Novel Neurotensin Analogues for Radioisotope Targeting to

Jul 17, 2009 - Vrije Universiteit Brussel. .... peptides (Table 1) were prepared by solid-phase peptide synthesis as described in detail elsewhere (10...
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Bioconjugate Chem. 2009, 20, 1602–1610

Novel Neurotensin Analogues for Radioisotope Targeting to Neurotensin Receptor-Positive Tumors Faisal Alshoukr,†,‡,§ Cedric Rosant,†,‡,§ Veronique Maes,⊥ Jallane Abdelhak,†,‡,§ Olivier Raguin,†,‡,§ Samuel Burg,†,‡,§ Laure Sarda,†,‡,§ Jacques Barbet,§,| Dirk Tourwe´,⊥ Didier Pelaprat,†,‡,§ and Anne Gruaz-Guyon*,†,‡,§ Inserm, U773, team 11, Paris, F-75018, France, Universite´ Denis Diderot-Paris 7, UMR S773 Paris, F-75018, France, CNRS, GDR Antibodies and therapeutic targeting 3260, Tours, F-37032, France, Centre de Recherche en Cance´rologie de Nantes-Angers, Inserm, Universite´ de Nantes, U892, Nantes, F-44000, France, and Department of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. Received April 6, 2009; Revised Manuscript Received June 19, 2009

The increased expression of the neurotensin (NT) receptor NTS1 by different cancer cells, such as pancreatic adenocarcinoma and ductal breast cancer cells, as compared to normal epithelium, offers the opportunity to target these tumors with radiolabeled neurotensin analogues for diagnostic or therapeutic purposes. The aim of the present study was to design and synthesize new neurotensin radioligands and to select a lead molecule with high in vivo tumor selectivity for further development. Two series of neurotensin analogues bearing DTPA were tested: a series of NT(8-13) analogues, with DTPA coupled to the R-NH2, sharing the same peptide sequence with analogues previously developed for radiolabeling with technetium or rhenium, as well as an NT(6-13) series in which DTPA was coupled to the ε-NH2 of Lys6. Changes were introduced to stabilize the bonds between Arg8Arg9, Pro10-Tyr11, and Tyr11-Ile12 to provide metabolic stability. Structure-activity studies of NT analogues have shown that the attachment of DTPA induces an important loss of affinity unless the distance between the chelator and the NT(8-13) sequence, which binds to the NTS1 receptor, is increased. The doubly stabilized DTPA-NT20.3 exhibits a high affinity and an elevated stability to enzymatic degradation. It shows specific tumor uptake and high tumor to blood, to liver, and to intestine activity uptake ratios and affords high-contrast planar and SPECT images in an animal model. The DTPA-NT-20.3 peptide is a promising candidate for imaging neurotensin receptor-positive tumors, such as pancreatic adenocarcinoma and invasive ductal breast cancer. Analogues carrying DOTA are being developed for yttrium-90 or lutetium-177 labeling.

INTRODUCTION Pancreatic adenocarcinoma, the tenth most common human cancer, grows extremely rapidly, disseminates early, and occult metastases are frequent. Noninvasive staging modalities have shown limited ability to detect local invasion or small volume metastatic disease. 18F-labeled 2-deoxy-2-fluoro-D-glucose (18FFDG), which has greatly improved the diagnosis and staging of numerous tumors, does not significantly increase the accuracy of the preoperative determination of resectability of pancreatic adenocarcinoma (1). Therefore, a noninvasive method to improve preoperative staging would be extremely useful. It has been shown that 75-88% of ductal pancreatic adenocarcinomas express neurotensin (NT) receptors (2, 3). These receptors have been proposed as new markers for this tumor, since they were not detected in normal pancreas and chronic pancreatitis (2). NT receptors were also identified in other tumor cells as, for example, Ewing’s sarcoma, meningiomas, small cell lung carcinoma, and colon adenocarcinoma (4-6). In patients with invasive ductal breast cancers, 91% of tumors are positive for * To whom correspondence should be addressed: Anne GruazGuyon, Inserm, U773, Faculte´ de Me´decine Xavier Bichat, 16 rue Henri Huchard, BP 416, F-75018, France; E-mail: [email protected], Phone: (33) 1 57 27 75 55, Fax (33) 1 57 27 83 21. † Inserm, U773. ‡ Universite´ Denis Diderot-Paris 7. § CNRS, GDR Antibodies and therapeutic targeting 3260. ⊥ Vrije Universiteit Brussel. | Centre de Recherche en Cance´rologie de Nantes-Angers, Inserm, Universite´ de Nantes.

the neurotensin high-affinity receptor (NTS1), while it is poorly expressed or absent in normal cells (7). This recent work suggests that neurotensin contributes to human breast cancer progression and points out to the therapeutic potential of molecules targeting NTS1 receptor. Three NT receptor subtypes have been cloned to date. Most NT biological effects are mediated by NTS1. NT(8-13) is the minimal sequence that mimics the effects of full-length NT (8). Numerous neurotensin analogues containing the (NRHis)Ac chelator and labeled with 99mTc or 188Re have been synthesized (9-15). Since neurotensin is rapidly degraded in vivo by peptidases, changes were introduced to protect the three major sites of enzymatic cleavage, the Arg8-Arg9, Pro10-Tyr11, and Tyr11-Ile12 bonds (9), to stabilize these molecules. The recently reported analogue NT-XIX with the three enzymatic cleavage sites stabilized displayed the highest tumor uptake described in the literature for radiolabeled neurotensin analogues, with low uptake in normal organs and particularly in kidneys (12, 13). Very encouraging therapy results of 50% reduction of tumor growth were obtained in nude mice with 188Re-labeled NT-XIX. Significant activity detected in the intestines may nevertheless be a source of radiotoxicity (12). Peptides and proteins coupled to diethylenetriaminepentaacetic acid (DTPA) have been used successfully to deliver indium-111, gallium-67, and gallium-68 for imaging applications (SPECT and TEP) (16-18). By contrast, yttrium-90 needs macrocyclic chelators such as 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) to prevent leakage and bone marrow toxicity in targeted radionuclide therapy (19). Lutetium-177 has also

