Development and Validation of an LC-MS/MS Method for the Quantitation of JNJ-64619178 (JNJ) in Mouse Plasma: Characterization of In Vitro and In Vivo Pharmacokinetic Properties

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article titled “Development and validation of a LC-MS/MS method for the quantitation of JNJ-64619178 (JNJ) in mouse plasma: Characterization of in vitro and in vivo pharmacokinetic properties” describes the development and validation of an LC-MS/MS method alongside in vitro and in vivo pharmacokinetic studies to characterize the systemic exposure and brain distribution of the PRMT5 inhibitor JNJ-64619178 in the context of medulloblastoma.

The introduction appropriately outlines the relevance of Group 3/MYC-driven medulloblastoma and PRMT5 as a therapeutic target. However, the novelty of this work could be stated more clearly. In particular, it would help to explain how the presented LC-MS/MS method and pharmacokinetic analysis extend beyond previously reported or partially described data for JNJ-64619178.

The choice of ESI positive mode, column conditions, and MRM transitions is explained adequately. However, the selection of oxibendazole as the internal standard would benefit from a short justification, particularly regarding its physicochemical similarity and chromatographic behavior relative to the analyte.

The validation experiments appear to follow FDA and ICH M10 guidance and include appropriate assessment of sensitivity, linearity, recovery, matrix effects, accuracy, precision, and stability. One aspect that may require additional clarification is the recovery values above 100% observed at MQC levels for both the analyte and internal standard. These results could indicate matrix-related signal enhancement and would benefit from additional discussion rather than simply being considered acceptable.

The conclusion that the compound does not undergo enzymatic hydrolysis or esterase-mediated degradation require further validation as additional controls, such as heat-inactivated matrices or esterase inhibitors are missing.

The in vitro DMPK characterization includes microsomal stability, protein binding, solubility, blood-to-plasma ratio, PAMPA permeability, and Caco-2 transport studies. While these data are useful, the discussion could better integrate them into a clearer ADME interpretation. For example, linking solubility and protein binding data to the observed oral bioavailability and tissue distribution would help strengthen the mechanistic understanding.

In the microsomal stability results, the relationship between in vitro predictions and the observed in vivo clearance could be explored further. A comparison between predicted hepatic clearance (from in vitro scaling) and measured systemic clearance would provide useful context and may help identify possible extrahepatic or transporter-related contributions.

The manuscript notes the very high protein binding in both plasma and brain (>94%). While this is acknowledged, its implications for free drug exposure are not fully examined. Presenting estimated unbound plasma and brain concentrations relative to the in vitro IC50 would help clarify the translational significance of the exposure levels.

The permeability data from PAMPA and Caco-2 assays suggest moderate passive permeability without strong efflux. However, the discussion could more clearly separate intrinsic permeability limitations from solubility or formulation-related constraints on oral absorption.

The in vivo pharmacokinetic analysis uses standard non-compartmental methods. The reported parameters are internally consistent with the in vitro stability findings. Nevertheless, the rationale for the selected doses (2.5 mg/kg IV and 10 mg/kg PO) could be described more clearly, especially in relation to expected therapeutic exposure levels in medulloblastoma models.

The key observation that brain concentrations remain below the in vitro IC₅₀ despite adequate systemic exposure is important. This point could be strengthened by presenting quantitative comparisons such as time above IC₅₀ in plasma versus brain, as well as explicit Kp,uu,brain values.

An interesting observation is that Kp,uu,brain appears low at 2 h but higher at 24 h. This pattern is somewhat unexpected and deserves further discussion. Possible explanations could include slow equilibration, binding kinetics in brain tissue, or analytical variability.

At several points, the manuscript alternates between reporting total and unbound concentrations without always clearly distinguishing between them. Consistent terminology and units would improve clarity, particularly when comparing in vivo exposure with in vitro potency values.

The discussion highlights the robustness of the analytical method but could include more critical comparison with other PRMT5 inhibitors or brain-penetrant epigenetic modulators. Such context would help readers better understand the relevance of these findings within the broader field.

The conclusion that solubility-limited absorption and low brain penetration represent major challenges is reasonable. However, this interpretation could be strengthened with more explicit quantitative support and consideration of other potential factors, such as blood-brain barrier transport mechanisms not captured in the current assays.

The translational implications for medulloblastoma could also be expressed more cautiously. Since the study does not evaluate pharmacodynamic effects or therapeutic efficacy in tumor models, the conclusions may be better framed in terms of enabling exposure assessment rather than supporting therapeutic validation.

