METABOLICALLY STABLE PEPTIDE ANALOGS

Abstract

The present invention relates to metabolically stable and non-immunogen analogs completely hydrosoluble at physiological pH, and their use for the prevention or treatment of diseases mediated by G-protein coupled receptor (GPCR), in particular (Central Nervous System) CNS and cardiovascular diseases or disorders, or their use in diagnostic methods.

Claims

1. A metabolically stable and non-immunogenic peptide analog completely hydrosoluble at physiological pH comprising a peptide covalently linked to a fluorocarbon group, directly or through a linker selected from the group consisting of a PEG or a peptide having from 1 to 6 amino acids, either on the alpha-amino or the epsilon-amino group of at least one lysine of said peptide, when the linker is a lysine, the fluorocarbon group is directly linked to the epsilon-amino group of said linker; but excluding an apelin analog having the following peptide of formula (I): TABLE-US-00028 (I) (SEQ ID NO: 1) Lys-Phe-Xaa1-Arg-Xaa2-Arg-Pro-Arg-Xaa3-Ser-Xaa4-  Lys-Xaa5-Pro-Xaa6-Pro-Xaa7 wherein said peptide of formula (I) is linked to a fluorocarbon group, an acetyl group, or an acyl group —C(O)R where R is a C.sub.7-30 alkyl, directly or through a linker selected from the group consisting of PEG, lysine and arginine, either on the alpha-amino or the epsilon-amino group of at least one lysine of the peptide formula (I), and when the linker is a lysine, the fluorocarbon group, acetyl or acyl group is directly linked either on the epsilon-amino group of said linker and wherein : Xaa1 is arginine (R) or D-isomer arginine (R.sub.D); Xaa2 is glutamine (Q) or D-isomer glutamine (Q.sub.D) Xaa3 is leucine (L) or D-isomer leucine (L.sub.D); Xaa4 is histidine (H) or α-aminoisobutyric acid (Aib); Xaa5 is alanine (A) or D-isomer alanine (A.sub.D) or glycine (G); Xaa6 is methionine (M) or norleucine (Nle); Xaa7 is phenylalanine (F) of 4-Br phenylalanine (4-BrF).

2. The peptide analog according to claim 1, wherein said peptide is further covalently linked to an acetyl group and/or an acyl group —C(O)R where R is a C.sub.7-30 alkyl.

3. The peptide analog according to claim 1, wherein said peptide covalently linked to the fluorocarbon group is no more than 13 amino acid residues.

4. The peptide analog according to claim 1, wherein said fluorocarbon group linked to said peptide has the following formula (II):
C.sub.mF.sub.n—C.sub.yH.sub.x(L)   (II) wherein m=3 to 30, n≤2m+1, y=0 to 2, x≤2y, (m+y)=3 to 30, and L, which is optional, is a linker selected from the group consisting of a PEG or a peptide having from 1 to 6 amino acids.

5. The peptide analog according to claim 1, wherein said acyl group has the following formula (III):
CH.sub.3—C.sub.yH.sub.x—C(O)—  (III) wherein y=7 to 30, x=2y.

6. The peptide analog according to claim 1, which is selected from: i) an apelin analog having said peptide of the formula (IV): TABLE-US-00029 (IV) (SEQ ID NO: 2) Xaa1-Arg-Pro-Xaa2-Leu-Xaa3-Xaa4-Xaa5-Gly-Pro-Xaa6- Pro-Xaa7   and wherein Xaa1 is L- or D-glutamine (QL or QD) or alanine (A); Xaa2 is arginine (R) or lysine (K) or D-norleucine (Nle); Xaa3 is serine (S) or alanine (A); Xaa4 is histidine (H) or alanine (A); Xaa5 is lysine (K) or norleucine (Nle); Xaa6 is methionine (M), leucine (L), phenylalanine (F) or norleucine (Nle); Xaa7 is phenylalanine (F), 4-Br phenylalanine (4-BrF), 4-(O-benzyl)phenylalanine (4-ObnF) or p-benzoyl phenylalanine (Bpa); and ii) an apelin analog with an amino acid sequence having at least 80% identity with the sequence of (i).

