A New Rapid in Vitro Assay for Assessing Reactivity of Acyl Glucuronides S

Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2015/08/17/dmd.115.066159.DC1

1521-009X/43/11/1711–1717$25.00 http://dx.doi.org/10.1124/dmd.115.066159 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:1711–1717, November 2015 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics A New Rapid In Vitro Assay for Assessing Reactivity of Acyl Glucuronides s

Sheng Zhong, Russell Jones, Wenzhe Lu, Simone Schadt, and Giorgio Ottaviani

Pharmaceutical Sciences, Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China (S.Z., W.L., G.O.) and Roche Innovation Center Basel, Basel, Switzerland (R.J., S.S.)

Received July 2, 2015; accepted August 12, 2015

ABSTRACT Idiosyncratic drug toxicity is a major challenge for the pharmaceutical selection of low-risk drug candidates in the drug discovery phase. industry since complex and multifactorial steps are involved, the Twenty marketed compounds with a wide range of half-lives were dose-dependency is unclear, and its occurrence is not reliably tested, their acyl glucuronide migration rates were determined and Downloaded from predictable. Whereas the exact mechanisms leading to idiosyncratic compared with the half-lives of the respective acyl glucuronides. toxicity remain elusive in many cases, there are often hints at the Ranking of acyl glucuronide stability using this method compared well involvement of reactive metabolites, such as acyl glucuronides with the results from existing methodologies. With this method, formed by conjugation of carboxylic acids with glucuronic acid. migration rates >20% would indicate higher risk of reactivity. This Because the patient-related susceptibilities leading to idiosyncratic simpler approach using the acyl glucuronide migration rate is not

toxicity are not sufficiently understood, the best option for the dependent on authentic standards, therefore eliminating the require- dmd.aspetjournals.org pharmaceutical industry is to minimize drug-related risk factors such ment for either lengthy chemical synthesis or in vitro biosynthesis and as potential acyl glucuronide formation. Here, we describe a rapid in purification of the 1-O-b-glucuronide. This methodology provides vitro assay for the assessment of the reactivity of acyl glucuronides, a rapid in vitro assay to assess acyl glucuronide stability and reactivity on the basis of acyl glucuronide migration, that can support the that is well suited for use early in the drug discovery phase. at ASPET Journals on September 26, 2021 Introduction the drug moiety migrates from the original 1-O position of the Idiosyncratic drug toxicity (IDT) is a major challenge for the glucuronic acid to the 2-O-,3-O-, and 4-O-positions. During this pharmaceutical industry because of its poor predictability, unclear migration process there is the potential to produce chemically reactive dose-dependency, and complex, multifactorial steps. Whereas the exact species that can covalently interact with proteins and other biomole- mechanisms leading to idiosyncratic toxicity remain elusive in many cules. This chemical reactivity can therefore be linked to the stability of cases, there are often hints that reactive metabolites and/or the adaptive the acyl glucuronide, such that the greater the migration rate the greater immune system play a role (Kaplowitz, 2013). One group of potentially the potential reactivity of the acyl glucuronide. (Bailey and Dickinson, reactive metabolites is acyl glucuronides formed by the conjugation of 2003). Generally, two types of reaction can occur, starting with the b carboxylic acids with glucuronic acid. Reactive acyl glucuronides can initial 1-O- -isomer, that can be investigated to draw conclusions about bind covalently to proteins, thus potentially disrupting protein function, the reactivity of the acyl glucuronide: first, direct reaction with changing cellular signaling pathways or acting as haptens (Bailey and nucleophiles, such as cysteine residues in human serum albumin Dickinson, 2003). Formation of new antigens by haptenization is (HSA), or with peptides, leading to displacement of the acyl residue; considered to be the first step in the immune response cascade, which or second, acyl migration to the 2-, 3-, and 4-acyl isomers, resulting in subsequently can lead to an idiosyncratic toxicity-like drug-induced formation of a potentially reactive aldehyde group on the ring-open liver injury (DILI), skin reactions (e.g., Steven-Johnson syndrome), or tautomer of the glucuronic acid moiety, which can subsequently anaphylaxis. Examples of carboxylic acid drugs that form acyl condense with primary amino functional groups of proteins (Fig. 1; glucuronides and that had to be withdrawn from the market include Fenselau, 1994, Bailey and Dickinson, 2003). benoxaprofen, ibufenac, and zomepirac (Boelsterli, 2002; Sawamura The stability and reactivity of acyl glucuronides varies widely; for b et al., 2010). Since the patient-related susceptibilities leading to example, the 1-O- - acyl glucuronide of tolmetin has a very short first- idiosyncratic toxicity are currently not sufficiently understood, the best order half-life of 0.26 hours, whereas the valproic acid acyl glucuronide option for the pharmaceutical industry is to minimize drug-related risk has a long half-life of 79 hours (Fenselau, 1994). The extent of covalent factors like the potential to form reactive acyl glucuronides. binding to HSA and the apparent first-order degradation rate of acyl Acyl glucuronide conjugates are able to undergo migration. This is glucuronides have been shown to correlate well (Benet et al., 1993). It an enzyme-independent intramolecular rearrangement process in which has been further demonstrated that the chemical stability of the acyl glucuronides correlates with the IDT risk, the shorter the half-life of the acyl glucuronide formed the higher the risk of induced IDT (Sawamura dx.doi.org/10.1124/dmd.115.066159. et al., 2010). Therefore, given this relationship between the rate of s This article has supplemental material available at dmd.aspetjournals.org. degradation of 1-O-b-acyl glucuronides and the extent of in vitro

