Jesús Novalbos

Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells

Abstract Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, Vh=–80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell IBa could be fractionated into various subcomponents. Thus, IBa had a 25% fraction sensitive to 1 µM nifedipine (L-type channels), 21% sensitive to 1 µM ω-conotoxin GVIA (N-type channels), and 60% sensitive to 2 µM ω-agatoxin IVA (P/Q-type channels). The activation of IBa was considerably slowed down, and the peak current was inhibited upon superfusion with 10 µM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of IBa was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5–50%. The results also provide strong support for the hypothesis that Ca2+ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates.

Effects of CYP2D6 Genotype on the Pharmacokinetics, Pharmacodynamics, and Safety of Risperidone in Healthy Volunteers

The objective of this study was to analyze the relationship between CYP2D6 genotype and pharmacokinetics and pharmacodynamics of risperidone. Seventy-one healthy volunteers (36 women and 35 men) received a 1-mg single oral dose of risperidone. Six major CYP2D6 polymorphisms (CYP2D6*3, *4, *5, *6, *7, and *9) and the duplication were detected. Subjects were classified into 4 phenotypic groups: 6 ultrarapid (UMs), 34 extensive (EMs), 25 intermediate (IMs), and 6 poor metabolizers (PMs). There was a clear relationship between the number of active alleles and the pharmacokinetic parameters for risperidone and 9-hydroxyrisperidone, but there were no differences for total active moiety. Area under the curve and half-life of risperidone were significantly higher in PMs and IMs compared with EMs and UMs, which showed higher area under the curve of 9-hydroxyrisperidone. Risperidone produced a small decrease in blood pressure, a mild increase in QTc and a quick increase in prolactin, without significant differences between groups. Surprisingly, the incidence of adverse reactions was lower in PMs (50%) than in other subjects (78%). In conclusion, metabolism of risperidone depends on the number of active CYP2D6 alleles. So, PM subjects show higher concentrations of risperidone and very low concentrations of 9-hydroxyrisperidone. On the contrary, EM and UM subjects show low concentrations of risperidone and high concentrations of 9-hydroxyrisperidone. However, no major pharmacodynamic differences are observed between CYP2D6 genotypes, presumably because of the similar pharmacological activity of parent drug and metabolite.

L-type calcium channels in enterochromaffin cells from guinea pig and human duodenal crypts: An in situ study

BACKGROUND & AIMS This study has investigated stimulus-secretion coupling of enterochromaffin cells by studying the cellular location and function of voltage-gated Ca(2+) channels within small intestinal crypts. METHODS Digital fluorescence imaging and electrochemical detection were used to measure intracellular Ca(2+) responses and serotonin (5-hydroxytryptamine [5-HT]) secretion in intact crypts isolated from guinea pig and human duodenum. RESULTS In fluo-3-loaded crypts, electrical depolarization with high K(+) solution increased cytosolic free [Ca(2+)] only in single cells subsequently identified by immunocytochemistry as enterochromaffin cells. In guinea pig enterochromaffin cells, the L-type Ca(2+) channel agonist FPL 64176 (3 micromol/L) did not change resting intracellular [Ca(2+)] but potentiated the depolarization-evoked increase in [Ca(2+)] (298 +/- 72 nmol/L) by 19 +/- 3-fold. In the majority of human enterochromaffin cells, FPL 64176 alone increased resting [Ca(2+)] by 423 +/- 171 nmol/L. Secretion studies in guinea pig crypts showed that high K(+) and FPL 64176 caused a 12-fold increase in 5-HT release. Noradrenaline caused increases in both enterochromaffin cell [Ca(2+)] and 5-HT release. CONCLUSIONS Using this approach, we have found that in duodenal crypts, enterochromaffin cells, but not other epithelial cells, contain L-type voltage-gated Ca(2+) channels involved in regulating 5-HT secretion. These data have implications for the pharmacological control of intestinal disorders involving enterochromaffin cell dysfunction.

Influence of CYP2C8 and CYP2C9 polymorphisms on pharmacokinetic and pharmacodynamic parameters of racemic and enantiomeric forms of ibuprofen in healthy volunteers.