10.1021/bc900151z CCC: $40.75  2009 American Chemical Society Published on Web 07/17/2009

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Table 1. Peptide Sequence and Analytical Data peptide

sequence

% purity

M+H+ MALDI-TOF

M+H+ calculated

>95b

2048.16b

2048.32b

>96 95 97 92 97

1192.23 1150.12 1150.33 1319.11 1346.50

1192.62 1150.65 1150.65 1318.73 1346.76

>99 >97 >99

1459.78 1459.77 1473.83

1459.78 1459.78 1473.80

NT(1-13) Analogues NT [Lys6(DTPA)]-NT

pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH pGlu-Leu-Tyr-Glu-Asn-Lys(DTPA)-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH

NT(8-13) DTPA-NT(8-13) DTPA-NT-VI DTPA-NT-XI DTPA-Ahx-NTXIIa DTPA-Ahx-NT-XIXa

H-Arg-Arg-Pro-Tyr-Ile-Leu-OH DTPA-Arg-Arg-Pro-Tyr-Ile-Leu-OH DTPA-Lys-Ψ(CH2-NH)-Arg-Pro-Tyr-Ile-Leu-OH DTPA-Lys-Ψ(CH2-NH)-Arg-Pro-Tyr-Tle-Leu-OH DTPA-Ahx-Arg-Me-Arg-Pro-Tyr-Tle-Leu-OH DTPA-Ahx-Arg-Me-Arg-Pro-Dmt-Tle-Leu-OH

NT-20.1 NT-20.2 NT-20.3 DTPA-NT-20.1 DTPA-NT-20.2 DTPA-NT-20.3

Ac-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH Ac-Lys-Pro-Arg-Arg-Pro-Tyr-Tle-Leu-OH Ac-Lys-Pro-Me-Arg-Arg-Pro-Tyr-Tle-Leu-OH Ac-Lys(DTPA)-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH Ac-Lys(DTPA)-Pro-Arg-Arg-Pro-Tyr-Tle-Leu-OH Ac-Lys(DTPA)-Pro-Me-Arg-Arg-Pro-Tyr-Tle-Leu-OH

NT(8-13) Analogues

NT(6-13) Analogues

a

Ahx: aminohexanoic acid. b Results already described (22).

been used with DOTA-substituted peptides and antibodies. However, with previously described DTPA/DOTA neurotensin analogues, tumor uptake was limited and accumulation of radioactivity in kidneys resulted in low tumor to kidney uptake ratios (20-22). In the present study, since DTPA is a suitable chelator for 111In scintigraphy, new neurotensin analogues carrying DTPA have been synthesized to further investigate the peptide structural parameters influencing tumor targeting and normal tissue uptake and to select a lead molecule for further development. They were evaluated with regard to binding affinity, stability to enzymatic degradation, internalization rate, and biodistribution. Their properties were compared to those of a reference peptide [Lys6(DTPA(111In))]NT in which DTPA was coupled to the Lys6 ε-NH2 of NT (22).

MATERIALS AND METHODS Cells. Experiments were performed with the human colorectal carcinoma cell line HT29 (ATCC, Rockville, USA). Cells were grown in DMEM (Gibco, France) supplemented with 10% fetal calf serum, 2 mM glutamine, and 50 µg/mL gentamycin at 37 °C in 5% CO2. Synthesis of the DTPA-NT Analogues. All reagents used for the synthesis were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France, or Bornem, Belgium), Novabiochem (La¨ufelfingen, 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 DiscoveryBIO 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 mL/min on a Waters apparatus. 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 (10, 13, 23). Tris-tBu-DTPA (3 equiv) (24) was coupled to the resin-bound neurotensin analogue in a mixture of DMF/CH2Cl2 using 2-1H(benzotriazol-1-yl)1,1,3,3-tetramethylureum tetrafluoroborate (TBTU), 1-hydroxybenzotriazole (HOBt), and diisopropylethylamine (DIPEA) during 4 h. The acetylated NT(6-13) analogues were synthesized by NeoMPS (Strasbourg, France) and DTPA was coupled to the lysine ε-NH2 as already described (25). Radiolabeling. The DTPA-NT analogues were labeled with indium-111 (111InCl3, 60 or 180 MBq, CIS bio International, France) in 100 mM acetate, 10 mM citrate, buffer pH 5, for

