1521-009X/12/4009-1825–1833$25.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
DMD 40:1825–1833, 2012
Vol. 40, No. 9
46508/3790668
Characterization of SAGE Mdr1a (P-gp), Bcrp, and Mrp2 Knockout
Rats Using Loperamide, Paclitaxel, Sulfasalazine, and
Carboxydichlorofluorescein Pharmacokinetics
Maciej J. Zamek-Gliszczynski, David W. Bedwell, Jing Q. Bao, and J. William Higgins
Drug Disposition, Lilly Research Laboratories, Indianapolis, Indiana
Received April 27, 2012; accepted June 18, 2012
ABSTRACT:
in Mdr1a knockout rats. Sulfasalazine oral bioavailability was
markedly increased 21-fold in Bcrp knockouts and, as expected,
was also 2- to 3-fold higher in P-gp and Mrp2 knockout rats. The
sulfapyridine metabolite/parent ratio was decreased 10-fold in rats
lacking Bcrp after oral, but not intravenous, sulfasalazine administration. Carboxydichlorofluorescein biliary excretion was obliterated in Mrp2 knockout rats, resulting in 25% decreased systemic
clearance and 35% increased half-life. In contrast, carboxydichlorofluorescein renal clearance was not impaired in the absence of
Mrp2, Bcrp, or P-gp. In conclusion, SAGE Mdr1a, Bcrp, and Mrp2
knockout rats generally demonstrated the expected phenotypes
with respect to alterations in pharmacokinetics of relevant probe
substrates; therefore, these knockout rats can be used as an
alternative to murine models whenever a larger species is practically advantageous or more relevant to the drug discovery/development program.
Introduction
studies. Rats are far easier than mice to manipulate surgically, their
total blood volume is sufficient for thorough characterization of
pharmacokinetics within a single animal, and they also yield adequate
amounts of urine and bile for routine drug excretion studies using
metabolism cages (Davies and Morris, 1993). The recent development
of zinc finger nuclease technology enabled rapid generation of targeted gene knockout rats (Geurts et al., 2009). Mdr1a zinc finger
nucleases were used to develop a proprietary P-gp knockout Wistar
rat, which exhibited the expected alterations in the pharmacokinetics
and CNS distribution of substrate drugs (Chu et al., 2012). Mdr1a,
Bcrp, and Mrp2 knockout rats recently became commercially available in the Sprague-Dawley strain, which is also commonly used in
toxicology, pharmacokinetic, and excretion studies (Mdr1a, Bcrp, and
Mrp2 Knockout Rat Data Sheets, 2011; http://www.sageresearchmodels.
com).
Because of the relevance of rats to drug discovery and development, as well as experimental convenience over mice, commercially
available Mdr1a, Bcrp, and Mrp2 knockout rats are potentially important preclinical models. However, to date, their phenotypic characterization is largely lacking. In a recent study, SAGE Mdr1a knock-
Transporter knockout mice have been used extensively to study the role of
transporters in drug pharmacokinetics, distribution, and excretion (Klaassen
and Lu, 2008). Murine models are well suited for the study of drug distribution into key organs; for example, the importance of P-gp in limiting
blood-brain barrier penetration of substrate drugs was first recognized after
Mdr1a knockout mice exhibited 87-fold greater CNS exposure to the P-gp
substrate ivermectin, resulting in 100-fold enhanced neurotoxicity (Schinkel
et al., 1994). Although mice are practical for routine measurement of drug
distribution typically at a single time point at steady state, they are not optimal
for sample-intensive determination of a pharmacokinetic time course or for
the study of drug excretion because of their small size and limited volume of
relevant bodily fluids (Davies and Morris, 1993). Thus, a larger species, such
as rat, is preferred for the conduct of pharmacokinetic and excretion studies
in the drug discovery and development setting.
Rats are the most common rodent toxicology and pharmacokinetic
species and are also commonly used in preclinical pharmacology
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
http://dx.doi.org/10.1124/dmd.112.046508.
ABBREVIATIONS: P-gp, P-glycoprotein; Mdr, multidrug resistance; CNS, central nervous system; Bcrp, breast cancer resistance protein; Mrp,
multidrug resistance-associated protein; LC, liquid chromatography; MS/MS, tandem mass spectrometry; Oatp/OATP, organic anion-transporting
polypeptide.
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Transporter gene knockout rats are practically advantageous over
murine models for pharmacokinetic and excretion studies, but
their phenotypic characterization is lacking. At present, relevant
aspects of pharmacokinetics, metabolism, distribution, and excretion of transporter probes [P-glycoprotein (P-gp): loperamide and
paclitaxel; breast cancer resistance protein (Bcrp): sulfasalazine;
and multidrug resistance-associated protein 2 (Mrp2): carboxydichlorofluorescein] were studied systematically across SAGE P-gp,
Bcrp, and Mrp2 knockout rats. In Mdr1a knockout rats, loperamide
and paclitaxel oral bioavailability was 2- and 4-fold increased,
respectively, whereas clearance was significantly reduced (40–
42%), consistent with the expected 10- to 20-fold reduction in
paclitaxel excretion. N-Desmethyl-loperamide pharmacokinetics
were not altered in any of the three knockouts after oral loperamide. In rats lacking P-gp, paclitaxel brain partitioning was significantly increased (4-fold). This finding is consistent with observations of loperamide central nervous system opioid pharmacology
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ZAMEK-GLISZCZYNSKI ET AL.
Materials and Methods
Chemicals. Paclitaxel from Taxus yannanensis, loperamide, and 5-(and
6)-carboxy-2⬘,7⬘-dichlorofluorescein (mixed isomers) were purchased from
Sigma-Aldrich (St. Louis, MO). Sulfasalazine and sulfapyridine were purchased from MP Biomedicals (Solon, OH). N-Desmethyl-loperamide was
purchased from Toronto Research Chemicals (North York, ON, Canada). All
other chemicals were of reagent grade and were readily available from commercial sources.
Animals. Male Sprague-Dawley and Wistar wild-type rats (250 –350 g)
were purchased from Harlan (Indianapolis, IN). Male Sprague-Dawley Mdr1a,
Bcrp, and Mrp2 knockout rats were purchased from SAGE (St. Louis, MO).
