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. Author manuscript; available in PMC: 2019 Jun 1.
Published in final edited form as: Biopharm Drug Dispos. 2018 Jun;39(6):315–318. doi: 10.1002/bdd.2135

CBR1 rs9024 genotype status impacts the bioactivation of loxoprofen in human liver a

Adolfo Quiñones Lombraña 1,†, Nasi Li 2,†, Virginia del Solar 2,†, G Ekin Atilla-Gokcumen 2, Javier G Blanco 1,*
PMCID: PMC6078805  NIHMSID: NIHMS972328  PMID: 29851133

Abstract

Loxoprofen is an anti-inflammatory drug that requires bioactivation into the trans-OH metabolite to exert pharmacological activity. Evidence suggests that carbonyl reductase 1 (CBR1) is important during the bioactivation of loxoprofen. Here, we examined the impact of the functional single nucleotide polymorphism CBR1 rs9024 on the bioactivation of loxoprofen in a collection of human liver samples. The synthesis ratios of trans-OH loxoprofen/cis-OH loxoprofen were 33% higher in liver cytosols from donors homozygous for the CBR1 rs9024 G allele in comparison to the ratios in samples from donors with heterozygous GA genotypes. Complementary studies examined the impact of CBR1 rs9024 on the bioactivation of loxoprofen in lymphoblastoid cell lines. CBR1 rs9024 genotype status impacts the synthesis of the bioactive trans-OH metabolite of loxoprofen in human liver.

Keywords: Loxoprofen, CBR1, CBR1 rs9024

1. INTRODUCTION

Loxoprofen is a nonsteroidal anti-inflammatory drug with analgesic and anti-pyretic properties (Naruto, Tanaka, Hayashi, & Terada, 1984; Tanaka, Nishikawa, & Hayashi, 1983; Tanaka, Nishikawa, Matsuda, Yamazaki, & Hayashi, 1984). Loxoprofen was initially marketed as an oral drug, and a topical formulation is also available and widely used in Japan since 2006 (Loxonin® gel 1%, Daiichi Sankyo Co., Ltd, Tokyo, Japan) (Sawamura, Kazui, Kurihara, & Izumi, 2015; Sawamura et al., 2015). Loxoprofen is a prodrug that undergoes enzymatic reduction into the pharmacologically active trans-OH metabolite. The trans-OH metabolite of loxoprofen, not the parent drug or the cis-OH isomer, is a strong and non-selective inhibitor of pro-inflammatory cyclooxygenase 2 (COX-2) (Riendeau et al., 2004; Sugimoto, Kojima, Asami, Iizuka, & Matsuda, 1991). An early report by Ohara et al. noted that the bioactivation of loxoprofen is catalyzed by hepatic carbonyl reductases (CBRs) (Ohara, Miyabe, Deyashiki, Matsuura, & Hara, 1995). More recently, Sawamura et al. showed that carbonyl reductase 1 (CBR1) is the predominant enzyme involved in the formation of the trans-OH metabolite in human skin. The immunodepletion of CBR1 in skin homogenates decreased the reduction of loxoprofen to its trans-OH form by more than 80% (Sawamura, Sakurai, et al., 2015).

CBR1 is a cytosolic NADPH dependent enzyme that catalyzes the reduction of endogenous and exogenous carbonyl substrates (Malatkova, Maser, & Wsol, 2010; Shi & Di, 2017). Evidence suggests that genetic polymorphisms in CBR1 impact the metabolism of carbonyl substrates (Shi & Di, 2017). A functional single nucleotide polymorphism (SNP) in the 3′-untranslated region of CBR1 (rs9024, G>A) impacts CBR1 expression in liver and myocardium through mechanisms involving the binding of specific microRNAs to the polymorphic site (Kalabus et al., 2012; Gonzalez-Covarrubias et al., 2009, Quiñones-Lombraña et al., 2014). Liver cytosols with CBR1 rs9024 homozygous GG genotypes showed higher maximal rates of doxorubicinol synthesis (1.5-fold) compared with samples with heterozygous GA genotypes (Gonzalez-Covarrubias et al., 2009). The variant CBR1 rs9024 allele exhibits various frequencies in DNA variation panels (https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=9024). To date it is not known whether CBR1 rs9024 impacts the bioactivation of loxoprofen in liver. The aim of this study was to investigate whether CBR1 rs9024 genotype status impacts the metabolism of loxoprofen in a collection of human liver samples.

2. MATERIAL AND METHODS

2.1. Chemicals

β-NADPH tetrasodium salt hydrate, loxoprofen sodium, and loxoprofen reduced stable isotopes (cis-OH form of loxoprofen-d3 and trans-OH form of loxoprofen-d3) were purchased from Toronto Research Chemicals (Toronto, Canada). The structures of loxoprofen and its trans-OH and cis-OH metabolites are shown in Figure 1a. Loxoprofen and NADPH stock solutions were prepared in 0.1 M potassium phosphate buffer (pH 7.4).

