Original Article 647
Authors
T. K. Kim, Y. S. Ji, M. H. Park, C. H. Kim
Affiliation
College of Science & Engineering, Jungwon University, Chungbuk, South Korea
Key words ▶ SK3497 ● ▶ sildenafil ● ▶ bioavailability ● ▶ pharmacokinetics ● ▶ rats ●
Abstract
▼
In this study, a sensitive and reliable method for the quantitation of SK3497 in rat plasma was developed and validated using high performance liquid chromatography. The plasma samples were prepared by deproteinization, and sildenafil was used as an internal standard. Chromatographic separation was achieved using a reversed-phase (C18) column. The mobile phase, 0.02 M ammonium acetate buffer:acetonitrile (45:55, v/v), was run at a flow rate of 1.0 mL/min, and the column eluent was monitored using an
Introduction
▼
received 31.03.2014 accepted 09.05.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1376996 Published online: June 11, 2014 Drug Res 2014; 64: 647–650 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence T. K. Kim College of Science & Engineering Jungwon University 85 Munmu-ro Geosan-gun Chungbuk 367-700 South Korea Tel.: + 82/43/830 8619 Fax: + 82/43/830 8579 [email protected]
Male erectile dysfunction, the present inability to achieve or maintain an erection for satisfactory sexual performance, is a common and important medical problem [1]. The erectile dysfunction is known to be associated with various diseases, including diabetes, lower urinay tract symptoms, depression, hypertension, astherosclerosis, and cardiovascular diseases [2]. Recently, phosphodiesterase type 5 (PDE-5) inhibitors are used to improve erectile dysfunction by binding cyclic guanosine monophosphate and maintaining sufficient cellular levels in the smooth muscles [3, 4]. However, PDE-5 inhibitors have common adverse reactions such as headache, flushing, nasal congestion, and dyspepsia ▶ Fig. 1), a new PDE-5 inhibi[5]. Thus, SK3497 (● tor, was developed to alleviate drawbacks of above common side effects of PDE-5 inhibitors. SK3497 appears to be safe and effective in the treatment of male erectile dysfunction. The protein binding of SK3497 to 4 % HSA was independent of SK3497 concentrations ranging from 0.5 to 100 μg/mL; the mean binding value was 95.5 %. The purposes of this study were to develop and validate a sensitive and reliable high-performance liquid chromatographic (HPLC) method.
ultraviolet detector at 254 nm at room temperature. The retention times of sildenafil (an internal standard), and SK3497 were approximately 5.6 and 8.3 min, respectively. The detection limit of SK3497 in rat plasma was 0.03 μg/mL. Pharmacokinetic parameters of SK3497 was evaluated after intravenous (i. v.; at doses of 15 mg/kg) and oral (p.o.; at doses of 30 mg/kg) administration of SK3497 in rats. After p.o. administration (30 mg/ kg) of SK3497, F-value was approximately 53.0 %. The protein binding of SK3497 to 4 % human serum albumin were also described.
Based on the developed method, we determined the pharmacokinetic properties of SK3497 after intravenous (i. v.) injection of SK3497 at a dose of 15 mg/kg or oral (p.o.) administration of SK3497 at a dose of 30 mg/kg to rats to evaluate the absolute p.o. bioavailability (F) of SK3497 in rats.
Materials and Methods
▼
Materials SK3497 were supplied from SK Chemicals. Sildenafil, an internal standard for the high-performance liquid chromatographic (HPLC) analysis of SK3497, salicylic acid, and dextran (m.w. 65 000) were purchased from Sigma-Aldrich Corporation (St. Louis, MO). Acetonitrile and methanol were products from Burdick & Jackson (Muskegon, MI, USA). HSA (20 %) was obtained from SK Chemical Pharmaceutical Company (Seongnam, South Korea). Polyethylene glycol 400 (PEG 400) was a product from Showa Chemical Company (Tokyo, Japan). Other chemicals were of reagent grade or HPLC grade.
Animal experiments Male Sprague-Dawley rats, 6–8 week old and weighing 220–300 g, were purchased from the
Kim TK et al. Determination of SK3497 in Rat Plasma … Drug Res 2014; 64: 647–650
Downloaded by: NYU. Copyrighted material.
