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Numerous different molecules of prostate-specific membrane antigen (PSMA) ligands are used to detect prostate cancer (PCa); most approaches utilize gallium PET and a few reports describe the role of SPECT/CT. [ 99m Tc]Tc-PSMA-T4 is a new radiopharmaceutical designed for the diagnosis of patients with PCa. We conducted a single site, prospective, preliminary case series study that included 31 patients with PCa; all had undergone clinical, biochemical or imaging examination and exhibited clear or suspicious active disease or clinical/biochemical recurrence of PCa. Whole-body (WB) SPECT/CT after i.v. administration of [ 99m Tc]Tc-PSMA-T4 was utilized; acquisition images were obtained at three time points. The clinical value of the images was assessed in regard to the evaluation of tumor extent in patients with confirmed PC that qualified for initial therapy and the evaluation of tumor recurrence; both provided encouraging results. The late acquisition of WB-SPECT resulted in better lesions delineation. The results of the analysis of the sensitivity/specificity were: 92%/100% in cases of primary cancer, 83%/100% in terms of pelvic lymph nodes disease, 100%/95% in other lymph nodes and soft tissue involvement, respectively, and bone mets were both 100%. An oncotropic SPECT [ 99m Tc]Tc-PSMA-T4 can help in selecting a rational therapeutic strategy for a patient with an initial diagnosis of PCa by assessing the extent of cancer and also after complex radical or palliative therapy in case of biochemical recurrence for re-staging.

Pre-targeting approaches based on the inverse-electron-demand Diels-Alder (iEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have emerged in recent years as valid alternatives to classic targeted strategies to improve the diagnostic and therapeutic properties of radioactive probes. To explore these pre-targeting strategies based on in vivo click chemistry, a small family of clickable chelators was synthesized and radiolabelled with medically relevant trivalent radiometals. The structure of the clickable chelators was diversified to modulate the pharmacokinetics of the resulting [ 111 In]In-radiocomplexes, as assessed upon injection in healthy mice. The derivative DOTA-Tz was chosen to pursue the studies upon radiolabelling with 90 Y, yielding a radiocomplex with high specific activity, high radiochemical yields and suitable in vitro stability. The [ 90 Y]Y-DOTA-Tz complex was evaluated in a prostate cancer PC3 xenograft by ex-vivo biodistribution studies and Cerenkov luminescence imaging (CLI). The results highlighted a quick elimination through the renal system and no relevant accumulation in non-target organs or non-specific tumor uptake. Furthermore, a clickable bombesin antagonist was injected in PC3 tumor-bearing mice followed by the radiocomplex [ 90 Y]Y-DOTA-Tz, and the mice imaged by CLI at different post-injection times (p.i.). Analysis of the images 15 min and 1 h p.i. pointed out an encouraging quick tumor uptake with a fast washout, providing a preliminary proof of concept of the usefulness of the designed clickable complexes for pre-targeting strategies. To the best of our knowledge, the use of peptide antagonists for this purpose was not explored before. Further investigations are needed to optimize the pre-targeting approach based on this type of biomolecules and evaluate its eventual advantages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 D'Onofrio, Silva, Gano, Karczmarczyk, Mikołajczak, Garnuszek and Paulo.)

Metastatic castration-resistant prostate cancer (mCRPC) is a progressive and incurable disease with poor prognosis for patients. Despite introduction of novel therapies, the mortality rate remains high. An attractive alternative for extension of the life of mCRPC patients is PSMA-based targeted radioimmunotherapy. In this paper, we extended our in vitro study of 223 Ra-labeled and PSMA-targeted NaA nanozeolites [ 223 RaA-silane-PEG-D2B] by undertaking comprehensive preclinical in vitro and in vivo research. The toxicity of the new compound was evaluated in LNCaP C4-2, DU-145, RWPE-1 and HPrEC prostate cells and in BALB/c mice. The tissue distribution of 133 Ba- and 223 Ra-labeled conjugates was studied at different time points after injection in BALB/c and LNCaP C4-2 tumor-bearing BALB/c Nude mice. No obvious symptoms of antibody-free and antibody-functionalized nanocarriers cytotoxicity and immunotoxicity was found, while exposure to 223 Ra-labeled conjugates resulted in bone marrow fibrosis, decreased the number of WBC and platelets and elevated serum concentrations of ALT and AST enzymes. Biodistribution studies revealed high accumulation of 223 Ra-labeled conjugates in the liver, lungs, spleen and bone tissue. Nontargeted and PSMA-targeted radioconjugates exhibited a similar, marginal uptake in tumour lesions. In conclusion, despite the fact that NaA nanozeolites are safe carriers, the intravenous administration of NaA nanozeolite-based radioconjugates is dubious due to its high accumulation in the lungs, liver, spleen and bones.

Competing Interests: The authors declare no conflict of interest.

A new PSMA ligand (PSMA-D4) containing the Glu-CO-Lys pharmacophore connected with a new linker system (L-Trp-4-Amc) and chelator DOTA was developed for radiolabeling with therapeutic radionuclides. Herein we describe the synthesis, radiolabeling, and preliminary biological evaluation of the novel PSMA-D4 ligand. Synthesized PSMA-D4 was characterized using TOF-ESI-MS, NMR, and HPLC methods. The novel compound was subject to molecular modeling with GCP-II to compare its binding mode to analogous reference compounds. The radiolabeling efficiency of PSMA-D4 with 177 Lu, 90 Y, 47 Sc, and 225 Ac was chromatographically tested. In vitro studies were carried out in PSMA-positive LNCaP tumor cells membranes. The ex vivo tissue distribution profile of the radioligands and Cerenkov luminescence imaging (CLI) was studied in LNCaP tumor-bearing mice. PSMA-D4 was synthesized in 24% yield and purity >97%. The radio complexes were obtained with high yields (>97%) and molar activity ranging from 0.11 to 17.2 GBq mcmol -1 , depending on the radionuclide. In vitro assays confirmed high specific binding and affinity for all radiocomplexes. Biodistribution and imaging studies revealed high accumulation in LNCaP tumor xenografts and rapid clearance of radiocomplexes from blood and non-target tissues. These render PSMA-D4 a promising ligand for targeted therapy of prostate cancer (PCa) metastases.

