J. vet. Pharmacol. Therap. 33, 50–55, doi: 10.1111/j.1365-2885.2009.01101.x.

Canine CYP2B11 metabolizes and is inhibited by anesthetic agents often

co-administered in dogs

M. T. BARATTA*
M. J. ZAYA
J. A. WHITE &
C. W. LOCUSON
*Pfizer Global Research and Development,
Pharmacokinetics, Dynamics, and
Metabolism, Creve Coeur, MO, USA;
Pfizer Animal Health, Veterinary Medicine
Research and Development, Metabolism and
Safety, Kalamazoo, MI, USA
Baratta, M. T., Zaya, M. J., White, J. A., Locuson, C. W. Canine CYP2B11
metabolizes and is inhibited by anesthetic agents often co-administered in dogs.
J. vet. Pharmacol. Therap. 33, 50–55.
Medetomidine is a well-established sedative and analgesic for dogs and cats. As
a premedicant for anesthesia regimens that also include other agents,
medetomidine can also provide a dose-sparing effect. While there are likely
several reasons for the dose-sparing effect of medetomidine, the role of
metabolic drug–drug interactions at the single enzyme level has not yet been
examined. Using a panel of individually expressed canine cytochromes P450
cloned from beagle liver, this report demonstrates that medetomidine is an
extremely potent CYP2B11 inhibitor (IC50 < 10 nM) and that ketamine and
midazolam are CYP2B11 substrates with high intrinsic clearances. These
in vitro findings suggest that under some circumstances, medetomidine (i.e.
‘perpetrator’) may inhibit the metabolic clearance of some high metabolic
clearance drugs (i.e. ‘victims’) with which it is commonly co-administered via
the CYP2B11 pathway. However, as the dose-sparing effect of medetomidine
premedication commonly results in anesthetic dose reduction, any increased
plasma concentrations of victim drugs caused by medetomidine would still be
lower than if no dose reduction had been made. Further studies are needed to
characterize whether medetomidine possesses the potential to cause pharma-
cokinetic interactions. In conclusion, the ability of recombinant P450s to define
canine-specific drug clearance pathways and P450 inhibitors should prove
useful in identifying drug combinations that may require dose adjustments in
dogs.
(Paper received 4 February 2009; accepted for publication 29 April 2009)
Charles W. Locuson, Pfizer Animal Health, Veterinary Medicine Research and
Development, Metabolism and Safety, 333 Portage Street, KZO-300-421NW,
Kalamazoo, MI 49001, USA. E-mail: charles.w.locuson@pfizer.com

INTRODUCTION
The growing companion animal health market will likely
encourage veterinary pharmaceutical companies to expand their
development of therapeutics for dogs (Evans & Chapple, 2002).
Assuming that instances of polypharmacy in dogs will increase
with more drugs being developed for companion animals, the
Drug Metabolism ⁄ Pharmacokinetics Scientist will require more
tools to assess the potential risks of concomitant medications
altering drug exposure in dogs. Currently, there are few
pharmaceuticals that have been adequately characterized with
canine enzymes to support their use as specific inhibitors or
substrates to use in dog interaction studies. For example, one can
find pharmacokinetic drug interaction studies, but the actual
enzyme(s) implicated have generally been inferred by analogy to
human enzymes. One tool that is increasingly being used to link
pharmacokinetic drug–drug interactions to specific metabolic
pathways is rP450 (recombinant, expressed cytochrome P450)
(Crespi & Miller, 1999; McGinnity et al., 2008; Youdim et al.,
2008). However, the availability of expressed canine P450
(cytochrome P450) isoforms has been limited and the relative
importance of the canine P450s to canine drug metabolism as a
whole is largely unknown, in part, because of the lack of
characterization of canine P450s.
Recently, several of the major hepatic canine P450s were
co-expressed with canine P450 oxidoreductase in Escherichia coli
by the authors (Locuson et al., 2009). During inhibition profiling
with a panel of the canine rP450s, the authors identified the
anesthetic medetomidine as a potent inhibitor of CYP2B11.
While the risk of drug–drug interactions involving human
CYP2B enzymes appears to be low because of minimal involve-
ment in drug oxidation and low hepatic expression (Walsky
et al., 2006), canine CYP2B11 has shown surprisingly high
concentrations of activity in vitro towards drugs used in dogs,
such as benzodiazepines (Shou et al., 2003). Hence, inhibition of
CYP2B11 by chloramphenicol (Ciaccio et al., 1987), loperamide,

