WO2016070815A1 - 一类新型生长激素释放激素类似肽及其在制备治疗不孕不育药物中的应用 - Google Patents

一类新型生长激素释放激素类似肽及其在制备治疗不孕不育药物中的应用 Download PDF

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WO2016070815A1
WO2016070815A1 PCT/CN2015/093814 CN2015093814W WO2016070815A1 WO 2016070815 A1 WO2016070815 A1 WO 2016070815A1 CN 2015093814 W CN2015093814 W CN 2015093814W WO 2016070815 A1 WO2016070815 A1 WO 2016070815A1
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peptide
hghrh
group
ghrh
peptides
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French (fr)
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唐松山
张娟辉
游娟
张旭东
周东
李红枝
李秋莹
郭小元
唐婧晅
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广东药学院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/25—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention belongs to the field of medical biology, and specifically relates to four novel growth hormone releasing hormone analog peptides and the application thereof in preparing medicine for treating infertility.
  • hGHRH Human growth hormone releasing hormone
  • hGH human growth hormone
  • the maturation of the preproprotein hGHRH (1-107/8) precursor is performed by a series of proteolytic processing steps.
  • the final mature forms are hGHRH(1–44)NH 2 , hGHRH(1–37)OH and hGHRH (1–40). OH.
  • hGHRH the shortest sequence with 51% biological activity is hGHRH (1–29), which is called a core peptide.
  • the homology of human hypothalamic hGHRH with GHRH in pigs, Chinese hamsters, cattle, golden hamsters, goats, guinea pigs, rats and mice was 93%, 93%, 89%, 89%, 86%, 73, respectively. %, 71% and 61%.
  • hGHRH(1-44)NH 2 has the highest molecular activity.
  • GHRH peptides have broad-spectrum activity in vitro, such as promoting wound healing, protecting cardiomyocyte apoptosis, improving sleep quality, reducing obesity in diabetes or AIDS, and improving neurocognitive function.
  • a first object of the invention is to provide a novel class of growth hormone releasing hormone analog peptides.
  • novel growth hormone releasing hormone-like peptide is a 2D, 2E, 2F or 2Y dimer peptide
  • the amino acid sequence of the 2D dimer peptide is:
  • the amino acid sequence of the 2E dimer peptide is:
  • the amino acid sequence of the 2F dimer peptide is:
  • the amino acid sequence of the 2Y dimer peptide is:
  • the present inventors have found through experiments that the above 2D, 2E, 2F or 2Y dimer peptides form two by oxidizing monomers D, E, F or Y in vitro. Polymer.
  • the dimeric peptide was tested by pituitary incubation and found to have high pituitary GH release activity and pituitary hormone release specificity.
  • the 2F dimer peptide has the greatest binding to pituitary receptor activity through pituitary GHRH receptor binding reaction and pituitary cell fluorescence staining analysis.
  • the 2F dimer peptide was used as a representative for the treatment of male infertility model. Compared with the saline group and the cyclophosphamide control group alone, the cells in the testicular seminiferous tubules of the 2F dimer peptide group were arranged neatly. The spermatogonia, spermatocytes, sperm cells and mature spermatozoa were significantly increased, the volume of seminiferous tubules became thicker, and the seminiferous tubules became smaller or even disappeared, and the efficacy was dose-dependent.
  • the 2F dimer peptide is representative of the treatment of female infertility model.
  • the pregnancy rate statistics show that compared with the cyclophosphamide group alone, the 2F group has a significant increase in both birth rate and pregnancy rate, and positive gonadotropin.
  • the HMG group there were multiple birth mice in the 2F group, and multiple mid-pregnancy pregnant mice in the HMG group.
  • the number of follicles showed that the number of mature follicles in the 2F group was higher than that in the other groups, while the primary and secondary follicles in the HMG group increased significantly.
  • HE staining showed that compared with the normal saline control group, the cells in the cyclophosphamide model group were disordered, lost normal gland structure, uneven staining, and nucleus pyknosis.
  • the cells were arranged neatly, with normal glandular structure and uniform staining, indicating that the peptide has obvious protective effect on ovarian structure.
  • a large number of mature follicles were observed in the ovary of 2F group, which had obvious follicular maturation, and was dose-dependent.
  • GHRH dimer peptides represented by 2F dimer peptides such as 2D, 2E and 2Y dimers
  • 2F dimer peptides such as 2D, 2E and 2Y dimers
  • a third object of the present invention is to provide a medicament for treating infertility, characterized in that the novel growth hormone releasing hormone-like peptide 2D, 2E, 2F or 2Y dimer peptide is used as an active ingredient.
  • 2D, 2E, 2F or 2Y dimer peptides described herein are abbreviated as 2D, 2E, 2F or 2Y peptides, respectively.
  • the 2D, 2E, 2F or 2Y peptides of the invention can be used to treat infertility in men and women, thus providing a drug candidate for the treatment of infertility.
  • Figure 1 is a HPLC purity analysis of the S1 peptide and a molecular weight identification map of MS;
  • Figure 2 is a HPLC purity analysis of the S2 peptide and a molecular weight identification map of MS;
  • Figure 3 is a HPLC purity analysis of the A peptide and a molecular weight identification map of MS;
  • Figure 4 is a HPLC purity analysis of B peptide and a molecular weight identification map of MS
  • Figure 5 is a HPLC purity analysis of the C peptide and a molecular weight identification map of MS
  • Figure 6 is a HPLC purity analysis of D peptide and a molecular weight identification map of MS
  • Figure 7 is a HPLC purity analysis of the E peptide and a molecular weight identification map of MS
  • Figure 8 is a HPLC purity analysis of the F peptide and a molecular weight identification map of MS
  • Figure 9 is a SDS-PAGE diagram of 2D, 2E, 2F and 2Y dimer peptides. Markers: low molecular weight protein standard 4.6-66 KDa; 2D, 2E, 2F and 2Y represent 2D, 2E, 2F and 2Y dimer peptides, respectively, D, E, F represent D, E, F monomer peptides, respectively.
  • Figure 10 is a graph showing the results of rat growth hormone releasing activity of various GHRH-like peptides and dimeric peptides;
  • Figure 11 is a graph showing the results of in vitro ACTH hormone releasing activity of various GHRH-like peptides and dimeric peptides;
  • Figure 12 is a graph showing the results of in vitro PRL hormone releasing activity of various GHRH-like peptides and dimeric peptides;
  • Figure 13 is a graph showing the results of in vitro LH hormone releasing activity of various GHRH-like peptides and dimeric peptides;
  • Figure 14 is a growth hormone release inhibition experiment.
  • I 3 -I 5 incubation 0.482 or 1.927 ⁇ M of GHIH was added to the 1.927 ⁇ M GHRH analog.
  • P 2 without peptide was used as a blank, and S peptide was used as a standard.
