Synthesis and antimicrobial evaluation of two peptide LyeTx I derivatives modified with the chelating agent HYNIC for radiolabeling with technetium-99m

Background Current diagnostic methods and imaging techniques are not able to differentiate septic and aseptic inflammation. Thus, reliable methods are sought to provide this distinction and scintigraphic imaging is an interesting option, since it is based on physiological changes. In this context, radiolabeled antimicrobial peptides have been investigated as they accumulate in infectious sites instead of aseptic inflammation. The peptide LyeTx I, from the venom of Lycosa erythrognatha, has potent antimicrobial activity. Therefore, this study aimed to synthesize LyeTx I derivatives with the chelating compound HYNIC, to evaluate their antimicrobial activity and to radiolabel them with 99mTc. Methods Two LyeTx I derivatives, HYNIC-LyeTx I (N-terminal modification) and LyeTx I-K-HYNIC (C-terminal modification), were synthesized by Fmoc strategy and purified by RP-HPLC. The purified products were assessed by RP-HPLC and MALDI-ToF-MS analysis. Microbiological assays were performed against S. aureus (ATCC® 6538) and E. coli (ATCC® 10536) in liquid medium to calculate the MIC. The radiolabeling procedure of LyeTx I-K-HYNIC with 99mTc was performed in the presence of co-ligands (tricine and EDDA) and reducing agent (SnCl2. 2H2O), and standardized taking into account the amount of peptide, reducing agent, pH and heating. Radiochemical purity analysis was performed by thin-layer chromatography on silica gel strips and the radiolabeled compound was assessed by RP-HPLC and radioactivity measurement of the collected fractions. Data were analyzed by ANOVA, followed by Tukey test (p-values < 0.05). Results Both LyeTx I derivatives were suitably synthesized and purified, as shown by RP-HPLC and MALDI-ToF-MS analysis. The microbiological test showed that HYNIC-LyeTx I (N-terminal modification) did not inhibit bacterial growth, whereas LyeTx I-K-HYNIC (C-terminal modification) showed a MIC of 5.05 μmol.L−1 (S. aureus) and 10.10 μmol.L−1 (E. coli). Thus, only the latter was radiolabeled with 99mTc. The radiochemical purity analysis of LyeTx I-K-HYNIC-99mTc showed that the optimal radiolabeling conditions (10 μg of LyeTx I-K-HYNIC; 250 μg of SnCl2. 2H2O; pH = 7; heating for 15 min) yielded a radiochemical purity of 87 ± 1 % (n= 3). However, RP-HPLC data suggested 99mTc transchelation from LyeTx I-K-HYNIC to the co-ligands (tricine and EDDA). Conclusions The binding of HYNIC to the N-terminal portion of LyeTx I seems to affect its activity against bacteria. Nevertheless, the radiolabeling of the C-terminal derivative, LyeTx I-K-HYNIC, must be better investigated to optimize the radiolabeled compound, in order to use it as a specific imaging agent to distinguish septic and aseptic inflammation.(AU)

Synthesis and antimicrobial evaluation of two peptide LyeTx I derivatives modified with the chelating agent HYNIC for radiolabeling with technetium-99m

