Terbium-149 production: a focus on yield and quality improvement towards preclinical application.

Terbium-149 (T1/2 = 4.1 h, Eα = 3.98 MeV (16.7%), 28 µm range in tissue) is a radionuclide with potential for targeted alpha therapy. Due to the negligible emission of α-emitting daughter nuclides, toxicity to healthy tissue may be reduced in comparison with other α-particle emitters. In this study, terbium-149 was produced via 1.4 GeV proton irradiation of a tantalum target at the CERN-ISOLDE facility. The spallation products were mass separated and implanted on zinc-coated foils and, later, radiochemically processed. Terbium-149 was separated from the co-produced isobaric radioisotopes and the zinc coating from the implantation foil, using cation-exchange and extraction chromatographic techniques, respectively. At the end of separation, up to 260 MBq terbium-149 were obtained with > 99% radionuclidic purity. Radiolabeling experiments were performed with DOTATATE, achieving 50 MBq/nmol apparent molar activity with radiochemical purity > 99%. The chemical purity was determined by inductively coupled plasma-mass spectrometry measurements, which showed lead, copper, iron and zinc only at ppb level. The radiolabeling of the somatostatin analogue DOTATATE with [149Tb]TbCl3 and the subsequent in vivo PET/CT scans conducted in xenografted mice, showing good tumor uptake, further demonstrated product quality and its ability to be used in a preclinical setting.

Cyclotron production and radiochemical purification of terbium-155 for SPECT imaging.

BACKGROUND: Terbium-155 [T1/2 = 5.32 d, Eγ = 87 keV (32%) 105 keV (25%)] is an interesting radionuclide suitable for single photon emission computed tomography (SPECT) imaging with potential application in the diagnosis of oncological disease. It shows similar decay characteristics to the clinically established indium-111 and would be a useful substitute for the diagnosis and prospective dosimetry with biomolecules that are afterwards labeled with therapeutic radiolanthanides and pseudo-radiolanthanides, such as lutetium-177 and yttrium-90. Moreover, terbium-155 could form part of the perfect "matched pair" with the therapeutic radionuclide terbium-161, making the concept of true radiotheragnostics a reality. The aim of this study was the investigation of the production of terbium-155 via the 155Gd(p,n)155Tb and 156Gd(p,2n)155Tb nuclear reactions and its subsequent purification, in order to obtain a final product in quantity and quality sufficient for preclinical application. The 156Gd(p,2n)155Tb nuclear reaction was performed with 72 MeV protons (degraded to ~ 23 MeV), while the 155Gd(p,n)155Tb reaction was degraded further to ~ 10 MeV, as well as performed at an 18 MeV medical cyclotron, to demonstrate its feasibility of production. RESULT: The 156Gd(p,2n)155Tb nuclear reaction demonstrated higher production yields of up to 1.7 GBq, however, lower radionuclidic purity when compared to the final product (~ 200 MBq) of the 155Gd(p,n)155Tb nuclear reaction. In particular, other radioisotopes of terbium were produced as side products. The radiochemical purification of terbium-155 from the target material was developed to provide up to 1.0 GBq product in a small volume (~ 1 mL 0.05 M HCl), suitable for radiolabeling purposes. The high chemical purity of terbium-155 was proven by radiolabeling experiments at molar activities up to 100 MBq/nmol. SPECT/CT experiments were performed in tumor-bearing mice using [155Tb]Tb-DOTATOC. CONCLUSION: This study demonstrated two possible production routes for high activities of terbium-155 using a cyclotron, indicating that the radionuclide is more accessible than the exclusive mass-separated method previously demonstrated. The developed radiochemical purification of terbium-155 from the target material yielded [155Tb]TbCl3 in high chemical purity. As a result, initial cell uptake investigations, as well as SPECT/CT in vivo studies with [155Tb]Tb-DOTATOC, were successfully performed, indicating that the chemical separation produced a product with suitable quality for preclinical studies.

