Accepted Manuscript Title: Bio-distribution and Pharmacokinetics of Nobiliside A-loaded Liposome Following Intravenous Administration in Rats Author: Xiong Yang Chen Jianming Guo Dan PII: DOI: Reference:
S1570-0232(14)00232-3 http://dx.doi.org/doi:10.1016/j.jchromb.2014.03.039 CHROMB 18870
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
22-12-2013 21-3-2014 26-3-2014
Please cite this article as: X. Yang, C. Jianming, G. Dan, Bio-distribution and Pharmacokinetics of Nobiliside A-loaded Liposome Following Intravenous Administration in Rats, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.03.039 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.
Bio-distribution and Pharmacokinetics of Nobiliside A-loaded Liposome Following Intravenous Administration in Rats Xiong Yanga*†, Chen Jianming b†, Guo Danc† Department of Pharmaceutical Science ，Zhejiang Chinese Medical
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a
b
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University, Hangzhou, Zhejiang, 310053, China
Department of Pharmaceutical Science, School of Pharmacy, Second
These authors contributed equally to this work.
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†
Shanghai Institute for food and drug control, Shanghai 201203, China
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c
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Military Medical University, Shanghai 200433, China
*Correspondence author:
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Dr. Xiong Yang,
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Department of Pharmaceutical Science, Zhejiang Chinese Medical
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University, Hangzhou, Zhejiang, 310053, P. R. China Tel: 011-86-571-86613524 Fax: + 011-86-571-86613606
Email：[email protected]
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Abstract: Nobiliside A (Nob) is a new triterpenoid saponin separated from Holothuria noblilis. In this article, a liquid chromatography–electrospray
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ionization-tandem mass spectrometry method was established to quantify
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Nob, a hemolytic saponin, in rat blood and tissue homogenates. Standard curves were linear (r=0.9988–0.9995) over the range 50~5000 ng/mL in
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blood and 100~10000 ng/g in tissues. The lower limit of quantification
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(LLOQ) was 50 ng/mL for Nob. The novel method was rapid, accurate, highly sensitive and highly selective. Using this method, the
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pharmacokinetics and biodistribution of Nob liposome and Nob solution in Sprague-Dawley rats after a single intravenous dose of 1mg/kg were
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then investigated. Nob was cleared slowly from circulation. There was no
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significant difference of the pharmacokinetic parameters in blood between
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Nob solution and Nob liposome. The highest AUC of Nob was observed in liver for the two groups, followed by spleen, lungs, kidney and heart. Compared with Nob solution, Nob liposome showed much higher AUC in liver and spleen and much lower AUC in kidney, heart and lung, which might be one important reason for the decreased toxicity of Nob.
Key words：Nobiliside A，liposome，LC/MS/MS，pharmacokinetics， bio-distribution
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1. Introduction Nobiliside A (Nob) is a new triterpenoid saponin separated from Holothuria noblilis with chemical molecular formula of C54H87O26SNa
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and stereotaxic configuration (see Fig.1A for chemical structure),
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determined by the modern spectral analysis, and especially advanced 2D nuclear magnetic resonance spectrum analysis. Extracorporeal antitumor
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test shows that Nob has obvious inhibitory effect on many kinds of tumor
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cell strains, such as P388 mouse lymphoma, A549 human lung cancer and so on. Furthermore, Nob is a dual-acting anticancer agent with both
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cytotoxic and angiogenesis inhibiting effects [1, 2]. Unfortunately, in our previous study (data not published), the bioavailability of Nob was low
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after oral administration due to the hydrolysis and enzymolysis of Nob in
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the artificial simulation gastric juices. While for intravenous injection,
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Nob was highly toxic, mainly because it could cause hemolysis of blood cells and heart toxicity [3].
Therefore, liposome was used as its carrier
to reduce the hemolysis and toxicity of Nob after intravenous administration [4].
How about the bio-distribution and pharmacokinetics of Nob
solution and Nob liposome after intravenous administration? Would the reduction of toxicity partly related with the change of the bio-distribution and pharmacokinetics? Up to now, there is no report answering these questions. 3
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In this work, liquid chromatography tandem mass spectrometry methods with multiple reaction monitoring (MRM) were developed and validated for determination of Nob in rat whole blood and tissues, using
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lovastatin acid as the analytical internal standard (see Fig.1B for chemical
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structure). Based on the analytical results, bio-distribution and pharmacokinetics of Nob and Nob liposome following intravenous
an
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administration in Sprague–Dawley(SD) rats were evaluated.
2.1 Materials and animals
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2. Materials and methods
Nobiliside A (Nob, purity >98%) was provided by Research Center
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for Marine Drugs, College of Pharmacy, Second Military Medical
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University (Shanghai, China). Internal standard lovastatin acid was
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purchased from the Shanghai Institute of Pharmaceutical Industry (Shanghai, China). Phospholipid was purchased from Tai-wei-yao-ye Ltd (Shanghai, China). Cholesterol was purchased from Shanghai Chemical Reagent Company (Shanghai, China). Methanol (Tedia Inc., CA, USA) was HPLC grade. Water used was
from a Millipore Milli-Q Plus 185 ultra-pure water system (Millipore, MA, USA).
All other chemicals were analytical grade or better.
SD rats(200±20g) were provided by Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Animal experiments were performed 4
Page 4 of 34
according to the Guiding Principles for the Care and Use of Experiment Animals in Shanghai Medicilon Inc., and were carried out in accordance
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with the SOPs of the facility. 2.2 Preparation of Nob liposome
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Nob liposome was prepared by thin-film ultrasonication technique [4]. Briefly, phospholipids and cholesterol at the proportion of 2∶0.9
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were co-dissolved in chloroform and evaporated to form a thin lipid film
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under reduced pressure. The thin film was hydrated with an aqueous Nob solution (0.5 mg·mL-1), and the lipids/drug ratio (w/w) was 40. The
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resulting mixture was sonicated for 30 min and further processed by probe sonication for 1-min cycle (3 s working and 3 s rest) at 200 W
d
(Ningbo Xinzhi Bio-tech Co. Ltd., China). The resulting liposome
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suspension was extruded through sterile Millipore Express (PES,
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Millipore, USA) with 0.22 µm of pore size. 2.3. LC/MS/MS determination 2.3.1 Apparatus and chromatographic conditions Apparatus and chromatographic conditions were the same with
which described in one article we published before [5]. Briefly, The LC system comprised an isocratic pump (1100 series), and autosampler (1100 series) and a degasser (1100 series) (Agilent Technologies Inc. Palo Alto, CA, USA). Mass spectrometric analysis was performed using an API3000 (triple-quadrupole) instrument with an ESI interface with 5
Page 5 of 34
TurboIon spray (Applied Biosastems, Foster City, CA, USA). Data were acquired and processed by Analyst 1.4 software from ABI Inc. The analytical column used was an Agilent Zorbax SB-Cl8 column (5 μm, 4.6
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mm ×15 mm). The mobile phase was a mixture of methanol and water
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75:25 (v/v) for blood samples and 80:20 (v/v) for tissues samples. The mobile phase was degassed automatically using the electronic degasser
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system. The column was equilibrated and eluted under isocratic
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conditions utilizing a flow rate of 1 mL/min at ambient temperature. Negative ion mass spectrometry was performed using the following
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conditions: nebulize gas: 8 L/min; curtain gas: 8 L/min; collision gas: 4
2.3.2 Assay method
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L/min; ion spray voltage: −4500 V; temperature: 500 ℃.
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Whole blood samples were collected from SD rats into vials
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containing sodium heparin and stored at −20 ℃ until use. Thawed blank blood was spiked with Nob stock solution to calibration standards (CS) and quality control (QC) samples. Predetermined volumes (20 μL) of
Nob stock solution and 500 μL of internal standard lovastatin acid (49.4
ng/mL) were added to obtain CS samples at Nob concentrations equal to 50, 100, 250, 500, 1000, 2500, 5000 ng/mL, and QC samples at concentrations equal to 75, 750, 4000 ng/mL, and then vortexed for 1 min. After centrifugation at 15000 × g for 5 min, 100 μL of the supernatant liquid was transferred to a sample vial and 5 μL was injected into the 6
Page 6 of 34
LC/MS/MS for quantitative analysis. 100 mg tissues (heart, liver, spleen, lungs and kidney) from SD rats were collected respectively, and then homogenized in 300 μL normal
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saline (0.9% NaCl). Homogenates were aliquoted (100 μL) ，
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predetermined volumes (20μL) of Nob stock solution and 500 μL of
internal standard lovastatin acid (49.4 ng/mL) was added to obtain CS
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samples at Nob concentrations equal to 100, 250, 500, 1000, 2500, 5000,
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10000 ng/g, and QC samples at concentrations equal to 200，2000，8000 ng/g, and then vortexed for 1 min. After centrifugation at 15000 × g for 5
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min, 100 μL of the supernatant liquid was transferred to a sample vial and 5 μL was injected into the LC/MS/MS for quantitative analysis.
