Antagonizing the corticotropin-releasing hormone receptor 1 with an orally bioavailable drug reduces endometriosis pain and associated adhesions†

At the time of sacrifice, none of the sham-operated animals developed lesions at the suture sites. In rats with induced endometriosis, the percent of developed lesions was calculated as [(number developed lesions / total number implants) × 100%] and then averaged per treatment group. Lesions developed at 80.00% of suture sites in the endometriosis vehicle-treated group. In the drug-treated groups, the percentage of developed vesicles was 68.75%, 77.27%, and 72.72%, for endo-pexacerfont-acute, endo-pexacerfont-chronic, and endo-elagolix groups, respectively. Nonparametric Kruskal–Wallis test showed no difference in the percentage of lesion development among the drug-treated groups compared to the vehicle group [ Figure 2A ; H(4) = 0.814, P  > 0.05]. We also measured the total weight and area of developed lesions per animal and averaged within the treatment groups ( Figure 2B and C ). One-way ANOVA showed no significant differences in lesion weight [F(3,28) = 0.48, P  > 0.1)] or area [F(3,38) = 0.07, P  > 0.1] between the experimental groups. Please refer to Supplemental Table S1 for specific information about endometriosis lesion development. The grading of lesions based on their length is shown in Figure 2D . Animals in the endometriosis vehicle group exhibited lesions sized 6.0 mm or larger (grade 5) in 15% of cases. In contrast, those in the endometriosis pexacerfont acute group showed this in only 1% of cases. There was a trend toward fewer grade 3 lesions in the endometriosis pexacerfont acute and endometriosis elagolix chronic groups compared to the endometriosis vehicle group. Histological visualization of endometriosis lesions revealed a cystic structure with a fibrous or glandular stroma with no gross differences across treatment groups. In some instances, we observed a mixed inflammatory infiltrate, specifically in one endo-pexacerfont acute and one endo-pexacerfont chronic rat ( Supplemental Figure S1 ). Pexacerfont did not alter the growth of endometriotic lesions. Endometriosis was induced in rats and animals were treated with vehicle, acute pexacerfont, chronic pexacerfont, or chronic elagolix. (A) Percentage of implanted uterine fragments that developed into lesions (lesions) in each rat. (B) Total weight of developed lesions per rat. (C) Total cross-sectional area of developed lesions per rat. (D) Distribution of lesion histological grades (1 = least severe, 5 = most severe). Bars represent mean ± SEM; individual symbols represent single animals ( n  = 7–13 per group). We performed a semiquantitative analysis of the staining area and intensity for NGF and VEGF in formalin-fixed endometriosis lesions using immunohistochemical staining. For VEGF, groups with endometriosis receiving acute and chronic pexacerfont showed significantly lower VEGF intensity than those in the endometriosis vehicle group [ Figure 3A ; F(3,14.7) = 8.48, P  < 0.01; Dunnett’s: P  < 0.01 and P  = 0.05, respectively]. Similarly, the VEGF area was significantly lower in the endometriosis pexacerfont acute and chronic groups compared to the endometriosis vehicle group [ Figure 3B ; F(3,16.2) = 8.43, P  < 0.01; Dunnett’s P   0.1 for both intensity and area). Effects of pexacerfont on VEGF, NGF, and Ki-67 in endometriotic lesions. Immunohistochemistry and immunofluorescence were used to evaluate angiogenic and neuronal markers. (A, B) Semi-quantitative intensity and area scoring of VEGF staining. (C, D) Semi-quantitative scoring of NGF staining intensity and area. (E) Integrated optical density of Ki-67 immunofluorescence, a marker of cell proliferation. Each symbol represents a single rat; bars show mean ± SEM. Asterisks indicate statistical significance versus vehicle ( * P  < 0.05; ** P  < 0.01). (F–H) Representative images of VEGF and NGF immunohistochemistry (dark precipitate) and Ki-67 immunofluorescence (bright punctae nuclei) in vehicle-treated animals. We observed significant decreases in staining intensity and area with acute pexacerfont for NGF ( Figure 3C and D ) . ANOVA followed by Dunnett’s post hoc showed a significant main effect for intensity [F(3,7.8) = 10.23, P  < 0.01] and area [F(3,8.5) = 10.48, P  < 0.01], with the respective post hoc test revealing a difference between the acute pexacerfont group and vehicle for both measures ( P   0.1). Using immunofluorescence, we also quantified the integrated density (area x intensity) for Ki-67 as a cell proliferation marker. Analysis revealed that endometriosis animals that received chronic pexacerfont had a significantly lower Ki-67 density compared to the endometriosis vehicle group [F(4,12.1) = 6.22, P  < 0.01; Dunnett’s multiple comparison, P  < 0.05]. While the endometriosis group that received elagolix also showed a decrease in Ki-67, this difference did not reach statistical significance ( P  = 0.06). Using the Von Frey test, we measured allodynia at three different time points during the experiment: before endometriosis induction (baseline), before the first drug administration (Day 24, pre-drug), and before sacrifice (Day 59, pre-sacrifice). A two-way ANOVA analysis of force normalized to sham comparison found a statistically significant difference in endo-pexacerfont acute at pre-surgery vs. pre-sacrifice [main effect of time: F(2,116) = 23.24, P  < 0.001, post hoc, P  < 0.01] and pre-drug vs. pre-sacrifice ( P  < 0.01). In endo-pexa chronic and endo-elagolix chronic, we saw a significant difference in pre-drug vs. pre-sacrifice ( P  < 0.05 and P  < 0.01, respectively). These results show that animals that received chronic pexacerfont or elagolix have less pain since they can resist more force (have higher pain thresholds) ( Figure 4A ). Using the Hot Plate test, we measured thermal pain or hyperalgesia. A two-way ANOVA