EP2432800A2

EP2432800A2 - Marked peptides and use thereof for assaying circulating irap

Info

Publication number: EP2432800A2
Application number: EP10728786A
Authority: EP
Inventors: Serge Bottari; Georges Vauquelin; Dirk Tourwe
Original assignee: Vrije Universiteit Brussel VUB; Universite Libre de Bruxelles ULB; Universite Joseph Fourier Grenoble 1
Current assignee: Vrije Universiteit Brussel VUB; Universite Libre de Bruxelles ULB; Universite Joseph Fourier Grenoble 1
Priority date: 2009-05-20
Filing date: 2010-05-19
Publication date: 2012-03-28
Also published as: US20120115163A1; JP2012527437A; FR2945808A1; WO2010133806A2; FR2945808B1; WO2010133806A3; CA2762525A1

Abstract

The invention relates to a method for assaying the circulating extracellular portion of the IRAP ("insulin-responsive aminopeptidase") protein, including at least one step of quantitatively assaying the released and purified extracellular portion of the IRAP using at least one marked peptide, said marked peptide specifically interacting with said extracellular portion of the IRAP.

Description

PEPTIDES BRANDS AND THEIR USE FOR DOSING

CIRCULATING IRAP

The present invention relates to labeled peptides and their use for the assay of circulating IRAP protein

The protein IRAP (Insulin-Regulated Amino Peptidase, EC 3.4.11.3) also known as Placental Leucine Amino Peptidase (P-LAP) and Leucine-cystinyl aminopeptidase (L-CAP) is a transmembrane zinc metalloproteinase protein that exists under three isoforms (Swiss-Prot: Q9UIQ6: 1, 2 and 3, respectively represented by SEQ ID NO: 1 to 3).

When inserted into the plasma membrane, in its native form, its extracellular domain can be cleaved and secreted. The secreted portion of an unspecified isoform of the IRAP protein was assayed in the blood by an enzymatic method using a non-specific synthetic substrate, L-leucine-nitroanilide in the presence of methionine (Mizutani, S., Yoshino, M., and Oya, M. (1976) Clin Biochem 9 (1), 16-18).

Using this Mizutani method, Oya and Tomoda demonstrated a cystinyl leucine aminopeptidase. This aminopeptidase is essentially of placental origin, for which reason it has been called P-LAP (Tsujimoto, M., Mizutani, S., Adachi, H., Kimura, M., Nagazato, H., and Tomoda, Y. (1992) Arch Biochem Biophys 292 (2), 388-392) and corresponds to the secreted domain of the protein.

This enzyme degrades oxytocin (Naruki, M., et al., (1996) Peptides 17 (2), 257-261), vasopressin (Wallis, M. Cet (2007) Am J Physiol Endocrinol Metab 293 (4), E1092-1102), angiotensin II and III (Matsumoto, H., et al (2000) Eur J Biochem 267 (1), 46-52) as well as a series of other peptides (Albiston , AL, et al (2007) Pharmacol Ther 116 (3), 417-427). Serum concentrations of this aminopeptidase have never been reported in humans. On the occasion of its cloning, it appeared that P-LAP corresponds to the IRAP protein as well as to the angiotensin IV receptor (Keller, SR, et al., (1995) J Biol Chem 270 (40), 23612 Rogi, T., et al (1996) Biol Chem 271 (1), 56-61, Albiston, AL, et al (2001) J Biol Chem 276 (52), 48623-48626). International Application WO 2005/038462 discloses a reagent for the diagnosis and / or prognostic evaluation of carcinomas which comprises a polyclonal anti-P-LAP antibody, obtained by immunization with the entire P-LAP protein. Given the homology between the different aminopeptidases, the antibody is probably not specific for IRAP and will also recognize other aminopeptidases. It should therefore not be possible to diagnose a pathology specifically related to a change in the expression or plasma concentration of IRAP.

A relation between the IRAP protein and the development of chemoresistance to anticancer drugs has been shown (Kondo C. et al., Int J. Cancer, 118, 1390-1394, 2006). According to this article, IRAP indeed reduces sensitivity to anticancer drugs by inhibiting the expression of the triggering factor of apoptosis and increases the expression of the factor of inhibition of apoptosis.

The work of Lukaszuk et al. (J. Med Chem 2008, 51, pp: 2291-2296) have shown that peptides derived from angiotensin IV were able to bind specifically to the IRAP protein located on the surface of cells.

To date, there is no method for measuring and quantifying the IRAP protein in its native form, that is to say uncleaved and anchored in the plasma membrane.

The extracellular domain of IRAP is cleaved by metalloproteases belonging probably to the ADAM family including ADAM9 and ADAM 12 (Ito, N., et al., (2004) Biochem Biophys Res Commun 314 (4), 1008-1013). and released into the bloodstream. ADAM9 (MDC9) is expressed particularly in the brain, liver, heart, kidneys and lungs (Hotoda, N., et al., (2002) Biochem Biophys Res Commun 293 (2), 800-805) and several others. members of this family are expressed in muscle and adipose tissue. To date, the only relationships established between the concentration of the extracellular domain of IRAP in a biological medium and a pathology concern severe preeclampsia as well as the threat of premature labor.

Thus, it appears necessary to accurately and quantitatively determine the circulating extracellular domain of the IRAP protein. To date, the circulating IRAP protein has never been quantified in the serum.

The problem to be solved is even more complicated than that of the IRAP assay in its native form, partly because of the presence, in the serum or the plasma, of many elements. especially proteases liable to degrade the potential ligands of IRAP, and secondly the need to implement relatively heavy means.

Thus, it is necessary to have a means of assaying the circulating IRAP protein in serum or plasma by means of compounds capable of resisting degradation caused by the proteases contained in the serum or the plasma.

It is also necessary to have specific, stable compounds which can be used for the implementation of a quantitative and reliable assay of the IRAP protein, in a biological sample containing no cells.

Thus, the purpose of the invention is to provide a reliable, specific and quantitative method for measuring the amount of circulating IRAP protein.

Another object of the invention is to provide circulating IRAP ligands for its detection.

Thus, the invention relates to a method for assaying the circulating extracellular portion of the IRAP protein ("insulin responsive aminopeptidase") comprising at least one step of quantitatively assaying said extracellular, secreted, purified part of IRAP, by means of at least one labeled peptide, said labeled peptide interacting specifically with said extracellular part of IRAP, said labeled peptide preferably being labeled with at least one radioactive isotope, said labeled peptide being an IRAP ligand, and in particular either a labeled substrate of the IRAP protein, an inhibitory peptide labeled with the IRAP protein, said peptide being optionally modified by the presence of at least one non-natural amino acid, and / or at least one β-homo amino acid, whether natural or not, subject to that said unmodified labeled peptide is different from angiotensin IV, and in particular that said unmodified labeled peptide is different from the SEQ ID NO. 21 (Val-Tyr-Ile-His-Pro-Phe), and provided that said modified peptide, labeled with an iodine atom ¹²⁵ I, is different from the Nle-Tyr-Ile-His- Pro-Phe (SEQ ID NO. 23). The invention relates to a method for assaying the circulating extracellular portion of the protein IRAP ("insulin responsive aminopeptidase") comprising at least one step of quantitatively assaying said extracellular, secreted, purified part of IRAP by means of less a modified and labeled peptide, said labeled peptide interacting specifically with said extracellular part of IRAP, said peptide being modified by the presence of at least one non-natural amino acid, and / or at least one natural β-homo amino acid, or not, said labeled peptide being preferentially labeled with at least one radioactive isotope, provided that - said peptide is not an antibody and

said modified peptide, labeled with a ¹²⁵ I iodine atom, is different from the Nle-Tyr-Ile-His-Pro-Phe sequence (SEQ ID No. 23).

The present invention is based on the unexpected finding made by the inventors that labeled peptides allow effective quantitative detection of the extracellular portion of circulating IRAP.

The "extracellular portion" of the IRAP protein is defined in the invention as the domain, or chain of amino acids, of the IRAP protein that is exposed outside the cell when the IRAP protein is anchored. in the plasma membrane of cells.

The "circulating extracellular portion" of the IRAP protein is defined in the invention as the extracellular portion of the IRAP protein as defined above which is no longer covalently bound to the transmembrane portion of the IRAP protein. This extracellular part is called circulating because, after its cleavage, it is found in the extracellular environment, and can be found in particular in plasma, serum, lymph, cerebrospinal fluid or urine.

Also, the extracellular part of circulating IRAP is called the extracellular part of secreted IRAP when it is found in the general circulation of the body, that is to say plasma, serum, lymph, cerebrospinal fluid. or urine. In what follows and the above, the terms "circulating extracellular portion of IRAP" and "extracellular portion of circulating IRAP" both refer to the extracellular domain of the IRAP protein which is no longer associated with the membrane portion of IRAP, and therefore no longer associated with the cell membrane. The IRAP protein that is the subject of the invention is represented by the amino acid sequence SEQ ID No. 1.

By "peptide" is meant any peptide chain corresponding to a covalent succession of at least two amino acids, or amino acid derivatives, linked by an amide function (-CO-NH-), said amino acids being natural or not and said amino acid derivatives being natural or non-naturally occurring amino acid derivatives.

By "labeled peptide" is meant in the invention a peptide as defined above having a molecule or an atom allowing its detection other than by conventional detection methods of proteins or peptides, that is to say -describe methods other than immunological methods.