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A thorough and complete method validation for assaying Nob in rat
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blood and tissue homogenate samples was done following the US Food
Ac ce p
and Drug Administration(FDA) guidelines[6]. The validation parameters included specificity, linearity, accuracy, precision and stability. All animal studies were approved by IACUC of Shanghai Medicilon
Inc. and carried out in accordance with the SOPs of the facility. 2.3.3 Pharmacokinetic study SD rats were randomly divided into two groups (5 per group). Group 1 was treated with Nob solution (dissolved in 5% glucose aqueous solution) whilst group 2 treated with Nob liposome, and the concentration of Nob in its both solution and liposome suspension was 0.5 mg/mL. 7
Page 7 of 34
Each preparation was injected through the tail vein at the Nob dose of 1mg/kg, at which dose Nob had shown therapeutic anticancer effect in pre-training administration.
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Blood samples about 0.3 mL were taken by terminal retro-orbital
cr
bleeding at various times (at 0.04, 0.083, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12,
24 h) into micro-tubes containing sodium heparin as an anticoagulant.
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Then a 100 μL aliquot of each blood sample was combined with 20 μL of
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methanol and 500 μL of internal standard lovastatin acid (49.4 ng/mL), and vortexed for 1 min. The methods for Nob to be extracted from the
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blood and to be determined were the same as described in Section 2.3.2. All samples were made in triplicate. Blood samples from untreated rats
d
were used as blank samples.
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2.3.4 Tissue distribution study
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SD rats were randomly divided into two groups (15 per group). Nob solution (0.5mg/mL) and Nob liposome suspension (0.5mg/mL) were injected through the tail vein at the dose of 1 mg/kg, respectively. Five rats per group were sacrificed at various times ( at 0.083 h，1 h，8 h) and tissues (heart, liver, spleen, lungs and kidneys) were obtained. All tissues were weighed and 100 mg of tissues was collected respectively, and then homogenized in 300 μL normal saline (0.9% NaCl). Homogenates were aliquoted (100 μL), and mixed with 20 μL of methanol and 500 μL of internal standard lovastatin acid (49.4 ng/mL). 8
Page 8 of 34
The methods for Nob to be extracted from the homogenates and to be determined were the same as described in Section 2.3.2.
untreated rats were used as blank samples respectively.
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2.4 Date analysis
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All samples were carried out in triplicate. Tissue samples from
Pharmacokinetic parameters for Nob were calculated by PK analysis
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software DAS 2.0(Gaosi Data Analysis Inc., Wuhu, China) and the
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noncompartmental model was applied. Trapezoidal rule was used to
3. Results and discussion 3.1 Assay method
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calculate the area under the curve (AUC).
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Chromatograms obtained from blood and blank tissue homogenate
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of the liver (as a representative sample) spiked with Nob and the internal
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standard lovastatin acid were shown in Fig. 2 and Fig. 3 respectively. Nob exhibited a strong mass response in negative ESI mode due to the efficiency of ionization of the analyte. ESI showed that nobiliside A (MW: 1206) and IS (MW: 422) formed predominately molecular ions [M−Na]− atm/z 1183.4, and mainly deprotonated molecular ions [M−H]−
at m/z 421.2 in full scan mass spectra. Parameters were tuned according to the MS signal response of the target compound. The dominant product ions for quantitative detection of Nob and IS were m/z 96.90 and 101.00 respectively. Under the described chromatographic conditions, there was 9
Page 9 of 34
not any endogenous interference from the blank rat blood or tissues, which in turn ensured a high specificity of the LC/MS/MS method. Under the described chromatographic conditions, the retention time was
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about 1.9 and 3.1 min for Nob and lovastatin acid in blood sample, and
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about 1.4 and 2.1 min for nobiliside A and lovastatin acid in tissue samples respectively.
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Blood quality control samples and tissue homogenate quality control
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samples were determined by LC/MS/MS. The standard curves between the peak area of Nob in quality control samples and Nob concentration
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were evaluated by least squares regression analysis. Standard curves were linear (r=0.9988~0.9995) over the range 50~5000 ng/mL in blood and
d
100~10000 ng/g in tissues, which were shown in Tab. 1.
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The precision was assessed by analyzing quality control samples at
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three concentrations in five duplicates. Tab. 2 showed the between-run, within-run precision, extraction efficiency for blood samples respectively. The stability of Nob in extract and whole blood using weighed-in quality control samples also studied (Tab.2). Tab. 3 showed the between-run, within-run precision and extraction efficiency of samples in tissue homogenates respectively. The lowest limit of qualification (LLOQ) for Nob was 50 ng/mL. The method was sufficiently sensitive for the determination of bio-distribution and pharmacokinetic analysis of Nob in vivo. 10
Page 10 of 34
3.2 Pharmacokinetics study The blood concentration–time curves of Nob in rats after intravenous administration of a single 1mg/kg dose of Nob solution and
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Nob liposome were shown in Fig. 4. The non-compartmental
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pharmacokinetic parameters calculated by DAS 2.0 software were shown in Tab.4. Based on the analysis of the models and parameters, it was
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concluded that the mean blood concentration-time profiles for both Nob
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solution and Nob liposome were described by a three-compartment model with a weight of 1/C2.
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Following intravenous administration of Nob solution at a dose of 1 mg/kg, the mean values of Cmax and Tmax were 3985.37±850.94 ng/mL
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and 0.04 h respectively. The systemic clearance was 0.191±0.06 L/h/kg;
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and AUC (0-∞) was 5712.06±1778.73 (μg/L·h). The volume of distribution
Ac ce p
at terminal phase was 0.20±0.08 L/kg(Tab. 4). For Nob liposome, Cmax (4818.25±508.7) was achieved at 0.04 h
(Tmax). The systemic clearance was 0.20±0.12 L/h/kg, and AUC(0-∞) was 6038.70±2633.32 (μg/L*h). The volume of distribution at terminal phase
was 0.18±0.02L/kg (Tab. 4). There was no significant difference of the pharmacokinetic parameters between Nob solution and Nob liposome and both of them were eliminated slowly in vivo. The blood concentration at 24 h was not much lower than that at 8 h. 11
Page 11 of 34
3.3 Bio-distribution study The levels of Nob in tissues including heart, liver, spleen, lungs, and kidneys determined at 0.083 h, 1 h and 8 h after intravenous
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administration of a single 1mg/kg dose of Nob solution and Nob
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liposome were shown in Tab.5, respectively.
After systemic administration, Nob was rapidly distributed into all
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tissues studied. The AUC of Nob in tissues after intravenous
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administration of Nob solution and Nob liposome to rats was shown in Fig. 5. In the two groups, the highest AUC of Nob was observed in liver,
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followed by spleen, lungs, kidneys and heart.
Tab. 5 showed Nob concentration in different tissues at different time
d
after dosing. There were same trends of Nob concentration of the two
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groups in the same tissue. In liver and spleen, the concentration of Nob
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firstly increased during 0.083 h to 1 h, and then decreased at a very slow speed in 24 h. However, in heart, lungs and kidneys, the concentration of Nob fell off quickly in the first 1 h and decreased slowly during 1 h to 8 h after administration.
Intravenously injecting liposome could be used for passive targeting of liver and spleen. In order to explore the passive target activity of Nob liposome used in this study, the ratio of AUCNob liposome/ AUC Nob solution
was introduced，which demonstrated the relative efficacy of the two delivery systems toward the same tissue [7]. The highest ratio was 12
Page 12 of 34
observed in spleen with the value of 1.45, followed by liver with the value of 1.27, indicating that Nob liposome was more efficient in delivering Nob to spleen and liver compared with the solution. The ratios
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for kidney, heart, and lung were 0.50, 0.46, and 0.15 respectively. Nob
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has many toxic effects, such as causing hemolysis, prohibiting respiration, damaging the heart and kidneys. Therefore, besides decreaseing
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hemolysis[4], Nob liposome might also alleviate other side effects, such
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as that on the heart and kidneys after intravenous administration. 4. Discussion and conclusions
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Nob is a water soluble saponin. However, there was no significant difference of the pharmacokinetic parameters between Nob solution and
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Nob liposome and both of them were eliminated slowly in vivo. Besides,
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for the same tissue, the concentration–time curves of the two groups had
Ac ce p
similar variation trends. Why? According to our previous study [8], there was a strong interaction between Nob and cholesterol. Nob and cholesterol in plasma and blood cells could be combined and then form the non-soluble molecule compounds, and then the pharmacokinetic parameters of the compounds would be similar to Nob liposome. From the concentration–time curve, Nob solution group and liposome group were metabolized slowly within the body, and could still be detected at 24 h. Slight difference between Nob concentration at 8 h and 24 h was found. As described in other literatures, the process of saponins 13
Page 13 of 34
metabolized in vivo was relatively slow. For example, at a dose of 5 mg/kg for intravenous administration, the concentration of Ginsenoside Rb1 in serum was (83.8±12.9) µg· mL-1 at 5 min; after 1 h, the
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concentration of Rb1 decreased rapidly, and remains (1.1±0.03) µg· mL-1
was 11.6 min for phase α and 14.5 h for phase β[9].
