AU8225191A

AU8225191A - Derivatives of pituitary posterior lobe hormones

Info

Publication number: AU8225191A
Application number: AU82251/91A
Authority: AU
Inventors: Michal Lebl; Per Melin; Jerzy Trojnar
Original assignee: Ferring AB
Current assignee: Ferring AB
Priority date: 1990-07-09
Filing date: 1991-07-04
Publication date: 1992-02-04
Also published as: FI930013A0; SE9002384D0; MC2291A1; PL297600A1; JPH05508849A; FI930013A7; CA2085603A1; IE912378A1; WO1992000996A1; EP0538367A1; IL98737A0

Description

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 positions 1, 2 , 4 and optionally 6.

Background 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 increased receptor density.

We have previously shown (EP-A-0 112 809) that modification of the vasotocin molecule in the positions 1, 2, 4 and 8 gives analogues which strongly inhibit uterine contractions both in animal and human tests (Melin et al, J. Endocrinol. Ill, 125, 1986). These analogues have been shown in this context to antagonize oxytocin or vasopressin induced contractions, and in clinical trials one analogue has been shown to counteract excessive uterus contractions in connec¬ tion with painful menstruations and premature labour. (Akerlund, Acta Obst. 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 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-0 182 627 discloses a large number of structurally similar vasopressin analogues, which are said to be basic Vi-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-0 182 627.

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 vasotocin derivatives disclosed in EP-A-0 112 809.

Vasotocin derivatives of the invention are comprised by the following formula:

A - B - He - C - Asn - D - E - F 1 2 3 4 5 6 7 8

I I

wherein

A = Mpa (3-mercaptopropionyl residue; -S-CH2-CH2-CO-) or Hmp (2-hydroxy-3-mercaptopropionyl residue;

OH

I

-S-CH₂-CH-C0-) B ^» D-Tyr, D-Tyr (Et ) , D-Phe , D-Phe (p-Et ) or D-Trp

C = Thr, Val or Hgn ( homoglutamine )

D = Cys or ot-Abu ( -HN-CH-CO- )

I

E = Pro or a peptide bond

F = a L- or D-residue of the fomula NH-(CH2)_m -CH-L

I

(CH₂)_r

NH

I K

wherein m = 0-6, preferably 0-4 n = 0-6, preferably 2-4 L = H, COOH, CO-NH₂ or CH₂-OH K = H, C=NH or a D- or L-amino acid residue

NH₂

with the provisio 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 pharmaceutically acceptable additives 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 side effects are minimized.

Short description of the drawings

Figure 1 presents antagonistic effect on rat uterus in vivo, following intranasal administration of a reference substance (Peptide 1) and a vasotocin derivative according to the invention (Peptide 2).

Figure 2 presents antagonistic effect on rat uterus in vivo, following intraintestinal administration of the same peptides as 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 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 according to the invention

The vasotocin derivatives according to the invention can be prepared —l 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 in accordance with the technique reported by Law, H.B. & Du Vigneaud, V. in the Journal of the American Chemical Society 12 (1960) 4579-4581, Zhuze, A.L., Jost, K., Kasafirek, E. & Rudinger, J. in the Collection of Czechoslovak Chemical Communications 2£, (1964), 2648-2662, and modified by Larsson, L.-E., Lindeberg, G., Melin, P. / Pliska, V. in the Journal of Medicinal Chemistry ___, (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 followin- examples, use has been made of this so-called solid phase technique in accordance with what has been reported by Merrifield, R.B., J. Am. Chem. Soc (1963), 8 2149, Merrifield, R.B. Bio¬ chemistry (1964), 3_, 1385 and Kδnig, W., Geiger, R. , Chem. Ber. (1970), 101 788. 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 Kromasil®, 13 u, 100 A, C₁₈ (EKA Nobel, Sweden) and the mobile phase was acetonitrile/water having 0.1 % trifluoroacetic 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, amino acid analysis and FAB-MS.

