Drug Testing
and Analysis
Research article
Received: 9 November 2014
Revised: 18 March 2015
Accepted: 8 June 2015
Published online in Wiley Online Library: 1 September 2015
(www.drugtestinganalysis.com) DOI 10.1002/dta.1834
Disposition of isoflupredone acetate in plasma,
urine and synovial fluid following intra-articular
administration to exercised Thoroughbred
horses
Heather K. Knych,a,b* Linda M. Harrison,c Alexandria Whitea
and Daniel S. McKemiea
The use of isoflupredone acetate in performance horses and the scarcity of published pharmacokinetic data necessitate further
study. The objective of the current study was to describe the plasma pharmacokinetics of isoflupredone acetate as well as timerelated urine and synovial fluid concentrations following intra-articular administration to horses. Twelve racing-fit adult Thoroughbred horses received a single intra-articular administration (8 mg) of isoflupredone acetate into the right antebrachiocarpal
joint. Blood, urine and synovial fluid samples were collected prior to and at various times up to 28 days post drug administration.
All samples were analyzed using liquid chromatography-Mass Spectrometry. Plasma data were analyzed using a population pharmacokinetic compartmental model. Maximum measured plasma isoflupredone concentrations were 1.76 ± 0.526 ng/mL at 4.0 ±
1.31 h and 1.63 ± 0.243 ng/mL at 4.75 ± 0.5 h, respectively, for horses that had synovial fluid collected and for those that did
not. The plasma beta half-life was 24.2 h. Isoflupredone concentrations were below the limit of detection in all horses by 48 h
and 7 days in plasma and urine, respectively. Isoflupredone was detected in the right antebrachiocarpal and middle carpal joints
for 8.38 ± 5.21 and 2.38 ± 0.52 days, respectively. Results of this study provide information that can be used to regulate the use of
intra-articular isoflupredone in the horse. Copyright © 2015 John Wiley & Sons, Ltd.
Keywords: corticosteroid; isoflupredone; horse, intra-articular; racehorse
Introduction
Drug Test. Analysis 2016, 8, 141–147
* Correspondence to: Heather K. Knych, K.L. Maddy Equine Analytical Chemistry
Laboratory, School of Veterinary Medicine, University of California, Davis, 620
West Health Science Drive, 95616 CA, USA.
E-mail: [email protected]
a K.L. Maddy Equine Analytical Chemistry Laboratory, School of Veterinary
Medicine, University of California, Davis 95616, CA, USA
b Department of Veterinary Molecular Biosciences, School of Veterinary Medicine,
University of California, Davis 95616, CA, USA
c Willow Oak Equine, Woodland 95695, CA, USA
Copyright © 2015 John Wiley & Sons, Ltd.
141
Corticosteroids are potent anti-inflammatory agents and as such
are commonly used to prevent and treat performance-related
injuries. Due to their ability to affect performance and their potential to allow a horse to compete when it otherwise should
not, corticosteroid use is regulated in performance horses. As part
of a concerted effort to establish standardized regulatory thresholds and withdrawal times, our laboratory has previously reported on plasma, urine, and synovial fluid concentrations as
well as the pharmacokinetics of intra-articular triamcinolone
acetonide[1] and methylprednisolone acetate[2] in horses. Based
on these studies, the Association of Racing Commissioners
International (ARCI) has recommended regulatory thresholds of
100 pg/mL in blood with corresponding withdrawal times of 7
and 21 days for triamcinolone acetonide and methylprednisolone
acetate, respectively. The extended withdrawal time for methylprednisolone acetate in particular has led some veterinarians to
seek out alternative corticosteroids, such as isoflupredone acetate.
While the ARCI has recommended a similar plasma threshold concentration (100 pg/mL) for intra-articular isoflupredone acetate,
comparable published pharmacokinetic data is not currently available for this drug.
There is only one published report describing the use of
isoflupredone acetate in the horse.[3] In that study, using an immunoassay, investigators reported detectable concentrations of
isoflupredone and isoflupredone acetate (IPA) equivalents for up
to 12 and 72 h post administration in blood and synovial fluid, respectively. The subsequent development of more sensitive liquid
chromatography-mass spectrometry (LC-MS) analytical assays warrants additional investigation of isoflupredone concentrations in biological samples in order to confirm the recommended regulatory
threshold and withdrawal time.
Blood concentrations are often used as surrogate markers of
drug concentrations at the site of pharmacologic effect. However,
for intra-articular triamcinolone acetonide and methylprednisolone acetate, plasma or serum concentrations do not appear to
be reflective of the concentration of drug within the joint.[1,2]
While drug was detected for only 7 days in blood samples collected from horses receiving a single intra-articular administration
of 9 mg of triamcinolone acetonide in a single joint, drug was still
detected in synovial fluid for up to 35 days post administration.
Drug Testing
and Analysis
H. K. Knych et al.
Methylprednisolone was detected in synovial fluid for up to 77
days post administration of a single dose of 100 mg methylprednisolone acetate but was only detectable in blood for 14 days.