Thus, the labeled peptides according to the invention can be coupled to either fluorescent molecules (cyanines, coumarins, rhodamines, xanthenes, quantum dots (nanocrystals of semiconductor materials), etc.), or can contain at least one, one or more radioactive atom (s), said radioactive atom being also called radioactive isotope. Among the radioactive isotopes which may be used in the invention, mention may be made of tritium ( ³ H), carbon 14 ( ¹⁴ C), iodine 131 ( ¹³¹ I) and iodine 125 ( ¹²⁵ I). or phosphate 32 or 33 ( ³² P OR ³³ P). Hereinafter, the peptides according to the invention are labeled with one or more ¹²⁵ I ( ¹²⁵ I) atoms, preferably with a single ¹²⁵ I ( ¹²⁵ I) atom.

The radioactive isotopes are capable of disintegrating and emitting particles in the form of radiation, said radiation being measurable by means for example of scintillation counters or not, and said proportional radiation being the amount of isotope. In the assay described in the invention, the quantification of the extracellular portion of IRAP is performed from the extracellular portion of IRAP that has been purified.

Methods for purifying the circulating IRAP protein may be any method of purifying proteins known to those skilled in the art. A preferred, but not limiting, method is an immunological method and in particular the immunoprecipitation or immunocapture of the circulating IRAP protein, by means of a specific antibody directed against the extracellular portion of the IRAP protein. The immunological immunocapture method of the extracellular portion of circulating IRAP is described in the examples. In the invention, the term "quantitative assay" means the action of precisely determining the amount of circulating IRAP protein contained in a biological sample of an individual or an animal.

The terms "said labeled peptide interacting specifically with said extracellular portion of IRAP" used in the invention mean that at least one labeled peptide according to the invention and the purified extracellular portion of circulating IRAP form a complex of high affinity. The order of magnitude of the affinity between said peptides and said circulating extracellular portion of IRAP is between about 10 ^-10 M to 10 ^-5 M, preferably between about 0.1 nM and about 100 nM. The terms are used to include the measurement variability of said affinity, said variation being generally from 5 to 10% error on the measurement.

The intensity of the interaction is measured for example as described in Lukaszuk et al. (J. Med Chem 2008, 51, pp: 2291-2296). The interaction is said to be specific, which means that the labeled peptide according to the invention is capable of forming a complex with the circulating extracellular portion of the IRAP protein, but said labeled peptide is not capable of forming a complex of the same affinity at the same concentrations with another protein having an amino acid sequence different from the amino acid sequence of said extracellular portion of IRAP. The labeled peptide of the invention is an IRAP ligand, and in particular is a labeled substrate of the IRAP protein, or an inhibitory peptide labeled with the IRAP protein, or any peptide whose affinity for the extracellular portion of circulating IRAP. is less than or equal to 10 μM (≤ 10 nM).

According to the invention, the peptide defined above can be modified by the presence of at least one non-natural amino acid, and / or at least one β-homo amino acid, natural or non-natural. By "non-natural amino acid" is meant in the invention amino acids that are not found naturally in proteins when synthesized in a living organism, or acellular system using the natural machinery of protein synthesis (mRNA , TRNA, ribosomes ...). The natural amino acids are the 20 amino acids known to those skilled in the art and whose correspondence with the triplets of nucleotides is given by the genetic code.

The non-natural amino acids used in the invention include, but are not limited to, amino acids derived from leucine such as cycloleucine (CIe), norleucine (Nie) or tert-leucine (ITe). By "β-homo amino acid", it is defined in the invention an amino acid having an additional carbon. In other words, the amino acids correspond to 2- (or α) amino 2- [side chain (R)] acetic acids, and the β homo amino acids correspond to 3 - (or β homo) 2 - [Side chain (R)] amino propanoic acids (β ² homo amino acid), or 3 - (or β homo) 3- [Side chain (R)] amino propanoic acids (β ³ homo amino acid). The formulas of the β ² homo amino acid and the β ³ homo amino acid are as follows:

β ² homo amino acid β ³ homo amino acid where R represents any one of the residues determining the amino acids.

The invention does not relate to the use of an unmodified labeled peptide which would be angiotensin IV, and in particular human Angiotensin IV of sequence SEQ ID NO: 21

(Val-Tyr-Ile-His-Pro-Phe).

The invention does not relate to the use of a modified peptide, labeled with an Iodine ¹²⁵ I atom, corresponding to Angiotensin IV or valine in position 1 is replaced by a Nie and represented by the sequence SEQ ID NO : 23 (Nle-Tyr-Isle-His-Pro-Phe).

In the foregoing and the following, by convention, the specific sequences will reveal natural amino acids, non-natural amino acids, and β ² or β ³ derivatives of said natural amino acids or not. On the other hand, the labeling does not appear in the sequence, for which reason the labeled peptide of sequence X will be represented by the sequence SEQ ID NO X unlabeled, and the type of labeling is specified. For example, the peptide X labeled with radioactive iodine will be indicated as follows: peptide of sequence SEQ ID NO X labeled with iodine ¹²⁵ I.

Advantageously, the subject of the invention is a method for assaying the circulating extracellular portion of IRAP as defined above, wherein said labeled peptide is not modified, provided that said unmodified labeled peptide is different from the angiotensin IV, and in particular that said unmodified labeled peptide is different from the sequence SEQ ID No. 21 (VaI-Tyr-Ile-His-Pro-Phe). These unmodified labeled peptides have the advantage of being similar or equivalent to the unlabeled peptides, and therefore of having a high affinity for the extracellular portion of the circulating IRAP protein.

According to another advantageous embodiment, the invention relates to a method for assaying the circulating extracellular portion of IRAP as defined above, wherein said labeled peptide is modified by the presence of at least one non-natural amino acid, and / or at least one β-homo amino acid, natural or non-natural, said modified labeled peptide interacting specifically with said extracellular part of IRAP, provided that said modified peptide, labeled with an iodine atom ¹²⁵ I, is different from the Nle-Tyr-Ile-His-Pro-Phe sequence (SEQ ID No. 23).

These modified labeled peptides have the advantage of having a high affinity for the extracellular portion of the circulating IRAP protein, and of having a high stability, especially in a sample containing numerous proteases or peptidases. The stability of the peptides according to the invention may in particular be measured by evaluating the inhibitory properties exerted by said peptides on the aminopeptidase activity of IRAP. This measure of stability is illustrated in the examples.

The invention also relates, in an advantageous embodiment, to a method for assaying the circulating extracellular portion of IRAP as defined above, comprising at least:

A step of purifying said circulating extracellular portion of IRAP, and

A step of quantifying said circulating extracellular portion purified in the preceding step, by means of at least one labeled modified peptide. Thus, the method according to the invention comprises at least one step of purifying the extracellular portion of IRAP in a biological sample. This purification step is followed by a step of quantification of said extracellular portion of IRAP purified in the previous step. This second step is implemented using at least one modified peptide as defined above.

As indicated above, the purification of the circulating extracellular portion of IRAP can be carried out by an immunological method, that is to say a method using an antibody directed against the IRAP protein, in particular immunoprecipitation or immunocapture. This method is specific when it uses a specific antibody that recognizes only the extracellular portion of the IRAP protein, and makes it possible to obtain an extracellular portion of the pure IRAP protein. The degree of contamination by other proteins can be evaluated by a conventional method of protein separation (SDS-PAGE) coupled with a protein detection method (silver staining, colloidal blue staining, etc.). methods known to those skilled in the art. Immunoprecipitation or immunocapture allows a purification to a very widely acceptable degree (> 90%), and in particular makes it possible to isolate a protein from its environment, for example contaminating proteases.

The invention relates, in another advantageous embodiment, to a method for assaying the circulating extracellular portion of IRAP as defined above, in which the said inhibitory peptide labeled with the IRAP protein is modified and is chosen from: Angiotensin IV labeled and modified and the labeled and modified LVV-hemorphin-7, or said labeled substrate of the IRAP protein is modified and is selected from: the labeled and modified [Arg] vasopressin, the modified and modified oxytocin, MeWLeu-enkephaline labeled and modified, labeled and modified Somatostatin, labeled and modified CCK-8, labeled and modified Neurokinin A, labeled and modified Neuromedin B, labeled and modified Lys-bradykinin and modified and modified modified Dynorphin A.

The modified labeled peptides that may be used in the invention correspond to either substrates of the IRAP protein or to peptide inhibitors of the IRAP protein. The inhibitory peptides are chosen from LVV-hemorphin-7 or angiotensin IV. These inhibitory peptides are modified and labeled.

The IRAP protein is a protease, so it is able to cleave proteins, said proteins being IRAP substrates. The aforementioned proteins as IRAP substrate are therefore proteins susceptible to be cleaved by IRAP.

Like any enzyme, the catalytic activity of IRAP can be blocked by compounds known as inhibitors. Generally, the inhibitors bind to the active site of an enzyme, are not metabolized by said enzyme, and prevent it from accessing its substrates. This kind of inhibition is a competitive inhibition. LVV-hemorphin-7 or angiotensin IV are peptides that inhibit IRAP activity. In another advantageous embodiment, the invention relates to a method for assaying the circulating extracellular portion of IRAP as defined above, wherein said purification of the circulating portion of the IRAP protein is carried out by means of at least an antibody specific for said extracellular portion of IRAP, in particular by immunoprecipitation or immunocapture.

Thus, in order to purify the extracellular portion of circulating IRAP in a biological sample, the invention proposes to use an antibody specifically recognizing the extracellular portion of IRAP, and not recognizing the transmembrane or intracellular portion of said IRAP protein.

This purification method may be an immunoprecipitation where an antibody specific for the extracellular portion of IRAP is attached to sugar polymer beads (agarose, sepharose). The purification method may also be an immunocapture in the context of an ELISA or an immunomagnetic radio test (IRMA) in which the said specific antibodies are fixed on a support, in particular the wall of a tube, and immobilize IRAP on this support. this.

