Human Liver Microsomal Incubations Research Articles

Liquid chromatography–tandem mass spectrometry method for measurement of nicotine N-glucuronide: A marker for human UGT2B10 inhibition

Nicotine is considered to be a specific substrate for UGT2B10, an isoform of human uridine diphosphate glucuronosyltransferase (UGT). In the present study, a sensitive and selective liquid chromatography/tandem mass spectrometry (LC–MS–MS) method for quantification of nicotine N-glucuronide in pooled human liver microsomal incubates was developed and validated. Proteins in a 200 μL aliquot of incubation solution were precipitated by adding 40 μL 35% perchloric acid. The overall extraction efficiency was greater than 98%. Nicotine N-glucuronide and internal standard were recorded using selected reaction monitoring in positive ion electrospray with ion transitions of m/ z 339–163 and m/ z 342–166, respectively. The linear calibration curve was obtained over the concentration range of 10–1000 nM, with a lower limit of quantification of 10 nM. The intra-day and inter-day precision (% CV) and accuracy (% bias) of the method were within 15% at all quality control levels. Nicotine glucuronide in processed samples was stable for 24 h at room temperature and 48 h at 4 °C based on the stability experiments performed in this study. This established method was employed to evaluate the inhibitory effects of five target compounds including amitriptyline, hecogenin, imipramine, lamotrigine, and trifluoperazine on enzymatic activity of UGT2B10. IC 50 values for inhibition of nicotine N-glucuronidation by amitriptyline, imipramine, lamotrigine, and trifluoperazine were calculated. Trifluoperazine was found to be a non-substrate inhibitor for human UGT2B10.

Identification of Amiodarone Metabolites in Human Bile by Ultraperformance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry

Amiodarone is recognized as an effective drug in the treatment of arrhythmias. Previous experiments demonstrated that mono-N-desethylamiodarone (MDEA) was the major circulating metabolite in humans. In addition, dealkylation, hydroxylation, and deamination were minor metabolic pathways. The purpose of this study was to identify the metabolites of amiodarone in the bile obtained from patients with T-tube drainage after oral drug administration. Amiodarone metabolism in vitro was also investigated using human liver microsomes (HLMs) and S9 fraction. Ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) revealed 33 metabolites in human bile, including 22 phase I and 11 phase II metabolites. The major metabolites were MDEA (M7) and ω-carboxylate amiodarone (M12). Metabolite M12 was isolated from human bile, and the chemical structure was confirmed using UPLC-Q/TOF MS and ¹H NMR. Moreover, the authentic standards of two hydroxylated metabolites, 2-hydroxylamiodarone and 3'-hydroxylamiodarone, were obtained through microbial transformation. Several novel metabolic pathways of amiodarone in human were proposed, including ω-carboxylation, deiodination, and glucuronidation. The in vitro study demonstrated that incubation of HLMs with amiodarone did not give rise to any carboxyl metabolites. In contrast, M12 and its metabolites were detected in human liver S9 incubation samples, and the production of these metabolites were inhibited almost completely by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, suggesting the involvement of alcohol dehydrogenase in the ω-carboxylation of amiodarone. Overall, UPLC-Q/TOF MS analysis leads to the discovery of several novel amiodarone metabolites in human bile and underscores the importance of bile as an excretion pathway.

A Refined Cytochrome P450 IC50 Shift Assay for Reliably Identifying CYP3A Time-Dependent Inhibitors

A refined cytochrome P450 (P450) enzyme IC₅₀ shift assay for more accurately screening CYP3A time-dependent inhibitors (TDIs) is presented. In contrast to the regular IC₅₀ shift assay, in which only one pair of P450 inhibition curves is generated, this modified method generates two pairs of inhibition curves; one pair of curves is created from human liver microsomal incubations with the test article in the presence or absence of NADPH (curves 1 and 2) (same as the traditional assay), and the other pair is created from new microsomal incubations with extract (compound/metabolites) of previous incubations (curves 3 and 4). To assess the true CYP3A time-dependent inhibition, we propose a new parameter, the vertical IC₅₀ curve shift (VICS), represented by vertical shift difference between the two sets of curves divided by inhibitor concentration at which maximal vertical shift of curves 1 and 2 is observed. A shift in the curves 1 and 2 could mean a time-dependent inhibition or formation of a more active inhibitory metabolite(s). The new method provides more reliable characterization of the shift as a result of a true TDI- or metabolite-mediated reversible inhibition. Nine known TDI drugs were evaluated using this refined shift assay. The derived VICS values correlated well with the reported k(inact)/K(I) values derived via the conventional dilution assay method. Thus, the refined assay can be used to identify a true TDI and quantitatively assess the inactivation potential of TDIs in a high-throughput fashion. This assay can be invaluable to screen for true P450 TDIs in the early drug discovery.

