Powder suspensions in non-aqueous vehicles for delivery of therapeutic proteins

Protein powder suspensions in non-aqueous vehicles provide an interesting alternative to conventional formulations such as aqueous solutions and lyophilizates and potentially feature a variety of advantages for different applications. Besides powder manufacturing, vehicle selection is key for such preparations. Powders for the use in such formulations can be prepared in several ways including milling of lyophilizates and spray-drying. Chapter III aimed to get a deeper understanding on how milling of model monoclonal antibody (mAb) lyophilizates can be used as a technique to generate protein powders for suspensions in non-aqueous vehicles. It was of interest, if wet media milling can yield protein powder suspensions without damage to the protein. An alternative is dry milling in absence of a suspension vehicle. The chapter aimed to identify critical process parameters and to optimize the milling process. Studied process parameters included the milling ball size, milling frequency, ball to powder mass ratio, milling duration as well as application of cooling. Furthermore, it was tested whether excipients or the protein to stabilizer ratio influence protein stability during milling. With the obtained powder, suspensions were prepared and their viscosities and glide forces were tested. Highly concentrated protein formulations currently play an important role in the pharmaceutical industry, but face different challenges such as decreased protein stability and increased glide forces needed for injection. Protein powder suspensions in non-aqueous vehicles may represent a beneficial alternative and can yield low viscosity formulations depending on the selected suspension vehicle. Traditionally used non-aqueous liquids for injection, such as vegetable oils, are not suitable for high concentration suspension formulations due to their high inherent viscosity. Consequently, low viscosity alternatives are of particular interest. Thus, the goal of Chapter IV was to test various potential vehicles regarding their ability to yield high concentration low viscosity formulations. Promising candidates included semifluorinated alkanes (SFAs), due to their low viscosity, good biocompatibility and inertness towards biopharmaceuticals. In contrary to the previous chapter, spray-drying was used to prepare the protein powder. It was of interest how SFA based suspensions perform with regard to rheology and injection characteristics compared to aqueous solutions. In order to select a suitable syringe design, prediction of glide forces using mathematical models and equations was evaluated. Finally, with commercial manufacturability in mind, the feasibility of the vial filling process of the developed suspensions, using a peristaltic pump, was tested and evaluated. Chapter V provides more detailed insight into long term physical suspension stability of lysozyme, mAb and bevacizumab powder suspensions in different suspension vehicles including SFAs, medium chain triglycerides (MCT) and ethyl oleate (EO). Physical suspension stability includes resuspendability, particle size constancy and injectability. Both excipients and the protein to stabilizer ratio might influence the stability. Furthermore, the application of an additional drying step was tested to improve the physical suspension stability. Suspensions were stored in vials, as well as in prefillable syringes, which provide easier administration. Not only suspension physical stability, but also protein stability has to be maintained over storage time. In the case of protein powder suspensions in non-aqueous vehicles, protein instabilities can have two different causes. First the inherent stability of the powder has to be considered. Secondly, the influence of the suspension vehicle on protein stability might play a role. The focus of Chapter VI lied on the influence of different potential vehicles and powder compositions on protein stability in non-aqueous powder suspensions. For this purpose, stability of a mAb and bevacizumab in SFAs, MCT and EO were studied. Combined with investigations on suspension physical stability this chapter should give valuable information on the applicability of the non-aqueous powder suspension approach for protein formulations. Besides subcutaneous injection, topical ocular protein delivery to the cornea is of interest, especially after the recent approval of a protein drug for the treatment of neurotrophic keratitis. This application route faces poor drug penetration and short residence time as important challenges. In Chapter VII formulations for improved corneal protein delivery based on protein powder suspensions in non-aqueous vehicles, considering the addition of penetration enhancers, were investigated. A main focus lied on the performance of protein powder suspension in SFAs, as previous investigations on topical ocular delivery of small molecule drugs in SFAs showed promising results. The overall goal of the thesis was to study the various opportunities of powder suspensions in non-aqueous vehicles for protein formulation with a focus on SFAs. The various chapters present pathways for successfully developing, characterizing and manufacturing such preparations, which may provide superior suspension and protein stability also at high protein concentration

