Heptapeptide analogues or pharmaceutically acceptable salts thereof consist of a hexapeptide moiety S and a C-terminal .beta.-aminoalcohol residue Z bound to the moiety S by an amide bond, wherein the .beta.-aminoalcohol Z is --NR--CH(Q)--CH.sub.2 OH, Q is (CH.sub.2).sub.n --NH--A is H or --C(.dbd.NH)NH.sub.2, and R is CH.sub.3 or C.sub.2 H.sub.5, and the moiety S wherein H is a D-aromatic .alpha.-aminoacid and Y is an aliphatic .alpha.-aminoacid and have oxytocin antagonist activity. Also disclosed is: a method of their synthesis; pharmaceutical compositions containing these analogues; the synthesis of such compositions; a method of control of uterine contractions.

FIELD OF THE INVENTION 
The present invention relates to new heptapeptide analogues (i.e. 
heptapeptides in which the N-terminal residue is deaminated and the 
C-terminus is reduced to an alcohol) exhibiting oxytocin antagonist 
activity useful, inter alia, for decreasing or blocking uterus muscle 
contraction associated with pre-term labour and menstrual pain. The 
invention also relates to pharmaceutical compositions containing these 
peptide analogues and to their use. 
BACKGROUND OF THE INVENTION 
Oxytocin is a peptide hormone. It stimulates contraction of the uterine 
muscles. For this reason, it is believed to be involved in the etiology of 
pre-term labour and menstrual pain. It is further believed that oxytocin 
antagonists would be useful in the control of these conditions. Oxytocin 
antagonist peptides of adequate potency and selectivity of therapeutic use 
are known. They are often intended for administration in aqueous solution. 
The manufacture of ready-for-use doses of such antagonists requires that 
the solutions be stable for extended periods, which is not always true. In 
such cases, the medicament must be prepared immediately prior to use from, 
for instance, the freeze-dried peptide or its pharmaceutically acceptable 
salt. This sort of manipulation is inconvenient and entails the risk of 
contamination. 
OBJECTS OF THE INVENTION 
It is an object of the invention to provide new oxytocin antagonists which 
are heptapeptide analogues having improved stability in aqueous media 
while retaining adequate potency and selectivity for therapeutic efficacy. 
It is a second object of the invention to provide pharmaceutical 
compositions containing said new heptapeptide-analogue oxytocin 
antagonists and having improved stability and, therefore, shelf-life. 
It is a further object of the invention to provide for a method of 
treatment of a medical condition associated with excess or inappropriate 
uterine contraction, which method is the administration of a 
pharmaceutical composition containing said heptapeptide analogue. 
SUMMARY OF THE INVENTION 
The invention comprises a class of compounds which are heptapeptide 
analogues, pharmaceutical compositions containing such analogues, and a 
use for these compositions which is the treatment of uterine contractions, 
particularly in the context of pre-term labour and menstrual pain. 
The heptapeptide analogues of the invention have an N-terminal hexapeptide 
moiety S and a C-terminal .beta.-aminoalcohol Z, which is considered 
hereinafter to be the formal equivalent of the seventh amino acid of the 
heptapeptide. The moiety S has the structure: 
##STR1## 
wherein Mpa, Ile, Asn and Abu have the following meanings: 
______________________________________ 
Mpa 3-mercaptopropionic acid residue (otherwise called desamino- 
cysteine) 
Ile isoleucine residue 
Asn asparagine residue 
Abu .alpha.-aminobutyric acid residue; 
______________________________________ 
and wherein X is an aromatic D-.alpha.-amino acid and Y is an aliphatic 
.alpha.-amino acid. 
The aminoalcohol Z has the structure: 
##STR2## 
wherein R is methyl or ethyl, and 
Q is --(CH.sub.2).sub.n --NH--A, where n is 1-6 and A is H or 
--C(.dbd.NH)NH.sub.2. 
The compounds of the invention can form acid addition salts, and to the 
extent that these salts are pharmaceutically acceptable they are included 
within the scope of the invention. 
The compounds can be incorporated into either solid or liquid formulations. 