ABBREVIATIONS: HSA, human serum albumin; IDT, idiosyncratic drug toxicity; LC-MS, liquid chromatography coupled to mass spectrometry; UGT, UDP-glucuronosyltransferase.

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Fig. 1. Acyl glucuronide migration, starting with the initial 1-O-b-isomer: 1) direct reaction with nucleophiles, such as cysteine residues in HSA or peptides, leading to dmd.aspetjournals.org displacement of the acyl residue; 2) acyl migration to the 2-, 3-, and 4-acyl isomers resulting in the formation of a potentially reactive aldehyde group on the ring-open tautomer of the glucuronic acid moiety, which can subsequently condense with primary amino functional groups of proteins. covalent binding (and IDT risk), the direct assessment of the stability of (Dallas, TX). Pooled human liver microsomes were obtained from Gentest the 1-O-b-acyl glucuronide provides a simple indication of the potential (Woburn, MA). Acetic acid glacial and ammonium acetate were ordered from reactivity of the acyl glucuronide product. Tedia (Fairfield, OH). Gradient-grade acetonitrile was purchased from Merck Multiple assays for the assessment of acyl glucuronide stability and KGaA (Darmstadt, Germany). All the other chemicals and reagents used in this at ASPET Journals on September 26, 2021 reactivity are described in the literature. The gold-standard methods work were of analytical grade. Acyl Glucuronide Formation and Acyl Migration. The acyl migration used to investigate acyl glucuronide stability typically assess the half- protocol was adapted from previously published methods (Bolze et al., 2002; life of an acyl glucuronide in buffer (Ebner et al., 1999; Ebner et al., Chen et al., 2007; Jinno et al., 2013). The initial mixture contained potassium 2010), buffer with human serum albumin (Benet et al., 1993), or plasma phosphate buffer (100 mM, pH 7.4), pooled human liver microsome (2 mg/ml), (Sawamura et al., 2010). These methods require authentic standard of MgCl2 (1 mM), and test compound (100 mM). After preincubation at 37Cin the 1-O-b-acyl glucuronide. Compound optimization early in drug a shaker for 5 minutes, the reaction was initiated by adding UDPGA (4 mM). The discovery requires rapid assessment methods that do not depend on final reaction volume was 1 ml. The reaction was incubated at 37C with shaking authentic standard, since its generation and purification is too time- at 1000 rpm for 1.5 hours (Supplemental Method 1, Supplemental Table 2, and consuming for application to multiple compounds. Therefore, methods Supplemental Fig. 1), and then the incubation solution was divided into two using acyl glucuronides produced by liver microsomes for determining equal fractions (A and B). m m the half-lives in buffer, HSA solution, or fresh plasma have been Fraction A (500 l) was stopped by adding 500 l of ice-cold acetonitrile containing 10% (v/v) of acetic acid, as acyl migration may be stabilized under an developed and previously documented (Bolze et al., 2002; Chen et al., acidic condition (pH , 4.0) according to previous publications (Walker et al., 2007; Jinno et al., 2013). 2007; Di Meo et al., 2013). The sample was mixed thoroughly and then Here, we describe a simpler approach using the acyl glucuronide centrifuged at 16,000g at 4C for 10 minutes. The supernatant obtained from migration rate in human liver microsomes to assess their reactivity in fraction A was directly injected onto a liquid chromatography coupled to mass a rapid screening assay that can support the selection of low-risk drug spectrometry (LC-MS) system for analysis. candidates in the drug discovery phase. Twenty marketed compounds Fraction B (500 ml) was quenched by adding the same volume of ice-cold (Supplemental Table 1) with a wide range of half-lives were tested, and acetonitrile. Neutral acetonitrile without acid was used to quench the sample for their migration rates were determined. 1-O-b-acyl glucuronide identification (fraction B) as the efficiency of the following b-glucuronidase hydrolysis study may be compromised in a low pH environment. The centrifugation conditions described above were applied. The Materials and Methods supernatant from fraction B was vacuum-evaporated at 25C and the residue