7. A peptide analog according to claim 1, which is selected from the group consisting of: i) an angiotensin II analog having said peptide of the formula (V): TABLE-US-00030 (V) (SEQ ID NO: 3) Xaa1-Arg-Val-Tyr-Ile-His-Pro-Xaa2 and wherein Xaa1 is aspartic acid (D) or sarcosine; Xaa2 is phenylalanine or OH; and ii) an angiotensin II analog with an amino acid sequence having at least 80% identity with the sequence of (i).

8. A peptide analog according to claim 1, which is selected from the group consisting of: i) an oxytocin analog having said peptide of the formula (VI): TABLE-US-00031 (VI) (SEQ ID NO: 4) Cys-Tyr-Ile-Xaa1-Asp-Cys-Xaa2-Xaa3-Gly and wherein Xaa1 is glutamine or threonine; Xaa2 is proline or glycine; Xaa3 is leucine, lysine or proline; ii) an oxytocin analog with an amino acid sequence having at least 80% identity with the sequence of (i).

9. A method of treating a disease, comprising administering the peptide analog according to claim 1.

10. The method of claim 9, wherein the disease is a GPCR-related disease selected from: cardiovascular disease: heart failure, kidney diseases, hypertension, pulmonary hypertension, cirrhosis, atherosclerosis, pulmonary emphysema, pulmonary oedema, stroke, brain ischemia, myocardial impairment in sepsis; the syndrome of inappropriate antidiuretic hormone (SIADH); metabolic diseases: obesity, anorexia, hyperphagia, polyphagia, hypercholesterolemia, hyperglyceridemia, hyperlipemia; various types of dementia: senile dementia, cerebrovascular dementia, dementia due to genealogical denaturation degenerative disesases, dementia resulting from infectious diseases, dementia associated with endocrine diseases, metabolic diseases, or poisoning, dementia caused by tumors, and dementia due to traumatic diseases, depression, hyperactive child syndrome, disturbance of consciousness, anxiety disorder, schizophrenia, phobia; pain and hyperalgesia.

11. A pharmaceutical composition comprising a peptide analog according to claim 1, and one or more pharmaceutically acceptable excipient.

12. A diagnostic method comprising contacting a sample with a peptide analog according to claim 1.

Description

EXAMPLES

Example 1: General Material and Methods

[0092] Reagents were obtained from commercial source and used without any further purification. Fmoc-L-amino acids were purchased from Novabiochem, Polypeptides and Iris Biotech. Fmoc-protected Rink Amide NovaGel® resin was purchased from Novabiochem and the overall yields for the solid-phase syntheses were calculated based on the initial loadings provided by the supplier (0.7 mmol/g). Fmoc-protected Wang NovaGel® resin was purchased from Novabiochem and the overall yields for the solid-phase syntheses were calculated based on the initial loadings provided by the supplier (0.1 mmol/g).

Analytical Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) Analysis

[0093] Analysis were performed either on a C18 Sunfire column (5 μm, 4.6 mm×150 mm) using a linear gradient (5% to 95% in 20 min, flow rate of 1 mL.min.sup.-1) of solvent B (0.1% TFA in CH.sub.3CN, v/v) in solvent A (0.1% TFA in H.sub.2O, v/v). Detection was set AT 220 nm and 254 nM.

Semi-Preparative RP-HPLC Chromatography Purifications

[0094] Purifications were performed on Sunfire C18 column (5 μm, 19×150 mm) on Gilson PLC2020 with absorption detection. The separation was achieved using successive isocratic and linear gradients (5 min at 5%; 5% to 60% in 30 min, 60% to 100% in 10 min, flow rate of 20 mL.min.sup.-1) of solvent B (0.1% TFA in CH.sub.3CN, v/v) in solvent A (0.1% TFA in H.sub.2O, v/v).

Liquid Chromatography Mass Spectra (LC-MS) Analysis

[0095] Analysis were obtained on a ZQ (Z quadripole) Waters/Micromass spectrometer equipped with an X-Terra C18 column (0.5 μm, 4.6 mm×50 mm) using electrospray ionization mode (ESI).

