Novel aspects of drug metabolism and transport

The 16th International Symposium on Microsomes and Drug Oxidations (MDO2006) in Budapest, Hungary, had almost 400 attendees and was nicely organized by Laszlo Vereczkey and colleagues. The meeting had a very high standard in the field of drug metabolism, drug transport and related areas and in addition, the social events were much appreciated. At the meeting 70 invited lectures were presented in plenary sessions and in three parallel symposia sessions, and about 178 posters were shown, among them 26 posters in the young investigators workshop. The review herein is given of a majority (57) of the lectures presented at the Symposium.     

The effect of dopamine upon oxidative metabolism of brown fat adipocytes

The effect of dopamine upon the oxygen consumption (QO2) of brown fat adipocytes was measured by polarography. The results showed a marked stimulation of QO2 by dopamine, similar to that induced by noradrenaline. The dopamine response could be blocked by haloperidol, butaclamol, propranolol and by high concentrations of tolazoline. These results suggest a role for dopamine in energy release from brown fat.     

Enantioselective and diastereoselective aspects of the oxidative metabolism of metoprolol

Enantio- and diastereoselective aspects of oxidative metabolism of metoprolol (1) were examined in the presence of rat liver and human liver microsomes using a pseudoracemate of 1, made up of equal molar (2R)-1-d0 and (2S)-1-d2, as substrate. Both O-demethylation and alpha-hydroxylation showed only slight enantioselectivity, 2R/2S ratios being 1.18 and 0.93 for these pathways in rat liver microsomes and 1.09 and 0.92 in human liver microsomes. In the presence of the rat liver microsomal fraction, alpha-hydroxylation yielded predominantly the 1'R-hydroxy product, 1'R/1'S ratio greater than 12, regardless of the stereochemistry of the side chain. In humans (extensive metabolizers) administered a single 50 mg oral dose of pseudoracemic metoprolol tartrate, urinary alpha-hydroxymetoprolol (2) accounted for 9.3 +/- 2.4% of the dose, 2R/2S ratio 0.85 +/- 0.14, and the carboxylic acid metabolite 4, accounted for 52.7 +/- 6.8% of the dose, 2R/2S ratio 1.15 +/- 0.09. The data suggested that preferential O-demethylation of the (2R)-enantiomer of 1 could contribute to the 2S greater than 2R plasma ratio of metoprolol enantiomers observed in this population.     

First pass metabolism and in vitro metabolism of a new orally active dopamine prodrug, N-(N-acetyl-L-methionyl)-O,O-bis(ethoxycarbonyl)dopamine in dogs.

To elucidate the first pass metabolism of the dopamine prodrug N-(N-acetyl-L-methionyl)-O,O-bis(ethoxycarbonyl)dopamine (TA-870) in the small intestine and liver of dogs, the blood and plasma concentrations of unchanged TA-870 and metabolites in the gastroduodenal vein, portal vein, hepatic vein, and abdominal aorta were measured by HPLC after intraduodenal administration of TA-870. In addition, an in vitro metabolic study of TA-870 was carried out using liver, small intestinal wall, and blood homogenates of dogs. The order of maximal concentration (Cmax 0-30 min) in gastroduodenal blood or plasma was unchanged TA-870 greater than deethoxycarbonylated TA-870 (DEC-TA-870) greater than conjugated dopamine greater than free dopamine (DA) greater than free homovanillic acid (HVA) greater than free 3,4-dihydroxyphenylacetic acid (DOPAC). This result showed that the main pathway of metabolism in the small intestine is catechol ester hydrolysis and minor pathways are amido hydrolysis, oxidative deamination, and catechol 3-O-methylation. The order of Cmax in the hepatic vein and abdominal aorta was conjugated DA greater than free DEC-TA-870 greater than free HVA greater than free DA greater than free DOPAC greater than conjugated DOPAC greater than unchanged TA-870. The main metabolic pathways in the liver were hydrolyses of ester and amido-linkage of TA-870 and conjugation of DA. In the in vitro studies, the main metabolites of TA-870 in liver, small intestinal wall, and blood homogenates were DEC-TA-870, 3- or 4-mono-deethoxycarbonylated TA-870, DA, and an unknown compound.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneous effects of ethylenebisdithiocarbamate (EBDC) pesticides on oxidative metabolism of xenobiotics

