{"paper_id":"2c4027cd-039d-41d2-94b3-57311685141e","body_text":"Ben Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 1 of 33\nMedicine\nImmunology and Inflammation\nKynurenine monooxygenase blockade\nreduces endometriosis-like lesions,\nimproves visceral hyperalgesia, and\nrescues mice from a negative\nbehavioural phenotype in\nexperimental endometriosis\nBen Higgins, Ioannis Simitsidellis, Xiaozhong Zheng, Frances Collins, Natalie ZM Homer, Scott G Denham,\nJoanna P Simpson, Mike Millar, Lyndsey Boswell, Hee Y Lee, Yeon G Kim, Kyung H Park, Larry C Park,\nPatrick J Sweeney, Gerard Feraille, Alessandro Taddei, David Chagras, Thierry Alvarez, Scott P Webster,\nAndrew Horne, Philippa TK Saunders, Damian J Mole\nCentre for Inflammation Research, Institute for Regeneration and Repair, Edinburgh Bioquarter, The University of\nEdinburgh, Edinburgh, UK • EXPPECT Edinburgh, Institute for Regeneration and Repair, Edinburgh Bioquarter, The\nUniversity of Edinburgh, Edinburgh, UK • MRC Centre for Reproductive Health, Institute for Regeneration and\nRepair, Edinburgh Bioquarter, The University of Edinburgh, Edinburgh, UK • Mass Spectrometry Core, Edinburgh\nClinical Research Facility, The University of Edinburgh, Edinburgh, UK • Centre for Cardiovascular Science, Queen’s\nMedical Research Institute, The University of Edinburgh, Edinburgh, UK • SuRF Molecular Histology Facility,\nQueen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK • Naason Science, Inc., Cheongju,\nRepublic of Korea • Syneos Health France, Biot, France • Clinical Surgery, The University of Edinburgh, Edinburgh,\nUK\nhttps://en.wikipedia.org/wiki/Open_access\nCopyright information\neLife Assessment\nThis useful study holds importance within the focused scope of endometriosis\ntreatment, providing initial evidence of a potential new therapeutic target. The\nstrength of the evidence is solid, as the methods, data, and analyses support the\nauthors' conclusions regarding the specific aims. The study provides promising\npreliminary evidence of KMO implication in endometriosis, but it falls short of\nestablishing a strong rationale for proposing KNS898 as a treatment for\nendometriosis given the limitations in evidence and mechanistic insights.\nhttps://doi.org/10.7554/eLife.99226.2.sa3\nAbstract\nReviewed Preprint\nv2 • November 11, 2024\nRevised by authors\nReviewed Preprint\nv1 • August 2, 2024\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 2 of 33\nSummary\nEndometriosis is a common and debilitating neuro-inflammatory disorder that is associated\nwith chronic pain. Definitive diagnosis is based on the presence of endometrial-like tissue\n(lesions) in sites outside the uterus. Kynurenine monooxygenase (KMO) is a mitochondrial\nenzyme of tryptophan metabolism that regulates inflammation and immunity. Here, we show\nthat KMO is expressed in epithelial cells in human endometriosis tissue lesions and in\ncorresponding lesions in a mouse model of endometriosis. In mice, oral treatment with the\npotent KMO inhibitor KNS898 induced a biochemical state of KMO blockade with\naccumulation of kynurenine, diversion to kynurenic acid and ablation of 3-\nhydroxykynurenine production. In the mouse model of endometriosis, KMO inhibition\nimproved histological outcomes and endometriosis pain-like behaviours, even when KNS898\ntreatment commenced one week after initiation of lesions. Taken together, these results\nsuggest that KMO blockade is a promising new non-hormonal therapeutic modality for\nendometriosis.\nIntroduction\nEndometriosis is a life-altering condition that affects approximately 10% of females. It is an\noestrogen-dependent neuroinflammatory disorder associated with debilitating pelvic pain,\nexcessive fatigue, gastrointestinal and urinary symptoms, and infertility1     . Worldwide, 200\nmillion prevalent cases are forecast by 2026. Endometriosis is defined by the presence of\nendometrial-like tissue (‘lesions’) outside the uterus. Physiological hormonal fluctuations in\nwomen induce cyclical episodes of cell proliferation, inflammation, injury, and repair within\nlesions that favour fibroblast to myofibroblast differentiation and fibrosis1     . We and others have\nidentified metabolic dysfunction in cells associated with development of endometriosis lesions2     .\nAt present, therapeutic options are largely limited to surgery (that often needs to be repeated) or\nmedical therapies that target hormonal activity with resultant side effects (block conception,\nmenopausal symptoms)1     . Patient surveys consistently show frustration with the lack of\navailable treatments that can give long term relief from symptoms including pain, low mood and\nbloating3     . Analysis of recent clinical trials directed at endometriosis1      has highlighted an\nunmet need for new, non-hormonal approaches to symptom relief. The studies in the current\npaper have addressed this need by focusing on an enzyme that is known to play a key role in\ninflammatory processes that are implicated in the aetiology of endometriosis, but which has not\npreviously been investigated as a target.\nOur proposed solution to this unmet medical need is by targeting the enzyme kynurenine 3-\nmonooxygenase (KMO). KMO is a critical regulator of inflammation at multiple organ sites that\nacts by altering metabolic flux through the kynurenine pathway of tryptophan metabolism4     .\nKMO is known to be expressed in non-pathological endometrium5     , but whether KMO is over-\nexpressed in endometriosis lesions and linked to the severity of inflammation remains to be\ndetermined. KMO has been identified as a critical step in converting kynurenine to the cytotoxic\nmetabolite, 3-hydroxykynurenine (3HK), that is an oxidative stressor, causes protein cross-linking,\nand regulates the immune-metabolic interface4     . Although there is no specific information about\na direct role of KMO in endometriosis, there is evidence of dysregulated tryptophan metabolism in\na recent study using a preclinical non-human primate model of endometriosis6     , and increased\nkynurenine pathway flux at the immune-metabolic interface between stromal cells and NK\nimmune cells in endometriosis lesions7     .\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 3 of 33\nAt present, there is a scientific rationale for KMO inhibition in endometriosis, but it remains to be\nshown in preclinical experiments whether KMO inhibition is efficacious in decreasing lesion\nvolume or behavioural symptoms which are used as a surrogate for pain responses in model\nsystems. KNS898 is a highly specific small molecule KMO inhibitor with potential for use by\nwomen with endometriosis, based on favourable characteristics for oral development in terms of\nbioavailability and predicted half-life8     ,9     . KNS898 is a competitive inhibitor of kynurenine\nsubstrate at the active site of KMO with a pIC50 of 8.88     –10     . We propose that KMO inhibition is a\nnovel therapeutic strategy for endometriosis and, if successful, we will make a significant positive\nimpact for women with this painful, disabling condition. The aim of this project was to obtain\nproof-of-concept for KMO inhibition as a novel therapy for endometriosis. Specifically, we sought\nto explore the expression of KMO in biobanked human endometrial and endometriosis lesion\ntissues, confirm target inhibition of KMO by KNS898 in mice, and define the preclinical efficacy of\nKNS898 in improving clinical features of disease (specifically hyperalgesia and altered cage\nbehaviour) and reducing endometriosis lesion volume in an experimental mouse model of\nendometriosis.\nResults\nKMO is expressed in human eutopic endometrium\nand human endometriosis tissue lesions\nTo explore whether we could detect variations between expression of KMO in endometrium\n(eutopic) within the uterus and a variety of lesions obtained from patients, we conducted detailed\nimmunohistochemistry with a primary antibody specific for KMO. On fixed tissue sections of\nnormal human endometrium KMO expression was most striking in epithelial cells lining the\nglands (Figure 1a     , insert B) with lower levels in the luminal layer (insert C). Notably expression\nin the glands was not uniform (Figure 1a     ). KMO was also strongly immunopositive in human\nperitoneal endometriosis lesions (Fig. 1d      to Fig. 1g     ), and evidently mostly localised to the\nepithelial tissues surrounding the distended endometrial gland-like structures (DEGLS) (Fig. 1e     \nand Fig. 1g     ). Expression in the stromal compartment appeared variable. In human ovarian\nendometriosis lesions, KMO was present at low expression levels in the mesothelial layers (Fig.\n1h      and Fig. 1i     ). Duplex immunohistochemistry with cell phenotype markers CD68\n(macrophages) did not show KMO colocalising with these immune cells (data not shown)\nOral KNS898 inhibits KMO in mice\nNext, we established that oral dosing of KNS898 by gavage in mice resulted in inhibition of KMO.\nUsing n=3 mice per group, we administered KNS898 at 0.01 mg/kg, 5 mg/kg, and 25 mg/kg twice\ndaily (b.d.) in vehicle for seven days, as described. Plasma drug levels and metabolite\nconcentrations are shown in Figure 2     . KNS898 dosed at 0.01 mg/kg b.d. resulted in a mean (±\nS.E.M.) plasma drug level of 0.18 ± 0.01 ng/mL, 5 mg/kg resulted in 88.8 ± 22.6 μg/mL, and 25 mg/kg\ngave 483.9 ± 84.0 μg/mL. The difference between groups was statistically significant by one-way\nANOVA with post hoc Tukey’s test (P = 0.001) (Fig. 2a     ). KMO blockade with KNS898 was clearly\nmeasurable. A backlog in the KMO substrate KYN was evident: KNS898 dosed at 0.01 mg/kg b.d.\nresulted in a mean (± S.E.M.) plasma level of KYN of 339 ± 39 ng/mL, 5 mg/kg resulted in 4940 ± 483\nng/mL, and 25 mg/kg gave 3682 ± 634 ng/mL. The difference between groups was statistically\nsignificant by one-way ANOVA with post hoc Tukey’s test (P = 0.001). The increase in KYN at\nmaximal inhibition was approximately 14-fold compared to the level seen after KNS898 0.01 mg/kg\n(Fig. 2b     ). Excess KYN was metabolised to KA by kynurenine aminotransferase: KNS898 dosed at\n0.01 mg/kg b.d. resulted in a mean (± S.E.M.) plasma level of KA of 629 ± 103 ng/mL, 5 mg/kg\nresulted in 14399 ± 3394 ng/mL, and 25 mg/kg gave 15965 ± 789 ng/mL. The difference between\ngroups was statistically significant by one-way ANOVA with post hoc Tukey’s test (P = 0.001). The\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 4 of 33Ben Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 4 of 33\nFigure 1.