analysis of the latency to withdraw the forepaw revealed a main effect of testing days—Baseline, Day 24, Day 29—[F(2,112) = 3.19, P  < 0.05]. Still, post hoc analyses showed no difference among groups ( Figure 4B ) . Inflammatory pain was measured only once on Day 59 of the experiment, right before sacrifice, using the formalin test. An ordinary one-way ANOVA showed a statistically significant difference in the CPS change using the sham (no-endometriosis)-vehicle group as the normal behavioral baseline [F(3,46) = 4.36, P  < 0.01]. Higher CPS values indicate more inflammatory pain responses, and lower CPS values indicate the opposite. Specifically, the endo-pexacerfont chronic group showed significantly lower CPS compared to the endo vehicle (Dunnett’s post-hoc, P  < 0.05). However, none of the other treatment groups showed significant differences from vehicle-treated animals ( Figure 4C ). Pexacerfont reduces mechanical and inflammatory pain behaviors. Behavioral tests were performed at baseline, Day 24 (pre-drug), and Day 59 (post-drug). (A) Von Frey test: mechanical withdrawal force normalized to baseline. (B) Hot-plate latency normalized to baseline. (C) Formalin test: percentage change in CPS relative to sham controls. Groups were sham + vehicle, endometriosis + vehicle, endometriosis + acute pexacerfont, endometriosis + chronic pexacerfont, and endometriosis + chronic elagolix. Bars represent mean ± SEM; symbols correspond to individual animals. Color scales indicate increasing pain intensity. Statistical significance is denoted by * P  < 0.05; ** P  < 0.01; *** P  < 0.001. To assess if the drug treatment altered the gonadal hormones, we monitored the estrous cycle before surgical induction of endometriosis, during the first drug treatment week (days 25–31), and during the subsequent weeks of drug administration in groups with chronic drug treatment (days 39–45 and 53–59). All animals had normal estrous cyclicity prior to surgery ( Figure 5A ); animals also continued to cycle normally regardless of the treatment. We also quantified serum GnRH, LH, and FSH levels on the day of sacrifice. GnRH levels were unaffected by treatments ( Figure 5B ). However, when we calculated the FSH/LH ratio, the ratio decreased significantly in rats treated with elagolix compared with the vehicle group ( Figure 5C ; dark symbols represent rats in estrus). Nonparametric analysis showed a main effect of treatment [H(5) = 14.4, P  < 0.01, Dunns post hoc P  < 0.05]. This decrease in the hormonal ratio was not observed for any of the other treatment groups in comparison to the vehicle-treated group. Estrous cycling and gonadotrophin secretion. (A) Percentage of time animals spent in estrous cycle stages (estrus, diestrus, proestrus, and metestrus) before surgery and during drug-treatment intervals (days 25–31, 39–45, and 53–59). (B) Serum GnRH concentrations. (C) Serum follicle-stimulating hormone to luteinizing hormone (FSH/LH) ratio. Groups were sham + vehicle, endometriosis + vehicle, endometriosis + acute pexacerfont, endometriosis + chronic pexacerfont, and endometriosis + chronic elagolix. Data are mean ± SEM; symbols denote individual animals. Asterisks indicate significant differences ( * P  < 0.05). Total macroscopic score assessment included adhesions, diarrhea, colon thickness, and colon hyperemia at the time of sacrifice. Statistical analyses revealed a significant main effect of treatment [ Table 2 ; F(5,35) = 11.76, P  < 0.0001]. Post hoc analyses showed that endometriosis increased macroscopic scores compared with sham controls (vehicle-sham vs. vehicle-endometriosis, P  < 0.0001). The increase in macroscopic score was controlled with both acute and chronic pexacerfont since both groups’ averages were lower than the vehicle-endometriosis group ( P  < 0.01 for both groups). The endo-elagolix group also showed a decrease in the macroscopic score compared to endo-vehicle ( P  < 0.05), but of weaker magnitude than the endo-pexacerfont chronic group. When we observe each component of the macroscopic score in the table shown in Table 1 , we can see that adhesion scores were 37% and 51% less in the endo-pexacerfont acute and endo-pexacerfont chronic compared to the endo-vehicle group. The colon in rats with endometriosis showed decreased thickness in response to acute and chronic pexacerfont compared to endo-vehicle of 34% and 28%, respectively. Diarrhea was seldom observed, while changes in hyperemia were more variable. Colonic macroscopic score parameters per treatment group * Represents a significant difference from control. Using qRT-PCR, we quantified three inflammatory cytokines within the endometriosis lesions ( Figure 6A–C ). Nonparametric statistical comparisons revealed a main difference between groups for IL-6 [H(4) = 11.52, P  < 0.01], with the endo-pexacerfont chronic and the endo-elagolix chronic showing a significant decrease compared to vehicle (Dunn’s post hoc, P  < 0.05 for both comparisons). No statistical differences were observed for IL-1β. However, TNF-α showed a similar pattern to that observed for IL-6, where groups with chronic pexacerfont and elagolix administration had a decrease in mRNA expression within the lesions [H(4) = 12.64, P  < 0.01; post hoc, P  = 0.05 and P  < 0.002, respectively]. Pexacerfont modulates pro-inflammatory cytokine gene expression in lesions. Quantitative PCR was used to assess mRNA expression of (A) interleukin-6 (IL-6), (B) interleukin-1 β (IL-1β), and (C) tumor necrosis factor-α (TNF-α) in endometriotic lesions collected on Day 60. Data are expressed as ΔΔCt values normalized to GAPDH housekeeping gene and to vehicle controls. Bars represent mean ± SEM; points represent individual lesions ( n  = 6–10 per group). Asterisks denote significant differences vs. vehicle ( * P  < 0.05; ** P  < 0.01). We also quantified the same cytokines in peritoneal fluid using a multiplex plate to