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1521-009X/43/5/669–678$25.00 http://dx.doi.org/10.1124/dmd.114.062000
DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:669–678, May 2015
Copyright ª2015 by The American Society for Pharmacology and Experimental Therapeutics
Renal Tubular Secretion of Tanshinol: Molecular Mechanisms,
Impact on Its Systemic Exposure, and Propensity for Dose-Related
Nephrotoxicity and for Renal Herb-Drug Interactions
s
Weiwei Jia, Feifei Du, Xinwei Liu, Rongrong Jiang, Fang Xu, Junling Yang, Li Li, Fengqing Wang,
Olajide E. Olaleye, Jiajia Dong, and Chuan Li
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai (W.J., F.D., X.L., R.J., F.X., J.Y., L.L., F.W., O.E.O.,
J.D., C.L.); Graduate School, Tianjin University of Traditional Chinese Medicine, Tianjin (W.J.); Institute of Chinese Materia Medica,
China Academy of Chinese Medical Sciences, Beijing (C.L.), People’s Republic of China
Received November 7, 2014; accepted February 20, 2015
ABSTRACT
Tanshinol has desirable antianginal and pharmacokinetic properties
and is a key compound of Salvia miltiorrhiza roots (Danshen). It is
extensively cleared by renal excretion. This study was designed to
elucidate the mechanism underlying renal tubular secretion of tanshinol
and to compare different ways to manipulatesystemicexposuretothe
compound. Cellular uptake of tanshinol was mediated by human
organic anion transporter 1 (OAT1) (K
m
, 121 mM), OAT2 (859 mM), OAT3
(1888 mM), and OAT4 (1880 mM) and rat Oat1 (117 mM), Oat2 (1207 mM),
and Oat3 (1498 mM). Other renal transporters (human organic anion-
transporting polypeptide 4C1 [OATP4C1], organic cation transporter 2
[OCT2], carnitine/organic cation transporter 1 [OCTN1], multidrug and
toxin extrusion protein 1 [MATE1], MATE2-K, multidrug resistance-
associated protein 2 [MRP2], MRP4, and breast cancer resistance
protein [BCRP], and rat Oct1, Oct2, Octn1, Octn2, Mate1, Mrp2, Mrp4,
and Bcrp) showed either ambiguous ability to transport tanshinol or no
transport activity. Rats may be a useful model, to investigate the
contribution of the renal transporters on the systemic and renal
exposure to tanshinol. Probenecid-induced impairment of tubular
secretion resulted in a 3- to 5-fold increase in the rat plasma area
under the plasma concentration-time curve from 0 to infinity (AUC
0–‘
)
of tanshinol. Tanshinol exhibited linear plasma pharmacokinetic
properties over a large intravenous dose range (2–200 mg/kg) in rats.
Thedosageadjustmentcouldresult in increases in the plasma AUC
0–‘
of tanshinol of about 100-fold. Tanshinol exhibited very little dose-
related nephrotoxicity. In summary, renal tubular secretion of tanshinol
consists of uptake from blood, primarily by OAT1/Oat1, and the
subsequent luminal efflux into urine mainly by passive diffusion. Dosage
adjustment appears to be an efficient and safe way to manipulate
systemic exposure to tanshinol. Tanshinol shows low propensity to
cause renal transporter-mediated herb-drug interactions.
Introduction
Herbal medicines normally contain many constituents. It is hypothesized
that only a few constituents with favorable drug-like properties, rather than
all the constituents present, are responsible for the pharmacologic effects of
an herbal medicine (Lu et al., 2008). An herbal constituent can be defined
as drug-like if it possesses the desired pharmacologic potency, a wide
safety margin, appropriate pharmacokinetic (PK) properties, and adequate
content in the medicine dosed. Recent multicompound PK studies have
indicated that human subjects and laboratory animals are considerably
exposed to only a few constituents of an herbal medicine after dosing
(Lu et al., 2008; Liu et al., 2009; Li et al., 2012a; Chen et al., 2013; Cheng
et al., 2013; Hu et al., 2013; Jiang et al., 2015; Li et al., 2015). Such PK
studies provide information for pharmacologists regarding which herbal
compounds merit further evaluation. Follow-up evaluations of PK studies
should focus on the potentially important herbal compounds that exhibit
the desired pharmacologic properties and have considerable body exposure
after dosing. Understanding the molecular mechanisms underlying the
major elimination pathways of key herbal compounds is a goal of such
studies. This helps to identify the factors influencing the compound
concentration after dosing and to predict the potential for compound-related
herb–drug interactions.
This work was supported by grants from the National Natural Science Fund of
China for Distinguished Young Scholars [Grant 30925044], the National Science
and Technology Major Project of China “Key New Drug Creation and
Manufacturing Program”[Grant 2009ZX09304-002], and the National Basic
Research Program of China [Grant 2012CB518403].
Part of this work was previously presented as follows: Jia W-W et al. Renal organic
anion transporter 1 (Oat1) as a determinant of rat systemic exposure to tanshinol of
Salvia miltiorrhiza.Posterpresentationatthe2nd Annual Shanghai Symposium on
Chemical and Pharmaceutical Solutions through Analysis (CPSA); 2011 Apr 13–16;
Shanghai, People’s Republic of China.
dx.doi.org/10.1124/dmd.114.062000.
sThis article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUC
0–‘
, area under the plasma concentration-time curve
from zero to infinity; BCRP, breast cancer resistance protein; BUN, blood urea nitrogen; CL
int
, intrinsic clearance; CL
R
, renal clearance; CL
R-cr
, renal
clearance of endogenous creatinine; CL
R,c-u
, renal clearance by the cellular efflux into urine across the apical brush border membrane; CL
tot,p
, total
plasma clearance; C
5min
, concentration at 5 minutes after dosing; Cum.A
e-U
, cumulative amount excreted into urine; f
e-U
, fraction of dose excreted
into urine; f
u
, unbound fraction in plasma; GFR, glomerular filtration rate; HEK-293, human embryonic kidney cell line; IC
50
, half maximal inhibitory
concentration; K
m
, Michaelis constant; LC-MS/MS, liquid chromatography with tandem mass spectrometry; MATE, multidrug and toxin extrusion
protein; MC, mock cells; MRP, multidrug resistance-associated protein; OAT, organic anion transporter; OATP, organic anion-transporting
polypeptide; OCT, organic cation transporter; OCTN, carnitine/organic cation transporter; PK, pharmacokinetic; sCr, serum creatinine; t
1/2
,
elimination half-life; TC, transfected cells; V
max
, maximum velocity; V
SS
, apparent volume of distribution at steady state.
669
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Supplemental material to this article can be found at:
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Salvia miltiorrhiza roots (Danshen) are used extensively in the
treatment of angina pectoris in the People’s Republic of China (Zhou
et al., 2005; Cheng, 2007). Emerging antianginal therapies are facilitating
its long-term use as they appear to have a low incidence of side effects
(Jia et al., 2012). Danshen therapies are given orally or intravenously.
Polyphenols are believed to be a class of major pharmacologically rele-
vant constituents of Danshen. A PK study of cardiotonic pills, a Danshen-
based formulation, in human subjects and laboratory animals has
indicated that tanshinol was the only Danshen polyphenol that exhibited
considerable systemic exposure after dosing (Lu et al., 2008). The other
polyphenols, including salvianolic acids A, B, and D, rosmarinic acid,
lithospermic acid, and protocatechuic aldehyde, were either poorly
absorbed from the gastrointestinal tract or were extensively metabolized,
which resulted in their poor detection in plasma after dosing.
Tanshinol was found to be the most abundant Danshen polyphenol in
clinically important Danshen-based intravenous injections (details
pending publication elsewhere). In a recent PK study of DanHong
injections—a Danshen-based intravenous formulation in human
subjects and laboratory animals—tanshinol exhibited the most signif-
icant systemic exposure of the Danshen polyphenols after dosing
(Li et al., 2015). The preceding PK studies of the Danshen polyphenols
found after dosing with Danshen-based formulations suggest that
tanshinol deserves additional attention and more investigation.
Studies have shown that tanshinol exhibited vasodilatory properties,
elevated serum nitric oxide levels and action of endothelial nitric oxide
synthase, protected endothelial cells from homocysteine-induced injury
and H
2
O
2
-induced apoptosis, and exerted antioxidant effects (Chan
et al., 2004; Lam et al., 2007; Zhao et al., 2008; Yang et al., 2009; Wang
et al., 2013). Tanshinol also decreased blood pressure in spontaneously
hypertensive rats and attenuated cardiac hypertrophy, venular throm-
bosis, and methionine-induced hyperhomocysteinemia in rats (Cao
et al., 2009; Wang et al., 2009; Yang et al., 2010; Tang et al., 2011a,b).
The preceding pharmacologic studies were either cell- or isolated-tissue-
based, which provided effective concentrations of tanshinol, or whole
animal-based, which provided effective doses of tanshinol. The reported
effective concentrations are higher (3–100 times) than human maximum
plasma concentrations of tanshinol after dosing the Danhong injections
at clinical dose level; the reported effective doses in rats also exceed (5–
10 times) the rat dose of tanshinol derived from the clinical dose.
Because tanshinol is an antianginal and a major PK constituent in
Danshen-based therapies, matching its levels of systemic exposure after
dosing to its effective concentration for antianginal activities most likely
results in better translation of its pharmacologic properties to the overall
antianginal effect of Danshen therapy. This requires having a way to
manipulate the postdose concentration of tanshinol. Dosage adjustment
and drug combination are commonly used, either alone or in concert, to
changeadrug’s concentration in the blood. These may, however, raise
some safety concerns. A therapeutically useful method should be both
effective (large potential to increase concentration) and safe (such as
with very little or no dose-related toxicity). Elimination is often a major
determinant of drug concentration after dosing. Renal excretion is the
predominant route of elimination of tanshinol in humans and rats,
attributed mainly to active tubular secretion (Lu et al., 2008). Our study
was designed to elucidate the molecular mechanisms underlying the
renal tubular secretion of tanshinol, to compare different ways of
manipulating systemic exposure to tanshinol, and to predict possible
renal transporter-mediated herb-drug interactions related to tanshinol.
