Some aspects of interindividual variations in the metabolism of xenobiotics.

Differences in drug metabolism among individuals are caused by numerous factors: differences in production and stability of mRNA of xenobiotic metabolizing enzymes, differences in the rate of enzyme synthesis and degradation, or enzyme inhibition. One of the most important reasons is genetic polymorphism of cytochrome P450 genes or cytochrome P450 regulatory factors. Nuclear receptors play great role in the regulation of these genes. The presence of the ligand induces the nuclear receptor to bind to the dimerisation partner and as a hetero/homodimer it can activate the DNA responsive element. In addition, several co-activators, co-repressors and other factors can modulate the effect of nuclear receptors. Hepatic levels of cytochrome P450 enzymes are reduced in multiple models of inflammation or infection. Cytochrome P450 enzymes of four families (CYP1-CYP4) are known to be involved in xenobiotic metabolism. Their genetic polymorphism and regulation are discussed in this review.

The effect of dexamethasone on P450 activities in regenerating rat liver.

The aim of our study was to detect four P450s (CYP1A, CYP2B, CYP2E1, CYP3A) on the basis of selective enzyme activities and protein amount, and to investigate the effect of dexamethasone treatment during liver regeneration. Partial hepatectomy of rats resulted in the loss of CYP1A, CYP2B, CYP2E1, and CYP3A activities. The reduction of enzyme activities and the loss of enzyme protein of CYP2B1/2, CYP2E1, and CYP3A1/2 were the most pronounced. In the case of CYP1A1, only slight decrease was observed. Dexamethasone treatment seems to counteract this loss mainly in the first 12 h.

Effect of alpha-methyldopa on pentoxyresorufin O-dealkylation in liver microsomes from rats treated with phenobarbital.

Loss of pentoxyresorufin O-dealkylation (PROD) was observed when microsomes from PB-treated rats were preincubated in the presence of NADPH. PROD proved to be quite sensitive towards inactivation. Decrease in cytochrome P450 (CYP) dependent activity was accompanied by simultaneous formation of thiobarbituric acid reactive substances (TBARS) indicating the occurrence of lipid peroxidation. The presence of 50 microM alpha-methyldopa (AMD) during preincubation with NADPH resulted in complete protection against enzyme activity loss and the extent of lipid peroxidation was also diminished. Addition of ascorbate or GSH in combination with AMD reduced the protective effect of the drug on PROD. AMD probably exerts its effect by scavenging reactive oxygen species but chelation of ferric ions can also contribute to the protective effect of the drug on PROD activity.

[About paracetamol again]. / Ismét a paracetamolról.

The mechanism of hepatotoxicity caused by paracetamol (acetaminophen) overdose and the treatment of patients is reviewed. Paracetamol is widely used over-the-counter drug with analgesic and antipyretic properties. Although it is considered to be safe at therapeutic doses, the incidence of hepatotoxicity caused by overdose or inadvertent application has been increasing lately. N-acetyl-p-benzoquinonimine, one of the metabolites formed from paracetamol is responsible for the hepatotoxicity. Until now there is no complete therapeutic strategy for the effective treatment of hepatotoxicity caused by paracetamol. Gut decontamination, N-acetylcysteine antidote administration and enhancement of elimination is used for the management of paracetamol overdose. Those with severe hepatotoxicity and neurological symptoms can benefit from removal of necrotic liver and undergo transplantation.

In vitro-in vivo correlation of the pharmacokinetics of vinpocetine.

Vinpocetine is extensively metabolized in rats, dogs and humans, and the plasma clearance approximates the hepatic plasma flow in each of the species. In vitro degradation studies with hepatocytes have shown that the activity of human hepatocytes is about one order of magnitude higher than the activity of dog hepatocytes, and two orders of magnitude higher than that of rat hepatocytes. These differences can explain the differences in bioavailabilities of vinpocetine in the three species (52% in rats, 21.5+/-19.3% in dogs and 6.2+/-1.9% in humans). In dogs and humans, the compound seems to be metabolized exclusively in the liver whereas in rats extrahepatic metabolism seems also to be important. The in vivo clearance predicted from the activity of hepatocytes is in good agreement with the values measured in vivo in the case of humans and dogs. The estimated values for bioavailability showed good correlation with in vivo data in each species if the free drug ratio was assumed to equal 1.

Effect of phenobarbital and spironolactone treatment on the oxidative metabolism of antipyrine by rat liver microsomes.

