Metabolism of Xenobiotics by Aldehyde Oxidase
UNIT 4.41
Deepak Dalvie1 and Michael Zientek1 1
Pfizer Global Research and Development, LaJolla Laboratories, San Diego
Aldehyde oxidase (AO) is a cytosolic molybdoflavoprotein whose contribution to the metabolism and clearance of xenobiotics-containing heterocyclic rings has attracted increased interest in recent years. This unit details methods for identification and confirmation of AO as a metabolic pathway as well as a C 2015 method for estimating clearance of compounds that are AO substrates. by John Wiley & Sons, Inc. Keywords: aldehyde oxidase (AO) r heteroaromatic rings r liver cytosol r liver S9 fraction
How to cite this article: Dalvie, D. and Zientek, M. 2015. Metabolism of Xenobiotics by Aldehyde Oxidase. Curr. Protoc. Toxicol. 63:4.41.1-4.41.13. doi: 10.1002/0471140856.tx0441s63
INTRODUCTION Aldehyde oxidase (AO) is a cytosolic molybdoflavoprotein that belongs to a family of structurally related molybdenum-containing enzymes (Garattini et al., 2003; Garattini et al., 2008; Garattini et al., 2009; Mendel, 2009; Garattini and Terao, 2011; Garattini and Terao, 2012). It is homologous to xanthine oxidase (XO), another mammalian molybdoflavoprotein, and both AO and XO show a remarkable degree of similarity in their amino acid sequence. AO is distributed across several tissues but the liver and adrenal gland have by far the highest expression of AO in all species including humans. AO has been known as a metabolizing enzyme for decades and is involved in the oxidation of nitrogen-containing heterocyclic compounds to lactams, as well as oxidation of aldehydes (as the name implies) and reduction reactions of N-O and NS bonds (Kitamura et al., 2006; Pryde et al., 2010). However, the contribution of this enzyme to the metabolism and clearance of drugs containing heteroaromatic rings appears to have attracted increased interest in recent years (Pryde et al., 2010; Hutzler et al., 2013). Drugs containing pyridines, pyrimidines, pyrazines, and their fused-ring analogues are all typical targets for oxidation by AO. For example, drugs such as brimonidine, carbazeran, N-[(2-diethylamino)ethyl]-acridine-4-carboximide (DACA), famciclovir, zaleplon, and zoniporide (Fig. 4.41.1) belong to a category of compounds that are primarily metabolized by AO (Pryde et al., 2010; Barr et al., 2014). This unit describes a method to identify AO as one of the enzymes responsible in the metabolism of xenobiotics (see Basic Protocol 1) (Fig. 4.41.2). Basic Protocol 2 describes the chemical inhibition of AO to further confirm the specificity of AO in the metabolism of a compound of interest. Basic Protocol 3 describes methods for assessment of hepatic intrinsic clearance (Clint ) for compounds that are AO substrates. In this unit, zoniporide has been used as an example to illustrate the utility of the methods in these protocols. However, these protocols are general enough to be used for other xenobiotics to assess the role and contribution of AO in their metabolism.
Current Protocols in Toxicology 4.41.1-4.41.13, February 2015 Published online February 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471140856.tx0441s63 C 2015 John Wiley & Sons, Inc. Copyright
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Figure 4.41.1
Structures of some drugs that are substrates for aldehyde oxidase (AO).
Figure 4.41.2
Conversion of zoniporide to its oxidative metabolite, 2-oxozoniporide (M1).
IDENTIFICATION OF AO AS AN ENZYME IN METABOLISM OF ZONIPORIDE After a non-P450 metabolic lability has been identified, follow-up experiments are generally performed, including incubation of the lead compound with liver cytosolic or S-9 fractions (fraction obtained from a liver homogenate by centrifuging 20 min at 9000 × g in a suitable medium; this fraction contains both cytosol and microsomal fractions) in the absence of NADPH (the co-factor commonly used for P450 and FMO oxidation reactions). This protocol describes a method that will help to identify AO as an enzyme in the metabolism of zoniporide, using liver S9 fractions to identify AO in the metabolism of zoniporide.
Materials Human liver S9 (pooled, mixed gender, ten donors; 23 mg/ml protein concentration; CelsisIVT) 100 mM potassium phosphate buffer, pH 7.4 (see recipe) 1000 mM MgCl2 (see recipe) 3 mM zoniporide stock (see recipe) HPLC-grade acetonitrile (Sigma-Aldrich) Nitrogen source HPLC-grade water with formic acid (see recipe) Metabolism of Xenobiotics by Aldehyde Oxidase
1.5-ml microcentrifuge tubes 37°C shaking water bath
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Vortex Tabletop centrifuge Pyrex disposable centrifuge tube, capacity 15-ml × 126-mm (Sigma-Aldrich) TurboVap evaporator 1-ml HPLC vials Agilent HP-1100 HPLC system (Agilent Technologies) ThermoFinnigan LCQ Deca XP ion trap mass spectrometer (ThermoScientific) Xcalibur v1.4 software (ThermoScientific) Perform reaction 1. Thaw human liver S9 fraction by placing the tube in cool water until the contents are defrosted and then place on ice. 2. Combine the following reagents in a 1.5-ml microcentrifuge tube (1.0 ml total volume for one reaction):
786.7 μl 100 mM potassium phosphate buffer, pH 7.4 10 μl 1000 mM MgCl2 stock (final 3 mM) 100 μl 20 mg/ml human liver S9 fraction (final 2.0 mg/ml) 3. Pre-incubate 2 min at 37°C. 4. Initiate reaction by adding 3 μl of 3 mM zoniporide stock solution (final 10 μM). 5. Incubate 60 min in a 37°C water bath with gentle shaking. 6. Terminate reaction by adding 1 ml acetonitrile and vortex for 5 sec. 7. Centrifuge 10 min at 3200 rpm at room temperature. 8. Transfer supernatant to a Pyrex disposable centrifuge tube. 9. Evaporate to dryness under nitrogen in a TurboVap under a stream of nitrogen at 35°C.
