生物活性

Evolocumab (Repatha, AMG 145) is a fully human monoclonal antibody againstPCSK9, significantly reduces low-density lipoprotein-cholesterol (LDL-C) levels. Evolocumab blocks huPCSK9 binding to the LDLR, with anIC50 of2.08 ± 1.21 nM.

体外研究

体内研究

激酶实验

Binding Affinity

Binding of mAb1 (evolocumab) to human andcynomolgus monkey PCSK9 was examined via KinExA 3000. Briefly, Reacti-Gel (6x) wasprecoated with each PCSK9 protein and blocked with BSA. mAb1 (10 or 100 pM) wasincubated with various concentrations of PCSK9 (0.1pMto10 nM) at roomtemperature for 8 h before incubation with the PCSK9-coated beads. The amountof bead-bound mAb1 was quantified by fluorescent Cy5-labeled goat antihuman IgG(H+L) antibody. The equilibrium dissociation constant (KD) was obtained fromnonlinear regression of the competition curves using a one-site homogeneousbinding model in KinExA Pro software. Binding of mAb1 to mouse PCSK9 was testedin a BIAcore solution equilibrium binding assay on T100.

细胞实验

动物实验

Studies in Mice

All mice were group housed, maintainedunder standard environmental conditions, and given murine chow and water adlibitum.

C57BL/6 mice and LDLR^-/- mice expressing huPCSK9 by AAV (huPCSK9-C57BL/6) were generated by injection(i.v. at 5 mL/kg) of ~5 x 10¹² pfu of a genetically engineeredversion of AAV5, which provided expression of the human protein. Animals werethen screened for serum non-HDL-C (defined as the sum of LDL-C and very-lowdensitylipoprotein cholesterol), HDL-C, and TC levels and were assigned to treatmentgroups with similar average values for each of these parameters.

For each experiment, cohorts of mice wereadministered a single i.v. dose of either anti-KLH antibody (control) or mAb1(evolocumab) (10 mg/kg in C57BL/6 and LDLR^-/- mice, and 10 or 30mg/kg in huPCSK9-C57BL/6 mice). At various time points after injection, animalswere killed, and blood and liver were collected. Designated groups of untreatedmice were killed at T_0 to establish baseline cholesterol and PCSK9 levels.

Studies in Monkeys

Male cynomolgus macaques were maintained atMPI Research under standard environmental conditions in individual cages. The animalswere fed twice daily, and water was provided ad libitum. All animal care andexperimental procedures were approved by the IACUC at MPI Research. Afterbaseline blood collection, monkeys were administered a single i.v. dose ofeither 3 mg/kg anti-KLH control antibody or 3 mg/kg mAb1. At specific timepoints after injection, blood was collected.

Serum Lipid, PCSK9, and mAb1 Analyses

Mouse and monkey serum was obtained fromwhole blood by centrifugation. Serum cholesterol analysis was performed using aHitachi 912 or a Cobas Integra 400 chemistry analyzer. To assess changes inserum cholesterol in LDLR^-/- mice, FPLC fractionation of sera wasperformed using a Superose 6 10/300 GL column. The TC content for eachlipoprotein particle population was determined by assaying individual FPLCfractions using the TC Infinity kit.

Levels of PCSK9 and serum antibodyconcentrations were determined using a specific sandwich ELISA in each case.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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