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There has been considerable interest in developing a monoclonal antibody (mAb) against-CD-20 (for example, Rituximab) modified by bifunctional chelating agents (BCA) for non-Hodgkin's lymphoma radioimmunotherapy. Therefore, many researchers have modified this monoclonal antibody by attaching different BCA moieties and evaluated their biological activities in terms of in vitro study and in vivo study in healthy and tumor xenografted rodents. This mini-perspective reviews the in vitro studies, the immunoreactivity and physiological distribution studies: organ-to-blood and the tumor-to-organ ratio of conjugates with different numbers of chelators per mAb. We set up a null hypothesis that states there is no statistical significance between the biological activity of monoclonal antibody (Rituximab) and the number of conjugated bifunctional chelators. Overall, we have concluded that there is no strong evidence for this hypothesis. However, the literature data should be questioned due to the potential lack of uniform study methodology.

Background Targeted radionuclide therapy with [177Lu]Lu-PSMA I&T (zadavotide guraxetan) has proven high efficacy and safety in treating patients with advanced prostate cancer worldwide. Several methods to determine the radiochemical purity have been reported but also limitations in the HPLC analysis due to retention of the sample and tailing effects when using standard gradients containing trifluoroacetic acid (TFA). We here report on the validation of a method for quality control of [177Lu]Lu-PSMA I&T including determination of radiochemical purity, identity testing and limit test for PSMA I&T by HPLC using a Phosphate buffer /Acetonitrile gradient system, complemented with a TLC system with 0.1N Citrate buffer pH 5 as mobile phase including validation of the methods, batch and stability data as well as identification of the main radiochemical impurity by mass spectrometry. Results The described HPLC method met the defined acceptance criteria in terms of accuracy, specificity, robustness, linearity, range and LOQ. HPLC analysis revealed symmetrical peaks and quantitative recovery from the column. Batch data showed a radiochemical purity > 95% as determined by HPLC, stability data a pronounced degradation due to radiolysis, which could be limited by addition of ascorbic acid, dilution and storage at low temperatures. The main radiochemical impurity was found to be the de-iodinated form of [177Lu]Lu-PSMA I&T. TLC analysis allowed to determine the amount of free Lu-177 even in the presence of DTPA in the final formulation. Conclusion Overall the described combination of HPLC and TLC provides a reliable tool for quality control of [177Lu]Lu-PSMA I&T.

Several gastrin-releasing peptide receptor (GRPR) antagonists with improved in vivo behavior have been recently developed and tested in the clinic. However, despite the generally mild side effects of peptide receptor radionuclide therapy (PRRT), toxicity has been observed due to high doses delivered to nontarget tissues, especially in the kidneys and pancreas. Previous experiences with radiolabeled peptides opened a unique opportunity to explore GRPR pretargeting using clickable bombesin antagonists. Toward this goal, we used clickable DOTA-like radiocomplexes which have been previously evaluated by our group. We functionalized a potent GRPR antagonist with a clickable TCO moiety using two different linkers. These precursors were then studied to select the compound with the highest GRPR binding affinity and the best pharmacokinetics to finally explore the advantages of the devised pretargeting approach. Our results provided an important proof of concept toward the development of bioorthogonal approaches to GRPR-expressing cancers, which are worth investigating further to improve the in vivo results. Moreover, the use of clickable GRPR antagonists and DOTA/DOTAGA derivatives allows for fine-tuning of their pharmacokinetics and metabolic stability, leading to a versatile synthesis of new libraries of (radio)conjugates useful for the development of theranostic tools toward GRPR-expressing tumors.

International audience; AbstractIn the frame of “precision medicine”, the scandium radionuclides have recently received considerable interest, providing personalised adjustment of radiation characteristics to optimize the efficiency of medical care or therapeutic benefit for particular groups of patients. Radionuclides of scandium, namely scandium-43 and scandium-44 (43/44Sc) as positron emitters and scandium-47 (47Sc), beta-radiation emitter, seem to fit ideally into the concept of theranostic pair. This paper aims to review the work on scandium isotopes production, coordination chemistry, radiolabeling, preclinical studies and the very first clinical studies. Finally, standardized procedures for scandium-based radiopharmaceuticals have been proposed as a basis to pave the way for elaboration of the Ph.Eur. monographs for perspective scandium radionuclides.