Overall, the study provides a foundational analytical and pharmacokinetic foundation for investigating JNJ-64619178. Strengthening the discussion of unbound exposure, providing a clearer interpretation of brain penetration, and clarifying the novelty of the work relative to existing data would further improve the manuscript and its relevance to CNS-targeted drug development.

Comments for author File: Comments.pdf

Author Response

Reviewer 1: Comments and Suggestions for Authors

The article titled “Development and validation of a LC-MS/MS method for the quantitation of JNJ-64619178 (JNJ) in mouse plasma: Characterization of in vitro and in vivo pharmacokinetic properties” describes the development and validation of an LC-MS/MS method alongside in vitro and in vivo pharmacokinetic studies to characterize the systemic exposure and brain distribution of the PRMT5 inhibitor JNJ-64619178 in the context of medulloblastoma.

The introduction appropriately outlines the relevance of Group 3/MYC-driven medulloblastoma and PRMT5 as a therapeutic target. However, the novelty of this work could be stated more clearly. In particular, it would help to explain how the presented LC-MS/MS method and pharmacokinetic analysis extend beyond previously reported or partially described data for JNJ-64619178.

Response:

Thank you for the suggestion. The Introduction has been revised in lines 76-80 to better highlight the novelty of the work, comparing with the previously reported data.

The choice of ESI positive mode, column conditions, and MRM transitions is explained adequately. However, the selection of oxibendazole as the internal standard would benefit from a short justification, particularly regarding its physicochemical similarity and chromatographic behavior relative to the analyte.

Response:

We have added a justification describing physicochemical similarity, extraction recovery, and chromatographic behavior alignment with the analyte in lines 261-264.

The validation experiments appear to follow FDA and ICH M10 guidance and include appropriate assessment of sensitivity, linearity, recovery, matrix effects, accuracy, precision, and stability. One aspect that may require additional clarification is the recovery values above 100% observed at MQC levels for both the analyte and internal standard. These results could indicate matrix-related signal enhancement and would benefit from additional discussion rather than simply being considered acceptable.

Response:

That’s a great observation. We agree and have included additional discussion in lines 291-296 to address matrix-induced signal enhancement. This interpretation is supported by the matrix effect evaluation, where similar findings were observed. Importantly, the use of an internal standard compensates for such variability, and the method showed acceptable accuracy and precision across all QC levels (Table 2), indicating that the assay remains reliable despite this effect.

The conclusion that the compound does not undergo enzymatic hydrolysis or esterase-mediated degradation require further validation as additional controls, such as heat-inactivated matrices or esterase inhibitors are missing.

Response:

We removed our initial discussion on hydrolysis or esterase-mediated degradation in line 141, giving thought to the compound's structure. Esterase-mediated hydrolysis typically occurs when compounds contain ester bonds (–COOR) or sometimes amides (–CONH–). JNJ (Figure 6) lacks ester functionality and is more heterocyclic. This further aligns with the low metabolic clearance in the metabolic stability study discussed in line 149.

The in vitro DMPK characterization includes microsomal stability, protein binding, solubility, blood-to-plasma ratio, PAMPA permeability, and Caco-2 transport studies. While these data are useful, the discussion could better integrate them into a clearer ADME interpretation. For example, linking solubility and protein binding data to the observed oral bioavailability and tissue distribution would help strengthen the mechanistic understanding.

Response:

In section 2.5 (line 222) we have linked the in vivo clearance finding with the in vitro microsomal data. Also in the discussion part, we have mechanistically pointed out the correlation of solubility and permeability with the oral bioavailability result in lines 314-322.

In the microsomal stability results, the relationship between in vitro predictions and the observed in vivo clearance could be explored further. A comparison between predicted hepatic clearance (from in vitro scaling) and measured systemic clearance would provide useful context and may help identify possible extrahepatic or transporter-related contributions.

Response:

This is a great comment to add additional details. We have calculated predicted hepatic clearance using the relevant equation and provided the value in Table 4. We made major changes in the methods (lines 483-489), results (lines 158-160) and discussion (lines 308-313), highlighting the comparison between predicted hepatic clearance and measured systemic clearance in PK data.

The manuscript notes the very high protein binding in both plasma and brain (>94%). While this is acknowledged, its implications for free drug exposure are not fully examined. Presenting estimated unbound plasma and brain concentrations relative to the in vitro IC50 would help clarify the translational significance of the exposure levels.

Response:

We thank the reviewer for this insightful comment. We agree that unbound drug concentrations are critical for assessing pharmacological activity. However, the in vitro IC₅₀ used in this study was determined under cell culture conditions where protein binding is present and therefore reflects total drug concentrations rather than unbound (free) concentrations. We acknowledge that determining the protein binding in the culture media would suffice as the justification, and it is a study limitation.

