Development of in vitro assays for the evaluation of cyclooxygenase inhibitors and application for predicting the selectivity of NSAIDs in the cat

Department of Veterinary Basic Sciences, Royal Veterinary College, Hawkshead Campus, North Mymms, Hatfield, Hertfordshire AL9 7TA, UK.
American Journal of Veterinary Research (Impact Factor: 1.34). 05/2005; 66(4):700-9. DOI: 10.2460/ajvr.2005.66.700
Source: PubMed
ABSTRACT
To develop and validate in cats suitable in vitro assays for screening and ranking nonsteroidal antiinflammatory drugs (NSAIDs) on the basis of their inhibitory potencies for cyclooxygenase (COX)-1 and COX-2.
10 cats.
COX-1 and COX-2 activities in heparinized whole blood samples were induced with calcium ionophore and lipopolysaccharide, respectively. For the COX-2 assay, blood was pretreated with aspirin. The COX-1 and COX-2 assays were standardized, such that time courses of incubation with the test compounds and conditions of COX expression were as similar as possible in the 2 assays. Inhibition of thromboxane B2 production, measured by use of a radioimmunoassay, was taken as a marker of COX-1 and COX-2 activities. These assays were used to test 10 to 12 concentrations of a COX-1 selective drug (SC-560) and of 2 NSAIDs currently used in feline practice, meloxicam and carprofen. Selectivities of these drugs were compared by use of classic 50% and 80% inhibitory concentration (ie, IC50 and IC80) ratios but also with alternative indices that are more clinically relevant.
These assay conditions provide a convenient and robust method for the determination of NSAID selectivity. The S(+) enantiomeric form of carprofen was found to be COX-2 selective in cats, but meloxicam was only slightly preferential for this isoenzyme.
In vitro pharmacodynamic and in vivo pharmacokinetic data predict that the COX-2 selectivity of both drugs for cats will be limited when used at the recommended doses. This study provides new approaches to the selection of COX inhibitors for subsequent clinical testing.

Full-text

Available from: Jerome M Giraudel, Jun 13, 2015
700 AJVR, Vol 66, No. 4, April 2005
N
onsteroidal anti-inflammatory drugs (NSAIDs)
are commonly used for the treatment of pain and
inflammation in many species of veterinary interest.
Several licensed drugs exist that can be used long-term
in dogs. It is now recognized that a major requirement
also exists for well-tolerated drugs in feline practice for
short- and long-term use.
It is accepted that the therapeutic effects of
NSAIDs are attributable mainly to inhibition of the
inducible prostaglandin endoperoxide synthase
cyclooxygenase (COX)-2, whereas toxic effects on the
stomach and kidney and bleeding complications are
primarily or solely attributed to COX-1.
1,2
Identifying
these 2 COX isoforms has stimulated the development
of several in vitro models for evaluating the potency
and selectivity of existing NSAIDs. This concept has
also provided a rationale for the development and test-
ing of specific COX-2 inhibitors with assumed
improved gastrointestinal tolerability.
As discussed by Brideau et al,
3
increasing evidence
exists that COX expression and activity (and therefore
NSAID potency for COX inhibition) differ among
species, despite the similarity of structure of these
enzymes. Transposing results for potency and selectiv-
ity of NSAIDs from other species to cats is therefore not
possible.
According to Griswold and Adams,
4
in vitro
enzyme assays are not sufficiently accurate to deter-
mine selectivity and potency for time-dependent
inhibitors of COX-2. Cellular and whole animal mea-
surements of potency and selectivity are therefore
required to confirm the results of isolated enzyme
assays. Whole blood assays allow for this time-depen-
dent inhibition because they involve preincubation of
the blood cells with the drug being tested. These assays
also have other advantages when the aim of the study
is to investigate the clinical relevance of selective COX-
2 inhibition.
5
Indeed, the cells used are target cells for
the anti-inflammatory effects (monocytes) and adverse
effects (platelets) of NSAIDs. Moreover, whole blood
samples for COX-1 and COX-2 assays can be taken
from the same animal at the same time, and
prostaglandin synthesis, thromboxane synthesis, or
both is measured from arachidonic acid released from
endogenous stores.
6
Finally, whole blood assays take
into account the binding of a drug to plasma proteins
that occurs in vivo and that, for NSAIDs, can exceed
99% of total plasma concentration.
The purposes of the study reported here were to
develop and validate for cats suitable in vitro assays for
screening and ranking NSAIDs on the basis of their
inhibitory potencies for COX-1 and COX-2. Because
whole blood is used for both COX assays, this test sys-
Received April 5, 2004.
Accepted June 28, 2004.
From the Department of Veterinary Basic Sciences, Royal Veterinary
College, Hawkshead Campus, North Mymms, Hatfield,
Hertfordshire AL9 7TA, UK. Dr. Giraudel’s present address is
Novartis Santé Animale S.A.S., 14 Bd Richelieu, BP 430, 92845
Rueil Malmaison Cedex, France. Dr. Toutain’s present address is the
Ecole Nationale Vétérinaire de Toulouse, UMR 181
Physiopathologie et Toxicologie Expérimentales INRA/ENVT, 23
chemin des Capelles, BP 87614, 31076 Toulouse Cedex 03, France.
Supported by Novartis Animal Health Incorporated, Basel,
Switzerland.
The authors thank Katey Gardner and Mike Andrews for technical
assistance.
Address correspondence to Dr. Giraudel.
Development of in vitro assays for the
evaluation of cyclooxygenase inhibitors
and predicting selectivity of nonsteroidal
anti-inflammatory drugs in cats
Jérôme M. Giraudel, DVM, PhD; Pierre-Louis Toutain, DVM, PhD, DSc; Peter Lees, BPharm, PhD, DSc
Objective—To develop and validate in cats suitable in
vitro assays for screening and ranking nonsteroidal anti-
inflammatory drugs (NSAIDs) on the basis of their
inhibitory potencies for cyclooxygenase (COX)-1 and
COX-2.
Animals—10 cats.
Procedure—COX-1 and COX-2 activities in heparinized
whole blood samples were induced with calcium
ionophore and lipopolysaccharide, respectively. For the
COX-2 assay, blood was pretreated with aspirin. The
COX-1 and COX-2 assays were standardized, such that
time courses of incubation with the test compounds
and conditions of COX expression were as similar as
possible in the 2 assays. Inhibition of thromboxane B
2
production, measured by use of a radioimmunoassay,
was taken as a marker of COX-1 and COX-2 activities.
These assays were used to test 10 to 12 concentra-
tions of a COX-1 selective drug (SC-560) and of 2
NSAIDs currently used in feline practice, meloxicam
and carprofen. Selectivities of these drugs were com-
pared by use of classic 50% and 80% inhibitory con-
centration (ie, IC
50
and IC
80
) ratios but also with alter-
native indices that are more clinically relevant.
Results—These assay conditions provide a convenient
and robust method for the determination of NSAID
selectivity. The S(+) enantiomeric form of carprofen
was found to be COX-2 selective in cats, but meloxi-
cam was only slightly preferential for this isoenzyme.
