Liver Microsomal P-450 Research Articles

Evidence for the formation of 6PPD-quinone from antioxidant 6PPD by cytochrome P450

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) as a rubber antioxidant has attracted global concern, since its ozone-oxidation product 6PPD-quinone (6PPDQ) was found to be the primary toxicant responsible for urban runoff mortality syndrome in coho salmon. However, the biotransformation fate and associated toxicological mechanism of 6PPD have not received much study yet. In this work, the in vitro assays showed 6PPD can be transformed into 6PPDQ by cytochromes P450 (CYP450) in human liver microsomes (HLMs) with 0.98 % production rate, and the adducts of 6PPDQ with calf thymus DNA and the N-N coupling product between 6PPD and 6PPDQ were further identified after 6PPD incubation in HLMs. Further evidence for the 6PPDQ formation can be obtained from the in vivo assays that the 6PPDQ-DNA adducts and 6PPD-N-N-6PPDQ dimer were detected in mice by oral gavage with 6PPD, and the latter dimer species was detected as well in 6PPD exposure to zebrafish larvae. Especially, the bioaccumulation property and high reactivity of 6PPDQ result in the continuous formation of the significant DNA adducts and 6PPD-N-N-6PPDQ dimer even in case of low production rate of biotransformation of 6PPD to 6PPDQ, which may provide potentially effective biomarkers for such process. DFT computations revealed the formation mechanism of 6PPDQ is the (N)H-abstraction of 6PPD by CYP450, followed by amino radical rebound at the nearby ortho-carbon, yielding a quinol intermediate due to spin delocalization, that might readily undergo further oxidation by CYP450 into 6PPDQ.

Characterization of the metabolism of eupalinolide A and B by carboxylesterase and cytochrome P450 in human liver microsomes.

Eupalinolide A (EA; Z-configuration) and eupalinolide B (EB; E-configuration) are bioactive cis-trans isomers isolated from Eupatorii Lindleyani Herba that exert anti-inflammatory and antitumor effects. Although one pharmacokinetic study found that the metabolic parameters of the isomers were different in rats, metabolic processes relevant to EA and EB remain largely unknown. Our preliminary findings revealed that EA and EB are rapidly hydrolyzed by carboxylesterase. Here, we investigated the metabolic stability and enzyme kinetics of carboxylesterase-mediated hydrolysis and cytochrome P450 (CYP)-mediated oxidation of EA and EB in human liver microsomes (HLMs). We also explored differences in the hydrolytic stability of EA and EB in human liver microsomes and rat liver microsomes (RLMs). Moreover, cytochrome P450 reaction phenotyping of the isomers was performed via in silico methods (i.e., using a quantitative structure-activity relationship model and molecular docking) and confirmed using human recombinant enzymes. The total normalized rate approach was considered to assess the relative contributions of five major cytochrome P450s to EA and EB metabolism. We found that EA and EB were eliminated rapidly, mainly by carboxylesterase-mediated hydrolysis, as compared with cytochrome P450-mediated oxidation. An inter-species difference was observed as well, with faster rates of EA and EB hydrolysis in rat liver microsomes. Furthermore, our findings confirmed EA and EB were metabolized by multiple cytochrome P450s, among which CYP3A4 played a particularly important role.

2-Oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole, a contributor to the odour of boar-tainted pork meat, mediated by porcine liver microsomal cytochromes P450 1A2, 2A19, 2E1, and 3A22

The 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole (a contributor to boar taint) mediated by minipig liver microsomes and recombinant P450 enzymes expressed in bacterial membranes were investigated. At low substrate concentrations of 10 µM, the formation rates of indole-3-carbinol, 6-hydroxyskatole, and the sum of 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole in male minipig liver microsomes were significantly lower than those in female minipig liver microsomes. Compensatory 3-methyloxindole and indole-3-carbinol formation in minipig liver microsomes, which lack 6-hydroxyskatole formation in males, was mediated partly by liver microsomal P450 1A2 and P450 1A2/2E1, respectively. These enzymes were suppressed by typical P450 inhibitors in female minipig liver microsomes. Among the 14 pig P450 forms evaluated, P450 2A19 was the dominant form mediating 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole formation from skatole at substrate concentrations of 100 µM. Positive cooperativity was observed in 3-methyloxindole formation from skatole mediated by male minipig liver microsomes and by pig P450 3A22 with Hill coefficients of 1.2–1.5. These results suggest high skatole 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation activities of pig P450 2A19 and compensatory skatole oxidations mediated by pig P450 1A2, 2E1, or 3A22 in male minipig liver microsomes.

