Journal of Chromatography B, 942 (2013) 77–82
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Pharmacokinetics and tissue distribution of a novel marine fibrinolytic compound in Wistar rat following intravenous administrations Tongwei Su a,1 , Wenhui Wu c,d,1 , Ting Yan a , Chaoyan Zhang a , Quangang Zhu b,∗ , Bin Bao d a
College of food Science and Technology, Shanghai Ocean University, Shanghai 201306, China Department of Pharmacy, Yueyang Hospital of Integrated Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China c Shanghai Engineering Research Center of Aquatic Product Processing & Preservation; Shanghai Ocean University, Institutes of Marine Science, Shanghai 201306, China d Institutes of Marine Science, Shanghai Ocean University, Shanghai 201306, China b
a r t i c l e
i n f o
Article history: Received 7 August 2013 Received in revised form 11 October 2013 Accepted 18 October 2013 Available online 25 October 2013 Keywords: Fibrinolytic compound Pharmacokinetic characters Tissue distribution HPLC
a b s t r a c t We investigated a novel marine fibrinolytic compound for use in thrombolytic therapy. Pharmacokinetics and the tissue distribution of this novel marine fibrinolytic compound, FGFC12 (fungi fibrinolytic compound 1), were investigated in Wistar rats after intravenous (IV) bolus administration of two concentrations (10 and 20 mg/kg). Plasma FGFC1 and tissue extracts were measured using HPLC with UV detection. FGFC1 was detected using a C18 column with a gradient eluted mobile phase of acetonitrile–water (0.1% trifluoroacetic acid), 1.0 mL/min. Chromatograms were monitored at 265 nm (column temperature: 40 ◦ C). Pharmacokinetic data indicate that FGFC1 fitted well to a twocompartment model. Elimination half-lives (t1/2 ) of FGFC1 were 21.51 ± 2.17 and 23.22 ± 2.11 min for 10 and 20 mg/kg, respectively. AUC0 - t were 412.19 ± 19.09, 899.09 ± 35.86 g/mL min, systemic clearance (CL) was 0.023 ± 0.002, 0.022 ± 0.002 ((mg/kg)/(g/mL)/min) and the mean residence time (MRT) was 10.15 ± 0.97, 9.65 ± 1.40 min at 10 and 20 mg/kg, respectively. No significant differences were observed in the systemic clearance and mean residence time at the tested doses, suggesting linear pharmacokinetics in rats. Tissue distribution data reveal that FGFC1 distributed rapidly in most tissues except the brain and that the highest concentration of the drug was in the liver. In the small intestine, FGFC1 initially increased and then declined, but remained comparatively high 60 min after administration, suggesting that enterohepatic circulation may exist © 2013 Published by Elsevier B.V.
1. Introduction Venous thromboembolic disease (pulmonary embolism and deep vein thrombosis) is a common cause of premature death and morbidity [1,2]. As emerging evidence accumulates about how to prevent thrombi formation, thrombolytic agents are extensively used in the clinic to mitigate thrombi that have already formed and are life-threatening [3]. Large studies have demonstrated the efficacy and safety of thrombolytic agents in the treatment of
∗ Corresponding author. 999 Huchenghuan Road, Pudong New District, Shanghai, China. Tel.: +86 021 6190 0388; fax: +86 021 6190 0364. E-mail addresses: [email protected] (Q. Zhu), [email protected] (B. Bao). 1 These authors contributed equally to this work. 2 FGFC1: Fungi fibrinolytic compound 1,2,5-bis-[8-(4,8-dimethyl-nona-3,7dienyl)-5,7-dihydroxy-8-methyl-3-keto-1,2,7,8-tertahydro-6H-pyran[a]isoindol2-yl]-pentanoic acid, a novel pyran-isoindolone derivative with bioactivity isolated from marine microorganism in our laboratory. 1570-0232/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jchromb.2013.10.031