observe changes in the peritoneal environment ( Figure 7A and B ). No significant changes in IL-6 were noted. However, the expression of IL-1 β differed between groups [F(5,27) = 2.70, P  < 0.05], with only the endo-vehicle group showing a significantly increased expression compared to the sham group (post hoc, P  < 0.05). TNF-α levels were undetectable in the peritoneal fluid samples and, thus, not shown. Peritoneal fluid IL-6 and IL-1β levels. Day-60 plasma concentrations of (A) interleukin-6 (IL-6) and (B) interleukin-1 β (IL-1β) were measured in sham + vehicle, endometriosis + vehicle, endometriosis + acute pexacerfont, endometriosis + chronic pexacerfont, and endometriosis + chronic elagolix groups. Bars represent mean ± SEM; symbols indicate individual animals. * P  < 0.05 versus endometriosis + vehicle. Using the multiplex, we measured leptin in the rats’ peritoneal fluid, which has been reported to play a role in endometriosis development [ 29–32 ]. It is well described that increased fat content results in increased leptin release. Using the animal’s body weight as a proxy for increased fat, we analyzed the relationship between peritoneal leptin and body weight at sacrifice for all the treatment groups ( Figure 8A–D ). There were no significant differences in peritoneal leptin levels among groups ( Figure 8A ). Similarly, the rats’ body weight at sacrifice was comparable across groups, averaging (± SEM) 271 ± 2.8 grams ( Figure 8B ). Leptin increased with increasing weight in both the sham and vehicle groups, as physiologically expected [ 33 ]. However, when we used simple linear regression analysis between body weight and peritoneal leptin levels, we observed a negative correlation for the endo-elagolix endometriosis group (slope = −0.16) compared to all other groups (Sham = 0.38, endo-vehicle = 0.28, endo-pexacerfont acute = 0.15, endo-pexacerfont chronic = 0.45). This suggests a de-regulation of the hormonal peritoneal environment resulting from elagolix treatment ( Figure 8C and 8D ). Leptin levels, body weight, and their correlation. (A) Plasma leptin concentrations on Day 60 for all treatment groups. (B) Body weight at sacrifice. (C) Scatter plot showing leptin versus body weight for sham and endometriosis vehicle groups. (D) Scatter plot showing leptin versus body weight for acute pexacerfont, chronic pexacerfont, and chronic elagolix groups. Trend lines were fitted by linear regression. Bars depict mean ± SEM; symbols show individual animals. There were no significant differences in leptin or body weight across groups. Linear regressions showed a negative correlation of leptin to body weight for elagolix, which was not observed for any of the other groups.

This report presents solid evidence that antagonizing the CRHR1 receptor with pexacerfont decreased mechanical and inflammatory pain responses, proliferative activity within lesions, and decreased colonic macroscopic score, including peritoneal adhesions. Together with previous findings from our team indicating that antalarmin—a highly potent and selective CRHR1 antagonist decreases endometriosis lesion size and development [ 18 ], the CRH signaling pathways in the uterus present as a robust therapeutic target for endometriosis. The results also highlighted the superiority of pexacerfont over the currently commercially available elagolix for alleviating inflammatory pain and reducing peritoneal adhesion formation and colonic macroscopic score without altering the FSH to LH ratio. Moreover, the intermittent dosing schedule selected might represent an advantage for the clinical application, since women will not have to take a daily dose of the drug to control their endometriosis-associated pain or inflammation. Targeting CRHR1 may be a more convenient and acceptable alternative to current medications on the market, without the serious side effects. Alleviation of endometriosis-associated pain (of any type) is a primary clinical goal when treating patients with endometriosis. Nevertheless, pain management in this clinical population is significantly complicated by other types of pain, such as bladder pain syndrome, pelvic myalgia, and vulvodynia, among others [ 34 ]. Pain management in endometriosis also needs to consider neural mechanisms, including peripheral, central, and cross-sensitization, in these women. Therefore, focusing on signaling cascades that may impact all these factors in the peripheral and central nervous system is desirable. CRH is uniquely positioned to act at both central and peripheral (paracrine) sites, including reproductive tissues [ 15 ]. CRH serves as the principal regulator of stress by activating the HPA axis [ 35 ]. Beyond this established pathway, chronic pain is documented to disrupt limbic structures, such as the amygdala and hippocampus, through CRH activity [ 36–38 ]. Conversely, it is recognized that the paracrine activity of CRH in uterine tissue contributes to endometrial inflammatory responses, and the activation of CRH can be stimulated by prostaglandins [ 39 ]. Central and peripheral CRH activities signal painful stimuli. Despite CRH’s role in pain modulation, this is the first study to demonstrate an analgesic effect in a well-validated endometriosis model. In contrast to our previous findings with antalarmin, pexacerfont did not decrease endometriosis lesion size [ 18 ]. However, there are several notable differences between our previous study and this one. First, antalarmin was administered intraperitoneally, rather than orally, in the current study. Second, the dose of antalarmin administered was 20 mg/kg, which is double the dose administered in the experiments herein. This is also paralleled by increased potency for antalarmin, with a reported IC50 of 0.04 nM [ 40 ], compared to 7.2 nM for pexacerfont for the human CRHR1 [ 41 ]. However, bioavailability for pexacerfont via the oral route has been reported at 