Effect of Panax notoginseng saponins on the pharmacokinetics of aspirin in rats

Accepted Manuscript Title: Effect of Panax notoginseng saponins on the pharmacokinetics of aspirin in rats Author: Zhihao Tian Huanhuan Pang Shouying Du Yang Lu Lin Zhang Huichao Wu Shuang Guo Min Wang Qiang Zhang PII: DOI: Reference:

S1570-0232(16)31375-7 http://dx.doi.org/doi:10.1016/j.jchromb.2016.12.007 CHROMB 20375

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

31-10-2016 29-11-2016 5-12-2016

Please cite this article as: Zhihao Tian, Huanhuan Pang, Shouying Du, Yang Lu, Lin Zhang, Huichao Wu, Shuang Guo, Min Wang, Qiang Zhang, Effect of Panax notoginseng saponins on the pharmacokinetics of aspirin in rats, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2016.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The effect of PNS to ASA in vitro and vivo experiment

Effect of Panax notoginseng saponins on the pharmacokinetics of aspirin in rats ZhihaoTian Huanhuan Pang Shouying Du* Yang Lu* Lin Zhang Huichao Wu Shuang Guo Min Wang Qiang

Zhang

School of chinese Materia Medica, Beijing University of Chinese Medicine, chaoyang District, Beijing, People’s republic of China *

Corresponding author: Pro. Shouying Du and Yang Lu, School of Chinese Materia Medica, Beijing University of Chinese Medicine, 6#, WangjingZhonghuanNanlu, Chaoyang District, Beijing 100102, China.

Tel: +0086-10-84738615

E-mail: [email protected] and [email protected]

1

The effect of PNS to ASA in vitro and vivo experiment

Highlights 1. In summary, this is the first report to evaluate the drug-drug interaction between aspirin and Panax Notoginseng Saponins about pharmacokinetics in the rats, which was considered to be the proper model to simulate human model. 2. The pharmacokinetic curve and Cmax of salicylic acid show marked increase when the drugs were taken together. 3. Cell experiment is also used to study the absorption of aspirin and salicylic acid. The result of cell experiment is consistent with the pharmacokinetic study.

2

The effect of PNS to ASA in vitro and vivo experiment

Abstract Aspirin (ASA) is widely used to treat fever, pain, inflammation and cerebral infarction in clinic. Panax Notoginseng Saponins (PNS) is the extracts of Panax Notoginseng (PN)-a traditional Chinese medicine extensively used in cardiovascular diseases. Panax notoginseng saponins and ASA are both widely used to treat cerebral infarction in China. Good results in clinical practice have been achieved when the two drugs were taken together. To investigate the effect of PNS on ASA in vivo, the concentrations of salicylic acid (SA) in blood were measured after oral administration of ASA or ASA combined with PNS by UPLC-MS/MS. Sample preparation was carried out by the protein precipitation technique with an internal Saikosaponin A（Fig.1）standard. The separation of two components was achieved by using an ACQUITY UPLC ®BEH C18 Column (1.7μm 2.1×100mm) by gradient elution using water (containing 0.2% formic acid) and acetonitrile (containing 0.2% formic acid) as the mobile phase at a flow rate of 0.2 mL/min. The pharmacokinetic parameters were determined by using non-compartmental analysis. The results suggested that drug-drug interaction in vivo existed between PNS and ASA. The concentration of the SA was increasing when the two drugs were administered together. The transport of ASA and SA in MDCK -MDR1 cell monolayer was used to verify this conclusion. The values of 3

The effect of PNS to ASA in vitro and vivo experiment

apparent permeability coefficients (Papp) were significantly increased when the two drugs were used together. This result suggested PNS could increase the gastrointestinal tract absorption of ASA and SA. These findings provide more insight for wise use of two drugs to treat or prevent cardiovascular diseases. Key words: Aspirin, Panax Notoginseng Saponins, Drug-drug interaction, Pharmacokinetic, Transport 1. Introduction ASA (Fig.1), also known as acetylsalicylic acid, is a classic drug in the history of medicine and widely used to treat pain [1], fever [2] and inflammation [3]. It is usually used to treat or prevent heart attacks and stroke

[4].

ASA is

one

of

the

most

common

OTC

drugs.

The consumption of aspirin is about 40-50 tons worldwide each year [5]. PNS which mainly contained Notoginsenoside R1(NGR1), ginsenoside Rg1(GRg1), Rb1(GRb1), Re(GRe), Rd(GRd)（Fig.1） and are extracted from

Panax notoginseng

(Burk.)

F.H.

Chen

(Sanqi)

and

also

extensively used in cardiovascular disease [6]. It has been widely used for over 400 years and still holds a unique position in today’s regional market, with remarkable annual sales of 5000 tons in China alone [7]. The elderly people, a special crowd with the decline of heart, liver and kidney function, are easier to contract a variety of drugs than the other groups [8, 9]. Due to

4

The effect of PNS to ASA in vitro and vivo experiment

this phenomenon, combination drug therapies were found to be used in the treatment of many diseases for elderly people [10, 11]. According to the reports

in

American

Medical

Association

(JAMA),

the incidence of adverse reactions was 6% to 50% respectively, when two to five drugs were taken together [12,13,14,15]. In China, the combination use of ASA and PNS is often to be found to treat or prevent cardiovascular diseases [16].

