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LETTER

RESPONSE TO THE LETTER TO THE EDITOR BY DRS. N. PICARD AND P. MARQUET REGARDING A PUBLICATION: BASU ET AL., DRUG METABOLISM AND DISPOSITION 32: 768-773 (2004)[Abstract/Free Full Text]

National Institutes of Health, Bethesda, Maryland

(Received September 15, 2004; accepted September 16, 2004)

Thin-layer chromatography (TLC) is a poor analytical tool? Although high-pressure liquid chromatography is preferred for analysis of products present in trace or low amounts, radioactive MPA-glucuronide formation with [¹⁴C]UDP-glucuronic acid (0.012 µCi/nmol) by enzymes that reach saturation kinetics between 1.6 and 2.4 mM MPA followed by TLC resolution easily overcomes this objection. We describe base hydrolysis to remove acylglucuronide according to Shipkova et al. (with MPA-7-O-glucuronide analyzed by HPLC) compared with our [¹⁴C]MPA-7-O-glucuronide resolved by TLC with quantitation of radioactivity. Moreover, ß-glucuronidase hydrolysis confirms that the products are glucuronides (Fig. 3B). We also cite studies by other investigators who analyzed radiolabeled glucuronides following similar TLC resolution (Mackenzie, 2000; Martineau et al., 2004).

Why carry out differential pH measurements on UGT isozymes? Our aim was to conduct a biochemical study of the individual isozymes using optimal in vitro conditions. The overlapping curves allow the reader to draw his/her own conclusions over a pH range. Although some investigators prefer the "physiological pH condition" for in vitro studies, the common practice is the determination of optimal pH condition(s) for in vitro studies.

What is the meaning of atypical kinetics? We consider it appropriate and reasonable to use "atypical metabolism" and "atypical increases in activity with increasing concentrations" to describe the results in this study. We have not used "atypical kinetics" in this report. We believe that the high rate of MPA glucuronide formation that increases with MPA concentrations up to millimolar levels described in this study likely contributes to the high dosage requirement. This phenomenon was not known before this report. One can argue, therefore, that the obvious is not necessarily true: high in vivo MPA-glucuronide concentrations cause high dosage requirement. Furthermore, it is not understood why MPA-glucuronides accumulate to such high levels in the blood, adversely affecting free MPA blood levels? What about transporters... etc. etc.? Although the pharmacokinetics of MPA-glucuronide clearance from the blood has likely been addressed, it has not been easy to locate that aspect of the literature?

What is the significance of a number of in vivo related points with respect to our results? It is not clear that the authors are justified in attempting to pose a number of in vivo related questions that we have not posed or addressed in such an in vitro study. It is not certain that the authors' extrapolations to the existing literature can answer all their questions. Most likely, specific experiments are required. It is noted that inhibition by curcumin (Fig. 4B, Basu et al., 2004b) represents mouse in vivo data which warrant further investigation.

	Footnotes

 
doi:10.1124/dmd.104.002345.

ABBREVIATIONS: MPA, mycophenolic acid; UGT, UDP-glucuronosyltransferase; TLC, thin-layer chromatography.

	References

Top References

 


Basu NK, Ciotti M, Huang MS, Kole L, Mitra PS, Cho TW, and Owens IS (2004a) Differential and special properties of the major human UGT1-encoded gastrointestinal UDP-glucuronosyltransferases enhance potential to control chemical uptake. J Biol Chem 279: 1429-1441.[Abstract/Free Full Text]

Basu NK, Kole L, Kubota S, and Owens IS (2004) Human UDP-glucuronosyltransferases show atypical metabolism of mycophenolic acid and inhibition by curcumin. Drug Metab Dispos 32: 768-773.

Mackenzie PI (2000) Identification of uridine diphosphate glucuronosyltransferases involved in the metabolism and clearance of mycophenolic acid. Ther Drug Monit 22: 10-13.[CrossRef][Medline]

Martineau I, Tchernof A, and Bélanger A (2004) Amino acid residue Ile211 is essential for the enzymatic activity of human UDP-glucuronosyltransferase 1A10 (UGT1A10). Drug Metab Dispos 32: 455-459.[Abstract/Free Full Text]

Shipkova M, Armstrong VM, Wieland E, Niedmann PD, Schütz E, Brenner-Weiss G, Voihsel M, Braun F, and Oellenrich M (1999) Identification of glucoside and carboxyl-linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil. Br J Pharmacol 126: 1075-1082.[CrossRef][Medline]

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