Untargeted plasma metabolomics by LC-HRMS is increasingly used to explore candidate biomarkers in neurodegenerative disease, yet the analytical output and downstream biological interpretability remain highly dependent on end-to-end workflow choices. This limits reproducibility and cross-study comparability in parkinsonian syndromes where objective fluid biomarkers for differential diagnosis are still lacking. Here, we implemented a QC-anchored, untargeted LC-HRMS plasma platform to compare sample preparation strategies and to explore disease-associated metabolic patterns across Parkinson’s disease (PD), multiple system atrophy–parkinsonian subtype (MSA-P), and progressive supranuclear palsy–parkinsonism (PSP-P) in a prospective single-center cohort (n = 102; 57 PD, 19 MSA-P, 26 PSP-P). Three protein precipitation-based workflows were compared within a harmonized analytical framework: tube precipitation, 96-well precipitation-filtration, and 96-well phospholipid removal. Integrated preparation QCs, system QCs, and blanks supported performance monitoring, while MS2 annotation enabled interpretation of disease-associated signals. Phospholipid removal provided the most favorable trade-off for discovery and quantitation, delivering reduced phospholipid-related chromatographic background, improved quantitative behavior, and a higher density of MS2-supported annotations despite a modest reduction in raw feature counts. Differential abundance was assessed using empirical Bayes moderated linear models implemented in limma. Pairwise contrasts were defined for PD vs. MSA-P, PD vs. PSP-P, and MSA-P vs. PSP-P, and P values were adjusted using the Benjamini–Hochberg false-discovery rate (FDR). Across workflows, the most robust exploratory disease-associated pattern was observed for PD versus PSP-P, whereas MSA-P comparisons yielded limited FDR-significant features. Because of the absence of MSI Level 1 annotation, the hypothesis-generating pathway-level was based on exploratory metabolomics interpretation, requiring targeted confirmation.

Background: Plasma levels of the catecholamine norepinephrine (NE) has emerged as a useful tool to help differentiate pre- and post-ganglionic disorders in patients with cardiovascular autonomic failure (AF). However, data on intrasubject reliability in individuals with these conditions are limited. We evaluated the intrasubject reproducibility of supine plasma NE levels drawn across two consecutive time points under controlled conditions during head-up table testing in a large cohort of patients with alpha-synucleinopathies and both pre- and post-ganglionic cardiovascular AF. Methods: Antecubital venous blood drawn via an indwelling cannula with the subject supine was assayed for plasma level of catecholamines. We collected two consecutive samples, the first after 20 min of supine rest (NE1) and the second 5 min later (NE2), from a group of 279 participants including 57 with Parkinson's disease/Lewy body dementia (44 M; 65.5 +/- 11.1 y), 131 with multiple system atrophy (81 M; 63.2 +/- 8.5 y), 41 with pure autonomic failure (25 M, 65.1 +/- 9.3 y), and 50 healthy controls (27 M; 46.7 +/- 19.4 y). Results: We found no difference between NE1 and NE2 (p = 0.645), with a mean intrasubject reproducibility (NE maximum - NE minimum) x 100 / NE maximum) of 11.5 % +/- 10.64. This finding was confirmed when controlling for diagnosis (p = 0.669), gender (p = 0.493), age (p = 0.865), disease duration (p = 0.596) or considering all factors together (p = 0.527). Conclusions: We found excellent test-retest reliability of consecutive supine NE measurements in patients with alpha-synucleinopathies and cardiovascular AF, independent of age, gender and disease duration. This lends evidence to support the use of a single supine NE measurement in these conditions.

