Investigating the Contribution of Major Drug-Metabolising Enzymes to Possum-Specific Fertility Control

Abstract

The potential to improve the effectiveness and efficiency of potential oestrogen-based oral contraceptives (fertility control) for possums was investigated by comparing the inhibitory potential of hepatic CYP3A and UGT2B catalytic activity using a selected compound library (CYP450 inhibitor-based compounds) in possums to that of three other species (mouse, avian, and human). The results showed higher CYP3A protein levels in possum liver microsomes compared to other test species (up to a 4-fold difference). Moreover, possum liver microsomes had significantly higher basal p-nitrophenol glucuronidation activity than other test species (up to an 8-fold difference). However, no CYP450 inhibitor-based compounds significantly decreased the catalytic activity of possum CYP3A and UGT2B below the estimated IC₅₀ and 2-fold IC₅₀ values and were therefore not considered to be potent inhibitors of these enzymes. However, compounds such as isosilybin (65%), ketoconazole (72%), and fluconazole (74%) showed reduced UGT2B glucuronidation activity in possums, mainly at 2-fold IC₅₀ values compared to the control (p < 0.05). Given the structural features of these compounds, these results could provide opportunities for future compound screening. More importantly, however, this study provided preliminary evidence that the basal activity and protein content of two major drug-metabolising enzymes differ in possums compared to other test species, suggesting that this could be further exploited to reach the ultimate goal: a potential target-specific fertility control for possums in New Zealand.

Keywords: Trichosurus vulpecula; brushtail possum; drug-metabolising enzymes (CYP3A and UGT2B); fertility control.

Figure 1

Basal hepatic CYP3A activity and protein levels in mice, possums, avians, and humans. Liver microsomes of each species were used to determine CYP3A activity via erythromycin N-demethylation. (A). Values are expressed in nmol/mg/min. Bars represent the mean ± SEM of n = 6 in triplicate (mouse and possum), n = 3 in triplicate (avian), and n = 2 in duplicate (pooled human liver microsomes). # Significantly different compared to possum and avian, p < 0.05. The protein content for CYP3A4 in liver microsomes from 4 species was determined by Western blotting (load of protein in each well was 2 µg). Hepatic microsomes were also subjected to CYP3A4 Western blotting and the scanning densitometry results were normalised to the housekeeping protein GAPDH. (B) The bars represent the mean ± SEM of optical density of n = 3 (except pooled human microsomes, n = 1). (*_a) Significantly different compared to avians and (*_b) significantly different compared to mice, p < 0.05. Representative Western blot (C).

Figure 2

Effect of CYP450 inhibitor-based compounds on CYP3A catalytic activity in possum and mouse liver microsomes. Possum (A) and mouse (B) liver microsomes were incubated with 0.1 M phosphate buffer and CYP450 inhibitor-based compounds, and erythromycin N-demethylation was determined as an indicator of CYP3A inhibitory catalytic activity at estimated IC₅₀ and 2-fold IC₅₀ values. The dotted line shows the 50% control of CYP3A enzymes. The bars represent the mean ± SEM of n = 3 in duplicate in percent control. * Significantly different from the respective solvent control. Abbreviated compound names refer to WAY-325945 (WAY-3259), ketoconazole (Keto), cobicistat (Cobi), terfenadine (Terf), benzbromarone (Benz), WAY-657644 (WAY-6576), ritonavir (Rito), gentiopicroside (Genti), WAY-325412 (WAY-3254), chlorzoxazone (Chlor), galeterone (Gale), and alizarin (Ali).

Figure 2

Effect of CYP450 inhibitor-based compounds on CYP3A catalytic activity in possum and mouse liver microsomes. Possum (A) and mouse (B) liver microsomes were incubated with 0.1 M phosphate buffer and CYP450 inhibitor-based compounds, and erythromycin N-demethylation was determined as an indicator of CYP3A inhibitory catalytic activity at estimated IC₅₀ and 2-fold IC₅₀ values. The dotted line shows the 50% control of CYP3A enzymes. The bars represent the mean ± SEM of n = 3 in duplicate in percent control. * Significantly different from the respective solvent control. Abbreviated compound names refer to WAY-325945 (WAY-3259), ketoconazole (Keto), cobicistat (Cobi), terfenadine (Terf), benzbromarone (Benz), WAY-657644 (WAY-6576), ritonavir (Rito), gentiopicroside (Genti), WAY-325412 (WAY-3254), chlorzoxazone (Chlor), galeterone (Gale), and alizarin (Ali).

Figure 3

Effect of CYP450 inhibitor-based compounds on CYP3A catalytic activity in mouse and possum liver microsomes. Possum and mouse liver microsomes were incubated with 0.1 M phosphate buffer and CYP450 inhibitor-based compounds, and erythromycin N-demethylation was determined at a higher concentration (10 µM) as an indicator of CYP3A inhibitory catalytic activity. The bars represent the mean ± SEM of n = 3 in duplicate. Data were analysed using one-way ANOVA and none were statistically different compared to controls. Abbreviated compound names refer to alizarin (Ali), galeterone (Gale), cobicistat (Cobi), gentiopicroside (Genti), chlorzoxazone (Chlor), and benzbromarone (Benz).

Figure 4

P-nitrophenol glucuronidation activity in mouse, possum, avian and human liver microsomes. Liver microsomes of each species were incubated with 0.1 M phosphate buffer and <10% DMSO, and the p-nitrophenol glucuronidation activity was determined as an indicator of UGT glucuronidation activity (A). Values are expressed as µmol/mg/min. Bars represent the mean ± SEM of n = 6 in triplicate for possum and mouse, n = 3 in triplicate for avian, and n = 2 in duplicate for pooled human liver microsomes. (*_a) Significantly different compared to avian and (*_b) significantly different from mouse, p < 0.05. The protein content for UGT2B4 in hepatic microsomes from 4 species was determined by Western blotting (load of protein in each well was 2 µg). UGT2B4 Western blots (B) and scanning densitometry results were normalised to the housekeeping protein GAPDH (C). The bars represent the mean ± SEM of optical density from n = 3 (except for pooled human liver microsomes, n = 1). * Significantly different compared to solvent control, p < 0.05.

Figure 5

Compound-mediated inhibition of p-nitrophenol glucuronidation activity in possum and mouse liver microsomes. Liver microsomes of possums (A) and mice (B) were incubated with 0.1 M phosphate buffer and inhibitory compounds and p-nitrophenol glucuronidation activity was determined as an indicator of UGT glucuronidation activity. * Significantly different compared to solvent control, p < 0.05. The bars represent the mean ± SEM of n = 3 in duplicate in percent control. Abbreviated compound names refer to isosilybin (Isosi), ketoconazole (Keto), fluconazole (Fluco), and cyclosporin A (Cyclo).