WO1991016343A1 - (hyp 9) lhrh, its derivatives or fragments, production method and uses as a drug or in an assay - Google Patents

Abstract

A peptide exhibiting a substantial sequence analogy with LHRH and having the formula (I), wherein X may be a GLY, D-ALA, D-SER, D-TRP or D-HIS residue and Y may be a -GLY-CO-NH2 residue or ethylamide. Fragments of peptides according to the formula with 4-10, 5-10, 6-10 or 7-10 amino acids. These peptides may display agonist or antagonist activity for LHRH and may be used for treating afflictions related to gynecology or reproduction endocrinology, cancerous afflictions affecting the gonads or the secondary sexual organs, and afflictions of a psychiatric nature in part involving behaviour disorders leading to sexual aggressiveness. These peptides can also be used for exploring the hypothalamic-pituitary axis, and in a radioimmunoassay or biological assay method for assaying biological fluids, or in a tissue biopsy method.

Description

(Hyp 9) LHRH, ITS DERIVATIVES OR FRAGMENTS, THEIR PROCESS FOR OBTAINING AND APPLICATION AS A MEDICINAL PRODUCT OR IN A DOSAGE PROCESS

The subject of the invention is a new LHRH derivative, hydroxyproline 9 LHRH [(Hyp 9) LHRH], the process for obtaining it and its application as a medicament.

 LHRH (Luteinizing Hormone Releasing Hormone) also called GnRH (Mammalian Gonadotropin-Releasing Hormone) was isolated for the first time from hypothalamic extracts of pig (Matsuo et al. 1971, Biochem.Biophys. Res. Commun. 43, 1334-1339; Schally et al. 1971. Biochem. Biophys. Res. Commun. 43, 393-399) and ovine (Amoss et al. 1971, Biochem. Biophys. Res. Commun. 44, 205-210). Multiple molecular forms not associated with precursor molecules, but corresponding to amino acid substitutions in the LHRH sequence at positions 3, 5, 6, 7 and 8, have been described in the brains of species n ' not belonging to mammals (Sherwood, 1987, Trends Neurosci. 10, 129-132). In these non-mammalian species, at least two distinct forms of LHRH can coexist in the brain.

 This peculiarity has not been described in mammals since only one LHRH sequence is known and only one gene coding for it. precursor of LHRH, as has been shown in the human placenta (Seeburg et al. 1984, Nature, 83, 179-183) and in the rat and human hypothalamus (Adelman et al. 1986, Proc. Natl. Acad Sci. USA, 83, 179-183).

Molecular heterogeneity in the immunoreactivity of mammalian LHRH (or DA. APRIL 17, 1991 mLHRH) has however already been described in the pineal gland (Piekut et al. 1981, J. Histochem. Cytochem., 29, 616-622; Piekut et al. 1982, J. Histochem. Cytochem., 30, 106-110; Millar et al. 1981, Neuropeptides Biochemical and Physiological Studies, 263-271; King et al. 1981, J. Endocrinol. 91, 405-414) in the placenta (Currie et al. 1981, Biochem, Biophys. Res. Commun. , 99, 332-338; Nowak et al. 1984, Biol. Repro. 31, 67-75; Tan et al. 1982, Biochem. Biophys. Res. Commun., 109, 1061-1071; Siler-Khodr, 1986, Biol. Repro. 34, 245-254 and 255-264; Siler-Khodr. 1986, Biol. Repro. 35, 312-319; Mathialagan et al. 1986, Biochem. Internat. 13, 757-765; Gautron et al. 1989), in the gonads (Dutlow et al. 1981. Biochem. Biophys. Res. Commun. 101, 486-494; Bhasin et al. 1983. Endocrinology, 112, 1144-1146; Hedger et al. 1985, Mol. Cell Endocrinol, 42, 163-174) and in the hypothalamus (Fawcett et al. 1975, Endocrinology, 96, 1311-1314; Anthony et al., 1987; Brain. Res. 424, 258-26 3; King et al. 1988, Endocrinology, 122, 2742-2752; Stopa et al. 1988. Peptides, 9, 419-423).

 We also tested (Heber and Odell, Biochem. Biophys. Res. Commun. 82, 67-73, 1978). The ability of LHRH derivatives, including (Hyp 9) LHRH to bind to pituitary receptors. This in vitro study shows that (Hyp 9) LHRH does not bind to these receptors.

From these various observations, it was therefore possible that in mammals there are at least two molecules of the LHRH type, as is the case in non-mammalian species. In this case, the difference (s) between the peptide sequence of the mLHRH and the sequence (s) of unknown molecules of LHRH type could also be too large or insufficient to allow the detection of distinct genes using oligodeoxynucleotide probes synthesized according to the sequence of LHRH (Seeburg et al. 1984, Nature, 83, 179-183).

 Furthermore, post-transcriptional enzymatic modifications of the LHRH sequence such as those suggested in the pineal gland (Millar et al. 1981 previously cited; King et al. 1981, J. Endocrinol, 91, 405-414) could also explain the apparent heterogeneity of the immunoreactivity of mLHRH, even with a simple discomfort coding for the precursor of mLHRH.

In previous studies (Gautron et al. 1981, Mol.Cell. Endocrinol ,; 24, 1-15; Drouva et al. 1986, Neuroendocrinol, 43, 32-37), it has been shown that hypothalamic rat LHRH co -eluted with synthetic LHRH by filtration through molecular sieve but is not quantified in the same way by three different anti-LHRH sera. The three anti-LHRH sera used in these studies are called N, C and W, and respectively recognize the N terminal fragments of mLHRH as well as other known LHRH molecules, except lamprey LHRH - the C terminal fragments of the mLHRH, but not other forms of LHRH, and the conformation of the mLHRH molecule (W antibody). The presence of a pyroGlu residue at the N end is essential for detection by the antibody N. On the other hand, the glycine-amide residue located at the end C is essential for the recognition by the antibody C. The W antibody quant he does not accept modification of the pyroGlu ₁ residue and the Glyamide ₁₀ residue. However, this latter antibody recognizes the other LHRH molecules except the lamprey LHRH molecule. The difference in immunoreactivity demonstrated in these preliminary studies was first attributed to the presence of co-eluted LHRH fragments, better recognized by one of the antibodies than by the other two. Additional studies have not confirmed this hypothesis.

 The present invention has made it possible to surprisingly show that two molecules of the LHRH type coexist in the hypothalamus of mammals, in particular in humans and rats, and these two molecules have been characterized.

 More particularly, the Applicant has demonstrated the molecular species of LHRH type, which showed a different immunoreactivity with respect to the three groups of antibodies previously mentioned compared to mLHRH. The Applicant has also developed a method for the preparation and preparative separation of these two forms, and in particular of the modified LHRH form.

The subject of the invention is therefore peptides exhibiting significant sequence homology with mLHRH, characterized in that they exhibit a high reactivity with respect to antibodies directed against the N-terminal end of LHRH and a low reactivity towards antibodies directed against the C-terminal end of LHRH or directed against antigenic determinants linked to the tertiary three-dimensional structure of LHRH, by comparison with the respective reactivities of the LHRH.

 These peptides can have, in mass spectrometry, a band corresponding to a molecular weight of approximately 1198.8 Da.

 More particularly, the subject of the invention is a peptide characterized in that it has the following sequence:

 1 2 3 4 5 6 7 8 9 10

pyroGLY-HIS-TRP-SER-TYR-X-LEU-ARG-HYP-Y

in which, relative to the native LHRH molecule, the proline at position 9 has been hydroxylated.

