Elsevier

Journal of Hepatology

Volume 34, Issue 6, June 2001, Pages 865-872
Journal of Hepatology

Identification of cyclosporine A and tacrolimus glucuronidation in human liver and the gastrointestinal tract by a differentially expressed UDP-glucuronosyltransferase: UGT2B7

https://doi.org/10.1016/S0168-8278(01)00040-XGet rights and content

Abstract

Background/Aims: The oral administration of the major transplant immunosuppressants cyclosporine A and tacrolimus leads to unpredictable drug levels requiring drug monitoring. Hepatic and extrahepatic metabolism of cyclosporine A and tacrolimus by cytochrome P450 proteins has been analyzed but metabolism and inactivation by glucuronidation has not been investigated.

Methods: Cyclosporine A and tacrolimus glucuronidation was measured in hepatic and gastrointestinal microsomal protein, and with 11 recombinant hepatic and extrahepatic family 1 and 2 UDP-glucuronosyltransferases. UDP-glucuronosyltransferase transcripts were determined by polymerase chain reaction.

Results: Significant cyclosporine and tacrolimus glucuronidation activity was present in endoplasmic reticulum from liver, duodenum, jejunum, ileum and colon, but was absent in stomach. Specific cyclosporine A glucuronidation activity was highest in liver and colon, tacrolimus glucuronidation was highest in liver. Analyses using recombinant UDP-glucuronosyltransferases identified UGT2B7 as a human UDP-glucuronosyltransferase with specific activity toward cyclosporine A and tacrolimus. The hepato-gastrointestinal distribution of immunosuppressant glucuronidation activity corresponded to the differential expression pattern of UGT2B7 mRNA.

Conclusions: This study provides conclusive evidence of hepatic and extrahepatic immunosuppressant glucuronidation by human UGT2B7 which was identified to be differentially expressed in the human hepatogastrointestinal tract. Hepatic and extrahepatic glucuronidation may influence the therapeutic efficacy of transplant immunosuppressants.

Introduction

The allogenic transplantation of organs is dependent upon immunosuppression to prevent allograft rejection [1]. The clinical introduction of cyclosporine A (CsA) and tacrolimus (FK506) has led to a significant increase of graft survival and a decrease of morbidity. Both compounds are water insoluble fungal metabolites. CsA is a lipophylic cyclic peptide of 11 residues produced by Tolypocladium inflatum[2], and FK506 is a macrolide lactone derived from Streptomyces tsukubaensis[3]. Although chemically unrelated, both share a remarkably similar mode of action on calcium dependent signal transduction leading to T-cell inhibition [4], [5].

Cyclosporine A and FK506 are usually administered orally and are characterized by an unpredictably variable absorption rate which requires monitoring of drug levels [6]. Extensive metabolism by hepatic cytochrome P450s (CYP) has been demonstrated for both drugs [7], [8], [9]. Extrahepatic CsA metabolism by CYP in human intestine has been demonstrated [10], [11]. The identification of CsA [8] and FK506 [12] glucuronides demonstrates that conjugation also influences the metabolism of these immunosuppressants which has not been analyzed in humans.

UDP-glucuronosyltransferases (UGT) are a superfamily of proteins localized in the endoplasmic reticulum [13]. The UGTs glucuronidate hydrophobic compounds rendering them more water soluble and facilitating subsequent elimination in bile and urine [14]. UGTs are divided into two families, UGT1 and UGT2 [13]. The UGT2 genes are encoded on chromosome 4 [15]. From liver, four UGT2 cDNAs have been cloned and characterized, UGT2B4 [16], UGT2B7 [17], UGT2B10 [18], and UGT2B15 [19], [20]. UGT2B4 is not expressed in small intestine [21], [22]. In contrast, UGT1Agenes are encoded on chromosome 2. A linear array of 12 variable exon-1 cassettes at the 5′ end of the UGT1A locus are followed by 4 common exons (exon 2–5) [23], [24]. Individual UGT1A gene products are generated by a strategy of exon sharing which combines unique exon 1 sequences with the common exons 2–5. This process potentially leads to the formation of nine functional UGT transcripts [23].

UGT1A gene products are expressed in a tissue-specific fashion [25], [26]. The analysis of liver tissue has led to the characterization of UGT1A1 [27], UGT1A3 [28], UGT1A4 [27], UGT1A6 [29], and UGT1A9 RNA [30]. An analysis of the human gastrointestinal tract has identified 3 extrahepatic UGT1A transcripts: UGT1A7, UGT1A8 and UGT1A10 [22], [25], [26], [33], [34]. Tissue-specific regulation of the UGT1A genes represents a biochemical basis for tissue-specific glucuronidation in the human digestive system [25], [35].

In the present study, we examined the glucuronidation of CsA and FK506 using human hepatic, and extrahepatic microsomal protein in addition to 11 recombinant UGT proteins to identify the anatomical site and responsible UGTs of immunosuppressant glucuronidation. Extrahepatic human tissues of the gastrointestinal tract were analyzed to test the hypothesis of mucosal inactivation of CsA and FK506 as a contributor to prehepatic metabolism.