Current diagnostic methods and imaging techniques are not able to differentiate septic and aseptic inflammation. Thus, reliable methods are sought to provide this distinction and scintigraphic imaging is an interesting option, since it is based on physiological changes. In this context, radiolabeled antimicrobial peptides have been investigated as they accumulate in infectious sites instead of aseptic inflammation. The peptide LyeTx I, from the venom of Lycosa erythrognatha, has potent antimicrobial activity. Therefore, this study aimed to synthesize LyeTx I derivatives with the chelating compound HYNIC, to evaluate their antimicrobial activity and to radiolabel them with 99mTc. Methods Two LyeTx I derivatives, HYNIC-LyeTx I (N-terminal modification) and LyeTx I-K-HYNIC (C-terminal modification), were synthesized by Fmoc strategy and purified by RP-HPLC. The purified products were assessed by RP-HPLC and MALDI-ToF-MS analysis. Microbiological assays were performed against S. aureus (ATCC® 6538) and E. coli (ATCC® 10536) in liquid medium to calculate the MIC. The radiolabeling procedure of LyeTx I-K-HYNIC with 99mTc was performed in the presence of co-ligands (tricine and EDDA) and reducing agent (SnCl2. 2H2O), and standardized taking into account the amount of peptide, reducing agent, pH and heating. Radiochemical purity analysis was performed by thin-layer chromatography on silica gel strips and the radiolabeled compound was assessed by RP-HPLC and radioactivity measurement of the collected fractions. Data were analyzed by ANOVA, followed by Tukey test (p-values 0.05). Results Both LyeTx I derivatives were suitably synthesized and purified, as shown by RP-HPLC and MALDI-ToF-MS analysis. The microbiological test showed that HYNIC-LyeTx I (N-terminal modification) did not inhibit bacterial growth, whereas LyeTx I-K-HYNIC (C-terminal modification) showed a MIC of 5.05 mol.L1 (S. aureus) and 10.10 mol.L1 (E. coli). Thus, only the latter was radiolabeled with 99mTc. The radiochemical purity analysis of LyeTx I-K-HYNIC-99mTc showed that the optimal radiolabeling conditions (10 g of LyeTx I-K-HYNIC; 250 g of SnCl2. 2H2O; pH = 7; heating for 15 min) yielded a radiochemical purity of 87 ± 1 % (n= 3). However, RP-HPLC data suggested 99mTc transchelation from LyeTx I-K-HYNIC to the co-ligands (tricine and EDDA). Conclusions The binding of HYNIC to the N-terminal portion of LyeTx I seems to affect its activity against bacteria. Nevertheless, the radiolabeling of the C-terminal derivative, LyeTx I-K-HYNIC, must be better investigated to optimize the radiolabeled compound, in order to use it as a specific imaging agent to distinguish septic and aseptic inflammation.(AU)

Synthesis and antimicrobial evaluation of two peptide LyeTx I derivatives modified with the chelating agent HYNIC for radiolabeling with technetium-99m

Current diagnostic methods and imaging techniques are not able to differentiate septic and aseptic inflammation. Thus, reliable methods are sought to provide this distinction and scintigraphic imaging is an interesting option, since it is based on physiological changes. In this context, radiolabeled antimicrobial peptides have been investigated as they accumulate in infectious sites instead of aseptic inflammation. The peptide LyeTx I, from the venom of Lycosa erythrognatha, has potent antimicrobial activity. Therefore, this study aimed to synthesize LyeTx I derivatives with the chelating compound HYNIC, to evaluate their antimicrobial activity and to radiolabel them with 99mTc. Methods Two LyeTx I derivatives, HYNIC-LyeTx I (N-terminal modification) and LyeTx I-K-HYNIC (C-terminal modification), were synthesized by Fmoc strategy and purified by RP-HPLC. The purified products were assessed by RP-HPLC and MALDI-ToF-MS analysis. Microbiological assays were performed against S. aureus (ATCC® 6538) and E. coli (ATCC® 10536) in liquid medium to calculate the MIC. The radiolabeling procedure of LyeTx I-K-HYNIC with 99mTc was performed in the presence of co-ligands (tricine and EDDA) and reducing agent (SnCl2. 2H2O), and standardized taking into account the amount of peptide, reducing agent, pH and heating. Radiochemical purity analysis was performed by thin-layer chromatography on silica gel strips and the radiolabeled compound was assessed by RP-HPLC and radioactivity measurement of the collected fractions. Data were analyzed by ANOVA, followed by Tukey test (p-values 0.05). Results Both LyeTx I derivatives were suitably synthesized and purified, as shown by RP-HPLC and MALDI-ToF-MS analysis. The microbiological test showed that HYNIC-LyeTx I (N-terminal modification) did not inhibit bacterial growth, whereas LyeTx I-K-HYNIC (C-terminal modification) showed a MIC of 5.05 mol.L1 (S. aureus) and 10.10 mol.L1 (E. coli). Thus, only the latter was radiolabeled with 99mTc. The radiochemical purity analysis of LyeTx I-K-HYNIC-99mTc showed that the optimal radiolabeling conditions (10 g of LyeTx I-K-HYNIC; 250 g of SnCl2. 2H2O; pH = 7; heating for 15 min) yielded a radiochemical purity of 87 ± 1 % (n= 3). However, RP-HPLC data suggested 99mTc transchelation from LyeTx I-K-HYNIC to the co-ligands (tricine and EDDA). Conclusions The binding of HYNIC to the N-terminal portion of LyeTx I seems to affect its activity against bacteria. Nevertheless, the radiolabeling of the C-terminal derivative, LyeTx I-K-HYNIC, must be better investigated to optimize the radiolabeled compound, in order to use it as a specific imaging agent to distinguish septic and aseptic inflammation.

