Predizione delle interazioni tra farmaci e alimenti con i citocromi P450: analisi in vitro

Prediction of cytochrome P450-based food-drug interactions from in vitro data Introduction Cytochromes P450 are a super family of heme proteins involved in the phase I metabolism of drugs and xenobiotics. Many studies have shown the adverse effects of drug-drug interactions that are costly both in terms of human life and investment (withdrawal of drugs from the market). Although less publicised, drug-food interactions can also cause an increase or decrease in the oral drug bioavailability when co-administered, the most well-known case being that of grapefruit juice and nifedipine (high blood pressure drug), which resulted in death. Cytochrome P450 2E1 represents about 10% of all cytochrome in the liver, and it is involved in 4% of drug metabolism. Three different food components namely tannic acid (polyphenol present in tea and coffee), resveratrol (present in red wine) and grape fruit juice were investigated for their ability to inhibit P450 2E1-mediated metabolism using p-nitrophenol as the substrate. Methods P450 2E1 and P450 2E1-BMR were heterologously expressed in E. coli DH5a and the resulting protein was purified using three chromatographic steps including an ion exchange DEAE column followed by a hydroxyapatite and finally a Ni-chelating sepharose affinity column. Inhibition studies were performed with the P450 2E1-BMR enzyme in solution and immobilised on glassy carbon electrodes. The IC50 values were calculated by plotting the residual activity (%) as function of the logarithm of the substrate concentration added. Results The inhibition of P450 2E1-BMR was investigated using the three different food components mentioned above. The IC50 values (the inhibitor concentration which reduced the metabolism of the P450 2E1-BMR probe substrate (p-nitrophenol) by 50%) measured for each of the food components with the enzyme in solution and at 37 ºC, was 21.2 ± 1.1 μM, 3 ± 1 μM and 7.7% ± 1.1% for tannic acid, resveratrol and grape fruit juice, respectively. The same inhibition studies were carried out with the enzyme immobilised on the surface of glassy carbon electrodes by entrappment in the cationic polymer poly (diallyldimethylammonium) (PDDA). Chronoamperometry was carried out with an applied potential bias of -0.60 V at 37 ºC for 30 min. Subsequently, the presence of the p-nitrocathecol product was measured spectrophotometrically at 515 nm. The electrochemically measured IC50 values of 11± 1.1 μM for tannic acid and 1.5 ± 1 μM for resveratrol were obtained. The IC50 values measured using the two different methods i.e. in solution and electrochemically, are in good agreement with results already published in literature using the microsomal P450 2E1. Conclusion These preliminary results could lead to the possibility of constructing a high throughput assay system for food-drug interactions of this important class of enzymes.