Enhanced in Vivo Targeting of Radiolabelled Peptides With the Means of Enzyme Inhibitors

Abstract

The present invention relates to a compound that inhibits the activity of a degrading enzyme for use in combination with a therapeutic or diagnostic compound, preferably a moiety conjugated peptide, in the diagnosis and/or treatment of a disease, in particular cancer, to enhance targeting of the therapeutic or diagnostic compound to the disease site.

Claims

1-5. (canceled)

6. A composition comprising: a radiolabeled biodegradable peptide analog and a neutral endopeptidase (NEP) inhibitor; a radiolabeled biodegradable peptide analog and an angiotensin-converting enzyme (ACE) inhibitor, or a radiolabeled biodegradable peptide analog and both an NEP inhibitor and an ACE inhibitor, wherein the peptide-portion of the radiolabeled biodegradable peptide analog is selected from the group consisting of somatostatins, gastrins, CCKs, neurotensins, bombesins, neuromedin C or GRP analogs, melanocyte stimulating hormones, chemotactic peptides, and neuropeptides, and wherein the composition is a therapeutic or diagnostic compound.

7. The composition of claim 6, wherein the neutral endopeptidase (NEP) inhibitor comprises phosphoramidon or racecadotril.

8. The composition of claim 6, wherein the angiotensin-converting enzyme (ACE) inhibitor comprises lisinopril.

9. The composition according to claim 6, wherein the radiolabeled biodegradable peptide analog is a radiometallated peptide-chelator conjugate or a peptide radiohalogenated via a prosthetic group.

10. The composition according to claim 6, wherein the radiolabeled biodegradable peptide analog comprises a radioactive label selected from the group consisting of Tc, Re, In, Ga, Cu, F, I, Lu, Y, Bi, and Ac.

11. A composition comprising a radiolabeled biodegradable peptide analog and a neutral endopeptidase (NEP) inhibitor or both an NEP inhibitor and an ACE inhibitor, wherein the composition comprises a combination selected from the group consisting of: [(X-DOTA)Ala.sup.1]SS-14 with a neutral endopeptidase inhibitor; [(X-DOTA)Ala.sup.1,DTrp.sup.8]SS-14 with a neutral endopeptidase inhibitor; X-DOTA-MG11 with a neutral endopeptidase inhibitor; X-Demotensin 6 with a neutral endopeptidase inhibitor; X-Demotensin 1 with a neutral endopeptidase inhibitor and an angiotensin converting enzyme inhibitor; X-SAR-NC1 with a neutral endopeptidase inhibitor; X-SAR-NC6 with a neutral endopeptidase inhibitor; X-Demobesin 4 with a neutral endopeptidase inhibitor; X-Pansarbesin 1 with a neutral endopeptidase inhibitor; X-Demobesin 1 with a neutral endopeptidase inhibitor; X-JMV4168 with a neutral endopeptidase inhibitor; X-SP-1 with a neutral endopeptidase inhibitor and an angiotensin converting enzyme inhibitor; X-SP-2/3 with a neutral endopeptidase inhibitor; X-MSH-1/2 with a neutral endopeptidase inhibitor; and X-CTP-1 with a neutral endopeptidase inhibitor, wherein X is a radionuclide, and wherein the composition is a therapeutic or diagnostic composition.

12. The composition according to claim 11, wherein the composition comprises a combination selected from the group consisting of: [(.sup.111In-DOTA)Ala.sup.1]SS14 with a neutral endopeptidase inhibitor; [(.sup.111In-DOTA)Ala.sup.1,DTrp.sup.8]SS14 with a neutral endopeptidase inhibitor; [.sup.99mTc]demotensin 6 with a neutral endopeptidase inhibitor; [.sup.99mTc]demotensin 1 with a neutral endopeptidase inhibitor and an angiotensin converting enzyme inhibitor; [.sup.99mTc]SAR-NC1 with a neutral endopeptidase inhibitor; [.sup.99mTc]SAR-NC6 with a neutral endopeptidase inhibitor; [.sup.99mTc]Demobesin 4 with a neutral endopeptidase inhibitor; [.sup.111In]Pansarbesin 1 with a neutral endopeptidase inhibitor; [.sup.99mTc]Demobesin 1 with a neutral endopeptidase inhibitor; [.sup.111In]JMV4168 with a neutral endopeptidase inhibitor; [.sup.111In]SP-1 with a neutral endopeptidase inhibitor and an angiotensin converting enzyme inhibitor; [.sup.111In]SP-2/3 with a neutral endopeptidase inhibitor; [.sup.111In]MSH-1/2 with a neutral endopeptidase inhibitor; and [.sup.111In]CTP-1 with a neutral endopeptidase inhibitor, wherein the composition is a therapeutic or diagnostic composition.

13. (canceled)

14. A method of treating and/or diagnosing cancer the method comprising co-administering: a radiolabeled biodegradable peptide analog with a neutral endopeptidase (NEP) inhibitor; a radiolabeled biodegradable peptide analog with an angiotensin converting enzyme (ACE) inhibitor; or a radiolabeled biodegradable peptide analog with both an NEP inhibitor and an ACE inhibitor, wherein the peptide-portion of the radiolabeled biodegradable peptide analog is selected from the group consisting of somatostatins, gastrins, CCKs, neurotensins, bombesins, neuromedin C or GRP analogs, melanocyte stimulating hormones, chemotactic peptides, and neuropeptides, and wherein the composition is a therapeutic or diagnostic compound.

15. The method according to claim 14, wherein the neutral endopeptidase inhibitor comprises phosphoramidon or racecadotril.

16. The method according to claim 14, wherein the angiotensin converting enzyme (ACE) inhibitor comprises lisinopril.

17. The method according to claim 14, wherein the administration of the radiolabeled biodegradable peptide analog is intravenous.

18. The method according to claim 14, wherein the radiolabeled biodegradable peptide analog is a radiometallated peptide-chelator conjugate or a peptide modified with a prosthetic group for incorporation of radiohalogens.

19. The method according to claim 14, wherein the radiolabeled biodegradable peptide analog comprises a radioactive label selected from the group consisting of Tc, Re, In, Ga, Cu, F, I, Lu, Y, Bi, and Ac.

20. The method according to claim 14, wherein the co-administration comprises: (i) administering the radiolabeled biodegradable peptide analog at the same time as the neutral endopeptidase (NEP) inhibitor, the angiotensin converting enzyme (ACE) inhibitor, or both the NEP inhibitor and an ACE inhibitor; (ii) first administering the therapeutic radiolabeled biodegradable peptide analog, followed by administering the neutral endopeptidase (NEP) inhibitor, the angiotensin converting enzyme (ACE) inhibitor, or both the NEP inhibitor and an ACE inhibitor; or (iii) first administering neutral endopeptidase (NEP) inhibitor the angiotensin converting enzyme (ACE) inhibitor, or both the NEP inhibitor and an ACE inhibitor, followed by administering the radiolabeled biodegradable peptide analog.

