Question

Is treatment with tetramethylpyrazine nitrone (TBN) safe and effective for improving functional outcomes in patients with amyotrophic lateral sclerosis (ALS)?

Findings

In this randomized clinical trial of 155 patients who were given low-dose tetramethylpyrazine nitrone, high-dose tetramethylpyrazine nitrone, or placebo, the occurrence of adverse events was comparable among all groups. The primary measure of effectiveness, the ALS Functional Rating Scale–Revised score, did not show a significant improvement with tetramethylpyrazine nitrone treatment, while high-dose tetramethylpyrazine nitrone showed a significant slowing of the decline in grip strength, especially in patients younger than 65 years with slowly progressing ALS.

Meaning

These findings demonstrate that tetramethylpyrazine nitrone is safe and may have potential benefits in slowing the decline in grip strength, although its impact on the primary outcome measure was not statistically significant.

Importance

Tetramethylpyrazine nitrone has exhibited promising results in improving motor dysfunction in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS).

Objective

To evaluate the safety and efficacy of orally administered tetramethylpyrazine nitrone in patients with ALS.

Design, Setting, and Participants

This phase 2, multicenter, double-masked, placebo-controlled, randomized clinical trial was conducted from December 24, 2020, through July 14, 2023, in 11 centers in China, with a 180-day follow-up. Patients aged 45 to 70 years, with ALS onset within 2 years, ALS Functional Rating Scale–Revised (ALSFRS-R) scores of at least 2 points on each item, and forced vital capacity (FVC) of at least 80% were included. Patients experienced a 1- to 4-point decrease in ALSFRS-R score during a 3-month screening period.

Interventions

Patients were randomly assigned 1:1:1 to receive low-dose tetramethylpyrazine nitrone (600 mg twice daily), high-dose tetramethylpyrazine nitrone (1200 mg twice daily), or placebo (twice daily) for 180 days.

Main Outcomes and Measures

The primary outcome was change in ALSFRS-R score (range of 0-48, with lower scores indicating worse function) from baseline to 180 days. The secondary outcomes were changes in FVC, grip strength, ALS Assessment Questionnaire-40 (ALSAQ-40) score, and end point events. Safety outcomes included adverse events.

Results

A total of 155 patients (mean [SD] age, 55.0 [6.5] years; 115 men [74.2%]) were randomized (51 [32.9%] to the low-dose tetramethylpyrazine nitrone group, 52 [33.6%] to the high-dose tetramethylpyrazine nitrone group, and 52 [33.6%] to the placebo group). No significant differences were observed in ALSFRS-R score changes between low-dose tetramethylpyrazine nitrone (least squares [LS] mean difference, −0.89 points; 95% CI −3.25 to 1.48 points) and high-dose tetramethylpyrazine nitrone (LS mean difference, −0.20 points; 95% CI −2.48 to 2.07 points) compared with placebo. High-dose tetramethylpyrazine nitrone showed a significantly slower decline in grip strength at day 180 (LS mean difference, 2.46 kg; 95% CI, 0.15-4.76 kg). In a subgroup of patients younger than 65 years with slower disease progression, tetramethylpyrazine nitrone significantly attenuated the decline in grip strength (LS mean difference, 3.63 kg; 95% CI, 0.84-6.41 kg), bulbar scores (LS mean difference, 0.66 points; 95% CI, 0.03-1.29 points), and respiratory scores (LS mean difference, 0.54 points; 95% CI, 0.03-1.06 points). Adverse events were mostly mild or moderate, with no severe treatment-related adverse events or deaths.

Conclusions and Relevance

This randomized clinical trial demonstrates that tetramethylpyrazine nitrone is safe and well-tolerated in patients with ALS. There was no difference in the primary end point across the low-dose, high-dose, and placebo groups, with significant benefits in a subgroup of younger patients with slower disease progression.