Analyze by HPLC-MS 10. Reconstitute residue in 200 μl mixture of 30% acetonitrile and 70% water containing 0.1% formic acid and vortex for 5 min. 11. Transfer 100 μl reconstituted mixture to a 1-ml HPLC vial. 12. Analyze 25 μl of the mixture by reversed-phase HPLC/MS/MS using mobile phase A and mobile phase B. HPLC conditions are described in Table 4.41.1 and mass spectrometer conditions are depicted in Table 4.41.2.
13. Analyze data obtained in the total ion chromatogram using Xcalibur v1.4 software. 14. Detect the ions for the metabolite and zoniporide and interpret the structure of the metabolite from the mass spectrum obtained from data-dependent scanning.
CONFIRMATION OF AO METABOLISM Involvement of AO can be confirmed by carrying out the same incubation in the presence of an AO inhibitor such as raloxifene in liver cytosolic or S9 fractions. Since raloxifene does not inhibit XO to a significant extent, the procedure confirms the role of AO in its metabolism. The following experiment was conducted by preincubating liver S9 fractions with raloxifene for phenotyping AO as an enzyme in the metabolism of zoniporide.
BASIC PROTOCOL 2
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Table 4.41.1 Reversed-Phase HPLC Conditions for Separation of Zoniporide and its Metabolite, 2-Oxo-zoniporide
Conditions
Information
Instrumentation
Type used
Column
Luna C4 100 A (3-μm, 150 × 2.0–mm, Phenomenex)
Pump
HP-1100 binary gradient pump
Autoinjector
HP-1100 autoinjector
Mobile phase Solvent A
0.1% formic acid in water
Solvent B
Acetonitrile
Gradient Time (min)
Solvent A
Solvent B
0-5
99
1
5-25
70
30
25-35
50
50
35-40
10
90
40-45
10
90
45-47
99
1
47-50
99
1
Detection
LCQ Deca XP mass spectrometer
Zoniporide
(M + H) 321
Oxidative metabolite
(M + H) 337
Materials 1 mM raloxifene stock (Sigma) DMSO 12 × 75–mm borosilicate glass tubes For additional reagents and equipment, see Basic Protocol 1. 1. Prepare a stock solution of zoniporide in a 12 × 75–mm borosilicate tube by weighing 1.0 mg of zoniporide powder and then dissolving it in 104 μl of DMSO and 938 μl of acetonitrile. 2. Prepare a stock solution of raloxifene in a 12 × 75–mm borosilicate tube by weighing 1 mg of raloxifene powder and then dissolving it in 211 μl of DMSO and 1900 μl of acetonitrile. 3. Thaw human liver S9 fraction by placing the tube in cool water until the contents are defrosted and then place on ice. 4. Combine the following reagents in a 1.5-ml microcentrifuge tube (1.0 ml total volume for one reaction):
Metabolism of Xenobiotics by Aldehyde Oxidase
786.7 μl 100 mM potassium phosphate buffer, pH 7.4 10 μl 1000 mM MgCl2 stock (final 3 mM) 100 μl 20 mg/ml human liver S9 fraction (final 2.0 mg/ml) 10 μl 1 mM raloxifene solution (final concentration 10 μM)
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Table 4.41.2 Mass Spectrometer Settings Used in Conducting the Experiment
Mass spectrometer settings
Value
Mass spectrometer
LCQ Deca XP
Source
Electrospray ionization source
Polarity
ES positive
Capillary temperature (ºC)
350
Sheath gas flow
30
Auxillary gas flow
5
Source voltage (kV)
4.5
Source current
100
Capillary voltage (V)
39
Tube lens (V)
70
Ions mointored
m/z 185–1000
Full scan parameters Automatic Gain Control
5 × 108
number of microscan
1
maximum ion time (msec)
10
Activation Q
0.25
Activation time
30
n
MS scanning Automatic gain control
1.0 × 108
Maximum ion time (msec)
100
Activation Q
0.250
Activation time
30
5. Pre-incubate for 2 min at 37°C. 6. Initiate the reaction by adding 3 μl of 3 mM zoniporide stock solution (final 10 μM). 7. Incubate for 60 min in a 37°C water bath with gentle shaking. 8. Terminate the reaction by adding 1 ml acetonitrile and vortex for 5 sec. 9. Centrifuge 10 min at 3000 × g, room temperature. 10. Transfer the supernatant to the Pyrex disposable centrifuge tube. 11. Evaporate to dryness under nitrogen in a TurboVap at 35°C. 12. Perform analysis on the products by performing HPLC and mass spectrometry using conditions in Tables 4.41.1 and 4.41.2 and described in Basic Protocol 1.
ESTIMATING HEPATIC INTRINSIC CLEARANCE OF COMPOUNDS METABOLIZED BY AO
BASIC PROTOCOL 3
Zientek et al. (2010) have attempted the clearance prediction of AO substrates. The following protocol describes the method estimating intrinsic clearance of zoniporide.
Materials Human liver S9 (pooled, mixed gender of ten donors; protein concentration, 23 mg/ml; CelsisIVT)
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0.1 M potassium phosphate buffer, pH 7.4 (see recipe) 1 mM MgCl2 (see recipe) 50 μM zoniporide stock (see recipe) Buspirone powder (see recipe) HPLC-grade (Milli-Q) water (Sigma Aldrich) 12 ml × 75 mm borosilicate tube 96-well polypropylene PCR plates (e.g., Axygen) 37°C heating block 96-well caps (Nalge Nunc) Vortexer Benchtop centrifuge Synergi Polar RP (2 × 30–mm, 4-μm, Phenomenex) CTC PAL autoinjector (Leap Technologies) Shimadzu HPLC pumps (Shimadzu Scientific Instruments) API 4000-triple quadrupole mass spectrometer (Applied Biosystems/MDS Sciex) MDS Sciex Analyst Software v.1.4.1 Perform reaction 1. Thaw human liver S9 fraction by placing tube in cool water until the contents are defrosted and then place on ice. 2. Combine the following reagents in a 12 ml × 75 mm borosilicate tube (total volume for about nine reactions):
1510 μl 100 mM potassium phosphate buffer, pH 7.4 200 μl 10 mM MgCl2 stock (final 1 mM) 250 μl of 20 mg/ml human liver S9 fraction (final 2.5 mg/ml) 3. Dispense 196 μl of the premix prepared in step 2 to each incubation well of a 96-well plate. 4. Prewarm entire plate for 3 min on a 37°C heating block. 5. Initiate reaction by adding 4 μl of 50 μM zoniporide stock solution (final 1 μM; final organic vehicle concentration 0.01% DMSO/0.6% acetonitrile). 6. Remove a 25-μl aliquot of the incubation mixture at time 0 min and add it to the 96-well plate containing 75 μl of ice-cold acetonitrile containing 10 μM buspirone (as an internal standard). 7. Repeat step 6 five more times at 5, 15, 30, 45, and 60 min. 8. Place cap on the plate and vortex for 5 min. 9. Centrifuge 15 min at 3200 rpm at room temperature. 10. Remove a 50-μl aliquot of the supernatant and transfer to a clean 96-well shallow plate. 11. Add 50 μl of Milli-Q water, mix, and analyze by HPLC/MS/MS. 12. Run the reaction in duplicate.