Advantageously, it can be added to the sample of serum, plasma, lymph, cerebrospinal fluid or urine in which the extracellular concentration of IRAP must be measured at least one of the following products: - chelators of cations such as: EDTA, EGTA, 1,10-orthophenantroline, dimercaprol, lipoic acid, BAPTA (1,2-bis (o-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid), DTPA (diethylene acid) triamine pentaacetic), DMPS (2,3-dimercapto-1-propanesulfonic acid), DMSA (dimercaptosuccinic acid), penicillamine, deferroxamine, sulfosalicylic acid, citric acid, oxalic acid, tartaric acid, N ₅ N-Dimethyldecylamine N-oxide, acid nitrilotriacetic acid, pyromellitic acid, EDTMP (ethylenediamine tetra (methylene phosphonic acid)), ferric ferrocyanide, 2,2'-bipyridine, N, IΨ, N'-tris (2-pyridylmethyl) -cώ, cώ-1,3, 5, -triaminocyclohexane (tachpyr) and its analogues, azobenzene and its analogues, diethyldithiocarbamate, TPEN (N, N, N ', N'-tetrakis (2-pyridylmethyl) ethylenediamine), 1,2-cyclohexylenedinitrilotetraacetic acid, maltol, thiom- maltol, ethylmaltol, ethylthio maltol, 2-mercaptopyridine oxide and other bidenated chelators ,

inhibitors of proteases and peptidases such as: AEBSF (4- (2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), 6-aminohexanoic acid, antipain, aprotinin, benzamidine dine, bestatin, chymostatin, E-64, leupeptin, pepstatin, phosphoramidon, trypsin inhibitor, diisopropyl fluorophosphate, PMSF, p-chloromercuribenzoic acid, diethyl pyrocarbonate, 2-Mercaptoethylamine, Apstatin, Phebestin, Bromoenol lactone, Ecotin (acid 1, 4 , 5,6-tetrahydro-2-methyl-4-pyrimidine carboxilic acid), N-acetyl-eglin C, Gabexate mesylate, N-Tosyl-L-phenylalanine chloromethyl ketone, Na-T-Boc-Deacetylleupeptin, 3,4 - Dichloroisocoumarin, Amastatin, (2S, 3R) -3-Amino-2-hydroxy-4- (4-nitro-phenyl) butanoyl-L-leucine, Actinonin, Epiamastatin, N - [(2S) - (Methoxycarbonylmethyl) -4 Methylpentanoyl] -L-tryptophan-methylamide, 2,2'-Dipyridyl, L-Leucinethil oxidized, Epibestatin - other compounds: methionine, dithiotreitol, dithioerythritol, β-mercaptoethanol, TCEP (tm (2-carboxyethyl) phosphine), cysteine, N-ethylmaleimide, mercaptoethylamine, nonionic detergents (Triton X-100, Tween20, ...)

All of these products are known to those skilled in the art and can be used at standard concentrations.

In another advantageous embodiment, the invention relates to a method for assaying the circulating extracellular portion of IRAP defined above, wherein said IRAP protein is represented by

the IRAP protein of sequence SEQ ID NO 1, or a protein homologous to the IRAP protein having a sequence identity of at least 80% with the sequence SEQ ID NO 1, provided that said homologous protein is capable of interacting specifically with the IRAP ligands or the IRAP inhibitory peptides as defined above, or an isoform of the IRAP protein, said iso form being the product of the alternative splicing of the RNA encoding the IRAP protein SEQ ID NO 1 provided that said isoform is capable of interacting specifically with the IRAP ligands or the IRAP inhibitory peptides as defined above, said iso forms of the IRAP protein SEQ ID NO 1 being in particular represented by the SEQ proteins ID NO 2 or 3.

In the invention, the homologs of the IRAP protein correspond to proteins having a sequence homologous to the sequence SEQ ID No. 1 but having amino acid substitutions.

The isoforms of the IRAP protein defined in the invention correspond to products of the alternative splicing of the product of the gene encoding the IRAP protein, so that the protein The resulting isoforms of the invention correspond to truncated proteins in the aminoterminal portion of several amino acids.

Another advantageous embodiment of the invention relates to an assay method defined above, where

Said variants of the IRAP protein have an amino acid sequence chosen from the sequences SEQ ID NO 4 to SEQ ID NO 7, and

Said isoforms of the IRAP protein have an amino acid sequence selected from the sequences SEQ ID NO 8 to SEQ ID NO 15.

Also, according to another advantageous embodiment, the invention relates to an assay method as defined above, wherein said circulating extracellular portion of the IRAP protein has an amino acid sequence represented by the sequences chosen from the following sequences: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

Another advantageous embodiment of the invention relates to a previously defined assay method, wherein said labeled modified peptide is labeled with at least one tritium atom or at least one ¹²⁵ I iodine atom, that is to say an iodine atom or more, preferably a single ¹²⁵ I iodine atom.

The invention also relates in a preferred embodiment to a previously defined assay method, wherein said modified modified peptide is represented by the following general formula (III):

(III) where a, b, c, d, e, f, g, h, i, and j may be 0 or 1, such that (a + b) ≤l, (c + d) ≤l, (e + f) ≤l, (g + h) ≤l and (i, j) ≤l, (a, b), (c, d), (e, f), (g, h) and (i, j ) being independent of each other, where (R ₁ , R ₁ ') are such that:

R 1 is chosen from: a group -CH (CH ₃ ) ₂ , a group -CH ₂ -CH (CH ₃ ) 2, a group -CH (CH ₃ ) -CH ₂ -CH ₃ , a group - (CH ₂ ) ₃ -CH ₃ and a group C (CH ₃ ) ₃ , and R 1 'is a hydrogen atom, or R 1 and R 1' together with the carbon which carry them a cyclopentyl, where (R ₂ , R ₂ ') are such that: if R2 is chosen from: a group -CH ₂ -CH (CH ₃ ) ₂ , a group -CH (CH ₃ ) -CH ₂ -CH ₃ , a group - (CH ₂ ) ₃ -CH ₃ and a group C (CH ₃ ) ₃ , R 2 'is a hydrogen atom, or

R2 and R2 'together with the carbon which carries them a cyclopentyl, the pairs (R ₁ , R ₁ ') and (R ₂ , R ₂ ') being chosen independently of each other, where A is selected from the following groups

said modified modified peptide being in the form of a racemate, any of its enantiomers, or any of the various tautomers corresponding to said racemates and enantiomers,

provided that if A is represented by the formula IHa and if a, b, c, d, e, f, g, h, i and j are 0,

* if (R _h R R ') = (-CH (CH ₃ ) ₂ ; H) then (R ₂ , R ₂ ') is different from (-CH (CH ₃ ) -CH ₂ -CH ₃ ; H), and

if (R ₂ , R ₂ ') = (- CH (CH ₃ ) -CH ₂ -CH ₃ ; H) then (Ri ₅ Ri') is different from (-CH (CH ₃ ) ₂ ; H) and under reserve that if said modified peptide is labeled with an iodine atom ¹²⁵ I, if a, b, c, d, e, f, g, h, i and j are equal to 0, then (Ri ₅ Ri ') is different of (- (CH ₂ ) ₃ -CH ₃ ; H).

In other words, the modified modified peptide corresponds to one of the following three general formulas:

formula I or IHaI

formula HIb 1, or

The invention also relates in a preferred embodiment to a previously defined assay method, wherein said modified modified peptide is represented by the following general formula (I):

where a, b, c, d, e, f, g, h, i, and j may be 0 or 1, such that (a + b) ≤l, (c + d) ≤l, (e + f) ≤l, (g + h) ≤l and (i, j) ≤l, (a, b), (c, d), (e, f), (g, h) and (i, j ) being independent of one another, where (Ri, Ri ') are such that:

R2 and R2 'together with the carbon which carries them a cyclopentyl, the pairs (R ₁ , R ₁ ') and (R ₂ , R ₂ ') being chosen independently of one another, said modified modified peptide being in the form of a racemate, any of its enantiomers, or any of the different tautomers corresponding to said racemates and enantiomers, provided that if a, b, c, d, e, f, g , h, i and j are 0, * if (R _h R R ') = (-CH (CH ₃ ) ₂ ; H) then (R ₂ , R ₂ ') is different from (-CH (CH ₃ ) -CH ₂ -CH ₃ ; H), and * if (R ₂ , R ₂ ') = (- CH (CH ₃ ) -CH ₂ -CH ₃ ; H) then (Ri ₅ Ri') is different from (- CH (CH ₃ ) ₂ ; H) and provided that if said modified peptide is labeled with an iodine atom ¹²⁵ I, if a, b, c, d, e, f, g, h, i and j are equal to 0, then (Ri ₅ Ri ') is different from (- (CH ₂ ) ₃ -CH ₃ ; H).

In other words, the peptide of formula (Ia) below

is excluded from the use according to the invention, whatever its marking. Is also excluded from the use according to the invention, the peptide of formula (Ib) below

labeled with an atom at least one iodine atom 125 (CL25 _τ I), especially marked by an iodine atom on the aromatic ring 125 4-hydroxy benzyl residue. Thus, the following two peptides of formula IbI and Ib2 are excluded:

In an advantageous embodiment, the invention relates to a previously defined assay method, in which the labeled modified peptide is chosen from the following peptides:

The invention also relates to an assay method as defined above, wherein said labeled and modified peptide consists of the following sequence:

X1-X2-X3-His-X4-X5 (SEQ ID NO. 22) where

X1 can be a Valine (Val), a Leucine (Leu), an Isoleucine (Ile), a Norleucine (Nie), a Cycloleucine (CIe) or a tert-leucine (TIe), or a derivative β ² or β ³ of one of these amino acids,

X2 may be a tyrosine, or a β ² or β ³ derivative of tyrosine,

X3 can be a Leucine (Leu), an Isoleucine (Ile), a Norleucine (Nie), a Cycloleucine (CIe) or a tert-leucine (TIe), or a derivative β ² or β ³ of one of these amino acids ,

X4 may be a proline or a β ² or β ³ derivative of proline,

X5 may be a phenylalanine, or a β ² or β ³ derivative of phenylalanine, and (His-X4) may be

an aba-gly of formula lia or aia-gly of formula Hb (Hb),

at least one of the amino acids X1 to X5 is a non-natural amino acid, and / or a β-homo amino acid, natural or otherwise provided that the sequence SEQ ID NO 22 labeled with an iodine atom ¹²⁵ I is different from the Nle-Tyr-Ile-His-Pro-Phe sequence (SEQ ID NO 23).