Materials and Methods
Tanshinol (sodiated form .98.0%) was obtained from the National Institutes for
Food and Drug Control (Beijing, People’s Republic of China). Para-aminohippuric
acid, prostaglandin F
2a
, estrone-3-sulfate, estradiol-17b-D-glucuronide,
tetraethylammonium, methotrexate, probenecid, cimetidine, verapamil, indometh-
acin, novobiocin, creatinine, puromycin, and cisplatin were obtained from Sigma-
Aldrich (St. Louis, MO). Inside-out membrane vesicles [5 mg protein/ml; prepared
from insect cells expressing human multidrug resistance-associated protein (MRP)
2, human MRP4, human breast cancer resistance protein (BCRP), rat Mrp2, rat
Mrp4, or rat Bcrp] were purchased from Genomembrane (Kanazawa, Japan).
Cellular Transport Assays. Human embryonic kidney 293 (HEK-293) cells
(American Type Culture Collection, Manassas, VA) were grown, at 37Cand5%
CO
2
,inDulbecco’s modified Eagle’s medium, which was fortified with 10% fetal
bovine serum, 1% minimal essential medium nonessential amino acids, and 1%
antibiotic-antimycotic solution. Full open reading frames of cDNA for human
organic anion transporter (OAT) 1, human OAT2, human OAT3, human OAT4,
human organic anion-transporting polypeptide (OATP) 4C1, human organic
cation transporter (OCT) 2, human carnitine/organic cation transporter (OCTN) 1,
human multidrug and toxin extrusion protein (MATE) 1, human MATE2-K, rat
Oat1, rat Oat2, rat Oat3, rat Oct1, rat Oct2, rat Octn1, rat Octn2, and rat Mate1
were synthesized and subcloned into pcDNA 3.1(+) expression vectors. The
inserts of the pcDNA 3.1(+)-transporter constructs were sequenced and aligned
according to the GenBank accession numbers NM_004790, NM_006672,
NM_004254, NM_018484, NM_180991, NM_003058, NM_003059, NM_018242,
NM_152908, NM_017224, NM_053537, NM_031332, NM_012697, NM_031584,
NM_022270, NM_019269, and NM_001014118, respectively (www.ncbi.nlm.
nih.gov/genbank/).
The pcDNA 3.1(+)-transporter constructs and the empty vector were in-
troduced separately into the HEK-293 cells with Lipofectamine 2000 transfection
reagent (Invitrogen, Carlsbad, CA). This produced transporter-expressing cells
and mock-transfected cells, respectively. Before being used, the transfected cells
were validated functionally using positive substrates para-aminohippuric acid (for
OAT1 and Oat1), prostaglandin F
2a
(OAT2 and Oat2), estrone-3-sulfate (OAT3,
OAT4, OATP4C1, and Oat3), and tetraethylammonium (OCT2, OCTN1,
MATE1, MATE2-K, Oct1, Oct2, Octn1, Octn1, and Mate1) and using positive
inhibitors probenecid (for OAT1, OAT2, OAT3, OAT4, OATP4C1, Oat1, Oat2,
and Oat3), cimetidine (OCT2, MATE1, MATE2-K, Oct1, Oct2, and Mate1), and
verapamil (OCTN1, Octn1, and Octn2).
Transport studies were performed in 24-well poly-D-lysine-coated plates with
cells 48 hours after transfection. After they were washed twice with Krebs-
Henseleit buffer (containing 118 mM NaCl, 4.83 mM KCl, 1.53 mM CaCl
2
,0.96
mM KH
2
PO
4
,23.8mMNaHCO
3
, 1.2 mM MgSO
4
, 5 mM glucose, and 12.5 mM
HEPES, pH 7.4; 500 ml per wash; the second wash involving 10 minutes of
preincubation at 37C, pH 7.4), the transfected cells and the mock-transfected
cells were incubated with tanshinol in the presence or absence of the positive
inhibitor. After incubation for 10 minutes, the transport was terminated by
removing the medium from the wells and rapidly rinsing the cells 3 times with
ice-cold Krebs-Henseleit buffer (500 ml per rinse). Unlike the other transporters,
the transport studies with human MATE and rat Mate used a buffer (containing
145mMNaCl,3mMKCl,1mMCaCl
2
,0.5mMMgCl
2
, 5 mM glucose, 5 mM
HEPES,and30mMNH
4
Cl, pH 7.4) for cell washing and preincubation. The
buffer (removing 30 mM NH
4
Cl, pH 8.4) was used for incubation.
The cells were lysed with water (150 ml) using a freeze-thaw and ultra-
sonication. Aliquots (100 ml) of the resulting lysates were precipitated with ice-
cold acetonitrile (300 ml). After centrifugation at 21,100gfor 10 minutes, the
supernatants (5 ml) were analyzed by liquid chromatography with tandem mass
spectrometry (LC-MS/MS). The total amount of protein in the lysate was
measured using a method first described by Bradford (1976). The transport rate
in pmol/mg protein/min was calculated using the following equation:
Transport ¼ðCCVCÞ=T=WC
where C
C
is the concentration of the test compound in cellular lysates (mM), V
C
is the volume of the lysates (ml), Tis the incubation time (10 minutes), and W
C
is the cellular protein amount of each well (mg). The differential uptake
between the transfected cells (TC) and the mock cells (MC) was defined as the
net transport ratio (Transport
TC
/Transport
MC
ratio); a net transport ratio .3
suggested a positive result.
The kinetics of cellular uptake of tanshinol as mediated by human OAT1,
human OAT2, human OAT3, human OAT4, rat Oat1, rat Oat2, and rat Oat3
were assessed with respect to the Michaelis constant (K
m
), maximum velocity
(V
max
), and intrinsic clearance (CL
int
). The incubation conditions were the same
670 Jia et al.
at ASPET Journals on March 25, 2015dmd.aspetjournals.orgDownloaded from
as those for the preceding transport study except for using an incubation time of
5 minutes. The incubation time was optimized to ensure that the assessment
was performed under linear uptake conditions. The concentrations of tanshinol
in the incubation medium were 25–1600 mM for OAT1 and Oat1, and 156–
5000 mM for OAT2, OAT3, OAT4, Oat2, and Oat3. The background
accumulation of tanshinol was also determined in the mock-transfected cells.
The inhibitory effect of probenecid on the cellular uptake activity of tanshinol
as mediated by OAT1, OAT2, OAT3, OAT4, Oat1, Oat2, and Oat3 was
measured with respect to half-maximal inhibitory concentration (IC
50
); the
tanshinol concentrations were equal to the respective K
m
values for the
transporters. The IC
50
values of tanshinol against para-aminohippuric acid
(10 mM), prostaglandin F
2a
(10 mM), estrone-3-sulfate (10 mM), and estrone-3-
sulfate (10 mM) for OAT1, OAT2, OAT3, and OAT4, respectively, were also
measured.
Vesicular Transport Assays. Membrane vesicles expressing one of the
transporters—human MRP2, human MRP4, human BCRP, rat Mrp2, rat Mrp4,
or rat Bcrp—were tested with tanshinol using a rapid filtration method. Before
use, these membrane vesicles were functionally validated using estradiol-17b-D-
glucuronide and methotrexate. To start the transport, preincubated membrane
vesicle suspension (10 ml) was combined with preincubated tanshinol/ATP or
tanshinol/AMP medium (50 ml). After incubation for 10 minutes, the transport
wasterminatedbyadding200ml of an ice-cold buffer (containing 40 mM MOPS
and 70 mM KCl; adjusted to pH 7.0 with 1.7 M Tris-base) followed by
immediate transfer of the mixture into a Millipore MultiScreen-FB filtration plate
(0.65 mm; Billerica, MA). Five washes of the membrane vesicles with the ice-
cold terminating buffer (200 ml per wash) were performed, and the filters that
retained membrane vesicles were transferred to 1.5-ml polypropylene tubes. The
membrane vesicles were lysed and extracted with 200 ml of 80% methanol per
sample. After centrifugation at 21,100gfor 10 minutes, the supernatants (5 ml)
were analyzed by LC-MS/MS.
The transport rate in pmol/mg protein/min was calculated using the following
equation:
Transport ¼ðCVVVÞ=T=WV
where C
V
is the concentration of the compound in vesicular lysates superna-
tant (mM), V
V
isthevolumeofthelysates(ml), Tis the incubation time
(10 minutes), and W
V
is the amount of vesicle protein amount per well (0.05 mg).
Positive results for ATP-dependent transport were defined as a net transport
ratio (Transport
ATP
/Transport
AMP
ratio) .2.
Rat Pharmacokinetic Studies. The use and treatment of rats were in
compliance with the 2006 Guidance for Ethical Treatment of Laboratory Animals
(Ministry of Science and Technology of the People’s Republic of China). Three
rat PK studies were conducted, all according to protocols approved by the
Institutional Animal Care and Use Committee at the Shanghai Institute of Materia
Medica (Shanghai, People’s Republic of China). Male Sprague-Dawley rats
(230–270 g) were obtained from Sino-British SIPPR/BK Laboratory Animal
(Shanghai, People’s Republic of China). The femoral arteries were cannulated for
blood sampling, and the rats were allowed to regain their preoperative body
weight before use. The rats were euthanatized with CO
2
gas after use.
The aim of the first rat PK study was to determine the impact of tubular
secretion on systemic exposure to and renal excretion of tanshinol after i.v.
administration of tanshinol. Four rats were individually housed in rat metabolic
cages, and the urine collection tubes were kept at 220C. The study was conducted
in a two-period, two-sequence crossover fashion with a 2-day washout between
periods 1 and 2. In sequence 1, two of the rats received an i.v. bolus dose of
a tanshinol solution (0.4 mg tanshinol/ml) and an i.v. bolus dose of a probenecid-
tanshinol solution (0.4 mg tanshinol/ml + 20 mg probenecid/ml) at 5 ml/kg through
the tail veins in periods 1 and 2, respectively. In sequence 2, the other two rats
received these solutions in reverse order. Before and after each dosing, serial blood
samples (80 ml; 0, 5, 15, and 30 minutes and 1, 2, and 4 hours) were collected,
heparinized, and centrifuged to produce the plasma fractions. Urine samples were
also collected 0–4, 4–12, and 12–24 hours after dosing and weighed. The rat study
was repeated once.