Metabolism of Xenobiotics by Aldehyde Oxidase
Analyze by HPLC-MS 13. Analyze a 20-μl aliquot of all samples by reversed-phase HPLC/MS/MS using mobile phase A and mobile phase B and HPLC conditions described in Table 4.41.3 and mass spectrometer conditions depicted in Table 4.41.4.
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Table 4.41.3 Reversed-Phase HPLC Conditions for Separation of Zoniporide and the Internal Standard, Buspirone
Conditions
Information
Instrumentation
Type used
Column
Synergi Polar-RP (4-μm, 30 × 2.0–mm)
Pump
Shimadzu LC-10ADvp (Shimadzu Scientific Instruments)
Autosampler
CTC PAL (Leap Technologies)
Mobile phase Solvent A
0.1% formic acid in water
Solvent B
Acetonitrile with 0.1% formic acid
Flow rate
0.8 ml/min
Injection volume
20 μl
Gradient Time (min)
Solvent A
Solvent B
0 – 0.3
99
1
0.3 – 1.2
1
99
1.2 – 1.9
1
99
1.9 – 1.95
99
1
Detector
Sciex API 4000 mass spectrometer
Retention times
Zoniporide
Buspirone
0.88 min
1.3
Table 4.41.4 Mass Spectrometer Settings Used in Conducting the Experiment
Mass spectrometer settings
Value
Source
Electrospray ionization source
Polarity
ES positive
Turbo-ion voltage (kV)
4
Auxillary gas temperature (ºC)
400
Gas used
N2
Electron multiplier (V)
2100
Declustering potential (V)
56
Collision energy (V)
23
Collision cell entrance potential (V)
10
Collision cell exit potential (V)
18
Dwell time (msec)
150
Multiple reaction monitoring transitions
Q1 (m/z)
Q2 (m/z)
Zoniporide
321
262
Buspirone
386
122
14. Detect analyte (zoniporide) and internal standard (buspirone) by monitoring their transitions (Table 4.41.4) and assess their peak area. The peak area ratio of the analyte to the internal standard is determined for each injection and used to measure the substrate depletion.
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REAGENTS AND SOLUTIONS Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Acetonitrile containing 0.1% formic acid Add 1 ml of formic acid to 500 ml of HPLC-grade acetonitrile (Sigma-Aldrich) in a 1000-ml volumetric flask. Mix solution well and bring to 1000 ml with HPLC-grade acetonitrile. Mix thoroughly. Store solution up to 1 year at room temperature. Buspirone To prepare a 10 mM stock solution of buspirone (mol. wt. = 385.5), weigh out 3.85 mg of buspirone (Sigma) and then dissolve it in 1.0 ml of DMSO. To achieve a 2 mM concentration of buspirone, take 200 μl of 10 mM buspirone solution and add 800 μl DMSO. To prepare a 10 μM buspirone solution in acetonitrile, add 10 μl of 2 mM buspirone solution to 2000 μl of acetonitrile. Store at 4°C until use.
Magnesium chloride hexahydrate (MgCl2 ·6H2 O) stock solution, 1000 mM Weigh 203.30 g MgCl2 .6H2 O (mol. wt. = 203.30) and dissolve it in 800 ml HPLCgrade water (Sigma-Aldrich) in a 1-liter volumetric flask, bring up volume with HPLC-grade water (Sigma-Aldrich) and mix thoroughly. Store up to 6 months at 5°C. Potassium phosphate buffer (100 mM), pH 7.4 Mix the 100 mM dibasic and monobasic potassium phosphate stock solutions (see recipes) together in a 10:3 ratio to the desired volume. Adjust the pH by adding dibasic or monobasic stock solutions as needed to obtain a pH of 7.40. Check and adjust the pH daily. Store up to 1 week at room temperature. Potassium phosphate, dibasic (K2 HPO4 ) stock solution, 100 mM Add 17.4 g K2 HPO4 (Sigma-Aldrich) to a 1-liter volumetric flask, bring up volume with HPLC-grade water (Sigma-Aldrich) and mix thoroughly. Store up to 6 months at 5°C. Potassium phosphate, monobasic (KH2 PO4 ) stock solution, 100 mM Add 13.6 g KH2 PO4 (Sigma-Aldrich) to a 1-liter volumetric flask and bring up volume with HPLC-grade water (Sigma-Aldrich) and mix thoroughly. Store up to 6 months at 5°C. Water containing 0.1% formic acid Add 1 ml of formic acid to 500 ml of HPLC-grade water in 1000-ml volumetric flask. Mix solution well and bring to 1000 ml with HPLC-grade water (Sigma-Aldrich). Mix thoroughly. Store solution up to 1 year at room temperature. Zoniporide To prepare a 3 mM stock solution of zoniporide (mol. wt. = 320.2) in a 12 × 75–mm borosilicate tube, weigh out 1.0 mg of zoniporide powder (Pfizer internal compound library) and then dissolve it in 104 μl of DMSO and 938 μl of acetonitrile.
