ISSX 2010, Poster 146

Published:  01 September 2010

In vitro inhibition and induction of human liver cytochrome P450 enzymes by NTBC and its metabolism in human liver microsomes

2-(2-Nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC, also known as nitisinone and marketed as Orfadin®) is an inhibitor of 4-hydroxyphenylpyruvate dioxgenase (HPPD) that is used to prevent the liver and kidney toxicity associated with tyrosinemia type 1, a metabolic disorder in the tyrosine catabolism caused by fumarylacetoacetate hydrolase (FAH) deficiency. Genetically modified mice deficient in FAH [Fah-/-/II2rg-/-Rag2-/- (FRG) mouse strain] can be repopulated with human hepatocytes to support, among other applications, studies of drug metabolism and disposition. To sustain mouse hepatocellular function prior to (and during) the repopulation with human hepatocytes, FRG mice are treated with NTBC to inhibit the formation of hepatotoxic levels of fumarylacetoacetate (FAA). In the present study, we investigated the metabolism of NTBC in human liver microsomes (HLM) and the potential for NTBC to inhibit or induce human cytochrome P450 (CYP) enzymes. NTBC (1, 10 and 100 μM) was incubated with multiple concentrations of NADPH-fortified pooled HLM (0.5, 1 and 2 mg /mL) for multiple incubation times (0, 30, 60, 120 and 240 min). Little-to-no loss of NTBC was observed, suggesting that NTBC undergoes little or no oxidative metabolism by human liver CYP enzymes and little-to-no ketone reduction by microsomal carbonyl reductase. These results are consistent with the long clinical plasma half-life (t1/2 ~ 52 –54 h) reported for NTBC. In CYP inhibition experiments, performed with pooled HLM (0.1 mg/mL), NTBC caused direct inhibition of CYP2C9 (IC50 11 μM). NTBC caused no direct inhibition of CYP1A2, 2B6, CYP2C8, 2C19, 2D6 and 3A4/5. Furthermore, NTBC caused no metabolism dependent inhibition of any of the CYP enzymes evaluated...

ISSX 2010, Poster LB43

Published:  01 September 2010

Accurate mass spectrometry elucidates a misleading metabonate formed from amine-containing drugs in reactive metabolite screening assays

The propensity of drug candidates to undergo cytochrome P450 (CYP)-dependent metabolic activation to reactive electrophilic meta-bolites is often evaluated with NADPH-fortified human liver microsomes (HLM) in the presence of a soft nucleophile (like glutathione, GSH) or a hard nucleophile (like cyanide, CN–) with analysis by liquid chromatography tandem mass spectrometry (LC/MS/MS). In the present study, diltiazem, nordiltiazem and di-nordiltiazem (100 μM), compounds containing a tertiary, secondary and primary amine, respectively, were incubated for 15 min with NADPH-fortified HLM (1 mg/mL) in the presence of KCN or GSH (1 mM). Zero-cofactor and zero-min incubations were included as negative controls. Samples were analyzed by UltraPerformance LC tandem accurate MS on a Synapt G2 quadrupole time-of-flight mass spectrometer and data were processed with compound-specific mass defect filtering (± 35 mDa) and optimal parameters for detecting trapped reactive metabolites. Under these conditions, all three compounds were converted to cyanide-trapped components, as summarized below.

ISSX 2010, Poster 181

Published:  01 September 2010

In situ evaluation of CYP1A2, CYP2B6 and CYP3A4/5 induction in cultured human hepatocytes with a cocktail of probe substrates: A comparison of percent positive control values

It has previously been demonstrated that a cocktail of probe substrates can be effectively used to screen simultaneously for induction of CYP1A2, CYP2B6 and CYP3A4/5 with in situ (cell-based) incubations based on the magnitude of induction relative to appropriate positive controls. Industry guidelines recommend that a compound be considered an inducer in vitro and therefore warrant further investigation in vivo if it elicits an increase in cytochrome P450 (CYP) enzyme activity that is equal to or greater than 40% of that caused by a suitable enzyme inducer (percent positive control). The objective of this study was to determine the percent of positive control value of known CYP inducers (at varying concentrations) compared with the US FDA-preferred positive controls, omeprazole (100 μM, CYP1A2), phenobarbital (750 μM, CYP2B6) and rifampin (10 μM, CYP3A4/5) based on in situ incubations of primary cultures of human hepatocytes with three marker substrates (namely, 100 μM phenacetin, 500 μM bupropion, and 100 μM midazolam) either individually or as a cocktail. Freshly isolated human hepatocytes (n=3) were cultured in a sandwich configuration and treated for three days with vehicle control (dimethyl sulfoxide, DMSO; 0.1%, v/v), the aforementioned positive controls or one of the following enzyme inducers: β-naphthoflavone (1-100 μM), lansoprazole (1-100 μM), dexamethasone (1-100 μM), phenytoin (1-100 μM), carbamazepine (1-100 μM) , and efavirenz (0.25-25 μM). CYP induction was measured by LC/MS/MS based on phenacetin O-dealkylation (CYP1A2), bupropion hydroxylation (CYP2B6) or midazolam 1´-hydroxylation (CYP3A4/5). The results indicate that...

