[Simulation of bioequivalence study on the base of dissolution curves]. / Bioekvivalencia vizsgálat szimulációja kioldódási görbék alapján.

A computer method based on the in vitro dissolution of drug preparations has been elaborated for the estimation of bioequivalence. The method generates a "dissolutaion surface" from the parameters of time (X-axis), from pH (Y-axis) and from the dissolved amount (A) in % of the drug. This dissolution surface allows the determination of the general dissolution curve of the test and reference preparations. By supposing that the absorption rate constant is known from the literature, the change of the amount of dissolved drug as the function of time can be determined. On the base of this function the maximum amount of the dissolved drug in the gastrointestinal tract and the AUC can be calculated. and the test/reference ratio can be determined. In the case of linear pharmacokinetics these ratios are identical to the ratios of parameters that can be calculated in the circulation. By generating parameters between the allowed biological limits the dissolved drug-time curves of "volunteers" in the necessary number are created with the randomly generated "residence times" and their confidence intervals can be determined, i.e., on the base of dissolution curves bioequivalence can be estimated.

Differential inhibitory effect of cyclosporin A and bosentan on taurocholate uptake in human and rat hepatocytes as a function of culturing time.

Bile salt transport across hepatocytes requires a coordinate action of transporters, which is thought to be a target for drug-induced cholestasis. Hepatocytes provide the most competent in vitro model to predict transporter-related toxic drug effects. The aim of this study was to show a correlation between inhibitory potential of drugs and the change of rate, as well as of the active to passive ratio of taurocholate uptake in these cells. In rat hepatocytes, along with a significant decrease of uptake (86.4% by 72h), and the shift of saturable/unsaturable transport (from 92/8 to 55/45 in a 24-72h time interval), the efficacy of taurocholate uptake inhibition was highly reduced (IC(50) cyclosporin A 3.9 to >100µM, and bosentan 9.1-49.8µM at 1 and 72h, respectively). In contrast, 5-day-old human hepatocytes preserved 70% of their taurocholate uptake capacity with a 2-fold higher active than passive transport, which resulted in a more efficient inhibition by drugs (IC(50) cyclosporin A, 2.4 to ∼10µM and bosentan 28.9-45.5µM at 1h and 5days, respectively). Our results support that reliable drug interaction studies might be performed in 5-day-old human hepatocyte cultures, while experiments using rat hepatocytes at more than 24h after seeding will highly underestimate the probability of drug interaction.

Contribution of high basolateral bile salt efflux to the lack of hepatotoxicity in rat in response to drugs inducing cholestasis in human.

Intrahepatic bile acid accumulation due to inhibition of the bile salt export pump (BSEP) has been proposed as a mechanism for drug-induced cholestasis. Many cholestatic drugs do not initiate hepatotoxicity in rats, although they inhibit rat Bsep and cause elevated serum bile acid concentration. In this study, we examined changes in the taurocholate (TC) transport in response to cholestatic drug treatments in human and rat sandwich-cultured hepatocytes. Our experimental setup allows studying the basolateral and canalicular efflux simultaneously, thus comparing drug-induced changes in the vectorial efflux of TC. We found that TC elimination highly differs in human and rat hepatocytes. In human hepatocytes, an equal fraction of TC(uptake) was eliminated by basolateral (34.8%) and canalicular (34.4%) transporters and remained in the cells (30.5%), while in the case of rats, the basolateral transport was dominant (71.7%) and intracellular TC accumulation was negligible (6.9%). The inhibition of BSEP/Bsep resulted in significantly higher intracellular TC(conc) in humans than in rats. The 15-fold difference in intracellular TC(conc) of control in human versus rat hepatocytes was increased 25-fold by troglitazone treatment. MK571 and indomethacin decreased the basolateral efflux and significantly increased the intracellular TC(conc) in rats. In rat hepatocytes, the highest intracellular TC(conc) was observed with cyclosporine A and glibenclamide, which inhibited TC elimination in both directions. Nevertheless, the basolateral transport remained dominant. We conclude that in rats, the higher rate of basolateral bile salt efflux represents an additional protective mechanism in cholestasis, which contributes to species differences in response to hepatotoxic drugs.

