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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

Immunohistochemistryof liver sections from female BN rats; blank control, NVP treatment(150 mg/kg/day × 7 days in food), DNVP treatment (150 mg/kg/day× 7 days in food), ABT treatment (50 mg/kg/day × 28 daysby gavage), or NVP (150 mg/kg/day) + ABT (50 mg/kg/day) × 28days by gavage. Slides were incubated with 1:100 dilution of primaryantisera and 1:2000 dilution of the secondary antisera. The slideswere counterstained with Mayer’s hematoxylin; magnification20×.
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fig5: Immunohistochemistryof liver sections from female BN rats; blank control, NVP treatment(150 mg/kg/day × 7 days in food), DNVP treatment (150 mg/kg/day× 7 days in food), ABT treatment (50 mg/kg/day × 28 daysby gavage), or NVP (150 mg/kg/day) + ABT (50 mg/kg/day) × 28days by gavage. Slides were incubated with 1:100 dilution of primaryantisera and 1:2000 dilution of the secondary antisera. The slideswere counterstained with Mayer’s hematoxylin; magnification20×.

Mentions: Hepatic covalent bindingof NVP and DNVP was greatest in the centrilobular area (Figure 5). The pattern of binding was dramatically differentin rats treated with a combination of NVP and the P450 inhibitor aminobenzotriazole;specifically, treatment with ABT blocked binding in the centrilobulararea and shifted it to the periportal area. Co-treatment with ABTalso changed the pattern of binding by Western blot, although therewas still significant binding (data not shown). Clearance of NVP dependson oxidative metabolism, and so even if P450 is inhibited, it causesan increase in blood levels, but ultimately NVP is oxidized.


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)

Immunohistochemistryof liver sections from female BN rats; blank control, NVP treatment(150 mg/kg/day × 7 days in food), DNVP treatment (150 mg/kg/day× 7 days in food), ABT treatment (50 mg/kg/day × 28 daysby gavage), or NVP (150 mg/kg/day) + ABT (50 mg/kg/day) × 28days by gavage. Slides were incubated with 1:100 dilution of primaryantisera and 1:2000 dilution of the secondary antisera. The slideswere counterstained with Mayer’s hematoxylin; magnification20×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Immunohistochemistryof liver sections from female BN rats; blank control, NVP treatment(150 mg/kg/day × 7 days in food), DNVP treatment (150 mg/kg/day× 7 days in food), ABT treatment (50 mg/kg/day × 28 daysby gavage), or NVP (150 mg/kg/day) + ABT (50 mg/kg/day) × 28days by gavage. Slides were incubated with 1:100 dilution of primaryantisera and 1:2000 dilution of the secondary antisera. The slideswere counterstained with Mayer’s hematoxylin; magnification20×.
Mentions: Hepatic covalent bindingof NVP and DNVP was greatest in the centrilobular area (Figure 5). The pattern of binding was dramatically differentin rats treated with a combination of NVP and the P450 inhibitor aminobenzotriazole;specifically, treatment with ABT blocked binding in the centrilobulararea and shifted it to the periportal area. Co-treatment with ABTalso changed the pattern of binding by Western blot, although therewas still significant binding (data not shown). Clearance of NVP dependson oxidative metabolism, and so even if P450 is inhibited, it causesan increase in blood levels, but ultimately NVP is oxidized.

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