Examples of such formulations include tablets, capsules, solutions and 
suspensions. Other components of such formulations can include, for 
example, diluents, dispersants, preservatives, buffering agents, 
flavouring agents and osmotic pressure regulating agents. Solid 
formulations are particularly suitable for oral administration, while 
solutions are most useful for injection (i.v., i.m. or s.c.) or intranasal 
administration. A particular merit of the compounds of the invention is 
that their solutions are more stable on prolonged storage than those of 
previously known compounds of comparable potency. 
The formulated pharmaceutical is useful in the control of uterine 
contractions. Two indications where such control is likely to be required 
are pre-term labour and menstrual pain. When used in the management of 
pre-term labour, the pharmaceuticals can be used as acute tocolytic agents 
following the onset of labour and as maintenance therapy for preventing 
the recurrence of such episodes. 
DETAILED DESCRIPTION OF THE INVENTION 
According to the present invention are disclosed heptapeptide analogues 
exhibiting therapeutically useful oxytocin antagonist activity and having 
improved stability in aqueous media. 
The heptapeptide analogues of the invention are characterised by a 
structure which comprises an N-terminal hexapeptide analogue moiety S and 
a C-terminal .beta.-aminoalcohol moiety Z. The structure of the 
.beta.-aminoalcohol Z is: 
##STR3## 
wherein Q is --(CH.sub.2).sub.n --NH--A, n is 1-6 and A is H or 
--C(.dbd.NH)NH.sub.2, and wherein R is CH.sub.3 or CH.sub.2 H.sub.5 ; 
and the moiety S is 
##STR4## 
wherein Mpa, Ile, Asn and Abu have the following meanings: 
______________________________________ 
Mpa 3-mercaptopropionic acid residue (otherwise called desamino- 
cysteine) 
Ile isoleucine residue 
Asn asparagine residue 
Abu .alpha.-aminobutyric acid residue; 
______________________________________ 
and wherein 
X is a D-aromatic .alpha.-amino acid; and 
Y is an aliphatic .alpha.-amino acid. 
By an aromatic .alpha.-amino acid is meant an .alpha.-amino acid wherein 
the side chain includes an aromatic ring system. Such a system may be 
carbocyclic or heterocyclic, monocyclic or fused. Examples of aromatic 
.alpha.-amino acids include (but are not limited to) phenylalanine, 
tyrosine, (O-ethyl)tyrosine, tryptophan, .beta.-(2-naphthyl)alamine and 
phenylglycine. It will be noted that the residue X is of the unnatural 
D-configuration in the compounds of the invention. 
By an aliphatic .alpha.-amino acid is meant an .alpha.-amino acid wherein 
the side chain has only carbon and hydrogen atoms. Such side chains will 
include alkyl and cycloalkyl groups. They may be unsaturated, but may not 
include aromatic residues. Side chains of 1 to 12 carbon atoms are 
included, although the preferred range is for 3-7 carbon atoms. Examples 
of aliphatic .alpha.-amino acids include (but are not limited to) alanine, 
valine, leucine, cyclohexylglycine and adamantylalanine. The residue Y has 
the natural L-configuration. 
In the structure of the hexapeptide analogue moiety S, the line joining the 
Mpa and Abu residues has its conventional meaning. It signifies that there 
is a covalent bond linking the ends of the side chains of these two 
residues. In this case, the sulphur atom of the Mpa residue is joined by a 
covalent bond to the .gamma.- (or 4-) carbon atom of the Abu residue. 
The aminoalcohol moiety Z includes a stereogenic centre and so can exist in 
two epimeric forms, R and S, corresponding to the D and L isomers of the 
related amino acids. Heptapeptide analogues with either of these isomers 
are included within the scope of the invention, as are mixtures of 
epimers. Preferably, the aminoalcohol moiety is present as a single 
epimer, and preferably it has the S configuration. 
In the context of the present invention, the Mpa residue and the 
aminoalcohol Z are considered to be formal equivalents of .alpha.-amino 
acids, and the compounds of the invention are termed heptapeptide 
analogues accordingly. 
In a preferred embodiment of the invention, X is either a D-tryptophan 
residue or a .beta.-(2-naphthyl)-D-alanine residue. 
In another preferred embodiment of the invention, Y is a residue of one of 
valine, leucine, isoleucine, alloisoleucine, cyclohexylalanine and 
(.beta.,.beta.-diethyl)alanine. 
