Arch. Pharm. Res. DOI 10.1007/s12272-014-0350-4
RESEARCH ARTICLE
Absolute bioavailability and metabolism of aceclofenac in rats Keumhan Noh • Beom Soo Shin • Kwang-il Kwon • Hwi-yeol Yun • Eunyoung Kim • Tae Cheon Jeong • Wonku Kang
Received: 10 December 2013 / Accepted: 27 January 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Aceclofenac is one of the most popular analgesic and anti-inflammatory drugs used for the relief of pain, rheumatoid arthritis, and osteoarthritis. To date, no intravenous preparation of aceclofenac has been developed because of its poor water solubility. In this study, to investigate its absolute bioavailability and metabolism in rats, aceclofenac was dissolved in a sterile aqueous solution containing urea (20 %) and trisodium citrate (10 %), and administered via oral (20 mg/kg) and intravenous (10 mg/kg) routes. Blood samples were taken serially, and aceclofenac and its three major metabolites (40 -hydroxydiclofenac, 40 -hydroxyaceclofenac, and diclofenac) were measured by HPLC–MS/MS. The absolute oral bioavailability of aceclofenac was approximately 15 %. Diclofenac and 40 -hydroxydiclofenac were the main metabolites in rats, in contrast to 40 -hydroxyaceclofenac in humans. The low bioavailability of aceclofenac is likely due to extensive
Keumhan Noh and Beom Soo Shin have equally contributed to this work. K. Noh T. C. Jeong (&) College of Pharmacy, Yeungnam University, Kyoungsan, Kyoungbuk 712-749, South Korea e-mail: [email protected] B. S. Shin College of Pharmacy, Catholic University of Daegu, Kyoungbuk 712-702, South Korea K. Kwon H. Yun College of Pharmacy, Chungnam National University, Taejon 305-764, South Korea E. Kim W. Kang (&) College of Pharmacy, Chung-Ang University, Seoul 156-756, South Korea e-mail: [email protected]
metabolism, and bioavailability may be even lower if the drug were administered as a tablet, considering its low water solubility. This study provides complete time profiles of the plasma concentrations of aceclofenac and its metabolites in rats and highlights the difference in drug metabolism between rats and humans. Keywords Aceclofenac Absolute bioavailability Metabolism Rat
Introduction Aceclofenac is one of the most popular analgesic and antiinflammatory drugs used for the relief of pain, rheumatoid arthritis, and osteoarthritis. A typical dose of 100 mg is given via the oral route twice a day. To date, an intravenous preparation of aceclofenac has not been developed because of its poor water solubility. However, Maheshwari and Indurkhya recently demonstrated enhanced water solubility of aceclofenac using a mixed hydrotropic solubilization technique (Maheshwari and Indurkhya 2010). Urea and sodium citrate were mixed at different combinations to dissolve aceclofenac in water, resulting in remarkably increased water solubility of aceclofenac, up to 50 mg/mL at injectable pHs (8–8.5). In humans, aceclofenac is dominantly metabolized to 40 -hydroxyaceclofenac via cytochrome P450 2C9 (CYP2C9) (Bort et al. 1996a). Diclofenac is generated by hydrolysis, but its plasma level is 100 times lower compared with that of the parent drug (Kang and Kim 2008). CYP2C9 also mediates the hydroxylation of diclofenac and the hydrolysis of 40 -hydroxyaceclofenac to yield 40 -hydroxydiclofenac (Bort et al. 1996a). In contrast, diclofenac is the major metabolite of aceclofenac in rats: the area
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under the plasma concentration–time curve (AUC) for diclofenac was 20 times that for aceclofenac, although the time course of the plasma aceclofenac concentration was not fully profiled owing to limited detection sensitivity. Neither 40 -hydroxyaceclofenac nor 40 -hydroxydiclofenac was detected in rat plasma, although the latter was detected in rat urine (Bort et al. 1996b). In the present work, we investigated the absolute bioavailability and metabolism of aceclofenac in rats, based on a sensitive determination method using tandem mass spectrometry (Bort et al. 1996a). The pharmacokinetic profiles of aceclofenac and its three major metabolites following oral and intravenous administration in rats were clarified.
a concentration of 20 mg/mL in a sterile aqueous solution containing urea (20 %) and trisodium citrate (10 %) (Maheshwari and Indurkhya 2010). After an overnight fast, heparinized blood (150 lL) was collected from the subclavicular vein prior to treatment. One group of rats was administered 10 mg/kg aceclofenac intravenously via the tail vein, and blood samples (150 lL) were collected at 2, 5, 15, 30, and 45 min, and 1, 2, 4, and 8 h afterward. The other group of rats was administered a single oral dose of 20 mg/kg aceclofenac, and blood samples (150 lL) were collected at 5, 15, and 30 min, and 1, 2, 4, 8, 10, and 12 h afterward. The plasma was separated by centrifugation of the samples at 12,000 rpm for 3 min and stored frozen until analyzed.
