CYP1A1 and 1B1-mediated metabolic pathways of dolutegravir, an HIV integrase inhibitor

Abstract

Dolutegravir (DTG), a potent integrase inhibitor, is part of a recommended initial regimen for the treatment of human immunodeficiency virus (HIV). Prior reports demonstrated that the clearance of DTG was higher in current smokers than non-smokers, but the mechanism remains unclear. Using a metabolomic approach, M4 (an aldehyde) was identified as a novel metabolite of DTG. In addition, the formation of M4 was found to be mediated by cytochrome P450 (CYP) 1A1 and 1B1, the enzymes that can be highly induced by cigarette smoking. CYP1A1 and 1B1 were also identified as the major enzymes contributing to the formation of M1 (an N-dealkylated metabolite of DTG) and M5 (an aldehyde). Furthermore, the production of M1 and M4 was significantly increased in the lung of mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin, an inducer of CYP1A1 and 1B1. In summary, the current study uncovered the CYP1A1 and 1B1-mediated metabolic pathways of DTG. These data suggest that persons with HIV infection receiving DTG should be cautious to cigarettes, and drugs, or exposure to environmental chemicals that induce CYP1A1 and 1B1.

Keywords: CYP1A1; CYP1B1; Dolutegravir; HIV; Metabolism.

Fig. 1.. Metabolomic screening of DTG metabolites in human liver microsomes (HLM).

Three groups of DTG incubations were conducted: HLM_NADPH, DTG_NADPH, and DTG_HLM_NADPH. All incubation samples were analyzed by UPLC-QTOFMS. (A) Separation of incubation samples in a PLS-DA score plot. (B ) A loading S-plot generated by the PLS-DA analysis. The top ranking ions were identified as DTG metabolites (M1, M2 and M3). (C) Chromatograms and structures of M1, M2 and M3.

Fig. 2.. Identification of M1-M3.

These metabolites were identified by UPLC- QTOFMS in the incubation of DTG with HLM. (A) MS/MS spectrum of M1. (B) MS/MS spectrum of M2. (C) MS/MS spectrum of M3. The structures of M1-M3 with fragmental patterns are inlaid in the spectra.

Fig. 3.. Metabolomic screening of DTG metabolites in HLM using NH₂OMe as a trapping regent.

Four groups of DTG incubations were conducted: DTG_NH₂OMe_NADPH, DTG_HLM_NADPH, DTG_HLM_NH₂OMe, and DTG_HLM_NH₂OMe_NADPH. All incubation samples were analyzed by UPLC- QTOFMS. (A) Separation of incubation samples in a PLS-DA score plot. (B) A trend plot of M4-NH₂OMe generated by the PLS-DA analysis. (C) Chromatogram of M4- NH₂OMe. (D) MS/MS spectrum and structure of M4-NH₂OMe.

Fig. 4.. Identification of M4 by UPLC-QTOFMS.

(A) The scheme for the formation of oximes M4-NH₂OMe and M4-semicarbazide from aldehyde M4 using NH₂OMe and semicarbazide as trapping agents, respectively. The aldehyde group reacted with the amine group to form an imine product. (B) MS/MS spectrum of M4-semicarbazide. The structure of M4-semicarbazide with fragmental patterns is inlaid in the spectrum.

Fig. 5.. Role of CYPs in Ml formation.

(A, B) Ml production in the incubations of DTG at 30 μM (A) and 5 μM (B) with recombinant CYPs, respectively. The relative abundance of M1 from the incubation with CYP1A1 was set as 100%. (C) Enzymatic kinetic plots of CYP1A1, 1B1 and 3A4 for M1 formation. The concentrations of DTG were 0, 5, 15, 30, 100, 200, and 300 μM. (D) The enlarged CYP3A4 kinetic plot for M1 production. (E) Effect of TMS on M1 formation in the incubations with CYP1A1 and 1B1. TMS was used as an inhibitor of CYP1A1 and 1B1. The relative abundance of M1 in control group was set as 100%. The enzymatic kinetic study was conducted in duplicate and other incubations were conducted in triplicate. All data are expressed as means ± S.D. **p < 0.05, ***p < 0.001 vs control.

Fig. 6.. Role of CYPs in formation of M2(A) and M3 (B).

The incubations were conducted with DTG (30 μM) and recombinant CYPs. The relative abundance of M2 and M3 from the incubation with CYP3A4 was set as 100%. All the data are expressed as means ± S.D. (n = 3).

Fig. 7.. Identification of M5 by UPLC-QTOFMS.

(A) The scheme for the formation of oxime M5-INH from aldehyde M5 using the trapping agent INH. The hydrazine group of INH can react with the aldehyde group of M5. (B) MS/MS spectrum of M5-INH. (C) The relative abundance of M5-INH in the incubation of INH (100 μM) with different concentration of M5 (0, 1, 10, and 100 nM). N.D., not detected. (D, E) Comparison of the productions of M5-INH (D) and M1 (E) in the incubations of DTG with control, CYP1A1, 1B1 and 3A4. The relative abundance from the incubation with CYP1A1 was set as 100%. All the data are expressed as means (n = 2).

Fig. 8.. Role of CYPs in M4 formation.

(A) M4-NH₂OMe production in the A incubations of DTG (30 μM) with individual CYP (15 pmol) in the presence of NADPH (1 mM) and NH₂OMe (2.5 mM). The relative abundance of M4-NH₂OMe from CYP1A1 was set as 100%. (B) Enzymatic kinetics plots of CYP1A1 and 1B1 for M4-NH₂OMe formation. (C) Effect of TMS on M4-NH₂OMe formation. The relative abundance of M4-NH₂OMe in control group was set as 100%. The enzymatic kinetic study was conducted in duplicate and other incubations were conducted in triplicate. All data are expressed as means ± S.D.. ***p < 0.001 vs control.

Fig. 9.. Effects of CYP1A1 and 1B1 induction on the production of M1 and M4.

All data are expressed as means ± S.D. (n = 4). (A) Cyp1a1 and 1b1 expression in the lung of mice treated with vehicle or TCDD. mRNAs of Cyplal and lbl were analyzed by qPCR. The expression of Cyp1a1 or 1b1 in vehicle group was set as 1. ***p < 0.001 vs vehicle group. (B, C) The formation of M1 (B) and M4-NH₂OMe (C) in the incubation of DTG with lung S9 of mice treated with vehicle or TCDD, and the inhibitory effects of TMS on these two metabolic pathways. M1 and M4-NH₂OMe were determined by UPLC-QTOFMS. The relative abundance of M1 and M4- NH₂OMe in control group was set as 100%. **p < 0.05, ***p < 0.001 vs control.

Fig. 10.. Roles of CYP1A1 and 1B1 in DTG metabolism.

CYP1A1 and 1B1 contribute to two metabolic pathways of DTG to produce M1, M4 and M5. Both M4 and M5 are aldehydes that can be trapped by NH₂OMe and INH, respectively.