In another preferred embodiment of the invention, n is in the range 2-4. 
In a more preferred embodiment of the invention, X is either a D-tryptophan 
residue or a .beta.-(2-naththyl)-D-alanine residue and Y is a residue of 
one of valine, leucine, isoleucine, alloisoleucine, cyclohexylalanine and 
(.beta.,.beta.-diethyl)alanine. 
A particularly preferred embodiment of the invention is a peptide analogue 
chosen from: 
##STR5## 
wherein the following further abbreviations have been used: 
______________________________________ 
D-Trp D-tryptophan residue 
alloIle alloisoleucine residue 
Ala(3,3-diethyl) (.beta.,.beta.-diethyl)alanine residue 
D-Nal .beta.-(2-naphthyl)-D-alanine residue 
Leu leucine residue 
Val valine residue 
Cha .beta.-cyclohexylalanine residue. 
______________________________________ 
A most preferred embodiment of the invention is the peptide analogue: 
##STR6## 
The compounds of the invention contain a basic site (amine or guanidine) 
and so can form salts with acids, which salts retain the pharmacological 
properties of the free bases. Accordingly, such salts are included within 
the scope of the invention. Examples of such salts include (but are not 
limited to) the hydrochloride, hydrobromide, sulphate, acetate, citrate, 
benzoate, trifluoroacetate and methanesulphonate. 
Also disclosed according to the invention are pharmaceutical compositions 
which include a pharmacologically effective amount of at least one of the 
oxytocin antagonist heptapeptide analogues described above. The 
composition may also include pharmaceutically acceptable additives such as 
preservatives, diluents, dispersing agents, agents to promote mucosal 
absorption (examples of which are disclosed by Merkus, F. W. H. M. et al., 
J. Controlled Release 24, 201-208, 1993, and which include surfactants, 
bile acids, fusidates, phospholipids and cyclodextrins), buffering agents 
and flavourings. Such compositions may be formulated as solids (for 
example as tablets, capsules or powders) or liquids (for example as 
solutions or suspensions), which is here taken to include creams and 
ointments, for oral or parenteral administration. Oral (including 
sublingual and buccal), intranasal, pulmonary, transdermal, rectal, 
vaginal, subcutaneous, intramuscular and intravenous administration may 
all be suitable routes for dosing. 
A preferred composition according to the invention is a sterile aqueous 
solution of a heptapeptide analogue as described, and particularly an 
isotonic saline solution suited to intranasal administration or 
intravenous injection. The solution may contain a buffering agent to 
maintain the pH of the solution in the range 0.3-7.0, and preferably in 
the range 3.5-5.5. The buffer is, for example, a phosphate/citrate buffer. 
Another preferred composition according to the invention is a tablet for 
oral administration. Particularly preferred is a tablet which is coated 
with a substance that is substantially insoluble at low pH such as is 
present in the stomach, but which dissolves at the more neutral pH of the 
small intestine to release the peptide analogue for absorption. Examples 
of such coatings are disclosed in PCT/SE94/00244 and in PCT/SE95/00249, 
which are incorporated into this specification by reference. 
A further disclosure of the invention is a method of reducing or stopping 
unwanted contractions of the uterine muscles. This method is the 
administration to the subject of an effective amount of one of the 
oxytocin antagonist heptapeptide analogues of the invention, preferably 
formulated as a composition as described above. It will be evident that 
this disclosure of the invention is equally the disclosure of a use for 
the compounds and formulations of the invention. 
A particularly preferred embodiment of the invention is a method of 
stopping the contractions of the uterus in pre-term labour. Following the 
initial intervention, which will involve a period of 1-3 days, the 
treatment may be continued to a prevent a recurrence of labour until such 
time as the attending physician sees fit. Thus there are two aspects to 
this embodiment, an acute tocolytic use and a maintenance therapeutic use. 
Another preferred embodiment of the invention is a method of reducing 
painful contractions of the uterus associated with menstruation. 
The amount of heptapeptide analogue which constitutes a therapeutically 
effective dose will depend on a number of factors. The route of 
administration will be an important consideration. Intravenous injection 
is likely to be the most efficient route of delivery, while intranasal 
administration can be expected to be efficient than oral dosing. 