ABCC2/Abcc2: a multispecific transporter with dominant excretory functions.

ABCC2/Abcc2 (MRP2/Mrp2) is expressed at major physiological barriers, such as the canalicular membrane of liver cells, kidney proximal tubule epithelial cells, enterocytes of the small and large intestine, and syncytiotrophoblast of the placenta. ABCC2/Abcc2 always localizes in the apical membranes. Although ABCC2/Abcc2 transports a variety of amphiphilic anions that belong to different classes of molecules, such as endogenous compounds (e.g., bilirubin-glucuronides), drugs, toxic chemicals, nutraceuticals, and their conjugates, it displays a preference for phase II conjugates. Phenotypically, the most obvious consequence of mutations in ABCC2 that lead to Dubin-Johnson syndrome is conjugate hyperbilirubinemia. ABCC2/Abcc2 harbors multiple binding sites and displays complex transport kinetics.

Biliary efflux transporters involved in the clearance of rosuvastatin in sandwich culture of primary rat hepatocytes.

Rosuvastatin (a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) has been shown to be excreted mostly unchanged into the bile; interactions on the level of hepatic apical efflux transporters may represent a risk of liver toxicity. So far, controversial and insufficient data are available concerning transporters involved in the elimination process. This study was designed to elucidate, which transporters take part in the biliary clearance of rosuvastatin using sandwich-cultured primary rat hepatocytes. The canalicular efflux of rosuvastatin was measured in the presence of inhibitors: Ko 134, mitoxanthrone, novobiocin for breast cancer resistance protein (Bcrp); verapamil for multidrug resistance protein (Mdr1); benzbromarone, sulfasalazine, probenecid for multidrug resistance associated protein (Mrp 2); and cyclosporine A, glibenclamide, troglitazone for bile salt export pump (Bsep). Mrp2 inhibitors decreased the biliary efflux of rosuvastatin most potently by 78.9%, 35%, 54.1%; benzbromarone, probenecid, sulfasalazine, respectively, while Bcrp and Bsep inhibitors showed much less effect (29.1%, 23.0% ,30.0%; Ko 134, mitoxanthrone, novobiocin, respectively, and 32.6%, 29.3%, 20.6%, glibenclamide, cyclosporine A, troglitazone, respectively). The marked decline of canalicular transport by Mrp2 inhibitors suggests major role of Mrp2 in this process; however, Bcrp and Bsep might also contribute to the biliary elimination of rosuvatatin in sandwich-cultured rat hepatocytes.

Interspecies differences in acetaminophen sensitivity of human, rat, and mouse primary hepatocytes.

Most of the experiments studying acetaminophen (APAP) induced hepatotoxicity were performed using moue as model specie, right because its high sensitivity. While the toxic responses can be called forth easily in mice, the human relevancy of these results is questionable. In this study human, rat, and mouse primary hepatocytes were treated with increasing concentrations of APAP, and cell viability was measured by MTT cytotoxicity assay. Pronounced interspecies differences were obtained in cell viability following 24h of APAP treatment starting at 24h after seeding (EC50: 3.8mM, 7.6mM, and 28.2mM, in mouse, rat, and human hepatocyte culture, respectively). The longer time of culturing highly increased the resistance of hepatocytes of all species investigated. In rat hepatocyte culture EC50 values were 6.0mM, 12.5mM, and 18.8mM, when starting APAP treatment after 24, 48, and 72 h of seeding. Although N-acetylbenzoquinoneimine, a minor metabolite of APAP, which is mainly formed by CYP2E1 at high APAP concentration in every species studied, is thought to initiate the toxic processes, no correlation was found between CYP2E1 activities and hepatocyte sensitivity of different species. We conclude that the toxicity induced by APAP overdose highly depends on the animal model applied.

Limited applicability of 7-methoxy-4-trifluoromethylcoumarin as a CYP2C9-selective substrate.

