WO1992002542A1 - Peptides et compositions pharmaceutiques presentant une activite agoniste ou antagoniste des hormones glycoproteiques - Google Patents

Peptides et compositions pharmaceutiques presentant une activite agoniste ou antagoniste des hormones glycoproteiques Download PDF

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WO1992002542A1
WO1992002542A1 PCT/NL1991/000139 NL9100139W WO9202542A1 WO 1992002542 A1 WO1992002542 A1 WO 1992002542A1 NL 9100139 W NL9100139 W NL 9100139W WO 9202542 A1 WO9202542 A1 WO 9202542A1
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amino acid
peptide
well
sequence
tdsds
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PCT/NL1991/000139
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English (en)
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Marjolein Hage-Van Noort
Wouter Cornelis Puijk
Robert Hans Meloen
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Stichting Centraal Diergeneeskundig Instituut
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    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to peptides having glycoprotein hormone agonistic or antagonistic activity, in particular peptides having a human follicle-stimulating hormone (human FSH) agonistic or antagonistic activity and to pharmaceutical compositions containing such peptides.
  • human FSH human follicle-stimulating hormone
  • LH glycoprotein hormones luteinizing hormone
  • FSH follicle-stimulating hormone
  • LH and FSH are both produced and secreted by the adenohypophysis.
  • CG chorionic gonadotropin
  • PMSG pregnant mare gonadotropin
  • eCG equine chorionic gonadotropin
  • the concentration of the gonadotropins in the blood and at the target cells is regulated by the hormone produced in the hypothalamus LH releasing hormone (LHRH; also called luliberine) and via a feedback mechanism in which substances produced by the target cells under the influence of LH/CG and FSH and delivered to the blood, such as steroid hormones and the protein inhibine, regulate the production and release of LH/CG and FSH (by inhibine) in the adenohypophysis.
  • LHRH hypothalamus LH releasing hormone
  • glycoprotein hormones act via binding to specific receptors on the plasma membrane of a target cell which, via so-called G proteins, are coupled to systems in the cell where second messenger molecules are made (such as cAMP, cGMP, diacyl glycerol, inositol triphosphate). Via a cascade of reactions then occurring in the cell
  • mice/female humans the granulosa cells and theca cells, are located in the wall of the follicle in the ovary.
  • the interaction between the adenohypophyseal hormones, gonadal hormones (which act both endocrinologically and locally) and in some cases external factors (such as day length, ambient temperature, etc.) finally leads to ovulation of an adult
  • pre-ovulatory follicle also called graafian follicle
  • the target cells for FSH and LH in male animals/male humans are the Sertoli cells and the Leydig cells,
  • ICSH which is also used for LH in male humans.
  • Sertoli and Leydig cells are both located in the testis.
  • the developing sperm cells themselves have no receptors for FSH or LH.
  • TSH glycoprotein hormone
  • FSH and LH can both be used in the regulation of
  • FSH biologically active FSH
  • FSH agonists may also be used. In the treatment of the
  • PCO polycystic ovarian syndrome
  • hMG menopausal gonadotropin
  • LHRH LHRH agonists
  • An increased amount of biologically active LH in a short phase of the cylce may lead to luteinization of the follicles and therefore also has a possible application in the induction of superovulation, in the treatment cf women with fertility problems such as anovulation, in which a urinary hCG preparation is being used up to now, in the treatment of anestrus or postpartum anestrus in animals and in in vitro fertilization.
  • an FSH antagonist optionally in combination with an LH agonist.
  • an FSH antagonist optionally in combination with an LH agonist.
  • cows and horses there is nymphomania in which, due co a high FSH and a low LH concentration in the blood, no luteinization of the follicle occurs but a very large amount of estrogens is produced by this follicle. This leads to a disturbance of the cycle (infertility).
  • the cow is then continuously in heat, thus effecting unrest among the other cows .
  • the mare is then continuously in heat and therefore very hard to handle.
  • the treatment of nymphomania hitherto consists in the manual luteinization of the follicle, which is not always successful and may lead to complications.
  • an LH agonist could be administered once a cycle.
  • An FSH antagonist, an LH antagonist or a combination of both could be used as an alternative "steroid-free" pill to block ovulation.
  • FSH farnesoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoido.
  • An FSH agonist, an LH agonist or a combination of both can also be used to improve the sperm production in male agricultural domestic animals, such as horses, bulls and boars kept for breeding.
