Experience of the NPC Brazil network with a comprehensive program for the screening and diagnosis of Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is a lysosomal disorder caused by impaired cholesterol metabolism. Levels of lysosphingomyelin 509 (LysoSM509) have been shown elevated in dried blood spots (DBS) of NPC and acid sphingomyelinase deficiency patients. In this study, we report our experience using a two-tier approach (1st tier is the quantification of lysoSM509 by ultra-performance liquid chromatography tandem mass spectrometry followed by the 2nd tier with next-generation sequencing of the NPC1 and NPC2 genes). DBS samples from 450 suspected patients were received by the NPC Brazil network. Of these, 33 samples had elevated levels of lysoSM509, and in 25 of them, variants classified as pathogenic, likely pathogenic, or of unknown significance were identified in the NPC1 or NPC2 genes by next-generation sequencing. The quantification of lysoSM509 in DBS as a first-tier test for the diagnosis of NPC followed by molecular analysis of the NPC1 and NPC2 genes almost doubled the detection rate when compared to the performance of chitotriosidase activity as a first-tier biomarker, and it could likely be increased with the addition of a third tier with MLPA of the two genes involved. This strategy seems suitable for the neonatal screening (NBS) of NPC if this disease is eventually adopted by NBS programs

Follow-up of pre-motor symptoms of Parkinson’s disease in adult patients with Gaucher disease type 1 and analysis of their lysosomal enzyme profiles in the CSF

Background: Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide. Its classic motor symptoms may be preceded by non-motor symptoms (NMS). Population studies have identified GBA variants as risk factors for idiopathic PD. The increased risk of PD has also been suggested in other Lysosomal Storage Disorders (LSDs). Objective: To assess the evolution of the prevalence of NMS compatible with PD in a cohort of South Brazilian adult patients with Gaucher Disease (GD) type 1, already evaluated 3 years ago (2018). Cerebrospinal Fluid (CSF) was collected to assess the levels of LSD enzymes (beta-hexosaminidases, beta-glucuronidase) and biomarker of macrophage activation (chitotriosidase, ChT), compared to controls (metachromatic leukodystrophy, MLD). Cognition was evaluated by the Montreal Cognitive Assessment (MoCA) questionnaire, daytime sleepiness by the Epworth Sleepiness Scale (ESS), depression by Beck´s Inventory, constipation by the Unified Multiple System Atrophy Rating Scale (UMSARS) scale, and REM sleep behavior disorder by the single-question screen. Hyposmia was assessed with Sniffin’ Sticks (SST). Results Nineteen patients completed the follow-up (mean age of the sample was 44 years; range, 26–71). The patient with the highest number of NMS at the baseline (4 including the lowest SST score) was diagnosed with PD four years later. Apart from an improvement in the ESS score, no other statistical significance was found between the number of NMS between the first and second evaluation, nor between patients with one L444P variant (n = 5) and the rest of the cohort. CSF was collected in five patients (mean age of the sample was 40 years, range 30–53. A significant difference was found in the mean CSF activity levels of beta-hexosaminidases and beta-glucuronidase between GD1 and MLD patients. Mean ChT (CSF) was 62 nmol/h/mL in GD patients and 142 in MLD (n = 6) patients. Conclusions: The patient with the highest number of NMS in our 2018 cohort was the one that developed PD, corroborating with the importance of this longitudinal follow-up. CSF and plasma analysis might allow a better understanding of the neurodegenerative processes connecting PD and the lysosomal environment. Further analysis is needed to understand this relationshi

Quantificação por cromatografia líquida de ultra eficiência de marcadores presentes no extrato aquoso de Ageratum conyzoides L. (Asteraceae)