The Metamorphosis of Radionuclide Production and Development at Paul Scherrer Institute.

Radionuclide production and development has a long history at the Paul Scherrer Institute (PSI) and dates back to the founding times of its forerunner institutions: the Federal Institute for Reactor Research and the Swiss Institute for Nuclear Research. The facilities used for this purpose have evolved substantially over the last five decades. Many radiometals in use today, as radiopharmaceuticals, are for the diagnosis and treatment of disease, with the most popular means of detection being Positron Emission Tomography. These positron emitters are easily produced at low proton energies using medical cyclotrons, however, developments at these facilities are lacking. Currently, the fixed 72 MeV proton beam at PSI is degraded at IP2 irradiation station to provide the desired energy to irradiate targets to produce the likes of 44Sc, 43Sc and 64Cu as a proof of principle, which are of great interest to the nuclear medicine community. This development work can then be implemented at facilities containing medical cyclotrons. A history of the development of radionuclides at PSI, along with current development and projects with partner institutions, is described.

Advancing Biomarker Development Through Convergent Engagement: Summary Report of the 2nd International Danube Symposium on Biomarker Development, Molecular Imaging and Applied Diagnostics; March 14-16, 2018; Vienna, Austria.

Here, we report on the outcome of the 2nd International Danube Symposium on advanced biomarker development that was held in Vienna, Austria, in early 2018. During the meeting, cross-speciality participants assessed critical aspects of non-invasive, quantitative biomarker development in view of the need to expand our understanding of disease mechanisms and the definition of appropriate strategies both for molecular diagnostics and personalised therapies. More specifically, panelists addressed the main topics, including the current status of disease characterisation by means of non-invasive imaging, histopathology and liquid biopsies as well as strategies of gaining new understanding of disease formation, modulation and plasticity to large-scale molecular imaging as well as integrative multi-platform approaches. Highlights of the 2018 meeting included dedicated sessions on non-invasive disease characterisation, development of disease and therapeutic tailored biomarkers, standardisation and quality measures in biospecimens, new therapeutic approaches and socio-economic challenges of biomarker developments. The scientific programme was accompanied by a roundtable discussion on identification and implementation of sustainable strategies to address the educational needs in the rapidly evolving field of molecular diagnostics. The central theme that emanated from the 2nd Donau Symposium was the importance of the conceptualisation and implementation of a convergent approach towards a disease characterisation beyond lesion-counting "lumpology" for a cost-effective and patient-centric diagnosis, therapy planning, guidance and monitoring. This involves a judicious choice of diagnostic means, the adoption of clinical decision support systems and, above all, a new way of communication involving all stakeholders across modalities and specialities. Moreover, complex diseases require a comprehensive diagnosis by converging parameters from different disciplines, which will finally yield to a precise therapeutic guidance and outcome prediction. While it is attractive to focus on technical advances alone, it is important to develop a patient-centric approach, thus asking "What can we do with our expertise to help patients?"

Enhanced sensitivity to drugs of abuse and palatable foods following maternal overnutrition.

Epidemiological studies have shown an association between maternal overnutrition and increased risk of the progeny for the development of obesity as well as psychiatric disorders. Animal studies have shown results regarding maternal high-fat diet (HFD) and a greater risk of the offspring to develop obesity. However, it still remains unknown whether maternal HFD can program the central reward system in such a way that it will imprint long-term changes that will predispose the offspring to addictive-like behaviors that may lead to obesity. We exposed female dams to either laboratory chow or HFD for a period of 9 weeks: 3 weeks before conception, during gestation and lactation. Offspring born to either control or HFD-exposed dams were examined in behavioral, neurochemical, neuroanatomical, metabolic and positron emission tomography (PET) scan tests. Our results demonstrate that HFD offspring compared with controls consume more alcohol, exhibit increased sensitivity to amphetamine and show greater conditioned place preference to cocaine. In addition, maternal HFD leads to increased preference to sucrose as well as to HFD while leaving the general feeding behavior intact. The hedonic behavioral alterations are accompanied by reduction of striatal dopamine and by increased dopamine 2 receptors in the same brain region as evaluated by post-mortem neurochemical, immunohistochemical as well as PET analyses. Taken together, our data suggest that maternal overnutrition predisposes the offspring to develop hedonic-like behaviors to both drugs of abuse as well as palatable foods and that these types of behaviors may share common neuronal underlying mechanisms that can lead to obesity.

