(12) United States Patent (10) Patent No.: US 8,642,729 B2 Rougeot (45) Date of Patent: Feb

USOO8642729B2

(12) United States Patent (10) Patent No.: US 8,642,729 B2 Rougeot (45) Date of Patent: Feb. 4, 2014

(54) OPORPHINPEPTIDE DERVATIVES AS (56) References Cited POTENT INHIBITORS OF ENKEPHALN-DEGRADING U.S. PATENT DOCUMENTS ECTOPEPTIDASES 5,859,189 A * 1/1999 Rosinski-Chupin et al. , 530/330 6,184,205 B1* 2/2001 Sparks et al...... 514,75 (75) Inventor: Catherine Rougeot, Chevreuse (FR) FOREIGN PATENT DOCUMENTS (73) Assignee: Institut Pasteur, Paris (FR) WO 98.37100 8, 1998 (*) Notice: Subject to any disclaimer, the term of this WO O2/O51435 T 2002 patent is extended or adjusted under 35 WO 2005/090386 9, 2005 U.S.C. 154(b) by 776 days. WO 2008/096276 8, 2008 OTHER PUBLICATIONS (21) Appl. No.: 12/419,426 Rougeot, Catherine (European Journal of Biochemistry (1994), (22) Filed: Apr. 7, 2009 219(3), 765-73).* U.S. Appl. No. 12/936,591, filed Oct. 6, 2010, Rougeot. (65) Prior Publication Data U.S. Appl. No. 13/131,470, filed May 26, 2011, Rougeot, et al. U.S. Appl. No. 13/102,367, filed May 6, 2011, Rougeot, et al. US 2009/O253639 A1 Oct. 8, 2009 International Search Report issued in the corresponding PCT/ Related U.S. Application Data EP2009/054171 dated Aug. 9, 2009. (60) Provisional application No. 61/042.922, filed on Apr. * cited by examiner 7, 2008. Primary Examiner — David Lukton (51) Int. C. (74) Attorney, Agent, or Firm — Oblon, Spivak, C07K 9/00 (2006.01) McClelland, Maier & Neustadt, L.L.P. C07K 5/10 (2006.01) (52) U.S. C. (57) ABSTRACT USPC ...... 530/330; 514/21.8 The present invention relates to modified opiorphin peptides (58) Field of Classification Search as new inhibitors of metallo-ectopeptidases. USPC ...... 530/330; 514/21.8 See application file for complete search history. 23 Claims, 23 Drawing Sheets U.S. Patent Feb. 4, 2014 Sheet 1 of 23 US 8,642,729 B2 Fig. 1

PeptidoMimetics Effect on AP

QHNPR (sialorphin)

QRFSR (Opiorphin) QRFS(O-C8)R QRFPR QRePR CQRFSR 30 Time (min) U.S. Patent Feb. 4, 2014 Sheet 2 of 23 US 8,642,729 B2

Fig. 2 PeptidoMimetic effect on NEP-Endopeptidase Activity

it. AS QHNPR Sialorphin 3.

QRFTR QRFSR Opiorphin Acetyl-QRFSR

Substrate without Enzyme

1000 2O 3O AO Time (min) U.S. Patent Feb. 4, 2014 Sheet 3 of 23 US 8,642,729 B2

Fig. 3 PeptidoMimetic Effect on NEPCDP1 Activity

Vehicle (Red)

QRFSR peluRFSR

CQRFSR

Substrate without Enzyme

Time (min) U.S. Patent Feb. 4, 2014 Sheet 4 of 23 US 8,642,729 B2

Fig. 4 PeptidoMimetic Effect on NEP-CDP2

peluRFSR QRFSR Opiorphin

CQRFSR

Substrate without Enzyme

Time (min) U.S. Patent Feb. 4, 2014 Sheet 5 of 23 US 8,642,729 B2

Fig. 5

Inhibition of Enkephalin-inactivating human ectopeptidases, 100. QRFSR-CONH2, 50 uM QRGPR, 50 uM 90. QHNPR, 50 uM 80%l QR(4BrFSR, 50 uM QRFPR, 50 uM 70% 60%|

40,

30,

20,

10,

O% UI ...... : ||

(10).

2 t us 2. U.S. Patent Feb. 4, 2014 Sheet 6 of 23 US 8,642,729 B2

Fig. 6

Inhibition of enkephalin-inactivating human ectopeptidases, 90%.

80% O Y(PE12)QRFSR, 50 M y(PE6)QRFSR, 50 uM 70% yoRFSR, 50 uM

60.

50.

40.

30.

20%

10,

O. I s g s s 2. i ep eM y y y U.S. Patent Feb. 4, 2014 Sheet 7 of 23 US 8,642,729 B2

Fig. 7 O CQRFSR, 50 uk C-CQRFSR2, 50 M (non purifié) Inhibition of enkephalin-inactivating human ectoenzymes, . OO

sox

80.

60.

50%-

40%-

3O.

2O,

10%-

O U.S. Patent Feb. 4, 2014 Sheet 8 of 23 US 8,642,729 B2

Fig. 8 O QRFs(C8)R, 50 uM (C8)QRFSR, 50 uM Inhibition of enkephalin-inactivating human ectopeptidases, AccRFSR.A 50 M 100%-

90.

80.

60% 50%

30%-

20%-

O 0%-

(10% U.S. Patent Feb. 4, 2014 Sheet 9 of 23 US 8,642,729 B2

as CORFSR (% inh NEP CDP2) COORFSR (%lrah NEP endo) CORFSR (% inh NEP-MicaBK) O- CORFSR (%inh APN) Inhibition of FRET-substrate Hydrolysis by human ectopeptidases, 7.

1OO90 - 4

1. O CQRFSR) uw FIG.9 U.S. Patent Feb. 4, 2014 Sheet 10 of 23 US 8,642,729 B2

v. CRFSO-C8R (% inh NEP CDP2) CRFSO-CBR (% inh NEP endo) CRFSO-C8)R (% inh NEP-MicaBK) or ORFSO-C8)R (%lnh APN) Inhibition of FRET-substrate Hydrolysis by human ectopeptidases, 7.

1. O (CRRFS(O-C8)RuM

FIG.10 U.S. Patent Feb. 4, 2014 Sheet 11 of 23 US 8,642,729 B2

Inhibition of FRET-substrate Hydrolysis by human ectopeptidases, 7.

0.5 O 15 2.0 Logy (C12)QRFSR) M O

YC2)QRFSR (Inh APN) A y(C2)QRFSR (, Inh NEP-COP2) YC2)QRFSR (, Irth NEP-Endo) v y(C12)QRFSR (Inh NEP-Mico8 K2)

FIG.11 U.S. Patent Feb. 4, 2014 Sheet 12 of 23 US 8,642,729 B2

Fig. 12

CQRFSR Effect on hapM 2OOOO role |- &

Vehicle

-- (->) Temp. 28°C

o OOOO

arrier wrestrass

Time (min) U.S. Patent Feb. 4, 2014 Sheet 13 of 23 US 8,642,729 B2

Fig. 13

CQRFSR effect on NEP CDP-MicaBK2 g7so-T-rr

H -- (-x) Temp.

10 IM

20 uw

30 IM.

50 LM

Control: Substrate without enzyme

O 2.5 5 7.5 10 Time (min) U.S. Patent Feb. 4, 2014 Sheet 14 of 23 US 8,642,729 B2

Fig. 14 CQRFSR Effect on NEP-CDP specific activity g750 - 8:48.8%sexes:Xss

-- (-x) Temp. Vehicle 1 IM 2 u. 3 IM 4 IM 5 M

10 IM.

20 IM

30 IM

50 uM

Control: Substrate without enzyme

O O 2O 30 40 Time (min) U.S. Patent Feb. 4, 2014 Sheet 15 of 23 US 8,642,729 B2

Fig. 15

CQRFSR effect on NEP endopeptidase Activity

SOOO wramminennessessity ro- on- -- sourismates -- (-X) Temp.

Vehicle 1 IM 2 IM 3 M 4 M

10 LM

2O IM

3OuW 50 iM Control: Substrate without enzyme s O O 2O 30 Time (min) U.S. Patent Feb. 4, 2014 Sheet 16 of 23 US 8,642,729 B2

Fig. 16

QRFSO-C8)REffect on NEP CDP specific Activity

1250

Vehicle 1 IM 2 plw 3 M 4 IM 5 pilw

10 IM

Control: Substrate without enzyme

Time (min) U.S. Patent Feb. 4, 2014 Sheet 17 Of 23 US 8,642,729 B2 Fig. 17

QRFSOC8)R Effect on NEP Endopeptidase

5000 rocessessor int-in-curu -- (->) Temp.

AOO

Vehicle

1 IM

2 IM 3 IM 4. LIM 5 M

10 IM

2O Lilw 30 IM. 50 in

O O 2O 30 Time (min) U.S. Patent Feb. 4, 2014 Sheet 18 of 23 US 8,642,729 B2

Fig. 18 Inhibition of FRET-substrate Hydrolysis by human ectopeptidases, 7%

80.

60.

-2 1. O 2 CORFSRuM (log scale)

e CORFSR (%irth APN) v CORFSR (% inh NEP-CDP1-MicaBK) D CORFSR (% inh NEP-ando) A CCRFSR (% inh NEP-CDP2) U.S. Patent Feb. 4, 2014 Sheet 19 Of 23 US 8,642,729 B2

Fig. 19 Inhibition of FRET-substrate Hydrolysis by human ectopeptidases, 7.

0.5 O .5 2.0 S. logy(CI2)QRFSR M. -1

to Y(C12)QRFSR (%Inh APN) y(C12)QRFSR (7. Inh NEP-Endopeptidase) A y(C2)QRFSR (%. Inh NEP-carboxypeptidase1) Y YC12)QRFSR (%. Inh NEP-carboxypeptidase2) U.S. Patent Feb. 4, 2014 Sheet 20 of 23 US 8,642,729 B2

Fig. 20 Inhibition of FRET-substrate Hydrolysis

by human ectopeptidases, 2.

Loq ICC6QRFSR M ... O

CC6XQRFSR (Inh APN) C(C6)ORFSR (%inh NEP-CDP2) A C(C6)ORFSR (%inh NEP-endo) A C(C6)CRFSR (%inh NEP-COP1-Mca) U.S. Patent Feb. 4, 2014 Sheet 21 of 23 US 8,642,729 B2

Fig. 21 Inhibition of FRET-substrate Hydrolysis by human ectopeptidases, 2. OO

-2 2 COPE12)QRFSR, M (log scale) O Inhibition haPN, 7. A Inhibition NEP1-COP, . O Inhibition NEP-endopeptidase, . U.S. Patent Feb. 4, 2014 Sheet 22 of 23 US 8,642,729 B2

Fig. 22 Inhibition of FRET-substrate Hydrolysis by human ectopeptidases, 2.

1.5 a 1.0 0.5 0.0 O.S O CPE6)QRF(S-O-C8)R, uM (log scale)

O Inhibition of haP-N,. Inhibition of NEP1-endopeptidase, 7. A Inhibition of NEP1-CDP, U.S. Patent Feb. 4, 2014 Sheet 23 of 23 US 8,642,729 B2

Fig. 23 Inhibition of Substance P hydrolysis by cell surface hNEP expressed by human epithelial LNCaP cells, 4. OO

