Advance in methods studying the pharmacokinetics of polyphenols.

Significant advances have been achieved during the past decade concerning the metabolism of polyphenol compounds in vitro, but scarce data has been presented about what really happens in vivo. Many studies on polyphenols to date have focused on the bioactivity of one specific molecule in aglycone form, often at supraphysiological doses, whereas foods contain complex, often poorly characterized mixtures with multiple additive or interfering activities. Whereas most studies up to the middle-late 1990s measured total aglycones in plasma and urine, after chemical or enzymatic deconjugation, or both, several recent works now report the polyphenol conjugate composition of plasma, urine, feces and/or tissues, after the administration of pure polyphenols or polyphenol-rich matrices. HPLC methods with electrochemical, mass spectrometric and fluorescence detection have adequate sensitivity. LC/UV-Vis methods have also been widely reported, but they are much less sensitive. Compared with electro-chemical and fluorescence detection, MS can quantify analytes without chromatographic separation, which leads to high throughput, presenting itself as the best choice to date. Regarding the experimental model to monitor the bioavailability of phenolic compounds, most published studies are based on human and animal models, with the majority using rodents, primates and recently the nematode Caenorhabditis elegans. This review focuses on the fundamentals of pharmacokinetic methods from the last 15 years and how the results are evaluated and validated. The types of analytical methods, animal models and biological matrices were used to better elucidate pharmacokinetics of polyphenols.

Correlation between total nitrite/nitrate concentrations and monoamine oxidase (types A and B) and semicarbazide-sensitive amine oxidase enzymatic activities in human mesenteric arteries from non-diabetic and type 2 diabetic patients

The aim of this study was to determine the correlation between total nitrite/nitrate concentrations (NOx) and the kinetic parameters of monoamine oxidase enzymes (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) in human mesenteric arteries. Arteries were from non-diabetic and type 2 diabetic patients with sigmoid or rectum carcinoma for whom surgery was the first option and who were not exposed to neo-adjuvant therapy. Segments of human inferior mesenteric arteries from non-diabetic (61.1 ± 8.9 years old, 7 males and 5 females, N = 12) and type 2 diabetic patients (65.8 ± 6.2 years old, 8 males and 4 females, N = 12) were used to determine NOx concentrations and the kinetic parameters of MAO-A, MAO-B and SSAO by the Griess reaction and by radiochemical assay, respectively. The NOx concentrations in arteries from diabetic patients did not differ significantly from those of the non-diabetic group (10.28 ± 4.61 vs 10.71 ± 4.32 nmol/mg protein, respectively). In the non-diabetic group, there was a positive correlation between NOx concentrations and MAO-B parameters: Km (r = 0.612, P = 0.034) and Vmax (r = 0.593, P = 0.042), and a negative correlation with the SSAO parameters: Km (r = -0.625, P = 0.029) and Vmax (r = -0.754, P = 0.005). However, in the diabetic group no correlation was found between NOx concentrations and the three kinetic parameters of the enzymes. These results suggest an important function of sympathetic nerves and vascular NOx concentrations in arteries of non-diabetic patients. Thus, these results confirm the importance of a balance between oxidants and antioxidants in the maintenance of vascular homeostasis to prevent oxidative stress.

Correlation between total nitrite/nitrate concentrations and monoamine oxidase (types A and B) and semicarbazide-sensitive amine oxidase enzymatic activities in human mesenteric arteries from non-diabetic and type 2 diabetic patients.

The aim of this study was to determine the correlation between total nitrite/nitrate concentrations (NOx) and the kinetic parameters of monoamine oxidase enzymes (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) in human mesenteric arteries. Arteries were from non-diabetic and type 2 diabetic patients with sigmoid or rectum carcinoma for whom surgery was the first option and who were not exposed to neo-adjuvant therapy. Segments of human inferior mesenteric arteries from non-diabetic (61.1 ± 8.9 years old, 7 males and 5 females, N = 12) and type 2 diabetic patients (65.8 ± 6.2 years old, 8 males and 4 females, N = 12) were used to determine NOx concentrations and the kinetic parameters of MAO-A, MAO-B and SSAO by the Griess reaction and by radiochemical assay, respectively. The NOx concentrations in arteries from diabetic patients did not differ significantly from those of the non-diabetic group (10.28 ± 4.61 vs 10.71 ± 4.32 nmol/mg protein, respectively). In the non-diabetic group, there was a positive correlation between NOx concentrations and MAO-B parameters: Km (r = 0.612, P = 0.034) and Vmax (r = 0.593, P = 0.042), and a negative correlation with the SSAO parameters: Km (r = -0.625, P = 0.029) and Vmax (r = -0.754, P = 0.005). However, in the diabetic group no correlation was found between NOx concentrations and the three kinetic parameters of the enzymes. These results suggest an important function of sympathetic nerves and vascular NOx concentrations in arteries of non-diabetic patients. Thus, these results confirm the importance of a balance between oxidants and antioxidants in the maintenance of vascular homeostasis to prevent oxidative stress.

