Biomarker-Based Pharmacological Characterization of ENX-102, a Novel α2/3/5 Subtype-Selective GABAA Receptor Positive Allo-Steric Modulator: Translational Insights from Rodent and Human Studies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript, “ Biomarker-based pharmacological characterization of ENX-102, 4 a novel α2/3/5 subtype-selective GABAA receptor positive allosteric modulator: translational insights from rodent and human studies,” by Nettesheim et al., submitted to Cells, examines the behavioral and pharmacokinetic and pharmacodynamic effects of ENX-102 in rodents and humans. The findings are novel and provide more information on ENX-102 as a possible therapeutic agent for anxiety.

However, there are some concerns.

 

Methods: The methods section needs to be reorganized for better clarity and smoother flow. For example, on Page 4, the elevated maze section is the first behavioral test for animals, but it lacks detailed information. However, when you read the  Quantitative EEG  section, some details are included. There should be consistency of information provided in the method sections.   

 

• On Page 4, the elevated maze section should have included the animals' weights, how many animals per treatment group,  what is the vehicle was made of and is both ENX-102, and chlordiazepoxide were dissolved or mixed in the same vehicle, and how the vehicle, ENX-102, and chlordiazepoxide were administered to the animals.

 

• On page 4, Quantitative EEG section:  Are these the same animals used in the Elevated maze, or a different group?  If they are different groups, then this needs to be stated. The doses of the ENX-102 should be included.  

 

• On page 5, Study design section: The authors state that 40 male and female participants were involved. Is it 40 males and 40 females for a total of 80, or is it a total of 40 subjects altogether? The author should include the exact number of males and the exact number of females in the study.

 

 

• On Page 6, Dosing scheme:  The author needs to include how many days the drug was administered to participants with the dose escalation method.  The number of post-administration days should also be included, and the measurements that will be taken during the post-administration.

 

• On pages 6-7, for safety assessments and blood draws, were the participants housed in a clinical setting to get recordings, blood draws,  and NeuroCart CNS test battery measurements at the 10- and/or 12-hour time points? The clinical setting and the duration of patient stay in the clinical setting need to be included in the study population section.

 

 

Results:

• The authors need to include F-values and p-values for all the result sections.

 

• Fig 3. The authors should include the bar graphs for the subchronic effects of ENX-102 on EPM.

 

• The graphs are blurry, making some difficult to read. For instance, Fig. 4 has very small legends that remain hard to see even when zoomed to 200%. The authors should provide clearer, more legible versions of all the graphs.

 

• On pages 11-12, lines 433-436, the Participant disposition and demographics paragraph should be moved to the study population section in the methods.

 

• On page 15, line 513, Figure 7A needs to be changed to Figure 8A for SPV.

 

• On page 15, line 519, Figure 8 for adaptive tracking should be changed to Figure 8B. Plus, Figure S3B is qEEG in rats, not adaptive tracking.  The authors should double-check the text and all graphs to ensure the correct graph is being discussed and that the information in the text agrees with what is shown in the graphs.

 

• On page 15, the authors state that a 5 mg dose of ENX-102 5.0 mg statistically significantly increased SRT, but none of the graphs show Saccadic Reaction Time (SRT). Figure 8C only shows body sway. 

 

• On page 15, the authors state that a 5 mg dose of ENX-102 5.0 mg statistically significantly increased body sway on day 1, but Figure 8C does not have an asterisk that denotes this significance.

 

• On page 15, the authors state that a 1.5mg dose of ENX-102  on day 12 significantly increased body sway, but the graph 8C shows that the 1.5mg dose of ENX-102 appears to further decrease body sway compared to the vehicle.  

 

• In Figures 3, 4,5, and 8, the figure legends authors should add a brief statement about what the Asterisk indicates in the graph or what is being compared (e.g., * p < 0.05  indicates ENX-102  vs vehicle).  

 

• On Page 15, 537-541, the authors need to indicate where the reader can find the data for VVLT results.

 

• On Page 16, the Quantitative EEG: the authors state that detailed results of qEEG can be found in Table S6. However, 9  is not mentioned in the text. The authors need to clarify what data in the Quantitative EEG section is depicted in Figure 9 vs Table S6.

 

• Missing a table for the participants' demographics.

Discussion:

• On page 17, lines 591-594, add Figure 3 after (human equivalent dose - HED ~3 mg), so the readers are directed to EPM data.  

