human EP4 receptor ( Ki = 13 nM ); rat EP4 receptor ( Ki = 20 nM ); EP4 ( Ki = 24 nM ); EP4 ( IC50 = 35 nM )

Grapiprant (0-50 mg/kg; oral administration; every 24 hours; for 9 months; beagles) is safe to give orally to dogs on a long-term basis. Further research is required to determine whether Grapiprant is effective in treating dogs with osteoarthritis[3].

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In a rat postoperative (paw incision) pain model, oral administration of grapiprant at a dose of 133.3 mg/kg, given 30 minutes before surgery, significantly reduced weight-bearing deficit (a measure of pain) at 3 and 4 hours post-surgery compared to the vehicle group. The mean plasma concentration of grapiprant in satellite animals at 4.5 hours after this oral dose was 378 ng/mL. [2]
In the same rat model, continuous subcutaneous infusion of grapiprant via osmotic pump at rates of 150, 300, and 600 µg/h, starting 24 hours before surgery, also significantly and dose-dependently reduced weight-bearing deficit at 3 and 4 hours post-surgery. The steady-state plasma concentrations achieved with these infusion rates were approximately 335-364 ng/mL (150 µg/h), 773-859 ng/mL (300 µg/h), and 1413-1646 ng/mL (600 µg/h) at 24 and 28 hours post-pump implantation. [2]
Based on these results, the efficacious plasma concentration of grapiprant for relieving acute surgical pain in rats was estimated to be approximately 350 ng/mL. [2]

Canine EP4 Receptor Binding Assay: Membranes were prepared from HEK293 cells stably expressing a cloned canine EP4 receptor. For competitive binding assays, the membranes were incubated with 1 nM of [³H]-PGE₂ and various concentrations of grapiprant. The IC₅₀ value for displacing [³H]-PGE₂ binding was determined. The equilibrium dissociation constant (Kd) for PGE₂ binding to the receptor was also measured using Scatchard plot analysis. The inhibition constant (Ki) for grapiprant was calculated from the IC₅₀ value using the Cheng-Prusoff equation. The assay was performed in three independent experiments, with each data point in duplicate. [2]
Serum Protein Binding Assay (Equilibrium Dialysis): The unbound fraction of grapiprant in serum from dogs, rats, and humans was determined using an equilibrium dialysis method. Fresh serum was fortified with grapiprant at nominal concentrations of 200 and 1000 ng/mL. Dialysis cells were assembled with a semi-permeable membrane separating a chamber filled with 1 mL of fortified serum from a chamber filled with 1 mL of phosphate-buffered saline. The cells were immersed in a water bath at 37°C with constant rotation for 5 hours to reach equilibrium. After dialysis, the concentrations of grapiprant in both the serum and buffer chambers were quantified using a validated LC/MS/MS method. The unbound fraction (fub) was calculated using a specific formula comparing the drug concentrations in both chambers at equilibrium. [2]

36 beagles of both sexes (9-month-old)
0 mg/kg, 1 mg/kg, 6 mg/kg, or 50 mg/kg
Oral administration; every 24 hours; for 9 months

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Pharmacokinetic Study in Dog: A single healthy male Beagle dog was administered a single intravenous injection of grapiprant at a dose of 0.5 mg/kg body weight. The drug was first dissolved in ethanol (10 mg/mL), then diluted with sterile water for injection at a 9:1 (v:v) ratio (ethanol:water), and immediately injected into the right jugular vein at a rate of 5 mL/min. Blood samples (2-3 mL) were collected via a catheter inserted in the left jugular vein at predetermined times: 0 (pre-dose), 15, 30, 45 minutes and 1, 2, 4, 6, 8, 10, and 24 hours post-dose. Blood was collected into lithium heparin tubes, centrifuged within 30 minutes, and the harvested plasma was frozen immediately at -20°C until analysis. [1]

