JPS6047248B2 - Method for promoting luteinizing hormone secretion for livestock production - Google Patents

Description

[Detailed description of the invention] Luteinizing hormone secretion stimulating hormone (LH-RH) is
It is a type of hormone derived from the hypothalamus and has the effect of stimulating the secretion of luteinizing hormone (LH) from the anterior pituitary gland.

LH-RH is thought to exist not only in mammals, but also in birds and fish, but those derived from pigeons and sheep were isolated and purified, and their chemical structures were determined in 1971 and 197. Peptide (L-pyroglutamyl-L-histidyl-L-tryptopyl-L-seryl-L
-Tyrosyl-glycyl-L-leucyl-L-arginyl-L-prolyl-glycinamide). This decapeptide was soon synthesized in many research labs, and it was revealed that it has strong physiological activity not only in the native pigtails and sheep, but also in many other mammals, birds, fish, etc. However, it has become clear that this LH-RH decapeptide stimulates and promotes the secretion of not only luteinizing hormone (LH) but also follicle stimulating hormone (FSH) in various animal species. Since LH-RH is a substance related to reproduction that originally exists in the body, it was expected to be developed as a high-quality medicine and livestock medicine. The two obstacles are that it has a half-life of 2 to 4 minutes) and that it stimulates both LH and FSH secretion at the same time, making it difficult to emphasize only one.
At present, it has not been widely applied as an effective medicine or livestock medicine. For example, in order to maintain the concentration levels of both LH and FSH hormones in the body for the time required to promote ovulation and spermatogenesis, methods such as continuous injection must be used, and follicular cysts, which occur frequently in domestic animals, have recently been reported. In order to treat reproductive disorders such as these, it is necessary to individually increase the LH level and FSH level, making it difficult to use LH-RH. This time we will discuss LH-RH, which is a biological component,
! ■, L-3,4-dihydroxyphenylalanine (L
We have discovered that when a combination of (dopa) or its derivatives is administered to a living body, LH release activity is significantly increased compared to when LH-RH is administered alone, but there is almost no change in FSH release activity. .

This result is unexpected, as administration of L-dopa alone does not affect LH-releasing activity in normal animals. For example, as a basic study, in an experiment using young male rats as a model in which the effects of LH-RH can be expressed without the influence of other sex hormones, L
Although the H concentration increased significantly when L-dopa was administered in combination compared to when LH-RH was administered alone, no particular change was observed in the FSH concentration. Furthermore, when ovulation was induced during the diestrus phase of adult female rats exhibiting a normal cycle, a significant enhancing effect was observed when L-dopa was used in combination with LH-RH. LH-
Although it is desirable to administer RH and L-dopa at the same time, almost the same effect was found when L-dopa was administered 6 times before or after LH-RH administration.

The administration method is by intravenous, subcutaneous, etc. injection, or orally. This effect is caused by LH-R instead of LH-RH.
It is also observed when using LH-RH analogues having H action. In addition, substances that can suppress the decomposition of L-dopa in the body and increase its long-lasting effect in the body1, such as L-α-hydrazino-3,4-dihydroxy-α-methyl-β-phenylpropionic acid (carbidopa ) and N
-DL-seryl-N''-(2,3,4-trihydroxybenzyl)hydrazine, etc., can be used in combination to significantly enhance the effect. Conventionally, reproductive disorders in livestock have been treated with high levels of chorionic gonadotropin (PMS), etc. Molecular protein hormone preparations and the like have been used, but they have the disadvantage that their effects weaken over a short period of time due to their antigenicity.

Since LH-RH and L-dopa included in the fifth aspect of the present invention are both low-molecular biological components, there is no such concern. Therefore, not only ovarian function decline, which is the main cause of reproductive disorders, such as ovarian hypoplasia, ovarian atrophy, ovarian rest, luteinization deficiency, and ovulatory disorders (delayed ovulation, anovulation, etc.)4, but also ovarian cysts (ovarian follicles). It can be safely used for cysts, corpus luteum cysts), and for synchronizing the sexual cycle. The dosage of the composition according to the present invention varies depending on the type of livestock, its weight, age, symptoms, etc., but generally, for example, in cattle, LH-RH is 1.
~20m9 or so, and L-dopa approximately 1~20y is good.

For parenteral administration, subcutaneous, intradermal or intramuscular injections are preferred, but oral administration may also be used. In addition, substances that promote the long-lasting effects of L-dopa used in combination, such as L-α-hydrazino-3,4-dihydroxy-α-
Methyl-β-phenylpropiozoic acid is preferably administered orally or parenterally in an amount of about 5% to 30% of the amount of L-dopa.

As mentioned above, the method of the present invention is applicable not only to LH-RH but also to LH-RH.
It can also be applied to H-RH analogs.

LH-RH analogs are those in which one or more of the constituent amino acid residues of the LH-RH decapeptide have been converted to amino acids, amines, or organic acid residues different from those of the original LH-RH. This refers to substances that have LH-RH-like effects.For example, they are as shown below. (1) Acetyl-N-methylglycyl-L-histidyl-L-tryptopyl-L-seryl-L-tyrosyl-D-alanyl-L
-Leucyl L-Arginyl L-Proline Ethylamide (2) L-Pyroglutamyl L-Histidyl L-Tryptopyl L-Seryl L-Tyrosyl D-Leucyl L-Leucyl L-Arginyl L-Prolyl Glycinamide (3) L-Pyroglutamyl L -Histidyl-L-Tryptophyl-L-Seryl-L-Tyrosyl-D-Alanyl-L-Leucyl-L-Arginyl-L-Prolineethylamide Also, regarding L-dopa used in combination with LH-RH, not only L-dopa but also its derivatives can be used. Often, the following L-dopa derivatives can be used, for example:

