Description: Purpose Sepantronium bromide (YM155) is a hydrophilic quaternary compound that cannot be administered orally due to its low oral bioavailability; it is furthermore rapidly eliminated via the kidneys. The current study aims at improving the pharmacokinetic profile of YM155 by its formulation in immunoliposomes that can achieve its enhanced delivery into tumor tissue and facilitate uptake in neuroblastoma cancer cells. Methods PEGylated YM155 loaded liposomes composed of DPPC, cholesterol and DSPE-PEG 2000 were prepared via passive film-hydration and extrusion method. Targeted (i.e. immuno-)liposomes were prepared by surface functionalization with SATA modified monoclonal anti-disialoganglioside (GD2) antibodies. Liposomes were characterized based on their size, charge, antibody coupling and YM155 encapsulation efficiency, and stability. Flow cytometry analysis and confocal microscopy were performed on IMR32 and KCNR neuroblastoma cell lines. The efficacy of developed formulations were assessed by in-vitro toxicity assays. A pilot pharmacokinetic analysis was performed to assess plasma circulation and tumor accumulation profiles of the developed liposomal formulations. Results YM155 loaded immunoliposomes had a size of 170 nm and zeta potential of −10 mV, with an antibody coupling efficiency of 60% andYM155 encapsulation efficiency of14%. Targeted and control liposomal formulations were found to have similar YM155 release rates in a release medium containing 50% serum. An in-vitro toxicity study on KCNR cells showed less toxicity for immunoliposomes as compared to free YM155. In-vivo pharmacokinetic evaluation of YM155 liposomes showed prolonged blood circulation and significantly increased half-lives of liposomal YM155 in tumor tissue, as compared to a bolus injection of free YM155. Conclusions YM155 loaded immunoliposomes were successfully formulated and characterized, and initial in-vivo results show their potential for improving the circulation time and tumor accumulation of YM155.

Description: Purpose This study aimed to further explore the mechanisms behind the ability of certain linear polyamidoamines (PAAs) to transfect cells with minimal cytotoxicity. Methods The transfection efficiency of DNA complexed with a PAA of a molecular weight over 10 kDa or 25 kDa branched polyethyleneimine (BPEI) was compared in A549 cells using a luciferase reporter gene assay. The impact of endo/lysosomal escape on transgene expression was investigated by transfecting cells in presence of bafilomycin A1 or chloroquine. Cytotoxicity caused by the vectors was evaluated by measuring cell metabolic activity, lactate dehydrogenase release, formation of reactive oxygen species and changes in mitochondrial membrane potential. Results The luciferase activity was ~3-fold lower after transfection with PAA polyplexes than with BPEI complexes at the optimal polymer to nucleotide ratio (RU:Nt). However, in contrast to BPEI vectors, PAA polyplexes caused negligible cytotoxic effects. The transfection efficiency of PAA polyplexes was significantly reduced in presence of bafilomycin A1 while chloroquine enhanced or decreased transgene expression depending on the RU:Nt. Conclusions PAA polyplexes displayed a pH-dependent endo/lysosomal escape which was not associated with cytotoxic events, unlike observed with BPEI polyplexes. This is likely due to their greater interactions with biological membranes at acidic than neutral pH.

Description: Drug bioavailability to the developing brain is a major concern in the treatment of neonates and infants as well as pregnant and breast-feeding women. Central adverse drug reactions can have dramatic consequences for brain development, leading to major neurological impairment. Factors setting the cerebral bioavailability of drugs include protein-unbound drug concentration in plasma, local cerebral blood flow, permeability across blood-brain interfaces, binding to neural cells, volume of cerebral fluid compartments, and cerebrospinal fluid secretion rate. Most of these factors change during development, which will affect cerebral drug concentrations. Regarding the impact of blood-brain interfaces, the blood-brain barrier located at the cerebral endothelium and the blood-cerebrospinal fluid barrier located at the choroid plexus epithelium both display a tight phenotype early on in embryos. However, the developmental regulation of some multispecific efflux transporters that also limit the entry of numerous drugs into the brain through barrier cells is expected to favor drug penetration in the neonatal brain. Finally, drug cerebral bioavailability is likely to be affected following perinatal injuries that alter blood-brain interface properties. A thorough investigation of these mechanisms is mandatory for a better risk assessment of drug treatments in pregnant or breast-feeding women, and in neonate and pediatric patients.

