LC-MS/MS analysis of the plasma concentrations of a cocktail of 5 cytochrome P450 and P-glycoprotein probe substrates and their metabolites using subtherapeutic doses.

Drug transporters and CYP enzymes are important sources of pharmacokinetics (PK) variability in drug responses and can cause various pharmacological and toxicological consequences, leading to either toxicity or an insufficient pharmacological effect. In recent years, the cocktail approach was developed to determine in vivo CYP and transporters activities, but these approaches are somewhat limited. We described the development and validation of three sensitive and specific LC-MS/MS assays for the determination of P-gp and major human CYP isoenzyme activities following oral administration of a drug cocktail of subtherapeutic doses (lower than 10 times) of caffeine (CAF), omeprazole (OME), losartan (LOS), midazolam (MDZ), metoprolol (METO) and fexofenadine (FEX) in healthy volunteers. The three validated methods were selective for all tested analytes. No interference or matrix effect was observed for the mass transition and retention times for all compounds monitored. Additionally, assays were linear over a wide range, and limits of quantification varied between 0.01-5 ng/mL plasma. The coefficients of variation obtained in the precision studies and the inter- and intra-assay accuracies were less than 15%, guaranteeing the reproducibility and repeatability of the results. All substrates and metabolites were stable in plasma during freeze-thaw cycles. Three healthy volunteers were selected based on genotyping for CYP2C9, CYP2C19 and CYP2D6. One volunteer was genotyped as an extensive metabolizer (EM) for all tested CYP isoforms, one volunteer was genotyped as a poor metabolizer (PM) for the CYP2C9 isoform (CYP2C9*3/*3), and one volunteer was genotyped as a PM for the CYP2D6 isoform (CYP2D6*4/*4). The methods allowed the quantification of all analytes over the entire sampling period (12 h) in all studied genotypes. Thus, the analytical methods described here were sufficiently sensitive for use in low-dose pharmacokinetic studies.

Pharmacokinetics and Transplacental Transfer of Fluoxetine Enantiomers and Their Metabolites in Pregnant Women.

Considering that fluoxetine (FLX) is used to treat depressive states during pregnancy and that it is a cytochrome P450 (CYP)2D6 inhibitor, which is involved in the metabolism of both of its enantiomers, this study aims to describe the enantioselective distribution and metabolism of FLX and of its metabolite norfluoxetine (NorFLX) following a single oral dose. Nine healthy pregnant women received 20 mg FLX at 32 weeks of gestation and later at the day of delivery. The apparent clearance of (S)-(+)-FLX (1.45 vs. 0.66 L/hour/kg) and the area under the plasma concentration vs. time curve (AUC) of the (S)-(+)-NorFLX (AUC0-∞ 942.7 vs. 498.6 ng hour/mL) were higher (P < 0.05) than those of the respective (R)-(-) enantiomers, indicating that the (S)-(+)-FLX enantiomer is preferentially metabolized to (S)-(+)-NorFLX. The placental transfer (umbilical vein/maternal vein) of FLX and NorFLX is low (30-40%), with the predominant transfer of (S)-(+)-FLX (44 vs. 33%). The distribution of the enantiomers of FLX and NorFLX to amniotic fluid is low (< 10%).

Effect of type 2 diabetes mellitus on the pharmacokinetics and transplacental transfer of nifedipine in hypertensive pregnant women.

AIMS: Diabetes mellitus can inhibit cytochrome P450 3A4, an enzyme responsible for the metabolism of nifedipine, used for the treatment of hypertension in pregnant women. We aimed to assess the effect of type 2 diabetes mellitus (T2DM) on the pharmacokinetics, placental transfer and distribution of nifedipine in amniotic fluid in hypertensive pregnant women. METHODS: The study was conducted in 12 hypertensive pregnant women [control group (CG)] and 10 hypertensive pregnant women with T2DM taking slow-release nifedipine (20 mg, 12/12 h). On the 34th week of gestation, serial blood samples were collected (0-12 h) after administration of the medication. At delivery, samples of maternal and fetal blood and amniotic fluid were collected for determination of nifedipine distribution in these compartments. RESULTS: The median pharmacokinetic parameters of CG were: peak plasma concentration (Cmax ) 26.41 ng ml-1 , time to reach Cmax (tmax ) 1.79 h, area under the plasma concentration vs. time curve from 0-12 h (AUC0-12 ) 235.99 ng.h ml-1 , half-life (t½) 4.34 h, volume of distribution divided by bioavailability (Vd/F) 560.96 l, and ClT /F 84.77 l h-1 . The parameters for T2DM group were: Cmax 23.52 ng ml-1 , tmax 1.48 h, AUC0-12 202.23 ng.h ml-1 , t½ 5.00 h, Vd/F 609.40 l, and apparent total clearance (ClT /F) 98.94 l h-1 . The ratios of plasma concentrations of nifedipine in the umbilical vein, intervillous space and amniotic fluid to those in the maternal vein for CG and T2DM were 0.53 and 0.44, 0.78 and 0.87, respectively, with an amniotic fluid/maternal plasma ratio of 0.05 for both groups. The ratios of plasma concentrations in the umbilical artery to those in the umbilical vein were 0.82 for CG and 0.88 for T2DM. CONCLUSIONS: There was no influence of T2DM on the pharmacokinetics or placental transfer of nifedipine in hypertensive women with controlled diabetes.