Metabolism of Xenobiotics by Aldehyde Oxidase
To prepare 10 mM stock solution of zoniporide in a 12 ml × 75–mm borosilicate tube, dissolve 3.20 mg of zoniporide powder in 1 ml DMSO. Add 2.5 μl of 10 mM zoniporide stock solution into a mixture of 147.5 μl of acetonitrile and 350 μl of 0.1 M potassium phosphate buffer (see recipe) to give a final concentration of 50 μM.
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COMMENTARY Background Information Attention to AO has increased in recent years with the recognition that extensive AO metabolism has led to clinical failures, either due to higher-than-predicted clearance in humans yielding unacceptable PK properties, or to safety-related findings (Pryde et al., 2010; Hutzler et al., 2013; Barr et al., 2014). Since AO/XO is a cytosolic enzyme, normal liver microsomal assays are not useful in the identification of these enzymes. A disconnect in the turnover rates of a compound by the two in vitro incubation systems hepatocytes and liver microsomes, with faster turnover in the hepatocyte incubations, is a good indicator that a compound is a substrate for a non-P450 enzyme. Furthermore, the identification of a hydroxylated metabolite unique to the hepatocyte incubation (absent in liver microsomes) points toward the contribution of either AO or XO in the biotransformation of a compound. Subsequent turnover of a xenobiotic in liver cytosolic and/or S9 fractions followed by identification of unique oxygenated product (oxidation on the carbon atom in the heteroatom in the ring) in the incubation mixture will confirm the presence of AO or XO (Hutzler et al., 2013). Once the role of molybdenum hydrolase in the metabolism of a xenobiotic has been confirmed, the role of AO in the metabolism of that xenobiotic can be discerned by adding specific AO inhibitor to the incubation mixture and observing a reduction in metabolite production. Also, the compound structure can be used as a prospective indicator of possible AO metabolism. Some inhibitors specific to AO have been identified. These include raloxifene (Obach, 2004), hydralazine (Johnson et al., 1985; Strelevitz et al., 2012), phenothiazine drugs, estradiol, and menadione (Obach et al., 2004). The most commonly used inhibitor is raloxifene (Dalvie et al., 2010). Observation that there is inhibition of turnover of the substrate with raloxifene is a satisfactory confirmation that AO (and not XO) is the enzyme responsible for its metabolism. Since the ultimate goal of a drug metabolism study is to project clearance (CL) of a compound with high confidence and make accurate dose predictions, it is important that its rate of metabolism by AO relative to other enzymes be evaluated. The Clint values determined using the method described in Basic Protocol 3 is only an estimate. Projecting CL using this estimate will result in under-prediction of the value observed in vivo.
However, this estimate is useful in rank ordering compounds with respect to other substrates. In Zientek et al. (2010), the rank order of the compounds was preserved, this permitted cutoff values for the in vitro data wherein a compound could be classified as a high-, medium-, or low-clearance compound. The authors suggest that in practice, one should calibrate the system using high, medium, and low Clint AO substrates. Once the in vitro system is calibrated, a Clint can be estimated for a new compound and benchmarked against the chosen controls.
Critical Parameters and Troubleshooting Levels of DMSO up to 1% to 2% have been shown to be compatible with AO. There is no decrease in AO activity at these levels (Obach, 2004; Choughule et al., 2013). In addition to S9 fractions described, all experiments can also be conducted using liver cytosolic fractions. It is also important to note that AO can be a contaminant in the microsomal preparations (due to the presence of some cytosol). Hence, non-NADPH (control) experiments in liver microsomes can sometimes hint at the AO-mediated metabolism of the compound. The protocol for evaluating the role of AO in other tissues is also very similar to those described for liver (see Basic Protocols 1 through 3) except that subcellular fractions of those respective tissues should be used. A similar assay can also be used in cell-based systems such as cryopreserved hepatocytes from humans or any other preclinical species by switching the potassium phosphate buffer used in S9 fraction experiment, for a hepatocyte medium, such as Williams’ E medium or InVitroGro KHB after the hepatocytes have undergone the thawing process (Hutzler et al., 2012; Strelevitz et al., 2012; Akabane et al., 2012a; Akabane et al., 2012b; Hutzler et al., 2014). For identification of AO-mediated metabolites, the assay is very reproducible irrespective of the tissue fractions used. However, the rate of turnover of the substrate depends on the lot and the source of subcellular fraction used. Hence, it is advisable to use a positive control that is primarily metabolized by AO (such as zoniporide) to assess and validate the rates of new candidates being tested in this assay. Chromatography conditions as well as incubation times can also vary depending upon the nature of the substrate. The sensitivity of the assay depends upon the turnover of the
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Figure 4.41.3 Total ion chromatogram zoniporide and its metabolite following incubation with human liver S9 fraction.
Figure 4.41.4
Metabolism of Xenobiotics by Aldehyde Oxidase
Mass spectrum of zoniporide and its oxidative metabolite.
compound by AO. It will also depend on the type of detector used in analyzing the unchanged substrate or metabolite. Generally, analysis is conducted using LC-MS/MS (as described in the protocols); however, other detectors can also be used to determine the levels of the analyte. Given that AO-mediated oxidation of compounds do not require any particular co-factors, such as in the case of P450 assays, it is important that the reaction be followed by incubation in the presence of specific inhibitors of AO such as raloxifene or mena-
dione to assess the contribution of AO in the turnover of the compound. For phenotyping studies (see Basic Protocol 2), although the liver fractions can be preincubated with raloxifene to obtain substantial inhibition, adding the substrate and raloxifene together achieves the required degree of inhibition because of the high potency of raloxifene in inhibiting AO. In kinetic studies (see Basic Protocol 3), maintaining protein concentrations as low as possible will reduce the effects of non-specific binding to cytosolic proteins and/or S9 fractions.
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Figure 4.41.5
Mass spectrum of metabolite M1.
