Figures and data
Immunohistochemistry of KMO expression in human endometrium and distended endometriosis gland-like lesions.
Fixed tissue sections were stained with anti-KMO antibody (1:500 dilution) and visualized with DAB as described in the Methods section. Panel A. Normal human endometrium (200 X magnification); B1 and C1 insets denote areas shown in panels B and C at higher magnification. KMO expression is demonstrated as dark brown DAB-positive staining and is most intense in the epithelial cells lining the glands. Panels D through G. Human peritoneal endometriosis tissue lesions stained with anti-KMO antibody visualized with DAB. D/E PIN3652, Stage II, F/G PIN3306 Stage I. Note intense staining of cells lining glandular structures. Panel H. Ovarian-type endometriosis tissue lesion with higher magnification inset (I1) showing KMO expression present but at lower intensity in the mesothelial tissue surface (this sample does not have epithelial cells in the lesion).
KNS898 plasma levels and pharmacodynamic effect of KMO blockade.
Mice (n=3 per group, individual data shown) were given KNS898 twice daily by gavage at the doses shown. After 7 days, blood was sampled at euthanasia and KNS898 levels and kynurenine pathway metabolite levels measured by LC-MS/MS. A. KNS898 drug levels. B. Kynurenine. C. Kynurenic acid. D. 3-hydroxykynurenine (logarithmic scale). Comparison between groups by one 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 the kynurenine pathway showing the key step catalyzed by KMO.
Therapeutic effect of KNS898 in an experimental mouse model of endometriosis.
A. Experimental design. Ovariectomized (OVX) donor mice were hormonally stimulated as shown (E2 estradiol, P4 progesterone). At Day 19, donor mouse endometrial fragments were inoculated into recipient mice in a 1:1 ratio. KNS898 treatment 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 cases continued for 2 weeks. Groups were G1: n=10, control mice; G2: n=10, sham-operated control mice; G3: n=15, endometriosis + vehicle; G4: n=15, endometriosis with KNS898 commenced at Day 19; G5: n=15, endometriosis with KNS898 commenced at Day 26. B. KNS898 drug levels (relative concentrations). C. Kynurenine. D. 3-hydroxykynurenine. E. Kynurenic acid. Individual data are shown in panels B through E. Panels F and G. Enumerated distended endometriosis gland-like structures (DEGLS) in recipient mice by treatment group. F. Total number of DEGLS per group. G. Total number of DEGLS per animal for all animals in the group; bars show mean with s.e.m. (G). Comparison between groups by one way ANOVA with post hoc Tukey’s test. *P <0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s. not statistically significant.
Quantification of KMO expression in mouse model distended endometriosis gland-like structure (DEGLS) lesions.
Sections were visualized at 200 X magnification (A, B, C) with higher magnification insets shown (D, E, F). Panel A and D. Fixed tissue sections were stained with anti-KMO antibody (1:500 dilution) and visualized with DAB as described in the Methods section. B and E. QuPath was used to identify the epithelial tissue layers (yellow arrows denote the boundary) which were quantified by thickness. C and F. KMO expression intensity quantified and heat map expression values are overlayed. G. KMO expression staining intensity per unit area of endometriosis DEGLS epithelium, categorized by treatment group (G3 endometriosis + vehicle; G4 endometriosis + KNS898 from D19; G5 endometriosis + KNS898 from D26. Individual data points shown. Comparison between groups by one way ANOVA with post hoc Tukey’s test. *P <0.05.. H, I and J. Representative micrographs from G3 (H), G4 (I) and G5 (J) showing KMO expression in the epithelial cells lining each DEGLS.
Effect of KNS898 on mechanical allodynia and illness behaviour in an experimental mouse model of endometriosis.
A. Hind paw Von Frey filament test showing effect of endometriosis and KNS898 treatment in groups G3, G4 and G5 B. Bladder Von Frey filament test C. Home Cage Analysis of motility showing a daytime motility deficit in mice with endometriosis compared to control mice, and clear restitution of normal motility in KNS898 treated groups. D. Nighttime home cage motility analysis showing the benefit of KNS898 treatment on normalizing the motility deficit seen in mice with endometriosis. Data are mean with s.e.m. For A and B, statistical comparison between groups was by two-way ANOVA to compare Group effect and Time effect, with multiple group comparison using Dunnett’s T3 test. Asterisks represent treatment group effect statistical significance of the Dunnett’s T3 test comparing treatment group G5 to G3 vehicle control *P <0.05, **P <0.01. For C and D, Welch’s ANOVA with multiple group comparison using Dunnett’s T3 test was used. Although the ANOVA was statistically significant, the post hoc Dunnett’s T3 was not, therefore no asterisks are marked.