  • Statistical significance (*P ⁇ 0.05 or **P ⁇ 0.01) was obtained by comparing data for different doses of one GHRH dimer at the same time I 3 -I 5 incubation.
  • Figure 15 is a Scatchard plot of GHRH dimeric peptide receptor binding to pituitary homogenate
  • Figure 16 is a GHRH receptor fluorescent staining assay of rat pituitary tissue (mag. 100 x 10 fold).
  • FITC FITC-labeled S, 2D, 2E, 2F, 2Y peptide staining
  • DAPI nuclear fluorescent staining
  • Merged FITC and DAPI images combined. The yellow part indicates cell positive;
  • Figure 17 is a H-E staining of the testicular tissue of the male model group in the 2F group (magnification 4 ⁇ 10);
  • Figure 18 is a H-E staining of ovarian tissue sections of a female model rat in the 2F group (magnification 4 ⁇ 10);
  • Figure 19 is a fluorescent staining of the testicular tissue with FITC-hGHRH(1-44)NH 2 peptide (magnification 10 ⁇ 10). Yellow is FITC-hGHRH(1-44) NH 2 peptide positive fluorescence, blue is nuclear DAPI fluorescence;
  • FIG 20 FITC-hGHRH (1-44) NH 2 Peptide staining of ovarian tissue (magnification of 4 ⁇ 10). Yellow is FITC-hGHRH(1-44) NH 2 peptide positive fluorescence, blue is nuclear DAPI fluorescence;
  • the monomer peptide synthesis process manual chemical solid phase peptide synthesis (SYMPHONY type 12 channel peptide synthesizer, software version Version.201, Protein Technologies Inc.), the operation steps:
  • Resin swelling Put Wang resin (purchased from Tianjin Nankai Synthetic Technology Co., Ltd.) into the reaction pot, add dichloromethane (DCM, Dikma Technologies Inc.) 15ml/g in Dawang resin, oscillate 30min.
  • DCM dichloromethane
  • the first amino acid remove the solvent by sand core filtration, add 3 millimoles of C-terminal first Fmoc-AA amino acid (all Fmoc-amino acids provided by Suzhou Tianma Pharmaceutical Group Fine Chemicals Co., Ltd.), and then Add 10 mmol of 4-dimethylaminopyridine (DMAP) and N,N'-dicyclohexylcarbodiimide (DCC), and finally add dimethylformamide (DMF) (purchased from Dikma Technologies) Inc.) dissolved and shaken for 30 min. Block with acetic anhydride.
  • DMAP 4-dimethylaminopyridine
  • DCC N,N'-dicyclohexylcarbodiimide
  • DMF dimethylformamide
  • Detection During the synthesis process, random detection is carried out to monitor whether the synthesis is smooth: remove the solvent, take more than ten resins, wash three times with ethanol, add one drop of ninhydrin, KCN and phenol solution, 105-110 °C Heating for 5 min, darkening blue is a positive reaction.
  • wash the resin wash twice with DMF (10 ml/g), wash twice with methanol (10 ml/g), and wash twice with DMF (10 ml/g).
  • the purified solution is freeze-dried into a powder (freeze dryer Freezone Plus 6 model, LABCONCO product) to obtain a finished product.
  • Peptide species 11 GHRH-like monomeric peptides (AF, Y, D L , E L , F L , Y L ) synthesized according to the above method, three hGHRH standard peptides (S1, S2 and S2 L peptides) and The amino acid sequences of the eight dimeric peptides (2D, 2E, 2F, 2Y, 2D L , 2E L , 2F L , 2Y L peptide) are shown in Tables 1 and 2. The amino acid sequences of the S1, S2, AF, and Y peptides are shown in SEQ ID NO. 1-9, respectively.
  • All glass tubes are kept for 5h (P 1 , P 2 , I 3 , I 4 and I 5 , representing different periods of 5 hours of heat preservation, each period represents 1 hour), while gently shaking every 5 minutes, every 1 hour Gently aspirate the buffer for pituitary GH, ACTH, PRL and LH hormone tests while adding 1 ml of fresh LRB without or with active GHRH-like peptide, hGHRH peptide or dimeric peptide to the pituitary. After pre-incubation for the first 2 hours (P 1 , P 2 ), followed by incubation at 3 hours (I 3 , I 4 and I 5 ), the active polypeptide was added.
  • the net GH release level is (I 3 + I 4 + I 5 ) - P 2 .
  • P 2 without peptide was used as a blank control and the hormone values of S1 and S2 were used as positive controls.
  • Rat pituitary hormones were tested by using the Rat Growth Hormone ELISA kit (Millipore Co., USA) and the pituitary ACTH, LH, PRL ELISA kit (Shanghai Yanhui).
  • Rat growth hormone release activity results, as shown in Figure 10 and Table 3, in the 5h pituitary incubation test, once S, 2D, 2E, 2F, or 2Y peptide was added to LRB buffer, pituitary growth hormone levels Significantly improved.
  • GH release with the addition of peptides 2F I 3 I 5 hatched or hatching 2Y added peptide exhibited higher than the peptide added S I 5 I 3 or the value (P ⁇ 0.01) (FIG. 10).
  • the peak level of GH release is in the third (I 3 ) (2D, 2E, 2F), fourth (I 4 ) (S) or fifth (I 5 ) (2Y) holding period.
  • the activities of the 2D, 2E, 2F and 2Y peptides covered 102 ⁇ 9.2%, 95 ⁇ 22.2%, 110 ⁇ 18.2% and 108 ⁇ 15.5% of hGHRH(1-44) NH 2 activity, respectively.
  • the GHRH dimer peptide activity is at least 40-87% higher than the corresponding GHRH monomer, and the 2F activity is the highest.
  • the growth hormone increase of 2F was 5.04 times that of 2D (15.84 ng/ml) (the second highest peptide).
  • Example 3 Inhibition of rat pituitary growth hormone release:
  • the growth hormone release inhibition method can be determined, and in the I 3 -I 5 incubation, 0.482 or 1.927 ⁇ M of growth hormone inhibition is added, respectively.
  • the hormone (GHIH) was in a tube containing 1.927 ⁇ M GHRH dimer.
  • Peptide-free P 2 incubation was used as a blank and S2 peptide as a standard control.
  • Rat pituitary growth hormone levels were determined using a rat growth hormone ELISA kit.
  • Example 4 GHRH dimer peptide and pituitary GHRH receptor binding reaction in vitro.
  • the fresh pituitary of 30 rats was ground in 10 ml of pre-cooled homogenization buffer (50 mM HEPES, 7 mM MgCl 2 , 5 mM EDTA, 50 ug/ml PMSF, 2 mg/ml BSA, pH 7.4) for 5 min, centrifuged at 12000 rpm for 2 min, and discarded. The supernatant. Resuspend in 10.8 ml homogenization buffer and measure the protein concentration by Bradford method.
