Optimised labeling, preclinical and initial clinical aspects of CCK-2 receptor-targeting with 3 radiolabeled peptides.

Medullary thyroid carcinoma (MTC) expresses CCK-2 receptors. (111)In-labeled DOTA-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH(2) (DOTA-MG11), DOTA-DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH(2) (DOTA-CCK), and (99m)Tc-labeled N(4)-Gly-DGlu-(Glu)(5)-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH(2) ((99m)Tc-Demogastrin 2) are analogs developed for CCK-2 receptor-targeted scintigraphy. All 3 radiolabeled analogs were selected on the basis of their high CCK-2 receptor affinity and their good in vitro serum stability, with in vitro serum t(1/2) values of several hours. Radiolabeling of DOTA-peptides with (111)In requires a heating procedure, typically in the range of 80 degrees -100 degrees C up to 30 min. Following this procedure with DOTA-MG11 resulted in a >98 % incorporation of (111)In, however, with a radiochemical purity (RCP) of <50 %. The decrease in RCP was found to be due to oxidation of the methionine residue in the molecule. Moreover, this oxidized compound lost its CCK-2 receptor affinity. Therefore, conditions during radiolabeling were optimised: labeling of DOTA-MG11 and DOTA-CCK with (111)In involved 5 min heating at 80 degrees C and led to an incorporation of (111)In of >98 %. In addition, all analogs were radiolabeled in the presence of quenchers to prevent radiolysis and oxidation resulting in a RCP of >90 %. All 3 radiolabeled analogs were i.v. administered to 6 MTC patients: radioactivity cleared rapidly by the kidneys, with no significant differences in the excretion pattern of the 3 radiotracers. All 3 radiolabeled analogs exhibited a low in vivo stability in patients, as revealed during analysis of blood samples, with the respective t(1/2) found in the order of minutes. In patient blood, the rank of radiopeptide in vivo stability was: (99m)Tc-Demogastrin 2 (t(1/2) 10-15 min)>(111)In-DOTA-CCK (t(1/2) approximately 5-10 min)>(111)In-DOTA-MG11 (t(1/2)<5 min).

Differential regulation of somatostatin receptor type 2 (sst 2) expression in AR4-2J tumor cells implanted into mice during octreotide treatment.

Octreotide is a somatostatin analogue that is widely used for cancer therapy and tumor imaging. Its efficacy in tumors depends mainly on the expression of the somatostatin receptor type 2 (sst 2). Desensitization and down-regulation of sst 2 after agonist exposure can have important consequences for patients under ongoing octreotide therapy because it may induce temporary tumor unresponsiveness and impair sst 2-based tumor scintigraphy. Therefore, we have investigated the effect of octreotide on sst 2 expression in vitro, as well as in a tumor mouse model. In vitro, short exposure to octreotide induced rapid dose-dependent down-regulation of sst 2 in the rat pancreatic AR4-2J cell line. Within 0.5 h, 80% of sst 2 had disappeared from the cell surface. A total recovery required 24 h and was shown to depend on protein synthesis, but not on new sst 2 mRNA transcription, indicating that sst 2 was probably degraded during the down-regulation process. Similar results were obtained in vivo. On the other hand, long-term continuous release of octreotide for 7 days, as achieved with octreotide-containing osmotic minipumps, caused sst 2 up-regulation in vivo, but not in vitro. Furthermore, this up-regulation of sst 2 in tumor-bearing scid mice was shown to depend on constant exposure of the animals to octreotide, as it was not observed when octreotide was given discontinuously in two s.c. daily injections. These results demonstrate that the continuous release of a small amount of octreotide, which in cancer therapy may be achieved with long-acting release formulations of the peptide, can induce sst 2 up-regulation on cancer cells. This may improve the efficacy of both tumor imaging and long-term octreotide therapy.

Cellular processing of copper-67-labeled monoclonal antibody chCE7 by human neuroblastoma cells.

