AU2015342341B2 - Novel Growth Hormone-Releasing Hormone Analogs and Application Thereof in Preparation of Drugs for Treating Infertility - Google Patents

Abstract

Disclosed in the present invention is a class of new type growth hormone releasing hormone-like peptide. The growth hormone releasing hormone-like peptide is in the form of homodimer and has a relatively high releasing activity of pituitary GH and specificity of pituitary hormone release, which can be used to prepare a drug for treating infertility.

Description

Field of the Invention

The present invention relates to the field of biomedicine, and exactly to four novel growth hormone-releasing hormone homodimers and an application thereof in the preparation of drugs for treating infertility.

Background of the Invention

Human growth hormone-releasing hormone (hGHRH), secreted by hypothalamus, is a neuroendocrine peptide that stimulates human growth hormone (hGH) production and release in the pituitary. The maturation of preproprotein hGHRH( 1-107/8) precursor comprises a series of proteolytic processing steps, and the final mature forms include hGHRH(l-44)NH2, hGHRH(l-37)OH, and hGHRH(l-40)OH. Within these proteins, the shortest sequence that shows 51% biological activity is hGHRH(l-29), which is called “core peptide”. The hGHRH from human hypothalamus shows homological identity of 93%, 93%, 89%, 89%, 86%, 73%, 71%, or 61% with those of pig, Chinese hamster, cattle, golden hamster, goat, guinea pig, Rattus norvegicus, or Mus musculus, respectively.

Ling N et al reported that, D-¹Tyr-hGHRH(l-40)OH, ‘Phe-hGHRHCMOjOH, ‘Trp-hGHRHCl-dOjOH, ¹His-hGHRH(l-40)OH, ‘Ala-hGHRHQ-dOjOH, ‘Tyr-hGHRHCMOjOH, ⁹Arg-hGHRH(l-40)OH, and ⁹Ala-hGHRH(l-40)OH had activities of 0.022, 0.038, 0.003, 0.351, 0.010, 0.032, 0.002, and 0.007 compared with that of hGHRH(l-40)OH. (3-Me)¹His-hGHRH(l-44)NH2 and (O-Me)¹Tyr-hGHRH(l-44)NH2 had activities of 0.132 and 0.001 compared with hGHRH(l-44)NH₂. Piquet G et al and Esposito P et al coupled the C-terminus of GHRH analog with polyethylene glycol (PEG) to obtain GHRH-PEG molecule with relative high stability. The GHRH analog with C-terminal amidation had double activities. Izdebski J et al synthesized JI-34, JI-36, and JI-38 GHRH agonists (JI series) by modifying hGHRH(l-29)NH₂ at the positions 29 (agmatine, Agm), 1 (desaminotyrosine, Dat), 27 (norleucine, Nle), and 15 (L-alpha-aminobutyric acid, Abu) of the backbone. Hassan HA et al modified hGHRH at the position of Tyr and/or Met, and the activities of ¹His,²⁷Nle-hGHRH(l-32)NH2, ¹His,mono-I-¹⁰Tyr,²⁷Nle-hGHRH(l-32)NH2, ²⁷Nle-hGHRH(l-29)NH2, D-¹Tyr-hGHRH(l-32)NH2, and des-¹Tyr-hGHRH(2-32)NH₂ were 3.2, 4.9, 1.4, 0.74, and 0.02 times higher than that of hGHRH(l-40)OH, respectively. Garcia JM et al reported that, Macimorelin (formerly known as AEZS-130, ARD-07, or EP-01572) was a novel GH secretagogue with good stability and oral bioavailability, but there was far less potent than hGHRH(l-29)NH₂ in stimulating hGH release. Cai R et al reported the incorporation of N-Me-Tyr into N-terminus, or methyl/ethyl-amide or Apa30-/Gab30-NH₂ into C-terminus of JI series, and the resulting compounds, N-Me-¹ Tyr-JI-3 8, N-Me-¹ Tyr,D-² Ala-JI-3 8, N-Me-¹ Tyr,D-² Ala,⁸ Asn-JI-3 8,

N-Me-¹ Tyr,D-² Ala,²⁹ Arg-NHCH3-JI-3 8, N-Me-¹ Tyr,²⁹ Arg-NHCH3-JI-3 8,

N-Me-¹ Tyr,D-²Ala,⁸Asn,²⁹Arg-NHCH3-JI-3 8, N-Me-¹ Tyr,D-²Ala,⁸Thr,²⁹Arg-NHCH3-JI-3 8, N-Me-¹Tyr,D-²Ala,²⁹Arg,³⁰Apa-NH₂-JI-38, and

D-²Ala,5F-⁶Phe,²⁸Ser,²⁹Arg,³⁰Gab-NH₂-JI-38, had activities 1-7.01 times higher than that of hGHRH(l-29)OH.

hGHRH(l-44)NH₂ shows the highest activity among all GHRH agonists reported so far.

Recent research discovered that, GHRH peptides had broad spectrum extrapituitary activities, for example, in promotion of wound healing, protection cardiomyocyte with apoptosis, improvement of sleep quality, reduction of obesity in diabetes or AIDS, and improvement of neurocognitive function.

Though hGHRH analogs have tremendous potentials in biomedical field, their application has been limited by their short half-life periods and low activities. We previously discovered that, the N-terminal 'Tyr-*Pro replacement and/or C-terminal GGC extension of hGHRH(l-44)OH effectively regulated the activities of the hormone. Recently, we have discovered that, the hGHRH(l-44) homodimers, with N-terminal Pro-'Pro- or 'Pro-extension/replacement and C-terminal GGC extension, surprisingly show higher GH-releasing activities than that of hGHRH(l-44)NH₂. The synthesis, biological evaluations and potencies of four novel hGHRH homodimers with high activity are reported in the present description.

Summary of the Invention

The first objective of the present invention is to provide a class of novel growth hormone-releasing hormone analogs.

The novel growth hormone-releasing hormone analogs are described to be a homodimeric peptide 2D, 2E, 2F, or 2Y.

The amino acid sequence of said dimeric peptide 2D is:

(H)PPYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC(OH)-(

OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRYSNTFIADAYPP(H).

The amino acid sequence of said dimeric peptide 2E is:

(H)PYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC(OH)-(

OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRYSNTFIADAYP(H).

The amino acid sequence of said dimeric peptide 2F is:

(H)PADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC(OH)-(O

H)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRYSNTFIADAP(H).

The amino acid sequence of said dimeric peptide 2Y is:

(H)YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC(OH)-(O

H)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRYSNTFIADAY(H).

In the above amino acid sequences of the dimeric peptides 2D, 2E, 2F, and 2Y, the capital letters are abbreviations for amino acids, which is common general knowledge. Furthermore, the “H” represents the amino group at N-terminus and the “OH” represents the carboxyl group at C-terminus, which is common general knowledge as well.

