Expression of EP4 receptors in human embryonic kidney cell line
The complementary DNA (cDNA) clone of the human EP4 receptor (hEP4R; NM_000958.2) was obtained from Thermo Fisher Scientific (Ultimate ORF Clone Collection, clone ID IOH46525; Waltham, MA, USA). The coding sequence was subcloned in expression vector pcDNATM6.2/V5-DEST by using Invitrogen Gateway technology (Thermo Fisher Scientific). Human embryonic kidney cells 293 (HEK293) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and transfected with expression vector for hEP4R according to the method described in the FuGENE 6 Transfection Reagent technical manual (Promega, Madison, WI, USA). The clones overexpressing the protein were selected for Blasticidin S HCl (Thermo Fisher Scientific) resistance. The selected clone was grown in DMEM containing 10% FBS and 10 μg/ml Blasticidin S HCl (selection medium) at 37 °C in a humidified atmosphere of 5% CO2 in air.
For rodent EP4 receptor cloning, the cDNA encoding the full-length rat (NM_032076) or mouse (NM_001136079) EP4 receptors cloned in pCMV6 entry was provided by OriGene Inc. (TrueORF Gold, catalogue numbers RN206839 and MC216813; OriGene, Rockville, MD, USA). Expression of rat and mouse receptors was obtained by transfecting HEK293 cells with the respective vector using Lipofectamine 3000 reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.
In vitro evaluation of immunomodulatory and anti-angiogenic potential
Gene expression analysis of inflammatory mediators in THP-1 cells differentiated to macrophages
The human monocyte cell line THP-1 was obtained from the ATCC and was grown according to the instructions provided. THP-1 monocytic cells were differentiated to macrophages with 100 nM phorbol 12-myristate 13-acetate (PMA) for 4 days. Macrophages were then stimulated with lipopolysaccharide (LPS) 10 ng/ml and PGE2 10 nM for 3 or 24 h. Total RNA was purified using the ABI Prism 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA, USA) and retrotranscribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). RT-PCR analysis was performed using the Applied Biosystems 7500 Fast Real-Time PCR System using specific TaqMan assays (number Hs00174131_m1 for IL-6, number Hs00900054_m1 for vascular endothelial growth factor A [VEGFA]; Thermo Fisher Scientific) and, as an endogenous control, the 18S Pre-Developed TaqMan® Assay (Thermo Fisher Scientific). The data analysis, with normalisation on 18S amplified values, was done following Thermo Fisher Scientific’s specific instructions for gene expression relative quantification. All individual data are the result of at least three different analyses for each sample.
Source of human peripheral blood mononuclear cells
Whole blood obtained from healthy volunteers upon receipt of their informed consent was used to prepare peripheral blood mononuclear cells (PBMCs). Because the study presented no material ethical issues (samples anonymised and discarded immediately after analysis), the procedure was approved by the internal research ethics advisor.
IL-23 release from human dendritic cells
PBMCs were purified using Histopaque (Sigma-Aldrich, St. Louis, MO, USA). CD14+ cells were positively selected in a magnetic field (magnetic-activated cell sorting [MACS]; Miltenyi Biotec, Bergisch Gladbach, Germany). Purified CD14+ cells were subsequently cultured for 7 days in differentiation medium (RPMI 1640 + 10% FBS, granulocyte-macrophage colony-stimulating factor 50 ng/ml and IL-4 100 ng/ml) and then stimulated with LPS (10 ng/ml), R848 2.5 μg/ml and PGE2 10 nM for 24 h in the presence of CR6086 (0.01–30 μM) or naproxen (1–30 μM). IL-23 release was measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA).
IL-17 release from human Th17 cells
PBMCs were purified using Histopaque. CD4+CD45+ naive T cells were indirectly selected in a magnetic field (MACS; Miltenyi Biotec). Purified CD4+ cells were subsequently cultured for 7 days in differentiation medium (RPMI 1640 + FBS 10%, IL-6 30 ng/ml, transforming growth factor [TGF]-β 2.25 ng/ml, IL-23 30 ng/ml, IL-1β 20 ng/ml, anti-interferon (IFN)-γ 1 ng/ml and anti-IL-4 2.5 ng/ml). Th17 cell expansion was stimulated by addition of anti-CD3/anti-CD8/anti-CD28 beads to the medium with a cell-to-beads ratio of 1:2. Th17 cells were stimulated with IL-6 30 ng/ml, TGF-β 2.25 ng/ml, IL-23 30 ng/ml, IL-1β 20 ng/ml and PGE2 10 nM for 48 h in the presence of CR6086 (0.03–10 μM) or naproxen (1–10 μM). IL-17 release was measured by ELISA (Abcam, Cambridge, UK).