 In this molecule, the amino acids in positions 1 to 5 and 7 to 9 are in the L configuration, and X can represent a residue GLY, D-ALA, D-SER, D-TRP, or D-HIS.

Y can represent a residue -GLY-CO- NH ₂ or an ethylamide group.

 X and Y cannot be GLY residues simultaneously.

 These peptides can also exhibit agonist or antagonist activity with respect to LHRH.

 The present invention also relates to fragments of these peptides, or of the (Hyp 9) LHRH and in particular to the fragments corresponding to the peptide sequence comprised between amino acids 4 and 10, amino acids 5 and 10, amino acids 6 and 10 or amino acids 7 and 10.

The N-terminal end of these fragments can advantageously carry a protective group, such as an acetyl, ter-butyl-oxycarbonyl group. (tBoc) or benzyl-oxycarbonyl (Z). These peptides or peptide fragments are advantageously obtained by extraction from a tissue or a biological fluid which can in particular be the hypothalamus of mammals and in particular of the rat or of man. They can also be obtained by synthesis, in particular by chemical synthesis.

 The invention also relates to a process for obtaining these peptides and peptide fragments or (Hyp 9) LHRH from tissues or biological fluids comprising:

 - at least one step of overall protein extraction;

 - at least one cation exchange chromatography;

 - And / or at least a high performance liquid chromatography (HPLC) in reverse phase.

 More particularly, this process of obtaining will be implemented on tissues or fluids forming part of the hypothalamus of mammals and in particular of the hypothalamus of man or of rat.

 Another object of the invention is the use of these peptides and peptide fragments and of the (Hyp

9) LHRH in the treatment of conditions which may be linked to LHRH relating to gynecology and / or endocrinology of reproduction, in particular in the treatment of infertility and syndromes linked to precocious or delayed puberty. These peptides and peptide fragments and (Hyp 9) LHRH can also be used in the treatment of cancerous conditions affecting the gonads (ovaries, testes) or the secondary sexual organs

(mammary glands, prostate) and in treatment psychiatric conditions, in particular behavioral disorders resulting in sexual aggression, or libido-related disorders.

 These peptides and peptide fragments and the (Hyp 9) LHRH can also be used for the functional exploration of the hypothalamic-pituitary axis, in particular by measurement by radioimmunological assays of the plasma and urinary levels of LH, FSH and steroids before and after administration of the peptide, or for radioimmunological or biological assays of biological fluids and tissue biopsies.

 The present invention also relates to medicaments containing as active principle at least one of these peptides and peptide fragments and the (Hyp 9) LHRH, and in particular medicaments for the treatment of conditions which can be linked to the LHRH pertaining to the reproductive gynecology or endocrinology, for the treatment of cancerous conditions affecting the gonads or secondary sexual organs, or for the treatment of psychiatric conditions and in particular behavioral disorders resulting in sexual aggression, or libido-related disorders (frigidity, impotance).

In addition, the subject of the invention is pharmaceutical compositions characterized in that they contain at least one of the peptides, peptide fragments previously described or the (Hyp 9) LHRH in combination with one or more diluents or adjuvants compatible and pharmaceutically acceptable. These compositions are especially intended for the treatment of conditions which may be related to LHRH related to reproductive gynecology and / or endocrinology, to the treatment of cancerous conditions affecting the gonads and / or secondary sexual organs, or to the treatment of psychiatric conditions, in particular behavioral disorders resulting in sexual aggression, or libido-related disorders.

 These compositions can be presented for parenteral or enteral administration.

 The present invention also relates to a radioimmunological or biological assay method of biological fluid and tissue biopsy characterized in that its implementation requires the use of a peptide or peptide fragment as described above.

 The invention will be further illustrated without being in any way limited by the examples below and in particular by the following figures:

 FIG. 1 represents the immunological specificity of the three types of anirops N, C, W, directed respectively against the N and C ends of the LHRH and against the three-dimensional structure of the

LHRH.

 Figure 2 shows the elution profiles of

HPLC on cation exchanger (2A) and on reverse phase (2B) of the synthetic and radioactive mLHRH (1 ²⁵ I) which was subjected to the extraction process in the presence of rat hypothalamus. The elution positions of radioactive non-extracted LHRH,

Non-radioactive LHRH and its N- and C-terminal fragments are indicated. In these two figures, the ordinate represents the quantity of radioactivity in counts per minute.

 FIGS. 3 and 4 respectively represent the elution profiles on a column of cation-exchange HPLC of hypothalamic extracts of rats and humans. The columns were calibrated as indicated respectively in FIGS. 3A and 4A by

Mammalian LHRH (mLHRH), chicken LHRH

(cLHRH-I), salmon LHRH (sLHRH) and lamprey LHRH (1LHRH).

 FIGS. 3B and 4B respectively represent the chromatographies of the extracts of 1000 rat hypothalamus and 6 human hypothalamus. Peaks 1 and 2 of FIGS. 3B and 4B are rechromatographed separately. The results of these second peak 1 chromatographies are shown in Figures 3C and 4C. Identical results are obtained for peak 2.

 Figures 3D and 4D show the chromatography of peaks B in Figures 3C and 4C.

 FIG. 5A represents the calibration of the reverse phase HPLC column (micro-Bondapak column) with synthetic mLHRH.

 FIGS. 5B and 5C respectively represent the elution on a micro-Bondapak column of the peaks B of FIGS. 4C and 3C. The profiles inserted in frames represent the peaks corresponding approximately to fraction 40, on a larger scale of representation.

FIGS. 6A and 6B respectively represent the elution on a phenyl micro-Bondapak column of the peaks C and D represented in FIG. 5C. The elution of the Synthetic mLHRH is shown in these figures. The optical density at 280 nm is shown on the ordinate.

 FIGS. 7A and 7B respectively represent the filtration on a molecular sieve of the synthetic mLHRH and of the peaks C and D of FIG. 5C.

 FIG. 7C represents the elution volume (on the ordinate) as a function of the logarithm of the molecular weight of peaks D and C and of various molecular weight markers.

 FIG. 8 represents the mass spectrum of the peak C represented in FIG. 5C.

 Figure 9 shows the trypsin and clostripain cleavage sites of mLHRH, LHRH derivatives and peak C shown in Figure 5C.

 FIG. 10 represents the chromatographic analysis on cation exchanger and on reverse phase of molecules of LHRH type after digestion with trypsin or clostripaine. The results presented are obtained with the antibody N.

 FIGS. 10A to 10E represent the chromatographic analysis on cation exchanger of the mLHRH (10A), the (Hyp 9) LHRH (10B), the (Leu 9) LHRH (10C), the (Lys 8) LHRH (10D) and of peak C represented in FIG. 5C (10E), after digestion with trypsin (non-filled circles) or with clostripaine (filled circles).

Figures 10F to 101 represent the reverse phase chromatographic analysis of fractions 25 to 30 isolated on a cation exchange column and generated respectively for Figures 10F to 101 from mLOHRH, (Hyp 9) LHRH, (Leu 9) LHRH and of rat peak C. In Figure 101, the solid circles represent the analysis of the material resulting only from the cut of the rat C peak, and the non-solid circles correspond to the combined analysis of the materials originating from the cuts of the rat C peak and of the mLHRH .

 FIG. 11 represents the chromatographic analysis on cation exchanger and on reverse phase of various molecules of LHRH type digested by pyroglutamate-aminopeptidase. The results presented are obtained with the antibody C.

 FIGS. 11A to 11D represent the analysis on a cation exchange column respectively of the mLHRH of the (Hyp 9) LHRH, of the (Leu 9) LHRH and of the rat C peak.

