Study design
A single-arm, open-label study was conducted. All subjects received pantethine during the 24-week period of treatment. This study was approved by the Ethics Committee of Peking University First Hospital and registered at the Chinese Clinical Trial Registry (ChiCTR1900021076). All the parents or legal guardians of children signed the written informed consent forms before any procedure was done.
Participants
Patients who met all the following criteria were enrolled: (1) aged 3–16 years who showed progressive dystonia; (2) “eye-of-the-tiger” sign in brain magnetic resonance imaging; (3) pathogenic or likely pathogenic variants identified in PANK2. Patients were excluded if they met any of the following criteria: (1) patients who participated in other clinical trials; (2) with severe liver damage (alanine aminotransferase or aspartate aminotransferase > 3 times the upper limit of normal value);(3) severe renal damage with glomerular filtration rate < 60 ml/min;(4) with cardiovascular system diseases, including cardiac dysfunction or arrhythmia; (5) abnormalities in blood system (hemoglobin < 60 g/L, platelet < 50 × 109/L, neutrophil < 1.0 × 109/L); and (6) patients who underwent DBS in the past 6 months.
Procedure
The study lasted for 24 weeks. Patients received pantethine through oral administration (capsules, 450 mg per serving, Jarrow Formulas, USA). The dose started at 20 mg/kg per day, gradually increased to 60 mg/kg per day within 4 weeks and maintained at 60 mg/kg per day (divided into two dosage) until the end of the study. During the study, the dosage of other drugs taken by patients at baseline remained unchanged. Patients were contacted weekly by phone to find out if there were any drug related adverse events in the first 5 weeks and were visited at week 12 (W12) and 24 (W24).
The Unified Parkinson’s Disease Rating Scale I-III (UPDRS I-III) and Fahn–Marsden Scale (FM) at W-24 (24 weeks before treatment) were retrospectively assessed at baseline. At baseline(W0), UPDRS I-III and FM scores, Activities of daily living (ADL) and Pediatric Quality of Life Inventory (PedsQL) were scaled, motion videos were recorded, and serum CoA levels were measured. At W12 and W24, the UPDRS I-III and FM scores were re-scaled. ADL, PedsQL, parental clinical impression evaluation and blinded video rating were scaled at W24 (Fig. 1).
Assessments of the efficacy and safety
Assessments of efficacy were conducted using the following measures: (1) UPDRS I-III was used to assess motor symptoms, including mental behavior, daily activity, and motor sections by generating individual scores and a total score (0–124); (2) FM scale was used to measure the severity of dystonia in nine body regions (eyes, mouth, neck, trunk, swallowing, speech, and each upper and lower extremities) by generating individual scores and a total score (0–120); (3) ADL scale was used to measure the patients’ ability of daily living, the higher the score, the better the ability of self-care; (4) PedsQL scale was used to measure quality of life, which includes PedsQL4.0 (Generic Core Scales), PedsQL3.0 (Neuromuscular Module), and PedsQL2.0 (Family Impact Module). The first two were used to assess patients’ quality of life. PedsQL2.0 was used to evaluate the quality of life of the families; (5) Parental clinical impression scale, which is a subjective instrument consists of a single question, was used to ask parents to rate their children’s condition on a scale from 1 (markedly better) to 5 (markedly worse) compared with how they felt at the baseline; (6) Blinded video rating was independently performed by two pediatric neurologists. Conflicts in opinions were resolved by consulting another neurologist to make a decision. Patients’ condition at W24 was rated on a scale from 1 (markedly better) to 5 (markedly worse) compared with how they felt at baseline through watching the videos (the actions included opening and closing eyes, opening and closing mouth, turning head, eye movements, tapping the index finger with the thumb, stretching and bending arms, placing both arms flexed at the elbow in front of the chest, stamping the foot, finger–nose test, turning body, walking, eating something, writing, and speaking).
Safety assessments were conducted at each visit through data collection of adverse events and clinical laboratory tests including liver function, renal function, and complete blood count.
The primary endpoints were the change of UPDRS I-III and FM scores from baseline to W24 after pantethine treatment. The secondary endpoints were changes in ADL score, PedsQL score, parental clinical impression scale, as well as blinded video rating from baseline to week 24. The increase rates of UPDRS I-III and FM scores 24 week after and before pantethine treatment were also compared.
Measurement of serum CoA
Blood was extracted at baseline and W24. The serum was immediately centrifuged (4000 rpm/min, 10 min) and stored at − 20 °C until CoA was measured. Serum CoA was measured using a colorimetric-based kit (Abcam, ab102504) in accordance with the manufacturer’s instructions. A CoA standard curve was generated on the basis of optical density and serial dilution of CoA standard. CoA levels in test samples were calculated using a calibration curve.
Measurement of sodium pantothenate
Blood was extracted two and five hours after taking pantethine at W12. Sodium pantothenate concentrations were determined by high-performance liquid chromatography coupled with mass spectrometry approach (LC-MS/MS), with ceftiofur as an internal standard. Negative electrospray ionization tandem mass spectrometry with multiple reaction monitoring mode was choose for the detection. Protein precipitation was applied with acetonitrile in the extraction process. The calibration curve was linear over the range of 0.05 to 50 μg/mL. The lower limit of quantification (LLOQ) was 0.05 μg/mL. The accuracy of low, middle and high concentration controls was 101.9, 114.1 and 114.2%, respectively.
Statistical analysis
Descriptive statistics were used to summarize the collected data. Categorical variables were summarized on the basis of frequencies and percentages. Continuous variables were summarized on the basis of mean, median, standard deviations, and minimum and maximum values. The change of UPDRS I-III, FM, ADL, and PedsQL scores from baseline to week 24 were compared through paired t test. The increase rate of UPDRS I-III and FM scores before and after treatment were compared using the Wilcoxon rank sum test. The correlation between blinded video rating and initial UPDRS I-III/FM scores at baseline was tested through one-way ANOVA. Spearman’s correlation was used to compare the correlation between sodium pantothenate concentrations and change of UPDRS I–III/FM scores. The correlation between parental clinical impression scale/ blinded video rating by doctors and the change of UPDRS I–III/FM scores during treatment of pantethine was tested through Kendall’s tau-b correlation coefficient. All statistical analyses were conducted on SPSS (version 20.0). P < 0.05 was considered statistically significant.