J. vet. Pharmacol. Therap. 38, 336--343. doi: 10.1111/jvp.12180.
Single- and multiple dose pharmacokinetics and multiple dose pharmacodynamics of oral ABT-116 (a TRPV1 antagonist) in dogs S. NIYOM* , † K. R. MAMA* D. L. GUSTAFSON* & M. L. REZENDE* *Department of Clinical Sciences, College of Veterinary Medicine and Biomedical sciences, Colorado State University, Fort Collins, CO, USA; †Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand
Niyom, S., Mama, K. R., Gustafson, D. L., Rezende, M. L. Single- and multiple dose pharmacokinetics and multiple dose pharmacodynamics of oral ABT116 (a TRPV1 antagonist) in dogs. J. vet. Pharmacol. Therap. 38, 336–343. Six dogs were used to determine single and multiple oral dose pharmacokinetics of ABT-116. Blood was collected for subsequent analysis prior to and at 15, 30 min and 1, 2, 4, 6, 12, 18, and 24 h after administration of a single 30 mg/kg dose of ABT-116. Results showed a half-life of 6.9 h, kel of 0.1/h, AUC of 56.5 lgh/mL, Tmax of 3.7 h, and Cmax of 3.8 lg/mL. Based on data from this initial phase, a dose of 10 mg/kg of ABT-116 (no placebo control) was selected and administered to the same six dogs once daily for five consecutive days. Behavioral observations, heart rate, respiratory rate, temperature, thermal and mechanical (proximal and distal limb) nociceptive thresholds, and blood collection were performed prior to and 4, 8, and 16 h after drug administration each day. The majority of plasma concentrations were above the efficacious concentration (0.23 lg/mL previously determined for rodents) for analgesia during the 24-h sampling period. Thermal and distal limb mechanical thresholds were increased at 4 and 8 h, and at 4, 8, and 16 h respectively, postdosing. Body temperature increased on the first day of dosing. Results suggest adequate exposure and antinociceptive effects of 10 mg/kg ABT-116 following oral delivery in dogs. (Paper received 11 June 2014; accepted for publication 18 September 2014) Khursheed R. Mama, Department of Clinical Sciences, Colorado State University, Fort Collins, CO 80523, USA. E-mail: [email protected]
INTRODUCTION The transient receptor potential channel vanilloid subfamily member 1 (TRPV1) is a calcium-permeable nonselective cation channel, located in neuronal tissue including the brain, sensory nerves, dorsal root and trigeminal ganglia, and in non-neuronal tissue including the urinary bladder and skin (Birder et al., 2001; Bodo et al., 2004; Stander et al., 2004; Immke & Gavva, 2006). The TRPV1 is considered an essential component of nociception in the pain pathway (Julius & Basbaum, 2001; Ji et al., 2002) responding to thermal and chemical noxious stimuli (Caterina et al., 1997; Tominaga et al., 1998). For example, TRPV1 knockout mice showed attenuation of nociceptive responses in models of inflammatory hyperalgesia (Caterina et al., 2000; Davis et al., 2000; Keeble et al., 2005; Barton et al., 2006). Recently, a number of TRPV1 antagonists have been synthesized and evaluated for analgesic efficacy (Walker et al., 2003; Gavva et al., 2005; Ghilardi et al., 2005; Honore et al., 2005, 2009; Culshaw et al., 2006; Rajpal et al., 2007). Only a few, however, possess sufficient safety and efficacy profiles to progress into clinical trials (Szallasi et al., 2007).
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ABT-116 is one of the TRPV1 antagonists being evaluated for use as an analgesic with demonstrated analgesic efficacy in rodent models of osteoarthritis, postoperative and inflammatory pain and suitable physiochemical and pharmacokinetic properties to warrant further investigation (Brown et al., 2010). Recently ABT-116 has been shown to transiently increase mechanical nociceptive thresholds (Niyom et al., 2012), attenuate lameness in dogs with sodium urate-induced synovitis (Cathcart et al., 2012), and decrease administration of rescue analgesic medication in client-owned dogs with hip osteoarthritis (Malek et al., 2012). Despite these favorable experimental and clinical outcomes, to the author’s knowledge, there is no information regarding the pharmacokinetic profile of ABT-116 in dogs. This manuscript describes the pharmacokinetic profile of a single (30 mg/kg) orally administered dose of ABT-116 and subsequent use of pharmacokinetic data to develop and evaluate a multiple day dosage regimen in dogs. The specific objective of this latter or multiple dose aspect of the study was to assess drug exposure and analgesic efficacy at the derived daily dose (10 mg/kg) of ABT-116.
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Pharmacokinetics and pharmacodynamics of ABT-116 in dogs 337
MATERIALS AND METHODS Animals This study protocol was approved by the Institutional Animal Care and Use Committee at Colorado State University. Six (three male and three female, 7–9 month old) purpose bred, healthy Walker hounds weighing 20.3 2.8 kg (mean SD) during phase 1 and 22 2.9 kg (mean SD) during phase 2 were studied. Dogs were considered healthy based on physical examination and laboratory evaluation including a complete blood count and serum chemistry profile. Each dog was housed in an individual run with fresh water and commercial dry dog food provided ad libitum during the entire study period (phase 1 and 2). Dogs interacted daily with study personnel and each other and were acclimatized to the nociceptive devices and the study environment for a week prior to each phase of the study. Experimental design The study was divided into two phases. Phase 1 was designed to determine the pharmacokinetics of a single orally administered dose (30 mg/kg) of ABT-116. This information was then used to simulate daily dosing of 5, 10, and 20 mg/kg and selected a dose that best targeted plasma concentrations previously shown to be consistent with analgesic efficacy (0.23 lg/mL; obtained from rodent models of acute inflammatory and osteoarthritis pain, unpublished data Abbott Animal Health, North Chicago, IL, USA) for Phase 2 of the study; data relative to efficacious plasma concentrations for dogs were not available when this study was initiated. This second phase was designed to verify simulated results and evaluate the analgesic efficacy of the dosage regimen. Phase 1 On the morning of the study, a 16-gauge long-term catheter (MILA International, Inc. Erlanger, KY, USA) was aseptically placed into the jugular vein of unsedated dogs and secured with a suture material and an elastic bandage. This was used to facilitate blood sampling during this phase of the study. Capsules of ABT-116 were administered within 1–2 h of a small meal (Canned puppy food; Hill’s Pet Nutrition Inc., Topeka, KS, USA) to facilitate absorption as recommended by the manufacturer. Blood was sampled prior to (baseline) and at 15 and 30 min and 1, 2, 4, 6, 12, 18, and 24 h postadministration, processed, and stored for subsequent measurement of plasma drug concentrations. At each time point 6 mL of blood was withdrawn to clear the catheter prior to collection of a 7 mL blood sample. The initially withdrawn blood was returned intravenously to the dog and the catheter flushed with heparinized saline. Behavioral and physiological observations and evaluation of the antinociceptive effects of ABT-116 in dogs were concurrently observed. These results have been previously reported (Niyom et al., 2012). © 2014 John Wiley & Sons Ltd
Phase 2 This was initiated approximately six weeks after the conclusion of phase one. Each dog received a capsule containing 10 mg/kg of ABT-116 orally once daily within 1 h of a small meal for five consecutive days. Blood (7 mL) was collected via direct cephalic venipuncture prior to (baseline) and 4, 8, and 16 h after the drug administration each day, processed, and stored for subsequent measurement of plasma drug concentrations. These values were used verify that the simulation based dosing schedule gave rise to plasma concentrations shown to be consistent with analgesic efficacy (as previously noted) throughout most of the dosing interval while limiting maximal and overall exposure to limit toxicity. Response to nociceptive threshold testing, behavioral and physiological (heart rate, respiratory rate, rectal temperature) observations, and the presence of urine, feces, vomit, and saliva were also recorded at these time points. Heart rate was obtained by femoral pulse palpation and respiratory rate by observation of thoracic excursions, both over a 30-sec interval. To avoid the influence of thermometer insertion on other parameters, rectal temperature was obtained after all other behavioral and physiological evaluations. Analgesic efficacy was assessed using a thermal nociceptive threshold device and two mechanical nociceptive threshold devices. The thermal and proximal mechanical nociceptive testing instruments were validated for dogs prior to the study (Niyom et al., 2011). The distal mechanical nociceptive testing instrument had been used by investigators previously (Niyom et al., 2012) and as with the other devices were calibrated daily prior to use. The thermal testing instrument (Topcat Metrology Ltd, Ely, Cambridgeshire, England) consisted of a thermal probe that was applied to the laterodorsal aspect of each dog’s shaved thorax. Baseline skin temperature (°C) was measured and the probe then remotely heated at a fixed rate of rise. The peak temperature at which the dog first responded was considered the threshold. The difference between baseline skin temperature and the threshold was calculated for each measurement. The proximal mechanical nociceptive threshold device (Topcat Metrology Ltd, Ely, Cambridgeshire, England) utilized in this study was applied to the dorsolateral aspect of the radius of each dog. A sham was placed in a similar location on the other limb. A single blunt ended probe was connected to a force sensor and remotely activated with a fixed rate of rise. The threshold (proximal nociceptive threshold) was recorded in Newtons as the value at which the dog first responded. The distal mechanical nociceptive threshold device (C-clamp, Colorado State University, Fort Collins, CO, USA) was manually applied in a dorsopalmar fashion distal to the large foot pad and a 1cm2 force transducer connected to an electronic recorder quantified the force (lb/cm2) at which the dog first responded. The measured force was subsequently converted to Newtons. Each measurement was repeated 2–3 times at manufacturer recommended intervals (for thermal and proximal mechanical
