1,721,092 research outputs found
Complement activation as a bioequivalence issue relevant to the development of generic liposomes and other nanoparticulate drugs
Full text linkLiposomes are known to activate the complement (C) system, which can lead in vivo to a hypersensitivity syndrome called C activation-related pseudoallergy (CARPA). CARPA has been getting increasing attention as a safety risk of i.v. therapy with liposomes, whose testing is now recommended in bioequivalence evaluations of generic liposomal drug candidates. This review highlights the adverse consequences of C activation, the unique symptoms of CARPA triggered by essentially all i.v. administered liposomal drugs, and the various features of vesicles influencing this adverse immune effect. For the case of Doxil, we also address the mechanism of C activation and the opsonization vs. long circulation (stealth) paradox. In reviewing the methods of assessing C activation and CARPA, we delineate the most sensitive porcine model and an algorithm for stepwise evaluation of the CARPA risk of i.v. liposomes, which are proposed for standardization for preclinical toxicology evaluation of liposomal and other nanoparticulate drug candidates
Targeted inhibition of cancer-inflammation
No full textThe new paradigm in cancer treatment that aims to inhibit the smoldering inflammatory response in tumors is explored to develop new anticancer treatments. It appears that targeted drug delivery is essential in this concept as high local levels of anti-inflammatory agents are needed to observe the beneficial effects of inflammation inhibition. An overview of classes of anti-inflammatory drugs and drug delivery systems that so far have been studied to treat cancer-inflammation is given. This thesis falls apart in two sections: the first section studies and evaluates the antitumor efficacy of targeted glucocorticoids, whereas the second part focuses on the development and preclinical evaluation of antitumor effects of different anti-inflammatory compounds encapsulated in long-circulating liposomes. In the first section, it is shown that the antitumor effects of liposomal prednisolone phosphate are not limited to tumor-graft models since this potent anti-inflammatory nanomedicine also inhibits tumor growth in a spontaneous mouse tumor model (which more closely resembles the slow progressive chronic inflammatory human disease). When the glucocorticoids is targeted to tumors with core-crosslinked polymeric micelles, the observed antitumor effects do not differ substantially compared to long-circulating liposomes, however the versatility of the polymeric system on drug release profile may assist on the optimization of glucocorticoids release for maximal antitumor effect. In the second section of this thesis, liposomal formulations of different natural (resveratrol, caffeic acid, pterostilbene, carvacrol, etc) or synthetic (pravastatin) anti-inflammatory compounds were developed, characterized and evaluated for antitumor efficacy. Interestingly, only targeted nanomedicines of multi-targeted and potent anti-inflammatory compounds seem to induce antitumor effects in experimental tumor models. The targeted delivery of anti-inflammatory drugs to tumors as anticancer strategy is more complex that foreseen at the outset of these studies and involves several critical factors that have been pinpointed with these studies. Finally, the findings presented in this thesis and suggestions for future work are discussed in a final summarizing discussion
Design and recombinant production of combinatorial peptide libraries for gene delivery
No full textGene therapy can be defined as the introduction of exogenous nucleic acids into cells with the intention of altering gene expression to prevent, halt or reverse a pathological process. It forms an attractive approach for therapeutic intervention of a wide range of diseases, including genetic diseases, metabolic disorders, infectious diseases, chronic illnesses, cardiovascular diseases and cancer. In order for gene-based therapeutics to become effective, the therapeutic nucleic acids must be delivered into target cells and have to reach their site of action within the cell. However, due to the high charge density and large molecular weight, nucleic acids are generally impermeable to cellular membranes and require assistance in order to reach their target site. To facilitate the uptake by target cells and delivery of nucleic acids at their target site, a sophisticated delivery system is required which must be capable of targeting the diseased cell, facilitate uptake and intracellular trafficking of the nucleic acid cargo to their site-of-action. Gene delivery systems can be divided into two broad classes: viral vectors and non-viral (synthetic) vectors. Viral delivery systems are derived from viruses, whereas non-viral systems are based on macromolecular complexes. Viruses are highly complex and are adepted to infecti cells and deliver their RNA/DNA cargo. The transfection efficiencies of viral vectors remain unprecedented and outperform their non-viral counterparts. However, viral vectors have several drawbacks, including their immunotoxicity and the chances for insertional mutagenesis. Subsequently, non-viral delivery systems have emerged as potential alternatives to viral vectors. In general, non-viral vectors lack the major safety issues associated with their viral counterparts. However, compared to viral vectors, the gene delivery efficiency of non-viral vectors is poor. Peptide-based gene delivery systems may offer a versatile platform for efficient gene