Accepted Manuscript

Title: Detection and quantification of the selective EP4receptor antagonist CJ-023423 (grapiprant) in canine plasmaby HPLC with spectrofluorimetric detection

Author: Virgina De Vito Alessandro Saba Hong-Ki Lee HelenOwen Amnart Poapolathep Mario Giorgi

PII: S0731-7085(15)30224-7DOI: http://dx.doi.org/doi:10.1016/j.jpba.2015.11.004Reference: PBA 10327

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 26-5-2015Revised date: 2-11-2015Accepted date: 3-11-2015

Please cite this article as: Virgina De Vito, Alessandro Saba, Hong-Ki Lee, HelenOwen, Amnart Poapolathep, Mario Giorgi, Detection and quantification of the selectiveEP4 receptor antagonist CJ-023423 (grapiprant) in canine plasma by HPLC withspectrofluorimetric detection, Journal of Pharmaceutical and Biomedical Analysishttp://dx.doi.org/10.1016/j.jpba.2015.11.004

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Detection and quantification of the selective EP4 receptor antagonist CJ-023423 (grapiprant) in

canine plasma by HPLC with spectrofluorimetric detection

Virgina De Vito,a Alessandro Saba,b Hong-Ki Lee,c Helen Owen,d Amnart Poapolathep,e Mario

Giorgif*

a Department of Veterinary Medicine, University of Sassari, Via Vienna 2, 07100, Sassari, Italy

b Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Italy

c School of Veterinary Science, University of Queensland, Gatton Campus, Gatton, QLD, 4343,

Australia

d College of Veterinary Medicine, Chungnam National University, Daejeon, South Korea

c Department of Veterinary Pharmacology, Faculty of Veterinary Medicine, University of Kasetsart,

Bangkok, Thailand

e Department of Veterinary Sciences, University of Pisa, Via Livornese (lato monte), 56122 San Piero a

Grado, Pisa, Italy

*Corresponding author. Tel.: +39 50 2210154; fax: +39 50 2210182.

E-mail address: mario.giorgi@unipi.it (M. Giorgi)

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Graphical Abstract

Grapiprant

3

Highlights

Grapiprant is a new active ingredient under development for the control of pain and inflammation

A validated HPLC method with spectrofluorimetric detection has been developed

The chromatographic runs were specific with no interfering peaks at the retention times of the analyte

and IS

LOD and LLOQ were 3 and 10 ng/ml

This method is suitable for pharmacokinetic investigations such as guiding dose adjustment

4

Abstract

Grapiprant, a novel pharmacologically active ingredient, acts as a selective EP4 receptor antagonist

whose physiological ligand is prostaglandin E2 (PGE2). It is currently under development for use in

humans and dogs for the control of pain and inflammation associated with osteoarthritis. The aim of the

present study was to develop an easy and sensitive method to quantify grapiprant in canine plasma and

to apply the method in a canine patient. Several parameters, both in the extraction and detection method

were evaluated. The final mobile phase consisted of ACN:AcONH4 (20 mM) solution, pH 4 (70:30,

v/v) at a flow rate of 1 mL/min. The elution of grapiprant and IS (metoclopramide) was carried out in

isocratic mode through a Synergi Polar-RP 80A analytical column (150 mm × 4.6 mm). The best

excitation and emission wavelengths were 320 and 365 nm, respectively. Grapiprant was extracted

from the plasma using CHCl3, which gave a recovery of 88.1 ± 10.22% and a lower limit of

quantification (LLOQ) of 10 ng/mL. The method was validated in terms of linearity, limit of detection

(LOD), LLOQ, selectivity, accuracy and precision, extraction recovery, stability, and inter-laboratory

cross validation, according to international guidelines. The chromatographic runs were specific with no

interfering peaks at the retention times of the analyte and IS, as confirmed by HPLC-MS experiments.