Chemicals and Reagents. Uridine 59-diphosphoglucuronic acid trisodium was reconstituted into 400 mlofNH4OAc buffer (0.8 M, pH 5). The reconstituted salt (UDPGA), b-glucuronidase from bovine, zomepirac sodium salt, diclofenac solution was divided into two equal fractions (200 ml) and both incubated at sodium salt, ibuprofen sodium salt, telmisartan, furosemide, gemfibrozil, 37C for 120 minutes without or in parallel with 200 mlofb-glucuronidase flufenamic acid, repaglinide, indomethacin, mefenamic acid, R-naproxen, pro- (2.0 mg/ml) in the NH4OAc buffer. Reaction was subsequently quenched with benecid, clofibric acid, valproic acid, S-naproxen, and diflunisal were purchased 400 ml of ice-cold acetonitrile containing 10% (v/v) of acetic acid. After centri- from Sigma-Aldrich (St. Louis, MO). Montelukast sodium hydrate and sodium fugation at 16,000g and 4C for 10 minutes, the supernatant was analyzed by an meclofenamate monohydrate were obtained from Tokyo Chemical Industry LC-MS system for data comparison. (Chuo-ku, Tokyo, Japan). (RS)-Benoxaprofen, dabigatran, levofloxacin hemi- LC-MS Analysis of Acyl Glucuronide. The separation and qualitative hydrate, and ibufenac were ordered from Toronto Research Chemicals (Toronto, determination of the primary acyl glucuronide (1-O-b-acyl glucuronide) and its ON, Canada). Tolmetin sodium was purchased from Santa Cruz Biotechnology isomeric products in the incubated samples were performed using an Agilent Assessment of Acyl Glucuronide Reactivity 1713