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in serum at 72 h. The half-life period of disappearance of Rb1 in blood
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Since there was a strong interaction between Nob and cholesterol, it
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was not strange to find that the highest AUC of Nob solution was observed in liver and followed by spleen. Firstly, more cholesterol exists
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in liver and spleen than in other tissues. Secondly, parts of the Nob form compounds with cholesterol in vivo and then the compounds could target
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to liver and spleen passively, just like the characters of liposomes or
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nanoparticles. However, now that Nob in liposome had a strong
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interaction with cholesterol in lipid membrane [8], it should have little interaction with cholesterol in vivo, so the Nob in liposome enriched in liver and spleen might be attributed to the fact that nanoparticles with proper particle size were recognized and trapped by the RES which abound in special tissues and organs, such as liver and spleen [10]. Particularly worth mentioning was, as seen from Tab.5 , Nob concentration of both the liposome group and solution group firstly increased during 0.083h to 1h(tmax), and then decreased at a very slow
speed during 1h to 24h. While in other tissues like heart, lungs, kidneys, 14
Page 14 of 34
Nob concentration of both groups reached tmax at 0.083h, and then decreased at a very slow speed in 24h. We speculated that may be part of free Nob released from liposome could firstly be combined with
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by the RES in liver and spleen, which need a little time.
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cholesterol in blood, and then most of them was recognized and trapped
There were similar reports about the distribution of other saponins in
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animals. After intragastric administration of total saponins of tribulus to
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rats, some hecogenin glycosides were hydrolyzed into hecogenin, which were still detected in the liver at 32 h [11]. Li K, et al. reported the
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distribution characteristics of dioscin after intravenous and intragastric administration in rats[12].The results showed that dioscin is mostly
d
stored in liver, less in lung and least in kidneys (no report about its
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distribution in spleen or heart). In addition, the elimination of dioscin was
Ac ce p
quite slow. For example, after intravenous administration of a single dose of 1 mg· kg-1, drug concentration in liver decreased by 37%, 70% and
78% at 24 h, 72 h and 120 h respectively. Dioscin belongs to spirostane saponin, which has large molecular weight and high surface activity, can be combined with cholesterol in cells or tissues. Just for its similar structure and properties as Nob, its activities just as elimination and metabolism in the body may be related to cholesterol content in tissues. Comparing with some other compound analyzed by HPLC/MS/MS, the method was less sensitive. There would be mainly because of the 15
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characteristic of the drug itself. Firstly, Nob is a triterpenoid saponin with high molecular（MW：1206）, it has lower ionization efficiency than other small molecular compounds, thus the responding value is relatively low.
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We tried many times to optimize its chromatographic and mass
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spectrographic conditions, but we found it was hardly to improve its sensitivity. Moreover, we found Nob had a strong selectivity to its
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analytical column. We ever tried C-18 column, C-8 column and polarity
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column to analyse Nob by HPLC/ELSD for in-vitro studies, but got bad results, such as unsightly chromatographic peak which has a tailing or
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furcation, stagnation of Nob on the column thus caused very low sensitivity and so on. While using SBC-18 column could get the best
d
effect including sensitivity and peak shape. Secondly, in this test, we
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found that the main fragment peak at m/z 96.90 has the most stability
Ac ce p
with also strong response, and thus got good linearity and precision in the range of 50–5000 ng/mL, which was hardly to be obtained by using other fragment peak. So it was selected to be quantative fragment ion. But as we know, the smaller is the m/z, the greater is the baseline noise and thus the lower of sensitivity because of the obvious background interference (such as mobile phase, endogenous substances and so on) between 30-300 m/z. Of course, although the low limit of this method was 50 ng/mL, it is sufficiently sensitive for the study on the bio-distribution and pharmacokinetics of Nob. So we think this method could be used in this 16
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test. Few people will research the hemolytic saponins for intravenous injection, so no paper is found about the pharmacokinetics and tissues
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distribution following intravenous injection about this kind of saponins
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and their preparations. This is the first paper describing a fast, sensitive, and specific method for blood and tissues quantification of Nob, a
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hemolytic saponin, by LC-ESI/MS/MS method. And it is also the first
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paper comparing biodistribution and pharmacokinetics of Nob and its liposome following intravenous administration in rats.
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Acknowledgements
This work was financially supported by project supported by
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National Science Foundation for Young Scientists of China
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(No. 81202926), National High Technology Research and Development
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Program of China (863 Program) (No. 2006AA090304), and also supported by Science Technology Department of Zhejiang Province(No. 2012R10044-09), Authors are grateful to Dr.Yanghua Yi, Second Military Medical University, China, for providing Nobliside A.
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Reference: [1]Wu, J., Yi, Y.H., Wu, H.M., He, Q.S., Zhang, S.L. Studies on the in vitro antifungai and antitumor activities of nobiliside A from the sea cucumber Holothuria nobilis
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Selenka. Chin. Pharmacol. Bull., 23(2007)139-140
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[2] Yi, Y.H., Ding, J., Zhang X.W., Wu, J., Chen, Y., Liu, B.S., Tian, F., 2006.
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Antitumor compound nobiliside A from Holathuria nobilis and its monoacetylated derivative. CN Patent CN1740185, 1 May.
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[3]Hu, M., Konoki, K., Tachibana, K., 1996. Cholesterol-independent membrane disruption caused by triterpenoid saponins. Biochim. Biophys. Acta, 1299, 252-258.
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[4]Xiong Y., Guo D., Wang L.L.，Zhen X.L., Xu L.Y., Chen J.M. Development of
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Pharm., 371 (2009) 197-203.
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nobiliside A loaded liposomal formulation using response surface methodology. Int J
[5]Guo D., Xiong Y., Zhang Y., et al. Development and validation of a LC/MS/MS
Ac ce p
method for quantification of Nobiliside-A in rat plasma. J Chromatogr B. 877 (2009) 323-327.
[6]FDA; 2001 Available from:WWW.fda.gov/dowloads/Drugs/ Guidance Compliance
Regulatory Information/Guidances/ucm070107.pdf. [7]Chen Z.P.,Zhu J.B., Chen H.X.,et al.. Distribution of liposomal bifdendate in liver following intravenous injection in mice. J Drug Target. 18(2010) 627-636. [8]Xiong Y., Chen J.M., Guo D. Interaction Between Nobiliside-A and Lipid Bilayers，Lat. Am. J. Pharm. 31 (2012) 800-807. [9]Odani T., Tanizawa, H., Takino, Y. Studies on the absorption, distribution, excretion and metabolism of ginseng saponins. III. The absorption, distribution and 18
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excretion of ginsenoside Rb1 in the rat. Chem Pharm Bull. 31(1983)1059-1066． [10]Mizushima Y., Hamano T., Yokoyama K. Tissue distribution and antiinflammatory activitu of corticosteroids incorporated in lipid emulsion. Ann Rheum
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Dis. 41(1982)263-267. [11]Chen Y.G., Qu W.J., Yang N.Y., et al. Metabolism and distribution of Hecogenin
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in rats after intragastric infusion with saponins of Tribulus terristris L. Nat Prod Res
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Dev. 18(2006)927-931.
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te
d
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dioscin in rat. Steroids, 70(2005) 525–530.
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[12]Li K., Tang Y.B., Fawcett J. P., et al. Characterization of the pharmacokinetics of
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Figure Legends
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Fig.1 Chemical structure of (A) nobiliside A and (B)internal standard lovastatin acid
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Fig.2 Representative MRM chromatograms from nobiliside A (m/z1183.4→96.9amu)
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channel and internal standard lovastatin acid (m/z421.2→101.0amu)channel of extracts from (A) blank whole blood ,(B) blank whole blood with Nob and IS, and (C)blood
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sample after intravenous injection
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Fig.3 Representative MRM chromatograms from nobiliside A(m/z1183.4→96.9) channel and internal standard lovastatin acid (m/z421.2→101.0))channel of extracts
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from (A) blank control liver homogenate, (B) blank liver homogenate with nobiliside A
Ac ce p
and IS, and (C)liver sample after intravenous injection
Fig.4 The blood concentration-time profiles of Nob solution and Nob liposomes after intravenous administration of a single 1mg/kg dose to rats (Each point represents the mean±SD of 5 rats)
Fig. 5 Tissues AUC of Nob solution and Nob liposomes in rats during 0-8h after intravenous administration at the dose of 1mg/kg
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*Highlights (for review)
LC /MS/MS is established for biodistribution and pharmacokinetics of Nobliside A. Biodistribution and pharmacokinetics of Nobliside A and its liposome are compared. Nobliside A and its liposome eliminate slowly in rat after intravenous injection. Similar behavior of Nobliside A and its liposome appears in whole blood of rat.
