1,721,159 research outputs found
Product related factors influencing the immunogenicity of interferon beta-1b
No full textTherapeutic interferon beta is the first line treatment of relapsing remitting Multiple Sclerosis. However, despite their success in improving patient wellbeing, all IFNβ products encounter a significant problem: immunogenicity. In some patients, IFNβ products induce the formation of antidrug antibodies that undermine treatment efficacy and may even lead to treatment failure. These patients not only have a substantial health risk due to failing treatment, but are being treated with expensive products that no longer work. We thus urgently need to lower immunogenicity of therapeutic interferon beta products. In this thesis, IFNβ-1b, the most immunogenic IFNβ product, was studied. The first step in lowering immunogenicity is to understand why a product is immunogenic. Here, there are two possibilities: (1) IFNβ-1b is immunogenic because of epitopes, the foreign peptide sequences that induce a classical immune response, and (2) immunogenicity is caused by aggregates present in the products, which multiple researchers have identified as a risk factor for immunogenicity (Van Beers et al., 2010a; Haji Abdolvahab et al., 2016). Research described in this thesis aims to determine which of these potential triggers is causing immunogenicity. Furthermore, this thesis also describes investigation into another possible product related factor in immunogenicity of this drug, the formulation of IFNβ-1b. Given ethical and practical restraints, I have used the tolerant mouse models described earlier to investigate human interferon beta. Further studies will be required to provide additional insight into the mechanisms underlying the immunogenicity of therapeutic interferon beta, and to develop a product with lower immunogenicity
Biosimilars: linking quality data to clinical outcomes
No full textThe aim of this study was to establish a link between quality attributes of biosimilars and potential clinical outcomes with regards to safety and immunogenicity. As we have access to multiple biosimilar and copy biologic products as well to patient data, the research involved linking comparative qualitative data of filgrastim and epoetin to clinical safety and immunogenicity. Common assays, which included sodium dodecyl sulfate polyacrylamide gel electrophoresis in combination with western blot, high performance size-exclusion chromatography, asymmetrical flow field-flow fractionation, capillary zone electrophoresis, iso-electric focusing, host cell protein impurities, endotoxin contamination, and potency testing were used to characterize biosimilar, copy and innovator products. With respect to filgrastim, except for the specific activities of the two copy filgrastim products, we found no clinically significant differences in product quality between the copy, the biosimilar, and the innovator filgrastim products. Our study also demonstrates comparable quality of biosimilar epoetin products compared to the innovator with some degree of variations in epoetin content, isoform profiles and potency found between products as well as among batches of single products. In contrast, some of the copy epoetin products from the Thai drug market did differ significantly from the innovator product, with higher impurities e.g. aggregation and host cell protein observed. Our clinical data indicate a higher incidence of pure red cell aplasia among patients administered these copies. These results suggest a potential link between quality attributes of biosimilars and potential clinical outcomes. More importantly, this may contribute to a future in which the biosimilars will be increasingly considered as classical generics
Pharmacy preparations: Back in the limelight? Pharmacists make up your mind!
