Accepted Manuscript Title: The effect of cephalexin in influencing the pharmacokinetics of a novel drug —5'-valyl-cytarabine hydrochloride Author: Xiaotong Song, Yinghua Sun, Chenyao Zhao, Zhonggui He PII: DOI: Reference:
S1818-0876(16)30074-5 http://dx.doi.org/doi: 10.1016/j.ajps.2016.08.003 AJPS 400
To appear in:
Asian Journal of Pharmaceutical Sciences
Received date: Revised date: Accepted date:
15-3-2016 13-6-2016 22-8-2016
Please cite this article as: Xiaotong Song, Yinghua Sun, Chenyao Zhao, Zhonggui He, The effect of cephalexin in influencing the pharmacokinetics of a novel drug —5'-valyl-cytarabine hydrochloride, Asian Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/doi: 10.1016/j.ajps.2016.08.003. 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.
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Title page
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The effect of cephalexin in influencing the pharmacokinetics of a novel
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drug —5’-valyl-cytarabine hydrochloride
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Xiaotong Songa, Yinghua Suna, Chenyao Zhaoa, Zhonggui He a,*
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a
Shenyang Pharmaceutical University, No.103, Wenhua Road, Shenyang 110016, China
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Corresponding author:
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Corresponding author: Zhonggui He a,*
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Mailing address: Shenyang Pharmaceutical University, No.103, Wenhua Road, Shenyang
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110016, China
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Tel.: +86-24-23986321; Fax: +86-24-23986321
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E-mail:
[email protected]
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Graphical Abstract
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The bioavailability of 5’-valyl-cytarabine hydrochloride (OPC) was investigated when
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OPC was co-administration with cephalexin. The results showed that the bioavailability of
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OPC could be reduced when co-administration with cephalexin, suggesting that the efficacy
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of a novel drug might be reduced when it came to combination use of β-lactam antibiotics.
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Abstract:
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The aim of this study is to investigate the pharmacokinetics of 5’-valyl-cytarabine
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hydrochloride (OPC) when co-administration with cephalexin, which are both the substrates
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of PepT1. The drugs were administered orally by gavage. Blood samples were collected from
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the orbital plexus of the rats after oral administration of drug solutions. A new
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high-performance liquid chromatographic method was validated and used for determination
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of the two drugs. Pharmacokinetic parameters were calculated using DAS 2.1.1 software with
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noncompartmental analysis. After oral administration of OPC and co-administration of OPC
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and cephalexin, there were significant differences in the main pharmacokinetic parameters.
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The main pharmacokinetic parameters for OPC group and co-administrative group were as
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follows: AUC0-10 (18168.7 ± 2561.4) ng·h/ml and (13448.5 ± 2544.73) ng·h/ml, AUC0-∞
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(18683.1 ± 3066.5) ng·h/ml and (13721.1 ± 2683.0) ng·h/ml, Cmax (6654.8 ± 481.3) ng/ml
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and (4765.1 ± 928.9) ng/ml, respectively. The results showed that the bioavailability of OPC
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could be reduced when co-administration with cephalexin, suggesting that the efficacy of a
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novel drug might be reduced when it came to combination use of β-lactam antibiotics.
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Keywords: 5’-valyl-cytarabine hydrochloride (OPC); Cephalexin; Pharmacokinetics;
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PepT1
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1. Introduction
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Drug transporters are very important in the process of oral drug absorption, distribution,
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metabolism, and elimination. Two major uptake transporters are two solute carrier (SLC and
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SLCO) superfamilies[1]. There are two kinds of POT having transport activity. One is PepT1,
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which is mainly expressed in small intestine and also in the proximal tubules of kidney[2, 3].
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The other is PepT2, which is predominantly located in kidney[4]. Cytarabine
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(1-β-D-arabinofuranosylcytosine) is used in approximately 70% of cases of acute
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myelogenous leukemia (AML)[5]. Combination using of cytarabine and interferon-alpha
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shows great efficiency in the treatment of chronic myeloid leukemia (CML)[6]. Cytarabine is
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also an alternative drug to many cancers (stomach cancer, pancreatic cancer, liver cancer,
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colon cancer, lung cancer, breast cancer, uterine cancer, etc.) It has a quite short half-life
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which necessitates the administrative way of continuous infusion to maintain therapeutic
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plasma level, which is not convenient and time-consuming. 5’-valyl-cytarabine hydrochloride
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(see Fig.1.), is a novel 5’-amino acid ester prodrug of cytarabine. It is the substrate of PepT1,
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which increases the oral bioavailability of cytarabine. In vivo pharmacokinetics study showed
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that the oral absolute bioavailability of rats increased 40% compared with that of cytarabine.