Accordingly, comparison of total in vivo concentrations with the reported IC₅₀ provides a relevant assessment of pharmacological exposure. Nevertheless, to provide additional context, we have estimated unbound plasma and brain concentrations (Table 8) and discussed their potential impact on pharmacological interpretation in lines 337-341.

The permeability data from PAMPA and Caco-2 assays suggest moderate passive permeability without strong efflux. However, the discussion could more clearly separate intrinsic permeability limitations from solubility or formulation-related constraints on oral absorption.

Response:

We agree that distinguishing intrinsic permeability from solubility- or formulation-related limitations is important for accurately interpreting oral absorption. In our revised discussion, we have rearranged the sentence in line 316 and clarified that the observed low oral bioavailability is more likely driven by solubility-limited absorption or due to formulation properties rather than permeability constraints in line 320.

The in vivo pharmacokinetic analysis uses standard non-compartmental methods. The reported parameters are internally consistent with the in vitro stability findings. Nevertheless, the rationale for the selected doses (2.5 mg/kg IV and 10 mg/kg PO) could be described more clearly, especially in relation to expected therapeutic exposure levels in medulloblastoma models.

Response:

We selected 2.5 mg/kg IV and 10 mg/kg PO dose levels based on standard practices for exploratory pharmacokinetic studies in mice (Typical doses: IV 1 mg/kg, PO 5 mg/kg), as well as prior reports of JNJ-64619178 in preclinical models (Brehmer et al.). These dose levels are within commonly used ranges for early PK characterization and were intended to ensure measurable plasma concentrations across the sampling period. In addition, the oral dose was selected to achieve plasma concentrations within or approaching the range associated with in vitro activity in MYC-driven medulloblastoma cell lines.

The key observation that brain concentrations remain below the in vitro IC₅₀ despite adequate systemic exposure is important. This point could be strengthened by presenting quantitative comparisons such as time above IC₅₀ in plasma versus brain, as well as explicit Kp,uu,brain values.

Response:

We thank the reviewer for this valuable suggestion. In the revised analysis, we incorporated explicit comparisons of plasma and brain concentrations relative to the in vitro IC₅₀, as well as calculated K_p,uu,brain values in Table 8.

An interesting observation is that Kp,uu,brain appears low at 2 h but higher at 24 h. This pattern is somewhat unexpected and deserves further discussion. Possible explanations could include slow equilibration, binding kinetics in brain tissue, or analytical variability.

Response:

Added discussion based on the suggestion in lines 335-336.

At several points, the manuscript alternates between reporting total and unbound concentrations without always clearly distinguishing between them. Consistent terminology and units would improve clarity, particularly when comparing in vivo exposure with in vitro potency values.

Response:

This is an important observation. We agree that a consistent distinction between total and unbound concentrations is essential for clarity. We have clarified this throughout the manuscript.  In this study, total plasma and brain concentrations were measured using the validated solid phase extraction based LC-MS/MS method, which quantifies total drug concentration.

For comparison with in vitro potency, total concentrations were used in line 207, as the IC₅₀ values were determined in cell culture media where protein binding is present and therefore reflect total drug exposure rather than purely unbound concentrations.

In contrast, unbound concentrations were used when discussing brain distribution (e.g., Kp,uu,brain), as this parameter specifically reflects the pharmacologically active fraction available for tissue partitioning. We have explicitly mentioned “unbound” when unbound concentrations were used in the discussion, otherwise “concentration” simply indicated total concentration.

To improve clarity, we have revised section 2.5 and the discussion in line 327 and in 338 to distinguish between total and unbound concentrations.

The discussion highlights the robustness of the analytical method but could include more critical comparison with other PRMT5 inhibitors or brain-penetrant epigenetic modulators. Such context would help readers better understand the relevance of these findings within the broader field.

Response:

We added a contextual comparison in the discussion section in lines 342-349, contrasting JNJ with other PRMT5 inhibitors and highlighting its limitations in crossing the BBB.

The conclusion that solubility-limited absorption and low brain penetration represent major challenges is reasonable. However, this interpretation could be strengthened with more explicit quantitative support and consideration of other potential factors, such as blood-brain barrier transport mechanisms not captured in the current assays.

Response:

That’s an excellent point to add to the discussion. In response, we have revised the manuscript to (i) incorporate quantitative context where available (e.g., comparison of systemic and brain exposure relative to in vitro potency and estimated brain distribution), and (ii) acknowledge the potential contribution of blood–brain barrier (BBB) transport mechanisms, including active efflux processes, which are not fully captured by the current in vitro assays (PAMPA and Caco-2) (line 330-333 discussion)

The translational implications for medulloblastoma could also be expressed more cautiously. Since the study does not evaluate pharmacodynamic effects or therapeutic efficacy in tumor models, the conclusions may be better framed in terms of enabling exposure assessment rather than supporting therapeutic validation.

