Benzyl [11C]Hippurate as an Agent for Measuring the Activities of

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Benzyl [ C]Hippurate as an Agent for Measuring the Activities of Organic Anion Transporter 3 in the Brain and Multidrug Resistance-Associated Protein 4 in the Heart of Mice Tatsuya Kikuchi, Toshimitsu Okamura, Maki Okada, Masanao Ogawa, Chie Suzuki, Hidekatsu Wakizaka, Joji Yui, Toshimitsu Fukumura, Antony D Gee, and Ming-Rong Zhang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00454 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on June 4, 2016

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Journal of Medicinal Chemistry

Benzyl [11C]Hippurate as an Agent for Measuring the Activities of Organic Anion Transporter 3 in the Brain and Multidrug Resistance-Associated Protein 4 in the Heart of Mice

Tatsuya Kikuchi†,‡,*, Toshimitsu Okamura†, Maki Okada†, Masanao Ogawa†,§, Chie Suzuki†,ǁ, Hidekatsu Wakizaka†, Joji Yui†, Toshimitsu Fukumura†, Antony D. Gee‡ and Ming-Rong Zhang†

†

Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa,

Inage-ku, Chiba 263-8555, Japan ‡

Division of Imaging Sciences and Biomedical Engineering, King’s College London, 4th

Floor Lambeth Wing, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, United Kingdom §

SHI Accelerater Service Co. Ltd., 5-9-1 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686,

Japan ǁ

Preeminent Medical Photonics Education Research Center, Hamamatsu University School

of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan

ACS Paragon Plus Environment


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*Corresponding author: Tatsuya Kikuchi, Molecular Probe Program, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan Tel: +81-43-206-3191; Fax: +81-43-206-3261; E-mail: [email protected]


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Abstract Multidrug resistance-associated protein 4 (MRP4) and organic anion transporter 3 (OAT3) mediate the efflux of organic anions from the brain and heart. In this study, we have developed a probe for estimating the activity of these transporters in these tissues using positron emission tomography. Several

11

C-labeled hippuric acid ester derivatives were

screened with the expectation that they would be hydrolyzed in situ to form the corresponding 11C-labeled organic acids in target tissues. Among the compounds screened, benzyl [11C]hippurate showed favorable hydrolysis rate and uptake properties in the target tissues of mice. Subsequent evaluation using transporter knockout mice revealed that radioactivity was retained in the brain and heart of Oat3–/– and Mrp4–/– mice, respectively, compared with control mice after the intravenous administration of benzyl [11C]hippurate. Benzyl [11C]hippurate could therefore be used as a probe for estimating the activities of OAT3 and MRP4 in mouse brain and heart, respectively.



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INTRODUCTION Membrane transporters involved in the excretion of endogenous substances and xenobiotics from cells and tissues play important roles in drug kinetics and homeostasis. Given that changes in the activities of efflux transporters can cause diseases, enhance the side-effects of certain drugs and lead to drug resistance,1,2 methods for the in vivo assessment of the activities of efflux transporters are highly desired. Radiolabeled probes for positron emission tomography (PET) and single photon emission computed tomography (SPECT) have therefore been developed to assess the activities of several efflux transporters in the tissues including brain, tumor, lung and liver.3–7 Among the many different efflux transporters, research in our group has focused on organic anion transporters, including multidrug resistance-associated protein 4 (MRP4, ABCC4) and organic anion transporter 3 (OAT3, SLC22A8). MRP4, which is an ATP-binding cassette transporter, and OAT3, which is a solute carrier, are both expressed at the blood– brain barrier, where they prevent small molecule therapeutic agents accumulating in the brain.8,9 MRP4 is also considered to play an important role in homeostasis, because it is involved in the transportation of several important endogenous substances, including cAMP, cGMP, ADP, prostaglandins and leukotrienes.10 It has also been suggested that MRP4


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regulates cAMP levels in cardiac myocytes by extruding cAMP.11 The development of a small molecule probe capable of measuring the in vivo activities of MRP4 and OAT3 in the brain and heart would therefore be valuable for investigation of the kinetic properties of therapeutic agents. These probes could also be used to develop a deeper understanding of the relationship between these transporters and specific diseases in these organs. Kuwahara and co-workers recently developed a method capable of altering the expression of specific proteins in brain capillary endothelial cells.12 In this particular study, OAT3 was used as a marker to evaluate the feasibility of their newly developed method, and a probe capable of measuring the in vivo activity of OAT3 in the brain would therefore be useful for evaluating the effectiveness of this method. The activities of P-glycoprotein and breast cancer resistance protein have been assessed by PET using a radiolabeled lipophilic substrates,3 and the results revealed that reduced transporter activity led to an increase in the accumulation of radioactivity in the target tissues. However, this strategy would be unsuitable for assessing the activities of MRP4 and OAT3, which typically transport hydrophilic substances, because the membrane permeability of the hydrophilic substrates of MRP4 and OAT3 would be limited. This would result in the accumulation of very low levels of radioactivity in the target tissues,