High Resolution Mass Spectra (HR-MS) Analysis

[0096] Analysis were acquired on a Bruker MicroTof mass spectrometer, using electrospray ionization (ESI) and a time-of-flight analyzer (TOF) or on an Autoflex II TOF/TOF Bruker mass spectrometer using matrix-assisted laser desorption/ionization technique (MALDI) and a time-of-light analyzer (TOF).

General Protocol for Standard Automated Solid-Phase Peptide Synthesis (SPPS)

[0097] Standard automated solid-phase peptide synthesis (SPPS) were performed on an Applied Biosystem ABI 433A synthesizer (Appelar, France). The elongation was carried out by coupling a 10-fold excess of Fmoc-L-amino acid derivatives, using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt), and diisopropylethylamine (Hünig's base) (DIPEA) as coupling reagents in N,N-dimethylformamide (DMF) as solvent. At the end of the peptide sequence synthesis, the resin was washed with CH.sub.2Cl.sub.2 and MeOH and then dried in vacuo. After each coupling step, Fmoc deprotection was performed by treatment with piperidine monitored by UV at 301 nm.

General Protocol for Peptide Elongation by Manual SPPS

[0098] Peptide elongation was performed starting from fried resin previously synthetized by standard automated SPPS. Non-automated SPPS were performed in polypropylene tubes equipped with polyethylene frits and polypropylene caps using an orbital agitator shaking device.

[0099] The Fmoc-protected resin (1 equiv) was swollen 1 hin DMF and the excess solvent was removed by filtration. Cleavage of the Fmoc protecting group was performed in a solution of 20% (v/v) piperidine in DMF (2 times for 15 min). The piperidine solution was drained off and the resin was washed with successively DMF, CH.sub.2Cl.sub.2 and MeOH (3×0.5 mL).

[0100] All Fmoc-protected amino acids (4 equiv) were coupled in N,N-dimethylformamide (DMF) for 45 min using HBTU (3.8 equiv) and HOBt (4 equiv) with N,N-diisopropylethylamine (DIEA) (12 equiv) as activating agents. The excess solvent was removed by filtration and the resin was washed with successively DMF, CH.sub.2Cl.sub.2 and MeOH (3×0.5 mL).

[0101] The cycle of coupling, washing and deprotection were repeated until the targeted peptides were obtained. The completion of couplings and Fmoc deprotections were monitored with ninhydrin test and TNBS test: [0102] Ninhydrin test (for primary amines) : Resin beads were suspended in 2 drops of a solution containing 5 g of ninhydrin dissolved in 100 mL of ethanol, 2 drops of a solution containing 80 g of liquefied phenol in 20 mL of ethanol, and 2 drops of a 0.001 M aqueous solution of potassium cyanide to 98 mL pyridine. The mixture was heated at 100° C. for 1 min. The color positive test (presence of free amino groups). A yellow or blue solution and yellow beads indicate a negative test. [0103] TNBS test (for primary amines) : Resin beads were suspended in 2 drops of a solution containing 10% (v/v) DIPEA in DMF and 2 drops of a solution containing 2,4,6-trinitrobenzenesulfonic acid (TNBS) in DMS. The color of the solution and the beads were observed. A yellow-red solution and red beads indicate a positive test. A yellow-red solution and yellow beads indicate a negative test.
General Protocol for Peptide Elongation with a Perfluoroalkyl Chain

[0104] The resin containing the peptide sequence of interest (1 equiv) was swollen in DMF, and the excess solvent was removed by filtration. A solution of piperidine in DMF (20% v/v-0.5 mL) was added, and the mixture was shaken at room temperature for 15 min. The solution was drained, and the operation was repeated for 15 min. The solution was drained, and the resin was washed with DMF and CH.sub.2Cl.sub.2. In a separate vial, DIEA (8 equiv) was added to a solution of 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11-heptadecafluoroundecanoic acid (2 equiv), HBTU (2 equiv), and HOBt (1.9 equiv) in DMF (0.5 mL). The mixture was stirred at room temperature for 1 min and was added to the resin. The mixture was shaken at room temperature for 2h. The solution was drained and the resin was washed with DMF, CH.sub.2Cl.sub.2, and MeOH the dried in vacuo.