  • An increase in the amount of developing sperm cells takes place when FSH is administered to intact adult cynomolgus apes (Van Alphen et al, 1988).
  • An FSH agonist may possibly (also) provide a restoration (reinitiation) of spermatogenesis when the spermatogenic cell population has been reduced to
  • TSH regulating the production of thyroid hormones a possible application substantially resides in humans, in particular in individuals suffering from Graves' disease or from Hashimoto's disease. Both diseases are autoimmune diseases in which antibodies are formed directed against the TSH receptor on the thyroid gland cell. Antibodies stimulating the thyroid gland cell after binding to the receptor (Graves' disease) cause a serious overproduction of thyroid hormones and a disturbance of metabolism. Binding of these antibodies could be avoided by administering TSH antagonists. In
  • Hashimoto's disease a disturbance of in particular the growth of thyroid gland occurs because here there are antibodies preventing the binding of TSH to the receptor (blocking antibodies).
  • TSH agonist (s) having a high affinity for the receptor could take place.
  • FSH FSH
  • LH and TSH also parts of FSH, LH and TSH (peptides) could be used in principle for the above applications.
  • active or passive immunization synthetic peptides which correspond with parts of the glycoprotein hormones, in particular of gonadotropins.
  • Antibodies, polyclonal or monoclonal, thus obtained are used in different test kits (enzyme immunoassays, radioimmunoassays, etc.) to determine the concentration of different hormones in the blood, e.g., in case of fertility disorders.
  • test kits enzyme immunoassays, radioimmunoassays, etc.
  • a problem with diagnostics is that the amount of hormone determined via immunological techniques often does not equal the amount of functionally active hormone, in particular because loose ⁇ - and ⁇ -subunits of the hormones can circulate and the hormones can be
  • Synthetic peptides may be used to generate antibodies directed against defined parts of a hormone which may lead to the development of better defined test kits.
  • active or passive immunization against gonadotropins may also be considered. Fertilization or pregnancy can thus be avoided, either actively by vaccination with the hormone itself or with a synthetic peptide corresponding to a part of the hormone coupled to a carrier protein, either passively with respect to antibodies directed against the hormone itself or against a synthetic peptide derived therefrom. Reversibility
  • the pregnancy hormone hCG plays an important role in the achievement and maintenance of pregnancy. Immunization against hCG could lead to termination of pregnancy (i.e. a "morning-after” vaccine) without administering a large dose of steroid hormone, as in case of the present "morning-after” pill.
  • Immunization can also be applied in the treatment of prostate cancer and breast cancer which are both steroid hormone (androgen or estrogen) dependent in a certain stage of tumour development. These steroids are formed in the sex organs under the influence of LH, hCG and FSH.
  • a 100% effective immunization against the gonadotropins could be used for the sterilization of, e.g., small pets, such as tomcats and pusses, or for the treatment of aggressiveness with male dogs, instead of drastic surgeries, such as castration and ovariectomy.
  • small pets such as tomcats and pusses
  • drastic surgeries such as castration and ovariectomy.
  • a disadvantage of vaccination with the hormone itself is that antibodies are often not specific for this hormone (e.g., hCG) but also react with other glycoprotein hormones (such as FSH, LH, TSH) because these hormones have the same ⁇ -chain.
  • Vaccination with the hormone specific ⁇ -subunit requires separation of this subunit from a highly purified hormone preparation or production of this subunit via the recombinant DNA technology, which are both relatively expensive techniques.
  • Vaccination with a synthetic peptide consisting of a plurality of amino acids of a hormone hat the advantage that the selectivity can be guaranteed while the expenses are relatively low.
  • the immunogenicity of a peptide has been found better for a synthetic peptide corresponding to a conformational epitope of the hormone comprising both the ⁇ - and the hormone specific ⁇ -subunit, namely the peptide hCG ⁇ -(50-59)- hCG ⁇ -(106-116) coupled to tetanus toxoid or keyhole limpet haemocyanin (KLH, Bidart et al, 1990, Science 248, 736-739).
  • Antibodies a synthetic peptide corresponding to a conformational epitope of the hormone comprising both the ⁇ - and the hormone specific ⁇ -subunit, namely the peptide hCG ⁇ -(50-59)- hCG ⁇ -(106-116) coupled to tetanus toxoid or keyhole limpet haemocyanin (KLH, Bidart et al, 1990, Science 248, 736-739).
