{"paper_id":"b64a7901-5cf7-43ab-ad31-6a784177abb6","body_text":"Ben Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 1 of 22\nMedicine\nImmunology and Inflammation\nKynurenine monooxygenase blockade\nreduces endometriosis-like lesions,\nimproves visceral hyperalgia, 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, UK • EXPPECT Edinburgh, Institute for Regeneration and Repair, Edinburgh Bioquarter, The University\nof Edinburgh, UK • MRC Centre for Reproductive Health, Institute for Regeneration and Repair, Edinburgh\nBioquarter, The University of Edinburgh, UK • Mass Spectrometry Core, Edinburgh Clinical Research Facility, The\nUniversity of Edinburgh, UK • Centre for Cardiovascular Science, Queen’s Medical Research Institute, The\nUniversity of Edinburgh, UK • SuRF Molecular Histology Facility, Queen’s Medical Research Institute, The University\nof Edinburgh, UK • Naason Science, Inc., Republic of Korea • Syneos Health France, Les Templiers, 2400 route des\nColles, 06410 Biot, Sophia-Antipolis, France • Clinical Surgery, The University of Edinburgh, UK\nhttps://en.wikipedia.org/wiki/Open_access\nCopyright information\nAbstract\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.\nReviewed Preprint\nv1 • August 2, 2024\nNot revised\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 2 of 22\neLife assessment\nThe findings presented by the authors are useful within the focused scope of\nendometriosis treatment, providing a potential new therapeutic approach. The\nstrength of the evidence is, however, incomplete, as the main claims are only\npartially supported by the authors' data. The research nevertheless offers promising\ninitial evidence for KMO inhibition as a novel non-hormonal therapy for\nendometriosis, but further studies are needed to confirm efficacy and address any\npotential side effects.\nhttps://doi.org/10.7554/eLife.99226.1.sa2\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-HK, that is an oxidative stressor, causes protein cross-linking, and regulates the\nimmune-metabolic interface4     . Although there is no specific information about a direct role of\nKMO in endometriosis, there is evidence of dysregulated tryptophan metabolism in a recent study\nusing a preclinical non-human primate model of endometriosis6     , and increased kynurenine\npathway flux at the immune-metabolic interface between stromal cells and NK immune cells in\nendometriosis lesions7     .\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\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 3 of 22\nbioavailability and predicted half-life8     ,9     . KNS898 is a competitive inhibitor of kynurenine\nsubstrate at the active site of KMO8     ,9     . We propose that KMO inhibition is a novel therapeutic\nstrategy for endometriosis and, if successful, we will make a significant positive impact for women\nwith this painful, disabling condition. The aim of this project was to obtain proof-of-concept for\nKMO inhibition as a novel therapy for endometriosis. Specifically, we sought to define the\nexpression of KMO in biobanked human endometrial and endometriosis lesion tissues and\ncorrelate KMO expression with clinical features of inflammation (specifically hyperalgia and\naltered cage behaviour), confirm target inhibition of KMO by KNS898 in mice, and define the\npreclinical efficacy of KNS898 in reducing inflammation, endometriosis lesion volume and pain\nbehaviours in a experimental mouse model of endometriosis.\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\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\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 4 of 22Ben Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 4 of 22\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. Panels D\nthrough G. Human peritoneal endometriosis tissue lesions stained with anti-KMO antibody visualized with DAB. E and G are\ninsets shown in higher magnification. Panel H. Ovarian-type endometriosis tissue lesion with higher magnification inset (I1)\nshowing KMO expression present but at lower intensity in the mesothelial tissue surface.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 5 of 22\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.\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     ), blockade of 3HK production (Fig.\n3d     ) 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%) (Fig. 3f     ).