Dual mechanisms suppress meloxicam bioactivation relative to sudoxicam

Abstract

Thiazoles are biologically active aromatic heterocyclic rings occurring frequently in natural products and drugs. These molecules undergo typically harmless elimination; however, a hepatotoxic response can occur due to multistep bioactivation of the thiazole to generate a reactive thioamide. A basis for those differences in outcomes remains unknown. A textbook example is the high hepatotoxicity observed for sudoxicam in contrast to the relative safe use and marketability of meloxicam, which differs in structure from sudoxicam by the addition of a single methyl group. Both drugs undergo bioactivation, but meloxicam exhibits an additional detoxification pathway due to hydroxylation of the methyl group. We hypothesized that thiazole bioactivation efficiency is similar between sudoxicam and meloxicam due to the methyl group being a weak electron donator, and thus, the relevance of bioactivation depends on the competing detoxification pathway. For a rapid analysis, we modeled epoxidation of sudoxicam derivatives to investigate the impact of substituents on thiazole bioactivation. As expected, electron donating groups increased the likelihood for epoxidation with a minimal effect for the methyl group, but model predictions did not extrapolate well among all types of substituents. Through analytical methods, we measured steady-state kinetics for metabolic bioactivation of sudoxicam and meloxicam by human liver microsomes. Sudoxicam bioactivation was 6-fold more efficient than that for meloxicam, yet meloxicam showed a 6-fold higher efficiency of detoxification than bioactivation. Overall, sudoxicam bioactivation was 15-fold more likely than meloxicam considering all metabolic clearance pathways. Kinetic differences likely arise from different enzymes catalyzing respective metabolic pathways based on phenotyping studies. Rather than simply providing an alternative detoxification pathway, the meloxicam methyl group suppressed the bioactivation reaction. These findings indicate the impact of thiazole substituents on bioactivation is more complex than previously thought and likely contributes to the unpredictability of their toxic potential.

Keywords: Bioactivation; Kinetics; Liver toxicity; Meloxicam; Sudoxicam; Thiazole.

Fig. 1.. Thiazole test compounds with epoxidation model predictions.

Epoxidation predictions made using the Xenosite epoxidation model 2.0 for sudoxicam (Panel A) and meloxicam (Panel B). Individual bonds are scored from 0 to 1, with higher scores indicating higher likelihood of epoxidation.

Fig. 2.. Epoxidation model outputs versus electron density of sudoxicam C5 substituted derivatives.

Sudoxicam derivatives were digitally constructed with 34 different substituents at the C5 position of the thiazole ring. Panel A: Epoxidation model outputs (y-axis) were grouped based on electron-donating/withdrawing strength of the C5 thiazole substituent (x-axis). Panel B: QM model predicted C4-C5 bond orders (y-axis) were grouped based on electron-donating/withdrawing strength of the C5 thiazole substituent (x-axis). Panel C: pKa predictions for the thiazole nitrogen atom (y-axis) were grouped based on electron-donating/withdrawing strength of the C5 thiazole substituent (x-axis). Panel D: Epoxidation model outputs (y-axis) were plotted as a function of QM model bond order predictions (x-axis) with points grouped based on identity of the alpha position atom. Panel E: Epoxidation model outputs (y-axis) were plotted as a function of pKa predictions for the thiazole nitrogen atom (x-axis) with points grouped based on identity of the alpha position atom. Panel F: QM model predictions (y-axis) and pKa values (x-axis) were plotted for comparison.

Fig. 3.. Chemical inhibition of bioactivation pathways.

As described in Experimental Procedures, alpha-dicarbonyl metabolite formation rates were measured for substrate reactions with human liver microsomes. Sudoxicam (Panel A) and meloxicam (Panel B) reactions at 40 μM (pink) and 400 μM (red) concentrations were conducted with 1 μM elaidamide for microsomal epoxide hydrolase inhibition. Coincubation with 1 mM 1-aminobenzotriazole was conducted with 400 μM sudoxicam (Panel C) and meloxicam (Panel D) for general cytochrome P450 inhibition. Each reaction was conducted in six to nine replicates. Alpha-dicarbonyl formation rates are reported as percentages normalized to rates in uninhibited reactions. Average non-inhibited rates (in pmol/min/mg enzyme) were 12 for 40 μM sudoxicam, 21 for 400 μM sudoxicam, 5.0 for 40 μM meloxicam, and 7.6 for 400 μM meloxicam. Significant differences between inhibited reaction rates and the controls were determined based on p < 0.05 calculated using Student’s t-test. Error bars denote standard error.

Fig. 4.. Steady state kinetic profiles for metabolites of sudoxicam and meloxicam.

Alpha-dicarbonyl metabolites from the thiazole bioactivation pathway were observed for sudoxicam and meloxicam reactions by human liver microsomes. Glyoxal (red circles) from sudoxicam (Panel A) and methylglyoxal (red squares) from meloxicam (Panel B) were detected using DMB labeling. The 5-hydroxymethyl-meloxicam metabolite (blue squares) from the meloxicam detoxification pathway (Panel C) was measured by mass detection (m/z 368). Metabolites were quantitated using standard curves to calculate initial rates (pmol/min/mg protein) as a function of substrate concentration (μM). Data for all reactions were fit best to a biphasic Michaelis-Menten equation or a single Michaelis-Menten equation using Akaike information criterion for small sample sizes (AICc), and the corresponding constants are reported in Table 2. Experimental reactions for each substrate concentration were replicated between nine and twenty-four times. Analysis was conducted as described in Materials and Methods.

Scheme 1.. Metabolic pathways for meloxicam and sudoxicam.

Both meloxicam and sudoxicam undergo a bioactivation pathway (red) at the thiazole group initiated by epoxidation of the C4-C5 double bond, followed by hydrolysis and ring cleavage forming the protoxin thioamide and an alpha-dicarbonyl. Meloxicam undergoes a unique detoxification pathway (blue) in which its C5 methyl group is hydroxylated. The metabolite can be directly eliminated or further oxidized and eliminated as a carboxylic acid.