Site-Specific Conjugation of Monomethyl Auristatin E to Anti-CD30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models.

Antibody-drug conjugates (ADCs) have demonstrated clinical benefits that have led to the recent FDA approval of KADCYLA and ADCETRIS. Most ADCs that are currently in clinical use or development, including ADCETRIS, are produced by chemical conjugation of a toxin via either lysine or cysteine residues, inevitably leading to heterogeneous products with variable drug-to-antibody ratios (DARs). Here, we describe the in vitro and in vivo characterization of four novel ADCs that are based on the anti-CD30 antibody cAC10, which has the same polypeptide backbone as ADCETRIS, and compare the results with the latter. Bacterial transglutaminase (BTG) was exploited to site-specifically conjugate derivatives of monomethyl auristatin E (all comprising a cleavable linker) to the glutamine at positions 295 and 297 of cAC10, thereby yielding homogeneous ADCs with a DAR of 4. In vitro cell toxicity experiments using two different CD30-positive cell lines (Karpas 299 and Raji-CD30(+)) revealed comparable EC50 values for ADCETRIS (1.8 ± 0.4 and 3.6 ± 0.6 ng/mL, respectively) and the four cAC10-based ADCs (2.0 ± 0.4 to 4.9 ± 1.0 ng/mL). Quantitative time-dependent in vivo biodistribution studies (3-96 h p.i.) in normal and xenografted (Karpas 299 cells) SCID mice were performed with a selected (125)I-radioiodinated cAC10 ADC and compared with that of (125)I-ADCETRIS. The chemo-enzymatically conjugated, radioiodinated ADC showed higher tumor uptake (17.84 ± 2.2% ID/g 24 h p.i.) than (125)I-ADCETRIS (10.5 ± 1.8% ID/g 24 h p.i.). Moreover, (125)I-ADCETRIS exhibited higher nontargeted liver and spleen uptake. In line with these results, the maximum tolerated dose of the BTG-coupled ADC (>60 mg/kg) was significantly higher than that of ADCETRIS (18 mg/kg) in rats. These results suggest that homogeneous ADCs display improved pharmacokinetics and better therapeutic indexes compared to those of chemically modified ADCs with variable DARs.

Multimodal imaging of pancreatic beta cells in vivo by targeting transmembrane protein 27 (TMEM27).

AIMS/HYPOTHESIS: Non-invasive diagnostic tools specific for pancreatic beta cells will have a profound impact on our understanding of the pathophysiology of metabolic diseases such as diabetes. The objective of this study was to use molecular imaging probes specifically targeting beta cells on human samples and animal models using state-of-the-art imaging modalities (fluorescence and PET) with preclinical and clinical perspective. METHODS: We generated a monoclonal antibody, 8/9-mAb, targeting transmembrane protein 27 (TMEM27; a surface N-glycoprotein that is highly expressed on beta cells), compared its expression in human and mouse pancreas, and demonstrated beta cell-specific binding in both. In vivo imaging was performed in mice with subcutaneous insulinomas overexpressing the human TMEM27 gene, or transgenic mice with beta cell-specific hTMEM27 expression under the control of rat insulin promoter (RIP-hTMEM27-tg), using fluorescence and radioactively labelled antibody, followed by tissue ex vivo analysis and fluorescence microscopy. RESULTS: Fluorescently labelled 8/9-mAb showed beta cell-specific staining on human and mouse pancreatic sections. Real-time PCR on islet cDNA indicated about tenfold higher expression of hTMEM27 in RIP-hTMEM27-tg mice than in humans. In vivo fluorescence and PET imaging in nude mice with insulinoma xenografts expressing hTMEM27 showed high 8/9-mAb uptake in tumours after 72 h. Antibody homing was also observed in beta cells of RIP-hTMEM27-tg mice by in vivo fluorescence imaging. Ex vivo analysis of intact pancreas and fluorescence microscopy in beta cells confirmed these findings. CONCLUSIONS/INTERPRETATION: hTMEM27 constitutes an attractive target for in vivo visualisation of pancreatic beta cells. Studies in mouse insulinoma models and mice expressing hTMEM27 demonstrate the feasibility of beta cell-targeted in vivo imaging, which is attractive for preclinical investigations and holds potential in clinical diagnostics.

