Evandro José Cesarino

Enantioselective analysis of metoprolol in plasma using high-performance liquid chromatographic direct and indirect separations: applications in pharmacokinetics

Direct enantioselective separation on chiral stationary phases and indirect separation based on the formation of diastereomeric derivatives were developed and compared for the HPLC analysis of R(+) and S(-)-metoprolol in human plasma. Plasma samples prepared using solid-phase extraction columns or liquid-liquid extraction were directly analyzed on a Chiralpack AD or on a Chiralcel OD-H columns, respectively. S-(-)-menthyl choroformate was also used to yield diastereomeric derivatives resolved on a RP-8 column. The methods were employed to determine plasma concentrations of metoprolol enantiomers in a pharmacokinetic study of single dose administration of racemic metoprolol to a healthy Caucasian volunteer phenotyped as extensive metabolizer of debrisoquine. The correlation coefficients among enantioselective metoprolol plasma concentrations (5-223 ng/ml) obtained by the three methods were equal or higher than 0.99. The direct method that employed the chiral column Chiralpak AD may be considered the most sensitive, although the three methods demonstrated interchangeable use in the pharmacokinetic investigation.

Physical exercise improves cardiac autonomic modulation in hypertensive patients independently of angiotensin-converting enzyme inhibitor treatment

We investigated the influence of angiotensin-converting enzyme inhibitor (ACEi) treatment and physical exercise on arterial pressure (AP) and heart rate variability (HRV) in volunteer patients with hypertension. A total of 54 sedentary volunteers were divided into three groups: normotensive (NT Group), hypertensive (HT Group) and HT volunteers treated with ACEi (ACEi Group). All volunteers underwent an aerobic physical-training protocol for 15 weeks. HRV was investigated using a spectral analysis of a time series of R–R interval (RRi) that was obtained in a supine position and during a tilt test. Physical training promoted a significant reduction in the mean arterial pressure of the HT group (113±3 vs. 106±1 mm Hg) and the ACEi group (104±2 vs. 98±2 mm Hg). Spectral analysis of RRi in the supine position before physical training demonstrated that the NT and ACEi groups had similar values at low frequency (LF; 0.04–0.15 Hz) and high frequency (HF; 0.15–0.5 Hz) oscillations. The HT group had an increase in LF oscillations in absolute and normalized units and a decrease in HF oscillations in normalized units compared with the other groups. The HT group had the lowest responses to the tilt test during LF oscillations in normalized units. Physical training improved the autonomic modulation of the heart rate in the supine position only in the HT group. Physical training promoted a similar increase in autonomic modulation responses in the tilt test in all groups. Our findings show that aerobic physical training improves cardiac autonomic modulation in HT volunteers independently of ACEi treatment.

Enantioselectivity in the steady-state pharmacokinetics of metoprolol in hypertensive patients

In the present study we investigated the enantioselectivity in the pharmacokinetics of metoprolol administered in a multiple-dose regimen as the racemate. The study was conducted on 10 patients of both sexes with mild to severe essential hypertension, aged 28 to 76 years, with normal hepatic and renal function and phenotyped as extensive metabolizers of debrisoquine (urine debrisoquine to 4-hydroxydebrisoquine ratios of 0.28 to 6.56). The patients were treated with racemic metoprolol (two 100 mg tablets every 24 h) for 7 days. Serial blood samples were collected at times zero, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 20, 22, and 24 h and urine at each 6 h period until 24 h after metoprolol administration. The plasma concentrations of the (-)-(S)- and (+)-(R)-metoprolol enantiomers were determined by HPLC using a chiral stationary phase (Chiralpak AD, 4.6 x 250 mm) and fluorescence detection. The enantiomeric ratios differing from one were evaluated by the paired t test and the results are reported as means (95% CI). No differences were observed between metoprolol enantiomers in half-lives and absorption, distribution and elimination rate constants. However, the following differences (p < 0.05) were observed between the (-)-(S) and (+)-(R) enantiomers: maximum plasma concentration, C(max), 179.99 (123. 33-236.64) versus 151.30 (95.04-207.57) ng/mL; area under the plasma concentration versus time curve, AUC(0-24)(SS), 929.85 (458.02-1401. 70) versus 782.11 (329.80-1234.40) ng h/mL; apparent total clearance, Cl(T)/f, 1.70 (0.79-2.61) versus 2.21 (1.06-3.36) L/h/kg, apparent distribution volume, Vd/f, 10.51 (6.35-14.68) versus 13.80 (6.93-20. 68) L/kg, and renal clearance, Cl(R), 0.06 (0.05-0.08) versus 0.07 (0.05-0.09) L/kg. The enantiomeric ratios AUC((-)-(S))/AUC((+)-(R)) ranged from 1.14 to 1.44, with a mean of 1.29. The data obtained demonstrate enantioselectivity in the kinetic disposition of metoprolol, with plasma accumulation of the pharmacologically more active (-)-(S)-metoprolol enantiomer in hypertensive patients phenotyped as extensive metabolizers of debrisoquine.