80mmam Maximal inhibition

Threshold inhibition

O 2 3 4. 5 CPE6)QRFS-O-C8)R, nM (log scale) US 8,642,729 B2 1. 2 OPORPHINPEPTDE DERVATIVES AS ronmental changes in rat. Ratsialorphin is an endocrine pep POTENT INHIBITORS OF tide signal whose expression is activated by androgen regu ENKEPHALN-DEGRADING lation and whose secretion is stimulated under adrenergic ECTOPEPTIDASES mediated response to environmental stress in male rats. It is a physiological inhibitor of the membrane-anchored rat NEP The instant application claims the benefit of U.S. provi activity and is a powerful inhibitor of pain sensation in rats sional application Ser. No. 61/042.922 which was filed on (Rougeot, C. Rosinski-Chupin, I., Namkepo, E & Rougeon, Apr. 7, 2008, and which is incorporated by reference. F. (1994) EurJ Biochem 219,765-773; Rougeot, C, Vienet, R, Cardona, A. Le Doledec, L. Grognet, J M & Rougeon, F. SEQUENCE LISTING 10 (1997) Am J Physiol 273, R1309-R1320.: Rougeot, C, Ros inski-Chupin, I & Rougeon, F. (1998) in Biomedical Reviews The instant application contains a Sequence Listing which eds. Chaldakov, GN & Mathison, R. (Bulgarian-American has been submitted in ASCII format via EFS-Web and is Center, Varna, Bulgaria.) Vol 9, pp. 17-32: Rosinski-Chupin, hereby incorporated by reference in its entirety. Said ASCII I, Huaulme, J. F. Rougeot, C & Rougeon, F. (2001) Endocri copy, created on May 9, 2013, is named 340443US0 SL.txt 15 nology 142, 4550-4559; Rougeot, C. Messaoudi, M. Her and is 12,700 bytes in size. mitte, V. Rigault, A G. Blisnick, T. Dugave, C. Desor, D & Rougeon, F. (2003) Proc Natl AcadSci USA 100,8549-8554). BACKGROUND OF THE INVENTION Previously we demonstrated that human Opiorphin native peptide (QRFSR-peptide (SEQID NO: 43)), the first charac 1. Field of the Invention terized in human to date, is an efficient dual inhibitor of two The present invention relates to modified opiorphin pep enkephalin-inactivating ectopeptidases, neutral endopepti tides as new inhibitors of metallo-ectopeptidases. dase NEP (EC 3.4.24.11) and aminopeptidase AP-N (EC 2. Description of the Related Art 3.4.11.2) (Wisner et al. Proc Natl AcadSci USA, November Zinc metal ectopeptidases control the receptor-dependent 2006, 103(47): 17979-84). activity of neural and hormonal mediators involved in the 25 Opiorphin peptide derivatives have been described previ regulation of important physiological functions in mammals. ously, see, e.g., Wisneretal. Proc Natl AcadSci USA, Novem They are located at the surface of cells in nervous and sys ber 2006, 103(47): 17979-84 and has the basic peptide temic tissues and catalyze postsecretory processing or sequence as QRFSR (SEQID NO: 1). metabolism of neuropeptides and regulatory peptides Thus, this sequence and the sequence defined by the fol (Rogues, BP, Noble, F. Dauge, V. Fournie-Zaluski, M C & 30 lowing formula may be modified: X1-X2-Arg-Phe-Ser-Arg Beaumont, A. (1993) Pharmacol Rev 45,87-146. Turner, AJ, (SEQ ID NO: 2), wherein X1 represents H atom or a Tyr Isaac, RE & Coates, D. (2001) BioEssays 23, 261-269). amino acid, X2 represents Glin or Glp when X1 is H, or X2 Prominent among these neuronal and/or hormonal peptide represents Gln when X1 is Tyr or Cys. Preferred is QRFSR signals are substance P (SP) and enkephalins, which are (SEQID NO: 1),YQRFSR (SEQID NO:3), and/or CQRFSR implicated in the receptor-dependent modulation of behav 35 (SEQ ID NO: 4) with QRFSR (SEQ ID NO: 1) most pre ioral adaptive responses to stressful or threatening environ ferred. It is understood that Glp is pyroglutamate, Tyr or Y is mental stimuli. They notably regulate spinal processing of Tyrosine, Gln or Q is glutamine, Arg or R is Arginine, Phe or nociceptive information and analgesic mechanisms, emo F is Phenylalanine, Ser or S is Serine, and Cys or C for tional and/or motivational responses, anxiety, aggression, and Cysteine. These peptides have been described in the interna neuroimmune inflammatory phenomena (Dickenson, A H. 40 tional patent application published as WO2005090386. (1995) Br J Anaesth 75, 193-200. Sora, I, Takahashi, N, Funada, M., Ujike, H. Revay, R S, Donovan, D M, Miner, SUMMARY OF THE INVENTION L. L & Uhl, G. R. (1997) Proc Natl AcadSci USA 94, 1544 1549; Konig, M. Zimmer, A M, Steiner, H., Holmes, P V. The invention provides new Opiorphin derivatives and in Crawley, JN, Brownstein, MJ & Zimmer, A. (1996) Nature 45 vitro functional characterization by using highly selective 383,535-538; Filliol, D, Ghozland, S, Chluba, J, Martin, M, biochemical assays. Matthes, H W. Simonin, F. Befort, K, Gaveriaux-Ruff, C, Dierich, A & LeMeur, M, et al. (2000) Nat Genet. 25, 195 BRIEF DESCRIPTION OF THE DRAWINGS 200). Because of the physiological importance and the critical 50 FIGS. 1-4. The kinetic of hydrolysis of corresponding role of Zinc ectopeptidases in modulating the functional FRET-peptide substrates by recombinant hNEP or hAP-N in potency of downstream neuronal and hormonal signals, it is the presence of vehicle or in the presence of 50 uMQRFSR essential to focus on what controls their activity and, as a peptide analogs (SEQID NO: 1). Each point represents the consequence, the overall regulatory cascade. The discovery intensity of the signal expressed in RFU (Relative Fluores of upstream regulators of ectopeptidase activity also is excit 55 cence Unit), which was proportional to the quantity of ing from physiopathological and therapeutic points of view metabolites formed, as function of reaction time (min). FIG. because of the potential for developing new candidate drugs. 1 discloses SEQ ID NOS 17, 34, 10, 43, 6, 26, 9 and 4, A brain-specific heptapeptide named spinorphin was iso respectively, in order of appearance. FIG. 2 discloses SEQID lated and characterized from bovine spinal cord based on its NOS 10.9, 31, 43, 12, 36,38, 17, 32, 6 and 4, respectively, in inhibitory activity toward enkephalin-degrading ectoen 60 order of appearance. FIG.3 discloses SEQID NOS 1, 17,32. Zymes, such as neutral endopeptidase (NEP: EC 3.4.24.11 6 and 4, respectively, in order of appearance. FIG. 4 discloses ECI) and aminopeptidase N (AP-N; EC 3.4.11.2 (ECI) SEQ ID NOS 9, 17, 43, 32, 6 and 4, respectively, in order of (Nishimura, K & Hazato, T. (1993) Biochem Biophy's Res appearance. Commun 194, 713-719. Yamamoto, Y, Ono, H., Ueda, A, FIG. 5: Inhibition by QRFSR-peptide analogs (SEQ ID Shimamura, M. Nishimura, K & Hazato, T. (2002) Curr 65 NO: 1) of hydrolysis of corresponding FRET-peptide sub Protein Pept Sci 3, 587-599). In addition, we characterized rat strates by pure recombinant human hNEP or AP-N. Each bar sialorphin, a peptide mediator involved in adaptation to envi represents the percentage of intact Substrate recovered and US 8,642,729 B2 3 4 calculated as: percentage of velocity without inhibitor-veloc strate recovered and calculated as: percentage of Velocity ity in presence of inhibitor/velocity without inhibitor, which without inhibitor-velocity in presence of inhibitor/velocity was measured in the absence or in the presence of 50 uM of without inhibitor, which was measured in the absence or in QRFSR-peptide analogs (SEQ ID NO: 1). FIG. 5 discloses the presence of various concentrations of CRFSR-peptide SEQID NOS 8-11 and 26, respectively, in order of appear (SEQID NO: 5) plotted in LM (log-scale). aCC. FIG. 19: Concentration-dependent Inhibition by YPE12 FIG. 6: substrates by pure recombinant human hNEP or QRFSR-COOH peptide (i.e. Y-(-HN-(CH)— AP-N. Each bar represents the percentage of intact substrate CO-)-QRFSR) (SEQ ID NO:32) of hydrolysis of corre recovered and calculated as: percentage of Velocity without sponding FRET-peptide substrates by pure recombinant inhibitor-velocity in presence of inhibitor/velocity without 10 human hNEP or AP-N. Each point represents the percentage inhibitor, which was measured in the absence or in the pres of intact Substrate recovered and calculated as: percentage of ence of 50 uM of QRFSR-peptide analogs (SEQID NO: 1). velocity without inhibitor-velocity in presence of inhibitor/ FIG. 6 discloses SEQID NOS 32, 38 and 3, respectively, in velocity without inhibitor, which was measured in the order of appearance. absence or in the presence of various concentrations of FIGS. 7-8: Inhibition by QRFSR-peptide analogs (SEQID 15 Y(PE12)QRFSR COOH peptide (SEQID NO:32) plotted NO: 1) of hydrolysis of corresponding FRET-peptide sub in LM (log scale). strates by pure recombinant human hNEP or AP-N. Each bar FIG. 20: Concentration-dependent Inhibition by CIPE6 represents the percentage of intact Substrate recovered and QRFSR-COOH peptide (i.e. C ( HN-(CH) calculated as: percentage of velocity without inhibitor-veloc CO-)-QRFSR) (SEQ ID NO: 33) of hydrolysis of corre ity in presence of inhibitor/velocity without inhibitor, which sponding FRET-peptide substrates by pure recombinant was measured in the absence or in the presence of 50 uM of human hNEP or AP-N. Each point represents the percentage QRFSR-peptide analogs (SEQ ID NO: 1). FIG. 7 discloses of intact Substrate recovered and calculated as: percentage of “CQRFSR as SEQ ID NO. 4 and the core peptide in the velocity without inhibitor-velocity in presence of inhibitor/ dimer “C CQRFSR2 as SEQ ID NO: 4. FIG. 8 discloses velocity without inhibitor, which was measured in the “QRFS(O C8)R’ as SEQID NO: 6, “(C8)QRFSR as SEQ 25 absence or in the presence of various concentrations of ID NO:36, and “AcQRFSR as SEQID NO: 12. CPE6QRFSR COOH peptide (SEQID NO:33) plotted in FIG. 9: Concentration-dependent Inhibition by CQRFSR uM (log scale). peptide (SEQ ID NO: 4) of hydrolysis of corresponding FIG. 21: Concentration-dependent Inhibition by C(PE12) FRET-peptide substrates by pure recombinant human hNEP QRFSR-COOH peptide (i.e. C ( HN-(CH) or AP-N. Each point represents the percentage of intact sub 30 CO-)-QRFSR) (SEQ ID NO. 34) of hydrolysis of corre strate recovered and calculated as: percentage of Velocity sponding FRET-peptide substrates by pure recombinant without inhibitor-velocity in presence of inhibitor?velocity human hNEP or AP-N. Each point represents the percentage without inhibitor, which was measured in the absence or in of intact Substrate recovered and calculated as: percentage of the presence of various concentrations of CRFSR-peptide velocity without inhibitor-velocity in presence of inhibitor/ (SEQ ID NO: 5) plotted in LM (log-scale). 35 velocity without inhibitor, which was measured in the FIG.10: Concentration-dependent Inhibition by QRFSIO absence or in the presence of various concentrations of octanoylR peptide (SEQID NO: 6) of hydrolysis of corre C(PE12)QRFSR COOH peptide (SEQID NO:34) plotted sponding FRET-peptide substrates by pure recombinant in LM (log scale). human hNEP or AP-N. Each point represents the percentage FIG. 22: Concentration-dependent Inhibition by CPE6 of intact Substrate recovered and calculated as: percentage of 40 QRFS O-Octanoyl)R peptide (i.e. C ( HN-(CH) velocity without inhibitor-velocity in presence of inhibitor/ CO-)-QRF S(O-octanoyl)-R) (SEQ ID NO: 35) of velocity without inhibitor, which was measured in the hydrolysis of corresponding FRET-peptide substrates by pure absence or in the presence of various concentrations of QRFS recombinant human hNEP or AP-N. Each point represents the O-octanoyl R-peptide (SEQ ID NO: 6) plotted in uM (log percentage of intact Substrate recovered and calculated as: scale). 45 percentage of velocity without inhibitor-velocity in presence FIG. 11: Concentration-dependent Inhibition by Y(PE12) of inhibitor/velocity without inhibitor, which was measured QRFSR (i.e. Y-NH-(CH2), CO-QRFSR) peptide in the absence or in the presence of various concentrations of (SEQ ID NO: 32) of hydrolysis of corresponding FRET CPE6QRFS-O-Octanoyl)R-peptide (SEQ ID NO: 35) peptide substrates by pure recombinant human hNEP or plotted in M (log scale). AP-N. Each point represents the percentage of intact sub 50 FIG. 23: Concentration-dependent Inhibition by CIPE6 strate recovered and calculated as: percentage of Velocity QRFIS O-OctanoylR peptide (i.e. C ( HN—(CH) without inhibitor-velocity in presence of inhibitor/velocity CO-)-QRF S(O-octanoyl)-R) (SEQ ID NO: 35) of without inhibitor, which was measured in the absence or in hydrolysis of the substance P physiological NEP-substrate by the presence of various concentrations of Y(PE12)QRFSR membrane-bound human NEP expressed by LNCaP epithe peptide (SEQID NO:32) plotted in LoguM. 55 lial cells in culture. Each point represents the percentage of FIGS. 12-17: Kinetics of hydrolysis of corresponding intact Substrate recovered and calculated as: percentage of FRET-peptide substrates by recombinant hNEP or hAP-N in velocity without inhibitor-velocity in presence of inhibitor/ the presence of vehicle or in the presence of 1 to 50 uM of velocity without inhibitor, which was measured in the CQRFSR (SEQID NO: 4) or QRFSIO-octanoyl)R (SEQID absence or in the presence of various concentrations of NO: 6) Opiorphin-peptide analogs. Each point represents the 60 CPE6QRFS-O-Octanoyl)R-peptide (SEQ ID NO: 35) intensity of the signal expressed in RFU (Relative Fluores plotted in nM (log scale). cence Unit), which was proportional to the quantity of metabolites formed, as function of reaction time (min). DETAILED DESCRIPTION OF THE PREFERRED FIG. 18: Concentration-dependent Inhibition by CQRFSR EMBODIMENTS peptide (SEQ ID NO: 4) of hydrolysis of corresponding 65 FRET-peptide substrates by pure recombinant human hNEP The invention here provides modified versions of these or AP-N. Each point represents the percentage of intact sub peptides where each or combinations of the amino acid resi

