EP0000764A1 - Anticonceptive agent containing a peptide - Google Patents

Abstract

The use of LHRH peptide derivatives in which in the 6-position D-serine tert. butyl ether or D-glutamic acid - cyclohexylamide is included as an anti-conception agent with repeated daily dosing, while the same compounds as described in DOS 2 438 350 have the reverse effect after a single administration, i.e. cause the release of gonadotropins from the anterior pituitary.

Description

in der X für D-Serin-tert-butyläther (la) oder D-Glutaminsdure-γ-cyelohexylamid (Ib) steht, als Antikonzeptiva.The invention relates to the use of peptides of the general formula I

in which X stands for D-serine-tert-butyl ether (la) or D-glutamic acid-γ-cyelohexylamide (Ib), as anticonceptives.

The compounds of formula I are prepared according to DOS 2 438 350 or the earlier application P 26 17 646.5. After a single dose in doses of 1.4-20 meg (this corresponds to 20-300 ng / kg) intravenously (iv) subcutaneously (sc) or intramuscularly (im) or 50-1000 mcg (which corresponds to 100-5000 ng / kg) ) intranasal (in) in test subjects the release of gonadotropins from the anterior pituitary lobe.

It has now been found that with repeated daily dosing of at least 4 days of more than 2.5 mcg iv, se or in (daily dose) or more than about 100 mcg in the effect does not increase, but surprisingly reverses, so that with appropriate dosage these compounds can be used as anticonceptives.

The dosage is e.g. for the connection Ia 3 x 5 meg s.e., i.v. or i.m., but can be safely increased to a multiple, since the peptides according to the invention are non-toxic. In practice one moves between 5-500 mcg daily. In intranasal use, this dose has to be increased by about 100 times because of the absorption of only approx. 1%.

The compounds according to the invention can be used in men to temporarily suppress testosterone production and thus spermatogenesis, and in women to suppress ovulation. The application for women begins e.g. on the first day of the cycle and is continued for about 14 days. In this way, ovulation is definitely prevented during the treatment period. The LH level drops to basal values and stimulation of the pituitary gland is no longer possible. With short-term treatment, ovulation can be postponed; with longer treatment, it is suppressed. The latter may also apply to the nidation, since both processes depend on the level of the FSH and LH levels.

So far, attempts have been made to suppress LH and FSH release by using competitive inhibitors of the LH-RH. However, this was insufficient and at concentrations which are about a thousand times higher than that of the compounds according to the invention.

In addition, the residue and metabolism problems to be taken into account, for example in the case of steroid-containing contraceptives, are eliminated. The preparations used according to the invention contain poptides which are easily broken down into amino acids in the body excreted or metabolized via these amino acids in a physiological manner.

Compounds of formula I can be used e.g. can be administered intravenously intramuscularly or subcutaneously in physiological saline. A particularly valuable form of medicinal use is particularly suitable for long-term treatment with aqueous or oily preparations for intranasal application. In the form of a suppository preparation, rectal or vaginal use is also possible.

a) Observation on men

3 men aged 30-55 years received 50 meg compound Ia subcutaneously in aqueous preparation daily for 10 days. Testosterone levels were measured by radioimmunoassay. There was a reduction in the plasma testosterone level to approximately 1/3 of the initial value. Testosterone levels were measured again 6 weeks after stopping treatment. They were back to normal.

The same results were obtained when 2 mg daily in oily preparation was administered intranasally for 10 days.

b) Observation on women

4 women aged 22-30 years received 3 x 5 mcg compound Ia subcutaneously in aqueous preparation daily from the beginning of the cycle. A few days after treatment, the plasma levels of luteinizing and follicle-stimulating hormone had dropped to basal levels and the pituitary no longer stimulated.

With short-term treatment, the ovulation was postponed, while with long-term treatment it was suppressed.

c) Observation on animals

Two groups of five sexually mature, pure-bred beagle males, weight 6-8 kg, received 2.5 mcg / kg of compound Ia subcutaneously in isotonic saline once a day. Testosterone levels dropped to 30% of baseline after 1 month and to 6% of baseline after 3 months.

The treatment was continued for 6 months. Significant testicular atrophy occurred during treatment, but testicular size increased significantly 8 weeks after treatment was discontinued and the animals showed normal sex drive.

The preparation of appropriate preparations is described in the following examples.

example 1

500 mg of compound Ia are dissolved in 100 liters of physiological saline. It is filtered sterile and filled into ampoules of 1 ml each. This preparation can be used for intravenous, intrasmuscular or subcutaneous administration of the compound Ia or more generally of a compound of the general formula I.

Example 2

600 mg of compound Ib are dissolved in 100 l of isotonic aqueous mannitol solution and further treated as in Example 1. The ampouled solution is freeze-dried for use again in 1 ml of dist. Water dissolved.

Example 3

9 1 dest. Water is heated to boiling and 20 g of methyl 4-hydroxybenzoate are dissolved in it. The mixture is cooled to about -30 ° C., 89.6 g of Na ₂ HFO ₄ , 13.5 g of citric acid, 10 g are added Sodium chloride and 250 g of mannitol and then dissolve 100 g of compound Ia in it. Then you fill with dist. Water to 10 1 and filtered.

1 ml solution is e.g. filled into a container that is equipped with a dispensing metering valve that releases 0.05 ml of the solution per application.

Example 4

10 g of benzyl alcohol are made up to 0.990 l with 2-octyldodecanol. 10 g of microfine ground compound Ia are added and the mixture is homogenized.

