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Preclinical Comparison in AR4–2J Tumor-Bearing Mice of Four Radiolabeled 1,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetraacetic Acid-Somatostatin Analogs for Tumor Diagnosis and Internal Radiotherapy¹

Department of Research-ZLF (S.F., A.N.E., A.-M.M., M.H.), Department of Radiology and Radiopharmacy Unit (A.H., M.B., P.P., H.R.M.), and Department of Internal Medicine (C.B.); University Hospital and University Children’s Hospital, CH-4031 Basel, Switzerland

Address all correspondence and requests for reprints to: Dr. S. Froidevaux, Department of Research, Kantonsspital Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland. E-mail: sylvie.froidevaux{at}unibas.ch

	Abstract

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Somatostatin analogs labeled with radionuclides are of considerable interest in nuclear oncology as diagnostic or therapeutic tools for somatostatin receptor (SSTR)-expressing tumors. We investigated the suitability of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as a replacement for the widely used diethylenetriaminepentaacetic acid, to enable stable labeling of somatostatin analogs with both therapeutic (⁹⁰Y) and diagnostic (¹¹¹In) radionuclides. The three clinically relevant somatostatin agonists, octreotide, vapreotide, and lanreotide, together with the newly designed Tyr³-octreotide (TyrOc), were conjugated to DOTA and labeled with ⁹⁰Y or ¹¹¹In. For all DOTA-somatostatin analogs tested, irrespective of the incorporated radionuclide, we observed favorable biodistribution profiles in AR4–2J tumor-bearing mice: 1) a rapid clearance from all SSTR-negative tissues except kidney; 2) a specific uptake in SSTR-positive tissues, including tumor; and 3) an excellent tumor penetration. The main route of excretion was via the kidneys. Nevertheless, DOTATOC was clearly superior to the other DOTA-somatostatin analogs tested, as well as OctreoScan, as indicated by the highest tumor-to-nontarget-tissue ratio, including the tumor-to-SSTR-positive-tissue ratios. The presence of different SSTR subtypes in the SSTR-positive tissues possibly contributes to these differential uptakes. We assume that the very favorable behavior of DOTATOC in our mouse model makes this radioligand very promising for future applications in nuclear oncology.

	Introduction

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SOMATOSTATIN IS A peptide hormone, initially found in the hypothalamus (1) and pancreas, which has a wide spectrum of actions on multiple organs such as brain, gastrointestinal tract, pancreas, thyroid, and pituitary gland (2). The effect of somatostatin is mediated by specific receptors in the target organs. High-affinity somatostatin receptors (SSTRs) are overexpressed by a variety of neuroendocrine tumors and their metastases (3, 4), making this peptide an attractive candidate for tumor targeting. Because the native peptide is biologically unstable and thereby not useful for in vivo applications, somatostatin analogs that are more resistant to enzymatic degradation were developed, e.g. the octapeptide octreotide (5). This peptide was the first successful ligand for imaging SSTR-positive tumors in patients by camera scintigraphy after radiolabeling either with ¹²³I after substitution of Phe³ by Tyr, or with ¹¹¹In after conjugation of the chelator diethylenetriaminepentaacetic acid (DTPA) to octreotide, yielding DTPA-octreotide (OctreoScan) (6, 7, 8, 9, 10).

This success prompted us and others to envisage the use of radiolabeled somatostatin analogs for selective internal radiotherapy (9, 11, 12, 13, 14). For this purpose, the choice of the appropriate radionuclide is critical. Among potential candidates, ⁹⁰Y is of special interest because of its well-developed coordination chemistry, its ready availability from a ⁹⁰Sr/⁹⁰Y generator, and its physical characteristics (including a 64.1-h half-life and a pure ß-emission of high energy. ¹¹¹In can be another suitable radionuclide because, besides its -emitting properties, it is also an Auger emitter that makes it interesting for treatment of micrometastases if one can direct it into the cell nucleus.

