Development and validation of liquid chromatography-tandem mass spectrometry method to quantify dasatinib in plasma and its application to a pharmacokinetic study

Abstract Dasatinib, a potent oral multi-targeted kinase inhibitor against Src and Bcr-Abl, can decrease inflammatory response in sepsis. A simple and cost-effective method for determination of an effective dose dasatinib was established. This method was validated in human plasma, with the aim of reducing the number of animals used, thus, avoiding ethical problems. Dasatinib and internal standard lopinavir were extracted from 180 uL of plasma using liquid-liquid extraction with methyl tert-butil ether, followed by liquid chromatography coupled to triple quadrupole mass spectrometry in multiple reaction monitoring mode. For the pharmacokinetic study, 1 mg/kg of dasatinib was administered to mice with and without sepsis. The method was linear over the concentration range of 1-98 ng/mL for DAS in mice and human plasma, with r2>0.99 and presented intra- and interday precision within the range of 2.3 - 6.2 and 4.3 - 7.0%, respectively. Further intra- and interday accuracy was within the range of 88.2 - 105.8 and 90.6 - 101.7%, respectively. The mice with sepsis showed AUC0-t = 2076.06 h*ng/mL and Cmax =102.73 ng/mL and mice without sepsis presented AUC0-t = 2128.46 h*ng/mL. Cmax = 164.5 ng/mL. The described analytical method was successfully employed in pharmacokinetic study of DAS in mice.

Hemoglobin metabolism by-products are associated with an inflammatory response in patients with hemorrhagic stroke. / Subprodutos do metabolismo da hemoglobina se associam com resposta inflamatória em pacientes com acidente vascular cerebral hemorrágico.

OBJECTIVE: To evaluate the relationships of brain iron and heme with the inflammatory response of the systemic and central nervous systems and to investigate the role of defensive systems against the toxicity of iron and heme in the central nervous system. METHODS: We assessed a prospective cohort of patients presenting with intracerebral and subarachnoid hemorrhage. We assayed plasma and cerebrospinal fluid samples for the presence of iron, heme, hemopexin, haptoglobin, enolase, S100-ß and cytokines for the first three days following hemorrhagic stroke. We also analyzed the dynamic changes in these components within both fluids and their relationship with early mortality rates. RESULTS: Hemopexin and haptoglobin concentrations were nearly negligible in the brain after intracerebral and subarachnoid hemorrhage. Cerebrospinal fluid iron and heme concentrations correlated with a pro-inflammatory response in the central nervous system, and plasmatic and cerebrospinal fluid inflammatory profiles on the third day after hemorrhagic stroke were related to early mortality rates. Interleukin 4 levels within the cerebrospinal fluid during the first 24 hours after hemorrhagic stroke were found to be higher in survivors than in non-survivors. CONCLUSION: Iron and heme are associated with a pro-inflammatory response in the central nervous system following hemorrhagic stroke, and protections against hemoglobin and heme are lacking within the human brain. Patient inflammatory profiles were associated with a poorer prognosis, and local anti-inflammatory responses appeared to have a protective role.

Subprodutos do metabolismo da hemoglobina se associam com resposta inflamatória em pacientes com acidente vascular cerebral hemorrágico / Hemoglobin metabolism by-products are associated with an inflammatory response in patients with hemorrhagic stroke

RESUMO Objetivo: Avaliar o relacionamento entre os níveis cerebrais de ferro e heme e a resposta inflamatória sistêmica e no sistema nervoso central, assim como o papel dos sistemas de defesa contra a toxicidade do ferro e do heme, no sistema nervoso central. Métodos: Avaliamos uma coorte prospectiva de pacientes com quadro de hemorragia intracraniana e subaracnóidea. Realizamos ensaios em amostras de plasma e líquido cefalorraquidiano quanto à presença de ferro, heme, hemopexina, haptoglobina, enolase, S100-β e citocinas nos primeiros 3 dias após um acidente vascular cerebral hemorrágico. Analisamos também as alterações dinâmicas em todos os componentes de ambos os líquidos e seu relacionamento com as taxas de mortalidade precoce. Resultados: As concentrações de hemopexina e haptoglobina foram quase desprezíveis no cérebro após hemorragia intracraniana e subaracnóidea. As concentrações de ferro e heme no líquido cefalorraquidiano se correlacionaram com resposta pró-inflamatória no sistema nervoso central, e os perfis inflamatórios no líquido cefalorraquidiano no terceiro dia após acidente vascular cerebral hemorrágico se correlacionaram com as taxas de mortalidade precoce. Identificamos que os níveis de interleucina 4 no líquido cefalorraquidiano durante as primeiras 24 horas após acidente vascular cerebral hemorrágico foram mais altos nos sobreviventes do que nos que não sobreviveram. Conclusão: Os níveis de ferro e heme se associaram com resposta pró-inflamatória no sistema nervoso central após acidente vascular cerebral hemorrágico, e o cérebro humano não tem proteção contra hemoglobina e heme. Os perfis inflamatórios dos pacientes se associaram com prognósticos piores, e as respostas inflamatórias locais pareceram ter um papel protetor.

