Bentamapimod (JNK Inhibitor AS602801) Induces Regression of Endometriotic Lesions in Animal Models

Abstract

Endometriosis is an estrogen (ER)-dependent gynecological disease caused by the growth of endometrial tissue at extrauterine sites. Current endocrine therapies address the estrogenic aspect of disease and offer some relief from pain but are associated with significant side effects. Immune dysfunction is also widely believed to be an underlying contributor to the pathogenesis of this disease. This study evaluated an inhibitor of c-Jun N-terminal kinase, bentamapimod (AS602801), which interrupts immune pathways, in 2 rodent endometriosis models. Treatment of nude mice bearing xenografts biopsied from women with endometriosis (BWE) with 30 mg/kg AS602801 caused 29% regression of lesion. Medroxyprogesterone acetate (MPA) or progesterone (PR) alone did not cause regression of BWE lesions, but combining 10 mg/kg AS602801 with MPA caused 38% lesion regression. In human endometrial organ cultures (from healthy women), treatment with AS602801 or MPA reduced matrix metalloproteinase-3 (MMP-3) release into culture medium. In organ cultures established with BWE, PR or MPA failed to inhibit MMP-3 secretion, whereas AS602801 alone or MPA + AS602801 suppressed MMP-3 production. In an autologous rat endometriosis model, AS602801 caused 48% regression of lesions compared to GnRH antagonist Antide (84%). AS602801 reduced inflammatory cytokines in endometriotic lesions, while levels of cytokines in ipsilateral horns were unaffected. Furthermore, AS602801 enhanced natural killer cell activity, without apparent negative effects on uterus. These results indicate that bentamapimod induced regression of endometriotic lesions in endometriosis rodent animal models without suppressing ER action. c-Jun N-terminal kinase inhibition mediated a comprehensive reduction in cytokine secretion and moreover was able to overcome PR resistance.

Keywords: JNK inhibitor; MMP; MPA.; bentamapimod; cytokine; endometriosis; progesterone.

Figure 1.

Chemical structure of bentamapimod/AS602801-HCl/PGL5001-HCl; MW 595.

Figure 2.

Regression of endometriotic lesions established in nude mice with biopsies from normal volunteers (BNV) or biopsies from women with endometriosis (BWE). A, Biopsies from normal volunteers (BNV) were prepared for injection to establish lesions; 10 days after disease was induced, treatments began. Estradiol was delivered by implants, and bentamapimod (JNKi) was administered orally. Medroxyprogesterone acetate (MPA) was delivered by twice-weekly injection as described in the Methods section. Results are mean ± standard error from 2 independent experiments; n = 3 to 4 mice/group/experiment. B, Regression of endometriotic lesions established in nude mice with BWE. Results are mean ± standard error from 2 to 4 independent experiments; n = 3 to 4 mice/group/experiment. Asterisks represent analysis of variance (ANOVA) followed by Tukey test for comparisons with 17β-estradiol (E2) + progesterone (PR) (P < .05). Comparisons with E2 alone were not significant (P > .1).

Figure 3.

Western blots of MMP-3 and MMP-7 in conditioned media of explant cultures of human endometrium from normal volunteers (A) or patients with endometriosis (B). Biopsies for the uterine tissue were cut into small pieces and pretreated with estradiol for 24 hours. Organ cultures were challenged with various factors for further 72 hours in tissue culture inserts. Media were stored at −70°C until analyzed by Western blot. A, Bentamapimod (JNKi) reduces expression of MMP-3, and MMP-7, and markers of endometriosis in endometrial explant cultures obtained from volunteers. B, Bentamapimod (JNKi) restores progesterone regulation of MMP-3 and MMP-7 expression in endometrial explant cultures obtained from women with endometriosis. MMP indicated matrix metalloproteinase.

Figure 4.

Bentamapimod (AS-01) causes reduction in the expression of genes previously associated with endometriosis in endometriotic-like lesions established with biopsies from patients with disease. TaqMan quantitative polymerase chain reaction was performed on lesions from each treatment group within an experiment. The lesions from 4 independent experiments were analyzed, and the mean responses are represented. Gene expression analysis was conducted on a TaqMan low-density array. The responses are grouped according to proinflammatory cytokines (A), matrix proteins (B), and steroidogenic pathway (C). The results are normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT1) expression, and error bars represent standard deviation determind by analysis of variance (ANOVA) followed by Tukey test. Significance (P < .05) is represented by asterisks relative to control groups receiving estrogen alone (dotted line at 1.0).

Figure 5.

Bentamapimod at both 30 mg/kg twice daily (BID) and 60 mg/kg BID causes regression of surgically induced autologous endometriotic-like foci in rats. Uterine segments from each rat were used to establish lesions. Three weeks later, animals with confirmed endometriotic foci were assigned to treatment groups. A sham-treated group received autologous uterine segment removal and adipose transplant near the uterus; they did not develop lesions (not shown). Bentamapimod (AS-01; orally) was administered BID for 9 days, and Antide (subcutaneous injection) was delivered as a single injection. Data are mean ± standard error of the mean (SEM), **P < .05, ***P < .01, analysis of variance (ANOVA) followed by Tukey test.

Figure 6.

Histological analysis of rat endometriotic foci treated with vehicle (A, D, G, L), bentamapimod (AS602801) 60 mg/kg (B, E, H, M), and Antide 2 mg/kg (C, F, I, N). These sections have been stained for anti-total-c-Jun antibody (A, B, C), anti-phospho-c-Jun antibody (Ser73; D, E, F), TUNEL assay for apoptotic cell detection (G, H, I), and anti-CD45 antibody for leukocyte infiltration evaluation (L, M, N).

Figure 7.

Concentrations of proinflammatory cytokines in endometriotic-like foci (A) and in unaffected sites on contralateral uterine horn (B). Cytokines were measured in lysates (per 100 μg protein) prepared from endometriotic foci or unaffected sites (contralateral horn) for each animal; n = 3 samples/rat/group; *P < .05 and **P < .01, analysis of variance (ANOVA) followed by Tukey test. Bentamapimod = AS602801.

Figure 8.

Cytotoxic activity of natural killer (NK) cells from splenocytes. Splenocytes isolated from endometriotic rat after their treatment were subjected to lytic activity of the target cells as described in the Methods section. Data are mean ± standard deviation (SD), n = 3, P < .05; Dunnett multiple comparison test, endometriotic versus sham and endometriotic versus endometriotic + JNKi treated. Bentamapimod = AS602801.