Fluorometric substrates selective for various cytochrome P450 isoforms (P450s) have great advantages in in vitro enzyme inhibition and induction studies because they are highly sensitive and suitable for rapid screening. 7-Methoxy-4-trifluoromethylcoumarin (MFC) has been reported as a CYP2C9-selective substrate. The present study investigated the relative catalytic selectivity of several human P450s in the O-demethylation of MFC and the applicability of MFC as a probe substrate for CYP2C9. The CYP2C9-selectivity in liver microsomes was not supported by the correlation analysis within a series of microsomes from individual donors or by studies using chemical inhibitors. MFC O-demethylation of microsomes did not correlate with tolbutamide 4-hydroxylation, the classical CYP2C9-marker activity, suggesting the possible participation of some of the other P450s. Results of inhibition studies using model P450 inhibitors also brought the CYP2C9-selectivity of MFC O-demethylation into question. In microsomes containing cDNA-expressed individual P450s, CYP2B6 and CYP2E1 seemed to be the most active in the O-demethylation of MFC. Our results support the participation of several P450 enzymes (CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4) in MFC O-dealkylation. Therefore, MFC cannot be considered a suitable probe substrate in models that express several P450s, such as liver microsomes or primary hepatocytes. Moreover, MFC is a more potent fluorogenic substrate for CYP2B6 and CYP2E1 than for CYP2C9 in microsomes containing cDNA-expressed P450s.

Modulation of sinusoidal and canalicular elimination of bilirubin-glucuronides by rifampicin and other cholestatic drugs in a sandwich culture of rat hepatocytes.

AIM: Drug-induced hyperbilirubinemia has been shown to often be derived from modulation of the expression and activity of hepatobiliary transporters. In this study we examined the interactions of some therapeutic agents, which have been shown to cause cholestasis, with the elimination of bilirubin-glucuronides, in order to clarify whether these drugs modify the activity of Mrp2 and Mrp3 directly. METHODS: The modulation of bilirubin-glucuronide elimination with rifampicin, probenecid, indomethacin and benzbromarone was assayed in sandwich cultured rat hepatocytes. RESULTS: All the drugs studied decreased the canalicular transport, but modified the sinusoidal efflux differently. Rifampicin and probenecid stimulated the sinusoidal efflux, shifting the elimination of bilirubin-glucuronides to the sinusoidal domain (biliary excretion index: 3.9 +/- 1.2; 22.7 +/- 7.4 vs. 56.6 +/- 1.5 and 56.8 +/- 5.5). However, the overall elimination of bilirubin-glucuronides did not change significantly. In contrast, indomethacin and benzbromarone inhibited bothtransport processes, resulting in the decrease of the overall bilirubin-glucuronide elimination (61 +/- 22; 56 +/- 5% of the control). Rifampicin, indomethacin and benzbromarone decreased 5,(6)-carboxy-2',7'-dichlorofluorescein transport by multidrug resistance-associated protein (Mrp)2 as visualized by confocal laser microscopy and in vesicular transport experiments. Interestingly, rifampicin decreased the MRP3 activity in vesicular transport experiments using 17-beta-estradiol-17-beta-D-glucuronide as substrate, in contrast to that observed in bilirubin-glucuronide transport experiments. CONCLUSION: Here we show that the interactions of drugs on hepatobiliary transporter proteins may be identified in vitro in a sandwich culture of hepatocytes, in which canalicular and sinusoidal transport can be studied separately.

Effect of mirtazapine on the CYP2D activity in the primary culture of rat hepatocytes.

Our previous studies carried out on rats showed that mirtazapine given intraperitoneally at a dose of 3 mg/kg, twice a day for two weeks, increased the activity of CYP2D measured as ethylmorphine O-deethylation in liver microsomes. The aim of the present work was to find out whether the mirtazapine-induced increase in the CYP2D activity observed in vivo is connected with the central action of mirtazapine or the drug acts directly on hepatocytes. For this purpose, we studied the influence of pharmacological concentrations of mirtazapine (0.1, 1.0, 10 microM for 96 h) on the activity of CYP2D measured as the rates of ethylmorphine O-deethylation and dextromethorphan O-demethylation in the primary culture of rat hepatocytes. Additionally, we tested the ability of CYP isoforms to catalyze ethylmorphine O-deethylation, using cDNA-expressed CYPs and CYP inhibitors applied to liver microsomes. The obtained results indicate that mirtazapine applied at pharmacological concentrations can moderately increase the activity of rat CYP2D in hepatocytes, and CYP2D2 isoform contributes mostly to this effect. Similar result was previously obtained after in vivo administered mirtazapine in liver microsomes, but not in brain microsomes, the latter containing mainly CYP2D4 isoform. Mirtazapine appears to act directly on hepatocytes and its effect does not seem to depend on the central pharmacological action of the antidepressant. CYP2D2 is the main isoform catalyzing ethylmorphine O-deethylation while CYP2A2, CYP2C6 and CYP2C11 are of minor importance.