\nImmunohistochemistry of KMO expression in human\nendometrium and distended endometriosis gland-like lesions.\nFixed tissue sections were stained with anti-KMO antibody (1:500 dilution) and visualized with DAB as described in the\nMethods section. Panel A. Normal human endometrium (200 X magnification); B1 and C1 insets denote areas shown in\npanels B and C at higher magnification. KMO expression is demonstrated as dark brown DAB-positive staining and is most\nintense in the epithelial cells lining the glands. Panels D through G. Human peritoneal endometriosis tissue lesions stained\nwith anti-KMO antibody visualized with DAB. D/E PIN3652, Stage II, F/G PIN3306 Stage I. Note intense staining of cells lining\nglandular structures. Panel H. Ovarian-type endometriosis tissue lesion with higher magnification inset (I1) showing KMO\nexpression present but at lower intensity in the mesothelial tissue surface (this sample does not have epithelial cells in the\nlesion).\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 5 of 33\nfold increase in KA at maximal inhibition was approximately 25-fold compared to the level seen\nafter KNS898 0.01 mg/kg (Fig. 2c     ). KMO blockade resulted in a statistically-significant reduction\nof 3HK in plasma: KNS898 dosed at 0.01 mg/kg b.d. resulted in a mean (± S.E.M.) plasma level of\n3HK of 27.7 ± 7.2 ng/mL, 5 mg/kg resulted in 4.2 ± 0.3 ng/mL, and 25 mg/kg gave 0.9 ± 0.4 ng/mL.\nThe difference between groups was statistically significant by one-way ANOVA with post hoc\nTukey’s test (P = 0.001). The fold decrease in 3HK at maximal inhibition was approximately 30-fold\ncompared to the level seen after KNS898 0.01 mg/kg (Fig. 2d     ). Overall, there was a clear dose\nresponse to KNS898 administration leading to maximal KMO blockade at 25 mg/kg b.d. This dose\nwas therefore selected for efficacy experiments going forward. A diagrammatic representation of\nthe kynurenine pathway is shown as Figure 2e      for reference.\nKMO blockade reduces endometrial gland-like lesion\nburden in experimental endometriosis in mice\nThe experimental design for the mouse model of endometriosis is shown in Figure 3a     . The\npharmacological effect of KNS898 therapy showed appropriate levels of KNS898 detected in\nplasma (Fig 3b     ), with accumulation of kynurenine (Fig. 3c     ), inhibition of 3HK production\n(Fig. 3d     ) and diverted metabolism of accumulated kynurenine to kynurenic acid (Fig. 3e     ). All\nrecipient mice inoculated with donor tissue (groups G3, G4, and G5) developed distended\nendometrial gland-like structures (DEGLS). The incidence of DEGLS formation was enumerated at\nautopsy, and the axial length of each DEGLS was measured after excision from the surrounding\ntissue. In G3 (endometriosis + vehicle), 8 of 15 (53%) of the inoculated animals had developed\nDEGLS. In KNS898-treated group G4 (endometriosis + treatment from Day 19), DEGLS formed in 4\nof 15 mice (26.7%) and in G5 (Endo + treatment start on Day 26) in 6 of 15 mice (40%). As expected,\nno DEGLS were formed in the non-inoculated control and sham groups. The total number of\nDEGLS per animal in each group was highest in G3 with an average of 4.0 per animal with DEGLS\n(total = 32 DEGLS in 8 mice in G3). Mice with endometriosis receiving KNS898 from the time of\ninoculation (G4) had an average of 2.0 DEGLS per animal with DEGLS (total = 8 DEGLS in 4 mice in\nG4) and those receiving KNS898 1 week after inoculation (G5) had an average of 1.8 DEGLS per\nanimal (total = 11 DEGLS in 6 mice in G5) (Figs. 3f      and 3g     ). Statistical analysis by ANOVA\nshowed a significant difference in endometriosis DEGLS burden between groups (P = 0.0295 for\nDEGLS per animal; P = 0.004 for DEGLS per group). DEGLS axial length and derived volume did not\ndiffer between groups (Supplementary Fig. 1a and b). All recipient mice inoculated with donor\ntissue lost body weight following inoculation which then gradually recovered. After recovery, body\nweight of all three inoculated groups was lower compared to the control groups for the duration of\nthe study. Overall, there was no significant difference in body weight between G3 and the KNS898\ntreatment groups G4 and G5 (endometriosis + treatment from Day 26) (Suppl. Fig. 1c).\nKMO is expressed in experimental endometriosis in mice\nHistological examination of DEGLS identified them as containing cystic structures lined with\nepithelial layers identifiable as columnar epithelium, pseudostratified epithelium, squamous\nepithelium, and cuboidal epithelium, with goblet cells. These DEGLS were considered to represent\nendometriosis-like lesions derived from the implanted basal endometrial/myoepithelial layers of\nthe donor mice uteri (Suppl. Fig 2). Immunohistochemistry using an antibody to KMO showed\nKMO protein expression localised mainly to the epithelial cells lining of the DEGLS, with a lesser\ndegree of KMO positive staining in the closest surrounding connective tissue, in keeping with the\npreviously observed KMO expression pattern in human endometriosis lesion tissue (Fig. 4a      and\n4d      and Supplementary Fig S3). The thickness (area divided by length) of the KMO positive\nepithelial layer was quantified for each DEGLS section using QuPath and there was no difference\nbetween groups G3, G4 and G5 (Fig. 4b      and 4e     ). However, quantification of KMO expression\nconfirmed the high intensity of KMO staining in the epithelial lining layers (Fig. 4c      and 4f     ),\nbut also showed a clear and statistically significant reduction in KMO expression intensity in those\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 6 of 33Ben Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 6 of 33\nFigure 2.\nKNS898 plasma levels and pharmacodynamic effect of KMO blockade.\nMice (n=3 per group, individual data shown) were given KNS898 twice daily by gavage at the doses shown. After 7 days, blood\nwas sampled at euthanasia and KNS898 levels and kynurenine pathway metabolite levels measured by LC-MS/MS. A. KNS898\ndrug levels. B. Kynurenine. C. Kynurenic acid. D. 3-hydroxykynurenine (logarithmic scale). Comparison between groups by\none way ANOVA with post hoc Tukey’s test. *P <0.05, **P<0.01, ***P<0.001, n.s. not statistically significant. E. A diagram of\nthe kynurenine pathway showing the key step catalyzed by KMO.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 7 of 33Ben Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 7 of 33\nFigure 3.\nTherapeutic effect of KNS898 in an experimental mouse model of endometriosis.\nA. Experimental design. Ovariectomized (OVX) donor mice were hormonally stimulated as shown (E2 estradiol, P4\nprogesterone). At Day 19, donor mouse endometrial fragments were inoculated into recipient mice in a 1:1 ratio. KNS898\ntreatment 25 mg/kg twice daily by oral gavage was commenced at Day 19 or after a 1 week interval on Day 26 and in both\ncases continued for 2 weeks. Groups were G1: n=10, control mice; G2: n=10, sham-operated control mice; G3: n=15,\nendometriosis + vehicle; G4: n=15, endometriosis with KNS898 commenced at Day 19; G5: n=15, endometriosis with KNS898\ncommenced at Day 26. B. KNS898 drug levels (relative concentrations). C. Kynurenine. D. 3-hydroxykynurenine. E. Kynurenic\nacid. Individual data are shown in panels B through E. Panels F and G. Enumerated distended endometriosis gland-like\nstructures (DEGLS) in recipient mice by treatment group. F. Total number of DEGLS per group. G. Total number of DEGLS per\nanimal for all animals in the group; bars show mean with s.e.m. (G). Comparison between groups by one way ANOVA with\npost hoc Tukey’s test. *P <0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s. not statistically significant.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 8 of 33\nareas in DEGLS removed from mice treated with the KMO inhibitor KNS898 (Fig. 4g     ; P = 0.008).\nRepresentative micrographs from each of groups G3, G4 and G5 are presented in Figures 4h, 4i     \nand 4j     .\nKMO inhibition reduces mechanical\nallodynia in experimental endometriosis\nClinical endometriosis is associated with visceral hyperalgesia and central sensitisation to\npain11     . Visceral and central hyperalgesia may be tested in rodents using the Von Frey filament\ntest12     . Baseline reaction values for hind paw and bladder Von Frey tests showed no significant\ndifference in mechanical allodynia before inoculation. In established endometriosis without\ntreatment (group G3), the mechanical allodynia threshold in the hind paw was statistically\nsignificantly lower compared to baseline for the group. When compared to the control groups at\nthe corresponding time point beginning 1 week after inoculation and continuing until the end of\nthe study. Day 26 KNS898-treated group (G5) showed a statistically-significant improvement in\nmechanical allodynia in the hind paw using the Von Frey test compared to mice in G3 with\nuntreated endometriosis given vehicle control (Two-way ANOVA, Group effect P = 0.003, time\neffect P < 0.0001; Dunnett’s multiple comparison test G5 vs G3 P=0.001) (Fig. 5a     ). The mechanical\nallodynia threshold for the bladder reflex also was lower in mice with endometriosis compared to\nbaseline throughout the study, and KNS898 treatment commencing at D26 (G5) was associated\nwith a statistically significant improvement in bladder mechanical allodynia threshold at D42\ncompared to mice with untreated endometriosis given vehicle control (G3)(Two-way ANOVA,\nGroup effect P = 0.038, time effect P < 0.001; Dunnett’s multiple comparison test G5 vs G3 P=0.021)\n(Fig. 5b     ).\nKMO inhibition rescues impaired cage exploration\nbehaviour and mobility in mice with endometriosis\nHCA peripheral moving speed, time at cage edge, and illness behaviour, including temperature,\nmotility and cage exploration behaviour was quantified using Home Cage Analysis (HCA). Baseline\nHCA was recorded before inoculation and at the end of the experiment. Mice with endometriosis\nwithout treatment showed an overall reduction in activity in moving distance and moving speed\nrelative to baseline, and compared to sham-operated control groups, indicating a negative effect\non behaviour due to endometriosis. Importantly, mice with endometriosis treated with KNS898\nshowed marked improvement in motility and cage exploration behaviour compared to untreated\nendometriosis mice, and although this difference between groups was statistically significant by\nWelch’s one-way ANOVA, post hoc testing (Dunnet’s T3) was not significant between groups. This\nqualitative difference of time spent exploring the periphery of the cage being lower for mice with\nendometriosis treated with vehicle control was seen in both the day and night phases (Figs. 5c     \nand 5d     ). A similar improvement with KNS898 treatment compared to vehicle control mice with\nendometriosis was seen for total moving distance, total moving speed, and peripheral distance,\nbut not for total moving or climbing time, for which no difference between groups was detected.