EP2951200A1 - Composés agonistes du récepteur kiss1 et leur utilisation pour induire l'ovulation chez les mammifères - Google Patents
Composés agonistes du récepteur kiss1 et leur utilisation pour induire l'ovulation chez les mammifèresInfo
- Publication number
- EP2951200A1 EP2951200A1 EP14702555.5A EP14702555A EP2951200A1 EP 2951200 A1 EP2951200 A1 EP 2951200A1 EP 14702555 A EP14702555 A EP 14702555A EP 2951200 A1 EP2951200 A1 EP 2951200A1
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- European Patent Office
- Prior art keywords
- peptide
- peptide compound
- compound
- compound according
- group
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/07—Tetrapeptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/02—Linear peptides containing at least one abnormal peptide link
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention is in the field of induction and synchronization of ovulation in mammals. More particularly, it relates to a peptide compound agonist of the KISS1 R receptor capable of inducing / synchronizing such ovulation, as well as the use of such a compound as a medicament, in particular for inducing this ovulation, and a veterinary composition and / or pharmaceutical container.
- the compound according to the invention has applications in the field of animal husbandry, in particular with a view to programming breeding throughout the year, for example sheep, goats, or cattle. Although the latter species is not of the spawning type, dairy cows nevertheless show a decline in fertility and a KISS1 R receptor agonist could be used to restore better fertility.
- the compound according to the invention also has applications in the therapeutic field, in particular for reducing the problems of infertility, and in particular in the human clinic for the treatment of reproductive pathologies, in particular in the context of the implementation of medically assisted procreation techniques.
- this compound is the treatment of other pathologies, such as hypothalamic amenorrhea and delayed puberty, and all other pathologies that require an increase in the secretion of GnRH (for Gonadotropin Releasing Hormone) and gonadotropins.
- GnRH for Gonadotropin Releasing Hormone
- the estrogen hormones used are pollutants that are not easily degraded, accumulate in the soil and water, and pose a threat to human health.
- the treatment of animals with such hormones also presents important constraints for the breeders, who are obliged to respect very strict specifications.
- the methods currently used to induce ovulation in sheep provide for the implantation for 12 to 24 days of a vaginal sponge containing progesterone, combined with co-treatment with an injection. intramuscular serum gonadotropin PMSG at the time of removal of the sponge. Such treatment is binding to implement.
- KISS1 R also called KISS1 receptor, or GPR54
- GPR54 GeneBank accession number, for the human receptor: NM-032551 .4, Gl: 189163516
- kisspeptides or kisspeptins
- KISS1 Endogenous ligands, called kisspeptides (or kisspeptins)
- KISS1 Endogenous ligands, called kisspeptides (or kisspeptins)
- KISS1 GenBank accession number, for the human peptide: NM_002256.3, Gl: 1 16829963; NP_002247.3 , Gl: 16829964; for the murine peptide: AB666166.1, Gl: 384367966; BAM1 1250.1, Gl: 384367967; for the ovine peptide: AFW03832.1, Gl: 41 1 100741), which are neurotransmitters having the ability to to stimulate KISS1 R (Kotani et al.,
- the abbreviation KISS1 R will be used to designate both the human receptor and the receptor of other species (indicated by the abbreviation Kissl r in the official nomenclature).
- a complete list of synonyms used to designate the KISS1 R is available on the IUPHAR website.
- the kisspeptide (kisspeptin) essentially responsible for the biological stimulation activity of KISS1 R is the decapeptide named kisspeptin-10, or KP10.
- the sequences of the mouse-derived kisspeptin-10 (mKP10, of sequence SEQ ID NO: 1, in which the C-terminal end is modified by amidation) and of the human-derived kisspeptin-10 are known ( hKP10, of sequence SEQ ID NO: 2, in which the C-terminal end is modified by amidation).
- the kisspeptin-10 derived from the ewe is in particular identical to mKP10.
- KP10 induces a very powerful stimulatory effect on the release of hormones LH and FSH in mammals, this effect resulting from an increase in the secretion of GnRH (for Gonadotropin Releasing Hormone) (Caraty and Franceschini, 2008).
- GnRH for Gonadotropin Releasing Hormone
- KP10 administered to ewes intravenously at the end of the follicular phase, is capable of inducing a peak of LH which is followed, 21 hours later, by ovulations synchronized to the hour (Caraty et al., 2007).
- KP10 Compared to steroid hormones, KP10 has the advantages of being very quickly eliminated from the body and easily destroyed in the natural environment, leaving only amino acids as residues. Moreover, it presents a mechanism of action, resulting in a triggering of the secretion of the hormone LH, hormone necessary for the induction of ovulation, completely different from that of current treatments, and allowing a more localized action and fine by avoiding unwanted side effects.
- KP10 is rapidly degraded and excreted by the body, it has a limited duration of action.
- the present inventors have therefore aimed to develop compounds derived from KP10 which have a prolonged and controlled duration of action in the body, while maintaining similar environmental degradability properties to those of KP10.