 

• On page 18, the authors state, “ ENX-102 1.0 mg and 5.0 mg reduced 667 SPV in the ranges of –52 to –76 and –45 to –50 degrees per second following single doses 668 and at steady state exposures, respectively.” Graph 8C and table S5 show that SPV  for a 1.0 mg dose stays basically the same (Day 1 =  46  and Day 12 =  51.59, as shown in Table S5). Plus, in table S5, the 5.0 mg dose day 12 data = 51.39, so it should be rounded up to 51.  The authors need to double-check the data.

 

The manuscript needs to be thoroughly checked for inconsistencies in results, graphs, etc.

 

Author Response

Dear reviewer,

Thank you for the constructive feedback on our manuscript titled “Biomarker-based pharmacological characterization of ENX-102, a novel α2/3/5 subtype-selective GABAA receptor positive allosteric modulator: translational insights from rodent and human studies.”

We have carefully reviewed all comments and suggestions provided by the reviewers and have revised the manuscript accordingly. Below, we address each of the concerns raised, along with our responses

Methods: The methods section needs to be reorganized for better clarity and smoother flow. For example, on Page 4, the elevated maze section is the first behavioral test for animals, but it lacks detailed information. However, when you read the  Quantitative EEG  section, some details are included. There should be consistency of information provided in the method sections.   

1. Reviewers comment: On Page 4, the elevated maze section should have included the animals' weights, how many animals per treatment group,  what is the vehicle was made of and is both ENX-102, and chlordiazepoxide were dissolved or mixed in the same vehicle, and how the vehicle, ENX-102, and chlordiazepoxide were administered to the animals.

Answer: The additional information has been added to the manuscript: “Rats (~ 260-300 grams prior to treatment) were randomly assigned to one of five treatment groups: vehicle, chlordiazepoxide (5 mg/kg, intraperitoneally (i.p.), positive control), or ENX-102 (0.1, 0.3, or 1.0 mg/kg, per os (p.o.)). Each group consisted of 10 rats, a sample size that was selected to ensure adequate statistical power. ENX-102 was formulated in 0.5% Tylose MH 300 solution and administered at a dose volume of 1.0 ml/kg; chlordiazepoxide was dissolved in water and administered at a dose volume”

2. Reviewers Comment: On page 4, Quantitative EEG section:  Are these the same animals used in the Elevated maze, or a different group?  If they are different groups, then this needs to be stated. The doses of the ENX-102 should be included.  

Answer: Different animal groups were used in the qEEG experiment vs. the Elevated plus maze experiment, this is now explicitly stated in the manuscript. Furthermore, the corresponding doses have been included in the figures (3A and 3B).

3. Reviewers comment: On page 5, Study design section: The authors state that 40 male and female participants were involved. Is it 40 males and 40 females for a total of 80, or is it a total of 40 subjects altogether? The author should include the exact number of males and the exact number of females in the study.

Answer: A short paragraph was added in the manuscript to clarify this: “Participant disposition and demographics: A total of 104 potential participants underwent screening, of whom 40 (18 female and 22 male) were randomized and completed the study. The consort diagram of randomized participants is shown in Figure S1. The mean (range) age and BMI were 30.5 years (19-52) and 25.2 kg/m2 (18.5 -34.7), respectively. Find more details on the demographics in Table S1.”

4. Reviewers comment: On Page 6, Dosing scheme:  The author needs to include how many days the drug was administered to participants with the dose escalation method.  The number of post-administration days should also be included, and the measurements that will be taken during the post-administration.

Answer: The drug was administered 12 days. This is now stated in the first sentence: “”All doses were administered as once daily as p.o. capsules for 12 days, with a fixed amount of 240 mL water intake in a fasted state (≥ 10h fasting).” Another sentence was moved and adjusted to make it clear that the escalation took place between each cohort and not within a cohort: “In the current MAD study, doses were subsequently escalated from 0.5 mg to 1.0 mg, 1.5 mg, 2.0 mg, and 5.0 mg during dedicated dose escalation meetings, following blinded review of all available PK, PD and safety data of each cohort.”

5. Reviewers comment: On pages 6-7, for safety assessments and blood draws, were the participants housed in a clinical setting to get recordings, blood draws,  and NeuroCart CNS test battery measurements at the 10- and/or 12-hour time points? The clinical setting and the duration of patient stay in the clinical setting need to be included in the study population section.

