Benzydamine plays a role in limiting inflammatory pain induced by neuronal sensitization.

Benzydamine is an active pharmaceutical compound used in the oral care pharmaceutical preparation as NSAID. Beside from its anti-inflammatory action, benzydamine local application effectively reliefs pain showing analgesic and anaesthetic properties. Benzydamine mechanism of action has been characterized on inflammatory cell types and mediators highlighting its capacity to inhibit pro-inflammatory mediators' synthesis and release. On the other hand, the role of benzydamine as neuronal excitability modulator has not yet fully explored. Thus, we studied benzydamine's effect over primary cultured DRG nociceptors excitability and after acute and chronic inflammatory sensitization, as a model to evaluate relative nociceptive response. Benzydamine demonstrated to effectively inhibit neuronal basal excitability reducing its firing frequency and increasing rheobase and afterhyperpolarization amplitude. Its effect was time and dose-dependent. At higher doses, benzydamine induced changes in action potential wavelength, decreasing its height and slightly increasing its duration. Moreover, the compound reduced neuronal acute and chronic inflammatory sensitization. It inhibited neuronal excitability mediated either by an inflammatory cocktail, acidic pH or high external KCl. Notably, higher potency was evidenced under inflammatory sensitized conditions. This effect could be explained either by modulation of inflammatory and/or neuronal sensitizing signalling cascades or by direct modulation of proalgesic and action potential firing initiating ion channels. Apparently, the compound inhibited Nav1.8 channel but had no effect over Kv7.2, Kv7.3, TRPV1 and TRPA1. In conclusion, the obtained results strengthen the analgesic and anti-inflammatory effect of benzydamine, highlighting its mode of action on local pain and inflammatory signalling.

Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties.

Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively. AncFMO1, sharing 89.5% sequence identity with human FMO1, was successfully expressed as a functional enzyme. It displayed typical FMO activities as demonstrated by oxygenating benzydamine, tamoxifen, and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the flavin adenine dinucleotide and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2'-phosphate being pushed inside the NADP+ binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.

Drug metabolite synthesis by immobilized human FMO3 and whole cell catalysts.

BACKGROUND: Sufficient reference standards of drug metabolites are required in the drug discovery and development process. However, such drug standards are often expensive or not commercially available. Chemical synthesis of drug metabolite is often difficulty due to the highly regio- and stereo-chemically demanding. The present work aims to construct stable and efficient biocatalysts for the generation of drug metabolites in vitro. RESULT: In this work, using benzydamine as a model drug, two easy-to-perform approaches (whole cell catalysis and enzyme immobilization) were investigated for the synthesis of FMO3-generated drug metabolites. The whole cell catalysis was carried out by using cell suspensions of E. coli JM109 harboring FMO3 and E. coli BL21 harboring GDH (glucose dehydrogenase), giving 1.2 g/L benzydamine N-oxide within 9 h under the optimized conditions. While for another approach, two HisTrap HP columns respectively carrying His6-GDH and His6-FMO3 were connected in series used for the biocatalysis. In this case, 0.47 g/L benzydamine N-oxide was generated within 2.5 h under the optimized conditions. In addition, FMO3 immobilization at the C-terminal (membrane anchor region) significantly improved its enzymatic thermostability by more than 10 times. Moreover, the high efficiency of these two biocatalytic approaches was also confirmed by the N-oxidation of tamoxifen. CONCLUSIONS: The results presented in this work provides new possibilities for the drug-metabolizing enzymes-mediated biocatalysis.

Expression and metabolic activity of flavin-containing monooxygenase 1 in cynomolgus macaque kidney.

Flavin-containing monooxygenase 1 (FMO1) largely remains to be characterized in cynomolgus macaque kidney. Immunoblotting showed expression of cynomolgus FMO1 in kidneys where activities of FMO1 (benzydamine N-oxygenation) were detected. No sex differences were observed in their contents or activities. These results suggest the functional role of cynomolgus FMO1 in kidney.

Hepatic Flavin-containing Monooxygenase and Aldehyde Oxidase Activities in Male Domestic Pigs at Different Ages.