3,4-diacyl form of IL-dopa and its salts such as (1) 3,4-diacetyloxy-L-phenylalanine hydrochloride (2) 3,4-dipivalyloxy-L-phenylalanine perchlorate ■ L-dopa or Parts 3 and 4
- diacyl carboxyesters and salts thereof, such as (1) 3,4-dihydroxy-L-phenylalanine-methyl ester hydrochloride (2) 3,4-diacetyloxy-L-phenylalanine-methyl ester hydrochloride: 3)
3,4-dihydroxy-L-phenylalanine-benzyl ester hydrochloride (4) 3,4-diacetyloxy-L
-Phenylalanine-benzyl ester hydrochloride (5) Glycyl-3,4-diacetyloxy-L-phenylalanine-methyl ester hydrochloride 1L-dopa or its 3
, 4-diacyl enamine derivatives such as (1) 3,4-dipivalyloxy-N(1-methyl-2
-acetylvinyl) on-phenylalanine-pivalyloxymethyl ester (3) 3,4-diacetyloxy-N-(1-methyl-2-acetylvinyl) on-phenylalanine potassium salt ■L-dopa or its 3, 4-diacyl form. Amide derivatives such as (1) N-formyl-3,4-dipivalyloxy-L-
Phenylalanine (2) N-formyl-3,4-dipivalyloxy-L-phenylalanine-pivalyloxy 3) N-formyl-3,4-diacetyloxy-L-phenylalanine 1) N-formyl-3,4-diacetyloxy-L -Phenylalanine potassium salt) glycyl-3,4-diacetyloxy-L-phenylalanine hydrochloride (613,4-diacetyloxy-L-phenylalanine-glycine hydrochloride) Dipeptides of VL-dopa and their derivatives, such as (1) 3,4-dihydroxy-L-phenylalanine hydrochloride enylalanyl-3,4-dihydroxy-L-phenylalanine-hydrochloride 1213,4-diacetyloxy-L-
Phenylalanyl-3,4-diacetyloxy-L-phenylalanine methyl ester hydrochloride (3) 3,4-diacetyloxy-L-phenylalanine-3,4-diacetyloxy-L-phenylalanine benzyl ester hydrochloride VlL-Dopa and others Dipetides with natural a-amino acids, such as (1) L-tyrosyl-L-dopa (2) glycyl-3,4-diacetyloxy-L-phenylalanine benzyl ester hydrochloride (3) L-leucil-L-dopa (4) L- Dorper D-Alanine (Example 1) Adult female Wistar Kondo rats (body weight around 250f) were kept at a room temperature of 23±1°C under artificial lighting with lights on at 5 a.m. and lights off at 7 p.m., and a 4-day oestrous cycle was observed. The ovulation-inducing effect was examined using only those individuals who had undergone the test exactly three times.

At 4:00 p.m. during the diestrus phase of the animals used, L-dopa 2.5
m9, LH-RH each dose (500, 1000, 2000
py) was simultaneously dissolved in 1 mt of physiological saline and administered intravenously under mild ether anesthesia. The next day, between noon and 1 p.m., the eggs were sacrificed and the oviducts were searched for eggs under a microscope. Furthermore, LH-RH alone administration (500, 1000, 2000, 4
000μy), L-dopa alone administration (5m9/rat)
A similar experiment was conducted for LH-RH(7)ED5O was calculated using 5 animals for each dose of LH-RH. LH-RH single administration ED, O = 1800 + 1 g/rat
'-Jmu ●υ L-dopa 5Tng administration No ovulation (Example 2) Male rats aged 24-27 days were used as basic model animals, and LH-RHI:. The effect of combined use of L-dopa was investigated.

Serum LH and FSH concentrations were determined by radioimmunoassay using the two-antibody method. The results are shown in the tables below. Table 2 Table 3 (Example 3) LH-RH (7) The most standard Ramirez-McCann method was performed to examine the LH release effect.

Four weeks after birth, ovariectomized female rats (same lot) were used 8 weeks after the surgery. An oil solution containing 50 m9 of progesterone and 0.1 mg of extraterogen was used on the 3rd day after subcutaneous injection. A sample dissolved in 1 ml of physiological saline was administered intravenously, and after 1 minute, blood was collected, serum was separated, and LH concentration was measured by radioimmunoassay. Three to five rats were used in each group. The results are shown in Table 4. Table 4 From Table 4, rib lower part - pa (
It can be seen that the release of LH is further promoted when a substance that can increase the internal properties of L-dopa is used in combination.

(Example 4) Acetyl-N-methylglycyl-L-histidyl-L
- When tryptopyl-L-seryl-L-tyrosyl-D-alanyl-L-leucyl-L-arginyl-L-proline ethylamide (LH-RH analogue) is used in combination with L-dopa.

The above two compounds were simultaneously administered subcutaneously to rats under the same conditions as those used in Example 2, blood was collected after the third and sixth test, and LH and FSH in the serum were quantified by radioimmunoassay.

Claims (1)

[Scope of Claims] 1. A method for promoting luteinizing hormone secretion for livestock production, which comprises administering L-dopa or a derivative thereof in combination with a luteinizing hormone secretion-stimulating hormone or an analog thereof. 2. Luteinizing hormone secretion for livestock production, which is characterized in that luteinizing hormone secretion stimulating hormone or its analogue is administered in combination with L-dopa or its derivatives and a substance capable of increasing the internal efficacy of L-dopa. Promotion method.