FSH compositions consist of relatively acidic isohormone fractions.
  • glycoprotein isoforms with isoelectric points in between and extending from the range 4.8 to 4.2 , having follicle stimulating activity which consists for more than 15% of isoforms with isoelectric points above 4.8 and for less than 30% of isoforms with isoelectric points below 4.2 , when used in the same clinical settings, exert a better effect than the known glycoprotein compositions.
  • glycoprotein isohormone mixture according to the invention contains a relatively high proportion of relatively basic isoforms.
  • This can be established by having glycoprotein isoform compositions having more than 15% of the isoforms with isoelectric points above 4.8 and less than 30% below 4.2 , also preferably, isoforms mixtures can be used wherein more than 15% of the isoforms have isoelectric points above 4.8 and less than 25% below 4.2 , also preferably more than 25% of the isoforms have isoelectric points above 4.8 and less than 25% below 4.2 , also preferably more than 25% of the isoforms have isoelectric points above 4.8 and less than 20% below 4.2
  • the percentage of the isoforms as used herein is defined as the relative amount of immunoreactive isoforms recovered after chromatofocussing.
  • protein contents can be determined by colorimetric assays.
  • glycoproteins according to this invention may be derived from urinary origin.
  • the number and relative amount of each isoform species depends on the source
  • the glycoprotein might also be a recombinant glycoprotein.
  • recombinant FSH FSH
  • the charge heterogeneity is determined by the host cell-line chosen for its production as well as cell culture conditions.
  • glycoproteins such as FSH
  • CHO Chinese Hamster Ovary
  • a recombinant FSH preparation according to this invention can be produced by a CHO cell line stably transfected with a plasmid containing the two subunit genes encoding human FSH (hFSH).
  • Such a CHO cell line produces intact, glycosylated hFSH that is secreted into the culture medium, which is the source for further purification. Batches like this can be prepared as described (van Wezenbeek et al, 1990, in: From Clone to Clinic, Kluwer Academic Publishers, 245-251). Previous preclinical studies demonstrated that the receptor binding affinity and in vitro and in vivo efficacy of recFSH are compatible to those of FSH isolated from natural sources (Mannaerts et al., 1991, Endocrinology, 129, 2623-2630).
  • FSH preparations according to the invention can be selected based upon their chromatofocusing profile.
  • the profile can be influenced by a particular host cell line choice or by adaptation of culture conditions.
  • glycoprotein isoform mixtures according to the invention can be isolated from cell lines the glycoprotein gene expression of which has been turned on by site specific targeting of a regulatory sequence to the glycoprotein gene.
  • basic isohormones can be obtained by expression of recombinant FSH in cell lines which are impaired in glycosylation.
  • a cell line might be e.g. a cell line deficient in the enzyme N-acetylglucosamine transferase or in sialic acid transport into the Golgi (Galway et al., 1990, Endocrinology, 127, 93-100).
  • inventions are basic glycoprotein isoforms mixtures obtained by enzymatic or chemical modification. With such a treatment parts of the carbohydrate chains can be removed without affecting the amino acid sequence. Glycoprotein batches can e.g. be treated with HF (Chen et al, 1982, J.Biol.Chem., 257, 14446- 14452). Partial desialylation can be performed by enzymatic hydrolysis with neuraminidase (Vaitukaitis and Ross, 1971, J.Clin.Endocrinol.- Metab., 55, 308-311).
  • basic glycoprotein hormone mixtures having biological i.e. follicle stimulating activity which are prepared by removing one or more N-linked oligosaccharide chains from either one of the subunits. These chains can be removed by site- directed mutagenesis of the gene encoding the glycoprotein. In this way a single amino acid change can be obtained in such a way that the carbohydrate attachment site is no longer present. Consequently, one or several carbohydrate chains cannot be attached on the a or ⁇ subunit (Matzuk and Boime, 1988, J.Cell Biol., 106, 1049-1059).
