Abstract
Doxorubicin (DOX) is an essential component in chemotherapy, and
Astragali Radix (AR) is a widely used tonic herbal medicine. The
combination of DOX and AR offers widespread, well-documented advantages
in treating cancer, e.g., reducing the risk of adverse effects. This
study mainly aims to uncover the impact of AR on DOX disposition in
vivo. Rats received a single intravenous dose of 5 mg/kg DOX following
a single-dose co-treatment or multiple-dose pre-treatment of AR (10
g/kg × 1 or × 10). The concentrations of DOX in rat plasma and six
tissues, including heart, liver, lung, kidney, spleen, and skeletal
muscle, were determined by a fully validated LC-MS/MS method. A
network-based approach was further employed to quantify the
relationships between enzymes that metabolize and transport DOX and the
targets of nine representative AR components in the human
protein–protein interactome. We found that short-term (≤10 d) AR
administration was ineffective in changing the plasma pharmacokinetics
of DOX in terms of the area under the concentration–time curve (AUC,
1303.35 ± 271.74 μg/L*h versus 1208.74 ± 145.35 μg/L*h, p > 0.46), peak
concentrations (C[max], 1351.21 ± 364.86 μg/L versus 1411.01 ± 368.38
μg/L, p > 0.78), and half-life (t[1/2], 31.79 ± 5.12 h versus 32.05 ±
6.95 h, p > 0.94), etc. Compared to the isotype control group, DOX
concentrations in six tissues slightly decreased under AR
pre-administration but only showed statistical significance (p < 0.05)
in the liver. Using network analysis, we showed that five of the nine
representative AR components were not localized to the vicinity of the
DOX disposition-associated module. These findings suggest that AR may
mitigate DOX-induced toxicity by affecting drug targets rather than
drug disposition.
Keywords: Adriamycin, drug disposition, combination therapy, LC-MS/MS,
network
1. Introduction
Doxorubicin (DOX), also known as Adriamycin, is widely used in clinics
for the treatment of many different types of cancer, such as acute
myeloid leukemia and breast cancer [[36]1]. DOX stops or slows the
growth of cancer cells by damaging DNA and chromatin, but it also kills
normal cells, resulting in a series of side effects such as arrhythmia
and bruising [[37]2,[38]3]. The clinical application of DOX is limited
by its severe side effects. For example, to prevent cardiotoxicity,
elderly cancer patients with heart problems are often excluded from
chemotherapy regimens containing DOX. Combination therapy using
multiple drugs to improve clinical outcomes offers a strategy to
mitigate DOX-induced toxicity with efficacy [[39]4,[40]5].
Astragali Radix (AR) is a common, traditional Chinese medicine that has
long been used as an anti-oxidative and anti-inflammatory herbal
prescription for multiple complex diseases, from diabetes to asthma and
cancer [[41]6]. Evidence suggests that AR has a therapeutic potential
to alleviate the DOX-induced toxicity of the heart, liver, and kidney
[[42]7,[43]8]. While cell death is a unifying feature of DOX-induced
toxicity, it is not clear how AR could simultaneously interfere with
the death programs in different organs. The protective effects of AR
probably involve pleiotropic mechanisms, including regulation of
enzymes and metabolites that fuel energy metabolism, inhibition of
pro-inflammatory cytokines, and suppression of oxidative stress
[[44]9,[45]10,[46]11].
DOX administered as a conventional injection is widely distributed in
the plasma and tissues but can hardly cross the blood–brain barrier
[[47]12]. There are three main metabolic routes of DOX in mammals:
one-electron reduction, two-electron reduction, and deglycosylation
[[48]13]. Previous studies have shown that lowering the level of DOX in
normal organs is beneficial in reducing toxicity. For example, the
liposomal formulation of DOX, which reduces myocardial drug
accumulation, has provided a significant reduction in the risk of
cardiotoxicity [[49]14,[50]15]. When DOX is combined with dandelion to
treat breast cancer, dandelion can reduce the intracellular
accumulation of DOX by activating the drug efflux transporter
P-glycoprotein, thereby reducing DOX-induced cardiotoxicity [[51]16].