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2016.A espécie Ageratum conyzoides L., conhecida popularmente como mentrasto, é amplamente utilizada na medicina popular de regiões tropicais e subtropicais do mundo devido as suas propriedades anti-inflamatória, antinociceptiva e antibacteriana. Entre os metabólitos secundários identificados nesta espécie estão flavonoides, cumarinas, cromenos, alcaloides e ácidos fenólicos. Com a finalidade de avaliar a composição qualitativa e quantitativa da preparação de uso medicinal (chá) de A. conyzoides, o presente trabalho teve por objetivo desenvolver e validar um método analítico por CLUE-DAD para quantificação das principais classes de substâncias presentes no extrato aquoso desta espécie. As análises qualitativas permitiram a identificação de dezessete substâncias no extrato aquoso: alcaloides pirrolizidínicos, ácidos fenólicos, cumarinas e metoxiflavonas. O método analítico foi desenvolvido utilizando coluna Acquity UPLC BEH C18 (150 mm x 2,1 mm i.d.; 1,7 µm), 25 ºC, fluxo de 0,25 ml/min com detecção DAD monitorada em 275 e 330 nm. A fase móvel foi composta por água acidificada (0,1% de ácido fórmico) e acetonitrila em sistema gradiente com tempo de análise de 25 minutos. O método foi validado segundo normas oficiais, demonstrando ser específico, linear, preciso, exato e sensível para a quantificação de ácido clorogênico, ácido cumárico, cumarina (1,2-benzopiranona), 5,6,7,3',4',5'-hexametoxiflavona, nobiletina, 5?-metoxinobiletina e eupalestina no extrato aquoso de A. conyzoides. Além disso, o método apresentou-se indicativo de estabilidade para os marcadores testados nas condições do estudo de degradação forçada. Por conseguinte, foi possível desenvolver e validar um simples e rápido método analítico para quantificação dos principais marcadores do extrato aquoso de A. conyzoides, podendo ser aplicado no controle de qualidade de preparações desta espécie.<br>Abstract : Ageratum conyzoides L. is a plant widely used in traditional medicine in tropical and subtropical regions of the world due to its anti-inflammatory, antinociceptive and antibacterial properties. Among the secondary metabolites identified in this specie are flavonoids, coumarins, chromenes, alkaloids and phenolic acids. In order to evaluate the qualitative and quantitative composition of the preparation for medicinal use (tea) from A. conyzoides, the present work had the aim of develop and validate an analytical method by UPLC-PDA for quantification of the mains classes of compounds found in the aqueous extract of this specie. The qualitative analyses allow the identification of seventeen compounds in the aqueous extract: pyrrolizidine alkaloids, phenolic acids, coumarins and methoxyflavones. The analytical method was developed using an Acquity UPLC BEH C18 column (150 mm x 2.1 mm i.d.; 1.7 µm), 25 ºC, at a flow rate of 0.25 ml/min with PDA detection on 275 and 330 nm. The mobile phase consisted of 0.1% formic acid aqueous solution and acetonitrile in a gradient system, with 25 minutes analysis. The method was validated according to official guidelines, demonstrating to be specific, linear, precise, accurate and sensitive for the quantification of chlorogenic acid, coumaric acid, coumarin (1,2-benzopyranone), 5,6,7,3',4',5'-hexamethoxyflavone, nobiletin, 5?-methoxynobiletin and eupalestin in the aqueous extract of A. conyzoides. Furthermore, the method proved to be stability-indicating for the tested markers at the forced degradation study conditions. Therefore, it was possible to develop and validate a simple and rapid analytical method to quantify the mains markers in A. conyzoides aqueous extract, which can be applied at the quality control of these specie preparations

Investigação farmacológica em dor inflamatória da fração padronizada de polimetoxiflavonas de Ageratum conyzoides L. (Asteraceae) e avaliação farmacocinética do marcador 5'-metoxinobiletina