Analysis of nifedipine in human plasma and amniotic fluid by liquid chromatography-tandem mass spectrometry and its application to clinical pharmacokinetics in hypertensive pregnant women.

Nifedipine is a dihydropyridine calcium channel blocker used for the treatment of hypertension in pregnant women. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for analysis of nifedipine in human plasma and amniotic fluid. Separation of nifedipine and nitrendipine (IS) was performed using a LiChroCART(®) RP-Select B column and a mixture of water:acetonitrile:glacial acetic acid (30:70:0.5 v/v) as the mobile phase. Aliquots of 500µL of biological samples were extracted at pH 13 using dichloromethane:n-pentane (3:7 v/v). The validated method was applied to a study of the pharmacokinetics of nifedipine in human plasma and amniotic fluid samples collected up to 12h after administration of the last slow-release nifedipine (20mg/12h) dose to 12 hypertensive pregnant women. The estimated pharmacokinetic parameters of nifedipine showed a mean AUC(0-12) of 250.2ngh/mL, ClT/F of 89.2L/h, Vd/F of 600.0L and t1/2 5.1h. The mean amniotic fluid/plasma concentration ratio was 0.05. The methods proved to be highly sensitive by showing a lower quantification limit of 0.1ng/mL for both matrices. And this study reports for the first time the complete development and validation of the method to quantify nifedipine in amniotic fluid using LC-MS-MS.

Transplacental Distribution of Lidocaine and Its Metabolite in Peridural Anesthesia Administered to Patients With Gestational Diabetes Mellitus.

BACKGROUND: Neonatal effects of drugs administered to mothers before delivery depend on the quantity that crosses the placental barrier, which is determined by the pharmacokinetics of the drug in the mother, fetus, and placenta. Diabetes mellitus can alter the kinetic disposition and the metabolism of drugs. This study investigated the placental transfer of lidocaine and its metabolite monoethylglycinexylidide (MEGX) in pregnant women with gestational diabetes mellitus (GDM) submitted to peridural anesthesia. PATIENTS AND METHODS: A total of 10 normal pregnant women (group 1) and 6 pregnant women with GDM (group 2) were studied, all at term. The patients received 200 mg 2% lidocaine hydrochloride by the peridural locoregional route. Maternal blood samples were collected at the time of delivery and, after placental expulsion, blood samples were collected from the intervillous space, umbilical artery, and vein for determination of lidocaine and MEGX concentrations and analysis of the placental transfer of the drug. RESULTS: The following respective lidocaine ratios between the maternal and the fetal compartments were obtained for groups 1 and 2: umbilical vein/maternal peripheral blood, 0.60 and 0.46; intervillous space/maternal blood, 1.01 and 0.88; umbilical artery/umbilical vein, 0.77 and 0.91; and umbilical vein/intervillous space, 0.53 and 0.51. The following MEGX ratios for groups 1 and 2 were, respectively, fetal/maternal, 0.43 and 0.97; intervillous space/maternal blood, 0.64 and 0.90; umbilical artery/umbilical vein, 1.09 and 0.99; and umbilical vein/intervillous space, 0.55 and 0.78. CONCLUSION: Gestational diabetes mellitus did not affect the transplacental transfer of lidocaine but interfered with the transfer of MEGX, acting as a mechanism facilitating the transport of the metabolite.

[Clinical and laboratory characteristics of pregnant women with preeclampsia versus gestational hypertension]. / Características clínicas e laboratoriais de gestantes com pré-eclâmpsia versus hipertensão gestacional.

PURPOSE: To compare clinical and laboratory characteristics, obstetric and perinatal outcomes of patients with pre-eclampsia versus gestational hypertension. METHODS: A retrospective study was carried out to analyze medical records of patients diagnosed with pre-eclampsia and gestational hypertension whose pregnancies were resolved within a period of 5 years, for a total of 419 cases. We collected clinical and laboratory data, obstetric and perinatal outcomes. Comparisons between groups were performed using the test suitable for the variable analyzed: unpaired t test, Mann-Whitney U test or χ2 test, with the level of significance set at p<0.05. RESULTS: Were evaluated 199 patients in the gestational hypertension group (GH) and 220 patients in the pre-eclampsia group (PE). Mean body mass index was 34.6 kg/m2 in the GH group and 32.7 kg/m2 in the PE group, with a significant difference between groups. The PE group showed higher systolic and diastolic blood pressure and higher rates of abnormal values in the laboratory tests, although the mean values were within the normal range. Cesarean section was performed in 59.1% of cases of PE and in 47.5% of the GH group; and perinatal outcomes in terms of gestational age and birth weight were significantly lower in the PE group. CONCLUSION: Women with gestational hypertension exhibit epidemiological characteristics of patients at risk for chronic diseases. Patients with pre-eclampsia present clinical and laboratory parameters of greater severity, higher rates of cesarean delivery and worse maternal and perinatal outcomes.

