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Bioactivation of nevirapine to a reactive quinone methide: implications for liver injury.

Sharma AM, Li Y, Novalen M, Hayes MA, Uetrecht J - Chem. Res. Toxicol. (2012)

Bottom Line: An analogue of NVP in which the methyl hydrogens were replaced by deuterium also produced less covalent binding than NVP.These data provide strong evidence that covalent binding of NVP in the liver is due to a quinone methide formed by oxidation of the methyl group.Liver histology in these animals showed focal areas of complete necrosis, while most of the liver appeared normal.

View Article: PubMed Central - PubMed

Affiliation: Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 3M2.

ABSTRACT
Nevirapine (NVP) treatment is associated with a significant incidence of liver injury. We developed an anti-NVP antiserum to determine the presence and pattern of covalent binding of NVP to mouse, rat, and human hepatic tissues. Covalent binding to hepatic microsomes from male C57BL/6 mice and male Brown Norway rats was detected on Western blots; the major protein had a mass of ~55 kDa. Incubation of NVP with rat CYP3A1 and 2C11 or human CYP3A4 also led to covalent binding. Treatment of female Brown Norway rats or C57BL/6 mice with NVP led to extensive covalent binding to a wide range of proteins. Co-treatment with 1-aminobenzotriazole dramatically changed the pattern of binding. The covalent binding of 12-hydroxy-NVP, the pathway that leads to a skin rash, was much less than that of NVP, both in vitro and in vivo. An analogue of NVP in which the methyl hydrogens were replaced by deuterium also produced less covalent binding than NVP. These data provide strong evidence that covalent binding of NVP in the liver is due to a quinone methide formed by oxidation of the methyl group. Attempts were made to develop an animal model of NVP-induced liver injury in mice. There was a small increase in ALT in some NVP-treated male C57BL/6 mice at 3 weeks that resolved despite continued treatment. Male Cbl-b(-/-) mice dosed with NVP had an increase in ALT of >200 U/L, which also resolved despite continued treatment. Liver histology in these animals showed focal areas of complete necrosis, while most of the liver appeared normal. This is a different pattern from the histology of NVP-induced liver injury in humans. This is the first study to report hepatic covalent binding of NVP and also liver injury in mice. It is likely that the quinone methide metabolite is responsible for NVP-induced liver injury.

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(A) Comparison of covalent binding of 12-OH-NVP (lanes2 and 5) and DNVP (lane 3, 6) with that of NVP (lanes 4 and 7) aftera 30 or 60 min incubation with male BN rat microsomes (1 mg/mL protein)at a drug concentration of 1 mM. For comparison, covalent bindingto hepatic proteins is shown after 8 days of treatment of female ratswith 12-OH-NVP (159 mg/kg/day, lane 9) or NVP (150 mg/kg/day, lane10). Protein loading was 15 μg for lanes 1–7 and 20 μgfor lanes 8–10. (B) Comparison of covalent binding of 12-OH-NVP(lanes 2, 5) and DNVP (lanes 3 and 6) with that of NVP (lanes 4 and7) at a concentration of 1 mM after a 30 or 60 min incubation withmicrosomes (1 mg/mL protein) from male C57BL/6 mice. For comparison,covalent binding to hepatic proteins is shown after 6 weeks of treatmentof C57BL/6 mice with NVP at a dose of 950 mg/kg/day in food. Proteinloading was 13 μg for lanes 1–7 and 20 μg for lanes8–9. (C) Comparison of covalent binding of NVP to hepatic microsomesfrom male C57BL/6 mice (lanes 2–4) or male BN rats (lanes 6–8)after a 15, 30, or 60 min incubation at a drug concentration of 1mM and microsome concentration of 1 mg/mL protein. Protein loadingwas 20 μg per lane. The primary antiserum dilution was 1:500,and that of the secondary antisera was 1:5000.
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fig2: (A) Comparison of covalent binding of 12-OH-NVP (lanes2 and 5) and DNVP (lane 3, 6) with that of NVP (lanes 4 and 7) aftera 30 or 60 min incubation with male BN rat microsomes (1 mg/mL protein)at a drug concentration of 1 mM. For comparison, covalent bindingto hepatic proteins is shown after 8 days of treatment of female ratswith 12-OH-NVP (159 mg/kg/day, lane 9) or NVP (150 mg/kg/day, lane10). Protein loading was 15 μg for lanes 1–7 and 20 μgfor lanes 8–10. (B) Comparison of covalent binding of 12-OH-NVP(lanes 2, 5) and DNVP (lanes 3 and 6) with that of NVP (lanes 4 and7) at a concentration of 1 mM after a 30 or 60 min incubation withmicrosomes (1 mg/mL protein) from male C57BL/6 mice. For comparison,covalent binding to hepatic proteins is shown after 6 weeks of treatmentof C57BL/6 mice with NVP at a dose of 950 mg/kg/day in food. Proteinloading was 13 μg for lanes 1–7 and 20 μg for lanes8–9. (C) Comparison of covalent binding of NVP to hepatic microsomesfrom male C57BL/6 mice (lanes 2–4) or male BN rats (lanes 6–8)after a 15, 30, or 60 min incubation at a drug concentration of 1mM and microsome concentration of 1 mg/mL protein. Protein loadingwas 20 μg per lane. The primary antiserum dilution was 1:500,and that of the secondary antisera was 1:5000.

