Thesis title: Natural supplements and doping drugs: possible interaction at level of the P-glycoprotein (P-gp) transport system.
Some recent studies reported that natural substances, such as isoflavones and ecdysteroids, could produce interesting effects on urinary steroid and gonadotropic profiles.
Overall, a good number of other natural substances are known to influence positively the production of endogenous hormones and could be used by athletes to support and mask doping with anabolic agents. Beside the well-known drug-drug interactions (DDIs), many herb-drug interactions (HDIs) are now studied and reported in literature, even if further investigations are needed.
One of the most important metabolic system, involved both in DDIs and HDIs, is represented by the P- glycoprotein transport system (P-gp/ ABCB1/ MDR1).
P-gp is an ATP-dependent cell membrane drug transporter which acts as an efflux pump for xenobiotics: it removes unidirectionally neutral or weakly basic amphipathic substances from the cytoplasm. It is widely studied because of its role in drug resistance of chemotherapy drugs.
Despite being extensively studied, the mechanism involving substrates binding to P-gp, including specificity, is still largely to be clarified.
Some natural substances and steroid hormones are both substrates of P-gp and this protein, depending on its localization and relative expression, is variously implicated in metabolic pathways.
The aim of this project was to investigate the possible interactions resulted from the associations between the three most abused anabolic drugs (testosterone, DHEA and stanozolol) and three natural products present in dietary supplements and largely diffused (cordycepin, gingerol and trigonelline), at the level of the P-gp dependent transport system.
We developed, optimized and validated an analytical method in luminescence which determines the induced variations of the ATPase activity on a membrane extract expressing P-gp. Each substance was characterized firstly isolated, then in association with verapamil (reference substrate and stimulator of P-gp ATPase activity), finally associated as described.
At the same time, docking studies, with the goal to provide additional insights on the type of interactions ligand/P-gp and to define the best ranked (most probable) configuration in energetic terms, were performed. Data obtained with docking were used to explain and discuss results obtained with ATPase activity determinations.
Our study confirmed that the three anabolic agents considered (testosterone, dehydroepiandrosterone, stanozolol) and the selected natural substances (cordycepin, gingerol, trigonelline), all interact with P-gp in different ways, both isolated and in association. When considering the isolated substances, all of them act as stimulators of P-gp ATPase activity.
Maximal activation occurred between 1 and 20 M and corresponds to an increase of 3/6 – fold the basal ATPase activity. The values of Km calculated are between 0,04 and 5,01 M. Significantly, natural modulators
showed lower values of Km than anabolic steroids.
When in association, we observed various effects, always depending on concentration, both of steroid and natural modulator. Competitive, non-competitive and uncompetitive inhibition were more frequent at the higher concentration of the natural modulator, while at the lower, we observed also cooperative behavior. The main metabolites of the considered steroids were also analyzed.
Docking studies have supported our interpretations of ATPase determination results and confirmed the presence of three main interaction regions on P-gp, as reported by other authors.
All the determined effects, given the wide distribution of P-gp in the body, may variously impact in metabolic processes and raise the risk of HDIs.
Further research to determine which of the considered ligands is also substrate of P-gp is needed, performing specific assays on cellular-mediated transport of the protein.
More experiments, testing membranes derived from different cellular types and so expressing variously P-gp, should be performed to verify our conclusion.
Furthermore, it is worth to extend the number of anabolic steroids to the more recurrent (clostebol, for
IV
example). Then, the study could be extended also to other categories of banned substances, starting with those provided of an anabolic effect and proceeding with the others.
With regard of natural substances, the first to be tested should be isoflavones and ecdysteroids, reported for their hormonal-like effects.
Finally, docking studies could be also supported by molecular dynamics studies which could explore the mechanism of interaction with P-gp in a more realistic and complete way.