EP0541663A1 - Peptides and pharmaceutical compositions having glycoprotein hormone agonistic or antagonistic activity - Google Patents

Abstract

Peptide exhibiting (ant)agonist activity of glycoprotein hormones and comprising at least two partial peptides selected from: (1) a partial peptide having an amino acid sequence comprising TDSDS based on the amino acids (92, 88, 89, 90 and 91) of the beta chain of the glycoprotein hormone hFSH, or a corresponding sequence of another glycoprotein hormone and/or another animal species; (2) a partial peptide having an amino acid sequence included in the alpha chain of glycoprotein hormones and comprising SRAY; (3) a partial peptide having an amino acid sequence comprising TRDL based on the amino acids (34-37) of the beta chain of the glycoprotein hormone hFSH, or a corresponding sequence of another glycoprotein hormone and/or of another animal species; and (4) a partial peptide having an amino acid sequence comprising AHASTA, derived from amino acids (82-87) of the alpha chain of human glycoprotein hormones by replacing cysteines with alanines, or a corresponding sequence of a glycoprotein hormone from another animal species; said partial peptides capable of being linked to each other by bridging groups. Variants and derivatives exhibiting agonist or antagonist activity of glycoprotein hormones have also been provided.

Description

Title: Peptides and pharmaceutical compositions having glycoprotein hormone agonistic or antagonistic activity.

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.

The regulation of fertility in male and female animals takes place substantially by a combined action between the glycoprotein hormones luteinizing hormone (LH; also called lutropin or, in male animals, interstitial cell-stimulating hormone (ICSH) ) and follicle-stimulating hormone (FSH; also called follitropin). The hormones LH and FSH are both produced and secreted by the adenohypophysis. Moreover, in primates (inter alia, man) a chorionic gonadotropin (CG) is produced by the placenta which has an LH-like effect in this species. With horses (equines) there is the, hitherto known exceptional, production of pregnant mare gonadotropin (PMSG) , also called equine chorionic gonadotropin (eCG) which is produced by cells of the fetus that have lodged in the uterus of the mother during a certain period of pregnancy (trophoblast cells). This PMSG/eCG substantially has an FSH-like effect but also partly an LH-like effect in case of injection into other animal species. Because these hormones produce their effect on the sex organs (the gonads), they are also designated as

gonadotropins.

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. The glycoprotein hormones (first messenger molecules) 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

production and release of a plurality of substances takes place (such as, inter alia, steroid hormones and inhibine).

The target cells for FSH and LH/CG in female

animals/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

(primary) oocyte from a 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,

respectively (located in the interstitial tissue, hence the name ICSH which is also used for LH in male humans). The

Sertoli and Leydig cells are both located in the testis.

Regulation of the initiation and maintenance of the sperm cell production takes place via these cells. The developing sperm cells themselves have no receptors for FSH or LH.

TSH, thyroid(= thyroid gland)-stimulating hormone, also called thyrotropin, is also a glycoprotein hormone (beside LH and FSH) produced and secreted in the hypophysis. TSH

stimulates the thyroid gland to form thyroid hormones

(substantially T3 and T4) which, in turn, provide regulation of a large number of processes in the body, together

controlling the basal metabolic activity of an organism (such as effects on growth and behaviour, on heart and muscle function, digestion, oxygen absorption in tissues and

disturbances in the immune system).

FSH and LH can both be used in the regulation of

fertility in humans and animals, both to improve fertility and to prevent fertility.

Here follow number of examples:

In female animals/female humans:

- Treatment with an FSH agonist, an LH agonist or a

combination of both.

An increased amount of biologically active FSH may lead to the development of several follicles and may therefore be used in in vitro fertilization, induction of superovulation and treatment of anovulation. An increase in the FSH

concentration via administering additional (exogenous) FSH leads, via feedback mechanisms, to an increased amount of inhibine so that the release of FSH by the hypophysis

(endogenous release) is reduced (so that more exogenous FSH is to be administered again, etc.). Treatment with an FSH agonist in combination with immunization against inhibine or treatment with an inhibine antagonist (in other words, a substance blocking the inhibine effect) therefore appears to be a promising combination. In the treatment of anestrus or

postpartum anestrus in animals or fertility problems in humans FSH agonists may also be used. In the treatment of the

polycystic ovarian syndrome (PCO; in animals this is also called COD = cystic ovarian disease), for example, use is now often being made of human menopausal gonadotropin (hMG; has FSH and LH activity) or LHRH agonists with the side effects (because not only FSH but also LH is increased) of forming several cysts and/or multiple pregnancy. 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.

- Application of an FSH antagonist, optionally in combination with an LH agonist. With 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). Moreover, the cow is then continuously in heat, thus effecting unrest among the other cows . Also 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. Alternatively, 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.

In malp animals/male humans:

A prolonged blockade of the effect of FSH on Sertoli cells could reduce fertility without affecting the

testosterone production and thus the secondary sex

characteristics and the libido. Experimental research with animals has shown that the effect of FSH ist most evident in the testes of non-adult or hypophysectomized animals. However, there are also indications that in adult animals FSH is required to maintain a quantitatively good production of sperm cells and to restore sperm cell production. 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

spermatogonia as a result of radiation or medicaments

(Matsumoto et ai, 1986). Application of TSH, its agonists or antagonists

With 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). Here treatment with TSH agonist (s) having a high affinity for the receptor could take place.

In addition to FSH, LH and TSH also parts of FSH, LH and TSH (peptides) could be used in principle for the above applications. For the following reasons it seems advantageous to use for active or passive immunization synthetic peptides which correspond with parts of the glycoprotein hormones, in particular of gonadotropins.

The generation of antibodies against gonadotropins for regulation of the fertility or for diagnostics thereof takes place substantially by applying the hormones themselves coupled to a carrier protein. 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. 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 α- and β-subunits of the hormones can circulate and the hormones can be

glycosylated to a different degree. 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.

In addition to the use of antagonists of gonadotropins to reduce fertility, active or passive immunization against gonadotropins (immune contraception) 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

(restoration of fertility will be desired in some cases) seems more probable in the case of passive immunization. Some examples are as follows.

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.

An effective immunization against FSH could lead to a contraceptive for the male human, with maintenance of the libido (Moudgal N.R. and Rao A.J., 1984, In: Methods for the regulation of male fertility, Eds. Kumar T.C.A. and Waites G.M.H., 31-37). Effective immunization against FSH or LH can also be used as a steroid-free contraceptive for women.

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.

In veterinary medicine 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. Other

conceivable objects of immunization against gonadotropins are prevention of heat with dogs and prevention of unrest when fattening calves.

A disadvantage of vaccination with the hormone itself (e.g., hCG) 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 α-chain. Vaccination with the hormone specific β-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.

In the case of hCG attempts have been made to vaccinate with a synthetic peptide consisting of 37 amino acids of the carboxy-terminal of the β-subunit coupled to diphteria toxin. It turned out that this peptide was not very antigenic and immunogenic (Stevens V.C., 1986, Immunol. Today 7, 369).

The immunogenicity of a peptide has been found better for a synthetic peptide corresponding to a conformational epitope of the hormone comprising both the α- and the hormone specific β-subunit, namely the peptide hCG α-(50-59)- hCG β-(106-116) coupled to tetanus toxoid or keyhole limpet haemocyanin (KLH, Bidart et al, 1990, Science 248, 736-739). Antibodies

generated against this peptide also proved to prevent the binding of hCG to the LH receptor in a test system consisting of membranes from the testis of the rat. A synthetic peptide corresponding to the amino acids α-(50-59)-β-(106-116) of rat LH coupled to tetanus toxoid was found to be immunogenic in the rat itself.

The use as an immunogen of synthetic peptides with an agonistic or antagonistic activity, as described above, seems very promising in view of the fact that in this case

antibodies are generated against a part of the hormone binding to the receptor. Binding of such antibodies to the hormone will therefore prevent binding of the hormone to the receptor. An effective vaccination will thus become possible.

The design of such peptides requires a thorough knowledge of FSH, LH and TSH (or related hormones: hCG, eCG) .

FSH, LH, CG and TSH belong to a group of related

glycoprotein hormones. These hormones consist of two non- covalently bonded unequal subunits designated as the α- and β-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 α-subunit (about 92 amino acids) of these hormones is identical within one species for all these

hormones, which will be commonly designated below as

glycoprotein hormones; the β-subunit (about 115 amino acids; hCG 145 amino acids) is specific for FSH, TSH and LH; the first 115 amino acids of the β-subunit of hCG are 85%

homologous to LH, for eCG this homology is 100%. Within 1 species the hormones FSH, LH, CG and TSH therefore consist of the same α-chain but of different β-chains. The three-dimensional (3D) structure of the subunits and the quaternary structure of the hormones (including the interaction between the α- and β-subunit) are not known. It has been found that both subunits in glycosylated form are required for a

completely active hormone. If the sugar groups are removed, the hormones act as antagonists. The loose subunits can also bind to the receptor but require higher concentrations.

With the aid of both polyclonal and monoclonal antibodies and using synthetic peptides a search is being made for the sites important for hormone activity and receptor interaction so as to be able to develop synthetic analogues that could be applied as agonists, antagonists or peptide vaccines in a regulation of fertility. In view of the homology between CG, LH, FSH and TSH knowledge obtained about one of the hormones is probably immediately useful for the development of

agonists, antagonists and peptide vaccines for the other hormones. It is known that at least four regions in human LH and CG, two at the α- and two at the β-subunit, show binding to the receptor: α₁-(25-46), α₂-(76-92), β₁-(38-57) (also called the "Keutmann loop") and β₂-( 93-100) (also called the "determinant loop") having an affinity (Kd) for the receptor of 10^-4-10^-5 M (Charlesworth et al, 1987; Gordon and Ward, 1985; Keutmann et al, 1987) . Important amino acid residues in the region

αχ-(25-46) seem to be the residues 29 through 35 and in

particular cysteine-31, phenylalanine-33 and arginine-35 (Reed et al, 1990).

In the presence of hCG synthetic peptides corresponding to these regions as for amino acid sequence act as

antagonists; the hCG induced production of testosterone in a Leydig cell culture is inhibited. The peptide β-(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 α₁-(30-45) and α₂-(71-85). These peptides can stimulate the production of testosterone in a Leydig cell culture; the EC50 is about 10^-4M (Erickson et al, 1990).