Design, synthesis, and evaluation of inhibitors for severe acute respiratory syndrome 3C-like protease based on phthalhydrazide ketones or heteroaromatic esters Targeting zoonotic viruses: structure-based inhibition of the 3C-like protease from bat coronavirus HKU4 - the likely reservoir host to the human coronavirus that causes Middle East Respiratory Syndrome (MERS) Discovery of N-(benzo[1,2,3]triazol-1-yl)-N-(benzyl)acetamido) phenyl) carboxamides as severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro inhibitors: identification of ML300 and noncovalent nanomolar inhibitors with an induced-fit binding Ligand-induced dimerization of Middle East Respiratory Syndrome (MERS) coronavirus nsp5 protease (3CL pro) [Extracted from the article] Copyright of Archives of Medical Science is the property of Termedia Publishing House and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission However, users may print, download, or email articles for individual use This abstract may be abridged No warranty is given about the accuracy of the copy Users should refer to the original published version of the material for the full abstract (Copyright applies to all Abstracts )

Background: This work aimed to evaluate the influence of two chelators: DOTA(SCN) and DOTA(NHS) on radioimmunotherapy using 177Lu-DOTA-Rituximab preparations in murine lymphoma xenograft models. Su...

Broad availability and cost-effectiveness of 99Mo/99mTc generators worldwide support the use, and thus the development, of novel 99mTc-labelled radiopharmaceuticals. In recent years, preclinical and clinical developments for neuroendocrine neoplasms patient management focused on somatostatin receptor subtype 2 (SST2) antagonists, mainly due to their superiority in SST2-tumour targeting and improved diagnostic sensitivity over agonists. The goal of this work was to provide a reliable method for facile preparation of a 99mTc-labelled SST2 antagonist, [99mTc]Tc-TECANT-1, in a hospital radiopharmacy setting, suitable for a multi-centre clinical trial. To ensure successful and reproducible on-site preparation of the radiopharmaceutical for human use shortly before administration, a freeze-dried three-vial kit was developed. The final composition of the kit was established based on the radiolabelling results obtained during the optimisation process, in which variables such as precursor content, pH and buffer, as well as kit formulations, were tested. Finally, the prepared GMP-grade batches met all predefined specification parameters together with long-term kit stability and stability of the product [99mTc]Tc-TECANT-1. Furthermore, the selected precursor content complies with micro-dosing, based on an extended single-dose toxicity study, where histopathology NOEL was established at 0.5 mg/kg BW, being more than 1000 times higher than the planned human dose of 20 µg. In conclusion, [99mTc]Tc-TECANT-1 is suitable to be advanced into a first-in-human clinical trial. Nasl. z nasl. zaslona. Opis vira z dne 9. 3. 2023. Št. članka: 885. Bibliografija: str. 15-16. Abstract. ERA PerMed, Novel 99mTc-labelled somatostatin receptor antagonists in the diagnostic algorithm of neuroendocrine neoplasms—a feasibility study” (TECANT), TECANT Austrian Science Fund FWF National Centre for Research and Development Jagiellonian University Medical College National Centre for Nuclear Research, MIZS (Ministry of Education, Science and Sport), Razvoj inovativnega, z gama sevalcem tehnecijem-99m označenega antagonista somatostatinskih receptorjev za diagnostiko nevroendokrinih novotvorb - raziskava izvedljivosti, TECANT

Background: 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. [99mTc]Tc-PSMA-T4 is a new radiopharmaceutical designed for the diagnosis of patients with PCa. Methods: 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 [99mTc]Tc-PSMA-T4 was utilized, acquisition images were obtained at three time points. Results: 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%. Conclusion: An oncotropic SPECT [99mTc]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.