Accordingly, less compound will be required for a single intravenous dose 
than for a single intranasal dose, and more compound will be required for 
a single oral dose. The attending physician will also need to take into 
account factors such as the age, weight and state of health of the 
patient. The management of menstrual pain is also likely to require less 
compound than is pre-term labour. The amount of compound which constitutes 
a single effective dose for intravenous treatment of an average woman in 
pre-term labour is from about 0.1 mg to about 500 mg, and preferably from 
about 1 mg to about 200 mg, in a period of 24 hours. 
The heptapeptide analogues of the present invention selectively inhibit 
uterine muscle contractions while lacking undesirable oxytocin agonist 
properties. They also have little or no antidiuretic, hypotensive or 
hypertensive effect, which might potentially be side effects of analogues 
of oxytocin and the related hormone vasopressin. They are comparable in 
potency to those compounds known in the art which they most resemble 
structurally. They differ from these compounds, which are disclosed in 
WO95/02609, in the nature of the C-terminal residue. The known compounds 
have a carboxamide function (--CONH.sub.2) where the compounds of the 
present invention have a primary alcohol (--CH.sub.2 OH). The compounds of 
the present invention are superior to those of WO95/02609 in respect of 
their stability, particularly in aqueous media. This is clearly an 
advantage when the compound is to be formulated as an aqueous solution 
which will consequently have a longer shelf-life and have less stringent 
requirements for refrigeration, but it is also an advantage in the 
manufacturing and formulating processes, which will involve periods when 
the compound is in solution even if the final composition is a solid. 
While it has been emphasised above that the compounds of the invention are 
particularly useful in the control of uterine muscle contractions, it will 
be appreciated by one familiar with the art that other therapeutic uses 
for oxytocin antagonists are possible. For example, another target of 
oxytocin action is the mammary gland, where it promotes milk ejection. The 
compounds of the invention might therefore be used to control 
inappropriate lactation. They might also be useful in the control of 
certain tumours, particularly mammary tumours and secondary metastases 
derived from a primary mammary tumour. Hyperplasia of the prostate might 
be a further therapeutic target. It has also been suggested that oxytocin 
is involved in luteal development and the facilitation of post-coital 
sperm transport. From this it can be inferred that the compounds of the 
invention might be useful as contraceptive or fertility-regulating agents. 
Another peripheral target of oxytocin is the immune system. Oxytocin 
antagonists are therefore potentially useful as immunomodulating and 
anti-inflammatory agents. Oxytocin is also present in the brain, where it 
has been suggested to have a role in the etiology of such diverse 
conditions as psychogenic erectile dysfunction, schizophrenia and 
alcohol-induced neuropsychological deficiencies. In some species it has 
been shown to have an effect on complex social behaviour. Accordingly, the 
compounds of the invention might be used as, for example, anti-psychotic 
or cognition-enhancing agents. The use of the compounds of the invention 
in any of these therapeutic situations is intended to fall within the 
scope of this disclosure. 
In the following, the invention will be described in general and by means 
of specific examples. It should be understood that this description is not 
intended to limit the scope of the invention, and that such variations as 
are known in the art and which the practitioner would consider to be 
equivalent fall equally within that scope. 
GENERAL METHODS FOR SYNTHESIS 
The chemical transformations necessary to effect the synthesis of the 
compounds of the invention are well known in the art. The techniques of 
peptide chemistry both in solution and an solid supports are particularly 
relevant. Solution phase methods are described in the following 
references: 
Law, H. B. and Du Vigneaud, V., J. Am. Chem. Soc. 82, 4579-4581, 1960; 
ZhuZe, A. L. et al., Coll. Czech. Chem. Comm. 29, 2648-2662, 1964; and 
Larsson, L.-E. et al., J. Med. Chem. 21, 352-356, 1978. 
Solid phase methods are discussed in: 
Merrifield, R. B., J. Am. Chem. Soc. 85, 2149, 1963; 
Merrifield, R. B., Biochemistry 3, 1385, 1964; and 
Konig, W. and Geiger, R., Chem. Ber. 103, 788, 1970. 
