- Email: [email protected]
Gender-related pharmacokinetics and bioavailability of a novel anticancer chalcone, cardamonin, in rats determined by liquid chromatography tandem mass spectrometry
Accepted Manuscript Title: Gender-related pharmacokinetics and bioavailability of a novel anticancer chalcone, cardamonin, in rats determined by liquid chromatography tandem mass spectrometry Author: Swati Jaiswal Abhisheak Sharma Mahendra Shukla Jawahar Lal PII: DOI: Reference:
S1570-0232(15)00081-1 http://dx.doi.org/doi:10.1016/j.jchromb.2015.01.041 CHROMB 19307
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
8-12-2014 26-1-2015 30-1-2015
Please cite this article as: S. Jaiswal, A. Sharma, M. Shukla, J. Lal, Gender-related pharmacokinetics and bioavailability of a novel anticancer chalcone, cardamonin, in rats determined by liquid chromatography tandem mass spectrometry, Journal of Chromatography B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.01.041 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.
Highlights 1. First reported LC-MS/MS method for quantification of anticancer chalcone, cardamonin, in rat serum. 2. A sensitive, accurate and precise bioanalytical method with a short chromatographic samples per day) bioanalysis.
Ac ce p
te
d
M
an
us
cr
3. First reported pre-clinical pharmacokinetics of cardamonin.
ip t
run time of 3 min which will provide benefit in high throughput (more than 400
1 Page 1 of 30
Gender-related pharmacokinetics and bioavailability of a novel anticancer chalcone, cardamonin, in rats determined by liquid chromatography tandem mass spectrometry
Pharmacokinetics & Metabolism Division, CSIR-Central Drug Research Institute, Lucknow-
cr
a
ip t
Swati Jaiswala,b,# , Abhisheak Sharmaa,b,# , Mahendra Shuklaa,b, Jawahar Lala,b,*
226031, India b
an
us
Academy of Scientific and Innovative Research, New Delhi, India
*Corresponding author: Dr. Jawahar Lal, Pharmacokinetics & Metabolism Division, CSIR-
M
Central Drug Research Institute, Jankipuram Extension, Sitapur Road, Lucknow - 226031, India; Tel.: +91-522-2772450, Ext. 4841; Fax: +91-522-2771941; E-mail: [email protected]
te
d
Authors contributed equally
Ac ce p
#
2 Page 2 of 30
Abstract A reversed phase liquid chromatography tandem mass spectrometry method was developed and validated for quantification of cardamonin, a potential anticancer chalcone, in
ip t
rat serum. Curcumin was used as an internal standard. Following liquid-liquid extraction using n-hexane and ethyl acetate (60:40, v/v), the processed samples were chromatographed
cr
on a C18 column using acetonitrile and ammonium acetate buffer (0.01 M, pH 4.5) (85:15, v/v) as mobile phase at a flow rate of 0.6 mL min-1. Mass spectrometric detection was
us
performed in the negative electrospray ionization mode by multiple reaction monitoring (m/z
an
269→122 and 367→217 for cardamonin and curcumin, respectively). The method was validated in terms of selectivity, accuracy, precision, sensitivity, reproducibility, dilution
M
integrity and stability. The linearity was established in the range of 1 to 200 ng mL-1 (r ≥0.999). The recovery of cardamonin from spiked serum was always >90%. The intra- and
d
inter-day precision (%RSD) and accuracy (%bias) were well within the acceptable limits. The
te
method was applied for single oral and intravenous dose pharmacokinetics in male and female Sprague Dawley rats. Following oral dose, cardamonin showed peak serum
Ac ce p
concentration that occurred at ~2 h with very low bioavailability in both male (0.6%) and female (4.8%) rats. Cardamonin exhibited a significant gender influence on pharmacokinetics and bioavailability in rats.
Keywords: cardamonin; pharmacokinetics, bioavailability, liquid chromatography, tandem mass spectrometry.
Abbreviations: ANOVA, analysis of variance; CAD, collision activated dissociation; CHOP, CAAT/enhancer binding protein homologous protein; CRD, cardamonin; CE, collision energy; CRC, curcumin; CXP, collision cell exit potential; DP, declustering potential; DDI,
3 Page 3 of 30
drug-drug interactions; EP, entrance potential; IS, internal standard; MAPK, mitogenactivated protein kinases; RSD, relative standard deviation; ROS, reactive oxygen species;
Ac ce p
te
d
M
an
us
cr
ip t
TDM, therapeutic drug monitoring; TRAIL, TNF-related apoptosis-inducing ligand.
4 Page 4 of 30
1. Introduction Cardamonin (CRD; (2E)-1-(2,4-Dihydroxy-6-methoxyphenyl)-3-phenyl-2-propen-1one), a chalcone, is a potential and promising anticancer compound. It is found in several
ip t
medicinal plants of Zingiberaceae family including Alpinia katsumadai, Alpinia conchigera, Alpinia rafflesiana, Amomum subulatum and Boesenbergia pandurata. CRD is reported as
cr
the most potent chalcone-type anti-tumor agent [1]. Recent studies on CRD illustrated that it augments apoptotic effects of TRAIL (TNF-related apoptosis-inducing ligand) against human
us
colon, pancreatic, myeloid leukaemia, multiple myeloma and prostate adenocarcinoma cell
an
lines [2]. The mechanism includes downregulation of anti-apoptotic proteins; ROS-CHOP (reactive oxygen species-CAAT/enhancer binding protein homologous protein) mediated
M
upregulation of TRAIL death receptors and decreased expression of decoy receptors and cell survival proteins. Several other studies on CRD suggested that it suppresses proliferation and
d
induces apoptosis in myeloma cells by inhibiting NF-kappaB (nuclear factor kappa-light-
te
chain-enhancer of activated B cells) pathway [3,4]. Moreover, CRD inhibit the breast cancer or multiple myeloma cell or RANKL induced bone osteoclastogenesis by suppressing NF-
Ac ce p
kappaB and MAPK (mitogen-activated protein kinases) pathway [5]. It has also shown cytotoxicity against leukemia K562 cells and human hepatoma SMMC-7721 cells [4]. Hitherto, CRD has also shown a variety of other appreciable pharmacological activities like anti-platelet, anti-HIV, vasorelaxant, insecticidal, anti-melanogenic, anti-mutagenic and antiinflammatory activity [6-14]. For the advanced development of a compound as a drug candidate along with pharmacological activities, pharmacokinetic properties must be explored [15]. For understanding and estimating the pharmacokinetics of any drug and/or its metabolites, quantification of that compound in suitable bio-matrices (blood, serum or plasma) is a prerequisite, which can be performed by developing a sensitive and selective bioanalytical method. With this rationale, the present investigation was carried out which
5 Page 5 of 30
involved bioanalytical method development and preclinical pharmacokinetic profiling of CRD in support of its development as a potential candidate drug. Until now, to the best of our information, there is no bioanalytical method available
ip t
for quantification of CRD in any biological matrix. Also, the knowledge concerning the pharmacokinetics of CRD is lacking. Herein, we report for the first time the development and
cr
validation of a sensitive reversed phase LC-MS/MS method with short chromatographic run time (3.0 min) for quantification of CRD in rat serum and then employed the method to
us
investigate the pharmacokinetic characteristics of CRD in male and female Sprague Dawley
an
rats following single oral and intravenous administration to explore the gender influence on
M
the pharmacokinetics of CRD.
2. Materials and Method
d
2.1. Chemicals and reagents
te
Cardamonin (99.5% pure) and curcumin (CRC) were procured from Sigma Aldrich (St. Louis, USA). CRC was used as an internal standard (IS). HPLC grade acetonitrile was
Ac ce p
purchased from Sigma Aldrich (Dombivli, India). HPLC grade n-hexane, ethyl acetate and analytical grade ammonium acetate were purchased from Spectrochem Pvt. Ltd. (Mumbai, India). HPLC grade glacial acetic acid was procured from J.T. Baker (Phillipsburg, USA). Ultra pure water was from a Milli-Q Plus PF (Billerica, USA) water purification system. Drug-free whole blood was collected from adult and healthy male and female SpragueDawley rats provided by the Laboratory Animal Services Division of the institute and serum was separated after centrifuging the collected blood at 3000 × g for 10 min. The serum samples from male and female rats were pooled separately and stored at -80°C till use. All experiments, euthanasia and disposal of carcasses were carried out as per the guidelines of local ethics committee for animal experimentation.
6 Page 6 of 30
2.2. Liquid Chromatography and tandem mass spectrometry conditions
ip t
The HPLC system (Shimadzu, Kyoto, Japan) consisted of a pump (LC-20 AD) with
us
cr
online degasser (DGU-20A3), column oven (CTO‐10AS) and auto injector (SIL-HTc, fixed
with a 100 μL loop) with a temperature-controlled pelteir-tray. Chromatographic separation
an
of the CRD and IS were achieved on a Supelco Discovery HS C18 column (5 μm, 50 x 4.6 mm, id) preceded with a Supelguard Discovery HS C18 column (5 μm, 20 x 4.0 mm, id)
M
packed with the same material. The mobile phase consisted of acetonitrile and ammonium acetate buffer (AAB; 0.01 M, pH 4.5) (85:15, v/v) and was delivered isocratically at a flow
d
rate of 0.6 mL min-1. The freshly prepared AAB was filtered through 0.22 µm membrane
te
filter (Millipore, USA). The mobile phase was degassed by ultrasonication for 15 min before
Ac ce p
use. Prior to commencement of analysis, LC-MS/MS system was equilibrated for 20 min. Aliquots (10 μL) of processed sample were injected onto the column and the column oven temperature was set at 40°C. Other LC parameters like needle rinsing speed and volume, needle stroke, sampling speed, purge time and rinse dip time were set at 35 µL s-1, 800 µL, 52 mm, 5 µL s-1, 0.5 min and 10 s, respectively. Acetonitrile: Milli-Q water (50:50, v/v) was used as rinsing solution and rinsing mode was set to after and before aspiration to minimize carryover, if any. The quantification of the compound was carried out using Q Trap 4000 MS/MS system (Applied Biosystems, Toronto, Canada) which included a hybrid triple quadrupole/LIT (linear ion trap) mass spectrometer and a Turbo V™ Ion Source. Sample
7 Page 7 of 30
introduction and ionization was performed by electrospray ionization in negative ion mode. The mass spectrometry operation was conducted under multiple reaction monitoring (MRM) mode (m/z 269→122 for CRD and m/z 367→217 for IS). The source parameters, namely,
ip t
temperature, curtain gas, ion spray voltage, nebulizer gas, heater gas, were set at 400 °C, 10 psi, -3000 V, 45 psi, 40 psi, respectively. Collision activated dissociation (CAD) gas was set
cr
at medium mode. The declustering potential (DP) for CRD and IS was set at -81 V. Other compound parameters, namely entrance potential (EP) and collision cell exit potential (CXP)
us
were set at -10 V for both, the compound and IS. The collision energy (CE) was set at -35
an
and -28 V for CRD and IS, respectively. Dwell time was set at 150 ms. Nitrogen was used as curtain and collision gas. Zero air was used as source gas. All the operations, acquisition and
M
analysis of data were controlled by AnalystTM (version 1.4.2) software (Applied Biosystems,
d
Toronto Canada).
