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Research ArticleArticle

Aldehyde Oxidase Activity in Donor-Matched Fresh and Cryopreserved Human Hepatocytes and Assessment of Variability in 75 Donors

J. Matthew Hutzler, Young-Sun Yang, Caitlin Brown, Scott Heyward and Timothy Moeller
Drug Metabolism and Disposition June 2014, 42 (6) 1090-1097; DOI: https://doi.org/10.1124/dmd.114.057984

Abstract

Studies were conducted to evaluate the impact of time and cryopreservation on aldehyde oxidase (AO) activity in human hepatocytes isolated from 10 donor livers, using O6-benzylguanine as a probe substrate. In addition, variability in activity was assessed using cryopreserved hepatocytes from 75 donors. Substantial donor-dependent loss in AO activity within 24 hours after isolation of hepatocytes was observed (average loss of 42%, range 15%–81%). Meanwhile, AO activity in cryopreserved hepatocytes more closely represented the activity observed in fresh hepatocytes that were incubated immediately after isolation for the same donors (within 81% of fresh, range 48%–100%). Activity of AO in cryopreserved hepatocytes from 75 donors varied by at least 17-fold (≤ 5.4 to 90 ml/minute per kilogram of body weight), with 63% of the donors having higher activity than a pooled 19-donor lot (34.2 ml/minute per kilogram). Comparison of demographics such as gender, body mass index, age, and ethnicity showed no statistically significant correlations with activity. Evaluation of medical histories revealed that three of the five donors with no measurable activity had immediate histories of extensive alcohol abuse. Meanwhile, two single nucleotide polymorphisms (SNPs) for AOX1 (rs3731772 and rs55754655) were detected in our donor pool and showed allelic frequencies similar to those reported from other cohort studies. However, these SNPs did not correlate with a statistically significant difference in intrinsic clearance compared with wild-type donors. With a general lack of clarity about what causes highly variable AO activity, prescreening donors for AO activity and creating a custom high-activity pooled lot of cryopreserved hepatocytes are advised to minimize underpredictions of clearance.

Introduction

Aldehyde oxidase (AO) is a cytosolic molybdenum-containing hydroxylase that has rapidly ascended in recent years to be a drug-metabolizing enzyme of high relevance within drug discovery and development (Pryde et al., 2010; Garattini and Terao, 2011, 2012; Hutzler et al., 2013). This enzyme comprises two identical ∼150-kDa subunits, is active as a homodimer, and demonstrates broad substrate selectivity, oxidizing a variety of aldehydes and azaheterocyclic-containing drug molecules (Beedham, 1987; Kitamura et al., 2006; Garattini et al., 2008). Because of the subcellular location of this enzyme, metabolic stability investigations using human liver microsomes fail to identify the contribution of AO-mediated metabolism to total metabolic clearance, which has led to early clinical failures for otherwise promising drug candidates. In particular, poor pharmacokinetic properties were observed after dosing of humans in phase 1 studies for drugs such as carbazeran, BIBX1382 (N8-(3-chloro-4-fluorophenyl)-N2-(1-methylpiperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, 2HCl), and FK3453 (6-(2-amino-4-phenylpyrimidine-5-yl)-2-isopropylpyridazin-3(2H)-one) (Kaye et al., 1984; Dittrich et al., 2002; Akabane et al., 2011). In addition, an AO-mediated metabolite of SGX523 (6-(6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-ylthio)quinoline) not identified preclinically was reported to cause acute renal toxicity as a result of extremely poor solubility and crystallization in the kidney (Diamond et al., 2010). Unfortunately, all these aforementioned examples of early clinical failures can be attributed to a lack of complete understanding of metabolic clearance mechanisms because in vitro metabolism studies were only conducted in microsomal liver fractions.

In the drug discovery setting, use of human hepatocytes has become more commonplace for predicting total metabolic clearance, as this in vitro system theoretically contains the full complement of drug-metabolizing enzymes and cofactors in the liver. In addition, advancement of cryopreservation techniques has enabled easier access to hepatocytes. However, cytochrome P450 activities are the primary focus of enzyme characterization using selective probe substrates. Activities for non-P450 enzymes such as aldehyde oxidase have typically not been sufficiently characterized. Using a number of probe substrates of AO, including O6-benzylguanine (O6-BG), our laboratory recently demonstrated that pooled cryopreserved human hepatocytes contain reasonably high AO activity and may be suitable in a drug discovery setting to screen out compounds that undergo rapid metabolism by AO (Hutzler et al., 2012). Despite these findings, some reports in the literature suggest that the AO enzyme may be unstable (Duley et al., 1985), which may contribute to observed underpredictions of clearance using hepatocytes (Akabane et al., 2012a), as well as other in vitro systems (e.g., cytosol and S-9 fractions) where AO is present (Zientek et al., 2010). An additional complication impeding progress toward scaling AO-mediated clearance is reported high donor-to-donor variability in AO activity. For example, AO activity as measured by DACA (N-[2-(dimethylamino)ethyl]acridone-4-carboxamide) hydroxylation varied 18-fold (Vmax/Km method) across 13 donors when using liver cytosol as the source of enzyme (Al-Salmy, 2001). Similarly, methotrexate 7-hydroxylase activity assayed in liver cytosol from six human donors varied 48-fold (Kitamura et al., 1999). Meanwhile, the variability of AO activity has been reported only in cryopreserved human hepatocytes using vanillin as the probe substrate across five donors, with a 5-fold range of activity observed (Sahi et al., 2008). Although these and other reports of variable activity (summarized in Table 1) highlight a question around potential clinical variability in pharmacokinetics for substrates of AO, analysis of a larger donor set is necessary to attempt to draw conclusions as to what donor characteristics and factors contribute to the observed variability in activity.