Male Mrp2-deficient Wistar rats (TR⫺ rats) were purchased from RRRC
(Columbia, MO). Femoral artery and femoral vein cannulations were performed for blood sampling and intravenous dosing, respectively. In biliary
excretion studies, bile ducts were also cannulated. Surgeries on wild-type rats
were performed by Harlan, and gene knockout and TR⫺ rat surgeries were
performed by Covance, Inc. (Greenfield, IN). In all experiments, knockout rats
and relevant wild-type controls were matched for age and body weight. Rats
were allowed to recover for a minimum of 2 days after surgeries (for wild-type
rats, a minimum of 4 days was allowed because of additional postshipment
acclimation). The Institutional Animal Care and Use Committees at Covance
and Harlan approved all animal procedures.
Loperamide Pharmacokinetics. The plasma concentration time course
was determined over 24 h in an intravenous/oral crossover study with a 3-day
washout period between drug administration. On day 0, loperamide was admin-
istered by intravenous bolus injection (1 mg/kg; 2 ml/kg saline with 0.2 M
equivalents of HCl) and on day 3 by oral gavage (10 mg/kg; 10 ml/kg water with
0.2 M equivalents of HCl). Arterial blood samples were collected at the following
times after dose administration: 0 (oral arm only), 0.08 (intravenous arm only),
0.25, 0.5, 1, 2, 4, 8, 12, and 24 h, and the resulting plasma was analyzed for both
loperamide and N-desmethyl-loperamide metabolite.
Paclitaxel Pharmacokinetics. The plasma concentration time course was
determined over 24 h in an intravenous/oral crossover study with a 3-day
washout period between drug administration. On day 0, paclitaxel was administered by intravenous bolus injection (1 mg/kg; 2 ml/kg 50% Cremophor EL
in ethanol) and on day 3 by oral gavage (5 mg/kg; 10 ml/kg 50% Cremophor
EL in ethanol). Arterial blood samples were collected at the following times
after dose administration: 0 (oral arm only), 0.08 (intravenous arm only), 0.25,
0.5, 1, 2, 4, 8, 12, and 24 h. In a separate study after oral dosing of paclitaxel
(5 mg/kg; 10 ml/kg 50% Cremophor EL in ethanol), rats were sacrificed at 1 and
4 h to collect brain, liver, kidneys, and blood. In a separate study, after intravenous
bolus injection of paclitaxel (1 mg/kg; 2 ml/kg 50% Cremophor EL in ethanol) to
bile duct-cannulated rats housed in metabolism cages, urine, bile, and feces were
collected in toto for 0 to 24 h, cages were washed and fluid was collected for
analysis at 24 h, and arterial plasma was sampled at the following times after dose
administration: 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h.
Sulfasalazine Pharmacokinetics. The plasma concentration time course
was determined over 24 h in an intravenous/oral crossover study with a 3-day
washout period between drug administration. On day 0, sulfasalazine was
administered by intravenous bolus injection (5 mg/kg; 2 ml/kg water with pH
adjusted to 9 –10 with 5 N NaOH) and on day 3 by oral gavage (20 mg/kg; 10
ml/kg 0.5% carboxymethyl cellulose in phosphate-buffered saline, pH 7.4).
Arterial blood samples were collected at the following times after dose administration: 0 (oral arm only), 0.08 (intravenous arm only), 0.25, 0.5, 1, 2, 4,
8, 12, and 24 h, and the resulting plasma was analyzed for both sulfasalazine
and sulfapyridine metabolite.
Carboxydichlorofluorescein Pharmacokinetics. The plasma concentration time course was determined after intravenous bolus injection of carboxydichlorofluorescein (3 mg/kg; 2 ml/kg 50 mM phosphate buffer, pH 8).
Arterial blood samples were collected at the following times after dose administration: 2.5, 5, 7.5, 10, 15, 20, 30, 40, 50, 60, 75, 90, 120, 150, 180, and
240 min. Rats were housed in metabolic cages over the duration of the 4-h
study to enable in toto 0 to 4 h collection of urine and feces. In a separate study
after bolus injection of carboxydichlorofluorescein (3 mg/kg; 2 ml/kg 50 mM
phosphate buffer, pH 8) to bile duct-cannulated rats housed in metabolism
cages, urine, bile, and feces were collected in toto for 0 to 4 h, cages were
washed and fluid was collected for analysis at 4 h, and arterial plasma was
sampled at the following times after dose administration: 2.5, 5, 7.5, 10, 15, 20,
30, 40, 50, 60, 75, 90, 120, 150, 180, and 240 min.
Carboxydichlorofluorescein In Vitro Studies. Carboxydichlorofluorescein (1 ␮M) unbound fraction in rat plasma was determined by equilibrium
dialysis as described previously (Zamek-Gliszczynski et al., 2012). Metabolic
stability was assessed in mouse and rat liver microsomes and hepatocytes. In
brief, carboxydichlorofluorescein was incubated in microsome suspension (0.5
mg/ml microsomal protein; 2 ␮M carboxydichlorofluorescein concentration;
0.5 h) or cryopreserved hepatocyte suspension (0.5 million cells/ml; 2 ␮M
carboxydichlorofluorescein; 4-h time course) at 37°C. Incubations were
stopped by addition of an equal volume of acetonitrile; parent concentrations
were determined by LC-MS/MS, and, in addition, samples were profiled for
parent-related metabolite(s) using LC-MS/MS.
Bioanalysis. Loperamide, N-desmethyl-loperamide, paclitaxel, sulfasalazine, sulfapyridine, and carboxydichlorofluorescein concentrations in relevant
matrices (plasma, bile, urine, feces, cage wash, and tissue homogenates of
brain, kidney, or liver) were quantified by LC-MS/MS. All samples were
mixed with an organic internal standard solution to precipitate protein and
centrifuged, and the resulting supernatants were directly analyzed. Analytes
were separated using reverse-phase chromatography with gradient elution from
the following columns: for paclitaxel, Betasil C18 (2.1 ⫻ 20 mm, 5 ␮m;
Thermo Fisher Scientific, Waltham, MA); for loperamide, N-desmethyl-loperamide, and carboxydichlorofluorescein, Betasil Javelin C18 (2 ⫻ 20 mm, 5
␮m; Thermo Fisher Scientific); and for sulfasalazine and sulfapyridine, Atlantis T3 (2.1 ⫻ 50 mm, 5 ␮m; Waters, Milford, MA). All analytes were
detected in positive ion mode, except that carboxydichlorofluorescein was
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out rats demonstrated good agreement with an established murine
model in the extent of P-gp-limited CNS exposure of seven probe
substrates (Bundgaard et al., 2012); however, phenotypic validation of
these Mdr1a knockout rats in terms of P-gp-limited intestinal absorption, distribution in other tissues, and excretion remains to be demonstrated. Finally, functional characterization of SAGE Bcrp and
Mrp2 knockout rats is completely absent in the literature.