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even in absence of the efflux transporters. With this in mind, we adopted the so-called metabolite extrusion method (MEM)13 as an alternative approach to design a PET probe for measuring the activities of MRP4 and OAT3. According to the MEM, a PET probe is metabolized to a specific radioactive substrate for the target transporter in a tissue of interest. The activity of the transporter can then be estimated by measuring the efflux rate of radioactivity only after the radiometabolite has been observed in the target tissue. Figure 1 shows a kinetic model based on the MEM for estimating the activities of MRP4 and OAT3 using radiolabeled hippuric acid ester. Hippuric acid was selected as a lead candidate because it is considered to be a substrate of MRP4 and OAT3.14,15 It was also envisaged that hippuric acid esters would form hippuric acid in tissue by hydrolysis. Any hippuric acid formed in the blood would be hindered from reaching the target tissue and be cleared rapidly from the blood. Furthermore, it was envisaged that this molecule could be easily labeled or derivatized with a radionuclide for PET/SPECT such as 11C, 18F or 123I. The development of a new probe for estimating the in vivo activity of efflux transporters using the MEM would require the probe to exhibit efficient metabolism and uptake in the tissues of interest to generate observable levels of a labeled substrate for target transporter engagement. Herein, we describe the design, synthesis and evaluation of eight hippuric acid


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esters and their derivatives, which were all labeled with

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C (1a–4b, Scheme 1). These

compounds were designed with the aim of identifying a promising candidate with appropriate properties in terms of its hydrolysis rate and uptake in the tissues of interest. The most promising of these candidate compounds, which showed an appropriate hydrolysis rate and suitable levels of uptake in the brain and heart tissues of control mice, was also evaluated in transporter knockout mice (i.e., Mrp4–/– and Oat3–/– mice).

RESULTS Preparation of 1a–4b. Hippuric acid esters 1a and 1b were synthesized by the N-acylation of corresponding glycine esters with [carbonyl-11C]benzoyl chloride (Scheme 1). The hippuric acid ester derivatives 2a–4b were synthesized by tetrabutylammonium fluoride-promoted N- or O-methylation of the corresponding precursors with [11C]methyl iodide. All of the 11

C-labeled compounds were synthesized in sufficient yields (5–20%, based on [11C]carbon

dioxide, uncorrected for decay), thereby avoiding the necessity for the optimization of the reaction conditions. The specific activities of 1a and 1b at the end of the synthesis (around 30 GBq/µmol) were high enough for the purposes of this study but lower than those of 2a–



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4b (>70 GBq/µmol). The radiochemical purities of the products prepared as injectable solutions were greater than 98% in all cases. It is noteworthy that the injectable solutions were prepared in the presence of ascorbic acid to avoid the decomposition of the radiolabeled product by radiolysis during the evaporation of the HPLC solvent. Indeed, radiolysis was observed during a preliminary study in the absence of ascorbic acid. [amide-11C]Hippuric acid ([11C]hippuric acid), which could be formed in the blood by the hydrolysis of 1b following its intravenous injection, was also synthesized to confirm its limited uptake in the brain and heart. [11C]Hippuric acid was synthesized in good yield (8±2%, based on [11C]carbon dioxide, uncorrected for decay) by the addition of an aqueous alkaline solution of glycine to neat [carbonyl-11C]benzoyl chloride (Scheme 1). It is noteworthy that the addition of a THF solution of [carbonyl-11C]benzoyl chloride to an aqueous alkaline solution of glycine resulted in the precipitation of the starting materials, which led to a low radiochemical yield of [11C]hippuric acid (~2%, based on [11C]carbon dioxide, uncorrected for decay). Hydrolysis rates of 1a–4b in mice tissues. The use of MEM to measure the activity of an efflux transporter requires a radiolabeled probe that can be rapidly converted into a single radiometabolite that is a specific substrate


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for the target transporter. With this in mind, we measured the hydrolysis rates of the candidate compounds in the cerebrum, heart and whole blood of control mice (n=3) as an initial screening exercise (Figures 2 and 3). The results revealed that the ester bonds of these compounds were hydrolyzed in a first-order manner (r2>0.97) to form the corresponding [11C]hippuric acid metabolites, with no other metabolites being detected (Figure 2). The coefficients of variation for the hydrolysis rates estimated by regression analysis were less than 11%. Furthermore, the spontaneous hydrolysis of the compounds in phosphate buffer (0.1 M, pH 7.4, 37 °C) was very slow (

Benzyl [11C]Hippurate as an Agent for Measuring the Activities of

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