Atrazine Metabolite Screening in Human Microsomes: Detection of Novel Reactive Metabolites and Glutathione Adducts by LC-MS

Atrazine (ATZ), one of the most widely used herbicides worldwide, has been the subject of several scientific studies associated with its human and ecological risks. In order to study atrazine's toxicity, the formation of its metabolites and the result of their exposure must be assessed. This relies on our ability to detect and identify all of atrazine's metabolites; however, no previous untargeted screening method has reported the detection of all known metabolites and glutathione conjugates at once. In this study, a compound-specific, postacquisition metabolic screening method was employed following a generic HPLC separation coupled with high resolution time-of-flight mass spectrometry (TOF-MS) to detect Phase I metabolites and glutathione conjugates generated by in vitro human liver microsomal incubations. Our method was designed to be unbiased and applicable to a wide variety of compounds since methods that can detect a broad range of metabolites with high sensitivity are of great importance for many types of experiments requiring thorough metabolite screening. On the basis of incubations with atrazine and three closely related analogues (simazine, propazine, and cyanazine), we have proposed a new Phase I metabolism scheme. All known Phase I transformations of atrazine were successfully detected, as well as a new N-oxidation product. Novel reactive metabolites were also detected as well as their glutathione conjugates. These newly detected species were produced via imine formation on the N-ethyl group, a biotransformation not previously observed for atrazine or its analogues.

Chemical inhibitors of cytochrome P450 isoforms in human liver microsomes: a re-evaluation of P450 isoform selectivity

The majority of marketed small-molecule drugs undergo metabolism by hepatic Cytochrome P450 (CYP) enzymes (Rendic 2002). Since these enzymes metabolize a structurally diverse number of drugs, metabolism-based drug-drug interactions (DDIs) can potentially occur when multiple drugs are coadministered to patients. Thus, a careful in vitro assessment of the contribution of various CYP isoforms to the total metabolism is important for predicting whether such DDIs might take place. One method of CYP phenotyping involves the use of potent and selective chemical inhibitors in human liver microsomal incubations in the presence of a test compound. The selectivity of such inhibitors plays a critical role in deciphering the involvement of specific CYP isoforms. Here, we review published data on the potency and selectivity of chemical inhibitors of the major human hepatic CYP isoforms. The most selective inhibitors available are furafylline (in co-incubation and pre-incubation conditions) for CYP1A2, 2-phenyl-2-(1-piperidinyl)propane (PPP) for CYP2B6, montelukast for CYP2C8, sulfaphenazole for CYP2C9, (-)-N-3-benzyl-phenobarbital for CYP2C19 and quinidine for CYP2D6. As for CYP2A6, tranylcypromine is the most widely used inhibitor, but on the basis of initial studies, either 3-(pyridin-3-yl)-1H-pyrazol-5-yl)methanamine (PPM) and 3-(2-methyl-1H-imidazol-1-yl)pyridine (MIP) can replace tranylcypromine as the most selective CYP2A6 inhibitor. For CYP3A4, ketoconazole is widely used in phenotyping studies, although azamulin is a far more selective CYP3A inhibitor. Most of the phenotyping studies do not include CYP2E1, mostly because of the limited number of new drug candidates that are metabolized by this enzyme. Among the inhibitors for this enzyme, 4-methylpyrazole appears to be selective.