It is possible that AR protective effects involve a similar mechanism.
However, there has been no report on the effect of AR on the
pharmacokinetics and tissue distribution of DOX.
Here, we hypothesized that, in addition to acting on DOX targets, AR
might mitigate the side effects by reducing DOX exposure in vivo. To
test this hypothesis, we developed and validated a simple, specific,
and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS)
method for the determination of DOX in rat plasma and six tissues
(heart, liver, lung, kidney, spleen, and skeletal muscle), allowing us
to analyze the effects of AR co-treatment and pre-treatment on DOX
disposition. Moreover, enzymes that metabolize and transport DOX are
not randomly scattered in the human protein–protein interactome (PPI),
but tend to cluster in the same neighborhood, known as the DOX
disposition-associated module [[52]17]. The targets (usually proteins)
of AR components do this as well. If AR does change the in vivo
metabolism or efflux of DOX, some AR components must be localized to
the vicinity of the module related to DOX disposition [[53]18].
Therefore, we further proposed a network-based measure that helped us
quantify the topological relationship between nine representative AR
components and DOX disposition, offering a novel approach to
understanding the potential mechanism.
2. Results
2.1. Method Validation
The representative extracted ion chromatograms of DOX and IS in rat
plasma and tissues are illustrated in [54]Supplementary Figure S1. DOX
showed good linearity (r^2 > 0.995) in all biological samples with
broad dynamic quantification ranges, from two to three orders of
magnitude ([55]Table 1). Intra-day and inter-day accuracy and precision
were appropriate for all determinations within the linear range.
Precision, measured as RSD, was below 10.6% and accuracy ranged from
89.15% to 104.26% ([56]Table 2). Recovery of DOX was over 77.32% in
plasma and most tissues, except for the spleen, which ranged from
66.34% to 72.23% ([57]Supplementary Table S1). The possible matrix
effects were also studied, and no significant suppression or
enhancement was observed ([58]Supplementary Table S1). Moreover, DOX
was found to be stable in all biological matrices under different
conditions, including autosampler, short- and long-term storage, and
three freeze-thaw cycles ([59]Supplementary Table S2). These results
demonstrate that the LC-MS/MS method is sensitive and accurate for the
reliable quantification of DOX present in rat plasma and six tissues.
Table 1.
The linear regression parameters for doxorubicin in rat plasma and six
tissues.
Biological Matrix Linear Range Calibration Curve r^2 LLOQ
Plasma 5–5000 ng/mL Y = 0.0116263X + 0.0132798 0.9993 5 ng/mL
Liver 20–2400 ng/g Y = 0.00274442X + 0.00861329 0.9988 20 ng/g
Heart 20–2400 ng/g Y = 0.00283048X + 0.0101572 0.9975 20 ng/g
Kidney 50–6000 ng/g Y = 0.00107142X + 0.00120827 0.9978 50 ng/g
Spleen 20–2400 ng/g Y = 0.00244706X − 0.00562543 0.9950 20 ng/g
Lung 20–2400 ng/g Y = 0.00276342X + 0.00405469 0.9979 20 ng/g
Skeletal muscle 20–2400 ng/g Y = 0.00282242X + 0.0109277 0.9977 20 ng/g
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Table 2.
The precision and accuracy for doxorubicin in rat plasma and six
tissues.