50  2009 Blackwell Publishing Ltd


ketoconazole, diazepam, and fluoxetine (Aidasani et al., 2008)
may raise legitimate drug interaction questions in canine
medicine. Canine CYP2B11 is also expressed at reasonable
concentrations in dog liver, estimated at 10% of total P450
(Eguchi et al., 1996), which would allow it to contribute to drug
clearance more readily in the dog vs. human CYP2B isoforms.
It is reported here that midazolam and ketamine, which are
often co-administered with the CYP2B11 inhibitor medetomi-
dine, demonstrated clearance by rCYP2B11 according to
phenotyping experiments. These findings may provide a partial
explanation for the dose-sparing effect of medetomidine preme-
dication on ketamine and other maintenance anesthetics
[reviewed by Cullen (1996)]. The results also demonstrate how
rP450s can be used in drug–drug interaction assessment in
veterinary medicine and reinforce the hypothesis that CYP2B11
may be an important clearance pathway in dogs.
MATERIALS AND METHODS
Materials
The isomers of medetomidine were synthesized at Pfizer (Sand-
wich, UK). Commercial formulations of medetomidine (Domi-
tor), ketamine (Ketaset), and atipamezole (Antisedan) were
purchased from Pfizer research veterinarians. Midazolam, prop-
ofol, dimethyl sulfoxide (DMSO), b-NADPH, and monobasic and
dibasic potassium phosphate were purchased from Sigma-
Aldrich (St Louis, MO, USA). Formic acid (88%) was purchased
from Mallinckrodt Baker, Inc. (Paris, KY, USA). High-perfor-
mance liquid chromatography grade solvents were purchased
from Honeywell Burdick & Jackson (Morristown, NJ, USA).
Canine rP450 Bactosomes (Cypex Ltd., Dundee, Scotland, UK)
were prepared as described (Locuson et al., 2009). Beagle liver
microsomes purchased from Xenotech, LLC (Lenexa, KS, USA)
were mixed gender from a pool of 23 subjects.
IC50 determination
Inhibitor concentration (IC50) values for medetomidine and
atipamezole were determined against phenacetin de-ethylation,
temazepam N-demethylation, diclofenac 4¢-hydroxylation, buf-
uralol 1¢-hydroxylation, and midazolam 1¢-hydroxylation by the
canine rP450s 1A1, 2B11, 2C21, 2D15, and 3A12, respectively.
The incubations were conducted as previously described with 12
inhibitor concentrations and substrate concentrations equal to
their Km values (Aidasani et al., 2008). Tolbutamide (50 ng ⁄ mL)
was used as the internal standard in the acetonitrile quenching
solution.
LC ⁄ MS ⁄ MS analysis
Liquid chromatography (LC) and mass spectrometry (MS)
detection of all test compounds analyzed for assay endpoints
was accomplished using a Perkin Elmer Series 200 micro-pump
system (Waltham, MA, USA) and a Leap Technologies HTC PAL
 2009 Blackwell Publishing Ltd
Canine CYP2B11 metabolizes anesthetic agents 51
Autosampler (Canboro, NC, USA) connected to a Applied
Biosystems MDS ⁄ Sciex API 4000 triple quadrupole mass spec-
trometer (Foster City, CA, USA). Detection was accomplished by
injection of 10 lL of supernatant onto a Phenomenex Syner-
gi 2.1 · 50 mm Polar-RP 80 Å analytical column (Torrance,
CA, USA) with mobile phases of 0.1% formic acid in water
(mobile phase A) and acetontrile (mobile phase B) using a step
gradient (10% B for 0.4 min, to 90% B in 0.1 min) at a flow rate
of 0.6 mL ⁄ min. The effluent from the LC was introduced into the
API 4000 mass spectrometer via a turbo ionspray source. Mass
spectrophotometric analysis was performed in positive ion mode
using reaction monitoring at unit resolution for the transitions of
178.8 fi 101.0, 239.0 fi 126.0, 200.9 fi 115.1,
326.9 fi 291.2, 212.8 fi 183.1 and 343.0 fi 230.0 for
propofol, ketamine, medetomidine, midazolam, atipamezole, and
triazolam (internal standard), respectively.
CLint,app determination
In vitro intrinsic clearance (CLint,app) was determined using a
substrate depletion approach as described (Youdim et al., 2008),
