J. vet. Pharmacol. Therap. doi: 10.1111/jvp.12306
Pharmacokinetic comparison of oral tablet and suspension formulations of grapiprant, a novel therapeutic for the pain and inflammation of osteoarthritis in dogs L. C. RAUSCH-DERRA* L. RHODES* L. FRESHWATER † & R. HAWKS ‡ *Aratana Therapeutics, Inc., Leawood, KS, USA; †BioSTAT Consultants, Inc., Portage, MI, USA; ‡Ricerca Biosciences, LLC, Concord, OH, USA This is an open access article under the terms of the Creative Commons AttributionNonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
Rausch-Derra, L. C., Rhodes, L., Freshwater, L., Hawks, R. Pharmacokinetic comparison of oral tablet and suspension formulations of grapiprant, a novel therapeutic for the pain and inflammation of osteoarthritis in dogs. J. vet. Pharmacol. Therap. doi: 10.1111/jvp.12306. A new anti-inflammatory drug for pain (grapiprant) was recently shown to have minimal side effects following chronic (9-month) daily oral dose of 6 or 50 mg/kg suspension. The current study compares the pharmacokinetics of the formulation used in the chronic safety study to those of the tablet formulation that will be marketed upon FDA approval. Sixteen Beagle dogs were randomized to receive single doses of either 6 or 50 mg/kg grapiprant as both suspension and table formulations within a cross-over design with a 15day washout. Clinical observations were vomiting in one high-dose suspension dog and loose stools in two dogs, one in each 6 mg/kg formulation group. For both formulations, grapiprant reached a maximum concentration within two hours. The tablet formulation had better bioavailability, with AUClast values 34% higher at 6 mg/kg and 64% higher at 50 mg/kg compared to the suspension. Results on Day 0 were similar to those reported on Day 15, suggesting little to no accumulation. Using conversion factors of 1.34 and 1.64, these findings suggest that the 6 and 50 mg/kg suspension doses are equivalent to 4.5 and 30 mg/kg tableted doses, respectively. Combining these findings with the 9-month safety study demonstrates that safety was evaluated at doses approximately 15-fold above the demonstrated therapeutic dose of 2 mg/kg and 10-fold over the ‘safety dose’, defined as the maximum dose a dog of any body weight could receive when dosed at 2 mg/kg with whole or half-tablets. (Paper received 15 September 2015; accepted for publication 26 February 2016) Lesley C. Rausch-Derra, Aratana Therapeutics, Inc., Leawood, KS, USA. E-mail:
[email protected]
INTRODUCTION Canine osteoarthritis is a progressive condition that leads to joint inflammation, cartilage damage, pain, and disability. Treatment is focused on the management and control of disease progression and clinical signs (McLaughlin, 2000; Roush et al., 2010). For many years, the mainstay of medical therapy has been nonsteroidal anti-inflammatory drugs (NSAIDs), which act by inhibiting the cyclooxygenase (COX) enzymes needed to produce prostanoids (Bergh & Budsberg, 2005; Curry et al., 2005; Innes et al., 2010), particularly prostaglandin E2 (PGE2) that plays a pivotal role in the development of joint inflammation and pain (Lin et al., 2006). Unfortunately, COXinhibiting NSAIDs inhibit the production of other important prostanoids as well as PGE2, which may result in unintended
adverse effects (Wright, 2002; Bergh & Budsberg, 2005; Curry et al., 2005; Innes et al., 2010). The nonspecific action of cyclooxygenase inhibition is not necessary for efficacy, as the pain and inflammatory effects of PGE2 in arthritis are mediated by binding of PGE2 to the EP4 receptor, one of four G-proteincoupled receptors that mediate the action of PGE2 (Lin et al., 2006; Clark et al., 2008). Grapiprant is a new drug that only targets the EP4 receptor of PGE2, thereby limiting the potential for adverse effects caused by wider COX inhibition (Nakao et al., 2007). Grapiprant is in development for the control of pain and inflammation associated with osteoarthritis in dogs at a dose of 2 mg/kg (Rausch-Derra et al., 2016). Recent research has shown grapiprant to cause minimal side effects during chronic (9-month) administration to dogs at doses up to 50 mg/kg (Rausch-Derra et al., 2015).
© 2016 The Authors. Journal of Veterinary Pharmacology and Therapeutics Published by John Wiley & Sons Ltd.
1
2 L. C. Rausch-Derra et al.
However, that study was conducted using a liquid formulation, not the tablet that will comprise the marketed product. The current study was designed to explore the pharmacokinetics of these two formulations, thereby bridging the relative bioavailability between suspension and tablet preparations and by extension defining the tableted dose that is comparable to the suspension dose. (The efficacy of grapiprant in dogs with osteoarthritis will be addressed in future publications.)
