Grapiprant: A snapshot of the current knowledge

Abstract Grapiprant is the pioneer member of the novel piprant class, a potent and specific antagonist of the prostaglandin E2 receptor 4. It has been approved in veterinary medicine for the control of pain and inflammation associated with osteoarthritis in dogs at the dose regimen of 2 mg/kg once a day by the FDA and EMA (for pain only) in 2016 and 2018, respectively. The aim of this narrative review was to report the analytical methods, pharmacokinetics, pharmacodynamics and safety of grapiprant in several animal species using the best available published scientific evidence. In conclusion, most of the analytical methods proposed for grapiprant detection are simple, reliable, sensitive and validated. The pharmacokinetics show discrepancies between animal species. The therapeutic efficacy seems more suited to chronic rather than acute pain.


| INTRODUC TI ON
In the present day, animal care management has drastically improved compared to the situation at the end of last century.
Companion animals are now living longer and so are more commonly manifesting age-related and/ or pain-associated diseases of medical importance such as cancer, arthritis and metabolic disorders (Giorgi, 2012).
The most commonly used drugs in the treatment of acute and chronic canine pain belong to the class of non-steroidal anti-inflammatory drugs (NSAIDs). Nonselective or preferential NSAIDs inhibit both COX isoforms (COX-1/COX-2) to different extents. The second generation of NSAIDs (coxibs) is highly selective for the COX-2 enzyme, reducing but not eliminating the risk of some adverse effects and maintaining anti-inflammatory and analgesic actions that would alleviate inflammation and pain states. Different members of the coxib class are available for the treatment of canine OA including cimicoxib, deracoxib, firocoxib, mavacoxib and robenacoxib Kim & Giorgi, 2013).
Osteoarthritis (OA) is a progressive condition that leads to joint inflammation, cartilage damage, pain and disability (Rychel, 2010). A number of approved NSAIDs are available for the treatment of pain associated with this chronic joint condition (Johnston et al., 2008;Kim & Giorgi, 2013;McLaughlin, 2000). The administration is usually life long and some of the above-mentioned drugs may produce side effects. For this reason, there is the need for new drugs effective for OA pain relief with a better safety and effectiveness profiles.

Grapiprant (Galliprant ® ) is an analgesic and anti-inflammatory drug.
This compound is the pioneer member of the novel piprant class. It is a potent and specific antagonist of the prostaglandin E2 (PGE2) receptor 4 (EP4). It was approved in veterinary medicine by the FDA in 2016 for the control of pain and inflammation and by the EMA in 2018 for the control of pain associated with OA in dogs . It is sold as 20, 60 and 100 mg tablets. The recommended clinical dose is 2 mg/kg once a day in fasting conditions. Grapiprant (C26H29N5O3S,pyridin-1-yl}phenyl)ethyl]-1-(4-methylbenzenesulfonyl) urea) is a small molecule with a molecular weight of 491.61 g/mol.

| PHYS ICO -CHEMIC AL FE ATURE S
It is characterized by a logP of 4.56 and a melting point >136°C. It appears as a white to off-white crystalline powder soluble in DMSO, but with a very poor water solubility (0.041 mg/L). Its molecular structure is depicted in Figure 1.

| ANALY TIC AL ME THODS
A number of analytical methods for the quantification of grapiprant in different biological matrices have been published (Baralla et al., 2018;Cox et al., 2020;De Vito et al., 2016Heit et al., 2018;Knych et al., 2018;Nagahisa & Okumura, 2017). The devices used for the quantification of grapiprant are HPLC and LC-MS/MS. The sample clean up mainly uses liquid-liquid or solid-phase extractions and the limits of quantification range from 50 to 0.005 ng/ml. The most important details of each analytical method are reported in Table 1.

| PHARMACOKINE TIC S
The pharmacokinetics of grapirant have mainly been described in the canine species although a few pioneer studies have been published in cats, horses and rabbits (Table 2). Other unpublished studies (at the time of submission of this review) are ongoing in small ruminants and birds.

