Pharmacological antagonism of EP2 receptor does not modify basal cardiovascular and respiratory function, blood cell counts, and bone morphology in animal models.

Compared to vehicle, mice treated with TG11-77.HCl (57 mg/kg/day) showed lower % increase in body weight over the 5 weeks of treatment (7.64% increase versus 10.7%, Fig 1B ). However, this observed difference was not statistically significant (p=0.11). In female mice, the weight increase in vehicle versus TG11-77.HCl treated mice was 9.83% versus 6.15% (p=0.054), whereas in male mice vehicle treated animals showed an increase of 11.6% versus 9.13% in TG11-77.HCl treated mice (p=0.73) ( Fig 1C ). It is worth noting that mice on vehicle treatment trended an increase of 0.8% after one week, whereas TG11-77.HCl treated mice trended a decrease of 1.32% body weight ( Fig. 1D ), but these observations are also statistically non-significant (P > 0.05, two-way ANOVA). To determine the effect on cardiovascular system, we used a tail-cuff method on mice to measure the systolic blood pressure (SBP) and HR every week. After five weeks of treatment with TG11-77.HCl (57 mg/kg/day), there was no significant difference in the blood pressure of vehicle treated mice versus TG11-77.HCl treated mice ( Fig. 1E ). Furthermore, no significant difference was observed in BP of mice treated with either vehicle or TG11-77.HCl, on any given week during the treatment ( Supplementary Figure S1A ). Similarly, mice treated with TG11-77.HCl for 5 weeks showed no significant difference in HR compared to vehicle treated mice ( Fig. 1F ). There was a significant increase in heart rate of mice after one week of treatment, but this increase in heart rate was similar in cohort treated with TG11-77.HCl and vehicle, suggesting the increase in heart rate might be a vehicle (pH 3.5 water) effect ( Supplementary Figure S1B ). To test the effectiveness of our experimental procedure in measuring changes in BP and HR we used pilocarpine (12 mg/kg) as a positive control. Mice showed a significant increase in BP (95.2 mm Hg versus 105 mm Hg, p<0.05) ( Fig 2A ) within 30min of pilocarpine injection. Treatment with pilocarpine resulted in significant reduction of HR (724 bpm versus 419 bpm, p<0.0001) ( Fig 2C ). However, in mice treated with vehicle versus EP2 antagonist TG11-77.HCl, there was no significant difference in the pilocarpine-induced change in BP ( Fig 2B ) and HR ( Fig 2D ). These results together indicate that EP2 antagonist TG11-77.HCl does not alter the cardiac response of mice, either under basal conditions or after pilocarpine. As shown in Figure 1A , we used the same cohort of mice, but switched them from regular diet to high-salt diet on day 41 and continued until Day 55-59 on high-salt diet with vehicle or EP2 antagonist treatment. Mice treated with a diet containing 10-fold elevated sodium showed a significant increase in their systolic blood pressure (SBP) after 14-19 days, compared to pre-HSD systolic blood pressure (109 mmHg versus 102 mmHg, p<0.01) ( Fig 2E ). When analyzed separately, this difference in systolic blood pressure observed in males was not statistically significant (113 mmHg versus 109 mmHg, p=0.25) ( Fig 2F ), but in females, HSD resulted in significant increase in systolic blood pressure (105 mmHg versus 96.1 mmHg, p<0.05) ( Fig 2G ). However, TG11-77.HCl (57 mg/kg/day) did not alter the effect of a high salt diet on systolic blood pressure ( Fig 2H ). There was a significant elevation in HR of mice post-HSD versus pre-HSD (735 bpm versus 684 bpm, p<0.0001) ( Fig 2I ). This increase in post HSD HR was independent of sex with significant changes observed in both male (741 bpm versus 708 bpm, p<0.05) ( Fig 2J ) and female (729 bpm versus 662 bpm, p=0.0001) ( Fig 2K ). However, change in HR of mice treated with vehicle versus TG11-77.HCl, pre- versus post-HSD was not significantly different (ΔHR post versus pre-HSD 55.9 bpm versus 45.3 bpm, p=0.54) ( Fig 2L ). These data indicate that the cardiac effects of the global EP2 KO ( Kennedy et al., 1999 ; Tilley et al., 1999 ) are not replicated by chronic exposure of adult mice to an EP2 antagonist. A separate cohort of mice (cohort 2, n=24) was used to ask whether chronic administration of this EP2 antagonist adversely affects overall health, and to determine if therapeutic drug levels are found in the plasma and the brain. In this study, treatment with TG11-77.HCl (62 mg/kg/day) for 4 weeks did not have significant effect on body weight ( Fig 3A ) or motor performance of mice when compared to vehicle as determined by the rotarod test by measuring latency to fall ( Fig 3B ). Similarly, 4 weeks of TG11-77.HCl treatment did not change the weight of internal organs such as heart, spleen, kidney, and