Although many clinical data indicated a drug–drug

interaction between PNS and ASA in vivo process, clinical researchers only focused on the observation of pharmacodynamical effects at present [17, 18, 19, 20]. Pharmacokinetic study is indispensable to confirm the alleged effect of PNS on ASA, and potentially provides some clues regarding its underlying mechanism. The dose of aspirin is from 75 to 300mg to treat or prevent cardiovascular diseases each day in China [21]. As reported, the t1/2 of ASA is about 15-20 min [22]. ASA is rapidly decomposed into salicylic

acid

(SA)

in

the

blood

and

gastrointestinal

tract

after giving medicine [23, 24,25]. At present, pharmacokinetics study of ASA is often replaced by SA. So we selected SA to study the pharmacokinetics of ASA. The reported bioanalytical methods for the determination of SA in biological matrices were mainly based on HPLC[26,27], GC[28] and LC-MS/MS[29,30]. Comparare to other methods, LC-MS/MS was able to achieve lower limit of quantitation in

5

The effect of PNS to ASA in vitro and vivo experiment

pharmacokinetic study. Based on this, SA was determined by using LC-MS/MS in this study. The reported HPLC and Mass spectrometry operating conditions of LC-MS/MS used acetonitrile, water with formic acid, negative electrospray ionization mode and multiple reaction monitoring (MRM) mode to separate and determined SA. Peaks which were sharp, symmetrical and good separation were got by these conditions. The Caco-2 cells (the human colonic adenocarcinoma cell line), MDCK cells (Madin-Darby canine kidney cell line), which have absorption properties of human intestinal tissue, are widely used to study intestinal absorption [31, 32]. Like Caco-2, MDCK cells into

a

showed

columnal highly

morphological semi permeable

epithelium

functionalized

to

epithelial

and biochemical membrane.

form

have

tight barrier

similarity

differentiated

junctions, with

when

and

remarkable

cultured

on

MDCK cells grow faster than Caco-2,

which significantly shorten experimental period [33, 34, 35]. Besides, compared with as Caco-2, MDCK cells express fewer subtypes. As a consequence, the results showed higher reproducibility [36, 37]. In a word, MDCK cells were selected to study the transport of ASA and SA.

6

The effect of PNS to ASA in vitro and vivo experiment

Fig.1 Chemical structures of ASA, Sa, NR1, GRg1, GRb1, GRd, GRe, Saikosaponin A.

7

The effect of PNS to ASA in vitro and vivo experiment

2. Materials and methods 2.1. Chemicals SA, ASA and Saikosaponin A were got from the National Institute for Food and Drug Control (Beijing, China). PNS were obtained from Yunnan Baiyao

Group

Co.,

Ltd.

PNS

contents

were

determined

as:

Notoginsenoside R1 (NGR1), 6.9%; GinsenosideRg1 (GRg1), 28.0%; Ginsenoside Rb1 (GRb1), 29.7%; Ginsenoside Re (GRe), 3.8%; Ginsenoside Rd (GRd), 7.3%. Raw material medicine of SA, ASA were purchased from Xi'an Yue Lai Medicine Technology Co., Ltd. Culture flasks (25 cm2growtharea),polyester (PET) cell culture inserts(12 mm diameter, 0.4μm pore size), 96-well plates, Costar 12-well plates and Transwell were got from Corning Costar Corporation (MA, USA) and untreated white opaque multi well plates were got from NUNC Denmark. Acetonitrile (HPLC grade) was got from Fisher Scientific. HPLC-quality water was obtained using a Cascada™ IX-water. All other chemicals used in this experiment were analytical grade. 2.2 Animals and cell experiment Male Sprague-Dawley (SD) rats (240-260 g) were supplied by Vital River Laboratory Animal Technology Co. Ltd. (Beijing, People’s Republic of China). Animals were housed in the Beijing University of Chinese Medicine Laboratory Rats were acclimatized for 7 days before the start of 8

The effect of PNS to ASA in vitro and vivo experiment

experiments. Animals were kept in a controlled environment with consistent temperature and humidity, 12-hour light/12-hour dark cycle, as well as abundant food and water. All the animal studies were performed under the Guidelines for the Care and Use of Laboratory animals and the experimental protocols were approved by the institutional animal experimentation committee of Beijing University of Chinese Medicine. MDCK-MDR1 cells were got from Dr. Zeng (Zhejiang University, People’s Republic of China) cultured in DMEM with 10% heat-inactivated fetal bovine serum (FBS) as well as 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific). All cell lines were maintained at 37℃ under a humidified atmosphere containing 5% CO2. For transport experiments, MDCK-MDR1 cells with passage numbers of 77-83 were seeded at a density of 2.5×105 cells/cm2 on PET inserts. Then the cells were maintained at 37℃ with 5% CO2 for five to seven days to reach confluence. Fresh media were changed every other day. 2.3 Instruments and conditions 2.3.1 Instruments and conditions for Pharmacokinetics Liquid chromatography The UPLC system consisted of a solvent delivery unit (Nexera X2 LC-30AD; Shimadzu (China), an operating system software (LC solution; Shimadzu). The separation of all samples was obtained with an ACQUITY 9