QTOF 6530 high resolution mass spectrometer (Agilent Technologies, Santa Clara, CA) coupled to an Agilent 1290 Infinity series HPLC system (Agilent Technologies, Waldbronn, Germany). Sample acquisition and data processing were carried out using MassHunter Workstation Data Acquisition version B.04.00 and MassHunter Workstation Qualitative Analysis revision B.04.00 (Agilent Technologies, Santa Clara), respectively. The Dual AJS ESI ionization was performed in positive (tolmetin, zompirac, telmisartan, montelukast, repaglinide, dabigatran, and levofloxacin) or negative (diclofenac, ibuprofen, furosemide, gemfibrozil, flufenamic acid, indomethacin, mefenamic acid, R-naproxen, probenecid, meclofenamate, (RS)-benoxaprofen, ibufenac, clofibric acid, valproic acid, S-naproxen, and diflunisal) ionization mode. The isomeric acyl glucuronides and the corresponding aglycone were detected by monitoring their molecular ions under full scan mode (m/z 100–1000). The HPLC column used to conduct separation was a core-shell C18 column (Kinetex 2.6-mm XB-C18 100Å, 2.1 mm 100 mm; Phenomenex, Torrance, CA). Mobile phase A was water and mobile phase B was acetonitrile, both containing 10 mM of ammonium acetate. The flow rate was 0.5 ml/min. The HPLC solvent gradients were well optimized for each individual drug to maintain the separation of 1-O- b -acyl glucuronide from the corresponding isomers as well as the corresponding Downloaded from aglycone (for gradients, please see Supplemental Table 1). All primary acyl glucuronides (1-O-b-acyl glucuronide) and their isomeric products from the incubated samples were completely resolved within a 20-minutes run. Data Analysis. The migration rate of the acyl group from the 1-O-b-acyl glucuronide to its isomers was determined by the following equation:

total peak area of isomers Acyl migration rate ¼ 100% dmd.aspetjournals.org total peak area of isomers þ peak area of 1 2 O 2 b 2 AG ð1Þ

The glucuronidation rate of parent drug was also evaluated:

total peak area of AG metabolites Glucuronidation rate ¼ 100% total peak area of AG metabolites þ peak area of parent ð2Þ

All the peak areas used in this study were obtained from mass spectrometry data at ASPET Journals on September 26, 2021 as previously discussed. The correlation between acyl migration rates versus reported half-lives of 1-O-b-acyl glucuronide of selected carboxylic acid–containing drugs was evaluated by linear regression analysis (GraphPad Prism 6.04 software; Graph- Pad Software Inc., La Jolla, CA).

Results Identification of Glucuronides of Selected Carboxylic Acid– Containing Drugs. Twenty-three carboxyl acid–containing drugs were incubated with pooled human liver microsomes. Glucuronidation was confirmed as the major metabolic reaction in these incubations as only 1-O-b-acyl glucuronide and its isomeric products were detected. The 1-O-b-acyl glucuronide was identified from its isomeric products by treatment with or without the b-glucuronidase. The peak that significantly decreased or disappeared in the presence of b-glucuronidase was identified as 1-O-b-acyl glucuronide (Wang et al., 2004). For tolmetin, telmisartan and ibuprofen, reference standards of the 1-O-b-acyl glucuronide were available. Representative chromatograms of gemfibrozil incubated with or without b-glucuronidase are presented in Fig. 2. In these illustrations, three acyl glucuronides were detected from the gemfibrozil incubation samples that were quenched by acetonitrile containing 10% (v/v) of Fig. 2. Representative chromatograms of gemfibrozil acyl glucuronide samples. (A) acetic acid (Fig. 2A). By comparing the chromatograms of samples Gemfibrozil acyl glucuronide samples. (B) Gemfibrozil acyl glucuronide samples incubated without (Fig. 2B) and with b-glucuronidase (Fig. 2C), it was incubated without b-glucuronidase. (C) Gemfibrozil acyl glucuronide samples confirmed that the peak at retention time of 5.22 minutes was from the incubated with b-glucuronidase. 1-O-b-acyl glucuronide, whereas those at retention times of 6.76 minutes and 7.15 minutes were from the isomeric products of the 1-O- b-acyl glucuronide, as determined by the disappearance of the peak at corresponding isomers in the incubation samples. The detailed LC 5.22 minutes when incubated in the presence of b-glucuronidase. separation results are shown in Table 1. LC gradients for each individual test compound were optimized to All compounds incubated with human liver microsomes and obtain baseline separation of the 1-O-b-acyl glucuronide from the UDPGA showed a number of detected acyl glucuronide metabolites 1714 Zhong et al.