40% to 59% in rats, dogs, and chimpanzees, whereas antalarmin has an oral bioavailability of only 19.3% in macaques [ 41 , 42 ]. Lacking a measurable effect on endometriosis lesion gross morphology does not necessarily represent a lack of effectiveness, as our results showed a decrease in NGF and a trend towards decreased VEGF. This suggests reduced angiogenesis within the lesions [ 43 ], and potentially decreased sensitization overall [ 44 ], however, this still needs to be determined within our model. CRHR1 antagonists such as pexacerfont have been previously tested in the clinical scenario for irritable bowel syndrome (IBS) [ 45 ] and explored for use in chronic disease models of the abdominal and pelvic organs [ 40 ]. This is the first time that the effectiveness of pexacerfont against endometriosis-associated GI damage has been reported, specifically to adhesions, colon thickness, and hyperemia. It has been documented that endometriosis is frequently comorbid with IBS [ 46–48 ], leading to misdiagnosis and delays in obtaining appropriate treatment. The percentage of women with endometriosis who display functional gastrointestinal disturbances is as high as 77% [ 49 ]. Parallel findings reported that women with endometriosis have up to five times higher incidence of IBS [ 50 ]. A 2021 report in Australia explored the association between allergic and non-allergic food sensitivities in women with endometriosis, showing that there is substantial crossover of symptoms between endometriosis and gastrointestinal disturbances, including abdominal/pelvic pain, dyschezia, bloating, nausea, vomiting, flatulence, and diarrhea [ 51 ]. Our findings represent a significant advantage over current treatment options for endometriosis, given that GnRH modulators exacerbate gastrointestinal dysmotility and alter the regular enteric nervous system physiology [ 52 , 53 ]. The novel finding that pexacerfont reduces the macroscopic score, particularly peritoneal adhesions, is clinically significant for the treatment of endometriosis. Adhesions can develop when endometrial lesions bleed into the surrounding tissue, triggering an inflammatory response that forms bands of tissue that connect two organs. Reports show that adhesions associated with endometriosis can range from thin, filmy, and transparent to thick, dense, and opaque. In severe cases, pelvic adhesions can lead to a potentially life-threatening condition known as “frozen pelvis” [ 54 ]. Moreover, adhesions can impact the fertility potential of women with endometriosis [ 45 ]. Up to 74% of women present endometriosis-associated adhesions before any surgical intervention [ 55 ], and repeated surgical interventions significantly exacerbate this problem [ 56 ]. The presence of dense adhesions in endometriosis patients is associated with increased severe complications, such as ureteral and rectal injuries, as well as voiding dysfunction [ 57 ]. To our knowledge, no current oral pharmacological treatment is available to mitigate endometriosis-associated adhesions. Directly targeting adhesions in endometriosis could reduce laparoscopic complications, resulting in greater savings in direct healthcare costs. Our data support the clinical testing of pexacerfont as an add-on treatment after surgical excision or ablation of endometriosis to minimize adhesion formation. Further, postoperative peritoneal adhesions remain a challenge for intra-abdominal surgeries in indications beyond endometriosis and are a source of complications following general gynecological surgeries [ 58 ]. Physical barrier methods developed to cover tissues during surgery remain limited in efficacy and have limited uptake among surgeons [ 59 ]. Pexacerfont may represent an additional tool for modifying the economic burden and symptoms associated with complications arising from adhesions and accompanying intestinal inflammation. Pexacerfont had a negligible impact on the gonadal axis, as evidenced by no effect on the estrous cycle and no alterations in the FSH/LH ratio. Low FSH/LH ratios in the clinical setting (less than 1.34), as observed for elagolix herein, are associated with an increase in follicular phase length (+2.4 days) and a lower ovulatory rate, without changes in luteal phase length nor progesterone levels [ 51 ]. In parallel, leptin has been established as a potent modulator of fertility in animals and humans [ 60–62 ]. Our experiments showed the expected positive correlation between peritoneal leptin levels and higher animal body weights. However, the group that received elagolix showed a completely reversed linear correlation with a negative slope. While lower leptin levels might initially appear favorable for fertility, they could also impair immune function and cause irregularities in menstrual cycles [ 54 , 63 ]. Taken together, targeting CRH activity with pexacerfont appears to be a safer alternative to the GnRH pathway, as it has minimal effects on the gonadal axis, which could reduce undesired side effects in the clinical setting. The team is aware that preclinical observations may not always translate into clinical results. This study utilized the autotransplantation model of endometriosis in female rats, which has been validated at the molecular level by our team [ 64 ]. While the model has a strong validity for clinical endpoints [ 64 ], it will never replicate the clinical condition in its complexity. To our knowledge, no ovarian endometriosis or deep infiltrating endometriosis models exist. However, the experimental approach showed benefits in lesion histology, disease extent, inflammation, and pain, which strongly aligns with predictions of human responses in a well-characterized animal model. We are not reporting a complete mechanistic approach to how pexacerfont exerts its effects, as this would require significantly more research, including additional targeted experiments and resources. Future