Elsevier

Biochemical Pharmacology

Volume 67, Issue 11, 1 June 2004, Pages 2093-2102
Biochemical Pharmacology

Quaternary ammonium-linked glucuronidation of tamoxifen by human liver microsomes and UDP-glucuronosyltransferase 1A4

https://doi.org/10.1016/j.bcp.2004.02.014Get rights and content

Abstract

Tamoxifen (TAM), a nonsteroidal antiestrogen, is the most widely used drug for chemotherapy of hormone-dependent breast cancer in women. In the present study, we found a new potential metabolic pathway of TAM via N-linked glucuronic acid conjugation for excretion in humans. TAM N+-glucuronide was isolated from a reaction mixture consisting of TAM and human liver microsomes fortified with UDP-glucuronic acid (UDPGA) and identified with a synthetic specimen by high-performance liquid chromatography-electrospray ionization-mass spectrometry. However, no TAM-glucuronidating activity was detected in microsomes from rat, mouse, monkey, dog, and guinea pig livers. A strong correlation (r2=0.92) was observed between N-glucuronidating activities toward TAM and trifluoperazine, a probe substrate for human UDP-glucuronosyltransferase (UGT) 1A4, in human liver microsomes from eight donors (five females, three males). However, no correlation (r2=0.02) was observed in the activities between 7-hydroxy-4-(trifluoromethyl)coumarin and TAM. Only UGT1A4 catalyzed the N-linked glucuronidation of TAM among recombinant UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in insect cells. Apparent Km values for TAM N-glucuronidation by human liver microsomes and recombinant UGT1A4 were 35.8 and 32.4 μM, respectively. These results strongly suggested that UGT1A4 could play a role in metabolism and excretion of TAM without Phase I metabolism in human liver. TAM N+-glucuronide still had binding affinity similar to TAM itself for human estrogen receptors, ERα and ERβ, suggesting that TAM N+-glucuronide might contribute to the biological activity of TAM in vivo.

Introduction

Tamoxifen (TAM, 1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) is a nonsteroidal triphenylethylene antiestrogen that has been widely used for chemotherapy and chemoprevention of breast cancer, one of the major causes of cancer-related death in women [1]. The antiestrogenic activity of TAM is based on competing activity with β-estradiol for estrogen receptors (ERs). However, TAM is not a pure antiestrogen and has agonistic properties in some estrogen target tissues [2]. The paradoxical activity of TAM remains unclear. Also, TAM was shown to have higher binding affinity to one isoform of human ER, ERα, than to another isoform, ERβ [3].

The metabolism and pharmacokinetics of TAM have been extensively studied in female patients and animals. In the human, orally administered TAM is converted to several metabolites, such as N-desmethyl-TAM, 4-hydroxytamoxifen (4-HO-TAM), N-desdimethyl-TAM, 4-hydroxy-N-desmethyl-TAM, TAM N-oxide and the primary alcohol named metabolite Y [4], [5], [6], [7]. Among these Phase I metabolites, 4-HO-TAM has been considered to be an active metabolite of TAM because of its higher affinity toward ERs than the parent drug and other side-chain metabolites [8]. Interestingly, 4-HO-TAM was partially converted in vivo to its geometrical isomer, cis-4-HO-TAM, a much less potent antiestrogen that may have weak estrogenic properties [9].

Our previous study on Phase II metabolism of 4-HO-TAM showed that the geometrical isomers of 4-HO-TAM were selectively glucuronidated in the manner of cistrans by human liver microsomes and sulfated in the manner of transcis by human liver cytosol [10]. We also demonstrated through the use of recombinant enzymes that such sulfation and glucuronidation were catalyzed by human sulfotransferase isoform SULT1A1 and UDP-glucuronosyltransferase (UGT) isoform UGT2B15, respectively [10].