View this table:
TABLE 1

Summary of literature reports demonstrating high variability of aldehyde oxidase activity in various in vitro systems

In an effort to address these knowledge gaps, AO activity was measured in fresh human hepatocytes from 10 individual donors over 24 hours immediately after the isolation of hepatocytes from intact livers, as well as in cryopreserved hepatocytes prepared from the same donors (75 total donors). In addition, characteristics of tested donors were examined to understand the impact of demographic variables such as age, gender, ethnicity, medical history, and common single nucleotide polymorphisms (SNPs) on AO activity. AO enzymatic activity was estimated using O6-BG as the probe substrate (Fig. 1). O6-BG has been reported to be exclusively metabolized by AO-mediated oxidation to the 8-oxoguanine metabolite in cancer patients, where the area under the concentration versus time curve of the metabolite was 12- to 29-fold greater than parent O6-BG (Dolan et al., 1998). In addition, we have demonstrated in our laboratory that when hydralazine, a selective inhibitor of AO (Strelevitz et al., 2012), is coincubated with O6-BG in cryopreserved hepatocytes, metabolism is effectively inhibited (Hutzler et al., 2012). Thus, O6-BG appears to be a suitable probe substrate for estimating AO activity in hepatocytes.

Fig. 1.
Fig. 1.

Structure of aldehyde oxidase probe substrate O6-benzylguanine.

Materials and Methods

Chemical and Biologic Reagents.

Both O6-BG and tolbutamide were purchased from Sigma Chemicals (St. Louis, MO). Human hepatocytes (fresh, cryopreserved individual donor, and pooled cryopreserved mixed gender, 19-donor, Lot OOO), hepatocyte media (InVitroGro HT thawing and InVitroGro KHB for incubation), and additional cell culture reagents were obtained from BioreclamationIVT (Baltimore, MD). All other reagents and chemicals were of the highest purity available.

Hepatocyte Isolation Procedure.

Human livers from 10 donors that were not suitable for transplantation for various reasons (demographic constraints; lifestyle history; anatomic concerns such as size, vascularization, fat content, etc.) were obtained through organ procurement organizations from donors with proper consent. The livers were flushed at the time of harvest with Viaspan or similar preservation solution and transported on wet ice to BioreclamationIVT between 12 and 23 hours after harvest. A two-step collagenase isolation with a differential centrifugation procedure was used to isolate the hepatocytes (Li et al., 1999). Three separate 10-million-hepatocyte suspensions from each donor, stored in a Dulbecco’s modified Eagle’s medium–based medium (25% Viaspan) were prepared for incubation and assessment of AO activity immediately after isolation (within 2 hours), ∼24 hours post-isolation (incubations conducted at BioreclamationIVT), and ∼24 hours post-isolation after shipment of hepatocytes to Boehringer-Ingelheim (a common practice when ordering “fresh” hepatocytes from a vendor). Hepatocyte suspensions for ∼24-hour post-isolation incubations conducted at BioreclamationIVT were stored at 4°C without mixing to mimic shipping conditions. The remainder of the suspension from each donor was cryopreserved according to a protocol used by BioreclamationIVT. Donor demographics are shown in Table 2.

View this table:
TABLE 2

Characteristics for 10 human donors used to assess impact of time and cryopreservation on aldehyde oxidase activity in suspended hepatocyte incubations

Hepatocyte Incubations.