In vitro–in vivo extrapolation of CYP2C8-catalyzed paclitaxel 6α-hydroxylation: effects of albumin on in vitro kinetic parameters and assessment of interindividual variability in predicted clearance

This study aimed to characterize the effects of bovine serum albumin (BSA) on the kinetics of CYP2C8-catalyzed paclitaxel 6α-hydroxylation in vitro; determine whether the addition of BSA to incubations improves the prediction of paclitaxel hepatic clearance via this pathway in vivo; and assess interindividual variability in predicted clearance. The kinetics of paclitaxel 6α-hydroxlation by human liver microsomes (HLM) and recombinant CYP2C8 were characterized in incubations performed with and without BSA (2% w/v) supplementation, and the in vitro kinetic data were extrapolated to provide estimates of in vivo clearances. The Simcyp population-based ADME simulator was used to determine interindividual variability in the predicted clearances. Supplementation of incubations of HLM with BSA resulted in a 3.6-fold increase in the microsomal intrinsic clearance for paclitaxel 6α-hydroxylation, due mainly to a reduction in K(m) (7.08 ± 2.50 to 2.26 ± 0.39μM), while addition of BSA to incubations of recombinant CYP2C8 resulted in an approximate doubling of intrinsic clearance. Mean values of predicted in vivo hepatic clearance were in good agreement with clinical data when in vitro data obtained in the presence of BSA were used for IV-IVE. Simcyp predicted 20- to 30-fold interindividual variability in in vivo paclitaxel hepatic clearance via the 6α-hydroxylation pathway. Human liver microsomal K(m) and intrinsic clearance values are over- and underpredicted, respectively, when incubations of the CYP2C8 substrate paclitaxel are performed without BSA supplementation. IV-IVE based on kinetic parameters generated in the presence of BSA improves the accuracy of predicted paclitaxel hepatic clearance.

Atazanavir Metabolism According to CYP3A5 Status: An In Vitro-In Vivo Assessment

The current study was a follow-up to an in vivo study in which atazanavir oral clearance was shown to be dependent on genetically determined CYP3A5 expression status, but only in non-African Americans. The aim of this study was to identify atazanavir metabolites generated by CYP3A5 and to evaluate this metabolite pattern in the African-American versus non-African-American CYP3A5 expressors from the previous study. First, the in vitro metabolism of atazanavir was evaluated using human liver microsomes (HLM) and CYP3A4 and CYP3A5 isoforms. Second, the metabolite pattern generated by CYP3A5 was evaluated in human plasma samples from the previous study. Atazanavir metabolites were analyzed using liquid chromatography-tandem mass spectrometry methods. Metabolite areas under the time-concentration curves (AUCs) were normalized to atazanavir AUC to generate an AUC ratio. Sixteen metabolites were observed in human liver microsomal incubations representing five "phase I" biotransformation pathways. Mono-oxidation products (M1 and M2) were formed by CYP3A5 at a faster rate than CYP3A4 by 32- and 2.6-fold, respectively. This finding was replicated in HLM from a genetically determined CYP3A5 expressor versus nonexpressor. In the in vivo samples, the M1 and M2 AUC ratios were approximately 2-fold higher in CYP3A5 expressors versus nonexpressors (P < 0.05), and the difference was similar in African Americans and non-African Americans. Thus, CYP3A5 produced a unique metabolite "signature" for atazanavir in vitro and in vivo, independent of race. Therefore, other pharmacological factors are likely to explain the apparent lack of effect of genetically determined CYP3A5 expressor status on atazanavir oral clearance in African Americans from the previous study.

Strategy of using microsome-based metabolite production to facilitate the identification of endogenous metabolites by liquid chromatography mass spectrometry