Monoclonal antibody chCE7, an internalizing neuroblastoma-specific chimeric antibody, was derivatized with the macrocyclic amine ligand 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid tetrahydrochloride and labeled with the potential therapeutic nuclide 67Cu. Using pulse labeling and an acid elution endocytosis assay, 67Cu-chCE7 was found to be internalized into human neuroblastoma (SKN-AS) cells at a similar rate and to a similar extent as 125I-labeled chCE7. Uptake of 67Cu-chCE7 and 125I-chCE7 into the acid stable (intracellular) pool proceeded with similar kinetics during the first 2 h of internalization. However, in contrast to 125I-chCE7-loaded cells, at later times intracellular radioactivity kept increasing in the case of 67Cu-chCE7-loaded cells. It was shown that this effect is due to the intracellular accumulation of a low M(r) degradation product consisting of the 67Cu-4[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid complex, possibly with a short peptide attached to it. Degradation of both 125I-chCE7 and 67Cu-chCE7 was inhibited by chloroquine, indicating endosomal or lysosomal degradation, and a 43,000 M(r) fragment was found to be the major high M(r) degradation product in both cases. Although at times between 4 and 6 h of internalization intracellular breakdown of 67Cu-chCE7 was found to proceed more slowly, the major difference between the two immunoconjugates resides in the prolonged cellular retention of the 67Cu-chCE7 metabolite.

Comparison of (111)In-labeled somatostatin analogues for tumor scintigraphy and radionuclide therapy.

We evaluated the following (111)In-labeled somatostatin (SS) analogues (diethylenetriaminepentaacetic acid, DTPA; tetraazacyclododecanetetraacetic acid, DOTA): [DTPA0]octreotide, [DTPA0,Tyr3]octreotide, [DTPA0,D-Tyr1]octreotide, [DTPA0,Tyr3]octreotate [Thr(ol) in octreotide replaced with Thr], and [DOTA0,Tyr3]octreotide, in vitro and in vivo. In vitro, all compounds showed high and specific binding to SS receptors in mouse pituitary AtT20 tumor cell membranes, and IC50s were in the nanomolar range. Furthermore, all compounds showed specific internalization in rat pancreatic tumor cells; uptake of [(111)In-DTPA0,Tyr3]octreotate was the highest of the compounds tested, and that of [(111)In-DTPA0,D-Tyr1]octreotide was the lowest. Biodistribution experiments in rats showed that, 4, 24, and 48 h after injection of [(111)In-DTPA0,Tyr3]octreotide, [(111)In-DTPA0,Tyr3]octreotate, and [(111)In-DOTA0,Tyr3]octreotide, radioactivity in the octreotide-binding, receptor-expressing tissues and tumor-to-blood ratios were significantly higher than those after injection of [(111)In-DTPA0]octreotide. Uptake of [(111)In-DTPA0,Tyr3]octreotate in the target organs was also, in vivo, the highest of the radiolabeled peptides tested, whereas that of [(111)In-DTPA0,D-Tyr1]octreotide was the lowest. Uptake of [(111)In-DTPA0,Tyr3]octreotide, [(111)In-DTPA0,Tyr3]octreotate, and [(111)In-DOTA0,Tyr3]octreotide in target tissues was blocked by >90% by 0.5 mg of unlabeled octreotide, indicating specific binding to the octreotide receptors. Blockade of [(111)In-DTPA0,D-Tyr1]octreotide was >70%. In conclusion, radiolabeled [DTPA0,Tyr3]octreotide and, especially, [DTPA0,Tyr3]octreotate and their DOTA-coupled counterparts are most promising for scintigraphy and radionuclide therapy of SS receptor-positive tumors in humans.

Locoregional regulatory peptide receptor targeting with the diffusible somatostatin analogue 90Y-labeled DOTA0-D-Phe1-Tyr3-octreotide (DOTATOC): a pilot study in human gliomas.

Human gliomas, especially of low-grade type, have been shown to express high-affinity somatostatin receptor type 2 (J-C. Reubi et al., Am. J. Pathol, 134: 337-344, 1989). We enrolled seven low-grade and four anaplastic glioma patients in a pilot study using the diffusible peptidic vector 90Y-labeled DOTA0-D-Phe1-Tyr3-octreotide (DOTATOC) for receptor targeting. The radiopharmakon was locoregionally injected into a stereotactically inserted Port-a-cath. DOTATOC competes specifically with somatostatin binding to somatostatin receptor type 2 in the low nanomolar range as shown by a displacement curve of 125I-[Tyr3]-octreotide in tumor tissue sections. Diagnostic (111)In-labeled DOTATOC-scintigraphy following local injection displayed homogeneous to nodular intratumoral vector distribution. The cumulative activity of regionally injected peptide-bound 90Y amounted to 370-3300 MBq, which is equivalent to an effective dose range between 60 +/- 15 and 550 +/- 110 Gy. Activity was injected in one to four fractions according to tumor volumes; 1110 MBq of 90Y-labeled DOTATOC was the maximum activity per single injection. We obtained six disease stabilizations and shrinking of a cystic low-grade astrocytoma component. The only toxicity observed was secondary perifocal edema. The activity:dose ratio (MBq:Gy) represents a measure for the stability of peptide retention in receptor-positive tissue and might predict the clinical course. We conclude that SR-positive human gliomas, especially of low-grade type, can be successfully targeted by intratumoral injection of the metabolically stable small regulatory peptide DOTATOC.

