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MS method for quantification of eggmanone in rat plasma and its application to pharmacokinetics
Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 37–43
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
A simple and sensitive HPLC–MS/MS method for quantification of eggmanone in rat plasma and its application to pharmacokinetics Chen Xie a , Ana Ramirez a,b , Zhijun Wang c,d , Moses S.S. Chow d , Jijun Hao a,e,∗ a
College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766, USA Department of Biology, California State Polytechnic University, Pomona, CA 91768, USA Department of Pharmaceutical Sciences, Marshall B. Ketchum University, Fullerton, CA 92831, USA d College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766, USA e Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA, 91766, USA b c
a r t i c l e
i n f o
Article history: Received 3 October 2017 Received in revised form 28 December 2017 Accepted 7 January 2018 Available online 11 February 2018 Keywords: Eggmanone Plasma HPLC–MS/MS Pharmacokinetics
a b s t r a c t Allosteric phosphodiesterase 4 (PDE4) inhibitors are highly sought after due to their important antiinflammatory and anti-cancer therapeutic effects. We recently identified Eggmanone, an extraordinarily selective allosteric PDE4 inhibitor displaying favorable drug properties. However, a specific analytic method of Eggmanone in serum and its pharmacokinetics have not been reported yet. In this study, we developed a rapid and sensitive high performance liquid chromatography–mass spectrometric (HPLC–MS/MS) method to determine Eggmanone concentrations in rat plasma. This assay method was validated in terms of specificity, linearity, sensitivity, accuracy, precision, matrix effect, recovery and stability, and was applied to a pharmacokinetic study in rats following intravenous injection of Eggmanone at doses of 1 and 3 mg/kg. The lower limit of quantification (LLOQ) of this assay was 5 ng/mL and the linear calibration curve was acquired with R2 > 0.99 between 5 and 1000 ng/m. The intra-day and inter-day precision was evaluated with the coefficient of variations less than 11.09%, whereas the mean accuracy ranged from 98.38% to 105.13%. The assay method exhibited good recovery and negligible matrix effect. The samples were stable under all the experimental conditions. The plasma concentrations of Eggmanone were detected and quantified over 24 h with the terminal elimination half-live of 3.57 ± 1.80 h and 5.92 ± 3.34 h for the low dose (1 mg/kg) and high dose (3 mg/kg) respectively. In summary, the present method provides a robust, fast and sensitive analytical approach for quantification of Eggmanone in plasma and was successfully applied to a pharmacokinetic study in rats. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Phosphodiesterase-4 (PDE4) hydrolyzes cyclic adenosine monophosphate (cAMP), a key secondary messenger in controlling crucial cellular functions [1]. Blocking cAMP hydrolysis by targeting PDE4 has shown anti-inflammatory responses in chronic obstructive pulmonary disease (COPD), asthma, allergic rhinitis and idiopathic pulmonary fibrosis [2–5]. In addition, PDE4 inhibition also displays anti-neoplastic effects on human malignancies including leukemia, colon cancer, glioma and prostate cancer [6–10]. Consequently, significant efforts have been made to develop PDE4 inhibitors. However, currently available PDE4 inhibitors are mainly competitive inhibitors which are associated with emesis and other side effects [1]. It is believed that allosteric
∗ Corresponding author at: College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766, USA. E-mail address: [email protected] (J. Hao). https://doi.org/10.1016/j.jpba.2018.01.009 0731-7085/© 2018 Elsevier B.V. All rights reserved.
inhibitors targeting the non-active site of PDE4 may overcome the side effects of the competitive PDE inhibitors [11–13]. In an in-vivo zebrafish embryo-based screening [14], we and colleagues have recently identified an allosteric PDE4 inhibitor named Eggmanone which specifically targets the upstream conserved region 2 (UCR2) of PDE4 [14] (Fig. 1A). Eggmanone displays extraordinarily selectivity to PDE4 without any known off-targets in counter-screens of 442 kinases, 158 G protein–coupled receptors, and 21 phosphatases [14]. Importantly, our discovery of Eggmanone also disclosed a new role of PDE4 in sonic Hedgehog (sHH) signaling, and this was further supported by another two recent reports that PDE4 inhibition down-regulated the sHH signaling in tumor growth [15,16]. As both available sHH signaling inhibitor drugs (Vismodegib and Sonidegib) target a Smoothened (SMO) protein that often acquires mutations conferring drug resistance in patients, Eggmanone which targets downstream of SMO in the sHH pathway may represent a viable new therapy to overcome clinical resistance problem of the current SMO inhibitor drugs. Additionally, Eggmanone was picked up in an in-vivo zebrafish
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C. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 37–43
Fig. 1. Chemical Structures of (A) Eggmanone and (B) internal standard testosterone.
embryo-based screening, and found to effectively improve systolic function of mice with failing hearts [14,17], suggesting it may exhibit favored drug properties. However, to date, neither a bio-analytical assay to determine Eggmanone in plasma nor its pharmacokinetics has been reported in the literature. Here, we developed a rapid and sensitive HPLC–MS/MS method to measure Eggmanone concentrations in rat plasma which can be applied to a pharmacokinetic study of Eggmanone in rats. 2. Materials and methods 2.1. Chemicals and reagents Eggmanone was purchased from Milipore (Bedford, MA, USA). Testosterone (internal standard, IS) was obtained from SigmaAldrich Co. (St. Louis, MO, USA). Formic acid was from Millipore (Bedford, MA, USA). Methanol (MeOH) and acetronitrile (ACN) were purchased from Fisher Scientific (Tustin, CA, USA). Distilled and deionized water (ddH2 O) was prepared from Mili-Q water purification system from Milipore (Bedford, MA, USA). 2.2. Apparatus The LC/MS/MS system consisted of an API 3200 LC/MS/MS system (Sciex, Framingham, MA, USA) and two Shimadzu LC-20AD Prominence Liquid Chromatography pumps equipped with an SIL20A Prominence auto-sampler (Shimadzu, Columbia, MD, USA). Chromatographic separation was performed using a ZORBAX SBC18 column (2.1 × 150 mm, 5 m, Agilent, Santa Clara, CA).