21. The method according to claim 14, wherein the combination of a radiolabeled biodegradable peptide analog and a neutral endopeptidase (NEP) inhibitor or both an NEP inhibitor and an ACE inhibitor is selected from the group consisting of: [(X-DOTA)Ala.sup.1]SS-14 with a neutral endopeptidase inhibitor; [(X-DOTA)Ala.sup.1,DTrp.sup.8]SS-14 with a neutral endopeptidase inhibitor; X-DOTA-MG11 with a neutral endopeptidase inhibitor; X-Demotensin 6 with a neutral endopeptidase inhibitor; X-Demotensin 1 with a neutral endopeptidase inhibitor and an angiotensin converting enzyme inhibitor; X-SAR-NC1 with a neutral endopeptidase inhibitor; X-SAR-NC6 with a neutral endopeptidase inhibitor; X-Demobesin 4 with a neutral endopeptidase inhibitor; X-Pansarbesin 1 with a neutral endopeptidase inhibitor; X-Demobesin 1 with a neutral endopeptidase inhibitor; X-JMV4168 with a neutral endopeptidase inhibitor; X-SP-1 with a neutral endopeptidase inhibitor and an angiotensin converting enzyme inhibitor; X-SP-2/3 with a neutral endopeptidase inhibitor; X-MSH-1/2 with a neutral endopeptidase inhibitor; X-CTP-1 with a neutral endopeptidase inhibitor, wherein X is a radionuclide, and wherein the composition is a therapeutic or diagnostic composition.

22. The method according to claim 14, wherein the combination comprises a combination selected from the group consisting of: [(.sup.111In-DOTA)Ala.sup.1] SS14 with a neutral endopeptidase inhibitor; [(.sup.111In-DOTA)Ala.sup.1,DTrp.sup.8]SS14 with a neutral endopeptidase inhibitor; .sup.111In-DOTA-MG11 with a neutral endopeptidase inhibitor; [.sup.99mTc]demotensin 6 with a neutral endopeptidase inhibitor; [.sup.99mTc]demotensin 1 with a neutral endopeptidase inhibitor and an angiotensin converting enzyme inhibitor; [.sup.99mTc]SAR-NC1 with a neutral endopeptidase inhibitor; [.sup.99mTc]SAR-NC6 with a neutral endopeptidase inhibitor; [.sup.99mTc]Demobesin 4 with a neutral endopeptidase inhibitor; [.sup.111In]Pansarbesin 1 with a neutral endopeptidase inhibitor; [.sup.99mTc]Demobesin 1 with a neutral endopeptidase inhibitor; [.sup.111In]JMV4168 with a neutral endopeptidase inhibitor; [.sup.111In]SP-1 with a neutral endopeptidase inhibitor and an angiotensin converting enzyme inhibitor; [.sup.111In]SP-2/3 with a neutral endopeptidase inhibitor; [.sup.111In]MSH-1/2 with a neutral endopeptidase inhibitor; and [.sup.111In]CTP-1 with a neutral endopeptidase inhibitor, wherein the composition is a therapeutic or diagnostic composition.

23. The composition according to claim 6, wherein the composition comprises a radiolabeled biodegradable peptide analog and a neutral endopeptidase (NEP) inhibitor.

24. The method according to claim 14, wherein the method comprises co-administering a radiolabeled biodegradable peptide analog and a neutral endopeptidase (NEP) inhibitor.

25. The composition according to claim 6, wherein the radiolabeled biodegradable peptide analog comprises a radionuclide metal or halogen selected from the group comprising .sup.111In, .sup.99mTc, .sup.94mTc, .sup.67Ga, .sup.66Ga, .sup.68Ga, .sup.52Fe, .sup.69Er, .sup.72As, .sup.97Ru, .sup.203Pb, .sup.62CU, .sup.64Cu, .sup.67Cu, .sup.186Re, .sup.188Re, .sup.86Y, .sup.90Y, .sup.51Cr, .sup.52mMn, .sup.157Gd, .sup.177Lu, .sup.161Tb, .sup.169Yb, .sup.175Yb, .sup.105Rh, .sup.166Dy, .sup.166Ho, .sup.153Sm, .sup.149Pm, .sup.151Pm, .sup.172Tm, .sup.121Sn, .sup.177mSn, .sup.213Bi, .sup.142Pr, .sup.143Pr, .sup.198Au, .sup.199Au, .sup.18F, .sup.123I, .sup.124I, .sup.131I, .sup.75Br, .sup.76Br, .sup.77Br, and .sup.82Br.

26. The method according to claim 14, wherein the radiolabeled biodegradable peptide analog comprises a radionuclide metal or halogen selected from the group comprising .sup.111In, .sup.99mTc, .sup.94mTc, .sup.67Ga, .sup.66Ga, .sup.68Ga, .sup.52Fe, .sup.69Er, .sup.72As, .sup.97Ru, .sup.203Pb, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.186Re, .sup.188Re, .sup.86Y, .sup.90Y, .sup.51Cr, .sup.52mMn, .sup.157Gd, .sup.177Lu, .sup.161Tb, .sup.169Yb, .sup.175Yb, .sup.105Rh, .sup.166Dy, .sup.166Ho, .sup.153Sm, .sup.149Pm, .sup.151Pm, .sup.172Tm, .sup.121Sn, .sup.177mSn, .sup.213Bi, .sup.142Pr, .sup.143Pr, .sup.198Au, .sup.199Au, .sup.18F, .sup.123I, .sup.124I, .sup.131I, .sup.75Br, .sup.76Br, .sup.77Br, and .sup.82Br.

Description

[0058] The invention will be further illustrated in the Examples that follow and that are not intended to limit the invention in any way. In the Examples reference is made to the following figures:

[0059] FIG. 1

[0060] A: Radiochromatogram of ex vivo mouse blood 5 min after injection of [(.sup.111In-DOTA)Ala.sup.1]Somatostatin-14 alone (upper panel) or with co-injection of phosphoramidon (PA 300 g)(middle panel) or 45 min after ip injection of racecadotril (race 2.5 mg) (lower panel). The percentage of parent peptide remaining intact by PA treatment increased from 2% to 85% and by race pretreatment to >65%.

[0061] B: Biodistribution of [(.sup.111In-DOTA)Ala]Somatostatin-14 in SCID mice bearing AR4-2J tumors (rsst.sub.2.sup.+) at 4 h pi. Bars represent average uptake as % injected dose per gram (% ID/g) of at least 4 animals with standard deviation (control at 4 h, 2.sup.nd bars); three additional groups of animals received either excess [Tyr.sup.3]octreotate (Tate, blockedfirst bars) or PA (3.sup.rd bars), or 2.5 mg race ip 40 min prior to radioligand injection (race.sub.4th bars). Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines, Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas, Fe=femur, Ad=adrenals and Tu=AR4-2J tumor. In the PA treated group, animals showed an uptake of 13.872.4% ID/g in the experimental tumor vs. 0.670.1% ID/g in the non-treated controls, while in the race group these values were 3.510.2% ID/g.