In human TSH the same regions have beenn found on the α-subunit showing a binding to TSH receptors. A synthetic peptide corresponding to the amino acids α-(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. There have been identified peptides that contain parts of the "Keutmann loop" at the β-subunit of TSH: β₁-(31-47) and β₁-(41- 55) which inhibit the binding of TSH to TSH receptors with an EC₅₀ of 2 × 10^-4 M. Also peptides near the "determinant loop" were found, namely β₂- (81-95) EC50 1 × 10^-3 M, β- (71-85) EC₅₀ 1 × 10^-4 M, probably β- (81-85) is the active component. In TSH two additional regions were found having a receptor binding activity: β-(1-15) EC50 3 × 10^-4 M and β-(101-112) EC₅₀ 8 × 10^-5 M (Morris et al, 1990).

For FSH the following is known. A synthetic peptide corresponding to the residues β- (33-53) in human FSH (Jameson et al, 1988), which corresponds to the "Keutmann loop" β₁-(37- 58) in hCG, binds to the FSH receptor having an affinity constant of about 5 × 10^-5 M. In a 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). Of two tetrapeptides from this part of the β-chain, TRDL β₁-(40-43) and KTCT β₁- (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). For the tetrapeptide TRDL an antagonistic activity in a Sertoli cell culture has also been described (50% inhibition of estradiol production at a concentration of about 2 × 10^-4 M). A synthetic peptide corresponding to the residues β-(81-95) in human FSH shows no antagonistic activity but an agonistic activity in the absence of FSH: the

conversion of androstenedione to estradiol was increased by 60% after addition of 80 μM of this peptide. At a

concentration of 10-20 μM this peptide, as regards the estradiol synthesis, also acted synergistically with 30 ng/ml ovine FSH (Santa Coloma et al, 1990). 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 α-subunit has not been shown so far (Ryan, 1988).

Summarizing, the following loose functional active peptides have been described (which are certainly less effective by 100,000 times than the hormones themselves in in vitro test svstems):

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.

In the various articles describing the activity of synthetic peptides which as for amino acid sequence correspond to a part of the α- or β-chain of one of the glycoprotein hormones it is indicated that the antagonistic and/or

agonistic activity of the peptides described could be improved by:

1. increasing the peptide by addition of flanking amino acids present in the native (hormone) sequence and

2. combining different parts from the α- and β-chains, it often oeing supposed that the receptor binding site consists of several parts of both the α- and the β-chain.

In this connection it is not indicated which amino acids from tne different parts of the α- and β-chains should be combined and how these ammo acids should be combined.

Recently, it has been shown that both LH and FSH have an intrinsic thioredoxin activity (Reicnert and Dattatreyamurty, 1989; Boniface and Reichert, 1990). In the test system used reactivation of reduced and denatured ribonuclease (rRNAse), LH was 300 times and FSH 60 times more active than thioredoxin itself.

The amino acid sequence of thioredoxin and thioredoxin reductase originating from a large number of prokaryotic and eukaryotic species has been explained (see Holmgren, 1985, 1989). The active disulfide bridge - responsible for the function - is excellently preserved with the consensus sequence: VDFXAXWCGPC (K) (M) (I) XP, X standing for any amino acid and K, M, and I sometimes being absent. Boniface and Reichert (1990) have postulated that in the hormones LH/CG and FSH an active disulfide bridge is formed between the amino acids β-(84-87) having the sequence CGKC in the β-chain of FSH and the amino acids β-(90-93) having the sequence CGPC in the β-chain of LH/CG, in view of the homology of these

tetrapeptides with the tetrapeptide CGPC in thioredoxin. No mention is made of a homology between thioredoxin and the α-chain of FSH and LH/CG. In view of the very fact that

glycoprotein hormones are made up of an α- and a β-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 β- and the α-chains of FSH, LH/CG, TSH and thioredoxin and homologous proteins such as thioredoxin reductase, glutaredoκin and glutaredoxin reductase via a totally different alignment (see Fig. 1). On the basis thereof, it was found possible to build a hypothetically spacial model of FSH. With the aid thereof, peptides have been constructed which mimic the receptor binding site of FSH.

These peptides are made up of amino acids from the earlier described domains α₁, α₂, β₁ and β₂. 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

(1) a partial peptide having an amino acid sequence comprising either the amino acid sequence TDSDS based on the amino acids

92, 88, 89, 90 and 91 of the β-chain of the glycoprotein hormone human FSH or a corresponding amino acid sequence of another glycoprotein hormone and/or another animal species,

(2) a partial peptide having an amino acid sequence occurring in the α-chain of glycoprotein hormones and comprising the amino acid sequence SRAY,

(3) a partial peptide having an amino acid sequence comprising either the amino acid sequence TRDL based on the amino acids 34-37 of the β-chain of the glycoprotein hormone human FSH or a corresponding amino acid sequence of another glycoprotein hormone and/or another animal species, and

(4) a partial peptide having an amino acid sequence comprising the amino acid sequence AHASTA, derived from the amino acids 82-87 of the α-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,

as well as sequence, substitution, deletion and insertion variants having a glycoprotein hormone agonistic or

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 β-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 β-chain of the glycoprotein hormone human FSH or a corresponding amino acid sequence of another glycoprotein hormone and/or another animal species. In order to determine which amino acid sequence of another glycoprotein hormone and/or another animal species corresponds to the amino acid sequence TDSDS based on the amino acids 92, 88, 89, 90 and 91 of the β-chain of the glycoprotein hormone human FSH, the sequences of the β-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 β-chain of human FSH corresponds to the following sequences of other glycoprotein hormones:

human/porcine FSH TDSDS

human TSH SNTDY

human LH SRRST

human CG TRRST

rat LH SRLSS

bovine/ovine LH TRLSS

porcine LH SRLSS

equine LH and CG TZIKT

Partial peptide (2) must consist of an amino acid

sequence occurring in the α-chain of glycoprotein hormones and comprising the amino acid sequence SRAY. In the α-chain of the human hormones, such as human FSH, 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 α-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. For that matter, this partial peptide is an example of a substitution variant since the natural sequence in the α-chain of the human glycoprotein hormones is QCMGCCFSRAY. The partial peptide (3) based on the β-chain has an amino acid sequence comprising either the amino acid sequence TRDL based on the amino acids 34-37 of the β-chain of the

glycoprotein hormone hunr^n FSH or a corresponding amino acid sequence of another glycoprotein hormone and/or another animal species. In order to determine which amino acid sequence of another glycoprotein hormone and/or another animal species corresponds to the amino acid sequence TRDL based on the amino acids 34-37 of the β-chain of the glycoprotein hormone human FSH, the sequences of the β-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 β-chain of human FSH corresponds to the following sequences of other glycoprotein hormones :

human FSH TRDL

porcine FSH TRGDL

human TSH TRDI

human LH TMMR

human CG TMTR

rat LH SMVR

bovine/ovine LH SMKR

porcine LH SMRR

equine LH and CG SMVR

The partial peptide (4) based on the α-chain has an amino acid sequence comprising the amino acid sequence AHASTA, derived from the amino acids 82-87 of the α-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. In the α-chain of the human hormones, such as human FSH, 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 α-chain of other animals, such as rat, bovine, ovine and porcine; in equine, however, the sequence is CYCSTC. In order to prevent undesirable disulfide bridges from being formed, the 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

optionally 4, may show agonistic activity, whereas the peptides comprising only two out of the said partial peptides act antagonistically. Without wishing to be tied down to this theory, it is assumed that a glycoprotein hormone antagonistic activity is caused by the fact that the peptides have a specific binding affinity for the receptor of the glycoprotein hormone and this to the extent that the binding of the glycoprotein hormone to the receptor is inhibited. In this theory, peptides having glycoprotein hormone agonistic activity also 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.

Since it is plausible that some modifications of the amino acid sequences will have no substantially negative effect on the agonistic or antagonistic activity of the peptides, the invention extends to 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 β-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 β-chain of the glycoprotein hormone human FSH. Analogously, there are also sequence variants for the other glycoprotein hormones. An example of an insertion variant is a peptide in which the amino acid sequence TRDL based on the amino acids 34-37 of the β-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. An example of a

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.

For comparable reasons 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 -CONH₂).

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) :

[X¹TDSDSX²-a-]_p [X³SRAYX⁴-b-]_q [X⁵TRDLX⁶-C-]_r [X⁷AHASTAX⁸]_s in which p, q, r and s independently of each other are equal to 0 or 1, but at least two of them are equal to 1,

a, b and c represent a bridge group, if any,

χ¹ - χ⁸ each represent one or more additional amino acids, if any.

as well as variants having another sequence of the partial peptides,

as well as sequence, substitution, deletion and insertion variants naving a human FSH agonistic or antagonistic

activity,

as well as derivatives in which the free amino group of the amino-terminal amino acid, the free carboxyl group of the carboxy-terminal amino acid, or both, are blocked or otherwise modified.

For the purpose of the invention, 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). This not only applies to peptides out of four partial peptides (ρ=q=r=s=1), but also to peptides out of three or two partial peptides, such as in particular those in which p=q=r=1 and s=0, or p=q=1 and r=s=0, or q=r=1 and p=s=0. Although the bridge groups present between the partial peptides are in themselves not subject to special

restrictions, they are preferably groups of the general formula -NH- (CH₂) _z-CO- in which z is a positive integer, most preferably having a value of 1-15. Thus a preferred embodiment of the peptides according to the invention is characterized by the general formula (2) :

[X¹TDSDSX²-NH- (CH₂) _d-CO-] _p [X³SRAYX⁴-NH- (CH₂) _e-CO-] _q

[X⁵TRDLX⁶-NH- (CH₂) _f-CO-] _r [X⁷AHASTAX⁸ ] _s

in which p, q, r and s independently of each other are equal to 0 or 1, but at least two of them are equal to 1,

d, e and f represent integers having a value ranging from 1 to 15,

X₁ - X₈ each represent one or more additional amino acids, if any,

as well as variants having another sequence of the partial peptides,

as well as sequence, substitution, deletion and insertion variants having a human FSH agonistic or antagonistic

activity, as well as derivatives in which the free amino group of the amino-terminal amino acid, the free carboxyl group of the carboxy-terminal amino acid, or both, are blocked or otherwise modified.

In general, it applies to each of the partial peptides that it preferably has no more than two flanking amino acids on both sides of the core sequence and most preferably

consists of the core sequence only (partial peptide 2 is an exception since the sequence QCMGCAFSRAY gives better results than the core sequence SRAY) . Thus a special preferred

embodiment of the peptides according to the invention is characterized by the general formula (3) :

[TDSDS-NH-(CH₂)_d-CO-]_p [X³SRAY-NH- (CH₂) _e-CO-] _q [TRDL-NH-(CH₂)_f-CO-]_r [AHASTA]s

in which p, q, r and s independently of each other are equal to 0 or 1, but at least two of them are equal to 1,

d, e and f represent integers having a value ranging from 1 to 15, X₃ represents an additional amino acid sequence QCMGCAF, if any,

as well as variants having another sequence of the partial peptides,

as well as sequence, substitution, deletion and insertion variants having a human FSH agonistic or antagonistic

activity, as well as derivatives in which the free amino group of the amino-terminal amino acid, the free carboxyl group of the carboxy-terminal amino acid, or both, are blocked or otherwise modified.

