CA2085603A1

CA2085603A1 - Derivatives of pituitary posterior lobe hormones

Info

Publication number: CA2085603A1
Application number: CA002085603A
Authority: CA
Inventors: Jerzy Trojnar; Michal Lebl; Per Melin
Original assignee: Individual
Current assignee: Ferring AB
Priority date: 1990-07-09
Filing date: 1991-07-04
Publication date: 1992-01-10
Also published as: IL98737A0; AU8225191A; EP0538367A1; FI930013A7; MC2291A1; SE9002384D0; JPH05508849A; WO1992000996A1; FI930013A0; PL297600A1; IE912378A1

Abstract

Derivatives of pituitary posterior lobe hormones, more precisely new vasotocin derivatives, are disclosed. The new vasotocin derivatives are comprised by formula (I), wherein A = Mpa or Hmp, B =
D-Tyr, D-Tyr(Et), D-Phe, D-Phe(p-Et) or D-Trp, C = Thr, Val or Hgn, D = Cys or .alpha.-Abu, E = Pro or a peptide bond, F = (a), wherein m = 0-6, n = 0-6, L = H, C00H, CO-NH2 or CH2-OH, K = H, (b) or a D- or L-amino acid residue with the proviso that L is not H when A is Mpa, C is Thr or Val and D is Cys. Further, pharmaceutical compositions including the new vasotocin derivatives and being preferably in a form suitable for intravenous, intranasal or intraintestinal administration, are described. The compositions are especially intended for use in the therapeutic treatment of excessive uterus muscle contractions.

Description

-. W092/00996 2 O 8 5 6 0 3 PCT/SE91/OW77 DERIVATIVES OF PITUITARY POSTERIOR LOBE HORMONES .

The present invention relates to derivatives of pituitary posterior lobe hormones, more precisely to new vasotocin derivatives which are competitive oxytocin receptor antagonists and inhibit excessive uterus contractions.

The new vasotocin derivatives are modified in relation to ~-the vasotocin molecule by having shortened and optionally even modified C-terminal along with alterations in the position~ 1, 2, 4 and optionally 6.
!
Backaround and prior art Oxytocin is a pituitary posterior lobe hormone, and receptors specific for said hormone are found in the body of mammals including humans. The sites of the receptors are hitherto found in the uterus, the lactation ducts and the ovaries.
Oxytocin receptor antagonists may thus be used as competitive receptor blockers in case of increased endogenous oxytocin secretion or increas-d receptor density.

We have previously shown ~EP-A-0 112 809) that modification of the vasotocin molecule in the posi~ions 1, 2, 4 and 8 gives analogues which ~trongly inhibit uterine contractions both in animal and human tests (Melin et al, J. Endocrinol.
111, 125, 1986). These analogues have been shown in this context to antagonize oxytocin or va60pressin induced contraction6, and in clinical trials one analogue ha~ been shown to counteract excessive uterus contractions in connec-tion with painful men6truations and premature labour.
(Akerlund~ Acta Ob6t. Gynecol, Scand, 66, 459, 1987, Akerlund et al, Br. J. Obst. Gynecol, 94, 1040, 1987). Our earlier vasotocin derivatives, which have been disclosed in the above mentioned EP-A, seem to lack side effects, but they have a limited half-life and thus give a rather short effect duration. The enzymatic stability of the molecule, and - thus the duration of the effect, is of major clinical W092/00996 ~ PCT/SE9l/~77 importance at a single administration. Since the effect duration of our previous vasotocin derivatives is compara- -tively short and the therapeutic dose is rather high, they have hitherto been administered intravenously in hospitals ~ -only.

Thus, there is a need of inhibitors with prolonged effect duration and enhanced potency, and also of other administra-tion routs, so that these substances can be utilized in non-institutional care.

EP-A-O 182 627 discloses a large number of structurally similar vasopressin analogues, which are said to be basic V1-Vasopressin antagonists and may be useful in the treatment of hypertension and cardiac ischaemic diseases. The compounds of the invention are represented by a general structural -formula which has been restricted by a provisio, to delimit the compounds from the compounds of the EP-A-O 182 627.
.
Description of the invention The present invention provides new derivatives of pituitary ;
posterior lobe hormones which are vasotocin derivatives having enhanced potency and longer effect duration than our prior v~sotocin derivatives disclosed in EP-A-O 112 809.
Vasotocin deriv~tives of the invention are comprised by the following formula:

A - B - Ile - C - Asn - D - E - F
l 2 3 4 5 6 7 8 .~ -wherein A = Mpa (3-mercaptopropionyl residue; -S-CH2-CH2-CO-) or Hmp (2-hydrqxy-3-mercaptopropionyl residue;
OH

-S-CH2-CH-CO- ) .~r- W092t00996 2 0 ~ ~ 6 ~ 3 PCT/SE91/0~77 B = D-Tyr, D-Tyr(Et), D-Phe, D-Phe~p-Et) or D-Trp C = Thr, Val or Hgn (homoglutamine) D = Cys or ~-Abu (-HN-CH-CO-) ~ .
: ~-.. '' ' :
CH2 ~:

CH2 -:

E = Pro or a peptide bond .~ .
. . . . ..
F z a L- or D-residue of the fomula NH-(CH2)m CH-L

(CH2)n : .
.
NH

R : .
:, ' '' :
wherein m ~ 0-6, preferably 0-4 .. :.
n = 0-6, preferably 2-4 :
L ~ H, COOH, C~-NH2 or CH2-OH
. ,.. ~, .. .
~ = H, C~NH or a D- or L-amino acid residue ;~, .. . , ' ~' .

