Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
This study explores the therapeutic potential of KMO inhibition in endometriosis, a condition with limited treatment options.
Strengths:
KNS898 is a novel specific KMO inhibitor and is orally bioavailable, providing a convenient and non-hormonal treatment option for endometriosis. The promising efficacy of KNS898 was demonstrated in a relevant preclinical mouse model of endometriosis with pathological and behavioural assessments performed.
Weaknesses:
(1) The expression of KMO in human normal endometrium and endometrial lesions was not quantified. Western blot or quantification of IHC images will provide valuable insight.
Given the differential expression of KMO in luminal epithelial cells lining the endometrial glands compared to the other parts of the endometrium, a general endometrial Western Blot prep is not going to be additionally helpful or accurate in addressing this question, without e.g. laser capture microdissection or single cell quantitative proteomics. Furthermore, KMO is a flavin-dependent monooxygenase and the activity, especially generating the oxidative stressor product 3-hydroxykynurenine is far more dependent on kynurenine substrate availability than it is on actual enzyme abundance - although it is important to show (as we have done), that KMO is present in the human endometrial glands and in human distended endometrial gland-like structures (DEGLS).
If KMO is not overexpressed in diseased tissues i.e. it may have homeostatic roles, and inhibition of KMO may have consequences on general human health and wellbeing.
KMO certainly does have important homeostatic roles, for example as key step in the repletion of NAD+ through de novo synthesis. Although with good nutrition and sufficient NAD+ precursors in the diet e.g. niacin, that specific role may be partially redundant. KMO knockout mice exhibit normal fertility and fecundity and do not show a survival deficit compared to littermate wildtype controls (e.g. Mole et al Nature Medicine 2016). To further develop KNS898 towards clinical use, preclinical GLP safety and toxicology studies and human Phase 1 clinical trials will of course need to be completed, but that is standard for the development of any new drug
In addition, KMO expression in control mice was not shown or quantified.
Control mice that were not inoculated intraperitoneally with endometrial fragments did not develop DEGLS and therefore there is nothing to show or quantify.
Images of KMO expression in endometriosis mice with treatments should be shown in Figure 4.
We have now included a representative KMO immunohistochemistry image from each endometriosis group and included all KMO immunohistochemistry images in Supplementary Information.
The images showing quantification analysis (Figure 4A-F) can be moved to supplementary material.
This recommendation contradicts the emphasis placed by the same reviewer earlier regarding quantification, so we have elected to keep it where it is.
(2) Figure 1 only showed representative images from a few patients. A description of whether KMO expression varies between patients and whether it correlates with AFS stages/disease severity will be helpful. Images from additional patients can be provided in supplementary material.
We have added extra information to the Figure legend to clarify the disease stage of the superficial peritoneal lesions which were illustrated (Stage I/II) and to link them to the information in supplementary Table S1. In total we examined 11 peritoneal lesions and 5 ovarian lesions (stage III/IV) – in every sample examined immunopositive staining was most intense in epithelial cells lining gland-like structures. Sections illustrated were chosen to illustrate this key finding.
(3) For Home Cage Analysis, different measurements were performed as stated in methods including total moving distance, total moving time, moving speed, isolation/separation distance, isolated time, peripheral time, peripheral distance, in centre zones time, in centre zones distance, climbing time, and body temperature. However, only the finding for peripheral distance was reported in the manuscript.
This was indeed a large amount of output, which we rationalised for the benefit of a concise paper. The paper now includes a description of which parameters showed a difference with drug treatment.
(4) The rationale for choosing the different dose levels of KNS898 - 0.01-25mg/kg was not provided. What is the IC50 of a drug?
KNS898 dosing has been extensively characterised by us in multiple species, and the pIC50 has already been published (e.g. Hayes et al Cell Reports 2023 and elsewhere). We now include the pIC50 in the present manuscript to save the reader from having to search through another reference.
(5) Statistical significance:
(a) Were stats performed for Fig 3B-E?
Now included, thank you.
(b) Line 141 - 'P = 0.004 for DEGLS per group'
However, statistics were not shown in the figure.
Thanks, now displayed on figure.
(c) Line 166 - 'the mechanical allodynia threshold in the hind paw was statistically significantly lower compared to baseline for the group'
However, statistics were not shown in the figure.
(d) Line 170 - 'Two-way ANOVA, Group effect P = 0.003, time effect P < 0.0001' The stats need to be annotated appropriately in Figure 5A as two separate symbols.