Full text linkIn this contribution to the theme issue recognizing prof. Florence's achievements as editor -in-chief of the Int. J. Pharmaceutics, we analyze the future of pharmacy preparations (also known as extemporaneous preparations or compounded products). Pharmacy preparations, long considered as an endangered part of the pharmacy profession on its way to extinction, may be at the brink of a revival. Drivers of this revival are a set of changes related to new clinical concepts and supply shortages. Moreover, new production and IT paradigms are being developed that facilitate the preparation processes and provide the necessary quality management systems. Finally, more detailed legislation (EU) and guidelines (US) gets a better hold on preparation in pharmacies. The question is now: is the pharmacy profession willing to accept preparation of high quality medicines in the pharmacy as an integral part of its professional tasks? If so, institutions for pharmacy education should provide the required competences to the pharmacy student. If not, alternative scenarios with other disciplines taking the lead should be considered. Whatever the choice made, the 'Physicochemical principles of pharmacy: in manufacture, formulation and clinical use' by Florence and Attwood (2016); will be on the engineer/pharmacy student's desk
Biomedical Applications of Self-Assembling Peptides
Full text linkSelf-assembling peptides have gained increasing attention as versatile molecules to generate diverse supramolecular structures with tunable functionality. Because of the possibility to integrate a wide range of functional domains into self-assembling peptides including cell attachment sequences, signaling domains, vaccine epitopes, and even therapeutic moieties, complex nanostructures can be obtained with a wide range of applications in the biomedical field. The first part of this Review provides a concise overview of how peptide primary and secondary structure dictate the way such self-assembling peptides organize into higher ordered, supramolecular structures. Next, an overview of the literature will be given on recent studies on peptide self-assembly for application in drug delivery, vaccination, and tissue engineering
Polymeric micelles for drug delivery: from synthesis to in vivo studies
No full textDrug delivery systems have been extensively utilized to increase water-solubility of hydrophobic chemotherapeutic drugs and target the drugs to tumors, which enhances the efficacy of chemotherapy and simultaneously decreases non-specific disposition of cytostatic drugs in healthy organ/tissues, and consequently avoids or minimizes toxicity/adverse effects. Among different nano-sized drug delivery systems, polymeric micelles are one of the most successful formulations applied on anti-cancer drugs, as evidenced from the fact that several micellar formulations for chemotherapeutic drugs have entered clinical trials. Polymeric micelles are extensively utilized for this purpose for the following reasons: (1) polymeric micelles possess a hydrophobic core suitable to accommodate hydrophobic (anti-cancer) compounds; (2) the hydrophilic micellar corona can provide ‘stealth’ properties and avoid their rapid removal by the reticuloendothelial system (RES); (3) polymeric micelles have a substantially lower critical micelle concentration (CMC) and therefore a better stability compared to micelles composed of small molecule surfactants; (4) micelle-forming polymers can be chemically tailored to increase the stability of polymeric micelles by introducing physical/chemical interactions between the polymer chains; (5) stimuli-sensitive micelles can be developed which release their payloads upon a certain physical trigger or upon degradation at their site of action; (6) polymeric micelles can be decorated with targeting moieties, which may potentially improve the therapeutic response by an increased uptake of drug-loaded polymeric micelles by cancer cells. Although polymeric micelles are attractive systems for tumor-targeted drug delivery, several hurdles hamper their applicability. For example, many micellar systems suffer from low stability in the blood circulation, which causes fast elimination of drug-loaded polymeric micelles after systemic administration and severely limits the tumor targeting efficacy of the systems, which is one of the main issues that the work described in this thesis aimed to address. Furthermore, applications of polymeric micelles other than tumor-targeted drug delivery have been proposed, including polymeric micelles based imaging-guided drug delivery and photodynamic therapy. This aim of this thesis is to further advance polymeric micelles as tumor-targeted carrier systems regarding the following aspects: (1) enhance the stability of polymeric micelles and drug retention in the blood circulation by means of chemical/physical interactions between the micelle forming polymer chains; (2) investigate the possibilities for the synthesis of better defined amphiphilic polymers by controlled/living polymerization, i.e., Reversible Addition Fragmentation Chain Transfer (RAFT) polymerization; (3) validate the concept of image-guided drug delivery based on fluorescently-labeled polymeric micelles; (4) study the pharmacokinetics, biodistribution and therapeutic efficacy of stabilized polymeric micelles in tumor models; (5) investigate the suitability of polymeric micelles for photodynamic therapy