Monoamine oxidase and semicarbazide-sensitive amine oxidase kinetic analysis in mesenteric arteries of patients with type 2 diabetes.

Monoamine oxidase (MAO, type A and B) and semicarbazide-sensitive amine oxidase (SSAO) metabolize biogenic amines, however, the impact of these enzymes in arteries from patients with type 2 diabetes remains poorly understood. We investigated the kinetic parameters of the enzymes to establish putative correlations with noradrenaline (NA) content and patient age in human mesenteric arteries from type 2 diabetic patients. The kinetic parameters were evaluated by radiochemical assay and NA content by high-performance liquid chromatography (HPLC). The activity of MAO-A and SSAO in type 2 diabetic vascular tissues was significantly lower compared to the activity obtained in non-diabetic tissues. In the correlation between MAO-A (K(m)) and NA content, we found a positive correlation for both the diabetic and non-diabetic group, but no correlation was established for patient age. In both groups, MAO-B (V(max)) showed a negative correlation with age. The results show that MAO-A and SSAO activities and NA content of type 2 diabetic tissues are lower compared to the non-diabetic tissues, while MAO-B activity remained unchanged. These remarks suggest that MAO-A and SSAO may play an important role in vascular tissue as well as in the vascular pathophysiology of type 2 diabetes.

The maximal electroshock seizure (MES) model in the preclinical assessment of potential new antiepileptic drugs.

The choice of appropriate animal models for the initial in vivo testing of potential anticonvulsant compounds is one of the most important steps in the successful search for new antiepileptic drugs. The purpose of this paper is to describe the most important aspects to take into account when performing the maximal electroshock seizure (MES) test in the routine laboratory screening of new antiepileptics: the conventional and threshold MES test experimental procedures, the factors affecting experimental data (laboratory conditions, administration vehicles and drug formulations, time after drug administration, and stimulus duration and site of stimulation) and the assessment of anticonvulsant activity are discussed.

Neuropharmacokinetic characterization of lamotrigine after its acute administration to rats.

The purpose of this study is to characterize the neuropharmacokinetics of lamotrigine following a single intraperitoneal dose. Adult male Wistar rats were given lamotrigine dose of 5, 10, or 20 mg/kg. Blood and brain samples were obtained at predetermined times over 120 h and analyzed by HPLC. The overall characteristics of plasma curves were determined by noncompartmental analysis with WINNONLIN. The kinetic characterization of lamotrigine distribution between plasma and brain was performed by indirect numerical deconvolution with MULTI(FILT). A linear disposition kinetics was observed within 5-20 mg/kg. The lamotrigine concentrations in brain homogenate were approx. twofold higher than in plasma. The following pharmacokinetic parameters were obtained for lamotrigine 5, 10, and 20 mg/kg, respectively: clearance of distribution from plasma to brain normalized with the volume of the brain, CL/V(h(-1)) = 4.64, 2.47, 2.40; brain-to-plasma partition coefficient, P = 0.40, 0.37, 0.34; first-order transfer rate constant from the brain to the plasma, K(h(-1)) = 11.68, 6.68, 5.96; single-pass mean transit time in the brain, MTT(h) = 0.086, 0.150, 0.168. These results indicate that lamotrigine plasma levels may be good indicators of lamotrigine levels in the brain and that higher response intensities could be expected with higher doses of lamotrigine, since efficacious concentrations are maintained for a longer period.

Lamotrigine pharmacokinetic/pharmacodynamic modelling in rats.