te
2.3. Preparation of standards and quality control samples Mother stock solutions (1 mg mL-1) of the reference standard (CRD) and IS were
Ac ce p
separately prepared by dissolving 10 mg of the compound in 10 mL methanol. Working standard solutions (100 µg mL-1) of CRD and IS were separately prepared by appropriately diluting mother stock solution with methanol. Spiking standard solutions of CRD were prepared in methanol from working standard solutions by serial dilution method in the range of 0.02 to 4 µg mL-1, which were utilized for preparation of calibration standards in rat serum. Separate mother stock solution, working standard solution and spiking standard solutions were prepared in methanol for preparation of quality control (QC) standards of CRD containing 0.02, 0.06, 1.8 and 3.6 µg mL-1 for lower limit of quantification, low, medium and high concentrations, respectively. The solutions were vortex-mixed and stored at
8 Page 8 of 30
4°C. Also, spiking standard solution containing 1 µg mL-1 of IS was prepared by spiking 0.1 mL of the working standard solution in 9.9 mL of methanol. Blank pooled rat serum was pre-screened to assure the absence of any endogenous
ip t
interference at the retention time of the CRD and IS. The calibration standards were prepared individually by spiking 95 µL of drug-free rat serum with 5 µL of spiking standard solutions
cr
to obtain 1, 2.5, 10, 25, 50, 100, 190 and 200 ng mL-1. Similarly, QC standards were prepared at lower limit of quantification (LLOQ), low (LQC), medium (MQC) and high concentrations
us
(HQC) containing 1, 3, 90 and 180 ng mL-1 of CRD. Similarly, the dilution integrity quality control (DIQC) samples were prepared at concentration of 3.6 µg mL-1 by spiking 3.6 µL of
an
100 µg mL-1 to 96.4 µL drug-free rat serum. A 20-fold dilution of the DIQC (n = 6) was
M
prepared in drug-free rat serum.
d
2.4. Sample cleanup
te
Extraction of the CRD and IS was carried out by a simple liquid-liquid extraction technique using the extraction solvent (60% n-hexane in ethyl acetate). Aliquots (10 µL) of
Ac ce p
each calibration standard, QC and test samples were separately transferred in 5 mL glass extraction tubes. To this, 10 µL of spiking standard solution of IS (1 µg mL-1) was added while vortexing followed by addition of 2 mL of extraction solvent through the bottle top dispenser. After that, extraction tubes containing this mixture were vortex-mixed for 1 min and centrifuged at 3000 rpm for 10 min at 4°C. The organic layer was transferred to another tube by snap freezing the aqueous layer in liquid nitrogen and evaporated to dryness at 40°C in Turbo vap LX concentrator (Caliper, Massachusetts, USA). The dried residues were reconstituted in 100 µL mobile phase and 10 µL was injected on to LC-MS/MS system.
2.5. Method validation
9 Page 9 of 30
The present bioanalytical method was validated for selectivity, sensitivity, linearity, accuracy, precision, recovery, matrix effect, stability, dilution integrity and robustness as per US Food and Drug Administration (US FDA) guidelines [16]. Selectivity was determined by
ip t
analyzing blank serum from six different rats. Each of these blank samples was tested for the presence of any endogenous serum components eluting at the retention times of CRD and IS.
cr
Selectivity was guaranteed if the peak response from endogenous serum components was less than 20% of the mean peak response of CRD at LLOQ and less than 5% of the mean peak
us
response of IS. For evaluation of the method sensitivity, replicates of blank serum spiked
an
with CRD were processed and analyzed. The limit of detection (LOD) was determined based on analyte’s signal to noise (S/N) ratio of 3:1. The lower limit of quantification (LLOQ) was
M
defined as the lowest concentration with S/N ratio greater than 10 and accuracy and precision within 20% of the nominal concentration of the analyte. The suppression of MS/MS detector
d
response owing to matrix components associated with rat serum was assessed by extracting
te
six different lots of blank rat serum without IS. Post extraction, dried residues were reconstituted with analytical standard solutions at low (3 ng mL-1, n = 6) and high
Ac ce p
concentrations (180 ng mL-1, n = 6) of CRD. The matrix effect was calculated by comparing the peak area responses of CRD and IS in the post-extracted spiked serum samples with the peak area of the corresponding concentration in analytical standard. Calibration curve consisted of eight non-zero standards (sample processed with
analyte and IS). A blank sample (without analyte and IS) and a zero sample (with IS only) were also assayed along with the calibration standards. All samples were prepared and processed as mentioned in sections 2.3 and 2.4. For checking linearity, the calibration curve was constructed by subjecting the peak area ratio of CRD to that of IS against the nominal concentration of the CRD to least-squares linear regression analysis and a weighted leastsquares regression analysis.
10 Page 10 of 30
Extraction recovery of CRD were calculated by comparing the peak area response obtained from processed spiked rat serum sample with that of peak area response obtained from the extracted drug-free serum reconstituted with analytical standard solution at four
ip t
different concentrations (LLOQ, LQC, MQC and HQC; each n=6). The recovery of IS was also determined similarly at a concentration of 100 ng mL-1. The recovery was calculated
cr
from: Recovery (%) = (Peak area of analyte after extraction / peak area of analytical standard solution) × 100.
us
The precision and accuracy of the method (both inter- and intra-day) were evaluated by analyzing LLOQ (1 ng mL-1) and QC samples at three different concentration levels (3, 90
an
and 180 ng mL-1) in replicates on five different days. Intra- and inter-batch accuracy was
M
calculated as %bias from the formula: %Bias = [(observed concentration - nominal concentration) / nominal concentration] × 100. Intra- and inter-batch precision, in terms of
d
percent relative standard deviation (%RSD), was calculated by subjecting the data to one-way
te
analysis of variance (ANOVA). The dilution integrity experiment was also conducted to evaluate the accurate quantification of CRD in samples with concentration greater than the
Ac ce p
upper limit of quantitation (ULOQ). An acceptance limit of ≤15% was applied for all QC standards except LLOQ (≤20%) for validation [16]. The stability of CRD in serum was tested by analyzing the QC samples at low,
medium and high concentrations (each n = 6) stored under different conditions (long term stability: stored in - 80°C for 30 days; bench-top stability: at room temperature for 6 h). For long term storage stability of the analyte, one set of the spiked control serum samples (each n = 6) were processed as outlined in section 2.4 and analyzed on the day of preparation (reference). The remaining two sets of the samples were stored at -80 °C. These samples were assayed after 15 and 30 days of storage and their concentrations were determined with a calibration curve constructed with the freshly prepared calibration standards and compared
11 Page 11 of 30
with the reference. For bench top stability of the analyte, two sets of the spiked control serum samples were prepared. One set (each n = 6) was processed and assayed immediately (reference) and another set was stored on the bench top for 6 h at room temperature. The
ip t
remaining set was processed and analyzed along with freshly prepared calibration standards and concentrations were determined and compared with the reference. Furthermore, auto
cr
sampler stability was determined by placing the processed samples in auto sampler for 12 h. Two sets of QC were prepared, processed and placed in auto sampler. One set of QC samples
us
(each n = 6) were injected immediately to the HPLC column and analyzed on MS/MS and
an
served as the reference while another set was injected and analyzed after 12 h of storage in auto sampler. The samples were considered stable when the results were within ±15% of the
M
reference values. Freeze-thaw stability of CRD in spiked serum samples was determined up to three freeze-thaw cycles. Replicates of the spiked QC samples at low and high
d
concentrations of CRD were prepared. One set of the QC samples (n = 6 at each
te
concentration) was assayed on the day of preparation, which served as the reference. The remaining sets of samples were analyzed after one, two and three freeze-thaw cycles.
Ac ce p
Thawing was achieved by keeping the samples at room temperature for 30 min. The change in concentration during the freeze-thaw cycles was determined by comparing the concentrations after thawing with the reference concentration and is expressed as %deviation. The stability of CRD and IS in stock/standard solution for storage (45 days at 4°C) was evaluated. The peak area responses of CRD and IS in analytical standards from freshly prepared stock/standard solutions and the stored stock/standard solutions were compared. The samples were considered stable when the difference between the two was <5%. The stability of CRD in whole blood was also determined at ambient room temperature for 2 h at low and high concentrations. Freshly collected whole blood was spiked with CRD, allowed to stand at room temperature and assayed at 0, 0.25, 0.5, 1 and 2 h. The concentration of CRD at 0 h was
12 Page 12 of 30
considered as reference (100% CRD remained) and the subsequent results (% CRD remained) were compared and are expressed as %deviation.
ip t
2.6. Pharmacokinetic studies The pharmacokinetic studies of CRD was carried out in young and healthy male and
cr
female Sprague Dawley rats (b. weight, 225 ± 25 g) obtained from Laboratory Animal Division of the institute. The animals were acclimatized at least a week prior to
us
commencement of the study and were maintained on standard pelleted laboratory chow
an
(Goldmohar Laboratory Animal Feed, Lipton India Ltd, Chandigarh, India) and water ad libitum. The oral dose was administered after overnight fasting (12-16 h). Serial sampling
M
was performed in all rats and samples were withdrawn till 24 h.
A suspension formulation of CRD was prepared by triturating the weighed quantity of
d
CRD in a dry mortar. To this, gum acacia (1 %, w/v) was added and mixed by grinding. The
te
suspension was prepared by drop wise addition of water. This suspension was orally administered by gavage to rats (3 male and 3 female) in a single dose of 5 mg/kg, in a volume
Ac ce p
of approximately 1 mL/250 g rat and the time of dosing was recorded. The tail of each rat was nicked with a scalpel and blood samples (≈ 50 µL) were collected from the caudal vein into micro-centrifuge tubes at 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h post dose. A solution formulation (20 mg/mL) of CRD was prepared by dissolving 2 mg of CRD in 0.1 mL dimethyl sulfoxide. The formulation was administered intravenously via the femoral vein to conscious rats (3 male and 3 female) at a dose of 1 mg/kg. Blood samples (≈ 50 µL) were carefully collected at 0.08, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12 and 24 h post dose. Total collected blood volume from each rat was less than 10% of the total blood volume. The blood was allowed to clot by keeping the tubes on a slant for approximately 30 min. Then, it was centrifuged at 3000 x g for 10 min at 4°C; the serum was separated into clean and labelled
13 Page 13 of 30
tubes and stored at -80°C until analysis. Test samples (10 µL) were assayed along with the calibration standards and QC samples prepared in rat serum and the levels of CRD was
ip t
calculated using Analyst software.