General Protocol for Resin Cleavage

[0105] The dried resin was treated with TFA/Phenol/Thioanisole/1,2-Ethanedithiol/water (10 mL/0.75 g/0.5 mL/0.25 mL/0.5 mL) and the mixture was shaken at room temperature for 3 h. The filtrate was collected in a cold diethyl ether solution and the beads washed with TFA. The solution was centrifuged at 3000 rpm for 2 min. The precipitate was washed in a cold diethyl ether solution and centrifuged at 3000 rpm for 2 min. The diethyl ether solution was eliminated and the precipitate was dried in vacuo. The crude product was purified by semi-preparative RP-HPLC and lyophilized.

Example 2: Peptide Analogs Synthesized and Their Characterization

Apelin Analogs

Synthesis of LE-122 and LE-124

[0106] ##STR00004##

[0107] Fmoc-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-Wang resin (16 μmol), wherein Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe is SEQ ID NO:2 with X1=Q, X2=R, X3=S, X4=H, X5=K, X6=M, and X7=F, 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11-heptadecafluoroundecanoic acid (2 equiv), HBTU (2 equiv), HOBt (1.9 equiv) and DIEA (8 equiv) were reacted according the general protocol, affording the title compound (18.9 mg, 47.7%) as a white solid. t.sub.R=11.05 min (>95% purity at 220 nm); HRMS (ESI) calcd for C.sub.80H.sub.114F.sub.17N.sub.23O.sub.17S: 2023.82123; found: 2023.82752.

##STR00005##

[0108] Fmoc-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-Wang resin (16 μmol), wherein Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe is SEQ ID NO:2 with X1=Q, X2=R, X3=S, X4=H, X5=K, X6=M, and X7=F, Boc-Lys(Fmoc)-OH (4 equiv), HBTU (3.8 equiv), HOBt (4 equiv) and DIEA (12 equiv) were reacted according the general procedure. Then, 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11-heptadecafluoroundecanoic acid (2 equiv), HBTU (2 equiv), HOBt (1.9 equiv) and DIEA (8 equiv) were reacted according the general protocol, affording the title compound (14.5 mg, 35%) as a white solid. t.sub.R=10.68 min (>95% purity at 220 nm); HRMS (ESI) calcd for C.sub.86H.sub.126F.sub.17N.sub.25O.sub.18S: 2151.91619; found: 2151.91393.

Characterization

Affinity

[0109] Evaluation of the agonist activity of compounds at the human APJ receptor expressed in transfected CHO cells, determined by measuring their effects on cAMP modulation using the HTRF detection method. Experimental protocol : The cells are suspended in HBSS buffer (Invitrogen) complemented with 20 mM HEPES (pH 7.4) and 500 μM IBMX, then distributed in microplates at a density of 1.5×104 cells/well in the presence of either of the following: HBSS (basal control), the reference agonist at 30 nM (stimulated control) or various concentrations (EC50 determination), or the test compounds. Thereafter, the adenylyl cyclase activator NKH 477 is added at a final concentration of 0.3 μM. Following 10 min incubation at 37° C., the cells are lysed and the fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (anti-cAMP antibody labeled with europium cryptate) are added. After 60 min at room temperature, the fluorescence transfer is measured at λex=337 nm and λem=620 and 665 nm using a microplate reader (Envison, Perkin Elmer). The cAMP concentration is determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio). The results are expressed as a percent of the control response to 30 nM apelin-13. The standard reference agonist is apelin-13, which is tested in each experiment at several concentrations to generate a concentration-response curve from which its EC50 value is calculated.

IC50

[0110] Evaluation of the affinity of compounds for the human apelin receptor in transfected CHO cells determined in a radioligand binding assay. Experimental protocol : Cell membrane homogenates (1 μg protein) are incubated for 120 min at 22° C. with 0.03 nM [125I](Glp65, Nle75, Tyr77)-apelin-13 in the absence or presence of the test compound in a buffer containing 50 mM Hepes/NaOH (pH 7.4), 100 mM NaCl, 10 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.04% bacitracin and 0.1% BSA. Nonspecific binding is determined in the presence of 1 μM apelin-13. Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is apelin-13, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.