[0062] FIG. 2

[0063] A: Radiochromatogram of ex-vivo mouse blood 5 min after injection of [(.sup.111In-DOTA)Ala.sup.1,DTrp.sup.9]Somatostatin-14 alone (upper panel) or with PA (300 g, lower panel). The percentage of parent peptide remaining intact by PA treatment is raised from 6% to 95%.

[0064] B: Biodistribution of [(.sup.111In-DOTA)Ala.sup.1, DTrp.sup.8]Somatostatin-14 in SCID mice bearing AR4-2J tumors (rsst.sub.2+) at 4 h pi. Bars represent average uptake as mean % injected dose per gram (% ID/g) of at least 4 animals with standard deviation (control at 4 h 2.sup.nd bars); two additional groups of animals received either excess Tate (100 g, blockedfirst bars) or PA (300 g PA3.sup.rd bars) or were ip pre-injected with 2.5 mg race 1 h prior to radiotracer injection (race4.sup.th bars). Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines, Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas, Fe=femur and Tu=AR4-2J tumor. In the PA treated group, animals showed an uptake of 9.063.57% ID/g in the experimental tumor while in the race pretreated animals tumor uptake was 4.182.28% ID/g vs. 1.820.36% ID/g in the non-treated controls.

[0065] FIG. 3

[0066] A: Radiochromatogram of ex vivo mouse blood 5 min after injection of [(.sup.111In-DOTA) DGlu.sup.10]Minigastrin (10-17) (.sup.111In-DOTA-MG11), a truncated des-(Glu).sub.5-minigastrin analog, alone (upper panel) or with PA (300 g PAmiddle panel), or were pretreated with race (2.5 mg ip 1 h beforelower panel). The percentage of parent peptide remaining intact by PA treatment is raised from <5% to >70%.

[0067] B: Biodistribution of .sup.111In-DOTA-MG11 in SCID mice bearing AR4-2J tumors (rCCK2R.sup.+) at 4 h pi. Bars represent average uptake as mean % injected dose per gram (% ID/g) of at least 4 animals with standard deviation (control at 4 h1.sup.st bars); two additional groups of animals simultaneously received PA (600 g2.sup.nd bars) or were pretreated with race (2.5 mg ip 1 h before4.sup.th bars). Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines, Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas, Fe=femur and Tu=AR4-2J tumor. In the PA treated group, animals showed an uptake of 11.123.09% ID/g in the experimental tumor and in the race-pretreated group an uptake of 6.792.02% ID/g vs. 1.220.06% ID/g in the non-treated controls.

[0068] FIG. 4

[0069] A: Radiochromatogram of ex vivo mouse blood 5 min after injection of [.sup.99mTc]Demotensin 6 ([(.sup.99mTcN.sub.4) (Ala.sup.7, Dab.sup.9, Tle.sup.12]NT(7-13), .sup.99mTcN.sub.4-(Ala-Arg-Dab-Pro-Tyr-Tle-Leu-OH) alone (upper panel) or with PA (lower panel).

[0070] The percentage of parent peptide remaining intact by PA treatment is raised from 52% to >90%; scaling down the dose from 300 to 30 to 3 g did not significantly affect the protective action of PA on the peptide. However, by lowering the dose to 0.3 and 0.03 g PA the percentage of intact peptide dropped to >60% and >55%, respectively. It is interesting to observe that co-injection of the NEP-inhibitor PA (300 g) and the ACE-inhibitor Lisinopril (Lis250 g) together with the radioligand did not further increased stability versus coinjection of the radioligand with PA alone, suggesting that ACE is not involved in the catabolism of partially stabilized [.sup.99mTc]Demotensin 6

[0071] B: Biodistribution of [.sup.99mTc]Demotensin 6 in SCID mice bearing WiDr tumors (hNTS1.sup.+) at 4 h pi. Bars represent average uptake as mean % injected dose per gram (% ID/g) of at least 4 animals with standard deviation (control at 4 h 2.sup.nd bars); three additional groups of animals received either excess NT(1-13) and PA (100 g blocker and 300 g PAfirst bars), or PA (300 g PA3.sup.rd bars) or PA and Lis (300 g PA+250 g Lis4.sup.th bars). Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines, Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas, Fe=femur and Tu=WiDr tumor. In the PA treated group, animals showed an uptake of 3.560.38% ID/g in the experimental tumor while in the PA+Lis co-injected animals tumor uptake remained at this level 3.500.34% ID/g vs. 1.610.42% ID/g in the non-treated controls. It is interesting to note that blockade was very effective in the presence of PA (0.380.15% ID/g).

[0072] FIG. 5

[0073] A: Radiochromatogram of ex vivo mouse blood 5 min after injection of [.sup.99mTc]Demotensin 1 ([(.sup.99mTcN.sub.4)Gly.sup.7]NT(7-13), .sup.99mTcN.sub.4-Gly-Arg-Arg-Pro-Tyr-Ile-Leu-OH) alone (upper panel) or with PA (600 g or 300 g PA, lower panels). The percentage of parent peptide remaining intact by PA treatment is raised from <1% to >25%; scaling down the PA dose to 30 g resulted in only 4.5% of the original peptide surviving the first 5 min after entry into circulation. Co-injection of the radiopeptide with a cocktail of the NEP-inhibitor PA (+300 g) and the ACE inhibitor Lis (+300 g) raised this percentage to 56%, implying a role for ACE in the catabolism. Co-injection of Lis (+300 g) alone increased the amount of surviving radiopeptide to only just above 16%. On the other hand pretreatment with race (2.5 mg ip 40 min prior to radioligand injection) raised the intact peptide percentage to 37%.

[0074] B: Biodistribution of [.sup.99mTc]Demotensin 1 in SCID mice bearing WiDr tumors (hNTS1) at 4 h pi. Bars represent average uptake as mean % injected dose per gram (% ID/g) of at least 4 animals with standard deviation (control at 4 h 2.sup.nd bars); three additional groups of animals received either excess NT and PA (100 g and 300 g PA, respectivelyfirst bars), or PA (300 g PA3.sup.rd bars) or PA and Lis (300 g PA+300 g Lis4.sup.th bars). Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines, Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas, Fe=femur and Tu=WiDr tumor. In the PA treated group, animals showed an uptake of 4.580.47% ID/g in the experimental tumor while in the PA+Lis co-injected animals tumor uptake was raised to 7.711.19% ID/g vs. 1.200.21% ID/g in the non-treated controls. It is interesting to note that blockade was very effective in the presence of PA (0.230.09% ID/g).

[0075] FIG. 6

[0076] A: Radiochromatogram of ex vivo mouse blood 5 min after injection of [.sup.99mTc]SAR-NC1 ([(.sup.99mTcN.sub.4)Gly.sup.1]NMC, [(.sup.99mTcN.sub.4-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2), alone (upper panel) or with PA (300 glower panel). The percentage of parent peptide remaining intact by PA treatment is raised from 30% to 68%.