ABSTRACT Objective: To evaluate the relationships of brain iron and heme with the inflammatory response of the systemic and central nervous systems and to investigate the role of defensive systems against the toxicity of iron and heme in the central nervous system. Methods: We assessed a prospective cohort of patients presenting with intracerebral and subarachnoid hemorrhage. We assayed plasma and cerebrospinal fluid samples for the presence of iron, heme, hemopexin, haptoglobin, enolase, S100-β and cytokines for the first three days following hemorrhagic stroke. We also analyzed the dynamic changes in these components within both fluids and their relationship with early mortality rates. Results: Hemopexin and haptoglobin concentrations were nearly negligible in the brain after intracerebral and subarachnoid hemorrhage. Cerebrospinal fluid iron and heme concentrations correlated with a pro-inflammatory response in the central nervous system, and plasmatic and cerebrospinal fluid inflammatory profiles on the third day after hemorrhagic stroke were related to early mortality rates. Interleukin 4 levels within the cerebrospinal fluid during the first 24 hours after hemorrhagic stroke were found to be higher in survivors than in non-survivors. Conclusion: Iron and heme are associated with a pro-inflammatory response in the central nervous system following hemorrhagic stroke, and protections against hemoglobin and heme are lacking within the human brain. Patient inflammatory profiles were associated with a poorer prognosis, and local anti-inflammatory responses appeared to have a protective role.

Hepatic microvascular dysfunction and increased advanced glycation end products are components of non-alcoholic fatty liver disease.

BACKGROUND: This study aimed to investigate the pathophysiology of hepatic microcirculatory dysfunction in non-alcoholic fatty liver disease (NAFLD). METHODS: In Wistar rats, NAFLD model was induced by 20 weeks of high-fat diet (HFD) feeding. Rolling and adhesion of leukocytes and tissue perfusion in hepatic microcirculation were examined using in vivo microscopic and laser speckle contrast imaging (LSCI), respectively. Oxidative stress and inflamatory parameters were analysed by TBARs, catalase enzyme activity, RT-PCR and ELISA. The participation of advanced glycation end-products (AGE) and its receptor RAGE was evaluated by the measurement of gene and protein expression of RAGE by RT-PCR and Western-blot, respectively and by liver and serum quantification of fluorescent AGEs. RESULTS: Wistar rats fed high-fat diet (HFD) showed increase in epididymal and abdominal fat content, systolic arterial blood pressure, fasting blood glucose levels, hepatic triglycerides and cholesterol, and impairment of glucose and insulin metabolisms. Liver histology confirmed the presence of steatosis and ultrasound analysis revealed increased liver size and parenchymal echogenicity in HFD-fed rats. HFD causes significant increases in leukocyte rolling and adhesion on hepatic microcirculation and decrease in liver microvascular blood flow. Liver tissue presented increase in oxidative stress and inflammtion. At 20 weeks, there was a significantly increase in AGE content in the liver and serum of HFD-fed rats and an increase in RAGE gene expression in the liver. CONCLUSION: The increase in liver AGE levels and microcirculatory disturbances could play a role in the pathogenesis of liver injury and are key components of NAFLD.

Oleic acid induces lung injury in mice through activation of the ERK pathway.

Oleic acid (OA) can induce acute lung injury in experimental models. In the present work, we used intratracheal OA injection to show augmented oedema formation, cell migration and activation, lipid mediator, and cytokine productions in the bronchoalveolar fluids of Swiss Webster mice. We also demonstrated that OA-induced pulmonary injury is dependent on ERK1/2 activation, since U0126, an inhibitor of ERK1/2 phosphorylation, blocked neutrophil migration, oedema, and lipid body formation as well as IL-6, but not IL-1ß production. Using a mice strain carrying a null mutation for the TLR4 receptor, we proved that increased inflammatory parameters after OA challenges were not due to the activation of the TLR4 receptor. With OA being a Na/K-ATPase inhibitor, we suggest the possible involvement of this enzyme as an OA target triggering lung inflammation.

Platelet-activating factor acetylhydrolase (PAF-AH) activity in patients with type 1 diabetes mellitus.