Results from both of these studies suggest that, in the case of
intra-articular corticosteroid administration, blood is not the best
means by which to evaluate drug concentrations at the site of
action. However, since measuring drug concentrations within the
joint is not a feasible alternative, regulatory recommendations
based on blood levels should be prolonged. Of even greater importance, these results suggest that intra-articular corticosteroids may
still have anti-inflammatory effects after drug concentrations have
fallen below the limit of detection (LOD) in blood.
Widespread use of corticosteroids in racing as well as a lack of information regarding the effects and fate of this class of drugs in the
horse necessitates further investigation and study. Isoflupredone
acetate is gaining in popularity as an intra-articular corticosteroid
and the apparent dearth of knowledge with respect to the disposition and pharmacologic effects of this drug warrant further study of
this drug in the horse. The study reported here characterizes
plasma, synovial fluid and urine concentrations as well as the pharmacokinetics of isoflupredone administration following intraarticular administration to the horse.
Experimental
Animals
142
Twelve university-owned, exercised adult Thoroughbred horses including 6 geldings and 6 mares (age: 4–8 years; weight: 492–600
kg) were studied. Prior to the study, horses were exercised five
days a week. The general exercise protocol was meant to simulate
the strenuous exercise of race training. The exercise regimen for
these horses consists of three days per week on an Equineciser
(Centaur Horse Walkers Inc., Mira Loma, CA, USA) (5 min at walk;
30 min trot; 5 min walk) and two days per week on a high speed
treadmill (Mustang 2200, Graber AG, Switzerland, Fahrwangen)
(Day 1: 5 min @1.6 m/s; 5 min @ 4 m/s; 5 min @ 7 m/s; 5 min @
1.6 m/s all at 6% incline. Day 2: 3 min @ 1.6 m/s; 4 min @ 4.0
m/s; 2 min @ 7.0 m/s; 2 min @ 11.0 m/s and 5 min @1.6 m/s all
at 3% incline). All horses were subject to regular fitness testing, including weekly heart rate measurements and calculation of V200
(running velocity that elicited a heart rate 200 bpm) and monthly
measurements of end-run plasma lactate concentrations, as a
means by which to ensure that the fitness level of the horses used
in this study were as comparable as possible to the average racehorse. As horses were subject to repeated arthrocentesis during
the study, the exercise regimen was modified during that time.
On the day of synovial fluid collection, horses were not exercised.
The day following synovial fluid collection, horses were allowed to
freely exercise in a round pen and two days after collections they
returned to their normal exercise regimen.
Before beginning the study, horses were determined healthy by
physical examination, complete blood count and a serum biochemistry panel that included aspartate aminotransferase, creatinine phosphokinase, alkaline phosphatase, total bilirubin, sorbital
dehydrogenase, blood urea nitrogen, and creatinine. Blood analyses were performed by the Clinical Pathology Laboratory of the
William R. Pritchard Veterinary Medical Teaching Hospital of the
University of California, Davis, using standard protocols. Horses
did not receive any other medications for at least two weeks prior
to commencement of the study. This study was approved by the
wileyonlinelibrary.com/journal/dta
Institutional Animal Care and Use Committee of the University of
California, Davis.
Instrumentation and drug administration
A 14-gauge catheter was placed in the external jugular vein for
blood sampling. Each horse was weighed immediately prior to drug
administration. Immediately prior to drug administration, the area
over the antebrachiocarpal joint was scrubbed with povidoneiodine (Betadine, Agri Laboratories Ltd, St Joseph, MO, USA) and
70% isopropyl alcohol, the joint flexed and a total dose of 8 mg
of isoflupredone acetate (Predef® 2X, Zoetis, Florham Park, NJ,
USA) administered aseptically into the right antebrachiocarpal joint.
The dose chosen for this study was based upon a published survey
of the most commonly used dose of intra-articular isoflupredone
acetate by equine practitioners.[4]
Sample collection
Blood samples for drug concentration determination were collected at time 0 (prior to drug administration) and at 15, 30, and
45 min, and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 18, 24, 36, 48, 72, and 96
h post administration. Subsequent samples were collected on day
7, 9, 11, and 13 post drug administration. Samples on day 13 were
analyzed, as described later, to ensure that drug concentrations
were no longer detectable before termination of sample collection.
Prior to drawing each sample of blood for analysis of drug concentrations, 10 mL of blood was aspirated and discarded from the catheter and T-Port extension set (combined internal volume < 2 mL).
The catheter was flushed with 10 mL of a dilute heparinized saline
solution (10 IU/mL) following each sampling time. Catheters were
removed following collection of the 18-h sample and the remaining
samples collected by direct venipuncture. Blood samples were collected into EDTA blood tubes (Kendall/Tyco Healthcare, Mansfield,
MA, USA) and stored on ice until centrifugation at 3000 x g for 10
min at -4 C. Plasma was then immediately transferred into storage
cryovials (Phenix Research Products, Chandler, NC, USA) and stored
at -20 C until analysis (approximately 2 weeks following collection
of the final sample).