  • FSH, LH, CG and TSH belong to a group of related
  • glycoprotein hormones consist of two non- covalently bonded unequal subunits designated as the ⁇ - and ⁇ -chain.
  • the primary structure of both subunits of the different glycoprotein hormones is known from a number a species, inter alia man (Pierce and Parsons, 1981; Ryan et al, 1988; Jameson et al, 1988).
  • the ⁇ -subunit (about 92 amino acids) of these hormones is identical within one species for all these
  • glycoprotein hormones the ⁇ -subunit (about 115 amino acids; hCG 145 amino acids) is specific for FSH, TSH and LH; the first 115 amino acids of the ⁇ -subunit of hCG are 85%
  • the loose subunits can also bind to the receptor but require higher concentrations.
  • cysteine-31 particular cysteine-31, phenylalanine-33 and arginine-35 (Reed et al, 1990).
  • the peptide ⁇ -(38-57) has a low agonistic activity in the absence of hCG, it can stimulate the production of testosterone (Keutmann et al, 1987).
  • a low agonistic activity has recently also been described for the peptides ⁇ 1 -(30-45) and ⁇ 2 -(71-85). These peptides can stimulate the production of testosterone in a Leydig cell culture; the EC50 is about 10 -4 M (Erickson et al, 1990).
  • TSH TSH receptors
  • a synthetic peptide corresponding to the amino acids ⁇ -(25-46) as for amino acid sequence not only inhibits the binding of TSH itself to TSH receptors but also the binding of autoimmune antibodies.
  • peptides near the "determinant loop” were found, namely ⁇ 2 - (81-95) EC50 1 ⁇ 10 -3 M, ⁇ - (71-85) EC 50 1 ⁇ 10 -4 M, probably ⁇ - (81-85) is the active component.
  • TSH two additional regions were found having a receptor binding activity: ⁇ -(1-15) EC50 3 ⁇ 10 -4 M and ⁇ -(101-112) EC 50 8 ⁇ 10 -5 M (Morris et al, 1990).
  • FSH FSH the following is known.
  • a synthetic peptide corresponding to the residues ⁇ - (33-53) in human FSH (Jameson et al, 1988), which corresponds to the "Keutmann loop" ⁇ 1 -(37- 58) in hCG, binds to the FSH receptor having an affinity constant of about 5 ⁇ 10 -5 M.
  • Sertoli cell culture a biological activity of FSH was partly inhibited (antagonistic activity in the presence of FSH), namely the stimulation of the enzyme aromatase which catalyzes the conversion of androstenedione to estradiol.
  • the peptide itself also had a low agonistic activity in the absence of FSH, the conversion of androstenedione to estradiol was significantly increased after addition of this peptide (Santa Coloma et al, 1989).
  • TRDL ⁇ 1 -(40-43) and KTCT ⁇ 1 - (55-58) it has been described before that at very high concentrations of peptide inhibition of the FSH binding to calf testis membranes occurs (respectively 27% inhibition at 8 ⁇ 10 -3 M and 70% inhibition at 8.8 ⁇ 10 -3 M; Sluss et al, 1986).
  • This synergistic peptide comprises the "determinant loop", 6 amino acids N-terminal of this loop and 1 amino acid C-terminal of this loop, namely the sequence QCHCGKCDSDSTDCT (underlined is the "determinant loop"). Inhibition of FSH binding to the FSH receptor on calf testis membranes by means of synthetic peptides corresponding to parts of the ⁇ -subunit has not been shown so far (Ryan, 1988).
  • anta antagonistic activity, binds to the receptor without leading in the cell to formation of cAMP and/or steroids
  • agon agonistic activity, binds to the receptor and leads to formation of cAMP and/or steroids.
  • LH and FSH have an intrinsic thioredoxin activity (Reicnert and Dattatreyamurty, 1989; Boniface and Reichert, 1990).
  • rRNAse reduced and denatured ribonuclease
  • glycoprotein hormones are made up of an ⁇ - and a ⁇ -chain the homology postulated by Boniface and Reichert (1990) is insufficient to develop a spacial model for glycoprotein hormones.
  • the present invention is based on a relation found by us between both the ⁇ - and the ⁇ -chains of FSH, LH/CG, TSH and thioredoxin and homologous proteins such as thioredoxin reductase, glutaredo ⁇ in and glutaredoxin reductase via a totally different alignment (see Fig. 1).
