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D Ratanasavanh

Publications and source records attributed to D Ratanasavanh.

At least 19 recordsLinked to original sources
PubMed2001

Metabolism of methoxymorpholino-doxorubicin in rat, dog and monkey liver microsomes: comparison with human microsomes.

The morpholino anthracycline, methoxymorpholino-doxorubicin (MMDx) is a novel anticancer agent. The metabolism of this highly lipophilic doxorubicin analogue is not fully elucidated. MMDx is metabolically activated in vivo, resulting in an 80-fold increase in potency over the parent drug. In this study, MMDx in vitro metabolism was compared in rat, dog, monkey and human liver microsomes. When microsomal fractions were incubated with MMDx, 6-8 metabolites were formed depending on the species and on the substrate concentrations. Among these eight metabolites, three comigrated with authentic standards, namely MMDx-ol, PNU156686 and PNU159682, and the five others are in the process of being characterized. Quantitatively, monkey and human metabolize MMDx with a higher rate than rat and dog. Qualitatively, MMDx metabolic profile in dog microsomes was different from the three other species. MMDx-ol was predominant in dog and only minor in other species. In conclusion, MMDx metabolism was species-different. Rat and monkey liver microsomes may be used as models to study MMDx metabolism in humans. Dog liver microsomes may be a good model for studying the formation of MMDx-ol.

Animals↗
PubMed2000

Stereoselective biotransformation of cicletanine in cultured rat and human hepatocytes.

Cicletanine [(+/-)-C] is a racemic furopyridine derivative used as an antihypertensive agent. Pharmacokinetic and metabolic studies have shown that cicletanine is rapidly and almost fully metabolized into sulfo- and glucuro-conjugated metabolites. However, the stereoselective metabolism of cicletanine is not well-known in humans. In the present study, the stereoselective aspect of cicletanine metabolism was investigated in cultured hepatocytes from humans and rats. The two enantiomers of cicletanine were both strongly metabolized in rat hepatocytes. So, after 24 h of incubation, very low amounts of free cicletanine were found [1.2% for (+)-C and 2.7% for (-)-C], respectively. In addition (+/-)-C was mainly biotransformed into conjugated metabolites: (-)-C mainly transformed into (-)-C-glucuronide (78.3+/-6.4%) and (+)-C mainly into (+)-C-sulfate (87.4+/-3.3%). In human hepatocytes, inter-individual variations were more marked than in rat hepatocytes. In addition (+/-)-C biotransformation in human was lower than the one observed in rat. (-)-C enantiomer was more metabolized than (+)-C. After a 24-h incubation the percentages of free (+)-C and (-)-C were 32.3+/-16.4 and 8.2+/-10.3, respectively. On the contrary to rat hepatocytes, both cicletanine enantiomers in humans were mainly metabolized into glucuroconjugated metabolites. These results clearly demonstrated that cicletanine underwent stereospecific metabolism in both rat and human hepatocytes.

Animals↗
PubMed1999

Effects of oxazepam and acetaminophen on cicletanine metabolism in rat hepatocytes and liver microsomes.

Cicletanine, a racemic furopyridine derivative synthesized as racemate, is used as an antihypertensive agent. Its two enantiomers are involved in the pharmacological effects of the drug. Cicletanine is metabolized by conjugation enzyme systems (phase II) into sulfoconjugated or glucuroconjugated enantiomers. As oxazepam and acetaminophen are widely prescribed, especially to elderly patients, these two drugs may be co-administered with cicletanine. The metabolic profile and the kinetics of biotransformation were studied by using rat hepatocytes and liver microsomes. Cicletanine was extensively metabolized by rat hepatocytes. More than 80% of the drug was biotransformed after a 3 h incubation. The formation of glucuroconjugated metabolites was characterized by the following kinetic parameters, i.e. Vmax = 2.05 +/- 0.21 nmol/min/mg protein and Km = 287 +/- 6.7 microM for (-)-cicletanine, and Vmax = 1.44 +/- 0.12 nmol/min/mg protein and K(m) = 171 +/- 4.1 microM for (+)-cicletanine. Oxazepam inhibited the glucuronidation of cicletanine in both rat hepatocytes and liver microsomes with a competitive-type inhibition, i.e. K(i) = 129 +/- 7.5 and 152 +/- 19.7 microM for (-)-cicletanine and (+)-cicletanine, respectively. The co-incubation of acetaminophen with cicletanine showed that only sulfoconjugation was inhibited in rat hepatocytes. Glucuronidation was not modified by acetaminophen. As natriuric activity is due to sulfoconjugated (+)-cicletanine, acetaminophen could potentially modulate in vivo the pharmacological effect of cicletanine. The data of the in vitro study reported here suggested an interaction between cicletanine and oxazepam or cicletanine and acetaminophen. However, the clinical impact of such a drug interaction needs further evaluation.