[0077] B: Biodistribution of [.sup.99mTc]SAR-NC1 in SCID mice bearing human prostate adenocarcinoma PC-3 xenografts (GRPR.sup.+) at 4 h pi. Bars represent average uptake as % injected dose per gram (% ID/g) of at least 4 animals with standard deviation (control at 4 h2.sup.nd bars); two additional groups of animals received either PA (300 g3.sup.rd bar) or excess [Tyr.sup.4]BBN (100 gfirst bars) along with the radioligand. Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines, Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas, Fe=femur and Tu=PC-3 tumor. In the PA treated group, animals showed an uptake of 28.348.05% ID/g in the experimental tumor vs. 6.511.91% ID/g in the non-treated controls.

[0078] FIG. 7

[0079] A: Radiochromatogram of ex vivo mouse blood 5 min after injection of [.sup.99mTc]SAR-NC6 ([(.sup.99mTcN.sub.4)Gly.sup.1, Sar.sup.7]NMC, [(.sup.99mTcN-Gly-Asn-His-Trp-Ala-Val-Sar-His-Leu-Met-NH.sub.2), alone (upper panel) or with PA (300 glower panel). The percentage of parent peptide remaining intact by PA treatment is raised from 35% to 70%.

[0080] B: Biodistribution of [.sup.99mTc]SAR-NC6 in SCID mice bearing human prostate adenocarcinoma PC-3 xenografts (GRPR.sup.+) at 4 h pi. Bars represent average uptake as % injected dose per gram (% ID/g) of at least 4 animals with standard deviation (control at 4 h2.sup.nd bars); two additional groups of animals received either PA (300 g3.sup.rd bar) or excess [Tyr.sup.4]BBN (100 gfirst bars) along with the radioligand. Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines, Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas, Fe=femur and Tu=PC-3 tumor. In the PA treated group, animals showed an uptake of 27.583.47% ID/g in the experimental tumor vs. 9.221.40% ID/g in the non-treated controls.

[0081] FIG. 8

[0082] A: Radiochromatogram of ex vivo mouse blood 5 min after injection of [.sup.99mTc]Demobesin 4 (.sup.99mTcN.sub.4-Pro-Gln-Arg-Tyr-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH.sub.2), alone (upper panel) or after iv co-injection of PA (30, 3 and 0.3 g) (following panels). The percentage of parent peptide remaining intact by PA treatment is raised from 26% to >77%, >63% and 30%, respectively.

[0083] B: Biodistribution of [.sup.99mTc]Demobesin 4 in SCID mice bearing human prostate adenocarcinoma PC-3 xenografts (GRPR.sup.+) at 4 h pi. Bars represent average uptake as % injected dose per gram (% ID/g) of at least 4 animals with standard deviation (control at 4 hfirst bars); an additional group of animals received PA (300 g2.sup.nd bar) along with the radioligand. Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines, Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas, Fe=femur and Tu=PC-3 tumor. In the PA treated group, animals showed an uptake of 35.507.50% ID/g in the experimental tumor vs. 11.261.81% ID/g in the non-treated controls.

[0084] FIG. 9

[0085] A: Radiochromatogram of ex vivo mouse blood 5 min after injection of [.sup.111In]PanSarbesin 1 ([(.sup.111In-DOTA-PEG.sub.2-DTyr-Gln-Trp-Ala-Val-Ala-His-Phe-Nle-NH.sub.2) alone (upper panel) or with PA (300 glower panel). The percentage of parent peptide remaining intact by PA treatment is raised from 13% to 80%.

[0086] B: Biodistribution of [.sup.111In]PanSarbesin 1 in SCID mice bearing human prostate adenocarcinoma PC-3 xenografts (GRPR.sup.+) at 4 h pi. Bars represent average uptake as % injected dose per gram (% ID/g) of at least 4 animals with standard deviation (control at 4 h2.sup.nd bars); two additional groups of animals received either PA (300 g3.sup.rd bar) or excess [Tyr.sup.4]BBN (100 g) in addition to PA (300 gfirst bars) along with the radioligand. Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines, Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas, Fe=femur and Tu=PC-3 tumor. In the PA treated group, animals showed an uptake of 20.962.58% ID/g in the experimental tumor vs. 3.750.73% ID/g in the non-treated controls. It is interesting to note that with co-injection of the blocker and PA (1.sup.st bar) tumor values were minimal (0.690.03% ID/g).

[0087] FIG. 10

[0088] A: Radiochromatogram of ex vivo mouse blood 5 min after injection of [.sup.99mTc]Demobesin 1 ([(.sup.99mTcN.sub.4) (p-aminobenzyl-diglycolic acid)-[DPhe.sup.6, LeuNHEt.sup.13]BBN (6-13), alone (upper panel) or after iv co-injection of PA (300 gmiddle panel) or 45 min after ip injection of PA (600 glower panel). The percentage of parent peptide remaining intact by PA treatment (either iv or ip 45 min in advance) is raised from 61% to >85%. Similar effect is achieved by injection of [.sup.99mTc]Demobesin 1 1 h after ip injection of race (2.5 mg) (lower panel, FIG. 10A-2) vs injection of [.sup.99mTc]Demobesin 1 alone (upper panel, FIG. 10A-2)

[0089] B: Biodistribution of [.sup.99mTc]Demobesin 1 in SCID mice bearing human prostate adenocarcinoma PC-3 xenografts (GRPR.sup.+) at 4 h pi. Bars represent average uptake as % injected dose per gram (% ID/g) of at least 4 animals with standard deviation (control at 4 hfirst bars); an additional group of animals received PA (300 g2.sup.nd bars) along with the radioligand. Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines, Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas, Fe=femur and Tu=PC-3 tumor. In the PA treated group, animals showed an uptake of 18.590.95% ID/g in the experimental tumor vs. 12.730.93% ID/g in the non-treated controls.

[0090] FIG. 11

[0091] A: Radiochromatogram of ex vivo mouse blood 5 min after injection of [.sup.111In]JMV4168 ([.sup.111In]DOTA-Ala-Ala-JMV594, [.sup.111In]DOTA-Ala-Ala-DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH.sub.2) alone (upper panel) or with PA (lower panel) The percentage of parent peptide remaining intact by PA treatment is raised from 64% to 98%.

[0092] B: Biodistribution of [.sup.111In]JMV4168 in SCID mice bearing human prostate adenocarcinoma PC-3 xenografts (GRPR.sup.+) at 4 h pi. Bars represent average uptake as % injected dose per gram (% ID/g) of at least 4 animals with standard deviation (control 4 h, first bars); an additional group of animals received PA (300 g2.sup.nd bars) along with the radioligand. Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines, Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas, Fe=femur and Tu=PC-3 tumor. In the PA treated group, animals showed an uptake of 23.3111.07% ID/g in the experimental tumor vs. 10.222.40% ID/g in the non-treated controls.