delivery. Peptides are biodegradable, biocompatible and various peptides have been identified that can perform several basic functions for gene delivery, such as DNA condensation or membrane disruption. By assembling different functional peptides required for effective gene delivery into a single-chain, the ideal gene delivery system can be created, thereby eliminating compositional variations, facilitate pharmaceutical formulation, and achieve reproducibility at the molecular level. The aim of this thesis is to set up a high-throughput screening method to select out of a large library of multimodular peptides those candidates that are able to efficiently deliver therapeutic nucleic acids into target cells at their site of action. To achieve this, we propose a design strategy that follows a random, integrative approach selecting multimodular peptides containing combinations of functional traits that are optimal for efficient gene transfer. By randomly combining peptides with properties needed for gene delivery (e.g. DNA condensing peptides and membrane disrupting peptides), a combinatorial library encoding multimodular peptides will be generated. This library will be used for the recombinant production and screening of these multimodular peptides for their transfection efficiency. Several rounds of screening and selection will be performed to obtain multimodular peptides for effective gene deliver
Design of kidney-targeted drug-carrier conjugates for the inhibition of profibrotic signaling cascades
No full textIn the past decades, the worldwide number of patients with chronic kidney disease (CKD) has increased remarkably and it is expected that this increasing trend will continue during the coming years (1). At this moment, therapy is focused on the prevention of progressive CKD by treatment of the underlying causes, such as hypertension, diabetes and obesity. CKD is asymptomatic in the initial stage and is often diagnosed in a stage at which treatment of the underlying cause alone is not sufficient to stop loss of kidney function. As a consequence, CKD will gradually progress to end-stage renal disease (ESRD) and renal dialysis or kidney transplantation are then the only possibilities for patients to survive. Because of the lack of clinically available drugs that can halt the progression of CKD and the seriousness of the disease, there is a high need for novel drugs. This thesis focuses on the intracellular delivery of drugs into the proximal tubular cells of the kidneys. Tubulointerstitial fibrosis is one of the common hallmarks of progressive CKD and renal drug targeting to the proximal tubular cells can be of great value in the development of therapeutics that halt or reverse tubulointerstitial fibrosis
Targeted liposomes for drug delivery across the blood-brain barrier
No full textOur brain is protected by the blood-brain barrier (BBB). This barrier is formed by specialized endothelial cells of the brain vasculature and prevents toxic substances from entering the brain. The downside of this barrier is that many drugs that have been developed to cure brain diseases cannot cross this barrier and do not reach the brain in therapeutic concentrations. An innovative way to help these drugs to reach the brain is by encapsulating them into nanoparticles (e.g. liposomes). A targeting ligand on the outside of the particle induces specific uptake of the drug-loaded particle into the endothelial cells of the BBB, after which the drug can be released into the brain. In this thesis, liposomes were coupled to brain-targeting ligands and the potential of these liposomes to reach the brain was studied. Several of the targeting ligands that were used were already described in literature for their brain-targeting potential. These ligands were compared to each other to identify which one was the best. In vivo experiments showed that an anti-transferrin receptor antibody was the best targeting ligand in our studies. Next to these already existing targeting ligands, a new brain-targeting ligand was identified in this thesis by using the technique of phage display. In vitro studies showed that this ligand could significantly enhance the binding of liposomes to brain endothelial cells after it had been fused to a phage coat protein. Therefore, this ligand has the potential to facilitate the brain uptake of therapeutics, and could benefit the treatment of severe brain diseases such as Alzheimer’s disease, brain cancer, or psychotic disorders
Synthetic scaffolds based on biodegradable, functionalized polyesters for tissue engineering applications
No full textThe aim of this thesis was to investigate the possibility of using a novel hydroxyl-functionalized polyester [poly(hydroxymethylglycolide-co-ε-caprolactone), pHMGCL] (Fig.9) to fabricate scaffolds for tissue engineering applications. Degradable polymers that are frequently used for tissue engineering applications are ‘normal’ aliphatic polyesters such as PCL, PLA and PLGA. One of the drawbacks of these polyesters is their hydrophobicity and lack of functional groups, which limits cell adhesion which is an important factor when constructing polymeric scaffolds. Another drawback is their slow hydrolytic degradation (in case of PCL and PLA) due to high extent of crystallinity and hydrophobicity, which makes these materials only suitable for long term implantable devices. Although several functionalized polyesters have been synthesized in recent years, only a limited number has been evaluated for tissue engineering applications so far. Thus, there is a strong need for development of novel materials with improved physical, chemical, and biological properties for tissue engineering application