More particularlu preferred are peptides according to the invention having a human FSH agonistic or antagonistic activity, characterized by the general formula (4) :

[TDSDS-NH-(CH₂)_d-CO-]p [SRAY-NH- (CH₂)_e-CO-]_q [TRDL-NH-(CH₂)_f-CO-]_r [AHASTAJs

or by the general formula (5) :

[TDSDS-NH-(CH₂)_d-CO-]_p [QCMGCAFSRAY-NH- (CH₂) _e-CO-]_q [TRDL-NH- (CH₂)_f-CO-]_r [AHASTA]s

in which p, q, r and s independently of each other are equal to 0 or 1, but at least two of them are equal to 1,

d, e and f represent integers having a value ranging from 1 to

15,

as well as variants having another sequence of the partial peptides,

as well as sequence, substitution, deletion and insertion variants having a human FSH agonistic or antagonistic

activity, as well as derivatives in which the free amino group of the amino-terminal amino acid, the free carboxyl group of the carboxy-terminal amino acid, or both, are blocked or otherwise modified.

For such peptides it has been established according to model and proved by way of experiment that for optimum results 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. For bridge groups - NH-(CH₂)_d-CO-, -NH-(CH₂)_e-CO- and -NH- (CH₂) _f-CO- this

corresponds to values of d, e and f of respectively 4-6, 4-6, and 9-11, most preferably 5, 5, and 11, respectively. In case a partial peptide contains more amino acids than the core sequence shown, other lengths of the bridge groups may be optimal. Thus, in case of the partial peptide (2) having an amino acid sequence QCMGCAFSRAY 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, however, may vary within broad limits, a value of

2-14 being acceptable to all, although e is preferably always at least 4.

Concrete examples of preferred peptides according to the invention are the peptides of formula (6) :

TDSDS -NH-(CH₂)₂-CO- QCMGCAFSRAY -NH- (CH₂) ₄-CO- TRDL -NH- (CH₂)n-CO- AHASTA

or formula (7) :

TDSDS -NH-(CH₂)₂-CO- QCMGCAFSRAY -NH- (CH₂) ₄-CO- TRDL or formula (8) :

TDSDS -NH-(CH₂)₅-CO- SRAY -NH- (CH₂) ₄-CO- TRDL -NH-(CH₂)₁₁- CO- AHASTA

or formula (9) :

TDSDS -NH-(CH₂)₅-CO- SRAY -NH- (CH₂) ₄-CO- TRDL

having a human FSH agonistic activity, as well as the peptides of formula (10) :

TDSDS -NH-(CH₂)₅-CO- SRAY

or formula (11) :

SRAY -NH-(CH₂)₄-CO- TRDL

or formula (12) :

TDSDS -NH-(CH₂) ₅-CO- SRAYTRDL

or formula (13) :

TDSDS -NH-(CH₂)₅-CO- SRAY -NH- <CH₂)₁-CO- TRDL

or formula (14) :

TDSDS -NH-(CH₂)₅-CO- SRAY -NH- (CH₂) ₂-CO- TRDL

or formula (15) :

TDSDSSRAY -NH-(CH₂)₄-CO- TRDL

or formula (16) :

TDSDSSRAYTRDL

having a human FSH antagonistic activity. 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. The adjuvants, carriers and diluents suitable and acceptable for

pharmaceutical compositions are known to those skilled in the art and will therefore not be listed here. Suitable routes of administration for the preparations are, e.g., intranasal, intraperitoneal (i.p.), intramuscular (i.m.), transdermal (suppository), oral, intravenous (i.v.).

Examples of pharmaceutical 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.

More in particular this invention provides a

pharmaceutical 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,

and at least one pharmaceutically acceptable adjuvant, carrier or diluent;

as well as a pharmaceutical 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.

For the objects and methods of using these pharmaceutical compositions according to the invention, reference is made to the possibilities listed in the introduction.

This invention will now be further explained on the basis of experimental data.

EXPERIMENTAL DATA

Synthesis of the peptides

All peptides were synthesized according to the Solid Phase method (Barany and Merrifield, 1980) . The synthesis of a number of peptides was carried out according to the tert- butyloxycarbonyl (Boc) /benzyl protection tactics. The resin used was the 4-methylbenzhydrylamine resin. The Boc amino acid derivatives (Bissendorf Biochemicals GmbH) were coupled by means of BOP reagent (BOP = benzotriazol-1-yl-oxy-tris- (dimethylamine) -phosphonium hexafluorophosphate; Castro et al, (1975) in dimethylformamide (DMF) as described by Fournier et al (1988). 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. Also used were the synthetic amino acids 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).

Other peptides were synthesized according to the

fluorenylmethyloxycarbonyl (Fmoc) /tert-butyl protecting tactics. 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 μl 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 amino acids D (Asp), S (Ser), T (Thr), Y (Tyr), R (Arg), H (His), C (Cys) and Q (Gin) were protected in the side chains, there being used respectively: Fmoc-Asp (O^tBu), Fmoc-Ser (^tBu), Fmoc-Thr (^tBu), Fmoc-Tyr (^tβu), Fmoc-Arg(Pmc), Fmoc-His (Trt ), Fmoc-Cys (Trt) and Fmoc-Gln (Trt). 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 β-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

trifluoroacetic acid / phenol / ethanedithiol / thioanisole / water in a ratio of 10 ml / 0.75 g / 0.25 ml / 0.5 ml / 0,5 ml. For 300 mg peptide resin 5 ml reagent was used.

A third series of peptides was likewise synthesized via the Fmoc tactics on an Applied Biosystems Inc. 430A

Synthesizer according to the FASTMOC method (User Bulletin 2 , 1990) on 0.1 mmol scale, likewise with a "Rink" resin.

Separation took place by the above-described Fmoc synthesis.

For purification of the peptides (up to about 90%) there was used a Polygosil 10C18 column (20 × 250 mm, Bischoff) in a HPLC (HP1082B) apparatus. About 35 mg peptide were purified using a gradient of methanol/water + 0.1% trifluoroacetic acid (TFA).

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 an analytic HPLC column (Supelcosil LC-18-DB 5 μm; 15 x 0.4 cm) in a gradient of methanol/water + 0.1% TFA.

Effects of the synthesized peptides in vitro

(a) Method

For studying the effects of the synthesized peptides use was made of the FSH inducible production of cAMP in the

Sertoli cells of 21 days old rats (Verhoeven et al, 1980; Oonk et al, 1985). Sertoli cells 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% CO₂ 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

incubation was started in medium with protein addition but with an inhibitor of the breakdown of the second messenger molecule cAMP, isobutylmethyl-xanthine (MIX; 22.3 mg/250 ml medium). This substance inhibits the activity of the enzyme phosphodiesterase and therewith the breakdown of cAMP. The antagonistic or agonistic activity of the peptides was studied by measuring the amount of cAMP in the cultured cells 1 hour after addition of different concentrations of peptide to the medium (MEM + L-glutamine + MIX) in the presence of absence of a solid concentration (500 ng/ml) ovine FSH (NIH FSH S-16 obtained from the "Endocrinological Study Section of the

National Institute of Health", Bethesda, MD, USA). This concentration of FSH maximally stimulates the production of cAMP in the cells.

There was also used a very pure human FSH preparation obtained from Organon, Oss, purified by Diosynth from human urine (GT-HMG-52-7, con. 2.5 mg/50 ml, frozen in portions of 150 μl at -80°C until use). As for the inhibition by the synthetic peptides, there were found to be no essential differences between this human FSH preparation and the ovine FSH preparation.

Any occurring aspecific effects of the peptides were studied by also adding 10^-5 M forskolin in addition to the peptide. Forskolin directly and maximally stimulates, i.e.

without using the receptor, the formation of cAMP (Dahl et al,, 1988). If the forskolin-induced cAMP response had not changed after addition of the peptide, aspecific effects were considered nil.

Cell death occurring after addition of peptides was checked by measuring the cellular ATP amount.

(b) Measurement of cAMP and ATP

For measurement of ATP the incubation of the Sertoli cells was terminated by removing the medium on ice and adding ice-cold 5% perchloric acid (PCA) to the cells. After 5 min there was centrifuged at 3000 × g for 1 min. Then the

supernatant was neutralized with KOH (about 3 M in 0.5 M

Tris). 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 μl 50% PCA are aαded on ice to the cells and the incubation medium (500 μl) until a final concentration of about 5% PCA. ( c ) Results

Unless otherwise mentioned, the amount of cAMP found in pmol per 100 μg 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.

Abbreviations used:

-2- -NH-(CH₂)₂-CO- β-alanine

-3- -NH-(CH₂)3-CO- Boc-4-aminobutanoic acid

-4- -NH-(CH₂)₄-CO- Boc-5-aminopentanoic acid -5- -NH-(CH₂)₅-CO- Boc-6-aminohexanoic acid

-7- -NH-(CH₂)₇-CO- Boc-8-aminooctanoic acid

-11- -NH-(CH₂)₁₁-CO- Boc-12-aminododecanoic acid Example 1

Addition of the peptide

Ac-TDSDS-NH-(CH₂)₅-CO-SRAY-NH-(CH₂)4-CO-TRDL-NH₂

made up of three partial peptides and purified over a C18 column (further specified als the 3-domain peptide) to Sertoli cells led to an increase in the amount of cAMP in the cells (Table 1). One hour after addition of the purified 3-domain peptide the amount of cAMP produced per 100 μg cellular protein was as high as that after addition of a maximally stimulable dose of FSH to the cells. The 3-domain peptide was effective until a concentration of 10^-7 M, i.e. at a 100 times lower concentration than that hitherto described for other agonistic peptides (Santa Coloma et al, 1989, 1990). The purified

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.

Example 2

The peptides Ac-TDSDS-NH-(CH₂)₅-CO-SRAY-NH₂ and

Ac-SRAY-NH-(CH₂)₄-CO-TRDL-NH₂ 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-(CH₂)₅-CO-SRAY-NH₂ 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.

