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Amprenavir is a human immunodeficiency virus-1 (HIV-1) protease inhibitor intended to be used to treat HIV-infected children. Although a pediatric dosage is proposed by the manufacturer, no data are currently available on the pharmacokinetics of amprenavir in neonates and infants. Amprenavir being primarily eliminated after oxidative biotransformation, we explored its in vitro metabolism by cytochrome P450 (P450)-dependent monooxygenases. In our conditions, five metabolites were formed in vitro and subsequently analyzed by liquid chromatography-mass spectrometry; P450-dependent oxidations occurred either on the tetrahydrofuran ring (M3 and M4), the aniline ring (M5), and the aliphatic chain (M2) or resulted from the N-dealkylation and loss of the tetrahydrofuran ring (M1). The two major metabolites, respectively M3 and M2 were formed by human liver microsomes with K(m) between 10 and 70 microM. CYP3A4 and to a lesser extent CYP3A5 were major contributors for the formation of M2, M3, and M5 metabolites, whereas CYP3A7 had no or little activity. This assumption was confirmed by inhibition with ketoconazole and ritonavir (two potent inhibitors of CYP3A) whereas sulfaphenazole (2C9 inhibitor) and quinidine (2D6 inhibitor) were inefficient. The metabolism of amprenavir was negligible in microsomes from either fetuses or neonates and steadily increased after the first weeks of life in relation with the maturation of CYP3A4/5. In conclusion, results demonstrated that the capacity of the human liver to oxidize amprenavir is low during the first weeks after birth and that dosage could be substantially reduced during the early neonatal period.

A modification of the human monooxygenase system have been previously associated with the sudden infant death syndrome (SIDS): the hepatic CYP2C content was markedly enhanced and resulted from an activation of CYP2C gene transcription. To determine the possible consequence of the up-regulation of CYP2C in SIDS, we examined the metabolism of arachidonic acid (AA) an endogenous substrate of CYP2C involved in the physiologic regulation of vascular tone. The overall AA metabolism was extremely low during the fetal period and rose after birth to generate 14,15 epoxyeicosatrienoic acid (EET), 11,12 EET and the sum of 5,6 dihydroxyeicosatrienoic acid (diHETE)+omega/omega-1 hydroxy AA. In SIDS, the accumulation of CYP2C proteins was associated with a significant increase in the formation of 14,15 and 11,12 diHETE, which were shown to be supported by individually expressed CYP2C8 and 2C9 and HETE1 (presumably 15 HETE). This increase was markedly inhibited by addition of sulfaphenazole, a selective inhibitor of CYP2C9. So, we propose that the higher CYP2C content in SIDS stimulates the production of EETs and diHETEs and might have severe pathologic consequences in children.

The ontogenesis of CYP1A proteins was investigated in a human liver bank composed of fetal, neonatal and adult samples. In immunoblots, a polyclonal antibody raised against rat CYP1A1, cross-reacted with cDNA-expressed human CYP1A1 and CYP1A2. In adult liver microsomes, this antibody reacted with a single band identified as the CYP1A2 protein, while no CYP1A1 could be detected. CYP1A2 protein was absent in microsomes prepared from fetal and neonatal livers and its levels increased in infants aged 1-3 months to attain 50% of the adult value at one year. Enzymatic activities supported by CYP1A proteins were assayed on these samples. Methoxyresorufin demethylase supported by the CYP1A2 recombinant protein followed the same ontogenic profile as the CYP1A2 protein. In liver microsomes, the demethylation of imipramine was essentially due to CYP1A2 and to a smaller extent to CYP3A. In fetuses and early neonates, CYP3A proteins were responsible for the low demethylation of imipramine (3-4% of the adult activity) before the onset of CYP1A2 and the subsequent rise of activity. Immunodetection and enzymatic activities were consistent with the absence of CYP1A1 and the late expression of CYP1A2 in the human liver, compared to the early rise of CYP3A4, CYP2C, CYP2D6, and CYP2E1 proteins.