2.7. Pharmacokinetic analysis
cr
All the experimental data and pharmacokinetic parameters are expressed as the mean ± standard error mean (SEM). From the observed concentration versus time data, the peak
us
serum concentration (Cmax) and the time to reach Cmax (Tmax) were obtained directly. The
an
concentration - time data were subjected to noncompartmental approach using Phoenix WinNonlin (version 6.3; Certara Inc, Missouri, USA). The area under the serum
M
concentration-time curve from time zero to the time of final measurable sample (AUClast) was calculated using the linear trapezoidal method [17]. Mean residence time (MRT), defined as
d
the time taken for 63.2% of the administered dose eliminated, was calculated by
te
AUMClast/AUClast, where AUMClast is the area under the moment curve from the time of dosing to the last measurable concentration calculated by linear trapezoidal method. The
Ac ce p
absolute bioavailability (F) of CRD in different gender was calculated individually and determined as F = [(AUClast, oral/AUClast, intravenous) × (Doseintravenous /Doseoral)] × 100 [17]. To compare the pharmacokinetic parameters unpaired student t-test was applied using GraphPad Prism software (version 5.01). A p value of <0.05, <0.01 and <0.001 were considered as statistically significant, very significant and highly significant difference, respectively.
3. Results and Discussion 3.1. LC-MS/MS Method development Optimization of mass spectrometric ionization and fragmentation of CRD and IS was performed through continuous infusion (10 µL min-1) of methanolic solution of CRD and IS
14 Page 14 of 30
(each 100 ng mL-1) into the electrospray source using a syringe pump (Harvard Pump 11plus, Holliston, USA). Tuning of mass parameters was performed both in positive and negative ionization mode. Good response was found in the negative ionization mode for both CRD
ip t
and IS. Identification of precursor ion of each analyte and IS was performed in the full scan mode by recording mass transition from m/z 50 to 600. Both CRD and IS showed only one
cr
prominent ion, which corresponds to the deprotonated [M-H]- ion. So, for CRD and IS, [MH]- ion was selected as the parent ion in Q1 spectrum and used as precursor ion to obtain Q3
us
product ion spectrum. The product ion mass spectrum of CRD and IS is presented in Fig. 1.
an
MRM technique was selected for the assay development and the parameters were optimized to maximize the response for CRD. The most sensitive mass transition was monitored from
M
m/z 269 to 122 and 367.1 to 216.9 for CRD and IS, respectively.
Among several chromatographic conditions and columns (cyano, C8, C18) tried, the
d
Supelco Discovery HS C18 column (5 μm, 50 x 4.6 mm) provided good selectivity, sensitivity
te
and better peak shape for CRD and IS in a short run time. Several combinations of solvents (acetonitrile and methanol) and buffers (ammonium acetate, ammonium formate and formic
Ac ce p
acid) were evaluated to select suitable mobile phase. The mobile phase comprising acetonitrile : AAB (0.01 M, pH 4.5) (85:15, v/v) at a flow rate of 0.6 mL min-1 was found most appropriate. A simple liquid-liquid extraction technique using various combinations of extraction solvents were tried for maximum recovery of CRD and IS from rat serum. A mixture of 60% n-hexane in ethyl acetate provided excellent recovery. Representative chromatograms of extracted analyte/IS-free and analyte/IS-fortified serum are illustrated in Fig. 2. The retention times of CRD (1.8 min) and IS (1.5 min) were low enough to allow a short run time of 3.0 min.
3.2. Selectivity and matrix effect
15 Page 15 of 30
The developed method was selective as no significant interference from endogenous serum constituents was observed at the retention times of CRD and IS in chromatograms derived from processed blank serum samples from six different rats (Fig. 2). No significant
ip t
matrix effect was observed in post-extraction spiked serum samples for the analyte at LQC, MQC and HQC concentrations. The results showed that the co-eluting endogenous serum
cr
components did not suppress or enhance the ionization of CRD and IS. A processed zero sample (sample processed with IS only) injected after the highest calibration standard showed
us
that the peak areas of the eluting peak at the retention times of CRD was less than 5% of the
M
3.3. Calibration curve, linearity and sensitivity
an
peak area of the CRD at LLOQ indicating an insignificant carryover effect.
The eight point calibration curves were constructed on each day of the 5-day
d
validation period and a weighted (1/x2) least squares linear regression analysis was applied to
te
the data. Linear responses were observed for the CRD in the range of 1 to 200 ng mL-1. All the calibration standards were within the acceptance criteria (accuracy of ±20% for LLOQ
Ac ce p
and ±15% for non-LLOQ standards) and the regression coefficient values were always greater than 0.999. The limit of detection (S/N ratio >3) was 0.2 ng mL-1 for CRD in rat serum. The LLOQ was selected as 1 ng mL-1 as the response at this concentration was more than 10-times higher than that of blank (S/N ratio >10). Sensitivity was tested at LLOQ and the accuracy and precision at LLOQ were within the acceptable limits (Table 1).
3.4. Extraction recovery Simple liquid-liquid extraction technique using the extraction solvent (n-hexane: ethyl acetate, 60:40, v/v) provided excellent and consistent recovery of CRD and IS from spiked serum samples. Moreover, the extraction solvent provided clean extracts with minimal
16 Page 16 of 30
endogenous serum components resulting in negligible matrix effect. The recovery of CRD was determined by analyzing sextuplet samples at LQC, MQC and HQC for five consecutive days. Mean recoveries of CRD from spiked QC standards were always more than 90% (Table
ip t
1) and found to be concentration independent and consistent. The recovery of the IS averaged
cr
to 50%.
3.5. Accuracy, precision and dilution integrity
us
The accuracy and precision were determined by analyzing sextuplet samples at
an
LLOQ, LQC, MQC and HQC for five consecutive days. The values of the intra-day and inter-day accuracy and precision in serum QC samples are summarized in Table 1. Intra-day
M
precision, as indicated by %RSD, varies from 4.0 to 12.4. However, inter-day precision evaluated at the same concentrations was ≤11.8. The accuracy of the method (%bias) ranged
d
from -9.1 to 4.9. The results demonstrated that all the value of accuracy and precision were
te
well within the acceptable limit of variation at LQC, MQC and HQC. A 20-fold dilution of DIQC samples by blank drug free-serum prior to extraction was used for determining dilution
Ac ce p
integrity. Sextuplet of DIQC samples were extracted and analyzed along with one of the validation batches. The results, mean accuracy (%bias) and precision (%RSD) of 10.4 and 0.2, respectively, demonstrated that the samples with concentrations greater than the ULOQ can be analyzed following dilution to obtain precisely accurate results.
3.7. Stability studies
The stability of CRD in serum matrix was investigated as described in section 2.5 and the results are presented in Table 2. In different stability experiments namely, bench-top stability, freeze-thaw stability, long term stability and auto sampler stability, the changes (%deviation) from the reference concentrations were within the acceptable limit (≤ ±15%)
17 Page 17 of 30
demonstrating that CRD was stable in serum and the processed samples. Moreover, CRD was found to be stable in whole blood up to 2 h as percent CRD remained at 2 h was within the acceptable limit (Fig. 3). Also, CRD in stock/standard solutions was found to be stable for 45
ip t
days at 4°C as the difference between the peak responses of CRD in analytical standards
cr
prepared from fresh and stored stock/standard solution was found to be 3.8 ± 0.1%.
3.8. Pharmacokinetic studies
us
The validated LC-MS/MS method was successfully applied for characterizing gender
an
influence on the pharmacokinetics of cardamonin in Sprague Dawley rats (male and female, each n = 3) following oral (5 mg/kg) and intravenous (1 mg/kg) administration. The
M
compound was quantified in test serum samples upto 24 h. The mean serum concentration time profiles and the pharmacokinetic parameters of CRD in male and female rats after oral
d
and intravenous administration are shown in Fig. 4 and Table 3. Although, the systemic
te
availability of CRD after oral administration is quite low in both male and female rats, but CRD has comparatively higher bioavailability in female (4.8%) than in male rats (0.6%).
Ac ce p
Following both oral and intravenous administration, CRD showed Vss higher than the total blood volume (0.064 L/kg; [18]) of the rat indicating extravascular distribution. The Cmax and the absolute bioavailability was higher in females than in males after oral dosing suggesting that the CRD has better absorption in females than in males. Moreover, comparatively higher Vss and CL were observed in females than in males after both intravenous and oral administrations, which demonstrates that CRD is distributed and eliminated more in females. As CRD is lipophilic in nature, higher Vss in female rats may be attributed to higher percentage of body fat as compared to that in males. Following both oral and intravenous administration, a 10-times higher CL was observed in females than in males. In accordance to this, MRT values were found higher in males than that in females. Based on significant
18 Page 18 of 30
difference in CL, it may be inferred that the activity of enzymes that metabolize the cardamonin might differ between the sexes. Conclusively, it can be stated that gender exert
ip t
significant effect on the pharmacokinetics of cardamonin in rats.
4. Conclusion
cr
To the best of our knowledge, this is the first paper to report preclinical pharmacokinetics and LC-MS/MS method for quantification of CRD. The LC-MS/MS assay
us
reported in this paper is sensitive and specific for quantification of CRD in rat serum. All the
an
regulatory requirement of method validation were followed and achieved. The extraction method provides excellent, consistent and reproducible recoveries of CRD. The simplicity of
M
the assay, use of liquid-liquid extraction, cost effectiveness and sample turnover rate of 3 min per sample, makes it attractive and applicable procedure for future studies (stability in
d
simulated body fluids, protein binding, dose escalation, tissue distribution and excretion) of
te
CRD. The method was successfully implemented for single dose pharmacokinetic studies of CRD in male and female rats. The study revealed that CRD is poorly bioavailable which
Ac ce p
might be due to poor solubility and/or hepatic first pass metabolism. Moreover, CRD exhibited significant difference in its pharmacokinetic parameters and bioavailability between male and female rats that could be due to gender related differences in the enzyme activity. Further studies are required for better understanding of low oral bioavailability and gender difference in its pharmacokinetic behavior. The study provides useful insight in devise of appropriate formulation, dosage regimen and route of administration for optimal pharmacological efficacy to aid in its development as a potential anticancer drug candidate.