Conclusions and Clinical Relevance—In vitro phar-
macodynamic and in vivo pharmacokinetic data pre-
dict that the COX-2 selectivity of both drugs for cats
will be limited when used at the recommended
doses. This study provides new approaches to the
selection of COX inhibitors for subsequent clinical
testing. (
Am J Vet Res
2005;66:700–709)
04-04-0144r.qxp 3/15/2005 9:55 AM Page 700
Page 1
tem can also provide information on the in vivo blood
concentrations of NSAIDs required for substantial
inhibition of COX-2. Validated assays are required for
the evaluation of new molecules and to establish crite-
ria that can be expected to improve the selection of
new drugs for subsequent clinical testing. In addition
to establishing general validation procedures, a final
necessary validation step involves testing compounds
with known activities in other species. In this study, a
COX-1 selective drug (SC-560) and 2 NSAIDs current-
ly used in feline practice, meloxicam and carprofen,
were investigated.
Materials and Methods
Animals—Blood samples were obtained from 10 healthy
domestic shorthair cats of both sexes. Cats were maintained
as a colony in a temperature-controlled (20 ± 2
o
C), loose-
housing environment. Weights and ages of cats ranged from
4.1 to 5.6 kg and 1 to 2 years, respectively.
Blood sample collection—The maximum blood volume
obtained on any occasion was 7 mL/kg. Food was withheld
from cats and blood samples were collected from the jugular
veins following sedation (IM administration of midazolam
a
[0.2 mg/kg] and ketamine
b
[10 mg/kg]). Blood was with-
drawn into 10-mL prefilled heparinized syringes (20 U hepar-
in
c
/mL of blood) via a 21-gauge butterfly catheter
d
that was
maintained in the jugular vein for the duration of sample col-
lection. The Royal Veterinary College Ethics and Welfare
Committee approved this study. For each sample, blood from
a single donor was mixed and then divided into 500-µL
aliquots in loosely capped polypropylene tubes.
e
Half of these
tubes were used for the COX-1 assay and half for the COX-2
assay. For 5 cats, the amount of blood collected was sufficient
to test the inhibitory actions of meloxicam
f
and the S(+) enan-
tiomeric form of carprofen (S-carprofen)
g
on the same blood.
For the remaining 5 cats, the only drug tested was SC-560.
h
COX-1 and COX-2 assays—Assays were adapted from
those described by Young et al
7
but with the following 2 main
differences: 1) pretreatment of blood with aspirin
i
in the
COX-2 assay was undertaken to avoid the contribution of
platelet and monocyte COX-1 to thromboxane B
2
(TxB
2
)
production, and 2) the COX-1 and COX-2 assays were stan-
dardized, such that time courses of incubation with the test
compounds, endogenous supply of substrate, and conditions
of COX expression were as similar as possible in the 2 assays
(Appendix). Concentrations and incubation times for
aspirin, calcium ionophore
i
(A23187), and lipopolysaccha-
ride (LPS)
j
were selected on the basis of results of prelimi-
nary studies. At the end of incubation, placing the tubes on
ice stopped eicosanoid production. Plasma was collected fol-
lowing centrifugation at 2,000 X g for 10 minutes at 4
o
C and
frozen at –20
o
C. Inhibition of TxB
2
production was taken as
a measure of inhibition of COX-1 and COX-2 activity.
Validation of assays and procedures—The first valida-
tion step was the determination of precision and accuracy of
the analytic procedure, commencing with pipetting the plas-
ma sample up to the reading given by the radioimmunoassay,
with each sample requiring 7 to 8 pipetting steps. Eicosanoid-
free plasma was prepared by charcoal stripping as follows:
plasma was stirred during 24 hours with 10% charcoal at 4
o
C,
centrifuged at 2,000 X g for 10 minutes, and then passed
through a filter with 0.2-µm-diameter pores. Plasma samples
were subsequently spiked with various concentrations of
TxB
2
i
and processed. The second validation procedure
addressed the question of stability of TxB
2
that was added to
the blood 4 hours before the end of the incubation. Some
authors have indeed experienced a large decrease in TxB
2
con-
centration in the hours following the peak production.
7
Test compounds—The NSAIDs tested were SC-560, a
highly COX-1 selective compound
8
; meloxicam, suggested to
be COX-2 preferential in dogs and humans in whole blood
assay studies
3,9-11
; and carprofen, a COX-2 preferential or
selective drug depending on the assay conditions used.
3,11-13
For this latter drug only, the S(+) enantiomer was tested
because it is the relevant enantiomer to be considered for
COX inhibition.
13
Inhibition of COX-1 and COX-2 was
assessed by adding various concentrations of the test com-
pound dissolved in dimethyl sulfoxide (DMSO)
i
to the
blood. Various concentrations of SC-560 (COX-1 assay,
0.0002, 0.0005, 0.002, 0.005, 0.01, 0.02, 0.05, 0.2, 0.5, and
2µM; COX-2 assay, 0.005, 0.01, 0.02, 0.05, 0.2, 0.5, 2, 5, 20,
50, and 100µM), meloxicam (COX-1 assay, 0.02, 0.05, 0.1,
0.2, 0.5, 1, 2, 5, 20, 50, 200, and 500µM; COX-2 assay, 0.005,
0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 20, 50, and 200µM), and S-
carprofen (COX-1 assay, 0.2, 0.5, 2, 5, 10, 20, 50, 100, 200,
500, 1,000, and 2,000µM; COX-2 assay, 0.02, 0.05, 0.2, 0.5,
1, 2, 5, 10, 20, 50, 200, and 500µM) were tested. It was nec-
essary to use 10 to 12 concentrations of each drug to estab-
lish the entire concentration-effect relationship.
Data analysis—Plasma concentrations of TxB
2
were
determined in duplicate as described by Higgins and Lees
14
by radioimmunoassay without extraction after dilution of
samples in assay buffer (1:10, 1:30, 1:100, or 1:500, depend-
ing on the concentration of TxB
2
). For each blood sample
collection period, 4 standard curves were prepared with
known concentrations of TxB
2
(20, 50, 100, 200, 500, 1,000,
2,000, 5,000, and 10,000 pg/mL), with each standard curve
incorporating 1:10, 1:30, 1:100, or 1:500 TxB
2
-free plasma.
For each sample, 2 different dilutions were prepared to
ensure at least 1 reading on the linear part of the standard
curve. The detection limit was 20 pg/mL (ie, 0.2 ng/mL for
the lowest dilution).
Enzyme inhibition was expressed as a percentage of the
control value (test compound, 0µM). Percent inhibition in
relationship to the test compound blood concentration was
fitted with a software program
k
by use of the following Hill
equation:
% Inhibition = Io +
where % inhibition is the test compound inhibition
expressed as a percentage of the control value; C is the test
compound concentration (as an independent variable); IC
50
(expressing potency) is the test compound concentration
giving 50% of maximum inhibition (Imax); Imax + baseline
inhibition (Io), as an expression of efficacy, is the maximal
response achieved by the test compound; and n is the Hill
coefficient (expressing sensitivity) that results in the slope of
the concentration-effect relationship.