The protein concentration and incubation time specified in the protocol should lie well within the linear range, with the substrate depletion being
UNIT 4.41
Deepak Dalvie1 and Michael Zientek1 1
Pfizer Global Research and Development, LaJolla Laboratories, San Diego
Aldehyde oxidase (AO) is a cytosolic molybdoflavoprotein whose contribution to the metabolism and clearance of xenobiotics-containing heterocyclic rings has attracted increased interest in recent years. This unit details methods for identification and confirmation of AO as a metabolic pathway as well as a C 2015 method for estimating clearance of compounds that are AO substrates. by John Wiley & Sons, Inc. Keywords: aldehyde oxidase (AO) r heteroaromatic rings r liver cytosol r liver S9 fraction
How to cite this article: Dalvie, D. and Zientek, M. 2015. Metabolism of Xenobiotics by Aldehyde Oxidase. Curr. Protoc. Toxicol. 63:4.41.1-4.41.13. doi: 10.1002/0471140856.tx0441s63
INTRODUCTION Aldehyde oxidase (AO) is a cytosolic molybdoflavoprotein that belongs to a family of structurally related molybdenum-containing enzymes (Garattini et al., 2003; Garattini et al., 2008; Garattini et al., 2009; Mendel, 2009; Garattini and Terao, 2011; Garattini and Terao, 2012). It is homologous to xanthine oxidase (XO), another mammalian molybdoflavoprotein, and both AO and XO show a remarkable degree of similarity in their amino acid sequence. AO is distributed across several tissues but the liver and adrenal gland have by far the highest expression of AO in all species including humans. AO has been known as a metabolizing enzyme for decades and is involved in the oxidation of nitrogen-containing heterocyclic compounds to lactams, as well as oxidation of aldehydes (as the name implies) and reduction reactions of N-O and NS bonds (Kitamura et al., 2006; Pryde et al., 2010). However, the contribution of this enzyme to the metabolism and clearance of drugs containing heteroaromatic rings appears to have attracted increased interest in recent years (Pryde et al., 2010; Hutzler et al., 2013). Drugs containing pyridines, pyrimidines, pyrazines, and their fused-ring analogues are all typical targets for oxidation by AO. For example, drugs such as brimonidine, carbazeran, N-[(2-diethylamino)ethyl]-acridine-4-carboximide (DACA), famciclovir, zaleplon, and zoniporide (Fig. 4.41.1) belong to a category of compounds that are primarily metabolized by AO (Pryde et al., 2010; Barr et al., 2014). This unit describes a method to identify AO as one of the enzymes responsible in the metabolism of xenobiotics (see Basic Protocol 1) (Fig. 4.41.2). Basic Protocol 2 describes the chemical inhibition of AO to further confirm the specificity of AO in the metabolism of a compound of interest. Basic Protocol 3 describes methods for assessment of hepatic intrinsic clearance (Clint ) for compounds that are AO substrates. In this unit, zoniporide has been used as an example to illustrate the utility of the methods in these protocols. However, these protocols are general enough to be used for other xenobiotics to assess the role and contribution of AO in their metabolism.
Current Protocols in Toxicology 4.41.1-4.41.13, February 2015 Published online February 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471140856.tx0441s63 C 2015 John Wiley & Sons, Inc. Copyright
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Figure 4.41.1
Structures of some drugs that are substrates for aldehyde oxidase (AO).
Figure 4.41.2
Conversion of zoniporide to its oxidative metabolite, 2-oxozoniporide (M1).
IDENTIFICATION OF AO AS AN ENZYME IN METABOLISM OF ZONIPORIDE After a non-P450 metabolic lability has been identified, follow-up experiments are generally performed, including incubation of the lead compound with liver cytosolic or S-9 fractions (fraction obtained from a liver homogenate by centrifuging 20 min at 9000 × g in a suitable medium; this fraction contains both cytosol and microsomal fractions) in the absence of NADPH (the co-factor commonly used for P450 and FMO oxidation reactions). This protocol describes a method that will help to identify AO as an enzyme in the metabolism of zoniporide, using liver S9 fractions to identify AO in the metabolism of zoniporide.
Materials Human liver S9 (pooled, mixed gender, ten donors; 23 mg/ml protein concentration; CelsisIVT) 100 mM potassium phosphate buffer, pH 7.4 (see recipe) 1000 mM MgCl2 (see recipe) 3 mM zoniporide stock (see recipe) HPLC-grade acetonitrile (Sigma-Aldrich) Nitrogen source HPLC-grade water with formic acid (see recipe) Metabolism of Xenobiotics by Aldehyde Oxidase
1.5-ml microcentrifuge tubes 37°C shaking water bath
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Vortex Tabletop centrifuge Pyrex disposable centrifuge tube, capacity 15-ml × 126-mm (Sigma-Aldrich) TurboVap evaporator 1-ml HPLC vials Agilent HP-1100 HPLC system (Agilent Technologies) ThermoFinnigan LCQ Deca XP ion trap mass spectrometer (ThermoScientific) Xcalibur v1.4 software (ThermoScientific) Perform reaction 1. Thaw human liver S9 fraction by placing the tube in cool water until the contents are defrosted and then place on ice. 2. Combine the following reagents in a 1.5-ml microcentrifuge tube (1.0 ml total volume for one reaction):
786.7 μl 100 mM potassium phosphate buffer, pH 7.4 10 μl 1000 mM MgCl2 stock (final 3 mM) 100 μl 20 mg/ml human liver S9 fraction (final 2.0 mg/ml) 3. Pre-incubate 2 min at 37°C. 4. Initiate reaction by adding 3 μl of 3 mM zoniporide stock solution (final 10 μM). 5. Incubate 60 min in a 37°C water bath with gentle shaking. 6. Terminate reaction by adding 1 ml acetonitrile and vortex for 5 sec. 7. Centrifuge 10 min at 3200 rpm at room temperature. 8. Transfer supernatant to a Pyrex disposable centrifuge tube. 9. Evaporate to dryness under nitrogen in a TurboVap under a stream of nitrogen at 35°C.