  • the mixture of relatively basic isohormone forms can be used according to the invention for the manufacture of a medicament to be used e.g. in the treatment of ovulation induction or controlled ovarian stimulation.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising these glycoprotein isoforms admixed with pharmaceutically acceptible auxiliairies.
  • Methods for making preparations and admixtures are disclosed in Remingtons 's Pharmaceutical Sciences, pp. 1463-1497 (16th ed. 1980, Mack Publ. Co of Easton, Pa, USA).
  • ampoules containing the pharmaceutical composition according to the invention may contain 1 to 1000 ⁇ g of the glycoprotein mixture (e.g. 75 IU is considered a therapeutic amount).
  • the invention also relates to an improved method to induce follicular growth and maturation wherein the pharmaceutical composition containing the mixture of basic glycoprotein isoforms as described above is used. According to the present invention there is no need to include in the treatment schedule the administration of more acidic isoform mixtures.
  • Another improvement of the method consists of a repeated administration of pharmaceutical compositions containing a mixture of isoforms of a glycoprotein with follicle stimulating activity, each composition having identical, i.e. having the same isoform profile. Thus, during a treatment the administration of only relatively basic isoforms is sufficient.
  • Such mixtures can also be prepared by isolation of only basic isoforms e.g. by preparative chromatofocussing. Preferably such isoforms have an isoelectric point above 4.2 . It will be clear that fractionation of isoform mixtures can be performed on glycoprotein batches obtained from different origins such as preparations isolated from urine or recombinant DNA cell lines which may or may not be chemically or enzymatically modified.
  • compositions according to this invention can be used in clinical treatments in combination with e.g. GnRH antagonists or agonists and/or LH activity e.g. HCG or LH to induce superovulation.
  • GnRH antagonists or agonists and/or LH activity e.g. HCG or LH to induce superovulation.
  • LH activity e.g. HCG or LH to induce superovulation.
  • hFSH isoelectric point
  • Chromatofocusing was performed in the range of pH 6-3 on a fast protein liquid chromatography (FPLC) column HR 5/20 (Pharmacia, Woerden, The Netherlands) packed with polybuffer exchanger 94
  • FSH immunoreactivity was measured in a two-site sandwich enzyme immunoassay, using a ⁇ -directed capturing antibody (monoclonal antibody 4B) and an ⁇ -directed HRP-labeled detection antibody (monoclonal antibody 116B) as described previously (Mannaerts et al., 1991, Endocrinology, 129, 2623-2630). This assay recognises only intact dimers and was found to detect all FSH isoforms equally well.
  • the assay sensitivity in terms of IS 70/45 was 0.4 IU/1 and the intra- and interassay coefficients of variations were 7% and 8%, respectively.
  • the cross-reactivity with hLH and hCG was ⁇ 0.01 % and ⁇ 0.01 % , respectively
  • the Org 32489 and Metrodin clinical preparations used to compare the clinical efficacy showed the same difference in the relative contribution of basic and acidic isoforms, i.e. the Org 32489 CP's contained more basic isoforms than the Metrodin CP's (17.120.2 % vs 12.7 % if pi > 4.8).
  • the study was designed as a multicentre, randomized, assessor- blind, group-comparative study, in which safety and efficacy of Org 32489 and Metrodin R were compared in infertile pituitary-suppressed subjects undergoing in vitro fertilisation (IVF) and embryo transfer (ET). Approximately one thousand subjects were included in this study with a ratio between subjects treated with Org 32489 and with Metrodin R of 3:2. The study period covered no more than 3 treatment cycles. Analysis of efficacy included first treatment cycles only.