At present, relevant aspects of loperamide, paclitaxel, sulfasalazine,
and carboxydichlorofluorescein pharmacokinetics, metabolism, distribution, and excretion were systematically studied across SAGE
Mdr1a, Bcrp, and Mrp2 knockout rats. These studies not only examined the relevant knockout phenotype but also investigated potentially
unexpected compensatory changes. Loperamide and paclitaxel were
used as P-gp probe substrates. In Mdr1a knockout models, loperamide
intestinal permeability was increased 3.1-fold, CNS partitioning was
markedly increased 22- to 107-fold, and intravenous exposure was
1.3- to 2-fold higher (Adachi et al., 2003; Chu et al., 2012). Likewise,
absorption, distribution, and excretion of paclitaxel are affected by
P-gp; in Mdr1a knockout mice oral bioavailability was increased
3.1-fold, clearance was decreased 50%, consistent with decreased
excretion, and CNS distribution was on average increased 2-fold
(Sparreboom et al., 1997; Gallo et al., 2003). Sulfasalazine was used to
investigate Bcrp function, because it has low oral bioavailability limited
by intestinal apical efflux primarily via Bcrp (Zaher et al., 2006). In Bcrp
knockout mice, sulfasalazine bioavailability was increased 9-fold (4%
versus 37%); intestinal absorption was also modestly increased 1.6-fold
in P-gp knockout mice and 3-fold by inhibition of Mrp2 (Zaher et al.,
2006; Dahan and Amidon, 2009). Carboxydichlorofluorescein was used
as a probe for in vivo Mrp2 function, because it is metabolically stable
and Mrp2-mediated biliary excretion is a substantial contributor to overall
systemic clearance (Zamek-Gliszczynski et al., 2003; Nezasa et al.,
2006).
In the present studies, SAGE Mdr1a, Bcrp, and Mrp2 knockout rats
generally demonstrated the expected phenotypes with respect to alterations in pharmacokinetics of relevant probe substrates. The data overall
support the use of these knockout rats as an alternative to murine
models whenever a larger species is practically advantageous or
more relevant.
PK CHARACTERIZATION OF P-gp, Bcrp, AND Mrp2 KNOCKOUT RATS
recovered by the 6-h time point. Loperamide central effects were more
pronounced after oral administration. One to 2 h after dosing, Mdr1a
knockout rats were hypoactive, exhibited eye protrusion and lacrimation, as well as shallow breathing, and would respond only when
handled. One of the Mdr1a knockout animals was found dead at the
4-h time point, whereas all the others were in a stupor, and so all these
animals were discontinued from the study. No central opioid effects
were noted in wild-type, Bcrp and Mrp2 knockout rats after either
intravenous or oral dosing. These findings are difficult to explain
solely by the 2- to 4-fold increased loperamide exposures in Mdr1a
knockout rats, because no central effects were observed in P-gpcompetent rats receiving oral loperamide despite 6- to 8-fold higher
exposures compared to intravenous administration. Oral exposure in
P-gp-competent rats was up to 4.2-fold higher than intravenous exposure in Mdr1a knockouts, in which central opioid effects were
noted. The observed opioid pharmacology in Mdr1a knockout rats is
therefore consistent with enhanced CNS distribution of loperamide.
Paclitaxel pharmacokinetic and excretory parameters are summarized in Table 2, and brain/plasma concentration ratios are presented
in Fig. 2. Paclitaxel clearance was 40% lower in Mdr1a knockout rats.
On average, 6.2 ⫾ 2.1 and 0.35 ⫾ 0.21% of the paclitaxel intravenous
dose was recovered over 24 h in excreta of bile duct-cannulated
wild-type and Mdr1a knockout rats, respectively. Decreased excretion
of paclitaxel is conceptually consistent with the decreased systemic
clearance in Mdr1a knockout rats; however, the 24-h excreta collection period was too short relative to the 7.2- to 12-h half-life to be
representative of mass balance (Sparreboom et al., 1997). Oral absorption was more extensive in Mdr1a knockout rats with a 4- to
5-fold increase in bioavailability and Cmax. Paclitaxel brain partitioning was increased 4-fold in rats lacking P-gp, consistent with observations of loperamide central effects. Despite decreased biliary and
urinary recovery of paclitaxel, liver and kidney partitioning was not
altered significantly in Mdr1a knockout rats (Fig. 3). Brain, liver, and
Results
Pharmacokinetics of loperamide and its N-desmethyl metabolite are
summarized in Fig. 1 and Table 1. In Mdr1a knockout rats, loperamide clearance was on average 40% reduced, and oral absorption was
more extensive and protracted (2.2–2.7-fold increase in bioavailability, Cmax, and Tmax). N-Desmethyl-loperamide pharmacokinetics were
not significantly altered in any of the three knockouts after oral
loperamide. Mdr1a knockout rats exhibited hallmark opioid central
effects. After intravenous administration of loperamide, independent
veterinary staff noted that all animals corresponding to the Mdr1a
knockout group became lethargic but were alert and responsive and
A
B
Loperamide (ng/mL)
Loperamide (ng/mL)
100
10
1
100
10
1
0
2
4
6
0
8
4
8
16
20
24
D
N-Desmethyl-Loperamide (ng/mL)
C
Loperamide (ng/mL)
12
Time (h)
Time (h)
100
10
1
0
1
2
Time (h)
3
4
100
10
1
0
1
2
Time (h)
3
4
FIG. 1. Loperamide concentration-time profiles in wild-type (F), Mdr1a knockout (E),
Bcrp knockout (䡺), and Mrp2 knockout (‚)
Sprague-Dawley male rats after administration of a 1 mg/kg intravenous bolus dose (A)
or a 10 mg/kg oral dose (B). Parent (C) and
N-desmethyl metabolite (D) concentrationtime profiles after administration of a 10
mg/kg loperamide oral dose; plots C and D
are truncated at 4 h, when Mdr1a knockout
rats became completely unresponsive, and
blood sampling was stopped. Data are
means ⫾ S.D. n ⫽ 3 to 6.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
detected in negative ion mode, using selected reaction monitoring (Sciex API
4000 triple quadrupole mass spectrometer equipped with a TurboIonSpray
interface; Applied Biosystems/MDS, Foster City, CA): paclitaxel 854.3 3
286.3 m/z, loperamide 477.1 3 266.1 m/z, N-desmethyl-loperamide 463.2 3
252.2 m/z, sulfasalazine 399.1 3 119.0 m/z, sulfapyridine 250.1 3 65.1 m/z,
and carboxydichlorofluorescein 442.9 3 362.9 m/z. The dynamic range of the
assays was 1 to 5000 ng/ml in all matrices for all analytes, except for
carboxydichlorofluorescein (25–50,000 ng/ml). Samples with analyte concentrations above the upper limit of quantification were diluted with matrix to
within the assay range; concentrations below the lower limit of quantification
were reported as such. All bioanalytical assays met prespecified criteria for
accuracy (⬍⫾30% relative error) and precision (⬍30% relative S.D.).