  • FSH FSH
  • LH/CG LH/CG
  • TSH thioredoxin
  • homologous proteins such as thioredoxin reductase, glutaredo ⁇ in and glutaredoxin reductase via a totally different alignment (see Fig. 1).
  • peptides have been constructed which mimic the receptor binding site of FSH.
  • peptides are made up of amino acids from the earlier described domains ⁇ 1 , ⁇ 2 , ⁇ 1 and ⁇ 2 . In a preferred embodiment they are coupled together in a special manner (special distance and sequence) which can only be derived from the model.
  • the invention provides a peptide having a glycoprotein hormone agonistic or antagonistic activity, which peptide comprises at least two partial peptides selected from
  • a partial peptide having an amino acid sequence comprising either the amino acid sequence TRDL based on the amino acids 34-37 of the ⁇ -chain of the glycoprotein hormone human FSH or a corresponding amino acid sequence of another glycoprotein hormone and/or another animal species, and
  • a partial peptide having an amino acid sequence comprising the amino acid sequence AHASTA, derived from the amino acids 82-87 of the ⁇ -chain of the human glycoprotein hormones by replacing the cysteine residues by alanine residues, or a corresponding amino acid sequence of a glycoprotein hormone of another animal species, which partial peptides can be linked together by bridge groups,
  • antagonistic activity as well as derivatives in which either the free amino group of the amino-terminal amino acid or the free carboxyl group of the carboxy-terminal amino acid, or both, are blocked or otherwise modified.
  • the peptides according to the invention are made up of two, three or four, preferably 3 or 4, most preferably 4, partial peptides, said partial peptides preferably being linked together via bridge groups.
  • Partial peptide (1) is based on the ⁇ -chain of a
  • glycoprotein hormone It has an amino acid sequence comprising either the amino acid sequence TDSDS based on the amino acids 92, 53, 39, 90 and 91 cf the ⁇ -chain of the glycoprotein hormone human FSH or a corresponding amino acid sequence of another glycoprotein hormone and/or another animal species.
  • TDSDS amino acid sequence
  • the sequences of the ⁇ -chains of the different hormones must be aligned in the manner shown in Fig. 1.
  • the sequence TDSDS based on the amino acids 92, 88, 89, 90 and 91 of the ⁇ -chain of human FSH corresponds to the following sequences of other glycoprotein hormones:
  • Partial peptide (2) must consist of an amino acid
  • this sequence is on sites 34-37. It is a highly conserved sequence, as shown in Fig. 1. Exactly the same sequence occurs in the ⁇ -chain of other animals, such as rat, bovine, porcine, equine and carp.
  • Partial peptide (2) may consist both of these four amino acids SRAY alone and of a longer sequence. In different preferred embodiments of the invention this partial peptide consists of sequence SRAY or of sequence QCMGCAFSRAY. Most preferred is, in connection with the very strong effect of the peptide as agonist or as antagonist, the use of sequence
  • QCMGCAFSRAY as partial peptide.
  • this partial peptide is an example of a substitution variant since the natural sequence in the ⁇ -chain of the human glycoprotein hormones is QCMGCCFSRAY.
  • the partial peptide (3) based on the ⁇ -chain has an amino acid sequence comprising either the amino acid sequence TRDL based on the amino acids 34-37 of the ⁇ -chain of the
  • glycoprotein hormone hunr ⁇ n FSH or a corresponding amino acid sequence of another glycoprotein hormone and/or another animal species.
  • amino acid sequence TRDL based on the amino acids 34-37 of the ⁇ -chain of the glycoprotein hormone human FSH
  • sequences of the ⁇ -chains of the different hormones must be aligned in the manner shown in Fig. 1.
  • Tne sequence TRDL based on the amino acids 34-37 of the ⁇ -chain of human FSH corresponds to the following sequences of other glycoprotein hormones :
  • the partial peptide (4) based on the ⁇ -chain has an amino acid sequence comprising the amino acid sequence AHASTA, derived from the amino acids 82-87 of the ⁇ -chain of the human glycoprotein hormones by replacing the cysteine residues by alanine residues, or a corresponding amino acid sequence of a glycoprotein hormone of another animal species.
  • the sequence CHCSTC is on sites 82-87. It is a highly conserved sequence, as shown in Fig. 1. Exactly the same sequence occurs in the ⁇ -chain of other animals, such as rat, bovine, ovine and porcine; in equine, however, the sequence is CYCSTC.