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 radiotherapy.

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.

Comparison of 111In-DOTA-Tyr3-octreotide and 111In-DTPA-octreotide in the same patients: biodistribution, kinetics, organ and tumor uptake.

UNLABELLED: Scintigraphy with [111In-diethylenetriamine pentaacetic acid0-D-Phe1]-octreotide (DTPAOC) is used to demonstrate neuroendocrine and other somatostatin-receptor-positive tumors. Despite encouraging results, this 111In-labeled compound is not well suited for peptide-receptor-mediated radiotherapy of somatostatin-receptor-positive tumors. Another somatostatin analog, [1,4,7,10-tetraazacyclododecane-N,N',N",N'''-tetraacetic acid0, D-Phe1, Tyr3]-octreotide (DOTATOC), can be labeled with the beta-emitter 90Y in a stable manner. METHODS: We compared the distribution, kinetics and dosimetry of 111In-DTPAOC and 111In-DOTATOC in eight patients to predict the outcomes of these parameters in patients who will be treated with 90Y-DOTATOC. RESULTS: Serum radioactivity levels for the radiopharmaceuticals did not differ significantly 2-24 h after injection (P>0.05). Up to 2 h postinjection they were slightly, but significantly, lower after administration of 111In-DOTATOC (P < 0.01 at most time points). The percentage of peptide-bound radioactivity in serum did not differ after administration of either compound. Urinary excretion was significantly lower after administration of 111In-DOTATOC (P < 0.01). The visualization of known somatostatin-receptor-positive organs and tumors was clearer after administration of 111In-DOTATOC than after administration of 111In-DTPAOC. This was confirmed by significantly higher calculated uptakes in the pituitary gland and spleen. The uptake in the tumor sites did not differ significantly (P > 0.05), although in three of the four patients in whom tumor uptake could be calculated, it was higher after administration of 111In-DOTATOC. CONCLUSION: The distribution and excretion pattern of 111In-DOTATOC resembles that of 111In-DTPAOC, and the uptake in somatostatin-receptor-positive organs and most tumors is higher for 111In-DOTATOC. If 90Y-DOTATOC shows an uptake pattern similar to 111In-DOTATOC, it is a promising radiopharmaceutical for peptide-receptor-mediated radiotherapy in patients with somatostatin-receptor-positive tumors.

Gallium-67/gallium-68-[DFO]-octreotide--a potential radiopharmaceutical for PET imaging of somatostatin receptor-positive tumors: synthesis and radiolabeling in vitro and preliminary in vivo studies.

UNLABELLED: When labeled with gamma-emitting radionuclides, somatostatin analogs have the potential to localize somatostatin receptor-positive tumors using gamma camera scintigraphy. We present a somatostatin analog, [DFO]-octreotide (SDZ 216-927), that comprises desferrioxamine B coupled to octreotide via a succinyl linker. This conjugate can be labeled with either 67Ga for gamma scintigraphy or 68Ga for PET imaging. The 67Ga-labeled conjugate is stable in vitro to autoradiolysis over a 24-hr period. METHODS: Rats bearing a somatostatin receptor-positive pancreatic islet cell tumor were injected with 20 MBq of 67Ga[DFO]-octreotide (33 GBq 67Ga/mumole). RESULTS: After 1 hr, the accumulation of 67Ga[DFO]-octreotide was 0.38 +/- 0.08 %ID/g and the tumor-to-nontumor ratios for blood, muscle, liver and intestine were 2.5, 7.4, 1.9 and 1.6, respectively. PET studies with 68Ga[DFO]-octreotide recorded a very rapid accumulation at the tumor and a subsequent residence half-life of about 6 hr. CONCLUSION: Gallium-68-[DFO]-octreotide can be used in PET studies to diagnose receptor-positive tumors such as gastroenteropancreatic, small-cell lung and breast tumors.