Data Analysis. Noncompartmental pharmacokinetic parameters were calculated using Watson (version 7.4; Thermo Fisher Scientific). Renal clearance
values were calculated as the ratio of total amount excreted in urine and
systemic exposure. Excel 2007 (Microsoft, Redmond, WA) was used for all
statistical analyses. The Student’s two-tailed t test with the Bonferroni correction for multiple comparisons was used to assess statistical significance between knockouts and relevant wild-type controls. In cases in which variance
was different between compared groups (F-test, p ⬍ 0.05), the unequal
variance t test with the Bonferroni correction for multiple comparisons was
used. The minimal criterion for significance was p ⬍ 0.05. All data are
reported as means ⫾ S.D.
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ZAMEK-GLISZCZYNSKI ET AL.
TABLE 1
Loperamide and N-desmethyl-loperamide pharmacokinetic parameters
Data are means ⫾ S.D. n ⫽ 3 to 6.
Parameter
Wild Type
Mdr1a KO
Bcrp KO
Mrp2 KO
131 ⫾ 12
7.7 ⫾ 0.7
9.0 ⫾ 1.4
1.0 ⫾ 0.1
243 ⫾ 85
4.5 ⫾ 1.4*
7.9 ⫾ 0.9
1.5 ⫾ 0.5
161 ⫾ 40
6.6 ⫾ 1.8
11.5 ⫾ 3.9
1.5 ⫾ 0.1*
122 ⫾ 53
10.0 ⫾ 5.2
21.2 ⫾ 15.4
1.1 ⫾ 0.1
806 ⫾ 397
90 ⫾ 33
0.3 ⫾ 0.1
13 ⫾ 11
43 ⫾ 18
3.5-fold1a
226 ⫾ 81*
0.8 ⫾ 0.3*
N.A.
2.2-fold1*,b
980 ⫾ 542
69 ⫾ 45
0.7 ⫾ 0.7
15 ⫾ 7
40 ⫾ 15
1020 ⫾ 293
60 ⫾ 17
0.4 ⫾ 0.1
13 ⫾ 7
60 ⫾ 17
2.4 ⫾ 0.9
1875 ⫾ 932
158 ⫾ 59
1.6 ⫾ 1.4
13 ⫾ 13
55%2c
1.5-fold1d
193 ⫾ 49
2.2 ⫾ 1.3
N.A.
3.3 ⫾ 0.1
3256 ⫾ 1762
150 ⫾ 48
1.4 ⫾ 0.8
14 ⫾ 7
3.5 ⫾ 0.7
3658 ⫾ 1587
109 ⫾ 12
10 ⫾ 5
24 ⫾ 17
KO, knockout; AUCinf, area under the curve extrapolated to infinity; CL, clearance; VD, SS, volume of distribution; t1/2, half-life; Cmax, maximum concentration; Tmax, time to maximum
concentration; F, bioavailability; M/P, metabolite/parent ratio; N.A., not available.
* p ⬍ 0.05.
a
Mdr1a KO rat oral loperamide exposure was measured only to 4 h, when Mdr1a KO rats became completely unresponsive. AUC0 – 4 h was 651 ⫾ 249 versus 184 ⫾ 51 ng 䡠 h/ml in Mdr1a KO
versus wild-type rats.
b
Mdr1a KO rat bioavailability was estimated using oral and intravenous AUC0 – 4 h values and was 34 ⫾ 6 versus 16 ⫾ 5% in Mdr1a KO versus wild-type rats.
c
Mdr1a KO rat M/P ratio was estimated using AUC0 – 4 h values and was 1.0 ⫾ 0.2 versus 2.3 ⫾ 0.3 in Mdr1a KO versus wild-type rats.
d
Mdr1a KO rat N-desmethyl-loperamide exposure was measured only to 4 h when Mdr1a KO rats became completely unresponsive. AUC0 – 4 h was 635 ⫾ 151 versus 427 ⫾ 156 ng-eq 䡠 h/ml
in Mdr1a KO versus wild-type rats.
kidney partitioning of paclitaxel was not altered in Bcrp knockout rats, but,
surprisingly, liver partitioning was 61% decreased in rats lacking Mrp2.
Sulfasalazine and sulfapyridine metabolite pharmacokinetics are
summarized in Fig. 4 and Table 3. Oral bioavailability was 21-fold
higher in Bcrp knockout rats and was also modestly increased 2- to
3-fold in P-gp and Mrp2 knockout rats. The sulfapyridine metabolite/
parent ratio was decreased 10-fold in rats lacking Bcrp after oral, but
not intravenous, sulfasalazine. After intravenous administration, sulfasalazine exposure was 28% increased in Bcrp knockout rats despite
2.4-fold increased volume of distribution, consistent with the 25% decrease in clearance and 56% decrease in the sulfapyridine/parent ratio,
neither of which achieved statistical significance. In Mrp2 knockout rats,
the volume of distribution was increased 7.7-fold, resulting in decreased
intravenous exposure and longer half-life. To establish potential perturbations in systemic pharmacokinetics of sulfasalazine and sulfapyridine
in an established model lacking Mrp2, studies were also conducted in
Mrp2-deficient Wistar (TR⫺) rats (Fig. 5; Table 4). Although a number
of relatively modest, but statistically significant, differences were observed, the most notable change was observed in the volume of distribution, which was increased 3.4-fold in TR⫺ rats, resulting in lower sulfasalazine exposures and longer half-life.
