Biocompatibility, absorption and safety of protein nanoparticle-based delivery of doxorubicin through oral administration in rats.

Doxorubicin, a potent anticancer drug associated with cardiotoxicity and low oral bioavailability, was loaded into apotransferrin nanoparticles to improve its pharmacological performance. Here, doxorubicin (doxo)-loaded apotransferrin nanoparticles were termed as Apodoxonano, and they were prepared by sol-oil chemistry. The pH-dependent stability of nanoparticles in simulated fluids was evaluated, and the in vitro release was investigated in phosphate-buffered saline. The pharmacokinetic and toxicity studies were conducted in Wistar rats. Nanoparticles have an average size of 75 nm, with 63% entrapment efficiency, at 10 mg w/w of apotransferrin. The particles displayed good pH-dependent stability in the pH range 1.1-7.4, but sensitive at endosomal pH of 5.5, thus facilitating intracellular drug release in endosomes. Multiplex assay showed high transport ability of nano form across epithelial cells (caco-2) when compared to doxo. Moreover, during oral administration, Apodoxonano localizes significantly in esophagus, stomach and small intestine, suggesting that it was absorbed in GI tract through epithelial lining. The drug localization was shown to be significantly lower in the heart reflecting its decreased cardiotoxic nature. The Apodoxonano with a longer bioavailability and a negligible cardiotoxicity can serve as an effective and safe vehicle of drug delivery.

Diphtheria toxin-based targeted toxin therapy for brain tumors.

Targeted toxins (TT) are molecules that bind cell surface antigens or receptors such as the transferrin or interleukin-13 receptor that are overexpressed in cancer. After internalization, the toxin component kills the cell. These recombinant proteins consist of an antibody or carrier ligand coupled to a modified plant or bacterial toxin such as diphtheria toxin (DT). These fusion proteins are very effective against brain cancer cells that are resistant to radiation therapy and chemotherapy. TT have shown an acceptable profile for toxicity and safety in animal studies and early clinical trials have demonstrated a therapeutic response. This review summarizes the characteristics of DT-based TT, the animal studies in malignant brain tumors and early clinical trial results. Obstacles to the successful treatment of brain tumors include poor penetration into tumor, the immune response to DT and cancer heterogeneity.

Brain delivery and cellular internalization mechanisms for transferrin conjugated biodegradable polymersomes.

Transferrin conjugated biodegradable polymersomes (Tf-PO) were exploited for efficient brain drug delivery, and its cellular internalization mechanisms were investigated. Tf-PO was prepared by a nanoprecipitation method with an average diameter of approximately 100 nm and a surface Tf molecule number per polymersome of approximately 35. It was demonstrated that the uptake of Tf-PO by bEnd.3 was mainly through a clathrin mediated energy-dependent endocytosis. Both the Golgi apparatus and lysosomes are involved in intracellular transport of Tf-PO. Thirty minutes after a 50mg/kg dose of Tf-PO or PO was injected into rats via the tail vein, fluorescent microscopy of brain coronal sections showed a higher accumulation of Tf-PO than PO in the cerebral cortex, the periventricular region of the lateral ventricle and the third ventricle. The brain delivery results proved that the blood-brain barrier (BBB) permeability surface area product (PS) and the percentage of injected dose per gram of brain (%ID/g brain) for Tf-PO were increased to 2.8-fold and 2.3-fold, respectively, as compared with those for PO. These results indicate that Tf-PO is a promising brain delivery carrier.

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.

Tyrphostin-8 enhances transferrin receptor-mediated transcytosis in Caco-2- cells and inreases hypoglycemic effect of orally administered insulin-transferrin conjugate in diabetic rats.

PURPOSE: To investigate the effect of tyrphostin 8 (T-8), a GTPase inhibitor, on transferrin receptor (TfR)-mediated transcytosis of insulin-transferrin (In-Tf) conjugate in cultured enterocyte-like Caco-2 cells and on gastrointestinal (GI) absorption of In-Tf in streptozotocin (STZ)-induced diabetic rats. METHODS: Caco-2 cells and diabetic rats were used as in vitro and in vivo models, respectively. TfR-mediated transcytosis was measured using 125I-In-Tf. The absorption of insulin in diabetic rats was demonstrated by the hypoglycemic effect. Rat blood glucose level was determined using a ONE TOUCH blood glucose monitoring system. RESULTS: T-8 increased apical-to-basolateral transport of In-Tf conjugate by enhancing TfR-mediated transcytosis in filter-grown Caco-2 cell monolayer, and this enhancement was higher and faster than the previously reported brefeldin A (BFA)-induced effect. The measurement of transepithelial electrical resistance (TEER) during the transport study showed that T-8 was less destructive on the cell tight junction than BFA. The GI absorption of In-Tf was evaluated by its hypoglycemic effect after oral administration in STZ-induced diabetic rats. The glucose-lowering effect of orally administered In-Tf in STZ-induced diabetic rats was improved by either T-8 or BFA. However, the effect of T-8 was more potent than that of BFA, especially at 7 h after administration. Either non-conjugated insulin or insulin-human serum albumin (In-HSA) conjugate by itself or in combination with T-8 did not show any hypoglycemic effect after oral administration, indicating that T-8-enhanced hypoglycemic activity of In-Tf was due to a selective enhancement of TfR-mediated transcytosis. CONCLUSIONS: Our data indicated that T-8 could be used to increase the GI absorption of insulin as a transferrin conjugate. T-8, as an enhancer of TfR-mediated transcytosis, is better than the previously reported BFA. T-8 produces a higher increase on the transport of In-Tf and a lower toxicity on epithelial cells. Our findings provide an alternative approach to promote the GI absorption of insulin, as well as other peptide or protein drugs.

Tumor regression with regional distribution of the targeted toxin TF-CRM107 in patients with malignant brain tumors.

We investigated regional therapy of recurrent malignant brain tumors with transferrin-CRM107, a conjugate of human transferrin (Tf) and a genetic mutant of diphtheria toxin (CRM107) that lacks native toxin binding. Physiological barriers to delivering proteins to tumor and surrounding infiltrated brain were circumvented with high-flow interstitial microinfusion. At least a 50% reduction in tumor volume on magnetic resonance imaging (MRI) occurred in 9 of 15 patients who could be evaluated (60%), including two complete responses. Peritumoral toxicity developed 1-4 weeks after treatment in three of three patients at 1.0 microg/ml, but in zero of nine patients treated at lower concentrations. No symptomatic systemic toxicity occurred. Regional perfusion with Tf-CRM107 produces tumor responses without systemic toxicity in patients with malignant brain tumors refractory to conventional therapy. Direct interstitial infusion can be used successfully to distribute a large protein in the tumor and infiltrated brain surrounding the tumor.