Investigation of nanobody-decorated albumin nanoparticles for tumor targeting
No full textCancer is still the worldwide leading cause of death. Cancer cells are similar to and different from healthy cells and this makes the eradication of these cells difficult without harming the healthy ones. They are similar in the sense that they share the same cellular machinery with the healthy cells (such as cell division) and different from healthy cells where the feedback mechanisms of controlled cell division and proliferation cease to exist. Conventional cancer chemotherapy aims at blocking the cell division by interfering with the cell cycle, provided that tumor cells divide faster than healthy cells. However the chances of harming fast dividing healthy cells and fast dividing tumor cells are equal. Indeed, the common toxicities observed for cytotoxic drugs are observed mostly in healthy cells that divide fast, such as cells of the gastrointestinal tract and hair follicles. Mutations in tumor cells that lead to higher expression levels of growth factor receptors, for instance, enable those cells to survive as they are more capable of proliferating and are more resistant to apoptosis. The tumor pathophysiology (at a tissue level) is characterized by leaky vasculature and impaired lymphatic drainage. The combination of these two lead to the enhanced permeation and retention at the tumor site, commonly referred to as the EPR effect. The field of “nanomedicine” exploits this EPR effect in order to treat or diagnose cancer. Nanomedicine, in simple terms, refers to nano-sized carriers that can retain a drug throughout its circulation in the blood stream and limit its whole body distribution as opposed to systemic intravenous injection of drugs. Drug carrying nanoparticles cannot easily cross the endothelial barrier and therefore avoid most healthy tissues. In contrast, these nano-sized carriers can pass through the leaky tumor blood vessels, provided that they can circulate long and are small enough to pass through the endothelial gaps. Nanoparticulate carriers will eventually accumulate at the tumor site, Lastly, since the lymphatic drainage at the tumor site is impaired, they are usually retained in the tumor. The initial part of this thesis describes the reformulation of alternative drugs used in the treatment of cancer in addition to conventional cell cycle inhibitors, such as molecularly targeted small molecule drugs into albumin nanoparticles. Overexpressed growth factor receptors (i.e EGFR and c-MET) are good druggable proteins. They can both be targeted with small molecule receptor kinase inhibitors that can inhibit the intracellular part of the protein and antibodies/nanobodies that can block the extracellular epitopes, resulting in the inhibition of signaling cascades. The nanobody-decorated albumin nanoparticles loaded with kinase inhibitors are evaluated for their cellular binding, uptake, trafficking and eventually efficacy on tumor cells. They are shown to be taken up only by the tumor cells where it exerts its effect. The second part deals with understanding the haemacompatibility and biodistribution of these nanoparticles. Nanoparticles showed haemacompatible properties whereas the tumor accumulation was dominated by liver uptake as is commonly observed for nanoparticulate drug carriers
In vitro confirmation of the quantitative differentiation of liposomal encapsulated and non-encapsulated prednisolone (phosphate) tissue concentrations by murine phosphatases
No full textThe quantitative differentiation of liposomal encapsulated and non-encapsulated drug tissue concentrations is desirable, since the efficacy and toxicity are only related to the level of non-encapsulated drug. However, such separate concentration profiles in tissues have still not been reported due to lacking analytical methodology. The encapsulation of prodrugs like prednisolone phosphate (PP) in liposomes offers new, analytical opportunities. Instantaneous dephosphorylation of PP into prednisolone (P) by phosphatases after its release from the liposome in vivo makes it possible to differentiate between the encapsulated and the non-encapsulated drug for such preparations of liposomal PP: PP represents the encapsulated drug, while P represents the non-encapsulated drug. In the here described study, the instantaneous dephosphorylation of PP by murine liver and kidney phosphatases has been verified by incubation of PP in liver and kidney homogenates followed by estimation of the dephosphorylation rate constants k and the dephosphorylation time of the expected maximal in vivo non-encapsulated drug concentrations. In vitro PP has been rapidly converted into P in the presence of homogenate from the excretory organs. The calculated values for k have shown that the liver contains more active sites per gram of tissue than the kidneys. However, the dephosphorylation of PP by these active sites is slower compared with the kidneys. Compared with other pharmacokinetic processes of P, the estimated dephosphorylation times of the expected maximal in vivo non-encapsulated drug concentrations in the liver and the kidneys are considered to be instantaneous. This enables the separate determination of the encapsulated and non-encapsulated drug concentrations in the excretory organs after administration of liposomal PP in mice generating the first pharmacokinetic profile of a liposomal preparation, in which the in vivo encapsulated and free drug tissues concentrations are measured separately. This can also gain important insights into the pharmacokinetics of liposomal formulations in general