High-performance liquid chromatographic determination of mexiletine enantiomers in plasma using direct and indirect enantioselective separations.

Two methods were developed for the determination of mexiletine enantiomers in plasma samples suitable for studies on the stereoselective disposition of this drug. Both methods used fluorescence detection to improve sensitivity and selectivity. The direct enantioselective separation was based on the chiral resolution of mexiletine-2-naphthamide derivatives on a Chiralcel OJ column. The calibration curves were linear over the concentration range 50-500 ng/ml for each enantiomer; therefore the method can be used only for therapeutic monitoring, drug interaction and multiple dose pharmacokinetic studies. The indirect method was based on the formation of diastereomers using o-phthaldialdehyde and N-acetyl-L-cysteine reagents. The diastereomers were resolved on a reversed-phase RP-18 column. The method proved to be suitable for single or multiple dose pharmacokinetic studies based on the low quantification limit (1 ng/ml) and the broader linear range (1-1000 ng/ml) obtained.

Prevention of hypertension in patients with pre-hypertension: protocol for the PREVER-prevention trial

BackgroundBlood pressure (BP) within pre-hypertensive levels confers higher cardiovascular risk and is an intermediate stage for full hypertension, which develops in an annual rate of 7 out of 100 individuals with 40 to 50 years of age. Non-drug interventions to prevent hypertension have had low effectiveness. In individuals with previous cardiovascular disease or diabetes, the use of BP-lowering agents reduces the incidence of major cardiovascular events. In the absence of higher baseline risk, the use of BP agents reduces the incidence of hypertension. The PREVER-prevention trial aims to investigate the efficacy, safety and feasibility of a population-based intervention to prevent the incidence of hypertension and the development of target-organ damage.MethodsThis is a randomized, double-blind, placebo-controlled clinical trial, with participants aged 30 to 70 years, with pre-hypertension. The trial arms will be chlorthalidone 12.5 mg plus amiloride 2.5 mg or identical placebo. The primary outcomes will be the incidence of hypertension, adverse events and development or worsening of microalbuminuria and of left ventricular hypertrophy in the EKG. The secondary outcomes will be fatal or non-fatal cardiovascular events: myocardial infarction, stroke, heart failure, evidence of new sub-clinical atherosclerosis, and sudden death. The study will last 18 months. The sample size was calculated on the basis of an incidence of hypertension of 14% in the control group, a size effect of 40%, power of 85% and P alpha of 5%, resulting in 625 participants per group. The project was approved by the Ethics committee of each participating institution.DiscussionThe early use of blood pressure-lowering drugs, particularly diuretics, which act on the main mechanism of blood pressure rising with age, may prevent cardiovascular events and the incidence of hypertension in individuals with hypertension. If this intervention shows to be effective and safe in a population-based perspective, it could be the basis for an innovative public health program to prevent hypertension in Brazil.Trial RegistrationClinical Trials NCT00970931.