US 8,642,729 B2 7 C—dO-RF S(O-octanoyl)-dR; C—(—HN (CH) CO )-QRFS-dR; dC-QRF S(O-octanoyl)-dR; CB2-QRF S(O-octanoyl)-B3R (SEQID NO: 16); CQRFSR (core peptide disclosed as SEQID NO: 4): Glp-RFSR (SEQID NO: 17); QRYSR (SEQID NO: 18); 10

QR-F4F-SR (SEQ ID NO: 19), wherein - F4F is a phenylalanine, the phenyl group of which is Substituted in the para position by a fluoro atom; QR-F4Br-SR (SEQID NO: 11), wherein—F4Br—is a phenylalanine, the phenyl group of which is substituted in the para position by a bromo atom; 15 QKFSR (SEQ ID NO: 20): QRFSK (SEQ ID NO: 21); C ( HN (CH) CO )-QRFSR (SEQID NO: 33): C—(—HN (CH) CO )-QRF S(O-octanoyl)-R (SEQ ID NO:35); C(—HN—(CH) CO )QRF S(O-octanoyl)-R (SEQ ID NO: 41); C ( HN-(CH), CO )-QRFS-dR; 25 C ( HN-(CH), CO )-QRF S(O-octanoyl)-33R (SEQ ID NO: 22); C )—HN-(CH) (SEQ ID NO: 42), 30 wherein: CB2 is H.N( CH, SH) CH, CO : |B3R is NH-CH C-(CH), NH C(NH)(NH)- COOH: 35 —S(—O-octanoyl) means a serine, the hydroxyl group of which is substituted by an octanoyl group; —S(—O-dodecanoyl) means a serine, the hydroxyl group of which is substituted by a dodecanoyl group. The following “S(OAlk)' means a serine, the hydroxyl group of which is definitions are used throughout the instant application. 40 Substituted by a linear or branched alkyl group having from 1 to 20 carbon atoms (i.e. “Alk”), preferably an octanoyl or a A "peptide' is a molecule comprised of a linear array of dodecanoyl group. In the present application, S(O—C8-poly amino acid residues connected to each other in the linear array by peptide bonds. Such linear array may optionally be cyclic, ethylene) or S(O C8) means a serine, the hydroxyl group of i.e., the ends of the linear peptide or the side chains of amino which is substituted by an octanoyl group. acids within the peptide may be joined, e.g., by a chemical 45 "C-linker-means Cys-NH (CH), CO , wherein bond. Such peptides may further include secondary, tertiary n is an integer between 1 and 20, preferably between 4 and 15, or quaternary structures, as well as intermolecular associa 6, 8 or 12 being particularly preferred. In the present appli tions with other peptides or other non-peptide molecules. cation, “C (C6-polyethylene) or “CPE6' means Cys Such intermolecular associations may be through, without NH-(CH) CO : “C (C8-polyethylene)” O limitation, covalent binding (e.g., through disulfide linkages), 50 “CPE8 means Cys-NH-(CH) CO-; and “C or through chelation, electrostatic interactions, hydrophobic (C12-polyethylene)” or “CPE12” means Cys-NH interactions, hydrogen bonding, ion-dipole interactions, (CH2)2 CO-. “Y-linker-” means Tyr-NH (CH), CO—, wherein dipole-dipole interactions, or any combination of the above. n' is an integer between 1 and 20, preferably between 4 and Any aminoacid of the following peptides may be in L-con 55 15, 6, 8 or 12 being particularly preferred. In the present figuration or D-configuration. In particular, the entire peptide application, “Y (C6-polyethylene)” or “YPE6” means may be either in L or D configuration. The hydrocarbon chain Tyr-NH-(CH) CO-; “Y (C8-polyethylene)” or can optionally contain a further methylene group compared to “YPE8” means Tyr-NH-(CH) CO-; and “Y - the natural aminoacid, so that the aminoacids can also be B (C12-polyethylene)” or “YPE12” means Tyr-NH aminoacids, more precisely 32 or B3 aminoacids. For 60 (CH2)2 CO-. example, R may represent one of the following aminoacid: 1R “Inhibitory potency” of a peptide derivative of the inven (i.e. L-Arg), dR (i.e. D-Arg), B3R or B2R. Any aminoacid of tion towards a particular peptidase can readily be assessed by the following peptides may also be an aza-aminoacid or a the one skilled in the art, e.g. either by measuring Ki values or B-aza-aminoacid. ICso values. In particular, where ICso values are determined, “FCX) means a phenylalanine, the phenyl group of which 65 the relative inhibitory potencies of two modified opiorphin is Substituted by one or more halogenatoms, preferably fluor, peptides can be compared by measuring the ICso value of the preferred F(X) having one the following formulae: each modified opiorphin peptide towards a given peptidase, in US 8,642,729 B2 10 given experimental conditions (same buffer, same concentra In a preferred embodiment, when is a hydroxyquinoline, tions of peptidase and substrate), and by comparing said ICso the peptide derivative has the following formula: values of the modified opiorphin peptides with the ICs value of opiorphin in the same experimental conditions. The invention concerns a peptide derivative of formula (I): OH O N -AA-AA-AA-AA-AAs-OH (I), N AA-AA-AA-AA-AAs-OH wherein: 2 is hydrogen atom, tyrosine, Y-linker- or a Zn chelating 10 group, Such as cysteine, C-linker-, N-acetyl-cysteine, Peptide derivatives of formula (I) are modified opiorphin N-mercaptoacetyl (HS CH CO ), hydroxamic peptides which advantageously have inhibitory potency acid (HO NH CO ) or an optionally substituted against neutral endopeptidase NEP and/or aminopeptidase hydroxyquinoline, 15 AP-N. AA is Q or Glp, Preferred peptide derivatives of formula (I) are: AA is K, R or H, preferably R. CQRFSR (SEQID NO: 4), QRFCX)SR (SEQID NO: 25), AA is Y. G., N, For F(X), preferably For F(X), QRGPR (SEQ ID NO:9), AA is P S or S(OAlk), preferably S or S(OAlk), QHNPR (SEQ ID NO: 10), AAs is Kor R, preferably R, QRFPR (SEQID NO: 26), QRFS(OAlk)R (SEQ ID NO: 27), preferably QRFS(O-oc C-linker-meaning Cys-NH. (CH), COI-, wherein n tanoyl)R (SEQID NO: 6), is an integer between 1 and 20, n being preferably 6, 8 or C-linker-QRFSR (SEQ ID NO: 1), preferably C NH 12, 25 (CH) CO-QRFSR (SEQ ID NO: 33), C -NH Y-linker-meaning Tyr-NH-(CH), CO—, wherein (CH) CO-QRFSR (SEQID NO:39), n' is an integer between 1 and 20, n' being preferably 6, or C–NH-(CH) CO-QRFSR (SEQID NO:34), 8 or 12, QRYSR (SEQID NO: 18), F(X) meaning a phenylalanine, the phenyl group of which 30 QKFSR (SEQID NO: 20), is Substituted by one or more halogen atoms, QRFSK (SEQID NO: 21), S(OAlk) meaning a serine, the hydroxyl group of which is C-linker-QRFSR (SEQ ID NO: 1), preferably C NH Substituted by a linear or branched alkyl group having (CH) CO-QRFSR (SEQ ID NO: 33), C -NH from 1 to 20 carbon atoms, (CH) CO-QRFSR (SEQ ID NO. 39), or CNH 35 (CH), CO-QRFSR (SEQID NO:34), said AA. AAAAAA, and AAs may be independently CQRFSR (core peptide disclosed as SEQID NO: 4), either in the L-configuration or D-configuration, and any C–NH (CH) CO-QRFS(OAlk)R (SEQ ID NO. 44), one of AAAAAAAA, and AAs may be optionally preferably C NH-(CH) CO-QRFS(O-octanoyl)R a Baminoacid, an aza-aminoacid or a B-aza-aminoacid; (SEQID NO:35) and wherein if the peptide derivative comprises a cysteine, said 40 Y NH (CH) CO-QRFSR (SEQ ID NO:32). peptide derivative is optionally a dimer, with the proviso that In an embodiment, the invention is related to a peptide the peptide is not QRFSR (SEQID NO: 1), QHNPR (SEQID derivative of formula (II): NO: 10), QRGPR (SEQID NO:9), YQRFSR (SEQID NO: 3) or GlpRFSR (SEQID NO: 17). '-Q-AA-AA-P-R-OH (II), 45 Generally, in the peptide derivatives according to the wherein: invention, AA, AA AA AA, and AAs are independently is a hydrogen atomor a Zn chelating group, such as either in the L-configuration or D-configuration, and any one cysteine, C-linker-, N-acetyl-cysteine, N-mercap of AAAAAAAA, and AAs is optionally a faminoacid, toacetyl (HS CH CO ), hydroxamic acid (HO an aza-aminoacid or a B-aza-aminoacid. 50 NH CO ) or an optionally substituted hydrox yduinoline, In an embodiment, at least one of AA, AA, AA, AA, AA' is R or H, preferably R. and AAs is a Baminoacid. In another embodiment, all of AA, AA's is G., N, For F(X), preferably F, AAAAAA, and AAs are faminoacids. said Q, AAAA. P. and R may be independently either in In an embodiment, at least one of AA, AA AA AA, 55 the L-configuration or D-configuration, and any one of and AAs is an aza-aminoacid. In another embodiment, all of Q, AA, AA. P. and R may be optionally a faminoacid, AA, AA, AA, AA, and AAs are an aza-aminoacids. an aza-aminoacid or a (B-aza-aminoacid; In an embodiment, at least one of AA, AA, AA, AA, wherein if the peptide derivative comprises a cysteine, said and AAs is a f-aza-aminoacid. In another embodiment, all of peptide derivative is optionally a dimer, AA, AA, AA, AA, and AAs are B-aza-aminoacid. 60 with the proviso that the peptide is not QHNPR (SEQID NO: 10) or QRGPR (SEQID NO: 9). In one embodiment, the peptide derivative comprises a Said peptide derivative of formula (II) is a modified opior cysteine and is a dimer wherein two sulfur atoms of two phin peptide and is advantageously a human AP-N inhibitor. cysteines are bound with a disulfur bond. For example, In particular it may have an inhibitory potency towards AP-N CQRFSR (core peptide disclosed as SEQID NO: 4) is the 65 higher (preferably at least 6 fold, 8 fold, or 10 fold) than its dimer of the peptide derivative CQRFSR (SEQID NO: 4), the inhibitory potency towards NEP endopeptidase and/or car cysteine aminoacids of which are linked with a disulfur bond. boxydipeptidase activity(ies). US 8,642,729 B2 11 12 Preferred peptide derivatives of formula (II) are: Peptide derivatives of formula (IV) are modified opiorphin QRGPR (SEQ ID NO:9), peptides which are advantageously dual human NEP and QHNPR (SEQID NO: 10), and human AP-N inhibitors, as they display significative inhibi QRFPR (SEQID NO: 26). tory potency towards both human NEP and human AP-N. In an embodiment, the invention is related to a peptide Preferred peptide derivatives of formula (IV) are: derivative of formula (III): CQRFSR (SEQID NO: 4), "-AA-AA-AA-AA-AA's-OH (III), CQRFSR (core peptide disclosed as SEQID NO: 4), C (-HN-(CH), CO-)-QRFSR (SEQID NO:34): wherein: C ( HN (CH) CO )-QRFSR (SEQID NO:33): " is a hydrogen atom, tyrosine or Y-linker-, 10 AA is Q or Glp, preferably Q. C-linker-QRF S(O-octanoyl)-R (SEQ ID NO: 6), prefer AA" is K or R, preferably R. ably C ( HN (CH) CO-)-QRF S(O-octanoyl)-R AA" is Y. For F(X), preferably For F(X), (SEQ ID NO: 35), C ( HN-(CH) CO )-QRF S AA is S or S(OAlk), (O-octanoyl)-R (SEQID NO: 40) or C ( HN (CH)— AA's is K or R, preferably R. 15 CO-)-QRF S(O-octanoyl)-R (SEQ ID NO: 41); said AA. AAAAAA, and AAs may be independently QRFCX)SR (SEQID NO: 25) and either in the L-configuration or D-configuration, and any QRFS(OAlk)R (SEQ ID NO: 27), preferably QRFS(O-oc one of AAAAAAAA, and AAs may be optionally tanoyl)R (SEQID NO: 6). a Baminoacid, an aza-aminoacid or a B-aza-aminoacid; Preferably, the peptide derivatives of the invention here are with the proviso that the peptide is not QRFSR (SEQID NO: modified opiorphin peptides and have inhibitory potency 1), QHNPR (SEQID NO: 10), YQRFSR (SEQID NO:3) or against neutral endopeptidase NEP and/or aminopeptidase GlpRFSR (SEQ ID NO:17). AP-N. Most preferably, the modified opiorphin peptides of Said peptide derivative of formula (III) is a modified opi the invention have inhibitory potency against endopeptidase orphin peptide and is advantageously a human NEP inhibitor. NEP and aminopeptidase AP-N. In particular, it may have an inhibitory potency towards NEP 25 The peptide derivatives according to the present invention endopeptidase and/or carboxydipeptidase activity(ies) higher may be prepared in a conventional manner by peptide Syn (preferably at least 6 fold, 8 fold, or 10 fold than its inhibitory thesis in liquid or solid phase by Successive couplings of the potency towards AP-N. different amino acid residues to be incorporated (from the In one embodiment, the invention is related to a peptide N-terminal end to the C-terminal end in liquid phase, or from derivative of formula (IIIa): 30 the C-terminal end to the N-terminal end in solid phase) AA-R-AA-S(OAlk)-AA's-OH (IIIa), wherein the N-terminal ends and the reactive side chains are previously blocked by conventional groups. wherein: For solid phase synthesis the technique described by Mer AA is Q or Glp, rifield may be used in particular. Alternatively, the technique AA", is For F(X), 35 AA's is K or R, preferably R. described by Houbenweyl in 1974 may also be used. with the proviso that the peptide is not QRFSR (SEQID NO: The peptide derivatives according to the present invention 1), QHNPR (SEQID NO: 10), YQRFSR (SEQID NO:3) or may also be obtained using genetic engineering methods, GlpRFSR (SEQ ID NO:17). provided they consist of natural aminoacids. Preferred peptide derivatives of formula (III) or (IIIa) are: 40 Modifications to the opiorphin peptides include chemical QRYSR (SEQID NO: 18), (e.g., containing additional chemical moieties, such as QRFCX)SR (SEQID NO: 25), methyl, polyethylene C. C. C. Cs Co., C and polyethyl QKFSR (SEQ ID NO: 20), eneglycol thereof, and/or glycosylated forms, and peptidomi QRFSK (SEQ ID NO: 21), metics (e.g., a low molecular weight compound that mimics a QRFS(OAlk)R (SEQ ID NO: 27), preferably QRFS(O-oc 45 peptide in structure and/or function (see, e.g., Abell, tanoyl)R (SEQ ID NO: 6) and Y-NH-(CH) CO Advances in Amino Acid Mimetics and Peptidomimetics, QRFSR (SEQID NO:32). London: JAI Press (1997); Gante, Peptidmimetica—massge In an embodiment, the invention is related to a peptide Schneiderite Enzyminhibitoren Angew. Chem. 106:1780-1802 derivative of formula (IV): (1994); and Olson et al., J. Med. Chem. 36: 3039-3049 50 (1993)). Other modifications which may be introduced in the pep wherein: tide derivatives of the invention include using unnatural " is a hydrogen atom, or a Zn chelating group, such as amino acids, Damino acid, Bamino acid, 3 amino acid, AZa cysteine, C linker-, N-acetyl-cysteine, N-mercap amino acid or B-AZa amino acid instead of L amino acid, toacetyl (HS CH CO ), hydroxamic acid (HO— 55 conformational restraints, isosteric replacement, cyclization, NH CO ) or an optionally substituted hydrox or other modifications. Other examples are where one or more yduinoline, amide bond is replaced by a non-amide bond, and/or one or AA" is For F(X), preferably F(X), more amino acid side chain is replaced by a different chemi AA" is S or S(OAlk), preferably S or S(OAlk), cal moiety, or one or more of the N-terminus, the C-terminus said Q, R, AAAA, and R may be independently either in 60 or one or more side chain is protected by a protecting group, the L-configuration or D-configuration, and any one of and/or double bonds and/or cyclization and/or Stereospecific Q, R, AAAA, and R may be optionally a Baminoacid, ity is introduced into the amino acid chain to increase rigidity an aza-aminoacid or a B-aza-aminoacid; and/or binding affinity. wherein if the peptide derivative comprises a cysteine, said Based on the crystal structure of the binding domain of the peptide derivative is optionally a dimer, 65 metallo-ectopeptidase targeted by the peptide derivative of with the proviso that the peptide is not QRFSR (SEQID NO: the invention, mimetics can also be obtained by means of 1) or YORFSR (SEQID NO:3). computer-assisted drug design development (Oefner et al. J. US 8,642,729 B2 13 14 Mol. Biol. (2000) 296(2):341-9; Gomeni et al. Eur. J. Pharm. derivative, of the present invention subcutaneously to New Sci. (2001) 13(3):261-70; Kan, Curr Top Med Chem (2002), Zealand white rabbits which have first been bled to obtain 2(3):247–69). pre-immune serum. The antigens can be injected at a total Still other modifications include: volume of 50 ul per site at ten different sites or at least five protecting the NH and COOH hydrophilic groups by different sites. The rabbits are then bled five weeks after the esterification (COOH) with lipophilic alcohols or by first injection and periodically boosted with the same antigen amidation (COOH) and/or by acetylation (NH) or administered subcutaneously at five fold lower concentration added carboxyalkyl or aromatic hydrophobic chain at than the primary injection at maximum depending on quality the NH terminus; of the immune response three times every six weeks. A retroinversion isomers of the CO. NH amide bonds or 10 sample of serum is then collected every 10 days after each methylation (or ketomethylene, methyleneoxy, boost. Polyclonal antibodies are then recovered from the hydroxyethylene) of the amide groups; serum by affinity chromatography using the corresponding insertion of HN (CH), CO – moieties between antigen to capture the antibody. This and other procedures for two aminoacids, n being an integer from 1 to 20, pref raising polyclonal antibodies are disclosed in E. Harlow, et. erably 6, 8 or 12. 