2.4. Preparation of stock and working solutions, calibration standards and quality control samples Stock solutions of Eggmanone and testosterone (IS) were prepared in DMSO and 50% methanol at 0.5 mg/mL and 1 mg/mL, respectively. The working solutions of Eggmanone were prepared by sequential dilution with DMSO to final concentrations ranging from 50 ng/mL to 10 g/mL. The IS working solution was diluted with 50% methanol to give a final concentration of 5 g/mL. All solutions were stored at −20 ◦ C and brought to room temperature before use. Calibration standard samples were prepared by spiking working solution of Eggmanone and IS to the plasma followed by the extraction process to yield calibration standards of 5, 10, 25, 50, 100, 250, 500 and 1000 ng/mL. Quality control (QC) samples were prepared as the calibration standards to give nominal Eggmanone concentrations at high (HQC), medium (MQC), and low (LQC) concentrations including lower limit of quantification (LLOQ). 2.5. Sample extraction procedure To 100 L of the rat plasma, 5 L of the internal standard were added (5 g/mL testosterone). Rat plasma was extracted with 400 L of ethyl acetate in a 1.5-mL polyethylene tube for 3 min by vortexing, followed by centrifugation at 10,000g for 5 min at 4 ◦ C. Clear organic layer was transferred to new tube and dried in a centrifuge-evaporator, and the residue dissolved in 100 L of mobile phase, mixed for 3 min and then centrifuged at 10,000g for 5 min. The supernatant was transferred to the sample vial and 5 L was injected into the LC/MS/MS system for analysis.
2.3. HPLC–MS/MS conditions 2.6. Method validation An isocratic elution method was applied for chromatographic separation of Eggmanone with two mobile phases: (A) 0.1% formic acid (0.075 mL/min) and (B) ACN (0.3 mL/min). The injection volume was kept at 5 L. After each injection, a needle wash with 50% MeOH (v/v) was performed. The temperatures of analytical column and auto-sampler were both set at room temperature. The effluent was ionized by positive ion mode by ESI and detected by mass spectrometry. Typical mass spectrometric conditions were: gas 1, nitrogen (40 psi); gas 2, air (20 psi); ion spray voltage, 4500 V; ion source temperature, 550 ◦ C; curtain gas, nitrogen (25 psi). The compound parameters, eg, declustering potential (DP), entrance potential (EP), collision energy (CE), collision cell exit potential (CEP) and collision cell exit potential (CXP), for Eggmanone and testosterone were 56, 10, 18, 45, and 4 eV and 61, 95, 18, 39 and 2 eV, respectively. Data were recorded in multiple reaction monitoring (MRM) mode. Eggmanone dissolved in DMSO and IS dissolved in methanol were used for the MS/MS optimization. Detection of the ions was performed in MRM mode, with the transition of m/z (Q1/Q3) 417.146/251.096 for Eggmanone and 289.206/97.027 for internal standard testosterone.
The developed method was validated for linearity and sensitivity, specificity, accuracy, precision, recovery and stability based on the guidelines for industry, Bioanalytical Method Validation published by the U.S. Food and Drug Administration (2001). The calibration curve for Eggmanone ranging from 5 to 1000 ng/mL was generated by plotting the peak area ratios of the analyte to IS versus the nominal concentration and fitted by a weighted linear least-squares linear regression with a weighing factor of 1/y, where y is the peak area ratio of Eggmanone versus IS. Sensitivity of the method was evaluated in terms of LLOQ which was determined based on the two criteria as follows: (1) the analyte response at the LLOQ be at least 5 times the response compared to blank response and (2) analyte peak be identifiable, discrete, and reproducible with an accuracy (relative error) of 80–120% and precision (relative standard deviation, RSD) within 20%. The specificity of the method was evaluated by comparing the chromatography of rat blank plasma samples with that of blank rat plasma spiked with standards at high, medium, low concentrations including LLOQ and rat plasma samples after intravenous
C. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 37–43
administration of Eggmanone. The intra-day accuracy and precision were determined by analyzing five replicated of the QC samples at high, medium, low and LLOQ concentrations within one day, while the inter-day accuracy and precision were conducted by determining five replicates of four levels of QCs on 3 separate days. The assay accuracy was determined as the percentage of the measured concentration to the nominal concentration. The precision was represented as the relative standard deviation (RSD) of the replicates. The matrix effect was determined by dividing the peak areas of Eggmanone spiked into extracted blank plasma by that of analyte spiked in mobile phase. Extraction recovery of Eggmanone and IS were assessed by comparing the peak area of the extracted QC samples to the peak area of analyte spiked to the extracted plasma. In order to determine the stability of Eggmanone in rat plasma, bench-top stability, freeze-thaw stability, auto-injector stability and long-term stability studies were carried out by using three replicates of the QC samples. For the bench-top stability during the handing process, the QC samples were prepared and kept at room temperature for 6 h. Freeze (−80 ◦ C) – thaw (room temperature) stability of the analytes in rat plasma was tested undergone three freeze-thaw cycles. The stability of sample in auto-sampler was determined by analyzing the extracted QC samples after kept in an auto-sampler at room temperature for 12 h. For storage stability, the QC samples were prepared and stored at −80 ◦ C for 30 days. 2.7. Application to pharmacokinetic study in rats Healthy Sprague-Dawley rats were obtained from Envigo and quarantined at least for 5 days. The animal room was at a controlled temperature of 21–23 ◦ C and a 12 h light-dark cycle. All experiments were approved by the Institutional Animal Care and Use Committee of Western University of Health Sciences (Pomona, CA, USA) and performed in accordance with the Guide for the Care and Use of Laboratory Animals. Eggmanone was dissolved in DMSO/PEG400 (1:3). Eight jugular cannulated rats were divided into 2 groups (2 male and 2 female each) receiving doses of Eggmanone (1 mg/kg and 3 mg/kg) via tail vein. Blood samples were collected at 0, 5, 15, 30 min, 1, 2, 4, 8 and 24-h post-dose. The blood sample (around 0.3 mL) for each time point was drawn via cannula and collected in a heparinized tube. The plasma was separated from the blood by centrifuging at 10,000 rpm for 5 min. All the plasma samples were stored at −80 ◦ C before analysis. The pharmacokinetics of Eggmanone was fitted using compartmental models with aid of Phoenix WinNonlin (version 7.0, CERTARA, Princeton, NJ). Either a 2-compartmental (equation of C = A·e−␣t + B·e−t ) or 3-compartmental (equation of C = A·e−␣t + B·e−t + C·e−␥t where A, B, C, ␣, , and ␥ are the hybrid constants) model was utilized to fit the experimental data. The Akaike information criterion (AIC) was used to determine the compartment model that can best fit the data. The 3-compartment model with the minimum AIC value was selected, and the major pharmacokinetic parameters (including half-life, volume distribution of the central compartment (Vp ) and at steady state (Vdss ), elimination constant of the central compartment (k), clearance and AUC) were calculated. 3. Results and discussion 3.1. Mass spectrometric detection and chromatographic separation The mass spectrometric detection of Eggmanone and internal standard (IS) testosterone was conducted in positive ion mode (Figs. 1 and 2). Testosterone was chosen as the IS since the same extraction procedure from rat plasma can be well applied to both testosterone and Eggmanone, and the endogenous testosterone in
