Boosting In-Vivo Anti-Tumor Immunity with an Oral Microparticulate Breast Cancer Vaccine and Low-Dose Cyclophosphamide.

Tumor cells express antigens that should induce immune-mediated rejection; however, spontaneous rejection of established tumors is rare. Recent evidence suggests that patients suffering from cancer exhibit an elevation in regulatory T cells population, a subset of CD4+ T cells, which suppress tumor recognition and elimination by cytotoxic T cells. This study investigates immunotherapeutic strategies to overcome the immunosuppressive effects exerted by regulatory T cells. A novel immunotherapeutic strategy was developed by simultaneous administration of oral microparticulate breast cancer vaccines and cyclophosphamide, a regulatory T cell inhibitor. Breast cancer vaccine microparticles were prepared by spray drying, and administered orally to female mice inoculated with 4TO7 murine breast cancer cells in combination with a low dose of intraperitoneally administered cyclophosphamide. Mice receiving the combination of vaccine microparticles and cyclophosphamide exhibited maximal tumor regression and the highest survival rate compared with the control groups. This study highlights the importance of cancer vaccination along with regulatory T cell depletion in cancer therapy, and suggests that a low dose of cyclophosphamide that specifically and significantly depletes regulatory T cells may be a highly effective immunotherapeutic strategy for the treatment of cancer.

Harnessing T-cell activity against prostate cancer: A therapeutic microparticulate oral cancer vaccine.

Prostate Cancer specific immunotherapy in combination with immune stimulating adjuvants may serve as a viable strategy for facilitating tumor regression and preventing recurrence. In this study, an oral microparticulate vaccine encapsulating tumor associated antigens (TAA) extracted from a murine prostate cancer cell line, TRAMP-C2, was formulated with the help of a spray dryer. Microparticles were characterized in vitro to determine their physicochemical properties and antigenicity. Formulated microparticles had an average size of 4.92 ±â€¯0.5 µm with a zeta potential of 7.92 ±â€¯1.2 mV. In order to test our formulation for its ability to demonstrate adequate antigen presentation and co-stimulation, microparticles were tested in vitro on murine dendritic cells. In vitro biological characterization demonstrated the activation of specific immune system markers such as CD80/86, CD40, MHC-I and MHC-II. Following in vitro characterization, in vivo anti-tumor efficacy of the oral microparticulate vaccine was evaluated in C57BL/6 male mice. Combination therapy of vaccine microparticles with cyclophosphamide and granulocyte macrophage-colony stimulating factor (GM-CSF) demonstrated a five-fold reduction in tumor volume as compared to non-vaccinated mice. At the cellular level, cyclophosphamide and GM-CSF augmented the vaccine response as indicated by the reduced tumor volume and significant elevation of cytotoxic T-cell (CTL) CD8+ and (T-helper) CD4+ T-cells compared to mice receiving vaccine microparticles alone. Furthermore, our studies indicate a significant reduction in T-regulatory cells (T-regs) in mice receiving vaccine along with GM-CSF and cyclophosphamide, one of the immune escape mechanisms linked to tumor growth and progression. Thus, oral microparticulate vaccines have the potential to trigger a robust anti-tumor cellular response, and in combination with clinically relevant agents, significantly resist tumor growth and progression.

Nintedanib-cyclodextrin complex to improve bio-activity and intestinal permeability.

Cyclodextrin complex of nintedanib was prepared aiming for increased bio-activity and improved transport across intestinal membrane with reduced p-glycoprotein (p-gp) efflux. Based on preliminary phase solubility studies and molecular modeling, sulfobutyl ether derivative of ß-cyclodextrin (SBE-ß-CD, Captisol®) was selected to prepare inclusion complex. Complexation was confirmed using FTIR, 1H NMR, DSC, and XRD. Bioactivity of the formed complex was tested using lung fibroblast cells, WI-38 for anti-proliferative activity and effect on collagen deposition and cells migration. In-vitro permeability studies were performed using epiIntestinal tissue model to assess the effect of complexation on transport and p-gp efflux. Results of the study demonstrated that cyclodextrin complexation increased stability of nintedanib in PBS (pH 7.4) and simulated intestinal fluid (SIF). Further, bioactivity of nintedanib also improved. Interestingly, complexation has increased transport of nintedanib across intestinal membrane and reduced efflux ratio, suggesting the role of cyclodextrin complexation in modulating p-gp efflux.

Nanoparticle formulations that allow for sustained delivery and brain targeting of the neuropeptide oxytocin.

Oxytocin is a promising candidate for the treatment of social-deficit disorders such as Autism Spectrum Disorder, but oxytocin cannot readily pass the blood-brain barrier. Moreover, oxytocin requires frequent dosing as it is rapidly metabolized in blood. We fabricated four polymeric nanoparticle formulations using poly(lactic-co-glycolic acid) (PLGA) or bovine serum albumin (BSA) as the base material. In order to target them to the brain, we then conjugated the materials to either transferrin or rabies virus glycoprotein (RVG) as targeting ligands. The formulations were characterized in vitro for size, zeta potential, encapsulation efficiency, and release profiles. All formulations showed slightly negative charges and sizes ranging from 100 to 278 nm in diameter, with RVG-conjugated BSA nanoparticles exhibiting the smallest sizes. No formulation was found to be immunogenic or cytotoxic. The encapsulation efficiency was ≥75% for all nanoparticle formulations. Release studies demonstrated that BSA nanoparticle formulation exhibited a faster initial burst of release compared to PLGA particles, in addition to later sustained release. This initial burst release would be favorable for clinical dosing as therapeutic effects could be quickly established, especially in combination with additional sustained release to maintain the therapeutic effects. Our size and release profile data indicate that RVG-conjugated BSA nanoparticles are the most favorable formulation for brain delivery of oxytocin.

Role of In Vitro Release Methods in Liposomal Formulation Development: Challenges and Regulatory Perspective.

In the past few years, measurement of drug release from pharmaceutical dosage forms has been a focus of extensive research because the release profile obtained in vitro can give an indication of the drug's performance in vivo. Currently, there are no compendial in vitro release methods designed for liposomes owing to a range of experimental challenges, which has created a major hurdle for both development and regulatory acceptance of liposome-based drug products. In this paper, we review the current techniques that are most often used to assess in vitro drug release from liposomal products; these include the membrane diffusion techniques (dialysis, reverse dialysis, fractional dialysis, and microdialysis), the sample-and-separate approach, the in situ method, the continuous flow, and the modified United States Pharmacopeia methods (USP I and USP IV). We discuss the principles behind each of the methods and the criteria that assist in choosing the most appropriate method for studying drug release from a liposomal formulation. Also, we have included information concerning the current regulatory requirements for liposomal drug products in the United States and in Europe. In light of increasing costs of preclinical and clinical trials, applying a reliable in vitro release method could serve as a proxy to expensive in vivo bioavailability studies. Graphical Abstract Appropriate in-vitro drug release test from liposomal products is important to predict the in-vivo performance.