A pharmacokinetic study in humans showed that the major route of TAM excretion was via the feces [11]. In females, more than 60% of the administered radiolabeled TAM was excreted as unchanged drug, with most of the remaining fecal radioactivity due to Phase II conjugated metabolites. Only 9–14% of radiolabeled TAM was eliminated into urine. Lien et al. [12] reported in an extensive study on the distribution of TAM and its metabolites in human biological fluids that bile and urine were rich in hydroxylated, conjugated metabolites (4-HO-TAM, 4-HO-N-desmethyl-TAM, and metabolite Y), whereas unconjugated 4-HO-TAM and unmetabolized TAM were the predominant species in feces. However, treatment of the fecal extract from one patient with β-glucuronidase increased the concentration of TAM and TAM metabolites, indicating the existence of glucuronic acid conjugates [12]. They also noted that significant amounts of “conjugated TAM,” which released TAM by treatment of β-glucuronidase, could be recovered from bile, although they provided no further information on conjugated TAM.

Entero-hepatic circulation of unmetabolized TAM as well as hydroxylated TAM metabolites in patients who were administered TAM was observed [11]. Therefore, we hypothesized a possible excretion pathway of TAM whereby TAM could be excreted into bile via TAM N+-glucuronide. N-Glucuronidation has been known to produce N-linked glucuronide as urinary and biliary metabolites for more than 30 nitrogen-containing drugs and chemicals [13]. In particular, N-glucuronidation has been extensively studied in the metabolism of H1 antihistamine and antidepressant drugs with an aliphatic tertiary amine group, such as chlorpheniramine, diphenhydramine, ketotifen, amitriptyline, and imipramine [14], [15], [16], [17]. Luo et al. [14] reported fecal excretion of cyclizine N+-glucuronide and suggested possible entero-hepatic circulation of the drug via N+-glucuronide metabolites. However, although TAM has an N,N-dimethylaminoalkyl side chain in its structure, there is no information on N-glucuronidation of TAM and TAM metabolites.

In the present study, we investigated whether human liver microsomes and recombinant UGT isoforms were capable of catalyzing N-glucuronidation of TAM to reveal a new potential excretion pathway of the anti-cancer drug. Binding affinity of TAM N+-glucuronide to human estrogen receptors, ERα and ERβ, was also investigated.

Section snippets

Chemicals

Bovine serum albumin (BSA), β-estradiol, eugenol, β-glucuronidase (Type VII-A from Escherichia coli, 100 U/ml), d-saccharic acid 1,4-lactone, TAM and UDP-glucuronic acid (UDPGA) were purchased from Sigma Chemicals Co. 7-Hydroxy-4-(trifluoromethyl)coumarin (HFC) was purchased from Kanto Chemicals Co. Alamethicin, trifluoperazine and [14C]UDPGA (300 mCi/mmol) were purchased from ICN Pharmaceuticals, Inc. Microsomes prepared from insect cells expressing recombinant human UGT1A1, UGT1A3, UGT1A6 and

Chemical synthesis of TAM N+-glucuronide

A simple reaction of TAM with methyl(2,3,4-tri-O-acetyl-α-d-glucopyranosyl bromide)uronate in dichloromethane gave the N-linked glucuronide. NMR spectrum of the glucuronide showed the signal for the anomeric proton on the sugar ring at δ 4.64 ppm as a doublet with a coupling constant of 8.8 Hz. The chemical shift and coupling constant were characteristic of N+-linked β-glucuronides [13], [14], [17]. Separation of N,N-dimethyl proton signals as singlets at δ 3.12 and 3.18 ppm indicated that these

Discussion

The present study provides the first evidence of TAM N-glucuronidation in human liver microsomes in vitro. TAM N+-glucuronide formed in the reaction mixture consisting of human liver microsomes and TAM in the presence of UDPGA was identified with the synthetic specimen by HPLC-ESI-TOF-MS. Substrate specificity of nine isoforms of human hepatic UGTs indicated that N-glucuronidation in human liver microsomes was catalyzed only by UGT1A4. Also revealed was that the activity of TAM N

Acknowledgements

We gratefully acknowledge Dr. Yasuo Shida and Dr. Chiseko Sakuma of the Analysis Center, Tokyo University of Pharmacy and Life Science, for expert technical assistance with mass and NMR spectrometry.

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