Solubility Study

[0111] The solubility of each fluoropeptide was evaluated after dissolution in water to reach 100 μM. The resulting solution was vortexed 1 min following by 1 min in bath sonication. Solubility was then assessed by visual observation of the resulting dispersion (Clear/Cloudy and presence of particulates).

Human Plasma Stability

[0112] This procedure is designed to determine the stability of a test compound in blood or plasma from human or animal species in a 96-well plate format. The test compound is quantified at 5 time points by HPLC-MS/MS analysis.

[0113] Test concentration: 1 μM with a final DMSO concentration of 0.5%.

[0114] Experimental protocol : Blood or plasma are pre-warmed at 37° C. water bath for 5 min, followed by addition of the test compound. The incubation is performed in a 37° C. water bath for 2 h. An aliquot of the incubation mixture is transferred to acetonitrile at 0, 0.5, 1, 1.5 and 2 h, respectively. Samples are then mixed and centrifuged. Supernatants are used for HPLC-MS/MS analysis. Reference compounds Propoxycaine and propantheline are tested simultaneously with the test compound in each assay. Analytical methods Samples are analyzed by HPLC-MS/MS using selected reaction monitoring. The HPLC system consists of a binary LC pump with autosampler, a C-18 column, and a gradient. Conditions may be adjusted as necessary. Data analysis Peak areas corresponding to the test compound are recorded. The compound remaining (%) is calculated by comparing the peak area at each time point to time zero. The half-life is calculated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming first order kinetics

[0115] The results are presented in the table below:

TABLE-US-00025 human APJ (apelin) receptor Human Affinity IC50 Solubility plasma % of inhibition cAMP in water stability Peptide at 10 μM (nM) (μM) (t.sub.1/2, min) pE13F 101.2 24 >100 5 LE-122 100.0 2.7 >100 1241 LE-124 101.3 1.3 — 357

[0116] These results demonstrate that the incorporation of the fluorocarbon chain has no impact on both the binding affinity and the functional activity of the resulting peptides, regardless of the location of the fluorocarbon chain, either on the epsilon or on the alpha-amino group of the apelin peptide. Noteworthy, the solubility of both fluoropeptide is above 100 μM in water. Finally, the location of the chain has an impact on the metabolic stability of the fluoropeptide in human plasma. Indeed, the more stable construct is obtained when the fluorocarbon chain is introduced on the alpha-amino part of the peptide (LE-122, t.sub.1/2>1241 min).

Angiotensin II Analogs

Synthesis of LE-120

[0117] ##STR00006##

[0118] Fmoc-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-Wang resin (50 μmol), wherein Asp-Arg-Val-Tyr-Ile-His-Pro-Phe is SEQ ID NO:3 with X1=D and X2=F, 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11-heptadecafluoroundecanoic acid (2 equiv), HBTU (2 equiv), HOBt (1.9 equiv) and DIEA (8 equiv) were reacted according the general protocol, affording the title compound (41.4 mg, 47%) as a white solid. t.sub.R=12.94 min (>95% purity at 220 nm); HRMS (ESI) calcd for C.sub.61H.sub.74F.sub.17N.sub.13O.sub.13: 15109.52576; found: 1519.52309.