The aim of this study was to perform a pharmacokinetic/pharmacodynamic (PK/PD) modelling of lamotrigine following its acute administration to rats. Adult male Wistar rats were given 10 mg/kg of lamotrigine intraperitoneally. Plasma and brain samples were obtained at predetermined times over 120 h post-dose and analysed by liquid chromatography. The anticonvulsant profile against maximal electroshock seizure stimulation was determined over 48 h after dosing. As a linear relationship between lamotrigine plasma and brain profiles was observed, only the plasma data set was used to establish the PK/PD relationship. To fit the effect-time course of lamotrigine, the PK/PD simultaneous fitting link model was used: the pharmacokinetic parameters and dosing information were used in the one-compartment first-order model to predict concentrations, which were then used to model the pharmacodynamic data with the sigmoid Emax model, in order to estimate all the parameters simultaneously. The following parameters were obtained: Vd = 2.00 L/kg, k(abs) = 8.50 h(-1), k(el) = 0.025 h(-1), k(e0) = 3.75 h(-1), Emax = 100.0% (fixed), EC50 = 3.44 mg/L and gamma = 8.64. From these results, it can be stated that lamotrigine is extensively distributed through the body, its plasma elimination half-life is around 28 h and a lamotrigine plasma concentration of 3.44 mg/L is enough to protect 50% of the animals. When compared with humans, the plasma concentrations achieved with this dose were within the therapeutic concentration range that had been proposed for epileptic patients. With the present PK/PD modelling it was possible to fit simultaneously the time-courses of the plasma levels and the anticonvulsant effect of lamotrigine, providing information not only about the pharmacokinetics of lamotrigine in the rat but also about its anticonvulsant response over time. As this approach can be easily applied to other drugs, it becomes a useful tool for an explanatory comparison between lamotrigine and other antiepileptic drugs.

Lamotrigine kidney distribution in male rats following a single intraperitoneal dose.

As it has been previously shown that lamotrigine (LTG) accumulates in the kidney of male rats, the purpose of the present investigation was to characterize the kidney profiles of LTG and its kidney distribution pattern in male rats, in order to confirm if a preferential distribution exists and to analyse if it does or does not affect the LTG systemic pharmacokinetics. Adult male Wistar rats were intraperitoneally injected with 5, 10 and 20 mg/kg of LTG. The concentration-time profiles of LTG in plasma and whole kidney were determined over 120 h postdose. The distribution of LTG in the rat kidney was investigated in another group of rats by measuring LTG levels in the renal cortex and medulla. The LTG plasma concentration-time profiles revealed a linear relationship with dose. However, a slight increase in the LTG elimination half-life with dose was observed. In contrast, a nonlinear relationship was established between LTG kidney levels and the dose administered. Consequently, nonparallel patterns were observed between LTG plasma and kidney profiles. The LTG kidney distribution pattern revealed an accumulation of LTG in the renal cortex. The present study demonstrated that LTG distributes preferentially to the kidneys of the male rat in a dose-dependent manner and suggests that such distribution may slightly affect the systemic kinetics of the drug.

Influence of administration vehicles and drug formulations on the pharmacokinetic profile of lamotrigine in rats.

Given that administration vehicles and drug formulations can affect drug bioavailability, their influence on the pharmacokinetic profile of lamotrigine (LTG), a new-generation anti-epileptic drug, was studied in rats. Three different formulations administered intraperitoneally at a dose of 10 mg/kg were used: (1) LTG suspended in a 0.25% methylcelulose solution, (2) LTG dissolved in a 50% propylene glycol solution, and (3) LTG isethionate dissolved in distilled water. Plasma and brain homogenate levels were determined in order to evaluate vehicle-dependent drug absorption. The results demonstrated rapid absorption of LTG when it was administered as an aqueous solution, in contrast to a slower and more erratic absorption after the injection of either the lipophilic solution or the suspension. A plasma peak was achieved 15 min post-dose with the aqueous solution, with a brain peak being achieved 15 min later, while with the other formulations both plasma and brain homogenate peaks were reached 2 h after LTG administration. This study suggests that LTG isethionate dissolved in distilled water is the most suitable formulation for successful LTG pharmacokinetic studies in rats.

Prejunctional alpha2A-autoreceptors in the human gastric and ileocolic arteries.