15 al., editors, Antibodies: A Laboratory Manual, Cold Spring The nucleic acids, also named polynucleotides, such as Harbor Laboratory, New York (1988). DNA or RNA molecules, that encode the peptides, including A "monoclonal antibody in its various grammatical forms peptide derivatives containing natural amino acids, defined refers to a population of antibody molecules that contain only above are also part of the invention, while taking into account one species of antibody combining site capable of immunore the degeneration of the genetic code. Preferably, the nucleic acting with a particular epitope. A monoclonal antibody thus acid comprises a sequence incoding a peptide derivative typically displays a single binding affinity for any epitope which consists of natural aminoacids. with which it immunoreacts. A monoclonal antibody may The nucleic acids of the invention include sequences that therefore contain an antibody molecule having a plurality of are hybridizable to any of the above sequences or their antibody combining sites, each immunospecific for a differ complementary sequences under high stringency hybridiza 25 ent epitope, e.g. a bispecific monoclonal antibody. tion conditions which refers to hybridization and/or washing Laboratory methods for preparing monoclonal antibodies conditions at 68°C. in 0.2xSSC, at 42°C. in 50% formamide, are well known in the art (see, for example, Harlow et al., 4xSSC, or under conditions that afford levels of hybridization supra). Monoclonal antibodies (Mabs) may be prepared by equivalent to those observed under either of these two condi immunizing a mammal, e.g. a mouse, rat, rabbit, goat, human tions. 30 and the like, against the peptide derivatives of the present The present invention further relates to vectors for cloning invention, including conjugated peptide derivatives. The anti and/or expression comprising a nucleic acid sequence of the body-producing cells in the immunized mammal are isolated invention and to host cell comprising the nucleic acid or said and fused with myeloma or heteromyeloma cells to produce vector, i.e. a host cell wherein at least one of these vectors was hybrid cells (hybridoma). The hybridoma cells producing the transferred. The expression vector according to the invention 35 monoclonal antibodies are used as a source of the desired comprises a nucleic acid sequence encoding a peptide, monoclonal antibody. including a peptide derivative, or protein of the invention, said While Mabs can be produced by hybridoma culture, the nucleic acid sequence being operably linked to elements invention is not to be so limited. Also contemplated is the use allowing its expression. The vector advantageously contains a of Mabs produced by an expressing nucleic acid cloned from promoter sequence, signals for initiation and termination of 40 a hybridoma. That is, the nucleic acid expressing the mol translation, as well as appropriate regions for regulation of ecules secreted by a hybridoma can be transferred into translation. Its insertion into the host cell may be transient or another cell line to produce a transformant. The transformant stable. The vector may also contain specific signals for secre is genotypically distinct from the original hybridoma but is tion of the translated protein. also capable of producing antibody molecules of this inven Host cells may be prokaryotic or eukaryotic, including but 45 tion, including immunologically active fragments of whole not limited to bacteria, yeasts, plant cells, insect cells, mam antibody molecules, corresponding to those secreted by the malian cells, including cell lines which are commercially hybridoma. In addition, the literature provides methods for available. Preferred examples for host cells are COS-1, HEK forming chimeric antibodies, humanized antibodies, single cells, 293 cells, or CHO cells. chain antibodies and the like variations on a basic immunore The present invention further provides antibodies, mono 50 active antibody fragment. All of these are considered within clonal or polyclonal, or fragments thereof specifically the scope of the invention insofar as a class and specificity of directed against (i.e. that specifically recognizes) the modi antibody is disclosed and claimed, regardless of the precise fied opiorphin peptides described herein. Such antibodies variant structure that one skilled in the art may construct. may be useful for instance for studying pharmacokinetic The modified opiorphin peptides can be formulated in properties of the peptide derivatives of the invention. 55 pharmaceutical compositions in association with a pharma The term “antibody” in its various grammatical forms is ceutically acceptable carrier. For instance, the pharmaceuti used hereinto refer to immunoglobulin molecules and immu cal compositions are suitable for a topical, oral, Sublingual, nologically active portions of immunoglobulin molecules, parenteral, intranasal, intravenous, intramuscular, Subcutane i.e., molecules that contain an antibody combining site or ous, transcutaneous or intraocular administration and others. paratope. Exemplary antibody molecules are intact immuno 60 The modified opiorphin peptides described above are use globulin molecules, Substantially intact immunoglobulin ful in the treatment of diseases or disorders, wherein a modu molecules and portions of an immunoglobulin molecule, lation of the activity of a membrane metallo-ectopeptidase is including those portions known in the art as Fab, Fab'. F(ab')2 sought, more particularly a membrane-zinc metallopepti and F(v). dase, such as NEP and AP-N. Procedures for raising polyclonal antibodies are also well 65 Natural NEP substrates are mainly the peptide hormones: known. Typically, Such antibodies can be raised by adminis Enkephalins, Substance P. Bradykinin, Angiotensin II and tering the peptide derivative, including conjugate peptide Atrial Natriuretic Peptide which play key role in the control of US 8,642,729 B2 15 16 central and peripheral pain perception, inflammatory phe Inhibition of ECE has a significant application in the treat nomena, mineral exchange and/or arterial tone (Rogues et al., ment of hypertension and the prevention and treatment of Pharmacol Rev. 1993: 45(1):87-146). atherosclerosis. More particularly, neutral endopeptidase, NEP 24-11, is Inhibition of AP-N in conjunction with NEP has significant distributed both in nervous and peripheral tissues of mam application in the treatment of pain and depression, anxiety, mals, and in the periphery it is particularly abundant in the sedation and Socio-Sexual emotional disorders. kidney and placenta. In these tissues the cell-surface metal Inhibition of related membrane metallopeptidases has lopeptidase NEP participates in the postsecretory processing therapeutic effects in the treatment of tumors, namely ova and metabolism of neuropeptides, systemic immunoregula rian, colorectal, brain, lung, pancreas, gastric and melanoma tory peptides and peptide-hormones. By controlling the 10 cancers, and reducing the incidence of metastasis, atheroscle active levels of circulating or secreted regulatory peptides, rosis and/or hypertension. NEP modulates their physiological receptor-mediated action. Inhibition of related membrane metallopeptidases has Hence, the membrane-anchored NEP is involved in regulat effects in pain relief. Such antinociceptive effects on acute ing the activity of potent vasoactive peptides such as Sub 15 pain are analgesic effects but also effects on chronic inflam stance P. Bradykinin (BK), Atrial Natriuretic peptide (ANP), matory pain such as arthritis or inflammatory bowel disease, and Angiotensin II (AII); potent inflammatory/immunoregu post-operative injury and neuropathic severe pain associated latory peptides such as Substance P and BK and fMet-Leu or not with cancer and/or cancer therapy. Phe (fMLP); potent opioid neuropeptides such as Met and Furthermore, inhibition of bacterial or viral metallopepti Leu-Enkephalins (Enk) and potent mineral exchange and dase is expected to have anti-infection effects. fluid homeostasis regulatory peptides such as ANP, C-type Metallopeptidases playing an important role in pathogen Natriuretic Peptide (CNP) and B-type Natriuretic Peptide host tissue invasion and immunological and inflammatory (BNP). However the levels of these peptides are changed processes, for example those of Streptococcus pyogenes, through the NEP-induced formation/degradation only in Pseudomonas aeruginosa, Porphyromonas gingivalis and regions where they are tonically released or where their 25 Legionella pneumophila. release is triggered by a stimulus. Furthermore, bacterial metallopeptidases, especially zinc From an integrative point of view, the NEP biological metallopeptidases play an important role in the diseases activity is to control the active levels of peptidergic signals caused by proteolytic toxins. Such as the toxins of B. anthra involved in arterial tension regulation, in inflammatory phe cis (Anthrax Lethal factor) and the neurotoxins of C. tetanum nomena, emotional states and in water-mineral homeostasis, 30 and botulinum. as well as, in the control of pain processing. From a clinical Other metallopeptidases play an important role in various point of view, this substantiates the fact that NEP is an impor infections such as infections caused by HIV (FR 2707169). tant drug target in various disease states. For example, by The importance of proteinase inhibitors for the treatment inhibiting NEP and APN, thereby increasing the levels and of bacterial or viral diseases may be found in J. Potempa and duration of action of central or peripheral endogenous opio 35 J. Travis. ids, an analgesic effect or an anti-depressant or psychoStimu The different roles of metallopeptidases are disclosed in lant effect could be obtained. The main advantage of modi Turner et al., 2001; Kenny et al., 1977; Kenny et al., 1987: fying the concentrations of endogenous regulatory peptides Beaumont et al., 1996. by use of NEP and/or AP-N inhibitors is that the pharmaco One object of the present invention is providing an analge logical effects are induced only at receptor sites activated by 40 sia or anti-depressant therapy with the modified opiorphin the natural ligands, and are critically dependent on their tonic peptides which acts by inhibiting NEP and AP-N at periph or stimulus-evoked release happening upon specific environ eral, spinal and/or Supraspinal levels and thereby increasing mental, behavioral and physiopathological stressful situa the levels and duration of action of central or peripheral tions (Rogues et al., 1993). endogenous opioids, including enkephalins. Examples of mammalian membrane metallopeptidases 45 The treatment of pain, especially acute and chronic pain, besides NEP are NEP2, ECE (Endothelin-Converting visceral inflammatory and neuropathic pain, is contemplated. Enzymes), in particular ECE1 and ECE2, the erythrocyte The treatment of any hydro-mineral imbalance is also an cell-surface antigen KELL and the product of PEX gene aim of the invention. Among target disorders one may cite associated with X-linked hypophosphatemic rickets and bone, teeth, kidney, parathyroid, pancreas, intestine, stomach AP-N (Aminopeptidase N). 50 mucosa, prostate, and salivary gland disorders that are caused NEP2 is distributed specifically in the nervous system and by hydro-mineral imbalance. genital tissues. In particular, the disorder may be selected from hyper or AP-N is a ubiquitous enzyme present in a wide variety of hypo-parathyroidism, osteoporosis, pancreatitis, Subman human organs, tissues and cell types (endothelial, epithelial, dibular gland lithiasis, nephrolithiasis and osteodystrophy. fibroblast, leukocyte) and is in particular abundant in the 55 The treatment of impaired interpersonal and behavioural kidneys and central nervous system. Identified Substrates disorders is of further interest. Various mental disorders are include Angiotensin III (Ang III); neuropeptides, including described in WO 02/051434. enkephalins and endorphins; and hormones, including kal In particular the invention is drawn at any disorder selected lidan and somatostatin. AP-N is a multifunctional enzyme, from the group consisting of avoidance disorder, decreased related with tumorigenesis, immune system, pain, regulation 60 awareness disorder, autistic disorder, attention deficit hyper of arterial blood pressure etc. AP-N is also involved in the activity disorder, arousal disorder, hospitalism, impaired trimming of antigen and the process of antigen presentation. interpersonal functioning and relationship to the external These functions facilitate the modulation of bioactive peptide world, Schizoid personality disorder, Schizophrenia, depres responses (pain management, vasopressin release) and influ sive disorder, decreased interest in environment, impaired ence immune functions and major biological events (cell 65 Social activity linked to sexuality, and impaired sexual behav proliferation, secretion, invasion, angiogenesis) thereby pro iour, including untimely ejaculation, hyperactive sexuality viding treatment options for many kinds of diseases. and erectile dysfunction. US 8,642,729 B2 17 18 The invention also relates to the use of the peptides or 071 filed on Sep. 15, 2006, the contents of which are peptide derivatives according to the invention as psycho incorporated herein by reference. stimulating agents. Accordingly the prevention or treatment of a narcolepsy, hypersomnia, obsessional compulsive EXAMPLES troubles, mood disorders such as depressive disorders or 5 major depressive disorder, either major depressive disorder Example 1 single episode or major depressive disorder recurrent, type I or II bipolar disorder, dysthymic disorder and cyclothymic Methods for Biochemical Assays disorder. Diseases wherein a modulation of a membrane metal 10 Formal kinetic analysis was performed for each assay lopeptidase is sought also include hypertension, aterosclero using real-time fluorescence monitoring of specific Substrate sis, tumor, inflammatory arthritis and bowel disease. hydrolysis. For each 96-well adapted fluorimetric model, all Treatment of infections is also encompassed. Especially, parameters allowing the analysis of human NEP and human the importance of proteinase inhibitors for the treatment of 15 AP-N enzyme activity were defined under conditions of ini bacterial or viral diseases may be found in J. Potempa and tial Velocity measurement. Travis. 1—Sources of the Human Ectopeptidases, hNEP and The modified opiorphin peptides described above are also hAP-N useful for controlling immuno-inflammatory responses. Recombinant human NEP and recombinant human AP-N The modified opiorphin peptides as defined above are also (devoid of their respective N-terminal cytosol and transmem useful as a natriuretic agent or a diuretic agent. brane segment) that were purchased from R&D Systems, Thus, the present invention advantageously provides pep were used as pure source of peptidase. tide derivatives as defined above for use in the treatment of a 2—Substrates and Synthetic Inhibitors disease or disorder selected from the group consisting of pain, In vitro, Amino-, Carboxyl Di- and Endo-Peptidase activi depressive disorders, impaired social activity linked to sexu 25 ties were assayed by measuring the breakdown of the follow ality, and impaired sexual behaviour. ing synthetic selective Substrates: Another object of the present invention is the use of the Abz-dR-G-L-EDDnp FRET-peptide that is an internally above described peptide derivatives or nucleic acids as a quenched fluorescent substrate specific for NEP-En Substitute in the treatment of drug abuse, notably morphine doPeptidase activity, was synthesized by Thermo-Fisher drug abuse. 30 Scientific (Germany). Indeed, studies have suggested that the Vulnerability to Abz-R-G-F-K-DnpOH FRET-peptide (SEQ ID NO: 28) drug abuse and the development of reward and drug depen that is an internally quenched fluorescent Substrate spe dence is at least in part, a result of pre-existent or induced cific for NEP-Carboxyl DiPeptidase activity, was synthe modifications and/or defect of the endogenous opioid system. sized by Thermo-Fisher Scientific (Germany) In this regard, using modified opiorphin peptides or nucleic 35 Mca-R P P-G-F S-A-F-K-(Dnp)-OH FRET-peptide acid to potentiate the effects of endogenous enkephalins will (Mca-BK2)(SEQIDNO: 29) that is an intramolecularly reduce the various side-effects (somatic signs of withdrawal) quenched fluorogenic peptide structurally related to produced by interruption of chronic morphine or heroin bradykinin, which is a selective Substrate for measuring administration. 40 NEP and ECE activity, was purchased from R&D Sys Preferred diseases or disorders which may be treated with temS. the peptide derivatives of the invention include pain, depres FRET is the distance-dependant transfer of energy from a sive disorders, impaired social activity linked to sexuality, donor fluorophore (Abzortho-aminobenzoyl or Mica=7- and impaired sexual behaviour. methoxycoumarin-4-yl-acetyl) to an acceptor fluorophore The invention further relates to the use of an agent that 45 (DnpOH-2,4-dinitrophenyl or EDDnp=2,4-dinitrophenyl modulates the interaction between endogenous BPLP protein ethylenediamine). or maturation product, e.g. natural opiorphin QRFSR, and a L-alanine-Mca, Ala-Mca, a fluorogenic Substrate for mea membrane metallopeptidase for the preparation of a thera Suring aminopeptidase activity was purchased from peutic composition for preventing or treating diseases Sigma. wherein a modulation of the activity of said membrane met 50 Measuring the hydrolysis rate of these substrates by allopeptidase is sought. soluble ectopeptidases in the presence and absence of differ The invention provides an in vitro method for Screening ent available selective synthetic peptidase inhibitors assessed compounds for their ability to bind to the NEP and/or APN the specificity of each enzyme assay:- Thiorphan (NEP binding site for the BPLP protein or a the QRFSR peptide inhibitor) (Bachem), Bestatin (AP inhibitor) (Calbiochem). 