Determination of SK3497 in Rat Plasma and Its Application in a Pharmacokinetic Study of SK3497
648 Original Article
Preparation of calibration standards and quality control samples Stock solutions of SK3497 (1 mg/mL) were prepared in methanol. Appropriate dilutions of the stock solutions of SK3497 were made with methanol (0.003, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 mg/mL). Standard solutions of SK3497 in rat plasma were
prepared by spiking with an appropriate volume (10 μL/mL of plasma) of the diluted stock solutions, giving final concentrations of 0.03, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, or 10 μg/mL for plasma. The IS working solution was prepared by dissolving sildenafil in acetonitrile to give a final concentration of 10 μg/mL.
Preparation of plasma samples A 50-μL aliquot of sample was deproteinized with a 75-μL of acetonitrile containing 10 μg/mL sildenafil (an IS). After vortexmixing and centrifugation at 3 000 rpm for 10 min, the supernatant was transferred into a vial and a 20-μL aliquot was injected directly onto the HPLC column.
HPLC analysis The HPLC system consisted of a Gilson-234 autosampler (Gilson, Middleton, WI, USA), a Gilson 307 pump (Gilson), a Capcell PACK (C18) column (250 mm × 4.6 mm, i.d.; particle size, 5 μm; Shiseido, Tokyo, Japan), a model UV-118 UV/VIS detector (Gilson), and a model Gilson unipoint system software (Gilson). The mobile phase, 0.02 M ammonium acetate buffer:acetonitrile (45:55, v/v), was run at a flow rate of 1.0 mL/min, and the column eluent was monitored using an ultraviolet detector at 254 nm at room temperature. The retention times of sildenafil (an internal standard) and SK3497 were approximately 5.6 and 8.3 min, respectively.
Analytical method validation The analytical method was validated with regards to its specificity, linearity, intra- and interday precision and accuracy, matrix effect, and stability according to the US Food and Drug Administration’s “Guidance for Industry, Bioanalytical Method Validation, 2001 [6].”
Pharmacokinetic and statistical analyses SK3497
The total area under the plasma concentration-time curve to the last time (AUClast), the maximum plasma concentration (Cmax), the time to reach Cmax (Tmax), and the half-life (T1/2) were estimated using noncompartmental calculations carried out within
Sildenafil (IS)
Fig. 1 Chemical structures of SK3497 and sildenafil (an IS).
a
b
c
0.005 AUFS
Time (min)
Kim TK et al. Determination of SK3497 in Rat Plasma … Drug Res 2014; 64: 647–650
Fig. 2 Representative HPLC chromatograms after deproteinization of drug-free rat plasma a, drugfree rat plasma spiked with 0.03 μg/mL (LLOQ) of SK3497 and 10 μg/mL of IS b, plasma collected 4 h after oral administration of 30 mg/kg of SK3497 to a male Sprague-Dawley rat c. Peaks: (1): SK3497 (8.3 min); (2): IS (5.7 min). The detector’s sensitivity was set at 0.005 AUFS.
Downloaded by: NYU. Copyrighted material.
Samtako Bio Korea (Osan, South Korea). Rats were maintained in a Clean room at a temperature of between 23 ± 2 °C with 12-h light (07:00–19:00) and dark (19:00–07:00) cycles, and a relative humidity of 55 ± 5%. Rats were housed in metabolic cages (Tecniplast, Varese, Italy) under filtered pathogen-free air and with food (Sam Yang Company, Pyeongtaek, South Korea) and water available ad libitum. The rats were fasted overnight before drug administration and for 4 h after dosing. SK3497 was dissolved in PEG400:distilled water = 1:1 (v/v) to make a concentration of 5 mg/mL. The rats were placed in a restrainer and were intravenously and orally administered a dose of 15 and 30 mg/kg with a catheter, respectively. Blood was collected in a heparinized tube at the pre-dose stage, and at 0.083, 0.17, 0.33, 0.5, 0.75, 1, 2, 3, 4, 6, 8, and 12 h after i. v. administration and at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 12 h after p.o. administration. Plasma was harvested after centrifugation at 3 000 rpm and 4 °C for 10 min and stored frozen at − 70 °C until it was analyzed.