Biological fate and interaction with cytochromes P450 of the nanocarrier material, d-α-tocopheryl polyethylene glycol 1000 succinate

d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS, also known as vitamin E-TPGS) is a biodegradable amphiphilic polymer prepared by esterification of vitamin E with polyethylene glycol (PEG) 1000. It is approved by the US Food and Drug Administration (FDA) and has found wide application in nanocarrier drug delivery systems (NDDS). Fully characterizing the in vivo fate and pharmacokinetic behavior of TPGS is important to promote the further development of TPGS-based NDDS. However, to date, a bioassay for the simultaneous quantitation of TPGS and its metabolite, PEG1000, has not been reported. In the present study, we developed such an innovative bioassay and used it to investigate the pharmacokinetics, tissue distribution and excretion of TPGS and PEG1000 in rat after oral and intravenous dosing. In addition, we evaluated the interaction of TPGS with cytochromes P450 (CYP450s) in human liver microsomes. The results show that TPGS is poorly absorbed after oral administration with very low bioavailability and that, after intravenous administration, TPGS and PEG1000 are mainly distributed to the spleen, liver, lung and kidney before both being slowly eliminated in urine and feces as PEG1000. In vitro studies show the inhibition of human CYP450 enzymes by TPGS is limited to a weak inhibition of CYP3A4. Overall, our results provide a clear picture of the in vivo fate of TPGS which will be useful in evaluating the safety of TPGS-based NDDS in clinical use and in promoting their further development.

Different substrate elimination rates of model drugs pH-dependently mediated by flavin-containing monooxygenases and cytochromes P450 in human liver microsomes

The predicted contributions of flavin-containing monooxygenase 3 (FMO3) to drug candidate N-oxygenations can be estimated using classic base dissociation constants of the N-containing moiety. In this study, metabolic clearance values in human liver microsomes were experimentally determined for available model drugs. Typical metabolic clearance values (34–96 μL/min/mg protein) at pH 8.4 of trimethylamine, benzydamine, and itopride were two-to fourfold higher than those at pH 7.4. In contrast, the metabolic clearance of control drug midazolam at pH 8.4 was half that at pH 7.4. The ratios of clearance values at pH 8.4 to those at pH 7.4 and the substrate pKa (base) values of reported metabolic N-oxygenation sites of trimethylamine, benzydamine, clomipramine, chlorpromazine, tamoxifen, itopride, loxapine, xanomeline, tozasertib, dasatinib, and clozapine were significantly correlated (r = 0.60, p < 0.05, n = 11). These results suggested that the simple comparison of metabolic clearance values at pH 8.4 and at pH 7.4 could be useful for predicting the contributions of FMO3 to the N-oxygenations of new drug candidates. This method, along with in silico pKa (base) values > 8.4, could prove useful for predicting the contributions of FMO3 to N-oxygenations as part of drug development.

Stereostructure-activity mechanism of cyproconazole by cytochrome P450 in rat liver microsomes: A combined experimental and computational study

Cyproconazole (CPZ), representing the chiral triazole fungicides, is widely used in the pharmaceutical and agricultural fields. To clarify its potential adverse effects on the generalized CYP-mediated processes within mammalian, a comparative experimental and computational approach was employed to investigate the CYP-mediated metabolism processes of CPZ stereoisomers in rat liver microsomes (RLMs). The depletion rate of CPZ stereoisomers in vitro incubation system with RLMs followed the order RR-> SS-> SR-> RS-CPZ. The results of kinetic assays were in line with the depletion rate results. Further inhibition assay confirmed the stereoselective metabolism of CPZ stereoisomers by different CYP isoforms. Molecular dynamics (MD) simulation revealed the stereoselective metabolism mechanism. Several hydrogen bonds and π-stacking restrict the position of CPZ isomers in the active cavity of CYPs so that the 4′-nitrogen on the triazole ring can bind closely to the heme of CYP, which results in the metabolism of CPZ isomers. By combining the computational and experimental approaches, the structure-activity relationship of CPZ and CYP was elucidated, and this method can be further applied to predict the degree of uncertainty in the process of xenobiotic biotransformation of triazole fungicides and serve as a basis for risk assessment.