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nociceptive devices) at each time point. Attempts were made to have 2–3 readings within 10% of each other, and the average value was used in subsequent analyses. It should be noted that during the acclimatization period, individual dogs were observed to respond differently to the devices. Some would turn their head toward the stimulus, a few would attempt to bite the device, and others would attempt to move away from the stimulus or show a definitive skin twitch (thermal) or leg lift (mechanical). An evaluator familiar with these animals and their typical responses was responsible for all assessments. Cut-off values of 60 °C, 20 Newtons, and 20 lb/cm2 were set for the thermal threshold, proximal, and distal limb threshold devices, respectively to minimize tissue damage in the absence of a nociceptive response. Animal averages for the thresholds at each time point and each day were subsequently compared. Blood samples were collected after nociceptive threshold testing at each time point to minimize any potential influence this may have on behavioral and physiological parameters. LC/MS/MS determination of ABT-116 in dog plasma ABT-116 was measured using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The analysis was carried out in the Pharmacology Core at the Colorado State University Veterinary Medical Center. The LC/MS/MS-based assay was developed and validated for the analysis of ABT-116 in dog plasma. For sample preparation, a 100 lL aliquot of plasma was transferred to a 1.5 mL polypropylene microcentrifuge tube followed by protein precipitation by the addition of 400 lL of acetonitrile and vortexing for 10 min. Samples were centrifuged at 14 500 g for 5 min and the resulting supernatant collected. Standards and quality assurance/control samples were constructed by spiking 100 lL of blank dog plasma with known amounts of ABT-116 and processing them as described. Guidelines for batch acceptance and assay validation were based on accepted practices for quantitative chromatographic assays (Viswanathan et al., 2007), and the assay accuracy and precision were calculated to be 91.9% 7.7%. The reference standards ranged from 10–10 000 ng/mL; quality control samples of 50, 500, and 5000 ng/mL were used. Positive ion electrospray ionization mass spectra were obtained with a 3200 Q-TRAP triple quadrupole mass spectrometer (Applied Biosystems Inc., Foster City, CA, USA) with a turbo ion spray source interfaced to a 1200 Series Binary Pump SL liquid chromatography system and HTC-PAL autosampler (Leap Technologies, Carrboro, NC, USA). Chromatography was performed on a Phenomenex Phenyl (Phenomenex, Torrance, CA, USA), 5 lm, 4.6 9 5 mm column using an isocratic gradient of 60% acetonitrile and 40% trifluoroacetic acid (0.1%). The flow rate was 500 lL/min, injection volume, 10 lL, and the total analysis time, 4 min. The mass spectrometer settings were as follows: turbo ion spray temperature, 450 °C; ion spray voltage, 2000 V; declustering potential, 41 V; entrance potential, 6.0 V; collision energy, 31 V; collision cell exit potential, 4 V; collision cell entrance potential,
16 V; collision gas, N2, medium; curtain gas, N2, 10 units; nebulizer gas, N2, 40 units; and auxiliary gas, N2, 20 units. Samples were quantified by area of the signal obtained by monitoring the transition m/z 433.2 ? 266.2 for the analyte ABT-116 and comparison to a standard curve constructed using known standards. Pharmacokinetic modeling Modeling of plasma concentration vs. time data for ABT-116 was carried out using WinNonlin version 4.1 (Pharsight Corp., Mountain View, CA, USA) software. Simulation and testing of alternative multiday dosing strategies were carried out using Excel software (Microsoft Corp., Redmond, WA, USA). Parameter values were derived from compartmental modeling specifically a standard one-compartment open model with a firstorder input equation (Wagner, 1993). In order to determine a dosage regimen that would result in ABT-116 plasma concentrations that exceed 0.23 lg/mL or the concentration previously determined to be efficacious in rodent models while minimizing maximal and total drug exposure concentrations to limit deleterious side effects, ABT-116 plasma concentrations were simulated using the pharmacokinetic parameters obtained from the 30 mg/kg single dose for prediction of steady-state at 5, 10, and 20 mg/kg/day doses. The accumulation factor (AF) at steady-state was calculated using the equation: AF ¼
1 1 ekel T
where kel is the elimination rate constant and Τ is the dosing interval (24 h). The average AF calculated from the single-dose study was 1.10 0.03. When using individual dog pharmacokinetic parameters 4/6, 3/6, and 1/6 dogs had plasma concentrations above the target concentration with 20, 10 and 5 mg/kg/day dosing, respectively when drug concentrations were extrapolated to steady-state. Statistical analysis The sample size (n = 6) was determined based on data from a previous study using the same testing methodology for which clinically important and statistically significant differences were detected (Niyom et al., 2012). Physiological and nociceptive threshold data were summarized as mean SD. Data were analyzed using commercially available statistical software (SAS/STAT software, version 9.2, SAS Institute Inc, Cary, NC, USA). Comparisons between pre-administration (baseline) data and those at the other time points on a given day and at a given time point between days were analyzed by use of a mixed model repeated-measures analysis of variance. The repeated-measures factors were time point and day. Residuals from the analysis of variance were plotted to confirm normality and independence. A linear contrast was used to test for a trend over days. Pair-wise comparisons of least square means © 2014 John Wiley & Sons Ltd
Pharmacokinetics and pharmacodynamics of ABT-116 in dogs 339
were further assessed by use of t-tests. Values of P < 0.05 were considered statistically significant. RESULTS Phase 1: pharmacokinetics and modeling of data following oral ABT-116 (30 mg/kg) ABT-116 plasma concentration vs. time curves were fit to both single and multiple compartment models and the resulting output analyzed to determine which model to use for further analysis. The one-compartment model was chosen over the two-compartment model due to 6/6 curves fitting this model with acceptable correlation coefficients (>0.9) as opposed to the two-compartment fit where only 5/6 curves could be resolved. Lower Akaike information criteria (AIC) values were also associated with the one-compartment model. Pharmacokinetic parameters calculated using the one-compartment model are shown in Table 1. Individual modeling simulations for each dog along with the actual data (mean values and standard deviation [SD] values are shown in Fig. 1 for the single 30 mg/kg dose. As shown in Fig. 2, simulated mean plasma concentrations from all dogs exceeded the 0.23 lg/mL concentration throughout the dosing interval with daily dosing of 10 and 20 mg/kg/ day; plasma concentrations after a simulated dose of 5 mg/kg/ day remained above this concentration for approximately 75% of a given 24-h period. These results were used to justify going forward with daily dosing studies using a dose of 10 mg/kg/ day to verify simulation results and concurrently test analgesic efficacy and assess dogs for potential side effects. Phase 2 As shown in Fig. 3, the multiple dose simulations were consistent with the sample measurements as each of the measured plasma concentrations fell within the 95% confidence interval of the predicted concentrations based on pharmacokinetic parameters from each of the six dogs used in the studies. The calculated drug accumulation on days 3–5, which presumably
should represent steady-state levels based on the predicted elimination half-life of 6.9 h, was 1.17 0.23 similar to the 1.10 0.03 as predicted from the single-dose data. At steadystate, all of the dogs had plasma drug concentrations above concentrations previously determined to be efficacious through the 16 h time point. Drug concentrations >0.23 lg/mL were measured in 3 of 6 dogs prior to the next dosing (24 h from prior dose). Analgesic efficacy and side effects. Analgesic efficacy assessment using the distal limb mechanical device included data from five dogs. Data collection for this assessment tool was not performed in dog 6 as the dog appeared agitated with attempts
Fig. 1. Mean SD of measured plasma ABT-116 concentration (ng/ mL) in 6 dogs at 15, 30 min and 1, 2, 4, 6, 12, 18, and 24 h after oral administration of a single dose (30 mg/kg) of ABT-116. The 0 time on the x-axis represents values collected at baseline prior to ABT-116 administration. Each model simulation line represents the one-compartment model fit (simulation) for each dog. The data were best fit by including a time lag (20 min) for drug absorption.