TABLE 1 Retention time of parent drug and its corresponding acyl glucuronide metabolites

Retention Time (Min) of Metabolites Detected Test Compound M0 1-O-b-AG Isomer-01 Isomer-02 Isomer-03 Isomer-04 Valproic acid 8.21 11.39 11.68 ——— Gemfibrozil 17.10 5.22 6.76 7.15 —— Telmisartan 6.06 5.98 6.47 7.00 —— Montelukast 12.00 4.81 3.07 ——— Meclofenamate 8.18 4.35 4.96 5.78 —— Mefenamic acid 7.28 3.84 5.12 ——— Levofloxacin 12.20 ————— Repaglinide 5.78 4.02 4.54 ——— Clofibric acid 10.10 14.84 16.59 17.12 —— Flufenamic acid 8.14 4.48 5.22 5.80 6.33 6.74 Furosemide 7.52 6.88 ———— Ibuprofen 10.30 9.51 9.94 10.66 10.94 11.27 S-Naproxen 8.56 11.48 12.93 13.30 13.70 — Indomethacin 9.97 9.78 11.24 11.54 —— R-naproxen 8.64 11.67 9.82 12.75 13.37 — (RS)-Benoxaprofen 12.20 4.35 4.96 5.78 —— Downloaded from Dabigatran 8.63 ————— Ibufenac 8.14 8.42 9.51 9.9 —— Diclofenac 8.58 9.73 4.04 6.2 7.79 — Diflunisal 13.9 1.90 7.67 10.91 11.93 13.05 Tolmetin 8.98 11.07 7.09 10.05 11.90 13.01 Zomepirac 8.27 9.73 6.52 8.71 10.39 11.17 Probenecid 7.38 4.58 7.42 7.94 9.07 — dmd.aspetjournals.org

in the incubations, with the exception of those incubations with Compound Selection. In this study, migration rates of 23 carboxylic furosemide, dabigatran, and levofloxacin. For furosemide only the 1-O-b- acid–containing drugs covering a broad chemical space and different acyl glucuronide was detected, whereas for dabigatran and levofloxacin indications were evaluated. no acyl glucuronide was detected in incubation samples. Half-life data of 1-O-b-acyl glucuronide in phosphate buffer at pH

The migration rate and glucuronidation rate of the different car- 7.4 of tested compounds were collected from the literature (Fenselau, at ASPET Journals on September 26, 2021 boxylic acid drugs were calculated with eqs. 1 and 2, respectively, under 1994; Williams and Dickinson, 1994; Ebner et al., 2010; Sawamura the assumption that all isomeric acyl glucuronides and corresponding et al., 2010) and ranged from 0.3 to 79.0 hours. Detailed information on aglycone have similar ionization efficiency on the MS (Table 2). tested drugs is listed in Tables 1, 2, and 3.

TABLE 2 Migration and glucuronidation rate of carboxylic acid–containing drugs Samples were prepared in triplicate; the values represent mean 6 S.D. (n = 3).

