An enantioselective CE method was used to identify the ability of CYP450 enzymes and their stereoselectivity in catalyzing the transformation of propafenone (PPF) to 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF). Using in vitro incubations with single CYP450 enzymes (SUPERSOMES), 5OH-PPF is shown to be selectively produced by CYP2D6 and N-dealkylation is demonstrated to be mediated by CYP2D6, CYP3A4, CYP1A2, and CYP1A1. For the elucidation of kinetic aspects of the metabolism with CYP2D6 and CYP3A4, incubations with individual PPF enantiomers and racemic PPF were investigated. With the exception of the dealkylation in presence of R-PPF only, which can be described by the Michaelis-Menten model, all CYP2D6-induced reactions were found to follow autoactivation kinetics. For CYP3A4, all NOR-PPF enantiomer formation rates as function of PPF enantiomer concentration were determined to follow substrate inhibition kinetics. The formation of NOR-PPF by the different enzymes is stereoselective and is reduced significantly when racemic PPF is incubated. Clearance values obtained for CYP3A4 dealkylation are stereoselective whereas those of CYP2D6 hydroxylation are not. This paper reports the first investigation of the PPF hydroxylation and dealkylation kinetics by the CYP2D6 enzyme and represents the first report in which enantioselective CE data provide the complete in vitro kinetics of metabolic steps of a drug.

A robust, inexpensive, and fully validated CE method for the simultaneous determination of the enantiomers of propafenone (PPF), 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF) in serum and in in vitro media is described. It is based upon liquid-liquid extraction at alkaline pH followed by analysis of the reconstituted extract by CE in presence of a pH 2.0 running buffer composed of 100 mM sodium phosphate, 19% methanol, and 0.6% highly sulfated beta-CD. For each compound, the S-enantiomers are shown to migrate ahead of their antipodes, and the overall run time is about 30 min. Enantiomer levels between 25 and 1000 ng/mL provide linear calibration graphs, and the LOD for all enantiomers is between 10 and 12 ng/mL. The assay is shown to be suitable for the determination of the enantiomers of PPF and its metabolites in in vitro incubations comprising human liver microsomes or single CYP450 enzymes (SUPERSOMES). Incubations with CYP2D6 SUPERSOMES revealed, for the first time, the simultaneous formation of the enantiomers of 5OH-PPF and NOR-PPF with that enzyme. CE data can be used for the evaluation of the enzymatic N-dealkylation and hydroxylation rates.

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Background Fumaria species (Fumariacea) has traditionally been used in wound healing in Iranian folk medicine. However, with the discovery of newer agents, its use has faded off into total obscurity. This study explored the wound healing potential of a gel containing 10% Fumaria vaillantii Loisel through topical application of total extract in a model of excisional as well as incisional wound healing in albino Wistar rats. Methods Rats were anesthetized, and excisional skin wound was established using a sterilized surgical scissors. The animals were then treated with 10% F.vaillantii topical gel formulation along with the gel base. The treatments were administered once a day after the injury for 21 days. For topical treatment, the hydrogel was formulated and evaluated for chemical and physical characteristics. Histopathological analysis with hematoxylin and eosin (H&E) was used for microscopic examination of the skin tissues on 21-day-old sections of excision wound. To verify collagen formation, hydroxyproline determination was performed 21 days post wound healing. Breaking strength was determined in a 10-day-old incision wound by the uniaxial tensile test. Results Topical administration of F.vaillantii gel formulation significantly enhanced skin wound closure on the 6th post-wounding day compared to both gel base and the negative control, indicating an accelerated wound healing process, while a significant difference was observed on 10th and 14th post –wound days in F.vaillantii treatment compared to the negative control groups. Gel formulation prepared with a 10% F. vaillantii extract exhibited a response in terms of wound epithelialization, angiogenesis and number of hair follicles at wound area better than the gel base on the 21st post-wound day. Application of gel base produced further advantages by increasing hydroxyproline content and collagen fiber thickness. Our results on incision wound model were supported by histopathological data indicating the role of gel base in the enhancement of breaking strength. Conclusion Traditional use of Fumaria species in the skin diseases was justified in this study by revealing the increase in wound healing activity after hydrogel containing F. vaillantii total extract administration. Graphical abstract

Propafenone is a potent antiarrhythmic drug, which is widely used in the treatment of ventricular and supraventricular arrhythmias. 1 It blocks the fast inward sodium current in all cardiac and other excitable tissues, like the central nervous system. Propafenone also has some β-blocking action, much less than that of propranolol, and a weak calcium-channelblocking effect. 2 Propafenone undergoes extensive first-pass metabolism by the liver to form several metabolites. The main metabolic pathway of propafenone is ring hydroxylation to 5-OH-propafenone, being primarily mediated by CYP2D6. This pathway is polymorphically expressed in humans and is under genetic control, co-segregating with a well-described debrisoquine oxidation polymorphism, 3 such that poor metabolizers can be distinguished from extensive metabolizers. 4 An additional route of propafenone metabolism is N-demethylation to N-despropylpropafenone, which is primarily mediated via CYP3A4 and CYP1A2.5 Five hydroxy-propafenone has been shown to exert pharmacologic activity comparable to that of the parent drug. 6Although N-despropylpropafenone exerted antiarrhythmic activity in animal experiments, 7 relatively little is known about its pharmacokinetics and pharmacodynamics in humans. The metabolic pathway of propafenone is illustrated in Fig. 1.The specific metabolism of propafenone, its potential drug-drug interactions and its widespread use have stimulated efforts to develop routine assays for this drug and its metabolites in human serum. Several chromatographic methods have been reported for the determination of propafenone and 5-OH-propafenone in biological matrices, including highperformance liquid chromatography (HPLC) [8][9][10][11][12][13][14][15]18 and gas chromatography. 16,17 For HPLC assays, different modes of detection have been employed, including UV 8-15 and fluorescence with precolumn derivatization. 18Most of these methods have only been validated for propafenone, itself, or propafenone and 5-OH-propafenone. The resolution of N-despropylpropafenone, which is probably co-eluted under reported procedures, was rarely investigated.Among the methods described so far, only a few allow for the determination of the serum or plasma concentrations of propafenone and its main metabolites in a single run. A rapid and specific HPLC method has been developed and validated for the simultaneous determination of propafenone, an antiarrhythmic agent, and its major metabolites in human serum. The sample preparation was a simple deproteinization with a mixture of ZnSO4 and methanol, yielding almost 100% recoveries of three compounds. Separation was developed on a reverse-phase tracer excel C18 column (25 × 0.46 cm i.d., 5 µm), using an acetonitrilephosphate buffer gradient at a flow rate of 1.7 ml min -1 , and UV detection of 210 nm. The calibration curves were linear (r 2 > 0.999) in the concentration range of 10 -750 ng ml -1 . The lower limit of quantification was 10 ng ml -1 for all of the compounds studied. The within and between day precisions in th...