Drug Metabolism Letters - Volume 2, Issue 4, 2008

UDP-glucuronosyltransferase1A1 (UGT1A1) plays a key role to conjugate bilirubin and preventing jaundice, but there is no report showing the induction of human UGT1A1 (UGT1A1) by bilirubin. In this report, we show findings of the induction of the reporter gene (-3475/+14) of UGT1A1 in HepG2 cells by bilirubin at 50 μM, 100 μM, with human aryl hydrocarbon receptor (hAhR). We confirmed that induction of the reporter gene by bilirubin is dependent on the position of the xenobiotic responsive element (XRE) (-3328/-3319) of UGT1A1, because the XRE deletion UGT1A1 gene did not respond to stimulation by a complex of bilirubin and hAhR. α-Naphthoflavone (α-NF) of a typical AhR antagonist at 50 μM inhibited induction by bilirubin, suggesting that bilirubin stimulates through binding with hAhR. Meanwhile, bilirubin itself did not stimulate the induction of AhR, because we detected no-elevation of the mRNA level of AhR by RTPCR. These results indicate that the induction of UGT1A1 by bilirubin-AhR did not depend on the elevation of AhR but on ligand binding. From this result, we considered that high bilirubin in neonates must induce the elevation of UGT1A1 after birth to prevent jaundice, and bilirubin in adults also regulates the level of UGT1A1. This is the first report showing direct induction of UGT1A1 by a bilirubin through AhR pathway.

We studied endogenous substrates for P-glycoprotein (P-gp) in an oxidative reaction mixture of ceramides, phospholipids, sphingolipids, or GM1-gangliosides (GM1-G). Extracts from the reaction mixture of galactocerebrosides (GalCer), sphingomyelin (SM) , lactocerebrosides (LactoCer), and asolectine (AS) with 0.3% hydrogen peroxide exhibited significant ATPase activity of P-gp of 7.6, 7.8, 5.3, and 4.7 nmol/min/mg protein, respectively, at a concentration of 10 μg equivalent/ml, but not GalCer, SM, LactoCer, and AS themselves. Meanwhile, both GM1-G and its oxidized product showed ATPase activity of 3.7 nmol/min/mg protein at a concentration of 0.75 μM. Phosphatidylcholine, phosphatidylethanolamine, phophatidylserine, triglyceride, and cholesterol did not show P-gp activity. When reactive oxygen species, such as hydrogen peroxide, exceed the ability of antioxidant defense systems to remove it from living cells, SM, GalCer, LactoCer, and AS could react with it; therefore, it is possible for these oxidized lipids to play as substrates for Pgp in living cells. This finding should be a milestone to search a new physiological P-gp function.

Chemical analysis of fingerprint deposits from methadone-maintained opioid-dependent patients by UPLCMS/ MS show detectable levels of methadone and its metabolite, EDDP, demonstrating intake of the drug.

To evaluate the effect of endotoxin administration on the glucuronidation of multiple substrates in a rat model. In addition, the effect of endotoxin treatment on selected UGT mRNA levels in the liver and the kidney was also investigated. Male Sprague-Dawley rats (n= 6) received endotoxin (1.6 mg/kg) by intraperitoneal injection. The animals were sacrificed at 6, 12, 24, 48 hr after treatment, and the liver and the kidneys were harvested. Glucuronidation of various substrates (i.e., acetaminophen, estradiol, testosterone, and morphine) was evaluated using prepared tissue microsomes. Real- Time PCR was used to determine mRNA levels of UGT1A1, 1A6, 2B1, and 2B3 in the harvested organs. UGT1A and UGT2B family isoforms were differentially affected by endotoxin treatment. The greatest reduction in glucuronide formation was observed for estradiol-17-glucuronide (-37%) in hepatic microsomes 48 hr after endotoxin administration. Estradiol- 3-glucuronide formation was reduced in liver microsomes (466.3±71.5 pmol/min/mg vs. 346.9±23.2 pmol/min/mg, p < 0.05) but was not significantly altered in the kidney. Similarly, acetaminophen glucuronide formation was decreased in liver but not kidney microsomes. Significant reductions in glucuronide formation were also observed for morphine (-26%) and testosterone (-30%) in septic rats compared to controls. Endotoxin administration was associated with a timedependent decrease in UGT1A1, UGT1A6, UGT2B1, and UGT2B3 mRNA levels in liver and kidney tissues. These findings demonstrate that both mRNA and activity of UGT isoforms in rats are decreased following endotoxin treatment, although tissue specific effects do also exist.

Virtual screening docking-based approach has been employed in order to select novel HIV-1 integrase (IN) potential inhibitors in large databases. Toxicity, metabolism and drug-like properties have been analyzed for the most promising compounds, using computational chemistry techniques. Results were compared and discussed with that obtained for a known HIV-1 (IN) inhibitor reported in the literature.

Andrographolide is a component of a famous traditional Chinese herbal medicine Andrographis paniculate (Burm) Nees. In this study, the metabolites of andrographolide in human urine after oral administration were investigated. Four urea adducts were isolated by chromatography methods and identified by high-resolution mass spectra, 1 D and 2D NMR spectroscopy.

