PRECLINICAL BEHAVIORAL AND PHARMACOLOGICAL CHARACTERISTICS OF TN-001, A NOVEL, NON-HALLUCINOGENIC NEUROPLASTOGEN FOR THE TREATMENT OF MAJOR DEPRESSIVE DISORDER

Background

Psychedelics have recently demonstrated significant clinical promise for the treatment of psychiatric illnesses including major depressive disorder (MDD) and posttraumatic stress disorder (PTSD) [1,2]. However, their hallucinogenic and cardiotoxic properties present unique challenges in the clinical setting that may limit their widespread use. Neuroplastogens are an emerging class of compounds that promote neuroplasticity, similar to psychedelics, without inducing hallucinations. Here we present data on TN-001, a novel neuroplastogen for the treatment of MDD.

Methods

Agonist and antagonist activity of TN-001 was assessed using PI hydrolysis and PathHunter® β-arrestin2 assays, and responses were normalized to serotonin. For receptor occupancy studies, C57BL/6J mice received intraperitoneal (i.p.) injections of vehicle, psilocybin, or TN-001 and cortical membranes were prepared after 60 minutes. 5-HT2A occupancy was determined using [3H]Cimbi-36. Neural plasticity was assessed using primary culture of mature cortical neurons from Wistar rat embryos, which were incubated with vehicle, psilocin, BDNF, or TN-001 on Day 11 of culture for 3 days. Cells were fixed on Day 14 and immunostained for synaptophysin (SYN), postsynaptic density protein 95 (PSD-95), and microtubule-associated protein 2 (MAP-2). Images were captured using Operetta® CLS (PerkinElmer) and analyzed using Harmony™ software (PerkinElmer). Behavioral assays were performed in C57BL/6J mice. For head twitch response (HTR) and locomotor activity assays, mice received an i.p. injection of vehicle, psilocybin, or TN-001. HTRs were scored by a blinded experimenter and locomotor activity was recorded as distance moved in cm using Ethovision® (Noldus). In the forced swim test (FST), mice were placed into a cylinder of water 1 hour or 24 hours following i.p. injection with vehicle, fluoxetine, or TN001, and immobility time was measured using video tracking software.

Results

TN-001 is a Gq-biased partial agonist of human 5-HT2A receptor with an EC50 of 68 nM and Emax of 67% (relative to serotonin response) and an antagonist of human 5-HT2B receptor with an IC50 of 142nM and Imax of 97%. Similar potency was observed for mouse 5HT2A receptor (106 nM, 45%). In ex vivo occupancy studies, TN-001 showed robust occupancy of cortical 5-HT2A receptors which exceeded that of psilocin at the same doses. TN-001 showed comparable binding affinities for both mouse and human cortical 5-HT2A receptors (55 nM and 79 nM, respectively). TN-001 induced significant dose-dependent increases in the number of MAP-2 positive neurons, number of MAP-2 positive neurites in µm, and overlap in SYN/PSD95/MAP-2 staining in µm^2. These observations represent increases in the number of neurons, total neurite network, and number/area of synapses, respectively. In vivo, TN-001 does not induce HTR and does not impair locomotor activity at relevant doses. In FST, TN-001 significantly reduced immobility time compared to vehicle.

Conclusion

TN-001 is a potent dual 5-HT2A partial agonist and 5-HT2B antagonist that demonstrates structural neuroplasticity ex vivo and antidepressant-like activity in mice without inducing HTR or locomotor impairments. While agonism of 5-HT2B is a common off-target activity among many 5-HT2A agonists, and confers significant risk of cardiac valvular hypertrophy, TN-001 is an antagonist at 5-HT2B receptors and therefore is unlikely to carry this risk. These results provide a promising avenue toward the safe application of 5-HT2A-mediated neuroplasticity to treat MDD and other neuropsychiatric conditions. TN-001 is an investigational compound and not approved by the Food and Drug Administration for human use.