liver along with no differences observed in weight of brain tissues cerebral cortex, hippocampus, and striatum ( Fig 3C ), indicating no overt toxicity by chronic treatment with the EP2 antagonist. We analyzed the concentration of TG11-77.HCl in plasma and brain of the mice used in this study and found about 3-fold higher total concentration of the TG11-77 in plasma, and 4.5-fold higher in the brain than its Schild potency against EP2 receptor (Schild K B = 9.7 nM) ( Amaradhi et al., 2020 ) with brain-to-plasma ratio of 1.4. ( Fig 3D ). To determine whether chronic administration of TG11-77.HCl altered the cellular composition of blood in mice, we investigated a separate cohort of mice (cohort 3, n=56) after treatment with TG11-77.HCl. Interestingly, TG11-77.HCl treatment (58 mg/kg/day) for 12 weeks did not produce any significant changes in complete blood counts, namely, red blood cells (RBCs), lymphocytes, monocytes, neutrophils and platelets ( Fig 4A ), although monocyte counts trended lower. Similarly, hemoglobin, hematocrit levels, red cell distribution width (RDWc) and platelet cell distribution width (PWDs) were unaltered ( Fig 4B ). These observations suggest that chronic dosing of EP2 antagonist has no adverse effect on cellular composition of blood. The above results indicate that chronic treatment of mice with the EP2 antagonist produced no effects on BP and HR. To generalize this conclusion we investigated a second EP2 antagonist, TG6-10-1, which displayed anti-inflammatory and neuroprotective efficacy in multiple models of brain injuries ( Jiang et al., 2013 ; Jiang et al., 2019 ; Kang et al., 2017 ; Li et al., 2020 ; Qiu et al., 2019 ; Rojas et al., 2015 ; Rojas et al., 2020 ; Yu and Jiang, 2020 ). Here we utilized rats (n=12) as animal species and used acute high dose administration of TG6-10-1 to study the effect on cardiovascular function. We studied the effects of TG6-10-1 on blood pressure (systolic, diastolic, and mean), pulse pressure, heart rate, and body temperature in conscious Sprague Dawley rats instrumented with a radiotelemetry transmitter. Acute administration by single intraperitoneal injection of TG6-10-1 at 50 mg/kg showed no significant change in cardiovascular function parameters of rats including heart rate (HR) ( Fig 5A ), systolic blood pressure (SBP) ( Fig 5B ), diastolic blood pressure (DBP) ( Fig 5C ), mean arterial pressure (MAP) ( Fig 5D ), pulse pressure (PP) ( Fig 5E ) as well as body temperature (BT) ( Fig 5F ). This dose is 5-fold higher than the efficacious dose ( Rojas et al., 2015 ; Rojas et al., 2016 ). Moreover, when animals after 7 days of high sodium diet (HSD) were injected with TG6-10-1, it had no effect on any of the cardiovascular parameters measured ( Fig 5 ). As expected, the positive control pilocarpine (12 mg/kg, IP injection) significantly decreased HR and BT ( Fig 5A and 5F ), significantly increased SBP ( Fig 5B ), DBP ( Fig 5C ), MAP ( Fig 5D ) and PP ( Fig 5E ) in rats irrespective of their diet. To study the effect of EP2 antagonist on respiratory function, respiratory assessment was performed following single intraperitoneal injection of TG6-10-1 in plethysmograph-restrained Sprague Dawley rats (separate cohort, n=32) that were treated with vehicle and three different doses of TG6-10-1. There was no significant effect observed on respiratory function of rats determined by breaths per minute after single injection of TG6-10-1 at the doses 5 mg/kg, 20 mg/kg, and 50 mg/kg when compared to vehicle injected rats (see methods for details). All three parameters of respiratory function, respiratory frequency ( Fig 6A ), tidal volume ( Fig 6B ), and minute volume ( Fig 6C ) showed similar values in 15-minute intervals at different doses of TG6-10-1 for up to 60 minutes after injection ( Fig 6 ). Given the beneficial effect of an EP2 agonist in a model of bone-injury ( Mitsui et al., 2011 ; Paralkar et al., 2003 ), we asked whether pharmacological antagonism of EP2 has any detrimental impact on bone volume and density in healthy mice. We used a separate cohort of mice (cohort 4, n=30) and tested the effect of chronic TG11-77.HCl treatment on bone phenotype. Both male and female C57BL/6 mice were treated with vehicle or TG11-77.HCl (134 mg/kg /day) in drinking water for 4 weeks. The tibia and femur from hind limbs were subjected to Microcomputed Tomography (μCT) scan to analyze the bone mass through diaphyseal scan and trabecular network through metaphyseal scan ( Supplementary Figure S2 ). The diaphysis of 0.6 mm volume-of-interest (VOI) at the mid shaft of tibia and femur did not show any difference in bone volume and density between TG11-77.HCl and vehicle treated mice in both males ( Fig 7A - C ) and females ( Fig 7D - F ). The