  • cysteine residues (C) in the peptides according to the invention are replaced by alanine residues (A). It has been established by way of experiment that peptides comprising the partial peptides 1, 2, 3 and
  • peptides having glycoprotein hormone agonistic activity may show such an activity inhibiting binding of the glycoprotein hormone to the receptor but, in addition, due to their own binding to the receptor, they are capable of producing an effect which is also produced by the binding of the glycoprotein hormone.
  • sequence, substitution, deletion and insertion variants which also show a glycoprotein hormone agonistic or antagonistic activity.
  • An example of such a sequence variant is a peptide in which the amino acid sequence TDSDS based on the amino acids 92, 88, 89, 90 and 91 of the ⁇ -chain of the glycoprotein hormone human FSH has been replaced by the amino acid sequence DSDST based on the amino acids 88-92 of the ⁇ -chain of the glycoprotein hormone human FSH.
  • sequence variants for the other glycoprotein hormones are also sequence variants for the other glycoprotein hormones.
  • an insertion variant is a peptide in which the amino acid sequence TRDL based on the amino acids 34-37 of the ⁇ -chain of human FSH has been replaced by the amino acid sequence TRGDL based on the corresponding sequence in the porcine. Peptides containing this insertion variant have been found as effective as the peptides containing the sequence TRDL.
  • substitution variant according to the invention is a peptide comprising a partial peptide (2) in which the arginine has been replaced by an alanine so that the sequence is SAAY instead of SRAY.
  • the invention also extends to derivatives in which either the free amino group of the aminoterminal amino acid or the free carboxyl group of the carboxyterminal amino acid, or both, are blocked or otherwise modified.
  • a particular example may be an acylated amino group of the amino-terminal amino acid (a group RCONH-, e.g., an acetylamino group) and an amidated carboxyl group of the carboxy-terminal amino acid (a carboxamide group -CONH 2 ).
  • Preferred embodiments of peptides according to the invention are peptides having a human FSH agonistic or antagonistic activity, which are characterized by the general formula (1) :
  • a, b and c represent a bridge group, if any
  • ⁇ 1 - ⁇ 8 each represent one or more additional amino acids, if any.
  • variants having another sequence of the partial peptides are peptides in which the sequence is not (1) - (2)- (3)- (4) , but (2) - (3) - (4) - (1) , or (3)-(4)-(1)-(2), or (4)-(1)-(2)-(3).
  • the bridge groups present between the partial peptides are in themselves not subject to special
  • d, e and f represent integers having a value ranging from 1 to 15,
  • X 1 - X 8 each represent one or more additional amino acids, if any,
  • each of the partial peptides preferably has no more than two flanking amino acids on both sides of the core sequence and most preferably
  • d, e and f represent integers having a value ranging from 1 to 15, X 3 represents an additional amino acid sequence QCMGCAF, if any,
  • peptides according to the invention having a human FSH agonistic or antagonistic activity, characterized by the general formula (4) :
  • d, e and f represent integers having a value ranging from 1 to
  • the lengths of the bridge groups between partial peptides 1 and 2, 2 and 3, and 3 and 4 are most preferably in the order of at least 7A, 7A and 13A, respectively.
  • d corresponds to values of d, e and f of respectively 4-6, 4-6, and 9-11, most preferably 5, 5, and 11, respectively.
  • a partial peptide contains more amino acids than the core sequence shown, other lengths of the bridge groups may be optimal.
  • the value of d will preferably be lower than 5, e.g., d will be equal to 2.
  • the values of d, e and f may vary within broad limits, a value of
  • This invention also comprises monoclonal and polyclonal antibodies generated against peptides according to the invention. Such antibodies can be obtained by immunizing otherwise known per se processes with a peptide according to the invention.
  • the invention further extends to pharmaceutical
  • compositions for active or passive immunization against a glycoprotein hormone or for influencing the activity of a glycoprotein hormone comprising one or more peptides according to the invention, as defined above, or antibodies generated thereagainst, as well as at least one pharmaceutically acceptable adjuvant, carrier or diluent.
  • Suitable routes of administration for the preparations are, e.g., intranasal, intraperitoneal (i.p.), intramuscular (i.m.), transdermal (suppository), oral, intravenous (i.v.).
  • compositions according to the invention are pharmaceutical compositions having a
  • glycoprotein hormone agonistic or antagonistic activity vaccine preparations and preparations for passive immunization against a glycoprotein hormone.