, with the provi6io that L is not H when A is Mpa, C is Thr or Val and D is Cys.

, : In the present specification and claims conventional three-letter abbreviations have been used for the amino acids.

The present invention also comprises pharmaceutical composi-tions, which include at least one vasotocin derivative according to the invention as an active ingredient in combination with pharmaceutica!ly acceptable additives - .
: -. .

W092/00996 2 0 8 ~ ~ O ~ PCT/SE91/~7 ~

and/or diluents. For the pharmaceutically acceptable diluent, preferably isotonic saline solution may be used. As to the other pharmaceutically acceptable additives, such can be found in the literature, e.g. the US Pharmacopoeia, and these additives can be chosen in conformity with the specific form of the composition for a specific administration rout. A
composition of the invention can be in a form which is suitable for intravenous, intranasal or intraintestinal administration. A form which is suitable for intraintestinal administration may be a tablet which is taken orally and which preferably is coated with a layer which is at least not completely dissolved in the stomach but primarily in the intestines so that the active ingredient can be resorbed through the intestinal mucous membrane.
The vasotocin derivatives according to the present invention totally lack agonistic effect as well as antidiuretic effect and blood-pressure effect, resulting in that possible clinical 6ide effects are minimized.
Short description of the drawinas Figure 1 presents antagonistic effect on rat uterus in vivo, following intr8nasal administration of a re~erence substance ~Peptide 1) and a vasotocin derivative according to the invention (Peptide 2).

Figure 2 pre6ents antagonistic effect on rat uterus in vivo, following intrainte6tinal administration of the same peptides a6 in Figure 1.

Figure 3 presents antagonistic effect on rat uterus in vivo, following intravenous administration of the same peptides as in Figure 1.
Figure 4 presents antagonistic effect on rat uterus in vivo, -~ -following intravenous administration of a reference substance (Peptide 1~ in a dose of 10-8 mole/kg and a vasotocin . .

~ W O 92/00996 2 0 8 ~ 6 0 3 P~r/SE91/00477 derivative according to the invention (Peptide 3) in a dose of 2 x 10-9 mole/kg (i.e. one fifth of the dose of the reference).
: ':
Figure 5 presents antagonistic effect on rat uterus in vivo, following intraintestinal administration of a dose of 8 x 10-6 mole/kg of the same peptides as in Figure 4.

Preparation of the vasotocin derivatives accordina to the invention , The vasotocin deri~atives according to the invention can be prepared in analogy with processes well known in the peptide field.
For example, the compounds according to the invention may be prepared in conventional manner by incremental coupling of ^~
amino acids to one another in the liquid phase, for instance h in accordance with the technique reported by Law, H.B. & Du Vigneaud, V. in the Journal of the American Chemical Society 82, (1960) 4579-4581, Zhuze, A.L., Jost, ~ asafirek, E.
Rudinger, '. in the Collection of Czechoslovak Chemical Communications 29, ~1964), 2648-2662, and modified by Larsson, L.-E., Lindeberg, G., Melin, P. / Pliska, V. in the Journal of Medicinal Chemistry 2~, ~1978), 352-356. The coupling of the amino acids to one another, whereby so-called -peptide bonds are formed, may also be carried out by starting with a solid phase (usually a resin) to which the C-terminal of the first amino acid is coupled, whereupon the C-terminal of the next amino acid is coupled to the N-terminal of the first amino acid etc. Finally, the built-up peptide is released from the solid phase. In the following examples, use has been made of chis so-called solid phase technique in accordance with what has been reported by Merrifield, R.B., J. Am. Chem. Soc (1963), 85, 2149, Merrifield, R.B. Bio- ;
chemistry (1964), 3, 1385 and Konig, W., Geiger, R., Chem.
Ber. (1970), 103, 788.
' '' ' W092/00996 2 ~ 8 5 ~ O ~ PCT/SE91/00477~7 ' General description of synthesis -The peptides disclosed in the following examples were synthesized using the solid phase technique (J. M. Stewart, J.D. Young. Solid Phase Peptide Synthesis, Pierce Chemical Company).