Synovial fluid was collected from 8 of the 12 horses. Horses from
which synovial fluid was collected were selected by the use of a
computerized random number generator. Prior to collection of synovial fluid, the area over the right and left carpi was scrubbed with
povidone-iodine solution. Immediately prior to collection, the area
over the joints was wiped multiple times with alcohol saturated
gauze pads. Synovial fluid samples were collected from the right
and left antebrachiocarpal and middle carpal joints by aspiration
with a 21G 1½ inch needle at 24, 48, 72, 96, and 120 h post drug administration. Additional synovial fluid samples were collected once
a week starting on day 7 until 28 days post administration. Synovial
fluid samples were tested to ensure that isoflupredone was no longer detected prior to termination of sample collection. Synovial
fluid was separated into aliquots in storage cryovials and stored at
-20°C until analysis of drug concentrations (approximately one
week following collection of the final sample).
Urine samples were collected from all horses via free catch for
measurement of isoflupredone concentrations. Samples were collected on Day 0 (prior to drug administration) and on days 1, 2, 3,
4, 6, 7, 10, and 13, post isoflupredone administration. All samples
were tested to ensure that isoflupredone was no longer detected
prior to termination of sample collection. All samples were stored
Copyright © 2015 John Wiley & Sons, Ltd.
Drug Test. Analysis 2016, 8, 141–147
Drug Testing
and Analysis
Disposition of isoflupredone acetate in plasma, urine and synovial fluid
at -20°C until analysis (approximately one week following administration of the final sample).
Determination of isoflupredone concentrations
Drug Test. Analysis 2016, 8, 141–147
Pharmacokinetic calculations
Determination of pharmacokinetic parameters for isoflupredone in
both plasma and synovial fluid was conducted using commercially
available software (Phoenix WinNonlin Version 6.3, Pharsight, Cary,
NC) and compartmental analysis. In order to obtain the best fit for
the data, a population pharmacokinetic model was utilized. The
AUC was calculated using the log-linear trapezoidal rule and was
extrapolated to infinity using the last measured plasma concentration divided by the terminal slope λz. Statistical analyses were used
to assess significant differences in plasma and urine concentrations
as well as plasma pharmacokinetic parameters between horses in
which synovial fluid was collected (Group 1) and those in which it
was not (Group 2). Data were analyzed using a Student’s non-paired
t-test based on the differences between the two parameters and a
non-parametric (Wilcoxon signed rank) test. Significance was set at
p <0.05. Pharmacokinetic parameters, synovial fluid concentrations
and urine concentrations for isoflupredone acetate are reported as
mean ± SD, median and range.
Results
The response for isoflupredone plasma calibrators was linear from
0.05 to 10 ng/mL and gave correlation coefficients (R2) of 0.99 or
better. The intra-assay and inter-assay linearity (R2) average was
0.996. The intra-assay, inter-assay, analyst-to-analyst precision, and
accuracy of the assay were determined by assaying quality control
(QC) samples in replicates (n = 6) for isoflupredone at three concentration levels within the curve including a QC level at three times
the LOQ (3XLOQ). The liquid/liquid extraction recovery was 99%.
Accuracy was reported as percent nominal concentration and precision was reported as percent relative standard deviation (Table 1).
The technique was optimized to provide a limit of quantitation
(LOQ) of 0.05 ng/mL and an LOD of approximately 0.04 ng/mL for
isoflupredone in plasma and urine. The synovial fluid LOQ was 0.5
ng/mL. Six sources of control plasma showed no interferences.
Mass spectra of isoflupredone from administration samples were
consistent with those of the standard as evidence by relative ion
ratios within ± 20% of those of the standard.
Copyright © 2015 John Wiley & Sons, Ltd.
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143
The analytical reference standard isoflupredone was obtained from
Steraloids (Steraloids, Newport, RI, USA) and internal standard
d7-triamcinolone acetonide (d7-TA) was obtained from CDN
Isotopes (CDN Isotopes, Pointe-Claire, Quebec, Canada).
Isoflupredone working solutions were prepared by dilution of
the 1 mg/mL stock solutions with methanol to concentrations of
0.01, 0.1, 1, and 10 ng/μL. Plasma calibrators were prepared by dilution of the working solutions with drug free equine plasma, urine or
synovial fluid collected from non-treated research horses, to concentrations of 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 20 ng/mL. Additional calibrators for synovial fluid included 20, 50, 100, 200, 500,
1000, 2000, 3000, 4000, and 5000 ng/mL. Calibration curves and
negative control samples were prepared fresh for each quantitative
assay. In addition, quality control samples (equine control plasma
fortified with analyte at three concentrations within the standard
curve) were included with each sample set as an additional check
of accuracy.