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Table 1 Intra- and inter-day accuracy and precision for the determination of Eggmanone in rat plasma (n = 5). Nominal Value (ng/mL)
Calculated concentration (ng/mL)
Accuracy (%)
Precision (RSD, %)
Intra-day 980 200 15 5 (LLOQ)
972.29 ± 32.11 197.43 ± 7.64 14.96 ± 0.47 5.26 ± 0.38
99.21 98.72 99.72 105.13
3.30 3.87 3.13 7.18
Inter-day 980 200 15 5 (LLOQ)
964.12 ± 63.29 196.83 ± 10.70 15.02 ± 1.06 5.13 ± 0.57
98.38 98.41 100.10 102.52
6.56 5.44 7.08 11.09
the blank plasma of the male rats was very minimum and could not be detected under our current MS condition for Eggmanone (Supplementary Fig. S1 in the online version at DOI: 10.1016/j. jpba.2018.01.009). The protonated molecular ion peaks ([M+H]+ ) of Eggmanone and testosterone were observed at m/z 417.146 and 289.206, respectively. In the product ion scan, the most abundant and stable fragment ions were observed at m/z 251.096 for Eggmanone and m/z 97.027 for testosterone. Therefore, the mass transitions chosen were m/z 417.146/251.096 for Eggmanone and m/z 289.206/97.027 for testosterone to detect ions in the MRM mode (Fig. 2). Both Eggmanone and IS were well separated under the described chromatographic conditions at retention times of ∼3.3 min and ∼1.7 min, respectively (Fig. 2). 3.2. Linearity, sensitivity and specificity Calibration standards were prepared and analyzed at eight different concentrations from 5 to 1000 ng/mL. The calibration curve for Eggmanone displayed good linearity relationship in the concentration range (y = 0.00264x - 0.00754, r2 = 0.9954). The LLOQ of the developed assay for Eggmanone was 5 ng/mL. Representative selective reaction monitoring chromatograms of blank rat plasma, the quality control samples of Eggmanone at concentrations of 980 ng/mL (high), 200 ng/mL (medium), 15 ng/mL (low) and 5 ng/mL (LLOQ) and testosterone at 5 g/mL are shown in Fig. 3A–F. No interference was observed at retention times of Eggmanone (∼ 3.3 min) and IS (∼1.7 min) in any MRM chromatograms of blank plasma samples from six different sources. Eggmanone at high, medium, low and LLOQ concentrations can be clearly detected. No interferences at the retention time of Eggmanone and the IS were observed for the plasma samples obtained from rat plasma after intravenous administration of Eggmanone, indicating a good selectivity and specificity (Fig. 3G and H). 3.3. Accuracy and precision The intra-day and inter-day accuracy and precision are shown in Table 1. Using QC samples at a high concentration (980 ng/mL), a medium concentration (200 ng/mL), a low concentration (15 ng/m) and LLOQ (5 ng/mL), the intra-day and inter-day precision were evaluated with RSD ranged from 3.13–7.18% (intra-day) and 5.44–11.09% (inter-day), whereas the mean accuracy ranged from 98.72% to 105.13% (intra-day) and 98.38–102.52% (inter-day).These results demonstrated that this assay possessed good accuracy and precision for detecting Eggmanone in rat plasma. 3.4. Matric effect on extraction recovery As shown in Table 2, the matrix effects for Eggmanone at concentrations of HQC, MQC and LQC were 98.72 ± 5.99, 99.13 ± 7.42
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C. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 37–43
Fig. 2. Representative multiple reaction monitoring chromatogram and Product ion mass spectra of Eggmanone and internal standard testosterone. Presentative multiple reaction monitoring chromatogram for Eggmanone and internal standard (IS) testosterone (A). The mass transitions chosen are (B) m/z 417.146/251.096 for Eggmanone and (C) m/z 289.206/97.027 for testosterone to detect ions in multiple reaction monitoring mode.
C. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 37–43
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Fig. 3. Sample multiple reaction monitoring chromatogram. (A) blank plasma, (B) 980 ng/mL Eggmanone, (C) 200 ng/mL Eggmanone, (D) 15 ng/mL Eggmanone, (E) 5 ng/mL Eggmanone (LLOQ), (F) 5 g/mL testestrone (IS). (G) Plasma samples obtained from a rat at 5 min post dosing at 3 mg/kg Eggmanone and (H) 1 mg/kg Eggmanone.
Table 2 Matrix effect and recovery of Eggmanone and testosterone in rat plasma (n = 5). Concentration (ng/mL)
Matrix effect (Mean ± SD)
Recovery (Mean ± SD)
980 200 15
98.72 ± 5.99 99.13 ± 7.42 97.52 ± 3.22
99.41 ± 8.94 99.24 ± 9.44 96.34 ± 7.57
and 97.52 ± 3.22% (n = 5), respectively. Therefore the matrix effect for Eggmanone was considered to be negligible using this method. The assay method also exhibited good recovery with 96.34–99.41% for Eggmanone at three QC levels.
3.5. Stability The stability of Eggmanone in rat plasma was evaluated at high, medium and low levels of QC concentrations (980, 200, and 15 ng/mL) under all experimental conditions. As shown in Table 3, Eggmanone was found to be stable in rat plasma at room temperature for 6 h (recovery > 101.05% and precision < 8.50%). Eggmanone also showed good stability in 12 h in the autosampler (recovery > 101.79% and precision < 8.81%) and 24 h at −80 ◦ C then exposed to 3 freeze and thaw cycles (recovery > 98.12% and precision < 5.65%). In addition, the Eggmanone was found to be stable
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C. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 37–43
Table 3 Stability of Eggmanone under different conditions (n = 4). Time
Sample Conc. (ng/mL)
Measured Conc. (ng/mL)
CV (%)
Stability (%)
6 h at room temperature
980 200 15 980 200 15 980 200 15 980 200 15
1098.71 ± 93.39 202.11 ± 4.89 15.26 ± 0.53 1066.57 ± 31.02 205.27 ± 4.71 15.27 ± 1.34 971.45 ± 15.56 196.24 ± 7.05 15.79 ± 0.89 906.30 ± 50.81 189.40 ± 21.65 14.24 ± 0.94
8.50 2.42 3.47 2.91 2.29 8.81 1.60 3.59 5.65 5.61 11.43 6.60
112.11 101.05 101.75 108.83 102.63 101.79 99.13 98.12 105.24 92.48 94.70 94.92
12 h in the autosampler
24 h at −80 ◦ C then exposed to 3 freeze and thaw cycles 1 month at −80 ◦ C
Table 4 Pharmacokinetic parameters following intravenous dose of Eggmanone. Parameters
T1/2␣ (h) T1/2 (h) T1/2␥ (h) k (h−1 ) Vp (L/kg) Vdss (L/kg) Clearance (mL/h/kg) AUC0-∞ (ng/ml h)
Fig. 4. Rat plasma concentration-time course after receiving 1 mg/kg and 3 mg/kg Eggmanone by intravenous injection (Mean ± SE, n = 4).