deep vein thrombosis and pulmonary embolism as well as better short- and long-term clinical outcome as compared to conventional anticoagulation therapy [4–6]. Thrombolytic agents such as plasminogen activators catalyze the conversion of endogenous plasminogen to plasmin, which cleaves fibrin fibers within thrombi [7]. The thrombolytic agents streptokinase (SK), urokinase (UK), and tissue-type plasminogen activator (t-PA) [8,9] are chiefly used and all have some limitations and risks associated with their use. Aside from bleeding, SK therapy can lead to the formation of both humoral and cellular antibodies against the streptococcal origin of the thrombolytic agent. These antibodies can bind SK and significantly reduce its activity [10]. Also, urokinase is rapidly cleared, as is t-PA [11,12]. Thus, better thrombolytics derived from natural resources with predictable response and/or satisfactory pharmacokinetics in thrombolytic therapy are urgent needed. To address this need, effective thrombolytic agents have been identified and characterized from snake venom [13], the vampire bat [14], insects [15–17], the earthworm [18,19], marine green algae
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T. Su et al. / J. Chromatogr. B 942 (2013) 77–82
[20] and various microorganisms [21,22]. In a similar fashion, we are investigating novel thrombolytic drugs derived from fungi. FGFC1 (fungi fibrinolytic compound 1), 2,5-bis-[8-(4,8dimethyl-nona-3,7-dienyl)-5,7-dihydroxy-8-methyl-3-keto1,2,7,8-tertahydro-6H-pyran[a]isoindol-2-yl]-pentanoic acid, is a novel marine fibrinolytic compound (MW: 869) isolated from a fungal culture of Stachybotrys longispora (CCTCCM 2012272) [23]. We previously demonstrated fibrinolytic activity for this isolate through FITC-fibrin (fluorescein isothiocyanate-fibrin) degradation in a classical rat acute pulmonary embolism model [24]. To investigate FGFC1 as a potential thrombolytic agent, we should characterize the FGFC1 pharmacokinetics in animals. To this end, we developed an HPLC method for quantifying FGFC1 in rat plasma and tissues. High-performance liquid chromatographic technology was one of the main methods for quantifying drugs in plasma and tissues [25,26]. With the demand for proving the feasibility of the HPLC method, we verified the precision, accuracy, recovery, and stability of FGFC1 in vitro, and then applied the HPLC method to the pharmacokinetics and tissue distribution study in vivo. These data will serve as a foundation for preclinical investigations into potentially promising new thrombolytic compound. 2. Materials and methods 2.1. Chemicals and reagents FGFC1 (purity >98%), was extracted and purified in our laboratory. The minimal effective dose of FGFC1 is 2.5 mg/kg (LD50 = 250 mg/kg) in the rat. The structure of FGFC1 is displayed in Fig. 1. HPLC-grade acetonitrile was purchased from J&K Scientific Co., Ltd (Shanghai, China), and all other analytical grade reagents were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Ultrapure water was used throughout the study. 2.2. Instrument and HPLC conditions The HPLC system (Hitachi Corp., Tokyo, Japan) consisted of a Hitachi L-2130 pump, a Hitachi L-2200 auto-sampler, a Hitachi L2400 ultraviolet detector, and a Hitachi D-2000 HPLC workstation. The analytical column was a Sepax HP-C18 column (4.6 × 250 mm, 5 m; Sepax Technologies, Inc., Delaware, DE). Ultrapure water was prepared by Millipore Direct-Q 3 (Millipore Corp., Billerica, MA). The mobile phase was acetonitrile–water (0.1% trifluoroacetic acid) and the gradient was 45:55 (v/v) to 85:15 (v/v) over 30 min (1 mL/min). Chromatograms were monitored at 265 nm and the column temperature was maintained at 40 ◦ C.