Freshly isolated human hepatocytes (as described already herein) were gently mixed before centrifugation at 50g for 5 minutes at 4°C. The supernatant was discarded, and the hepatocyte pellet was resuspended in InVitroGro KHB. Pooled mixed-gender (lot number OOO, 19 mixed-gender donors); individual donor cryopreserved human hepatocytes were stored in liquid nitrogen until use. Immediately before experiments, sufficient aliquots of hepatocytes were thawed rapidly (∼2 minutes) in a shaking water bath at 37°C. The contents of each vial were diluted 1:50 in prewarmed (37°C) InVitroGro HT thawing medium and gently mixed before centrifugation at 50g for 5 minutes at room temperature. The hepatocyte pellet was resuspended in InVitroGro KHB. The cell number and viability were determined using a hemocytometer after staining with trypan blue. Viabilities for each hepatocyte experiment were ≥70%. Cell suspension was diluted to provide a 2× stock and prewarmed at 37°C for 15 minutes. Incubations were initiated by the addition of O6-BG substrate solution (250 μl) to the hepatocyte suspension (250 μl, 250,000 viable cells) in a 48-well polypropylene incubation plate (1 µM final substrate concentration, 0.02% dimethylsulfoxide, 0.5% acetonitrile), followed by gentle swirling in an atmosphere of 5% CO2 and 95% relative humidity (n = 4 replicates). Reactions were terminated at 0, 5, 15, 30, 60, and 120 minutes by aliquotting 50 µl of incubate into 150 µl of cold acetonitrile (0.1% acetic acid) containing internal standard (0.25 μM tolbutamide). Quench plates were then centrifuged at 3000g for 5 minutes, and supernatants were transferred to 96-well plates for bioanalysis by liquid chromatography-tandem mass spectrometry (LC/MS/MS) (for incubations conducted at Boehringer-Ingelheim) or stored at −80°C and then shipped on dry ice to Boehringer-Ingelheim for tandem LC/MS/MS bioanalysis (for incubations conducted at BioreclamationIVT).

Bioanalytical Procedures.

Quantitation of all analytes was performed using an AB Sciex (Foster City, CA) API-5000 triple-quadrupole MS equipped with electrospray ionization interface in positive ion mode and connected in-line to a Waters Acquity ultraperformance liquid chromatography system (Waters, Milford, MA). Separation of analytes was performed using a Waters Acquity BEH high-pressure C18 1.7-µm (2.1 × 50 mm) column held at 50°C. Mobile phase was flowed at 0.5 ml/minute with a rapid gradient starting at 95% A (0.1% formic acid in water) and 5% B (0.1% formic acid in acetonitrile), held for 0.5 minute, ramped linearly to 5:95 A:B, and held for 1.4 minutes, followed by returning to initial conditions (2 minutes total run time). The multiple reaction monitoring transitions for each analyte were as follows: O6-BG (m/z 242.0 > 199.1), and tolbutamide (m/z 271.3 > 91.1). Standard curves for O6-benzylguanine were prepared and found to be linear within concentration range of 0.025–2.0 μM. The limit of quantitation for O6-benzylguanine was determined to be 0.07 µM. All data were analyzed using AB Sciex Analyst 1.4.2 software.

Intrinsic Clearance Estimates.

In vitro intrinsic clearance (Clint) was estimated (ml/min per kilogram) after incubations in human hepatocytes for 2 hours using substrate depletion methods and eq. 1:Embedded Image(1)where t1/2 is the observed in vitro substrate depletion half-life (min) as determined by linear regression. Scaling factors for intrinsic clearance calculations were as follows: 25.7 g liver weight per kilogram of body weight and 120 × 106 hepatocytes per gram of liver.

Single Nucleotide Polymorphism Analysis.

Genomic DNA was prepared from cryopreserved hepatocytes (n = 71 donors) using the Qiagen QIAamp DNA Mini kit (Gaithersburg, MD). TaqMan Genotyping SNP assays and TaqMan Genotyping Master Mix were obtained from Life Technologies (Carlsbad, CA) and were used in reactions according to the manufacturer’s instructions. Reactions were run on an ABI7500 Fast Real-Time polymerase chain reaction system, and data were captured and analyzed using SDS v1.3 software. All SNP markers detected were in Hardy-Weinberg equilibrium as tested by Fisher’s exact test. Statistical analysis was performed using GraphPad Prism (v5.02) (La Jolla, CA).

Results

Effect of Time and Cryopreservation on Intrinsic Clearance in Hepatocytes from 10 Donors.

In vitro intrinsic clearance was determined using O6-BG as a marker for AO activity immediately after isolation of fresh hepatocytes from intact livers (the process for isolation and preparing for incubation takes ≤2 hours from receipt of liver), as well as ∼24 hours post-isolation and after cryopreservation. Donors included seven males and three females, with ages ranging from 4 to 74 years (Table 2). Intrinsic clearance data for the 10 donors are presented in Fig. 2. Overall, the data suggest that substantial loss in AO activity within the first 24 hours after isolation of hepatocytes is common (average loss of 42%, range 15%–81% for 7 of 10 donors). Interestingly, this downward trend in activity was not observed for donors D3, D4, and D7, which suggests that loss of activity is likely donor-dependent. Comparison of AO activity 24 hours post-isolation (stored at 4°C) with 24 hours in hepatocytes that were shipped overnight (mimicking process for how “fresh” hepatocytes are typically acquired) showed intrinsic clearance values that were comparable for 5 of 10 donors, with measurably higher activity in donors D1, D2, D8, and D10 (Fig. 2). Lastly, the effect of cryopreservation was evaluated using the same 10 donors. Overall, activity in cryopreserved hepatocytes represented fairly well the activity observed in fresh hepatocytes that were isolated and incubated immediately for the same donors (within 81%, range 48%–100%), which suggests that cryopreserved hepatocytes may be preferable from an activity standpoint compared with fresh hepatocytes that typically are at least 24 hours old before conduct of in vitro metabolism studies. Lastly, comparing activity across the 10 donors showed high donor-to-donor variability (5- to 8-fold), regardless of condition.