The following abbreviations have been used:

Boc = t-butyloxycarbonyl

D-Tyr(Et) = O-ethyl-D-tyrosyl

Fmoc = 9-fluorenylmethoxycarbonyl

FmocONSu = 9-fluorenylmethoxycarbonyl-N-hydroxy- succinimide 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-Bu) = O-tert.butyl-threonyl

DCC = dicyclohexyl carbodiimide

HOBt = hydroxybenzotriazole

Synthesis of the dicyclohexylammonium salt of Fmoc-Orn(Cbz)OH

NS-benzyloxycarbonyl ornithine (5.3 g, 20 mole) was suspended in a mixture of water (50 ml) and acetonitrile (100 ml). Diisopropylethylamine (3.4 ml) and Fmoc-ONSu (7.4 g 22 mmole) were added. Following agitation for 2 hours at room temperature, the acetonitrile was distilled 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 Nd-tert-butyloxycarbonyl-D.L-p-ethyl-phenyl- alanine pentafluorophenyl ester and N*τ-tert-butyloxycarbonyl- -O-ethyl-D-tyrosine pentafluorophenyl ester

N -tert-butyloxycarbonyl-D,L-p-ethylphenylalanine (A.L. Zhuse, K. Jost, E. Kaεafirek, J. Rudinger. Collection Czechoslov Chem Commun. Vol 2£, 2648 (1964) (0.88 g, 3 mmole), pentafluorophenol (0.61 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 was concentrated, diluted with hexane and cooled. The product was filtered off and washed with hexane. Yield 0.87 g (63%). Mp: 95-96°C

The N r-tert-butyloxycarbonyl-0-ethyl-D-tyrosine pentafluoro- phenyl ester was synthesized in a corresponding manner from NβV-tert-butyloxycarbonyl-O-ethyl-D-tyrosine. Yield 77%. Mp: 103-104°C. Scheme for the synthesis of the amino acid derivatives I-III

CH₂C0₂P² P¹-NHCHC0 P³

I I CH₂ CH₂

I I

S CH₂

I P¹ = H, P² = t-Bu, P³ = H

II P¹ = Fmoc, P² = t-Bu, P³ = H

III P¹ = 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 solution was decolourized) and the ammonia was evaporated. The residue was dissolved in water

(120 ml, 0°C) 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), aceto¬ nitrile (15 ml), diisopropylethylamine (1.7 ml) and Fraoc-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 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 g (52 %). Mp. : 198-201°C.

S^ynthesis of the amino acid derivative III

The dicyclohexylammonium salt of the derivative II (3.4 g, 5 mmole) was suspended in ethyl acetate and shaken with 0.5 M H2SO4. 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 dicyclohexyl 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: 58-59°C.

EXAMPLE 1

Preparation of Peptide 1 (reference = peptide according to EP 0 112 809^

Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH₂ I _ _J

The peptide was synthesized 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/HOBt and the group Nβ- Boc was removed with 50 % trifluoroacetic acid in methylene chloride. The resin was of methylbenzhydryl type with the 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 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/1. This solution was treated with 0.1 M iodine solution in methanol until a faint yellowish brown colour persisted. Then, Dowex 1x8 ion exchange resin in acetate form (10 g/1) was added and the mixture was filtered. The filtrate 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%.

EXAMPLE 2

Preparation of the Peptide 2

Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys-Pro-0rn-NH₂

L i

The peptide was synthesized and purified in accordance with the methods used for the peptide in Example 1. Yield: 25 mg. Purity (HPLC): ∑ 99%.

EXAMPLE 3

Preparation of the Peptide 3

0 0

II II

CH₂C-D-Tyr(Et)-lle-Thr-Asn-NHCHC-Pro-0rn-NH₂

CH₂ CH₂

^•CH₂

Scheme for the preparation of the intermediates IV and V:

II

CH₂C0 P² Boc-D-Tyr(Et)-Ile-Thr(t-Bu)-Asn-NHCHC-Pro-Orn(Cbz)-Z

I l CH₂ CH₂

I I

S- — CH₂

IV z = 0-resin, P² = t-Bu, V Z - NH2, P² = ammonium group

Scheme for the ring closure of the intermediate V to the intermediate VI and the deblocking of the Peptide 3:

0 0

II II

CH₂C-D-Tyr(Et)-Ile-Thr-Asn-NHCHC-Pro-Orn(P⁴)-NH₂

I ^' i CH₂ CH₂

I I

S CH₂ 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 KB, loading 0.09 mmole/g) and 2.2 g was used in every synthesis. Fmoc- -0rn(Cbz)-0H was coupled to the resin as a symmetric an¬ hydride. The other amino acid derivatives were coupled as active esters (4 eqv) . The following derivatives were used:

Fmoc-Pro-OPfp, derivative III, Fmoc-Aεn-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 chloride/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 μl) and K HP0₄ (110 mg) in dimethylformamide (25 ml) at 0°C for 24 hours.

The reaction solution was evaporated. The residue was treated with water. The water was decanted off and the residal 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. The hydrogene fluoride was distributed in ethyl acetate and water. The aqueous phase, which contained the peptide 3, was freeze-dried. The peptide was purified according to the above method. Yield 38 mg. Purity (HPLC): ∑ 99%.

EXAMPLE 4 Preparation of the Peptide 4

0 0

CH₂C II-D-Phe(p-Et)-Ile-Thr-Asn-NHCHC II-Pro-Orn-NH₂

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 Boc-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 was used. At the purification (in accordance with 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%. EXAMPLE 5

Preparation of the Peptide 5

0 0

II II

For the synthesis, the same method as disclosed 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%.

EXAMPLE 6

Preparation of the Peptide 6 (known = peptide accordin^g to EP 0 112 809^

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

The peptide 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%.

EXAMPLE 7

Preparation of Peptide 7

Mpa-D-Trp-Ile-Thr-Asn-Cys-Pro-0rn-NH2 1

The peptide was synthesized and purified in accordance with the methods used for the peptide in the Example 2, with the exception of D-Tyr(Et) which was replaced by D-Trp in position

2.

Yield: 24 mg. Purity (HPLC): ∑ 99%.

EXAMPLE 8

Preparation of the Peptide 8

0 0

II II CH₂C-D-Trp-Ile-Thr-Asn-NHCHC-Pro-0rn-NH

I I

CH₂ CH₂

S - -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. Yield: 45 mg. Purity: ∑ 99%.

EXAMPLE 9

Preparation of the Peptide 9 (known = peptide according to

EP 0 112 809

Mρa-D-Tyr(Et)-Ile-Val-Asn-Cys-Pro-Orn-Gly-NH₂

I 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 10 ' Preparation of the Peptide 10

Mpa-D-Tyr(Et)-Ile-Val-Asn-Cys-Pro-Orn-NH2

L. I 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). Tissue pieces from pregnant women were excised from the isthmus part of the uterus. Isometric contractions were measured on isolated tissue (2x2x20 mm), and the recording of the contractions were performed with the aid of a Grass force transduser (F03) and polygraph (P 08) at a resting tension of 10 mN. Krebs-Ringer (1.5 mmole/1) buffer was used as buffer at 37°C. A dose of the agonist (oxytocin) was given to concentration (0.1 jιmol/1) 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. 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 (Stϋrmer 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: NaCl 153, KC1 5.63, CaCl₂ 0.541, NaHC0₃ 5.95 and glucose 2.78). The solution was gaεed with 5% C0₂ in oxygen at 30°C. The uterine contractions were allowed to stabilize for 30 minutes. The 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. & Krejci, I. Experientia 18, (1962), 585-588). pA₂ is a measure of the inhibitory property of the peptide and was defined by Schild (Schild, H.O. British Journal of Pharmacology, 2, (1947), 189-206) as the negative logarithm of the molar concentration of an antagonist which reduces 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, cr -esponding 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 (250 g) in natural estrous were anaes^¬ thetized with Inactin (0.5 mg/100 g body weight i.p.). Myometrial activity was recorded by means of a catheter fixed in the uterine cavity and filled with 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