Prior to analysis, 1 mL of plasma was diluted with 100 μL of water
containing d7-TA at 0.5 ng/μL and the samples were vortexed
briefly to mix. Five millilitres of MTBE was added to each plasma
sample, and the samples were mixed by rotation (Glas-Col, Terre
Haute, IN, USA) for 20 min at 40 revolutions per minute (rpm). After
rotation, samples were centrifuged at 3300 rpm (2260 g) for 5 min
at 4°C and the top organic layer was transferred to a 12x75 mm
glass tube. Samples were dried under nitrogen and dissolved in
150 μL of 5% acetonitrile in water, both with 0.2% formic acid.
Urine samples were diluted with 100 μL of water containing 0.5
ng/μL of d7-TA internal standard and 0.4 mL of β-glucuronidase enzyme (Patella Vulgata; Sigma Aldrich, St Louis, MO, USA) at 10 000
Units/mL in pH 5, 1.6 M acetate buffer. The pH of samples were adjusted to 5 ± 0.5 with 2 N NaOH or 2 N HCl, as necessary, and heated
and sonicated in a water bath at 65 °C for 2 h with 99 min of sonication. After cooling to room temperature, 5 mL of MTBE was
added to each urine sample, and the samples were mixed by rotation (Glas-Col, Terre Haute, IN, USA) for 20 min at 40 rpm. After rotation, samples were centrifuged at 3300 rpm (2260 g) for 5 mins
at 4° C and the top organic layer was transferred to a 12X75 mm
glass tube. Samples were dried under nitrogen and dissolved in
150 μL of 5% acetonitrile in water, both with 0.2% formic acid.
The extraction method for synovial fluid samples was the similar
to plasma, utilizing a 0.1 mL aliquot for analysis. Samples were dried
under nitrogen and dissolved in 120 μL of 5% acetonitrile in water,
both with 0.2% formic acid.
The concentration of isoflupredone was measured in plasma by
liquid chromatography-tandem mass spectrometry (LC-MS/MS)
using positive heated electrospray ionization (HESI(+)). Quantitative
analysis of plasma, urine, and synovial fluid (SF) were performed on
a triple quadrupole mass spectrometer (TSQ Vantage triple quadrupole mass spectrometer, Thermo Scientific, San José, CA, USA)
coupled with a turbulent flow chromatography system (TFC TLX4,
Thermo Scientific, San José, CA, USA) having 1100 series liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA) and
operated in laminar flow mode. The spray voltage was 3500V, the
vaporizer temperature was 350°C, the capillary temperature was
300°C and the sheath and auxiliary nitrogen gas were 40 and 30 respectively (arbitrary units). Product masses and collision energies of
each analyte were optimized by infusing the standards into the LC-
MS/MS system. Chromatography employed an ACE3 C18 10cm x
2.1mm 3μm column (Mac-Mod Analytical, Chadds Ford, PA, USA)
and a linear gradient of acetonitrile (ACN) in water, both with
0.2% formic acid, at a flow rate of 0.35 mL min-1. The initial ACN concentration was held at 20% for 0.5 min, ramped to 40% over 5 min,
ramped to 95% over 0.5 min, held at 95% for 1 min, before reequilibrating for 4.5 min at initial conditions. The injection volume
was 30, 40, and 40 μL into the LC-MS/MS system for plasma, urine,
and synovial fluid, respectively.
Detection and quantification was conducted using selective reaction monitoring (SRM) of initial precursor ion for isoflupredone
(mass to charge ratio (m/z) 379.181) and the internal standard d7TA (m/z 442.3). The response for the product ions for isoflupredone
(plasma: m/z 237.1, 265.2, 341.2, 359.2; urine: m/z 341.2, 359.2; synovial fluid: m/z 237.1, 265.2) and the internal standard d7-TA (m/z
404.3, 422.3) were plotted and peaks at the proper retention time
integrated using Quanbrowser software (Thermo Scientific, San
José, CA, USA). Quanbrowser software was used to generate calibration curves and quantitate isoflupredone in all samples by linear
regression analysis. A weighting factor of 1/X was used for all calibration curves.
Drug Testing
and Analysis
H. K. Knych et al.
Table 1. Accuracy and precision values for LC-MS/MS analysis of isoflupredone in equine plasma, urine and synovial fluid
Plasma
Urine
Synovial Fluid
Concentration
(ng/mL)
Intra-assay accuracy
(% nominal conc)
Intra-assay precision
(% relative SD)
Inter-day accuracy
(% nominal conc)
Inter-day precision
(% relative SD)
0.15
2.0
9.0
0.15
2.0
9.0
3.0
750.0
4000.0
94.0
90.0
98.0
96.0
107.0
104.0
89.0
98.0
109.0
10.0
4.0
2.0
20.0
8.0
11.0
20.0
5.0
18.0
98.0
90.0
97.0
96.0
107.0
104.0
99.0
108.0
101.0
9.0
5.0
5.0
20.0
8.0
11.0
12.0
6.0
11.0
Autosampler stability was assayed by re-injecting a calibration
curve after sitting for 25 h. The average recovery after this time
was 104%. Room temperature stability was determined by
extracting and analyzing plasma with a known concentration of
isoflupredone after sitting for 0, 1, 4, and 6 h. The triplicate replicate
average recovery was 98% after 1 h, 94% after 4 h, and 87% after 6
h. To determine if EDTA in the blood collection tubes affected
isoflupredone concentrations, known amounts of isoflupredone
was added to EDTA tubes containing blood collected from nondosed horses. Recovery after a 1–6 h incubation was comparable
to the initial concentrations, suggesting that EDTA does not affect
isoflupredone drug concentrations.