The peptides were purified by liquid chromatography (reversed phase). The stationary phase was composed of ~romasil~, 13 u, loO A, C18 (EKA Nobel, Sweden) and the mobile phase was !,"
acetonitrile/water having 0.1 ~ tri'luoroacetic acid. Those fractions containing pure product (HPLC analysis) were pooled, evaporated and the product freeze-dried from water.

Purity and structure of the peptides were determined by the use of HPLC, a~ino acid analysis and FAB-MS.

The following abbreviations have been used:

9OC = t-butyloxycarbonyl D-Tyr~Et) = O-ethyl-D-tyrosyl Fmoc = 9-fluorenyImethoxycarbonyl FmocONSu = 9-fluorenylmethoxycarbonyl-N-hytroxy-succinimide 25 D-Phe~p-Et) ~ p-ethyl-D-phenylalanyl Mpa = 3-mercaptopropionic acid Cbz = carbobenzoxy Pfp = pentafluorophenyl --ODhbt = 3,4-dihydro-4-oxo-benzotriazine-3-oxy-Thr(t-~u) = O-tert.butyl-threonyl DCC = dicyclohexyl carbodiimide -HO3t = hydroxybenzotriazole Synthesis of the dicyclohexylammonium salt of Fmoc-Orn(Cbz?OH

N~-benzyloxycarbonyl ornithine (5.3 g, 20 mmole) was suspended in a mixture of water ~S0 ml) and acetonitrile ~100 ml). Diisopropylethylamine (3.4 ml) and Tmoc-ONSu ~7.4 g 22 :
~ W092t00996 2 ~ 8 ~ ~ O ~ PCT/SE91/00477 mmole) were added. Following agitation f(~r 2 hours at room temperature, the acetonitrile was distil _d off. The residue was acidified with 1 M hydrochloric acid and the product was extracted into ethyl acetate. The ethyl acetate phase was washed with water, dried with sodium sulphate, filtered and evaporated. The residue was dissolved in ethyl acetate, dicyclohexylamine ( 4 ml, 20 mmole) was added and the solution was diluted with hexane and cooled. The crystals were filtered off and washed with hexane. Yield 11.5 g ~82~).
. .~
Synthesis of No,tert-butyloxycarbonyl-D L-p-ethyl-~henyl-alanine pentafluorophenyl ester and N~.tert-butyloxvcarbonyl--O-ethyl-D-tyrosine pentafluoro~henyl ester lS
N~-tert-butyloxycarbonyl-D,L-p-ethylphenylalanine (A.L.
Zhuse, ~. Jost, E. Kasafirek, J. Rudinger. Collection Czechoslov Chem Commun. Vol 29, 2648 (1964) (O.88 g, 3 mmole), pentafluorophenol tO.61 g, 3.3 mmole) and dicyclo-hexyl carbodiimide (0.68 g, 3.3 mmole) were dissolved in 6 ml ethyl acetate. Following agitation at room temperature for 1 hour the reaction mixture was cooled on ice for 2 hours, whereupon the dicyclohexyl urea was filtered off. The filtrate wag concentrated, diluted with hexane and cooled. :
The product was filtered off and washed with hexane.
Yield 0.87 g (63~i). Mp: 95-96C
. " ',", ' The N~rtert-butyloxycarbonyl-O-ethyl-D-tyrosine pentafluoro- -~
phenyl ester was syn~hesized in a corresponding manner from Nd~tert-butyloxycarbonyl-O-ethyl-D-tyrosine. :
Yield 77S. Mp: 103-104C.
-~,', -W092/00996- 2 0 8 ~ ~ ~ PcT/s~9~ 77 Scheme for the synthesis of the amino acid derivatives I~

CH2C02P2 Pl-NHCHCo2P3 S CH2 CH2 .. ~.

I ~1 = H, p2 = t-Bul P3 = H

II pl = Fmoc, p2 = t-Bu, P3 = H
III pl = Fmoc, p2 = t-Bu, P3 = Pfp Synthesis of the amino acid derivative I

Homocystine (8.06 g, 30 mmole) was reduced with sodium in liquid ammonia (300 ml). The excess sodium was destroyed with ammonium chloride (the olution was decolourized) and the ammonia was evaporated. The residue was dissolved in water (120 ml, 0C) which had been flushed with helium gas. The pH
was adjusted to 8.1 with 1 M hydrochloric acid. Tert-butyl-acrylate ~17.4 ml, 120 mmole) was added within 15 minutes, ;
whereupon the solution was allowed to stand under agitation and hydrogen atmosphere over night. The precipitated product(I) was filtered off, washed with ethanol and ether, and dried.
Yield 6.2 g (41 ~) Synthesis of amino acid derivative II
.

The derivative I (2.5 g, 10 mmole)~ water (15 ml)l aceto-nitrile (15 ml)~ diisopropylethylamine (1.7 ml~ and Fmoc-ONSu ~ -(3.74 g, 11 mmole) were allowed to stand under agitation for 30 minutes. The acetonitrile was distilled off and the residue was acidified with 1 M hydrochloric acid. The product was extracted into ethyl acetate. The ethyl acetate phase was washed with water, dried with sodium sulphate, filtered and ' ' `. ''. . '' ' ' . '.'. . . ' ~ ' ' . ~ ~ " . ' , " . ' ' . ' ,, ' . ' ' .