The second rat PK study was designed to assess changes in the tissue dis-
tribution of tanshinol caused by the probenecid-induced impairment of tubular
secretion. Rats were randomized into two groups. They received an i.v. bolus
dose of the tanshinol solution (0.4 mg tanshinol/ml) at 5 ml/kg or an i.v. bolus
dose of the probenecid-tanshinol solution (0.4 mg tanshinol/ml + 20 mg
probenecid/ml) at 5 ml/kg through the tail veins. The rodents (under isoflurane
anesthesia) were killed by bleeding from the abdominal aorta at 0, 5, and
30 minutes and 1 and 2 hours after dosing (three rats per point in time). The collected
blood samples were heparinized and centrifuged to produce the plasma fractions.
Selected tissues, including the heart, lungs, brain, liver, and kidneys, were
excised, rinsed in ice-cold saline, blotted, weighed, and homogenized in 4-fold
volumes of ice-cold saline. The rat study was repeated once.
The third rat PK study was a single ascending dose study. It was performed to
determine the influence of dose on level of systemic exposure to and renal
disposition of tanshinol. Rats were randomly divided into five groups (12 rats per
group) and each group received an i.v. bolus dose of tanshinol solution (0.4, 1, 3,
10, or 40 mg tanshinol/ml) at 5 ml/kg through the tail veins. Three rats from each
group were randomly selected and received an i.v. dose of tanshinol at their group
dose level. Urine samples were collected 0–24 hours after dosing. After the urine
sampling was completed, the rats were returned to their group enclosures and
received a 2-day washout. Thereafter, all the rats in the different groups received
an i.v. dose of tanshinol at the designated level. Each group of rats was further
divided randomly, and the rats were killed under isoflurane anesthesia by
bleeding from the abdominal aorta at 5 and 30 minutes and 1 and 2 hours after
dosing (three rats per point in time). The blood samples were heparinized and
centrifuged to prepare plasma fractions. The kidneys and livers were excised,
rinsed, and homogenized. All the rat samples were stored at 270C until analysis.
According to dose normalization by body surface area (Reagan-Shaw et al.,
2008), the i.v. dose of tanshinol of 2 mg/kg given to rats was close to the clinical
daily dose of tanshinol from Danshen injection (a solution prepared from
Danshen extract available as a sterile, nonpyrogenic parenteral dosage form for i.
v. injection in the treatment of angina pectoris; People’s Republic of China Food
and Drug Administration ratification no. GuoYaoZhunZi-Z33020177). Accord-
ingly, the i.v. dose of tanshinol of 200 mg/kg given to rats was 100 times as much
as the clinical daily dose. Plasma PK and urine excretion profile of probenecid
were evaluated in rats receiving an i.v. bolus dose at 100 mg/kg.
Assessment of Tanshinol-Induced Nephrotoxicity. Rats were randomly
divided into four treatment groups (four or five rats per group) and given
subchronic doses of tanshinol (i.v. through the tail veins; 200 mg/kg/d), saline
(i.v. through the tail veins; 5 ml/kg/d), cisplatin (i.p. into the lower abdomen
above the left leg; 1 mg/kg/d), and puromycin (i.p.; 40 mg/kg/d) for 14
consecutive days. On day 15, the rats were anesthetized with isoflurane and
killed by bleeding from the abdominal aorta. The collected blood samples were
centrifuged to produce serum fractions for measurement of levels of blood urea
nitrogen (BUN), creatinine (sCr), alanine aminotransferase (ALT), and
aspartate aminotransferase (AST). The kidneys and livers were excised,
weighed, and placed in 10% neutral buffered formalin for histopathologic
evaluation. The tissues were fixed for over 24 hours, processed, and embedded
in paraffin. The embedded tissues were sectioned at 4–6mm and were stained
with H&E. The histopathologic examinations of the tissue sections were
conducted by a veterinary pathologist and subjected to peer review.
Quantification of Tanshinol and Other Test Compounds in Biologic
Samples. Validated LC/MS/MS-based bioanalytic methods were used to
measure the concentrations of tanshinol, para-aminohippuric acid, prostaglan-
din F
2a
, estrone-3-sulfate, estradiol-17b-D-glucuronide, methotrexate, tetrae-
thylammonium, probenecid, and creatinine in biologic samples. A TSQ
Quantum mass spectrometer (Thermo Fisher, San Jose, CA) was interfaced via
an electrospray ionization-probe with an Agilent 1100 series liquid chromato-
graph (Waldbronn, Germany). Chromatographic separation was achieved on
a5mm Gemini C
18
column (50 mm 2.0 mm i.d.; Phenomenex, Torrance,
CA). Tetraethylammonium and creatinine levels were analyzed using a 3-mm
Luna Hilic column (100 mm 3.0 mm i.d.; Phenomenex). The mobile phase,
which consisted of solvent A (water/acetonitrile, 98:2, v/v, containing 1 mM
formic acid) and solvent B (water/acetonitrile, 2:98, v/v, containing 1 mM
formic acid), was delivered at 0.35 ml/min, except for in the analysis of
creatinine at 0.7 ml/min. A pulse gradient elution method was used in the
measurement of the compounds (except for creatinine), with an analyte-
dependent start proportion (0–50% solvent B) and analyte-independent elution
proportion (100% solvent B), elution proportion segment (1.5 minutes), and
column equilibrium segment (3.5 minutes) (Wang et al., 2007); for creatinine,
the gradient parameters were 100% solvent B and 80% solvent B, 1 minute and
7 minutes, respectively.
Renal Tubular Secretion of Tanshinol 671
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The tandem mass spectrometry measurement was performed in the negative
ion mode with the precursor-product ion pairs for multiple-reaction-monitoring
of tanshinol, para-aminohippuric acid, prostaglandin F
2a
, estrone-3-sulfate,
estradiol-17b-D-glucuronide, and probenecid at m/z 197→135, 193→93,
353→193, 349→269, 447→271, and 281→140, respectively. Measurement
of tetraethylammonium, methotrexate, and creatinine in the positive ion mode
was performed at m/z130→86, 445→135, and 114→44, respectively. For
measurement of tanshinol in the rat samples, 2 M hydrochloride acid (5 ml)
was used to acidify the samples (15 ml) before extraction with ethyl acetate
(500 ml). For measurement of creatinine in the rat samples, methanol (40 ml) was
used to precipitate the samples (10 ml). Matrix-matched calibration curves were
constructed using weighted (1/X) linear regression of the analyte area (Y)against
the corresponding nominal analyte concentration (X,mM).
Data Processing. GraFit software (version 5; Surrey, United Kingdom) was
used to determine K
m
and V
max
by nonlinear regression analysis of initial
transport rates as a function of tanshinol concentration. The IC
50
for inhibition
of transport activity was obtained from a plot of percentage activity remaining
(relative to control) versus log
10
inhibitor concentration.
Plasma PK parameters were determined using noncompartmental analysis with
a Kinetica software package (version 5.0; Thermo Scientific, Philadelphia, PA).
The renal clearance (CL
R
) was calculated by dividing the cumulative amount
excreted into urine (Cum.A
e
) by the area under the plasma concentration-time
curve from 0 to infinity (AUC
0–‘
). The kidney clearance by the cellular efflux
into urine across the apical brush border membrane (CL
R,c-u
) was calculated by
dividing the Cum.A
e
by the kidney homogenate AUC
0–‘
(Imaoka et al., 2007).
The glomerular filtration rate (GFR) of the rats was estimated in terms of renal
clearance of endogenous creatinine (CL
R-cr
; Takahashi et al., 2007). Dose
proportionality was assessed using the regression of log-transformed data (the
Power model), with the criteria calculated according to a method by Smith et al.
(2000). All data are expressed as mean 6S.D. Statistical analysis was performed
with SPSS Statistics Software (version 19.0; IBM, Chicago, IL). P,0.05 was
considered to be the minimum level of statistical significance.
Results
In Vitro Interactions between Tanshinol and Human Renal
Transporters. There was significantly more uptake of tanshinol into
human OAT1-expressing HEK-293 cells than into the mock-transfected
cells, suggesting that tanshinol was a substrate of OAT1. Tanshinol was
also taken up by cells expressing human OAT2, OAT3, and OAT4. The
relevant net transport ratios are shown in Table 1. OAT1-mediated
uptake of tanshinol was saturable with K
m
,V
max
,andCL
int
values
shown in Table 2. OAT2, OAT3, and OAT4 exhibited lower affinity for
tanshinol than OAT1 (Table2). The uptake of tanshinol mediated by
OAT1, OAT2, OAT3, and OAT4 was considerably inhibited by
probenecid, and the IC
50
values are shown in Table 2. Human
OATP4C1, OCT2, OCTN1, MATE1, and MATE2-K did not exhibit
any transport activities for tanshinol (Table 1).
Human MRP2 and MRP4 had low in vitro transport activities for
tanshinol; reliable kinetic parameters were difficult to find. Human
BCRP had no statistically significant transport activity. Tanshinol
exhibited low inhibition potency toward the OAT transporters; its IC
50
values against para-aminohippuric acid for OAT1, against prosta-
glandin F
2a
for OAT2, against estrone-3-sulfate for OAT3, and
against estrone-3-sulfate for OAT4 are shown in Table 2. Tanshinol
(1 mM) did not exhibit any statistically significant inhibitory activity
toward OATP4C1, OCT2, OCTN1, MRP2, MRP4, BCRP, MATE1,
or MATE2-K (Table 1).
TABLE 1
Comparative net transport ratios for a variety of human and rat renal transporters mediating in vitro transport of tanshinol
The concentrations of methotrexate for BCRP and Bcrp were 100 mM. The concentration of probenecid to inhibit OAT2- and Oat2-mediated transport of prostaglandin F
2a
was 10 mM. Net
transport ratios represent the mean 6S.D. (n = 3). When the net transport ratio was .3 (for the solute carrier family transporters) or .2 (for the ABC transporters), there were statistically significant
differences between Transport
TC
and Transport
MC
or between Transport
ATP
and Transport
AMP
(P,0.05).