Radiolabeling of methanol extracts of yarrow (Achillea millefolium l) in rats / Radiomarcação do extrato metanólico de yarrow (Achillea millefolium l) em ratos

PURPOSE: Current study is focused on extraction with methanol, purification, labeling with 131I using iodogen method of the yarrow plant and investigating in vivo biological activity using biodistribution and imaging studies on healthy animal models. The aim of the study is to contribute plant extracts to discover new drugs in the diagnosis and treatment of several diseases. METHODS: Nine female and nine male healthy Wistar albino rats, which were approximately 100-150 g in weight, were used for biodistribution studies. For imaging studies four healthy male Balb-C mice were used. Quality control studies were done utilizing thin layer radio chromatography (TLRC) and high performance liquid chromatography (HPLC) methods. For biodistribution studies, 131I radiolabeled Peak 7 (131I-Peak 7) was sterilized and injected into the tail veil of rats and imaging studies were obtained using Kodak FX PRO in vivo Imaging System. RESULTS: The radiolabeling yield of each purified the bioactive extracts of the yarrow plant, seven peaks was between 79 and 92%. The highest radiolabeling yield was calculated for 131I radiolabeled seventh peak (131I-Peak 7) (92.78±5.04, n=5). For this reason the biodistribution and imaging studies were done for 131I-Peak 7. That's why; these studies with Peak 7 were carried out. CONCLUSION: Peak 7 was radiolabeled with 131I in high yield for using imaging and therapeutic studies in nuclear medical applications.(AU)

OBJETIVO: O atual estudo tem por objetivo a extração com metanol, purificação, marcação com I131 usando o método direto de marcação da planta Achillea, para investigar in vivo a atividade biológica usando biodistribuição e estudos de imagem em modelos animais saudáveis. O objetivo do estudo é contribuir com extratos de plantas para descobrir novas drogas para o diagnóstico e tratamento de várias doenças. MÉTODOS: Nove fêmeas e nove machos ratos Wistar albino saudáveis, com aproximadamente 100 a 150g de peso foram usados para estudos de biodistribuição. Para estudos de imagem, quatro camundongos Balb-C machos e saudáveis foram usados. Estudos de controle de qualidade foram realizados usando métodos de cromatografia de camada fina e cromatografia líquida de alta performance. Para estudos de biodistribuição, pico 5 radiografado com I131 (I131-Peak 7) foi esterilizado e injetado na veia da cauda dos ratos e estudos de imagem foram obtidos usando Sistema de Imagem Kodak FX PRO in vivo. RESULTADOS: O retorno radiomarcado de cada extrato bioativo purificado da planta Achillea sete picos estavam entre 79 e 92%. O retorno com maior marcação foi calculado para I131 sétimo pico (I131-Peak 7) (92,78±5,04, n=5). Por esta razão os estudos de biodistribuição e de imagem foram feitos para I131-Peak 7. CONCLUSÃO: Peak 7 foi radiomarcado com I131 em alto retorno para uso em estudos terapêuticos e de imagens nas aplicações médicas nucleares.(AU)

Effects of broccoli extract on biodistribution and labeling blood components with 99mTc-GH / Efeitos do extrato de brócolis na biodistribuição e marcação dos componentes do sangue com 99mTc-GH

PURPOSE: People consume vegetables without the knowledge of the side effects of the biological and chemical contents and interactions between radiopharmaceuticals and herbal extract. To this end, current study is focused on the effects of broccoli extract on biodistribution of radiolabeled glucoheptonate (99mTc-GH) and radiolabeling of blood components. METHODS: GH was labeled with 99mTc. Quality control studies were done utilizing TLC method. Biodistribution studies were performed on male rats which were treated via gavage with either broccoli extract or SF as control group for 15 days. Blood samples were withdrawn from rats' heart. Radiolabeling of blood constituents performed incubating with GH, SnCl2 and 99m Tc. RESULTS: Radiochemical yield of 99mTc-GH is 98.46±1.48 % (n=8). Biodistribution studies have shown that according to the control, the treated group with broccoli has approximately 10 times less uptake in kidney. The percentage of the radioactivity ratios of the blood components is found to be same in both groups. CONCLUSIONS: Although there is no considerable effect on the radiolabeling of blood components, there is an outstanding change on the biodistribution studies especially on kidneys. The knowledge of this change on kidney uptake may contribute to reduce the risk of misdiagnosis and/or repetition of the examinations in Nuclear Medicine.(AU)