Influence of the molecular charge on the biodistribution of bombesin analogues labeled with the [99mTc(CO)3]-core.

The overexpression of Bombesin (BBS) receptors on a variety of human cancers make them interesting targets for tumor imaging and therapy. Analogues of the neuropeptide BBS have been functionalized with the (NalphaHis)- chelator for labeling with the 99mTc-tricarbonyl core. The introduction of a betaAla-betaAla linker between the stabilized BBS binding sequence and the chelator led to increased tumor uptake but still rather unfavorable in ViVo properties. Novel polar linkers, with different charge, have been introduced in the molecule and tested for their influence on the biodistribution. The new analogues showed a shift in hydrophilicity from a Log D=0.9 to Log D values between 0.4 and -2.2. All compounds kept the increased stability in both human plasma (t(1/2)>16 h) and in tumor cells (t(1/2)=30-40 min). The compounds with Log D values between +1 and -1 showed the highest binding affinities with Kd values of <0.5 nM, as well as the highest cellular uptake. However, higher hydrophilicity (Log D < -1.8) led to lower affinity and a substantial decrease of internalization. The introduction of a positive charge (beta3hLys) resulted in unfavorable biodistribution, with increased kidney uptake. The introduction of an uncharged hydroxyl group (beta3hSer) improved the biodistribution, resulting in significantly better tumor-to-tissue ratios. The compound with one single negative charge (beta3hGlu) showed a significant increase in the tumor uptake (2.1+/-0.6% vs 0.80+/-0.35% ID/g in comparison to the betaAla-betaAla analogue) and also significantly higher tumor-to-tissue ratios. The specificity of the in ViVo uptake was confirmed by coinjection with natural BBS. Moreover, the analogue provided a much clearer image of the tumor xenografts in the SPECT/CT studies. The introduction of a single negative charge may be useful in the development of new BBS analogues to obtain an improved biodistribution profile, with increased tumor uptake and better imaging.

Production and separation of ''non-standard'' PET nuclides at a large cyclotron facility: the experiences at the Paul Scherrer Institute in Switzerland.

Radioimmuno-positron emission tomography (PET) combined with radioimmunotherapy is attractive to assess tumor targeting and quantitate the radiation dose to tumor and normal tissues. For this purpose, PET radionuclides with adequate physical half-lives matching those of the targeting molecule (e.g. antibodies) are preferable. Copper-64 (T(1/2)=12.7 h, E(beta+max)=653 keV) and Zirconium-89 (T(1/2)=78.4 d, E(beta+max)=901 keV) are attractive isotopes for this purpose. The 72 MeV cyclotron at the Paul Scherrer Institute provides the infrastructure for the production of a wide variety of radionuclides for diagnostic and therapeutic purposes. (64)Cu and (89)Zr are currently evaluated at the Center for Radiopharmaceutical Science (CRS) of the Paul Scherrer Institute (PSI) in combination with the L1 cam targeting antibody chCE7 and various protein formats thereof. A second focus of the CRS is the radiolabeling of small, tumor targeting molecules with technetium. The PET isotope (94m)Tc offers potential alternative to its widely used (99m)Tc SPECT counterpart. In this report, the development, optimization and pitfalls of (64)Cu, (89)Zr and (94m)Tc production/separation are reported and discussed.

