Elsevier

Biochemical Pharmacology

Volume 73, Issue 10, 15 May 2007, Pages 1683-1691
Biochemical Pharmacology

Glucuronidation of fenamates: Kinetic studies using human kidney cortical microsomes and recombinant UDP-glucuronosyltransferase (UGT) 1A9 and 2B7

https://doi.org/10.1016/j.bcp.2007.01.030Get rights and content

Abstract

Mefenamic acid, a non-steroidal anti-inflammatory drug (NSAID), is used commonly to treat menorrhagia. This study investigated the glucuronidation kinetics of flufenamic, mefenamic and niflumic acid using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7. Using HKCM Michaelis–Menten (MM) kinetics were observed for mefenamic (Kmapp 23 μM) and niflumic acid (Kmapp 123 μM) glucuronidation, while flufenamic acid exhibited non-hyperbolic (atypical) glucuronidation kinetics. Notably, the intrinsic renal clearance of mefenamic acid (CLint 17 ± 5.5 μL/min mg protein) was fifteen fold higher than that of niflumic acid (CLint 1.1 ± 0.8 μL/min mg protein). These data suggest that renal glucuronidation of mefenamic acid may result in high intrarenal exposure to mefenamic acyl-glucuronide and subsequent binding to renal proteins. Diverse kinetics were observed for fenamate glucuronidation by UGT2B7 and UGT1A9. Using UGT2B7 MM kinetics were observed for flufenamic (Kmapp 48 μM) and niflumic acid (Kmapp 135 μM) glucuronidation and atypical kinetics with mefenamic acid. Similarity in Kmapp between HKCM and UGT2B7 suggests that UGT2B7 may be the predominant renal UGT isoform catalysing niflumic acid glucuronidation. In contrast, UGT1A9 glucuronidation kinetics were characterised by negative cooperativity with mefenamic (S50 449 μM, h 0.4) and niflumic acid (S50 7344 μM, h 0.4) while atypical kinetics were observed with flufenamic acid. Additionally, potent inhibition of the renal glucuronidation of the UGT substrate ‘probe’ 4-methylumbelliferone by flufenamic, mefenamic and niflumic acid was observed. These data suggest that inhibitory metabolic interactions may occur between fenamates and other substrates metabolised by UGT2B7 and UGT1A9 in human kidney.

Introduction

The continued and widespread use of prescribed and over-the-counter (OTC) non-steroidal anti-inflammatory drugs (NSAIDs) remains an important cause of morbidity and mortality, and represents a significant and costly health problem. In the USA, NSAIDs alone account for approximately 70 million prescriptions and 30 billion OTC sales [1]. Substantial use of prescribed and OTC NSAIDs occurs in the treatment of heavy menstrual bleeding (menorrhagia). NSAIDs commonly used for this condition include aspirin, diclofenac, flurbiprofen, ibuprofen, indomethacin, meclofenamic acid, mefenamic acid and naproxen. Although, it is generally assumed that clinical efficacy is similar between NSAIDs, mefenamic acid is one of the most effective first-line drugs with demonstrated benefit in comparison to placebo in the treatment of menorrhagia [2]. Additionally, on a cost-benefit scale, mefenamic acid is considered an economical choice [3].

In the year 2000, mefenamic acid was listed in the top twenty five most used prescription pharmaceuticals in the English National Health Service by weight accounting for 14,522 kg and 544,000 prescriptions [4]. A comparable number of prescriptions (424,900) were dispensed for mefenamic acid in the UK in 2005 [5]. Interestingly, detection of mefenamic acid in the influent and effluent from wastewater treatment plants in Switzerland (17,275 kg sold/year) [6], southern England [7] and Japan [8] suggests substantial human consumption.