The effect of PNS to ASA in vitro and vivo experiment

UPLC ®BEH C18 (1.7μm 2.1×100mm Column) reversed-phase column. The auto-sampler was set at 16℃, and the gradient elution was employed with 0.2% formic acid acetonitrile solution as solvent A and 0.2% formic acid aqueous solution as solvent B. The gradient program was as follows: 0-3 min, 20-20% A; 3-5min, 20-50% A; 5-7 min, 50-100% A; 7-8 min, 100-100% A; 8-8.01min, 100-20% A; 8.01-12 min, 20-20% A. The flow rate was set at 0.2 mL·min-1, and the injection volume was 10µL. The total run time was 12 min for each sample. Mass spectrometric conditions Mass

spectrometric detection

was

performed

on 4500 AB

QTRAP-LC/MS/MS (Palo Alto, CA, USA) equipped with an electrospray ionization source (ESI). The ESI source was set in positive ionization mode with the capillary voltage set at 3500 V. The dwell time was 100 ms, and the other parameters in the source were set as the following: source temperature 350℃; desolvation gas flow 10 L/min; nebulizer gas (N2) pressure 40 psi. The mass spectrometer scanned in multiple reactions monitoring (MRM) mode. The mass spectrometer was operated in negative ion mode using MRM (multiple reaction monitoring) to assess SA and IS: m/z 137.0→92.0 for SA, m/z 779.4→617.3 for IS. The optimized collision energies were -56 eV and -55 eV respectively. 2.3.2 Instruments and conditions for Cell experiment 10

The effect of PNS to ASA in vitro and vivo experiment

ASA and SA in the samples were analyzed by HPLC using a Hibar 250-4,6 Purospher STAR RP-18e LP（5 μm）. The conditions of HPLC for ASA were as follows: The mobile phase consisted of 60:40 (v/v) acetonitrile: 0.1% phosphoric acid pumping at a flow rate of 1.0 mL/min and the samples were analyzed with UV detection (λ= 276 nm).The injected volume was 10 µL and the retention time of ASA was about 7 min. The conditions of HPLC for SA were as follows: The mobile phase consisted of 60:40(v/v) acetonitrile: 1% formic acid pumping at a flow rate of 1.0 mL/min and the samples were analyzed with UV detection (λ= 303 nm).The injected volume was 10 µl and the retention time of SA was about 8 min. 2.4 Sample preparation Pharmacokinetic samples: The protein precipitation with acetonitrile was finally chosen to prepare samples with comprehensive consideration. Plasma samples which were diluted to a certain concentration with blank blood, 200 μL were treated with 600 μL ethyl acetate containing the IS (Saikosaponin A) (50 ng/mL). The mixture was vortexed for 3 min and then centrifuged at 14,000 rpm for 10 min at 4 ℃. The supernatant was transferred into a 1.5 mL centrifuge tube and evaporated to dryness. The residue was reconstituted in 200μL of ethyl acetate/water (2:8, v/v), vortexed for 3min and then 11

The effect of PNS to ASA in vitro and vivo experiment

centrifuged at 14,000 rpm for 10 min at 4℃. After centrifugation for twice, the supernatants were transferred to vials, and 10μL of each was injected into the 4500 AB QTRAP-LC/MS/MS system for analysis at 4 ℃. Cell samples These samples were centrifuged at 14,000 rpm for 10 min. The supernatant fluid was transferred to vials, and 10μL of each was injected into the HPLC-UV. 2.5 Method validation 2.5.1 Method validation for UPLC-MS/MS The method for this study was validated according to the USA Food and Drug Administration (FDA) bioanalytical method validation guidance. Specificity Mass spectra of blank plasma, drug-containing plasma samples obtained from rats after oral administration ASA and blank plasma spiked with analyse and IS (50 ng/mL) were compared to investigate the specificity and shown in Fig. 2.

12

The effect of PNS to ASA in vitro and vivo experiment

Fig.2 Representative Mass spectra (daughter ion scans) and MRM chromatograms of the analytes and internal standards: of (A) SA; (B) internal standard (Saikosaponin A); (C) a blank plasma sample spiked with the analytes; (D) a drug-containing plasma sample;

(E) a blank

plasma sample. Linearity and LLOQ The calibration curves were obtained by determining consisted of at least seven points for SA. These samples were prepared by adding 20 µL of internal standard solution and 180 µL working solutions in blood samples and submitted to the same analytical procedure described for sample preparation. The peak-area ratios of SA to internal standard were calculated and plotted with fitted by least-square regression using 1/x2 as the weighting factor. The lower limit of quantification (LLOQ) was determined at the lowest detectable concentration, taking into consideration a 1:10 base-line noise-calibration point ratio. It was repeated six times for

13

The effect of PNS to ASA in vitro and vivo experiment

confirmation of precision and accuracy less than 20%. Precision and accuracy The intra-day precision and accuracy were assessed by determining the concentrations of QC samples of plasma at three levels using six replicates during the same day. The concentration of each QC sample was obtained by using the calibration curves prepared that day. Three batches of QC samples were analyzed on three consecutive days to measure the inter-day precision and accuracy. The precision was defined as RSD. The accuracy was expressed as relative error (RE). Extraction recovery rates and matrix effects Extraction recovery was calculated by comparing the responses of QC samples at three levels that were spiked with analytes prior to extraction with the response of those that were spiked with blank plasma. The matrix effect was assessed in a similar way. Analytes for the SA were added to the extract of precipitated blank plasma to achieve three concentration levels. These peak areas were compared with those obtained by adding the same concentration of analytes in 20% acetonitrile-water solution, and it was considered negligible if values below ±15% were observed. Stability The stability was tested by analyzing QC samples at three 14