In another advantageous embodiment, the invention relates to an aforementioned assay method, wherein said labeled and modified peptide consists of the following sequence: X1-X2-X3-His-X4-X5 (SEQ ID NO 22) where

X1 can be a Valine (Val), a Leucine (Leu), an Isoleucine (Ile), a Norleucine (Nie), a Cycloleucine (CIe) or a tert-leucine (TIe), or a derivative β ² or β ³ of one of these amino acids, X2 may be a tyrosine, or a β ² or β ³ derivative of tyrosine,

X4 can be proline or a β ² or β ³ derivative of proline,

X5 may be a phenylalanine, or a β ² or β ³ derivative of phenylalanine, at least one of amino acids X 1 to X 5 being a non-natural amino acid, and / or a β-homo amino acid, natural or non-natural. provided that the sequence SEQ ID No. 22 labeled with a ¹²⁵ I iodine atom is different from the Nle-Tyr-Ile-His-Pro-Phe sequence (SEQ ID No. 23).

According to the invention, the modified modified peptide consisting of the amino acid sequence SEQ ID No. 22 corresponds to the peptide of general formula (I) defined above, in which all the amino acids are in an L configuration according to the L nomenclature. / D known to those skilled in the art.

The name as well as the structural formula of the various natural amino acids or not, and the β 2 or β 3 derivatives of said natural or non-natural amino acids, are shown in Table 1 below. efore. Column N indicates the nature of the corresponding amino acid: natural (O) or non-natural (N). propioni

An advantageous embodiment of the invention relates to a previously defined assay method, wherein said labeled and modified peptide consists of the following sequence:

Xl-X2-X3-His-X4 -X5 (SEQ ID No. 22) and wherein at least one of amino acids X1 to X5 is a β-homo amino acid, natural or not. In other words, at least one of the amino acids X1, or X2, or X3, or X4 or X4 or X5 corresponds to an amino acid chosen from: β2homo valine, β3homo valine, β2homo leucine, β3homo leucine, β2homo isoleucine, β3homo isoleucine, β2homo tyrosine, β3homo tyrosine, β2homo proline, β3homo proline, β2homo phenylalanine, β3homo phenylalanine, β2 homo norleucine, β3 homo norleucine, β2 homo tertleucine, β 3 homo tertleucine, β 2 homo cycloleucine and β 3 homo cycloleucine.

In another advantageous embodiment, the invention relates to a previously defined assay method, wherein said labeled modified peptide is labeled

By at least one ¹²⁵ I iodine atom on tyrosine X 2, preferably one single ¹²⁵ I iodine atom, in particular on the phenyl group, or

By replacing at least one of the hydrogen atoms with a ³ H tritium atom.

In cases where the modified labeled peptide of the invention is labeled with an iodine atom 125, said peptide has at least one ¹²⁵ I atom, or two ¹²⁵ I atoms, in the meta position on the benzyl ring of tyrosine, or in the meta position on the benzyl ring of the β ² homo tyrosine or β ³ homo tyrosine derivatives.

Preferably, the peptide is labeled with a single ¹²⁵ I iodine atom.

Another advantageous embodiment of the invention relates to a previously defined assay method, wherein said labeled modified peptide is selected from the following peptides: β ² hVal-Tyr-Ile-His-Pro-Phe- (SEQ ID NO 24 ), of formula:

β ² hVal-Tyr-Ile-His-Pro-β ³ hPhe- (SEQ ID No. 25), of formula

β2HLeu-Tyr-Ile-His-Pro-β3hPhe- (SEQ ID No. 26), of formula

Val-Tyr-Ile-His-β ² hPro-Phe- (SEQ ID No. 27), of formula

β ² hVal-Tyr-Ile-Aba-Gly-Phe- (SEQ ID No. 28), of formula

β ² hVal-Tyr-Ile-Aia-Gly-Phe- (SEQ ID No. 29), of formula

preferably peptide SEQ ID NO 25, said modified modified peptide being iodinated.

The above peptides have a high affinity for the circulating extracellular portion of IRAP, are stable, and easily detectable by counting the radioactivity emitted by ¹²⁵ I iodine.

Advantageously, the invention relates to a previously defined assay method, wherein said labeled modified peptide is selected from the following peptides: - β2hVal-Tyr-Ile-His-Pro-Phe- (SEQ ID NO 24),

- β2hVal-Tyr-Ile-His-Pro-β3hPhe- (SEQ ID No. 25), - β2HLeu-Tyr-Ile-His-Pro-β3hPhe- (SEQ ID No. 26),

Val-Tyr-Ile-His-β ² hPro-Phe- (SEQ ID No. 27),

β ² hVal-Tyr-Ile-Aba-Gly-Phe- (SEQ ID No. 28), and

- β ² hVal-Tyr-Ile-Aia-Gly-Phe- (SEQ ID NO 29), preferably the peptide SEQ ID NO 25, said labeled modified peptide being tritiated.

The above peptides have a high affinity for the circulating extracellular portion of IRAP, are stable, and easily detectable by counting the radioactivity emitted by tritium H.

Another advantageous embodiment of the invention relates to an assay method defined above, comprising

A step of purifying said circulating extracellular portion of IRAP, and

A step of quantification of said circulating extracellular portion purified in the preceding step, by means of at least one modified peptide of amino acid sequence SEQ ID No. 25 labeled with radioactive iodine ¹²⁵ I, preferably a single radioactive iodine atom 125 _T I.

Another advantageous embodiment of the invention relates to a previously defined assay method, comprising: a step of purifying said circulating extracellular portion of IRAP, and

A step of quantification of said circulating extracellular portion purified in the preceding step, by means of at least one modified peptide of amino acid sequence SEQ ID No. 25, tritiated.

The invention also relates to a labeled modified peptide represented by the following general formula (III):

if (R ₂ , R ₂ ') = (- CH (CH ₃ ) -CH ₂ -CH ₃ ; H) then (Ri ₅ Ri') is different from (-CH (CH ₃ ) ₂ ; H) and under reserve that if said modified peptide is labeled with an iodine atom ¹²⁵ I, if a, b, c, d, e, f, g, h, i and j are equal to 0 then (Ri ₅ Ri ') is different of (- (CH ₂ ) ₃ -CH ₃ ; H).

The invention also relates to a labeled modified peptide represented by the following general formula (I):

said peptide possibly modified by the presence of at least one non-natural amino acid, and / or at least one β-homo amino acid, natural or not, where a, b, c, d, e, f, g, h, i and j may be equal to 0 or 1, such that (a + b) ≤l, (c + d) ≤l, (e + f) ≤l, (g + h) ≤l and (i, j) ≤1, (a, b), (c, d), (e, f), (g, h) and (i, j) being independent of each other, where (Ri, Ri ') are such than :

R1 is chosen from: a group -CH (CH ₃ ) ₂ , a group -CH ₂ -CH (CH ₃ ) _2i a group -CH (CH ₃ ) -CH ₂ -CH ₃ , a group - (CH ₂ ) ₃ -CH ₃ and a group C (CH ₃ ) ₃ , and R 1 'is a hydrogen atom, or - R 1 and R 1' together with the carbon which bears them a cyclopentyl, where (R ₂ , R ₂ ') are such as : - if R2 is chosen from: a group -CH ₂ -CH (CH ₃ ) 2, a group -CH (CH ₃ ) -CH ₂ -CH ₃ , a group - (CH ₂ ) ₃ -CH ₃ and a group C (CH ₃ ) ₃ , R2 'is a hydrogen atom, or

R2 and R2 'together with the carbon which carries them a cyclopentyl, the pairs (R ₁ , R ₁ ') and (R ₂ , R ₂ ') being chosen independently of one another, said labeled peptide being under the form of a racemate, any of its enantiomers, or any of the different tautomers corresponding to said racemates and enantiomers, provided that if a, b, c, d, e, f, g, h, i and j are 0, if (R ₁ R ') = (-CH (CH ₃ ) ₂ ; H) then (R ₂ , R ₂ ') is other than (-CH (CH ₃ ) -CH ₂ -CH ₃ ; H) and if (R ₂ , R ₂ ') = (- CH (CH ₃ ) -CH ₂ -CH ₃ ; H) then (R 1, R 1') is other than (- CH ( CH ₃ ),; H) and provided that if said labeled peptide is labeled with an iodine atom ¹²⁵ I, if a, b, c, d, e, f, g, h, i and j are equal to 0 then (Rl, Rl ') is different from (- (CH ₂ ) ₃ -CH ₃ ; H).

Previous peptides are new.