Acknowledgements
19 Page 19 of 30
Author AS is thankful to Indian Council of Medical Research, New Delhi, for financial support in the form of his fellowship for the Central Drug Research Institute
ip t
Communication (324/2014/JL).
Conflict of interest
us
cr
There is no potential conflict of interest.
References
A. Murakamia, A. Kondoab, Y. Nakamuraa, H. Ohigashia & K. Koshimizua, Possible
an
[1]
Anti-tumor Promoting Properties of Edible Plants from Thailand, and Identification of
Biochem. 57 (1993) 1971-3.
V.R. Yadav, S. Prasad, B.B. Aggarwal, Cardamonin sensitizes tumour cells to TRAIL
d
[2]
M
an Active Constituent, Cardamonin, of Boesenbergia pandurata, Biosci. Biotech.
te
through ROS- and CHOP-mediated up-regulation of death receptors and downregulation of survival proteins, Br. J. Pharmacol. 165 (2012) 741-53. Y. Qin, C.Y. Sun, F.R. Lu, X.R. Shu, D. Yang, L. Chen, X.M. She, N.M. Gregg, T.
Ac ce p
[3]
Guo, Y. Hu, Cardamonin exerts potent activity against multiple myeloma through blockade of NF-kappaB pathway in vitro, Leuk. Res. 36 (2012) 514-20.
[4]
J. Tang, N. Li, H. Dai, K. Wang, Chemical constituents from seeds of Alpinia katsumadai, inhibition on NF-kappaB activation and anti-tumor effect, Zhongguo. Zhong. Yao. Za. Zhi. 35 (2010) 1710-4.
[5]
B. Sung, S. Prasad, V.R. Yadav, S.C. Gupta, S. Reuter, N. Yamamoto, A. Murakami, B.B. Aggarwal, RANKL signaling and osteoclastogenesis is negatively regulated by cardamonin, PLoS One 8 (2013) e64118.
20 Page 20 of 30
[6]
S. Hatziieremia, A.I. Gray, V.A. Ferro, A. Paul, R. Plevin, The effects of cardamonin on lipopolysaccharide-induced inflammatory protein production and MAP kinase and NFkappaB signalling pathways in monocytes/macrophages, Br. J. Pharmacol. 149
ip t
(2006) 188-98. [7]
D.A. Israf, T.A. Khaizurin, A. Syahida, N.H. Lajis, S. Khozirah, Cardamonin inhibits
cr
COX and iNOS expression via inhibition of p65NF-kappaB nuclear translocation and Ikappa-B phosphorylation in RAW 264.7 macrophage cells, Mol. Immunol. 44 (2007)
Y.L. Chow, K.H. Lee, S. Vidyadaran, N.H. Lajis, M.N. Akhtar, D.A. Israf, A.
an
[8]
us
673-9.
Syahida, Cardamonin from Alpinia rafflesiana inhibits inflammatory responses in
M
IFN-gamma/LPS-stimulated BV2 microglia via NF-kappaB signalling pathway, Int. Immunopharmacol. 12 (2012) 657-65.
J.H. Lee, H.S. Jung, P.M. Giang, X. Jin, S. Lee, P.T. Son, D. Lee, Y.S. Hong, K. Lee,
d
[9]
te
J.J. Lee, Blockade of nuclear factor-kappaB signaling pathway and anti-inflammatory activity of cardamomin, a chalcone analog from Alpinia conchigera, J. Pharmacol.
Ac ce p
Exp. Ther. 316 (2006) 271-8.
[10]
H. Ko, Y.J. Kim, E.C. Amor, J.W. Lee, H.C. Kim, H.J. Kim, H.O. Yang, Induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma HCT116 and LOVO cells, J. Cell. Biochem. 112 (2011) 2471-9.
[11]
M. Cho, M. Ryu, Y. Jeong, Y.H. Chung, D.E. Kim, H.S. Cho, S. Kang, J.S. Han, M.Y. Chang, C.K. Lee, M. Jin, H.J. Kim, S. Oh, Cardamonin suppresses melanogenesis by inhibition of Wnt/beta-catenin signaling, Biochem. Biophys. Res. Commun. 390 (2009) 500-5.
[12]
H. Doug, S.X. Chen, H.X. Xu, S. Kadota, T. Namba, A new antiplatelet diarylheptanoid from Alpinia blepharocalyx, J. Nat. Prod. 61 (1998) 142-4.
21 Page 21 of 30
[13]
S. Tewtrakul, S. Subhadhirasakul, J. Puripattanavong, T. Panphadung, HIV-1 protease inhibitory substances from the rhizomes of Boesenbergia pandurata Holtt, Songklanakarin. J. Sci. Technol. 25 (2003) 503-8. G. Trakoontivakorn, K. Nakahara, H. Shinmoto, M. Takenaka, M. Onishi-Kameyama,
ip t
[14]
H. Ono, M. Yoshida, T. Nagata, T. Tsushida, Structural analysis of a novel
cr
antimutagenic compound, 4-Hydroxypanduratin A, and the antimutagenic activity of flavonoids in a Thai spice, fingerroot (Boesenbergia pandurata Schult.) against
us
mutagenic heterocyclic amines, J. Agric. Food. Chem. 49 (2001) 3046-50.
J. Hodgson, ADMET turning chemicals into drugs, Nat. Biotechnol. 19 (2001) 722-6.
[16]
FDA,
Guidance
for
Industry:
an
[15]
Bioanalytical
Method
Validation.
[17]
M
http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf [08 October 2014] M. Gibaldi, D. Perrier, Pharmacokinetics, second ed., Informa Healthcare, New York,
N. Rangaraj, K. Vaghasiya, S. Jaiswal, A. Sharma, M. Shukla, J. Lal, Do Blood
te
[18]
d
1982.
Ac ce p
Sampling Sites Affect Pharmacokinetics?, Chem. Biol. Interface. 4 (2014) 176-191.
22 Page 22 of 30
Legend for figures Fig. 1. Product ion spectra (MS-MS) of cardamonin and curcumin (IS) Fig. 2. Typical MRM chromatograms of cardamonin (left panel) and curcumin (right panel;
ip t
IS) (a-b) drug-free and IS-free rat serum, (c-d) drug-free and IS (100 ng mL-1) fortified rat serum, (e-f) serum containing 1 ng mL-1 (LLOQ) of cardamonin and 100 ng mL-1 of IS, and
cr
(g-h) serum sample taken at 2 h from a female rat treated with 5 mg/kg oral dose of
Fig. 3. Stability of cardamonin in rat whole blood
us
cardamonin.
an
Fig. 4. Mean serum concentration-time profile of cardamonin in male and female Sprague Dawley rats (each n=3) after a single (A) 5 mg/kg oral and (B) 1 mg/kg intravenous
Ac ce p
te
d
M
administration. Bar represents SEM.
23 Page 23 of 30
Table 1 Recovery, accuracy and precision of the proposed assay method for cardamonin quantification (n = 6 at each concentration)
(ng mL-1)
Mean
Accuracy (%Bias)
CV (%)
Intra-day
Inter-
Intra-
Inter-
day
day
day
93.1
8.7
-2.2
-9.1
3
98.2
8.1
2.2
4.9
90
108.0
7.8
0.0
180
110.6
13.6
12.4
11.8
4.6
3.8
8.0
8.0
4.0
7.5
us
1
an
-0.2 2.6
Ac ce p
te
d
M
4.5
Precision (%RSD)
ip t
Absolute recovery
cr
Concentration
24 Page 24 of 30
Table 2 Stability of cardamonin under different storage conditions in serum (n = 6 at each concentration) Measured
Measured mean
concentration
concentration
concentration in
in QC samples
in Reference#
stability QC
(ng mL-1)
QC samples
samples (ng
(ng mL-1)
mL-1)
3
3.1 ± 0.3
3.3 ± 0.1
stability
90
86.9 ± 13.8
(room
180
183.7 ± 2.8
Freeze-thaw
3
stability
90
86 ± 14.1
0.5
-0.7
169.3 ± 4.9
5.7
-7.8
3.1 ± 0.2
2.9 ± 0.1
3.3
-4. 5
95.5 ± 8.7
1.6
1.9
180.5 ± 16.3
177.6 ± 3.2
1.1
-1.6
M
temperature, 6 h)
cycles,
-
180
te
3
97.3 ± 8.8
Ac ce p
(after
cr
6.0
us
4.1
an
top
%deviation*
d
Bench
%CV
ip t
Nominal
Stability test
80°C) Long
term
3
3.1 ± 0.1
2.8 ± 0.1
6.6
-8.6
stability (30
90
90.5 ± 1.3
92.0 ± 6.6
1.2
1.7
days, -80°C)
180
172.7 ± 5.7
196.4 ± 1.3
8.5
13.7
Auto
3
2.8 ± 0.1
2.9 ± 0.1
1.8
2.6
sampler
90
97.3 ± 7.7
87.9 ± 2.2
7.2
-9.7
stability (12
180
166.5 ± 5.6
167.0 ± 5.4
0.2
0.3
h, 10°C) *%deviation in comparison to reference concentration, #freshly prepared and immediately analyzed set of quality control samples 25 Page 25 of 30
Table 3 Pharmacokinetic parameters of cardamonin after oral (5 mg/kg) and intravenous (1 mg/kg) administrations in rats.
Oral administration
Female
Male
1285.3 ± 33.0
1498.3 ± 611.0
12.9 ± 1.8*
52.6 ± 20.7*
2.3 ± 0.9
2.2 ± 0.9
5184.7 ±
803.5 ±
193.9 ± 18.8
565.8**
227.0**
us
tmax (h)
Female
cr
Cmax (ng/ml)
Male
ip t
Intravenous administration Parameters
1.9 ± 0.6
4.7 ± 1.7
1.1 ± 0.1**
7.1 ± 0.8**
CL (L/h/kg)
0.17 ± 0.01*
1.37 ± 0.30*
0.11 ± 0.01**
1.08 ± 0.14**
MRT (h)
6.4 ± 0.2***
1.9 ± 0.4***
10.0 ± 0.3*
6.8 ± 1.1*
-
0.6***
4.8***
-
d
Bioavailability (%)
an
Vss (L/kg)
157.4 ± 24.1
M
AUClast (ng h/ml)
te
Values of PK parameters are mean ± SEM (n=3); Abbreviations: AUClast = area under the serum concentration-time curve up to last sampling
Ac ce p
time, Cmax = serum peak concentration, tmax = time to Cmax, Vss = volume of distribution at steady-state, CL= Clearance, MRT= mean residence time; *= p<0.05 significant difference, **= p<0.01, very significant difference, ***= p<0.001, highly significant difference.