experiments could be designed to better understand the molecular and immunological mechanisms at play. The intermittent dosing paradigm used herein, while it does not directly correlate with the rat’s cycle, was designed with the rationale that the menstrual cycle in women exhibits hormonal variations, which could be advantageous for targeting specific stages of the cycle to reduce endometriosis. Additionally, we are taking advantage of the long half-life of pexacerfont, 2–4 weeks in humans [ 65 ], allowing us to reduce daily dosing. This approach responds to voice of customer data obtained by the team showing that a substantial proportion of women with endometriosis prefer not to take medication every day. However, intermittent dosing has some limitations, such as fluctuating drug levels and the possibility of reduced efficacy, and it might require increased monitoring by health providers. We decided to administer elagolix on the same schedule as pexacerfont for a more direct comparison. Additionally, the doses used herein directly translate to clinically relevant doses of both pexacerfont and elagolix, as calculated using the Food and Drug Administration’s human-equivalent dosing [ 66 ]. A previous report has shown that pexacerfont may result in maternal hyperthyroidism during pregnancy but when administered at 300 mg/kg/day, which is 30-times the mean AUC in humans at 100 mg/day [ 67 ]. The team recognizes that well-controlled preclinical studies do not always translate to clinical effectiveness, and further testing beyond the scope presented here may be needed.

Antagonizing CRHR1 with pexacerfont offers a promising non-gonadotrophic therapeutic strategy for endometriosis, effectively reducing pain and disease-associated adhesions with minimal impact on reproductive hormone balance. Unlike current hormonal treatments, pexacerfont targets inflammatory and proliferative pathways, addressing key unmet needs in endometriosis management. Together with our team’s previous findings on the benefits of antalarmin in endometriosis, these findings support further clinical evaluation of CRHR1 antagonists as safer, patient-centered alternatives to improve quality of life for those affected by endometriosis.

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Ponce Health Sciences University (PHSU), protocol number 2307143471. Female Sprague Dawley Rats, aged 60 days and with an average weight of 180 grams, were procured from the Animal House at Ponce Research Institute (PHSU, Ponce, Puerto Rico). Rats were housed in pairs in a controlled environment with a room temperature of 23°C and a 12-h light–dark cycle. Standard laboratory chow and tap water were provided ad libitum, and bedding was changed twice weekly. To minimize experimenter-induced stress, rats were handled for 7 days before endometriosis induction. Additionally, vaginal smears were collected to monitor the regularity of the estrous cycle. Experiments were conducted in the morning to minimize the influence of circadian rhythms. Animals were randomly allocated to five treatment groups: endo-vehicle acute ( n  = 15), endo-pexacerfont acute ( n  = 13), endo-pexacerfont chronic ( n  = 11), endo-elagolix chronic ( n  = 11), and sham-vehicle acute ( n  = 11). Acute administration refers to a single week of drug administration, while chronic administration refers to 7 consecutive days of drug administration starting on days 25, 39, and 53 post-endometriosis induction surgery for a total of 21 days of drug administration. Please refer to Figure 1 for details. Experimental timeline for the endometriosis study. Rats underwent surgery on Day 0 to induce endometriosis or serve as sham controls. Pre-surgery and pre-drug handling, smears, and baseline pain tests were performed before endometriosis induction surgery and on Day 24. Starting Day 25, animals received one of three oral treatments: pexacerfont (10 mg/kg) or vehicle for 7 days (acute regimen), pexacerfont (10 mg/kg) or vehicle for three 7-day cycles separated by 7-day drug-free intervals (chronic regimen), or elagolix (14 mg/kg; positive control) given on the same schedule as chronic pexacerfont. Rats were left undisturbed, except for weekly weighing between treatment cycles. Pain assessments (Von Frey, Hot-plate) were conducted at baseline, Day 24, and Day 59. Formalin test was conducted on Day 59 only. Tissues and endometriotic lesions were collected on Day 60. Colored boxes denote drug-administration periods; arrows mark time points for pain testing and tissue collection. The surgical procedures for inducing endometriosis in rats were conducted following the Vernon & Wilson model [ 26 ]. Rats were initially anesthetized in a chamber with 4% isoflurane and maintained at 2–3% using an open mask once positioned supine over a warm pad to maintain adequate body temperature. Under sterile conditions, a midline laparotomy was performed to expose the right uterine horn. A 2 cm segment of the distal portion was excised and placed in a sterile culture medium. Subsequently, the endometrium was exposed by carefully opening the right uterine horn, and four pieces measuring 2 mm ×2 mm were excised. These endometrial implants were sutured onto the mesentery of the small intestine, with the serosal surface facing the mesentery and positioned adjacent to the vessels. For the sham procedure, rats underwent a simulated surgery in which the right uterine horn was gently massaged for 2 min with fingertips to mimic mechanical handling without implantation. Additionally, four sutures were placed on the mesentery without the inclusion of uterine implants. The peritoneal cavity was irrigated throughout the surgery with copious amounts of sterile saline solution to minimize adhesions. Consistent with previous research protocols, endometriosis was allowed to progress for a total of 60 days before rats were euthanized. Before surgery, baseline