Muise Eric S, Chute Ian C, Claveau David, Masson Paul, Boulet Louise, Tkalec Lydia, Pon Douglas J, Girard Yves, Frenette Richard, Mancini Joseph A
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Quebec, Canada H9R 4P8.
Biochem Pharmacol. 2002 Apr 15;63(8):1527-35. doi: 10.1016/s0006-2952(02)00903-6.
Phosphodiesterase 4 (PDE4) inhibitors elevate cyclic adenosine 5'-monophosphate (cAMP), and this elevation has been shown to inhibit inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Using TNF-alpha as a biomarker, we have developed transcription-based assays to examine inhibition of PDE4 activity in human and guinea pig whole blood. In vitro inhibition by PDE4 inhibitors was measured using quantitative PCR (qPCR) analysis of TNF-alpha mRNA levels in whole blood stimulated with lipopolysaccharide (LPS). The kinetics of human TNF-alpha mRNA production were analyzed and shown to be highest 4 hr following LPS stimulation. The guinea pig displayed kinetics of TNF-alpha transcription similar to those of the human. Analysis of inhibition of human TNF-alpha protein production was performed by immunoassay and shown to correlate with inhibition of transcription for three of the four compounds tested. Roflumilast was found to be 9-fold more potent for TNF-alpha inhibition in the qPCR assay than in the protein assay. The potencies of L-826,141 and roflumilast were determined in human and guinea pig whole blood by qPCR, with IC(50) values of 270 and 20 nM, respectively, in humans and 100 and 10 nM, respectively, in guinea pigs. These results show that the potency of PDE4 inhibitors can be monitored in whole blood using a transcription-based assay, and that this type of assay can be adapted to various species provided the TNF-alpha nucleotide sequence is known. The in vitro whole blood IC(50) for TNF-alpha inhibition was compared to inhibition in the ovalbumin-challenged guinea pig model of bronchoconstriction. Obtaining plasma levels at the IC(50) determined in vitro for L-826,141 and roflumilast provides significant inhibition of bronchoconstriction. This suggests that TNF-alpha can be used as a whole blood biomarker in the guinea pig for PDE4 inhibition in this inflammatory model.
磷酸二酯酶4（PDE4）抑制剂可提高环磷酸腺苷（cAMP）水平，且已证实这种升高可抑制炎性细胞因子，如肿瘤坏死因子-α（TNF-α）。以TNF-α作为生物标志物，我们开发了基于转录的检测方法，以检测人及豚鼠全血中PDE4活性的抑制情况。通过对脂多糖（LPS）刺激的全血中TNF-α mRNA水平进行定量PCR（qPCR）分析，测定PDE4抑制剂的体外抑制作用。分析了人TNF-α mRNA产生的动力学，结果显示在LPS刺激后4小时达到最高。豚鼠TNF-α转录的动力学与人相似。通过免疫测定法分析了人TNF-α蛋白产生的抑制情况，结果显示在所测试的四种化合物中的三种中，其与转录抑制相关。在qPCR检测中，发现罗氟司特对TNF-α抑制的效力比蛋白检测中高9倍。通过qPCR测定了L-826,141和罗氟司特在人及豚鼠全血中的效力，在人中的IC50值分别为270 nM和20 nM，在豚鼠中分别为100 nM和10 nM。这些结果表明，可使用基于转录 的检测方法在全血中监测PDE4抑制剂的效力，并且只要已知TNF-α核苷酸序列，这种检测方法就可适用于各种物种。将体外全血中TNF-α抑制的IC50与卵清蛋白激发的豚鼠支气管收缩模型中的抑制情况进行了比较。在体外测定的L-826,141和罗氟司特的IC50水平下获得血浆水平，可显著抑制支气管收缩。这表明在该炎性模型中，TNF-α可作为豚鼠全血中PDE4抑制的生物标志物。