Original Article 649
Results
▼
Development and validation of the HPLC method Representative chromatograms of the deproteinized drug-free rat plasma, drug standards in rat plasma spiked with 0.03 μg/mL (LLOQ) of SK3497 and 10 μg/mL of sildenafil, an IS, and plasma collected 4 h after oral administration (plasma concentration is about 0.8 μg/mL) of 30 mg/kg of SK3497 to a male Sprague-Daw▶ Fig. 2. No interferences from endoley rat were shown in ● genous substances were observed in the blank rat plasma samples. The retention times of sildenafil and SK3497 were 5.7 and 8.3 min, respectively. The analytical method used was linear over the range of 0.03–10 μg/mL, with correlation coefficients (r values) greater than 0.9997. The lower limit of quantitation was 0.03 μg/mL with relative standard deviation (RSD) values less than 20 % and relative errors within ± 20 %. Intra- and inter-day accuracies (as relative error values) ranged between 0.3 % and 9.6 % and intra- and inter-day precision (as RSDs) were 1.8–9.3 % for all QC samples, with the result that they all met the criteria ▶ Table 1). SK3497 was found for bioanalysis method validation (● to be stable under various conditions, whether in the plasma or in the stock solutions. The recovery of SK3497 from the processed plasma was 97.1 % indicating that there is no significant matrix effects. In summary, the HPLC method developed in the current study was found to be suitable for the quantitation of SK3497 in rat plasma with acceptable specificity, linearity, accuracy, precision, and stability. On the basis of this HPLC method, SK3497 concentrations in rat plasma were determined.
Pharmacokinetics of SK3497 after intravenous (i. v.) or oral (p.o.) administration of SK3497 to rats For the i. v. administration of SK3497 at doses of 15 mg/kg to rats, the mean arterial plasma concentration-time profiles of SK3497 ▶ Fig. 3, and relevant pharmacokinetic parameters are shown in ● ▶ Table 2. After i. v. administration of SK3497, the are listed in ● plasma concentrations of SK3497 declined in polyexponential manners with a half-life of 2.89 h. The estimated total body clearance was 13.1 mL/min/kg. For the p.o. administration of SK3497 at a dose of 30 mg/kg to rats, the mean arterial plasma ▶ Fig. 3, concentration-time profiles of SK3497 are shown in ● and the relevant pharmacokinetic parameters are listed ▶ Table 2. After p.o. administration of SK3497, absorption of in ● SK3497 from the rat gastrointestinal tract was rapid; SK3497 was detected in plasma from the first or second blood sampling time (15 or 30 min) and rapidly reached Tmax (at 30 min). SK3497 was well absorbed, and showed that Cmax was 5.94 μg/mL and AUC0–12 was 20.15 μg · h/mL. Hence, the F-value at a SK3497 dose of 30 mg/kg was calculated to be about 53.0 %.
Table 1 Intra- and inter-day precision and accuracy for SK3497 in rat plasma QC samples. Nominal conc.
Measured conc.
Coefficient of
Relative error
(ng/mL)
(ng/mL)
variation ( %)
( %)
5.6 4.1 1.8 2.3
− 4.3 0.3 9.6 5.4
9.3 8.4 3.4 3.2
3.7 8.2 8.3 4.6
Intra-day (n = 6) 30 28.7 ± 1.6 100 100.3 ± 4.1 1 000 1 095.5 ± 20.2 7 500 7 905.4 ± 180.7 Inter-day (n = 18, 6 runs per day) 30 31.1 ± 2.9 100 108.2 ± 9.1 1 000 1 083.3 ± 36.8 7 500 7 847.6 ± 250.4 Data represent mean ± SD
Coefficient of variation ( %) = (SD/mean) × 100 Relative error ( %) = ((Measured conc. – Nominal conc.)/Nominal conc.) × 100
Fig. 3 Mean arterial plasma concentration-time profiles of SK3497 after i. v. (▲) injection of SK3497 at a dose of 15 mg/kg to rats and mean arterial plasma concentration-time profiles of SK3497 after oral (∆) administration of SK3497 at a dose of 30 mg/kg to rats. Bars represent S.D.