Transporter
Positive Substrate (10 mM) Tanshinol (100 mM)
Substrate Not Treated with
Any Inhibitor
Treated with 1 mM
Positive Inhibitor
Treated with 1 mM
Tanshinol as Inhibitor
Not Treated with
Any Inhibitor
Treated with 1 mM
Positive Inhibitor
Human renal SLC
transporters
OAT1 Para-aminohippuric acid 146.9 622.0 1.7 60.1 (Probenecid) 15.1 62.7 164.0 64.3 4.6 60.4 (Probenecid)
OAT2 Prostaglandin F
2a
68.0 64.6 7.6 60.8 (Probenecid) 23.9 61.6 80.7 63.1 6.7 60.7 (Probenecid)
OAT3 Estrone-3-sulfate 23.9 61.8 1.9 60.3 (Probenecid) 10.3 62.8 4.6 60.4 1.2 60.1 (Probenecid)
OAT4 Estrone-3-sulfate 14.1 61.0 3.0 60.6 (Probenecid) 8.3 61.5 7.3 61.5 0.9 60.3 (Probenecid)
OATP4C1 Estrone-3-sulfate 5.7 60.7 2.3 60.4 (Probenecid) 5.4 60.2 1.2 60.2 ―
OCT2 Tetraethylammonium 103.2 610.7 46.1 64.7 (Cimetidine) 105.7 68.9 1.4 60.2 ―
OCTN1 Tetraethylammonium 7.9 60.2 0.5 60.1 (Verapamil) 7.4 60.2 1.5 60.2 ―
MATE1 Tetraethylammonium 60.9 62.0 0.5 60.1 (Cimetidine) 63.2 61.0 0.8 60.2 ―
MATE2-K Tetraethylammonium 3.9 60.4 0.6 60.1 (Cimetidine) 5.3 60.6 1.9 60.6 ―
Human renal ABC
transporters
MRP2 Estradiol-17b-D-glucuronide 24.2 60.5 3.7 60.1 (indomethacin) 17.1 60.5 2.3 60.4 ―
MRP4 Estradiol-17b-D-glucuronide 20.2 60.7 4.4 60.8 (indomethacin) 15.9 60.3 1.7 60.2 ―
BCRP Methotrexate 17.7 60.1 4.1 60.3 (novobiocin) 17.9 60.3 1.0 60.3 ―
Rat renal SLC
transporters
Oat1 Para-aminohippuric acid 58.8 64.1 1.7 60.1 (Probenecid) 7.8 60.9 198.2 622.0 3.4 60.1 (Probenecid)
Oat2 Prostaglandin F
2a
22.1 61.6 1.8 60.4 (Probenecid) 20.0 60.7 17.9 63.8 2.5 60.7 (Probenecid)
Oat3 Estrone-3-sulfate 27.0 67.3 1.3 60.6 (Probenecid) 9.7 61.9 5.0 60.3 0.9 60.2 (Probenecid)
Oct1 Tetraethylammonium 102.1 610.5 46.9 63.2 (Cimetidine) 99.1 69.8 1.6 60.7 ―
Octn1 Tetraethylammonium 6.1 60.1 0.7 60.1 (Verapamil) 6.4 60.2 1.3 60.3 ―
Octn2 Tetraethylammonium 59.2 63.0 1.3 60.1 (Verapamil) 61.6 61.1 2.3 60.6 ―
Oct2 Tetraethylammonium 95.2 610.1 41.2 67.4 (Cimetidine) 93.3 68.3 1.3 60.3 ―
Mate1 Tetraethylammonium 22.1 60.3 0.6 60.1 (Cimetidine) 19.3 60.5 1.0 60.2 ―
Rat renal ABC
transporters
Mrp2 Estradiol-17b-D-glucuronide 10.2 63.2 ――1.8 60.1 ―
Mrp4 Estradiol-17b-D-glucuronide 11.4 61.3 ――2.2 60.2 ―
Bcrp Methotrexate 43.4 610.2 ――1.0 60.2 ―
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In Vitro Interactions between Tanshinol and Rat Renal
Transporters. Rat Oat1, Oat2, and Oat3 are orthologs of human
OAT1, OAT2, and OAT3, respectively. To delineate and extrapolate the
in vivo regulatory role of the human renal transporters in the plasma
pharmacokinetics and renal disposition of tanshinol, the rat transporters
were examined with respect to saturability and affinity. Tanshinol was
taken up by cells expressing rat Oat1, Oat2, and Oat3; the relevant K
m
,
V
max
,andCL
int
values are shown in Table 2. The results indicated that
Oat1, Oat2, and Oat3 exhibited in vitro saturability and affinity for
tanshinol similar to their human counterparts, except that the V
max
of rat
Oat3 was considerably greater than that of human OAT3 but
comparable to that of human OAT2. Probenecid inhibited the Oat1-,
Oat2-, and Oat3-mediated transport of tanshinol with IC
50
values shown
in Table 2. Rat Oct1, Oct2, Octn1, Octn2, and Mate1 exhibited no
transport activities for tanshinol (Table 1). Rat Mrp2 and Mrp4 had low
in vitro transport activities for tanshinol, and rat Bcrp had no significant
transport activity. Taken together, the rat renal transporters exhibited
interaction profiles with tanshinol similar to the human transporters.
Impact of Probenecid-Impaired Tubular Secretion on Plasma
Pharmacokinetics and Disposition of Tanshinol in Rats. To de-
termine the impact of the renal transporter-mediated tubular secretion
on systemic exposure to and renal disposition of tanshinol, an i.v. bolus
of probenecid (100 mg/kg) was used to impair the rat tubular secretion
of tanshinol by inhibiting Oat1 and Oat3. Probenecid exhibited
concentration-dependent binding to rat plasma protein; its unbound
fraction in plasma (f
u
) increased from 28% to 60% as the plasma
concentrations increased from 200 mM to 2000 mM. Probenecid
exhibited unbound plasma concentrations at 5 minutes (unbound
C
5min
) and 4 hours after dosing (unbound C
4h
) that were 100 and
14 times, respectively, as much as its IC
50
against tanshinol for rat Oat1,
respectively (Supplemental Table 1). The unbound C
5min
and C
4h
were
60 and 9 times, respectively, as much as the IC
50
for rat Oat3. These
data suggest that probenecid treatment could impair Oat1/Oat3-
mediated tubular secretion of tanshinol in rats. Probenecid had a total
plasma clearance (CL
tot,p
) of 0.09 l/h/kg in rats. Its renal excretion was
poor, with a CL
R
of 0.001 l/h/kg; the fraction of the dose excreted into
urine (f
e-U
) was only 1.5%.
After an i.v. bolus of tanshinol (2 mg/kg), the systemic exposure to
tanshinol in probenecid-treated rats was significantly enhanced as
compared with that in the same rats when they were not given pro-
benecid treatment (Fig. 1). As shown in Table 3, probenecid treatment
resulted in 3- to 5-fold increases in plasma AUC
0–‘
of tanshinol (P=
0.0001), 1.6- to 2.3-fold elevations in C
5min
(P= 0.00001), and 1.7- to
2.3-fold increases in elimination half-life (t
1/2
)(P= 0.00001).
Probenecid treatment led to a notable decrease in CL
R
of tanshinol,
only 20%–34% of that in the same rats when given no probenecid (P=
0.00004).
To rule out differences in glomerular function as a confounding
factor, endogenous creatinine excretion was measured in the rats during
both the probenecid treatment period and the probenecid-free period.
Probenecid treatment was not found to significantly change the renal
clearance of endogenous creatinine (P= 0.158) or the f
u
values of
tanshinol (P= 0.795) (Table 3). Accordingly, the probenecid-induced
changes in the CL
R
of tanshinol may be caused predominantly by
decreases in Oat-mediated tubular secretion. Noncompartmental PK
analysis also revealed probenecid-induced abnormalities in total plasma
TABLE 2
Comparative kinetic parameters for human OAT and rat Oat transporters mediating in vitro transport of tanshinol
Values represent the mean 6S.D.(n = 3).
Transporter K
m
V
max
CL
int
IC
50
Probenecid against Tanshinol Tanshinol against Positive Substrate
mM pmol/mg protein/min ml/mg protein/min mM
Positive substrate (in parentheses)
Human
OAT1 (Para-aminohippuric acid) 60.3 65.5 1379 639 22.9 ―98 67
OAT2 (Prostaglandin F
2a
) 45.5 612.4 360 654 7.91 ―1528 651
OAT3 (Estrone-3-sulfate) 28.1 63.7 73.1 64.4 2.60 ―2803 6150
OAT4 (Estrone-3-sulfate) 49.9 65.3 232 69 4.64 ―4079 6410
Rat
Oat1 (Para-aminohippuric acid) 102 617 2299 6138 22.5 ――
Oat2 (Prostaglandin F
2a
) 27.0 610.3 511 6113 18.9 ――
Oat3 (Estrone-3-sulfate) 17.1 64.1 161 611 9.41 ――
Tanshinol as substrate
Human
OAT1 121 613 1414 640 35.4 4.57 60.30 ―
OAT2 859 670 4555 6146 5.30 393 6118 ―
OAT3 1888 6395 182 617 0.0963 10.9 62.9 ―
OAT4 1880 6555 169 622 0.0899 134 650 ―
Rat
Oat1 117 624 599 635 5.12 6.19 60.18 ―
Oat2 1207 6470 1751 6297 1.45 108 631 ―
Oat3 1498 6225 1030 663 0.688 9.51 61.52 ―
Fig. 1. Plasma concentrations (A) and urinary excretion (B) of tanshinol over time
after an i.v. bolus of tanshinol at 2 mg/kg in rats not treated with probenecid (s) and
in the same rats treated with probenecid (d). The details of the rat PK study are
described in Materials and Methods (the first rat PK study). The plasma PK and
renal excretion parameters of tanshinol are shown in Table 3. The data represent the
mean and S.D. from two independent rat experiments where each treatment was
performed tetraplicate.
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clearance (CL
tot,p
) of tanshinol, demonstrating 61%–78% reductions
(P= 0.00004), and in apparent volume of distribution at steady state
(V
SS
), demonstrating 5%–38% reductions (P= 0.006).
The effects of probenecid on rat tissue distribution of tanshinol were
further determined by measuring tanshinol concentrations in the tissue
homogenates of rats after dosing. As with the systemic exposure, pro-
benecid treatment led to heart, lung, brain, and liver C
max
and AUC
0–‘
levels of tanshinol that were higher than those in the normal rats not
treated with probenecid (P= 0.000005–0.005) (Table 4). However,
probenecid treatment did not cause a significant change in the average
kidney AUC
0–‘
level (P= 0.309); the average maximum kidney
concentration after dosing (C
max
) in the probenecid-treated rats was
markedly lower than that in the normal rats (P= 0.001). These data
suggest that the reduced V
SS
of tanshinol by probenecid treatment
resulted, at least in part, from the change in kidney exposure to the
compound.