Acetaminophen↗
PubMed1997

Both cytochromes P450 2E1 and 3A are involved in the O-hydroxylation of p-nitrophenol, a catalytic activity known to be specific for P450 2E1.

4-Nitrophenol 2-hydroxylation activity was previously shown to be mainly catalyzed by P450 2E1 in animal species and humans. As this chemical compound is widely used as an in vitro probe for P450 2E1, this study was carried out to test its catalytic specificity. First, experiments were carried out on liver microsomes and hepatocyte cultures of rat treated with different inducers. Liver microsomes from pyrazole- and dexamethasone-treated rats hydroxylated p-nitrophenol with a metabolic rate increased by 2.5- and 2.7-fold vs control. Dexamethasone treatment increased the hepatic content of P450 3A but not that of P450 2E1. Two specific inhibitors of P450 3A catalytic activities, namely, ketoconazole and troleandomycin (TAO), inhibited up to 50% of 4-nitrophenol hydroxylation in dexamethasone-treated rats but not in controls. Hepatocyte cultures from dexamethasone-treated rats transformed p-nitrophenol into 4-nitrocatechol 7.8 times more than controls. This catalytic activity was inhibited by TAO. Similarly, hepatocyte cultures from pyrazole-treated rats hydroxylated p-nitrophenol with a metabolic ratio increased by about 8-fold vs control. This reaction was inhibited by diethyl dithiocarbamate and dimethyl sulfoxide, both inhibitors of P450 2E1. Second, the capability of human P450s other than P450 2E1 to catalyze the formation of 4-nitrocatechol was examined in a panel of 13 human liver microsomes. Diethyl dithiocarbamate and ketoconazole reduced 4-nitrophenol hydroxylase activity by 77% (+/- 11) and 13% (+/- 16), respectively. Furthermore, the residual activity following diethyl dithiocarbamate inhibition was significantly correlated with seven P450 3A4 catalytic activities. Finally, the use of human cell lines genetically engineered for expression of human P450s demonstrated that P450 2E1 and 3A4 hydroxylated 4-nitrophenol with turnovers of 19.5 and 1.65 min-1, respectively. In conclusion, P450 3A may make a significant contribution to 4-nitrophenol hydroxylase activity in man and rat.

Animals↗
PubMed1997

Glucuronidation of SN-38, the active metabolite of irinotecan, by human hepatic microsomes.

We have investigated the glucuronidation in vitro of SN-38, the active metabolite of irinotecan, a semi-synthetic anticancer drug derived from 20(S)camptothecin. Preparations of human hepatic microsomes (final concentration : 1 mg prot./ml), were incubated for 1 hr in 0.1 M Tris buffer, pH 7.4, containing 10 mM MgCl2, in the presence of UDP-glucuronic acid (4 mM), saccharolactone (4 mM), and a detergent. Microsomes from five livers were studied individually or as a pooled preparation. SN-38, either in its lactone or its carboxylate form, was added at a range of concentrations. The SN-38 beta-glucuronide formed was measured by HPLC with fluorometric detection. The glucuronidation reaction appeared linear over 1 hr in these conditions and Brij 35 at 0.5 mg/mg prot. was the best activator. The apparent parameters of the reaction were independent of the molecular form of the substrate. The half-saturation constant was 17-20 microM and Vmax was 60-75 pmol/min./mg prot. The interindividual variation of SN-38 glucuronidation was relatively low (ratio of 1.8 between extreme values). In addition, the effect of twelve drugs currently associated with irinotecan in clinics was evaluated in this system (drug concentration: 100 microM; SN-38 concentration: 5 microM). These produced little if any interference with SN-38 glucuronidation. Therefore, major interferences of this transformation by comedications are unlikely to occur in vivo.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Metabolism and toxicity of coumarin on cultured human, rat, mouse and rabbit hepatocytes.