TG11-77.HCl treatment also did not show any difference in the bone volume and density in metaphyseal scan at the growth plates of tibia and femur in both sexes, revealing there was no adverse effect of the TG11-77.HCl treatment in these mice ( Fig 7A - F ). Interestingly, the metaphyseal scan of the trabecular network showed a significant decrease in trabecular numbers upon TG11-77.HCl treatment only in males without altering the trabecular thickness and spacing ( Supplementary Fig S3 ). In contrast to the males, the trabecular numbers in females were found to be moderately increased but, as in males, no changes were observed in trabecular thickness and spacing ( Supplementary Fig S3 ). To evaluate further the effect of TG11-77HCl on trabecular network, in particular in trabecular number in male mice, we sectioned the fixed and decalcified samples of tibia and femur and analyzed by histology using H&E staining. We found no difference in cancellous bone density between the groups treated with vehicle or EP2 antagonist ( supplementary Fig S4 ). This result is discordant with the finding from microCT ( Fig S3 A - B ). Overall, we conclude that treatment with the EP2 antagonist at high dose for up to 1 month is unlikely to have a detrimental effect on bone density of healthy animals.

EP2 antagonists TG11-77.HCl and TG6-10-1 (chemical structures in supplementary Table S1 ) were synthesized and purified in our laboratory ( Amaradhi et al., 2020 ; Jiang et al., 2013 ). Pilocarpine was purchased from Sigma-Aldrich, prepared in saline and injected at dose of 12 mg/kg to induce changes in heart rate and blood pressure. Regular diet, containing 0.3% NaCl, was provided by Emory University DAR, for high salt diet the Teklad diet containing 3.15% NaCl was used. All animal procedures were approved by the Emory University Animal Care and Use Committee and conformed to the guidelines of the National Institutes of Health. Animals, C57Bl/6 mice (8-16 weeks old) and SD rats (14-16 weeks old) used in this study were purchased from Charles River Laboratories. Multiple cohorts of animals were used in this study as shown in Table 1 . We have shown that EP2 antagonist TG6-10-1 is anti-inflammatory, neuroprotective and prevents cognitive deterioration in rats after status epilepticus at a dose of 10 mg/kg via intraperitoneal injection ( Rojas et al., 2015 ; Rojas et al., 2016 ), therefore to assess the safety of this EP2 antagonist we selected 50 mg/kg (5-fold more than efficacious dose). Moreover, we have also shown that EP2 antagonist TG11-77.HCl.2H2O is anti-inflammatory in a mouse model of Alzheimer’s disease at 10 (free base 8.4) mg/kg I.P., and can be administered orally by supplanting in drinking water, therefore we used a projected oral dose of 100 (free base 84) mg/kg (>5-fold more than efficacious dose) in this study. We used pilocarpine (a muscarinic receptor agonist) at 12 mg/kg in the blood pressure and heart rate assays to verify the sensitivity of the instrument. TG11-77.HCl was dissolved in water at pH 2.8-3.5 at concentration of either 0.5 or 1.5 mg/ml with an aim to achieve a dosage of either 100 or 300 (free base 84 or 252) mg/kg/day respectively. Either water or water containing TG11-77.HCl was provided to cages containing 3 mice each for drinking. Consumption in ml was measured once a week and the average amount of TG11-77.HCl utilized for each mouse was calculated as mg/kg/day. The actual drug dose delivered was determined taking into account the 92.5% recovery from the drinking water on 7th day at room temperature, measured by HPLC. The resulting doses of drug are shown in supplementary Table S1 and mentioned in the results section for individual experiments. EP2 antagonist TG6-10-1 (a free base) has low aqueous solubility so was dissolved in vehicle and administered via intraperitoneal (IP) injection (see details below). The antagonist TG6-10-1 (EP2 K B = 17.8 nM, > 10-fold selective against other prostanoid receptors) has low water solubility ( 300-fold selective against other prostanoid receptors) has plasma half-life of 2.4 hrs. brain-to-plasma ratio 0.4 by oral delivery, and high water solubility (2.52 mM), which facilitate chronic dosing in drinking water at the strength of 0.5 mg/mL ( Amaradhi et al., 2020 ). These parameters guided us to administer TG6-10-1 acutely by IP-injection and TG11-77.HCl by oral dosing via drinking water. Moreover, the choice of the rodent is based on the technical considerations and test compound requirement for the planned experiments. For example, we preferred mice over rats for chronic dosing of antagonist due to limited amount of compound needed to treat the animals for nearly 2 months ( Fig 1A ). A total of 24 mice (cohort 1) were used in this study, n=12 (6 males and 6 females) in vehicle group and EP2 antagonist TG11-77.HCl group. On day 0 (D0), after measuring body weight (BW), blood pressure (BP) and heart rate (HR) using a tail-cuff method, mice were randomized and separated in cages containing either vehicle (water at pH 3.5) or TG11-77.HCl (0.5 mg/ml) resulting in a measured average consumption of 57 mg/kg/day free base (see supplementary Table S1 ). This dose is 7 to 14-fold higher than the dose that achieves anti-inflammatory efficacy in an acute brain injury model of status epilepticus (Varvel et al., in preparation). BP, HR, and BW were measured weekly for 5 weeks. On D39, mice were injected intraperitoneally with 12 mg/kg of pilocarpine, a muscarinic receptor agonist that has been shown to elevate blood pressure and reduce heart rate ( Moreira et al., 2003 ; Szeto and Hinman, 1990 ; Trendelenburg, 1961 ), and BP and HR were measured as a test of the sensitivity of the apparatus. On D41 mice were switched to a high salt diet (HSD) for 14-19 days with their BP, HR and BW measured weekly and at the end of study ( Fig 1 A ). Systolic blood pressure (SBP) and heart rate (HR) of mice were measured using a tail-cuff method ( Williams et al., 2019 ) with BP-2000 blood pressure analysis system (Visitech Systems), which measures BP and HR by transmission photoplethysmography. A total of n=24 animals, 12 males and 12 females were used in the study; animals were randomized into vehicle arm or TG11-77.HCl treatment arm (57 mg/kg/day), with both groups consisting of 6 males and 6 females. On the day of measurement, mice were acclimatized to the procedure room for 30 minutes before experiment. Then, 6 animals at a time were immobilized on the heated platform (37 °C) using adapters specific to mice. The mouse tail was put into tail-cuff and fixed using tape. Once immobilized, mice were given 10 minutes to get accustomed to being restrained on the platform before measurements were commenced. BP and HR were measured a total of 20 times with each recording episode being 45 sec, with 5 trial readings followed by 15 experimental readings. For data to be considered for final analysis, at least 10 out of 15 readings had to be recorded, with a standard deviation of <10%. If an animal’s readings did not meet the criteria, it was rested for a minimum of 30 minutes and the measurements were redone. In case of pilocarpine injection, if an animal’s readings did not meet the criteria it was excluded from final analysis, as it was not possible to reinject them and redo the measurements. Out of 24 animals, 6 were excluded for BP and 5 were excluded for HR. Output of the experiment was given by the software in the form of individual readings and average reading for BP and HR. Paired or Unpaired t-test were used for analysis depending on the requirement of the data set. GraphPad Prism was used for analysis. This study was performed by SRI Biosciences following our instructions. The objective of this study was to measure the effects of IP injection of TG6-10-1 on blood pressure (systolic, diastolic, and mean), pulse pressure, heart rate, and body temperature in conscious Sprague Dawley rats instrumented with a radiotelemetry transmitter. Pilocarpine was again used as a positive control compound. A total of n=24 animals were randomized and used in this study. Pilocarpine (12 mg/kg) and TG6-10-1 (50 mg/kg) in its vehicle (10% DMSO, 50% PEG400, and 40% DI water) were administered as a single dose by intraperitoneal injection into male Sprague Dawley rats following maintenance at low and high salt diets. Heart rate, arterial blood pressure (systolic, diastolic, pulse, and mean arterial pressure), and body temperature were collected every 2 min for at least 24 hours prior to dietary change or treatment on study days 7, 10, 11, 18, and 21. The same 12 animals from each group were dosed on study days 7 (normal salt diet) and 18 (high salt diet) with TG6-10-1 and on study days 10 and 21 with pilocarpine. The 24-hour analysis period was divided into three different phases, analysis phase 1 (0-2 hours after drug injection), analysis phase 2 (2-14 hours) and analysis phase 3 (14-24 hours). Data are reported from analysis phase 1 as the measured parameters returned to basal levels by 2 hours. On all other days, telemetry data were collected for 5 minutes every 60 minutes through the dosing period. Clinical observations were collected once daily beginning with administration of the regular diet (study day 0). Physical examination (piloerection, appearance, scabbing, presence of any anomalies on nose or neck, hair loss) were performed once weekly, and individual body weights were collected on each day of dosing (prior to dosing) and once weekly. Analysis was performed for a