OBJETIVO: As pessoas consomem verduras sem o conhecimento dos efeitos colaterais dos conteúdos biológicos e químicos e interações entre os medicamentos radiofarmacêuticos e os extratos vegetais. Para este fim, o estudo atual é focado sobre os efeitos do extrato de brócolis na biodistribuição do fármaco glucoheptonato (99mTc-GH) e da marcação de componentes do sangue. MÉTODOS: GH foi marcado com 99mTc. Estudos de controle de qualidade foram feitos utilizando o método do TLC. Os estudos de biodistribuição foram realizados em ratos machos que foram tratados por gavagem com um extrato de brócolis ou SF como grupo controle para 15 dias. Amostras de sangue foram retiradas do coração de ratos. Marcação de constituintes sanguíneos realizados incubação com SnCl2 GH e 99mTc. RESULTADOS: Radioquímica rendimento de 99mTc-GH é 98,46 ± 1,48% (n = 8). Os estudos de biodistribuição mostraram que de acordo com o controle, o grupo tratado com brócolis tem aproximadamente 10 vezes menor absorção no rim. O percentual do ratio de radioatividade dos componentes do sangue é encontrado para ser igual nos dois grupos. CONCLUSÕES: Embora não haja nenhum efeito considerável sobre a marcação dos componentes do sangue há uma mudança notável na biodistribuição especialmente nos rins. O conhecimento desta mudança na captação de rim pode contribuir para reduzir o risco de erro diagnóstico e/ou a repetição dos exames de Medicina Nuclear.(AU)

Comparison of two purified toxic fractions from Mesobuthus eupeus scorpion venom

Iranian scorpions belong mainly to the Buthidae and Scorpionidae families, distributed into 16 genera and 25 species. In Iran, similar to other parts of the world, there are a few known species of scorpions responsible for severe envenoming; amongst which Mesobuthus eupeus is the most common. Its venom contains several toxin fractions that may affect the ion channel. In the present study purification, labeling and biological evaluation of M. eupeus venom are described. For separation, soluble venom was loaded on a chromatography column packed with Sephadex G-50 gel. Subsequently, the fractions were collected according to UV absorption at a wavelength of 280 nm. Toxic fraction (F3) was loaded on an anionic ion exchanger resin and then on a cationic resin. Finally, toxic subfractions F3.1.6 and F3.1.9 were labeled with 99mTc and injected into normal mice to distinguish excretion pathway. The venom toxic fraction was successfully obtained in its purified form. Radiolabeling of toxic fractions was performed at high specific activity with radiochemical purity of more than 97 and 95 percent respectively for F3.1.6 and F3.1.9. Biodistribution studies in normal mice with two toxic fractions usually show rapid clearance of the compounds from blood and tissue except for kidneys. Since tissue distribution studies are very important for clinical purpose, the present findings suggest that 99mTc labeling of venom is a useful tool for in vivo studies and comprises an excellent approach to monitoring the process of biodistribution and kinetics of toxins.(AU)

Comparison of two purified toxic fractions from Mesobuthus eupeus scorpion venom

Iranian scorpions belong mainly to the Buthidae and Scorpionidae families, distributed into 16 genera and 25 species. In Iran, similar to other parts of the world, there are a few known species of scorpions responsible for severe envenoming; amongst which Mesobuthus eupeus is the most common. Its venom contains several toxin fractions that may affect the ion channel. In the present study purification, labeling and biological evaluation of M. eupeus venom are described. For separation, soluble venom was loaded on a chromatography column packed with Sephadex G-50 gel. Subsequently, the fractions were collected according to UV absorption at a wavelength of 280 nm. Toxic fraction (F3) was loaded on an anionic ion exchanger resin and then on a cationic resin. Finally, toxic subfractions F3.1.6 and F3.1.9 were labeled with 99mTc and injected into normal mice to distinguish excretion pathway. The venom toxic fraction was successfully obtained in its purified form. Radiolabeling of toxic fractions was performed at high specific activity with radiochemical purity of more than 97 and 95% respectively for F3.1.6 and F3.1.9. Biodistribution studies in normal mice with two toxic fractions usually show rapid clearance of the compounds from blood and tissue except for kidneys. Since tissue distribution studies are very important for clinical purpose, the present findings suggest that 99mTc labeling of venom is a useful tool for in vivo studies and comprises an excellent approach to monitoring the process of biodistribution and kinetics of toxins.(AU)