Methods
Measurement of aceclofenac and its three metabolites in rat plasma
Materials Aceclofenac, diclofenac, and 40 -hydroxydiclofenac were kindly donated by Novartis Pharma AG (Basel, Switzerland). Flufenamic acid (used as an internal standard), urea, and trisodium citrate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). HPLC-grade acetonitrile was purchased from Merck Co. (Darmstadt, Germany). Formulation of intravenous preparation Based on a previous report by Maheshwari and Indurkhya, we dissolved aceclofenac in a sterile aqueous solution containing urea (20 %) and trisodium citrate (10 %). Aceclofenac was prepared to a concentration of 20 mg/mL, and 1 mL/kg and 0.5 mL/kg were administered by oral and intravenous routes, respectively. Pharmacokinetic study Ten male Sprague–Dawley rats weighing 32–340 g were supplied by OrientBio, Ltd. (Suwon, Korea). The animal room was maintained at a temperature of 23 ± 3 °C, relative humidity of 50 ± 10 % with 10–20 air changes/h, and light intensity of 150–300 Lux with a 12-h light/dark cycle. This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Yeungnam University (Kyoungsan, Korea). All animals used in this study were cared for in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The rats were randomized into two groups for oral and intravenous administrations. Aceclofenac was dissolved to
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The plasma concentrations of aceclofenac and its three major metabolites (diclofenac, 40 -hydroxydiclofenac, and 40 -hydroxyaceclofenac) were quantified by high-performance liquid chromatography-tandem mass spectrometry using an API 4000 LC/MS/MS system (Sciex Division of MDS, Inc., Toronto, Canada) equipped with an electrospray ionization interface. As an internal standard, 60 lL of flufenamic acid in acetonitrile (10 ng/mL) were mixed with 20 lL of each plasma sample, followed by centrifugation at 13,200 rpm for 10 min. A 5-lL sample of the supernatant was injected onto a reversed-phase C18 column (Atlantis; internal diameter, 100 9 2.1 mm; particle size, 3 lm; Waters, Milford, MA, USA) and separated isocratically with a mobile phase consisting of acetonitrile/0.1 % formic acid(aq) (9:1, v/v). The column was heated to 40 °C, and the mobile phase was eluted at 0.3 mL/min using an HP 1100 series pump (Agilent, Wilmington, DE, USA). Quantitation was performed by selective reaction monitoring of the protonated precursor ion and the related product ion for aceclofenac and its three metabolites, using an internal standard method with peak area ratios. The mass transitions used for aceclofenac, 40 -hydroxyaceclofenac, diclofenac, 40 -hydroxydiclofenac, and flufenamic acid were m/z 352.9 ? 74.9, 368.9 ? 74.9, 296.1 ? 251.7, 311.8 ? 267.7, and 279.9 ? 235.9, respectively (Kang and Kim 2008). The coefficients of variation of the precision of the intra- and inter-day validation were less than 5.3 and 6.5 %, respectively. The accuracy of the method ranged from 93 to 110 %. The ion suppressions due to the matrix components were about 15, 18, and 48 % for aceclofenac, diclofenac, and 40 -OH-diclofenac, respectively. The limits of quantification were 2 ng/mL for aceclofenac and 0.2 ng/mL for diclofenac and 40 -OH-diclofenac at a signal-to-noise (S/N) ratio of 5.
Absolute bioavailability and metabolism
Data analysis Pharmacokinetic parameters were calculated by modelindependent analysis. The time courses of the plasma concentrations of aceclofenac and its three major metabolites were used to determine the maximum plasma concentration (Cmax) and time to reach Cmax of each compound (Tmax), and the volume of distribution (Vd) of aceclofenac. The elimination rate constant (ke) was obtained by linear regression of the terminal phase, and the elimination halflife (t1/2) was calculated as 0.693/ke. The area under the plasma concentration–time curve (AUClast) was calculated using the trapezoidal rule and extrapolated to infinity for AUC (AUCinf). Total clearance (Cl) was calculated as dose/ AUC. Absolute bioavailability was represented by the dose normalized AUCinf, po/AUCinf, iv.
