RESUMEN
UNLABELLED: The α(ν)ß(3) integrin is over-expressed in the tumor neovasculature and the tumor cells of glioblastomas. The HIV Tat-derived peptide has been used to deliver various cargos into cells. The aim of this research was to synthesize and assess the in vitro and in vivo uptake of (99m)Tc-N2S2-Tat(49-57)-c(RGDyK) ((99m)Tc-Tat-RGD) in α(ν)ß(3) integrin positive cancer cells and compare it to that of a conventional (99m)Tc-RGD peptide ((99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2). METHODS: The c(RGDyK) peptide was conjugated to a maleimidopropionyl (MP) moiety through Lys, and the MP group was used as the branch position to form a thioether with the Cys(12) side chain of the Tat(49-57)-spacer-N2S2 peptide. (99m)Tc-Tat-RGD was prepared, and stability studies were carried out by size exclusion HPLC analyses in human serum. The in vitro affinity for α(v)ß(3) integrin was determined by a competitive binding assay. In vitro internalization was determined using glioblastoma C6 cells. Biodistribution studies were accomplished in athymic mice with C6 induced tumors that had blocked and unblocked receptors. Images were obtained using a micro-SPECT/CT. RESULTS: (99m)Tc-Tat-RGD was obtained with a radiochemical purity higher than 95%, as determined by radio-HPLC and ITLC-SG analyses. Protein binding was 15.7% for (99m)Tc-Tat-RGD and 5.6% for (99m)Tc-RGD. The IC50 values were 6.7 nM ((99m)Tc-Tat-RGD) and 4.6 nM ((99m)Tc-RGD). Internalization in C6 cells was higher in (99m)Tc-Tat-RGD (37.5%) than in (99m)Tc-RGD (10%). Biodistribution studies and in vivo micro-SPECT/CT images in mice showed higher tumor uptake for (99m)Tc-Tat-RGD (6.98% ± 1.34% ID/g at 3h) than that of (99m)Tc-RGD (3.72%±0.52% ID/g at 3h) with specific recognition for α(v)ß(3) integrins. CONCLUSIONS: Because of the significant cell internalization (Auger and internal conversion electrons) and specific recognition for α(v)ß(3) integrins, the hybrid (99m)Tc-N2S2-Tat(49-57)-c(RGDyK) radiopharmaceutical is potentially useful for the imaging and possible therapy of tumors expressing α(v)ß(3) integrins.
Asunto(s)
Diseño de Fármacos , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Integrina alfaVbeta3/metabolismo , Oligopéptidos/química , Compuestos de Organotecnecio , Fragmentos de Péptidos/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Animales , Transporte Biológico , Línea Celular Tumoral , Glioma/diagnóstico por imagen , Glioma/patología , Humanos , Masculino , Ratones , Imagen Multimodal , Compuestos de Organotecnecio/química , Compuestos de Organotecnecio/metabolismo , Compuestos de Organotecnecio/farmacocinética , Cintigrafía , Radiofármacos/química , Radiofármacos/metabolismo , Radiofármacos/farmacocinéticaRESUMEN
The human cathelicidin hCAP18/LL-37 is an antimicrobial protein consisting of a conserved N-terminal prosequence called the cathelin-like domain and a C-terminal peptide called LL-37. This peptide contains 37 amino acid residues, and several truncated variants obtained from natural sources or by chemical synthesis differ in their capability to damage Gram positive and Gram negative bacteria as well as Candida albicans. KR-12 is the shortest peptide (12 amino acids) of LL-37 that has conserved antibacterial activity. In addition to LL-37, other active cathelicidin-derived peptides have been reported; for instance, the peptides KR-20, a 20-aa derivative of LL-37, and KS-30, a 30-aa derivative of LL-37, have been found in human sweat. Both peptides exhibit an overall increased antibacterial and antifungal activity when compared with LL-37. We investigated the effect of LL-37 and three peptides derived from this antimicrobial molecule, KR-12, KR-20 and KS-30, on the integrity of Entamoeba histolytica trophozoites. The four peptides showed effects on E. histolytica integrity and viability in the concentration range of 10-50 µM. The peptides KR-12, KR-20, KS-30 and LL-37 differed in their capability to damage the parasite integrity, with KR-20 being the most effective and with KR-12 and LL-37 being less active. These results demonstrate the ability of antimicrobial peptides derived from human cathelicidin to damage Entamoeba trophozoites. Moreover, it was shown that the integrity of the peptides is altered in the presence of an ameba soluble fraction with cysteine protease activity.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Antiprotozoarios/farmacología , Entamoeba histolytica/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/metabolismo , Antiprotozoarios/química , Antiprotozoarios/aislamiento & purificación , Antiprotozoarios/metabolismo , Catelicidinas/aislamiento & purificación , Catelicidinas/metabolismo , Catelicidinas/farmacología , Proteasas de Cisteína/metabolismo , Entamoeba histolytica/enzimología , Entamoeba histolytica/crecimiento & desarrollo , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Hemólisis , Humanos , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Trofozoítos/efectos de los fármacos , Trofozoítos/crecimiento & desarrolloRESUMEN
C-Br bond activation followed by a C-C coupling reaction of the 2-bromo-pyridyl unit of [1-phenyl-2-(6-bromopyridin-2-yl)-benzoimidazole] was performed by Pd(CH(2)CMe(2)-o-C(6)H(4))(η(4)-COD). Two new seven membered palladacycles were obtained. A combined experimental and theoretical DFT study elucidates the mechanism for this reaction.