Our experiments discovered that, the above dimeric peptide 2D, 2E, 2F, or 2Y, was produced through the in vitro oxidation of monomer D, E, F, or Y. The dimeric peptides were discovered to have significant pituitary GH-releasing activities and pituitary hormone-releasing specificities. Through pituitary GHRH receptor-binding assays and fluorescent staining analysis of pituitary cells, the dimeric peptide 2F was found to have the highest pituitary receptor binding activity.

By treating male infertility models with the dimeric peptide 2F as a representative of the four GHRH homodimers, the inventors discovered that, compared with the saline group and the single cyclophosphamide group, the tubular cells in the 2F groups arranged normally. There were significant increases in spermatogonia, spermatocytes, spermoblasts, and sperms in the tubules, and the seminiferous tubules become bigger in size but their lumens became smaller or even disappeared. The 2F potency showed the dose-dependency.

By treating female infertility models with the dimeric peptide 2F as a representative of the four GHRH homodimers, the inventors discovered that, compared with the single cyclophosphamide group, the statistic results of pregnancy rates showed the significant higher birth and pregnancy rates in the 2F groups; in comparison with the positive gonadotropin HMG group, more hamsters in the 2F groups gave birth, whereas there were more mid- or late-pregnant hamsters in the HMG group. The statistic results of ovarian follicular numbers showed that, the hamsters of the 2F groups had more mature ovarian follicles than those of other groups, while the HMG group showed a significant increase in primary and secondary ovarian follicles. The results of H-E staining showed that, compared with the saline group, the single cyclophosphamide group presented the irregular arrangement of cells, abnormal glandular structures, inhomogeneous staining, and pyknosis. After treated with peptide 2F, the groups presented the regular arrangement of cells, normal glandular structures, and homogeneous staining, indicating that the peptide had significant effect in protecting the ovary. A large amount of mature ovarian follicles in the 2F groups were observed in the cortical area of ovary, indicating a significant promotion in ovarian follicles maturation on the dose-dependent manner. Through the fluorescent staining of FITC-hGHRH(l-44)NH2 peptide, significant yellow fluorescent labeled cells were observed in the primary and secondary ovarian follicles and the ovarian tissues, indicating the distribution of GHRH receptors. The results demonstrated that the novel GHRH peptides promote the ovum proliferation and maturation, and finally promote pregnancy by upregulating GHRH receptors.

These results demonstrated that, taking the dimeric peptide 2F as a representative of the four GHRH homodimers, the GHRH dimeric peptides such as 2D, 2E, and 2Y, had significant activities in stimulating the proliferation and maturation of sperm/ovum, which can promote fertility, and thus can be used in the drugs for treating infertility.

Therefore, the second objective of the present invention is to provide an application of the dimeric peptide 2D, 2E, 2F, or 2Y in the preparation of drugs for treating male and female infertility.

The third objective of the present invention is to provide a drug for treating infertility, comprising the novel growth hormone-releasing hormone analog, dimeric peptide 2D, 2E, 2F, or 2Y, as an active ingredient.

Said dimeric peptides 2D, 2E, 2F, and 2Y are abbreviated as peptides 2D, 2E, 2F, and 2Y, respectively.

The peptides 2D, 2E, 2F, and 2Y of the present invention can be used in treating male and female infertility, which makes them drug candidates for treating infertility.

Brief Description of the Figures

FIG. 1 shows the HP EC purity analysis and MS molecular weight determination of peptide

SI.

FIG. 2 shows the HPFC purity analysis and MS molecular weight determination of the peptide S2.

FIG. 3 shows the HPFC purity analysis and MS molecular weight determination of the peptide A.

FIG. 4 shows the HPFC purity analysis and MS molecular weight determination of the peptide B.

FIG. 5 shows the HPFC purity analysis and MS molecular weight determination of the peptide C.

FIG. 6 shows the HPFC purity analysis and MS molecular weight determination of the peptide D.

FIG. 7 shows the HPFC purity analysis and MS molecular weight determination of the peptide E.

FIG. 8 shows the HPFC purity analysis and MS molecular weight determination of the peptide F.

FIG. 9 shows the SDS-PAGE analyses of the dimeric peptides 2D, 2E, 2F, and 2Y. Marker: low molecular weight standard proteins 4.6-66 KDa. The lanes 2D, 2E, 2F, and 2Y represent the dimeric peptides 2D, 2E, 2F, and 2Y, respectively, and the lanes D, E, and F represent the monomeric peptides D, E, and F respectively.

FIG. 10 shows the activities of the GHRH analogs and dimeric peptides in stimulating rat growth hormone release.

FIG. 11 shows the activities of the GHRH analogs and dimeric peptides in stimulating rat ACTH hormone release in vitro.

FIG. 12 shows the activities of the GHRH analogs and dimeric peptides in stimulating rat PRL hormone release in vitro.

FIG. 13 shows the activities of the GHRH analogs and dimeric peptides in stimulating rat LH hormone release in vitro.

FIG. 14 shows the growth hormone-releasing inhibition test of the dimeric peptides. During the incubations I3-I5, 0.482 or 1.927 μΜ of GHIH (growth hormone inhibiting hormone) was added into 1.927 μΜ of the GHRH analog. The group P2 without any peptide was taken as a blank control while that with the peptide S as a standard. The statistical significance (*P<0.05 or **P<0.01) was determined by comparing the data of a specific GHRH dimer in different doses obtained during the incubations /3-/5.

FIG. 15 shows the Scatchard plots for the binding of the GHRH dimeric peptide with pituitary homogenate.

FIG. 16 shows the fluorescent staining analysis of GHRH receptor in rat pituitary tissue (Magnification 100x10 times). FITC: FITC-labeled peptides S, 2D, 2E, 2F, and 2Y. DAPI: nucleus staining. Merged: FITC merged with DAPI. Yellow staining indicates positive cells.

FIG. 17 shows the H-E staining of testicular tissue of male model in the 2F groups (Magnification 4x10 times).

FIG. 18 shows the H-E staining of ovarian tissue section of female model in the 2F groups (Magnification 4x10 times).

FIG. 19 shows the fluorescent staining of testicular tissue with the peptide FITC-hGHRH(l-44)NH2 (Magnification 10^χ10 times). Yellow color indicates positive staining in the FITC-hGHRH(l-44)NH2 and blue color is the DAPI nucleus staining.

FIG. 20 shows the fluorescent staining of ovarian tissue with the peptide FITC-hGHRH(l-44)NH2 (Magnification 4x10 times). Yellow color indicates positive staining in the FITC-hGHRH( 1-44)18¾ and blue color is the DAPI nucleus staining.

Detailed Description of the Embodiments

The following embodiments are used for further describing this invention rather than limiting the invention.