Results Figure 1 shows the mean plasma concentration–time profiles of aceclofenac in rats after a single intravenous or oral administration. Table 1 presents the pharmacokinetic parameters of aceclofenac and its metabolites following a single intravenous dose of 10 mg/kg or oral dose of 20 mg/ kg. After intravenous administration, the plasma concentration of aceclofenac demonstrated three exponential decay components. The half-life of the terminal phase was approximately 3.2 ± 0.8 h, and the mean apparent volume of distribution and total clearance were 157.5 ± 25.5 mL/kg and 1.1 ± 0.2 L/h/kg, respectively. AUCinf was 9.23 ± 2.03 lg h/mL. Diclofenac and 40 -hydroxydiclofenac were rapidly generated and then disappeared bi-exponentially. The
Fig. 1 Time course for the plasma concentration of aceclofenac after a single intravenous (open circle 10 mg/kg) or oral (filled circle 20 mg/kg) administration in rats (n = 5)
systemic exposure of 40 -hydroxyaceclofenac was very weak (Fig. 2a). Following oral administration of 20 mg/kg aceclofenac, the plasma concentration of aceclofenac showed two peaks, one at 0.17 ± 0.06 h (4.6 ± 2.2 lg/mL) and the other at 4 h (*0.2 lg/mL), and it mono-exponentially decayed with a half-life of 1.92 ± 0.72 h. The AUCinf was 2.7 ± 0.7 lg h/mL, representing *15 % absolute bioavailability (BA). Total clearance (1.17 ± 0.35 L/h/kg), taking into account the absolute BA, was similar to that achieved with intravenous administration (Fig. 2b). The time courses of the plasma concentrations of diclofenac, 40 -hydroxydiclofenac, and 40 -hydroxyaceclofenac showed the same pattern as that of aceclofenac. The half-lives of diclofenac and 40 -hydroxydiclofenac were similar to that of the parent drug, whereas their AUCinf values were about 4.1-fold and 33.3-fold that of aceclofenac, respectively. The systemic exposure of 40 -hydroxyaceclofenac was only about one-fifth that of aceclofenac (Fig. 2b; Table 1).
Discussion Poor water solubility limits the development of intravenous preparations of some drugs, especially Biopharmaceutical Classification System class II (high permeable and low soluble) drugs such as aceclofenac. Many researchers have tried to improve the solubility of aceclofenac by using solubilizers and surfactants. Initially, we used different combinations of dimethylsulfoxide, polyethyleneglycol, ethanol, and sterile saline at tolerable concentrations for intravenous aceclofenac injection (data not shown). However, all mixtures failed to dissolve aceclofenac, even at doses as low as 1 mg/mL. Maheshwari and Indurkhya recently introduced an intravenous formulation of aceclofenac developed using the mixed hydrotropic solubilization technique (Maheshwari and Indurkhya 2010). With this methodology, the solubility of a solute is increased by the addition of high concentrations of alkaline metal salts of organic acids. Maheshwari and Indurkhya used two hydrotropic agents, urea and trisodium citrate, to reduce the concentration of each hydrotropic agent and minimize its undesirable effects. Urea, the main nitrogen-containing substance in the urine, is important in the metabolism of nitrogen-containing compounds and in the maintenance of plasma osmolarity. It is highly soluble in water and practically non-toxic: lethal doses in dogs and hamsters following intravenous administration were 3 and 4–8 g/kg, respectively (Abderhalden 1935). Sodium citrate is used mainly as a food additive, for flavor or as a preservative. Nevertheless, the safety of sodium citrate in humans may be a concern. Although sodium citrate was used at 0.5 g/kg body weight in college runners to improve running performance (Oo¨pik et al. 2003), recent
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K. Noh et al. Fig. 2 Time courses for the plasma concentrations of diclofenac (filled triangle), 40 hydroxydiclofenac (open triangle), and 40 hydroxyaceclofenac (open circle) after a single (a) intravenous (10 mg/kg) or (b) oral (20 mg/kg) administration of aceclofenac in rats (n = 5)
Table 1 Pharmacokinetic parameters of aceclofenac and its three metabolites in rat after single-dose oral (20 mg/kg) or intravenous (10 mg/kg) administration of aceclofenac (mean ± SD, n = 5) Parameter
Intravenous
Oral
Aceclofenac
Aceclofenac
Diclofenac
40 -Hydroxydiclofenac
40 -Hydroxyaceclofenac
AUCinf (lg h/mL)
9.23 ± 2.03
2.70 ± 0.66
11.18 ± 2.22
90.00 ± 128.18
0.54 ± 0.15
Cmax (lg/mL)
–
4.59 ± 2.21
10.09 ± 4.10
5.53 ± 2.21
0.25 ± 0.06
Tmax (h)
–
0.17 ± 0.06
0.33 ± 0.16
0.25 ± 0.00
0.32 ± 0.17
Vd (mL/kg)
157.5 ± 25.5
–
–
–
–
ke (1/h)a
0.24 ± 0.08
0.36 ± 0.14
0.33 ± 0.17
0.37 ± 0.14
0.21 ± 0.07
t1/2 (h)
3.24 ± 0.83
1.92 ± 0.72
2.10 ± 1.20
1.92 ± 0.65
3.34 ± 1.16
Cl (L/h/kg)
1.10 ± 0.24
1.17 ± 0.35b
–
–
–
F (%)
15
–
–
–
–
a