Thermosensitive release systems for image guided local drug delivery
No full textNanosized drug delivery systems are developed to improve the therapeutic efficacy and to reduce unwanted side effects of existing drugs as well as drug candidates. Liposomes are the most intensively studied drug delivery systems and a number of studies showed that encapsulation of doxorubicin (DOX) in liposomes resulted in an increased therapeutic index particularly due to a significant reduction in unwanted side effects. Nevertheless, the concentration of free drug in the tumor is relatively low due to the slow and uncontrolled release of the drug from the liposomes and as a consequence, cytotoxic free drug concentrations are not always obtained in the tumor. In order to optimize the free drug concentration in the tumor, liposomes should be capable of releasing drugs at the target site in response to a specific stimulus. Therefore, in this thesis temperature sensitive drug delivery systems were explored that release their content in a fast manner at mild hyperthermia while remaining stable at body temperature. Temperature sensitive liposomes with tunable release characteristics were developed that release their content at elevated temperatures. Temperature-sensitive N-(2-hydroxypropyl)methacrylamide mono/dilactate polymers were incorporated into liposomes via a cholesterol anchor (chol-pHPMAlac). The onset-temperature of the DOX release was dependent on the characteristics (copolymer composition and molecular weight) of the chol-pHPMAlac used. It was also shown that these liposomes released their DOX content quantitatively upon exposure to High Intensity Focused Ultrasound. The results of different in vitro assays suggested that chol-pHPMAlac liposomes are not cleared rapidly from the blood circulation after i.v. administration and these liposomes do most likely not generate serious complications e.g. the formation of thrombi after injection. Besides nanoparticles, also micro-sized drug delivery systems are often used for local drug delivery in tumors. This thesis describes the development and characterization of alginate microspheres that combine embolization with on-demand triggered drug release. For that purpose, alginate microspheres loaded with temperature sensitive liposomes (TSL-Ba-ms) were developed, which release their payload after mild hyperthermia. These liposomes contained DOX and [Gd(HPDO3A)(H2O)], a T1 MRI contrast agent, for real time monitoring of the release by MRI. Empty alginate microspheres were crosslinked with holmium ions (T2* MRI contrast agent) to allow microsphere visualization (Ho-ms). TSL-Ba-ms had a mean diameter of 76 µm and released DOX quantitatively within 3 minutes at 42 °C in 50% serum. The deposition of the microspheres and [Gd(HPDO3A)(H2O)] release after i.a. co-administration of TSL-Ba-ms and Ho-ms was monitored in the VX2 tumor model in the auricle of a New Zealand White rabbit. Microsphere clusters appeared on the T2*-wt image after the co-administration of TSL-Ba-ms and Ho-ms and these clusters remained visible after applying mild hyperthermia. The release of [Gd(HPDO3A)(H2O)] from the TSL-Ba-ms was visualized on the T1-wt image. The location of Ho-ms overlapped with the location of [Gd(HPDO3A)(H2O)] released from TSL-Ba-ms making Ho-ms a suitable marker for TSL-Ba-ms
Optical Molecular Imaging of Ultrasound-mediated Drug Delivery
No full textThe goal of this PhD project was to develop optical molecular imaging methods to study drug delivery facilitated by ultrasound waves (US) and hyperthermia. Fibered confocal fluorescence microscopy (FCFM), together with dedicated image analysis, was used in vitro on a cell monolayer, and in vivo at the tissue scale, to monitor in real time and assess model drug and drug distribution. To this end, setups were designed that allowed ultrasound exposure or hyperthermia conditions, and that would present geometrical constraints in conventional optical imaging systems. However, these were largely overcome by the fiber based design of the microscope. In chapter 2, the feasibility to monitor in real-time US- and microbubble-mediated uptake of a cell-impermeable fluorescent model drug, i.e., SYTOX Green, is evaluated. An in vitro setup was designed that combined a mono-element US transducer, a cell culture chamber containing a monolayer of tumor cells together with SonoVue® microbubbles, and FCFM. The sequences showed a remaining plasma membrane permeability after the end of US exposure. To improve the accuracy of uptake kinetic parameter estimates of SYTOX Green, a post-processing method including cell tracking was presented in chapter 3. Using a two-compartment model representing the extracellular space and the cellular compartment, separated by a plasma membrane, the statistical analysis of the population kinetic data showed a median time