Despite significant advances in nuclear medicine for diagnosing and treating prostate cancer (PCa), research into new ligands with increasingly better biological properties is still ongoing. Prostate-specific membrane antigen (PSMA) ligands show great potential as radioisotope carriers for the diagnosis and therapy of patients with metastatic PCa. PSMA is expressed in most types of prostate cancer, and its expression is increased in poorly differentiated, metastatic, and hormone-refractory cancers; therefore, it may be a valuable target for the development of radiopharmaceuticals and radioligands, such as urea PSMA inhibitors, for the precise diagnosis, staging, and treatment of prostate cancer. Four developed PSMA-HYNIC inhibitors for technetium-99m labeling and subsequent diagnosis were subjected to preclinical in vitro and in vivo studies to evaluate and compare their diagnostic properties. Among the studied compounds, the PSMA-T4 (Glu-CO-Lys-L-Trp-4-Amc-HYNIC) inhibitor showed the best biological properties for the diagnosis of PCa metastases. [99mTc]Tc-PSMA-T4 also showed effectiveness in single-photon emission computed tomography (SPECT) studies in humans, and soon, its usefulness will be extensively evaluated in phase 2/3 clinical trials.

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 [111In]In-radiocomplexes, as assessed upon injection in healthy mice. The derivative DOTA-Tz was chosen to pursue the studies upon radiolabelling with 90Y, yielding a radiocomplex with high specific activity, high radiochemical yields and suitable in vitro stability. The [90Y]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 [90Y]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.

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 223Ra-labeled and PSMA-targeted NaA nanozeolites [223RaA-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 133Ba- and 223Ra-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 223Ra-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 223Ra-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.

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 177Lu, 90Y, 47Sc, and 225Ac 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 &gt, 97%. The radio complexes were obtained with high yields (&gt, 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.

Background: The choice of mice strain can significantly influence the physiological distribution and may lead to an inadequate assessment of the radiopharmaceutical properties. Objective: This work aims to present how the legal requirements that apply to radiopharmaceuticals contained in the various guidelines determine the choice of the mouse strain for quality control and preclinical studies and affect the results of physiological distribution. Methods: Swiss and BALB/c mice were chosen as commonly used strains in experiments for research and quality control purposes. Radiopharmaceuticals, i.e., preparations containing one or more radioactive isotopes in their composition, are subject to the same legal regulations at every stage of the research, development and routine quality control as all other medicines. Therefore, in vivo experiments are to be carried out to confirm the pharmacological properties and safety. Moreover, if a radiopharmaceutical's chemical structure is unknown or complex and impossible to be determined by physicochemical methods, an analysis of physiological distribution in a rodent animal model needs to be performed. Results: In our studies, thirty-six mice (Swiss n=18, BALB/c n=18) were randomly divided into six groups and injected with the following radiopharmaceuticals: [99mTc]Tc-Colloid, [99mTc]Tc-DTPA and [99mTc]Tc-EHIDA. Measurement of physiological distribution was conducted following the requirements of European Pharmacopoeia (Ph. Eur.) monograph 0689, internal instructions and the United States Pharmacopeia (USP) monograph. Additionally, at preclinical studies, ten mice (Swiss n=5, BALB/c n=5) were injected with the new tracer [99mTc]Tc-PSMA-T4, and its physiological distribution has been compared. The p-value Conclusion: We claim that mice strain choice can significantly influence the physiological distribution and may lead to inaccurate quality control results and incomprehensible interpretation of the results from preclinical in vivo studies of a new radiopharmaceutical.

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, eHPLC-DAD = 48 × 103M−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.