Description: Purpose The purpose was to evaluate DSF for high throughput screening of protein thermal stability (unfolding/ aggregation) across a wide range of formulations. Particular focus was exploring PROTEOSTAT® – a commercially available fluorescent rotor dye – for detection of aggregation in surfactant containing formulations. Commonly used hydrophobic dyes (e.g. SYPRO™ Orange) interact with surfactants, complicating DSF measurements. Methods CRM197 formulations were prepared and analyzed in standard 96-well plate rT-PCR system, using SYPRO™ Orange and PROTEOSTAT® dyes. Orthogonal techniques (DLS and IPF) are employed to confirm unfolding/aggregation in selected formulations. Selected formulations are subjected to non-thermal stresses (stirring and shaking) in plate based format to characterize aggregation with PROTEOSTAT®. Results Agreement is observed between SYPRO™ Orange (unfolding) and PROTEOSTAT® (aggregation) DSF melt temperatures across wide range of non-surfactant formulations. PROTEOSTAT® can clearly detect temperature induced aggregation in low concentration (0.2 mg/mL) CRM197 formulations containing surfactant. PROTEOSTAT® can be used to explore aggregation due to non-thermal stresses in plate based format amenable to high throughput screening. Conclusions DSF measurements with complementary extrinsic dyes (PROTEOSTAT®, SYPRO™ Orange) are suitable for high throughput screening of antigen thermal stability, across a wide range of relevant formulation conditions – including surfactants –with standard, plate based rT-PCR instrumentation.

Description: Purpose Oral drug delivery using NPs is a current strategy for poorly absorbed molecules. It offers significant improvement in terms of bioavailability. However, the encapsulation of proteins and peptides in polymeric NPs is a challenge. Firstly, the present study focused on the double emulsion process in order to encapsulate the OXY peptide. Then the technique was challenged by a one-step simplified process, the simple emulsion. Methods In order to study the influence of formulation and process parameters, factorial experimental designs were carried on. The responses observed were the NP size (〈200 nm in order to penetrate the intestinal mucus layer), the suspension stability (ZP 〈 |30| mV) and the OXY loading. Results It was thus found that the amount and the nature of surfactant, the ratio between the phases, the amount of PLA-PEG polymer and OXY, the presence of a viscosifying agent, and the duration of the sonication could significantly influence the responses. Finally, OXY-loaded NPs from both processes were obtained with NP size of 195 and 226 nm and OXY loading of 4 and 3.3% for double and simple emulsions, respectively. Conclusion The two processes appeared to be suitable for OXY encapsulation and comparable in term of NP size, peptide drug load and release obtained.

Description: Purpose Biometals such as zinc and copper have been shown to affect tight junction expression and subsequently blood-brain barrier (BBB) integrity. Whether these biometals also influence the expression and function of BBB transporters such as P-glycoprotein (P-gp) however is currently unknown. Methods Using the immortalised human cerebral microvascular endothelial (hCMEC/D3) cell line, an in-cell western assay (alongside western blotting) assessed relative P-gp expression after treatment with the metal ionophore clioquinol and biometals zinc and copper. The fluorescent P-gp substrate rhodamine-123 was employed to observe functional modulation, and inductively coupled plasma mass spectrometry (ICP-MS) provided information on biometal trafficking. Results A 24-h treatment with clioquinol, zinc and copper (0.5, 0.5 and 0.1 μM) induced a significant upregulation of P-gp (1.7-fold) assessed by in-cell western and this was confirmed with western blotting (1.8-fold increase). This same treatment resulted in a 23% decrease in rhodamine-123 accumulation over a 1 h incubation. ICP-MS demonstrated that while t8his combination treatment had no effect on intracellular zinc concentrations, the treatment significantly enhanced bioavailable copper (4.6-fold). Conclusions Enhanced delivery of copper to human brain microvascular endothelial cells is associated with enhanced expression and function of the important efflux pump P-gp, which may provide therapeutic opportunities for P-gp modulation.

