The role of human umbilical cord tissue-derived mesenchymal stromal cells (UCX®) in the treatment of inflammatory arthritis.

BACKGROUND: ECBio has developed proprietary technology to consistently isolate, expand and cryopreserve a well-characterized population of stromal cells from human umbilical cord tissue (UCX® cells). The technology has recently been optimized in order to become compliant with Advanced Medicine Therapeutic Products. In this work we report the immunosuppressive capacity of UCX® cells for treating induced autoimmune inflammatory arthritis. METHODS: UCX® cells were isolated using a proprietary method (PCT/IB2008/054067) that yields a well-defined number of cells using a precise proportion between tissue digestion enzyme activity units, tissue mass, digestion solution volume and void volume. The procedure includes three recovery steps to avoid non-conformities related to cell recovery. UCX® surface markers were characterized by flow cytometry and UCX® capacity to expand in vitro and to differentiate into adipocyte, chondrocyte and osteoblast-like cells was evaluated. Mixed Lymphocyte Reaction (MLR) assays were performed to evaluate the effect of UCX® cells on T-cell activation and Treg conversion assays were also performed in vitro. Furthermore, UCX® cells were administered in vivo in both a rat acute carrageenan-induced arthritis model and rat chronic adjuvant induced arthritis model for arthritic inflammation. UCX® anti-inflammatory activity was then monitored over time. RESULTS: UCX® cells stained positive for CD44, CD73, CD90 and CD105; and negative for CD14, CD19 CD31, CD34, CD45 and HLA-DR; and were capable to differentiate into adipocyte, chondrocyte and osteoblast-like cells. UCX® cells were shown to repress T-cell activation and promote the expansion of Tregs better than bone marrow mesenchymal stem cells (BM-MSCs). Accordingly, xenogeneic UCX® administration in an acute carrageenan-induced arthritis model showed that human UCX® cells can reduce paw edema in vivo more efficiently than BM-MSCs. Finally, in a chronic adjuvant induced arthritis model, animals treated with intra-articular (i.a.) and intra-peritoneal (i.p.) infusions of UCX® cells showed faster remission of local and systemic arthritic manifestations. CONCLUSION: The results suggest that UCX® cells may be an effective and promising new approach for treating both local and systemic manifestations of inflammatory arthritis.

Targeted and intracellular triggered delivery of therapeutics to cancer cells and the tumor microenvironment: impact on the treatment of breast cancer.

Limiting tumor invasion to the surrounding healthy tissues has proven to be clinically relevant for anticancer treatment options. We have demonstrated that, within a solid tumor, it is possible to achieve such a goal with the same nanoparticle by intracellular and triggered targeted drug delivery to more than one cell population. We have identified the nucleolin receptor in endothelial and cancer cells in tissue samples from breast cancer patients, which enabled the design of a F3-peptide-targeted sterically stabilized pH-sensitive liposome. The clinical potential of such strategy was demonstrated by the successful specific cellular association by breast cancer cells harvested from tumors of patients submitted to mastectomy. In vitro, the nanoparticle targeted the nucleolin receptor on a cell and ligand-specific manner and improved cytotoxicity of doxorubicin (used as a model drug) towards breast cancer and endothelial cells by 177- and 162-fold, respectively, relative to the commercially available non-targeted non-pH-sensitive liposomes. Moreover, active accumulation of F3-targeted pH-sensitive liposomes into human orthotopic tumors, implanted in the mammary fat pad of nude mice, was registered for a time point as short as 4 h, reaching 48% of the injected dose/g of tissue. Twenty-four hours post-injection the accumulation of the dual-targeted pH-sensitive nanoparticle in the tumor tissue was 33-fold higher than the non-targeted non-pH-sensitive counterpart. In mice treated with the developed targeted nanoparticle significant decrease of the tumor viable rim area and microvascular density, as well as limited invasion to surrounding healthy tissues were observed (as opposed to other tested controls), which may increase the probability of tumors falling in the category of "negative margins" with reduced risk of relapse.

Synthesis, stability, biochemical and pharmacokinetic properties of a new potent and selective 4-oxo-ß-lactam inhibitor of human leukocyte elastase.

The 4-oxo-ß-lactams (azetidine-2,4-diones) are potent acylating agents of the human leukocyte elastase (HLE), a neutrophil serine protease that plays a key role in several inflammatory diseases. A novel 4-oxo-ß-lactam containing a N-(4-(phenylsulphonylmethyl)phenyl) group, 3, was designed as a potential mechanism-based inhibitor capable of undergoing elimination of phenylsulphinate upon Ser-195 acylation. Compound 3 was found to be a potent slow-tight binding inhibitor of HLE, presenting a remarkable second-order rate constant of 1.46 x 106 M⁻¹s⁻¹ and displaying selectivity over the proteinase 3 and cathepsin G. However, liberation of phenylsulphinate was not observed in the hydrolysis of 3 in both pH 7.4 phosphate buffer and human plasma. The C(max) values of 1207 µg/total blood, 179 µg/g spleen and 106 µg/g lung were determined by HPLC, following a single 30 mg/kg dose of 3 given intraperitoneally to NMRI mice, suggesting that the inhibitor distributes well into tissues. Although being a powerful selective inhibitor of HLE, 4-oxo-ß-lactam 3 has a limited stability, being susceptible to off-target reactions (plasma and liver enzymes).

Evaluation of the growth-inhibitory effect of trifluralin analogues on in vitro cultured Babesia bovis parasites.

Bovine babesiosis, caused by Babesia bovis, is a global tick borne hemoprotozoan parasite disease characterized by fever, anemia, weight losses and ultimately death. Several babesicidal drugs that have been in use in cattle for years have proven to be only partially effective and the development of alternative chemotherapeutics that are highly specific and have low toxicity against babesiosis is needed. Trifluralin derivatives specifically bind alpha-tubulin in plants and protozoa parasites causing growth inhibition. A set of 12 trifluralin analogues (TFLA) has previously been shown to be inhibitory for the growth of Leishmania species. The conservation of several key amino acids involved in the trifluralin binding site of alpha-tubulin among Leishmania sp. and B. bovis provides rationale for testing these compounds also as babesiacides. The previously tested Leishmania inhibitory, TFLA 1-12 minus TFLA 5, in addition to three novel TFLA (termed TFLA 13-15), were tested against in vitro cultured B. bovis parasites. While all of the TFLA tested in the study showed inhibition of B. bovis growth in vitro TFLA 7, TFLA 10 and TFLA 13, were the most effective inhibitors with estimated IC50 (µM) at 72 h of 8.5 ± 0.3; 9.2 ± 0.2; 8.9 ± 0.7, respectively for the biologically attenuated cloned B. bovis Mo7 strain, and 13.6 ± 1.5; 18.7 ± 1.6; 10.6 ± 1.9, respectively for the virulent B. bovis T3Bo strain. The differences found between the two strains were not statistically significant. Importantly, these drugs displayed low levels of toxicity for the host erythrocytes and bovine renal arterial endothelial cells at the doses tested. The demonstrated ability of trifluralin analogues to inhibit in vitro growth of B. bovis parasites combined with their low toxicity for host cells suggests that these compounds may be further developed as novel alternatives for the treatment of bovine babesiosis.