Arguably the far more important comparison in this figure is whether there is any effect of treatment, and to mark multiple statistical comparisons on the figure would make it difficult to understand. Instead, the figure legend and results text have been clarified on this point.
(e) Figure 5B - multiple comparisons of two-way ANOVA are needed. G4 does not look different to G3 at D42.
Multiple comparison testing (Dunnett’s T3) was done and the results have been clarified in the text and figure legends.
(f) Line 565 - 'non-significant improvement in KNS898 treated groups'. However, ** was annotated in Figure 5A.
Thank you. This is an error that has been checked and corrected.
(6) Discussion is very light. No reference to previous publications was made in the discussion. Discussion on potential mechanistic pathways of KYR/KMO in the pathogenesis of endometriosis will be helpful, as the expression and function of KMO and/or other metabolites in endometrial-related conditions.
The discussion is deliberately concise and focussed. The paper has 21 references to previous publications. A speculative discussion is generally not favoured by us.
The findings in this study generally support the conclusion although some key data which strengthen the conclusion eg quantification of KMO in normal and diseased tissue is lacking.
We differ from the reviewer here and do not think that those data would materially affect the likelihood of KMO inhibition being efficacious in human endometriosis in Phase 2/3 clinical trials.
Before KMO inhibitors can be used for endometriosis, the function of KMO in the context of endometriosis should be explored eg KMO knockout mice should be studied.
We take the view that before KMO inhibitors can be used for endometriosis in patients there are multiple other regulatory and clinical development steps that are required that would be a priority. While using a KMO knockout mouse might be an interesting scientific experiment, it would not impact on the critical path in a material way.
Reviewer #2 (Public Review):
Summary:
The authors aim to address the clinical challenge of treating endometriosis, a debilitating condition with limited and often ineffective treatment options. They propose that inhibiting KMO could be a novel non-hormonal therapeutic approach. Their study focuses on:
• Characterising KMO expression in human and mouse endometriosis tissues.
• Investigating the effects of KMO inhibitor KNS898 on inflammation, lesion volume, and pain in a mouse model of endometriosis.
• Demonstrating the efficacy of KMO blockade in improving histological and symptomatic features of endometriosis.
Strengths:
• Novelty and Relevance: The study addresses a significant clinical need for better endometriosis treatments and explores a novel therapeutic target.
• Comprehensive Approach: The authors use both human biobanked tissues and a mouse model to study KMO expression and the effects of its inhibition.
• Clear Biochemical Outcomes: The administration of KNS898 reliably induced KMO blockade, leading to measurable biochemical changes (increased kynurenine, increased kynurenic acid, reduced 3-hydroxykynurenine).
Weaknesses:
• Limited Mechanistic Insight: The study does not thoroughly investigate the mechanistic pathways through which KNS898 affects endometriosis. Specifically, the local vs. systemic effects of KMO inhibition are not well differentiated.
While we agree that this is not a comprehensive mechanistic analysis, given that the ultimate therapy would be almost certainly a once daily oral dosing i.e. systemic administration, we do not consider differentiating local vs systemic effects of KMO inhibition to be critical to therapeutic development in this scenario.
• Statistical Analysis Issues: The choice of statistical tests (e.g., two-way ANOVA instead of repeated measures ANOVA for behavioral data) may not be the most appropriate, potentially impacting the validity of the results.
The selection of two-way ANOVA (time and group) is sufficient and correct for this experimental analysis and its use does not invalidate the results. We agree that repeated measures ANOVA could be a valid alternative.
• Quantification and Comparisons: There is insufficient quantitative comparison of KMO expression levels between normal endometrium and endometriosis lesions,
Please see response above to quantification question raised by Reviewer 1.
and the systemic effects of KNS898 are not fully explored or quantified in various tissues.
Please see earlier responses. KNS898 has been thoroughly explored in multiple tissues, species and experimental models, but those data do not need rehearsed here.
• Potential Side Effects: The systemic accumulation of kynurenine pathway metabolites raises concerns about potential side effects, which are not addressed in the study.
As discussed above (response to Reviewer 1), KMO knockout mice exhibit normal fertility and fecundity and do not show a survival deficit compared to littermate wildtype controls (e.g. Mole et al Nature Medicine 2016). To further develop KNS898 towards clinical use, preclinical GLP safety and toxicology studies and human Phase 1 clinical trials will naturally need to be completed, but this is standard for the development of any new drug.
Achievement of Aims:
• The authors successfully demonstrated that KMO is expressed in endometriosis lesions and that KNS898 can induce KMO blockade, leading to biochemical changes and improvements in endometriosis symptoms in a mouse model.
