Neurotensin analogues for radioisotope targeting to neurotensin receptor-positive tumors

Abstract

The invention relates to a new neurotensin analogue, or a salt thereof, useful for targeting to neurotensin receptor-positive tumors, like ductal pancreatic adenocarcinoma, exocrine pancreatic cancer, invasive ductal breast cancers, colon adenocarcinoma, small cell lung carcinoma, Ewing sarcoma, meningioma, medulloblastoma and astrocytoma.

Claims

1. A neurotensin analogue, or a salt thereof, of formula (I)
X-L-Aa8-Aa9-(L)Pro-Aa11-Aa12-(L)Leu  (I) wherein Aa8 is NMe-(L)Arg, Nme-(D)Arg, NMe-(L)Lys or NMe-(D)Lys, and the sequence Aa9-(L)Pro-Aa11-Aa12-(L)Leu differs from (L)Arg-(L)Pro-(L)Tyr-(L)Ile-(L)Leu by at least one substitution in the amino acid sequence, the substitution(s) being selected from: Aa9 is selected from the group consisting of (L)Lys, ψ(CH.sub.2—NH)-(L)Arg, and ψ(CH.sub.2—NH)-(L) Lys, Aa11 is selected from the group consisting of (D)Tyr, (L)Dmt and (D)Dmt, and Aa12 is selected from the group consisting of (L)Tle, (L)Leu and (L)Val, X is a poly(aminocarboxylate) chelating moiety selected from the group consisting of: i) diethylenetriamine pentaacetic acid (DTPA) and its derivatives, ii) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and its derivatives, iii) 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and its derivatives, iv) 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA) and its derivatives, v) 1,4,7,10-tetraazacyclotridecane-N,N′,N″,N′″-tetracetic acid (TITRA) and its derivatives, vi) triethylenetetramine hexaacetic acid (TTHA) and its derivatives, vii) 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) and its derivatives, and viii) 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA) and its derivatives, and L is a linker which separates X and Aa8, and said linker L is selected from the group consisting of -Aa6-Aa7-, wherein X is coupled to Aa6 via the ε-NH.sub.2 of the lateral chain of Aa6, or -L1-Aa6-Aa7-, wherein X is coupled to L1, and L1 is coupled to Aa6 via the ε-NH.sub.2 of the lateral chain of Aa6 or via the α-NH2 of Aa6, wherein Aa6 is selected from the group consisting of (D)Lys, and (L)Lys, Aa7 is selected from the group consisting of (L)Pro, and (D)Pro, and L1 is —NH—(CH.sub.2).sub.n—CO— wherein n is from 1 to 5, and an acetyl group is coupled to the remaining NH.sub.2 of Aa6, and wherein said analogue, when in solution at physiological pH and at physiological temperature, has at most two positive charges.

2. The neurotensin analogue, or the salt thereof, according to claim 1, wherein Aa8 is NMe-(L)Arg, and Aa12 is (L)Tle.

3. The neurotensin analogue, or the salt thereof, according to claim 1, wherein the neurotensin analogue is selected from the group consisting of the following formulas:
Ac-(L)Lys.sup.6(DTPA)-(L)Pro.sup.7-NMeArg.sup.8-(L)Arg.sup.9-(L)Pro.sup.10-(L)Tyr.sup.11-(L)Tle.sup.12-(L)Leu.sup.13
Ac-(L)Lys.sup.6(DOTA)-(L)Pro.sup.7-NMe(L)Arg.sup.8-(L)Arg.sup.9-(L)Pro.sup.10-(L)Tyr.sup.11-(L)Tle.sup.12-(L)Leu.sup.13
Ac-(L)Lys.sup.6(Ahx-DOTA)-(L)Pro.sup.7-NMeArg.sup.8-(L)Arg.sup.9-(L)Pro.sup.10-(L)Dmt.sup.11-(L)Tle.sup.12-(L)Leu.sup.13.

4. A pharmaceutical composition comprising the neurotensin analogue according to claim 1 and a pharmaceutically acceptable carrier.

5. The neurotensin analogue according to claim 1, further comprising a detectable element which forms a complex with the poly(aminocarboxylate) chelating moiety X, wherein the detectable element is selected from the group consisting of .sup.111In, .sup.67Ga, .sup.68Ga, .sup.64Cu and .sup.44Sc.

6. The neurotensin analogue according to claim 5, wherein said detectable element is Gd.sup.3+.

7. The neurotensin analogue according to claim 5, wherein said detectable element is Eu.sup.3+.

8. The neurotensin analogue according to claim 1, further comprising a cytotoxic element that forms a complex with the chelating moiety X, wherein said cytotoxic element is selected from the group consisting of .sup.90Y, .sup.177Lu, .sup.67Cu, .sup.47Sc, .sup.212Bi, .sup.213Bi, .sup.226Th, .sup.111In and .sup.67Ga.

9. The neurotensin analogue, or the salt thereof, according to claim 1, wherein X represents a poly(aminocarboxylate) chelating moiety selected from the group consisting of: diethylenetriamine pentaacetic acid (DTPA), S-2(4-Aminobenzyl)-diethylenetriamine pentaacetic acid (p-NH2-Bn-DTPA), (R)-2-Amino-3-(4-aminophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid, [(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid, and 2-(4-Isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-Bn-DTPA).

10. The neurotensin analogue, or the salt thereof, according to claim 1, wherein X represents a poly(aminocarboxylate) chelating moiety selected from the group consisting of: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclo-dodecane tetraacetic acid (p-aminobenzyl-DOTA), and S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-tetraacetic acid (p-SCN-Bn-DOTA).

11. The neurotensin analogue, or the salt thereof, according to claim 1, wherein X represents a poly(aminocarboxylate) chelating moiety selected from the group consisting of: 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), S-2-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-NH2-Bn-NOTA) and S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: In vivo serum stability of DTPA(.sup.111In)-peptides: representative C18 HPLC chromatograms of plasma samples collected 15 minutes post-injection to mice. A: reference peptide and NT(8-13) analogues, B: NT(6-13) analogues. Arrows show the intact peptide retention time. Mean percent radioactivity associated to intact peptide and individual values (between brackets) are indicated.

(2) FIG. 2: Degradation kinetics of DTPA(.sup.111In)-peptides in human serum. Peptides (2 pmol) were incubated with human serum (100 μL) at 37° C.: [Lys.sup.6(DTPA(In))]-NT open triangle, DTPA(In)-NT-20.1 black triangle, DTPA(In)-NT-20.2 open square, DTPA(In)-NT-20.3 black square (mean±sem, three independent experiments).

(3) FIG. 3: Internalisation of DTPA(.sup.111In)-NT20.3 in HT29 cells. Results are expressed as the ratio between internalized and specifically bound radioactivity (I/B, mean±sem, 3 experiments in triplicate).

(4) FIG. 4: DTPA(.sup.111In)-NT-20.3 planar images of a male nude mouse grafted with HT29 cells. A: photograph, B: planar anterior acquisition performed from 0 to 60 min post-injection under anaesthesia, C: dynamic series of images of 5 min each computed from the recorded scintigraphy data. B1: Bladder, K: Kidney, T: Tumor. Tumor weight: 240 mg.

(5) FIG. 5: SPECT/CT imaging of a male nude mice mouse grafted with HT29 cells in the right flank 2.5 h post-injection of DTPA(.sup.111In)-NT-20.3. Left: CT; center: SPECT, right: SPECT/CT fused images. Frames: A: coronal, B: axial, C: sagittal. Abbreviations as in FIG. 5 and r: right, l: left, a: anterior, p: posterior. Tumor weight: 498 mg.