Volumetric absorptive microsampling (VAMS) is increasingly proposed as a clinically reliable therapeutic drug monitoring (TDM) sampling methodology. The study aimed to establish the reliability and real-life feasibility of patient self-collected capillary VAMS for TDM of antiseizure medication (ASMs), using plasma ASMs concentrations from venous blood as a reference standard. Nurses collected venous and capillary blood samples using VAMS. Afterward, persons with epilepsy (PWE) performed VAMS sampling by themselves. All samples were analyzed by UHPLC-MS/MS. We performed a cross-validation study, comparing ASMs concentrations obtained by VAMS nurses and patients' self-collected versus plasma through Bland-Altman analysis and Passing-Bablok regression. We enrolled 301 PWE (M: F 42.5%:57.5%; mean age 44±16 years), treated with 13 ASMs, providing a total of 464 measurements. Statistical analysis comparing VAMS self-collected versus plasma ASMs concentrations showed a bias close to zero and slope and intercept values indicating a good agreement for CBZ, LCS, LEV, LTG, OXC, PB, and PHT, while a systematic difference between the two methods was found for VPA, PMP, TPM and ZNS. This is the first study showing the reliability and feasibility of the real-world application of PWE self-collected VAMS for most of the ASMs considered, giving a promising basis for at-home VAMS applications.

A volumetric absorptive microsampling (VAMS) combined with UHPLC-MS/MS method was validated for the analysis of lyso-Gb3 in capillary blood. Lyso-Gb3, a biomarker for Fabry disease (FD), was extracted from VAMS device with methanol/acetonitrile/water mixture, in a temperature-controlled ultrasonic bath and the solution was analysed on a Kinetex C18 in gradient elution with MRM detection. The VAMS method was fully validated according to EMA guidelines. No hematocrit effect was observed over a range of 20-70 %. The method showed linearity from 0.25 to 100.00 ng/mL, the lower limit of quantitation was 0.25 ng/mL, both precision and accuracy were <= 15 % and the average extraction recovery was 94.6 %. The VAMS UHPLC-MS/MS method was successfully compared with a reference plasma method in a series of 13 Fabry patients and 12 healthy volunteers. This is the first validated UHPLC-MRM VAMS-based method, hematocrit-independent, for capillary blood lysoGb3 quantification in FD patients.

Dopamine agonist (DA) use is considered the main risk factor for impulse control disorder (ICD) development in Parkinson’s disease (PD). Besides DAs, personality traits and cognitive features may represent risk factors for ICDs. The primary aim of this study was to investigate differences in DA plasma concentrations in PD patients with and without a positive screening for ICDs according to validated tools. The secondary aim was to compare the psychological profile between ICD positive and negative screened patients. PD patients receiving chronic DA therapy were screened for ICDs according to the Questionnaire for Impulsive-Compulsive Disorders in Parkinson’s Disease (QUIP). Blood samples for measurement of DA (pramipexole, ropinirole, rotigotine) trough plasma concentrations were drawn in the morning, at mean 16–19 h from the last DA dose. Patients’ psychological profile was investigated by Millon Clinical Multiaxal Inventory III and Barratt Impulsiveness Scale (BIS-11). One hundred and five PD patients were enrolled. Forty-one patients (39

Treatment of freezing of gait (FoG) and other Parkinson disease (PD) axial symptoms is challenging. Systematic assessments of axial symptoms at progressively increasing levodopa doses are lacking. We sought to analyze the resistance to high levodopa doses of FoG, posture, speech, and altered gait features presenting in daily-ON therapeutic condition. We performed a pre-/postinterventional study including patients treated with levodopa/carbidopa intestinal gel infusion (LCIG) with disabling FoG in daily-ON condition. Patients were evaluated at their usual LCIG infusion rate (T1), and 1 h after 1.5× (T2) and 2× (T3) increase of the LCIG infusion rate by quantitative outcome measures. The number of FoG episodes (primary outcome), posture, speech, and gait features were objectively quantified during a standardized test by a blinded rater. Changes in motor symptoms, dyskinesia, and plasma levodopa concentrations were also analyzed. We evaluated 16 patients with a mean age of 69 ± 9.4 years and treated with LCIG for a mean of 2.2 ± 2.1 years. FoG improved in 83.3% of patients by increasing the levodopa doses. The number of FoG episodes significantly decreased (mean = 2.3 at T1, 1.7 at T2, 1.2 at T3; p  = 0.013). Posture and speech features did not show significant changes, whereas stride length ( p  = 0.049), turn duration ( p  = 0.001), and turn velocity ( p  = 0.024) significantly improved on doubling the levodopa infusion rate. In a short-term evaluation, the increase of LCIG dose can improve "dopa-resistant" FoG and gait issues in most advanced PD patients with overall good control of motor symptoms in the absence of clinically significant dyskinesia.