Analyze by HPLC-MS 10. Reconstitute residue in 200 μl mixture of 30% acetonitrile and 70% water containing 0.1% formic acid and vortex for 5 min. 11. Transfer 100 μl reconstituted mixture to a 1-ml HPLC vial. 12. Analyze 25 μl of the mixture by reversed-phase HPLC/MS/MS using mobile phase A and mobile phase B. HPLC conditions are described in Table 4.41.1 and mass spectrometer conditions are depicted in Table 4.41.2.
13. Analyze data obtained in the total ion chromatogram using Xcalibur v1.4 software. 14. Detect the ions for the metabolite and zoniporide and interpret the structure of the metabolite from the mass spectrum obtained from data-dependent scanning.
CONFIRMATION OF AO METABOLISM Involvement of AO can be confirmed by carrying out the same incubation in the presence of an AO inhibitor such as raloxifene in liver cytosolic or S9 fractions. Since raloxifene does not inhibit XO to a significant extent, the procedure confirms the role of AO in its metabolism. The following experiment was conducted by preincubating liver S9 fractions with raloxifene for phenotyping AO as an enzyme in the metabolism of zoniporide.
BASIC PROTOCOL 2
Techniques for Analysis of Chemical Biotransformation
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Table 4.41.1 Reversed-Phase HPLC Conditions for Separation of Zoniporide and its Metabolite, 2-Oxo-zoniporide
Conditions
Information
Instrumentation
Type used
Column
Luna C4 100 A (3-μm, 150 × 2.0–mm, Phenomenex)
Pump
HP-1100 binary gradient pump
Autoinjector
HP-1100 autoinjector
Mobile phase Solvent A
0.1% formic acid in water
Solvent B
Acetonitrile
Gradient Time (min)
Solvent A
Solvent B
0-5
99
1
5-25
70
30
25-35
50
50
35-40
10
90
40-45
10
90
45-47
99
1
47-50
99
1
Detection
LCQ Deca XP mass spectrometer
Zoniporide
(M + H) 321
Oxidative metabolite
(M + H) 337
Materials 1 mM raloxifene stock (Sigma) DMSO 12 × 75–mm borosilicate glass tubes For additional reagents and equipment, see Basic Protocol 1. 1. Prepare a stock solution of zoniporide in a 12 × 75–mm borosilicate tube by weighing 1.0 mg of zoniporide powder and then dissolving it in 104 μl of DMSO and 938 μl of acetonitrile. 2. Prepare a stock solution of raloxifene in a 12 × 75–mm borosilicate tube by weighing 1 mg of raloxifene powder and then dissolving it in 211 μl of DMSO and 1900 μl of acetonitrile. 3. Thaw human liver S9 fraction by placing the tube in cool water until the contents are defrosted and then place on ice. 4. Combine the following reagents in a 1.5-ml microcentrifuge tube (1.0 ml total volume for one reaction):
Metabolism of Xenobiotics by Aldehyde Oxidase
786.7 μl 100 mM potassium phosphate buffer, pH 7.4 10 μl 1000 mM MgCl2 stock (final 3 mM) 100 μl 20 mg/ml human liver S9 fraction (final 2.0 mg/ml) 10 μl 1 mM raloxifene solution (final concentration 10 μM)
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Table 4.41.2 Mass Spectrometer Settings Used in Conducting the Experiment
Mass spectrometer settings
Value
Mass spectrometer
LCQ Deca XP
Source
Electrospray ionization source
Polarity
ES positive
Capillary temperature (ºC)
350
Sheath gas flow
30
Auxillary gas flow
5
Source voltage (kV)
4.5
Source current
100
Capillary voltage (V)
39
Tube lens (V)
70
Ions mointored
m/z 185–1000
Full scan parameters Automatic Gain Control
5 × 108
number of microscan
1
maximum ion time (msec)
10
Activation Q
0.25
Activation time
30
n
MS scanning Automatic gain control
1.0 × 108
Maximum ion time (msec)
100
Activation Q
0.250
Activation time
30
5. Pre-incubate for 2 min at 37°C. 6. Initiate the reaction by adding 3 μl of 3 mM zoniporide stock solution (final 10 μM). 7. Incubate for 60 min in a 37°C water bath with gentle shaking. 8. Terminate the reaction by adding 1 ml acetonitrile and vortex for 5 sec. 9. Centrifuge 10 min at 3000 × g, room temperature. 10. Transfer the supernatant to the Pyrex disposable centrifuge tube. 11. Evaporate to dryness under nitrogen in a TurboVap at 35°C. 12. Perform analysis on the products by performing HPLC and mass spectrometry using conditions in Tables 4.41.1 and 4.41.2 and described in Basic Protocol 1.
ESTIMATING HEPATIC INTRINSIC CLEARANCE OF COMPOUNDS METABOLIZED BY AO
BASIC PROTOCOL 3
Zientek et al. (2010) have attempted the clearance prediction of AO substrates. The following protocol describes the method estimating intrinsic clearance of zoniporide.
Materials Human liver S9 (pooled, mixed gender of ten donors; protein concentration, 23 mg/ml; CelsisIVT)
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0.1 M potassium phosphate buffer, pH 7.4 (see recipe) 1 mM MgCl2 (see recipe) 50 μM zoniporide stock (see recipe) Buspirone powder (see recipe) HPLC-grade (Milli-Q) water (Sigma Aldrich) 12 ml × 75 mm borosilicate tube 96-well polypropylene PCR plates (e.g., Axygen) 37°C heating block 96-well caps (Nalge Nunc) Vortexer Benchtop centrifuge Synergi Polar RP (2 × 30–mm, 4-μm, Phenomenex) CTC PAL autoinjector (Leap Technologies) Shimadzu HPLC pumps (Shimadzu Scientific Instruments) API 4000-triple quadrupole mass spectrometer (Applied Biosystems/MDS Sciex) MDS Sciex Analyst Software v.1.4.1 Perform reaction 1. Thaw human liver S9 fraction by placing tube in cool water until the contents are defrosted and then place on ice. 2. Combine the following reagents in a 12 ml × 75 mm borosilicate tube (total volume for about nine reactions):
1510 μl 100 mM potassium phosphate buffer, pH 7.4 200 μl 10 mM MgCl2 stock (final 1 mM) 250 μl of 20 mg/ml human liver S9 fraction (final 2.5 mg/ml) 3. Dispense 196 μl of the premix prepared in step 2 to each incubation well of a 96-well plate. 4. Prewarm entire plate for 3 min on a 37°C heating block. 5. Initiate reaction by adding 4 μl of 50 μM zoniporide stock solution (final 1 μM; final organic vehicle concentration 0.01% DMSO/0.6% acetonitrile). 6. Remove a 25-μl aliquot of the incubation mixture at time 0 min and add it to the 96-well plate containing 75 μl of ice-cold acetonitrile containing 10 μM buspirone (as an internal standard). 7. Repeat step 6 five more times at 5, 15, 30, 45, and 60 min. 8. Place cap on the plate and vortex for 5 min. 9. Centrifuge 15 min at 3200 rpm at room temperature. 10. Remove a 50-μl aliquot of the supernatant and transfer to a clean 96-well shallow plate. 11. Add 50 μl of Milli-Q water, mix, and analyze by HPLC/MS/MS. 12. Run the reaction in duplicate.