- the invention also aims that these compounds are capable of effectively inducing ovulation in a female mammal, including in off-season, by a small number of administrations, in particular by a single injection.
- a further object of the invention is that these compounds have a low cost of preparation.
- peptide compound agonist KISS1 R selected from:
- ⁇ [ ⁇ ] represents a 1,2,3-triazole-1,4-disubstituted group of formula (I):
- Xaa1 represents Gly or Ala
- Xaa2 represents Leu or a similar aliphatic ⁇ -amino acyl residue, such as Ile, Val, Ala (cPr), Nie or Nval
- Xaa3 represents Arg, whose -NH 2 function is optionally substituted by a methyl group, or a positively-charged pos-like cc-amino acyl residue, such as Arg (asymme 2 ), Lys, optionally substituted, or Orn, where appropriate substituted
- Xaa4 represents Tyr, Phe, Trp or a cc-amino acyl residue of similar aryl alanine type, such as 1 -, 2- or 3-naphthylalanine, 2- or 3-thienylalanine, 2- or 3-membered -furylalanine, 2-, 3- or 4-fluorophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or 4-bromorophenylalanine, 2-,
- the disubstituted 1,2,3-triazole ring is advantageously an inexpensive and easily accessible isostere, so that the process for synthesizing such a compound is advantageously inexpensive to implement.
- This synthesis can also be easily achieved by conventional peptide synthesis techniques, for example solid phase in Fmoc / tBu strategy.
- the peptide compound according to the invention advantageously has, with respect to KP10, a prolonged lifespan in the body, more particularly in the blood serum, while retaining a stimulating efficiency of KISS1 R that is as important, or even better, as well as minimal environmental remanence. It makes it possible in particular to significantly increase the blood level of the LH hormone in mammals, including during the anestrus period, by a limited number of administrations, moreover by a route of administration which is particularly compatible with use in breeding, in particular by means of a single injection, for example intramuscular or subcutaneous.
- C-terminal region will be used to denote the region of the peptide compound of sequence SEQ ID NO: 3, and by the term “N-terminal region", the remaining region of the compound.
- Xaa1 represents Gly
- Xaa2 represents Leu
- Xaa3 represents Arg, whose -NH 2 function is optionally substituted with a methyl group.
- the peptide compound according to the invention may have a size of from 5 to 54 amino acids, preferably from 5 to 16 amino acids, more preferably from 7 to 12 amino acids, and preferably 9, 10 or 1 1 amino acids.
- the peptide compound has one or more of the following characteristics:
- N-terminal amine function is modified by substitution with a group chosen from linear alkanoyls, in particular C 1 -C 6, preferably C 2 -C, benzoyl groups and tetramethylguanidinium groups,
- N-terminal amino function is replaced by an azide function, or by a 1,4-disubstituted 1,2,3-triazole group of formula (I);
- At least one amino acid, more particularly the N-terminal region is linked to one or more polyalkylene glycol chains, preferably polyethylene glycol, and / or to one or more lipid chains, such as a palmitoyl group, and / or groups binding to serum albumin.
- polyalkylene glycol chains preferably polyethylene glycol, and / or to one or more lipid chains, such as a palmitoyl group, and / or groups binding to serum albumin.
- at least one amino acid is linked to a polyethylene glycol chain, linear or branched, of molecular weight between 5 and 40 kDa; at least the N-terminal amine functional group is substituted by an alkyl group, especially chosen from linear, preferably C 1 -C 6, alkyls, in particular by a methyl group, or by a benzyl group.
- the peptide compound according to the invention can be cyclized, by any conventional cross-linking in itself between the side chains of two amino acids of its N-terminal region and / or its N-terminal amine function, in particular by formation of a amide, a triazole, an alkene, a disulfide bridge, a bis-thioether, etc.
- the peptide compound is chosen from:
- pseudo-peptide of sequence SEQ ID NO: 4 capable of binding KISS1 R
- one or more modifications at the level of the N-terminal region in particular by addition, substitution, by a natural or non-natural amino acid, including D enantiomers, and / or deletion, of one or more acids amines, and / or by a modification of the N-terminus and / or of any functional group carried by the side chain of an amino acid, and / or by the replacement of a peptide bond by an isosteric bond, any in retaining the ability to bind KISS1 R.
- the ability of peptide compounds to bind KISS1 R can be controlled in different ways. It may in particular be controlled by an in vivo test, by measuring the concentration of LH in ewe blood samples to which the test molecule has been injected, for example according to the protocol described hereinafter in the present description. An increase in this concentration, relative to an untreated control, indicates an ability of the analog to bind KISS1 R.
- this ability can be controlled by in vitro test, by measuring the amount of intracellular calcium in a cell line expressing KISS1 R after incubation with the test molecule.
- the stimulation of KISS1 R leads to the activation of two distinct intracellular pathways, which induce the increase of the intracellular concentration of calcium ions: by release of the intracellular stores, following the production of IP3, and by entry following the opening of the ion channels (for example TRPC) of the plasma membrane of the cell, calcium ions present in the extracellular medium.
- An example of such a test, called calcium mobilization is described in detail hereinafter in the present description.