Biological Matrix Concentration Intra-Day (n = 5) Inter-Day (n = 15)
Accuracy
(%) Precision
(RSD%) Accuracy
(%) Precision
(RSD%)
Plasma 5 ng/mL 96.08 6.44 97.12 5.46
10 ng/mL 94.82 6.12 95.70 6.53
500 ng/mL 100.59 4.30 101.79 5.48
4000 ng/mL 98.15 3.54 100.75 6.13
Liver 20 ng/g 95.66 6.22 97.25 5.85
40 ng/g 90.33 4.56 93.10 5.45
400 ng/g 92.23 6.50 95.75 5.67
2000 ng/g 93.88 4.05 98.00 5.89
Heart 20 ng/g 99.82 6.85 95.99 8.79
40 ng/g 103.21 4.28 100.30 5.15
400 ng/g 96.43 3.47 100.41 5.54
2000 ng/g 104.26 5.39 100.07 6.06
Spleen 20 ng/g 96.44 6.36 97.96 6.39
40 ng/g 91.86 5.61 95.55 6.06
400 ng/g 89.15 2.11 95.04 7.75
2000 ng/g 92.44 4.49 96.44 5.98
Kidney 50 ng/g 99.50 5.46 98.78 5.90
100 ng/g 98.54 6.05 95.03 7.72
1000 ng/g 102.07 5.90 94.98 7.68
5000 ng/g 100.59 4.86 96.87 6.11
Lung 20 ng/g 90.87 10.16 95.79 9.23
40 ng/g 91.56 2.22 99.08 7.90
400 ng/g 99.31 6.76 97.93 5.50
2000 ng/g 103.38 4.95 101.80 4.40
Skeletal muscle 20 ng/g 96.81 9.01 97.02 8.55
40 ng/g 91.89 1.17 92.36 3.78
400 ng/g 95.22 3.69 99.25 5.23
2000 ng/g 98.20 4.39 100.40 4.93
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2.2. Effects of Astragali Radix on Doxorubicin Disposition
The current LC-MS/MS method was successfully applied for investigating
the plasma pharmacokinetics and tissue distribution of DOX in rats
after co-treatment with single-dose AR or pre-treatment with
multiple-dose AR ([62]Figure 1). The mean plasma concentration–time
curves of DOX are shown in [63]Figure 2A,B, and all 14 pharmacokinetic
parameters are summarized in [64]Table 3. For the two isotype control
groups that received only DOX, the pharmacokinetic parameters of the
maximum experimental concentration (C[max]), the total area under the
concentration–time curve (AUC[0-t]), and the half-life (t[1/2]) were in
the range of 838.4 ng/mL to 2157.8 ng/mL, 1054.7 µg/L*h to 1940.1
µg/L*h, and 25.0 h to 39.3 h, respectively. These results were
consistent with previously reported animal experiment data
[[65]19,[66]20]. Compared with the DOX groups, we found no significant
differences in all pharmacokinetic parameters (e.g., C[max], AUC[0-t],
t[1/2], and MRT) after AR co- or pre-treatment. Furthermore, we
explored the variation patterns of these 14 pharmacokinetic parameters
among the four groups of rats using principal component analysis (PCA).
As illustrated in [67]Figure 2C, no clear separation is observed
between the four groups, although there are two discrete samples. These
results indicate that AR co- or pre-treatment could not change the drug
disposition of DOX in vivo.
Figure 1.
[68]Figure 1
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Schematic of animal experiments. (A) Mice received 5.0 mg/kg DOX via
the tail vein after a single dose of 10 g/kg AR administration. (B)
Mice received 5.0 mg/kg DOX via the tail vein after 10 days
pre-treatment with AR (10 g/kg/d). DOX: doxorubicin, AR: Astragali
Radix.
Figure 2.
[70]Figure 2
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Astragali Radix (AR) does not affect the plasma pharmacokinetics of
doxorubicin (DOX). (A) Mean plasma concentration–time curves of DOX
after intravenous administration of DOX (5 mg/kg) alone and the
co-treatment with AR (10 g/kg). (B) Mean plasma concentration–time
curves of DOX after intravenous administration of DOX (5 mg/kg) alone
and the pre-treatment with AR (10 g/kg × 10). (C) The principal
component analysis score plot shows no clear separation among four
groups of rats. All data are presented as mean ± SD.
Table 3.
The pharmacokinetic parameters after intravenous administration of 5
mg/kg doxorubicin (DOX) following the treatment with Astragali Radix
(AR) (mean ± SD).