Biotransformation of deramciclane in primary hepatocytes of rat, mouse, rabbit, dog, and human.

The metabolic fate of deramciclane [(1R,2S,4R)-(-)-2-phenyl-2-(2'-dimethylamino-ethoxy)-1,7,7-trimethyl-bicyclo[2.2.1]heptane], a new anxiolytic drug candidate, has been determined in rat, mouse, rabbit, dog, and human hepatocytes. Rat and rabbit cells were the most active, whereas the rate of metabolism was quite slow in human hepatocytes. During biotransformation, deramciclane underwent side chain modification and oxidation at several positions of the molecule. The side chain modification led to the formation of N-desmethyl deramciclane and phenylborneol. The oxidation of deramciclane resulted in several hydroxy-, carboxy-, and N-oxide derivatives. The hydroxylation took place at primary or secondary carbons of the camphor ring as well as at the side chain; furthermore, dihydroxylated derivatives were also found. The side chain-modified metabolites were also oxidized to hydroxy- or carboxy-derivatives. Conjugation of phase I metabolites, as a route of elimination, was also observed in rat, rabbit, and dog hepatocytes. Although there were some species differences in biotransformation of deramciclane, it was concluded that phase I metabolism in human liver cells seemed to be similar to the metabolism in the hepatocytes isolated from rat. With careful approach, the rat model may be considered to be predictive for human metabolism of deramciclane.

The spectrum of enzymes involved in activation of 2-aminoanthracene varies with the metabolic system applied.

The aim of this study was to estimate the involvement of cytochrome P450s (CYPs) in the metabolic activation of 2-aminoanthracene (2AA) by use of metabolic systems such as liver S9 or hepatocytes from untreated and beta-naphthoflavone (BNF)- or phenobarbital (PB)-treated rats. Metabolic activation was determined in the Salmonella reverse mutation assay (Ames test). Unexpectedly, both enzyme inducers, BNF and PB, significantly decreased the mutagenicity of 2AA activated by S9 fractions. 2AA mutagenicity was detected in the presence of cytochrome P450 inhibitors such as alpha-naphthoflavone (ANF), clotrimazole and N-benzylimidazole to study the contribution of CYP isoenzymes to the activation process. ANF significantly decreased the activation of 2AA by S9 from untreated rats. In contrast, ANF significantly increased the metabolic activation of 2AA by S9 from BNF- and PB-treated rats. The enhanced mutagenicity was not altered by co-incubation with clotrimazole and ANF. Pre-incubation of 2AA in the presence of N-benzylimidazole significantly increased the activation of 2AA by S9 from BNF- and PB-treated rats, which suggests that CYPs play minor role in 2AA metabolic activation by rat liver S9 fractions. In contrast with the results described above, BNF treatment of rats significantly enhanced the activation of 2AA by hepatocytes. ANF attenuated the extent of this activation suggesting that different enzymes play a major role in the activation processes in these metabolic systems. Our results indicate that identification of mutagenic hazard by use of the Ames test may depend on the metabolic system applied.

Canalicular and sinusoidal disposition of bilirubin mono- and diglucuronides in sandwich-cultured human and rat primary hepatocytes.