The route used by the inventors is discussed in outline below and then 
exemplified in detail. It will be apparent to one familiar with the 
practice of peptide chemistry that the order in which some of the 
transformations are performed can be varied. It is not the intention of 
the inventors that such obvious variations should be excluded from the 
scope of the invention. 
Most frequently, the starting material will be a protected N-alkyl amino 
acid of general formula 1. 
##STR7## 
R and n are chosen from the possibilities outlined previously. P.sup.1 is a 
nitrogen protecting group. One particularly favoured choice of P.sup.1 is 
9-fluorenylmethyloxycarbonyl (Fmoc). 
When A in the target compound is to be H then either P.sup.2 is a nitrogen 
protecting group distinguishable from P.sup.1 (for example 
benzyloxycarbonyl) and P.sup.3 is H or the same as P.sup.2, or P.sup.2 and 
P.sup.3 together are a divalent protecting group for nitrogen (for example 
phthaloyl). 
When A is to be --C(.dbd.NH)NH.sub.2 then P.sup.2 is either H or a 
protecting group as above and P.sup.3 is --C(.dbd.NP.sup.4)NH.sub.2, where 
P.sup.4 is a protecting group, and is preferably the same as P.sup.2. It 
will be apparent to the practitioner that these protected guanidines can 
exist as tautomers and positional isomers. Although --(CH.sub.2).sub.n 
--N(P.sup.2)P.sup.3 has been defined as 2.sup.A, isomers 2.sup.B, 2.sup.C 
and 2.sup.D can all be considered as equivalent to this structure for the 
purposes of this description. 
##STR8## 
When the protected N-alkyl amino acids of general formula 1 are not 
commercially available they can be prepared by methods described in the 
literature, or by methods analogous to them. 
Assuming that solid phase methods are to be used, the amino acid 1 is 
attached to a suitable resin to give 3 as a first intermediate. 
##STR9## 
where Res represents the polymeric resin. 
P.sup.1 is a cleaved and FmocAbu(SCH.sub.2 CH.sub.2 CO.sub.2 t-Bu)OH is 
coupled to give 4. 
##STR10## 
The peptide is extended by sequential coupling with FmocAsn, FmocY, FmocIle 
and then BocX. When X is D-Trp it is advantageous to protect the indole 
nitrogen as its formyl derivative. The use of Boc-protection for this 
amino acid allows for simultaneous cleavage of the t-butyl ester and the 
N-terminal protecting group. At this stage, intermediate 5 is present. 
##STR11## 
The peptide is cleaved from the resin using appropriate standard conditions 
and then esterified, for example by treatment with benzyl bromide to give 
the benzyl ester 6. 
##STR12## 
The Boc group and the t-butyl ester are cleaved by acid treatment, and the 
resulting amine and acid groups are condensed to form the macrocycle. The 
benzyl ester is then reduced to give the primary alcohol of the target 
compound, for example by reaction with sodium borohydride in aqueous 
isopropanol. Conveniently, the removal of the remaining protecting groups 
can also be achieved during this conversion. If that is not the case, a 
final deprotection step is necessary. The product is isolated and purified 
using standard techniques. 
The following specific examples were prepared according to this general 
outline. They are representative of the compounds of the present 
invention. The following abbreviations are used: 
______________________________________ 
TBTU 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetra- 
fluoroborate 
Boc tert-butyloxycarbonyl 
Fmoc 9-fluorenylmethyloxycarbonyl 
TFA trifluoroacetic acid 
DMF dimethylformamide 
DBU 1,8-diazabicyclo[5.4.0]undec-7-ene 
Bn benzyl 
Orn ornithine 
Pht phthaloyl 
______________________________________ 
Protected amino acids were obtained as follows: 
FmocAbu(SCH.sub.2 CH.sub.2 CO.sub.2 t-Bu) prepared according to Prochazka, 
E. et al., Coll. Czech. Chem. Comm. 57, 1335, 1992; 
FmocN.sup..alpha. MeOrn(Pht) prepared by analogy to the route used for the 
Lysine derivative by Freidinger, R. M. et al., J. Org. Chem. 48, 77, 1983; 
Fmoc-alloIle prepared according to Ten Kortenaar, P. B. W. et al., Int. J. 
Peptide Protein Res. 27, 398, 1986; 
FmocAla(3,3-diethyl) prepared according to Eisler, K. et al., Coll. Czech. 