Parameters ^# Unit AR Co-Treatment (n = 6 per Group) AR Pre-Treatment
(n = 7 per Group)
DOX DOX + AR
(10g/kg × 1) p-Value ^$ DOX DOX + AR
(10g/kg × 10) p-Value ^$
AUC[(0-t)] μg/L*h 1212.08 ± 107.82 1265.88 ± 226.98 0.642 1303.35 ±
271.74 1208.74 ± 145.35 0.467
AUC[(0-∞)] μg/L*h 1561.04 ± 147.02 1773.14 ± 288.55 0.174 1763.3 ±
339.93 1626.07 ± 231.54 0.430
AUMC[(0-t)] h*h*μg/L 13,438.17 ± 881.77 18,109.93 ± 6093.09 0.148
17,173.75 ± 3436.44 15,248.33 ± 1891.35 0.252
AUMC[(0-∞)] h*h*μg/L 46,004.25 ± 8764.93 67427.17 ± 25,880.54 0.129
60,856.88 ± 14,612.33 55,604.05 ± 15,045.69 0.551
MRT[(0-t)] h 11.13 ± 0.74 14.38 ± 3.67 0.105 13.24 ± 1.11 12.69 ± 1.51
0.486
MRT[(0-∞)] h 29.34 ± 4.25 37.7 ± 11.6 0.161 34.59 ± 6.48 33.81 ± 5.55
0.826
VRT[(0-t)] h^2 197.67 ± 4.35 209.5 ± 15.52 0.154 212.17 ± 6.72 211.82 ±
7.21 0.933
VRT[(0-∞)] h^2 1766.66 ± 538.86 2213.68 ± 1222.38 0.472 2022.61 ±
650.14 2067.43 ± 786.43 0.916
λz 1/h 0.023 ± 0.004 0.022 ± 0.004 0.621 0.022 ± 0.003 0.023 ± 0.005
0.830
C_last μg/L 7.86 ± 0.8 10.85 ± 3.98 0.156 10.04 ± 2.17 8.99 ± 1.02
0.307
t[1/2] h 30.52 ± 4.47 32.59 ± 7.48 0.607 31.79 ± 5.12 32.05 ± 6.95
0.944
V L/kg 141.38 ± 18.15 135.98 ± 35.78 0.770 133.85 ± 28.5 142.17 ± 25.7
0.605
CL L/h/kg 3.23 ± 0.33 2.89 ± 0.43 0.189 2.92 ± 0.43 3.13 ± 0.38 0.390
C[max] μg/L 1667.94 ± 304.04 1211.41 ± 764.75 0.243 1351.21 ± 364.86
1411.01 ± 368.38 0.782
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^# Abbreviations: AUC, area under the concentration–time curve; AUMC,
area under the first moment curve; MRT, mean residence time; VRT,
variance of mean residence time; C_last, the predicted last
concentration; CL, plasma clearance; V, apparent volume of
distribution; Cmax, maximum plasma concentration. ^$ Student t-test.
At 48 h after injection, DOX concentrations in various tissues, namely
heart, liver, lung, kidney, spleen, and skeletal muscle, were detected
at levels above the LOQ. Extensive distribution was seen in some
organs, such as the kidney, spleen, and heart, whereas relatively low
levels were detected in the lung and skeletal muscle ([73]Supplementary
Table S3), which was in accordance with the known disposition property
of DOX [[74]21]. As seen in [75]Figure 3, the concentrations of DOX in
these tissues were not affected by the co-treatment with a single dose
of AR. In contrast, the pre-treatment with multiple doses of AR reduced
DOX tissue exposure but only showed statistical significance (p < 0.05)
in the liver. These data suggest that the short-term (≤10 d)
administration of AR appears not to change the tissue distribution of
DOX.
Figure 3.