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Nobliside A liposome shows much higher AUC in liver and spleen than its solution.
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Tables
Tab. 1 Regression parameters of standard curves for the determination of Nob in rat blood (concentration: 50～5000 ng·g-1) and tissues (concentration: 100～10000 ng·g-1) Blood or Tissue
R
Intercept（*10-4） 1.54
0.9989
Heart
25.8
3.56
0.9995
Liver
9.47
6.83
0.9987
Spleen
6.47
1.67
0.9986
Lung
8.24
3.41
Kidney
8.71
2.51
ip t
blood
Slope (*10-6） 9.36
0.9988
cr
0.9995
Within-day accuracy (%)
us
Tab.2 Precision, extraction recovery and stability data of the method of Nob in rat blood by LC/MS/MS (n=5) 75 ng·mL-1
750 ng·mL-1
4000 ng·mL-1
100.78±4.18
101.24±2.55
99.01±1.58
an
(n=5) Between-day accuracy
102.76±8.77
(%) (n=5) 91.51±4.11
Stability
for 24h(ARa) ARa of the blood at 4℃
107.09±8.88
92.17±8.70
84.58±6.86
98.92±5.19
103.48±8.80
99.51±3.53
94.82±1.50
ce pt
for 48h a
102.25±6.11
ed
ARa of the extract at 25℃
101.89±4.44
M
Extraction recovery(%)
100.34±1.99
AR: analytical recovery was determined as the mean assayed concentration expressed as a percentage
Ac
of the weighed-in concentration.
Page 31 of 34
Tab.3 Precision and extraction recovery of the method of Nob in rat tissues by LC/MS/MS (n=5) Extraction recovery(%)
98.68±2.24
93.28±3.04 〔%〕
2000
1981.19±113.79
99.06±5.69
95.00±0.13
8000
7481.80±21.21
93.52±0.27
99.55±4.78
200
193.79±6.93
96.90±3.46
89.24±4.11
2000
1854.23±40.83
92.71±2.04
97.36±5.12
8000
7343.92±301.46
91.80±3.77
95.93±4.08
200
236.81±8.56
118.41±4.28
Spleen
86.52±4.96
2000
2192.25±15.85
109.61±0.79
104.91±3.53
8000
8258.63±485.21
103.23±6.07
105.26±1.64
200
221.13±9.58
110.57±4.79
88.43±2.11
Lung
2000
1794.16±3.95
89.71±0.20
93.01±2.17
8000
8255.13±305.68
103.19±3.82
98.21±4.00
199.02±6.97
99.51±3.49
85.38±1.76
1861.45±83.70
93.07±4.19
95.51±2.02
8311.99±783.62
103.89±9.79
97.88±1.27
200 Kidney
cr
us
Liver
an
Heart
ip t
Analytical recovery (%)
200
Observed concentration(ng·g-1) (ng· g-1) 197.35±4.48
Nominal concentration( ng·g-1)
M
Tissue
2000
ed
8000
ce pt
Tab.4 Pharmacokinetic parameters of Nob after intravenous administration of Nob solution and Nob liposome to rats (n=5) Unit
Nob solution
Nob lipisomes
P
h
0.08±0.04
0.11±0.03
0.35
h
1.80±1.00
1.29±1.03
0.57
h
52.33±21.82
55.85±23.32
0.86
h
0.04
0.04
μg/L
3985.37±850.94
4818.25±508.7
0.49
V1
L/kg
0.20±0.08
0.18±0.02
0.66
CL
L/h/kg
0.191±0.06
0.20±0.12
0.88
AUC(0-t)
ug/L*h
4612.42±1471.35
4498.21±1860.17
0.94
AUC(0-∞)
ug/L*h
5712.06±1778.73
6038.70±2633.32
0.87
K10
1/h
1.08±0.53
1.14±0.67
0.91
K12
1/h
7.71±6.27
3.08±0.33
0.27
K21
1/h
3.02±2.89
2.22±1.31
0.68
K31
1/h
0.05±0.02
0.06±0.04
0.89
K13
1/h
1.25±0.81
1.29±1.08
0.96
Parameter t1/2α t1/2β t1/2γ Tmax
Ac
Cmax
Page 32 of 34
Tab.5 Nob concentration (ng· g-1) in different tissues after intravenous administration of Nob solution or Nob liposome to rats (Dose: 1 mg·kg-1, n=5) 1h( ng· g-1)
Tissue Heart
Nob solution
Nob liposome
8h( ng· g-1)
Nob solution
Nob liposome 309.24±27.24 10793.94±976.36
Nob solution
Nob liposome
169.81±33.51
176.84±28.60
6863.36±678.75
8952.81±1633.83
ip t
-1
0.083h( ng· g )
4360.17±971.36
896.56±200.37 8332.67±1454.25
Spleen
4111.13±576.35
3799.68±696.22
6403.13±1196.89
9734.93±1506.91
3058.36±871.50
4273.04±451.45
Lung
14449.43±2260.31
1445.77±9.20
7311.24±1101.94
625.27±136.57
467.65±134.82
623.68±196.09
595.25±149.99
917.38±119.19
451.57±91.31
672.62±205.76
385.12±18.30
us
1768.71±290.51
Ac
ce pt
ed
M
an
Kidney
cr
1379.07±193.02
Liver
2829.75±331.86 6172.54±920.33
Page 33 of 34
Tab.6 Extraction recovery of Nob in rat tissues by LC/MS/MS (n=3) Extraction recovery〔%〕
Added(ng·g-1)
Heart
Liver
Spleen
Lung
Kidney
93.28±3.04
89.24±4.11
86.52±4.96
88.43±2.11
85.38±1.76
2000
95.00±0.13
97.36±5.12
104.91±3.53
93.01±2.17
95.51±2.02
8000
99.55±4.78
95.93±4.08
105.26±1.64
98.21±4.00
97.88±1.27
ip t
200
cr
Tab.7 Pharmacokinetic parameters of Nob after intravenous administration of Nob solution and Nob liposome to rats (n=5) Unit
Nob solution
Nob lipisomes
P
t1/2α
h
0.08±0.04
0.11±0.03
0.35
t1/2β
h
1.80±1.00
1.29±1.03
0.57
t1/2γ
h
52.33±21.82
55.85±23.32
0.86
Tmax
h
0.04
Cmax
μg/L
3985.37±850.94
4818.25±508.7
0.49
V1
L/kg
0.20±0.08
0.18±0.02
0.66
CL
L/h/kg
0.191±0.06
0.20±0.12
0.88
AUC(0-t)
ug/L*h
4612.42±1471.35
4498.21±1860.17
0.94
AUC(0-∞)
ug/L*h
5712.06±1778.73
6038.70±2633.32
0.87
K10
1/h
1.08±0.53
1.14±0.67
0.91
K12
1/h
7.71±6.27
3.08±0.33
0.27
K21
1/h
3.02±2.89
2.22±1.31
0.68
1/h
0.05±0.02
0.06±0.04
0.89
1/h
1.25±0.81
1.29±1.08
0.96
an
M
Ac ce p
K13
0.04
d
te
K31
us
Parameter
Tab.8 Nob concentration (ng· g-1) in different tissues after intravenous administration of Nob solution to rats (Dose: 1 mg·kg-1, n=5)
T(h)
Heart
Liver
Spleen
Lung
Kidney
0.083
2829.75±331.86
6172.54±920.33
4111.13±576.35
14449.43±2260.31
1768.71±290.51
1
896.56±200.37
8332.67±1454.25
6403.13±1196.89
7311.24±1101.94
917.38±119.19
8
169.81±33.51
6863.36±678.75
3058.36±871.50
467.65±134.82
672.62±205.76
Tab. 9 Nob concentration (ng· g-1) in different tissues after intravenous administration of Nob liposome to rats (Dose: 1 mg· kg-1, n=5) T(h)
Heart
Liver
Spleen
Lung
Kidney
0.083
1379.07±193.02
4360.17±971.36
3799.68±696.22
1445.77±9.20
595.25±149.99
1
309.24±27.24
10793.94±976.36
9734.93±1506.91
625.27±136.57
451.57±91.31
8
176.84±28.60
8952.81±1633.83
4273.04±451.45
623.68±196.09
385.12±18.30
Page 34 of 34
S1570-0232(14)00232-3 http://dx.doi.org/doi:10.1016/j.jchromb.2014.03.039 CHROMB 18870
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
22-12-2013 21-3-2014 26-3-2014
Please cite this article as: X. Yang, C. Jianming, G. Dan, Bio-distribution and Pharmacokinetics of Nobiliside A-loaded Liposome Following Intravenous Administration in Rats, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.03.039 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.