PET imaging of somatostatin receptors using [68GA]DOTA-D-Phe1-Tyr3-octreotide: first results in patients with meningiomas.

UNLABELLED: Imaging of somatostatin receptors (SSTRs) using [111In]diethylenetriaminepentaacetic-acid-octreotide (DTPAOC) has proven to be helpful in the differentiation of meningiomas, neurinomas or neurofibromas, and metastases as well as in the follow-up of meningiomas. A drawback of the SPECT method is its limited sensitivity in detecting small meningiomas. Because of PET's increased spatial resolution and its ability to absolutely quantify biodistribution, a PET tracer for SSTR imaging would be desirable. METHODS: 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic-acid-D-Phe1-Tyr3-octreotide (DOTATOC) was labeled using the positron-emitting generator nuclide 68Ga. We acquired dynamic PET images over 120 min after intravenous injection of 175 MBq [68Ga]DOTATOC in 3 patients suffering from 8 meningiomas (WHO I degrees; 7- to 25-mm diameter). Patients' heads had been fixed using individually shaped fiber masks equipped with an external stereotactic localizer system to match PET, CT, and MRI datasets. RESULTS: [68Ga]DOTATOC was rapidly cleared from the blood (half-life alpha, 3.5 min; half-life beta, 63 min). Standardized uptake values (SUVs) of meningiomas increased immediately after injection and reached a plateau 60-120 min after injection (mean SUV, 10.6). No tracer could be found in the surrounding healthy brain tissue. All meningiomas (even the 3 smallest [7- to 8-mm diameter]) showed high tracer uptake and could be visualized clearly. Tracer boundaries showed a good correspondence with the matched CT and MRI images. PET provided valuable additional information regarding the extent of meningiomas located beneath osseous structures, especially at the base of the skull. CONCLUSION: According to our initial experiences, [68Ga]DOTATOC seems to be a very promising new PET tracer for imaging SSTRs even in small meningiomas, offering excellent imaging properties and a very high tumor-to-background ratio.

Synthesis and characterisation of [90Y]-Bz-DTPA-oct: a yttrium-90-labelled octreotide analogue for radiotherapy of somatostatin receptor-positive tumours.

An investigation into the in vitro behaviour of two yttrium-90-labelled somatostatin analogues was performed. Further in vivo characterisation was performed with the most promising agent. A new DTPA-octreotide analogue (Bz-DTPA-oct) was synthesised by coupling a bifunctional DTPA chelator to the N-terminal amine of the D-Phe1 of Tyr3-octreotide. This new SRIF analogue and DTPA-octreotide (OctreoScan) were radiolabelled with 90Y prior to serum stability being evaluated. Receptor binding assays were also performed on the two radioligands using rat cortex membranes. The [90Y]-Bz-DTPA-oct was further evaluated in vivo using tumour-bearing rats. The first conjugate (DTPA-octreotide) bound with a high affinity to SRIF receptors and the 90Y complex was relatively stable in human serum (t1/2 3.8 d for 90Y lost to serum proteins). The second conjugate (Bz-DTPA-oct) also exhibited a high binding affinity to SRIF receptors, but it demonstrated an even slower loss of 90Y to serum proteins (t1/2 12.1 d). The in vivo evaluation of the more stable [90Y]-Bz-DTPA-oct showed a very rapid and high accumulation in somatostatin receptor-positive tumours, which after 1 h resulted in tumour/nontumour ratios of 3.8, 21, and 4.9 (for blood, muscle, and liver, respectively). These tumour/nontumour ratios increased, and were by 24 h postinjection 138, 285, and 6.1 (for blood, muscle, and liver). Yttrium-90-labelled Bz-DTPa-oct is rapidly and selectively accumulated in somatostatin receptor-positive tissue. Octadentate Bz-DTPA-oct could be ligand for 90Y radiotherapy of somatostatin receptor-positive tumours and their metastases.

NMR spectroscopy of Group 13 metal ions: biologically relevant aspects.