  • this invention provides a
  • composition having a human FSH agonistic activity comprising a peptide of one of formulae 6-9, or a derivative thereof, in which the free amino group of the amino-terminal amino acid is converted to an acetylamino group and/or the free carboxyl group of the carboxy-terminal amino acid is converted to an amido group,
  • composition having a human FSH antagonistic activity comprising a peptide of one of formulae 10-16, or a derivative thereof, in which the free amino group of the amino-terminal amino acid is converted to an
  • acetylamino group and/or the free carboxyl group of the carboxy-terminal amino acid is converted to an amido group, and at least one pharmaceutically acceptable adjuvant, carrier or diluent.
  • the amino acids K (Lys), D (Asp), Y (Tyr), R (Arg), S (Ser) , T (Thr) and C (Cys) were protected in the side chains, there being used respectively: Boc-Lys (2-Br-Z), Boc-Asp(Ochx), Boc-Tyr (2-Br-Z), Boc-Arg (Tos), Boc-Ser(Bzl) and Boc-Cys (MeOBzl).
  • the protecting groups used are designated by the conventional abbreviations, such as 2-Br-Z for 2-bromobenzyloxycarbonyl, and Ochx for cyclohexylester.
  • Boc-8-amino-octanoic acid Boc-6-amino-hexanoic acid, Boc-5-amino-pentanoic acid and Boc-4-amino-butanoic acid.
  • the peptides were separated from the resin by means of hydrogen fluoride (HF) with 10% anisol worked up and freeze-dried (see Houghten, 1986).
  • the resin used was the "Rink” resin (Rink, 1987).
  • the Fmoc amino acids were coupled in DMF by successively adding to the resin (0.13 mmol) 0.5 mmol Fmoc amino acid, 0.5 mmol HOBt and 300 ⁇ l DIEA. After 1 hour reaction time the resin was rinsed twice with DMF. Then the Fmoc group was separated in 50% piperidine/DMF (2 ⁇ 5 min) , after which the resin was rinsed with DMF (5 times).
  • the protecting groups used are indicated by the conventional abbreviations, such as Pmc for 2, 2, 5, 7, 8-pentamethylchroman-6-sulfonyl, and Trt for triphenylmethyl. Also used were the synthetic amino acids ⁇ -alanine, Fmoc-4-aminobutanoic acid, Fmoc-5-aminopentanoic acid, Fmoc-6-aminohexanoic acid, Fmoc-8-aminooctanoic acid and Fmoc-12-aminododecanoic acid. The peptides were separated from the resin by means of
  • the peptides were freeze-dried and their amino acid preparation was controlled by means of a Waters Pico-Tag system. The purity was determined an analytic HPLC column (Supelcosil LC-18-DB 5 ⁇ m; 15 x 0.4 cm) in a gradient of methanol/water + 0.1% TFA.
  • Sertoli cells of 21 days old rats were isolated from the testis of 21 days old Wistar rats according to Oonk et al (1985) and then cultured for 2 days at 32°C and 5% CO 2 in Eagles' minimal essential medium (MEM) with 5 mM L-glutamine, antibiotics and 1% (v/v) fetal calf serum (FCS). After 2 days spermatogenic cells were removed from the culture by means of a hypotonic shock (10% MEM in water; Oonk and Grootegoed, 1987). The cells were then cultured for 1 day in MEM without FCS but with 0.1% bovine serum albumin (BSA, FrV Sigma). The following day the cells were washed twice with medium, after which the hypotonic shock (10% MEM in water; Oonk and Grootegoed, 1987). The cells were then cultured for 1 day in MEM without FCS but with 0.1% bovine serum albumin (BSA, FrV Sigma). The following day the cells were washed twice with medium, after which the
  • ATP was determined in the neutralized supernatant using a firefly luciferin-luciferase reaction (Lumac) as described by Grootegoed et al (1984). Cyclic AMP (cAMP) was measured by means of a cAMP assay kit (Amersham International TRK 432, Amersham, U.K.). Since cAMP is partly separated in the medium, the removal of the medium is omitted. Instead thereof, 40 ⁇ l 50% PCA are a ⁇ ded on ice to the cells and the incubation medium (500 ⁇ l) until a final concentration of about 5% PCA. ( c ) Results
  • the amount of cAMP found in pmol per 100 ⁇ g protein is indicated in the tables as an average ⁇ standard deviation SD.
