[Heterogeneous expression of human CYP and its application in early stage of new drug research and development].

Human cytochrome P450 (CYP) has a pivotal role on metabolism of xenobiotics and endogenous substances in clinical practice. Since the CYP from human tissue is very complex, and the human tissue itself is not easy to obtain, investigators begin to use all kinds of expression system to heterogeneously express the CYP. The single CYP expressed was then used for drug metabolism and drug-drug interaction research, to improve the efficiency of high-throughput drug screening greatly. Besides, since the polymorphism of drug-metabolizing enzymes makes efficacy variance for some drugs in different population, the heterogeneous expression and drug metabolizing research of certain CYP mutants will be helpful to guide the optimization of therapeutic regimen and conduct the personalized medication.

[Optimization of tri-expression of human CYP3A4 with POR and cyt b5 in Sf 9 cells].

OBJECTIVE: To investigate the optimal conditions of tri-expression of CYP3A4, POR and cyt b5 in Sf 9 cells. METHODS: The Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks. The optimized conditions, including the temperature and rotation speed, the culture volume, the amount of surfactant and the culture time were studied. The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined. RESULTS: When the temperature and rotation speed of the shaker were 27 degree and 90 r/min, the cell density and culture volume were 5X105 cells/ml and 80-120 ml per 250 ml shaker flasks, respectively. When Pluronic F-68 was 0.1% and the culture time was 72 h, the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins. When the ratio of the volume of three added viruses was 1:1:1, the expression condition was optimal, under which the Km, Vmax, and CLint for testosterone metabolism were 119.6 µmol/L,0.52 µmol/(min*g protein) and 4.34 ml/(min*g protein), respectively. CONCLUSION: The conditions of tri-expressing of CYP3A4, POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.

Antitumor activity of a 5T4 targeting antibody drug conjugate with a novel payload derived from MMAF via C-Lock linker.

Antibody-drug conjugates (ADCs) belong to a promising class of biopharmaceuticals in which target-killing of tumor cells was achieved by marrying the potency of the cytotoxic payload with the tumor specificity of the antibody. Here we developed a novel ADC (ZV0508) that targets 5T4 oncofetal antigen, which is overexpressed in many carcinomas on both bulk tumor cells and cancer stem cells. A novel cytotoxic payload called Duostatin-5 (Duo-5) which was derived from monomethyl auristatin F (MMAF) was attached to a 5T4 targeting antibody (ZV05) by interchain cysteine cross-linking conjugation via a disubstituted C-Lock linker. We have investigated the antitumor efficacy of ZV0508 by in vitro and in vivo studies, and compared its antitumor activity with ZV05-mcMMAF (ZV0501), in which MMAF was linked via a conventional noncleavable maleimidocaproyl linker. As results, ZV0508 exhibited ideal antiproliferative effects through blocking cell cycle and inducing cell apoptosis. The in vivo studies revealed that both ZV0501 and ZV0508 exhibited excellent antitumor activities even at a single dose. Although ZV0508 was inferior to ZV0501 in vitro, it elicited more durable antitumor responses than ZV0501 in vivo. The superior in vivo activity of ZV0508 may be due to the combined use of the disubstituted C-Lock linker and the novel payload Duo-5, resulting in a more stable and potent ADC. Taken together, these data suggest ZV0508 is a worthy candidate for the treatment of 5T4 positive cancers.

Novel conjugation of tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) with monomethyl auristatin E for efficient antitumor drug delivery.

Monomethyl auristatin E (MMAE) is conjugated with TNF-related apoptosis-inducing ligand (TRAIL) via a linker that is stable in extracellular fluid, while it is cleaved by cathepsin once the conjugate has entered a tumor cell, thus activating the antimitotic mechanism of MMAE. The TRAIL-MMAE conjugate is a conceptually viable therapeutic strategy with improved in vitro antitumor activity, cell circle arrest and specific accumulation in tumor to treat TRAIL-resistant tumors.