OBJECTIVE: To evaluate platelet-activating factor acetylhydrolase (PAF-AH) activity and its relationship with clinical and demographic variables, metabolic control, apolipoprotein A and B levels and the susceptibility of low-density lipoprotein (LDL) to in vitro oxidation in patients with type 1 diabetes mellitus (DM 1). METHODS: Forty two patients with DM 1 (27 females) and 48 control subjects (16 females) matched for gender, age and body mass index (BMI) were evaluated. The following tests were performed: fast plasma glucose (FG) and postprandial plasma glucose (PPG), lipid profile, uric acid (UA), glycosylated hemoglobin (HbA1c), and low-density lipoprotein (LDL) oxidation rate using colorimetric assay. The PAF-AH activity was analyzed using colorimetric assay (Cayman Chemical). RESULTS: The analysis of PAF-AH activity showed a higher enzyme activity in patients with DM 1 than in control subjects (0.0150 +/- 0.0051 vs. 0.0116 +/- 0.0041; p < 0.001). In patients with DM 1, a direct correlation between PAF-AH activity and age and LDL, and an inverse correlation between PAF-AH and HbA1c and high-density lipoprotein (HDL) were found. CONCLUSION: In the sample studied, PAF-AH showed a higher activity in patients with DM 1, a factor that may be related to the higher risk of developing cardiovascular diseases observed in these patients. Further studies are necessary to evaluate the real participation of this enzyme in the risk of development of atherosclerotic diseases in patients with DM 1.

Atividade da enzima acetil-hidrolase do fator ativador de plaquetas (PAF-AH) em pacientes com diabete melito tipo 1 / Platelet-activating factor acetylhydrolase (PAF-AH) activity in patients with type 1 diabetes mellitus

OBJETIVO: Avaliar a atividade da acetil-hidrolase do fator ativador de plaquetas (PAF-AH) e sua relação com variáveis clinicodemográficas, com o controle metabólico, os níveis de apolipoproteínas A e B e a suscetibilidade da lipoproteína de baixa densidade (LDL) à oxidação in vitro em pacientes com DM tipo 1 (DM 1). MÉTODOS: Foram avaliados 42 pacientes com DM1 (27 mulheres) e 48 não-diabéticos (16 mulheres), pareados por sexo, idade e índice de massa corporal (IMC). Os exames realizados foram: glicemia de jejum (GJ) e pós-prandial (GPP), lipidograma, ácido úrico (AU), hemoglobina glicosilada (HbA1c) e coeficiente de oxidação da lipoproteína de baixa densidade (LDL) por espectrofotometria. A análise da atividade da PAF-AH foi realizada por espectrofotometria (Cayman Chemical). RESULTADOS: A análise da atividade da PAF-AH mostrou haver maior atividade enzimática nos pacientes com DM 1 do que nos não-diabéticos (0,0150 ± 0,0051 versus 0,0116 ± 0,0041; p < 0,001). Nos pacientes com DM 1, encontramos correlação direta entre a atividade da PAF-AH e a idade e a LDL, e inversa entre a PAF-AH e a HbA1c e a lipoproteína de alta densidade (HDL). CONCLUSÃO: A PAF-AH, na amostra estudada, apresentou maior atividade nos pacientes com DM 1, fator que pode estar relacionado ao maior risco de desenvolver doenças cardiovasculares apresentados por portadores dessa doença. Ainda são necessários novos estudos para avaliar a real participação dessa enzima no risco de desenvolvimento das doenças ateroscleróticas nos pacientes com DM 1.

OBJECTIVE: To evaluate platelet-activating factor acetylhydrolase (PAF-AH) activity and its relationship with clinical and demographic variables, metabolic control, apolipoprotein A and B levels and the susceptibility of low-density lipoprotein (LDL) to in vitro oxidation in patients with type 1 diabetes mellitus (DM 1). METHODS: Forty two patients with DM 1 (27 females) and 48 control subjects (16 females) matched for gender, age and body mass index (BMI) were evaluated. The following tests were performed: fast plasma glucose (FG) and postprandial plasma glucose (PPG), lipid profile, uric acid (UA), glycosylated hemoglobin (HbA1c), and low-density lipoprotein (LDL) oxidation rate using colorimetric assay. The PAF-AH activity was analyzed using colorimetric assay (Cayman Chemical). RESULTS: The analysis of PAF-AH activity showed a higher enzyme activity in patients with DM 1 than in control subjects (0.0150 ± 0.0051 vs. 0.0116 ± 0.0041; p < 0.001). In patients with DM 1, a direct correlation between PAF-AH activity and age and LDL, and an inverse correlation between PAF-AH and HbA1c and high-density lipoprotein (HDL) were found. CONCLUSION: In the sample studied, PAF-AH showed a higher activity in patients with DM 1, a factor that may be related to the higher risk of developing cardiovascular diseases observed in these patients. Further studies are necessary to evaluate the real participation of this enzyme in the risk of development of atherosclerotic diseases in patients with DM 1.