Kinetics of metabolism of deltamethrin and cis- and trans-permethrin in vitro. Studies using rat and human liver microsomes, isolated rat hepatocytes and rat liver cytosol

The kinetics of metabolism of deltamethrin (DLM) and cis- and trans-permethrin (CPM and TPM) was studied in male Sprague-Dawley rat and human liver microsomes. DLM metabolism kinetics was also studied in isolated rat hepatocytes, liver microsomes and cytosol. Apparent intrinsic clearance (CLint) values for the metabolism of DLM, CPM and TPM by cytochrome P450 (CYP) and carboxylesterase (CES) enzymes in rat and human liver microsomes decreased with increasing microsomal protein concentration. However, when apparent CLint values were corrected for nonspecific binding to allow calculation of unbound (i.e., corrected) CLint values, the unbound values did not vary greatly with microsomal protein concentration. Unbound CLint values for metabolism of 0.05-1 μM DLM in rat liver microsomes (CYP and CES enzymes) and cytosol (CES enzymes) were not significantly different from rates of DLM metabolism in isolated rat hepatocytes. This study demonstrates that the nonspecific binding of these highly lipophilic compounds needs to be taken into account in order to obtain accurate estimates of rates of in vitro metabolism of these pyrethroids. While DLM is rapidly metabolised in vitro, the hepatocyte membrane does not appear to represent a barrier to the absorption and hence subsequent hepatic metabolism of this pyrethroid.

Metabolism of a New Antiaggregant, Indolinone Derivative.

Cytochrome p450-mediated metabolism of GRS (indolinone antiaggregant) and its effects on activities of cytochrome p450 isoenzymes were studied. Inhibition of 6 isomers of cytochrome p450 in human liver microsomes was studied with the use of specific substrates. It was found that human liver cytochrome p450 enzymes could not induce degradation of GRS and that GRS was not an inductor or inhibitor of cytochrome p450 family members 1A2, 2C9, 2C19, 2D6, 2C8, and 3A4. Hence, clinical use of the prospective antiaggregant would not involve the risk of uncontrolled fluctuations in GRS concentrations in the organism because of interactions between the drugs.

P75 - In vitro inhibitory effects of ZIZIPHI SPINOSI semen extract on cytochrome P450 and UDP-glucuronosyltransferase enzymes activities in human liver microsomes

P75 - In vitro inhibitory effects of ZIZIPHI SPINOSI semen extract on cytochrome P450 and UDP-glucuronosyltransferase enzymes activities in human liver microsomes

Phase I metabolic profiling of the synthetic cannabinoids THJ-018 and THJ-2201 in human urine in comparison to human liver microsome and cytochrome P450 isoenzyme incubation.

Despite the increasing relevance of synthetic cannabinoids as one of the most important classes within "New Psychoactive Substances", there is still a lack of knowledge concerning their metabolism in humans. Due to the extensive metabolism of synthetic cannabinoids, metabolites are necessarily the best target analytes in urine, posing additional challenges to forensic analysis. The aims of this study were to identify appropriate urinary targets indicating intake of THJ-018 or THJ-2201 as well as to elucidate the most important cytochrome P450 isoenzymes within the metabolism of THJ-018 and THJ-2201 in vitro. For this purpose, the in vitro metabolism of THJ-018 and THJ-2201 was initially established using pooled human liver microsomes. The results obtained were compared to previously published in vitro results as well as to the results of the metabolic profiles from selected recombinant cytochrome P450 isoenzymes and from 23 urine samples from forensic cases. LC-HRMS was used to conduct product ion scans and to examine the metabolite spectra. For THJ-018, 17 different metabolite groups containing 33 different metabolites and isomers were detected after microsomal incubation, with the major metabolic pathways being monohydroxylation at the pentyl chain and of the naphthyl moiety as well as dihydroxylation of both residues. For THJ-2201, 19 different metabolite groups and 46 different metabolites and isomers were observed. The major metabolic pathways were monohydroxylation at the naphthyl moiety and oxidative defluorination. Significant contribution to the in vitro metabolism of THJ-018 and THJ-2201 originated from CYP2B6, CYP2C19, CYP3A4, and CYP3A5. As several cytochrome P450 isoenzymes are involved in the metabolism of these synthetic cannabinoids, a co-consumption with other drugs is unlikely to have an impact on their metabolism.

