LRF Antagonists

The present invention relates to peptides which inhibit the release of gonadotropins by the pituitary gland in mammalians, including humans and to methods of preventing ovulation and/or inhibiting the release of steroids. More particularly, the present invention is directed to peptides which inhibit gonadal function and the release of the steroidal hormones, progesterone and testosterone.

The pituitary gland is attached by a stalk to the region in the base of the brain known as the hypothalamus. The pituitary gland has two lobes, the anterior and the posterior lobes. The posterior lobe of the pituitary gland stores and passes onto the general circulation two hormones manufactured in the hypothalamus, these being vasopressin and oxytocin. The anterior lobe of the pituitary gland secretes a number of hormones, which are complex protein or glyco-protein molecules that travel through the bloodstream to various organs and which, in turn, stimulate the secretion into the blood stream of other hormones from the peripheral organs. In particular, follicle stimulating hormone (FSH) and luteinizing hormone (LH), sometimes referred to as gonadotropins or gonadotropic hormones, are released by the pituitary gland. These hormones, in combination, regulate the functioning of the gonads to produce testosterone in the testes and progesterone and estrogen in the ovaries, and also regulate the production and maturation of gametes.

The release of a hormone by the anterior lobe of the pituitary gland usually requires a prior release of another class of hormones produced by the hypothalamus. One of the hypothalamic hormones acts as a factor that triggers the release of the gonadotropic hormones, particularly LH. The hypothalamic hormone which acts as a releasing factor for LH is referred to herein as LRF although it has also been referred to as LH-RH and as GnRH. LRF has been isolated and characterized as a decapeptide having the following structure:

Peptides are compounds which contain two or more amino acids in which the carboxyl group of one acid is linked to the amino group of the other acid. The formula for LRF, as represented above, is in accordance with conventional representation of peptides where the amino group appears to the left and the carboxyl group to the right. The position of the amino acid residue is identified by numbering the amino acid residues from left to right. In the case of LRF, the hydroxyl portion of the carboxyl group of glycine has been replaced with an amino group (NH.sub.2). The abbreviations for the individual amino acid residues above are conventional and are based on the trivial name of the amino acid; where p-Glu is pyroglutamic acid, His is histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, Gly is glycine, Leu is Leucine, Arg is arginine, Asp is asparagine, Cys is cysteine and Pro is proline. Except for glycine, amino acids of the peptides of the invention are of the L-configuration unless noted otherwise. Acetyl is abbreviated as Ac.

It is known that the substitution of D-amino acids for Gly in the 6-position of the LRF decapeptide provides a peptide material having from about 1 to 35 times greater potency than does LRF to effect the release of LH and other gonadotropins by the pituitary gland of mammalians. The releasing effect is obtained when the LRF analog is introduced into the bloodstream of a mammalian.

It is also known that substitution of various amino acids for His (or the deletion of His) at the 2-position of the LRF decapeptide produces analogs having an inhibitory effect on the release of LH and other gonadotropins by the pituitary gland of mammalians. In particular, varying degrees of inhibition of the release of LH are obtained when His is deleted (des His) or replaced by Asp, Cys, D-Ala, D-Phe or Gly. The inhibitory effect of such peptides modified at the 2-position can be further enhanced when a D-amino acid is substituted for Gly in the 6-position of the decapeptides. For example, the peptide: pGlu-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH.sub.2 is 3 times more potent as an inhibitor for the release of gonadotropins than is the same peptide where Gly is present in the 6-position rather than D-Ala.

Some female mammalians who have no ovulatory cycle and who show no pituitary or ovarian defect begin to secrete normal amounts of the gonadotropins LH and FSH after the appropriate administration of LRF. Thus, the administration of LRF is considered suitable for the treatment of those cases of infertility where a functional defect resides in the hypothalamus.

There are also reasons for desiring to prevent ovulation in female mammalians, and the administration of LRF analogs that are antagonistic to the normal function of LRF have been used to prevent ovulation. For this reason, analogs of LRF which are antagonistic to LRF are being investigated for their potential use as a contraceptive or for regulating conception periods. It is desired to provide peptides which are strongly antagonistic to endogenous LRF and which prevent secretion of LH and the release of steroids by the gonads of mammals.

The present invention provides peptides which inhibit the release of gonadotropins in mammalians, including humans, and also provides methods for inhibiting the release of steroids by the gonads of male and female mammalians. The improved LRF analogs are antagonistic to LRF and have an inhibitory effect on the reproduction processes of mammalians and may accomplish this through direct gonadal effects.

Generally, in accordance with the present invention, peptides have been synthesized which strongly inhibit the secretion of gonadotropins by the pituitary gland of mammalians, including humans, and/or inhibit the release of steroids by the gonads. These peptides inhibit ovulation of female mammals when administered at very low levels at proestrous and also are effective to cause resorption of fertilized eggs if administered shortly after conception.

The peptides of the present invention are represented by the following formula:

wherein R.sub.1 is selected from the group consisting of dehydro Pro, dehydro D-Pro, acetyl dehydro Pro and acetyl dehydro D-Pro; R.sub.2 is selected from the group consisting of D-Phe, Phe, N.alpha.Me-Phe, His, D-His, D-Trp, Trp and N.alpha.Me-Leu; R.sub.3 is selected from the group consisting of D-Trp, Trp, D-Phe, Phe, Pro and D-His; R.sub.4 is selected from the group consisting of Gly, D-Trp, D-Phe, imBzl D-His and D-Tyr; R.sub.5 is selected from the group consisting of Leu and N.alpha.Me-Leu; and R.sub.6 is selected from the group consisting of Pro-Gly-NH.sub.2 and Pro-NHCH.sub.2 CH.sub.3.