Introduction: The information on the presence of cold metal complexes in radiolabeled DOTA-TATE or DOTA-TOC is important in assessing the cause of the radiolabeling failure, poor radiolabeling yield and/or low effective molar activity. DOTA-peptide complexes are detectable using UV-Vis detector. The main limitation in the quantitative analysis is the limited availability of standard substances and the lack of data on their molar absorption coefficients. The aim of our study was development and validation of HPLC method enabling RCP analysis and identification and quantification of metal complexes impurities in the radiopharmaceutical preparations of DOTA-chelated peptides., Methods: Complexes of DOTA-TATE and DOTA-TOC with several metals, were prepared. Their molar absorption coefficients at 220 nm were determined. The developed HPLC method has been validated in terms of quantitative determination of non-complexed DOTA-TATE and DOTA-TOC and their respective complexes with metallic individuals., Results: Good chromatographic separation of the individual metal-DOTA-peptide complexes was achieved. The resolution between peaks of interest in radioactive preparations (complexes with: yttrium-90, lutetium-177, gallium-68) and metallic impurities was well above 1.5 (except gallium-68 DOTA-TOC preparations). Limits of detection and quantification were determined based on the parameters of the calibration curves. Based on the spectrophotometric and HPLC-DAD studies and statistical analysis of the results obtained, the average molar absorption coefficient was determined for studied DOTA-TATE and DOTA-TOC complexes, ε HPLC-DAD  = 48 × 10 3 M -1  cm -1 . With the use of the determined molar absorption coefficient the method enabled quantitative determination of non-labelled peptide in the radioactive preparation in the linearity range of 0.5-100 μg/mL for DOTA-TATE(net) and 0.5-100 μg/mL for DOTA-TOC(net)., Conclusion: The developed HPLC method is suitable for RCP determination of radiolabelled DOTA-TATE and DOTA-TOC preparations. Determination of the average molar absorption coefficient for DOTA-TATE and DOTA-TOC complexes allows assessment of the total content of the peptide in radiopharmaceutical preparation regardless of its chemical form (free ligand, associated with radionuclide, in the form of a complex with metal ions being the impurity) using the HPLC method with UV detection., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recently, radiolabelled antagonists targeting somatostatin receptors subtype 2 (SST2) in neuroendocrine neoplasms demonstrated certain superior properties over agonists. Within the ERA-PerMED project "TECANT" two 99m Tc-Tetramine (N4)-derivatized SST2 antagonists (TECANT-1 and TECANT-2) were studied for the selection of the best candidate for clinical translation. Receptor-affinity, internalization and dissociation studies were performed in human embryonic kidney-293 (HEK293) cells transfected with the human SST2 (HEK-SST2). Log D , protein binding and stability in human serum were assessed. Biodistribution and SPECT/CT studies were carried out in nude mice bearing HEK-SST2 xenografts, together with dosimetric estimations from mouse-to-man. [ 99m Tc]Tc-TECANT-1 showed higher hydrophilicity and lower protein binding than [ 99m Tc]-TECANT-2, while stability was comparable. Both radiotracers revealed similar binding affinity, while [ 99m Tc]Tc-TECANT-1 had higher cellular uptake (>50%, at 2 h/37 °C) and lower dissociation rate (<30%, at 2 h/37 °C). In vivo, [ 99m Tc]Tc-TECANT-1 showed lower blood values, kidney and muscles uptake, whereas tumour uptake was comparable to [ 99m Tc]Tc-TECANT-2. SPECT/CT imaging confirmed the biodistribution results, providing the best tumour-to-background image contrast for [ 99m Tc]Tc-TECANT-1 at 4 h post-injection (p.i.). The estimated radiation dose amounted to approximately 6 µSv/MBq for both radiotracers. This preclinical study provided the basis of selection of [ 99m Tc]Tc-TECANT-1 for clinical translation of the first 99m Tc-based SST2 antagonist.

Background: This work aimed to evaluate the influence of two chelators: DOTA(SCN) and DOTA(NHS) on radioimmunotherapy using 177 Lu-DOTA-Rituximab preparations in murine lymphoma xenograft models. Subsequently, based on animal data, the organ radiation-absorbed doses were extrapolated to humans (adult male). Materials and Methods: Therapeutic efficacy of 177 Lu-DOTA-Rituximab was evaluated in male nude mice bearing either Raji (B lymphocyte, CD20 + ) and Jurkat (T lymphocyte, CD20) xenografts, utilizing an anti-CD20 antibody-Rituximab conjugate with either DOTA(SCN) or DOTA(NHS). The DOTA-Rituximab conjugates were prepared in the form of freeze-dried kits. Results: All radioimmunoconjugates were obtained with high radiolabeling yield (radiochemical purity, RCP > 95%) and specific activity of ca. 0.5 GBq/mg. Therapeutic effects of 177 Lu-DOTA-Rituximab were observed in animals regardless whether DOTA(SCN) or DOTA(NHS) were used for conjugation. Importantly, therapy involving 177 Lu-DOTA-Rituximab was more effective than use of Rituximab alone. Conclusions: The degree of antitumor efficacy was dependent on the type of applied bifunctional chelators conjugated to mAb. However, this difference was not statistically significant. Dosimetry calculations showed that the absorbed radiation doses extrapolated to humans were very low for osteogenic cells regardless of the conjugates. Organs like the liver and spleen, treated with 177 Lu-DOTA(SCN)-Rituximab, showed similar radiation absorbed doses when compared with 177 Lu-DOTA(NHS)-Rituximab.

Endohedral structures with La 0 or La 3+ encapsulated in chiral (1,16)C 58 N 2 or achiral (1,4)C 58 N 2 diazafullerenes were studied at the B3LYP/G-31G*/SDD level. Two stable locations of La 0 and La 3+ are possible in each cage but only with La 0 @(1,16)C 58 N 2 can the two isomers coexist. We found that an AIM determined hapticity of the endohedral species selectively differentiates the systems. We predict that there will always exist IR and Raman bands which allow for them to be identified in the presence of the parent cage. For the La 0 @(1,16) C 58 N 2 molecules and the parent diazafullerene, the Raman spectra are likely to reveal a pre-resonance effect even at 785 nm and it seems possible to selectively excite only one isomer. The calculated electronic spectra suggested a chance to determine the less populated diazafullerene in the presence of the more populated one, be it chiral or achiral. For the chiral endohedral isomers, the calculated VCD spectra are quite dissimilar and the two endohedral isomers and the parent heterofullerene seem to be easily detected. Eventually, we defined the endohedral isomerism as follows: The endohedral isomerism is the phenomenon whereby an internal individuum captured in a cage can occupy more than one stable position without changing the cage connectivity., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

With the development of ever more radiopharmaceuticals suitable for theranostic applications, translation of novel compounds from the preclinical stage towards clinical application becomes a bottleneck for the advances in Nuclear Medicine. This review article summarizes the current regulatory framework for clinical trials with radiopharmaceuticals in the European Union, provides a general overview of the documentation required, and addresses quality, safety, and clinical aspects to be considered. By using a recent successful example of translating a theranostic peptide radioligand, namely 111 In-CP04, which targets receptors expressed in medullary thyroid carcinoma, the pathway from the preclinical development over establishing the required pharmaceutical documentation to designing and submitting a clinical trial is reviewed. Details regarding preclinical data, generation of the documentation, and final successful application are described. This article should provide an insight in an ever more complex process to bring innovations in the field of radiopharmaceuticals into patients., (© 2019 The Authors. Journal of Labelled Compounds and Radiopharmaceuticals Published by John Wiley & Sons Ltd.)