Dose-Dependent Changes in Levels of Systemic and Renal
Exposure to Tanshinol in Rats. Changes in systemic and renal
exposure to tanshinol were evaluated in a single ascending dose study
in rats. As shown in Table 5 and Fig. 2A–C, systemic exposure to
tanshinol increased as a function of the dose (i.v.; 2–200 mg/kg).
Plasma C
5min
of tanshinol exhibited a dose-proportional increase; the
slope of ln(plasma AUC
0–‘
) versus ln(dose) was 1.04 (Table 6). There
were dose-independent trends in plasma t
1/2
(P= 0.280–0.542), CL
tot,p
(P= 0.193–0.842), and V
SS
(P= 0.196–0.568) of tanshinol (Table 5).
Meanwhile, kidney C
5min
and AUC
0–‘
of tanshinol also increased
as the dose increased (Table 5 and Fig. 2D–F). The slopes of ln(kidney
C
5min
) and ln(kidney AUC
0–‘
) versus ln(dose) were 0.98 and 1.07,
respectively. Over the dose range, the kidney t
1/2
of tanshinol was also
dose-independent (P= 0.066–0.417). The kidney C
5min
and AUC
0–‘
of tanshinol at each dose level were 6.9–11.0 and 5.1–6.7 times,
respectively, as high as the corresponding plasma data.
It is worth mentioning that the rat Oat1/Oat3-mediated basolateral
uptake is expected to result in the real concentration of tanshinol in the
tubular epithelium being considerably higher than the associated
kidney homogenate concentration. Tanshinol exhibited concentration-
independent renal clearance by luminal efflux into urine (CL
R,c-u
;P=
0.548–0.956); no evidence of saturation of CL
R,c-u
suggested that the
luminal efflux of tanshinol into urine probably did not involve
transporter-mediated mechanism (Table 5).
Taken together, tanshinol exhibited a linear plasma pharmacoki-
netics over a wide range of i.v. doses in rats, and the change in
systemic exposure to tanshinol by dosage adjustment (about 100 times)
was substantially greater than that by probenecid-impaired tubular
secretion (about 3–5 times). A similar scenario is expected to take place
in humans. Oat1/Oat3-mediated tubular uptake resulted in a level of
kidney exposure to tanshinol considerably higher than the level of sys-
temic exposure. This raised concerns regarding the risk of dose-related
nephrotoxicity of tanshinol.
Lack of Nephrotoxicity after Tanshinol Overdose in Rats. As
with saline-treated rats (the negative controls), both the renal tubules
and glomeruli of rats given 14 consecutive days of subchronic
treatment with tanshinol at an i.v. dose of 200 mg/kg per day
(equivalent to 100 times the clinical daily dose) were histologically
normal on day 15 (Fig. 3). No evidence of toxicity to the liver was
observed in tanshinol-treated rats (data not shown). Consistent with
these histopathological observations, the rats undergoing multiple-
dose treatment with tanshinol showed serum markers of renal function
(BUN, 4.6–6.3 mM; sCr, 20–24 mM) within the normal ranges (4.2–
7.8 mM for BUN and 16–31 mM for sCr) (Fig. 3).
TABLE 3
Comparative plasma pharmacokinetics and renal excretion of tanshinol after an
i.v. bolus dose of tanshinol at 2 mg/kg in rats not treated with probenecid and
in the same rats treated with probenecid
The details of the rat PK study are described in Materials and Methods (the first rat PK study).
No obvious effect of tanshinol was observed on the plasma pharmacokinetics or renal disposition
of probenecid. The data represent mean 6S.D. from two independent experiments where each
treatment was performed in tetraplicate (total n = 8).
PK Parameter
Rat Treatment with Probenecid
Not treated Treated (Same Rats)
Plasma data
C
5min
(mM) 11.8 61.9 23.2 63.6
a
AUC
0–‘
(mM×h) 3.19 60.57 10.6 63.1
a
t
1/2
(h) 0.196 60.055 0.441 60.062
a
MRT (h) 0.173 60.015 0.450 60.052
a
CL
tot, p
(l/h/kg) 3.26 60.64 1.01 60.24
a
V
SS
(l/kg) 0.538 60.087 0.428 60.077
a
Urine data
Cum.A
e
(mmol) 1.51 60.09 1.28 60.14
a
CL
R
(l/h/kg) 1.94 60.38 0.503 60.096
a
f
e-U
(%) 59.7 63.8 50.7 65.7
a
CL
R
/(GFRf
u
) 6.56 1.72
Plasma protein binding data
f
u
(%; at 0.5–50 mM) 98.6 61.1 96.5 62.5
GFR data
CL
R-cr
(l/h/kg) (Before dosing) 0.295 60.043 0.315 60.036
CL
R-cr
(l/h/kg) (6 h after dosing) 0.314 60.060 0.288 60.037
Cum.A
e
, cumulative amount excreted; MRT, mean residence time.
a
P,0.05 when compared with the rat group not treated with probenecid.
TABLE 4
Comparative tissue distribution of tanshinol after an i.v. bolus dose of tanshinol at 2
mg/kg in rats not treated with probenecid and in rats treated with probenecid
The rat blood and tissue samples were collected at 0, 5, and 30 minutes and 1 and 2 hours after
dosing. The details of the rat PK study are described in Materials and Methods (the second rat PK
study). AUC
0–‘
, area under the plasma concentration-time curve from zero to infinity; C
5min
,
concentration at 5 min after dosing; MRT, mean residence time; t
1/2
, elimination half-life; No
obvious effect of tanshinol was observed on the plasma pharmacokinetics or renal disposition of
probenecid. The data represent the mean 6S.D. from two independent experiments where each
treatment was performed in triplicate (total n = 6).
PK parameter
Rat Treatment with Probenecid
Not Treated Treated
Heart data
C
5min
(mM) 4.18 61.06 7.38 61.49
a
AUC
0–‘
(mM×h) 1.16 60.10 3.64 60.50
a
t
1/2
(h) 0.235 60.027 0.429 60.138
a
MRT (h) 0.353 60.05 0.588 60.115
a
Lung data
C
5min
(mM) 2.80 60.29 4.25 60.54
a
AUC
0–‘
(mM×h) 0.72 60.09 2.05 60.12
a
t
1/2
(h) 0.15 60.00 0.32 60.02
a
MRT (h) 0.26 60.01 0.50 60.03
a
Brain data
C
5min
(mM) 0.212 60.101 0.309 60.068
a
AUC
0–‘
(mM×h) 0.0729 60.0177 0.173 60.055
a
t
1/2
(h) 0.223 60.116 0.363 60.077
a
MRT (h) 0.343 60.19 0.543 60.091
a
Liver data
C
5min
(mM) 0.601 60.298 1.35 60.32
a
AUC
0–‘
(mM×h) 0.227 60.143 0.644 60.206
a
t
1/2
(h) 0.124 60.025 0.293 60.018
a
MRT (h) 0.201 60.065 0.442 60.037
a
Kidney data
C
5min
(mM) 127 618 68.0 616.7
a
AUC
0–‘
(mM×h) 23.6 63.2 26.5 65.7
t
1/2
(h) 0.225 60.032 0.286 60.032
a
MRT (h) 0.216 60.012 0.378 60.031
a
Plasma data
C
5min
(mM) 13.3 62.5 28.3 65.1
a
AUC
0–‘
(mM×h) 4.17 60.62 12.6 61.5
a
t
1/2
(h) 0.287 60.057 0.396 60.095
a
MRT (h) 0.238 60.020 0.416 60.034
a
a
P,0.05 when compared with the rat group not treated with probenecid.
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In contrast, the positive controls of cisplatin and puromycin caused
considerable renal injury in rats. The lesions observed in cisplatin-
treated rats were characterized by dilated tubules filled with necrotic
tubular epithelial cells, cellular debris, and proteinaceous casts, but the
glomeruli were histologically normal. The histopathologic evaluation
of the kidneys from the puromycin-treated rats demonstrated renal
cellular degeneration, necrosis, and sloughing of proximal tubule
epithelium and vacuolation of glomerular podocytes. In rats, cisplatin
treatment led to the BUN (11–33 mM) and sCr levels (47–108 mM)
that exceeded the normal ranges. Abnormally elevated BUN and sCr
levels were also observed in the puromycin-treated rats: 103–131 mM
and 179–266 mM, respectively.
Collectively, tanshinol exhibited very little dose-related nephrotox-
icity in rats. A similar scenario is expected to take place in humans.
Discussion
Tanshinol is a carboxyl acid and cleared predominantly by renal
excretion. Many organic anions are substrates of renal organic anion
transporters (Masereeuw and Russel, 2010). Renal excretion of
tanshinol mainly involves active tubular secretion. This suggests that
the transporters influence the systemic exposure to and renal disposition
of tanshinol via mediating the tubular secretion. To test this hypothesis,
a comprehensive investigation of interactions between renal transporters
TABLE 5.
Comparative plasma and kidney pharmacokinetics of tanshinol in rats receiving an i.v. bolus dose of tanshinol solution
(2–200 mg/kg)
The rat blood and kidney tissue samples were collected at 5 and 30 minutes and 1 and 2 hours after dosing. Urine samples were collected
0–24 hours after dosing. The details of the rat PK study are described in Materials and Methods (the third rat PK study).