Whatever the selected radiometal, the success of internal therapy depends on the accumulation of high amounts of radioactivity within the tumor and metastases, along with a rapid clearance of radioactivity from the other tissues. This may be possible to achieve by the development of radiolabeled somatostatin analogs with very high affinity for SSTRs, favorable biodistribution profile regarding hepatic and kidney clearance, and sufficient stability to prevent nonspecific uptake in nontarget organs attributable to radiometal loss in vivo. Initial attempts to label antibodies or small molecules with ⁹⁰Y for radiotherapeutic applications, using DTPA or DTPA derivatives as the chelating agent, have been hampered by the dose-limiting myelotoxicity attributable to the in vivo instability of the ⁹⁰Y-chelator complex and subsequent uptake of the released ⁹⁰Y into the bone (15, 16, 17, 18).

To circumvent this problem, we used the macrocyclic DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as radiometal chelator. DOTA, as opposed to DTPA or DTPA-derivatives, has proved to form stable metal complexes with various metals when conjugated to monoclonal antibodies (16, 17, 19, 20). As somatostatin analog, we assessed the three clinically relevant somatostatin agonists, namely octreotide, vapreotide, and lanreotide. Furthermore, we designed a fourth somatostatin analog consisting of octreotide with Tyr substituted for Phe³, with the aim of: 1) increasing peptide hydrophilicity, thus presumably kidney excretion; and 2) allowing concomitant labeling, i.e. iodination of Tyr³, which can be of additional benefit in therapeutic applications if the Auger-emitter ¹²⁵I or the ß-emitter ¹³¹I is used. These somatostatin analogs show little difference in their binding properties, i.e. a high or intermediate affinity for SSTR2, SSTR3, and SSTR5 and no or very-low affinity for SSTR1 and SSTR4 (21). Therefore, they constitute attractive targeting peptides for cancer cells expressing SSTR2 that have been detected at high levels in a wide range of human tumors (22).

In this study, we compare the aforementioned somatostatin analog constructs, DOTA⁰-(D)Phe¹-octreotide (DOTAOc), DOTA⁰-(D)Phe¹-Tyr³-octreotide (DOTATOC), DOTA⁰-(D)ßNal¹-lanreotide (DOTALan), and DOTA⁰-(D)Phe¹-vapreotide (DOTAVap). They were evaluated with regard to their in vivo behavior in AR4–2J tumor-bearing mice after radiolabeling with either ⁹⁰Y or ¹¹¹In. This included biodistribution in the presence or absence of an excess of cold octreotide and tumor penetration, as indicated by the ratio tumor uptake to tumor size. Furthermore, because we observed intriguing differences in the uptakes of the four radiolabeled constructs in the SSTR-positive tissues (i.e. tumor; adrenals; and, to a lesser extent, pancreas), we investigated the pattern of SSTR subtype expression in these tissues, by reverse transcriptase PCR (RTPCR), to see whether there is a correlation between the retention of a somatostatin analog and the expression of a given SSTR subtype. Finally, we report the in vitro binding affinity of the four Y-DOTA-somatostatin analogs for AR4–2J tumor membranes.

Overall, our results show the superiority of radiolabeled DOTATOC over the other DOTA-somatostatin analogs for targeting SSTR2-expressing malignancies, as clearly indicated by the highest binding affinity for AR4–2J membranes correlated in vivo with the highest tumor-to-kidney uptake ratio. This makes this radioligand very promising for both internal radiotherapy and tumor diagnosis.

	Materials and Methods

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Radioligands
The structure of OctreoScan and DOTA-somatostatin analogs used in this study is shown in Fig. 1. Vapreotide and Lys⁵(BOC)-octreotide were received from Debiopharma (Lausanne, Switzerland) and Sandoz Pharmaceuticals Corp. Pharma (Basel, Switzerland), respectively. The DOTA⁰-(D)Phe¹-octreotide (DOTAOc), DOTA⁰-(D)ßNal¹-lanreotide (DOTALan), DOTA⁰-(D)Phe¹-Tyr³-octreotide (DOTATOC), and DOTA⁰-(D)Phe¹-vapreotide conjugates were synthesized as previously reported (23). Incorporation of the radiometals ⁹⁰Y or ¹¹¹In was performed as indicated in a previous publication (23). Briefly, the DOTA-somatostatin analogs were dissolved in sodium acetate buffer, pH 4.8–5.5, and heated, at 95 C for 25 min, after addition of either ¹¹¹InCl₃ (in 0.1 M HCl; Mallinckrodt, Inc., Petten, The Netherlands) or ⁹⁰YCl₃ (in 0.05 M HCl; Pacific Northwest National Laboratory, Hanford, WA). DTPA-(D)Phe¹-octreotide was labeled at room temperature. The resulting radioligands were of high purity, as indicated by HPLC quality controls, and the specific activity was always greater than 40 GBq/µmol.

