The present invention deals with LHRH antagonists which possess improved water solubility and while having the high antagonist potency of the basic peptides, are free of the edematogenic effects. These compounds are highly potent in inhibiting the release of gonadotropins from the pituitary gland in mammals, including humans. The compounds of this invention are represented by the formula EQU X--R.sup.1 --R.sup.2 --R.sup.3 --Ser--Tyr--R.sup.6 --Leu--Arg--Pro--R.sup.10 --NH.sub.2 wherein PA1 X is an acyl group derived from straight or branched chain aliphatic or alicyclic carboxylic acids having from 1 to 7 carbon atoms, or H.sub.2 N--CO, PA1 R.sup.1 is D-- or L--Pro, D-- or L--.DELTA..sup.3 --Pro, D--Phe, D--Phe(4--H1), D--Ser, D--Thr, D--Ala, D--Nal(1) or D--Nal (2), PA1 R.sup.2 is D--Phe or D--Phe(4--C1) PA1 R.sup.3 is D--Trp, D--Phe, D--Pal(3), D--Nal(1) or D--Nal(2), PA1 R.sup.6 is D--Cit, D--Hci, D--Cit(Q) or D--Hci(Q) and PA1 R.sup.10 is Gly or D--Ala where Q is lower alkyl of 1-3 carbon atoms and H1 is fluoro, chloro or bromo, and the pharmaceutically acceptable acid addition salts thereof and methods of use pertaining to these compounds.

BACKGROUND OF THE INVENTION 
The present invention relates to novel peptides which inhibit the release 
of gonadotropins by the pituitary gland in mammals without inducing 
edematous reactions. More specifically, the present invention relates to 
analogs of the luteinizing hormone releasing hormone (LHRH), which has the 
structure: 
EQU p--Glu--His--Trp--Ser--Tyr--Gly--Leu--Arg--Pro--Gly--NH.sub.2, 
salts thereof, and to pharmaceutical compositions and methods of use 
pertaining to these analogs. 
DISCUSSION OF THE PRIOR ART 
For more than 15 years, investigators have been searching for selective, 
potent antagonists of the LHRH decapeptide (M. Karten and J. E. Rivier, 
Endocrine Reviews, 7, 44-66 (1986)). The high degree of interest in such 
antagonists is due to their usefulness in the fields of endocrinology, 
gynecology, contraception and cancer. A large number of compounds have 
been prepared as potential LHRH antagonists. The most interesting 
antagonists to date have been compounds whose structure is a modification 
of the structure of LHRH. 
The first series of potent antagonists was obtained by introduction of 
aromatic amino acid residues into positions 1, 2, 3 and 6, or, 2, 3, and 
6. The compounds are expressed as LHRH modified by replacement of the 
original amino acid residues by others at the position indicated by the 
superscript numbers. The known antagonists include: 
[Ac-D-Phe(4-Cl).sup.1,2, D-Trp.sup.3,6 ] LHRH (D. H Coy, et al., In: Gross, 
E. and Meienhofer, J. (eds) Peptides, Proceedings of the 6th. American 
Peptide Symposium, pp. 775-779, Pierce Chem. Co., Rockville Ill., 1979); 
[Ac-Pro.sup.1, D-Phe(4-Cl).sup.2 D-Nal(2).sup.3,6 ] LHRH (U.S. Pat. No. 
4,419,347); and [Ac-.DELTA..sup.3 
Pro.sup.1,D-Phe(4-Cl).sup.2,D-Trp.sup.3,6 ]LHRH (J. L. Pineda, et al., J. 
Clin. Endocrinol. Metab. 56, 420, 1983). 
Later, in order to increase the water solubility of antagonists, basic 
amino acids, such as D-Arg, were introduced into position 6. For instance, 
[Ac-D-Phe(4-Cl).sup.1,2, D-Trp.sup.3, D-Arg.sup.6, D-Ala.sup.10 ]LHRH 
(ORG-30276) (D. H. Coy, et al., Endocrinology, 100, 1445, 1982); and 
[Ac-D-Nal(2).sup.1, D-Phe(4-F).sup.2, D-Trp.sup.3, D-Arg.sup.6 ]LHRH 
(ORF-18260) (J. E. Rivier, et al., In: Vickery B. H., Nestor, Jr. J. J., 
Hafez, E.S.E. (eds), LHRH and Its Analogs, pp. 11-22, MTP Press, 
Lancaster, UK, 1984). 