Answer: Two sentences were added to the section Study design section to clarify the reviewers comment: " Participants were admitted to the Centre for Human Drug Research (CHDR) in Leiden, the Netherlands, from Day -1 to Day 13. Safety, pharmacokinetic (PK), and pharmacodynamic (PD) assessments were performed during this period. Follow-up visits occurred on Day 19 and Day 26, during which only safety and PK assessments were conducted.”

 Results:

6. Reviewers comment: The authors need to include F-values and p-values for all the result sections.

Answer: We thank the reviewer for this valuable suggestion. We have now added the p-values, estimated differences, and corresponding 95% confidence intervals throughout the manuscript. While our initial intention was to refer readers to the relevant tables in order to maintain readability in the results section, we appreciate the importance of presenting key statistical outcomes directly in the text. We believe that reporting the estimated difference along with its 95% CI and p-value provides sufficient information for interpreting the results.

7. Reviewers comment: Fig 3. The authors should include the bar graphs for the subchronic effects of ENX-102 on EPM.

Answer: Fig 3. Has been updated to include the bar graphs for the subchronic effects of ENX-102 on EPM.

8. Reviewers comment: The graphs are blurry, making some difficult to read. For instance, Fig. 4 has very small legends that remain hard to see even when zoomed to 200%. The authors should provide clearer, more legible versions of all the graphs.

Answer: We thank the reviewer for bringing this to our attention. We have now updated this, and will submit all figures as high-resolution images.   

9. Reviewers comment: On pages 11-12, lines 433-436, the Participant disposition and demographics paragraph should be moved to the study population section in the methods.

Answer: The suggested changes by the reviewer have been implemented.

10. Reviewers comment: On page 15, line 513, Figure 7A needs to be changed to Figure 8A for SPV.

Answer: The suggested changes by the reviewer have been implemented.

11. Reviewers comment: On page 15, line 519, Figure 8 for adaptive tracking should be changed to Figure 8B. Plus, Figure S3B is qEEG in rats, not adaptive tracking.  The authors should double-check the text and all graphs to ensure the correct graph is being discussed and that the information in the text agrees with what is shown in the graphs.

Answer: The suggested changes by the reviewer have been implemented.

12. Reviewers comment:On page 15, the authors state that a 5 mg dose of ENX-102 5.0 mg statistically significantly increased SRT, but none of the graphs show Saccadic Reaction Time (SRT). Figure 8C only shows body sway. 

Answer: Figure 8C has been updated to include the SRT data.

13. Reviewers comment: On page 15, the authors state that a 5 mg dose of ENX-102 5.0 mg statistically significantly increased body sway on day 1, but Figure 8C does not have an asterisk that denotes this significance.

Answer: Figure 8C has been adjusted to reflect the significance.

14. Reviewers comment: On page 15, the authors state that a 1.5mg dose of ENX-102  on day 12 significantly increased body sway, but the graph 8C shows that the 1.5mg dose of ENX-102 appears to further decrease body sway compared to the vehicle.  

Answer: Thank you for pointing this out. The reviewer is correct - this should have been 'decreased' body sway. The manuscript has been corrected accordingly 

15. Reviewers comment: In Figures 3, 4,5, and 8, the figure legends authors should add a brief statement about what the Asterisk indicates in the graph or what is being compared (e.g., * p < 0.05  indicates ENX-102  vs vehicle).  

Answer: The legends of Figures 3, 4, 5, and 8 have been adjusted to reflect the reviewers request.

16. Reviewers comment: On Page 15, 537-541, the authors need to indicate where the reader can find the data for VVLT results.

Answer: The reference to Source Table S5 has been added in the manuscript.

17. Reviewers comment: On Page 16, the Quantitative EEG: the authors state that detailed results of qEEG can be found in Table S6. However, 9  is not mentioned in the text. The authors need to clarify what data in the Quantitative EEG section is depicted in Figure 9 vs Table S6.

Answer: Figure 9 is now described in the text: "Figure 9 illustrates the direction of EEG power               changes induced by ENX-102 in both humans and rats, measured under eyes-closed            conditions in the frontal cortex in rats and the fronto-central region in humans."

18. Reviewers comment: Missing a table for the participants' demographics.

Answer: Table demographics added as Supplementary Table S1.

Discussion:

19. Reviewers comment: On page 17, lines 591-594, add Figure 3 after (human equivalent dose - HED ~3 mg), so the readers are directed to EPM data.  