  • pre-cooled homogenization buffer 50 mM HEPES, 7 mM MgCl 2 , 5 mM EDTA, 50 ug/ml PMSF, 2 mg/ml BSA, pH 7.4
  • Example 5 Fluorescent staining analysis of rat pituitary tissue
  • the 2F peptide high, medium and low dose groups were injected with 8, 4 and 2 ⁇ g/g of the hind leg muscle respectively, and the HMG group was 0.2 unit/g body weight.
  • Others used physiological saline as a control. It is administered twice a week. In the second half of the fifth week, the model hamster and the normal hamster were in the same cage. After that, only the therapeutic drug was given twice a week for 5 weeks. The physiological changes (spirit, activity, secretion, etc.) during the estrus of the mouse were observed and the mice were observed. Whether pregnant. In the tenth week after the start of the experiment, the hamsters were removed from the squirrels, serum, testes and ovaries were taken, and tissue sections were stained to analyze the reproductive ability.
  • Mode of administration intraperitoneal injection of cyclophosphamide, 2F peptide and HMG peptide intramuscular injection.
  • test indicators Second, the test indicators:
  • test group N First pregnancy rate Pregnancy rate Late pregnancy rate Birth rate Total pregnancy rate Simple cyclophosphamide group 9 33.3 0.0 0.0 0.0 33.3 High 2F group 10 0.0 40.0 0.0 20.0 ** 60.0 ** Medium 2F group 7 28.6 0.0 0.0 0.0 28.6 Low 2F group 10 10.0 10.0 10.0 ** 40.0 * HMG group 6 33.3 33.3 16.7 0.0 83.3 ** Saline group 10 0 0 10 90 100
  • test group N First pregnancy rate Pregnancy rate Late pregnancy rate Birth rate
  • the number of early pregnancy in each group may be due to CTX being metabolized, losing toxicity, causing testicular function recovery, because the heterosexual cage time is 35 days, and the Chinese hamster reproductive cycle is 18-21 days, so the real pregnancy The number should be from the mid-pregnancy to the number of births.
  • Fig. 17 shows that spermatocytes and spermatogonia in the seminiferous tubules of the 2F dimer peptide group were significantly proliferated.
  • the seminiferous tubule cells were arranged neatly, the volume became thicker, and the seminiferous tubules became smaller or even disappeared, which was dose-dependent.
  • Table 9 compares the area of 70 semi-concave seminiferous tubules (short * long axis). It is found that the effect of increasing the seminiferous tubules in the 2F group is statistically significant.
  • test group Primary follicle Secondary follicle Mature follicle Simple cyclophosphamide group 45 6 10 High 2F group 18 0 31 ** Medium 2F group 31 13 21 ** Low 2F group twenty four 5 16 * HMG group 34 twenty one 22 ** Saline group 5 6 28
  • 3GHRH receptor using FITC-hGHRH(1-44)NH 2 peptide, fluorescent staining of testicular or ovarian sections ( Figures 19 and 20): Semen cells, mother cells and sperm cells are evident in the seminiferous tubules of the testis The distribution of GHRH receptors showed no difference in the expression of spermatogonial cells, but the expression of heads of sperm cells or mature spermatozoa increased significantly.
  • GHRH receptor positive staining is present in primary, secondary, and mature follicles.
  • the 2F peptide Compared to the GH level of the P 2 phase, the 2F peptide continuously caused an increase in GH throughout the peptide incubation period (I 3 -I 5 ), and the total GH-increment (79.77 ng/ml, compared with S2) was 2D ( 15.84 ng/ml, 5.04 times the second strongest peptide compared to S2. This means that the 2F peptide has the strongest and longest effect on prolonging the half-life, not only because 1 Pro-GHTH is more potent than 1 Tyr-GHRH, but also because of the release of GH with a circular amino acid at the N-terminus. Induction.
  • hGHRH dimers as a standard hGHRH(1-44)NH 2 peptide have good functional selectivity and species specificity, the dimers show rACTH and / or rLH, rPRL enhanced activity, which shows These GHRH peptides slightly regulate the release of other pituitary hormones.
  • the inhibitory effect of GHIH on GH release was dose/time dependent. In the presence of 1.927 ⁇ M GHRH dimer peptide, 1.927 ⁇ M GHIH significantly inhibited growth hormone secretion compared to 0.482 ⁇ M GHIH, and the inhibitory effect increased with increasing incubation time.
  • the S, 2D, 2E, 2F and 2Y peptides are specific for the binding of the pituitary of SD rats. From the relationship of B/F and Bmax in the Scatchard plot, the order of maximum affinity (Bmax value) of these GHRH dimers (2F>2D>2Y>2E) or dissociation constant (KD) (2F ⁇ 2D ⁇ 2E) ⁇ 2Y), both showed that the 2F peptide and the pituitary homogenate expressed the strongest binding ability, which was basically consistent with the activity result.
  • Fluorescence staining of FITC-labeled GHRH dimers showed cell membrane distribution, and the order of fluorescence staining intensity (2F>2D>2E>2Y>S) also showed that 2F peptide had the most abundant distribution in pituitary cells, indicating that 2F peptide has pituitary The maximum affinity of the cell membrane.