Background: 44Sc is becoming attractive as a PET radionuclide due to its decay characteristics. It can be produced from 44Ca present in natural calcium with 2.08% abundance., Materials and Methods: The targets were mostly prepared from natural CaCO3 or metallic calcium in the form of pellets. After irradiation they were dissolved in 3 M hydrochloric acid and 44Sc was separated from excess of calcium by precipitation of scandium hydroxide using ammonia. Alternatively, targets were dissolved in 11 M hydrochloric acid and 44Sc was separated by extraction chromatography on UTEVA resin. As the next step, in both processes 44Sc was further purified on a cation exchange resin. Initially, the separation procedures were developed with 46Sc as a tracer. Gamma spectrometry with a high purity germanium detector was used to determine the separation efficiency. Finally, the CaCO3 pellet with 99.2% enrichment in 44Ca was activated with protons via 44Ca(p,n)44Sc nuclear reaction., Results: Altogether twenty two irradiations and separations were performed. The working procedures were developed and the quality of separated 44Sc solution was confirmed by radiolabeling of DOTATATE. The chemical purity of the product was sufficient for preclinical experiments. At the end of around 1 hour proton beam irradiation of CaCO3 pellet with 99.2% enrichment in 44Ca the obtained radioactivity of 44Sc was more than 4.8 GBq., Conclusion: 44Sc can be produced inexpensively with adequate yields and radionuclidic purity via 44Ca(p,n)44Sc nuclear reaction in small cyclotrons. The recovery yield in both investigated separation methods was comparable and amounted above 90%. The obtained 44Sc was pure in terms of radionuclide and chemical purity, as shown by the results of peptide radiolabeling.

This work presents a comparative biological evaluation of 90 Y- and 177 Lu- labelled DOTA-SCN and DOTA-NHS conjugated to Rituximab in tumour-bearing mice. Two DOTA derivatives, p-SCN-Bn-DOTA and DOTA-NHS-ester were conjugated to Rituximab and then freeze-dried kit formulations were prepared, as previously described (1). Tissue distribution was investigated in tumour-bearing (Raji s.c.) male Rj: NMRI-Foxn1 nu /Foxn1 nu mice at different time points after administration of 177 Lu-DOTA-Rituximab or 90 Y-DOTA-Rituximab (6 MBq/10 μg per mouse). In addition, tumour images were acquired with a PhotonIMAGER TM after injection of 90 Y-DOTA (SCN)-Rituximab. All radioimmunoconjugates were obtained with high radiolabelling yield (RCP > 98%) and specific activity of ca. 0.6 GBq/mg. The conjugates were stable in human serum and in 0.9% NaCl; however, progressive aggregation was observed with time, in particular for DOTA -(SCN) conjugates. Both 177 Lu- and 90 Y-DOTA -(SCN)-Rituximab revealed slow blood clearance. The maximum tumour uptake was found 72 h after injection of 177 Lu-DOTA -(SCN)-Rituximab (9.3 ID/g). A high radioactivity uptake was observed in liver and spleen, confirming the hepatobiliary excretion route. The results obtained by the radioactive optical imaging harmonize with those from the biodistribution study.

Background: Technetium Tc99m Exametazime (99mTc-HMPAO) is currently used as a radiopharmaceutical for determining regional cerebral blood flow and for the labelling of autologous leucocytes for infection and inflammation imaging. The HMPAO ligand exists in two diastereomeric forms: d,l and meso. Usually, the substance is obtained in low chemical yield in a time consuming procedure. Furthermore, the final product still contains some amounts of the meso-form. The aim of this study was to develop the efficient, reliable and fast method for isolation of the d,l-HMPAO, which would provide the ligand with high purity and free from the meso-diastereomer., Material and Methods: The mixture of the meso- and d,l-HMPAO was synthesized in two-steps by condensation of propanediamine with keto-oxime and the reduction of the obtained bisimine. The d- and l-enantiomers were separated individually directly from this mixture by repeated crystallizations from ethanol as their tartrate salts and pooled together in equal proportions. That substance was characterized for its identity and isomeric purity using IR, HPLC and GC methods. The meso-free d,l-HMPAO was used for the preparation of the radiopharmaceutical freeze-dried kit for technetium-99m radiolabelling. Quality assessment of obtained 99mTc-d,l-HMPAO complex was performed according to the current Ph.Eur. monograph 1925 and USP monograph - Technetium Tc99m Exametazime Injection. To verify its biological activity, the kit-prepared 99mTc-d,l-HMPAO has been used for the white blood cell (WBC) labelling., Results: According to the proposed synthesis route the d,l-HMPAO was obtained with around 18-20% yield in the total time of 10 days. The ligand identity was confirmed and the HPLC analysis revealed more than 99% chemical purity. The undesired meso-form was not detected. Freeze dried kit formulation for 99mTc-labelling of d,l-HMPAO has been established and four batches of kits were manufactured. The radiochemical purity of 99mTc-d,l-HMPAO complex was high (> 95% of lipophilic technetium-99m exametazime). Brain uptake in rats reached 2.1 ± 0.3%. The in vitro labelling of WBC resulted in 68.3 ± 6.6% yield., Conclusion: A new synthesis method of d,l-HMPAO, drug substance for technetium-99m exametazime preparation has been developed.

Introduction: From a series of radiolabelled cholecystokinin (CCK) and gastrin analogues, (111)In-CP04 ((111)In-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) was selected for further translation as a diagnostic radiopharmaceutical towards a first-in-man study in patients with medullary thyroid carcinoma (MTC). A freeze-dried kit formulation for multicentre application has been developed. We herein report on biosafety, in vivo stability, biodistribution and dosimetry aspects of (111)In-CP04 in animal models, essential for the regulatory approval of the clinical trial., Materials and Methods: Acute and extended single dose toxicity of CP04 was tested in rodents, while the in vivo stability of (111)In-CP04 was assessed by HPLC analysis of mouse blood samples. The biodistribution of (111)In-CP04 prepared from a freeze-dried kit was studied in SCID mice bearing double A431-CCK2R(±) xenografts at 1, 4 and 24h pi. Further 4-h animal groups were either additionally treated with the plasma expander gelofusine or injected with (111)In-CP04 prepared by wet-labelling. Pharmacokinetics in healthy mice included the 30min, 1, 4, 24, 48 and 72h time points pi. Dosimetric calculations were based on extrapolation of mice data to humans adopting two scaling models., Results: CP04 was well-tolerated by both mice and rats, with an LD50>178.5μg/kg body weight for mice and a NOAEL (no-observed-adverse-effect-level) of 89μg/kg body weight for rats. After labelling, (111)In-CP04 remained >70% intact in peripheral mouse blood at 5min pi. The uptake of (111)In-CP04 prepared from the freeze-dried kit and by wet-labelling were comparable in the A431-CCK2R(+)-xenografts (9.24±1.35%ID/g and 8.49±0.39%ID/g, respectively; P>0.05). Gelofusine-treated mice exhibited significantly reduced kidneys values (1.69±0.15%ID/g vs. 5.55±0.94%ID/g in controls, P<0.001). Dosimetry data revealed very comparable effective tumour doses for the two scaling models applied, of 0.045 and 0.044mSv/MBq., Conclusion: The present study has provided convincing toxicology, biodistribution and dosimetry data for prompt implementation of the freeze-dried kit formulation without or with gelofusine administration in a multicentre clinical trial in MTC patients., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: A variety of radiolabelled minigastrin analogues targeting the cholecystokinin 2 (CCK2) receptor were developed and compared in a concerted preclinical testing to select the most promising radiotracer for diagnosis and treatment of medullary thyroid carcinoma (MTC). DOTA-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (CP04) after labelling with (111)In displayed excellent characteristics, such as high stability, receptor affinity, specific and persistent tumour uptake and low kidney retention in animal models. Therefore, it was selected for further clinical evaluation within the ERA-NET project GRAN-T-MTC. Here we report on the development of a pharmaceutical freeze-dried formulation of the precursor CP04 for a first multi-centre clinical trial with (111)In-CP04 in MTC patients., Materials and Methods: The kit formulation was optimised by adjustment of buffer, additives and radiolabelling conditions. Three clinical grade batches of a final kit formulation with two different amounts of peptide (10 or 50 μg) were prepared and radiolabelled with (111)In. Quality control and stability assays of both the kits and the resulting radiolabelled compound were performed by HPLC analysis., Results: Use of ascorbic acid buffer (pH4.5) allowed freeze-drying of the kit formulation with satisfactory pellet-formation. Addition of methionine and gentisic acid as well as careful selection of radiolabelling temperature was required to avoid extensive oxidation of the Met(11)-residue. Trace metal contamination, in particular Zn, was found to be a major challenge during the pharmaceutical filling process in particular for the 10 μg formulation. The final formulations contained 10 or 50 μg CP04, 25mg ascorbic acid, 0.5mg gentisic acid and 5mg L-methionine. The radiolabelling performed by incubation of 200-250 MBq (111)InCl3 at 90 °C for 15 min resulted in reproducible radiochemical purity (RCP) >94%. Kit-stability was proven for >6 months at +5 °C and at +25 °C. The radiolabelled product was stable for >4h at +25 °C., Conclusion: A kit formulation to prepare (111)In-CP04 for clinical application was developed, showing high stability of the kit as well as high RCP of the final product., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