[0093] C: A Static SPECT-CT image 1 h (upper panels) and 4 h (lower panels) after injection of [.sup.111In]JMV4168 alone (left images) or together with PA (right images). The hGRPR.sup.+PC295 tumor on the shoulder(s) is/are excellently delineated in the PA-treated animals, whereas in the non-treated controls the uptake is significantly poorer.

[0094] FIG. 12

[0095] [.sup.111In]SP-1 control; +300 g PA; +300 g PA+300 g Lis; SP-1:[(DOTA)Arg1]Substance P; SP-1: (DOTA)Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2.

[0096] FIG. 13

[0097] FIG. 13: [111In]SP-2 control; +300 g PA; SP-2: [(DOTA)Arg1,Met(O2)11]Substance P; SP-2: (DOTA)Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met(O2)-NH2.

[0098] FIG. 14

[0099] FIG. 14: [111In]SP-3 control; +300 g PA; SP-3:[(DOTA)Arg1,Sar9,Met(O2)11]Substance P; SP-3: (DOTA)Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Sar-Leu-Met (O2)-NH2.

[0100] FIG. 15

[0101] FIG. 15: [111In]MSH-1 control; +300 g PA; MSH-1: [(DOTA)Ser1,Nle4]-MSH; MSH-1: (DOTA)Ser-Tyr-Ser-Nle-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2.

[0102] FIG. 16

[0103] FIG. 16: [111In]MSH-2 control; +300 g PA; MSH-2: [(DOTA)Ser1,Nle4,DPhe7]-MSH; MSH-2: (DOTA)Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2.

[0104] FIG. 17

[0105] FIG. 17: [111In]CTP-1 control; +300 g PA; CTP-1: For-Nle-Leu-Phe-Nle-Tyr-Lys(DOTA)-OH (Chemotactic peptide-1).

EXAMPLE 1

Somatostatin-14

[0106] Native somatostatin-14 elicits its physiological effects after binding to somatostatin receptors comprising five subtypes, sst.sub.1-5. Due to the high density expression of sst.sub.2 in neuroendocrine tumors synthetic stable sst.sub.2-preferring radioligands have been developed, while the in vivo application of SS14 was abandoned due to its rapid in vivo degradation. Interest for the development of a pansomatostatin-like analog (one binding to all five sst.sub.1-5 with a high affinity) was revived by the fact that sst.sub.1-5 are expressed alone or in various combinations in more types of human tumors. The inventors have recently developed SS14 analogs derivatized at the N-terminus with DOTA to allow for trivalent radiometal binding, such as .sup.111In.

In Vivo Stability

[0107] To study in vivo stability and the effect of vasopeptidase inhibition in prolonging biological half life of .sup.111In[(DOTA)Ala.sup.1]SS14 and .sup.111In[(DOTA) Ala.sup.1,DTrp]SS14, each radiopeptide was injected in the tail vein of Swiss albino mice alone or with the NEP inhibitor phosphoramidon (PA, 300 g). Whole blood was collected 5 min postinjection (pi), blood cells were removed and major proteins were precipitated and then the supernatant was analyzed by RP-HPLC coupled to a gamma detector.

[0108] Alternatively, the dual NEP and ACE inhibitor racecadotril (race; 2.5 mg) was ip injected 45-60 min prior to radioligand injection and the same procedure was followed as described above.

[0109] Representative radiochromatograms are shown in FIG. 1A and FIG. 2A. Indeed, both radiolabeled analogs showed very poor stability in vivo despite the common N-terminus modification and the Trp.sup.8 substitution by DTrp.sup.8 in the second analog, with the percentages of intact peptides not exceeding 2% and 6%, respectively. By PA co-injection these percentages spectacularly rose above 85% and 95%, respectively. Pretreatment with race provoked similar but less pronounced stabilization effects, with the respective percentage rising to >65%.

Tumor Uptake

[0110] Of great importance is the direct translation of PA- (or race) induced radiopeptide stabilization into meaningful and dramatic increase of tumor uptake in animals. Indeed, uptake in the rsst.sub.2.sup.+ AR4-2J tumor after injection of .sup.111In[(DOTA)Ala.sup.1]SS14 reached 13.872.4% ID/g in the PA-treated group at 4 h pi vs. 0.670.1% ID/g in the non-treated controls, while in the race pre-treated mice tumor values reached 3.510.2% ID/g, as presented in FIG. 1B. The corresponding values for the DTrp.sup.8-analog, shown in FIG. 2B, reach 9.063.57% ID/g (PA group), 4.182.28% ID/g (race group) and 1.820.36% ID/g (non-treated controls).

[0111] The above unexpected findings are of great significance for the applicability of radiolabeled SS14 analogs as pansomatostatin-like diagnostic and therapeutic tools. More so, in view of the fact that the pharmacological character of the native hormone is preserved. Recent efforts to develop synthetic pansomatostatin-like analogs led to radioligands that bind differently to some or all sst-subtypes, or do not efficiently internalize or show disappointingly poor pharmacokinetics. Accordingly, the combination of SS14 based radioligands with PA provides new molecular tools of potentially higher diagnostic sensitivity and therapeutic efficacy, given the inherent capacity of SS14 per se to most efficiently interact with all five sst.sub.1-5.

EXAMPLE 2

Gastrin

[0112] The overexpression of cholecystokinin-2 receptors (CCK2R) in many human tumors, such as in medullary thyroid cancer (MTC), small cell lung cancer, ovarian cancer and others, renders them attractive molecular targets for CCK2R-targeted diagnosis and therapy with radiolabeled CCK- and gastrin-derived probes. Most of the gastrin based radioligands show high CCK2R-affinity and metabolic stability but display undesirable kidney accumulation. On the other hand, the non-kidney accumulating radiolabeled CCKs or des(Glu).sub.5-truncated gastrins suffer from very rapid in vivo degradation and/or lower CCK2R affinity. As a result, the search for clinically useful radiolabeled CCKs and gastrins for CCK2R-targeted diagnosis and therapy is currently intense. (.sup.111In-DOTA) DGlu.sup.10]Minigastrin(10-17) (.sup.111In-DOTA-MG11) was identified as one of the most rapidly degraded des(Glu).sub.5-minigastrin radioligands, unable to achieve satisfactory CCK2R-targeting in mouse models.

In Vivo Stability

[0113] To test the in vivo stability, .sup.111In-DOTA-MG11 was injected in the tail vein of Swiss albino mice and 5 min afterwards blood was collected, blood cells were removed and major proteins were precipitated and then the supernatant was analyzed by RP-HPLC coupled to a gamma detector. As shown in the radiochromatogram of FIG. 3A, the major part of .sup.111In-DOTA-MG11 was consumed during this period (<5% intact peptide still detected), in agreement with previous reports. Further, .sup.111In-DOTA-MG11 was co-injected together with PA or injected 1 h after ip injection of race (2.5 mg) in more mice and the same protocol was followed as above. Amazingly, the percentage of .sup.111In-DOTA-MG11 found intact in the blood of the PA-treated mice was raised from <5% to 75% and in the race-pretreated mice to 73%.