Elimination rate constant obtained from the terminal phase
b
Bioavailability was taken into account
in vitro results suggest that trisodium citrate catheter locking solution should not be used at concentrations greater than 10 % for safety reasons, as concentrations above 12 % caused plasma proteins to precipitate inside hemodialysis catheters (Schilcher et al. 2012). Using mixtures of 15 and 20 % trisodium citrate with 30 % urea, Maheshwari and Indurkhya achieved aceclofenac solubilities of 65.6 and 73.5 mg/mL, respectively. However, considering safety issues and the pH of the vehicle, we selected an aqueous mixture of 20 % urea and 10 % trisodium citrate to prepare aceclofenac at 20 mg/mL. To our knowledge, the present work represents the first report of the absolute bioavailability of aceclofenac and the
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complete time profiles of aceclofenac and its major metabolites in rats. Only about 15 % of the oral aceclofenac dose reached the systemic circulation, and this may be attributable to its extensive metabolism. As the drug was given by the oral route as well as by intravenous injection, its poor water solubility should not have affected its absorption in the gastrointestinal tract. However, when administered orally as a suspension or a tablet in clinical practice, aceclofenac may have much lower bioavailability than indicated by our results. The major metabolite of aceclofenac in humans is 40 -hydroxyaceclofenac, which has systemic exposure similar to that of the parent drug. In contrast, the metabolites
Absolute bioavailability and metabolism
diclofenac and 40 -hydroxydiclofenac were present in rat plasma at concentrations that were about 4- and 33-fold the aceclofenac concentration. The species difference in aceclofenac metabolism stems from significant differences between rats and humans with respect to the capacity of hepatocytes for oxidative and ester hydrolysis (Bort et al. 1996b). In a previous metabolism study of aceclofenac in rats (Bort et al. 1996a), determined that the AUC for diclofenac was 14-fold that for aceclofenac. The large difference between our finding (fourfold) and theirs may be caused by different sensitivities of the determination methods. Previously, HPLC with UV detection was used to determine aceclofenac concentrations; however, this method may not be sensitive enough to measure the time course of plasma aceclofenac concentrations and might have resulted in an underestimation of the AUC for aceclofenac. The second aceclofenac peak observed after oral administration in the present study might have originated from the enterohepatic circulation, which could have slightly, but not significantly, prolonged the half-life. In summary, we determined that the absolute oral bioavailability of aceclofenac was *15 %. This relatively low absorption of aceclofenac is likely the result of its extensive metabolism. The bioavailability of aceclofenac would be expected to be even lower if the drug were given as a tablet in clinical practice. This study also highlights the importance of considering species differences in drug metabolism between animals and humans.
Acknowledgments This research was supported by the Yeungnam University Research Grants in 2010. Conflict of interest
The authors declared no conflict of interest.
References Abderhalden, E. 1935. Handbuch der biologischen Arbeitsmethoden, vol. IV, 1353. Berlin: Urgan & Schwarzenberg. Bort, R., X. Ponsoda, E. Carrasco, M.J. Go´mez-Lecho´n, and J.V. Castell. 1996a. Comparative metabolism of the nonsteroidal antiinflammatory drug, aceclofenac, in the rat, monkey, and human. Drug Metabolism and Disposition 24: 834–841. Bort, R., X. Ponsoda, E. Carrasco, M.J. Gomez-Lecheon, and J.V. Castell. 1996b. Metabolism of aceclofenac in humans. Drug Metabolism and Disposition 24: 969–975. Kang, W., and E. Kim. 2008. Simultaneous determination of aceclofenac and its three metabolites in plasma using liquid chromatography-tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 46: 587–591. Maheshwari, R.K., and A. Indurkhya. 2010. Formulation and evaluation of aceclofenac injection made by mixed hydrotropic solubilization technique. Iranian Journal of Pharmaceutical Research 9: 233–242. Oo¨pik, V., I. Saaremets, L. Medijainen, K. Karelson, T. Janson, and S. Timpmann. 2003. Effects of sodium citrate ingestion before exercise on endurance performance in well trained college runners. British Journal of Sports Medicine 37: 485–489. Schilcher, G., H. Scharnagl, J.H. Horina, A.R. Rosenkranz, T. Stojakovic, and H. Polaschegg. 2012. Trisodium citrate induced protein precipitation in haemodialysis catheters might cause pulmonary embolism. Nephrology, Dialysis, Transplantation 27: 2953–2957.