We compared the cytotoxic effect of coumarin and its derivatives, 7-hydroxycoumarin (7-OHC), 4-hydroxycoumarin (4-OHC), o-hydroxyphenyl acetic acid (OHPAA) and o-coumaric acid (CA), on cultured hepatocytes from human, rat, mouse and rabbit liver. At 10(-5) and 5 x 10(-5) M, coumarin and its derivatives did not give rise to any signs of toxicity on cultured hepatocytes of the four species. At 10(-4) M, coumarin, but not its derivatives, induced release of lactate dehydrogenase (LDH) into the medium, especially in rat hepatocyte cultures. Intracellular LDH activities were correspondingly reduced. The cytotoxic effect of coumarin in cultured rat hepatocytes was evidenced on morphological examination and from the results of the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium (MTT) reduction test. At higher concentrations (5 x 10(-4) M), 7-OHC and CA were also found to be cytotoxic in cultured rat hepatocytes. The cytotoxic effect of coumarin (5 x 10(-4) M) was decreased in the presence of SKF 525-A, a cytochrome P450 inhibitor. Interspecies comparisons showed that rat hepatocytes were the most sensitive to the toxicity of coumarin and its derivatives, whereas human hepatocytes were the most resistant. Our results suggest that the cytotoxicity of coumarin is metabolism and species-dependent. Thus, the rat may not be a suitable model for evaluating the pharmacological hazards of coumarin in humans.

4-Hydroxycoumarins↗
PubMed1992

Modulation of multidrug resistance gene expression in rat hepatocytes maintained under various culture conditions.

P-glycoprotein (P-gp), the multidrug resistance gene product, is overexpressed in normal adult rat hepatocytes under standard culture conditions. We have studied the modulation of P-gp expression in this in vitro model in the presence of both epidermal growth factor and pyruvate, which favor hepatocyte growth, as well as in the presence of either dimethyl sulfoxide (DMSO) or nicotinamide, which favors maintenance of differentiated functions. P-gp overexpression, estimated by northern blotting and doxorubicin-mediated drug efflux analyses, was similarly observed during culture in both standard and proliferating conditions, while it was delayed, but not inhibited, in the presence of DMSO or nicotinamide. These results suggest that the functional P-gp overexpression occurring in rat hepatocytes when exposed to an unfamiliar environment is at least partly not related to cell proliferation or the degree of cell differentiation in vitro.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Overexpression of the multidrug resistance gene product in adult rat hepatocytes during primary culture.

Expression of P-glycoprotein (P-gp), the product of multidrug resistance gene(s), was investigated in primary cultures of normal adult rat hepatocytes. Levels of P-gp mRNAs determined by Northern blotting and of P-gp measured by immunoblotting increased in parallel with time in culture. As in normal liver, P-gp was found to be localized on the membrane of bile canaliculus-like structures. This increased expression of P-gp was associated with decreased intracellular retention of doxorubicin, which could be restored by compounds such as verapamil and cyclosporin; doxorubicin (and also vincristine) was more cytotoxic to early than to late cultures. As in preneoplastic and neoplastic liver, overexpression of P-gp in cultured hepatocytes was associated with differential changes in drug-metabolizing enzymes, including increased glutathione S-transferase 7-7. Functional P-gp over-expression was observed in the absence of xenobiotic exposure or cell division; it could be linked to cellular stress occurring during cell isolation and plating. Increased expression of P-gp was blocked by actinomycin D, indicating its dependence on increased transcription, while cycloheximide led to a superinduction suggesting negative regulation by a protein factor.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

In vitro assessment of stereoselective hepatic metabolism of disopyramide in humans: comparison with in vivo data.