The use of [(18)F]labelled nortropane derivative 2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-fluoroethyl)-nortropane (FECNT) as a dopamine transporter ligand for PET imaging is dependent on efficient radiosynthesis method. Herein, the automated synthesis of [(18)F]FECNT from its chlorinated precursor in commercially available SynChrom [(18)F] R&D module has been developed. The synthesis unit was readily configured for the one-step synthesis from corresponding chlorinated precursor. The radiolabeling process involved a classical [(18)F]fluoride nucleophilic substitution performed at 110 °C for 12 min and finally HPLC and SPE purification. Crude [(18)F]FECNT was obtained with a radiolabeling yield of 59 ± 12% (n = 5). The average uncorrected amount of [(18)F]FECNT in the final formulated dose was 2.0 ± 0.5 GBq (32 ± 7% overall decay-corrected yields) obtained with radiochemical purity over 99% and specific activity of 55 GBq/µmol. The total duration of the procedure was 80-90 min. An automated radiosynthesis of [(18)F]FECNT with high radiochemical purity may provide a simple and robust method of radiopharmaceutical preparation for routine clinical applications., (Copyright © 2016 John Wiley & Sons, Ltd.)

Background: Preliminary clinical evaluation of 99mTc-EDDA/HYNIC-Met14-Exendin-4 showed that the complex offers new diagnostic possibilities for insulinoma and MTC. Exendin-4 contains methionine at position 14 in the amino acid chain, which may be oxidized to methionine sulfoxide and, from the pharmaceutical point of view, the oxidized moiety becomes an undesired impurity in the final radioactive preparation. Therefore, the aim of this study was to investigate the influence of commonly used methods to eliminate the effect of methionine oxidation in peptides, i.e. the replacement of methionine by norleucine (Nle) and the addition of L-methionine, on the in vitro stability and the biodistribution., Material and Methods: 99mTc-EDDA/HYNIC-Met14-Exendin-4, 99mTc-EDDA/HYNIC-Nle14-Exendin-4, 99mTc-EDDA/HYNIC-Met14-Ex-endin-4 with the addition of L-methionine and an oxidized form of Exendin-4, i.e. 99mTc-EDDA/HYNIC-Met14(ox)-Exendin-4 were compared in vivo with 68Ga-NODAGA-Nle14-Exendin-4 in normal Wistar rats. The stability and lipophilicity were determined in vitro., Results: Biodistribution studies confirmed the specific uptake of all tested complexes in the GLP-1 positive organs: lungs, pancreas and stomach. The uptake of 99mTc-EDDA/HYNIC-Met14-Exendin-4 with the addition of L-methionine and for 68Ga-NODAGA-Nle14-Exendin-4 at 1h p.i. was around 2-fold higher than that of 99mTc-EDDA/HYNIC-Met14-Exendin-4 and 99mTc-EDDA/HYNIC-Nle14-Exendin-4., Conclusion: Although the substitution of methionine by norleucine in the HYNIC-Exendin-4 did not result in improved bio-distribution, the use of L-methionine, as the excipient that inhibits the oxidation of methionine in the peptide chain resulted in higher lung/blood and stomach/blood uptake ratios. Our results confirmed that methionine at position 14 of amino acid chain of Exendin-4 plays an important role in the interaction with GLP-1 receptor positive tissue.

Background: The minigastrin analogue - CP04: DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 has been developed for CCK2R targeting. This analogue can be radiolabelled with 111In or 68Ga for imaging, or with 90Y and 177Lu for therapy. However, affinity of the chelator-peptide conjugates to the cell membrane receptors may vary depending on the metal incorporated into the complex. So far, there are no such studies for the ligands of gastrin/cholecystokinin receptor CCK2R. It is supposed that the reason for the differentiation of receptor affinity to the respective receptors is in the changes of structure of chelating system and their influence on the bioactive conformations of the metal conjugated peptides. Herein, we report on the radiolabeling of CP04 with 90Y, 177Lu and 68Ga and synthesis of cold CP04 complexes with respective stable metals for further structural and physico-chemical and biological studies., Materials and Methods: From 200 to 600 MBq of 90Y, 177Lu or 68Ga were used for radiolabelling of 20 μg of CP04 dissolved in ascorbic acid solution (50 mg/mL, pH 4.5). Non-radioactive complexes with Lu and Ga were synthesized in milligram amounts starting from 0.5 mg up to 5 mg of CP04 dissolved in ascorbic acid solution (50 mg/mL, pH 4.5) when using 2-molar excess of the metal ions. Complex formation needed 5 min in microwave oven or 12 min in thermo-block at 95°C. RP-HPLC isocratic method (Kinetex 150/4.6 mm; 25% AcN/0.1% TFA, 1 mL/min) with UV/Vis and radiometric detection was developed for investigation of the radiolabelled and "cold" complexes. For LC-MS investigations, HPLC method was modified replacing TFA by formic acid., Results and Discussion: Yields of CP04 radiolabelling were greater than 90% for all three radionuclides. The HPLC method enabled identification of these radio-complexes based on comparison to their non-radioactive equivalents. In all cases, chromatograms revealed peaks that could be attributed to the metal-CP04 complexes and to impurities (including methionine oxidation). LC-MS analysis of Ga and Lu complexes revealed conformity of the observed molecular ions to the predicted formulas (m/z 2116 and 2220 Da for Ga and Lu, respectively). Different chromatographic behaviour observed for Ga-CP04 complex comparing to Lu- and Y- labelled peptide (relative retention to CP04: 1.08, 0.86 and 0.85, respectively) suggest different coordination of the metal ions. Therefore, further studies are planned using the non-radioactive complexes in order to assess their structural conformations.