under the storage conditions (−80 ◦ C) for at least 30 days (recovery > 92.48% and precision < 11.43%). 3.6. Pharmacokinetic study in rats The validated assay method was successfully applied to a pharmacokinetic study of Eggmanone in rat plasma following intravenous doses of 1 mg/kg and 3 mg/kg. To avoid the potential gender-associated pharmacokinetics difference, we recruited an equal number of male and female rats (2 male and 2 female rats in each group) receiving Eggmanone at doses of 1 mg/kg and 3 mg/kg
Dose 1 mg/kg Eggmanone
3 mg/kg Eggmanone
0.05 ± 0.02 0.21 ± 0.09 3.57 ± 1.80 4.8 ± 1.4 0.42 ± 0.20 1.7 ± 1.4 1.55 ± 0.32 639.5 ± 160.7
0.08 ± 0.08 0.26 ± 0.04 5.92 ± 3.34 6.4 ± 5.9 0.27 ± 0.18 1.5 ± 0.5 2.22 ± 1.08 1636.3 ± 823.2
in this pharmacokinetic study. It was found that no gender-related difference for pharmacokinetic profiles of Eggmanone. The Eggmanone plasma concentrations were fitted using both 2-compartmental and 3-compartmental models, while the best fitting was achieved by the 3-compartmental model with average AIC values of 64.29 and 43.31 for the high dose group and low dose group, respectively. The AIC values from the 3-compartmental model were lower than those obtained from the 2-compartmental model (Fig. 4). The major pharmacokinetic parameters are shown in Table 4. The total clearance values adjusted to body weight were calculated to be 1.55 ± 0.32 L/h/kg and 2.22 ± 1.08 L/h/kg for doses of 1 mg/kg and 3 mg/kg respectively. The average values for distribution at steady state (Vss) adjusted to body weight were 1.7 L/kg and 1.5 L/kg, respectively, and the values of the mean terminal elimination half-life (t1/2␥ ) were 3.57 ± 1.80 h and 5.92 ± 3.34 h for the low and high doses respectively. The values for the areas under the curve were 639.5 ± 160.7 ng h/ml and 1636.3 ± 823.2 ng h/ml for the two doses. Although a large elimination constant values were observed in the central compartment (k = 4.8 ± 1.4 h−1 and 6.4 ± 5.9 h−1 for low and high dose groups respectively), the wide distribution into deep compartment maintains a reasonable drug exposure for its in-vitro activity.
4. Conclusion Eggmanone is a novel PDE4 inhibitor which has important therapeutic potential. In this study, we developed and validated a rapid, reliable and sensitive HPLC–MS/MS method which was successfully applied to a pharmacokinetic study of Eggmanone in rats. The sample preparation was simple and the assay displayed excellent linearity, sensitivity, accuracy and precision. The current assay methodology of Eggmanone and its application to the pharmacokinetic profile could facilitate future pharmacodynamics studies of this interesting compound, providing important knowledge of its potential therapeutics.
C. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 37–43
Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the intramural fund of Western University of Health Sciences (J. Hao). The authors would like to acknowledge ChemAxon (http://www.chemaxon.com) for providing an academic license to their software. We thank Dr. Yilong Zhang (Ph.D) for his input in the pharmacokinetic model analysis. References [1] D.H. Maurice, et al., Advances in targeting cyclic nucleotide phosphodiesterases, Nat. Rev. Drug Discov. 13 (4) (2014) 290–314. [2] T.J. Torphy, Phosphodiesterase isozymes: molecular targets for novel antiasthma agents, Am. J. Respir. Crit. Care Med. 157 (2) (1998) 351–370. [3] K.F. Rabe, Update on roflumilast: a phosphodiesterase 4 inhibitor for the treatment of chronic obstructive pulmonary disease, Br. J. Pharmacol. 163 (1) (2011) 53–67. [4] H. Tenor, et al., Pharmacology, clinical efficacy, and tolerability of phosphodiesterase-4 inhibitors: impact of human pharmacokinetics, Handb. Exp. Pharmacol. 204 (2011) 85–119. [5] L.M. Fabbri, et al., Roflumilast in moderate-to-severe chronic obstructive pulmonary disease treated with longacting bronchodilators: two randomised clinical trials, Lancet 374 (9691) (2009) 695–703. [6] D.C. Lin, et al., Genomic and functional characterizations of phosphodiesterase subtype 4D in human cancers, Proc. Natl. Acad. Sci. U. S. A. 110 (15) (2013) 6109–6114.
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[7] E.P. Rahrmann, et al., Identification of PDE4D as a proliferation promoting factor in prostate cancer using a sleeping beauty transposon-based somatic mutagenesis screen, Cancer Res. 69 (10) (2009) 4388–4397. [8] P. Goldhoff, et al., Targeted inhibition of cyclic AMP phosphodiesterase-4 promotes brain tumor regression, Clin. Cancer Res. 14 (23) (2008) 7717–7725. [9] D.H. Kim, A. Lerner, Type 4 cyclic adenosine monophosphate phosphodiesterase as a therapeutic target in chronic lymphocytic leukemia, Blood 92 (7) (1998) 2484–2494. [10] R. Ogawa, et al., Inhibition of PDE4 phosphodiesterase activity induces growth suppression: apoptosis, glucocorticoid sensitivity, p53, and p21(WAF1/CIP1) proteins in human acute lymphoblastic leukemia cells, Blood 99 (9) (2002) 3390–3397. [11] A.B. Burgin, et al., Design of phosphodiesterase 4D (PDE4D) allosteric modulators for enhancing cognition with improved safety, Nat. Biotechnol. 28 (1) (2010) 63–70. [12] M.D. Houslay, P. Schafer, K.Y.J. Zhang, Phosphodiesterase-4 as a therapeutic target, Drug Discov. Today 10 (22) (2005) 1503–1519. [13] O. Bruno, et al., New selective phosphodiesterase 4D inhibitors differently acting on long: short, and supershort isoforms, J. Med. Chem. 52 (21) (2009) 6546–6557. [14] C.H. Williams, et al., An in vivo chemical genetic screen identifies phosphodiesterase 4 as a pharmacological target for hedgehog signaling inhibition, Cell Rep. 11 (1) (2015) 43–50. [15] G.L. Powers, et al., Phosphodiesterase 4D inhibitors limit prostate cancer growth potential, Mol. Cancer Res. 13 (1) (2015) 149–160. [16] X. Ge, et al., Phosphodiesterase 4D acts downstream of Neuropilin to control Hedgehog signal transduction and the growth of medulloblastoma, Elife (2015) 4. [17] Tromondae K. Feaster, et al., Discovery of a novel pharmaceutical class as potential heart failure treatment, Circ. Res. 117 (2015) A66.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