Fig. 2.
Fig. 2.

Bar chart demonstrating aldehyde oxidase activity (O6-benzylguanine intrinsic clearance) for n = 10 human donors over initial 24 hours after hepatocyte isolation from intact liver, as well as activity after cryopreservation of the same donor hepatocytes. Clearance values are mean of n = 4 replicates, with error bars = S.D. See Materials and Methods for incubation procedures.

Donor Variability in AO Activity.

Given that cryopreserved hepatocytes appear to maintain reasonable activity for AO compared with fresh human hepatocytes that are incubated in suspension immediately after isolation, intrinsic clearance for 75 individual donors was subsequently assessed in cryopreserved human hepatocytes using O6-BG. Donor characteristics for the 75 donors were collected and can be found in the supplemental information (Supplemental Table 1). The data for O6-BG intrinsic clearance across 75 donors are presented graphically in Fig. 3. First, it is worth noting that the activity observed in a lot of 19 pooled mixed-gender donors (Clint = 34.2 ± 1.4 ml/minute per kilogram (n = 4 replicates), dotted line in Fig. 3) was less than ∼63% the donors in the current 75 donor-pool population. Also, the first five donors had unmeasurable AO activity as determined by substrate depletion. The cutoff used for extrapolation of half-life was ∼790 minutes (e.g., half-life ≥790 minutes), which is derived from estimating that at a minimum, ≥10% of substrate must be consumed by end of the 120-minute incubation time to conclude measurable enzyme activity. This scales to an estimated intrinsic clearance of ≤5.4 ml/minute per kilogram of body weight (eq. 1). Overall, activity for the 75 tested donors varied by at least 17-fold, with the lowest activity being ≤5.4 ml/minute per kilogram and highest being 90 ml/minute per kilogram. The average activity across 70 donors (not including the five donors that had unmeasurable activity) was 39.9 ml/minute per kilogram, with a high S.D. of 19 ml/minute per kilogram (solid line in Fig. 3), indicating the donor variability. Evaluation of the medical history information of the donors showed that three of the five low-activity donors had histories of extensive alcohol abuse up until time of death (Supplemental Table 1). Meanwhile, donor GTV was a “social drinker,” but quantitative information was not available. Two donors with higher AO activity than what was observed in the pooled cryopreserved lot had medical histories of alcohol abuse, but this abuse had ended years before their death. Overall, comparison of demographics such as gender, body mass index, ethnicity (African American, Hispanic, and Caucasian) and age (range 1–88) revealed no statistically significant differences with respect to activity when using two-tailed t test and one-way analysis of variance analysis (Fig. 4).

Fig. 3.
Fig. 3.

Variability in intrinsic clearance (ml/minute per kilogram) for O6-benzylguanine oxidation in cryopreserved hepatocytes from 75 individual donors. Dotted line represents observed intrinsic clearance for O6-benzylguanine in a pooled cryopreserved hepatocyte lot consisting of 19 mixed-gender donors. Clearance values for all are mean of n = 4 replicates, with error bars = S.D. See Supplemental Material for donor characteristics, including medical histories.

Fig. 4.
Fig. 4.

Whisker plots comparing AO activity (O6-benzylguanine Clintrinsic) across 75 donors stratified by (A) gender, (B) body mass index, (C) ethnic background, and (D) age groups. Black bar indicates mean; error bars = S.D. No statistically significant differences were observed between gender (A, P = 0.3653) and body mass index (B, P = 0.4189) groups using two-tailed t test. In addition, no significant differences in activity were observed between race (C) and various age groups (D) using 1-way analysis of variance with Bonferroni’s multiple comparison test (P > 0.05).

With respect to disease factors, a history of diabetes was noted for 15 of 75 donors (20%), but it was a disease characteristic in donors spanning the entire range of activities (Supplemental Table 1). No additional trends were identified.

Single Nucleotide Polymorphism Analysis.

Single nucleotide polymorphism (SNP) data for four donors were unable to be collected because of a lack of sufficient cells for DNA isolation after the initial metabolism studies. Thus, known SNPs for AOX1 were analyzed in the donor population (n = 71) using Taqman polymerase chain reaction assays (Table 3). Four of the six SNPs tested were not detected in our donor population. Two SNPs for AOX1 (rs3731772 and rs55754655) were detected and showed allelic frequencies similar to those reported from other cohort studies such as the National Heart, Lung, and Blood Institute Exome Sequencing Project (Table 4). However, the presence of these mutant alleles did not correlate with a statistically significant difference in intrinsic clearance values for rs55754655 (P = 0.2502) or rs3731722 (P = 0.0514) compared with the wild-type group using a two-tailed t test (Fig. 5).

