Jouko Uusitalo

STABLE EXPRESSION, ACTIVITY AND INDUCIBILITY OF CYTOCHROMES P450 IN DIFFERENTIATED HepaRG CELLS

HepaRG cells possess the unique property to differentiate in vitro and to express various functions of mature hepatocytes, including the major cytochromes P450 (P450s). In the present study, we carefully analyzed mRNA expression and activity of the major P450s and their responsiveness to three prototypical inducers, phenobarbital, rifampicin, and omeprazole, in differentiated HepaRG cell cultures over a 4-week period after low and high seeding. Only minor differences were observed in P450 activities when measured by two cocktails of probe substrates, probably related to the choice and/or concentration of substrates. Similar results were obtained from the two cell seeding conditions. Expression and activities of several P450s were dimethyl sulfoxide-dependent. However, basal P450 expression and activities as well as their responsiveness to the prototypical inducers were well maintained over the 4-week period, and a good correlation was observed between transcript levels and corresponding activities. Thus, CYP1A2, CYP2B6, and CYP3A4 were found to accurately respond to their respective prototypical inducers, i.e., omeprazole, phenobarbital, and rifampicin. Likewise, basal expression of several phase II enzymes, transporters, and nuclear receptors, and response to inducers were also well preserved. More genes were found to be induced in HepaRG cells than in primary human hepatocytes, and no marked variation was noticed between the different passages. Taken together, these data support the conclusion that HepaRG cells represent a promising surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies.

Functional expression, inhibition and induction of CYP enzymes in HepaRG cells

Practically all human hepatocyte cell lines are deficient in major cytochrome P450 (CYP)-related enzyme activities, making them unrepresentative of in vivo hepatocytes. We have used the recently developed HepaRG cell line to determine the spectrum of most important CYP enzyme activities involved in xenobiotic metabolism (CYP1A1/2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) and the effect of the prototypical CYP-inducer phenobarbital and a panel of known CYP-selective inhibitors on these activities. Comparison of these activities was carried out with two human primary hepatocyte populations. We show that excluding CYP2A6 and CYP2E1, HepaRG cells express high functional levels of most of the major xenobiotic metabolising CYPs. These activities were found to be selectively inhibited and induced by prototypical CYP-selective inhibitors and inducer at comparable levels to primary hepatocytes. In conclusion, HepaRG cells may be a promising cell line for various applications, which currently employ hepatic subcellular preparations or cultured primary hepatocytes.

Estrogen receptor alpha genotype confers interindividual variability of response to estrogen and testosterone in mesenchymal-stem-cell-derived osteoblasts

Hormone replacement therapy is effectively used to prevent postmenopausal bone loss. Variation in response to the therapy is, however, frequently seen. In addition, the direct effects of sex steroids on isolated human bone marrow stromal cells have been reported to vary depending on the donor, but the biological mechanisms are not understood. The aim of this study was to investigate the effects of 17beta-estradiol (E2) and testosterone in human-bone-marrow-derived mesenchymal stem cell (MSC) cultures from both female and male donors of various ages. The osteoblast differentiation capacity and activity of the MSCs were quantified in vitro by measuring alkaline phosphatase activity and calcium deposition. We show here that also the osteoblast responses of MSCs to sex hormones vary widely depending on the donor. When the results from all donors were analyzed together, treatment with E2 increased calcium deposition significantly by MSCs of both sexes but ALP activity only in the male MSCs. Testosterone had no effect on ALP activity nor calcium deposition in either sex. To further characterize the individual variation, we investigated estrogen receptor alpha PvuII restriction site polymorphism with PCR. Restriction fragment-length polymorphism was assigned as P or non-P, P signifying the absence of the restriction site. Our results indicate that higher basal osteoblast differentiation capacity of MSCs is associated with the presence of the P allele in females, whereas higher response to sex steroids treatment is associated with the non-P allele. These results could help explain the contradictory effects of E2 on osteoblasts in vitro and might also provide new insights to understanding the differences in responses to hormone replacement therapy.