An advantageous embodiment of the invention relates to a labeled peptide as defined above, represented by the general formula Ic, Id, Ie, If, Ig, Ih, Ii, Ij, Ik, II, Im, In, Io, IHd or IHe as defined above. The invention also relates, in an advantageous embodiment, to a labeled and modified peptide consisting of the following sequence:

X1-X2-X3-His-X4-X5 (SEQ ID NO. 22) where

X2 may be a tyrosine, or a β ² or β ³ derivative of tyrosine,

X3 can be a Leucine (Leu), an Isoleucine (Ile), a Norleucine (Nie), a Cycloleucine (CIe) or a tert-leucine (TIe), or a derivative β ² or β ³ of one of these amino acids , X4 may be a proline or a β ² or β ³ derivative of proline,

X5 may be a phenylalanine, or a β ² or β ³ derivative of phenylalanine, and (His-X4) may be an aba-gly of formula lia or

aia-gly of formula Hb

In another aspect, the invention relates to the aforementioned modified unlabeled peptides, in particular the peptides of formulas IbbI and IIIc that follow:

formula HIb 1, or

Hc formula 1

An advantageous aspect of the invention relates to a labeled peptide defined above, said peptide being labeled with at least one radioactive isotope consisting of the following sequence: X1-X2-X3-His-X4-X5 (SEQ ID NO. 22) ) said modified peptide, where

X1 can be Valine (Val), Leucine (Leu), Isoleucine (Ile), or an unnatural amino acid derived from leucine, in particular Norleucine (Nie), Cyclo leucine (CIe) or tert- leucine (TIe), or a β ² or β ³ derivative of either these natural or unnatural amino acids,

X2 may be a tyrosine, or a β ² or β ³ derivative of tyrosine,

X3 may be Leucine (Leu), Isoleucine (Ile), or an unnatural amino acid derived from leucine, in particular Norleucine (Nie), Cycloleucine (CIe) or tert-leucine (TiE), or a derivative thereof β ² or β ³ of one of these natural or unnatural amino acids, X4 can be proline or a β ² or β ³ derivative of proline, X5 can be a phenylalanine, or a β ² or β ³ derivative of phenylalanine and provided that said unmodified labeled peptide is different from angiotensin IV, and in particular that said unmodified labeled peptide is different from SEQ ID NO. 21 (Val-Tyr-Ile-His-Pro-Phe), provided that the sequence SEQ ID No. 22 labeled with a ¹²⁵ I iodine atom is different from the Nle-Tyr-Ile-His-Pro-Phe sequence (SEQ ID No. 23).

Advantageously, the invention relates to a modified modified peptide defined above, consisting of the following sequence:

X1-X2-X3-His-X4-X5 (SEQ ID NO 22), said peptide being modified such that at least one amino acid X1 to X5 is a β-homo amino acid, natural or not.

Another advantageous aspect of the invention relates to a modified modified peptide defined above, said modified labeled peptide being labeled:

By at least one ¹²⁵ I iodine atom on the tyrosine X 2, in particular on the phenyl group, or by replacing at least one of the hydrogen atoms with a ³ H tritium atom. In another advantageous aspect, the invention relates to a modified modified peptide mentioned above, wherein said modified labeled peptide is selected from the following peptides:

- β2HVal-Tyr-Ile-His-Pro-Phe- (SEQ ID NO. 24),

- β2hVal-Tyr-Ile-His-Pro-β3hPhe- (SEQ ID NO: 25), - β2HLeu-Tyr-Ile-His-Pro-β3hPhe- (SEQ ID No. 26),

Val-Tyr-Ile-His-β ² hPro-Phe- (SEQ ID No. 27),

β ² hVal-Tyr-Ile-Aba-Gly-Phe- (SEQ ID No. 28), and

- β ² hVal-Tyr-Ile-Aia-Gly-Phe- (SEQ ID No. 29), preferably the peptide SEQ ID NO 25, said modified modified peptide being iodinated, preferably with a single ¹²⁵ I iodine atom.

In another advantageous aspect, the invention relates to a modified modified peptide mentioned above, wherein said modified labeled peptide is selected from the following peptides:

- β2HVal-Tyr-Ile-His-Pro-Phe- (SEQ ID NO: 24), - β2HVal-Tyr-Ile-His-Pro-β3hPhe- (SEQ ID NO: 25),

- β2HLeu-Tyr-Ile-His-Pro-β3hPhe- (SEQ ID No. 26),

Val-Tyr-Ile-His-β ² hPro-Phe- (SEQ ID No. 27),

β ² hVal-Tyr-Ile-Aba-Gly-Phe- (SEQ ID No. 28), and

The invention is illustrated by Figures IA-Q, Figures 2A-Q and the following examples, without being limited to said examples.

Legend of figures:

FIGS. 1A-Q represent the absorption spectra at 215 nm of elutions on reverse phase HPLC in the presence of water / acetonitrile solvent, the mobile phase containing 0.1% of TFA. The standard gradient corresponds to a 20 min migration from 3% to 97% acetonitrile, at a rate of 1 mL / min.

FIG. 1A represents the UV absorption spectrum as a function of time of the peptide H-β3hVal-Tyr-Ile-His-Pro-Phe-OH. FIG. 1B shows the UV absorption spectrum as a function of time of the peptide H-VaI-β3hTyr-Ile-His-Pro-Phe-OH.

FIG. 1C represents the UV absorption spectrum as a function of time of the H-VaI-peptide.

Tyr-Pro β3hIle-His-Phe-OH. FIG. 1D shows the UV absorption spectrum as a function of time of the H-VaI-peptide.

Tyr-Ile-His-Phe-OH-β3hPro.

FIG. 1E represents the UV absorption spectrum as a function of time of the H-VaI-peptide.

Tyr-Ile-His-Pro-OH-β3hPhe.

Figure IF shows the UV absorption spectrum as a function of time of the first diastereoisomer of the peptide H-β2hVal-Tyr-Ile-His-Pro-Phe-OH.

FIG. 1G shows the UV absorption spectrum as a function of time of the peptide of the second diastereoisomer of the peptide H-β2hVal-Tyr-Ile-His-Pro-Phe-OH.

FIG. 1H shows the UV absorption spectrum as a function of time of the mixture of the H-Val-β2hTyr-Ile-His-Pro-Phe-OH peptide racemates. FIG. II represents the UV absorption spectrum as a function of time of the first diastereoisomer of the peptide H-Val-Tyr-β2hLeu-His-Pro-Phe-OH

FIG. 1J represents the UV absorption spectrum as a function of time of the mixture of the H-Val-Tyr-β2hLeu-His-Pro-Phe-OH peptide racemates

FIG. 1K represents the UV absorption spectrum as a function of time of the peptide H-VaI-Tyr-Ile-His-β2hPro-Phe-OH

FIG. 11 represents the UV absorption spectrum as a function of time of the mixture of the H-Val-Tyr-Ile-His-Pro-β2hPhe-OH peptide racemates.

FIG. 1I shows the UV absorption spectrum as a function of time of the peptide H-β2hVal-Tyr-Ile-His-Pro-β3hPhe-OH. FIG. 1N represents the UV absorption spectrum as a function of time of the peptide.

NH2->^:> CO-Tyr-Ile-His-Pro-Phe-OH

FIG. 10 represents the UV absorption spectrum as a function of time of the mixture of the H-β2hNle-Tyr-Ile-His-Pro-Phe-OH peptide racemates.

FIG. 1P represents the UV absorption spectrum as a function of time of the mixture of the H-β2HLeu-Tyr-Ile-His-Pro-Phe-OH peptide racemates.

FIG. 10 represents the UV absorption spectrum as a function of time of the mixture of the H-β2hLeu-Tyr-Ile-His-Pro-β3hPhe-OH peptide racemates FIGS. 2A-Q show the absorption spectra at 215 nm of elutions on reverse phase HPLC in the presence of water / methanol solvent, the mobile phase containing 0.1% of TFA.

The standard gradient corresponds to a 20 min migration from 3% to 97% methanol, at a rate of 1 mL / min.

FIG. 2A represents the UV absorption spectrum as a function of time of the H-β3hVal-Tyr-Ile-His-Pro-Phe-OH peptide.

FIG. 2B represents the UV absorption spectrum as a function of time of the peptide H-VaI-β3hTyr-Ile-His-Pro-Phe-OH. FIG. 2C represents the UV absorption spectrum as a function of time of the H-VaI-peptide.

Tyr-His-Pro β3hIle-Phe-OH.

FIG. 2D represents the UV absorption spectrum as a function of time of the H-VaI-peptide.

Tyr-Ile-His-Phe-OH-β3hPro.

FIG. 2E represents the UV absorption spectrum as a function of time of the peptide H-VaI-Tyr-Ile-His-Pro-β3hPhe-OH.

FIG. 2F represents the UV absorption spectrum as a function of time of the first diastereoisomer of the peptide H-β2hVal-Tyr-Ile-His-Pro-Phe-OH.

FIG. 2G represents the UV absorption spectrum as a function of time of the peptide of the second diastereoisomer of the peptide H-β2hVal-Tyr-Ile-His-Pro-Phe-OH. FIG. 2H shows the UV absorption spectrum as a function of time of the mixture of the H-Val-β2hTyr-Ile-His-Pro-Phe-OH peptide racemates.

FIG. 21 represents the UV absorption spectrum as a function of time of the first diastereoisomer of the H-Val-Tyr-β2hLeu-His-Pro-Phe-OH peptide;

FIG. 2 J represents the UV absorption spectrum as a function of time of the mixture of the H-Val-Tyr-β2hLeu-His-Pro-Phe-OH peptide racemates.

FIG. 2K represents the UV absorption spectrum as a function of time of the H-VaI-peptide.

Tyr-Ile-His-Phe-OH-β2hPro

FIG. 2L shows the UV absorption spectrum as a function of time of the mixture of the H-Val-Tyr-Ile-His-Pro-β2hPhe-OH peptide racemates. FIG. 2M represents the UV absorption spectrum as a function of time of the peptide H- β2hVal-Tyr-Ile-His-Pro-β3hPhe-OH FIG. 2N represents the UV absorption spectrum as a function of time of the peptide

NH2 ^v ^ CO-Tyr-Ile-His-Pro-Phe-OH

FIG. 20 represents the UV absorption spectrum as a function of time of the mixture of the H-β2hNle-Tyr-Ile-His-Pro-Phe-OH peptide racemates. FIG. 2P represents the UV absorption spectrum as a function of mixing time of the H-β2hLeu-Tyr-Ile-His-Pro-Phe-OH peptide racemates

FIG. 20 shows the UV absorption spectrum as a function of time of the mixture of the H-β2hLeu-Tyr-Ile-His-Pro-β3hPhe-OH peptide racemates

Figures 3A-D show HPLC profiles of iodination of peptides # 6, 11, and 17 and angiotensin IV. The x-axis represents the retention time in minutes, and the y-axis represents the intensity expressed in mV / I *.