Test Compound Migration Rate Glucuronidation Rate Half-Life of 1-O-b-AGa

% % hours Valproic acid 0.3 6 0.1 11.4 6 0.4 79.0 (Fenselau, 1994) Gemfibrozil 0.5 6 0.2 42.3 6 4.8 71.4 (Sawamura et al., 2010) Telmisartan 0.3 6 0.0 0.5 6 0.2 45.6 (Sawamura et al., 2010) Montelukast 6.8 6 0.5 0.3 6 0.1 37.5 (Sawamura et al., 2010) Meclofenamate 3.5 6 0.6 29.4 6 1.6 28.1 (Sawamura et al., 2010) Mefenamic acid 5.6 6 0.4 48.6 6 3.8 17.0 (Sawamura et al., 2010) Levofloxacin NA 0.0 6 0.0 16.1 (Sawamura et al., 2010) Repaglinide 3.3 6 0.1 8.7 6 0.2 11.5 (Sawamura et al., 2010) Clofibric acid 13.1 6 1.2 1.8 6 0.9 7.3 (Fenselau, 1994) Flufenamic acid 10.6 6 0.6 9.7 6 1.6 7.2 (Sawamura et al., 2010) Furosemide 0.0 6 0.0 0.7 6 0.1 3.2 (Sawamura et al., 2010) Ibuprofen 40.3 6 2.2 10.7 6 1.0 2.7 (Sawamura et al., 2010) S-Naproxen 11.7 6 2.1 80.1 6 2.0 2.2 (Sawamura et al., 2010) Indomethacin 9.8 6 0.5 22.0 6 1.6 1.7 (Sawamura et al., 2010) R-Naproxen 27.2 6 3.5 79.2 6 2.1 1.1 (Sawamura et al., 2010) (RS)-Benoxaprofen 42.3 6 0.6 18.7 6 0.5 1.05b (Sawamura et al., 2010) Dabigatran NA 0.0 6 0.0 1.0 (Ebner et al., 2010) Ibufenac 50.9 6 3.1 5.2 6 0.3 0.8 (Sawamura et al., 2010) Diclofenac 98.1 6 0.2 18.5 6 3.8 0.7 (Sawamura et al., 2010) Diflunisal 25.2 6 5.1 0.2 6 0.0 0.67 (Williams and Dickinson, 1994) Tolmetin 54.9 6 0.8 0.5 6 0.0 0.4 (Sawamura et al., 2010) Zomepirac 61.9 6 0.2 0.9 6 0.0 0.4 (Sawamura et al., 2010) Probenecid 76.3 6 0.4 0.5 6 0.0 0.3 (Sawamura et al., 2010)

aData from previous publications. bHalf-life of (RS)-benoxaprofen was calculated as an average of half-lives of the R- and S- isomers. Assessment of Acyl Glucuronide Reactivity 1715

TABLE 3 Indications of selected carboxylic acid–containing drugs

Indication Drug Name Seizure Valproic acid Hyperlipidemia Gemfibrozil, clofibric acid Hypertension Telmisartan, furosemide Asthma Montelukast Inflammation Meclofenamate, mefenamic acid, flufenamic acid, ibuprofen, S-naproxen, indomethacin, R-naproxen, benoxaprofen, ibufenac, diclofenac, diflunisal, tolmetin, zomepirac Diabetes Repaglinide Hyperuricemia Probenecid Infection Levofloxacin Stroke Dabigatran

Migration Rate and Stability of 1-O-b-Acyl Glucuronides. The objective of this study was to evaluate the correlation between the Downloaded from migration rates of primary acyl glucuronide in carboxylic acid– containing drugs to the gold-standard half-life method of the 1-O- b-acyl glucuronides reference standard in phosphate buffer (Sawamura et al., 2010). The half-lives obtained from buffer with HSA (Castillo and Smith, 1995; Bolze et al., 2002) or buffer with fresh human plasma

(Castillo and Smith, 1995; Chen et al., 2007) can also be used to dmd.aspetjournals.org categorize the acyl glucuronide reactivity of carboxylic acid–containing at ASPET Journals on September 26, 2021

Fig. 4. Correlation between reciprocals of migration rate normalized with glucuronidation rate to half-lives of 1-O-b-acyl glucuronides. (A) Regression curve in linear scale. (B) Regression curve in logarithmic scale; origin was set at (–1.0, –2.5).