Section snippets

Gastrointestinal tissue samples

Tissue samples were obtained from the Department of Abdominal and Transplant Surgery, Hannover Medical School, Hannover, Germany. Macroscopically and histologically normal liver tissue was obtained from a male patient receiving a liver transplantation for hepatocellular carcinoma. Gastric tissue was from a male patient undergoing gastrectomy for gastric adenocarcinoma. Normal duodenal and jejunal tissue was collected from two male patients receiving a Whipple's procedure for carcinoma of the

Expression and activity of recombinant UGT proteins

Baculovirus expression of UGT cDNAs in Sf9 cells have been optimized to produce abundant catalytically active UGT protein [36], [37]. Previous data and control experiments confirm the presence of the UGT proteins employed in this study by Western blot analysis. In Fig. 1, examples of the activities from expressed UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A9, UGT1A10, as well as liver microsomal protein are shown. The activity of UGT2B4 and UGT2B15 have been previously characterized [20], [22]

Discussion

Recent studies have identified a tissue-specific expression of the human UDP-glucuronosyltransferases (UGT) in gastrointestinal tissues [22], [25], [26], [31], [34]. The epithelia of the gastrointestinal tract represent a metabolically active barrier capable of specific extrahepatic glucuronidation and may serve as protection from cytotoxic and genotoxic compounds [22], [33], [35], [39], [40], [41]. These tissues would therefore be capable of metabolizing orally administered drugs during or

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft Grant STR493/3-1 (to C.P.S.) and USPHS grant GM49135 (to R.H.T). The authors wish to thank Professor J. Klempnauer and Dr R. Raab, Department of Abdominal and Transplant Surgery, for the assistance with tissue sample procurement, and J. Topp, M.S., for technical assistance with the PCR analysis.

References (41)

  • B. Mojarrabi et al.

    cDNA cloning and characterization of the human UDP glucuronosyltransferase, UGT1A3

    Biochem Biophys Res Commun

    (1996)
  • Z. Cheng et al.

    Cloning and expression of human UDP-glucuronosyltransferase (UGT) 1A8

    Arch Biochem Biophys

    (1998)
  • B. Mojarrabi et al.

    Characterization of two UDP glucuronosyltransferases that are predominantly expressed in human colon

    Biochem Biophys Res Commun

    (1998)
  • B. Mojarrabi et al.

    The human UDP glucuronosyltransferase, UGT1A10, glucuronidates mycophenolic acid

    Biochem Biophys Res Commun

    (1997)
  • C.P. Strassburg et al.

    UDP-glucuronosyltransferase activity in human liver and colon

    Gastroenterology

    (1999)
  • C.P. Strassburg et al.

    Autoantibodies against glucuronosyltransferases differ between viral hepatitis and autoimmune hepatitis

    Gastroenterology

    (1996)
  • K.W. Bock

    UDP-glucuronosyltransferases and their role in metabolism and disposition of carcinogens

    Adv Pharmacol

    (1994)
  • J.F. Borel et al.

    Biological effects of cyclosporin A: a new antilymphocytic agent

    Agents Actions

    (1976)
  • T. Kino et al.

    FK-506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics

    J Antibiot (Tokyo)

    (1987)
  • W.M. Flanagan et al.

    Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A

    Nature

    (1991)
  • Cited by (34)

    • Influence of Absorption, Distribution, Metabolism, and Excretion Genomic Variants on Tacrolimus/Sirolimus Blood Levels and Graft-versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation

      2016, Biology of Blood and Marrow Transplantation
      Citation Excerpt :

      Tacrolimus distributes from plasma to red blood cells rapidly, with a half-life of approximately 12 hours [11], and is extensively metabolized in the liver, mainly by enzymes of the cytochrome P450 system [12], including CYP3A5 [13], CYP3A4 [14], CYP2E1 [15], CYP2A6 [15], and CYP2D6 [16]. In addition, other enzymes affect tacrolimus metabolism, such as UGT2B7 [17] and P-glycoprotein (or ABCB1) [18]. Sirolimus has a half-life of approximately 60 hours and is primarily metabolized via the cytochrome P450 system (CYP3A5 and CYP3A4) [10], with additional metabolic effects by P-glycoprotein [19], SLCO1B1-OATP1B1, and SLCO1B3-OATP1B3 [20].

    • From gut to kidney: Transporting and metabolizing calcineurin-inhibitors in solid organ transplantation

      2013, International Journal of Pharmaceutics
      Citation Excerpt :

      Analyses using a selection of recombinant UGT's identified UGT2B7 as the most important isoform in relation to CNI metabolism. This was in agreement with the differential hepato-gastrointestinal distribution of CNI glucuronidation activity and the expressional pattern of UGT2B7 mRNA (Strassburg et al., 2001). Laverdiere et al. confirmed the presence of CNI glucuronidation in liver and intestines, but could not reproduce the dominant role for UGT2B7.

    • The influence of UGT polymorphisms as biomarkers in solid organ transplantation

      2012, Clinica Chimica Acta
      Citation Excerpt :

      Among the currently available immunosuppressive agents, cyclosporine, tacrolimus and mycophenolic acid are in vitro substrates of the UGT1A and 2B families of UGTs. For cyclosporine, glucuronidation is generally considered a minor pathway [7]. Tacrolimus undergoes glucuronidation via UGT1A4 in vitro but the significance of this pathway and its contribution to inter-patient pharmacokinetic variability is not known [8].

    • Glucuronidation in therapeutic drug monitoring

      2005, Clinica Chimica Acta
      Citation Excerpt :

      The CYP450 system obtains much attention for its contribution to this phenomenon, whereas a possible impact of glucuronidation is commonly underestimated in practice. However, Strassburg et al. [16] have recently shown with microsomal preparations obtained from gut tissue that a significant cyclosporine A and tacrolimus glucuronidation activity is present in duodenum, jejunum, ileum and colon, that may also influence the therapeutic efficacy. If the glucuronides are formed in the liver, they can be excreted by either renal or biliary elimination.

    View all citing articles on Scopus
    View full text