Bio-distribution and Pharmacokinetics of Nobiliside A-loaded Liposome Following Intravenous Administration in Rats Xiong Yanga*†, Chen Jianming b†, Guo Danc† Department of Pharmaceutical Science ，Zhejiang Chinese Medical
ip t
a
b
cr
University, Hangzhou, Zhejiang, 310053, China
Department of Pharmaceutical Science, School of Pharmacy, Second
These authors contributed equally to this work.
M
†
Shanghai Institute for food and drug control, Shanghai 201203, China
an
c
us
Military Medical University, Shanghai 200433, China
*Correspondence author:
d
Dr. Xiong Yang,
te
Department of Pharmaceutical Science, Zhejiang Chinese Medical
Ac ce p
University, Hangzhou, Zhejiang, 310053, P. R. China Tel: 011-86-571-86613524 Fax: + 011-86-571-86613606
Email：[email protected]
1
Page 1 of 34
Abstract: Nobiliside A (Nob) is a new triterpenoid saponin separated from Holothuria noblilis. In this article, a liquid chromatography–electrospray
ip t
ionization-tandem mass spectrometry method was established to quantify
cr
Nob, a hemolytic saponin, in rat blood and tissue homogenates. Standard curves were linear (r=0.9988–0.9995) over the range 50~5000 ng/mL in
us
blood and 100~10000 ng/g in tissues. The lower limit of quantification
an
(LLOQ) was 50 ng/mL for Nob. The novel method was rapid, accurate, highly sensitive and highly selective. Using this method, the
M
pharmacokinetics and biodistribution of Nob liposome and Nob solution in Sprague-Dawley rats after a single intravenous dose of 1mg/kg were
d
then investigated. Nob was cleared slowly from circulation. There was no
te
significant difference of the pharmacokinetic parameters in blood between
Ac ce p
Nob solution and Nob liposome. The highest AUC of Nob was observed in liver for the two groups, followed by spleen, lungs, kidney and heart. Compared with Nob solution, Nob liposome showed much higher AUC in liver and spleen and much lower AUC in kidney, heart and lung, which might be one important reason for the decreased toxicity of Nob.
Key words：Nobiliside A，liposome，LC/MS/MS，pharmacokinetics， bio-distribution
2
Page 2 of 34
1. Introduction Nobiliside A (Nob) is a new triterpenoid saponin separated from Holothuria noblilis with chemical molecular formula of C54H87O26SNa
ip t
and stereotaxic configuration (see Fig.1A for chemical structure),
cr
determined by the modern spectral analysis, and especially advanced 2D nuclear magnetic resonance spectrum analysis. Extracorporeal antitumor
us
test shows that Nob has obvious inhibitory effect on many kinds of tumor
an
cell strains, such as P388 mouse lymphoma, A549 human lung cancer and so on. Furthermore, Nob is a dual-acting anticancer agent with both
M
cytotoxic and angiogenesis inhibiting effects [1, 2]. Unfortunately, in our previous study (data not published), the bioavailability of Nob was low
d
after oral administration due to the hydrolysis and enzymolysis of Nob in
te
the artificial simulation gastric juices. While for intravenous injection,
Ac ce p
Nob was highly toxic, mainly because it could cause hemolysis of blood cells and heart toxicity [3].
Therefore, liposome was used as its carrier
to reduce the hemolysis and toxicity of Nob after intravenous administration [4].
How about the bio-distribution and pharmacokinetics of Nob
solution and Nob liposome after intravenous administration? Would the reduction of toxicity partly related with the change of the bio-distribution and pharmacokinetics? Up to now, there is no report answering these questions. 3
Page 3 of 34
In this work, liquid chromatography tandem mass spectrometry methods with multiple reaction monitoring (MRM) were developed and validated for determination of Nob in rat whole blood and tissues, using
ip t
lovastatin acid as the analytical internal standard (see Fig.1B for chemical
cr
structure). Based on the analytical results, bio-distribution and pharmacokinetics of Nob and Nob liposome following intravenous
an
us
administration in Sprague–Dawley(SD) rats were evaluated.
2.1 Materials and animals
M
2. Materials and methods
Nobiliside A (Nob, purity >98%) was provided by Research Center
d
for Marine Drugs, College of Pharmacy, Second Military Medical
te
University (Shanghai, China). Internal standard lovastatin acid was
Ac ce p
purchased from the Shanghai Institute of Pharmaceutical Industry (Shanghai, China). Phospholipid was purchased from Tai-wei-yao-ye Ltd (Shanghai, China). Cholesterol was purchased from Shanghai Chemical Reagent Company (Shanghai, China). Methanol (Tedia Inc., CA, USA) was HPLC grade. Water used was
from a Millipore Milli-Q Plus 185 ultra-pure water system (Millipore, MA, USA).
All other chemicals were analytical grade or better.
SD rats(200±20g) were provided by Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Animal experiments were performed 4
Page 4 of 34
according to the Guiding Principles for the Care and Use of Experiment Animals in Shanghai Medicilon Inc., and were carried out in accordance
ip t
with the SOPs of the facility. 2.2 Preparation of Nob liposome
cr
Nob liposome was prepared by thin-film ultrasonication technique [4]. Briefly, phospholipids and cholesterol at the proportion of 2∶0.9
us
were co-dissolved in chloroform and evaporated to form a thin lipid film
an
under reduced pressure. The thin film was hydrated with an aqueous Nob solution (0.5 mg·mL-1), and the lipids/drug ratio (w/w) was 40. The
M
resulting mixture was sonicated for 30 min and further processed by probe sonication for 1-min cycle (3 s working and 3 s rest) at 200 W
d
(Ningbo Xinzhi Bio-tech Co. Ltd., China). The resulting liposome
te
suspension was extruded through sterile Millipore Express (PES,
Ac ce p
Millipore, USA) with 0.22 µm of pore size. 2.3. LC/MS/MS determination 2.3.1 Apparatus and chromatographic conditions Apparatus and chromatographic conditions were the same with
which described in one article we published before [5]. Briefly, The LC system comprised an isocratic pump (1100 series), and autosampler (1100 series) and a degasser (1100 series) (Agilent Technologies Inc. Palo Alto, CA, USA). Mass spectrometric analysis was performed using an API3000 (triple-quadrupole) instrument with an ESI interface with 5
Page 5 of 34
TurboIon spray (Applied Biosastems, Foster City, CA, USA). Data were acquired and processed by Analyst 1.4 software from ABI Inc. The analytical column used was an Agilent Zorbax SB-Cl8 column (5 μm, 4.6
ip t
mm ×15 mm). The mobile phase was a mixture of methanol and water
cr
75:25 (v/v) for blood samples and 80:20 (v/v) for tissues samples. The mobile phase was degassed automatically using the electronic degasser
us
system. The column was equilibrated and eluted under isocratic
an
conditions utilizing a flow rate of 1 mL/min at ambient temperature. Negative ion mass spectrometry was performed using the following
M
conditions: nebulize gas: 8 L/min; curtain gas: 8 L/min; collision gas: 4
2.3.2 Assay method
d
L/min; ion spray voltage: −4500 V; temperature: 500 ℃.
te
Whole blood samples were collected from SD rats into vials
Ac ce p
containing sodium heparin and stored at −20 ℃ until use. Thawed blank blood was spiked with Nob stock solution to calibration standards (CS) and quality control (QC) samples. Predetermined volumes (20 μL) of
Nob stock solution and 500 μL of internal standard lovastatin acid (49.4
ng/mL) were added to obtain CS samples at Nob concentrations equal to 50, 100, 250, 500, 1000, 2500, 5000 ng/mL, and QC samples at concentrations equal to 75, 750, 4000 ng/mL, and then vortexed for 1 min. After centrifugation at 15000 × g for 5 min, 100 μL of the supernatant liquid was transferred to a sample vial and 5 μL was injected into the 6
Page 6 of 34
LC/MS/MS for quantitative analysis. 100 mg tissues (heart, liver, spleen, lungs and kidney) from SD rats were collected respectively, and then homogenized in 300 μL normal
ip t
saline (0.9% NaCl). Homogenates were aliquoted (100 μL) ，
cr
predetermined volumes (20μL) of Nob stock solution and 500 μL of
internal standard lovastatin acid (49.4 ng/mL) was added to obtain CS
us
samples at Nob concentrations equal to 100, 250, 500, 1000, 2500, 5000,
an
10000 ng/g, and QC samples at concentrations equal to 200，2000，8000 ng/g, and then vortexed for 1 min. After centrifugation at 15000 × g for 5
M
min, 100 μL of the supernatant liquid was transferred to a sample vial and 5 μL was injected into the LC/MS/MS for quantitative analysis.