Table 1. Pharmacokinetic parameters following oral administration of ABT-116 (30 mg/kg) to six dogs PK parameter* AUC (lgh/mL) Cmax (lg/mL) ka (h1) Τmax (h) kel (h1) t1/2 (h)
Mean SD
Range
14.97–121.25 1.62–7.91 0.39–9.75 0.92–5.94 0.09–0.13 5.50–8.12
56.54 3.81 2.15 3.66 0.10 6.88
38.93 2.27 3.73 1.77 0.02 0.94
AUC, area under curve; Cmax, maximum concentration; ka, absorption rate constant; Tmax, time to reach Cmax; kel, elimination rate constant; t1/2, half-life. *Pharmacokinetic parameters were estimated using a one-compartment open model with first-order input and a time lag for drug absorption. © 2014 John Wiley & Sons Ltd
Fig. 2. Simulated mean plasma concentrations following 3 doses (5, 10, 20 mg/kg/day) of ABT-116. A time lag of 20 min for drug absorption was used in the simulations. The horizontal dotted line represents the efficacious concentration (0.23 lg/mL) previously determined for rodents.
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(a)
(b)
Fig. 3. Measured plasma drug concentrations of the six dogs prior to and at 4, 8, and 16 h after oral administration of 10 mg/kg of ABT116 once daily for five consecutive days. The shaded area represents the 95% confidence interval (CI) predicted from the dose simulation. The horizontal dotted line represents the efficacious concentration (0.23 lg/mL) previously determined for rodents.
to apply this testing modality during the acclimatization period. As shown in Fig. 4, the distal limb threshold averages at 4, 8, and 16 h (39.3, 37.5, and 34.4 Newton’s, respectively) postdrug over five days were significantly higher (P = 0.0001, P = 0.0007, and P = 0.0283, respectively) than at baseline (29.2 Newton’s). The proximal mechanical threshold testing on the other hand was unaltered. An increase in thermal threshold was observed at 4 and 8 h postdrug (12.6 and 12.3 °C; P = 0.0001 and P = 0.001, respectively) compared to the baseline value (10.9 °C). Average thermal thresholds on day 1 and 3 (12.3 and 12.3 °C) were higher than that on day 4 (11.1 °C; P = 0.038 and P = 0.029, respectively); the average value (12.5 °C) for day 2 was higher than values recorded on day 4 and 5 (11.1 and 11.2 °C; P = 0.015 and P = 0.028, respectively) (Fig. 5). During repeated day administration of ABT-116, heart and respiratory rates did not change significantly compared to preadministration values. Rectal temperature was elevated at 4, 8, and 16 h after the drug administration (all P < 0.0001) (Table 2). Mean rectal temperature on day 1 (39.9 °C) was higher than that on the other days (39.4, 39.3, 39.2, and 39 °C for day 2–5, respectively; all P < 0.0001). Day 2 and 3 values were higher than those obtained on day 5 (P = 0. 0008 and 0.0262, respectively). Diarrhea was observed in one male dog 4 h postdrug administration on day 5.
DISCUSSION Several TRPV1 antagonists have been developed since 1997 (Zicha et al., 2012). Approximately, fifteen compounds entered phase 1 clinical trials with about five progressing to phase 2 trials (Trevisani & Gatti, 2013) some of which have been
(c)
Fig. 4. Mean SD of thermal (a), proximal (b) and distal (c, n = 5) limb mechanical nociceptive thresholds in dogs prior to and at 4, 8, and 16 h after oral administration of 10 mg/kg ABT-116 once daily for five consecutive days. The 0 time on the x-axis represents values collected at baseline prior to ABT-116 administration. *Indicate differences (P < 0.05) of the nociceptive threshold compared to the corresponding baseline value.
suspended or terminated because of side effects, such as unmanageable hyperthermia. As mentioned previously, ABT-116 was targeted for evaluation in animal models due to favorable early results of its analgesic efficacy, pharmacokinetic properties, and safety profile relative to other vanilloid compounds (Gomtsyan et al., 2008; Brown et al., 2010; Maher et al., 2011). In the current study, despite following the recommendation of providing a small meal prior to drug administration to © 2014 John Wiley & Sons Ltd
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Fig. 5. Thermal nociceptive threshold in six dogs averaged from the four time points on each day. Different letters denote significant (P < 0.05) differences between days. Table 2. Mean SD values for heart rate, respiratory rate, and rectal temperature averaged at each time point over five days in six dogs receiving 10 mg/kg of ABT-116 orally
Heart rate (beats/min) Respiratory rate (breaths/ min) Temperature (°C)
Baseline
4h
8h
16 h
112 16
112 15
114 13
113 12
19 3
19 3
20 4
20 3
39.6 0.5*
39.6 0.3*
39.3 0.3*
39 0.3
*Significantly different (P < 0.05) from baseline. See manuscript for additional details.