\nTogether, these data indicate that KMO inhibition with KNS898 results in an improvement in well-\nbeing evidenced by improved cage exploration behaviour in addition to improved objective\nhistological measures of endometriosis disease burden.\nDiscussion\nIn this study, we set out to investigate the potential for KMO inhibition as a non-hormonal therapy\nfor endometriosis. First, we confirmed that KMO was expressed in human endometrium by\nimmunohistochemistry, and then showed that KMO was clearly expressed in the epithelial cells in\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 9 of 33Ben Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 9 of 33\nFigure 4.\nQuantification of KMO expression in mouse model distended\nendometriosis gland-like structure (DEGLS) lesions.\nSections were visualized at 200 X magnification (A, B, C) with higher magnification insets shown (D, E, F). Panel A and D.\nFixed tissue sections were stained with anti-KMO antibody (1:500 dilution) and visualized with DAB as described in the\nMethods section. B and E. QuPath was used to identify the epithelial tissue layers (yellow arrows denote the boundary) which\nwere quantified by thickness. C and F. KMO expression intensity quantified and heat map expression values are overlayed. G.\nKMO expression staining intensity per unit area of endometriosis DEGLS epithelium, categorized by treatment group (G3\nendometriosis + vehicle; G4 endometriosis + KNS898 from D19; G5 endometriosis + KNS898 from D26. Individual data points\nshown. Comparison between groups by one way ANOVA with post hoc Tukey’s test. *P <0.05.. H, I and J. Representative\nmicrographs from G3 (H), G4 (I) and G5 (J) showing KMO expression in the epithelial cells lining each DEGLS.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 10 of 33Ben Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 10 of 33\nFigure 5.\nEffect of KNS898 on mechanical allodynia and illness\nbehaviour in an experimental mouse model of endometriosis.\nA. Hind paw Von Frey filament test showing effect of endometriosis and KNS898 treatment in groups G3, G4 and G5 B.\nBladder Von Frey filament test C. Home Cage Analysis of motility showing a daytime motility deficit in mice with\nendometriosis compared to control mice, and clear restitution of normal motility in KNS898 treated groups. D. Nighttime\nhome cage motility analysis showing the benefit of KNS898 treatment on normalizing the motility deficit seen in mice with\nendometriosis. Data are mean with s.e.m. For A and B, statistical comparison between groups was by two-way ANOVA to\ncompare Group effect and Time effect, with multiple group comparison using Dunnett’s T3 test. Asterisks represent\ntreatment group effect statistical significance of the Dunnett’s T3 test comparing treatment group G5 to G3 vehicle control *P\n<0.05, **P <0.01. For C and D, Welch’s ANOVA with multiple group comparison using Dunnett’s T3 test was used. Although\nthe ANOVA was statistically significant, the post hoc Dunnett’s T3 was not, therefore no asterisks are marked.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 11 of 33\nhuman endometriosis lesions. Next, we demonstrated that the highly specific KMO inhibitor\nKNS898 was orally bioavailable when given twice daily by gavage in mice, and clearly blocked\nKMO activity in a dose-dependent manner at a dose of 25mg/kg. We therefore used that dose to test\nthe efficacy of KMO blockade with KNS898 in mice with experimentally-induced endometriosis.\nOne important finding of this project is that KMO blockade resulted in a reduction in\nendometriosis severity compared to untreated mice with endometriosis, specifically in terms of\nreducing i) the number of mice that developed endometriosis tissue lesions, and ii) the number of\nlesions per mouse in those that did develop lesions. The histopathology of the experimental\nendometriosis lesions was sufficiently similar macroscopically to that seen in the examined\nhuman DEGLS, and KMO was evidently highly expressed in the same tissue distribution in model\nlesions compared to human disease. KMO blockade also decreased lesion KMO expression.\nCritically, and importantly from a translational perspective, therapeutic blockade of KMO\nimproved visceral hyperalgesia measured by reduced mechanical allodynia and restored normal\ncage exploration behaviour and mobility in treated mice compared to untreated mice with\nendometriosis. Together, these data show that KMO is expressed in human and mouse\nendometriosis tissue lesions and that therapeutic KMO blockade reduces the number of\nendometriosis lesions and improves holistic metrics of disease behaviour in mice.\nThe model of endometriosis, using inoculation of endometrial tissue of ovariectomized donor\nmice, reliably induced the pathophysiological symptoms indicative of endometriosis in recipient\nmice. Test groups inoculated with endometrial tissue (G3-G5) showed significant growth of ectopic\nendometrial tissue. Groups treated with test article experienced significantly less DEGLS\ndevelopment (significantly fewer DEGLS were noted in treated groups when compared to vehicle\ntreated groups). Disease burden in the treatment group that had treatment starting immediately\nafter inoculation was lower than that of the vehicle-only treated group. It is not clear why mean\ncystic size and cystic volume in treated animals was not smaller in treated animals. We can only\nspeculate that KMO blockade may potentiate rapid involution of cysts, but this cannot be proven\nmechanistically here.\nMice that received inoculated endometrial tissue showed a measurable and increased visceral\nhyperalgic pain response (lower mechanical threshold) as measured by bladder response to von\nFrey filament testing, and improvement in a surrogate marker of central sensitisation to pain\nmeasured by hind paw Von Frey filament testing when compared to control mice. The mechanical\nthreshold of both treatment groups trended higher compared to the vehicle treated group. One\ninterpretation of these data is that KMO inhibition reduced responses to pain caused by the\npresence of endometriosis, i.e. improving visceral hyperalgesia.\nHome cage behaviour using HCA indicated a reduced overall activity in endo-inoculated mice\nwhen compared to control mice. It should also be noted that test article-treated animals in both\ngroups showed more locomotor behaviour and a seemingly better quality of life within the home\ncage environment when the home-cage dynamics were monitored. This supports the notion that\ntreated mice exhibit less propensity for behaviours that are, at times, typical of depressive and\nanxiety-like behaviour in home-cage, group housed conditions.\nUnder the experimental conditions imposed, treatment with KNS898 on two dosing schedules\nprovided a significant reduction in DEGLS formation within the inoculated mice, as well as a\nseemingly higher pain threshold. This attenuation of pain response was coupled with increased\nactivity levels in the home cage. Taken together, these data suggest a therapeutic effect to\nalleviation of certain salient symptoms of endometriosis, as well as a reduction in the number and\nsize of DEGLS.\nNon-pathological endometrium is a site of high KMO expression. Because endometriosis lesions in\nwomen are ‘endometrial-like’ tissue rather than normal endometrium, we tested, and\ndemonstrated expression of KMO in the epithelial layers of endometriosis lesions sampled from\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 12 of 33\nwomen undergoing surgery for endometriosis. First-line medical treatment for endometriosis is\nthe contraceptive pill or other ovarian steroid hormone suppressive drugs. Treatment failures are\nfrequent, side effects are common, all approaches are contraceptive. Many women opt for invasive\nsurgery to remove or ablate the endometriosis lesions.\nIn conclusion, KMO is expressed in human endometriosis tissue lesions and in a mouse model of\nendometriosis in the epithelial layers of distended endometrial gland-like structures. Oral KNS898\nreliably induced a biochemical state of KMO blockade with accumulation of kynurenine, diversion\nto kynurenic acid and ablation of 3-hydroxykynurenine production. KMO blockade improved\nhistological and symptomatic behavioural endometriosis disease features with an overall benefit,\neven when treatment commenced one week after establishment of the disease. KMO blockade is\ntherefore a promising avenue for a new non-hormonal therapeutic modality for endometriosis.\nMaterials and methods\nEthical approvals and permissions\nThe human tissue samples were obtained from participants who had given fully informed written\nconsent under ethical approval granted by Lothian Research Ethics Committee (LREC 11/AL/0376).\nHuman tissue samples were obtained with ethical approval and fully informed consent from\nindividuals attending the Royal Infirmary of Edinburgh as described below. Animal experiments\nconducted by NAASON Inc were carried out according to the National Institute of Health (NIH) &\nNational Institutes of Health Korea (NIHK) guidelines for the care and use of laboratory animals\nand approved by Naason Science in accordance with all applicable FELASA, IACUC and AAALAC\nguidelines. Animal experiments outsourced to Syneos Health were conducted with institutional\nethical approval.