Support of Conclusions:
• While the data supports the potential of KMO inhibition as a therapeutic strategy, the conclusions are somewhat overextended given the limitations in mechanistic insights and statistical analysis. The study provides promising initial evidence but requires further exploration to firmly establish the efficacy and safety of KNS898 for endometriosis treatment.
We do not agree that the conclusions are overextended based on the data presented, as expanded in the reply to the eLife editorial assessment at the beginning of this response. It is clear that additional preclinical, regulatory and clinical development work, and human clinical trials will be required to firmly establish the efficacy and safety of KN898 for endometriosis treatment.
Impact on the Field:
• The study introduces a novel therapeutic target for endometriosis, potentially leading to non-hormonal treatment options. If validated, KMO inhibition could significantly impact the management of endometriosis.
Utility of Methods and Data:
• The methods used provide a foundation for further research, although they require refinement. The data, while promising, need more rigorous statistical analysis and deeper mechanistic exploration to be fully convincing and useful to the community.
We believe that the data are a) convincing, and b) useful to the community. To be advanced effectively towards patients, KNS898 needs to follow the critical development path outlined above.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
(1) Change 'hyperalgia' to hyperalgesia throughout the manuscript including the title.
Done
(2) Line 69 - write '3-HK' in full.
Done
(3) Line 85 - the findings of the study include 'define the preclinical efficacy of KNS898 in reducing inflammation'. The inflammatory profile was not studied.
Changed to “disease”
(4) Line 259 - write 'EPHect' in full.
Done
(5) Line 260 - write 'AFS' in full. Also, abbreviate 'AFS' in the caption of Table S1.
Done
(6) 20 patients were listed in Table S1 but only 19 were accounted for in the methods section.
Apologies there was an error and has now been corrected in the methods section as one of the endometrial samples had not been included. Table S1 has also been changed to make it clear which samples were eutopic endometrium to differentiate them from the lesions.
(7) The location from which the endometrial lesion tissues were obtained should be provided in Table S1.
Table S1 has been changed to make it clear that the subtypes of lesions examined were classified as Stage I/II – superficial peritoneal subtype and Stage III/IV – endometrioma. The methods section has also been updated to reflect these subtypes (lines 272-277).
(8) Table S2 - G5 should be given compound 'A' not 'B'.
Thank you. Corrected.
(9) Figure 2E was not referenced in the text and no figure legend was provided.
Now referenced and the figure legend updated.
(10) Figure 3A - font needs to be enlarged. HCA baseline recording was annotated as performed twice in the protocol. When is the baseline taken and on what day was the Week 12 measurement taken (refer to Figures 5C and D)?
Font has been enlarged as requested. The second HCA baseline annotation in Fig 3A is a cut-and-paste error, now rectified and the time of second measurement annotated.
(11) Line 133 - 'In KNS898-treated group G4 (endometriosis + treatment from Day 19), DEGLS formed in 4 of 15 mice (26.7%) and in G5 (Endo + treatment start on Day 26) in 6 of 15 mice (40%) (Fig. 3f).'. The aforementioned data is not reflected in Figure 3F.
Thank you. This has been rectified.
(12) Line 137 - 'Mice with endometriosis receiving KNS898 from the time of inoculation (G4) had an average of 2.0 DEGLS per animal with DEGLS (total = 8 DEGLS in 4 mice in G4) and those receiving KNS898 1 week after inoculation (G5) had an average of 1.8 DEGLS per animal (total = 11 DEGLS in 6 mice in G5) (Figs. 3g and 3h).'
The aforementioned data is not reflected in Figure 3G. There is no Figure 3H shown.
Rectified as above.
(13) Provide a discussion of why KA levels were significantly lower in Figure 3E compared to Figure 2C.
(14) Figure legend for Figure 3 - G1 and G2 were noted as n=8. However, Figure S1 and Table S2 noted both groups as n=10.
Thank you. This is a typographical error. The legend for Fig 3 should indeed read n=10 for G1 and G2 and has been corrected.
(15) Line 181 - 'compared to non-operated and sham-operated control groups'. Only the sham group was shown in Figures 5C and D.
This text has been clarified to refer only to the data shown.
(16) Figure 1 images need scalebars. Same for Figure 4.
Now added
(17) Figure 3B - y-axis is fold change?
Relative concentration. Legend has been clarified.
(18) Figures 5A and B - are the last Von Frey measurements taken on Day 40 (as per Figure 3A) or 42?