- An increase in the amount of intracellular calcium, relative to an untreated control then testifies to the ability of the analog to bind KISS1 R.
- Particular analogues of the pseudo-peptide of sequence SEQ ID NO: 4, in particular having a half-life in the blood serum greater than that of KP10, correspond to one or more of the following characteristics: at least one acid
- the amine of Tyr1, Asn2 and Trp3 is substituted with a lysine, the amine function of which is preferably substituted by one or more groups selected from an alkanoyl group, in particular an acetyl group, a polyalkyleneglycol chain, preferably polyethylene glycol, and a chain.
- lipid such as a palmitoyl group or other group allowing interaction with serum albumin; at least the amino acid Tyr1 is replaced by its enantiomer D.
- Particular peptide compounds according to the invention are analogs of the pseudo-peptide of sequence SED ID NO: 4, differing from the latter but retaining a serine residue (Ser) two positions upstream of Xaa1, that is, in most cases, in position 5.
- Ser serine residue
- peptide compounds according to the invention have a threonine (Thr) residue at this position.
- Particular peptide compounds according to the present invention are: H-Tyr-Asn-Trp-Asn-Ser-Phe-G (Tz) Leu-Arg-Tyr-NH 2 (SEQ ID NO: 5)
- N-terminus is modified by acetylation
- particularly advantageous peptide compounds according to the present invention are such that at least one amino acid is linked to a unit capable of binding to serum albumin. It has been observed by the present inventors that, surprisingly, such a characteristic has the effect of very significantly increasing the duration of action of the peptide compound in the body.
- the unit capable of binding serum albumin is attached to an amino acid located in the N-terminal region of the peptide compound.
- a ⁇ -glutamyl spacer group between the amino acid and a group capable of binding to serum albumin such as a hexadecanoyl group (also called palmityl)
- increases the affinity of said group with serum albumin Kernudsen et al., 2000).
- Any other motif capable of binding serum albumin may also be used in the context of the invention, for example a acide-carboxylate fatty acid (Zarandi et al., 2006), Albu-tag (Dumelin et al., 2008) or a cyclopeptide (Dennis et al., 2002, Angelini et al., 2012).
- Examples of such units, and associated spacer arms for their binding to a residue of the peptide compound according to the invention are in particular TTDS- (y- (/ V-hexadecanoyl-Glu-OH)) or else 2- ( succinamido) -6- (4- (4-iodophenyl) butanamido) hexanoate.
- TTDS- y- (/ V-hexadecanoyl-Glu-OH)
- 2- ( succinamido) -6- (4- (4-iodophenyl) butanamido) hexanoate is an analogue of the pseudo-peptide of sequence SEQ ID NO: 4
- a unit able to bind serum albumin is introduced at least at position 1, at position 2 and / or in position 3 of the peptide compound, that is to say at the level of Tyr1, Asn2 and / or Trp3, or residues which are analogous thereto.
- the unit When the pattern capable of binding serum albumin is set at position 1, the unit is preferably attached to the N-terminal amino function.
- the residue concerned for example the residue Asn2 or the residue Trp3
- the residue concerned is preferably substituted by a lysine residue, on the chain. side of which is fixed the pattern capable of binding serum albumin.
- Another aspect of the invention is the use of a peptide compound having one or more of the above-described characteristics as a medicament, and more particularly for inducing ovulation in a female mammal.
- This mammal may especially be a farm animal such as a sheep, a goat, a bovine, a pig, an equine, etc., a pet, such as a dog or a cat, or, for example, a wild animal, as found in zoos and animal parks, etc. ; otherwise, this mammal can be a human being.
- the invention relates to the use of such a peptide compound to stimulate KISS1 R, in order to increase the secretion of GnRH, and consequently to stimulate the release of LH and / or FSH hormones in a patient. mammal.
- the peptide compound according to the invention may especially be used in the context of the treatment of pathological conditions resulting from low circulating levels of LH and FSH, for example pathological conditions resulting from insufficient pituitary stimulation. More generally, it can be used for the treatment of pathologies related to a reduction in the activity of the hypothalamic-pituitary-gonad axis, such as amenorrhea of hypothalamic origin or puberty delays.
- peptide compound according to the invention are in particular the treatment of certain forms of cancer, sensitive to steroid hormones, or the delay of aging by stimulating the secretion of GnRH (Zhang et al., 2013).
- the peptide compound according to the invention is preferably administered in the form of injection, which may for example be carried out intramuscularly, intravenously, subcutaneously, or intradermally.
- the treatment may consist of a single injection of the compound to the mammal to be treated.
- the administration can also be performed orally.
- the dose of the peptide compound administered according to the invention may be between 1 ⁇ 9 and 1 mg, in particular between 1 ⁇ g and 250 ⁇ g, depending on the mammal and the molecular mass of the compound. For example, for an ewe, the administered dose may be about 10 ⁇ g.
- the present invention relates to a veterinary or pharmaceutical composition, in particular for inducing ovulation in a female mammal, which contains a peptide compound corresponding to one or more of the above characteristics, in a pharmaceutically acceptable vehicle.