One of the challenges in metabolomic profiling of complex biological samples is to identify new and unknown compounds. Typically, standards are used to help identify metabolites, yet standards cannot be purchased or readily synthesized for many unknowns. In this work we present a strategy of using human liver microsomes (HLM) to metabolize known endogenous human metabolites (substrates), producing potentially new metabolites that have yet to be documented. The metabolites produced by HLM can be tentatively identified based on the associated substrate structure, known metabolic processes, tandem mass spectrometry (MS/MS) fragmentation patterns and, if necessary, accurate mass measurements. Once identified, these metabolites can be used as references for identification of the same compounds in complex biological samples. As a proof of principle, a total of 9 metabolites have been identified from individual HLM incubations using 5 different substrates. Each metabolite was used as a standard. In the analysis of human urine sample by liquid chromatography MS/MS, four spectral matches were found from the 9 microsome-produced metabolite standards. Two of them have previously been documented as endogenous human metabolites, the third is an isomer of a microsome-metabolite and the fourth compound has not been previously reported and is also an isomer of a microsome-metabolite. This work illustrates the feasibility of using microsome-based metabolism to produce metabolites of endogenous human metabolites that can be used to facilitate the identification of unknowns in biological samples. Future work on improving the performance of this strategy is also discussed.

Mechanistic Studies on a P450-Mediated Rearrangement of BMS-690514: Conversion of a Pyrrolotriazine to a Hydroxypyridotriazine

BMS-690514 ((3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4] triazin-5-yl)methyl)-3-piperidinol) is an oral oncologic agent being developed for the treatment of patients with advanced nonsmall cell lung cancer and breast cancer. The compound is metabolized via multiple metabolic pathways, including P450-mediated oxidation at one of the carbons of its pyrrolotriazine group. Oxidation at this site results in the formation of two metabolites, M1 and M37. Mass spectrometric and NMR analysis revealed that M1 underwent an unusual structural change, where the pyrrolotriazine moiety rearranged to yield a hydroxypyridotriazine group. In contrast, the structure of the pyrrolotriazine moiety remained intact in M37. In vitro experiments with liver microsomes and deuterated or tritiated BMS-690514 containing the isotopic label on the carbon that underwent oxidation indicated that during the formation of M1, the isotope label was retained at the site of hydroxylation, while the label was lost during the formation of M37. On the basis of these results, a mechanism for the formation of M1 was proposed as follows: BMS-690514 was first oxidized by P450 enzymes either via epoxidation or an iron-oxo addition pathway to form a zwitterionic intermediate. This was followed by opening of the pyrrolotriazine ring to form an aldehyde intermediate, which could be partially trapped with methoxyamine. The aldehyde intermediate then reacted with the secondary amine of the methoxyaniline group in the molecule to form the pyridotriazine moiety of M1. This mechanism is consistent with the observed retention of the isotope label in M1. Metabolite M37 may be formed either via a common zwitterionic intermediate, shared with M1, or through a direct insertion pathway. In in vitro human liver microsome incubations, the abundance of M1 was higher than M37, suggesting that breaking of the carbon-nitrogen bond to generate the aldehyde intermediate, a process similar to N-dealkylation, was a preferred pathway.

Studies on the metabolism of the α‐pyrrolidinophenone designer drug methylenedioxy‐pyrovalerone (MDPV) in rat and human urine and human liver microsomes using GC–MS and LC–high‐resolution MS and its detectability in urine by GC–MS

Since the late 1990s, many derivatives of the α-pyrrolidinophenone (PPP) drug class appeared on the drugs of abuse market. The latest compound was described in 2009 to be a classic PPP carrying a methylenedioxy moiety remembering the classic entactogens (ecstasy). Besides Germany, 3,4-methylene-dioxypyrovalerone (MDPV) has appeared in many countries in Europe and Asia, indicating its worldwide importance for forensic and clinical toxicology. The aim of the presented work was to identify the phase I and II metabolites of MDPV and the human cytochrome-P450 (CYP) isoenzymes responsible for its main metabolic step(s). Finally, the detectability of MDPV in urine by the authors' systematic toxicological analysis (STA) should be studied. The urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified after work-up by GC-MS and liquid chromatography (LC)-high-resolution MS (LC-HR-MS). The studies revealed the following phase I main metabolic steps in rat and human: demethylenation followed by methylation, aromatic and side chain hydroxylation and oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Using LC-HR-MS, most metabolite structures postulated according to GC-MS fragmentation could be confirmed and the phase II metabolites were identified. Finally, the formation of the initial metabolite demethylenyl-MDPV could be confirmed using incubation of human liver microsomes. Using recombinant human CYPs, CYP 2C19, CYP 2D6 and CYP 1A2 were found to catalyze this initial step. Finally, the STA allowed the detection of MDPV metabolites in the human urine samples.