 FIGS. 11E to 11H represent the analysis in reverse phase chromatography of the fractions 25 to 29 isolated on a cation exchange column and generated respectively for FIGS. 11E to 11H from mLHRH, (Hyp 9) LHRH, (Leu 9) LHRH and rat C peak.

 For FIG. 11H, the solid circles represent the analysis of the material resulting only from the cut of the rat C peak, and the non-solid circles correspond to the combined analysis of the materials originating from the cut of the rat C peak of the ( Hyp 9) LHRH.

FIG. 12 represents the elution by chromatography of the human and rat peaks and of the (Hyp 9) LHRH. In these figures, the solid circles represent the elution of the rat C peak and the non-solid circles represent the elution of the (Hyp 9) LHRH. The elution of mLHRH is also indicated by an arrow. FIGS. 12A, 12B, 12C respectively represent the elution after the stages of purification 1, 2 and 3, while FIG. 12E represents the elution of the rat peak C in the presence of (Hyp 9) LHRH after the stage of purification 3, and FIG. 12D represents the elution of the human peak C after the purification step 3.

 Figure 13 represents the diagram of the operations having led to the isolation of (Hyp 9) LHRH.

 FIG. 14 represents the variation in the secretion of FSH or LH, expressed in (ng / ml), as a function of the logarithm of the concentration of LHRH or in (Hyp 9) LHRH expressed in mole / l.

 FIG. 15 represents the kinetics of appearance of the fragment (1-9) during the incubation of LHRH or (Hyp 9) LHRH in the presence of homogenates of hypothalamus or hippocampus.

Figure 16 illustrates the effect of the (4-10) 9 CHRH Hyp, LHRH and (D-Ala6, Gly 10) LHRH ethylamide nonradioactive (10- ⁶ M) to the displacement of the (4 -10) Hyp 9 LHRH radioactive (10- ⁹ M) specifically linked to a hippocampal membrane preparation.

 Figure 17 illustrates the effect of (DAla6, Gly 10) LHRH ethylamide and (4-10) Hyp 9 non-radioactive LHRH on the displacement of radioactive (DAla 6, Gly 10) LHRH ethylamide

(10 ^-9 M) specifically bound to a membrane preparation of the hippocampus.

Figures 18A and 18B are reverse phase electrophoretic profiles (cLHP) of extracts Sertoli (18A) and Leydig (18B) cell acids.

 FIG. 19 is a CHLP chromatogram of hypothalamus (19A), olfactory bulb (19B) and hippocampus (19 C) extracts.

 FIG. 20 is a chromatogram of the immunoreactivity of the fractions 5-20 of the chromatography corresponding to FIG. 19 using a simple gradient of acetonitrile from hypothalamus extracts (20A) and the olfactory bulb (20B).

 Figure 21 is a chromatogram of the same fractions as for Figure 20 in a gradient composed of extracts of hypothalamus (21 A) and hippocampus (21 B).

EXAMPLE 1 Obtaining and Purifying LHRH-Type Peptides.

1. Tissue extraction process:

 6000 rat hypothalamus were collected from both sexes (60% females, 40% males) for 1 year on dry ice then freeze-dried and stored at - 80ºC. In parallel, fresh rat hypothalamus were immediately extracted, tested for their content in LHRH type molecules and chromatographies in the same way as lyophilized tissues. In addition, 6 human hypothalamus of both sexes (5 women, 1 man) were collected one day after their death.

In rats, the extraction is carried out according to the following method called "combined extraction process". For each extraction, 1000 rat hypothalamus, with a dry weight of between 12 + 0.05 grams were homogenized (10% weight / volume) in 2 M acetic acid (HOAc) at 4ºC using 'a "Teflon" mill and a glass homogenizer. The homogenate is centrifuged at 30,000 g for 90 minutes. The supernatant is stored and then mixed with the supernatant obtained after centrifugation at 30,000 g for 60 minutes of the pellet resuspended in 2 M HAc (half the volume of the initial homogenate). The mixed supernatants are then frozen to dryness and then lyophilized.

 The lyophilisates are resuspended (half of the initial homogenate) at 4ºC with acetic acid 2

M in methanol (MeOH) at 70% (volume / volume), then centralized at 10,000 g for 90 minutes. The resulting supernatant is then mixed as above with the volume of washing of the pellet. The methanol is then removed from the supernatants using a rotary vacuum evaporator at a maximum temperature of 30ºC. The remaining extracts are diluted in cold water, frozen then lyophilized and stored at -80ºC in the form of samples each corresponding to 200 hypotalamus.

 All of the human material (6.5 grams dry weight) was extracted according to the method described above.

The results in Table 1 show that the recovery of molecules detectable by anti-LHRH antibodies is the greatest after extraction of the tissues with 2 M acetic acid. Extraction with 70% methanol is also very effective. In all cases, the quantity of immunoreactive material of the LHRH type is systematically greater (12 to 30%) with the antibody N, in comparison with the evaluations obtained in the two other radioimmunological tests (W and C). The results of the combined extraction process are collated in Table 2. For human and rat hypothalamus, the antibody N always detects more immunoreactive material than the other two antibodies after combined extraction with 2 M acetic acid and with a mixture of 2 M acetic acid and 70% methanol . The methanol extraction step is necessary only because of the elimination of protein contamination. In addition, the content of LHRH-type molecules in the rat hypothalamus is identical in fresh or lyophilized tissues. A comparative study is not possible with human material due to the small amount of tissue available.

Table 3 shows the recovery of the radioactivity associated with LHRH iodine-labeled I ²⁵ I ( ¹²⁵ I-LHRH) added to the tissue before combined extraction with 2 M acetic acid and 70% methanol. The radioactivity is found with a yield of 90% in the supernatant. It is exclusively associated with LHRH molecules, as indicated by the cation exchanger and reverse phase chromatographies in FIG. 2. No additional radioactive peak is observed.

2. Purification process by HPLC:

 The entire process is controlled using HPLC Gilson equipment (France).

a) First step: HPLC by cation exchange: The total extract of human hypothalamus or a sample corresponding to 200 rat hypothalamus is resuspended in 50 ml of a 20 mM ammonium acetate buffer (NH ₄ Ac) , at pH 4.10, containing 30% (volume / volume) of acetonitrile (CH ₃ CN) (buffer of ), then centrifuged at 30,000 g for 30 minutes. The supernatant is chromatographed on a TSK SP 5 PW preparative column (21.5 x 300 mm, LKB). After a washing period of 60 minutes, the elution is carried out with a gradient of 120 minutes ranging from the starting buffer to 15% of 200 mM ammonium acetate buffer, pH 4.1 containing 30% of 'acetonitrile. The flow rate is 2 ml per minute and fractions of 4 ml are collected. A 10 ml loop is used for injections.

 The immunoreactive material eluted by five passages, corresponding to a total quantity of 1000 rat hypothalamus is separated into two groups (groups 1 and 2). The resulting immunoreactive material co-eluting with synthetic LHRH (peak B) is rechromatographed on the same column then is lyophilized and stored at -80ºC. This procedure is repeated six times (six times 1000 hypothalamus of rats).

 The same procedure is applied to human tissue.

 Chromatography of different amounts of synthetic mLHRH generates a single immunoreactive peak which is clearly separated from both the N- and C-terminal fragments of LHRH or from other known LHRH sequences as shown in Figures 3A and 4A. This peak is detected in the same way by the three antibodies

In FIGS. 3 and 4, the immunoreactivity profiles with the antibody W are not indicated to avoid confusion. These profiles are identical to those obtained with the antibody C.