Estimation of the IC
50
for both isoenzymes allows direct
comparison of the selectivity and biochemical potency of the
3 NSAIDs investigated. After log transformation of the data,
differences between test compounds were evaluated by use of
t tests.
l
The within-cat variance was taken into account for
comparison of NSAIDs tested on the same samples of blood
(meloxicam vs S-carprofen), and the overall between-cat
variance was taken into account for other comparisons.
Values of P < 0.05 were considered significant.
A so-called naïve pooled data analysis was also per-
formed with a software program.
k
For each drug, a mean
inhibition curve for COX-1 and COX-2 was determined by
fitting simultaneously the 5 data sets by use of the same Hill
equation described. The corresponding mean parameter val-
ues were used to calculate the selectivity indices.
AJVR, Vol 66, No. 4, April 2005 701
Imax X C
n
IC
n
+ C
n
50
,
04-04-0144r.qxp 3/15/2005 9:55 AM Page 701
Page 2
702 AJVR, Vol 66, No. 4, April 2005
Results
Preliminary results—Preliminary results with
feline blood revealed that background TxB
2
concentra-
tions (without stimulation) were high in both assays,
compared with other species. In the COX-2 assay, pre-
treatment with aspirin (10 µg/mL) led to a better con-
centration response curve for LPS stimulation (wider
range between background and stimulated concentra-
tions; Figure 1) and significantly decreased the back-
ground TxB
2
concentrations. With higher concentra-
tions of aspirin (up to 30 µg/mL), a decreased concen-
tration of TxB
2
in LPS-treated blood was observed but
with no additional decrease in background concentra-
tions of TxB
2
(data not shown). Even if only small
amounts of aspirin (< 10%) remain when COX-2 com-
mences synthesis (2.5 to 3 hours after aspirin addition
in this study), aspirin-mediated COX-2 inhibition with
high concentrations of aspirin cannot be completely
excluded.
7
Therefore, a 10 µg/mL concentration of
aspirin was added before addition of LPS, and this con-
centration was routinely used for all subsequent
COX-2 assays. Concentrations of LPS and A23187
were selected on the basis of previously published
results and preliminary concentration response curves
established for feline blood. As for LPS, the concentra-
tion response curve for A23187 was flat for high con-
centrations of the stimulus (TxB
2
concentrations of
6.0, 113.0, 162.8, and 119.1 ng/mL following a 4-hour
incubation in a shaking waterbath and an additional 4-
hour incubation with 0, 20, 50, and 100µM A23187
[n = 5], respectively).
The incubation times selected were chosen on the
basis of the following considerations and preliminary
findings (data not shown): 1) TxB
2
production
occurred quickly after A23187 addition to blood, with
TxB
2
concentrations increasing up to 3 hours and
maintaining a plateau for 3 hours, and 2) back-
ground concentrations of TxB
2
increased with time in
the COX-2 assay, and 24 hours after LPS addition, the
difference between TxB
2
concentrations in LPS-treated
and saline (0.9% NaCl) solution-treated samples was
< 2-fold. An incubation time of 4 hours for A23187
(50µM) and 6 hours for LPS (100 µg/µL) was finally
adopted to achieve optimal standardization of condi-
tions for both assays in terms of time allocated for each
enzyme to express its activity. Thus, for both assays,
TxB
2
production occurred for over approximately 4
hours, as COX-2 activity starts to be substantial
approximately 2 hours after LPS addition (Appendix).
Mean (± SD) TxB
2
production (n = 10) in the pres-
ence of a drug vehicle was 2.1 ± 1.9 ng/mL and 190.2 ±
132.4 ng/mL, respectively, after 1.5 and 8 hours of incu-
bation for the COX-1 assay. Mean TxB
2
production
(n = 10) in the presence of a drug vehicle was 1.1 ± 1.4
ng/mL and 8.0 ± 2.5 ng/mL, respectively, after 1.5 and 8
Figure 1—Thromboxane B
2
(TxB
2
) production (mean ± SD) in
whole blood samples from 5 cats after a 3-hour pretreatment
period with aspirin (10 µg/mL) followed by a 5-hour incubation
with increasing concentrations (0, 1, 10, 20, 50, and 100 µg/mL)
of lipopolysaccharide (LPS). Comparisons were performed after
log transformation of the data. **Significantly (
P
< 0.01) differ-
ent from control (LPS, 0 µg/mL) values. ***Significantly
(
P
< 0.001) different from control (LPS, 0 µg/mL) values.
Table 1—Thromboxane B
2
concentrations (mean values, precision, and accuracy) of quality control
samples (ie, TxB
2
added to eicosanoid-free plasma) assayed in replicates of 6 on 3 separate days.
Coefficients of variation (%)
TxB
2
concentration (ng/mL) Within-day Between-day
precision precision
Calibration curves* Known Measured (mean) (repeatability) (reproducibility) Accuracy (%)†
1:500 dilution (2 step)
500 616.4 7.4 25.6 23.3
250 298.8 7.9 15.6 19.5
50 52.3 14.8 18.5 4.6
1:100 dilution (1 step)
100 133.8 9.8 23.3 33.8
50 70.4 5.0 6.8 40.7
10 8.8 14.2 18.9 –12.5
1:30 dilution (2 step)
30 37.5 13.0 13.0 25.1
15 20.0 4.4 5.0 33.2
3 3.5 9.1 11.1 15.5
1:10 dilution (1 step)
10 10.2 8.7 8.7 2.2
5 5.1 10.0 16.4 2.6
1 0.9 12.0 31.0 –7.0
MMeeaann (( SSDD)) 99..77 (( 33..33)) 1166..22 (( 77..88)) 1188..33 (( 1133..11))
*Dilutions were performed as for unknown samples (ie, 1:100 + 1:5 for the 1:500 dilution and 1:10 + 1:3 for
the 1:30 dilution). †Accuracy (%) = ([measured – known]/known) X 100.
1
04-04-0144r.qxp 3/15/2005 9:55 AM Page 702
Page 3
hours of incubation for the COX-2 assay. In the COX-2
assay, TxB
2
concentrations at the time that the test com-
pound was added (t + 1.5 hours [ie, 1.5 hours after the
beginning of the incubation]) were relatively high in
comparison to final TxB
2
concentrations. Because this
TxB
2
production cannot be suppressed by the test com-
pound, TxB
2
concentrations at t + 1.5 hours were sub-
tracted from all of the final TxB
2
concentrations before
calculation of the percent inhibition values in the COX-
2 assay.
Validation of the assays—For each of the 4 cali-
bration curves, 3 quality-control samples were prepared
with eicosanoid-free plasma and a stock solution of
TxB
2
to provide concentrations in the high, medium,
and low regions of the linear section of the standard
curve (Table 1). Further samples were prepared to
make sure that 2 separate dilutions of the same sample
(calculated with 2 different standard curves) gave simi-
lar results (data not shown). For each concentration, 6
replicates were measured and the entire procedure was
repeated on 3 different days (on each day, new solutions
of reagents were prepared). Precision and accuracy were
assessed for each of these concentrations.
