The role of aldehyde oxidase and xanthine oxidase in the biotransformation of a novel negative allosteric modulator of metabotropic glutamate receptor subtype 5

Abstract

Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu₅) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu₅. Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (+16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of ¹⁸O-labeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because ¹⁸O was incorporated into M1 (+18 Da) as well as in a secondary metabolite (M2; +36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates.

Scheme 1.

Proposed metabolism of VU0409106 in vitro in rat (r), monkey (m), and human (h) and in vivo in rat plasma (R).

Fig. 1.

Comparison of ¹H NMR of M1 (A) and VU0409106 (B).

Fig. 2.

¹H-¹³C HSQC (A) and ¹H-¹H COSY (B) of M1 in DMSO-d₆ support oxidation at C-6 of the pyrimidine moiety.

Fig. 3.

Representative LC/MS total ion current (TIC) (A) of extracts from a rat hepatic S9 incubation of VU0409106 and LC-UV chromatograms (overlay) of extracts from rat (B), dog (C), monkey (D), and human (E) hepatic S9 incubations of VU0409106 showing principal metabolites and the species differences in metabolism. An LC-UV trace of rat plasma (F) shows additional metabolites not detected in vitro.

Scheme 2.

Metabolism of the methylated analog VU0465585 (A) and the fluoropyridine analog VU0458442 (B) in rat S9 indicates the sensitivity of AO and XO metabolism to pyrimidine substitution.

Fig. 4.

Raloxifene (100 μM, AO inhibitor), hydralazine (100 μM, AO inhibitor), and allopurinol (100 μM, XO inhibitor) inhibit the metabolism of VU0409106 metabolism in rat (A) and human (B) hepatic S9 (−NADPH), effectively reducing the formation of M1 (percentage of control).

Fig. 5.

The selective XO inhibitor allopurinol (100 μM), but not the AO inhibitors (raloxifene or hydralazine, 100 μM), prevent the biotransformation of M1 to M2 in rat hepatic S9 (M2 formation as percentage of control).

Fig. 6.

Mass spectra of M1 (A) and M2 (B) from rat hepatic S9 incubations performed in H₂¹⁸O indicate the incorporation of ¹⁸O into the respective metabolites. The insets to A and B are MS/MS spectra of ¹⁸O-labeled metabolites and provide evidence for regiochemical insertion of ¹⁸O into M1 and M2.