facilitate absorption, it took an average of 3.66 1.77 h to reach maximum plasma concentration (Tmax) after a single oral dose (30 mg/kg) of ABT-116. High variability of ka among subjects with values indicating a twenty-five-fold difference indicates highly variable absorption of ABT-116 in dogs after oral administration. The half-life of 6.88 h is approximately twice that reported in rats (3.1 h) after a single oral dose of ABT-116 (Brown et al., 2010) indicative of a higher elimination rate of ABT-116 in rats. Following multiple day dosing of ABT-116, the measured plasma concentrations were within the simulation based predicted 95% confidence intervals. Overall the results are suggestive of a positive correlation between measured plasma concentrations and both body temperature changes and analgesic efficacy assessed using thermal and distal limb nociceptive thresholds. Similar analgesic benefits are reported following multiple day administration of the same (10 mg/kg) orally administered dose of ABT-116 in dogs with sodium urate-induced synovitis (Cathcart et al., 2012) and in a rat model of osteoarthritic pain (Honore et al., 2009). Interestingly, we did not observe changes in nociceptive thresholds in the three dogs with measured plasma drug concentrations >0.23 lg/mL (or the targeted and previously described efficacious concentration in rodents) observed 24 h after the prior dose. Hence, while increases in both thermal and distal limb nociceptive thresholds in the current multiple dose phase of the study indicate that ABT-116 has the potential © 2014 John Wiley & Sons Ltd
to reduce nociceptive pain, other factors may play a role. It is possible that this is accounted for by individual variation, or that plasma drug concentrations associated with analgesia differ between dogs and rodents, or that effect site concentration (which were not measured in this study) is the more relevant end point. It has also been suggested that repeat dosing provides sustained blockade of TRPV1 receptors and additionally reduces the influence of substance P and calcitonin generelated peptide in nociceptive processing (Honore et al., 2009). In this study, we noted variations in response over time. For example, the thermal nociceptive threshold averages on day 4 and 5 tended to be lower than those obtained from the first three days of the multiple dosing. This is not in accordance with findings from studies with other TRPV1 antagonists such as A-995662 and ABT-102. In those studies, the analgesic efficacy was greater after repeated oral dosing of A-995662 and ABT-102 for 8 and 12 days, respectively when compared to the potency after a single oral dose administration in rat models of osteoarthritic pain (Honore et al., 2009; Puttfarcken et al., 2010). The variation may result from species and drug differences or as a result of the different models used to test analgesia. While we cannot rule out a temporal affect due to lack of a placebo control, dogs were acclimatized to the study devices and the study environment in an effort to standardize variables. Additionally, evaluation of the repeatability of thermal and mechanical nociceptive testing was previously performed at similar intervals in the same dogs over a three-day period (Niyom et al., 2011). Hyperthermia is a major side effect of TRPV1 antagonists and is reported in rats, dogs, monkeys, and humans (Swanson et al., 2005; Gavva et al., 2007a,b, 2008). Vasoconstriction and an increase in thermogenesis induced by TRPV1 blockade are proposed as mechanisms for the elevated body temperature (Gavva, 2008). In the present study, body temperature in dogs receiving the 10 mg/kg of ABT-116 was markedly increased on the first day of the multiple dose administration, but the increase was less notable during subsequent days. This attenuation of hyperthermia following multiple dosing is in accordance with reports from previous TRPV1 antagonist studies in rodents where prominently elevated temperatures were observed on the first day of administration with lesser elevations on subsequent days (Gavva et al., 2007a; Honore et al.,
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2009). Even though the mechanism of the attenuation of hyperthermia is unknown, the present data support an assumption proposed by Gavva in 2007 that there must be compensatory mechanisms controlling the homeostatic body temperature (Gavva et al., 2007a). On day 5, one male dog had diarrhea 4 h postdrug. It is possible this was an incidental finding, but we cannot rule out a potential cause and effect relationship of the drug and this effect as TRPV1 receptors expressed in gastric epithelial cells are reported to play a role in gastroprotection (Takeuchi et al., 1992; Kato et al., 2003; Holzer, 2004; Horie et al., 2004; Mozsik et al., 2005). Hence. it has been postulated that the TRPV1 antagonists may disrupt the mucus layer which surrounds the gastric epithelial lining resulting in irritation of the stomach and duodenum, and transient diarrhea. If attributed to the drug, it is likely that these gastrointestinal side effects are dose dependent; in the current lower repeated dose study, diarrhea was observed only once whereas in the prior single high-dose study, we observed this side effect within 4 h postdosing in a five of six dogs (Niyom et al., 2012). In conclusion, pharmacokinetic parameters derived from the oral administrations of a single (30 mg/kg) dose of ABT-116 in dogs were useful in predicting a multiple day administration regimen. As predicted, the majority of measured plasma drug concentrations were above the targeted previously described efficacious concentration for rodents through the postdrug sampling period for five consecutive days. Additionally, nociceptive threshold testing revealed the analgesic potential of this dosage regimen, while attenuating hyperthermia and gastrointestinal effects such as diarrhea which were considered significant side effects after single high dose administration of ABT-116.
ACKNOWLEDGMENTS The test compound (ABT-116) and funding for this study were provided by Abbott Animal Health. The authors gratefully acknowledge James R zumBrunnen for providing assistance with statistical analyses, and Ryan J. Hansen and Susan Hudachek for providing assistance with sample processing and analysis.
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Pharmacokinetics and pharmacodynamics of ABT-116 in dogs 343 Carruthers, N.I., Swanson, D., Kuskowski, M., Flores, C.M., Julius, D. & Mantyh, P.W. (2005) Selective blockade of the capsaicin receptor TRPV1 attenuates bone cancer pain. Journal of Neuroscience, 25, 3126–3131. Gomtsyan, A., Bayburt, E.K., Schmidt, R.G., Surowy, C.S., Honore, P., Marsh, K.C., Hannick, S.M., McDonal, H.A., Wetter, J.M., Sullivan, J.P., Jarvis, M.F., Faltynek, C.R. & Lee, C. (2008) Identification of (R)-1-(5-tert-butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl) urea (ABT-102) as a potent TRPV1 antagonist for pain management. Journal of Medicinal Chemistry, 51, 392–395. Holzer, P. (2004) TRPV1 and the gut: from a tasty receptor for a painful vanilloid to a key player in hyperalgesia. European Journal of Pharmacology, 500, 231–241. Honore, P., Wismer, C.T., Mikusa, J., Zhu, C.Z., Zhong, C., Gauvin, D.M., Gomtsyan, A., El Kouhen, R., Lee, C.H., Marsh, K., Sullivan, J.P., Faltynek, C.R. & Jarvis, M.F. (2005) A-425619 [1-isoquinolin-5yl-3-(4-trifluoromethyl-benzyl)-urea], a novel transient receptor potential type V1 receptor antagonist, relieves pathophysiological pain associated with inflammation and tissue injury in rats. Journal of Pharmacology and Experimental Therapeutics, 314, 410–421. Honore, P., Chandran, P., Hernandez, G., Gauvin, D.M., Mikusa, J.P., Zhong, C., Joshi, S.K., Ghilardi, J.R., Sevcik, M.A., Fryer, R.M., Segreti, J.A., Banfor, P.N., Marsh, K., Neelands, T., Bayburt, E., Daanen, J.F., Gomtsyan, A., Lee, C.H., Kort, M.E., Reilly, R.M., Surowy, C.S., Kym, P.R., Mantyh, P.W., Sullivan, J.P., Jarvis, M.F. & Faltynek, C.R. (2009) Repeated dosing of ABT-102, a potent and selective TRPV1 antagonist, enhances TRPV1-mediated analgesic activity in rodents, but attenuates antagonist-induced hyperthermia. Pain, 142, 27–35. Horie, S., Yamamoto, H., Michael, G.J., Uchida, M., Belai, A., Watanabe, K., Priestley, J.V. & Murayama, T. (2004) Protective role of vanilloid receptor type 1 in HCl-induced gastric mucosal lesions in rats. Scandinavian Journal of Gastroenterology, 39, 303–312. Immke, D.C. & Gavva, N.R. (2006) The TRPV1 receptor and nociception. Seminars in Cell & Developmental Biology, 17, 582–591. Ji, R.R., Samad, T.A., Jin, S.X., Schmoll, R. & Woolf, C.J. (2002) p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron, 36, 57–68. Julius, D. & Basbaum, A.I. (2001) Molecular mechanisms of nociception. Nature, 413, 203–210. Kato, S., Aihara, E., Nakamura, A., Xin, H., Matsui, H., Kohama, K. & Takeuchi, K. (2003) Expression of vanilloid receptors in rat gastric epithelial cells: role in cellular protection. Biochemical Pharmacology, 66, 1115–1121. Keeble, J., Russell, F., Curtis, B., Starr, A., Pinter, E. & Brain, S.D. (2005) Involvement of transient receptor potential vanilloid 1 in the vascular and hyperalgesic components of joint inflammation. Arthritis and Rheumatism, 52, 3248–3256. Maher, M.P., Bhattacharya, A., Swanson, H.A.N., Wu, N., Freedman, J., Kansagara, M., Scott, B., Li, D.H., Eckert, W.A. III, Liu, Y., Seppassi, K., Rizzolio, M., Fitzgerald, A., Liu, J., Branstetter, B.J., Rech, J.C., Lebsack, A.D., Breitenbucher, J.G. & Wickenden, A.D. (2011) Characterization of 2-(2,6-dichloro-benzyl)-thiazolo[5,4-d]pyrimidin-7-yl](4-trifluoromethyl-phenyl)-amine (JNJ-39729209) as a novel TRPV1 antagonist. European Journal of Pharmacology, 663, 40–50. Malek, S., Sample, S.J., Schwartz, Z., Nemke, B., Jacobson, P.B., Cozzi, E.M., Schaefer, S.L., Bleedorn, J.A., Holzman, G. & Muir, P. (2012) Effect of analgesic therapy on clinical outcome measures in a randomized controlled trial using client-owned dogs with hip osteoarthritis. BMC Veterinary Research, 8, 185. Mozsik, G., Szolcsanyi, J. & Racz, I. (2005) Gastroprotection induced by capsaicin in healthy human subjects. World Journal of Gastroenterology, 11, 5180–5184.