View this table:
TABLE 3

Summary of AOX1 single nucleotide polymorphisms (SNP) and allelic frequencies observed in 71 donors

View this table:
TABLE 4

Genotype prevalence by ethnic background for two most common AOX1 single nucleotide polymorphisms (SNPs)

The prevalence of each genotype is given in total and by race for 71 donors. A large cohort population from the National Center for Biotechnology Information dbSNP database is included for comparison.

Fig. 5.
Fig. 5.

Whisker plots comparing AO activity (O6-benzylguanine Clintrinsic) for subjects genotyped and grouped into wild-type (WT) or with either one or two G alleles (G). Black bar indicates mean with error bars indicating S.D. No statistically significant differences were observed between genotypes using a two-tailed t test: (A) rs55754655 (n = 58, 34 WT/24 G) P = 0.2502; (B) rs3731722 (n = 44, 34 WT/10 G) P = 0.0514.

Discussion

AO is a cytosolic drug-metabolizing enzyme that has emerged in recent years in the metabolism of azaheterocyclic drug molecules (Pryde et al., 2010; Garattini and Terao, 2013; Hutzler et al., 2013). The ideal in vitro system for comprehensively evaluating metabolic clearance is hepatocytes, which theoretically contain the full complement of drug-metabolizing enzymes. However, the activity of non-P450 metabolic enzymes, such as AO, has generally not been well characterized. Recently, we reported AO activity in two separate lots of pooled human cryopreserved hepatocytes (Hutzler et al., 2012). However, an appraisal of the activity of AO within the first 24 hours after isolation of hepatocytes from a human liver has not been reported. Acute loss of AO enzymatic activity over this time remains an open question, as underpredictions of clearance have been reported (Zientek et al., 2010; Akabane et al., 2011). To this end, in vitro studies were designed and conducted using fresh and donor-matched cryopreserved hepatocytes to evaluate the impact of time and cryopreservation on enzymatic activity. In addition, donor variability in the largest set of subjects reported to date (75 donors) was evaluated using cryopreserved human hepatocytes.

When a drug metabolism research laboratory orders fresh hepatocytes from a vendor, by the time they receive cells for incubations, they are at least 24 hours old (next day after isolation). Understanding the potential loss in AO activity over this period is important, especially if using metabolism data for scaling and prediction of human metabolic clearance. When in vitro intrinsic clearance was determined immediately after isolation of fresh hepatocytes from 10 donor livers (within 2 hours post-isolation) and then measured again in the same cells roughly 24 hours following isolation, substantial loss in AO activity was observed (average loss of 42%, range 15%–81%). Interestingly, this trend was dependent on the donor, as no activity loss was observed over this period for donors D3, D4, and D7 (Fig. 2). Evaluation of the available donor characteristics did not clarify why some donors displayed a loss in activity within the initial 24 hours, whereas others did not. Since all donor hepatocytes were treated the same, it is unlikely to be due to the process of cell isolation or storage over the initial 24 hours. One could plausibly suggest a link between the general health of the donor livers or cells and AO activity over time, but this is entirely speculative. Additional research using more donors would need to be conducted to provide convincing evidence. Comparison of the activity of AO 24 hours post-isolation (e.g., stored) with hepatocytes that were shipped overnight (mimicking the process for how “fresh” hepatocytes are typically acquired) showed intrinsic clearance values that were overall comparable but with a currently unexplained increase in activity for donors D1, D2, D8, and D10 after shipment and incubation (Fig. 2). The impact of cryopreservation was also evaluated using the same 10 donors. Overall, activity in cryopreserved hepatocytes fairly well represented the activity observed in hepatocytes that were isolated and incubated immediately for the same donors (within 81%, range 48%–100%), which suggests that cryopreserved hepatocytes would be preferable to fresh hepatocytes that commonly are at least 24 hours old before the conduct of in vitro metabolism studies. This is further rationalized when one considers the variability in activity loss observed over the initial 24 hours and the uncertainty around availability of fresh donor hepatocytes. A similar evaluation was undertaken by Akabane and colleagues (Akabane et al., 2012b), who compared activity for various non-P450 enzymes in fresh and cryopreserved hepatocytes. AO was among the non-P450 enzymes tested, and they used multiple probe substrates to estimate activity, including O6-BG. Their results were dependent on AO substrate; but with O6-BG as the marker, cryopreserved hepatocytes from the same donors (n = 4 donors) appeared to have higher activity than fresh hepatocytes (Akabane et al., 2012b). It is worth noting that the “fresh” hepatocytes used in their studies were acquired from multiple vendors in the traditional manner (e.g., ordered and shipped next day). Although speculative, it may be possible based on our observations that substantial AO activity was lost during the isolation and shipment time, which could explain the higher activity observed in cryopreserved hepatocytes from the same donor.