but using 0.1 M potassium phosphate buffer (pH 7.4),
150 pmol ⁄ mL of rP450s or 0.5 mg ⁄ mL beagle liver microsomes,
and 1 mM sodium b-NADPH rather than a NADPH regenerating
system. Drugs were dissolved DMSO to a final stock concentra-
tion of 10 mM and further diluted in buffer to achieve a final
incubation of substrate at 1.0 lM. Liquid handling was con-
ducted on a Beckman Coulter Biomek 2000 (Fullerton, CA, USA)
with a water-filled aluminum plate holder maintained at 37 C.
Incubations (0.7 mL) were carried out in a ‘one-pot’ method in a
1.0 mL capacity 96-deep-well titer plate with aliquots removed
to a quench plate containing 25 lL of formic acid (25 %) with
500 ng ⁄ mL of triazolam at 0, 5, 10, 20, 30, and 60 min.
Midazolam and ketamine reactions used sampling times of 0, 1,
2, 5, 10, and 15 min because of their short half-lives, and
regression was only conducted with the first three time points
because of the drug concentrations dropping below 1% remain-
ing. The elimination rate constant and CYP or microsomal
protein content was then used to calculate CLint,app as
lL ⁄ min ⁄ pmol rP450 or lL ⁄ min ⁄ mg dog liver microsomes.
Testosterone was used as a positive control for liver microsome
activity.
Inter-system extrapolation factor activity correction
The specific activity of each rP450 preparation might vary
because of different concentrations of redox partner proteins (e.g.
NADPH P450 reductase), the lack of inter-isoform P450-P450
protein interactions, and different concentrations of non-P450
proteins that change the nonspecific protein binding of drugs. To
‘normalize’ an observed rP450 activity to those of liver
microsomes using the known hepatic expression concentration
of each CYP isoform, inter-system extrapolation factor (ISEF)
methods were developed to increase the accuracy of in vitro–
in vivo extrapolation of metabolic clearance (Proctor et al.,
2004). Proctor et al. (2004) originally described three levels of
52 M. T. Baratta et al.
rigor in which an ISEF can be determined. We have used a
simple substrate depletion CLint,app approach rather than V or
V ⁄ Km, and did not factor in nonspecific protein binding. The
primary goal was to ensure that the relative clearance values for
a drug across the panel of rP450s did not greatly over- or
underestimate the role of a particular rP450 isoform. Equation 1
was used to determine the CLint ISEF value for each rP450
isoform with each substrate.
CLint DLMðlL/min/mg DLMÞ
CLint ISEF ¼
CLint rCYPðlL/min/pmol CYPÞ  DLM CYP abundanceðpmol/mg DLMÞ
Inter-system extrapolation factor values were then multiplied by
the rP450 CLint values to arrive at the corrected activities. Liver
expression concentrations of the dog CYP were taken from
published findings where immunoquantitation of CYP1A,
CYP2B, CYP2C, and CYP3A estimated liver concentrations of
17, 48, 160, and 69 pmol ⁄ mg, respectively (Eguchi et al.,
1996), and 160 pmol ⁄ mg for CYP2D (Sakamoto et al., 1995).
Therefore, it has been assumed that the average CYP expression
concentrations from pooled beagle liver microsomes are similar
between various sources of laboratory beagles even though the
expression variability has not been thoroughly characterized to
our knowledge. ISEF values bracketed unity with a range of
0.77–8.2.
Spectrophotometric studies
Spectral binding titration studies were carried out with CYP2B11
Bactosomes (1 lM P450) in 100 mM potassium phosphate buffer
pH 7.4. Incubation mixtures were evenly divided into 1.5 mL
10 mm black-walled cuvettes, and experiments were performed
at room temperature by titrating in 1 lL aliquots of Atipamezole
(DMSO solution) into a sample cuvette (0.02–20 lM), with an
equal aliquot of DMSO in the reference cuvette (DMSO < 1%
v ⁄ v). After each titration, cuvettes were mixed and allowed to sit