- composition is preferably in a form that can be administered intramuscularly, subcutaneously, intravenously or intradermally, or in an orally administrable form.
- FIGS. 2a to 2i are graphs showing the fluorescence intensity, in arbitrary units, as a function of the concentration of compound, obtained in an intracellular calcium mobilization test showing the activation of KISS1 R by the compound, for the following compounds according to the invention: FIG.
- FIG. 3 shows the concentration of LH measured in blood samples taken from ewes during the anoestrus period, at different times before and after the intravenous administration of physiological solutions containing respectively 5 nmol / ew of the compounds Comp.
- FIG. 4 shows the concentration of LH measured in blood samples taken from oestrus ewes, at different times before and after the intravenous administration of physiological saline containing respectively 5 nmol / ewe of the comparative compound Comp. 1, compounds according to the invention C1, C4 and C6, and physiological solution alone (T), the moment of administration being indicated by an arrow in the figure; said concentration of LH being expressed as a percentage with respect to the maximum concentration of LH measured after injection of 5 nmol / ew of Comp.1 taken as reference (% of the maximum stimulation of Comp.1);
- FIGS. 5a to 5i are graphs showing the fluorescence intensity, in arbitrary units, as a function of the concentration of compound, obtained in an intracellular calcium mobilization test showing the activation of KISS1 R by the compound, for the following compounds according to the invention: FIG. 5a, compound C8; Figure 5b, compound C9; Figure 5c, compound C10; Figure 5d, compound C1 1; Figure 5e, compound C12; Figure 5f, compound C13; Figure 5g, compound C14; Figure 5h, compound C15; Figure 5i, compound C16; and, in all the figures, for the comparative compound Comp.1;
- FIGS. 6a to 6d show the concentration of LH measured in blood samples taken from ewes during the anoestrus period, at different times before and after the intravenous administration of physiological solutions containing respectively 5 nmol / ewe of the compounds: Figure 6a, Comp.1; Figure 6b, Comp.3; Figure 6c, C1; Figure 6d, C2;
- FIG. 7 is a histogram showing the area under the curve (AUC) calculated for each curve of FIGS. 6a to 6d;
- FIGS. 8a and 8b show the concentration of LH measured in blood samples taken from ewes during the anoestrus period, at different times before and after the intravenous administration of physiological solutions respectively containing the compounds according to the invention: Figure 8a, C2; Figure 8b, C8, in concentrations of 1 nmol / ewe, 5 nmol / ewe and 15 nmol / ewe;
- FIGS. 9a and 9b are histograms showing the area under the curve (AUC) calculated for each of the curves respectively of FIGS. 8a and 8b;
- FIG. 10 shows the concentration of LH measured in blood samples taken from sheep during the oestrus period, at different times before and after the injection of a physiological solution containing the compound according to the invention C8 at a concentration 15 nmol / ewe, on the one hand, intravenously (iv), and on the other hand, intramuscularly (im).
- the peptide syntheses are carried out on solid phase in Fmoc / iBu strategy, at a scale of 0.1 mmol.
- the solid support used is a ChemMatrix® resin functionalized by a Rink amide arm.
- Deprotection of the Fmoc group is carried out using a 20% solution of piperidine in NMP.
- the protective groups of the side chains used are Arg (Pbf), Arg (Me, Pbf), Asn (Trt), Ser (iBu), Trp (Boc), Tyr (iBu), Hyp (iBu), Thr (Bu).
- 1,4-disubstituted, 1,2-triazole, 1,2-triazole ( ⁇ // [4- (1,2,3-triazol-1-yl)], abbreviated ⁇ [ ⁇ ]) pseudopeptide linkages are formed by copper (I) catalysed cycloaddition between an alkyne and an azide (CuAAC), in solution (route A) or on a solid support (route B), according to the general reaction schemes below:
- the formation of the first triazole is carried out on a solid support by cycloaddition with a / V-Fmoc-amino alkyne.
- the Fmoc group is then deprotected under standard conditions, then the amine is converted to azide with the aid of a diazo transfer reagent
- the formation of the second triazole is then carried out by cycloaddition with a ⁇ -Fmoc ⁇ -amino alkyne, according to the general reaction scheme below ( track C): way (C)
- the crude triazolopeptide is finally released from the resin with a solution of TFA / H 2 0 // Pr 3 SiH / phenol, 87.5 / 5 / 2.5 / 5 for 2 h.
- the peptide is precipitated in cold Et 2 0, centrifuged and then washed 3 times with Et 2 0.
- the pure triazolopeptide is analyzed by HPLC (using either a Nucleosil C1 8 300 ⁇ , 5 ⁇ , 4.6 ⁇ 250 mm, 1 mL / min column, or a Chromolith HighResolution RP-1.8 column, 4.6 ⁇ 100 mm. , 3mL / min) and MALDI-TOF mass spectrometry (matrix: cc-cyano-4-hydroxy cinnamic acid, instrument: Ultraflex, Bruker Daltonics, Germany).