BACKGROUND: Age has a significant impact on activities of hepatic metabolizing enzymes in humans and animals. Flavin-containing Monooxygenase (FMO) and Aldehyde Oxidase (AO) are two important hepatic enzymes. Understanding of the impact of age on these two enzymes is still limited in pigs. OBJECTIVE: The aim of this work was to assess hepatic FMO and AO activities of male domestic pigs at five different ages of 1 day, 2, 5, 10 and 20 weeks. METHODS: Porcine liver microsomes and cytosol were prepared from the livers of male domestic pigs at ages of 1 day, 2, 5, 10 and 20 weeks. FMO activity was assessed using N-oxidation of benzydamine in porcine liver microsomes and AO activity was evaluated using oxidation of O6-benzylguanine in the porcine liver cytosol. RESULTS: Porcine hepatic FMO activity was substantial at the age of 1 day, rapidly increased in 2 weeks, and remained high afterwards. Porcine hepatic AO activity was minimal at the age of 1 day and gradually increased to the maximum in 5 weeks and remained relatively constant to the age of 20 weeks. Porcine hepatic FMO activity is higher than other species, including humans. Age-dependent FMO developmental pattern in porcine liver is different from porcine hepatic CYP450 and human hepatic FMO. Porcine hepatic AO activity is much lower than humans although their developmental patterns are similar. CONCLUSION: Age impact on hepatic activities of both FMO and AO is obvious in domestic male pigs although age patterns of both enzymes are different.

Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice.

1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide. 3. Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.

Benzydamine N-oxygenation as an index for flavin-containing monooxygenase activity and benzydamine N-demethylation by cytochrome P450 enzymes in liver microsomes from rats, dogs, monkeys, and humans.

Benzydamine is an anti-inflammatory drug that undergoes flavin-containing monooxygenase (FMO)-dependent metabolism to benzydamine N-oxide; however, benzydamine N-demethylation is also catalyzed by liver microsomes. In this study, benzydamine N-oxygenation and N-demethylation mediated by liver microsomes from rats, dogs, monkeys, and humans were characterized comprehensively. Values of the maximum velocity/Michaelis constant ratio for benzydamine N-oxygenation by liver microsomes from dogs and rats were higher than those from monkeys and humans, despite roughly similar rates of N-demethylation in the four species. Benzydamine N-oxygenation by liver microsomes was extensively suppressed by preheating liver microsomes at 45 °C for 5 min or at 37 °C for 5-10 min without NADPH, and benzydamine N-demethylation was strongly inhibited by 1-aminbobenztriazole. Liver microsomal benzydamine N-oxygenation was inhibited by dimethyl sulfoxide and methimazole, whereas N-demethylation was inhibited by quinidine. High benzydamine N-oxygenation activities of recombinant human FMO1 and FMO3 and human kidney microsomes were observed at pH 8.4, whereas N-demethylation by cytochrome P450 2D6 was faster at pH 7.4. These results suggest that benzydamine N-oxygenation and N-demethylation are mediated by FMO1/3 and P450s, respectively, and that the contribution of FMO to metabolic eliminations of new drug candidates might be underestimated under certain experimental conditions suitable for P450 enzymes.

Potential for drug interactions mediated by polymorphic flavin-containing monooxygenase 3 in human livers.

Human flavin-containing monooxygenase 3 (FMO3) in the liver catalyzes a variety of oxygenations of nitrogen- and sulfur-containing medicines and xenobiotic substances. Because of growing interest in drug interactions mediated by polymorphic FMO3, benzydamine N-oxygenation by human FMO3 was investigated as a model reaction. Among the 41 compounds tested, trimethylamine, methimazole, itopride, and tozasertib (50 µM) suppressed benzydamine N-oxygenation at a substrate concentration of 50 µM by approximately 50% after co-incubation. Suppression of N-oxygenation of benzydamine, trimethylamine, itopride, and tozasertib and S-oxygenation of methimazole and sulindac sulfide after co-incubation with the other five of these six substrates was compared using FMO3 proteins recombinantly expressed in bacterial membranes. Apparent competitive inhibition by methimazole (0-50 µM) of sulindac sulfide S-oxygenation was observed with FMO3 proteins. Sulindac sulfide S-oxygenation activity of Arg205Cys variant FMO3 protein was likely to be suppressed more by methimazole than wild-type or Val257Met variant FMO3 protein was. These results suggest that genetic polymorphism in the human FMO3 gene may lead to changes of drug interactions for N- or S-oxygenations of xenobiotics and endogenous substances and that a probe battery system of benzydamine N-oxygenation and sulindac sulfide S-oxygenation activities is recommended to clarify the drug interactions mediated by FMO3.

Human flavin-containing monooxygenase 3 on graphene oxide for drug metabolism screening.

