RESUMEN
Alzheimer's disease (AD) is a devastating neurological disorder for which soluble oligomers of the peptide amyloid-ß (Aß) are now recognized as the neurotoxic species. Metal-based therapeutics are uniquely suited to target Aß, with ruthenium-based (Ru) complexes emerging as propitious candidates. Recently, azole-based Ru(III) complexes were observed to modulate the aggregation of Aß in solution, where the inclusion of a primary amine proximal to the ligand coordination site improved the activity of the complexes. To advance these structure-activity relationships, a series of oxazole-based Ru complexes were prepared and evaluated for their ability to modulate Aß aggregation. From these studies, a lead candidate, Oc, emerged that had superior activity relative to its azole predecessors in modulating the aggregation of soluble Aß and diminishing its cytotoxicity. Further evaluation of Oc demonstrated its ability to disrupt formed Aß aggregates, resulting in smaller amorphous species. Because altering both sides of the aggregation equilibrium for Aß has not been previously suggested for metal-based complexes for AD, this work represents an exciting new avenue for improved therapeutic success.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/antagonistas & inhibidores , Complejos de Coordinación/farmacología , Fármacos Neuroprotectores/farmacología , Oxazoles/farmacología , Rutenio/farmacología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Supervivencia Celular , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Conformación Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Oxazoles/química , Agregado de Proteínas/efectos de los fármacos , Ratas , Rutenio/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Alzheimer's disease (AD) is the most common form of dementia, where one of the pathological hallmarks of AD is extracellular protein deposits, the primary component of which is the peptide amyloid-ß (Aß). Recently, the soluble form of Aß has been recognized as the primary neurotoxic species, making it an important target for therapeutic development. Metal-based drugs are promising candidates to target Aß, as the interactions with the peptide can be tuned by ligand design. In the current study, 11 ruthenium complexes containing pyridine-based ligands were prepared, where the functional groups at the para position on the coordinated pyridine ligand were varied to determine structure-activity relationships. Overall, the complexes with terminal primary amines had the greatest impact on modulating the aggregation of Aß and diminishing its cytotoxicity. These results identify the importance of specific intermolecular interactions and are critical in the advancement of metal-based drugs for AD therapy.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Complejos de Coordinación/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Piridinas/uso terapéutico , Rutenio/uso terapéutico , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Relación Dosis-Respuesta a Droga , Humanos , Enlace de Hidrógeno , Ligandos , Estructura Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Piridinas/química , Ratas , Rutenio/química , Relación Estructura-ActividadRESUMEN
Quantitation of residual hydrogen peroxide (H(2)O(2)) and evaluation of the impact on product stability is necessary as unwanted H(2)O(2) can potentially be introduced during the manufacturing of pharmaceuticals, biologics, and vaccines. A sensitive and convenient microplate-based method with fluorescence detection for H(2)O(2) quantitation was recently reported (Towne et al., 2004, Anal Biochem 334: 290-296). This method was found to be highly robust and reproducible, with a level of detection of 0.015 ppm and a level of quantitation of 0.025 ppm (in water). The relatively small sample requirements and amenability for automation make this assay an attractive tool for detecting residual H(2)O(2) levels. Without additional manipulation, the assay can be conducted on heterogeneous solutions with significant degree of turbidity, such as the presence of suspensions or aluminum-containing adjuvants. The quantitation of H(2)O(2) and its decomposition kinetics was also studied in presence of two common vaccine preservatives (thimerosal and phenol) and eight commonly used excipients (polyols). Over time, there is a distinct, temperature dependent decrease in H(2)O(2) recovered in thimerosal and phenol containing samples versus non-preservative containing controls. Based on the half-life of spiked H(2)O(2), the decay rates in eight polyols tested were found to be: ribose > sucrose > (glycerol, glucose, lactose, mannitol, sorbitol, and xylose).
Asunto(s)
Excipientes/análisis , Peróxido de Hidrógeno/análisis , Fenol/análisis , Conservadores Farmacéuticos/análisis , Timerosal/análisis , Colorantes/farmacología , Relación Dosis-Respuesta a Droga , Estabilidad de Medicamentos , Peróxido de Hidrógeno/farmacología , Concentración de Iones de Hidrógeno , Cinética , Oxazinas/farmacología , Oxidación-Reducción , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Temperatura , Agua/químicaRESUMEN
The highly sensitive, convenient fluorescence assay, based on the oxidation of nonfluorescent 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) to highly fluorescent resorufin, is becoming increasingly popular for hydrogen peroxide quantitation. Yet, the intricacies of the horseradish peroxidase-catalyzed oxidation of the reductant substrate Amplex Red by hydrogen peroxide and the resulting resorufin could complicate the assay design and data interpretation. In particular, substrate inhibition and enzyme inactivation at higher hydrogen peroxide concentrations were known to affect the enzyme kinetics and end-point fluorescence. In addition, here we report the spontaneous transformation of resorufin to less or nonfluorescent product(s) in the absence of hydrogen peroxide and horseradish peroxidase. This spontaneous decay of resorufin fluorescence is most prominent in the pH range 6.2-7.7, likely due to general base-catalyzed de-N-acetylation and polymerization of resorufin. From a practical point of view, precautions for properly designing assays for hydrogen peroxide or characterizing hydrogen peroxide-generating systems are discussed based on the spontaneous transformation of resorufin to less fluorescent compound(s), substrate inhibition and enzyme inactivation at higher (>100 microM) hydrogen peroxide concentrations, and enzymatic oxidation of resorufin to nonfluorescent resazurin.