Correlation of intrinsic clearance estimates for erythromycin N‐demethylation (ERMND) and midazolam 1′‐hydroxylation (MDZOH) with CYP3A4 and CYP3A5 content using human liver microsomes (HLM)

Lyke, A.C.; Nelsen, Andrew C.; Paine, Mary F.; Rowland, Elizabeth; Kashuba, Angela D. M.; Lindley, Celeste; Clarke, Morris J.; Hawke, Roy L.

doi:10.1016/s0009-9236(03)90426-7

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“…Western blot analysis for CYP3A content. A panel of liver microsomes obtained from African American donors (n ϭ 10) was characterized as described previously (Lyke et al, 2003) to generate two pools of HLM that contained either CYP3A4 or CYP3A5 as the major CYP3A enzyme. Briefly, microsomes were diluted in sample buffer as described to yield a final concentration of 5 to 20 g/60 l. Reference standards were prepared similarly using the recombinant CYP3A enzymes (rCYP3A).…”

Section: Methodsmentioning

confidence: 99%

“…Erythromycin was dissolved in methanol to yield a 10 mM solution. To increase the sensitivity of the radiometric assay for determination of erythromycin N-demethylase activity (Lyke et al, 2003), [ 14 C]erythromycin was purified to remove trace amounts of [ 14 C]formaldehyde. In brief, a volume of the stock solution (in 100% ethanol) was diluted 1:10 in water, after which a saturated solution of sodium carbonate (0.5% v/v) was added.…”

Section: Methodsmentioning

confidence: 99%

“…For technical and cost reasons, the IC 50 , rather than K i , was determined. The highly sensitive radiometric assay for the determination of erythromycin N-demethylase activity has been described previously (Lyke et al, 2003) and was modified from a method by Riley and Howbrook (1997). The amount of [ 14 C]erythromycin required for an experiment (i.e., 0.2 Ci/incubation) was removed from the diluted purified stock solution and combined with an appropriate volume of the cold erythromycin solution (10 mM) to achieve a total erythromycin content of 10 nmol/incubation.…”

Section: Methodsmentioning

confidence: 99%

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The Influence of CYP3A5 Expression on the Extent of Hepatic CYP3A Inhibition Is Substrate-Dependent: An in Vitro-in Vivo Evaluation

Isoherranen

Ludington²,

Givens³

et al. 2008

Drug Metabolism and Disposition

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ABSTRACT:Despite several studies suggesting that CYP3A5 expression can influence the extent of hepatic CYP3A-mediated inhibition, a systematic in vitro-in vivo evaluation of this potential clinically important issue has not been reported. Using representative probes from two distinct CYP3A substrate subgroups (midazolam, erythromycin), the inhibitory potency of fluconazole was evaluated in pooled human liver microsomes (HLM) with a low or high specific CYP3A5 content, in recombinant CYP3A enzymes (rCYP3A), and in healthy volunteers lacking or carrying the CYP3A5*1 allele. Fluconazole was a slightly more potent inhibitor of CYP3A activity in CYP3A5؊ HLM than in CYP3A5؉ HLM with midazolam (K i of 15 and 25 M, respectively) but not with erythromycin (IC 50 of 70 and 54 M, respectively). In comparison, fluconazole was a much more potent inhibitor of rCYP3A4 than rCYP3A5 with both midazolam (K i of 7.7 and 54 M, respectively) and erythromycin (IC 50 of 100 and 350 M, respectively). As predicted from HLM, with i.v. midazolam, the average (؎ S.D.) in vivo K i (K i,iv ) was significantly higher in CYP3A5*1 carriers (24 ؎ 17 and 17 ؎ 8 M for homozygous and heterozygous groups, respectively) than in noncarriers (13 ؎ 6 M) (p ‫؍‬ 0.02). With the erythromycin breath test, the average K i,iv was not different between homozygous CYP3A5*1 carriers (30 ؎ 12 M) and noncarriers (58 ؎ 53 M). In conclusion, the effect of CYP3A5 on hepatic CYP3A-mediated inhibitory drug-drug interactions is substrate-dependent, and HLM, rather than rCYP3A, are the preferred in vitro system for predicting these interactions in vivo.Adverse interactions between two or more medications have been a longstanding problem in clinical practice. Such interactions frequently result from one drug impairing the elimination of another, which can lead to increased systemic concentrations of the affected drug and the potential for an adverse or toxic reaction. The cytochrome P450 3A (CYP3A) subfamily, consisting primarily of CYP3A4 and CYP3A5 in adults, is believed to participate in the metabolism of more than half of therapeutic agents that undergo oxidation (Wilkinson, 2005). Taken together, inhibition of CYP3A-mediated metabolism is a common mechanism underlying numerous drug-drug interactions. In addition, the extent of inhibition of CYP3A-mediated drug clearance by a comedicant often varies between individuals, making it difficult to predict the magnitude and severity of the interaction.Both CYP3A4 and CYP3A5 are expressed primarily in the liver and small intestine. Although more than 30 allelic variants in the CYP3A4 gene have been identified, low-variant frequencies, often combined with a lack of functional consequence, indicate a limited contribution by these variants to the large interindividual variation observed in CYP3A4 expression Wojnowski, 2004). In contrast, CYP3A5 expression is clearly polymorphic, and the frequency of robust expression of a functionally significant protein results from genetic mutations that vary among different ethnic groups Xie...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%