for 1 min to allow the samples to reach equilibrium. Difference
spectra were recorded between 350 and 500 nm using a Hitachi
(Danbury, CT, USA) U3300 dual-beam spectrophotometer and
data were analyzed using UV SOLUTIONS 1.2 software (Hitachi).
The difference in absorbance between the peak (430 nm) and
trough (390 nm) for type II spectra were then plotted vs.
titrant concentration and fit to a hyperbolic curve function for
estimation of Ks values using GRAPH PAD PRISM 4.0 software
(San Diego, CA, USA).
RESULTS AND DISCUSSION
As P450s are often involved in the clearance of drugs,
characterizing the specific P450 enzyme(s) that metabolize or
are inhibited by co-administered drugs is critical in assessing the
risk of pharmacokinetic drug interaction. The use of rP450s is
now routine in human drug metabolism phenotyping (Zhang
et al., 2007) and drug interaction screening (Trubetskoy et al.,
2005), but canine recombinant CYP1A, 2B, and 2D isoforms
were not previously available for veterinary use. Furthermore,
few canine isoform-specific inhibitors, which could be used to
characterize drug metabolism in liver microsomes, have been
defined. This led us to report on the cloning and preparation of
several canine rP450s (Locuson et al., 2009).
ð1Þ
Recently, in-house rP450 screening identified the alpha-2
adrenergic receptor agonist medetomidine as a particularly
potent CYP2B11 inhibitor of temazepam N-demethylation
(Table 1). The structurally similar reversal agent atipamezole
often used to antagonize the effects of medetomidine also
inhibited CYP2B11 much more than the other canine rP450s.
This was not unexpected as submicromolar IC50s for medetom-
idine were reported previously in human liver microsomes
(Kharasch et al., 1992), but the use of rP450s definitively
identified the P450 isoform inhibited in dogs. Both medetomidine
and atipamezole were found to induce type-II difference binding
spectra with rCYP2B11 (data not shown). This suggested that
these compounds likely derive some of their high affinity from
the imidazole group coordinating as the sixth ligand to the heme
prosthetic group (Jefcoate, 1978).
Many sedation ⁄ anesthetic agents like medetomidine are
almost always used in combination to provide balanced anes-
thesia. Therefore, the in vitro clearance of commonly employed
anesthetics was analyzed with rP450s to determine whether
medetomidine might inhibit their metabolism. Midazolam,
propofol, and ketamine were chosen because they are used
extensively in veterinary medicine (Hellebrekers & Sap, 1997;
Kojima et al., 2002; Seliskar et al., 2007), and several studies
have examined the effects of combining these anesthetics on
cardiac output, respiration, blood gases, and other physiological
parameters [reviewed by (Cullen (1996)]. Furthermore, there is
existing evidence that propofol (Hay Kraus et al., 2000) and
ketamine (Hijazi & Boulieu, 2002) are partly metabolized by
canine or human microsomal CYP2B isoforms and this allowed
the opportunity to compare these findings with canine rP450s.
Most of the drugs tested were substrates of canine CYP 2B11,
2C21, 2D15, and 3A12 to varying degrees. This suggestion is
based on the substrate depletion experiments conducted at a low
substrate concentration to mimic conditions of linear pharma-
cokinetics (Table 2). However, medetomidine, atipamezole, and
propofol were turned over much slower than midazolam and
ketamine with rP450s and dog liver microsomes. This is
consistent with other pathways like glucuronidation contribut-
ing to the clearance of medetomidine (Kaivosaari et al., 2002)
and propofol (Soars et al., 2001; Favetta et al., 2002). Propofol
4-hydroxylation by dog liver microsomes is likely to be carried
out by CYP2B11 (Hay Kraus et al., 2000), yet it is not clear why
 2009 Blackwell Publishing Ltd
 Canine CYP2B11 metabolizes anesthetic agents 53