View larger version (21K): [in this window] [in a new window]   Figure 1. Structure of OctreoScan and DOTA-somatostatin analogs used in this study.

HPLC analysis
HPLC was performed on a Hewlett Packard 1050 chromatography system (Hewlett Packard Co., Palo Alto, CA) connected to a flow-through Berthold LB506C1 -detector (Berthold Ltd., Bundoora, Australia) and a C18-RP (Hewlett Packard Co.) column (Vydac 218TP54, Western Analytical Products Inc., Murrieta, CA). The conditions were as followed: eluent A = 0.1% trifluoroacetic acid in water; eluent B = acetonitrile; gradient = 0–5 min at 100% A, 5–25 min at 100%–70% A, 25–35 min at 70% A, 35–40 min at 70%–0% A, 40–45 min at 0%–100% A; flow 1.5 ml/min.

In vitro binding assay
Octreotide was purchased from Novartis Pharmaceuticals (Basel, Switzerland), and Tyr³-octreotide was obtained from Mallinckrodt, Inc. (St Louis, MO). DOTAOc, DOTATOC, DOTALan, and DOTAVap were labeled with cold yttrium as described above. Their precise concentration was determined by OD measurement ( = 280 nm) after determination of the respective molar extinction coefficient by two methods: 1) assessment of the optical density at = 280 nm of a mixture of all amino acids plus chelator; and 2) labeling experiment using a defined excess of radiometal (M¹), in the presence of DTPA, followed by a comparison of the two peaks (M¹-DTPA vs. M¹-DOTA-peptide) obtained after injection in a HPLC system connected to a -detector. Stock solutions were prepared in 0.01 M acetic acid at 0.5–10 mM and further diluted in binding medium consisting of modified Eagle’s medium supplemented with 25 mM HEPES, 0.4% BSA and 1 mM 1,10-phenanthroline. The stock solutions, as well as the dilutions, were made in prelubricated microcentrifuge tubes (Costar, Cambridge, MA) to minimize loss by absorption. Competitive binding experiments were performed in BSA-precoated 96-well plates, as previously described (24), by using ¹²⁵I-Tyr³-octreotide as radioligand and AR4–2J tumor membranes as a source of SSTR2. IC₅₀ (inhibitory concentration 50%) was calculated using the Prism software (GraphPad Software, Inc., San Diego, CA).