CYP3A isoforms are responsible for the biotransformation of a wide variety of exogenous chemicals and endogenous steroids in human tissues. Two members of the CYP3A subfamily display developmentally regulated expression in the liver; CYP3A7 is expressed in the fetal liver, whereas CYP3A4 is the major cyrochrome P-450 isoform present in the adult liver. To gain insight into the descriptive ontogenesis of CYP3A isoforms during the neonatal period, we have developed several approaches to explore a neonatal liver bank. Although CYP3A4 and CYP3A7 are structurally closely related, they differ in their capacity to carry out monooxygenase reactions. We have cloned CYP3A4 and CYP3A7 and established stable transfectants in Ad293 cells to investigate their substrate specificities. The 16alpha hydroxylation of dehydroepiandrosterone is catalyzed by both proteins, but CYP3A7 has a higher affinity and maximal velocity than CYP3A4. Conversely, the conversion of testosterone into its 6beta derivative is essentially supported by CYP3A4. We used these two probes to determine the ontogenic evolution at the protein level; CYP3A7 was very active in the fetal liver and its activity was maximal during the first week following birth before to progressively decline and reached a very low level in adult livers. Conversely, the activity of CYP3A4 was extremely weak in the fetus and began to raise after birth to reach 30-40% of the adult activity after one month. CYP3A4 RNA accumulation displays a similar pattern of evolution; when probed with an oligonucleotide, its concentration increased rapidly after birth to reach a plateau as soon as the first week of age. These data supports the assumption that CYP3A4 expression is transcriptionally activated during the first week after birth and is accompanied by a simultaneous decrease of CYP3A7 expression, in such a way that the overall CYP3A protein content and the level of pentoxyresorufin dealkylase catalyzed by the two proteins remain nearly constant.

In the human liver, the major rise of the cytochrome P-450 isoform content occurs during the first months following birth (e.g., the high vulnerability period to sudden infant death syndrome (SIDS), a syndrome frequently associated with viral infection and drug hypersensitivity. We examined the expression of individual P-450 isoforms in liver samples collected postmortem from SIDS infants and compared values with those of control adults and children of the same age suffering from various pathologies. Hepatic microsomes were prepared and examined for their content in total P-450, the level of individual isoforms (CYP1A2, CYP2E1, CYP4A, CYP3A, and CYP2C) determined with specific antibodies and for their enzymatic activities. Total RNA was extracted and probed with several CYP cDNAs and oligomers. The overall hepatic P-450 content was not modified in SIDS infants. Among cytochrome P-450 isoforms, only CYP2C was markedly increased. This rise resulted from an accumulation of RNA encoding CYP2C and was associated with a stimulation of diazepam demethylation. The precocious expression of CYP2C in SIDS could result in a higher production of epoxyeicosatrienoic acids in the neonate, believed to act as relaxant of pulmonary smooth muscles. Its consequence might be the induction of fatal apnea in SIDS.

Cytochromes P-450 are responsible for the biotransformation of drugs and other hydrophobic molecules by the liver. Several isoforms coexist which display an asynchronous onset during the perinatal period suggesting the involvement of multiple mechanisms of regulation. In this paper, we have shown that the CYP2E1 protein and its associated activity could not be detected in the fetal liver and rise during the first few hours following birth independently of the gestational age (between 25-40 weeks). During this period, the CYP2E1 RNA content remains fairly low: the stabilization of the low amount of existing CYP2E1 protein by endogenous ketone bodies could explain the early neonatal rise of the protein level. From 1 month to 1 year, the protein content gradually increases and is accompanied by the accumulation of CYP2E1 RNA, suggesting a transcriptional activation of the gene during the late neonatal period. We examined the methylation status of CpG residues in the 5' flanking region, first exon and first intron of CYP2E1 gene cleaved with HpaII/MspI. Genomic DNA from fetal liver shows several hypermethylated spots in the first-exon-first-intron region, which progressively disappear in neonatal samples. We conclude that during the neonatal period, the accumulation of hepatic CYP2E1 RNA is correlated with the degree of methylation at the 5' end of the CYP2E1 gene.

A cDNA was isolated from a library prepared with 15-day-old rat liver RNA and was shown to correspond to CYP2C23 with some minor alterations which modified the open reading frame. The expression of CYP2C23 was examined in rat liver, kidney, lung and testis and during ontogenesis. An appreciable amount of CYP2C23 RNA was observed in liver and kidney but the age-dependent expression was quite different between these two tissues. In liver, expression reached its maximal value in early neonates and remained quite stable, while the increase was progressive in kidney before declining after 3 weeks of age. In the liver, classical inducers of cytochrome P-450 decreased the CYP2C23 RNA content. Experimental data confirm the classification of CYP2C23 as a constitutive member of the 2C subfamily and clearly establish its age- and tissue-dependent expression in rat.