The stability of TxB
2
in the COX-1 and COX-2
assays was investigated by measuring TxB
2
concentra-
tions of quality-control blood samples to which TxB
2
was added 4 hours after the start of incubation and that
were then left for 4 more hours in the waterbath at
38
o
C. Recovery, that is ([TxB
2
measured – TxB
2
added]/TxB
2
added) X 100, ranged from –27.4% to
29.9% for the small concentrations of TxB
2
(5 and
10 ng/mL) and from –8.7% to 7.1% when higher con-
centrations of TxB
2
were added to the blood (20 to
500 ng/mL). No linear decline with time occurred in
this study, compared with the investigation of Young et
al,
7
who attributed this decline to breakdown products
of TxB
2
binding less efficiently to the antibodies used
in the immunoassay or the radioimmunoassay.
Inhibition profiles of SC-560, meloxicam, and S-
carprofen—Several approaches were used to assess
COX inhibition by use of the 3 test compounds. The
simplest and most accurate approach was to analyze
individually the data from each of 5 cats and calculate
mean parameters for each NSAID (the so-called 2-stage
method; Table 2). Distribution of individual parame-
ters was close to log normality. Potency and selectivity
were therefore best described by the geometric mean
and corresponding confidence interval. A second
approach to data processing consisted of averaging the
percent inhibition values for each concentration of the
test compound and fitting it, as if it was the curve of a
single cat. This corresponds to the so-called naïve aver-
aging of data approach. In a third approach, simultane-
ous fitting of the 5 inhibition curves for each COX
isoenzyme, also known as the naïve pooled data analy-
sis, allowed calculation of a further set of mean para-
meters (Table 3) and estimation of several selectivity
and safety indices (Table 4). All approaches gave simi-
lar results, but only the 2-stage method and the naïve
pooled data analysis are presented; the naïve averaging
of data approach sometimes leads to a distortion of the
shape of the concentration-effect relationship.
Regardless of the approach used, efficacy for COX-
2 inhibition (Imax + Io) never exceeded 85%. A simi-
lar finding has been reported in another study,
12
sug-
gesting that this submaximal inhibition of COX-2 is
model-dependent. Therefore, percent inhibition values
for both isoenzymes were rescaled on a 0% to 100%
scale to compute selectivity indices. Another interest-
AJVR, Vol 66, No. 4, April 2005 703
Table 3—Mean parameters of the Hill equation as determined
via simultaneous fitting of the individual percent inhibition values
from whole blood samples of 5 cats.
Assay IC
X
ratio
Test compound parameters COX-1 COX-2 COX-1:COX-2
SC-560 (n = 5)
Io (%) –7.99 9.58 NA
Imax (%) 104.02 72.19 NA
Actual Imax (%) 96.03 81.77 NA
n 1.21 2.25 NA
IC
5
(µM) 0.0011 0.052 0.021
IC
50
(µM) 0.0125 0.191 0.065
IC
80
(µM) 0.0394 0.354 0.111
IC
95
(µM) 0.1432 0.709 0.202
Meloxicam (5)
Io (%) –11.20 0.80 NA
Imax (%) 119.14 71.66 NA
Actual Imax (%) 107.95 72.46 NA
n 0.58 3.27 NA
IC
5
(µM) 0.027 0.547 0.048
IC
50
(µM) 4.10 1.35 3.05
IC
80
(µM) 44.08 2.06 21.42
IC
95
(µM) 635.38 3.31 191.71
S-carprofen (5)
Io (%) 1.33 –7.39 NA
Imax (%) 99.09 75.90 NA
Actual Imax (%) 100.43 68.50 NA
n 1.03 3.34 NA
IC
5
(µM) 1.67 0.47 3.55
IC
50
(µM) 29.08 1.14 25.60
IC
80
(µM) 111.64 1.72 64.88
IC
95
(µM) 506.43 2.74 184.54
IC
X
= Test compound concentration producing a COX-1 or COX-
2 inhibition of X percent. Io = Baseline inhibition. Imax = Maximum
inhibition. Actual Imax = Imax + Io. n = Hill coefficient. NA = Not
applicable.
Table 2—Mean potency and selectivity of SC-560, meloxicam,
and the S(+) enantiomeric form of carprofen (S-carprofen) for
inhibition of cyclooxygenase (COX)-1 and COX-2 activity in
whole blood samples of 10 cats as determined by the so-called
2-stage method.
IC
50
(
µµ
M) IC
50
ratio
Test compound results COX-1 COX-2 COX-1:COX-2
SC-560 (n = 5)
Geometric mean 0.0072 0.088 0.0813
Lower 95% CI 0.0027 0.026 0.0293
Upper 95% CI 0.0192 0.298 0.2256
Meloxicam (5)
Geometric mean 4.0 1.2 3.5
Lower 95% CI 1.6 0.5 1.1
Upper 95% CI 10.2 2.5 10.4
S-carprofen (5)
Geometric mean 26.6 0.9 28.1
Lower 95% CI 14.4 0.5 11.9
Upper 95% CI 49.3 1.9 66.4
IC
50
= Test compound concentration giving 50% of maximum
inhibition. CI = Confidence interval.
04-04-0144r.qxp 3/15/2005 9:55 AM Page 703
Page 4
704 AJVR, Vol 66, No. 4, April 2005
ing finding was that the percent inhibition values for
the lowest concentrations of the test compounds were
usually negative, implying slight stimulation of TxB
2
production for these low concentrations. The mecha-
nism remains unknown, but this finding has been pre-
viously reported in another study.
15
As recommended by previous authors,
6
we calcu-
lated an IC
80
ratio of COX-1:COX-2 because this may
be more predictive of the clinical relevance of the COX-
2 selectivity. Because the inhibition curves for COX-1
and COX-2 were parallel for SC-560 (Figure 2), the
IC
50
and IC
80
ratios were similar (Table 3). For meloxi-
cam and S-carprofen, on the other hand, Hill coeffi-
cients (sensitivities) for COX-1 and COX-2 were dif-
ferent, so that IC ratios varied profoundly over the
range of inhibitory concentrations (Figure 3). Clearly,
for S-carprofen and meloxicam and probably for other
drugs also, it is not possible to find a single selectivity
index that accounts for the distance between the inhi-
bition curves of COX-1 and COX-2 over the entire
range of concentration. Nevertheless, if the aim of the
study is to consider the likely clinical relevance of
selective COX-2 inhibition, the indices describing the
selectivity of the test compound indicated in table 4,
are more informative than the classic IC
50
or IC
80
ratios. Another approach to predict the clinical rele-
vance of COX selectivity for NSAIDs was to plot the
percent inhibition of COX-2 against the corresponding
percent inhibition of COX-1 (Figure 4).
Table 4—Two categories of indices used to describe the selec-
tivity of SC-560, meloxicam, and S-carprofen as determined by
simultaneous fitting of the individual percent inhibition values
from whole blood samples of 5 cats.
Test compounds
Indices SC-560 Meloxicam S-carprofen
IC
X
:IC
Y
of COX-1:COX-2*
IC
1
:IC
99
0.0002 0.0003 0.075
IC
5
:IC
95
0.0015 0.0080 0.608
IC
10
:IC
90
0.0040 0.0361 1.571
IC
20
:IC
80
0.0112 0.1857 4.401
% Inhibition of COX-1 (0% to 100%)†
For 50% inhibition 96.42 34.28 3.42
of COX-2
For 80% inhibition 98.27 40.05 5.15
of COX-2
For 90% inhibition 98.87 43.58 6.52
of COX-2
For 95% inhibition 99.24 46.88 8.07
of COX-2
For 99% inhibition 99.69 54.24 12.76
of COX-2
*Selectivity can be defined as a safety factor, which is usually
expressed as a ratio of a cutoff concentration above which unac-
ceptable adverse effects occur over a concentration producing
maximal effect or therapeutic efficacy. †Percent inhibition by which
COX-1 is inhibited for a given percentage of COX-2 inhibition.
