In vitro assessment of drug-drug interaction potential of boceprevir associated with drug metabolizing enzymes and transporters.

The inhibitory effect of boceprevir (BOC), an inhibitor of hepatitis C virus nonstructural protein 3 protease was evaluated in vitro against a panel of drug-metabolizing enzymes and transporters. BOC, a known substrate for cytochrome P450 (P450) CYP3A and aldo-ketoreductases, was a reversible time-dependent inhibitor (k(inact) = 0.12 minute(-1), K(I) = 6.1 µM) of CYP3A4/5 but not an inhibitor of other major P450s, nor of UDP-glucuronosyltransferases 1A1 and 2B7. BOC showed weak to no inhibition of breast cancer resistance protein (BCRP), P-glycoprotein (Pgp), or multidrug resistance protein 2. It was a moderate inhibitor of organic anion transporting polypeptide (OATP) 1B1 and 1B3, with an IC(50) of 18 and 4.9 µM, respectively. In human hepatocytes, BOC inhibited CYP3A-mediated metabolism of midazolam, OATP1B-mediated hepatic uptake of pitavastatin, and both the uptake and metabolism of atorvastatin. The inhibitory potency of BOC was lower than known inhibitors of CYP3A (ketoconazole), OATP1B (rifampin), or both (telaprevir). BOC was a substrate for Pgp and BCRP but not for OATP1B1, OATP1B3, OATP2B1, organic cation transporter, or sodium/taurocholate cotransporting peptide. Overall, our data suggest that BOC has the potential to cause pharmacokinetic interactions via inhibition of CYP3A and CYP3A/OATP1B interplay, with the interaction magnitude lower than those observed with known potent inhibitors. Conversely, pharmacokinetic interactions of BOC, either as a perpetrator or victim, via other major P450s and transporters tested are less likely to be of clinical significance. The results from clinical drug-drug interaction studies conducted thus far are generally supportive of these conclusions.

Identification of human liver cytochrome P450 enzymes involved in the metabolism of SCH 530348 (Vorapaxar), a potent oral thrombin protease-activated receptor 1 antagonist.

Vorapaxar (SCH 530348), a potent oral thrombin protease-activated receptor 1 antagonist, is being developed as an antiplatelet agent for patients with established vascular disease. The objective of this study was to identify the human liver cytochrome P450 (P450) enzyme(s) responsible for the metabolism of SCH 530348. Human liver microsomes metabolized SCH 530348 to M19, an amine metabolite formed via carbamate cleavage, and M20 (monohydroxy-SCH 530348). Recombinant human CYP3A4 exhibited the most activity (11.5% profiled radioactivity) for the formation of M19, followed by markedly less substrate conversion with CYP1A1 and CYP2C19. Trace levels of M19, a major excreted human metabolite, were detected with CYP1A2, CYP3A5, and CYP4F3A. Formation of M19 by human liver microsomes was inhibited 89% by ketoconazole (IC(50), 0.73 µM), 34% by tranylcypromine, and 89% by anti-CYP3A4 monoclonal antibody. There was a significant correlation between the rate of M19 formation and midazolam 1'-hydroxylation (r = 0.75) or M19 formation and testosterone 6ß-hydroxylation (r = 0.92). The results of screening, inhibition, and correlation studies confirmed that CYP3A4 is the major P450 enzyme responsible for M19 formation from SCH 530348. In contrast, formation of M20, a major circulating human metabolite at steady state, was primarily catalyzed by CYP3A4 and CYP2J2. M20 is pharmacologically equipotent to SCH 530348, whereas M19 is an inactive metabolite. Formation of M20 by human liver microsomes was inhibited 89% by ketoconazole, 75% by astemizole (a CYP2J2 inhibitor), and 43% by CYP3A4 monoclonal antibody. These results suggest that CYP3A4 and CYP2J2 are both involved in the formation of M20 metabolite.

Characterization of human liver enzymes involved in the biotransformation of boceprevir, a hepatitis C virus protease inhibitor.