SSTR messenger RNA (mRNA) expression in tissues
Total RNA was extracted from adrenals, pancreas, or AR4–2J tumors implanted into female Swiss nude mice, in 10 vol TriZOL reagent (Life Technologies, Basel, Switzerland), according to the manufacturer’s protocol. Fifty micrograms of total RNA were subjected to a DNase RQ1 (Promega Corp., Madison, WI) digest, followed by a classical phenol/chloroform extraction. First-strand complementary DNA was produced by Moloney murine leukemia virus reverse transcriptase (Promega Corp.), using 1 µg total RNA and 200 ng oligo(dT)15, in a final vol of 20 µl. For each RNA preparation, genomic DNA removal was controlled by omitting reverse transcriptase. The first-strand complementary DNA (2 µl) was then amplified by PCR in 20-µl reaction volumes containing 0.05% Tween 20, 5% dimethylsulfoxide, 250 µM of each deoxynucleotide triphosphate, 1 U Taq DNA Polymerase (Boehringer, Mannheim, Germany), 1X Taq PCR reaction buffer (Boehringer), and 1 µM of each primer. Primer pair sequences were as followed: 5'-tcagctgggatgttccccaatg-3' and 5'-gtcgtcttgctcggcgaacacg-3' for mouse/rat SSTR1, 5'-caccgccccctctcccacct-3' and 5'-gtcgtcttgctcggcgaacacg-3' for rat SSTR1, 5'-tgggtgtcctctccatttg-3' and 5'-gattgatgccatctacagtc-3' for mouse SSTR2, 5'-gaggacacgatggcctgg-3' and 5'-cacgcgcggaactttga-3' for rat SSTR2, 5'-ctgggaacacatcctcgacc-3' and 5'-taggacagggcgttctgagc-3' for mouse SSTR3, 5'-tctcggcgagtacggagcca-3' and 5'-acagatggctcagcgtgctg-3' for rat SSTR3, 5'-gatgccactgtcaaccatgtgtccct-3' and 5'-gtcctacccccatggaggtg-3' for mouse SSTR4, 5'-gatgccactgtcaaccatgtgtccct-3' and 5'-acggagttgtccttggagccagtcag-3' for rat SSTR4, 5'-agatacatgtgctctggcat-3' and 5'-aggatgtacacattagtaac-3' for mouse SSTR5, 5'-gacacgcgtggtctggcacc-3' and 5'-aataatacgtcagccacggc-3' for rat SSTR5. After the initial denaturation step, at 95 C for 3 min, amplifications were carried out for 60 cycles, with a final 10-min elongation step at 72 C. Each cycle consisted of denaturation at 95 C for 45 sec; annealing at 62 C for SSTR1/SSTR3/rat SSTR5, 58 C for SSTR2/SSTR4, 55 C for mouse SSTR5 for 1 for 45 sec, and elongation at 72 C for either 90 sec (SSTR1) or 45 sec (SSTR2–5). As positive and species-specificity controls, 100 ng of either rat or mouse genomic DNA (CLONTECH Laboratories, Inc., Palto Alto, CA) were used, because SSTRs lack introns. Integrity of RNA was controlled with actin primers. PCR products were then visualized with ethidium bromide after electrophoresis on 1–2% agarose gel.

Biodistribution in AR4–2J tumor-bearing mice
All animal experiments were performed in compliance with the Swiss regulation for animal treatment.

Standard protocol. Female Swiss nude mice (6–10 weeks old, IFFA-CREDO, L’ Arbresle, France) were implanted sc with 5 million AR4–2J cells (ATCC, Manassas,), previously expanded in DMEM medium containing 10% FCS, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. One week later, 5 µCi radioligand, diluted in NaCl, 0.1% BSA (pH 7.5) was injected iv into the lateral tail vein, in 200 µl. For determination of nonspecific uptake, 50 µg cold octreotide was coinjected with the radioligand. The animals were killed at the indicated time points. Organs and tissues of interest were dissected and rinsed of excess blood, weighed, and assayed for radioactivity in a -counter. In the case of ⁹⁰Y, the organs were dissolved in 2 ml 1N HCl, and the Bremsstrahlung was measured in a -counter. Percent of injected dose per gram (I.D./g) was calculated for each tissue. The total counts injected per animal were calculated by extrapolation from counts of a standard taken from the injected solution for each animal.

Comparison of preblockage vs. coblockage. CB17 scid mice (6–7 weeks old, breeding pairs obtained from IFFA-CREDO) were implanted with AR4–2J cells as described above. One week later, the mice were divided into three groups. The first group was given 5 µCi radioligand iv; the second one, a mixture of 50 µg cold octreotide and 5 µCi radioligand; and the third one, 50 µg cold octreotide ip 30 min before 5 µCi radioligand iv. The animals were killed 4 h after radioligand administration and processed as indicated above.

Analysis of data
Unless otherwise stated, results are expressed as mean ± SEM. Statistical evaluation was performed using the one-way ANOVA test. When significant overall effects were obtained by ANOVA, multiple comparisons were made with the Bonferroni correction. A P value less than 0.05 was considered a statistically significant difference.

	Results

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Hydrophobicity of DOTA-somatostatin analogs
Peptide hydrophobicity can influence their biodistribution, particularly with regard to their excretion route; therefore, ¹¹¹In-labeled DOTA-somatostatin analogs were subjected to HPLC. The elution time order was DOTATOC (22.1 min) < DOTAOc (24.2 min) < DOTALan (26.5 min) < DOTAVap (27.3 min), which is indicative of a similar hydrophobicity order (data not shown).