(6) FIG. 6: DOTA(.sup.111In)-NT-20.3 and DOTA(.sup.111In)-LB119 planar images of male nude mice grafted with HT29 cells. Planar anterior acquisitions were performed from 0 to 1 h, 1 to 1.5 h, 4.5 to 5.5 h, 24 to 25 h and 48 to 49 h post-injection under anaesthesia. B1: Bladder, K: Kidney, T: Tumor.

(7) FIG. 7: TEP imaging of a male nude mouse, grafted with HT29 cells in the right flank, injected with DOTA(.sup.68Ga)-NT-20.3: coronal frame 47 minutes post injection, 10 min acquisition, tumor volume: 40 mm.sup.3. B1: Bladder, K: Kidney, T: Tumor.

EXAMPLES

(8) In the following examples DTPA-NT-20.3, DOTA-NT-20.3 and DOTA-LB119 are neurotensin analogues according to the invention. Other neurotensin analogues are presented for comparison.

(9) 1. Synthesis of the DTPA- and DOTA-NT Analogues

(10) 1.1 Synthesis of the DTPA-NT Analogues

(11) DTPA-NT-VI, DTPA-NT-XI, DTPA-Ahx-NT-XII, DTPA-Ahx-NT-XIX are DTPA-NT(8-13) analogues, that were stabilized against enzymatic degradation at the bonds between Arg.sup.8 and Arg.sup.9, Pro.sup.10 and Tyr.sup.11 or Tyr.sup.11 and Ile.sup.12 by changes introduced in the peptide sequence (Table 1).

(12) DTPA-NT-20.1, DTPA-NT-20.2 and DTPA-NT-20.3 are analogues of the 6-13 sequence of [Lys.sup.6(DTPA)]-NT. The N terminal end was acetylated. In these analogues DTPA was coupled to the ε-NH.sub.2 group of Lys.

(13) All reagents used for the synthesis were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France or Bornem, Belgium), Novabiochem (Läufelfingen, Switzerland), Bachem (Bubendorf, Switzerland) and RSP (Shirley, USA). The purity of the compounds was checked by HPLC on a Nucleosil C.sub.18 (5 μm, 100 Å, Shandon, France) reverse phase column or on a Discovery®BIO SUPELCO Wide Pore (5 μm, 300 Å, Sigma-Aldrich) column with a gradient of A: water (0.05% TFA) and B: CH.sub.3CN (0.05% TFA) at a flow rate of 1 mL/min on a Waters apparatus.

(14) The NT(8-13), NT-VI, NT-XI, NT-XII and NT-XIX peptides (Table 1) were prepared by solid phase peptide synthesis as described in detail elsewhere (Bruehlmeier et al., Nucl. Med. Biol. (2002) 29, 321-327; Maes et al., J. Med. Chem. (2006) 49, 1833-1836; Bergmann et al., Nucl. Med. Biol. (2002) 29, 61-72). Tris-tBu-DTPA (3 eq.) (Achilefu et al., J. Org. Chem. (2000) 65, 1562-1565) was coupled to the resin-bound neurotensin analog in a mixture of DMF/CH.sub.2Cl.sub.2 using 2-1H(benzotriazol-1-yl)-1,1,3,3-tetramethylureum tetrafluoroborate (TBTU), 1-hydroxybenzotriazole (HOBt) and diisopropylethylamine (DIPEA) during 4 h.

(15) The acetylated NT(6-13) analogues were synthesized by NeoMPS (Strasbourg, France). DTPA was coupled to the lysine ε-NH.sub.2 as already described (Janevik-Ivanovska et al., Bioconjug. Chem. (1997) 8, 526-533).

(16) All DTPA-peptides were purified to at least 92% purity and identified by mass spectrometry (Table 1).

(17) TABLE-US-00001 TABLE 1 Peptide sequence and analytical data M + H.sup.+ M + H Peptide Sequence % purity MALDI-TOF calculated NT(1-13) analogues NT pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile- Leu-OH [Lys6(DTPA)]-NT pGlu-Leu-Tyr-Glu-Asn-Lys(DTPA)-Pro-Arg-Arg-Pro- >95.sup.a  2048.16.sup.a  2048.32.sup.a Tyr-Ile-Leu-OH NT(8-13) analogues NT(8-13) H-Arg-Arg-Pro-Tyr-Ile-Leu-OH DTPA-NT(8-13) DTPA-Arg-Arg-Pro-Tyr-Ile-Leu-OH >96 1192.23 1192.62 DTPA-NT-VI DTPA-Lys-Ψ(CH.sub.2-NH)-Arg-Pro-Tyr-Ile-Leu-OH 95 1150.12 1150.65 DTPA-NT-Xi DTPA-Lys-Ψ(CH.sub.2-NH)-Arg-Pro-Tyr-Tle-Leu-OH 97 1150.33 1150.65 DTPA-Ahx-NTXII DTPA-Ahx-Arg-Me-Arg-Pro-Tyr-Tle-Leu-OH 92 1319.11 1318.73 DTPA-Ahx-NT-XIX DTPA-Ahx-Arg-Me-Arg-Pro-Dmt-Tle-Leu-OH 97 1346.50 1346.76 NT(6-13) analogues NT-20.1 Ac-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH NT-20.2 Ac-Lys-Pro-Arg-Arg-Pro-Tyr-Tle-Leu-OH NT-20.3 Ac-Lys-Pro-Me-Arg-Arg-Pro-Tyr-Tle-Leu-OH DTPA-NT-20.1 Ac-Lys(DTPA)-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH >99 1459.78 1459.78 DTPA-NT-20.2 Ac-Lys(DTPA)-Pro-Arg-Arg-Pro-Tyr-Tle-Leu-OH >97 1459.77 1459.78 DTPA-NT-20.3 Ac-Lys(DTPA)-Pro-Me-Arg-Arg-Pro-Tyr-Tle-Leu-OH >99 1473.83 1473.80 DOTA-NT-20.3 Ac-Lys(DOTA)-Pro-Me-Arg-Arg-Pro-Tyr-Tle-Leu-OH 98 1484.83 1484.85 DOTA-LB119.sup.b Ac-Lys(Ahx-DOTA)-Pro-Me-Arg-Arg-Pro-Dmt-Tle-Leu-OH >95 1627.08 1626.98 .sup.aResults already described (Hillairet De Boisferon et al., Bioconjug. Chem. (2002) 13, 654-662). .sup.bAhx: 6-aminohexanoic acid.

(18) The following peptides DTPA-NT-20.3, DOTA-NT-20.3, DOTA-LB119 are neurotensin analogues according to the invention.

(19) 1.2. Synthesis of the DOTA-NT Analogues

(20) All reagents used for the synthesis were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France or Bornem, Belgium), Macrocyclics (Dallas, USA), Novabiochem (Läufelfingen, Switzerland), Bachem (Bubendorf, Switzerland) and RSP (Shirley, USA). The purity of the compounds was checked by HPLC on a Nucleosil C18 (5 μm, 100 Å, Shandon, France) reverse phase column or on a Discovery®BIO SUPELCO Wide Pore (5 μm, 300 Å, Sigma-Aldrich) column with a gradient of A: water (0.05% TFA) and B: CH3CN (0.05% TFA) at a flow rate of 1.5 mL/min on a Waters apparatus.