Testosterone metabolism was studied in human adult and fetal liver microsomes. In fetal livers 6 beta-hydroxylase (6 beta OH) activity (1-2% of adult activity) and 2 alpha-hydroxylase (2 alpha OH) activity (about 40% of adult activity) were present. Also some fetal livers produced two unknown metabolites. Androstenedione was formed in all fetal livers studied (10-20% of adult activity). Testosterone hydroxylations at 6 beta-, 2 beta-, 15 alpha- and 15 beta-positions were associated with CYP3A isoform(s) in adult liver, because they were strongly inhibited by midazolam, a known substrate for CYP3A4 and by anti-CYP3A4 antibody. Fetal liver activities were consistently inhibited less than the activities in adult livers. The formation of androstenedione was not affected by these inhibitors in fetal or adult liver microsomes. Benzphetamine N-demethylase activity in the fetal livers was about 40% of adult activity. Anti-CYP3A4 antibody had no effect on that activity in fetal or in adult liver microsomes, whereas a monoclonal antibody 1-68-11 (generated against rat CYP2C11) slightly inhibited benzphetamine N-demethylase activity in adult liver. This study indicates that human fetal and adult liver are dissimilar in their testosterone metabolism pattern. The formation of androstenedione from testosterone in fetal liver may have a physiological role. Testosterone hydroxylases are less inhibited by anti-CYP3A4 antibody, midazolam and progesterone in fetal than in adult liver.

Cytochrome P-450 2C11 (CYP2C11) is the main isozyme present in adult male rat liver and specifically hydroxylates testosterone in positions 2 alpha and 16 alpha. In Gunn rats, this isozyme is recognized by the anti-CYP2C11 antibody but its activity towards testosterone is dramatically decreased. Moreover, peptide mapping, after digestion of microsomal fractions by V8 protease and probing with anti-CYP2C11 antibody, exhibit a pattern which differs from that obtained with Wistar rats. Taken together, data suggest that the protein sequence of CYP2C11 from the Gunn rat differs from that of the Wistar rat.

Gunn rats are characterized by hereditary hyperbilirubinemia and a decrease, when compared to Wistar rats, in hepatic heme pool size which could result from an alteration of mitochondrial functions. Unconjugated bilirubin present in Gunn rat liver did not modify either the ultrastructural morphology or oxidative metabolism of the mitochondria as compared to those in Wistar rat liver. However, 5-aminolevulinic acid synthase activity is reduced by nearly 40% in Gunn rat liver mitochondria, thus explaining the reduced size of the hepatic heme pool.

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of alpha 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.

The CYP2D6 protein is a polymorphic isoenzyme involved in the biotransformation of several drugs including the probe drug dextromethorphan. The rise in the protein concentration, immunochemically determined with a specific antibody, was shown to occur within the first week following birth, whatever the gestational age at birth. In fetuses, the concentration of hepatic CYP2D6 protein was very low or undetectable in 70% of samples tested. In the remaining 30%, its concentration was comparable to that of newborns aged 1-7 days. This early rise was associated with spontaneous abortion in 70% of positive samples, whereas in fetuses with an intermediate CYP2D6 protein concentration, 80% were from induced abortions. The rise in CYP2D6 protein was associated with the developmental onset of dextromethorphan O-demethylation, but not N-demethylation, even if activity was lower in fetal than in neonatal and in adult liver microsomes. Lastly, the CYP2D6 RNA is detectable earlier than the protein and exhibits a peak of hepatic accumulation in newborns, before declining in adulthood. A positive correlation between RNA accumulation and protein concentration can be demonstrated only in the adult. This suggest that regulation is primarily at the transcriptional level, but cannot rule out the participation of post-transcriptional events in the regulation process throughout ontogenesis.

This preliminary study reports the functional capacities of freshly isolated human hepatocytes in regard to their energetic metabolism and monooxygenase activities. Incubated for 30 or 60 min, isolated cells maintain their membrane integrity, ATP and reduced glutathione content and redox potential estimated by means of lactate to pyruvate and beta-hydroxypyruvate to acetoacetate ratios. Three monooxygenase activities, supported by different isoenzymes of cytochrome P-450 are determined by the accumulation of unconjugated metabolites: their relative magnitudes are similar to those observed in microsomes, indicating a good preservation of hydroxylase activities during cell isolation and incubation. Although incubations did not exceed 60 min, one can conclude that human hepatocytes maintain their viability and metabolic capacities after isolation and might be considered in transplantation process for the treatment of acute hepatic failure. Isolated human hepatocytes might be also used as a tool for studying biochemical and toxicological effects of a drug.