Boceprevir (SCH 503034), a protease inhibitor, is under clinical development for the treatment of human hepatitis C virus infections. In human liver microsomes, formation of oxidative metabolites after incubations with [(14)C]boceprevir was catalyzed by CYP3A4 and CYP3A5. In addition, the highest turnover was observed in recombinant CYP3A4 and CYP3A5. After a single radiolabeled dose to human, boceprevir was subjected to two distinct pathways, namely cytochrome P450-mediated oxidation and ketone reduction. Therefore, attempts were made to identify the enzymes responsible for the formation of carbonyl-reduced metabolites. Human liver S9 and cytosol converted ∼ 28 and ∼ 68% of boceprevir to M28, respectively, in the presence of an NADPH-generating system. Screening of boceprevir with recombinant human aldo-keto reductases (AKRs) revealed that AKR1C2 and AKR1C3 exhibited catalytic activity with respect to the formation of M+2 metabolites (M28 and M31). The formation of M28 was inhibited by 100 µM flufenamic acid (80.3%), 200 µM mefenamic acid (83.7%), and 100 µM phenolphthalein (86.1%), known inhibitors of AKRs, suggesting its formation through carbonyl reduction pathway. Formation of M28 was also inhibited by 100 µM diazepam (75.1%), 1 mM ibuprofen (70%), and 200 µM diflunisal (89.4%). These data demonstrated that CYP3A4 and CYP3A5 are primarily responsible for the formation of oxidative metabolites and the formation of M28 and M31, the keto-reduced metabolites, are most likely mediated by AKR1C2 and AKR1C3. Because the biotransformation and clearance of boceprevir involves two different enzymatic pathways, boceprevir is less likely to be a victim of significant drug-drug interaction with concomitant medication affecting either of these pathways.

A retention-time-shift-tolerant background subtraction and noise reduction algorithm (BgS-NoRA) for extraction of drug metabolites in liquid chromatography/mass spectrometry data from biological matrices.

A retention-time-shift-tolerant background subtraction and noise reduction algorithm (BgS-NoRA) is implemented using the statistical programming language R to remove non-drug-related ion signals from accurate mass liquid chromatography/mass spectrometry (LC/MS) data. The background-subtraction part of the algorithm is similar to a previously published procedure (Zhang H and Yang Y. J. Mass Spectrom. 2008, 43: 1181-1190). The noise reduction algorithm (NoRA) is an add-on feature to help further clean up the residual matrix ion noises after background subtraction. It functions by removing ion signals that are not consistent across many adjacent scans. The effectiveness of BgS-NoRA was examined in biological matrices by spiking blank plasma extract, bile and urine with diclofenac and ibuprofen that have been pre-metabolized by microsomal incubation. Efficient removal of background ions permitted the detection of drug-related ions in in vivo samples (plasma, bile, urine and feces) obtained from rats orally dosed with (14)C-loratadine with minimal interference. Results from these experiments demonstrate that BgS-NoRA is more effective in removing analyte-unrelated ions than background subtraction alone. NoRA is shown to be particularly effective in the early retention region for urine samples and middle retention region for bile samples, where the matrix ion signals still dominate the total ion chromatograms (TICs) after background subtraction. In most cases, the TICs after BgS-NoRA are in excellent qualitative correlation to the radiochromatograms. BgS-NoRA will be a very useful tool in metabolite detection and identification work, especially in first-in-human (FIH) studies and multiple dose toxicology studies where non-radio-labeled drugs are administered. Data from these types of studies are critical to meet the latest FDA guidance on Metabolite in Safety Testing (MIST).

HPLC-high-resolution mass spectrometry with polarity switching for increasing throughput of human in vitro cocktail drug-drug interaction assay.

AIM: Evaluation of HPLC-high-resolution mass spectrometry (HPLC-HRMS) full scan with polarity switching for increasing throughput of human in vitro cocktail drug-drug interaction assay. MATERIALS & METHODS: Microsomal incubates were analyzed using a high resolution and high mass accuracy Q-Exactive mass spectrometer to collect integrated qualitative and quantitative (qual/quant) data. RESULTS: Within assay, positive-to-negative polarity switching HPLC-HRMS method allowed quantification of eight and two probe compounds in the positive and negative ionization modes, respectively, while monitoring for LOR and its metabolites. CONCLUSION: LOR-inhibited CYP2C19 and showed higher activity for CYP2D6, CYP2E1 and CYP3A4. Overall, LC-HRMS-based nontargeted full scan quantitation allowed to improve the throughput of the in vitro cocktail drug-drug interaction assay.

Identification of unstable metabolites of Lonafarnib using liquid chromatography-quadrupole time-of-flight mass spectrometry, stable isotope incorporation and ion source temperature alteration.