drugs. However, it was shown (Sawamura et al., 2010) that the use of either HSA or fresh human plasma may overestimate the reactivity of 1-O-b-acyl glucuronide. Twenty-three carboxylic acid–containing drugs were analyzed and the corresponding migration rates were determined as described above. Furosemide showed a nonmeasureable migration rate (0%) because of its acyl glucuronide metabolite’s low signal intensity in MS analysis. With respect to levofloxacin and dabigatran, the migration rate could not be estimated mainly owing to the low glucuronidation of levofloxacin (Jinno et al., 2014) and potential low UDP-glucuronosyltransferase (UGT) activity to dabigatran. Accordingly, these three compounds were not included in the correlation analysis. Figure 3 shows that a good correlation (R2 = 0.727) was obtained with reciprocals of migration rate plotted versus half-lives of the 1-O- b-acyl glucuronide for the remaining 20 carboxylic acid–containing drugs; the correlation improved (R2 = 0.831) when data were converted into log-log units. Discussion FDA 2008 Guidance for Industry, Safety Testing of Drug Metabo- Fig. 3. Correlation between reciprocals of migration rate to half-lives of 1-O-b-acyl glucuronides. (A) Regression curve in linear scale. (B) Regression curve in lites for small-molecule metabolite safety testing (Food and Drug logarithmic scale; origin was set at (–0.6, 0). Administration, 2008) stated specifically that additional reactivity 1716 Zhong et al. evaluation of acyl glucuronide formed in human would be needed. The the basis of this cut-off value, half-lives of the 1-O-b-acyl glucuronide half-life approach is currently considered as the gold standard for acyl of safe drugs were larger than 7.2 hours (flufenamic acid) and that of glucuronide reactivity assessment (Benet et al., 1993; Sawamura et al., “warning” or “withdrawn” drugs were shorter than 3.2 hours (furosemide) 2010). Although this assay is robust and relatively easy to conduct, its (Table 2). Likewise, with some exceptions, the migration rate of the 1-O- application at early candidate selection stage is rather challenging as the b-acyl glucuronide of safe carboxylic acid compounds should be lower method requires the synthesis of the 1-O-b-acyl glucuronide authentic than 10%, whereas that of potentially unsafe compounds might be greater standard. Thus, the objective of this study was to develop a fast, robust than 20% on the basis of our results (Table 2). With respect to the and predictive screening method to rank and flag compounds on the migration rates of the 1-O-b-acyl glucuronide in carboxylic acid drugs basis of their acyl glucuronide reactivity without synthesizing the 1-O- that were tested in the range of 10 to 20%, additional consideration b-acyl glucuronide authentic standard. should be made for the reactivity assessment. This work demonstrated that the migration rate assay can nicely The methods used to investigate the acyl glucuronide stability predict the buffer stability of 1-O-b-acyl glucuronides in carboxylic acid– typically assess the half-life of an acyl glucuronide in buffer (Ebner containing drugs. Figure 3 shows that a good correlation (R2 =0.831) et al., 2010), buffer with human serum albumin (Benet et al., 1993), was obtained with reciprocals of migration rate versus half-lives of 1-O- or plasma (Sawamura et al., 2010), which require the authentic b-acyl glucuronides for the 20 carboxylic acid–containing drugs tested. standard of the 1-O-b-acyl glucuronide. However, for compound An in-depth analysis of the plot reveals that the correlations were be- optimization early in drug discovery, rapid methods are needed that yond the 95% confidence interval of regression curve for meclofenamate do not depend on authentic standards. Additionally, methods have Downloaded from and montelukast, and that reciprocals of migration rate of these two been documented in which the acyl glucuronide metabolite was first drugs underestimated the reported corresponding half-lives. It has been biosynthesized in vitro, and subsequently 1) incubated with human reported that the acyl migration played a more important role in serum albumin (Bolze et