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Niyom, S., Mama, K.R. & Rezende, M.L. (2011). Repeatability of thermal and mechanical nociceptive threshold testing devices in dogs. In, Proceedings of the Association of Veterinary Anaesthetists/ European College of Veterinary Anaesthesia and Analgesia Spring Meeting, p 113, Bari, Italy, 2011 Niyom, S., Mama, K.R. & De Rezende, M.L. (2012) Comparison of the analgesic efficacy of oral ABT-116 administration with that of transmucosal buprenorphine administration in dogs. American Journal of Veterinary Research, 73, 476–481. Puttfarcken, P.S., Han, P., Joshi, S.K., Neelands, T.R., Gauvin, D.M., Baker, S.J., Lewis, L.G., Bianchi, B.R., Mikusa, J.P., Koenig, J.R., Perner, R.J., Kort, M.E., Honore, P., Faltynek, C.R., Kym, P.R. & Reilly, R.M. (2010) A-995662 [(R)-8-(4-methyl-5-(4-(trifluoromethyl)phenyl) oxazol-2-ylamino)-1,2,3,4-tetrahydr onaphthalen-2-ol], a novel, selective TRPV1 receptor antagonist, reduces spinal release of glutamate and CGRP in a rat knee joint pain model. Pain, 150, 319–326. Rajpal, S., Gerovac, T.A., Turner, N.A., Tilghman, J.I., Allcock, B.K., McChesney, S.L., Miranpuri, G.S., Park, S.W. & Resnick, D.K. (2007) Antihyperalgesic effects of vanilloid-1 and bradykinin-1 receptor antagonists following spinal cord injury in rats. Journal of Neurosurgery Spine, 6, 420–424. Stander, S., Moormann, C., Schumacher, M., Buddenkotte, J., Artuc, M., Shpacovitch, V., Brzoska, T., Lippert, U., Henz, B.M., Luger, T.A., Metze, D. & Steinhoff, M. (2004) Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast cells, and epithelial cells of appendage structures. Experimental Dermatology, 13, 129–139. Swanson, D.M., Dubin, A.E., Shah, C., Nasser, N., Chang, L., Dax, S.L., Jetter, M., Breitenbucher, J.G., Liu, C., Mazur, C., Lord, B., Gonzales, L., Hoey, K., Rizzolio, M., Bogenstaetter, M., Codd, E.E., Lee, D.H., Zhang, S.P., Chaplan, S.R. & Carruthers, N.I. (2005) Identification and biological evaluation of 4-(3-trifluoromethylpyridin-2-yl)piperazine-1-carboxylic acid (5-trifluoromethylpyridin-2-yl)amide, a high affinity TRPV1 (VR1) vanilloid receptor antagonist. Journal of Medicinal Chemistry, 48, 1857–1872. Szallasi, A., Cortright, D.N., Blum, C.A. & Eld, S.R. (2007) The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of concept. Nature Reviews Drug Discovery, 6, 357–372. Takeuchi, K., Ueshima, K., Matsumoto, J. & Okabe, S. (1992) Role of capsaicin-sensitive sensory nerves in acid-induced bicarbonate secretion in rat stomach. Digestive Diseases and Sciences, 37, 737–743. Tominaga, M., Caterina, M.J., Malmberg, A.B., Rosen, T.A., Gilbert, H., Skinner, K., Raumann, B.E., Basbaum, A.I. & Julius, D. (1998) The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron, 21, 531–543. Trevisani, M. & Gatti, R. (2013) TRPV1 antagonists as analgesic agents. Open Pain Journal, 6, 108–118. Viswanathan, C.T., Bansal, S., Booth, B., DeStefano, A.J., Rose, M.J., Sailstad, J., Shah, V.P., Skelly, J.P., Swann, P.G. & Weiner, R. (2007) Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays. Pharmaceutical Research, 24, 1962–1973. Wagner, J.G. (1993) Simple linear models. In Pharmacokinetics for the Pharmaceutical Scientist. Ed. Wagner, J.G., pp. 1–14. Technomic Publishing Company Inc., Lancaster, PA. Walker, K.M., Urban, L., Medhurst, S.J., Patel, S., Panesar, M., Fox, A.J. & McIntyre, P. (2003) The VR1 antagonist capsazepine reverses mechanical hyperalgesia in models of inflammatory and neuropathic pain. Journal of Pharmacology and Experimental Therapeutics, 304, 56–62. Zicha, S., Radresa, O., Laplante, P., Morton, M., Jones, K., Main, M., Trivedi, S., Julien, R.P., Griffin, A., Labrecque, J., Ahmad, S. & Brown, W. (2012) Novel methodology to identify TRPV1 antagonists independent of capsaicin activation. Journal of Biomolecular Screening, 18, 544–555.
Single- and multiple dose pharmacokinetics and multiple dose pharmacodynamics of oral ABT-116 (a TRPV1 antagonist) in dogs S. NIYOM* , † K. R. MAMA* D. L. GUSTAFSON* & M. L. REZENDE* *Department of Clinical Sciences, College of Veterinary Medicine and Biomedical sciences, Colorado State University, Fort Collins, CO, USA; †Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand
Niyom, S., Mama, K. R., Gustafson, D. L., Rezende, M. L. Single- and multiple dose pharmacokinetics and multiple dose pharmacodynamics of oral ABT116 (a TRPV1 antagonist) in dogs. J. vet. Pharmacol. Therap. 38, 336–343. Six dogs were used to determine single and multiple oral dose pharmacokinetics of ABT-116. Blood was collected for subsequent analysis prior to and at 15, 30 min and 1, 2, 4, 6, 12, 18, and 24 h after administration of a single 30 mg/kg dose of ABT-116. Results showed a half-life of 6.9 h, kel of 0.1/h, AUC of 56.5 lgh/mL, Tmax of 3.7 h, and Cmax of 3.8 lg/mL. Based on data from this initial phase, a dose of 10 mg/kg of ABT-116 (no placebo control) was selected and administered to the same six dogs once daily for five consecutive days. Behavioral observations, heart rate, respiratory rate, temperature, thermal and mechanical (proximal and distal limb) nociceptive thresholds, and blood collection were performed prior to and 4, 8, and 16 h after drug administration each day. The majority of plasma concentrations were above the efficacious concentration (0.23 lg/mL previously determined for rodents) for analgesia during the 24-h sampling period. Thermal and distal limb mechanical thresholds were increased at 4 and 8 h, and at 4, 8, and 16 h respectively, postdosing. Body temperature increased on the first day of dosing. Results suggest adequate exposure and antinociceptive effects of 10 mg/kg ABT-116 following oral delivery in dogs. (Paper received 11 June 2014; accepted for publication 18 September 2014) Khursheed R. Mama, Department of Clinical Sciences, Colorado State University, Fort Collins, CO 80523, USA. E-mail: [email protected]
INTRODUCTION The transient receptor potential channel vanilloid subfamily member 1 (TRPV1) is a calcium-permeable nonselective cation channel, located in neuronal tissue including the brain, sensory nerves, dorsal root and trigeminal ganglia, and in non-neuronal tissue including the urinary bladder and skin (Birder et al., 2001; Bodo et al., 2004; Stander et al., 2004; Immke & Gavva, 2006). The TRPV1 is considered an essential component of nociception in the pain pathway (Julius & Basbaum, 2001; Ji et al., 2002) responding to thermal and chemical noxious stimuli (Caterina et al., 1997; Tominaga et al., 1998). For example, TRPV1 knockout mice showed attenuation of nociceptive responses in models of inflammatory hyperalgesia (Caterina et al., 2000; Davis et al., 2000; Keeble et al., 2005; Barton et al., 2006). Recently, a number of TRPV1 antagonists have been synthesized and evaluated for analgesic efficacy (Walker et al., 2003; Gavva et al., 2005; Ghilardi et al., 2005; Honore et al., 2005, 2009; Culshaw et al., 2006; Rajpal et al., 2007). Only a few, however, possess sufficient safety and efficacy profiles to progress into clinical trials (Szallasi et al., 2007).