Metabolic Mechanism of Aryl Phosphorus Flame Retardants by Cytochromes P450: A Combined Experimental and Computational Study on Triphenyl Phosphate

Understanding metabolic mechanisms is critical and remains a difficult task in the risk assessment of emerging pollutants. Triphenyl phosphate (TPHP), a widely used aryl phosphorus flame retardant (aryl-PFR), has been frequently detected in the environment, and its major metabolite was considered as diphenyl phosphate (DPHP). However, knowledge of the mechanism for TPHP leading to DPHP and other metabolites is lacking. Our in vitro study shows that TPHP is metabolized into its diester metabolite DPHP and mono- and dihydroxylated metabolites by cytochromes P450 (CYP) in human liver microsomes, while CYP1A2 and CYP2E1 isoforms are mainly involved in such processes. Molecular docking gives the conformation for TPHP binding with the active species Compound I (an iron IV-oxo heme cation radical) in specific CYP isoforms, showing that the aromatic ring of TPHP is likely to undergo metabolism. Quantum chemical calculations have shown that the dominant reaction channel is the O-addition of Compound I onto the aromatic ring of TPHP, followed by a hydrogen-shuttle mechanism leading to ortho-hydroxy-TPHP as the main monohydroxylated metabolite; the subsequent H-abstraction-OH-rebound reaction acting on ortho-hydroxy-TPHP yields the meta- and ipso-position quinol intermediates, while the former of which can be metabolized into dihydroxy-TPHP by fast protonation, and the latter species needs to go through type-I ipso-substitution and fast protonation to be evolved into DPHP. We envision that the identified mechanisms may give inspiration for studying the metabolism of several other aryl-PFRs by CYP.

Interaction between 3,4‑dichlorophenyl‑propenoyl‑sec.‑butylamine (3,4‑DCPB), an antiepileptic drug, and cytochrome P450 in rat liver microsomes and recombinant human enzymes in vitro

The compound 3,4‑dichlorophenyl‑propenoyl‑sec.‑butylamine (3,4‑DCPB) is an antiepileptic drug. The purpose of the present research was to identify cytochrome P450 (CYP450) responsible for the metabolism of 3,4‑DCPB and evaluate the effects of 3,4‑DCPB on the activities of CYP450 enzymes. 3,4‑DCPB was incubated with rat liver microsomes (RLMs) plus six CYP450 enzyme-specific inhibitors, or six recombinant human CYP450 enzymes (rhCYP450s). The concentrations of 3,4‑DCPB and six CYP450 enzyme-activities probe drugs were detected by high-performance liquid chromatographic (HPLC). The results showed that the prototype of 3,4‑DCPB was metabolized by multiple CYP450 enzymes into three metabolites, and the predominant isoforms were CYP2D6 (metabolite M1), CYP1A2 (M2), CYP2C19 and CYP3A4 (M3), respectively., in the presence of β-NADPH (1 mM) in RLMs or rhCYP450s. Compared with the control (PB−), phenobarbital pre-treatment (PB+) significantly enhanced levels (all of p < 0.01) of hydroxylmethytobutamide (CYP2C9), 4‑hydroxy‑mephenytoin (CYP2C19), acetaminophen (CYP1A2), 6‑hydroxychlorzoxazone (CYP2E1) and oxidized nifedipine (CYP3A4), respectively, in spite of dextrophan (CYP2D6) was not markedly enhanced in RLMs. Conversely, the inhibitory ratios of 3,4‑DCPB (16 μg/mL, 59 μM) on the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6 were 97.6%, 59.0%, 53.5% and 36.5%, respectively. However, CYP2E1 (both of PB− and PB+) and CYP3A4 (PB+) were not inhibited by 3,4‑DCPB in RLMs. In conclusion, the present study showed that 3,4‑DCPB was metabolized by multiple CYP450 enzymes. 3,4‑DCPB inhibited the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6, rather than that CYP2E1 and CYP3A4 enzymes, suggesting that the different effects of 3,4‑DCPB on the CYP450 enzymes might influence metabolic drug-drug interaction in antiepileptics therapy.