22 h at room temperature, and then free DTPA groups were saturated with nonradioactive InCl3 as already described (26). Determination of the NTS1 Binding Affinities of the DTPA-NT Analogues. Binding to HT29 Cell Membranes. Cell membranes (60 µg protein) were incubated for 45 min at room temperature in 250 µL buffer (50 mM Tris HCl, 5 mM MgCl2, 0.8 mM 1,10-phenanthroline, 0.2% BSA, pH 7.4) in the presence of 50 pM 125I-Tyr3-neurotensin (Perkin-Elmer) and increasing concentrations of nonradioactive DTPA(In)-NT analogues. Membrane-bound activity was recovered by filtration onto Whatman GF/B filters presoaked for 1 h with polyethyleneimine (0.2% in water) and rinsed twice with buffer. Nonspecific binding was evaluated in the presence of 10-6 M neurotensin. Radioactivity was counted, and results were analyzed with GraphPad Prism (GraphPad Software, Inc., San Diego, CA). All experiments were performed three times. Binding to LiVing HT29 Cells and Internalization. IC50 values for the binding to living HT29 cells were determined from competition experiments between [Lys6(DTPA(111In))]-NT (150 pM) and the peptides without DTPA or DTPA(In)-Ahx-NTXIX. For the other peptides, IC50 was evaluated using the labeled DTPA(111In)-peptide and increasing concentrations of the corresponding nonradioactive DTPA(In)-peptide. Cells were rinsed with 500 µL DMEM and 0.2% BSA, and incubated with DTPA(111In)-NT analogue (0.15 × 10-9 M, 300 µL DMEM, 0.2% BSA, 0.8 mM 1,10-phenanthroline, 60 min, 37 °C) in the presence of increasing concentrations of nonradioactive DTPA(In)-NT analogue. After washing the wells twice with icecold DMEM 0.2% BSA, cells were lysed in 500 µL 0.1 N NaOH, and radioactivity was counted. Nonspecific binding was evaluated in the presence of 10-6 M neurotensin. Competition curves were analyzed with the Equilibrium Expert software (27). All experiments were performed three times. Incubation for internalization studies was performed with 0.15 × 10-9 M DTPA(111In)-NT analogue 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 surfacebound ligand. Internalized activity was then counted after washing. Nonspecific binding and internalization were evaluated in the presence of 10-6 M neurotensin. Results are expressed as the ratio between internalized and specifically bound radioactivity. Metabolic Stability. In Human Serum. Serum from healthy donors (100 µL) was incubated with the DTPA(111In) analogues

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(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 C18 RP-HPLC as described above. Detection was performed with a radioactivity detector (HERM LB 500, Berthold, France). Elution was performed using, after 5 min 0% B, a linear 10 min gradient from 0% to 35% B and a linear 25 min gradient from 35% to 50%, flow rate 1.5 mL/min. The sample was also coinjected with the radioactive control to identify the peak corresponding to intact peptide. In ViVo Stability. Female BALB/c mice were injected in the tail vein with 111In-labeled DTPA-NT analogues (25 pmol). The mice were sacrificed 15 min after injection. Plasma and urine samples (50 µL) were added to 200 µL methanol and treated as above. Biodistribution and Imaging Studies. 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. HT29 cells (6.7 × 105 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 111Inlabeled-DTPA-NT analogues (20-25 pmol in 100 µL PBS) and sacrificed at different times. Blood, organs, and tumors were collected, weighed, and radioactivity counted. Injected activity was corrected for losses by subtraction of noninjected and subcutaneously injected material (remaining in the animal tail). In blocked experiments, each mouse received a coinjection of the labeled peptide and of its unlabeled counterpart (60 nmol of NT or 180 nmol of NT-20.3). 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. Scintigraphic imaging was performed under pentobarbital anesthesia after i.v. injection with DTPA(111In)-NT-20.3 (30-50 pmol, 9-13 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). A planar anterior acquisition was performed from 0 to 60 min postinjection, and a dynamic series of images of 5 min each were computed from the recorded scintigraphy data. At 2 h postinjection, 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 mm2) ratio was evaluated on planar images. Radioactivity excretion in urine was determined from activity at 1 h postinjection in the bladder.

RESULTS Synthesis of DTPA-NT Analogues. We synthesized two series of DTPA-bearing neurotensin analogues that were stabilized against enzymatic degradation at the bonds between Arg8 and Arg9, Pro10 and Tyr11, or Tyr11 and Ile12 by changes introduced in the peptide sequence (Table 1). DTPA-NT-VI, DTPA-NT-XI, DTPA-Ahx-NT-XII, and DTPA-Ahx-NT-XIX are DTPA-NT(8-13) analogues. They share the same peptide sequence with analogues carrying (NR-His)Ac for radiolabeling withtechnetiumorrheniumdescribedintheliterature(9-11,13,15). These analogues were prepared by solid-phase synthesis as described previously, and tris-tBu-DTPA was coupled to their N-terminus, followed by resin cleavage and HPLC purification. We also synthesized analogues of the 6-13 sequence of [Lys6(DTPA)]-NT, which provided encouraging in vivo tumor targeting results in earlier studies (22). The N terminal end was acetylated in order to decrease the positive charge that favors

Alshoukr et al. Table 2. Affinity of Peptides peptide

Ki (nM) membranes

IC50 (nM) cells

NT [Lys6(DTPA(In))]-NT NT(8-13) DTPA(In)-NT(8-13) DTPA(In)-NT-VI DTPA(In)-NT-XI DTPA(In)-Ahx-NTXII DTPA(In)-Ahx-NT-XIX NT-20.1 NT-20.2 NT-20.3 DTPA(In)-NT-20.1 DTPA(In)-NT-20.2 DTPA(In)-NT-20.3