Simultaneous determination of propafenone and 5-hydroxypropafenone enantiomers in plasma by chromatography on an amylose derived chiral stationary phase

An enantioselective liquid chromatography method was developed for the simultaneous determination of propafenone (PPF) and 5-hydroxypropafenone (PPF-5OH) enantiomers in plasma. After liquid liquid extraction with dichloromethane, the enantiomers were resolved on a Chiralpak AD column using hexane-ethanol (88:12, v/v) plus 0.1% diethylamine as the mobile phase and monitored at 315 nm. Under these conditions the enantiomeric fractions of the drug and of its metabolite were analysed within 20 min. The extraction procedure resulted in absolute recoveries of 62.9 and 61.3% for (R)- and (S)-PPF, respectively, and of 57.6 and 56.5% for (R)- and (S)-PPF-5OH, respectively. This procedure was efficient in removing endogenous interferents as well the interference of an other PPF metabolite, N-despropylpropafenone (PPF-NOR). The calibration curves were linear over the concentration range 25-1250 ng/ml. Low values of the coefficients of variation were demonstrated for both within-day and between day assays. The method described in this paper allows the determination of PPF and PPF-5OH enantiomers at plasma levels as low as 25 ng/ml and can be used in clinical pharmacokinetic studies.

Effectiveness of Chlorthalidone Plus Amiloride for the Prevention of Hypertension: The PREVER‐Prevention Randomized Clinical Trial

Background Prehypertension is associated with higher cardiovascular risk, target organ damage, and incidence of hypertension. The Prevention of Hypertension in Patients with PreHypertension (PREVER‐Prevention) trial aimed to evaluate the efficacy and safety of a low‐dose diuretic for the prevention of hypertension and end‐organ damage. Methods and Results This randomized, parallel, double‐blind, placebo‐controlled trial was conducted in 21 Brazilian academic medical centers. Participants with prehypertension who were aged 30 to 70 years and who did not reach optimal blood pressure after 3 months of lifestyle intervention were randomized to a chlorthalidone/amiloride combination pill or placebo and were evaluated every 3 months during 18 months of treatment. The primary outcome was incidence of hypertension. Development or worsening of microalbuminuria, new‐onset diabetes mellitus, and reduction of left ventricular mass were secondary outcomes. Participant characteristics were evenly distributed by trial arms. The incidence of hypertension was significantly lower in 372 study participants allocated to diuretics compared with 358 allocated to placebo (hazard ratio 0.56, 95% CI 0.38–0.82), resulting in a cumulative incidence of 11.7% in the diuretic arm versus 19.5% in the placebo arm (P=0.004). Adverse events; levels of blood glucose, glycosylated hemoglobin, creatinine, and microalbuminuria; and incidence of diabetes mellitus were no different between the 2 arms. Left ventricular mass assessed through Sokolow‐Lyon voltage and voltage‐duration product decreased to a greater extent in participants allocated to diuretic therapy compared with placebo (P=0.02). Conclusions A combination of low‐dose chlorthalidone and amiloride effectively reduces the risk of incident hypertension and beneficially affects left ventricular mass in patients with prehypertension. Clinical Trial Registration URL: http://www.ClinicalTrials.gov, www.ensaiosclinicos.gov. Unique identifiers: NCT00970931, RBR‐74rr6s.

Enantioselective analysis of mirtazapine, demethylmirtazapine and 8-hydroxy mirtazapine in human urine after solid-phase microextraction.

A selective and reproducible off-line solid-phase microextraction procedure was developed for the simultaneous enantioselective determination of mirtazapine (MRT), demethylmirtazapine and 8-hydroxymirtazapine in human urine. CE was used for optimization of the extraction procedure whereas LC-MS was used for method validation and application. The influence of important factors in the solid-phase microextraction efficiency is discussed, such as the fiber coatings, extraction time, pH, ionic strength, temperature and desorption time. Before extraction, human urine samples were submitted to enzymatic hydrolysis at 37 degrees C for 16 h. Then, the enzyme was precipitated with trichloroacetic acid and the pH was adjusted to 8 with 1 mol/L pH 11 phosphate buffer solution. In the extraction, the analytes were transferred from the aqueous solution to the polydimethylsiloxane-divinylbenzene fiber coating and then desorbed in methanol. The mean recoveries were 5.4, 1.7 and 1.0% for MRT, demethylmirtazapine and 8-hydroxymirtazapine enantiomers, respectively. The method was linear over the concentration range of 62-1250 ng/mL. The within-day and between-day assay precision and accuracy were lower than 15%. The method was successfully employed in a preliminary cumulative urinary excretion study after administration of racemic MRT to a healthy volunteer.

Physical exercise attenuates the cardiac autonomic deficit induced by nitric oxide synthesis blockade.