Background: Different studies, mostly with limited cohorts, have suggested the effects of patients' characteristics on levodopa (LD) pharmacokinetics. Objective: We primarily aimed at investigating in a large population the relationship between patients' features and LD kinetic variables, to assess the main demographic and clinical predictors of LD clinical pharmacokinetics. Methods: The study was retrospective, based on data collected from subjects with parkinsonism on chronic LD undergoing LD therapeutic monitoring (TM). LD TM includes serial quantitative motor tests and blood samples to measure plasma drug concentrations after each subject's chronically taken first-morning LD dose intake. Results: Five hundred patients, 308 males (61.6%), mean (SD) age of 65 (10.1) years were included. Parkinsonian symptoms and LD therapy lasted 5.5 (4.5) and 3.4 (3.9) years, respectively. MDS-UPDRS part III "off" score was 28.8 (15.2). LD dose was 348.2 (187.1) mg/day. From multiple linear regression analysis, test dose, sex, type of LD decarboxylase inhibitor, weight and MDS-UPDRS part III score were linear predictors of both LD peak plasma concentration (C-max) (R-2 = 0.52) and area under the 3-h plasma concentration-time curve (AUC) (R-2 = 0.71), while age was a further predictor only for AUC. Besides test dose, sex was the strongest independent contributing variable to LD AUC, which resulted 27% higher in females compared to males. Conclusion: This is the largest collection of data on the relationship between demographic and clinical-therapeutic variables and LD kinetics in patients with parkinsonian symptoms. As a main clinically practical finding, women might require a 25% reduced weight-normalized LD dose compared with men to achieve the same LD bioavailability.

Background and AimLimited data are available in clinical settings on the pharmacokinetics of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). We investigated the use of cannabis-based products in neurological practice, monitoring patients' steady-state cannabinoids (CBs) plasma concentrations matched with different preparations.MethodsThis was a prospective, single-center, observational study. Patients underwent venous blood withdrawal before the CBs' morning dose and then 2.5 h post-dosing. Spasticity or pain were patient self-assessed by the Numeric Rating Scale (NRS) before the morning CB's administration and 2.5 h post-dosing.ResultsThirty-three patients were enrolled. Main indications for CBs were spasticity and chronic pain. Sixteen patients were treated with oromucosal spray formulation Sativex® and 17 with oil-based solutions. Both CBs trough plasma concentrations were ≤ limit of detection (0.1 ng/ml) in 45% of patients. Intrasubject CB's plasma levels significantly increased over baseline values in patients treated with Bediol® oil (p < 0.05) and Sativex® (p < 0.01). Post-dosing CB's bioavailability did not significantly differ between oral oil and oromucosal spray. NRS scores decreased (p < 0.01), matching the increase (p < 0.01) in CB's plasma concentrations.ConclusionThis is the first study investigating CB's plasma concentrations of oral and oromucosal preparations in real-world neurological practice. Findings of similar bioavailability for both CBD and THC after galenic oil compared with oromucosal spray dosing may be clinically relevant and deserve additional research in larger cohorts.

