Original Paper
Design of a novel oral fluoropyrimidine carbamate, capecitabine, which generates 5-fluorouracil selectively in tumours by enzymes concentrated in human liver and cancer tissue

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Abstract

Capecitabine (N4-pentyloxycarbonyl-5′-deoxy-5-fluorocytidine) is a novel oral fluoropyrimidine carbamate, which is converted to 5-fluorouracil (5-FU) selectively in tumours through a cascade of three enzymes. The present study investigated tissue localisation of the three enzymes in humans, which was helpful for us to design the compound. Carboxylesterase was almost exclusively located in the liver and hepatoma, but not in other tumours and normal tissue adjacent to the tumours. Cytidine (Cyd) deaminase was located in high concentrations in the liver and various types of solid tumours. Finally, thymidine phosphorylase (dThdPase) was also more concentrated in various types of tumour tissues than in normal tissues. These unique tissue localisation patterns enabled us to design capecitabine. Oral capecitabine would pass intact through the intestinal tract, but would be converted first by carboxylesterase to 5′-deoxy-5-fluorocytidine (5′-dFCyd) in the liver, then by Cyd deaminase to 5′-deoxy-5-fluorouridine (5′-dFUrd) in the liver and tumour tissues and finally by dThdPase to 5-FU in tumours. In cultures of human cancer cell lines, the highest level of cytotoxicity was shown by 5-FU itself, followed by 5′-dFUrd. Capecitabine and 5′-dFCyd had weak cytotoxic activity only at high concentrations. The cytotoxicity of the intermediate metabolites 5′-dFCyd and 5′-dFUrd was suppressed by inhibitors of Cyd deaminase and dThdPase, respectively, indicating that these metabolites become effective only after their conversion to 5-FU. Capecitabine, which is finally converted to 5-FU by dThdPase in tumours, should be much safer and more effective than 5-FU, and this was indeed the case in the HCT116 human colon cancer and the MX-1 breast cancer xenograft models.

Introduction

Many strategies for selectively delivering anticancer drugs to tumours have been reported. Specifically, prodrug activation by enzymes located in tumour tissues has been discussed. We have previously shown that 5′-deoxy-5-fluorouridine (5′-dFUrd) is not itself cytotoxic but becomes effective only after conversion to the active drug 5-fluorouracil (5-FU) by pyrimidine nucleoside phosphorylase (PyNPase), which is preferentially located in tumour tissues[1]. Since 5′-dFUrd produces higher levels of 5-FU in tumours than in normal counterparts[2], it has been found to be more effective than 5-FU and other fluoropyrimidines in various studies with mouse transplantable tumour models, particularly in terms of therapeutic indices1, 3, 4. 5′-dFUrd is being marketed in Japan, China and Korea (Furtulon®) for the treatment of breast, colorectal, gastric and other cancers, while it is being clinically assessed in the EU.

The major drawback of cancer treatment with 5′-dFUrd, given orally, is its dose-limiting side-effect, diarrhoea[5]. When 5′-dFUrd passes through the intestinal mucosal membrane, 5′-dFUrd is thought to cause the intestinal toxicity of 5-FU generated in the intestine[6]. PyNPase exists predominantly as thymidine phosphorylase (dThdPase) in humans[7], while it is predominantly as uridine phosphorylase in rodents[8]. We, therefore, tried to identify a new fluoropyrimidine which could pass through the intestinal tract, by applying an approach of prodrug activation by additional enzymes. These studies created a novel fluoropyrimidine carbamate, capecitabine, which is sequentially converted to 5′-dFUrd by carboxylesterase and cytidine (Cyd) deaminase with unique tissue localisation in humans and then to 5-FU by dThdPase. In this report, we describe the tissue distribution of these enzymes for capecitabine activation. In addition, we describe that capecitabine and its intermediate metabolites were not themselves cytotoxic but became effective once they were converted to the active drug 5-FU.

Section snippets

Animals and tissues

Male and female BALB/c nu/nu mice were obtained from CLEA Japan Co., Ltd, (Tokyo, Japan). Male BDF1 mice were obtained from SLC Inc. (Hamamatsu, Japan). The mice were observed for at least 1 week and then used when 6 weeks old.

Chemicals

Capecitabine and 6-amino-5-chlorouracil (ACU, an inhibitor of dThdPase) were synthesised by methods described elsewhere9, 10. 5-FU and a combination drug of uracil and tegafur (UFT) were purchased from Kyowa Hakko K.K. and Taiho Pharma Co. (Tokyo, Japan), respectively,

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