Comparative evaluation of the acute effects of ethylenebisdithiocarbamate (EBDC) fungicides, mancozeb and zineb on microsomal mixed function oxidases (MFO) revealed marked substrate-dependent inhibition of oxidative metabolism of aminopyrine, p-nitroanisole and aniline in rats sacrificed 4 hr after oral administration of 100 mg mancozeb or zineb/kg body weight. Mancozeb inhibited p-nitroanisole O-dealkylase and aniline hydroxylase to a greater degree than zineb, whereas the inhibition of aminopyrine N-demethylase by the two fungicides was quantitatively comparable. Interestingly, aryl hydrocarbon hydroxylase (AHH) which exhibited maximum inhibition with mancozeb, remained unaffected following zineb administration. The time-course and dose-dependence of MFO inhibition examined 1 and 4 hr after a single oral dose of 100 mg or 250 mg zineb/kg was expressed as a dose-dependent decline in the rate of xenobiotic biotransformation at 4 hr. In vitro interaction of zineb with MFO resulted in slightly greater inhibition of aminopyrine, p-nitroanisole and aniline while AHH exhibited more pronounced decrease with mancozeb. The magnitude of inhibition of aminopyrine N-demethylase and AHH was independent of the time of preincubation of fungicides with the enzyme. Kinetic studies indicated the non-competitive nature of AHH inhibition. Chronic oral treatment with mancozeb and zineb at a dose of 250 mg/kg for 4 weeks did not modify xenobiotic biotransformations except for a slight induction of aminopyrine N-demethylase by mancozeb.     

Perturbation of dopamine metabolism by 3-amino-2-(4'-halophenyl)propenes leads to increased oxidative stress and apoptotic SH-SY5Y cell death

We have recently characterized a series of 3-amino-2-phenyl-propene (APP) derivatives as reversible inhibitors for the bovine adrenal chromaffin granule vesicular monoamine transporter (VMAT) that have been previously characterized as potent irreversible dopamine-beta-monooxygenase (DbetaM) and monoamine oxidase (MAO) inhibitors. Halogen substitution on the 4'-position of the aromatic ring gradually increases VMAT inhibition potency from 4'-F to 4'-I, parallel to the hydrophobicity of the halogen. We show that these derivatives are taken up into both neuronal and non-neuronal cells, and into resealed chromaffin granule ghosts efficiently through passive diffusion. Uptake rates increased according to the hydrophobicity of the 4'-substituent. More importantly, these derivatives are highly toxic to human neuroblastoma SH-SY5Y but not toxic to M-1, Hep G2, or human embryonic kidney 293 non-neuronal cells at similar concentrations. They drastically perturb dopamine (DA) uptake and metabolism in SH-SY5Y cells under sublethal conditions and are able to deplete both vesicular and cytosolic catecholamines in a manner similar to that of amphetamines. In addition, 4'-IAPP treatment significantly increases intracellular reactive oxygen species (ROS) and decreases glutathione (GSH) levels in SH-SY5Y cells, and cell death is significantly attenuated by the common antioxidants alpha-tocopherol, N-acetyl-l-cysteine and GSH, but not by the nonspecific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. DNA fragmentation analysis further supports that cell death is probably due to a caspase-independent ROS-mediated apoptotic pathway. Based on these and other findings, we propose that drastic perturbation of DA metabolism in SH-SY5Y cells by 4'-halo APP derivatives causes increased oxidative stress, leading to apoptotic cell death.     

8-OH-DPAT-induced place preference and place aversion: effects of PCPA and dopamine antagonists

Low doses of 8-OH-DPAT (62.5–250 µg/kg) were reinforcing in the place preference conditioning procedure, while a higher dose (1 mg/kg) supported a conditioned place aversion. The 5-HT synthesis inhibitor PCPA, and the DA antagonists pimozide and sulpiride, had no effect when administered alone, but abolished the 8-OH-DPAT-induced place preference. However, neither PCPA nor pimozide altered the 8-OH-DPAT-induced place aversion. The results are consistent with other evidence showing that 8-OH-DPAT acts through different mechanisms at low and high doses, and support the hypothesis that low doses of 8-OH-DPAT act through 5-HT neurons to disinhibit dopaminergic activity.     

医患矛盾产生原因的分析与防范

医疗纠纷的发生,不仅给医患双方带来许多不良影响,同时也间接地影响社会其他许多方面.医疗纠纷的解决,牵涉到许多单位和部门,引发出一系列需要探讨的理论问题.一方面是促使有关管理部门对工作加速有关理论问题的研究,尽快解决法律文件之间的矛盾冲突;另一方面增强人们的法制观念,使人们逐渐认识到用法律手段维护自己的合法权益.     