[76]Figure 3
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Astragali Radix (AR) has little effect on the tissue distribution of
doxorubicin (DOX). (A) Tissue distribution of DOX at 48 h after
intravenous injection of DOX (5 mg/kg) alone and the co-treatment with
AR (10 g/kg). (B) Tissue distribution of DOX at 48 h after intravenous
injection of DOX (5 mg/kg) alone and the pre-treatment with AR (10 g/kg
× 10). All data are presented as mean ± SD. Student t-test, * p < 0.05.
2.3. Network-Based Measures of Doxorubicin–Astragali Radix Relationship
To understand why AR failed to change the plasma pharmacokinetics and
tissue distribution of DOX, we turned to quantifying the network-based
relationship between the modules related to DOX disposition and AR
components. We found that five of the nine representative components of
AR were not localized to the vicinity of the DOX disposition-associated
module, of which the network-based distances (S[AB]) values were
greater than 0 ([78]Supplementary Table S4). For example, Astragaloside
IV (AsIV) and calycosin-7-O-glucoside (C7G), two quality control
markers of AR specified by Chinese Pharmacopoeia [[79]22], were
topologically separated from DOX with S[AB] values of 0.059 and 0.085,
respectively ([80]Figure 4A). Pathway enrichment analysis further
confirmed the results of the network-based evaluation. It was revealed
that 22 enzymes involved in DOX transport and metabolism were
significantly enriched in the nitric oxide-related pathways ([81]Figure
4B), while the enriched pathways of AsIV and C7G had few overlaps with
those of DOX ([82]Figure 4C). Altogether, it is not easy for AR to
directly interfere with the DOX disposition in vivo because of their
topological separation.
Figure 4.
[83]Figure 4
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Network-based relationship between Astragali Radix (AR) components and
doxorubicin (DOX) disposition. (A) A subnetwork of the protein–protein
interactome illustrating the network-based separation between DOX,
Astragaloside IV (ASIV), and calycosin-7-O-glucoside (C7G). (B) Top 10
enriched pathways related to DOX disposition. (C) Venn diagram showing
little overlap between significantly (FDR < 0.01) enriched pathways of
DOX, ASIV, and C7G.
3. Discussion
The combination of herbal medicine and chemotherapy offers widespread,
well-documented advantages in treating cancer [[85]23]. The main
objective of this study was to uncover the effects of co- and
pre-treatment of AR on the plasma pharmacokinetics and tissue
distribution of DOX in rats. To achieve this goal, we first developed
an LC-MS/MS analytical method for the reliable quantification of DOX in
different biological matrices, including rat plasma and six types of
tissues. The proposed method enabled the accomplishment of easy sample
preparation (one-step protein precipitation), high sensitivity (LOQ < 5
ng/mL or 20 ng/g), good repeatability (RSD < 15%), and wide dynamic
range (up to three orders of magnitude). Compared with recently
reported methods developed based on the UPLC system [[86]24,[87]25],
our method, which used HPLC, considerably reduced instrument
requirements while providing acceptable throughput (11 min/run).
Moreover, the method was fully validated in each of the seven
biological matrices, following the validation criteria established by
the FDA. The validation results not only demonstrate the robustness of
the LC-MS/MS method but also guarantee sufficient sensitivity and
specificity for the reliable quantification of DOX present in different
biological samples, as well as at different time points.
DOX, in clinics, is administered as a single high dose, frequent low
doses, or a continuous infusion [[88]1]. Here, we determined the
concentrations of DOX in rat plasma and six tissues following a single
intravenous dose of 5 mg/kg. Consistent with previous studies
[[89]12,[90]26], our data show that the intravenous bolus injection of
DOX produces high plasma concentrations, which fall quickly due to a
rapid and extensive distribution into tissues ([91]Figure 3). More
importantly, we found that short-term (≤10 d) administration of AR,
whether a single-dose co-treatment or multiple-dose pre-treatment, was
ineffective at changing the plasma pharmacokinetics of DOX ([92]Table
3). Meanwhile, the tissue exposure of DOX seemed to be slightly
affected by AR pre-treatment, showing a downward trend, but only a
significant difference was observed in the liver ([93]Figure 3). The
network-based measures revealed the unexpected weak overlapping between
nine representative AR components and DOX disposition. For example,
there was only one shared node (NOS3) between the network modules of
AsIV and DOX ([94]Figure 4). Previous network medicine studies have
demonstrated that, for a drug to have a therapeutic effect, the
drug-target module must overlap with the disease module [[95]18]. Thus,
our findings suggest that AR components are unlikely to act on the
enzymes that metabolize or transport DOX. In other words, AR may
mitigate DOX-induced toxicity by affecting drug targets rather than
drug disposition. Furthermore, AR is not only a herbal medicine but
also a legal dietary supplement in both China and the USA [[96]27].