26 Page 26 of 30
Ac ce p
te
d
M
an
us
cr
ip t
Figure 1
Page 27 of 30
Ac ce p
te
d
M
an
us
cr
ip t
Figure 2
Page 28 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 29 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure 4
Page 30 of 30
S1570-0232(15)00081-1 http://dx.doi.org/doi:10.1016/j.jchromb.2015.01.041 CHROMB 19307
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
8-12-2014 26-1-2015 30-1-2015
Please cite this article as: S. Jaiswal, A. Sharma, M. Shukla, J. Lal, Gender-related pharmacokinetics and bioavailability of a novel anticancer chalcone, cardamonin, in rats determined by liquid chromatography tandem mass spectrometry, Journal of Chromatography B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.01.041 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.
Highlights 1. First reported LC-MS/MS method for quantification of anticancer chalcone, cardamonin, in rat serum. 2. A sensitive, accurate and precise bioanalytical method with a short chromatographic samples per day) bioanalysis.
Ac ce p
te
d
M
an
us
cr
3. First reported pre-clinical pharmacokinetics of cardamonin.
ip t
run time of 3 min which will provide benefit in high throughput (more than 400
1 Page 1 of 30
Gender-related pharmacokinetics and bioavailability of a novel anticancer chalcone, cardamonin, in rats determined by liquid chromatography tandem mass spectrometry
Pharmacokinetics & Metabolism Division, CSIR-Central Drug Research Institute, Lucknow-
cr
a
ip t
Swati Jaiswala,b,# , Abhisheak Sharmaa,b,# , Mahendra Shuklaa,b, Jawahar Lala,b,*
226031, India b
an
us
Academy of Scientific and Innovative Research, New Delhi, India
*Corresponding author: Dr. Jawahar Lal, Pharmacokinetics & Metabolism Division, CSIR-
M
Central Drug Research Institute, Jankipuram Extension, Sitapur Road, Lucknow - 226031, India; Tel.: +91-522-2772450, Ext. 4841; Fax: +91-522-2771941; E-mail: [email protected]
te
d
Authors contributed equally
Ac ce p
#
2 Page 2 of 30
Abstract A reversed phase liquid chromatography tandem mass spectrometry method was developed and validated for quantification of cardamonin, a potential anticancer chalcone, in
ip t
rat serum. Curcumin was used as an internal standard. Following liquid-liquid extraction using n-hexane and ethyl acetate (60:40, v/v), the processed samples were chromatographed
cr
on a C18 column using acetonitrile and ammonium acetate buffer (0.01 M, pH 4.5) (85:15, v/v) as mobile phase at a flow rate of 0.6 mL min-1. Mass spectrometric detection was
us
performed in the negative electrospray ionization mode by multiple reaction monitoring (m/z
an
269→122 and 367→217 for cardamonin and curcumin, respectively). The method was validated in terms of selectivity, accuracy, precision, sensitivity, reproducibility, dilution
M
integrity and stability. The linearity was established in the range of 1 to 200 ng mL-1 (r ≥0.999). The recovery of cardamonin from spiked serum was always >90%. The intra- and
d
inter-day precision (%RSD) and accuracy (%bias) were well within the acceptable limits. The
te
method was applied for single oral and intravenous dose pharmacokinetics in male and female Sprague Dawley rats. Following oral dose, cardamonin showed peak serum
Ac ce p
concentration that occurred at ~2 h with very low bioavailability in both male (0.6%) and female (4.8%) rats. Cardamonin exhibited a significant gender influence on pharmacokinetics and bioavailability in rats.
Keywords: cardamonin; pharmacokinetics, bioavailability, liquid chromatography, tandem mass spectrometry.
Abbreviations: ANOVA, analysis of variance; CAD, collision activated dissociation; CHOP, CAAT/enhancer binding protein homologous protein; CRD, cardamonin; CE, collision energy; CRC, curcumin; CXP, collision cell exit potential; DP, declustering potential; DDI,
3 Page 3 of 30
drug-drug interactions; EP, entrance potential; IS, internal standard; MAPK, mitogenactivated protein kinases; RSD, relative standard deviation; ROS, reactive oxygen species;
Ac ce p
te
d
M
an
us
cr
ip t
TDM, therapeutic drug monitoring; TRAIL, TNF-related apoptosis-inducing ligand.
4 Page 4 of 30
1. Introduction Cardamonin (CRD; (2E)-1-(2,4-Dihydroxy-6-methoxyphenyl)-3-phenyl-2-propen-1one), a chalcone, is a potential and promising anticancer compound. It is found in several
ip t
medicinal plants of Zingiberaceae family including Alpinia katsumadai, Alpinia conchigera, Alpinia rafflesiana, Amomum subulatum and Boesenbergia pandurata. CRD is reported as
cr
the most potent chalcone-type anti-tumor agent [1]. Recent studies on CRD illustrated that it augments apoptotic effects of TRAIL (TNF-related apoptosis-inducing ligand) against human
us
colon, pancreatic, myeloid leukaemia, multiple myeloma and prostate adenocarcinoma cell
an
lines [2]. The mechanism includes downregulation of anti-apoptotic proteins; ROS-CHOP (reactive oxygen species-CAAT/enhancer binding protein homologous protein) mediated
M
upregulation of TRAIL death receptors and decreased expression of decoy receptors and cell survival proteins. Several other studies on CRD suggested that it suppresses proliferation and
d
induces apoptosis in myeloma cells by inhibiting NF-kappaB (nuclear factor kappa-light-
te
chain-enhancer of activated B cells) pathway [3,4]. Moreover, CRD inhibit the breast cancer or multiple myeloma cell or RANKL induced bone osteoclastogenesis by suppressing NF-
Ac ce p
kappaB and MAPK (mitogen-activated protein kinases) pathway [5]. It has also shown cytotoxicity against leukemia K562 cells and human hepatoma SMMC-7721 cells [4]. Hitherto, CRD has also shown a variety of other appreciable pharmacological activities like anti-platelet, anti-HIV, vasorelaxant, insecticidal, anti-melanogenic, anti-mutagenic and antiinflammatory activity [6-14]. For the advanced development of a compound as a drug candidate along with pharmacological activities, pharmacokinetic properties must be explored [15]. For understanding and estimating the pharmacokinetics of any drug and/or its metabolites, quantification of that compound in suitable bio-matrices (blood, serum or plasma) is a prerequisite, which can be performed by developing a sensitive and selective bioanalytical method. With this rationale, the present investigation was carried out which
5 Page 5 of 30
involved bioanalytical method development and preclinical pharmacokinetic profiling of CRD in support of its development as a potential candidate drug. Until now, to the best of our information, there is no bioanalytical method available
ip t
for quantification of CRD in any biological matrix. Also, the knowledge concerning the pharmacokinetics of CRD is lacking. Herein, we report for the first time the development and
cr
validation of a sensitive reversed phase LC-MS/MS method with short chromatographic run time (3.0 min) for quantification of CRD in rat serum and then employed the method to
us
investigate the pharmacokinetic characteristics of CRD in male and female Sprague Dawley
an
rats following single oral and intravenous administration to explore the gender influence on
M
the pharmacokinetics of CRD.
2. Materials and Method
d
2.1. Chemicals and reagents
te
Cardamonin (99.5% pure) and curcumin (CRC) were procured from Sigma Aldrich (St. Louis, USA). CRC was used as an internal standard (IS). HPLC grade acetonitrile was
Ac ce p
purchased from Sigma Aldrich (Dombivli, India). HPLC grade n-hexane, ethyl acetate and analytical grade ammonium acetate were purchased from Spectrochem Pvt. Ltd. (Mumbai, India). HPLC grade glacial acetic acid was procured from J.T. Baker (Phillipsburg, USA). Ultra pure water was from a Milli-Q Plus PF (Billerica, USA) water purification system. Drug-free whole blood was collected from adult and healthy male and female SpragueDawley rats provided by the Laboratory Animal Services Division of the institute and serum was separated after centrifuging the collected blood at 3000 × g for 10 min. The serum samples from male and female rats were pooled separately and stored at -80°C till use. All experiments, euthanasia and disposal of carcasses were carried out as per the guidelines of local ethics committee for animal experimentation.
6 Page 6 of 30
2.2. Liquid Chromatography and tandem mass spectrometry conditions
ip t
The HPLC system (Shimadzu, Kyoto, Japan) consisted of a pump (LC-20 AD) with
us
cr
online degasser (DGU-20A3), column oven (CTO‐10AS) and auto injector (SIL-HTc, fixed
with a 100 μL loop) with a temperature-controlled pelteir-tray. Chromatographic separation
an
of the CRD and IS were achieved on a Supelco Discovery HS C18 column (5 μm, 50 x 4.6 mm, id) preceded with a Supelguard Discovery HS C18 column (5 μm, 20 x 4.0 mm, id)
M
packed with the same material. The mobile phase consisted of acetonitrile and ammonium acetate buffer (AAB; 0.01 M, pH 4.5) (85:15, v/v) and was delivered isocratically at a flow
d
rate of 0.6 mL min-1. The freshly prepared AAB was filtered through 0.22 µm membrane
te
filter (Millipore, USA). The mobile phase was degassed by ultrasonication for 15 min before
Ac ce p
use. Prior to commencement of analysis, LC-MS/MS system was equilibrated for 20 min. Aliquots (10 μL) of processed sample were injected onto the column and the column oven temperature was set at 40°C. Other LC parameters like needle rinsing speed and volume, needle stroke, sampling speed, purge time and rinse dip time were set at 35 µL s-1, 800 µL, 52 mm, 5 µL s-1, 0.5 min and 10 s, respectively. Acetonitrile: Milli-Q water (50:50, v/v) was used as rinsing solution and rinsing mode was set to after and before aspiration to minimize carryover, if any. The quantification of the compound was carried out using Q Trap 4000 MS/MS system (Applied Biosystems, Toronto, Canada) which included a hybrid triple quadrupole/LIT (linear ion trap) mass spectrometer and a Turbo V™ Ion Source. Sample
7 Page 7 of 30
introduction and ionization was performed by electrospray ionization in negative ion mode. The mass spectrometry operation was conducted under multiple reaction monitoring (MRM) mode (m/z 269→122 for CRD and m/z 367→217 for IS). The source parameters, namely,
ip t
temperature, curtain gas, ion spray voltage, nebulizer gas, heater gas, were set at 400 °C, 10 psi, -3000 V, 45 psi, 40 psi, respectively. Collision activated dissociation (CAD) gas was set
cr
at medium mode. The declustering potential (DP) for CRD and IS was set at -81 V. Other compound parameters, namely entrance potential (EP) and collision cell exit potential (CXP)
us
were set at -10 V for both, the compound and IS. The collision energy (CE) was set at -35
an
and -28 V for CRD and IS, respectively. Dwell time was set at 150 ms. Nitrogen was used as curtain and collision gas. Zero air was used as source gas. All the operations, acquisition and
M
analysis of data were controlled by AnalystTM (version 1.4.2) software (Applied Biosystems,
d
Toronto Canada).