In spite of the fact that Group 13 metal ions (Al(3+), Ga(3+), In(3+) and Tl(+/3+)) show no main biological role, they are NMR-active nuclides which can be used in magnetic resonance spectroscopy of biologically relevant systems. The fact that these metal ions are quadrupolar (with the exception of thallium) means that they are particularly sensitive to ligand type and coordination geometry. The line width of the NMR signals of their complexes shows a strong dependence on the symmetry of coordination, which constitutes an effective tool in the elucidation of structures. Here we report published NMR studies of this family of elements, applied to systems of biological importance. Special emphasis is given to binding studies of these cations to biological molecules, such as proteins, and to chelating agents of radiopharmaceutical interest. The possibility of in vivo NMR studies is also stressed, with extension to (27)Al-based MRI (magnetic resonance imaging) experiments.

Receptor-mediated radionuclide therapy with 90Y-DOTA-D-Phe1-Tyr3-Octreotide: preliminary report in cancer patients.

Recent advances in receptor mediated tumor imaging led to the development of a new somatostatin analogue DOTA-D-Phe1-Tyr3-Octreotide. This new compound, named DOTATOC, has shown high affinity for somatostatin receptors, stable labeling with yttrium-90 (90Y) and favourable biodistribution in patients. The aim of this work was to evaluate acute and late toxicity and the response rate in cancer patients administered 90Y-DOTATOC. Twenty patients received three equal i.v. injections of 90Y-DOTATOC. Cohorts of 5 patients were treated starting with 1.1 GBq per cycle in escalating dosage (0.4 GBq increments) in subsequent groups. No patients showed acute or delayed major adverse reactions up to the dose of 2.2 GBq of 90Y-DOTATOC per cycle (6.6 GBq total). Maximum tolerated dose has not been determined yet. One patient, after 4.4 GBq total dose, developed delayed kidney grade II toxicity. Complete and partial tumor mass reduction (CR and PR) was measured in 25% of patients along with 55% showing stable disease (SD) and 20% progressive disease (PD). These results indicate that high activities of 90Y-DOTATOC can be administered with low risk of myelotoxicity, although the radiation doses to the kidneys require careful consideration. Tumor doses were high enough in most cases to obtain objective therapeutic responses.

Octreotide and octreotate derivatives radiolabeled with yttrium: pharmacokinetics in rats.

Distribution profiles and elimination pathways in rats of two new octreotate derivatives radiolabeled with yttrium, namely Y-DOTAGA-tate and Y-DOTA-t-GA-tate, were compared with those of Y-DOTA-octreotide and Y-DOTA-Tyr(3)-octreotide. All synthetic somatostatin analogues under study were rapidly cleared from the blood and most organs of rats. The main elimination pathway for all peptides under study was urine excretion. High and long-term uptakes of radioactivity in the kidneys and also in organs with high density of somatostatin receptors (the adrenals and pancreas) were found. Radioactivity concentrations in these somatostatin receptor-rich organs were substantially higher for octreotate derivatives in comparison with octreotide analogues; the highest values for Y-DOTAGA-tate were determined. The octreotate derivatives under study appear to be specific ligands for treatment of somatostatin receptor-positive tumors if some mechanism to decrease their kidney retention is provided.

Internalization of radiolabelled [DTPA0]octreotide and [DOTA0,Tyr3]octreotide: peptides for somatostatin receptor-targeted scintigraphy and radionuclide therapy.

We compared the internalization of [90Y-DOTA0,Tyr3]octreotide and [111In-DOTA0,Tyr3]octreotide with that of [125I-Tyr3]octreotide and [111In-DTPA0]octreotide in the subtype 2 somatostatin receptor (sst2)-positive rat pancreatic tumour cell lines CA20948 and AR42J and in the somatostatin receptor-negative human anaplastic thyroid tumour cell line ARO. We demonstrated that [111In-DTPA0]octreotide, [90Y-DOTA0,Tyr3]octreotide and [111In-DOTA0,Tyr3]octreotide are internalized by a receptor-specific, time- and temperature-dependent process. The amount of [90Y-DOTA0,Tyr3]octreotide internalized was higher than that of [111In-DOTA0,Tyr3]octreotide and [111In-DTPA0]octreotide.

A convenient synthesis of novel bifunctional prochelators for coupling to bioactive peptides for radiometal labelling.

New DOTA-based bifunctional prochelators, e.g., 1-(1-carboxy-3-carbotertbutoxypropyl)-4,7,10-(carbotertbutoxyme thyl)-1,4,7,10-tetraazacyclodode-cane (DOTAGA(tBu)4), (6d) for a broad application in the modification of biomolecules with metal ions were prepared. The five-step synthesis of 6d has an overall yield of about 20%. The coupling of 6d to a bioactive peptide on solid-phase was exemplified with use of a CCK-B (cholecystokinin) analogue.