period of 2 hours post injection (PI) and was compared to baseline (BL) values for the corresponding time point before injection. For BL, data was averaged for the first 2 hours and for PI data from every 30 minutes was used. 3-way ANOVA (time, diet, and treatment) with Tukey’s multiple comparison test was used to analyze the data. GraphPad Prism was used for analysis. This study was performed by SRI biosciences following our instructions. The objective of this study was to evaluate the acute respiratory effects of TG6-10-1 when given via intraperitoneal injection to Sprague Dawley rats. Vehicle (10% DMSO, 50% PEG400, and 40% DI water) or TG6-10-1 in the vehicle was administered as a single dose by intraperitoneal injection to 4 groups (Groups 1 through 4) of 8 male Sprague Dawley rats/group at 0, 5, 20, or 50 mg/kg, respectively (n=32). For measurement of respiratory function parameters, each rat was placed in a single-chamber, head-out, neck-sealed plethysmograph. Respiratory frequency, tidal volume, and calculated minute volume were collected for at least 60 minutes prior to dosing and for at least 5 hours postdosing. Analysis was performed for a period of 1 hour post injection (PI) and was compared to 0 mg/kg value for the corresponding time point post injection. 2-way ANOVA with Tukey’s multiple comparison test was performed for analyzing the data. GraphPad Prism was used for analysis. C57BL/6 male mice at 8 weeks of age (n=24) were subjected to either vehicle (i.e., drinking water at pH 3.5) or TG11-77.HCl (average of 62 mg/kg/day) treatment through drinking water. After 4 weeks of treatment, mice were perfused with ice cold PBS to harvest different organs. Before perfusion, blood was collected by cardiac puncture and stored in EDTA tubes for further processing to separate plasma. Heart, liver, kidney, and spleen were collected, and wet weights immediately measured. Brain tissues were dissected to separate into neocortex and hippocampus before measuring their weights. All organ weights were normalized to the body weights of the individual mice. For determination of compound in brain and plasma, blood samples were collected in microcentrifuge tubes containing K2EDTA as anticoagulant. Plasma was separated by centrifugation of whole blood and stored below −70°C until analysis. Whole brain samples were homogenized using phosphate buffered saline (pH 7.4) and the total homogenate volume was thrice the brain weight. All samples were processed for analysis by protein precipitation using acetonitrile (ACN) and analyzed by LC-MS/MS (lower limit of quantification (LLOQ) was determined to be 1.23 ng/mL). Waters Xterra MS C18 column was used with flow rate 0.8 ml/min using the mobile phase 0.1 % formic acid in acetonitrile (pump A) and 0.1 % formic acid in water (pump B). Internal standard glipizide (ret. time 1.60 min) was used to quantify the peaks and the ratio was used for concentration analysis in the brain and plasma. The bioanalysis of the samples was done at Sai-life (CRO company), India. Unpaired t-test with Bonferroni correction was applied between groups for different organ weights. GraphPad Prism was used for analysis. Rotarod test trials were performed using the Rotamex rotarod (Columbus Instruments) in an isolated room. The device was set in an accelerating mode with initial speed at 4 rpm and increasing linearly to 40 rpm over 5 minutes. On the testing days, mice (n=24) were transported to the rotarod room two hours before the trials started. Either TG11-77.HCl (62 mg/kg/day) or vehicle treated mice were placed on the moving rod (1.5-inch diameter) separated by plexiglass sheets and the latency to fall from the rod was recorded for each mouse using automated timers set at the platform. Each day mice were subjected to 4 trials for 5 minutes each and after each trial, they were housed in the resting cage for 20 minutes before their next trial. If the mice did not fall at the end of the trial, their latency to fall was measured as 300 seconds. Mice were tested on day 0, 7, 14, 21 and 28 and their average latency to fall over days was represented as an index of muscle coordination upon drug treatment. Two-way repeated measure ANOVA with Sidak's multiple comparisons test was applied for Rotarod analysis. GraphPad Prism was used for analysis. Samples of fresh arterial blood were analyzed for complete blood count (CBC) in 20-week-old males and females (n=56), treated with vehicle (drinking water at pH 3.5) or TG11-77.HCl (58 mg/kg/day) in drinking water for 12 weeks. CBC analysis was performed by the Quality Assurance & Diagnostic Lab, Division of Animal Resources, Emory University. Blood samples were collected into KEDTA tubes, stored at 4 °C and analyzed within 24H by a VetScan HM5 v2.3 