During the first passage and under the elution conditions used, the LHRH type materials, both human and of rats, elute in a single broad peak which is better recognized by the antibody N than by the antibodies C or W as shown in FIGS. 3B and 4B. This peak does not co-elute perfectly with synthetic mLHRH and does not co-elute with other known LHRH type molecules or with terminal N and C fragments of mLHRH.

 Secondary chromatographies of groups 1 and 2 of the large initial peaks generate two narrow immunoreactive peaks (peaks A and B) as shown in Figures 3C and 4C. Peak A eluted before synthetic mLHRH and is exclusively recognized by antibody C. Characterization of this peak has not been carried out.

 On the other hand, peak B co-elutes perfectly with synthetic mLHRH but is better recognized by the antibody N than by the two other antibodies.

 Repeated chromatography of peak B generates a single peak, which co-elutes with synthetic mLHRH as shown in Figures 3D and 4D. This peak is always better detected by the antibody N than by the other antibodies.

 b) Step 2: Reverse phase HPLC chromatography on a micro-Bondapak column.

The lyophilized peaks B, originating from stage 1 and corresponding to all the human and rat tissues extracted, are resuspended respectively in 2 ml and 10 ml of a 15% solution (volume / volume) of acetonitrile in water containing 0.1% (volume / volume) of trifluoroacetic acid (TFA) then chromatography on a micro-Bondapak C18 column (3.9 x 300 mm, Waters). Human and rat materials are fractionated by one and five column passes respectively. After an 11 minute wash, the elution is carried out for 60 minutes with a gradient ranging from 15 to 25% acetonitrile in water containing 0.1% trifluoroacetic acid. The flow rate is 1 ml per minute and fractions of 0.5 to 1 ml are collected. A 2 ml loop is used for injections.

 As shown in Figure 5A, the synthetic mLHRH generates a simple immunoreactive peak which is detected in the same way by the three antibodies. No additional peak is detected. Under the same elution conditions, the human or rat peak B, originating from the cation exchange column, generates two peaks, the peaks C and D shown in FIGS. 5B and 5C. Washing with 100% acetonitrile does not detect other immunoreactive peaks.

 Peak C is eluted before the synthetic mLHRH.

 Faster elution conditions or passage of large quantities of material affect the detection of this peak. Peak C is better detected by antibody N than by the other two antibodies

However, detection by antibodies C and W is identical.

 Peak D co-eluted with synthetic mLHRH. It is detected in the same way by the three antibodies.

 The peaks C and D represent respectively for the antibody N 12% and 88% of the total immunoreactivity of the molecules of the LHRH type recovered on the micro-Bondapak column, in comparison with 4% and 96% for the antibodies C and W.

The same distribution is observed in fractions prepared from rat and human hypothalamus as indicated in Table 4. c) Step 3: Reverse phase chromatography on a phenyl micro-Bondapak column.

 Only the immunoreactive material obtained from the hypothalamus of rats is subjected to this step. The immunoreactive peaks (peaks C and D) isolated during step 2 are resuspended entirely with 3 to 4 ml of acetonitrile at 14 and 15% (volume / volume) respectively in water containing 0.1% of TFA . The resuspended peaks C and D are then respectively chromatographed by three and four passages on a phenyl micro-Bondapak column (3.9 × 150 mm, Waters).

 After 5 minutes of washing, the elution is carried out for 60 minutes using a gradient ranging from 14 to 15% (peak C) and 15 to 16% (peak D). The flow rate is 1 ml per minute and fractions of 0.5 to 1 ml are collected. A 1 ml loop is used for injections.

 As shown in FIG. 6A, the rat peak C is eluted in a single peak which is always better recognized by the antibody N than by the two other antibodies. The immunoreactivity peak perfectly matches the UV peak. Synthetic mLHRH is eluted later under these chromatography conditions

Peak D is also eluted in a single peak (FIG. 6B) but in this case, it is always co-eluted with synthetic mLHRH and its detection is always identical with the three antibodies. The immunoreactivity peak also corresponds perfectly with the UV peak. Table 5 shows the data corresponding to the results of the different chromatographies. Peaks C and D are purified 75,000 times and

 78,000 times respectively following phenyl micro-Bondapak chromatography.

 3) Control of extraction and chromatographic processes.

 Identical amounts of rat hypothalamus of both sexes (15 females and 15 males) are homogenized in different extraction media: 0.2 M hydrochloric acid; 0.1% (volume / volume) of

TFA in water; 70% (volume / volume) of methanol in water; 0.2 M acetic acid and 2M acetic acid, and 2 M acetic acid and 70% methanol. In this last extraction condition, synthetic mLHRH (between 2 and 4 micrograms) is also added before homogenization of the tissues. The presence of LHRH type materials is checked in the various crude extracts and in the fractions obtained on a CHLP column under the chromatography conditions of step 2.

The recovery of ¹²⁵ I LHRH is also verified during extraction with 2 M acetic acid and 70% methanol. Synthetic radioactive LHRH is introduced into the medium before the tissue is homogenized. Radioactivity is monitored during extraction and identified in the two HPLC chromatography systems. The first step is similar to step 1 except that a cation exchange column TSK SP-5 PW (7.5 x 74 mm, LKB) is used in place of that used in step 1. In this case, the elution is carried out for 60 minutes with a gradient ranging from starting buffer at 20% of a 200 mm ammonium acetate buffer, at pH 4.10 containing 30% acetonitrile. The flow rate is 1 ml per minute and fractions of 1 ml are collected. The second CHLP system is that of step 2.

 All the chromatography steps are calibrated with synthetic mLHRH. The quantities of synthetic mLHRH used for these calibrations are identical to those used for endogenous LHRH type materials, the calibration is also carried out with LHRH I of salmon, lamprey and chicken, as well as with fragments N and C mLHRH terminals.

 After use, all the columns are washed with 100% acetonitrile (for columns in reverse phase) and with solutions of 0.2 M sodium hydroxide, 2 M acetic acid and 60% acetonitrile in water (for cation exchange columns). In addition, each purification step is preceded by a blank passage in which the starting buffer is injected in place of the sample in order to avoid any contamination.

4) Recovery of rat peaks C and D after various extraction modes.

Unlike the immunoreactivity of LHRH type molecules which has been described previously as dependent on the extraction procedure (Table 1), the ratio of peaks C and D remains constant after reverse phase chromatography, regardless of the initial medium. extraction system used (Table 6). The addition of synthetic mLHRH in amounts corresponding to the LHRH content of 300 to 600 rat hypothalamus during homogenization according to the combined extraction method (2 M acetic acid and 70% methanol) does not affect the immunoreactivity of peak C compared to that evaluated in the absence of exogenous synthetic mLHRH. By comparison, the immunoreactivity of peak D is considerably increased after the addition of synthetic peptide.

5) Description of the radio immunology (RIA) detection tests for LHRH type molecules:

Aliquots of the crude extracts and of HPLC fractions obtained on a CHLP column as well as synthetic mLHRH for the standard curves are lyophilized in the presence of 100 microliters of an RIA buffer (50 mM of a Na / Na ₂ phosphate buffer, pH 7, containing 0.9% (weight / volume) of NaCl and 0.1% (weight / volume) of gelatin in order to avoid the absorption of immunoreactive material.

The lyophilized samples are resuspended in the presence of one of the three antibodies and tested against LHRH monoiodee by iodine ¹²⁵ I as described previously (Gautron et al. 1989, Placenta, 10, 19-35).