2.3. Animals and ethical statement Female Wistar rats (SPF, 165 ± 10 g, 8 weeks-of-age) were provided by SIPPR-BK Experimental Animal Co., Ltd (Shanghai, China). All rats were fed with standard laboratory food and water ad libitum at least 5 days prior to the experiments in an environmentally controlled room (12 h dark–light cycle; GB14925-2001, ∼23 ◦ C, 60% humidity). The rats were housed separately. All protocols and procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Ocean University (Permit Number: 130012). The rats were sacrificed under ether anesthesia, and blood was collected from the fossa orbitalis vein. Every effort was made to minimize animal pain and suffering. 2.4. Experiments in vitro 2.4.1. Preparation of standard and quality control samples Stock FGFC1 solution was prepared by weighing and dissolving the compound in normal saline with NaHCO3 as a solvent (FGFC1/NaHCO3 , m/m, 1:1). The stock solution was serially diluted to 500, 200, 100, 50, 10, 1, and 0.5 g/mL. All solutions were extracted with methanol and filtered with a 0.22-m disposable syringe filter before HPLC detection. For method validation, quality control samples at three concentrations (2.8, 56, 560 g/mL) for plasma and tissue homogenates were prepared. For detection, the required volume of methanol was added to each sample which was centrifuged at 4000g for 15 min at 4 ◦ C. Then, the supernatant was collected and filtered through a 0.22-m disposable syringe filter and injected into the HPLC system. 2.4.2. Specificity Specificity was assessed by analyzing blank plasma and tissue homogenate samples, blank plasma and tissue homogenates samples spiked with FGFC1, and rat plasma and tissue homogenates samples after IV administration of FGFC1. All samples were extracted with methanol and centrifuged at 4000g for 15 min at 4 ◦ C. Then, supernatant was collected and filtered with a 0.22-m disposable syringe filter before HPLC detection. 2.4.3. Accuracy and precision Method accuracy was determined by calculating the percent deviation observed in the analysis of quality control samples and this is expressed by relative error. Intra-day precision was tested by analyzing quality control samples of plasma and tissue homogenates at three concentrations on the same day (n = 5). Interday precision was determined by repeated analysis quality control samples over three consecutive days (n = 15). All samples for this analysis were extracted with methanol and centrifuged at 4000g for 15 min at 4 ◦ C, and the supernatant was collected and filtered as described above prior to HPLC detection. 2.4.4. Recovery Recoveries were measured for high, medium, and low quality control samples. Extraction recovery was measured by comparing extracted FGFC1/corresponding standard FGFC1 peak area ratios obtained from extracted plasma samples and tissues homogenates. All samples were extracted with methanol and centrifuged at 4000g for 15 min at 4 ◦ C. Then supernatant was collected and filtered as described above prior to HPLC detection.
Fig. 1. Chemical structure of FGFC1.
2.4.5. Stability The stabilities were performed by evaluating small variations in three different conditions. All stability studies were assayed at three concentration levels. Short-term stability of FGFC1 in different quality control samples at room temperature (∼25 ◦ C) was
T. Su et al. / J. Chromatogr. B 942 (2013) 77–82
determined after 24 h. Long-term stability was studied by assaying samples following a period of 2 weeks of storage at −20 ◦ C. Freezethaw stability was evaluated at three consecutive freeze-thaw cycles (−20 ◦ C to room temperature). The results were expressed as the percentage of initial content of FGFC1 in the freshly treated samples. All samples were treated as described above and analyzed by HPLC.