Human flavin-containing monooxygenase 3 (hFMO3), a membrane-bound hepatic protein, belonging to the second most important class of phase-1 drug-metabolizing enzymes, was immobilized in its active form on graphene oxide (GO) for enhanced electrochemical response. To improve protein stabilization and to ensure the electrocatalytic activity of the immobilized enzyme, didodecyldimethylammonium bromide (DDAB) was used to mimic lipid layers of biological membranes and acted as an interface between GO nanomaterial and the hFMO3 biocomponent. Grazing angle attenuated total reflectance Fourier transform infrared (GATR-FT-IR) experiments confirmed the preservation of the protein secondary structure and fold. Electrochemical characterization of the immobilized enzyme with GO and DDAB on glassy carbon electrodes was carried out by cyclic voltammetry, where several parameters including redox potential, electron transfer rate, and surface coverage were determined. This system's biotechnological application in drug screening was successfully demonstrated by the N-oxidation of two therapeutic drugs, benzydamine (nonsteroidal anti-inflammatory) and tamoxifen (antiestrogenic widely used in breast cancer therapy and chemoprevention), by the immobilized enzyme.

Determination of psychostimulants and their metabolites by electrochemistry linked on-line to flowing atmospheric pressure afterglow mass spectrometry.

The flowing atmospheric pressure afterglow (FAPA) ion source operates in the ambient atmosphere and has been proven to be a promising tool for direct and rapid determination of numerous compounds. Here we linked a FAPA-MS system to an electrochemical flow cell for the identification of drug metabolites generated electrochemically in order to study simulated metabolic pathways. Psychostimulants and their metabolites produced by electrochemistry (EC) were detected on-line by FAPA-MS. The FAPA source has never been used before for an on-line connection with liquid flow, neither for identification of products generated in an electrochemical flow cell. The system was optimized to achieve the highest ionization efficiency by adjusting several parameters, including distances and angles between the ion source and the outlet of the EC system, the high voltage for plasma generation, flow-rates, and EC parameters. Simulated metabolites from tested compounds [methamphetamine (MAF), para-methoxy-N-methylamphetamine (PMMA), dextromethorphan (DXM), and benzydamine (BAM)] were formed in the EC cell at various pH levels. In all cases the main products were oxidized substrates and compounds after N-demethylation. Generation of such products and their thorough on-line identification confirm that the cytochrome P450 - driven metabolism of pharmaceuticals can be efficiently simulated in an electrochemical cell; this approach may serve as a step towards predictive pharmacology using a fast and robust design.

Drug oxygenation activities mediated by liver microsomal flavin-containing monooxygenases 1 and 3 in humans, monkeys, rats, and minipigs.

Liver microsomal flavin-containing monooxygenases (FMO, EC 1.14.13.8) 1 and 3 were functionally characterized in terms of expression levels and molecular catalytic capacities in human, cynomolgus monkey, rat, and minipig livers. Liver microsomal FMO3 in humans and monkeys and FMO1 and FMO3 in rats and minipigs could be determined immunochemically with commercially available anti-human FMO3 peptide antibodies or rat FMO1 peptide antibodies. With respect to FMO-dependent N-oxygenation of benzydamine and tozasertib and S-oxygenation of methimazole and sulindac sulfide activities, rat and minipig liver microsomes had high maximum velocity values (Vmax) and high catalytic efficiency (Vmax/Km, Michaelis constant) compared with those for human or monkey liver microsomes. Apparent Km values for recombinantly expressed rat FMO3-mediated N- and S-oxygenations were approximately 10-100-fold those of rat FMO1, although these enzymes had similar Vmax values. The mean catalytic efficiencies (Vmax/Km, 1.4 and 0.4 min(-1)µM(-1), respectively) of recombinant human and monkey FMO3 were higher than those of FMO1, whereas Vmax/Km values for rat and minipig FMO3 were low compared with those of FMO1. Minipig liver microsomal FMO1 efficiently catalyzed N- and S-oxygenation reactions; in addition, the minipig liver microsomal FMO1 concentration was higher than the levels in rats, humans, and monkeys. These results suggest that liver microsomal FMO1 could contribute to the relatively high FMO-mediated drug N- and S-oxygenation activities in rat and minipig liver microsomes and that lower expression of FMO1 in human and monkey livers could be a determinant factor for species differences in liver drug N- and S-oxygenation activities between experimental animals and humans.