Due to cholestasis or adverse drug effects, the excretion of bilirubin conjugates can decrease; therefore, the level of bilirubin (B) and bilirubin glucuronides (BGs) increases in the serum with the concomitant shift of bilirubin diversus monoglucuronide (BDG/BMG) equilibrium. The aim of this study was to utilize the collagen-sandwich culture of hepatocytes as an in vitro model for studying B conjugation and canalicular versus sinusoidal disposition of BGs. Canalicular and sinusoidal efflux of BMG and BDG obtained in sandwich-cultured rat primary hepatocytes was compared with that measured in human hepatocyte cultures. The BMG and BDG were separated by high-performance liquid chromatography and identified by mass spectrometry. The biliary excretion index (BEI) was estimated by measuring disposition of BGs into standard and Ca(2+), Mg(2+)-free medium. Significantly more BGs were excreted into the canalicular networks than into the medium in 96-h sandwich culture of both human and rat hepatocytes (BEI, 62.5 and 80.6, respectively). The BDG/BMG ratio in the medium versus that in the canalicular networks was 0.55/1.48, which is similar to the serum/bile values (0.6/1.5) observed in vivo by Mesa et al. [Mesa VA, De Vos R, and Fevery J (1997) J Hepatol 27:912-916]. In contrast, the BEI for p-nitrophenol glucuronide was 5.2. The low BEI value is in agreement with empirical observations, which suggest that molecules with low molecular weight are preferably excreted by the kidney. In conclusion, sandwich-cultured primary hepatocytes provide a useful in vitro method to differentiate between sinusoidal and canalicular disposition of BGs. Since the normal BDG/BMG ratio changes in hyperbilirubinemia, this model could be used to predict drug effects leading to hyperbilirubinemia.

[The role of drug metabolizing enzymes in the effect and side-effect of the drugs]. / A gyógyszer-metabolizáló enzimrendszerek szerepe a gyógyszerek hatásában, illetve mellékhatásában.

The authors give an overview on the role of drug metabolizing enzymes in the effect and side-effect of drugs. They describe the properties of cytochrome P450 and other phase I enzymes (hydrolases, flavin-monooxygenases), as well as of phase II. enzymes (glucuronyl-, sulfate-, glutathione-, acetyl-, methyltranferases), their polymorphism, inductive and inhibition properties. The possible drug-drug interactions are discussed in detail.

Role of CYP2E1 in deramciclane metabolism.

The aim of our study was to identify the form(s) of cytochrome P450 responsible for the metabolism of deramciclane, a new anxiolytic drug candidate. The main routes of biotransformation in hepatic microsomes were side chain modification (N-demethylation or total side chain cleavage) and hydroxylation at several points of the molecule. Although several cytochrome P450 forms were involved in the metabolism, the role of CYP2E1 should be emphasized, since it catalyzed almost all steps. Production of deramciclane metabolites was significantly inhibited by diethyl-dithiocarbamate and was elevated in liver microsomes of isoniazid-treated rats. Furthermore, cDNA-expressed rat CYP2E1 generated the metabolites formed by side chain modification and hydroxylation. Neither deramciclane nor its primary metabolite, N-desmethyl deramciclane were able to influence directly the activity of CYP2E1. However, during the biotransformation, one or more metabolites must have been formed which were potent inhibitors of CYP2E1.

The effect of synthetic glucocorticoid, dexamethasone on CYP1A1 inducibility in adult rat and human hepatocytes.

Glucocorticoids act synergistically with polycyclic aromatic hydrocarbons in increasing mRNA and protein levels of CYP1A1 in rat liver. The action of dexamethasone to modify CYP1A1 expression has been investigated in adult human hepatocytes. The effect of dexamethasone on the induction of CYP1A1 by 3-methylcholanthrene is different in rat and human liver cells. Dexamethasone potentiates the induction of CYP1A1 about 3- to 4-fold in rat cells. In human hepatocytes, it reduces CYP1A1 induction by 50-60% at enzyme protein level, while it does not have an effect on CYP1A1 mRNA amount.

