EGFR-targeting, ß-defensin-tailored fusion protein exhibits high therapeutic efficacy against EGFR-expressed human carcinoma via mitochondria-mediated apoptosis.

Defensins play an essential role in innate immunity. In this study, a novel recombinant ß-defensin that targets the epidermal growth factor receptor (EGFR) was designed and prepared. The EGFR-targeting ß-defensin consists of an EGF-derived oligopeptide (Ec), a ß-defensin-1 peptide (hBD1) and a lidamycin-derived apoprotein (LDP), which serves as the "scaffold" for the fusion protein (Ec-LDP-hBD1). Ec-LDP-hBD1 effectively bound to EGFR highly expressed human epidermoid carcinoma A431 cells. The cytotoxicity of Ec-LDP-hBD1 to EGFR highly expressed A431 cells was more potent than that to EGFR low-expressed human lung carcinoma A549 and H460 cells (the IC50 values in A431, A549, and H460 cells were 1.8 ± 0.55, 11.9 ± 0.51, and 5.19 ± 1.21 µmol/L, respectively); in addition, the cytotoxicity of Ec-LDP-hBD1 was much stronger than that of Ec-LDP and hBD1. Moreover, Ec-LDP-hBD1 suppressed cancer cell proliferation and induced mitochondria-mediated apoptosis. Its in vivo anticancer action was evaluated in athymic mice with A431 and H460 xenografts. The mice were administered Ec-LDP-hBD1 (5, 10 mg/kg, i.v.) two times with a weekly interval. Administration of Ec-LDP-hBD1 markedly inhibited the tumor growth without significant body weight changes. The in vivo imaging further revealed that Ec-LDP-hBD1 had a tumor-specific distribution with a clear image of localization. The results demonstrate that the novel recombinant EGFR-targeting ß-defensin Ec-LDP-hBD1 displays both selectivity and enhanced cytotoxicity against relevant cancer cells by inducing mitochondria-mediated apoptosis and exhibits high therapeutic efficacy against the EGFR-expressed carcinoma xenograft. This novel format of ß-defensin, which induces mitochondrial-mediated apoptosis, may play an active role in EGFR-targeting cancer therapy.

CYP3A4-Mediated oxygenation versus dehydrogenation of raloxifene.

Raloxifene was approved in 2007 by the FDA for the chemoprevention of breast cancer in postmenopausal women at high risk for invasive breast cancer. Approval was based in part on the improved safety profile for raloxifene relative to the standard treatment of tamoxifen. However, recent studies have demonstrated the ability of raloxifene to form reactive intermediates and act as a mechanism-based inhibitor of cytochrome P450 3A4 (CYP3A4) by forming adducts with the apoprotein. However, previous studies could not differentiate between dehydrogenation to a diquinone methide and the more common oxygenation pathway to an arene oxide as the most likely intermediate to inactivate CYP3A4. In the current work, (18)O-incorporation studies were utilized to carefully elucidate CYP3A4-mediated oxygenation versus dehydrogenation of raloxifene. These studies established that 3'-hydroxyraloxifene is produced exclusively via CYP3A4-mediated oxygenation and provide convincing evidence for the mechanism of CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide, while excluding the alternative arene oxide pathway. Furthermore, it was demonstrated that 7-hydroxyraloxifene, which was previously believed to be a typical O(2)-derived metabolite of CYP3A4, is in fact produced by a highly unusual hydrolysis pathway from a putative ester, formed by the conjugation of raloxifene diquinone methide with a carboxylic acid moiety of CYP3A4, or other proteins in the reconstituted system. These findings not only confirm CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide but also suggest a novel route of raloxifene toxicity.

A tandem scFv-based fusion protein and its enediyne-energized analogue show intensified therapeutic efficacy against lung carcinoma xenograft in athymic mice.

Gelatinases play important roles in tumor progression and are abundantly expressed in a variety of malignant tumors. Antibody targeting gelatinases is a possible avenue to fight against cancer. However, antibody alone can not achieve curative efficacy. Herein, we demonstrated the intensified targeting therapy of a tandem scFv-based fusion protein and its enediyne-energized analogue against gelatinases-overexpressed tumor. A fusion protein dFv-LDP, comprising a tandem scFv of anti-gelatinases linked to the apoprotein (LDP) of lidamycin, was generated and showed strong tumor targeting capability in three different tumor xenografts. In PG-BE1 lung carcinoma xenograft, the tumor inhibition rate was 77.5% by dFv-LDP versus 94.2% by dFv-LDP-AE, the product of dFv-LDP assembled with the active enediyne chromophore (AE) of lidamycin. Moreover, the combination of dFv-LDP with dFv-LDP-AE further augmented the therapeutic efficacy, producing initial tumor shrinkage in five of six mice. The microvessel density (P<0.05) and proliferation index (P<0.05) were also stepwise decreased in groups of dFv-LDP, dFv-LDP-AE and the combination. In conclusion, our results demonstrated that the antibody-based therapy against gelatinases was stepwise intensified in use of dFv-LDP, dFv-LDP-AE and dFv-LDP plus dFv-LDP-AE, and indicated that the combination of an antibody with its drug-armed analogue might be of interest as a new approach to augment antitumor efficacy.

Oligodendrogenesis in iron-deficient rats: effect of apotransferrin.