Reversible inhibition of three important human liver cytochrome p450 enzymes by tiliroside

Tiliroside, an active flavonoid extensively found in many medicinal plants including Helichrysum italicum, Geranium mexicanum and Helianthemum glomeratum, has been demonstrated to exert multiple biological effects including antiinflammatory, antimicrobial, antioxidant and antitumor activities. Cytochrome P450 (CYP) enzymes play an important role in the Phase I oxidation metabolism of a wide range of xenobiotics and inhibition of CYP isoforms might influence the elimination of drugs and induce serious adverse drug response. The inhibition of seven CYP isoforms (CYP3A4, CYP1A2, CYP2A6, CYP2D6, CYP2C9, CYP2C8 and CYP2E1) by tiliroside was investigated using in vitro human liver microsomal incubation assays. The results showed that tiliroside strongly inhibited the activity of CYP3A4 (IC(50) = 9.0 ± 1.7 μm), CYP2C8 (IC(50) = 12.1 ± 0.9 μm) and CYP2C9 (IC(50) = 10.2 ± 0.9 μm) with other CYP isoforms negligibly influenced. Further kinetic analysis showed that inhibition of these three CYP isoforms by tiliroside is best fit to a competitive way. The K(i) value was calculated to be 5.5 μm, 3.3 μm, 9.4 μm for CYP3A4, CYP2C9 and CYP2C8, respectively. The relatively low K(i) values suggested that tiliroside might induce drug-drug interactions with many clinically used drugs which are mainly metabolized by these three CYP isoforms. Therefore, attention should be given to the probable drug-drug interaction between tiliroside-containing herbs and substrates of CYP3A4, CYP2C9 and CYP2C8.

Plasma Stability-Dependent Circulation of Acyl Glucuronide Metabolites in Humans: How Circulating Metabolite Profiles of Muraglitazar and Peliglitazar Can Lead to Misleading Risk Assessment

Muraglitazar and peliglitazar, two structural analogs differing by a methyl group, are dual peroxisome proliferator-activated receptor-α/γ activators. Both compounds were extensively metabolized in humans through acyl glucuronidation to form 1-O-β-acyl glucuronide (AG) metabolites as the major drug-related components in bile, representing at least 15 to 16% of the dose after oral administration. Peliglitazar AG was the major circulating metabolite, whereas muraglitazar AG was a very minor circulating metabolite in humans. Peliglitazar AG circulated at lower concentrations in animal species than in humans. Both compounds had a similar glucuronidation rate in UDP-glucuronic acid-fortified human liver microsomal incubations and a similar metabolism rate in human hepatocytes. Muraglitazar AG and peliglitazar AG were chemically synthesized and found to be similarly oxidized through hydroxylation and O-demethylation in NADPH-fortified human liver microsomal incubations. Peliglitazar AG had a greater stability than muraglitazar AG in incubations in buffer, rat, or human plasma (pH 7.4). Incubations of muraglitazar AG or peliglitazar AG in plasma produced more aglycon than acyl migration products compared with incubations in the buffer. These data suggested that the difference in plasma stability, not differences in intrinsic formation, direct excretion, or further oxidation of muraglitazar AG or peliglitazar AG, contributed to the observed difference in the circulation of these AG metabolites in humans. The study demonstrated the difficulty in doing risk assessment based on metabolite exposure in plasma because the more reactive muraglitazar AG would not have triggered a threshold of concern based on the recent U.S. Food and Drug Administration guidance on Metabolites in Safety Testing, whereas the more stable peliglitazar AG would have.