208~0'~
-~ W092/00996 PCT/SE91/0~77 g evaporated. The residue was dissolved in ethyl acetate, dicyclohexylamine (2 ml, 10 mmole) was added and the solution ''~:"!' ' was diluted with ether. The crystals (derivative II) were filtered off (5.5 g, 84 %) and were recrystallized from 80 water/ethanol.
Yield 3.4 9 (52 %). Mp.: 198-201C.

Synthesis of the amino acid derivative III
The dicyclohexyldmmonium salt of the derivative II (3.4 g, 5 mmole) was suspended in ethyl acetate and shaken with 0.5 M ! ' ' H2S04. The phases were separated and the organic phase was washed with water, dried with sodium sulphate, filtered and evaporated. The residue was dissolved in ethyl acetate (10 ml)~ Pentafluorophenol (1.02 g, 5.5 mmole) and dicyclohexyl carbodiimide (1.13 g, 5.5 mmole) were added. Following :
agitation at room temperature for 1 hour, the reaction mixture was cooled with ice for 2 hours, whereupon the dicyclohoxyl urea was filtered off. The filtrate was concentrated, diluted with hexane and cooled. The product , (III) was filtered off and washed with hexane.
Yield 2.3 g ~70 ~). Mp: 58-59C.

~reparation of Peptide 1 ~reference = peptide according to EP 0 112 809) Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH2 , : , The peptide was synchesized according to the general description of synthesis by the use of Boc/benzyl methodol-ogy. The tiol groups in cysteine and mercaptopropionic acid ;~-were blocked with p-methoxy-benzyl groups. Activation of the amino acids was effectuated with DDC/HO~t and the group N~-3OC was removed with 50 ~ trifluo-oacetic acid in methylene chloride. The resin was of methylbenzhydryl type with the .

':: .:, . ' ' , : '., , . ' ', ,. , , ~: . , ,. . , ' ", ' ' `' ' ~ ' ' ': , W092/00996 ~O~ O ~ PCT/SE91/0~77.,:~-loading of 0.7 mmole/g. Resin in an amount of 0.7 g was used in every synthesis.

The peptide was deblocked and cleaved from the resin with S liquid hydrogen fluoride/anisole/ethylmethyl-sulphide in the ratio of 90:5:5. Following the evaporation of the hydrogen fluoride, the resin was suspended in ethyl acetate, filtered -and washed with additional ethyl acetate. The resin was triturated with acetic acid in order to yield the peptide.
The resin was filtered off and the filtrate diluted with 20 acetic acid in methanol, so that the peptide concentration became approximately 0.5 mmole/l. This solution was treated with 0.1 M iodine solution in methanol until a faint yellowish brown colour persisted. Then, ~owex lx8 ion exchange resin in acetate form (10 g/l) was added a~d the mixture was filtered. The fil rate was evaporated and the residue was freeze-dried from water. The product was purified according to the general description of synthesis.
Yield: 29 mg. Purity (HPLC): > 99%.

Pceparation of the Peptide 2 -.; :
Mpa-D-Tyr~t)-Ile-Thr-Asn-cys-~ro-or~-NH2 ~

The peptide was synthesized and purified in accordance with the methods used for the peptide in Example 1.
Yield: 25 m~. Purity (HPLC): > 99~. -.

. .
. : ' . W092/00996 2 0 8 ~ ~ O ~ PCT/SE91/00477 11 :.

Preparation of the Peptide 3 o o :: :
Il 11 ','''.. :, CH2c-D-Tyr(Et)-Ile-Thr-Asn-NHcHc-pro-orn-NH2 ~, , , ..... ..
S ~ CH2 . .

Scheme for the ~reoaration of the intermedlates IV and V: :
O
Il ' ., .
CH2C02P2 Boc-D-Tyr(Et)-Ile-Thr(t-8u)-Asn-NHCHC-Pro-Orn(Cbz)-Z
.;
CH2 CH2 ~
', S --- CH2 .