Table 2 Pharmacokinetic parameters of SK3497 after a single i. v. administration of SK3497 at a dose of 15 mg/kg and a single oral administration of SK3497 at a dose of 30 mg/kg to male rats. Parameters
Intravenous (n = 5)
Oral (n = 5)
dose (mg/kg) AUC0–12 (μg · h/mL) Cmax (μg/mL) Tmax (h) CL (mL/min/kg) T1/2 (h) F ( %)
15 19.02 ± 8.37 79.14 ± 36.40 0.08 ± 0.00 13.05 ± 5.17 – –
30 20.15 ± 8.87 5.94 ± 2.79 0.50 ± 0.31 – 1.84 ± 0.75 53.0
The values of kinetic parameters represent the mean ± SD (n = 5) AUC: Area under the curve to the collected time point (μg · h/mL) Cmax: Peak plasma concentration (μg/mL) Tmax: Time to reach peak plasma concentration (h) CL: Total body clearance (mL/min/kg) T1/2: Elimination half life (h) F: Fraction absorbed ( %)
Discussion
▼
The developed HPLC method is suitable for SK3497 quantification in plasma with acceptable specificity, linearity, accuracy, and precision. Based on this HPLC method, the SK3497 concen-
trations in rat plasma were determined and pharmacokinetic parameters were calculated. After i. v. administration of SK3497 at a dose of 15 mg/kg to rat, the CL value which was 13.1 mL/min/ kg based on plasma data was considerably smaller than the Kim TK et al. Determination of SK3497 in Rat Plasma … Drug Res 2014; 64: 647–650
Downloaded by: NYU. Copyrighted material.
WinNonlin™ 5.2 (Pharsight, Sunnyvale, CA, USA). The statistical significance was analyzed using Student’s t-tests carried out within SPSS (IBM, Yorktown Heights, NY, USA). A P-value of < 0.05 was considered statistically significant. All data are expressed as the mean ± standard deviation (SD).
650 Original Article
reported cardiac output of 295 mL/min/kg based on blood data [7] in rats. This suggests that the first-pass effect of SK3497 in the lung and heart could be almost negligible in rats. The F-value ▶ Table 2). After of SK3497 at a p.o. dose of 30 mg/kg was 53.0 % (● intraportal and i. v. administration of SK3497 at a dose of 15 mg/ kg to rats, the AUC of SK3497 after its i.p. administration was significantly smaller than that after its i. v. administration (unprecedented data), suggesting that the hepatic first-pass effect of SK3497 after its absorption into the portal was major in rats. The considerable hepatic first-pass effect of SK3497 is being tested based on the liver homogenate studies. In conclusion, after p.o. administration of SK3497 at a dose of 30 mg/kg to rats, the F-value was 53.0 % showing good bioavailability for entering preclinical studies. The major elimination pathway is thought to be hepatic and gastrointestinal first-pass effects.
References 1 Yoo HH, Kim NS, Im GJ et al. Pharmacokinetics and tissue distribution of a novel PDE5 inhibitor, SK-3530, in rats. Acta Pharmacol Sin 2007; 28: 1247–1253 2 Hatzimouratidis K, Hatzichristou DG. Looking to the future for erectile dysfunction therapies. Drugs 2008; 68: 231–250 3 Lincoln TM. CyClic GMP and phosphodiesterase 5 inhibitor therapies: what’s on the horizon. Mol Pharmacol 2004; 66: 776–784 4 Aversa A, Bruzziches R, Pili M et al. Phosphodiesterase 5 inhibitors in the treatment of erectile dysfunction. Curr Pharm Res 2006; 12: 3467–3484 5 Broderick GA. P.o. pharmacotherapy and the contemporary evaluation and management of erectile dysfunction. Rev Urol 2003; 5: S9–S20 6 http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070107.pdf 7 Davies B, Morris T. Physiological parameters in laboratory animals and humans. Pharm Res 1993; 10: 1009–1095
Conflict of Interest
▼
Downloaded by: NYU. Copyrighted material.
This research was sponsored by SK Chemicals (Seongnam, Korea).