Introduction Epilepsy is a chronic condition requiring consistent follow-up aimed at seizure control, and monitoring of anti-seizure medication (ASM) levels and side effects. Telemedicine (TM) offers invaluable support to patient follow-up, guaranteeing the prompt availability of a team of experts for persons with epilepsy (PWE) widely distributed across the country. Although many health institutions have endorsed the use of TM, robust data on effectiveness, safety and costs of TM applied to epilepsy are lacking. TELEmedicine for EPIlepsy Care (TELE-EPIC) will evaluate the effectiveness of video consultation (VC) via TM compared with usual care (UC) for the monitoring of PWE (TELE-EPIC_RCT). Moreover, TELE-EPIC will apply an innovative Volumetric Absorptive Microsampling (VAMS) device for quantitation of ASM through finger prick blood sampling as an alternative to venipuncture sampling (TELE-EPIC_VAMS). Methods and analysis TELE-EPIC_RCT is a multicentre, open, pragmatic two-arm randomised controlled trial prospectively including adult and paediatric outpatients with established diagnosis of epilepsy consecutively attending the Epilepsy Centres of Bologna and Rome, respectively. The primary outcome is the non-inferiority of VC on seizure control compared with UC after an 18-month follow-up. Secondary outcomes are adherence to treatment, ASM-related adverse events, quality of life, mood disorders, patient and caregiver satisfaction, safety and costs. TELE-EPIC_VAMS is a cross-validation study for blood ASM quantitation through a novel, VAMS-based device, comparing (1) VAMS versus plasma samples (reference standard method); and (2) nurse-collected versus self-collected blood by VAMS device. Ethics and dissemination The study has been approved by the local ethics committee (349-2019-SPER-AUSLBO). Complete information about the state of project, relevant events and results will be regularly updated on the project’s webpage on ClinicalTrials.gov. The project’s results and data on the potential impact of TM in epilepsy will be disseminated on social media. A closeout meeting will be convened for the communication and dissemination of the project, highlighting its main achievements and impacts. Trial registration number NCT04496310

Background: Differential diagnosis between Parkinson’s disease (PD) and atypical parkinsonisms (APs) may be difficult at disease onset. The response to levodopa (LD) is a key supportive feature but its definition is largely empirical. Studies evaluating this issue by quantitative tests are scanty. Objective: We aimed to assess the utility of a subacute low LD dose kinetic-dynamic test in the differential diagnosis between PD and APs. It was applied at the baseline of a prospective follow-up in patients with parkinsonian signs within three years of disease motor onset (“BoProPark” cohort) and eventually diagnosed as PD or APs according to consensus criteria. Methods: Patients under at least 3-month LD therapy received a first morning fasting dose of LD/benserazide or carbidopa (100/25 mg) and underwent simultaneous serial assessments of plasma LD concentration and alternate finger tapping frequency up to 3 h. The main outcome was the extent of LD motor response, calculated by the area under the 3 h tapping effect–time curve (AUC_ETap). A receiver operating characteristic (ROC) curve analysis was performed to establish the optimal AUC_ETap cut-off to differentiate PD and APs. Results: The first 100 consecutive “BoProPark” patients were analyzed. Forty-seven patients were classified as possible, 37 as probable PD and 16 as APs. AUC_ETap medians were similar in the PD subgroups but reduced to a third in APs (p < 0.001). The optimal AUC_ETap cut-off value was >2186 [(tap/min) x min], with a sensitivity of 92% and a specificity of 75%. Accuracy of the test was 0.85 (95% CI 0.74–0.95), p < 0.0001. Conclusion: The estimation of 3 h AUC_ETap after a subacute low LD dose proved a reliable, objective tool to assess LD motor response in our cohort of patients. AUC_ETap value rounded to ≥2200 supports PD diagnosis, while lower values may alert to AP diagnoses.

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by α-galactosidase A gene (GLA) mutations, resulting in loss of activity of the lysosomal hydrolase, α-galactosidase A (α-Gal A). As a result, the main glycosphingolipid substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3), accumulate in plasma, urine, and tissues. Here, we propose a simple, fast, and sensitive method for plasma quantification of lyso-Gb3, the most promising secondary screening target for FD. Assisted protein precipitation with methanol using Phree cartridges was performed as sample pre-treatment and plasma concentrations were measured using UHPLC-MS/MS operating in MRM positive electrospray ionization. Method validation provided excellent results for the whole calibration range (0.25–100 ng/mL). Intra-assay and inter-assay accuracy and precision (CV%) were calculated as <10%. The method was successfully applied to 55 plasma samples obtained from 34 patients with FD, 5 individuals carrying non-relevant polymorphisms of the GLA gene, and 16 healthy controls. Plasma lyso-Gb3 concentrations were larger in both male and female FD groups compared to healthy subjects (p < 0.001). Normal levels of plasma lyso-Gb3 were observed for patients carrying non-relevant mutations of the GLA gene compared to the control group (p = 0.141). Dropping the lower limit of quantification (LLOQ) to 0.25 ng/mL allowed us to set the optimal plasma lyso-Gb3 cut-off value between FD patients and healthy controls at 0.6 ng/mL, with a sensitivity of 97.1%, specificity of 100%, and accuracy of 0.998 expressed by the area under the ROC curve (C.I. 0.992 to 1.000, p-value < 0.001). Based on the results obtained, this method can be a reliable tool for early phenotypic assignment, assessing diagnoses in patients with borderline GalA activity, and confirming non-relevant mutations of the GLA gene.