IV Z z 0-resin, p2 = t-3u, V Z ~ NH2, p2 ~ ammonium group Scheme for the ring closure of the intermediate V to the intermediate VI and the deblockina of the Peptide 3:

:0 , O
;' ll 11 '' CH2c-D-Tyr(Et)-Ile-Thr-Asn-NHcHc-pro-orn(p4)-NH2 I
:~ 35CH2 CH2 W092/00996 2 ~ 8 ~ & ~ ~ 12 PCT/SE91/0~7 VI P4 = Cbz Peptide 3 P4 = H

The peptide was synthesized in accordance with the general description of synthesis by using Fmoc/t-butyl methodology.
The resin was of polyamide kiselguhr type (PepSyn RB, loading 0.09 mmole/g) and 2.2 g was used in every synthesis. Fmoc--Orn(Cbz)-OH was coupled to the resin as a symmetric an-hydride. The other amino acid deriva~ives were coupled as active esters (4 eqv)~ The following derivatives were used:
Fmoc-Pro-OPfp, derivative III, Fmoc-Asn-OPfp, Fmoc-Thr~t-Bu)--ODhbt, Fmoc-Ile-OPfp and Boc-D-Tyr~Et)-opfp~

Following the solid phase synthesis, the peptide resin IV was treated with trifluoroacetic acid/methylene chioride/anisole in the ratio of 45:45:10; (2x15 min.) Then, the peptide resin was washed with methylene chloride, 5 % diisopropylethylamine in methylene chloride and methylene chloride. The peptide was then cleaved from the resin with ammonia (100 ml) in methanol (50 ml)~ The resin was filtered off and the methanol solution was evaporated. The residue was dissolved in a small volume of methanol. The peptide (V) was precipitated with ether.
Yield 130 mg.

The peptide V ~100 mg) was cyclized with diphenyl-phosphoryl azide ~DPPA, 50 ~l~ and K2HPO4 (110 mg) in dimethylformamide t25 ml) at 0C for 24 hours.

The reaction solution was evaporated. The residue was treated with water. The water was decanted off and the residsl oil was treated with ether, which yielded a precipitate (peptide VI).
Yield 57 mg.

The protective group of ornithine in the peptide VI was removed with liquid hydrogene fluoride/anisole in the ratio -of 10:1. Thé hydrogene fluoride was distributed in ethyl acetate and water. The aqueous phase, which contained the ~ -peptide 3, was freeze-dried. The peptide was purified ~5&0~ ;
wo92/ooss6 PCT/SE91/~477 according to the above method.
Yield 38 mg. Purity (HPLC): > 99%.

Preparation of the Peptide 4 ; ;-O o Il 11 ~., .~, CH2C-D-Phe~p-Et)-Ile-Thr-Asn-NHCHC-Pro-Orn-NH2 CH2 CH2 .~
, ~
S - CH2 ~
':
15 For the synthesis, the same method as disclosed in Example 3 was used, with the exception that a racemate was used at the -coupling of the amino acid in the position 2 (Boc-D,L-Phe-(p-Et)-OPfp). Only 1.1 equivalents of 30c-D,L-Phe(p-Et)-OPfp was used in the first coupling, so that neither of the enantiomers would be favoured in the coupling. The period of coupling was increased to 4 hours compared to ordinarily 45 minutes. In the second coupling, 0.8 equivalents of Boc-D,L-Phe~p-Et)-opfp wa used. At the purification ~in accordance wlth the general description of synthesis) the peptide analogues were separated with D-Phe(p-Et) and L-Phe(p-Et), respectively. The configuration-of the amino acid (L- or D-form) in the position 2 was determined by use of a method which has been described in Houben-Weyl-Synthese von Peptiden, Band 15, Part 2, page 695.
Yield: 14 mg. Purity (HPLC): > 99%.

2 ~
W092/00g96 PCTtSE91/00477~-Preparation of the Peptide 5 :

o o , H2C-C-D-Tyr ~ Et ) - I le-Thr-Asn-NHCHC-Orn-NH2 ' 10 S --- . ................ CH2 For the synthesis, the same method as disciosed in Example 3 was used, with the exception of Fmoc-Pro-OPfp, which was not used due to the fact that Pro in position 7 is omitted. .
Yield: 48 mg. Purity (HPLC): > 99%.

Preparation of the Peptide 6 (known = peptide according to EP 0 112 809~

Mpa-D-Trp-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH2 , ... . .

The p~ptlde was synthesized and purified in accordance with the methods used for the peptide in the Example 1, with the .
exception of D-Tyr~Et~ which was replaces by D-Trp in position 2. ~ ::
Yield: 26 mg. Purity (HPLC): > 99~.
:~

Preparation of Peptide 7 .

Mpa-D-Trp-Ile-Thr-Asn-Cys-Pro-Orn-NH2 :~

The peptide was synthesized and pu-ified in accordance with the methods used for the peptide i-. the Example 2, with the ... . .. .. .. . . .

~ W092/~996 2 08 J60 3 PCT/SE91/00477 : - ' exception of D-Tyr(Et) which was replaced by D-Trp in position 2.
Yield: 24 mg. Purity (HPLC): > 99%.
' . . .
S EXAMPLE 8 : -Preparation of the Peptide 8 O o '' ' Il 11 : . ~
CH2C-D-Trp-Ile-Thr-Asn-NHCHC-Pro-Orn-NH2 , .' CH2 CH2 , '' .
The peptide was synthesized and purified in accordance with the methods used for the peptide in Example 3, with the exception of D-Tyr(Et~ which was replaced by D-Trp in position 2. :~
20 Yielt: 45 mg. Purity: ) 99%.