[Yttrium 90 DOTATOC: a new somatostatin analog for cancer therapy of neuroendocrine tumors]. / Yttrium-90-DOTATOC: Ein neues Somatostatinanalogon zur Krebstherapie von neuroendokrinen Tumoren.

Current concepts for the treatment of somatostatin receptor positive tumours have not been very motivating up to now. A promising alternative could be the new peptidic vector DOTA-D-Phe1-Tyr3-Octreotide (DOTATOC) recently developed in Basel. It may be labelled with the beta-emitter yttrium-90 (90Y) for internal radiotherapy after systemic application. Pilot therapy studies have shown convincing results with this new radiopharmaceutical. These studies are presented with regard to efficacy and possible toxicity. In summary, the new receptor-mediated 90Y-DOTATOC therapy led to tumour response in the majority of patients, and only in some receiving high cumulative doses of > 200 mCi per m2 body surface renal and hematological toxicity due to irradiation occurred. For the reduction of renal accretion, concepts with concomitant amino acid infusions containing L-lysine in a higher concentration are currently under way.

Affinity profiles for human somatostatin receptor subtypes SST1-SST5 of somatostatin radiotracers selected for scintigraphic and radiotherapeutic use.

In vivo somatostatin receptor scintigraphy using Octreoscan is a valuable method for the visualisation of human endocrine tumours and their metastases. Recently, several new, alternative somatostatin radioligands have been synthesised for diagnostic and radiotherapeutic use in vivo. Since human tumours are known to express various somatostatin receptor subtypes, it is mandatory to assess the receptor subtype affinity profile of such somatostatin radiotracers. Using cell lines transfected with somatostatin receptor subtypes sst1, sst2, sst3, sst4 and sst5, we have evaluated the in vitro binding characteristics of labelled (indium, yttrium, gallium) and unlabelled DOTA-[Tyr3]-octreotide, DOTA-octreotide, DOTA-lanreotide, DOTA-vapreotide, DTPA-[Tyr3]-octreotate and DOTA-[Tyr3]-octreotate. Small structural modifications, chelator substitution or metal replacement were shown to considerably affect the binding affinity. A marked improvement of sst2 affinity was found for Ga-DOTA-[Tyr3]-octreotide (IC50 2.5 nM) compared with the Y-labelled compound and Octreoscan. An excellent binding affinity for sst2 in the same range was also found for In-DTPA-[Tyr3]-octreotate (IC50 1.3 nM) and for Y-DOTA-[Tyr3]-octreotate (IC50 1.6 nM). Remarkably, Ga-DOTA-[Tyr3]-octreotate bound at sst2 with a considerably higher affinity (IC50 0.2 nM). An up to 30-fold improvement in sst3 affinity was observed for unlabelled or Y-labelled DOTA-octreotide compared with their Tyr3-containing analogue, suggesting that replacement of Tyr3 by Phe is crucial for high sst3 affinity. Substitution in the octreotide molecule of the DTPA by DOTA improved the sst3 binding affinity 14-fold. Whereas Y-DOTA-lanreotide had only low affinity for sst3 and sst4, it had the highest affinity for sst5 among the tested compounds (IC50 16 nM). Increased binding affinity for sst3 and sst5 was observed for DOTA-[Tyr3]-octreotide, DOTA-lanreotide and DOTA-vapreotide when they were labelled with yttrium. These marked changes in subtype affinity profiles are due not only to the different chemical structures but also to the different charges and hydrophilicity of these compounds. Interestingly, even the coordination geometry of the radiometal complex remote from the pharmacophoric amino acids has a significant influence on affinity profiles as shown with Y-DOTA versus Ga-DOTA in either [Tyr3]-octreotide or [Tyr3]-octreotate. Such changes in sst affinity profiles must be identified in newly designed radiotracers used for somatostatin receptor scintigraphy in order to correctly interpret in vivo scintigraphic data. These observations may represent basic principles relevant to the development of other peptide radioligands.

New octreotide derivatives for in vivo targeting of somatostatin receptor-positive tumors for single photon emission computed tomography (SPECT) and positron emission tomography (PET).