OBJECTIVES (i) To define the incidence of alleles CYP2C8*1 to *5 in a Spanish population; (ii) to test the impact of such alleles, and those of CYP2C9, on the metabolism of racemic ibuprofen, R-ibuprofen and S-ibuprofen; and (iii) to discern whether those metabolic alterations have safety implications. METHODS Data from three phase I clinical trials with 69 healthy volunteers taken ibuprofen were analyzed. Genotyping were performed by PCR. Pharmacokinetic parameters were determined in studies 1 and 2 by non-compartmental analysis. Levels of COX-1, COX-2, eNOS and iNOS were determined by Western Blots in gastric biopsies of study 3. RESULTS Allelic frequencies were 0.80, 0.02, 0.11, 0.07 and 0 for CYP2C8*1, *2, *3, *4 and *5. CYP2C9*3 allele had a decreased racemic ibuprofen metabolism, leading to a 30% augmentation of AUC(0-infinity) and a 30% reduction of clearance compared to CYP2C9*1 (p < 0.05). CYP2C8*3 had a 20% augmentation of clearance compared to CYP2C8*1 (p < 0.05) of R-ibuprofen. CYP2C9*3 had a 45% reduction of clearance, as well as a 87% and 47% augmentation of AUC(0-infinity) and t(1/2) with respect to CYP2C9*1 of S-ibuprofen and a 30% reduction of clearance of R-ibuprofen. A decreased iNOS expression was found in CYP2C8*3 compared to wild type (p < 0.05). Adverse events in CYP2C8*3 (20%) and *4 (20%) were fewer than in CYP2C8*1 (77%). CONCLUSIONS This study suggest an impaired metabolism of racemic, S-ibuprofen and R-ibuprofen in CYP2C9*3; an increased R-ibuprofen metabolism in CYP2C8*3; and fewer adverse events in CYP2C8*3 volunteers; that correlates with a decreased expression of iNOS.

DRD2 Taq1A polymorphism modulates prolactin secretion induced by atypical antipsychotics in healthy volunteers.

Hyperprolactinemia mediated by antagonism of dopaminergic neurotransmission in the pituitary gland is a common adverse effect of antipsychotics. Recent studies have suggested that polymorphisms of dopamine receptors can affect the therapeutic response to antipsychotics. Thus, our aim was to evaluate whether 2 such polymorphisms (DRD2 Taq1A and DRD3 Ser9Gly) modulate prolactin release in healthy volunteers (n = 119) receiving a single dose of quetiapine (25 mg, n = 26), olanzapine (5 mg, n = 57), or risperidone (1 mg, n = 36). The increases in maximum concentration and in area under the curve were calculated from plasma prolactin levels after subtraction of pretreatment levels. Multiple regression analyses revealed that prolactin increases in maximum concentration and in area under the curve depended on drug (quetiapine < olanzapine < risperidone; P < 0.001), sex (women > men; P < 0.001), and Taq1A polymorphism (A1+ > A2/A2; P < 0.05). Analysis of the individual drugs revealed that prolactin secretion was modulated by sex and Taq1A polymorphism in olanzapine and risperidone (P < 0.05); however, these factors were not linked to prolactin secretion in quetiapine.

Gender differences in angiotensin-converting enzyme (ACE) activity and inhibition by enalaprilat in healthy volunteers.

Abstract: This bioequivalence study was supported by Laboratorios Vita S.A (Barcelona). To study the existence of differences between sexes in the pharmacokinetic and pharmacodynamic of enalapril. A bioequivalence phase 1 clinical trial to compare two formulations of enalapril was carried out in twenty-four healthy volunteers (12 men and 12 women). Enalaprilat concentrations, plasma activity of ACE, and systolic and diastolic arterial pressure were determined. Basal activity of ACE and the maximum ACE inhibition were significantly smaller in women. No significant differences in the drug concentration required to produce 50% of Emax were observed. Women had lower systolic arterial pressures and ACE activities than men at any time, even when the maximum inhibition of the ACE activity was attained. Women at the follicular phase had a minimum activity of ACE significantly inferior than men. Healthy women had lower systolic arterial pressures and ACE activities than men.

Effects of dotarizine and flunarizine on chromaffin cell viability and cytosolic Ca2

Dotarizine (a novel piperazine derivative with antimigraine properties) and flunarizine (a Ca2+ channel antagonist) were compared concerning: first, their ability to cause chromaffin cell damage in vitro; second, the possible correlation of their octanol/water partition coefficients and those of another 28 compounds (i.e., Ca2+ channel antagonists, blockers of histamine H1 receptors, antimycotics, beta-adrenoceptor antagonists, neuroleptics), with their ability to cause cell damage; third, their capacity to protect the cells against the damaging effects of veratridine; and fourth, their capabilities to enhance the basal cytosolic Ca2+ concentration in fura-2-loaded single chromaffin cells, or to modify the pattern of [Ca2+]i oscillations elicited by veratridine. After 24-h exposure to 1-30 microM dotarizine, the viability of bovine adrenal chromaffin cells (measured under phase contrast or as lactate dehydrogenase, released into the medium) was similar to that of control, untreated cells; at 100 microM, 80% lactate dehydrogenase release was produced. At 1-3 microM flunarizine caused no cell damage; however 10 microM caused 20% lactate dehydrogenase release and 30 and 100 microM over 90% lactate dehydrogenase release. The time course of cell damage was considerably faster for flunarizine, in comparison to dotarizine. Out of 30 molecules tested (at 10 microM), having different octanol/water partition coefficients (log P), dotarizine was among the molecules causing no cell damage; flunarizine caused 20% cell loss, lidoflazine and verapamil over 50% cell loss, and penfluridol, draflazine, astemizole or nifedipine over 80% cell loss. No correlation was found between log P and cytotoxicity. Both dotarizine (10-30 microM) and flunarizine (3-10 microM) provided protection against veratridine-induced cell death; however, at 30 microM dotarizine afforded a pronounced protection while flunarizine enhanced the cytotoxic effects of veratridine. Dotarizine (30 microM) (but not flunarizine) caused a prompt transient elevation of the basal [Ca2+]i. Both compounds abolished the K+-induced increases of [Ca2+]i as well as the oscillations of [Ca2+]i induced by veratridine. The blocking effects of dotarizine were readily reversed after washout, while those of flunarizine were long-lasting. These differences might be relevant to the clinical use of dotarizine as an antimigraine drug.