A 99mTc(I)-postlabeled high affinity bombesin analogue as a potential tumor imaging agent.

The overexpression of neuropeptide receptors observed in many cancers provides an attractive target for tumor imaging and therapy. Bombesin is a peptide exhibiting a high affinity for the gastrin releasing peptide (GRP) receptor, which is overexpressed by a variety of tumors such as breast or prostate cancer. In the present study, we have evaluated if the bombesin analogue [N(alpha)-histidinyl acetate]bombesin(7-14), radiolabeled with the novel [99mTc(OH(2))(3)(CO)(3)]+, has the potential to be used as a diagnostic radiopharmaceutical. Receptor saturation studies, carried out on the GRP receptor-expressing PC-3 human prostate cancer cell line, revealed for [99mTc(CO)(3)-N(alpha)-histidinyl acetate]bombesin(7-14) K(d) values in the subnanomolar range. Competitive binding assays, using the cold rhenium(I)-labeled analogue as a surrogate for the 99mTc-conjugate, also showed high affinity binding. Incubation of the radioconjugate with PC-3 cells resulted in a rapid temperature- and time-dependent specific internalization. At 37 degrees C more than 70% was internalized within the first 15 min and remained constant up to 2 h. Despite the weak proteolytic stability of [99mTc(CO)(3)-N(alpha)-histidinyl acetate]bombesin(7-14) in vitro, biodistribution studies, performed in PC-3 tumor-bearing mice, showed low uptake in the tumor (0.89 +/- 0.27% ID/g 30 min pi) but high uptake into the pancreas (7.11 +/- 3.93% ID/g 30 min pi), a GRP receptor-positive organ. Blockade experiment (coinjection of 300 microg bombesin/mouse with the radioligand) showed specificity of the uptake. Despite the low tumor uptake, tumor-to-blood ratios of 2.0 and 2.7 and tumor-to-muscle ratios of 8.9 and 8.0 were obtained at 30 min and 1.5 h postinjection, respectively. The promising results merit the future in vivo investigation of 99mTc/188Re-tricarbonyl-labeled bombesin analogues.

Derivatization of glucose and 2-deoxyglucose for transition metal complexation: substitution reactions with organometallic 99mTc and Re precursors and fundamental NMR investigations.

Synthetic strategies for the bifunctionalization of glucose and 2-deoxyglucose at position C-1 for transition metal coordination are reported. In particular organometallic technetium and rhenium complexes for potential use in diagnostic nuclear medicine were synthesized and investigated. Specifically, a common iminodiacetic acid (IDA) moiety was O-glycosidically connected through an ethylene spacer group to produce the pure alpha- (in case of 2-deoxyglucose) and beta-anomer (in case of glucose). Reaction of the sugar derivatives with the organometallic precursor [M(H2O)3(CO)3]+ (M = 99mTc, Re) produced single products in high yield, which are water-soluble and water-stable. The displacement of the three water molecules of the metal precursor and thus the tridentate coordination of the metal-tricarbonyl core exclusively via the amine and the two carboxylic acid functionalities of the IDA chelate was verified by means of 1D and 2D 1H NMR spectroscopy, mass spectrometry, and IR spectroscopy. The radioactive-labeled products (99mTc) proved their excellent stability in vitro in physiological phosphate buffer (pH = 7.4) and human plasma over a period of 24 h at 37 degrees C.

Development of novel water-soluble, organometallic compounds for potential use in nuclear medicine: synthesis, characterization, and (1)H and (31)P NMR investigations of the complexes fac-[ReBr(CO)3L] (L=bis(bis(hydroxymethyl)phosphino)ethane, bis(bis(hydroxymethyl)phosphino)benzene).

