CS272678B1 - Analog of salmon hypothalamic gnrh and method of its preparation and utilization - Google Patents

Abstract

The invention concerns a new analogue of salmon hypothalamic GnRH of formula I PGlu-His-Trp-Ser-Tyr-(D-Glu(NH-Ad))-Trp-Leu-Pro-GlyNH2, where NH-Ad is adamantyl-l-amide. The method of its preparation is based on the synthesis of the peptide chain on the polymer carrier in the solid phase, subsequent release of the protective groups splitting-off of the carrier by liquid hydrogen fluoride and superimposition of the product. This analogue of the salmon trigger factor of gonadotrophin can be used for induced ovulation of female tench fish in a dose of 5 - 25 micrograms/kg of weight of the fish with a one-off intramuscular injection application.

Description

and a process for its preparation (57) The present invention relates to a novel salmon hypothalamic GnRH analog of formula I PGlu-His-Trp-Ser-Tyr- / D-Glu / NH-Ad // - Trp-Leu-Pro-GlyNHg, wherein NH-Ad is adamantyl-1-amld, a process for its preparation starting from the synthesis of a peptide chain on a polymeric solid phase support, subsequent release of protective gaps, cleavage of the support with liquid hydrogen fluoride, and purification of the product. This salmon trigger factor analog of gonadotropin can be used to induce ovulation of tench eggs at a dose of 5-25 µg / kg of the markers by a single injection by intramuscular injection.

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The present invention relates to a salmon hypothalamic GnRH analogue, a process for its preparation and use.

For induced spawning of eggs from some species, fish are commonly used in carp hypophyses or recently also luliberin, which is a trigger factor for luteising and follicle-stimulating hormone of mammalian origin (cf. Czech Patent Certificate No. 214369). In view of the fact that triggering factors from different animal species have been gradually isolated (N. Sherwood; Trends in řfeurosciences 10, 129 (1987)) and it has been shown that there are structurally different factors in the individual developmental series (the structure of salmon LH-RH is different from the mammalian one), the focus was on structural variations of the fish factor. In the study of structure-effect dependence, it has been shown in a number of mammalian LH-RH analogues that a change in position 6 / ie substitution of glycine with various non-proteinogenic amino acids of configuration D and their particularly lipophilic and sterically intensive derivatives leads to highly active, high agonist and that is, gonadotropic activity.

By replacing the glycine at position 6 with the adamantylamide derivative of D-glutamic acid, which was newly prepared and used inter alia in the preparation of peptides with adjuvant effects (cf. no. induced spawning of certain fish species of the general formula I pGlu-His-Trp-Ser-Tyr-D-GluKK-AdJ-Trp-Leu-Erq-Gly-IIHg (i),

wherein 1 H-Ad is adamantyl-1-amide. Abbreviations for amino acids have this meaning pGlu pyroglutamic acid His histldin Trp tryptophan Ser serine Tyr tyrosine D-Glu • adamantyl-1-amide linked to the carboxyl group in the • N -D-glutamic acid position Leu leucine For proline GlyKH ₂ glycinamide

This new substance - biologically highly effective decapeptide - is a synthetic analogue of the natural, so-called trigger factor for luteinizing hormone.

The process for preparing this salmon hypothalamic GnRH analogue of Formula I consists in the stepwise construction of a peptide chain from suitably protected amino acids on a solid phase polymeric support, followed by release of the protecting groups and cleavage of the chain from the support by liquid hydrogen fluoride.

The solid phase method was used to synthesize the peptides of the invention (J, J, f, Stewart, J.D, Young, Pierce Chem, Comp, 1984). We used a methylbenzhydrylamine polyraeric carrier / amino substitution of 0.51 mmol / g. The tert-butyloxycarbonyl residue was used as a temporary protecting group and the following derivatives were used:

BOO-Trp (Por), BOC-D-Glu (NH-Ad), BOC-Tyr (Z), BOC-Ser (Bzl), BOO-His (BOC).

CS 272678 Bl

Condensations were carried out in a 1: 1 mixture of dimethylformamide and dichloromethane,

Vo / Vo using dicyclohexylcarbodiimide and N-hydroxybenztriazole in the so-called flow arrangement (V. Krchňák et al .: Tetrahedron Letters 28, 4469 (1987)). Upon completion of the synthesis, the peptide chain was cleaved from the polymeric support by the liquid hydrogen fluoride method of Lowhing Tam, JP, et al., J. Am.Chem. Soc. 105, 442 (1983). The salmon hypothalamic GnRH analog was purified by dextran gel chromatography and by high performance liquid chromatography.