Background The cholecystokinin receptor subtype 2 (CCK-2R) is an important target for diagnostic imaging and targeted radionuclide therapy (TRNT) due to its overexpression in certain cancers (e.g., medullary thyroid carcinoma (MTC)), thus matching with a theranostic principle. Several peptide conjugates suitable for the TRNT of MTC have been synthesized, including a very promising minigastrin analogue DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (CP04). In this contribution, we wanted to see whether CP04 binding affinity for CCK-2R is sensitive to the type of the complexed radiometal, as well as to get insights into the structure of CP04-CCK2R complex by molecular modeling. Results In vitro studies demonstrated that there is no significant difference in CCK-2R binding affinity and specific cellular uptake between the CP04 conjugates complexed with [68Ga]Ga3+ or [177Lu]Lu3+. In order to investigate the background of this observation, we proposed a binding model of CP04 with CCK-2R based on homology modeling and molecular docking. In this model, the C-terminal part of the molecule enters the cavity formed between the receptor helices, while the N-terminus (including DOTA and the metal) is located at the binding site outlet, exposed in large extent to the solvent. The radiometals do not influence the conformation of the molecule except for the direct neighborhood of the chelating moiety. Conclusions The model seems to be in agreement with much of structure-activity relationship (SAR) studies reported for cholecystokinin and for CCK-2R-targeting radiopharmaceuticals. It also explains relative insensitivity of CCK-2R affinity for the change of the metal. The proposed model partially fits the reported site-directed mutagenesis data.

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.

Endohedral structures with La

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.

Endohedral structures with La0 or La3+ encapsulated in chiral (1,16)C58N2 or achiral (1,4)C58N2 diazafullerenes were studied at the B3LYP/G-31G*/SDD level. Two stable locations of La0 and La3+ are possible in each cage but only with La0@(1,16)C58N2 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 La0@(1,16) C58N2 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.

This work presents a comparative biological evaluation of

Medullary thyroid carcinoma (MTC) is one of the most challenging cancers. Epidemiological studies have shown that during the past 30 years neither a change in stage at diagnosis nor a significant improvement in survival has been achieved. Therefore, new diagnostic and therapeutic strategies are needed for early detection of metastases or disease recurrence and tumor growth control.

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 111In 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 111In-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 111In. 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 (pH 4.5) 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 Met11-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, 25 mg ascorbic acid, 0.5 mg gentisic acid and 5 mg l -methionine. The radiolabelling performed by incubation of 200–250 MBq 111InCl3 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 > 4 h at + 25 °C. Conclusion A kit formulation to prepare 111In-CP04 for clinical application was developed, showing high stability of the kit as well as high RCP of the final product.

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.

Rhenium-188 is a radionuclide of high therapeutic potential for nuclear medicine. Because of its short half-life (t??= 16.98h), logistic problem appears with its delivery to customers. This can be solved by delivering a 188W/188Re generator as a source of 188Re. A number of 188W/188Re generator systems have been described. Due to chemical similarity of this pair to 99Mo/99mTc pair, the alumina – based generators are most common. The technology for producing sterile, isotonic solution of sodium perrhenate-188Re, useful for medical applications, has been developed and implemented by the elution of an alumina based 188W/188Re generator, at the Radioisotope Centre POLATOM. The eluted 188Re was used for the labeling of a Bombesin analog (BN1.1) which has been prior labeled, in high yields, with 99m Tc and gave promising results of its in vitro and in vivo evaluation The Bombesin analog under study BN1.1 has the aminoacid sequence GGCAca- QRLGNQWAVGHLM-CONH2. The peptide was labeled with 188Re, through a Re-gluconate complex via an exchange reaction. We studied the conditions for optimal labeling yield and specific activity of the resulted labeled peptide. 188Re-BN1.1 with specific activity 20.4-34.0 GBq 188Re/μmol BN1.1, radiochemical yield > 95, and pH < 1.0 was obtained. 188Re-BN1.1 was stable when the pH was adjusted to 5.0-6.0. Preliminary biodistribution study in normal mice showed specific uptake in GRP positive tissues, fast blood clearance, but also a significant uptake in the stomach indicating dissociation of the metal from the ligand core and its reoxidation.

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] RD 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.