In rats, iron deficiency produces an alteration in myelin formation. However, there is limited information on the effects of this condition on oligodendroglial cell (OLGc) proliferation and maturation. In the present study, we further analyzed the hypomyelination associated with iron deficiency by studying the dynamics of oligodendrogenesis. Rats were fed control (40 mg Fe/kg) or iron-deficient (4 mg Fe/kg) diets from gestation day 5 until postnatal day 3 (P3) or 11 (P11). OLGc proliferation, migration and differentiation were investigated before and after an intracranial injection of apotransferrin at 3 days of age (P3). The proliferating cell population was evaluated at P3. Iron-deficient (ID) animals showed an increase in the oligodendrocyte precursors cell (OPC) population in comparison with controls. The overall pattern of migration of cells labeled with BrdU was investigated at P11. Iron deficiency increased the amount of BrdU(+) cells in the corpus callosum (CC) and decreased OLGc maturation and myelin formation. Changes in nerve conduction were analyzed by measuring visual evoked potentials. Latency and amplitude were significantly disturbed in ID rats compared with controls. Both parameters were substantially normalized when animals were treated with a single intracranial injection of 350 ng apotransferrin (aTf). The current results give support to the idea that iron deficiency increases the number of proliferating and undifferentiated cells in the CC compared with the control. Treatment with aTf almost completely reverted the effects of iron deficiency, both changing the migration pattern and increasing the number of mature cells in the CC and myelin formation.

Effect of transferrin on hypomyelination induced by iron deficiency.

We have used a model of iron deficiency in the rat to analyze the effects of a disruption in iron availability on oligodendroglial cell (OLGc) maturation and myelinogenesis and to explore the possible beneficial influence of an intracranial injection (ICI) of apotransferrin (aTf) at 3 days of age on this process. Studies carried out on postnatal days 17 and 24 showed that iron deficiency produced a decrease in myelin proteins and lipids at 24 days of age. Immunohistochemistry showed that in untreated iron-deficient (ID) rats, the immunoreactivity of anti-adenomatous polyposis coli (APC) and anti-MBP antibodies decreased markedly with reference to normal controls, whereas in ID rats treated with an ICI of aTf, the immunoreactivity of these markers increased. A similar situation occurred with the immunoreactivity of H-ferritin. In primary OLGc cultures from ID rats, there was a high number of cells positive to the antibody against the polysialylated form of the cell surface glycoprotein NCAM (PSA-NCAM) compared with in OLGc cultures prepared from normal controls or from ID animals treated with aTf. The number of MBP+ cells in cultures from ID rats increased after treatment with aTf. The presence of lipid rafts evaluated with a specific anti-protein prion cellular (PrPc) antibody showed a smaller number of PrPc-positive structures in ID rat cultures. Treatment of the ID animals with a single ICI of aTf stimulated myelination, producing a significant correction in the different biochemical parameters affected by ID.

Effect of repeated apotransferrin administrations on serum iron parameters in patients undergoing myeloablative conditioning and allogeneic stem cell transplantation.

Myeloablative conditioning prior to allogeneic stem cell transplantation causes a rapid increase in transferrin saturation and potentially toxic non-transferrin-bound iron (NTBI) in plasma. We have studied the ability of repeatedly administered apotransferrin to maintain this iron in a transferrin-bound form. Twenty adult patients undergoing myeloablative conditioning and allogeneic stem cell transplantation were enrolled to receive apotransferrin with one of three dosage regimens. Ten consecutive patients with the same preconditioning were studied as controls. At the highest dose level, full transferrin saturation and appearance of NTBI were prevented in five of the eight patients. Serum iron increased significantly more in the patients receiving apotransferrin than in the controls and remained elevated until erythropoietic recovery. From the increment of iron saturation and the amount of endogenous and administered apotransferrin, an average 180 mumol of iron per day was bound to transferrin during the first 4 d after the start of the conditioning therapy. Thereafter, iron accumulation levelled off in most patients. The results suggested that about half of the amount of iron normally transported to erythropoiesis was initially released to plasma after induction of the erythroid arrest. Complete iron binding with apotransferrin would apparently require very high apotransferrin doses.

Development of a pharmaceutical apotransferrin product for iron binding therapy.

High-dose chemotherapy of patients with haematological malignancies results in extracellular iron accumulation and appearance of non-transferrin-bound iron, which is thought to predispose the patients to septic infections and contribute to organ toxicity. We describe the development of a human plasma-derived apotransferrin product for iron binding therapy. The product is purified from Cohn fraction IV of human plasma by two ion exchange chromatography steps and ultrafiltration. The process comprises solvent detergent treatment as the main virus inactivation step and 15 nm virus filtration and polyethylene glycol precipitation as removal steps for physico-chemically resistant infectious agents. Product characterization by electrospray and MALDI-TOF mass spectrometry indicated no other chemical modifications than N-linked glycan chains and disulphide bonds, except minor oxidation. The purity of the product was more than 98%, main impurities being IgG, IgA and hemopexin. The product had intact iron binding capacity and native conformation. A stable liquid formulation for the finished product was developed. The product has proved safe and well tolerated in early clinical trials in iron binding therapy.

[Contact factors in severe sepsis]. / Facteurs de contact au cours des états septiques graves.

Coagulation and fibrinolysis are not activated in an isolated system but it involves numerous interrelations with the kininogen-kinin pathway and the complement. In severe sepsis, substances released by microorganisms, notably lipopolysaccharides, can activate the contact system, and particularly in such circumstances, contact activation probably plays a role in the occurrence of haemodynamic changes and consumption coagulopathy. Evidence of kininogen-kinin pathway activation as assessed by biological investigations in patients with severe sepsis, could lead to the therapeutical use of natural or synthetic protease inhibitors.