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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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Covalent binding of NVP to expressed male rat CYP2C11 (A) or CYP3A1(B) in vitro. Protein concentration for each incubationwas 0.8 mg/mL with 0.5 mM of drug. For immunoblots, protein loadingwas 9 μg and 7.5 μg per lane for A and B, respectively.(+) indicates incubations containing NVP, while (−) indicatesincubations lacking NVP. Proteins were resolved on 12% gels with 1:100dilution of primary antiserum followed by 1:2000 dilution of secondaryantisera. Comparison of covalent binding of 12-OH-NVP (lanes 2 and5) or DNVP (lanes 3 and 6) with that of NVP (lanes 4 and 7) to humanCYP3A4 with a drug concentration of 1 mM and protein concentrationin each incubation of 1 mg/mL (C). Proteins (10 μg/lane) wereresolved on an 8% gel. Dilutions of antisera were 1:500 for the primaryantiserum and 1:5000 for the secondary antiserum.
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fig3: Covalent binding of NVP to expressed male rat CYP2C11 (A) or CYP3A1(B) in vitro. Protein concentration for each incubationwas 0.8 mg/mL with 0.5 mM of drug. For immunoblots, protein loadingwas 9 μg and 7.5 μg per lane for A and B, respectively.(+) indicates incubations containing NVP, while (−) indicatesincubations lacking NVP. Proteins were resolved on 12% gels with 1:100dilution of primary antiserum followed by 1:2000 dilution of secondaryantisera. Comparison of covalent binding of 12-OH-NVP (lanes 2 and5) or DNVP (lanes 3 and 6) with that of NVP (lanes 4 and 7) to humanCYP3A4 with a drug concentration of 1 mM and protein concentrationin each incubation of 1 mg/mL (C). Proteins (10 μg/lane) wereresolved on an 8% gel. Dilutions of antisera were 1:500 for the primaryantiserum and 1:5000 for the secondary antiserum.

Mentions: Incubation of NVP with expressed rat CYP2C11 (Figure 3A) or CYP3A1 Supersomes (Figure 3B),the dominant forms of P450 in male rats14−16 led to covalent bindingwith major bands produced at ∼50 kDa and ∼52 kDa, respectively.In the absence of NVP as indicated in the figures, there is a smallartifact band. Binding to 2C11 and 3A4 was strongest at 30 min; adecrease in the intensity of the P450 band was observed from 30 to120 min.


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)

Covalent binding of NVP to expressed male rat CYP2C11 (A) or CYP3A1(B) in vitro. Protein concentration for each incubationwas 0.8 mg/mL with 0.5 mM of drug. For immunoblots, protein loadingwas 9 μg and 7.5 μg per lane for A and B, respectively.(+) indicates incubations containing NVP, while (−) indicatesincubations lacking NVP. Proteins were resolved on 12% gels with 1:100dilution of primary antiserum followed by 1:2000 dilution of secondaryantisera. Comparison of covalent binding of 12-OH-NVP (lanes 2 and5) or DNVP (lanes 3 and 6) with that of NVP (lanes 4 and 7) to humanCYP3A4 with a drug concentration of 1 mM and protein concentrationin each incubation of 1 mg/mL (C). Proteins (10 μg/lane) wereresolved on an 8% gel. Dilutions of antisera were 1:500 for the primaryantiserum and 1:5000 for the secondary antiserum.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Covalent binding of NVP to expressed male rat CYP2C11 (A) or CYP3A1(B) in vitro. Protein concentration for each incubationwas 0.8 mg/mL with 0.5 mM of drug. For immunoblots, protein loadingwas 9 μg and 7.5 μg per lane for A and B, respectively.(+) indicates incubations containing NVP, while (−) indicatesincubations lacking NVP. Proteins were resolved on 12% gels with 1:100dilution of primary antiserum followed by 1:2000 dilution of secondaryantisera. Comparison of covalent binding of 12-OH-NVP (lanes 2 and5) or DNVP (lanes 3 and 6) with that of NVP (lanes 4 and 7) to humanCYP3A4 with a drug concentration of 1 mM and protein concentrationin each incubation of 1 mg/mL (C). Proteins (10 μg/lane) wereresolved on an 8% gel. Dilutions of antisera were 1:500 for the primaryantiserum and 1:5000 for the secondary antiserum.
Mentions: Incubation of NVP with expressed rat CYP2C11 (Figure 3A) or CYP3A1 Supersomes (Figure 3B),the dominant forms of P450 in male rats14−16 led to covalent bindingwith major bands produced at ∼50 kDa and ∼52 kDa, respectively.In the absence of NVP as indicated in the figures, there is a smallartifact band. Binding to 2C11 and 3A4 was strongest at 30 min; adecrease in the intensity of the P450 band was observed from 30 to120 min.

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