The bidentate, water-soluble phosphine ligands, bis(bis(hydroxymethyl)phosphino)benzene (HMPB, 1) and bis(bis(hydroxymethyl)phosphino)ethane (HMPE, 2) were reacted with the organometallic precursor fac-[ReBr(3)(CO)(3)](2-), 3, to produce the complexes fac-[Re(OH(2))(CO)(3)L](+) and fac-[ReBr(CO)(3)L] (L = HMPE, HMPB), respectively, in good yields. The rhenium complexes fac-[ReBr(CO)(3)HMPB], 5, and fac-[ ReBr(CO)(3)HMPE], 8, were characterized using (1)H and (31)P NMR spectroscopy. The structure of fac-[ReBr(CO)(3)HMPB] was confirmed by single-crystal X-ray spectroscopy. The substitution reactions of HMPE/HMPB with the rhenium precursor 3 in aqueous solution were monitored using time-dependent (31)P NMR techniques. A significant discrepancy in the reaction kinetics and the substitution mechanism between the two bidentate ligands could be observed presumably due to the different chemical backbones.

Influence of the denticity of ligand systems on the in vitro and in vivo behavior of (99m)Tc(I)-tricarbonyl complexes: a hint for the future functionalization of biomolecules.

Functionalization of biologically relevant molecules for the labeling with the novel fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) precursor has gained considerable attention recently. Therefore, we tested seven different tridentate (histidine L(1)(), iminodiacetic acid L(2)(), N-2-picolylamineacetic acid L(3)(), N, N-2-picolylaminediacetic acid L(4)()) and bidentate (histamine L(5)(), 2-picolinic acid L(6)(), 2,4-dipicolinic acid L(7)()) ligand systems, with the potential to be bifunctionalized and attached to a biomolecule. The ligands allowed mild radiolabeling conditions with fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) (30 min, 75 degrees C). The ligand concentrations necessary to obtain yields of >95% of the corresponding organometallic complexes 1-7 ranged from 10(-)(6) to 10(-)(4) M. Complexes of the general formula "fac-[(99m)TcL(CO)(3)]" (L = tridentate ligand) and "fac-[(99m)Tc(OH(2))L'(CO)(3)]" (L' = bidentate ligand), respectively, were produced. Challenge studies with cysteine and histidine revealed significant displacement of the ligands in complexes 5-7 but only little exchange with complexes 1-4 after 24 h at 37 degrees C in PBS buffer. However, no decomposition to (99m)TcO(4)(-) was observed under these conditions. All complexes showed a hydrophilic character (log P(o/w) values ranging from -2.12 to 0.32). Time-dependent FPLC analyses of compounds 1-7 incubated in human plasma at 37 degrees C showed again no decomposition to (99m)TcO(4)(-) after 24 h at 37 degrees C. However, the complexes with bidentate ligands (5-7) became almost completely protein bound after 60 min, whereas the complexes with tridentate coordinated ligands (1-4) showed no reaction with serum proteins. The compounds were tested for their in vivo stability and the biodistribution characteristics in BALB/c mice. The complexes with tridentate coordinated ligand systems (1-4) revealed generally a good and fast clearance from all organs and tissues. On the other hand, the complexes with only bidentate coordinated ligands (5-7) showed a significantly higher retention of activity in the liver, the kidneys, and the blood pool. Detailed radiometric analyses of murine plasma samples, 30 min p.i. of complex fac-[(99m)TcL(1)(CO)(3)], 1, revealed almost no reaction of the radioactive complex with the plasma proteins. By contrast, in plasma samples of mice, which were injected with complex fac-[(99m)Tc(OH(2))L(5)(CO)(3)](+), 5, the entire radioactivity coeluded with the proteins. On the basis of these in vitro and in vivo experiments, it appears that functionalization of biomolecules with tridentate-chelating ligand systems is preferable for the labeling with fac-[(99m)Tc(OH(2))(3)(CO)(3)](+), since this will presumably result in radioactive bioconjugates with better pharmacokinetic profiles.