Structural characterization of unstable metabolites and other drug-derived entities poses a serious challenge to the analytical chemist using instrumentation such as LC-MS and LC-MS/MS, and may lead to inaccurate identification of metabolite structures. The task of structural elucidation becomes even more difficult when an analyte is unstable in the ion source of the mass spectrometer. However, a judicious selection of the experimental conditions and the advanced features of new generation mass spectrometers can often overcome these difficulties. We describe here the identification of three drug-derived peaks (A, B and C) that were detected from a Schering-Plough developmental compound (Lonafarnib) following incubation with cDNA-expressed human CYP3A4. Definitive characterization was achieved using (1) accurate mass measurement, (2) stable isotope incorporation, (3) reduced ion source temperature, (4) alkali ion attachment and (5) MS/MS fragmentation studies. The protonated ions of compounds A and B fragmented almost completely in the source, yielding ions of the same mass-to-charge ratio (m/z) as that of protonated C (CH+). Fortunately, the presence of Na+ and K+ adducts of A and B provided information crucial to distinguishing AH+ and BH+ from their fragment ions. Metabolite A was shown to be an unstable hydroxylated metabolite of Lonafarnib. The metabolite C was shown to be a dehydrogenated metabolite of Lonafarnib (Lonafarnib-2H), unstable in the presence of protic solvents. Finally, B was artifactually formed most likely from C by the solvolytic addition of methanol during sample preparation. MS/MS fragmentation experiments assisted in identifying the site of metabolism in A and chemical modification in B. A and C readily interconvert through hydration/dehydration, and B and C through addition/elimination of methanol present in the sample-processing solvents. Finally, NMR experiments were performed to confirm the structures of A and C.

Metabolism of loratadine and further characterization of its in vitro metabolites.

The present study demonstrated that in addition to CYP3A4 and CYP2D6, the metabolism of loratadine is also catalyzed by CYP1A1, CYP2C19, and to a lesser extent by CYP1A2, CYP2B6, CYP2C8, CYP2C9 and CYP3A5. The biotransformation of loratadine was associated with the formation of desloratadine (DL) and further hydroxylation of both DL and the parent drug (loratadine). Based on the inhibition and correlation studies contribution of CYP2C19 in the formation of the major circulating metabolite DL seems to be minor. Reported clinical results suggest that the steady state mean (%CV) plasma Cmax and AUC(24hr) of loratadine were 4.73 ng/ml (119%) and 24.1 ng.hr/ml (157%), respectively, after dosing with 10 mg loratadine tablets for 10 days. High inter-subject variability in loratadine steady-state data is probably due to the phenotypical characteristics of CYP2D6, CYP2C19, and CYP3A4. The relative abundance of CYP3A4 in the human liver exceeds that of CYP2C19 and CYP2D6 and therefore the contribution of CYP3A4 in the metabolism of loratadine should be major (approximately 70%).

Identification of human liver cytochrome P450 enzymes involved in biotransformation of vicriviroc, a CCR5 receptor antagonist.

Vicriviroc (SCH 417690), a CCR5 receptor antagonist, is currently under investigation for the treatment of human immunodeficiency virus infection. The objective of this study was to identify human liver cytochrome P450 enzyme(s) responsible for the metabolism of vicriviroc. Human liver microsomes metabolized vicriviroc via N-oxidation (M2/M3), O-demethylation (M15), N,N-dealkylation (M16), N-dealkylation (M41), and oxidation to a carboxylic acid metabolite (M35b/M37a). Recombinant human CYP3A4 catalyzed the formation of all these metabolites, whereas CYP3A5 catalyzed the formation of M2/M3 and M41. CYP2C9 only catalyzed the formation of M15. There was a high correlation between the rates of formation of M2/M3, M15, and M41, which was determined using 10 human liver microsomal samples and testosterone 6beta-hydroxylation catalyzed by CYP3A4/5 (r > or = 0.91). Ketoconazole and azamulin (inhibitors of CYP3A4) were potent inhibitors of the formation of M2/M3, M15, M41, and M35b/M37a from human liver microsomes. A CYP3A4/5-specific monoclonal antibody (1 microg/microg of protein) inhibited the formation of all metabolites from human liver microsomes by 86 to 100%. The results of this study suggest that formation of the major vicriviroc metabolites in human liver microsomes is primarily mediated via CYP3A4. CYP2C9 and CYP3A5 most likely play a minor role in the biotransformation of this compound. These enzymology data will provide guidance to design clinical studies to address any potential drug-drug interactions.