Metabolism of disopyramide (DP) enantiomers has been investigated in primary cultures of adult human hepatocytes. Results were compared with in vivo data obtained from a previous pharmacokinetic study (Le Corre et al. Drug Metab. Dispos. 16:858-864 1988). Metabolism of DP enantiomers as a function of incubation time showed constant velocity over time. The intracellular/extracellular distribution of both DP and mono-N-desisopropyldisopyramide did not appear to be stereoselective. Metabolism of DP enantiomers as a function of substrate concentration followed a first order kinetics. The average fractions of (-)-(R)-DP and (+)-(S)-DP metabolized in vitro (4.7 +/- 2.7 and 7.1 +/- 4.2%, respectively, n = 4) were about 5-fold lower than the fractions metabolized in vivo (26.0 +/- 6.0 and 40.2 +/- 8.8%, respectively, n = 6). The stereoselective index [(+)-(S)/(-)-(R)] of the N-dealkylation pathway obtained in vitro (1.51 +/- 0.11, n = 4) was very close to the one obtained in vivo (1.55 +/- 0.10, n = 6). These results highlight the interest of hepatocyte cultures in the evaluation of drug metabolism and especially in the assessment of stereoselectivity.

Adult↗
PubMed1991

Metabolism of doxorubicin, daunorubicin and epirubicin in human and rat hepatoma cells.

The metabolism of three anthracyclines, namely daunorubicin, doxorubicin and epirubicin, was studied in two human (HepG2 and HBG2) and one rat (FAO) hepatoma cell lines. Both species and substrate differences in anthracycline metabolism were retained by these hepatoma cell lines with regard to the aldoketoreductase pathway. However, glucuronidation of epirubicin normally found in vivo and in human hepatocyte cultures was not recovered in human hepatoma cells, further indicating that these cells express only some of the liver functions. Nevertheless, these cell lines could be a suitable model to investigate the still poorly understood aldoketoreductase activity.

Animals↗
PubMed1991

Intralobular distribution and quantitation of cytochrome P-450 enzymes in human liver as a function of age.

We have used immunohistochemical, immunoblotting and messenger RNA blotting approaches to study the distribution and quantitation of three cytochrome P-450 enzymes, namely P-450 IA2, P-450 IIC and P-450 IIIA and, for comparison, epoxide hydrolase and NADPH-cytochrome P-450 reductase in human liver. Age-related changes in both the amounts and the intralobular distributions of these enzymes were demonstrated, and the enzymes differ in these regards: In fetal liver, P-450 IA2 and P-450 IIC were very low, when present at all, whereas P-450 IIIA, epoxide hydrolase and the reductase were already abundant and found in all the hepatocytes. During the postnatal period, P-450 IIC dramatically increased and was observed in all hepatocytes, the centrilobular ones being more intensely stained. P-450 IIIA was restricted to centrilobular and midzonal hepatocytes in normal adult liver. P-450 IA2 showed this same intralobular distribution; however, its presence was detected only several weeks or months after birth as judged both by immunohistochemical and immunoblotting techniques. Epoxide hydrolase and NADPH-cytochrome P-450 reductase were easily visualized in all hepatocytes regardless of the age of the donor; in child and adult livers, centrilobular hepatocytes were more intensely stained. Immunoreactive protein contents and corresponding messenger RNA levels correlated well with immunohistochemical observations. No major modification was seen in fibrotic liver, whereas both positive and negative cells were observed in cirrhotic liver nodules for all enzymes studied.

Aging↗
PubMed1990

Expression of cytochrome P-450 enzymes in cultured human hepatocytes.