The effect of PNS to ASA in vitro and vivo experiment

concentration levels exposed to different conditions: at room temperature for 4 h before sample preparation (short-term stability), at room temperature for 12 h in the autosampler after sample preparation (autosampler stability), after three freeze/thaw cycles from -20℃ to ambient temperature (freeze-thaw stability) and at -20 ℃for four weeks (long-term stability). The obtained results were compared with the freshly prepared QC samples and the percentage concentration deviation was calculated to evaluate stability. 2.5.2 Method validation for HPLC-DAD Linearity, limits of detection and quantification were tested to validate the HPLC-DAD method. Precision, reproducibility and stability were tested to study the method of HPLC-DAD. Precision includes intra- and inter-day reproducibility. The standard solution was determinated at different times (a single day and three consecutive days, respectively). Six independent sample solutions were tested for repeatability evaluation. Stability evaluation which was used a sample solution was tested within 12 h. Recovery was calculated by using the formula: recovery (%) = (observed amount -original amount)/spiked. 2.6 Pharmacokinetics study Twelve male SD rats (240-260 g) were used in the pharmacokinetic study, and all rats were divided into two groups. Group One: Six rats were 15

The effect of PNS to ASA in vitro and vivo experiment

orally administered the ASA suspended in 0.5% carboxymethyl cellulose sodium aqueous solution at 20.83 mg/kg. Group two: Six rats were orally administered the ASA and PNS suspended in 0.5% carboxymethyl cellulose sodium aqueous solution at 20.83 mg/kg and 30.25 mg/mL. Rats were assigned to each group representing 0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h and 24 h. The blood samples were drawn from the retro-orbital plexus of each rat and placed into heparinized microfuge tubes. All of the collected blood samples were immediately centrifuged at 14,000 rpm for 10 min to obtain plasma, which was labeled and frozen at -80 ℃ until analysis. Transport study In order to further study the effect of PNS on the permeability of ASA and SA penetration amount, cell monolayers were used to analyze the transport of SA and ASA. MDCK-MDR1 cells were seeded at a density of 1×105 cells·cm-2 onto 12 mm Transwells with 0.4μm poresize collagen-coated clear polyester membranes and formed the monolayers (TRE reached about 250-350Ω·cm-2). Before each experiment, the cells were washed three times with HBSS and equilibrated for 30 min at 37°C. 0.5mL of drug solution was added in A side and 1.5 mL of HBSS in B side to measure A→B transporter. Cells were incubated in a 37℃ shaking incubator. 600 μL of samples were collected from the basal side at 30, 60,

16

The effect of PNS to ASA in vitro and vivo experiment

90, 120, 150 and 180 min. Samples (200 μL) were collected from the A side at the same time and measured with the same HPLC method used to assess A→B transport. 2.7 Data analysis The pharmacokinetic parameters were calculated using drug and Statistics 3.0 (DAS 3.0, Mathematical Pharmacology Professional Committee of China, and Shanghai, China). The data were analyzed with one-way analysis of variance to compare differences between combined drugs group and the control group (SPSS 16.0 software, USA). Significance was set at P<0.05. 3 Results 3.1 Results for Pharmacokinetic Method validation Specificity A good separation of the analyses was achieved and no significant endogenous peak in plasma to interfere with SA and Saikosaponin A, was observed. Linearity and LLOQ Good linearity (r>0.9900) and wide linear range were obtained

17

The effect of PNS to ASA in vitro and vivo experiment

according to the established method. The LLOQ of SA was acceptable, with RSD of less than 20%. Table 1 The linearity and LLOQ of SA Analytes

SA

Linear equation

r

y = 0.020x + 0.453

0.9970

Linear range

LLOQ

(ng·mL-1)

(ng·mL-1)

1-1000

1

Accuracy and precision The intra-day (n = 6) and inter-day (n = 6) accuracies and precisions of three analytes were evaluated by testing three concentrations of QC samples (4, 50, 500 ng/mL). The results were shown in Table 2. All results of tested samples were within the acceptable range of ±15%.

18

RSD(%)

2.3

The effect of PNS to ASA in vitro and vivo experiment

Table 2 Accuracy and precision of the SA at three QC concentrations in rat plasma (n=6). Precision (RSD %)

Concentration Analyte

Accuracy (RE %) (ng/mL)

SA

Intra-day

Inter- day

4.00

2.20

3.02

2.20

50.00

7.80

3.28

2.70

500.00

-9.10

2.83

2.80

Abbreviations: RSD, relative standard deviation; RE, relative error; Extraction recovery and matrix effect The extraction recovery (n=6) of SA at three QC levels (4, 50, 500 ng/mL) was calculated by comparing the signal of PNS in blank blood samples spiked before extraction with that of samples to which PNS had been added post-extraction, at the same concentrations. The Matrix effects at three QC Levels were evaluated by comparing the peak areas of samples spiked with pure standard solutions to those containing equivalent concentrations of SA. Table 3 Extraction recovery and IS normalized matrix effect of SA at three QC concentrations in rat plasma (n=6). Spiked

Extraction recovery (%

Matrix

Analyte C(ng/mL)

± SD/%)

19

(% ± SD/%)

The effect of PNS to ASA in vitro and vivo experiment

SA

4.00

104.28±9.31

107.79 ± 5.38

50.00

98.29 ± 9.23

103.58 ± 2.84

500.00

98.72 ± 5.11

105.40±5.53

Abbreviations: SD, standard deviation; RE, relative error. Stability The stability of the three analytes in rat plasma under different conditions was presented in Table 4. Comparing with those in freshly spiked plasma, the concentrations of SA in plasma under different conditions deviated less than 15%. The results indicated a good stability. Table 4 Stability of SA at three QC concentrations in rat plasma (n = 6).