Embodiment 1: Synthesis of the novel growth hormone-releasing hormone homodimers.

1. Syntheses of monomeric peptides. The monomeric peptides were synthesized by solid phase polypeptide synthesis (SYMPHONY, 12-channel polypeptide synthesizer, Software Version 201, Protein Technologies Inc.). The following steps contained.

(1) Swelling the resin. Wang resin (purchased from Tianjin Nankai Synthetic Co., Ltd) was added in a reactor. Dichloromethane (DCM, Dikma Technologies Inc.) was added to the Wang resin (15ml of DCM per gram of resin), and then shaken for 30 min.

(2) Incorporation of the first amino acid. The solvent was removed by suction filtration with a sand core funnel. The residual was added with 3 mmol of Fmoc-amino acid (Fmoc-AA) which was the first one from the C-terminus (all Fmoc-amino acids were provided by Suzhou Tianma Medicine Group Fine Chemicals Co., Ltd), and then 10 mmol of 4-(dimethylamino) pyridine (DMAP) and Ν,Ν’-dicyclohexylcarbodiimide (DCC) were added. They were then solved in dimethylformamide (DMF, purchased from Dikma Technologies Inc.) and shaken for 30 min, followed by capping with acetic anhydride.

(3) Deprotection. After DMF was removed, the residual was added in 5ml of 20% piperidine-DMF solution (15ml per gram of resin) and kept for 5 min, and then the solvent was removed by filtration. Then the residual was added in 15 ml of 20% piperidine-DMF solution (15 ml per gram of resin) and kept for 15 min. The piperidine was provided by Shanghai Chemical Reagent Inc.

(4) Test. During the synthesis, the test was performed randomly to monitor the process. After the solvent was removed, a dozen of the resin particles were collected, and washed in ethanol for three times. One drop of ninhydrin, KCN, and phenol were added respectively in the particles, and then maintained at 105-110°C for 5 min. Dark bluish color indicated positive reaction.

(5) Washing the resin. The resin was washed twice in DMF (10 ml per gram of resin), twice in methanol (10 ml per gram of resin), and twice in DMF (10 ml per gram of resin).

(6) Condensation. 3 mmol of Fmoc-AA (from the second amino acid at the C-terminus to the N-terminal amino acid) and 3 mmol of 2-( 1 H-benzotriazol-1 -yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, Suzhou Tianma Medicine Group Fine Chemicals Co., Ltd) were solved in DMF of an volume as little as possible, and then added into the reactor. And tenfold of N-methylmorpholin (NMM, Suzhou Tianma Medicine Group Fine Chemicals Co., Ltd) was added into the reactor immediately and allowed to react for 30 min. The test was performed and the result was negative.

(7) Washing the resin. The resin was washed once in DMF (10 ml per gram of resin), twice in methanol (10 ml per gram of resin), and twice in DMF (10 ml per gram of resin).

(8) The steps (2)-(7) were repeated to incorporate all the amino acids in the order from the right terminus to the left terminus according to the amino acid sequences as shown in Tables 1 and 2.

(9) After the FMOC group of the last FMOC-'Pro or 'Tyr was removed, the test was performed and the result was positive. Then the solvent was removed, and the residual was reserved.

(10) The resin was washed twice in DMF (10 ml per gram of resin), twice in methanol (10 ml per gram of resin), twice in DMF (10 ml per gram of resin), and twice in DCM (10 ml per gram of resin). The resin was washed for 10 min each time and then the solvent was removed.

(11) Cleaving the polypeptides from the resin. Preparation of the cleavage solution (10 ml per gram of resin): 94.5% of TFA (J.T.Baker Chemical Company), 2.5% of water, 2.5% of ethanedithiol (EDT, Sigma-Aldrich Chemistry), and 1% of triisopropylsilane (TIS, Sigma-Aldrich Chemistry). Cleavage time: 120 min.

(12) Drying and Washing. The resulting mixture was dried by purging it with nitrogen gas to remove the cleavage solution as much as possible, washed six times in diethyl ether, and then dried by evaporation at room temperature.

(13) The purifications of polypeptides in HPLC. The crude peptide was solved in pure water or a small volume of acetonitrile, and purified under the following condition:

Equipments: High performance liquid chromatograph (software: Class-VP. Sevial

System; manufacturer: SHIMADZU, Japan) and Venusi MRC-ODS C18 column (30^x250mm, Bonna-Agela Technologies, Tianjin). Mobile phase A: 0.1% trifluoroacetic acid-water solution. Mobile phase B: 80% acetonitrile solved in the Mobile Phase A (acetonitrile was purchased from Fisher Scientific). Flow rate: 1.0 ml/min. Sample volume: 30 μΐ. Detection wavelength: 220 nm. Elution program: 0-5 gradient min: 90% A/10% B; 5-30min gradient: 90% A/ 10% B—>20% A/80% B.

(14) The purified solution was lyophilized to powder (Freeze dryer, Freezone Plus 6, FABCONCO), and thereby a product was obtained.

(15) Identification. A small amount of the product was subjected to molecular weight determination by ESI MS and purity identification by C18-HPFC.

(16) The polypeptide in powder form was stored in sealed package at 20°C in the dark.

2. Formations of dimeric peptides. The monomeric peptide of the peptide D, E, F, or Y as shown in Table 1, or, the peptide D^L, E^L, F^L, or Y^L as shown in Table 2 was incubated in ammonia solution (pH=11.5) at a concentration of 0.5 mg/ml at 37°C for 60 hours, and then 100% of the dimeric peptide 2D, 2E, 2F, 2Y, 2D^L, 2E^L, 2F^L, or 2Y^L was formed. The SDS-PAGE analysis of the peptides was as shown in FIG. 9. The amino acid sequences of the peptides were as shown in Tables 1 and 2.

3. Types of the peptides. As shown in Tables 1 and 2 were the amino acid sequences of eleven monomeric GHRH analogs (A-F, Y, D^L, E^L, F^L, and Y^L), three hGHRH standard peptides (SI, S2, and S2^L), and eight dimeric peptides (2D, 2E, 2F, 2Y, 2D^L, 2E^L, 2F^L, and 2Y^L), which were synthesized according to the above protocol. The amino acid sequences of the peptides SI, S2, A-F, and Y, were shown in SEQ ID NO. 1-9. Their purities were determined to be more than 95% in HPFC (as shown in FIG. 1-8 were the HPFC purity analysis and MS molecular weight determination of the peptides A-F, SI, and S2). The FITC-labeled peptides thereof were as shown in Table 2. The peptides SI and S2 served as the positive controls. The Shanghai Qiangyao Biotechnology Co, Ftd was authorized to synthesize all the GHRH analogs and hGHRH peptides as shown in Tables 1 and 2.