d
A thorough and complete method validation for assaying Nob in rat
te
blood and tissue homogenate samples was done following the US Food
Ac ce p
and Drug Administration(FDA) guidelines[6]. The validation parameters included specificity, linearity, accuracy, precision and stability. All animal studies were approved by IACUC of Shanghai Medicilon
Inc. and carried out in accordance with the SOPs of the facility. 2.3.3 Pharmacokinetic study SD rats were randomly divided into two groups (5 per group). Group 1 was treated with Nob solution (dissolved in 5% glucose aqueous solution) whilst group 2 treated with Nob liposome, and the concentration of Nob in its both solution and liposome suspension was 0.5 mg/mL. 7
Page 7 of 34
Each preparation was injected through the tail vein at the Nob dose of 1mg/kg, at which dose Nob had shown therapeutic anticancer effect in pre-training administration.
ip t
Blood samples about 0.3 mL were taken by terminal retro-orbital
cr
bleeding at various times (at 0.04, 0.083, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12,
24 h) into micro-tubes containing sodium heparin as an anticoagulant.
us
Then a 100 μL aliquot of each blood sample was combined with 20 μL of
an
methanol and 500 μL of internal standard lovastatin acid (49.4 ng/mL), and vortexed for 1 min. The methods for Nob to be extracted from the
M
blood and to be determined were the same as described in Section 2.3.2. All samples were made in triplicate. Blood samples from untreated rats
d
were used as blank samples.
te
2.3.4 Tissue distribution study
Ac ce p
SD rats were randomly divided into two groups (15 per group). Nob solution (0.5mg/mL) and Nob liposome suspension (0.5mg/mL) were injected through the tail vein at the dose of 1 mg/kg, respectively. Five rats per group were sacrificed at various times ( at 0.083 h，1 h，8 h) and tissues (heart, liver, spleen, lungs and kidneys) were obtained. All tissues were weighed and 100 mg of tissues was collected respectively, and then homogenized in 300 μL normal saline (0.9% NaCl). Homogenates were aliquoted (100 μL), and mixed with 20 μL of methanol and 500 μL of internal standard lovastatin acid (49.4 ng/mL). 8
Page 8 of 34
The methods for Nob to be extracted from the homogenates and to be determined were the same as described in Section 2.3.2.
untreated rats were used as blank samples respectively.
cr
2.4 Date analysis
ip t
All samples were carried out in triplicate. Tissue samples from
Pharmacokinetic parameters for Nob were calculated by PK analysis
us
software DAS 2.0(Gaosi Data Analysis Inc., Wuhu, China) and the
an
noncompartmental model was applied. Trapezoidal rule was used to
3. Results and discussion 3.1 Assay method
M
calculate the area under the curve (AUC).
d
Chromatograms obtained from blood and blank tissue homogenate
te
of the liver (as a representative sample) spiked with Nob and the internal
Ac ce p
standard lovastatin acid were shown in Fig. 2 and Fig. 3 respectively. Nob exhibited a strong mass response in negative ESI mode due to the efficiency of ionization of the analyte. ESI showed that nobiliside A (MW: 1206) and IS (MW: 422) formed predominately molecular ions [M−Na]− atm/z 1183.4, and mainly deprotonated molecular ions [M−H]−
at m/z 421.2 in full scan mass spectra. Parameters were tuned according to the MS signal response of the target compound. The dominant product ions for quantitative detection of Nob and IS were m/z 96.90 and 101.00 respectively. Under the described chromatographic conditions, there was 9
Page 9 of 34
not any endogenous interference from the blank rat blood or tissues, which in turn ensured a high specificity of the LC/MS/MS method. Under the described chromatographic conditions, the retention time was
ip t
about 1.9 and 3.1 min for Nob and lovastatin acid in blood sample, and
cr
about 1.4 and 2.1 min for nobiliside A and lovastatin acid in tissue samples respectively.
us
Blood quality control samples and tissue homogenate quality control
an
samples were determined by LC/MS/MS. The standard curves between the peak area of Nob in quality control samples and Nob concentration
M
were evaluated by least squares regression analysis. Standard curves were linear (r=0.9988~0.9995) over the range 50~5000 ng/mL in blood and
d
100~10000 ng/g in tissues, which were shown in Tab. 1.
te
The precision was assessed by analyzing quality control samples at
Ac ce p
three concentrations in five duplicates. Tab. 2 showed the between-run, within-run precision, extraction efficiency for blood samples respectively. The stability of Nob in extract and whole blood using weighed-in quality control samples also studied (Tab.2). Tab. 3 showed the between-run, within-run precision and extraction efficiency of samples in tissue homogenates respectively. The lowest limit of qualification (LLOQ) for Nob was 50 ng/mL. The method was sufficiently sensitive for the determination of bio-distribution and pharmacokinetic analysis of Nob in vivo. 10
Page 10 of 34
3.2 Pharmacokinetics study The blood concentration–time curves of Nob in rats after intravenous administration of a single 1mg/kg dose of Nob solution and
ip t
Nob liposome were shown in Fig. 4. The non-compartmental
cr
pharmacokinetic parameters calculated by DAS 2.0 software were shown in Tab.4. Based on the analysis of the models and parameters, it was
us
concluded that the mean blood concentration-time profiles for both Nob
an
solution and Nob liposome were described by a three-compartment model with a weight of 1/C2.
M
Following intravenous administration of Nob solution at a dose of 1 mg/kg, the mean values of Cmax and Tmax were 3985.37±850.94 ng/mL
d
and 0.04 h respectively. The systemic clearance was 0.191±0.06 L/h/kg;
te
and AUC (0-∞) was 5712.06±1778.73 (μg/L·h). The volume of distribution
Ac ce p
at terminal phase was 0.20±0.08 L/kg(Tab. 4). For Nob liposome, Cmax (4818.25±508.7) was achieved at 0.04 h
(Tmax). The systemic clearance was 0.20±0.12 L/h/kg, and AUC(0-∞) was 6038.70±2633.32 (μg/L*h). The volume of distribution at terminal phase
was 0.18±0.02L/kg (Tab. 4). There was no significant difference of the pharmacokinetic parameters between Nob solution and Nob liposome and both of them were eliminated slowly in vivo. The blood concentration at 24 h was not much lower than that at 8 h. 11
Page 11 of 34
3.3 Bio-distribution study The levels of Nob in tissues including heart, liver, spleen, lungs, and kidneys determined at 0.083 h, 1 h and 8 h after intravenous
ip t
administration of a single 1mg/kg dose of Nob solution and Nob
cr
liposome were shown in Tab.5, respectively.
After systemic administration, Nob was rapidly distributed into all
us
tissues studied. The AUC of Nob in tissues after intravenous
an
administration of Nob solution and Nob liposome to rats was shown in Fig. 5. In the two groups, the highest AUC of Nob was observed in liver,
M
followed by spleen, lungs, kidneys and heart.
Tab. 5 showed Nob concentration in different tissues at different time
d
after dosing. There were same trends of Nob concentration of the two
te
groups in the same tissue. In liver and spleen, the concentration of Nob
Ac ce p
firstly increased during 0.083 h to 1 h, and then decreased at a very slow speed in 24 h. However, in heart, lungs and kidneys, the concentration of Nob fell off quickly in the first 1 h and decreased slowly during 1 h to 8 h after administration.
Intravenously injecting liposome could be used for passive targeting of liver and spleen. In order to explore the passive target activity of Nob liposome used in this study, the ratio of AUCNob liposome/ AUC Nob solution
was introduced，which demonstrated the relative efficacy of the two delivery systems toward the same tissue [7]. The highest ratio was 12
Page 12 of 34
observed in spleen with the value of 1.45, followed by liver with the value of 1.27, indicating that Nob liposome was more efficient in delivering Nob to spleen and liver compared with the solution. The ratios
ip t
for kidney, heart, and lung were 0.50, 0.46, and 0.15 respectively. Nob
cr
has many toxic effects, such as causing hemolysis, prohibiting respiration, damaging the heart and kidneys. Therefore, besides decreaseing
us
hemolysis[4], Nob liposome might also alleviate other side effects, such
an
as that on the heart and kidneys after intravenous administration. 4. Discussion and conclusions
M
Nob is a water soluble saponin. However, there was no significant difference of the pharmacokinetic parameters between Nob solution and
d
Nob liposome and both of them were eliminated slowly in vivo. Besides,
te
for the same tissue, the concentration–time curves of the two groups had
Ac ce p
similar variation trends. Why? According to our previous study [8], there was a strong interaction between Nob and cholesterol. Nob and cholesterol in plasma and blood cells could be combined and then form the non-soluble molecule compounds, and then the pharmacokinetic parameters of the compounds would be similar to Nob liposome. From the concentration–time curve, Nob solution group and liposome group were metabolized slowly within the body, and could still be detected at 24 h. Slight difference between Nob concentration at 8 h and 24 h was found. As described in other literatures, the process of saponins 13
Page 13 of 34
metabolized in vivo was relatively slow. For example, at a dose of 5 mg/kg for intravenous administration, the concentration of Ginsenoside Rb1 in serum was (83.8±12.9) µg· mL-1 at 5 min; after 1 h, the
ip t
concentration of Rb1 decreased rapidly, and remains (1.1±0.03) µg· mL-1
was 11.6 min for phase α and 14.5 h for phase β[9].