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2.5.2. Data processing Data were collected and analyzed with Microsoft Excel 2010 and SPSS V17.0. Pharmacokinetic parameters and the compartment model were calculated using pharmacokinetics program software PKsolver V2.0 edited by China Pharmaceutical University (Nanjing, China) [27]. 3. Results and discussion
2.5. In vivo experiments
3.1. Method validation
2.5.1. Drug administration and sampling FGFC1 injection was prepared as follows: FGFC1 was weighed and dissolved in normal saline with NaHCO3 as a solvent (FGFC1/NaHCO3 , m/m, 1:1), and agitated overnight on a magnetic stirrer at room temperature. For the pharmacokinetic study, 10 rats were evenly and randomly divided into two groups. Then, FGFC1 was administered as an IV bolus via the caudal vein (10, 20 mg/kg, injection volume keep the same, ∼0.5 mL). Blood samples (100 L) were withdrawn from the fossa orbitalis vein at 1, 5, 10, 15, 30, 45, 60, 120 min after drug treatment, and immediately stored in 2 mL heparinized tubes with 900 L methanol. Samples were mixed and centrifuged at 4000g for 15 min, and supernatants were filtered with a 0.22-m disposable syringe filter and injected into the HPLC system for analysis. For the tissue distribution study, 15 rats were evenly and randomly divided into three groups. FGFC1 was administered as an IV bolus via the caudal vein (20 mg/kg). Then, the brain, heart, liver, spleen, lung, kidney, small intestine (emptied of gastric contents), stomach (emptied of gastric contents) were collected at 15, 30, and 60 min after drug administration. Tissues were flushed with normal saline, dried, weighed, and then homogenized in 5 mL methanol. Tissue samples were centrifuged at 4000g, and supernatants were filtered with a 0.22-m disposable syringe filter and injected into the HPLC system for analysis.
3.1.1. Specificity The degree of endogenous interference was assessed by inspection of chromatograms derived from blank samples, samples spiked with FGFC1 and samples after IV administration of FGFC1. In Fig. 2, typical chromatograms of blank plasma and liver samples and plasma and liver samples spiked with FGFC1 after organ removal, and rat plasma and liver samples after IV administration of FGFC1. Under the described HPLC conditions, FGFC1 was eluted at ∼29.0 min, and the FGFC1 target peak was identified by comparison with HPLC data from standard FGFC1 samples under the same HPLC conditions. There was no interfering peak at the FGFC1retention time. 3.1.2. Linearity of standard curve The lower limit of quantification (LLOQ) was defined as the lowest concentration at which both precision and accuracy were less than or equal to 20%, and this was
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Pharmacokinetics and tissue distribution of a novel marine fibrinolytic compound in Wistar rat following intravenous administrations Tongwei Su a,1 , Wenhui Wu c,d,1 , Ting Yan a , Chaoyan Zhang a , Quangang Zhu b,∗ , Bin Bao d a
College of food Science and Technology, Shanghai Ocean University, Shanghai 201306, China Department of Pharmacy, Yueyang Hospital of Integrated Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China c Shanghai Engineering Research Center of Aquatic Product Processing & Preservation; Shanghai Ocean University, Institutes of Marine Science, Shanghai 201306, China d Institutes of Marine Science, Shanghai Ocean University, Shanghai 201306, China b
a r t i c l e
i n f o
Article history: Received 7 August 2013 Received in revised form 11 October 2013 Accepted 18 October 2013 Available online 25 October 2013 Keywords: Fibrinolytic compound Pharmacokinetic characters Tissue distribution HPLC
a b s t r a c t We investigated a novel marine fibrinolytic compound for use in thrombolytic therapy. Pharmacokinetics and the tissue distribution of this novel marine fibrinolytic compound, FGFC12 (fungi fibrinolytic compound 1), were investigated in Wistar rats after intravenous (IV) bolus administration of two concentrations (10 and 20 mg/kg). Plasma FGFC1 and tissue extracts were measured using HPLC with UV detection. FGFC1 was detected using a C18 column with a gradient eluted mobile phase of acetonitrile–water (0.1% trifluoroacetic acid), 1.0 mL/min. Chromatograms were monitored at 265 nm (column temperature: 40 ◦ C). Pharmacokinetic data indicate that FGFC1 fitted well to a twocompartment model. Elimination half-lives (t1/2 ) of FGFC1 were 21.51 ± 2.17 and 23.22 ± 2.11 min for 10 and 20 mg/kg, respectively. AUC0 - t were 412.19 ± 19.09, 899.09 ± 35.86 g/mL min, systemic clearance (CL) was 0.023 ± 0.002, 0.022 ± 0.002 ((mg/kg)/(g/mL)/min) and the mean residence time (MRT) was 10.15 ± 0.97, 9.65 ± 1.40 min at 10 and 20 mg/kg, respectively. No significant differences were observed in the systemic clearance and mean residence time at the tested doses, suggesting linear pharmacokinetics in rats. Tissue distribution data reveal that FGFC1 distributed rapidly in most tissues except the brain and that the highest concentration of the drug was in the liver. In the small intestine, FGFC1 initially increased and then declined, but remained comparatively high 60 min after administration, suggesting that enterohepatic circulation may exist © 2013 Published by Elsevier B.V.