Response:

As we don’t have pharmacodynamic effects or therapeutic efficacy in tumor models, we have modified the text in line 324 – replace “therapeutically relevant exposure” by “exposure relative to IC₅₀ values”. In conclusion (line 583) we removed the “therapeutic translation”, framed the text in terms of achieving adequate exposure.

Overall, the study provides a foundational analytical and pharmacokinetic foundation for investigating JNJ-64619178. Strengthening the discussion of unbound exposure, providing a clearer interpretation of brain penetration, and clarifying the novelty of the work relative to existing data would further improve the manuscript and its relevance to CNS-targeted drug development.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript describes the validation of a bioanalytical LC-MS/MS method for the quantitation of a drug that is under investigation for the treatment of medulloblastoma.

The validated method was then applied to derive pharmacokinetic properties.

The experiments are well described and the discussion puts the results into perspective.

The authors can take into account some minor remarks for improvement of the text:

Please write “in vivo” and “in vitro” in italic font throughout the manuscript.

Please avoid the terminology “parent” ion throughout the manuscript, and replace by “product” ion.

Please avoid the terminology “protonated molecular ion” throughout the manuscript, and replace by “protonated molecule”.

Please write m/z in italic font throughout the manuscript.

Please make sure that the r2 value is called a determination coefficient; r is called a correlation coefficient.

The shoulder that can be observed in the peak of JNJ (Figure 1) raises the question whether the authors have determined the peak purity of this peak?

In Figure 2 the trace of the HLM Negative control (pink triangle) does not seem to be visible.

Line 168: add “a” before kinetic.

Please improve the phrasing of the sentence on line 213.

Line 322: the lowest working stock concentration mentioned here is 2 ng/mL, but the range reaches ten times lower.

Line 342: please define DL.

Author Response

Reviewer 2: Comments and Suggestions for Authors

This manuscript describes the validation of a bioanalytical LC-MS/MS method for the quantitation of a drug that is under investigation for the treatment of medulloblastoma.

The validated method was then applied to derive pharmacokinetic properties.

The experiments are well described and the discussion puts the results into perspective.

The authors can take into account some minor remarks for improvement of the text:

We thank the reviewer for all the comments and suggestions.

Please write “in vivo” and “in vitro” in italic font throughout the manuscript.

Response: Thank you for the suggestion. We have italicized throughout the manuscript and highlighted in the text.

Please avoid the terminology “parent” ion throughout the manuscript, and replace by “product” ion.

Response: We have modified the terminology to “precursor ion” and “product ion” marked as track changes.

Please avoid the terminology “protonated molecular ion” throughout the manuscript, and replace by “protonated molecule”.

Response: We have corrected it throughout the manuscript.

Please write m/z in italic font throughout the manuscript.

Response: Thank you for the comment. We have italicized m/z throughout the manuscript.

Please make sure that the r2 value is called a determination coefficient; r is called a correlation coefficient.

Response: changed to determination coefficient

The shoulder that can be observed in the peak of JNJ (Figure 1) raises the question whether the authors have determined the peak purity of this peak?

Response: We thank the reviewer for the comment. We agree that at LQC level, a slight rise or bump was observed. But, the minor shoulder observed is likely attributable to chromatographic effects rather than co-eluting species, as no corresponding signal was detected in blank matrices. In addition, the selected MRM transition was highly specific, and retention time was consistent across all samples. The observed peak shape, including the minor shoulder, did not impact peak integration or quantitation.

In Figure 2 the trace of the HLM Negative control (pink triangle) does not seem to be visible.

Response: Corrected the figure, and new figure has been inserted in line 163.

Line 168: add “a” before kinetic.

Response: Added

Please improve the phrasing of the sentence on line 213.

Response: We thank the reviewer for the observation. We modified the text (line 227).

Line 322: the lowest working stock concentration mentioned here is 2 ng/mL, but the range reaches ten times lower.

Response: Working stock solutions were subsequently diluted during spiking into matrix to keep the final dynamic range of quantitation from 0.2-500 ng/mL in matrix (10 µL spiked to the 100 µL matrix, thus 10X diluted and the final conc. mentioned is in the matrix.  This has been clarified in line 376.

Line 342: please define DL.

Response: Defined

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all comments from the previous reviewers, reflecting careful attention to detail and a commitment to manuscript improvement. The work now meets the expected standards of scientific quality and rigor for publication.

Author Response

We have now revised the manuscript as suggested. There is no SD in Table 8.  

Thank you, 

Nagendra