Effects of salinity acclimation on the pesticide-metabolizing enzyme flavin-containing monooxygenase (FMO) in rainbow trout (Oncorhynchus mykiss).

Thioether-containing pesticides are more toxic in certain anadromous and catadromous fish species that have undergone acclimation to hypersaline environments. Enhanced toxicity has been shown to be mediated through the bioactivation of these xenobiotics by one or more flavin-containing monooxygenases (FMOs), which are induced by hyperosmotic conditions. To better understand the number of FMO genes that may be regulated by hyperosmotic conditions, rainbow trout (Oncorhynchus mykiss) were maintained and acclimated to freshwater (<0.5 g/L salinity) and to 18 g/L salinity. The expression of 3 different FMO transcripts (A, B and C) and associated enzymatic activities methyl p-tolyl sulfoxidation (MTSO) and benzydamine N-oxigenation (BZNO) were measured in four tissues. In freshwater-acclimated organisms FMO catalytic activities were as follows: liver>kidney>gills=olfactory tissues; in hypersaline-acclimated animals activities were higher in liver>gills>olfactory tissues>kidney. Acclimation to 18 g/L caused a significant induction in the stereoselective formation of R-MTSO in gill. In olfactory tissues, stereoselective (100%) formation of S-MTSO was observed and was unaltered by acclimation to hypersaline water. When specific transcripts were evaluated, salinity-acclimation increased FMO A in liver (up to 2-fold) and kidney (up to 3-fold) but not in olfactory tissues and gills. FMO B mRNA was significantly down-regulated in all tissues, and FMO C was unchanged by hypersaline acclimation. FMO B and C failed to correlate with any FMO catalytic activity, but FMO A mRNA expression linearly correlated to both FMO catalytic activities (MTSO and BZNO) in liver (r(2)=0.92 and r(2)=0.88) and kidney microsomes (r(2)=0.93 and r(2)=90). FMO A only correlated with MTSO activity in gills (r(2)=0.93). These results indicate unique tissue specific expression of FMO genes in salmonids and are consistent with salinity-mediated enhancement of thioether-containing pesticide bioactivation by FMO which may occur in liver or kidney after salinity acclimation.

Entrapment of human flavin-containing monooxygenase 3 in the presence of gold nanoparticles: TEM, FTIR and electrocatalysis.

BACKGROUND: Nanosized particles of gold are widely used as advanced materials for enzyme catalysis investigations. In some bioanalytical methods these nanoparticles can be exploited to increase the sensitivity by enhancing electron transfer to the biological component i.e. redox enzymes such as drug metabolizing enzymes. METHODS: In this work, we describe the characterization of human flavin-containing monooxygenase 3 (hFMO3) in a nanoelectrode system based on AuNPs stabilized with didodecyldimethylammonium bromide (DDAB) on glassy carbon electrodes. Once confirmed by FTIR spectroscopy that in the presence of DDAB-AuNPs the structural integrity of hFMO3 is preserved, the influence of AuNPs on the electrochemistry of the enzyme was studied by cyclic voltammetry and square wave voltammetry. RESULTS: Our results show that AuNPs improve the electrochemical performance of hFMO3 on glassy carbon electrodes by enhancing the electron transfer rate and the current signal-to-noise ratio. Moreover, the electrocatalytic activity of hFMO3-DDAB-AuNP electrodes which was investigated in the presence of two well known substrates, benzydamine and sulindac sulfide, resulted in K(M) values of 52µM and 27µM, with V(max) of 8nmolmin(-1)mg(-1) and 4nmolmin(-1)mg(-1), respectively, which are in agreement with data obtained with the microsomal enzyme. CONCLUSIONS: The immobilization of hFMO3 protein in DDAB stabilized AuNP electrodes improves the bioelectrochemical performance of this important phase I drug metabolizing enzyme. GENERAL SIGNIFICANCE: This bio-analytical method can be considered as a promising advance in the development of new techniques suitable for the screening of novel hFMO3 metabolized pharmaceuticals.

In vitro drug metabolism by C-terminally truncated human flavin-containing monooxygenase 3.