(21) The acetylated NT(6-13) analogue NT-20.3 (Ac-Lys-Pro-Me-Arg-Arg-Pro-Tyr-Tle-Leu-OH) was synthesized by NeoMPS (Strasbourg, France). 1,4,7,10-Tetraazacyclododecane-1,4,7-tris(acetic acid)-10-acetic acid mono(N-hydroxysuccinimidyl ester) (DOTA-NHS ester) (Macrocyclics, Dallas, Tex., USA) (5 eq.) was coupled to the lysine ε-NH2 of NT-20.3 (1 eq.) as described (1). This DOTA-NT20.3 was purified by C18 reverse phase chromatography (5 μm, 100 Å, Nucleosil, Shandon, France) using a linear 150-minute gradient (Flow: 2 mL/min, A: H2O/TFA(0.05%), B: acetonitrile/TFA(0.05%)) from 0% to 37% B. Coupling yield was approximately 85% for DOTA-NT-20.3.

(22) DOTA-LB119 was obtained starting from Ac-Lys(Dde)-Pro-MeArg(Pbf)-Arg(Pbf)-Pro-Dmt(Trt)-Tle-Leu-OWang resin. After deprotection of the Dde protection using NH.sub.2OH.HCl/imidazole, (Brans et al., Chemical Biology & Drug Design, (2008) 72, 496-506). Fmoc-Ahx was coupled to the free ε-NH2 group of Lys (DIC/HOBt) followed by Fmoc deprotection and coupling of DOTA(OtBu).sub.3 using HATU. The peptide was cleaved from the resin using TFA/H2O/thioanisole/phenol/ethanedithiol (82.5:5:5:5:2.5), and purified by HPLC.

(23) All DOTA-peptides were purified to at least 95% purity and identified by mass spectrometry (Table 1).

(24) 2. Radiolabeling

(25) The DTPA-NT analogues were labeled with indium-111 (.sup.111InCl.sub.3, 60 or 180 MBq, CIS bio International, France) in 100 mM acetate, 10 mM citrate, buffer pH 5 during 22 h at room temperature, then free DTPA groups were saturated with non-radioactive InCl.sub.3 as already described (Raguin et al., Angew. Chem. Int. Ed. Engl. (2005) 44, 4058-4061). The DOTA-NT (1 nmol) analogues were labeled with indium-111 (.sup.111InCl.sub.3, 10-20 MBq, CIS bio International, France) in 270 mM acetate, 27 mM citrate, buffer pH 4.5 during 25 minutes at 95° C.

(26) 3. Determination of the NTS1 Binding Affinities and Internalization Studies.

(27) 3.1. Materials and Methods

(28) 3.1.1. Binding to HT29 Cell Membranes.

(29) Cell membranes (60 μg protein), were incubated for 45 min at room temperature in 250 μL buffer (50 mM Tris HCl, 5 mM MgCl.sub.2, 0.8 mM 1,10-phenanthroline, 0.2% BSA, pH 7.4), in the presence of 50 pM .sup.125I-Tyr.sup.3-neurotensin (Perkin-Elmer) and increasing concentrations of non-radioactive DTPA(In)-NT analogues. Membrane bound activity was recovered by filtration onto Whatman GF/B filters presoaked for 1 hour with polyethyleneimine (0.2% in water) and rinsed twice with buffer. Non-specific binding was evaluated in the presence of 10.sup.−6 M neurotensin. Radioactivity was counted and results were analyzed with GraphPad Prism® (GraphPad Software, Inc. San Diego, Calif.). All experiments were performed three times in triplicate.

(30) 3.1.2. Binding to Living HT29 Cells and Internalization

(31) IC50 for the binding to living HT29 cells were determined from competition experiments between [Lys.sup.6(DTPA(.sup.111In))]-NT and the peptides without DTPA or DTPA(In)-Ahx-NT-XIX. For the other DTPA-peptides IC50 was evaluated using the labeled DTPA(.sup.111In)-peptide and increasing concentrations of the corresponding non radioactive DTPA(In)-peptide. IC 50 of non radioactive DOTA(metal)-peptide complex (DOTA(Me)-peptide with Me:In; Y or Lu) was determined using trace amounts of .sup.125I-NT and increasing concentrations of the DOTA(Me)-peptide. For non radioactive metal chelation the DOTA-peptides (150 nmol in 150 μL water) were incubated (25 min 95° C.) with a solution of non radioactive InCl.sub.3, YCl.sub.3, or GaCl.sub.3 (1.5 mmol in 150 μL acetate 100 mM, citrate 10 mM, buffer).

(32) Cells were rinsed by 500 μL DMEM, 0.2% BSA, and incubated with the labeled analogue (DTPA(.sup.111In)-NT analogue 150 pM or .sup.125I-NT 40 pM, 300 μL DMEM, 0.2% BSA, 0.8 mM 1,10-phenanthroline, 60 min, 37° C.) in the presence of increasing concentrations of non-radioactive DTPA(In)-NT analogue or DOTA(Me)-peptide. After washing the wells twice with ice-cold DMEM 0.2% BSA, cells were lysed in 500 μL 0.1N NaOH and radioactivity was counted. Non-specific binding was evaluated in the presence of 10.sup.−6 M neurotensin. Competition curves were analyzed with the “Equilibrium Expert” software (Raguin et al., Anal. Biochem. (2002) 310, 1-14). All experiments were performed three times in triplicate.

(33) Incubation for internalization studies was performed with 0.15×10.sup.−9 M DTPA(.sup.111In)-NT analogue or 0.5×10.sup.−9M DOTA(.sup.111In)-NT20.3 or DOTA(.sup.111In)-LB119 as above except for the use of twelve-well plates (600 μL). At selected times the total binding was evaluated as above. To determine the amount of internalized radioactivity wells were incubated in DMEM/0.2% BSA, pH 2.0 for 15 min at 4° C., to dissociate the surface-bound ligand. Internalized activity was then counted after washing. Non-specific binding and internalization was evaluated in the presence of 10.sup.−6 M neurotensin. Results are expressed as the ratio between internalized and specifically bound radioactivity.

(34) 3.2. Binding and Internalization Results

(35) K.sub.i values for binding to HT29 cell membranes and IC50 for binding to cells were used to evaluate affinity (Table 2). K.sub.i values for binding to HT29 membranes were, for most peptides, about 10 times lower than the IC50 for the binding to HT29 cells. This can be attributed to the decreased affinity for binding to the NTS1 induced by sodium (Kitabgi et al., Peptides (2006) 27, 2461-2468) and to the effects of internalization and externalization of radioactivity in cells.

(36) DTPA(In) coupled to the NH.sub.2-α of NT(8-13) induced an important decrease in the affinity for membranes and for cells (by a factor of 31 and 32 respectively) as compared to NT(8-13). This loss of affinity is less important when the distance between the receptor-binding (8-13) sequence and DTPA is larger. When coupling DTPA to the ε-NH.sub.2 of Lys.sup.6 of NT, the affinity loss is only a factor of 6 for membranes and of 10 for cells. Similarly the affinity loss in DTPA(In)-NT-20.1 is only a factor of 9 and 8 as compared to NT-20.1. As a result, the affinity of DTPA(In)-NT-20.1 was two fold higher than that of DTPA(In)-NT(8-13), even though NT(8-13) displayed an affinity slightly higher than that of NT-20.1.