Metabolism of Xenobiotics by Aldehyde Oxidase
Analyze by HPLC-MS 13. Analyze a 20-μl aliquot of all samples by reversed-phase HPLC/MS/MS using mobile phase A and mobile phase B and HPLC conditions described in Table 4.41.3 and mass spectrometer conditions depicted in Table 4.41.4.
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Table 4.41.3 Reversed-Phase HPLC Conditions for Separation of Zoniporide and the Internal Standard, Buspirone
Conditions
Information
Instrumentation
Type used
Column
Synergi Polar-RP (4-μm, 30 × 2.0–mm)
Pump
Shimadzu LC-10ADvp (Shimadzu Scientific Instruments)
Autosampler
CTC PAL (Leap Technologies)
Mobile phase Solvent A
0.1% formic acid in water
Solvent B
Acetonitrile with 0.1% formic acid
Flow rate
0.8 ml/min
Injection volume
20 μl
Gradient Time (min)
Solvent A
Solvent B
0 – 0.3
99
1
0.3 – 1.2
1
99
1.2 – 1.9
1
99
1.9 – 1.95
99
1
Detector
Sciex API 4000 mass spectrometer
Retention times
Zoniporide
Buspirone
0.88 min
1.3
Table 4.41.4 Mass Spectrometer Settings Used in Conducting the Experiment
Mass spectrometer settings
Value
Source
Electrospray ionization source
Polarity
ES positive
Turbo-ion voltage (kV)
4
Auxillary gas temperature (ºC)
400
Gas used
N2
Electron multiplier (V)
2100
Declustering potential (V)
56
Collision energy (V)
23
Collision cell entrance potential (V)
10
Collision cell exit potential (V)
18
Dwell time (msec)
150
Multiple reaction monitoring transitions
Q1 (m/z)
Q2 (m/z)
Zoniporide
321
262
Buspirone
386
122
14. Detect analyte (zoniporide) and internal standard (buspirone) by monitoring their transitions (Table 4.41.4) and assess their peak area. The peak area ratio of the analyte to the internal standard is determined for each injection and used to measure the substrate depletion.
Techniques for Analysis of Chemical Biotransformation
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REAGENTS AND SOLUTIONS Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Acetonitrile containing 0.1% formic acid Add 1 ml of formic acid to 500 ml of HPLC-grade acetonitrile (Sigma-Aldrich) in a 1000-ml volumetric flask. Mix solution well and bring to 1000 ml with HPLC-grade acetonitrile. Mix thoroughly. Store solution up to 1 year at room temperature. Buspirone To prepare a 10 mM stock solution of buspirone (mol. wt. = 385.5), weigh out 3.85 mg of buspirone (Sigma) and then dissolve it in 1.0 ml of DMSO. To achieve a 2 mM concentration of buspirone, take 200 μl of 10 mM buspirone solution and add 800 μl DMSO. To prepare a 10 μM buspirone solution in acetonitrile, add 10 μl of 2 mM buspirone solution to 2000 μl of acetonitrile. Store at 4°C until use.
Magnesium chloride hexahydrate (MgCl2 ·6H2 O) stock solution, 1000 mM Weigh 203.30 g MgCl2 .6H2 O (mol. wt. = 203.30) and dissolve it in 800 ml HPLCgrade water (Sigma-Aldrich) in a 1-liter volumetric flask, bring up volume with HPLC-grade water (Sigma-Aldrich) and mix thoroughly. Store up to 6 months at 5°C. Potassium phosphate buffer (100 mM), pH 7.4 Mix the 100 mM dibasic and monobasic potassium phosphate stock solutions (see recipes) together in a 10:3 ratio to the desired volume. Adjust the pH by adding dibasic or monobasic stock solutions as needed to obtain a pH of 7.40. Check and adjust the pH daily. Store up to 1 week at room temperature. Potassium phosphate, dibasic (K2 HPO4 ) stock solution, 100 mM Add 17.4 g K2 HPO4 (Sigma-Aldrich) to a 1-liter volumetric flask, bring up volume with HPLC-grade water (Sigma-Aldrich) and mix thoroughly. Store up to 6 months at 5°C. Potassium phosphate, monobasic (KH2 PO4 ) stock solution, 100 mM Add 13.6 g KH2 PO4 (Sigma-Aldrich) to a 1-liter volumetric flask and bring up volume with HPLC-grade water (Sigma-Aldrich) and mix thoroughly. Store up to 6 months at 5°C. Water containing 0.1% formic acid Add 1 ml of formic acid to 500 ml of HPLC-grade water in 1000-ml volumetric flask. Mix solution well and bring to 1000 ml with HPLC-grade water (Sigma-Aldrich). Mix thoroughly. Store solution up to 1 year at room temperature. Zoniporide To prepare a 3 mM stock solution of zoniporide (mol. wt. = 320.2) in a 12 × 75–mm borosilicate tube, weigh out 1.0 mg of zoniporide powder (Pfizer internal compound library) and then dissolve it in 104 μl of DMSO and 938 μl of acetonitrile.
