FR2661180A1 - (HYDROXYPROLINE 9) LHRH, ITS PROCESS FOR OBTAINING AND APPLYING AS A MEDICINE. - 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

The subject of the invention is a new derivative of the
LHRH, 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. Common. 43, 393-399) and sheep (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 brain 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 the 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).

However, molecular heterogeneity in the immunoreactivity of mammalian LHRH (or mLHRH) has 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. nineteen eighty one,
Neuropeptides Biochemical and Physiological Studies, 263271; 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. Boarding school. 13, 757-765
Gautron et al. 1989), in the gonads (Dutlow et al. 1981.

Biochem. Biophys. Res. Common. 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, 258-263; King et al. 1988 ,
Endrocrinology, 122, 2742-2752; Stopa et al. 1988.

Peptides, 9, 419-423).

 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 the LHRH type could also be too large or insufficient to allow the detection of distinct genes using the oligodeoxynucleotide probes synthesized in function of the LHRH sequence (Seeburg et al. 1984, Nature, 83, 179-183).

 Furthermore, posttranscriptional 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 gene coding for the precursor of mLHRH.

In previous studies (Gautron et al. 1981,
Mol. Cel. 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 sieves 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 C end is essential for recognition by the antibody C.

As for the antibody W, it does not accept modification of the pyroGlu1 residue and of the Glyamide10 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 the m LHRH, characterized in that they exhibit a high reactivity with respect to antibodies directed against the N-terminus of the LHRH and a low reactivity towards antibodies directed against the C-terminal end of LHRH or directed against antigenic determinants linked to the three-dimensional tertiary structure of LHRH, in comparison with the respective reactivities of 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, in relation to the native LHRH molecule, the proline in 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
NH2 or an ethylamide group.

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

 These peptides 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 from tissues or biological fluids comprising - at least one step of overall protein extraction - at least one chromatography by cation exchange - and / or at least high performance liquid chromatography
formance (HPLC) in reverse phase.

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

 Another object of the invention is the use of these peptides and more particularly of (Hyp 9) LHRH in the treatment of conditions which can be linked to LHRH relating to gynecology and / or endocrinology of reproduction. , especially in the treatment of infertility and syndromes related to early or delayed puberty. These peptides can also be used in the treatment of cancerous conditions affecting the gonads (ovaries, testes) or secondary sexual organs (mammary glands, prostate) and in the treatment of psychiatric conditions, in particular behavioral disorders resulting in sexual aggression.

These peptides can also be used for functional exploration of the hypothalamic-pituitary axis, in particular by measurement by radioimmunological assays of the plasma and urinary LH levels,
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 more particularly (Hyp 9) LHRH, and in particular medicaments for the treatment of conditions which can be linked to LHRH relating to gynecology. or reproductive 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.

 In addition, the subject of the invention is pharmaceutical compositions characterized in that they contain at least one of the peptides previously described, in particular (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 linked to LHRH relating to gynecology and / or endocrinology of reproduction, to the treatment of cancerous conditions affecting the gonads and / or secondary sexual organs, or to the treatment of psychiatric disorders, in particular behavioral disorders resulting in sexual aggression.

 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 as previously described.

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 antibody 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 (125I) which was subjected to the extraction process in the presence of rat hypothalamus. Elution positions of radioactive non-extracted LHRH, non-radioactive LHRH and its fragments
N- and C- terminals are indicated.

 In these two figures, the ordinate represents the quantity of radioactivity in counts per minute.

 Figures 3 and 4 respectively represent the elution profiles on an HPLC cation exchange column of hypothalamic extracts of rat and human. The columns were calibrated as shown in Figure 3A and 4A respectively by mammalian LHRH (mLHRH), chicken LHRH (cLHRH-I), salmon LHRH (sLHRH) and lamprey LHRH ( lLHRH).

 Figures 3B and 4B respectively represent the chromatographies of the extracts of 1000 rat hypothalamus and 6 human hypothalamus. Peaks 1 and 2 of Figures 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 4 show the chromatography of peaks B in Figures 3C and 4C.