In Vitro Inhibitory Effects of Synthetic Cannabinoid EAM-2201 on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

EAM-2201, a synthetic cannabinoid, is a potent agonist of the cannabinoid receptors that is widely abused as an illicit recreational drug in combination with other drugs. To evaluate the potential of EAM-2201 as a perpetrator of drug–drug interactions, the inhibitory effects of EAM-2201 on major drug-metabolizing enzymes, cytochrome P450s (CYPs) and uridine 5′-diphospho-glucuronosyltransferases (UGTs) were evaluated in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry (LC-MS/MS). EAM-2201 at doses up to 50 µM negligibly inhibited the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and five UGTs (1A1, 1A4, 1A6, 1A9 and 2B7) in human liver microsomes. EAM-2201 exhibited time-dependent inhibition of CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP2C19-catalyzed [S]-mephenytoin 4′-hydroxylation and CYP3A4-catalyzed midazolam 1′-hydroxylation with Ki values of 0.54 µM (kinact: 0.0633 min−1), 3.0 µM (kinact: 0.0462 min−1), 3.8 µM (kinact: 0.0264 min−1) and 4.1 µM (kinact: 0.0250 min−1), respectively and competitively inhibited UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, with a Ki value of 2.4 µM. Based on these in vitro results, we conclude that EAM-2201 has the potential to trigger in vivo pharmacokinetic drug interactions when co-administered with substrates of CYP2C8, CYP2C9, CYP2C19, CYP3A4 and UGT1A3.

Progesterone hydroxylation by cytochromes P450 2C and 3A enzymes in marmoset liver microsomes

1. Common marmosets (Callithrix jacchus) are potentially useful nonhuman primate models for preclinical drug metabolism studies. However, the roles of marmoset cytochrome P450 (P450) isoforms in the oxidation of endobiotic progesterone have not been fully investigated. In this study, the roles of marmoset P450 isoforms in progesterone hydroxylation were extensively determined.2. The activities of liver microsomes from individual marmosets with respect to progesterone 21/17α- and 16α/6β-hydroxylation were significantly correlated with those for flurbiprofen 4-hydroxylation and midazolam 1′-hydroxylation, respectively, as similar correlations have been found in humans. Anti-P450 2 C and 3 A antibodies suppressed progesterone 21/17α- and 16α/6β-hydroxylation, respectively, in marmoset liver microsomes.3. Recombinant marmoset P450 2C58 and 2C19 catalyzed progesterone to form 21-hydroxyprogesterone and 16α-hydroxyprogesterone, respectively, as major products with high maximum velocity/Km values of 0.53 and 0.089 mL/min/nmol, respectively. Recombinant marmoset P450 3A4/90 oxidized progesterone to form 6β-hydroxyprogesterone as a major product with homotropic cooperativity (>1 of Hill coefficients).4. These results indicate that the overall activities and roles of liver microsomal P450 enzymes in marmoset livers are similar to those in humans, especially for progesterone 21/17α- and 16α/6β-hydroxylation by marmoset P450 2 C and 3 A enzymes, respectively, suggesting important roles for these P450 enzymes in the metabolism of endobiotics in marmosets.

Inhibitory Effects of 12 Ginsenosides on the Activities of Seven Cytochromes P450 in Human Liver Microsomes

Ginseng, a traditional herbal drug, has been used in Eastern Asia for more than 2000 years. Various ginsenosides, which are the major bioactive components of ginseng products, have been shown to exert numerous beneficial effects on the human body when co-administered with drugs. However, this may give rise to ginsenoside-drug interactions, which is an important research consideration. In this study, acassette assay was performed the inhibitory effects of 12 ginsenosides on seven cytochrome P450 (CYP) isoforms in human liver microsomes (HLMs) using LC-MS/MS to predict the herb-drug interaction. After incubation of the 12 ginsenosides with seven cocktail CYP probes, the generated specific metabolites were quantified by LC-MS/MS to determine their activities. Ginsenoside Rb1 and F2 showed strong selective inhibitory effect on CYP2C9-catalyzed diclofenac 4′-hydroxylation and CYP2B6-catalyzed bupropion hydroxylation, respectively. Ginsenosides Rd showed weak inhibitory effect on the activities of CYP2B6, 2C9, 2C19, 2D6, 3A4, and compound K, while ginsenoside Rg3 showed weak inhibitory effects on CYP2B6. Other ginsenosides, Rc, Rf, Rg1, Rh1, Rf, and Re did not show significant inhibitory effects on the activities of the seven CYPs in HLM. Owing to the poor absorption of ginsenosides after oral administration in vivo, ginsenosides may not have significant side effects caused by interaction with other drugs.

