Abstract
Glucuronidation is a Phase 2 metabolic pathway responsible for the metabolism and excretion of testosterone to a conjugate testosterone glucuronide. Bioavailability and the rate of anabolic steroid testosterone metabolism can be affected upon UGT glucuronidation enzyme alteration. However, there is a lack of information about the in vitro potential assessment of UGT2B17 inhibition by salicylic acid. The purpose of this study is to investigate if UGT2B17 enzyme activity is inhibited by salicylic acid. A UGT2B17 assay was developed and validated by HPLC using a C18 reversed phase column (SUPELCO 25 cm × 4.6 mm, 5 μm) at 246 nm using a gradient elution mobile phase system: (A) phosphate buffer (0.01 M) at pH = 3.8, (B) HPLC grade acetonitrile and (C) HPLC grade methanol. The UGT2B17 metabolite (testosterone glucuronide) was quantified using human UGT2B17 supersomes by a validated HPLC method. The type of inhibition was determined by Lineweaver–Burk plots. These were constructed from the in vitro inhibition of salicylic acid at different concentration levels. The UGT2B17 assay showed good linearity (R2 > 0.99), acceptable recovery and accuracy (80–120%), good reproducibility and acceptable inter and intra-assay precision (<15%), low detection (6.42 and 2.76 μM) and quantitation limit values (19.46 and 8.38 μM) for testosterone and testosterone glucuronide respectively, according to ICH guidelines. Testosterone and testosterone glucuronide were found to be stable up to 72 h in normal laboratory conditions. Our investigational study showed that salicylic acid uncompetitively inhibited UGT2B17 enzyme activity. Thus, drugs that are substrates for the UGT2B17 enzyme have negligible potential effect of causing interaction with salicylic acid in humans.
Highlights
Published: 21 July 2021By definition, UGT enzymes are glycoproteins, i.e., Phase 2 drug-mediated enzymes that can glucuronidate biologically anabolic steroids, such as testosterone, xenobiotic and phenolic compounds, into more lipophobic molecules [1,2]
The High Performance Liquid Chromatography (HPLC) method for UGT2B17 assay was systematically developed using a mobile phase of phosphate buffer solution at pH = 3.8, methanol and acetonitrile in a gradient elution mode system
The current study underlines the assessment of UGT2B17 enzyme activity inhibition by salicylic acid in human supersomes
Summary
UGT enzymes are glycoproteins, i.e., Phase 2 drug-mediated enzymes that can glucuronidate biologically anabolic steroids, such as testosterone, xenobiotic and phenolic compounds, into more lipophobic molecules [1,2]. Fatty acid derivatives or steroids into hydrophilic group moieties, they are excreted by the liver or bile [4]. These biological transformations are known as conjugation reactions [5] and are mediated by various processes, such as amino acid, glutathione conjugation, sulfation, acetylation, glucuronidation and methylation [6].
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