In vitro receptor-binding studies
To characterize the receptor-binding properties of the somatostatin analogs, competitive binding experiments were performed using ¹²⁵I-Tyr³-octreotide as radioligand and AR4–2J membranes. Because some peptides were only available in tiny amounts, a limited number of experiments could be performed; therefore these results have to be considered as preliminary studies. Table 1 lists the IC₅₀ for the four DOTA-somatostatin analogs labeled with cold yttrium. As controls, OC, TyrOc, and unlabeled DOTATOC were included. We used Y-complexed somatostatin analogs because the metal by itself can influence the binding affinity of the complex. The IC₅₀ values were all in the nanomolar range, but Y-DOTATOC showed the highest affinity to SSTR2. The comparison of the IC₅₀ obtained for TyrOc, DOTATOC, and Y-DOTATOC (0.33, 2.44, and 2.37 nM; respectively) showed that DOTA influenced the binding affinity of the somatostatin analog but that incorporation of yttrium did not.

Tumor uptake, as compared with kidney, liver, muscle, and blood uptakes
To better evaluate the potential suitability of the four radiolabeled somatostatin analogs for tumor targeting, the ratios tumor-to-liver, tumor-to-muscle, tumor-to-kidney, and tumor-to-blood were calculated 4 h post injection (Fig. 2). The values obtained for the two radiometals were pooled, because they were statistically similar. These ratio values clearly demonstrated the superiority of DOTATOC, not only over OctreoScan but also over the other DOTA-somatostatin analogs. Indeed, the tumor-to-liver, tumor-to-muscle, tumor-to-kidney, and tumor-to-blood ratios calculated for DOTATOC were 23 x, 44 x, 9.2 x, and 86 x higher than those found with OctreoScan and 8 x, 13 x, 4 x, and 12 x higher than those obtained for the second-best somatostatin analog, i.e. DOTAOc, respectively. It is to be noted, however, that DOTATOC was the only DOTA-somatostatin analog giving a tumor-to-kidney ratio superior to 1.

View larger version (23K): [in this window] [in a new window]   Figure 2. Tumor-to-tissue ratios, 4 h post injection. A, Tumor-to-liver; B, tumor-to-muscle; C, tumor-to-kidney; D, tumor-to-blood ratios. For each DOTA-somatostatin analog, the values obtained for the two radiometal conjugates were pooled. Results are expressed as mean ± SEM. *, P < 0.05 vs. DOTATOC. N, Number of mice.

Specific uptake in tissues
Tumor-bearing mice were either coinjected or preinjected with 50 µg octreotide to determine the specific uptake of DOTATOC in tissues 4 h post injection. As shown in Fig. 3, the uptake of DOTATOC in SSTR-rich tissues, such as tumor or pancreas, represented mainly specific receptor-mediated uptake, because radioligand accumulation was decreased by 93% in the tumor and 81% in the pancreas in SSTR-blocked (compared with unblocked) mice. This injection of octreotide had no statistically significant effects on radioligand uptake in tissues such as liver or small intestine. Stomach and lung uptake was only partially displaceable, indicating that only a fraction of the observed uptake was presumably receptor-mediated. Both blockage protocols, i.e. preblockage or coblockage, were essentially equivalent for all tissues, including SSTR-rich tissues (exemplified by tumor and pancreas), with the exception of the kidney. Surprisingly, whereas preinjection of octreotide did not change kidney uptake of DOTATOC significantly, its coinjection resulted in a 1.7-fold increase in kidney uptake. Similar kidney uptake increases were also observed with the three other DOTA-somatostatin analogs when octreotide was coinjected with the radioligand (not shown).

View larger version (27K): [in this window] [in a new window]   Figure 3. Specific uptake of 111In-DOTATOC in tissues, 4 h post injection. 111In-DOTATOC was injected at + 30 min (preblockage, open columns) or 0 min (coblockage, hatched columns), relative to the injection of an excess of cold octreotide. As control, no octreotide was injected (filled columns). Results are expressed as percentage of control (mean ± SEM, of 3 mice). *, P < 0.05 vs. control; +, P < 0.05 vs. preblockage.

