A Brazilian Rare-Disease Center's Experience with Glucosylsphingosine (lyso-Gb1) in Patients with Gaucher Disease: Exploring a Novel Correlation with IgG Levels in Plasma and a Biomarker Measurement in CSF.

Gaucher disease (GD, OMIM 230800) is one of the most common lysosomal disorders, being caused by the deficient activity of the enzyme acid ß-glucocerebrosidase (Gcase). Three clinical forms of Gaucher's disease (GD) are classified based on neurological involvement. Type 1 (GD1) is non-neuronopathic, while types 2 (GD2) and 3 (GD3) are neuronopathic forms. Gcase catalyzes the conversion of glucosylceramide (GlcCer) into ceramide and glucose. As GlcCer accumulates in lysosomal macrophages, it undergoes deacylation to become glycosylsphingosine (lyso-Gb1), which has shown to be a useful and reliable biomarker for the diagnosis and monitoring of treated and untreated patients with GD. Multiple myeloma (MM) is one of the leading causes of cancer-related death among patients with GD and monoclonal gammopathy of undetermined significance (MGUS) is a non-neoplastic condition that can be a telltale sign of a B clonal proliferation caused by the chronic activation of B cells. This study aimed to quantify Lyso-Gb1 levels in dried blood spots (DBS) and cerebrospinal fluid (CSF) as biomarkers for Gaucher disease (GD) and discuss the association of this biomarker with other clinical parameters. This is a mixed-methods study incorporating both cross-sectional and longitudinal elements within a cohort design with a convenience-sampling strategy. Data collection took place from January 2012 to March 2023. Lyso-Gb1 extraction from DBS involved the use of a methanol-acetonitrile-water mixture, followed by incubation and centrifugation. Analysis was performed using UPLC-MS/MS with MassLynx software version 4.2 and the control group for the DBS measurements included general newborns. CSF Lyso-Gb1 was extracted using ethyl acetate, analyzed by UPLC-MS/MS with a calibration curve, and expressed in pmol/L. Lysosomal activity in CSF was assessed by measuring chitotriosidase (Cht), and other lysosomal enzyme activities were assessed as previously described in the literature. Patients with metachromatic leukodystrophy (MLD) were used as controls. Thirty-two treated patients (twenty-nine GD1 and three GD3, all on ERT except for one GD type on SRT with eliglustat) and three untreated patients (one GD1, one GD2, and one GD3) were included. When analyzing only the treated GD1 group, a significant correlation was found between lyso-Gb1 and age (rho = -0.447, p = 0.001), ChT, and IgG levels (rho = 0.73, p < 0.001; and rho = 0.36, p = 0.03, respectively). Five GD1 patients (three females, mean age 40 years) also had their CSF collected and analyzed. The average measurement of lyso-Gb1 in CSF was 94 pmol/L (range: 57.1-157.9 pmol/L) versus <6.2 pmol/L in the control group (MLD). This is the first time, to the best of our knowledge, that lyso-Gb1 has been associated with IgG levels. While this finding reflects a risk for MGUS or MM and not only chronic plasma B-cell activation, it still requires further studies. Moreover, the analysis of CSF lyso-Gb1 levels in GD1 patients was demonstrated to be significantly higher than the control group. This raises the hypothesis that CSF lyso-Gb1 may serve as a valuable indicator for neurological involvement in GD, providing insights into the potential implications for neurological manifestations in GD, including GD1. The correlation between lyso-Gb1 and ChT levels in treated GD1 patients further underscores the interconnectedness of lysosomal markers and their relevance in monitoring.

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 relationship.

Preclinical Pharmacokinetic and Pharmacodynamic Investigation of 5'-Methoxynobiletin from Ageratum conyzoides: In vivo and In silico Approaches.

PURPOSE: 5'-methoxynobiletin (5'-MeONB), a polymethoxyflavone isolated from A. conyzoides, has shown anti-inflammatory property. Nevertheless, the antinociceptive activity and pre-clinical pharmacokinetics (PK) characteristics of 5'-MeONB remain unknown. Considering the anti-inflammatory potential of the 5'-MeONB, this study aimed to investigate the pre-clinical PK behavior of 5'-MeONB, as well as its time course antinociceptive activity. METHODS: 5'-MeONB plasma concentrations were determined in Wistar rats after intravenous (i.v.) (10 mg/kg) and oral (50 mg/kg) administration, and in Swiss mice after oral administration (100 mg/kg). Plasma samples were deproteinization and 5'-MeONB quantified by a validated UPLC-MS method. Additionally, the antinociceptive activity of 5'-MeONB was evaluated after 15, 30, 60, 180 and 360 min following oral administration on the acute nocifensive behavior of mice induced by formalin. RESULTS: 5'-MeONB rats and mice plasma concentration-time profiles were best one-compartment model. After i.v. administration to rats, a short half-life, a high clearance and moderate volume of distribution at steady state were observed. Similar results were obtained after oral administration. The oral bioavailability ranged from 8 to 11%. Additionally, 5'-MeONB exhibited antinociceptive activity in both formalin phases, especially in the inflammatory phase of the model, inhibiting 68% and 91% of neurogenic and inflammatory responses, respectively, after 30 min of oral administration. CONCLUSIONS: The results described here provide novel insights on 5'-MeONB pharmacokinetics and pharmacodynamic effect, serving as support for future studies to confirm this compound as anti-nociceptive and anti-inflammatory effective agent.