The RIA test is implemented with some modifications according to the method of Nett et al. (1973) Clin. Endocrinol. Metab. 36, 880-885). The incubations are carried out in the presence of 100 microliters of normal rabbit serum (dilution 1.60) and 10 microliters of a pH indicator (universal indicator solution, pH 4-10, Merck) to check the presence of interfering substances at the level of the pH in the crude extracts and the fractions collected from HPLC chromatography. The antigen-antibody complex is precipitated with 1 ml of absolute ethanol after 48 hours of reaction at 4ºC. EXAMPLE 2 Characterization of the peaks obtained in Example 1.

 1) Filtration on molecular sieve.

 The purified peptides and eluted in peaks C and D of rat are resuspended in 200 μl of 20 mM ammonium acetate buffer, pH 4.2 containing guanidine hydrochloride 6 M. These peptides are then chromatographed under HPLC condition on a column TSK G 2000 SW (7.5 x 300 mm, Interchim.) Eluted with the same buffer used for the resuspension of the samples. The column flow rate is 0.5 ml per minute and the volume of the fractions collected is 0.1 to 0.5 ml. A loop of 200 microliters is used for the injection. Guanidine is removed from the fractions collected by passage through SEP PAK C 18 cartridges (Waters).

 The column is calibrated with mLHRH and N- and C- terminal fragments as well as with the following peptides: the intestinal vasoactive peptide (VIP), human beta endorphin, neuropeptide Y (NPY), somatostatin- 14 and -28, human angiotensins 1 and 2 and beta casomorphine (1,4-amide).

 FIG. 7 represents the elution profiles of peaks C and D of rats. After several chromatographies, the molecular weights of the two peptides eluted in peaks C and D are estimated between 1086 and 1188 Daltons.

2) Mass spectrum:

420 picomoles of the rat peak C peptide are dissolved in a mixture of dithiothreitol and dithioerythritol (5: 1 by weight) for analyzes (Neosystems Laboratories) and the peak was analyzed between 1000 and 1400 m / z. The results in FIG. 8 represent the mass spectrum obtained. An intensity at 1198.8 m / z corresponds to the mass of the peptide eluted in peak C. Under the same experimental conditions, the mass of the synthetic peptide mLHRH is determined at 1182.8 m / z.

 The peptide contained in the rat peak C and the synthetic peptide LHRH can be considered to be different from 16 Daltons.

3) Analysis of the amino acid composition:

 One hundred picomoles of purified peptides eluted in peaks C and D of rat are hydrolyzed in 6 M hydrochloric acid at 110 ºC for 22 hours in the absence of mercapto-sulfonic acid. The hydrolysates are dried and the amino acids are determined by the PITC method (Phenyl Iso Thio

Cyanate).

 Amino acid analysis shows that the peptide eluted in peak D corresponds to mLHRH (Table 7).

The analysis is less clear for peak C due to significant contamination. A comparison between the amino acid composition of peaks C and D suggests that their composition is roughly similar. The main difference concerns the apparent absence of proline in the peak C peptide, while it could on the contrary contain an additional leucine and / or serine.

 The difference in mass (16.04 Daltons) due to the replacement of proline by leucine is the value closest to the difference in mass (16

Daltons) between the molecular weight obtained by mass spectrometry from the peptide whose molecular weight is determined (1188.8 Daltons) and the mLHRH (1182.8 Daltons). However, the perfect match is only obtained by replacing the proline with a hydroxyproline (Hyp) which elutes near the serine during the amino acid analysis. This may explain the high level of serine in the analysis of this peptide.

 In the context of the present invention, we therefore synthesized (Leucine 9) LHRH and (Hyp 9) LHRH and compared their sensitivity to enzymatic digestion as well as their behavior in chromatography by comparison with the endogenous peptide eluted in peak C .

4) Synthesis of (Hyp 9) LHRH and (Leu 9) LHRH:

 LHRH-type sequences have been synthesized by Neosystem Laboratories. The purity of the peptides is verified by HPLC and confirmed by amino acid analysis. The synthesis of HYP-9-LHRH is carried out with 4-hydroxyproline.

5) Enzymatic digestion:

 5 ng of synthetic mLHRH, of (Lys 8) LHRH, of (Leu 9) LHRH and of (Hyp 9) LHRH as well as of purified rat peptide are digested with 0.25 ng of trypsin (7500 to 9000 units per mg of protein) treated with DPCC (diphenylcarbamyl chloride), or with 10 ng of clostripaine (215 units per mg of protein) or with 5 mg of pyroglutamate aminopeptidase (PGA) (160 units per mg of protein).

The digestion reactions are carried out in 50 microliters of 25 mM Na / Na2 phosphate buffer, at pH 7.5, and containing either 2.5 mM of dithiothreitol (DTT) for clostripain or 2.5 mM of DTT and 2 mM d 'acid ethylenedimain tetraacetic in the case of digestion with pyroglutamate aminopeptidase. The reaction times are 180, 120 and 25 minutes for trypsin, clostripaine and pyroglutamate aminopeptidase. These reactions are stopped by heating to 90ºC.

 The digestion products are chromatographed on the HPLC ion exchange column described above. For the products of digestion with trypsin and clostripaine, the elution is carried out for 60 minutes with a gradient ranging from the starting buffer to 20% of a buffer containing 200 mM of ammonium acetate, pH 4.10, and 30% acetonitrile. The product of digestion with PGA is eluted for 30 minutes with a gradient ranging from 50 to 70% in 200 mM ammonium acetate, pH 4.10, containing 30% acetonitrile in the starting buffer.

 The isolated peptide fragments are then analyzed on a Novapak column (7.5 x 75 mm, Waters) with a gradient for 30 minutes ranging from 15 to 20 or 25% acetonitrile in water containing 0.1% TFA. The flow rate is 1 ml per minute and the fractions collected are 0.5 ml.

The results of these digestions are as follows: trypsin cuts the peptide bond at the level of the carboxyl grouping of the arginine and lysine residues, with the exception of the sequences Arg-pro (or Hyp) and Lys-pro (or Hyp), the first of these sequences being only sensitive to clostripaine. Under these experimental conditions, trypsin and clostripaine cut the peptide bond of arginine in different synthetic peptides of the mLHRH type with the exception of Lys-8-LHRH and cut also the enogenous peptide eluted in peak C (peak C peptide) which may or may not generate the common fragment (1-8) mLHRH. The cuts are shown in Figure 9.

 Synthetic mLHRH, (Hyp 9) LHRH and (Lys 8) LHRH as well as the peak C peptide are not hydrolyzed by trypsin. On the other hand, the (Leu 9) LHRH generates an N-immunoreactive fragment, which has a chromatographic behavior identical to that of the N-terminal fragments of LHRH on a cation exchange column as shown in FIG. 10.

 After digestion with clostripaine, a comparable N immunoreactivity is obtained from the synthetic molecules of LHRH and from the peptide of peak C with an exception for the (Lys 8) LHRH. Analysis by reverse phase chromatography which follows shows that these N-immunoreactivities resulting from hydrolysis correspond to a common peptide which coelutes with synthetic (1-8) mLHRH, which indicates that the cuts in the sequences take place exclusively after arginine.

 Digestion with pyroglutamate aminopeptidase of mLHRH, (Hyp 9) LHRH, and (Leu 9) LHRH generates immunoreactivity C which has a chromatographic behavior identical to that of synthetic (2-10) mLHRH, which is well separated from the other C-terminal LHRH fragments on a cation exchange column, as shown in FIG. 11. Comparable C immunoreactivity is generated by the peak C peptide.

Reverse phase chromatographic analysis shows that the immunoreactivity C generated from mLHRH co-eluted with synthetic (2-10) mLHRH, which which indicates that the cut takes place exclusively at the level of the pyro Glu ¹ -His ² link under the experimental conditions described above. On the contrary, the immunoreactivities C generated from (Hyp 9) LHRH and (Leu 9) LHRH corresponding to the respective fragments (2-10) elute respectively before and after the (2-10) mLHRH synthetic. On the other hand, the immunoreactivity C generated from the peptide of peak C co-eluted with the hydrolysis product of (Hyp 9) LHRH.