Comparing activity across the 10 donors, regardless of whether comparing the intrinsic clearance values in fresh or cryopreserved hepatocytes, we observed donor variability (5- to 8-fold). Several reports in the literature demonstrate high donor-to-donor variability for AO activity, although the magnitude appears dependent on the substrate and method (e.g., metabolite formation or substrate depletion) used (Table 1). Given that cryopreserved hepatocytes from the 10 donors tested appear to possess comparable activity to hepatocytes that were immediately incubated after isolation, the use of cryopreserved hepatocytes was determined to be suitable for evaluating variation in AO activity across a larger donor pool (n = 75 donors), where characteristics (e.g., gender, age, ethnicity, and medical history) could be evaluated in an effort to elucidate the potential causes for this variation. To our knowledge, this represents the largest donor pool from which variability in activity has been assessed, in particular, using human hepatocytes. Overall, activity for the 75 tested donors varied by at least 17-fold, with the lowest activity being ≤5.4 ml/minute per kilogram of body weight (five donors) and the highest being 90 ml/minute per kilogram (average intrinsic clearance, 39.9 ± 19 ml/minute per kilogram). No significant trends emerged from comparison of activities across ethnic backgrounds (African American, Hispanic, and Caucasian). In addition, when comparing gender, age and body mass index, no significant differences were observed with respect to activity (Fig. 4). Interestingly, evaluation of the medical history data of the donors revealed that three of the five low-activity donors had histories of extensive alcohol abuse up until time of death. This acute effect of alcohol abuse was also recently reported by scientists at Pfizer (Fu et al., 2013). The exact mechanism of this alcohol effect on AO activity was not elucidated, although it was found that AOX1 protein expression level appeared unaffected, despite significant decreases in enzyme activity (Fu et al., 2013). Thus, the presence of AO protein apparently does not correlate with activity, which complicates the assessment of variability factors. Fu and colleagues (2013) suggest that several additional factors may contribute to variable or low AO activity, including disruption of active homodimer formation, and molybdenum cofactor deficiency. Meanwhile, in our studies, other donors with a history of alcohol abuse who abstained from alcohol years before death demonstrated moderate (45.3 ± 4.9 ml/minute per kilogram) to high (77 ± 13 ml/minute per kilogram) AO activity, suggesting that the alcohol effects may be more acute. This finding is worthy of additional research, as there could be pharmacokinetic/pharmacodynamic drug interaction implications for alcoholics taking any drug that has a predominant clearance mechanism through AO.

In addition to donor demographics and medical histories, a potential contributor to variable AO activity is the presence of SNPs, which are known and have been reported (Smith et al., 2009; Hartmann et al., 2012; Kurzawski et al., 2012). Whereas the two variants observed in our donor population (rs55754655 and rs3731722) have been reported to have clinical implications in use of azathioprine (Smith et al., 2009), the intrinsic clearance differences in our cohort did not reach statistical significance, despite a trend toward higher activity compared with wild-type individuals (Fig. 5). The limited number of subjects and the high variability of the activity may require a larger cohort to establish this connection. Hartmann and colleagues (2012) reported that both AOX1 SNPs rs55754655 (N1135S) and rs3731722 (H1297R) led to 2- to 4-fold higher catalytic activities, but this conclusion was derived using purified expressed AO enzyme, and thus was independent of additional donor variable factors. Considering all the aforementioned factors involved in impacting AO activity (e.g., molybdenum deficiency, etc.), it is unlikely that genotype alone dictates the phenotypic activity of AO. Although this SNP analysis was limited in scope, this premise is supported by a recent report where variability in XK469 clearance was found to be independent of AOX1 polymorphisms (Ramirez et al., 2014).

In summary, it has been demonstrated that “fresh” hepatocytes obtained from a vendor that are in reality at least 24 hours old may have lost substantial AO activity, depending on the donor, whereas cryopreserved hepatocytes appear to better represent the activity in a donor liver when incubations are conducted immediately after isolation of hepatocytes. Thus, cryopreserved human hepatocytes appear to be a suitable in vitro model for determining intrinsic clearance activity for substrates of AO as part of an integrated approach to understanding total metabolic clearance. In addition, there is substantial donor-to-donor variability for AO activity, which requires careful characterization of donors or donor pools when selecting hepatocyte lots that contain adequate activity for the multitude of drug-metabolizing enzymes. Since it appears that multiple factors may contribute to variable or low AO activity, use of hepatocytes from a single donor to estimate AO activity is not recommended, especially if alcohol abuse is noted in the donor’s medical history. Instead, prescreening donors for AO activity and creating a custom high-activity pooled lot of cryopreserved hepatocytes is advised to minimize underprediction of metabolic clearance in human.

Acknowledgments

The authors thank Matthew A. Cerny for helpful comments and thoughtful review of our manuscript.

Authorship Contributions

Participated in research design: Hutzler, Yang, Moeller, Brown, Heyward.