There was not a statistically significant difference in plasma or
urine concentrations at any of the time points sampled or in any
of the calculated plasma pharmacokinetic parameters between
horses from which synovial fluid was collected (Group 1) and those
from which synovial fluid was not collected (Group 2). Mean
isoflupredone plasma concentration over time curves are depicted
in Figure 1. Mean (±SD) isoflupredone plasma concentrations are
listed in Table 2. Isoflupredone was below the LOD (0.05 ng/mL)
in plasma by 39 h (mean) (range 36–48 h) for horses that had synovial fluid removed and 36 h (mean) for the horses that did not.
Based on coefficient of variation, Akaike Information Criterion[5]
and visual inspection of the residual plots, a two-compartment
population model (Cp = Ae-αt + Be-βt) with a multiplicative error
gave the best fit to the isoflupredone concentration data.
Pharmacokinetic parameters following isoflupredone acetate administration are listed in Table 3. As there was not a statistically significant difference (p <0.05) in pharmacokinetic parameters
between horses having synovial fluid removed and those that did
not, the mean (± SE) values reported include both groups of horses.
There was not a significant difference in urine isoflupredone concentrations between horses that had synovial fluid collected and
those that did not. The maximum measured urine concentration
was 3.36 ± 1.06 ng/mL and occurred at 24 h post drug administration. Isoflupredone was below the LOD (0.05 ng/mL) in urine by
72 h in 11/12 horses and by day 7 in the horse. Synovial fluid was
collected from 8 of the 12 horses studied and removal of drug
during collection of synovial fluid does not appear to have a significant effect on the plasma or urine elimination or detection
time or any of the determined pharmacokinetic parameters. Individual synovial fluid isoflupredone concentration over time curves
is depicted in Figure 2. In addition to the right antebrachiocarpal
joint, isoflupredone was detected in the right middle carpal joint.
Mean ± SD synovial fluid concentrations of isoflupredone following
intra-articular administration are listed in Table 4. Isoflupredone was
below the LOD (0.15 ng/mL) in synovial fluid collected from the
right antebrachiocarpal and middle joints between days 4 and 21
(8.38 ± 5.21 (mean ± SD)) and 2 and 3 (2.38 ± 0.52 (mean ± SD)), respectively. Selected pharmacokinetic parameters for isoflupredone
acetate in synovial fluid of the right antebrachiocarpal joint are
listed in Table 4. The AUC, λz and t1/2 λ for synovial isoflupredone
concentrations differed significantly (p <0.05) from those calculated for plasma isoflupredone concentrations. No drug was detected in either the left antebrachiocarpal or middle carpal joint.
Discussion
144
Figure 1. Average (± SD) plasma isoflupredone concentration over time
curves following intra-articular administration of 8 mg of isoflupredone
acetate (Predef 2X®) into the right antebrachipal joint of 12 exercised
Thoroughbred horses, Group 1 and Group 2 include horses that had
synovial fluid collected and those thhat did not, respectively.
wileyonlinelibrary.com/journal/dta
There are a limited number of reports in the literature describing
the pharmacokinetics of intra-articular isoflupredone acetate in
the horse. Therefore, the primary goal of the current study was to
describe the pharmacokinetics of isoflupredone acetate following
a single intra-articular administration to horses. Secondarily, we
sought to describe synovial fluid concentrations of isoflupredone
relative to plasma and urine concentrations following intra-articular
administration.
In the presently reported study, measurement of drug concentrations within the joint, necessitated removal of synovial fluid and
presumably removal of some amount of drug from the joint by a
route other than diffusion into the systemic circulation. As this
has the potential to affect the pharmacokinetics of a drug administered via the intra-articular route, in the current study, an additional
Copyright © 2015 John Wiley & Sons, Ltd.