te
2.3. Preparation of standards and quality control samples Mother stock solutions (1 mg mL-1) of the reference standard (CRD) and IS were
Ac ce p
separately prepared by dissolving 10 mg of the compound in 10 mL methanol. Working standard solutions (100 µg mL-1) of CRD and IS were separately prepared by appropriately diluting mother stock solution with methanol. Spiking standard solutions of CRD were prepared in methanol from working standard solutions by serial dilution method in the range of 0.02 to 4 µg mL-1, which were utilized for preparation of calibration standards in rat serum. Separate mother stock solution, working standard solution and spiking standard solutions were prepared in methanol for preparation of quality control (QC) standards of CRD containing 0.02, 0.06, 1.8 and 3.6 µg mL-1 for lower limit of quantification, low, medium and high concentrations, respectively. The solutions were vortex-mixed and stored at
8 Page 8 of 30
4°C. Also, spiking standard solution containing 1 µg mL-1 of IS was prepared by spiking 0.1 mL of the working standard solution in 9.9 mL of methanol. Blank pooled rat serum was pre-screened to assure the absence of any endogenous
ip t
interference at the retention time of the CRD and IS. The calibration standards were prepared individually by spiking 95 µL of drug-free rat serum with 5 µL of spiking standard solutions
cr
to obtain 1, 2.5, 10, 25, 50, 100, 190 and 200 ng mL-1. Similarly, QC standards were prepared at lower limit of quantification (LLOQ), low (LQC), medium (MQC) and high concentrations
us
(HQC) containing 1, 3, 90 and 180 ng mL-1 of CRD. Similarly, the dilution integrity quality control (DIQC) samples were prepared at concentration of 3.6 µg mL-1 by spiking 3.6 µL of
an
100 µg mL-1 to 96.4 µL drug-free rat serum. A 20-fold dilution of the DIQC (n = 6) was
M
prepared in drug-free rat serum.
d
2.4. Sample cleanup
te
Extraction of the CRD and IS was carried out by a simple liquid-liquid extraction technique using the extraction solvent (60% n-hexane in ethyl acetate). Aliquots (10 µL) of
Ac ce p
each calibration standard, QC and test samples were separately transferred in 5 mL glass extraction tubes. To this, 10 µL of spiking standard solution of IS (1 µg mL-1) was added while vortexing followed by addition of 2 mL of extraction solvent through the bottle top dispenser. After that, extraction tubes containing this mixture were vortex-mixed for 1 min and centrifuged at 3000 rpm for 10 min at 4°C. The organic layer was transferred to another tube by snap freezing the aqueous layer in liquid nitrogen and evaporated to dryness at 40°C in Turbo vap LX concentrator (Caliper, Massachusetts, USA). The dried residues were reconstituted in 100 µL mobile phase and 10 µL was injected on to LC-MS/MS system.
2.5. Method validation
9 Page 9 of 30
The present bioanalytical method was validated for selectivity, sensitivity, linearity, accuracy, precision, recovery, matrix effect, stability, dilution integrity and robustness as per US Food and Drug Administration (US FDA) guidelines [16]. Selectivity was determined by
ip t
analyzing blank serum from six different rats. Each of these blank samples was tested for the presence of any endogenous serum components eluting at the retention times of CRD and IS.
cr
Selectivity was guaranteed if the peak response from endogenous serum components was less than 20% of the mean peak response of CRD at LLOQ and less than 5% of the mean peak
us
response of IS. For evaluation of the method sensitivity, replicates of blank serum spiked
an
with CRD were processed and analyzed. The limit of detection (LOD) was determined based on analyte’s signal to noise (S/N) ratio of 3:1. The lower limit of quantification (LLOQ) was
M
defined as the lowest concentration with S/N ratio greater than 10 and accuracy and precision within 20% of the nominal concentration of the analyte. The suppression of MS/MS detector
d
response owing to matrix components associated with rat serum was assessed by extracting
te
six different lots of blank rat serum without IS. Post extraction, dried residues were reconstituted with analytical standard solutions at low (3 ng mL-1, n = 6) and high
Ac ce p
concentrations (180 ng mL-1, n = 6) of CRD. The matrix effect was calculated by comparing the peak area responses of CRD and IS in the post-extracted spiked serum samples with the peak area of the corresponding concentration in analytical standard. Calibration curve consisted of eight non-zero standards (sample processed with
analyte and IS). A blank sample (without analyte and IS) and a zero sample (with IS only) were also assayed along with the calibration standards. All samples were prepared and processed as mentioned in sections 2.3 and 2.4. For checking linearity, the calibration curve was constructed by subjecting the peak area ratio of CRD to that of IS against the nominal concentration of the CRD to least-squares linear regression analysis and a weighted leastsquares regression analysis.
10 Page 10 of 30
Extraction recovery of CRD were calculated by comparing the peak area response obtained from processed spiked rat serum sample with that of peak area response obtained from the extracted drug-free serum reconstituted with analytical standard solution at four
ip t
different concentrations (LLOQ, LQC, MQC and HQC; each n=6). The recovery of IS was also determined similarly at a concentration of 100 ng mL-1. The recovery was calculated
cr
from: Recovery (%) = (Peak area of analyte after extraction / peak area of analytical standard solution) × 100.
us
The precision and accuracy of the method (both inter- and intra-day) were evaluated by analyzing LLOQ (1 ng mL-1) and QC samples at three different concentration levels (3, 90
an
and 180 ng mL-1) in replicates on five different days. Intra- and inter-batch accuracy was
M
calculated as %bias from the formula: %Bias = [(observed concentration - nominal concentration) / nominal concentration] × 100. Intra- and inter-batch precision, in terms of
d
percent relative standard deviation (%RSD), was calculated by subjecting the data to one-way
te
analysis of variance (ANOVA). The dilution integrity experiment was also conducted to evaluate the accurate quantification of CRD in samples with concentration greater than the
Ac ce p
upper limit of quantitation (ULOQ). An acceptance limit of ≤15% was applied for all QC standards except LLOQ (≤20%) for validation [16]. The stability of CRD in serum was tested by analyzing the QC samples at low,
medium and high concentrations (each n = 6) stored under different conditions (long term stability: stored in - 80°C for 30 days; bench-top stability: at room temperature for 6 h). For long term storage stability of the analyte, one set of the spiked control serum samples (each n = 6) were processed as outlined in section 2.4 and analyzed on the day of preparation (reference). The remaining two sets of the samples were stored at -80 °C. These samples were assayed after 15 and 30 days of storage and their concentrations were determined with a calibration curve constructed with the freshly prepared calibration standards and compared
11 Page 11 of 30
with the reference. For bench top stability of the analyte, two sets of the spiked control serum samples were prepared. One set (each n = 6) was processed and assayed immediately (reference) and another set was stored on the bench top for 6 h at room temperature. The
ip t
remaining set was processed and analyzed along with freshly prepared calibration standards and concentrations were determined and compared with the reference. Furthermore, auto
cr
sampler stability was determined by placing the processed samples in auto sampler for 12 h. Two sets of QC were prepared, processed and placed in auto sampler. One set of QC samples
us
(each n = 6) were injected immediately to the HPLC column and analyzed on MS/MS and
an
served as the reference while another set was injected and analyzed after 12 h of storage in auto sampler. The samples were considered stable when the results were within ±15% of the
M
reference values. Freeze-thaw stability of CRD in spiked serum samples was determined up to three freeze-thaw cycles. Replicates of the spiked QC samples at low and high
d
concentrations of CRD were prepared. One set of the QC samples (n = 6 at each
te
concentration) was assayed on the day of preparation, which served as the reference. The remaining sets of samples were analyzed after one, two and three freeze-thaw cycles.
Ac ce p
Thawing was achieved by keeping the samples at room temperature for 30 min. The change in concentration during the freeze-thaw cycles was determined by comparing the concentrations after thawing with the reference concentration and is expressed as %deviation. The stability of CRD and IS in stock/standard solution for storage (45 days at 4°C) was evaluated. The peak area responses of CRD and IS in analytical standards from freshly prepared stock/standard solutions and the stored stock/standard solutions were compared. The samples were considered stable when the difference between the two was <5%. The stability of CRD in whole blood was also determined at ambient room temperature for 2 h at low and high concentrations. Freshly collected whole blood was spiked with CRD, allowed to stand at room temperature and assayed at 0, 0.25, 0.5, 1 and 2 h. The concentration of CRD at 0 h was
12 Page 12 of 30
considered as reference (100% CRD remained) and the subsequent results (% CRD remained) were compared and are expressed as %deviation.
ip t
2.6. Pharmacokinetic studies The pharmacokinetic studies of CRD was carried out in young and healthy male and
cr
female Sprague Dawley rats (b. weight, 225 ± 25 g) obtained from Laboratory Animal Division of the institute. The animals were acclimatized at least a week prior to
us
commencement of the study and were maintained on standard pelleted laboratory chow
an
(Goldmohar Laboratory Animal Feed, Lipton India Ltd, Chandigarh, India) and water ad libitum. The oral dose was administered after overnight fasting (12-16 h). Serial sampling
M
was performed in all rats and samples were withdrawn till 24 h.
A suspension formulation of CRD was prepared by triturating the weighed quantity of
d
CRD in a dry mortar. To this, gum acacia (1 %, w/v) was added and mixed by grinding. The
te
suspension was prepared by drop wise addition of water. This suspension was orally administered by gavage to rats (3 male and 3 female) in a single dose of 5 mg/kg, in a volume
Ac ce p
of approximately 1 mL/250 g rat and the time of dosing was recorded. The tail of each rat was nicked with a scalpel and blood samples (≈ 50 µL) were collected from the caudal vein into micro-centrifuge tubes at 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h post dose. A solution formulation (20 mg/mL) of CRD was prepared by dissolving 2 mg of CRD in 0.1 mL dimethyl sulfoxide. The formulation was administered intravenously via the femoral vein to conscious rats (3 male and 3 female) at a dose of 1 mg/kg. Blood samples (≈ 50 µL) were carefully collected at 0.08, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12 and 24 h post dose. Total collected blood volume from each rat was less than 10% of the total blood volume. The blood was allowed to clot by keeping the tubes on a slant for approximately 30 min. Then, it was centrifuged at 3000 x g for 10 min at 4°C; the serum was separated into clean and labelled
13 Page 13 of 30
tubes and stored at -80°C until analysis. Test samples (10 µL) were assayed along with the calibration standards and QC samples prepared in rat serum and the levels of CRD was
ip t
calculated using Analyst software.