Carboxydichlorofluorescein pharmacokinetic and excretory parameters are summarized in Table 5, and the corresponding concentrationtime profiles are presented in Fig. 6. Carboxydichlorofluorescein
biliary excretion was obliterated in Mrp2 knockout rats, resulting in
25% decreased systemic clearance and 32 to 35% increased exposure
and half-life. Surprisingly in both Bcrp and Mdr1a knockout rats,
carboxydichlorofluorescein exposure was decreased 28% due to significantly increased clearance, although dose recovery in excreta was
not affected. Carboxydichlorofluorescein renal clearance was on average 0.64 to 1.5 ml 䡠 min⫺1 䡠 kg⫺1, which is above the unbound
glomerular filtration rate of 0.39 ml 䡠 min⫺1 䡠 kg⫺1 (glomerular
TABLE 2
Paclitaxel pharmacokinetic and excretory parameters
Data are means ⫾ S.D. n ⫽ 5 to 9 (n ⫽ 6 to 12; bile duct-cannulated rats).
Parameter
Paclitaxel (1 mg/kg i.v.)
AUCinf, ng 䡠 h/ml
CL, l 䡠 h⫺1 䡠 kg⫺1
VD, SS, l/kg
t1/2, h
Paclitaxel (1 mg/kg i.v.; bile duct-cannulated rats)
Xurine0–24 h, % dose
Xbile0–24 h, % dose
Xfeces0–24 h, % dose
Paclitaxel (5 mg/kg p.o.)
AUCinf, ng 䡠 h/ml
Cmax, ng/ml
Tmax, h
t1/2, h
F, %
Wild Type
Mdr1a KO
Bcrp KO
Mrp2 KO
172 ⫾ 67
6.7 ⫾ 2.6
30.3 ⫾ 8.6
7.2 ⫾ 3.1
303 ⫾ 193
4.0 ⫾ 1.4*
38.1 ⫾ 12.7
12.2 ⫾ 5.4
171 ⫾ 40
6.1 ⫾ 1.5
29.1 ⫾ 9.7
6.4 ⫾ 2.2
375 ⫾ 211
9.1 ⫾ 16
28.6 ⫾ 18.0
8.1 ⫾ 3.4
3.1 ⫾ 1.6
0.5 ⫾ 0.5
2.3 ⫾ 1.3
0.14 ⫾ 0.06*
0.00 ⫾ 0.00*
0.21 ⫾ 0.17*
2.0 ⫾ 0.8
0.1 ⫾ 0.1
1.0 ⫾ 0.8
4.5 ⫾ 1.1
0.2 ⫾ 0.1
2.8 ⫾ 0.7
88 ⫾ 28
30 ⫾ 6
0.8 ⫾ 0.3
4.4 ⫾ 3.6
12 ⫾ 4
746 ⫾ 649*
145 ⫾ 77*
1.2 ⫾ 1.1
10.0 ⫾ 3.2*
47 ⫾ 21*
113 ⫾ 52
40 ⫾ 14
0.9 ⫾ 0.2
5.3 ⫾ 3.2
13 ⫾ 4
622 ⫾ 394
151 ⫾ 18*
0.9 ⫾ 1.6
9.4 ⫾ 4.0
27 ⫾ 9
KO, knockout; AUCinf, area under the curve extrapolated to infinity; CL, clearance; VD, SS, volume of distribution; t1/2, half-life; X, amount excreted; Cmax, maximum concentration; Tmax, time
to maximum concentration; F, bioavailability.
* p ⬍ 0.05.
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Loperamide (1 mg/kg i.v.)
AUCinf, ng 䡠 h/ml
CL, l 䡠 h⫺1 䡠 kg⫺1
VD, SS, l/kg
t1/2, h
Loperamide (10 mg/kg p.o.)
AUCinf, ng 䡠 h/ml
Cmax, ng/ml
Tmax, h
t1/2, h
F, %
N-Desmethyl-loperamide (10 mg/kg p.o. loperamide)
M/P
AUCinf, ng-eq 䡠 h/ml
Cmax, ng-eq/ml
Tmax, h
t1/2, h
PK CHARACTERIZATION OF P-gp, Bcrp, AND Mrp2 KNOCKOUT RATS
Paclitaxel Brain Kp
4
*
3
2
1
0
1h
4h
Time (h)
filtration rate ⫽ 5.24 ml 䡠 min⫺1 䡠 kg⫺1; plasma fraction unbound ⫽
7.4%), consistent with a contribution from an active secretory process
in addition to passive filtration (Davies and Morris, 1993). However,
carboxydichlorofluorescein urinary excretion was unchanged in all
knockouts, indicating that Mrp2, Bcrp, and P-gp are not involved in
the active secretory process. Carboxydichlorofluorescein metabolic
stability was confirmed in vitro using liver microsomes and hepatocytes, in which time-dependent loss of parent compound or formation
of metabolite(s) related to the parent structure were not observed.
Discussion
Independent characterization of SAGE transporter knockout rats
has not been reported to date, and validation is necessary before this
model can be applied to address novel questions. Unpublished preliminary studies conducted by the vendor were consistent with the
expected phenotypes, but these studies were very limited in scope.
Oral digoxin concentrations were increased one order of magnitude in
Mdr1a knockout rats; however, digoxin is not a specific P-gp probe
and is also transported by Oatp’s and Ost␣/␤, as well as undergoing
appreciable metabolism in rodents (Lage and Spratt, 1965; KullakUblick et al., 2001; van Montfoort et al., 2002; Seward et al., 2003;
Mdr1a Knockout Rat Data Sheet, 2011, http://www.sageresearchmodels.com). CNS distribution of dantrolene was enhanced 3.7-fold in Bcrp
knockout rats (Enokizono et al., 2008; Bcrp Knockout Rat Data Sheet,
2011, http://www.sageresearchmodels.com). Mrp2 knockout rats exhibited the expected hereditary conjugated hyperbilirubinemia; serum and
B
4
3
2
1
*
0
Mdr1a-KO
Bcrp-KO
Mrp2-KO
Paclitaxel Kidney Kp KO/WT Ratio
Paclitaxel Liver Kp KO/WT Ratio
A
urine total bilirubin levels were increased 86- and 50-fold, respectively
(Jansen et al., 1985; Mrp2 Knockout Rat Data Sheet, 2011, http://
www.sageresearchmodels.com). More recently, enhancement of CNS
distribution of seven P-gp substrates was compared between Mdr1a
knockout mice and SAGE rats, and generally good agreement was
observed between these two species (Bundgaard et al., 2012).