Evaluation of the Relationship between Sex, Polymorphisms in CYP2C8 and CYP2C9, and Pharmacokinetics of Angiotensin Receptor Blockers

Angiotensin II receptor blockers (ARBs) are used to treat hypertension. Most ARBs are metabolized by CYP2C9. The aim of this study is to evaluate the possible association between sex, polymorphisms in the CYP2C8 and CYP2C9 genes, and the pharmacokinetics of losartan, valsartan, candesartan, and telmisartan. The study population comprised 246 healthy volunteers from seven single-dose clinical trials: 64 from two candesartan studies, 43 from a telmisartan study, 36 from a losartan study, and 103 from three valsartan studies. DNA was extracted from blood samples and single-nucleotide polymorphisms in the CYP2C8 (CYP2C8*2, CYP2C8*3, CYP2C8*4, CYP2C8*5) and CYP2C9 (CYP2C9*2, CYP2C9*3) genes were evaluated using real-time polymerase chain reaction. Sex only affected telmisartan pharmacokinetics, since women showed a higher telmisartan Cmax than men (590.5 ± 75.8 ng/ml versus 282.1 ± 30.8 ng/ml; P ≤ 0.01). CYP2C9 variants were associated only with losartan pharmacokinetics: the half-life of losartan was higher in CYP2C9*3 allele carriers (3.1 ± 0.4 hours) than in volunteers with the wild-type genotype (2.3 ± 0.1 hours) (P ≤ 0.05). CYP2C8 polymorphisms were associated only with valsartan pharmacokinetics, since *2 allele carriers showed faster clearance (1.07 ± 0.57 l/h·kg) than those with the wild-type genotype (0.48 ± 0.72 l/h·kg; P ≤ 0.01) and carriers of the *3 allele (0.35 ± 0.49 l/h·kg; P ≤ 0.001). These results suggest that genotypes for CYP2C9 and CYP2C8 are relevant to the pharmacokinetics of losartan and valsartan, respectively, but not the pharmacokinetics of candesartan or telmisartan.

Influence of sex and CYP2D6 genotype on mirtazapine disposition, evaluated in Spanish healthy volunteers

AIMS To evaluate the influence of sex and CYP2D6 genotype on mirtazapine disposition within two bioequivalence studies in healthy volunteers. METHODS Seventy-two healthy volunteers were included in two standard 2 x 2 crossover bioequivalence trials. Subjects received a single 30-mg oral dose of each mirtazapine formulation in each study period. Plasma concentrations were measured from 0 to 96 or 120 h by a HPLC with coupled mass spectrometry validated method. CYP2D6 genotyping was available for 68 subjects that were classified into three phenotypic groups depending on the number of active gene copies: extensive/ultrarapid metabolizers (UM-EM), intermediate (IM) and poor metabolizers (PM). To evaluate the influence of sex and genotype on mirtazapine disposition we performed a linear mixed model for repeated measures. Pharmacokinetic data were log-transformed and AUC and C(max) adjusted to the administered dose/weight. Factors included in the model were centre, formulation, period, sequence, sex and genotype as fixed effects, and subject nested sequence x sex x genotype as random one. A second model was also performed adding the interaction sex x genotype to the previous model. RESULTS Mirtazapine disposition evaluated as AUC(0-infinity) is influenced by sex (p=0.007) and CYP2D6 phenotype group (p=0.01). Attending to the theoretical figures provided by the model, mean (95% CI) dose/weight adjusted AUC(0-infinity) (ng h/ml)/(mg/kg) is 1516.62 (1411.27-1628.22) in EM/UM, 1613.63 (1482.14-1758.55) in IM and 2049.28 (1779.78-2357.24) in PM. In the case of C(max) these figures also show a trend to higher values in PM, but it did not reach statistical significance. Females show a lower dose/weight adjusted AUC(0-infinity): 1594.39 (1477.70-1720.28) vs. 1837.65 (1694.67-1992.70). On the contrary dose/weight adjusted C(max) is higher in females than in males: 38.33 (34.79-42.28) vs. 32.66 (29.44-36.21). CONCLUSIONS Both CYP2D6 genotype group and sex influence the disposition of mirtazapine in healthy volunteers and confirm reported data in the literature obtained by different methods. No sex-by-genotype interaction could be detected.

Effect of polymorphisms on the pharmacokinetics, pharmacodynamics, and safety of risperidone in healthy volunteers

To identify genetic markers capable of predicting the pharmacokinetics, pharmacodynamics, and adverse effects of risperidone.