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RESEARCH ARTICLE
Absolute bioavailability and metabolism of aceclofenac in rats Keumhan Noh • Beom Soo Shin • Kwang-il Kwon • Hwi-yeol Yun • Eunyoung Kim • Tae Cheon Jeong • Wonku Kang
Received: 10 December 2013 / Accepted: 27 January 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Aceclofenac is one of the most popular analgesic and anti-inflammatory drugs used for the relief of pain, rheumatoid arthritis, and osteoarthritis. To date, no intravenous preparation of aceclofenac has been developed because of its poor water solubility. In this study, to investigate its absolute bioavailability and metabolism in rats, aceclofenac was dissolved in a sterile aqueous solution containing urea (20 %) and trisodium citrate (10 %), and administered via oral (20 mg/kg) and intravenous (10 mg/kg) routes. Blood samples were taken serially, and aceclofenac and its three major metabolites (40 -hydroxydiclofenac, 40 -hydroxyaceclofenac, and diclofenac) were measured by HPLC–MS/MS. The absolute oral bioavailability of aceclofenac was approximately 15 %. Diclofenac and 40 -hydroxydiclofenac were the main metabolites in rats, in contrast to 40 -hydroxyaceclofenac in humans. The low bioavailability of aceclofenac is likely due to extensive
Keumhan Noh and Beom Soo Shin have equally contributed to this work. K. Noh T. C. Jeong (&) College of Pharmacy, Yeungnam University, Kyoungsan, Kyoungbuk 712-749, South Korea e-mail: [email protected] B. S. Shin College of Pharmacy, Catholic University of Daegu, Kyoungbuk 712-702, South Korea K. Kwon H. Yun College of Pharmacy, Chungnam National University, Taejon 305-764, South Korea E. Kim W. Kang (&) College of Pharmacy, Chung-Ang University, Seoul 156-756, South Korea e-mail: [email protected]
metabolism, and bioavailability may be even lower if the drug were administered as a tablet, considering its low water solubility. This study provides complete time profiles of the plasma concentrations of aceclofenac and its metabolites in rats and highlights the difference in drug metabolism between rats and humans. Keywords Aceclofenac Absolute bioavailability Metabolism Rat
Introduction Aceclofenac is one of the most popular analgesic and antiinflammatory drugs used for the relief of pain, rheumatoid arthritis, and osteoarthritis. A typical dose of 100 mg is given via the oral route twice a day. To date, an intravenous preparation of aceclofenac has not been developed because of its poor water solubility. However, Maheshwari and Indurkhya recently demonstrated enhanced water solubility of aceclofenac using a mixed hydrotropic solubilization technique (Maheshwari and Indurkhya 2010). Urea and sodium citrate were mixed at different combinations to dissolve aceclofenac in water, resulting in remarkably increased water solubility of aceclofenac, up to 50 mg/mL at injectable pHs (8–8.5). In humans, aceclofenac is dominantly metabolized to 40 -hydroxyaceclofenac via cytochrome P450 2C9 (CYP2C9) (Bort et al. 1996a). Diclofenac is generated by hydrolysis, but its plasma level is 100 times lower compared with that of the parent drug (Kang and Kim 2008). CYP2C9 also mediates the hydroxylation of diclofenac and the hydrolysis of 40 -hydroxyaceclofenac to yield 40 -hydroxydiclofenac (Bort et al. 1996a). In contrast, diclofenac is the major metabolite of aceclofenac in rats: the area
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under the plasma concentration–time curve (AUC) for diclofenac was 20 times that for aceclofenac, although the time course of the plasma aceclofenac concentration was not fully profiled owing to limited detection sensitivity. Neither 40 -hydroxyaceclofenac nor 40 -hydroxydiclofenac was detected in rat plasma, although the latter was detected in rat urine (Bort et al. 1996b). In the present work, we investigated the absolute bioavailability and metabolism of aceclofenac in rats, based on a sensitive determination method using tandem mass spectrometry (Bort et al. 1996a). The pharmacokinetic profiles of aceclofenac and its three major metabolites following oral and intravenous administration in rats were clarified.
a concentration of 20 mg/mL in a sterile aqueous solution containing urea (20 %) and trisodium citrate (10 %) (Maheshwari and Indurkhya 2010). After an overnight fast, heparinized blood (150 lL) was collected from the subclavicular vein prior to treatment. One group of rats was administered 10 mg/kg aceclofenac intravenously via the tail vein, and blood samples (150 lL) were collected at 2, 5, 15, 30, and 45 min, and 1, 2, 4, and 8 h afterward. The other group of rats was administered a single oral dose of 20 mg/kg aceclofenac, and blood samples (150 lL) were collected at 5, 15, and 30 min, and 1, 2, 4, 8, 10, and 12 h afterward. The plasma was separated by centrifugation of the samples at 12,000 rpm for 3 min and stored frozen until analyzed.