FIG. 3A represents the profile of the peptide of angiotensin IV,

FIG. 3B represents the profile of ligand 6, FIG. 3C represents the profile of ligand 11, and

Figure 3D shows the profile of ligand 16.

Figure 4 depicts HPLC profiles of iodination of peptide No. 17 mono iodine. The x-axis represents the retention time in minutes, and the y-axis represents the intensity expressed in mV / I *.

Figure 5 shows the binding of angiotensin IV and peptide derivatives (ligands 6,11 and 17) to the recombinant IRAP-His protein. The abscissa axis represents the amount of recombinant IRAP expressed in μg / mL, the ordinate axis represents the radioactivity of iodine 125 expressed in B / Bmax. EXAMPLES

Example 1 Peptide synthesis

The synthesis of all the peptides was carried out by solid phase peptide synthesis using amino acids protected at the amino terminal position with tert-butoxycarbonyl (Boc) or β-9-fluorenylmethoxycarbonyl (Fmoc).

The peptides were synthesized on a Boc-Phe Merrified Resin (0.57 mmol / g), a Wang Fmoc-Phe resin (0.76 mmol / g) or a 2-chlorotrityl chloride resin (1.5 mmol / g). ). The side chain protection groups were Tyr (t-Bu), β3-hTyr (t-Bu), and His (Trt) for Fmoc synthesis, and Tyr (2,6-di-Cl-Bzl) and His ( Tos) for Boc synthesis. The Fmoc protection groups were removed with a solution of 20% piperidine in dimethyl formamide (DMF) (2 x 5 min), and the cleavage of the Boc protection groups was carried out in a solution of 50% trifluoroacetic acid ( TFA) in dichloromethane (DCM) (2 x 10 min) and a solution of 10% triethylamine (TEA) in DCM (2 x 5 min) was used for the neutralization. The amino acid coupling was carried out in DMF / DCM (Iv / Iv) using O- (Benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate (TBTU) (3 equivalents) and diisopropylethylamine (DIPEA) (6 equivalents). The synthesis protocols were as follows: Standard Fmoc cycle:

The 3 eq of amino acids (relative to the resin) and 6 eq of DIPEA (relative to the resin) were dissolved in CH ₂ Cl ₂ (10 ml per 1 g of resin), containing, if necessary a small amount of DMF to facilitate the solubility of the amino acid. The 2-chlorotritilchloride resin was pre-stabilized in CH ₂ Cl ₂ for 1 h, and after adding the mixture the resin was mixed for 30-120 min. Later, the resin was washed three times with CH ₂ Cl ₂ / MeOH / DIPEA (17: 2: 1), then twice with DMF and three times with CH ₂ Cl ₂ .

Standard Boc cycle

The amino-Boc acid was dissolved in EtOH (2 mL / mmol) and at 0. 5 mL / mmol, the pH was adjusted to 7 with 2M aqueous CS2CO3 solution. The solution was evaporated by drying, the residues were evaporated twice with dioxane. The Merrified resin was pre-stabilized in CH ₂ Cl ₂ for 1 h and washed with DMF. The cesium salt of amino acid (1.2 eq) in DMF was added to the resin, and the whole heated at 50 ⁰ C overnight. At the end of the reaction, the resin was washed three times in DMF, three times in DMF / water (1: 1), three times in DMF, three times in CH ₂ Cl ₂ , and three times in MeOH. In both strategies (Boc and Fmoc), 3 eq. of amino acid, 3 eq. TBTU and 6 eq. DI PEA were mixed and left for 3 min to allow activation. The solution is then added to the resin and stirred for two hours or overnight.

The colorimetric test corresponds to a Kaiser test. This test consists of 3 solutions: A: 5 g of ninhydrin in 100 ml of n-BuOH; B: 80 g of phenol in 20 ml of n-BuOH; C: 2 mL 0.001 M aqueous KCN in 98 mL of pyridine.

Some resin beads are placed in a test tube. 3 drops of each of the solutions are added and the tube is heated at 100 ^° C. for 5 minutes. When the resin or solution is colorless or yellowish, the result is negative. A blue color indicates the presence of a primary amine, and a brown / red color indicates the presence of a secondary amine.

The peptides were cleaved from the resin by treatment with a TFA / H 2 O / triethylsylan (TES) mixture (95: 2.5: 2.5) for 2 h, either with HF hydrofluoric acid, or a TF A / trifluoromethanesulphonic acid mixture ( TFMSA) / TES (20: 2: 3) in the case of a Merrifïeld resin. In both cases ethanol is added to precipitate the peptides. The precipitates are washed with EtOH.

The peptides were purified by reverse phase HPLC on a C18 Supelco Disco-BIO Wide Pore column. Each peptide was at least 98% pure after thin layer chromatography (TLC) and reversed phase HPLC analysis in water / acetonitrile solvent (FIGS. IA-IQ), or in a water / methanol solvent (FIGS. 2A-2Q). ). Molecular weights were confirmed by mass spectrometry-electrospay / ionization (ESI-MS).

The following table summarizes the properties of the peptides synthesized, M cal corresponds to the predicted mass, ESI MS corresponds to the mass obtained by mass spectrometry, R corresponds to the yield in percent relative to the crude peptides, Time Retl corresponds to the time at the time of elution water / acetonitrile, purity% l 1 corresponds to the purity of the product obtained after elution water / acetonitrile Time Ret2 corresponds to the retention time when eluting water / methanol, purity% 2 corresponds to the purity of the product obtained after elution water / methanol.

During the synthesis, all the β2 homo amino acids used were in the form of racemates, and after synthesis, two diastereoisomers were obtained. The separation of these diastereomers is indicated by the presence of the letters "a" and "b". When the diastereoisomers could not be separated, they are represented by "ab". By convention, the "a" diastereoisomers are eluted before the "b" diastereoisomers during reverse phase HPLC. Example 2 Tritium Marking

The neosynthesized and unpurified peptides were tritiated by catalytic saturation according to the procedure described in Tόth G et al. (1997 Methods Mol Biol Vol 73, pp: 219-30). The tritiated gaseous dihydrogen gas ³ H ₂ used is obtained from Technobexport, (Russia) and contains a tritium content greater than or equal to 98%.

The radioactivity of the crude peptides was measured using a TRI-CARB 2100TR scintillation counter in a scintillant composed of a toluene-Triton X-100 mixture. The measurement of the radioactivity was approximately 10OmCi (3.7 GBq)

The tritiated peptides were purified by HPLC using a Grace Vydac 218TP54 C18 column, and detection of the scintillation fluid was performed on a Canberra Packard Radiomatic 505TR Flow Radiochromatography detector using Ultima-Flo M scintillant. Specific activity tritiated purified peptides were measured by HPLC using a standard curve. The specific activity obtained was between about 30.0 Ci mmol- ¹ and

45.0 Ci mmol ^-1 .

Example 3: Iodine 125 Marking ( ¹²⁵ I)

The iodination of the peptides can be carried out according to several protocols known to those skilled in the art. The principle of protein iodination is based on the conversion of IT (NaI) to I ⁺ or I ^{3 "} in the presence of oxidizing agents such as chloramine-T, iodogen (1,3,4,6-tetrachloro) - 3α, 6α-diphenyl-glycoluril), the JV-chloro-benzenesulfonamide or the lactoperoxidase I ⁺ or I ^{3 "} are reactive species that attack the aromatic ring tyrosines meta position as described in" Antibodies: a laboratory manual " E. Harlow and D. Lane, CoId Spring Harbor Laboratory Press, 1988 pp. 324 - 339.

Material for iodination by JV-chloro-benzenesulfonamide

¹²⁵ I radioactive iodine is used as Na ¹²⁵ I in a basic solution at about 1 mCi (37 MBq) / 10 μL IODINE-125 (Amersham, IMS30).

Reagents

• 0.1 M phosphate buffer, pH 7.0 • Tris-buffered saline buffer (TBS)

• 5% BSA in TBS

• Stabilization solution (10% sodium thiosulfate + 0.1N NaOH) • IODO-BEAD ™ (PIERCE): grafted with an oxidizing agent (N-chlorobenzenesulfonamide)

• Hamilton microsyringe (model 702, 25μl, 22S needle, style 2)

• desalting column (Bio-rad DG-IO etc.) Procedure

1. In a plastic tube, 200 μl of 0.1 M phosphate buffer, pH 7 are mixed with 500 μCi of Na ¹²⁵ I (5 μL) and 1 at two doses of IODO-BEADs.

2. the mixture is incubated at room temperature for 5 min.

3. The peptides are added to the solution (between 1 to 100 μg of peptide)

4. the new mixture is incubated at room temperature for 10-25 min

5. the supernatant is removed and placed on a desalting column to remove salts, Na ¹²⁵ I and I ⁺ or I ^{3 "} free.

The fractions of the column are collected, and the radioactivity of each of the fractions is measured, using a scintillation counter.

7- In the radioactive fractions, ¹ A of the volume of a 5% BSA solution is added to prevent radiation decomposition.