Composition

RE (%)

Spiked Short-term(4h at Room

Post-treatment(24

Three-thaw

Long-term

Temperature)

h at sample pool)

cycles

(1 month at

(ng/mL)

-80 ℃)

SA

4.00

-9.08

-8.63

-7.32

6.45

50.00

-0.16

3.50

-6.46

3.23

500.00

-3.37

-13.47

-5.67

-4.12

Abbreviations: RE, relative error. Pharmacokinetic study 20

The effect of PNS to ASA in vitro and vivo experiment

The validated method was successfully applied to the pharmacokinetic studies of SA in rat plasma after oral administration of ASA (20.83 mg/kg) and ASA (20.83 mg/kg) combined with PNS (30.25 mg/kg). The plasma concentration-time curves were shown in Fig. 3, and the pharmacokinetic parameters were presented in Table 5. 40000

Concentracion (ng/mL)

35000 30000 25000 20000

ASA

15000

ASA+PNS

10000 5000 0 0

5

10

T (h)

15

20

25

Fig. 3 Plasma concentration-time curves of SA in rat plasma after oral administration of PNS and PNS combined with ASA (n=6).

21

30

The effect of PNS to ASA in vitro and vivo experiment

Table 5 Pharmacokinetic parameters for SA after oral administration of ASA and ASA combined with PNS (n=6) SA

ASA

ASA+ PNS

Tmax(h)

4.00±0.00.

3.00±1.10

Cmax(ng/mL)

19803.33±819.21

33960.00.±3875.28

AUC0-t(ng/mL*h)

105453.95±3625.86

202300.23±10121.54

MRT(h)

4.56±0.07

4.92±0.20

T1/2(h)

4.40±0.05

3.74±0.03

Cl（L/h/kg）

0.20±0.01

0.04±0.00

Fr

100%

191.84%

Notes: Cmax, maximum concentration; AUC, area under the curve; Cl, clearance; MRT0-t, mean residence time; Tmax, time for peak concentration; T1/2, elimination half-time. Fr, relative bioavailability.

Abbreviations: SD, standard deviation. The plasma concentration–time curves of SA showed marked change

22

The effect of PNS to ASA in vitro and vivo experiment

between ASA and PNS combined with ASA in present study. Then significant pharmacokinetic differences were also found between PNS and PNS combined with ASA. Comparing with oral administration of ASA, the pharmacokinetic parameters such as Cmax (maximum concentration), AUC0-t (area under the curve) were significantly different after oral administration of PNS combined with ASA (p < 0.05). ASA combined with PNS -administration markedly improved the systemic levels of them in vivo, with AUC0-t and Cmax increasing from 105453.95 to 202300.23 h·ng/mL

and

from

19803.33

to

33960.00

ng/mL,

respectively.

It is generally known that a series of side effects can be caused by the increasing of serum concentration of ASA and SA [38]. This phenomenon should be paid more attention in clinical practice. 3.2 Results for Transport study Method validation The RSD of HPLC-DAD method study, such as linearity, limits of detection and quantification, precision, reproducibility, recovery and stability were deviated less than 15%. The results indicated that the validated method could be successfully applied to the transport of ASA and SA in MDCK-MDR1. Measurement of the change of cell TEER after exposed with different compounds 23

The effect of PNS to ASA in vitro and vivo experiment

Cell TEER was used to investigate the changes of tight junctional function for observing the cell membrane integrity in MDCK-MDR1. After the cells formed monolayer, drug solution (0.5 mL) and Hank’s balanced salt solution (HBSS, 1.5 mL) were added in apical (A) side and basolateral (B) side to simulated A→B transporter, respectively. B→A transporter was evaluated adding drug solution (1.5 mL) in B side and HBSS (0.5 mL) of in A side. TEER of untreated cells and cells treated with SA or ASA were determined at 30 min, 60 min, 90 min, 120 min, 150 min and 180 min. The measured TEER before the experiment was set as 100% and all other values were calculated according to this. Then the relative TEER in each time point was compared to the control group and statistically analyzed. The TEER value gradually decreased, whereas the values of more than ninety-five percent. This phenomenon suggested that cell membrane was relatively stable during the test period. The situation of SA and ASA transport across MDCK-MDR1 cells The Papp for ASA and SA were calculated according to the following Eq. (1): Papp=

(dQ/dt) CS

(1), where dQ/dt is the apparent appearance rate of

ASA or SA in the receiver side calculated using linear regression of amounts in the receiver chamber versus time, C is the ASA or SA concentration in the donor chamber and S is the surface area of the polyester membrane of Transwell. The efflux ratio (ER) was calculated 24

The effect of PNS to ASA in vitro and vivo experiment

according to the following Eq. (2): ER=

Papp (B→A) Papp (A→B)

(2).