Methods
Measurement of aceclofenac and its three metabolites in rat plasma
Materials Aceclofenac, diclofenac, and 40 -hydroxydiclofenac were kindly donated by Novartis Pharma AG (Basel, Switzerland). Flufenamic acid (used as an internal standard), urea, and trisodium citrate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). HPLC-grade acetonitrile was purchased from Merck Co. (Darmstadt, Germany). Formulation of intravenous preparation Based on a previous report by Maheshwari and Indurkhya, we dissolved aceclofenac in a sterile aqueous solution containing urea (20 %) and trisodium citrate (10 %). Aceclofenac was prepared to a concentration of 20 mg/mL, and 1 mL/kg and 0.5 mL/kg were administered by oral and intravenous routes, respectively. Pharmacokinetic study Ten male Sprague–Dawley rats weighing 32–340 g were supplied by OrientBio, Ltd. (Suwon, Korea). The animal room was maintained at a temperature of 23 ± 3 °C, relative humidity of 50 ± 10 % with 10–20 air changes/h, and light intensity of 150–300 Lux with a 12-h light/dark cycle. This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Yeungnam University (Kyoungsan, Korea). All animals used in this study were cared for in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The rats were randomized into two groups for oral and intravenous administrations. Aceclofenac was dissolved to
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The plasma concentrations of aceclofenac and its three major metabolites (diclofenac, 40 -hydroxydiclofenac, and 40 -hydroxyaceclofenac) were quantified by high-performance liquid chromatography-tandem mass spectrometry using an API 4000 LC/MS/MS system (Sciex Division of MDS, Inc., Toronto, Canada) equipped with an electrospray ionization interface. As an internal standard, 60 lL of flufenamic acid in acetonitrile (10 ng/mL) were mixed with 20 lL of each plasma sample, followed by centrifugation at 13,200 rpm for 10 min. A 5-lL sample of the supernatant was injected onto a reversed-phase C18 column (Atlantis; internal diameter, 100 9 2.1 mm; particle size, 3 lm; Waters, Milford, MA, USA) and separated isocratically with a mobile phase consisting of acetonitrile/0.1 % formic acid(aq) (9:1, v/v). The column was heated to 40 °C, and the mobile phase was eluted at 0.3 mL/min using an HP 1100 series pump (Agilent, Wilmington, DE, USA). Quantitation was performed by selective reaction monitoring of the protonated precursor ion and the related product ion for aceclofenac and its three metabolites, using an internal standard method with peak area ratios. The mass transitions used for aceclofenac, 40 -hydroxyaceclofenac, diclofenac, 40 -hydroxydiclofenac, and flufenamic acid were m/z 352.9 ? 74.9, 368.9 ? 74.9, 296.1 ? 251.7, 311.8 ? 267.7, and 279.9 ? 235.9, respectively (Kang and Kim 2008). The coefficients of variation of the precision of the intra- and inter-day validation were less than 5.3 and 6.5 %, respectively. The accuracy of the method ranged from 93 to 110 %. The ion suppressions due to the matrix components were about 15, 18, and 48 % for aceclofenac, diclofenac, and 40 -OH-diclofenac, respectively. The limits of quantification were 2 ng/mL for aceclofenac and 0.2 ng/mL for diclofenac and 40 -OH-diclofenac at a signal-to-noise (S/N) ratio of 5.
Absolute bioavailability and metabolism
Data analysis Pharmacokinetic parameters were calculated by modelindependent analysis. The time courses of the plasma concentrations of aceclofenac and its three major metabolites were used to determine the maximum plasma concentration (Cmax) and time to reach Cmax of each compound (Tmax), and the volume of distribution (Vd) of aceclofenac. The elimination rate constant (ke) was obtained by linear regression of the terminal phase, and the elimination halflife (t1/2) was calculated as 0.693/ke. The area under the plasma concentration–time curve (AUClast) was calculated using the trapezoidal rule and extrapolated to infinity for AUC (AUCinf). Total clearance (Cl) was calculated as dose/ AUC. Absolute bioavailability was represented by the dose normalized AUCinf, po/AUCinf, iv.