Figure 2—Mean concentration response curves for cyclooxyge-
nase (COX)-1 and COX-2 inhibition by SC-560 in whole blood
assays. The mean (± SEM) percent inhibition of TxB
2
production
was calculated by averaging, for each concentration of SC-560,
individual percent inhibition values of 5 cats. For each whole
blood sample, inhibition was expressed as a percentage of the
individual control value (SC-560, 0µM). By use of the Hill equa-
tion, fitted inhibition curves were obtained via simultaneous fit-
ting of the individual percent inhibition values of 5 cats.
Figure 3—Mean concentration response curves for COX-1 and
COX-2 inhibition by meloxicam and S-carprofen in whole blood
assays. For each whole blood sample, inhibition was expressed
as a percentage of the individual control value (test compound,
0µM). By use of the Hill equation, fitted inhibition curves were
obtained via simultaneous fitting of the individual percent inhibi-
tion values of 5 cats.
Figure 4—Percent inhibition of COX-2 and the corresponding
percent inhibition of COX-1 by SC-560, meloxicam, and S-carpro-
fen in whole blood samples from 10 cats. Parameters of the Hill
equations describing COX-1 and COX-2 inhibition by use of the
3 test compounds were obtained via simultaneous fitting of the
individual percent inhibition values of 5 cats. Derived mean fit-
ted percents, initially ranging from baseline inhibition (Io) to max-
imum inhibition (Imax + Io) were rescaled on a 0% to 100%
scale. For given percent inhibition values of COX-2, correspond-
ing test compound concentrations were calculated by use of the
Hill equation for COX-2 and these concentrations were then
incorporated in the Hill equation for COX-1 to calculate corre-
sponding COX-1 inhibitions. Two of the dotted lines indicate cut-
off values for inhibition of both isoenzymes (ie, 20% inhibition of
COX-1 is considered the percent value above which there may
be a risk of adverse effects; 80% inhibition of COX-2 is consid-
ered the percent value above which a good therapeutic effect is
expected).
04-04-0144r.qxp 3/15/2005 9:56 AM Page 704
Page 5
AJVR, Vol 66, No. 4, April 2005 705
Statistical comparisons of potency (IC
50
) and iso-
form inhibition selectivity (ie, IC
50
ratio of COX-
1:COX-2) lead to the following conclusions: 1) SC-560
is a more potent inhibitor than meloxicam and S-
carprofen for COX-1 and COX-2 and is selective for
COX-1; 2) S-carprofen has a much lower potency for
COX-1 inhibition than meloxicam and similar potency
for COX-2 inhibition and is therefore significantly
more selective for COX-2; and 3) no significant differ-
ences exist in the efficacy (Imax + Io) for COX-1 and
COX-2 inhibition between the 3 drugs tested.
Considering the alternative selectivity indices
(Table 4), it is not possible to choose a selectivity index
that is most appropriate for in vivo clinical conditions
because plasma concentration will change with time.
Therefore, several percentages of COX-1 inhibition for
fixed percentages of COX-2 inhibition (ranging from
50% to 99% inhibition) can be considered. For meloxi-
cam, these ranged from 34.3% to 54.2%, respectively,
and corresponding values for S-carprofen ranged from
3.4% to 12.8%.
Because drug protein binding is taken into
account, the use of whole blood assays allows compar-
ison of IC
50
, IC
80
, and IC
95
values obtained in vitro
with therapeutically relevant concentrations (Table 5).
Assuming that these NSAIDs do not penetrate RBCs,
the multiplying factor for the transformation of whole
blood concentrations into plasma concentrations was
approximately 1.5 when the Hct for a cat is 35%.
10
Predicted in vivo doses were calculated with these
transformed concentrations by use of the following
equation:
Dose Total clearance X Target concentration
Dosing interval F
where dose divided by dosing interval is the dose to be
given in vivo to provide, in steady-state conditions, a
mean concentration over the dosing interval equal to
the target concentration. Total clearance is the total
plasma clearance of the test compound, F is the
bioavailability, and target concentration is the plasma
concentration of interest (in terms of beneficial or
adverse effects) and is usually an in vivo concentration
that is considered therapeutically efficacious. In the
study reported here, the target concentration was an in
vitro–determined concentration that has been multi-
plied by 1.5 for the transformation of whole blood con-
centrations into plasma concentrations.
Discussion
In our study, special attention was paid to standard-
izing the classic whole blood assays in terms of time
courses of incubation with the test compounds and con-
ditions of COX expression to make the COX-1 and the
COX-2 assays as similar as possible. Some authors
16,17
have proposed that prostaglandin E
2
is preferable to
TxB
2
as a marker of COX-2 activity. However, TxA
2
is
the major COX-derived metabolite of arachidonic acid
in LPS-stimulated rat alveolar macrophages and human
monocytes, as assessed by radioimmunoassay of
TxB
2
.
1,18
Moreover, LPS has no effect on TxA
2
synthase
of pretreated monocytes and liver macrophages but
induces a strong expression of prostaglandin E
2
syn-
thase, which seems to be coupled more efficiently to
COX-1 than to COX-2 in liver macrophages.
1,19
The cou-
pling of COX enzymes to the downstream synthases
might differ depending on the cell type, but these find-
ings raise the question of whether the induction of the
prostaglandin E
2
synthase can bias the assessment of
COX-2 activity as measured by prostaglandin E
2
pro-
duction. Furthermore, because of the early onset of TxB
2
production, compared with prostaglandin E
2
produc-
tion, it was possible in our studies to allocate equal incu-
bation times with the test drug for each COX isoform
assay. Different incubation times are indeed believed to
skew the data towards either beneficial effects
(enhanced COX-2 potency) or inhibition of COX-1. For
these reasons, we chose to use TxB
2
as a measure of
COX-2 as well as COX-1 activity.
=
Table 5—In vitro-to-in vivo extrapolation.
Test compounds
Parameters Meloxicam S-carprofen
In vitro determined IC
80
for COX-2 inhibition (µM)* 2.06 1.72
Predicted in vivo dose for 80% inhibition of COX-2 (mg/kg/24h) 0.165 0.318
In vitro determined IC
95
for COX-2 inhibition (µM) 3.31 2.74
Predicted in vivo dose for 95% inhibition of COX-2 (mg/kg/24h) 0.266 0.507
Manufacter’s recommended dose (mg/kg) 0.3 4 (RS)
Corresponding in vivo plasma Cmax concentration (µM) † 3.95 35.11
% Inhibition of COX-1 corresponding to Cmax 43.6 44.4
% Inhibition of COX-2 corresponding to Cmax 90.0 100.0
Time above IC
50
for COX-2 (h) 23.0 72.0
Time above IC
80
for COX-2 (h) 8.8 57.0
Time above IC
95
for COX-2 (h) 0 42.2
Time above IC
10
for COX-1 (h) 109.5 36.2
Time above IC
20
for COX-1 (h) 64.3 20.2
*All in vitro determined concentrations were calculated using a naïve pooled data analysis. The concentra-
tions displayed in this table were then multiplied by 1.5 (for the transformation of whole blood into plasma con-
centrations) to calculate corresponding predicted in vivo doses. †In vivo concentrations and pharmacokinetic
parameters were obtained from other studies (Taylor et al
25
for S-carprofen; unpublished data for
meloxicam).
