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

Recommendations for In Vitro and In Vivo Testing of Magnetic Nanoparticle Hyperthermia Combined with Radiation Therapy

Magnetic nanoparticle (MNP)-mediated hyperthermia (MH) coupled with radiation therapy (RT) is a novel approach that has the potential to overcome various practical difficulties encountered in cancer treatment. In this work, we present recommendations for the in vitro and in vivo testing and application of the two treatment techniques. These recommendations were developed by the members of Working Group 3 of COST Action TD 1402: Multifunctional Nanoparticles for Magnetic Hyperthermia and Indirect Radiation Therapy (“Radiomag”). The purpose of the recommendations is not to provide definitive answers and directions but, rather, to outline those tests and considerations that a researcher must address in order to perform in vitro and in vivo studies. The recommendations are divided into 5 parts: (a) in vitro evaluation of MNPs; (b) in vitro evaluation of MNP-cell interactions; (c) in vivo evaluation of the MNPs; (d) MH combined with RT; and (e) pharmacokinetic studies of MNPs. Synthesis and characterization of the MNPs, as well as RT protocols, are beyond the scope of this wor

Development of ready to use kit formulation for trastuzumab radioimmunoconjugates and identification of radiochemical purity as the first step in quality control of the final product

Aim The aim of this study is to present the part of our project dedicated to obtaining a stable, ready to use freeze dried kit formulation of antibody radioimmunoconjugates (trastuzumab immunoconjugates labelled with 90Y and 177Lu). As the first step in on-going in vitro stability of the final product and radiochemical purity determination, we used ITLC-SG method with different mobile phases. Methods Radioactive labelling of trastuzumab was performed with 90Y and 177Lu via DOTA, DTPA and 1B4M-DTPA in molar ratio 1:20. The specific activity of 1.425 mCi (90Y) and 8.150 mCi (177Lu) was achieved, using a solutions of 0.04 M HCl. Radiolabeling is performed by adding 8.5 µL of 90Y at pH 4.5-5 and 5 µL 177Lu at pH 6. Solutions with Tr-DTPA and Tr-1B4M-DTPA were incubated at room temperature for 30 min, while Tr-DOTA was incubated at 40 °C for 1 hour. Radiochemical purity of radioisotopes was tested with ITLC-SG using three mobile phases: 0.9% NaCl, 0.4 M methanol/sodium-acetate (1:1) and 0.1 M acetic buffer. The stability of radioimmunoconjugates was tested in 0.9% NaCl (177Lu) and 0.4 M methanol/sodium-acetate (1:1) (90Y), after incubation at room temperature for 1, 24, 48 and 72h. Results After choosing the most suitable mobile phase for determination of radiochemical purity by ITLC-SG of conjugates labeled with 90Y (99.87%) we used 0.4 M methanol/sodium acetate (1:1), and those with 177Lu (100%) with 0.9%. NaCl. Examination of radiochemical yield of radioimmunoconjugates showed the presence of radioactivity only at the start of the strip, due to the high Mw of Tr. The absence of radiolabeled fragments of the antibody, as well as radiolabeled chelators and free radioisotopes, proved that the stable radioimmunoconjugates were formulated. The highest yield of labeling with 90Y (>96%) is achieved in 0.4 M methanol/sodium-acetate (1:1), while with 177Lu (>99%) in 0.9% NaCl. Test stability after 24h showed the highest stability of 90Y-DOTA-Tr (>92.40%) and 177Lu-DOTA-Tr (>99.14%), with minimum released 90Y3+ (84.90%) and 177Lu-DOTA-Tr (>98.52%), with minimum released 90Y3+ (<15.10%) and 177Lu3+ (<1.48%). Conclusions After obtaining the final ready to use kit formulation, the results of the determination of radiochemical purity using ITLC-SG show a high radiolabeling efficiency (>95%), using both isotopes. However, radioactive yield with 177Lu (99%) was higher compared with 90Y (>96%). This method was used to monitor the stability of radiolabeled conjugates and after 72 hours of incubation, a small amount of free radioisotopes was released from radioimmunoconjugates (<5% of 177Lu and <25% of 90Y). The next planed step includes in vivo examinations in healthy mice and in a mouse model of HER2 positive breast tumor after the i.v. injection of radiolabeled trastuzumab radioimmunoconjugates in order to monitor and determine their pharmacokinetics and biodistribution in the whole body and critical organs/tumor