55 3—Measurement of Peptidase Activities Using 96-Well (SEQID NO: 1). Details of this procedure are described, e.g., Adapted Fluorimetric Assays in U.S. application Ser. No. 10/593,071 filed on Sep. 15, According to conditions of initial Velocity measurement: 2006, also published as WO 2005/090386, as well as in the the time, pH and temperature of incubation as well as enzyme international patent application PCT/EP2009/050567, the and Substrate concentrations were defined for each assay. contents of which are incorporated herein by reference. 60 Hydrolysis of substrates was measured by real-time monitor Another object of the present invention is a process for ing their metabolism rate by the two peptidases in the pres determining the relative affinity of the modified opiorphin ence and absence of tested inhibitory compound (concentra peptides that specifically bind to the NEP and/or to the AP-N tions ranging from 1 to 50 uM). These were added to the binding sites for the BPLP protein, or maturation products, preincubation medium. The background rate of Substrate (or the peptide that retains the binding specificity or the physi 65 autolysis representing the fluorescent signal obtained in the ological activity of the BPLP protein or of its matured prod absence of enzyme was subtracted to calculate the initial ucts) as described in e.g., in U.S. application Ser. No. 10/593, velocities in RFU (Relative Fluorescent Unit)/min. US 8,642,729 B2 19 20 Measurement of NEP-Endopeptidase Activity. Using Other FRET-peptide substrates for assaying NEP carboxy FRET Specific Peptide-Substrate, Abz-dR-G-L-EDDmp. dipeptidase activity have been described in Barros et al. Biol. Using black half-area 96 well microplate, the standard Chem., 2007, 388:447-455. reaction consisted of enzyme (12.5 ng) in 100 mM Tris-HCl Example 2 pH 7 containing 200 mM NaCl (100 ul final volume). The 5 substrate (15uM final concentration) was added after prein Results of Biochemical Assays cubation for 10 min at 28°C. and the kinetics of appearance of the fluorescent signal (RFU) was directly analyzed for 40 1—Screening of Opiorphin Peptide-Derivatives on hNEP mM at 28° C. (2.3 mM-interval successive measures) by and hAP-N using a fluorimeter microplate reader (monochromator Infi Compounds were tested in triplicate at 50 uM final con nite 200-Tecan) at 320 nm and 420 nm excitation and emis centration. sion wavelengths, respectively. FIGS. 1 to 9 display the results of biochemical assays on Under conditions of initial velocity measurement, the NEP endopeptidase and carboxydipeptidase activities and intensity of the signal was directly proportional to the quan AP-N activity with various peptide derivatives of the inven tity of metabolites formed during the 20-40 min time-period tion. of the reaction. Thus, in absence of inhibitor, the initial veloc 15 Various Opiorphin peptido-analogs were tested for their inhibitory potency towards the two membrane-anchored ity of rhNEP-mediated specific endoproteolysis of Abz-dR ectoenzymes, NEP (specific endopeptidase and carboxy G-L-EDDnp, was calculated from the linear regression dipeptidase activities) and APN by using selective fluores (slope=NEP activity in presence of vehicle/incubation time) cence-based enzyme assays: the FRET-based Enzyme in as 8218+2878 RFU/min/ug rhNEP. n=3 independent deter Vitro Models previously developed and validated in the labo minations. ratory (PCT application PCT/filed Jan. 18, 2009, incorpo Measurement of NEP-Carboxyl DiPeptidase Activity. Using rated herein by reference). FRET Specific Peptide-Substrate Abz-R-G-F-K-DnpOH The following compounds were analyzed. In the com (SEQ ID NO:28). Using black half-area 96 well micro pounds below, NH2-represents the terminal amine of the plate, the standard reaction consisted of enzyme (2.5 ng) in peptide and COOH the terminal carboxylic acid of the pep 100 mM Tris-HCl pH 6.5 containing 50 mM NaCl (100 ul 25 tide. final volume). The substrate (4 uM final concentration) was NH2-QRFSR CONH2 (Opiorphin) (SEQ ID NO: 8): added after preincubation for 10 min and the kinetics of NH2-QRGPR COOH (SEQ ID NO: 9); NH2-QH appearance of the fluorescent signal (RFU) was directly ana NPR COOH (SEQ ID NO: 10); NH2-QR(4BromoF) lyzed for 40 min at 28°C. (2.3 min-interval successive mea SR COOH (SEQID NO: 11) (i.e. QRF(4Br)SR) (SEQ sures) by using the fluorimeter reader at 320 nm excitation ID NO: 11); NH2-QRFPR COOH (SEQID NO: 26): and 420 nm emission wavelengths. Under these conditions of 30 they displayed inhibitory preference for human AP-N initial velocity measurement, human NEP-mediated specific and a weak inhibitory potency towards human NEP hydrolysis of Abz-R-G-F-K-DnpOH (SEQ ID NO: 28) was N-(Acetyl)QRFSR COOH (SEQID NO: 12); N-(C8 evaluated at 59796+18685 RFU/min/ug rhNEP. n=4 indepen polyethylene)QRFSR-COOH (SEQID NO:36): dent determinations. NEP-Carboxyl DiPeptidase activity N-(biotine-C6-polyethylene)QRFSR COOH (SEQID NO: assayed with the substrate Abz-R-G-F-K-DnpOH (SEQ ID 35 37): they displayed weak inhibitory potency towards human NO: 28) is referred to as “NEP-CDP1 activity” in FIGS. 5-8. AP-N and NEP with a preference for NEP endopeptidase In addition, the intramolecularly quenched fluorogenic activity NH2-dRdSdFdRdO-COOH (retroinversion D-enanti peptide, Mca-BK2 (10 uM), was submitted to hydrolysis by 5 omere): It did not display significant inhibitory activity ng rhNEP under the same experimental conditions as those towards human AP-N and NEP with a preference for described behind. Under these conditions the hNEP-enzyme 40 AP-N activity acted upon Mca-R-P-P-G-F-S-A-F-K-(Dnp)-OH (SEQ ID NH2-YQRFSR COOH (SEQID NO:3); NH2-Y (C6 NO: 29) mainly as a Carboxydipeptidase preferentially polyethylene)QRFSR-COOH (SEQID NO:38); cleaving A-F bond but also as an EndoPeptidase cleaving NH2-Y (C12-polyethylene)QRFSR COOH (SEQ ID the G—F bond. Under conditions of initial velocity measure NO: 32): they displayed inhibitory preference for human ment, human NEP-mediated specific hydrolysis of Mca-BK2 45 NEP endopeptidase activity in particular Y—(C12-polyeth was evaluated at 121910+24755 RFU/min/ug rhNEP. n=3 ylene)QRFSR (SEQ ID NO:32) independent determinations. NEP-Carboxyl DiPeptidase NH2-QRFIS O C8-polyethyleneR-COOH (SEQID activity assayed with the substrate Abz-R-G-F-K-DnpOH NO: 6): NH2-CQRFSR-COOH (SEQID NO:4): They (SEQ ID NO: 28) is referred to as “NEP-CDP2 activity” in were potent dual inhibitors of human NEP (specific FIGS. 5-8. 50 endopeptidase and carboxydipeptidase activities) and Measurement of AP-N-Ectopeptidase Activity Using Ala AP-N. Mca Substrate. Other modified opiorphin peptides are: Using black half-area 96 well microplate the standard reac NH2-CQRFIS O C8-polyethyleneR-COOH (SEQ tion consisted of enzyme (4 ng) in 100 mM Tris-HCl pH 7.0 ID NO: 7); (100 ul final volume). The Ala-Mca substrate (25 nM final 55 NH2-CQRFIS O C12-polyethylene R-COOH (SEQ ID NO:30) concentration) was added after preincubation for 10 min at NH2-C (C8-polyethylene)QRFSR COOH (SEQ ID 28°C. and the kinetics of appearance of the signal was moni NO:39); tored for 40 min at 28°C. by using the fluorimeter reader at NH2-C (C12-polyethylene)QRFSR-COOH (SEQ ID 380 nm excitation and 460 nm emission wavelengths. The NO:34) intensity of the signal was directly proportional to the quan 60 NH2-C (C8-polyethylene)QRFIS O C8-polyethyl tity of metabolites formed during the 10-40 min time-period eneR COOH (SEQ ID NO: 40) of the reaction. Under these conditions of initial velocity NH2-CB2IQRFIS O-C8-polyethyleneR-COOH measurement, the human AP-N-mediated aminoproteolysis (SEQID NO: 14) of Ala-Mca was directly calculated (from the slope: AP-N NH2-C (C8-polyethylene)QRFSB3R-COOH (SEQ activity in absence of inhibitor in function of incubation time) 65 ID NO: 15) as 147042+44657 RFU/min/ug rhAP-N, n=3 independent NH2-CIdORFS O C8-polyethylenedR-COOH determinations. NH2-C (C8-polyethylene)QRFSdR US 8,642,729 B2 21 22 NH2-doGRFS O C8-polyethylenedR-COON cleavage mediated by the rhAP-N activity: r=0.99, n=20 NH2-CB2IQRFIS O C8-polyethylene B3R)-COOH determination points, with a IC50 at 122+20 uM. (SEQ ID NO: 16) Y(PE12)QRFSR COOH peptide (SEQID NO:32) pre CQRFSR2 (core peptide disclosed as SEQID NO: 4) as vented in a concentration dependent manner the Mca-R P cystine-dipeptide through disulfide linkage. P-G-F-S-A-F-K-(Dnp)-OH FRET-peptide (Mca-BK2) (SEQ 2 Concentration-Dependent Inhibition of Selected Opi ID NO: 29) cleavage mediated by the rhNEP activity: orphin Peptide-Derivatives on hNEP and hAP-N r=0.99, n=18 determination points, with a IC50 at 31+2 uM. CQRFSR COOH Peptide (SEQID NO:4) (FIGS. 9 and Thus the inhibitory preference for human NEP endopeptidase 18) activity ofY (C12-polyethylene)QRFSR (SEQID NO:32) The CQRFSR-COOH peptide (SEQID NO: 4) prevented 10 was confirmed in a concentration dependent manner the AbZ-dR-G-L- CPE6QRFSR Peptide (SEQ ID NO: 33) (PE6-amino EDDnp cleavage mediated by the rhNEP-Endopeptidase hexanoic-acid) (FIG. 20) activity: r=0.97, n=18 determination points, with a ICso at CPE6QRFSR-COOH peptide (SEQ ID NO: 33) pre 7+3 uM. vented in a concentration dependent manner the Abz-dR-G- CQRFSR COOH (SEQID NO: 4) peptideprevented in a 15 L-EDDnp cleavage mediated by the rhNEP-Endopeptidase concentration dependent manner the Abz-R-G-F-K-DnpOH activity: r=0.98, n=18 determination points, with a IC50 at (SEQ ID NO: 28) cleavage mediated by the rhNEP activity: 40+5 puM. r=0.99, n=18 determination points, with a IC50 at 6+1 uM. CPE6QRFSR-COOH peptide (SEQ ID NO: 33) pre CQRFSR COOH peptide (SEQID NO: 4) prevented in a vented in a concentration dependent manner the Abz-R-G-F- concentration dependent manner the Ala-AMC cleavage K-DnpOH (SEQID NO: 28) cleavage mediated by therhNEP mediated by the rhAP-N activity: r=0.99, n=30 determina activity: r=0.99, n=18 determination points, with a IC50 at tion points, with a IC50 at 0.8+0.1 uM. about 217. CQRFSR COOH peptide (SEQID NO: 4) prevented in a CPE6QRFSR-COOH peptide (SEQ ID NO: 33) pre concentration dependent manner the Mca-R P P-G-F vented in a concentration dependent manner the Ala-AMC S-A-F-K-(Dnp)-OH FRET-peptide (Mca-BK2) (SEQ ID 25 cleavage mediated by the rhAP-N activity: r=0.99, n=20 NO: 29) cleavage mediated by the rhNEP activity: r=0.99, determination points with a IC50 at 0.8+0.1 uM. n=16 determination points, with a IC50 at 17+2 uM. CPE6QRFSR-COOH peptide (SEQ ID NO: 33) pre Therefore, modification of the N-terminal position of the vented in a concentration dependent manner the Mca-R P NH2-QRFSR peptide (SEQID NO: 1) by formation of amide P-G-F S-A-F-K-(Dnp)-OH FRET-peptide (Mca-BK2) link with a Cysteine amino acid (the thiol functional group is 30 (SEQ ID NO: 29) cleavage mediated by the rhNEP activity: a strong Zn-chelating group) led to a compound (CQRFSR r=0.99, n=18 determination points, with a IC50 at about 227 peptide (SEQ ID NO: 4)) displaying reinforced inhibitory uM. potency towards hAP-N (10-fold compared to QRFSR native C—amino-dodecanoic-acid-spacer-QRFSR peptide peptide (SEQID NO: 43)) and towards hNEP (5-fold). (SEQID NO. 34) (FIG. 21) QRFS O-Octanoyl RPeptide (SEQID NO: 6) (FIG.10) 35 CPE12QRFSR COOH peptide (SEQID NO:34) pre QRFS O C8R peptide (SEQID NO: 6) prevented in a vented in a concentration dependent manner the Abz-dR-G- concentration dependent manner the Abz-dR-G-L-EDDnp L-EDDnp cleavage mediated by the rhNEP-Endopeptidase cleavage mediated by the rhNEP-Endopeptidase activity: activity: r=0.96, n=24 determination points, with a IC50 at r=0.98, n=18 determination points, with a IC50 at 2.8+0.2 5.7+0.5 uM. uM. 40 CPE12QRFSR COOH peptide (SEQID NO:34) pre QRFS O C8R peptide (SEQID NO: 6) prevented in a vented in a concentration dependent manner the Abz-R-G-F- concentration dependent manner the Abz-R-G-F-K-DnpOH K-DnpOH (SEQID NO: 28) cleavage mediated by therhNEP (SEQ ID NO: 28) cleavage mediated by the rhNEP activity: activity: r=0.97, n=30 determination points, with a IC50 at r=0.99, n=18 determination points, with a IC50 at 12+5uM. 19+2 uM. QRFS O C8R peptide (SEQID NO: 6) prevented in a 45 CPE12QRFSR COOH peptide (SEQID NO:34) pre concentration dependent manner the Ala-AMC cleavage vented in a concentration dependent manner the Ala-AMC mediated by the rhAP-N activity: r=0.99, n=20 determina cleavage mediated by the rhAP-N activity: r=0.98, n=27 tion points, with a IC50 at 10+1 uM. determination points, with a IC50 at 0.8+0.1 uM. QRFS O C8R peptide (SEQID NO: 6) prevented in a C—amino-hexanoic-acid spacer-QRFSIO-OctanoylR concentration dependent manner the Mca-R-P-P-G-F-S-A- 50 Peptide (SEQID NO: 35) (FIG. 22) F-K-(Dnp)-OH FRET-peptide (Mca-BK2) (SEQID NO: 29) CPE6QRFIS O-Octanoyl)R peptide (SEQID NO:35) cleavage mediated by the rhNEP activity: r=0.99, n=18 prevented in a concentration dependent manner the AbZ-dR determination points, with a IC50 at 14+4 uM. G-L-EDDnp cleavage mediated by the rhNEP-Endopepti Y-amino-dodecanoic-acid spacer-QRFSR Peptide (SEQ dase activity: r=0.99, n=27 determination points, with a ID NO:32) (FIGS. 11 and 19) 55 IC50 at 759.56 nM. Y(PE12)QRFSR COOH peptide (SEQID NO:32) pre CPE6QRFIS-O-Octanoyl)R peptide (SEQ ID NO: 35) vented in a concentration dependent manner the Abz-dR-G- prevented in a concentration dependent manner the AbZ-R- L-EDDnp cleavage mediated by the rhNEP-Endopeptidase G-F-K-DnpOH (SEQID NO: 28) cleavage mediated by the activity: r=0.98, n=18 determination points, with a IC50 at rhNEP activity: r=0.99, n=27 determination points, with a 10+1 uM. 60 IC50 at 84858 nM. Y(PE12)QRFSR COOH peptide (SEQID NO:32) pre CPE6QRFIS O-Octanoyl)R peptide (SEQID NO:35) vented in a concentration dependent manner the Abz-R-G-F- prevented in a concentration dependent manner the Ala-AMC K-DnpOH (SEQID NO: 28) cleavage mediated by therhNEP cleavage mediated by the rhAP-N activity: r=0.99, n=24 activity: r=0.99, n=18 determination points, with a IC50 at determination points, with a IC50 at 1.7+0.1 uM. 24+2 uM. 65 In a biologically relevant in vitro assay, using the Sub Y(PE12)QRFSR COOH peptide (SEQID NO:32) pre stance P physiological NEP-substrate and human cell mem vented in a concentration dependent manner the Ala-AMC branes as source of hNEP, the CIPE6QRFS O-Oc US 8,642,729 B2 23 24 tanoyl)R derivative peptide (SEQID NO:35) prevented in a hNEP and haP-N peptidase activities. Indeed, as compared to concentration dependent manner the Substance P cleavage Opiorphin (QRFSR (SEQ ID NO: 43)) native peptide, sub mediated by the membrane-bound hNEP-Endopeptidase stitution of Tyr residue for Phe (QRYSR (SEQ ID NO: 18)) activity: r2=0.95, n=13 determination points, with a IC50 at led to a compound displaying a 6-fold decrease in inhibitory 1.6+0.4 uM (at least 5-fold increase inhibitory potency com potency for haP-N and a light reduction of inhibitory potency pared to Opiorphin native peptide for the same test) (FIG. 23). towards human NEP. CQRFSR (SEQID NO: 4) Dipeptide (CQRFSR), (Core However, interestingly substitution of 4-Fluoro-Phe resi Peptide Disclosed as SEQID NO: 4)) due for Phe (QR4FFSR (SEQ ID NO: 19)) led to a com CQRFSR (SEQID NO: 4) dipeptide prevented in a con pound displaying only a 2-fold decrease in inhibitory potency centration dependent manner the Abz-dR-G-L-EDDmp cleav 10 towards hNEP and a equivalent inhibitory potency towards age mediated by the rhNEP-Endopeptidase activity: r=0.87, haP-N compared to Opiorphin-QRFSR native peptide (SEQ n=24 determination points, with a IC50 at 1.4+0.5 uM. ID NO: 43). Conversely, substitution of (4Bromo-Phe) resi CQRFSR (SEQID NO: 4) dipeptide prevented in a con due or Phe (QR4BrFSR (SEQ ID NO: 11)) led to a com centration dependent manner the Abz-R-G-F-K-DnpOH pound displaying a weak inhibitory potency towards human (SEQ ID NO: 28) cleavage mediated by the rhNEP activity: 15 NEP r’=0.99, n=27 determination points, with a IC50 at 3.3+0.2 Modification of the RFS central residues of the QRFSR CQRFSR (SEQID NO: 4) dipeptide prevented in a con peptide (SEQ ID NO: 1) affects the inhibitory potency of centration dependent manner the Ala-AMC cleavage medi Opiorphin towards hNEP, since, as compared to Opiorphin ated by the rhAP-N activity: r=0.99, n=24 determination QRFSR native peptide (SEQ ID NO: 43), the compounds points, with a IC50 at 0.8+0.1 uM. QRGPR (SEQ ID NO: 9), QHNPR (SEQ ID NO: 10), and TABLE 1. Summary of IC50 values of various peptides and peptide derivatives on NEP and AP-N activities IC50, IM towards IC50, M towards IC50, M NEP- NEP Opiorphin derivatives towards hAP-N Endopeptidase CarboxyDiPeptidase Y-PE12QRFSR 12220 10 - 1 24, 231 + 2 (SEQID NO:32) QRFSO-C8)R 10 - 1 2.8 O2 125,144 (SEQID NO: 6) CQRFSR 0.8.0.1 73 6 - 1,172 (SEQID NO:4) C-PE6-QRFSR O.801 405 ~220 (SEQID NO:33) C-PE12-QRFSR O.801 5.7 O.S 19 - 2 (SEQID NO:34) C-PE6-QRFSO-C8)R 17 O.1 O.76 O.O6 O.85 - 0.06 (SEQID NO:35) CQRFSR2 O.801 1.4 - O.S 3.30.2 (core peptide disclosed as SEQID NO:4)