Pharmacokinetics, biodistribution, and anti-angiogenesis efficacy of diamino propane tetraiodothyroacetic acid-conjugated biodegradable polymeric nanoparticle

The anti-angiogenic agent, diamino propane tetraiodothyroacetic acid (DAT), is a thyro-integrin (integrin alpha v beta 3) antagonist anticancer agent that works via genetic and nongenetic actions. Tetraiodothyroacetic acid (tetrac) and DAT as thyroid hormone derivatives influence gene expression after they transport across cellular membranes. To restrict the action of DAT to the integrin alpha v beta 3 receptors on the cell surface, we used DAT-conjugated PLGA nanoparticles (NDAT) in an active targeting mode to bind to these receptors. Preparation and characterization of NDAT is described, and both in vitro and in vivo experiments were done to compare DAT to NDAT. Intracellular uptake and distribution of DAT and NDAT in U87 glioblastoma cells were evaluated using confocal microscopy and showed that DAT reached the nucleus, but NDAT was restricted from the nucleus. Pharmacokinetic studies using LC-MS/MS analysis in male C57BL/6 mice showed that administration of NDAT improved the area under the drug concentration curve AUC(()(0-)(48 h)) by 4-fold at a dose of 3 mg/kg when compared with DAT, and C-max of NDAT (4363 ng/mL) was 8-fold greater than that of DAT (548 ng/ mL). Biodistribution studies in the mice showed that the concentrations of NDAT were higher than DAT/Cremophor EL micelles in heart, lung, liver, spleen, and kidney. In another mouse model using female NCr nude homozygous mice with U87 xenografts, tumor growth was significantly decreased at doses of 1 and 3 mg/kg of NDAT. In the chick chorioallantoic membrane (CAM) assay used to measure angiogenesis, DAT (500 ng/CAM) resulted in 48% inhibition of angiogenesis levels. In comparison, NDAT at low dose (50 ng/CAM) showed 45% inhibition of angiogenesis levels. Our investigation of NDAT bridges the study of polymeric nanoparticles and anti-angiogenic agents and offers new insight for the rational design of anti-angiogenic agents.Pharmaceutical Research Institute (PRI)NanoPharmaceuticals LLC (Rensselaer, NY, USA

Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

A dramatic increase in knowledge regarding the molecular biology of brain tumors has been established over the past few years. In particular recent new avenues regarding the role of stem cells and microRNAs along with further understanding of the importance of angiogenesis, immunotherapy and explanations for the resistance of the tumors to chemotherapeutic agents and radiation therapy has been developed. It is hopeful that this new information will lead to efficacious treatment strategies for these tumors which remain a challenge. In this book a review of the latest information on these topics along with a variety of new therapeutic treatment strategies with an emphasis on molecular targeted therapies is provided

Development of saporin-based therapeutic options for the treatment of solid tumors.

Up to now, cancer represents one of the most challenging disease to treat, mostly due to its ability to adapt and negatively respond to current available therapies. Over the past 30 years, Ribosome Inactivating Proteins (RIPs) have attracted great interest in the scientific community for their therapeutic potential. Indeed, such toxins have been extensively used as potent and versatile therapeutic weapons due to important advantages compared to chemotherapeutics: for instance, they act independently form cell cycle, killing both quiescent and dividing cells, limiting the development of cancer resistance. However, the successful application of toxins-based therapeutics to solid tumors remains to be demonstrated. In this study, we explored the potential use of the plant-RIP saporin (SAP) for the treatment of solid tumors. In particular, we propose two different strategies, in which SAP is selectively and safely conveyed to malignant cells either in the form of recombinant protein through genetically fused targeting moieties, or throughout cell-derived extracellular vesicles as vehicles. Overall our data indicate that SAP-based recombinant proteins are promising antitumoral therapeutic options. Applied as single or combined treatment, as well as used together with traditional therapeutics, they appropriately address both intra and inter- tumor heterogeneity. In addition, the use of exosomes as SAP nanocarriers is a promising strategy to improve safety and drug delivery to tumor cells