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Abstract

本发明公开了一类新型生长激素释放激素类似肽，所述生长激素释放激素类似肽为同源二聚体形式并且具有较高的垂体GH释放活性和垂体激素释放特异性，可用于制备治疗不孕不育的药物。

Description

一类新型生长激素释放激素类似肽及其在制备治疗不孕不育药物中的应用 技术领域：
本发明属于医药生物领域，具体涉及四种新型生长激素释放激素类似肽及其在制备治疗不孕不育药物中的应用。
背景技术
人生长激素释放激素(hGHRH)，由下丘脑分泌，促进垂体合成和释放人生长激素(hGH)的神经内分泌肽。前原蛋白hGHRH(1-107/8)前体的成熟由一系列蛋白水解加工步骤，最终的成熟形式是hGHRH(1–44)NH2,hGHRH(1–37)OH和hGHRH(1–40)OH。在这些GHRH分子中，具有51％生物活性的最短序列是hGHRH(1–29)，被称为核心肽。人下丘脑hGHRH与猪、中华地鼠、牛、金黄地鼠、山羊、豚鼠、大鼠和小鼠的GHRH分子同源性分别有93％,93％,89％,89％,86％,73％,71％和61％。
Ling N等报告，相对于hGHRH(1-40)OH，D-1Tyr-hGHRH(1-40)OH,1Phe-hGHRH(1-40)OH,1Trp-hGHRH(1-40)OH,1His-hGHRH(1-40)OH,1Ala-hGHRH(1-40)OH,1Tyr-hGHRH(1-40)OH,9Arg-hGHRH(1-40)OH和9Ala-hGHRH(1-40)OH分别具有0.022,0.038,0.003,0.351,0.010,0.032,0.002和0.007的活性。以hGHRH(1-44)NH2作基准，(3-Me)1His-hGHRH(1-44)NH2和(O-Me)1Tyr-hGHRH(1-44)NH2分别具有0.132和0.001的活性。Piquet G等和Esposito P等将GHRH类似物的C-末端与聚乙二醇(PEG)耦合以获得具有较高稳定性的GHRH-PEG分子。带有C-末端酰胺化的GHRH类似物具有翻倍活性。Izdebski J等在hGHRH(1–29)NH2分子骨架的29位(胍基丁胺Agm),1位(脱氨酪氨酸Dat),27位(亮氨酸Nle)和15位(L-a-氨基丁酸Abu)改构，合成了JI-34,JI-36和JI-38(JI系列)的GHRH激活剂。Hassan HA等在hGHRH分子的1Tyr和/或27Met位改构，1His,27Nle-hGHRH(1-32)NH2,1His,mono-I-10Tyr,27Nle-hGHRH(1-32)NH2，27Nle-hGHRH(1-29)NH2，D-1Tyr-hGHRH(1-32)NH2和des-1Tyr-hGHRH(2-32)NH2的活性分别比hGHRH(1-40)OH高3.2,4.9,1.4,0.74和0.02倍。Garcia JM等报道，Macimorelin(以前叫AEZS-130,ARD-07或EP-01572)是具有很好稳定性和口服生物利用率的新GH分泌剂，但是它的hGH释放效果远远低过hGHRH(1-29)NH2的活性。Cai R等报道，在JI系列的N端N-Me-Tyr,C端methyl-或ethyl-amide,Apa30-或Gab30-NH2改构，这些改构化合物N-Me-1Tyr-JI-38,N-Me-1Tyr,D-2Ala-JI-38,N-Me-1Tyr,D-2Ala,8Asn-JI-38,N-Me-1Tyr,D-2Ala,29Arg-NHCH3-JI-38,N-Me-1Tyr,29Arg-NHCH3-JI-38,N-Me-1Tyr,D-2Ala,8Asn,29Arg-NHCH3-JI-38,N-Me-1Tyr,D-2Ala,8Thr,29Arg-NHCH3-JI-38.N-Me-1Tyr,D-2Ala,29Arg,30Apa-NH2-JI-38和D-2Ala,5F-6Phe,28Ser,29Arg,30Gab-NH2-JI-38的活性是hGHRH(1-29)OH的1-7.01倍。Hassan HA等在hGHRH分子中的位置1Tyr 和/或27Met位改构，1His,27Nle-hGHRH(1-32)NH2,1His,mono-I-10Tyr,27Nle-hGHRH(1-32)NH2，27Nle-hGHRH(1-29)NH2，D-1Tyr-hGHRH(1-32)NH2和des-1Tyr-hGHRH(2-32)NH2的活性分别为hGHRH(1-40)OH的3.2,4.9,1.4,0.74和0.02倍。
到现在为止，所有被报道的GHRH激活剂中，hGHRH(1-44)NH2分子活性最高。
最近研究发现，GHRH肽具有垂体外的广谱活性，如促进伤口愈合、保护心肌细胞凋亡、提高睡眠质量、减少糖尿病或艾滋病中的肥胖和改善神经认知功能等。
虽然hGHRH类似物在生物医学上具有巨大的潜在应用，但其半衰期短和活性低是主要限制。我们以前的工作发现，N端1Tyr→Pro替换和/或hGHRH(1–44)OH的C-末端GGC扩展可有效调控这各激素的活性。最近发现，在hGHRH(1–44)分子的N-端Pro-1Pro-或1Pro-延伸或代换，和C端GGC延伸的同源二聚体意外表现出比hGHRH(1–44)NH2更高的生长激素释放活性。本说明书报道这四个新性高活性hGHRH类似物的合成、生物评价和药效。
发明内容
本发明的第一个目的是提供一类新型生长激素释放激素类似肽。
所述的新型生长激素释放激素类似肽为2D、2E、2F或2Y二聚体肽；
所述的2D二聚体肽的氨基酸序列为：
Figure PCTCN2015093814-appb-000001
所述的2E二聚体肽的氨基酸序列为：
Figure PCTCN2015093814-appb-000002
所述的2F二聚体肽的氨基酸序列为：
Figure PCTCN2015093814-appb-000003
所述的2Y二聚体肽的氨基酸序列为：
Figure PCTCN2015093814-appb-000004
上述2D、2E、2F或2Y二聚体肽的氨基酸序列中的大写字母为氨基酸的简写，这属于公知常识。其中H代表氨基位于N端，OH代表羧基COOH位于C端，这也属于本领域的公知常识。
本发明通过实验发现，上述2D、2E、2F或2Y二聚体肽通过体外氧化单体D、E、F或Y形成二 聚体。二聚体肽经过垂体保温测试，发现具有很高的垂体GH释放活性和垂体激素释放特异性。经过垂体GHRH受体结合反应和垂体细胞荧光染色分析，2F二聚体肽具有最大结合垂体受体活性。
以2F二聚体肽为代表对对雄性不孕不育模型治疗发现：与生理盐水组和单纯环磷酰胺对照组比较，2F二聚体肽组的睾丸曲细精管中细胞排列整齐，管中的精原细胞、精母细胞、精子细胞和成熟精子明显增加，曲细精管体积变粗，曲细精管腔变小，甚至消失，药效呈现剂量依赖性。