In conclusion, this was a simple and effective method using HPLC-FL to detect grapiprant in plasma,

which may be useful for future pharmacokinetic studies.

Abbreviation: Prostaglandin E2 (PGE2); Internal Standard (IS); Acetonitrile (ACN); Methanol

(MeOH); Dichloromethane (CH2Cl2); Diethyl ether (Et2O); Chloroform (CHCl3); 2-propanol (C3H8O);

Ethyl acetate (AcOEt); Ammonium acetate (AcONH4); Hydrochloric acid (HCl); Sodium hydroxide

(NaOH); Electrospray ionization source (ESI); Positive ion mode (Ps); Negative ion mode (Ng); Limit

of detection (LOD); Lower limit of quantification (LLOQ); Quality control (QC); Relative error (RE);

Standard deviation (SD); Probability level (P); Percentage relative standard deviation (RSD); The area

under the concentration versus time curve (AUC0-).

Keywords: CJ-023,423; Grapiprant, Fluorescence; HPLC; Dog

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1. Introduction

CJ-023423, 1-[2-[4-(2-ethyl-4,6-dimethylimidazo[4,5-c]pyridin-1-yl)phenyl]ethyl]-3-(4-

methylphenyl)sulfonylurea (Fig. 1), is a novel pharmacologically active ingredient. It has a molecular

weight of 491.61 g/mol, a predicted octanol-water partitioning coefficient of 4.56 and a very poor water

solubility (0.041 mg/L). CJ-023423, also called grapiprant, works as selective EP4 receptor antagonist

[1] whose physiological ligand is prostaglandin E2 (PGE2). The EP4 receptor is one of four G-protein

coupled receptors (EP1, EP2, EP3 and EP4) that mediate the action of PGE2. The EP4 receptor

mediates PGE2-elicited sensitization of sensory neurons [2] and studies have demonstrated that EP4 is

a major receptor in mediating pain associated with both rheumatoid and osteoarthritis [3, 4] and in

inflammation [1, 5]. Grapiprant is currently under development for use in humans and dogs for the

control of pain and inflammation associated with osteoarthritis [6]. Very few data are available on this

active ingredient so far and no data is reported concerning its quantification in biological matrices.

Since grapiprant is an under development drug at advanced stage [7] an easy and accurate method for

its detection in plasma is needed for pharmacokinetic studies. The aim of the present study was two-

fold: i) to develop an easy and sensitive method to quantify grapiprant in canine plasma, ii) to apply the

optimized method for the detection of its pharmacokinetics in a dog.

2. Materials and methods

2.1. Chemicals and reagents

Pure grapiprant analytical standard (> 99.0% purity) was obtained from Cayman Chemical (Ann

Arbor, MI, USA). The Internal Standard (IS) metoclopramide powder (> 99.0% purity) was supplied by

Sigma-Aldrich (St. Louis, MO, USA). Other compounds tested as IS were flupirtine, sulpiride and

trazodone, supplied in powder form by Sigma-Aldrich (St. Louis, MO, USA).

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HPLC grade acetonitrile (ACN), methanol (MeOH), dichloromethane (CH2Cl2), diethyl ether

(Et2O), chloroform (CHCl3), 2-propanol (C3H8O) and ethyl acetate (AcOEt) were purchased from Merck

(Darmstadt, Germany). Ammonium acetate (AcONH4) was purchased from Carlo Erba (Milano, Italy).

Hydrochloric acid (HCl) and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich (St. Louis,

MO, USA). Deionised water was produced by a Milli-Q Millipore Water System (Millipore, MA, USA).

All the other reagents and materials were of analytical grade and supplied from commercial sources. The

aqueous and organic components of the mobile phase, degassed under pressure, were mixed by the

HPLC. The LC mobile phases were filtered through 0.2 μm cellulose acetate membrane filters (Sartorius

Stedim Biotech S.A., Aubagne Cedex, France) with a solvent filtration apparatus. Canine control plasma

samples (containing the same anti-coagulant used in samples from the treated dog) were supplied by the

blood bank of the Veterinary hospital at University of Pisa.