Kinetics of tumor and kidney uptake
Tumor and kidney clearance in tumor-bearing mice of the four DOTA-somatostatin analogs and OctreoScan, from 4–48 h post injection, are presented in Fig. 4. Maximum tumor uptake was observed 4 h post injection for all the radioligands tested. At that time, only DOTATOC and DOTALan showed a statistically higher tumor uptake than that of OctreoScan. DOTALan was then cleared rapidly from the tumor (25% of maximum remaining at 24 h), resulting in a tumor uptake that was essentially equivalent to that of OctreoScan by 24 and 48 h. In contrast, DOTATOC showed slower tumor clearance (62% of maximum remaining at 24 h); and therefore, its retention in the tumor remained the highest during the observation period of 48 h.

View larger version (40K): [in this window] [in a new window]   Figure 4. Tumor and kidney distributions, 4, 24, and 48 h post injection. Tissue radioactivity in tumor (A) and kidney (B). For each DOTA-somatostatin analog, the values obtained for the two radiometal conjugates were pooled. Results are expressed as % I. D./g (mean ± SEM, of 5–12 mice). P < 0.05 vs. OctreoScan (a), DOTATOC (b), DOTAOc (c), DOTALan (d), DOTAVap (e).

A good indicator for the suitability of the DOTA-somatostatin analogs for internal radiotherapy is the ratio of the area under the curve of tumor-to-kidney. These ratios were calculated for the period ranging from 4–48 h and were as follows: 1.15 for DOTATOC, 0.33 for DOTAOc, 0.29 for DOTALan, 0.22 for DOTAVap, and 0.16 for OctreoScan, indicating again the most favorable behavior for DOTATOC.

Thus, the kinetics of tumor and kidney uptakes strongly argued in favor of DOTATOC as the best candidate for tumor targeting, even though kidney uptake remained high.

Tumor penetration
Tumor penetration was evaluated for each DOTA-somatostatin analog by plotting tumor uptake as a function of tumor size (ranging from 8–2300 mg) (Fig. 5). Tumor uptake was normalized to 100% for each radioligand to compare the four DOTA-somatostatin analogs. For all radioligands, there was no statistically significant linear relationship between tumor size and tumor uptake. This indicates that tumor penetration was excellent for the four DOTA-somatostatin analogs.

View larger version (18K): [in this window] [in a new window]   Figure 5. Tumor uptake as a function of tumor sizes, 4 h post injection. The mean tumor uptake was calculated for each radiolabeled DOTA-somatostatin analog and set as 100%. Individual values are then expressed as a percentage of the mean value calculated for the group. Each symbol represents a single mouse. The slope of the linear correlation curve = -0.0003, R2 = 0.0001, and P = 0.95.

Thus, mouse adrenals expressed mRNA for SSTR2, SSTR3, and SSTR5; and mouse pancreas for SSTR2 and SSTR3. The rat AR4–2J tumor contained only SSTR2 mRNA but was infiltrated by host cells expressing SSTR1and SSTR2 mRNA.

	Discussion

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The major goal of this project was to evaluate several DOTA-somatostatin analogs with regard to their potential use in nuclear oncology. Because DOTA enables the stable chelation of a multitude of 2+ and 3+ charged metals, the DOTA-somatostatin analogs should be suitable for various applications such as single photon emission tomography (e.g. ¹¹¹In), positron emission tomography (e.g. ⁶⁸Ga), and peptide receptor-mediated internal radiotherapy (e.g. ⁹⁰Y). All the DOTA-somatostatin analogs were tested in the same in vitro- and in vivo model to make a reliable comparison of their performances. The SSTR-positive cancer cell line used in this study was the rat pancreatic AR4–2J cell line that was previously shown to express exclusively SSTR2 (26). Given that this SSTR subtype is most abundant in human SSTR-expressing malignancies (27) and that the rat SSTR2 exhibits a 95% homology with the human SSTR2 (28), a considerable clinical relevance can be foreseen.