assessments of mechanical and thermal pain were conducted in rats ( Figure 1 ). Following the induction of endometriosis but before drug administration, assessments were repeated. We utilized an electronic Von Frey apparatus (DCA Software, Ugo Basile) to evaluate mechanical pain or allodynia. Rats were acclimatized for 15 min in separate compartments (17 cm × 69 cm × 14 cm), followed by an additional 15 min of acclimatization with the investigator. A vinyl filament was applied to the left hind paw of each rat three times, followed by three applications to the right hind paw. Force and time were given by the apparatus and recorded. We employed a hot plate analgesia meter to assess thermal pain or hyperalgesia. Each rat was acclimatized to the hot plate apparatus at room temperature for 5 min prior to testing. The plate was preheated to 52°C before use. Animals were placed on the hot plate for 40 s, and the rat was recorded the whole time. Reaction time was later analyzed from the video recordings. Evoked pain due to injury, involving inflammatory, neurogenic, and central mechanisms of nociception, was measured using the formalin test. This test was conducted only once, the day prior to euthanasia. A 2.5% formalin solution was prepared by diluting 1 ml of 10% neutral buffered formalin with 3 ml of water. Each rat received a 0.05 ml injection of the formalin solution into the right hind paw. Animals were placed in individual modules and recorded for 1 h. For analysis, we used the recorded videos for scoring as follows: an observer blinded to treatment quantified behaviors by watching the video every 15 s for 20 observations over 5 min, then assigning a score to the most frequently repeated behavior in that 5-min block. This was repeated every 5 min for the duration of the video. The scoring scale is based on the following criteria: “0” if the injected paw is not favored, “1” if the injected paw has little or no weight, “2” if the injected paw is elevated and is not in contact with any surfaces, and “3” if the injected paw is licked, bitten, or shaken. The sum of each of the 12 scores was obtained and the cumulative pain score (CPS) formula was used for statistical comparison between treatments. Food-grade methylcellulose 0.5% diluted in water served as the vehicle for the experiment. Animals receiving pexacerfont, a corticotropin-releasing hormone antagonist, were administered a concentration of 10 mg/ml diluted in 0.5% methylcellulose solution. Elagolix, a hormonal therapeutic commonly used in patients with endometriosis, was used as a positive control at a concentration of 14 mg/ml, diluted in 0.5% methylcellulose in phosphate buffered saline (PBS). Pexacerfont was purchased primarily from Biotechne R&D Systems (Minneapolis, MN, USA) and subsequently from Biosynth International, Inc. (San Diego, CA, USA). Elagolix was purchased from MedChem Express (Cat. No.: HY-14789; Monmouth Junction, NJ, USA). Rats undergoing acute treatment were administered the compounds starting 25 days after induction of endometriosis and continued for 7 consecutive days. Chronic treatment regimens were initiated on days 25, 39, and 53 post-endometriosis induction ( Figure 1 ). All treatments were administered orally to the animals by placing the drug or vehicle solution into oyster crackers [ 27 ]. Full ingestion of the crackers was observed in each rat to ensure adherence to the treatments. The regular cycling pattern of the rats was confirmed by analyzing morphological changes indicative of the different phases of the estrous cycle (diestrus, proestrus, estrus, and metestrus) over several days. Vaginal smears were collected 7 days prior to endometriosis induction to ensure baseline regularity in the cycling pattern. On the day of surgery and during behavioral assessments, rats underwent vaginal cytological smears to verify consistent estrous cyclicity. Similarly, on days of drug administration, smears were obtained to assess the treatments’ impact on reproductive cyclicity, if any. Finally, all animals underwent vaginal smear analysis on the day of sacrifice to evaluate estrous cycle phases and their potential influence on experimental outcomes. After 60 days of endometriosis progression, animals were euthanized to evaluate disease severity and collect tissues. A one (1) ml solution of pentobarbital (65 mg/kg) was administered intraperitoneally to each rat before laparotomy. Upon verification of the appropriate plane of anesthesia, a terminal laparotomy was conducted to assess the extent of endometriosis and retrieve relevant tissues. We meticulously examined for the presence of lesions and the original sutures. Measurements of the lesions’ longest length and width were obtained using a digital caliper, and their growth was graded using a modified scoring system shown in Table 1 [ 24 ]. Lesion scoring parameters based on length The entire colon was excised and examined for macroscopic damage defined as the presence of adhesions, ulceration, diarrhea, and colon thickness, using a previously established scoring system [ 28 ]. Colon and uterine tissue segments were weighed and fixed in 10% formalin. Lesions were rapidly frozen in liquid nitrogen (for mRNA extraction) or fixed in formalin (for immunolabeling). Lesions were randomly selected for each procedure, aiming to maintain equal proportions of tissues across procedures. Serum and peritoneal fluid were collected and stored at −80°C until further assay. After embedding in paraffin blocks, lesions and uterine tissue segments of 4 μm were stained with hematoxylin and eosin to determine glandular epithelium and collagen deposition. Formalin-fixed and paraffin-embedded endometriotic lesions were cut at 4 μm thickness with a microtome (Microm HM340 E, Microm International) and mounted on positively charged glass slides. Tissue sections were deparaffinized with xylene, two changes, 15 min