Oxidation of R- and S-omeprazole stereoselectively mediated by liver microsomal cytochrome P450 2C19 enzymes from cynomolgus monkeys and common marmosets

Racemic omeprazole has been used for clinically treating gastric acid-related diseases and also as a typical human cytochrome P450 (P450) 2C19 probe substrate in preclinical studies. S-Omeprazole has been developed as a single enantiomer medicine, which has been reported not to be associated with polymorphic human P450 2C19 phenotypes. In this study, 5-hydroxylation and sulfoxidation activities, with respect to stereoselective R- and S-omeprazole oxidations by liver microsomes from experimental animals including non-human primates and humans, were investigated in vitro. Liver microsomes from humans, cynomolgus monkeys, and mice preferentially mediated R-omeprazole 5-hydroxylations, however those from marmosets, minipigs, dogs, and rats preferentially mediated S-omeprazole 5-hydroxylations. High catalytic activities were observed for recombinant human P450 2C19 in R-omeprazole 5-hydroxlations, cynomolgus monkey P450 2C19 in both R- and S-omeprazole 5-hydroxlations, and marmoset P450 2C19 in S-omeprazole 5-hydroxlations. On the other hand, human, cynomolgus monkey, and marmoset P450 3A enzymes preferentially mediated S-omeprazole sulfoxidations. Correlation and kinetic analyses revealed a high affinity of polymorphic cynomolgus monkey and marmoset liver microsomal P450 2C19 enzymes with respect to R- and S-omeprazole 5-hydroxylations, respectively, and a high capacity of cynomolgus monkey and marmoset liver microsomal P450 3A4 for omeprazole 5-hydroxylations and sulfoxidations. R-and S-omeprazole 5-hydroxylation activities in cynomolgus monkey and marmoset liver microsomes were significantly different among wild-type, heterozygous, and homozygous animals genotyped for cynomolgus monkey P450 2C19 p.[(Phe100Asn; Ala103Val; Ile112Leu)] and for marmoset P450 2C19 p.[(Phe7Leu; Ser254Leu; Ile469Thr)], respectively. The results of this study demonstrate polymorphic cynomolgus monkey and marmoset P450 2C19-dependent omeprazole oxidation activities with individual variations similar to humans.

A Focal Adhesion Kinase-Derived Peptide Binds the Src SH3 Domain in Two Orientations, As Demonstrated Using Paramagnetic Nuclear Magnetic Resonance.

SH3 binding peptides contain polyproline helices and are classified according to their binding orientations as N-to-C-terminal or C-to-N-terminal. We have tested the hypothesis that such a peptide binds in both orientations but with different populations. A focal adhesion kinase (FAK)-derived peptide was tested for its binding orientation on the Src SH3 domain. Paramagnetic tags were introduced at several positions on the SH3 domain, and on the basis of the paramagnetic relaxation enhancements (PREs) of the amide protons, the positions of the paramagnetic centers were determined. Two peptides were synthesized with (13)C-enriched Ala or Pro, at the N-terminal or C-terminal side of the peptide, and the intermolecular PREs were measured. The results provide compelling evidence that the FAK-derived peptide binds the SH3 domain in two orientations. In the major state, the SH3 domain binds the peptide in the N-C orientation, whereas 20% of the time, the peptide binds in the C-N orientation. We conclude that the distinction between N-C and C-N orientations, which is based on crystal structures, might be artificial. The pseudosymmetric nature of the polyproline helix might allow for binding in both orientations in the solution state.

Further Characterization of the Metabolism of Desloratadine and Its Cytochrome P450 and UDP-glucuronosyltransferase Inhibition Potential: Identification of Desloratadine as a Relatively Selective UGT2B10 Inhibitor.