Cell viability
Viability was evaluated by Trypan Blue vital dye count, ViaCount assay (EMD Millipore, Billerica, MA, USA) or MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay after 24- and 48-h exposure to the test drug.
Animals, drugs and treatments
Male Lewis rats (Charles River Laboratories, Wilmington, MA, USA) aged 7 weeks and male DBA/1 mice (Charles River Laboratories) aged 6–7 weeks were housed with access to food and water ad libitum in a temperature-controlled room with a 12-h/12-h light/dark cycle at least 1 week before the beginning of the experiment. Experimental procedures were carried out in compliance with national and international laws and policies (Italian Legislative Decree 116/1992, and 26/2014 that transposed the European Union Directive 86/609, and 2010/63/EU on the protection of animals used for scientific purposes) and as authorised by the Italian Ministry of Health (Decr. Min. number 60/2009-B, Decr. Min. number 214/2013-B, and Decr. Min. number 561/2017-PR).
CR6086 (synthesised at Rottapharm Biotech, Monza, Italy), rofecoxib (Sigma-Aldrich) and naproxen (Sigma-Aldrich) were dissolved in distilled water. Tofacitinib (Sigma-Aldrich) was suspended in hydroxypropyl methylcellulose 0.5% Tween 80/0.1% phosphate buffer (150 mM), pH 7. Etanercept (Enbrel; Amgen, Thousand Oaks, CA, USA) and methotrexate (MTX) (Sigma-Aldrich) were dissolved in sterile PBS and sterile saline (NaCl 0.9%), respectively. CR6086, rofecoxib, naproxen and tofacitinib were administered orally; etanercept and MTX were injected intraperitoneally. Comparators were used at doses or dose ranges reaching their maximum pharmacological effects. Volumes of administration were 5 ml/kg and 10 ml/kg for rats and mice, respectively. Naive, sham and control animals always received the appropriate vehicle.
Collagen-induced arthritis in rats
Male Lewis rats were immunised by intradermal injection at the base of the tail with an emulsion containing 150 μg of bovine collagen type II (bCII) in complete Freund’s adjuvant (CFA) or received an injection with incomplete Freund’s adjuvant (sham group). Three weeks after the first immunisation, animals were boosted with the same procedure. Three days after booster injection, arthritis had developed in all animals but the sham group. Oedema was assessed via plethysmometer (Ugo Basile, Varese, Italy), and the animals were then randomised to treatment groups. Oedema measurement was performed again after 7 and 14 days of treatment. Animals were blindly scored by two different investigators for clinical signs of arthritis (redness, swelling, pain of hind limb joints) as follows: 0 = normal; 1 = mild but definite redness and swelling of the ankle or wrist or individual digits, regardless of the number of affected digits; 2 = moderate redness and swelling of ankle or wrist; 3 = severe redness and swelling of the entire paw including digits; and 4 = maximally inflamed limb with involvement of multiple joints. The mean of the scores assigned by the investigators was calculated.
Three different experiments in rat CIA are reported: a dose-response study, as well as two comparative studies vs the COX-2 inhibitor rofecoxib and the Janus kinase (JAK) inhibitor tofacitinib, respectively. The number of animals per experimental group is reported in the figure legends.
In the comparative experiment vs rofecoxib, mechanical hyperalgesia was assessed at the end of treatment by using a digital pressure application measurement (PAM) device (Ugo Basile). Increasing pressure was applied to the lateral side of the knee until the animal attempted to escape or vocalised. The force required to elicit this response was measured in grams (gram-force). An optimal stimulus increase rate of 100 g/second was used, and mechanical thresholds were reached after approximately 10 seconds. This increase was visualised with a rate meter on the built-in display, so the pressure applied was visually controlled. At the end of the experiment, animals were killed, and their hind paws were explanted and processed for histology.
Histological procedures
Briefly, the paws were fixed in 10% neutral buffered formalin (Bio-Optica, Milan, Italy) and subsequently decalcified in EDTA for 4 weeks. Samples were dehydrated in an ethanol series and embedded in paraffin. At least three non-consecutive sections for each paw were blindly scored at the level of the tarsus, metatarsus and calcaneus for the following histological features: synovial oedema, pannus, synovial inflammatory cell infiltrate, cartilage damage, osteolysis and woven bone. Based on these histological features, a synovial score (synovial oedema, plus synovial inflammatory cell infiltrate, plus pannus), a bone score (woven bone plus osteolysis) and a total score were calculated. Two independent pathologists performed histopathology on blinded slides. The data of right and left hind limbs were summed for each rat.