Mentions: When microsomes produced from male BN ratswere incubated with NVP, 12-OH-NVP, or DNVP, the greatest covalentbinding observed was with NVP, and the stongest band was at ∼55kDa (Figure 2A), which corresponds to the massof the male dominant P450 2C11/3A1 isoforms.14 Incubation of mouse liver microsomes with NVP produced a band ofslightly higher mass, ∼57 kDa (Figure 2B), corresponding to the mass of the dominant murine P450 3A11.9 Significant covalent binding of 12-OH-NVP wasnot observed with rat microsomes, and DNVP produced a much fainterband at 55 kDa than NVP in both rodent species tested. Invivo experiments with either species displayed a wide rangeof covalently modified bands that were much more intense than from in vitro experiments. The covalent binding of DNVP to bothrat and mouse hepatic microsomes was also much less than that of NVPby almost 5-fold as determined by densitometry (data not shown). Theamount of binding did not increase significantly beyond 15 min (Figure 2C).


Bioactivation of nevirapine to a reactive quinone methide: implications for liver injury.

Sharma AM, Li Y, Novalen M, Hayes MA, Uetrecht J - Chem. Res. Toxicol. (2012)

(A) Comparison of covalent binding of 12-OH-NVP (lanes2 and 5) and DNVP (lane 3, 6) with that of NVP (lanes 4 and 7) aftera 30 or 60 min incubation with male BN rat microsomes (1 mg/mL protein)at a drug concentration of 1 mM. For comparison, covalent bindingto hepatic proteins is shown after 8 days of treatment of female ratswith 12-OH-NVP (159 mg/kg/day, lane 9) or NVP (150 mg/kg/day, lane10). Protein loading was 15 μg for lanes 1–7 and 20 μgfor lanes 8–10. (B) Comparison of covalent binding of 12-OH-NVP(lanes 2, 5) and DNVP (lanes 3 and 6) with that of NVP (lanes 4 and7) at a concentration of 1 mM after a 30 or 60 min incubation withmicrosomes (1 mg/mL protein) from male C57BL/6 mice. For comparison,covalent binding to hepatic proteins is shown after 6 weeks of treatmentof C57BL/6 mice with NVP at a dose of 950 mg/kg/day in food. Proteinloading was 13 μg for lanes 1–7 and 20 μg for lanes8–9. (C) Comparison of covalent binding of NVP to hepatic microsomesfrom male C57BL/6 mice (lanes 2–4) or male BN rats (lanes 6–8)after a 15, 30, or 60 min incubation at a drug concentration of 1mM and microsome concentration of 1 mg/mL protein. Protein loadingwas 20 μg per lane. The primary antiserum dilution was 1:500,and that of the secondary antisera was 1:5000.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475366&req=5