2.2. Standard solutions

Singular stock solutions of grapiprant and IS in MeOH were prepared, each with a concentration

of 1,000 μg/mL by using volumetric flasks. These were stored at -80° C.

To obtain a final concentration of 100 μg/mL, appropriate dilutions of stock standard solutions were

prepared by diluting 1 mL of each solution to 10 mL. Successively, these solutions of grapiprant and IS

were diluted in glass tubes (10 mL), to reach final concentrations of 10, 5 and 1 μg/mL. These were

stored at -20° C.

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2.3. Instrumentation and chromatographic conditions

2.3.1. HPLC-FL

The HPLC system was an LC Jasco (Como, Italy) consisting of quaternary gradient system (PU

980) and an in-line multilambda fluorescence detector (FP 1520). The chromatographic separation assay

was performed with a Synergi Polar-RP 80A analytical column (150 mm × 4.6 mm inner diameter, 4 µm

particle size [Phenomenex, Bologna, Italy]) preceded by a security guard column with the same

stationary phase [Phenomenex, Bologna, Italy]. The system was maintained at 25° C. A range of diverse

aqueous phases (compatible with the subsequent use of the triple quadrupole mass spectrometer) were

tested (10, 20, 50 mM AcONH4). Once the optimal aqueous phase was detected a range of pH (3.5, 4.0,

5.0 and 6.0) was assayed to optimize the chromatographic separation. The flow rate was tested in the

range 0.7-1.2 mL/min. The final mobile phase consisted of ACN:AcONH4 (20 mM) solution, pH 4

(70:30, v/v) at a flow rate of 1 mL/min. The elution of grapiprant and IS was carried out in isocratic

mode. The best excitation and emission wavelengths were found after scanning a 2 µg/mL grapiprant

solution by the fluorescence detector. They were set as 320 and 365 nm for excitation and emission,

respectively.

2.3.2. HPLC-MS

HPLC-MS chromatographic separation was performed by an Agilent Technologies (Santa Clara,

CA, USA) 1290 HPLC system which consisted of a high pressure pump, auto-sampler and column oven,

coupled to an AB-Sciex (Concord, Ontario, Canada) API 4000 triple quadrupole mass spectrometer,

equipped with Turbo V electrospray ionization source (ESI). Main parameters were as follows: mass

range m/z 50-700 Th; source temperature, 600° C; ionspray voltage in positive ion mode (Ps), 5.5 kV;

ionspray voltage in negative ion mode (Ng), -4.2 kV; declustering potential in Ps, 50 V; declustering

8

potential in Ng, -50 V. HPLC runs were carried out by using the same parameters used for HPLC-FL

and reported in the previous section.

2.4. Sample extraction

The procedure was performed in a 15 mL snap cap polypropylene tube. A 0.5 mL aliquot of

plasma sample was added to 100 μL of IS (25 μg/mL). After vortexing for 30 sec, 4 mL of CHCl3 was

added, and the sample was vortexed (30 sec), shaken (60 osc/min, 10 min) and centrifuged at 21,913 g

for 10 min at 25° C. Three mL of the supernatant was collected in a separate clean snap cap polypropylene

tube. The organic phase was evaporated under a gentle stream of nitrogen and reconstituted with 500 μL

of mobile phase. 50 μL of this latter solution was injected onto the HPLC-FL.