以2F二聚体肽为代表对对雌性不孕不育模型治疗发现：怀孕率统计显示，与单纯环磷酰胺组比较，2F组不论是出生率还是怀孕率都有明显增多，与阳性促性腺激素HMG组比较，2F组有多个出生小鼠，而HMG组有多个中晚期怀孕鼠。卵泡数统计显示，2F组的成熟卵泡数比其它各组都多，而HMG组的初级和次级卵泡增加明显。H-E染色法显示：与正常生理盐水对照组比较，单纯环磷酰胺模型组细胞排列紊乱，失去正常腺体结构，染色不均，细胞核固缩。经过2F肽治疗后，细胞排列较整齐，有正常腺体结构，染色均匀，显示该肽对卵巢结构具有明显保护作用。2F组卵巢边缘可见大量成熟卵泡，有明显的促卵泡成熟现象，具有剂量依赖性。利用FITC-hGHRH(1-44)NH2肽的荧光染色显示，在初级和次级卵泡和卵巢组织中，有许多细胞有明显FITC黄色荧光标记，显示有GHRH受体分布。这些结果证实新型GHRH激素肽通过GHRH受体发挥作用，促进卵细胞的增殖和成熟，促进受孕。
这些说明，以2F二聚体肽为代表的GHRH二聚体肽，如2D、2E和2Y二聚体，具有明显刺激精、卵细胞增殖和成熟作用，从而促进生殖，因此能够用于男女不孕不育药物。
因此本发明的第二个目的是提供2D、2E、2F或2Y二聚体肽在制备治疗不孕不育药物中应用。
本发明的第三个目的是提供一种治疗不孕不育药物，其特征在于，以所述的新型生长激素释放激素类似肽2D、2E、2F或2Y二聚体肽作为活性成份。
文中所述的2D、2E、2F或2Y二聚体肽其简称分别为2D、2E、2F或2Y肽。
本发明的2D、2E、2F或2Y肽能够用于治疗男女不孕不育，因此为治疗不孕不育提供了候选药物。
附图说明：
图1是S1肽的HPLC纯度分析和MS的分子量鉴定图；
图2是S2肽的HPLC纯度分析和MS的分子量鉴定图；
图3是A肽的HPLC纯度分析和MS的分子量鉴定图；
图4是B肽的HPLC纯度分析和MS的分子量鉴定图；
图5是C肽的HPLC纯度分析和MS的分子量鉴定图；
图6是D肽的HPLC纯度分析和MS的分子量鉴定图；
图7是E肽的HPLC纯度分析和MS的分子量鉴定图；
图8是F肽的HPLC纯度分析和MS的分子量鉴定图；
图9是2D、2E、2F和2Y二聚体肽的SDS-PAGE图。Markers：低分子量蛋白质标准4.6-66KDa；2D、2E、2F和2Y分别代表2D、2E、2F和2Y二聚体肽，D、E、F分别代表D、E、F单体肽。
图10是各种GHRH类似肽和二聚体肽的大鼠生长激素释放活性结果图；
图11是各种GHRH类似肽和二聚体肽的大鼠体外ACTH激素释放活性结果图；
图12是各种GHRH类似肽和二聚体肽的大鼠体外PRL激素释放活性结果图；
图13是各种GHRH类似肽和二聚体肽的大鼠体外LH激素释放活性结果图；
图14是生长激素释放抑制实验。在I3-I5孵育中，0.482或1.927μM的GHIH被添加于1.927μM的GHRH类似物中。无肽的P2作为空白，S肽作为标准。统计学显著性(*P<0.05或**P<0.01)通过比较不同剂量的一个GHRH二聚体在I3-I5孵育同一时间的数据获得。
图15是GHRH二聚体肽受体结合垂体匀浆的Scatchard图；
图16是大鼠垂体组织(mag.100×10倍)的GHRH受体荧光染色分析。FITC：FITC标记的S、2D、2E、2F、2Y肽染色；DAPI：核荧光染色；Merged：FITC和DAPI图像合并。黄色部分表示细胞阳性；
图17是2F组治疗雄性模型组睾丸组织H-E染色(放大倍数4×10)；
图18是2F组治疗雌性模型鼠卵巢组织切片H-E染色(放大倍数4×10)；
图19是FITC-hGHRH(1-44)NH2肽对睾丸组织的荧光染色(放大倍数10×10)。黄色为FITC-hGHRH(1-44)NH2肽阳性荧光，蓝色为细胞核DAPI荧光；
图20FITC-hGHRH(1-44)NH2肽对卵巢组织的荧光染色(放大倍数4×10)。黄色为FITC-hGHRH(1-44)NH2肽阳性荧光，蓝色为细胞核DAPI荧光；
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：新型生长激素释放激素类似物的合成：
一、单体肽合成过程：手工化学固相多肽合成(SYMPHONY型12通道多肽合成仪，软件版本Version.201，Protein Technologies Inc.)，操作步骤：
1、树脂溶涨：将大王树脂(wang resin，购自天津市南开合成科技有限公司)放入反应锅中，加二氯甲烷(DCM，Dikma Technologies Inc.)15ml/g在大王树脂中，振荡30min。
2、接第一个氨基酸：通过沙芯抽滤除去溶剂，加入3毫摩尔的C端第一个Fmoc-AA氨基酸(所有的Fmoc-氨基酸由苏州天马医药集团精细化学品有限公司提供)，再加入10毫摩尔量的4-二甲氨基吡啶(DMAP)和N,N'-二环己基碳二亚胺(DCC)，最后加入二甲基甲酰胺(DMF)(购自Dikma Technologies  Inc.)溶解，振荡30min。用醋酸酐封闭。
3、脱保护：去掉DMF，加20％哌啶-DMF溶液(15ml/g)5min，过滤去掉溶剂，再加20％哌啶-DMF溶液(15ml/g)15min(哌啶由国药集团上海化学试剂公司提供)。
4、检测：在合成过程中，随机进行检测，以便监控合成是否顺利：抽掉溶剂，取十几粒树脂，用乙醇洗三次，加入茚三酮、KCN和苯酚溶液各一滴，105-110℃加热5min，变深蓝色为阳性反应。
5、洗树脂：依次加入DMF(10ml/g)洗两次，甲醇(10ml/g)洗两次，DMF(10ml/g)洗两次。
6、缩合：3毫摩尔的Fmoc-AA(从C端第二个氨基酸到N末端氨基酸)和3毫摩尔的2-(7-偶氮苯并三氮唑)-四甲基脲六氟磷酸酯(HBTU，苏州天马医药集团精细化学品有限公司)，均用尽量少DMF溶解，加入反应锅中。立刻加入十倍量的N-甲基吗啉(NMM，苏州天马医药集团精细化工有限公司)，反应30min，检测呈阴性。
7、洗树脂：依次DMF(10ml/g)洗一次，甲醇(10ml/g)洗两次，DMF(10ml/g)洗两次。
8、重复二至六步操作，如表1和2的氨基酸序列所示，从右到左依次连接相应氨基酸。
9、脱除最后Pro的FMOC保护基，检测呈阳性，溶液抽干备用。
10、按照下列方法洗树脂，依次DMF(10ml/g)两次，甲醇(10ml/g)两次，DMF(10ml/g)两次，DCM(10ml/g)两次，10min抽干。