Simple and fast UPLC-MS method for quantifying the anti-inflammatory candidate 5'-methoxynobiletin in rat plasma: Validation and application in a preliminary pharmacokinetic study.

This study presents the development and validation of a fast and simple bioanalytical ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method intended for quantifying the anti-inflammatory candidate 5'-methoxynobiletin (5'-MeONB) in rat plasma. Standard of 5'-MeONB was purified from A. conyzoides extract by using preparative HPLC. After a pretreatment of plasma samples with acetonitrile, chromatographic separations were efficiently achieved with a C18 column using a 9 min gradient system of 0.1% aqueous formic acid and acetonitrile as eluent. Drug candidate 5'-MeONB and chrysin (internal standard, IS) detection were carried out using ESI+ through the extracted ion chromatograms approach, monitored at m/z 433.1494 (for 5'-MeONB, tR:1.78 min) and m/z 255.0657 (for IS, tR:1.57 min). Method was validated according to US FDA guidelines, presenting linearity (R2 > 0.999) over concentration range of 30-750 ng/mL. Relative standard deviation (RSD) of repeatability and intermediary precision respectively ranged between 1.93-3.65% and 2.16-7.54%, considering lower limit of quantitation (30 ng/mL) and quality control (90, 360 and 600 ng/mL) samples, while accuracy was between 82.51 and 109.44%. Moreover, no interference from plasma endogenous substances, no carryover effect, and no influence of extraction method even in hemolyzed blood samples were observed. Sample stability in auto-sampler and long-term -80 °C storage, as well as matrix effect were within acceptable limits. For the first time, using the validated UPLC-MS bioanalytical method, the plasma pharmacokinetics of 5'-MeONB following 2 mg/kg intravenous bolus dosing to Wistar rats was characterized allowing the determination of the parameters describing drug distribution and elimination.

Phytochemical profile of different anatomical parts of jambu (Acmella oleracea (L.) R.K. Jansen): A comparison between hydroponic and conventional cultivation using PCA and cluster analysis.

Jambu [Acmella oleracea (L.) R.K. Jansen] is an edible plant with a wide range of constituents of biological interest. In this study, the chemical composition of leaves, flowers and stems of jambu cultivated in hydroponic and conventional systems was investigated. In both crop systems, the leaves showed the highest total phenolic content, total flavonoid content and in vitro antioxidant capacity. The extracts were characterized by determining 45 compounds, including phenolic acids, glycosylated flavonoids, alkamides and fatty acids, by LC-MS analysis. Of these compounds, 31 are described for the first time in this species, five of which are reported for the first time in the literature. The PCA and cluster analysis results distinguished different anatomical parts (PC1 and PC2) and cultivation systems (PC3) into well-defined groups.

A single-step isolation by centrifugal partition chromatography of the potential anti-inflammatory glaucolide B from Lepidaploa chamissonis.

Sesquiterpene lactones (SL) are commonly found in Asteraceae and present a promising anti-inflammatory activity. Previously described in Lepidaploa genus, glaucolide B has never been investigated for its anti-inflammatory potential. This study aimed to establish an efficient process for the extraction of glaucolide B (1) from Lepidaploa chamissonis leaves and to develop a simple and fast method for its purification by using centrifugal partition chromatography (CPC), as well as to investigate in vitro the anti-inflammatory effects of glaucolide B. Thus, an optimized washing extractive process performed on L. chamissonis leaves allowed to obtain a SL enriched extract (4.11 g). After a successful defatting pretreatment of the crude extract, the glaucolide B enriched ethyl acetate portion (2.00 g) was fractionated by CPC affording, in a single-step isolation, compound 1 (1.04 g) in great yield (25%) and purity (97%). Cytotoxicity effect of 1 on RAW 264.7 macrophages was determined by using MTT assay, revealing a CC10 of 14.11 µM. Compound 1 at 1, 3 and 10 µM inhibited the nitrite/nitrate (NOx) metabolites production and the pro-inflammatory interleukin 6 (IL-6) secretion on lipopolysaccharide-stimulated RAW 264.7 cells. The extractive process used turned to be selective for SL and CPC technique proved a simple and effective tool for the isolation of 1 within few hours. Isolated for the first time from L. chamissonis leaves, glaucolide B presented a significant inhibitory effect on both NO and IL-6 secretion under non-toxic concentrations.