RESUMEN
Integrin α(V)ß(3) plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic RGD peptides can be used for noninvasive imaging of α(V)ß(3) expression. The aim of this research was to prepare a multimeric system of technetium-99m-labeled gold nanoparticles conjugated to c[RGDfK(C)] and to evaluate its biological behavior as a potential radiopharmaceutical for molecular imaging of tumor angiogenesis. Hydrazinonicotinamide-GGC (HYNIC-GGC) and c[RGDfK(C)] peptides were synthesized and conjugated to gold nanoparticles (AuNP, 20 nm) by means of spontaneous reaction of the thiol groups of cysteine. The nanoconjugate was characterized by TEM, FT-IR, UV-vis, XPS, and Raman spectroscopy. To obtain (99m)Tc-HYNIC-GGC-AuNP-c[RGDfK(C)] ((99m)Tc-AuNP-RGD), the (99m)Tc-HYNIC-GGC radiopeptide was first prepared and added to 1.5 mL of AuNP solution (1 nM) followed by c[RGDfK(C)] (10 µL, 50 µM) at 18 °C with stirring for 15 min. Radiochemical purity (RP) was determined by size-exclusion HPLC and ITLC-SG analyses. In vitro binding studies were carried out in α(V)ß(3) receptor-positive C6 glioma cancer cells. Biodistribution studies were accomplished in athymic mice with C6-induced tumors with blocked and nonblocked receptors, and images were obtained using a micro-SPECT/CT. TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with peptides. RP was 96 ± 2% without postlabeling purification. (99m)Tc-AuNP-RGD showed specific recognition for α(V)ß(3) integrins expressed in C6 cells, and 3 h after i.p. administration in mice, the tumor uptake was 8.18 ± 0.57% ID/g. Micro-SPECT/CT images showed evident tumor uptake. (99m)Tc-AuNP-RGD demonstrates properties suitable for use as a target-specific agent for molecular imaging of tumor α(V)ß(3) expression.
Asunto(s)
Glioma/metabolismo , Oro , Integrina alfaVbeta3/análisis , Nanopartículas del Metal , Imagen Molecular , Compuestos de Organotecnecio , Péptidos Cíclicos , Animales , Línea Celular Tumoral , Femenino , Glioma/patología , Oro/química , Oro/farmacocinética , Humanos , Integrina alfaVbeta3/biosíntesis , Marcaje Isotópico , Nanopartículas del Metal/química , Ratones , Ratones Desnudos , Estructura Molecular , Compuestos de Organotecnecio/química , Compuestos de Organotecnecio/farmacocinética , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacocinética , Distribución TisularRESUMEN
UNLABELLED: The aim of this research was to prepare a multifunctional system of technetium-99m-labelled gold nanoparticles conjugated to HYNIC-GGC/mannose and to evaluate its biological behaviour as a potential radiopharmaceutical for sentinel lymph node detection (SLND). METHODS: Hydrazinonicotinamide-Gly-Gly-Cys-NH(2) (HYNIC-GGC) peptide and a thiol-triazole-mannose derivative were synthesized, characterized and conjugated to gold nanoparticles (AuNP, 20 nm) to prepare a multifunctional system of HYNIC-GGC-AuNP-mannose by means of spontaneous reaction of the thiol (Cys) present in HYNIC-GGC sequence and in the thiol-mannose derivative. The nanoconjugate was characterized by transmission electron microscopy (TEM), IR, UV-Vis, Raman, fluorescence and X-ray photoelectron spectroscopy (XPS). Technetium-99m labelling was carried out using EDDA/tricine as coligands and SnCl(2) as reducing agent with further size-exclusion chromatography purification. Radiochemical purity was determined by size-exclusion HPLC and ITLC-SG analyses. In vitro binding studies were carried out in rat liver homogenized tissue (mannose-receptor positive tissue). Biodistribution studies were accomplished in Wistar rats and images obtained using a micro-SPECT/CT system. RESULTS: TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with HYNIC-GGC-NH(2) and thiol-mannose through interactions with thiol groups and the N-terminal amine of cysteine. Radio-chromatograms showed radiochemical purity higher than 95%. (99m)Tc-EDDA/HYNIC-GGC-AuNP-mannose ((99m)Tc-AuNP-mannose) showed specific recognition for mannose receptors in rat liver tissue. After subcutaneous administration of (99m)Tc-AuNP-mannose in rats (footpad), radioactivity levels in the popliteal and inguinal lymph nodes revealed that 99% of the activity was extracted by the first lymph node (popliteal extraction). Biodistribution studies and in vivo micro-SPECT/CT images in Wistar rats showed an evident lymph node uptake (11.58 ± 1.98 %ID at 1 h) which was retained during 24 h with minimal kidney accumulation (0.98 ± 0.10 %ID) and negligible uptake in all other tissues. CONCLUSIONS: This study demonstrated that (99m)Tc-AuNP-mannose remains within the first lymph node during 24 h and therefore might be useful as a target-specific radionanoconjugate for SLND using "1-day" or "2-day" conventional protocols.