  • the number of incubations is given in curved brackets, the stimulation factor or the inhibition in terms of percentage relative to the average is given in square brackets.
  • NE not examined; -: not detectable.
  • 3-domain peptide had no effect on the FSH or the forskolin-induced cAMP production of the cells.
  • the purified 3-domain peptide had no effect on the ATP amount (11.22 ⁇ 3.98; 30 tests) in the Sertoli cells.
  • Ac-SRAY-NH-(CH 2 ) 4 -CO-TRDL-NH 2 made up of two partial peptides (further indicated as 2-domain peptides) act in vitro as antagonists of FSH activity until a concentration of about 10 -8 M (Table 2).
  • the peptide Ac-TDSDS-NH-(CH 2 ) 5 -CO-SRAY-NH 2 has also been tested in vivo by injection into a rat model system. In view of the fact that nothing is still known of the half-value time of this peptide in the body, a rather high dose is used (2 mg/rat). At this dose this peptide also in vivo seems to act as an antagonist of FSH.
  • ULO in the adult cyclic rat may lead to a functional compensation of follicle maturation and ovulation in the remaining ovary for the rest of the cycle.
  • the compensatory mechanism comprises increase in the FSH concentration in the blood 6-18 h after ULO (Welschen et al, 1978).
  • physiological salt (0.9% NaCl in H 2 O) .
  • the other rats were injected (subcutaneously) with 2 mg peptide (dissolved in physiological salt) per rat. Twenty-four hours after ULO, on diestrus-3 between 9.00-10.00 h, the rats were killed and the uterus weight of each rat was determined.
  • the body weight of the rats treated with peptide was equal to that of rats injected with physiological salt. Examples 4 -41
  • Tables 4-41 show the results of experiments in conformity with Examples 1-2 with different peptides according to the
  • the peptide Ac-DSDST-5-SRAY-4TRDL-NH 2 has the same effect as the peptide Ac-TDSDS-5- SRAY-4-TRDL-NH 2 , namely agonistic up to a concentration of 10 -7 M.
  • the peptide Ac- TDSDSSRAYTRDL-NH 2 works less effective than the peptide Ac-TDSDS-5-SRAY-NH 2 ; in other words, 3-domain peptide without spacers is less effective (antagonistic) than 2-domain peptide with spacers (10 -7 M) .
  • the peptide Ac-SRAY-4-TRDI-NH 2 has the same effect (antagonistic up to 10 -7 M) as the peptide Ac-SRAY-4-TRDL-NH 2 .
  • Replacement of a leucine by an isoleucine has no effect on the antagonistic activity of the peptide. Since the sequence TRDL occurs in FSH and TRDI in TSH, the peptide Ac-SRAY-4-TRDL-NH 2 is probably not very useful as a specific antagonist.
  • cysteines could be linked in a bridge and in interaction with the receptor they could play a role in either activation or additional binding energy.
  • the cysteines could be linked in a bridge and in interaction with the receptor they could play a role in either activation or additional binding energy.
  • Of the 3 cysteines from the original sequence 1 has changed to an alanine (peptides 6 and 7, respectively).
  • Peptide 6 has been found ineffective both agonistically and antagonistically.
  • Peptide 7 has been found agonistic to a lower concentration (at least 10 -8 M) than the 3-domain peptide Ac-TDSDS-5-SRAY-4-TRDL-NH 2 .
  • this peptide also seems to have a low antagonistic activity.
  • F-moc synthesis gives a 2-domain antagonistic peptide Ac-TDSDS-5-SRAY-NH 2 which is as effective as peptide obtained via Boc synthesis.
  • Peptide fraction 4 has been found toxic as measured at suppression of the forskolin induced cAMP production.
  • Peptide Ac-TDSDS-2-SRAY-4-TRDL-NH fr2 seems as effective (agonistic) as peptide Ac-TDSDS-5-SRAY-4-TRDL-NH 2 .
  • Reduction of spacer 1 between the 1st and the 2nd domain from NH-(CH 2 )s-CO to NH- (CH 2 ) 2 -CO (b-alanine) seems to be allowed.
  • TDADA leads to a peptide having a low antagonistic activity at 10 -4 M (instead of an agonist at 10 -7 M) .
  • Peptide Ac-DSDST-5-SRAY-NH 2 has the same effect as the "original" 2-domain peptide Ac- TDSDS-5-SRAY-NH 2 , namely antagonistic up to a concentration of 10 -8 M.