Example 3

Effects of the peptide Ac-TDSDS-NH- (CH₂)₅-CO-SRAY-NH₂ in vivo

Method

Fourteen female young-adult rats (2.5-3 months; Wistar substrain R-Amsterdam) were kept at a controlled temperature of 22-25°C for a light-dark period of respectively 14 hours - 10 hours. Rats of this strain normally have a 5-day cycle (Osman et al, 1985; Welschen et al, 1975) . Via daily vaginal smears the stage of the cycle was determined and with 14 rats 1 ovary was removed on diestrus-2 (= 3rd day of the cycle; day 1 of the cycle = estrus) at 9.00 a.m. (unilateral ovariectomy, abridσed below as ULO). 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).

Four hours after ULO. 7 rats were injected with

physiological salt (0.9% NaCl in H₂O) . 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.

Results (see table 3)

In the rats injected with physiological salt the

increased amount of FSH after ULO in the rats provides an increased estrogen production in the remaining ovary. An increased amount of estrogens in the blood then leads to an increase in the uterus weight until 320 ± 11.4 mg (average ± SEM, n=7). in the rats injected with the peptide

Ac-TDSDS-NH-(CH₂)₅-CO-SRAY-NH₂ there is established a

significantly decreased uterus weight (246 ± 6.2 mg (n=7);

average ± SEM) with respect to the rats injected with

physiological salt.

The most plausible explanation is that the peptide

Ac-TDSDS-NH-(CH₂)5-CO-SRAY-NH₂ in vivo acts as FSH antagonist; upon administration of the peptide

Ac-TDSDS-NH-(CH₂)₅-CO-SRAY-NH₂ (2 mg/rat) 4 hours after ULO the increased FSH concentration in the blood caused by ULO

apparently cannot or can only partly lead, for a period of time that is not yet defined exactly, to stimulation of the production of estrogens in the remaining ovary.

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

invention.

REFERENCES

Applied Bio Systems (1990) User Bulletin 32, 1-40

Barany and Merrifield (1980) Solid Phase Peptide Synthesis, In: The Peptides, Analysis, Synthesis, Biology 2, Eds. Gross and Meienhofer, 1-284

Bidart et al. (1990) Science 248, 736-739

Boniface and Reichert Jr. (1990) Science 247, 61-64

Castro et al., (1975) Tetrahedron letters 14, 1219-1222

Channabasavaiah and Stewart (1979) Biochem. & Biophys. Res. Commun. 86, 1266

Charlesworth et al. (1987) J. Biol. Chem. 262, 13409-13416 Erickson et al . (1990) Endocrinology 126, 2555-2560

Fournier et al. (1988) Int. J. Pept . & Prot . Res. 31, 86-97 Gordon and Ward (1985) In: Ascoli (ed) Luteinizing hormone action and receptors, CRC Press, Boca Raton, Florida, 173 e.v. Holmgren (1985) Ann. Rev. Biochem. 54, 237-271

Houghten (1985) Proc. Natl. Acad. Sci . U.S.A. 82, 5231-5135 Houghten et al. (1986) Int. J. Pept. & Prot. Res. 27, 673-678 Houghten et al. (1986) Bio Techniques 4, 522-528

Jameson et al. (1988) Molec. Endocrinol. 2, 806-815

Keutmann et al. (1987) PNAS U.S.A. 84, 2083-2042

Morris et al. (1990) J. Biol. Chem. 265, 1881-1884

Moudgal and Rao (1984) In: Methods for the regulation of male fertility, Eds. Kumar and Waites, 31-37

Oonk and Grootegoed (1987) Mol. Cell. Endocrinol. 49, 51-62 Oonk et al . (1985) Mol. Cell. Endocrinol. 42, 39-48

Osman (1985) J. Reprod. Fert . 73, 261-270

Pierce and Parsons (1981) Annu . Rev. Biochem. 50, 465-495 Reed et al. (1990) The Endocrine Society 72nd Annual Meeting, Atlanta, GA, U.S.A., No. 755 Reichert Jr. and Dattatreyamurty (1989) Biol. Reprod. 40, 13-26

Rink (1987) Tetrahedron Lett. 28, 3787

Ryan et al. (1987) Rec. Progr. Horm. res. 43, 383-422

Ryan et al. (1988) Faseb J. 2, 2661-2669

Santa Coloma et al. (1990 a) Biochemistry 29, 1194-1200 Santa Coloma and Reichert Jr. (1990 b) J. Biol. Chem. 265, 5037-5042

Schneyer et al. (1988) Biochemistry 27, 666-671

Sluss et al. (1986) Biochemistry 25, 2644-2649

Stevens (1986) Immunol. Today 7, 369

Verhoeven et al. (1980) Mol. Cell. Endocrinol. 20, 113-116 Welschen et al. (1978) Biol. Reprod. 18, 421-427

Table 1.

Effects of the purif ied 3-domain pept ide on the amount of cAMP formed by Sertoli cells which were incubated in the presence of the phosphodiesterase inhibitor MIX. The peptide was added for 1 hour

Ac-TDSDS-5-SRAY-4-TRDL-NH₂

Basal o-FSH 500 ng/ml forskolin (10-5M)

0 18 , 6± 5 .7 (12) 115.9±17.6 (12) [6.2] 126.0+29.6 (12)

10^_3M 91 ,6± 3 .3 ( 6) [4.9] NE

10^-4M 90 .9±11 .8 (12) [4.9] 113.0±17.9 ( 9) 106.5±17.7 ( 9)

10^-5M 93 .5+ 4 .3 ( 9) [5.0]

10^-6M 85 .7±11 .0 (12) [4.6]

10^-7M 78 .6± 7 .1 (12) [4.2]

10^-8M 17 .1± 2 .0 (12) [ - 1

10^-9M 19 .7+ 2 .1 ( 3) [ - 1

Table 2

Effects of the purified peptides Ac-TDSDS-NH- (CH₂) ₅-CO-SRAY-NH₂ and Ac-SRAY-NH- (CH₂) ₄-CO-TRDL-NH₂ on the amount of cAMP in Sertoli cells which were incubated for 1 hour in the presence of the

phosphodiesterase inhibitor MIX and in the presence or absence of 500 ng/ml ovine FSH or 10^-5 M forskolin .

Ac-TDSDS- -5-SRAY-NH2

Basal o-FSH 500 ng/ml forskolin (10-5M)

0 18.6 ± 5. 7 (12) 115 .9 ± 17, .6 (12) 126.0 ± 29.6 (12)

10^-3M NE 42 .1 ± 7, .0 (12) [64%] 112.8 ± 11.8 (12)

10^-4M 18.5 ± 4. 0 (12) 43 .1 ± 7, .1 (12) [63%] 106.9 ± 21.2 (12)

10^-5M 41 .1 ± 3, .7 (12) [65%]

10^-6M 40 .3 ± 8, .0 (12) [65%]

10^-7M 39 .9 ± 13, .1 (12) [66%]

10^-8M 77 .7 ± 15 .1 (12) [33%]

10^-9M 100 .2 ± 21, .4 (12) [ - ]

AC-SRAY-4-TRDL-NH2

Basal o-FSH 500 ng/ml forskolin (10-5M)

0 18 6 ± 7 (12) 115, ± 17 (12) 126.0 ± 29.6 (12)

10^-3M 11 0 ± 9 ( 6) 37, ± 7 (12) [67%] 120.1 ± 15.6 ( 6)

10^-4M 17.3 ± 3 (12) 43, ± 19 (12) [63%] 113.3 ± 23.0 (12)

10^-5M 48, ± 8 (12) [58%]

10^-6M 56, ± 21 (12) [51%]

10^-7M 71, ± 15 (12) [38%]

10^-8M 89.7 ± 13 (12) [ - 1

10^-9M 93.6 ± 20 (12) [ - ]

Table 3

Phys .salt Ac-TDSDS-NH- (CH₂) ₅-CO-SRAY- -NH₂ (2 mg/rat)

Uterus weight (mg)

rat No. 1 324 rat No. 8 219

2 353 9 261

3 337 10 230

4 271 11 258

5 311 12 245

6 296 13 248

7 352 14 263

320 + 11.4 246 + 6.2*

Body weight (gram)

rat No. 1 160 rat No. 8 165

2 177 9 184

3 178 10 162

4 196 11 161

5 199 12 191

6 180 13 167

7 189 15 180

182.7 + 5.0 172.9 + 4.5

Effects of administration of the peptide Ac-TDSDS-NH-(CH₂)₅-CO-SRAY-NH₂ after ULO on uterus weight and body weight of 7 rats (Nos. 8-14) v/ith respect to rats injected with physiological salt (Nos.

1-7). The values of each rat are given as well as the average ± SEM. There is no verlap in uterus weights between the physiological salt group rats and the rats injected with peptide.

* = significantly different from rats treated with physiological salt according to the Wilcoxen two sample test p < 0.0001.

Table 4

Ac-DSDST-5-SRAY-4-TRDL-NH₂ basal o-FSH (500 ng/ml) forskolin (10^-5M)

0 21.9 ± 5 (4 129.8 ± 14, 0 (4) [5.9] 135.1 ± 28.7 (4)

10^{- 3}M 86 ± 12 (4 [3.9] 118 1 ± + 7, 9 (4) 128.0 ± 31.0 (4)

10^{- 4}M 90 ± 5 (4 [4.1] NE NE

10^{- 5}M 82 ± 10 (4 [3.8]

10-⁶M 89 ± 15 (4 [4.1]

10-⁷M 79 ± 15 (4 [3.6]

10^-8M 19, + 4 (4 [ - 1

10^-9M NE

Conclusion: The peptide Ac-DSDST-5-SRAY-4TRDL-NH₂ has the same effect as the peptide Ac-TDSDS-5- SRAY-4-TRDL-NH₂, namely agonistic up to a concentration of 10^-7M.

Table 5

Ac-TDSDSSRAYTRDL-NH₂

basal o-FSH (500 ng/ml) forskolin (10^-5M)

21.9 ± 5.3 (4) 129.8 ± 14.0 (4) 135.1 ± 28.7 (4)

10^-3M 32.8 + 10.1 (4) 55.1 ± 7.2 (4) [58%] 108.3 ± 20.3 (4)

10^-4M 18.3 ± 7.9 (4) 48.9 ± 9.8 (4) [62%] 110.2 ± 19.5 (4)

10^-5M NE 47.1 ± 10.1 (4) [64%] NE

10^-6M '' 62.3 ± 8.7 (4) [52%] ''

10^-7M '' 115.3 ± 7.1 (4) [ - ] ''

10^-8M NE 121.1 ± 9.3 (4) [ - ]

Conclusion : The removal of the spacers from the peptide Ac-TDSDS-5-SRAY-4-TRDL-NH₂ changes the

effect from agonistic (up to 10^-7M) to antagonistic (up to 10^-6M). The peptide Ac- TDSDSSRAYTRDL-NH₂ works less effective than the peptide Ac-TDSDS-5-SRAY-NH₂; in other words, 3-domain peptide without spacers is less effective (antagonistic) than 2-domain peptide with spacers (10^-7M) .