BACKGROUND: The minigastrin analogue — CP04: DOTA-(DGlu) 6 -Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH 2 has been developed for CCK2R targeting. This analogue can be radiolabelled with 111 In or 68 Ga for imaging, or with 90 Y and 177 Lu 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 radiolabelling of CP04 with 90 Y, 177 Lu and 68 Ga 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 90 Y, 177 Lu or 68 Ga 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.

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 90 min 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.

New complexes of platinum(II) with histamine (Hist) and iodohistamine (IHist) have been synthesised and characterised by elemental analysis, mass spectrometry and chromatographic techniques. The structure of PtCl2Hist complex has been determined by single-crystal X-ray diffraction technique. The intermolecular NH⋯Pt and CH⋯Pt hydrogen bonds were observed. Radioactive analogues of PtCl2Hist or PtCl2IHist labelled with isotopes of iodine, i.e. 125I or 131I were prepared with the intention of potential application for concomitant anticancer radio-chemotherapy of solid tumours.

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.

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 purity99% 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.

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The structure of mono-iodohistamine cation was determined by the 1 H, 13 C NMR, supported by ab initio calculations. Theoretical NMR spectra of the two mono-iodohistamine tautomers were calculated by using the CHF-GIAO approach. The N3–H tautomer of 4-I-histamine cationic form has been predicted to be the most stable, and its potential energy surface has been scanned at the HF/3-21G ∗∗ level. Reasons for higher stability of the 4-I-histamine comparing to the other iodohistamine isomers are also discussed.

Stabilization energies ΔE (kcal/mol) of various substitution isomers of the two tautomeric iodo-histamine forms have been calculated at the RHF/3-21G∗∗, MP2/3-21G∗∗ and HF/Sadlej-PTZ levels. The most stable forms are produced by iodo-substitution of histamine in the side chain, ca. 95% of the 5-(2′-amino-2′-iodoethyl)-3H-imidazole and ca. 5% of the 5-(2′-amino-2′-iodoethyl)-1H-imidazole isomer. If iodo-substituted is the imidazole ring only, then the 4-iodo-histamine and 2-iodo-histamine N(1)-H tautomers are predicted to coexist is a mole ratio of ca. 95:5. The only stable form of the deprotonated histamine anion is that with both the imidazole nitrogen atoms deprotonated.

Synthesis, characteristics and radioiodination of the new carboxylic derivatives of dithizone are described in this paper. We have applied the carboxy dithizones for preparation of radioactive compounds by coupling with [131I]-histamine. Preliminary biological studies of the new radiodithizone were done in rats after two different application routs: peripheral i.v. injection and direct injection to splenic artery.Biodistribution of the carboxy dithizone-[131I]-histamine conjugate (i.v. injection) was quite different than that for free [131I]-histamine. However, uptake of activity in pancreas was low (0.81% g−1 of tissue). Direct application of the conjugate to splenic artery resulted in high activity retention in pancreas after 30 and 45 min post injection (respectively 8.8 and 12.4% g−1 of tissue) indicating potential usefulness of the new radiodithizone for in vivo monitoring of pancreas.

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.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.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.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 Tc-99m 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 Tc-99m-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 Tc-99m-labelling was optimized using various amounts of HYNIC rhIL-2, Tc-99m, SnCl2, 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 Tc-99m-HYNIC rhIL-2 are formed during radiolabelling. At optimized conditions of wet radiolabel ling, the Tc-99m-HYNIC rhIL-2 monomer was obtained with radiochemical purity >99%, specific activity of ca. 4 GBq/mg rhIL-2 and overall yield lea. 65%. The two-vial freeze-dried kit was prepared: the first vial contained 30 mu g HYNIC rhIL-2, coligands, buffer and antioxidant; the second vial contained tricine and SnCl2. The monomer of Tc-99m-HYNIC rhIL-2 was obtained by gel chromatography on a PD-ID 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 Tc-99m 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 Tc-99m-HYNIC(tricine,NA)-rhIL-2 within 1 h. (C) 2010 Elsevier Inc. All rights reserved.