PK Parameter
Dosage
2 5 15 50 200
mg/kg
Plasma data
C
5min
(mM) 15.5 61.5 36.7 62.9 98.7 617.9 388 648 1403 698
AUC
0–‘
(mM×h) 4.33 60.45 11.0 61.0 31.2 65.5 125 623 504 661
t
1/2
(h) 0.278 60.017 0.285 60.010 0.260 60.027 0.269 60.022 0.259 60.014
MRT (h) 0.272 60.030 0.252 60.004 0.237 60.031 0.288 60.002 0.328 60.009
V
SS
(l/kg) 0.635 60.023 0.582 60.049 0.578 60.052 0.595 60.097 0.662 60.067
CL
tot, p
(l/h/kg) 2.35 60.25 2.31 60.20 2.48 60.45 2.07 60.34 2.02 60.26
Urine data
Cum.A
e
(mmol) 1.48 60.06 3.75 60.29 12.3 61.1 44.2 62.9 192 618
f
e-U
(%) 61.5 63.6 59.7 66.8 66.3 67.5 73.3 65.7 77.9 66.7
CL
R
(l/h/kg) 1.44 60.10 1.37 60.05 1.62 60.11 1.50 60.15 1.56 60.06
Kidney data
C
5min
(mM) 118 637 278 679 1083 6410 3199 61046 9671 61536
AUC
0–‘
(mM×h) 22.7 66.9 67.9 618.0 268 697 899 6309 3552 6518
t
1/2
(h) 0.277 60.010 0.250 60.031 0.238 60.003 0.267 60.016 0.256 60.007
MRT (h) 0.232 60.011 0.197 60.028 0.172 60.008 0.214 60.010 0.254 60.011
CL
R,c-u
(l/h/kg) 0.289 60.075 0.282 60.064 0.261 60.061 0.269 60.062 0.272 60.017
AUC
0–‘
, area under the plasma concentration-time curve from 0 to infinity; CL
R,c-u
, renal clearance by the cellular efflux into urine
across the apical brush border membrane; CL
R
, renal clearance; CL
tot,p
, total plasma clearance; C
5min
, concentration at 5 min after dosing;
Cum.A
e
, cumulative amount excreted; MRT, mean residence time; t
1/2
, elimination half-life; V
SS
, apparent volume of distribution at steady
state.
Fig. 2. Plasma concentration (A) and kidney concentration (D) of tanshinol over time in rats receiving an i.v. bolus dose of tanshinol at 2 (u), 5 (s), 15 (m), 50 (j), and 200
mg/kg (d). Correlations of plasma C
5min
, plasma AUC
0–‘
, kidney C
5min
, and kidney AUC
0–‘
of tanshinol with the dose are also shown in (B), (C), (E), and (F), respectively.
The details of the rat PK study are described in Materials and Methods (the third rat PK study). The plasma and kidney PK parameters of tanshinol are shown in Table 5.
Renal Tubular Secretion of Tanshinol 675
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and tanshinol was undertaken. This resulted in an understanding of the
mechanistic tubular secretion of tanshinol and enabled us to sub-
sequently explore the impact of tubular secretion on systemic exposure
to tanshinol and propensity of the compound for dose-related
nephrotoxicity and for renal transporter-mediated herb-drug interactions.
Cellular uptake of tanshinol could be mediated by human OAT1,
OAT2, OAT4, and OAT3 (in decreasing order of affinity for
tanshinol), rather than human OATP4C1, OCT2, OCTN1, MATE1,
and MATE2-K. The transporters OAT1, OAT2, and OAT3 are
expressed at the basolateral membrane of renal proximal tubules and
play roles in uptake of tanshinol from blood in tubular secretion
(Enomoto et al., 2002; Motohashi et al., 2002). OAT1 and OAT3 are
major renal transporters, and OAT2 probably expresses at a lower
level. OAT2 and OAT3 exhibited an in vitro CL
int
that was 39% and
1%, respectively, of the OAT1 efficiency.
OAT4 has a major role in the tubular reabsorption of organic anions
from urine (Ekaratanawong et al., 2004). OAT4 exhibited a CL
int
that
was only 1% of the OAT1 value. 1) The low affinity of OAT4 for
tanshinol, 2) its relatively low expression in the kidney, and 3) the short
residence time of tanshinol in luminal filtrate (a couple of seconds)
indicate that OAT4 had limited contribution to renal excretion of
tanshinol.
The apically located MRP2, MRP4, BCRP, MATE1, MATE2-K,
and OCTN1 transporters that support luminal efflux from proximal
renal tubules (Masereeuw and Russel, 2010) exhibited ambiguous or
no in vitro ability to transport tanshinol. A similar scenario was
observed with the rat orthologs at the apical membrane. Rats exhibited
dose-independent trends in kidney t
1/2
and CL
R,c-u
of tanshinol over
the i.v. dose range 2–200 mg/kg; its C
5min
in kidney homogenate
increased from 118 to 9671 mM as the dose increased (Table 5).
A Caco-2 cell-based study revealed that tanshinol had favorable
membrane permeability for intestinal absorption (Lu et al., 2008). The
concentration of tanshinol in the rat epithelia of proximal tubules at the
dose 200 mg/kg should be much higher than 10 mM. Such a high
intracellular concentration is expected to exceed the K
m
and results in
the saturation of possible transporter-mediated efflux. According to
the Michaelis-Menten equation for membrane permeation, when
a drug compound with high membrane permeability has a concentra-
tion significantly higher than K
m
, concentration-gradient-driven
passive transcellular transport can be the dominating mechanism
(Sugano et al., 2010). Accordingly, the luminal efflux of tanshinol into
urine was most likely based on a passive diffusion mechanism.
In rats, Oat1 and Oat3 are highly expressed at the basolateral mem-
brane of renal proximal tubules, and Oat2 is at the apical membrane
(Kojima et al., 2002). The roles of Oat1 and Oat3 are to support the
basolateral uptake of organic anions from blood. Rat Oat2 has a major
role in tubular reabsorption of organic anions from urine. Tanshinol was
a substrate of Oat1, Oat2, and Oat3 but not of Oct1 and Oct2. The CL
int
values of Oat2 and Oat3 were 29% and 14% that of Oat1, respectively.
Rat Mrp2, Mrp4, Bcrp, and Mate1 are expressed at the apical
membrane. The apical membrane efflux transporters exhibited limited
(Mrp2 and Mrp4) or no (Bcrp, Octn1, Octn2, and Mate1) affinity for
tanshinol.
Based on these results, rats were used to investigate the impact of renal
transporters on systemic and renal exposure to tanshinol. For this purpose,
probenecid was used to impair Oat1/Oat3-mediated tubular secretion of
tanshinol. However, it also exhibits inhibitory activity against rat Oatp1a1/
Oatp1a4, Mrp2/Mrp3, and UDP-glucuronosyltransferases (Sugiyama
et al., 2001; Horikawa et al., 2002; Uchaipichat et al., 2004). For
tanshinol, transport mediated by rat Mrp2 and human MRP3 is very poor,
and metabolism via glucuronidation is limited (details pending publication
elsewhere). Tanshinol (molecular weight 198 Da) is not a substrate of rat
TABLE 6.
Summary of results from dose proportionality assessment of a single ascending dose
study in rats receiving an i.v. bolus dose of tanshinol solution (2–200 mg/kg)
Critical intervals were 0.952–1.048 for the systemic exposure data of tanshinol from the single
ascending dose study of rat given an i.v. bolus dose of tanshinol (2–200 mg/kg). The term
r denotes the correlation coefficient. Correlations were statistically significant with a P ,0.05. The
term ”linear”was concluded statistically if the 90% confidence interval (90% CI) for slope was
contained completely within the critical interval; inconclusive was concluded statistically if the
90% CI lay partly within the critical interval; nonlinear was concluded statistically if the 90% CI
was entirely outside the critical interval.
PK Parameter rP Slope (90% CI) Conclusion
Plasma
C
5min
0.998 2.31 10
216
0.989 (0.955–1.023) Linear
AUC
0–‘
0.997 2.57 10
216
1.038 (1.002–1.075) Inconclusive
Kidney
C
5min
0.985 2.73 10
211
0.978 (0.893–1.062) Inconclusive
AUC
0–‘
0.990 1.66 10
212
1.070 (0.996–1.144) Inconclusive
Fig. 3. Comparative kidney histology (A–F) and serum biochemistry (G and H) in
rats receiving subchronic treatment of 14 consecutive days of saline (negative
control), tanshinol (i.v., 200 mg/kg/d), puromycin (i.p., 40 mg/kg/d; positive
control), and cisplatin (i.p., 1 mg/kg/d; positive control). The rat blood samples were
collected before (open bars) and after (solid bars) 15 days of treatment for
assessment of blood urea nitrogen (BUN) and serum creatinine (sCr). The rat kidney
tissues were sampled and processed for H&E staining to evaluate tubular damage,
glomerular damage, and histology. *P,0.05 versus the negative control. Stain:
H&E; original magnification, 200.
676 Jia et al.
at ASPET Journals on March 25, 2015dmd.aspetjournals.orgDownloaded from
Oatp1a1 or Oatp1b2. Therefore, inhibition of Oatp1a1/Oatp1a4, Mrp2/
Mrp3, and UDP-glucuronosyltransferases by probenecid probably had
negligible effect on the rat pharmacokinetics of tanshinol.
Probenecid-induced impairment of tubular secretion resulted in 61%–
78% reductions in the CL
tot,p
of tanshinol, and the CL
R
/(GFRf
u
) ratio
of tanshinol was reduced from 6.6 to 1.7. Although probenecid
treatment led to 3 to 5 times the enhancement of systemic exposure to
the compound (AUC
0–‘
), the kidney exposure to tanshinol was reduced.
It is worth mentioning that probenecid treatment also resulted in
a decrease in nonrenal clearance (CL
tot,p
2CL
R
;from1.32to0.51
l/h/kg). This probably resulted, at least in part, from inhibition of hepatic
Oat2 by probenecid, unbound plasma C
5min
and C
4h
,whichwere6and
0.8 times, respectively, its IC
50
against tanshinol.
Tanshinol exhibited a linear plasma pharmacokinetics over the
i.v. dose range 2–200 mg/kg in rats; dosage adjustment could cause
approximately 100-fold increases in the plasma AUC
0–‘
and C
5min
of
tanshinol. For tanshinol, Oat1 exhibited a higher affinity and CL
int
than
Oat3. When the doses were 2–15 mg/kg, all the unbound plasma C
5min
of tanshinol (15–97 mM) was lower than the K
m
for Oat1. This
suggested that the basolateral uptake for tubular secretion of tanshinol
was mainly mediated by Oat1. As the dose increased, the initial
unbound plasma concentrations of tanshinol, particularly at 200 mg/kg,
exceeded the K
m
for Oat1 (Fig. 2A). Tanshinol pharmacokinetics
remained linear with the increasing dose, and the CL
R
was not markedly
saturated over 2–200 mg/kg, suggesting that, at a higher concentration,
Oat3 supplements Oat1 in mediating renal uptake. This was evidenced
by the K
m
for Oat3, which was higher than the unbound plasma
concentrations of tanshinol at the 200 mg/kg dose.