Características clínicas e laboratoriais de gestantes com pré-eclâmpsia versus hipertensão gestacional / Clinical and laboratory characteristics of pregnant women with preeclampsia versus gestational hypertension

OBJETIVO: Comparar as características clínicas e laboratoriais, os resultados maternos e perinatais de gestantes com pré-eclâmpsia versus hipertensão gestacional. MÉTODOS: Análise retrospectiva dos prontuários médicos de pacientes com diagnóstico de pré-eclâmpsia e hipertensão gestacional, cujas gestações foram resolvidas em um período de cinco anos. Foram coletadas informações laboratoriais, resultados obstétricos e perinatais. As comparações entre os grupos foram realizadas com o uso do teste adequado para a variável analisada: teste t não pareado, teste U de Mann-Whitney, ou teste do χ2. Consideramos p<0,05 como nível de significância estatística. RESULTADOS: Foram avaliadas 199 pacientes no grupo com hipertensão gestacional (HG) e 220 pacientes no grupo com pré-eclâmpsia (PE). No grupo HG o índice de massa corpórea médio foi 34,6 kg/m2 e no grupo PE, 32,7 kg/m2, com diferença significativa. O grupo PE apresentou valores de pressão arterial sistólica superiores ao grupo HG. Em relação aos exames laboratoriais, a média de valores denotou, de uma forma geral, maior gravidade no grupo PE. Pacientes submetidas à cesárea foram 59,1% dos casos no grupo PE e 47,5% no grupo HG. Em relação aos resultados perinatais, a idade gestacional e o peso ao nascer foram significativamente inferiores no grupo PE. CONCLUSÃO: As mulheres com hipertensão gestacional apresentam características epidemiológicas de pacientes com risco de doenças crônicas. As pacientes com pré-eclâmpsia apresentam parâmetros clínicos e laboratoriais de maior gravidade, taxas superiores de cesárea e piores resultados maternos e perinatais. .

PURPOSE: To compare clinical and laboratory characteristics, obstetric and perinatal outcomes of patients with pre-eclampsia versus gestational hypertension. METHODS: A retrospective study was carried out to analyze medical records of patients diagnosed with pre-eclampsia and gestational hypertension whose pregnancies were resolved within a period of 5 years, for a total of 419 cases. We collected clinical and laboratory data, obstetric and perinatal outcomes. Comparisons between groups were performed using the test suitable for the variable analyzed: unpaired t test, Mann-Whitney U test or χ2 test, with the level of significance set at p<0.05. RESULTS: Were evaluated 199 patients in the gestational hypertension group (GH) and 220 patients in the pre-eclampsia group (PE). Mean body mass index was 34.6 kg/m2 in the GH group and 32.7 kg/m2 in the PE group, with a significant difference between groups. The PE group showed higher systolic and diastolic blood pressure and higher rates of abnormal values in the laboratory tests, although the mean values were within the normal range. Cesarean section was performed in 59.1% of cases of PE and in 47.5% of the GH group; and perinatal outcomes in terms of gestational age and birth weight were significantly lower in the PE group. CONCLUSION: Women with gestational hypertension exhibit epidemiological characteristics of patients at risk for chronic diseases. Patients with pre-eclampsia present clinical and laboratory parameters of greater severity, higher rates of cesarean delivery and worse maternal and perinatal outcomes. .

Analysis of rocuronium in human plasma by liquid chromatography-tandem mass spectrometry with application in clinical pharmacokinetics.

Rocuronium (ROC) is a neuromuscular blocking agent used in surgical procedures which is eliminated primarily by biliary excretion. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for analysis of ROC in human plasma. Separation of ROC and IS (verapamil) was performed using an endcapped C-18 column and a mixture of water:acetonitrile:trifluoracetic acid (50:50:0.1, v/v) as mobile phase. Aliquots of 100 µL of human plasma were extracted at pH 3, using dichloromethane. The lower limit of quantification of 5 ng/mL shows the high sensitivity of this method. Intra- and inter-assay precision (as relative standard deviation) was all ≤14.2% and accuracy (as relative standard error) did not exceed 10.1%. The validated method was successfully applied to quantify ROC concentrations in patients under surgical procedures up to 6h after the administration of the 0.4-0.9 mg/kg ROC. The pharmacokinetic parameter estimations of ROC showed AUC/dose of 563 µg min/mL, total clearance of 2.5 mL/min/kg, volume of distribution at steady state of 190 mL/kg and mean residence time of 83 min.