\nAs expected, no DEGLS were formed in the non-inoculated control and sham groups. The total\nnumber of DEGLS per animal in each group was highest in G3 with an average of 4.0 per animal\nwith DEGLS (total = 32 DEGLS in 8 mice in G3). Mice with endometriosis receiving KNS898 from\nthe time of inoculation (G4) had an average of 2.0 DEGLS per animal with DEGLS (total = 8 DEGLS\nin 4 mice in G4) 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     ). Statistical analysis by\nANOVA showed a significant difference in endometriosis DEGLS burden between groups (P =\n0.0295 for DEGLS per animal; P = 0.004 for DEGLS per group). DEGLS axial length and derived\nvolume did not differ between groups (Supplementary Fig. 1a and b). All recipient mice\ninoculated with donor tissue lost body weight following inoculation which then gradually\nrecovered. After recovery, body weight of all three inoculated groups was lower compared to the\ncontrol groups for the duration of the study. Overall, there was no significant difference between\nG3 and the KNS898 treatment groups G4 and G5 (endometriosis + treatment from Day 26) (Suppl.\nFig. 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     ). The thickness (area divided by length) of the KMO positive epithelial layer was quantified\nfor each DEGLS section using QuPath and there was no difference between groups G3, G4 and G5\n(Fig. 4b      and 4e     ). However, quantification of KMO expression confirmed the high intensity of\nKMO staining in the epithelial lining layers (Fig. 4c      and 4f     ), but also showed a clear and\nstatistically significant reduction in KMO expression intensity in those areas in DEGLS removed\nfrom mice treated with the KMO inhibitor KNS898 (Fig. 4g     ; P = 0.008).\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 6 of 22Ben Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 6 of 22\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.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 7 of 22Ben Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 7 of 22\nFigure 3.\nTherapeutic effect of KNS898 in an experimental mouse model of endometriosis.\nA. Experimental design. Ovariectomized donor mice were hormonally stimulated as shown. At Day 19, donor mouse\nendometrial fragments were inoculated into recipient mice in a 1:1 ratio. KNS898 treatment 25 mg/kg twice daily by oral\ngavage was commenced at Day 19 or after a 1 week interval on Day 26 and in both cases continued for 2 weeks. Groups were\nG1: n=8, control mice; G2: n=8, sham-operated control mice; G3: n=15, endometriosis + vehicle; G4: n=15, endometriosis with\nKNS898 commenced at Day 19; G5: n=15, endometriosis with KNS898 commenced at Day 26. B. KNS898 drug levels. C.\nKynurenine. D. 3-hydroxykynurenine. E. Kynurenic acid. Panels F through H. 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. Individual data are shown in b through e; bars show counts (F) or mean with s.e.m. (G\nand H)). Comparison between groups by one way ANOVA with post hoc Tukey’s test. *P <0.05.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 8 of 22Ben Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 8 of 22\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.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 9 of 22\nKMO inhibition reduces mechanical\nallodynia in experimental endometriosis\nClinical endometriosis is associated with visceral hyperalgia and central sensitisation to pain10     .\nVisceral and central hyperalgesia may be tested in rodents using the Von Frey filament test11     .\nBaseline reaction values for hind paw and bladder Von Frey tests showed no significant difference\nin mechanical allodynia before inoculation. In established endometriosis without treatment\n(group G3), the mechanical allodynia threshold in the hind paw was statistically significantly\nlower compared to baseline for the group. When compared to the control groups at the\ncorresponding time point beginning 1 week after inoculation and continuing until the end of the\nstudy. KNS898-treated groups G4 and 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) (Fig. 5a     ). The mechanical allodynia threshold for the bladder reflex also was\nlower in mice with endometriosis compared to baseline throughout the study, and KNS898\ntreatment (G4 and G5) was associated with a statistically significant improvement in bladder\nmechanical allodynia threshold compared to mice with untreated endometriosis given vehicle\ncontrol (G3) (Two-way ANOVA, Group effect P = 0.038, time effect P < 0.001)(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 non-operated and sham-operated control groups, indicating\na negative effect on behaviour due to endometriosis. Importantly, mice with endometriosis treated\nwith KNS898 showed marked improvement in motility and cage