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ABT-116 is one of the TRPV1 antagonists being evaluated for use as an analgesic with demonstrated analgesic efficacy in rodent models of osteoarthritis, postoperative and inflammatory pain and suitable physiochemical and pharmacokinetic properties to warrant further investigation (Brown et al., 2010). Recently ABT-116 has been shown to transiently increase mechanical nociceptive thresholds (Niyom et al., 2012), attenuate lameness in dogs with sodium urate-induced synovitis (Cathcart et al., 2012), and decrease administration of rescue analgesic medication in client-owned dogs with hip osteoarthritis (Malek et al., 2012). Despite these favorable experimental and clinical outcomes, to the author’s knowledge, there is no information regarding the pharmacokinetic profile of ABT-116 in dogs. This manuscript describes the pharmacokinetic profile of a single (30 mg/kg) orally administered dose of ABT-116 and subsequent use of pharmacokinetic data to develop and evaluate a multiple day dosage regimen in dogs. The specific objective of this latter or multiple dose aspect of the study was to assess drug exposure and analgesic efficacy at the derived daily dose (10 mg/kg) of ABT-116.
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MATERIALS AND METHODS Animals This study protocol was approved by the Institutional Animal Care and Use Committee at Colorado State University. Six (three male and three female, 7–9 month old) purpose bred, healthy Walker hounds weighing 20.3 2.8 kg (mean SD) during phase 1 and 22 2.9 kg (mean SD) during phase 2 were studied. Dogs were considered healthy based on physical examination and laboratory evaluation including a complete blood count and serum chemistry profile. Each dog was housed in an individual run with fresh water and commercial dry dog food provided ad libitum during the entire study period (phase 1 and 2). Dogs interacted daily with study personnel and each other and were acclimatized to the nociceptive devices and the study environment for a week prior to each phase of the study. Experimental design The study was divided into two phases. Phase 1 was designed to determine the pharmacokinetics of a single orally administered dose (30 mg/kg) of ABT-116. This information was then used to simulate daily dosing of 5, 10, and 20 mg/kg and selected a dose that best targeted plasma concentrations previously shown to be consistent with analgesic efficacy (0.23 lg/mL; obtained from rodent models of acute inflammatory and osteoarthritis pain, unpublished data Abbott Animal Health, North Chicago, IL, USA) for Phase 2 of the study; data relative to efficacious plasma concentrations for dogs were not available when this study was initiated. This second phase was designed to verify simulated results and evaluate the analgesic efficacy of the dosage regimen. Phase 1 On the morning of the study, a 16-gauge long-term catheter (MILA International, Inc. Erlanger, KY, USA) was aseptically placed into the jugular vein of unsedated dogs and secured with a suture material and an elastic bandage. This was used to facilitate blood sampling during this phase of the study. Capsules of ABT-116 were administered within 1–2 h of a small meal (Canned puppy food; Hill’s Pet Nutrition Inc., Topeka, KS, USA) to facilitate absorption as recommended by the manufacturer. Blood was sampled prior to (baseline) and at 15 and 30 min and 1, 2, 4, 6, 12, 18, and 24 h postadministration, processed, and stored for subsequent measurement of plasma drug concentrations. At each time point 6 mL of blood was withdrawn to clear the catheter prior to collection of a 7 mL blood sample. The initially withdrawn blood was returned intravenously to the dog and the catheter flushed with heparinized saline. Behavioral and physiological observations and evaluation of the antinociceptive effects of ABT-116 in dogs were concurrently observed. These results have been previously reported (Niyom et al., 2012). © 2014 John Wiley & Sons Ltd
Phase 2 This was initiated approximately six weeks after the conclusion of phase one. Each dog received a capsule containing 10 mg/kg of ABT-116 orally once daily within 1 h of a small meal for five consecutive days. Blood (7 mL) was collected via direct cephalic venipuncture prior to (baseline) and 4, 8, and 16 h after the drug administration each day, processed, and stored for subsequent measurement of plasma drug concentrations. These values were used verify that the simulation based dosing schedule gave rise to plasma concentrations shown to be consistent with analgesic efficacy (as previously noted) throughout most of the dosing interval while limiting maximal and overall exposure to limit toxicity. Response to nociceptive threshold testing, behavioral and physiological (heart rate, respiratory rate, rectal temperature) observations, and the presence of urine, feces, vomit, and saliva were also recorded at these time points. Heart rate was obtained by femoral pulse palpation and respiratory rate by observation of thoracic excursions, both over a 30-sec interval. To avoid the influence of thermometer insertion on other parameters, rectal temperature was obtained after all other behavioral and physiological evaluations. Analgesic efficacy was assessed using a thermal nociceptive threshold device and two mechanical nociceptive threshold devices. The thermal and proximal mechanical nociceptive testing instruments were validated for dogs prior to the study (Niyom et al., 2011). The distal mechanical nociceptive testing instrument had been used by investigators previously (Niyom et al., 2012) and as with the other devices were calibrated daily prior to use. The thermal testing instrument (Topcat Metrology Ltd, Ely, Cambridgeshire, England) consisted of a thermal probe that was applied to the laterodorsal aspect of each dog’s shaved thorax. Baseline skin temperature (°C) was measured and the probe then remotely heated at a fixed rate of rise. The peak temperature at which the dog first responded was considered the threshold. The difference between baseline skin temperature and the threshold was calculated for each measurement. The proximal mechanical nociceptive threshold device (Topcat Metrology Ltd, Ely, Cambridgeshire, England) utilized in this study was applied to the dorsolateral aspect of the radius of each dog. A sham was placed in a similar location on the other limb. A single blunt ended probe was connected to a force sensor and remotely activated with a fixed rate of rise. The threshold (proximal nociceptive threshold) was recorded in Newtons as the value at which the dog first responded. The distal mechanical nociceptive threshold device (C-clamp, Colorado State University, Fort Collins, CO, USA) was manually applied in a dorsopalmar fashion distal to the large foot pad and a 1cm2 force transducer connected to an electronic recorder quantified the force (lb/cm2) at which the dog first responded. The measured force was subsequently converted to Newtons. Each measurement was repeated 2–3 times at manufacturer recommended intervals (for thermal and proximal mechanical
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nociceptive devices) at each time point. Attempts were made to have 2–3 readings within 10% of each other, and the average value was used in subsequent analyses. It should be noted that during the acclimatization period, individual dogs were observed to respond differently to the devices. Some would turn their head toward the stimulus, a few would attempt to bite the device, and others would attempt to move away from the stimulus or show a definitive skin twitch (thermal) or leg lift (mechanical). An evaluator familiar with these animals and their typical responses was responsible for all assessments. Cut-off values of 60 °C, 20 Newtons, and 20 lb/cm2 were set for the thermal threshold, proximal, and distal limb threshold devices, respectively to minimize tissue damage in the absence of a nociceptive response. Animal averages for the thresholds at each time point and each day were subsequently compared. Blood samples were collected after nociceptive threshold testing at each time point to minimize any potential influence this may have on behavioral and physiological parameters. LC/MS/MS determination of ABT-116 in dog plasma ABT-116 was measured using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The analysis was carried out in the Pharmacology Core at the Colorado State University Veterinary Medical Center. The LC/MS/MS-based assay was developed and validated for the analysis of ABT-116 in dog plasma. For sample preparation, a 100 lL aliquot of plasma was transferred to a 1.5 mL polypropylene microcentrifuge tube followed by protein precipitation by the addition of 400 lL of acetonitrile and vortexing for 10 min. Samples were centrifuged at 14 500 g for 5 min and the resulting supernatant collected. Standards and quality assurance/control samples were constructed by spiking 100 lL of blank dog plasma with known amounts of ABT-116 and processing them as described. Guidelines for batch acceptance and assay validation were based on accepted practices for quantitative chromatographic assays (Viswanathan et al., 2007), and the assay accuracy and precision were calculated to be 91.9% 7.7%. The reference standards ranged from 10–10 000 ng/mL; quality control samples of 50, 500, and 5000 ng/mL were used. Positive ion electrospray ionization mass spectra were obtained with a 3200 Q-TRAP triple quadrupole mass spectrometer (Applied Biosystems Inc., Foster City, CA, USA) with a turbo ion spray source interfaced to a 1200 Series Binary Pump SL liquid chromatography system and HTC-PAL autosampler (Leap Technologies, Carrboro, NC, USA). Chromatography was performed on a Phenomenex Phenyl (Phenomenex, Torrance, CA, USA), 5 lm, 4.6 9 5 mm column using an isocratic gradient of 60% acetonitrile and 40% trifluoroacetic acid (0.1%). The flow rate was 500 lL/min, injection volume, 10 lL, and the total analysis time, 4 min. The mass spectrometer settings were as follows: turbo ion spray temperature, 450 °C; ion spray voltage, 2000 V; declustering potential, 41 V; entrance potential, 6.0 V; collision energy, 31 V; collision cell exit potential, 4 V; collision cell entrance potential,
