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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) Plasma ALT levels in male Cbl-b–/– mice fed NVP orally for 14 days (950 mg/kg/day). Values are basedon the mean of triplicate readings per time point per animal ±SD, n = 5 treated mice or n = 4control mice. Unpaired t test, 7 d.f., p < 0.05. (B) Covalent binding of NVP in the livers of the sameCbl-b–/– mice. (C) Plasma ALT levels in NVP-treated(950 mg/kg/day) female Cbl-b–/– mice, n = 4 treated or n = 4 control mice. Valuesare based on the mean of triplicate readings per time point per animal± SD, n = 5 treated mice or n = 4 control mice. Unpaired t test, 6 d.f., p < 0.05. (D) Covalent binding of NVP in the livers ofthe same mice. Protein loading was 25 μg per lane. Samples wereresolved on 10–20% gradient gels. A 1:500 dilution of primaryantisera followed by 1:5000 dilution of secondary antisera was used.
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fig8: (A) Plasma ALT levels in male Cbl-b–/– mice fed NVP orally for 14 days (950 mg/kg/day). Values are basedon the mean of triplicate readings per time point per animal ±SD, n = 5 treated mice or n = 4control mice. Unpaired t test, 7 d.f., p < 0.05. (B) Covalent binding of NVP in the livers of the sameCbl-b–/– mice. (C) Plasma ALT levels in NVP-treated(950 mg/kg/day) female Cbl-b–/– mice, n = 4 treated or n = 4 control mice. Valuesare based on the mean of triplicate readings per time point per animal± SD, n = 5 treated mice or n = 4 control mice. Unpaired t test, 6 d.f., p < 0.05. (D) Covalent binding of NVP in the livers ofthe same mice. Protein loading was 25 μg per lane. Samples wereresolved on 10–20% gradient gels. A 1:500 dilution of primaryantisera followed by 1:5000 dilution of secondary antisera was used.

Mentions: There was no change in plasma ALT in BN rats treatedwith NVP (data not shown). Various strains of mice were treated withNVP to determine if it causes liver damage, covalent binding, and/orhistological changes. Male BALB/c mice treated with NVP had no increasein ALT (data not shown), while there was an increase in ALT in maleC57BL/6 mice at 3 weeks followed by normalization of ALT levels (Figure 7A). Immunoblots revealed no significant differencesbetween the pattern and degree of binding in these two strains (Figure 7B). ALT levels in both male and female Cbl-b–/– mice increased at week 2, with a somewhatgreater increase in male mice (Figure 8A) thanfemale mice (Figure 8C). The animals with thelargest ALT increase displayed areas of gross hepatic necrosis evidentas areas of white on the surface of the liver upon sacrifice at 2weeks. Immunoblot analysis showed the presence of a wide range ofmodified hepatic protein in both male (Figure 8B) and female (Figure 8D) mice. Animals withgross necrosis appeared to have slightly more binding of NVP to lowermolecular mass proteins (Figure 8B,D).


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) Plasma ALT levels in male Cbl-b–/– mice fed NVP orally for 14 days (950 mg/kg/day). Values are basedon the mean of triplicate readings per time point per animal ±SD, n = 5 treated mice or n = 4control mice. Unpaired t test, 7 d.f., p < 0.05. (B) Covalent binding of NVP in the livers of the sameCbl-b–/– mice. (C) Plasma ALT levels in NVP-treated(950 mg/kg/day) female Cbl-b–/– mice, n = 4 treated or n = 4 control mice. Valuesare based on the mean of triplicate readings per time point per animal± SD, n = 5 treated mice or n = 4 control mice. Unpaired t test, 6 d.f., p < 0.05. (D) Covalent binding of NVP in the livers ofthe same mice. Protein loading was 25 μg per lane. Samples wereresolved on 10–20% gradient gels. A 1:500 dilution of primaryantisera followed by 1:5000 dilution of secondary antisera was used.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: (A) Plasma ALT levels in male Cbl-b–/– mice fed NVP orally for 14 days (950 mg/kg/day). Values are basedon the mean of triplicate readings per time point per animal ±SD, n = 5 treated mice or n = 4control mice. Unpaired t test, 7 d.f., p < 0.05. (B) Covalent binding of NVP in the livers of the sameCbl-b–/– mice. (C) Plasma ALT levels in NVP-treated(950 mg/kg/day) female Cbl-b–/– mice, n = 4 treated or n = 4 control mice. Valuesare based on the mean of triplicate readings per time point per animal± SD, n = 5 treated mice or n = 4 control mice. Unpaired t test, 6 d.f., p < 0.05. (D) Covalent binding of NVP in the livers ofthe same mice. Protein loading was 25 μg per lane. Samples wereresolved on 10–20% gradient gels. A 1:500 dilution of primaryantisera followed by 1:5000 dilution of secondary antisera was used.
Mentions: There was no change in plasma ALT in BN rats treatedwith NVP (data not shown). Various strains of mice were treated withNVP to determine if it causes liver damage, covalent binding, and/orhistological changes. Male BALB/c mice treated with NVP had no increasein ALT (data not shown), while there was an increase in ALT in maleC57BL/6 mice at 3 weeks followed by normalization of ALT levels (Figure 7A). Immunoblots revealed no significant differencesbetween the pattern and degree of binding in these two strains (Figure 7B). ALT levels in both male and female Cbl-b–/– mice increased at week 2, with a somewhatgreater increase in male mice (Figure 8A) thanfemale mice (Figure 8C). The animals with thelargest ALT increase displayed areas of gross hepatic necrosis evidentas areas of white on the surface of the liver upon sacrifice at 2weeks. Immunoblot analysis showed the presence of a wide range ofmodified hepatic protein in both male (Figure 8B) and female (Figure 8D) mice. Animals withgross necrosis appeared to have slightly more binding of NVP to lowermolecular mass proteins (Figure 8B,D).

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