Comparative antagoni£t_tests. Oxytocin was infused intra¬ venously (0.05 μg/min/100 g body weight). When a regular contraction pattern had been obtained, the antagonist (0.8-8.0 Hg/100 9 body weight) was administered intravenously in a 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 B Antac£oni£rt_teεts._£_ inhibitory_Dose I^D^) . [I.D. = that antagonist dose 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 μmole/kg) was carried out. Such an oxytocin dose (2 x) is selected that gives an effect corresponding to an intraluminar contraction pressure of 10-30 mg Hg and that lies on the linear part of the dose-effect curve. The effects are measured as the net values of the integrated curve recorded over 15 minutes after injection.

Then, the effect (eff x) of the agonist 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 corresponding to the effect (eff x) of the agonist dose (x), i.e. the I.D. dose, is obtained. The results are shown in Table I, Column B.

HS ϊ ς>d C An£asonist_testSj_ eff_ect_duration. Such a dose of the agonist is selected (5*10^~4 - 5*10^~3 μmole/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).

Then 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 agonist dose is then injected at 20 minutes intervals, the inhibition effect successively declining.

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

Method D &n£agonist_t£s£Sj. in£ranasal_adrninistration.

Principle: A measure of the bioavailability of the peptide is obtained if the inhibitory effect of the intranasal ad¬ ministration is compared to the effect after intravenous administratio .

Oxytocin (OT) is infused intravenously (0.5 μg/ in/kg) to elicit an agonist effect. When a regular contraction pattern was obtained, the antagonist (0.01-0.1 μg/kg) was administered intravenously in a volume of 0.2 ml. The effects of two different doses were investigated. When the effects had ceased the peptide was administered, after a period of 15 minutes, intranasaly in a single dose (0.1-1.0 μg/kg). In this connection the peptide was administered in a volume of 10 μl 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 introduced into the esophagus. The results are shown in Figure 1.

Method E

Antasoni^t_t^sts_fol]_iowing_intrainte^ti.nal_a^inistration. The method and the calculation of the bioavailability of the peptide corresponds to the Method D, with the exception of the intraintestinal administration:

The peptide was administered via a catheter into an approxi- raately 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 section 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 effectuated via the distal intestinal tube in 1 ml volume of saline solution per 10 cm intensine. The results are shown in Figures 2 and 5.

Method F

Antagoniat_tgsts_following_in.traverιous_administration. (for calculation of bioavailability of the peptides). The method corresponds to the Method D, with the exception of the dose of the Peptides used, which was 10"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. TABLE 1

Pharmacological data for derivatives of vasotocin (VT)

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 1) and the known Peptides 6 and 9 having the normal length of the molecule. 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 prolonged 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 1, 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. 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 10), 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 1, 6 and 9). The Peptides 3 and 4, which both are< -Abu⁶ derivatives, give, in analogy with what has been previously known from pituitary posterior lobe agonists, a further prolonged effect duration. It is remarkable that the Peptides 3 and 7 additionally give a substantially enhanced inhibitory effect in regard to isolated human myometrium, which indicates an enhanced 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

A - B - He - C - Asn - D - E - F 1 2 3 4 5 6 7 8 I

wherein

A = Mpa (3-mercaptopropionyl residue; -S-CH2-CH2-CO-) or Hmp (2-hydroxy-3-mercaptopropionyl residue; OH

-S-CH₂-CH-CO-)

B = D-Tyr, D-Tyr(Et), D-Phe, D-Phe(p-Et) or D-Trp

C = Thr, Val or Hgn (homogluta ine)

D = Cys or_.βt-Abu (o(.-Aminobutyric acid residue; -HN-CH-CO-)

CH₂ i

CH₂

I

E = Pro or a peptide bond

F = a L- or D-residue of the formula NH-(CH₂)m -CH-L

I

<CH₂)π

I

NH

K I wherein m = 0-6 n - 0-6

L = H, COOH, CO-NH₂ or CH₂-OH K = H, C=NH or a D- or L-amino acid residue

I

NH₂

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(Et), C - Thr,

E = 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 = oς-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.