We have generated a humanized anti-cocaine monoclonal antibody (mAb), which is at an advanced stage of pre-clinical development. We report here in vitro binding affinity studies, and in vivo pharmacokinetic and efficacy studies of the recombinant mAb. The overall aim was to characterize the recombinant antibody from each of the three highest producing transfected clones and to select one to establish a master cell bank. In mAb pharmacokinetic studies, after injection with h2E2 (120 mg/kg iv) blood was collected from the tail tip of mice over 28 days. Antibody concentrations were quantified using ELISA. The h2E2 concentration as a function of time was fit using a two-compartment pharmacokinetic model. To test in vivo efficacy, mice were injected with h2E2 (120 mg/kg iv), then one hour later injected with an equimolar dose of cocaine. Blood and brain were collected 5 minutes after cocaine administration. Cocaine concentrations were quantified using LC/MS. The affinity of the antibody for cocaine was determined using a [3H] cocaine binding assay. All three antibodies had long elimination half-lives, 2–5 nM Kds for cocaine, and prevented cocaine’s entry into the brain by sequestering it in the plasma. Pharmacokinetic and radio-ligand binding assays supported designation of the highest producing clone (85) as the master cell bank candidate. Overall, the recombinant h2E2 showed favorable binding properties, pharmacokinetics, and in vivo efficacy.
The complicated coding of tumorous cells requires emerging molecular biology tools for early diagnosis and for the better understandings of cancer progression. In spite of the advancement in diagnostic and therapeutic approaches to hit breast cancer, it is still a second major cause of cancerous deaths among women globally. In therapeutic vibes, deregulation in the expression of genes such as BRCA1, BRCA2, PIK3, RB, MDM2, TPK53 and HER2 involved in breast cancer prevalence. Furthermore, microRNAs such as miR-200, miR-27a, miR-182 and let-7 have been demonstrated that hold some potential hope to extricate from the complexity of cancerous cells. This review elaborates the different risk factors that lead to breast cancer progression and the current therapeutic approaches ongoing to tackle the tangled cellular behavior of cancerous cell. Moreover, for the better understandings of various signaling cascades involved in breast cancer development and to design effective therapies, researchers further needed to take interest in upcoming approaches including biophysics and nanotechnology based gene therapy and drug delivery.
BackgroundOur previous studies demonstrated that growth and migration of medulloblastoma (MB), the most common malignant brain tumor in children, are stimulated by 17β-estradiol. The growth stimulating effects of estrogens are mediated through ERβ and insulin-like growth factor 1 signaling to inhibit caspase 3 activity and reduce tumor cell apoptosis. The objective of this study was to determine whether estrogens decreased sensitivity of MB cells to cytotoxic actions of chemotherapeutic drugs.MethodsUsing in vitro cell viability and clonogenic survival assays, concentration response analysis was used to determine whether the cytoprotective effects of estradiol protected human D283 Med MB cells from the cytotoxic actions of the MB chemotherapeutic drugs cisplatin, vincristine, or lomustine. Additional experiments were done to determine whether the ER antagonist fulvestrant or the selective ER modulator tamoxifen blocked the cytoprotective actions of estradiol. ER-selective agonists and antagonists were used to define receptor specificity, and the impacts of the soy-derived phytoestrogens genistein, daidzein, and s-equol on chemosensitivity were evaluated.ResultsIn D283 Med cells the presence of 10 nM estradiol increased the IC50 for cisplatin-induced inhibition of viability 2-fold from ~5 μM to >10 μM. In clonogenic survival assays estradiol decreased the chemosensitivity of D283 Med cells exposed to cisplatin, lomustine and vincristine. The ERβ selective agonist DPN and low physiological concentrations of the soy-derived phytoestrogens genistein, daidzein, and s-equol also decreased sensitivity of D283 Med cells to cisplatin. The protective effects of estradiol were blocked by the antiestrogens 4-hydroxytamoxifen, fulvestrant (ICI 182,780) and the ERβ selective antagonist PPHTP. Whereas estradiol also decreased chemosensitivity of PFSK-1 cells, estradiol increased sensitivity of Daoy cell to cisplatin, suggesting that ERβ mediated effects may vary in different MB celltypes.ConclusionsThese findings demonstrate that E2 and environmental estrogens decrease sensitivity of MB to cytotoxic chemotherapeutics, and that ERβ selective and non-selective inhibition of estrogen receptor activity blocks these cytoprotective actions. These findings support the therapeutic potential of antiestrogen adjuvant therapies for MB, and findings that soy phytoestrogens also decrease sensitivity of MB cells to cytotoxic chemotherapeutics suggest that decreased exposure to environmental estrogens may benefit MB patient responses to chemotherapy.
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