RESUMEN
Ethyl succinic anhydride and its d5-labeled version have been synthesized and applied to quantitative analysis of peptides in combination with MALDI or ESI mass spectrometry. These modifiers react with amino groups in the N-termini and lysine side chains in proteins, and therefore the combination of these modifiers was shown to be a useful tool for quantification of peptides and hence for proteomics research.
Asunto(s)
Espectrometría de Masas/métodos , Péptidos/química , Anhídridos Succínicos/síntesis química , Proteómica , Anhídridos Succínicos/químicaRESUMEN
The present study was undertaken to evaluate the effects of l-DOPA pre-loading on the uptake of BPA using the F98 rat glioma and the murine B16 melanoma models. In vitro pretreatments of F98 glioma and B16 melanoma cells with l-DOPA, followed by exposure to BPA increased boron uptake, as determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES). Based on this, in vivo studies were initiated in F98 glioma bearing rats. Initially, the l-DOPA dosing paradigm was evaluated. Maximum tumor boron uptake was observed following i.p. administration of l-DOPA (50mg/kg) followed 24h later by BPA (31.8±8.9 vs. 17.2±6.3µg/g for BPA alone). Next, the effect of l-DOPA pre-loading as a function of the route of administration of BPA was evaluated in F98 glioma bearing rats. The greatest increase in uptake was seen following i.v. administration of BPA, while in contrast no significant increase was seen following intracarotid (i.c.) administration (38.6±12.4 vs. 34.2±10.9). Cellular localization of the F98 glioma, as determined by secondary ion mass spectrometry (SIMS) boron imaging revealed equivalent tumor boron concentrations following l-DOPA pre-loading. In vivo studies in B16 melanoma bearing mice showed equivalent tumor boron values in treated and untreated mice, suggesting that the effects of l-DOPA pre-loading may depend both on the histologic type of tumor and its anatomic site.
Asunto(s)
Compuestos de Boro/farmacocinética , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Levodopa/administración & dosificación , Melanoma/metabolismo , Fenilalanina/análogos & derivados , Premedicación/métodos , Animales , Compuestos de Boro/administración & dosificación , Neoplasias Encefálicas/radioterapia , Sinergismo Farmacológico , Femenino , Glioma/radioterapia , Melanoma/radioterapia , Tasa de Depuración Metabólica/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos/efectos de los fármacos , Fenilalanina/administración & dosificación , Fenilalanina/farmacocinética , Distribución Tisular/efectos de los fármacos , Resultado del TratamientoRESUMEN
Unnatural cyclic amino acids (UNAAs) are a new class of boron delivery agents that are in a pre-clinical stage of evaluation. In the present study, the biodistribution of racemic forms of the cis- and trans-isomers of the boronated UNAA 1-amino-3-boronocyclopentanecarboxylic acid (ABCPC) and 1-amino-3-boronocycloheptanecarboxylic acid (ABCHC) were evaluted in B16 melanoma bearing mice and this was compared to l-p-boronophenylalanine (BPA). Boron concentrations were determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES) at 2.5h following intraperitoneal (i.p.) injection of the test agents at a concentration equivalent to 24mg/B/kg. While all compounds attained comparable tumor boron concentrations, the tumor/blood (T/Bl) boron concentration ratios were far superior for both cis-ABCPC and cis-ABCHC compared to BPA (T/Bl=16.4, and 15.1 vs. 5.4). Secondary ion mass spectrometry (SIMS) imaging revealed that the cis-ABCPC delivered boron to the nuclei, as well as the cytoplasm of B16 cells. Next, a biodistribution study of cis-ABCPC and BPA was carried out in F98 glioma bearing rats following i.p. administration. Both compounds attained comparable tumor boron concentrations but the tumor/brain (T/Br) boron ratio was superior for cis-ABCPC compared to BPA (6 vs. 3.3). Since UNAAs are water soluble and cannot be metabolized by tumor cells, they could be potentially more effective boron delivery agents than BPA. Our data suggest that further studies are warranted to evaluate these compounds prior to the initiation of clinical studies.