\nHuman Patients and Samples\nTissue samples were collected from patients undergoing a diagnostic laparoscopy for suspected\nendometriosis following Endometriosis Phenome and Biobanking Harmonisation Project (EPHect)\nguidelines13     . Patient summary characteristics are presented in Supplementary Table S1. Note\nthere was a range of disease stages assigned at time of surgery according to American Fertility\nSociety (AFS) criteria14     . Cycle stage was determined by measuring hormones in blood according\nto standard protocols and assessment of eutopic endometrial tissue histology when such samples\nwere available15     . Lesions were recovered from 17 patients, of these n=10 were recovered from\nthe peritoneal side wall consistent with classification as superficial peritoneal endometriosis\nlesions and n=5 from the cysts of ovarian disease (endometrioma) (n=2 on hormones). Eutopic\nendometrium was from 4 patients n=3 of which had no lesions at time of surgery (noted as stage\n0). General histology of samples was assessed using H&E staining.\nImmunohistochemistry (human endometrium\nand human endometriosis tissue lesions)\n5 μm sections of formalin-fixed paraffin-embedded tissue blocks were mounted on SuperFrost\nPlus adhesion slides (Thermo Fisher Scientific). Sections were deparaffined with xylene and\nrehydrated prior to heat-induced antigen retrieval using Instant Pot: Tris-EDTA pH916     . Sections\nwere washed with tap water and incubated in phosphate buffered saline (PBS) for 5 minutes.\nEndogenous peroxidase was blocked with 0.3% hydrogen peroxide in 70% v/v methanol for 30\nmins at room temperature then washed in PBS prior to blocking in Normal Goat Serum\n(NGS)/PBS/bovine serum albumin (BSA)(5%) for 30 mins and streptavidin for 15 mins. Sections\nwere washed twice in PBS and then blocked with biotin for 15 mins and washed in PBS. The\nprimary antibody to KMO (KMO Rabbit polyclonal, Proteintech, Catalog Number:10698-1-AP)17     \nwas diluted to a final concentration of 1:1000 in NGS/PBS/BSA and incubated overnight at 4°C in a\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 13 of 33\nhumidity chamber. Sections were washed twice with PBS/Tween 0.05% (1ml Tween in 2L PBS) for\n5 mins. The secondary detection antibody Goat Anti-Rabbit Biotinylated (Vector Cat number: BA-\n1000) was diluted in NGS/PBS/BSA (1:500) and incubated for 30 mins, prior to washing twice in\nPBS/Tween 0.05%, for 5 mins before adding the detection system reagent Streptavidin-HRP (DAKO\nCat Number P0397) 1:500 in PBS for 30 min, washed and stained with DAB (DAKO Cat Number\nK3468) as per manufacturer’s directions and incubated for 5 mins before a final wash with tap\nwater. Sections were counterstained with Haematoxylin, dehydrated through graded ethanol and\nmounted. Sections of stained slides were scanned on a Zeiss Axioscan Z1 slide scanner and\nexported as TIFF files: images were evaluated for stromal, epithelial and immune cell content.\nKNS898 preparation for oral administration\nKNS898 powder was weighed and dissolved at the required concentrations in a final vehicle of 2%\nDMSO, 20% PEG200, 78% 0.15M NaCl by volume. Brief sonication on ice was done to facilitate\ndisolution.\nIn vivo confirmation of KMO inhibition by KNS898 in mice\nThis experiment was outsourced to Syneos Health (Les Templiers, 2400 route des Colles, 06410\nBiot, Sophia-Antipolis, France). A formal pharmacokinetic/pharmacodynamic study was not\nrequired at this stage. Female C5Bl/6J mice aged 12 weeks were purchased from Charles River\nLaboratories, maintained on standard 12 hour light-dark cycle, given free access to water and\nstandard chow before being randomised to one of three dose levels of KNS898 (n=3 per group,\ntotal n=9 mice). Dose levels tested were 0.01mg/kg, 5mg/kg and 25mg/kg. Mice were gavaged with\n0.5 mL of drug in vehicle twice daily for 7 days before euthanasia and plasma sampling.\nPlasma samples\nBlood was sampled into Sarstedt Microvette CB K2EDTA 300 μL tubes and centrifuged at 5,000 rpm\n(2380 RCF) for 3 mins. Plasma was aliquoted, frozen on dry ice and transferred to storage at −80°C\nprior to temperature-controlled shipping.\nLC-MS/MS analysis of plasma drug\nlevels and kynurenine metabolites\nPlasma samples (100 μL) were diluted at a 1:1 ratio with 4% phosphoric acid and enriched with 50\nng 13C6-kynurenine, 13C6-3-hydroxykynurenine (Sigma Aldrich, custom synthesis) and d5-\nkynurenic acid (CDN isotopes). 12-point calibration standards (0.1 to 100 ng) were prepared for\nKNS898, kynurenine (KYN), kynurenic acid (KA), and 3-hydroxykynurenine (3HK) and extracted\nalongside samples using solid phase extraction plates (Waters Oasis HLB, 10 mg sorbent, 30 μm\nparticle size). Extracts were dried down under nitrogen and reconstituted in LC-MS grade water\n(100 μL). 10 μL was injected onto a column (Ace C18-PFP column; 100 x 2.1 mm internal diameter\n1.7 μm; HiChrom (VWR, Lutterworth)) using an Acquity I-Class UPLC liquid chromatography\nsystem (Waters) linked to a QTRAP 6500+ mass spectrometer (AB Sciex)10     . The flow rate was set\nat 0.4 mL/min with a column temperature of 40°C. Separation was carried out using a gradient\nmobile phase system of A – 0.1% aqueous formic acid and B – 0.1% formic acid in methanol,\nstarting at 15%B, rising to 85%B over 6 mins and returning to 15%B by 9 mins. Mass spectrometry\nsettings were for positive mode electrospray (5.5 kV, 700°C) and multiple reaction monitoring m/z\n209.0 → 192.2 for KYN, m/z 189.9 → 144.1 for KA m/z 225.0 → 208.0 for 3HK and m/z 361.1 → 120.1\nfor KNS898 and for internal standards were m/z 231.0 → 214.0 for 13C6-3HK, m/z 195.1 → 177.2 for\nd5-KA and m/z 215.0 → 197.8 for 13C6-kynurenine. Retention times for KYN, KA, 3HK and KNS898\nwere 1.8, 3.2, 1.2 and 6.5 mins, respectively and 3.2 mins for d5KA, 1.8 mins for 13C6-3HK and 1.2\nmins for 13C6-KYN. Data were acquired by Analyst 1.7.1 software (AB Sciex) and linear regression\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 14 of 33\nanalysis was carried out on MultiQuant 3.0.3 software (AB Sciex) where peak integrations and\namounts of each kynurenine metabolite and KNS898 were calculated using the peak area ratio of\ncompound/internal standard, with data further handled in Microsoft Excel 2016 as described18     .\nExperimental mouse model of endometriosis\nThis experiment was outsourced to Naason Science Inc., Osong, Korea (KBIO New Drug\nDevelopment Center #506, Chungbuk, Korea, 28160) using protocols originally developed by the\nSaunders team in Edinburgh19     ,20     . Experimental design and groups are shown in Figure 3a     \nand Supplementary Table S2. There were 5 groups of mice with n=10-15/group):25 mg/kg KNS898\nwas administered twice a day via oral gavage in two of the groups of mice. Group 4 received\nKNS898 from the time of endometrial tissue inoculation (Day 19; G4); group 5 commenced dosing 1\nweek after inoculation (Day 26; G5). Group 3 received vehicle (2% DMSO, 20% PEG200, 78% 0.15M\nNaCl) in the same regimen.\nTo perform the mouse model of endometriosis, donor female C57Bl/6 mice aged 6 weeks were\nacclimatized for 2 weeks prior to surgery. Ovariectomy (Day 0) was performed at 8 weeks of age\nunder general anaesthesia with monitoring, with analgesia that extended to the post-operative\nperiod with buprenorphine (0.03 ml) (Veterges ic® 3 mg/ml, Ceva Inc., Korea) subcutaneously. To\nprepare donor tissue that would best replicate menstrual-like tissue in women, ovariectomized\n(OVX) mice were primed with daily s.c. injections of 100 ng 17β-estradiol (E2) on days 7, 8 and 9.\nOn days 13 – 19 a silastic progesterone (P4) pellet was implanted subcutaneously. These animals\nwere injected once daily with E2 (5 ng in sesame oil) on days 13, 14 and 15. Decidualization was\ninduced in one uterine horn with an injection of 20 µl sesame oil 4 hours after the last E2 injection.\nOn day 19 (4 days after induction of decidual response), donor mice were killed 4 hours after\nremoval of the P4 pellet. Endometrial tissue was then scraped from the myometrial layer of the\ndecidualized uterine horn, suspended in 500 μl PBS and injected via a XG needle sprayed into the\nlower abdominal cavity of the recipient mouse under general anaesthesia with monitoring and\npost-operative analgesia as described. The ratio of donor to recipient mouse was 1:1 (from one\ndonor to one recipient). Recipient mice had intact ovaries to ensure ongoing hormonal stimulation\nof the injected tissue: group allocations are shown in Supplementary Table S2.\nMechanical allodynia test by the Von Frey method\nAbdominal and hind paw Von Frey tests were performed in the recipient animals before\ninoculation (baseline), and 1, 2, and 3 weeks after inoculation. Mechanical threshold was\nmeasured using Von Frey filaments. For the hind-paw, 15, 8, 6, 4, 2, 1.4, 0.6, 0.4 g filaments were\nused, and for the bladder reflex to filament application to the lower abdomen, 60, 26, 10, 8, 6, 4, 2,\n1 g filaments were used. The experimenter was blind to the group allocation in order to reduce\nbias.\nCage exploration and behavioural assay using Home Cage Analysis\nHome Cage Analysis (HCA) was performed in the recipient animals before inoculation (baseline),\nand at the late stage of treatment. Recipient mice were randomly housed using a Monte Carlo\nrandomization. All animals had a micro-chip (BioMark, USA) inserted to the abdomen prior to\nbeing placed in the home-cage. This procedure does not cause undue discomfort or hamper, in any\nway, animal movement. Total moving distance, total moving time, moving speed,\nisolation/separation distance, isolated time, peripheral time, peripheral distance, in centre zones\ntime, in centre zones distance, climbing time, and body temperature were tracked automatically\nby an ActualHCA™ Home Cage Analyzer (ActualAnalytics Ltd., Edinburgh, UK) and processed with\nproprietary machine learning and artificial intelligence algorithms.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 15 of 33\nEndpoint tissue and plasma sampling\nOn experimental Day 40, all recipient mice were euthanized, and blood collected via cardiac\npuncture. Whole blood was collected into heparinised tubes, and plasma was separated by\ncentrifugation (3000 rpm for 15 min) at 4°C. Separated plasma was collected in Eppendorf\nmicrotubes, frozen on dry ice and stored at −80°C. Photographs of the abdominal cavity were\nobtained. Lesions from the abdominal cavity were harvested. DEGLS were dissected from the\nsurrounding abdominal tissue and measured for size and volume. DEGLS volume was measured\nusing the following formula21     : Volume = long diameter × (short diameter/2)2 × π. DEGLS were\nfixed in a 4% paraformaldehyde solution and prepared for standard H&E. If more than one DEGLS\nwas present in an animal, DEGLS that were not used for H&E were snap-frozen in liquid nitrogen\nand stored at −80oC.