2.5. Animal treatment and sampling

Blood samples were obtained from one healthy male Beagle dog administered with an

intravenous injection of grapiprant (0.5 mg/kg) in the right jugular vein. The drug was prior dissolved on

ethanol (10 mg/mL), then diluted with sterile water for injection (9:1 v:v) and immediately injected

(injection rate 5 mL/min). The blood (2 to 3 mL) was collected via catheter, previously inserted in the

left jugular vein, at assigned times (0, 15, 30, 45 min and 1, 2, 4, 6, 8, 10 and 24 h). The blood was

immediately placed into collection tubes containing lithium heparin. The samples were centrifuged at

1,006 g within 30 min of collection and the harvested plasma was frozen immediately and stored at -20°

C. Samples were analysed within 1 week from the collection. Immediately prior to the analysis, the

samples were thawed at room temperature. Standard animal care and handling were performing

according to the Directive 2010/63/UE. The dog was fed with standard food (Hill's Science Diet Pet

Food, Topeka, KS) to avoid potential food impurities (i.e. preservatives) in the blood.

9

2.6. Bioanalytical method validation

The described method was validated in terms of linearity, limit of detection (LOD), lower limit

of quantification (LLOQ), selectivity, accuracy and precision, extraction recovery, stability, and inter-

laboratory cross validation, according to international guidelines on the bioanalytical method validation

[8].

2.6.1. Calibration curves

The peak area ratios of grapiprant (10, 25, 50, 75, 100, 150, 250 and 500 ng/mL) to IS were

plotted against corresponding nominal concentrations of grapiprant. Accuracy (percentage relative

error) for each calibration sample and correlation coefficient were determined in three assay batches.

Linearity of the regression curve in the range 10-500 ng/mL was assessed on the base of the residual

plot, the fit test and the back calculation (within 20% of known amount).

2.6.2. Selectivity and potential impact of breeds

Drug-free blank canine plasma from six individuals of 4 different breeds was extracted and then

assayed. The chromatograms were inspected for any endogenous peaks that could interfere with

retention time of grapiprant or IS. Since nothing is known about the metabolism of this drug in canine

species, in order to assess potential interferences due to metabolites formed in vivo, HPLC-MS analyses

were performed (see 2.4 HPLC-MS).

2.6.3. Accuracy and precision

Intra- and inter-batch reproducibility was examined by assessing accuracy and precision using

quality control (QC) samples (LLOQ, 10 ng/mL; LQC, 50 ng/mL; MQC, 200 ng/mL; HQC, 500

ng/mL; six replicates/concentration) that cover the calibration range. The relative error (RE) and

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percentage relative standard deviation (RSD) were calculated to assess accuracy and precision,

respectively. The acceptance criteria for RE and RSD were ± 15 and 15%, respectively, for LQC,

MQC, and HQC samples, and ± 20 and 20% for the LLOQ samples. Dilution integrity was investigated

using QC samples at 500 and 1000 ng/mL. The QC samples (six replicates/concentration) diluted by

10- and 50-fold, respectively, with blank canine plasma were extracted and then assayed. Accuracy and

precision at each dilution were determined to find whether RE and RSD did not exceed ± 15 and 15%,

respectively.

2.6.4. Extraction recovery

Extraction recovery was evaluated at three levels using LQC (50 ng/mL), MQC (200 ng/mL)

and HQC (500 ng/mL) samples for grapiprant and 5 g/mL for the IS. Extraction recovery was

determined by dividing the peak area of the analytes spiked to blank plasma before extraction by that

spiked after extraction. Then mean recovery at each concentration was represented from three

replicates.

2.6.5. Stability

Stability of grapiprant in canine plasma was assessed at two levels using the LQC and HQC

samples (three replicates/concentration). Bench-top stability in plasma up to 4 h, freeze–thaw stability

from -20° C to ambient temperature in plasma (up to three cycles), the stability in processed samples at

10° C for 23 h and the stability of grapiprant and the IS in stock solution at both 4° C (for 37 and 36

days for grapiprant and the IS, respectively) and ambient temperature (for 17 h), were evaluated.

Samples were considered stable when accuracy was within ± 15%.