Table 1. Inhibition of canine rP450 isoforms

Enzyme IC50 (lM)*
by medetomidine and atipamezole
Inhibitor CYP1A1 CYP2B11 CYP2C21 CYP2D15 CYP3A12

rac-Medetomidine 1.1 ± 0.2 0.0048 ± 0.0006 22 ± 4 1.1 ± 0.3 15 ± 4

S-Medetomidine ND 0.034 ± 0.003 ND ND ND
Atipamezole 0.60 ± 0.09 0.0072 ± 0.0013 18 ± 5 0.089 ± 0.014 5.5 ± 1.9

ND, not determined. *Values determined by fitting a titration of twelve inhibitor concentrations
conducted in triplicate to a sigmoidal function; ± values represent the standard error of the fit.

Table 2. Turnover of anesthetics by canine rP450s and liver microsomes

In vitro CLint,app (lL ⁄ min ⁄ pmol, rP450) or (lL ⁄ min ⁄ mg, microsomes)*

Substrate CYP1A1 CYP2B11 CYP2C21 CYP2D15 CYP3A12 DLM

Midazolam ND 2.72 1.08 ND ND 1066

rac-Ketamine ND 26.7 1.24 ND 16.5 1009
Propofol ND ND ND ND ND ND
Atipamezole ND 0.082 0.123 0.141 ND 61.1
S-medetomidine ND 0.047 0.091 0.200 ND 65.5
R-medetomidine ND 0.628 0.262 0.968 ND 104

Half-lives were not extrapolated more than two-fold greater than the value of the last incubation time point of 15 or 60 min (<0.15 lL ⁄ min ⁄ pmol and
<0.039 lL ⁄ min ⁄ pmol). DLM, dog liver microsomes; ND, not detected. *Calculated from a mean elimination rate constant as described in Materials and
methods.

propofol clearance was not demonstrated in our studies since the
rP450s and liver microsomes were active with other substrates.
Low rates of metabolism can prevent measuring substrate
disappearance because of the difficulty in monitoring small
changes in large mass spectrometry signals (e.g. drug concen-
trations near 1 lM). It is speculated that had we monitored
formation of hydroxy-propofol it would have been detected, but
upon calculating rates, would lead to the same result of low
metabolic conversion. In case the rP450 clearances differed from
microsome clearances because of varying redox partner protein
concentrations and P450–P450 protein interactions, the
CLint,app ISEF method was applied (Proctor et al., 2004).
According to both corrected and uncorrected in vitro clearance
values, medetomidine and atipamezole behaved as CYP2B11
inhibitors because of their high affinity and low turnover.
Conversely, midazolam and ketamine were excellent CYP2B11
substrates according to high turnover rates (Tables 2 and 3).
The above in vitro findings suggest medetomidine and atipa-
mezole might alter the pharmacokinetics of co-administered
midazolam and ketamine via inhibition of CYP2B11. A significant
portion of midazolam and ketamine clearance is determined by
P450 metabolism (Kaka & Hayton, 1980; Thummel et al., 1996).
However, the authors are not aware of any in vivo studies that have
examined plasma concentrations of midazolam and ketamine
when dogs are co-administered with medetomidine or atipamez-
ole. Studies have documented dose-sparing effects of medetomi-
dine premedication on propofol and ketamine (as maintenance
agents; Cullen, 1996), and with inhalation anesthetics (Bloor
et al., 1992; Gomez-Villamandos et al., 2008). In these cases, the
reduced general anesthesia requirements have previously been
attributed to the intended pharmacology of medetomidine or
changes in pharmacokinetics that are unrelated to metabolism.
Despite the high affinity of medetomidine and atipamezole for
CYP2B11, there are also several factors that are likely to
minimize their potential for pharmacokinetic interactions with
benzodiazepines and ketamine. One factor that likely reduces
pharmacokinetic interactions is simply the careful study of
anesthesia regimens. Doses of anesthetics given i.v. or by
inhalation are dosed to achieve the desired pharmacodynamic
effect. This ensures any unpredicted concentration of respiratory,
cardiovascular, or central nervous system depression achieved
with varying combinations of premedication and induction
agents is not exacerbated by overdosing the maintenance agent.
Medetomidine is also extremely potent and requires only low
lg ⁄ kg doses, and all of the sedatives ⁄ anesthetics discussed here
have short half-lives minimizing the time over which P450-
mediated interactions can occur in vivo. Finally, CYP3A12 and
2C21 would be expected to compensate for ketamine and ⁄ or
midazolam clearance in the event of CYP2B11 inhibition in vivo
(Tables 2 and 3).
It is worth noting that the active isomer dexmedetomidine
(S-isomer) provides the same efficacy as medetomidine at half the
dose (Gomez-Villamandos et al., 2006; Granholm et al., 2007).
Since levomedetomidine (R-isomer) is largely devoid of alpha-2
adrenergic receptor activity (MacDonald et al., 1991; Kuusela
et al., 2001), the use of dexmedetomidine (Dexdomitor, Orion
Pharma, Espoo, Finland) would be expected to further decrease
the risk for CYP2B11 inhibition in vivo vs. medetomidine.
Dexmedetomidine was even measured to be a weaker inhibitor
than medetomidine in agreement with findings in human liver
microsomes where levomedetomidine was slightly more potent