Tumor Targeting

[0114] The unpredictable in vivo protection of .sup.111In-DOTA-MG11 conveyed by PA or race, albeit not full, translated into a surprisingly huge increase of CCK2R-targeting in an experimental tumor in mice. Biodistribution results in SCID mice bearing rCCK2R+AR4-2J tumors 4 h after injection of .sup.111In-DOTA-MG11 alone or together with PA or 1 h after ip injection of race are shown in FIG. 3B. Most astonishingly, tumor values reached 11.113.09% ID/g in the PA-receiving group vs. 1.220.06% ID/g in the untreated controls, whereas in the race-treated group this value reached 6.792.02% ID/g.

Kidney Uptake

[0115] Of particular relevance is the fact that PA administration, while causing an astounding >9 fold increase in the tumor, exerted absolutely no effect on kidney uptake. The same observation was made for the race group as well. As a result, unprecedented tumor-to-kidney ratios were achieved after injection of .sup.111In-DOTA-MG11 by PA or race treatment.

[0116] This finding fulfils an important prerequisite for effective radionuclide therapy of CCK2R.sup.+-tumors. This perspective is especially relevant for MTC patients, who are lacking therapeutic options at an advanced and disseminated state of the disease.

EXAMPLE 3

Neurotensin (NT)

[0117] The expression of neurotensin subtype 1 receptor (NTS1R) in human cancers, such as Ewing's sarcomas, ductal exocrine pancreatic carcinomas, colorectal cancer and meningiomas has been well documented. Especially for exocrine pancreatic cancer there is an urgent need for new effective clinical tools for its early diagnosis and therapy due to its high prevalence and very poor prognosis. Accordingly, several new neurotensin (NT) analogs have been developed and radiolabeled with .sup.99mTc or with trivalent metals (Maes V et al, 2006; Maina T et al, 2007; De Visser M et al 2003) with a few already evaluated in the clinic. However, results have been disappointing so far and one of the main reasons suspected for sub-optimal targeting is poor radioligand in vivo stability.

[0118] As stability has been so far exclusively investigated in blood plasma in vitro and stabilized radiolabeled NTs performed poorly in patients, the inventors decided to test stability in vivo using one such analog, [.sup.99mTc]Demotensin 6 (.sup.99mTcN.sub.4-Ala-Arg-Dab-Pro-Tyr-Tle-Leu-OH). This analog, while stable in plasma of mouse and patients in vitro failed to delineate NTS1R cancers in vivo in man despite its good affinity and internalization capacity.

[0119] For ex vivo blood analysis, [.sup.99mTc]Demotensin 6 was injected in the tail vein of Swiss albino mice and blood collected 5 min thereafter was analyzed as previously described. The effect of PA on stability was studied by co-injection of PA along with [.sup.99mTc]Demotensin 6. Furthermore, a PA dose study was performed with the PA injected dose ranging from 300 g down to 0.03 g and results are summarized in FIG. 4A.

[0120] Unexpectedly and while [.sup.99mTc]Demotensin 6 was found >90% stable during in vitro incubation with mouse and human plasma (Maina T et al, 2007; Gabriel M et al, 2011), in vivo it is degraded by half (52%) within 5 min already. Most significantly, the inventors were able to observe a direct effect of PA on the in vivo stability of the radioligand, which varied with the administered PA-dose implying a major role of NEP in its catabolism.

[0121] Thus, the percentage of [.sup.99mTc]Demotensin 6 remaining intact by PA 300 g co-injection rose to >90% and remained at this high level even by scaling down the PA dose to 30 g initially and then to 3 g. However, by further reducing the dose to 0.3 g and finally 0.03 g PA the percentage of intact peptide dropped to >60% and >55%, respectively. It is interesting to observe that co-administration of the ACE inhibitor Lis (300 g) and PA (300 g) showed identical results as when PA (300 g) was injected alone.

[0122] In FIG. 4B the effects of such regimens on SCID mice bearing the human colon adenocarcinoma WiDr tumor (a hNTS1R tumor) on enhancing in vivo tumor targeting are presented. Thus, in the group receiving 300 g PA along with the radioligand, animals showed an uptake of 3.560.38% ID/g in the experimental tumor, while in the PA+Lis co-injected animals tumor uptake remained at this level 3.500.34% ID/g vs. 1.60.42% ID/g in the non-treated controls, suggesting that NEP is only implicated in the catabolism of [.sup.99mTc]Demotensin 6 and the radiopeptide is stable against ACE. It is also interesting to note that blockade was very effective in the presence of PA (0.380.15% ID/g), showing that the PA-induced increase on the tumor is specific and NTS1R-mediated.

[0123] Similarly to the doubly stabilized [.sup.99mTc]Demotensin 6 radiotracer, the inventors were further interested to investigate the effects of PA on [.sup.99mTc]Demotensin 1 ([(.sup.99mTC-N.sub.4)Gly.sup.7]NT(7-13), .sup.99mTcN.sub.4-Gly-Arg-Arg-Pro-Tyr-Ile-Leu-OH) wherein the original NT(8-13) peptide fragment is preserved. As shown in FIG. 5A less than 1% intact peptide survived 5 min in vivo as opposed to the 52% found after administration of [.sup.99mTc]Demotensin 6 demonstrating the clearly positive impact of strategic Arg.sup.9 and Ile.sup.12 replacement by Dab and Tle, respectively, on the in vivo stability of [.sup.99mTc]Demotensin 6. Co-injection of 600 g or 300 g PA increased the percentage of [.sup.99mTc]Demotensin 1 detected in mouse blood to 28% and 25%, respectively.

[0124] In contrast to [.sup.99mTc]Demotensin 6, co-injection of [.sup.99mTc]Demotensin 1 and 30 g PA failed to sufficiently stabilize the radiopeptide, with only 4.5% still found in mouse blood. This finding suggests that other enzymes in addition to NEP are involved in the in vivo catabolism of [.sup.99mTc]Demotensin 1.

[0125] In order to elucidate such involvement, the inventors have co-injected 300 g PA and 300 g of the ACE inhibitor Lis along with the radioligand. This inhibitor combination resulted in an overall 56% of intact peptide surviving in mice during 5 min whereas co-injection of 300 g PA alone led to only 25% intact peptide under the same experimental protocol. It is interesting to note that Lis alone was able to protect [.sup.99mTc]Demotensin 1 only up to 16%. Furthermore, the pattern of metabolites found by PA treatment significantly differs from the metabolic pattern after Lis treatment (FIG. 5A) indicating that the two enzymes attack different bonds of the peptide chain. When ip pre-injecting race (2.5 mg) to the animals prior to [.sup.99mTc]Demotensin 1 injection an overall of 37% peptide remains intact in the circulation.