Drug-metabolizing enzyme activities were determined in liver microsomes from six kidney-transplant donors, one tricyclic antidepressant-treated and five untreated donors. The tricyclic antidepressant treatment modifies neither the overall cytochrome P-450 content of the liver, nor enzymatic activities of 4-nitroanisole demethylase, aniline hydroxylase, epoxide hydrolase and glutathione S-transferase. Only benzphetamine and ketotifen demethylation and conjugation of bilirubin with UDP-glucuronic acid are markedly augmented (more than two-fold). Separation of the different cytochrome P-450 fractions on a DEAE cellulose column indicates a modification of the elution pattern: the fraction increased by tricyclic antidepressants is responsible for the enhanced monooxygenase activity towards benzopyrene and benzphetamine.

In neonatal and, to a lesser extent, in fetal rat liver, 9-hydroxyellipticine was able to promote the induction of cytochrome P-450, supporting especially aryl hydrocarbon hydroxylase (AHH) but not aldrin epoxidase activity. The examination of benzopyrene metabolites by high performance liquid chromatography (HPLC) or by benzopyrene-DNA adducts formation shown that, as in adult animals, the formation of hydroxylated metabolites in position 9,10 was enhanced. In primary fetal liver cells culture, similar effects were observed. Furthermore, the presence of glucocorticoids in the culture medium was not required for the induction of AHH by 9-hydroxyellipticine (9-OHE).

The expression of phenobarbital-, pregnenolone 16 alpha-carbonitrile- and polycyclic aromatic hydrocarbon-inducible cytochromes P-450 and of phenobarbital-inducible UDP-glucuronosyltransferase was examined in developing rat liver. RNAs coding for these proteins were present in fetal rat liver and their respective concentrations remained quite stable in non-induced animals. Inducers differently affected the concentration of RNAs: clofibrate had no action, whereas methylcholanthrene was highly active in fetal liver. Induction by phenobarbital gradually increased during ontogenesis, in parallel with the augmentation of the number of hepatocyte cells in the liver. Our contribution definitively demonstrates that the ability of phenobarbital to enhance P-450 and UDPGT RNAs is strictly restricted to hepatocytes and remains roughly unchanged throughout ontogenesis. In addition, phenobarbital was also able to potentiate the inducing capacity of methylcholanthrene (i.e., raising the TCDD-binding protein) exclusively in hepatocytes. This is the first direct evidence that the number of hepatocytes in the liver, rather than a biochemical maturation, controls the expression of phenobarbital-inducible genes. Pregnenolone 16 alpha-carbonitrile was also effective as inducer in fetal and neonatal rats and its maximal effect was observed in 5-d-old neonates, suggesting a regulation mechanism temporally different from that of phenobarbital.

Drug-metabolizing enzyme activities were assayed in human fetuses and neonates and compared with activities in adults. Among these enzymes, only UDP-glucuronosyltransferase exhibited a very low activity, whereas cytochromes P-450, epoxide hydrolase and glutathion S-transferase reached about 50% of adult values as soon as 15-20 weeks of gestational age. Multiple cytochromes P-450 were present in human adults: isoenzymes involved in the metabolism of exogenous substrates such as ethylmorphine, hexobarbital, aniline and diazepam, and endogenous substrates were present and active in the fetus, whereas isoenzyme(s) permitting the hydroxylation of polycyclic aromatic hydrocarbons exhibited a very low activity. A low number of transplacental inductions have been enzymatically studied; only correlations between the pharmacokinetics of a drug in neonates (full-term or premature) and drug intake of the mother during gestation were observed and showed that a treatment with barbiturates significantly increased drug elimination in neonates. Thus, in human fetus and neonate, drug-metabolizing enzymes are present and active, except UDP-glucuronosyltransferase which develops only after birth.

Twenty-two-day-old fetal and five-day-old newborn rats were pretreated with phenobarbital and its hydroxylated metabolites. Drug-metabolizing enzymes (cytochrome P450, epoxide hydrolase, UDP-glucuronosyltransferase, and glutathione-S-transferase) and microsomal ribonuclease were not modified in fetuses treated with 80 or 400 mg . kg-1 of p-hydroxyphenobarbital, in spite of its accumulation in fetal liver. At fetal age, phenobarbital was a poor inducer of drug-metabolizing enzymes. In five-day-old newborns, p-hydroxyphenobarbital provoked a proliferation of endoplasmic reticulum without enzyme induction, whereas phenobarbital induced some drug-metabolizing enzymes. Thus, the effects of p-hydroxyphenobarbital and phenobarbital are retained in five-day-old rats, but undetectable in the fetuses.

