Preliminary radiochemical and biological studies on the liposome encapsulated platinum-[125I]iodohistamine complex

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- [125I]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-[125I]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

Radiopharmaceuticals in cardiology

Myocardial perfusion studies are among the most often performedinvestigations in Nuclear Medicine. However, the developmentof radiopharmaceuticals for cardiology is an emergingdiscipline and several other radiotracers have been proven to beuseful. Although the myocardial perfusion studies have a wellestablishedrole in the management of cardiac disorders, stilla number of radiopharmaceuticals are under development fora variety of specific cardiac indications and their eventual clinicalrole remains to be seen. The paper provides a short overviewof currently used radiopharmaceuticals and potential molecularimaging radiotracers applicable in cardiology

The radiometal makes a difference. Synthesis and preliminary characterisation of DOTA-minigastrin analogue complexes with Ga, Lu and Y

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

Identification of transplanted pancreatic islet cells by radioactive Dithizone-[131I]-Histamine conjugate. Preliminary report

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-[131I]-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-[131I]-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-[131I]-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

Oxidation of methionine — is it limiting the diagnostic properties of 99mTc-labeled Exendin-4, a Glucagon-Like Peptide-1 receptor agonist?

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-Ex­endin-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 bio­distribution, 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

Uptake of radiolabelled modified fragment of human alfa-fetoptrotein by experimental mammary adenocarcinoma: in vitro and in vivo studies

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

Comparison of chromatographic methods for quality control of DMSA complexes with 99mTc and 188Re at (III) and (V) oxidation states

BACKGROUND: The reliable method for determination ofidentity and radiochemical purity (RCP) is of great importancein radiopharmaceutical development. This is especially relevantwhen more than one form of radiometal/ligand complex can beformed during radiolabelling, such as complexes of 99mTc or 188Rewith meso-2,3-dimercaptosuccinic acid (DMSA), where dependingon the pH, metal can occur either at +3 or +5 oxidation state.The aim of our study was to evaluate possibilities for optimizationof chromatographic systems leading to specific and reliableanalytical method for determination of the identity and RCP ofDMSA 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 complexeswere prepared by addition of NaHCO3 to the kit vial prior to99mTc-eluate to obtain pH ~8. 188Re(V)-DMSA was prepared eitherdirectly or using intermediate 188Re(III)-EDTA complex addedto DMSA. RCP was evaluated by TLC using: ITLC-SG developedin methylethylketon, SG60 coated plates developed in:n-BuOH/H2O/CH3COOH and n-PrOH/H2O/CH3COOH systems,and in H2O. Comparative biodistribution studies were performedin normal Wistar rats.RESULTS: Using silica gel plates and n-PrOH, H2O and aceticacid in the developing solution, we observed that 99mTc/188Re(III)-DMSA and 99mTc/188Re(V)-DMSA complexes could be wellseparated from each other and from the impurities in the formof free pertechnetate/perrhenate. In vivo studies showed quitedifferent biodistribution of 99mTc(III)- and 99mTc(V)-DMSA. Thetrivalent complex accumulated mainly in kidneys (>40%ID),while 99mTc(V)-DMSA revealed high excretion with urine andrelatively 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 prepareddirectly was almost identical to that of 99mTc(V)-DMSA. Biodistributionresults of the 188Re preparation obtained using 188Re(III)-EDTA intermediate indicated that the preparation contained themixture of penta- and trivalent 188Re complexes. The quite highaccumulation of radioactivity in kidneys (23 %ID) gave evidenceof the presence of 188Re(III)-DMSA in this preparation, what wasalso confirmed by the results of TLC analysis performed usingsilica gel plate and n-propanol/water/acetic acid as developingsystem. CONCLUSIONS: Based on our study, we have made recommendationon the suitable methods for investigations of RCP ofDMSA complexes, i.e.: SG60 plates developed in the mixtureof n-propanol/water/acetic acid, which enable determination of the tri- and pentavalent DMSA complexes, as well as, thepertechnetate/perrhenate impurity, and developed in water fordetermination of the colloidal residue.BACKGROUND: The reliable method for determination ofidentity and radiochemical purity (RCP) is of great importancein radiopharmaceutical development. This is especially relevantwhen more than one form of radiometal/ligand complex can beformed during radiolabelling, such as complexes of 99mTc or 188Rewith meso-2,3-dimercaptosuccinic acid (DMSA), where dependingon the pH, metal can occur either at +3 or +5 oxidation state.The aim of our study was to evaluate possibilities for optimizationof chromatographic systems leading to specific and reliableanalytical method for determination of the identity and RCP ofDMSA 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 complexeswere prepared by addition of NaHCO3 to the kit vial prior to99mTc-eluate to obtain pH ~8. 188Re(V)-DMSA was prepared eitherdirectly or using intermediate 188Re(III)-EDTA complex addedto DMSA. RCP was evaluated by TLC using: ITLC-SG developedin methylethylketon, SG60 coated plates developed in:n-BuOH/H2O/CH3COOH and n-PrOH/H2O/CH3COOH systems,and in H2O. Comparative biodistribution studies were performedin normal Wistar rats.RESULTS: Using silica gel plates and n-PrOH, H2O and aceticacid in the developing solution, we observed that 99mTc/188Re(III)-DMSA and 99mTc/188Re(V)-DMSA complexes could be wellseparated from each other and from the impurities in the formof free pertechnetate/perrhenate. In vivo studies showed quitedifferent biodistribution of 99mTc(III)- and 99mTc(V)-DMSA. Thetrivalent complex accumulated mainly in kidneys (>40%ID),while 99mTc(V)-DMSA revealed high excretion with urine andrelatively 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 prepareddirectly was almost identical to that of 99mTc(V)-DMSA. Biodistributionresults of the 188Re preparation obtained using 188Re(III)-EDTA intermediate indicated that the preparation contained themixture of penta- and trivalent 188Re complexes. The quite highaccumulation of radioactivity in kidneys (23 %ID) gave evidenceof the presence of 188Re(III)-DMSA in this preparation, what wasalso confirmed by the results of TLC analysis performed usingsilica gel plate and n-propanol/water/acetic acid as developingsystem.CONCLUSIONS: Based on our study, we have made recommendationon the suitable methods for investigations of RCP ofDMSA complexes, i.e.: SG60 plates developed in the mixtureof n-propanol/water/acetic acid, which enable determination of the tri- and pentavalent DMSA complexes, as well as, thepertechnetate/perrhenate impurity, and developed in water fordetermination of the colloidal residue

Radiopharmaceutical Precursors for Theranostics

Due to the complex nomenclature used in various regulations and guidance documents, the understanding of radiopharmaceutical precursor’s definition might be challenging. Depending on the context it could be interpreted as the substance which becomes a radiopharmaceutical after radiolabeling with a radionuclide of choice or a radionuclide which is used for radiolabeling of that substance. In this Chapter we present and discuss the requirements for precursors which are used in the preparation of theranostic radiopharmaceuticals, in particular for preparation of new radiopharmaceuticals for clinical trials within the EU. In discussion on the available methods for assessing the quality of radiopharmaceutical precursors and on the specified limits the reference to Ph. Eur. is made. Since the EANM guidelines for in-house preparation of radiopharmaceuticals also specify the need for testing the quality of radiopharmaceutical precursors, information provided herein might help the radiopharmacist working on the development of new theranostic agents to adequately define identity, strength, quality, purity and stability of the final radiopharmaceutical preparation

New synthesis route of active substance d,l-HMPAO for preparation Technetium Tc99m Exametazime

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

Improved procedures of Sc(OH)3 precipitation and UTEVA extraction for 44Sc separation

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