0.28 ( 0.05 1.77 ( 0.39 0.044 ( 0.009 1.36 ( 0.39 3.20 ( 0.81 8.11 ( 1.03 5.26 ( 1.24 67 ( 11 0.072 ( 0.019 0.26 ( 0.07 0.16 ( 0.03 0.66 ( 0.1 1.55 ( 0.42 2.24 ( 0.21

1.67 ( 0.40 17.3 ( 4.3 0.68 ( 0.04 21.7 ( 5.1 14.7 ( 1.6 101 ( 17 132 ( 44 626 ( 30 0.82 ( 0.08 2.46 ( 0.79 2.20 ( 0.31 6.73 ( 0.31 41.2 ( 6.2 15.9 ( 1.7

renal accumulation of radiometals (28, 29) and to protect it against amino-peptidases. These analogues were obtained by coupling DTPA to the free ε-NH2 group of Lys. Coupling yields were approximately 80% except for DTPA-NT-20.2 (48%). All DTPA-peptides were purified to at least 92% purity (92-99%) and identified by mass spectrometry (Table 1). Radiolabeling. 111In-labeling of all DTPA-NT was performed with >90% indium-111 incorporation. Specific activities between 220 and 260 MBq/nmol could be obtained and were used for in vitro and imaging studies. Lower specific activities were used for biodistribution studies. Binding Affinity Profile. Affinity of the peptides for NTS1 was evaluated using HT29 cells. These cells from which NTS1 has been first cloned (30) have been used to evaluate most labeled NT analogues described in the literature. Ki values for binding to membranes and IC50 values for binding to cells were used to evaluate affinity. Ki values for binding to HT29 membranes were, for most peptides, about 10 times lower (5-27) than the IC50 for the binding to HT29 cells (Table 2). This can be attributed to the decreased affinity for binding to the NTS1 induced by sodium (31) and to the effects of internalization and externalization of radioactivity in cells. Nevertheless, the affinities of the different peptides for membranes and living cells show a similar rank order. Shortening the NT(1-13) sequence increased the affinity of the NR-acetyl-NT(6-13) analogue NT-20.1 as already reported for NR-acetyl-Lys6(acetyl)-NT(6-13) (8). DTPA(In) coupled to the NH2-R 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-NH2 of Lys6 of NT, the affinity loss is only a factor of 6 for membranes and 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. A lower decrease in affinity (by a factor of 3) was observed in a larger peptide described in previous studies: DTPA(In)-(Gly0-Glu1[AcLys6]NT) (22). As a result, the affinity of DTPA(In)-NT20.1 was 2-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. In the NT(8-13) series containing the stabilized Lys-Ψ(CH2NH)-Arg bond (13), introduction of a tertiary leucine (Tle12) reduced the affinity by a factor of 3 for membranes and 7 for living HT29 cells, resulting in the lower affinity of DTPA(In)NT-XI. N-Methylation of the Arg8-Arg9 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.

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Table 3. In Vitro and in Vivo Stability of Peptides peptide

in vitro stability (t1/2 h)a

in vivo stability (% intact peptide)b

[Lys6(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

25 ( 2 ND ND 0.4 ( 0.02 4.4 ( 0.6 257 ( 71

4 (3-5) 10 (5-15) 47 (40-53) 0.8 (0.8-0.8) 10 (6-14) 26.5 (26-27)

a In vitro stability is expressed as the degradation half-life in human serum at 37 °C. b In vivo stability is expressed as the % intact peptide (mean (individual values)) recovered in plasma 15 min after tracer injection.

Replacement of Tyr11 by 2′,6′-dimethyltyrosine in DTPA(In)Ahx-NT-XIX led to an additional loss of affinity. Introduction of a Tle12 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 Pro7-Arg8 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 analogue resulted in a high affinity, for membranes and for living cells, similar to those of the reference peptide [Lys6(DTPA(In))]-NT. The peptides exhibiting the highest affinities, [Lys6(DTPA(In))]NT, DTPA(In)-NT-VI, DTPA(In)-NT-XI, DTPA(In)-NT-20.1, DTPA(In)-NT-20.2, and DTPA(In)-NT-20.3, were further evaluated for stability and tumor targeting in vivo. In Vivo and in Vitro Peptide Catabolism. The fraction of radioactivity associated to the intact DTPA(111In)-peptide in serum and in urine was determined 15 min after iv injection to BALB/c mice (Table 3). Samples were analyzed by C18 RPHPLC chromatography. Metabolites eluted at shorter retention times than the full-length radioactive peptide. The nonstabilized peptides [Lys6(DTPA(In))]-NT and DTPA(111In)-NT-20.1 were rapidly catabolized (Figure 1, Table 3). Protection of Arg8-Arg9 (DTPA(111In)-NT-VI) or Tyr11-Ile12 (DTPA(111In)-NT-20.2) bonds improved slightly the stability. Doubly stabilized peptides were much more resistant. Higher amounts of intact tracer were recovered in serum and about 20% of the intact peptide was excreted in urine. The in vitro stability in human serum was also evaluated for 111 In-labeled NT(6-13) analogues and for the reference peptide (Figure 2, Table 3). In agreement with the in vivo results, the unprotected peptide DTPA(111In)-NT-20.1 was very rapidly degraded and DTPA(111In)-NT-20.3 was more stable than DTPA(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 [Lys6(DTPA(111In))]NT displayed an in vitro stability higher than that of the monostabilized DTPA(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 (9). In Vitro Internalization. As shown in Figure 3, DTPA(111In)NT-20.3 internalized rapidly in HT29 cells, reaching a 86 ( 3% plateau with a t1/2 of 2.1 ( 0.4 min. Similar internalization curves were obtained with [Lys6(DTPA(111In))]-NT (t1/2 4.2 ( 1.1 min, plateau: 88 ( 6%) and DTPA(111In)-NT-VI (t1/2: 3.8 ( 1.2 min, plateau: 82 ( 6%). Biodistribution and Imaging Studies. The results of biodistribution studies performed in female nude mice grafted with HT29 cells are presented in Tables 4 and 5. They are expressed as the percentage of injected dose per gram of tissue (%ID/g). DTPA(111In)-NT-20.3 displayed the highest tumor uptake, about 3-fold higher than that of [Lys6(DTPA(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). The difference observed between