al., 2002), or with a small peptide (Wang reactivity compared with the hydrolysis of acyl glucuronide to the et al., 2004) to monitor covalent adducts; 2) incubated with plasma aglycone (Baba and Yoshioka, 2009; Di Meo et al., 2013). Both re- (Chen et al., 2007) or buffer (Jinno et al., 2013) to assess the half-life actions were comprehensively monitored by the half-life approach, of the acyl glucuronide. Our simpler approach relying on the acyl whereas the migration process was the only reaction evaluated in the glucuronide migration rate to evaluate reactivity of 1-O-b-acyl dmd.aspetjournals.org acyl migration method. This discrimination may lead to underestima- glucuronide can be used to support the selection of low-risk drug tion of half-life values of some drugs for which the hydrolysis of 1-O- candidates in the drug discovery phase. b-acyl glucuronide to aglycone is the dominant pathway. Therefore the application of the acyl migration method to assess reactivity of those Acknowledgments compounds for ranking and selection may provide misestimated results. The authors thank Jon K. Bodnar for language editing and David Moore for However, the acyl migration method was still applicable to a majority of helpful discussions. drugs that have acyl glucuronide issues, as it has been reported that acyl at ASPET Journals on September 26, 2021 migration was identified as the predominant reaction occurring in vitro Authorship Contributions (Walker et al., 2007). More importantly, the acyl migration method can Participated in research design: Zhong, Lu, Ottaviani, Jones, Schadt. provide preliminary reactivity information in a rapid screening assay Conducted experiments: Zhong. that can be used to support the selection of low-risk drug candidates in Performed data analysis: Zhong, Lu, Ottaviani. the drug discovery phase that do not depend on authentic standards. Wrote or contributed to the writing of the manuscript: Zhong, Lu, Ottaviani, Because of the metabolic properties or potentially poor UGT enzyme Jones, Schadt. activity in pooled human liver microsome, trace or no acyl glucuronide metabolite was detected for some drugs. This could also lead to References underestimation of migration rate. Baba A and Yoshioka T (2009) Structure-activity relationships for the degradation reaction of We also evaluated whether there is benefit in factoring in compound- 1-b-O-acyl glucuronides. Part 3: Electronic and steric descriptors predicting the reactivity of b aralkyl carboxylic acid 1-b-O-acyl glucuronides. Chem Res Toxicol 22:1998–2008. specific determinants affecting the 1-O- -acyl glucuronide formation Bailey MJ and Dickinson RG (2003) Acyl glucuronide reactivity in perspective: biological (e.g., UGT enzyme binding rate, test compound concentration, UGT consequences. Chem Biol Interact 145:117–137. Benet LZ, Spahn-Langguth H, Iwakawa S, Volland C, Mizuma T, Mayer S, Mutschler E, and Lin isoform activity, etc.). Therefore, the migration rate data for each ET (1993) Predictability of the covalent binding of acidic drugs in man. Life Sci 53: compound was also normalized by the respective glucuronidation rate PL141–PL146. Boelsterli UA (2002) Xenobiotic acyl glucuronides and acyl CoA thioesters as protein-reactive calculated using eq. 2 (Fig. 4). However, the correlation of normalized metabolites with the potential to cause idiosyncratic drug reactions. Curr Drug Metab 3: 2 migration rates versus half-lives (R = 0.543 in linear scale) showed no 439–450. improvement when compared with the migration rates calculated using Bolze S, Bromet N, Gay-Feutry C, Massiere F, Boulieu R, and Hulot T (2002) Development of an 2 in vitro screening model for the biosynthesis of acyl glucuronide metabolites and the assess- eq. 1 (R = 0.727 in linear scale). The possible explanations of this ment of their reactivity toward human serum albumin. Drug Metab Dispos 30:404–413. observation could be: 1) Non-cytochrome P450–dependent drug me- Castillo M and Smith PC (1995) Disposition and reactivity of ibuprofen and ibufenac acyl glucuronides in vivo in the rhesus monkey and in vitro with human serum albumin. 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