RESUMO
The Bacterial Endotoxin Test (BET) is a method for exclusion of endotoxin-related pyrogen contamination in pharmaceutical products, as an alternative to the Rabbit Pyrogen Test (RPT). However, BET does not detect a broad range of biologically relevant pyrogens, and interferences can limit its practical use for different medical products. This work aimed to scope the evidence in the scientific literature for case-by-case validity assessments of BET in different uses for medical products. A search strategy was conducted in PubMed, Scopus, and Web of Science in April 2020, according to the PRISMA-ScR statement. Twenty-two references were included, evaluating medical products for endotoxin contamination through both BET and RPT according to standardized protocols. A critical appraisal was performed through ToxRTool, followed by data extraction and qualitative synthesis of outcomes and methodological issues. Four classes of products assessed by BET were identified, including nanoparticles, drugs, blood and biological products. A considerable variation was observed on the BET methods used. Collectively, the evidence indicates different factors influencing the outcome of BET, including the chemical nature of samples that may cause interference depending on the selected method. While some applications to medical products appear adequate, others, such as nanoparticles, may require the use of different in vitro pyrogen testing methods, reinforcing the need for case-by-case validation for each BET method and type of medical product.
Assuntos
Endotoxinas/análise , Pirogênios/análise , Alternativas aos Testes com Animais , Animais , Bioensaio , CoelhosRESUMO
BACKGROUND: The blood brain barrier (BBB) is the bottleneck of brain-targeted drug development. Due to their physico-chemical properties, nanoparticles (NP) can cross the BBB and accumulate in different areas of the central nervous system (CNS), thus are potential tools to carry drugs and treat brain disorders. In vitro systems and animal models have demonstrated that some NP types promote neurotoxic effects such as neuroinflammation and neurodegeneration in the CNS. Thus, risk assessment of the NP is required, but current 2D cell cultures fail to mimic complex in vivo cellular interactions, while animal models do not necessarily reflect human effects due to physiological and species differences. RESULTS: We evaluated the suitability of in vitro models that mimic the human CNS physiology, studying the effects of metallic gold NP (AuNP) functionalized with sodium citrate (Au-SC), or polyethylene glycol (Au-PEG), and polymeric polylactic acid NP (PLA-NP). Two different 3D neural models were used (i) human dopaminergic neurons differentiated from the LUHMES cell line (3D LUHMES) and (ii) human iPSC-derived brain spheroids (BrainSpheres). We evaluated NP uptake, mitochondrial membrane potential, viability, morphology, secretion of cytokines, chemokines and growth factors, and expression of genes related to ROS regulation after 24 and 72 h exposures. NP were efficiently taken up by spheroids, especially when PEGylated and in presence of glia. AuNP, especially PEGylated AuNP, effected mitochondria and anti-oxidative defense. PLA-NP were slightly cytotoxic to 3D LUHMES with no effects to BrainSpheres. CONCLUSIONS: 3D brain models, both monocellular and multicellular are useful in studying NP neurotoxicity and can help identify how specific cell types of CNS are affected by NP.
Assuntos
Encéfalo/efeitos dos fármacos , Ouro/toxicidade , Nanopartículas Metálicas/toxicidade , Modelos Biológicos , Poliésteres/química , Esferoides Celulares/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Sistemas de Liberação de Medicamentos , Expressão Gênica/efeitos dos fármacos , Ouro/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Poliésteres/metabolismo , Polietilenoglicóis/química , Citrato de Sódio/química , Esferoides Celulares/metabolismo , Propriedades de SuperfícieRESUMO
Several initiatives have recently taken place in Brazil in order to foster the creation of centers dedicated to alternatives to animal testing. In 2008, Vanessa Sá-Rocha organized a meeting with Brazilian regulatory authorities and the major stakeholders in the field of testing to foster discussions on the process of funding, development, and validation of alternative methods in Brazil. Octavio Presgrave published a scientific article on "The Need for the Establishment of a Brazilian Centre for the Validation of Alternative Methods." Also in 2008, Jadir Nunes, together with Dermeval de Carvalho, prepared and presented a proposal to the Brazilian National Agency of Health Surveillance (ANVISA) for the creation of a Centre for the Validation of Alternative Methods. ECVAM and other European stakeholders have been involved in the initiatives. Furthermore, also in 2008, a new legislation has been adopted in Brazil regarding the use of animals for scientific purposes ("lei Arouca"). The legislation establishes, among other provisions, the task of monitoring and evaluating the introduction of alternative methods. However, the legislation does not provide for promotion of or information about, existing alternative methods to the larger Brazilian scientific community. In order to streamline the different activities, Chantra Eskes acted as a facilitator by establishing a new joint proposal with the current Brazilian stakeholders, aimed at setting up a Brazilian Center on Alternative Test Methods.
Assuntos
Alternativas aos Testes com Animais/organização & administração , Bem-Estar do Animal , Avaliação Pré-Clínica de Medicamentos/métodos , Alternativas aos Testes com Animais/métodos , Animais , Brasil , Estudos de Validação como AssuntoRESUMO
The recently described family of Toll-like receptors (TLRs) plays a major role in innate immunity by mediating inflammatory reactions against a wide array of pathogens. TLR-2 is reported to interact with various bacterial partial structures including lipoproteins, peptidoglycan, and lipoteichoic acid. Two polymorphisms of the TLR-2 gene have recently been described: Arg753Gln, correlated with the incidence of sepsis in a white population, and Arg677Trp, correlated with the incidence of lepromatous leprosy in an Asian population. Both polymorphisms, when inserted into expression vectors encoding for human TLR-2, reduced stimulation of Chinese hamster ovary cells by synthetic lipopeptides. We furthermore developed a rapid and inexpensive method for the detection of both single nucleotide polymorphisms based on restriction fragment length polymorphism. While no individuals carrying the Arg677Trp SNP were identified in a large group of whites, 9.4% of the study population were found to be heterozygous for the Arg753Gln polymorphism. This ratio is significantly higher than previously reported, and therefore detection of this polymorphism among patients may yield important information for the assessment of risk profiles regarding susceptibility to bacterial infections.