The preceding linear pharmacokinetics mainly depended on Oat1/3-
mediated tubular secretion. For matching levels of systemic exposure to
tanshinol after dosing to its effective concentrations for the antianginal
activities, dosage adjustment was a more effective way to manipulate
exposure, because the change in the systemic exposure to tanshinol via
dosage adjustment (about 100 times) was statistically significantly greater
than that via probenecid-induced drug interaction (about 3 to 5 times).
Despite the Oat-mediated tubular secretion mechanism, tanshinol
exhibited very little dose-related nephrotoxicity. This suggests that dosage
adjustment probably is also a safe way to manipulate exposure to
tanshinol, and it did not need to be used in concert with drug combination.
Gao et al. (2009) and Li et al. (2009) reported that tanshinol exhibited
very little dose-related toxicity in rats, mice, or dogs. Tanshinol is expected
to have a similar linear plasma PK property over a large i.v. dose range in
humans. This is because, like the rat Oat transporters, the human OAT
transporters also exhibit low affinities and high transport capacities for
tanshinol. Similar to rat Oat1, humanOAT1playedakeyroleinmediating
the renal uptake of tanshinol at low concentrations; like rat Oat3, human
OAT2 and OAT3 supplemented OAT1, to mediate the tanshinol uptake at
high concentration.
Herb-drug interactions are an important safety concern (Li et al.,
2012b). Rat Oat transporters was found to influence systemic exposure
to tanshinol; likewise, human OAT transporters are expected to be
clinically important. Human OAT1, OAT3, and OCT2 are major renal
transporters with a broad range of substrates; renal drug interactions
often occurred in relation to their actions (Giacomini et al., 2010). Both
the K
m
and IC
50
data shown in Table 2 indicated that tanshinol had low
affinity for OAT1. After an i.v. infusion daily dose of DanHong
injection (40 mL containing around 55 mg of tanshinol) in human
subjects, the average maximum plasma concentration of tanshinol was
measured as about 2.5 mM (Li et al., 2015). Tanshinol exhibits a f
u
of
85% in human plasma and a short t
1/2
of 1.1–1.3 hours (Lu et al., 2008).
According to the equation (drug-drug interaction index = unbound
C
max
/IC
50
), the OAT1-mediated drug-drug interaction index was
calculated for tanshinol as 0.02. This suggests that tanshinol has
a low propensity to act as an inhibitory perpetrator in OAT1-mediated
drug interactions when DanHong injection is used at a clinically
relevant dose. Compared with OAT1, the renal transporters OAT2,
OAT3, and OAT4 exhibited higher K
m
and IC
50
values for tanshinol
(Table 2), suggesting a lower potential for these transporter-mediated
herb-drug interactions. In addition, tanshinol had no inhibition potency
toward human OATP4C1, OCT2, OCTN1, MRP2, MRP4, BCRP,
MATE1, or MATE2-K. In rats, the probenecid-impaired tubular
secretion resulted in 1.6- to 4.5-fold elevations in systemic exposure
to tanshinol, suggesting that tanshinol could be a substrate victim on Oat
transporters. A similar scenario is expected to take place in humans.
However, the change in systemic exposure is probably not clinically
relevant, because tanshinol exhibits very little dose-related toxicity.
Wang and Sweet (2013) reported that the Danshen polyphenols
rosmarinic acid, lithospermic acid, and salvianolic acid A exhibited
strong inhibitory activities against human OAT1 or OAT3 (K
i
, 0.16–
0.59 mM). Like probenecid, these Danshen polyphenols, concurrently
present in Danshen-based i.v. injections, may influence systemic and
renal exposure to tanshinol after dosing. Studying the PK matrix
effects will help more accurately define and predict the exposure level
and pharmacokinetics of tanshinol.
Understanding the mechanisms governing systemic exposure to
tanshinol helps with matching the exposure levels after dosing to the
effective concentrations for its antianginal activities; this most likely
results in enhanced efficacy of Danshen-based therapy. In summary,
renal tubular secretion of tanshinol involves the basolateral uptake from
blood primarily by human OAT1 and rat Oat1, and the subsequent
luminal efflux into urine, mainly by passive diffusion (Fig. 4). Human
OAT2/OAT3 and rat Oat3 are also important for the basolateral uptake
at high tanshinol concentrations in blood. Human OAT4- and rat Oat2-
mediated tubular reabsorption of tanshinol may have limited contribu-
tion to renal excretion. Tanshinol shows low propensity to cause renal
transporter-mediated herb-drug interactions. Tanshinol exhibits linear
pharmacokinetics properties over a large i.v. dose range and very little
dose-related nephrotoxicity in rats. Dosage adjustment appears to be an
efficient, safe way to manipulate its systemic exposure. Additional
safety studies are under way to define the risk of hyperhomocysteinemia
related to dose-dependent tanshinol methylation.
Acknowledgments
The authors thank X.-M Gao and Y Zhu for their stimulating discussions and
D-D Wang for technique assistance. The histopathologic evaluation was
performed by Center for Drug Safety Assessment at the Second Military
Medical University (Shanghai, People’s Republic of China).
Authorship Contributions
Participated in research design: C. Li, Jia.
Conducted experiments: Jia, Du, Liu, Jiang, Xu, Wang, Olaleye, Dong.
Performed data analysis: C. Li, Jia, Yang, L. Li.
Wrote or contributed to the writing of the manuscript: C. Li, Jia.
Fig. 4. Tubular secretion of tanshinol mediated by human (h) and rat (r) organic
anion transporters (OAT/Oat).
Renal Tubular Secretion of Tanshinol 677
at ASPET Journals on March 25, 2015dmd.aspetjournals.orgDownloaded from
References
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.
Cao Y-G, Chai J-G, Chen Y-C, Zhao J, Zhou J, Shao J-P, Ma C, Liu X-D, and Liu X-Q (2009)
Beneficial effects of danshensu, an active component of Salvia miltiorrhiza, on homocysteine
metabolism via the trans-sulphuration pathway in rats. Br J Pharmacol 157:482–490.
Chan K, Chui SH, Wong DYL, Ha WY, Chan CL, and Wong RNS (2004) Protective effects of
Danshensu from the aqueous extract of Salvia miltiorrhiza (Danshen) against homocysteine-
induced endothelial dysfunction. Life Sci 75:3157–3171.
Chen F, Li L, Xu F, Sun Y, Du F-F, Ma X-T, Zhong C-C, Li X-X, Wang F-Q, and Zhang N-T,
et al. (2013) Systemic and cerebral exposure to and pharmacokinetics of flavonols and terpene
lactones after dosing standardized Ginkgo biloba leaf extracts to rats via different routes of
administration. Br J Pharmacol 170:440–457.
Cheng C, Liu X-W, Du F-F, Li M-J, Xu F, Wang F-Q, Liu Y, Li C, and Sun Y (2013) Sensitive
assay for measurement of volatile borneol, isoborneol, and the metabolite camphor in rat
pharmacokinetic study of Borneolum (Bingpian) and Borneolum syntheticum (synthetic
Bingpian). Acta Pharmacol Sin 34:1337–1348.
Cheng TO (2007) Cardiovascular effects of Danshen. Int J Cardiol 121:9–22.
Enomoto A, Takeda M, Shimoda M, Narikawa S, Kobayashi Y, Kobayashi Y, Yamamoto T,
Sekine T, Cha SH, and Niwa T, et al. (2002) Interaction of human organic anion transporters 2
and 4 with organic anion transport inhibitors. J Pharmacol Exp Ther 301:797–802.
Ekaratanawong S, Anzai N, Jutabha P, Miyazaki H, Noshiro R, Takeda M, Kanai Y, Sophasan S,
and Endou H (2004) Human organic anion transporter 4 is a renal apical organic anion/
dicarboxylate exchanger in the proximal tubules. J Pharmacol Sci 94:297–304.
Gao Y-L, Liu Z-F, Li G-S, Li C-M, Li M, and Li B-F (2009) Acute and subchronic toxicity of
danshensu in mice and rats. Toxicol Mech Methods 19:363–368.
Giacomini KM, Huang SM, Tweedie DJ, Benet LZ, Brouwer KLR, Chu X, Dahlin A, Evers R,
Fischer V, and Hillgren KM, et al.; International Transporter Consortium (2010) Membrane
transporters in drug development. Nat Rev Drug Discov 9:215–236.
Horikawa M, Kato Y, Tyson CA, and Sugiyama Y (2002) The potential for an interaction
between MRP2 (ABCC2) and various therapeutic agents: probenecid as a candidate inhibitor of
the biliary excretion of irinotecan metabolites. Drug Metab Pharmacokinet 17:23–33.
Hu Z-Y, Yang J-L, Cheng C, Huang Y-X, Du F-F, Wang F-Q, Niu W, Xu F, Jiang R-R, and Gao
X-M, et al. (2013) Combinatorial metabolism notably affects human systemic exposure to
ginsenosides from orally administered extract of Panax notoginseng roots (Sanqi). Drug Metab
Dispos 41:1457–1469.
Imaoka T, Kusuhara H, Adachi M, Schuetz JD, Takeuchi K, and Sugiyama Y (2007) Functional
involvement of multidrug resistance-associated protein 4 (MRP4/ABCC4) in the renal elimi-
nation of the antiviral drugs adefovir and tenofovir. Mol Pharmacol 71:619–627.
Jia Y-L, Huang F-Y, Zhang S-K, and Leung SW (2012) Is danshen (Salvia miltiorrhiza) dripping
pill more effective than isosorbide dinitrate in treating angina pectoris? A systematic review of
randomized controlled trials. Int J Cardiol 157:330–340.
Jiang R-R, Dong J-J, Li X-X, Du F-F, Jia W-W, Xu F, Wang F-Q, Yang J-L, Niu W, and Li C
(2015) Molecular mechanisms governing different pharmacokinetics of ginsenosides and potential
for ginsenoside-perpetrated herb-drug interactions onOATP1B3. Br J Pharmacol 172:1059–1073.