  • Inclusion criteria At least 18 and at most 39 years of age at the time of screening;
  • Infertility caused by endocrine abnormalities such as hyperprolactinaemia, polycystic ovary syndrome, and absence of ovarian function;
  • Male infertility defined according to the following criteria: ⁇ 10x10 ⁇ sperms per mL and/or ⁇ 40% normal morphology and/or 40% normal motility;
  • Buserelin treatment was started on the first day of the menstrual cycle.
  • the first day of the menstrual cycle was defined as the first day the subject waked up with menstrual bleeding.
  • Treatment with Org 32489 (75 IU/ampoule) or Metrodin R (75 IU/ampoule) was started 14 to 18 days after the onset of the buserelin intake when this treatment had resulted in an hypogonadotropic state (i.e. , serum E2 ⁇ 50 pg/mL). In case this state had not been achieved, Org 32489 or Metrodin treatment was postponed and the dose of buserelin was increased to 4 x 300 ⁇ g/day.
  • HCG (10.000 IU) was administered when at least three follicles > 17 mm were present. No more than three embryos were replaced and frozen embryos were replaced in natural cycles or stimulated cycles. A cycle was considered cancelled if no embryo transfer had taken place.
  • the luteal phase was monitored by progesterone assessments.
  • Luteal phase support e.g. three injections of 1500 IU hCG or at least 50 mg progesterone i.m. per day or 400 mg progesterone intravaginally per day was given for at least two weeks after the injection of 10.000 IU hCG.
  • the total number of oocytes recovered in the first treatment cycle was a primary efficacy variable.
  • Other analysed variables in the first treatment cycle were the number of FSH ampoules administered for ovarian stimulation and the duration of FSH treatment.
  • the outcome of these parameters were analysed by means of Cochran's method of combining individual centre results.
  • the Wilcoxon rank sum test and an analysis of variance (ANOVA) were applied to consolidate the outcome of Cochran's method.
  • the number of oocytes recovered in each treatment group per centre is presented in Table 3.
  • the overall mean number of oocytes was 10.84 oocytes in the Org 32489 group and 8.95 oocytes in the Metrodin group, resulting in a treatment difference of 1.89 oocytes in favour of Org 32489.
  • the difference of 1.89 oocytes is more than 5 times its standard error and highly significant (P ⁇ 0.0001).
  • the resulting 95% confidence interval indicated that on the average subjects treated with Org 32489 end up with at least 1.2 and at most 2.6 oocytes more than those treated with Metrodin R . Exclusion of immature oocytes from the total number of recovered oocytes did not influence the treatment effect (see Table 4).
  • the total number of ampoules administered to punctured subjects is presented per centre in Table 5.
  • the total dosage of Org 32489 administered was in 14 out of the 18 centres lower than the total dosage of Metrodin .
  • the overall mean FSH dosage was 28.5 ampoules in the Org 32489 group and 31.8 ampoules in the Metrodin group, resulting in a treatment difference of 3.3 ampoules which is highly statistically significant (P ⁇ 0.0001).
  • the duration of FSH treatment of punctured subjects is presented per centre in Table 6.
  • the treatment duration was in 13 out of 18 centres shorter in the Org 32489 group than in the Metrodin group.
  • the overall mean treatment duration (weighted over centres) was 10.7 days in the Org 32489 group and 11.3 days in the Metrodin group, resulting in a treatment difference of 0.6 days which is highly statistically significant (P ⁇ 0.0001).
  • the resulting 95% confidence interval indicated that on the average for subjects treated with Org 32489 the treatment period is minimally 0.3 day and maximally 0.9 day shorter than for those treated with Metrodin R .
  • Percentage basic isoforms defined as FSH immunoreactivity at pi > 4.8
  • percentage acidic isoforms defined as FSH immunoreactivity at pi ⁇ 4.2 of the Org 32489 and Metrodin R CP's used to compare the clinical efficacy (see Example 2).