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2.6.6. Inter-laboratory cross-validation

Sample analysis of grapiprant across the dog trial was performed in two different laboratories

(Veterinary Pharmacology and Chromatography, both at the University of Pisa). As a cross-validation

study is required to compare grapiprant concentrations in plasma across clinical trials as per the

bioanalytical guidance from the European Medicines Agency [8] and US Food and Drug

Administration [9], grapiprant concentrations in 9 plasma samples were determined at the two

laboratories. Percentage bias was calculated using the following equation:

% bias = (Clab1 − Clab2)

(Clab1 + Clab2)2

x 100

where Clab1 and Clab2 are plasma grapiprant levels in laboratory 1 and 2, respectively.

3. Theory and calculation

3.1. Quantification

When unknown samples were assayed, a control and a fortified blank sample were processed

simultaneously for quality control. LOD and LLOQ were determined as analyte concentrations giving

signal-to-noise ratios of 3 and 10, respectively.

3.2. Statistical analysis and pharmacokinetic evaluation

The statistical analyses were evaluated using the student-t test. The results were presented as

mean ± standard deviation (SD). All the analyses were conducted using GraphPad InStat (GraphPad

Software, Inc, La Jolla CA, USA). In all the experiments, differences were considered significant if the

12

associated probability level (P) was lower than 0.05.

The pharmacokinetic calculations were carried out using WinNonlin v 5.3.1 (Pharsight Corp,

Sunnyvale, CA, USA). The area under the concentration versus time curve (AUC0-) was calculated

using the linear trapezoidal rule. Changes in plasma concentrations of grapiprant were evaluated using

the standard bi-compartmental analysis, and the relative pharmacokinetic parameters were determined

using standard compartmental equations [10].

4. Results and discussion

4.1. Detection method development

Derivation of the mobile phase was achieved using mobile phases that were suitable for the triple

quadrupole mass spectrometer. The ACN:AcONH4 (20 mM) 30:70 v/v, provided the best separation of

grapiprant from the matrix impurities. A range of buffer pH (3.5, 4.0, 5.0 and 6.0) was assayed to optimise

the chromatographic separation and analytes fluorescence intensity. The highest fluorescence intensity

of grapiprant was shown at pH 4.0, while retention times increased with increasing buffer pH. pH 5 and

6 buffers showed peak of grapiprant with both long retention time and broad shape. At pH 3.5 the peak

of grapiprant resulted partially overlapped to a matrix impurities, reducing the selectivity of the method.

The optimal pH value was 4.0 (Fig. 2).

Different flow rates between 0.7-1.2 mL/min were tested using Luna C18 (150x4.6 mm) with 3

µm particle size and Synergi Polar-RP 80A (150x4.6 mm) with 4 µm particle size as stationary phase.

The Synergi analytical column provided the best peak resolution. The final mobile phase resulted in

ACN:AcONH4 (20 mM) pH 4.0 (30:70 v/v) with a 1 mL/min flow rate. It was an excellent compromise

in terms of sensitivity and peak separation. Excitation and emission wavelengths were tested within the

ranges 200-300 and 310-450 nm, respectively. The wavelength values of 320 and 365 nm resulted in

13

optimal excitation and emission, respectively.

For the IS, four compounds (metoclopramide, sulpiride, flupirtine and trazodone) with amenable

chemical features were evaluated. Among these, flupirtine showed a little extraction recovery (36 ± 9%)

while sulpiride showed a short retention time (about 2 min) co-eluting with the matrix impurities.

Metoclopramide and trazodone resulted two good options. Trazodone showed a good recovery and a

good sensitivity to the wave lengths used in the final method, but partially overlapped to an impurity

peak. Metoclopramide was the best candidate with a similar extraction recovery of grapiprant, an

excellent resolution and a short retention time (3.52 ± 0.2 min).

4.2. Optimization of the extraction method

The influence of both the kind of solvent (important tool for the selectivity of the method) and

number of extraction cycles, were studied in order to find the optimal extraction protocol for grapiprant.