The peptides of the present invention can be synthesized by a solid phase technique using a chloromethylated resin for those peptides wherein R.sub.6 is Pro-NHCH.sub.2 CH.sub.3 and a benzhydrylamine (BHA) resin for those peptides wherein R.sub.6 is Pro-Gly-NH.sub.2. The synthesis is conducted in a manner to stepwise add the amino acids in the chain in the manner set forth in detail in the afore-mentioned U.S. Patent, the disclosure of which is incorporated herein by reference. Side-chain protecting groups, as are well known in the art, are added to Ser, Tyr and His before these amino acids are coupled to the chain being built up upon the resin.

Such a method provides the fully protected intermediate peptidoresin. The fully protected peptide can be cleaved from the resin support by ammonolysis, as is well known in the art, to yield the fully protected amide intermediate. The intermediates of the invention may be represented as:

where X.sup.1 represents hydrogen or an .alpha.-amino protecting group; X.sup.2 is a protecting group for Ser and preferably is benzyl ester (OBzl); X.sup.3 is a protecting group for Tyr selected from the group consisting of OBzl and 2-6 dichlorobenzyl; and X.sup.4 is selected from dimethylamine, alkylamine of 1 to 5 carbon atoms, phenethylamine, O-CH.sub.2 -[resin support] or Gly-O-CH.sub.2 [resin support] or Gly-NH [resin support].

The criterion for selecting side chain protecting groups for X.sup.1 -X.sup.4 are that the protecting group must be stable to the reagent under the reaction conditions selected for removing the .alpha.-amino protecting group at each step of the synthesis, the protecting group must not be split off under coupling conditions and the protecting group should generally be removable upon completion of the synthesis of the desired amino acid sequence under reaction conditions that will not alter the peptide chain. When R.sub.1 includes acetyl, it may be employed as the protecting group for the .alpha.-amino group of proline, in which case it is preferably added to proline before it is coupled to the peptide chain. Alternatively, the peptide on the resin may be reacted with acetic acid in the presence of dicyclohexyl carbodiimide (DCC) or with acetic anhydride.

The group --O--CH.sub.2 [resin support] of Gly-O-CH.sub.2 -[resin support] defining X.sup.4 in the intermediates of the invention represents the ester moiety of one of the many functional groups of the polystyrene resin support. The group Gly-NH-[resin support] defining X.sup.4 in the intermediates of the invention represents the amide bond of Gly to benzhydrylamine resin.

Deprotection of the peptides as well as cleavage of the peptide from the benzhydrylamine resin takes place at 0.degree. C. with hydrofluoric acid (HF). Anisole is added to the peptide prior to treatment with HF. After the removal of HF, under vacuum, the cleaved, deprotected peptide is treated with ether, decanted, taken in dilute acetic acid and lyophilized.

Purification of the peptide is effected by ion exchange chromotography on a CMC column, followed by partition chromotography using the elution system: n-butanol; 0.1 N acetic acid (1:1 volume ratio) on a column packed with Sephadex G 25. The peptides of the invention are effective at levels of 5 milligrams (in some instances even less) per kilogram of body weight, when administered at about noon on the day of proestrous, to prevent ovulation of female mammalian eggs. It is preferred to use dosage levels in the range of from about 0.1 to about 10 milligrams per kilogram of body weight. Higher levels can be used but no significant benefit is attained through use of higher levels. These antagonists are also effective as contraceptives when administered to male mammals on a regular basis.

The following examples further illustrate various features of the invention but are intended to in no way limit the scope of the invention which is defined in the appended claims.

The following peptides having the formula

are prepared by the solid phase procedure referred to above.

The peptides set forth in the foregoing table are assayed in vitro and in vivo. The in vitro assay is made using four day old primary culture of dispersed rat pituitary cells. The levels of LH mediated in response to the application of peptides is assayed by specific radioimmunoassay for rat LH. Control dishes of cells only receive a measure which is 3 nanomolar in LRF: experimental dishes receive a measure 3 nanamoles of LRF and a concentration of test peptide ranging from 1 to 100 nanomolar. The amount of LH secreted in the samples treated only with LRF is compared with that secreted by the samples treated with the peptide plus LRF. Results are calculated and expressed in Table II (In Vitro column) as the molar concentration ratio of test peptide required to reduce the amount of LH released by the 3 nanomolar LRF to 50 percent of the control value (ICR.sub.50).

Several of the peptides described hereinabove are used to determine effectiveness to prevent ovulation in female rats. In this test, a number of mature female Sprague-Dawley rats, each having a body weight from 225 to 250 grams, are injected with 0.02 milligram of peptide in corn oil at about noon on the day of proestrous. Proestrous is the afternoon before esterous (ovulation). A separate female rat group is used as a control to which the peptide is not administered. Each of the control rat females has ovulation at estrous. As indicated in the In Vivo column, the peptides are significantly effective to prevent ovulation of female rats at a very low dosage, and all of the peptide compositions are considered to be totally effective at a dose of one milligram.

These peptides can be administered to mammals intravenously, subcutaneously, intramuscularly or orally to achieve fertility inhibition and/or control. Effective dosages will vary with the form of administration and the particular species of mammal being treated. An example of one typical dosage form is a physiological saline solution containing the peptide which solution is administered to provide a dose in the range of about 5 to 20 mg/kg of body weight. Oral administration of the peptide may be given in either solid form or liquid form.

Although the invention has been described with regard to its preferred embodiments, it should be understood that changes and modifications as would be obvious to one having the ordinary skill in this art without departing from the scope of the invention which is set forth in the claims which are appended hereto. For example, other substitutions known in the art which do not significantly detract from the effectiveness of the peptides may be employed in the peptides of the invention.