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

 Figures 5B and 5C respectively represent the elution on a micro-Bondapak column of the peaks B of Figures 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 synthetic mLHRH is shown in these Figures. The optical density at 280 nm is shown on the ordinate.

 Figures 7A and 7B respectively represent the molecular sieve filtration of the synthetic mLHRH and peaks C and D of Figure 5C.

 Figure 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.

Figure 8 shows the mass spectrum of the peak
C shown in Figure 5C.

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

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

 Figures 10A to 10E represent the chromatographic analysis on cation exchanger of the mLHRH (lOA), the (Hyp 9) LHRH (lOB), the (Leu 9) LHRH (1OC), the (Lys 8) LHRH (10D) and of peak C represented in FIG. 5C (10E), after digestion with trypsin (non-solid circles) or with clostripaine (solid circles).

 Figures 10F to 10I represent the reverse phase chromatographic analysis of fractions 25 to 30 isolated on a cation exchange column and generated respectively for Figures 10F to 10I from mLHRH, (Hyp 9) LHRH, (Leu 9) LHRH and of rat peak C.

 In Figure 10O, 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 .

 Figure 11 represents the chromatographic analysis on cation exchanger and on reverse phase of various molecules of the 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.

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

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

Figure 12 shows the elution by chromatography of human and rat C peaks and of LHRH (Hyp 9). In these Figures, the solid circles represent the elution of the peak
C of rat and the non-filled circles represent the elution of the (Hyp 9) LHRH. The elution of mLHRH is also indicated by an arrow.

FIGS. 12A, 12B, 12C represent respectively the elution after the stages of purification 1, 2 and 3, while FIG. 12E represents the elution of the peak C of rat in the presence of (Hyp 9) LHRH after the stage of purification 3, and Figure 12D shows the elution of the peak
Human C after the purification step 3.

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

 Figure 14 represents the variation of the secretion of FSH or LH, expressed in (ng / ml), according to the logarithm of the concentration of LHRH or in (Hyp 9) LHRH expressed in mole / l.

EXAMPLE 1 Obtaining and Purifying the Peptides of
type LHRH 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 -800C. At the same time, fresh rat hypothalamus were immediately extracted, tested for their content in LHRH type molecules and chromatographed 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 40C using 'a "Teflon" grinder 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 40C with 2 M acetic acid in methanol (MeOH) at 70% (volume / volume), then centrifuged 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 300C. The remaining extracts are diluted in cold water, frozen and then lyophilized and stored at -800C 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 of Table 1 show that the recovery of molecules detectable by anti antibodies
LHRH is most important after extraction of tissues with 2 M acetic acid. Extraction with 70% methanol is also very effective. In all cases, the quantity of LHRH-type immunoreactive material 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 radioactivity associated with 125 I-labeled LHRH (1251
LHRH) added to the tissue before extraction combined 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 stage: HPLC by exchange of cations
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 (NH4Ac), at pH 4.10, containing 30% (volume / volume) of acetonitrile (CH3CN) (starting buffer), then centrifuged at 30,000 g for 30 minutes. The supernatant is chromatographed on a preparative column TSK SP 5 PW (21.5 X 300 mm, LKB). After a 60-minute washing period, the elution is carried out with a 120-minute gradient 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 amount 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 -800C. 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 Figures 3 and 4, the immunoreactivity profiles with antibody W are not shown to avoid confusion. These profiles are identical to those obtained with the antibody C.

 During the first pass and under the elution conditions used, both human and rat LHRH type materials elute in a single broad peak which is better recognized by the antibody N than by the antibodies C or W as shown Figures 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
micro-Bondapak column
The lyophilized peaks B, originating from stage I 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 chromatographed on a micro column
Bondapak C18 (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 single 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 represented on the
Figures 5B and 5C. Washing with 100% acetonitrile does not make it possible to detect other immunoreactive peaks.

Peak C is eluted before 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.