 6) Chromatographic behavior of the Pic C peptide and the (Hyp 9) LHRH.

 The chromatographic behaviors of the rat peak C peptide and the (Hyp 9) LHRH were found to be strictly identical at different stages of the purification process. In all cases the endogenous rat peptide as well as the synthetic peptide elute at the same position as a single peak when they are chromatographed separately, as can be seen in Figures 12. Figures 12A, 12B and 12C correspond to the resulting peptides purification steps 1, 2 and 3. The same result is obtained with the human peak C under the conditions of step 3

(Figure 12D). Co-elution is confirmed when the rat peak C and the (Hyp 9) LHRH are chromatographed together (FIG. 11E).

 In these two examples, the determination of the protein concentration is carried out according to Lowry et al. [(1951) J. Biol. Chem. 193, 165-275].

The previous results clearly show the presence of two LHRH-type molecules in extracts of both murine and human hypothalamus: mammalian LHRH, already known, and (hydroxyproline 9) LHRH which had not been demonstrated.

 On the other hand, the controls carried out during these manipulations showed that the (hydroxyproline 9) LHRH was not due to a transformation of the LHRH during the manipulations, but pre-existed in the extracts. This was particularly highlighted during the chromatography of pure and synthetic LHRH.

 The results obtained in mass spectrometry, by measurement of the molecular weight, and after cutting by trypsin, clostripaine or pyroglutamate aminopeptidase confirmed that this second form of molecule of the LHRH type is indeed a (hydroxyproline 9) LHRH.

EXAMPLE 3 Biological activity of (Hyp 9) LHRH.

In in vitro systems using dispersed pituitary cells, (Hyp 9) LHRH induces the release of LH and FSH in proportions comparable to that of LHRH itself. In molar concentration, the activity of (Hyp 9) LHRH on pituitary secretions, as well as that calculated on the LHRH receptor by measuring the displacement of radioactive LHRH by iodine ¹²⁵ I linked to membrane preparations pituitary, is fifteen times weaker than that of LHRH. This lesser apparent activity is however compensated by a prolonged bioavailability.

 FIG. 14 represents the variation of the secretion of FSH or LH, expressed in (ng / ml) as a function of the logarithm of the concentration of LHRH or in (Hyp 9) LHRH expressed in mole / l.

 EXAMPLE 4 Catabolism of LHRH and (Hyp 9)

LHRH: formation of C-terminal fragments. LHRH is mainly degraded by two enzymes at the level of the Pro (9) - GlyNH ₂ (10) and Tyr (5) -Gly (6) peptide bonds. The cleavage of the first bond which is carried out by the Post Proline Cleaving Enzyme (PPCE), conditions the rate of hydrolysis of the second bond.

 In the presence of dithiothreitol, the rate of hydrolysis of the Tyr (5) -Gly (6) bond is considerably reduced and it is then possible to observe the accumulation of the fragment (1-9), product of hydrolysis of the LHRH by PCAP. Under these conditions it is observed that the (Hyp 9) LHRH does not generate a fragment (1-9) Hyp9 LHRH in contact with a homogenate of the hypothalamus or of the hippocampus (FIG. 15). On the contrary, the fragment (1-9) LHRH accumulates very rapidly during the incubation of LHRH in the presence of homogenate of the two brain structures. This accumulation is twice as great for the hippocampus (1.44 ng / μg Prot / min) than for the hypothalamus (0.73 ng / μg Prot / min). This is the result of greater PCAP activity in the hippocampus.

 As a result, the lifespan of the C-terminal fragments of LHRH is shorter than that of the C-terminal fragments of (Hyp 9) LHRH. In addition, this lifespan, for the C-terminal fragments of LHRH, is considerably reduced in the hippocampus following a higher PPCE activity.

In conclusion, only endogenous C-terminal fragments of the (Hyp 9) LHRH are present in the hippocampus. The greater stability, the result of greater resistance to degradation, C-terminal fragments of (Hyp 9) HRH make these fragments the only possible endogenous ligands for possible specific receptors in the hippocampus. EXAMPLE 5 Demonstration of receptors and C-terminal fragments of (Hyp 9) LHRH in the hippocampus.

 A C-terminal fragment of the (Hyp 9) LHRH, the (4- 10) Hyp9 LHRH labeled with iodine 125 is linked to a membrane preparation of the hippocampus and specifically displaced by the (4-10) Hyp9 LHRH not radioactive (Figure 16). The displacement obtained is maximum. On the other hand, it is very weak with LHRH or with an LHRH super agonist, (DAla6, des Gly10) LHRH ethylamide.

 Conversely, the radioactive (DAla6, Gly10) LHRH ethylamide linked to the same membrane preparation of the hippocampus is only displaced by the non-radioactive (HRa ethylamide LHRH ethylamide) (Figure 17). Non-radioactive (4-10) Hyp9LHRH is ineffective under these conditions.

 In conclusion, the binding sites or the receptors recognizing LHRH (demonstrated by the (DAla6, desGly10) LHRH ethylamide) and the C-terminal fragments of (Hyp9) LHRH are not the same in the hippocampus. There are therefore receptors or a subclass of "LHRH" receptors specific only for C-terminal fragments of (Hyp9) LHRH in the hippocampus.

EXAMPLE 6 Demonstration of (Hyp 9) LHRH and LHRH in various tissues.

 A) In the hypothalamus, the hippocampus and the olfactory bulb.

1) Experimental protocol a) Tissue extraction processes.

 The hypothalamus, the hippocampus and the olfactory bulbs of the spleen are collected on dry ice and then lyophilized and stored at -30 ° C.

 The extraction begins with a homogenization of the tissues (10% dry weight / volume) in 2M acetic acid (AcOH) at 4ºC using a Teflon grinder and a glass potter. The homogenate is centrifuged at 30,000 g for 90 minutes. The supernatant is then removed and lyophilized.

 The lyophilisate is resuspended (half the volume of the initial homogenate) at 4ºC with 2M AcOH in 70% methanol (volume / volume) then centrifuged at 6000 g for 90 minutes. The supernatant is removed and subjected to vacuum evaporation to remove the methanol. The remaining extract is diluted in water then lyophilized and stored at -80ºC.

 b) High performance liquid chromatography (HPLC)

 ) Step 1:

The methanolic extracts corresponding to 1 g of dry weight of tissue are resuspended in 10 ml of a 15% solution (volume / volume) of acetonitrile (CH ₃ CN) in water containing 0.1% (volume / volume) of trifluoroacetic acid (TFA), then centrifuged at 30,000 g for 30 minutes. The supernatants are then chromatographed on a preparative micro-Bondapak C 18 column. After injection of the sample, the column is eluted for 20 minutes with 15% CH ₃ CN and then for 120 minutes with a gradient ranging from 15 to 25% CH ₃ CN. The flow rate is 2ml per minute and fractions of 2ml are collected. A 10 ml loop is used for injections. 2nd step:

The fractions (tubes 5 to 20) collected during step 1 and corresponding to 2 g of dry weight of tissue are dried and then combined by resuspension in 10 ml of water (H ₂ O) containing 0.025% TFA (volume / volume) or of a 1.5% solution of CH ₃ CN (volume / volume) in H ₂ O containing 0.1% TFA (volume / volume). The samples are then chromatographed on the preparative column described above. After injection of the samples, the column is eluted for 10 minutes with either 1.5% CH ₃ CN or H ₂ O containing 0.025% TFA, then for 120 minutes with either 1 simple gradient ranging from 1.5% to 25% CH ₃ CN is a gradient composed of three stages: 1) H ₂ O containing 0.02% TFA up to 9% CH ₃ CN for 70 minutes, 2) stationary phase at 9% CH ₃ CN for 40 minutes and 3) 9% up to 15% CH ₃ CN for 10 minutes. The flow rate is 2 ml per minute and fractions of 2 ml are collected. A 10 ml loop is used for injections.