Conducted experiments: Yang, Moeller, Brown, Heyward.

Performed data analysis: Yang, Hutzler, Moeller, Brown, Heyward.

Wrote or contributed to the writing of the manuscript: Hutzler, Yang, Moeller, Brown, Heyward.

Footnotes

Abbreviations

AO
aldehyde oxidase
Clint
intrinsic clearance
LC-MS/MS
liquid chromatography-tandem mass spectrometry
O6-BG
O6-benzylguanine
SNP
single nucleotide polymorphism

References

  1. ↵
    1. Akabane T,
    2. Gerst N,
    3. Masters JN, and
    4. Tamura K
    (2012a) A quantitative approach to hepatic clearance prediction of metabolism by aldehyde oxidase using custom pooled hepatocytes. Xenobiotica 42:863–871.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Akabane T,
    2. Gerst N,
    3. Naritomi Y,
    4. Masters JN, and
    5. Tamura K
    (2012b) A practical and direct comparison of intrinsic metabolic clearance of several non-CYP enzyme substrates in freshly isolated and cryopreserved hepatocytes. Drug Metab Pharmacokinet 27:181–191.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Akabane T,
    2. Tanaka K,
    3. Irie M,
    4. Terashita S, and
    5. Teramura T
    (2011) Case report of extensive metabolism by aldehyde oxidase in humans: pharmacokinetics and metabolite profile of FK3453 in rats, dogs, and humans. Xenobiotica 41:372–384.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Al-Salmy HS
    (2001) Individual variation in hepatic aldehyde oxidase activity. IUBMB Life 51:249–253.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Beedham C
    (1987) Molybdenum hydroxylases: biological distribution and substrate-inhibitor specificity. Prog Med Chem 24:85–127.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Diamond S,
    2. Boer J,
    3. Maduskuie TP Jr.,
    4. Falahatpisheh N,
    5. Li Y, and
    6. Yeleswaram S
    (2010) Species-specific metabolism of SGX523 by aldehyde oxidase and the toxicological implications. Drug Metab Dispos 38:1277–1285.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Dittrich Ch,
    2. Greim G,
    3. Borner M,
    4. Weigang-Köhler K,
    5. Huisman H,
    6. Amelsberg A,
    7. Ehret A,
    8. Wanders J,
    9. Hanauske A, and
    10. Fumoleau P
    (2002) Phase I and pharmacokinetic study of BIBX 1382 BS, an epidermal growth factor receptor (EGFR) inhibitor, given in a continuous daily oral administration. Eur J Cancer 38:1072–1080.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Dolan ME,
    2. Roy SK,
    3. Fasanmade AA,
    4. Paras PR,
    5. Schilsky RL, and
    6. Ratain MJ
    (1998) O6-benzylguanine in humans: metabolic, pharmacokinetic, and pharmacodynamic findings. J Clin Oncol 16:1803–1810.
    OpenUrlAbstract
  9. ↵
    1. Duley JA,
    2. Harris O, and
    3. Holmes RS
    (1985) Analysis of human alcohol- and aldehyde-metabolizing isozymes by electrophoresis and isoelectric focusing. Alcohol Clin Exp Res 9:263–271.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Fu C,
    2. Di L,
    3. Han X,
    4. Soderstrom C,
    5. Snyder M,
    6. Troutman MD,
    7. Obach RS, and
    8. Zhang H
    (2013) Aldehyde oxidase 1 (AOX1) in human liver cytosols: quantitative characterization of AOX1 expression level and activity relationship. Drug Metab Dispos 41:1797–1804.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Garattini E,
    2. Fratelli M, and
    3. Terao M
    (2008) Mammalian aldehyde oxidases: genetics, evolution and biochemistry. Cell Mol Life Sci 65:1019–1048.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Garattini E and
    2. Terao M
    (2011) Increasing recognition of the importance of aldehyde oxidase in drug development and discovery. Drug Metab Rev 43:374–386.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Garattini E and
    2. Terao M
    (2012) The role of aldehyde oxidase in drug metabolism. Expert Opin Drug Metab Toxicol 8:487–503.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Garattini E and
    2. Terao M
    (2013) Aldehyde oxidase and its importance in novel drug discovery: present and future challenges. Expert Opin Drug Discov 8:641–654.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Hartmann T,
    2. Terao M,
    3. Garattini E,
    4. Teutloff C,
    5. Alfaro JF,
    6. Jones JP, and
    7. Leimkühler S
    (2012) The impact of single nucleotide polymorphisms on human aldehyde oxidase. Drug Metab Dispos 40:856–864.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Hutzler JM,
    2. Obach RS,
    3. Dalvie D, and
    4. Zientek MA
    (2013) Strategies for a comprehensive understanding of metabolism by aldehyde oxidase. Expert Opin Drug Metab Toxicol 9:153–168.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Hutzler JM,