Human flavin-containing monooxygenase 3 (hFMO3) is a microsomal drug-metabolizing monooxygenase that catalyzes the NADPH-dependent oxygenation of a wide range of drugs and xenobiotics which contain a soft-nucleophiles, usually sulfur or nitrogen. As the release from the microsomal membranes can facilitate the in vitro experimental determination of drug metabolism by hFMO3, in this work we identified and eliminated the membrane anchoring sequence without affecting the activity of the enzyme and producing a soluble active enzyme. The truncated hFMO3 carrying a C-terminal deletion of 17 amino acids (tr-hFMO3) was expressed and purified from the cytosolic fraction. The tr-hFMO3 proves to be detached from the membrane, properly folded and fully active towards well-known marker substrates such as benzydamine and sulindac sulfide with measured apparent K(m) values of 45 ± 8 µM and 25 ± 4 µM, respectively. Its activity was further tested with newly discovered Aurora kinase inhibitors, Tozasertib and Danusertib, and compared to those of the wild type enzyme. The use of this soluble form of the hFMO3 enzyme as opposed to the usual microsomal preparations is advantageous for in vitro drug metabolism studies that are a requirement in the early phases of drug development by pharmaceutical industry.

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

Benzydamine (BZ), a weak base and an indazole derivative with analgesic and antipyretic properties used in human and veterinary medicine, is metabolized in human, rat, cattle and rabbit to a wide range of metabolites. One of the main metabolites, BZ N-oxide (BZ-NO), is produced in the liver and brain by flavin-containing monooxygenases (FMOs), by liver and brain enzymes. To evaluate the suitability of BZ as an FMO probe in veterinary species, BZ metabolism was studied in vitro using liver microsomes from bovine, rabbit and swine. Kinetic parameters, K(m) and V(max), of BZ-NO production, were evaluated to corroborate the pivotal role of FMOs. Inhibition studies were carried out by heat inactivation and by specific FMO chemical inhibitors: trimethylamine and methimazole. The results confirmed the presence of FMO activity in the liver and the role of BZ as a suitable marker of FMO enzyme activities for the veterinary species considered.

Direct electrochemistry of drug metabolizing human flavin-containing monooxygenase: electrochemical turnover of benzydamine and tamoxifen.

This communication reports on the first electrochemical study of the human flavin-containing monooxygenase 3 (hFMO3) either absorbed or covalently linked to different electrode surfaces. Glassy carbon and gold electrodes gave reversible electrochemical signals of an active hFMO3. The midpoint potential measured for the immobilized enzyme on a glassy carbon electrode was -445 +/- 8 mV (versus Ag/AgCl). A monolayer coverage was obtained on gold functionalized with dithio-bismaleimidoethane that covalently linked surface accessible cysteines of hFMO3. A structural model of the enzyme was generated to rationalize electrochemistry results. The turnover of the active enzyme was measured with two specific drugs: tamoxifen and benzydamine. For tamoxifen, 1.7 and 8.0 microM of its N-oxide product were formed by the enzyme immobilized on glassy carbon and gold electrodes, respectively. In the case of benzydamine, a K(M) of 44 +/- 5 microM was measured upon application of a -600 mV bias to the enzyme immobilized on the glassy carbon electrode that is in good agreement with the values published for microsomal hFMO3 where NADPH is the electron donor.

Inter-individual variation in flavin-containing monooxygenase 3 in livers from Japanese: correlation with hepatic transcription factors.

Human flavin-containing monooxygenase 3 (FMO3)-mediated microsomal oxygenation activity, levels of FMO3 protein and FMO3 mRNA and modifications were investigated in Japanese livers genotyped for the FMO3 gene. Significant correlations were observed for benzydamine N-oxygenation or methyl p-tolyl sulfide S-oxygenation activity (in the range of approximately 20- to approximately 40-fold) and FMO3 levels determined immunochemically in liver microsomes (r(2)=0.73-0.75, p<0.0001, n=16). Preincubation with the reducing agent ascorbate revealed that FMO3 activity in some liver samples is suppressed. Microsomal FMO3 protein content (approximately 40-fold) was correlated with FMO3 mRNA levels (r(2)=0.55, p=0.0010, n=16), but FMO3 haplotypes did not affect FMO3 mRNA expression (approximately 100-fold) under the conditions used. FMO3 mRNA levels were multivariately correlated with trans-acting factors, i.e. hepatic nuclear factor 4 (HNF-4) mRNA and nuclear factor Y box-binding protein (NF-Y) mRNA (r(2)=0.31, p=0.0017, n=37). These results suggest that considerable individual differences in FMO3 levels may exist in Japanese livers. The liver-enriched transcription factor HNF-4 appears to be a determinant of FMO3 expression in livers, as well as the ubiquitous factor NF-Y.