Metabolism of Ebracteolata Compound B Studied In Vitro with Human Liver Microsomes, HepG2 Cells, and Recombinant Human Enzymes

Ebracteolata compound B (ECB) is one major active component of both Euphorbia ebracteolata and Euphorbia fischeriana, which have been extensively used as a tuberculocide in the Asian countries. The aim of our present study was to characterize ECB metabolism in human liver microsomes, HepG2 cells, and recombinant human enzymes. One monohydroxylation metabolite, determined by mass spectrometry to be 1-(2,4-dihydroxy-6-methoxy-3-methylphenyl)-2-hydroxyethanone, and one monoglucuronide, isolated and determined by hydrolysis with β-glucuronidase, mass spectrometry, and (1)H NMR to be 2-hydroxy-6-methoxy-3-methyl-acetophenone-4-O-β-glucuronide, were observed in human liver microsomal incubates in the presence of NADPH or UDP-glucuronic acid (UDPGA), respectively. However, the mixed incubation of ECB with human liver microsomes in the presence of both NADPH and UDPGA showed the monoglucuronide to be the most major metabolite, indicating that glucuronidation was probably the major clearance pathway of ECB in humans. No glucuronide and only trace monohydroxylation metabolite were observed in HepG2 cells. The cytochrome P450 and UDP-glucuronosyltransferase (UGT) isoenzymes were identified by using selective chemical inhibition and recombinant human enzymes. The results indicated that CYP3A4 was probably involved in ECB oxidative metabolism and UGT1A6 and UGT1A9 were important catalytic enzymes in ECB glucuronidation. The results from enzymatic kinetic analysis showed the oxidative metabolism in human liver microsomes; the glucuronidation in human liver microsomes and recombinant UGT1A6 exhibited a typical Michaelis-Menten pattern, but the glucuronidation in UGT1A9 exhibited a substrate inhibition pattern. UGT1A6 had the highest affinity compared with human liver microsomes and UGT1A9, indicating its important role in ECB glucuronidation.

Evaluation of Cytochrome P450 Inhibition Assays Using Human Liver Microsomes by a Cassette Analysis /LC-MS/MS

In vitro inhibition assays are used to screen new chemical entities (NCEs) for inhibition studies by using human liver microsomes. High-throughput inhibition assays using pooling methods have been developed to keep pace with screening requirements at the lead ADME stage. This method can determine IC(50) NCEs using microsomes from various organs from any species. A cassette analysis inhibition assay for five of the major CYP enzymes (phenacetin for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, bufurarol for CYP2D6 and midazolam, nifedipine and atorvastatin for CYP3A4) in pooled human liver microsomes using ultraperformance liquid chromatography tandem mass spectrometry (LC/MS/MS) were developed. The major metabolites of seven CYP-specific probe substrates for the five P450 isoforms were monitored and quantified to determine IC(50) values. Human liver microsomal incubation samples at each test compound concentration were combined and analyzed simultaneously by the LC/MS/MS method. The incubation was performed using the selective CYP inhibitors for each isoform: fluvoxamine (CYP1A2), sulphaphenazole (CYP2C9), ticlopidine (CYP2C19), quinidine (CYP2D6), and Ketoconazole (CYP3A4). Similar IC(50) results were obtained using the cassette analysis and discrete analysis method. The IC(50) values determined for typical CYP inhibitors were reproducible and consistent with those reported in the literature. The assay was fully automated in 96 well plate formats using Microlab 4000 series (Hamilton) coupled with two termomixer comfort (Eppendorf). An overall 70% time savings was achieved by pooling four isoforms samples (1A2, 2C9, 2C19 and 2D6) into one sample and also by pooling three CYP 3A4 substrate samples (MDZ, ATR, NIF) into one sample. Cassette analysis minimized the number of injections on LC/MS/MS analysis which results in a decrease in the LC/MS/MS analysis time.

A Novel Doxorubicin Prodrug with Controllable Photolysis Activation for Cancer Chemotherapy