FIGURE 1.

FIGURE 1

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(a) Ion chromatograms of loxoprofen, trans-OH form of loxoprofen, and cis-OH form of loxoprofen in human liver. (b) CBR1 mRNA expression in liver samples (n = 24). (c) CBR1 protein content in liver samples (n = 24). Inset shows a representative immunoblot for cytosolic CBR1, GAPDH, and the recombinant CBR1 standards. (d) Impact of CBR1 rs9024 genotype status on loxoprofen reductase activity in liver tissue (n = 24), and (e) lymphoblastoid cell lines (n = 9). Each symbol represents the average of individual samples analyzed in triplicates. Horizontal lines indicate group means.

2.2. Human Liver Samples

The Institutional Review Board of the State University of New York at Buffalo, NY, approved this research. Human liver tissues (n = 24) were processed at St. Jude Children’s Research Hospital (Memphis, TN) and were provided by the Liver Tissue Procurement and Distribution System (National Institutes of Health Contract N01-DK-9-2310) and by the Cooperative Human Tissue Network (http://chtn.nci.nih.gov/). Liver samples were processed following standardized procedures to isolate DNA, RNA, and protein. CBR1 mRNA expression and CBR1 rs9024 genotype status of selected liver samples were reported by us (Gonzalez-Covarrubias, Zhang, Kalabus, Relling, & Blanco, 2009). Hepatic CBR1 protein expression was determined by quantitative immunoblotting with a specific anti-CBR1 antibody and recombinant CBR1 protein standards as previously described (Gonzalez-Covarrubias, et al., 2009).

2.3. Human Lymphoblastoid Cell Lines

Lymphoblastoid cell lines (GM10853, GM10845, GM10857, GM10858, GM10860, GM17240, GM16654, GM16688, and GM16689) derived from donors with European (N = 6) or Chinese (N = 3) ancestries were purchased from the Coriell Institute for Medical Research (Camden, NJ). Cultures were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA). Cultures were grown using standard incubation conditions at 37°C, 5% CO2, and 95% relative humidity.

2.4. Quantification of trans- and cis-OH forms of loxoprofen

Cytosolic protein extraction was performed with NE-PER Nuclear and Cytoplasmic Extraction Reagents kits (Thermo Fisher Scientific). Validation experiments with liver and lymphoblastoid cell cytosols were performed to determine the oxidation rate of NADPH using a method similar to that reported previously (Gonzalez-Covarrubias, et al., 2009). Our results showed that 150 μM loxoprofen [S] ensured conditions of Vmax - i.e., maximal CBR activity, zero-order kinetics -. Incubation mixtures (50 μL) containing potassium phosphate buffer (pH 7.4, 0.1 M), cytosolic protein (15 μg), NADPH (400 μM), and loxoprofen (150 μM) were incubated at 37°C for 4 hours (Koo et al., 2005; Mordente et al., 2003). Reactions were quenched by adding 1.1 mL of methanol and incubated again at 37°C for 30 minutes. Samples were then centrifuged, and 1 mL of supernatant was dried under vacuum and resuspended in 100 μL of methanol. Samples were spiked with the cis-OH and trans-OH forms of loxoprofen-d3 at 1 μM each. The generation of trans-OH and cis-OH forms of loxoprofen was monitored by liquid chromatography–mass spectrometry (LC-MS) in positive ionization mode ([M+NH4]+, m/z = 266.1751). The LC-MS method is summarized in table 1, and a representative chromatogram is shown in Figure 1a. Calibration curves were linear over the 1.00 – 20.00 pmol range for the cis-OH form of loxoprofen-d3 (r2= 0.9998) and the 0.10 – 20.00 pmol range for the trans-OH form of loxoprofen-d3 (r2= 0.9992). The lower limits of quantification (LLOQ) were 0.50 pmol for the trans-OH form and 2.50 pmol for the cis-OH form. Quantifications were carried out in triplicates.

Table 1.

LC-MS method for the simultaneous determination of loxoprofen, trans-OH form of loxoprofen, and cis-OH form of loxoprofen.

LC-MS Method
HPLC Agilent 1260
Mass spectrometer Agilent 6530 Series Accurate-Mass Quadrupole TOF
Mobile Phase A 90:10 water/acetonitrile
Mobile Phase B 10:90 water/acetonitrile
Additives 0.1% (v/v) formic acid and 5 mM ammonium formate
Gradient Time (min) Mobile Phase A (%) Mobile Phase B (%) Flow Rate (mL/min)
0 – 3 100 0 0.1
3 – 20 62.5 37.5 0.5
20 – 27 0 100 0.5
27 – 35 100 0 0.5
Column Gemini C18 reversed-phase column (5 μm, 4.6 mm × 50 mm)
Injection Volume (μL) 10
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2.5. Statistical Analysis

Excel 2007 (Microsoft Office; Microsoft, Redmond, WA) and GraphPad Prism version 4.03 (GraphPad Software Inc., La Jolla, CA) were used for statistical data analysis. The D’Agostino & Pearson test was used to analyze the normality of data sets. The Student’s t test or the Mann Whitney test were used to compare group means.