Description: Purpose Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs were found to be able to target cells that express Toll-like receptor 9 to modulate innate and adaptive immune reactions. But their in vivo application in immunotherapy against cancer has not been successful. We attempted in this study to examine polyethylene-glycol (PEG) conjugated CpG ODNs and investigated their mechanism of immune modulation in anti-cancer therapy. Methods CpG-PEG conjugates with different PEG lengths were synthesized. In vitro activity as well as in vivo pharmacokinetics and pharmacodynamics properties were evaluated. Results CpG-PEG20Ks were found to be able to persist longer in circulation and activate various downstream effector cells. After intravenous injection, they resulted in higher levels of IL-12p70 in the circulation and lower M-MDSC infiltrates in the tumor microenvironment. Such activities were different from those of CpG ODNs without PEGylation, suggesting different PK-PD profiles systemically and locally. Conclusions Our data support the development of CpG-PEGs as a new therapeutic agent that can be systemically administered to modulate immune responses and the microenvironment in tumor tissues.

Description: ABSTRACT Purpose To understand hydrolysis and alcoholysis of polyvinylpyrrolidone-co-vinylacetate (PVPVA) during formulation and storage, elucidate the reaction mechanism, establish an intrinsic kinetic model, and apply this model coupled with GastroPlus™ modeling to predict the amount of PVPVA degradation in vivo . Methods The experimental approach includes the detection of the polymer reaction by solution nuclear magnetic resonance (NMR) and the measurement of reaction product concentration via gas chromatography (GC). The theoretical approach includes the establishment of the intrinsic kinetic model and the application of GastroPlus™ to predict the degree of PVPVA degradation. Results The kinetic model established is a first order reaction between PVPVA and 2-propanol (IPA) or water under an acidic condition. The application of this kinetic model shows that between 1.7 and 6.8 mg of degradant is formed in the GI tract for a 850 mg dose of PVPVA. Conclusions The results from this application provide valuable input for process development and the risk analysis of the degradation of PVPVA.

Description: Purpose Studies were conducted to investigate dilute solutions of the monoclonal antibody (mAb) bevacizumab, mAb fragment ranibizumab and fusion protein aflibercept, develop common procedures for formulation of low concentration mAbs and identify a stabilizing formulation for anti-VEGF mAbs for use in in vitro permeation studies. Methods Excipient substitutions were screened. The most stabilizing formulation was chosen. Standard dilutions of bevacizumab, ranibizumab and aflibercept were prepared in PBS, manufacturer’s formulation, and the new formulation. Analysis was by SE-HPLC and ELISA. Stability, disaggregation and pre-exposure tests were studied. Results When Avastin, Lucentis and Eylea are diluted in PBS or manufacturer’s formulation, there is a 40–50% loss of monomer concentration and drug activity. A formulation containing 0.3% NaCl, 7.5% trehalose, 10 mM arginine and 0.04% Tween 80 at a pH of 6.78 stabilized the mAbs and minimized the drug loss. The formulation also disaggregates mAb aggregation while preserving the activity. Degassing the formulation increases recovery. Conclusions We developed a novel formulation that significantly stabilizes mAbs under unfavorable conditions such as low concentration or body temperature. The formulation allows for tissue permeation experimentation. The formulation also exhibits a disaggregating effect on mAbs, which can be applied to the manufacture/packaging of mAbs and bioassay reagents.

Description: Purpose A multidrug resistance (MDR) modulator, disulfiram (DSF), was incorporated into pure paclitaxel (PTX) nanoparticles to construct a smart paclitaxel-disulfiram nanococrystals (PTX-DSF Ns) stabilized by β-lactoglobulin (β-LG), with the aim to reverse MDR and therefore enhnce cytotoxicity towards Taxol-resistant A549 cells (A549/TAX). Method PTX-DSF Ns was prepared by antisolvent precipitation method. Flow cytometry was used to determine the cell uptake, drug efflux inhibition, cell cycle phase arrest and apoptosis. MDR-1 gene expression level was detected by real time quantitative PCR and gel electrophoresis. Results PTX-DSF Ns prepared from the optimized formulation had an optimum diameter of 160 nm, was stable and had a high drug-loading capacity. Importantly, the uptake of PTX-DSF Ns in A549/TAX cells was 14-fold greater than the uptake of PTX Ns. Furthermore, PTX-DSF Ns promoted 5-folds increase in apoptosis, enabled 7-folds reduction in the IC 50 , and rendered 8.9-fold decrease in the dose compared with free PTX. Conclusion PTX-DSF Ns with a precise mass ratio offer efficient cytotoxicity against Taxol-resistant cells and a novel approach for codelivery and sensitizing MDR cancer to chemotherapy. In addition, the use of nanosuspensions as a combined treatment provides a new research avenue for nanosuspensions.