Table 6

AC-SRAY-4-TRDI

basal o-FSH (500 ng/ml) forskolin (10^-5M)

21.9 ± 5.3 (4) 129.8 ± 14.0 (4) 135.1 ± 28.7 (4)

10^-3M 19.3 ± 8.2 (4) 46.3 ± 9.8 (4) [64%] 115.1 ± 17.3 (4)

10^-4M 15.1 ± 7.9 (4) 55.1 ± 10.22 (4) [58%] 141.4 + 30.2 (4)

10^-5M NE 61.0 ± 7.9 (4) [53%] NE

10^-6M '' 59.8 ± 8.3 (4) [54%]

10^-7M '' 71.2 ± 9.0 (4) [45%]

10^-8M '' 83.1 ± 17.1 (4) [36%]

10^-9M " 110.3 ± 15.5 (4) [ - ]

Conclusion : The peptide Ac-SRAY-4-TRDI-NH₂ has the same effect (antagonistic up to 10^-7M) as the peptide Ac-SRAY-4-TRDL-NH₂. 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-NH₂ is probably not very useful as a specific antagonist.

Table 7

Ac-TRDL-7-SDS-4-SRAY-3-KS-NH₂

basal o-FSH (500 ng/ml) forskolin (10^-5M)

0 21.9 ± 5.3 (4) 129.8 ± 14.0 (4) 135.1 ± 28.7 (4)

10^-3M 96.8 ± 15.3 (4) [4.4] 121.7 ± 8.3 (4) 109.3 ± 15.6 (4)

10^-4M 97.3 ± 8.2 (4) [4.4] 115.6 ± 19.2 (4) 121.9 ± 35.2. (4)

10^-5M 73.2 + 7.3 (4) [3.3] NE NE

10^-6M 25.3 ± 7.0 (4) [ - ] "

10^-7M 15.0 ± 8.2 (4) [ - ] "

10^-8M NE "

10^-9M " " "

Conclusion: The 4-domain peptide Ac-TRDL-7-SDS-4-SRAY-3-KS-NH₂ is less effective (agonistic) than the 3-domain peptide Ac-TDSDS-5-SRAY-4-TRDL-NH₂.

Table 8

Ac-TDSDS-5-SAAY-4-TRDL-NH₂

basal o-FSH (500 ng/ml) forskolin (10^-5M)

0 21.9 ± 5.3 (4) 129.8 ± 14.0 (4) 135.1 ± 28.7 (4)

10^-3M 25.0 ± 12.1 (4) 65.8 ± 13.1 (4) [49%] 127.0 ± 35.2 (4)

10^-4M 29.1 ± 3, .2 (4) 53.5 ± 7 [59%] 116.2 ± 19.5 (4)

10^-5M 35.1 ± 8, .3 (4) 57.8 ± 10 [55%] NE

10^-6M 37.0 ± 19, .1 (4) 52.1 ± 8 [60%]

10^-7M 27.1 ± 7, .5 (4) 69.7 ± 15 [46%]

10^-8M NE 76.8 ± 13 [41%]

10^-9M 135.2 ± 21.8 (4) [ - ]

Conclusion : The change of an arginine (R) to an alanine (A) in the 2nd domain (SRAY->SAAY) of the domain peptide changes the effect of this peptide from an agonist (10^-7M) to an antagonist (10^-8M) . In comparison with the 2-domain peptide Ac-TDSDS-5-SRAY-NH₂ this antagonistic 3-domain peptide is equally effective (i.e. not better by adding the 3rd domain).

Remark : From here Fmoc synthesis, instead of Boc synthesis, is applied for solid-phase peptide synthesis.

Table 9

Ac-TDSDS-2-QCMGACFSRAY-4-TRDL-NH₂ basal o-FSH (500 ng/ml) forskolin (10^-5M)

0 23.5 ± 11 .5 (11) 148.9 ± 29.1 (11) [6.3] 126.2 ± 21.7 (6)

10^-4M 26.5 ± 6 .0 ( 3) 118.5 ± 23 ( 3) 147.0 ± 7.5 (3)

10^-6M 28.0 ± 2 .5 ( 3) 169.5 ± 16 ( 3) NE

10^-8M 15.5 ± 3 .5 ( 3) NE NE

Table 10

Ac-TDSDS-2-QCMGCAFSRAY-4-TRDL-NH₂

basal o-FSH (500 ng/ml) forskolin (10^-5M)

10^-4M 93.0 ± 16.0 (3) [4.0] 99.3 ± 5.5 (3) [33%] 117.5 ± 13.5 (3)

10^-6M 119.0 ± 10.0 (3) [5.1] 116.5 ± 23.5 (3) [ - ] NE

10^-8M 104 ± 6.0 (3) [4.4] NE NE

Remark : In the sequence around SRAY in FSH the following amino acids are normally present:

QCMGCCFSBAY . 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. We have each time allowed 2 cys to form a bridge with each other (oxidation over weekend, check with Ellman test on free SH was found negative, all cysteine present in pairs). Of the 3 cysteines from the original sequence 1 has changed to an alanine (peptides 6 and 7, respectively).

Conclusion : Peptide 6 has been found ineffective both agonistically and antagonistically. Peptide 7 has been found agonistic to a lower concentration (at least 10^-8M) than the 3-domain peptide Ac-TDSDS-5-SRAY-4-TRDL-NH₂. However, this peptide also seems to have a low antagonistic activity.

Table 11

Ac-TDSDS-2-QCMGCAFSRAY-4-TRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 10.26 ± 5.23 (13) 123.99 ± 16.64 (12) [12.1] 113.90 ± 25.79 (10)

10^-4M 74.0 ± 11.0 ( 3) [7.2] 128.0 ± 25.25 ( 3) 129.75 ± 33.5 ( 3)

10^-5M 54.0 ± 3.2 ( 3) [5.3]

10^-6M 50.8 ± 1.02 ( 3) [5 ]

10^-7M 45.0 ± 3.1 ( 3) [4.4]

10^-8M 48.1 ± 15.3 ( 3) [4.7]

10^-9M 27.0 ± 10.0 ( 3)

10^-10M 30.5 ± 19.5 ( 3)

10^-11M 18.25 ± 2.5 ( 3)

10^-12M 29.75 ± 5.5 ( 3)

Conclusion : Addition of the sequence QCMGCAF N-terminal of the 2nd domain (SRAY) leads to an agonistic peptide which is 10 times more effective than the 3-domain peptide (10^-7M)

Table 12

Ac-TDSDS-5-SRAY-4-TRDL-11-AHASTA-NH₂

basal o-FSH (500 ng/ml) forskolin (10^-5M)

0 23.5 ± 11.5 (11) 148.9 ± 29.1 (11) 126.2 ± 21.7 (6)

10^-4M 114.0 ± 17.0 ( 3) [4 .9] 134.0 ± 25.0 ( 3) 167 ± 8.0 (3)

10^-6M 110 ± 4.0 ( 3) [4 .7] 146.5 ± 27 ( 3) NE

10^-8M 103 ± 35 ( 3) [4 .4] NE NE

Remark : In the sequence of FSH the following amino acids are normally present: CHCSTC. The C is replaced here by an A.

Conclusion : Attachment of a 4th domain to the 3-domain peptide leads to an at least 1 ¹⁰log scale more effective (agonistic) peptide.

Table 13

AC-TDSDS-5-SRAY-4-TRDL-11-AHASTA

basal o-FSH 500 ng/ml forskolin 10^-5M

0 10.26 ± 5. 23 (13) 123.99 ± 16.64 (12) [12.1] 113.90 ± 25.79 (10)

10^-4M 61.0 ± 39.2 ( 3) [ 5. 9] 164.25 ( 2) 92.5 ( 2)

10^-5M 73, ± 10. 5 ( 3) [ 7.1]

10^-6M 100, ± 18.0 ( 3) [ 9.8]

10^-7M 119, ± 22.3 ( 3) [11.6]

10^-8M 87 . 0 ± 15.0 ( 3) [ 8.5]

10^-9M 22 . 75 ± 5.25 ( 3) [ - ]

10^-10M 9 . 20 ± 5.0 ( 3)

10^--1M 12 . 50 ± 0.5 ( 3)

10^_12M 10 . 01 ± 7.8 ( 3)

Conclusion : Addition of a 4th domain C-terminal to the 3-domain peptide leads to an agonistic peptide which is 10 times more effective than the 3-domain peptide (10^-7M).

Table 14

Ac-TDSDS-5-SRAY-NH₂ (F-moc)

basal o-FSH (500 ng/ml) forskolin (10^-5M)

0 23.5 ± 11.5 (11) 148.9 ± 29.1 (11) 126.2 ± 21.7 (6)

10^-4M 20.8 ± 1.5 ( 3) 41.5 ± 7.0 ( 3) [73%] 127 ± 17 (3)

10^_6M 26.5 ± 2.3 ( 3) 51.1 ± 11.4 ( 3) [66%]

10^~8M NE 68.3 ( 2) [54%] of . Boc synthesized product

Ac-TDSDS-5-SRAY-NH₂

basal o-FSH (500 ng/ml) forskolin (10^-5M)

10^_4M 23.0 ± 5.1 (3) 45.1 ± 7.1 (3) [70%] 135.8 ± 30.2 (3)

10^{- 6}M NE 48.3 ± 18.9 (3) [68%]

10^-8M NE 69.8 ± 15.4 (3) [53%]

Conclusion : F-moc synthesis gives a 2-domain antagonistic peptide Ac-TDSDS-5-SRAY-NH₂ which is as effective as peptide obtained via Boc synthesis.

Table 15

Ac-TDSDS-2-SRAY-4-TRDL-NH₂

basal o-FSH (500 ng/ml) forskolin (10^-5M) fr2 0 23.5 ± 11.5 (11) 148.9 ± 29.1 (11) 126.2 ± 21.7 (6)

10^-4M 104 ± 17 ( 3) [4 .4] 102.0 ± 12.5 ( 3) 126 (2)

10^-6M 106 ± 5 ( 3) [4 .5] 126 ± 6 ( 3)

10^-8M 44 ( 2) 146.5 ( 2)

fr2 10^-4M 39.5 ± 8.5 ( 3) 39.5 ± 8.5 ( 3) 42 ( 2 ) [ 67 % ]

10^-6M 34.5 ± 10.5 ( 3) 33.0 ± 3.5 ( 3)

10^-8M 33.5 ( 2) 25.5 ( 2)

Conclusion : Of this peptide 2 equally large fractions were present on HPLC, fraction 2 and fraction

4. Both have been tested. 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-NH₂. Reduction of spacer 1 between the 1st and the 2nd domain from NH-(CH₂)s-CO to NH- (CH₂) ₂-CO (b-alanine) seems to be allowed.

