Assessing the Impact of Anabolic-Androgenic Steroids on Drug- Drug Interactions: In-Vitro Evaluation of Metabolic Clearance of Common Illicit and Medicinal Compounds

Introduction: Drug clearance plays a vital role in determining the efficacy and safety of a drug, and can be influenced by drug-drug interactions (DDIs). DDIs occur when multiple drugs are present simultaneously in the body and the pharmacology of one or more is altered by another. Metabolic DDI can occur through various mechanisms, including competition for, or inhibition of, metabolic enzymes or transporters responsible for drug clearance and distribution. This can result in slowed drug clearance or altered metabolite formation.
Illicit use of anabolic-androgenic steroids (AASs) has increased in recent years, including polydrug use with cocaine and MDMA. AAS misuse has been linked with greater psychopharmacological treatment, while recent research has also detected the presence of AASs alongside synthetic cannabinoid receptors agonists. Currently there is a lack of information regarding the impact of DDIs on AASs and analyte clearance.

Objectives: To evaluate the impact metandienone, nandrolone and testosterone have on the metabolic clearance of ADB-FUBIATA, CH-PIATA, cocaine, MDMA, and quetiapine via in-vitro human liver microsome (HLM) incubations.
Methods: Intrinsic clearance of test compounds was measured in HLM incubations both with and without AASs. HLM (final concentration 0.5 mg/mL microsomal protein) solutions were prepared in potassium phosphate buffer (50 mM, pH 7.4) and, where appropriate, pre-incubated with each AAS (each at 10 μM, consistant with levels of exogenous use) as potential inhibitors (5 min, 37 °C, shaking at 100 rpm). Test compounds and verapamil (positive control) (each at 0.5 μM) were added to incubations, and then 50 μL of NADPH (8 mg/mL) was used to initiate the reaction (final incubation volume 500 μL). Samples were incubated (37 °C, shaking at 100 rpm) and 50 μL samples were transferred to 200 μL acetonitrile (containing donepezil internal standard (10 ng/mL)) to stop the reaction at 0, 3, 6, 9, 15, 30, 45 and 60 min(s). Following incubation, 80 μL dH2O was added to each sample before centrifugation (3750 rpm, 20 °C, 10 min). UPLC-MS/MS analysis was achieved by gradient elution using a Waters Acquity UPLC (column: Acquity BEH C18 (2.1 x 50mm, 1.7μm) coupled with a Waters Xevo TQ-S MS. Peak area ratio was plotted against incubation time to determine half-lives (t1/2) and intrinsic clearance rates (CLint) of parent analytes with and without the presence of AAS as potential inhibitors.

Results: Clearance of cocaine, MDMA and quetiapine were unaffected showing that their phase I metabolism by HLM is not significantly affected by the presence of these AASs. ADB-FUBIATA was also unaffected, however, we can report here for the first time the experimental HLM clearance of this compound in isolation (t1/2 = 9.82 ± 1.38 min). The intrinsic clearance of CH-PIATA was t1/2 = 4.04 ± 0.47 min. CH-PIATA was found to be cleared at a slower rate in the presence of AASs, with CH-PIATA clearance as follows; with metandienone, t1/2 = 12.42 ± 3.84 mins; with nandrolone, t1/2 = 8.71 ± 2.56 mins; with testosterone, t1/2 = 9.09 ± 2.48 mins.

Discussion: The co-incubation of metandienone with CH-PIATA resulted in, on average, a 3-fold reduction in CH-PIATA intrinsic clearance, while nandrolone and testosterone caused an average 2-fold reduction compared to CH-PIATA alone. This preliminary data suggests that high-dose AAS use may significantly affect CH-PIATA clearance in vivo.