(37) TABLE-US-00002 TABLE 2 Affinity of peptides for binding to HT29 cells or cell membranes. K.sub.i (nM) Peptide membranes IC50 (nM) cells NT 0.28 ± 0.05  1.67 ± 0.40 [Lys.sup.6(DTPA(In))]-NT 1.77 ± 0.39 17.3 ± 4.3 NT(8-13) 0.044 ± 0.009  0.68 ± 0.04 DTPA(In)-NT(8-13) 1.36 ± 0.39 21.7 ± 5.1 DTPA(In)-NT-VI 3.20 ± 0.81 14.7 ± 1.6 DTPA(In)-NT-XI 8.11 ± 1.03 101 ± 17 DTPA(In)-Ahx-NTXII 5.26 ± 1.24 132 ± 44 DTPA(In)-Ahx-NT-XIX 67 ± 11 626 ± 30 NT-20.1 0.072 ± 0.019  0.82 ± 0.08 NT-20.2 0.26 ± 0.07  2.46 ± 0.79 NT-20.3 0.16 ± 0.03  2.20 ± 0.31 DTPA(In)-NT-20.1 0.66 ± 0.1   6.73 ± 0.31 DTPA(In)-NT-20.2 1.55 ± 0.42 41.2 ± 6.2 DTPA(In)-NT-20.3 2.24 ± 0.21 15.9 ± 1.7 DOTA(In)-NT-20.3 ND 14.9 ± 1.1 DOTA(Ga)-NT-20.3 ND 13.9 ± 2.2 DOTA(Y)-NT-20.3 ND  7.0 ± 0.7 DOTA(In)LB119 ND 14.1 ± 0.7 DOTA(Ga)LB119 ND  7.5 ± 0.7 DOTA(Y)LB119 ND  9.9 ± 0.4

(38) N-methylation of the Arg.sup.8-Arg.sup.9 bond and introduction of an aminohexanoic acid spacer between DTPA and the 8-13 receptor binding sequence did not improve the affinity of DTPA(In)-Ahx-NT-XII as compared to DTPA(In)-NT-XI. Replacement of Tyr.sup.11 by 2′,6′-dimethyltyrosine in DTPA(In)-Ahx-NT-XIX led to an additional loss of affinity.

(39) Introduction of a Tle.sup.12 in the NT(6-13) series induced a decrease in affinity similar to that observed in the DTPA(In)-NT(8-13) series. N-methylation of the Pro.sup.7-Arg.sup.8 bond had little effect on affinity. Because the affinity of NT-20.1 was higher than that of NT, DTPA coupling and sequence modifications to the doubly-stabilized DTPA(In)-NT-20.3, the only peptide of this series which is a neurotensin analogue according to the invention, resulted in a high affinity, for membranes and for living cells, similar to those of the reference peptide [Lys.sup.6(DTPA(In))]-NT.

(40) The DTPA(In)-peptides exhibiting the highest affinities, [Lys.sup.6(DTPA(In))]-NT, DTPA(In)-NT-VI, DTPA(In)-NT-XI, DTPA(In)-NT-20.1, DTPA(In)-NT-20.2, DTPA(In)-NT-20.3, were further evaluated for stability and tumor targeting in vivo.

(41) DOTA coupling had similar effects as DTPA since DOTA(In)-NT20.3 affinity to cells was similar to that of its DTPA(In)-counterpart. The substitution of Tyr by Dmt and introduction of an aminohexanoic acid spacer between DOTA and the ε-NH2 of Lys.sub.6 in DOTA(In)-LB119 had no effect on affinity.

(42) The gallium chelate of DOTA-NT-20.3 exhibited an affinity similar to that of the indium complex, in opposition to the affinity increase of the yttrium chelate. Unexpectedly the gallium complexe of DOTA-LB119 displayed an affinity increase similar to that of the yttrium complexe as compared to the indium one. The high affinities observed for the complexes of DOTA-NT20.3 and DOTA-LB-119 with gallium and yttrium suggest that these peptides are suitable for in vivo targeting of their radioisotopes.

(43) DTPA(.sup.111In)-NT-20.3 and DOTA(.sup.111In)-NT-20.3 internalized rapidly in HT29 cells, reaching a 86±3% and a 84±1% internalization plateau with a t.sub.1/2 of 2.1±0.4 and 4.8±0.1 min respectively (Table 3). DOTA(.sup.111In)-LB119 internalization t.sub.1/2 was significantly lower.

(44) TABLE-US-00003 TABLE 3 Peptide internalization in HT29 cells t.sub.1/2 Plateau Peptide (min) (%) [Lys.sup.6(DTPA(.sup.111In))]-NT 4.2 ± 1.1 88 ± 6 DTPA(.sup.111In)-NT-VI 3.8 ± 1.2 82 ± 6 DTPA(.sup.111In)-NT-20.3 2.1 ± 0.4 86 ± 3 DOTA(.sup.111In)-NT-20.3 4.8 ± 0.1 84 ± 1 DOTA(.sup.111In)-LB119 19.3 ± 0.7  93 ± 1
4. Metabolic Stability
4.1. In Human Serum

(45) Serum from healthy donors (100 μL) was incubated with the DTPA(.sup.111In) analogues (2 pmol, 37° C.). Samples were collected at different time points and proteins were precipitated with methanol and filtered. Then methanol was evaporated under vacuum and the sample was analyzed by C.sub.18 RP-HPLC. Detection was performed with a radioactivity detector (HERM LB 500, Berthold, France). Elution was performed using, after 5 min 0% B, a linear 10-minute gradient from 0% to 35% B and a linear 25-minute gradient from 35% to 50%, flow rate 1.5 mL/min. The sample was also co-injected with the radioactive control to identify the peak corresponding to intact peptide.

(46) The in vitro stability in human serum was evaluated for .sup.111In-labeled DTPA-NT(6-13) analogues and for the reference peptide (FIG. 2, Table 4). In agreement with the in vivo results, the unprotected peptide DTPA(.sup.111In)-NT-20.1 was very rapidly degraded and DTPA(.sup.111In)-NT-20.3, a neurotensin analogue according to the invention, was more stable than DTPA(.sup.111In)-NT-20.2. These results confirmed the stabilizing effect of the two modifications. By contrast to the rapid degradation observed in vivo, the unprotected [Lys.sup.6(DTPA(.sup.111In))]-NT displayed an in vitro stability higher than that of the mono-stabilized DTPA(.sup.111In)-NT-20.2. These results point out the discrepancies that could occur between in vitro and in vivo degradation even when low tracer amounts are used in vitro in order to avoid saturation of peptidases (Garcia-Garayoa et al., Nucl. Med. Biol. (2001) 28, 75-84).

(47) 4.2. In Vivo Stability

(48) Female BALB/c mice were injected in the tail vein with .sup.111In-labeled DTPA-NT analogues (25 pmol) or with .sup.111In-labeled DOTA-NT analogues (50 pmol). The mice were sacrificed 15 minutes after injection. Plasma and urine samples (50 μL) were added to 200 μL methanol and treated as above except for the DOTA-peptides for which elution was performed using, after 5 min 0% B, a linear 15-minute gradient from 0% to 35% B and a linear 25-minute gradient from 35% to 50%, flow rate 1.5 mL/min.