Table 16

Ac-TDSDS-5-SR*AY-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 12 ± 6.5 (16) 93.5 ± 11.6 (15) [7.8] 96 ± 12.5 (15)

10^-4M NE 70.8 ± 7.4 ( 3) 70 ± 13 ( 3)

10^-6M NE 86.6 ± 7.8 ( 3) NE

Remark : In the HPLC analysis this peptide (F-moc synthesized) was found improperly deprotected.

The retention time was too high (30 min, instead of 10 min, with peptide No. 9 and "original" Boc synthesized 2-domain peptide). Most probably, the R is still protected with the PMC group. Reactive compounds formed during TFA separation may also lead to binding with the tyrosine (Y).

Conclusion : This improperly deprotected 2-domain peptide in which, most probably, a protective group is attached to the R, is not effective as antagonist. The R seems to be an important amino acid (see also conclusion at peptide No. 5: Ac-TDSDS-NH-5-SAAY-4-TRDL-NH₂, in which the R has been replaced by an A). However, it is not quite impossible that the R is deprotected indeed but a reactive compound of the PMC group has reacted with the Y. This leads to the conclusion that the Y is an important amino acid (see also conclusion at peptide 16) .

Table 17

Ac-TDSDS-3-SRAY-4-TRDL-NH₂

basal oo--]FSH 500 ng/ml forskolin 10^-5M

0 12 ± 6.5 (16) 93 5 ± 11.6 (15) [7.8] 96 ± 12.5 (15)

fr3 10^-4M 15 ( 2) NE 8 ± 3 ( 3) [92%]

10^-6M 19 ( 2) NE NE .

10^-8M NE NE NE

fr5 10^-4M 28 ( 2) [2.3] 74 ( 2) 94 ± 2 (3)

10^-6M 89 ( 2) [7.4] NE NE

10^-8M 37 ( 2) [3.1] NE NE

Remark : Of this peptide 2 equally large fractions were present on HPLC, fraction 3 and fraction

5. Both have been tested.

Conclusion : Peptide fraction 3 has been found toxic as measured at suppression of the forskolin induced cAMP production. Fraction 5 gives a stimulation of the basal cAMP production, although this is low at 10^-4M and seems better at 10^-6M. Reduction of spacer 1 between the 1st and the 2nd domain from NH-(CH₂)s-CO to NH-(CH₂)₃-CO seems to be allowed; it turns out that the agonistic effect of the peptide is maintained.

Table 18

Ac-TDSDS-4 -SRAY-4-TRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

f r4 0 12 ± 6.5 (16) 93.5 ± 11.6 (15) 96 ± 12.5 (15)

10-⁴M 12.5 ± 5 ( 3) 72.7 ( 2) 112 ( 2)

10^{- 6}M 10.0 ± 1.0 ( 3) 86.1 ( 2)

10^-8M 5.0 ± 7.5 ( 3) 76.9 ( 2)

f r5 basal o-FSH 500 ng/ml forskolin 10^-5M

0 10.26 ± 5.23 (13) 123.99 ± 16.64 (12) [12.1] 113.90 ± 25.79 (10)

10^{- 4}M 40 ± 6 ( 3) [3.9] 106.50 ± 12.50 ( 3) 82.0 ( 2)

10^-6M 39 ± 1 ( 3) [3.8] 122.75 ± 12.50 ( 3)

10^{- 8}M 16.25 ( 2) 135.5 ( 2)

Rema r k : Of this peptide 2 equally large fractions were present on HPLC, fraction 4 and fraction

5. Both have been tested. In amino acid analysis [T] has been found too low in both. Conclusion: Peptide fraction 4 is ineffective. However, peptide fraction 5 is agonistically less stimulation than FSH itself. Reduction of spacer 1 between the 1st and the 2nd domain from 5 to 4 seems to be allowed; it turns out that the agonistic effect of the peptide is maintained.

Table 19

Ac-TDSDS-ll-SRAY-4-TRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 12 ± 6.5 (16) 93.5 ± 11.6 (15) [7.8] 96 ± 12 . 5 ( 15)

10^-4M 44 ± 11 ( 3) [3.7] 89 ± 4 ( 3) 87 ± 1 . 9 ( 3 )

10-⁶M 46 ± 16 ( 3) [3.8] 96 ± 9.4 ( 3) NE

10^-8M 15 ± 9.9 ( 3) NE NE

Conclusion : Increasing spacer 1 between the 1st and the 2nd domain from NH-(CH₂)₅-CO to NH-(CH₂)n- CO leads to an agonistic peptide which, however, induces a less high cAMP production than the original 3-domain peptide Ac-TDSDS-5-SRAY-4-TRDL-NH₂.

Table 20

Ac-TDSDS-5-SRAY-4-ARDL-NH₂

basal o-FSH 500 ng/ml forskol in 10^-5M

0 12 ± 6.5 (16) 93.5 ± 11.6 (15) [7.8] 96 ± 12.5 (15)

10^-4M 80 ± 7.6 ( 3) [6.7] 87.2 ± 6.9 ( 3) 82 ± 12 ( 3)

10^-6M 60 ± 28 ( 3) [5 ] 82 + 10 ( 3) NE

10^-8M 5.7 ± 1.5 ( 3) NE NE

Conclusion : Changing a threonine (T) to an alanine (A) in the 3rd domain (TRDL->ARDL) of the 3- domain peptide has no effect on the activity of this peptide (at least up to 10^-6M agonistic).

Table 21

Ac-TDSDS-5-SRAA-4-TRDL-NH₂

basal o-FSH 5oo ng/ml forskolin 10^-5M

0 12 ± 6.5 (16) 93.5 ± 11.6 (15) 131 ± 16 (3)

10^-4M 11.2 ± 2.8 ( 3) 47.6 ± 8.9 ( 3) [49%]

10^-6M 14.4 ± 3.6 ( 3) 91.2 ± 12.8 ( 3)

10^-8M 15.2 ± 1.4 ( 3)

Conclusion : Changing a tyrosine (Y) to an alanine (A) in the 2nd domain (SRAY->SRAA) of the 3-domaiN peptide changes the activity of this peptide from an agonist (10^-7M) to an antagonist (10^-4M) . (The tyrosine seems to be an important amino acid residue for agonistic

activity.) In comparison v/ith the 2-domain peptide Ac-TDSDS-5-SRAY-NH₂ this antagonistiC 3-domain peptide is much less potent. (The tyrosine also seems an important amino acid residue for antagonistic activity.)

Table 22

Ac-TDSDS-5-TRDL-4-SRAY-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 12 ± 6.5 (16) 93.5 ± 11.6 (15) 96 ± 12.5 (15)

10^-4M 5.8 ± 0.3 ( 3) 47.2 ± 5.15 ( 3) [50%] 82 ± 13 ( 3)

10^-6M 6.0 ± 0.7 ( 3) 103.6 ± 10.1 ( 3)

10^-8M 7.2 + 2.6 ( 3)

Conclusion : Exchanging domains 2 and 3 in the 3-domain peptide leads to an antagonistic peptide

(10^-4M) which is not better than a 1-domain peptide Ac-TRDL-NH₂ or Ac-SRAY-NH₂.

Table 23

Ac-TDSDA-5-SRAY-4-TRDL-NH₂ basal o-FSH 500 ng/ml forskolin 10^-5M

0 29.3 ± 6 (6) 128.5 ± 13.48 (6) [4.4] 152.67 ± 18.91 (6) [5.2]

10^-4M 35.33 ± 7.97 (3) 111.83 ± 27.71 (3) 103.33 ± 30.09 (3)

10^-6M 35.67 ± 9.44 (3) 106.67 ± 21.13 (3) NE

10^-8M 36.33 ± 4.62 (3) NE NE

Conclusion : Replacing the last serine (S) in 1st domain by an alanine (A) (TDSDS -> TDSDA) leads to a peptide which has neither agonistic nor antagonistic activity.

Table 24

Ac-TDADA-5-SRAY-4-TRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 29.3 ± 6 (6) 128.5 ± 13.48 (6) [4.4] 152.67 ± 18.91 (6) [5.2]

10^-4M 36.8 ± 2.84 (3) 80.97 ± 15.19 (3) [37%] 115.33 ± 18.84 (3)

10^-6M 33.83 ± 10.68 (3) 112.17 ± 18.91 (3) [ - ] NE

10^-8M NE NE NE

Conclusion : Replacing the first and the second serine (S) in 1st domain by an alanine (A) (TDSDS ¬

TDADA) leads to a peptide having a low antagonistic activity at 10^-4M (instead of an agonist at 10^-7M) .

Table 25

Ac-TASDA-5-SRAY-4-TRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 29.3 ± 6.0 (6) 128.5 ± 13.48 (6) [4.4] 152.67 ± 18.91 (6) [5.2]

10^-4M 39.17 ± 11.34 (3) 99.83 ± 10.32 (3) 123.0 ± 2.65 (3)

10^-6M 45.5 ± 1.5 (3) [1.55] 104.83 ± 21.87 (3) NE

10^-8M 39.33 ± 5.97 (3) NE NE

Conclusion : Replacing both the first aspartate (D) and the second serine (S) in an alanine (A) in the first domain (TDSDS -> TASDA) leads to an ineffective peptide (having neither agonistic nor antagonistic activity).

Table 26

Ac-ADSDA-5-SRAY-4-TRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 29.3 ± 6.0 (6) 128.5 ± 13.48 (6) [4.4] 152.67 ± 18.91 (6) [5.2]

10^-4M 39.3 ± 6.51 (3) 152.0 ± 22.34 (3) 114.5 ± 18.03 (3)

10^-6M 39.67 ± 3.2 (3) 101.5 (2) NE

10^-8M 42.83 ± 2.25 (3) [1.46] NE NE

Conclusion : Replacing both the first threonine (T) and the last serine (S) in the 3-domain peptide by an alanine (A) leads to an ineffective peptide (having neither agonistic nor

antagonistic activity).