: p~e~ar8tion of the Peptide 9 (known = peptide acco~ding to ~P 0.112 Mpa-D-Tyr(Et)-Ile-Val-Asn-Cys-Pro-Orn-Gly-NH2 . . - :
I

The peptide was synthesized and purified in accordance with the methods used for the peptide in Example 1, with the exception of Thr which was replaced by Val in position 4.
Yield: 28 mg. Purity (HPLC): > 99 EXAMPLE lO
~Preparation of the Peptide 10 Mpa-D-Tyr(Et)-Ile-val-Asn-cys-pro-ûrn-NH2 .

W092/00996 2 ~ g ~ 6 0 ~ PCT/SE91/~77 -~

The peptide was synthesized and purified in accordance with : -the methods used for the peptide in Example 2, with the exception of Thr which was replaced by Val in position 4.
Yield: 25 mg. Purity (HPLC): > 99~. -Pharmacological tests :~

The compounds according to the invention were investigated with regard to uterotonic potency on isolated rat uterus and myometric tissue from woman, using oxytocin (OT) as agonist.
The antagonistic properties of the compounds were also evaluated with the aid of this preparation. Also, rat uterus in vivo tests using oxytocin as the agonist were carried out, the results being compared to those obtained with a reference substance, Peptide 1; a compound according to EP-A-0 112 809).

In vitro tests woman Tissue from the myometrium was obtained at Caesarean sections ~from the University Clinic of Lund, Sweden). Tis~ue pieces from pregnant women were excised from the isthmus part of the uteru~. Isometric contractions were measured on isolated ti6sue ~2x2x20 mm)l and the recording of the contractions were performed with the aid of a Gra5s Sorce transduser (F03) and polygraph (P 08) at a re6ting tension of 10 mN. Rrebs-Ringer (1.5 mmole/1) buffer was used as buffer at 37C. A dose of the agonist (oxytocin) was given to concentration ~0.1 ~mol~
either alone or 2 minutes after a dose of reference substance the Peptide 1 or the Peptides 2-4 according to the invention.
At least 2 doses each of the reference and test preparations were administered to the same tissue preparation in a randomized manner, and 4 tissue preparations were tested in parallel. The effects on the myometrium was measured by integrating the registration curves during 10 minutes after the addition of the agonist. Inhibition was expressed as per cent of the average effect following administration of agonist only at the beginning and the end of the experiment.

wo 92/00g96 2 0 8 ~ G 0 3 PCT/SE91/~77 .: ' The per cent inhibition of each inhibitor (the Peptides 2-4) -~
was compared with the reference substance (the Peptide 1) according to a so-called 4-point test (StUrmer 1968). The results are given in Table 1, Column a.

In vitro tests rat Sprague Dawley rats (body weight approximately 250 g) in natural estrous were selected by vaginal smears. An ap-proximately 20 mm long segment was cut from the middle of a uterine horn and mounted in an organ bath containing 10 ml of a modified Locke~s solution of the following composition (mM: NaZl 153, KC1 5.63, CaC12 0.541, NaHC03 5.95 and glucose 2.78). ~he solution was gased with 5% C02 in oxygen at 30C.
The uterine contractions were allowed to stabilize for 30 minutes. ~he contractions were recorded isometrically at a loading of 1.5 g with the aid of a Grass force transduser (Ft.03). The antagonistic potency of the analogues were calculated as their pA2-values (Rudinger, J. & Rrejci, I.
Experiontia 18, ~19623, 5~5-588). pa2 is a measure of the inhibitory property of the peptide and was defined by Schild ~Schild, HØ British Journal of Pharmacology, ~, ~1947), 189-206) as the negative logarithm of the molar concentration of an antagonist which retuces the effect of a dose of agonist to that of half the dose. The possible agonistic effects of the antagonists were investigated by adding to the bath containing the uterus preparation a varying amount of peptide, corresponding at most to a concentration of 4 nmole/ml. No agonistic effect was observed in any of the cases. The results are shown in Table I, Column b.

In vivo tests. rat ~, ,' Sprague Dawley rats (2s0 g) in natural estrous were anaes- --theti2ed with Inactin ~0.5 mg/100 c body weight i.p.).
Myometrial activity was recorded by means of a catheter fixed in the uterine cavity and filled w th modified Locke's ' :''-: ' ' ' ' ~ . ' .
.: . .: ' ' . .. ~ . . ' ,, , .: .

solution. The catheter was connected to a Statham P23d transduser, and the concentrations were recorded on a Grass polygraph (model 7D). The tests were performed by 6 different methods, called Method A-F.
Method A
Comp3r~t ve anta~onist_tests. Oxytocin was infused intra-venously (0.05 ~g/min/100 g body weight). ~hen a regular contraction pattern had been obtained, the antagonist (0.8-8.0 pg/loo g body weight) was administered intravenously in à volume of 0.2 ml. The recorded curve was integrated over a 15 minutes period immediately before and after injection of the antagonist. The inhibition of the increase in the magnitude of the uterus contractions caused by oxytocin infusion was compared with the inhibition caused by the reference Peptide 1, which was given the value 100. The results are shown in Table 1, Column A. -~