Asunto(s)
Aminoácidos Cíclicos/farmacocinética , Aminoácidos Cíclicos/uso terapéutico , Compuestos de Boro/farmacocinética , Compuestos de Boro/uso terapéutico , Terapia por Captura de Neutrón de Boro/métodos , Glioma/metabolismo , Melanoma/metabolismo , Aminoácidos Cíclicos/química , Animales , Compuestos de Boro/química , Línea Celular Tumoral , Portadores de Fármacos , Femenino , Glioma/radioterapia , Melanoma/radioterapia , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Ratas , Ratas Endogámicas F344 , Distribución Tisular , Resultado del TratamientoRESUMEN
The development of new boron-delivery agents is a high priority for improving the effectiveness of boron neutron capture therapy. In the present study, 1-amino-3-borono-cyclopentanecarboxylic acid (cis-ABCPC) as a mixture of its L- and D-enantiomers was evaluated in vivo using the B16 melanoma model for the human tumor and the F98 rat glioma as a model for human gliomas. A secondary ion mass spectrometry (SIMS) based imaging instrument, CAMECA IMS 3F SIMS Ion Microscope, was used for quantitative imaging of boron at 500 nm spatial resolution. Both in vivo and in vitro studies in melanoma models demonstrated that boron was localized in the cytoplasm and nuclei with some cell-to-cell variability. Uptake of cis-ABCPC in B16 cells was time dependent with a 7.5:1 partitioning ratio of boron between cell nuclei and the nutrient medium after 4 hrs. incubation. Furthermore, cis-ABCPC delivered boron to cells in all phases of the cell cycle, including S-phase. In vivo SIMS studies using the F98 rat glioma model revealed an 8:1 boron partitioning ratio between the main tumor mass and normal brain tissue with a 5:1 ratio between infiltrating tumor cells and contiguous normal brain. Since cis-ABCPC is water soluble and can cross the blood-brain-barrier via the L-type amino acid transporters (LAT), it may accumulate preferentially in infiltrating tumor cells in normal brain due to up-regulation of LAT in high grade gliomas. Once trapped inside the tumor cell, cis-ABCPC cannot be metabolized and remains either in a free pool or bound to cell matrix components. The significant improvement in boron uptake by both the main tumor mass and infiltrating tumor cells compared to those reported in animal and clinical studies of p-boronophenylalanine strongly suggest that cis-ABCPC has the potential to become a novel new boron delivery agent for neutron capture therapy of gliomas and melanomas.
Asunto(s)
Aminoácidos , Compuestos de Boro , Terapia por Captura de Neutrón de Boro/métodos , Neoplasias Encefálicas , Glioma , Melanoma , Neoplasias Experimentales , Espectrometría de Masa de Ion Secundario , Aminoácidos/síntesis química , Aminoácidos/química , Aminoácidos/farmacocinética , Aminoácidos/farmacología , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Compuestos de Boro/síntesis química , Compuestos de Boro/química , Compuestos de Boro/farmacocinética , Compuestos de Boro/farmacología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , Glioma/diagnóstico por imagen , Glioma/metabolismo , Glioma/patología , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patología , Melanoma/radioterapia , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Experimentales/radioterapia , Radiografía , Ratas , Ratas Endogámicas F344 , Fase S/efectos de los fármacos , Fase S/efectos de la radiaciónRESUMEN
Two cysteine-specific modifiers we reported previously, N-ethyl maleimide (NEM) and iodoacetanilide (IAA), have been applied to the labeling of cysteine residues of peptides for the purpose of examining the enhancement of ionization efficiencies in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The peak intensities of the peptides as a result of modification with these modifiers were compared with the peak intensities of peptides modified with a commercially available cysteine-specific modifier, iodoacetamide (IA). Our experiments show significant enhancement in the peak intensities of three cysteine-containing synthetic peptides modified with IAA compared to those modified with IA. The results showed a 4.5-6-fold increase as a result of modification with IAA compared to modification with IA. Furthermore, it was found that IAA modification also significantly enhanced the peak intensities of many peptides of a commercially available proteins, bovine serum albumin (BSA), compared to those modified with IA. This significant enhancement helped identify a greater number of peptides of these proteins, leading to a higher sequence coverage with greater confidence scores in identification of proteins with the use of IAA.
Asunto(s)
Cisteína/química , Fragmentos de Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Alquilación , Secuencia de Aminoácidos , Animales , Bovinos , Cisteína/metabolismo , Yodoacetamida/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismoRESUMEN
Benzoyloxysuccinimide and its d(5)-labeled version, which react with amino groups in the N-termini and lysine side chains in proteins, were synthesized and applied to quantitative analysis of peptides and a commercially available protein in combination with a MALDI mass spectrometer.
Asunto(s)
Péptidos/análisis , Proteínas/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Succinimidas/química , Hormona Adrenocorticotrópica/análisis , Angiotensinas/análisis , Humanos , Marcaje Isotópico , Lisina/química , Succinimidas/síntesis químicaRESUMEN
Boron delivery characteristics of cis and trans isomers of a boronated unnatural amino acid, 1-amino-3-boronocyclopentanecarboxylic acid (ABCPC) were tested in the B16 mouse model for human melanoma. Both ABCPC isomers delivered comparable boron to B16 melanoma tumor cells as L-p-boronophenylalanine (BPA). Secondary ion mass spectrometry (SIMS) analysis revealed the presence of boron throughout the tumor from these compounds, and a near homogeneous distribution between the nucleus and cytoplasm of B16 cells grown in vitro. These encouraging observations support further studies of these new boron carriers in BNCT.