\nEndometriosis lesion histology\nEndometriosis lesion tissue blocks were sectioned at a uniform thickness of 5 μm and were\nmounted onto a microscope slide. The slide then underwent deparaffination and hydration.\nParaffin was removed from the slide using xylene, then hydration through graded ethanol and\nwashing were performed. Slides were then stained with Harris haematoxylin and alcoholic eosin\nY and mounted after dehydration and clearing with xylene. The H&E-stained images were\nvisualized using a Slide Scanner (Panoramic scan, 3D HISTECH).\nImmunohistochemistry (mouse DEGLS)\nImmunohistochemistry to detect KMO in mouse DEGLS tissue was performed on a Leica Bond III\nautomated immunostaining robot. 5 μm thick sections obtained from FFPE (formalin Fixed\nParaffin Embedded) samples mounted on superfrost plus slides were stained as follows. Heat\ninduced epitope retrieval (HIER) was performed using Epitope Retrieval Solution 1 (Leica, ER1 pH\n6.0 citrate based solution) for 20 minutes at 990C. Tissue sections were then incubated for 10\nminutes in hydrogen peroxide to block endogenous hydrogen peroxidase activity followed by 10\nminute blocking with normal goat serum. The primary antibody against KMO (Proteintech 10698-\n1-AP @1:1000 Rabbit)17      was incubated for 1 hour, then incubated with a goat anti-rabbit\nperoxidase conjugated secondary antibody for 30 minutes prior to visualisation with\ndiaminobenzoate (DAB) using standard protocols.\nDigital slide scanning\nWhole sections were scanned using a Zeiss Axioscan Z1 whole slide scanner. The image files (.czi)\nwere batch converted to Tif format for image analysis and quantification, acquisition and batch\nexport used Carl Zeiss Zen v2.5 software.\nQuantitative image analysis\nImage analysis was carried out using QuPath v0.4.2. Analysis was standardised by using multiple\nregions of interest from individual slides and collating them into a training image. To improve\nstain contrast, overlapping DAB and haematoxylin staining was deconvoluted by manual\noptimisation of the stain vectors. Individual slide epithelia were annotated, with relative DAB\noptical densities and annotation shape measurements taken. Epithelial thickness and area were\ncalculated to enable correlation with KMO intensity. To generate the heat map of cell KMO\nexpression, a cell detection was carried out on haematoxylin staining using standard parameters,\na 5-100um2 area range, cell expansion of 1um and a threshold of 0.14.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 16 of 33\nStatistical analysis\nPower calculations were performed using G*Power (v3.1.9.4) software. Input parameters were\nused: 2-tailed, α-error probability = 0.05, and power (1-β error probability) = 0.80. Continuous\nvariable data were tested for Normality of distribution with a one sample Kolmogorov-Smirnov\ntest. Normally distributed data were analysed by one-way ANOVA with post-hoc Dunnett’s T3 for\nmultiple groups. Data comparing treatment group effects at multiple time-points were analysed by\ntwo-way ANOVA with multiple comparison testing by Dunnett’s method. Data comparing multiple\ngroups with unequal variances were analyzed with Welch’s ANOVA. Data not following the Normal\ndistribution were analysed with non-parametric Kruskal-Wallis test. Categorical and proportions\ndata were analysed by Fisher’s exact test. Data were visualised with GraphPad Prism.\nAcknowledgements\nWe would like to thank the University of Edinburgh MRC Confidence in Concept award team:\nAndrew McBride, Lorraine Jackson. We thank Susan Bodie, Dave Pritchard from Edinburgh\nInnovations. We thank all staff and support team members at Syneos Health and NAASON Science\nInc.\nAdditional information\nFunding\nUKRI Medical Research Council Confidence-in-Concept grant MRC/CIC8/73 (DJM, SPW, PTKS, AH)\nUKRI Medical Research Council Senior Clinical Fellowship MR/P008887/1 (DJM)\nAuthor contributions\nConceptualization: DJM, SPW, PTKS, AH\nMethodology: BH, IS, NZMH, SGD, JPS, MM, LB, LCP, PJS, AT, SPW, AH, PTKS, DJM\nInvestigation: BH, IS, XZ, FC, SGP, JPS, LB, HYL, YGK, KHP, LCP GF, AT, DC, TA\nVisualization: BH, IS, KHP, LCP, DJM\nFunding acquisition: DJM, SPW, PTKS, AH.\nProject administration: DJM, XZ, LCP, PES, AT, PTKS, AH, NZMH\nSupervision: MM, LCP, AT, PTKS, DJM\nWriting – original draft: DJM, LCP\nWriting – review & editing: BH, IS, XZ, FC, NZMH, SGD, JPS, MM, LB, LCP, PJS, AT, SPW, AH, PTKS,\nDJM.\nDeclaration of interests\nThe following authors have interests to declare: S.P.W., D.J.M. are co-founders of Kynos\nTherapeutics Ltd.. D.J.M. is a Board Member of Kynos. The University of Edinburgh controls\nPatents WO2015/091647, WO2016/097144, WO2016/188827 that relate to inhibitors of KMO\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 17 of 33\ninhibitors, and include the compound used in this paper. The remaining authors declare no\ncompeting interests.\nData and materials availability\nAll data are available in the main text or the supplementary materials.\nKNS898 availability is restricted under a Material Transfer Agreement. Please contact the\ncorresponding author in the first instance.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 18 of 33\nReferences\nSaunders P.T.K., Horne A.W. (2021) Endometriosis: Etiology, pathobiology, and therapeutic\nprospects Cell 184:2807–2824https://doi.org/10.1016/j.cell.2021.04.041\nDorning A., Dhami P., Panir K., Hogg C., Park E., Ferguson G.D., Hargrove D., Karras J., Horne\nA.W., Greaves E. 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(2016) Silencing Prion Protein in\nHT29 Human Colorectal Cancer Cells Enhances Anticancer Response to Fucoidan\nAnticancer Res 36:4449–4458https://doi.org/10.21873/anticanres.10989\nAuthor information\nBen Higgins\nCentre for Inflammation Research, Institute for Regeneration and Repair, Edinburgh\nBioquarter, The University of Edinburgh, Edinburgh, UK\nIoannis Simitsidellis\nCentre for Inflammation Research, Institute for Regeneration and Repair, Edinburgh\nBioquarter, The University of Edinburgh, Edinburgh, UK, EXPPECT Edinburgh, Institute for\nRegeneration and Repair, Edinburgh Bioquarter, The University of Edinburgh, Edinburgh,\nUK, MRC Centre for Reproductive Health, Institute for Regeneration and Repair, Edinburgh\nBioquarter, The University of Edinburgh, Edinburgh, UK\n13.\n14.\n15.\n16.\n17.\n18.\n19.\n20.\n21.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 20 of 33\nXiaozhong Zheng\nCentre for Inflammation Research, Institute for Regeneration and Repair, Edinburgh\nBioquarter, The University of Edinburgh, Edinburgh, UK\nFrances Collins\nCentre for Inflammation Research, Institute for Regeneration and Repair, Edinburgh\nBioquarter, The University of Edinburgh, Edinburgh, UK, EXPPECT Edinburgh, Institute for\nRegeneration and Repair, Edinburgh Bioquarter, The University of Edinburgh, Edinburgh,\nUK, MRC Centre for Reproductive Health, Institute for Regeneration and Repair, Edinburgh\nBioquarter, The University of Edinburgh, Edinburgh, UK\nNatalie ZM Homer\nMass Spectrometry Core, Edinburgh Clinical Research Facility, The University of Edinburgh,\nEdinburgh, UK, Centre for Cardiovascular Science, Queen’s Medical Research Institute, The\nUniversity of Edinburgh, Edinburgh, UK\nScott G Denham\nMass Spectrometry Core, Edinburgh Clinical Research Facility, The University of Edinburgh,\nEdinburgh, UK\nJoanna P Simpson\nMass Spectrometry Core, Edinburgh Clinical Research Facility, The University of Edinburgh,\nEdinburgh, UK\nMike Millar\nSuRF Molecular Histology Facility, Queen’s Medical Research Institute, The University of\nEdinburgh, Edinburgh, UK\nLyndsey Boswell\nSuRF Molecular Histology Facility, Queen’s Medical Research Institute, The University of\nEdinburgh, Edinburgh, UK\nHee Y Lee\nNaason Science, Inc., Cheongju, Republic of Korea\nYeon G Kim\nNaason Science, Inc., Cheongju, Republic of Korea\nKyung H Park\nNaason Science, Inc., Cheongju, Republic of Korea\nLarry C Park\nNaason Science, Inc., Cheongju, Republic of Korea\nPatrick J Sweeney\nNaason Science, Inc., Cheongju, Republic of Korea\nGerard Feraille\nSyneos Health France, Biot, France\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 21 of 33\nAlessandro Taddei\nSyneos Health France, Biot, France\nDavid Chagras\nSyneos Health France, Biot, France\nThierry Alvarez\nSyneos Health France, Biot, France\nScott P Webster\nCentre for Cardiovascular Science, Queen’s Medical Research Institute, The University of\nEdinburgh, Edinburgh, UK\nAndrew Horne†\nEXPPECT Edinburgh, Institute for Regeneration and Repair, Edinburgh Bioquarter, The\nUniversity of Edinburgh, Edinburgh, UK, MRC Centre for Reproductive Health, Institute for\nRegeneration and Repair, Edinburgh Bioquarter, The University of Edinburgh, Edinburgh, UK\n†Senior authors\nPhilippa TK Saunders†\nEXPPECT Edinburgh, Institute for Regeneration and Repair, Edinburgh Bioquarter, The\nUniversity of Edinburgh, Edinburgh, UK, MRC Centre for Reproductive Health, Institute for\nRegeneration and Repair, Edinburgh Bioquarter, The University of Edinburgh, Edinburgh, UK\nORCID iD: 0000-0001-9051-9380\n†Senior authors\nDamian J Mole†\nCentre for Inflammation Research, Institute for Regeneration and Repair, Edinburgh\nBioquarter, The University of Edinburgh, Edinburgh, UK, Clinical Surgery, The University of\nEdinburgh, Edinburgh, UK\nORCID iD: 0000-0001-6884-7302\nFor correspondence: damian.mole@ed.ac.uk\n†Senior authors\nEditors\nReviewing Editor\nOmowumi Kayode\nMountain Top University, Makogi Oba, Nigeria\nSenior Editor\nBenoît Kornmann\nUniversity of Oxford, Oxford, United Kingdom\nReviewer #1 (Public review):\nSummary:\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 22 of 33\nThis study serves as a proof of concept for KMO inhibition as a new non-hormonal treatment\nfor endometriosis. The authors investigated KMO expression in human endometrial and\nendometriosis lesion tissues, confirmed that KNS898 effectively inhibits KMO and alleviates\nmanifestations of endometriosis in mice - reduced endometriosis lesions and improved\nhyperalgesia and cage behaviour.\nStrengths:\n(1) Inhibition of KMO may present as a promising first-in-class non-hormonal therapeutic\nagent for patients suffering from endometriosis and the side-effects of hormonal treatments.\n(2) The expression of KMO in endometrial tissues was demonstrated in both human (multiple\npatients per AFS stage of disease) and mice tissues.