Elsevier

Biochemical Pharmacology

Volume 97, Issue 2, 15 September 2015, Pages 215-223
Biochemical Pharmacology

Metabolic mapping of A3 adenosine receptor agonist MRS5980

https://doi.org/10.1016/j.bcp.2015.07.007Get rights and content

ABSTRACT

(1S,2R,3S,4R,5S)-4-(2-((5-Chlorothiophen-2-yl)ethynyl)-6-(methylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-carboxamide (MRS5980) is an A3AR selective agonist containing multiple receptor affinity- and selectivity-enhancing modifications and a therapeutic candidate drug for many inflammatory diseases. Metabolism-related poor pharmacokinetic behavior and toxicities are a major reason for drug R&D failure. Metabolomics with UPLC–MS was employed to profile the metabolism of MRS5980 and MRS5980-induced disruption of endogenous compounds. Recombinant drug-metabolizing enzymes screening experiment were used to determine the enzymes involved in MRS5980 metabolism. Analysis of lipid metabolism-related genes was performed to investigate the reason for MRS5980-induced lipid metabolic disorders. Unsupervised principal components analysis separated the control and MRS5980 treatment groups in feces, urine, and liver samples, but not in bile and serum. The major ions mainly contributing to the separation of feces and urine were oxidized MRS5980, glutathione (GSH) conjugates and cysteine conjugate (degradation product of the GSH conjugates) of MRS5980. The major ions contributing to the group separation of liver samples were phosphatidylcholines. In vitro incubation experiments showed the involvement of CYP3A enzymes in the oxidative metabolism of MRS5980 and direct GSH reactivity of MRS5980. The electrophilic attack by MRS5980 is a minor pathway and did not alter GSH levels in liver or liver histology, and thus may be of minor clinical consequence. Gene expression analysis further showed decreased expression of PC biosynthetic genes choline kinase a and b, which further accelerated conversion of lysophosphatidylcholine to phosphatidylcholines through increasing the expression of lysophosphatidylcholine acyltransferase 3. These data will be useful to guide rational design of drugs targeting A3AR, considering efficacy, metabolic elimination, and electrophilic reactivity.

Introduction

Drug efficiency, toxicity, and metabolic elimination are the major properties determining whether a drug candidate can successfully enter clinical trials [1]. Through combining in vitro incubation mixtures and in vivo animal models, the metabolic pathways of drugs and drug candidates have been elucidated, such as ifosfamide [2], noscapine [3] and aryl hydrocarbon receptor antagonist GNF-351 [4]. Metabolomics can be used to phenotype the alteration of endogenous substances, which can directly reflect the disruption of metabolic pathways by a drug and serve as biomarkers for efficacy and toxicity. For example, serum metabolomics was employed to explain the adverse effects of gemfibrozil through analyzing the alteration of lysophosphatidylcholine and bile acid components in gemfibrozil-treated mice [5].

The A3 adenosine receptor (AR), a G protein-coupled receptor, is a therapeutic target for many diseases (e.g., hepatocellular carcinoma, autoimmune inflammatory diseases, chronic neuropathic pain, etc.), and development of A3AR selective agonists as drug candidates has been a major goal to treat these diseases [6], [7]. (1S,2R,3S,4R,5S)-4-(2-((5-Chlorothiophen-2-yl)ethynyl)-6-(methylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-carboxamide (MRS5980) is a recently developed A3AR selective agonist that contains multiple receptor affinity- and selectivity-enhancing modifications [8]. This nucleoside derivative contains a rigid ribose carbocyclic substitution ([3.1.0]bicyclohexane) that maintains a receptor-preferred conformation. The 2-arylethynyl substituent of sterically constrained nucleoside analogues is predicted to induce an adjustment of the position of the second transmembrane helical domain of the A3AR, which contributes to its lack of affinity at the A2AAR [8]. The present study used metabolomics to evaluate MRS5980 metabolism and interactions, including the metabolic elimination of MRS5980, electrophilic reactivity and influence on lipid profiles of MRS5980 in the body.

Section snippets

Chemicals and reagents

MRS5980 was synthesized as described [8]. β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt (NADPH) and reduced glutathione were purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were of the highest grade commercially available. Livers from untreated 6- to 8-week-old male C57BL/6J mice were used to prepare mouse liver microsomes (MLMs) as previously described [4]. Human liver microsomes (HLMs), recombinant cytochromes P450 (CYP) 1A1, 1A2, 1B1, 2A6, 2B6,

Metabolomics analysis to find biomarkers for exposure to MRS5980

The chromatographic behavior of MRS5980 showed elution at 5.6 min (Fig. 1A), and MRS5980 showed doublet m/z 459.104 and 461.102 ions (Fig. 1B). MS was performed in the positive-ion mode, because compounds containing halogens (especially chlorine and bromine) can produce very distinct isotope peaks [11]. The MS/MS spectrum of MRS5980 (m/z = 459.104) showed the major fragment ions 428 and 290, and the corresponding fragmentation patterns were also given (Fig. 1C). UPLC-ESI-QTOFMS analysis coupled

Discussion

Developing safer drugs and drug candidates has been a major goal of the pharmaceutical industry, and its importance has been intensified by the withdrawal of many drugs (e.g., Ximelagatran, Rofecoxib, Lumiracoxib, etc.) from the market and a decrease of new chemical entities (NCEs) introduced into the market [13]. In preclinical efficacy testing, in the chronic constriction injury (CCI, sciatic nerve) model in mice, the ED50 value of MRS5980 at its peak effect following oral administration was

Conflicts of interest

The authors report no conflict of interest.

Acknowledgements

This study was funded by the Intramural Research Programs of the Center for Cancer Research, National Cancer Institute and the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, the National Natural Science Foundation of China (No. 81202586), and Tianjin Project of Thousand Youth Talents.

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    Present address: College of Pharmaceutical Sciences, Shandong University, Shandong 250012, China.

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