This study was undertaken to determine the subtype of prejunctional alpha2-autoreceptors in human blood vessels. Segments of gastric and ileocolic arteries were incubated with [3H]noradrenaline and subsequently perifused with modified Krebs-Henseleit solution containing cocaine (12 microM). Five periods of electrical stimulation (S1-S5) were applied (1 Hz, 1 ms, 50 V for 1 min). Concentration-response curves for the facilitatory effect of eight alpha-adrenoceptor antagonists [rauwolscine, 2-[2-(2-methoxy-1,4-benzodioxanyl)] imidazoline (RX821002), yohimbine, phentolamine, idazoxan, 2-(2',6'-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxan (WB4101), spiroxatrine and prazosin] were determined. All antagonists enhanced the stimulation-evoked overflow of tritium, indicating the existence of alpha2-autoreceptors. The EC30% values of the antagonists (concentrations that increased the evoked overflow of tritium by 30%) were taken as a measure of affinity to the autoreceptors. Correlations between the pEC30% values obtained in the present study and the pKi values of the same antagonists at cloned human alpha2A-, alpha2B-, alpha2C-adrenoceptors expressed in Chinese hamster lung cells and at alpha2D-adrenoceptors in the rat submaxillary gland or the bovine pineal gland showed that the alpha2-autoreceptors in the human gastric and ileocolic arteries resemble most closely the alpha2A-subtype.

The role of MAO-A and MAO-B in the metabolic degradation of noradrenaline in human arteries.

1. Segments of human cystic, gastric and ileocolic arteries were obtained from patients undergoing surgery. 2. Segments of arterial tissues, the noradrenaline content of which ranged between 0.27 and 0.52 microg g(-1), were incubated with 0.1 micromol l(-1) [3H]-noradrenaline for 30 min and the accumulation of the amine as well as the formation of metabolites was measured. 3. In all the arteries, oxidative deamination predominated over O-methylation; the mean values of the deaminated and O-methylated metabolites formed for the three arteries were 247.6 and 82.4 pmol g(-1) tissue, respectively. Dihydroxymandelic acid (DOMA) was the most abundant metabolite. 4. Both clorgyline (a selective MAO-A inhibitor) and selegiline (a selective MAO-B inhibitor) reduced the formation of dihydroxyphenylglycol (DOPEG), DOMA and O-methylated-deaminated metabolites (OMDA), and increased that of normetanephrine (NMN). However, clorgyline depressed the formation of DOPEG more than that of DOMA, while selegiline depressed the formation of DOMA more than that of DOPEG. 5. In conclusion, three major differences distinguish the metabolism of noradrenaline by human arteries from that observed in other species: (1) the large predominance of deamination over O-methylation; (2) the extremely high formation of DOMA; and (3) the relative lack of selectivity of clorgyline and selegiline for MAO-A and B, respectively. Since the arterial vessels used were collected from patients older than 60 years, the morphological changes depending on age may explain the increase in DOMA formation.

Monoamine oxidase activities in human cystic and colonic arteries--influence of age.

The deamination of 5-hydroxytryptamine, phenylethylamine and benzylamine by monoamine oxidases (MAO-A and B) and semicarbazide sensitive amine oxidase (SSAO) respectively has been studied in homogenates of human cystic and colonic arteries by radiochemical assays. In cystic artery the deamination is mainly carried out by SSAO with a lower participation of MAO-B. The kinetic parameters were: to MAO-B the Vmax = 15.11 +/- 0.51 nmol/mg protein.h and the Km = 78.51 +/- 5.16 microM (+/- SE) and to SSAO the Vmax = 211.70 +/- 8.75 nmol/mg protein.h and the Km = 211.51 +/- 23.27 microM (+/- SE). We could not measure MAO-A activity in our experimental conditions and also the levels of catecholamines are very low and the histological studies show a poor innervation in these tissues. In colonic artery the kinetic parameters were: to MAO-B the Vmax = 5.09 +/- 0.31 nmol/mg protein.h and the Km = 29.12 +/- 4.55 microM (+/- SE) and to SSAO the Vmax = 273.67 +/- 8.35 nmol/mg protein.h and the Km = 197.89 +/- 21.81 microM (+/- SE). In this artery we could find MAO-A in five among the nine samples studied and the kinetic parameters were: the Vmax = 14.48 +/- 0.82 nmol/mg protein.h and the Km = 136.40 +/- 25.46 microM. As we have performed the experiments with human vessels from donors with different age we could not find any relationship between the activity or affinity, in MAO-B and SSAO, with age. Nevertheless, the results show in cystic artery an increase in the affinity of MAO-B with age when we consider the female group which suggests a possible role of the hormonal condition in this behaviour.

Influence of experimental hypercholesterolemia on the monoamine oxidase activity in rabbit arteries.

Using a model of experimental atherogenesis in New Zealand rabbits we found a lower noradrenaline level in the aorta than in the femoral artery. The activity of monoamine oxidase was decreased in the femoral artery and increased in the aorta of the cholesterol-fed animals when compared with controls.