Solvents such as CH2Cl2, AcOEt, Et2O, C3H8O and CHCl3 were examined. Variations in the proportions

of the selected extraction solvents (CH2Cl2:Et2O 3:7; AcOEt:CH2Cl2 3:7, 7:3 v/v; CHCl3:C3H8O 5:1;

AcOEt; CHCl3) were also assessed in terms of recovery and selectivity. CHCl3 was selected as the most

suitable organic solvent in terms of analyte extraction and minimization of matrix components

(interferents). The CHCl3 showed the best recovery of analyte (88.1 ± 10.22%) and IS (84.63 ± 9.36%).

In order to increase the recovery, acidic (0.1 N HCl) and alkaline (0.1 N NaOH) conditions were tested

in the CHCl3 extraction method. No improvement of selectivity and recovery was observed (Table 1).

Moreover, the influence of the number of extraction cycles on the extraction efficiency was also

evaluated. The extraction time was set to 10 min and the number of extraction cycles was varied from

one to two. A protocol using two extraction cycles did not significantly increase the recovery. Finally, a

single extraction cycle was selected to ensure an efficient extraction of grapiprant and IS.

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4.3. Method validation

It was necessary to validate each step in the analytical method because to date, methodology for

the determination of grapiprant from plasma samples through a fluorescence detector is yet to be

published.

4.3.1. Linearity

The calibration curve was linear in the investigated range of concentrations (10-500 ng/mL).

Linearity of the regression curve was assessed on the base of the residual plot, the fit test and the back

calculation. The linear regression equation is reported in Table 2.

According to EMA guidelines [8], limit of detection (LOD) and lower limit of quantification (LLOQ)

were calculated based on signal-to-noise approach. The LOD and LLOQ were 3 and 10ng/mL (Table 2),

suggesting a good sensitivity of the method.

4.3.2. Selectivity and potential interference of the breed

Representative chromatograms of grapiprant and IS are shown in Fig. 3. No endogenous

interference was observed at the elution time of grapiprant and IS in canine plasma from six individuals

of 4 different breeds (data not shown). The retention times of grapiprant and the IS were 9.75 ± 0.23 and

3.52 ± 0.2 min, respectively. No matrix impurities were found at the grapiprant and IS retention times.

In addition, HPLC-MS analyses carried out by full scan acquisitions, both in positive and negative ion

mode, evaluated that no impurities due to potential metabolites co-eluted with grapiprant and IS peaks.

In particular, full scan acquisitions in positive ion mode exhibited peaks at 9.85 and 3.77 min, associated

to MS spectra containing [M+H]+ ions at m/z 492.2 and 300.2 Th, and attributable to grapiprant and IS,

respectively. On the contrary, acquisitions in negative ion mode did not provide any chromatographic

peak at the same retention times. The absence of any impurities co-eluting with IS and grapiprant was

15

confirmed by the accurate comparison between the MS spectra from authentic standards and the spectra

from the above mentioned peaks. The MS spectra relative to grapiprant are shown in Fig. 4.

4.3.3. Accuracy and Precision

The intra- (n=6) and inter-batch (n=3) precision and accuracy for the grapiprant assay are shown

in Table 3. Four QC levels (LLOQ, LQC, MQC and HQC) were evaluated to determine RE (accuracy)

and RSD (precision). The intra-batch RE and RSD of LLOQ samples ranged from 5.1 to 8.2% and from

4.1 to 4.9%, respectively; the inter-batch RE and RSD were 8.4 and 6.9%, respectively. For LQC, MQC

and HQC samples, the intra-batch RE and RSD did not exceed 6.6 and 3.5%, respectively, and the inter-

batch RE and RSD were within 7.2 and 6.1%, respectively. These results were within the acceptance

criteria recommended by the bioanalytical guidelines of European Medicines Agency (2012) and US

Food and Drug Administration [9]. QC samples of grapiprant diluted 10-fold showed RE and RSD values

of -3.7 and 2.1%, respectively, and the RE and RSD values of 50-fold diluted samples were -1.96 and

7.3%, respectively.

