Selective mPGES-1 Inhibitor Ameliorated Adjuvant-Induced Arthritis in the Rat Model

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Introduction
Infammation is one of the complex biological responses of body tissues to harmful stimuli such as pathogens, injured cells, or irritants.It is also a process by which body's immune systems protect our body from these stimuli [1,2].Tere are two main types of infammation: acute and chronic [3][4][5].As compared to acute infammation characterized by 5 cardinal signs such as pain, redness, loss of function, swelling, and heat, chronic infammation is associated with various diseases such as cancer, heart disease, diabetes, asthma, arthritis, and Alzheimer's disease [5].
Prostaglandin E 2 (PGE 2 ) plays an important role in the physiology of the mammalian body but is a principal mediator of infammation [6].In cells exposed to diverse harmful stimuli, excess of arachidonic acid is converted into upregulated PGE 2 via the coupled action of both inducible cyclooxygenase-2 (COX-2) and terminal PGE synthase, particularly microsomal PGE synthase-1 (mPGES-1) [7].Terefore, nonsteroidal anti-infammatory drugs (NSAIDs) and selective cyclooxygenase-2 (COX-2) inhibitors reduce PGE 2 production to relieve infammation.However, these inhibitors have adverse efects such as the gastrointestinal bleeding of NSAIDs and the risk of serious cardiovascular events of COX-2 inhibitors [8,9].Tus, selective inhibition of mPGES-1, which is the terminal enzyme responsible for the conversion of PGH 2 into PGE 2 , has been suggested as a safer therapeutic strategy without afecting the normal production of other prostaglandins related to the body's homeostasis [10,11].Despite the high number of inhibitors identifed so far [12], however, only a few mPGES-1 inhibitors such as GRC-27864 (zaloglanstat) and GS-248 (vipoglanstat) have been tested in humans (Figure 1) [13,14].
Continuing our research for the discovery of novel mPGES-1 inhibitors, we recently reported that phenylsulfonyl hydrazide derivative MPO-0144 inhibited LPSinduced PGE 2 production (IC 50 : 41.77 nM) in RAW 264.7 cells via the inhibition of murine mPGES-1 enzyme (IC 50 : 1.16 nM) as shown in Figure 1 [15][16][17].MPO-0144 exhibited in vitro and in vivo strong neuroprotective efects in the animal model of Parkinson's disease via this mechanism of action [17].Based on in vitro and in vivo results, herein, we investigated the anti-infammatory properties of MPO-0144 in adjuvant-induced arthritis (AIA) rodent models.

Synthesis of MPO-0144.
Te synthetic method of phenylsulfonyl hydrazide derivative MPO-0144 was recently reported by our group [17].

Adjuvant-Induced Arthritis (AIA) Mode. AIA was set up by injection of Freund's complete adjuvant containing
Mycobacterium butyricum into the right tibiotarsal ankle joint (10 mg/mL, 100 μL per rat) [18].MPO-0144 (10 mg/kg, p.o.) and indomethacin (2.5 mg/kg, p.o.) were orally administered at the time of adjuvant injection on day 0 for 14 days.Paw volumes were measured by using a plethysmometer just before induction of AIA and every third day for 14 days.Paw volumes were individually normalized as percentages of change from their values at day 0 and then averaged for each treatment group.On the last day, animals were euthanized.Te rat plasma and stomach were obtained and freshly frozen (−80 °C) for the biochemical analysis.

Gastric Mucosal Injury
Test in AIA Rats.Te rats were sacrifced, their stomachs were removed, and the gastric mucosa was photographed using a digital camera.Te area of the lesions was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).Ulcer scoring in the indomethacin-treated AIA mice (n � 8) was determined using the procedures described by [20].Briefy, stomach lesion width (mm) and length (mm) were determined using a vernier caliper.

Determination of pKa and LogP.
Tese values were evaluated according to potentiometric acid-base titration using Sirius T3 (Pion Inc, Riverside, UK).For pKa, the pH, when the ionized state and the neutral state are the same at a 50% ratio from the titration curve, estimated by acid-base titration in the pH range of 2.0-12.0 was measured.LogP was measured for the partition coefcient by acid-base titration using the same method as pKa in a dual-phase solvent system of octanol and water [24].

Determination of Solubility.
Te solubility of MPO-0144 was determined by the following methods.Stock solutions of MPO-0144 were prepared at 10 mM in 5% DMSO (dimethyl sulfoxide) and then diluted in 99% phosphate-bufered saline (PBS, pH 7.4) bufer.As a result, the diluted compound had a fnal concentration of 100 µM.Te volume of the test compound in a 96-well plate was set to be 250 µL, and the solubility was measured by NEPHELOstar ® , which is a fully automated laser-based microplate nephelometer that measures forward light scattering [25].