See
Table 3 for key.
04-04-0144r.qxp 3/15/2005 9:56 AM Page 705
Page 6
As has been previously discussed,
20
TxB
2
can be
used as a marker of COX-2 activity in LPS-stimulated
monocytes if these cells have been pretreated with
aspirin. Despite addition of aspirin to our COX-2 assay,
TxB
2
production in saline solution–treated samples
was relatively high, compared with that obtained in
human blood (< 5% of the TxB
2
production in LPS-
treated samples).
7
Even if some COX-1 contribution in
the COX-2 assay cannot be completely excluded, it is
believed that this background production is the result
of some degree of COX-2 induction in these samples.
A similar finding has been described in other studies
1,21
in which COX synthesis was investigated. One expla-
nation is that blood collection, vortexing of the blood
after compound additions, and blood incubation in a
shaking waterbath may also cause some degree of
COX-2 induction (the magnitude of which may be
species specific) through mechanical stimulation of the
cells. In our study, TxB
2
concentrations in saline solu-
tion–treated samples were therefore not subtracted
from the TxB
2
production of the LPS-treated samples.
Despite pretreatment with aspirin (10 µg/mL) in
the COX-2 assay, some degree of COX-1 activity
remained, as determined with pretreated blood stimu-
lated with A23187. A higher concentration of aspirin
(ie, 30 µg/mL) was more effective in eliminating COX-
1 activity (data not shown), but it is also important to
ensure that pretreatment with aspirin does not inter-
fere with COX-2 activity. The aspirin concentration
chosen (ie, 10 µg/mL) provides the best compromise
between these conflicting considerations, enabling
good inactivation of COX-1 in the COX-2 assay with
almost no effect on subsequent induced COX-2. This
was also determined in another study, in which < 10%
of the initial amount of aspirin remained after 3 hours
of incubation in whole blood, which corresponds
approximately to the time when COX-2 induction is
likely to commence in our study.
The potency of the COX-1 inhibitor SC-560 for
COX-2 inhibition was rather high in our study. This
resulted in selectivity for COX-1 that was 19- and 45-
fold less than that described in 2 in vitro assays in
which human monocytes in culture medium
8
or
recombinant human enzymes were used.
22
Different
assay conditions may account for differences in selec-
tivity, but potencies for COX-1 were surprisingly simi-
lar despite the variability in experimental conditions
(0.007µM in our study vs 0.009µM in that of Smith et
al
22
and 0.005µM as determined by Kato et al
8
).
Meloxicam has recently been tested in 2 canine
whole blood assays, and the data were consistent with
the slight COX-2 selectivity observed in feline blood
(IC
50
ratio of COX-1:COX-2 of 10
3
and 2.72,
11
com-
pared with 3.5 in our study). Such degrees of selectiv-
ity are usually insufficient to clearly separate the effects
of NSAIDs on the 2 isoenzymes (especially when the
inhibition curve for COX-1 is shallow). This has been
confirmed for meloxicam by use of results of ex vivo
studies
6,7,10
in humans, which indicate that this small
degree of in vitro COX-2 selectivity did not translate
into therapeutically relevant selectivity. For such
drugs, the selected dosage regimen will often be asso-
ciated with plasma concentrations producing a sub-
stantial inhibition of both isoforms, unless a major
contribution of a separate mechanism of anti-inflam-
matory action exists that requires lower plasma con-
centrations.
In whole blood assays, the potency of the drug
being tested is often limited by drug binding to plasma
proteins, which makes such assays most suitable when
extrapolating to in vivo situations, and the presence of
arachidonic acid at the time of COX-2 induction. This
reduction in COX-2 potency in whole blood assays can
explain the discrepancy, also observed for carprofen,
when comparing results from whole blood assays with
data obtained from assays without binding of the test
compound to plasma proteins.
13,m
S-carprofen had nev-
ertheless high COX-2 selectivity, which proves that, at
least in vitro and in cats, it may be classified as a COX-
2 preferential or even selective agent. The COX-2
selectivity for S-carprofen in our study was approxi-
mately twice as high as values reported for whole blood
assays in dogs.
11,m
In another study, Brideau et al
3
pub-
lished data indicating selectivity of carprofen to be 6.5
in dogs and 5.5 in cats on the basis of IC
50
ratios.
However, their potencies for COX-1 and COX-2 in cats
(8.93 and 1.64µM for the IC
50
of COX-1 and COX-2,
respectively) can be compared with values of 26.6 and
0.9µM obtained in our study. The apparently 5-fold
lower selectivity in the study of Brideau et al
3
might be
an artefact explained in part by their use of a racemic
mixture. S-enantiomers of 2-arylpropionates usually
have a higher potency for inhibition of COX enzymes,
with R-enantiomers being nearly devoid of
prostaglandin synthesis inhibition in vitro.
23
The
R-enantiomer present in the racemic mixture is there-
fore likely to produce an apparent decrease in COX-2
potency and selectivity.
13
This might be even more pro-
nounced for carprofen in vivo because enantioselective
pharmacokinetics after administration of the racemate
result in predominance of R-carprofen in biological flu-
ids in all species investigated, including cats.
24-27
Interestingly, an increase in the selectivity of carpro-
fen between the IC
50
and IC
80
ratios has been described
by Streppa et al,
11
who reported an increase from 16.8 to
101.2 for dogs. In a human whole blood assay, Warner
et al
6
reported an IC
50
ratio for COX-1:COX-2 of 0.020
(implying that racemic carprofen is COX-1 selective),
whereas the IC
80
ratio was 0.256, indicating only slight
selectivity for COX-1, which again indicates major
species differences and dependence of selectivity on the
level of IC chosen for comparison.
A number of indices have been used to describe
the differential inhibition of COX-1 and COX-2 by var-
ious compounds, the classic approach being to calcu-
late IC
50
or IC
80
ratios.
6,17
These ratios are dependent
on the relative slopes of the inhibition profiles of the
test compound for COX-1 and COX-2. Alternative
indices are more clinically relevant because they com-
pare segments of the inhibition curves for COX-1 and
COX-2 that correspond to target concentrations for
therapeutic and adverse effects (Table 4). An IC
80
ratio
of COX-1:COX-2, for example, does not indicate the
degree of inhibition of COX-1 attained with therapeu-
tically efficacious concentrations (assumed to corre-
spond to the IC
80
or even the IC
95
for COX-2). In con-
706 AJVR, Vol 66, No. 4, April 2005
04-04-0144r.qxp 3/15/2005 9:56 AM Page 706
Page 7
trast, an IC
10
COX-1:IC
90
COX-2 ratio that is close to
1, as obtained for S-carprofen, indicates that for 90%
inhibition of COX-2, there will be only 10% of inhibi-
tion of COX-1.
