Discussion
ALSFRS-R, neuroinflammation, ALS
DISEASE PROGRESSION, CHRONIC INFLAMMATION, DISEASE, SECONDARY, EVENTS
This clinical trial measured the biological, clinical and safety effects of rapamycin on patients affected by ALS. Unfortunately, the primary outcome measure could not be satisfied also due to the reduced number of samples that could be analyzed at week 18, to which the COVID-19 pandemic significantly contributed. Notwithstanding our expectations for an inevitable loss in samples at treatment end, this led to a final sample size of 50 on which the primary endpoint was assessed. Therefore, we could not demonstrate a significant effect of the study drug on Treg lymphocytes, and the question as to why some subjects did not show the expected Tregs increase in response to rapamycin remains.Inter-individual variation amongst ALS patients in the ability to increase Tregs in response to treatment have been recently observed in other RCTsFurthermore, increased expression of the mTOR-interacting Raptor protein, increased phosphorylation of Akt, and activation of growth-related transcriptional factor AP-1 may induce rapamycin resistanceAmong secondary biological outcome measures we observed changes on B and T cell subpopulations, monocytes, and on IL-18, which could be suggestive of a rapamycin-mediated effect on neuroinflammation in ALS, where misfolded protein aggregates may activate a cascade of events that drives chronic inflammation and secretion of proinflammatory cytokines, among which IL-18, that finally leads to tissue damage and cell deathTogether with the above mentioned limitation, the increase in classical monocytes and the reduction in intermediate monocytes in patients treated with rapamycin, is even more difficult to interpret because of the uncertainty on their role in ALS, and if monocyte subsets and activation profiles are altered depending on the stage of the disease (i.e., the changes are a response to disease and their changes suggest that the immune system becomes more activated as the disease progresses) or if they are instead pathogenetic deserves further studiesSerial measurements of neurofilament showed a constant decrease in placebo-arm patients, not observed in the treatment arms, where we found stable neurofilament levels. The different behavior of neurofilament in treatment and placebo arms may have several explanations since longitudinal behavior of neurofilament during ALS and/or in clinical trials is still being studied with conflicting results. Some studies found fast disease progression to be associated with neurofilament decrease over timeThis is a non-profit, exploratory study on rapamycin action in ALS patients, that was designed to investigate if this drug warrants further research in patients with ALS, considering also its safety profile. Rapamycin in combination with riluzole, was safe and well tolerated by ALS patients. AEs and SAEs were equally distributed between treatment and placebo arms, reassuring about safety, provided that a drug plasma dosage monitoring was performed. It is also reassuring that ~24% of the participants discontinued the trial, that is in line with recent data from other clinical studiesThis study was not powered to test an effect on clinical measures, but we could not observe a slowing in the ALSFRS-R decline nor an effect on quality of life, even after excluding SOD1 patients from enrollment. If the effect on Treg cells and other immunological outcome measures was more evident for patients treated with rapamycin 1 mg/mGiven the reported detrimental effects in SOD1-ALS miceThe main potentiality of this trial stands on immediate availability of the drug for patients use, should an effect be found by larger trials, and by the existence of newer and interesting molecules i.e., rapalogs, who offer the possibility to cross the blood brain barrier perhaps deserving further properties towards autophagy and neuroinflammation. In fact, we could not find the drug in the CSF of patients treated for 18 weeks, and although we cannot exclude rapamycin free fraction values below the detection limit of LC-MS/MS, it is probable that rapamycin cannot cross blood brain barrier. If this may be of secondary importance for the drug immunological effects, the concentration at which rapamycin or any rapalog are found able to penetrate the blood-brain barrier might be a matter of uttermost relevance for the sake of fostering autophagyLimitations of this early trial of rapamycin in ALS were the small number of participants and the short duration of treatment, that were mainly due to safety concerns. Furthermore, the expected effect size of the primary outcome measure was perhaps too optimistic and, together with the difficulties in obtaining blood samples at treatment end, this led to miss the primary outcome. The choice of the primary outcome measure, as a binary response, has to be acknowledged as the main drawback of this study, together with the fact that Treg function, rather than their number would have been relevant for treatment effect. Indeed, we did not plan to study Treg suppressive function instead of their number alone and we were not able to assess Treg suppressor function as a post-hoc analysis due to lack of available samples. Before further studies in ALS, assessment of Treg suppressor function in response to different doses of rapamycin would be critical. In addition, some data on the primary outcome were not measured due to the COVID-19 pandemic, which may introduce a risk of bias into the results. However, the double-blind design of the study and the random missing mechanisms should have limited this risk. Other drawbacks are represented by the imbalance of some factors at baseline (such as in edaravone treatment), and the exploratory nature of the clinical outcomes. Finally, the results on biological outcomes should be interpreted cautiously and require confirmation in larger studies, considering that we did not apply corrections for multiple tests.In conclusion, this trial demonstrated that treatment with a low dose of rapamycin is safe in patients with ALS, but it failed to demonstrate an effect of the drug on Treg cells. Further trials focused on different outcome measures are necessary to better understand rapamycin biological and clinical effects in ALS.