SA and ASA transport was studied at three different concentrations (50, 100 and 200 μg·mL-1) across MDCK-MDR1. The result was shown in Table 6. The effects of PNS on ASA or SA transport in MDCK-MDR1 cells were shown in Table 6. The effects of PNS on ASA and SA were studied at 150, 100 and 50 μg·mL-1 concentrations in the presence of 50 μg·mL-1 ASA or SA. After combined with different concentrations of PNS, Papp (A→B and B→A) values did differ significantly from the control group (P > 0.05). So the result indicated that PNS could give rise to the change of the fluxes of ASA or SA. These results were consistent with the pharmacokinetic study. Drug-drug interaction between ASA and PNS occurred mainly in absorption process. Table 6 Increasing concentration of ASA or SA transport across MDCK-MDR1 cells monolayer. Papp(10-6cm•s-1)

Concentration Group

ER -1

ASA

（µg•mL ）

AP→BL

BL→AP

50

1.49±0.090

2.33±0.225

1.56

100

2.27±0.059

3.37±0.146

1.48

200

1.99±0.086

2.44±0.103

1.49

25

The effect of PNS to ASA in vitro and vivo experiment

ASA：PNS

SA

SA：PNS

50：50

3.21±0.310**

4.12±0.253**

1.28

50：100

3.02±0.249**

4.18±0.223**

1.38

50：150

2.91±0.210**

3.38±0.0.236**

1.29

50

2.51±0.069

4.12±0.460

1.35

100

3.28±0.483

4.38±0.734

1.34

200

4.71±0.209

6.25±0.159

1.33

50：50

7.01±0.450**

10.46±0.253**

1.49

50：100

4.56±0.249**

6.78±0.280**

1.48

50：150

5.44±0.210**

6.51±0.0.366**

1.20

*P<0.05 **P<0.01 Notes: Papp, permeability; A, apical side; B, basolateral side. Values shown as mean ± SD (n=3). Abbreviations: ER, efflux ratio; SD, standard deviation. Discussion Drug exposure is a crucial determinant of drug response and therefore its efficacy and safety. In the current study, we compared the systemic exposure to ASA and ASA combined with PNS. In this study, pharmacokinetics and cross-membrance transportation of aspirin indicated that good membrane permeability is the primary factor limiting systemic exposure to ASA and its metabolite. The absorption rate of SA was also increasing when the two drugs were taken together. This phenomenon was

26

The effect of PNS to ASA in vitro and vivo experiment

consistent with the pharmacodynamical effects which were found by clinical researchers [39]. However, a series of side effects might be caused by the increasing of serum concentration of SA and ASA especially for eldly people [40, 41]. Transepithelial fluxes of SA and ASA were investigated in both transport directions (A→B and B→A) to determine whether its transport was polarized. The sample contains high concentration nonvolatile inorganic salts, so the HPLC-UV was selected to determine the SA or ASA. The values of Papp (A→B) of SA and ASA were between 1.49 and 2.27×10−6 cm·s-1 and between 2.51 and 3.28×10−6 cm·s-1 in MDCK-MDR1 cells. The value of Papp (B→A) of ASA and SA were between 2.33 and 3.37×10−6 cm·s-1 and between 4.12 and 6.25×10−6 cm·s-1 in MDCK-MDR1 cells. The efflux ratios (ER) [42, 43] of each concentration of ASA and SA were 0.5-2 in MDCK-MDR1 cells. These results suggested ASA and SA were easy to be absorbed by the small intestine and non-active transport. However, intestinal absorption is a complicated and comprehensive process. Therefore, this study is not a complete description of this type of transport, and further research is required. A selective and sensitive UPLC–ESI–MS/MS method for the determination of SA was established in this study. Even though there are many HPLC, GC and LC-MS/MS methods of , there are many challenges

27

The effect of PNS to ASA in vitro and vivo experiment

for bioanalytical sample analysis of SA. Interference with the analyte peak and high levels of noise are the main difficult to determine SA in biological matrices [44]. This method which we established has shortened detection time of 12.0 min and lower LLOQ value of 1ng/mL and overcomes the above difficulties. A successful analysis of plasma over 150 samples also proved the method’s efficiency. Conclusion In summary, this is the first report to evaluate the drug-drug interaction between ASA and PNS about pharmacokinetics in the rats, which was considered to be the proper model to simulate human model. A rapid, reliable and sensitive UPLC–MS/MS method was also built for quantitative analysis of SA in plasma. Following a single oral administration of the ASA, part of the ASA is rapidly decomposed into SA in blood. The pharmacokinetic curve and Cmax of AS showed marked increase when the drugs were taken together. Cell experiment was used to study the absorption of ASA and SA. The result of cell experiment is consistent with the pharmacokinetic study. In a word, these results for the pharmacokinetics and cell experiment must be useful for the clinical application of the ASA and PNS, which also can provide reliable scientific data for ameliorating drug treatment regimens. Acknowledgement

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The effect of PNS to ASA in vitro and vivo experiment

This work was supported, in part, by National Natural Science Foundation of China (No. 53703596). Reference 1. Pursnani A, Celeng C, Schlett CL. Use of Coronary Computed Tomographic Angiography Findings to Modify Statin and AspirinPrescription in Patients With Acute Chest Pain. Am J Cardiol., 117(2016), pp.319-324. 2.