A simple and sensitive HPLC–MS/MS method for quantification of eggmanone in rat plasma and its application to pharmacokinetics Chen Xie a , Ana Ramirez a,b , Zhijun Wang c,d , Moses S.S. Chow d , Jijun Hao a,e,∗ a
College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766, USA Department of Biology, California State Polytechnic University, Pomona, CA 91768, USA Department of Pharmaceutical Sciences, Marshall B. Ketchum University, Fullerton, CA 92831, USA d College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766, USA e Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA, 91766, USA b c
a r t i c l e
i n f o
Article history: Received 3 October 2017 Received in revised form 28 December 2017 Accepted 7 January 2018 Available online 11 February 2018 Keywords: Eggmanone Plasma HPLC–MS/MS Pharmacokinetics
a b s t r a c t Allosteric phosphodiesterase 4 (PDE4) inhibitors are highly sought after due to their important antiinflammatory and anti-cancer therapeutic effects. We recently identified Eggmanone, an extraordinarily selective allosteric PDE4 inhibitor displaying favorable drug properties. However, a specific analytic method of Eggmanone in serum and its pharmacokinetics have not been reported yet. In this study, we developed a rapid and sensitive high performance liquid chromatography–mass spectrometric (HPLC–MS/MS) method to determine Eggmanone concentrations in rat plasma. This assay method was validated in terms of specificity, linearity, sensitivity, accuracy, precision, matrix effect, recovery and stability, and was applied to a pharmacokinetic study in rats following intravenous injection of Eggmanone at doses of 1 and 3 mg/kg. The lower limit of quantification (LLOQ) of this assay was 5 ng/mL and the linear calibration curve was acquired with R2 > 0.99 between 5 and 1000 ng/m. The intra-day and inter-day precision was evaluated with the coefficient of variations less than 11.09%, whereas the mean accuracy ranged from 98.38% to 105.13%. The assay method exhibited good recovery and negligible matrix effect. The samples were stable under all the experimental conditions. The plasma concentrations of Eggmanone were detected and quantified over 24 h with the terminal elimination half-live of 3.57 ± 1.80 h and 5.92 ± 3.34 h for the low dose (1 mg/kg) and high dose (3 mg/kg) respectively. In summary, the present method provides a robust, fast and sensitive analytical approach for quantification of Eggmanone in plasma and was successfully applied to a pharmacokinetic study in rats. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Phosphodiesterase-4 (PDE4) hydrolyzes cyclic adenosine monophosphate (cAMP), a key secondary messenger in controlling crucial cellular functions [1]. Blocking cAMP hydrolysis by targeting PDE4 has shown anti-inflammatory responses in chronic obstructive pulmonary disease (COPD), asthma, allergic rhinitis and idiopathic pulmonary fibrosis [2–5]. In addition, PDE4 inhibition also displays anti-neoplastic effects on human malignancies including leukemia, colon cancer, glioma and prostate cancer [6–10]. Consequently, significant efforts have been made to develop PDE4 inhibitors. However, currently available PDE4 inhibitors are mainly competitive inhibitors which are associated with emesis and other side effects [1]. It is believed that allosteric
∗ Corresponding author at: College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766, USA. E-mail address: [email protected] (J. Hao). https://doi.org/10.1016/j.jpba.2018.01.009 0731-7085/© 2018 Elsevier B.V. All rights reserved.
inhibitors targeting the non-active site of PDE4 may overcome the side effects of the competitive PDE inhibitors [11–13]. In an in-vivo zebrafish embryo-based screening [14], we and colleagues have recently identified an allosteric PDE4 inhibitor named Eggmanone which specifically targets the upstream conserved region 2 (UCR2) of PDE4 [14] (Fig. 1A). Eggmanone displays extraordinarily selectivity to PDE4 without any known off-targets in counter-screens of 442 kinases, 158 G protein–coupled receptors, and 21 phosphatases [14]. Importantly, our discovery of Eggmanone also disclosed a new role of PDE4 in sonic Hedgehog (sHH) signaling, and this was further supported by another two recent reports that PDE4 inhibition down-regulated the sHH signaling in tumor growth [15,16]. As both available sHH signaling inhibitor drugs (Vismodegib and Sonidegib) target a Smoothened (SMO) protein that often acquires mutations conferring drug resistance in patients, Eggmanone which targets downstream of SMO in the sHH pathway may represent a viable new therapy to overcome clinical resistance problem of the current SMO inhibitor drugs. Additionally, Eggmanone was picked up in an in-vivo zebrafish
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Fig. 1. Chemical Structures of (A) Eggmanone and (B) internal standard testosterone.
embryo-based screening, and found to effectively improve systolic function of mice with failing hearts [14,17], suggesting it may exhibit favored drug properties. However, to date, neither a bio-analytical assay to determine Eggmanone in plasma nor its pharmacokinetics has been reported in the literature. Here, we developed a rapid and sensitive HPLC–MS/MS method to measure Eggmanone concentrations in rat plasma which can be applied to a pharmacokinetic study of Eggmanone in rats. 2. Materials and methods 2.1. Chemicals and reagents Eggmanone was purchased from Milipore (Bedford, MA, USA). Testosterone (internal standard, IS) was obtained from SigmaAldrich Co. (St. Louis, MO, USA). Formic acid was from Millipore (Bedford, MA, USA). Methanol (MeOH) and acetronitrile (ACN) were purchased from Fisher Scientific (Tustin, CA, USA). Distilled and deionized water (ddH2 O) was prepared from Mili-Q water purification system from Milipore (Bedford, MA, USA). 2.2. Apparatus The LC/MS/MS system consisted of an API 3200 LC/MS/MS system (Sciex, Framingham, MA, USA) and two Shimadzu LC-20AD Prominence Liquid Chromatography pumps equipped with an SIL20A Prominence auto-sampler (Shimadzu, Columbia, MD, USA). Chromatographic separation was performed using a ZORBAX SBC18 column (2.1 × 150 mm, 5 m, Agilent, Santa Clara, CA).