Rotenone-induced PC12 cell toxicity is caused by oxidative stress resulting from altered dopamine metabolism.

Rotenone is a widely used pesticide. Administration of rotenone can induce biochemical and histological alterations similar to those of Parkinson's disease in rats, leading to the selective loss of dopaminergic neurons in the substantia nigra pars compacta. However, it remains unclear why rotenone seems to affect preferentially dopaminergic cells. To address this question, we studied the effects of rotenone on dopamine distribution and metabolism to determine the role of endogenous dopamine in rotenone-induced PC12 cells toxicity. Results showed that cell viability was decreased and intracellular dopamine concentration was increased with rotenone administration in a dose-dependent manner. Rotenone exposure led to changes of proteins and enzymes associated with dopamine synthesis and transportation in PC12 cells. Tyrosine hydroxylase (TH) and vesicular monoamine transporter 2 (VMAT(2)) were markedly down-regulated, and dopamine transporter (DAT) was up-regulated in the cells. The activity of monoamine oxidase (MAO) was also increased. In addition, rotenone increased ROS formation, which was clearly inhibited by the pretreatment of GSH. Similar inhibitions of ROS formation were also observed in PC12 cells pretreated with the classical dopamine transporter inhibitor of GBR-12909 and the MAO inhibitor l-deprenyl. Moreover, opposite effects were observed in PC12 cells pretreated with the specific VMAT(2) inhibitor reserpine. These results suggest that rotenone administration may interfere with dopamine distribution and metabolism, leading to dopamine accumulated in the cytoplasm of PC12 cells, which may contribute to the ROS formation and cell death. Therefore, the endogenous dopamine resulted from the altered dopamine metabolism and redistribution may play an important role in rotenone toxicity in dopamine neurons.     

The metabolism of dopamine, NN-dialkylated dopamines and derivatives of the dopamine agonist 2-amino-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN) by catechol-O-methyltransferase

A variety of dopamine derivatives and analogues were investigated to assess their potential to act as catechol-O-methyltransferase (COMT) substrates using purified, homogeneous pig liver enzyme. This enabled accurate kinetic constants to be determined as opposed to previous in-vivo studies (Rollema et al 1980; Horn et al 1981; Costall et al 1982; Feenstra et al 1983). 2-Amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (A-6,7-DTN) proved to be a far better substrate (Km = 0.082 mM; Vmax = 300 mu mg-1 protein) than its 5,6-dihydroxy isomer (Km = 2.60 mM; Vmax = 113.9 mu mg-1 protein). This result supports evidence suggesting that differences in brain concentration of these isomers are due to their differential susceptibility to O-methylation by COMT (Rollema et al 1980). A similar result was obtained with a series of NN-di-n-alkyl substituted ADTN derivatives: the same pattern of preferential O-methylation of A-6,7-DTN derivatives over the corresponding A-5,6-DTN isomers was observed. However, increasing the length of the alkyl chain increased the susceptibility of both isomers to metabolism by COMT as shown by a decline in Km. An homologous series of NN-di-n-alkylated dopamines showed a similar trend implying that more hydrophobic compounds are better COMT substrates.     

Effects of ultrafine diesel exhaust particles on oxidative stress generation and dopamine metabolism in PC-12 cells

A major constituent of urban air pollution is diesel exhaust, a complex mixture of gases, chemicals, and particles. Recent evidence suggests that exposure to air pollution can increase the risk of a fatal stroke, cause cerebrovascular damage, and induce neuroinflammation and oxidative stress that may trigger neurodegenerative diseases, such as Parkinson's disease. The specific aim of this study was to determine whether ultrafine diesel exhaust particles (DEPs), the particle component of exhaust from diesel engines, can induce oxidative stress and effect dopamine metabolism in PC-12 cells. After 24 h exposure to DEPs of 200 nm or smaller, cell viability, ROS and nitric oxide (NO₂) generation, and levels of dopamine (DA) and its metabolites, (dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)), were evaluated. Results indicated cell viability was not significantly changed by DEP exposure. However, ROS showed dramatic dose-dependent changes after DEP exposure (2.4 fold increase compared to control at 200 μg/mL). NO₂ levels were also dose-dependently increased after DEP exposure. Although not in a dose-dependent manner, upon DEP exposure, intracellular DA levels were increased while DOPAC and HVA levels decreased when compared to control. Results suggest that ultrafine DEPs lead to dopamine accumulation in the cytoplasm of PC-12 cells, possibly contributing to ROS formation. Further studies are warranted to elucidate this mechanism.     