These analogs not only possessed the expected improved water solubility but 
also showed increased antagonistic activity. However, these highly potent, 
hydrophilic analogs containing D-Arg and other basic side chains at 
position 6 proved to produce transient edema of the face and extremities 
when administered subcutaneously in rats at 1.25 or 1.5 mg/kg (F. Schmidt, 
et al., Contraception, 29, 283, 1984; J. E. Morgan, et al., Int. Archs. 
Allergy Appl. Immun. 80, 70, (1986). Since the occurrence of edematogenic 
effects after administration of these antagonists to rats cast doubts on 
their safety for the use in humans and delayed the introduction of these 
drugs for clinical use, it is desirable to provide antagonistic peptides 
which are free of these side effects. 
SUMMARY OF THE INVENTION 
The present invention deals with LHRH antagonists which possess an improved 
water solubility and high antagonist potency of the basic peptides, and 
are free of the edematogenic effects. These compounds are highly potent in 
inhibiting the release of gonadotropins from the pituitary gland in 
mammals, including humans. 
The compounds of this invention are represented by formula I 
X--R.sup.1 --R.sup.2 --R.sup.3 --Ser--Tyr--R.sup.6 
--Leu--Arg--Pro--R.sup.10 --NH.sub.2 I 
wherein 
X is an acyl group derived from straight or branched chain aliphatic or 
alicyclic carboxylic acids having from 1 to 7 carbon atoms, or a carbamyl 
(H.sub.2 N--CO) group. 
R.sup.1 is D-- or L--Pro, D-- or L--.DELTA..sup.3 --Pro, D--Phe, 
D--Phe(4--Hl), D--Ser, 
D--Thr, D--Ala, D--Nal(1) or D--Nal(2), 
R.sup.2 is D--Phe or D--Phe(4--Cl) 
R.sup.3 is D--Trp, D--Phe, D--Pal(3), D--Nal(1) or D--Nal(2), 
R.sup.6 is D--Cit, D--Hci, D--Cit(Q) or D--Hci(Q) and 
R.sup.10 is Gly or D--Ala 
where Q is lower alkyl of 1-3 carbon atoms and Hl is fluoro, chloro or 
bromo, and the pharmaceutically acceptable acid addition salts thereof. 
The compounds of Formula I are synthesized by any suitable method. For 
example, exclusively solid-phase technique, partial solid-phase technique 
or by classical solution couplings Preferably, the compounds of Formula I 
are prepared by a known solid-phase technique. Such method provides 
intermediate peptides and/or intermediate peptide-resins of Formula II. 
EQU X.sup.1 --R.sup.1 --R.sup.2 --R.sup.3 
--Ser(X.sup.4)--Tyr(X.sup.5)--R.sup.*6 
(X.sup.6)--Leu--Arg(X.sup.8)--Pro--R.sup.10 --NH--X.sup.10 II 
wherein 
X.sup.1 is an acyl group derived from straight and branched chain aliphatic 
or alicyclic carboxylic acids having from 1 to 7 carbon atoms, t-Boc, 
carbamyl or hydrogen, 
X.sup.4 is hydrogen or a protecting group for the Ser hydroxyl group, 
X.sup.5 is hydrogen or a protecting group for the Tyr phenolic hydroxyl 
group, 
X.sup.6 is hydrogen or a protecting group for the Lys or Orn side chain 
amino group, 
X.sup.8 is hydrogen or a protecting group for the Arg guanidino group, 
X.sup.10 is hydrogen or a resin support containing benzhydryl or 
methylbenzhydryl groups 
R.sup.1 is D-- or L--Pro, D-- or L--.DELTA..sup.3 --Pro, D--Phe, 
D--Phe(4--Hl), D--Ser, D--Thr, D--Ala, D--Nal(1) or D--Nal(2), 
R.sup.2 is D--Phe or D--Phe(4--Cl), 
R.sup.3 is D--Trp, D--Phe, D--Pal(3), D--Nal(1) or D--Nal(2), 
R.sup.*6 is D--Lys or D--Orn, D--Cit and D--Hci 
and R.sup.10 is Gly or D--Ala 
where Hl is fluoro, chloro or bromo. 