All together data showed the importance of the N-terminal QRFPR (SEQ ID NO: 26) displayed equivalent inhibitory C. amine-group of the NH2-QRFSR peptide (SEQID NO: 1) potency for human AP-N and a weak inhibitory potency for the inhibitory potency of Opiorphin towards human 45 towards human NEP. AP-N. The results further demonstrate the importance of the Indeed, as compared to Opiorphin-QRFSR native peptide guanidium ion of the Arg residue at position 2 of the QRFSR (SEQ ID NO: 43), its acetylation (Ac-QRFSR (SEQID NO: peptide (SEQID NO: 1) for the inhibitory potency of Opior 12)) or cyclisation (pGlu-RFSR (SEQ ID NO: 17)), its 50 phin towards hAPN. As compared to Opiorphin-QRFSR octanoylation (C8-QRFSR (SEQ ID NO:36)) or biotinyla native peptide (SEQID NO: 43), substitution of a c-amine of tion (biotine-C6)QRFSR (SEQ ID NO: 37), led to com Lys residue (QKFSR (SEQ ID NO: 20)) for Arg2 led to a pounds displaying a weak inhibitory potency towards hAP-N. compound displaying more than a 10-fold decrease in inhibi However, compared to Opiorphin-QRFSR native peptide tory potency towards hAP-N and a light reduction in inhibi (SEQID NO: 43), the two compounds: pGlu-RFSR (SEQID 55 tory potency towards human NEP. NO: 17) and C8-QRFSR (SEQID NO:36) displayed at least Similarly the guanidium ion of the Arg residue at position equivalent if not better inhibitory potency for human hNEP. 5 of the QRFSR peptide (SEQID NO: 1) is important for the These data further demonstrate the importance of the free inhibitory potency of Opiorphin towards hAPN, as compared carboxyl terminus of the QRFSR-COOH peptide (SEQ ID to Opiorphin-QRFSR native peptide (SEQID NO: 43), sub NO: 1) for its inhibitory potency towards hNEP, especially 60 stitution of a e-amine of Lys residue for Arg5 (QRFSK (SEQ towards the Carboxyl DiPeptidase activity of NEP as com ID NO: 21)) led to a compound displaying a 10-fold decrease pared to Opiorphin (QRFSR (SEQID NO: 43)) native pep in inhibitory potency for haP-N and an equivalent inhibitory tide, amidation (QRFSR CONH2 (SEQID NO: 8)) led to a potency for human NEP. compound displaying a weak inhibitory potency towards Interestingly, esterification of the hydroxyl group of the hNEP activity. 65 serine residue of the QRFSR peptide (SEQ ID NO: 1) by The Phe residue of the QRFSR peptide (SEQID NO: 1) is octanoic acid (QRFoctanoyl-SerineR (SEQ ID NO: 6)) led important for the inhibitory potency of Opiorphin towards to a compound displaying equivalent inhibitory potency US 8,642,729 B2 25 26 towards hAP-N compared to Opiorphin-QRFSR native pep or C12 spacers would allow to increase its biological in vivo tide (SEQ ID NO: 43) and reinforced inhibitory potency adsorption potency towards membranes. Among all com towards hNEP-Endo and Carboxyl Di-peptidase activities (at pounds tested, NH2-QRFIS-O-Octanoyl)R-COOH (SEQ least 10-fold more inhibitory potency than Opiorphin native ID NO: 6). NH2-Y(PE12)QRFSR COOH (SEQ ID NO: is 32) (PE=CH2n: PE12-amino-dodecanoic-acid) were peptide) (see FIG. 19). shown to be potent dual inhibitors of human NEP (specific Endopeptidase and Carboxyl DiDeptidase activities) and Example 3 AP-N activities 2 Potential Gain in Metabolic Stability: Cystine-Dipep tide: CQRFSR), (Core Peptide Disclosed as SEQID NO: 4) Identification of InVitro Potent Bioactive 10 CQRFSR (SEQID NO: 4) di-peptide displayed at least Peptido-Mimetics of Opiorphin that would 2-fold increase inhibitory potency towards hNEP-Endo and Potentially Displaying In Vivo Bioavailability Carboxyl Di-peptidase activities compared to CORFSR Properties Superior to the Native Peptide monomer peptide (SEQID NO: 4). The di-peptide sequence should allow to protect the derivative compound against deg 15 radation by circulating aminopeptidase. 1. Potential Gain in Biological Adsorption: Trans-Mem Obviously, numerous modifications and variations of the brane Transport (Epithelial and Endo-Epithelial Cell Mem present invention are possible in light of the above teachings. brane Passage) It is therefore to be understood that within the scope of the Modifications to Opiorphin-peptide by addition of chemi appended claims, the invention may be practiced otherwise cal hydrophobic moieties such as polyethylene C6, C8, C10 than as specifically described herein.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 44