Table 1 Amino acid sequences of the GHRH analogs, hGHRH peptides and dimeric peptides

Peptide	Sequence
SI	hGHRH(l-40)	YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGA
S2	hGHRH(l-44)-NH2	yadaiftnsyrkvlgqlsarkllqdimsrqqgesnqergararl-nh2
A	Pro-Pro-hGHRH(l-44)	PPYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL
B	Pro-hGHRH(l-44)	PYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL
C	‘Pro-hGHRH(2-44)	PADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL
D	Pro-Pro-hGHRH(l-44)-G	PPYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLG
GC	GC
E	Pro-hGHRH(l-44)-GGC	PYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGG C
F	‘Pro-hGHRH(2-44)-GGC	PADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC
Y	hGHRH(l-44)-GGC	YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC
	Pro-Pro-hGHRH(l-44)-G	(H)PPYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARAR
2D	GC-CGG-hGHRH(44-1)-	LGGC(OH)-(OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVK
	Pro-Pro	RYSNTFIADAYPP-(H)
2E	Pro-hGHRH(l-44)-GGC- CGG-hGHRH(44-l)-Pro	(H)PYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL GGC(OH)-(OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKR YSNTFIADAYP(H)
	‘Pro-hGHRH(2-44)-GGC	(H)PADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLG
2F	-	GC(OH)-(OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRY
	CGG-hGHRH(44-2)- ‘Pro	SNTFIADAP(H)
2Y	hGHRH(l-44)-GGC- CGG-hGHRH(44-l)	(H)YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLG GC(OH)-(OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRY SNTFIADAY(H)

Table 2 Amino acid sequences of the FITC-labeled GHRH analogs, hGHRH peptides and dimeric peptides

Peptide	Sequence
S2 l	YADAIFTNSYRKVLGQLSARK-(FITC)LLQDIMSRQQGESNQERGARARL-NH2
dl	PPYADAIFTNSYRKVLGQLSARK-(FITC)LLQDIMSRQQGESNQERGARARLGGC
el	PYADAIFTNSYRKVLGQLSARK-(FITC)LLQDIMSRQQGESNQERGARARLGGC
fl	PADAIFTNSYRKVLGQLSARK-(FITC)LLQDIMSRQQGESNQERGARARLGGC
2Dl	(H)PPYADAIFTNSYRKVLGQLSARK-(FITC)LLQDIMSRQQGESNQERGARARLGGC(OH)-(O H)CGGLRARAGREQNSEGQQRSMIDQLL(FITC)KRASLQGLVKRYSNTFIADAYPP(H)
2El	(H)PYADAIFTNSYRKVLGQLSARK-(FITC)LLQDIMSRQQGESNQERGARARLGGC(OH)-(OH) CGGLRARAGREQNSEGQQRSMIDQLL(FITC)-KRASLQGLVKRYSNTFIADAYP(H)
2Fl	(H)PADAIFTNSYRKVLGQLSARK-(FITC)LLQDIMSRQQGESNQERGARARLGGC(OH)-(OH)C GGLRARAGREQNSEGQQRSMIDQLL(FITC)-KRASLQGLVKRYSNTFIADAP(H)
2Yl	(H)YADAIFTNSYRKVLGQLSARK-(FITC)LLQDIMSRQQGESNQERGARARLGGC(OH)-(OH) CGGLRARAGREQNSEGQQRSMIDQLL(FITC)-KRASLQGLVKRYSNTFIADAY(H)

Embodiment 2: In vitro hormone-releasing activity

1. Method of in vitro pituitary stimulation. The female S-D rats (about 8-week old, body weight 200±20g) were purchased from Faboratory Animal Centre of Guangzhou University of Chinese Medicine. All experiments were performed in accordance with the Institutional Guidelines for the Care and Use of Faboratory Animals. The rats were housed on a 12:12-hr of light: dark cycle at 26±1°C. The rats were sacrificed in cervical dislocation, and their pituitaries were harvested in 30 minutes. After rinsing with sterilized lactated Ringer’s buffer (FRB), the pituitaries were placed in 1 ml of FRB in lx 10 cm glass tubes immediately, and maintained at 37°C. All glass tubes were maintained at 37°C for 5 hours (the 5-h period was divided into 5 intervals, 1 hour each, which were represented by P\, P2, /3, A, and /5) with a gentle shake every 5 minutes. Buffer samples were collected each one hour and assayed for pituitary hormones GH,

ACTH, PRL, and FH. Then 1 ml of fresh FRB, with or without the active GHRH analogs, hGHRH peptides, or dimeric peptides, was added to the pituitary. After the initial 2 hours (Pi and P₂) of preincubation, the active polypeptides were added during the subsequent 3 hours of incubation (/3, /4, and I5). The net GH release was calculated as (Ρ+Ρ+Ιρ-Ρτ. The samples of P₂ without the peptides served as the blank control. The samples of SI and S2 served as the positive controls. The rat pituitary hormones were assayed using ELISA kits for Rat Growth Hormone (Millipore Co., USA), and for ACTH, LH, and PRL (Shanghai Yanhui Co.).

2. The results of the rat growth hormone-releasing activity assay were as shown in FIG. 10 and Table 3. In the 5-hour pituitary incubation, once the LRB buffer was added with the peptide S, 2D, 2E, 2F, or 2Y, the pituitary GH level increased significantly. The incubation I3 in which the peptide 2F was added, and the incubation I₅ in which the peptide 2Y was added, presented higher GH release than the incubation I3 or I5 in which the peptide S was added (P<0.01, FIG. 10). The peak values of GH release were observed in the third incubation (/3) (2D, 2E, or 2F), the fourth incubation (/4) (S), or the fifth incubation (/₅) (2Y). Interestingly, the GH release increased during the whole incubation period (/3-/5) when the peptide 2F was added, suggesting that the peptide 2F has the highest rGH-releasing activity. For the peptide 2Y, the GH release of the incubation /3, /4, or /5 was not higher than that of P₂, indicating a relatively lower activity of the peptide 2Y. Compared with the P₂ value, the net GH release values of the peptides showed statistical difference (P<0.01). The activities of the peptides 2D, 2E, 2F, and 2Y were 102±9.2%, 95±22.2%, 110±18.2%, and 108±15.5% of that of hGHRH(l-44)NH₂. The activities of the GHRH dimeric peptides were at least 40-87% higher than that of the corresponding monomer, wherein the peptide 2F had the highest activity. According to the net GH release of the peptide S2, the GH increase of the peptide 2F (79.77ng/ml) was 5.04 times of that of the peptide 2D (the second highest, 15.84ng/ml).