11、从树脂上切割多肽：配制切割液(10毫升/g)：TFA 94.5％(J.T.Baker Chemical Company)；水2.5％，乙二硫醇ethanedithiol(EDT,Sigma-Aldrich Chemistry)2.5％和三异丙基硅烷triisopropylsilane(TIS,Sigma-Aldrich Chemistry)1％。切割时间：120min。
12、吹干洗涤：将裂解液用氮气尽量吹干，用乙醚洗六次，然后常温挥干。
13、用HPLC仪纯化多肽：将粗肽用纯水或者加少量乙腈溶解，按照下列条件纯化：
高效液相色谱仪(制备型和分析型；软件Class-VP.Sevial System；厂商日本SHIMADZU)和Venusi MRC-ODS C18色谱柱(30x250mm，天津Bonna-Agela Technologies)。流动相A液：0.1％三氟乙酸水溶液，流动相B液：0.1％三氟醋酸-乙腈溶液(乙腈Fisher Scientific公司购买)。流速：1.0ml/min，上样体积30μl，检测波长220nm。洗脱程序：0～5min：90％A液+10％B液；5～30min梯度：90％A液/10％B液→20％A液/80％B液。
14、最后将纯化后的溶液冻干成粉末(冻干机Freezone Plus 6型号，LABCONCO产品)，即得到成品。
15、鉴定：分别取少量的成品多肽，做ESI MS的分子量鉴定和C18-HPLC分析的纯度鉴定。
16、将粉末状的多肽，密封包装，-20度避光保存。
二、二聚体肽的形成：当带有GGC的单体肽(表1所示的D肽、E肽、F肽、Y肽或表2所示的 DL肽、EL肽、FL肽、YL肽)在0.5mg/ml浓度下，在pH＝11.5氨水溶液中，37℃保温60小时，形成100％的二聚体肽2D、2E、2F、2Y或2DL、2EL、2FL、2YL等二聚体肽，它们的SDS-PAGE图如图9所示。其氨基酸序列如表1和表2所示。
三、肽种类：按照上述方法合成的11个GHRH类似单体肽(A-F、Y，DL、EL、FL、YL)、三个hGHRH标准肽(S1、S2和S2L肽)和八个二聚体肽(2D、2E、2F、2Y，2DL、2EL、2FL、2YL肽)的氨基酸序列如表1和2所示。S1、S2、A-F、Y肽的氨基酸序列分别如SEQ ID NO.1-9所示。其HPLC纯度>95％(A-F以及S1和S2肽的HPLC纯度鉴定图以及分子量鉴定图如图1-图8所示)。它们FITC标记的肽如表2所示。S1和S2被用作阳性对照。表1和2所示的所有GHRH类似肽和hGHRH肽委托上海强耀生物科技有限公司合成。
表1：GHRH类似肽、hGHRH肽和二聚体肽的氨基酸序列
Figure PCTCN2015093814-appb-000005
表2：FITC标记的GHRH类似肽、hGHRH肽和二聚体肽的氨基酸序列
Figure PCTCN2015093814-appb-000006
实施例2：体外激素释放活性：
1.体外垂体刺激方法：S-D雌鼠(大约8周龄，体重200±20g)购自广州中医药大学实验动物中心。所有实验动物的饲养和使用的制度指导方针一致。在饲养中，大鼠12:12小时明暗交替饲养，饲养温度26±1℃。大鼠经过脱臼猝死，在30分钟摘取全部脑垂体。在用灭菌的lactated Ringer’s buffer(LRB)冲洗后，垂体立即放到内有1ml LRB的1×10cm玻璃管中，37℃保温。所有的玻璃管总保温5h(P1,P2,I3,I4和I5，代表5小时保温的不同时段，每个时段代表1小时)，同时每5min轻轻晃动，每1小时后，轻轻吸出缓冲液进行垂体GH,ACTH,PRL和LH激素测试，同时加入1毫升新鲜的不带有或带有活性GHRH类似肽、hGHRH肽或二聚体肽的LRB到垂体中保温。在前2小时(P1、P2)预保温后，随后在第3小时保温(I3、I4和I5)中，开始增加活性多肽。净GH释放水平为(I3+I4+I5)-P2。未加肽的P2做为空白对照和S1、S2的激素值做为阳性对照。大鼠垂体激素通过使用Rat Growth Hormone ELISA试剂盒(Millipore Co.,USA)和垂体ACTH,LH,PRL ELISA 试剂盒(上海研辉公司)分别进行测试。
2.大鼠生长激素释放活性结果，如图10和表3所示，在5h垂体孵化试验中，一旦S，2D，2E，2F，或2Y肽添加于LRB缓冲液中，垂体生长激素水平即明显提高。GH的释放在添加了2F肽的I3孵化或添加了2Y肽的I5孵化中表现出比添加S肽在I3或I5中的值更高(P<0.01)(图10)。GH释放的峰值水平在第三(I3)(2D，2E，2F)，第四(I4)(S)或第五(I5)(2Y)保温期。有趣的是，生长激素 的释放在添加了2F肽的整个保温期(I3-I5)都增加，这意味着2F肽具有最高的rGH释放活性，而2Y肽在I3，I4或I5中的GH值并不比在P2中的高，显示2Y肽活性较弱。相较于P2的值，这些肽的净GH值显示统计学显著性差异(P<0.01)。2D，2E，2F和2Y肽的活性分别覆盖hGHRH(1-44)NH2活性的102±9.2％,95±22.2％,110±18.2％和108±15.5％。GHRH二聚体肽活性比相应的GHRH单体高至少40-87％的GH活性，2F活性最高。根据S2肽的净生长激素释放值，2F的生长激素增加值(79.77ng/ml)为2D(15.84ng/ml)(第二最高肽)的5.04倍。
表3：GHRH类似肽、hGHRH肽和二聚体肽在体外刺激垂体释放GH的水平
Figure PCTCN2015093814-appb-000007
bP净GH vs P2值.
3、大鼠体外垂体ACTH,PLR,LH激素释放活性结果：如图11、12和13所示，与P2值相比，虽然S，2D，2E，2F或2Y肽对ACTH，PRL或LH释放有一些刺激作用，但是在P2和I3-I5之间没有显示显著性差异。这表明，和标准hGHRH(1-44)NH2肽一样，这些GHRH二聚体肽可能轻微调节垂体促肾上腺皮质激素，催乳素或LH的释放。
实施例3：大鼠垂体生长激素释放抑制分析：
根据垂体GH释放活性试验(P1，P2，I3，I4，I5)，可确定生长激素释放抑制方法，在I3-I5保温中， 分别添加0.482或1.927μM的生长激素抑制激素(GHIH)于含有1.927μM GHRH二聚体的管中。无肽P2保温做空白和S2肽做标准对照。大鼠垂体生长激素水平用大鼠生长激素ELISA试剂盒测定。
根据5-h垂体培养测定，在I3-I5孵育中，有1.927μM GHRH肽出现的情况下，0.482或1.927μM GHIH肽的添加，表现出明显抑制效果，其抑制效应与GHIH肽剂量和保温时间成正比(图14)。在I3-I5中，1.927μM GHIH的抑制显著超过0.482μM GHIH肽的抑制(P<0.05或0.01)，延长培养时间可使抑制效果得到加强，这个抑制效应在hGHRH二聚体肽之间没有统计上的显著性差异。
实施例4：GHRH二聚体肽和垂体GHRH体外受体结合反应.
