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Effects of Age on the Pharmacokinetics of Piroxicam in Rats
Effects of Age on the Pharmacokinetics of Piroxicam in Rats SALLY
G.BOUDINOT, ERICD. FUNDERBURG, AND F. DOUGLASBOUDINOT~
Received October 15, 1991, from the Department of Pharmaceutics, College of Pharmacy, University of Georgia, Athens, GA 30602. Accepted for publication July 15, 1992. This study examined the effects of age on the pharmacokinetics of piroxicam in rats. Two groups of rats, aged 5 and 24 months, were administered 1 mg of piroxicam per kg intravenously, and blood samples were withdrawn for up to 120 h. Protein binding studies, with pooled serum from each age group were also performed. Piroxicam concentrationswere determined by HPLC analysis, and pharmacokinetic parameters were' characterized by area-moment analysis. Plasma piroxicam concentrations declined in both age groups in a biexponential fashion,with half-lives of 5.9 2 0.7 h (mean ? SD) in the young rats and 30.6 f 9.9 h in the old rats. Total clearance in the young rats was 0.048 f 0.012 Uh/kg, whereas that in the old rats was 0.021 f 0.003 Uhlkg. The steady-statevolume of distributionin the young rats was 0.42 2 0.05 Ukg, and that in the old rats was 0.56 f 0.10 Ukg. There was a statistically significant difference between these parameters calculated for each age group. Piroxicam is a highly plasma protein-bounddrug; the fraction unbound in the young rats was determined to be 0.067 f 0.022, and that in the old rats was determined to be 0.134 f 0.065, or twice that in the young rats. Differences in protein binding were due, in part, to a 20% decreased albumin concentration in the old rats; however, there was also a decrease in the number of binding sites and/or the binding affinity with aging. Clearance of free drug in the young rats was 0.73 2 0.18 Uhikg, and that in the old rats was 0.15 ? 0.02 Uh/kg; the steady-state volumes of distribution for unbound drug were determined to be 6.27 & 0.78 Ukg for the young rats and 4.18 f 0.75 Ukg for the old rats. These differences were significantly different. Thus, piroxicam exhibited an age-related disposition in rats. Abstract
Piroxicam, a nonsteroidal antiinflammatory drug (NSAID), was the third most widely prescribed antiarthritis drug in 1990.1 The long half-life and once-a-day dosing of this drug make it quite acceptable in terms of patient compliance.2 Piroxicam is used extensively in the elderly, in part because of the convenient dosing. Concern has surfaced, however, with regard to the safety of piroxicam, particularly in the elderly.3 Piroxicam has been associated with such side effects as renal disease and electrolyte disturbances,G gastrointestinal ulcers and blood loss,7-9 and aplastic anemia.8 These side effects appear to be age related, with an increased incidence in the elderly.2 The increased occurrence of adverse reactions in the elderly may be due to alterations in the disposition of the drug in this population of patients; however, results of clinical studies have been somewhat conflicting.2 Patients in some studies were on concurrent medications, and other factors may have contributed to the lack of a definitive relationship between pharmacokinetic changes and aging. Woolf et al.10 found no correlation between age and the half-life and systemic clearance of piroxicam, but the majority of the geriatric patients participating in the study had a concurrent illness, such as cardiac failure, diabetes, Parkinson's disease, myxedema, and urinary tract infection. Protein binding determinations were not made, and protein binding has been shown to have a profound effect on the pharmacokinetics of NSAIDs.11 Richardson et al.12 found a n increase in half-life among elderly females compared with young females but only a slight increase in half-life among older men compared with young men. A subsequent article by Verbeeck et al.13 ac254 I Journal of Pharmaceutical Sciences Vol. 82,No. 3, March 1993
knowledged a great variation in elimination kinetics between individuals but found little variation within a n individual. In a study by Blocka et al.,14 who examined the effects of age on the disposition of piroxicam in patients with rheumatoid arthritis, the total clearance of piroxicam increased with the age of the patients, whereas the total volume of distribution at steady state decreased with age. The results of that study showed no change in the unbound fraction of piroxicam, despite decline in albumin concentration in the elderly, possibly as a result of the change in albumin composition found in patients with rheumatoid arthritis. All clinical studies used the oral route of administration of piroxicam, and the effect of bioavailability was not assessed. Animal models, particularly rats, have often been used to study the pharmacokinetics of NSAIDS.lS-20Fischer 344 rats have been studied as a model for aging, and their aging process closely resembles that of hurnans.21-22 Roskos and Boudinotlg examined the effect of dose on the pharmamkinetics of piroxicam in rats and established that drug disposition was independent of dose over the range of 0.5-5 mgkg. The results of that study also indicated that rats represented a useful laboratory animal model for investigating piroxicam disposition. The purpose of this study was to assess the effects of age on the pharmacokinetics of piroxicam in rats, with the elimination of concomitant factors that could possible affect the results. In addition, piroxicam was administered intravenously to better examine the effects of age on drug distribution and elimination without the confounding factor of bioavailability .
