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Disposition of Isoflupredone Acetate in Plasma, Urine
Disposition of Isoflupredone Acetate in Plasma, Urine
and Analysis
Research article
Received: 9 November 2014
Introduction
141
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.
Experimental
Animals
142
Twelve university-owned, exercised adult Thoroughbred horses including 6 geldings and 6 mares (age: 48 years; weight: 492600
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
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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 1 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
-20C 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
Drug Testing
and Analysis
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 z. 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 Students 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.
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143
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
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, z and t1/2 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
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Drug Testing
and Analysis
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)
(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.
Plasma
(n=12)
Synovial Fluid
(n=8)
Cmax (ng/mL)
Tmax (h)
Tlast (d)
AUC (h*ng/mL)
A (ng/mL)
B (ng/mL)
(1/h)
(1/h)
T1/2 (h)
T1/2 (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
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145
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
146
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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 ( half-life)
was determined to be approximately 24.2 h, which is much shorter
than that previously reported for methylprednisolone acetate[2,3,810]
and triamcinolone acetonide,[1,1113] 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.
Drug Testing
and Analysis
[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
147
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