People, such as elderly adults who feel weak, may take AR as a health
product on a daily basis. Future work is needed to explore whether the
effects of AR on DOX disposition become pronounced under long-term
(months or even years) treatment.
Our study has several strengths. We used a clinically relevant regimen
of low DOX and AR doses to establish the animal model. We used an
LC-MS/MS method that was well-validated and possessed good quantitative
capability and reproducibility. The network-based approach contributed
to identifying mechanisms of the combination usage of DOX and AR.
However, there are several limitations to the current analysis. Some in
vivo metabolites of DOX (i.e., doxorubicinol) are cytotoxic, while
others (i.e., doxorubicinone) are not [[97]28]. Although AR shows a
weak effect on the concentrations of DOX in rat plasma and tissues, the
production of each metabolite of DOX may vary. To increase the coverage
of analytes, we may update the LC-MS/MS method to further determine
both DOX and its primary metabolites simultaneously. Although DOX
levels in each tissue were successfully quantified at 48 h after
intravenous administration, adding two to three sampling time points,
such as at 0.5 h, 1 h, and 4 h, may be more appropriate to fully
characterize the tissue distribution of DOX, as well as the effects of
AR. In addition, owing to the complex combination of DOX and AR dosing
regimens, further experiments on the multiple doses and long-term
administration are warranted.
4. Materials and Methods
4.1. Chemicals and Reagents
Doxorubicin hydrochloride injection was obtained from Shenzhen Main
Luck Pharmaceuticals Inc. (Shenzhen, China). Astragali Radix crude
slices were purchased from Gansu Longmaotong Chinese Herbal Medicine
Trading Co., Ltd. (Lanzhou, China). Chemical standards, including DOX
and daunorubicin, were purchased from Shanghai Yuanye Biotechnology
Co., Ltd. (Shanghai, China). LC-MS-grade methanol was bought from Merck
KGaA (Darmstadt, Germany) and HPLC-grade formic acid was obtained from
ROE Scientific Inc. (Newark, DE, USA). Water was purified with a
Milli-Q system (Millipore Corporation, Bedford, MA, USA). Isoflurane, a
general inhalation anesthetic, was purchased from Shenzhen Reward Life
Technology Co., Ltd. (Shenzhen, China) and heparin sodium was purchased
from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China).
4.2. Preparation of Astragali Radix Water Extract
The AR water extract was prepared by the procedure in our previous
study [[98]9]. Briefly, AR crude slides were accurately weighed and
boiled twice in deionized water (1:10, w/v) for 2 h each time. The two
filtrates were pooled together and then lyophilized to obtain a
freeze-dried powder. To ensure the quality of the AR extract, the
contents of 9 components in the powder were determined, including
Astragaloside I, Astragaloside II, Astragaloside III, Astragaloside IV,
Isoastragaloside II, calycosin, calycosin-7-O-glucoside, formononetin,
and ononin ([99]Supplementary Methods).
4.3. Sample Preparation
Frozen tissue samples were thawed at 4 °C. Each 100 mg of tissue was
added with 400 μL of pre-cold saline, except for the kidney sample, to
which 1000 µL of saline was added. Then, tissues were homogenized seven
times (6.5 m/s for 10 s) with 30 s intervals between the homogenization
steps. Tubes were centrifuged at 6000× g for 15 min at 4 °C, and
supernatants were transferred to clean tubes. A 50 μL aliquot of plasma
or tissue homogenate was added with 10 μL of internal standard (IS)
solution (daunorubicin, 500 ng/mL) and 200 μL of methanol. The mixture
was thoroughly vortexed for 5 min and centrifugated twice at 19,000× g
for 10 min at 4 °C. The supernatant was transferred for further
LC-MS/MS analysis.