Rituximab when radiolabelled with (177)Lu or (90)Y has been investigated for the treatment of patients with Non-Hodgkin's Lymphoma. In this study, we optimized the preparation of antibody conjugates with chelating agent in the freeze-dried kit. It shortens procedures needed for the successful radiolabeling with lutetium-177 and yttrium-90 and assures reproducible labelling yields. Various molar ratios of Rituximab:DOTA (from 1:5 to 1:100) were used at the conjugation step and different purification method to remove unbound DOTA were investigated (size-exclusion chromatography, dialysis, ultrafiltration). The final monoclonal antibody concentration was quantified by Bradford method, and the number of DOTA molecules was determined by radiolabeling assay using (64)Cu. The specific activity of (177)Lu-DOTA-Rituximab and (90)Y-DOTA-Rituximab were optimized using various amounts of radiometal. Quality control (SE-HPLC, ITLC) and stability study were performed. An average of 4.2 ± 0.8 p-SCN-Bz-DOTA molecules could be randomly conjugated to a single molecule of Rituximab. The ultrafiltration system was the most efficient for purification and resulted in the highest recovery efficiency (77.2%). At optimized conditions the (177)Lu-DOTARituximab and (90)Y-DOTA-Rituximab were obtained with radiochemical purity >99% and specific activity ca. 600 MBq/mg. The radioimmunoconjugates were stable in human serum and 0.9% NaCl. After 72 h of incubation the radiochemical purity of (177)Lu-DOTA-Rituximab decreased to 94% but it was still more than 88% for (90)Y-DOTA-Rituximab. The radioimmunoconjugate showed stability after six months storage at 2 - 8(0)C, as a lyophilized formulation. Our study shows that Rituximab-DOTA can be efficiently radiolabeled with (177)Lu and (90)Y via p-SCN-Bn-DOTA using a freezedried kit.

Background: The reliable method for determination of identity and radiochemical purity (RCP) is of great importance in radiopharmaceutical development. This is especially relevant when more than one form of radiometal/ligand complex can be formed during radiolabelling, such as complexes of 99mTc or 188Re with meso-2,3-dimercaptosuccinic acid (DMSA), where depending on the pH, metal can occur either at +3 or +5 oxidation state. The aim of our study was to evaluate possibilities for optimization of chromatographic systems leading to specific and reliable analytical method for determination of the identity and RCP of DMSA complexes with 99mTc or 188Re., Material and Methods: The commercial DMSA kits (POLATOM) were used for preparation of technetium-99m (III) and (V) complexes with DMSA. 99mTc(V)-DMSA complexes were prepared by addition of NaHCO3 to the kit vial prior to 99mTc-eluate to obtain pH ~8. 188Re(V)-DMSA was prepared either directly or using intermediate 188Re(III)-EDTA complex added to DMSA. RCP was evaluated by TLC using: ITLC-SG developed in methylethylketon, SG60 coated plates developed in: n-BuOH/H2O/CH3COOH and n-PrOH/H2O/CH3COOH systems, and in H2O. Comparative biodistribution studies were performed in normal Wistar rats., Results: Using silica gel plates and n-PrOH, H2O and acetic acid in the developing solution, we observed that 99mTc/188Re(III)-DMSA and 99mTc/188Re(V)-DMSA complexes could be well separated from each other and from the impurities in the form of free pertechnetate/perrhenate. In vivo studies showed quite different biodistribution of 99mTc(III)- and 99mTc(V)-DMSA. The trivalent complex accumulated mainly in kidneys (>40%ID), while 99mTc(V)-DMSA revealed high excretion with urine and relatively high concentration in osseous tissue (ca. 2 %ID/g). Accumulation of this complex in kidneys was very low (ca. 2.5 %ID). Biodistribution pattern of 188Re(V)-DMSA prepared directly was almost identical to that of 99mTc(V)-DMSA. Biodistribution results of the 188Re preparation obtained using 188Re(III)-EDTA intermediate indicated that the preparation contained the mixture of penta- and trivalent 188Re complexes. The quite high accumulation of radioactivity in kidneys (23 %ID) gave evidence of the presence of 188Re(III)-DMSA in this preparation, what was also confirmed by the results of TLC analysis performed using silica gel plate and n-propanol/water/acetic acid as developing system., Conclusions: Based on our study, we have made recommendation on the suitable methods for investigations of RCP of DMSA complexes, i.e.: SG60 plates developed in the mixture of n-propanol/water/acetic acid, which enable determination of the tri- and pentavalent DMSA complexes, as well as, the pertechnetate/perrhenate impurity, and developed in water for determination of the colloidal residue.

Myocardial perfusion studies are among the most often performed investigations in Nuclear Medicine. However, the development of radiopharmaceuticals for cardiology is an emerging discipline and several other radiotracers have been proven to be useful. Although the myocardial perfusion studies have a well established role in the management of cardiac disorders, still a number of radiopharmaceuticals are under development for a variety of specific cardiac indications and their eventual clinical role remains to be seen. The paper provides a short overview of currently used radiopharmaceuticals and potential molecular imaging radiotracers applicable in cardiology.