Characterization

Affinity

[0119] Evaluation of the agonist activity of compounds at the human AT1 receptor expressed in transfected HEK-293 cells, determined by measuring their effect on cytosolic Ca2+ ion mobilization using a fluorimetric detection method. Experimental protocol: The cells are suspended in DMEM buffer (Invitrogen), then distributed in microplates at a density of 4.104 cells/well. The fluorescent probe (Fluo4 Direct, Invitrogen) mixed with probenicid in HBSS buffer (Invitrogen) complemented with 20 mM Hepes (Invitrogen) (pH 7.4) is then added into each well and equilibrated with the cells for 60 min at 37° C. then 15 min at 22° C. Thereafter, the assay plates are positioned in a microplate reader (CellLux, PerkinElmer) which is used for the addition of the test compound, reference agonist or HBSS buffer (basal control), and the measurements of changes in fluorescence intensity which varies proportionally to the free cytosolic Ca2+ ion concentration. For stimulated control measurements, angiotensin-II at 30 nM is added in separate assay wells. The results are expressed as a percent of the control response to 30 nM angiotensin-II. The standard reference agonist is angiotensin-II, which is tested in each experiment at several concentrations to generate a concentration-response curve from which its EC50 value is calculated.

IC50

[0120] Evaluation of the affinity of compounds for the human angiotensin-II AT1 receptor in transfected HEK-293 cells determined in a radioligand binding assay. Experimental protocol : Cell membrane homogenates (8 μg protein) are incubated for 120 min at 37° C. with 0.05 nM [125I][Sar1-IIe8]angiotensin-II in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA and 0.1% BSA. Nonspecific binding is determined in the presence of 10 μM angiotensin II. Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is saralasin, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.

Solubility Study

[0121] The solubility of the fluoropeptide was evaluated after dissolution in water to reach 100 μM. The resulting solution was vortexed 1 min following by 1 min in bath sonication. Solubility was then assessed by visual observation of the resulting dispersion (Clear/Cloudy and presence of particulates).

Human Plasma Stability

[0122] This procedure is designed to determine the stability of a test compound in blood or plasma from human or animal species in a 96-well plate format. The test compound is quantified at 5 time points by HPLC-MS/MS analysis.

[0123] Test concentration: 1 μM with a final DMSO concentration of 0.5%. Experimental protocol: Blood or plasma are pre-warmed at 37° C. water bath for 5 min, followed by addition of the test compound. The incubation is performed in a 37° C. water bath for 2 h. An aliquot of the incubation mixture is transferred to acetonitrile at 0, 0.5, 1, 1.5 and 2 h, respectively. Samples are then mixed and centrifuged. Supernatants are used for HPLC-MS/MS analysis. Reference compounds Propoxycaine and propantheline are tested simultaneously with the test compound in each assay. Analytical methods Samples are analyzed by HPLC-MS/MS using selected reaction monitoring. The HPLC system consists of a binary LC pump with autosampler, a C-18 column, and a gradient. Conditions may be adjusted as necessary. Data analysis Peak areas corresponding to the test compound are recorded. The compound remaining (%) is calculated by comparing the peak area at each time point to time zero. The half-life is calculated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming first order kinetics

[0124] The results are presented in the table below:

TABLE-US-00026 human AT1R Human Affinity EC50 Ca.sup.2+ Solubility plasma % of inhibition production in water stability Peptide at 10 μM (nM) (μM) (t.sub.1/2, min) Angiotensin II 100.2 14 >100 85 LE120 66.4 460 >100 >1440

[0125] These results indicate that the incorporation of the fluorocarbon chain has an impact on both the affinity and the functional activity compared to the native peptide. Indeed, the affinity of LE120 is significantly decreased altogether with the efficacy from 14 vs 460 nM, respectively. However, as previously described for apelin-13, the human plasma stability is greatly improved from 85 min for the native peptide to >1440 min for the fluoropeptide, again demonstrating the beneficial effect of the fluorocarbon chain on the metabolic stability.

Oxytocin Analogs

Synthesis of SR-11-74

[0126] ##STR00007##

[0127] Fmoc-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Lys-Gly-NH.sub.2-Rink resin (165 μmol), wherein Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Lys-Gly is SEQ ID NO:4 with X1=Q, X2=P, and X3=, 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11-heptadecafluoroundecanoic acid (2 equiv), HBTU (2 equiv), HOBt (1.9 equiv) and DIEA (8 equiv) were reacted according the general protocol, affording the title compound (21.1 mg, 8%) as a white solid. t.sub.R=12.78 min (>95% purity at 220.8 nm); HRMS (ESI) calcd for C.sub.54H.sub.70F.sub.17N.sub.13O.sub.13S.sub.2: 1495.4386; found: 1495.43437.

