Summary of studies to define the therapeutic index (TI) of Org 214007-0.

A) THP-1: Microarray (m.a.) analysis of mRNA isolated from THP-1 cells incubated for 6 hours with either 1 μM prednisolone or 1 μM Org 214007-0 without (ind) or with IFNγ (220 ng/ml)/TNFα (374 ng/ml) ( = I/T), (rep) The mean percentage repression or induction of genes compared to that by prednisolone (set at 100%) is indicated. B) THP-1 – rep: Repression of gene expression in THP-1 cells. I/T – MCP-1, IL-6, IL-8  =  TNFα (60 ng/ml)/IFNγ (40 ng/ml) induced MCP-1, IL-6 or IL-8 release. C) THP-1 – ind: Induction of FK506 binding protein 51 (FKBP51), glucocorticoid induced leucine zipper (GILZ) and dual specificity phosphatase 1 (DUSP1) in THP-1 cells. D) hWB – rep: Inhibition of LPS-induced (LPS) TNFα release or PMA/anti-CD28 (P/28) induced IL-5 release by primary human whole blood cells and hWB – ind: enhancement of PMA/anti-CD28/compound (P/28) induced G-CSF release by primary human whole blood cells. E) CASM3C – rep: Inhibition of MCP-1 release of coronary artery smooth muscle cells ( = CASM3C) stimulated with a cytokine mixture of IL-1β (1 ng/ml), IFNγ (100 ng/ml) and TFNα (5 ng/ml) ( = 1/I/T) by 1 μM prednisolone or 1 μM Org 214007-0. CASM3C – ind: Induction of serum amyloid A (SAA) of the cells mentioned above by 1 μM prednisolone or 1 M Org 214007-0. F) HDF3CGF – rep: inhibition of matrix metalloproteinase (MMP-1) release of human neonatal foreskin fibroblasts stimulated with the cytokine mixture mentioned above plus required growth factors ( = HDF3CGF). HDF3CGF – ind: Activation of plasminogen activator inhibitor-1 (PAI-1) by cells mentioned above by 1 μM prednisolone or 1 μM Org 214007-0. G) CIA mus.: Microarray (m.a.) analysis of mRNA isolated from muscle cells isolated at day 21, 2.5 hours after the final oral administration of either 1.5 mg/kg prednisolone or 0.3 mg/kg Org 214007-0 in the mouse CIA experiment. The mean percentage repression (rep) or induction (ind) of genes compared to that by prednisolone (set at 100%) is indicated.
IC50 or EC50 values represent the mean concentration of compound (±SD) required to resp. inhibit or effect the response to 50%. Maximal efficacy (Max. eff.) is expressed as the mean relative maximal effect (±SD) compared to the maximal effect by prednisolone (set at 100%). A relative therapeutic index (TIrel) is calculated by the ratio of (the mean) % maximal efficacy in repression and (the mean) % maximal efficacy in induction of genes by Org 214007, where that of prednisolone is set at 1 (100%/100%). All assays (except for the microarray experiments) are performed at least two times.

A) THP-1细胞系：采用基因芯片（microarray，缩写m.a.）技术分析经1 μM泼尼松龙（prednisolone）或1 μM Org 214007-0处理6小时的THP-1细胞中提取的信使RNA（mRNA），实验分组分为：未添加干扰素γ（IFNγ，220 ng/ml）/肿瘤坏死因子α（TNFα，374 ng/ml），以及添加上述细胞因子（记为I/T）；其中(rep)代表基因表达抑制率、(ind)代表基因表达诱导率，所有效应均以泼尼松龙处理组的水平设为100%，给出相对百分比的均值。
B) THP-1细胞系（rep组）：检测THP-1细胞的基因表达抑制效应。其中I/T组指经肿瘤坏死因子α（TNFα，60 ng/ml）/干扰素γ（IFNγ，40 ng/ml）诱导单核细胞趋化蛋白1（MCP-1）、白细胞介素6（IL-6）、白细胞介素8（IL-8）释放的组别。
C) THP-1细胞系（ind组）：检测THP-1细胞中FK506结合蛋白51（FKBP51）、糖皮质激素诱导亮氨酸拉链蛋白（GILZ）及双特异性磷酸酶1（DUSP1）的表达诱导效应。
D) 人类全血（hWB）rep组：检测原代人类全血细胞中脂多糖（lipopolysaccharide，缩写LPS）诱导的肿瘤坏死因子α（TNFα）释放，以及佛波酯（PMA）/抗CD28抗体（P/28）诱导的白细胞介素5（IL-5）释放的抑制效应；hWB ind组：检测原代人类全血细胞中佛波酯/抗CD28抗体/受试化合物（P/28）诱导的粒细胞集落刺激因子（G-CSF）释放的增强效应。
E) 冠状动脉平滑肌细胞（CASM3C）rep组：经白细胞介素1β（IL-1β，1 ng/ml）、干扰素γ（IFNγ，100 ng/ml）及肿瘤坏死因子α（TNFα，5 ng/ml）细胞因子混合物（记为1/I/T）刺激的CASM3C细胞，经1 μM泼尼松龙或1 μM Org 214007-0处理后，其单核细胞趋化蛋白1（MCP-1）释放的抑制效应；CASM3C ind组：上述细胞经1 μM泼尼松龙或1 μM Org 214007-0处理后，血清淀粉样蛋白A（SAA）表达的诱导效应。
F) 人类新生儿包皮成纤维细胞（HDF3CGF）rep组：经上述细胞因子混合物及必要生长因子（记为HDF3CGF）刺激的HDF3CGF细胞，其基质金属蛋白酶1（MMP-1）释放的抑制效应；HDF3CGF ind组：上述细胞经1 μM泼尼松龙或1 μM Org 214007-0处理后，纤溶酶原激活物抑制剂-1（PAI-1）的激活效应。
G) 胶原诱导性关节炎（CIA）小鼠肌肉组织：在小鼠CIA实验中，于末次口服给药1.5 mg/kg泼尼松龙或0.3 mg/kg Org 214007-0后的2.5小时，采集第21天的肌肉细胞并提取信使RNA（mRNA），采用基因芯片（microarray，缩写m.a.）技术进行分析；基因的平均抑制百分比或诱导百分比以泼尼松龙处理组的水平设为100%，给出相对百分比的均值。
半数抑制浓度（IC50）与半数效应浓度（EC50）指受试化合物分别使反应被抑制50%或产生50%效应所需的平均浓度，结果以均值±标准差（±SD）表示。最大效应（Max. eff.）以泼尼松龙的最大效应为基准（设为100%），给出受试化合物的平均相对最大效应，结果以均值±标准差表示。相对治疗指数（TIrel）通过Org 214007的基因表达抑制最大效应百分比均值与基因表达诱导最大效应百分比均值的比值计算得到，其中泼尼松龙的相对治疗指数设为1（即100%/100%）。除基因芯片实验外，所有实验均至少重复2次。