Effect of genetic variants of the human flavin-containing monooxygenase 3 on N- and S-oxygenation activities.

The decreased capacity of the flavin-containing monooxygenase 3 (FMO3) to oxygenate xenobiotics including trimethylamine is believed to contribute to metabolic disorders. The aim of this study was to functionally characterize FMO3 variants recently found in a Japanese population and compare them with selective functional activity of other FMO3 variants. Recombinant Glu158Lys and Glu158Lys-Glu308Gly FMO3 expressed in Escherichia coli membranes showed slightly decreased N-oxygenation of benzydamine and trimethylamine. Selective functional S-oxygenation of these variants by methyl p-tolyl sulfide or sulindac sulfide was comparable to that of wild-type FMO3. The Glu158Lys-Thr201Lys-Glu308Gly and Val257Met-Met260Val variants showed significantly decreased oxygenation of typical FMO3 substrates (i.e., approximately one-tenth of the V(max)/K(m) values). Val257Met FMO3 had a lower catalytic efficiency for methyl p-tolyl sulfide and sulindac sulfide S-oxygenation. However, compared with wild-type FMO3, Val257Met FMO3 showed a similar catalytic efficiency for N-oxygenation of benzydamine and trimethylamine. The catalytic efficiency for benzydamine and trimethylamine N-oxygenation by Arg205Cys FMO3 was only moderately decreased, but it possessed decreased sulindac sulfide S-oxygenation activity. Kinetic analysis showed that Arg205Cys FMO3 was inhibited by sulindac in a substrate-dependent manner, presumably because of selective interaction between the variant enzyme and the substrate. The results suggest that the effects of genetic variation of human FMO3 could operate at the functional level for N- and S-oxygenation for typical FMO3 substrates. Genetic polymorphism in the human FMO3 gene might lead to unexpected changes of catalytic efficiency for N- and S-oxygenation of xenobiotics and endogenous materials.

Benzydamine N-oxygenation as a measure of flavin-containing monooxygenase activity.

Benzydamine is a nonsteroidal anti-inflammatory drug that undergoes flavin-containing monooxygenase (FMO)-dependent metabolism to a stable N-oxide. This metabolite can be quantified with high specificity and sensitivity by using a simple reverse-phase high-performance liquid chromatography (HPLC) assay with fluorescence detection. Studies with recombinant FMO enzymes demonstrate that FMOI and FMO3 are the primary catalysts of benzydamine N-oxygenation, with minimal contributions from cytochrome P450 enzymes. Investigations conducted with human liver microsomes confirm that FMO3, in large part, is responsible for benzydamine N-oxide formation in this tissue. These features render benzydamine a useful in vitro probe for FMO activity in a wide range of tissues and cell types. In addition, benzydamine appears to be a suitable in vivo probe for human liver FMO3. This chapter provides a detailed account of the experimental protocol for determining rates of formation of benzydamine N-oxide by FMO-containing enzyme fractions.

Effect of lipophilic counter-ions on membrane diffusion of benzydamine.

Many topically applied drugs are ionized molecules that exhibit poor penetration across the lipid domains of the stratum corneum. Reduction of the charge on the molecule would be expected to enhance skin penetration. The objective of this study was to investigate the interaction of the non-steroidal anti-inflammatory drug benzydamine hydrochloride with suitable counter-ions including ibuprofen sodium. The influence of pH of the donor solution and hence degree of ionization on partitioning between n-octanol:buffer and the flux of benzydamine hydrochloride across polydimethyl siloxane (PDMS) membrane and human epidermis was determined. The maximum flux was determined at pH 7.6 when the fraction unionized was 2.51%, rather than at pH 9 when the fraction unionized was 38.7%. This suggests that at higher pH, although the permeability coefficient is increased, the decrease in solubility and therefore concentration of dissolved benzydamine in the medium results in a decrease in flux across the PDMS membrane. Ion-pair formation or interaction with each of the counter-ions was confirmed by NMR spectroscopy. Significant increases in logP and flux across PDMS membrane were determined for the ion-pairs (0.087, 12.54, 11.31, 0.121 microg cm(-2)h(-1) for benzydamine hydrochloride and ion-pairs with ibuprofen sodium, sodium benzoate and sodium octane sulfonate respectively). This study shows that it is possible to significantly enhance the flux of salts across a lipophilic membrane in the presence of counter-ions, resulting from intermolecular interaction and/or ion-pair formation.