| Dogs
A study in a tightly controlled research environment was conducted with Beagle dogs (Nagahisa & Okumura, 2017). Grapiprant was administered to animals in accordance with the exact body weight (methyl cellulose suspension of grapiprant at 1, 3 and 10 mg/kg PO and 1 mg/kg IV). The plasma concentration of grapiprant after 1, 3 and 10 mg/kg PO administration rapidly increased with T max of F I G U R E 1 Chemical structure of grapriprant (1-[2-[4-(2ethyl-4,6-dimethylimidazo[4,5-c] 0.42-0.67 h. The plasma levels of grapiprant increased in a dosedependent manner but not in linear fashion (AUC 0-24 were 1800, 7600 and 31,000 ng h/ml for 1, 3 and 10 mg/kg, respectively). This behaviour might be due to the small animal sample size of the study (3 animals for group). The serum protein binding of grapiprant was high (95%). The grapiprant t 1/2λz range was relatively broad 3.7-6.1 h. Surprisingly, the absolute bioavailability values were 62%, 90% and 110% at the doses of 1, 3 and 10 mg/kg, respectively. The apparent increase in F% with increasing doses was speculated to be due to saturation of efflux pumps and/or enzymatic metabolism.
The bioavailability of grapiprant after oral administration was estimated in fasted and fed Labrador retriever dogs (Łebkowska-Wieruszewska, Barsotti, et al., 2017). The IV dose at 0.5 mg/kg, freshly dissolved in ethanol at 50 mg/ml, was administered to two animals. The plasma concentrations vs. time curves of the two dogs were almost identical. The oral dose was 2 mg/kg. This dose was prepared by micronizing the pure grapiprant powder manually in a mortar and putting the required dose into empty gelatine capsules.
Animals (n = 6) received PO grapiprant after both fasting for 12 h overnight and in fed conditions. In this latter status, animals were fed one hour prior to drug administration. After oral administration of grapiprant to fed dogs, there was a delay in absorption, but this varied between dogs. The fed condition resulted in a reduced C max (614 vs. 1598 ng/ml) and delayed T max (3 vs. 1 h) compared to fasting dogs.
This result differs from that presented by the pharmaceutical company (Anonymous, 2016b). The t 1/2λz was similar to that observed in the oral fasted and IV groups. The estimated absolute bioavailability in the fed group was 66.19% while it was almost twice that in fasting animals. Despite this large difference, the decrease in concentrations of grapiprant in plasma triggered by the fed condition mostly affected C max but had negligible effects on the time when concentrations were greater than the theoretical canine minimal effective concentration (MEC) (Nagahisa & Okumura, 2017).
Sixteen Beagle dogs were randomized to receive single doses of either 6 or 50 mg/kg grapiprant as both suspension and tablet formulations within a cross-over design with a 15-day washout in order to evaluate its pharmacokinetics .
Grapiprant rapidly achieved elevated concentrations in the sera, with mean T max of <2 h regardless of formulation. Half-lives ranged between approximately 3 and 11 h. As expected, the higher dose resulted in substantially higher C max and AUC last values compared to the lower dose. However, this increase did not correlate with the dose in a linear fashion as it had in the study of Nagahisa and Okumura (2017). A likely explanation for this discrepancy is the nonlinearity of clearance and volume of distribution with dose, as speculated by Łebkowska-Wieruszewska, Barsotti, et al. (2017). A short note reported the pharmacokinetics of grapiprant in Mdr1-deficient collies (Heit et al., 2018). Eight adult collie dogs homozygous for the Mdr1 mutation were treated with approximately, but no less than, 2 mg/kg grapiprant orally once daily for 28 days after an overnight fast. Grapiprant was readily absorbed with a C max of approximately 5000 ng/ml occurring at a median T max of 1 h. Although no difference was seen in t 1/2λz (mean 4.6 h), the C max and AUC (16,000 ng ×h/ml) were approximately fourfold and sixfold greater, respectively, than those reported in normal Beagles. It was concluded that Mdr1-deficient dogs are expected to have higher AUC and C max values compared to normal dogs treated with the same dose.

TA B L E 3 Main pharmacokinetic parameters of grapiprant found in the literature in different veterinary species
The main pharmacokinetic parameters of grapiprant reported in the above-mentioned studies are summarized in Table 3.
The variability in plasma concentrations and in some PK parameters found in the above-mentioned studies might be due to the small animal sample size used. A remarkable result is that oral absorption of grapiprant is decreased by feeding (Łebkowska-Wieruszewska, Barsotti, et al., 2017), this finding is not in line with the report issued by the pharmaceutical company (Anonymous, 2016a(Anonymous, , 2016b. However, the instruction sheet of the drug (Anonymous, 2016b) does not recommend administration in the fed state. Indeed, it has been reported that the time range in which grapiprant plasma con-

| Cats
The pharmacokinetics of grapiprant after 2 mg/kg administra-  (Toutain & Bousquet-Mélou, 2004) values in cats were in the range 1.5%-3.7%. These values are considered low and suggest that the contribution of the liver and kidney in the clearance of grapiprant might be limited.
The safety and toxicokinetic profiles associated with daily oral administration of grapiprant were evaluated (Rausch-Derra & Rhodes, 2016). Cats (both gender) were randomly assigned (3 × group) to receive a placebo capsule or grapiprant at 3, 9 or 15 mg/kg, administered PO once daily for 28 days. Very broad variation was found in the pharmacokinetic parameters after the last grapiprant dose. C max ranged from 683 to 4950 ng/ml with T max within 1-4 h. Half-lives ranged from 2 to 14 h. The comparison between the AUC after the first dose and that at the 27th indicated that no drug accumulation was likely. No apparent differences were reported in the pharmacokinetic parameters between sexes.

