大环内酯类抗生素对人肠壁代谢的抑制作用：克拉霉素对细胞色素P450 3A4/5活性及表达的影响

Inhibition of human intestinal wall metabolism by macrolide antibiotics: effect of clarithromycin on cytochrome P450 3A4/5 activity and expression.

作者信息

Pinto Amar G, Wang Ying-Hong, Chalasani Naga, Skaar Todd, Kolwankar Dhanashri, Gorski J Christopher, Liangpunsakul Suthat, Hamman Mitchell A, Arefayene Million, Hall Stephen D

机构信息

Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

出版信息

Clin Pharmacol Ther. 2005 Mar;77(3):178-88. doi: 10.1016/j.clpt.2004.10.002.

DOI:10.1016/j.clpt.2004.10.002

PMID:15735612

Abstract

BACKGROUND

Clarithromycin increases both hepatic and intestinal availability of the selective cytochrome P450 (CYP) 3A probe midazolam. This study was designed to identify determinants of variability in the extent of intestinal wall CYP3A inhibition by clarithromycin, such as CYP3A5 genotype, and the mechanism of inhibition.

METHODS

Ten healthy volunteers received 500 mg oral clarithromycin twice a day for 7 days. Before and after administration of clarithromycin, small-bowel mucosal biopsy specimens were obtained endoscopically. Intestinal CYP3A activity was determined from the rate of 1'-hydroxymidazolam and 4-hydroxymidazolam formation by incubation of small-bowel homogenate with midazolam (25 micromol/L) and NADPH for 5 minutes. Intestinal CYP3A4 and CYP3A5 messenger ribonucleic acid was quantified by real-time reverse transcriptase-polymerase chain reaction. Intestinal CYP3A4 and CYP3A5 protein concentrations were determined by immunoblotting. Serum and homogenate concentrations of midazolam, clarithromycin, and metabolites were determined by liquid chromatography-mass spectrometry. CYP3A5 genotype was determined by real-time polymerase chain reaction.

RESULTS

The formation of 1'-hydroxymidazolam (1.36 +/- 0.46 pmol . min(-1) . mg(-1) at baseline versus 0.35 +/- 0.16 pmol . min(-1) . mg(-1) after administration) and 4-hydroxymidazolam (0.39 +/- 0.12 pmol . min(-1) . mg(-1) at baseline versus 0.12 +/- 0.05 pmol . min(-1) . mg(-1) after administration) was significantly (P < .001) reduced after clarithromycin administration. Clarithromycin administration did not result in a significant change in intestinal CYP3A4 and CYP3A5 messenger ribonucleic acid and protein expression. All subjects had detectable serum clarithromycin concentrations after 7 days of clarithromycin (3.71 +/- 2.43 micromol/L). The mean concentration of clarithromycin in the intestinal biopsy homogenate was 1.2 +/- 0.7 nmol/L (range, 0.42-2.39 nmol/L). Compared with CYP3A5 nonexpressers, subjects with at least 1 CYP3A5*1 allele (CYP3A5 expressers) had greater inhibition of intestinal CYP3A activity after treatment with clarithromycin. There was a strong linear relationship between the decrease in intestinal CYP3A activity and baseline catalytic activity (R(2) = 0.9).

CONCLUSION

Baseline intestinal activity of CYP3A4 was a key determinant of variability of the inhibitory effect of clarithromycin among individuals. CYP3A5*1 alleles were associated with greater baseline intestinal CYP3A activity and, therefore, greater extent of inhibition. The primary in vivo mechanism was not rapidly reversible competitive or irreversible inhibition but was likely formation of metabolic intermediate complexes.

摘要

背景

克拉霉素可提高选择性细胞色素P450（CYP）3A探针咪达唑仑在肝脏和肠道的可用性。本研究旨在确定克拉霉素对肠壁CYP3A抑制程度变异性的决定因素，如CYP3A5基因型以及抑制机制。

方法

10名健康志愿者每天口服500 mg克拉霉素，分两次服用，共7天。在服用克拉霉素前后，通过内镜获取小肠黏膜活检标本。通过将小肠匀浆与咪达唑仑（25 μmol/L）和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）孵育5分钟，根据1'-羟基咪达唑仑和4-羟基咪达唑仑的生成速率来测定肠道CYP3A活性。通过实时逆转录聚合酶链反应对肠道CYP3A4和CYP3A5信使核糖核酸进行定量。通过免疫印迹法测定肠道CYP3A4和CYP3A5蛋白浓度。通过液相色谱-质谱法测定血清和匀浆中咪达唑仑、克拉霉素及其代谢产物的浓度。通过实时聚合酶链反应确定CYP3A5基因型。

结果

克拉霉素给药后，1'-羟基咪达唑仑的生成量（基线时为1.36±0.46 pmol·min⁻¹·mg⁻¹，给药后为0.35±0.16 pmol·min⁻¹·mg⁻¹）和4-羟基咪达唑仑的生成量（基线时为0.39±0.12 pmol·min⁻¹·mg⁻¹，给药后为0.12±0.05 pmol·min⁻¹·mg⁻¹）显著降低（P <.001）。克拉霉素给药并未导致肠道CYP3A4和CYP3A5信使核糖核酸及蛋白表达的显著变化。克拉霉素服用7天后，所有受试者血清中克拉霉素浓度均可检测到（3.71±2.43 μmol/L）。肠道活检匀浆中克拉霉素的平均浓度为1.2±0.7 nmol/L（范围为0.42 - 2.39 nmol/L）。与CYP3A5无表达者相比，至少有1个CYP3A5*1等位基因的受试者（CYP3A5表达者）在接受克拉霉素治疗后肠道CYP3A活性受到更大抑制。肠道CYP3A活性的降低与基线催化活性之间存在强线性关系（R² = 0.9）。