1 _- 2 ml of this suspension are filled into small containers. The containers are equipped with a sampling valve that releases 0.05 ml of the suspension per application.

Example 5

2 kg of commercially available suppository masses are heated to approx. 60 ° C. 2 g of microfine ground compound Ia are added and the mixture is homogenized. The mass is poured into molds that release suppositories weighing 2 g each after cooling.

Example 6

a) Z-D-Gln (cyclohexyl) -OBzl

(c = 1, in Eisessig)

6 g Z-D-Gln (cyclohexyl)-OBzl werden in 100 ml Dioxan/ Wasser (4:1) suspendiert und mit 1n NaOH titriert (Thymolphthalein als Indikator, Verbrauch: 14 ml 1n NaOH). Anschließend wird mit 1n HC1 neutralisiert und i. Vak. eingeengt. Der Rückstand wird zwischen Essigester und 1n HC1 verteilt. Die Essigesterphase wird mit Wasser gewaschen, getrocknet und eingeengt. Der Rückstand wird mit Äther verrieben und abgesaugt. Ausb. 4.3 g Schmp. 112 - 175⁰,

= + 6.2° (c = 1, in Methanol)

To a solution of 12.3 g (33.1 mmol) ZD-Glu-OBzl, 4.06 ml (33.1 mmol) cyclohexylamine and 4.47 g 1-hydroxybenzotriazole (33.1 mmol) in 50 ml abs. Tetrah'ydrofu- ran are added at 0 ° C 7:35 g DCC, 2 leaves h at 0 ⁰ C and stand overnight at room temperature. The next day, the precipitate is suctioned off, washed with tetrahydrofuran and the filtrate is concentrated. The residue is triturated with saturated NaHCO ₃ solution ben, sucked off and washed well with water and dried. Educ. 14.5 g, mp 105 ° C. After recrystallization from isopropanol / petroleum ether: 6 g mp 163 °,

(c = 1, in glacial acetic acid)

6 g ZD-Gln (cyclohexyl) -OBzl are suspended in 100 ml dioxane / water (4: 1) and titrated with 1N NaOH (thymolphthalein as indicator, consumption: 14 ml 1N NaOH). Then it is neutralized with 1n HC1 and i. Vac. constricted. The residue is distributed between ethyl acetate and 1N HC1. The ethyl acetate phase is washed with water, dried and concentrated. The residue is triturated with ether and suction filtered. Educ. 4.3 g mp 112 - 175 ⁰ ,

= + 6.2 ° (c = 1, in methanol)

To a solution of 1.89 g (5 mmol) ZD-Gln (cyclohexyl) -OH, 3.6 g (5 mmol) H-Leu-Arg-Pro-NH-C ₂ H ₅ -ditosylate and 1.35 g (10 mmol) 1- Hydroxybenzotriazole in 20 ml of dimethylformamide is added at 0 ° C to 1.3 ml of N-ethylmorpholine and 1.1 g of DCC. The mixture is stirred at 0 ° C. for 1 h and left at room temperature overnight. The reaction product precipitates together with the dicyclohexylurea. The reaction mixture is mixed with ether and the precipitate is filtered off with suction. The filter cake is triturated with saturated NaHCO ₃ solution and suction filtered. Educ. 3.2 g of amorphous mass, which is catalytically hydrogenated in methanol with a Pd catalyst analogous to Example 1d. After the hydrogenation has ended, the catalyst is filtered off with suction, the filtrate is concentrated and the residue is triturated with ether. Educ. 2.2 g of an amorphous mass, which without further purification with 1.5 g of Z-Ser-Tyr (Bzl) -OH and 405 mg of 1-hydroxybenzotriazole in a little dimethylformamide is solved. 0.78 ml of N-ethylmorpholine and 660 mg of DDC at 0 ° C. are added to this solution. The precipitate is filtered off and the filtrate is concentrated. The residue is triturated with saturated NaHC0 ₃ solution, suction filtered and washed with water. Educ. 2.6 g of an amorphous mass which is catalytically hydrogenated without purification in a mixture of dimethylformamide and methanol (1: 1). After the hydrogenation has ended, the catalyst is filtered off with suction and the filtrate is concentrated. The residue is triturated with ether and then purified by distribution chromatography. Educ. 420 mg of a thin layer chromatographically uniform ninhydring positive compound with correct amino acid analysis. (Solvent 1).

diacetate

= - 33.4° (c = 1, in Wasser).215 mg of Glu-His-Trp-NH-NH ₂ are reacted analogously to Example 1f with 420 mg of H-Ser-Tyr-D-Gln (cyclohexyl) -Leu-Arg-Pro-NH-C ₂ H ₅ '2HCl and into the Acetate transferred. The compound is purified by gradient elution on a carboxymethyl cellulose column (11 x 1.5 cm) with 0.002-0.01 N ammonium acetate buffer as the eluent. 128.3 mg of pure product by thin layer chromatography (solvent 1) were isolated. Peptide base content according to UV spectrum and amino acid analysis 80%. The rest is water (10%) and acetic acid (10%).

= - 33.4 ° (c = 1, in water).

Amino acid analysis (hydrolysis 68 h in 6n HCl at 110 ° C): Ser (0.7), Glu (2.0), Pro (0.9), Leu (1.0), Tyr (0.9), His (1.0), Arg (1.0). The Trp content is determined by the UV absorbance (1.0).

Claims (1)

Use of peptides of the general formula I

in which X stands for D-serine tert-butyl ether or D-glutamic acid-γ-cyclohexylamide, as aniticone septives.