Acute Oral Toxicity Testing
In Vivo.Te acute oral toxicity test was carried out in ICR mice (9 weeks old, each group n � 6, 3 male and 3 female mice) at doses of 5.5, 17.5, 55, 175, 550, and 1,000 mg/kg p.o. of MPO-0144 (total volume � 10 mL : DMSO 20% and olive oil 80%).Te animals were observed for signs of toxicity for 2 days.As no dead animals were observed up to 1,000 mg/kg, the 2 nd toxicity test was performed at two high doses of 1,000 and 2,000 mg/kg (n � 6, 3 male and 3 female mice).Te animals were continuously observed for signs of toxicity for 7 days.Observations were conducted twice daily, including morality, injury, and abnormal behavior.Besides, body weights for all mice were recorded on study days 1, 3, 5, and 7. On study day 7, all the animals were sacrifced.

Statistical Analysis.
Results are presented as the means ± SD of triplicate experiments.In the animal study, the data were expressed as the means ± SD (n � 8).Statistically signifcant values were compared using one-way ANOVA and Dunnett's post hoc test.P values less than 0.05 were considered signifcant.All statistics were performed using GraphPad Prism 5 statistical software (GraphPad Software Inc., CA, USA).(c) Te inhibition activity of MPO-0144 against human recombinant COX-2 enzyme.Dup-697 (100 nM) was used as a positive control of COX-2 inhibition.Tis IC 50 value (IC 50 � 0.03 μM) was obtained by the extrapolation method.Values are indicated as the mean ± SEM (n � 3).Statistically signifcant values were compared using one-way ANOVA and Dunnett's post hoc test.* P < 0.05; * * P < 0.01; * * * P < 0.001 vs. the vehicle group.All statistics were performed using GraphPad Prism 5 statistical software (GraphPad Software Inc., CA, USA).

Results and Discussion
To evaluate the antiarthritic efects of MPO-0144, we investigate its therapeutic efects in the AIA rat model.AIA was set up by injection of Freund's complete adjuvant containing Mycobacterium butyricum into the right tibiotarsal ankle joint [18].Te induction of arthritis signifcantly enhanced the arthritic paw edema from the frst day onwards and attained maximum edema at 14 days.MPO-0144 (10 mg/kg) or indomethacin (2.5 mg/kg, a positive control) treatment signifcantly attenuated AIA-induced the paw swelling as displayed in Figure 2. At the incipient stage, the treatment with MPO-0144 exhibited a lower inhibitory activity than indomethacin but almost the same potency to indomethacin in suppressing paw swelling from the 11th day.Tis observation showed that MPO-0144-treated rats did not develop severe arthritis, indicating that MPO-0144 exhibited potential immune-modulating activity.
In order to evaluate the selectivity of MPO-0144 for mPGES-1, MPO-0144 was assayed for its potential inhibitory activities against COX-1 and COX-2, respectively, together with each positive control such as sc-560 and Dup-697 using enzyme immunoassay (EIA) kits in a cell-free assay.As seen in Figures 3(b) and 3(c), MPO-0144 moderately inhibited COX-1 (IC 50 � 0.83 μM) but signifcantly inhibited COX-2 (IC 50 � 0.03 μM, which value was calculated by the extrapolation method).Compared with its highest inhibition against rat mPGES-1 enzyme in Figure 3A (IC 50 � 1.16 nM, which value was previously reported by our group [16]), nevertheless, MPO-0144 was selective for the mPGES-1 enzyme over both COX-1 (selectivity index: >700) and COX-2 (selectivity index: >25).Tese overall data suggest a possibility that MPO-0144 could downregulate PGE 2 production by the combined inhibition of potent mPGES-1 and weak COX-2, thus attenuating the paw swelling in AIA rat models.Interestingly, MPO-0144 did not show any inhibitory efects on human mPGES-1 enzyme prepared from IL-1β-stimulated A549 human lung cancer cells at a high concentration of 1 μM (the data were not shown here) [32].
In order to evaluate the acute toxicity of MPO-0144 in the GI tract, we also investigated the real morphological data obtained by dissecting the stomach interior of each group.No hemorrhagic lesions were observed in the MPO-0144treated group compared with indomethacin-treated groups in the gastric mucosa (ulcerative score: 3.5 ± 0.6) as shown in Figure 4(a).To estimate whether the MPO-0144-treated group has any adverse efects on their organs, we have measured plasma parameters such as GOT/GPT (liver injury marker), BUN (kidney function marker), and troponin I   (sulfaphenazole, 94.3% @10 μM), CYP2C19 (amitriptyline, 88.9% @100 μM), CYP2D6 (quinidine, 95.7% @10 μM), and CYP3A4 (ketoconazole, 96.8% @10 μM).c Oral rat acute toxicity.d ALD (approximate lethal dose).6 Journal of Chemistry (heart attack marker) levels using EIA kits.As shown in Figure 4(b), the levels of all biomarkers were not signifcantly afected compared to those in the vehicle-treated AIA group.We also explored the brief drug-like properties of MPO-0144 through the in vitro ADMET study using the reported standard protocol [33].Preliminary results of the in vitro ADMET study are listed in Table 1.With regard to absorption of compounds, the evaluated parameters showed that MPO-0144 has a pK a value of 2.96 (an ionized form exists at physiological pH) and a logP value of 6.77, which resulted in its high hydrophobicity, lower water solubility, and medium membrane permeability as shown in Table 1.MPO-0144 showed a little inhibitory efect (2.13% inhibition) on P-glycoprotein (gp) at 10 μM concentration, not causing P-gp-mediated drug-drug interaction (DDI) compared with verapamil as a positive control (99.74% at 10 μM).Te inhibitory activity of MPO-0144 against CYP450 was also evaluated at 10 μM concentration.MPO-0144 showed a little or no inhibitory efect on CYP1A2 and CYP2D6 enzymes (21.5 and 4.1% inhibition, respectively) but could moderately inhibit the other three CYP isozymes (∼50% inhibition).Terefore, MPO-0144 had a relatively favorable profle on CYP isoforms involved in the drug-drug interaction.In the hERG inhibition, we observed an intermediate range of activity (1 μM < IC 50 < 10 μM) of hERG potassium channels possibly corresponding to cardiotoxicity with an IC 50 value of 7.37 μM.In the acute toxicity study, a single administration of MPO-0144 was performed orally to the ICR rats at the single maximum doses of 1,000 (1 st test) and 2,000 mg/kg (2 nd test), and they were then monitored for possible side efects, mortality, or behavioral changes up to 2 and 7 days, respectively.It was observed that MPO-0144 did not cause any behavioral alterations at a high dose of 2,000 mg/kg.No mortality was recorded for the period of 7 days, and after necropsy, no macroscopic pathological alterations caused by MPO-0144 were found in all mice, indicating that the lethal dose of MPO-0144 is above 2,000 mg/kg in rats (LD 50 : >2,000 mg/ kg).We also screened for potential mutagenic activities of MPO-0144 using the Ames test.An Ames test was carried out on two strains of Salmonella typhimurium (TA98 and TA100) in the absence and presence of metabolic activation (S9).As a result, MPO-0144 showed no mutagenic activity regardless of metabolic activation in both tester strains.Tese overall ADMET profles disclosed that MPO-0144 can be considered to be less harmful compound for further in vivo therapeutic studies.