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Cytarabine could be released from the prodrug rapidly[7]. This improvement suggested that
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OPC might become a promising oral drug in dealing with AML, CML and stomach cancer,
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etc. A number of drugs have been reported as the substrates of PepT1, including β-lactam
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antibiotics, ACE-inhibitors, rennin inhibitors, thrombin inhibitors, bestatin and prodrugs of
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acyclovir, ganciclovir[8-15].
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Cytarabine is mainly used in the treatment of acute myelogenous leukemia and
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non-Hodgkin's lymphoma. It is also used with other chemotherapy agents when people
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suffered chronic myelogenous leukemia, multiple myeloma, Hodgkin's lymphoma and
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non-Hodgkin's lymphomas[16]. Therefore, cytarabine plays an important role in cancer
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chemotherapy. Patients are susceptible to infections, due to compromised immune system
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resulting from chemotherapy. Neutropenia and fever are very common life-threatening
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complications in cancer chemotherapy patients. Oral antibiotics can be an alternative
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approach for low risk cancer patients. Up to date, the best oral regimen is combination of
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quinolone and amoxicillin/clavulanate[17]. Cephalexin, is a kind of broad-spectrum, oral,
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antimicrobial agent. Cephalexin and amoxicillin are both β-lactam antibiotics, they share
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similar functional group. Previously, the pharmacokinetic interaction between cephalexin and
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quinapril-a substrate of PepT1 was investigated, the pharmacokinetic interaction between
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cephalexin and JBP485-another substrate of PepT1 was also studied. Cephalexin was chosen
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as a typical kind of β-lactam antibiotics for study of interaction between substrates of
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PepT1[18, 19].
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Previously, the study of the absorption of OPC in the presence of cephalexin by using
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single pass perfusion model was completed in our laboratory. The results showed that the
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absorption of OPC was greatly inhibited by cephalexin. However, the intraluminal
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environment is quite complicated and there are many differences between in vivo experiments
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and in situ single pass perfusion. Further study has to be carried out to elucidate the
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pharmacokinetic interaction in rats between OPC and cephalexin which are both the
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substrates of PepT1. Pharmacokinetic interaction between the two drugs is of great
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importance to clinical application of OPC.
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2. Materials and methods
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2.1. Materials
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OPC was provided by Kunming Jida Pharmaceutical Co., Ltd. (Kunming, China) with a
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purity of 99.8%. Cytarabine was purchased from Langrb Technology Co., Ltd. (Beijing,
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China) with a purity of 99.9%. Cephalexin with a purity of 98% was purchased from HMC
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Chemical Technology Co., Ltd. (Beijing, China). Lamivudine with a purity of 99.7% was
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purchased from Longze Pharmaceutical Co., Ltd. (Shijiazhuang, China). Tetrahydrouridine
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was provided by Toronto Research Chemicals Inc. The rest of chemicals were of analytical
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grade.
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Male, Sprague–Dawley (SD) rats weighing 220–250 g were purchased from the
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Experimental Animal Center (Shenyang Pharmaceutical University, Shenyang, China). The
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experimental protocol were evaluated and approved by the University Ethics Committee for
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the use of experimental animals and conformed to the Guide for Care and Use of Laboratory
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Animals.
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2.2. Preparation of solutions for oral administration
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The cephalexin solution was prepared by dissolving 125 mg cephalexin in 25 ml
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aqueous solution to obtain a concentration of 5 mg/ml; The OPC solution was prepared by
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dissolving 64 mg OPC in 25 ml aqueous solution to obtain a concentration of 1.5 mg/ml
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(calculated as cytarabine); The mixed solution of cephalexin and OPC was prepared by
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dissolving 125 mg cephalexin and 64 mg OPC in 25 ml aqueous solution.
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2.3. Preparation of standard solution
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Standard solutions of two drugs at concentrations of 0.05, 0.1, 0.2, 0.25, 0.5, 1.0, 5.0, 8.0,
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10.0 μg/ml for cytarabine and 0.2, 0.4, 0.8, 1.0, 2.0, 4.0, 20.0, 32.0, 40.0 μg/ml for cephalexin
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were prepared. The internal standard solution was prepared by dissolving 30 mg lamivudine
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in 100 ml distilled water to obtain a stock solution. 1 ml stock solution was diluted with water
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to obtain 100 ml solution at concentration of 3 μg/ml.
