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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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Related in: MedlinePlus

ELISAanalysis showing (A) binding of the anti-NVP-NAC-KLH antiserum tothe NVP-NAC-BSA conjugate, KLH, or BSA, and (B) the effect of preincubationof the antiserum with NVP or its metabolites on the binding of theantisera to the NVP-NAC-BSA conjugate. Data represent the mean ±SD from 3 incubations.
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fig1: ELISAanalysis showing (A) binding of the anti-NVP-NAC-KLH antiserum tothe NVP-NAC-BSA conjugate, KLH, or BSA, and (B) the effect of preincubationof the antiserum with NVP or its metabolites on the binding of theantisera to the NVP-NAC-BSA conjugate. Data represent the mean ±SD from 3 incubations.

Mentions: ELISA analysis showed that the anti-NVP-NAC-KLH antiserum recognizedthe NVP-NAC-BSA conjugate or KLH but not BSA alone (Figure 1A). The binding of the antisera to the NVP-NAC-BSAconjugate was inhibited by preincubating the anti-NVP-NAC-KLH antiserumwith NVP or its metabolites (Figure 1B). Inhibitionwas much less with 2-OH-NVP, 3-OH-NVP, and 4-COOH-NVP (the metabolitein which the methyl group has been oxidized to a carboxylic acid).Binding could still be detected at an antiserum dilution of 1/1,000,000.


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)

ELISAanalysis showing (A) binding of the anti-NVP-NAC-KLH antiserum tothe NVP-NAC-BSA conjugate, KLH, or BSA, and (B) the effect of preincubationof the antiserum with NVP or its metabolites on the binding of theantisera to the NVP-NAC-BSA conjugate. Data represent the mean ±SD from 3 incubations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: ELISAanalysis showing (A) binding of the anti-NVP-NAC-KLH antiserum tothe NVP-NAC-BSA conjugate, KLH, or BSA, and (B) the effect of preincubationof the antiserum with NVP or its metabolites on the binding of theantisera to the NVP-NAC-BSA conjugate. Data represent the mean ±SD from 3 incubations.
Mentions: ELISA analysis showed that the anti-NVP-NAC-KLH antiserum recognizedthe NVP-NAC-BSA conjugate or KLH but not BSA alone (Figure 1A). The binding of the antisera to the NVP-NAC-BSAconjugate was inhibited by preincubating the anti-NVP-NAC-KLH antiserumwith NVP or its metabolites (Figure 1B). Inhibitionwas much less with 2-OH-NVP, 3-OH-NVP, and 4-COOH-NVP (the metabolitein which the methyl group has been oxidized to a carboxylic acid).Binding could still be detected at an antiserum dilution of 1/1,000,000.

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