Timolol Metabolism in Human Liver Microsomes Is Mediated Principally by CYP2D6

Timolol has mainly been used topically for the treatment of glaucoma. It has been suggested that the drug is metabolized by cytochrome P450 CYP2D6. The matter has not, however, been extensively studied. The aim here was to tentatively identify timolol metabolites and to determine the P450-associated metabolic and interaction properties of timolol in vitro. Four metabolites were identified, the most abundant being a hydroxy metabolite, M1. The Km value for the formation of M1 was 23.8 μM in human liver microsomes. Metabolism of timolol with recombinant P450s and correlation analysis have confirmed the conception that the drug is metabolized principally by CYP2D6, CYP2C19 being only a minor contributor (<10%) to the intrinsic microsomal clearance. The CYP2D6 inhibitor quinidine proved a potent competitive inhibitor of timolol metabolism, with an in vitro Ki value of 0.08 μM. Fluvoxamine, an inhibitor of CYP2C19, inhibited timolol metabolism to a lesser extent, confirming its minor contribution. Timolol itself did not inhibit CYP2D6-catalyzed dextromethorphan O-demethylation. Judging from the disappearance of timolol in human liver homogenate, the in vivo half-life was extrapolated to be about 3 h, an estimate close to the half-life of about 2 to 5 h observed in vivo. In conclusion, the inhibition of timolol metabolism by quinidine should be taken into account when patients are treated with timolol. However, when plasma timolol concentrations in patients remain low (≤0.2 μg/l), it is suggested that such interaction is of minor clinical relevance.

Metabolic and Efflux Properties of Caco-2 Cells Stably Transfected with Nuclear Receptors

PurposeTo characterise in detail the patterns of expression and functional activities of CYP and efflux pump genes in Caco-2 cells stably transfected with human Pregnane X Receptor or murine Constitutive Androstane Receptor.Materials and MethodsCell lines transfected with nuclear receptors were treated with established ligands, and gene expression of CYP and efflux pump genes were quantified by qRT-PCR and Western blot. P-glycoprotein activity was assessed by measuring calcein-AM accumulation and bidirectional permeability coefficients of digoxin and quinidine. CYP activities were measured with both fluorescent and non-fluorescent substrates.ResultshPXR and mCAR upregulated some CYP and efflux pump genes ligand dependently. P-glycoprotein level was increased, but CYP3A4 protein remained below the limit of detection. P-glycoprotein activity was markedly elevated in Caco/mCAR cells and more modestly in Caco/hPXR cells. CYP3A4 activity remained lower than that in vitamin D-treated Caco-2 cells.ConclusionsNuclear receptors can modulate the expression of metabolic genes in Caco-2 cells, but the overall level of metabolism could not be efficiently controlled. P-glycoprotein activity increased, but CYP activities remained very low.

Ophthalmic timolol in a hydrogel vehicle leads to minor inter-individual variation in timolol concentration in aqueous humor

Ophthalmic timolol has been used for decades in the treatment of glaucoma and ocular hypertension, traditionally in aqueous 0.5% eye drops. Recently a timolol 0.1% hydrogel has been developed to improve systemic safety. The aim of the present study was to compare aqueous humor timolol concentrations after administration of 0.1% hydrogel and aqueous 0.5% timolol in patients scheduled for a cataract operation. The concentration in the aqueous humor was 210+/-175 ng/ml (mean+/-S.D.) 2h after administration of timolol 0.1% hydrogel and 538+/-304 ng/ml after aqueous 0.5% timolol. In the aqueous 0.5% timolol group more patients had unnecessarily high concentrations of timolol in the aqueous humor. beta(1)-receptors and beta(2)-receptors were practically 100% occupied after administration of both products. The hydrogel proved to be an excellent formulation in giving smaller inter-individual variation in penetration of timolol into the aqueous humor. Only a weak correlation was seen between corneal thickness and the aqueous humor concentration of timolol in the aqeuous 0.5% timolol group. In conclusion, in contrast to the conventional aqueous 0.5% timolol, 0.1% timolol hydrogel caused only slight inter-individual variation in timolol concentration in the aqueous humor.

Regional and habitat differences in 7-methyljuglone content of Finnish Drosera rotundifolia

The concentration of 7-methyljuglone was studied in the round-leaved sundew Drosera rotundifolia L. collected from different regions in Northern Finland. Samples for analysis were collected from peat bogs and sandpit habitats. The mean concentration of 7-methyljuglone varied from 1.0 to 2.3% of dry weight. Variation between years in the amount of 7-methyljuglone was significant in plants growing on sand, and in the northernmost region studied. Overall, the variation in the production of 7-methyljuglone among different populations of round-leaved sundew in Northern Finland was rather low. The variation between years in the production of 7-methyljuglone was more significant.

Ospemifene metabolism in humans in vitro and in vivo: metabolite identification, quantitation, and CYP assignment of major hydroxylations.