4.3.4. Extraction recovery

The extraction recovery of grapiprant from canine plasma was 87.2, 92.2 and 89.3% at LQC,

MQC and HQC levels, respectively, and the recovery of the IS was 84.6%. A similar percentage

recovery (average 88.1 ± 10.22) was achieved between grapiprant across all concentrations tested and

the IS.

4.3.5. Stability

Stability studies were performed to ensure good reproducibility of the method. Results in all the

stability assessment showed that the accuracy was ≤ ± 8.5%. Bench-top stability in plasma was ensured

16

for 4 h at ambient temperature and grapiprant was stable even after three freeze–thaw cycles from -20°

C to ambient temperature. Grapiprant in processed samples for injection was stable for 23 h at 10° C.

The stability of grapiprant and the IS in standard solution was ensured up to 37 and 36 days,

respectively, at 4° C, and for at least 17 h at ambient temperature. These findings indicated that the

storage of grapiprant in plasma samples at -20° C is adequate, and no stability-related problems would

be expected during routine analyses for pharmacokinetic study.

4.3.6. Cross-validation

Comparison of grapiprant levels in plasma for the inter-laboratory cross-validation study

showed that the two laboratories gave comparable results with bias ≤ ± 10.9%. These findings suggest

that the cross-validation study was successful and data obtained in the two different laboratories can be

compared. These results demonstrate that the method enables accurate quantification of grapiprant. The

validation parameters were in agreement with the EMA guidelines [8].

4.4. Application of the method

The applicability of this method has been verified by determining grapiprant concentration in

canine plasma after intravenous treatment of 0.5 mg/kg of grapiprant. HPLC analysis of the plasma

confirmed the presence of grapiprant in time-related amounts (Fig. 5). The amounts of the drug in plasma

ranged between 38.9 ng/mL (10 h) and 779 ng/mL (15 min). This latter sample was found to be outside

the linear range of the calibration curve (10-500 ng). It was subsequently diluted with mobile phase and

re-injected. The described method allowed monitoring of the concentration versus time curves of the

analyte and the calculation of the basic pharmacokinetic parameters (Table 4). The pharmacokinetic

profile showed a two-compartment elimination phase. One faster up to 4h (steep) and one slower 4 to 10

h (shallow). These data have to be carefully evaluated because the drug was administered as pure

17

substance in a single dog. Additional studies with larger animal sample size administered with the

marketed drug formulation are necessary to obtain a sound pharmacokinetic evaluation.

Conclusion

The analytical method described in this work provides selective and accurate analysis of

grapiprant without the need for expensive clean up steps, solvent consuming flows or expensive devices.

The low LLOQ shows that the method could be useful for drug measurement even when administered in

sub-clinical doses. These features make the described method suitable for pharmacokinetic investigations

including drug-drug interaction, and potential future applications such as guidance for dose adjustment.

In summary, this is the first time that the HPLC-FL technique is reported detecting grapiprant.

This method (extraction, separation and applied techniques) is simple and efficacious for the

determination of grapiprant in canine plasma.

Acknowledgements

None of the authors has any financial or personal relationships that could inappropriately

influence or bias the content of the paper. This work was supported by athenaeum funds (University of

Pisa) PRA 2016 (3_PRA_2016_8). The preparation of manuscript was not supported by any external

funding.

18

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20

Figure captions

Fig. 1. Molecular structure of grapiprant.

Fig. 2. Effect of the pH on the retention time (spheres) and fluorescence intensity (bars) values.

Fig. 3. Chromatographic curves by HPLC-FL. (A) Chromatographic curve from canine control plasma.

(B) Chromatographic curve from a fortified sample (Grapiprant 100 ng/mL; IS 5 µg/mL). (C)

Chromatographic curve from the plasma sample collected in a treated dog (45 min). (D)

Chromatographic curve from a fortified sample (Grapiprant 10 ng/mL; IS 5 µg/mL).