Method 3 ~ntago~i6t_tes_6, ~nhibito~y_DQse ~I.D.). [I.D. = that antagoni6t do6e which inhibits an agonist dose (2 x) to an effect corresponding to the effect of half the agonist dose ~ x ) ~

At first, the dose-effect curve for oxytocin (2 10-4 - 5 10-3 ~mole/kg) wa6 carried out. Such an oxytocin dose ~2 x) is selected that gives sn effect corresponding to an intraluminar contraction pre6sure of 10-30 mg Hg and that lies on the ~ -linear part of the do6e-effect curve. The effects are measured ;
as the net vslues of the integrated curve recorded over 15 ~ -minutes after injection.
.~.: "
Then, the effect (eff x) of the agoni6t for its half dose (x) is calculated. Thereafter at least two doses of antagonist (peptide 1-4) are injected in combination with the agonist dose (2 x). By interpolating the dose-effect curve for the - inhibition, the antagonist dose correspondlng to the effect , " ' :., :, .. :, . ~ . , . , . - . . :

;- W092/00996 2 0 8 5 & O ~ PCT/SE91/~77 (eff x) of the agonist dose (x)~ i.e. the I.D. dose, is obtained. The results are shown in Table I, Column B.

Method C
Anta~onist_tests, effect duration. Such a dose of the agonist is selected (5-10-4 - 5 10-3 ~mole/kg) that gives an effect ~the effect was measured over a 15 minutes period after agonist and antagonist administration, respectively, the contraction curve being integrated) corresponding to approximately 50 ~ of the maximum effect (ED50).

~hen a dose of the antagonist (Peptide 1-10) 0.8 - 4 10-8 mole/kg, which in combination with the agonist dose gives at least 50 % inhibition of the effect of the agonist dose only, is administered. Only the agonis~ dose is then injected at 20 minutes intervals, the inhibition effect successively declining.

~y interpolating the period of time from administration of the inhibitor to when 75 ~ of the inhibition of the agonist effect has ceased, is obtained. ~he results are shown in Table I, Column C.

Method D
anta~onist_tggts, intran~sal_a~m~nistr~t~on.
Princ~pIe: A mea~ure of the bioavailability of the peptide is obtained if the inhibitory effect of the intranasal ad-ministration is compared to the effect sfter intravenou6 administration.
Oxytocin (OT) is infused intravenously (0.5 ~ug/min/kg~ to elicit an agonist effect. When a regular contraction pattern ~ was obtained, the ant~agonist (0.01-0.i ~g/kg) was a~ministered -~ intravenou61y in a volume of 0.2 ml. The effects of two ~- 35 different doses were investigated. When the effects had ceased the peptide~was administered, afte a period of 15 minutes, ~- :
intranasaly in a single dose (0.1-1.0 ~g/kg). In this connection the peptide was adminis~ered in a volume of 10 ~l '. . ,,; ,.. ',,.. ., . ,. : . .. , , .,:, ,.. '.`,, .. ,,, ~`'-,, . - . .. , :

wo 92/00996 2 0 ~ ~ G 0 3 PCT/SE91/00477 in isotonic saline solution via a fine tube, the tip of which being 10 mm inside the nasal cavity of the rat. Rinsing of this was performed by perfusion with saline solution ( 20 ml/h) over 10 minutes via an additional tube, which had been S introduced into the esophagus. The results are shown in Figure 1.

Method E
~ntagonist_tests_following_intraintestinal_administratloa-The method and the calculation of the bioavailability of thepeptide corresponds to the Method D, with the exception of the intraintestinal administration:
. .
The peptide was administered via a catheter into an approxi-mately 20 cm long segment of the small intestine, which had been ligated from the rest of the intestine by ligatures at both ends of the 6ection of the intestine, and a tube had been fixed to each of these ends for rinsing and peptide .
administration. The rinsing liquid was driven out by air prior to the administration of the peptide, which was effectusted via the dista} intestinal tube in 1 ml volume of saline solution per 10 cm intensine. The results are shown in Figures 2 and S.

~ethod F
~nta~onist_tests_fQllowing_intrayenous_a_ministration (for calculation of bioavailability of the peptides)~ The method corre6ponds to the Method D, with the exception of the dose of the Peptides used, which was lo-8 mole/kg in Figure 3 and 10-8 (reference) and 2 x 10-9 (Peptide 3) in Figure 4. The results are shown in Figures 3 and 5.