Figure 1. In vivo serum stability of DTPA(111In)-peptides: representative C18 HPLC chromatograms of plasma samples collected 15 min postinjection to mice. (A) reference peptide and NT(8-13) analogues, (B) NT(6-13) analogues. Arrows show the intact peptide retention time identified by a coinjection of the sample with the radioactive control. Results are expressed as the fraction of radioactivity (%) which eluted with the intact peptide.

Figure 2. Degradation kinetics of DTPA(111In)-peptides in human serum. Peptides (2 pmol) were incubated with human serum (100 µL) at 37 °C: ∆, [Lys6(DTPA(In))]-NT; 2, DTPA(In)-NT-20.1; 0, DTPA(In)-NT-20.2; 9, DTPA(In)-NT-20.3 (mean ( sem, three independent experiments).

tumor retention at 1 h and 3 h postinjection for DTPA(111In)NT-20.3 was not statistically significant, indicating a slow washout of radioactivity from the tumor, confirmed by the 0.33 ( 0.04%ID/g tumor uptake observed 100 h postinjection (Figure 4). Radioactivity uptake of other peptides in tumor was lower than that of [Lys6(DTPA(111In))]-NT, but the difference was not significant. Tumor uptake of [Lys6(DTPA(111In))]-NT or DTPA(111In)NT-20.3 was receptor mediated, since it was significantly reduced by coinjection of their unlabeled counterpart (78% reduction, P ) 0.02; and 94% reduction, P < 0.0001; respectively). Radioactivity in blood at 1 h postinjection was significantly higher for DTPA(111In)-NT-20.3 and [Lys6(DTPA(111In))]-NT

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of higher activity uptake in tumor and lower accretion in kidney for DTPA(111In)-NT-20.3. Accumulation of DTPA(111In)-NT-20.3 was clearly observed in tumors in planar at early (30 to 60 min, Figure 5) and late time points, 24, 48, and 100 h (not shown), postinjection, and in tomographic images recorded in male mice (Figure 6). Kidneys and bladder were the only other sites of activity accumulation. 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). Figure 3. Internalization of DTPA(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).

than for other peptides. It decreased rapidly with time for both peptides. Radioactivity excretion in urine was fast and amounted to 69 ( 4% of the injected dose 1 h after injection for DTPA(111In)-NT-20.3. Low activity accretion was observed in normal tissues for all peptides except in kidneys and, particularly for DTPA(111In)-NT-20.3, in gastrointestinal tract. Nevertheless, for DTPA(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 at 1 h and 3.5 ( 0.6 at 3 h), and colon (8.3 ( 0.8 at 1 h and 3.0 ( 0.5 at 3 h). We investigated the basis of the gastrointestinal uptake of DTPA(111In)-NT-20.3 (Table 5). In contrast to colon uptake, which was significantly decreased by coinjection of the unlabeled analogue (P ) 0.004), stomach and small intestine uptakes were not significantly reduced by the coinjection, 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, coinjection of DTPA(111In)-NT-20.3 with its unlabeled counterpart significantly decreased uptake at 3 h postinjection 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. Kidney uptake of DTPA(In)-NT-20.3 in female nude mice was significantly lower than that of [Lys6(DTPA(In))]-NT and significantly higher than that of other tested peptides at 1 and 3 h postinjection, with the exception of DTPA(111In)-NT-XI for which the difference was not significant. As already reported in the literature for labeled octreotide analogues (32), kidney uptake of DTPA(111In)-NT-20.3 was lower in males than in females. The difference was not significant at 6 h (2.8 ( 0.3% ID/g vs 3.6 ( 0.4%) but was highly significant at 100 h (0.83 ( 0.05% ID/g vs 0.48 ( 0.08, P ) 0.002) (Figure 4). Therefore, tumor to kidney uptake ratio was higher in male than in female mice: at 6 h post injection (0.72 ( 0.09 vs 0.45 ( 0.03) and at 100 h (0.86 ( 0.13 vs 0.40 ( 0.04). DTPA(111In)-NT-20.1 displayed the lowest renal accretion of the peptides tested in this study with 1.4 ( 0.25%ID/g as soon as 1 h post injection, but tumor uptake was also lower. For DTPA(111In)-NT-20.3, tumor to normal tissues uptake ratios were elevated for most organs; in particular, tumor/ pancreas ratio was 17.5 ( 0.8 and 68.2 ( 6.5 at 1 and 3 h postinjection, respectively. They were markedly improved as compared to [Lys6(DTPA(111In))]-NT, particularly tumor/blood (60.5 ( 6.8 vs 10.9 ( 1.7 P < 0.0001 at 3 h postinjection), 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) ratios. Tumor to kidney uptake ratio was also improved about 5-fold (0.49 ( 0.04 vs 0.11 ( 0.01 P < 0.0001, 3 h postinjection) as a result