2.4. Preparation of stock and working solutions, calibration standards and quality control samples Stock solutions of Eggmanone and testosterone (IS) were prepared in DMSO and 50% methanol at 0.5 mg/mL and 1 mg/mL, respectively. The working solutions of Eggmanone were prepared by sequential dilution with DMSO to final concentrations ranging from 50 ng/mL to 10 g/mL. The IS working solution was diluted with 50% methanol to give a final concentration of 5 g/mL. All solutions were stored at −20 ◦ C and brought to room temperature before use. Calibration standard samples were prepared by spiking working solution of Eggmanone and IS to the plasma followed by the extraction process to yield calibration standards of 5, 10, 25, 50, 100, 250, 500 and 1000 ng/mL. Quality control (QC) samples were prepared as the calibration standards to give nominal Eggmanone concentrations at high (HQC), medium (MQC), and low (LQC) concentrations including lower limit of quantification (LLOQ). 2.5. Sample extraction procedure To 100 L of the rat plasma, 5 L of the internal standard were added (5 g/mL testosterone). Rat plasma was extracted with 400 L of ethyl acetate in a 1.5-mL polyethylene tube for 3 min by vortexing, followed by centrifugation at 10,000g for 5 min at 4 ◦ C. Clear organic layer was transferred to new tube and dried in a centrifuge-evaporator, and the residue dissolved in 100 L of mobile phase, mixed for 3 min and then centrifuged at 10,000g for 5 min. The supernatant was transferred to the sample vial and 5 L was injected into the LC/MS/MS system for analysis.
2.3. HPLC–MS/MS conditions 2.6. Method validation An isocratic elution method was applied for chromatographic separation of Eggmanone with two mobile phases: (A) 0.1% formic acid (0.075 mL/min) and (B) ACN (0.3 mL/min). The injection volume was kept at 5 L. After each injection, a needle wash with 50% MeOH (v/v) was performed. The temperatures of analytical column and auto-sampler were both set at room temperature. The effluent was ionized by positive ion mode by ESI and detected by mass spectrometry. Typical mass spectrometric conditions were: gas 1, nitrogen (40 psi); gas 2, air (20 psi); ion spray voltage, 4500 V; ion source temperature, 550 ◦ C; curtain gas, nitrogen (25 psi). The compound parameters, eg, declustering potential (DP), entrance potential (EP), collision energy (CE), collision cell exit potential (CEP) and collision cell exit potential (CXP), for Eggmanone and testosterone were 56, 10, 18, 45, and 4 eV and 61, 95, 18, 39 and 2 eV, respectively. Data were recorded in multiple reaction monitoring (MRM) mode. Eggmanone dissolved in DMSO and IS dissolved in methanol were used for the MS/MS optimization. Detection of the ions was performed in MRM mode, with the transition of m/z (Q1/Q3) 417.146/251.096 for Eggmanone and 289.206/97.027 for internal standard testosterone.
The developed method was validated for linearity and sensitivity, specificity, accuracy, precision, recovery and stability based on the guidelines for industry, Bioanalytical Method Validation published by the U.S. Food and Drug Administration (2001). The calibration curve for Eggmanone ranging from 5 to 1000 ng/mL was generated by plotting the peak area ratios of the analyte to IS versus the nominal concentration and fitted by a weighted linear least-squares linear regression with a weighing factor of 1/y, where y is the peak area ratio of Eggmanone versus IS. Sensitivity of the method was evaluated in terms of LLOQ which was determined based on the two criteria as follows: (1) the analyte response at the LLOQ be at least 5 times the response compared to blank response and (2) analyte peak be identifiable, discrete, and reproducible with an accuracy (relative error) of 80–120% and precision (relative standard deviation, RSD) within 20%. The specificity of the method was evaluated by comparing the chromatography of rat blank plasma samples with that of blank rat plasma spiked with standards at high, medium, low concentrations including LLOQ and rat plasma samples after intravenous
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administration of Eggmanone. The intra-day accuracy and precision were determined by analyzing five replicated of the QC samples at high, medium, low and LLOQ concentrations within one day, while the inter-day accuracy and precision were conducted by determining five replicates of four levels of QCs on 3 separate days. The assay accuracy was determined as the percentage of the measured concentration to the nominal concentration. The precision was represented as the relative standard deviation (RSD) of the replicates. The matrix effect was determined by dividing the peak areas of Eggmanone spiked into extracted blank plasma by that of analyte spiked in mobile phase. Extraction recovery of Eggmanone and IS were assessed by comparing the peak area of the extracted QC samples to the peak area of analyte spiked to the extracted plasma. In order to determine the stability of Eggmanone in rat plasma, bench-top stability, freeze-thaw stability, auto-injector stability and long-term stability studies were carried out by using three replicates of the QC samples. For the bench-top stability during the handing process, the QC samples were prepared and kept at room temperature for 6 h. Freeze (−80 ◦ C) – thaw (room temperature) stability of the analytes in rat plasma was tested undergone three freeze-thaw cycles. The stability of sample in auto-sampler was determined by analyzing the extracted QC samples after kept in an auto-sampler at room temperature for 12 h. For storage stability, the QC samples were prepared and stored at −80 ◦ C for 30 days. 2.7. Application to pharmacokinetic study in rats Healthy Sprague-Dawley rats were obtained from Envigo and quarantined at least for 5 days. The animal room was at a controlled temperature of 21–23 ◦ C and a 12 h light-dark cycle. All experiments were approved by the Institutional Animal Care and Use Committee of Western University of Health Sciences (Pomona, CA, USA) and performed in accordance with the Guide for the Care and Use of Laboratory Animals. Eggmanone was dissolved in DMSO/PEG400 (1:3). Eight jugular cannulated rats were divided into 2 groups (2 male and 2 female each) receiving doses of Eggmanone (1 mg/kg and 3 mg/kg) via tail vein. Blood samples were collected at 0, 5, 15, 30 min, 1, 2, 4, 8 and 24-h post-dose. The blood sample (around 0.3 mL) for each time point was drawn via cannula and collected in a heparinized tube. The plasma was separated from the blood by centrifuging at 10,000 rpm for 5 min. All the plasma samples were stored at −80 ◦ C before analysis. The pharmacokinetics of Eggmanone was fitted using compartmental models with aid of Phoenix WinNonlin (version 7.0, CERTARA, Princeton, NJ). Either a 2-compartmental (equation of C = A·e−␣t + B·e−t ) or 3-compartmental (equation of C = A·e−␣t + B·e−t + C·e−␥t where A, B, C, ␣, , and ␥ are the hybrid constants) model was utilized to fit the experimental data. The Akaike information criterion (AIC) was used to determine the compartment model that can best fit the data. The 3-compartment model with the minimum AIC value was selected, and the major pharmacokinetic parameters (including half-life, volume distribution of the central compartment (Vp ) and at steady state (Vdss ), elimination constant of the central compartment (k), clearance and AUC) were calculated. 3. Results and discussion 3.1. Mass spectrometric detection and chromatographic separation The mass spectrometric detection of Eggmanone and internal standard (IS) testosterone was conducted in positive ion mode (Figs. 1 and 2). Testosterone was chosen as the IS since the same extraction procedure from rat plasma can be well applied to both testosterone and Eggmanone, and the endogenous testosterone in