Kim TK et al. Determination of SK3497 in Rat Plasma … Drug Res 2014; 64: 647–650
Authors
T. K. Kim, Y. S. Ji, M. H. Park, C. H. Kim
Affiliation
College of Science & Engineering, Jungwon University, Chungbuk, South Korea
Key words ▶ SK3497 ● ▶ sildenafil ● ▶ bioavailability ● ▶ pharmacokinetics ● ▶ rats ●
Abstract
▼
In this study, a sensitive and reliable method for the quantitation of SK3497 in rat plasma was developed and validated using high performance liquid chromatography. The plasma samples were prepared by deproteinization, and sildenafil was used as an internal standard. Chromatographic separation was achieved using a reversed-phase (C18) column. The mobile phase, 0.02 M ammonium acetate buffer:acetonitrile (45:55, v/v), was run at a flow rate of 1.0 mL/min, and the column eluent was monitored using an
Introduction
▼
received 31.03.2014 accepted 09.05.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1376996 Published online: June 11, 2014 Drug Res 2014; 64: 647–650 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence T. K. Kim College of Science & Engineering Jungwon University 85 Munmu-ro Geosan-gun Chungbuk 367-700 South Korea Tel.: + 82/43/830 8619 Fax: + 82/43/830 8579 [email protected]
Male erectile dysfunction, the present inability to achieve or maintain an erection for satisfactory sexual performance, is a common and important medical problem [1]. The erectile dysfunction is known to be associated with various diseases, including diabetes, lower urinay tract symptoms, depression, hypertension, astherosclerosis, and cardiovascular diseases [2]. Recently, phosphodiesterase type 5 (PDE-5) inhibitors are used to improve erectile dysfunction by binding cyclic guanosine monophosphate and maintaining sufficient cellular levels in the smooth muscles [3, 4]. However, PDE-5 inhibitors have common adverse reactions such as headache, flushing, nasal congestion, and dyspepsia ▶ Fig. 1), a new PDE-5 inhibi[5]. Thus, SK3497 (● tor, was developed to alleviate drawbacks of above common side effects of PDE-5 inhibitors. SK3497 appears to be safe and effective in the treatment of male erectile dysfunction. The protein binding of SK3497 to 4 % HSA was independent of SK3497 concentrations ranging from 0.5 to 100 μg/mL; the mean binding value was 95.5 %. The purposes of this study were to develop and validate a sensitive and reliable high-performance liquid chromatographic (HPLC) method.
ultraviolet detector at 254 nm at room temperature. The retention times of sildenafil (an internal standard), and SK3497 were approximately 5.6 and 8.3 min, respectively. The detection limit of SK3497 in rat plasma was 0.03 μg/mL. Pharmacokinetic parameters of SK3497 was evaluated after intravenous (i. v.; at doses of 15 mg/kg) and oral (p.o.; at doses of 30 mg/kg) administration of SK3497 in rats. After p.o. administration (30 mg/ kg) of SK3497, F-value was approximately 53.0 %. The protein binding of SK3497 to 4 % human serum albumin were also described.
Based on the developed method, we determined the pharmacokinetic properties of SK3497 after intravenous (i. v.) injection of SK3497 at a dose of 15 mg/kg or oral (p.o.) administration of SK3497 at a dose of 30 mg/kg to rats to evaluate the absolute p.o. bioavailability (F) of SK3497 in rats.
Materials and Methods
▼
Materials SK3497 were supplied from SK Chemicals. Sildenafil, an internal standard for the high-performance liquid chromatographic (HPLC) analysis of SK3497, salicylic acid, and dextran (m.w. 65 000) were purchased from Sigma-Aldrich Corporation (St. Louis, MO). Acetonitrile and methanol were products from Burdick & Jackson (Muskegon, MI, USA). HSA (20 %) was obtained from SK Chemical Pharmaceutical Company (Seongnam, South Korea). Polyethylene glycol 400 (PEG 400) was a product from Showa Chemical Company (Tokyo, Japan). Other chemicals were of reagent grade or HPLC grade.
Animal experiments Male Sprague-Dawley rats, 6–8 week old and weighing 220–300 g, were purchased from the
Kim TK et al. Determination of SK3497 in Rat Plasma … Drug Res 2014; 64: 647–650
Downloaded by: NYU. Copyrighted material.