Hematology Analyzer. Multiple unpaired t-test with Bonferroni correction for multiple comparisons was applied between groups. p=0.05 was selected as the threshold for change. GraphPad Prism was used for analysis. C57BL/6 mice of both sexes at 10 weeks of age (n=30) were randomly subjected to either vehicle (drinking water with adjusted pH 2.8) or TG11-77.HCl (134 mg/kg/day) in vehicle. After 4 weeks of treatment, mice were sacrificed by overdose of isoflurane and bones were collected immediately from both hind limbs. Tibia and femur were separated, and the muscles and ligaments were carefully removed using a tweezer. They were then fixed in 4% paraformaldehyde solution in PBS overnight and stored in 70% ethanol at 4°C till further analyzed. Bone samples were shipped to the Microcomputed Tomography (μCT) Core at Georgia Tech, Atlanta for bone phenotyping analysis. Tibia and femur pairs were immersed vertically in specimen holders and supported by foam pieces to maintain orientation. μCT scans of the long bone mid-diaphysis and the metaphysis near the knee joint were performed using a Scanco CT40 (6 μm voxels, 300ms). For the diaphyseal scans, the length of the long bone was estimated from scout view images and 158 slices (6 μm) straddling the approximate mid-point were acquired. For tibial metaphyseal scans, 368 slices starting at the proximal end of the tibia were acquired to encompass the proximal metaphysis. Similarly, for femoral metaphyseal scans, 525 slices including the distal end of the femur were acquired to encompass the distal metaphysis. Each mid-diaphyseal scan was manually contoured at the initial slice around the outer margins of the cortex and then automatically adjusted with the native software for best fit around the cortex. The contouring was then automatically progressed to the remaining slices. Bone volume and density parameters were estimated from all mid-diaphyseal scans. For both metaphyseal scans, all images were first run through an auto-contouring script to delineate the boundary of the trabecular region within the cortical shell. Subsequently, 100 slices starting at a fixed number of slices distal to the end of the growth plate (and moving towards the mid-diaphysis, away from the knee joint) were analyzed after further manual adjustments of the contours as needed to better define the trabecular regions or margins. Total bone volume, density, and trabecular network parameters were estimated for all samples ( Supplementary Fig S2 ). Unpaired t -test with Holm-Sidak correction for multiple comparisons was applied between groups. p=0.05 was selected as the threshold for change. GraphPad Prism was used for analysis. The bone samples from the bone phenotyping experiment were used for this analysis. Tibia and femur samples were fixed in 10% neutral-buffered formalin and then stored in 70% ethanol until processed. Samples were decalcified then processed for standard histology and stained with hematoxylin and eosin. Histological sections were examined under a light microscope (Olympus BX41) by a board-certified veterinary pathologist. Representative images were prepared using a digital camera (Olympus DP25) and a connected imaging software (Olympus cellSens Standard). Samples were analyzed blindly. Cancellous bone density for each bone section was qualitatively scored on a scale of 0 to 5 (0 = most depleted cancellous bone density and 5 = normal density of cancellous bone). Unpaired t -test with Holm-Sidak correction for multiple comparisons was applied between groups. p=0.05 was selected as the threshold for change. GraphPad Prism was used for analysis.

A number of inflammatory CNS and peripheral diseases are exacerbated by excessive PGE 2 induction and its actions in particular via activation of EP2 receptor. Therefore, EP2 receptor antagonism with a small molecule has a therapeutic value. However, PGE 2 and EP2 are also had a physiological role in relaxing the vascular, respiratory and other smooth muscles, therefore, de-risking experiments with small molecules targeting physiological EP2 actions in naïve animals were necessary to advance a small molecule antagonist for clinical use for any disease indication. In this study, we provide experimental evidence that use of a small molecule EP2 antagonist by acute and chronic delivery should be safe for cardiovascular, respiratory, basic immune response, and bone density in healthy animals. Our results alleviate long-held reservations, which were merely derived from the studies of EP2-global knockout animals, against targeting EP2 and paving a path for EP2 antagonist to use against array of diseases. Further studies with EP2 antagonist in disease specific models are required to promote EP2 antagonist as a clinical agent.