 Answer: The reviewers request has been implemented in the manuscript.

20. Reviewers comment: On page 18, the authors state, “ ENX-102 1.0 mg and 5.0 mg reduced SPV in the ranges of –52 to –76 and –45 to –50 degrees per second following single doses and at steady state exposures, respectively.” Graph 8C and table S5 show that SPV  for a 1.0 mg dose stays basically the same (Day 1 =  46  and Day 12 =  51.59, as shown in Table S5). Plus, in table S5, the 5.0 mg dose day 12 data = 51.39, so it should be rounded up to 51.  The authors need to double-check the data.

Answer: We appreciate the reviewer’s feedback. The manuscript indeed contained an incorrect statement. The reduction in SPV is greater on Day 1 compared to Day 12 only for dose levels ≥ 1.0 mg. As correctly noted, the SPV for the 1.0 mg dose remains unchanged. Moreover, statistically significant reductions in SPV on both Day 1 and Day 12 are observed only for the ENX-102 2.0 mg and 5.0 mg dose levels. This section has been revised accordingly in the manuscript:“SPV reductions following single doses on Day 1 were larger than those demonstrated at steady state exposures on Day 12 for ENX-102 1.5 mg and higher. For instance, ENX-102 2.0 mg and 5.0 mg reduced SPV in the range of –65.5 to –76.2 degrees per second following single doses, while at steady state exposures ENX-102 2.0 mg and 5.0 mg reduced SPV with –44.1 and –51.4 degrees per second, respectively.”

We believe these revisions address all reviewer concerns and improve the clarity and scientific rigor of the manuscript. Please find the revised manuscript and response document attached.

We appreciate the opportunity to revise our work and look forward to your feedback.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Review on the manuscript of Nettesheim P et al., (cells-3817231): “Biomarker-based pharmacological characterization of ENX-102, a novel α2/3/5 subtype-selective GABAA receptor positive allosteric modulator: translational insights from rodent and human studies”.

In this study, the Authors utilized a rat model and human subjects to pharmacologically characterize the positive allosteric modulator of the GABA_A receptor, ENX-102. They found that ENX-102 produced dose-dependent anxiolytic-like effects in rats following both acute and sub-chronic administration. It also induced a unique quantitative electroencephalography (qEEG) spectral signature, distinct from that of the benzodiazepine lorazepam. In a double-blind, placebo-controlled, multiple ascending dose study in healthy human subjects, ENX-102 increased the incidence of somnolence, reduced saccadic peak velocity (SPV), and affected measures of alertness and body sway. Additionally, qEEG data showed increased β-band power and decreased δ- and θ-band activity, differentiating ENX-102 from non-selective positive allosteric modulators of the GABA_A receptor.

Overall, I find this topic to be of great interest, as benzodiazepines are widely used and represent one of the most used therapies in the treatment of anxiety, insomnia, and related disorders. However, their broad action on GABA_A​ receptor subtypes is associated with significant side effects such as sedation, and cognitive impairment. As such, developing novel GABA_A​ receptor positive allosteric modulators with greater subtype selectivity offers the potential to retain therapeutic benefits as anxiolytics while minimizing the undesired effects.

I believe the Authors have addressed the primary question posed. Below, I indicate the issues identified in the current version of the manuscript. I hope the Authors find the following comments and suggestions helpful.

1 - In the Introduction section, the Authors include original results related to the molecule studied in this manuscript. I would kindly suggest moving these findings to the Results section, as it is more appropriate to present new data there. Typically, the Introduction should be reserved for background information and context, rather than describing original results.

2 - In general, the second part of the Introduction section (starting from “The pharmacodynamic (PD) characteristics of non-selective GABAAR agonists”) contains a high level of detail that could potentially be condensed. Reducing this section may help reduce the overall length of the Introduction, which could be beneficial.

3 - In the Materials and Methods section (subsection “Study population”), the Authors mention that individuals with a history of psychiatric disorders or convulsions were excluded from the study. However, there are several other important factors that should be considered. For instance, did the participants present with any significant medical conditions? Were they taking any medications, and if so, what types? What wre the inclusion criteria? These details should be clearly documented in the manuscript. Additionally, the reported BMI range of 18 to 35 suggests that some participants may have been classified as obese. It would be helpful to clarify whether these participants were evenly distributed across the different study groups.