Peaks C and D represent respectively for the antibody N 12% and 88% of the total immunoreactivity of the LHRH type molecules recovered on the micro column
Bondapak, compared to 4% and 96% for 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 column chromatography
phenyl micro-Bondapak.

 Only the immunoreactive material obtained from the hypothalamus of rats is subjected to this step. The immunoreactive peaks s (peaks C and D) isolated during step 2 are respectively resuspended in full with 3 to 4 ml of acetonitrile at 14 and 15% (volume / volume) 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 corresponds perfectly with the W peak. The synthetic mLHRH is eluted later under these chromatography conditions.

 Peak D is also eluted in a single peak (Figure 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 perfectly matches the W 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 chromatography on phenyl micro-Bondapak.

3) Control of extraction and chromatoqra processes
phiques:
Identical amounts of rat hypothalamus of both sexes (15 females and 15 males) are homogenized in different extraction media: hydrochloric acid 0.2
M; 0.1% (volume / volume) of TFA in water; 70% (volume / volume) of methanol in water; 0.2 M acetic acid and 2 M 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 column
CHLP under the chromatography conditions of step 2.

 The recovery of 125I 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. The radioactivity is monitored during the 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, LRB) is used in place of that used in step 1. In in this case, the elution is carried out for 60 minutes with a gradient ranging from the starting buffer to 20% of a 200 mm ammonium acetate buffer, at a 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 terminals of the mLHRH.

 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 modes
extraction
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. 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 l 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 immunolo radio detection tests
qie (RIA) of LHRH tvpe 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 / Na2 phosphate buffer, pH 7, containing 0.9% (weight / volume) of NaC1 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 monoiodized with 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 verify the presence of interfering substances at the level 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 40C.

EXAMPLE 2 Characterization of the peaks obtained in Example
ple 1 1) Filtration on molecular sieve
The purified peptides and eluted in peaks C and
D rats 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 conditions on a TSK G 2000 SW column (7.5 × 300 mm,
Interchim.) Eluted with the same buffer used for 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 200 microliters loop is used for 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.

 Figure 7 shows 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 analysis (Neosystem
Laboratories) and the peak was analyzed between 1000 and 1400 m / z. The results in Figure 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 rat peaks C and D are hydrolyzed in 6 M hydrochloric acid at 1100C 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 mass difference (16.04 daltons) due to the replacement of proline by leucine is the value closest to the mass difference (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 (Hvp 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) Enzyme disestion
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 ethylenediamine tetraacetic acid 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 9O0C.

 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 ammonium acetate, pH 4, 10, and 30% acetonitrile. The product of digestion by
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 × 75 mm, Waters) with a gradient for 30 minutes ranging from 15 to 20 or 25% in acetonitrile in water containing 0.1% of 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 also cut the enogenous peptide eluted in peak C (peak peptide C) which may or may not generate the common fragment (1-8) m LHRH. The cuts are shown in Figure 9.

Synthetic mLHRH, (Hyp 9) LHRH and (Lys 8)
LHRH and 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 the
LHRH on cation exchange column as shown on the
Figure 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 the hydrolysis correspond to a common peptide which co-elutes with the synthetic (1-8) mLHRH, which indicates that the cleavages in the sequences have takes place exclusively after arginine.

 Digestion with pyroglutamate amminopeptidase 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 other LHRH Cterminal fragments on a cation exchange column, as shown in Figure 11. A 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 indicates that the cleavage takes place exclusively at the pyro Glul-His2 bond in the experimental conditions previously described. 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, immunoreactivity C generated from the peak C peptide co-eluted with the hydrolysis product of (Hyp 9) LHRH.

6) Chromatographic behaviors of the peak C peptide
and the (Hvp 9! LHRH
The chromatographic behaviors of the rat C peak 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. The
Figures 12A, 12B and 12C correspond to the peptides resulting from the 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 (Figure 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 unambiguously 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 with trypsin, clostripaine or pyroglutamate amminopeptidase confirmed that this second form of molecule of the LHRH type is indeed a (hydroxyproline 9) LHRH.