3. RESULTS AND DISCUSSION

The relative expression of CBR1 mRNA in liver samples from donors with CBR1 rs9024 homozygous GG genotype was significantly higher than in samples from donors with heterozygous GA genotype (CBR1 mRNArs9024GG = 6.7 ± 4.7 vs. CBR1 mRNArs9024GA = 3.4 ± 2.5. Mann Whitney test, p = 0.022. Figure 1b). Livers from donors with CBR1 rs9024 homozygous GG genotype exhibited higher cytosolic CBR1 protein content than samples from donors with heterozygous GA genotype (CBR1 proteinrs9024GG = 9.4 ± 2.1 nmol/g cytosolic protein vs. CBR1 proteinrs9024GA = 7.6 ± 2.5 nmol/g cytosolic protein. Student’s t test, p = 0.030. Figure 1c). On average, the synthesis ratios of trans-OH/cis-OH forms of loxoprofen were 33% higher in liver cytosols from donors homozygous for the CBR1 rs9024 GG allele in comparison to the ratios in samples from donors with heterozygous GA genotypes (CBR1 rs9024 GGmole of trans-OH form/mole of cis-OH form: 4.85 ± 1.49 vs. CBR1 rs9024 GAmole of trans-OH form/mole of cis-OH form: 3.66 ± 1.23. Student’s t test p = 0.044. Figure 1d). Pearson’s correlation analysis showed no linear correlation between CBR1 protein content and trans-OH/cis-OH loxoprofen ratios (rp = 0.258, p = 0.224). The reason for this discrepancy is unclear. It is known that CBR1 is subjected to posttranslational modifications - e.g., S-glutathionylation in cysteine 226 - that impact catalytic activity towards specific substrates (Hartmanova et al., 2013). Thus, studies with more robust sample sizes coupled to protein content analysis of CBR1 isoforms are needed to further define the contribution of CBR1 expression to the synthesis of relevant metabolites such as trans-OH loxoprofen in human liver.

The impact of CBR1 rs9024 on the synthesis of trans-OH and cis-OH forms of loxoprofen was also investigated in lymphoblastoid cell lines with known CBR1 rs9024 genotype status. None of the lymphoblastoid cell lines synthetized quantifiable levels of the cis-OH metabolite (LLOQcis-OH loxoprofen: 2.50 pmol). There was a modest trend towards increased synthesis of trans-OH form in cytosols from cells with CBR1 rs9024 GG genotype in comparison to cells with heterozygous GA and homozygous A genotypes, respectively (CBR1 rs9024 GG: 1.62 ± 0.06 pmol of trans-OH form of loxoprofen/μg of cytosolic protein; CBR1 rs9024 GA: 1.33 ± 0.22 pmol of trans-OH form of loxoprofen/μg of cytosolic protein; CBR1 rs9024 AA: 1.41 ± 0.39 pmol of trans-OH form of loxoprofen/μg of cytosolic protein. Student’s t test, GG vs. GA, p ~ 0.096, and GG vs. AA, p ~ 0.405. Figure 1e).

This study is limited by the lack of samples from individuals homozygous for the CBR1 rs9024 A allele. While the frequency of the CBR1 rs9024 A allele is relatively low in individuals of European and African ancestries (e.g., q = 0.087 in Europeans, and q = 0.006 in Africans), the A allele is relatively more common in individuals of Japanese ancestry (q = 0.322. https://www.pharmgkb.org/variant/PA166155972). Our data suggest that CBR1 rs9024 genotype status impacts the hepatic synthesis of bioactive trans-OH form of loxoprofen in a manner consistent with the magnitude and direction of previously documented CBR1 genotype-phenotype associations for the carbonyl substrates menadione and daunorubicin (Gonzalez-Covarrubias, et al., 2009; Quinones-Lombrana et al., 2014). Data from the pilot study in lymphoblastoid cells suggest that CBR1 rs9024 genotype status may impact the bioactivation of loxoprofen in blood cells. Koo et al. characterized the pharmacokinetics of loxoprofen in rats after intragastric, intravenous, and intramuscular administration (Koo, et al., 2005). The authors concluded that the extrahepatic metabolism of loxoprofen by blood cells could be of pharmacological relevance irrespective of the route of administration (Koo, et al., 2005; Sawamura, Sakurai, et al., 2015). This study provides a foundation to investigate the contribution of CBR1 rs9024 to the pharmacokinetics and pharmacodynamics of loxoprofen in humans.

Footnotes

a

This study was supported by the National Institute of General Medical Sciences (award GM073646).

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest.

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