4 - I kindly suggest that the Authors consider including the values corresponding to clordiazepoxide in the table shown in Figure 3B.

5 - In the manuscript, three different benzodiazepines were used in distinct sections. It would be helpful to understand whether there is a specific rationale for selecting different compounds. If possible, using a single benzodiazepine for all conditions might provide greater consistency. I kindly suggest that the Authors clarify their choice and provide a brief explanation in the text.

6 - I kindly suggest that the Authors consider increasing the size of the graphs shown in Figure 4. Additionally, in the current format, it is somewhat difficult to clearly distinguish the colors representing each condition (this issue could be bypassed by providing high-resolution figures).   

7 - I kindly suggest that the Authors consider renumbering the supplementary figures to align with the order in which they are mentioned in the text. For instance, the figure currently labeled as Figure S2 is cited first and might be more appropriately labeled as Figure S1. Additionally, Figure S5 is not cited in the text. I kindly recommend that the Authors clarify these issues.

8 - In the animal study, it is somewhat unexpected that the 0.1 mg/kg dose resulted in a Tmax of 4 hours, while the 0.32 mg/kg dose led to a shorter Tmax of 1.5 hours. Interestingly, increasing the dose further to 1 mg/kg brought the Tmax back to 4 hours (Table S1). The result observed at the 0.32 mg/kg dose is particularly intriguing. I kindly suggest that the Authors provide further clarification and, if possible, propose a hypothesis to help explain this finding.

9 - The left part of Table S3 is absent, making it difficult to fully assess its content. I kindly recommend that the Authors address this issue.

10 - In the Results section, the Authors mention: “Five participants experienced moderate AEs, accounting for one event each of gastroenteritis (reported 6 days after final dosing on Day 12), headache (reported 9 days after final dosing 444 on Day 12), balance disorder (reported on Day 1 of dosing), and muscle pain (reported on 445 Day 7 of dosing), and two events of somnolence (reported on Day 1 and Day 6 of dosing in two different participants), which together represents approximately 1.5% of the total 447 number of reported AEs”. I believe this sentence is not clear. For example, based on Table S3, 20 individuals experienced headaches. I kindly recommend that the Authors clarify these issues.

11 - Based on the data presented in Table S3 and Figure 6, there is a noticeable increase in somnolence among individuals treated with ENX-102, with 27 out of 30 participants (90%) reporting this effect, compared to 5 out of 10 participants (50%) in the placebo group. Given this difference, the columns in Table S3 referring to “All Subjects” may be less informative in this context. It may be more helpful to include a column showing the percentage of individuals in the ENX-102 groups who experienced each symptom. I kindly suggest that the Authors consider revising the table to reflect this.

12 - The graph shown in panel 7A may be somewhat misleading and could benefit from clarification. On Day 1, there is a clear increase in ENX-102 plasma concentrations, reflecting its absorption into the bloodstream, followed by a gradual decrease (consequent to its metabolism/elimination). However, from Day 2 to Day 12, the data is represented as a straight line, which does not accurately reflect the expected pharmacokinetic fluctuations due to ongoing absorption and elimination. Since there are no data points available for this interval, I kindly suggest that the Authors consider removing the connecting line between Day 2 and Day 12, as it may unintentionally give the impression of a linear and steady plasma concentration over time.

13 - Based on the data presented in Figure 8, ENX-102 on Day 1 was associated with decreases in SPV, body sway, and VAS alertness, which are indicative of sedative effects. These effects generally appeared to resolve by Day 12. This pattern might suggest that the benefits of ENX-102 are more pronounced with prolonged use rather than acute administration, despite the initial increase in symptom severity observed at the beginning of treatment. I kindly recommend that the Authors consider discussing these findings with this perspective in mind.

14 - I would kindly suggest that the Authors include the reference for the approval of the studies with animal and humans.

15 - The human study included only six subjects per group (the only exception is the placebo group that included 10 subjects), and there appears to be considerable heterogeneity among participants, particularly in terms of body mass index, which ranged from 18 to 35 kg/m². These factors represent important limitations that I kindly suggest the Authors address more thoroughly in the manuscript.

Author Response

We sincerely thank the reviewer for their thoughtful and encouraging feedback on our manuscript. We are pleased to hear that the topic and findings were found to be of interest, and we greatly appreciate the recognition of the potential impact of ENX-102 in advancing anxiolytic therapies with improved selectivity and safety profiles.