\n(3) Measurement of multiple substrates/analytes of the KMO regulatory pathway was\nperformed and demonstrated strong correlation to each other in response to KMO inhibition.\n(4) The aims of study (as proof-of-concept) were achieved in the study and the results support\ntheir conclusions.\nWeaknesses:\nIf any dysregulation in the KMO/tryptophan metabolic activity, expression and/or pathway in\nendometriosis can be shown, this will strengthen the rationale for the use of KMO inhibitor in\nthe disease.\nhttps://doi.org/10.7554/eLife.99226.2.sa2\nReviewer #2 (Public review):\nSummary:\nThe authors aim to address the clinical challenge of treating endometriosis, a debilitating\ncondition with limited and often ineffective treatment options. They propose that inhibiting\nKMO could be a novel non-hormonal therapeutic approach. Their study focuses on:\n• Obtaining proof-of-concept for KMO inhibition as a novel therapy for endometriosis.\n• Characterising KMO expression in human and mouse endometriosis tissues.\n• Demonstrating the efficacy of KMO inhibition in improving histological and symptomatic\nfeatures of endometriosis.\nStrengths:\n• Novelty and Relevance: The study addresses a significant clinical need for better\nendometriosis treatments and explores a novel therapeutic target.\nWeaknesses:\n• Limited Mechanistic Insight: The study lacks a comprehensive investigation of the\nmechanistic pathways through which KNS898 affects endometriosis. The dysregulation of\nKMO activity and the kynurenine pathway in endometriosis remains poorly characterized,\nboth in the human condition and the experimental model. While the authors present\npreliminary evidence that kynurenine metabolites (KYN, 3HK, and KYNA) are not\ndysregulated in the experimental model of endometriosis, they show that KMO inhibition\nmodulates these metabolite levels and leads to some improvement in disease features.\nHowever, these findings do not significantly close the existing knowledge gap or provide a\nstrong rationale for targeting KMO as a therapeutic approach for endometriosis. Further\nmechanistic insights are necessary to justify the potential of KMO inhibition in this context.\nAchievement of Aims:\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 23 of 33\n• The authors demonstrated that KMO is expressed in endometriosis lesions and that KNS898\ncan induce KMO inhibition, leading to biochemical changes and improvements in few\nendometriosis features in a mouse model. Therefore, the authors addressed the proposed\nspecific aims. However, fail to provide a clear rationale for proposing KMO inhibition as a\nnovel therapy for endometriosis.\nSupport of Conclusions:\n• The conclusions are somewhat overextended given the limitations in mechanistic insights to\nexplain how KMO inhibition result in improvment of histological and symptomatic features\nof experimental endometriosis. The study provides promising initial evidence but requires\nfurther exploration to firmly establish the efficacy of KNS898 for endometriosis treatment.\nImpact on the Field:\n• The study introduces a novel therapeutic target to be explored for endometriosis, potentially\nleading to non-hormonal treatment options.\nUtility of Methods and Data:\n• The methods used provide a foundation for further research, although they require\nrefinement. The data, while promising, need more rigorous investigation and deeper\nmechanistic exploration to be fully convincing and useful to the community.\nhttps://doi.org/10.7554/eLife.99226.2.sa1\nAuthor response:\nThe following is the authors’ response to the original reviews.\nPublic Reviews:\nReviewer #1 (Public Review):\nSummary:\nThis study explores the therapeutic potential of KMO inhibition in endometriosis, a\ncondition with limited treatment options.\nStrengths:\nKNS898 is a novel specific KMO inhibitor and is orally bioavailable, providing a\nconvenient and non-hormonal treatment option for endometriosis. The promising\nefficacy of KNS898 was demonstrated in a relevant preclinical mouse model of\nendometriosis with pathological and behavioural assessments performed.\nWeaknesses:\n(1) The expression of KMO in human normal endometrium and endometrial lesions was\nnot quantified. Western blot or quantification of IHC images will provide valuable insight.\nGiven the differential expression of KMO in luminal epithelial cells lining the endometrial\nglands compared to the other parts of the endometrium, a general endometrial Western Blot\nprep is not going to be additionally helpful or accurate in addressing this question, without\ne.g. laser capture microdissection or single cell quantitative proteomics. Furthermore, KMO is\na flavin-dependent monooxygenase and the activity, especially generating the oxidative\nstressor product 3-hydroxykynurenine is far more dependent on kynurenine substrate\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 24 of 33\navailability than it is on actual enzyme abundance - although it is important to show (as we\nhave done), that KMO is present in the human endometrial glands and in human distended\nendometrial gland-like structures (DEGLS).\nIf KMO is not overexpressed in diseased tissues i.e. it may have homeostatic roles, and\ninhibition of KMO may have consequences on general human health and wellbeing.\nKMO certainly does have important homeostatic roles, for example as key step in the\nrepletion of NAD+ through de novo synthesis. Although with good nutrition and sufficient\nNAD+ precursors in the diet e.g. niacin, that specific role may be partially redundant. KMO\nknockout mice exhibit normal fertility and fecundity and do not show a survival deficit\ncompared to littermate wildtype controls (e.g. Mole et al Nature Medicine 2016). To further\ndevelop KNS898 towards clinical use, preclinical GLP safety and toxicology studies and\nhuman Phase 1 clinical trials will of course need to be completed, but that is standard for the\ndevelopment of any new drug\nIn addition, KMO expression in control mice was not shown or quantified.\nControl mice that were not inoculated intraperitoneally with endometrial fragments did not\ndevelop DEGLS and therefore there is nothing to show or quantify.\nImages of KMO expression in endometriosis mice with treatments should be shown in\nFigure 4.\nWe have now included a representative KMO immunohistochemistry image from each\nendometriosis group and included all KMO immunohistochemistry images in Supplementary\nInformation.\nThe images showing quantification analysis (Figure 4A-F) can be moved to\nsupplementary material.\nThis recommendation contradicts the emphasis placed by the same reviewer earlier\nregarding quantification, so we have elected to keep it where it is.\n(2) Figure 1 only showed representative images from a few patients. A description of\nwhether KMO expression varies between patients and whether it correlates with AFS\nstages/disease severity will be helpful. Images from additional patients can be provided\nin supplementary material.\nWe have added extra information to the Figure legend to clarify the disease stage of the\nsuperficial peritoneal lesions which were illustrated (Stage I/II) and to link them to the\ninformation in supplementary Table S1. In total we examined 11 peritoneal lesions and 5\novarian lesions (stage III/IV) – in every sample examined immunopositive staining was most\nintense in epithelial cells lining gland-like structures. Sections illustrated were chosen to\nillustrate this key finding.\n(3) For Home Cage Analysis, different measurements were performed as stated in\nmethods including total moving distance, total moving time, moving speed,\nisolation/separation distance, isolated time, peripheral time, peripheral distance, in\ncentre zones time, in centre zones distance, climbing time, and body temperature.\nHowever, only the finding for peripheral distance was reported in the manuscript.\nThis was indeed a large amount of output, which we rationalised for the benefit of a concise\npaper. The paper now includes a description of which parameters showed a difference with\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 25 of 33\ndrug treatment.\n(4) The rationale for choosing the different dose levels of KNS898 - 0.01-25mg/kg was not\nprovided. What is the IC50 of a drug?\nKNS898 dosing has been extensively characterised by us in multiple species, and the pIC50\nhas already been published (e.g. Hayes et al Cell Reports 2023 and elsewhere). We now\ninclude the pIC50 in the present manuscript to save the reader from having to search through\nanother reference.\n(5) Statistical significance:\n(a) Were stats performed for Fig 3B-E?\nNow included, thank you.\n(b) Line 141 - 'P = 0.004 for DEGLS per group'\nHowever, statistics were not shown in the figure.\nThanks, now displayed on figure.\n(c) Line 166 - 'the mechanical allodynia threshold in the hind paw was statistically\nsignificantly lower compared to baseline for the group'\nHowever, statistics were not shown in the figure.\n(d) Line 170 - 'Two-way ANOVA, Group effect P = 0.003, time effect P < 0.0001' The stats\nneed to be annotated appropriately in Figure 5A as two separate symbols.\nArguably the far more important comparison in this figure is whether there is any effect of\ntreatment, and to mark multiple statistical comparisons on the figure would make it difficult\nto understand. Instead, the figure legend and results text have been clarified on this point.\n(e) Figure 5B - multiple comparisons of two-way ANOVA are needed. G4 does not look\ndifferent to G3 at D42.\nMultiple comparison testing (Dunnett’s T3) was done and the results have been clarified in\nthe text and figure legends.\n(f) Line 565 - 'non-significant improvement in KNS898 treated groups'. However, ** was\nannotated in Figure 5A.\nThank you. This is an error that has been checked and corrected.\n(6) Discussion is very light. No reference to previous publications was made in the\ndiscussion. Discussion on potential mechanistic pathways of KYR/KMO in the\npathogenesis of endometriosis will be helpful, as the expression and function of KMO\nand/or other metabolites in endometrial-related conditions.\nThe discussion is deliberately concise and focussed. The paper has 21 references to previous\npublications. A speculative discussion is generally not favoured by us.\nThe findings in this study generally support the conclusion although some key data which\nstrengthen the conclusion eg quantification of KMO in normal and diseased tissue is\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 26 of 33\nlacking.