1. Introduction Venous thromboembolic disease (pulmonary embolism and deep vein thrombosis) is a common cause of premature death and morbidity [1,2]. As emerging evidence accumulates about how to prevent thrombi formation, thrombolytic agents are extensively used in the clinic to mitigate thrombi that have already formed and are life-threatening [3]. Large studies have demonstrated the efficacy and safety of thrombolytic agents in the treatment of
∗ Corresponding author. 999 Huchenghuan Road, Pudong New District, Shanghai, China. Tel.: +86 021 6190 0388; fax: +86 021 6190 0364. E-mail addresses: [email protected] (Q. Zhu), [email protected] (B. Bao). 1 These authors contributed equally to this work. 2 FGFC1: Fungi fibrinolytic compound 1,2,5-bis-[8-(4,8-dimethyl-nona-3,7dienyl)-5,7-dihydroxy-8-methyl-3-keto-1,2,7,8-tertahydro-6H-pyran[a]isoindol2-yl]-pentanoic acid, a novel pyran-isoindolone derivative with bioactivity isolated from marine microorganism in our laboratory. 1570-0232/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jchromb.2013.10.031
deep vein thrombosis and pulmonary embolism as well as better short- and long-term clinical outcome as compared to conventional anticoagulation therapy [4–6]. Thrombolytic agents such as plasminogen activators catalyze the conversion of endogenous plasminogen to plasmin, which cleaves fibrin fibers within thrombi [7]. The thrombolytic agents streptokinase (SK), urokinase (UK), and tissue-type plasminogen activator (t-PA) [8,9] are chiefly used and all have some limitations and risks associated with their use. Aside from bleeding, SK therapy can lead to the formation of both humoral and cellular antibodies against the streptococcal origin of the thrombolytic agent. These antibodies can bind SK and significantly reduce its activity [10]. Also, urokinase is rapidly cleared, as is t-PA [11,12]. Thus, better thrombolytics derived from natural resources with predictable response and/or satisfactory pharmacokinetics in thrombolytic therapy are urgent needed. To address this need, effective thrombolytic agents have been identified and characterized from snake venom [13], the vampire bat [14], insects [15–17], the earthworm [18,19], marine green algae
78
T. Su et al. / J. Chromatogr. B 942 (2013) 77–82
[20] and various microorganisms [21,22]. In a similar fashion, we are investigating novel thrombolytic drugs derived from fungi. FGFC1 (fungi fibrinolytic compound 1), 2,5-bis-[8-(4,8dimethyl-nona-3,7-dienyl)-5,7-dihydroxy-8-methyl-3-keto1,2,7,8-tertahydro-6H-pyran[a]isoindol-2-yl]-pentanoic acid, is a novel marine fibrinolytic compound (MW: 869) isolated from a fungal culture of Stachybotrys longispora (CCTCCM 2012272) [23]. We previously demonstrated fibrinolytic activity for this isolate through FITC-fibrin (fluorescein isothiocyanate-fibrin) degradation in a classical rat acute pulmonary embolism model [24]. To investigate FGFC1 as a potential thrombolytic agent, we should characterize the FGFC1 pharmacokinetics in animals. To this end, we developed an HPLC method for quantifying FGFC1 in rat plasma and tissues. High-performance liquid chromatographic technology was one of the main methods for quantifying drugs in plasma and tissues [25,26]. With the demand for proving the feasibility of the HPLC method, we verified the precision, accuracy, recovery, and stability of FGFC1 in vitro, and then applied the HPLC method to the pharmacokinetics and tissue distribution study in vivo. These data will serve as a foundation for preclinical investigations into potentially promising new thrombolytic compound. 2. Materials and methods 2.1. Chemicals and reagents FGFC1 (purity >98%), was extracted and purified in our laboratory. The minimal effective dose of FGFC1 is 2.5 mg/kg (LD50 = 250 mg/kg) in the rat. The structure of FGFC1 is displayed in Fig. 1. HPLC-grade acetonitrile was purchased from J&K Scientific Co., Ltd (Shanghai, China), and all other analytical grade reagents were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Ultrapure water was used throughout the study. 