2.7. Pharmacokinetic analysis
cr
All the experimental data and pharmacokinetic parameters are expressed as the mean ± standard error mean (SEM). From the observed concentration versus time data, the peak
us
serum concentration (Cmax) and the time to reach Cmax (Tmax) were obtained directly. The
an
concentration - time data were subjected to noncompartmental approach using Phoenix WinNonlin (version 6.3; Certara Inc, Missouri, USA). The area under the serum
M
concentration-time curve from time zero to the time of final measurable sample (AUClast) was calculated using the linear trapezoidal method [17]. Mean residence time (MRT), defined as
d
the time taken for 63.2% of the administered dose eliminated, was calculated by
te
AUMClast/AUClast, where AUMClast is the area under the moment curve from the time of dosing to the last measurable concentration calculated by linear trapezoidal method. The
Ac ce p
absolute bioavailability (F) of CRD in different gender was calculated individually and determined as F = [(AUClast, oral/AUClast, intravenous) × (Doseintravenous /Doseoral)] × 100 [17]. To compare the pharmacokinetic parameters unpaired student t-test was applied using GraphPad Prism software (version 5.01). A p value of <0.05, <0.01 and <0.001 were considered as statistically significant, very significant and highly significant difference, respectively.
3. Results and Discussion 3.1. LC-MS/MS Method development Optimization of mass spectrometric ionization and fragmentation of CRD and IS was performed through continuous infusion (10 µL min-1) of methanolic solution of CRD and IS
14 Page 14 of 30
(each 100 ng mL-1) into the electrospray source using a syringe pump (Harvard Pump 11plus, Holliston, USA). Tuning of mass parameters was performed both in positive and negative ionization mode. Good response was found in the negative ionization mode for both CRD
ip t
and IS. Identification of precursor ion of each analyte and IS was performed in the full scan mode by recording mass transition from m/z 50 to 600. Both CRD and IS showed only one
cr
prominent ion, which corresponds to the deprotonated [M-H]- ion. So, for CRD and IS, [MH]- ion was selected as the parent ion in Q1 spectrum and used as precursor ion to obtain Q3
us
product ion spectrum. The product ion mass spectrum of CRD and IS is presented in Fig. 1.
an
MRM technique was selected for the assay development and the parameters were optimized to maximize the response for CRD. The most sensitive mass transition was monitored from
M
m/z 269 to 122 and 367.1 to 216.9 for CRD and IS, respectively.
Among several chromatographic conditions and columns (cyano, C8, C18) tried, the
d
Supelco Discovery HS C18 column (5 μm, 50 x 4.6 mm) provided good selectivity, sensitivity
te
and better peak shape for CRD and IS in a short run time. Several combinations of solvents (acetonitrile and methanol) and buffers (ammonium acetate, ammonium formate and formic
Ac ce p
acid) were evaluated to select suitable mobile phase. The mobile phase comprising acetonitrile : AAB (0.01 M, pH 4.5) (85:15, v/v) at a flow rate of 0.6 mL min-1 was found most appropriate. A simple liquid-liquid extraction technique using various combinations of extraction solvents were tried for maximum recovery of CRD and IS from rat serum. A mixture of 60% n-hexane in ethyl acetate provided excellent recovery. Representative chromatograms of extracted analyte/IS-free and analyte/IS-fortified serum are illustrated in Fig. 2. The retention times of CRD (1.8 min) and IS (1.5 min) were low enough to allow a short run time of 3.0 min.
3.2. Selectivity and matrix effect
15 Page 15 of 30
The developed method was selective as no significant interference from endogenous serum constituents was observed at the retention times of CRD and IS in chromatograms derived from processed blank serum samples from six different rats (Fig. 2). No significant
ip t
matrix effect was observed in post-extraction spiked serum samples for the analyte at LQC, MQC and HQC concentrations. The results showed that the co-eluting endogenous serum
cr
components did not suppress or enhance the ionization of CRD and IS. A processed zero sample (sample processed with IS only) injected after the highest calibration standard showed
us
that the peak areas of the eluting peak at the retention times of CRD was less than 5% of the
M
3.3. Calibration curve, linearity and sensitivity
an
peak area of the CRD at LLOQ indicating an insignificant carryover effect.
The eight point calibration curves were constructed on each day of the 5-day
d
validation period and a weighted (1/x2) least squares linear regression analysis was applied to
te
the data. Linear responses were observed for the CRD in the range of 1 to 200 ng mL-1. All the calibration standards were within the acceptance criteria (accuracy of ±20% for LLOQ
Ac ce p
and ±15% for non-LLOQ standards) and the regression coefficient values were always greater than 0.999. The limit of detection (S/N ratio >3) was 0.2 ng mL-1 for CRD in rat serum. The LLOQ was selected as 1 ng mL-1 as the response at this concentration was more than 10-times higher than that of blank (S/N ratio >10). Sensitivity was tested at LLOQ and the accuracy and precision at LLOQ were within the acceptable limits (Table 1).
3.4. Extraction recovery Simple liquid-liquid extraction technique using the extraction solvent (n-hexane: ethyl acetate, 60:40, v/v) provided excellent and consistent recovery of CRD and IS from spiked serum samples. Moreover, the extraction solvent provided clean extracts with minimal
16 Page 16 of 30
endogenous serum components resulting in negligible matrix effect. The recovery of CRD was determined by analyzing sextuplet samples at LQC, MQC and HQC for five consecutive days. Mean recoveries of CRD from spiked QC standards were always more than 90% (Table
ip t
1) and found to be concentration independent and consistent. The recovery of the IS averaged
cr
to 50%.
3.5. Accuracy, precision and dilution integrity
us
The accuracy and precision were determined by analyzing sextuplet samples at
an
LLOQ, LQC, MQC and HQC for five consecutive days. The values of the intra-day and inter-day accuracy and precision in serum QC samples are summarized in Table 1. Intra-day
M
precision, as indicated by %RSD, varies from 4.0 to 12.4. However, inter-day precision evaluated at the same concentrations was ≤11.8. The accuracy of the method (%bias) ranged
d
from -9.1 to 4.9. The results demonstrated that all the value of accuracy and precision were
te
well within the acceptable limit of variation at LQC, MQC and HQC. A 20-fold dilution of DIQC samples by blank drug free-serum prior to extraction was used for determining dilution
Ac ce p
integrity. Sextuplet of DIQC samples were extracted and analyzed along with one of the validation batches. The results, mean accuracy (%bias) and precision (%RSD) of 10.4 and 0.2, respectively, demonstrated that the samples with concentrations greater than the ULOQ can be analyzed following dilution to obtain precisely accurate results.
3.7. Stability studies
The stability of CRD in serum matrix was investigated as described in section 2.5 and the results are presented in Table 2. In different stability experiments namely, bench-top stability, freeze-thaw stability, long term stability and auto sampler stability, the changes (%deviation) from the reference concentrations were within the acceptable limit (≤ ±15%)
17 Page 17 of 30
demonstrating that CRD was stable in serum and the processed samples. Moreover, CRD was found to be stable in whole blood up to 2 h as percent CRD remained at 2 h was within the acceptable limit (Fig. 3). Also, CRD in stock/standard solutions was found to be stable for 45
ip t
days at 4°C as the difference between the peak responses of CRD in analytical standards
cr
prepared from fresh and stored stock/standard solution was found to be 3.8 ± 0.1%.
3.8. Pharmacokinetic studies
us
The validated LC-MS/MS method was successfully applied for characterizing gender
an
influence on the pharmacokinetics of cardamonin in Sprague Dawley rats (male and female, each n = 3) following oral (5 mg/kg) and intravenous (1 mg/kg) administration. The
M
compound was quantified in test serum samples upto 24 h. The mean serum concentration time profiles and the pharmacokinetic parameters of CRD in male and female rats after oral
d
and intravenous administration are shown in Fig. 4 and Table 3. Although, the systemic
te
availability of CRD after oral administration is quite low in both male and female rats, but CRD has comparatively higher bioavailability in female (4.8%) than in male rats (0.6%).
Ac ce p
Following both oral and intravenous administration, CRD showed Vss higher than the total blood volume (0.064 L/kg; [18]) of the rat indicating extravascular distribution. The Cmax and the absolute bioavailability was higher in females than in males after oral dosing suggesting that the CRD has better absorption in females than in males. Moreover, comparatively higher Vss and CL were observed in females than in males after both intravenous and oral administrations, which demonstrates that CRD is distributed and eliminated more in females. As CRD is lipophilic in nature, higher Vss in female rats may be attributed to higher percentage of body fat as compared to that in males. Following both oral and intravenous administration, a 10-times higher CL was observed in females than in males. In accordance to this, MRT values were found higher in males than that in females. Based on significant
18 Page 18 of 30
difference in CL, it may be inferred that the activity of enzymes that metabolize the cardamonin might differ between the sexes. Conclusively, it can be stated that gender exert
ip t
significant effect on the pharmacokinetics of cardamonin in rats.
4. Conclusion
cr
To the best of our knowledge, this is the first paper to report preclinical pharmacokinetics and LC-MS/MS method for quantification of CRD. The LC-MS/MS assay
us
reported in this paper is sensitive and specific for quantification of CRD in rat serum. All the
an
regulatory requirement of method validation were followed and achieved. The extraction method provides excellent, consistent and reproducible recoveries of CRD. The simplicity of
M
the assay, use of liquid-liquid extraction, cost effectiveness and sample turnover rate of 3 min per sample, makes it attractive and applicable procedure for future studies (stability in
d
simulated body fluids, protein binding, dose escalation, tissue distribution and excretion) of
te
CRD. The method was successfully implemented for single dose pharmacokinetic studies of CRD in male and female rats. The study revealed that CRD is poorly bioavailable which
Ac ce p
might be due to poor solubility and/or hepatic first pass metabolism. Moreover, CRD exhibited significant difference in its pharmacokinetic parameters and bioavailability between male and female rats that could be due to gender related differences in the enzyme activity. Further studies are required for better understanding of low oral bioavailability and gender difference in its pharmacokinetic behavior. The study provides useful insight in devise of appropriate formulation, dosage regimen and route of administration for optimal pharmacological efficacy to aid in its development as a potential anticancer drug candidate.
Acknowledgements
19 Page 19 of 30
Author AS is thankful to Indian Council of Medical Research, New Delhi, for financial support in the form of his fellowship for the Central Drug Research Institute
ip t
Communication (324/2014/JL).