Effect of acute and chronic stress restraint on amphetamine-associated place preference: involvement of dopamine D(1) and D(2) receptors

Fenfluramine + phentermine was a widely used combination for weight loss. Fenfluramine and phentermine are believed to act via serotonin and catecholamines, respectively. To what extent these drugs interact has not been well-established. We compared the anorectic efficacy of a range of doses of the combination (using dexfenfluramine instead of fenfluramine) relative to a range of doses of the individual drugs in 90 min sweetened milk intake tests in deprived and nondeprived rats. Results were plotted on isobolograms to determine whether the anorectic effects of the combination were either additive or synergistic. Collectively, the isobolographic analysis revealed that: (1) under acute conditions, dexfenfluramine and phentermine interact for the most part in a synergistic manner, and (2) with the exception of phentermine alone, deprivation state at time of testing did not alter the efficacy of the anorectics. These findings suggest that combined drug treatment for obesity is a theoretical approach that merits further investigation.     

The metabolism of debrisoquine in man: (1) regioselectivity of hydroxylation and (2) aberrant oxidative metabolism in two sibling patients with carbimazole-induced agranulocytosis

The regioselectivity of the metabolic hydroxylation of debrisoquine has been determined in 43 healthy British white volunteers and the priority was found to be in the order 4 greater than 7 greater than 6 greater than 5 greater than 8. The order of preference for hydroxylation position was independent of debrisoquine 4-hydroxylation phenotype. The extent of total aromatic hydroxylation varied widely between individuals and was largely independent of the extent of 4-hydroxylation, and thus of the influence of the DH/DL locus. Two sisters and their blood relations all excreted comparatively large amounts of the phenolic metabolites in their urine, indicating some genetic basis for the control of aromatic oxidation of debrisoquine in man. These same two sisters had previously developed agranulocytosis in association with carbimazole therapy.     

Effect of oxidative polymorphism (debrisoquine/sparteine type) on hepatic first-pass metabolism of bufuralol

Summary Bufuralol is a beta-adrenoceptor blocking drug whose oxidative metabolism is under the same genetic control as debrisoquine and sparteine. The pharmacokinetics of bufuralol were studied in 10 healthy subjects (7 extensive and 3 poor metabolizers of debrisoquine) after oral and intravenous administration. In extensive metabolizers the systemic availability of bufuralol was 43%. Poor metabolizers were characterized by a considerable increase in systemic availability due to a corresponding decrease in hepatic first-pass metabolism. After oral administration of bufuralol non-linear kinetics may occur.     

Changes in the oxidative glucose brain metabolism under long-term xantinol-nicotinate medication (author's transl)

8 patients suffering from dementia were treated with xantinol-nicotinate for 4 weeks. At the beginning and at the end of the treatment cerebral blood flow and cerebral metabolic rates of oxygen, glucose and lactate were measured with the N2O-method. With regard to the initial level of each parameter CBF, CMR glucose and CMR oxygen increased significantly, if it had been decreased at the first measurement, while primarily normal or even increased parameters did not change. Seming contradictions to other authors are discussed.     

The dopamine D(3) receptor-preferring partial agonist BP 897 dose-dependently attenuates the expression of amphetamine-conditioned place preference in rats

Previously we reported that systemic administration of the dopamine D3 receptor-preferring partial agonist BP 897 blocked the expression, but not the acquisition, of amphetamine-conditioned activity. This suggested the hypothesis that BP 897 would block the expression, but not the acquisition, of amphetamine-conditioned place preference (CPP). Thus, during preconditioning rats had access to two chambers connected by a tunnel for three 15-min sessions. During eight conditioning days with the tunnel blocked, one chamber was paired with drug administration for four 30-min sessions, alternating with pairing of the other chamber with saline administration. In a drug-free test session, time on the drug-paired side was compared to time spent there in preconditioning; a significant increase was defined as a place preference. Systemic amphetamine (2.0 mg/kg) or amphetamine+BP 897 (1.0, 2.0 mg/kg) during conditioning produced a significant place preference, while administration of BP 897 (1.0 or 2.0 but not 0.5 mg/kg) during the test blocked the amphetamine-CPP. There was no evidence that BP 897 produced a conditioned aversion. Results supported the hypothesis that BP 897 would block expression, but not acquisition, of amphetamine-CPP.