exploration behaviour compared\nto untreated endometriosis mice, and although this difference between groups was statistically\nsignificant by Welch’s one-way ANOVA, post hoc testing (Dunnet’s T3) was not significant between\ngroups. This qualitative difference of time spent exploring the periphery of the cage being lower\nfor mice with endometriosis treated with vehicle control was seen in both the day and night\nphases (Figs. 5c      and 5d). Together, these data indicate that KMO inhibition with KNS898 results\nin an improvement in well-being evidenced by improved cage exploration behaviour in addition\nto improved objective histological 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\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\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 10 of 22Ben Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 10 of 22\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 in groups GS, G4 and G5 and a non-significant\nimprovement in KNS898 treated groups. B. Bladder Von Frey filament test C. Home Cage Analysis of motility showing a\ndaytime motility deficit in mice with endometriosis compared to control mice, and clear restitution of normal motility in\nKNS898 treated groups. D. Nighttime home cage motility analysis showing the benefit of KNS898 treatment on normalizing\nthe motility deficit seen in mice with endometriosis. Data are mean with s.e.m. For A and B, statistical comparison between\ngroups was by two-way ANOVA to compare Group effect and Time effect. Asterisks represent treatment group effect\nstatistical significance *P <0.05, **P <0.01. For C and D, Welch’s ANOVA was used.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 11 of 22\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 hyperalgia 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 hyperalgia.\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\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\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 12 of 22\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 EPHect guidelines12     . Patient summary characteristics are presented in\nSupplementary Table S1. Note there was a range of disease stages assigned at time of surgery\naccording to AFS criteria13     . Cycle stage was determined by measuring hormones in blood\naccording to standard protocols and assessment of eutopic endometrial tissue histology when such\nsamples were available14     . Three patients did not have a lesion at time of surgery (noted as stage\n0) and 6 samples of ovarian disease (endometrioma) were recovered (n=2 on hormones) with all\nother samples being considered characteristic of peripheral endometriosis lesions (n=10). General\nhistology 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 pH915     . 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)16     \nwas diluted to a final concentration of 1:1000 in NGS/PBS/BSA and incubated overnight at 4°C in a\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.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 13 of 22\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 13     C6-kynurenine, 13     C6-3-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)17     . 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 13     C6-3HK, m/z 195.1 → 177.2\nfor d5-KA and m/z 215.0 → 197.8 for 13     C6-kynurenine. Retention times for KYN, KA, 3HK and\nKNS898 were 1.8, 3.2, 1.2 and 6.5 mins, respectively and 3.2 mins for d5KA, 1.8 mins for 13     C6-\n3HK and 1.2 mins for 13     C6-KYN. Data were acquired by Analyst 1.7.1 software (AB Sciex) and\nlinear regression analysis was carried out on MultiQuant 3.0.3 software (AB Sciex) where peak\nintegrations and amounts of each kynurenine metabolite and KNS898 were calculated using the\npeak area ratio of compound/internal standard, with data further handled in Microsoft Excel 2016\nas 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\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 14 of 22\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.\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.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 15 of 22\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)16      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.\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. Data comparing multiple groups with unequal variances were analyzed with\nWelch’s ANOVA. Data not following the Normal distribution were analysed with non-parametric\nKruskal-Wallis test. Categorical and proportions data were analysed by Fisher’s exact test. Data\nwere visualised with GraphPad Prism.