Background: Purified cannabidiol (CBD) was administered to highly refractory patients with Dravet (DS) or Lennox–Gastaut (LGS) syndromes in an ongoing expanded access program (EAP). Herein, we report interim results on CBD safety and seizure outcomes in patients treated for a 12-month period. Material and Methods: Thirty centers were enrolled from December 2018 to December 2019 within the open-label prospective EAP up to a maximum of 25 mg/kg per day. Adverse effects and liver function tests were assessed after 2 weeks; 1, 3, and 6 months of treatment; and periodically thereafter. Seizure endpoints were the percentage of patients with ≥50 and 100% reduction in seizures compared to baseline. Results: A total of 93 patients were enrolled and included in the safety analysis. Eighty-two patients [27 (32.9%) DS, 55 (67.1%) LGS] with at least 3 months of treatment have been included in the effectiveness analysis; median previously failed antiseizure medications was eight. Pediatric and adult patients were uniformly represented in the cohort. At 3-month follow-up, compared to the 28-day baseline period, the percentage of patients with at least a 50% reduction in seizure frequency was 40.2% (plus 1.2% seizure-free). Retention rate was similar according to diagnosis, while we found an increased number of patients remaining under treatment in the adult group. CBD was mostly coadministered with valproic acid (62.2%) and clobazam (41.5%). In the safety dataset, 29 (31.2%) dropped out: reasons were lack of efficacy [16 (17.2%)] and adverse events (AEs) [12 (12.9%)], and one met withdrawal criteria (1.1%). Most reported AEs were somnolence (22.6%) and diarrhea (11.9%), followed by transaminase elevation and loss of appetite. Conclusions: CBD is associated with improved seizure control also in a considerable proportion of highly refractory patients with DS and LGS independently from clobazam use. Overall, CBD safety and effectiveness are not dose-related in this cohort.

Background and Aim: Data on the clinical pharmacokinetics of cannabidiol (CBD) are scanty. We explored the effect of demographic and clinical variables on plasma concentrations of purified CBD in patients with Dravet (DS) and Lennox–Gastaut syndrome (LGS). Methods: The study design was an open, prospective, multicenter expanded access program (EAP). Venous blood samples were drawn from patients between 8 and 9 am, before the CBD morning dose, 12 h apart from the last evening dose, and then 2.5 h after their usual morning dose. Results: We collected 127 plasma samples (67-morning pre-dosing and 60 post-dosing) from 43 patients (24 females, 19 males), 27 with LGS and 16 with DS. Mean ± standard deviation age was 26 ± 15 years. Duration of CBD treatment averaged 4.2 ± 2.9 months at 13.2 ± 4.6 mg/kg/day. CBD median trough plasma concentration was 91 ng/ml; it doubled to 190 ng/ml 2.5 h post-dosing (p < 0.001). Cannabidiol trough plasma concentrations were linearly related to daily doses (r = 0.564, p < 0.001). Median trough CBD plasma concentration-to-weight-adjusted dose ratio (C/D) was 32% higher (p < 0.02) in plasma samples from subjects aged 18 and over than in those under 18. Sex and concomitant antiseizure medications (ASMs) were not associated with significant variations in CBD C/D, but caution is required due to the potential influence of confounders. Conclusion: These are the first data on CBD pharmacokinetics in children and adults with LGS or DS in a real-world setting. The most relevant finding was the higher CBD C/D in adults. In practice, reduced weight-normalized doses might be required with aging to achieve the same CBD plasma levels.