Chem. Comm. 31, 4563, 1966; 
Boc-D-Trp(CHO); Boc-D-Nal; FmocAsn; FmocIle; FmocVal; FmocLeu; FmocCha all 
from Bachem (CH and USA).

EXAMPLE I 
##STR13## 
Peptide Ia was synthesised using solid phase methodology on o-chlorotrityl 
resin and with the Fmoc strategy. 
The first amino acid, FmocN.sup..alpha. MeOrn(Pht)OH, was attached to the 
resin. Cleavage of the Fmoc group was achieved with 2% DBU in DMF. Other 
residues were coupled sequentially, finishing with Boc-D-Trp(CHO)OH. The 
resin-bound peptide was treated with a mixture of acetic 
acid/trifluoroethanol/dichloromethane (1:2:7), then the mixture was 
filtered and the filtrate was evaporated and freeze dried. The resulting 
peptide acid was esterified by reaction with benzyl bromide (2 eq.) and 
diisopropylethylamine (2.5 eq) in DMF for 27 h. The solvent was evaporated 
and the residue was freeze dried from acetic acid. 
##STR14## 
The N-terminal Boc group and the t-butyl ester of Ia were cleaved by 
treatment with 95% TFA/2.5% anisole/2.5% water for 1.5 h at room 
temperature. TFA was evaporated and the produce was precipitated by the 
addition of diethyl ether. The peptide was cyclised by treatment with TBTU 
(1 eq) and N-methylmorpholine (17 eq) in DMF at room temperature. The 
solvent was evaporated and the peptide Ib was purified by reversed phase 
chromatography. 
##STR15## 
The purified benzyl ester Ib was treated with NaBH.sub.4 (7 eq) in solution 
in a mixture of isopropanol/water (6:1) at room temperature for 22 h under 
an inert gas atmosphere. Acetic acid (18 eq) was added and the mixture was 
heated at 80.degree. C. for 6 h. The solvent was evaporated and the 
product peptide Ic (.tbd."peptide I") was purified by reversed phase 
liquid chromatography: stationary phase: mobile phase; acetonitrile/0.1% 
TFA in water. Yield 14 mg. Mass spectrometry (electrospray ionisation, ion 
trap analysis, positive mode) indicated a molecular mass in agreement with 
the proposed structure (found m/z=830.5 [MH.sup.+ ]; calc. for [C.sub.40 
H.sub.63 N.sub.9 O.sub.8 S'H.sup.+ ] m/z=830.5). 
EXAMPLES II-VII 
Using the same method as for Example I, and by substituting the appropriate 
protected amino acids for Boc-D-Trp)CHO)OH and Fmoc-alloIleOH, the 
peptides listed in Table 1 were prepared. 
TABLE 1 
______________________________________ 
Mass spectroscopy data 
#STR16## 
Mass spec. 
Peptide X Y Calc. MH.sup.+ 
Found 
______________________________________ 
II D-Trp Leu 830.5 830.4 
III D-Trp Val 816.4 816.4 
IV D-Trp Cha 870.5 870.5 
V D-Trp Ile 830.5 830.5 
VI D-Nal alloIle 841.5 841.5 
VII D-Trp Ala(3,3-diethyl) 844.5 844.5 
______________________________________ 
A number of reference peptide amides were prepared according to the methods 
disclosed in WO95/02609 in order to compare the properties of the present 
invention with those known in the art. These reference peptides are listed 
in Table 2. 
TABLE 2 
______________________________________ 
Reference peptides 
#STR17## 
Peptide X Y 
______________________________________ 
r-IX D-Trp alloIle 
r-X D-Trp Val 
r-XI D-Nal alloIle 
______________________________________ 
EXAMPLE VIII 
Using the method of Example I, but substituting N.sup..alpha. 
-ethyl-ornithine for the N.sup..alpha. -methyl amino acid, the following 
peptide (VIII) was prepared (found m/z=855.1 [MH.sup.+ ]; calc. for 
[C.sub.41 H.sub.65 N.sub.9 O.sub.8 S+H.sup.+ ] m/x=855.5). 