cr
in serum at 72 h. The half-life period of disappearance of Rb1 in blood
us
Since there was a strong interaction between Nob and cholesterol, it
an
was not strange to find that the highest AUC of Nob solution was observed in liver and followed by spleen. Firstly, more cholesterol exists
M
in liver and spleen than in other tissues. Secondly, parts of the Nob form compounds with cholesterol in vivo and then the compounds could target
d
to liver and spleen passively, just like the characters of liposomes or
te
nanoparticles. However, now that Nob in liposome had a strong
Ac ce p
interaction with cholesterol in lipid membrane [8], it should have little interaction with cholesterol in vivo, so the Nob in liposome enriched in liver and spleen might be attributed to the fact that nanoparticles with proper particle size were recognized and trapped by the RES which abound in special tissues and organs, such as liver and spleen [10]. Particularly worth mentioning was, as seen from Tab.5 , Nob concentration of both the liposome group and solution group firstly increased during 0.083h to 1h(tmax), and then decreased at a very slow
speed during 1h to 24h. While in other tissues like heart, lungs, kidneys, 14
Page 14 of 34
Nob concentration of both groups reached tmax at 0.083h, and then decreased at a very slow speed in 24h. We speculated that may be part of free Nob released from liposome could firstly be combined with
cr
by the RES in liver and spleen, which need a little time.
ip t
cholesterol in blood, and then most of them was recognized and trapped
There were similar reports about the distribution of other saponins in
us
animals. After intragastric administration of total saponins of tribulus to
an
rats, some hecogenin glycosides were hydrolyzed into hecogenin, which were still detected in the liver at 32 h [11]. Li K, et al. reported the
M
distribution characteristics of dioscin after intravenous and intragastric administration in rats[12].The results showed that dioscin is mostly
d
stored in liver, less in lung and least in kidneys (no report about its
te
distribution in spleen or heart). In addition, the elimination of dioscin was
Ac ce p
quite slow. For example, after intravenous administration of a single dose of 1 mg· kg-1, drug concentration in liver decreased by 37%, 70% and
78% at 24 h, 72 h and 120 h respectively. Dioscin belongs to spirostane saponin, which has large molecular weight and high surface activity, can be combined with cholesterol in cells or tissues. Just for its similar structure and properties as Nob, its activities just as elimination and metabolism in the body may be related to cholesterol content in tissues. Comparing with some other compound analyzed by HPLC/MS/MS, the method was less sensitive. There would be mainly because of the 15
Page 15 of 34
characteristic of the drug itself. Firstly, Nob is a triterpenoid saponin with high molecular（MW：1206）, it has lower ionization efficiency than other small molecular compounds, thus the responding value is relatively low.
ip t
We tried many times to optimize its chromatographic and mass
cr
spectrographic conditions, but we found it was hardly to improve its sensitivity. Moreover, we found Nob had a strong selectivity to its
us
analytical column. We ever tried C-18 column, C-8 column and polarity
an
column to analyse Nob by HPLC/ELSD for in-vitro studies, but got bad results, such as unsightly chromatographic peak which has a tailing or
M
furcation, stagnation of Nob on the column thus caused very low sensitivity and so on. While using SBC-18 column could get the best
d
effect including sensitivity and peak shape. Secondly, in this test, we
te
found that the main fragment peak at m/z 96.90 has the most stability
Ac ce p
with also strong response, and thus got good linearity and precision in the range of 50–5000 ng/mL, which was hardly to be obtained by using other fragment peak. So it was selected to be quantative fragment ion. But as we know, the smaller is the m/z, the greater is the baseline noise and thus the lower of sensitivity because of the obvious background interference (such as mobile phase, endogenous substances and so on) between 30-300 m/z. Of course, although the low limit of this method was 50 ng/mL, it is sufficiently sensitive for the study on the bio-distribution and pharmacokinetics of Nob. So we think this method could be used in this 16
Page 16 of 34
test. Few people will research the hemolytic saponins for intravenous injection, so no paper is found about the pharmacokinetics and tissues
ip t
distribution following intravenous injection about this kind of saponins
cr
and their preparations. This is the first paper describing a fast, sensitive, and specific method for blood and tissues quantification of Nob, a
us
hemolytic saponin, by LC-ESI/MS/MS method. And it is also the first
an
paper comparing biodistribution and pharmacokinetics of Nob and its liposome following intravenous administration in rats.
M
Acknowledgements
This work was financially supported by project supported by
d
National Science Foundation for Young Scientists of China
te
(No. 81202926), National High Technology Research and Development
Ac ce p
Program of China (863 Program) (No. 2006AA090304), and also supported by Science Technology Department of Zhejiang Province(No. 2012R10044-09), Authors are grateful to Dr.Yanghua Yi, Second Military Medical University, China, for providing Nobliside A.
17
Page 17 of 34
Reference: [1]Wu, J., Yi, Y.H., Wu, H.M., He, Q.S., Zhang, S.L. Studies on the in vitro antifungai and antitumor activities of nobiliside A from the sea cucumber Holothuria nobilis
ip t
Selenka. Chin. Pharmacol. Bull., 23(2007)139-140
cr
[2] Yi, Y.H., Ding, J., Zhang X.W., Wu, J., Chen, Y., Liu, B.S., Tian, F., 2006.
us
Antitumor compound nobiliside A from Holathuria nobilis and its monoacetylated derivative. CN Patent CN1740185, 1 May.
an
[3]Hu, M., Konoki, K., Tachibana, K., 1996. Cholesterol-independent membrane disruption caused by triterpenoid saponins. Biochim. Biophys. Acta, 1299, 252-258.
M
[4]Xiong Y., Guo D., Wang L.L.，Zhen X.L., Xu L.Y., Chen J.M. Development of
te
Pharm., 371 (2009) 197-203.
d
nobiliside A loaded liposomal formulation using response surface methodology. Int J
[5]Guo D., Xiong Y., Zhang Y., et al. Development and validation of a LC/MS/MS
Ac ce p
method for quantification of Nobiliside-A in rat plasma. J Chromatogr B. 877 (2009) 323-327.
[6]FDA; 2001 Available from:WWW.fda.gov/dowloads/Drugs/ Guidance Compliance
Regulatory Information/Guidances/ucm070107.pdf. [7]Chen Z.P.,Zhu J.B., Chen H.X.,et al.. Distribution of liposomal bifdendate in liver following intravenous injection in mice. J Drug Target. 18(2010) 627-636. [8]Xiong Y., Chen J.M., Guo D. Interaction Between Nobiliside-A and Lipid Bilayers，Lat. Am. J. Pharm. 31 (2012) 800-807. [9]Odani T., Tanizawa, H., Takino, Y. Studies on the absorption, distribution, excretion and metabolism of ginseng saponins. III. The absorption, distribution and 18
Page 18 of 34
excretion of ginsenoside Rb1 in the rat. Chem Pharm Bull. 31(1983)1059-1066． [10]Mizushima Y., Hamano T., Yokoyama K. Tissue distribution and antiinflammatory activitu of corticosteroids incorporated in lipid emulsion. Ann Rheum
ip t
Dis. 41(1982)263-267. [11]Chen Y.G., Qu W.J., Yang N.Y., et al. Metabolism and distribution of Hecogenin
cr
in rats after intragastric infusion with saponins of Tribulus terristris L. Nat Prod Res
us
Dev. 18(2006)927-931.
Ac ce p
te
d
M
dioscin in rat. Steroids, 70(2005) 525–530.
an
[12]Li K., Tang Y.B., Fawcett J. P., et al. Characterization of the pharmacokinetics of
19
Page 19 of 34
Figure Legends
ip t
Fig.1 Chemical structure of (A) nobiliside A and (B)internal standard lovastatin acid
cr
Fig.2 Representative MRM chromatograms from nobiliside A (m/z1183.4→96.9amu)
us
channel and internal standard lovastatin acid (m/z421.2→101.0amu)channel of extracts from (A) blank whole blood ,(B) blank whole blood with Nob and IS, and (C)blood
an
sample after intravenous injection
M
Fig.3 Representative MRM chromatograms from nobiliside A(m/z1183.4→96.9) channel and internal standard lovastatin acid (m/z421.2→101.0))channel of extracts
te
d
from (A) blank control liver homogenate, (B) blank liver homogenate with nobiliside A
Ac ce p
and IS, and (C)liver sample after intravenous injection
Fig.4 The blood concentration-time profiles of Nob solution and Nob liposomes after intravenous administration of a single 1mg/kg dose to rats (Each point represents the mean±SD of 5 rats)
Fig. 5 Tissues AUC of Nob solution and Nob liposomes in rats during 0-8h after intravenous administration at the dose of 1mg/kg
20
Page 20 of 34
*Highlights (for review)
LC /MS/MS is established for biodistribution and pharmacokinetics of Nobliside A. Biodistribution and pharmacokinetics of Nobliside A and its liposome are compared. Nobliside A and its liposome eliminate slowly in rat after intravenous injection. Similar behavior of Nobliside A and its liposome appears in whole blood of rat.