Determination of SK3497 in Rat Plasma and Its Application in a Pharmacokinetic Study of SK3497
648 Original Article
Preparation of calibration standards and quality control samples Stock solutions of SK3497 (1 mg/mL) were prepared in methanol. Appropriate dilutions of the stock solutions of SK3497 were made with methanol (0.003, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 mg/mL). Standard solutions of SK3497 in rat plasma were
prepared by spiking with an appropriate volume (10 μL/mL of plasma) of the diluted stock solutions, giving final concentrations of 0.03, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, or 10 μg/mL for plasma. The IS working solution was prepared by dissolving sildenafil in acetonitrile to give a final concentration of 10 μg/mL.
Preparation of plasma samples A 50-μL aliquot of sample was deproteinized with a 75-μL of acetonitrile containing 10 μg/mL sildenafil (an IS). After vortexmixing and centrifugation at 3 000 rpm for 10 min, the supernatant was transferred into a vial and a 20-μL aliquot was injected directly onto the HPLC column.
HPLC analysis The HPLC system consisted of a Gilson-234 autosampler (Gilson, Middleton, WI, USA), a Gilson 307 pump (Gilson), a Capcell PACK (C18) column (250 mm × 4.6 mm, i.d.; particle size, 5 μm; Shiseido, Tokyo, Japan), a model UV-118 UV/VIS detector (Gilson), and a model Gilson unipoint system software (Gilson). The mobile phase, 0.02 M ammonium acetate buffer:acetonitrile (45:55, v/v), was run at a flow rate of 1.0 mL/min, and the column eluent was monitored using an ultraviolet detector at 254 nm at room temperature. The retention times of sildenafil (an internal standard) and SK3497 were approximately 5.6 and 8.3 min, respectively.
Analytical method validation The analytical method was validated with regards to its specificity, linearity, intra- and interday precision and accuracy, matrix effect, and stability according to the US Food and Drug Administration’s “Guidance for Industry, Bioanalytical Method Validation, 2001 [6].”
Pharmacokinetic and statistical analyses SK3497
The total area under the plasma concentration-time curve to the last time (AUClast), the maximum plasma concentration (Cmax), the time to reach Cmax (Tmax), and the half-life (T1/2) were estimated using noncompartmental calculations carried out within
Sildenafil (IS)
Fig. 1 Chemical structures of SK3497 and sildenafil (an IS).
a
b
c
0.005 AUFS
Time (min)
Kim TK et al. Determination of SK3497 in Rat Plasma … Drug Res 2014; 64: 647–650
Fig. 2 Representative HPLC chromatograms after deproteinization of drug-free rat plasma a, drugfree rat plasma spiked with 0.03 μg/mL (LLOQ) of SK3497 and 10 μg/mL of IS b, plasma collected 4 h after oral administration of 30 mg/kg of SK3497 to a male Sprague-Dawley rat c. Peaks: (1): SK3497 (8.3 min); (2): IS (5.7 min). The detector’s sensitivity was set at 0.005 AUFS.
Downloaded by: NYU. Copyrighted material.
Samtako Bio Korea (Osan, South Korea). Rats were maintained in a Clean room at a temperature of between 23 ± 2 °C with 12-h light (07:00–19:00) and dark (19:00–07:00) cycles, and a relative humidity of 55 ± 5%. Rats were housed in metabolic cages (Tecniplast, Varese, Italy) under filtered pathogen-free air and with food (Sam Yang Company, Pyeongtaek, South Korea) and water available ad libitum. The rats were fasted overnight before drug administration and for 4 h after dosing. SK3497 was dissolved in PEG400:distilled water = 1:1 (v/v) to make a concentration of 5 mg/mL. The rats were placed in a restrainer and were intravenously and orally administered a dose of 15 and 30 mg/kg with a catheter, respectively. Blood was collected in a heparinized tube at the pre-dose stage, and at 0.083, 0.17, 0.33, 0.5, 0.75, 1, 2, 3, 4, 6, 8, and 12 h after i. v. administration and at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 12 h after p.o. administration. Plasma was harvested after centrifugation at 3 000 rpm and 4 °C for 10 min and stored frozen at − 70 °C until it was analyzed.