Results Figure 1 shows the mean plasma concentration–time profiles of aceclofenac in rats after a single intravenous or oral administration. Table 1 presents the pharmacokinetic parameters of aceclofenac and its metabolites following a single intravenous dose of 10 mg/kg or oral dose of 20 mg/ kg. After intravenous administration, the plasma concentration of aceclofenac demonstrated three exponential decay components. The half-life of the terminal phase was approximately 3.2 ± 0.8 h, and the mean apparent volume of distribution and total clearance were 157.5 ± 25.5 mL/kg and 1.1 ± 0.2 L/h/kg, respectively. AUCinf was 9.23 ± 2.03 lg h/mL. Diclofenac and 40 -hydroxydiclofenac were rapidly generated and then disappeared bi-exponentially. The
Fig. 1 Time course for the plasma concentration of aceclofenac after a single intravenous (open circle 10 mg/kg) or oral (filled circle 20 mg/kg) administration in rats (n = 5)
systemic exposure of 40 -hydroxyaceclofenac was very weak (Fig. 2a). Following oral administration of 20 mg/kg aceclofenac, the plasma concentration of aceclofenac showed two peaks, one at 0.17 ± 0.06 h (4.6 ± 2.2 lg/mL) and the other at 4 h (*0.2 lg/mL), and it mono-exponentially decayed with a half-life of 1.92 ± 0.72 h. The AUCinf was 2.7 ± 0.7 lg h/mL, representing *15 % absolute bioavailability (BA). Total clearance (1.17 ± 0.35 L/h/kg), taking into account the absolute BA, was similar to that achieved with intravenous administration (Fig. 2b). The time courses of the plasma concentrations of diclofenac, 40 -hydroxydiclofenac, and 40 -hydroxyaceclofenac showed the same pattern as that of aceclofenac. The half-lives of diclofenac and 40 -hydroxydiclofenac were similar to that of the parent drug, whereas their AUCinf values were about 4.1-fold and 33.3-fold that of aceclofenac, respectively. The systemic exposure of 40 -hydroxyaceclofenac was only about one-fifth that of aceclofenac (Fig. 2b; Table 1).
Discussion Poor water solubility limits the development of intravenous preparations of some drugs, especially Biopharmaceutical Classification System class II (high permeable and low soluble) drugs such as aceclofenac. Many researchers have tried to improve the solubility of aceclofenac by using solubilizers and surfactants. Initially, we used different combinations of dimethylsulfoxide, polyethyleneglycol, ethanol, and sterile saline at tolerable concentrations for intravenous aceclofenac injection (data not shown). However, all mixtures failed to dissolve aceclofenac, even at doses as low as 1 mg/mL. Maheshwari and Indurkhya recently introduced an intravenous formulation of aceclofenac developed using the mixed hydrotropic solubilization technique (Maheshwari and Indurkhya 2010). With this methodology, the solubility of a solute is increased by the addition of high concentrations of alkaline metal salts of organic acids. Maheshwari and Indurkhya used two hydrotropic agents, urea and trisodium citrate, to reduce the concentration of each hydrotropic agent and minimize its undesirable effects. Urea, the main nitrogen-containing substance in the urine, is important in the metabolism of nitrogen-containing compounds and in the maintenance of plasma osmolarity. It is highly soluble in water and practically non-toxic: lethal doses in dogs and hamsters following intravenous administration were 3 and 4–8 g/kg, respectively (Abderhalden 1935). Sodium citrate is used mainly as a food additive, for flavor or as a preservative. Nevertheless, the safety of sodium citrate in humans may be a concern. Although sodium citrate was used at 0.5 g/kg body weight in college runners to improve running performance (Oo¨pik et al. 2003), recent
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K. Noh et al. Fig. 2 Time courses for the plasma concentrations of diclofenac (filled triangle), 40 hydroxydiclofenac (open triangle), and 40 hydroxyaceclofenac (open circle) after a single (a) intravenous (10 mg/kg) or (b) oral (20 mg/kg) administration of aceclofenac in rats (n = 5)
Table 1 Pharmacokinetic parameters of aceclofenac and its three metabolites in rat after single-dose oral (20 mg/kg) or intravenous (10 mg/kg) administration of aceclofenac (mean ± SD, n = 5) Parameter
Intravenous
Oral
Aceclofenac
Aceclofenac
Diclofenac
40 -Hydroxydiclofenac
40 -Hydroxyaceclofenac
AUCinf (lg h/mL)
9.23 ± 2.03
2.70 ± 0.66
11.18 ± 2.22
90.00 ± 128.18
0.54 ± 0.15
Cmax (lg/mL)
–
4.59 ± 2.21
10.09 ± 4.10
5.53 ± 2.21
0.25 ± 0.06
Tmax (h)
–
0.17 ± 0.06
0.33 ± 0.16
0.25 ± 0.00
0.32 ± 0.17
Vd (mL/kg)
157.5 ± 25.5
–
–
–
–
ke (1/h)a
0.24 ± 0.08
0.36 ± 0.14
0.33 ± 0.17
0.37 ± 0.14
0.21 ± 0.07
t1/2 (h)
3.24 ± 0.83
1.92 ± 0.72
2.10 ± 1.20
1.92 ± 0.65
3.34 ± 1.16
Cl (L/h/kg)
1.10 ± 0.24
1.17 ± 0.35b
–
–
–
F (%)
15
–
–
–
–
a