Conflict of interest
us
cr
There is no potential conflict of interest.
References
A. Murakamia, A. Kondoab, Y. Nakamuraa, H. Ohigashia & K. Koshimizua, Possible
an
[1]
Anti-tumor Promoting Properties of Edible Plants from Thailand, and Identification of
Biochem. 57 (1993) 1971-3.
V.R. Yadav, S. Prasad, B.B. Aggarwal, Cardamonin sensitizes tumour cells to TRAIL
d
[2]
M
an Active Constituent, Cardamonin, of Boesenbergia pandurata, Biosci. Biotech.
te
through ROS- and CHOP-mediated up-regulation of death receptors and downregulation of survival proteins, Br. J. Pharmacol. 165 (2012) 741-53. Y. Qin, C.Y. Sun, F.R. Lu, X.R. Shu, D. Yang, L. Chen, X.M. She, N.M. Gregg, T.
Ac ce p
[3]
Guo, Y. Hu, Cardamonin exerts potent activity against multiple myeloma through blockade of NF-kappaB pathway in vitro, Leuk. Res. 36 (2012) 514-20.
[4]
J. Tang, N. Li, H. Dai, K. Wang, Chemical constituents from seeds of Alpinia katsumadai, inhibition on NF-kappaB activation and anti-tumor effect, Zhongguo. Zhong. Yao. Za. Zhi. 35 (2010) 1710-4.
[5]
B. Sung, S. Prasad, V.R. Yadav, S.C. Gupta, S. Reuter, N. Yamamoto, A. Murakami, B.B. Aggarwal, RANKL signaling and osteoclastogenesis is negatively regulated by cardamonin, PLoS One 8 (2013) e64118.
20 Page 20 of 30
[6]
S. Hatziieremia, A.I. Gray, V.A. Ferro, A. Paul, R. Plevin, The effects of cardamonin on lipopolysaccharide-induced inflammatory protein production and MAP kinase and NFkappaB signalling pathways in monocytes/macrophages, Br. J. Pharmacol. 149
ip t
(2006) 188-98. [7]
D.A. Israf, T.A. Khaizurin, A. Syahida, N.H. Lajis, S. Khozirah, Cardamonin inhibits
cr
COX and iNOS expression via inhibition of p65NF-kappaB nuclear translocation and Ikappa-B phosphorylation in RAW 264.7 macrophage cells, Mol. Immunol. 44 (2007)
Y.L. Chow, K.H. Lee, S. Vidyadaran, N.H. Lajis, M.N. Akhtar, D.A. Israf, A.
an
[8]
us
673-9.
Syahida, Cardamonin from Alpinia rafflesiana inhibits inflammatory responses in
M
IFN-gamma/LPS-stimulated BV2 microglia via NF-kappaB signalling pathway, Int. Immunopharmacol. 12 (2012) 657-65.
J.H. Lee, H.S. Jung, P.M. Giang, X. Jin, S. Lee, P.T. Son, D. Lee, Y.S. Hong, K. Lee,
d
[9]
te
J.J. Lee, Blockade of nuclear factor-kappaB signaling pathway and anti-inflammatory activity of cardamomin, a chalcone analog from Alpinia conchigera, J. Pharmacol.
Ac ce p
Exp. Ther. 316 (2006) 271-8.
[10]
H. Ko, Y.J. Kim, E.C. Amor, J.W. Lee, H.C. Kim, H.J. Kim, H.O. Yang, Induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma HCT116 and LOVO cells, J. Cell. Biochem. 112 (2011) 2471-9.
[11]
M. Cho, M. Ryu, Y. Jeong, Y.H. Chung, D.E. Kim, H.S. Cho, S. Kang, J.S. Han, M.Y. Chang, C.K. Lee, M. Jin, H.J. Kim, S. Oh, Cardamonin suppresses melanogenesis by inhibition of Wnt/beta-catenin signaling, Biochem. Biophys. Res. Commun. 390 (2009) 500-5.
[12]
H. Doug, S.X. Chen, H.X. Xu, S. Kadota, T. Namba, A new antiplatelet diarylheptanoid from Alpinia blepharocalyx, J. Nat. Prod. 61 (1998) 142-4.
21 Page 21 of 30
[13]
S. Tewtrakul, S. Subhadhirasakul, J. Puripattanavong, T. Panphadung, HIV-1 protease inhibitory substances from the rhizomes of Boesenbergia pandurata Holtt, Songklanakarin. J. Sci. Technol. 25 (2003) 503-8. G. Trakoontivakorn, K. Nakahara, H. Shinmoto, M. Takenaka, M. Onishi-Kameyama,
ip t
[14]
H. Ono, M. Yoshida, T. Nagata, T. Tsushida, Structural analysis of a novel
cr
antimutagenic compound, 4-Hydroxypanduratin A, and the antimutagenic activity of flavonoids in a Thai spice, fingerroot (Boesenbergia pandurata Schult.) against
us
mutagenic heterocyclic amines, J. Agric. Food. Chem. 49 (2001) 3046-50.
J. Hodgson, ADMET turning chemicals into drugs, Nat. Biotechnol. 19 (2001) 722-6.
[16]
FDA,
Guidance
for
Industry:
an
[15]
Bioanalytical
Method
Validation.
[17]
M
http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf [08 October 2014] M. Gibaldi, D. Perrier, Pharmacokinetics, second ed., Informa Healthcare, New York,
N. Rangaraj, K. Vaghasiya, S. Jaiswal, A. Sharma, M. Shukla, J. Lal, Do Blood
te
[18]
d
1982.
Ac ce p
Sampling Sites Affect Pharmacokinetics?, Chem. Biol. Interface. 4 (2014) 176-191.
22 Page 22 of 30
Legend for figures Fig. 1. Product ion spectra (MS-MS) of cardamonin and curcumin (IS) Fig. 2. Typical MRM chromatograms of cardamonin (left panel) and curcumin (right panel;
ip t
IS) (a-b) drug-free and IS-free rat serum, (c-d) drug-free and IS (100 ng mL-1) fortified rat serum, (e-f) serum containing 1 ng mL-1 (LLOQ) of cardamonin and 100 ng mL-1 of IS, and
cr
(g-h) serum sample taken at 2 h from a female rat treated with 5 mg/kg oral dose of
Fig. 3. Stability of cardamonin in rat whole blood
us
cardamonin.
an
Fig. 4. Mean serum concentration-time profile of cardamonin in male and female Sprague Dawley rats (each n=3) after a single (A) 5 mg/kg oral and (B) 1 mg/kg intravenous
Ac ce p
te
d
M
administration. Bar represents SEM.
23 Page 23 of 30
Table 1 Recovery, accuracy and precision of the proposed assay method for cardamonin quantification (n = 6 at each concentration)
(ng mL-1)
Mean
Accuracy (%Bias)
CV (%)
Intra-day
Inter-
Intra-
Inter-
day
day
day
93.1
8.7
-2.2
-9.1
3
98.2
8.1
2.2
4.9
90
108.0
7.8
0.0
180
110.6
13.6
12.4
11.8
4.6
3.8
8.0
8.0
4.0
7.5
us
1
an
-0.2 2.6
Ac ce p
te
d
M
4.5
Precision (%RSD)
ip t
Absolute recovery
cr
Concentration
24 Page 24 of 30
Table 2 Stability of cardamonin under different storage conditions in serum (n = 6 at each concentration) Measured
Measured mean
concentration
concentration
concentration in
in QC samples
in Reference#
stability QC
(ng mL-1)
QC samples
samples (ng
(ng mL-1)
mL-1)
3
3.1 ± 0.3
3.3 ± 0.1
stability
90
86.9 ± 13.8
(room
180
183.7 ± 2.8
Freeze-thaw
3
stability
90
86 ± 14.1
0.5
-0.7
169.3 ± 4.9
5.7
-7.8
3.1 ± 0.2
2.9 ± 0.1
3.3
-4. 5
95.5 ± 8.7
1.6
1.9
180.5 ± 16.3
177.6 ± 3.2
1.1
-1.6
M
temperature, 6 h)
cycles,
-
180
te
3
97.3 ± 8.8
Ac ce p
(after
cr
6.0
us
4.1
an
top
%deviation*
d
Bench
%CV
ip t
Nominal
Stability test
80°C) Long
term
3
3.1 ± 0.1
2.8 ± 0.1
6.6
-8.6
stability (30
90
90.5 ± 1.3
92.0 ± 6.6
1.2
1.7
days, -80°C)
180
172.7 ± 5.7
196.4 ± 1.3
8.5
13.7
Auto
3
2.8 ± 0.1
2.9 ± 0.1
1.8
2.6
sampler
90
97.3 ± 7.7
87.9 ± 2.2
7.2
-9.7
stability (12
180
166.5 ± 5.6
167.0 ± 5.4
0.2
0.3
h, 10°C) *%deviation in comparison to reference concentration, #freshly prepared and immediately analyzed set of quality control samples 25 Page 25 of 30
Table 3 Pharmacokinetic parameters of cardamonin after oral (5 mg/kg) and intravenous (1 mg/kg) administrations in rats.
Oral administration
Female
Male
1285.3 ± 33.0
1498.3 ± 611.0
12.9 ± 1.8*
52.6 ± 20.7*
2.3 ± 0.9
2.2 ± 0.9
5184.7 ±
803.5 ±
193.9 ± 18.8
565.8**
227.0**
us
tmax (h)
Female
cr
Cmax (ng/ml)
Male
ip t
Intravenous administration Parameters
1.9 ± 0.6
4.7 ± 1.7
1.1 ± 0.1**
7.1 ± 0.8**
CL (L/h/kg)
0.17 ± 0.01*
1.37 ± 0.30*
0.11 ± 0.01**
1.08 ± 0.14**
MRT (h)
6.4 ± 0.2***
1.9 ± 0.4***
10.0 ± 0.3*
6.8 ± 1.1*
-
0.6***
4.8***
-
d
Bioavailability (%)
an
Vss (L/kg)
157.4 ± 24.1
M
AUClast (ng h/ml)
te
Values of PK parameters are mean ± SEM (n=3); Abbreviations: AUClast = area under the serum concentration-time curve up to last sampling
Ac ce p
time, Cmax = serum peak concentration, tmax = time to Cmax, Vss = volume of distribution at steady-state, CL= Clearance, MRT= mean residence time; *= p<0.05 significant difference, **= p<0.01, very significant difference, ***= p<0.001, highly significant difference.
26 Page 26 of 30
Ac ce p
te
d
M
an
us
cr
ip t
Figure 1
Page 27 of 30
Ac ce p
te
d
M
an
us
cr
ip t
Figure 2
Page 28 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 29 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure 4
Page 30 of 30