each, and hydrated in descending grades of ethanol for 3 min each to distilled water for 1 min. This was followed by a 3% hydrogen peroxide (Sigma-Aldrich) incubation for 15 min to block endogenous peroxidase and a 5-min PBS wash. After antigen retrieval (0.01 M Citrate-EDTA buffer, pH 6.0, 95–99°C for 40 min), slides were cooled for 20 min at room temperature, rinsed with two changes of distilled water for 2 min, and placed in PBS for 5 min. Slides were blocked with normal serum (HK112-9 K Bio Genex) for 15 min and followed by an overnight incubation with primary antibody (rabbit polyclonal nerve growth factor (NGF) antibody catalog # sc-548 Santa Cruz Biotechnology dilution 1:100, vascular endothelial growth factor (VEGF) antibody catalog # sc-7269 Santa Cruz Biotechnology, Inc. dilution 1:50 used, respectively). A negative control with PBS instead of primary antibody was run on each slide. On the second day, slides were washed with PBS for 5 min. A multi-link was used as the secondary antibody for 20 min, followed by PBS wash for 5 min. The slides were incubated with Streptavidin Peroxidase for 20 min (LP000-ULE Bio Genex). For development, one drop of 3,3′ Diaminobenzidine (DAB) (HK153-5KE Bio Genex) was used on each tissue, and the exposure was monitored for 3 min under a light microscope. Then, the slides were dipped in distilled water, counterstained with hematoxylin for 15 s, washed with running water for 5 min, dehydrated through graded alcohol, cleared with xylene, and mounted with cytoseal 60 (Epredia, Cat # 23-244256 Fisher Scientific). Four distinct areas of the lesions were photographed and evaluated independently by three blinded observers. Staining intensity was graded using a scoring system ranging from 0 (no staining) to 3 (strongest staining). Additionally, each area was assessed for the extent of staining: 0 for 0–10% of tissue stained, 1 for 10–40%, 2 for 40–70%, and 3 for 70–100%. We stained for Ki-67, a marker for cell proliferation, on segmented endometriotic lesions cut at 4 μm. Briefly, formalin-fixed paraffin-embedded tissue sections were deparaffinized with xylene-substitute, hydrated with descending grades of alcohols, boiled in Citrate-EDTA for 40 min, and blocked with normal goat serum. Tissues were incubated with primary antibodies (Ki-67 1:50 dilution cat#550609 BD Pharmingen) overnight at 4°C in a humidifying chamber. After washing with PBS, the tissues were incubated with highly cross-absorbed secondary antibodies (Molecular Probes by Thermo Fischer Scientific, Inc.) for 30 min at room temperature in a humidifying chamber. All tissues were counterstained with a nuclear dye, DAPI ( R37606 Invitrogen by Thermo Fisher Scientific) for 5 min. A tissue section on each slide was used as a negative control, receiving PBS instead of the primary antibody. Tissues were visualized with Nikon Confocal Microscopy. The integrated immunofluorescence density of three representative areas for each lesion tissue was analyzed using ImageJ Software. The Milliplex Rat Pituitary Magnetic Bead Panel (RPTMAG86K, MilliporeSigma) was used according to the manufacturer’s protocol to measure the concentration of Luteinizing Hormone (LH) and Follicle-Stimulating Hormone (FSH). The Milliplex Rat Adipocyte Panel (RADPCMAG-82K, MilliporeSigma) was used according to the manufacturer’s protocol to measure the concentration of IL-6, IL-1 β, and TNF- α in the rat’s peritoneal fluid. Both serum and peritoneal fluid were collected at the time of sacrifice. All samples were run in duplicate in the Luminex MAGPIX system, and concentrations were expressed in pg/ml. Rat (GnRH) Gonadotropin-Releasing Hormone ELISA Kit ( EKF57934 , BIOMATIK) has high sensitivity and excellent specificity for detecting GnRH. We measured GnRH in the rats’ serum in a 1:10 dilution following the manufacturer’s protocol. All samples were run in duplicate in the MultiSkan Reader, and concentrations were expressed in pg/mL. All serum samples were collected in the morning. Lesions that were frozen in liquid nitrogen were used for RNA extraction. Thirty milligrams of tissue were weighed and transferred to mRNAse-free micro centrifuge tubes filled with beads and homogenizing solution. Tissue samples were homogenized for 5 min using the Bullet blender (Advance Co. Troy, NY). Samples were extracted using RNeasy Mini Kit (QIAGEN Cat. No.: 74106). After extraction, RNA concentration and quality were verified using Nanodrop 2000 (Thermo Scientific Cat No.: ND2000). Then, 1 μg from the mRNA was converted to complementary DNA using iScript cDNA synthesis kit (BioRad Cat. No.: 1708891BUN). qPCR was performed using iQ SYBR Green Supermix (cat # 1708882, BioRad) and primers for IL-1 β (QIAGEN Cat. #:330001- PPR06480B-200), IL-6 (cat # 330001- PPR06483B-200 Qiagen), and TNF-α (Cat. #: 330001-PPH00341F-200 Qiagen). The cytokines IL-1 β, IL-6, and TNF-α are frequently reported in patients with endometriosis. Data were reported as fold change compared to control lesions (from vehicle-treated rats) using the equation 2-ΔCT. GAPDH was used as the housekeeping gene. Statistical analyses were conducted to compare the effects of the different drug treatments on the measured outcomes. Data were expressed as mean ± (SEM) unless otherwise noted. Normality was assessed using the Shapiro–Wilk test, and homogeneity of variances was evaluated with Barlett’s test. For normally distributed data, a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed to compare groups. Behavioral measures were analyzed using a two-way ANOVA, with time as a repeated measure variable. The Kruskal–Wallis test was used for non-normally distributed data, followed by Dunnett’s post hoc test. Simple linear regressions were used to examine the relationship between two continuous variables. Statistical significance was set at P  < 0.05. Analyses were performed using GraphPad Prism Software V. 10.4 (Boston, MA).