##STR18## 
EXAMPLE IX 
Biological Evaluation of Compounds 
The compounds of the present invention can be assessed in a number of in 
vitro and in vivo biological systems. These test systems are chosen to be 
as relevant to the intended human patient as possible. 
i) Oxytocin receptor binding assay 
Recombinant human oxytocin receptors were expressed in either CHO or HEK293 
cells using standard molecular biological techniques. A membrane fraction 
was prepared and incubated in the presence of [.sup.125 I]-oxytocin and 
varying concentrations of heptapeptide analogue. Membranes were then 
isolated by filtration and counted for radioactivity to determine oxytocin 
binding. An inhibition constant K.sub.i was determined for the analogue. 
The results obtained are presented in Table 3. 
TABLE 3 
______________________________________ 
Oxytocin receptor assay; inhibition constants K.sub.i 
Peptide K.sub.i (nM); mean .+-. SEM 
______________________________________ 
I 0.25 .+-. 0.16 
II 3.2 .+-. 0.75 
III 0.80 .+-. 0.30 
IV 7.0 .+-. 1.85 
V 1.4 .+-. 0.15 
VI 0.1 .+-. 0.0 
VII 2.4 .+-. 0.85 
______________________________________ 
ii) Antagonist effect in vitro in a human uterus model 
Uterus muscle tissue from women in late pregnancy undergoing Caesarean 
section was cut into strips which were mounted in a tissue bath filled 
with Krebs-Ringer buffer and oxygenated with carbogen (95% O.sub.2 +5% 
CO.sub.2) gas. Changes in isometric muscle tension detected with a tension 
transducer were recorded on a Grass polygraph. 
The concentration-effect curve of oxytocin was recorded. The effect 
measured in this case corresponds to the net value of the integrated 
contraction curve during the 10 minute period following agonist (i.e. 
oxytocin) administration. A concentration of oxytocin giving at least 
half-maximal response was selected. This concentration of agonist was 
administered to the tissue in the presence of different concentrations of 
antagonist heptapeptide analogue and the response was recorded. The 
concentration of antagonist required to reduce the response to 50% of its 
control value was determined by regression analysis and is given here as 
an IC.sub.50 value. [IC.sub.50 value=concentration of antagonist required 
to reduce the effect of a given agonist dose by 50%]. The results are 
given in Table 4. 
TABLE 4 
______________________________________ 
Inhibition of agonist effect (uterus model) 
Peptide IC.sub.50, human uterus model (nM) 
______________________________________ 
I 5 .+-. 1 
r-VIII 18 .+-. 3 
______________________________________ 
iii) In vivo rat model 
Sprague Dawley rats (ca. 250 g) in natural estrus were anaesthetised with 
Inactin (0.5 mg/100 g body weight, i.p.). The activity of the myometrium 
was measured with the aid of a catheter fixed in the uterine cavity and 
filled with modified Lockes solution. The catheter was connected to a 
Statham P23D force transducer and the contractions were recorded on a 
Grass polygraph (model 7D). 
The dose-response curve for oxytocin (2.times.10.sup.-4 -5.times.10.sup.-3 
.mu.mol/kg) was recorded. In this case the response was quantified by 
integration of the curve during the 15 minutes following agonist 
injection. A dose of oxytocin giving an effect corresponding to an 
intralumenar contraction pressure of 10-30 mm Hg and within the linear 
section of the curve was selected. This dose of oxytocin was administered 
to the animal in conjunction with at least two different doses of 
antagonist and the effect was recorded. The dose of antagonist which 
reduces the effect of the agonist to 50% of tis control value was 
determined by interpolation and is given here as an ID.sub.50 value. 
[ID.sub.50 value=dose of antagonist required to reduce the effect of a 
given agonist dose by 50%]. The results are given in Table 5. 
The duration of action of the antagonists was also determined in this 
model. An oxytocin dose was selected (2.times.10.sup.-4 -5.times.10.sup.-3 
.mu.mol/kg) giving an effect corresponding to half the maximum effect 
(this dose is the ED.sub.50). The effect determined here is the same as 
for the ID.sub.50 determination defined above. An antagonist dose was 
selected (8.times.10.sup.-4 -4.times.10.sup.-3 .mu.mol/kg) so as to give 
at least 50% inhibition of the response to the agonist. At the beginning 
of the experiment, single doses of the agonist and antagonist were 
co-administered. At 20 minute intervals thereafter, doses of the agonist 
alone were administered and the response was measured. The time taken for 
the inhibition of the agonist effect to decline to 25% of its starting 
value was determined by interpolation and is given here as the t.sub.75 
value. [t.sub.75 value=time period required for the effectiveness of a 
single dose to decline by 75%]. The results are presented in Table 5. 