Drug Test. Analysis 2016, 8, 141–147
Drug Testing
and Analysis
Disposition of isoflupredone acetate in plasma, urine and synovial fluid
Table 2. Mean (± SD) plasma isoflupredone concentrations following intra-articular administration of 8 mg of isoflupredone acetate (Predef® 2X) in the
right antebrachiocarpal joint of 12 exercised Thoroughbred horses
Plasma [Isoflupredone] (ng/mL) Mean ± SD
Baseline
0.25 h
0.5 h
0.75 h
1h
1.5 h
2h
2.5 h
3h
4h
5h
6h
8h
12 h
18 h
24 h
36 h
48 h
72 h
96 h
Day 7
Day 9
Day 11
Day 13
Significance
Group 1 (n=8)
Group 2 (n=4)
Both Groups (n=12)
(p-value)
0
0.33 ± 0.15
0.64 ± 0.34
0.37 ± 0.53
0.80 ± 0.48
0.83 ± 0.39
1.16 ± 0.52
1.26 ± 0.55
1.51 ± 0.62
1.64 ± 0.55
1.58 ± 0.42
1.34 ± 0.43
1.13 ± 0.29
0.54 ± 0.11
0.20 ± 0.04
0.10 ± 0.03
0.04 ± 0.02
<LOD
ND
ND
ND
ND
ND
ND
0
0.45 ± 0.11
0.70 ± 0.16
0.90 ± 0.17
0.83 ± 0.24
0.95 ± 0.25
1.15 ± 0.30
1.23 ± 0.36
1.36 ± 0.26
1.50 ± 0.33
1.61 ± 0.21
1.21 ± 0.16
0.94 ± 0.18
0.38 ± 0.12
0.16 ± 0.04
0.08 ± 0.01
0.03 ± 0.01
ND
ND
ND
ND
ND
ND
ND
0
0.37 ± 0.15
0.66 ± 0.29
0.79 ± 0.32
0.81 ± 0.40
0.87 ± 0.34
1.16 ± 0.45
1.25 ± 0.48
1.46 ± 0.52
1.59 ± 0.47
1.59 ± 0.35
1.29 ± 0.36
1.07 ± 0.26
0.49 ± 0.13
0.19 ± 0.04
0.09 ± 0.03
0.04 ± 0.02
<LOD
ND
ND
ND
ND
ND
ND
--0.20
0.73
0.42
0.90
0.60
0.97
0.94
0.66
0.64
0.91
0.58
0.28
0.05
0.19
0.09
0.41
---------------
Group 1 – synovial fluid samples collected; Group 2 – synovial fluid samples not collected; Samples collected until Day 13-all ND; significance (p-value)
represents comparison between group 1 and Group 2. ND, not detected.
Table 3. Pharmacokinetic parameters for plasma and synovial fluid
following a single intra-articular (8 mg) administration of isoflupredone
acetate (Predef® 2X) in the right antebrachiocarpal joint of exercised
Thoroughbred horses (n=12). All values in this table were generated
using compartmental analysis.
Parameter
Plasma
(n=12)
Synovial Fluid
(n=8)
Cmax (ng/mL)
Tmax (h)
Tlast (d)
AUC (h*ng/mL)
A (ng/mL)
B (ng/mL)
α (1/h)
β (1/h)
T1/2α (h)
T1/2β (h)
1.53
3.34
36
16.3
8.81
0.087
0.240
0.029
2.89
24.2
—
—
14
768.4
12.4
4.58
0.056
0.008
12.3
82.9
Drug Test. Analysis 2016, 8, 141–147
group of horses were studied in which plasma pharmacokinetics
and detection time were determined but synovial fluid was not collected. Based on the results of the current study, collection of
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145
Cmax, maximal measured plasma concentration; Tmax, time of maximal
measured plasma concentration; Tlast, time of last measured
concentration; coefficient for distribution phase; B, coefficient for
elimination phase; α, exponential term for distribution phase; β,
exponential term for elimination phase; T1/2α, distribution half-life; T1/2
β, elimination half-life; AUC, area under the plasma concentration time
curve
Figure 2. Individual antebrachiocarpal joint synovial fluid concentration
over time curves following intra-articular administration of 8 mg of
isoflupredone acetate (Predef 2X®) into the right antebrachiocarpl joint of
8 exercised Thoroughbred horses.
Drug Testing
and Analysis
H. K. Knych et al.
Table 4. Mean (± SD) synovial fluid isoflupredone concentrations following a single intra-articular administration of 8 mg of isoflupredone acetate
(Predef® 2X) in the right antebrachiocarpal joint to exercised Thoroughbred horses (n=8)
[Isoflupredone] (ng/mL)
Baseline
Day 1
Day 2
Day 3
Day 4
Day 7
Day 14
Day 21
Day 28
Day 35
Day 42
Right
Antebrachiocarpal Joint
Right Middle
Carpal Joint
Left Antebrachiocarpal
Joint
Left Middle
Carpal Joint
ND
647 ± 273
37.2 ± 33.6
8.86 ± 15.9
0.55 ± 0.41
6.43 ± 0.0
1.77 ± 0.0
ND
ND
ND
ND
ND
5.20 ± 3.26
0.28 ± 0.22
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND, not detected.
146
synovial fluid did not appear to affect the clearance, elimination or
length of detection of isoflupredone from plasma.
As part of an attempt to establish uniform regulatory recommendations for drugs administered to racehorses, the Racing Medication and Testing Consortium and the ARCI have recommended a
regulatory threshold of 100 pg/mL with a corresponding withdrawal time of 7 days for intra-articular administered isoflupredone.