Table 27

Ac-TDSDS-5-ARAY-4-TRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 12.44 ± 4.23 (13) 165.29 ± 15.45 (13) [13.3] 12.96 ± 6.01 (12)

10^-4M 70 ± 22.75 ( 3) [5.6] 150.0 ( 2) 22.5 ( 2)

10^-6M 60.45 ± 17.05 ( 3) [4.9] 148.25 ± 12.5 ( 3)

10^-8M 11.25 ( 2) 162.75 ( 2)

Remark : Forskolin has not worked in this test.

Conclusion : Changing a serine (S) to an alanine (A) in the 2nd domain of the 3-domain peptide (SRAY-

> ARAY) does not lead to changing the properties of this peptide (agonist 10^-6M,

>10^-8M) .

Table 28

Ac-TDSDSSRAY-4-TRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 12.44 ± 4.23 (13) 165.29 ± 15.45 (13) 12.96 ± 6.01 (12)

10^-4M 9.75 ± 2.75 ( 3) 99.25 ± 10.25 ( 3) [40%] 9.25 ( 2)

10^-6M 8.75 ± 3.75 ( 3) 108 ( 2) [35%]

10^-8M 7.25 ( 2) 148.25 ( 2)

Remark : Forskolin has not worked in this test.

Conclusion : Removal of the spacer between domains 1 and 2 of the 3-domain peptide changes this peptide to an antagonist (10^-6M, >10^-8M) which, however, is less effective than the antagonist Ac-TDSDS-5-SRAY-NH₂ (2-domain peptide, 10^-8M) but as effective as the antagonist Ac-SRAY-4-TRDL-NH₂.

Table 29

Ac-TDSDS-5-SRAYTRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 12.44 ± 4.23 (13) 165.29 ± 15.45 12.96 ± 6.01

10^-4M 11.50 ± 0.6 ( 3) 66.38 ± 6.50 (3) [60%] 18.0 (2)

10^-6M 19.0 ± 1.0 ( 3) 64.88 ± 13.13 (3) [61%]

10^-8M 5.0 117.50 (2) [29%]

Remark : Forskolin has not worked in this test.

Conclusion : Removal of the spacer between the 2nd and the 3rd domain changes the peptide to an antagonist (10^-8M) which seems as effective as the antagonist Ac-TDSDS-5-SRAY-NH₂.

Table 30

Ac-TDSDS-5-SRAY-2-TRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 12.44 ± 4123 (13) 165.29 ± 15.45 12.96 ± 6.01

10^-4M 19.5 ± 3.0 ( 3) 54.88 ± 2.50 (3) [67%] 28.5 (2)

10^-6M 24.5 ± 7.0 ( 3) 66.38 ± 7.13 (3) [60%]

10^-8M 35.5 ( 2) 71.63 (2) [57%]

Remark : Forskolin has not worked in this test.

Conclusion : Reduction of the spacer between the 2nd and the 3rd domain to -2- (b-alanine) changes the peptide to an antagonist (10^-8M) which seems as effective as the antagonist Ac- TDSDS-5-SRAY-NH₂.

Table 31

Ac-TRDL-5-TDSDS-4-SRAY-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 12.44 ± 4.23 (13) 165.29 ± 15.45 (13) [13.3] 12.96 ± 6.01 (12)

10^-4M 15.25 ± 2.25 ( 3) 106.25 ± 12.75 ( 3) [36%] 12.0 ( 2)

10^-6M 19.5 ± 4.25 ( 3) 133.0 ± 6.75 ( 3) [20%]

10^-8M 18.0 ( 2) 135.25 ( 2) [18%]

Remark : Forskolin has not worked in this test.

Conclusion : Exchanging the domains 1, 2 and 3 to 3-1-2 leads to a low antagonistic peptide (10^-4M) which is not better than a 1-domain peptide Ac-TRDL-NH₂ or Ac-SRAY-NH₂.

Table 32

Ac-DSDST-5-SRAY-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 12.44 ± 4.23 (13) 165.29 ± 15.45 (13) [13.3] 12.96 ± 6.01 (12)

10^-4M 19.75 ± 1.25 ( 3) 74.25 ± 2.13 ( 3) [55%] 23.0 ( 2)

10^-6M 20.75 ± 2.00 ( 3) 88.63 ± 7.25 ( 3) [46%]

10^-8M 22.25 ( 2) 83.75 ( 2) [49%]

Remark : Forskolin has not worked in this test.

Conclusion: Peptide Ac-DSDST-5-SRAY-NH₂ has the same effect as the "original" 2-domain peptide Ac- TDSDS-5-SRAY-NH₂, namely antagonistic up to a concentration of 10^-8M.

Table 33

Ac-TDSDS-5-SRAY-3-TRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 12.44 ± 4.23 (13) 165.29 ± 15.45 (13) 12.96 ± 6.01 (12)

10^-4M 14.5 ± 0.5 ( 3) 68.75 ± 3.38 ( 3) [58%] 12.75 ( 2)

10^-6M 14.25 ± 5.75 ( 3) 62.63 ± 0.5 ( 3) [62%]

10^-8M 17.5 171.75 ( 2)

Remark : Forskolin has not worked in this test.

Conclusion : Reduction of the spacer between the 2nd and the 3rd domain from 4 to 3 changes this

peptide to an antagonist (10^-6M, >10^-8M) which is less effective than the antagonist Ac- TDSDS-5-SRAY-NH₂ or those peptides in which the spacer is 0 and 2, respectively. In order to obtain an agonist ic peptide, the spacer between domains 2 and 3 must be at; least. 4. In order to thus obtain an antagonistic peptide (10^-8M), the spacer must, bo 2 or 0 or domain 3 must be omitted.

Table 34

Ac-TDSDS-2-QCMGCAFSRAY-4-TRDL-ll-AHASTA-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 10.26 ± 5 23 13) 123.99 ± 16.64 (12) [12.1] 113.90 ± 25.79 (10)

10^{- 4}M 90 00 ± 12 3) [8.8] 142.0 ± 20.75 ( 3) 71.50 ± 7.5 ( 3)

10^-5M 76 20 + 30 5 3) [7.4]

10^-6M 52 30 ± 18 0 3) 1]

10^-7M 94 50 ± 28 .3 3) 2]

10^-8M 94 50 ± 20 .0 3) 2]

10^-9M 54 0 ± 14 ,5 3) 3]

10^-10M 25.0 ± 10 ,0 3) 1

10^-11M 19.0 ± 2 ,5 3)

10^-12M 14.50 ± 9 ,5 3)

Conclusion : Addition of the sequence QCMGCAF N-terminal of the 2nd domain (SRAY) and addition of a

4th domain C-terminal to the 3-domain peptide leads tυ an agonistic peptide which is 100 times more effective than the 3-domain peptide (10^-7M).

Table 35

Ac-TDSDS-5-SRAY-4-TADL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 20.40 ± 7.2 (13) 110.0 ± 15.2 (14) [5.4] 73.57 ± 13.08 (14)

10^-4M 12.4 ± 1.2 ( 3) 49.70 + 5.1 ( 3) [55%] 59.8 ( 2)

10^-6M 14.6 ± 3.8 ( 3) 48.0 ± 20 ( 3) [56%]

10^-8M 15.0 50.2 ( 2) [54%]

Conclusion : Changing an arginine (R) to an alanine (A) in the 3rd domain of the 3-dornain peptide changes the activity of this peptide from an agonist to an antagonist (10^-8M).

Table 36

Ac-TDSDS-5-SRAY-4-TRDL-NH₂ basal O-FSH 500 ng/ml forskolin 10^-5M

0 20.40 ± 7.2 (13) 110.0 ± 15.2 (14 ) [5.4] 73.57 ± 13.08 (14)

10^-4M 93.2 ± 14.0 ( 3) [4.6] NE NE

Boc 10^-6M 95.4 ± 9.3 ( 3) [4.7]

10^-8M 15.0

Fmoc 10^-4M 90.0 ± 17 (3) 107.2 (2) 107 (2)

10^-6M 97.0 ± 7.0 (3)

10^-8H 19.4 (2)

Conclusion: Fmoc synthesized 3-domain peptide = Boc synthesized 3-domain peptide

Table 37

Ac-TDSDS-5-SRAY-ll-TRDL-NH₂

baiial o-FSH 500 ng/ml forskolin 10^-5M

0 20.40 ± 7.2 (13) 110.0 ± 15.2 (14) [5.4] 73.57 ± 13.08 (14)

10^-4M 75 ± 14 ( 3) [3.8] 87.87 ± 3.31 ( 3) 67.7 ( 2)

10^-6M 95.2 ± 11.5 ( 3) [4.7] 100.4 ± 10.8 ( 3)

10^-8M 18.46 ( 2) 135.5 ( 2)

Conclusion : Increasing spacer 2 between the 2nd and the 3rd domain from NH-(CH₂)₄-CO to NH-(CH₂)₁₁- CO leads to an agonistic peptide (10^-6M, <10^-8M) which is as effective as the 3-domain peptide (10^-7M) .

Table 38

Ac-TDSDS-5-SRAY-5-TRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 20.40 ± 7.2 (13) 110.0 ± 15.2 (14) [5.4] 73.57 + 13.08 (14)

10^-4M 136.0 ± 8.0 ( 3) [6.7 ] 96.67 ± 5.62 ( 3) 58.73 ± 4.45 ( 3)

10^-6M 134.0 ± 16.52 ( 3) [6.6 ] 137.40 ± 43.0 ( 3)

10^-8M 120.67 ± 3.51 ( 3) [5.92]

Conclusion : Increasing spacer 2 between the 2nd and the 3rd domain from NH-(CH₂)₄-CO to NH-(CH₂)s leads to an agonistic peptide (10^-8M) which is more effective by a factor of 10 than the 3-domain peptide (10^-7M) .

Table 39

Ac-TDSDS-5-SRAY-4-TRAL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 20.40 ± 7.2 (13) 110.0 ± 15.2 (14) [5.4] 73.57 ± 13.08 (14)

10^-4M 131.0 ( 2) [6.4] 70.2 (89.8-50.6) 89.52 ( 2)

10^-6M 117.67 ± 14.64 ( 3) [5.8] 109.13 ± 17.26 ( 3)

10^-8M 35.0 ( 2) 76.82 ± 40.17 ( 3) ConcIusion: Changing an aspartate (D) to an alanine (A) in the 3rd domain of the 3-domain peptide does not lead to changing the agonistic properties of this peptide (agonist 10^-6M, >10^-8M), however, it seems that now a partial antagonist has been formed (inhibition of FSH-induced cAMP response), the role of the D is still uncertain.