Fig. 4. Comparison between MS spectra at tR = 9.85 from (A) authentic standard of grapiprant and (B)

the plasma sample collected in a treated dog at 45 min.

Fig. 5. Observed values of plasma concentrations of grapiprant (--) following a single intravenous

dose (0.5 mg/kg BW) in one adult beagle dog.

Figure 1

N

N

N

N N

S

HH

O

O O

Grapiprant (C26H29N5O3S)

FW: 491.6

Cl

NH2

OCH3

HN

N CH3

O

CH3

Metoclopramide (C14H22ClN3O2)

FW: 299.8

Figure 2

2

4

6

8

10

12

0

6000000

12000000

18000000

3.5 4.0 5.0

Ret

enti

on t

ime

(min

) nn

Flu

ore

scen

ce i

nte

nsi

ty n

n

pH

Figure 3

Matrix

Matrix

IS

Grapiprant

IS

Matrix

Grapiprant

IS

Matrix

Grapiprant

A

B

C

D

Figure 4

A

0

2.0e6

4.0e6

Inte

nsity,

cp

s

492.2

100 150 200 250 300 350 400 450 500 550 600 700

0

2.0e6

3.8e6

Inte

nsity,

cp

s

492.2

m/z, Da

B

412.1214.0

219.0 256.6

214.0

219.0256.6

412.1

Figure 5

10

100

1000

0 2 4 6 8 10

Conce

ntr

atio

n (

ng/m

L)

Time (hours)

21

Table 1

Single extraction recovery percentage (± SD) of grapiprant and IS spiked at 100 ng/mL and 5 g/mL,

respectively with different organic solvents

Organic solvents v/v ratio Recovery

Grapiprant IS

CH2Cl2 : Et2O 3:7 46.2±4.6 58.3±3.4

AcOEt:CH2Cl2 3:7 53.4±9.3 51.3±9.8

AcOEt:CH2Cl2 7:3 58.2±2.4 54.2±8.3

CHCl3:C3H8O 5:1 64.1±11.8 52.2±9.2

AcOEt 51.3±10.1 67.1±7.8.4

CHCl3 88.1±10.22 84.6±9.3

CHCl3/HCl 0.1 N 80.22±9.6 71.2±5.4

CHCl3/NaOH 0.1 N 74.3±8.2 86.4±7.4

22

Table 2

Summary of validation parameters

Property Grapiprant

Linear range (ng/mL) 10-500

Calibration

equation y=0.013X-0.42

Correlation coefficient (r2) 0.99

LOQ (ng/mL) 10

LOD (ng/mL) 3

Recovery (%) 88.10±10.22

23

Table 3

Intra- and inter-batch accuracy (% RE) and precision (% RSD) of grapiprant in canine plasma

Quality

control

samples

Nominal

concentration

(ng/mL)

Intra-

batch

Inter-

batch

1 2 3

LLOQ 10 RE (%) 7.3 5.1 8.2 8.4

RSD

(%) 4.5 4.1 4.9 6.9

LQC 50

RE

(%) 6.4 6.6 4.9 7.2

RSD

(%) 3.5 3.2 3 6.1

MQC 200

RE

(%) 4.1 3.2 3.2 7

RSD

(%) 2.4 1.5 1.5 3.2

HQC 500

RE

(%) 3.3 3.5 2.9 6.5

RSD

(%) 1.1 1.1 0.7 2.9

24

Table 4

Predicted pharmacokinetic parameters

Parameters Grapiprant

R2 0.99

AUC (h ng/mL) 1339

HL (h) 6.07

Cl (mL Kg/h) 373

Vss (mL/Kg) 2162

R2, correlation between observed/predicted points; AUC0-

, area under the plasma concentration–time curve

extrapolated to infinity; HL, half life; Cl, total body

clearance; Vss, estimate of the volume of distribution at

steady state.