Desloratadine (Clarinex), the major active metabolite of loratadine (Claritin), is a nonsedating antihistamine used for the treatment of seasonal allergies and hives. Previously we reported that the formation of 3-hydroxydesloratadine, the major human metabolite of desloratadine, involves three sequential reactions, namely N-glucuronidation by UGT2B10 followed by 3-hydroxylation by CYP2C8 followed by deconjugation (rapid, nonenzymatic hydrolysis of the N-glucuronide). In this study we assessed the perpetrator potential of desloratadine based on in vitro studies of its inhibitory effects on cytochrome P450 and UDP-glucuronosyltransferase (UGT) enzymes in human liver microsomes (HLM). Desloratadine (10 µM) caused no inhibition (<15%) of CYP1A2, CYP2C8, CYP2C9, or CYP2C19 and weak inhibition (32-48%) of CYP2B6, CYP2D6, and CYP3A4/5. In cryopreserved human hepatocytes (CHH), which can form the CYP2C8 substrate desloratadine N-glucuronide, desloratadine did not inhibit the CYP2C8-dependent metabolism of paclitaxel or amodiaquine. Assessment of UGT inhibition identified desloratadine as a potent and relatively selective competitive inhibitor of UGT2B10 (Ki value of 1.3 μM). Chemical inhibition of UGT enzymes in HLM demonstrated that nicotine (UGT2B10 inhibitor) but not hecogenin (UGT1A4 inhibitor) completely inhibited the conversion of desloratadine (1 µM) to 3-hydroxydesloratadine in HLM fortified with both NADPH and UDP-glucuronic acid. 3-Hydroxydesloratadine formation correlated well with levomedetomidine glucuronidation (UGT2B10 marker activity) with a panel of individual CHH (r(2) = 0.72). Overall, the results of this study confirm the role of UGT2B10 in 3-hydroxydesloratadine formation and identify desloratadine as a relatively selective in vitro inhibitor of UGT2B10.

In Vitro Metabolism and Stability of the Actinide Chelating Agent 3,4,3-LI(1,2-HOPO)

The hydroxypyridinonate ligand 3,4,3-LI(1,2-HOPO) is currently under development for radionuclide chelation therapy. The preclinical characterization of this highly promising ligand comprised the evaluation of its in vitro properties, including microsomal, plasma, and gastrointestinal fluid stability, cytochrome P450 inhibition, plasma protein binding, and intestinal absorption using the Caco-2 cell line. When mixed with active human liver microsomes, no loss of parent compound was observed after 60 min, indicating compound stability in the presence of liver microsomal P450. At the tested concentrations, 3,4,3-LI(1,2-HOPO) did not significantly influence the activities of any of the cytochromal isoforms screened. Thus, 3,4,3-LI(1,2-HOPO) is unlikely to cause drug-drug interactions by inhibiting the metabolic clearance of coadministered drugs metabolized by these enzymes. Plasma protein-binding assays revealed that the compound is protein-bound in dogs and less extensively in rats and humans. In the plasma stability study, the compound was stable after 1 h at 37°C in mouse, rat, dog, and human plasma samples. Finally, a bidirectional permeability assay demonstrated that 3,4,3-LI(1,2-HOPO) is not permeable across the Caco-2 monolayer, highlighting the need to further evaluate the effects of various compounds with known permeability enhancement properties on the permeability of the ligand in future studies.

Effect of Yajieshaba, a preparation of Dai indigenous medicine, on enhanced liver detoxification

ObjectiveTo explore the mechanistic effects of Yajieshaba (YJSB) on enhanced liver detoxification. MethodsThe effects of YJSB on alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed in five acute chemical liver injury models [carbon tetrachloride (CCl4), D-galactosamine (D-Glan), 4-acetamidophenol (AAP), thioacetamide (TAA) and 1-naphthyl isothiocyanate (ANIT)]. Sleep latency and sleep time of pentobarbital sodium were tested in control mice and CCl4 model miceafter oral YJSB administration. The effects of YJSB on drug metabolism enzymes of liver microsomes were tested in control rats and CCl4 model rats. The levels of cytochrome P450 (CYP450) and Cyt b5 in liver microsomes were assayed using the method by Omura and Sato, and activities of erythromycin N-demethylase (ERD) and aminopyrine N-demethyl (ADM) were evaluated by Nash colorimetry. Probe substrate-based high performance liquid chromatography (HPLC) methods were established for CYP3A4 and CYP1A2. ResultsThe level of serum ALT was reduced by YJSB at 3.51 g/kg in the five models as follows: CCl4 > D-Glan, AAP, ANIT > TAA. YJSB treatment did not reduce the level of serum AST. YJSB at 3.51 g/kg prolonged the sleep latency in control mice and shortened the sleep time of control mice and CCl4 model mice. For control rats, YJSB at 2.43 g/kg increased the levels of CYP450 and Cyt b5 and induced the activities of ERD and ADM; for liver injuries induced by CCl4 in rats, YJSB at 2.43 g/kg increasedthe levels of CYP450 and Cyt b5. These results suggest that YJSB at 2.43 g/kg induces CYP3A4 and CYP1A2. ConclusionThese results suggest that YJSB enhanced liver detoxification and the mechanisms may be partially related to CYP3A4 and CYP1A2 induction.