One process comprises reacting a peptide of Formula II wherein R.sup.*6 is 
D-Lys or D-Orn and X.sup.6 is hydrogen, with a source of cyanate to yield 
a peptide of Formula III: 
EQU X.sup.1 --R.sup.1 --R.sup.2 --R.sup.3 
--Ser(X.sup.4)--Tyr(X.sup.5)--R.sup.**6 --Leu--Arg(X.sup.8)--Pro--R.sup.10 
--NH--X.sup.10 III 
wherein 
EQU X.sup.1, R.sup.1, R.sup.2, R.sup.3, X.sup.4, X.sup.5, X.sup.8, R.sup.10 and 
X.sup.10 are as defined above, and 
R.sup.**6 is Cit or Hci. Suitably, the reaction is carried out when X.sup.1 
is acyl and all other X moieties are hydrogen. Suitable cyanate sources 
are alkali metal cyanates, e.g., potassium cyanate, or an N-alkyl 
isocyanate, e.g., N-ethyl-isocyanate. The peptide of Formula II are 
preferably synthesized by a known solid phase technique. 
Alternatively and preferably, peptides of Formula I wherein X is an acyl or 
carbamyl group, are directly obtained by cleavage and deprotection of 
intermediate peptide-resins of Formula II wherein X.sup.1 is an acyl or 
carbamyl group and R.sup.*6 is D-Cit or D-Hci. Peptides of Formula I 
wherein X is carbamyl (H.sub.2 N--CO) group are also obtained from 
peptide-resins of Formula II wherein X.sup.1 is hydrogen or Boc by 
cleavage and deprotection followed by carbamoylation. 
A gonadotropin antagonizing pharmaceutical composition is provided by 
admixing the compound of Formula I with a pharmaceutically acceptable 
carrier including microcapsules (microspheres) for delayed delivery. 
There is also provided a method for relieving complications resulting from 
the physiological availability of amounts of pituitary gonadotropins in a 
mammal, in excess of the desired amount, which involves administering to 
the mammal a gonadotropin antagonizing dose of the compound of Formula I. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The nomenclature used to define the peptides is that specified by the 
IU-IUB Commission on Biochemical Nomenclature (European J. Biochem., 
1984, 138, 9-37), wherein in accordance with conventional representation 
the amino groups at the N-terminus appears to the left and the carboxyl 
group at the C-terminus to the right. By natural amino acid is meant one 
of the common, naturally occurring amino acids found in proteins 
comprising Gly, Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, 
Gln, Cys, Met, Phe, Tyr, Pro, Trp and His. The abbreviations for the 
individual amino acid residues are based on the trivial name of the amino 
acid and are Ala, alanine; Arg, arginine; Cit, citrulline, Gly, glycine; 
Hci, homocitrulline; Leu, leucine; Lys, lysine; Pal(3), 
3-(3-pyridyl)alanine; Nal(1), Nal(2), 3-(1-naphtyl)alinine, 
3-(2-naphthyl)alanine; Orn, ornithine; Phe, phenylalanine; Phe(4-Cl), 
4-chlorophenylalanine; Phe (4-F), 4-fluorophenylalanine; Pro, proline; 
Ser, serine; Trp, tryptophan and Tyr, tyrosine. All amino acids described 
herein are of the L-series unless stated otherwise, e.g., D-Trp represents 
D-tryptophan and D-Nal(2) represents 3 -(2-naphthyl)-D-alanine. 