Table 3: In vitro pituitary GH release stimulated by the GHRH analogs, hGHRH peptides and dimeric peptides

GHRH Peptide	Dose (μΜ)	GH (Mean±SE, ng/mL buffer)
Total GH (I3+I4+I5)	Blank (A)b	Net GH (I3+I4+Is-P2)	Pvs.-b
SI	1.92	1095.891±163.86	396.696±15.28	699.156±148.43	P<0.05
S2	1.92	1221.638±9.87	407.765±17.38	813.872±31.54	RcO.OOOl
A	1.92	767.243±78.85	346.447±31.43	420.796±69.93	P>0.05
B	1.92	695.477±74.95	340.560±24.07	354.917±46.87	P>0.05
C	1.92	894.316±103.47	339.979±22.72	554.337±81.45	P<0.05
D	1.92	640.916±69.16	300.054±31.36	340.862±35.44	P>0.05
E	1.92	788.056±70.25	308.263±51.96	479.793±154.26	P>0.05
F	1.92	962.93U168.15	335.558±86.59	627.264±70.96	P<0.05
2D	1.92	1244.689±46.54	414.979±25.87	829.710±20.67	RcO.OOOl
2E	1.92	1201.426±115.18	408.636±39.48	792.790±75.69	P<0.01
2F	1.92	1303.188±109.77	409.548±42.03	893.640 U67.74	RcO.OOl
2Y	1.92	1350.19±32.42	476.404±23.88	873.787±55.11	P<0.01

^bP, Net GH vs P2 value.

3. The results of in vitro activity test in stimulating the release of rat pituitary hormones

ACTH, PLR, and LH. As shown in FIGs. 11, 12, and 13, compared with the values of P2, though the peptide S, 2D, 2E, 2F, or 2Y had certain stimulation effect on the release of hormone ACTH, PRF, or FH, there was no statistical difference between the incubations P2 and /3-/5. The results indicated that, just like the standard peptide hGHRH(l-44)NH2, the GHRH dimeric peptides might slightly regulate the release of pituitary ACTH, prolactin, or FH.

Embodiment 3: Analysis of rat pituitary growth hormone-releasing inhibition

The method of growth hormone-releasing inhibition was established according to the method of pituitary GH-releasing stimulation (Pi, P2, /3, /4, and /5). During the incubations /3-/5,

0.482 or 1.927 μΜ of growth hormone-inhibiting hormone (GHIH) was added into the tubes containing 1.927 μΜ of the GHRH dimer. The samples of P₂ without the peptides served as the blank control. The samples of S2 served as the standard control. The rat pituitary GH level was assayed using the Rat Growth Hormone ELISA kit.

The 5-hour assay showed that, during the incubations /3-/5, in the presence of 1.927 μΜ of GHRH peptide, the addition of 0.482 or 1.927 μΜ of GHIH presented a significant inhibition, which was in positive correlation with the GHIH dose and incubation time (FIG. 14). During the incubations /3-/5, 1.927 μΜ of GHIH showed significantly higher inhibition than 0.482 μΜ of GHIH (F<0.05 or 0.01). The extension of incubation time could enhance the inhibiting effect, but there was no statistical difference in inhibition effect between the hGHRH dimeric peptides.

Embodiment 4: In vitro binding reaction between the GHRH dimeric peptides and pituitary GHRH receptor

Thirty pituitaries, harvested from female S-D rats, were homogenized in 10 ml of cold buffer (50 mM HEPES, 7 mM MgCl₂, 5 mM EDTA, 50 pg/mL PMSF, 2 mg/mL BSA, pH 7.4) on ice for 5 min and then followed by a centrifugation (12000 rpm) at 4°C for 2 min. The resulted pellet was collected and re-suspended in 10.8 ml of the cold homogenization buffer. In the binding assay, each plate well contained 100 μΐ of pituitary' homogenate (153 pg protein), 140 μΐ of the homogenization buffer, one of 5 concentrations (30, 60, 120, 240, 480 fluorescent quantity) of FITC-labeled hGHRH peptide (0.0186 μΜ) and the corresponding non-labeled hGHRH peptide (0.93 μΜ, 50-fold more than the FITC-labeled hGHRH peptide) with total volume of 300 μΐ. Each treatment had 2 replicates. The assay plates were incubated on a slow shaker at 30°C for 1 h and followed by a centrifugation of 6000 rpm at 4°C for 20 min. After removing supernatant, pellets were washed once with 200 μΐ of HEPES buffer and then fresh HEPES buffer (200 μΐ) was added to each well for fluorescent reading at wavelength of 490/525 nm using fluorescent detector. The results were shown in Table 4 and FIG. 15. The binding of the peptide S, 2D, 2E, 2F, or 2Y with rat pituitary homogenate was specific and a relative high specific binding was observed (total binding of over 50%). The Scatchard plots (P/F-Pmax) suggested that, the Rmax Value of dimeric peptide was ranked as 2F>2D>2Y>2E, and the Kd value was done as 2F<2D<2E<2Y, indicating the peptide 2F had the highest binding capacity with pituitary homogenate.

Table 4: Binding test of the hGHRH dimeric peptides with pituitary homogenate receptor (X±S, n=2)

hGHRH peptide	Kd (pmoL/L)	Rmax (pmoL/mg protein)
2D	1.020±0.13	2.60±0.31
2E	1.264±0.16	1.62±0.25
2F	0.607±0.92a	2.68±0.36b
2Y	1.630±0.20	1.79±0.44

^aP<0.05, compared with the Kd value of 2D or 2E; ^bP<0.05, compared with the Rmax value of 2E or 2Y.

Embodiment 5: Fluorescent staining analysis of rat pituitary tissue

5pm-thick formalin-fixed sections of SD rat pituitary tissue were stained with the following steps. The sections were dewaxed by regular method. Newly dewaxed sections were soaked three times in a HEPES buffer for 5 minutes. 200 μΐ of HEPES buffer containing 75.99±0.97 fluorescent intensity of FITC-GHRH dimer (S^L, 2D^L, 2E^L, 2F^L, or 2Y^L) was dropped onto each tissue section, and then the sections were incubated at 37°C for 1 hour. After the sections were washed twice for 5 minutes in HEPES buffer, DAPI-water solution (0.01 g/ml) was dropped onto the tissue section, and then incubated at 37°C for 15 minutes. The sections were washed twice for 5 minutes and a fluorescent mounting medium (Shanghai Biaoben Model Co., Ltd.) was mounted on the section, and thereby the staining was finished. The staining was performed in a dark environment.

The results showed obvious distribution and expression of rat pituitary GHRH receptor (FIG. 16). The staining of FITC-labeled GHRH dimer showed an obvious distribution in the cell membrane and the fluorescent staining intensity was ranked as 2F>2D>2E>2Y>S. The staining in the peptide 2F showed the most abundant expression in the pituitary cells, suggesting that the peptide 2F had the highest affinity with GHRH receptor in pituitary cells membrane.