We have carefully addressed the comments and suggestions and have revised the manuscript to enhance clarity and consistency. Below, we provide a detailed, point-by-point response outlining the changes made.

We are grateful for the opportunity to improve our work and hope the revised version meets the reviewer’s expectations.

1. Reviewers Comment: In the Introduction section, the Authors include original results related to the molecule studied in this manuscript. I would kindly suggest moving these findings to the Results section, as it is more appropriate to present new data there. Typically, the Introduction should be reserved for background information and context, rather than describing original results.

Answer: Original results were removed from the Introduction and incorporated into the Methods and Results sections.

2. Reviewers Comment: In general, the second part of the Introduction section (starting from “The pharmacodynamic (PD) characteristics of non-selective GABAAR agonists”) contains a high level of detail that could potentially be condensed. Reducing this section may help reduce the overall length of the Introduction, which could be beneficial.

Answer: The Introduction was streamlined to reduce length and improve readability.

3. Reviewers Comment: In the Materials and Methods section (subsection “Study population”), the Authors mention that individuals with a history of psychiatric disorders or convulsions were excluded from the study. However, there are several other important factors that should be considered. For instance, did the participants present with any significant medical conditions? Were they taking any medications, and if so, what types? What wre the inclusion criteria? These details should be clearly documented in the manuscript. Additionally, the reported BMI range of 18 to 35 suggests that some participants may have been classified as obese. It would be helpful to clarify whether these participants were evenly distributed across the different study groups.

Answer: The main Inclusion and exclusion criteria have been added; full inclusion criteria are extensive but can be shared in the supplementals if needed. A demographics table has been added (Table S2.)

4. Reviewers Comment: I kindly suggest that the Authors consider including the values corresponding to clordiazepoxide in the table shown in Figure 3B.

Answer: Repeated daily handling of animals is known to reduce anxiety levels over time; therefore, the elevated plus maze is primarily suited for use in acute treatment paradigms. Chronic treatment with chlordiazepoxide has not been validated in this assay and, accordingly, could not be included as a positive control. We appreciate the reviewer’s point and, to address it have updated the figure to include bar graphs illustrating the effects under subchronic conditions. We have also noted this limitation in the revised manuscript.

5. Reviewers comment: In the manuscript, three different benzodiazepines were used in distinct sections. It would be helpful to understand whether there is a specific rationale for selecting different compounds. If possible, using a single benzodiazepine for all conditions might provide greater consistency. I kindly suggest that the Authors clarify their choice and provide a brief explanation in the text.

Answer: We understand the reviewer’s concern regarding the use of different benzodiazepines in the study. In the various preclinical assays conducted, benzodiazepines were employed primarily as positive comparators to establish assay sensitivity. Each assay had been previously validated with the respective benzodiazepine; therefore, their use was intended as positive reference control rather than to enable cross-study comparisons. Furthermore, the dosing ranges for the positive control have been selected based on the clinical effect. For example, lorazepam at 1 mg/kg has been shown to reliably induce NREM sleep changes in rodents [doi:10.1093/sleep/31.3.355] and to exert significant pharmacological effects on EEG activity [doi:10.1152/jn.00539.2013]. Similarly, chlordiazepoxide at 5 mg/kg is a widely used and well-characterized dose in elevated plus maze (EPM) studies in rats, demonstrating consistent anxiolytic effects [doi:10.1097/FBP.0000000000000423; doi:10.1159/000118478].

6. Reviewers Comment: I kindly suggest that the Authors consider increasing the size of the graphs shown in Figure 4. Additionally, in the current format, it is somewhat difficult to clearly distinguish the colors representing each condition (this issue could be bypassed by providing high-resolution figures).  

Answer: We thank the reviewer for bringing this to our attention. We have now updated this, and all figures as high-resolution images.  

7. Reviewers Comment: I kindly suggest that the Authors consider renumbering the supplementary figures to align with the order in which they are mentioned in the text. For instance, the figure currently labeled as Figure S2 is cited first and might be more appropriately labeled as Figure S1. Additionally, Figure S5 is not cited in the text. I kindly recommend that the Authors clarify these issues.

Answer: The reviewers request has been implemented.

