RESUMEN
Despite progress in identifying molecular drivers of cancer, it has been difficult to translate this knowledge into new therapies, because many of the causal proteins cannot be inhibited by conventional small molecule therapeutics. RNA interference (RNAi), which uses small RNAs to inhibit gene expression, provides a promising alternative to reach traditionally undruggable protein targets by shutting off their expression at the messenger RNA (mRNA) level. Challenges for realizing the potential of RNAi have included identifying the appropriate genes to target and achieving sufficient knockdown in tumors. We have developed high-potency Dicer-substrate short-interfering RNAs (DsiRNAs) targeting ß-catenin and delivered these in vivo using lipid nanoparticles, resulting in significant reduction of ß-catenin expression in liver cancer models. Reduction of ß-catenin strongly reduced tumor burden, alone or in combination with sorafenib and as effectively as DsiRNAs that target mitotic genes such as PLK1 and KIF11. ß-catenin knockdown also strongly reduced the expression of ß-catenin-regulated genes, including MYC, providing a potential mechanism for tumor inhibition. These results validate ß-catenin as a target for liver cancer therapy and demonstrate the promise of RNAi in general and DsiRNAs in particular for reaching traditionally undruggable cancer targets.
Asunto(s)
Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , ARN Interferente Pequeño/genética , beta Catenina/genética , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/terapia , Masculino , Ratones , Nanopartículas/administración & dosificación , Nanopartículas/química , Interferencia de ARN , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/metabolismo , Carga Tumoral/genética , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/metabolismoRESUMEN
This study investigated the root cause behind an observed free fatty acid particle formation and resulting Polysorbate 20 (PS20) loss for a sulfatase drug product upon long-term storage at 5 ± 3°C. Reversed- phase chromatography with mass spectrometric analysis as well as charged aerosol detection was used to characterize the peaks associated with the intact and degraded PS20. Additionally, a proteomics study was undertaken to identify the residual host cell proteins in the sulfatase drug substance. PS20 stability studies were conducted in the presence of sulfatase, a sulfatase inhibitor, putative phospholipase B-like 2, and mock drug substance produced using a null cell line vector under experimental conditions optimized for PS20 degradation. This study provides the first published evidence where the residual host cell protein present in the drug substance was identified and experimentally shown to catalyze the breakdown of PS20 in a protein formulation over time, resulting in free fatty acid particles and PS20 loss. This study demonstrates the importance of early detection of potential impurities in the protein drug substance that may contribute to polysorbate degradation to make a judicious selection of the surfactant and its optimized concentration for the final drug product.
Asunto(s)
Ácidos Grasos no Esterificados/metabolismo , Lisofosfolipasa/metabolismo , Tamaño de la Partícula , Polisorbatos/metabolismo , Sulfatasas/metabolismo , Ácidos Grasos no Esterificados/análisis , Humanos , Lisofosfolipasa/análisis , Polisorbatos/análisis , Estabilidad Proteica , Proteómica/métodos , Sulfatasas/análisis , Espectrometría de Masas en Tándem/métodosRESUMEN
The year 2002 marked the 20th anniversary of the first successful product of modern biotechnology, the regulatory approval of recombinant insulin for biopharmaceutical applications. Insulin is also the first crystalline protein to be approved for therapeutic use. Over the past two decades, almost 150 biopharmaceuticals have gained marketing authorisation; however, insulin remains the only crystalline protein on the market. Significant research and development efforts have focused on the engineering of protein molecules, efficacy testing, model development, and protein production and characterisation. These advances have dramatically boosted the therapeutic applications of proteins, which now include treatments against acute conditions, such as cancer, cardiovascular disease and viral disease, and chronic conditions, such as diabetes, growth hormone deficiency, haemophilia, arthritis, psoriasis and Crohn's disease. Despite these successes, many challenges normally associated with biopharmaceuticals, such as poor stability and limited delivery options, remain. Protein crystals have shown significant benefits in the delivery of biopharmaceuticals to achieve high concentration, low viscosity formulation and controlled release protein delivery. This review will discuss challenges related to the broader utilisation of protein crystals in biopharmaceutical applications, as well as recent advances and valuable new directions that protein crystallisation-based technologies present.