Absorption, Distribution and Excretion
Animal (horse) studies showed that serum drug concentrations were >0.005 ng/ml 72 hours after the first 2 mg/kg dose. The drug is rapidly absorbed, with a reported Cmax of 31.9 ng/ml, Tmax of 1.5 hours, and AUC of 2000 ng·hr/ml. Regarding bioavailability, the mean bioavailability of glappiram is 39%. It has been reported that bioavailability, time to peak concentration, and maximum concentration are significantly reduced after food intake. Most of the drug is absorbed within 72 hours after oral administration. Animal (horse) studies showed that urinary drug concentrations were >0.005 ng/ml 96 hours after the first 2 mg/kg dose. Of the excreted dose, 55%, 15%, and 19% were excreted in bile, urine, and feces, respectively. Animal (cat) studies reported a volume of distribution of 918 ml/kg.
Animal studies (cats) reported a clearance of 173.2 ml/hr·kg.
Metabolism/Metabolites
In vitro canine microsomal studies identified four metabolites: one N-deamination metabolite (the major metabolite in urine and feces), two hydroxylated metabolites, and one N-oxidized metabolite.
Biological half-life
Animal studies (horses) reported an elimination half-life of 5.86 hours.
In a beagle dog, after a single intravenous injection of 0.5 mg/kg, the pharmacokinetic profile of grapiprant was described by a two-compartment model, with a rapid distribution/elimination phase lasting up to 4 hours and a slower terminal elimination phase lasting 4 to 10 hours. [1]
Key pharmacokinetic parameters derived from this single-dose study included: area under the concentration-time curve (AUC₀–∞) = 1339 h·ng/mL. [1]
Terminal half-life (HL) = 6.07 hours. [1]
Systemic clearance (Cl) = 373 mL/kg/h. [1]
Steady-state volume of distribution (Vss) = 2162 mL/kg. [1]
Plasma concentration ranged from 779 ng/mL at 15 minutes post-administration to 38.9 ng/mL at 10 hours post-administration. [1]

Protein Binding
Glappirena's serum protein binding rate is approximately 95%. The main protein binding to glappirena is albumin.

[1]. Detection and quantification of the selective EP4 receptor antagonist CJ-023423 (grapiprant) in canine plasma by HPLC with spectrofluorimetric detection. J Pharm Biomed Anal. 2016 Jan 25;118:251-8.

[2]. Pharmacology of grapiprant, a novel EP4 antagonist: receptor binding, efficacy in a rodent postoperative pain model, and a dose estimation for controlling pain in dogs. J Vet Pharmacol Ther. 2017 Jun;40(3):285-292.

[3]. Evaluation of the safety of long-term, daily oral administration of grapiprant, a novel drug for treatment of osteoarthritic pain and inflammation, in healthy dogs.Am J Vet Res. 2015 Oct;76(10):853-9.

Grapiprant, also known as AT-001 and CJ-023, is a pipronoid drug. These molecules are derived from acylsulfonamides and are characterized by a novel class of p-N-acylaminomethylbenzoic acid, known as prostaglandin receptor antagonists. Currently, these molecules are being developed for veterinary clinical use. The World Health Organization defined this class of drugs in 2013. Grapiprant received approval from the U.S. Food and Drug Administration's Center for Veterinary Medicine in March 2016 as a non-cyclooxygenase inhibitor (NOASI) for use in veterinary medicine. Grapiprant is an orally bioavailable prostaglandin E receptor 4 (EP4) antagonist with potential analgesic, immunomodulatory, and antitumor activities. After administration, grapiprant selectively binds to and inhibits the binding of prostaglandin E2 (PGE2), thereby preventing EP4 receptor activation. This inhibits PGE2-EP4 receptor-mediated signaling and prevents the proliferation of tumor cells with overactivated PGE2-EP4 signaling pathways. Furthermore, EP4 receptor inhibition modulates the immune system by inhibiting the production of interleukin-23 (IL-23) and IL-23-mediated Th17 cell expansion. Since EP4 is expressed in peripheral sensory neurons, blocking EP4-mediated signaling may produce analgesic effects. EP4 is a prostaglandin receptor subtype belonging to the G protein-coupled receptor family and is expressed in certain types of cancer. It promotes tumor cell proliferation and invasion.