8. Reviewers Comment: In the animal study, it is somewhat unexpected that the 0.1 mg/kg dose resulted in a Tmax of 4 hours, while the 0.32 mg/kg dose led to a shorter Tmax of 1.5 hours. Interestingly, increasing the dose further to 1 mg/kg brought the Tmax back to 4 hours (Table S1). The result observed at the 0.32 mg/kg dose is particularly intriguing. I kindly suggest that the Authors provide further clarification and, if possible, propose a hypothesis to help explain this finding.

Answer: It was indeed an interesting observation to note a faster Tmax at this dose, particularly since both the lower and higher doses demonstrated longer Tmax values. Data from our pharmacokinetic and toxicokinetic studies with ENX-102 indicate a Tmax range of 1–4 hours, and the observed Tmax of 1.5 hours falls well within this expected range. Moreover, we have generally observed that data from the low and high dose groups tend to show greater variability across studies, which may account for this finding. 

9. Reviewers Comment: The left part of Table S3 is absent, making it difficult to fully assess its content. I kindly recommend that the Authors address this issue.

Answer: Figure S3 has been updated to implement the reviewers request.

10. Reviewers Comment; In the Results section, the Authors mention: “Five participants experienced moderate AEs, accounting for one event each of gastroenteritis (reported 6 days after final dosing on Day 12), headache (reported 9 days after final dosing 444 on Day 12), balance disorder (reported on Day 1 of dosing), and muscle pain (reported on 445 Day 7 of dosing), and two events of somnolence (reported on Day 1 and Day 6 of dosing in two different participants), which together represents approximately 1.5% of the total 447 number of reported AEs”. I believe this sentence is not clear. For example, based on Table S3, 20 individuals experienced headaches. I kindly recommend that the Authors clarify these issues.

Answer: We clarified this part by rephrasing: These six moderate AEs represent approximately 1.5% of all post-dose AEs (6 of 408). To answer the reviewers question: We observed 6 moderate AEs (1x Gastro-enteritis, 1x balance disorder, 1x, 1x muscle ache, and 2x somnolence) in 5 participants. These are only the 6 moderate AEs, whereas Table S3 (in the updated manuscript table S4) shows all AE’s irrespective of mild or moderate severeness.

11. Reviewers Comment; Based on the data presented in Table S3 and Figure 6, there is a noticeable increase in somnolence among individuals treated with ENX-102, with 27 out of 30 participants (90%) reporting this effect, compared to 5 out of 10 participants (50%) in the placebo group. Given this difference, the columns in Table S3 referring to “All Subjects” may be less informative in this context. It may be more helpful to include a column showing the percentage of individuals in the ENX-102 groups who experienced each symptom. I kindly suggest that the Authors consider revising the table to reflect this.

Answer: The Table has been updated to implement the reviewers request.

12. Reviewers Comment:The graph shown in panel 7A may be somewhat misleading and could benefit from clarification. On Day 1, there is a clear increase in ENX-102 plasma concentrations, reflecting its absorption into the bloodstream, followed by a gradual decrease (consequent to its metabolism/elimination). However, from Day 2 to Day 12, the data is represented as a straight line, which does not accurately reflect the expected pharmacokinetic fluctuations due to ongoing absorption and elimination. Since there are no data points available for this interval, I kindly suggest that the Authors consider removing the connecting line between Day 2 and Day 12, as it may unintentionally give the impression of a linear and steady plasma concentration over time.

Answer: The graphs has been updated to implement the reviewers request.

13. Reviewers Comment: Based on the data presented in Figure 8, ENX-102 on Day 1 was associated with decreases in SPV, body sway, and VAS alertness, which are indicative of sedative effects. These effects generally appeared to resolve by Day 12. This pattern might suggest that the benefits of ENX-102 are more pronounced with prolonged use rather than acute administration, despite the initial increase in symptom severity observed at the beginning of treatment. I kindly recommend that the Authors consider discussing these findings with this perspective in mind.

Answer: A sentence is added in the discussion: "The fact that 12 days of dosing resulted in maintained SPV reduction is encouraging and supports further evaluation of ENX-102 for use in anxiety disorders, which are often persistent and require ongoing treatment."

14. Reviewers Comment: I would kindly suggest that the Authors include the reference for the approval of the studies with animal and humans.

Answer: The approval for the animal studies has been added.

15. Reviewers Comment: The human study included only six subjects per group (the only exception is the placebo group that included 10 subjects), and there appears to be considerable heterogeneity among participants, particularly in terms of body mass index, which ranged from 18 to 35 kg/m². These factors represent important limitations that I kindly suggest the Authors address more thoroughly in the manuscript.