The effects of pretreating rats with the inducers, phenobarbital or spironolactone, on the formation rate of the three major oxidative metabolites of antipyrine in vitro by hepatic microsomal fractions have been investigated. Both inducers reduced the rate of 3-methylhydroxylation of antipyrine by approximately 50%. In contrast, N-demethylation and 4-hydroxylation were enhanced 1.7-fold and 3.4-fold, respectively, in case of phenobarbital induction and 1.4-fold and 2.6-fold, respectively, following spironolactone treatment. To elucidate the role of some cytochrome P450 isoenzymes in the production of the three major metabolites of antipyrine, the effects of form selective enzyme inhibitors on antipyrine oxidation were also studied. Troleandomycin did not alter 3-methylhydroxylation but reduced both N-demethylation and 4-hydroxylation of antipyrine in microsomes from induced rat liver. Cimetidine and chloramphenicol decreased the rate of formation of all three metabolites in microsomes from induced and uninduced animal livers as well. Chloramphenicol seemed to be the most potent inhibitor of in vitro antipyrine oxidation. Alpha-methyldopa significantly enhanced the rate of formation of 4-hydroxyantipyrine and slightly reduced the rate of N-demethylation and 3-methylhydroxylation. According to the data obtained with microsomes from uninduced rat livers, the formation of the three major metabolites of antipyrine is extensively mediated by CYP2C11/C6. In microsomes from induced animal liver, CYP2B and CYP3A may contribute to both N-demethylation and 4-hydroxylation of antipyrine.

Glucuronidation of thyroxine in primary monolayer cultures of rat hepatocytes: in vitro induction of UDP-glucuronosyltranferases by methylcholanthrene, clofibrate, and dexamethasone alone and in combination.

Induction of UDP-glucuronosyltransferases (UGTs) toward thyroxine (T4) and p-nitrophenol (pNP) by 3-methylcholanthrene (MC), dexamethasone (DEX), clofibrate (Cl), and MC combined with DEX or Cl was studied in rat hepatocyte culture. We have developed a sensitive method for the measurement of glucuronide conjugates of the two substrates based on HPLC analysis of culture medium. MC, Cl, or DEX increased the activity of T4 UGT. Combination of MC and Cl showed additive effect, enzyme activity was enhanced compared with either MC or Cl treatment alone (617, 441, and 217% of the control, respectively). Combination of MC and DEX did not result in higher T4 UGT activity than MC treatment alone. Both MC and DEX enhanced the pNP UGT activity (182 and 162% of the control, respectively). Combination of MC with DEX resulted in additive effect. Cl treatment did not affect pNP conjugation either alone or in combination with MC. Western blot analysis revealed that only the amount of UGT1A1 was elevated by Cl and DEX. In contrast, concentration of UGT1A6 was increased by MC. Previous studies demonstrated that UGT1A1 inducers like phenobarbital have no effect on T4 conjugation (). Our results suggest that Cl, a known inducer of UGT1A1, enhances the activity of other enzyme(s) involved in T4 glucuronidation as well. It is well documented that DEX potentiates the inductory effect of polycyclic aromatic hydrocarbon on UGT1A6 (). In our study, MC increased the rate of T4 glucuronidation, and DEX had no additional effect on this reaction, suggesting that UGT1A6 is not the only enzyme inducible by MC that can catalyze T4 conjugation.

The in vitro biosynthesis and stability measurement with agyl-glycuronide isoformes of the main metabolite of ipriflavone.

The formation and stability of 1-beta-glucuronide conjugate of the main metabolite of ipriflavone [7-(1-carboxy-ethoxy)-isoflavone] (CI)--were studied by using liver microsomes, hepatocytes, and isolated perfused liver of untreated and 3-methylcholanthrene (MC) treated dog and rat, and human liver microsomes. MC treatment enhanced the rate of conjugation twice as much as that of the control in the microsomes of both dogs and rats. Conjugation of CI by microsomes results in two metabolites, both sensitive to pH and temperature. Other two glucuronide forms appeared in experiments with hepatocytes and perfused liver. Mass spectrometry supported. The conclusion, assumption that both metabolites produced by microsomes are glucuronide conjugate isoforms of CI, and that they could be distinguished according to the intensity of peaks on FAB-MIKE spectra. The beta-glucuronidase enzyme hydrolysed only the 1-beta-glucuronide isomer, the other, migrated form remained unchanged. D-saccharic-acid-1,4-lactone, a specific inhibitor of beta-glucuronidase enzyme, decreased the rate of enzymatic cleavage. Standard curves of CI were prepared by HPLC, and 1-beta-CI-glucuronide was quantified according to the amount of CI formed by hydrolysis. The stability of conjugates greatly depends on pH and temperature, and the rate of degradation and isomerization is sensitive to the value of both. Lowering the pH from 7.4 to 5.0 and the temperature from 37 degrees C to 18 degrees C increased the stability of glucuronides. Increasing the pH to 12.0 results in very rapid acyl migration and hydrolysis.

