Biotransformation of benzydamine by microsomes and precision-cut slices prepared from cattle liver

Abstract

1. Benzydamine (BZ), a non-steroidal anti-inflammatory drug used in human and veterinary medicine, is not licensed for use in food-producing species. Biotransformation of BZ in cattle has not been reported previously and is investigated here using liver microsomes and precision-cut liver slices. 2. BZ was metabolized by cattle liver microsomes to benzydamine N-oxide (BZ-NO) and monodesmethyl-BZ (Nor-BZ). Both reactions followed Michaelis-Menten kinetics (Km = 76.4 +/- 16.0 and 58.9 +/- 0.4 microM Vmax = 6.5 +/- 0.8 and 7.4 +/- 0.5 nmolmg(-1) min(-1) respectively); sensitivity to heat and pH suggested that the N-oxidation is catalysed by the flavin-containing monooxygenases. 3. BZ-NO and Nor-BZ were the most abundant products derived from liver slice incubations, and nine other BZ metabolites were found and tentatively identified by LC-MS. Desbenzylated and hydroxylated BZ-NO analogues and a hydroxylated product of BZ were detected, which have been reported in other species. Product ion mass spectra of other metabolites, which are described here for the first time, indicated the formation of a BZ N- -glucuronide and five hydroxylated and N+-glucuronidated derivatives of BZ, BZ-NO and Nor-BZ. 4. The results indicate that BZ is extensively metabolized in cattle. Clearly, differences in metabolism compared with, for example, rat and human, will need to be considered in the event of submission for marketing authorization for use in food animals.

Benzydamine as a useful substrate of hepatic flavin‐containing monooxygenase activity in veterinary species

“…As the K m value (27.18 μ m ) in bovine microsomes is similar to that of swine (20.1 μ m ), we could support the hypothesis of the presence of FMO1 also in the bovine liver, but more evidence is necessary. The K m value was different from that reported in our previous experiment (Santi et al. , 2002): this could be related to the more restricted BZ concentration range adopted (from 2.5 to 250 μ m instead of from 2.5 to 1000 μ m ) to perform the inhibition studies (Kakkar et al.…”

“…The microsome incubation protocol was based on the one previously described by Santi et al (2002). The incubation mixture contained 0.2 mg ⁄ mL microsomal protein, 1 mM NADPH and 20 lM MgCl 2 , BZ (2.5, 5, 10, 25, 50, 100 and 250 lM) in a final volume of 0.4 mL with 0.1 M phosphate buffer (pH 7.4).…”

“…FMOs involvement in drug metabolism was demonstrated in vitro using sheep liver microsomes (Demirdöğen & Adali, 2005; Virkel et al. , 2006), bovine liver subcellular fractions and precision‐cut slices (Santi et al. , 2002) and with rat, hamster, rabbit and dog primary hepatocytes, (Ubead et al ., 1999) and also using microsomes from the liver, kidney, brain cortex and upper portion of small intestine of camel (Raza et al.…”

“…Until now, FMOs had been purified from rabbit liver (FMO1, 2, 3) and lung (FMO2) and from pig liver (FMO1) and sheep liver (FMO1, 3), (Krueger & Williams, 2005) and from dog liver (FMO1 and FMO3) (Lattard et al, 2002;Lickteig et al, 2009). FMOs involvement in drug metabolism was demonstrated in vitro using sheep liver microsomes (Demirdögen & Adali, 2005;Virkel et al, 2006), bovine liver subcellular fractions and precision-cut slices (Santi et al, 2002) and with rat, hamster, rabbit and dog primary hepatocytes, (Ubead et al, 1999) and also using microsomes from the liver, kidney, brain cortex and upper portion of small intestine of camel (Raza et al, 2004). The results reported in those studies were obtained, in several cases, using indirect and nonstandardized analytical methods (Longin-Sauvageon et al, 1998;Raza et al, 2004;Demirdögen & Adali, 2005).…”

Flavin-Containing Monooxygenase Activity in Hepatocytes and Microsomes: In Vitro Characterization and In Vivo Scaling of Benzydamine Clearance

“…This strongly suggests that the heat lability observed for human liver FMO is a consistent characteristic in other preclinical species, and heat lability is a useful and general method for differentiating between FMO and P450 in microsomal incubations. This is used routinely for human liver microsomes (Grothusen et al, 1996), and has been reported for rat, mouse, and cow (Kedderis and Rickert, 1985;Venkatesh et al, 1992;Blake et al, 1995;Ring et al, 1999;Santi et al, 2002). We extend this analysis here for other traditional preclinical species (i.e., dog and monkey).…”