Metabolism of Xenobiotics by Aldehyde Oxidase
To prepare 10 mM stock solution of zoniporide in a 12 ml × 75–mm borosilicate tube, dissolve 3.20 mg of zoniporide powder in 1 ml DMSO. Add 2.5 μl of 10 mM zoniporide stock solution into a mixture of 147.5 μl of acetonitrile and 350 μl of 0.1 M potassium phosphate buffer (see recipe) to give a final concentration of 50 μM.
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COMMENTARY Background Information Attention to AO has increased in recent years with the recognition that extensive AO metabolism has led to clinical failures, either due to higher-than-predicted clearance in humans yielding unacceptable PK properties, or to safety-related findings (Pryde et al., 2010; Hutzler et al., 2013; Barr et al., 2014). Since AO/XO is a cytosolic enzyme, normal liver microsomal assays are not useful in the identification of these enzymes. A disconnect in the turnover rates of a compound by the two in vitro incubation systems hepatocytes and liver microsomes, with faster turnover in the hepatocyte incubations, is a good indicator that a compound is a substrate for a non-P450 enzyme. Furthermore, the identification of a hydroxylated metabolite unique to the hepatocyte incubation (absent in liver microsomes) points toward the contribution of either AO or XO in the biotransformation of a compound. Subsequent turnover of a xenobiotic in liver cytosolic and/or S9 fractions followed by identification of unique oxygenated product (oxidation on the carbon atom in the heteroatom in the ring) in the incubation mixture will confirm the presence of AO or XO (Hutzler et al., 2013). Once the role of molybdenum hydrolase in the metabolism of a xenobiotic has been confirmed, the role of AO in the metabolism of that xenobiotic can be discerned by adding specific AO inhibitor to the incubation mixture and observing a reduction in metabolite production. Also, the compound structure can be used as a prospective indicator of possible AO metabolism. Some inhibitors specific to AO have been identified. These include raloxifene (Obach, 2004), hydralazine (Johnson et al., 1985; Strelevitz et al., 2012), phenothiazine drugs, estradiol, and menadione (Obach et al., 2004). The most commonly used inhibitor is raloxifene (Dalvie et al., 2010). Observation that there is inhibition of turnover of the substrate with raloxifene is a satisfactory confirmation that AO (and not XO) is the enzyme responsible for its metabolism. Since the ultimate goal of a drug metabolism study is to project clearance (CL) of a compound with high confidence and make accurate dose predictions, it is important that its rate of metabolism by AO relative to other enzymes be evaluated. The Clint values determined using the method described in Basic Protocol 3 is only an estimate. Projecting CL using this estimate will result in under-prediction of the value observed in vivo.
However, this estimate is useful in rank ordering compounds with respect to other substrates. In Zientek et al. (2010), the rank order of the compounds was preserved, this permitted cutoff values for the in vitro data wherein a compound could be classified as a high-, medium-, or low-clearance compound. The authors suggest that in practice, one should calibrate the system using high, medium, and low Clint AO substrates. Once the in vitro system is calibrated, a Clint can be estimated for a new compound and benchmarked against the chosen controls.
Critical Parameters and Troubleshooting Levels of DMSO up to 1% to 2% have been shown to be compatible with AO. There is no decrease in AO activity at these levels (Obach, 2004; Choughule et al., 2013). In addition to S9 fractions described, all experiments can also be conducted using liver cytosolic fractions. It is also important to note that AO can be a contaminant in the microsomal preparations (due to the presence of some cytosol). Hence, non-NADPH (control) experiments in liver microsomes can sometimes hint at the AO-mediated metabolism of the compound. The protocol for evaluating the role of AO in other tissues is also very similar to those described for liver (see Basic Protocols 1 through 3) except that subcellular fractions of those respective tissues should be used. A similar assay can also be used in cell-based systems such as cryopreserved hepatocytes from humans or any other preclinical species by switching the potassium phosphate buffer used in S9 fraction experiment, for a hepatocyte medium, such as Williams’ E medium or InVitroGro KHB after the hepatocytes have undergone the thawing process (Hutzler et al., 2012; Strelevitz et al., 2012; Akabane et al., 2012a; Akabane et al., 2012b; Hutzler et al., 2014). For identification of AO-mediated metabolites, the assay is very reproducible irrespective of the tissue fractions used. However, the rate of turnover of the substrate depends on the lot and the source of subcellular fraction used. Hence, it is advisable to use a positive control that is primarily metabolized by AO (such as zoniporide) to assess and validate the rates of new candidates being tested in this assay. Chromatography conditions as well as incubation times can also vary depending upon the nature of the substrate. The sensitivity of the assay depends upon the turnover of the
Techniques for Analysis of Chemical Biotransformation
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Figure 4.41.3 Total ion chromatogram zoniporide and its metabolite following incubation with human liver S9 fraction.
Figure 4.41.4
Metabolism of Xenobiotics by Aldehyde Oxidase
Mass spectrum of zoniporide and its oxidative metabolite.
compound by AO. It will also depend on the type of detector used in analyzing the unchanged substrate or metabolite. Generally, analysis is conducted using LC-MS/MS (as described in the protocols); however, other detectors can also be used to determine the levels of the analyte. Given that AO-mediated oxidation of compounds do not require any particular co-factors, such as in the case of P450 assays, it is important that the reaction be followed by incubation in the presence of specific inhibitors of AO such as raloxifene or mena-
dione to assess the contribution of AO in the turnover of the compound. For phenotyping studies (see Basic Protocol 2), although the liver fractions can be preincubated with raloxifene to obtain substantial inhibition, adding the substrate and raloxifene together achieves the required degree of inhibition because of the high potency of raloxifene in inhibiting AO. In kinetic studies (see Basic Protocol 3), maintaining protein concentrations as low as possible will reduce the effects of non-specific binding to cytosolic proteins and/or S9 fractions.
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Figure 4.41.5
Mass spectrum of metabolite M1.
The protein concentration and incubation time specified in the protocol should lie well within the linear range, with the substrate depletion being