TABLE 5 
______________________________________ 
Inhibition of agonist effect (in-vivo rat model) 
Peptide ID.sub.50, rat model (nmol/kg) 
t.sub.75, rat model (min) 
______________________________________ 
I 2.9 .+-. 0.3 169 .+-. 2 
r-IX 2.9 .+-. 0.3 180 .+-. 9 
______________________________________ 
It is evident from the results presented in Tables 3-5 that the compounds 
of the present invention are at least as good as the earlier compounds in 
the rat model, both in terms of potency and duration of action, and that 
they are superior to the earlier compounds in the more relevant human 
model. 
EXAMPLE X 
Pharmaceutical Formulations 
Solution in isotonic buffered saline for i.v. injection. 
The following solutions were prepared: 
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Solution A (0.02M citric acid) 
citric acid monohydrate 0.42 g 
distilled water ad 100 ml 
Solution B (0.04M disodium hydrogen phosphate) 
Na.sub.2 HPO.sub.4.2H.sub.2 O 0.712 g 
distilled water ad 100 ml 
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To 27 ml of solution A is added 23 ml of solution B, 0.81 g of NaCl and 
0.322 g of peptide I acetate. The pH is adjusted to 4.5 with solution A, 
then distilled water is added to give a total volume of 100 ml. Finally, 
the mixture is filtered through Sterivex-GV 0.22 .mu.m. This gives an 
isotonic solution containing 0.3 mg/ml of peptide I (calculated as free 
base) suitable for intravenous injection. 
Tablets for oral administration 
The following components were combined: 
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Peptide I acetate 108 mg 
mannitol 7.7 g 
lactose 6.0 g 
microcrystalline cellulose 6.0 g 
crosslinked carboxymethyl cellulose 200 mg 
talcum 800 mg 
magnesium stearate 200 mg 
polyvinylpyrrolidone/ethanol 
to bind 
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The blended mixture was formed into tablets using standard methods. The 
mixture is sufficient to prepare 100 tablets containing 1 mg of peptide I 
(calculated as free base) each. 
Enteric tablet for oral administration 
25 of the aforementioned tablets were air-spray coated with 100 mg of 
cellulose acetate phthalate (4 mg per tablet). 
Also useful in the invention is the composition disclosed in WO 95/25534 in 
which the active agent (desmopressin, for instance), can be substituted by 
the compounds according to the present invention. The adaptation of the 
composition to handle the higher amounts of active agents required to be 
incorporated into the tablet is within the easy reach of the person 
skilled in the art. 
EXAMPLE XI 
Evaluation of Stability 
Solutions of the heptapeptide analogues of the present invention were 
prepared with compositions representative of aqueous formulations. The 
solutions were stored for several weeks at 50.degree. C. and aliquots were 
withdrawn periodically for analysis by HPLC. Reference peptides were 
studied in parallel. Degradation was determined as the loss of peptide (% 
by weight per week), independent of the nature of the decomposition 
products. The results are presented in Table 6. 
TABLE 6 
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Stability test 
Degradation at 
Peptide Composition of test solution 50.degree. C. (wt %/week) 
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I Isotonic citrate/phosphate buffer, pH 4.5 
0.4 
r-IX as above 2.9 
III as above 0.7 
r-X as above 4.3 
VI as above 1.2 
r-XI as above 4.0 
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The results presented above clearly indicate that the compounds of the 
invention are more stable in solution than the previously described 
compounds. In practice, this increased stability means that aqueous 
formulations will have a longer shelf-life and be less demanding in their 
need for refrigerated storage. Besides the financial savings which result, 
there will be benefits in both convenience and safety because the need to 
prepare individual doses immediately prior to administration will be 
reduced. 
The manufacturing process will also benefit, since the compounds will be 
more resistant to decomposition during and after purification.