Introduction: Interleukin-2 (IL-2) when radiolabelled with (99m)Tc has been proved useful in imaging the side of lymphocytic infiltration in patients with autoimmune disorders and plays a significant role as a T-cell imaging agent. However, the labelling procedures used so far appeared to be rather complex and laborious. The aim of present study was to develop an efficient procedure of (99m)Tc-labelling of recombinant human interleukin-2 (rhIL-2) via hydrazinonicotinamide (HYNIC) to develop a dry kit formulation., Methods: Various molar ratios of rhIL-2/HYNIC (from 1:2 to 1:12) were used at the conjugation step. The conjugates were purified on a PD-10 column to remove the excess of unbound HYNIC, as well as of any aggregates. The final peptide concentration was quantified by the BCA method, and the number of HYNIC molecules incorporated into a rhIL-2 molecule was determined based on the reaction with 2-sulfobenzaldehyde. The (99m)Tc-labelling was optimized using various amounts of HYNIC-rhIL-2, (99m)Tc, SnCl(2), tricine and nicotinic acid (NA). Quality control included GF-HPLC, ITLC, SDS-PAGE and biological assay. Biodistribution studies were performed in Swiss mice and Wistar rats., Results: Generally, the highest radiolabelling yields were achieved when the HYNIC-rhIL-2 conjugates of ca. 2-4 HYNIC molecule substitution ratios were used. The optimal pH of the reaction medium was found to be in the range of 6.5 to 7.0. GF-HPLC analysis indicated that monomer and aggregates of (99m)Tc-HYNIC-rhIL-2 are formed during radiolabelling. At optimized conditions of wet radiolabelling, the (99m)Tc-HYNIC-rhIL-2 monomer was obtained with radiochemical purity >99%, specific activity of ca. 4 GBq/mg rhIL-2 and overall yield of ca. 65%. The two-vial freeze-dried kit was prepared: the first vial contained 30 μg HYNIC-rhIL-2, co-ligands, buffer and antioxidant; the second vial contained tricine and SnCl(2). The monomer of (99m)Tc-HYNIC-rhIL-2 was obtained by gel chromatography on a PD-10 column. No differences between labelled and unlabelled IL2 in terms of biological activity were observed., Conclusions: Our study shows that rhIL-2 can be efficiently radiolabelled with (99m)Tc via HYNIC, with tricine and NA as co-ligands using a two-vial freeze-dried kit. This enables the preparation of sterile and ready-to-use (99m)Tc-HYNIC(tricine,NA)-rhIL-2 within 1 h., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Application of cross-linking agents such as SATA and 2-iminothiolane (2-IT) for radiochemical synthesis of new radioactive Pt(II) and Pt(IV) complexes with carnosine was investigated. The mixed-ligand Pt(II)([(125)I]Hist)(Carnosine) complex has been synthesized in a multi-step reaction. First, carnosine was modified by the attachment of SATA. After chromatographic purification, the conjugate was unprotected to form a reactive sulfhydryl functional group, and then the modified carnosine was substituted to PtCl(2)[(125)I]Hist complex. The Pt(II)(IT-[(125)I]Carnosine) and Pt(IV)(IT-[(131)I]Carnosine) complexes were synthesized in a three-step reaction. First, carnosine was labeled with iodine radionuclide ((125)I or (131)I), followed by conjugation with 2-IT. The modified IT-[*I]Carnosine was complexed with tetrachloroplatinate or hexachloroplatinate. Comparative biodistribution studies were performed in normal Wistar rats and in Lewis rats with implanted (s.c.) rat pancreatic tumor cells (AR42J). The HPLC analysis showed a relatively fast formation of the new mixed-ligand Pt([(125)I]Hist)(Carnosine) complex (yield ca. 50% after 20h). Reaction of K(2)PtCl(4) with [(125)I]Carnosine modified by 2-IT proceeded rapidly and with a high yield (>95% after 2h). The synthesis of the Pt(IV)IT-[*I]Carnosine complex was the slower reaction in comparison to the analogous synthesis of the Pt(II) complex (yield ca. 70% after 12h), thus a purification step was necessary. The biodistribution study proved the in vivo stability of the newly synthesized complexes (a low accumulation in thyroid gland and in GIT) and showed that the conjugation of the modified carnosine changes significantly biodistribution scheme of the Pt complexes comparing to the reference Pt(II)[*I]Hist and Pt(IV)([*I]Hist)(2) complexes. The mixed-ligand complex was rapidly excreted in urine and revealed the highest accumulation in kidneys (>5%ID/g). A very high concentration in blood and in liver was observed for the Pt(II)(IT-[(125)I]Carnosine) complex; however, at the same time the lowest concentration in kidneys was noted. Preliminary studies in the rat's tumor model indicated for this complex a favorable tumor to muscle ratio. In the case of Pt(IV)(IT-[*I]Carnosine) apart from ca. 12-times decrease of the liver accumulation, additional 4-times decrease of an accumulation in kidneys was observed in comparison to the Pt(IV)([*I]Hist)(2) complex. Our study showed that the short peptides can be efficiently substituted to the platinum core via the reactive sulfhydryl group introduced by SATA or 2-IT. The new radioactive platinum complexes with carnosine possess favorable biodistribution schemes, which make them potential candidates for radio-chemotherapeutical agents., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Introduction: The aim of the study was to compare in vitro and in vivo a novel DOTA-chelated bombesin (BN) analog of the amino acid sequence, QRLGNQWAVGHLM-CONH(2) (BN[2-14]NH(2)), labeled with (90)Y and (177)Lu, for its potential use in targeted radiotherapy of tumors expressing gastrin releasing peptide (GRP) receptors. The same amino acid sequence, but with different chelator, referred as BN1.1 (Gly-Gly-Cys-Aca-QRLGNQWAVGHLM-CONH(2)), has already been studied and reported; however, the DOTA-chelated one, suitable for labeling with M(+3) type radiometals, was not yet described., Methods: The conditions for labeling of DOTA-BN[2-14]NH(2) with noncarrier added (90)Y and with (177)Lu [specific activity (SA), 15 Ci/mg Lu] were investigated and optimized to provide (90)Y-DOTA-BN[2-14]NH(2) and (177)Lu-DOTA-BN[2-14]NH(2) of high SA. The stability of the radiolabeled compounds in human serum was evaluated over a period of 24 h. The human prostate cancer cell line PC-3, known to express GRP receptors, was used for in vitro evaluation of radiolabeled peptide affinity to GRP receptors and for assessment of cytotoxicity of both nonlabeled and radiolabeled peptide. Biodistribution accompanied by receptor blocking was studied in normal Swiss mice., Results: (90)Y-DOTA-BN[2-14]NH(2) and (177)Lu-DOTA-BN[2-14]NH(2) were obtained with radiochemical yield >98% and high SA (67.3 GBq (90)Y/mumol and 33.6 GBq (177)Lu/mumol, respectively). They were stable when incubated in human serum for up to 24 h. The binding affinities of DOTA-BN[2-14]NH(2) and both (nat)Y- and (nat)Lu-labeled analogs to GRP receptors were high (IC(50)=1.78, 1.99, and 1.34 nM, respectively), especially for the (nat)Lu-DOTA-BN[2-14]NH(2) complex. The cytotoxicity study of DOTA-BN[2-14]NH(2) to PC-3 cells revealed an IC(50)=6300 nM after 72 h of exposition, while the labeled derivatives showed no significant cytotoxic effect. The internalization rate to PC-3 cells was more rapid for (177)Lu-labeled peptide (84.87%) than for the (90)Y-labeled one (80.79%), while the efflux rate was slower for (177)Lu-DOTA-BN[2-14]NH(2) (46.8% vs. 61.74%). The biodistribution study of both derivatives in normal mice revealed a specific binding to GRP receptor-positive tissues, which could be blocked by coinjection of cold peptide. The effect of receptor blockage in vivo was also more pronounced for the (177)Lu-labeled peptide than that for the (90)Y-labeled (81% vs. 42%, respectively)., Conclusions: Our studies demonstrated that DOTA-BN[2-14]NH(2) can be labeled with (90)Y (NCA) and (177)Lu (CA) with high radiochemical yields. The in vitro and in vivo comparison between (90)Y-DOTA-BN[2-14]NH(2) and (177)Lu-DOTA-BN[2-14]NH(2) indicated that the change of radiometal in the complex from Y to Lu influence the binding affinity to the GRP receptors with preference to the (177)Lu-labeled derivative.