Embodiment 6: Treating infertility models with the dimeric peptide 2F

1. Method of infertility modeling assay male Chinese hamsters and 60 female Chinese hamsters (provided by Sichuan Experimental Animal Center) were divided into six groups [high-, middle-, and low-dose of peptide 2F groups, human menopausal gonadotropin (HMG) group, cyclophosphamide control group, and saline group without cyclophosphamide (CTX)] respectively, according to an indifferent body weight in statistics by /-test. For each group, the number of the hamsters was as n=10. The hamsters were injected intraperitoneally (ip) with 20mg/kg of cyclophosphamide (National Medicine Permission Number: H32020857, Ratification No. 12032925, Jiangsu Henrui Pharmaceutical Co, Ftd) once in a week for five times. After the third cyclophosphamide injection, the hamsters of the high-, middle-, and low-dose of peptide 2F groups were respectively administered with 8, 4, and 2 pg/g of peptide 2F, the hamsters of the HMG group done with 0.2 units/g of HMG, and the rest done with saline. The intramuscular injection at the hind leg was performed twice in a week. During the 6 -10^th week experimental period, each male hamster model was housed with a normal female hamster in the same cage and only the drug (dimer peptide or HMG) for treatment was injected twice in a week. The spirit, activity, and secretion of the hamster models were observed during oestrum and whether the female hamsters were pregnant. Ten weeks later, all the hamsters were sacrificed in cervical dislocation and their blood, testes, livers, and ovaries were collected.

Evaluation of pregnancy: Early-pregnant hamster: successive acicular feta (very small) can be observed in the ovarian duct. Mid-pregnant hamster: the successive beaded feta in the ovarian duct presented as the match head-like shape, with middle particle sizes. Fate-pregnant hamster: The red feta in the ovarian duct can be observed clearly, with bigger size. New born hamster: bom before the end of the ten weeks.

Approach of administration: Cyclophosphamide, in intraperitoneal injection; peptides 2F and HMG, in intramuscular injection at hind legs.

2. Test parameters

2.1. Weight change of the hamsters. There was no statistical significance in body weight between the hamster models before cyclophosphamide administration. Compared with the hamsters of the saline group, the hamsters of the modeling groups decreased significantly in body weight after administrated with cyclophosphamide. After injected intramuscularly with 2F or HMG, the decreases in body weight were significantly contained. Since the hamster models occurred in certain mortality rate due to the cyclophosphamide toxicity, the survivals of the hamsters in the experimental groups were 6-10 animals eventually (Tables 5 and 6).

Table 5: Body weight change of the female hamsters after CTX administration (n=6-10)

Week of experiment	Group (unit: g)
Singe CTX	High-dose of 2F	Middle-dose of 2F	Low-dose of 2F	HMG	Saline
1	18.6±2.4	18.7±2.3	18.7±1.6	18.7±2.4	18.7±3.3	18.7±3.0
2	21.4±3.2	22.4±2.2	21.2±2.4	22.1±2.7	22.8±2.6	19.H3.2
3	25.H4.0	25.0±5.0	25.H3.7	25.H3.7	25.0±2.5	23.2±4.6
4	25.7±4.2	26.8±5.6	25.H4.8	24.7±5.5	26.7±2.0	27.4±4.0
5	26.H4.1	28.6±6.3	25.6±5.1	27.4±5.2	27.8±2.4	27.3±3.9
6	26.0±3.1	28.0±5.6	25.H4.7	28.3±5.0	28.H1.9	27.4±4.5
7	26.9±3.1	27.4±5.5	25.9±4.2	28.H5.3	28.4±1.7	27.2±4.4
8	27.8±3.5	27.H6.8	27.5±4.9	29.4±5.1	28.8±3.8	28.8±5.2
9	31.6±3.7	26.8±9.1	30.4±6.5	30.H5.2	31.3±2.7	30.9±5.4

Table 6: Body weight change of the male hamsters after CTX administration (n=6-10)

Week of experiment	Group (unit: g)
Singe CTX	High-dose of 2F	Middle-dose of 2F	Low-dose of 2F	HMG	Saline
1	16.0±2.5	15.9±2.1	15.9±2.6	16.0±2.3	15.9±3.1	15.8±2.6
2	21.6 ±3.3	21.5±3.9	22.7±3.6	21.2±3.0	22.H4.2	23.0±2.9
3	26.6 ±4.9	26.7±4.9	26.7±3.4	26.5±5.9	26.7±6.5	29.H3.4

4	27.3 ±6.0	27.2±6.9	27.0±4.2	25.8±6.9	27.9±7.4	29.3±4.5
5	29.1 ±6.2	27.2±8.1	26.2±5.6	27.2±7.2	29.6±7.3	30.4±4.2
6	29.9 ±6.4	26.9±8.2	27.4±3.6	27.5±5.9	30.H6.1	30.2±3.9
7	32.6 ±4.7	30.8±6.6	27.4±3.8	28.2±5.2	31.0±4.5	30.H3.3
8	33.6 ±4.8	31.5±6.0	27.7±3.8	28.6±5.2	32.3±4.3	30.3±3.0
9	33.6 ±3.9	33.2±6.4	28.9±4.1	28.8±5.8	33.8±3.7	31.9±2.5

2.2. Pregnancy rate statistics. For the female models, compared to the single CTX group, the high or low 2F, and HMG group showed statistical significances in the birth rate and the total pregnancy rate. The saline group presented a pregnancy rate of 100%. (Table

7)

For the male models, compared to the single CTX group, the 2F and HMG groups showed statistical significances in the birth rate and the total pregnancy rate. (Table 8) Table 7: Pregnancy rate statistics of the female model groups

Group	N	Early-pregnancy	Mid-pregnancy	Late-pregnancy	Birth rate	Total pregnancy
Single CTX	9	33.3	0.0	0.0	0.0	33.3
High-dose of 2F	10	0.0	40.0	0.0	20.0**	60.0
Middle-dose of 2F	7	28.6	0.0	0.0	0.0	28.6
Low-dose of 2F	10	10.0	10.0	10.0	10.0**	40.0*
HMG	6	33.3	33.3	16.7	0.0	83.3**
Saline	10	0	0	10	90	100

*P<0.05, P<0.01**, vs the single CTX group, by X² chi-square test

Table 8: Pregnancy rate statistics of the male model groups

Group	N	Early-pregnancy	Mid-pregnancy	Late-pregnancy	Birth rate	Total pregnancy
Single CTX	8	30.0	0.0	0.0	0.0	30.0
High-dose of 2F	8	37.5	0.0	0.0	12.5**	50.0
Middle-dose of 2F	7	42.9	0.0	0.0	0.0	42.9*

Low-dose of 2F	9	33.3	22.2	0.0	0.0	55.5**
HMG	9	33.3	0.0	11.1	11.1”	55.5
Saline	10	0	0	0	100	100

*P<0.05, P<0.01**, vs the single CTX group, by X² chi-square test

It should be explained that, the early pregnancy resulted from the function recovery of testis due to CTX metabolized, since the male and female hamsters were kept together for 35 days while hamster had a reproductive cycle of 18-21 days. Thus the effective pregnancy rate should be calculated from the amount of the mid-pregnant number to the birth number.