Taken on Day 42. Fig 3A (the prospective protocol figure) has been clarified to reflect what actually happened (D42) as opposed to what was planned (D40) to pre-empt any further confusion.
(19) Symbols in Figure S1 need to be explained in the Figure legend.
Done
(20) Figures 2A and 2D should not be plotted in log scale to match the description of results in Line 106 and Line 118.
These particular results are plotted on a log scale to allow the reader to visualise that detectable levels of drug are measurable at very low doses and that there is no significant pharmacodynamic effect at that low dose. We choose to retain the present format.
Reviewer #2 (Recommendations For The Authors):
Comments and queries
Introduction/aims section:
Line 82 - 87: Clarify in the proposal aims what is being accessed and analysed in humans and/or in animal models (mice). Specifically state clearly the correlations with KMO expression. Were the correlations between KMO expression with features of inflammation performed only in mice or also in humans?
Thank you for this comment. The aims have been clarified in the Introduction.
Section - KMO is expressed in human eutopic endometrium and human endometriosis tissue lesions:
Was any quantitative or semi-quantitative method used to quantify the KMO expression in human tissues? Although the authors claimed that "KMO was strongly immunopositive in human peritoneal endometriosis lesions" by the representative figures it is not clear if KMO expression is similar, higher or lower between normal endometrium and peritoneal endometriosis lesions.
We have added extra information to the legend of Figure 1 to identify the PIN number of the superficial lesions illustrated. The key finding from the immunostaining with the antibody which had been previously validated as specific for KMO was that the most intense immunopositive response was in glandular epithelial cells and the samples illustrate this result.
Section - Oral KNS898 inhibits KMO in mice:
The authors clearly confirmed the target engagement of KNS898 in inhibiting KMO activity and, therefore, affecting upstream and downstream metabolites systemically in (peripheral fluid/ plasma) mice. Whether KNS898 effect is broad and targets systemic immune cells and whole body cells and tissue was not explored. It was also not explored if KNS898 is able to specifically inhibit KMO locally at the endometrium tissue by targeting epithelial and/or infiltrated immune cells, for example.
That is correct.
It would be interesting to measure (or if it was measured to report in this section and also in Figure 2) the levels of KYN, KA and 3HK in naïve animals that did not receive KNS898. It would help to understand the net effect of KNS898 on the levels of kynurenine pathway metabolites and, therefore, justify the dose chosen.
These data are already presented in Fig 3B-E, control group.
Perhaps then the chosen dose could be lower considering the possible substantial changes in kynurenine pathway metabolites levels, which are reported to exert an effect in many cells, tissues and systems and could, therefore, precipitate side effects. Even more considering that the values for these metabolites are expressed as ng/ml, which hinders the comparison of the metabolite levels with the one reported for naïve animals in the literature. I would also suggest expressing the metabolite levels as nM/L.
This is not a relevant method of determining dose-limiting toxicity or safety pharmacology/toxicology, either non-GLP or GLP. There are international guidelines on the proper conduct of those studies. This is also why it is important not to make claims about the safety or otherwise of an experimental compound in an in vivo setting that has not explicitly complied with those regulatory standards. With regard to the units recommendation, accepted units are ng/mL or nM, not usually nM/L.
Section - KMO blockade reduces endometrial gland-like lesion burden in experimental endometriosis in mice:
Line 130: It would be better to replace "blockade of 3HK production" with "reduction of 3HK production" to better reflect the results.
Changed to “inhibition of 3HK production”.
Line 140: In G5 (treatment starting at Day 26/ 1 week after inoculation), is the experimental model of endometriosis already established with all pathological and phenotypic features?
This was not specifically tested in this experiment.
Lines 146 - 148: It would be better to specify that "Overall, there was no significant difference IN BODY WEIGHT between G3 and the KNS898 treatment groups G4 and G5 (endometriosis + treatment from Day 26)". Otherwise, this last sentence might be interpreted as the overall conclusion of this result sub-section.
Thank you, a good point and has been corrected.
The authors demonstrated with an experimental approach that KMO blockade reduces a pathological measure of endometriosis i.e., endometrial gland-like lesion burden, in experimental endometriosis in mice when both administrated concomitant but also after the disease development. Although mechanistic insights about how reduced KMO activity can reduce the developed distended endometrial gland-like structures were not explored. Therefore, it remains to be investigated which (and how ) kynurenine pathway metabolites are directly linked to the beneficial effects of KMO blockade in the experimental model of endometriosis.
We agree.