In the current study, following administration of 8 mg in a single
joint, plasma isoflupredone concentrations were below the recommended threshold by 30 h in all horses studied. By 7 days post drug
administration (recommended withdrawal time), isoflupredone was
not detected in plasma samples from any horses. This is in agreement with a previous report, whereby plasma concentrations were
below the LOD of the analytical assay at 7 days post administration
of intra-articular isoflupredone doses ranging from 4 to 18 mg.[6]
To the authors’ knowledge, there is only one other published report describing plasma, urine and synovial fluid concentrations following intra-articular isoflupredone acetate in horses.[3] Plasma
pharmacokinetic parameters were not reported in that study, presumably due to the low sensitivity of the analytical assay employed
to measure drug concentrations. Using a one-step ELISA developed
for dexamethasone (reported LOD of 0.10 ng/mL), investigators reported plasma concentrations of isoflupredone and isoflupredone
acetate equivalents of less than 10 ng/mL.[3] Isoflupredone acetate
was detected in plasma for between 2 and 12 h post administration
with maximum concentrations noted at 10 h.[3] Further analysis by
a high performance liquid chromatography (HPLC) method with an
LOD of 10 ng/mL did not detect any isoflupredone acetate in
plasma samples. In the study reported here, isoflupredone was detected in plasma for between 24 and 36 h post administration, with
the maximum concentration (1.53 ng/mL) occurring at 3.34 h. The
discrepancy in detection time between the two studies is likely attributable to the differing sensitivities of the analytical assays
employed in each study. Although perhaps less likely, another possible explanation for the differences in plasma concentrations between the two studies is the joint that was utilized for drug
administration. In the current study, the antebrachiocarpal joint
was used while Lillich and colleagues[3] used the tarsocrural joint.
While information is limited in the horse, differences in blood flow
and rate of diffusion of drug from a joint to the systemic circulation
may affect measured plasma concentrations following intra-
wileyonlinelibrary.com/journal/dta
articular drug administration. Differences in maximal plasma concentrations have been reported previously following intra-articular
administration of triamcinolone acetonide into the tarsometatarsal
joint[7] and the antebrachiocarpal joint.[1] The same LC-MS assay
from the same laboratory was utilized for analysis of samples from
both of those studies and therefore the discrepancy in that case
cannot be explained by assay variability.
In the current study, the half-life associated with the terminal
phase of the plasma isoflupredone concentration curve (β half-life)
was determined to be approximately 24.2 h, which is much shorter
than that previously reported for methylprednisolone acetate[2,3,8–10]
and triamcinolone acetonide,[1,11–13] two other commonly used
intra-articular corticosteroids in horses. With respect to methylprednisolone acetate, the slow terminal phase has been attributed to a
prolonged rate of absorption (flip-flop pharmacokinetics), based on
a much lower slope compared to intravenous administration.[14]
While intravenous administration was not performed in the current
study and to the authors’ knowledge there are no studies describing
the pharmacokinetics of isoflupredone following intravenous administration, it is possible that similar to that reported from methylprednisolone acetate, the prolonged elimination phase in the current
study is actually representative of absorption.
As expected, the highest isoflupredone concentrations were detected in the injected joint (right antebrachiocarpal joint). Lower
concentrations were detected in the right middle carpal joint, presumably as a result of communication between the two joints. Although the administered dose was different, synovial fluid
isoflupredone concentrations at 24 h post administration in the
injected joint agreed with a previous report describing synovial
fluid concentrations in the horse.[3] Following administration of 4
mg of isoflupredone acetate into the tarsocrural joint, Lillich and
colleagues[3] reported a synovial fluid concentration of 679
ng/mL at 24 h post drug administration compared with a mean
concentration of 646.8 ng/mL in the current study. While synovial
fluid concentrations were comparable at 24 h, isoflupredone concentrations at 48 h differed more than 10-fold between the two
studies (Lillich et al.[3] 385 ng/mL; current study 37.2 ng/mL). Similar
to that described previously for plasma, the discrepancy between
the two studies is likely due to differences in analytical methods
(HPLC vs LC-MS/MS) or the rate of diffusion from the different joints
(antebrachiocarapal vs tarsocrural) into the systemic circulation.
Copyright © 2015 John Wiley & Sons, Ltd.
