Levamisole HCl is a drug we worked with for nearly 20 years. The treatment is used to resolve a lethal, neurodegenerative disease in horses. Levamisole HCl was first discovered in 1966 and it has a long, successful history as an immune modulator because it regulats dysregulated innate immune responses. It was developed for humans and marketed under the name Ergamisol®. Levamisole was even used for several neurodegenerative disease studies, but they were unsuccessful for various reasons. One annoying problem is that a metabolite of Ergamisol® is toxic to some people so FDA removed it from the human market.
Some therapeutic properties of levamisole HCl are due to its ability to bind the thymopoietin receptor, T5 (thymopentin), it mimics T5 in most, but not in all cellular pathways. Neurodegenerative disease research’s (NDR) goals were to exploit the thymopentin pathways in ALS by modifying levamisole thereby preventing the toxic metabolite or to extend the life of thymopentin. Thymopentin has an Achilles heel and that is its ultra-short half-life. We explored possibilities using the commercially available formulation Zadaxin®, a pro-molecule of thymopoietin, in studies dosing ALS mouse models SOD1 and TDP43 to get some answers (link to publication).
The wish for an extended T5 or a safe Ergamisol® was ever on our minds, but we were getting little traction. Thymopentin isn’t on the radar of the ALS researchers we talked to, and no human researchers had heard of levamisole HCl. Fortunately, our virtual paths crossed with Dr. Steve Pelech, Kinexus Bioinformatics, British Columbia. At the time, we were interested in a specific phosphokinase inhibitor and his name came up as a kinase expert. Dr. Pelech had once been very involved in ALS research, alas funding had sidelined his ambitions as is often the case in science, his proteomics approach was overshadowed by newer, molecular pursuits.
NDR was facilitating work to identify a CTMP inhibitor or a molecule that would increase Akt signaling by funding a project for Dr. Chandler Walker, Indiana University. In the first discussion with Dr. Pelech and Dr. Walker, Steve quickly found seventy-two small molecules and 3-4 peptide agonists that might fit the project. Steve thought it would be efficient to develop a binding assay use CTMP to look for Akt activity. The conversation led to varying amino acids one at a time to increase binding CTMP, but after a quick look at the three dimensional structure of the binding pockets, Steve felt that peptides would be less likely to break protein-protein interactions and ultimately a peptide would fail to result in a therapeutic. He suggested combining small molecules and that might work.
The opportunity to discuss thymopentin and the desire to design non-hydrolyzing analogs of T5 presented itself. Steve brought up the 3D-structure of thymopentin and its receptors binding pocket on the shared screen and after a quick look he suggested reversing the sequence of the pentapeptide and using D instead of L amino acids. You might consider attaching two pentapeptides, end to end he mused. We wasted no time enlisting Dr. Pelech to design an assay to optimize binding of the T5 receptor, he’d alter the chemistry of T5 as part of the 2020 NDR Kinase Inhibition Project. He explained the science of peptidomimetics to us (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC44897/). From this back-of-the-napkin analysis, TVALA® was born.
Many tried to modify the structure of thymopentin, in fact the retro-inverso analogs of T5 were the subject of a 1987 patent. The compounds in the patent possess the equivalent or enhanced biological activity and had substantially increased resistance to enzymatic degradation when compared to the parent compound. But no treatments were produced. There were patents on d-peptides, and again, no commercial treatments using T5. We would begin by investigating TVALA® and comparing it to the T5 and levamisole HCl work we had already finished.
We investigated the action of T5, AO-101 (a first pass at a modified levamisole HCl molecule) and Zadaxin® on ALS-derived adipose stem cells in assays developed by Dr. Lauren Kokai. She explored the effects on purinergic signaling and got some surprising results. In low doses some drugs were useful but when they were used in high doses, they were toxic. What was clear was that when the ALS cells were stressed extracellular ATP was measurably high and some of the drugs she tried in vitro restored homeostasis. Since microglial activation was discovered as an important mechanism of motor neuron death in ALS and extracellular ATP is a crucial neuron-to-glia alarm signal, the involvement of purinergic signaling in neuroinflammation has become evident and largely accepted (Cieslak 2019).
NDR funded more work: Dr. Sayuri Miyamoto, San Paulo, Brazil, observed the effect of the retro-enantiomer on erastin-induced ferroptotic cell death and Dr. Jessica Morrice measured the actions of our drugs of interest on ConA stimulated human peripheral mononuclear cells …a similar process we employed with levamisole HCl in horse cells in 2015. The horse paper published in 2019 (https://pubmed.ncbi.nlm.nih.gov/30693587/). NDR added TVALA® studies in ALS mouse models, dogs, mice, and rats and many in vitro assays.
TVALA® can be used orally and when given sub-cutaneously, the drug crosses into the CNS in potentially therapeutic levels, a requirement to target diseased microglial cells. Some of the TVALA® studies have been published and the rest will follow when our scientists are satisfied with their story. It’s time to move on.
The next step is testing the safety of TVALA® in clinically normal people. If all goes according to plan the Phase 1b trial will begin in ALS patients, the intended target of this long process. We will answer the question we posed in 2019, can modulating the innate inflammatory response slow the progress of ALS in people. The advantage of targeting this innate receptor is that thymopentin is pluripotent. It has multiple actions and depending on the class of receptors engaged, as well as the different combinations of receptors on different cell types, thymopentin elicits a versatile regulation of pathophysiological processes. Just as levamisole HCl was proven to do many years ago.
There are new studies conducted during and following the Pandemic, some using levamisole HCl and some using thymopentin in Covid19 patients. There was a study using T5 in a mouse model of human colitis model (2019) and another study in end-stage renal disease patients undergoing dialysis (2022), and yet another that predicts that T5 will bind NSP15 of SARS CoV 2 that could moderate viral infections (Vijayan 2021). We will be able to use Steve’s assay to test the action of TVALA® on the NF-кB/NLRP3 signaling pathway in vitro, comparing results to a 2023 study that poses T5 prevents lipopolysaccharide-induced neuroinflammation and dopaminergic neuron injury (Peng 2023). Pelech assessed the ability of TVALA® to bind the NSP15 protein of SARS CoV 2 and found it superior to T5, we are waiting to see if TVALA® prevents infections in cultured cells.
Our first steps are done and that is the pre-clinical data and cGMP manufacture of TVALA®. It took two and a half years from our discussion with Steve, practically overnight in the drug development world. Of course if successful it was not an overnight venture, we put in 10,000 hours of work in neurological disease, even if it was using levamisole HCl horses.