constant of 2 minutes 19 seconds. Using the setup described in chapter 2 and the post-processing pipeline developed in chapter 3, we investigated in chapter 4 whether endocytosis is involved in US- and microbubbles- mediated delivery of small molecules using chlorpromazine, an inhibitor of clathrin-mediated endocytosis, or genistein, an inhibitor of caveolae-mediated endocytosis. During the real-time monitoring of SYTOX Green uptake, the cells in the presence of SonoVue® microbubbles were exposed to 1.4 MHz US waves at a 0.2 MPa peak-negative pressure. Both inhibitors were observed to slow down the US-mediated uptake of SYTOX Green, with a significant 2.5-fold increase of the uptake time constant with chlorpromazine, and a 1.1-fold increase with genistein. The impact of photobleaching on uptake rate estimates measured by FCFM was evaluated in chapter 5 to correct for it and improve the accuracy of pharmacokinetic parameter estimates. To model photobleaching of SYTOX Green, a photobleaching rate was added to the current two-compartment model describing cell uptake. Using this three-compartment model, an uptake rate of 6.0 10-3 s-1, independent of the applied laser power, was measured. In chapter 6, a feasibility study is described to evaluate tissue penetration of doxorubicin after its intravascular release from the thermo-sensitive liposomes ThermodoxTM using FCFM, and in a normal physiological environment. To this end, rat R1 rhabdomyosarcoma tumor pieces were implanted subcutaneously in the hind leg of 9 nude rats. A setup combining a water bath with a platform was designed to create local hyperthermia and thus trigger the release of doxorubicin from ThermodoxTM injected intravenously. In the tumor microenvironment, real-time simultaneous monitoring of doxorubicin penetration (488-nm excitation channel) succeeded in 2 of the 9 rats. A strong heterogeneity of doxorubicin distribution in the tumor was observed, which likely limited the success rate of the real-time monitoring
Biosimilar monoclonal antibodies: The scientific basis for extrapolation
Full text linkIntroduction: Biosimilars are biologic products that receive authorization based on an abbreviated regulatory application containing comparative quality and nonclinical and clinical data that demonstrate similarity to a licensed biologic product. Extrapolation of safety and efficacy has emerged as an important way to simplify biosimilar development. Regulatory authorities have generally reached the consensus that extrapolation of similarity from one indication to other approved indications of the reference product can be permitted if it is scientifically justified. Areas covered: Recently, the first biosimilar, biosimilar infliximab (Remsima/Inflectra) to the innovator monoclonal antibody infliximab (Remicade), was approved in the European Union, Canada and South Korea; the USA subsequently approved its first biosimilar, a less complex molecule (filgrastim-sndz). Based on two clinical trials of biosimilar infliximab in patients with rheumatoid arthritis and ankylosing spondylitis, the European Medicines Agency allowed extrapolation to all eight approved indications for innovator infliximab, whereas Health Canada did not permit extrapolation to the indications for ulcerative colitis and Crohn's disease. These differing decisions on extrapolation of indications for biosimilar infliximab highlight important unanswered regulatory and scientific questions. Here, we propose substantive scientific considerations for indication extrapolation. Expert opinion: The preclinical and clinical criteria that are currently required to merit indication extrapolation have not been rigorously evaluated
Production and pharmaceutical formulation of plasmid DNA vaccines
No full textResearch leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papilloma viruses (HPV) 16 induced malignancies. Furthermore, this thesis focuses on the development of pDNA-polyplex formulations to further improve the transfection efficiency and immunogenicity of intradermally delivered DNA vaccines. The pharmaceutical development of the pDNA vaccine pVAX TTFC E7SH consisted of the improved upstream processing and downstream processing of pDNA. It was shown that the introduction of a transposon from a well-defined host cell in a pDNA vector can occur. In order to prevent costly rejection of clinical material, both the quality control of the source pDNA, the MCB and in-process controls during manufacture of pDNA bulk drug must be sensitive enough to detect a contamination as early as possible. We show that the freeze-dried pDNA formulation has a shelf life stability for more than 5 years when stored at -20°C. To determine if pDNA transfection can be further improved by formulating pDNA into polyplexes, we screened a broad panel of polymers with distinct differences in molecular structure and characteristics. We measured ex vivo human skin transfection efficiency and polymer characteristics (size, PDI, charge) for this panel of polyplex formulations. Based on the finding in this study, PAA-PEG based polyplexes are the most promising candidates for improving pDNA transfection