Other abbreviations used are: 
______________________________________ 
AcOH acetic acid 
AcOEt ethyl acetate 
Ac.sub.2 O acetic anhydride 
Boc- tert.butyloxycarbonyl- 
DIC diisopropylcarbodiimide 
DIEA diisopropylethylamine 
DMF dimethylformamide 
HOBt 1-hydroxybenzenetriazole hydrate 
HPLC high performance liquid chromatography 
MeOH methyl alcohol 
TEA triethylamine 
DCC dicyclohexylcarbodiimide 
MeCN acetonitrile 
IpOH isopropanol 
Z(2-Cl) 2-chloro-benzyloxycarbonyl 
DCB 2,6-dichlorobenzyl 
Tos p-toluenesulfonyl 
TFA trifluoroacetic acid 
Z benzyloxycarbonyl 
______________________________________ 
Especially preferred are LHRH analogs of Formula I wherein: 
X.sup.1 is acetyl or carbamyl 
R.sup.1 is Pro, D--Phe, D--Phe(4--Cl) or D--Nal(2), 
R.sup.2 is D--Phe(4--Cl) or D--Phe(4--F), 
R.sup.3 is D--Trp or D--Pal(3) 
R.sup.6 is D--Cit or D--Hci, and 
R.sup.10 is D--Ala. 
The peptides are synthesized by a suitable method, such as by exclusively 
solid-phase techniques, by partial solid-phase techniques, by fragment 
condensation or by classical solution phase synthesis (See M. Bodanszky, 
"Principles of Peptide Synthesis", Springer-Verlag, 1984). 
For example, the techniques of exclusively solid-phase synthesis are set 
forth in the textbook "Solid Phase Peptide Synthesis", J. M. Stewart and 
J. D. Young, Pierce Chem. Company, Rockford, Ill., 1984 (2nd. ed.), G. 
Barany and R. B. Merrifield, "The Peptides", Ch. 1, 1-285, pp. 1979, 
Academic Press, Inc. 
Classical solution synthesis is described in detail in the treatise 
"Methoden der Organischen Chemie (Houben-Weyl): Synthese von Peptiden", E. 
Wunsch (editor) (1974) Georg Thieme Verlag, Stuttgart, W. Germany. 
Common to such synthesis is the protection of the reactive side chain 
functional groups of the various amino acid moieties with suitable 
protecting groups which will prevent a chemical reaction from occurring at 
that site until the group is ultimately removed Usually also common is the 
protection of an alpha-amino group on an amino acid or a fragment while 
that entity reacts at the carboxyl group, followed by the selective 
removal of the alpha-amino protecting groups to allow subsequent reaction 
to take place at that location Accordingly, it is common that, as a step 
in the synthesis, an intermediate compound is produced which includes each 
of the amino acid residues located in its desired sequence in the peptide 
chain with side-chain protecting groups linked to the appropriate 
residues. 
In Formula II: 
R.sup.1, R.sup.2, and R.sup.3 are as defined hereinabove, 
X.sup.1 is hydrogen or an acyl group derived from straight or branched 
chain aliphatic or alicyclic carboxylic acids having from 1 to 7 carbon 
atoms, or an alpha-amino protecting group. The alpha-amino protecting 
groups contemplated by X.sup.1 are those well known to be useful in the 
art of step-wise synthesis of polypeptides. Among the classes of 
alpha-amino protecting groups which may be employed as X.sup.1 may be 
mentioned fluoroenylmethyloxycarbonyl (Fmoc) or t-butyloxycarbonyl (Boc). 
X.sup.4 may be a suitable protecting group for the hydroxyl group of Ser 
such as benzyl (Bzl), and 2,6-dichloro-benzyl (DCB). The preferred 
protecting group is Bzl. 
X.sup.5 may be a suitable protecting group for the phenolic hydroxyl group 
of Tyr, such as Bzl, 2-Br-Z and 2,6-dichloro-benzyl (DCB). The preferred 
protecting group is DCB. 
X.sup.6 is a suitable protecting group for the side chain amino group of 
Lys or Orn. Illustrative of suitable side chain amino protecting groups 
are benzyloxycarbonyl (Z), and 2-chloro-benzyloxycarbonyl ((Z-(2-Cl)). 
X.sup.8 is a suitable protecting group for the guanidino group of Arg, such 
as nitro, Tos, methyl-(t-butyl benzene)-sulfonyl, 
4-methoxy-2,3,6-trimethylbenzenesulfonyl; Tos is the preferred group. 
X.sup.10 is an amide protecting benzhydryl or methylbenzhydryl group 
incorporated into resin support; for the synthesis of peptide amides 98% 
styrene-2% divinylbenzene copolymers containing benzhydryl amine or 
methylbenzhydryl amine groups are preferred. 