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Table 1 Intra- and inter-day accuracy and precision for the determination of Eggmanone in rat plasma (n = 5). Nominal Value (ng/mL)
Calculated concentration (ng/mL)
Accuracy (%)
Precision (RSD, %)
Intra-day 980 200 15 5 (LLOQ)
972.29 ± 32.11 197.43 ± 7.64 14.96 ± 0.47 5.26 ± 0.38
99.21 98.72 99.72 105.13
3.30 3.87 3.13 7.18
Inter-day 980 200 15 5 (LLOQ)
964.12 ± 63.29 196.83 ± 10.70 15.02 ± 1.06 5.13 ± 0.57
98.38 98.41 100.10 102.52
6.56 5.44 7.08 11.09
the blank plasma of the male rats was very minimum and could not be detected under our current MS condition for Eggmanone (Supplementary Fig. S1 in the online version at DOI: 10.1016/j. jpba.2018.01.009). The protonated molecular ion peaks ([M+H]+ ) of Eggmanone and testosterone were observed at m/z 417.146 and 289.206, respectively. In the product ion scan, the most abundant and stable fragment ions were observed at m/z 251.096 for Eggmanone and m/z 97.027 for testosterone. Therefore, the mass transitions chosen were m/z 417.146/251.096 for Eggmanone and m/z 289.206/97.027 for testosterone to detect ions in the MRM mode (Fig. 2). Both Eggmanone and IS were well separated under the described chromatographic conditions at retention times of ∼3.3 min and ∼1.7 min, respectively (Fig. 2). 3.2. Linearity, sensitivity and specificity Calibration standards were prepared and analyzed at eight different concentrations from 5 to 1000 ng/mL. The calibration curve for Eggmanone displayed good linearity relationship in the concentration range (y = 0.00264x - 0.00754, r2 = 0.9954). The LLOQ of the developed assay for Eggmanone was 5 ng/mL. Representative selective reaction monitoring chromatograms of blank rat plasma, the quality control samples of Eggmanone at concentrations of 980 ng/mL (high), 200 ng/mL (medium), 15 ng/mL (low) and 5 ng/mL (LLOQ) and testosterone at 5 g/mL are shown in Fig. 3A–F. No interference was observed at retention times of Eggmanone (∼ 3.3 min) and IS (∼1.7 min) in any MRM chromatograms of blank plasma samples from six different sources. Eggmanone at high, medium, low and LLOQ concentrations can be clearly detected. No interferences at the retention time of Eggmanone and the IS were observed for the plasma samples obtained from rat plasma after intravenous administration of Eggmanone, indicating a good selectivity and specificity (Fig. 3G and H). 3.3. Accuracy and precision The intra-day and inter-day accuracy and precision are shown in Table 1. Using QC samples at a high concentration (980 ng/mL), a medium concentration (200 ng/mL), a low concentration (15 ng/m) and LLOQ (5 ng/mL), the intra-day and inter-day precision were evaluated with RSD ranged from 3.13–7.18% (intra-day) and 5.44–11.09% (inter-day), whereas the mean accuracy ranged from 98.72% to 105.13% (intra-day) and 98.38–102.52% (inter-day).These results demonstrated that this assay possessed good accuracy and precision for detecting Eggmanone in rat plasma. 3.4. Matric effect on extraction recovery As shown in Table 2, the matrix effects for Eggmanone at concentrations of HQC, MQC and LQC were 98.72 ± 5.99, 99.13 ± 7.42
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Fig. 2. Representative multiple reaction monitoring chromatogram and Product ion mass spectra of Eggmanone and internal standard testosterone. Presentative multiple reaction monitoring chromatogram for Eggmanone and internal standard (IS) testosterone (A). The mass transitions chosen are (B) m/z 417.146/251.096 for Eggmanone and (C) m/z 289.206/97.027 for testosterone to detect ions in multiple reaction monitoring mode.
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Fig. 3. Sample multiple reaction monitoring chromatogram. (A) blank plasma, (B) 980 ng/mL Eggmanone, (C) 200 ng/mL Eggmanone, (D) 15 ng/mL Eggmanone, (E) 5 ng/mL Eggmanone (LLOQ), (F) 5 g/mL testestrone (IS). (G) Plasma samples obtained from a rat at 5 min post dosing at 3 mg/kg Eggmanone and (H) 1 mg/kg Eggmanone.
Table 2 Matrix effect and recovery of Eggmanone and testosterone in rat plasma (n = 5). Concentration (ng/mL)
Matrix effect (Mean ± SD)
Recovery (Mean ± SD)
980 200 15
98.72 ± 5.99 99.13 ± 7.42 97.52 ± 3.22
99.41 ± 8.94 99.24 ± 9.44 96.34 ± 7.57
and 97.52 ± 3.22% (n = 5), respectively. Therefore the matrix effect for Eggmanone was considered to be negligible using this method. The assay method also exhibited good recovery with 96.34–99.41% for Eggmanone at three QC levels.
3.5. Stability The stability of Eggmanone in rat plasma was evaluated at high, medium and low levels of QC concentrations (980, 200, and 15 ng/mL) under all experimental conditions. As shown in Table 3, Eggmanone was found to be stable in rat plasma at room temperature for 6 h (recovery > 101.05% and precision < 8.50%). Eggmanone also showed good stability in 12 h in the autosampler (recovery > 101.79% and precision < 8.81%) and 24 h at −80 ◦ C then exposed to 3 freeze and thaw cycles (recovery > 98.12% and precision < 5.65%). In addition, the Eggmanone was found to be stable
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Table 3 Stability of Eggmanone under different conditions (n = 4). Time
Sample Conc. (ng/mL)
Measured Conc. (ng/mL)
CV (%)
Stability (%)
6 h at room temperature
980 200 15 980 200 15 980 200 15 980 200 15
1098.71 ± 93.39 202.11 ± 4.89 15.26 ± 0.53 1066.57 ± 31.02 205.27 ± 4.71 15.27 ± 1.34 971.45 ± 15.56 196.24 ± 7.05 15.79 ± 0.89 906.30 ± 50.81 189.40 ± 21.65 14.24 ± 0.94
8.50 2.42 3.47 2.91 2.29 8.81 1.60 3.59 5.65 5.61 11.43 6.60
112.11 101.05 101.75 108.83 102.63 101.79 99.13 98.12 105.24 92.48 94.70 94.92
12 h in the autosampler
24 h at −80 ◦ C then exposed to 3 freeze and thaw cycles 1 month at −80 ◦ C
Table 4 Pharmacokinetic parameters following intravenous dose of Eggmanone. Parameters
T1/2␣ (h) T1/2 (h) T1/2␥ (h) k (h−1 ) Vp (L/kg) Vdss (L/kg) Clearance (mL/h/kg) AUC0-∞ (ng/ml h)
Fig. 4. Rat plasma concentration-time course after receiving 1 mg/kg and 3 mg/kg Eggmanone by intravenous injection (Mean ± SE, n = 4).