 Control of stages.

 The chromatographic steps are calibrated with LHRH, (Hyp 9) LHRH and C-terminal fragments: (4-10), (5-10), (6-10), (7-10) and Ac

(5-10) of these two molecules. All of these molecules have been synthesized and identified (amino acid analysis) by Neosystem Laboratoires (Strasbourg). In order to avoid any misinterpretation of the elution profiles of endogenous LHRH immunoreactivities, the synthetic peptides were subjected to chromatography in the presence of tissues and then identified by radioimmunoassay. c) Radioimmunological assays (RIA) of molecules related to LHRH.

The fractions obtained on CHLP columns as well as the synthetic LHRH for the standard curve are lyophilized in the presence of 100 μl of the RIA buffer (50 mM Na / Na ₂ phosphate, pH 7, containing 0.9% (weight / volume) of NaCl and 0, 1% (weight / volume) of gelatin to avoid sticking of immunoreactive material on the walls of the tubes.

 The RIA assay used is an adaptation of the method described by Nett et al. (1973, Clin.

Endocrinol. Metab. 36, 880-885). The incubations are carried out in the presence of 100 μl of normal rabbit serum (1/60 dilution) and 10 μl of a pH indicator to check the presence of interfering substances at the pH level in the fractions collected by HPLC. The antigen-antibody complex is precipitated with 1 ml of absolute ethanol after 48 hours of reaction at 4ºC.

 d) Statistical analysis.

 Statistical analysis of the results was performed with Student's t-test.

 2) Results.

The LHRH-related immunoreactivity present in the hypothalamus (Figure 18 A), the olfactory bulb (Figure 18 B) and the hippocampus (Figure 18C) is distributed between three peaks after HPLC. The first is recognized exclusively by the antibody C and coeluted with C-terminal fragments of LHRH and (Hyp 9) LHRH. The second and third peaks co-elute with (Hyp 9) LHRH and LHRH respectively. The characterization of the second and third peaks detected in the olfactory bulb and the hippocampus was shown that the endogenous LHRH immunoreactivities eluted in these peaks correspond respectively to (Hyp 9) LHRH and to LHRH. With regard to the hypothalamus, the characterization of these two peaks has already been presented in Example 2.

 The total LHRH immunoreactivity is greater in the hypothalamus than in the olfactory bulb and in the hippocampus where it is substantially the same (Table 8). However, the proportions of the three immunoreactivities separated by HPLC are not the same in the three brain structures. LHRH represents 87% of the total immunoreactivity in the hypothalamus whereas it corresponds to 55% and 28% in the olfactory bulb and in the hippocampus. The situation is reversed for immunoreactivity C co-eluting with the C-terminal fragments. This immunoreactivity represents only 1% of the total LHRH immunoreactivity in the hypothalamus whereas it corresponds to 35% and 66% in the olfactory bulb and in the hippocampus. With regard to (Hyp 9) LHRH, its proportion does not change in the hypothalamus and in the olfactory bulb; but it practically halves in the hippocampus.

In certain tissues, the expression of the gene coding for LHRH, and therefore the quantity of LHRH, are regulated by sex steroids. Removal of these steroids (for example by means of castration) causes a decrease in the concentration of LHRH in the tissues. This is what we observe in the hippocampus, (table 9). Note, however, that only the immunoreactivities corresponding to (Hyp 9) LHRH and to the C- fragments terminals decrease significantly and concomitantly.

 Chromatography of immunoreactivity C (first peak, fractions 5 to 20) under slower elution conditions generates several peaks which, depending on the brain structure studied, coelute with C-terminal fragments of LHRH or of (Hyp 9) LHRH (Figures 19 and 20). For the hypothalamus, the majority C fragments come from the LHRH (FIGS. 19A and 20A). These are mainly (7-10), (6-10), (5-10) and (4-10) LHRH. The same fragments are also detected for (Hyp 9) LHRH. However, it should be noted that taking into account the different immunoreactivities of the C fragments of LHRH and of (Hyp 9) LHRH with respect to the antibody C, the description given above is only of a qualitative nature. For the olfactory bulb, the situation is practically the opposite of that observed in the hypothalamus, the majority fragment being (4-10) (Hyp9) LHRH (FIG. 19B). For the hippocampus, the only detectable fragments correspond to (7-10) and (6-10) (Hγp9) LHRH (Figure 20B). No LHRH fragment is detected.

 B) In the testicle.

 The (Hyp 9) LHRH and the LHRH are demonstrated by reverse phase HPLC with acid extracts in the Sertoli (FIG. 21A) and Leydig (21 B) cells eluted by an acetonitrile gradient.

The proportion of (Hyp 9) LHRH, compared to the total immunoreactivity related to LHRH, is not the same in the two cell types: 45% for Sertoli and 15% for Leydig. This difference, given the different regulations of Sertoli and Leydig cells as well as the different functions performed by these two cell types, suggests that (Hyp 9) LHRH or fragments of its molecule could play a role in the physiology of testicle, in particular on spermatogenesis and / or on the production of testosterone.

 CONCLUSION:

 Studies carried out on the biological activity of (Hyp 9) LHRH have made it possible to demonstrate a notable resistance of this peptide with respect to the degradation enzymes which very quickly inactivate LHRH itself. This resistance is thought to be due to the particular configuration of the (Hyp 9) LHRH in its C-terminal portion, which makes it a poor substrate for PCAP (post proline-cleaving enzyme or enzyme making a cut after proline). This resistance leads to a significant increase in the half-life of (Hyp 9) LHRH compared to LHRH and therefore of its pharmacokinetic properties. In in vivo models, it has thus been possible to show that the rise in plasma LH levels is maintained longer after administration of (Hyp 9) LHRH than of LHRH. In parallel, comparable doses of the two peptides lead to gonadotropic stimulation of the same amplitude, even though the affinity of (Hyp 9) LHRH for its receptor is less than that of LHRH.

On the other hand, in other tissues (in particular the ovary), the displacement of the binding of LHRH to its receptor can be obtained with equimolar concentrations of LHRH and (Hyp 9) LHRH. Those data, as well as those obtained on the existence of a heterogeneity of the LHRH receptors which would be expressed in certain tissues (pituitary) and not in others (brain, ovary) suggest that (Hyp 9) LHRH is a selective ligand d 'one of the receptor isoforms.

 It is the first time that the physico-chemical characterization of LHRH-related immunoreactivity has been described in the olfactory bulb and the hippocampus. Contrary to what is observed in the hypothalamus, LHRH and (Hyp 9) LHRH are not the only important molecular species in the olfactory bulb and the hippocampus, two brain structures involved in the control of sexual behavior. The present results indeed emphasize the C-terminal fragments which, in the case of the hippocampus, represent 70% of the total immunoreactivity related to LHRH.

 Such a distribution suggests that these fragments could have a biological activity within the central nervous system. This biological activity would correspond to that already described in the literature on sexual behavior.

