Safety, Tolerability and Pharmacokinetics of the Serotonin 5-HT6 Receptor Antagonist, HEC30654, in Healthy Chinese Subjects

Background and Objective: HEC30654 is a selective 5-HT6 receptor antagonist that was safe and well-tolerated in preclinical models of Alzheimer’s disease. The objective of this double-blind, randomized, placebo-controlled clinical trial was to evaluate the safety, tolerability, and pharmacokinetic profile of HEC30654 after single ascending doses in healthy Chinese subjects. Methods: Healthy volunteers received a single oral dose of HEC30654 (5, 10, 15, 30, 60 mg). Safety and tolerability assessments included adverse events, vital signs, and findings on electrocardiograms, electroencephalograms, physical examination, and clinical laboratory tests. Pharmacokinetic analysis of HEC30654 and its major metabolite HEC93263 were conducted in blood, urine, and fecal samples. Results: Single doses of HEC30654 up to 30 mg were generally safe and well tolerated, but dose escalation was terminated early as the 60 mg HEC30654 treatment group met the pre-defined stopping rules specified in the protocol. Median tmax of HEC30654 was 6 h (range, 4–12 h), t1/2 of 10–60 mg HEC30654 ranged from 52.1 to 63.8 h. Exposure to HEC30654 across the dose range explored in this study increased more than in proportion to dose. Metabolism of HEC30654 to HEC93263 was slow (<10%), and HEC30654 was mainly eliminated unchanged through feces. Conclusion: Single doses of HEC30654 up to 30 mg were generally safe and well tolerated. Based on preclinical efficacy in various models of cognition, HEC30654 may represent a therapeutic option for symptomatic treatment of cognitive disorders.


INTRODUCTION
Alzheimer's disease (AD) is a chronic neurodegenerative disease that manifests as a progressive impairment of memory and cognitive function, behavioral changes, and psychiatric symptoms, including depression and psychosis. The pathogenesis of AD remains to be elucidated, but involves amyloid plaques, neurofibrillary tangles, and profound neuronal loss (Upton et al., 2008;Ramírez, 2013;Folch et al., 2016;Sun et al., 2018). Currently available treatments for AD, such as acetylcholinesterase inhibitors (donepezil, rivastigmine, and galantamine) and the N-methyl-d-aspartate (NMDA) receptor antagonist memantine, help manage patients' symptoms but do not change or delay progression of the disease (Wicke et al., 2015;Ferrero et al., 2017). There remains a critical unmet clinical need for disease-modifying therapies (DMTs) that halt or slow the progression of AD. In preclinical studies, 5-HT6 receptor reportedly modulated the activity of cholinergic systems, and there is evidence suggesting that the blockade of 5-HT6 receptors induces acetylcholine release, it became apparent that 5-HT6 antagonism may be a promising approach to restore acetylcholine levels in a deteriorated cholinergic system (Ramírez, 2013).
Recently, 5-HT 6 receptor antagonists have emerged as potential treatment strategies in AD, with some 5-HT 6 receptor antagonists, such as Lu-AE58054 (idalopirdine) and masupirdine , being investigated in Phase II or Phase III clinical trials (Wilkinson et al., 2014;Khoury et al., 2018;Frölich et al., 2019;Nirogi et al., 2019;Nirogi et al., 2020). In these clinical studies, 5-HT 6 receptor antagonists were usually used as an adjunct to combination therapy (cholinesterase inhibitor and memantine) in patients with AD (Khoury et al., 2018). The results shown that idalopirdine was not effective for AD patients as a whole, although idalopirdine might be more effective at high doses and in moderate AD subgroups (Matsunaga et al., 2019). In a triple therapy with masupirdine added to background dual combination treatment with donepezil and memantine, the treatment significantly reduced the behavioral and psychological symptoms, with positive effect on cognition (Nirogi et al., 2020).
In rats and dogs (beagles), HEC30654 had a terminal half-life (t 1/2 ) of 4-8 h after intravenous administration, and bioavailability of HEC30654 after oral administration was 12.2% or 51%, respectively. In animals and humans, HEC30654 had a plasma protein binding rate of >93%. Previous studies revealed that phase 1 metabolism of HEC30654 may involve CYP3A4, CYP2D6, and CYP2B6. In rats, mean total recovery of radioactivity in urine and fecal samples after a single oral administration of 89.7 μCi/ 6.04 mg/kg [ 14 C] HEC30654 was 95.0%, with urine and feces accounting for 2.46 and 91.0%, respectively.
The present study aims to evaluate the safety, tolerability, and PK profile of HEC30654 after single ascending doses in healthy Chinese subjects.