DISCUSSION For imaging and therapy of neuroendocrine tumors, indisputable successes have been obtained with somatostatin analogues labeled with radiometals, such as 111In, 68Ga, 90Y, or 177Lu, PET imaging with 68Ga potentially providing higher diagnostic efficacy than SPECT (33, 34) and therapy with 90Y or 177Lu affording symptomatic improvement, prolonged survival, and better quality of life in some instances (35-37). NTS1 ligands bearing polyamino polycarboxylate chelators such as DTPA or macrocyclicchelatorssuchasDOTAhavealsobeendeveloped(20-22). DTPA is considered a very powerful binder of indium-111 for single-photon emission computed tomography (SPECT), and no data suggest any superiority of macrocyclic ligands for imaging with this radionuclide. By contrast, yttrium-90 has been shown to leak out of DTPA too rapidly for therapeutic applications (19). Macrocyclic polyamino polycarboxylates such as DOTA are suitable chelators for 90Y and 177Lu, which afford promising treatment options for patients with inoperable or metastasized neuroendocrine tumors using DOTA-substituted somatostatin analogues (35-37). The situation with gallium is less clear: DTPA was used as a chelating agent in different gallium-labeled molecules (16-18). In vitro, the exchange of 111 In and 67Ga from DTPA-albumin to transferrin occurs at approximately the same rate, which would be insignificant in studies with 68Ga, a short half-life (68 min) isotope (17). In addition, the DTPA-albumin gallium-68 complex was found stable enough in vivo over the time span of studies possible with 68Ga, since only 2% 68Ga is transferred to transferrin 1 h after injection (17). Nevertheless, gallium-labeled DTPAoctreotide provided somewhat disappointing results (38). Many publications also deal with gallium-labeled DOTA-substituted somatostatin analogues (38) prepared for therapeutic application with yttrium-90 in the first place. The aim of the present study was to design new polyamino polycarboxylic acid-substituded neurotensin radioligands providing higher tumor uptake and higher tumor to normal tissue uptake ratios, particularly for kidneys, than DTPA or DOTA neurotensin analogues previously described in the literature. To achieve this goal, intermediate objectives were (i) to better understand structure-activity relationships, particularly the effect on affinity induced by coupling the chelating agent and by amino-acid sequence modifications and (ii) to evaluate the relative impacts of affinity and stability on in vivo tumor uptake. In a first step, DTPA was the chelating agent since high radiolabeling efficiency under mild conditions can be obtained (19) and little difference is usually observed in terms of affinity, biodistribution, tumor accretion and tumor to nontumor uptake ratios between DTPA and DOTA-carrying peptides, when labeled with indium-111 (21). We synthesized a series of DTPApeptides with the same sequence as the 99mTc- or 188Re-labeled (NRHis)Ac-NT(8-13) derivatives, which have already been described and which have shown promising targeting properties and encouraging preclinical therapy results (10-13). We also synthesized analogues of the C-terminal (6-13) part of [Lys6(DTPA(In))]-NT, which provided encouraging in vivo tumor

NTS1 Radioisotope Targeting

Bioconjugate Chem., Vol. 20, No. 8, 2009 1607

Table 4. Tissue Distributions of [Lys6(DTPA(111In))]-NT and the DTPA(111In)-NT(8-13) Analogues in Female Nude Mice Grafted with HT29 Cells [Lys6(DTPA(111In))]-NT

DTPA(111In)-NT-VI

DTPA(111In)-NT-XI

b

uptake (%ID/g)a

1hn)6

3hn)9

3 h blocked n)8

blood lungs liver spleen stomachc small intestinec large intestinec muscle bone tumor kidney

0.63 ( 0.12 0.44 ( 0.06 0.22 ( 0.03 0.19 ( 0.02 2.46 ( 2.01 0.69 ( 0.09 0.16 ( 0.02 0.14 ( 0.03 0.13 ( 0.03 1.02 ( 0.26 12.50 ( 1.63

0.06 ( 0.01 0.07 ( 0.01 0.16 ( 0.07 0.07 ( 0.01 0.26 ( 0.15 0.59 ( 0.30 0.71 ( 0.16 0.03 ( 0.01 0.06 ( 0.01 0.71 ( 0.18 9.28 ( 0.73

0.04 ( 0.01 0.07 ( 0.01 0.09 ( 0.01 0.45 ( 0.37 0.15 ( 0.09 0.20 ( 0.09 1.05 ( 0.47 0.03 ( 0.01 0.03 ( 0.01 0.22 ( 0.02 7.18 ( 0.48

3.3 ( 2.1 5.8 ( 2.3 10.4 ( 4.8 0.08 ( 0.02

10.9 ( 1.7 9.3 ( 0.8 33.1 ( 4.1 0.11 ( 0.01

1hn)3

1hn)3

0.24 ( 0.13 0.21 ( 0.06 0.14 ( 0.06 0.10 ( 0.02 0.06 ( 0.02 0.28 ( 0.05 0.17 ( 0.04 0.11 ( 0.04 0.17 ( 0.05 0.62 ( 0.06 2.80 ( 0.37

0.28 ( 0.02 0.37 ( 0.02 0.19 ( 0.02 0.18 ( 0.01 0.14 ( 0.02 0.38 ( 0.07 0.19 ( 0.04 0.16 ( 0.08 0.13 ( 0.03 0.52 ( 0.23 3.90 ( 0.59

4.9 ( 2.5 6.2 ( 2.3 9.6 ( 5.6 0.20 ( 0.04

2.0 ( 0.9 3.0 ( 1.4 3.4 ( 1.6 0.16 ( 0.07

Tumor(T)/Organ T/blood T/liver T/muscle T/kidney

5.7 ( 0.5 2.8 ( 0.6 8.9 ( 1.3 0.03 ( 0.01

a Uptake is expressed as the percentage of injected dose per gram of tissue (%ID/g). b Blocked animals received a coinjection of the labeled peptide with neurotensin (60 nmol). c Organ with its content.