16 V; collision gas, N2, medium; curtain gas, N2, 10 units; nebulizer gas, N2, 40 units; and auxiliary gas, N2, 20 units. Samples were quantified by area of the signal obtained by monitoring the transition m/z 433.2 ? 266.2 for the analyte ABT-116 and comparison to a standard curve constructed using known standards. Pharmacokinetic modeling Modeling of plasma concentration vs. time data for ABT-116 was carried out using WinNonlin version 4.1 (Pharsight Corp., Mountain View, CA, USA) software. Simulation and testing of alternative multiday dosing strategies were carried out using Excel software (Microsoft Corp., Redmond, WA, USA). Parameter values were derived from compartmental modeling specifically a standard one-compartment open model with a firstorder input equation (Wagner, 1993). In order to determine a dosage regimen that would result in ABT-116 plasma concentrations that exceed 0.23 lg/mL or the concentration previously determined to be efficacious in rodent models while minimizing maximal and total drug exposure concentrations to limit deleterious side effects, ABT-116 plasma concentrations were simulated using the pharmacokinetic parameters obtained from the 30 mg/kg single dose for prediction of steady-state at 5, 10, and 20 mg/kg/day doses. The accumulation factor (AF) at steady-state was calculated using the equation: AF ¼
1 1 ekel T
where kel is the elimination rate constant and Τ is the dosing interval (24 h). The average AF calculated from the single-dose study was 1.10 0.03. When using individual dog pharmacokinetic parameters 4/6, 3/6, and 1/6 dogs had plasma concentrations above the target concentration with 20, 10 and 5 mg/kg/day dosing, respectively when drug concentrations were extrapolated to steady-state. Statistical analysis The sample size (n = 6) was determined based on data from a previous study using the same testing methodology for which clinically important and statistically significant differences were detected (Niyom et al., 2012). Physiological and nociceptive threshold data were summarized as mean SD. Data were analyzed using commercially available statistical software (SAS/STAT software, version 9.2, SAS Institute Inc, Cary, NC, USA). Comparisons between pre-administration (baseline) data and those at the other time points on a given day and at a given time point between days were analyzed by use of a mixed model repeated-measures analysis of variance. The repeated-measures factors were time point and day. Residuals from the analysis of variance were plotted to confirm normality and independence. A linear contrast was used to test for a trend over days. Pair-wise comparisons of least square means © 2014 John Wiley & Sons Ltd
Pharmacokinetics and pharmacodynamics of ABT-116 in dogs 339
were further assessed by use of t-tests. Values of P < 0.05 were considered statistically significant. RESULTS Phase 1: pharmacokinetics and modeling of data following oral ABT-116 (30 mg/kg) ABT-116 plasma concentration vs. time curves were fit to both single and multiple compartment models and the resulting output analyzed to determine which model to use for further analysis. The one-compartment model was chosen over the two-compartment model due to 6/6 curves fitting this model with acceptable correlation coefficients (>0.9) as opposed to the two-compartment fit where only 5/6 curves could be resolved. Lower Akaike information criteria (AIC) values were also associated with the one-compartment model. Pharmacokinetic parameters calculated using the one-compartment model are shown in Table 1. Individual modeling simulations for each dog along with the actual data (mean values and standard deviation [SD] values are shown in Fig. 1 for the single 30 mg/kg dose. As shown in Fig. 2, simulated mean plasma concentrations from all dogs exceeded the 0.23 lg/mL concentration throughout the dosing interval with daily dosing of 10 and 20 mg/kg/ day; plasma concentrations after a simulated dose of 5 mg/kg/ day remained above this concentration for approximately 75% of a given 24-h period. These results were used to justify going forward with daily dosing studies using a dose of 10 mg/kg/ day to verify simulation results and concurrently test analgesic efficacy and assess dogs for potential side effects. Phase 2 As shown in Fig. 3, the multiple dose simulations were consistent with the sample measurements as each of the measured plasma concentrations fell within the 95% confidence interval of the predicted concentrations based on pharmacokinetic parameters from each of the six dogs used in the studies. The calculated drug accumulation on days 3–5, which presumably
should represent steady-state levels based on the predicted elimination half-life of 6.9 h, was 1.17 0.23 similar to the 1.10 0.03 as predicted from the single-dose data. At steadystate, all of the dogs had plasma drug concentrations above concentrations previously determined to be efficacious through the 16 h time point. Drug concentrations >0.23 lg/mL were measured in 3 of 6 dogs prior to the next dosing (24 h from prior dose). Analgesic efficacy and side effects. Analgesic efficacy assessment using the distal limb mechanical device included data from five dogs. Data collection for this assessment tool was not performed in dog 6 as the dog appeared agitated with attempts
Fig. 1. Mean SD of measured plasma ABT-116 concentration (ng/ mL) in 6 dogs at 15, 30 min and 1, 2, 4, 6, 12, 18, and 24 h after oral administration of a single dose (30 mg/kg) of ABT-116. The 0 time on the x-axis represents values collected at baseline prior to ABT-116 administration. Each model simulation line represents the one-compartment model fit (simulation) for each dog. The data were best fit by including a time lag (20 min) for drug absorption.
Table 1. Pharmacokinetic parameters following oral administration of ABT-116 (30 mg/kg) to six dogs PK parameter* AUC (lgh/mL) Cmax (lg/mL) ka (h1) Τmax (h) kel (h1) t1/2 (h)
Mean SD
Range
14.97–121.25 1.62–7.91 0.39–9.75 0.92–5.94 0.09–0.13 5.50–8.12
56.54 3.81 2.15 3.66 0.10 6.88
38.93 2.27 3.73 1.77 0.02 0.94
AUC, area under curve; Cmax, maximum concentration; ka, absorption rate constant; Tmax, time to reach Cmax; kel, elimination rate constant; t1/2, half-life. *Pharmacokinetic parameters were estimated using a one-compartment open model with first-order input and a time lag for drug absorption. © 2014 John Wiley & Sons Ltd
Fig. 2. Simulated mean plasma concentrations following 3 doses (5, 10, 20 mg/kg/day) of ABT-116. A time lag of 20 min for drug absorption was used in the simulations. The horizontal dotted line represents the efficacious concentration (0.23 lg/mL) previously determined for rodents.
340 S. Niyom et al.
(a)
(b)
Fig. 3. Measured plasma drug concentrations of the six dogs prior to and at 4, 8, and 16 h after oral administration of 10 mg/kg of ABT116 once daily for five consecutive days. The shaded area represents the 95% confidence interval (CI) predicted from the dose simulation. The horizontal dotted line represents the efficacious concentration (0.23 lg/mL) previously determined for rodents.
to apply this testing modality during the acclimatization period. As shown in Fig. 4, the distal limb threshold averages at 4, 8, and 16 h (39.3, 37.5, and 34.4 Newton’s, respectively) postdrug over five days were significantly higher (P = 0.0001, P = 0.0007, and P = 0.0283, respectively) than at baseline (29.2 Newton’s). The proximal mechanical threshold testing on the other hand was unaltered. An increase in thermal threshold was observed at 4 and 8 h postdrug (12.6 and 12.3 °C; P = 0.0001 and P = 0.001, respectively) compared to the baseline value (10.9 °C). Average thermal thresholds on day 1 and 3 (12.3 and 12.3 °C) were higher than that on day 4 (11.1 °C; P = 0.038 and P = 0.029, respectively); the average value (12.5 °C) for day 2 was higher than values recorded on day 4 and 5 (11.1 and 11.2 °C; P = 0.015 and P = 0.028, respectively) (Fig. 5). During repeated day administration of ABT-116, heart and respiratory rates did not change significantly compared to preadministration values. Rectal temperature was elevated at 4, 8, and 16 h after the drug administration (all P < 0.0001) (Table 2). Mean rectal temperature on day 1 (39.9 °C) was higher than that on the other days (39.4, 39.3, 39.2, and 39 °C for day 2–5, respectively; all P < 0.0001). Day 2 and 3 values were higher than those obtained on day 5 (P = 0. 0008 and 0.0262, respectively). Diarrhea was observed in one male dog 4 h postdrug administration on day 5.