| Horses
The drug concentrations and the pharmacokinetics of grapiprant in exercised Thoroughbred horses administered with a 2 mg/kg oral dose of grapiprant were described (Knych et al., 2018).
A recent study determined if the approved dose of grapiprant in dogs would produce measurable concentrations in urine and plasma of horses (Cox et al., 2020). Six mares were administered one dose of 2 mg/kg grapiprant via nasogastric tube. Grapiprant plasma concentrations ranged from 71 to 149 ng/ml with the mean peak concentration (106 ng/ml) occurring at 30 minutes. This study also reported that oral administration of grapiprant (2 mg/kg) in horses did not achieve the MEC reported for dogs (Nagahisa & Okumura, 2017).

| Rabbits
The pharmacokinetics of grapiprant after 2 mg/kg IV administration

It does not inhibit the production of other prostanoids and does not
interfere with the maintenance of numerous normal homeostatic functions, preventing the classic adverse effects associated with NSAIDs (Giorgi, 2015;Kirkby Shaw et al., 2016).
EP4, along with EP1, EP2, EP3, is one of the target receptors for PGE2 (Woodward et al., 2011), is the most abundant PG in synovia and is a key mediator of inflammation and pain (Yokoyama et al., 2013). The EP4 receptor has been shown to regulate inflammatory nociception at the peripheral and CNS level. Particularly in inflammatory pain, a number of inflammatory mediators in the periphery such as PGs, nerve growth factor and bradykinin activate and/or sensitize neurons (Basbaum et al., 2009;Mizumura et al., 2009).
PGE2 is a typical inflammatory mediator released after induction of COX-2 in injured tissue and cells, mostly immune cells such as mast cells, basophils or macrophages, which accumulate in the area of injury. PGE2 binds to EP receptors with subsequent activation of intracellular signalling pathways resulting in an increase in intracellular cAMP and thus activation of protein kinases. The latter leads to phosphorylation of a number of target proteins including TRPV1 channels, voltage-gated Na+ or T-type calcium channels which contribute to sensitization mechanisms in the peripheral nervous system (Kawabata, 2011). This sensitization also occurs in the CNS by gene regulation in the pre-and post-synaptic neurons (Kuner, 2010).
COX-2 is constitutively expressed in neuronal and glial cells of the CNS and upregulated after ongoing nociceptive stimulation associated with augmented PG synthesis which leads to increased neurotransmitter release (Ferreira & Lorenzetti, 1996;Nishihara et al., 1995), enhanced calcium concentrations and activation of a number of pain-related genes (Figure 3). The consequence of peripheral and central sensitization is an increased sensitivity towards nociceptive stimuli (hyperalgesia) or a nociceptive response to non-noxious stimulation (allodynia). The canine EP4 receptor has approximately 90% homology to the human receptor (Castleberry et al., 2001), and it is a Gsα-(that stimulates the cAMP-dependent pathway by activating adenylyl cyclase) and Giα-(inhibits the cAMP-dependent pathway by inhibiting adenylyl cyclase) coupled protein (Yokoyama et al., 2013). EP4 signalling plays a variety of roles through stimulating adenylate cyclase leading to elevated levels of intracellular cAMP (Yokoyama et al., 2013). The EP4 receptor has been showed to be specifically involved in the pain and inflammation associated with experimentally induced arthritis in rodents (Boyd et al., 2011;Chen et al., 2010;Clark et al., 2008;Kabashima et al., 2007;Lin et al., 2006;McCoy et al., 2002;Nakao et al., 2007;Southall & Vasko, 2001;St-Jacques & Ma, 2013).

