Potential Assessment of UGT2B17 Inhibition by Salicylic Acid in Human Supersomes In Vitro

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.

Keywords: UGT2B17; glucuronidation; human supersomes; salicylic acid; testosterone.

Figure 1

Chemical structures of: salicylic acid (A), testosterone (B), and testosterone glucuronide (C).

Figure 2

Typical HPLC chromatogram of UGT2B17 components at 243 nm using gradient elution programming. The peaks marked are: (1) UGT2B17 enzyme (Rt = 2.184 min); (2) salicylic acid (100 µM) (Rt = 3.177 min); (3) phenacetin (50 µM) (Rt = 6.196 min); (4) testosterone glucuronide (100 µM) (Rt = 9.417 min); and (5) testosterone (200 µM) (Rt = 14.388 min), respectively.

Figure 3

Representative Lineweaver–Burk plot for the inhibition of UGT2B17-catalysed testosterone glucuronide (testosterone (25–200 µM)) with 0, 15, 25, and 40 µM salicylic acid (n = 3).