2.2. Instrument and HPLC conditions The HPLC system (Hitachi Corp., Tokyo, Japan) consisted of a Hitachi L-2130 pump, a Hitachi L-2200 auto-sampler, a Hitachi L2400 ultraviolet detector, and a Hitachi D-2000 HPLC workstation. The analytical column was a Sepax HP-C18 column (4.6 × 250 mm, 5 m; Sepax Technologies, Inc., Delaware, DE). Ultrapure water was prepared by Millipore Direct-Q 3 (Millipore Corp., Billerica, MA). The mobile phase was acetonitrile–water (0.1% trifluoroacetic acid) and the gradient was 45:55 (v/v) to 85:15 (v/v) over 30 min (1 mL/min). Chromatograms were monitored at 265 nm and the column temperature was maintained at 40 ◦ C.
2.3. Animals and ethical statement Female Wistar rats (SPF, 165 ± 10 g, 8 weeks-of-age) were provided by SIPPR-BK Experimental Animal Co., Ltd (Shanghai, China). All rats were fed with standard laboratory food and water ad libitum at least 5 days prior to the experiments in an environmentally controlled room (12 h dark–light cycle; GB14925-2001, ∼23 ◦ C, 60% humidity). The rats were housed separately. All protocols and procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Ocean University (Permit Number: 130012). The rats were sacrificed under ether anesthesia, and blood was collected from the fossa orbitalis vein. Every effort was made to minimize animal pain and suffering. 2.4. Experiments in vitro 2.4.1. Preparation of standard and quality control samples Stock FGFC1 solution was prepared by weighing and dissolving the compound in normal saline with NaHCO3 as a solvent (FGFC1/NaHCO3 , m/m, 1:1). The stock solution was serially diluted to 500, 200, 100, 50, 10, 1, and 0.5 g/mL. All solutions were extracted with methanol and filtered with a 0.22-m disposable syringe filter before HPLC detection. For method validation, quality control samples at three concentrations (2.8, 56, 560 g/mL) for plasma and tissue homogenates were prepared. For detection, the required volume of methanol was added to each sample which was centrifuged at 4000g for 15 min at 4 ◦ C. Then, the supernatant was collected and filtered through a 0.22-m disposable syringe filter and injected into the HPLC system. 2.4.2. Specificity Specificity was assessed by analyzing blank plasma and tissue homogenate samples, blank plasma and tissue homogenates samples spiked with FGFC1, and rat plasma and tissue homogenates samples after IV administration of FGFC1. All samples were extracted with methanol and centrifuged at 4000g for 15 min at 4 ◦ C. Then, supernatant was collected and filtered with a 0.22-m disposable syringe filter before HPLC detection. 2.4.3. Accuracy and precision Method accuracy was determined by calculating the percent deviation observed in the analysis of quality control samples and this is expressed by relative error. Intra-day precision was tested by analyzing quality control samples of plasma and tissue homogenates at three concentrations on the same day (n = 5). Interday precision was determined by repeated analysis quality control samples over three consecutive days (n = 15). All samples for this analysis were extracted with methanol and centrifuged at 4000g for 15 min at 4 ◦ C, and the supernatant was collected and filtered as described above prior to HPLC detection. 2.4.4. Recovery Recoveries were measured for high, medium, and low quality control samples. Extraction recovery was measured by comparing extracted FGFC1/corresponding standard FGFC1 peak area ratios obtained from extracted plasma samples and tissues homogenates. All samples were extracted with methanol and centrifuged at 4000g for 15 min at 4 ◦ C. Then supernatant was collected and filtered as described above prior to HPLC detection.