An increased methadone enantiomer ratio (R/S) was associated to both nevirapine (179%, n=5) and efavirenz (36%, n=9) treatments when compared with that of controls (n=52). Additionally, in four follow-up patients, both R- and S-methadone normalized concentrations decreased (19%-93%) while R/S increased (22%-314%) following nevirapine/ efavirenz treatment. R/S decreased (42%) after non-compliance with efavirenz treatment. Therefore, the methadonemaintenance- treatment outcome should be evaluated when patients are treated with drugs which are supposed to induce CYP3A4 and CYP2B6 isoforms.

In probing enhancement of the transdermal delivery of the anti-psychotic drug haloperidol, five prodrugs (ethanoate, propanoate, butanoate, octanoate and decanoate) were synthesised and their relative rates of hydrolysis determined in the presence of porcine liver esterase (PLE), a model for cutaneous esterases. ¹H NMR, MS and elemental analysis confirmed the successful synthesis of each prodrug in high purity, and each was found to hydrolyse in the presence of PLE with the hydrolytic rate reaching a maximum with haloperidol octanoate (C8) at 2.31 ± 0.06 nmol ml-¹ h-¹ (p < 0.001).

The serum concentration of valproic acid (VPA) in epilepsy patients decreased in the administration of carbapenem antibiotics (CP), such as meropenem, panipenem, biapenem or imipenem, to a sub-therapeutic level. The liver is the key organ for the decrease of VPA concentration by CP, because it has been reported that no decrease of the VPA level by CP was found in hepatectomized rats. This effect was also shown with monkey and rat liver slices. In this report, we show the results of in vitro inhibition of VPA-glucuronidase in human liver microsomes and cytosol by CP. We found the highest metabolic activity of VPA-glucuronide in human liver cytosol. The level in liver cytosol was 149 pmol/min/mg protein. The level in human liver microsomes (HLM) was one-fifth of that in cytosol and the level in serum was negligible. We found that this hydrolysis depends on VPA-glucuronidase in cytosol, because digestion was inhibited by D-saccharic acid 1,4-lactonemonohydrate of a specific inhibitor of β-glucuronidase, but not by phenylmethylsulfonylfluoride of an esterase inhibitor. We also found the inhibition of VPA-glucuronidase in cytosol by CP, and the maximum inhibition was found with panipenem (IC50 = 3 μM). We also found inhibition of VPA-glucuronidase in HLM by meropenem. These results showed that the inhibition in liver slices depended on the inhibition of VPA-glucuronidase by CP. We considered that the inhibition of VPA-glucuronidase by CP in cytosol is a key factor to decrease the plasma VPA level.

An earlier reported population pharmacokinetic study documented limited predictability of amikacin clearance (65%) at birth. An exercise in the first month of life resulted in extended predictability (92%) with similar clearance estimates after the first postnatal days, reflecting the impact of postnatal age on clearance predictability.

Increased heme oxygenase-1 (HO-1) expression improves vascular function by decreasing superoxide and increasing antioxidant levels. We therefore examined if HO-1 induction increased serum adiponectin levels and ameliorated vascular dysfunction in Type 1 diabetes. Administration of either carbon monoxide (CORM-3) or the HO-1 inducers, Resveratrol, and cobalt protoporphyrin (CoPP), increased serum levels of adiponectin (high molecular weight) in diabetic (streptozotocin; STZ-induced) Sprague Dawley rats. Resveratrol and CoPP administration increased HO-1 protein expression and HO activity in the aorta and significantly (p<0.05) increased serum adiponectin levels, compared to untreated diabetic rats. The results obtained with the CO releasing molecule, CORM-3, indicate a direct involvement of CO leading to increased levels of adiponectin. The increase in adiponectin was associated with a significant decrease in circulating endothelial cells (CEC) (p<0.002), decreased EC fragmentations and a significant increase in thrombomodulin (TM) and CD31+ cells (p<0.05). Increased adiponectin levels were associated with a decrease in TNF-α-induced ICAM-1 and VCAM-1 and caspase 3 activity in endothelial cells while phosphorylation of eNOS at Ser-1179 increased. The adiponectin mediated increase in peNOS and pAKT was prevented by the phosphatidylinositol-3 kinase inhibitor, LY294002. In conclusion, there appears to be a temporal HO-1-adiponectin relationship that has a key role in vascular protection in Type 1 diabetes via a mechanism that involves increased levels of carbon monoxide.

Evidence, obtained in rodent and primate models of Parkinson's disease (PD) and in preliminary clinical trials, indicates that adenosine A2A receptor antagonists might represent a promising non-dopaminergic therapeutic tool for the treatment of PD. Recently, we have reported the biological evaluation of 8-substituted 9-ethyladenines (ANR) as new A2A receptor antagonists, three of which (ANR 82, ANR 94, and ANR 152) showed high efficacy in in vivo models for Parkinson's. Understanding the metabolic pathways of new drug candidates is an important aspect of drug discovery. The ANR compounds have been investigated in order to clarify their activity on rat liver microsomes, and more specifically on recombinant human cytochrome P450 2D6 (CYP2D6). The metabolites of all three compounds were detected by liquid chromatography/tandem mass spectrometry (LC-MS/MS). The results indicate that this class of 9-ethyladenines is metabolized only to a fraction of 1.5-5%. These compounds also act as potent mechanism-based inhibitors of CYP450 and in particular of human isoform CYP2D6. Kinetic-analysis of enzyme inactivation was used to describe the effect of these time-dependent inhibitors and to derive the inhibition parameters Kinact and Ki defined with respect to the O-demethylation of dextromethorphan.