In the present study, relevant aspects of loperamide, paclitaxel,
sulfasalazine, and carboxydichlorofluorescein pharmacokinetics were
systematically studied across SAGE Mdr1a, Bcrp, and Mrp2 knockout
rats to confirm the relevant phenotype and investigate unexpected
compensatory changes. Loperamide and paclitaxel were used as P-gp
probes (Sparreboom et al., 1997; Kalvass et al., 2004). Although
loperamide transport interactions are limited to P-gp, paclitaxel is also
transported by OATP1B3 and OAT2; in addition MRP2, but not
MRP3 or MRP4, transported paclitaxel in vitro (Kool et al., 1999;
Zeng et al., 1999; Lee et al., 2000; Huisman et al., 2005; Kobayashi
et al., 2005; Smith et al., 2005). Sulfasalazine was used to investigate
potential changes in Bcrp activity, whereas carboxydichlorofluorescein was used as a probe for Mrp2 function (Zamek-Gliszczynski et
al., 2003; Zaher et al., 2006). Sulfasalazine undergoes extensive
intestinal Bcrp efflux but is also effluxed by P-gp and Mrp2 to a low
extent and is transported by OATP2B1 (Zaher et al., 2006; Dahan and
Amidon, 2009; Kusuhara et al., 2012). In addition to Mrp2, carboxydichlorofluorescein is also transported by Oatp1a1, Oatp1b2, and
Mrp3 (Zamek-Gliszczynski et al., 2003).
SAGE Mdr1a knockout rats demonstrated the expected changes in
loperamide and paclitaxel pharmacokinetics associated with the absence of P-gp in key drug disposition organs. Loperamide exposures
were increased due to a 42% reduction in clearance and 2.2-fold
increase in oral bioavailability. Clear opioid CNS effects were observed only in rats lacking P-gp. Overall, these findings are consistent
with alterations in loperamide pharmacokinetics in established P-gp
knockout models, which demonstrated 3.1-fold increased intestinal
permeability, 65-fold enhanced CNS partitioning, and 25 to 50%
reduction in systemic clearance (Adachi et al., 2003; Kalvass et al.,
2004; Chu et al., 2012). Likewise, paclitaxel exposures were increased
in Mdr1a knockout rats owing to 40% decreased clearance and a
4-fold increase in oral bioavailability, and brain partitioning was
increased 4-fold. These findings are generally consistent with paclitaxel disposition in established P-gp-deficient animal models, in
which oral bioavailability was increased 3.1-fold, clearance was decreased 50%, and CNS distribution was on average increased 2-fold
(Sparreboom et al., 1997; Gallo et al., 2003).
SAGE Bcrp knockout rats demonstrated the expected marked increase in sulfasalazine oral bioavailability (1.4% versus 30%), which
was in good agreement with the 4 to 37% increase reported in mice
(Zaher et al., 2006). The relatively modest 2- to 3-fold increase in
4
FIG. 3. A, wild-type (WT)/knockout (KO)
liver Kp ratio. Liver Kp was measured at 1
and 4 h after paclitaxel administration, but
Kp values were not significantly different
between these two time points and are therefore presented as a single value. B, wild-type/
knockout kidney Kp ratio at 1 h (f) and 4 h
(䡺) after paclitaxel intravenous administration. Kidney Kp was significantly different
between 1 and 4 h time points; thus, data are
shown separately. The dashed line of unity
represents no change from control. Data are
mean ⫾ S.D. n ⫽ 3 to 9. ⴱ, p ⬍ 0.05.
3
2
1
0
Mdr1a-KO
Bcrp-KO
Mrp2-KO
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
FIG. 2. Paclitaxel brain/plasma concentration ratio (Kp) in wild-type (f), Mdr1a
knockout (䡺), Bcrp knockout (s), and Mrp2 knockout (u) Sprague-Dawley male
rats. Data are means ⫾ S.D. n ⫽ 3 to 9. ⴱ, p ⬍ 0.05.
1829
1830
ZAMEK-GLISZCZYNSKI ET AL.
B
100000
10000
10000
1000
Sulfasalazine (ng/mL)
Sulfasalazine (ng/mL)
A
1000
100
10
1
0.1
100
10
1
0.1
0
2
4
6
8
10
12
0
2
4
Time (h)
6
8
10
12
Time (h)
C
D
10000
Sulfapyridine (ng/mL)
100
10
1
1000
100
10
1
0
4
8
12
16
20
24
0
4
8
Time (h)
12
16
20
24
Time (h)
sulfasalazine bioavailability observed in the Mdr1a and Mrp2 knockout rats is also consistent with previous studies (Zaher et al., 2006;
Dahan and Amidon, 2009). The 10-fold decrease in the sulfapyridine
metabolite/parent ratio after oral sulfasalazine administration to Bcrp
knockout rats is consistent with decreased exposure to intestinal
metabolism in the absence of Bcrp efflux (Zaher et al., 2006). Unlike
Bcrp knockout mice, in which sulfasalazine clearance was decreased
by one order of magnitude, the knockout rats demonstrated only a
25% decrease in clearance, which did not achieve statistical significance (Zaher et al., 2006). Although the discrepancy in the impact of
Bcrp ablation on sulfasalazine clearance is large, this drug is cleared
primarily by metabolism and secondly by urinary excretion via glo-
merular filtration (Azulfidine prescribing information; Pfizer, Inc.,
2011). Therefore, Bcrp knockout rats appear to better maintain sulfasalazine clearance than their murine counterpart.
Sulfasalazine and sulfapyridine pharmacokinetics in an established
Mrp2-deficient model are not reported in the literature and were
therefore investigated in the present study. Hepatic Bcrp expression
and function are decreased by one order of magnitude in TR⫺ rats, but
it is not known whether intestinal Bcrp is subject to the same downregulation (Yue et al., 2011). Of note, the TR⫺ rat did not exhibit
increased sulfasalazine bioavailability, indicating that unlike in the
liver, intestinal Bcrp activity is not decreased in these animals. As in
SAGE Mrp2 knockout rats, the largest effect on sulfasalazine phar-
TABLE 3
Sulfasalazine and sulfapyridine pharmacokinetic parameters in SAGE knockout rats
Data are means ⫾ S.D. n ⫽ 4 to 8.