(49) TABLE-US-00004 TABLE 4 In vitro and in vivo stability of DTPA-peptides. In vitro In vivo stability stability (% intact peptide).sup.b Peptide (t.sub.1/2 h).sup.a in plasma in urine [Lys.sup.6(DTPA(In))]-NT 25 ± 2 4 (3-5) 0 DTPA(In)-NT-VI ND 10 (5-15) 14.5 (15-14)   DTPA(In)-NT-XI ND  47 (40-53) 21 (26-16) DTPA(In)-NT-20.1  0.4 ± 0.02   0.8 (0.8-0.8) 0 DTPA(In)-NT-20.2  4.4 ± 0.6 10 (6-14) 0 DTPA(In)-NT-20.3 257 ± 71 26.5 (26-27)    24 (23-31-19) .sup.aIn vitro stability is expressed as the degradation half-life in human serum at 37° C. .sup.bIn vivo stability is expressed as the % intact peptide (mean (individual values)) recovered in plasma or urine 15 min after tracer injection

(50) TABLE-US-00005 TABLE 5 In vivo stability of DOTA-peptides. In vivo stability (% intact peptide).sup.a Peptide in plasma in urine DOTA(In)-NT-20.3 21 ± 2 26 ± 6 DOTA(In)-LB119 28 ± 3 ND .sup.aIn vivo stability is expressed as the % of radioactivity associated to intact peptide (mean ± sem) recovered in plasma 15 min after tracer injection (n = 3-4)

(51) The fraction of radioactivity associated to the intact .sup.111In-labeled peptide in serum and in urine determined 15 minutes after iv injection to BALB/c mice are presented in Table 4 and 5. Metabolites eluted by C.sub.18 RP-HPLC chromatography at shorter retention times than the radioactive full-length peptide. The non-stabilized peptides [Lys.sup.6(DTPA(In))]-NT and DTPA(.sup.111In)-NT-20.1 were rapidly catabolized (FIG. 1, Table 4). Protection of Arg.sup.8-Arg.sup.9 (DTPA(.sup.111In)-NT-VI) or Tyr.sup.11-Ile.sup.12 (DTPA(.sup.111In)-NT-20.2) bonds improved the stability. Peptides with two or three sequence modifications were much more resistant (Table 4-5). Higher amounts of intact tracer were recovered in serum and about 20% of the intact peptide was excreted in urine.

(52) 6. Biodistribution and Imaging Studies

(53) 6.1 Biodistribution and Imaging Studies: Materials and Methods

(54) All in vivo experiments were performed in compliance with the French guidelines for experimental animal studies and fulfill the UKCCCR guidelines for the welfare of animals in experimental neoplasia.

(55) HT29 cells (6.7×10.sup.5 cells) were injected subcutaneously in the flank of 6-8 week old athymic nu/nu mice, (Harlan, France). Two weeks later mice were i.v. injected with .sup.111In-labeled DTPA-NT analogues (20-50 pmol in 100 μL PBS) or DOTA-analogues (40-65 pmol, 0.5-0.7 MBq, except for mice dissected 49 h post injection: 500-900 pmol, 7-12 MBq) and sacrificed at different times. Blood, organs and tumors were collected, weighted and radioactivity was counted. Injected activity was corrected for losses by subtraction of non-injected and subcutaneously injected material (remaining in the animal tail). In blocked experiments each mouse received a co-injection of the labeled peptide and of its unlabeled counterpart (60 nmol of NT for [Lys.sup.6(DTPA(In))]-NT or 180 nmol of NT-20.3 for DTPA-NT-20.3, DOTA-NT-20.3 and of LB119 (Ac-Lys(Ahx)-Pro-Me-Arg-Arg-Pro-Dmt-Tle-Leu-OH) for DOTA-LB119). Statistical analysis of differences in the tissue uptake values was performed using unpaired t test for comparison between two groups, or ANOVA variance analysis followed by Newman-Keuls' test for multiple comparisons. Differences of p<0.05 were considered significant.

(56) Scintigraphic imaging was performed under pentobarbital anesthesia after iv injection of the .sup.11In-labeled analogue (DTPA(.sup.111In)-NT-20.3: 30-50 pmol, 9-13 MBq, DOTA-NT analogues: 500-900 pmol, 7-12 MBq) using a dedicated small animal Gamma Imager-S/CT system (Biospace Mesures) equipped with parallel collimators (matrix 128×128, with 15% energy windows centered on both indium-111 peaks at 171 and 245 KeV). SPECT images (1 h acquisition) were obtained after volume reconstruction using an iterative algorithm. Tumor to background activity (evaluated in a ROI symmetrical to that of the tumor, counts per mm.sup.2) ratio was evaluated on planar images. Radioactivity excretion in urine was determined from activity at 1 h post-injection in the bladder.

(57) 6.2 Results of Biodistribution and Imaging Studies of the DTPA-NT Series

(58) The results of biodistribution studies of the DTPA-NT analogues, at 1 h and 3 h post-injection, performed in female nude mice grafted with HT29 cells are presented in tables 6 and 7. Biodistribution results of DTPA(.sup.111In)-NT-20.3 in female nude mice and in male nude mice from 1 h to 100 h after injection are presented in table 8 and table 9 respectively. They are expressed as the percentage of injected dose per gram of tissue (% ID/g).

(59) TABLE-US-00006 TABLE 6 Tissue distributions of [Lys.sup.6(DTPA(.sup.111In))]-NT and the DTPA(.sup.111In)-NT(8-13) analogues in female nude mice grafted with HT29 cells. DTPA(.sup.111In)- DTPA(.sup.111In)- [Lys6(DTPA(.sup.111In))]-NT NT-VI NT-XI 1 h 3 h 3 h blocked.sup.b 1 h 1 h n = 6 n = 9 n = 8 n = 3 n = 3 Uptake (% ID/g).sup.a Blood 0.63 ± 0.12 0.06 ± 0.01 0.04 ± 0.01 0.24 ± 0.13 0.28 ± 0.02 Lungs 0.44 ± 0.06 0.07 ± 0.01 0.07 ± 0.01 0.21 ± 0.06 0.37 ± 0.02 Liver 0.22 ± 0.03 0.16 ± 0.07 0.09 ± 0.01 0.14 ± 0.06 0.19 ± 0.02 Spleen 0.19 ± 0.02 0.07 ± 0.01 0.45 ± 0.37 0.10 ± 0.02 0.18 ± 0.01 Stomach.sup.c 2.46 ± 2.01 0.26 ± 0.15 0.15 ± 0.09 0.06 ± 0.02 0.14 ± 0.02 Small intestine.sup.c 0.69 ± 0.09 0.59 ± 0.30 0.20 ± 0.09 0.28 ± 0.05 0.38 ± 0.07 Large intestine.sup.c 0.16 ± 0.02 0.71 ± 0.16 1.05 ± 0.47 0.17 ± 0.04 0.19 ± 0.04 Muscle 0.14 ± 0.03 0.03 ± 0.01 0.03 ± 0.01 0.11 ± 0.04 0.16 ± 0.08 Bone 0.13 ± 0.03 0.06 ± 0.01 0.03 ± 0.01 0.17 ± 0.05 0.13 ± 0.03 Tumor 1.02 ± 0.26 0.71 ± 0.18 0.22 ± 0.02 0.62 ± 0.06 0.52 ± 0.23 Kidney 12.50 ± 1.63  9.28 ± 0.73 7.18 ± 0.48 2.80 ± 0.37 3.90 ± 0.59 Tumor(T)/organ T/Blood 3.3 ± 2.1 10.9 ± 1.7  5.7 ± 0.5 4.9 ± 2.5 2.0 ± 0.9 T/Liver 5.8 ± 2.3 9.3 ± 0.8 2.8 ± 0.6 6.2 ± 2.3 3.0 ± 1.4 T/Muscle 10.4 ± 4.8  33.1 ± 4.1  8.9 ± 1.3 9.6 ± 5.6 3.4 ± 1.6 T/Kidney 0.08 ± 0.02 0.11 ± 0.01 0.03 ± 0.01 0.20 ± 0.04 0.16 ± 0.07 .sup.aUptake is expressed as the percentage of injected dose per gram of tissue (% ID/g). .sup.bBlocked animals received a co-injection of the labeled peptide with neurotensin (60 nmol). .sup.cOrgan with its content.