Trial Registration

ChiCTR Identifier:ChiCTR2000039689

Study Design and Patients

This phase 2, double-masked, placebo-controlled, randomized clinical trial was conducted from December 24, 2020, through July 14, 2023, at 11 centers in China. Details of the trial rationale, design, and methods have been described previously and are provided in the trial protocol (Supplement 1). The trial was conducted in conformity with the Declaration of Helsinki,²⁵ the Chinese Good Clinical Practice guidelines,²⁶ and local regulations. The institutional review board of each participating center approved the protocol, and all patients provided written informed consent. This study adhered to the Consolidated Standards of Reporting Trials (CONSORT) reporting guideline.

All patients had a diagnosis of definite or probable ALS per revised El Escorial criteria. Eligible patients were aged 45 to 70 years with an ALS duration of 2 years or less, a decrease in ALSFRS-R score of 1 to 4 points within a 3-month screening period, a score of at least 2 points on each ALSFRS-R item, normal respiratory function (4 points), and a forced vital capacity (FVC) of at least 80% at baseline. The ALSFRS-R is a 12-subdomain self-assessment for functionality scored on a scale of 0 to 48, with lower scores indicating worse function. The use of standard of care, such as riluzole, was permitted, but edaravone was contraindicated. Exclusion criteria included familial ALS, cognitive impairment, dysphagia, severe hepatic impairment (alanine aminotransferase or aspartate aminotransferase >3 times the upper limit of normal), severe kidney impairment (creatinine clearance <30 mL/min), cancer, hypersensitivity to the study drug or tetramethylpyrazine, or participation in other trials within 30 days. Coordinating centers and clinical site information are available in eTable 1 inSupplement 2. Detailed inclusion and exclusion criteria are shown in eTable 2 inSupplement 2.

Randomization and Masking

Dynamic randomization by minimization was used to balance sample sizes and prognostic factors across groups. Patients were stratified by ALS diagnosis (definite vs probable), change in ALSFRS-R score within the 3-month screening period (−1 or −2 vs −3 or −4), age (<65 vs ≥65 years), and riluzole use (yes vs no). Eligible patients were randomly assigned (1:1:1) to a low-dose tetramethylpyrazine nitrone group (600 mg orally twice daily), high-dose tetramethylpyrazine nitrone group (1200 mg orally twice daily), or placebo group using an interactive web response system, which only authorized personnel could access for randomization codes; treatment assignments followed these codes. The tetramethylpyrazine nitrone and placebo tablets were packaged using random codes. All patients and study personnel were masked to treatment assignments.

Procedures

Before randomization, potential participants entered a screening period of up to 3 months. Patients with a decrease in ALSFRS-R score of 1 to 4 points within this period were randomly assigned to the low-dose tetramethylpyrazine nitrone, high-dose tetramethylpyrazine nitrone, or placebo group. The tetramethylpyrazine nitrone or placebo tablets were administered on an empty stomach at least 1 hour before or 2 hours after meals. The double-masked treatment period was from baseline to day 180. Study patients had visits on day 30, 90, and 180 after the baseline visit, with a telephone interview done on days 7, 60, 120, and 150. Data on demographics, clinical characteristics, medical history, concomitant medications, adverse events (AEs), serious AEs, vital signs, physical examinations, electrocardiograms, and laboratory tests were collected and updated throughout the trial. All data were uploaded to an electronic data capture system.

Outcomes

The primary efficacy end point was the change in ALSFRS-R score from baseline to day 180. Secondary end points included the total score of the ALSFRS-R evaluated on days 30, 90, and 180, as well as the corresponding changes in grip strength (calculated as the mean of both hands) measured at these time points. Secondary end points included the proportion of end point events, changes in FVC, and variations in ALS Assessment Questionnaire-40 (ALSAQ-40) scores (scale of 0-500, with lower scores indicating better function). The prespecified end point events included death, tracheotomy, or invasive or persistent noninvasive ventilation (daily usage time ≥22 hours and duration ≥10 days). Safety outcomes included AEs, serious AEs, and changes in vital signs and laboratory data before and after treatment.