SEO ID NO 1 LENGTH: 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs SEQUENCE: 1 Glin Arg Phe Ser Arg 1. 5

SEO ID NO 2 LENGTH: 6 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE; NAME/KEY: MOD RES LOCATION: (1) . . (1) OTHER INFORMATION: May or may not be present FEATURE; NAME/KEY: MOD RES LOCATION: (2) ... (2) OTHER INFORMATION: Glin or Glp FEATURE; OTHER INFORMATION: See specification as filed for detailed description of substitutions and preferred embodiments

<4 OOs SEQUENCE: 2 Tyr Xaa Arg Phe Ser Arg 1. 5

SEO ID NO 3 LENGTH: 6 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs SEQUENCE: 3 US 8,642,729 B2 27 28 - Continued Tyr Glin Arg Phe Ser Arg 1. 5

SEQ ID NO 4 LENGTH: 6 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

SEQUENCE: 4 Cys Glin Arg Phe Ser Arg 1. 5

SEO ID NO 5 LENGTH: 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

SEQUENCE: 5 Cys Arg Phe Ser Arg 1. 5

SEQ ID NO 6 LENGTH: 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (4) ... (4) OTHER INFORMATION: SerO-octanoyl

SEQUENCE: 6 Glin Arg Phe Ser Arg 1. 5

SEO ID NO 7 LENGTH: 6 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (5) . . (5) OTHER INFORMATION: SerO-octanoyl

SEQUENCE: 7 Cys Glin Arg Phe Ser Arg 1. 5

SEQ ID NO 8 LENGTH: 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: OTHER INFORMATION: C-term CONH2

SEQUENCE: 8 US 8,642,729 B2 29 30 - Continued Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 9 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 9 Glin Arg Gly Pro Arg 1. 5

<210s, SEQ ID NO 10 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 10 Glin His Asn Pro Arg 1. 5

<210s, SEQ ID NO 11 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phe (43romo)

<4 OOs, SEQUENCE: 11 Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 12 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <223> OTHER INFORMATION: N-term acetylated <4 OOs, SEQUENCE: 12 Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 13 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: SerO-C12-polyethylene

<4 OOs, SEQUENCE: 13 US 8,642,729 B2 31 32 - Continued Cys Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 14 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Beta2-Cys 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: SerO-C8-polyethylene 22 Os. FEATURE: <223> OTHER INFORMATION: See specification as filed for detailed description of substitutions and preferred embodiments <4 OOs, SEQUENCE: 14 Cys Glin Arg Phe Ser Arg 1. 5

SEO ID NO 15 LENGTH: 7 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (2) ... (2) OTHER INFORMATION: Amino - octanoic acid FEATURE: NAME/KEY: MOD RES LOCATION: (7) . . (7) OTHER INFORMATION: Beta3-Arg FEATURE: OTHER INFORMATION: See specification as filed for detailed description of substitutions and preferred embodiments

SEQUENCE: 15 Cys Xaa Glin Arg Phe Ser Arg 1. 5

SEQ ID NO 16 LENGTH: 6 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (1) ... (1) OTHER INFORMATION: Beta2-Cys FEATURE: NAME/KEY: MOD RES LOCATION: (5) . . (5) OTHER INFORMATION: SerO-C8-polyethylene FEATURE: NAME/KEY: MOD RES LOCATION: (6) . . (6) OTHER INFORMATION: Beta3-Arg FEATURE: OTHER INFORMATION: See specification as filed for detailed description of substitutions and preferred embodiments SEQUENCE: 16 Cys Glin Arg Phe Ser Arg US 8,642,729 B2 33 34 - Continued

<210s, SEQ ID NO 17 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Glp <4 OOs, SEQUENCE: 17 Xaa Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 18 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 18 Glin Arg Tyr Ser Arg 1. 5

<210s, SEQ ID NO 19 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phe (4Fluoro)

<4 OOs, SEQUENCE: 19 Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 2 O &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 2O Glin Llys Phe Ser Arg 1. 5

<210s, SEQ ID NO 21 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 21 Glin Arg Phe Ser Lys 1. 5 US 8,642,729 B2 35 36 - Continued

SEQ ID NO 22 LENGTH: 7 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (2) ... (2) OTHER INFORMATION: Amino - dodecanoic acid FEATURE: NAME/KEY: MOD RES LOCATION: (6) . . (6) OTHER INFORMATION: SerO-C8-polyethylene FEATURE: NAME/KEY: MOD RES LOCATION: (7) . . (7) OTHER INFORMATION: Beta3-Arg FEATURE: OTHER INFORMATION: See specification as filed for detailed description of substitutions and preferred embodiments

SEQUENCE: 22 Cys Xaa Glin Arg Phe Ser Arg 1. 5

SEQ ID NO 23 LENGTH: 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: OTHER INFORMATION: C-term amidated

SEQUENCE: 23 Glin Arg Phe Ser Arg 1. 5

SEQ ID NO 24 LENGTH: 6 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (5) . . (5) OTHER INFORMATION: SerO- dodecanoyl