under the storage conditions (−80 ◦ C) for at least 30 days (recovery > 92.48% and precision < 11.43%). 3.6. Pharmacokinetic study in rats The validated assay method was successfully applied to a pharmacokinetic study of Eggmanone in rat plasma following intravenous doses of 1 mg/kg and 3 mg/kg. To avoid the potential gender-associated pharmacokinetics difference, we recruited an equal number of male and female rats (2 male and 2 female rats in each group) receiving Eggmanone at doses of 1 mg/kg and 3 mg/kg
Dose 1 mg/kg Eggmanone
3 mg/kg Eggmanone
0.05 ± 0.02 0.21 ± 0.09 3.57 ± 1.80 4.8 ± 1.4 0.42 ± 0.20 1.7 ± 1.4 1.55 ± 0.32 639.5 ± 160.7
0.08 ± 0.08 0.26 ± 0.04 5.92 ± 3.34 6.4 ± 5.9 0.27 ± 0.18 1.5 ± 0.5 2.22 ± 1.08 1636.3 ± 823.2
in this pharmacokinetic study. It was found that no gender-related difference for pharmacokinetic profiles of Eggmanone. The Eggmanone plasma concentrations were fitted using both 2-compartmental and 3-compartmental models, while the best fitting was achieved by the 3-compartmental model with average AIC values of 64.29 and 43.31 for the high dose group and low dose group, respectively. The AIC values from the 3-compartmental model were lower than those obtained from the 2-compartmental model (Fig. 4). The major pharmacokinetic parameters are shown in Table 4. The total clearance values adjusted to body weight were calculated to be 1.55 ± 0.32 L/h/kg and 2.22 ± 1.08 L/h/kg for doses of 1 mg/kg and 3 mg/kg respectively. The average values for distribution at steady state (Vss) adjusted to body weight were 1.7 L/kg and 1.5 L/kg, respectively, and the values of the mean terminal elimination half-life (t1/2␥ ) were 3.57 ± 1.80 h and 5.92 ± 3.34 h for the low and high doses respectively. The values for the areas under the curve were 639.5 ± 160.7 ng h/ml and 1636.3 ± 823.2 ng h/ml for the two doses. Although a large elimination constant values were observed in the central compartment (k = 4.8 ± 1.4 h−1 and 6.4 ± 5.9 h−1 for low and high dose groups respectively), the wide distribution into deep compartment maintains a reasonable drug exposure for its in-vitro activity.
4. Conclusion Eggmanone is a novel PDE4 inhibitor which has important therapeutic potential. In this study, we developed and validated a rapid, reliable and sensitive HPLC–MS/MS method which was successfully applied to a pharmacokinetic study of Eggmanone in rats. The sample preparation was simple and the assay displayed excellent linearity, sensitivity, accuracy and precision. The current assay methodology of Eggmanone and its application to the pharmacokinetic profile could facilitate future pharmacodynamics studies of this interesting compound, providing important knowledge of its potential therapeutics.
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Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the intramural fund of Western University of Health Sciences (J. Hao). The authors would like to acknowledge ChemAxon (http://www.chemaxon.com) for providing an academic license to their software. We thank Dr. Yilong Zhang (Ph.D) for his input in the pharmacokinetic model analysis. References [1] D.H. Maurice, et al., Advances in targeting cyclic nucleotide phosphodiesterases, Nat. Rev. Drug Discov. 13 (4) (2014) 290–314. [2] T.J. Torphy, Phosphodiesterase isozymes: molecular targets for novel antiasthma agents, Am. J. Respir. Crit. Care Med. 157 (2) (1998) 351–370. [3] K.F. Rabe, Update on roflumilast: a phosphodiesterase 4 inhibitor for the treatment of chronic obstructive pulmonary disease, Br. J. Pharmacol. 163 (1) (2011) 53–67. [4] H. Tenor, et al., Pharmacology, clinical efficacy, and tolerability of phosphodiesterase-4 inhibitors: impact of human pharmacokinetics, Handb. Exp. Pharmacol. 204 (2011) 85–119. [5] L.M. Fabbri, et al., Roflumilast in moderate-to-severe chronic obstructive pulmonary disease treated with longacting bronchodilators: two randomised clinical trials, Lancet 374 (9691) (2009) 695–703. [6] D.C. Lin, et al., Genomic and functional characterizations of phosphodiesterase subtype 4D in human cancers, Proc. Natl. Acad. Sci. U. S. A. 110 (15) (2013) 6109–6114.
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[7] E.P. Rahrmann, et al., Identification of PDE4D as a proliferation promoting factor in prostate cancer using a sleeping beauty transposon-based somatic mutagenesis screen, Cancer Res. 69 (10) (2009) 4388–4397. [8] P. Goldhoff, et al., Targeted inhibition of cyclic AMP phosphodiesterase-4 promotes brain tumor regression, Clin. Cancer Res. 14 (23) (2008) 7717–7725. [9] D.H. Kim, A. Lerner, Type 4 cyclic adenosine monophosphate phosphodiesterase as a therapeutic target in chronic lymphocytic leukemia, Blood 92 (7) (1998) 2484–2494. [10] R. Ogawa, et al., Inhibition of PDE4 phosphodiesterase activity induces growth suppression: apoptosis, glucocorticoid sensitivity, p53, and p21(WAF1/CIP1) proteins in human acute lymphoblastic leukemia cells, Blood 99 (9) (2002) 3390–3397. [11] A.B. Burgin, et al., Design of phosphodiesterase 4D (PDE4D) allosteric modulators for enhancing cognition with improved safety, Nat. Biotechnol. 28 (1) (2010) 63–70. [12] M.D. Houslay, P. Schafer, K.Y.J. Zhang, Phosphodiesterase-4 as a therapeutic target, Drug Discov. Today 10 (22) (2005) 1503–1519. [13] O. Bruno, et al., New selective phosphodiesterase 4D inhibitors differently acting on long: short, and supershort isoforms, J. Med. Chem. 52 (21) (2009) 6546–6557. [14] C.H. Williams, et al., An in vivo chemical genetic screen identifies phosphodiesterase 4 as a pharmacological target for hedgehog signaling inhibition, Cell Rep. 11 (1) (2015) 43–50. [15] G.L. Powers, et al., Phosphodiesterase 4D inhibitors limit prostate cancer growth potential, Mol. Cancer Res. 13 (1) (2015) 149–160. [16] X. Ge, et al., Phosphodiesterase 4D acts downstream of Neuropilin to control Hedgehog signal transduction and the growth of medulloblastoma, Elife (2015) 4. [17] Tromondae K. Feaster, et al., Discovery of a novel pharmaceutical class as potential heart failure treatment, Circ. Res. 117 (2015) A66.