Statistical Analysis

The sample size was not statistically predetermined, as this was an exploratory study on safety and efficacy, estimating 150 total patients and 50 per group. Analysis of covariance (ANCOVA) was used to analyze the primary end point in the full analysis set. The full analysis set was defined as randomized patients with at least 1 dose of investigational medicine product and 1 postbaseline efficacy assessment. The dependent variable was the change in ALSFRS-R score from baseline to day 180. Patients were included as a random effect in the analysis of the change in ALSFRS-R scores from baseline, which was evaluated on days 30, 90, and 180. Covariates included the baseline ALSFRS-R score, site, sex, ethnicity (Han Chinese, other), ALS diagnostic grade (clinical definite diagnosis vs clinical probable diagnosis), ALSFRS-R decline during screening (−1 or −2 vs −3 or −4), age (<65 vs ≥65 years), and riluzole use (yes vs no). The ethnicity of the participants was determined according to the Chinese citizenship registration system. In addition to Han Chinese patients, patients from Miao, Dong, and Manchu ethnic groups were also enrolled. Ethnicity was collected to show the high homogeneity of the population. Group assignment was the fixed effect. At the outset of this study, a prespecified fixed sequential testing approach was used. The comparison between the high-dose group and the placebo group was tested first, followed by the low-dose vs placebo comparison, contingent upon the success of the high-dose test. Least squares (LS) means and 95% CIs were calculated for each group. The primary efficacy end point, ie, the change in ALSFRS-R score from baseline to day 180, underwent Bonferroni correction to adjust for multiple comparisons. Analyses were also conducted on a per-protocol set using ANCOVA.

The ALSFRS-R subdomain scores followed the total score methodology. Secondary end points, including changes in ALSFRS-R scores on days 30, 90, and 180 from baseline and FVC and grip strength, were analyzed using a mixed model for repeated measures in the full analysis set. The ALSAQ-40 score was analyzed using ANCOVA. End point event proportions and time-to-event end points were analyzed using Fisher exact test and Wilcoxon rank sum test, respectively. Safety was assessed in patients receiving at least 1 dose of investigational medicine product. To address missing data, we used multiple imputation techniques to fill in missing values based on observed data. This approach ensures that the analysis remains robust and minimizes the impact of missing data on the study’s findings. Statistical analyses were performed using SAS, version 9.4 (SAS Institute Inc).P < .05 was set as the threshold for statistical significance (2-sided).

Randomization and Baseline Characteristics

A total of 259 patients with ALS were screened, and 155 (mean [SD] age, 55.0 [6.5] years; 115 men [74.2%] and 40 women [25.5%]; 152 of Han Chinese [98.1%] and 3 of other [1.9%] ethnicity) were randomly assigned to the low-dose tetramethylpyrazine nitrone group (51 patients [32.9%]), high-dose tetramethylpyrazine nitrone group (52 patients [33.6%]), or placebo group (52 patients [33.6%]). Among these 155 patients, 22 discontinued early due to voluntary withdrawal, AEs, or progressive disease. Ultimately, 42 patients (82.4%) in the low-dose tetramethylpyrazine nitrone group, 46 (88.5%) in the high-dose tetramethylpyrazine nitrone group, and 45 (86.5%) in the placebo group completed the 180-day treatment (Figure 1).

The demographic and baseline characteristics of the full analysis set (148 patients) were similar across groups except for prior riluzole and/or edaravone use (Table 1). Prior use of riluzole alone was the most common among all groups (39 patients [84.8%] in the low-dose tetramethylpyrazine nitrone group, 33 [63.5%] in the high-dose tetramethylpyrazine nitrone group, and 39 [78.0%] in the placebo group). Only 1 patient (2.0%) with a history of edaravone use alone was present in the placebo group. The number of patients with a history of using both riluzole and edaravone was different across the groups, with 3 (6.5%) in the low-dose tetramethylpyrazine nitrone group, 15 (28.8%) in the high-dose tetramethylpyrazine nitrone group, and 10 (20.0%) in the placebo group (Table 1). Most patients were male (110 [74.3%] vs 38 female [25.7%]) and younger than 65 years (130 [87.8%] vs 18 [12.2%] aged ≥65 years). The mean (SD) ALS duration was 14.4 (5.0) months. During screening, 111 patients (75.0%) had an ALSFRS-R score change of −1 or −2 points, and 37 (25.0%) had a change of −3 or −4 points. The mean (SD) baseline grip strength was 15.8 (10.2) kg, and mean (SD) baseline FVC was 98.4% (13.1%). The median duration of drug exposure for patients was 178.5 days (IQR, 170.0-181.0 days) in the low-dose tetramethylpyrazine nitrone group, 178.8 days (IQR, 173.5-181.5 days) in the high-dose tetramethylpyrazine nitrone group, and 178.0 days (IQR, 172.5-181.5 days) in the placebo group. During the trial, riluzole was administered to 45 patients (88.2%) in the low-dose tetramethylpyrazine nitrone group, 46 (88.5%) in the high-dose tetramethylpyrazine nitrone group, and 48 (92.3%) in the placebo group.