Kodela

R,

Chattopadhyay

M,

Velázquez-Martínez

CA.

NOSH-aspirin (NBS-1120), a novel nitric oxide- and hydrogen sulfide-releasing hybrid has enhanced chemo-preventive properties compared to aspirin, is gastrointestinal safe with all the classic therapeutic indications. Biochem Pharmacol., 98(2015), pp.564-72. 3. Romano M, Cianci E, Simiele F, Recchiuti A. Lipoxins and aspirin-triggered lipoxins in resolution of inflammation. Eur J Pharmacol., 760(2015), pp.49-63. 4. Xian Y, Wang TY, McCoy LA. Association of Discharge Aspirin Dose with Outcomes after Acute Myocardial Infarction: Insights From the Treatment with ADP Receptor Inhibitors: Circulation., 132(2015), pp.174-81. 5. Fuster V, Sweeny JM. Aspirin: a historical and contemporary therapeutic overview. Circulation, 123(2011), pp768–778. 6. Wang T, Guo R, Zhou G, Zhou X.Traditional uses, botany, phytochemistry, pharmacology and toxicology of Panax notoginseng(Burk.) F.H. Chen: A review. J Ethnopharmacol., 188(2016), pp.234-258. 7. Liu JJ, Wang Y T. QIU lan. Saponins of Panax notoginseng:chemistry, cellular

29

The effect of PNS to ASA in vitro and vivo experiment

targets and therapeutic opportunities incardiovascular diseases. Eepert Opiniv inv drug., 23 (2014), pp.523. 8. Agarwal S, Coakley M, Reddy K. Quantifying the effect of antiplatelet a

comparison

of

thromboelastography

the

platelet

(mTEG)

function

with

light

analyzer

(PFA-100)

transmission

platelet

and

therapy: modified

aggregometry.

Anesthesiology, 105(2006), pp.676-683. 9. Maree AO, Curtin RJ, Chubb A, et al．Cyclooxygenase-1 haplotype modulates platelet response to aspirin. J Thromb Haemost., 3(2005), pp.2340-2345. 10.

Antithrombotic

Trialists′Collaboration.

Collaborative

meta-analysis

of

randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ., 324(2002), pp.71-86. 11. Patrono C, Coller B, Fitz Gerald GA, et al. Platelet-active drugs:

the

relationships among dose, effectiveness, and side effects: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest.,126(2004): pp.234s-264s. 12. Feuring M, Schultz A, Losel R, et a1. Monitoring acetylsalicylic acid effects with the platelet function analyzer PFA-100. Semin Thromb Hemost,, 31(2005), pp.411-415. 13. Michelson AD. Methods for the measurement of platelet function. Am J Cardiol., 103(2009), pp.20-26. 14. Nicholson NS, Panzer-Knodle SG, Haas NF, et al. Assessment of platelet function assays. Am Heart J., 135(1998), pp.170-178.

30

The effect of PNS to ASA in vitro and vivo experiment

15. Pinto Slottow TL, Bonello L, Gavini R, et al. Prevalence of aspirin and clopidogrel resistance among patients with and without drug-eluting stent

thrombosis.

Am J Cardiol., 104(2009), pp.525-530. 16. Zhao HJ, LI K. Clinical Research of Panax Notoginseng Saposins combined with Aspirin on Neural Function and the Secondary Prophylaxis of Stroke, 19(2012), pp.453 -455. 17. Chen ZM, Jiang LX, Chen YP, et al. Addition of clopidogrel to aspirin in 45852 patients with acute myocardial infarction: randomised placebo-controlled trial. Lancet., 366(2005), pp.1607-1621． 18. Buchanan MR, Brister SJ. Individual variation in the effects of ASA on platelet function implications for the use of ASA clinically. Can J Cardiol., 11(1995), pp.221– 227. 19. Gurbel PA, Bliden KP, Hiatt BL, et al. Clopidogrel for coronary stentingres ponse variability, drug resistance, and the effect of pretreatment platelet reactivity. Circulation., 107(2003), pp.2908–2913. 20. Lev EI, Patel RT, Maresh KJ, et al．Aspirin and clopidogrel drug response in patients undergoing percutaneous coronary intervention: the role of dual drug resistance. J Am Coll Cardiol., 47(2006), pp.27-33. 21. Ikeda Y, Shimada K, Teramoto T, Uchiyama S, Yamazaki T, Oikawa S, Sugawara M, Ando K, Murata M, Yokoyama K, Ishizuka N

Low-dose aspirin for

primary prevention of cardiovascular events in japanese patients 60 years or older with