PurposeDoxorubicin (DOX) is a very effective anticancer agent. However, in its pure form, its application is limited by significant cardiotoxic side effects. The purpose of this study was to develop a controllably activatable chemotherapy prodrug of DOX created by blocking its free amine group with a biotinylated photocleavable blocking group (PCB).MethodsAn n-hydroxy succunamide protecting group on the PCB allowed selective binding at the DOX active amine group. The PCB included an ortho-nitrophenyl group for photo cleavability and a water-soluble glycol spacer arm ending in a biotin group for enhanced membrane interaction.ResultsThis novel DOX-PCB prodrug had a 200-fold decrease in cytotoxicity compared to free DOX and could release active DOX upon exposure to UV light at 350 nm. Unlike DOX, DOX-PCB stayed in the cell cytoplasm, did not enter the nucleus, and did not stain the exposed DNA during mitosis. Human liver microsome incubation with DOX-PCB indicated stability against liver metabolic breakdown.ConclusionsThe development of the DOX-PCB prodrug demonstrates the possibility of using light as a method of prodrug activation in deep internal tissues without relying on inherent physical or biochemical differences between the tumor and healthy tissue for use as the trigger.

524 Oesophageal cancer proliferation is mediated by cytochrome P450 2C9 (CYP2C9)

A preliminary report implicated cytochrome P450 (CYP) 2C9 in the human liver microsomal O-demethylation of S-naproxen, suggesting that this pathway may be suitable for investigation of human hepatic CYP2C9 in vitro. Kinetic and inhibitor studies with human liver microsomes and confirmatory investigations with cDNA-expressed enzymes were undertaken here to define the role of CYP2C9 and other isoforms in the O-demethylation of R- and S-naproxen. All studies utilised a newly developed sensitive and specific HPLC assay that measured the respective O-desmethyl metabolites of R- and S-naproxen in incubations of human liver microsomes and in COS cell lysates. Microsomal R- and S-naproxen O-demethylation kinetics followed Michaelis-Menten kinetics, with respective mean apparent Km values of 123 μM and 143 μM. Sulfaphenazole, a specific inhibitor of CYP2C9, reduced the microsomal O-demethylation of R-and S-naproxen by 43% and 47%, respectively, and the CYP1A2 inhibitor furafylline decreased R- and S-naproxen O-demethylation by 38% and 28%, respectively. R,S-Mephenytoin was a weak inhibitor of R- and S-naproxen O-demethylation, but other CYP isoform specific inhibitors (e.g., coumarin, diethyldithiocarbamate, quinidine, troleandomycin) had little or no effect on these reactions. cDNA-expressed CYP2C9 and CYP1A2 were both shown to O-demethylate R- and S-naproxen. Apparent Km values (92–156 μM) for the reactions catalysed by the recombinant enzymes were similar to those observed for human liver microsomal R- and S-naproxen O-demethylation. The data demonstrate that CYP2C9 and CYP1A2 together account for the majority of human liver R- and S-naproxen O-demethylation, precluding the use of either R- or S-naproxen as a CYP isoform-specific substrate in vitro and in vivo.

The relative importance of CYP26A1 in hepatic clearance of all-trans retinoic acid

All-trans retinoic acid (RA) is a critical signaling molecule and its concentration is tightly regulated. Several P450 enzymes including CYP26A1, CYP2C8, and CYP3A4 have been proposed to be responsible for RA clearance in the liver but their quantitative importance has not been demonstrated. To determine the contribution of CYP26A1 to hepatic clearance of RA, CYP26A1 protein was quantified in 37 human liver microsomes (HLMs). CYP26A1 expression ranged from not detectable to 2.80 pmol/mg microsomal protein. RA clearance by P450 enzymes abundant in human liver was measured in Supersomes ®. CYP2C8, CYP3A4, CYP3A5 and CYP3A7 metabolized RA with unbound K m values of 3.4–7.2 μM and V max values of 2.3–4.9 pmol/min/pmol P450, but were less efficient than CYP26A1 in clearing RA. Simulations performed for livers with varying P450 expression levels over a range of RA concentrations demonstrated that at both endogenous and therapeutic concentrations of RA, CYP26A1 is the primary enzyme responsible for 4-OH RA formation clearance. HLM incubation data showed that 4-OH RA formation velocity varied from 0.2 to 15.3 pmol/min/mg microsomal protein and velocity in HLMs was significantly correlated ( p < 0.01) to CYP26A1, CYP3A4, and CYP3A5 protein content, but not to CYP2C8. When experimental data were scaled to in vivo clearances, the predicted hepatic clearance of RA (0.07 L/min using combined Supersome ® data) was similar to the published in vivo clearance of RA. These findings suggest that CYP26A1 is the P450 isoform that should be targeted when designing RA metabolism blocking agents.

Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1′-hydroxymidazolam, and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC–MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1′-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D 5) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 μL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 μL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm × 100 mm, 3.5 μm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100–250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85–115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.

Cytochrome P450-Mediated Bioactivation of the Epidermal Growth Factor Receptor Inhibitor Erlotinib to a Reactive Electrophile

The epidermal growth factor receptor tyrosine kinase inhibitor erlotinib (ERL) is approved for treatment of non-small-cell lung cancer. Numerous reports of ERL-associated toxicities are consistent with immune-mediated toxicity, including drug-induced hepatitis, interstitial lung disease, Stevens-Johnson syndrome, and toxic epidermal necrolysis. Although the mechanism of toxicity has not been established, we present evidence that reactive intermediates are formed during the metabolism of ERL, which can covalently conjugate to the cysteine group of the peptide-mimetic GSH. Seven ERL-GSH conjugates were identified in incubations with hepatic microsomes. Cytochrome P450 (P450)-dependent adducts are proposed to be formed via reactive epoxide and electrophilic quinone-imine intermediates. In incubations of human liver microsomes, intestinal microsomes, pulmonary microsomes, and recombinant P450s, CYP3A4 was the primary enzyme responsible for the bioactivation of ERL; however, CYP1A1, CYP1A2, CYP3A5, and CYP2D6 were capable of catalyzing the bioactivation as well. During the metabolism of ERL, CYP3A4 and CYP3A5 are irreversibly inactivated by ERL in a time- and concentration-dependent manner. Inactivation was not dependent on oxidation of the ERL alkyne group to form a reactive oxirene or ketene, as shown by synthesizing analogs where the alkyne was replaced with a cyano group. CYP1A1, CYP1A2, and CYP2D6 were not inactivated despite catalyzing the formation of ERL-GSH adducts.

Characterisation and identification of the human N +-glucuronide metabolite of cediranib

Cediranib (4-[(4-fluoro-2-methyl-1 H-indol-5-yl)oxy]-6-methoxy-7-[3-(1-pyrrolidinyl)propoxy]quinazoline; RECENTIN™), a vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor (TKI) of all three VEGF receptors, is currently in Phase III clinical trials for the first-line treatment of colorectal cancer and the treatment of recurrent glioblastoma. During its clinical development a unique human metabolite, an N +-glucuronide, was identified as a major circulating metabolite and one of the major metabolites excreted into faeces. Given the possibility of four sites for the conjugation of the glucuronic acid moiety, determination of the location of the conjugation site on cediranib was warranted. A small quantity of the N +-glucuronide metabolite of cediranib was initially generated using recombinant human uridine glucuronosyltransferase 1A4 (UGT1A4) enzymes. The metabolite generated was characterised by HPLC-UV and mass spectrometric (HPLC–MS n ) detection and confirmed by 1H NMR spectroscopy. However, the exact site of conjugation could not be determined without generating more of the metabolite. Hence a subsequent biosynthetic scale-up experiment was devised to generate a sufficiently large quantity for full structural characterisation by 1H NMR spectroscopy. The identity of the N +-glucuronide metabolite generated in the UGT1A4 scale-up experiment was confirmed by HPLC–MS n and displayed the same retention time, molecular mass and mass fragmentation data as the metabolite generated in previous human liver microsomal and hepatocyte incubations. 1H NMR spectroscopy clearly showed the characteristic anomeric doublet at approximately 4.7 ppm, which, following irradiation during selective Rotating frame Overhauser Effect Spectroscopy (ROESY) experiments, enabled the site of glucuronidation to be confirmed on the pyrrolidine nitrogen. With the exception of the N +-glucuronide metabolite, all other human metabolites of cediranib were observed following incubation with hepatocytes from rat and cynomolgus monkey, the species used for toxicology testing of the drug [6]. As the N +-glucuronide was not detected in the preclinical species, it is suggested that its formation is more likely in human and higher primates (great apes), a finding widely supported in the literature.