Flufenamic acid (Fig. 1), one of a series of N-phenylanthranilic acid derivatives synthesised during the early 1960s, was the first fenamate to undergo clinical trial as an anti-inflammatory drug. It was followed closely by mefenamic acid (Fig. 1), meclofenamic acid and niflumic acid (Fig. 1) [9]. Within ten years of marketing, cases of non-oliguric renal failure were reported with mefenamic acid [10], [11], [12], [13]. Histological examination provided evidence of renal papillary necrosis (RPN) in some individuals [10] while in others nephrotoxicity was consistent with allergic interstitial nephritis [11], [13]. The latter is often characteristic of an immune mediated response to irreversible binding of either a drug or its metabolites to various cell proteins [14].

RPN was described originally in diabetic humans but it is also commonly associated with analgesic abuse [15]. Early reports implicated chronic consumption of OTC compound analgesics containing phenacetin as a major factor in RPN. Population surveys reported wide geographic variation in analgesic abuse [16] with Australia having both the highest consumption of phenacetin (40 g/year in 1976) and the highest incidence of RPN at autopsy (3.7–21.4%) [17]. Investigations into the mechanism of analgesic induced RPN led to the development of animal models of RPN using either N-phenylanthranilic acid (a close structural analogue of mefenamic acid) [18] or mefenamic acid [19].

Subsequently, clinical studies implicated a range of NSAIDs in the development of RPN including alclofenac, amidopyrine, antipyrine, aspirin, benoxaprofen, fenoprofen, flufenamic acid, ibuprofen, indomethacin, mefenamic acid, naproxen, phenazone and phenylbutazone [17], [20], [21], [22]. The exact mechanism of NSAID induced RPN is unknown but theories have centred on either a direct toxic effect on medullary interstitial cells from local generation of reactive drug metabolites or ischaemic injury through loss of production of renal protective vasodilatory prostaglandins [9], [20]. It is probable that more than one mechanism prevails.

Metabolism is the principal route of NSAID elimination in vivo and only a small fraction of the administered dose is excreted unchanged in urine [23]. Like many other carboxylic acid NSAIDs, the fenamates are metabolised extensively by glucuronidation as either the parent drug or oxidative metabolites. Various members of the UDP-glucuronosyltransferase (UGT) superfamily of enzymes catalyse the glucuronidation of NSAIDs. To date, of the nine human UGTs (UGT 1A3, 1A6, 1A9, 2B4, 2B7, 2B10, 2B11, 2B15, and 2B17) identified in the kidney, UGT 1A9 and 2B7, and to a lesser extent UGT1A3 are the predominant NSAID glucuronidating forms [24], [25]. Unlike other glucuronides, however, NSAID acyl-glucuronides are electrophilic and bind covalently to plasma and tissue proteins both in vitro and in vivo [26]. Covalent modification of macromolecules by acyl-glucuronides (the hapten theory) results in production of circulating antibodies, which cause an immune response and drug hypersensitivity. In the case of mefenamic acid, formation of an acyl-glucuronide is known to result in irreversible binding of mefenamic acid glucuronide to proteins in vitro and ex vivo [27]. The question arises whether there is any basis for considering intrarenal formation of fenamate acyl-glucuronides as a contributing factor in the development of RPN in humans. However, fenamate glucuronidation by human kidney has not been investigated fully to date, hence any relationship to fenamate-induced RPN is unknown. This study sought to investigate the glucuronidation kinetics of flufenamic, mefenamic and niflumic acid using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7.

As fenamates have been shown to inhibit human liver phenol sulfotransferases [28], [29], CYP2E1 [30] and mycophenolic acid glucuronidation by human liver and kidney [31] additional studies were undertaken to determine the universality of fenamates as potential inhibitors of renal glucuronidation using the UGT substrate ‘probe’ 4-methylumbelliferone (4-MU).

Section snippets

Materials

The following compounds were purchased from Sigma–Aldrich (Sydney, Australia): uridine diphosphate glucuronic acid (UDPGA), 4-MU, and 4-MU-β-d-glucuronide. Flufenamic and mefenamic acid were obtained from Parke-Davis & Co. (Sydney, Australia), and niflumic acid from E.R. Squibb & Sons Inc. (Princeton, NJ, USA) all as the free acid. Other chemicals and reagents were of the highest analytical grade available.