Inhibition of cytochrome P450 enzymes participating in p-nitrophenol hydroxylation by drugs known as CYP2E1 inhibitors.

p-Nitrophenol hydroxylation is widely used as a probe for microsomal CYP2E1. Several drugs are known as CYP2E1 inhibitors because of their capability to inhibit p-nitrophenol hydroxylation. Our results suggest further participation of CYP2A6 and CYP2C19 enzymes in p-nitrophenol hydroxylation. Moreover, CYP2A6 and CYP2C19 may be considered as the primary catalysts, whereas CYP2E1 can also contribute to the hydroxylation of p-nitrophenol. Further aim of our study was to evaluate the selectivity of p-nitrophenol hydroxylase inhibitors towards cytochrome P450 enzymes. The effects of antifungals: bifonazole, econazole, clotrimazole, ketoconazole, miconazole; CNS-active drugs: chlorpromazine, desipramine, fluphenazine, thioridazine; and the non-steroidal anti-inflammatory drug: diclofenac were investigated on the enzyme activities selective for CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4. None of the drugs could be considered as a potent inhibitor of CYP2E1. Strong inhibition was observed for CYP3A4 by antifungals with IC(50) values in submicromolar range. However, ketoconazole was the only imidazole derivative that could be considered as a selective inhibitor of CYP3A4. The CNS-active drugs investigated were found to be weak inhibitors of CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4. Diclofenac efficiently inhibited CYP2C9 and to a less extent CYP3A4 enzyme.

Comparative study in the Ames test of benzo[a]pyrene and 2-aminoanthracene metabolic activation using rat hepatic S9 and hepatocytes following in vivo or in vitro induction.

We studied the replacement of hepatic S9 with in vivo and in vitro induced hepatocytes as a metabolic activation system with the aim of broadening the possibilities of mutagenic assays. Rats were pretreated with beta-naphthoflavone (BNF), phenobarbital (PB), 3-methylcholanthrene (MC) and a combination of BNF and PB (BNF + PB). Mutagenic activation of benzo[a]pyrene (BP) and 2-aminoanthracene (2AA) by hepatic S9 and hepatocytes was determined in the Ames test. Primary rat hepatocytes were used for in vitro induction and were used as the activating system in the Ames test. In vivo BNF treatment greatly increased the metabolic activation capacity of hepatic S9 and hepatocytes towards BP. With regard to 2AA activation, S9 and hepatocytes showed different BNF induction profiles. PB treatment reduced the mutagenicity of both compounds. Although ethoxyresorufin O-dealkylase (EROD) activity of S9 from BNF + PB-treated animals was almost 30-fold greater than the control, its effectiveness in activation of 2AA was below the control level. A large part of the EROD activity of control cells was lost during culture, together with the ability to activate 2AA, however, 72 h of MC induction increased EROD activity to 200-fold of the control, which corresponds to 28% of that of in vivo induced hepatocytes. The mutagenic potential of BP activated by in vitro induced hepatocytes was 10-fold above the control, which is 47% of the mutagenicity detected following in vivo induction. In vitro induced hepatocytes increased 2AA mutagenicity to 14.6-fold over the control, which corresponds to 68% of in vivo induction. Our results suggest that primary culture of hepatocytes provides a useful model for the study of the role of metabolic activation processes concerning enzyme activity of cytochromes P450 and other metabolic enzymes and induction profiles of different inducers.

In vitro induction of cytochrome P450 enzymes in hepatocytes isolated from the regenerating rat liver.

Partial hepatectomy results in the loss of cytochrome P450 enzymes. During regeneration, the levels of cytochrome P450 activities, apoproteins and mRNA are reduced. Our present study investigated CYP1A, CYP2E1 and CYP3A induction in the cells of rat liver regenerating for 1, 3, 7, or 14 days. Hepatocytes were isolated from regenerating liver of hepatectomized rats and treated with enzyme inducers: 3-methylcholanthrene, imidazole and dexamethasone. CYP1A enzymes of the cells isolated from regenerating liver were inducible by 3-methylcholanthrene. The rate of induction of the cells from 3-day regenerating liver by 3-methylcholanthrene was three times higher than that of the hepatocytes of sham-operated rats. Dexamethasone caused about two- or three-fold stronger elevation of CYP3A in the cells of 1-, 3- and 7-day regenerating liver than in hepatocytes of sham-operated animals. However, the degree of CYP2E1 induction by imidazole was the same (about 2.5-fold) at each regenerating time as it was detected in the hepatocytes of sham-operated animals. In conclusion, the inducibility of the cells was retained at each regenerating time, but the degree of induction showed some differences.