Safety and Preliminary Efficacy of Aleeto in Amyotrophic Lateral Sclerosis

This study is a single-center, randomized, double-blind, placebo parallel-controlled, dose-escalation clinical study. The aim of this study was to evaluate the safety, tolerability, and preliminary effect of Aleeto in adult patients with ALS, and to provide an appropriate dose for the future clinical trial.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which is characterized by progressive loss of number and function of upper and lower motor neurons located in the brain and spinal cord, which leads to paralysis. ALS mainly begins in the limbs and face, where about one-third of patients have difficulty in speaking, chewing, or swallowing, then progressively affecting the pharyngeal muscles, attacking the sphincter in the advanced stages of the disease, and eventually affecting the trunk and respiratory function as the disease progresses.

"Aleeto" derived from cellular exosomes, is a group of specific microenvironment protein polymers secreted by stem cells under stressed conditions, which has the advantages of selective assembly, targeted delivery, efficient repair of damaged tissues, high safety, stable chemical properties, and easy preservation. What's more, Aleeto has a strong nerve repair function.

This study is a single-center, randomized, double-blind, placebo parallel-controlled, dose-escalation clinical study, aiming to evaluate the safety, tolerability, and preliminary effect of Aleeto in adult ALS patients, and provide an appropriate dose for the future clinical trial.

In this study, we plan to enroll 24 patients, and will be randomly assigned to 4 groups. The initial dose group consists of four subjects, out of which three subjects will receive intravenous administration and intrathecal administration of 0.5μg/kg Aleeto, while one subject will receive a placebo. Fourteen subjects will be randomly assigned to the 1μg/kg and 2μg/kg dose groups to receive intrathecal administration combined with intravenous administration, with one subject in each dose group randomly receiving placebo treatment. Six subjects will be randomly assigned to the 2μg/kg dose group to receive intravenous administration only. Data are collected in face-to-face interviews at baseline and Day 14, 30, 37, 60, 67, 90, 120 follow-up visits. All patients will be examined before and after treatment, and the results will be compared.

The primary outcomes are the incidence of adverse events (AEs) and serious adverse events (SAEs), and the incidence of abnormal changes in laboratory examination indicators, vital signs, neurological physical examination, and electrocardiogram within 120 days after the treatment. The secondary outcomes included changes in immunological indicators, pharmacokinetics, ALS Functional Rating Scale-Revised (ALSFRS-R), as well as the incidence of invasive mechanical ventilation and mortality rate .

The statistical analysis was performed by bilateral test, and the test level α was 0.05.

Baseline Equilibrium Analysis: Analysis of variance or Kruskal-Wallis H test is used to compare continuous data.The Chi-Squared test or Fisher's exact test is used to compare the categorical data.

Primary Efficacy Analysis: The comparability among different dosage groups in terms of incidence of adverse events (abnormal liver functions, hypersensitivity reactions, liver failure, dyspnea, etc.), severe adverse events, and other abnormal indicators will be analyzed using Chi-squared tests or Fisher's exact test. Changes in laboratory examination indicators and vital signs before and after treatment will be analyzed using paired t-tests or Wilcoxon signed rank sum test. The intergroup differences in laboratory examination indicators and changes in vital signs before and after treatment will be analyzed using t-test or Mann-Whitney U test.

Secondary Efficacy Analyses: The improvement of immunological indicators (lymphocyte subgroup analysis), pharmacokinetic indicators, and ALSFRS-R before and after treatment will be analyzed using paired t-tests or Wilcoxon signed rank sum test. The intergroup comparison of immunological indicators (lymphocyte subgroup analysis), pharmacokinetic indicators, and ALSFRS-R before and after treatment will be analyzed using t-tests or Mann-Whitney U tests. The intergroup significance of the incidence of invasive mechanical ventilation and mortality rate is determined using Chi-squared tests or Fisher's exact test.

Exploratory Analysis: Comparison of measurement data: The improvement of ALSAQ-40, EQ-5D-5L, modified Norris scale, forced lung capacity, muscle strength, electromyographic indicators, gait function, biomarker levels, FSS, imaging indicators, HAMA and HAMD scales will be analyzed using paired t-tests or Wilcoxon signed rank sum test. The above exploratory analysis indicators and the difference in survival time between two groups will be analyzed using t-test or Mann-Whitney U test. Analysis of variance or Kruskal Wallis H-test will be used for comparing the differences ａｍｏｎｇ different dosａｇｅ groups, and Bonferroni correction method will be used for multiple comparisons. Mixed linear models will be used for analyzing the differences in the trend of changes between different dose groups over a period of 120 days (the data which corresponds to skewed distribution will be transformed). LSmeans will be used for analyzing the differences of indicators between different groups at different times, as well as the differences within the same group at different times.

Comparison of enumeration data: Chi-square test or Fisher's exact test for enumeration data.

Eligibility Criteria:
Inclusion Criteria:

1. Subjects who are qualified for the clinical trial program cycle judged by well-trained physicians;
2. 18 years old≤ age≤ 75 years old, males or females;
3. Forced vital capacity ≥ 80% of predicted vital capacity during the screening period;
4. The total score of the ALSFRS-R scale ≥ 30 points and ≤40 points during enrollment, and the respiratory function item ≥ 3 points;
5. Diagnosis of confirmed or probable ALS in accordance with the revised EI Escorial diagnostic criteria for amyotrophic lateral sclerosis of the World Federation of Neurology;
6. Subjects or their legal representatives clearly understand and voluntarily participate in the study and sign the informed consent form;
7. Subjects (including male subjects) are willing to have no birth plan and voluntarily take effective contraceptive measures during the entire study period and within 3 months after the end of the study, and have no plan to donate sperm or eggs.

Exclusion Criteria:

1. Diagnosed with familial ALS (based on family history);
2. With obvious cognitive impairment (MMSE scale: ≤19 points in the illiteracy group，≤ 22 points in the primary school group, and ≤26 points in the junior high school group (more than 8 years of education);
3. Obvious dysphagia;
4. Positive HIV test or history of positive test;
5. Positive hepatitis C virus antibody or positive test history;
6. Hepatitis B active infection (hepatitis B surface antigen positive and/or serum HBV DNA positive or serum HBV DNA \> 2 × 108 IU/ml;
7. Have used other investigational drugs within 1 month or within 5 drug half-lives;
8. Diseases and deformities of the lumbar spine;
9. Have other conditions known to be associated with motor neuron dysfunction that may confuse or obscure an ALS diagnosis;
10. Other psychiatric disorders diagnosed according to DSM-V diagnostic criteria, or significant suicide intent;
11. With severe hepatic insufficiency, renal insufficiency or severe cardiac insufficiency (severe hepatic insufficiency refers to ALT value≥2.0 times the upper limit of normal value or AST value≥2.0 times the upper limit of normal value; severe renal insufficiency refers to CRE≥1.5 times the upper limit of normal value or eGFR\<40mL/min/1.73m2; severe cardiac insufficiency refers to NYHA class 3-4);
12. Permanently dependent on ventilator-assisted ventilation;
13. History of alcohol and drug abuse; Edaravone users;
14. Patients who are pregnant, breast-feeding, or who are likely to become pregnant and plan to become pregnant;
15. Patients participating in other clinical trials or using other biological agents, drugs, or devices under investigation;
16. Patients who have received any vaccinations within 28 days;
17. Contraindications to MRI (eg, claustrophobia);
18. Unable to be cooperative and complete the follow-up due to other reasons.