The anti-epileptic drug lamotrigine inhibits the CYP17A1 lyase reaction in vitro.

The potential endocrine disrupting effects of the commonly prescribed anti-epileptic drug lamotrigine (LAM) were investigated using the H295R steroidogenic in vitro assay and computational chemistry methods. The H295R cells were exposed to different concentrations of LAM, and a multi-steroid LC-MS/MS method was applied to quantify the amount of secreted steroid hormones. LAM affected several steroid hormones in the steroidogenesis at therapeutic concentrations. All progestagens as well as 11-deoxycorticosterone and corticosterone increased 100-200% with increasing concentrations of LAM suggesting a selective inhibitory effect of LAM on CYP17A1, in particular on the lyase reaction. Recombinant CYP17A1 assay confirmed the competitive inhibition of LAM toward the enzyme with IC50 values of 619 and 764 µM for the lyase and the hydroxylase reaction, respectively. Levels of androstenedione and testosterone decreased at LAM concentrations above the therapeutic concentration range. The ability of LAM to bind to CYP17A1, CYP19A1, and CYP21A2 was investigated using docking and molecular dynamics simulations. This in silico study showed that LAM was able to bind directly to the heme iron in the active site of CYP17A1, but not CYP21A2, thus supporting the results of the in vitro studies. The molecular dynamics simulations also suggested binding of LAM to the heme iron in the CYP19A1 active site. No inhibition of the aromatase enzyme was, however, observed in the H295R assay. This could be due to a sequential effect within the steroidogenesis caused by the inhibition of CYP17A1, which reduced the amounts of androgens available for CYP19A1.

Steroidogenic disruptive effects of the serotonin-noradrenaline reuptake inhibitors duloxetine, venlafaxine and tramadol in the H295R cell assay and in a recombinant CYP17 assay.

The aim of this study was to determine the steroidogenic endocrine disrupting effect of three widely used serotonin-noradrenaline reuptake inhibitors duloxetine, venlafaxine and tramadol, using two in vitro models, the H295R assay and a recombinant CYP17 enzyme assay. Steroid hormones were quantified using LC-MS/MS. Duloxetine showed endocrine disrupting effects at 5-20µM with CYP17 being the main target. Venlafaxine also affected the steroidogenesis, mainly by affecting the CYP17 lyase reaction, although at much higher concentrations i.e. 100µM. Tramadol only exerted minor effects on the steroidogenesis with the lowest observed effect at 314µM. Based on the H295R results, the inhibition of CYP17 by duloxetine and venlafaxine was investigated in a recombinant CYP17 assay with the use of the 4 major CYP17 substrates pregnenolone, progesterone, 17α-hydroxypregnenolone and 17α-hydroxyprogesterone. Both duloxetine and venlafaxine inhibited CYP17 enzyme activity, but duloxetine was most potent. IC50-values were in the range 5.3-21µM for duloxetine and 1318-2750µM for venlafaxine. Overall, results from the recombinant CYP17 assay confirmed the results from the H295R cell assay. Using testosterone as end point, the margin of safety (defined as NOAEL/Cmax) for duloxetine was 1.6 indicating that duloxetine may have endocrine disrupting effects. In contrast, venlafaxine and tramadol showed higher margins of safety (venlafaxine: 24; tramadol: 157) indicating a lower potential to disrupt the human steroidogenesis.

Structure-based optimisation of non-steroidal cytochrome P450 17A1 inhibitors.

Five new non-steroidal inhibitors for cytochrome P450 17A1 (CYP17A1) were identified by structure-based optimisation from a recently identified selective CYP17A1 inhibitor. The compounds are nanomolar inhibitors of steroidogenesis measured in recombinant CYP17A1 and in H295R cells.