| Dogs
The oral dose predicted to be efficacious in dogs was computed Grapiprant is a newly introduced drug, and so reports of testing under laboratory and field conditions have been limited to studies that are required for regulatory approvals (Rausch-Derra, Huebner, et al., 2016). Surprisingly two experimental (Budsberg et al., 2019;de Salazar Alcalá et al., 2019) (Anonymous, 2016a). Several studies have correlated the pain relief produced with this model with those reported in clinical trials attributable to OA in dogs (Borer et al., 2017;Cross et al., 1997;Doig et al., 2000). Unfortunately, only a single clinical trial has been carried out to evaluate the real clinical efficacy of this active ingredient (Rausch-Derra, Huebner, et al., 2016). Although results in general are encouraging, the efficacy compared to the placebo had a p value of 0.044, which is barely significant, and the efficacy response was only 41%-46%. Additional trials are warranted to confirm its efficacy.

| Rabbits
The thermal antinociceptive effect of grapiprant in the induced inflammatory pain model in the rabbit after a single IV injection of 2 mg/kg was evaluated and compared with that generated by 0.5 mg/kg meloxicam injected subcutaneously ).
An infrared thermal stimuli was applied to the plantar surface of the rabbits' hindlimbs to evaluate the thermal withdrawal latency (TWL).
The thermal antinociceptive effect was expressed as maximum possible response. The grapiprant-treated animals showed a significant increase in TWL from 1 h and up to 10 h after drug administration compared to the control and TWL was higher than meloxicam in the range 1-2 h and at 10 h. The difference in efficacy in the initial hours was supposed to be due to the different routes of administration of the two drugs. The increased efficacy shown at 10 h was speculated to be due to the different doses of drugs administered, to a wider counter-clockwise hysteresis loop or to a potential active metabolite The safety of long-term, daily oral administration of grapiprant was tested (Rausch-Derra et al., 2015). Thirty-six dogs were randomly assigned to four groups (8 dogs/group) that received grapiprant via oral gavage at 0, 1, 6 or 50 mg/kg, once a day for 9 months. All dogs received daily ophthalmologic, ECG, and laboratory evaluations before treatment began (baseline) and periodically afterwards. Dogs were euthanized at the end of the study for necropsy and histologic evaluation. All dogs remained clinically normal during treatment, with no apparent changes in appetite or demeanour. Emesis and soft or mucoid faeces that occasionally contained blood were observed in all groups, although these findings were more common in dogs that received grapiprant. In general, clinicopathologic findings remained within baseline ranges. Drug-related changes in serum total protein and albumin concentrations were detected, but differences were small and resolved during recovery. No drug-related gross or microscopic pathological changes were detected in tissue samples except mild mucosal regeneration in the ileum of one dog in the 50 mg/kg group. These findings suggested that long-term administration of grapiprant is safe in dogs. It might be suggested that if any other NSAID was administered once daily for 9 months at 25 times its clinical dose, the consequences would have been more serious than those reported for grapiprant.
To the best of Authors' knowledge, no study comparing the adverse effects of grapiprant and any other NSAID is available.
However, the pharmaceutical company reports that a dog group (n = 94) treated with 2 mg/kg once daily dose had similar incidence rates for adverse events (diarrhoea, vomiting and inappetence) to the placebo group (n = 88) (Anonymous, 2016a).
When an oral dose of 2 mg/kg was administered daily for 28 days to normal Beagles and Mdr1-deficient dogs, despite grapiprant exposure being greater in these latter animals, no differences in general health, clinical sign or body weight were found (Heit et al., 2018).

| Cats
A single oral dose of grapirant at 2 mg/kg (IV and PO) did not produce visible side effects in cats (Łebkowska-Wieruszewska, De Vito, et al., 2017).
The safety profile was evaluated after daily PO administration of 3, 9 or 15 mg/kg over a period of 4 weeks (Rausch-Derra & Rhodes, 2016). Grapiprant was well tolerated, and no adverse effects were detected at doses ≤15 mg/kg. No significant effects on body weight, food consumption, clinicopathologic variables, or gross or histologic necropsy findings were noted.

| CON CLUS ION
A number of studies have been carried out to assess diverse analytical methods for grapiprant quantification, its pharmacokinetics, pharmacodynamics and safety especially in the canine species. The pharmacokinetic profile seems well characterised in dogs. Although some data are conflicting (oral bioavailability fed vs. fasted), the differences in plasma concentrations might not generate appreciable variation in clinical efficacy. In terms of efficacy, grapiprant is barely better than placebo and inferior to other traditional NSAIDs. It may be an alternative to traditional NSAIDs because of its alternative mode of action, however, further clinical studies are warranted to clarify this point.

ACK N OWLED G EM ENT
Authors would like to express their gratitude to Dr H Owen (University of Queensland, Australia) for her English editing of the manuscript.

CO N FLI C T O F I NTE R E S T
Authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
MG and IS conceived of the presented idea. Both authors read and discussed the literarure and contributed to the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Please contact the author for any requests for access to the data.