Purpose: Antitumor activity of the dichloroplatinum(II)-histamine complexes labeled with I-125 or I-131 was investigated in a transplantable murine adenocarcinoma (MA) model., Methods: The tumor model was obtained in C3H/W female mice after subcutaneous inoculation of the tumor cells derived from the mice bearing a mammary tumor of spontaneous origin. Antitumor activities of the platinum-histamine complexes were investigated in three independent experiments, which differed in applied doses of preparations (PtCl(2)Hist, PtCl(2)[(125)I]Hist, PtCl(2)[(131)I]Hist, PtCl(2)Hist/PtCl(2)[(125)I]Hist and PtCl(2)Hist/PtCl(2)[(131)I]Hist), treatment schedules as well as stages of the disease progress in the animals used. Experiment 1 included long-term, multidose treatment with low single doses (treatment duration 31-32 days; 8-10 doses of ca. 0.25.MTD(Pt) each). Experiment 2 included short-term, multidose treatment with higher single doses (4 x ca. 0.5.MTD(Pt) up to Day 13 of the treatment). Experiment 3 included long-term concomitant multidose treatment with higher single doses (9x0.9-0.4.MTD(Pt) up to Day 33)., Results: The long-term treatment with the platinum-histamine preparations revealed inhibiting activity on the tumor growth and size in comparison to control groups. The most intensive and significant antitumor effects were observed for the radioactive complexes. The tumor growth delay factors (GDFs) observed in Experiment 1 were 0.4, 0.7, and 1.2 for PtCl(2)Hist, PtCl(2)Hist/PtCl(2)[(131)I]Hist, and PtCl(2)Hist/PtCl(2)[(125)I]Hist, respectively. Significant (P<.05) prolongations of median survivals (MS) were found in Experiment 2 following the treatment with higher single doses of PtCl(2)Hist and PtCl(2)His/PtCl(2)[(125)I]Hist (Ratio MS(tr)/MS(con) ca. 1.4). A slightly less potent activity was observed for PtCl(2)Hist/PtCl(2)[(131)I]Hist, and no survival improvement was found for the groups treated mostly with the radiation (PtCl(2)[(125)I]Hist and PtCl(2)[(131)I]Hist). The intensive and long-term concomitant scheduling of the radioactive platinum-histamine complexes labeled with I-125 and I-131 (Experiment 3) resulted in a significant inhibition of the tumor growth (GDF=1.9) and survival prolongation of the tumor-bearing mice (MS(tr)/MS(con)=1.5, P=.023). The treatment-related toxicity was mild., Conclusion: An enhancement of the antitumor activity due to the multidose concomitant treatment with a combination of cytotoxic/cytostatic dichloroplatinum(II)-histamine and the attached iodine radionuclides was shown in the murine model of experimental neoplasm.

Background: The aim of the study was to examine in vitro and in vivo binding of radiolabelled analogues of P149 peptide by experimental mammary adenocarcinoma with the intention of potential application for diagnosis and internal radiotherapy of tumours., Material and Methods: The 36-amino acid peptide (P149-QY) of 90% homology to 447-480 peptide fragment of hAFP was synthesised and radiolabelled with iodine-125. The biodistribution of P149-Q[125I]-Y was studied in experimental mammary tumours. For in vitro experiments, extract from mouse mammary tumours were prepared and incubated with radioiodinated P149-QY peptide in the presence of a cross-linking reagent., Results: The gel electrophoresis analysis (SDS-PAGE) showed that radioiodinated P149-QY peptide formed a complex with adenocarcinoma proteins of about 30 kDa. The biodistribution of P149-Q[125I]-Y studied in experimental mammary tumours revealed a higher pharmacokinetic rate in comparison with the whole radioiodinated AFP molecule. A moderate uptake of P149-Q[125I]-Y in the tumour tissue was observed (3.2% ID/g at 30-min p.i.v). However, a faster radioactivity clearance from blood and normal tissues resulted in an increase in the tumour/muscle (T/M) ratio, i.e. from 2.3 to 3.4 after 30 mins and 24 h p.i.v, respectively., Conclusions: The present study shows that radioiodinated P149-QY peptide reveals some positive features as the AFP receptor radioligand, however, some additional structural modifications of the initial peptide molecule are necessary for full retention of the ligand-receptor interaction of its radiolabelled forms.

A novel class of water-soluble Pt(IV) complexes with histamine (Hist) and radioiodinated histamine ([(125)I/(131)I]Hist) has been synthesised with the goal of potential application for concomitant anticancer radio-chemotherapy of solid tumours. The prepared complex of 1:2 metal:ligand stoichiometry ([Pt(IV)(Hist)(2)(OH)(2)]Cl(2)) was characterised by microanalysis, mass spectrometry, and chromatographic methods. Cytotoxic/cytostatic activities of the complex were examined by flow cytometry method using the MCF-7 cells line. A slightly lower cytotoxicity of the Pt(IV) complex comparing to cisplatin was found (IC(50) 59 and 48 microM, respectively). Both cisplatin and the histamine complex show a cytostatic activity by blocking MCF-7 cells in S-phase of cell cycle. Biodistribution studies in normal rats revealed the highest accumulation of the (131)I-labelled complex in liver and kidneys (41.3% and 12.4% ID after 24 h post-intravenous injection (p.i.v.)). The similar pharmacokinetics was observed in tumour-bearing C3H/W mice, however, a lower accumulation in liver was observed following an intraperitoneal comparing to an intravenous administration. A concentration of the complex in tumour increased with time post-intraperitoneal injection (1.2 and 2.5%ID/g after 2 and 24 h (p.i.), respectively). An increasing tumour/muscle ratio was also observed (2.2 and 4.5 after 2 and 24 h p.i., respectively), and that suggests a penetration of the complex into the tumour cells, and a permanent binding with some cellular components, probably with the DNA.

Background: Recently, a new PtCl2-histamine complex, and its radioactive analogues labelled with I-131 and I-125 have been synthesised and investigated both in vitro and in vivo. In this preliminary report the survival rate of radioactive platinum-[125I] histamine therapy in tumour-bearing mice is demonstrated., Material and Methods: A murine model of transplantable colon adenocarcinoma (C38) in C57BL/6 mice (15 days postimplantation) was used for the experiment. Three groups of animals were treated every 2-3 days with five intraperitoneal injections of the following preparations: PtCl2Hist (total dose of Pt--125 micromol/kg), PtCl2[125I]Hist (total dose of I-125--4.2 MBq; Pt--13 micromol/kg), and "Active/Cold"--PtCl2[125I]Hist/PtCl2Hist (I-125--4.2 MBq; Pt--125 micromol/kg). A solution of 15% dimethylformamide in saline was applied to the control group. A survival analysis with the Kaplan-Meier estimation of survival curves and a statistical comparison by a log-rank test was applied to evaluate the anticancer activity of the tested preparations., Results: Treatment of the animals with platinum-histamine preparations resulted in a significant prolongation of survivals, especially if the radioactive complex with carrier-added PtCl2Hist (p < 0.005) was applied. The highest, almost a 60% prolongation of survival was observed in the Active/Cold group (MStr/MScon ratio = 1.58, 95% CI 1.22-1.93). For this group there was the lowest risk of death (hazard ratio HR = 0.29), whereas HR = 0.45 and 0.47 were found in the animals treated with unattended PtCl2Hist and 125I-labelled complex, respectively., Conclusion: The significant enhancement of in vivo anti-cancer activity by a concomitant combination of the therapeutic factors, i.e. cytotoxic/cytostatic activity of the platinum(II)-histamine and the Auger electrons effects generated by the attached I-125 radionuclide, was found on the murine model of transplantable colon adenocarcinoma.