Abstract Background: The metabolism of ospemifene, a novel nonsteroidal selective estrogen receptor modulator, was investigated as part of its development. Methods: Metabolite identification, tentative quantitation, and CYP assignment of ospemifene were performed in human liver microsomes or homogenate incubations and in plasma samples from volunteer humans. The potential contributions of CYP enzymes were determined by recombinant human CYPs. Metabolite identification and tentative quantification were performed by liquid chromatography-mass spectrometry. Results: The relative abundances of metabolites produced were dependent on ospemifene concentration and liver preparation, but the largest quantities of 4- and 4′-hydroxy-ospemifene (and their glucuronides in smaller quantities) were produced in human liver microsomes at low ospemifene concentrations. Other metabolites were detected in in vitro incubation with human liver including a direct glucuronide of ospemifene and some metabolites with only minor abundance. In human plasma samples, 4-hydroxy-ospemifene was the most abundant metabolite, representing about 25% of the abundance of the parent compound. All the other metabolites detected in plasma, including 4′-hydroxy-ospemifene, represented <7% of the abundance of ospemifene. Several CYP enzymes participated in 4-hydroxylation, including CYP2C9, CYP2C19, CYP2B6, and CYP3A4, whereas CYP3A enzymes were the only ones to catalyze 4′-hydroxylation. Conclusions: In vitro incubations with liver preparations provided a rather reliable starting point in the search for potential metabolites in clinical settings. The in vitro metabolite profile is informative for the in vivo metabolite profile, especially regarding the major hydroxylated metabolites. However, it is anticipated that extended in vivo exposures may result in an increased production of more distal metabolites from major metabolites.

Effects of cytochrome P450 inhibitors and inducers on the metabolism and pharmacokinetics of ospemifene

Purpose: The objectives were to determine the cytochrome P450 (CYP) enzymes involved in the metabolism of ospemifene and its main hydroxylated metabolites and to examine the effects of CYP inhibitors and inducers on ospemifene pharmacokinetics. Methods: In vitro metabolism studies were conducted using human liver microsomes; CYP‐selective inhibitors and CYP‐specific substrates were used to determine the roles of nine CYP isoforms in ospemifene metabolism. Two Phase 1 clinical trials were conducted in healthy postmenopausal women; crossover designs examined the effects of pretreatment with the CYP modulators rifampicin, ketoconazole, fluconazole and omeprazole on ospemifene pharmacokinetics. Results: Although several CYP inhibitors decreased the in vitro formation of ospemifene metabolites, none of them completely blocked metabolism. Roles for CYP3A4, CYP2C9, CYP2C19 and CYP2B6 in the metabolism of ospemifene and its two main metabolites, 4‐hydroxyospemifene and 4′‐hydroxyospemifene, were confirmed. The in vivo experiments demonstrated that ospemifene serum concentrations were decreased by rifampicin pretreatment, increased by ketoconazole or fluconazole pretreatment, and minimally affected by omeprazole pretreatment. Conclusions: The clinical pharmacokinetic findings and in vitro data suggest that CYP3A4 is important for ospemifene metabolism, but other CYP isoforms and metabolic pathways also contribute. Strong CYP3A or CYP2C9 inducers (e.g. rifampicin) would be expected to decrease the exposure to ospemifene. Ospemifene should be used with caution when coadministered with the modest CYP3A inhibitor ketoconazole and should not be coadministered with the potent CYP3A/CYP2C9/CYP2C19 inhibitor fluconazole. The potent CYP2C19 inhibitor omeprazole is unlikely to cause clinically significant changes in ospemifene pharmacokinetics. Copyright

Variation in the Amount of Yield and in the Extract Composition Between Conventionally Produced and Micropropagated Peppermint and Spearmint

Abstract The quality and quantity of oil obtained from three different peppermint Mentha x piperita L. origins and the spearmint Mentha spicata L. were compared in micropropagated and conventionally propagated plants in northern Finland (64°40′N) in 1997–1998. Each block comprised M. piperita peppermint plants from three different origins; namely, the United States, Bulgaria and Egypt (Black Mitcham), and a spearmint, M. spicata of Egyptian origin. The micropropagated plantlets and conventionally propagated plants were transferred to the experimental field in June. In the first year, there were no differences in the dry leaf yield between the micropropagated plants and their conventionally produced counterparts, although the former had a higher leaf/stem ratio. In the second year, the dry leaf yield of micropropagated plants, which were left in 1997 in field and developed from the underground stolons, was higher than that of conventionally propagated plants. The percentage of oil was higher with conventionally propagated plants, but only in the first year. Significant differences were observed between mint origins in the different propagation methods. Moreover, conventionally propagated plants showed a higher menthol percentage. There was no significant difference in the isomenthone content of the plants. In spearmint, the dry yield was higher in the second year with micropropagated plants, but there were no significant differences in the percentage of carvone between the two propagation methods.