Cytochrome P 450 concentration, related monooxygenase activities (towards aniline, p-nitroanisole, benzphetamine, ethoxycoumarin, benzo(a)pyrene, testosterone, and dehydroepiandrosterone), epoxide hydrolase, and glutathione S-transferase activities were measured in the liver of human foetuses aged from 15 to 38 weeks and compared to adult activities. Different ontogenic patterns seem to exist between monooxygenase activities: if the overall cytochrome P 450 concentration, aniline hydroxylase, benzphetamine demethylase, epoxide hydrolase, and glutathione S-transferase activities reach about the half of adult values as early as 15-25 weeks of gestational age, the metabolism of benzo(a)pyrene, ethoxycoumarin, and testosterone in position 6 beta is very low in these foetuses, whereas the 16 alpha hydroxylation of dehydroandrosterone is higher than in adult human liver. Foetal and adult cytochromes P 450 were resolved by DEAE cellulose chromatography into three different fractions: in reconstitution experiments, the major fraction (A) was active toward aniline, whereas benzphetamine and ethoxycoumarin were mainly metabolized by the two other fractions (Ba and Bb). Results show that multiple cytochromes 450 are present in foetal liver and are able to catalyze, with a lower molecular activity, the same reactions as in adult human liver.

The overall cytochrome P-450 content and the immunochemically determined concentrations of constitutive isoenzymes UT-A and UT-I and isoenzymes induced by phenobarbital (PB-B), pregnenolone 16 alpha-carbonitrile (PCN-E), beta-naphthoflavone (BNF-B) and isosafrole (ISF-G) were investigated in liver microsomes from developing rats and tentatively correlated with mono-oxygenase activities. In fetuses of untreated rat, although the cytochrome P-450 was easily quantified spectrally, none of the isoenzymes tested could be immunochemically detected. After birth, each isoenzyme develops in untreated animals with its own pattern of evolution. Mono-oxygenase activities exhibited different developmental pictures. Benzphetamine-N-demethylase and lauric acid 11-hydroxylase activities progressively increased up to adult values, aniline hydroxylase activity was maximal in 15-day-old animals and benzopyrene hydroxylase and lauric acid 12-hydroxylation were increased after birth until 15 days of age in both males and females and underwent a second increase in males. Pretreatment with phenobarbital resulted in the precocious onset of PB-B in fetal and neonatal rat liver accompanied by an increase in classically associated monooxygenase activities. The PCN-E concentration was enhanced by phenobarbital pretreatment only at 15 days and in older animals. BNF-B and benzo(a)pyrene hydroxylase activity were significantly increased by 3-methylcholanthrene whatever the age considered, whereas this inductive effect upon ISF-G concentration became effective only after 2 weeks of age. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, UT-A was faint in early neonates and intensified at 15 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Schizandrin (Sin) A, B and C, Schizandrol (Sol) A and B and Schizandrer (Ser) A and B were isolated from Fructus Schizandrae chinensis, a traditional Chinese tonic. These components are the derivatives of dibenzo[a,c]cylooctene. Dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylenedioxy-biphenyl-2,2'-dicarboxylate (DDB) is an intermediate for synthesizing Sin C. The effect of these compounds on rat liver microsomal monooxygenases and epoxide hydrolase has been studied. Among these compounds, Sin B, Sin C, Sol B and DDB significantly increased rat liver cytochrome P-450 concentration, NADPH-cytochrome c reductase, benzphetamine and aminopyrene demethylase activities. The four compounds also markedly stimulated proliferation of smooth endoplasmic reticulum of liver cells. Metyrapone (1 mM) inhibited to a same extent (about 50%) the activity of aminopyrene demethylase of microsomes from rats treated by Sin B, Sin C, Sol B, DDB and phenobarbital (PB). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of liver microsomal preparations showed that Sin B and Sol B induce a major protein band of P-450 similar to that induced by PB. In addition, the effect of Sin B-, Sol B- and DDB-treated rat liver microsomes on [G-3H]-benzo[a]pyrene (BP) metabolism and covalent binding of reactive metabolites to DNA, in vitro, resembles that of PB. Dual induction of rats by Sol B and BP decreased mutagenicity of BP.