Endometriosis, characterized by the growth of endometrial-like tissue in ectopic locations, is a multifaceted, chronic condition with a highly heterogeneous presentation among individuals. Symptoms of endometriosis include chronic pelvic pain, dysmenorrhea (menstrual pain), dyspareunia (painful intercourse), dysuria (painful urination), dyschezia (painful defecation), metrorrhagia (intermenstrual bleeding), gastrointestinal disturbances, fatigue, and infertility necessitating lifelong management [ 1–4 ]. Current pharmacological treatments target the hypothalamic–pituitary–gonadal (HPG) axis and endogenous gonadal hormones through gonadotropin-releasing hormone (GnRH) agonists/antagonists, exogenous estrogens/progestins, and androgens, adversely affecting fertility. Due to serious side effects that include osteoporosis, increased liver enzymes, and suicidal ideations, GnRH modulators can only be taken for periods of 12 months [ 5 , 6 ]. This puts women at significant risk of relapse, which contributes to 70% of patients continuing to suffer from unresolved pain [ 7 ]. This represents an unmet need in the management of endometriosis, particularly among younger patients [ 8 ]. The complexity of treating endometriosis is exacerbated by the lack of correlation between disease severity, as determined by the American Society of Reproductive Medicine [ 9 ] or the Enzian classification [ 10 ] of surgical staging and pain severity. Multiple mechanisms, including inflammatory/immunological and neuropathic processes, are implicated, with local peripheral nerve activation leading to central sensitization [ 11–13 ]. The persistence and recurrence of symptoms despite hormonal treatments suggest that endometriosis involves additional physiological mechanisms beyond the gonadal/reproductive system. Recognizing significant unmet needs in endometriosis treatment, our team is shifting the therapeutic focus from the gonadal axis to the hypothalamic–pituitary–adrenal (HPA) axis, commonly known as the stress axis, which is known to be dysregulated in endometriosis [ 14 ]. The central signaling molecule of the HPA axis, corticotropin-releasing hormone (CRH; also known as corticotropin-releasing factor), is present in endometrial epithelial cells and stroma and is overexpressed in endometriotic tissues [ 15–18 ]. Despite CRH’s significant paracrine role in reproductive tissues and its involvement in inflammation, pain perception, neuroendocrine signaling, and hormonal regulation, its potential as a therapeutic target has been largely overlooked [ 19 , 20 ]. It is well known that estrogen facilitates the expression of CRH in endometrial tissue [ 21 ]. CRH, in turn, mediates inflammatory mechanisms and pain perception [ 22 , 23 ], making CRH a suitable target downstream of estrogen activity. Research from our team reveals the increased activity of the stress axis and the role of CRH receptor (CRHR) signaling in promoting the growth and development of endometriosis lesions [ 18 , 24 , 25 ]. The primary goal of this study was to explore the potential of CRHR antagonists in reducing pain perception and disease severity, with the long-term objective of developing and commercializing new therapeutic alternatives for endometriosis. Previous findings using the rat autotransplantation model of endometriosis demonstrated an increase in CRHR1 within endometriotic lesions and a 60% reduction in disease progression when CRHR1 was blocked with antalarmin, the first synthetic CRHR1 antagonist [ 18 ]. These findings suggested that antagonizing CRHR1 could alleviate endometriosis pain and decrease disease severity. This manuscript reports the results of pexacerfont, an orally bioavailable CRHR1 antagonist with a known safety profile, as a possible treatment for endometriosis-associated pain. CRHR1 antagonists were initially designed and clinically tested for the treatment of mood disorders and addictions. Still, they were never approved by the Food and Drug Administration due to the lack of effectiveness for the previously tested diseases. In the current manuscript, we tested and found that targeting the CRHR1 receptor with pexacerfont decreases proliferative activity within endometriosis lesions, making it a strong candidate for repositioning for gynecological disorders. We also observed minimal disruption of gonadal signaling compared with a currently available commercial gonadotropin-releasing hormone antagonist used for the treatment of endometriosis. Furthermore, the compound effectively minimizes endometriosis-associated adhesions, which are currently removed only by surgical intervention. By targeting CRHR1 signaling, this approach offers a fresh direction in the management of endometriosis, particularly for patients who are unresponsive to conventional hormonal therapies or are at risk of adverse effects. Continued research and clinical evaluation of CRHR1 antagonists, such as pexacerfont, may pave the way for more effective, safer, and patient-centered treatments, ultimately improving the quality of life for those affected by this challenging condition.