2.3. Histochemical staining. The H-E staining in FIG 17 showed that, compared with the saline group and the single CTX group, the peptide 2F or the other dimeric peptide 2D, 2E, or

2Y) had a significant effect on stimulating the proliferation and maturation of spermoblast / ovum, which can promote fertility. Thus they can be used as the drugs to treat infertility.

(1) Male hamster models. As shown in FIG. 17, in the dimeric peptide 2F group, the spermatocytes and spermatogonia in the seminiferous tubules proliferated significantly. The tubular cells in the seminiferous tubules were arranged normally. The seminiferous tubules become bigger in size but their lumens became smaller or even disappeared. The 2F potency showed a dose-dependency. As shown in Table 9, the areas of 70 seminiferous tubules (horizontal axisHongitudinal axis) were measured. Compared to the single CTX group, the effect of the 2F groups on seminiferous tubules showed statistical significance.

Table 9: Size analysis of seminiferous tubules of the male models (X±SD, n=70)

Group	2 Area of the seminiferous tubules (pm )	P*
Single CTX	309868±125964	—
High-dose of 2F	328238X125110	<0.05
Middle-dose of 2F	106845X37978	<0.001
Fow-dose of 2F	709288X2323533	<0.001
HMG	446250X171099	<0.001

Saline

183891±112497 <0.001 *P <0.05 or 0.001, vs the single CTX group, by /-test (2) Female hamster models. As shown in FIG. 18, compared with the single CTX group and positive control HMG group, the dimeric peptide 2F groups showed significant increase of mature ova on the dose-dependent manner. As shown in Table 10 was the statistic of the ovarian follicles in each period. Compared to the mature follicles in the single CTX group, the effect of the 2F groups on promoting the maturation of ovum showed statistical significance, indicating that the peptide had a significant stimulating effect on the maturation of ovarian follicles, which led to a significant increase of mature ovarian follicles, and promotion and increase of pregnancy (Table 10).

Table 10: Quantity analysis of ovarian follicles of the female hamster models

Group	Primary follicles	Secondary follicles	Mature follicles
Single CTX	45	6	10
High-dose of 2F	18	0	31**
Middle-dose of 2F	31	13	21**
Low-dose of 2F	24	5	16*
HMG	34	21	22
Saline	5	6	28

*P <0.05 or **P <0.001, vs the mature follicles in the single CTX group, by /-test (3) GHRH receptor. The fluorescent staining of the testicular tissues in FIG. 19 or ovarian tissues in FIG. 20 using FITC-hGHRH( 1-44)181¾ peptide showed that, there were obvious distributions of GHRH receptor in the spermatogonia, spermatocytes, and spermatoblasts in the seminiferous tubules. The receptor expression showed no difference in the spermatogonia and spermatocytes, but significant increase in the spermatoblasts or the heads of mature sperms.

In the ovarian tissue, positive staining of GHRH receptor could be observed in the primary, secondary, and mature ovarian follicles.

The results demonstrated that the novel GHRH dimeric peptides promote the proliferations of spermatogonium and oogonium, and finally promote pregnancy by upregulating GHRH receptors.

Discussion

Compared to the standard hGHRH(l-44)NH2, all the hGHRH dimers (2D, 2E, 2F, and 2Y) presented better rGH-releasing activities and pituitary hormone-releasing specificities. The peak values of rGH release were observed in the third incubation (/3) for the peptide 2D, 2E, or 2F, while in the fourth incubation (/4) and the fifth incubation (/₅) for the peptides S and 2Y. These results suggested that, the peptides 2D, 2E, and 2F worked rapidly, while the peptides S and 2Y worked slowly, which results from the different N-terminal structures of Pro-Pro- or 'Pro-GHTH or 'Tyr-GHRH. For the peptide 2F, the GH level increased during the incubations /3-/5 compared with that of the P₂ period. Moreover, the total increase of the 2F GH release (79.77ng/ml) was 5.04 times of that of the peptide 2D (15.84ng/ml, the second highest peptide) compared to the peptide S2. The results suggested that, the peptide 2F had the highest and longest-lasting effect on extending the half-life period, which was because that, the stimulation of'Pro-GHTH was stronger than the 'Tyr-GHRH, and the GH release required an aromatic amino acid at the N-terminus.

In our preliminary research, we discovered that the GHRH monomers (D, E, F, and Y) had activities of only 48.8-89.7% of that of hGHRH(l-44)OH, or 41.9-77.1% of that of hGHRH(l-44)NH₂ (data not given). The dimers 2D, 2E, and 2F had 102%, 98%, and 110% of that of hGHRH(l-44)NH₂. The dimers presented at least 40-87% higher than their corresponding monomers in GH-releasing activity. Compared with hGHRH(l-44)NH₂ which had only one N-terminus, all the dimers with two N-termini presented better GH-releasing activities. It was known that the N-terminus of GHRH molecule played an important role in the interaction with GHRH receptor for GH release. Thus, when the C-terminus was the same, the molecule with more N-termini presented larger action capacity.

Although the results showed that, the hGHRH dimers and standard peptide hGHRH(l-44)NH₂ had good functional selectivity and species specificity. The dimeric peptides presented enhancing activities in rACTH, and/or, rLH, and rPRL, indicating that they could slightly regulate the release of other pituitary hormones.

The inhibition of GHIH in GH release showed the dose/time-dependency. In the presence of 1.927 μΜ of GHRH dimeric peptide, 1.927 μΜ of GHIH showed significant inhibition in GH release compared with 0.482 μΜ of GHIH, and moreover with the extending the incubation time the inhibiting effect enhanced.

The binding of peptides S, 2D, 2E, 2F, and 2Y with S-D rat pituitary was specific. The Scatchard plots (B/F-Bmax) suggested that, the maximal binding affinity (Bmax Value) of the dimeric peptides was ranked as 2F>2D>2Y>2E, and the dissociation constant (Kd value) was ranked as 2F<2D<2E<2Y indicating the peptide 2F had the highest binding capacity to pituitary homogenate, indicating the consistence with the result of activity. The fluorescent staining of FITC-labeled GHRH dimer showed the distribution of GHRH receptor in the cell membrane, and the fluorescent staining intensity was ranked as 2F>2D>2E>2Y>S, indicating that the peptide 2F had the most abundant distribution of

GHRH receptor in the pituitary cells and the highest affinity with pituitary cells membrane.