METHODS A total of 16 Beagle dogs were randomized (four males and four females per group) to receive either 6 or 50 mg/kg grapiprant. Treatment with the suspension and tablet formulations was administered sequentially within a cross-over design within dose groups, with a 15-day washout period between doses (Table 1). Such an approach assured that a four-dog group received each dose and each formulation on each study day. Animals All dogs were laboratory-bred and had been purchased from a commercial supplier. All females were nulliparous, nonpregnant, and experimentally na€ıve. Five of the males were experimentally na€ıve, with three males having been used once previously (>3 months before) in an earlier pharmacokinetic study with grapiprant. Dogs were 11.4–15.3 months of age at the start of dosing and had body weights of 7.95–10.35 kg at the time of randomization. The health status of each dog was confirmed prior to randomization. Routine vaccinations and anthelmintic treatments were administered by the supplier prior to arriving at the laboratory. All dogs were housed at the test facility for a minimum of 4 days prior to randomization. Dogs were housed in individual steel cages within the same study room, which was well ventilated (10 air changes per hour) and maintained at 64–84 oF and 30–70% relative humidity. Dogs were provided a commercial ration once daily (removed after 8 h if not consumed) and ad lib water. On dosing days (Days 0 and 15), dogs were fasted overnight and fed approximately 4 h after dosing. All dogs were treated and handled in accordance with the guidelines recommended in Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Housing also conformed to the USDA Animal Welfare Act (9 CFR Parts 1, 2, and 3). Table 1. Cross-over design comparing two doses of grapiprant administered by two formulations each
Day 0
6 mg/kg (n = 8)
50 mg/kg (n = 8)
Tablet*
Tablet*
Suspension*
Washout Day 15
Suspension*
Suspension*
Washout Tablet*
Suspension*
*Each cell consists of two male and two female dogs.
Tablet*
Study materials The tablets contained either 60 or 100 mg of grapiprant within a flavored, chewable solid. Tablets were placed into sealed and labeled individual containers within 1 day prior to dosing. The tablet batches were tested for stability prior to delivery to the test facility and after study completion. The suspension was formulated to provide 1.2 or 10 mg grapiprant per mL within a 0.5% methylcellulose vehicle for the 6 and the 50 mg/kg dose groups, respectively. Fresh suspension was prepared on each day of dosing (Days 0 and 15) and kept stirred prior to gavage. Suspension homogeneity and stability were tested prior to dosing. Treatment The same number of tablets or volume of suspension was administered to each animal in a group, regardless of body weight, so as to achieve a nominal dose of either 6 or 50 mg/kg. Dogs were given either one 60 mg or five 100 mg tablets, depending on the dosage group. Each tablet was placed in the back of the animal’s mouth to be swallowed. The dogs were then given an oral 10 mL dose of water to aid ingestion. Suspension was administered as 50 mL of either 1.2 or 10 mg/L liquid, depending on the dose group, through a disposable gavage tube. The tube was then flushed with 10 mL water followed by a small amount of air to clear the tube prior to removal. Animals were observed for signs of vomiting at 15 min to 1 h after dosing. At 2 h postdosing, cage-side observations were recorded for general signs of clinical health, illness, distress, or unusual behavior. All animals were also checked for viability once in the morning and once in the afternoon. Pharmacokinetic assessment Following dosing on Days 0 and 15, blood samples were collected from the jugular vein of all animals at the approximate time points of 0.5, 1, 2, 4, 5, 8, 12, 24, and 36 h postdose. Predose samples were also taken at on Day 15 only. Approximately 1 mL of blood was collected into clot-activator tubes, which were then inverted several times and centrifuged under refrigeration (5 °C for 10 min at 2000 g) within 30–60 min of collection. Serum samples were analyzed with the addition of 10 M urea to the serum to a final concentration of 10% urea in each sample. This was performed to reduce carryover. Serum samples were placed in prelabeled tubes and stored frozen at 70 °C until analysis by at least one of two validated methods1 at the laboratory (Ricerca Biosciences, LLC, Concord, OH, USA). Both methods for the quantitation of grapiprant were validated in compliance with the 2001 FDA Guidance for Bioanalytical Method Validation. Drug concentrations of grapiprant were determined with a liquid chromatography triple quadruple mass spectrometry method. Authenticated reference standard was used for the serum standards and quality control 1
Data on file at Aratana; Ricerca studies 028321 and 030814.
© 2016 The Authors. Journal of Veterinary Pharmacology and Therapeutics Published by John Wiley & Sons Ltd.