Efficacy Outcomes

For the primary end point, the LS mean difference vs placebo for change from baseline in ALSFRS-R scores at 180 days was −0.89 points (95% CI, −3.25 to 1.48 points;P = .40) with low-dose tetramethylpyrazine nitrone and −0.20 (95% CI, −2.48 to 2.07;P = .84) with high-dose tetramethylpyrazine nitrone (Table 2). There were no significant differences in ALSFRS-R score changes between the tetramethylpyrazine nitrone and placebo groups. The ALSFRS-R score trends over time are shown inFigure 2A and further detailed in eTables 3 and 4 inSupplement 2. The ALSFRS-R subdomain scores also did not differ among groups at day 180 (Table 2). The eFigure inSupplement 2 shows the results of a prespecified subgroup analysis of change from baseline to day 180 in ALSFRS-R score. A prespecified subgroup analysis suggested potential treatment interactions favoring tetramethylpyrazine nitrone by day 180 in patients consistently using riluzole.

From baseline to day 180, the LS mean difference vs placebo in FVC was −1.43% (95% CI, −7.47% to 4.62%;P = .64) with low-dose tetramethylpyrazine nitrone and 0.16% (95% CI, −5.76% to 6.08%;P = .96) with high-dose tetramethylpyrazine nitrone (Table 2). In the full analysis set, grip strength changes from baseline to day 180 were not significantly different between the low-dose tetramethylpyrazine nitrone and placebo groups (difference, 1.06 kg; 95% CI, −1.31 to 3.44 kg;P = .38). However, the high-dose tetramethylpyrazine nitrone group showed a significant attenuation of the decline in grip strength compared with placebo (LS mean difference, 2.46 kg; 95% CI, 0.15-4.76 kg;P = .04) (Figure 2B). Similarly, in the per-protocol set, the high-dose tetramethylpyrazine nitrone group exhibited a significant attenuation of grip strength decline compared with placebo at day 180 (LS mean difference, 2.42 kg; 95% CI, 0.04-4.80 kg;P = .046) (eTable 5 inSupplement 2). Sensitivity analyses confirmed the robustness of the grip strength results. At day 180, the ALSAQ-40 scores increased slightly in the low-dose tetramethylpyrazine nitrone group compared with the placebo group (LS mean difference, 2.92 points; 95% CI, −31.91 to 37.75 points;P = .87) but decreased in the high-dose tetramethylpyrazine nitrone group (LS mean difference, −5.16 points; 95% CI, −38.37 to 28.05 points;P = .76); however, neither of these differences were statistically significant.

End point events occurred in 9 patients (19.6%) in the low-dose tetramethylpyrazine nitrone group, 5 (9.6%) in the high-dose tetramethylpyrazine nitrone group, and 7 (14.0%) in the placebo group, with no significant differences (Fisher exact testP = .35) (Table 2). The median time to end point events was 3.3 months (IQR, 3.0-5.3 months) in the placebo group, 5.0 months (IQR, 3.0-5.9 months) in the low-dose tetramethylpyrazine nitrone group, and 6.1 months (IQR, 5.1-6.2 months) in the high-dose tetramethylpyrazine nitrone group (Table 2). Compared with the placebo group, the time to end point events increased by 51.5% in the low-dose tetramethylpyrazine nitrone group (from 3.3 to 5.0 months) and by 84.8% in the high-dose tetramethylpyrazine nitrone group (from 3.3 to 6.1 months).