[Human drug metabolizing enzymes III. Epoxide hydrolases, esterases, and amidases]. / Human gyógyszermetabolizáló enzimek III. Epoxid hidrolázok, eszterázok és amidázok.

In this review we focus on human hydrolytic enzymes that participate in the metabolism of xenobiotics. Although hydrolysis in most of the cases result in detoxication, in some cases hydrolysis may lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity. We have summarised the data available on these enzymes concerning their catalytic profile and specificity, inhibition, induction properties, their possible role in the generation of toxic compounds, their importance in clinical practice and drug development.

Predicting the time needed to achieve steady state if absorption and elimination constants are equal.

A simple table is derived to facilitate the rapid estimation of the number of dose administrations needed to achieve a certain fraction of the steady-state plasma concentration in the case of one-compartment model with uniform multiple oral dosing and equal absorption and elimination constants.

[Human drug metabolizing enzymes. I. Oxidative enzymes]. / Human gyógyszermetabolizáló enzimek. I. Oxidációs enzimek.

In this review we focus on human oxidative enzymes that are responsible for the metabolism of xenobiotics. More and more publications prove that the reactions catalysed by these enzymes very often lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity (liver, neuro-, embryotoxicity, allergy, carcinogenecity). We have summarised the data available on these enzymes, concerning their catalytic profile and specificity, inhibition, induction properties, their possible role in the generation of toxic compounds, their importance in clinical practice and drug development.

[Human drug metabolizing enzymes. II. Conjugation enzymes]. / Human gyógyszermetabolizáló enzimek. II. Konjugációs enzimek.

In this review we focus on human conjugation enzymes (UDP-glucuronyltransferases, methyl-trasferases, N-acetyl-transferases, O-acetyl-transferases, Amidases/carboxyesterases, sulfotransferases, Glutation-S-transferases and the enzymes involved in the conjugation with amino acids) that participate in the metabolism of xenobiotics. Although conjugation reactions in most of the cases result in detoxication, more and more publications prove that the reactions catalysed by these enzymes very often lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity (liver, neuro-, embryotoxicity, allergy, carcinogenecity). We have summarised the data available on these enzymes concerning their catalytic profile and specificity, inhibition, induction properties, their possible role in the generation of toxic compounds, their importance in clinical practice and drug development.

[Mass spectrometry in the verification of pyrimethamine poisoning]. / Pyrimethamin intoxikáció tömegspektrometriás igazolása.

A 17 years old male patient with Pyrimethamin therapy was released from our department by emphasising the necessity of continuous control. A month later the patient was accepted again with serious anaemia. Since the patient did not follow the instructions Pyrimethamin intoxication was presumed, but it had to be proved. At last the drug in the plasma was identified and quantified by mass spectrometry. The plasma concentration of Pyrimethamin was five times higher than the therapeutic level. The rapid analysis (4 hours after taking of blood) and adequate treatment resulted in rapid improvement with the concomitant decrease of plasma Pyrimethamin concentration. During clinical treatment the level of Pyrimethamin in the plasma was followed by mass spectrometry.

Ipriflavone as an inhibitor of human cytochrome P450 enzymes.

1. Reduction of theophylline metabolism and elimination were observed in a theophylline-treated patient during ipriflavone administration. After withdrawal of ipriflavone, the serum theophylline level decreased to an extent similar to that found before administration of ipriflavone. The effects of ipriflavone and its major metabolites 7-hydroxy-isoflavone and 7-(1-carboxy-ethoxy)-isoflavone on cytochrome P450 activities were studied in vitro in human liver microsomes from three donors. 2. Ipriflavone and 7-hydroxy-isoflavone competitively inhibited phenacetin O-deethylase and tolbutamide hydroxylase activity. The parent compound and its dealkylated metabolite were strong inhibitors exhibiting Ki values around 10-20 microM, while 7-(1-carboxy-ethoxy)-isoflavone had no effect on the cytochrome P450 activities investigated. 7-Hydroxy-isoflavone is the only one that influenced nifedipine oxidase activity. It competitively inhibited this activity with a Ki value of 129.5 microM. 3. The steady state concentrations of ipriflavone and 7-hydroxy-isoflavone in plasma of patients receiving 3 x 200 mg daily doses of ipriflavone for 48 weeks were found to be 0.33 +/- 0.32 microM and 1.44 +/- 0.77 microM, respectively. 4. The results indicate that the decrease in theophylline metabolism observed in a patient treated with ipriflavone may be due to a competitive interaction of ipriflavone or its metabolite, 7-hydroxy-isoflavone with CYP1A2. On the other hand, our in vitro findings predict some more interaction with CYP2C9.