取30只大鼠的新鲜垂体，在10ml预冷匀浆缓冲液(50mM HEPES，7mM MgCl2，5mM EDTA,50ug/ml PMSF，2mg/ml BSA，pH 7.4)中研磨5min，12000rpm离心2min，弃上清。10.8ml匀浆缓冲液重悬，Bradford法测蛋白浓度。在测试中，取100μl垂体匀浆液(153μg蛋白)，加140μl匀浆buffer，5个浓度(30,60,120,240,480荧光强度)的荧光标记二聚体肽(2DL、2EL、2FL或2YL二聚体肽)(0.0186pM/μl)，加入对应的非标记GHRH二聚体(0.93μM,50倍的FITC标记的GHRH二聚体)，总体积300μl。放入30℃水浴箱保温一小时，6000rpm离心20min，弃上清，每个测试点加200μl匀浆缓冲液轻轻重悬，6000rpm离心20min，再加200μl匀浆缓冲液混匀，测荧光值。结果如表4和图15所示：S，2D，2E，2F或2Y肽对SD大鼠的垂体细胞的结合是特异性的，显示相对高的特异性结合(总结合大于50％)。通过B/F-Bmax的Scatchard图，表明，这些二聚体肽的Bmax或Kd值的排序分别为2F>2D>2Y>2E或2F<2D<2E<2Y，表明2F肽具有最高的垂体匀浆结合能力。
表4 hGHRH二聚体肽对垂体匀浆受体结合试验(X±S,n＝2)
Figure PCTCN2015093814-appb-000008
aP<0.05，与2D或2E的Kd值比较；bP<0.05，与2E或2Y的Bmax值比较
实施例5：大鼠垂体组织的荧光染色分析
5μm厚的福尔马林固定SD大鼠垂体组织切片染色，染色方法如下：将切片按常规方法脱蜡。新鲜脱蜡的玻片浸泡在HEPES缓冲液3次，每次5分钟。在组织切片上滴加200μl含有75.99±0.97荧光强度的FITC-GHRH二聚体(SL,2DL,2EL,2FL和2YL)的HEPES缓冲液，37℃下孵育1小时。载玻片 在HEPES缓冲液中每次5分钟洗涤两次后，在切片上滴加DAPI水溶液(0.01克/毫升)，37℃孵育15分钟。将切片每次5分钟洗涤两次，然后用荧光封固剂(上海Biaoben模型有限公司)封片，即获得染色结束。此染色过程在黑暗环境中进行。
染色结果可见，大鼠垂体GHRH受体的分布和表达水平明显可见(图16)。FITC标记GHRH二聚体染色有明显细胞膜分布，荧光染色强度排序为2F>2D>2E>2Y>S，2F肽染色在垂体细胞中表现最丰富，这表明在垂体细胞膜上2F肽和GHRH受体之间的亲和力最强。
实施例6：2F二聚体肽对不孕模型的治疗
一、不育不孕模型试验方法
取60只中华地鼠雄鼠和60只中华地鼠雌鼠(由四川省实验动物中心提供)，按无差异性体重t检验分组成六组(2F肽高、中和低剂量组，人促性腺激素HMG组，环磷酰胺对照组和无环磷酰胺生理盐水组)。每组n＝10，按20毫克/kg腹腔注射环磷酰胺(江苏恒瑞医药股份有限公司，国药准字H32020857，批号12032925)，每周一次，共五次。在第三次环磷酰胺注射后，2F肽高、中和低剂量组分别后腿肌注剂量8、4、2微克/g，HMG组为0.2单位/g体重，其它使用生理盐水作对照，每周给药两次。第五周下半周模型地鼠和正常地鼠异性同笼，此后只给予治疗药物，每周两次，持续5周，观察老鼠动情期间的生理变化(精神、活动、分泌物等)和观察老鼠是否怀孕。试验开始后第十周同时脱臼杀地鼠，取血清、睾丸和卵巢，组织切片染色分析生殖能力。
怀孕判断：初期怀孕鼠：输卵管能看到连珠针状颗粒，很小；中期怀孕鼠：输卵管内颗粒呈连珠火柴头状，能明显判断其怀孕，颗粒中度大小；末期怀孕鼠：输卵管内红色颗粒状物明显可见，且较大；新生鼠：在十周前已经出生。
给药方式：环磷酰胺腹腔注射，2F肽和HMG肽后腿肌内注射。
二、检测指标：
1、中华地鼠给药后体重变化：实验鼠给药前体重没有统计学差异。与生理盐水组比较，环磷酰胺给药后各模型组小鼠体重明显降低，当肌肉注射2F或HMG后，体重减低明显好于单纯环磷酰胺组。由于造模后有不同程度的地鼠死亡，所以最后各组动物数为n＝6～10。见表5和6)。
表5 雌鼠给药后体重变化(n＝6～10)
Figure PCTCN2015093814-appb-000009
Figure PCTCN2015093814-appb-000010
表6 雄鼠给药后体重变化(n＝6～10)
Figure PCTCN2015093814-appb-000011
2、怀孕率统计：与仅给单纯环磷酰胺比较，2F组不论是出生率，还是总怀孕率都有明显统计学意义，实验中生理盐水组怀孕率100％(表7和8所示)
表7 雌鼠模型组怀孕率统计
实验组 N 初怀孕率 中怀孕率 晚怀孕率 出生率 总怀孕率
单纯环磷酰胺组 9 33.3 0.0 0.0 0.0 33.3
高2F组 10 0.0 40.0 0.0 20.0** 60.0**
中2F组 7 28.6 0.0 0.0 0.0 28.6
低2F组 10 10.0 10.0 10.0 10.0** 40.0*
HMG组 6 33.3 33.3 16.7 0.0 83.3**
生理盐水组 10 0 0 10 90 100
*P<0.05和P<0.01**，与单纯环磷酰胺组比较，X2卡方检验。
表8.雄鼠模型组怀孕率统计
实验组 N 初怀孕率 中怀孕率 晚怀孕率 出生率 总怀孕率
单纯环磷酰胺组 8 30.0 0.0 0.0 0.0 30.0
高2F组 8 37.5 0.0 0.0 12.5** 50.0**
中2F组 7 42.9 0.0 0.0 0.0 42.9*
低2F组 9 33.3 22.2 0.0 0.0 55.5**
HMG组 9 33.3 0.0 11.1 11.1** 55.5**
生理盐水组 10 0 0 0 100** 100**
*P<0.05和P<0.01**，与单纯环磷酰胺组比较，卡方检验。
这里需说明的是，各组早孕数可能是由于CTX被代谢，失去毒性，引起睾丸功能恢复造成，因为异性同笼时间是35天，而中华地鼠生殖周期是18-21天，所以真实怀孕数应该是从中孕到出生数相加。
3、组织化学染色：H-E染色法显示(如图17)：与生理盐水组和单纯环磷酰胺对照组比较，2F二聚体肽(图17中的GHRH是指2F二聚体肽)或者这类GHRH肽(如2D、2E二聚体肽)具有明显刺激精卵细胞增殖和成熟作用，从而促进生殖，因此能够用于不孕不育药物。
⑴雄性模型鼠：图17显示，2F二聚体肽组的曲细精管中的精母细胞、精原细胞明显增生。曲细精管细胞排列整齐，体积变粗，曲细精管腔变小，甚至消失，具有明显剂量依赖性。表9量取70个横切面曲细精管面积(短*长轴)比较发现，2F组增加曲细精管的作用具有明显统计意义。
表9：雄性模型鼠曲细精管大小统计(X±SD，n＝70)
雄性模型鼠 曲细精管面积(μm2) P*
单纯环磷酰胺组 309868±125964 ---
高2F组 328238±125110 <0.05
中2F组 106845±37978 <0.001
低2F组 709288±2323533 <0.001
HMG组 446250±171099 <0.001