Background: "Impulse Control Disorders" are behavioral conditions (e.g., gambling, hypersexuality), which are increasingly reported as reactions to dopamine agonists in Parkinson's disease. The Questionnaire for Impulsive Compulsive Disorders in Parkinson's disease focuses only on 6 behaviors. Nonetheless, impulsivity could affect the entire range of human practices. Because of their heterogeneity and undefined boundaries, it is not clear what conditions should be considered as Impulse Control Disorders. This results in poorly standardized scientific literature and underdiagnosis. Objective: We aimed to create a comprehensive list of possible manifestations of drug-induced Impulse Control Disorders in Parkinson's disease and test it on pharmacosurveillance data. Methods: PubMed was used to identify reviews in English about Impulse Control Disorders in Parkinson's disease. Mentioned conditions were charted and translated to the lexicon of MedDRA, ICD-11, and DSM-5. The relevant MedDRA terms were used to test potential association with dopamine agonists on the FDA Adverse Event Reporting System. Results: 50 reviews published between 2001 and 2020 were identified. 66 conditions were collected as possible Impulse Control Disorders. Pathological gambling, shopping, eating and sexuality, dopamine dysregulation syndrome, hobbyism and punding were the most frequently mentioned, together with leisure activities, body focused compulsivity, disruptive, impulse control and conduct disorders, and substance abuse. All these conditions were disproportionately reported with dopamine agonists, except for substance abuse. Conclusions: We defined a potential extended list of ICDs, which, along with its conversion to international taxonomies, can support the identification of drug-induced conditions in pharmacovigilance archives, as well as monitoring processes in clinical practice.

PURPOSE:The purpose of the study was to investigate the potential correlation between plasma concentration of the newer antiseizure medication (ASM) perampanel (PMP) and both tolerability and seizure control in patients with epilepsy. METHODS:The study design was multicenter, open, and prospective. Plasma samples were collected in the morning 12 h apart from once-a-day bedtime PMP dose. Perampanel tolerability was assessed on the day of drug monitoring by clinical examination and patients' interview. Response to PMP was defined as ≥50% reduction from baseline seizure frequency (pretreatment). The main outcomes were the comparisons of PMP plasma concentration-to-weight-adjusted dose ratio (C/D) [(μg/mL)/(mg/kg/day)] between patients with and without PMP-related adverse effects (AEs) and between responders and nonresponders. RESULTS:Ninety-seven patients (54% men), mean ± SD age 36 ± 14 years were enrolled in the study. The mean PMP dose was 6.7 ± 2.3 mg, drug treatment averaged 46 ± 34 weeks. The mean plasma concentration was 360 ± 268 ng/mL (range: 37-1213 ng/mL). Forty patients (41%) reported at least one AE, mainly dizziness and behavioral changes. No significant difference was found in median PMP C/Ds between patients with (2.94) and without (2.76) AEs, otherwise comparable for clinical variables. Forty-four patients (45%) were responders, at a median PMP C/D of 3.10, similar to the value of 2.76 found in nonresponders. These two groups also overlapped for clinical characteristics. CONCLUSION:This is the first prospective real-life study to evaluate the relationship between PMP plasma concentrations, seizure control, and AEs. In line with the few real-world available data, we did not find any significant correlation between PMP plasma concentrations and both tolerability and seizure control.

A volumetric microsampling (VAMS) device (20 μL) was evaluated and validated for the analysis of gamma-hydroxybutyric acid (GHB) in venous blood using a simple ultra high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. GHB was extracted from VAMS device by acetonitrile, after a re-hydration step in a temperature controlled ultrasonic bath at 60°C for 10 min. Chromatographic analysis was carried out on a Kinetex C18 column using 0.1% formic acid in water and acetonitrile as binary gradient mobile phase (from 5 to 95% of acetonitrile from 1 to 2.5 min) at a flow rate of 0.3 mL/min. VAMS method was fully validated according to current guidelines with satisfactory results in terms of linearity, selectivity, precision, absolute recovery, matrix effect and stability. The linearity was determined from 0.5 to 200 μg/mL and the lower limit of quantitation (LLOQ) was 0.5 μg/mL. The novel VAMS-UHPLC-MS/MS method has been successfully compared with plasma-based method in a GHB-treated patient as a proof of concept.