SEQUENCE: 24 Cys Glin Arg Phe Ser Arg 1. 5

SEO ID NO 25 LENGTH: 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (3) ... (3) OTHER INFORMATION: Phe may be substituted by one or more halogen atoms

SEQUENCE: 25 Glin Arg Phe Ser Arg US 8,642,729 B2 37 38 - Continued

<210s, SEQ ID NO 26 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 26 Glin Arg Phe Pro Arg 1. 5

<210s, SEQ ID NO 27 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: SerOAlk) <4 OOs, SEQUENCE: 27 Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 28 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) 223 OTHER INFORMATION: AZ 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Lys-DnpOH

<4 OOs, SEQUENCE: 28 Xaa Arg Gly Phe Llys 1. 5

<210s, SEQ ID NO 29 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Mca-Arg 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Lys-DnpOH <4 OOs, SEQUENCE: 29 Arg Pro Pro Gly Phe Ser Ala Phe Lys 1. 5

<210s, SEQ ID NO 3 O US 8,642,729 B2 39 40 - Continued

LENGTH: 6 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (5) . . (5) OTHER INFORMATION: SerO - C12-polyethylene SEQUENCE: 3 O Cys Glin Arg Phe Ser Arg 1. 5

SEQ ID NO 31 LENGTH: 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

SEQUENCE: 31 Glin Arg Phe Thr Arg 1. 5

SEQ ID NO 32 LENGTH: 7 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (2) ... (2) OTHER INFORMATION: Amino - dodecanoic acid

SEQUENCE: 32 Tyr Xaa Glin Arg Phe Ser Arg 1. 5

SEQ ID NO 33 LENGTH: 7 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (2) ... (2) OTHER INFORMATION: Amino -hexanoic acid

SEQUENCE: 33 Cys Xaa Glin Arg Phe Ser Arg 1. 5

SEQ ID NO 34 LENGTH: 7 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (2) ... (2) OTHER INFORMATION: Amino - dodecanoic acid

SEQUENCE: 34 US 8,642,729 B2 41 42 - Continued

Cys Xaa Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 35 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Amino-hexanoic acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: SerO-Octanoyl

<4 OOs, SEQUENCE: 35 Cys Xaa Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 36 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: N-Amino - octanoic acid

<4 OOs, SEQUENCE: 36 Xaa Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 37 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: N-biotine-amino-hexanoic acid

<4 OO > SEQUENCE: 37 Xaa Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 38 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Amino-hexanoic acid

<4 OOs, SEQUENCE: 38 Tyr Xaa Glin Arg Phe Ser Arg 1. 5 US 8,642,729 B2 43 44 - Continued

<210s, SEQ ID NO 39 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Amino - octanoic acid

<4 OOs, SEQUENCE: 39 Cys Xaa Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 4 O &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Amino - octanoic acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: SerO-C8-polyethylene

<4 OOs, SEQUENCE: 4 O Cys Xaa Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 41 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Amino - dodecanoic acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: SerO-C8-polyethylene

<4 OOs, SEQUENCE: 41 Cys Xaa Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 42 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Amino - octanoic acid 22 Os. FEATURE: US 8,642,729 B2 45 46 - Continued <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: SerO-C8-polyethylene 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Beta3-Arg

<4 OOs, SEQUENCE: 42 Cys Xaa Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 43 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: Native Opiorphin peptide

<4 OOs, SEQUENCE: 43 Glin Arg Phe Ser Arg 1. 5

<210s, SEQ ID NO 44 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Amino-hexanoic acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: SerOAlk) <4 OOs, SEQUENCE: 44 Cys Xaa Glin Arg Phe Ser Arg 1. 5

The invention claimed is: said AAAAAAAA, and AAs may be independently 1. A peptide derivative of formula (I): either in the L-configuration or D-configuration, and any 45 one of AAAAAAAA, and AAs may be optionally -AA-AA-AA-AA-AAs-OH (I), a Baminoacid, an aza-aminoacid or a B-aza-aminoacid; wherein: wherein if the peptide derivative comprises a cysteine, said is hydrogen atom, tyrosine, Y-linker- or a Zn chelating peptide derivative is optionally a dimer, group selected from the group consisting of cysteine, with the proviso that the peptide is not QRFSR (SEQID NO: C-linker-, N-acetyl-cysteine, N-mercaptoacetyl 50 1), QRGPR (SEQ ID NO:9), YQRFSR (SEQ ID NO:3) or (HS CH CO ), hydroxamic acid (HO NH GlpRFSR (SEQID NO:17). CO—) and an optionally substituted hydroxyquinoline, 2. The peptide derivative according to claim 1, of formula AA is Q or Glp, (II): AA is K, R or H. 55 AA is Y, G, F or F(X), I-AA-AA-P-R-OH (II), AA is P S or S(OAlk), wherein: AAs is Kor R, is a hydrogen atom, or a Zn chelating group, C-linker-meaning Cys-NH (CH), CO , wherein AA' is R or H. n is an integer between 1 and 20, 60 AA is G, F or F(X), Y-linker- meaning Tyr-NH-(CH), CO , wherein C-linker-, F(X) and S(OAlk) being as defined in claim 1, n' is an integer between 1 and 20, said Q, AA, AA. P. and R may be independently either in F(X) meaning a phenylalanine, the phenyl group of which the L-configuration or D-configuration, and any one of is Substituted by one or more halogen atoms, Q, AA, AA. P. and R is optionally a Baminoacid, an S(OAlk) meaning a serine, the hydroxyl group of which is 65 aza-aminoacid or a B-aza-aminoacid; Substituted by a linear or branched alkyl group having wherein if the peptide derivative comprises a cysteine, said from 1 to 20 carbon atoms, peptide derivative is optionally a dimer, US 8,642,729 B2 47 48 with the proviso that the peptide is not or QRGPR (SEQ ID dC-QRF S(O-octanoyl)-dR; NO:9). CB2-QRF S(O-octanoyl)-B3R (SEQID NO: 16); 3. The peptide derivative according to claim 1, of formula CQRFSR) (SEQID NO: 4): (III): QRYSR (SEQ ID NO: 18); QR-F4F-SR (SEQ ID NO: 19), wherein—F4F– is a "-AA-AA-AA-AA-AA's-OH (III), phenylalanine, the phenyl group of which is substituted wherein: in the para position by a fluoro atom; " is a hydrogen atom, tyrosine or Y-linker-, QR-F4Br-SR (SEQID NO: 11), wherein—F4Br—is a AA is Q or Glp. phenylalanine, the phenyl group of which is substituted AA" is K or R, 10 in the para position by a bromo atom; AA" is Y. For F(X), AA is S or S(OAlk), QKFSR (SEQ ID NO: 20): AA's is K or R, QRFSK (SEQ ID NO: 21); Y-linker-, F(X) and S(OAlk) being as defined in claim 1, C ( HN (CH) CO )-QRFSR (SEQID NO:33): said AAAAAAAA, and AAs may be independently 15 C ( HN (CH) CO )-QRF S(O-octanoyl)-R either in the L-configuration or D-configuration, and any (SEQ ID NO:35); one of AA, AA, AA, AA, and AAs is optionally a B C(—HN—(CH) CO—)QRF S(O-octanoyl)-R aminoacid, an aza-aminoacid or a 3-aza-aminoacid; (SEQID NO: 41); with the proviso that the peptide is not QRFSR (SEQID NO: C—(—HN (CH) CO )-QRFS-dR; 1),YQRFSR (SEQID NO:3) or GlpRFSR (SEQID NO:17). C—(—HN (CH) CO )-QRF S(O-octanoyl)- 4. The peptide derivative according to claim 1, of formula B3R (SEQ ID NO: 22); (IV): C—(—HN (CH) CO )-QRF S(O-octanoyl)f3R (SEQID NO: 42): "-Q-R-AA-AA-R-OH (IV), wherein: wherein: 25 CB2 is HN(-CH SH)—CH2—CO—; and "is a hydrogenatom, ora Znchelating group, C-linker-, |B3R is NH-CH C-(CH), NH C(NH) N-acetyl-cysteine, N-mercaptoacetyl (HS CH (NH) COOH: CO ), hydroxamic acid (HO NH CO ) or an —S(—O-octanoyl) means a serine, the hydroxyl group of optionally substituted hydroxyquinoline, which is Substituted by an octanoyl group, AA", is For F(X), 30 —S(—O-dodecanoyl) means a serine, the hydroxyl group AA" is S or S(OAlk), of which is substituted by a dodecanoyl group. C-linker-, F(X) and S(OAlk) being as defined in claim 1, 6. The peptide derivative according to claim 1, which has said Q, R, AA, AA, and R may be independently either in inhibitory potency against neutral endopeptidase NEP and/or the L-configuration or D-configuration, and any one of aminopeptidase AP-N. Q, R, AA, AA, and R is optionally a faminoacid, an 35 7. The peptide derivative according to claim 2, which has aza-aminoacid or a B-aza-aminoacid; inhibitory potency against human AP-N. wherein if the peptide derivative comprises a cysteine, said 8. The peptide derivative according to claim 3, which has peptide derivative is optionally a dimer, inhibitory potency against human NEP. with the proviso that the peptide is not QRFSR (SEQID NO: 9. The peptide derivative according to claim 4, which is a 1). 40 dual neutral endopeptidase NEP and aminopeptidase AP-N 5. A peptide derivative selected from the group consisting inhibitor. of 10. A composition comprising at least one peptide deriva QR-F4Br-SR (SEQID NO: 11), wherein—F4Br—is a tive according to claim 1, and a pharmaceutically acceptable phenylalanine, the phenyl group of which is substituted carrier. in the para position by a bromo atom; 45 11. The peptide derivative according to claim 1, wherein QRFPR (SEQID NO: 26); AA is R. (Acetyl)-QRFSR (SEQID NO: 12): AA is For F(X), C ( HN (CH) CO )-QRFSR (SEQID NO:39); AA is S or S(OAlk), biotine-(-HN-(CH) CO-)-QRFSR (SEQ ID NO: AAs is R. 37): 50 12. The peptide derivative according to claim 4, wherein DArg-DSer-DPhe-DArg-DGln; AA" is F(X), Y ( HN (CH) CO )-QRFSR (SEQID NO:38); AA" is S or S(OAlk). Y ( HN (CH) CO )-QRFSR (SEQ ID NO: 13. A composition comprising at least one peptide deriva 32); tive according to claim 5, and a pharmaceutically acceptable QRF S(O-octanoyl)-R (SEQ ID NO: 6): 55 carrier. CQRF S(O-octanoyl)-R (SEQ ID NO: 7); 14. The peptide derivative according to claim 1, wherein CQRF S(O-dodecanoyl)-R (SEQID NO: 24); is a Zn chelating group selected from the group consisting of C ( HN (CH) CO )-QRFSR (SEQID NO:39); cysteine, C-linker-, N-acetyl-cysteine, N-mercaptoacetyl C ( HN-(CH) CO-)-QRFSR (SEQ ID NO: (HS CH CO ), hydroxamic acid (HO NH CO ) 34): 60 or an optionally Substituted hydroxyquinoline and wherein C ( HN-(CH) when the peptide derivative is CQRFSR (SEQID NO: 4), the (SEQID NO: 40); peptide derivative is a dimer. CB2IQRF S(O-octanoyl)-R (SEQID NO: 14); 15. The peptide derivative according to claim 2, wherein' C ( HN-(CH) CO-)-QRFS-B3R (SEQID NO: is a Zn chelating group selected from the group consisting of 15): 65 cysteine, C-linker-, N-acetyl-cysteine, N-mercaptoacetyl C dO-RF S(O-octanoyl)-dR; (HS CH CO ), hydroxamic acid (HO NH CO ) C ( HN-(CH) CO-)-QRFS dR; or an optionally Substituted hydroxyquinoline. US 8,642,729 B2 49 50 16. The peptide derivative according to claim 1, wherein AA is F: is C-linker wherein n is 8: AA is S(O-octanoyl); and AA is Q; AAs is D-Arg. AA is R; 20. The peptide derivative according to claim 5, wherein AA is F. 5 is D-Cys; AA is S(O-octanoyl); and AA is Q; AAs is R. AA is R; 17. The peptide derivative according to claim 1, wherein AA is F: is C—linker wherein n is 12: AA is S(O-octanoyl); and AA is Q; 10 AAs is R. AA is R; 21. The peptide derivative according to claim 2, wherein AA is F: AA' is R. AA is S(O-octanoyl); and AA's is F. AAs is R. 15 22. The peptide derivative according to claim 3, wherein 18. The peptide derivative according to claim 1, which is a AA is Q. dimer and wherein AA" is R. is C; AA" is For F(X), AA is Q; AA's is R. AA is R; 23. The peptide derivative according to claim 1, wherein AA is F. 2O is C-linker wherein n is 6: AA is S.; and AA is Q; AAs is R. AA is R; 19. The peptide derivative according to claim 5, wherein AA is F: is D-Cys; AA is S(O-octanoyl); and AA is Q; 25 AAs is R. AA is R;