 2009 Blackwell Publishing Ltd

54 M. T. Baratta et al.
Corrected in vitro CLint,app (lL ⁄ min ⁄ pmol)*
Substrate CYP1A1 CYP2B11 CYP2C21
Midazolam NC 22.2 6.66
rac-Ketamine NC 20.6 6.19
Propofol NC NC NC
Atipamezole NC 1.27 0.382
S-medetomidine NC 1.36 0.409
R-medetomidine NC 2.17 0.650
*Activities from Table 2 were corrected to provide clearance estimates that more closely mimic those
of liver microsomes using published expression concentrations for each CYP isoform. Not calculated
because of lack of detectable turnover.
than dexmedetomidine (Kharasch et al., 1992). Since our source
of medetomidine was the formulated product, we cannot rule out
an added inhibition effect of paraben formulation stabilizers. We
would point out, though, that the medetomidine formulation is
primarily aqueous and it is estimated that dilutions required for
IC50 analysis would result in paraben concentrations below
where inhibition effects would be expected (<1.0 lM) (van Gelder
et al., 2002). As for atipamezole, its interaction with other
anesthetics could theoretically slow patient recovery time, but
this has not been studied.
Although CYP2B11 has been implicated in propofol clearance
in dogs (Hay Kraus et al., 2000), liver microsomes and rP450s
suggested it is low. However, in this case, there is some evidence
that a pharmacokinetic interaction does not occur during co-
administration of propofol and medetomidine. One study has
demonstrated that the propofol half-life was actually reduced
when co-administered with medetomidine vs. a propofol-only
treatment because of a change in volume of distribution and no
change in plasma clearance (Hall et al., 1994). As expected, total
plasma propofol concentrations were lower in dogs receiving
both drugs as less propofol is required to maintain anesthesia
with medetomidine premedication. By this account, the potential
for a propofol–medetomidine interaction because of pharmaco-
kinetics is low.
In summary, in vitro studies with a panel of canine rP450s
suggest medetomidine and atipamezole are CYP2B11 inhibitors
and that ketamine and midazolam are CYP2B11 substrates.
Commonly used doses of medetomidine in dogs do result in
plasma drug concentrations near the CYP2B11 IC50 value, but
the exact-free hepatic medetomidine concentrations ‘seen’ by
P450s in vivo are difficult to estimate. Therefore, further
investigation is required to determine whether drug interactions
involving some anesthestics used for companion animal sedation
occur with CYP2B11, which then translate to altered pharma-
codynamics. In anesthesia, such a result could be viewed as a
double-edged sword – the dose-sparing effect can often improve
patient outcome, but may simultaneously increase the risk for
drug interactions when anesthetics or other surgical drugs are
combined without any knowledge of their drug interaction
potential. However, the dose-to-effect practice of anesthesia,
overlapping pharmacological activities of anesthetics, and
altered drug volume of distribution (Buhrer et al., 1994) are
currently the reported explanations for the dose-sparing effects of
Table 3. ISEF-corrected rP450 isoform activ-
ities
CYP2D15 CYP3A12
NC NC
NC 14.3
NC NC
0.382 NC
0.409 NC
0.650 NC
medetomidine that are expected to minimize metabolic
interactions.
ACKNOWLEDGMENTS
The authors thank Dr Scott Brown for support of this research,
Dr David Martin for providing anesthesia expertise, and Dr
Kuresh Youdim, Paul Guimond, and C. Locuson, CRNA for
helpful discussions. We also thank Dr Dawn Merritt and Drs M
Rinkinen, J Salonen, B McKusick, M Huhtinen, H Kaukinen, R
Virtanen, and J Aspegren at Orion Pharma for reviewing the
manuscript, Richard Nicholas for help with literature searches,
and Dr Steven Nederveld for providing anesthetics.
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