The success of both the diagnostic and therapeutic strategies relies on the quantity of radioactivity that can be accumulated in the tumor and on its rapid clearance from the other organs. For radiotherapeutic applications, additional criteria have to be taken into account, such as biological half-life of the radioligand in the tumor and tumor penetration, because homogenous radioligand distribution is necessary for complete elimination of the tumor, particularly with low- or medium-energy radionuclides. All DOTA-somatostatin analogs tested showed a comparably favorable behavior in vitro and in vivo: 1) They retained their capacity to bind SSTR2, with IC₅₀ in the nanomolar range, as indicated by displacement of ¹²⁵I-Tyr³-octreotide from AR4–2J tumor membranes; 2) They accumulated preferentially in SSTR-expressing tissues including the tumor; 3) Their main route of excretion was via the kidneys; 4) In contrast to radiolabeled antibodies that were shown to localize only in the periphery of the tumors (29), they diffused within the tumor, as evidenced by the absence of correlation between tumor size and tumor uptake. In addition, our study clearly demonstrated that the new chelator DOTA enables successful labeling of somatostatin analogs with both diagnostic and therapeutic radionuclides. Indeed, for all DOTA-somatostatin analogs tested, the biodistributions of ¹¹¹In- vs. ⁹⁰Y-labeled somatostatin analogs were comparable. The low amounts of radioactivity found in the bones, for the 48-h period of observation, suggested excellent ⁹⁰Y-DOTA complex stability in vivo, because dissociated ⁹⁰Y rapidly accumulates in bone (15). This strongly indicates that DOTA can hold ⁹⁰Y with high stability in vivo and, in that respect, is superior to the DTPA chelator that was reported to form unstable complexes with ⁹⁰Y and thereby caused some bone-marrow toxicity (15). DOTA seems, therefore, to be the chelator of choice, particularly if internal radiotherapy is envisaged.

Despite these overall similarities, one DOTA-somatostatin analog, i.e. DOTATOC, was clearly found to be far superior to the other DOTA-somatostatin analogs, for both diagnostic and therapeutic use. DOTATOC showed the highest tumor uptake at all time points tested. Its rapid clearance from nontarget tissues led to excellent tumor-to-background ratios, mostly superior to the ones obtained with OctreoScan or the other DOTA-somatostatin analogs. Moreover, its accumulation tended to be higher in the tumor than in the normal SSTR-positive organs, which is of importance for radiotherapeutic applications when high doses of radioactivity are injected. Retention of radioactivity in the kidneys was most favorable with DOTATOC. It was indeed the only radioligand tested, which resulted in higher radioactivity exposure of tumor cells than kidney cells, as indicated by a ratio of tumor-to-kidney area under the curve that was greater than 1. Taken together, the remarkable performances of DOTATOC in our mouse tumor model makes it very promising for treatment or imaging of SSTR2-expressing neuroendocrine tumors. It is very likely that renal accumulation, which is still problematic in the clinic, can be lowered by infusion of lysine, as reported previously for several other radiolabeled proteins and peptides (30, 31). Preliminary data confirm the usefulness of this radiolabeled peptide in internal radiotherapy (13, 14).

The highest tumor uptake of DOTATOC was correlated with the highest binding affinity to tumor membrane in vitro. For the other DOTA-somatostatin analogs, there was no direct correlation between binding activity in vitro and accumulation in the tumor. This is very well exemplified by DOTALan, which showed the lowest binding affinity in vitro despite being the second-best radiolabeled DOTAsomatostatin analog, with regard to tumor uptake in vivo. Obviously, other parameters influence tumor uptake of radiopeptides, such as in vivo stability, internalization, and tumor penetration. We demonstrated, in this study, that tumor diffusion of the four DOTA-somatostatin analogs was essentially identical. Radioligand stability has not yet been carefully evaluated, but it is not expected to differ markedly because the four peptides used for coupling with DOTA are stable in vivo. Internalization of DOTATOC, after binding to SSTRs, was described recently (32); but in the absence of data concerning the other DOTA-somatostatin analogs, it is hard to speculate further on the role of internalization in the observed tumor uptake.

An interesting observation arising from this study was that kidney uptake of DOTA-somatostatin analogs was increased when an excess of cold octreotide was coinjected, suggesting the presence of saturable carriers for DOTA-somatostatin analogs in kidney. At this stage of the investigation, we can only speculate on the type of saturable carriers involved in DOTA-somatostatin transport in this organ. A possible candidate is the P-glycoprotein that was shown to be expressed in the proximal tubules (33, 34), where it mediates transport of various compounds, including peptides (35, 36). Ongoing studies are focusing on the identification of DOTATOC carriers in kidney to set up strategies to lower its renal accumulation.