Identification of human liver cytochrome P450 enzymes responsible for the metabolism of lonafarnib (Sarasar).

Lonafarnib (Sarasar), a farnesyl transferase inhibitor, is currently under development for the treatment of solid tumors. Incubation of lonafarnib with human liver microsomes resulted in the formation of four oxidative metabolites (M1, M2, M3, and M4). Minor to trace levels of these metabolites were detected in humans after multiple-dose administration of lonafarnib. Liquid chromatography-mass spectrometry analyses exhibited a mass to charge ratio (m/z) for the (M+H)(+) ion of M1, M2, M3, and M4 at 653, 635, 669, and 653 Th, respectively. These metabolites, respectively, resulted from changes of +O, -2H, +2O, and +O relative to lonafarnib. Recombinant human CYP3A4 and CYP3A5 exhibited catalytic activity with respect to the formation of M1, M2, and M3, whereas CYP2C8 exhibited catalytic activity with respect to the formation of M4. There was a high correlation between the formation of M1, determined in 10 human liver microsomal samples, and 6beta-hydroxylation of testosterone catalyzed by CYP3A4/5 (r = 0.93). The IC(50) values of ketoconazole for inhibition of M1 and M2 were 0.61 and 0.92 microM, respectively. The formation of M4 by human liver microsomes was inhibited 72% by 50 microM quercetin, suggesting that the formation of M4 was mediated via CYP2C8. A CYP3A4/5-specific inhibitory monoclonal antibody inhibited the formation of M1, M2, and M3 by 85, 75, and 100%, respectively. In conclusion, the formation of metabolites M1, M2, and M3 from lonafarnib was mediated via CYP3A4 and CYP3A5.

Constitutive and inducible levels of CYP1A1 and CYP1A2 in rat cerebral cortex and cerebellum.

We examined the constitutive and inducible levels of microsomal cytochromes P450 1A1 and 1A2 (CYP1A) in rat cerebral cortex and cerebellum at the level of proteins by western blot analysis, and by catalytic activities via ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD). In the cerebral cortex, cytochrome P450 1A1 (CYP1A1) protein was more abundant than cytochrome P450 1A2 (CYP1A2) protein. Treatment with beta-naphthoflavone (beta-NF) caused a slight decrease in the level of the former but induced the latter 5.8-fold. In the cerebellum, in contrast to the cerebral cortex, CYP1A1 protein was less abundant than CYP1A2 protein in untreated rats, and while beta-NF treatment caused a 3.3-fold induction of CYP1A1 protein, it resulted in a 10-fold decrease in CYP1A2 protein. The CYP1A-preferential activity EROD was 2.3-fold higher in the cerebellum than in the cerebral cortex, and was induced 1.5-fold and 1.9-fold in the cerebellum and cerebral cortex, respectively, by beta-NF treatment. The CYP1A2-preferential activity MROD was 3-fold higher in the cerebellum than in the cerebral cortex, and was repressed 2.2-fold in the cerebellum but induced 3.7-fold in the cerebral cortex following beta-NF treatment. The results show that CYP1A1 and CYP1A2 proteins and catalytic activities are constitutively expressed in brain but are differentially inducible in the rat cerebral cortex and cerebellum.

Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes.

Cytochrome P450 (CYP) substrates that yield fluorescent metabolites were used for rapid screening of drug metabolism activities of 13 recombinant human cytochromes P450, human liver microsomes and human hepatocytes. Reproducible results were obtained using a fluorescent plate reader (CytoFluor) more expediently than those generated using conventional HPLC methods. Typically, results for 96 samples were obtained with the plate reader in less than 10 min as opposed to 15-35 min/sample required by conventional HPLC. The fluorescent substrates used to measure CYP activities were as follows: 3-cyano-7-ethoxycoumarin (CEC) for CYP1A1, CYP1A2, CYP2C9 and CYP2C19; 7-ethoxyresorufin (7-ER) for CYP1A1, CYP1A2 and CYP1B1; 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) for CYP2D6; dibenzylfluorescein (DBF) for CYP3A4, CYP3A5 and CYP2C8; 7-methoxy-4-trifluoromethylcoumarin (7-MFC) for CYP2E1, CYP2B6 and CYP2C18; and coumarin for CYP2A6. The chemical inhibition and correlation data indicated that the following substrates can be used as specific functional probes for individual cytochrome P450 present in human liver microsomes: coumarin for CYP2A6 (r=0.82), AMMC for CYP2D6 (r=0.83) and DBF for CYP3A4 (r=0.92). The fluorescent plate reader was found to be useful for the rapid assessment of CYP activities (positive control) in both intact cells and subcellular fractions.

Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of posaconazole (Noxafil).

Posaconazole (Noxafil, SCH 56592), an orally available broad-spectrum triazole antifungal, is currently in phase III clinical studies for treating serious opportunistic fungal infections. The major in vitro metabolite of posaconazole formed by human liver microsomes supplemented with uridine 5'-diphosphate-glucuronic acid was a glucuronide of posaconazole (m/z877). Screening of 10 cDNA-expressed recombinant human UDP-glucuronosyltransferase (UGT) enzymes showed that only UGT1A4 exhibited catalytic activity with respect to the formation of the glucuronide of posaconazole. The formation of glucuronide by human liver microsomes and UGT1A4 was inhibited by bilirubin, a known inhibitor of UGT1A4. There was a high correlation (r =0.90) between the rate of formation of glucuronide, determined in 10 human liver microsomal samples, and trifluoperazine glucuronidation catalyzed by UGT1A4. These results confirmed that the formation of major posaconazole-glucuronide produced from human liver microsomes was mediated via UGT1A4.

Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of ezetimibe (Zetia).

Ezetimibe [1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone] (Zetia; Schering-Plough, Kenilworth, NJ) is the first in a new class of cholesterol-lowering agents known as cholesterol absorption inhibitors. The objective of this study was to identify the isoform(s) of human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for the glucuronidation of ezetimibe. The main circulating metabolite of this drug in human plasma is SCH 60663, the phenolic glucuronide conjugate of ezetimibe. SCH 60663 [m/z = 584 Thompsons (Th)] is also the major in vitro metabolite formed by human liver microsomes supplemented with UDP glucuronic acid (UDPGA). In contrast to the liver, human jejunum microsomes supplemented with UDPGA converted ezetimibe to two glucuronides with the same mass (m/z = 584 Th) by liquid chromatography-mass spectrometry. One corresponds to the phenolic glucuronide (1-O-[4-trans-2S,3R)-1-(4-fluorophenyl)-4-oxo-3-[3(S)-hydroxy-3-(4-fluorophenyl)propyl]-2-azetidinyl]phenyl-beta-D-glucopyranuronic acid; SCH 60663) and the other was identified as the benzylic glucuronide of ezetimibe (1-O-[1(S)-(4-fluorophenyl)-3-[1-(4-fluorophenyl)-2(S)-(4-hydroxyphenyl)-4-oxo-3(R)-azetidinyl]propyl]-beta-D-glucopyranuronic acid; SCH 488128). Recombinant human UGT1A1, UGT1A3, and UGT2B15 all exhibited catalytic activity with respect to the formation of the phenolic glucuronide. However, UGT2B7 exclusively formed SCH 488128, a trace metabolite detected in dog and human plasma samples after oral administration of ezetimibe. In conclusion, the formation of SCH 60663 is mediated via UGT1A1, UGT1A3, and UGT2B15, and the formation SCH 488128 is mediated via UGT2B7.

Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of 3-hydroxydesloratadine.

Desloratadine is a non-sedating antihistamine recently approved for the treatment of seasonal allergic rhinitis. The major metabolite of desloratadine in human plasma and urine is the glucuronide conjugate of 3-hydroxydesloratadine. 3-Hydroxydesloratadine-glucuronide is also the major in vitro metabolite of 3-hydroxydesloratadine formed by incubation of 3-hydroxydesloratadine with human liver microsomes supplemented with uridine 5'-diphosphate-glucuronic acid (UDPGA). The metabolite structure was confirmed by LC-MS and LC-MS/MS. Out of ten recombinant human UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A3, UGT1A8 and UGT2B15 exhibited catalytic activity with respect to the formation of 3-hydroxydesloratadine-glucuronide. Inhibition studies with known inhibitors of UGT (diclofenac, flunitrazepam and bilirubin) confirmed the involvement of UGT1A1, UGT1A3 and UGT2B15 in the formation of 3-hydroxydesloratadine-glucuronide. The results from this study demonstrated that the in vitro formation of 3-hydroxydesloratadine-glucuronide from 3-hydroxydesloratadine was mediated via UGT1A1, UGT1A3 and UGT2B15 in human liver.