The selection of a side chain amino protecting group is not critical except 
that generally one is chosen which is not removed during deprotection of 
the alpha-amino groups during the synthesis. 
The peptides of Formula I may be from intermediate peptide-resins of 
Formula II by procedures known in the art. The solid phase systhesis of 
intermediate peptide-resins of Formula II is essentially carried out as 
described by Merrifield, J. Am. Chem. Soc., 85, p. 2149 (1963). Solid 
phase synthesis is commenced from the C-terminal end of the peptide by 
coupling a protected amino acid to a suitable resin Such a starting 
material can be prepared by attaching -amino protected Gly or D-Ala by an 
amide bond to a benzylhydrilamine resin. Such resin supports are 
commercially available and generally used when the desired polypeptide 
being synthesized has an carboxamide at the C-terminal. 
The selection of an appropriate coupling reagent is within the skill of the 
art. Particularly suitable as a coupling reagent is N,N'-diisopropyl 
carbodiimide (DIC). 
Each protected amino acid or amino acid sequence is introduced into the 
solid phase reactor in about a two-three fold excess, and the coupling may 
be carried out in a medium of DMF:CH.sub.2 Cl.sub.2 (1:1) or in CH.sub.2 
Cl.sub.2 alone. In cases where incomplete coupling occurs, the coupling 
procedure is repeated before removal of the alpha-amino protecting group 
prior to the coupling of the next amino acid. The success of the coupling 
reaction at each stage of the synthesis, is preferably monitored by the 
ninhydrin reaction, as described by E. Kaiser, et al., Anal. Biochem., 34, 
595 (1970). 
After the desired amino acid sequence of intermediates B has been 
completed, the terminal Boc group is removed and if desired, N-terminal 
acylation carried out using the appropriate acyl anhydride or acid 
chloride in 50-fold excess in a halogenated hydrocarbon solvent; suitably, 
acetic anhydride in methylene chloride for 30 minutes. The intermediate 
peptide can be removed from the resin support by treatment with a reagent 
such as liquid hydrogen fluoride, which not only cleaves the peptide from 
the resin but also cleaves all remaining side chain protecting groups 
X.sup.4, X.sup.5, X.sup.8, X.sup.10 and, if present, X.sup.6. 
When using hydrogen fluoride for cleaving, anisole or m-cresol, and, if 
desired, methylethyl sulfide are included as scavengers in the reaction 
vessel. 
Peptides of Formula II wherein R.sup.*6 is D-Lys or D-Orn and X.sup.6 is 
hydrogen, are converted into peptides of Formula I by treatment with 
cyanate, suitably an alkali metal cyanate, preferably potassium cyanate, 
or an N-alkylisocyanate, for instance, N-ethylisocyanate, in DMF or 
aqueous DMF. The latter reaction, i.e., transformation of Orn/Lys-peptides 
into the corresponding Cit/Hci-peptides can be readily followed by HPLC 
using MeCN-aqueous TFA systems because of a characteristic 2.6.+-.0.3 
minutes increase of the retention times of Cit/Hci--and, for example, 
Cit(Et)/Hci(Et)-peptides relative to the corresponding Orn/Lys-peptides 
respectively. 
When acylation is omitted, treatment of peptide-resins of Formula II with 
hydrogen fluoride yields decapeptides which have free omega-amino and/or 
alpha-amino groups and correspond to a Formula II where X.sup.1, X.sup.4, 
X.sup.5, X.sup.8, X.sup.10, and, if present, X.sup.6 are hydrogen. These 
free peptides are converted into peptides of Formula I wherein X is 
carbamyl by treatment with cyanate, suitably an alkali metal cyanate, 
preferably potassium cyanate. The latter reaction, i.e., transformation of 
H.sub.2 N into H.sub.2 N--CO--NH at the amino terminus of peptides and 
conversion of the Orn/Lys residues into the Cit/Hci residues, can be 
easily followed by HPLC using MeCN-aqueous TFA systems, because of a 
characteristic 2-3 min. increase of the retention times of carbamylated 
peptides, i.e., compounds with H.sub.2 N--CO--NH-- group, relative to 
their congeners with H.sub.2 N group. 