fig2: (A) Comparison of covalent binding of 12-OH-NVP (lanes2 and 5) and DNVP (lane 3, 6) with that of NVP (lanes 4 and 7) aftera 30 or 60 min incubation with male BN rat microsomes (1 mg/mL protein)at a drug concentration of 1 mM. For comparison, covalent bindingto hepatic proteins is shown after 8 days of treatment of female ratswith 12-OH-NVP (159 mg/kg/day, lane 9) or NVP (150 mg/kg/day, lane10). Protein loading was 15 μg for lanes 1–7 and 20 μgfor lanes 8–10. (B) Comparison of covalent binding of 12-OH-NVP(lanes 2, 5) and DNVP (lanes 3 and 6) with that of NVP (lanes 4 and7) at a concentration of 1 mM after a 30 or 60 min incubation withmicrosomes (1 mg/mL protein) from male C57BL/6 mice. For comparison,covalent binding to hepatic proteins is shown after 6 weeks of treatmentof C57BL/6 mice with NVP at a dose of 950 mg/kg/day in food. Proteinloading was 13 μg for lanes 1–7 and 20 μg for lanes8–9. (C) Comparison of covalent binding of NVP to hepatic microsomesfrom male C57BL/6 mice (lanes 2–4) or male BN rats (lanes 6–8)after a 15, 30, or 60 min incubation at a drug concentration of 1mM and microsome concentration of 1 mg/mL protein. Protein loadingwas 20 μg per lane. The primary antiserum dilution was 1:500,and that of the secondary antisera was 1:5000.
Mentions: When microsomes produced from male BN ratswere incubated with NVP, 12-OH-NVP, or DNVP, the greatest covalentbinding observed was with NVP, and the stongest band was at ∼55kDa (Figure 2A), which corresponds to the massof the male dominant P450 2C11/3A1 isoforms.14 Incubation of mouse liver microsomes with NVP produced a band ofslightly higher mass, ∼57 kDa (Figure 2B), corresponding to the mass of the dominant murine P450 3A11.9 Significant covalent binding of 12-OH-NVP wasnot observed with rat microsomes, and DNVP produced a much fainterband at 55 kDa than NVP in both rodent species tested. Invivo experiments with either species displayed a wide rangeof covalently modified bands that were much more intense than from in vitro experiments. The covalent binding of DNVP to bothrat and mouse hepatic microsomes was also much less than that of NVPby almost 5-fold as determined by densitometry (data not shown). Theamount of binding did not increase significantly beyond 15 min (Figure 2C).

Bottom Line: An analogue of NVP in which the methyl hydrogens were replaced by deuterium also produced less covalent binding than NVP.These data provide strong evidence that covalent binding of NVP in the liver is due to a quinone methide formed by oxidation of the methyl group.Liver histology in these animals showed focal areas of complete necrosis, while most of the liver appeared normal.

View Article: PubMed Central - PubMed

Affiliation: Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 3M2.

ABSTRACT
Nevirapine (NVP) treatment is associated with a significant incidence of liver injury. We developed an anti-NVP antiserum to determine the presence and pattern of covalent binding of NVP to mouse, rat, and human hepatic tissues. Covalent binding to hepatic microsomes from male C57BL/6 mice and male Brown Norway rats was detected on Western blots; the major protein had a mass of ~55 kDa. Incubation of NVP with rat CYP3A1 and 2C11 or human CYP3A4 also led to covalent binding. Treatment of female Brown Norway rats or C57BL/6 mice with NVP led to extensive covalent binding to a wide range of proteins. Co-treatment with 1-aminobenzotriazole dramatically changed the pattern of binding. The covalent binding of 12-hydroxy-NVP, the pathway that leads to a skin rash, was much less than that of NVP, both in vitro and in vivo. An analogue of NVP in which the methyl hydrogens were replaced by deuterium also produced less covalent binding than NVP. These data provide strong evidence that covalent binding of NVP in the liver is due to a quinone methide formed by oxidation of the methyl group. Attempts were made to develop an animal model of NVP-induced liver injury in mice. There was a small increase in ALT in some NVP-treated male C57BL/6 mice at 3 weeks that resolved despite continued treatment. Male Cbl-b(-/-) mice dosed with NVP had an increase in ALT of >200 U/L, which also resolved despite continued treatment. Liver histology in these animals showed focal areas of complete necrosis, while most of the liver appeared normal. This is a different pattern from the histology of NVP-induced liver injury in humans. This is the first study to report hepatic covalent binding of NVP and also liver injury in mice. It is likely that the quinone methide metabolite is responsible for NVP-induced liver injury.

Show MeSH
Related in: MedlinePlus