Subgroup Analysis

Since the rate of disease progression and patient age may affect drug response, we stratified patients according to their initial progression rate and age in a sensitivity analysis. The subset of individuals younger than 65 years who had a decline in ALSFRS-R score of 1 to 2 points within the 3-month screening period was designated as the slower progression subgroup. This subgroup included 90 of 155 (58.1%) patients, with 28 (18.1%) receiving low-dose tetramethylpyrazine nitrone, 29 (18.7%) receiving high-dose tetramethylpyrazine nitrone, and 33 (21.3%) receiving placebo. The primary and all secondary end points, excluding ALSAQ-40 and proportion of end point events, were evaluated within the slower progression subgroup.

For ALSFRS-R scores at day 180, the LS mean difference vs placebo was 0.75 points (95% CI, −1.40 to 2.90 points;P = .49) with low-dose tetramethylpyrazine nitrone and 1.83 points (95% CI, −0.29 to 3.95 points;P = .09) with high-dose tetramethylpyrazine nitrone (Table 2). Moreover, high-dose tetramethylpyrazine nitrone showed significant positive effects on ALSFRS-R bulbar subdomain scores compared with placebo (LS mean difference, 0.66 points; 95% CI, 0.03-1.29 points;P = .04). For the ALSFRS-R respiratory subdomain scores, the LS mean difference vs placebo was 0.54 points (95% CI, 0.03-1.06 points;P = .04) with low-dose tetramethylpyrazine nitrone and 0.61 points (95% CI, 0.09-1.12 points;P = .02) with high-dose tetramethylpyrazine nitrone. At day 180, grip strength also exhibited a significantly slower decline with high-dose tetramethylpyrazine nitrone (LS mean difference, 3.63 kg; 95% CI, 0.84-6.41;P = .01). Grip strength also exhibited a slower decline with low-dose tetramethylpyrazine nitrone (LS mean difference, 2.51 kg; 95% CI, −0.36 to 5.37 kg;P = .09), although the difference was not significant.

Safety Outcomes

Of the 155 randomized patients, 153 were included in the safety analysis set. Treatment-emergent AEs of any grade occurred in 115 patients (75.2%). As detailed inTable 3, 5 of 50 patients (10.0%) in the low-dose tetramethylpyrazine nitrone group, 3 of 52 (5.8%) in the high-dose tetramethylpyrazine nitrone group, and 3 of 51 (5.9%) in the placebo group experienced treatment-emergent AEs of grade 3 or higher. Treatment-related AEs were noted in 12 of 50 patients (24.0%) in the low-dose group, 18 of 52 (34.6%) in the high-dose group, and 9 of 51 (17.6%) in the placebo group and were mostly mild or moderate in intensity. No grade 3 or higher treatment-related AEs or deaths occurred. Treatment-related AEs leading to drug interruption were rare. Serious AEs were similar in incidence among all study groups, with none related to the investigational medicine product (Table 3).

Limitations

This study has several limitations. In SOD1^G93A ALS mice, tetramethylpyrazine nitrone mitigated the loss of spinal motor neurons, the glial response, muscle fiber denervation, and fibrosis.²³ Additionally, tetramethylpyrazine nitrone extended survival in TDP-43^M337V mice injected bilaterally during the early stages of ALS.²⁴ The objective of this phase 2 study was to maintain functionality and quality of life for patients with early-stage ALS, and long-term survival outcomes were not assessed due to the constraints on treatment and observation periods. Oxidative stress, mitochondrial dysfunction, and impaired autophagy are all potential factors contributing to ALS pathophysiology, from its initial to advanced stages. This study did not evaluate the effectiveness of tetramethylpyrazine nitrone in a broader cohort of patients with advanced-stage ALS. Furthermore, there was a lack of data on neurofilament light chains in cerebrospinal fluid or plasma, which would be necessary to assess the impact of tetramethylpyrazine nitrone on neurodegeneration.

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Supplementary Materials