4.4. LC-MS/MS Analysis
Chromatographic separation was performed in a Nexera UPLC system
(Shimadzu, Japan) equipped with a ZORBAX SB-C18 column (150 mm × 2.1
mm, 5 μm, Agilent, Santa Clara, CA, USA). The sample injection volume
was 5 µL. The mobile phase consisted of 0.1% formic acid in water (A)
and methanol (B) with a 0.3 mL/min flow rate. An 11 min elution
gradient was performed as follows: the proportion of B was set at 60%
for the first 1 min, increased linearly to 95% in the next 2.5 min, and
then maintained for 2 min; finally, the initial conditions were
restored within 1.5 min and kept for 4 min for column conditioning. The
temperatures of the column and autosampler were set at 30 °C and 4 °C,
respectively.
MS detection was performed using a Triple Quad^TM 8040 system
(Shimadzu, Japan) equipped with an electrospray ionization (ESI) source
working in the positive-ion mode. The optimized multiple reaction
monitoring (MRM) transitions of DOX and IS were m/z 544.1 → 397.1 and
m/z 528.1 → 320.9, respectively. Collision energy (CE) was set at 12 V
for DOX and 29 V for IS. The key instrument parameters were set as
follows: capillary voltage, 4.5 kV; nebulizing gas, 3 L/min; drying
gas, 15 L/min; heat block temperature, 400 °C; desolvation temperature,
250 °C. Raw data were obtained and processed using LabSolutions LCMS
software 5.86 (Shimadzu, Japan).
4.5. Method Validation
The LC-MS/MS method was developed following the compliance criteria
described by the FDA Bioanalytical Method Validation Guidance for
Industry, in terms of lower limit of detection (LLOD), lower limit of
quantification (LLOQ), linearity, precision, accuracy, recovery, matrix
effect, and stability.
Working solutions were spiked with blank plasma or tissue homogenate to
yield a calibration curve. The 8-point calibration curve was
constructed by plotting the peak area ratios of DOX to the IS against
the nominal concentrations of DOX. Each calibration curve was
statistically analyzed using weighted least squares. LLOD and LLOQ were
calculated at a signal-to-noise ratio (S/N) of at least 3 and 10,
respectively. The inter-day and intra-day accuracy and precision were
evaluated by analyzing four (LLOQ, low, medium, and high) levels of
quality control (QC) samples on three consecutive days, and each level
contained five replicates on the same day. Precision was expressed by
the relative standard deviation (RSD), with the acceptance criteria of
less than 20% for the LLOQ and 15% for the other concentrations.
Accuracy was calculated by the percentage ratio of measured
concentrations to the nominal value, with the acceptance criteria of
80–120% for the LLOQ and 85–115% for the other concentrations.
A recovery experiment was carried out at three (low, medium, and high)
levels of QCs, with five replicates at each level. Recovery was
calculated by comparing the analytical results of extracted samples
with corresponding extracts of blanks spiked with the analyte
post-extraction. Matrix effect (ME) was evaluated at two (low and high)
QC levels and calculated by comparing the peak areas of DOX and IS
spiked into the post-extracted biological matrix with the solution
containing the equivalent amounts of analytes. There was no matrix
effect if the ratio was in the range of 85–115%. The stability of DOX
was assessed in terms of autosampler (4 °C for 12 h), benchtop (room
temperature for 10 h), three freeze-thaw cycles, and long-term (−80 °C
for 30 d). Stability QCs were compared against freshly prepared QCs and
considered stable if the accuracy was within 85–115% and RSD ≤ 15%.