\nWe differ from the reviewer here and do not think that those data would materially affect the\nlikelihood of KMO inhibition being efficacious in human endometriosis in Phase 2/3 clinical\ntrials.\nBefore KMO inhibitors can be used for endometriosis, the function of KMO in the context\nof endometriosis should be explored eg KMO knockout mice should be studied.\nWe take the view that before KMO inhibitors can be used for endometriosis in patients there\nare multiple other regulatory and clinical development steps that are required that would be\na priority. While using a KMO knockout mouse might be an interesting scientific experiment,\nit would not impact on the critical path in a material way.\nReviewer #2 (Public Review):\nSummary:\nThe authors aim to address the clinical challenge of treating endometriosis, a\ndebilitating condition with limited and often ineffective treatment options. They propose\nthat inhibiting KMO could be a novel non-hormonal therapeutic approach. Their study\nfocuses on:\n• Characterising KMO expression in human and mouse endometriosis tissues.\n• Investigating the effects of KMO inhibitor KNS898 on inflammation, lesion volume, and\npain in a mouse model of endometriosis.\n• Demonstrating the efficacy of KMO blockade in improving histological and\nsymptomatic features of endometriosis.\nStrengths:\n• Novelty and Relevance: The study addresses a significant clinical need for better\nendometriosis treatments and explores a novel therapeutic target.\n• Comprehensive Approach: The authors use both human biobanked tissues and a mouse\nmodel to study KMO expression and the effects of its inhibition.\n• Clear Biochemical Outcomes: The administration of KNS898 reliably induced KMO\nblockade, leading to measurable biochemical changes (increased kynurenine, increased\nkynurenic acid, reduced 3-hydroxykynurenine).\nWeaknesses:\n• Limited Mechanistic Insight: The study does not thoroughly investigate the mechanistic\npathways through which KNS898 affects endometriosis. Specifically, the local vs. systemic\neffects of KMO inhibition are not well differentiated.\nWhile we agree that this is not a comprehensive mechanistic analysis, given that the ultimate\ntherapy would be almost certainly a once daily oral dosing i.e. systemic administration, we do\nnot consider differentiating local vs systemic effects of KMO inhibition to be critical to\ntherapeutic development in this scenario.\n• Statistical Analysis Issues: The choice of statistical tests (e.g., two-way ANOVA instead of\nrepeated measures ANOVA for behavioral data) may not be the most appropriate,\npotentially impacting the validity of the results.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 27 of 33\nThe selection of two-way ANOVA (time and group) is sufficient and correct for this\nexperimental analysis and its use does not invalidate the results. We agree that repeated\nmeasures ANOVA could be a valid alternative.\n• Quantification and Comparisons: There is insufficient quantitative comparison of KMO\nexpression levels between normal endometrium and endometriosis lesions,\nPlease see response above to quantification question raised by Reviewer 1.\nand the systemic effects of KNS898 are not fully explored or quantified in various tissues.\nPlease see earlier responses. KNS898 has been thoroughly explored in multiple tissues,\nspecies and experimental models, but those data do not need rehearsed here.\n• Potential Side Effects: The systemic accumulation of kynurenine pathway metabolites\nraises concerns about potential side effects, which are not addressed in the study.\nAs discussed above (response to Reviewer 1), KMO knockout mice exhibit normal fertility and\nfecundity and do not show a survival deficit compared to littermate wildtype controls (e.g.\nMole et al Nature Medicine 2016). To further develop KNS898 towards clinical use, preclinical\nGLP safety and toxicology studies and human Phase 1 clinical trials will naturally need to be\ncompleted, but this is standard for the development of any new drug.\nAchievement of Aims:\n• The authors successfully demonstrated that KMO is expressed in endometriosis lesions\nand that KNS898 can induce KMO blockade, leading to biochemical changes and\nimprovements in endometriosis symptoms in a mouse model.\nSupport of Conclusions:\n• While the data supports the potential of KMO inhibition as a therapeutic strategy, the\nconclusions are somewhat overextended given the limitations in mechanistic insights\nand statistical analysis. The study provides promising initial evidence but requires further\nexploration to firmly establish the efficacy and safety of KNS898 for endometriosis\ntreatment.\nWe do not agree that the conclusions are overextended based on the data presented, as\nexpanded in the reply to the eLife editorial assessment at the beginning of this response. It is\nclear that additional preclinical, regulatory and clinical development work, and human\nclinical trials will be required to firmly establish the efficacy and safety of KN898 for\nendometriosis treatment.\nImpact on the Field:\n• The study introduces a novel therapeutic target for endometriosis, potentially leading to\nnon-hormonal treatment options. If validated, KMO inhibition could significantly impact\nthe management of endometriosis.\nUtility of Methods and Data:\n• The methods used provide a foundation for further research, although they require\nrefinement. The data, while promising, need more rigorous statistical analysis and\ndeeper mechanistic exploration to be fully convincing and useful to the community.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 28 of 33\nWe believe that the data are a) convincing, and b) useful to the community. To be advanced\neffectively towards patients, KNS898 needs to follow the critical development path outlined\nabove.\nRecommendations for the authors:\nReviewer #1 (Recommendations For The Authors):\n(1) Change 'hyperalgia' to hyperalgesia throughout the manuscript including the title.\nDone\n(2) Line 69 - write '3-HK' in full.\nDone\n(3) Line 85 - the findings of the study include 'define the preclinical efficacy of KNS898 in\nreducing inflammation'. The inflammatory profile was not studied.\nChanged to “disease”\n(4) Line 259 - write 'EPHect' in full.\nDone\n(5) Line 260 - write 'AFS' in full. Also, abbreviate 'AFS' in the caption of Table S1.\nDone\n(6) 20 patients were listed in Table S1 but only 19 were accounted for in the methods\nsection.\nApologies there was an error and has now been corrected in the methods section as one of\nthe endometrial samples had not been included. Table S1 has also been changed to make it\nclear which samples were eutopic endometrium to differentiate them from the lesions.\n(7) The location from which the endometrial lesion tissues were obtained should be\nprovided in Table S1.\nTable S1 has been changed to make it clear that the subtypes of lesions examined were\nclassified as Stage I/II – superficial peritoneal subtype and Stage III/IV – endometrioma. The\nmethods section has also been updated to reflect these subtypes (lines 272-277).\n(8) Table S2 - G5 should be given compound 'A' not 'B'.\nThank you. Corrected.\n(9) Figure 2E was not referenced in the text and no figure legend was provided.\nNow referenced and the figure legend updated.\n(10) Figure 3A - font needs to be enlarged. HCA baseline recording was annotated as\nperformed twice in the protocol. When is the baseline taken and on what day was the\nWeek 12 measurement taken (refer to Figures 5C and D)?\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 29 of 33\nFont has been enlarged as requested. The second HCA baseline annotation in Fig 3A is a cut-\nand-paste error, now rectified and the time of second measurement annotated.\n(11) Line 133 - 'In KNS898-treated group G4 (endometriosis + treatment from Day 19),\nDEGLS formed in 4 of 15 mice (26.7%) and in G5 (Endo + treatment start on Day 26) in 6\nof 15 mice (40%) (Fig. 3f).'. The aforementioned data is not reflected in Figure 3F.\nThank you. This has been rectified.\n(12) Line 137 - 'Mice with endometriosis receiving KNS898 from the time of inoculation\n(G4) had an average of 2.0 DEGLS per animal with DEGLS (total = 8 DEGLS in 4 mice in\nG4) and those receiving KNS898 1 week after inoculation (G5) had an average of 1.8\nDEGLS per animal (total = 11 DEGLS in 6 mice in G5) (Figs. 3g and 3h).'\nThe aforementioned data is not reflected in Figure 3G. There is no Figure 3H shown.\nRectified as above.\n(13) Provide a discussion of why KA levels were significantly lower in Figure 3E compared\nto Figure 2C.\n(14) Figure legend for Figure 3 - G1 and G2 were noted as n=8. However, Figure S1 and\nTable S2 noted both groups as n=10.\nThank you. This is a typographical error. The legend for Fig 3 should indeed read n=10 for G1\nand G2 and has been corrected.\n(15) Line 181 - 'compared to non-operated and sham-operated control groups'. Only the\nsham group was shown in Figures 5C and D.\nThis text has been clarified to refer only to the data shown.\n(16) Figure 1 images need scalebars. Same for Figure 4.\nNow added\n(17) Figure 3B - y-axis is fold change?\nRelative concentration. Legend has been clarified.\n(18) Figures 5A and B - are the last Von Frey measurements taken on Day 40 (as per\nFigure 3A) or 42?\nTaken on Day 42. Fig 3A (the prospective protocol figure) has been clarified to reflect what\nactually happened (D42) as opposed to what was planned (D40) to pre-empt any further\nconfusion.\n(19) Symbols in Figure S1 need to be explained in the Figure legend.\nDone\n(20) Figures 2A and 2D should not be plotted in log scale to match the description of\nresults in Line 106 and Line 118.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 30 of 33\nThese particular results are plotted on a log scale to allow the reader to visualise that\ndetectable levels of drug are measurable at very low doses and that there is no significant\npharmacodynamic effect at that low dose. We choose to retain the present format.\nReviewer #2 (Recommendations For The Authors):\nComments and queries\nIntroduction/aims section:\nLine 82 - 87: Clarify in the proposal aims what is being accessed and analysed in humans\nand/or in animal models (mice). Specifically state clearly the correlations with KMO\nexpression. Were the correlations between KMO expression with features of\ninflammation performed only in mice or also in humans?