Our investigation of the pattern of SSTR subtype expression in adrenals, pancreas, and tumor (by RTPCR) revealed the presence of different SSTR subtypes, which can presumably explain the differential uptakes of the radioligands tested in SSTR-positive tissues. Adrenals were found to express mRNA for SSTR2, SSTR3, and SSTR5; whereas in the pancreas, only SSTR2 and SSTR3 mRNA were detected. The tumor cells per se contained exclusively SSTR2, but the tumor was infiltrated by cells of host origin expressing mRNA for SSTR1 and SSTR2. To our knowledge, no data are available regarding the distribution of mRNA encoding the five cloned SSTRs in peripheral tissues in mouse. Human adrenals were reported to contain mRNA encoding either all five SSTRs (37) or only SSTR1 and SSTR2 (38); whereas in human pancreas, SSTR1, SSTR2, and SSTR4 mRNA were identified (38). In rat pancreas, SSTR2 mRNA was found to be the most abundant (39), but the presence of SSTR3 mRNA was also noted (39). Taken together, our results obtained in mouse indicate an overall closer similarity with the rat, at least for the pancreas.

The finding that our rat tumor was infiltrated by host cells expressing SSTR1 and SSTR2 mRNA is interesting per se and can have some implications in the observed tumor uptake if these transcripts give rise to significant amounts of corresponding proteins. Additionally, the presence of SSTR mRNA of nontumor origin in tumors can strongly influence the interpretation of the results concerning the identification of SSTR subtype expression in human malignancies, by Northern blotting or RTPCR protocols. Similar host infiltration of SSTR2-mRNA-expressing cells was also observed in scid mice implanted with AR4–2J cells (not shown). The origin of these SSTR1- and SSTR2-mRNA-expressing cells is unclear. These two SSTR subtypes were identified in activated lymphocytes/thymocytes (40, 41, 42); but given the immunodeficient status of the nude/scid mice used as hosts, it is very unlikely that the implanted tumor was infiltrated by lymphocytes. Expression of SSTRs in blood vessels in the immediate vicinity of tumors has been described (43). Nevertheless, further investigations, such as in situ hybridization, would be necessary for further speculation.

Interestingly, based on the recently reported affinity profiles of the DOTA-somatostatin analogs for human SSTR subtypes (25), a good correlation was found between the uptake of DOTA-somatostatin analogs in adrenals, pancreas, and tumor and the observed pattern of SSTR subtype expression in these tissues. For instance, we found that adrenals differed from pancreas by the presence of SSTR5; and, interestingly, the two radioligands exhibiting the highest adrenal-to-pancreas uptake ratio, namely DOTALan and DOTAVap, were reported to display the highest affinity for SSTR5. Similarly, the difference between tumor and pancreas lay in the presence of SSTR3 in the latter and, again, the highest tumor-to-pancreas uptake ratio was obtained with the radioligands exhibiting the lowest SSTR3 affinity, DOTATOC and DOTALan. Clearly this interpretation is based on the assumption that DOTA-somatostatin analogs display a similar affinity profile for mouse and human SSTR subtypes; this needs further clarification, although mouse and human SSTRs share many similarities (between 85% and 98%, depending on the subtype).

In conclusion, our study clearly showed that the use of DOTA enables successful radiolabeling of various somatostatin analogs with both diagnostic and therapeutic radionuclides. DOTATOC is most promising and clearly superior to the other DOTA-somatostatin analogs tested and to the widely used OctreoScan, with regard to both potential applications in nuclear oncology, i.e. tumor localization and internal radiotherapy of SSTR2-expressing malignancies. Even though these results were obtained in an animal model and thus raise the question of whether we can extrapolate them to human, the high homology between the human and the rat SSTR2 (95%) together with the predominance of the expression of this subtype in human malignancies foresee a high clinical relevance.

	Acknowledgments

 
We thank Dr. J. Baumann for her critical review of the manuscript and Dr. I. Virgolini for providing DOTA-lanreotide.

	Footnotes

 
¹ This work was supported by the Swiss Cancer League, the Roche Research Foundation, and the Swiss National Science Foundation.

Received December 30, 1999.

	References

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