Ta b l e 40

Ac-TDSAS-5-SRAY-4 -TRDL-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 20.40 ± 7.2 (13) 110.0 ± 15.2 (14) [5.4] 73.57 ± 13.08 (14)

10^-4M 11.73 ± 2.03 ( 3) 126.66 ± 12.42 ( 3) 71.50 ( 2)

10^-6M 13.60 ± 1.13 ( 3) 116.40 ± 6.64 ( 3)

10^-8M 12.55 123.10 ( 2)

Conclusion : Changing the last aspartate (D) to an alanine (A) in the 1st domain of the 3-domain peptide leads to an ineffective (neither agonistic nor antagonistic) peptide.

Table 41

Ac-TDSDS-5-SRAY-4-TRDA-NH₂

basal o-FSH 500 ng/ml forskolin 10^-5M

0 20.40 ± 7.2 (13) 110.0 ± 15.2 (14) [5.4] 73.57 ± 13.08 (14)

10^-4M 89.66 ± 3.06 ( 3) [4.4] 101.33 ± 4.63 ( 3) 66.50 ( 2)

10^-6M 113.66 ± 17.62 ( 3) [5.6] 116.40 ± 9.31 ( 3)

10^-8M 21.50 ( 2) 116.33 ( 2)

Conclusion : Changing the leucine (L) tυ an alanine (A) in the 3rd domain of the 3-domain peptide does not lead to changing the agonistic properties of this peptide (agonist 10^-6M, >10^-8M).

Claims

1. A peptide having a glycoprotein hormone agonistic or antagonistic activity, said peptide comprising at least two partial peptides selected from

(1) a partial peptide having an amino acid sequence comprising either the amino acid sequence TDSDS based on the amino acids

92, 88, 89, 90 and 91 of the β-chain of the glycoprotein hormone human FSH or a corresponding amino acid sequence of another glycoprotein hormone and/or another animal species,

(2) a partial peptide having an amino acid sequence occurring in the α-chain of glycoprotein hormones and comprising the amino acid sequence SRAY,

(3) a partial peptide having an amino acid sequence comprising either the amino acid sequence TRDL based on the amino acids 34-37 of the β-chain of the glycoprotein hormone human FSH or a corresponding amino acid sequence of another glycoprotein hormone and/or another animal species, and

(4) a partial peptide having an amino acid sequence comprising the amino acid sequence AHASTA, derived from the amino acids 82-87 of the α-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,

as well as sequence, substitution, deletion and insertion variants having a glycoprotein hormone agonistic or

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.

2. A peptide according to claim 1 having a human FSH agonistic or antagonistic activity, characterized by the general formula (1) :

[X¹TDSDSX²-a-]_p [X³SRAYX⁴-b-]_q [X⁵TRDLX⁶-C-]_r [X⁷AHASTAX⁸ ]_s in which p, q, r and s independently of each other are equal to 0 or 1, but at least two of them are equal to 1,

a, b and c represent a bridge group, if any,

X¹ - X⁸ each represent one or more additional amino acids, if any.

as well as variants having another sequence of the partial peptides,

as well as sequence, substitution, deletion and insertion variants having a human FSH agonistic or antagonistic

activity,

as well as derivatives in which the free amino group of the amino-terminal amino acid, the free carboxyl group of the carboxy-terminal amino acid, or both, are blocked or otherwise modified.

3. A peptide according to claim 1 having a human FSH agonistic or antagonistic activity, characterized by the general formula (2) :

[X¹TDSDSX²-NH- (CH₂)_d-CO-]_p [X³SRAYX⁴-NH- (CH₂) -CO-]_q

[X⁵TRDLX⁶-NH-(CH₂)_f-CO-]_r [X⁷AHASTAX⁸] _s

in which p, q, r and s independently of each other are equal to 0 or 1, but at least two of them are equal to 1,

d, e and f represent integers having a value ranging from 1 to

15,

X₁ - X₈ each represent one or more additional amino acids, if any,

as well as variants having another sequence of the partial peptides,

as well as sequence, substitution, deletion and insertion variants having a human FSH agonistic or antagonistic

activity, as well as derivatives in which the free amino group of the amino-terminal amino acid, the free carboxyl group of the carboxy-terminal amino acid, or both, are blocked or otherwise modified.

4. A peptide according to claim 1 having a human FSH agonistic or antagonistic activity, characterized by the general formula (3) :

[TDSDS-NH-(CH₂)_d-CO-]_p [X³SRAY-MH- (CH₂)_e"CO-]_q [TRDL-MH-(CH₂)_f-CO-]_r [AHASTA] _s

in which p, q, r and ε independently of each other are equal to 0 or 1, but at least two of them are equal to 1,

d, e and f represent integers having a value ranging from 1 to 15,

X³ represents an additional amino acid sequence QCMGCAF, if any,

as well as va-iants having another sequence of the partial peptides,

as well as sequence, substitution, deletion and insertion variants having a human FSH agonistic or antagonistic

activity, as well as derivatives in which the free amino group of the amino-terminal amino acid, the free carboxyl group of the carboxy-terminal amino acid, or both, are blocked or otherwise modified.

5. A peptide according to claim 1 having a human FSH agonistic or antagonistic activity, characterized by the general formula (4):

characterized by the general formula (4) :

[TDSDS-NH-(CH₂)_d-CO-]_p [SRAY-NH- (CH₂) _e-CO-]_q

[TRDL-NH-(CH₂)_f-CO-]_r [AHASTA

or by the general formula (5) :

[TDSDS-NH-(CH₂)_d-CO-]_p [QCMGCAFSRAY-NH- (CH₂)_e-CO-]_q

[TRDL-NH-(CH₂)_f-CO-]_r [AHASTA]_s

in which p, q, r and s independently of each other are equal to 0 or 1, but at least two of them are equal to 1,

d, e and f represent integers having a value ranging from 1 to 15,

as well as variants having another sequence of the partial peptides,

as well as sequence, substitution, deletion and insertion variants having a human FSH agonistic or antagonistic

activity, as well as derivatives in which the free amino group of the amino-terminal amino acid, the free carboxyl group of the carboxy-terminal amino acia, or ooth, are blocked or otherwise modified.

6. A peptide according co any of claims 2-5, characterized in that p=q=r=s=1, or p=q=r=1 and s=0, or p=q=1 and r=s=0, or q=r=1 and p=s=0.

7. A peptide according co any of claims 2-5, characterized in that d, e and f are incegers having a value ranging from 2 to 14.

8. A peptide according to any of claims 2-5, characterized in that d is equal to 2-14, e is' equal to 4-14, and f is equal to 2-14.

9. A peptide according to claim 1 having a human FSH agonistic activity, characterized by formula (6) :

TDSDS -NH-(CH₂)₂-CO- QCMGCAFSRAY -NH- (CH₂) ₄-CO- TRDL

-NH- (CH₂)₁₁-CO- AHASTA

or formula (7) :

TDSDS -NH-(CH₂)₂-CO- QCMGCAFSRAY -NH- (CH₂) ₄-CO- TRDL or formula (8) :

TDSDS -NH-(CH₂)₅-CO- SRAY -NH- (CH₂)₄-CO- TRDL

-NH- (CH₂)₁₁-CO- AHASTA

or formula (9) :

TDSDS -NH-(CH₂)₅-CO- SRAY -NH-(CH₂)₄-CO- TRDL

as well as variants having another sequence of the partial peptides,

as well as sequence, substitution, deletion and insertion variants having a human FSH agonistic activity,

as well as derivatives in which the free amino group of the amino-terminal amino acid, the free carboxyl group of the carboxy-terminal amino acid, or both, are blocked or otherwise modified.

10. A peptide according to claim 1 having a human FSH antagonistic activity, characterized by formula (10) :

TDSDS -MH-(CH₂)₅-CO- SRAY

or formula (11) :

SRAY -NH-(CH₂)₄-CO- TRDL

or formula (12) :

TDSDS -MH- (CH₂)₅-CO- SRAYTRDL

or formula (13) :

TDSDS -NH- (CH₂) ₅-CO- SRAY -MH- (CH₂)₁-CO- TRDL or formula (14) :

TDSDS -NH-(CH₂) ₅-CO- SRAY -NH-(CH₂)₂-CO- TRDL

or formula (15) :

TDSDSSRAY -NH-(CH₂)₄-CO- TRDL

or formula (16) :

TDSDSSRAYTRDL

as well as variants having another sequence of the partial peptides,

as well as sequence, substitution, deletion and insertion variants having a human FSH antagonistic activity,

as well as derivatives in which the free amino group of the amino-terminal amino acid, the free carboxyl group of the carboxy-terminal amino acid, or both, are blocked or otherwise modified.

11. A peptide according to any of the preceding claims, in which the free amino group of the amino-terminal amino acid is converted to an acetylamino group -NHCOCH₃ and the free carboxyl group of the carboxy-terminal amino acid is converted to a carboxamide group -CONH₂.

12. Antibodies (monoclonal or polyclonal) generated against a peptide according to any of claims 1-11.

13. A pharmaceutical composition for active or passive immunization against a glycoprotein hormone or for affecting the activity of a giycoproteir. hormone, comprising one or more peptides according to any of claims 1-11 or antibodies according to claim 12, as well as at least one

pharmaceutically acceptable adjuvant, carrier or diluent.

14. A pharmaceutical composition having a glycoprotein hormone agonistic or antagonistic activity, comprising one or more peptides according to any of claims 1-11, as well as at least one pharmaceutically acceptable adjuvant, carrier or diluent.

15. A pharmaceutical composition having a human FSH

agonistic activity, comprising a peptide according to any of formulae 6-9, or a derivative thereof, in which the free amino group of the amino-cerminal amino acid is converted to an acetvlamino σrouo and/or the free carooxvl σrouo of the carboxy-terminal amino acid is converted to an amido group, and at least one pharmaceutically acceptable adjuvant, carrier or diluent.

16. A pharmaceutical composition having a human FSH

antagonistic activity, comprising a peptide according to any 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.

17. A vaccine preparation against a glycoprotein hormone, comprising one or more peptides according to any of claims 1-11, as well as at least one pharmaceutically acceptable adjuvant, carrier or diluent.

18. A preparation for passive immunization against a

glycoprotein hormone, comprising antibodies according to claim 12, as well as at least one pharmaceutically acceptable adjuvant, carrier or diluent.

19. The use of one or more peptides according to any of claims 1-11 or antibodies according to claim 12 for diagnostic purposes.

20. The use of one or more peptides according to any of claims 1-11 or antibodies according to claim 12 for

immunization purposes.

21. The use of one or more peptides according to any of claims 1-11 or antibodies according to claim 12 for

therapeutic purposes.