Human kidney tissue

Human kidney tissue from six male subjects (K1, K4-K7, and K11) undergoing radical

Kinetics of flufenamic, mefenamic and niflumic acid glucuronidation by HKCM

Flufenamic acid glucuronidation by HKCM (n = 3) exhibited atypical (i.e. non-hyperbolic) kinetics. Eadie-Hofstee plots were indicative of either the involvement of two enzymes or negative cooperativity (Fig. 2A). However, kinetic analyses were inconclusive; data were not adequately described by the kinetic models (2-enzyme Michaelis–Menten and Hill equations) normally used to describe these types of data. Although the goodness of fit was poor, the Hill coefficient was consistently <1 indicative

Discussion

A substantial number of clinically used NSAIDs from various chemical classes are metabolised by glucuronidation [37]. Although the majority of studies involve investigation of hepatic metabolism, the relative contribution of the kidney to the systemic clearance of NSAIDs is unknown. However, NSAIDs can cause renal dysfunction, ranging from electrolyte disturbances to acute renal failure and RPN. The latter is often associated with the use of the fenamate class of NSAIDs, in particular mefenamic

Acknowledgements

The authors acknowledge Dr. Nu Uchaipichat for expression of recombinant UGT proteins, Professor Anthony Thomas for histological examination of the renal tissue, Dr. Adrian Esterman and Ms. Kylie Lang for statistical advice. The research was supported by grants from the Flinders Medical Research Institute (K.M.K.) and the National Health and Medical Research Council of Australia (J.O.M.).

References (54)

  • J.O. Miners et al.

    Drug glucuronidation in humans

    Pharm Ther

    (1991)
  • M. Kurkela et al.

    Expression and characterisation of recombinant human UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than other UGTs and was purified as an active dimeric protein

    J Biol Chem

    (2003)
  • S. Gobec et al.

    Nonsteroidal anti-inflammatory drugs and their analogues as inhibitors of aldo-keto reductase AKR1C3: new lead compounds for the development of anticancer agents

    Bio Med Chem Lett

    (2005)
  • G.A. Green

    Understanding NSAIDs: from aspirin to COX-2

    Clin Cornerstone

    (2001)
  • Lethaby A, Augood C, Duckitt K. Nonsteroidal anti-inflammatory drugs for heavy menstrual bleeding. Cochrane Database of...
  • G. Khastgir et al.

    Choice of treatment of menorrhagia

  • Prescription Cost Analysis England 2005. NHS Health and Social Care Information Centre, Health Care Statistics 2006....
  • N. Nakada et al.

    Occurrence and fate of anti-inflammatory drugs in wastewater treatment plants in Japan

    Environ Sci

    (2005)
  • Winder CV, Kaump DH, Glazko AJ, Holmes EL. Pharmacology of the fenamates, experimental observations on flufenamic,...
  • P.L. Drury et al.

    Mefenamic acid nephropathy: further evidence

    Lancet

    (1981)
  • K.L. Woods et al.

    Mefenamic acid nephropathy

    Lancet

    (1981)
  • R.T. McCluskey et al.

    Immunological aspects of renal tubular and interstitial diseases

    Annu Rev Med

    (1978)
  • M.D. Griffin et al.

    Renal papillary necrosis—a sixteen-year clinical experience

    J Am Soc Nephrol

    (1995)
  • P. Kincaid-Smith

    Effects of non-narcotic analgesics on the kidney

    Drugs

    (1986)
  • R.S. Nanra

    Analgesic nephropathy in the 1990's—an Australian perspective

    Kidney Int

    (1993)
  • T.L. Hardy

    N-Phenyl anthranilic acid: an agent for inducing and studying renal papillary necrosis in the rat

    Br J Exp Pathol

    (1970)
  • N.T.K. Thanh et al.

    Urinary lipid changes during the development of chemically-induced renal papillary necrosis: a study using mefenamic acid and N-phenylanthranilic acid

    Biomarkers

    (2001)
  • Cited by (0)

    View full text