In spite of the breadth of preclinical data suggesting the value of EP2 antagonists ( Sluter et al., 2021 ), there are limited advances towards clinical development. This is in part due to two published results that indicated EP2 global deletion reduces reproductive capability and arterial blood pressure and induces salt-sensitive hypertension ( Kennedy et al., 1999 ; Tilley et al., 1999 ). Therefore, exacerbating salt-sensitive hypertension would effectively preclude the chronic use of EP2 antagonists in humans. However, these studies could not distinguish between pharmacologically (acute or chronic) diminished EP2 activation and a developmental defect in these mice. Other studies reported that EP2 agonists promote healing of bone fractures ( Paralkar et al., 2003 ), raising further doubts about potential adverse effects of EP2 antagonists. In brief we found no effects of high dose pharmacologic EP2 block on cardiovascular or respiratory parameters, bone structure or blood cell counts. We tested two EP2 antagonists in adult EP2-expressing rodents: one antagonist (TG11-77.HCl) by chronic oral administration in mice, and another (TG6-10-1) by acute injection into rats. It is worth emphasizing that the cardiovascular and fertility adverse effects were only reported in EP2 global KO mice, and beneficial effects were reported in normal mice with the use of EP2 agonist. We wanted to monitor potential adverse effects in more than one rodent species (mouse and rat), using more than one EP2 antagonist, and by chronic and acute dosing protocols so the results can be generalized. Chronic treatment with an EP2 antagonist showed no adverse effect on cardiovascular function in mice ( Fig 1 , 2 ). Moreover, acute administration of an EP2 antagonist at high dose in rats was without any effect on cardiovascular ( Fig 5 ). These results are in discord with earlier findings, which indicated that EP2 gene KO results in reduced systolic blood pressure in mice on regular diet in one study ( Tilley et al., 1999 ), increased baseline systolic blood pressure in mice with high-salt diet in another study ( Kennedy et al., 1999 ) . The discordance between our findings and previous reports using EP2 gene KO animals may be attributed to role of EP2 in development at prenatal and postnatal stage, which further complicates the co-morbidities such as cardiovascular, respiratory function and fertility efficacy. - Our experimental results show that acute IP administration of an EP2 antagonist does not have any adverse effect on normal respiratory function in rats ( Fig 6 ). However, our study is limited to use of healthy mice and rats, therefore, further research in animals with underlying respiratory disease, such as asthma, would be a logical next step to have a better understanding of the effects of EP2 antagonism on respiratory and cardiovascular functions. EP2 receptor activation is associated with acute and chronic inflammation and autoimmune diseases ( Tsuge et al., 2019 ). EP2 receptor’s involvement in inflammation and immune response is well established in both in-vitro ( Lee et al., 2019 ; Robb et al., 2018 ; Yao et al., 2013 ; Yao et al., 2009 ) and in-vivo models ( Kabashima et al., 2007 ; Lee et al., 2019 ). Therefore, we intended to determine whether chronic treatment EP2 would induce any immunogenic responses in the blood. Our study showed no difference in blood composition of healthy mice treated with either vehicle or EP2 antagonist, TG11-77.HCl ( Fig 4 ) -. This observation further enhances the chance of pharmacological inhibition of EP2 to be a viable therapeutic target. An EP2 agonist, when directly injected into the bone marrow, induced bone healing in canine model of bone fracture ( Paralkar et al., 2003 ), and administration of PGE 2 (agonist, via EP2) prevented degeneration of articular cartilage during the early stages of osteoarthritis in rabbits ( Mitsui et al., 2011 ), suggesting EP2 activation can play a role in bone repair and healing. On the other hand, activation of EP4 receptor, but not EP2, rescued the impaired bone healing ( Xie et al., 2009 ), and EP2 deletion in mouse cultures impairs osteoclastogenesis ( Li et al., 2000 ), suggesting EP2 antagonism might be beneficial. Considering these mixed results, we tested an EP2 antagonist for adverse effects on bone phenotype. A high dose of EP2 antagonist TG11-77.HCl (134 mg/kg/day freebase) in drinking water for 28 days did not have any effect except number of bone trabeculae in the marrow cavity in males that was significantly reduced in the treated group. This observation can be explained by the role of prostaglandins in bone resorption and formation. It has been shown that systemic injection of prostaglandins can increase bone resorption and formation, but bone formation usually exceeds resorption; therefore, prostaglandin receptor antagonists can induce bone resorption ( Blackwell et al., 2010 ). Furthermore, prostaglandin treatment results in increased bone mass in male rats. Therefore, EP2 receptor antagonists could cause bone loss in male rats ( Ke et al., 1992 ). This observation must be further investigated to understand the interplay between sex hormones and TG11-77.HCl. A limitation of our study is that we did not investigate EP2 antagonist efficacy in a fractured-bone model. Therefore, our results further warrant exploration of EP2 antagonist efficacy in rheumatological models such as osteoarthritis and rheumatoid arthritis, or injured bone-models to understand whether EP2 receptor signaling will promote the activity of cells that are involved in bone remodeling process.

