The interactions of Propafenone and its enantiomers with the major human forms of cytochrome P450 in terms of inhibition and metabolic rates

Carroll, Leigh (2010) The interactions of Propafenone and its enantiomers with the major human forms of cytochrome P450 in terms of inhibition and metabolic rates. Masters thesis, University of Central Lancashire.

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Propafenone is a class 1C antiarrhythmic drug used for the treatment of ventricular arrhythmias. Propafenone is a chiral compound which is normally administered as the racemate. The use of racemate therapy can be problematic as variability in both the pharmacodynamic and pharmacokinetic properties of the separate enantiomeric forms of the drug can exist. Propafenone enantiomers have been shown to have similar pharmacological properties, but studies are limited with regards to their disposition and interaction with drug metabolising enzymes. The aim of the study conducted here was to investigate the interaction of racaemic propafenone with the major cytochrome P450 isoforms (CYP2D6, CYP1A2, CYP3A4 and CYP2C19) to determine any stereospecific differences which may exist. This was conducted by measuring the in vitro metabolism of propafenone (racemate and enatiomers) and by developing CYP inhibition screens which could prove to be useful in providing information of potential drug interactions. In addition, stereospecific binding to human albumin was measured to investigate whether any enantiomeric differences in unbound drug exist.
For the inhibition studies propafenone (racemate and enatiomers) was incubated in 96 well plates with separate CYP isoforms in the presence of a NADPH regenerating system. CYP activity was monitored using the following fluorogenic substrates: CYP2C19/ CYP1A2 – CEC; CYP2D6 – AMMC; CYP3A4 - BFC. IC50 values were calculated and compared to those of control inhibitors (CYP2C19 – Tranycylpromine; CYP2D6 – Quinidine; CYP3A4 – Ketoconazole ; CYP1A2 - Furafylline). In vitro metabolism was conducted using human liver microsomes incubated with propafenone (racemate and enatiomers) and the degree of metabolism measured using hplc analysis. Protein binding was estimated for propafenone (racemate and enatiomers) by a chromatographic method utilising a chiral HSA column.
Inhibition studies showed that the lowest IC50 values were obtained when propafenone was co-incubated with CYP2D6 and CYP 3A4 which is to be expected as these CYP isoforms have been shown previously to be the major ones responsible for phase 1 metabolism of racaemic propafenone. There was a distinct stereospecific difference with these isoforms, with the R-enantiomer showing a higher degree of inhibition. This would suggest that there may be merit in considering single enantiomer therapy (in this instance using the S-enantiomer) to minimise the risk of any drug-drug interactions in vivo. However, the in vitro metabolism study showed that both single enantiomers were metabolised at a higher rate than the racaemic mixture. This may be explained in terms of the 2 enantiomers competing for metabolism and thus inhibiting the metabolism of each other. These results suggest a possible problem with single enantiomer therapy of propafenone as this drug has a short half life and increased metabolism would decrease this even further. Investigations into the plasma protein binding of propafenone showed that there is no difference in the binding of the separate enantiomers and therefore there would be no stereospecific differences in free drug concentration, although propafenone also binds to acid glycoprotein and so binding studies with separate enantiomers need to be conducted with this protein too.
Overall, this study shows how in vitro techniques can be utilised to investigate stereospecific differences in drug disposition. The work described here warrants further study on the metabolism/disposition of propafenone enantiomers in vivo to examine the clinical implications of enantiospecific therapy with this drug.

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