\nThank you for this comment. The aims have been clarified in the Introduction.\nSection - KMO is expressed in human eutopic endometrium and human endometriosis\ntissue lesions:\nWas any quantitative or semi-quantitative method used to quantify the KMO expression\nin human tissues? Although the authors claimed that \"KMO was strongly immunopositive\nin human peritoneal endometriosis lesions\" by the representative figures it is not clear if\nKMO expression is similar, higher or lower between normal endometrium and peritoneal\nendometriosis lesions.\nWe have added extra information to the legend of Figure 1 to identify the PIN number of the\nsuperficial lesions illustrated. The key finding from the immunostaining with the antibody\nwhich had been previously validated as specific for KMO was that the most intense\nimmunopositive response was in glandular epithelial cells and the samples illustrate this\nresult.\nSection - Oral KNS898 inhibits KMO in mice:\nThe authors clearly confirmed the target engagement of KNS898 in inhibiting KMO\nactivity and, therefore, affecting upstream and downstream metabolites systemically in\n(peripheral fluid/ plasma) mice. Whether KNS898 effect is broad and targets systemic\nimmune cells and whole body cells and tissue was not explored. It was also not explored\nif KNS898 is able to specifically inhibit KMO locally at the endometrium tissue by\ntargeting epithelial and/or infiltrated immune cells, for example.\nThat is correct.\nIt would be interesting to measure (or if it was measured to report in this section and\nalso in Figure 2) the levels of KYN, KA and 3HK in naïve animals that did not receive\nKNS898. It would help to understand the net effect of KNS898 on the levels of kynurenine\npathway metabolites and, therefore, justify the dose chosen.\nThese data are already presented in Fig 3B-E, control group.\nPerhaps then the chosen dose could be lower considering the possible substantial\nchanges in kynurenine pathway metabolites levels, which are reported to exert an effect\nin many cells, tissues and systems and could, therefore, precipitate side effects. Even\nmore considering that the values for these metabolites are expressed as ng/ml, which\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 31 of 33\nhinders the comparison of the metabolite levels with the one reported for naïve animals\nin the literature. I would also suggest expressing the metabolite levels as nM/L.\nThis is not a relevant method of determining dose-limiting toxicity or safety\npharmacology/toxicology, either non-GLP or GLP. There are international guidelines on the\nproper conduct of those studies. This is also why it is important not to make claims about the\nsafety or otherwise of an experimental compound in an in vivo setting that has not explicitly\ncomplied with those regulatory standards. With regard to the units recommendation,\naccepted units are ng/mL or nM, not usually nM/L.\nSection - KMO blockade reduces endometrial gland-like lesion burden in experimental\nendometriosis in mice:\nLine 130: It would be better to replace \"blockade of 3HK production\" with \"reduction of\n3HK production\" to better reflect the results.\nChanged to “inhibition of 3HK production”.\nLine 140: In G5 (treatment starting at Day 26/ 1 week after inoculation), is the\nexperimental model of endometriosis already established with all pathological and\nphenotypic features?\nThis was not specifically tested in this experiment.\nLines 146 - 148: It would be better to specify that \"Overall, there was no significant\ndifference IN BODY WEIGHT between G3 and the KNS898 treatment groups G4 and G5\n(endometriosis + treatment from Day 26)\". Otherwise, this last sentence might be\ninterpreted as the overall conclusion of this result sub-section.\nThank you, a good point and has been corrected.\nThe authors demonstrated with an experimental approach that KMO blockade reduces a\npathological measure of endometriosis i.e., endometrial gland-like lesion burden, in\nexperimental endometriosis in mice when both administrated concomitant but also after\nthe disease development. Although mechanistic insights about how reduced KMO activity\ncan reduce the developed distended endometrial gland-like structures were not explored.\nTherefore, it remains to be investigated which (and how ) kynurenine pathway\nmetabolites are directly linked to the beneficial effects of KMO blockade in the\nexperimental model of endometriosis.\nWe agree.\nAlthough the beneficial effects on the pathological measures are evident, Figure 3 shows\nan exorbitant accumulation of KYN and KA and also a substantial reduction in 3HK after\nthe treatment with KNS898, which then raises concerns about tolerability and side\neffects. Would this effective KNS898 dose be viable and translational as a therapeutic\napproach?\nPlease refer to comments above at multiple junctures about safety pharmacology and the\nclinical development critical path.\nSection - KMO is expressed in experimental endometriosis in mice:\nBy histological examination, the authors confirm that the treatment with KNS898\nspecifically reduced the KMO expression intensity in the DEGLS from mice. Therefore, the\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 32 of 33\neffect exerted by KNS898 locally on the KMO expression at the DEGLS could be, at least,\npartially responsible for the beneficial effects observed in Figure 3 i.e., the reduction of\npathological measures. Although remains to be explored whether the effect of KNS898 in\nother cells or tissues could also be accountable for the beneficial effects exerted by\nKNS898 on the animal model of endometriosis.\nThis is correct.\nFrom a logical experimental point of view, I would suggest switching the order of the\nresult subsection \"KMO blockade reduces endometrial gland-like lesion burden in\nexperimental endometriosis in mice\" and \"KMO is expressed in experimental\nendometriosis in mice\" as well as the respective Figures 3 and 4.\nWe do not agree. Fig 3 (and section) is the macroscopic enumeration of DEGLS, Fig 4 (and\nsection) is the microscopic and immunohistochemical evaluation of the lesions introduced in\nFig 3. The sequence as originally presented is the more logical.\nSections - KMO inhibition reduces mechanical allodynia in experimental endometriosis -\nand - KMO inhibition reduces mechanical allodynia in experimental endometriosis:\nThe authors suggested that the KMO inhibition with KNS898 exerts beneficial effects on\nbehavioural paradigms related to the experimental model of endometriosis. Based on\nthe statistical analysis performed for the author, KMO inhibition with KNS898 reduces\nmechanical allodynia, as well as rescues, impaired cage exploration behaviour and\nmobility in mice with endometriosis. However, I believe that the most indicated statistical\ntests for Von Frey (allodynia behaviour) and Home cage (illness behaviour) analyses over\ntime would be repeated measures ANOVA and paired t-test, respectively (and not two-\nway ANOVA as performed). Therefore for a more trustful analysis and interpretation of\nthis data set, I would suggest the authors modify the statistical analysis and report the\ncorresponding interpretation of these tests.\nThe selection of two-way ANOVA (time and group) is suitable for this experimental analysis\nand its use does not invalidate the results. We agree that repeated measures ANOVA could be\na valid alternative.\nOverall, the authors present a solid and useful case for KMO inhibition as a potential\ntherapeutic strategy for endometriosis. However, the study would benefit from more\ndetailed mechanistic insights, appropriate statistical analyses, and an evaluation of\npotential side effects. With these improvements, the research could have a significant\nimpact on the field and pave the way for new treatment modalities for endometriosis.\nWe thank the reviewer for the positive comments and we have responded to the criticisms\nabove.\nSpecific recommendations for improvement:\n• Mechanistic Studies: Conduct detailed studies to understand the local vs. systemic\neffects of KMO inhibition and its specific impacts on different cell types and tissues. If not\nfeasible here, the authors could include in the discussion section a detailed overview of\nthe possible mechanisms implicated.\nWhile we agree that this is not a comprehensive mechanistic analysis, given that the ultimate\ntherapy would be almost certainly a once daily oral dosing i.e. systemic administration, we do\nnot consider differentiating local vs systemic effects of KMO inhibition to be critical to\ntherapeutic development in this scenario. We do not think speculation about possible\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.2 33 of 33\nmechanisms that is not supported by experimental data should be included. Furthermore,\nthat notion (of statements not supported by data) has been given as a criticism by the\nreviewers, and therefore consistency on this point must be preferable.\n• Quantitative Analysis: Include more robust quantitative methods to compare KMO\nexpression levels in different tissues and assess the correlation between KNO expression\nand pathological and behavioural changes.\nAs discussed above, the pathophysiological importance of KMO is in its enzymatic activity, not\nin its abundance as a protein, and 3HK production is far more dependent on kynurenine\nsubstrate availability rather than KMO protein abundance.\n• Appropriate Statistics: Use the most suitable statistical tests for behavioural and other\nrepeated measures data to ensure accurate interpretation.\nAs discussed above\n• Side Effect Evaluation: Investigate potential side effects of systemic KMO inhibition,\nparticularly focusing on the long-term implications of altered kynurenine pathway\nmetabolites. If not feasible here, the authors could include in the discussion section a\ndetailed overview of the possible side effects associated as well as inform if KNS898 can\ncross the BBB and its implications.\nFor a novel small molecule therapeutic compound in preclinical/clinical development, there\nare strictly regulated preclinical and clinical development standards that need to be met. It\nwould not be responsible to publish or make claims about safety and potential adverse effect\nprofiles without conducting the proper panel of tests within a suitable regulatory framework.\nhttps://doi.org/10.7554/eLife.99226.2.sa0","source_license":"CC0","license_restricted":false}