Characterization

Affinity

[0128] Evaluation of the agonist activity of compounds at the human OT receptor endogenously expressed in ECV304 cells, determined by measuring their effect on cytosolic Ca2+ ion mobilization using a fluorimetric detection method. Experimental protocol : The cells are suspended in DMEM buffer (Invitrogen), then distributed in microplates at a density of 3.104 cells/well. The fluorescent probe (Fluo4 Direct, Invitrogen) mixed with probenicid in HBSS buffer (Invitrogen) complemented with 20 mM Hepes (Invitrogen) (pH 7.4) is then added into each well and equilibrated with the cells for 60 min at 37° C. then 15 min at 22° C. Thereafter, the assay plates are positioned in a microplate reader (CellLux, PerkinElmer) which is used for the addition of the test compound, reference agonist or HBSS buffer (basal control), and the measurements of changes in fluorescence intensity which varies proportionally to the free cytosolic Ca2+ ion concentration. For stimulated control measurements, oxytocin at 3 μM is added in separate assay wells. The results are expressed as a percent of the control response to 3 μM oxytocin. The standard reference agonist is oxytocin, which is tested in each experiment at several concentrations to generate a concentration-response curve from which its EC50 value is calculated.

IC50

[0129] Evaluation of the affinity of compounds for the human vasopressin Oxytocin receptor in transfected Chem-1 cells determined in a radioligand binding assay. Experimental protocol: Cell membrane homogenates (about 10 μg protein) are incubated for 120 min at 22° C. with 0.8 nM [3H]Oxytocin in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2 and 0.1% BSA. Nonspecific binding is determined in the presence of 1 μM Oxytocin. Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl and 0.1% BSA using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is Oxytocin, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.

Solubility Study

[0130] The solubility of the fluoropeptide was evaluated after dissolution in water to reach 100 μM. The resulting solution was vortexed 1 min following by 1 min in bath sonication. Solubility was then assessed by visual observation of the resulting dispersion (Clear/Cloudy and presence of particulates).

Human Plasma Stability

[0131] This procedure is designed to determine the stability of a test compound in blood or plasma from human or animal species in a 96-well plate format. The test compound is quantified at 5 time points by HPLC-MS/MS analysis.

[0132] Test concentration: 1 μM with a final DMSO concentration of 0.5%. Experimental protocol: Blood or plasma are pre-warmed at 37° C. water bath for 5 min, followed by addition of the test compound. The incubation is performed in a 37° C. water bath for 2 h. An aliquot of the incubation mixture is transferred to acetonitrile at 0, 0.5, 1, 1.5 and 2 h, respectively. Samples are then mixed and centrifuged. Supernatants are used for HPLC-MS/MS analysis. Reference compounds Propoxycaine and propantheline are tested simultaneously with the test compound in each assay. Analytical methods Samples are analyzed by HPLC-MS/MS using selected reaction monitoring. The HPLC system consists of a binary LC pump with autosampler, a C-18 column, and a gradient. Conditions may be adjusted as necessary. Data analysis Peak areas corresponding to the test compound are recorded. The compound remaining (%) is calculated by comparing the peak area at each time point to time zero. The half-life is calculated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming first order kinetics.

[0133] The results are presented in the table below:

TABLE-US-00027 human oxytocin receptor Human Affinity EC50 Ca.sup.2+ Solubility plasma % of inhibition production in water stability Peptide à 10 nM (nM) (μM) (t.sub.1/2, min) Oxytocin 102.2 42 >100 147 SR-11-74 91.3 <25% at 100 >100 239 nM

[0134] In the case of cyclic oxytocin, the incorporation of the fluorocarbon chain has a profound effect on the functional activity of the peptide (EC50 from 42 nM for the native peptide to >25% at 100 nM for the fluoro-ocytocin). In another hand, the presence of the fluorocarbon chain has a low impact on the human plasma stability (t.sub.1/2=147 min for the oxytocin vs 239 min for the fluoro-oxytocin).

REFERENCE LISTING

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