RESUMEN
Fibrosing chronic graft-versus-host disease (cGVHD) is a debilitating complication of allogeneic stem cell transplantation (alloSCT). A driver of fibrosis is the kynurenine (Kyn) pathway, and Kyn metabolism patterns and cytokines may influence cGVHD severity and manifestation (fibrosing versus gastrointestinal [GI] cGVHD). Using a liquid chromatography-tandem mass spectrometry approach on sera obtained from 425 patients with allografts, we identified high CXCL9, high indoleamine-2,3-dioxygenase (IDO) activity, and an activated Kyn pathway as common characteristics in all cGVHD subtypes. Specific Kyn metabolism patterns could be identified for non-severe cGVHD, severe GI cGVHD, and fibrosing cGVHD, respectively. Specifically, fibrosing cGVHD was associated with a distinct pathway shift toward anthranilic and kynurenic acid, correlating with reduced activity of the vitamin-B2-dependent kynurenine monooxygenase, low vitamin B6, and increased interleukin-18. The Kyn metabolite signature is a candidate biomarker for severe fibrosing cGVHD and provides a rationale for translational trials on prophylactic vitamin B2/B6 supplementation for cGVHD prevention.
Asunto(s)
Enfermedad Injerto contra Huésped/sangre , Ácido Quinurénico/sangre , Quinurenina/sangre , Riboflavina/sangre , Trasplante de Células Madre , Vitamina B 6/sangre , Adolescente , Adulto , Anciano , Quimiocina CXCL9/sangre , Quimiocina CXCL9/genética , Femenino , Fibrosis , Regulación de la Expresión Génica , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/patología , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/sangre , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Interleucina-18/sangre , Interleucina-18/genética , Quinurenina 3-Monooxigenasa/sangre , Quinurenina 3-Monooxigenasa/genética , Leucemia/genética , Leucemia/metabolismo , Leucemia/patología , Leucemia/terapia , Linfoma/genética , Linfoma/metabolismo , Linfoma/patología , Linfoma/terapia , Masculino , Redes y Vías Metabólicas/genética , Persona de Mediana Edad , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Transducción de Señal , Trasplante Homólogo , Triptófano/sangre , ortoaminobenzoatos/sangreRESUMEN
A new methodology based on Nuclear Magnetic Resonance (NMR) was developed to determine plasma protein binding (PPB) of drug candidates in drug discovery programs. A strong correlation was found between the attenuation of NMR signals of diverse drugs in the presence of different plasma concentrations and their fraction bound (fb) reported in the literature. Based on these results, a protocol for a rapid calculation of fb of small molecules was established. The advantage of using plasma instead of purified recombinant proteins and the possibility of pool analysis to increase throughput were also evaluated. This novel methodology proved to be very versatile, cost-effective, fast and suitable for automation. As a plus, it contemporarily provides a quality check and solubility of the compound.
Asunto(s)
Proteínas Sanguíneas/química , Descubrimiento de Drogas/métodos , Resonancia Magnética Nuclear Biomolecular , Preparaciones Farmacéuticas/sangre , Descubrimiento de Drogas/instrumentación , Evaluación Preclínica de Medicamentos , Humanos , Técnicas In Vitro , Preparaciones Farmacéuticas/química , Unión Proteica , Proteínas Recombinantes/química , Albúmina Sérica Humana/química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
This work describes a simple, sensitive and rapid liquid chromatography-high resolution mass spectrometry method for the quantitation of perhexiline and the simultaneous detection of perhexiline metabolites in C57bl/6 mice plasma. Only 5 µL of plasma was used for analysis. Pretreatment was limited to a 100-fold dilution ('dilute-and-shoot'). The analyte was detected by high resolution mass spectrometry (Orbitrap™ technology). Three scan events were performed over the entire chromatogram. Targeted single ion monitoring with data dependent acquisition was employed for perhexiline quantitation and confirmation, while full scan was used to perform untargeted detection of perhexiline phase I and phase II circulating metabolites. The calibration curve was linear (r(2)=0.990) ranging from 0.305 ng/mL (LLOQ) to 10000 ng/mL. Matrix effect was limited to 6.1%. The method was applied to a pharmacokinetic study of perhexiline in mouse plasma and the results obtained were compared to a standard sample preparation method based on protein precipitation and liquid chromatography-tandem mass spectrometry (MRM mode) detection. The new approach provided comparable results in terms of pharmacokinetics parameters estimate with a high sensitivity, additional information on perhexiline circulating metabolites and a low consumption of biological sample. The combination of the 'dilute-and-shoot' approach together with HRMS targeted and untargeted detection represents a suitable alternative to classic bioanalytical approaches in preclinical research.
Asunto(s)
Perhexilina/sangre , Perhexilina/farmacocinética , Espectrometría de Masas en Tándem/métodos , Animales , Cromatografía Liquida/métodos , Evaluación Preclínica de Medicamentos/métodos , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.
Asunto(s)
Fármacos Antiobesidad/síntesis química , Hipoglucemiantes/síntesis química , Neuropéptidos/síntesis química , Neuropéptidos/farmacología , Polietilenglicoles/farmacología , Albúmina Sérica/síntesis química , Animales , Fármacos Antiobesidad/farmacocinética , Fármacos Antiobesidad/farmacología , Glucemia , Línea Celular , Evaluación Preclínica de Medicamentos , Humanos , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuropéptidos/farmacocinética , Polietilenglicoles/farmacocinética , Receptores de Neurotransmisores/agonistas , Albúmina Sérica/farmacocinética , Albúmina Sérica/farmacología , Albúmina Sérica Humana , Pérdida de Peso/efectos de los fármacosRESUMEN
A very highly sensitive and highly selective liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed to evaluate and quantify a new drug candidate in different biological matrices. Following a simple plasma protein precipitation using acetonitrile, the post-treatment samples were analyzed on a C18 column interfaced with a new generation of triple-quadrupole mass spectrometer. The recently introduced triple-quadrupole mass spectrometer, the TSQ Quantum Ultra, with enhanced mass-resolution capability, demonstrated improved sensitivity (0.05 ng/mL), coupled with suitable accuracy and precision, over a broad linear dynamic range (0.05-1000 ng/mL). A comparison of the assay performance data (dynamic range, calibration curve equation, precision and accuracy) of the enhanced resolution method against a unit resolution method under optimized conditions showed the performance improvement of the enhanced mass resolution method for bioanalytical high-throughput applications. The enhanced mass resolution method herein described was successfully applied to the evaluation of the pharmacokinetic profile of a new drug candidate in rat, rabbit and dog plasma samples.