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

• 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).
• 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.
• 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).
• 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).
• 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 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;
• 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).
• 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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• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
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• 1995
  • 1995-01-20 JP JP7519345A patent/JPH09508114A/ja active Pending
  • 1995-01-20 AU AU15751/95A patent/AU1575195A/en not_active Abandoned
  • 1995-01-20 CA CA002181711A patent/CA2181711A1/en not_active Abandoned
  • 1995-01-20 WO PCT/EP1995/000212 patent/WO1995019991A1/en not_active Ceased
  • 1995-01-20 BR BR9506557A patent/BR9506557A/pt not_active Application Discontinuation
  • 1995-01-20 HU HU9601975A patent/HUT76660A/hu unknown
  • 1995-01-20 FI FI962923A patent/FI962923L/fi not_active Application Discontinuation
  • 1995-01-20 CN CN95191276.3A patent/CN1138864A/zh active Pending
  • 1995-01-20 EP EP95907574A patent/EP0739352A1/de not_active Withdrawn

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