\nList of supplementary materials\nFig. S1 and Fig. S2. Table S1 and Table S2.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 16 of 22\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.\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\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.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 17 of 22\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.99226.1 18 of 22\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. (2021) Bioluminescent imaging in induced mouse models of\nendometriosis reveals differences in four model variations Dis Model Mech 14 https://doi\n.org/10.1242/dmm.049070\nHorne A.W., Saunders P.T.K., Abokhrais I.M., Hogg L., (appendix), E.P.S.P.S.G (2017) Top ten\nendometriosis research priorities in the UK and Ireland Lancet 389:2191–2192https://doi\n.org/10.1016/S0140-6736(17)31344-2\nMole D.J. et al. (2016) Kynurenine-3-monooxygenase inhibition prevents multiple organ\nfailure in rodent models of acute pancreatitis Nat Med 22:202–209https://doi.org/10.1038\n/nm.4020\nThul P.J., Lindskog C. (2018) The human protein atlas: A spatial map of the human\nproteome Protein Sci 27:233–244https://doi.org/10.1002/pro.3307\nAtkins H.M., Bharadwaj M.S., O’Brien Cox A., Furdui C.M., Appt S.E., Caudell D.L. (2019)\nEndometrium and endometriosis tissue mitochondrial energy metabolism in a\nnonhuman primate model Reprod Biol Endocrinol 17 https://doi.org/10.1186/s12958-019-0513\n-8\nLiu X.T. et al. (2018) Indoleamine 2,3-dioxygenase suppresses the cytotoxicity of 1 NK cells\nin response to ectopic endometrial stromal cells in endometriosis Reproduction 156:397–\n404https://doi.org/10.1530/REP-18-0112\nLiddle J. et al. (2017) The discovery of potent and selective kynurenine 3-monooxygenase\ninhibitors for the treatment of acute pancreatitis Bioorg Med Chem Lett 27:2023–2028https:\n//doi.org/10.1016/j.bmcl.2017.02.078\nWalker A.L. et al. (2017) Development of a Series of Kynurenine 3-Monooxygenase\nInhibitors Leading to a Clinical Candidate for the Treatment of Acute Pancreatitis J Med\nChem 60:3383–3404https://doi.org/10.1021/acs.jmedchem.7b00055\nRyan A., Healey M., Cheng C., Dior U., Reddington C. (2022) Central sensitisation in pelvic\npain: A cohort study Aust N Z J Obstet Gynaecol 62:868–874https://doi.org/10.1111/ajo.13596\nDeuis J.R., Dvorakova L.S., Vetter I. (2017) Methods Used to Evaluate Pain Behaviors in\nRodents Front Mol Neurosci 10 https://doi.org/10.3389/fnmol.2017.00284\nRahmioglu N. et al. (2014) World Endometriosis Research Foundation Endometriosis\nPhenome and Biobanking Harmonization Project: III. Fluid biospecimen collection,\nprocessing, and storage in endometriosis research Fertil Steril 102:1233–1243https://doi\n.org/10.1016/j.fertnstert.2014.07.1208\n1.\n2.\n3.\n4.\n5.\n6.\n7.\n8.\n9.\n10.\n11.\n12.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 19 of 22\n(1997) Revised American Society for Reproductive Medicine classification of\nendometriosis: 1996 Fertil Steril 67:817–821https://doi.org/10.1016/s0015-0282(97)81391-x\nNoyes R.W., Hertig A.T., Rock J. 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Using MultiQuant and Excel software to evaluate and report multi-analyte\ntargeted LC-MS/MS data\nGreaves E., Cousins F.L., Murray A., Esnal-Zufiaurre A., Fassbender A., Horne A.W., Saunders\nP.T. (2014) A novel mouse model of endometriosis mimics human phenotype and reveals\ninsights into the inflammatory contribution of shed endometrium Am J Pathol 184:1930–\n1939https://doi.org/10.1016/j.ajpath.2014.03.011\nHorne A.W., Ahmad S.F., Carter R., Simitsidellis I., Greaves E., Hogg C., Morton N.M., Saunders\nP.T.K. (2019) Repurposing dichloroacetate for the treatment of women with\nendometriosis Proc Natl Acad Sci U S A 116:25389–25391https://doi.org/10.1073/pnas\n.1916144116\nYun C.W., Yun S., Lee J.H., Han Y.S., Yoon Y.M., An D., Lee S.H. (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\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:\nThis study explores the therapeutic potential of KMO inhibition in endometriosis, a condition\nwith limited treatment options.\n13.\n14.\n15.\n16.\n17.\n18.\n19.\n20.\n21.\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 20 of 22\nStrengths:\nKNS898 is a novel specific KMO inhibitor and is orally bioavailable, providing a convenient\nand non-hormonal treatment option for endometriosis. The promising efficacy of KNS898\nwas demonstrated in a relevant preclinical mouse model of endometriosis with pathological\nand behavioural assessments performed.