Original Article 649
Results
▼
Development and validation of the HPLC method Representative chromatograms of the deproteinized drug-free rat plasma, drug standards in rat plasma spiked with 0.03 μg/mL (LLOQ) of SK3497 and 10 μg/mL of sildenafil, an IS, and plasma collected 4 h after oral administration (plasma concentration is about 0.8 μg/mL) of 30 mg/kg of SK3497 to a male Sprague-Daw▶ Fig. 2. No interferences from endoley rat were shown in ● genous substances were observed in the blank rat plasma samples. The retention times of sildenafil and SK3497 were 5.7 and 8.3 min, respectively. The analytical method used was linear over the range of 0.03–10 μg/mL, with correlation coefficients (r values) greater than 0.9997. The lower limit of quantitation was 0.03 μg/mL with relative standard deviation (RSD) values less than 20 % and relative errors within ± 20 %. Intra- and inter-day accuracies (as relative error values) ranged between 0.3 % and 9.6 % and intra- and inter-day precision (as RSDs) were 1.8–9.3 % for all QC samples, with the result that they all met the criteria ▶ Table 1). SK3497 was found for bioanalysis method validation (● to be stable under various conditions, whether in the plasma or in the stock solutions. The recovery of SK3497 from the processed plasma was 97.1 % indicating that there is no significant matrix effects. In summary, the HPLC method developed in the current study was found to be suitable for the quantitation of SK3497 in rat plasma with acceptable specificity, linearity, accuracy, precision, and stability. On the basis of this HPLC method, SK3497 concentrations in rat plasma were determined.
Pharmacokinetics of SK3497 after intravenous (i. v.) or oral (p.o.) administration of SK3497 to rats For the i. v. administration of SK3497 at doses of 15 mg/kg to rats, the mean arterial plasma concentration-time profiles of SK3497 ▶ Fig. 3, and relevant pharmacokinetic parameters are shown in ● ▶ Table 2. After i. v. administration of SK3497, the are listed in ● plasma concentrations of SK3497 declined in polyexponential manners with a half-life of 2.89 h. The estimated total body clearance was 13.1 mL/min/kg. For the p.o. administration of SK3497 at a dose of 30 mg/kg to rats, the mean arterial plasma ▶ Fig. 3, concentration-time profiles of SK3497 are shown in ● and the relevant pharmacokinetic parameters are listed ▶ Table 2. After p.o. administration of SK3497, absorption of in ● SK3497 from the rat gastrointestinal tract was rapid; SK3497 was detected in plasma from the first or second blood sampling time (15 or 30 min) and rapidly reached Tmax (at 30 min). SK3497 was well absorbed, and showed that Cmax was 5.94 μg/mL and AUC0–12 was 20.15 μg · h/mL. Hence, the F-value at a SK3497 dose of 30 mg/kg was calculated to be about 53.0 %.
Table 1 Intra- and inter-day precision and accuracy for SK3497 in rat plasma QC samples. Nominal conc.
Measured conc.
Coefficient of
Relative error
(ng/mL)
(ng/mL)
variation ( %)
( %)
5.6 4.1 1.8 2.3
− 4.3 0.3 9.6 5.4
9.3 8.4 3.4 3.2
3.7 8.2 8.3 4.6
Intra-day (n = 6) 30 28.7 ± 1.6 100 100.3 ± 4.1 1 000 1 095.5 ± 20.2 7 500 7 905.4 ± 180.7 Inter-day (n = 18, 6 runs per day) 30 31.1 ± 2.9 100 108.2 ± 9.1 1 000 1 083.3 ± 36.8 7 500 7 847.6 ± 250.4 Data represent mean ± SD
Coefficient of variation ( %) = (SD/mean) × 100 Relative error ( %) = ((Measured conc. – Nominal conc.)/Nominal conc.) × 100
Fig. 3 Mean arterial plasma concentration-time profiles of SK3497 after i. v. (▲) injection of SK3497 at a dose of 15 mg/kg to rats and mean arterial plasma concentration-time profiles of SK3497 after oral (∆) administration of SK3497 at a dose of 30 mg/kg to rats. Bars represent S.D.