EXAMPLE 3 Biological activity of (Hvp 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 125I linked to preparations of pituitary membranes , is fifteen times weaker than that of the
LHRH. This less apparent activity is however compensated by a prolonged bioavailability.

 Figure 14 represents the variation of the secretion of FSH or LH, expressed in (ng / ml) according to the logarithm of the concentration of LHRH or in (Hyp 9) LHRH expressed in mole / l.

Preliminary studies carried out on the biological activity of (Hyp 9) LHRH have indeed made it possible to highlight a notable resistance of this peptide with respect to the degradation enzymes which very quickly inactivate the
LHRH itself. This resistance is due to the particular configuration of the (Hyp 9) LHRH in its C-terminal portion, which makes it a bad substrate for PCAP (post prolinecleaving enzyme or enzyme making a cut after proline). This resistance results in a significant increase in the half-life of (Hyp 9) LHRH compared to
LHRH and therefore 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 LHRH. In parallel, comparable doses of the two peptides lead to gonadotropic stimulation of the same amplitude and this although 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. These 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 for one of the receptor isoforms.

 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 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.

TABLE 1
RATE OF LHRH TYPE MOLECULES IN RAT HYPOTHALAMUSES
AFTER EXTRACTION BY VARIOUS PROCEDURES
pg LHRH / hypothalamus
C / N% W / N%
Extraction (1) ANTIBODY-N% ANTIBODY-C% ANTIBODY W%
(2) (2) (2) 2 M HOAc 5.924 # 138 100 5.236 # 210 100 3.973 # 134 100 88 67 0.2 M HOAc 4.087 # 134 69 5.560 # 131 68 2.848 # 94 72 87 70 0.2 M HCl 4.206 # 112 71 3.716 # 188 71 2.980 # 102 75 88 71 0.1% TFA in 4.265 # 156 72 3.770 # 100 72 3.155 # 125 79 88 74
H2O 70% MeOH 4,875 # 78 82 4,300 # 112 82 3,377 # 108 85 88 69 (1) extracts performed on groups of lyophilized hypothalamus of 15 males and 15 females (2) average # standard deviation on 3 extractions.

TABLE 2
LEHR TYPE MOLECULES IN RAT AND HUMAN HYPOTHALAMUS
AFTER COMBINED EXTRACTION
Pg LHRH / hypothalamus mg protein / hypothalamus
ANTIBODY-N ANTIBODY-C ANTIBODY-W
RAT (FRESH FABRIC)
(1) (1) 2M HOAc 6,600 # 255 4,855 # 227 NTNT

2M HOAc, 70% MeOH 6,694 # 245 4,940 210 N.T. N.T.

RAT (Lyophilized fabric)
(1) (1) (1) 2M HOAc 6.589 # 3634 5.436 # 378 4.842 # 185 2.515 # 0.095 2M HOAc, 70% MeOH 6.680 # 314 5.10 # 116 4.864 # 116 0.530 # 0.040
mg LHRH / hypothalamus
MAN (LYOPHILIZED FABRIC)
(2) (2) (2) 2M HOAc 45 36 33 32 2M HOAc, 70% MeOH 48 38 34 7 (1) AVERAGE # standard deviation calculated on 6 extractions (2) values corresponding to an extraction
NT = not tested.