Representing inhibition profiles for both isoenzymes
on the same graph is also a useful approach for assessing
selectivity of the test compound over the range of thera-
peutic concentrations (Figure 3). When the aim of a
study is to predict the clinical relevance of COX selectiv-
ity for NSAIDs, another approach consists of plotting the
percent inhibition of COX-2 against the corresponding
percent inhibition of COX-1 (Figure 4). By the addition
of cutoff values for both isoforms to the graph, it became
clear in our study that there is virtually no meloxicam
concentration for which COX-1 is not inhibited by at
least 20% and that in the predicted range of therapeutic
concentrations (> 80% of inhibition of COX-2), the lim-
ited selectivity of meloxicam against COX-2 is largely lost
(COX-1 inhibited by more than 40%).
By use of in vitro-to-in vivo extrapolations (Table
5) and an IC
50
for COX-2 inhibition as the lowest tar-
get concentration to be reached, the predicted dose of
meloxicam would be 0.11 mg/kg/24 h (or
0.17 mg/kg/24 h and 0.27 mg/kg/24 h if the target con-
centrations are the IC
80
and IC
95
, respectively) when
taking into account a value of 0.006 L/h/kg for plasma
clearance scaled by bioavailability in cats.
Applying the same calculation to S-carprofen, the
predicted therapeutic dose derived from IC
50
is
0.21 mg/kg/24 h (0.32 mg/kg/24 h if the target concen-
tration is IC
80
) when considering pharmacokinetic data
from the study of Taylor et al.
25
When an IC
95
for COX-
2 is used as the concentration to be reached, the pre-
dicted dose of 0.51 mg/kg/24 h is still only one fourth
of the currently recommended dose for cats (racemic
mixture [4 mg/kg] for the first injection; ie, S-enan-
tiomer [2 mg/kg]). For this dose, in vitro results predict
100% inhibition of COX-2 and 44% inhibition of COX-
1 at the maximum plasma concentration, so that for
these high concentrations, the selectivity of S-carprofen
is largely lost. For a single dose of 4 mg/kg racemic
carprofen, plasma concentrations of the S-enantiomer
would exceed an IC
20
for COX-1 for 20 hours (Table 5).
These predictions correlate well with ex vivo
results of TxB
2
inhibition obtained after SC adminis-
tration of racemate carprofen at doses of 0.7 and
4 mg/kg in cats.
25
Results of that study revealed that no
inhibition of serum TxB
2
was observed with the small-
est dose of carprofen but that with the higher dose, a
40% to 60% inhibition of TxB
2
occurred for approxi-
mately 24 hours. One explanation could be that COX
inhibition may not be the only mechanism of action of
carprofen. Some authors
27,28
have indeed shown that
carprofen is sparing for COX-1 and COX-2 in horses
and dogs. The COX-sparing effect of carprofen in these
species is compatible with the hypothesis of an addi-
tional mechanism of action of this drug.
26-31
In dogs, the maximum plasma concentration of
meloxicam at a dose of 0.2 mg/kg is only half the max-
imum plasma concentration after SC administration of
0.3 mg/kg in cats, and it was shown in vivo that no
inhibition of COX-1 occurred in dogs for the clinically
recommended dose.
32
Despite similar potencies for
COX-1 in both species, it is anticipated from the in
vitro data that only small doses (meloxicam
[< 0.03 mg/kg/24 h]) could result in mean concentra-
tions corresponding to < 20% inhibition of COX-1 in
cats. It is not known if a precise degree of COX-1 inhi-
bition exists below which there is virtually no possibil-
ity of adverse effects on the gastrointestinal tract.
However, if a plasma concentration corresponding to at
least IC
80
for COX-2 inhibition is required for a thera-
peutic effect and a concentration less than IC
20
for
COX-1 is required to avoid adverse effects, a single
dose of 0.3 mg/kg meloxicam in the cat would exceed
this safety margin for 64.3 hours and the therapeutic
concentration for 8.8 hours (Table 5).
It may be that no firm conclusions can be drawn
from our studies regarding the concentrations of
meloxicam and S-carprofen required in vivo for thera-
peutic effects. Participation of other mechanisms of
action in the anti-inflammatory effect of NSAIDs has
already been evoked, but other reasons exist as to why
in vitro data do not always translate readily into clinical
situations (eg, accumulation of the active compound in
the target cells or biotransformation leading to active
metabolites). A further consideration is the variation in
pharmacodynamics as reflected in variability of the
potency for COX-2 as well as interindividual differences
in pharmacokinetics. Nevertheless, for drugs in which
the main mechanism of action is believed to be inhibi-
tion of COX enzymes, whole blood assays in the species
of interest can be, as shown for meloxicam, a useful tool
for screening new molecules for therapeutic usefulness.
Ex vivo studies that use whole blood assays and
in vivo studies on tissue cages that mimic deep
inflammatory sites are also interesting approaches to
determine the degree and time course of inhibition of
COX isoforms and to investigate further the clinical
relevance of in vitro results.
25-28
Ultimately, in vivo
studies that involve the use of clinical indices need to
be performed to confirm in vitro and ex vivo findings.
Studies on dogs by Jones et al
32
(ie, in vivo assessment
of blood, gastric mucosal, and synovial fluid
prostanoid synthesis) and Toutain et al
33
(ie, determi-
nation of the concentration-effect relationship for
clinically relevant end points) are in this respect par-
ticularly relevant because they address in a quantita-
tive and objective manner beneficial and adverse
effects of 2 NSAIDs of veterinary interest.
a. Roche Products Ltd, Hertfordshire, UK.
b. Fort Dodge Animal Health, Southampton, UK.
c. CP Pharmaceuticals Ltd, Wrexham, UK.
d. Terumo Europe N.V., Leuven, Belgium.
e. Becton Dickinson Labware Europe, Le Pont De Claix, France.
f. Cipla, Mumbai, India.
g. Grampian Pharmaceuticals, Lancashire, UK.
h. Calbiochem-Novabiochem Corp, La Jolla, Calif.
i. Sigma-Aldrich Co Ltd, Dorset, UK.
j. Escherichia coli serotype 026:B6, Sigma-Aldrich Co Ltd,
Dorset, UK.
k. Scientist software program, MicroMath Research, St Louis, Mo.
l. SYSTAT, Systat Software Inc, Richmond, Calif.
m. Haven M, Seibel S, Petras CF, et al. In vitro and ex vivo whole
blood evidence of selective cyclooxygenase-2 inhibition by
Rimadyl (abstr), in Proceedings. 6th Int Cong Eur Assoc Vet
Pharmacol Toxicol 1998.