The Scientific Value of Non-Clinical Animal Studies in Drug Development
No full textAnimal studies are considered needed as predictive models to evaluate safety and efficacy of new pharmaceuticals and are required by law. However, the scientific basis of the current paradigm on the predictability of animal studies for the effects of drugs in man is under discussion. Therefore, in this thesis we evaluated the scientific basis of the current practices and guidelines for the use of animal studies in pharmaceutical development and assessed the consequences and implications for the regulatory guidelines when animal studies are not informative. Marketing authorization applications (MAA) were used to study whether new post marketing adverse effects of small molecule therapeutics could have been detected from non-clinical studies. Less than 20% of SARs had a true positive corollary in animal studies. For biopharmaceuticals, 50% of post marketing adverse effects had an animal counterpart, which were related to target pharmacology or immune responses. Biopharmaceutical drug development increasingly uses non-human primate (NHP) as the primary species because they are often the only species available that are pharmacologically responsive. Safety studies with monoclonal antibodies in NHP show most adverse effects are either related to the pharmacology of the mAb or an immune response. The quality and interpretability of NHP data could be reduced by methodological issues and immunogenicity, which could be severe. Animal studies submitted for biosimilar applications did not show relevant differences compared to the reference product unless quality issues were previously identified. This further stresses the use of a step-wise approach to demonstrating biosimilarity. Full characterization and similarity to the reference product should be demonstrated in terms of quality, manufacture and control, which should be the starting point for any subsequent studies. To study the value of non-clinical drug development, the use of the MAA has been invaluable. Making these data available for unrestricted and serious study is needed but increased transparency of regulatory authorities and the pharmaceutical industry by disclosing proprietary data is essential for future studies. More informative non-clinical animal studies can be achieved by introducing reporting guidelines and registries for non-clinical animal studies. A thorough revision of regulatory guidelines on non-clinical issues could identify unnecessary animal studies and quickly improve the efficiency of non-clinical drug development. Earlier dialogue with regulatory authorities through scientific advice may increase regulatory intelligence, leading to smaller but more informative study packages. Where no relevant model exists, a slow and cautious entry into fist-in-man studies, preceded by informative in vitro and in silico assays and micro-dosing studies, is the only scientifically relevant option. While regulatory authorities should rethink their reliance on the precautionary principle, the pharmaceutical industries should to invest in alternative technologies to provide informative data on the safety of new drugs. Governments have an important role in incentivizing these activities, which includes the use of political leverage as well as economic incentives, and promoting further research. This takes considerable effort and trust by pharmaceutical companies and regulatory authorities alike but the trade-off is an efficient non-clinical drug development process which is based on science
Plasma, blood and liver tissue sample preparation methods for the separate quantification of liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone
Full text linkBesides the development of sample preparation methods for the determination of separate liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone concentrations in murine plasma and blood, this article also presents the first description of an accurate sample preparation method for the determination of such separate concentrations in the murine liver. The quantitative differentiation is based on the immediate hydrolysis of prednisolone phosphate (PP) into prednisolone (P) after its release from the liposomes in vivo: PP represents the encapsulated drug, while P represents the non-encapsulated drug. The use of 10 ml methanol/g tissue during homogenization of liver tissue ensures complete liposome rupture, prevention of the dephosphorylation of PP released during homogenization, sufficient clean supernatants, excellent extraction of P and sufficient extraction of PP and excellent accuracies and precision complying with the internal guidelines for pre-clinical studies (80-120% and maximal 20%, respectively). Similarly, the matching sample preparation methods for plasma and blood involve protein precipitation with four equivalents of methanol also ensuring accuracies and precision complying with the internal guidelines for pre-clinical studies. Application of these sample preparation methods is going to generate the first pharmacokinetic (PK) profile of a liposomal preparation, in which the encapsulated and non-encapsulated drug concentrations in a tissue are measured separately. Such separated concentration profiles can gain important insights into the PKs of liposomal PP and probably also with regard to liposomal formulations in general, like the quantification of the in vivo drug release from the liposomes
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