Hepatocytes from adult and newborn humans were put into primary culture and exposed to phenobarbital, 3-methylcholanthrene, or rifampicin, three well-known inducers of cytochrome P-450 in animals. The expression of four cytochrome P-450 enzymes (or groups of enzymes, namely P-450 IIIA, P-450 IIC8/9/10, P-450 IIE1, and P-450 IA2) was investigated. These enzymes were found to remain expressed during the period of culture studied. Treatment with the inducers for three days resulted in different responses, depending upon the inducer and the enzyme. Phenobarbital and rifampicin increased P-450 IIC8/9/10 mRNA transcripts and the corresponding protein, while 3-methylcholanthrene was ineffective. Both P-450 IIIA mRNA and protein were strongly induced by rifampicin. All of the hepatocytes were found to synthesize P-450 IIIA in response to rifampicin, as shown by immunoperoxidase staining. P-450 IIIA expression was not affected by phenobarbital and was decreased by 3-methylcholanthrene. P-450s IA2 and IIE1 decreased to 25-50% of the initial level during these cultures. P-450 IA2 and ethoxyresorufin O-deethylase activity (which is a monooxygenase activity related to P-450 IA family) were increased only by 3-methylcholanthrene and did not respond to the other inducers. P-450 IIE1 was not induced by any of these compounds. P-450 IIC8/9/10 and P-450 IIIA mRNA levels were also measured in human hepatocytes from one newborn. P-450 IIC8/9/10 was barely expressed in freshly isolated cells but increased dramatically with time in culture. P-450 IIIA transcripts were abundant in both freshly isolated and cultured cells derived from a newborn. These results clearly demonstrate that human hepatocytes continue to express cytochrome P-450 enzymes and respond to inducers in culture. This model system provides a useful approach for investigating the effects of drugs on maturation and expression of drug-metabolizing enzymes in human liver.

Adult↗
PubMed1990

Methylcholanthrene but not phenobarbital enhances caffeine and theophylline metabolism in cultured adult human hepatocytes.

Biotransformation of caffeine and theophylline and the effect of two well-known inducers of P-450 isozymes, namely phenobarbital (PB) and methylcholanthrene (3-MC) were studied in cultured hepatocytes from six human adult donors. Hepatocytes co-cultured with rat liver epithelial cells maintained a higher metabolic capacity than pure cultures. PB treatment of cultured hepatocytes for 3 days slightly increased the rate of caffeine metabolism 1.4 +/- 0.5-fold (N = 6) vs controls, and theophylline metabolism 1.2 +/- 0.4-fold (N = 6), whereas 3-MC treatment increased metabolism markedly 5.8 +/- 2.3- and 3.3 +/- 1.1-fold (N = 6) vs controls for caffeine and theophylline, respectively. Paraxanthine and theophylline formations from caffeine were the most induced by 3-MC. Their increase was significantly correlated (rs = 0.89, P less than 0.007) but not with TB formation, suggesting that at least two isozymes of the P-450IA family are involved in the first demethylations of caffeine. In addition, the N-1 demethylation of theophylline (mean increase of 554% vs controls) was not correlated with the N-1 demethylation of caffeine (mean to increase 247% vs controls) for the same donor after 3-MC treatment, suggesting that these two demethylations are mediated by a different P-450.

Adult↗
PubMed1990

Changes in expression of mRNA coding for glutathione S-transferase subunits 1-2 and 7 in cultured rat hepatocytes.

mRNA hybridizing to probes for glutathione S-transferase (GST) subunits 1 and 2 (probe pGSTr 155) and subunit 7 (probe pGSTr 7) has been measured by Northern blot analysis in adult rat hepatocytes both in conventional monoculture and in co-culture with epithelial cells. In addition, several media conditions were used, namely with and without fetal calf serum (FCS) and with and without nicotinamide or dimethyl sulfoxide (DMSO). In monoculture, mRNA coding for subunits 1 and 2 was extensively reduced in the presence of FCS. In the absence of FCS, after an initial decrease, an increase of subunits 1 and 2 mRNA was noticed on day 6. When nicotinamide or DMSO was added to the medium, the GST subunits 1 and 2 mRNA level increased during the culture period. In co-culture, an initial reduction in levels of mRNA encoding subunits 1 and 2 was less marked and the values measured increased with co-culture time. Nicotinamide tended to reduce these mRNA levels, whereas DMSO increased them. In contrast, in conventional culture, mRNA encoding subunit 7 was expressed de novo and this induction was prevented by DMSO but not by nicotinamide. Similar results were obtained with co-culture.