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2.4. Collection and treatment of biological samples
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Serial blood samples (0.5 ml) were obtained from orbital plexus at 5, 15, 30, 45 min, 1, 4
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1.5, 2, 3, 4, 5, 6, 8, 10 h after oral administration separately. The blood samples were
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centrifuged at 13000 r/m for 10 min, plasma was then removed and stored at –80 ºC until
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later analysis. During sampling, rats were anesthetized with ether. All samples were placed
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into heparinized tubes containing the deaminase inhibitor, tetrahydrouridine (0.1 mM). A 100
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μl aliquot of internal standard solution and a 100 μl aliquot of distilled water were added to a
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100 μl aliquot of plasma and vortex-mixed for 3 min. Then, a 0.8 ml aliquot of acetonitrile
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was added, vortex-mixed for 3 min, centrifuged at 13000 r/m for 10 min, then the supernatant
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was transferred to a clean tube and dried under nitrogen gas at 37 ºC. The residue was
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dissolved in 100 μl distilled water, centrifuged for 10 min at 13000 r/m, 20 μl supernatant
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was injected into the HPLC column. The peak-area ratios of the biological sample to the
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internal standard were used to calculate drug concentrations at different time points.
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2.5. Chromatographic conditions
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HPLC analysis was carried out on a Waters liquid chromatography instrument equipped
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with a Waters 2489 UV/Visible Detector and e2695 Separations Module. A C18 column
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(ZORBAX SB-C18, 4.6 mm×250 mm, 5 μm, Agilent Technologies) was used; HPLC elution
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was carried out using phosphate buffer containing 0.005 M K2HPO4 and KH2PO4 and
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methanol. The elution gradient is listed in Table 1. Column temperature was 30 ºC, flow rate
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was 1.0 ml/min, Ultraviolet detection wavelength was 254 nm and injection volume was 20
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μl.
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2.6. Methodology verification
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2.6.1. Method specificity
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Under the chromatographic conditions described, there was no interference from the endogenous substances present in the plasma.
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2.6.2. Preparation of standard curves and quality control samples
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Different concentrations of standard solution were mixed with 100 μl aliquots of blank
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plasma to prepare biological sample solutions. The regression equation was obtained from the
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plot of the peak-area ratio of cephalexin or cytarabine to internal standard (As/Ai) as the 5
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Y-axis and the concentrations (C) as the X-axis. Cytarabine (0.1, 0.5, 8.0 μg/ml) and
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cephalexin (0.4, 2.0, 32 μg/ml) were added in blank plasma to prepare the low, medium and
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high levels of quality control (QC) samples. The spiked samples were then treated as the
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sample preparation procedure indicated in Section 2.4.
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2.6.3. Extraction recovery
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The extraction efficiency was determined by comparing the peak areas of extracted QC
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samples with peak areas of standards solution and IS solution added to blank plasma extract.
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The concentration of the IS solution was 3 μg/ml. Three concentration levels (0.1, 0.5, 8.0
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μg/ml for cytarabine and 0.4, 2.0, 32 μg/ml for cephalexin) in rat plasma were studied in
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recovery experiments.
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2.6.4. Precision experiment
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The intra-day and inter-day precision were determined at the same three high, medium
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and low concentrations of QC samples. The intra-day precision was obtained from 6
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replicates injected on the same day, while the inter-day precision was obtained from samples
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injected on 3 different days.
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2.6.5. Lower limit of quantification (LLOQ)
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The samples of LLOQ were made by using the method of preparing lowest point of
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standard curve with standard solution. The intra-day precision was obtained from 6 replicates
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injected on the same day. The accuracy was calculated by the mean deviation of all
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concentration from the theoretical value.
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2.6.6. Stability test
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The stability of the plasma samples at the high, medium and low concentrations were
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examined after storing at room temperature for 12 h, after repeated freeze-thawing (3 times)
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and after storing at –80 ºC for 14 d.
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2.7. Animal study 6
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Before the experiments, SD rats were fasted for 12 h with free access to water. They
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were then randomly divided into three groups, namely OPC group after an oral administration
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at 15 mg/kg (calculated as cytarabine), cephalexin group after an oral administration at 50
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mg/kg[18] cephalexin and combination group after an oral administration at 15 mg/kg
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(calculated as cytarabine) and 50 mg/kg cephalexin, with 6 rats per group. The drugs were
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administered orally by gavage. 0.5 ml blood samples were collected from the orbital plexus
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of the rats before and after oral administration of drug solutions, at time intervals of 0.08,
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0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 8 and 10 h. The rats were then sacrificed. The
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concentration of drugs in each sample was determined as described previously.
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2.8. Pharmacokinetics and statistical analysis
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The main pharmacokinetic parameters were calculated according to the DAS 2.1.1
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software with non-compartmental analysis. The statistical differences were tested using
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student t test at the P