It turned out that, not only could the monomeric hGHRH analogs with N or/and C-terminal modulation regulate their GH releases, but also could their homo-dimers more strongly improve rat GH release by enhancing their interactions with pituitary cell receptor.

Although the usefulness of hGHRH(lM4)NH2 and hGHRH(l-29)NH2 for medical application has been verified, there is still a great demand for analogs with higher stability and efficiency. The presented peptide 2F, [¹P-hGHRH(2-44)-GGC-CGG-hGHRH(44-2)-¹P], may be promising for future clinical trial.

Claims (9)

1. CLAIMS:

   1. A novel growth hormone-releasing hormone analog, characterized in that, the novel growth hormone-releasing hormone analog is a dimeric peptide 2D, 2E, 2F, or 2Y:

   the amino acid sequence of said dimeric peptide 2D is:

   (H)PPYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC(OH )-(OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRYSNTFIADAYPP(H);

   the amino acid sequence of said dimeric peptide 2E is:

   (H)PYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC(OH)

   -(OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRYSNTFIADAYP(H);

   the amino acid sequence of said dimeric peptide 2F is:

   (H)PADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC(OH)(OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRYSNTFIADAP(H); and the amino acid sequence of said dimeric peptide 2Y is:

   (H)YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC(OH)(OH)CGGLRARAGREQNSEGQQRSMIDQLLKRASLQGLVKRYSNTFIADAY(H).
2. 2. An application of the novel growth hormone-releasing hormone dimeric peptide 2D, 2E, 2F, or 2Y of claim 1 in the preparation of drugs for treating male and female infertility.
3. 3. A drug for treating infertility, characterized in that, it comprises the novel growth hormone-releasing hormone dimeric peptide 2D, 2E, 2F, or 2Y of claim 1 as an active ingredient.

   1/9

   Si ADAIFTNSYRKVLGQLSARKLLQD1MSRQQGESNQERGARARL

   ESI MS
4. 4-ΤΓ-Ϊ-.

   S2: ADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL-NH2 j I W «βι:*ΕΑΐα>ΚτΙ<ή5ΐΐΤ7!:-- -'•nil i>eeSc»>i»M>i

   FIG. 2

   A: PPYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL WVAlM<C>#SRaWrRP-HPLC

   ESI-MS

   FIG. 3

   2/9

   B: PYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL

   C:PADAIFTOSYRKVLGQLSARKLLQD!MSRQQGESNQERGARARL

   ESI-MS

   D: PPYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC

   FIG. 6

   ESI MS

   3/9

   E: PYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGGC

   FIG. 9

   4/9 g

   ei

   600 -ι P2 ·Ι3 ·Ι4 ·15 *3 >

   £» w

   ft, pa

   S 2D 2E 2F 2Y hGHRH analogs

   FIG. 10

   90 -i 80 - a s 70 J3 'k > 60 - X 50 - u < X 40 - 4- 3 * 30 - 3 '5. ** 20 - 10 -

   P2 ·Ι3 ·Ι4 ·Ι5

   S 2D 2E 2F 2Y hGHRH analogs

   FIG. 11
5. 5/9 hGHRH analogs FIG. 12 hGHRH analogs

   FIG. 13
6. 6/9

   600 P2 ·ί3 «14 !S

   Rat pituitary UH value (rtg/ml) hGHRH analogs Glilll conceDtration(uM)

   FIG. 14

   2D Peptide 0.15

   040

   0J5

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   025 fi

   020

   0.15

   0.10

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   0.00 ca lcwij ω

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   2F Peptide wo ojo oio

   U MJ U tli ω ¢2

   BlV

   R(lM

   B'pM

   FIG. 15
7. 7/9

   FITC DAPI Merged

   FIG. 16

   Single CTX Group

   High-dose of 2F Group

   Middle-dose of 2F Group

   Low-dose of 2F Group

   HMG Group

   FIG. 17

   Saline Group
8. 8/9

   I II III

   FIG. 18

   FIG. 19
9. 9/9

   • t. . ^v S - >’· ·· ? i v® . - *« 'i ·, * .. ® ' • . - ·'» & : ?», ,* ·;.<· r* * « · . ✓ ' ,*» . - 9 . *· · < , , * * * K * • · ·) ·» ' . , ' 9 % ; / · · Ά . - - »* ’i ' x<’ ‘&ζ . I » ' . 'V- - ♦ ’·

   Secondary ovarian follicle

   Primary' ovarian follicle

   FIG. 20

   KP17871011AU_Sequence list

   Sequence List <110> GUANGDONG PHARMACEUTICAL UNIVERSITY <120> Novel Growth Hormone-Releasing Hormone Analogs and Application Thereof in Preparation of Drugs for Treating Infertility <160> 9 <210> 1 <211> 40 <212> PRT <213> Artificial Sequence <400> 1

   Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly 1 5 10 15 Gln Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln 20 25 30 Gln Gly Glu Ser Asn Gln Glu Arg Gly Ala

   35 40 <210> 2 <211> 44 <212> PRT <213> Artificial Sequence

   <400> 2 Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly 1 5 10 15 Gln Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln 20 25 30 Gln Gly Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu

   35 40 44 <210> 3 <211> 46 <212> PRT <213> Artificial Sequence

   <400> 3 Pro Pro Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val 1 5 10 15 Leu Gly Gln Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser 20 25 30 Arg Gln Gln Gly Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg 35 40 45 Leu 46 <210> 4 <211> 45 <212> PRT <213> Artificial Sequence <400> 4 Pro Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu 1 5 10 15 Gly Gln Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg 20 25 30 Gln Gln Gly Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu 35 40 45 <210> 5

   Page 1

   KP17871011AU_Sequence list <211> 44 <212> PRT <213> Artificial Sequence <400> 5

   Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly 1 5 10 15 Gln Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln 20 25 30 Gln Gly Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu

   35 40 44 <210> 6 <211> 49 <212> PRT <213> Artificial Sequence <400> 6

   Pro Pro Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val 1 5 10 15 Leu Gly Gln Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser 20 25 30 Arg Gln Gln Gly Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg 35 40 45 Leu Gly Gly Cys

   <210> 7 <211> 48 <212> PRT <213> Artificial Sequence <400> 7

   Pro Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu 1 5 10 15 Gly Gln Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg 20 25 30 Gln Gln Gly Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu 35 40 45 Gly Gly Cys

   <210> 8 <211> 47 <212> PRT <213> Artificial Sequence <400> 8

   Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly 1 5 10 15 Gln Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln 20 25 30 Gln Gly Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu Gly 35 40 45

   Gly Cys 47 <210> 9 <211> 47 <212> PRT

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   KP17871011AU_Sequence list <213> Artificial Sequence <400> 9

   Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly 1 5 10 15 Gln Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln 20 25 30 Gln Gly Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu Gly 35 40 45

   Gly Cys 47

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