Complete Carbonylation of fac-

The rate constant of ligand exchange on the complex fac-[(99)Tc(H(2)O)(3)(CO)(3)](+) was determined by means of (13)C, (17)O, and (99)Tc NMR spectroscopy under pressurized conditions in aqueous media. After keeping the sample under CO pressure for an extended period, the formation of [(99)Tc(CO)(6)](+) could unambiguously be detected in the (13)C and (99)Tc NMR spectra.

In vitro and in vivo evaluation of bidentate, water-soluble phosphine ligands as anchor groups for the organometallic fac-[99mTc(CO)3]+-core.

The organometallic precursor fac-[99mTc(OH2)3(CO)3]+, 1a, was reacted with the bidentate, water-soluble phosphine ligands bis(bis(hydroxymethyl)phosphino)ethane (HMPE) and bis(bis(hydroxymethyl)phosphino)benzene (HMPB) in 0.9% saline to produce complexes in >95% yields. High performance liquid chromatography analyses indicate the initial formation of the complexes fac-[99mTcCl(CO)3L] (L = HMPE 2a, HMPB 3a). The neutral complexes ultimately lose the coordinated chloride to produce the cationic species fac-[99mTc(OH2)(CO)3L]+ 2b/3b. In vitro studies showed a high stability of 2b/3b over a wide pH range for >24 h. No decomposition or alteration of the complexes was observed even in the presence of excess histidine, cysteine, or human serum albumin. Experiments performed in normal mice demonstrated a fast clearance of the cationic compounds 2b/3b from the blood pool and clearance through the hepatobiliary and the urinary pathways.

Organometallic 99mTc-aquaion labels peptide to an unprecedented high specific activity.

UNLABELLED: A new peptide labeling method that uses the organometallic aquaion [99mTc(H2O)3(CO)3]+ has been developed. METHODS: A selection of amino acids was labeled at different concentrations with the organometallic aquaion, and the labeling yield was determined by high-performance liquid chromatography. This investigation has shown histidine to be a very potent ligand, with specific activities of up to 6 TBq/micromol (160 Ci/micromol) ligand. Histidine derivatives have been coupled to neurotensin(8-13) (NT[8-13]) and have been labeled with the aquaion, resulting in high specific activities with (N(alpha)-histidinyl)acetic acid-NT(8-13) similar to those with histidine. RESULTS: Histidine derivatives of NT(8-13) labeled using this approach fully retained their receptor affinity, showing KD values of all investigated NT analogs below 1 nmol/L on colon carcinoma HT29 cells. Biodistrbution experiments in BALB/c mice showed complete clearance of (N(alpha)-histidinyl)acetic acid-NT(8-13) from the blood after 24 h and no unwanted accumulation in any tissue. CONCLUSION: The novel labeling method using the organometallic 99mTc-aquaion combines the advantage of highest specific activities with minimal functionalization of proteins and peptides under retention of biologic affinity.

Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex.

We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99m Tc(I)-carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188 Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.

Catabolism of neurotensins. Implications for the design of radiolabeling strategies of peptides.

A major impact in diagnosis and treatment of cancer with peptide based radiopharmaceuticals is expected. Among others neurotensin is considered to be a promising candidate. However, most neurotensin analogues, which bind to the neurotensin receptor have a too short biological half live due to catabolism. Therefore, stabilized fragments have been prepared and labeled with the newly developed [Tc(CO)3](+)-moiety. A single histidine or a (N alpha-His)-Ac group coupled to the N-terminus of the neurotensin fragments were used as a bidentate or a tridentate ligand respectively, which coordinate the metal carbonyl efficiently. Affinity and binding studies of the 99mTc(I) radiolabeled neurotensin fragments revealed a behavior influenced by catabolism and properties of the metal complex.