Antiprotozoal investigation of 20 plant metabolites on Trypanosoma cruzi and Leishmania amazonensis amastigotes. Atalantoflavone alters the mitochondrial membrane potential.

The study aims to evaluate the antiprotozoal activities of 20 plant metabolites on Trypanosoma cruzi and Leishmania amazonensis amastigotes. Compounds 1-20 were obtained and identified by using chromatographic and spectroscopic techniques. The antiparasitic assays were performed on the intracellular form of T. cruzi and L. amazonensis using human leukaemic THP-1 cells as the host. The mechanism of action of the most active compounds was explored in silico by molecular docking using T. cruzi trypanothione reductase (TR) as a target, whereas the in vitro studies were performed by enzymatic assay using T. cruzi recombinant TR. In addition, the mitochondrial membrane potential was evaluated by flow cytometry. Two flavonoids, one triterpene and three acetogenins showed from high to moderate trypanocidal activities with IC50 values ranging 3.6-37.2 µm while three of the metabolites were moderately leishmanicidal. The molecular docking study revealed interactions between TR and the most trypanocidal compounds 1 (abyssinone IV) and 2 (atalantoflavone). In contrast, both showed no effect on TR in vitro. For the mitochondrial membrane potential assay, atalantoflavone (2) displayed a dose-dependent depolarization. On the basis of the aforementioned results, this compound's structure could be chemically explored in order to develop more potent trypanocidal derivatives.

Antinociceptive and anti-inflammatory activities of the Jatropha isabellei dichloromethane fraction and isolation and quantitative determination of jatrophone by UFLC-DAD.

CONTEXT: Jatropha isabellei Müll. Arg. (Euphorbiaceae) has been used in the traditional medicine to treat arthritis. OBJECTIVE: To evaluate the anti-inflammatory and antinociceptive activities of the dichloromethane fraction (DFJi) from underground parts of J. isabellei, and to develop an analytical method to quantify the diterpene jatrophone. MATERIALS AND METHODS: Anti-inflammatory and antinociceptive activities of the DFji were determined by an acute arthritis model through assessment of the paw elevation time (PET) and articular diameter (AD) of Wistar rats treated orally (50, 100 or 200 mg/kg in a single-dose), and intravenously (0.1, 1, 10, 25 or 50 mg/kg in a bolus administration). The isolation of jatrophone from the DFji was carried out and confirmed by spectroscopic techniques. A UFLC-DAD method was developed and validated. RESULTS: When orally administered, the highest dose (200 mg/kg) of DFJi was able to significantly reduce the PET to 24.8 ± 1.4 s (p < 0.01), when compared with the control group (33.7 ± 1.8 s). The administration of the intravenous dose of 10 mg/kg reduced the PET to 14.8 ± 0.3 s (p < 0.001). The oral and intravenous administration of the DFJi at dose of 200 and 10 mg/kg significantly prevented the formation of edema, reducing the AD in 25.3% and 32.5% (p < 0.01), respectively. The UFLC-DAD method allowed the quantification of jatrophone, which was found to be around 90 µg/mg of fraction. DISCUSSION AND CONCLUSION: The DFJi displayed antinociceptive and antiedematogenic activities, representing a promising plant product for the arthritis treatment.

Spectrophotometric determination of the total flavonoid content in Ocimum basilicum L. (Lamiaceae) leaves.

BACKGROUND: The chemical characterization is essential to validate the pharmaceutical use of vegetable raw materials. Ultraviolet spectroscopy is an important technique to determine flavonoids, which are important active compounds from Ocimum basilicum. OBJECTIVE: The objective of this work was to optimize a spectrophotometric method, based on flavonoid-aluminum chloride (AlCl3) complexation to determine the total flavonoid content (TFC) in leaves of O. basilicum (herbal material), using response surface methodology. MATERIALS AND METHODS: The effects of (1) the herbal material: Solvent ratio (0.02, 0.03, 0.05, 0.07, and 0.08 g/mL), (2) stock solution volume (0.8, 2.3, 4.4, 6.5, and 8.0 mL) and (3) AlCl3 volume (0.8, 1.0, 1.2, 1.4, and 1.6 mL) on the TFC were evaluated. The analytical performance parameters precision, linearity and robustness of the method were tested. RESULTS: The herbal material: Solvent ratio and stock solution volume showed an important influence on the method response. After choosing the optimized conditions, the method exhibited a precision (RSD%) lower than 6% for repeatability (RSD%) and lower than 8% for intermediate precision (on the order of literature values for biotechnological methods), coefficient of correlation of 0.9984, and no important influence could be observed for variations of the time of complexation with AlCl3. However, the time and temperature of extraction were critical for TFC method and must be carefully controlled during the analysis. CONCLUSION: Thus, this study allowed the optimization of a simple, fast and precise method for the determination of the TFC in leaves of O. basilicum, which can be used to support the quality assessment of this herbal material.