- HPLC using either a Nucleosil C1 8 300 ⁇ , 5 ⁇ , 4.6 ⁇ 250 mm, 1 mL / min column, or a Chromolith HighResolution RP-1.8 column, 4.6 ⁇ 100 mm. , 3mL / min
- MALDI-TOF mass spectrometry matrix: cc-cyano-4-hydroxy cinnamic acid, instrument: Ultraflex, Bruker Daltonics, Germany.
- the aqueous phase is acidified with an aqueous solution of 6M HCl to pH 2, then extracted with AcOEt (4 x 50 ml).
- the organic phases are combined, dried over MgSO 4 and then concentrated on a rotary evaporator.
- the product (5) is obtained as a pale green solid (3.01 g, 6.9 mmol, 98%), and is used as an automated SPPS building block without additional purification step.
- 1 H NMR (500 MHz, CDCl 3 ) ⁇ 7.79 - 7.72 (m, 3H), 7.59 - 7.50 (m,
- the alkyne compound (0.4 mmol, 4 eq.) And CuBr (Me 2 S) (82 mg, 0.4 mmol, 4 eq.) are dissolved in 10 ml of NMP under an atmosphere of argon. After addition of iPr 2 Net (70 ⁇ l, 0.4 mmol, 4 eq.), The solution is aspirated into a syringe provided with a sintered polypropylene, and containing the resin bearing the azide peptide (0.1 mmol). The reaction medium is stirred for 2 hours at room temperature.
- the resin is then washed successively with NMP (3 x 2 min), CH 2 Cl 2 (2 x 2 min), 1 M pyridinium chloride in CH 2 Cl 2 / MeOH 95: 5 (2 x 2 min). ), CH 2 Cl 2 (2 x 2 min) then DMF (2 x 2 min).
- NMP 3 x 2 min
- DMF 2 x 2 min
- the continuation of the elongation is carried out by standard SPPS.
- the resin bearing the modified peptide provided with an unprotected N-terminal amino function (50 ⁇ ) is coupled after the automated SPPS elongation with Fmoc-Glu-OiBu (10 equivalents).
- protected amino acid 9.5 equivalents of HCTU and 20 equivalents of di-isopropylethylamine in NMP.
- Deprotection of the group Fmoc is performed using a solution of 20% piperidine in NMP.
- the hexadecanoic acid is then coupled (10 equivalents of acid, 9.5 equivalents of HCTU and 20 equivalents of di-isopropylethylamine in a NMP / CH 2 CI 2 1: 4 mixture).
- the crude Y- (/ V-hexadecanoyl-L-glutamyl) -triazolopeptide is finally released from the resin with 5 ml of a solution of TFA / H 2 0 // Pr 3 Si H / phenol, 87.5 / 5/2, 5/5 for 2 hours.
- the resin is rinsed with TFA (2x5mL for 5 min), and the filtrates are concentrated at room temperature using a rotary evaporator to a volume of about 1 mL.
- the peptide is precipitated by dilution with 40 mL Et 2 0 precooled to -80 ° C, then centrifuged and washed 2 times with Et 2 0. It is then purified and analyzed according to standard protocols.
- a derivative of Fmoc-L-lysine provided with a Dde group on its side chain, Fmoc-Lys (Dde) -OH is introduced at position 2 or 3 during the SPPS.
- the resin bearing the modified peptide provided with an amine N-Dde function (50 ⁇ ) is treated with 10 ml of a 2% hydrazine solution in NMP (2 ⁇ 5 min) to remove the Dde group, then the resin carrying the modified peptide provided with an unprotected amine function (50 ⁇ ) is treated according to a protocol identical to that used for the introduction of the modification in position 1.
- the peptide compound Comp.1, of sequence SEQ ID NO: 1: H-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Tyr-NH 2 , of chemical formula: is prepared by standard SPPS.
- the peptide compound C1 of sequence SEQ ID NO: 5: H-Tyr-Asn-Trp-Asn-Ser-Phe-Gly ⁇ [Tz] Leu-Arg-Tyr-NH2, of chemical formula:
- Peptide compound C2 of sequence SEQ ID NO: 6: Ac-Tyr-Asn-Trp-Asn-Ser-Phe-G ⁇ [Tz] Leu-Arg-Tyr-NH 2 , of Chemical Formula:
- Peptide compound C3 of sequence SEQ ID NO: 7: Ac-Tyr-Asn-Trp-Asn-Ser-Phe-Gly ⁇ [Tz] Leu-Arg (Me) -Tyr-NH2, of chemical formula:
- Peptide compound C4 of sequence SEQ ID NO: 8:
- the first triazole (Glyi /> [Tz] Leu) is formed according to route B, introducing the azide function by automated coupling of -azido acid (1).
- the formation of triazole by CuAAC on a solid support is carried out with the alkyne (2), following the general procedure.
- the second triazole (Phei> [Tz] Gly) is formed according to route C.