Although the beneficial effects on the pathological measures are evident, Figure 3 shows an exorbitant accumulation of KYN and KA and also a substantial reduction in 3HK after the treatment with KNS898, which then raises concerns about tolerability and side effects. Would this effective KNS898 dose be viable and translational as a therapeutic approach?
Please refer to comments above at multiple junctures about safety pharmacology and the clinical development critical path.
Section - KMO is expressed in experimental endometriosis in mice:
By histological examination, the authors confirm that the treatment with KNS898 specifically reduced the KMO expression intensity in the DEGLS from mice. Therefore, the effect exerted by KNS898 locally on the KMO expression at the DEGLS could be, at least, partially responsible for the beneficial effects observed in Figure 3 i.e., the reduction of pathological measures. Although remains to be explored whether the effect of KNS898 in other cells or tissues could also be accountable for the beneficial effects exerted by KNS898 on the animal model of endometriosis.
This is correct.
From a logical experimental point of view, I would suggest switching the order of the result subsection "KMO blockade reduces endometrial gland-like lesion burden in experimental endometriosis in mice" and "KMO is expressed in experimental endometriosis in mice" as well as the respective Figures 3 and 4.
We do not agree. Fig 3 (and section) is the macroscopic enumeration of DEGLS, Fig 4 (and section) is the microscopic and immunohistochemical evaluation of the lesions introduced in Fig 3. The sequence as originally presented is the more logical.
Sections - KMO inhibition reduces mechanical allodynia in experimental endometriosis - and - KMO inhibition reduces mechanical allodynia in experimental endometriosis:
The authors suggested that the KMO inhibition with KNS898 exerts beneficial effects on behavioural paradigms related to the experimental model of endometriosis. Based on the statistical analysis performed for the author, KMO inhibition with KNS898 reduces mechanical allodynia, as well as rescues, impaired cage exploration behaviour and mobility in mice with endometriosis. However, I believe that the most indicated statistical tests for Von Frey (allodynia behaviour) and Home cage (illness behaviour) analyses over time would be repeated measures ANOVA and paired t-test, respectively (and not two-way ANOVA as performed). Therefore for a more trustful analysis and interpretation of this data set, I would suggest the authors modify the statistical analysis and report the corresponding interpretation of these tests.
The selection of two-way ANOVA (time and group) is suitable for this experimental analysis and its use does not invalidate the results. We agree that repeated measures ANOVA could be a valid alternative.
Overall, the authors present a solid and useful case for KMO inhibition as a potential therapeutic strategy for endometriosis. However, the study would benefit from more detailed mechanistic insights, appropriate statistical analyses, and an evaluation of potential side effects. With these improvements, the research could have a significant impact on the field and pave the way for new treatment modalities for endometriosis.
We thank the reviewer for the positive comments and we have responded to the criticisms above.
Specific recommendations for improvement:
• Mechanistic Studies: Conduct detailed studies to understand the local vs. systemic effects of KMO inhibition and its specific impacts on different cell types and tissues. If not feasible here, the authors could include in the discussion section a detailed overview of the possible mechanisms implicated.
While we agree that this is not a comprehensive mechanistic analysis, given that the ultimate therapy would be almost certainly a once daily oral dosing i.e. systemic administration, we do not consider differentiating local vs systemic effects of KMO inhibition to be critical to therapeutic development in this scenario. We do not think speculation about possible mechanisms that is not supported by experimental data should be included. Furthermore, that notion (of statements not supported by data) has been given as a criticism by the reviewers, and therefore consistency on this point must be preferable.
• Quantitative Analysis: Include more robust quantitative methods to compare KMO expression levels in different tissues and assess the correlation between KNO expression and pathological and behavioural changes.
As discussed above, the pathophysiological importance of KMO is in its enzymatic activity, not in its abundance as a protein, and 3HK production is far more dependent on kynurenine substrate availability rather than KMO protein abundance.
• Appropriate Statistics: Use the most suitable statistical tests for behavioural and other repeated measures data to ensure accurate interpretation.
As discussed above
• Side Effect Evaluation: Investigate potential side effects of systemic KMO inhibition, particularly focusing on the long-term implications of altered kynurenine pathway metabolites. If not feasible here, the authors could include in the discussion section a detailed overview of the possible side effects associated as well as inform if KNS898 can cross the BBB and its implications.
For a novel small molecule therapeutic compound in preclinical/clinical development, there are strictly regulated preclinical and clinical development standards that need to be met. It would not be responsible to publish or make claims about safety and potential adverse effect profiles without conducting the proper panel of tests within a suitable regulatory framework.