生理盐水组 183891±112497 <0.001
*P<0.05or 0.001显示差异性，与单纯环磷酰胺组比较，t检验。
⑵雌性模型鼠：图18显示，与单纯CTX组和阳性对照组HMG组比较，2F二聚体肽组的成熟卵 细胞明显增多，具有明显剂量依赖性。表10统计了各个时期的卵泡，比较发现，2F组增加卵细胞成熟作用具有明显统计意义，对卵泡具有明显促成熟作用，是成熟卵泡明显增加，促进受孕，增加怀孕(见表10)。
表10：雌性模型组卵泡统计(个)
实验组 初级卵泡 次级卵泡 成熟卵泡
单纯环磷酰胺组 45 6 10
高2F组 18 0 31**
中2F组 31 13 21**
低2F组 24 5 16*
HMG组 34 21 22**
生理盐水组 5 6 28
*P<0.05或**P<0.001，与单纯CTX组比较，t检验。
③GHRH受体：利用FITC-hGHRH(1-44)NH2肽，荧光染色睾丸或卵巢切片显示(图19和20)：在睾丸曲细精管中精原细胞、母细胞和精子细胞都有明显的GHRH受体分布，精原母细胞表达无差异，但精子细胞或成熟精子的头部表达明显增多。
在卵巢组织中，初级、次级和成熟卵泡中有GHRH受体阳性染色。
这些结果显示：新型GHRH激素肽通过GHRH受体发挥作用，促进精卵原细胞的增殖，促进受孕。
讨论：相对于标准品hGHRH(1-44)NH2，所有的hGHRH二聚体(2D，2E，2F，2Y)都表现出较好的rGH-释放活性和类似的垂体激素释放特异性。2D，2E，2F的GH峰值是在第三阶段(I3)，而S和2Y的是在第四(I4)和第五(I5)阶段，这表明2D，2E，2F起效快速，S和2Y作用慢慢，这是由于它们不同的N-末端结构-1Pro-GHTH或1Tyr-GHRH所造成。相比P2阶段的GH水平，2F肽连续引起了GH在整个肽孵育期间(I3-I5)的增加，而且GH-增量总值(79.77ng/ml，与S2比较)是2D(15.84ng/ml，与S2比较，第二最强肽)的5.04倍。这意味着2F肽对于延长半衰期具有最强和最长效的作用，这不仅是由于1Pro-GHTH比1Tyr-GHRH更能增强刺激，也是由为GH的释放需在N-末端有环形氨基酸诱导。
在我们初步的实验中，GHRH单体(D，E，F，Y)的活性仅是hGHRH(1-44)OH活性的48.8-89.7％，或者说它们仅是hGHRH(1-44)NH2活性的41.9-77.1％(数据略)。对于二聚体，2D，2E，2F的活性是hGHRH(1-44)NH2的102％，98％和110％。GHRH二聚体表现出比相应的GHRH单体高至少40-87％的GH释放活性。相比单一N末端的hGHRH(1-44)NH2，带有两个N末端的二聚体分子都表现出了更好的GH释放效果。已知GHRH分子的N末端在GHRH受体对GH释放的相互作用中起着关键作用，因此具有相同的C-末端时，具有多个N-末端的分子会具有更强的行动能力。
虽然实验表明，hGHRH二聚体作为标准品hGHRH(1-44)NH2肽具有良好的功能选择性和种属特异性，其中二聚体显示了rACTH和/或rLH,rPRL增强活性，这显示这些GHRH肽可轻微调节其他垂体激素的释放。
GHIH对GH释放的抑制作用呈剂量/时间依赖性。在1.927μM GHRH二聚体肽存在时，与0.482μM GHIH相比，1.927μM GHIH显著抑制生长激素的分泌，而且抑制效果随孵育时间的增加而增强。
S，2D，2E，2F和2Y肽对SD大鼠垂体的结合是特异性的。从Scatchard图中的B/F和Bmax的关系，这些GHRH二聚体的最大亲和力(Bmax值)排序(2F>2D>2Y>2E)或解离常数(KD)的排序(2F<2D<2E<2Y)，都显示2F肽与垂体匀浆表达了最强的结合能力，与活性结果基本一致。FITC-标记的GHRH二聚体的荧光染色呈细胞膜分布，荧光染色强度排序(2F>2D>2E>2Y>S)也显示2F肽在垂体细胞具有最丰富的分布，这表明2F肽具有与垂体细胞膜的最大亲和力。
事实表明，不仅带有N或/和C-端改构的hGHRH类似物能调节其生长激素释放，其同源二聚体也可以通过加强与垂体细胞受体的相互作用强力提高大鼠生长激素的释放。
虽然hGHRH(1–44)NH2和hGHRH(1–29)NH2在药学研究中有用性已被证实，但对于更稳定、降低剂量和给药频率的类似物仍不断需要探索。本文中的2F肽[1P-hGHRH(2-44)-GGC-CGG-hGHRH(44-2)-1P]或许是对未来临床实验的一个希望。
Figure PCTCN2015093814-appb-000012
Figure PCTCN2015093814-appb-000013
Figure PCTCN2015093814-appb-000014
Figure PCTCN2015093814-appb-000015

Claims (3)

  1. 一类新型生长激素释放激素类似肽，其特征在于，所述的新型生长激素释放激素类似肽为2D、2E、2F或2Y二聚体肽：
    所述的2D二聚体肽的氨基酸序列为：
    (H)PPYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC(OH)-(OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRYSNTFIADAY PP(H)；
    所述的2E二聚体肽的氨基酸序列为：
    (H)PYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC(OH)-(OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRYSNTFIADAYP(H)
    所述的2F二聚体肽的氨基酸序列为：
    (H)PADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC(OH)-(OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRYSNTFIADAP(H)；
    所述的2Y二聚体肽的氨基酸序列为：
    (H)YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC(OH)-(OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRYSNTFIADAY(H)。
  2. 权利要求1所述的新型生长激素释放激素类似肽2D、2E、2F或2Y二聚体肽在制备治疗不孕不育药物中的应用。
  3. 一种治疗不孕不育药物，其特征在于，以权利要求1中所述的新型生长激素释放激素类似肽2D、2E、2F或2Y二聚体肽作为活性成份。
PCT/CN2015/093814 2014-11-04 2015-11-04 一类新型生长激素释放激素类似肽及其在制备治疗不孕不育药物中的应用 WO2016070815A1 (zh)

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