- the resin is stirred for 1 hour with the transfer reagent of diazo 1 H-imidazole-1-sulfonyl azide.H 2 SO 4 (135 mg, 0.5 mmol, 5 eq), and K 2 CO 3 (140 mg, 1 mmol, 10 eq) dissolved in DMF / H 2 O (3/7, 3 mL).
- the transfer reagent of diazo 1 H-imidazole-1-sulfonyl azide.H 2 SO 4 (135 mg, 0.5 mmol, 5 eq)
- K 2 CO 3 140 mg, 1 mmol, 10 eq
- the resulting supported azidopeptide is engaged in a solid supported CuAAC reaction with the alkyne (4), following the general procedure. The rest of the synthesis is done by classical SPPS.
- the peptide compound C8 of sequence SEQ ID NO: 1 1:
- the peptide compound C9 of sequence SEQ ID NO: 12: ⁇ - ( ⁇ / - ⁇ 3 ⁇ 3 ⁇ - ⁇ - ⁇ ) - ⁇ - ⁇ 5 ⁇ - ⁇ - ⁇ 5 ⁇ -8 ⁇ - ⁇ - ⁇ [ ⁇ ⁇ ] ⁇ - ⁇ 9- ⁇ - ⁇ 2 , wherein y - (/ V-hexadecanoyl-Glu-OH) represents a unit of formula (II) above is prepared according to route B, introducing the azide function by automated coupling of ⁇ -azido acid (1) .
- the formation of triazole by CuAAC on a solid support is carried out with the alkyne (2), following the general procedure.
- the continuation of the elongation synthesis is continued by standard SPPS, then the modification y - (/ V-hexadecanoyl-Glu-OH) is introduced following the general procedure for position 1.
- the peptide compound C10 of sequence SEQ ID NO: 13: ⁇ - ( ⁇ / - ⁇ 3 ⁇ 3 ⁇ - ⁇ - ⁇ ) ⁇ - ⁇ 5 ⁇ - ⁇ - ⁇ 5 ⁇ -8 ⁇ - ⁇ ⁇ ⁇ - ⁇ [ ⁇ ⁇ ] ⁇ - ⁇ 9- ⁇ - ⁇ 2 , wherein y - (/ V-hexadecanoyl-Glu-OH) represents a unit of formula (II) above is prepared according to route B, introducing the azide function by automated coupling of -azido acid (1) .
- the formation of triazole by CuAAC on a solid support is carried out with the alkyne (2), following the general procedure. Further synthesis is continued by conventional SPPS, and then the modification y - (/ V-hexadecanoyl-Glu-OH) is introduced following the general procedure for position 1.
- the peptide compound C13 with the sequence SEQ ID NO: 16: ⁇ - ( ⁇ / - ⁇ 3 ⁇ 3 ⁇ - ⁇ - ⁇ ) ⁇ - ⁇ 5 ⁇ - ⁇ - ⁇ 5 ⁇ -8 ⁇ - ⁇ ⁇ ⁇ - ⁇ [ ⁇ ] ⁇ - ⁇ 9- ⁇ ⁇ ⁇ - ⁇ 2 , wherein y - (/ V-hexadecanoyl-Glu-OH) represents a unit of formula (II) above is prepared according to the B route, introducing the azide function by automated coupling of ⁇ -azido acid ( 1). The formation of triazole by CuAAC on a solid support is carried out with the alkyne (2), following the general procedure.
- TTDS is a 1,13-diamino-4,7,10-trioxatridecanesuccinic acid spacer arm and TTDS- ( ⁇ - (/ V-hexadecanoyl-Glu-OH)) represents a unit of formula (III):
- the resin bearing the modified peptide provided with an amino function N-Dde (50 ⁇ ) is treated with 10 mL of a 2% hydrazine solution in NMP (2 x 5 min) for remove the Dde group, then the resin bearing the modified peptide provided with an unprotected amino function (50 ⁇ ) is coupled with [1 - / V- (9-fluorenylmethoxycarbonyl) -1, 13-diamino-4 acid , 7,10-trioxatridecanesuccinamic acid (Fmoc-TTDS-OH, 3 equivalents, 3 equivalents of HATU (2- (7-aza-1 / - / - benzotriazol-1-yl) hexafluorophosphate -1, 1, 3 3-tetramethylaminium) and 6 equivalents of di-isopropylethylamine in NMP).
- the Fmoc group is removed by treatment with a solution of 20% piperidine in NMP.
- Fmoc-Glu-OiBu is then coupled (10 equivalents of protected amino acid, 9.5 equivalents of HCTU and 20 equivalents of di-isopropylethylamine in NMP).
- Deprotection of the Fmoc group is carried out using a 20% solution of piperidine in NMP.
- the hecadecanoic acid is then coupled (10 equivalents of acid, 9.5 equivalents of HCTU and 20 equivalents of di-isopropylethylamine in a NMP / CH 2 CI 2 1: 4 mixture).
- the crude TTDS- [Y- (/ V-hexadecanoyl-L-glutamyl)] triazolopeptide is finally released from the resin with 5 mL of a solution of TFA / H 2 0 // Pr 3 SiH / phenol, 87.5 / 5 / 2.5 / 5 for 2 hours.