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2021.Ageratum conyzoides é utilizada na medicina popular como analgésica e anti-inflamatória. Entre as substâncias de interesse identificadas nesta espécie estão as polimetoxiflavonas (PMF). Diante da necessidade do desenvolvimento de alternativas terapêuticas para o manejo de manifestações dolorosas e inflamatórias, este trabalho teve por objetivo obter e padronizar uma fração enriquecida em polimetoxiflavonas de A. conyzoides (FEPMF-Ac), avaliando sua efetividade farmacológica em modelos de nocicepção, assim como estabelecendo os parâmetros farmacocinéticos do marcador 5?-metoxinobiletina (5?-MeONB). A otimização das condições de extrações para a obtenção das PMF indicou como condições ideais o uso de n-hexano em aparato de Soxhlet. A otimização dos métodos de fracionamento cromatográfico do extrato bruto possibilitou a obtenção da FEPMF-Ac em apenas duas etapas de purificação, utilizando a cromatografia de partição centrífuga (CPC) como ferramenta inicial de enriquecimento da fração, seguida por filtração por cromatografia em coluna utilizando resina Sephadex® LH20 para a remoção das substâncias interferentes. A caracterização da FEPMF-Ac por UPLC-EM possibilitou a identificação de nove PMF, ageconiflavona C, 3'-hidroxi-5,6,7,8,4',5'-hexametoxiflavona, 7-hidroxi-5,6,8,5'-tetrametoxi-3',4'-metilenodioxiflavona, sinensetina, 5,6,7,3',4',5'-hexametoxiflavona, nobiletina, linderoflavona B, 5-MeONB e eupalestina. A quantificação do teor de PMF totais na FEPMF-Ac foi realizada em função de 5?-MeONB. A validação do método analítico desenvolvido em UPLC-EM demonstrou ser linear, exato e preciso para a quantificação deste marcador. Na investigação da atividade antinociceptiva em camundongos, utilizando o modelo de formalina, a FEPMF-Ac apresentou efeito antinociceptivo em diferentes doses, com maior inibição da nocicepção durante a fase inflamatória. Paralelamente, inibiu a nocicepção induzida por glutamato, bradicinina, histamina, assim como por substâncias ativadoras dos canais TRPV1 e ASIC e proteínas quinases C e A. Além disso, constatou-se que a 5?-MeONB contribui para este efeito desempenhado pela fração, visto que a mesma demonstrou atividade antinociceptiva. A biodisponibilidade oral em ratos foi de 8-11%. A compreensão dos parâmetros farmacocinéticos da 5?-MeONB contribui para o planejamento das próximas etapas incluídas no desenvolvimento de fármacos. Os resultados obtidos demonstram o potencial biológico das PMF de A. conyzoides para o alívio de quadros dolorosos inflamatórios.Abstract: Ageratum conyzoides is used in folk medicine as an analgesic and anti-inflammatory. Among the substances of biological interest identified in this species are polymethoxyflavones (PMF). In order to develop therapeutic alternatives for the management of painful and inflammatory manifestations, this study aimed to obtain and standardize an enriched fraction of polymethoxyflavones from A. conyzoides (EFPMF-Ac), evaluating its pharmacological effectiveness in nociception models, as well as establishing the pharmacokinetic parameters of the fraction marker 5'-methoxynobiletin (5?-MeONB). Conditions for the extraction of the PMF were optimized, indicating the ideal conditions as n-hexane in Soxhlet apparatus. Optimization of chromatographic fractionation methods of the crude extract has enabled to obtain EFPMF-Ac in two purification steps, using centrifugal partition chromatography (CPC) as the initial enrichment tool, followed by column chromatography filtration using Sephadex® LH20 resin for the removal of interfering substances. Characterization of EFPMF-Ac by UPLC-MS enabled the identification of nine PMF, ageconyflavone C, 3'-hydroxy-5,6,7,8,4',5'-hexamethoxyflavone, 7-hydroxy-5,6,8,5'-tetramethoxy-3',4'-metilenedioxyflavone, sinensetin, 5,6,7,3',4',5'-hexamethoxyflavone, nobiletin, linderoflavone B, 5-MeONB and eupalestina. Quantification of total PMF content in the EFPMF-Ac was performed using the major substance 5?-MeONB as reference. Validation of the UPLC-MS analytical method developed has proved to be linear, accurate and precise for the quantification of 5'-MeONB. EFPMF-Ac has showed antinociceptive effect at different doses, in the investigation of antinociceptive activity in mice using formalin, with greater inhibition of nociception during the inflammatory phase. In addition, it has been effective by inhibiting nociception induced by glutamate, bradykinin, histamine, as well as activating substances of TRPV1 and ASIC channels and protein kinases C and A. Similarly, it was found that 5'-MeONB contributes to the effect of EFPMF-Ac, since it has demonstrated antinociceptive activity. Characterization of the pharmacokinetic parameters of 5'-MeONB revealed an oral bioavailability in rats of around 8-11%. The understanding of the pharmacokinetic parameters of the marker contributes to planning the next steps included in the development of drugs. Results obtained demonstrate the biological potential of PMF from A. conyzoides to relief painful inflammatory conditions

Phytochemical analysis of Vernonanthura tweedieana and a validated UPLC-PDA method for the quantification of eriodictyol

AbstractVernonanthura tweedieana (Baker) H. Rob., Asteraceae, is used in the Brazilian folk medicine for the treatment of respiratory diseases. In this work the phytochemical investigation of its ethanol extracts as well as the development and validation of an UPLC-PDA method for the quantification of the eriodictyol from the leaves were performed. The phytochemical study for this species lead to the identification of ethyl caffeate, naringenin and chrysoeriol in mixture, eriodictyol from leaves, and the mixture of 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-propan-1-one and evofolin B, apigenin, the mixture of caffeic and protocatechuic acid and luteolin from stems with roots, being reported for the first time for V. tweedieana, except for eriodictyol. The structural elucidation of all isolated compounds was achieved by 1H and 2D NMR spectroscopy, and in comparison with published data. An UPLC-PDA method for quantification of the eriodictyol in leaves of V. tweedieana was developed and validated for specificity, linearity, precision (repeatability and intermediate precision), limit of detection (LOD) and limit of quantification (LOQ), accuracy and robustness. In this study, an excellent linearity was obtained (r2 = 0.9999), good precision (repeatability RSD = 2% and intermediate precision RSD = 8%) and accuracy (average recovery from 98.6% to 99.7%). The content of eriodictyol in the extract of leaves of V. tweedieana was 41.40 ± 0.13 mg/g. Thus, this study allowed the optimization of a simple, fast and validated UPLC-PDA method which can be used to support the quality assessment of this herbal material