Parameter
Sulfasalazine (5 mg/kg i.v.)
AUCinf, ng 䡠 h/ml
CL, ml 䡠 min⫺1 䡠 kg⫺1
VD, SS, ml/kg
t1/2, h
Sulfasalazine (20 mg/kg p.o.)
AUCinf, ng 䡠 h/ml
Cmax, ng/ml
Tmax, h
t1/2, h
F, %
Sulfapyridine (20 mg/kg sulfasalazine p.o.)
M/P
AUCinf, ng-eq 䡠 h/ml
Cmax, ng-eq/ml
Tmax, h
t1/2, h
Wild Type
Bcrp KO
Mdr1a KO
8401 ⫾ 1923
10.4 ⫾ 2.5
168 ⫾ 47
1.6 ⫾ 1.3
10,727 ⫾ 1088*
7.8 ⫾ 0.8
408 ⫾ 142*
1.4 ⫾ 0.5
6260 ⫾ 785
13.5 ⫾ 1.9
285 ⫾ 62*
1.0 ⫾ 0.3
4320 ⫾ 1772*
22.0 ⫾ 8.2
1298 ⫾ 499*
4.3 ⫾ 0.7*
562 ⫾ 247
185 ⫾ 74
0.25 ⫾ 0.00
2.6 ⫾ 1.2
1.4 ⫾ 0.7
13,104 ⫾ 6522*
3872 ⫾ 1600*
0.6 ⫾ 0.6
1.9 ⫾ 0.9
30 ⫾ 15*
711 ⫾ 338
213 ⫾ 65
0.3 ⫾ 0.1
2.3 ⫾ 1.1
2.8 ⫾ 1.2
635 ⫾ 181
320 ⫾ 392
0.25 ⫾ 0.00
3.8 ⫾ 1.1
4.0 ⫾ 1.7*
50 ⫾ 29
21,992 ⫾ 3398
4764 ⫾ 1777
5.0 ⫾ 1.9
2.3 ⫾ 0.8
4.6 ⫾ 10.2*
14,576 ⫾ 10,009
2843 ⫾ 2135
4.0 ⫾ 0.0
1.7 ⫾ 0.7
24 ⫾ 15
14,985 ⫾ 5891
2525 ⫾ 1218
7.0 ⫾ 2.0
1.3 ⫾ 0.5
Mrp2 KO
22 ⫾ 8
12,675 ⫾ 1198*
2499 ⫾ 368*
4.4 ⫾ 2.2
1.6 ⫾ 0.7
KO, knockout; AUCinf, area under the curve extrapolated to infinity; CL, clearance; VD, SS, volume of distribution; t1/2, half-life; Cmax, maximum concentration; Tmax, time to maximum
concentration; F, bioavailability; M/P, metabolite/parent ratio.
* p ⬍ 0.05.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
Sulfapyridine (ng/mL)
1000
FIG. 4. Sulfasalazine concentration-time
profiles in wild-type (F), Mdr1a KO (E),
Bcrp KO (䡺), and Mrp2 KO (‚) SpragueDawley male rats after administration of a
5 mg/kg intravenous bolus dose (A) or a
20 mg/kg oral dose (B). Corresponding
sulfapyridine concentration-time profiles
after intravenous (C) or oral (D) administration of sulfasalazine. Data are means ⫾
S.D. n ⫽ 4 to 8.
PK CHARACTERIZATION OF P-gp, Bcrp, AND Mrp2 KNOCKOUT RATS
A
B
10000
1000
Sulfasalazine (ng/mL)
Sulfasalazine (ng/mL)
100000
1000
100
10
1
100
10
1
0
2
4
6
8
10
12
0
2
4
Time (h)
6
8
10
12
Time (h)
C
D
10000
Sulfapyridine (ng/mL)
10
1
FIG. 5. Sulfasalazine concentration-time profiles in wild-type () and Mrp2-deficient TR⫺
(ƒ) Wistar male rats after administration of a 5
mg/kg intravenous bolus dose (A) or a 20
mg/kg oral dose (B). Corresponding sulfapyridine concentration-time profiles after intravenous (C) or oral (D) administration of sulfasalazine. Mean ⫾ S.D. n ⫽ 3.
1000
100
10
1
0
4
8
12
16
20
24
0
4
Time (h)
TABLE 4
Sulfasalazine and sulfapyridine pharmacokinetic parameters in Wistar TR⫺ rats
Data are means ⫾ S.D. n ⫽ 3.
Sulfasalazine (5 mg/kg i.v.)
AUCinf, ng 䡠 h/ml
CL, ml 䡠 min⫺1 䡠 kg⫺1
VD, SS, ml/kg
t1/2, h
Sulfasalazine (5 mg/kg p.o.)
AUCinf, ng 䡠 h/ml
Cmax, ng/ml
Tmax, h
t1/2, h
F, %
Sulfasalazine (20 mg/kg
sulfasalazine p.o.)
M/P
AUCinf, ng-eq 䡠 h/ml
Cmax, ng-eq/ml
Tmax, h
t1/2, h
12
16
20
24
Time (h)
macokinetics in TR⫺ rats was increased volume of distribution, leading to reduced drug concentrations. The increased volume is probably
the result of impaired cellular excretion of sulfasalazine in the absence
of Mrp2 and low hepatic Bcrp function (Yue et al., 2011).
Carboxydichlorofluorescein biliary excretion was obliterated in
Mrp2 knockout rats, leading to increased systemic exposure and
half-life due to reduced clearance. Carboxydichlorofluorescein is metabolically stable and Mrp2-mediated biliary excretion is a substantial
contributor to overall systemic clearance (Zamek-Gliszczynski et al.,
2003; Nezasa et al., 2006). Renal clearance was not reduced in Mrp2
Parameter
8
Wild-Type
Wistar Rat
TR⫺ Wistar Rat
12,300 ⫾ 1630
6.8 ⫾ 1.0
244 ⫾ 30
1.5 ⫾ 0.5
7730 ⫾ 1130*
10.9 ⫾ 1.5*
839 ⫾ 79*
4.0 ⫾ 0.3*
1810 ⫾ 215
490 ⫾ 108
0.5 ⫾ 0.0
2.1 ⫾ 0.2
3.8 ⫾ 0.9
462 ⫾ 19*
144 ⫾ 21*
0.25 ⫾ 0.00*
2.3 ⫾ 0.3
1.5 ⫾ 0.2*
16 ⫾ 1
29,403 ⫾ 3963
4315 ⫾ 320
4.0 ⫾ 0.0
2.3 ⫾ 0.2
34 ⫾ 3*
15,437 ⫾ 1080*
3244 ⫾ 139*
4.0 ⫾ 0.0
1.7 ⫾ 0.5
AUCinf, area under the curve extrapolated to infinity; CL, clearance; VD, SS, volume of
distribution; t1/2, half-life; Cmax, maximum concentration; Tmax, time to maximum concentration;
F, bioavailability; M/P, metabolite/parent ratio.