Asunto(s)
Oro/química , Ganglios Linfáticos/metabolismo , Manosa/metabolismo , Nanopartículas del Metal/química , Oligopéptidos/química , Oligopéptidos/metabolismo , Compuestos de Organotecnecio/química , Animales , Humanos , Lectinas Tipo C/metabolismo , Metástasis Linfática , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Microscopía Electrónica de Transmisión , Oligopéptidos/farmacocinética , Radioquímica , Ratas , Ratas Wistar , Receptores de Superficie Celular/metabolismo , Biopsia del Ganglio Linfático Centinela , Análisis Espectral , Compuestos de Sulfhidrilo/químicaRESUMEN
Antimicrobial peptides are widely distributed in nature; they play important roles in several aspects of innate immunity and may provide a basis for the design of novel therapeutic agents. In this study, C-amidated tritrpticin, a 13 amino acid tryptophan-rich antimicrobial peptide derived from a porcine cathelicidin, was tested against Trichomonas vaginalis, a protozoan that causes a serious non-viral sexually transmitted disease associated with preterm birth, low birth weight, and high risk of HIV-1 infection. Tritrpticin was selected due to its reasonably easy synthesis and because analogs with lower toxicity may be designed. Our results show that tritrpticin-NH(2) at either 100 or 200 µg/ml (52.5 or 105 µM) clearly reduces the viability and growth of Trichomonas vaginalis. Together with tritrpticin-NH(2), sodium bicarbonate further limited trichomonad growth. Additionally, a low concentration of metronidazole (5.8 µM), the most commonly used medication for Trichomonas vaginalis, was more effective against the growth of the parasite when it was combined with tritrpticin-NH(2).
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Antiprotozoarios/farmacología , Oligopéptidos/farmacología , Trichomonas vaginalis/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Metronidazol/farmacología , Viabilidad Microbiana/efectos de los fármacos , Pruebas de Sensibilidad Parasitaria , Bicarbonato de Sodio/farmacología , Trichomonas vaginalis/crecimiento & desarrollo , Trichomonas vaginalis/fisiologíaRESUMEN
The synthesis of new DOTA tetraamide (DOTAMR(4)) compounds is of great interest given their application in the formation of Ln(III) complexes as potential PARACEST contrast agents in MRI or fluorescent molecular probes. In this context amino acid and peptide DOTAMR(4) derivatives are particularly attractive since the amino-acid and/or peptide moiety can show responsive properties dependent on a given stimuli which might translate to changes in water exchange rates of the corresponding Ln(III) complex. Current synthesis of DOTAMR(4) derivatives is typically carried out by reacting haloacetamide intermediates with cyclen. However, this method fails to generate the tetra-substituted products when bulky substituents are present in the haloacetamide and in some cases this intermediate cannot be prepared by conventional acylation procedures limiting the number of DOTAMR(4) compounds available for study. As a solution to these limitations, an improved methodology for the synthesis of DOTAMR(4) by coupling DOTA to an appropriate amine containing reagent (i.e. protected amino-acids with the alpha-amino group free) is presented in this work. Several DOTAMR(4) derivatives which are difficult or impossible to prepare with the traditional methodologies were easily obtained starting with DOTA. A new protocol was derived using this methodology for the solution-phase synthesis of DOTA peptide derivatives. With this methodology, many other DOTAMR(4) peptide and non-peptide derivatives have been prepared in our laboratories with several of these new compounds showing interesting properties for molecular imaging.