DISCUSSION Several TRPV1 antagonists have been developed since 1997 (Zicha et al., 2012). Approximately, fifteen compounds entered phase 1 clinical trials with about five progressing to phase 2 trials (Trevisani & Gatti, 2013) some of which have been
(c)
Fig. 4. Mean SD of thermal (a), proximal (b) and distal (c, n = 5) limb mechanical nociceptive thresholds in dogs prior to and at 4, 8, and 16 h after oral administration of 10 mg/kg ABT-116 once daily for five consecutive days. The 0 time on the x-axis represents values collected at baseline prior to ABT-116 administration. *Indicate differences (P < 0.05) of the nociceptive threshold compared to the corresponding baseline value.
suspended or terminated because of side effects, such as unmanageable hyperthermia. As mentioned previously, ABT-116 was targeted for evaluation in animal models due to favorable early results of its analgesic efficacy, pharmacokinetic properties, and safety profile relative to other vanilloid compounds (Gomtsyan et al., 2008; Brown et al., 2010; Maher et al., 2011). In the current study, despite following the recommendation of providing a small meal prior to drug administration to © 2014 John Wiley & Sons Ltd
Pharmacokinetics and pharmacodynamics of ABT-116 in dogs 341
Fig. 5. Thermal nociceptive threshold in six dogs averaged from the four time points on each day. Different letters denote significant (P < 0.05) differences between days. Table 2. Mean SD values for heart rate, respiratory rate, and rectal temperature averaged at each time point over five days in six dogs receiving 10 mg/kg of ABT-116 orally
Heart rate (beats/min) Respiratory rate (breaths/ min) Temperature (°C)
Baseline
4h
8h
16 h
112 16
112 15
114 13
113 12
19 3
19 3
20 4
20 3
39.6 0.5*
39.6 0.3*
39.3 0.3*
39 0.3
*Significantly different (P < 0.05) from baseline. See manuscript for additional details.
facilitate absorption, it took an average of 3.66 1.77 h to reach maximum plasma concentration (Tmax) after a single oral dose (30 mg/kg) of ABT-116. High variability of ka among subjects with values indicating a twenty-five-fold difference indicates highly variable absorption of ABT-116 in dogs after oral administration. The half-life of 6.88 h is approximately twice that reported in rats (3.1 h) after a single oral dose of ABT-116 (Brown et al., 2010) indicative of a higher elimination rate of ABT-116 in rats. Following multiple day dosing of ABT-116, the measured plasma concentrations were within the simulation based predicted 95% confidence intervals. Overall the results are suggestive of a positive correlation between measured plasma concentrations and both body temperature changes and analgesic efficacy assessed using thermal and distal limb nociceptive thresholds. Similar analgesic benefits are reported following multiple day administration of the same (10 mg/kg) orally administered dose of ABT-116 in dogs with sodium urate-induced synovitis (Cathcart et al., 2012) and in a rat model of osteoarthritic pain (Honore et al., 2009). Interestingly, we did not observe changes in nociceptive thresholds in the three dogs with measured plasma drug concentrations >0.23 lg/mL (or the targeted and previously described efficacious concentration in rodents) observed 24 h after the prior dose. Hence, while increases in both thermal and distal limb nociceptive thresholds in the current multiple dose phase of the study indicate that ABT-116 has the potential © 2014 John Wiley & Sons Ltd
to reduce nociceptive pain, other factors may play a role. It is possible that this is accounted for by individual variation, or that plasma drug concentrations associated with analgesia differ between dogs and rodents, or that effect site concentration (which were not measured in this study) is the more relevant end point. It has also been suggested that repeat dosing provides sustained blockade of TRPV1 receptors and additionally reduces the influence of substance P and calcitonin generelated peptide in nociceptive processing (Honore et al., 2009). In this study, we noted variations in response over time. For example, the thermal nociceptive threshold averages on day 4 and 5 tended to be lower than those obtained from the first three days of the multiple dosing. This is not in accordance with findings from studies with other TRPV1 antagonists such as A-995662 and ABT-102. In those studies, the analgesic efficacy was greater after repeated oral dosing of A-995662 and ABT-102 for 8 and 12 days, respectively when compared to the potency after a single oral dose administration in rat models of osteoarthritic pain (Honore et al., 2009; Puttfarcken et al., 2010). The variation may result from species and drug differences or as a result of the different models used to test analgesia. While we cannot rule out a temporal affect due to lack of a placebo control, dogs were acclimatized to the study devices and the study environment in an effort to standardize variables. Additionally, evaluation of the repeatability of thermal and mechanical nociceptive testing was previously performed at similar intervals in the same dogs over a three-day period (Niyom et al., 2011). Hyperthermia is a major side effect of TRPV1 antagonists and is reported in rats, dogs, monkeys, and humans (Swanson et al., 2005; Gavva et al., 2007a,b, 2008). Vasoconstriction and an increase in thermogenesis induced by TRPV1 blockade are proposed as mechanisms for the elevated body temperature (Gavva, 2008). In the present study, body temperature in dogs receiving the 10 mg/kg of ABT-116 was markedly increased on the first day of the multiple dose administration, but the increase was less notable during subsequent days. This attenuation of hyperthermia following multiple dosing is in accordance with reports from previous TRPV1 antagonist studies in rodents where prominently elevated temperatures were observed on the first day of administration with lesser elevations on subsequent days (Gavva et al., 2007a; Honore et al.,
342 S. Niyom et al.
2009). Even though the mechanism of the attenuation of hyperthermia is unknown, the present data support an assumption proposed by Gavva in 2007 that there must be compensatory mechanisms controlling the homeostatic body temperature (Gavva et al., 2007a). On day 5, one male dog had diarrhea 4 h postdrug. It is possible this was an incidental finding, but we cannot rule out a potential cause and effect relationship of the drug and this effect as TRPV1 receptors expressed in gastric epithelial cells are reported to play a role in gastroprotection (Takeuchi et al., 1992; Kato et al., 2003; Holzer, 2004; Horie et al., 2004; Mozsik et al., 2005). Hence. it has been postulated that the TRPV1 antagonists may disrupt the mucus layer which surrounds the gastric epithelial lining resulting in irritation of the stomach and duodenum, and transient diarrhea. If attributed to the drug, it is likely that these gastrointestinal side effects are dose dependent; in the current lower repeated dose study, diarrhea was observed only once whereas in the prior single high-dose study, we observed this side effect within 4 h postdosing in a five of six dogs (Niyom et al., 2012). In conclusion, pharmacokinetic parameters derived from the oral administrations of a single (30 mg/kg) dose of ABT-116 in dogs were useful in predicting a multiple day administration regimen. As predicted, the majority of measured plasma drug concentrations were above the targeted previously described efficacious concentration for rodents through the postdrug sampling period for five consecutive days. Additionally, nociceptive threshold testing revealed the analgesic potential of this dosage regimen, while attenuating hyperthermia and gastrointestinal effects such as diarrhea which were considered significant side effects after single high dose administration of ABT-116.
ACKNOWLEDGMENTS The test compound (ABT-116) and funding for this study were provided by Abbott Animal Health. The authors gratefully acknowledge James R zumBrunnen for providing assistance with statistical analyses, and Ryan J. Hansen and Susan Hudachek for providing assistance with sample processing and analysis.
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