Table 5. Tissue Distributions of the DTPA(111In)-NT(6-13) Analogues in Female Nude Mice Grafted with HT29 Cells DTPA(111In)-NT-20.1

DTPA(111In)-NT-20.2

DTPA(111In)-NT-20.3

uptake (%ID/g)

1hn)3

3hn)6

1hn)5

3hn)5

1hn)6

3 h n ) 15

3 h blockedb n ) 4

blood lungs liver spleen stomach (with content) small intestine (with content) large intestine (with content) stomach (without content) small intestine (without content) large intestine (without content) muscle bone tumor kidney

0.19 ( 0.03 0.17 ( 0.01 0.11 ( 0.01 0.08 ( 0.01 0.13 ( 0.04

0.03 ( 0.00 0.04 ( 0.01 0.06 ( 0.01 0.05 ( 0.01 0.02 ( 0.01

0.31 ( 0.06 0.30 ( 0.04 0.14 ( 0.01 0.12 ( 0.01 0.42 ( 0.17

0.02 ( 0.01 0.10 ( 0.04 0.07 ( 0.01 0.06 ( 0.01 0.04 ( 0.01

0.70 ( 0.09 0.73 ( 0.04 0.39 ( 0.04 0.31 ( 0.01 0.66 ( 0.19

0.04 ( 0.01 0.17 ( 0.03 0.17 ( 0.05 0.11 ( 0.01 0.17 ( 0.04

0.04 ( 0.01 0.12 ( 0.01 0.08 ( 0.01 0.09 ( 0.01 0.14 ( 0.04

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

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

ND

ND

ND

ND

ND

0.21 ( 0.03

0.09 ( 0.02

ND

ND

ND

ND

ND

0.78 ( 0.10

0.10 ( 0.03

ND

ND

ND

ND

ND

0.45 ( 0.04

0.09 ( 0.01

0.07 ( 0.01 0.07 ( 0.01 0.46 ( 0.06 1.44 ( 0.25

0.01 ( 0.01 0.03 ( 0.01 0.49 ( 0.12 1.36 ( 0.10

0.07 ( 0.01 0.43 ( 0.22 0.93 ( 0.32 2.55 ( 0.24

0.01 ( 0.01 0.03 ( 0.01 0.46 ( 0.09 1.97 ( 0.26

0.16 ( 0.01 0.22 ( 0.05 3.27 ( 0.21 7.49 ( 0.54

0.03 ( 0.01 0.11 ( 0.02 2.38 ( 0.21 4.85 ( 0.25

0.04 ( 0.01 0.28 ( 0.11 0.14 ( 0.03 4.81 ( 0.63

T/blood T/liver T/muscle T/pancreas T/kidney

2.5 ( 0.3 4.3 ( 0.4 7.1 ( 1.9 ND 0.32 ( 0.02

18.8 ( 4.7 8.5 ( 1.7 35.6 ( 8.3 ND 0.37 ( 0.04

4.6 ( 2.8 6.6 ( 2.1 14.8 ( 6.7 ND 0.35 ( 0.08

5.6 ( 1.5 8.8 ( 0.6 20.8 ( 1.4 17.5 ( 0.8 0.44 ( 0.03

60.5 ( 6.8 19.1 ( 1.5 91.6 ( 8.6 68.2 ( 6.5 0.49 ( 0.04

3.7 ( 0.8 1.7 ( 0.2 4.2 ( 1.0 ND 0.03 ( 0.01

a

Tumor(T)/Organ 19.4 ( 3.7 6.5 ( 0.9 34.0 ( 8.0 ND 0.23 ( 0.01

a Uptake is expressed as the percentage of injected dose per gram of tissue (%ID/g). b Blocked animals received a coinjection of the labeled peptide with NT-20.3 (180 nmol).

targeting. The R-NH2 was acetylated to protect against aminopeptidases and to lower the number of positive charges, which may increase renal uptake (28, 29). By contrast to the 5-fold affinity increase for HT29 cells provided by the introduction of [99mTc(CO)3]-(NR-His)Ac at the N-terminal end of NT(8-13) (39), DTPA(In) coupling led to an important loss of affinity, which was minimized by increasing the distance between DTPA and the NT(8-13) sequence. This observation may be attributed to steric hindrance due to the DTPA(In) complex, but also to structural changes induced by DTPA: it was recently reported that coupling DOTA to a 13 aminoacid peptide induced an R-helix structure, which was not observed in the free peptide (40). This possibility remains to be explored with NT analogues.