Alternatively and preferably, peptides of Formula I wherein X is an acyl or 
carbamyl group, are directly obtained by cleavage and deprotection of 
intermediate peptide-resins of Formula II, where X.sup.1 is an acyl or 
carbamyl group and R.sup.*6 is D-Cit or D-Hci. 
Although an exclusively solid-phase synthesis and a partially solid-phase 
synthesis of compounds of Formula I are disclosed herein, the preparation 
of the compounds also can be realized by classical solution-phase methods. 
The synthetic peptides prepared as described in the Examples are compared 
with two of the most potent LHRH antagonists reported recently, i.e., 
[Ac-D-Phe(4-Cl).sup.1,2, D-Trp.sup.3, D-Arg.sup.6, D-Ala.sup.10 ] LHRH 
(ORG-30276) (Coy, et al., Endocrinology, 100, 1445, 1982) and 
[Ac-D-Nal(2).sup.1, D-Phe(4-F).sup.2, D-Trp.sup.3, D-Arg.sup.6 ] LHRH (ORF 
18260) (Rivier, et al., In: Vickery, B. H., Nestor, Jr., J. J. Hafez, E. 
S. E. (eds.), LHRH and Its Analogs, pp. 11-22, MTP Press, Lancaster, UK, 
1984), and are found to exert similarly high inhibitory activities both in 
vitro and in vivo, but, unlike to the control peptides, not to produce the 
in vivo edematous effects. 
Hormonal activities in vitro are compared in superfused rat pituitary cell 
systems (S. Vigh and A. V. Schally, Peptides. 5 suppl. 1: 241-247, 1984) 
in which the effectiveness of LHRH (and other releasing hormones) can be 
accurately evaluated since the amount of LH (or other pituitary hormones) 
secreted into the effluent medium is not only proportional to the 
hormone-releasing potency of the peptide applied but also measurable 
readily by well-characterized radioimmunoassays. 
To determine the potency of an LHRH antagonist, mixtures containing LHRH in 
a constant concentration (usually 1 nM) and the antagonist in varying 
concentrations are used for the superfusion in order to determine the 
molecular ratio of the antagonist to LHRH at which the action of LHRH is 
completely blocked. These ratios are about 5 for both peptides of the 
present invention and the control peptides when the rat pituitary cell 
system is preincubated with antagonists for 9 minutes. 
In an antiovulatory in vivo assay (A. Corbin and C. W. Beattie; Endocr. 
Res. Commun. 2, 1-23, 1975; D. H. Coy, et al., Endocrinology, 100, 1445, 
1982), the peptides of the present invention are also found to be about 
equipotent to the control antagonist, namely, 87.5-100% blockade of 
ovulation can be observed at a subcutaneous dose of 1-3 ug/rat for each 
peptide. 
In the edematogenic test of Schmidt, et al. (Contraception, 29, 283-289, 
1984), however, a marked difference can be found between the control 
peptides and the peptides of the present invention. The control 
administered subcutaneously in rats at doses of 1.25 or peptides produce 
edema of the face and extremities when 1.50 mg/kg. No such reaction can be 
observed with the peptides of the present invention when given at a 
subcutaneous dose of 1.5 mg/kg. 
In the tests as run, the rats were assigned to three groups of five rats 
per group per compound tested. Comparison was made with a known prior art 
compound designated ORG 30276 namely (N-Ac-D-p-Cl-Phe.sup.1,2,D-Trp.sup.3, 
D-Arg.sup.6,D-Ala.sup.10)-LHRH. The groups were injected subcuntaneously 
once a day on two consecutive days with the LHRH antagonists at a dose 
level of 1.5 mg/kg. One control group was injected with diluent only. The 
rats were observed during five hours each day. Reactions of the rats were 
classified as follows: NR no apparent reaction, PR partial responders: 
edema of the nasal and paranasal area, FR full responders: facial edema 
with edematous extremities. 
These results are summarized in Table 1 below. 