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~ SUBSTITI.1TE 5HE-~

W092/00996 ~~ ~ ~ PCT/SE9l/00477 It is evident from Table I that the antagonistic effect, measured as PA2 values, of the C-terminally shortened derivatives on rat uterus is essentially unchanged (Peptides 2-5, 7, 8 and 10) compared to the values for the reference substance (Peptide l) and the known Peptides 6 and 9 having the normal length of the moleGule. Effects on pregnant human uterus show a retained inhibitory activity for the Peptides 2, 4, 5, 8 and 10, but, significantly increased inhibitory potency for Peptides 3 and 7 compared to the respective peptides of the full length (Peptide 1, Peptide 6 and Peptide 9, respectively). .
.
In vivo,the potency increase up to five times with the Peptides 2-4, 7, 8 and 10, i.e. they can be injected in doses correspondingly lower compared to the corresponding known peptides of full length. The duration of the inhibitory effect is unchanged with the Peptide 5, increases at least two times with the Peptides 2 and 7, three times with the Peptide 3 and ~-four times or more with the Peptides 4 and 8.
From the Figures 1-5 it is evident that the Peptides 2 and 3 according to the present invention have further been compared to the reference substance Peptide 1, following intranasal, -intraintestinal and intravenous administration. It is evident the Peptides 2 and 3 have such a strongly p:olonged effect compared to the Peptide 1, that the inhibition becomes impossible to quantify. Further, it is evident that the antagonistic effect, following intravenous administration of the Peptides 2 and 3 and the Peptide 1, does not give a corresponding difference in the inhibitory effect.
- :.
Altogether these results show that the Peptides 2 and 3 according to the invention are more easily resorbed through -~
the nasal and intestinal mucous membranes compared to the Peptide l, which makes it possible to administer the peptides in a pharmaceutical composition which is suitable for intra-nasal or intraintestinal administration. Thus, therapeutic treatment in the non-institutional care is made available.
':

W092/00996 2 0 8 ~ 6 0 3 PCT/SE91/00477 .
As far as pituitary posterior lobe hormones are concerned, ~.
no one has hitherto shown that by simply removing the C- :
terminal glycine amino acid residue (Peptides 2, 7 and lO), :~ .
there is obtained an analogue which enhances the in vivo potency approximately tree times and the effect duration to -.
the double, compared to the corresponding analogue having the full molecular length (Peptides l, 6 and 9). The Peptides 3 and 4, which both are ~-Abu6 derivatives, give, in analogy with what has been previously ~nown from pituitary posterior lobe agonists, a further prolonged effect duration. It is.
remarkable that the Peptides 3 and ~ additionally give a substantially enhanced inhibitory effect in regard to :l isolated human myometrium, which indicates an enhanced . :
lS receptor affinity.

.. . .

,

Claims

1. Vasotocin derivative, c h a r a c t e r i z e d in that it has the formula wherein A = Mpa (3-mercaptopropionyl residue; -S-CH2-CH2-CO-) or Hmp (2-hydroxy-3-mercaptopropionyl residue;

B = D-Tyr, D-Tyr(Et), D-Phe, D-Phe(p-Et) or D-Trp C = Thr, Val or Hgn (homoglutamine) D = Cys or .alpha.-Abu (.alpha.-Aminobutyric acid residue; E = Pro or a peptide bond F = a L- or D-residue of the formula wherein m = 0-6 n = 0-6 L = H, COOH, CO-NH2 or CH2-OH
K = H, or a D- or L-amino acid residue with the provisio that L is not H when A is Mpa, C is Thr or Val and D is Cys.

2. Vasotocin derivative according to claim 1, c h a r a c t e r i z e d in that A = Mpa B = D-Tyr(Et), C = Thr, D = Cys, E = Pro and F = Orn-NH2.

3. Vasotocin derivative according to claim 1, c h a r a c t e r i z e d in that A = Mpa B = D-Tyr(E), C = Thr, D = .alpha.-Abu S = Pro and F = Orn-NH2

4. Vasotocin derivative according to claim 1, c h a r a c t e r i z e d in that A = Mpa B = D-Phe(p-Et), C = Thr, D = .alpha.-Abu E = Pro and F = Orn-NH2.

5. Pharmaceutical composition, c h a r a c t e r i z e d in that it comprises at least one vasotocin derivative according to claim 1, as an active ingredient, in combination with pharmaceutically acceptable additives and/or diluents.

6. Pharmaceutical composition according to claim 5, c h a r a c t e r i z e d in that the composition is in a form suitable for intravenous administration.

7. Composition according to claim 5, c h a r a c t e r i z e d in that it is in a form which is suitable for intranasal administration.

8. Composition according to claim 5, c h a r a c t e r i z e d in that it is in a form suitable for intraintestinal administration.

9. Composition according to any one of claims 5-8 for use in therapeutic treatment of excessive uterus muscle contractions.

10. A method of counteracting excessive uterus muscle contractions in which a pharmaceutical composition according to any one of claims 5-8 is administered in a therapeutically effective amount.