It has further been found to use an analog of formula I for induced ovine ovary ovulation, wherein the analog is administered at a dose of 5 to 25 µg / kg of egg cell weight by a single injection intramuscularly.

When determining biological activity, a significantly higher yield was observed at lower doses of this peptide compared to another salmon trigger factor analog of gonadotropin. The results are shown in Tables 1 and 2.

Example 1

Preparation and purification of the peptide;

peptide structure: substance I pGlu-His-Trp-Ser-Tyr-D-Glu (WH-Ad) -Trp-Leu-Pro-Gly-KH ₂ polymer carrier: methylbenzhydrylamine.polymer substitution 0.51 mmol / g-NHg amount reactor supports: 0.5 g

amino acid mol. mass weight (g) BOC-Gly 175.19 0.13 BOC-Pro 215.2 0.16 B0C-Leu.H ₂ 0 249.3 0.19 BOC-Trp 333.4 0.25 BOC-D-Glu 380.5 0.28 BOC-Tyr 415.5 0.32 BOC-Ser 295.13 0.22 BOC-Trp 333.4 0.25 BOC-His (BOC) DCHA 536.72 0.40 pGlu 111.1 0.10

All condensation took 45 min (negative, ninhydrin test)

Cleavage of tert-butyloxycarbonyl. groups were performed for 30 min.

neutralization 5 min.

In all steps, 3 equivalents of activated amino acid derivatives (0.75 mmol) were used. The methodology of flow, arrangement (quoted in the text) was used. P-cresol and thiocresol were used as preservatives in peptide cleavage in an amount corresponding to the above publication. After desalination on dextran column. gel (50% acetic acid elution) and lyophilization gave 55 mg of sur, peptide, which was further purified on a column of silica gel modified with octadecyl groups in the mobile phase system

CS 272678 B1 containing 50% methanol and 50% 0.1% trifluoroacetic acid in water. A total of 10 mg of peptides of the corresponding amino acid and elemental analysis was obtained (for performing control liquid, silica gel and thin layer chromatography).

According to analytical high-performance chromatography on an ODS-reversed phase column in a mobile system containing the same components as in the preparation of the substance, the trigger factor analog contained 97% of the amino acid peptides. Ingredients:

pGlu 1.0, His 1.1, Trp 1.7, Her 0.9, Tyr 1.2, Olu 1.0, Leu 1.2, Pro 1.0, Gly 1.1.

Example 2

Induction of ovulation of tench eggs

The fish caught by the VÚRH-Vodňany fish farm were sorted, weighed, labeled and distributed according to the individual experimental conditions (different doses of substance I) in laminated tanks with a water volume of 70 l with the inflow of recirculated water. Water temperature was raised to 22-24 ° C by the start of injection, compound I was dissolved in saline and diluted so that all eggs were injected with a 1 ml / kg fish weight solution. The dosage range ranged from 0.2 µg to 125 µg per kg fish weight. The control group of eggs was not injected. A comparative experiment was performed with another previously described salmon derivative GnRH - D-Tle GnRH - Kouril J, et al. Animal Production 10,901 (1987).

A few hours before the expected ovulation date, the eggs were started and checked at 1-2 hour intervals. When ovulation was detected, the eggs were artificially wiped out, the weight of the eggs spawned (to the nearest 1 kg) was determined, and the eggs were sampled for the determined average egg weights. The relative weight of spawned eggs and the relative fertility for the administration of Compound I and Compound II were calculated from the observed data (see Tables 1 and 2).

Compound II pGlu-His-Trp-Ser-iyr-D-Tle-Trp-Leu-Pro-GlyNHg, wherein D-Tle

D - tertiary leucine &

Traternal fertility of tench eggs after application of the substance

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Claims (1)

OBJECT OF THE INVENTION 1, An analogue of the salmon hypothalamic GnRH of Formula I pGlu-His-Trp-Ser-Tyr- (D-Glu) NH-Ad / - Trp-Leu-Pro-Gl, yNH2, short of NH-Ad is ndamantyl-1-nmide. ?. The method of claim 1, wherein the protonated peptide chain on the polymeric support in a solid phase is removed from the carrier, and the protective groups are released, the chain is cleaved from the support with liquid hydrogen fluoride, and the resulting product is purified by a combination of gel and high performance liquid chromatography