It should be observed that, taking into account the different reactivities of the fragments with respect to the antibody C, the C-terminal fragments of the (Hyp 9) LHRH are probably in majority compared to those of the LHRH. This is perfectly illustrated in the h'ppocampus where only C-terminal fragments of the (Hyp 9) LHRH are detected. On the other hand, these results show that castraction affects only and concomitantly the (Hyp 9) LHRH and its C-terminal fragments in the hippocampus. These last two results suggest that (Hyp 9) LHRH and more particularly its C-terminal derivatives play a privileged biological role in the central nervous system.

 These results therefore emphasize the presence of C-terminal fragments of (Hyp 9) LHRH in extrahypothalamic brain structures. These fragments include a modification of the C-terminus of the LHRH. This modification (replacement of proline by hydroxyproline in position 9) would make it possible to generate one or more ligands (C-terminal fragments) more capable, than those generated from LHRH, of binding to brain receptors, thus triggering the process or processes of sexual behavior. This hypothesis supposes that the cerebral distribution of the C-terminal fragments originating from (Hyp 9) LHRH and from LHRH is not identical. This is what the present results show. C-terminal fragments of the two neurohormones are present in the hypothalamus, although they have no known biological effect on the pituitary gland, the only target organ for (Hyp 9) LHRH and hypothalampic LHRH. On the other hand, in the hippocampus where receptors for peptides related to LHRH are present, only the C-terminal fragments of the (Hyp 9) LHRH representing 70% of the hippocampal LHRH immunoreactivity, are highlighted, which suggests that they are the endogenous ligands of these receptors.

The potential clinical indications for (Hyp 9) LHRH fall within the gynecology and endocrinology of reproduction. The main advantages of this molecule, compared to LHRH, are due to its bioavailability. Reduced activity compared to that of LHRH, associated with a prolonged half-life, are positive elements for progressive stimulation of gonadotropic function, by better controlling the risk of hyperstimulation.

 ND not detectable

- The values given are molar ratios. The numbers in parentheses for the D peaks are the closest whole ratios. The Trp residues are destroyed under the hydrolysis conditions described in the text.

Claims

 1. Peptide exhibiting significant sequence homology with LHRH, characterized in that it has a high reactivity with respect to antibodies directed against the N-terminal end of LHRH and a low reactivity with respect to antibodies directed against the C-terminus of LHRH or directed against antigenic determinants linked to the three-dimensional tertiary structure of LHRH, by comparison with the respective reactivities of LHRH.

 2. Peptide according to claim 1, characterized in that it has the following amino acid sequence in the L configuration for residues 1 to 5 and 7 to 9.

 1 2 3 4 5 6 7 8 9 10

pyroGLU-HIS-TRP-SER-TYR-X-LEU-ARG-HYP-Y

in which X and Y can only be GLY residues simultaneously.

 3. Peptide according to claim 2, characterized in that X represents a residue -GLY.

 4. Peptide according to claim 2, characterized in that Y represents a residue -GLY-CO-

NH ₂ .

 5. Peptide according to claim 2, characterized in that Y represents an ethylamide group.

 6. Peptide according to claim 2, characterized in that X represents a residue D-ALA, D- SER, D-TRP OR D-HIS.

7. Peptide according to claim 2, characterized in that it has spectrometry of mass a band corresponding to a molecular weight of approximately 1198.8 Da.

8. Peptide according to claim 7, characterized in that X represents a residue -GLY and Y represents a residue-GLY-CO-NH ₂ .

 9. Fragment of the peptide according to one of claims 2 to 8 or of (Hyp 9) LHRH.

 10.Fragment according to claim 9, corresponding to the peptide sequence between amino acids 4 and 10.

 11. Peptide fragment according to claim 9, corresponding to the peptide sequence between amino acids 5 and 10.

 12. Peptide fragment according to claim 9, corresponding to the peptide sequence between amino acids 6 and 10.

 13. Peptide fragment according to claim 9, corresponding to the peptide sequence between amino acids 7 and 10.

 14. Peptide fragment according to any one of claims 9 to 13, characterized in that their N-terminal end carries a protective group.

 15. Peptide fragment according to claim 14, characterized in that said protective group is an acetyl, tert-butyloxycarbonyl (t-Boc) or benzyl-oxycarbonyl (Z) group.

 16. Peptide or peptide fragment according to any one of claims 1 to 15, characterized in that it is obtained by extraction from a tissue or a biological fluid.

17. Peptide or peptide fragment according to claim 16, characterized in that it is obtained at from the mammalian hypothalamus, and in particular from the human or rat hypothalamus.

 18. Peptide or peptide fragment according to one of claims 1 to 15, characterized in that it is obtained by synthesis.

 19. Peptide or peptide fragment according to one of claims 1 to 18, characterized in that it has an agonist or antagonist activity with respect to LHRH.

 20. Method for obtaining from tissues or biological fluids a peptide or peptide fragment according to any one of claims 1 to 16, or the (Hyp 9) LHRH comprising the succession of the following steps:

 - at least one step of overall protein extraction,

 - at least one cation exchange chromatography,

 - And / or at least one high performance liquid chromatography in reverse phase.

 21. The method of claim 20, characterized in that the tissues or fluids are part of the mammalian hypothalamus and in particular of the human or rat hypothalamus.

 22. Use of a peptide or peptide fragment according to one of claims 1 to 18 or of (Hyp 9) LHRH in the treatment of conditions relating to gynecology and / or endocrinology of reproduction, in particular in the treatment of infertility and syndromes related to early or delayed puberty.

23. Use of a peptide or peptide fragment according to one of claims 1 to 18, or LHRH (Hyp 9) in the treatment of cancerous conditions affecting the gonads or secondary sexual organs.

 24. Use of a peptide or peptide fragment according to one of claims 1 to 18 or of (Hyp 9) LHRH, in the treatment of psychiatric disorders, in particular disorders linked to libido.

 25. Use of a peptide or peptide fragment according to one of claims 1 to 18 or of (Hyp 9) LHRH, for the functional exploration of the hypothalamic-pituitary axis.

 26. Use of a peptide or peptide fragment according to one of claims 1 to 18 or of (Hyp 9) LHRH, for radioimmunological or biological assays of biological fluids and tissue biopsies.

 27. Medicament containing at least one of the compounds according to any one of claims 1 to 18.

 28. Medicine containing LHRH (Hyp 9).

 29. Medicament according to one of claims 27 and 28, for the treatment of conditions relating to gynecology or endocrinology of reproduction, in particular for the treatment of infertility and syndromes related to precocious or delayed puberty .

 30. Medicament according to one of claims 27 and 28, for the treatment of cancerous conditions affecting the gonads or the secondary sexual organs.

31. Medicament according to one of claims 27 and 28, for the treatment psychiatric conditions and in particular libido-related disorders.

 32. Pharmaceutical composition characterized in that it contains an effective amount of at least one compound according to any one of claims 1 to 18 or of (Hyp 9) LHRH in association with one or more diluents or adjuvants compatible and pharmaceutically acceptable.

 33. Composition according to claim 32, characterized in that it is intended for the treatment of conditions relating to gynecology and / or endocrinology of reproduction, in particular to the treatment of sterility and of syndromes related to puberty early or delayed.

 34. Composition according to claim 32, characterized in that it is intended for the treatment of cancerous conditions affecting the gonads or the secondary sexual organs.

 35. Composition according to claim 32, characterized in that it is intended for the treatment of affections relating to psychiatry, in particular disorders linked to the libido.

 36. Pharmaceutical composition according to one of claims 32 to 35 presented for parenteral or enteral administration.

 37. A method of radioimmunological or biological assay of biological fluids and tissue biopsies, characterized in that its implementation requires the use of a peptide according to one of claims 1 to 18.