[0126] In FIG. 5B the effects of above regimens on SCID mice bearing WiDr tumors (hNTS1R+) on enhancing in vivo tumor targeting are shown. Thus, in the group receiving 300 g PA along with the radioligand, animals showed a tumor uptake of 4.580.47% ID/g, while in the PA+Lis co-injected animals tumor uptake was raised to 7.711.19% ID/g vs. 1.200.21% ID/g in the non-treated controls, suggesting that both NEP and ACE are implicated in the catabolism of the radiopeptide contrasting [.sup.99mTc]Demotensin 6 which is stable against ACE.

[0127] It is also interesting to note that blockade was very effective in the presence of PA and Lis (0.230.09% ID/g), showing that the PA+Lis-induced increase on the tumor is specific and NTS1R-mediated.

EXAMPLE 4

Neuromedin C (NMC)

[0128] The high density expression of gastrin releasing peptide receptors (GRPRs) in many frequently occurring human cancers, such as prostate and breast cancer, gastrinomas, small cell lung cancers and others, provides the opportunity for their diagnosis and therapy using radiolabeled bombesin-like analogs. Neuromedin C (NMC) is the C-terminal decapeptide fragment (H-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2), of native human GRP binding with a high affinity with GRPR. The inventors have recently developed a series of NMC analogs functionalized at the N-terminus with acyclic tetraamines for stable binding of .sup.99mTc, SAR-NCs, as candidates for the diagnostic imaging of GRPR-expressing tumors.

[0129] The in vivo stability of [.sup.99mTc]SAR-NC1 ([(.sup.99mTC-N.sub.4)Gly.sup.1]NMC) and of [.sup.99mTC]SAR-NC6 ([(.sup.99mTcN.sub.4)Gly.sup.1, Sar.sup.7]NMC) was tested as described above by collecting blood 5 min after injection of radioligand alone or together with 300 g PA in Swiss albino mice. As shown in FIG. 6A and FIG. 7A, respectively, the amount of in vivo surviving parent peptide was 30% and 35%. PA (300 g) treatment doubled this percentage (>68% and 70%, respectively) implicating NEP again in the catabolism of these analogs.

[0130] In FIG. 6B and FIG. 7B, the biodistribution of [.sup.99mTc]SAR-NC1 and [.sup.99mTc]SAR-NC6 in SCID mice bearing human prostate adenocarcinoma PC-3 xenografts (GRPR.sup.+) at 4 h respectively, after injection of the radioligand alone or with co-injection of PA (300 g) are shown. In the PA treated group, animals showed an uptake of 28.348.05% ID/g in the experimental tumor vs. 6.511.91% ID/g in the non-treated controls for [.sup.99mTc]SAR-NC1. In the case of [.sup.99mTc]SAR-NC6, the PA treated group, showed a tumor uptake of 27.583.47% ID/g vs. 9.221.40% ID/g in the non-treated controls.

EXAMPLE 5

Bombesin (BBN)

[0131] Targeting of GRPR.sup.+ human tumors has been attempted by quite a few bombesin analogs labeled with diagnostic and therapeutic radionuclides (Maina T et al, 2006; Smith C J et al, 2005; Lantry L E et al, 2006; Zhang H et al, 2004; Nock B et al, 2005; Schroeder R P et al, 2011; Ananias H J et al, 2008; Wild D et al, 2011). [.sup.99mTc]Demobesin 4 (.sup.99mTcN.sub.4-Pro-Gln-Arg-Tyr-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH.sub.2) is a bombesin analog radiolabeled with .sup.99mTc via an acyclic chelator coupled to its N-terminal Pro. [.sup.99mTc]Demobesin 4 showed promising characteristics in human biopsy specimens and in mice bearing human GRPR.sup.+ prostate cancer xenografts while its in vitro stability in mouse plasma was very high. The clinical value of [.sup.99mTc]Demobesin 4 as a diagnostic radiotracer is currently under study in prostate cancer patients.

[0132] To test the in vivo stability of [.sup.99mTc]Demobesin 4 in mice the same protocol as described above was applied. The radiotracer was injected alone or together with decreasing amounts of PA (30, 3 and 0.3 g) in mice. Results of 5 min ex vivo mouse blood analysis by HPLC are summarized in FIG. 8A. It is very clear to observe that the percentage of [.sup.99mTc]Demobesin 4 remaining intact by PA treatment is raised from 26% (control) to >77%, >63% and 30%, respectively.

[0133] This effect can be elegantly exploited to enhance tumor accumulation, and thereby diagnostic sensitivity, of [.sup.99mTc]Demobesin 4, as shown in FIG. 8B, whereby the biodistribution of [.sup.99mTc]Demobesin 4 in SCID mice bearing PC-3 xenografts is presented at 4 h after injection of the radioligand alone or with PA (300 g) It is interesting to note, that in the PA treated group, animals showed a tumor uptake of 35.507.50% ID/g vs. 11.261.81% ID/g in the non-treated controls.

[0134] In a second example illustrated in FIG. 9A, [.sup.111In]PanSarbesin 1 ([(.sup.111In-DOTA-PEG.sub.2-DTyr-Gln-Trp-Ala-Val-Ala-His-Phe-Nle-NH.sub.2) an .sup.111In-labeled DOTA-derivatized BBN analog with affinity to all human BBN-receptors (GRPR, NMBR and BB.sub.3R) was injected in Swiss albino mice either alone or or with PA (300 g) and blood was collected 5 min thereafter following the previously described protocol. The respective radiochromatograms (radioligand aloneupper panel) or with PA (300 glower panel) show that the percentage of parent peptide remaining intact by PA treatment is raised from 13% to 80%.

[0135] Translation of this effect in enhancement of tumor uptake is quite prominent as can be seen in FIG. 9B, including biodistribution of [.sup.111In]PanSarbesin 1 in SCID mice bearing PC-3 xenografts (GRPR.sup.+) at 4 h pi (control) or with coinjection of PA (300 g) or with co-injection of excess [Tyr.sup.4]BBN (100 g) in addition to PA (300 g) along with the radioligand. In the PA treated group, animals showed an uptake of 20.962.58% ID/g in the experimental tumor vs. 3.750.73% ID/g in the non-treated controls. It is interesting to note that with co-injection of the blocker and PA tumor values were minimal (0.690.03% ID/g) showing that PA provoked a GRPR-mediated increase in tumor uptake.

EXAMPLE 6

GRPR-Antagonists

[0136] While GRPR-agonists (and in general peptide receptor agonists) have been originally preferred for GRPR targeting of human tumors due to their internalization capacity in cancer cells, increasing evidence reveals superior characteristics of radiolabeled GRPR-antagonists (Nock B et al, 2003; Cescato R et al, 2008; Mansi R et al, 2009; Abd-Elgaliel W R et al, 2008). Given that antagonists do not elicit undesirable adverse reactions after binding to the GRPR they are much better tolerated after iv injection in humans than agonists. In addition, they seem to clear much more rapidly from background tissues, even from the strongly GRPR.sup.+ pancreas. This quality often leads to high tumor-to-background ratios after injection of radiolabeled GRPR-antagonists thereby favouring high contrast tumor imaging and high therapeutic efficacy.

