Effectiveness of levamisole HCl and thymalfasin in two mouse models of ALS

Many ALS rodent models are available for testing therapies. The SOD1 G93A transgenic mouse expresses the mutant human SOD1 gene and shows a mortality/aging phenotype with 50% survival at slightly over 100 days. The TDP43 transgenic mice express a mutant human TAR DNA binding protein, pathology in this model is associated with ubiquitin aggregates and a gastrointestinal pathology.

The purpose of this study using two test articles, thymalfasin (Zadaxin®) and levamisole HCl in the SOD1G93A and Prp-TDP43A315T models of ALS, was efficacy assessment on the neuromuscular phenotypes in vivo and ubiquitin load and neuroinflammation at necropsy. Three cohorts of six mice in each group were treated (thymalfasin, sub-cutaneous injection or levamisole HCl, gastric gavage) for 14 weeks.

In vivo measured parameters were body weights and neurological score in both models and compound muscle action potential (CMAP) in the SOD1 mice. In vitro analysis of spinal cord, brain, femoral nerve and six additional organs were collected at 14 weeks. Additionally, the colon of the TDP43 mice were collected and fixed for histology. The fixed spinal cord and brain tissues were stained with anti-ubiquitin, GFAP and Iba1 IHC; the femoral nerves were stained with Toluidine Blue, and the myenteric plexus of the colon was stained for acetylcholinesterase activity and ganglion cells were quantified.

Ten week CMAP measurement in SOD1G93A mice showed that the untreated mice had a muscle system genotype that was statistically different than the wild-type mice but there was no significant difference between the treatment. The RNS at 10 weeks was better in the levamisole HCl group. The mean survival in one cohort of the thymalfasin treated mice was encouraging, no disease was apparent in six mice, but this was not repeated in the other two cohorts treated with thymalfasin.

Ubiquitin staining in the SOD1 G93A model was affected by treatment, thymalfasin reduced ubiquitin in the spinal cord. Levamisole HCl and thymalfasin reduced ubiquitin in the brain. Overall, the effects of these drugs on ubiquitin were mild in this model. In the TDP43 model, the brain GFAP ratio showed a significant difference between untreated mice and thymalfasin, but beneficial effects were not found in the spinal cord. However, a significant treatment effect was seen in brain Iba1 for levamisole and thymalfasin but there was no treatment effect in spinal cord Iba1 in the TDP43 model.

Neither treatment extended the life significantly in either SOD1 G93A or the TDP43 model in this study. Although thymalfasin prevented disease in one cohort of six mice, it was not repeated in the other two cohorts. Levamisole HCl increased survival slightly in the TDP43 model. These studies would benefit from pharmacokinetic studies to determine dose, duration, and frequency to maximize the positive effects that were observed in this study. There were mild treatment effects observed in the ubiquitin staining in the SOD1 model. It is possible that the differences observed in the ubiquitin staining between the tissues were due to different anti-inflammatory pathways targeted by levamisole HCl and thymalfasin.

Modifications to both chemicals used in this study would improve their efficacy against the multiple pathologies seen in ALS. To approach an effective therapy levamisole HCl would need to be modified to prevent possible toxic metabolites that can be seen in a small number of people taking this medication. Thymalfasin would need modification to extend the half-life of the drug in people. Additional areas of investigation are determining the target of engagement for each molecule and slowing the progression of ALS in patients. An advantage to creating non-toxic levamisole HCl analogs are the eventual cost of production over a small peptide therapy. The advantage of both of these chemicals is that they are pleiotropic and could serve as adjunctive therapies in ALS and other diseases. NDR has multiple ongoing studies with both of these drugs that we feel may bring hope to those suffering from ALS.

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