TrkB is a tyrosine kinase receptor that is activated upon binding to brain-derived neurotrophic factor (BDNF). To date, the search for low-molecular-weight molecules mimicking BDNF’s action has been unsuccessful. Several molecules exerting antidepressive effects in vivo, such as 7,8-DHF, have been suggested to be TrkB agonists. However, more recent publications question this hypothesis. In this study, we developed a set of experimental procedures including the evaluation of direct interactions, dimerization, downstream signaling, and cytoprotection in parallel with physicochemical and ADME methods to verify the pharmacology of 7,8-DHF and other potential reference compounds, and perform screening for novel TrkB agonists. 7,8 DHF bound to TrkB with K_d = 1.3 μM; however, we were not able to observe any other activity against the TrkB receptor in SN56 T48 and differentiated SH-SY5Y cell lines. Moreover, the pharmacokinetic and pharmacodynamic effects of 7,8-DHF at doses of 1 and 50 mg/kg were examined in mice after i.v and oral administration, respectively. The poor pharmacokinetic properties and lack of observed activation of TrkB-dependent signaling in the brain confirmed that 7,8-DHF is not a relevant tool for studying TrkB activation in vivo. The binding profile for 133 molecular targets revealed a significant lack of selectivity of 7,8-DHF, suggesting a distinct functional profile independent of interaction with TrkB. Additionally, a compound library was screened in search of novel low-molecular-weight orthosteric TrkB agonists; however, we were not able to identify reliable drug candidates. Our results suggest that published reference compounds including 7,8-DHF do not activate TrkB, consistent with canonical dogma, which indicates that the reported pharmacological activity of these compounds should be interpreted carefully in a broad functional context.

Background: Phosphodiesterase 10A (PDE10A) is expressed almost exclusively in the striatum and its inhibition is suggested to offer potential treatment in disorders associated with basal ganglia. We evaluated the selectivity, cytotoxicity, genotoxicity, pharmacokinetics and potential adverse effects of a novel PDE10A inhibitor, CPL500036, in vivo.Methods: The potency of CPL500036 was demonstrated by microfluidic technology, and selectivity was investigated in a radioligand binding assay against 44 targets. Cardiotoxicity in vitro was evaluated in human ether-a-go-go related gene (hERG)-potassium channel-overexpressing cells by the patch-clamp method and by assessing key parameters in 3D cardiac spheroids. Cytotoxicity was determined in H1299, HepG2 and SH-SY5Y cell lines. The Ames test was used for genotoxicity analyses. During in vivo studies, CPL500036 was administered by oral gavage. CPL500036 exposure were determined by liquid chromatography–tandem mass spectrometry and plasma protein binding was assessed. The bar test was employed to assess catalepsy. Prolactin and glucose levels in rat blood were measured by ELISAs and glucometers, respectively. Cardiovascular safety in vivo was investigated in dogs using a telemetry method.Results: CPL500036 inhibited PDE10A at an IC₅₀ of 1 nM, and interacted only with the muscarinic M2 receptor as a negative allosteric modulator with an IC₅₀ of 9.2 µM. Despite inhibiting hERG tail current at an IC₂₅ of 3.2 μM, cardiovascular adverse effects were not observed in human cardiac 3D spheroids or in vivo. Cytotoxicity in vitro was observed only at > 60 μM and genotoxicity was not recorded during the Ames test. CPL500036 presented good bioavailability and penetration into the brain. CPL500036 elicited catalepsy at 0.6 mg/kg, but hyperprolactinemia or hyperglycemic effects were not observed in doses up to 3 mg/kg.Conclusion: CPL500036 is a potent, selective and orally bioavailable PDE10A inhibitor with a good safety profile distinct from marketed antipsychotics. CPL500036 may be a compelling drug candidate.

Background Drugs that inhibit phosphodiesterase 10A (PDE10A) activity have shown efficacy in animal models. However, clinical trials of PDE10A inhibitors in patients with schizophrenia have failed to provide clinically significant results up to this point. Our study investigated whether alofropodect (previously referred to as CPL’36 or CPL500036), a PDE10A inhibitor with an improved pharmacological profile compared with other PDE10A inhibitors, including a fast dissociation rate and high activity, could improve symptoms of acute schizophrenia. Methods In this phase 2, randomised, double-blind, placebo-controlled, parallel-group trial (11 centres, three countries), investigators randomly assigned hospitalised patients with acute schizophrenia to treatment or placebo groups. The main inclusion criteria were age 18–65 years, a documented diagnosis of schizophrenia for at least 2 years before screening and a positive and negative syndrome scale (PANSS) total score ≥ 80. Participants were randomly assigned (1:1:1) to receive oral alofropodect (20 mg or 40 mg) or placebo administered once daily in the morning for 4 weeks. The primary endpoint of the study was an improvement in the PANSS positive subscale at week 4. The secondary endpoints included improvements in PANSS total and negative subscale scores, along with other efficacy and safety assessments. Clinical responders were defined as those with a ≥ 30% reduction in PANSS total score from baseline at week 4. Results Between 24 May 2021 and 8 May 2024, investigators randomised 189 patients into three groups: 58 into the alofropodect 20 mg group, 66 into the alofropodect 40 mg group and 65 into the placebo group. The mean age was 41.7 years (standard deviation (SD) 10.1), and 119 (63%) participants were men, 70 (37%) were women. All participants were White. We measured the difference in PANSS positive subscale before and after treatment, and the change met the primary endpoints at week 4 with the group where alofropodect 20 mg was compared with placebo (− 3.70 [90% CI − 5.51 to − 1.89]; p < 0.001) and alofropodect 40 mg versus placebo (− 6.35 [90% CI − 8.12 to − 4.57]; p = 0.001). Both the 20-mg and 40-mg doses of alofropodect were effective in improving the total PANSS score compared with placebo and met most secondary efficacy endpoints. A clinical response was observed in 2 of 53 patients (3.8%) in the placebo group, 8 of 52 patients (15.4%) in the 20 mg group and 22 of 52 patients (42.3%) in the 40 mg group at week 4. The compound was well tolerated, with only a few serious adverse events. The discontinuation rates were similar in the placebo and treatment arms. Extrapyramidal symptoms (EPS) occurred only in the active treatment groups, but rates were low overall. Somnolence was also reported only in alofropodect groups. Vital signs and biochemical parameters remained unaffected by either dose at any time point. Specifically, blood glucose, total cholesterol and triglycerides did not increase at any dose compared with placebo within our study window. Conclusions Alofropodect was effective in the treatment of acute schizophrenia symptoms and was well-tolerated, warranting further investigation in larger clinical studies. Clinical Trial Registration Clinicaltrials.gov identifier: NCT05278156; registration date: 2022-02-09.