CR6086 pharmacokinetics and pharmacokinetic/pharmacodynamic correlation in rats
CR6086 concentrations in rat plasma were determined by high-performance liquid chromatography (Agilent 1200 series; Agilent Technologies, Santa Clara, CA, USA) coupled with a triple-quadrupole mass spectrometer (API 4000 QTRAP; SCIEX, Concord, ON, Canada). A ZORBAX SB-C18 2.0 × 50-mm, 3.5-μm column (Agilent Technologies) in isocratic conditions was used with mobile phase 0.1% formic acid in water/acetonitrile (68:32
vol/vol). The method was linear within the dynamic range 1.0–10,000 ng/ml (lower to upper limit of quantification). CR6086 plasma protein binding was determined in the 0.2–200 μM concentration range using [
14C]CR6086 and equilibrium dialysis. EP4 receptor occupancy (RO) was calculated on the basis of CR6086 concentrations according to the following formula:
$$ \mathrm{RO}\ \left(\%\right)=\mathrm{Cave}/\left(\mathrm{Cave}+\mathrm{Ki}\right)\times 100, $$
where Cave = AUC24/24.
Collagen-induced arthritis in mice
Male DBA/1 mice were immunised by intradermal injection at the base of the tail with an emulsion of 200 μg of bCII in CFA containing 3 mg/ml
Mycobacterium tuberculosis [
14,
18,
24,
25]. Non-immunised mice served as the negative control of disease. Animals were monitored by visual inspection for appearance of peripheral oedema. Arthritis onset occurred starting from day 20 after immunisation. Upon onset, animals were recruited and randomised. Recruitment was given a cut-off at day 40. Upon recruitment, arthritis clinical score was assigned, and oedema was measured via caliper. The number of animals per experimental group is reported in the figure legends.
In a first study, mice were randomised into the following treatment groups: vehicle, 30 mg/kg CR6086, 60 mg/kg CR6086, 60 mg/kg naproxen and 10 mg/kg etanercept. Animals received the test drugs for 10 days. CR6086 and naproxen were administered orally once daily, whereas etanercept was administered intraperitoneally every other day. Animals treated with vehicle, 60 mg/kg CR6086, naproxen and etanercept were additionally analysed for the proportion of populations of Th17 cells, Th1 cells, regulatory T cells, B cells, macrophages, neutrophils and dendritic cells by fluorescence-activated cell sorting (FACS) after collection of blood, draining lymph nodes and joints.
In a second study, mice were randomised into the following groups: vehicle, 30 mg/kg CR6086, 60 mg/kg CR6086 and 60 mg/kg naproxen. Animals received the test drugs once daily for 10 days. At the end of the study, serum was isolated for determination of different cytokine biomarkers (IL-6, tumour necrosis factor [TNF]-α, IL-10, IL-17, IFN-γ, IL-22 and IL-23) by multiplex analysis on the MSD platform (Artialis, Liège, Belgium).
In a third study, mice were randomised into the following treatment groups: naive, vehicle, 30 mg/kg CR6086, 1 or 3 mg/kg MTX, with the latter administered alone or in combination with 30 mg/kg CR6086. CR6086 was administered orally once daily. MTX was administered intraperitoneally three times per week (every third day). Mice were treated with test drugs for 16 days.
Oedema measurement was performed every day before treatment, and all animals were blindly scored for clinical signs of arthritis as follows: 0 = normal; 1 = slight swelling and/or erythema; 2 = pronounced oedematous swelling; and 3 = ankyloses and severe swelling. A score of 0.5 was given to swollen toe/toes or when inflammation was localised to one part of the foot. Each limb was measured separately, with a final score based on the sum of the scores from all four paws.
Because animals were recruited for treatment at disease onset, arthritis was already evident in terms of both oedema and clinical score. Therefore, the individual progress of signs was calculated for each animal as the AUC from randomisation (baseline) to the end of treatment.
At the end of the treatment period, animals were killed, and their paws were explanted and processed for histology. In the third study, the serum concentrations of immunoglobulin G (IgG) antibodies against bCII were measured by ELISA (catalogue number 2032; Chondrex, Redmond, WA, USA).
Histological procedures
In the second experiment, the signal limb (i.e., the limb that determined the onset of arthritis) was assessed. In the third experiment, instead, all four limbs were analysed, and a summed score for all limbs was calculated. Paws were processed according to the procedure described for rats. At least two non-consecutive sections for each paw, 4 μm thick, of the tarsus, metatarsus, calcaneus, carpal, metacarpal, ulna and radius were collected and assessed for cartilage degeneration, woven bone, osteolysis, synovial inflammation, synovial hyperplasia and pannus formation. Based on these histological features, a synovial score (synovial inflammation, plus synovial hyperplasia, plus pannus), a bone score (woven bone plus osteolysis) and a total score were calculated. Two independent pathologists performed histopathology on blinded slides.