\nWeaknesses:\n(1) The expression of KMO in human normal endometrium and endometrial lesions was not\nquantified. Western blot or quantification of IHC images will provide valuable insight. If KMO\nis not overexpressed in diseased tissues ie it may have homeostatic roles, and inhibition of\nKMO may have consequences on general human health and wellbeing. In addition, KMO\nexpression in control mice was not shown or quantified. Images of KMO expression in\nendometriosis mice with treatments should be shown in Figure 4. The images showing\nquantification analysis (Figure 4A-F) can be moved to supplementary material.\n(2) Figure 1 only showed representative images from a few patients. A description of whether\nKMO expression varies between patients and whether it correlates with AFS stages/disease\nseverity will be helpful. Images from additional patients can be provided in supplementary\nmaterial.\n(3) For Home Cage Analysis, different measurements were performed as stated in methods\nincluding total moving distance, total moving time, moving speed, isolation/separation\ndistance, isolated time, peripheral time, peripheral distance, in centre zones time, in centre\nzones distance, climbing time, and body temperature. However, only the finding for\nperipheral distance was reported in the manuscript.\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?\n(5) Statistical significance:\n(a) Were stats performed for Fig 3B-E?\n(b) Line 141 - 'P = 0.004 for DEGLS per group'\nHowever, statistics were not shown in the 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 need to\nbe annotated appropriately in Figure 5A as two separate symbols.\n(e) Figure 5B - multiple comparisons of two-way ANOVA are needed. G4 does not look\ndifferent to G3 at D42.\n(f) Line 565 - 'non-significant improvement in KNS898 treated groups'. However, ** was\nannotated in Figure 5A.\n(6) Discussion is very light. No reference to previous publications was made in the discussion.\nDiscussion on potential mechanistic pathways of KYR/KMO in the pathogenesis of\nendometriosis will be helpful, as the expression and function of KMO and/or other\nmetabolites in endometrial-related conditions.\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 lacking.\nBefore KMO inhibitors can be used for endometriosis, the function of KMO in the context of\nendometriosis should be explored eg KMO knockout mice should be studied.\nhttps://doi.org/10.7554/eLife.99226.1.sa1\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 21 of 22\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• Characterising KMO expression in human and mouse endometriosis tissues.\n• Investigating the effects of KMO inhibitor KNS898 on inflammation, lesion volume, and pain\nin a mouse model of endometriosis.\n• Demonstrating the efficacy of KMO blockade 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.\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 blockade,\nleading to measurable biochemical changes (increased kynurenine, increased kynurenic acid,\nreduced 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.\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, potentially\nimpacting the validity of the results.\n• Quantification and Comparisons: There is insufficient quantitative comparison of KMO\nexpression levels between normal endometrium and endometriosis lesions, and the systemic\neffects of KNS898 are not fully explored or quantified in various tissues.\n• Potential Side Effects: The systemic accumulation of kynurenine pathway metabolites raises\nconcerns about potential side effects, which are not addressed in the study.\nAchievement of Aims:\n• The authors successfully demonstrated that KMO is expressed in endometriosis lesions and\nthat KNS898 can induce KMO blockade, leading to biochemical changes and improvements in\nendometriosis 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 and\nstatistical analysis. The study provides promising initial evidence but requires further\nexploration to firmly establish the efficacy and safety of KNS898 for endometriosis 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 the\nmanagement of endometriosis.\nUtility of Methods and Data:\n\nBen Higgins et al., 2024 eLife. https://doi.org/10.7554/eLife.99226.1 22 of 22\n• The methods used provide a foundation for further research, although they require\nrefinement. The data, while promising, need more rigorous statistical analysis and deeper\nmechanistic exploration to be fully convincing and useful to the community.\nhttps://doi.org/10.7554/eLife.99226.1.sa0","source_license":"CC0","license_restricted":false}