[Some thoughts on Bateman's equation]. / Néhány gondolat a Bateman egyenletröl.

The authors have investigated the characteristics of one of the basic equations of pharmacokinetics, Bateman's equation in the function of the differences between the absorption and elimination constants.

[Validation of gas and liquid chromatographic methods in the bioanalytical laboratory]. / Folyadék és gázkromatográfiás módszerek validálása a bioanalitikai munka során.

The authors discuss the validation of bioanalytical methods used to generate data for bioavailability, bioequivalence and pharmacokinetics studies. The basis of this manuscript is a consensus on the requirements for bioanalytical validation, which has been reached by a panel of experts at the Washington Conference Report. In this paper it is attempted to suggest approaches to validation parameters both for the method and assay to be evaluated, namely specificity/selectivity, linearity, LOQ/LOD, accuracy, prescision, recovery and stability.

Human liver cytochrome P-450 and conjugating enzymes. Hungarian data.

Cytochrome P-450 and the conjugating enzymes of the liver are the most important enzymes in the detoxification of xenobiotics. In order to get direct data in the Hungarian human population the activity of liver cytochrome P-450 enzymes (CYP1A2, 2A6, 2C19, 2D6, E1, 3A4) and of the conjugating enzymes has been determined in 11 Hungarian tissue donors died in accidents or in brain vascular catastrophe, by using specific enzymic reactions. In the activity of cytochrome P-450 enzymes a wide range has been found, a ten-fold difference being rather common. In the activities of conjugating enzymes the differences seem to be less.

Species differences in metabolism of panomifene, an analogue of tamoxifen.

In vitro metabolism of panomifene (E-1,2-diphenyl-1--4-(2-(2-hydroxyethyl-amino)-ethoxy)-phenyl--3,3,3- trifluoropropene), a novel antiestrogen against hormone dependent tumors, has been investigated using liver microsomes from mouse, rat, dog, and human. Hydroxylation and side chain modifications were the routes of panomifene metabolism. Microsomal biotransformation showed some qualitative similarities, but several differences were observed in the metabolic profiles of the four species tested. Seven metabolites were detected in the incubation mixtures analyzed by thin layer chromatography and autoradiography, although there was only one produced by all species that had lost the side chain. Among the side chain shortened metabolites, the compound that had lost the hydroxyethyl-amino group was formed by the microsomal system of rodents, whereas the one that had lost the hydroxyethyl group was detected in the incubation mixtures with rat, dog, and human microsomes. Three metabolites (M1, M3, and M4) were produced exclusively by the dog. The structure of M3 was identified by mass spectroscopy as 4-hydroxy-panomifene. Furthermore, human liver microsomes formed a metabolite (M8) that was not detectable in the mixtures with mouse, rat, or dog microsomes. Its structure is suspected to be an oxidized form of panomifene with a double bound in the side chain. The structure of panomifene is analogous to tamoxifen, an antiestrogen currently used as a therapeutic agent against breast cancer, and there are some similar routes in their metabolism. The main difference is that the rate of tamoxifen biotransformation seems faster than that of panomifene. On the other hand, 4-hydroxy-panomifene is produced by only dog, while 4-hydroxylated derivative is one of the main metabolites of tamoxifen that has potent antiestrogenic activity and is considered to be responsible for the formation of DNA-adducts.

Ion-pair high-performance liquid chromatographic separation of two thyroxine glucuronides formed by rat liver microsomes.

A simple reversed-phase ion-pair high-performance liquid chromatographic separation method has been developed for thyroxine (T4) and its glucuronide metabolites formed by liver microsomes of untreated and 3-methylcholanthrene-treated rats. Besides the phenol-T4-glucuronide, another, probably acyl-T4-glucuronide, formation has been detected. The effect of pH and temperature on the stability of the acyl-T4-glucuronide was also investigated. The lowering of pH to 2 and cooling the samples to 5 degrees C is necessary to prevent the hydrolysis of acylglucuronide, while both pH and temperature do not affect the stability of the phenol-T4-glucuronide. The retention times of T4 and phenol-T4-glucuronide are highly influenced by the pH of the mobile phase, but not that of acyl-T4-glucuronide.