(60) TABLE-US-00007 TABLE 7 Tissue distributions of the DTPA(.sup.111In)-NT(6-13) analogues in female nude mice grafted with HT29 cells. DTPA(.sup.111In)-NT-20.1 DTPA(.sup.111In)-NT-20.2 DTPA(.sup.111In)-NT-20.3 1 h 3 h 1 h 3 h 1 h 3 h 3 h blocked.sup.b Uptake (% ID/g).sup.a n = 3 n = 6 n = 5 n = 5 n = 6 n = 15 n = 4 Blood 0.19 ± 0.03 0.03 ± 0.00 0.31 ± 0.06 0.02 ± 0.01 0.70 ± 0.09 0.04 ± 0.01 0.04 ± 0.01 Lungs 0.17 ± 0.01 0.04 ± 0.01 0.30 ± 0.04 0.10 ± 0.04 0.73 ± 0.04 0.17 ± 0.03 0.12 ± 0.01 Liver 0.11 ± 0.01 0.06 ± 0.01 0.14 ± 0.01 0.07 ± 0.01 0.39 ± 0.04 0.17 ± 0.05 0.08 ± 0.01 Spleen 0.08 ± 0.01 0.05 ± 0.01 0.12 ± 0.01 0.06 ± 0.01 0.31 ± 0.01 0.11 ± 0.01 0.09 ± 0.01 Stomach (with 0.13 ± 0.04 0.02 ± 0.01 0.42 ± 0.17 0.04 ± 0.01 0.66 ± 0.19 0.17 ± 0.04 0.14 ± 0.04 content) Small intestine 0.53 ± 0.20 0.18 ± 0.04 1.07 ± 0.44 0.16 ± 0.02 1.90 ± 0.22 1.30 ± 0.46 0.18 ± 0.05 (with content) Large intestine 0.09 ± 0.01 1.65 ± 0.99 0.11 ± 0.02 0.46 ± 0.09 0.42 ± 0.05 1.03 ± 0.14 0.15 ± 0.04 (with content) Stomach ND ND ND ND ND 0.21 ± 0.03 0.09 ± 0.02 (without content) Small intestine ND ND ND ND ND 0.78 ± 0.10 0.10 ± 0.03 (without content) Large intestine ND ND ND ND ND 0.45 ± 0.04 0.09 ± 0.01 (without content) Muscle 0.07 ± 0.01 0.01 ± 0.01 0.07 ± 0.01 0.01 ± 0.01 0.16 ± 0.01 0.03 ± 0.01 0.04 ± 0.01 Bone 0.07 ± 0.01 0.03 ± 0.01 0.43 ± 0.22 0.03 ± 0.01 0.22 ± 0.05 0.11 ± 0.02 0.28 ± 0.11 Tumor 0.46 ± 0.06 0.49 ± 0.12 0.93 ± 0.32 0.46 ± 0.09 3.27 ± 0.21 2.38 ± 0.21 0.14 ± 0.03 Kidney 1.44 ± 0.25 1.36 ± 0.10 2.55 ± 0.24 1.97 ± 0.26 7.49 ± 0.54 4.85 ± 0.25 4.81 ± 0.63 Tumor(T)/organ T/Blood 2.5 ± 0.3 18.8 ± 4.7  4.6 ± 2.8 19.4 ± 3.7  5.6 ± 1.5 60.5 ± 6.8  3.7 ± 0.8 T/Liver 4.3 ± 0.4 8.5 ± 1.7 6.6 ± 2.1 6.5 ± 0.9 8.8 ± 0.6 19.1 ± 1.5  1.7 ± 0.2 T/Muscle 7.1 ± 1.9 35.6 ± 8.3  14.8 ± 6.7  34.0 ± 8.0  20.8 ± 1.4  91.6 ± 8.6  4.2 ± 1.0 T/Pancreas ND ND ND ND 17.5 ± 0.8  68.2 ± 6.5  ND T/Kidney 0.32 ± 0.02 0.37 ± 0.04 0.35 ± 0.08 0.23 ± 0.01 0.44 ± 0.03 0.49 ± 0.04 0.03 ± 0.01 .sup.aUptake is expressed as the percentage of injected dose per gram of tissue (% ID/g). .sup.bBlocked animals received a co-injection of the labeled peptide with NT-20.3 (180 nmol).

(61) DTPA(.sup.111In)-NT-20.3, which is in the DTPA-neurotensin series the only neurotensin analogue according to the present invention, displayed the highest tumor uptake as compared to other DTPA-NT analogues, about 3 fold higher than that of [Lys.sup.6(DTPA(.sup.111In))]-NT at 1 h (3.3±0.2 vs 1.0±0.3% ID/g, P<0.001) and at 3 h (2.4±0.2 vs 0.7±0.2% ID/g, P<0.001). Radioactivity uptake of other peptides in tumor was much lower. Particularly, DTPA(.sup.111In)-NT-20.2 with only one sequence modification displayed low tumor uptake though the chelating agent was separated from Aa8 by a chain of 11 consecutive bonds.

(62) The difference observed between tumor retention at 1 h and 3 h post-injection for DTPA(.sup.111In)-NT-20.3 was not statistically significant, indicating a slow wash out of radioactivity from the tumor, confirmed by the 0.33±0.04% ID/g tumor uptake observed 100 h post-injection (Table 6).

(63) Tumor uptake of [Lys.sup.6(DTPA(.sup.111In))]-NT or DTPA(.sup.111In)-NT-20.3 was receptor mediated since it was significantly reduced by co-injection of their unlabeled counterpart (78% reduction, P=0.02 and 94% reduction, P<0.0001 respectively).

(64) Radioactivity in blood at 1 h post-injection was significantly higher for DTPA(.sup.111In)-NT-20.3 and [Lys.sup.6(DTPA(.sup.111In))]-NT than for other peptides. It decreased rapidly with time for both peptides. Radioactivity excretion in urine was fast and amounted 69±4% of the injected dose 1 h after injection for DTPA(.sup.111In)-NT-20.3. Low activity accretion was observed in normal tissues for all peptides except in kidneys and, particularly for DTPA(.sup.111In)-NT-20.3, in gastrointestinal tract. Nevertheless, for DTPA(.sup.111In)-NT-20.3, high uptake ratios were obtained between tumor and stomach (7.2±1.7 at 1 h and 30±7 at 3 h), small intestine (1.8±0.2 and 3.5±0.6) and colon (8.3±0.8 and 3.0±0.5).

(65) The basis of the gastrointestinal uptake of DTPA(.sup.111In)-NT-20.3 (Table 7) has been investigated. In contrast to colon uptake, which was significantly decreased by co-injection of the unlabeled analogue (P=0.004), stomach and small intestine uptakes were not significantly reduced by the co-injection, despite the expression of NTS1 in these organs. Most of the activity was associated to the content of the organs (stomach: 68±4%, small intestine: 59±6%, colon 73±6%) indicating an elimination by the gastrointestinal route. When organ content was removed, co-injection of DTPA(.sup.111In)-NT-20.3 with its unlabeled counterpart significantly decreased uptake at 3 h post-injection in stomach (P=0.04), in small intestine (P=0.001), and in colon (P=0.0002). These results suggest that some uptake in these tissues is receptor mediated, but most of the activity comes from gastrointestinal elimination.

(66) Kidney uptake of DTPA(In)-NT-20.3 in female nude mice was significantly lower than that of [Lys.sup.6(DTPA(In))]-NT and significantly higher than that of other tested peptides at 1 h and 3 h post-injection, with the exception of DTPA(.sup.111In)-NT-XI for which the difference was not significant. DTPA(.sup.111In)-NT-20.1 displayed the lowest renal accretion of the peptides tested in this DTPA series with 1.4±0.25% ID/g as soon as 1 h post injection.