Preparation and examination of 177Lu- and 90Y-labeled immunoconjugates of Trastuzumab

Trastuzumab is a monoclonal antibody for treatment of HER2 positive breast cancer. Labelled antibody with lutetium-177 and yttrium-90 has been investigated as potential radiopharmaceutical agent for use in radioimmunotherapy. In this study, the labeling was done via DOTA, DTPA and 1B4M-DTPA as a chelator in molar ratios of 1:20. A several chemical techniques have been used to characterize the stability and retained immunoreactivity of the antibody in the formulated immunoconjugates. A protein integrity and purity were proved by applying of reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Vibrational spectroscopy (Infra-red (IR) and Raman spectroscopy) provide molecular structure information and are convenient for verification of changes in the secondary structure. The number of chelating groups per one trastuzumab molecule was calculated by MALDI-TOF-MS. After conjugation, the freeze-drying process was performed in order to obtain stable immunoconjugates for further labeling. Quality control and stability were examined with ITLC using a three different mobile phases (0.9% saline solution, 0.4 M methanol:sodium acetate (1:1) and 0.1M acetic buffer). The same intensity of the fragments (25 kDa for light chain and 50 kDa for heavy chain) of lyophilized immunoconjugates and pure trastuzumab was indicated that there is no degradation of the antibody. The presence of characteristic amide bands in IR spectra (amide I (1700-1600 cm-1), amide II (1480-1575 cm-1) and amide III bands (1255-1244 cm-1) and Raman spectra (amide I band at ~1670 cm-1 and amide III band (1230-1300 cm-1) also have indicated that all samples have retained native secondary structure. An average of 3.92 p-SCN-Bn-DTPA, 3.69 p-SCN-Bn-DOTA and 4.43 1B4M-DTPA groups could be randomly conjugated to an antibody molecule, which is a good result for successful labeling. The immunoconjugates were labeled with 177Lu and 90Y with specific activity of 200 µCi/mL. The ITLC studies have shown stability of the radioimmunoconjugates in 0.9% NaCl after 72 h. The 0.4 M methanol:sodium acetate (1:1) was the most appropriate mobile phase for examination of 90Y labeled conjugates and the 0.9% NaCl for 177 Lu labeled. The stability studies after 72 h have shown that 177Lu-labeled trastuzumab is more stable (< 10% of the released 177Lu) than 90Y-labeled one (< 25% of released 90Y). Our study shows successful formulation of stable radioimmunoconjugates which makes this agent as potential use for in vivo investigations

Recommendations for In Vitro and In Vivo Testing of Magnetic Nanoparticle Hyperthermia Combined with Radiation Therapy.

Magnetic nanoparticle (MNP)-mediated hyperthermia (MH) coupled with radiation therapy (RT) is a novel approach that has the potential to overcome various practical difficulties encountered in cancer treatment. In this work, we present recommendations for the in vitro and in vivo testing and application of the two treatment techniques. These recommendations were developed by the members of Working Group 3 of COST Action TD 1402: Multifunctional Nanoparticles for Magnetic Hyperthermia and Indirect Radiation Therapy (?Radiomag?). The purpose of the recommendations is not to provide definitive answers and directions but, rather, to outline those tests and considerations that a researcher must address in order to perform in vitro and in vivo studies. The recommendations are divided into 5 parts: (a) in vitro evaluation of MNPs; (b) in vitro evaluation of MNP-cell interactions; (c) in vivo evaluation of the MNPs; (d) MH combined with RT; and (e) pharmacokinetic studies of MNPs. Synthesis and characterization of the MNPs, as well as RT protocols, are beyond the scope of this work