Elimination rate constant obtained from the terminal phase
b
Bioavailability was taken into account
in vitro results suggest that trisodium citrate catheter locking solution should not be used at concentrations greater than 10 % for safety reasons, as concentrations above 12 % caused plasma proteins to precipitate inside hemodialysis catheters (Schilcher et al. 2012). Using mixtures of 15 and 20 % trisodium citrate with 30 % urea, Maheshwari and Indurkhya achieved aceclofenac solubilities of 65.6 and 73.5 mg/mL, respectively. However, considering safety issues and the pH of the vehicle, we selected an aqueous mixture of 20 % urea and 10 % trisodium citrate to prepare aceclofenac at 20 mg/mL. To our knowledge, the present work represents the first report of the absolute bioavailability of aceclofenac and the
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complete time profiles of aceclofenac and its major metabolites in rats. Only about 15 % of the oral aceclofenac dose reached the systemic circulation, and this may be attributable to its extensive metabolism. As the drug was given by the oral route as well as by intravenous injection, its poor water solubility should not have affected its absorption in the gastrointestinal tract. However, when administered orally as a suspension or a tablet in clinical practice, aceclofenac may have much lower bioavailability than indicated by our results. The major metabolite of aceclofenac in humans is 40 -hydroxyaceclofenac, which has systemic exposure similar to that of the parent drug. In contrast, the metabolites
Absolute bioavailability and metabolism
diclofenac and 40 -hydroxydiclofenac were present in rat plasma at concentrations that were about 4- and 33-fold the aceclofenac concentration. The species difference in aceclofenac metabolism stems from significant differences between rats and humans with respect to the capacity of hepatocytes for oxidative and ester hydrolysis (Bort et al. 1996b). In a previous metabolism study of aceclofenac in rats (Bort et al. 1996a), determined that the AUC for diclofenac was 14-fold that for aceclofenac. The large difference between our finding (fourfold) and theirs may be caused by different sensitivities of the determination methods. Previously, HPLC with UV detection was used to determine aceclofenac concentrations; however, this method may not be sensitive enough to measure the time course of plasma aceclofenac concentrations and might have resulted in an underestimation of the AUC for aceclofenac. The second aceclofenac peak observed after oral administration in the present study might have originated from the enterohepatic circulation, which could have slightly, but not significantly, prolonged the half-life. In summary, we determined that the absolute oral bioavailability of aceclofenac was *15 %. This relatively low absorption of aceclofenac is likely the result of its extensive metabolism. The bioavailability of aceclofenac would be expected to be even lower if the drug were given as a tablet in clinical practice. This study also highlights the importance of considering species differences in drug metabolism between animals and humans.
Acknowledgments This research was supported by the Yeungnam University Research Grants in 2010. Conflict of interest
The authors declared no conflict of interest.
References Abderhalden, E. 1935. Handbuch der biologischen Arbeitsmethoden, vol. IV, 1353. Berlin: Urgan & Schwarzenberg. Bort, R., X. Ponsoda, E. Carrasco, M.J. Go´mez-Lecho´n, and J.V. Castell. 1996a. Comparative metabolism of the nonsteroidal antiinflammatory drug, aceclofenac, in the rat, monkey, and human. Drug Metabolism and Disposition 24: 834–841. Bort, R., X. Ponsoda, E. Carrasco, M.J. Gomez-Lecheon, and J.V. Castell. 1996b. Metabolism of aceclofenac in humans. Drug Metabolism and Disposition 24: 969–975. Kang, W., and E. Kim. 2008. Simultaneous determination of aceclofenac and its three metabolites in plasma using liquid chromatography-tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 46: 587–591. Maheshwari, R.K., and A. Indurkhya. 2010. Formulation and evaluation of aceclofenac injection made by mixed hydrotropic solubilization technique. Iranian Journal of Pharmaceutical Research 9: 233–242. Oo¨pik, V., I. Saaremets, L. Medijainen, K. Karelson, T. Janson, and S. Timpmann. 2003. Effects of sodium citrate ingestion before exercise on endurance performance in well trained college runners. British Journal of Sports Medicine 37: 485–489. Schilcher, G., H. Scharnagl, J.H. Horina, A.R. Rosenkranz, T. Stojakovic, and H. Polaschegg. 2012. Trisodium citrate induced protein precipitation in haemodialysis catheters might cause pulmonary embolism. Nephrology, Dialysis, Transplantation 27: 2953–2957.
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