Asunto(s)
Productos Biológicos/uso terapéutico , Proteínas/uso terapéutico , Animales , Productos Biológicos/administración & dosificación , Cristalización , Sistemas de Liberación de Medicamentos , Humanos , Proteínas/administración & dosificaciónRESUMEN
Development of ready-to-inject in situ formable controlled release gel systems for proteins is extremely challenging due to poor stability of proteins in the organic solvents typically used to fabricate these systems and because of the need of initial drying of proteins. The focus of the present study was to develop and characterize injectable controlled release systems composed of crystals of amylase, a model protein, suspended in solutions of polymeric and non-polymeric matrix materials in organic solvents. In this study, alpha-amylase derived from Aspergillus oryzae was crystallized and crystals were suspended in a poly(DL-lactide-co-glycolide) (PLGA) solution in acetonitrile (PLGA/acetonitrile), or in sucrose acetate isobutyrate (SAIB) plasticized with ethanol (SAIB/ethanol) systems. The results indicate that the protein crystals could be incorporated in these in situ formable gels without the need for initial drying. The crystals withstand organic solvents and water/organic solvent interfaces, and provide high protein loading (>30%) in these systems. Moreover, changing the morphology of the amylase crystals successfully modulated amylase release profiles. Study of long-term stability at 4 degrees C revealed a greater stability of crystalline protein compared to amorphous amylase. The above-mentioned data suggest that protein crystals might offer greater feasibility in developing sustained release injectable in situ formable protein depot systems.
Asunto(s)
Amilasas/administración & dosificación , Amilasas/química , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/química , Química Farmacéutica , Cromatografía Líquida de Alta Presión/métodos , Cristalización , Geles , InyeccionesRESUMEN
A method using multi-mode solid-phase extraction and ultra-high-performance liquid chromatography (UHPLC)-electrospray mass spectrometry was developed to quantify Dicer-substrate small interfering RNA (DsiRNA) directed against the hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene transcript in mouse liver tissue. The oligonucleotides were separated into sense and antisense strands using a UHPLC C(18) column with mobile phases containing 1,1,1,3,3,3-hexafluoro-2-propanol in both water (mobile phase A) and methanol (mobile phase B) with triethylamine as the ion pairing agent at a column temperature of 65°C. The lower limits of detection for the sense and antisense strands were ~1 ng/mg. The dynamic ranges for the sense and antisense strands were 5 ng/mg-1,000 ng/mg and 1 ng/mg-1,000 ng/mg, respectively. The lower limits of quantification for the sense and antisense strands were 5 ng/mg and 1 ng/mg, respectively, each with a relative standard deviation <15% over the range of calibration curve with sufficient precision and accuracy. Oligonucleotides were quantified at different time intervals after intravenous administration of living mice with lipid nanoparticle formulated HPRT1 DsiRNA and were detected as early as 15 min after administration, but not detected beyond the 24 h time point.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Hipoxantina Fosforribosiltransferasa/metabolismo , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/metabolismo , Manejo de Especímenes/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Femenino , Hígado/metabolismo , Ratones , Ribonucleasa III/genéticaRESUMEN
PURPOSE: The aim of the study is to solve a significant challenge of extending the half-life of therapeutic proteins using crystalline biopharmaceuticals and without redesigning the molecules. METHODS: Crystals of recombinant human growth hormone were coated with a monomolecular layer of positively charged poly(arginine). The pharmacokinetics and pharmacodynamics of this poly(arginine)-coated human growth hormone crystalline formulation were determined in hypophysectomized rats and monkeys. RESULTS: Here we have demonstrated for the first time that crystals of human growth hormone coated with positively charged poly(arginine) allowed for in vivo pharmacokinetic release profiles of over several days in animal models. The efficacy of this crystalline formulation injected subcutaneously once a week was found to be equivalent to seven daily soluble injections in the standard weight gain assay using the hypophysectomized rat model and in measurement of serum insulin-like growth factor in monkeys. The nonviscous nature of the suspension facilitated easy administration through a fine, 30-gauge needle and should provide for improved patient convenience and compliance. CONCLUSIONS: The approach described here offers an exciting possibility of being broadly applicable to other therapeutic proteins.