    2. Yang YS,
    3. Albaugh D,
    4. Fullenwider CL,
    5. Schmenk J, and
    6. Fisher MB
    (2012) Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes. Drug Metab Dispos 40:267–275.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Kaye B,
    2. Offerman JL,
    3. Reid JL,
    4. Elliott HL, and
    5. Hillis WS
    (1984) A species difference in the presystemic metabolism of carbazeran in dog and man. Xenobiotica 14:935–945.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Kitamura S,
    2. Sugihara K,
    3. Nakatani K,
    4. Ohta S,
    5. Ohhara T,
    6. Ninomiya S,
    7. Green CE, and
    8. Tyson CA
    (1999) Variation of hepatic methotrexate 7-hydroxylase activity in animals and humans. IUBMB Life 48:607–611.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Kitamura S,
    2. Sugihara K, and
    3. Ohta S
    (2006) Drug-metabolizing ability of molybdenum hydroxylases. Drug Metab Pharmacokinet 21:83–98.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Kurzawski M,
    2. Dziewanowski K,
    3. Safranow K, and
    4. Drozdzik M
    (2012) Polymorphism of genes involved in purine metabolism (XDH, AOX1, MOCOS) in kidney transplant recipients receiving azathioprine. Ther Drug Monit 34:266–274.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Li AP,
    2. Lu C,
    3. Brent JA,
    4. Pham C,
    5. Fackett A,
    6. Ruegg CE, and
    7. Silber PM
    (1999) Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential. Chem Biol Interact 121:17–35.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Pryde DC,
    2. Dalvie D,
    3. Hu Q,
    4. Jones P,
    5. Obach RS, and
    6. Tran TD
    (2010) Aldehyde oxidase: an enzyme of emerging importance in drug discovery. J Med Chem 53:8441–8460.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Ramírez J,
    2. Kim TW,
    3. Liu W,
    4. Myers JL,
    5. Mirkov S,
    6. Owzar K,
    7. Watson D,
    8. Mulkey F,
    9. Gamazon ER,
    10. Stock W,
    11. et al.
    (2014) A pharmacogenetic study of aldehyde oxidase I in patients treated with XK469. Pharmacogenet Genomics 24:129–132.
    OpenUrlCrossRefPubMed
    1. Rodrigues AD,
    2. Ferrero JL,
    3. Amann MT,
    4. Rotert GA,
    5. Cepa SP,
    6. Surber BW,
    7. Machinist JM,
    8. Tich NR,
    9. Sullivan JP,
    10. Garvey DS,
    11. et al.
    (1994) The in vitro hepatic metabolism of ABT-418, a cholinergic channel activator, in rats, dogs, cynomolgus monkeys, and humans. Drug Metab Dispos 22:788–798.
    OpenUrlAbstract
  26. ↵
    1. Sahi J,
    2. Khan KK, and
    3. Black CB
    (2008) Aldehyde oxidase activity and inhibition in hepatocytes and cytosolic fractions from mouse, rat, monkey and human. Drug Metab Lett 2:176–183.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Smith MA,
    2. Marinaki AM,
    3. Arenas M,
    4. Shobowale-Bakre M,
    5. Lewis CM,
    6. Ansari A,
    7. Duley J, and
    8. Sanderson JD
    (2009) Novel pharmacogenetic markers for treatment outcome in azathioprine-treated inflammatory bowel disease. Aliment Pharmacol Ther 30:375–384.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Strelevitz TJ,
    2. Orozco CC, and
    3. Obach RS
    (2012) Hydralazine as a selective probe inactivator of aldehyde oxidase in human hepatocytes: estimation of the contribution of aldehyde oxidase to metabolic clearance. Drug Metab Dispos 40:1441–1448.
    OpenUrlAbstract/FREE Full Text
    1. Sugihara K,
    2. Kitamura S,
    3. Tatsumi K,
    4. Asahara T, and
    5. Dohi K
    (1997) Differences in aldehyde oxidase activity in cytosolic preparations of human and monkey liver. Biochem Mol Biol Int 41:1153–1160.
    OpenUrlPubMed
    1. Tayama Y,
    2. Sugihara K,
    3. Sanoh S,
    4. Miyake K,
    5. Kitamura S, and
    6. Ohta S
    (2012) Developmental changes of aldehyde oxidase activity and protein expression in human liver cytosol. Drug Metab Pharmacokinet 27:543–547.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Zientek M,
    2. Jiang Y,
    3. Youdim K, and
    4. Obach RS
    (2010) In vitro-in vivo correlation for intrinsic clearance for drugs metabolized by human aldehyde oxidase. Drug Metab Dispos 38:1322–1327.
    OpenUrlAbstract/FREE Full Text

 

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Aldehyde Oxidase Activity in Human Hepatocytes

J. Matthew Hutzler, Young-Sun Yang, Caitlin Brown, Scott Heyward and Timothy Moeller
Drug Metabolism and Disposition June 1, 2014, 42 (6) 1090-1097; DOI: https://doi.org/10.1124/dmd.114.057984
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