efficiency. Because screening of a large library of polyplexes is a very time-consuming process, the High Content Screening (HCS) method was chosen as an application in our development study. We initially screened for the optimal polymer/pDNA ratio of Poly (amido amine)-polyplexes. In order to obtain a clinically feasible, stable pharmaceutical formulation we investigated the use of an improved buffer system. When formulated in l-histidine buffer, the transfection efficiency of PAA-polyplexes in vitro and in an ex vivo human skin model is enhanced compared to the same polyplexes dispersed in HEPES buffer. The better buffering capacity of l-histidine in the lower pH region of endosomal acidification, together with a higher concentration of cationic charge carriers (polymer and protonated histidine) is hypothesized to result in a more pronounced proton sponge effect and higher polymer-endosomal membrane interactions, giving rise to increased efficiency of endosomal escape and thus transfection. To investigate if lyophilization can preserve our PEGylated PAA-polyplexes, polyplexes formulated with potential lyoprotectants (trehalose, sucrose or HPßCD) and with or without l-histidine-buffer were assessed for physico-chemical characteristics (appearance, size, PDI and charge) as well as transfection efficiency before lyophilization and after reconstitution of the freeze-dried products. The PAA-PEG polyplex formulation with 10 % sucrose in WFI showed a comparable transfection efficiency before lyophilization and after reconstitution of the freeze-dried product. Apparently, the addition of an L-histidine buffer to the freeze-dried polyplexes instead of building it into the lyophilized formulation results in increased transfection efficiency
Decationized polyplexes for targeted delivery of nucleic acids: from carrier design to in vivo evaluation
No full textGene therapy is considered a promising treatment for current intractable diseases. However, the clinical applicability of gene therapy is highly dependent on the development of safe and efficient gene delivery vectors. So far, viral vectors have been used for clinical applications, but due to severe risks associated with viruses, cationic polymers have been evaluated as alternatives to viral vectors. Cationic polymers can easily form nanosized particles with plasmid DNA (pDNA), named polyplexes, via electrostatic interactions and possess an enormous chemical and structural flexibility. Polycation-based vectors have demonstrated high efficiency in vitro, however, they induce severe toxicity and possess suboptimal efficiencies in vivo, mainly due to their cationic nature, which significantly hampers their clinical applicability. With our work we have developed an alternative to conventional polycation-based polyplexes: decationized polyplexes. Unlike the cationic polymer based systems, decationized polyplexes are formed by hydrophilic and neutral polymers and can be obtained by an innovative 3-step process: polyplex formation by electrostatic interaction between pDNA and a polycationic precursor, structure stabilization by disulfide crosslinking, and finally removal of cationic charge - decationization. Structurally, decationized polyplexes consist of a disulfide-crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) core stably entrapping plasmid DNA (pDNA), surrounded by a shell of poly(ethylene glycol) (PEG). Retention of pDNA in the nanoparticles is exclusively based on physical entrapment given by the disulfide crosslinks, which provides an intracellularly triggered release profile, since disulfides are cleaved under the higher reducing environment present inside the cells. Through our study decationized polyplexes have demonstrated important advantages when compared to their cationic counterparts, such as much lower degree of nonspecific uptake and high degree cell specific uptake when decorated with targeting moieties as demonstrated by several cell uptake studies. Furthermore, in vitro studies showed lower cellular toxicity and in vivo nanotoxicity studies using a zebrafish model showed remarkable lower teratogenicity and mortality profile from decationized polyplexes. Stability evaluation in biological fluids a high stability for prolonged periods was found. Finally, in an in vivo biodistribution study, using tumor bearing mice, decationized polyplexes have shown greater retention in blood circulation and higher target tissue (tumor) accumulation. Given their important advantages, decationized polyplexes were also investigated and optimized for small interfering RNA (siRNA) delivery purposes, showing that decationized polyplexes can be used as a platform for different gene delivery modalities. In conclusion, decationzed polyplexes have demonstrated to be an important contribution for the development of safer polymeric gene delivery systems especially for targeted therapies. Importantly, the requirements for decationized polyplexes optimization have been identified, that will be the focus of future studies to further improve transfection efficiency and in vivo performance
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