Inflammation is the natural response of the immune system challenged with either sudden or chronic insult. Inflammation is associated with neurodegenerative diseases, cancer, diabetes, and cardiovascular diseases ( Ganesh, 2014 ; Tsuge et al., 2019 ). Signaling pathways involved in inflammation include p38 MAPK, IL-6/JAK/STAT3, PI3K, Hippo ( Yeung et al., 2018 ), and COX-2 ( Rojas et al., 2019b ). Activation of these inflammatory pathways is an essential step in restoring normalcy and maintaining homeostasis. However, prolonged activation leads to secondary damage in nearby cells and tissues. To prevent deleterious effects of prolonged inflammation, targeting the pathways with small molecule drugs could be an effective strategy. Prostaglandin-E 2 (PGE 2 ), a lipid mediator that is ubiquitously present in tissues, is synthesized from arachidonic acid by cyclooxygenase-1 (COX-1) or cyclooxygenase-2 (COX-2) as the rate limiting enzymes ( Tsuge et al., 2019 ). PGE 2 exerts its physiological and pathological effects via four G-protein coupled receptors (EP1, EP2, EP3, and EP4) ( Rojas et al., 2019a ; Tsuge et al., 2019 ). PGE 2 signaling via EP2 is associated with increased inflammation in animal models of status epilepticus ( Jiang et al., 2013 ; Jiang et al., 2015 ; Rojas et al., 2015 ; Rojas et al., 2016 ), Alzheimer’s disease ( Johansson et al., 2015a ; Johansson et al., 2015b ; Liang et al., 2005 ), Parkinson’s disease ( Jin et al., 2007 ), amyotrophic lateral sclerosis (ALS) ( Liang et al., 2008 ), colitis ( Sheibanie et al., 2007 ), arthritis ( Honda et al., 2006 ), skin inflammation ( Kabashima et al., 2007 ), endometriosis ( Greaves et al., 2017 ), sepsis (Jiang et al., 2020) cancer and tumor progression ( Brouxhon et al., 2007 ; Donnini et al., 2007 ; Jiang and Dingledine, 2013 ; Kamiyama et al., 2006 ; Qiu et al., 2019 ; Sung et al., 2006 ). Moreover, in cell culture and mouse models lacking EP2, activation of innate immunity is accompanied by reduced cerebral oxidative damage ( Montine et al., 2002 ), enhanced amyloid-β (Aβ) phagocytosis ( Shie et al., 2005a ), reduced neurotoxicity by Aβ-peptide ( Shie et al., 2005a ; Shie et al., 2005b ), and reduced Aβ-peptide accumulation ( Hoshino et al., 2007 ). Based on these observations, inhibiting EP2 receptor pharmacologically could be an effective therapeutic strategy for reducing inflammation in a variety of diseases ( Ganesh, 2014 ). While studies listed above encourage the development and clinical advancement of EP2 antagonists for therapeutic applications, two studies published in 1999 raised serious questions about the viability of this target. Namely, one report showed that global EP2 KO in mice causes reduced reproduction rate and significant elevation in blood pressure with high salt diet compared to mice on regular diet ( Tilley et al., 1999 ). Similarly, a study by Kennedy et al., showed that EP2 deletion in mice results in salt-sensitive hypertension ( Kennedy et al., 1999 ). Subsequently, two other studies using a selective EP2 agonist reported that activation of EP2 promotes healing of fractured bone in a canine model ( Paralkar et al., 2003 ) and prevents the degeneration of articular cartilage during the early stage of osteoarthritis in rabbits ( Mitsui et al., 2011 ), suggesting EP2 activation has beneficial roles in bone repair and healing. Furthermore, evidence suggests that EP2 receptor function is associated with protective effects against fibroproliferative lung disease, aspirin-exacerbated respiratory disease (AERD), rhinitis and asthma ( Cahill et al., 2016 ; Kay et al., 2013 ; Machado-Carvalho et al., 2016 ; White et al., 2005 ; Ying et al., 2006 ). However, it was not clear whether EP2 has any role in normal lung function, and if using an EP2 antagonist could lead to any adverse effects on respiratory function. To promote EP2 antagonists for preclinical and clinical use for any disease indication, we must determine whether the adverse effects seen in EP2 KO animals are replicated by the antagonists in healthy animals. EP2 KO mice developed high systolic blood pressure only when they were fed high-salt diet, but not normal diet. ( Kennedy et al., 1999 ; Tilley et al., 1999 ). Therefore, the overall goal of this study was to test whether two EP2 selective antagonists, TG6-10-1 and TG11-77.HCl, by acute and chronic administration, have any adverse effects on cardiovascular function, respiratory function, bone morphology and cellular composition of blood in normal, non-disease mouse and rat models with or without high salt diet (HSD). We used two structurally different EP2 antagonists with different pharmacological profile; both mice and rats; two dosing protocols acute vs chronic. Gratifyingly, the results presented in this study reveal no adverse effects, and we conclude that the adverse effects observed in EP2 KO animals are not present with pharmacological EP2 antagonism.