Study Subjects
Healthy Chinese volunteers were eligible to participate in this study. Key inclusion criteria were: 1) males and females (not pregnant or lactating) aged 18-45 years; 2) body mass index (BMI) 18-28 kg/m 2 (body weight: males, ≥ 50 kg, females, 45 kg); and 3) physical examination, medical history, and clinical laboratory test results showed no clinically significant abnormal findings at screening. Key exclusion criteria were: 1) long-term history of smoking or alcohol and/or drug abuse; 2) use of any medication during the 14 days prior to the initial dose of study drug or while participating in the study; 3) blood donation during the 3 months prior to the study; 4) exposure to any CYP3A4, P-gp or Bcrp inducers or inhibitors during the 3 months prior to the initial dose of study drug; or 5) participation in a clinical trial of another investigational drug during the 3 months prior to this study. This single ascending dose, double-blind, randomized, placebo-controlled clinical trial investigated the safety, tolerability, and PK of HEC30654 in healthy subjects. The study planned to randomize eligible healthy subjects to one of seven treatment groups. In the first treatment group, three sentinel subjects were to receive a single oral dose of 5 mg HEC30654. In the six remaining treatment groups, ten subjects were to receive a single oral dose of 10, 15, 30, 60, 90 or 120 mg HEC30654 (n 8 per group) or matching placebo (n 2 per group) after an overnight fast.

Study Design
Dose escalation was dependant on the safety and tolerability of the previous doses defined according to the National Cancer Frontiers in Pharmacology | www.frontiersin.org August 2021 | Volume 12 | Article 726536 Institute Common Terminology Criteria for the Classification of Adverse Events (NCI CTCAE) v.4.03. Dose escalation was stopped based on the occurrence of the following drug-related adverse events (AEs): >50% of subjects in the previous treatment group experienced Grade ≥2 drug-related toxicities, or >25% of subjects in the previous treatment group experienced Grade 3-4 drug-related toxicities, or one drug-related serious adverse event (SAE). The trial was terminated early with subjects in five treatment groups receiving a single dose of 5, 10, 15, 30, or 60 mg HEC30654.

Safety Assay
Safety and tolerability, including AEs, vital signs (body temperature, blood pressure at rest, heart rate, and respiratory rate), and findings on electrocardiograms, electroencephalograms, physical examination, and clinical laboratory tests (biochemistry, hematology, urinalysis, coagulation, and immune globulin) were evaluated according to NCI-CTCAE, 4.03. Incidence, severity, and relationship of AEs to study drug were recorded. Tolerability was evaluated on Day 2 and Day 6 for subjects administered 5, 10, 15, or 30 mg HEC30654, and Day 2, Day 6, and Day 10 for subjects administered 60 mg HEC30654.

Statistical Analysis
Standard non-compartmental methods using WinNonlin version 6.4 (Certara United States of America Inc,.) were used to analyze plasma PK data, including peak plasma concentration (C max ), time to peak plasma concentration (T max ), area under the plasma concentration-time curve from time 0 to the last timepoint with a quantifiable concentration (AUC 0-t ), AUC from time 0 to infinity (AUC 0-∞ ), t 1/2 , clearance (CL/F), and apparent volume of distribution (Vz/F).
Statistical analysis was performed using SAS software, version 9.4 (SAS Institute Inc., United States). Continuous variables were reported as number, mean with standard deviation, median, maximum, and minimum. Categorical variables, were reported as frequency and percentage. Dose proportionality was explored using a regression power model, relating log-transformed C max and AUC and log-transformed dose.

Baseline Characteristics
This study included a total of 43 healthy subjects enrolled into five treatment groups. Baseline demographic characteristics were well balanced across the treatment groups ( Table 1). All subjects completed the study and were included in the safety, tolerability, and PK analyses.
All the subjects in 60 mg group occurred grade ≥2 drug related AEs, and three of them occurred grade ≥3 drug related AEs ( Table 3). As the 60 mg HEC30654 treatment group met the predefined stopping rules specified in the protocol, the study was terminated early.
HEC30654 and HEC93263 were detected in urine and fecal samples. The percentage cumulative recovery of HEC30654 in the urine and feces from 0 to 216 h was 0.998 and 21.4%, respectively. The percentage cumulative recovery of HEC93263 in the urine and feces from 0 to 216 h was low (<0.1%) ( Table 5).

DISCUSSION
This Phase I clinical trial was designed to characterize the safety, tolerability, and PK of HEC30654 in healthy Chinese volunteers.
HEC30654 was safe and well-tolerated in preclinical animal studies. The non observed adverse effect level (NOAEL) in rat and dog were 34.5 mg/kg and 2.3 mg/kg, corresponding to maximum recommended starting doses (MRSD) of 29.2 and 6.3 mg in humans, considering a safety factor of 10. The initial effective dose in rats was 1.5 mg/kg/day, and according to physiologically based PK modeling, the initial effective dose in humans was 5 mg/ day. The maximal tolerated dose (MTD) in rat and dog were 690 mg/kg and 1,150 mg/kg, corresponding to MTDs of 6,624 mg   (h) 6 (6-6) 6 (6-8) 6 (6-12) 6 (6-8) 6 (4-6) C max (ng/ml) 0.937 (1.2) 2.65 (9.9) 3.24 ( T max , time to peak plasma concentration; C max , peak plasma concentration; AUC 0-t , area under the plasma concentration-time curve from time zero to time t; AUC 0-a , area under the plasma concentration-time curve from time zero to infinity; t 1/2 , terminal elimination half-life; Vz/F, apparent volume of distribution; CL/F, apparent clearance; CV%, percentage coefficient of variation.