Animals↗
PubMed1989

Regulation of glutathione S-transferase subunits 3 and 4 in cultured rat hepatocytes.

mRNA levels of glutathione S-transferase (GST) subunits 3 and 4 were measured with a specific cDNA probe in adult rat hepatocytes maintained either in conventional culture or in coculture with rat liver epithelial cells. Four media conditions were used, i.e. with or without fetal calf serum (FCS) and with nicotinamide or dimethylsulfoxide (DMSO). When FCS was present in the culture medium, GST subunit 3 and 4 mRNAs were expressed at a level close to that found in freshly isolated hepatocytes during the whole culture period both in conventional culture and in coculture. All other culture conditions resulted in an increase of GST 3 and 4 mRNA levels. After exposure to phenobarbital an increase in GST 3 and 4 mRNA levels was demonstrated in both culture systems. Comparison with previous findings on the expression of GST subunits 1, 2 and 7 in the same culture conditions indicates that the different classes of GST are regulated independently.

Animals↗
PubMed1989

Comparison of caffeine metabolism by slices, microsomes and hepatocyte cultures from adult human liver.

1. Caffeine was used as a molecular probe for hepatic monooxygenase activity in three in vitro models from human livers: slices, microsomes and hepatocyte cultures. 2. A h.p.l.c. method for determination of all possible metabolites of caffeine (16 compounds) is described. 3. Caffeine biotransformation by these three in vitro systems was low. However, metabolite formation was shown to proceed at a rate close to that calculated from in vivo caffeine elimination half-life. 4. Metabolic profiles were the same whatever the in vitro system used. The ratio of primary demethylated metabolites was similar to that measured by in vivo studies, i.e. the selectivity for caffeine N-3 demethylation to paraxanthine was retained. 5. Significant correlation (p less than 0.001) between 7-ethoxyresorufin O-deethylation and caffeine demethylations was demonstrated for the 12 human livers studied.

Adolescent↗
PubMed1988

Different cytotoxicity and metabolism of doxorubicin, daunorubicin, epirubicin, esorubicin and idarubicin in cultured human and rat hepatocytes.

Both cytotoxicity and metabolism of five anthracyclines, namely doxorubicin, daunorubicin, epirubicin, esorubicin and idarubicin, were investigated in primary cultures of both rat and human adult hepatocytes and, for comparison, in a rat liver epithelial cell line. Toxicity was assessed by morphological examination and measurement of lactate dehydrogenase leakage after 24 hr of treatment. The rank order of toxicity for both rat and human hepatocytes was esorubicin greater than doxorubicin = epirubicin greater than or equal to idarubicin greater than daunorubicin, and for rat epithelial cells: esorubicin greater than or equal to epirubicin greater than idarubicin = daunorubicin = doxorubicin. Human cells were around 2-fold less sensitive than rat hepatocytes to all anthracyclines. Anthracyclines and their metabolites were analyzed by HPLC. Differences in both the percentages and routes of metabolism were demonstrated between rat and human hepatocytes. The main metabolite was the 13-dihydro-derivative (-ol derivative) in both species from daunorubicin, idarubicin and esorubicin. Glucuronides of epirubicin and epirubicinol were found only in human hepatocytes. In addition, several unidentified metabolites were detected of esorubicin, idarubicin and daunorubicin in rat hepatocytes. In human hepatocytes, only one unknown metabolite from daunorubicin and doxorubicin was found to be formed by cells from a different donor. In spite of variations between individuals, human hepatocytes generally metabolized anthracyclines more actively than did rat hepatocytes. Rat liver epithelial cells were only able to convert daunorubicin and idarubicin, the two molecules which have the best affinity for the non-specific NADPH-dependent aldoketoreductase system. Three compounds (doxorubicin, epirubicin and esorubicin) were present in large amounts in the cells as the parent drug, another (idarubicin) as the 13-dihydro-derivative. This comparative study on cytotoxicity and metabolism of five anthracyclines in rat and human hepatocyte cultures emphasises species differences and the importance of this in vitro model system for further analysis of the metabolism and effect of anthracyclines.

Adolescent↗