[0137] Antagonists are synthetic compounds and in general expected to show higher metabolic stability than agonists. The inventors therefore decided to test the in vivo stability of [.sup.99mTc]Demobesin 1 ([(.sup.99mTcN.sub.4) (p-aminobenzyl-diglycolic acid)-[DPhe.sup.6,LeuNHEt.sup.13]BBN(6-13), the first radiolabeled antagonist shown to display superior pharmacokinetics as compared to similarly modified agonists. [.sup.99mTc]Demobesin 1 has shown high in vitro stability in mouse plasma, but by analysis of ex vivo blood 5 min pi the percentage of intact peptide was 60-65% (FIGS. 10A-1 and 10A-2).

[0138] Nevertheless, co-injection of 300 g PA increased this percentage to >85% and the same increase was observed when 600 g PA were ip administered 45 min prior to radioligand injection (FIG. 10A). By ip injection of 2.5 mg race 1 h before injection of [.sup.99mTc]Demobesin 1 the inventors observed again the same increase in the percentage of parent peptide (FIG. 10A-2).

[0139] The effect of PA-induced stabilization of [.sup.99mTc]Demobesin 1 on tumor uptake is illustrated in FIG. 10B, showing the biodistribution at 4 h pi after injection of [.sup.99mTc]Demobesin 1 in SCID mice bearing human PC-3 xenografts (GRPR.sup.+) (control), or with PA (300 g). In the PA treated group, animals showed an uptake of 18.590.95% ID/g in the experimental tumor vs. 12.730.93% ID/g in the non-treated controls.

[0140] Another characteristic example of the metabolic radioligand stabilization induced by PA is shown in FIG. 11A. The recently synthesized radiotracer [.sup.111In]JMV4168 ([.sup.111In]DOTA-Ala-Ala-JMV594, [.sup.111In]DOTA-Ala-Ala-DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH.sub.2) based on the potent GRPR-antagonist JMV594 (Tokita K et al, 2001) was injected in Swiss albino mice alone or together with PA and blood collected after 5 min by HPLC, was analyzed as previously detailed.

[0141] [.sup.111In]JMV4168 showed higher stability (64%) than GRPR-agonists, such as [.sup.99mTc]SAR-NCs (30-35%), or [.sup.99mTc]Demobesin 4 (26%), and comparable to the GRPR-antagonist [.sup.99mTc]Demobesin 1 (61-65%), it clearly profited by PA treatment with 98% remaining stable.

[0142] This in vivo prolongation of half-life translated into higher tumor uptake in mice bearing GRPR.sup.+ PC-3 xenografts. As shown in FIG. 11B, tumor values raised from 10.222.40% ID/g in the non-treated controls to 23.3111.07% ID/g in the PA-receiving mice. Of great interest is the fact, that pancreas uptake remained very low.

[0143] In FIG. 11C, the effect of PA co-injection with [.sup.111In]JMV4168 on the delineation of an hGRPR.sup.+ implanted tumor in a SPECT/CT image was observed. The hGRPR.sup.+ PC295 tumors on the shoulder(s) are excellently delineated in the PA-treated animals, whereas in the non-treated controls the uptake is significantly poorer. Furthermore, no evidence of pancreatic uptake is shown in any of the images, in agreement with the results of the respective biodistribution in hGRPR.sup.+ PC-3 tumor bearing mice.

[0144] This finding has dosimetric implications in the treatment of GRPR.sup.+ tumors with radiolabeled GRPR-antagonists. In fact, it is a very powerful modality to selectively enhance uptake on tumor lesions but not to GRPR-expressing tissues like the pancreas, thus sparing them from harmful radiation doses. Since the local enzymatic degradation differs between pancreas and tumor it is possible according to the invention to selectively stabilize radioligands in the tumor and peritumoral milieu only.

EXAMPLE 7

Alternative Peptides

[0145] Additional groups of radiopeptides with relevance for nuclear medicine applications have been studied for their in vivo stability. Radiopeptides were injected in the tail vein of healthy mice, either alone, or with a NEP inhibitor (PA300 g), or with a NEP (PA300 g) and an ACE (Lis300 g) inhibitor mixture; alternatively, another NEP inhibitor prodrug (race2.5 mg) was ip injected in the animals=45 min prior to radioligand injection. Blood was collected 5 min afterwards, and analyzed by RP-HPLC after suitable preparation, as previously described.

Peptides are grouped on the following categories:

[0146] Substance P analogs: SP-1 is the non-modified SP sequence with DOTA coupled to its N-terminus. The in vivo catabolism of [111In]SP-1 (FIG. 12upper panel) is extremely fast with only 7% surviving in this period. By co-injection of PA (300 g) this percentage raises to 32% (FIG. 12middle panel), while by co-injection of the NEP and ACE inhibitor cocktail (300 g PA+300 g Lis) the percentage roses to above 60% (FIG. 12lower panel), implying the combined role of NEP and ACE in the catabolism of [111In]SP-1 in vivo.

[0147] In SP-2, Met11 is oxidized to the corresponding sulfone, reported for its high affinity to the neurokinin-1 receptor subtype (NK1R). In this case inhibition of NEP by PA-treatment raises the percentage of surviving [111In]SP-2 from 24% (FIG. 13upper panel) to 80% (FIG. 13upper panel) showing that oxidation of Met11 conveys extra stability, especially against ACE.

[0148] In SP-3, an additional modification in the original peptide chain, and specifically Gly9 by Sar9 substitution, further increases in vivo stability, with 51% of [.sup.111In]SP-3 surviving in circulation (FIG. 14upper panel) and this percentage rising to almost 90% by PA-co-injection (FIG. 14lower panel)

[0149] MSH analogs: Two MSH analogs for MSH-receptor (MSHR) targeting are coupled to DOTA at their N-terminus and the stability of the respective 111In-radiopeptides studied, as described above. In FIG. 15 (upper panel) we observe that 52% of [111In]MSH-1 survives in mouse blood stream and by PA-co-injection (lower panel) this percentage rises above 90%. Substitution of Phe7 by DPhe7 substantially increases stability in [111In]MSH-2 to 77% (FIG. 16upper panel). However, PA-co-injection almost totally stabilizes the analog (97% intact peptide: FIG. 16 (lower panel).

[0150] Chemotactic peptide (CTP) analogs to target infection as exemplified by [111In] CTP-1. As shown in FIG. 17 (upper panel), only 2% of the radioligand remains intact 5 min after entry into circulation in healthy mice, whereas by PA-co-injection84% survives.

REFERENCES

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