Two new modifications of the somatostatin analog octreotide, designed to hold the gallium isotopes 67Ga and 68Ga (DFO-SMS, Fig. 1a) and 99mTc (PnAO-SMS, Fig. 1b) have been synthesized and evaluated in vitro and in vivo in tumor bearing rats. DFO-SMS can be labeled with 67Ga3+ and 68Ga3+ with high specific activity within less than 30 minutes in a "cold kit" type formulation. The labeled conjugate is stable under physiological conditions. Moreover the binding affinity to somatostatin receptors on rat brain cortex membranes was shown to be retained. In vivo fast tumor localization was demonstrated and the pharmacokinetics proved to be favourable as the main excretion route was via the kidneys. First PET studies with [68Ga]-DFO-SMS showed a rapid accumulation in the tumor and a residence half-life at the tumor site of about 6 hours. PnAO-SMS can be labeled with 99mTc with high radiochemical purity. In vivo the radiotracer accumulates well in the tumor but due to its high lipophilicity, its main excretion route is via the hepatobiliary system.

Experience with the iodine-123 and technetium-99m labelled anti-granulocyte antibody MAb47: a comparison of labelling methods.

Four different methods of radiolabelling the anti-granulocyte monoclonal antibody MAb47 were compared and their influence on diagnostic value studied. The best clinical images were obtained following labelling with iodine-123 by the Iodogen method and direct labelling with technetium-99m after tris-(carboxyethyl)-phosphine treatment of MAb47 to achieve disulphide bridge reduction. 99mTc labelling using a specific ligand (MAb47-mtp), or a second method involving direct reduction with mercaptoethanol, led to an increased background activity in clinical studies, thus impeding the diagnosis of chronic disease. Fresh infections were clearly localized by all four preparations. The elimination of the activity from the blood was slower in the case of the iodinated MAb47, while the collected urine samples showed an excretion of about 10% of the injected activity per day independent of the labelling method. The results in terms of sensitivity and specificity were rather similar for all labelling methods and ranged from 90% to 99%.

Internalization of [DOTA degrees,125I-Tyr3]Octreotide by somatostatin receptor-positive cells in vitro and in vivo: implications for somatostatin receptor-targeted radio-guided surgery.

We compared internalization of three radioiodinated octreotide (OCT) somatostatin (SS) analogs-[125I-Tyr3]OCT, [DTPA degrees, 125I-Tyr3]OCT, and [DOTA degrees,125I-Tyr3]OCT-by somatostatin receptor (SSR)-positive mouse AtT20 pituitary tumor cells and human insulinoma cells. The three SS analogs were internalized in a specific, time-dependent manner. Internalization was significantly inhibited by pertussis toxin (100 microg/l) by 38%, 43%, and 31%, and by an inhibitor of receptor-mediated endocytosis (phenyl arsine oxide; 10 microM) by 98%, 94%, and 92%, respectively. Binding affinities of the three radioligands were comparable (0.2, 0.2, and 0.3 nM, respectively). However, [DOTA degrees,125I-Tyr3]OCT was internalized in a five-fold higher amount in comparison with the two other radioligands. A comparably high uptake of [DOTA degrees, 125I-Tyr3]OCT was found in SSR-positive organs (pituitary, pancreas, and adrenals) in vivo in rats (a ten-fold, five-fold, and eight-fold higher uptake 4 hr post injection, respectively, compared with the two other radioligands). This resulted in very high target-background ratios for [DOTA degrees,125I-Tyr3]OCT 4 hr post injection amounting to 274, 566, and 623 in the pituitary, adrenals, and pancreas, respectively. Both in vivo and in vitro there was a rapid dissociation of radioactivity from the SSR-positive cells. Main conclusions are that: 1) coupling of chelating groups like DTPA or DOTA to the SS analog [Tyr3]OCT does not prevent the internalization of OCT after binding to SSRs; 2) [DOTA degrees, 125I-Tyr3]OCT is internalized in a significantly higher amount by AtT20 and human insulinoma cells and in vivo in rats in SSR-positive organs, in comparison with [DTPA degrees,125I-Tyr3]OCT and [125I-Tyr3]OCT; and 3) the very high target-background ratios in vivo make radioiodinated [DOTA degrees,Tyr3]OCT a very suitable ligand for SSR-targeted radioguided surgery of SSR-positive human neuroendocrine tumors.