4.6. Pharmacokinetics and Tissue Distribution Studies
Male Sprague-Dawley (SD) rats (220 ± 20 g) were purchased from Shanghai
Sippr-BK Lab Animal Co., Ltd. (Shanghai, China). Animals were housed in
a standard laboratory condition with controlled temperature (25 °C),
humidity (45 ± 5%), and dark/light cycle (12/12 h). All experimental
procedures were conducted according to the guidelines for the care and
use of laboratory animals and approved by the Animal Ethics Committee
of China Pharmaceutical University.
To uncover the effects of single-dose AR administration on DOX
pharmacokinetic behavior and tissue distribution, 12 rats were randomly
divided into two groups (n = 6 per group): DOX and DOX+AR co-treatment
groups. Animals in the DOX+AR co-treatment group received a single dose
of 5.0 mg/kg DOX via the tail vein immediately after 10 g/kg AR
intragastrical administration. In contrast, the DOX group was given the
same dose of DOX and an equal volume of water. An additional 14 rats
were employed to investigate the effects of multiple-dose AR
pre-treatment. Animals were randomly divided into two groups (n = 7 per
group) that were given a single dose of DOX (5.0 mg/kg, i.v.) after 10
days of pre-treatment with AR (10 g/kg/d, i.g.) or an identical volume
of water.
Approximately 0.2 mL of blood samples were collected from the jugular
vein catheter, at 0, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 24, 32, and
48 h, into centrifuge tubes containing heparin. Plasma samples were
obtained after centrifugation (2000× g for 10 min at 4 °C). A
corresponding volume of saline was supplemented after each blood
collection. All rats resumed eating 4 h after DOX administration and
were free to move around and drink during the pharmacokinetic
experiment. The rats were sacrificed by anesthesia at 48 h after DOX
administration. Tissue samples, including heart, liver, lung, kidney,
spleen, and skeletal muscle, were harvested. All plasma and tissue
samples were stored at −80 °C for further analysis.
Pharmacokinetic parameters were calculated using DAS software 3.2.8
(Mathematical Pharmacology Professional Committee of China, China) with
the noncompartmental method. Differences in continuous variables were
compared using Student’s t-test. p < 0.05 was considered significant.
4.7. Network Analysis
To evaluate the network-based relationship of DOX disposition (A) and
an AR component (B), we used a recently introduced separation measure
[[100]29]:
[MATH:
SAB≡〈dAB〉−〈dAA〉+〈
dBB〉2 :MATH]
where
[MATH:
〈dAA
〉 :MATH]
and
[MATH:
〈dBB
〉 :MATH]
are the shortest distances between proteins within the network modules
of DOX metabolism and an AR component, respectively;
[MATH:
〈dAB
〉 :MATH]
is the shortest distance between A-B protein pairs. For S[AB] < 0, the
proteins of DOX and an AR component are located in the same network
neighborhood, while for S[AB] ≥ 0, the two drug proteins are
topologically separated.
A state-of-the-art human PPI, including 18,198 unique proteins and
271,278 edges, was used as the background network in this study
[[101]30]. We collected 22 enzymes that play a role in DOX transport
and metabolism from PharmGKB ([102]www.pharmgkb.org/, accessed on 9
June 2022) and selected 9 quantifiable components as representatives of
AR, whose targets had been reported in our previous work
([103]Supplementary Data) [[104]9]. The network analysis was performed
using Mathematica software (version 13.0, Wolfram Research, USA).
5. Conclusions
In summary, we reported the effects of AR on the pharmacokinetics and
tissue distribution of DOX using a novel analytical method to determine
DOX in rat plasma and six tissues. Specifically, we observed that the
plasma pharmacokinetics of DOX was not affected by AR co- and
pre-treatment, whereas DOX concentrations in six tissues were slightly
decreased under AR pre-administration for 10 consecutive days. In
addition, network-based measures revealed that representative AR
components could hardly act on the enzymes involved in DOX metabolism
and transport. This study provided valuable data for understanding the
DOX–AR combination and might help uncover the mechanisms of AR in
alleviating DOX-induced toxicity.
Acknowledgments