Alternative procedure of iodination Reagents

^• Labeling buffer = 25mM ammonium acetate buffer pH 5.3

^• Conditioning buffer = PBS + 0.1% BSA

^• Purification buffer = 20% Acetonitrile + 0.5% TFA

^• HPLC purification, column Cl 8

^• reconstruction of labeling buffer ligands [ligand] = lOOμg / ml

Marking

Specific activity = 500μCi / μg or 18.5MBq / μg of ligand ^• 5μg (50μL) ligand

^• 92.5MBq of iodine

^• 15μg ChT

^• contact time = 60s

^• 15μg MBS

^• contact time = 30s

^• purification followed DO = reverse phase HPLC 275 nm according to the following gradient

Results: The HPLC profiles of 125 I-labeled IRAP synthetic ligands and angiotensin IV are shown in FIGS. 3A to 3D

^• Determination of the fraction "mono-iodinated" marking the step of ligand = 17 with iodine "cold" purification by HPLC analysis of fractions obtained by mass spectrometry, collecting the fraction "mono-iodinated" _ 2 step = Simultaneous injection of the "mono-iodine" fraction obtained during the stage and of the Fl fraction of Ligand 17 marked with iodine 125

3rd step = co-elution of the fraction F1 of the ligand labeled with TI125 and the fraction "monoiodée"

Figure 4 shows that the fraction F1 is "mono-iodinated".

Example 4: Specificity of peptides: measurement of labeled peptide binding / extracellular portion of IRAP.

Measurement of the binding between the labeled peptides and the extracellular domain of IRAP was performed by a binding assay adapted from the test described by Demaegdt et al. (Demaegdt et al., 2004, Biochem Pharmacol 68, 885-892). The extracellular domain of recombinant IRAP produced in affinity purified insect cells (High-Five) was taken up in a 50 mM Tris-HCl binding buffer (pH 7.4) containing 140 mM NaCl.

The incubations were performed in 24-well plates in a final volume of 300 μL comprising 100 μl of the extracellular portion of IRAP, 50 μl of an EDTA / 1,10-phenanthroline mixture with a final concentration of 500 μM of EDTA and 100 μM of 1,10-phenanthroline, and either 50 μl of binding buffer, or 50 μl of binding buffer containing the unlabeled peptides for the competition tests.

50 μl of labeled peptides were added at a final concentration of 0.5 to 0.8 nM for the saturation tests, ie at a concentration of 3nM for the other tests.

Incubation was performed at 37 ° C, at varying times (kinetics) up to 60 min.

Wells in which the extracellular portion of IRAP is adsorbed were then washed several times with incubation buffer to remove free radioactivity.

The radioactivity was then measured using a scintillation counter in the presence of ad hoc scintillation.

The affinity of the labeled peptides for the extracellular portion of IRAP has been evaluated between 1 and 10O nM

Example 5: Stability of Marked Peptides

In order to measure the stability of labeled peptides derived from angiotensin IV, their homologs labeled with cold iodine ( ¹²⁷ I) were incubated at a concentration of 100 μM for 40 min in a serum sample, which serum is not not previously treated with protease inhibitors.

The peptides were then separated from the serum by filtration gel and incubated at concentrations ranging from 0.1 nM to 1 .mu.M with the extracellular domain of IRAP purified in the presence of ^[125 I] - AnglV (0.1 - 1 nM) or [ ¹²⁵ I] -NiE-AnglV (0.1 - 1 nM) and peptidase inhibitors and proteases. The stability of the peptides in the serum sample is then measured indirectly by measuring the competition of the I-labeled peptides with the binding of [I] -AngIV or [I] -Nle-AnglV to the extracellular domain of IRAP (see A. Lukaszuk et al., Chem Chem 51, 2291-2226, 2008). Example 6: IRAP RIA

PRINCIPLE OF THE PROCEDURE The assay of the circulating extracellular portion of IRAP (extIRAPc) according to the invention uses an immunoradiometric method. The circulating extracellular portion of IRAP present in samples from patients where the standards is recognized by a monoclonal antibody (Mab) specific for the extracellular portion of IRAP, said monoclonal antibody being bound to the inner surface of the polystyrene tubes. . The addition of a peptide labeled with 125 I or tritium (peptide *), having a high affinity for extIRAPc induces the formation of a solid phase-bound sandwich complex (tube): Mab-extIRAPc - peptide * .

At the end of the incubation, the peptide fraction * not bound to the solid phase is removed by suction and washing. The formation of the complex is only realized in the presence of extIRAPc, the radioactivity related to the solid phase (tube) is directly proportional to the concentration of extIRAPc in the sample. A calibration curve makes it possible to determine, by interpolation, the concentration of extIRAPc of the samples to be assayed.

SAMPLE COLLECTION, PREPARATION AND STORAGE The assay can be performed on human serum. If the assay is performed within 24 hours of collection, samples may be stored at 2-8 ° C. Otherwise, they must be aliquoted and frozen at -20 ^° C. or at lower temperatures for a maximum of five months. If the samples have been frozen, wait until they are completely thawed and homogenize them before the assay. Avoid repeated freeze / thaw cycles.

If high extIRAPc levels are expected, it is necessary to dilute the sample with the zero standard (see below). It is recommended to use plastic tubes when preparing dilutions.

ASSAY PROCEDURE RECONSTITUTION OF LYOPHILIZED REAGENTS

• Reconstitute the zero standard with 5 mL of distilled water, the zero standard corresponding to a sample that does not have extIRAPc.

• Reconstitute standards 1 to 6 (serum samples for which the amount of extIRAPc is known) and controls Gl and G2 (Gl and G2 correspond to solutions containing extLRAPc) with 1 mL of distilled water.

Reconstitute reagents several minutes before use and mix gently (avoid foaming).

DILUTION OF THE WASH PAD: Add 900 mL of distilled water to 100 mL of concentrated wash buffer. Avoid foaming, (wash buffer: PBS, 0.1% albumin, pH 7.4).

ASSAY PROTOCOL Prior to use, coated tubes, standards, control sera and test sera are placed at room temperature (18-25 ° C) for at least 30 minutes; then their contents are mixed with the Vortex. It is recommended to run the assay in duplicate for both standards, control sera and samples. Carefully observe the order of use of the reagents: 1. Pipette 50 μL of each sample (standard, control serum and sample) and deposit as many tubes coated with the Mabs as samples taken, and in as many samples. uncoated tubes as samples taken.

2. Dispense 200 μL of peptide * into each of the tubes, including uncoated tubes for evaluation of total activity. 3. Incubate for 2 hours ± 5 minutes at room temperature (18-25 ° C) on a horizontal shaker (200-300 rpm).

4. Wash the tubes as follows: carefully aspirate the contents of the tubes (except those intended for evaluation of total activity), add 1 mL of diluted Wash Buffer to each tube (except those intended for evaluation). total activity) and aspirate the dressing solution. Repeat the washing a second time. To obtain reproducible and reliable results it is necessary to carry out the washing correctly. Careful observation of the incubation times and complete elimination of the washing solution are fundamental for a successful analysis.

5. Using a gamma counter, measure the activity of all tubes, including those intended for evaluation of total activity.

QUALITY CONTROL

It is recommended to include in each analysis control samples to verify the quality of the results obtained.

All samples should be treated in the same way and the results analyzed with an adequate statistical method. If the values of the control sera are not within the indicated acceptability range, the assay must be repeated. CALCULATION OF RESULTS

1. Calculate the average of the counts for each standard, control or sample.

2. Plot the calibration curve using the average counts of each standard plotted on the y-axis versus the respective insulin concentrations on the x-axis. 3. Calculate the concentrations of the controls and samples by interpolation of the calibration curve using the respective average counts, correcting, as appropriate, the dilution factor used.

Example of RIA * 4G6: Anti-IRAP monoclonal antibody (IgG3a) directs against the peptide sequence

QKKGKELFIQQER (peptide sequence of IRAP N ° 3)

* Tube: BSA-biotin-avidin-4G6-biot

* Recombinant protein IRAP-HIS (Iot712-CAP-O5p) at 410 μg / ml

Range between 0 and 12.5μg / ml Buffer range "IRAP-HIS": PBS (1 ImM PO4 + 140mM Nacl) + 1% BSA free protease

+ 0.1% proclin + 5mM EDTA

* Tracer Packaging Buffer = IRAP-HIS + 120-M Phenanthroline Buffer

Tracers, F1 fractions of ligands labeled at lμci / ml

* Ligand 6

* Ligand 11

* Ligand 17

* Angiotensin IV

^ Wash solution: H2O + 0.05% tween 20

Protocol

* 200μl / tube IRAP-His

Incubation 4h TA with shaking (750 rpm) * 2 washes = suction + (2 x 2ml / tube) * 200μl ligand * / tube Incubation one night at RT * 2 washes = suction + (2 x 2ml / tube) * count of radioactivity on lmn

The results are shown in Figure 5.

Claims

claims

1. Method for assaying the circulating extracellular portion of the protein IRAP ("insulin responsive aminopeptidase") comprising at least one step of quantitative assay of said extracellular, secreted, purified part of IRAP using at least one peptide modified and labeled, said labeled peptide interacting specifically with said extracellular part of IRAP, said peptide being modified by the presence of at least one non-natural amino acid, and / or at least one β-homo amino acid, natural or otherwise, said labeled peptide being preferentially labeled with at least one radioactive isotope, provided that

said peptide is not an antibody and

said modified peptide, labeled with a ¹²⁵ I iodine atom, is different from the NIe-Tyr-Ile-His-Pro-Phe sequence (SEQ ID No. 23).