Kojima R, Sekine T, Kawachi M, Cha SH, Suzuki Y, and Endou H (2002) Immunolocalization of
multispecific organic anion transporters, OAT1, OAT2, and OAT3, in rat kidney. J Am Soc
Nephrol 13:848–857.
Lam FFY, Yeung JHK, Chan KM, and Or PMY (2007) Relaxant effects of danshen aqueous
extract and its constituent danshensu on rat coronary artery are mediated by inhibition of
calcium channels. Vascul Pharmacol 46:271–277.
Li G-S, Gao Y-L, Li S-J, Li C-M, Zhu X-Y, Li M, and Liu Z-F (2009) Study on toxicity of
danshensu in beagle dogs after 3- month continuous intravenous infusion. Toxicol Mech Methods
19:441–446.
Li L, Zhao Y-S, Du F-F, Yang J-L, Xu F, Niu W, Ren Y-H, and Li C (2012a) Intestinal
absorption and presystemic elimination of various chemical constituents present in GBE50
extract, a standardized extract of Ginkgo biloba leaves. Curr Drug Metab 13:494–509.
Li M-J, Wang F-Q, Huang Y-H, Du F-F, Zhong C, Olaleye OE, Jia W-W, Li Y-F, Xu F, and Dong
J-J, et al. (2015) Systemic exposure to and dispos ition of catechols derived from Salvia miltiorr hiza
roots (Danshen) after intravenous administration of DanHong injection in human subjects, rats,
and dogs. Drug Metab Dispos 43:679–690
Li Y, Lu J, and Paxton JW (2012b) The role of ABC and SLC transporters in the pharmacoki-
netics of dietary and herbal phytochemicals and their interactions with xenobiotics. Curr Drug
Metab 13:624–639.
Liu H-F, Yang J-L, Du F-F, Gao X-M, Ma X-T, Huang Y-H, Xu F, Niu W, Wang F, and Mao
Y-T, et al. (2009) Absorption and disposition of ginsenosides after oral administration of Panax
notoginseng extract to rats. Drug Metab Dispos 37:2290–2298.
Lu T, Yang J-L, Gao X-M, Chen P, Du F-F, Sun Y, Wang F-Q, Xu F, Shang H-C, and Huang
Y-H, et al. (2008) Plasma and urinary tanshinol from Salvia miltiorrhiza (Danshen) can be used
as pharmacokinetic markers for cardiotonic pills, a cardiovascular herbal medicine. Drug
Metab Dispos 36:1578–1586.
Masereeuw R and Russel FGM (2010) Therapeutic implications of renal anionic drug trans-
porters. Pharmacol Ther 126:200–216.
Motohashi H, Sakurai Y, Saito H, Masuda S, Urakami Y, Goto M, Fukatsu A, Ogawa O, and Inui
K (2002) Gene expression levels and immunolocalization of organic ion transporters in the
human kidney. J Am Soc Nephrol 13:866–874.
Reagan-Shaw S, Nihal M, and Ahmad N (2008) Dose translation from animal to human studies
revisited. FASEB J 22:659–661.
Smith BP, Vandenhende FR, DeSante KA, Farid NA, Welch PA, Callaghan JT, and Forgue ST
(2000) Confidence interval criteria for assessment of dose proportionality. Pharm Res 17:
1278–1283.
Sugano K, Kansy M, Artursson P, Avdeef A, Bendels S, Di L, Ecker GF, Faller B, Fischer H,
and Gerebtzoff G, et al. (2010) Coexistence of passive and carrier-mediated processes in drug
transport. Nat Rev Drug Discov 9:597–614.
Sugiyama D, Kusuhara H, Shitara Y, Ab e T, Meier PJ , Sekine T, En dou H, Suzuk i H,
and Sugiyama Y (2001) Characterization of the efflux transport of 17b-estradiol-D-17b-
glucuronide from the brain across the blood-brain barrier. J Phar macol Exp Ther 298:
316–322.
Takahashi N, Boysen G, Li F, Li Y, and Swenberg JA (2007) Tandem mass spectrometry
measurements of creatinine in mouse plasma and urine for determining glomerular filtration
rate. Kidney Int 71:266–271.
Tang Y-Q, Wang M-H, Chen C-L, Le X-Y, Sun S-J, and Yin Y-M (2011a) Cardiovascular
protection with Danshensu in spontaneously hypertensive rats. Biol Pharm Bull 34:1596–1601.
Tang Y-Q, Wang M-H, Le X-Y, Meng J-N, Huang L, Yu P, Chen J, and Wu P (2011b) Anti-
oxidant and cardioprotective effects of Danshensu (3-(3, 4-dihydr oxyphenyl)-2-hydroxy-propanoic
acid from Salvia miltiorrhiza) on isoproterenol-induced myocardial hypertrophy in rats.
Phytomedicine 18:1024–1030.
Uchaipichat V, Mackenzie PI, Guo X-H, Gardner-Stephen D, Galetin A, Houston JB, and Miners
JO (2004) Human UDP-glucuronosyltransferases: isoform selectivity and kinetics of 4-meth-
ylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and inhibition by
diclofenac and probenecid. Drug Metab Dispos 32:413–423.
Wang D-D, Fan G-W, Wang Y-F, Liu H-T, Wang B-Y, Dong J, Zhang P, Zhang B-L, Karas RH,
and Gao X-M, et al. (2013) Vascular reactivity screen of Chinese medicine Danhong injection
identifies Danshensu as a NO-independent but PGI
2
-mediated relaxation factor. J Cardiovasc
Pharmacol 62:457–465.
Wang F, Liu Y-Y, Liu L-Y, Zeng Q-J, Wang C-S, Sun K, Yang J-Y, Guo J, Fan J-Y, and Han J-Y
(2009) The attenuation effect of 3,4-dihydroxy-phenyl lactic acid and salvianolic acid B on
venular thrombosis induced in rat mesentery by photochemical reaction. Clin Hemorheol
Microcirc 42:7–18.
Wang L, Sun Y, Du F-F, Niu W, Lu T, Kan J-M, Xu F, Yuan K-H, Qin T, and Liu C-X, et al.
(2007) ‘LC-electrolyte effects’improve the bioanalytical performance of liquid chromatography/
tandem mass spectrometric assays in supporting pharmacokinetic study for drug discovery. Rapid
Commun Mass Spectrom 21:2573–2584.
Wang L and Sweet DH (2013) Competitive inhibition of human organic anion transporters
1 (SLC22A6), 3 (SLC22A8) and 4 (SLC22A11) by major components of the medicinal herb
Salvia miltiorrhiza (Danshen). Drug Metab Pharmacokinet 28:220 –228.
Yang R-X, Huang S-Y, Yan F-F, Lu X-T, Xing Y-F, Liu Y, Liu Y-F, and Zhao Y-X (2010)
Danshensu protects vascular endothelia in a rat model of hyperhomocysteinemia. Acta Phar-
macol Sin 31:1395–1400.
Yang G-D, Zhang H, Lin R, Wang W-R, Shi X-L, Liu Y, and Ji Q-L (2009) Down-regulation of
CD40 gene expression and inhibition of apoptosis with Danshensu in endothelial cells. Basic
Clin Pharmacol Toxicol 104:87–92.
Zhao G-R, Zhang H-M, Ye T-X, Xiang Z-J, Yuan Y-J, Guo Z-X, and Zhao L-B (2008) Char-
acterization of the radical scavenging and antioxidant activities of Danshensu and salvianolic
acid B. Food Chem Toxicol 46:73–81.
Zhou L, Zuo Z, and Chow MSS (2005) Danshen: an overview of its chemistry, pharmacology,
pharmacokinetics, and clinical use. J Clin Pharmacol 45:1345–1359.
Address correspondence to: Dr. Chuan Li, Laboratory for DMPK Research of
Herbal Medicines, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 501 Haike Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People’s
Republic of China. E-mail: chli@simm.ac.cn
678 Jia et al.
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DMD # 62000
- 1 -
Renal Tubular Secretion of Tanshinol: Molecular Mechanism,
Impact on Its Systemic Exposure, and Propensity for Dose-Related
Nephrotoxicity and for Renal Herb-Drug Interactions
Weiwei Jia, Feifei Du, Xinwei Liu, Rongrong Jiang, Fang Xu, Junling Yang, Li Li, Fengqing Wang,
Olajide E. Olaleye, Jiajia Dong, and Chuan Li
Drug Metabolism and Disposition (DOI: 10.1124/dmd.114.062000)
Supplemental Table S1
Plasma pharmacokinetics and renal excretion of probenecid after an i.v. bolus dose at 100 mg/kg in rats
AUC0-∞, area under the plasma concentration-time curve from zero to infinity; CLR, renal clearance; CLR-cr, renal clearance
of endogenous creatinine; CLtot,p, total plasma clearance; C5min, concentration at 5 min after dosing; C4h, concentration at 4 h
after dosing; Cum.Ae, cumulative amount excreted; fe-U, fraction of dose excreted into urine; fu, unbound fractions in plasma;
MRT, mean residence time; t1/2, elimination half-life; VSS, apparent volume of distribution at steady state. The data represent
means ± standard deviations from two independent experiments where each rat group was performed in triplicate (total n = 6)
PK parameter Rats treated with probenecid
Plasma data
C5min (unbound C5min) [μM] 1281 ± 92 (602 ± 43)
C4h (unbound C4h) [μM] 298 ± 36 (83.4 ± 10.1)
AUC0-∞ [μM·h] 3414 ± 361
t1/2 [h] 2.59 ± 0.15
MRT [h] 3.77 ± 0.22
CLtot, p [l/h/kg] 0.0947 ± 0.0143
VSS [l/kg] 0.383 ± 0.045
Urine data
Cum.Ae [μmol] 1.41 ± 0.26
CLR [l/h/kg] 0.00143 ± 0.00048
fe-U [%] 1.49 ± 0.37
CLR/(GFR×fu) 0.007–0.013
Plasma protein binding data
fu (%; at 200 μM) 28.1 ± 0.3
fu (%; at 2000 μM) 59.7 ± 1.2
GFR data
CLR-cr [l/h/kg] (Before dosing) 0.301 ± 0.056
CLR-cr [l/h/kg] (6 h after dosing) 0.292 ± 0.021