Table 2 Pharmacokinetic parameters of SK3497 after a single i. v. administration of SK3497 at a dose of 15 mg/kg and a single oral administration of SK3497 at a dose of 30 mg/kg to male rats. Parameters
Intravenous (n = 5)
Oral (n = 5)
dose (mg/kg) AUC0–12 (μg · h/mL) Cmax (μg/mL) Tmax (h) CL (mL/min/kg) T1/2 (h) F ( %)
15 19.02 ± 8.37 79.14 ± 36.40 0.08 ± 0.00 13.05 ± 5.17 – –
30 20.15 ± 8.87 5.94 ± 2.79 0.50 ± 0.31 – 1.84 ± 0.75 53.0
The values of kinetic parameters represent the mean ± SD (n = 5) AUC: Area under the curve to the collected time point (μg · h/mL) Cmax: Peak plasma concentration (μg/mL) Tmax: Time to reach peak plasma concentration (h) CL: Total body clearance (mL/min/kg) T1/2: Elimination half life (h) F: Fraction absorbed ( %)
Discussion
▼
The developed HPLC method is suitable for SK3497 quantification in plasma with acceptable specificity, linearity, accuracy, and precision. Based on this HPLC method, the SK3497 concen-
trations in rat plasma were determined and pharmacokinetic parameters were calculated. After i. v. administration of SK3497 at a dose of 15 mg/kg to rat, the CL value which was 13.1 mL/min/ kg based on plasma data was considerably smaller than the Kim TK et al. Determination of SK3497 in Rat Plasma … Drug Res 2014; 64: 647–650
Downloaded by: NYU. Copyrighted material.
WinNonlin™ 5.2 (Pharsight, Sunnyvale, CA, USA). The statistical significance was analyzed using Student’s t-tests carried out within SPSS (IBM, Yorktown Heights, NY, USA). A P-value of < 0.05 was considered statistically significant. All data are expressed as the mean ± standard deviation (SD).
650 Original Article
reported cardiac output of 295 mL/min/kg based on blood data [7] in rats. This suggests that the first-pass effect of SK3497 in the lung and heart could be almost negligible in rats. The F-value ▶ Table 2). After of SK3497 at a p.o. dose of 30 mg/kg was 53.0 % (● intraportal and i. v. administration of SK3497 at a dose of 15 mg/ kg to rats, the AUC of SK3497 after its i.p. administration was significantly smaller than that after its i. v. administration (unprecedented data), suggesting that the hepatic first-pass effect of SK3497 after its absorption into the portal was major in rats. The considerable hepatic first-pass effect of SK3497 is being tested based on the liver homogenate studies. In conclusion, after p.o. administration of SK3497 at a dose of 30 mg/kg to rats, the F-value was 53.0 % showing good bioavailability for entering preclinical studies. The major elimination pathway is thought to be hepatic and gastrointestinal first-pass effects.
References 1 Yoo HH, Kim NS, Im GJ et al. Pharmacokinetics and tissue distribution of a novel PDE5 inhibitor, SK-3530, in rats. Acta Pharmacol Sin 2007; 28: 1247–1253 2 Hatzimouratidis K, Hatzichristou DG. Looking to the future for erectile dysfunction therapies. Drugs 2008; 68: 231–250 3 Lincoln TM. CyClic GMP and phosphodiesterase 5 inhibitor therapies: what’s on the horizon. Mol Pharmacol 2004; 66: 776–784 4 Aversa A, Bruzziches R, Pili M et al. Phosphodiesterase 5 inhibitors in the treatment of erectile dysfunction. Curr Pharm Res 2006; 12: 3467–3484 5 Broderick GA. P.o. pharmacotherapy and the contemporary evaluation and management of erectile dysfunction. Rev Urol 2003; 5: S9–S20 6 http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070107.pdf 7 Davies B, Morris T. Physiological parameters in laboratory animals and humans. Pharm Res 1993; 10: 1009–1095
Conflict of Interest
▼
Downloaded by: NYU. Copyrighted material.
This research was sponsored by SK Chemicals (Seongnam, Korea).
Kim TK et al. Determination of SK3497 in Rat Plasma … Drug Res 2014; 64: 647–650