* p ⬍ 0.05.
and other knockout rats, indicating that these three transporters are not
involved in carboxydichlorofluorescein renal clearance.
The present characterization studies revealed a few phenotypic
changes not directly attributed to the knocked-out transporters; however, these compensatory alterations are not unprecedented. The 61%
decrease in paclitaxel liver partitioning in Mrp2 knockout rats may
have resulted from compensatory up-regulation of metabolism. Naturally occurring Mrp2-deficient rats exhibit an increase in several
hepatic clearance mechanisms, including certain glucuronidation, sulfation, and glutathione conjugation activities (Zamek-Gliszczynski et
al., 2006a,b). Although the increase in paclitaxel clearance in Mrp2
knockout rats did not attain statistical significance, a consistent trend
of increased clearance of all non-Mrp2 probe substrates was observed
in rats lacking Mrp2 (loperamide 30%, paclitaxel 36%, sulfasalazine
212%, and sulfasalazine 60% in TR⫺ rats). In the absence of Mrp2,
the liver is unable to excrete conjugated bile acids across the canalicular membrane, resulting in compensatory hepatic adjustments for
the conjugated hyperbilirubinemia (Dubin and Johnson, 1954; Jansen
et al., 1985; Hirohashi et al., 1998).
The observation of increased carboxydichlorofluorescein clearance
in Bcrp and Mdr1a knockout rats is also not unprecedented. Carboxydichlorofluorescein is metabolically stable, and its hepatic clearance
occurs solely by biliary excretion via Mrp2 (Zamek-Gliszczynski et
al., 2003; Nezasa et al., 2006). In Bcrp knockout mice, a significant
increase in carboxydichlorofluorescein biliary clearance was reported
previously (Nezasa et al., 2006). Several explanations for this observation were postulated, including up-regulation of Mrp2 and/or downregulation of hepatic basolateral efflux via Mrp3; however, hepatic
Mrp2 and Mrp3 expression in Bcrp knockout mice was not altered,
and so indirect transporter trafficking and/or stimulation/suppression
by an endogenous Bcrp substrate were proposed instead (Nezasa et
al., 2006). Overall, these unexpected findings in Bcrp and P-gp
knockout rats do not diminish their general utility because the primary
importance of P-gp and Bcrp is in limiting intestinal absorption of
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Sulfapyridine (ng/mL)
1000
100
1831
1832
ZAMEK-GLISZCZYNSKI ET AL.
TABLE 5
Carboxydichlorofluorescein pharmacokinetic and excretory parameters
Data are means ⫾ S.D. n ⫽ 4 to 6.
Parameter
Carboxydichlorofluorescein (3 mg/kg i.v.; bile duct-intact rats)
AUCinf, ␮g 䡠 h/ml
CL, ml 䡠 min⫺1 䡠 kg⫺1
VD, SS, ml/kg
t1/2, h
Carboxydichlorofluorescein (3 mg/kg i.v.; bile duct-cannulated rats)
Xurine0–4 h, % dosea
Xbile0–4 h, % dose
Total0–4 h, % dose
Xurine0–4 h, % dosea
Xbile0–4 h, % dose
Total0–4 h, % dose
Wild Type
Mrp2 KO
Bcrp KO
Mdr1a KO
31.6 ⫾ 3.9
1.6 ⫾ 0.2
170 ⫾ 8
1.4 ⫾ 0.2
41.8 ⫾ 4.5*
1.2 ⫾ 0.1*
190 ⫾ 20
1.9 ⫾ 0.2*
22.8 ⫾ 4.2*
2.3 ⫾ 0.5*
184 ⫾ 22
1.1 ⫾ 0.1
22.6 ⫾ 4.0*
2.3 ⫾ 0.3*
192 ⫾ 18
1.2 ⫾ 0.1
30 ⫾ 14
56 ⫾ 21
86 ⫾ 23
34 ⫾ 11
20 ⫾ 11
54 ⫾ 5
61 ⫾ 24
0 ⫾ 0*
61 ⫾ 24
N/A
N/A
N/A
52 ⫾ 15
45 ⫾ 18
97 ⫾ 19
N/A
N/A
N/A
N/A
N/A
N/A
32 ⫾ 20
18 ⫾ 18
50 ⫾ 23
KO, knockout; AUCinf, area under the curve extrapolated to infinity; CL, clearance; VD, SS, volume of distribution; t1/2, half-life; X, amount excreted; N/A, not applicable. .
* p ⬍ 0.05.
a
Amount excreted in urine also includes urine dried on the side of the metabolic cage, which was recovered in the cage wash at the end of the study.
Carboxydichlorofluorescein (ng/mL)
100000
10000
1000
0
1
2
3
4
Time (h)
FIG. 6. Carboxydichlorofluorescein concentration-time profiles in wild-type (F),
Mdr1a knockout (E), Bcrp knockout (䡺), and Mrp2 knockout (‚) Sprague-Dawley
male rats after administration of a 3 mg/kg intravenous bolus dose. Data are
means ⫾ S.D. n ⫽ 10 to 12.
Acknowledgments
Kenneth J. Ruterbories is acknowledged for the development of LC-MS/MS
methods. Karen E. Sprague is acknowledged for contributing the carboxydichlorofluorescein plasma protein binding values.
Authorship Contributions
Participated in research design: Zamek-Gliszczynski, Bedwell, Bao, and
Higgins.
Conducted experiments: Bedwell and Bao.
Performed data analysis: Zamek-Gliszczynski, Bedwell, and Bao.
Wrote or contributed to the writing of the manuscript: Zamek-Gliszczynski,
Bedwell, Bao, and Higgins.
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