Answer: A demographic table was added (Table S1) to display the different dosing groups and a sentence was added to clarify the distribution of the dosing groups: “In total, ENX-102 was administered across five cohorts, each consisting of eight healthy participants in a 6:2 ratio of active treatment to placebo. This resulted in six participants receiving ENX-102 at each dose level, while the ten placebo participants across all cohorts were pooled for analysis.”. We agree that the small sample size and heterogeneity are important limitations. While the inclusion criteria allowed for a broad BMI range (18–35 kg/m²), the actual BMI distribution among enrolled participants was narrower, as reflected in the demographics table. The modest sample size is consistent with a Phase 1 study, where the primary focus is on characterizing safety, tolerability, and pharmacokinetics rather than efficacy

We sincerely thank the reviewer for the thorough evaluation and constructive feedback. The suggested revisions have greatly improved the clarity and presentation of our manuscript. We hope that the changes made satisfactorily address all concerns and we respectfully submit the revised version for further consideration.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, the authors have addressed the concerns and improved the manuscript.  However, the asterisks in Fig. 4 need to be clearer because they are too light in color.

Author Response

Comment reviewer: Overall, the authors have addressed the concerns and improved the manuscript.  However, the asterisks in Fig. 4 need to be clearer because they are too light in color.

Answer: We thank the reviewer for their positive feedback and valuable suggestion. In response, we have enhanced the visibility of the asterisks in Fig. 4 by adjusting their color and contrast. Additionally, the corresponding figure in the supplemental materials (Fig. S3) has been updated in the same manner.

Reviewer 2 Report

Comments and Suggestions for Authors

Second review on the manuscript of Nettesheim P et al., (cells-3817231): “Biomarker-based pharmacological characterization of ENX-102, a novel α2/3/5 subtype-selective GABAA receptor positive allosteric modulator: translational insights from rodent and human studies”.

In this study, the Authors utilized a rat model and human subjects to pharmacologically characterize the positive allosteric modulator of the GABA_A receptor, ENX-102. They found that ENX-102 produced dose-dependent anxiolytic-like effects in rats following both acute and sub-chronic administration. It also induced a unique quantitative electroencephalography (qEEG) spectral signature, distinct from that of the benzodiazepine lorazepam. In a double-blind, placebo-controlled, multiple ascending dose study in healthy human subjects, ENX-102 increased the incidence of somnolence, reduced saccadic peak velocity (SPV), and affected measures of alertness and body sway. Additionally, qEEG data showed increased β-band power and decreased δ- and θ-band activity, differentiating ENX-102 from non-selective positive allosteric modulators of the GABA_A receptor.

This represents the second version of the manuscript after peer review. After a careful reading of the revised document, I would like to congratulate the Authors on the clarifications and improvements introduced. There are only a few minor points that could still be corrected.

1 - Some sentences in the manuscript appear in bold. I kindly suggest that the Authors review and correct this formatting issue.

2 - In line 595, where it reads “For VVLT performance was comparable to placebo…”, it would be more appropriate to write “VVLT performance was comparable to placebo…”.

3 - In line 595, where it reads “A Serious adverse event”, it would be more appropriate to write “A serious adverse event”.

Author Response

Comment reviewer: 

This represents the second version of the manuscript after peer review. After a careful reading of the revised document, I would like to congratulate the Authors on the clarifications and improvements introduced. There are only a few minor points that could still be corrected.

1 - Some sentences in the manuscript appear in bold. I kindly suggest that the Authors review and correct this formatting issue.

2 - In line 595, where it reads “For VVLT performance was comparable to placebo…”, it would be more appropriate to write “VVLT performance was comparable to placebo…”.

3 - In line 595, where it reads “A Serious adverse event”, it would be more appropriate to write “A serious adverse event”.

Answer: We sincerely thank the reviewer for their thoughtful feedback and kind words regarding the revised manuscript. We appreciate the time and care taken in reviewing our work and are grateful for the constructive suggestions. We have addressed the remaining minor points as follows:

1 - Formatting issue: We have carefully reviewed the manuscript and corrected the formatting issues, including the unintended bold text.

2 - The sentence has been revised to read “VVLT performance was comparable to placebo…” as suggested.

3 - The phrase has been corrected to “A serious adverse event” to ensure proper capitalization.