BACKGROUND: The nitric oxide (NO) synthesis blockade is characterized by an increase in the cardiac sympathetic activity and the physical training promotes the decrease in the sympathetic activity. OBJECTIVE: We investigated the effect of the NO synthesis blockade on the autonomic cardiovascular control in rats submitted to aerobic exercises during a 10-week period. METHODS: Male Wistar rats were divided in four groups: control rats, treated with chow food and water ad libitum for 10 weeks (CR); control rats, treated with NG-nitro-L-arginine methyl ester (L-NAME) during the last week (CRL); rats trained during 10 weeks on an electrical treadmill (TR); rats trained for 10 weeks and treated with L-NAME during the last week (TRL). The autonomic cardiovascular control was investigated in all groups with the use of a double blockade with methylatropine and propranolol and analysis of variability. RESULTS: The CRL and TRL groups presented hypertension. The CRL group presented tachycardia and predominance of the sympathetic tonus in heat rate (HR) measurement after the pharmacological autonomic blockade. The TR group presented bradycardia and lower intrinsic HR when compared to the others. The evaluation of the HR variability showed lower absolute and normalized values in the low frequency (LF) band in the CRL group. On the other hand, the TRL presented an increase in the LF band in absolute values. The analysis of variability of the systemic arterial pressure (SAP) showed that the CRL and TRL groups presented higher values in the LF band. CONCLUSION: The previous physical exercise prevented the deficit in the autonomic cardiac control induced by the treatment with L-NAME, but did not prevent the increase in the SAP variability.BACKGROUND The nitric oxide (NO) synthesis blockade is characterized by an increase in the cardiac sympathetic activity and the physical training promotes the decrease in the sympathetic activity. OBJECTIVE We investigated the effect of the NO synthesis blockade on the autonomic cardiovascular control in rats submitted to aerobic exercises during a 10-week period. METHODS Male Wistar rats were divided in four groups: control rats, treated with chow food and water ad libitum for 10 weeks (CR); control rats, treated with N G-nitro-L-arginine methyl ester (L-NAME) during the last week (CRL); rats trained during 10 weeks on an electrical treadmill (TR); rats trained for 10 weeks and treated with L-NAME during the last week (TRL). The autonomic cardiovascular control was investigated in all groups with the use of a double blockade with methylatropine and propranolol and analysis of variability. RESULTS The CRL and TRL groups presented hypertension. The CRL group presented tachycardia and predominance of the sympathetic tonus in heat rate (HR) measurement after the pharmacological autonomic blockade. The TR group presented bradycardia and lower intrinsic HR when compared to the others. The evaluation of the HR variability showed lower absolute and normalized values in the low frequency (LF) band in the CRL group. On the other hand, the TRL presented an increase in the LF band in absolute values. The analysis of variability of the systemic arterial pressure (SAP) showed that the CRL and TRL groups presented higher values in the LF band. CONCLUSION The previous physical exercise prevented the deficit in the autonomic cardiac control induced by the treatment with L-NAME, but did not prevent the increase in the SAP variability.

Phagocytosis, Oxidative Burst, and Produced Reactive Species are Affected by Iron Deficiency Anemia and Anemia of Chronic Diseases in Elderly

Iron and oxidative stress have a regulatory interplay. During the oxidative burst, phagocytic cells produce free radicals such as hypochlorous acid (HOCl). Nevertheless, scarce studies evaluated the effect of either iron deficiency anemia (IDA) or anemia of chronic disease (ACD) on phagocyte function in the elderly. The aim of the present study was to determine the oxidative burst, phagocytosis, and nitric oxide (•NO) and HOCl, reactive species produced by monocytes and neutrophils in elderly with ACD or IDA. Soluble transferrin receptor, serum ferritin, and soluble transferrin receptor/log ferritin (TfR-F) index determined the iron status. The study was constituted of 39 patients aged over 60 (28 women and 11 men) recruited from the Brazilian Public Health System. Oxidative burst fluorescence intensity per neutrophil in IDA group and HOCl generation in both ACD and IDA groups were found to be lower (p < 0.05). The percentages of neutrophils and monocytes expressing phagocytosis in ACD group were found to be higher (p < 0.05). There was an overproduction of •NO from monocytes, whereas the fundamental generation of HOCl appeared to be lower. Phagocytosis, oxidative burst, and •NO and HOCl production are involved in iron metabolism regulation in elderly patients with ACD and IDA.