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Drug Indications
Glappiram has been investigated in the analgesic and anti-inflammatory fields due to its reported analgesic and anti-inflammatory effects. This molecule has been approved and widely accepted in the veterinary field for relieving arthritis pain. In humans, it has been studied for controlling pain and inflammation associated with osteoarthritis. The effects of glappiram can be explained by the function of prostaglandin E2 (PGE2), a key mediator of the typical symptoms of inflammation—swelling, redness, and pain. PGE2 exerts its effects through four receptors: EP1, EP2, EP3, and EP4, with EP4 being the primary mediator of PGE2-driven inflammation. It is used to treat mild to moderate osteoarthritis-related pain in dogs. Mechanism of Action Glapipram is an EP4 prostaglandin receptor antagonist; therefore, its activity is believed to be entirely related to its selective blocking of this receptor. It binds with high affinity to the EP4 prostaglandin receptor in humans and other mammals without interfering with other prostaglandin pathways essential for various physiological functions. Glapipram's binding blocks PGE2 binding, thereby inhibiting its biological effects associated with the pain and inflammation signaling cascade. Glapipram is widely used in veterinary medicine because its mechanism of action is to achieve targeted analgesia without affecting prostaglandin production and thus without interacting with other prostaglandin receptor pathways. Pharmacodynamics Preclinical studies have shown that glapipram effectively reduces acute and chronic pain and inflammation. The efficacy of grapiprant appears to be dose-dependent, comparable to that of rofecoxib and piroxicam. Grapiprant has been reported to be effective in relieving pain associated with canine arthritis.

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Grapiprant (CJ-023423) is a novel selective prostaglandin E2 (PGE2) EP4 receptor antagonist. It is currently being developed for the control of pain and inflammation associated with osteoarthritis in humans and dogs. [1]
Its molecular weight is 491.61 g/mol, the predicted octanol-water partition coefficient (logP) is 4.56, and its water solubility is extremely poor (0.041 mg/L). [1]
This study established and validated a high-performance liquid chromatography-fluorescence detection method (HPLC-FL) with high specificity, sensitivity, and accuracy for the quantitative analysis of grapiprant in canine plasma. The limit of quantification (LLOQ) of this method was 10 ng/mL, and the average extraction recovery rate was 88.1%. This method has been successfully applied to preliminary pharmacokinetic studies in dogs. [1]

C26H29N5O3S

491.61

491.199

C, 63.52; H, 5.95; N, 14.25; O, 9.76; S, 6.52

415903-37-6

11677589

White to off-white solid powder

1.3±0.1 g/cm3

>136 ºC (Grapiprant hydrochloride)

1.647

4.56

2

5

7

35

802

0

S(C1C([H])=C([H])C(C([H])([H])[H])=C([H])C=1[H])(N([H])C(N([H])C([H])([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])N1C(C([H])([H])C([H])([H])[H])=NC2C(C([H])([H])[H])=NC(C([H])([H])[H])=C([H])C1=2)=O)(=O)=O

HZVLFTCYCLXTGV-UHFFFAOYSA-N

InChI=1S/C26H29N5O3S/c1-5-24-29-25-19(4)28-18(3)16-23(25)31(24)21-10-8-20(9-11-21)14-15-27-26(32)30-35(33,34)22-12-6-17(2)7-13-22/h6-13,16H,5,14-15H2,1-4H3,(H2,27,30,32)

1-[2-[4-(2-ethyl-4,6-dimethylimidazo[4,5-c]pyridin-1-yl)phenyl]ethyl]-3-(4-methylphenyl)sulfonylurea

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 3 mg/mL (6.10 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3 mg/mL (6.10 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 3 mg/mL (6.10 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)