- the resin is rinsed with TFA (2 x 5 mL for 5 min), and the filtrates are concentrated at room temperature using a rotary evaporator to a volume of about 1 mL.
- the peptide is precipitated by dilution with 40 mL Et 2 0 previously cooled to - 80 ° C, then centrifuged and washed 2 times with Et 2 0. It is then purified and analyzed according to standard protocols.
- Peptide compound C15 of sequence SEQ ID NO: 18:
- the resin bearing the modified peptide provided with an amino function N-Dde (50 ⁇ ) is treated with 10 ml of a 2% hydrazine solution in NMP (2 x 5 min) to remove the Dde group, then the resin bearing the modified peptide provided with an unprotected amino function (50 ⁇ ) is treated with succinic anhydride (10 equivalents of anhydride and 20 equivalents of diisopropylethylamine in NMP).
- HD-Lys (Boc) -OiBu is then coupled (10 equivalents of protected amino acid, 10 equivalents of PyAOP (7-Azabenzotriazol-1-yloxy) hexafluorophosphate tripyrrolidinophosphonium) and 20 equivalents of di-isopropylethylamine in NMP).
- the crude triazolopeptide is finally released from the resin with 5 mL of a solution of TFA / H 2 0 // Pr 3 SiH / phenol, 87.5 / 5 / 2.5 / 5 for 2 h.
- the resin is rinsed with TFA (2x5 mL for 5 minutes), and the filtrates are concentrated at room temperature using a rotary evaporator to a volume of about 1 mL.
- the peptide is precipitated by dilution with 40 mL Et 2 0 previously cooled to 0 ° C, then centrifuged and washed 2 times with Et 2 0.
- the peptide is finally coupled in solution with 4 - (- p-iodophenyl) butanoic acid (3 equivalents) activated in the form of its N-hydroxysuccinimide ester as described in the publication by Trussel et al., 2009.
- the compound C15 thus obtained is then purified and analyzed according to the standard protocols.
- the peptide compound C16 of sequence SEQ ID NO: 19: ⁇ - ⁇ - ⁇ 5 ( ⁇ 5000) - ⁇ - ⁇ 5 ⁇ -5 ⁇ - ⁇ - ⁇ [ ⁇ ⁇ ] ⁇ ⁇ - ⁇ - ⁇ 2 , in which PEG5000 represents a motif of formula: COCH 2 CH 2 NH (CH 2 CH 2 0) nMe with -85 ⁇ n ⁇ -130, with an average molecular weight of about 5000 g / mol, is prepared according to route B, by introducing the azide function by automated coupling of ⁇ -azido acid (1).
- the formation of triazole by CuAAC on a solid support is carried out with the alkyne (2), following the general procedure.
- the peptide (2 equivalents) is finally coupled in solution with a commercial polymer (IRIS Biotech Gmbh) with an average mass of about 5000 g / mol activated in the form of its ester of / V-hydroxysuccinimide (5 mM peptide in a 50mM HEPES buffer mixture pH 8.5 and MeCN 9: 1).
- a commercial polymer IRIS Biotech Gmbh
- the compound C16 thus obtained is then purified and analyzed according to the standard protocols.
- each peptide compound to be analyzed and the internal calibrator (L-phenylalaninol) are dissolved in milliQ water (0.5 mM and 6.6 mM, respectively).
- the commercial protease inhibitor cocktail (Sigma, P8340) is supplied in solution in DMSO.
- Serum and stock solutions of peptide compounds are preheated to 39 ° C for 30 minutes.
- Each solution of peptide compound (50 ⁇ , ie a final concentration in the serum of 50 ⁇ ) and the internal calibrant solution (25 ⁇ ) are mixed with 425 ⁇ . of serum and incubated at 39 ° C.
- the monitoring of the kinetics of proteolytic degradation of the compounds is carried out by taking, at various given times, 75 ⁇ . of the solution, which are diluted with 150 ⁇ . of acetonitrile to precipitate the serum proteins.
- the suspension obtained is centrifuged at 14,000 rpm for 10 min at 4 ° C. 100 ⁇ . supernatant are diluted in 900 ⁇ .
- the value corresponding to 100% of intact peptide compound is obtained by mixing 420 ⁇ . of serum, 5 ⁇ . inhibitor cocktail and 25 ⁇ l of the phenylalaninol solution, followed by 50 ⁇ l. of peptide compound, then immediately treating the resulting solution according to the protocol described above. For all the compounds tested, the results obtained after 3 hours of incubation in the serum are shown in Table 1 below.
- the cell line HEK293A (ATCC, American Type Culture Collection, Manassas, Virginia, USA) was stably transfected with the receptor human KISS1 R (Genbank accession number: NM_032551)
- pcDNA3.1 vector Invitrogen, Cergy Pontoise, France
- the KISS1R receptor is coupled to Gq proteins and its activation produces an increase in intracellular calcium concentration.