Conclusions
Based on in vitro inhibitory activity of MPO-0144 against PGE 2 production, we evaluated its anti-infammatory effects using AIA rat models in vivo.Te oral treatment of MPO-0144 could strongly suppress arthritic paw edema in AIA rat models, while it did not afect the plasma levels of biomarkers representing liver, kidney, and heart toxicities.On the other hand, MPO-0144 exhibited the selectivity of mPGES-1 over both COX-1 (SI: >270) and COX-2 (SI: >25).Tese overall data suggest a mode of action that MPO-0144 could downregulate PGE 2 production by potent mPGES-1 and weak COX-2 inhibitory activities, thus reducing arthritis in rat models.In addition, MPO-0144 demonstrated favorable ADMET profles.However, MPO-0144 did not show any inhibitory efects on human mPGES-1 enzyme at a high concentration.Terefore, MPO-0144 represents a valuable pharmacological tool for the study of the regulation of inducible mPGES-1 in the only infammatory arthritis rat model.Now, we are performing the optimization study for the discovery of new compounds with inhibitory potency on both human and murine mPGES-1.

Figure 4 :
Figure 4: Analysis of MPO-0144 side efects.All data were collected from AIA rats, which had been treated with MPO-0144 for 14 days.(a) Representative images of gastric lesions in the corpus of the stomach following diferent treatments, and the blue-colored arrows indicated the indomethacin-induced gastric hemorrhagic regions (top).Ulcerative score of the indomethacin-treated group � 3.5 ± 0.6, which are means ± SD of n � 8 animals (bottom); (b) level of biomarkers representing liver, kidney, and heart toxicities.Values are means ± SD of n � 8 animals for each group.Statistically signifcant values were compared using one-way ANOVA and Dunnett's post hoc test.* P < 0.05, * * P < 0.01, and * * * P < 0.001 vs. the vehicle group.All statistics were performed using GraphPad Prism 5 statistical software (GraphPad Software Inc., CA, USA).