Drug Test. Analysis 2016, 8, 141–147
Drug Testing
and Analysis
Disposition of isoflupredone acetate in plasma, urine and synovial fluid
While not as prolonged, similar to methylprednisolone acetate[2]
and triamcinolone acetonide,[1] isoflupredone concentrations
remained high in synovial fluid for a longer period of time, relative
to plasma concentrations. Isoflupredone concentrations fell below
the LOD of the assay between 1.5 and 2 days in the plasma while
synovial fluid isoflupredone concentrations remained above the
LOD for between 4 and 7 days post administration. The elimination
half-life of isoflupredone from synovial fluid was 82.9 h, suggesting
a very slow rate of disappearance of drug from the joint. The reason
for the long persistence in the joint was beyond the scope of the
study reported here. However, the current study, as well as previous
reports,[3] support rapid hydrolysis of isoflupredone acetate to
isoflupredone suggesting that hydrolysis is not the rate-limiting
step. One possible explanation for the slow rate of disappearance
of isoflupredone from the joint is local precipitation of the acetate
formulation in the joint as has been described for methylprednisolone acetate.[14]
In summary, the current study sought to describe the pharmacokinetics and disposition of isoflupredone acetate in plasma, urine
and synovial fluid following intra-articular administration of 8 mg
into a single joint. Isoflupredone was detected for up to 36 h in
plasma and 48 h in urine. It is important to note that only a single
dose was studied in the current study and based on reports of other
corticosteroids in horses, the total dose administered as well as the
specific joint and number of joints treated can markedly affect the
time to reach the threshold concentration. Additionally, in the presently reported study, the synovial concentration remained above
the LOD for up to 14 days post administration in one horse, suggesting that urine and blood concentrations are not indicative of
synovial fluid concentrations and likely pharmacologic effect.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgements
[12]
Financial support for this study was provided by the Grayson Jockey
Club Research Foundation. The authors would like to acknowledge
Eugene Steffey, VMD, Scott Stanley, PhD, Stacy Steinmetz, Michelle
Mitchell, Nadia Chapman, Sandy Yim, Carley Corado and Sheena
Mouton for assistance.
[13]
[14]
References
[1] H.K. Knych, M.A. Vidal, H.C. Casbeer, D.S. McKemie. Pharmacokinetics of
triamcinolone acetonide following intramuscular and intra-articular
administration to exercised Thoroughbred horses. Equine Vet. J. 2013,
45, 715.
H.K. Knych, L.M. Harrison, H.C. Casbeer, D.S. McKemie. Disposition of
methylprednisolone acetate in plasma, urine, and synovial fluid
following intra-articular administration to exercised Thoroughbred
horses. J. Vet. Pharmacol. 2014, 37, 125.
J.D. Lillich, A.L. Bertone, L.M. Schmall, A.J. Ruggles, R.A. Sams. Plasma,
urine, and synovial fluid disposition of methylprednisolone acetate
and isoflupredone acetate after intra-articular administration in
horses. Am. J. Vet. Res. 1996, 57, 187.
D.J. Ferris, D.D. Frisbie, C.W. McIlwraith, C.E. Kawcak. Current joint
therapy usage in equine practice: a survey of veterinarians 2009.
Equine Vet. J. 2011, 43, 530.
K. Yamaoka, T. Nakagawa, T. Uno. Application of Akaike’s information
criterion (AIC) in the evaluation of linear pharmacokinetic equations.
J. Pharmacokinet. Biopharm. 1978, 6, 165.
H.K. Knych, J. Blea, R.A. Arthur. Clearance of corticosteroids following
intra-articular administration of clinical doses to racehorses (abstract).
AAEP Proceedings 2014, 60, 253.
H.K. Knych, K. Fleming, S. Van Pelt, J. Carter. Elimination of
triamcinolone acetonide after intra-articular administration to
performance horses (abstract). AAEP Proccedings. 2011, 57, 84.
A. Autefage, M. Alvinerie, P.L. Toutain. Synovial fluid and plasma
kinetics of methylprednisolone acetate in horses following intraarticular administration of methylprednidolone acetate. Equine Vet. J.
1986, 18, 193.
M.I. Menendez, M.A. Phelps, E.A. Hothem, A.L. Bertone.
Pharmacokinetics of methylprednisolone acetate after intra-articular
administration and subsequent suppression of endogenous
hydrocortisone secretion in exercising horses. Am. J. Vet. Res. 2012,
73, 1453.
L.R. Soma, C.E. Uboh, Y. Luo, F. Guan, P.J. Moate, R.C. Boston.
Pharmacokinetics of methylprednisolone acetate after intra-articular
administration and its effect on endogenous hydrocortisone and
cortisone secretion in horses. Am. J. Vet. Res. 2006, 67, 654.
C.L. Chen, J.A. Sailor, J. Collier, J. Wiegand. Synovial and serum levels of
triamcinolone following intra-articular administration of trimacinolone
acetonide in the horse. J. Vet. Pharmacol. Therap. 1992, 15, 240.
K. French, C.C. Pollitt, M.A. Pass. Pharmacokinetics and metabolic
effects of triamcinolone acetonide and their possible relationships to
glucocorticoid-induced laminitis in horses. J. Vet. Pharmacol. Ther.
2000, 23, 287.
L.R. Soma, C.E. Uboh, Y. You, F. Guan, R.C. Boston. Pharmacokinetics of
intra-articular, intravenous and intramuscular administration of
triamcinolone acetonide and its effects on endogenous plasma
hydrocortisone and cortisone concentrations in horses. Am. J. Vet.
Res. 2011, 72, 1234.
P.L. Toutain, M. Alvinerie, P. Fayolle, Y. Ruckebusch. Bovine plasma and
synovial fluid kinetics of methylprednisolone and methylprednisolone
acetate after intra-articular administration of methylprednisolone
acetate. J. Pharmacol. Exp. Ther. 1986, 236, 794.
147
Drug Test. Analysis 2016, 8, 141–147
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