Fig. 1. Chemical structure of FGFC1.
2.4.5. Stability The stabilities were performed by evaluating small variations in three different conditions. All stability studies were assayed at three concentration levels. Short-term stability of FGFC1 in different quality control samples at room temperature (∼25 ◦ C) was
T. Su et al. / J. Chromatogr. B 942 (2013) 77–82
determined after 24 h. Long-term stability was studied by assaying samples following a period of 2 weeks of storage at −20 ◦ C. Freezethaw stability was evaluated at three consecutive freeze-thaw cycles (−20 ◦ C to room temperature). The results were expressed as the percentage of initial content of FGFC1 in the freshly treated samples. All samples were treated as described above and analyzed by HPLC.
79
2.5.2. Data processing Data were collected and analyzed with Microsoft Excel 2010 and SPSS V17.0. Pharmacokinetic parameters and the compartment model were calculated using pharmacokinetics program software PKsolver V2.0 edited by China Pharmaceutical University (Nanjing, China) [27]. 3. Results and discussion
2.5. In vivo experiments
3.1. Method validation
2.5.1. Drug administration and sampling FGFC1 injection was prepared as follows: FGFC1 was weighed and dissolved in normal saline with NaHCO3 as a solvent (FGFC1/NaHCO3 , m/m, 1:1), and agitated overnight on a magnetic stirrer at room temperature. For the pharmacokinetic study, 10 rats were evenly and randomly divided into two groups. Then, FGFC1 was administered as an IV bolus via the caudal vein (10, 20 mg/kg, injection volume keep the same, ∼0.5 mL). Blood samples (100 L) were withdrawn from the fossa orbitalis vein at 1, 5, 10, 15, 30, 45, 60, 120 min after drug treatment, and immediately stored in 2 mL heparinized tubes with 900 L methanol. Samples were mixed and centrifuged at 4000g for 15 min, and supernatants were filtered with a 0.22-m disposable syringe filter and injected into the HPLC system for analysis. For the tissue distribution study, 15 rats were evenly and randomly divided into three groups. FGFC1 was administered as an IV bolus via the caudal vein (20 mg/kg). Then, the brain, heart, liver, spleen, lung, kidney, small intestine (emptied of gastric contents), stomach (emptied of gastric contents) were collected at 15, 30, and 60 min after drug administration. Tissues were flushed with normal saline, dried, weighed, and then homogenized in 5 mL methanol. Tissue samples were centrifuged at 4000g, and supernatants were filtered with a 0.22-m disposable syringe filter and injected into the HPLC system for analysis.
3.1.1. Specificity The degree of endogenous interference was assessed by inspection of chromatograms derived from blank samples, samples spiked with FGFC1 and samples after IV administration of FGFC1. In Fig. 2, typical chromatograms of blank plasma and liver samples and plasma and liver samples spiked with FGFC1 after organ removal, and rat plasma and liver samples after IV administration of FGFC1. Under the described HPLC conditions, FGFC1 was eluted at ∼29.0 min, and the FGFC1 target peak was identified by comparison with HPLC data from standard FGFC1 samples under the same HPLC conditions. There was no interfering peak at the FGFC1retention time. 3.1.2. Linearity of standard curve The lower limit of quantification (LLOQ) was defined as the lowest concentration at which both precision and accuracy were less than or equal to 20%, and this was