ISSX 2010, Poster 247

Published:  01 September 2010

The effect of multiple cryopreservation cycles on drug-metabolizing enzymes in human hepatocytes

Cryopreserved hepatocytes provide a convenient in vitro test system to study the phase-1 and phase-2 metabolism of new chemical entities; however cryopreservation (freeze-thaw cycles) can damage the cells.
Cryoinjury is associated with rapid dehydration of cells (osmotic effects) and formation of intracellular ice during the freezing process, which in turn leads to a disruption of cellular membranes, changes in protein conformation and nucleic acid damage (single and double DNA strand breaks). This study characterized the effects of multiple cryopreservation cycles on the activities of drug-metabolizing enzymes in individual and pooled samples of human hepatocytes. Hepatocytes isolated from four donors were cryopreserved once, twice or three times according to a stepwise protocol. Pooled hepatocytes (n=5) were prepared by two protocols: one involving the thawing of individual lots of frozen hepatocytes, followed by pooling and re-freezing, and the other by pooling cryopreserved hepatocyte pellets (CryostaX™) without thawing or re-freezing the cells. All cells were stored in the vapor phase of liquid nitrogen and were thawed under the same conditions. Viable hepatocytes were separated from non-viable cells by Percoll® gradient centrifugation. The following enzyme activities were measured in situ: CYP3A4 (testosterone 6β-hydroxylation), CYP1A2 (phenacetin O-dealkylation), CYP2B6 (bupropion hydroxylation), FAD-containing monooxygenase (FMO, benzydamine N-oxidation), UDP-glucuronosyltransferase (UGT), and sulfonotransferase (SULT, 7-hydroxycoumarin sulfonation)...

ISSX 2010, Poster 203

Published:  01 September 2010

Pitfalls in the Design of Metabolism-Dependent CYP Inhibition (MDI) Experiments With a Dilution Step: Inhibitor Depletion by Metabolism and/or Microsomal Binding Leads to Underestimation of the Shifted IC50 Value

We previously demonstrated that, when a dilution step is used to assess MDI potential (i.e., IC50 shift experiments), IC50 values for direct-inhibition and MDI should be processed based on the final, post-dilution concentration and the initial, pre-dilution concentration, respectively (Paris et al., 2009). When processed appropriately, the "shifted IC50 values" (those determined following a 30-min preincubation of the drug candidate with NADPH-fortified HLM) were notably higher for several known MDIs in experiments conducted with a 10-fold dilution step than those determined by a non-dilution method, suggesting less MDI occurs at the higher concentration of HLM. More than a two-fold difference in shifted IC50 values was observed between the dilution and non-dilution methods for five of ten inhibitors commonly used positive controls in MDI experiments; namely ticlopidine (CYP2B6), tienillic acid (CYP2C9), paroxetine (CYP2D6), S-fluoxetine (CYP2C19) and azamulin (CYP3A4). Experiments were performed to determine whether these discrepancies in shifted IC50 values between are attributable to 1) decreased free inhibitor concentration (fuinc) and/or 2) extensive metabolism of the inhibitor. In the case of S-fluoxetine, microsomal binding, not extensive metabolism, leads to the discrepancy...

ISSX 2010, Poster 197

Published:  01 September 2010

Identification of a novel carbamoyl glucuronide as a metabolism-dependent inhibitior of CYP2C8

Glucuronidation is a major route of drug biotransformation and detoxification, whereby a drug is conjugated with glucuronic acid in a reaction catalyzed by UDP glucuronosyltransferases (UGTs). However, previous reports suggest glucuronide conjugates can be inhibitors of Phase I metabolism. Of particular note is gemfibrozil glucuronide, which is oxidized by CYP2C8, causing clinically relevant irreversible inactivation of this enzyme (Ogilvie et al., 2006). In the present study, we evaluated the ability of Lu AA34893 and its carbamoyl glucuronide to inhibit in vitro the major drug metabolizing cytochrome P450 (CYP) enzymes. In NADPH-fortified human liver microsomes (HLM), Lu AA34893 was found to directly inhibit CYP2C19...