TABLE 1 
______________________________________ 
LHRH 1st Day 2nd Day 
Antagonist 
NR PR FR NR PR FR 
______________________________________ 
ORG 30276 
3 7 0 0 0 10 
Control 9 0 0 9 0 0 
EX III 8 0 0 8 0 0 
EX V 9 0 0 8 1* 0 
EX IV 9 0 0 9 0 0 
EX I 8 0 0 8 0 0 
EX XX 9 0 0 8 1* 0 
EX XXI 9 0 0 9 0 0 
EX XXVI 8 0 0 8 0 0 
EX XXVII 9 0 0 9 0 0 
______________________________________ 
*Very light edema of the face. 
LHRH secretion in vitro at some reasonable concentration, although most are 
slightly less potent than the present standard in vitro; however, these 
peptides are much more potent in vivo. 
This was shown by a test on histamine release in vitro from peritoneal mast 
cells carried out in accordance with the procedure of Morgan et al (Int. 
Archs. Allergy appl. Immun. 80, 70 1986). 
Histamine Release In Vitro 
In this test rats were anesthetized with ether and peritoneal exudate cell 
were harvested by washing with 12 ml. of mast cell medium (MCM) (150m M 
NaCl; 3.7m M KCl; 3.0m M Na.sub.2 HPO.sub.4 ; 3.5m M KH.sub.2 PO.sub.4, 
0.98m M CaCl; 5.6m M dextrose; 0.1% bovine serum albumin; 0.1% gelatin and 
10 units/ml heparin)[9]. Cells from 4 or 5 rats were pooled, centrifuged 
at 120 g, resuspended with MCM to a concentration of 0.5.times.10.sup.6 ml 
and 1 ml was aliquoted into 12.times.75 mm polyethylene tubes. Tubes were 
equilibrated to 37.degree. C. for 15 min and incubated alone (background 
histamine release), with 48/80 (positive control) (Sigma Chemicals, St. 
Louis, Mo.), or with appropriate concentrations (1 ng through 10 ug/ml) of 
LHRH antagonists for 60 min. The reaction was terminated by cooling the 
tubes to 4.degree. C. Tubes were centrifuged; supernatants were recovered 
and stored at -20.degree. C. until assayed for histamine. Assays were 
performed in duplicate. Total cell histamine was determined by boiling for 
10 min. Histamine released in reponse to antagonist was expressed as a 
percentage of total release. That concentration that released 50% of total 
mast cell histamine (HRD.sub.50 ug/ml) was determined for each antagonist. 
The results are summarized in FIG. 1. 
All of the peptides are considered to be effective to prevent ovulation of 
female mammals at very low dosages. The peptides of the invention are 
often administered in the form of pharmaceutically acceptable, nontoxic 
salts, such as acid addition salts. Illustrative of such acid addition 
salts are hydrochloride, hydrobromide, sulphate, phosphate, fumarate, 
gluconate, tannate, maleate, acetate, citrate, benzonate, succinate, 
alginate, pamoate, malate, ascorbate, tartrate, and the like. If the 
active ingredient is to be administered in tablet form, the tablet may 
contain a pharmaceutically acceptable diluent which includes a binder, 
such as tragacanth, corn starch or gelatin; a disintegrating agent, such 
as alginic acid and a lubricant, such as magnesium stearate. 
If administration in liquid form is desired, sweetening and/or flavoring 
may be used as part of the pharmaceutically-acceptable diluent, and 
intravenous administration in isotonic saline, phosphate buffer solutions 
or the like may be effected. 
The pharmaceutical compositions will usually contain the peptide in 
conjunction with a conventional, pharmaceutically-acceptable carrier. 
Usually, the dosage will be from about 1 to about 100 micrograms of the 
peptide per kilogram of the body weight of the host when given 
intravenously; oral dosages will be higher. Overall, treatment of subjects 
with these peptides is generally carried out in the same manner as the 
clinical treatment using other antagonists of LHRH. 
These peptides can be administered to mammals intravenously, 
subcutaneously, intramuscularly, orally, intranasally or intravaginally to 
achieve fertility inhibition and/or control and also in applications 
calling for reversible suppression of gonadal activity, such as for the 
management of precocious puberty or during radiation- or chemo-therapy. 
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 0.1 
to 2.5 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 
obvious to one having the ordinary skill in his art may be made without 
departing from the scope of the invention, which is set forth in the 
claims which are appended thereto. Substitutions known in the art which do 
not significantly detract from its effectiveness may be employed in the 
invention.