  • the spacer between domains 2 and 3 must be at; least. 4.
  • the spacer In order to thus obtain an antagonistic peptide (10 -8 M), the spacer must, bo 2 or 0 or domain 3 must be omitted.

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  • Health & Medical Sciences (AREA)
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  • Endocrinology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Peptide présentant une activité (ant)agoniste des hormones glycoprotéiques et comportant au moins deux peptides partiels sélectionnés parmi: (1) un peptide partiel possédant une séquence d'acides aminés comportant TDSDS à base des acides aminés (92, 88, 89, 90 et 91) de la chaîne β de l'hormone glycoprotéique hFSH, ou bien une séquence correspondante d'une autre hormone glycoprotéique et/ou d'une autre espèce animale; (2) un peptide partiel possédant une séquence d'acides aminés comprise dans la chaîne α des hormones glycoprotéiques et comportant SRAY; (3) un peptide partiel possédant une séquence d'acides aminés comportant TRDL à base des acides aminés (34-37) de la chaîne β de l'hormone glycoprotéique hFSH, ou bien une séquence correspondante d'une autre hormone glycoprotéique et/ou d'une autre espèce animale; et (4) un peptide partiel possédant une séquence d'acides aminés comportant AHASTA, dérivée des acides aminés (82-87) de la chaîne α des hormones glycoprotéiques humaines par le remplacement des cystéines par des alanines, ou bien une séquence correspondante d'une hormone glycoprotéique d'une autre espèce animale; lesdits peptides partiels pouvant être liés les uns aux autres par des groupes de pontage. On a également prévu des variantes et des dérivés présentant une activité agoniste ou antagoniste des hormones glycoprotéiques.
PCT/NL1991/000139 1990-07-27 1991-07-26 Peptides et compositions pharmaceutiques presentant une activite agoniste ou antagoniste des hormones glycoproteiques WO1992002542A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL9001709A NL9001709A (nl) 1990-07-27 1990-07-27 Peptiden en farmaceutische preparaten met een glycoprotene hormoon agonistische of antagonistische werking.
NL9001709 1990-07-27

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WO1992002542A1 true WO1992002542A1 (fr) 1992-02-20

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EP (1) EP0541663A1 (fr)
AU (1) AU8319391A (fr)
CA (1) CA2088162A1 (fr)
MX (1) MX9203582A (fr)
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4193915A (en) * 1978-04-13 1980-03-18 Mckerns Kenneth W Contraceptive, antibody generating, polypeptides
WO1989001943A1 (fr) * 1987-09-04 1989-03-09 Whitehead Institute For Biomedical Research Complexes de peptides a stabilite augmentee
EP0323769A1 (fr) * 1987-11-26 1989-07-12 Lafon Pharma S.A. Structures peptidiques, immunogènes les contenant et leurs applications au contrôle de la fertilité

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4193915A (en) * 1978-04-13 1980-03-18 Mckerns Kenneth W Contraceptive, antibody generating, polypeptides
WO1989001943A1 (fr) * 1987-09-04 1989-03-09 Whitehead Institute For Biomedical Research Complexes de peptides a stabilite augmentee
EP0323769A1 (fr) * 1987-11-26 1989-07-12 Lafon Pharma S.A. Structures peptidiques, immunogènes les contenant et leurs applications au contrôle de la fertilité

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biochemistry, volume 27, 1988, American Chemical Society, A.L. Schneyer et al.: "Identification of a receptor binding region on the beta subunit of human follicle-stimulating hormone", pages 666-671,see the whole article *
The FASEB Journal, volume 2, no. 11, August 1988, R.J. Ryan et al.: "The glycoprotein hormones: recent studies of structure-function relationships", pages 2661-2669, see the whole article *
The Journal of Biological Chemistry, volume 265, no. 9, 25 March 1990, The American Society for Biochemistry and Molecular Biology, Inc. (US), T.A. Santa Coloma et al.: "Identification of a follicle-stimulating hormone receptor-binding region in hFSH-beta-(81-95) using synthetic peptides", pages 5037-5042, see the whole article *

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CA2088162A1 (fr) 1992-01-28
NL9001709A (nl) 1992-02-17
AU8319391A (en) 1992-03-02
EP0541663A1 (fr) 1993-05-19
MX9203582A (es) 1992-07-01

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