AJVR, Vol 66, No. 4, April 2005 707
04-04-0144r.qxp 3/15/2005 9:56 AM Page 707
Page 8
708 AJVR, Vol 66, No. 4, April 2005
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Appendix
Time courses of incubation of whole blood with test compound and stimuli in the
cyclooxygenase (COX)-1 and COX-2 assays. Feline blood was stimulated with cal-
cium ionophore (A23187), which leads to platelet activation with subsequent for-
mation of thromboxane B
2
(TxB
2
) by COX-1 or with bacterial lipopolysaccharide
(LPS) after which TxB
2
production is mainly the result of COX-2 induction in mono-
cytes. All concentrations are final concentrations in blood; test compounds,
A23187, and aspirin were added in 1 µL dimethyl sulfoxide (DMSO) and LPS was
added in 10 µL saline (0.9% NaCl) solution.
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33. Toutain PL, Cester CC, Haak T, et al. A pharmacokinetic/phar-
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    • "The indicator in healthy animals of g.i.t. toxicity relative to efficacy proposed by Giraudel et al. (2005a Giraudel et al. ( , 2009), IC 20 COX-1: IC 80 COX-2, is of interest. It was, for mavacoxib and carprofen, almost identical, 1.92:1 and 1.95:1, respectively (Table 3, Lees et al., 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: Mavacoxib is a novel nonsteroidal anti-inflammatory drug (NSAID), with a preferential action on the cyclooxygenase (COX)-2 isoform of COX and a long duration of action. It is classified chemically as a member of the sulphonamide subgroup of coxibs. Mavacoxib is highly lipid but very poorly water soluble. In the dog, the pharmacokinetic (PK) profile comprises very slow body clearance, long elimination half-life and a relatively large distribution volume. Biotransformation and renal excretion are very limited, and elimination occurs primarily by biliary secretion and excretion of unchanged drug in faeces. The PK profile of mavacoxib differs quantitatively between young healthy dogs (Beagles and mongrels) and clinical cases with osteoarthritis (OA). In OA dogs, mavacoxib exhibits a much longer terminal half-life, associated principally with their greater median body weight compared with dogs used in preclinical studies. There is also some evidence of breed differences and a small effect of age on mavacoxib PK in the OA canine population. The pharmacodynamics (PD) of mavacoxib has been established: (i) in whole blood assays at the molecular level (inhibition of COX-1 and COX-2 isoforms); (ii) in preclinical models of inflammation and pain; and (iii) in clinical OA subjects treated with mavacoxib. The dosage schedule of mavacoxib for clinical use has been determined by owner and veterinary clinical assessments and is supported by integration of PK and PD preclinical data with clinical responses in canine disease models and in dogs with naturally occurring OA. The dosage regimen has been further confirmed by correlating levels of inhibition of COX isoforms in in vitro whole blood assays with plasma concentrations of mavacoxib achieved in OA dogs. In addition to the specific properties of mavacoxib, some general aspects of the PK and PD of other agents of the NSAID group, together with pathophysiological and clinical aspects of OA, are reviewed, as a basis for correlating with the safety and efficacy of mavacoxib in therapeutic use. Integration of PK and PD data suggests that the recommended dosage regimen of 2 mg/kg bw once for 14 days, followed by administration at monthly intervals, is optimal from both efficacy and safety perspectives and is further confirmed by clinical field studies.
    Full-text · Article · Nov 2014 · Journal of Veterinary Pharmacology and Therapeutics
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    • "To better define the selectivity , PGE2:TXB2 ratios were calculated at three different percentages of inhibition, namely, 20%, 50% and 80%. The IC20 TXB2:IC80 PGE2 ratio was 6.65:1 which was relatively high compared to that for other drugs tested in cats such as robenacoxib (4.23), diclofenac (0.48) and meloxicam (0.19) (Giraudel et al., 2005aGiraudel et al., , 2005b Schmid et al., 2010). For a more accurate evaluation, an ex vivo study was performed using blood collected at 0, 1, 2, 4, 10 and 24 h after PX injection during the pharmacokinetic study. "
    [Show abstract] [Hide abstract] ABSTRACT: Parecoxib (PX) is an injectable prodrug of valdecoxib (VX, which is a selective cyclo-oxyganase-2 (COX-2) inhibitor licensed for humans. The aim of the present study was to evaluate pharmacokinetics and in vitro/ex vivo cyclooxygenase selectivity of PX and VX in cats. In a whole blood in vitro study, PX did not affect either COX enzymes whereas VX revealed a COX-2 selective inhibitory effect in feline whole blood. The IC50 values of VX for COX-2 and COX-1 were 0.45 and 38.6 µM, respectively. Six male cats were treated with 2.5 mg/kg of PX by intramuscular injection. PX was rapidly converted to VX with a relatively short half-life of 0.4 h. VX achieved peak plasma concentration (2.79 ± 1.59 µg/mL) at 7 h following PX injection. The mean residence times for PX and VX were 0.43 ± 0.15 and 5.94 ± 0.88 h, respectively. In the ex vivo study, PX showed a COX-2 inhibition rate of about 70% in samples taken at 1, 2, 4 and 10 h after injection, with a significant difference compared to the control. In contrast, COX-1 was slightly inhibited, ranging from 0.7% to 9.7% of the control inhibition rate without any significant difference for 24 h after PX administration. The preliminary findings of the present research appear promising and encourage further studies to investigate whether PX can be successfully used in feline medicine.
    Full-text · Article · Oct 2014 · The Veterinary Journal
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    • "The animal species might have played a role in determining COX selectivity (Lees et al. 2004). Carprofen, which is not a coxib structure, may act as a COX-2 preferential or selective drug in the dog and cat rather than being a non-selective drug as in the horse (Brideau et al. 2001; Giraudel et al. 2005). Further studies to assess the selectivity of cimicoxib in horses are required. "
    [Show abstract] [Hide abstract] ABSTRACT: Aims: To determine the pharmacokinetics of cimicoxib and to assess the inhibition of cyclooxygenase (COX) after a 5 mg/kg, single oral administration in horses that were fasted or fed. Methods: The study was conducted using an open, single dose (5 mg/kg), two treatment (fasted and fed), two-period, crossover design with a 2-week interval between dosages. Six healthy mares received 5 mg/kg of cimicoxib via nasogastric tube after fasting for 12 hours, or 2 hours after feeding. After administration, blood samples were collected for up to 24 hours and plasma used for pharmacokinetic analysis. Additional serum and plasma samples were used to measure concentrations of thromboxane B2 (TXB2) and prostaglandin E2 (PGE2), to assess COX-1 and -2 inhibition, respectively. Results: Following cimicoxib administration, the mean maximum plasma concentration was 0.16 (SD 0.01) µg/mL and 0.14 (SD 0.03) µg/mL in fasted and fed groups, respectively. The mean time taken to reach maximum plasma concentration was longer in the fed group (5.91 (SD 3.23) hours) compared with the fasted group (3.25 (SD 1.17) hours), but this difference was not significant (p=0.12). The mean maximal inhibition of TXB2 was 62.4 (SD 13.8)% and 54.6 (SD 15.4)%, and of PGE2 was 72.1 (SD 43.3)% and 68.5 (SD 24.4)%, in fasted and fed horses, respectively. Conclusion: In the present study, although the COX-2 selective action of cimicoxib was not apparent, a relatively low concentration of cimicoxib resulted in both COX-1 and -2 inhibition in horses. Further investigations are required to establish an optimal dosage regimen and safety profile before clinical trials are initiated.
    Full-text · Article · Jul 2014 · New Zealand veterinary journal
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