In the DTPA(In)-NT(8-13) series, the influence on affinity of most of the sequence modifications is in agreement with previously reported results for the [99mTc(CO)3]-(NR-His)AcNT(8-13) analogues (10, 12, 13). On the contrary, the loss of affinity induced by a Tle12 substitution, also observed in the NT (6-13) peptides and already reported in the literature for DTPA analogues (20), was not observed with the 99mTc-labeled NT(8-13) derivatives. As a result of the effect of DTPA coupling and Tle12 substitution, DTPA-NT-XIX displayed a low affinity, in opposition to its [99mTc(CO)3]-(NR-His)Ac counterpart (13). Since the affinity of NT-20.1 was higher than that of NT, the double-stabilized DTPA(In)-NT-20.3 exhibited an affinity in the low nanomolar range despite DTPA coupling and sequence modifications.

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Figure 4. Tumor and kidney uptake in female and male nude mice, grafted with HT29 cells, after iv injection of DTPA((111In)-NT-20.3. Uptake is expressed as the percentage of injected dose per gram of tissue (%ID/g, mean ( SEM). **: P < 0.005.

Figure 6. SPECT/CT imaging of a male nude mice grafted with HT29 cells in the right flank 2.5 h postinjection of DTPA(111In)-NT-20.3. Left, CT; center, SPECT; right, SPECT/CT fused images. Frames: A, coronal; B, axial; C, sagittal. Abbreviations as in Figure 5 and r, right; l, left; a, anterior; p, posterior. Tumor weight: 498 mg.

Figure 5. DTPA(111In)-NT-20.3 planar images of a male nude mice grafted with HT29 cells. (A) photograph, (B) planar image, (C) 5 min frames, acquisition from 0 to 60 min postinjection under anesthesia. Bl, bladder; K, kidney; T, tumor. Tumor weight: 240 mg.

As expected, sequence modifications increased the in vitro and in vivo stability to enzymatic degradation and the peptides with two protected bonds were the most resistant. In vivo, increased stability had a major impact on tumor uptake in the DTPA-NT(6-13) series as already reported in the literature for bombesin analogues (41). Nevertheless, higher in vivo stability associated to lower affinity of DTPA(In)-NT-XI led to much lower tumor uptake as compared to DTPA(In)-NT-20.3. Charge and charge distribution of radiolabeled peptides may produce various effects on renal uptake, but in general, it is increased by positive charges (28, 29). One objective of the present study was to lower kidney uptake as compared to the reference peptide. [Lys6(DTPA(111In))]-NT may, after cleavage in the 1-6 N-terminal end, release labeled metabolites with a free positively charged R-NH2, which could contribute to the high kidney uptake. To avoid the formation of these metabolites, we deleted the 1-6 N-terminal part of the molecule and acetylated its N-terminal end to neutralize the positive charge. The difference between the remarkably low renal accretion of DTPA(111In)-NT-20.1, compared to DTPA(111In)-NT-20.3, cannot be related to their charge or charge distribution at physi-

ological pH which are identical. It may not be attributed to differences of hydrophobicity, as all peptides eluted with approximately the same acetonitrile percentage in HPLC (data not shown), indicative of similar hydrophobicity (29). It may only be ascribed to reabsorption and accumulation of metabolites. Cleavage of DTPA(111In)-NT-20.1 at the Arg8-Arg9 bond may produce labeled metabolites with only one positive charge (Arg8). The same is true for DTPA(111In)-NT-20.2, which also exhibits low renal uptake. In DTPA(111In)-NT-20.3, the Arg8Arg9 bond is stabilized, and it may be hypothesized that metabolites with two positively charged Arg are released that induce higher renal accumulation of radioactivity.

CONCLUSION In summary, structure-activity studies of new NT analogues carrying DTPA have shown that this polyamino polycarboxylate chelating agent induces an important loss of affinity unless the distance between the chelating agent and the NT(8-13) sequence that binds to the NTS1 receptor is increased. Altogether, it was not possible to design a peptide with both the lowest kidney retention and highest tumor uptake, but DTPA(111In)-NT-20.3 is a good compromise with improved uptake ratio between tumor and kidneys, superior to previously published DTPA and DOTA neurotensin analogues. In spite of some excretion of activity by the gastrointestinal route and some receptor-mediated binding of the tracer to stomach, small intestine and colon, ratios between uptakes in the tumor and in these organs was high. The tumor to intestines ratio was higher than that of the 99mTclabeled analogue NT-XIX described in the literature (12). In spite of a relatively high uptake in kidneys, DTPA(111In)NT-20.3 showed specific tumor uptake and afforded high-

NTS1 Radioisotope Targeting

contrast planar and SPECT tumor images in nude mice. It may be considered as a promising candidate for imaging neurotensin receptor positive tumors, such as pancreatic adenocarcinoma and invasive ductal breast cancer. If therapeutic application of this neurotensin analogue is developed, infusion of basic amino acids, gelofusin, and albumin fragments may be considered to prevent nephrotoxicity, as with radiolabeled somatostatin analogues (42, 43). Thus, an analogue carrying DOTA is being developed for therapy with 90 Y or 177Lu. 68Ga PET imaging with DOTA- and DTPANT20.3 will be evaluated.

ACKNOWLEDGMENT The authors are grateful to E. Treca, S. Mendes, H Medjoudoum, A Darbois, and A. Augereau for help in affinity determination and to Pr. M. A. Pocidalo for providing human serum. This work was supported by Inserm, by the Association pour la Recherche sur le Cancer (ARC), by the Cance´ropole Ile de France and by the Fund for Scientific Research Flanders (FWO). V. M. is a postdoctoral researcher of the Fund for Scientific Research-Flanders (FWO). We thank the Syrian government for awarding a research fellowship to F. A.

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