TABLE 3
RECOVERY OF RADIOACTIVITY
LINKED TO THE SYNTHETIC LHRH
AFTER EXTRACTION IN THE PRESENCE OF RAT HYPOTHALAMUS
RADIOACTIVITY%
CPM / TOTAL VOLUME
FIRST STAGE OF EXTRACTION
(2M HOAc)
(1)
HOMOGENATE 230 500 + 3898 100
SURNANTANT 221 200 + 5354 96
POCKET 1 153 + 165 0.5 SECOND EXTRACTION STEP
(2M HOAc / 70% MeOH)
(1)
HOMOGENATE 215 600 + 4565 100
SURNANTANT 215 208 + 3995 98
BASE 861 = 132 0.4 (1) AVERAGE + STANDARD DIFFERENCE ON 5 EXTRACTIONS TABLE 4
DISTRIBUTION OF LHRH TYPE MOLECULES DURING REVERSE PHASE HPLC CHROMATOGRAPHY
FROM HUMAN AND RAT PICS
G LHRH (1)
ANTIBODY-N% ANTIBODY-C% ANTIBODY W%
Rat
TOTAL (peaks C and D) 24,200 100 22,300 100 22,340 100 peak C 2,880 12 0.940 4 0.960 4 peak D 21.320 88 21.360 96 21.380 96
ng LHRH (2)
HUMAN
TOTAL (peaks C and D) 38,240 100 35,450 100 35,468 100 peak C 4,200 11 1,450 4 1,418 4 peak D 34,040 89 34,000 95 34,050 95 (1) Values corresponding to the extraction of 6000 rat hypotalamus.

(2) Values corresponding to the extraction of 6 human hypothalamus.

note: the same results are observed from fresh rat hypothalamus.

TABLE 5
DATA ON PURIFICATION PROCESSES
degree of
LHRH (1)% mg Protein gLHRH / mg Prot. purification (2)
EXTRACT 2M HOAc, 70% MeOH 40.164 100 3180 (3) 0.013 1
Cation exchanger (step 1)
Peak B 25.160 65 0.6530 (3) 38.530 2,964 Bondapak (step 2)
Peak C 2,880 7 0.0170 (4) 169.410 13,032
Peak D 21.320 53 0.0640 (4) 333.125 25 625
Phenyl Bondapak (step 3)
Pic C 2 350 6 0.0024 (4) 975.167 75 090
Peak D 19 188 48 0.0190 (4) 1010 77 690 (1) Immunoreactivity N for all of the rat hypothalamus extract (2) ratio g LHRH / mg protein of the given purification step compared to the crude extract (3) measured quantities of proteins (4) estimated quantities of proteins according to the UV spectra TABLE 6
RECOVERY OF PICS C AND D OF RATS AFTER INVERSE PHASE HPLC CHROMATOGRAPHY
(Step 2) AFTER DIFFERENT TISSUE EXTRACTION PROCEDURES
ng of LHRH type molecules / total fraction collected (1)
PIC extraction C% PI D% PIC C + PIC D%
(2) (2) (2) 2M HOAc / 70% MeOH 25 # 0.15 19 106 # 0.50 81 131 # 0.42 100
(100%) (100%) (100%) 0.2M HOAc 17 # 0.09 18 76 # 0.35 82 93 # 0.030 100
(68%) (71%) (71%) 0.2M HCl 18 # 0.11 18 81 # 0.31 82 99 # 0.25 100
(72%) (76%) (76%) 0.1% VAT 18 # 0.07 17 88 # 0.40 83 106 # 0.29 100
(72%) (83%) (81%) 70% MeOH 21 # 0.12 19 89 # 0.45 81 110 # 0.32 100
(84%) (81%) (84%) 2M HOAc / 70% MeOH 24 # 0.10 1.2 1995 # 2.50 98.8 2019 # 1.85 100 and 2 g mLHRH (3) (96% ) (1882%) (1541%) 2M HOAc / 70% MeOH 24 # 0.13 0.06 4090 # 2.75 99.4 4114 # 2.00 100 and 4 g mLHRH (3) (96%) (3858 %) (3140%) (1) N immunoreactivity of the fractions collected corresponding to the chromatography of extracts of 28 spleen hypotalamus (14 males, 14 females) (2) mean # standard deviation (3) the synthetic LHRH was added in the extraction medium before homogenization of the tissues.

