Elsevier

Biochemical Pharmacology

Volume 65, Issue 9, 1 May 2003, Pages 1441-1449
Biochemical Pharmacology

The effect of valproic acid on drug and steroid glucuronidation by expressed human UDP-glucuronosyltransferases

https://doi.org/10.1016/S0006-2952(03)00076-5Get rights and content

Abstract

Valproic acid glucuronidation kinetics were carried out with three human UGT isoforms: UGT1A6, UGT1A9, and UGT2B7 as well as human liver and kidney microsomes. The glucuronidation of valproic acid was typified by high Km values with microsomes and expressed UGTs (2.3–5.2 mM). The ability of valproic acid to interact with the glucuronidation of drugs, steroids and xenobiotics in vitro was investigated using the three UGT isoforms known to glucuronidate valproic acid. In addition to this the effect of valproic acid was investigated using two other UGT isoforms: UGT1A1 and UGT2B15 which do not glucuronidate valproic acid. Valproic acid inhibited UGT1A9 catalyzed propofol glucuronidation in an uncompetitive manner and UGT2B7 catalyzed AZT glucuronidation competitively (Ki=1.6±0.06 mM). Valproate significantly inhibited UGT2B15 catalyzed steroid and xenobiotic glucuronidation although valproate was not a substrate for this UGT isoform. No significant inhibition of UGT1A1 or UGT1A6 by valproic acid was observed. These data indicate that valproic acid inhibition of glucuronidation reactions is not always due to simple competitive inhibition of substrates.

Introduction

Valproic acid is a broad-spectrum antiepileptic drug that is also used in the treatment of bipolar disease and migraine headaches. Structurally, valproate is a short chain fatty acid, which is metabolized by the body in the same manner as endogenous fatty acids with greater than 97% eliminated by hepatic metabolism. Major pathways of valproate metabolism include glucuronidation by UGTs, mitochondrial β-oxidation and a minor cytochrome P450-dependent oxidation pathway and desaturation [1].

The UGTs are family of enzymes which catalyze the transfer of a glucuronic acid moiety from a donor co-substrate UDPGA to an aglycone [2]. The UGT family can be separated into two distinct families, the UGT1 family are all derived from a single gene by alternative splicing of four constant exons (exons 2–5) with a variable exon 1 which determines the substrate specificity of the isoform [3]. The UGT1 family members have demonstrated to be capable of glucuronidating a wide range of drugs, xenobiotics and endobiotics [4]. The UGT2 family of isoforms are encoded by single genes and are clustered on chromosome 4q [5]. This family have long been considered to be more involved in the glucuronidation of endobiotics including steroids and bile acids. UGT2 isoenzymes seem to favour these types of compound as substrates but can glucuronidate other chemical types.

Three UGT isoforms have been reported to be capable of glucuronidating valproic acid: UGT1A6 [6], UGT1A9 [7] and UGT2B7 [8]. Both UGT1A6 and UGT1A9 are capable of glucuronidating a wide range of drugs and xenobiotics [7]. UGT2B7 seems to be exceptional among the 2B family in its acceptance of drug molecules including morphine [9] and AZT [10], [11] as well as glucuronidating a range of endogenous molecules including 4-hydroxyestrone and the bile acid hyodeoxycholic acid.

Valproate has been demonstrated to inhibit a wide variety of hepatic enzymes in humans and animal species both in vivo and in vitro. These include cytochrome P450, UGTs and epoxide hydrolase [12]. Overall, the largest inhibitory effect of valproate appears to be on drugs metabolized by the UGTs. The glucuronidation of valproic acid in humans is thought to affect the metabolism of other drugs which are glucuronidated. Lorazepam undergoes reduced clearance in the presence of valproic acid [13] as does lamotrigine with a corresponding increase in half life [14]. An interaction was observed in humans in vivo with concurrent dosing of valproic acid and zidovudine (AZT) which was used in an attempt to modulate the pharmacokinetics of AZT in HIV sufferers [15]. Co-administration of valproic acid significantly decreased the normally extensive first pass metabolism of AZT to its glucuronide conjugate and increased the ratio of urinary AZT glucuronide to unconjugated AZT by 50%. Valproic acid also inhibits the glucuronidation of parahydroxyphenobarbital, the major metabolite of phenorbarbital. This is thought to occur by valproate competing with parahydroxyphenobarbital at the UGT active site; this interaction has been observed in both in vitro and in vivo studies in rat [16], [17]. Valproic acid has also been shown to inhibit the formation of 4-hydroxyandrostendione glucuronide, a synthetic steroid, in both in vitro and in vivo rat studies [18] an indication that valproic acid may also be able to interfere with the glucuronidation of steroids.

The aims of this study were to identify those UGT isoforms which could glucuronidate valproic acid and determine their kinetic constants to compare with those measured in human liver and kidney microsomal preparations. The effect of valproic acid on the glucuronidation of probe substrates, drugs and steroids known to be substrates for the human recombinant isoforms was to be investigated in order to assess the inhibitory potential of valproic acid on glucuronidation and whether metabolic interactions might be able to affect the glucuronidation of these compounds in vivo. To this end new analytical methods were developed to investigate the potential interactions between valproic acid and substrates for three UGT isoforms which glucuronidate valproic acid: UGT1A6, UGT1A9 and UGT2B7. In addition to this another two steroid glucuronidating UGT isoforms were included in the study. UGT1A1 has been reported to glucuronidate estriol and the synthetic steroid ethinylestradiol [19] and UGT2B15 which has been reported to glucuronidate a wide range of androgenic steroids [20].

Section snippets

Chemicals

Substrates, UDP-glucuronic acid, AZT, AZT glucuronide and other reagents used in the assays were purchased from Sigma, Aldrich, BDH and were of the highest grade available. [14C]UDPGA, [14C] dihydrotestosterone and were purchased from NEN Dupont. Penicillin, streptomycin and geneticin were purchased from Gibco Life Technologies.

Tissue culture and human microsomes

The cloning of the UDP-glucuronosyltransferase isoforms used and their expression in V79 Chinese hamster lung fibroblast recombinant UGT cell lines have been published

Glucuronidation of valproic acid by human UGTs

The kinetic parameters for valproic acid glucuronidation with expressed human UGTs and human tissue microsomes are listed in Table 1. No glucuronidation activity towards valproic acid could be measured for UGT1A1 or UGT2B15 but UGT1A6, UGT1A9 and UGT2B7 all clearly demonstrated significant activity towards this substrate. The Km values for all the expressed UGTs are high typically the millimolar range (2.1–5.2 mM) and are consistent with the Km values measured for valproic acid glucuronidation

Discussion

The results of this study highlight the inherent difficulties in extrapolating from in vitro data to the in vivo situation. It is clear that a simple comparison of Km values for two substrates metabolized by the same enzyme is insufficient when estimating potential for metabolic drug interactions.

Initially, simulations were run (Berkely Madonna software v. 8.0.1, Macey and Oster) making the assumption that Km=Ki using the equation for simple competitive inhibition substituting the kinetic

Acknowledgements

We thank the Wellcome Trust for grants supporting this work.

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