Ac
ce pt
ed
M
an
us
cr
ip t
Nobliside A liposome shows much higher AUC in liver and spleen than its solution.
Page 21 of 34
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pt
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M
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Figure 1
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pt
ed
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Figure 2A
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pt
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Figure 2B
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ed
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Figure 2C
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Figure 3A
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Figure 3B
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pt
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Figure 3C
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Figure 4
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Figure 5
Page 30 of 34
Tables
Tab. 1 Regression parameters of standard curves for the determination of Nob in rat blood (concentration: 50～5000 ng·g-1) and tissues (concentration: 100～10000 ng·g-1) Blood or Tissue
R
Intercept（*10-4） 1.54
0.9989
Heart
25.8
3.56
0.9995
Liver
9.47
6.83
0.9987
Spleen
6.47
1.67
0.9986
Lung
8.24
3.41
Kidney
8.71
2.51
ip t
blood
Slope (*10-6） 9.36
0.9988
cr
0.9995
Within-day accuracy (%)
us
Tab.2 Precision, extraction recovery and stability data of the method of Nob in rat blood by LC/MS/MS (n=5) 75 ng·mL-1
750 ng·mL-1
4000 ng·mL-1
100.78±4.18
101.24±2.55
99.01±1.58
an
(n=5) Between-day accuracy
102.76±8.77
(%) (n=5) 91.51±4.11
Stability
for 24h(ARa) ARa of the blood at 4℃
107.09±8.88
92.17±8.70
84.58±6.86
98.92±5.19
103.48±8.80
99.51±3.53
94.82±1.50
ce pt
for 48h a
102.25±6.11
ed
ARa of the extract at 25℃
101.89±4.44
M
Extraction recovery(%)
100.34±1.99
AR: analytical recovery was determined as the mean assayed concentration expressed as a percentage
Ac
of the weighed-in concentration.
Page 31 of 34
Tab.3 Precision and extraction recovery of the method of Nob in rat tissues by LC/MS/MS (n=5) Extraction recovery(%)
98.68±2.24
93.28±3.04 〔%〕
2000
1981.19±113.79
99.06±5.69
95.00±0.13
8000
7481.80±21.21
93.52±0.27
99.55±4.78
200
193.79±6.93
96.90±3.46
89.24±4.11
2000
1854.23±40.83
92.71±2.04
97.36±5.12
8000
7343.92±301.46
91.80±3.77
95.93±4.08
200
236.81±8.56
118.41±4.28
Spleen
86.52±4.96
2000
2192.25±15.85
109.61±0.79
104.91±3.53
8000
8258.63±485.21
103.23±6.07
105.26±1.64
200
221.13±9.58
110.57±4.79
88.43±2.11
Lung
2000
1794.16±3.95
89.71±0.20
93.01±2.17
8000
8255.13±305.68
103.19±3.82
98.21±4.00
199.02±6.97
99.51±3.49
85.38±1.76
1861.45±83.70
93.07±4.19
95.51±2.02
8311.99±783.62
103.89±9.79
97.88±1.27
200 Kidney
cr
us
Liver
an
Heart
ip t
Analytical recovery (%)
200
Observed concentration(ng·g-1) (ng· g-1) 197.35±4.48
Nominal concentration( ng·g-1)
M
Tissue
2000
ed
8000
ce pt
Tab.4 Pharmacokinetic parameters of Nob after intravenous administration of Nob solution and Nob liposome to rats (n=5) Unit
Nob solution
Nob lipisomes
P
h
0.08±0.04
0.11±0.03
0.35
h
1.80±1.00
1.29±1.03
0.57
h
52.33±21.82
55.85±23.32
0.86
h
0.04
0.04
μg/L
3985.37±850.94
4818.25±508.7
0.49
V1
L/kg
0.20±0.08
0.18±0.02
0.66
CL
L/h/kg
0.191±0.06
0.20±0.12
0.88
AUC(0-t)
ug/L*h
4612.42±1471.35
4498.21±1860.17
0.94
AUC(0-∞)
ug/L*h
5712.06±1778.73
6038.70±2633.32
0.87
K10
1/h
1.08±0.53
1.14±0.67
0.91
K12
1/h
7.71±6.27
3.08±0.33
0.27
K21
1/h
3.02±2.89
2.22±1.31
0.68
K31
1/h
0.05±0.02
0.06±0.04
0.89
K13
1/h
1.25±0.81
1.29±1.08
0.96
Parameter t1/2α t1/2β t1/2γ Tmax
Ac
Cmax
Page 32 of 34
Tab.5 Nob concentration (ng· g-1) in different tissues after intravenous administration of Nob solution or Nob liposome to rats (Dose: 1 mg·kg-1, n=5) 1h( ng· g-1)
Tissue Heart
Nob solution
Nob liposome
8h( ng· g-1)
Nob solution
Nob liposome 309.24±27.24 10793.94±976.36
Nob solution
Nob liposome
169.81±33.51
176.84±28.60
6863.36±678.75
8952.81±1633.83
ip t
-1
0.083h( ng· g )
4360.17±971.36
896.56±200.37 8332.67±1454.25
Spleen
4111.13±576.35
3799.68±696.22
6403.13±1196.89
9734.93±1506.91
3058.36±871.50
4273.04±451.45
Lung
14449.43±2260.31
1445.77±9.20
7311.24±1101.94
625.27±136.57
467.65±134.82
623.68±196.09
595.25±149.99
917.38±119.19
451.57±91.31
672.62±205.76
385.12±18.30
us
1768.71±290.51
Ac
ce pt
ed
M
an
Kidney
cr
1379.07±193.02
Liver
2829.75±331.86 6172.54±920.33
Page 33 of 34
Tab.6 Extraction recovery of Nob in rat tissues by LC/MS/MS (n=3) Extraction recovery〔%〕
Added(ng·g-1)
Heart
Liver
Spleen
Lung
Kidney
93.28±3.04
89.24±4.11
86.52±4.96
88.43±2.11
85.38±1.76
2000
95.00±0.13
97.36±5.12
104.91±3.53
93.01±2.17
95.51±2.02
8000
99.55±4.78
95.93±4.08
105.26±1.64
98.21±4.00
97.88±1.27
ip t
200
cr
Tab.7 Pharmacokinetic parameters of Nob after intravenous administration of Nob solution and Nob liposome to rats (n=5) Unit
Nob solution
Nob lipisomes
P
t1/2α
h
0.08±0.04
0.11±0.03
0.35
t1/2β
h
1.80±1.00
1.29±1.03
0.57
t1/2γ
h
52.33±21.82
55.85±23.32
0.86
Tmax
h
0.04
Cmax
μg/L
3985.37±850.94
4818.25±508.7
0.49
V1
L/kg
0.20±0.08
0.18±0.02
0.66
CL
L/h/kg
0.191±0.06
0.20±0.12
0.88
AUC(0-t)
ug/L*h
4612.42±1471.35
4498.21±1860.17
0.94
AUC(0-∞)
ug/L*h
5712.06±1778.73
6038.70±2633.32
0.87
K10
1/h
1.08±0.53
1.14±0.67
0.91
K12
1/h
7.71±6.27
3.08±0.33
0.27
K21
1/h
3.02±2.89
2.22±1.31
0.68
1/h
0.05±0.02
0.06±0.04
0.89
1/h
1.25±0.81
1.29±1.08
0.96
an
M
Ac ce p
K13
0.04
d
te
K31
us
Parameter
Tab.8 Nob concentration (ng· g-1) in different tissues after intravenous administration of Nob solution to rats (Dose: 1 mg·kg-1, n=5)
T(h)
Heart
Liver
Spleen
Lung
Kidney
0.083
2829.75±331.86
6172.54±920.33
4111.13±576.35
14449.43±2260.31
1768.71±290.51
1
896.56±200.37
8332.67±1454.25
6403.13±1196.89
7311.24±1101.94
917.38±119.19
8
169.81±33.51
6863.36±678.75
3058.36±871.50
467.65±134.82
672.62±205.76
Tab. 9 Nob concentration (ng· g-1) in different tissues after intravenous administration of Nob liposome to rats (Dose: 1 mg· kg-1, n=5) T(h)
Heart
Liver
Spleen
Lung
Kidney
0.083
1379.07±193.02
4360.17±971.36
3799.68±696.22
1445.77±9.20
595.25±149.99
1
309.24±27.24
10793.94±976.36
9734.93±1506.91
625.27±136.57
451.57±91.31
8
176.84±28.60
8952.81±1633.83
4273.04±451.45
623.68±196.09
385.12±18.30
Page 34 of 34