TABLE 7
AMINO ACID COMPOSITIONS OF PEPTIDES
ELECTED IN RATS PICS C AND D
IN THE CONDITIONS OF STEP 3
Residue Pic C Pic D (PIC C- PIC D)
Arg 1.16 0.96 (1) + 0.20
Ser 1.71 0.97 (1) + 0.74 Glx 1.18 0.83 (1) + 0.35
Gly 2.22 2.09 (2) + 0.13
Tyr 0.82 0.80 (1) + 0.02
Pro 0.23 1.08 (1) - 0.85
Leu 2 1 (1) + 1
His 0.98 0.96 (1) + 0.02
Trp NDNDND

Thr 0.14 - + 0.14
Ala 0.30 - + 0.30
Val 0.27 - + 0.27
Phe 0.19 - + 0.19
Island 0.16 - + 0.16
ND not detectable - The values given are molar ratios. The
numbers in parentheses for the D peaks are the
closest whole reports. Trp residues are
destroyed under the hydrolysis conditions described in
the text.

Claims (29)

 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 the LHRH or directed against antigenic determinants linked to the tertiary three-dimensional structure of the LHRH, in comparison with the respective reactivities of the LHRH.  2. Peptide according to 1A claim 1, characterized in that it has the following amino acid sequence in configuration L 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  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-NH2.  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 in mass spectrometry 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-NH2.  9. Peptide according to any one of claims 1 to 8, characterized in that it is obtained by extraction from a tissue or a biological fluid.  10. Peptide according to claim 9, characterized in that it is obtained from the hypothalamus of mammalian mothers, and in particular from the hypothalamus of humans or rats.  11. Peptide according to claim 1 to 8, characterized in that it is obtained by synthesis.  12. Peptide according to one of claims 1 to 11, characterized in that it has an agonist or antagonist activity vis-à-vis the LHRH.  13. A method of obtaining from tissues or biological fluids the peptide according to any one of claims 1 to 8, comprising the succession of the following steps - at least one step of overall protein extraction, - at least one chromatography by cation exchange, - and / or at least high performance liquid chromatography  reverse phase training.  14. The method of claim 13, characterized in that the tissues or fluids are part of the mammalian hypothalamus and in particular of the human or rat hypothalamus.  15. Use of a peptide according to one of claims 1 to 11 in the treatment of conditions relating to gynecology and / or endocrinology of reproduction, in particular in the treatment of sterility and syndromes related to precocious or delayed puberty.  16. Use of a peptide according to one of claims 1 to 11, in the treatment of cancerous conditions affecting the gonads or the secondary sexual organs.  17. Use of a peptide according to one of claims 1 to 11, in the treatment of psychiatric disorders, in particular behavioral disorders resulting in sexual aggression.  18. Use of a peptide according to one of claims 1 to 11, for the functional exploration of the hypothalamic-pituitary axis.  19. Use of a peptide according to one of claims 1 to 11, for radioimmunological or biological assays of biological fluids and tissue biopsies.  20. Medicament containing at least one of the compounds according to any one of claims 1 to 11.  21. The medicament as claimed in claim 20, for the treatment of conditions relating to reproductive gynecology or endocrinology, in particular for the treatment of infertility and of syndromes linked to precocious or delayed puberty.  22. The medicament as claimed in claim 20, for the treatment of cancerous conditions affecting the gonads or the secondary sexual organs.  23. The medicament as claimed in claim 20, for the treatment of psychiatric disorders and in particular behavioral disorders resulting in sexual aggression.  24. Pharmaceutical composition characterized in that it contains an effective amount of at least one compound according to any one of claims 1 to 11 in combination with one or more compatible and pharmaceutically acceptable diluents or adjuvants.  25. Composition according to claim 24, 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 infertility and of syndromes related to puberty early or delayed.  26. Composition according to claim 24, characterized in that it is intended for the treatment of cancerous affections affecting the gonads or the secondary sexual organs.  27. Composition according to claim 24, characterized in that it is intended for the treatment of affections relating to psychiatry, in particular behavioral disorders resulting in sexual aggression.  28. Pharmaceutical composition according to one of claims 24 to 27, presented for parenteral or enteral administration.  29. A method of radioimmunological or biological assay of biological fluids and tissue biposies, characterized in that its implementation requires the use of a peptide according to one of claims 1 to 11.