31

The effect of PNS to ASA in vitro and vivo experiment

atherosclerotic risk factors: a randomized clinical trial. JAMA., 312(2014), pp.2510–2520 22. Patrono C, Baigent C, Hirsh J, and Roth G. American College of Chest Physicians: antiplatelet drugs: American College of Chest Physicians evidence-based clinical practice guidelines (8th edition). Chest. 133(2008), pp.199–233. 23 D. Vijaya B, Kishore K.H, Pandu R, et al. Low dose aspirin estimation: an application to a human pharmacokinetic study, Biomed. Chromatogr. 27（2013）pp 89 – 598 24 Hervery PS,Goa KL. Extended -release dipyridamole /aspirin. Drugs, 58（1999） 469 -475. 25. Seegers J.M., Ollins M., Jager L.P. and Van Noord-Wijk J. Interactions of aspirin with acetaminophen and caffeine in rat stomach: Pharmacokinetics of absorption and accumulation in gastric mucosa. J. Pharm. Sci., 69(1980), pp.900–906. 26. Buskin JN, Upton RA and Willams RL. Improved liquid chromatography of aspirin, salicylate, and salicyluric acid in plasma, with a modi fi cation for determining aspirin metabolites in urine. Clin. Chem., 8(1982), pp.1200-1203. 27. Peng GW, Gadalla MA, Smith V, Peng A and Ghiou WL. Simple and rapid high-pressure liquid chromatographic simultaneous determination of aspirin, salicylic acid and salicyluric acid in plasma. J. Pharm. Sci., 67(1978), pp. 710 – 720. 28. Walter LJ, Biggs Df and Coutts RT. Simultaneous GLC estimation of salicylic acid and aspirin in plasma. J. Pharm. Sci., 63(1974), pp.1754-1758.

32

The effect of PNS to ASA in vitro and vivo experiment

29. Dams R, Huestis MA, Lambert WE and Murphy CM. Matrix effect in bioanalysis of illicit drugs with LC-MS/MS: in fluence of ionization type, sample preparation, and biofluid. J. Am. Soc. Mass Spectr., 14(2003), pp.1290-1294. 30.Nirogi R, Kandikere V, Mudigonda K, Ajjala D, Suraneni R and Thoddi P.Simultaneous extraction of acetylsalicylic acid and salicylic acid from human plasma and simultaneous estimation by liquid chromatography and atmospheric pressure chemical

ionization/tandemmass

spectrometry

detection.

Application

to

a

pharmacokinetic study. Arzneimittel-Forschung, 61(2011), pp.301-311. 31. Irvine JD, Takahashi L, Lockhart K, et al. MDCK (Madin-Darby canine kidney) cells: a tool for membrane permeability screening. J Pharm Sci., 88(1999), pp28-33. 32. Volpe DA. Variability in Caco-2 and MDCK cell-Based intestinal permeability assays. J Pharm Sci., 97(2008), pp.712-725. 33. Putnam WS, Ramanathan S, Pan L, H.et al. Functional characterization of monocarboxylic acid, large neutral amino acid, bile acid and peptide transporters, and P-glycoprotein in MDCK and Caco-2 cells. J Pharm Sci., 91(2002), pp.2622–2635. 34. Luo S, Wang Z, Kansara V, et al. Activity of a sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and mechanism of ascorbate uptake. Int J Pharm., 358(2008), pp.168-176. 35. Takahashi Y, Kondo H, Yasuda T, et al. Common solubilizers to estimate the Caco-2 transport of poorly water-soluble drugs. Int J Pharm., 246(2002), pp.85-94. 36. Wang Q, Rager JD, Weinstein K, et al. Evaluation of The MDCK-MDR cell line as a permeability screen for the blood-brain barrier. Int J Pharm., 288(2005), 33

The effect of PNS to ASA in vitro and vivo experiment

pp.349-359. 37. Madgula VL, Avula B, Reddy NVL, et al. Transport of decursin and decursinol angelate across Caco-2 and MDR-MDCK cell monolayers: in vitro models for intestinal and blood-brain barrier permeability. Planta Med.,73(2007), pp.330-335. 38. Zhong WQ, The retrospective research on the main adverse reactions of the long-term aspirin taken in elderly patients[D],China, Jilin University, 2015, pp.6-10. 39. Ke XL，Shen SJ，LI SM, et al. Effect and the Changes of Neurological Function of Aspirin Combined with Saponins of Panax Notoginseng in the Treatment of Cerebral Infarction Medical Innovation of China, 13（2016）, pp.13-16. 40. Baigent C, Blackwell L, Collins R, et al.Aspirin in the primary and secondary prevention of vascular disease:collaborative meta-analysis of individual participant data from randomised trials. Lancery, 373(2009), pp.1849-1860. 41. Masoudi FA, Wolfe P, Havranek EP, et al. with

Aspitin

use in

older patients

heart failure and coronary artery disease:national presctiption pattetns

and ralationship

with

outcomes. Am Coll Cardiol., 46 (2005), pp.955-962.

42. Cecchelli, R., Dehouck, B., Descamps, L., et al. In vitro model for evaluating drug transport across the blood brain barrier. Adv Drug Del Rev., 36(1999), pp.165-178. 43. Chen, Z.Z., Lu, Y., Du, S.Y., et al. Influence of borneol and muscone on geniposide transport through MDCK and MDCK-MDR1 cells as blood-brain barrier in vitro model. Int J Pharm., 456 (2013), pp.73-79. 44. D. Vijaya B*, Kishore KH, Pandu R, et al. Low dose aspirin estimation: an

34

The effect of PNS to ASA in vitro and vivo experiment

application to a human pharmacokinetic study. Biomedical Chromagraphy. 27(2013), pp.589-598.

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