A freeze-dried kit developed for formulation of ethylenediamine-tetramethylenephosphonic acid (EDTMP) chelates with "pain-palliation" radiolanthanides (e.g., 153Sm and 177Lu) or "diagnostic" 99mTc has been evaluated, meeting quality and safety criteria required for medicinal use. The EDTMP kit enables an instant one-step preparation of a radiopharmaceutical of high radiochemical purity (>99%) and has a sufficiently long shelf life. Comparative biodistribution studies of 177Lu-EDTMP and 99mTc-EDTMP prepared from the kit revealed similar tissue uptake and clearance to those obtained for pre-formulated 153Sm-EDTMP. The most significant difference was observed for 99mTc-EDTMP, which shows a high retention in kidney, reaching ca. 2% ID after 90min p.i.v.Although preliminary clinical evaluations suggest that 99mTc-EDTMP possesses limited value for bone scintigraphy, application of the radiopharmaceutical for specific diagnostic purposes may still be considered, e.g., investigation of bone metastases or ossification processes in inflammatory spondyloarthropathy.

Cisplatin chemotherapy in combination with external irradiation or with low-dose continuous internal radiotherapy produces significant supra-additive treatment effects towards several tumor cells. The purpose of our research is to develop a new class of platinum-based anticancer drugs containing moieties of synergistic potency such as platinum core and a radiotherapeutic isotope which, delivered directly to the tumorous cells by a specifically designed vectors, should produce a local enhancement of therapeutic dose. Thus, we have synthesized a new platinum-iodohistamine complex and its radioactive analogues labeled with I-125 and I-131. In the present study some biological properties of those compounds have been investigated. The in vitro screening study pointed out that non-radioactive platinum-iodohistamine complex possesses high cytostatic activity against COLO-205 cells, and moderate activity against HL-60 cell line. No cytotoxicity was observed against MOLT-4 and L-1210 cells, as well as against VERO normal cells. The biodistribution of intravenously administered radioactive platinum-[131I]-iodohistamine complex to normal rats revealed the highest accumulation in the liver (c.a. 40%ID). Intraperitoneal injections of the complex to tumor-bearing C3H mice resulted in scattering of the dose in the organs (mainly in GIT, liver, kidney). The retention of radioactive complex in neoplastic tissue was 3-4 times higher than in normal muscular tissue, although exhibited the tendency to decrease with time post injection. The results of the present study show promising features of the newly developed platinum-iodohistamine complexes and justify prospective investigation of in vivo anticancer potency on animal models of solid tumors.

Background: The platinum-iodohistamine complex with in vitro cytostatic activity toward colon and mammary cancer cells has been synthesised recently in our laboratory. The pharmacokinetics of radioactive complex analogues, labelled with I-131 and I-125, has been examined in murine model of spontaneous mammary adenocarcinoma. The present work is devoted to the examination of the potential use of liposomes as a carrier system for the radioactive platinum-[*I]iodohistamine complex in vivo., Material and Methods: Encapsulations of the Pt- [(125)I]iodohistamine were studied using a different molar ratio of the complex and liposomes with positive surface charge, as well as various incubation procedures. Biodistribution of the initial and the liposomal form of the complex were studied in C3H tumour-bearing mice with spontaneously developed and transplantable (16C) mammary adenocarcinoma., Results: Comparative biodistribution studies in C3H/16C mice and in mice with spontaneously developed mammary tumour have shown that in the former model pharmacokinetics of the Pt-[125I]iodohistamine complex is more predictable and more similar to that observed for cisplatin. Therefore, the transplantable tumour model is more advantageous for the complex and its liposomal form evaluation. In C3H/16C mice, significant differences in the biodistribution between the radioactive platinum complex and its liposomal form were observed. The concentration of the activity in blood after 2 h p.i.v. was two times lower for the encapsulated complex, and the uptake of the radioactivity by liver, spleen, and lungs was twice as high as that obtained for the free Pt-[(125)I]iodohistamine preparation. The radioactivity in tumour was almost constant for liposomal platinum complex (ca. 2% ID/g), although it was two times lower compared to the initial platinum complex., Conclusions: The results of the present study indicate that platinum-[*I]iodohistamine can be efficiently incorporated into cationic liposomes (c. 40%). However, the uptake of the encapsulated complex by the liver and spleen macrophages demands further modification of the lipid membrane.

Background: The unique mechanism of dithizone action in the interior of the viable pancreatic islet suggests the possible development of a specific radiopharmaceutical that may have a potential clinical application in the diagnosis of the pancreatic organ allografts or islets rejection. The radiodiagnostic properties of the newly developed radioactive analogue of dithizone, i.e. Dithizone-[(131)I]-Histamine conjugate have been evaluated in the present study., Methods: The four islet cells transplantation models were chosen for this purpose. The most important feature of the Dithizone-[(131)I]-Histamine conjugate is its possessed ability of zinc chelation. As was presented in the recent study, the conjugate stains pink-reddish the isolated pancreatic islets in vitro. Among the studied transplantation models, only the islets grafting under testis capsule enabled determination of the pancreatic islets in rats by radioactive Dithizone-[(131)I]-Histamine conjugate. The level of the radioactivity in the recipient testis (right) was almost two times higher compared to the controls (0.24 vs. 0.13% ID/g, respectively)., Conclusions: These preliminary data demonstrate the ability of the developed radioactive analogue of dithizone for in vivo identification of transplanted pancreatic islets, and suggests a potential clinical application of the radiodithizone in the diagnosis of the pancreatic islet rejection.

Transplantation of whole pancreas or pancreatic islets remains a promising approach to treatment of diabetes mellitus. Since there is no efficient method presently known for in vivo detection of pancreatic islet rejection, we have utilized dithizone [DTZ] to monitor the survival of transplanted islet allografts following the induction of tolerance by a new strategy of deliberate introduction of donor antigens into the adult thymus. In this study, we examined the morphology of islet allografts in vivo and in vitro following pretreatment with intrathymic (IT) inoculation of 2 mg soluble Ag obtained from 3M KCl extracts of resting T-cells with or without ALS immunosuppression in the WF-to-Lewis combination. Fresh isolated rat islets stained pink 3-5 minutes following exposure to medium containing 0.12 mM DTZ solution in DMSO. Intravenous (i.v.) injection of DTZ solution into unmodified recipients of islet allografts that had rejected their grafts showed massive degranulation of islets which did not stain pink with DTZ. This was confirmed by microscopic finding of fibrosis and lymphocytic infiltration. In contrast, i.v. injection of DTZ solution into long-term recipients of islet allografts at 50, 100, and 150 days after transplantation showed viable islet cells which stained crimson red with DTZ and the findings were confirmed with microscopic sections. This study demonstrates that DTZ is an effective means of in vivo and in vitro identification of transplanted pancreatic islets and suggests that this strategy may have potential clinical application in the diagnosis of the pancreatic islet rejection.