The method for assaying the circulating extracellular portion of IRAP according to claim 1, wherein said modified and labeled peptide is an IRAP ligand which is either a labeled substrate of the IRAP protein, selected from [Arg] -vasopressin marked and modified,

• marked and modified oxytocin,

• the marked and modified Met- / Leu-enkephalin,

• marked and modified Somatostatin,

• Marked and modified CCK-8, • Marked and modified Neurokinin A,

• marked and modified Neuromedine B,

• the lys-bradykinin labeled and modified, and

Modified and modified modified Dynorphin A, an inhibitory peptide labeled with the IRAP protein, selected from angiotensin IV, labeled and modified, and

• LVV-hemorphin-7 marked and modified,

The method for assaying the circulating extracellular portion of IRAP according to claim 1 or 2, comprising at least:

A step of purifying said circulating extracellular portion of IRAP, and

A step of quantification of said circulating extracellular portion purified in the preceding step, by means of at least one modified peptide labeled as defined in claim 1 or 2.

4. A method for assaying the circulating extracellular portion of IRAP according to any one of claims 1 to 3, wherein said purification of the circulating portion of the IRAP protein is carried out using at least one antibody specific for said extracellular portion. IRAP, in particular by immunoprecipitation or immunocapture.

5. A method for assaying the circulating extracellular portion of IRAP according to any one of claims 1 to 4, wherein said IRAP protein is represented by the IRAP protein of sequence SEQ ID No. 1, or a protein homologous to the IRAP protein. having a sequence identity of at least 80% with the sequence SEQ ID NO 1, provided that said homologous protein is capable of interacting specifically with the IRAP ligands or the IRAP inhibitory peptides, or - an iso form of the IRAP protein, said iso form being the product of the alternative splicing of the RNA encoding the IRAP protein SEQ ID NO 1, provided that said isoform is capable of interacting specifically with the IRAP ligands or the inhibitory peptides of IRAP, said isoform of the IRAP protein SEQ ID NO 1 being in particular represented by the proteins SEQ ID NO 2 or 3.

The assay method according to claim 5, wherein

The assay method according to any one of claims 1 to 6, wherein said circulating extracellular portion of the IRAP protein has a amino acid sequence. embedded image represented by the sequences selected from the following sequences: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

8. Assay method according to any one of claims 1 to 7, wherein said labeled modified peptide is labeled with at least one tritium atom or at least one iodine atom.

¹²⁵ I, preferably a single ¹²⁵ I iodine atom.

An assay method according to any one of 1 to 8, wherein said modified modified peptide is represented by the following general formula (III):

where a, b, c, d, e, f, g, h, i and j may be equal to 0 or 1, such that (a + b) ≤l, (c + d) ≤l,

(e + f) ≤l, (g + h) ≤l and (i, j) ≤l, (a, b), (c, d), (e, f), (g, h) and (i , j) being independent of one another, where (Ri, Ri ') are such that:

R1 is chosen from: a group -CH (CHs) ₂ , a group -CH ₂ -CH (CHs) ₂ , a group -CH (CH ₃ ) -CH ₂ -CH ₃ , a group - (CH ₂ ) ₃ - CH ₃ and a group C (CH ₃ ) ₃ , and R 1 'is a hydrogen atom, or R 1 and R 1' together with the carbon which carries them a cyclopentyl, where (R ₂ , R ₂ ') are such that : if R2 is chosen from: a group -CH ₂ -CH (CH ₃ ) ₂ , a group -CH (CH ₃ ) -CH ₂ -CH ₃ , a group - (CH ₂ ) ₃ -CH ₃ and a group C (CH ₃ ) ₃ , R2 'is a hydrogen atom, or

R2 and R2 'together with the carbon which carries them a cyclopentyl, the pairs (Ri, Ri') and (R ₂ , R ₂ ') being chosen independently of each other, where A is selected from groups following

if (R 1, R 1 ') = (-CH (CH ₃ ) ₂ ; H) then (R ₂ , R ₂ ') is different from (-CH (CH ₃ ) -CH ₂ -CH ₃ ; H), and

An assay method according to any one of claims 1 to 9, wherein said labeled and modified peptide consists of the following sequence: X1-X2-X3-His-X4-X5 (SEQ ID NO. 22) or

X2 may be a tyrosine, or a β ² or β ³ derivative of tyrosine,

X4 may be a proline or a β ² or β ³ derivative of proline,

an aba-gly of formula lia or

aia-gly of formula Hb

The assay method of claim 9 or 10, wherein said labeled and modified peptide consists of the following sequence:

Xl-X2-X3-His-X4-X5 (SEQ ID No. 22) and wherein at least one amino acid X1 to X5 is a β-homo amino acid, natural or not.

The assay method according to any one of claims 9 to 11, wherein said labeled modified peptide is labeled By at least one ¹²⁵ I iodine atom on tyrosine X 2, preferably one single ¹²⁵ I iodine atom, in particular on the phenyl group, or

By replacing at least one of the hydrogen atoms with a ³ H tritium atom.

The assay method of any one of claims 9 to 12, wherein said labeled modified peptide is selected from the following peptides:

β ² hVal-Tyr-Ile-His-Pro-Phe- (SEQ ID No. 24),

- β ² hVal-Tyr-Ile-His-Pro-β ³ hPhe- (SEQ ID NO 25), - β ² hLeu-Tyr-Ile-His-Pro-β ³ hPhe- (SEQ ID NO 26),

Val-Tyr-Ile-His-β ² hPro-Phe- (SEQ ID No. 27),

β ² hVal-Tyr-Ile-Aba-Gly-Phe- (SEQ ID No. 28), and

- β2HLeu-Tyr-Ile-His-Pro-β3hPhe- (SEQ ID No. 26),

Val-Tyr-Ile-His-β ² hPro-Phe- (SEQ ID No. 27),

β ² hVal-Tyr-Ile-Aba-Gly-Phe- (SEQ ID No. 28), and

The assay method according to any one of claims 1 to 14, comprising

A step of purifying said circulating extracellular portion of IRAP, and a step of quantifying said circulating extracellular portion purified in the preceding step, by means of at least one modified peptide of amino acid sequence SEQ ID NO Labeled with ¹²⁵ I radioactive iodine, preferably with a single ¹²⁵ I iodine atom.

16. Assay method according to any one of claims 1 to 14, comprising

A step of purifying said circulating extracellular portion of IRAP, and

A step of quantification of said circulating extracellular portion purified in the preceding step, by means of at least one modified peptide labeled with amino acid sequence SEQ ID No. 25 tritiated.

17. A modified modified peptide represented by the following general formula (III):

where a, b, c, d, e, f, g, h, i, and j may be 0 or 1, such that (a + b) ≤l, (c + d) ≤l, (e + f) ≤l, (g + h) ≤l and (i, j) ≤l, (a, b), (c, d), (e, f), (g, h) and (i, j ) being independent of each other, where (R ₁ , R ₁ ') are such that:

R1 is chosen from: a group -CH (CH ₃ ) ₂ , a group -CH ₂ -CH (CH ₃ ) 2, a group -CH (CHs) -CH ₂ -CH ₃ , a group - (CH ₂ ) ₃ -CH ₃ and a group C (CH ₃ ) ₃ , and R 1 'is a hydrogen atom, or

R1 and R1 'together with the carbon which carries them a cyclopentyl, where (R ₂ , R ₂ ') are such that:

- if R2 is chosen from: a group -CH ₂ -CH (CH ₃ ) ₂ , a group -CH (CH ₃ ) -CH ₂ -CH ₃ , a group - (CH ₂ ) ₃ -CH ₃ and a group C (CH ₃ ) ₃ , R2 'is a hydrogen atom, or

A modified labeled peptide, said peptide being labeled with at least one radioactive isotope consisting of the following sequence: X1-X2-X3-His-X4-X5 (SEQ ID NO. 22), said peptide being modified, wherein

X2 may be a tyrosine, or a β ² or β ³ derivative of tyrosine,

X3 may be Leucine (Leu), Isoleucine (Ile), or an unnatural amino acid derived from leucine, in particular Norleucine (Nie), Cycloleucine (CIe) or tert-leucine (TiE), or a derivative thereof β ² or β ³ of one these natural or unnatural amino acids,

X4 can be proline or a β ² or β ³ derivative of proline,

an aba-gly of formula lia

aia-gly of formula Hb and provided that said unmodified labeled peptide is different from angiotensin IV, and in particular that said unmodified labeled peptide is different from SEQ ID NO. 21 (Val-Tyr-Ile-His-Pro-Phe), under reserve that the sequence SEQ ID NO 22 labeled with a ¹²⁵ I iodine atom is different from the Nle-Tyr-Ile-His-Pro-Phe sequence (SEQ ID NO 23).

19. The modified modified peptide according to claim 18, consisting of the following sequence: X1-X2-X3-His-X4-X5 (SEQ ID No. 22), said peptide being modified such that at least one of amino acids X1 to X5 is a β-homo amino acid, natural or not.

The modified modified peptide according to claim 18 or 19, wherein said modified labeled peptide is labeled

By replacing at least one of the hydrogen atoms with a ³ H tritium atom.

A modified modified peptide according to any one of claims 18 to 20, wherein said modified labeled peptide is selected from the following peptides:

- β2HVal-Tyr-Ile-His-Pro-Phe- (SEQ ID NO. 24),

Val-Tyr-Ile-His-β ² hPro-Phe- (SEQ ID No. 27),

β ² hVal-Tyr-Ile-Aba-Gly-Phe- (SEQ ID No. 28), and

- β ² hVal-Tyr-Ile-Aia-Gly-Phe- (SEQ ID NO 29), preferably the peptide SEQ ID NO 25, said modified modified peptide being iodinated, preferably with a single 125 I _T iodine atom.

A modified modified peptide according to any one of claims 8 to 20, wherein said modified labeled peptide is selected from the following peptides:

- β2HLeu-Tyr-Ile-His-Pro-β3hPhe- (SEQ ID No. 26),

Val-Tyr-Ile-His-β ² hPro-Phe- (SEQ ID No. 27),

β ² hVal-Tyr-Ile-Aba-Gly-Phe- (SEQ ID No. 28), and