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  • Health & Medical Sciences (AREA)
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  • Endocrinology (AREA)
  • Organic Chemistry (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
PCT/EP1995/000212 1994-01-20 1995-01-20 New composition of glycoprotein isoforms having follicle stimulating activity WO1995019991A1 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP95907574A EP0739352A1 (en) 1994-01-20 1995-01-20 New composition of glycoprotein isoforms having follicle stimulating activity
BR9506557A BR9506557A (pt) 1994-01-20 1995-01-20 Composição farmacêutica e processo para induzir o crescimento e maturação folicular em individuos do sexo feminino
JP7519345A JPH09508114A (ja) 1994-01-20 1995-01-20 卵胞刺激活性を有する糖タンパク質イソ型新規組成物
AU15751/95A AU1575195A (en) 1994-01-20 1995-01-20 New composition of glycoprotein isoforms having follicle stimulating activity
FI962923A FI962923A (fi) 1994-01-20 1996-07-19 Uusi glykoproteiini-isomuotokoostumus, jolla on follikkelia stimuloiva aktiivisuus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP94200140 1994-01-20
EP94200140.5 1994-01-20

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WO1995019991A1 true WO1995019991A1 (en) 1995-07-27

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EP (1) EP0739352A1 (ja)
JP (1) JPH09508114A (ja)
CN (1) CN1138864A (ja)
AU (1) AU1575195A (ja)
BR (1) BR9506557A (ja)
CA (1) CA2181711A1 (ja)
FI (1) FI962923A (ja)
HU (1) HUT76660A (ja)
WO (1) WO1995019991A1 (ja)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000032222A1 (en) * 1998-12-01 2000-06-08 Akzo Nobel N.V. Improvement of folliculogenesis
US9642850B2 (en) 1997-02-24 2017-05-09 Purdue Pharma L.P. Method of providing sustained analgesia with buprenorphine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2300573A1 (fr) * 1975-02-17 1976-09-10 Serono Lab Composition pharmaceutique a base de gonadotrophine chorionique humaine partiellement desialylee
EP0322226A2 (en) * 1987-12-21 1989-06-28 Applied Research Systems ARS Holding N.V. Site-directed mutagenesis modified glycoprotein hormones and methods of use
EP0388223A2 (en) * 1989-03-17 1990-09-19 Applied Research Systems ARS Holding N.V. Novel method of ovulation induction in humans

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2300573A1 (fr) * 1975-02-17 1976-09-10 Serono Lab Composition pharmaceutique a base de gonadotrophine chorionique humaine partiellement desialylee
EP0322226A2 (en) * 1987-12-21 1989-06-28 Applied Research Systems ARS Holding N.V. Site-directed mutagenesis modified glycoprotein hormones and methods of use
EP0388223A2 (en) * 1989-03-17 1990-09-19 Applied Research Systems ARS Holding N.V. Novel method of ovulation induction in humans

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"MORE BASIC FORMS OF BOTH HUMAN FOLLLICLE-STIMULATING HORMONE AND LUTEINIZING HORMONE IN SERUM AT MIDCYCLE COMPARED WITH THE FOLLICULAR OR LUTEAL PHASE", THE JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM, vol. 76, PHILADELPHIA,USA, pages 885 - 889 *
FILE SERVER STN KARLSRUHE,FILE MEDLINE ABSTRACT NO.93209440 & FERTIL STERIL, (1993 APR) 59 (4) 738-42 *
FILE SERVER STN KARLSRUHE,FILE MEDLINE ABSTRACT NO.93346595 & HUM REPROD, (1993 JUN) 8 (6) 863-5 *
HARLIN ET AL: "MOLECULAR COMPOSITION OF LUTEINIZING HORMONE AND FOLLICLE-STIMULATING HORMONE IN COMMERCIAL GONADOTROPIN PREPARATIONS", FERTILITY AND STERILITY, vol. 46, BIRMINGHAM,ALA.,USA, pages 1055 - 1061 *
MATIKAINEN ET AL: "CIRCULATING BIOACTIVE AND IMMUNOREACTIVE RECOMBINANT HUMAN FOLLICLE STIMULATING HORMONE (ORG 32489) AFTER ADMINISTRATION TO GONADOTROPIN-DEFECIENT SUBJECTS", FERTILITY AND STERILITY, vol. 61, BIRMINGHAM,ALA.,USA, pages 62 - 69 *
PADMANABHAN ET AL: "MODULATION OF SERUM FOLLICLE-STIMULATING HORMONE BIOACTIVITY AND ISOFORM DISTRIBUTION BY ESTROGENIC STEROIDS IN NORMAL WOMEN AND IN GONADAL DYSGENESIS", THE JOURNAL OF CLINICAL ENDOCRINOLOGY AND METBOLISM, vol. 67, PHILADELPHIA,USA, pages 465 - 473 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9642850B2 (en) 1997-02-24 2017-05-09 Purdue Pharma L.P. Method of providing sustained analgesia with buprenorphine
WO2000032222A1 (en) * 1998-12-01 2000-06-08 Akzo Nobel N.V. Improvement of folliculogenesis
US6660717B1 (en) 1998-12-01 2003-12-09 Akzo Nobel, N.V. Folliculogenesis

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HUT76660A (en) 1997-10-28
HU9601975D0 (en) 1996-09-30
CA2181711A1 (en) 1995-07-27
FI962923A0 (fi) 1996-07-19
BR9506557A (pt) 1997-10-28
JPH09508114A (ja) 1997-08-19
CN1138864A (zh) 1996-12-25
EP0739352A1 (en) 1996-10-30
FI962923A (fi) 1996-07-19
AU1575195A (en) 1995-08-08

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