Pharmacokinetic comparison of formulations 3
samples in serum, and grapiprant-d5 was used as the internal standard. Standard curves and QCs were made in canine serum with an analytical range 5–5000 ng/mL for the low range curve and from 2000 to 100 000 ng/mL for the high range curve with coefficient of determination at least 0.99 for both ranges. Dilutions up to 10-fold were also validated. Analytical runs were accepted if at least 2/3 QCs (low, middle, and high) were within 15% of actual concentration. Pharmacokinetic analysis was conducted using WinNonlin version 6.2 (Pharsight, Mountain View CA, USA). Noncompartmental analysis was conducted using an extravascular administration model. The peak serum concentration (Cmax), time to achieve peak serum concentration (Tmax), half-life (T1/2), and area under the serum concentration–time curve at the last measured time point above the lower limit of quantification (AUClast) were calculated using individual animal grapiprant concentrations. Actual blood collection times were used for the calculation of pharmacokinetic parameters. Serum concentration values that were below the lower limit of quantitation were set to 0. Statistical analysis All analyses were generated using SAS version 9.3.1 (SAS Institute, Cary, NC, USA). Formulation differences were tested
using analysis of variance (ANOVA) for natural-log-transformed measures of Cmax, Tmax, and AUClast. The ANOVA models included sequence, formulation, and time period as fixed effects and subject nested within sequence as a random effect. Ratios of least-square means (LSM) were calculated for each dose using the exponentiation of the difference between formulation LSM based on natural-log-transformed Cmax, Tmax, and AUClast values. For each dose, these ratios were expressed as a ratio relative to the suspension formulation. Consistent with the two-one-sided tests for bioequivalence, 90% confidence intervals (CIs) for the ratios were derived for each dose. Where bioequivalence was not demonstrated, equivalent doses of tablet formulation compared to suspension formulation were calculated by dividing the tablet dose by the geometric relative bioavailability (AUC 0-last) ratio of the tablet to suspension.
RESULTS No dogs died or had any serious health effects. Adverse events were limited to mild gastrointestinal signs. One dog in the 50 mg/kg suspension group vomited at 9 and 11 min after dosing on Day 0. Two dogs in the 6 mg/kg group (one of each
Table 2. Pharmacokinetic ranges on Day 0 and Day 15 for tablet and suspension formulations at each dose Nominal dose
Parameter Tmax (h)
6 mg/kg
Half-life (h) Cmax (ng/mL) AUClast (hng/mL) Tmax (h)
50 mg/kg
Half-life (h) Cmax (ng/mL) AUClast (hng/mL)
Tablet
Suspension*
1–2 (Day 0) 1–2 (Day 15) 2.99–9.12 (Day 0) 2.95–5.42 (Day 15) 2620–11 400 (Day 0) 3520–9210 (Day 15) 10 600–27 600 (Day 0) 13 000–22 700 (Day 15) 1–4 (Day 0) 1–2 (Day 15) 5.00–6.88 (Day 0) 2.23–7.76 (Day 15) 79 900–120 000 (Day 0) 94 800–114 000 (Day 15) 337 000–556 000 (Day 0) 348 000–535 000 (Day 15)
1 (Day 0) 1 (Day 15) 3.14–8.28 (Day 0) 1.64–9.04 (Day 15) 2290–5780 (Day 0) 2610–6150 (Day 15) 10 300-19 100 (Day 0) 10 700-17 100 (Day 15) 1–2 (Day 0) 1 (Day 15) 4.41–11.00 (Day 0) 4.03–5.19 (Day 15) 83 500–122 000 (Day 0) 46 000–91 900 (Day 15) 258 000–621 000 (Day 0) 124 000–264 000 (Day 15)
*For 50 mg/kg group, excludes animal that vomited. Table 3. Relative bioavailability of grapiprant at nominal doses of 6 and 50 mg/kg
Nominal Dose
Parameter
6 mg/kg
AUC0-last Cmax Tmax AUC0-last Cmax Tmax
50 mg/kg
Unit
Grapiprant-flavored tablets GM N=8
ngh/mL ng/mL h ngh/mL ng/mL h
17 595 5020 1.297 436 198 97 721 1.834
Methylcellulose suspension GM N=7 13 164 3713 1.000 265 786 83 553 1.122
Formulation P-value
Geometric ratio
LCL
UCL
0.027 0.035 0.097 0.013 0.207 0.058
1.34 1.35 1.30 1.64 1.17 1.63
1.10 1.09 1.00 1.26 0.94 1.09
1.62 1.68 1.68 2.14 1.45 2.45
GM, geometric mean; LCL, lower confidence limit; UCL, upper confidence limit; Tmax, time to maximum plasma concentration (Cmax). © 2016 The Authors. Journal of Veterinary Pharmacology and Therapeutics Published by John Wiley & Sons Ltd.
4 L. C. Rausch-Derra et al.
DISCUSSION
Mean serum grapiprant concentration (ng/mL)
(a)
Tablet 1E+06 6 mg/kg 50 mg/kg 1E+05
1E+04
1000
100
10
1 0
8
16
24
32
40
Time (h)
(b) Mean serum grapiprant concentration (ng/mL)
formulation) developed soft or loose stools following the Day 0 dose, and two in the 50 mg/kg group (one of each formulation) developed mucoid stools after the Day 15 dose. Otherwise, all animals remained clinically normal. The Cmax and AUClast results for the dog that vomited twice within 11 min of dosing were approximately an order of magnitude below those of other dogs in the 50 mg/kg suspension group, so this dog’s values were excluded from pharmacokinetic analysis (Table 2). Grapiprant rapidly achieved elevated concentrations in the sera, with mean Tmax of