(67) Charge and charge distribution of radiolabeled peptides may produce various effects on renal uptake, but in general it is increased by positive charges (Akizawa et al., Nucl. Med. Biol. (2001) 28, 761-768; Froidevaux et al., J. Nucl. Med. (2005) 46, 887-895). One objective of the present invention was to lower kidney uptake as compared to the reference peptide. [Lys.sup.6(DTPA(.sup.111In))]-NT may, after cleavage in the 1-6 N-terminal end, release labeled metabolites with a free positively charged α-NH.sub.2, which could contribute to the high kidney uptake. To avoid the formation of these metabolites, the 1-6 N-terminal part of the molecule has been deleted and its N-terminal end has been acetylated to neutralize the positive charge. Cleavage of DTPA(.sup.111In)-NT-20.1 at the Arg.sup.8-Arg.sup.9 bond may produce labeled metabolites with only one positive charge (Arg.sup.8).

(68) The same is true for DTPA(.sup.111In)-NT-20.2, which also exhibits low renal uptake. In DTPA(.sup.111In)-NT-20.3, the Arg.sup.8-Arg.sup.9 bond is stabilized. Thus, a higher renal accumulation of radioactivity may be introduced by the release of metabolites with two positively charged Arg.

(69) For DTPA(.sup.111In)-NT-20.3, tumor to normal tissues uptake ratios were elevated for most organs, particularly tumor/pancreas ratio was 17.5±0.8 and 68.2±6.5 at 1 and 3 h post injection respectively. They were markedly improved as compared to [Lys.sup.6(DTPA(.sup.111In))]-NT particularly tumor/blood (60.5±6.8 vs 10.9±1.7 P<0.0001 at 3 h post-injection), tumor/liver (19.1±1.5 vs 9.3±0.8 P<0.0001) and tumor/muscle (91.6±8.6 vs 33.1±4.1 P<0.0001). Tumor to kidney uptake ratio was also improved about five fold (0.49±0.04 vs 0.11±0.01 P<0.0001, 3 h post-injection) as a result of higher radioactivity uptake in tumor and lower accretion in kidney for DTPA(.sup.111In)-NT-20.3.

(70) DTPA(.sup.111In)-NT-20.3, as compared to DTPA-neurotensin conjugates previously described in the literature provided in male mice higher tumor uptake and/or higher tumor to kidneys uptake ratios at early times post injection.

(71) Accumulation of DTPA(.sup.111In)-NT-20.3 was clearly observed in tumors in planar at early (FIG. 4) and late time points: 24, 48 and 100 h (not shown) post-injection and in tomographic images recorded in male mice (FIG. 5). Kidneys and bladder were the only other sites of activity accumulation. Tumor was detected as soon as 30 minutes post-injection on sequential 5 minutes acquisition images. Tumor-to-background ratio increased with time reaching 2.8±0.7 at 1 h and 4.5±1.0 at 24 h. At 24 h, the activity ratio between tumor and kidneys was 1.3±0.4 (tumor weight: 0.428±0.095 g).

(72) 6.3 Results of Biodistribution and Imaging Studies of the DOTA-NT Series

(73) Biodistribution studies of neurotensin analogues according to the invention DOTA(.sup.111In)-NT-20.3 (Table 10), the DOTA analogue of DTPA(.sup.111In)-NT-20.3, and DOTA(.sup.111In)-LB119 (Table 11) were also performed at various time post injection in male nude mice. No significant difference was observed between tumor accretion of DTPA(.sup.111In)-NT-20.3 and of DOTA(.sup.111In)-NT-20.3 at any time post-injection (Anova and Student-Newman-Keuls Multiple Comparisons Test), indicating similar tumor targeting efficacy of these two peptides. In the DOTA-NT series at early times post-injection DOTA(.sup.111In)-NT-20.3 displayed an higher tumor uptake than DOTA(.sup.111In)-LB119 (1 h and 3 h P<0.05), but DOTA(.sup.111In)-LB119 tumor uptake decreased slowly with time and from 6 h to 24 h no significant difference was observed between these two peptides.

(74) Renal accumulation of radioactivity was lower for DOTA(.sup.111In)-LB119 than for DOTA(.sup.111In)-NT20.3 at early times (P<0.05 from 1 to 6 h).

(75) DOTA(.sup.111In)-NT-20.3 and DOTA(.sup.111In)LB119 as compared to DOTA neurotensin conjugates previously described in the literature, provided in male mice higher tumor uptake and/or higher tumor to normal tissue uptake ratios, particularly higher tumor to kidneys uptake ratios at early times post injection.

(76) The efficacy of DOTA-NT-20.3 to target 68Ga in vivo to tumors expressing the NTSR1 receptor is shown by the TEP images recorded with this peptide (FIG. 7).

(77) TABLE-US-00008 TABLE 8 Tissue distributions of DTPA(.sup.111In)-NT(20.3) in female nude mice grafted with HT29 cells from 1 h to 100 h. DTPA- NT-20.3 1 h 3 h 3 h blocked 6 h 24 h 48 h 100 h Blood 0.70 ± 0.09 0.043 ± 0.005 0.039 ± 0.002 0.028 ± 0.002 0.012 ± 0.001 0.0055 ± 0.0007 0.0029 ± 0.0005 Lungs 0.73 ± 0.04 0.17 ± 0.03 0.12 ± 0.01 0.10 ± 0.01 0.10 ± 0.01 0.048 ± 0.007 0.029 ± 0.002 Liver 0.39 ± 0.04 0.17 ± 0.06 0.079 ± 0.008 0.10 ± 0.01 0.081 ± 0.006 0.082 ± 0.008 0.050 ± 0.002 Spleen 0.31 ± 0.01 0.11 ± 0.01 0.092 ± 0.008 0.10 ± 0.01 0.12 ± 0.02 0.089 ± 0.014 0.055 ± 0.003 Stomach 0.66 ± 0.19 0.17 ± 0.04 0.14 ± 0.04 0.14 ± 0.03 0.10 ± 0.02 0.047 ± 0.005 0.022 ± 0.004 Small 1.90 ± 0.22 1.30 ± 0.46 0.18 ± 0.05 0.42 ± 0.04 0.34 ± 0.04 0.23 ± 0.02 0.13 ± 0.01 intestine Large 0.42 ± 0.05 1.03 ± 0.14 0.15 ± 0.04 0.51 ± 0.19 0.36 ± 0.11 0.17 ± 0.03 0.23 ± 0.11 intestine Muscle 0.16 ± 0.01 0.029 ± 0.004 0.042 ± 0.014 0.021 ± 0.002 0.020 ± 0.002 0.011 ± 0.003 0.0095 ± 0.0012 Bone 0.22 ± 0.05 0.11 ± 0.03 0.27 ± 0.11 0.068 ± 0.010 0.065 ± 0.013 0.054 ± 0.009 0.028 ± 0.004 Tumor 3.27 ± 0.21 2.38 ± 0.21 0.14 ± 0.03 1.63 ± 0.19 1.41 ± 0.21 0.55 ± 0.07 0.33 ± 0.04 Kidney 7.49 ± 0.54 4.85 ± 0.25 4.81 ± 0.63 3.62 ± 0.38 3.13 ± 0.43 1.80 ± 0.38 0.83 ± 0.05 Pancreas 0.19 ± 0.01 0.032 0.001 tumor 0.48 ± 0.10  0.26 ± 0.046 0.0956 ± 0.028  0.49 ± 0.10  0.40 ± 0.041  0.1972 ± 0.02133  0.2327 ± 0.06114 weight

