In vitro fatty acid esterification of budesonide and other corticosteroids in human liver microsomes and human lung microsomes

Budesonide has been reported to undergo oleate esterification in rat and human airway tissue, via an oleate esterification process requiring the presence of ATP and coenzyme A. Two Acyltransferase enzyme acyl coenzyme A:cholesterol acyltransferase (ACAT) or acyl coenzyme A:estradiol acyltransferase (AEAT) have been inferred without experimental data to be responsible for the in vivo esterification of budesonide. The goal of this study is to identify whether ACAT or AEAT is the enzyme responsible for the in vitro oleate esterification of budesonide and to study the oleate esterification kinetics of budesonide in human liver microsomes (HLM) and human lung microsomes (HLUM) of smokers and non-smokers. Budesonide and other corticosteroids were incubated with the microsomes in the presence of oleoyl coenzyme A. Budesonide 21-oleate as a mixtured of R,S epimers and other corticosteroid 21-oleate were separated using normal-phase HPLC with a Varian microsorb-MV silica column (150x4.6 mm) and a mobile phase consisting of hexane/isopropanol: 97/3 (v/v). The oleate product of the corticosteroids were identified by Sciex API 4000 triple quadrupole mass spectrometer equipped with an Heated Nebulizer as LC/MS interface. Budesonide 21-oleate, triamcinolone acetonide 21-oleate and hydrocortisone 21-oleate standards were synthesized from the parent corticosteroid and oleoyl chloride, purified by silica column chromatography, and characterized by 1H-NMR and MS. The involvement of ACAT was evaluated using potent competitive inhibitors, CP-113818 and oleic acid-2,6-diisopropylanalide (ODA) coincubated with budesonide and oleoyl coenzyme A in HLM. No significantly inhibition was observed when both inhibitors were coincubated with budesonide whereas they completely inhibited the formation of cholesteryl oleate in the control reaction indicates that ACAT is not the enzyme responsible for the in vitro oleate esterification of budesonide. Moreover the in vitro oleate esterification of budesonide appears to be optimal at pH range of 7.0-7.4 unlike the optimal pH for AEAT at pH 5.0-5.5. Thus AEAT is also not involved in the oleate esterification of budesonide therefore the oleate esterification of budesonide is catalyzed by an unidentified microsomal acyl coenzyme A:acyltransferase, which shows the favorable site of esterification at C-21 hydroxyl group rather than C-11 hydroxyl group. This result indicates a substrate specificity of this acyl coenzyme A:acyltransferase for C-21 hydroxy steroids. The kinetic parameters for budesonide 21-oleate formation were: Km 97+/-8 muM and Vmax 409+/-13 pmol/mg/min for HLM; Km 76+/-10 muM and Vmax 1167+/-26 pmol/mg/min for HLUM from smokers; and Km 86+/-10 muM and Vmax 891+/-17 pmol/mg/min for HLUM from non-smokers. The kinetic parameters of the human acyl coenzyme A:acyltransferase catalyzing budesonide oleate esterification (ABAT) shows greater activity (higher Vmax) and affinity (lower K m) in human lung microsomes compared to human liver microsomes suggest that budesonide has a better chance of being esterified in human lung than in human liver supporting the reported sustained antiasthmatic activity of budesonide.