- HEK293A cells expressing the human KISS1 R receptor were seeded in a 96-well plate (clear plate black) at a concentration of 40,000 cells / well, and placed in an incubator at 37 ° C. After 48 hours, the medium was changed and the cells were incubated with Fluo-4NW fluorescent dye, as specified by the manufacturer (Molecular Probe). To avoid peptide-plastic adhesion, the compounds to be tested were prediluted in a so-called "non-binding" plate (Corning) at a concentration 20 times greater (20X) at the desired final concentration.
- Serum albumin, or PEG enhances the stimulatory potency of the human KISS1 R peptide compounds, with the exception of the compound C1 1, which remains active however, although less, and the compound C16, which shows a power similar to the Comp.1 comparative compound.
- the peptide compound to be tested was injected into the catheter at the desired dose (5 nmol / ewe), diluted in 1 ml of physiological saline solution. Heparin-containing saline (3 ml) was injected immediately after the peptide compound to rinse the catheter and bring all of the compound into the bloodstream of the animal.
- a negative control (T) consisting of physiological solution alone has also been realized.
- blood samples were taken at variable intervals between 10 min and 1 h, for a period of between 3 h and 30 h.
- the tests were carried out on Ile de France ewes during the anestrus period, as indicated above with reference to the 1 st Experimentation (no treatment with progesterone).
- a catheter was placed in the jugular vein of the animal.
- the peptide compound to be tested was injected into the catheter at the desired dose (5 nmol / ewe), diluted in 1 ml of physiological saline solution.
- FIGS. 6a to 6d The results obtained, in terms of LH concentration in each blood sample as a function of time, are shown in FIGS. 6a to 6d, respectively for compounds Comp.1, Comp.3, C1 and C2.
- the arrow, showing the time "0" indicates the moment of the injection.
- the duration of action of the compound concerned is symbolized by asterisks, the number of which is proportional to the duration of action of the compound.
- C2 and C8 which differ in the presence, in C8, of a motif capable of binding serum albumin at the 2-position of the peptide compound.
- the tests were performed on ewes Ile de France during the period of anoestrus, as indicated above in reference to the 1 st Experiment (without progesterone treatment).
- a catheter was placed in the jugular vein of the animal.
- the peptide compound to be tested was injected into the catheter at the desired dose, diluted in 1 ml of saline.
- the following three doses were tested: 1 nmol / ew, 5 nmol / ew and 15 nmol / ew.
- FIGS. 8a and 8b The results obtained, in terms of LH concentration in each blood sample as a function of time, for each injected dose, are shown in FIGS. 8a and 8b, respectively for compounds C2 and C8.
- the arrow, showing the time "0" indicates the moment of the injection.
- the compound according to the invention C8 differing from the compound C2 by the introduction of a pattern capable of binding serum albumin, has an action that is even more prolonged over time, and very significantly, that the compound C2, which has been shown above that it is itself much better than the Comp.1 and Comp.3 comparative compounds.
- the area on the curve (AUC) was calculated.
- the set of values obtained are shown in FIGS. 9a and 9b, respectively for the compounds C2 and C8. It is observed that the compound according to the invention C8 is even more active than the compound C2 as regards the secretion of LH.
- the compound according to the invention C8 was injected, on the one hand intravenously, and on the other hand intramuscularly, to sheep of Ile de France breed in oestrus period.
- the ewes were previously treated with a vaginal sponge containing 20 mg of fluorogestone acetate (Chronogest CR sponge, Intervet) to block the secretion of LH and simulate a luteal phase.
- test compound Before and after the injection of the test compound, blood samples were taken at variable intervals between 10 minutes and 1 hour, for a period of 10 hours.
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FR1350858A FR3001455B1 (fr) | 2013-01-31 | 2013-01-31 | Composes agonistes du recepteur kiss1r et leur utilisation pour induire l'ovulation chez les mammiferes |
PCT/EP2014/051886 WO2014118318A1 (fr) | 2013-01-31 | 2014-01-31 | Composés agonistes du récepteur kiss1 et leur utilisation pour induire l'ovulation chez les mammifères |
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EP2951200A1 true EP2951200A1 (fr) | 2015-12-09 |
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EP14702555.5A Pending EP2951200A1 (fr) | 2013-01-31 | 2014-01-31 | Composés agonistes du récepteur kiss1 et leur utilisation pour induire l'ovulation chez les mammifères |
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EP (1) | EP2951200A1 (fr) |
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FR3128958A1 (fr) | 2021-11-09 | 2023-05-12 | Institut National De Recherche Pour L'agriculture L'alimentation Et L'environnement | Composés peptidiques cycliques agonistes du récepteur kiss1r et leurs utilisations thérapeutiques |
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US9127038B2 (en) * | 2011-08-04 | 2015-09-08 | Merck Sharp & Dohme Corp. | Kisspeptide-pentasaccharide conjugates |
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BR112015018283A2 (pt) | 2017-08-22 |
US20150361138A1 (en) | 2015-12-17 |
WO2014118318A1 (fr) | 2014-08-07 |
FR3001455A1 (fr) | 2014-08-01 |
US9884891B2 (en) | 2018-02-06 |
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