(78) TABLE-US-00009 TABLE 9 Tissue distributions of DTPA(.sup.111In)-NT(20.3) in male nude mice grafted with HT29 cells from 1 h to 100 h. DTPA-NT-20.3 1 h 3 h 6 h 24 h 48 h 100 h Blood 0.13 ± 0.03 0.026 ± 0.004 0.023 ± 0.004 0.0076 ± 0.0008 0.0028 ± 0.0005 0.0017 ± 0.0006 Lungs 0.63 ± 0.35 0.11 ± 0.01 0.13 ± 0.04 0.044 ± 0.003 0.035 ± 0.003 0.18 ± 0.05 Liver 0.12 ± 0.01 0.093 ± 0.008 0.07 ± 0.01 0.048 ± 0.003 0.040 ± 0.002 0.063 ± 0.007 Spleen 0.13 ± 0.01 0.13 ± 0.02 0.10 ± 0.01 0.074 ± 0.004 0.064 ± 0.004 0.106 ± 0.017 Stomach 0.14 ± 0.03 0.076 ± 0.015 0.15 ± 0.05 0.27 ± 0.08 0.033 ± 0.008 0.018 ± 0.003 Small intestine 1.05 ± 0.37 0.47 ± 0.13 0.61 ± 0.09 0.38 ± 0.05 0.19 ± 0.01 0.098 ± 0.014 Large intestine 0.46 ± 0.15 0.23 ± 0.03 1.38 ± 0.23 0.81 ± 0.13 0.15 ± 0.03 0.037 ± 0.005 Muscle 0.52 ± 0.42 0.14 ± 0.09 0.023 ± 0.005 0.023 ± 0.007 0.016 ± 0.004 0.0083 ± 0.0027 Bone 0.20 ± 0.06  0.10 ± 0.020 0.083 ± 0.013 0.044 ± 0.003 0.027 ± 0.004 0.036 ± 0.005 Tumor 3.05 ± 0.36 1.99 ± 0.39 2.00 ± 0.24 0.86 ± 0.06 0.92 ± 0.13 0.38 ± 0.01 Kidney 7.79 ± 1.00 6.54 ± 1.69 2.84 ± 0.32 1.89 ± 0.33 1.50 ± 0.34 0.48 ± 0.08 tumor weight 0.26 ± 0.06 0.35 ± 0.04 0.40 ± 0.07  0.40 ± 0.042 0.65 ± 0.19 0.40 ± 0.07

(79) TABLE-US-00010 TABLE 10 Tissue distributions of DOTA(.sup.111In)-NT(20.3) in male nude mice grafted with HT29 cells from 1 h to 49 h. DOTA-NT-20.3 1 h 3 h 3 h blocked 4 h 30 6 h 24 h 49 h Blood 0.36 ± 0.06 0.033 ± 0.014 0.13 ± 0.01 0.015 ± 0.001 0.038 ± 0.012 0.0028 ± 0.0003 0.0028 ± 0.0004 Lungs 0.47 ± 0.04 0.14 ± 0.02 0.16 ± 0.01 0.12 ± 0.01 0.11 ± 0.01 0.061 ± 0.004 0.068 ± 0.011 Liver 0.21 ± 0.02 0.13 ± 0.02 0.12 ± 0.01 0.14 ± 0.01 0.12 ± 0.01 0.085 ± 0.002 0.072 ± 0.013 Spleen 0.19 ± 0.01 0.11 ± 0.01 0.10 ± 0.01 0.11 ± 0.01 0.11 ± 0.01 0.10 ± 0.01 0.16 ± 0.01 Stomach 0.13 ± 0.03 0.22 ± 0.12 0.081 ± 0.017 0.12 ± 0.03 0.092 ± 0.037 0.057 ± 0.013 0.020 ± 0.005 Small intestine 0.85 ± 0.10 0.52 ± 0.08 0.12 ± 0.02 0.58 ± 0.05 0.34 ± 0.06 0.32 ± 0.02 0.070 ± 0.003 Large intestine 0.39 ± 0.05 1.13 ± 0.30 0.18 ± 0.08 2.22 ± 0.96 1.47 ± 0.45 0.19 ± 0.02 0.058 ± 0.007 Muscle 0.10 ± 0.02 0.027 ± 0.008 0.053 ± 0.013 0.022 ± 0.005 0.042 ± 0.011 0.012 ± 0.001 0.008 ± 0.001 Bone 0.15 ± 0.02 0.10 ± 0.02 0.13 ± 0.03 0.056 ± 0.016 0.10 ± 0.01 0.030 ± 0.005 0.053 ± 0.002 Tumor 4.72 ± 0.76 2.48 ± 0.19 0.14 ± 0.02 2.40 ± 0.21 1.86 ± 0.20 1.26 ± 0.15 0.68 ± 0.09 Kidney 7.55 ± 0.85 4.89 ± 0.40 6.70 ± 0.23 4.07 ± 0.28 5.16 ± 0.47 2.50 ± 0.12 0.86 ± 0.08 pancreas 0.10 0.01 0.033 ± 0.010 0.028 ± 0.001 0.030 ± 0.002 tumor weight 0.20 ± 0.03 0.185 ± 0.036 0.055 ± 0.010 0.14 ± 0.03 0.11 ± 0.03 0.21 ± 0.03 0.148 ± 0.042

(80) TABLE-US-00011 TABLE 11 Tissue distributions of DOTA(.sup.111-In)-LB119 in male nude mice grafted with HT29 cells from 1 h to 49 h. DOTA-LB119 1 h 3 h 3 h blocked 6 h 24 h 49 h Blood 0.38 ± 0.05 0.023 ± 0.002 0.042 ± 0.008 0.0045 ± 0.0002 0.0074 ± 0.0018 0.0021 ± 0.0003 Lungs 0.36 ± 0.03 0.11 ± 0.01 0.086 ± 0.010 0.086 ± 0.014 0.060 ± 0.013 0.041 ± 0.010 Liver 0.20 ± 0.01 0.15 ± 0.01 0.077 ± 0.005 0.14 ± 0.02 0.080 ± 0.005 0.089 ± 0.026 Spleen 0.15 ± 0.01 0.087 ± 0.011 0.077 ± 0.007 0.076 ± 0.009 0.064 ± 0.003 0.095 ± 0.027 Stomach 0.28 ± 0.08 0.16 ± 0.04 0.37 ± 0.21 0.52 ± 0.46 0.080 ± 0.013 0.024 ± 0.004 Small intestine 1.11 ± 0.10 0.67 ± 0.08 0.50 ± 0.19 0.69 ± 0.14 0.35 ± 0.05 0.084 ± 0.005 Large intestine 0.44 ± 0.12 1.54 ± 0.53 0.17 ± 0.07 1.24 ± 0.81 0.16 ± 0.03 0.10 ± 0.01 Muscle 0.094 ± 0.013 0.021 ± 0.005 0.03 ± 0.01 0.049 ± 0.023 0.010 ± 0.003 0.015 ± 0.007 Bone 0.15 ± 0.03 0.047 ± 0.011 0.10 ± 0.05 0.068 ± 0.018 0.053 ± 0.010 0.049 ± 0.020 Tumor 1.83 ± 0.13 1.41 ± 0.05 0.12 ± 0.03 1.35 ± 0.18 0.98 ± 0.27 0.46 ± 0.06 Kidney 3.37 ± 0.20 2.40 ± 0.21 2.18 ± 0.19 2.15 ± 0.19 1.04 ± 0.07 0.64 ± 0.12 Pancreas 0.081 ± 0.008 0.022 ± 0.001 0.026 ± 0.006 0.018 ± 0.001 0.018 ± 0.001 0.013 ± 0.002 tumor weight 0.124 ± 0.019 0.306 ± 0.030 0.181 ± 0.018 0.121 ± 0.032 0.202 ± 0.095 0.195 ± 0.051