Experimental Section Animals-Male Fischer 344 rats were obtained from National Institute on Aging colonies (HarlanSprague Dawley, Inc., Indianapolis, IN). Young adult rats of 5 months of age and weighing 320.5 2 30.5 g (mean f SD) and senescent rats of 24 months of age and weighing407.5 2 18.5 gwere selected for the study. Both groups were housed identically, in a controlled-temperature (22 "C) and 12-h light-12-h dark-cycle environment. During the 1-week acclimation period, food (Purina Lab Chow 5001; Ralston Purina Co., St. Louis, MO) and water were given ad libitum. On the day before the study, the rats were subjected to light ether anesthesia, during which right external vein cannulas were inserted. The animals were subjected to fasting for 16 h before drug administration,and food was provided 12 h after drug administration. Experimental Design-Six youngand six old rats weregiven 1 mg of piroxicam (Sigma Chemical Co., St. Louis, MO)per kg intravenously via the cannulas, over 60 a. The drug solution was prepared by dissolving piroxicam in 0.2 M carbonate in physiologic saline. Blood samples (0.25 mL) were collected before and at 0.08,0.25, 0.5, 0.75, 1,2,3,4,6,8,12,24,30,36,48,54,60,72,84,96,108, and 120 h after drug administrationvia the cannulas into heparinized tubes, and the blood volume was replaced with saline. Preliminary investigations demonstrated a lack of difference in the dispositionof piroxicam when blood volume was replaced with whole blood rather than saline. Additionally,there was no observed decline in the hematocrit during the course of the study. Previous studies showed no binding of the drug to the cannula.19 Blood samples were centrifuged, and the plasma was frozen at -20 "C until analysis. 0022-3549/93/300-02~$02.50/0 0 1993,American Pharmaceutical Association
Protein Binding-Serum was obtained from piroxicam-free young and old rats and pooled for protein binding studies. Known amounts of piroxicam were added to serum samples to yield drug concentrations ranging from 1-100 jg/mL. Equilibrium dialysis was performed with acrylic plastic dialysis cells and Spectrapor I1 cellulose dialysis membranes (Spectrum Medical Industries, New York, Nn with a molecular weight cutoff of 12 000-14 000 as previously described.19 Serum samples of 0.5 mL were equilibrated for 16 h at 37 “C with gentle shaking against 0.5 mL of isotonic sodium phosphate buffer (pH 7.4). Postdialysis sample volumes were measured, and samples were frozen until analysis. Data were corrected for fluid shifts that occurred during dialysis.23 Procedures were performed in triplicate. Piroxicam Assay-Piroxicam concentrations were assayed by use of the slightly modified%&nique of Boudinot and b r a h i m a with HPLC. In brief, 0.1-mL plasma samples were prepared with the addition of 0.1 mL of the internal standard S’-methylpiroxicam (Pfner Corp., Gmton, CT) and 0.2 mL of 1.0 M phosphate buffer (pH 2.0). Methylene chloride (5mL) was added, and samples were shaken at 120cycleslminfor 10min and then centrifuged at 2000 x g for 10 min. The aqueous layer was aspirated, and methylene chloride was evaporated under a stream of nitrogen gas. Samples were reconstituted with 0.4 mL of methanol phosphate buffer (pH 8) (4060). Samples ranging in volume from 0.02-0.2 mL were injected into the HPLC column for piroxicam concentration determinations. The chromatographic assay was done with a CIS reversed-phase column [4.6 mm (inner diameter) X 25 cm; 5 - p particle size; Alltech Associates, Deerfield, IL]. A model M45 solvent delivery system, a model 710B WISP automatic injedor, and a model 481 W detector set at 360 nm (allfrom Waters Associates, Milford, MA) and a model 3390A reporting integrator (Hewlett-Packard, Avondale, PA) completed the HPLC system. The mobile phase consisted of 40% methanol-60% phosphate buffer (pH €9, and a flow rate of 1.0 mWmin was used. Piroxicam concentrations were determined from the slopes of calibration plots of the peak area ratios of phxicam/6’-methylpiroxicam versus standard piroxicam concentrations. The slopes were determined with a weighted linear least-squares regression analysis. Reciprocal peak area ratios (lly) used as weighting factors for the regression analysis generated a normal distribution of residuals around the fitted calibration line.25 Calibration curves were linear in the range of 10-50,OOO ng/mL, and the limit of quantitation was 10 ng/mL. Coefficients of variation for intraday and interday precision were <8%, and the accuracy was >97%. 5’-Hydroxypiroxicam,a major metabolite of piroxicam, can also be quantitated by this assay methodology. Protein Binding Analysis-The fraction of the drug unbound or free in plasma was calculated as D,JDt,where Df and D,represent the concentrations of unbound drug and total drug, respectively. Drug binding was linear over the concentration range studied; therefore, the concentration of bound drug as a function of the concentration of free drug can be described by the equation D, = nKPDf, where n is the number of binding sites, P is the protein (albumin)concentration, and K is the equilibrium association constant. Pooled serum samples from young and old rats were analyzed for total protein and albumin concentrations by a n autoanalyzer technique (Technicon, Tarrytown, NY).Both sides of the equation D, = nKPD, were divided by the measured albumin concentration to normalize for the decline in protein concentration with aging, yielding the equation DJP = nKDp The data were graphed to determine the slope, nK, for each age group by linear regression, assuming that there was error in both variables.26 Pharmacokinetic Analysis-Pharmacokinetic parameters were determined using aredmoment analysis. Area under the plasma concentration-time curve (AUC) and the area under the first moment curve (AUMC) from time zero to the last sample time point were calculated by Lagrange polynomial interpolation and integration,27,28with extrapolation to time infinity by use of the nonlinear least-squares29 terminal slope (Az). The half-life was calculated as 0.693/AZ.Total clearance was determined as dose/AUC. Mean residence time was calculated as AUMC/AUC. The total volume of distribution at steady state was determined as total clearance x mean residence time. The clearance and steady-state volume of distribution of unbound drug were calculated by dividing the total clearance and total volume of distribution, respectively, by the fraction of unbound drug. Statistical Analysis-Statistical analysis of the effect of age on pharmacokinetic parameters was performed by use of a t test, with a p value of <0.05 being statistically significant.
Results and Discussion Several clinical studies have assessed the effects of age on the pharmacokinetics of piroxicarn2JOJ2-14; however, the conclusions have been somewhat conflicting. Differences in study design, failure to assess plasma protein binding, patient selection, and oral drug administration have produced variable and inconclusive results. The present investigation was undertaken with Fischer 344 rats as an animal model of age to factor out age from numerous other variables that can influence drug disposition. Piroxicam was administered intravenously to eliminate uncertainties due to bioavailability. The results of this study render insight into the effects of age on the pharmacokinetics of piroxicam, without the implication of other confounding factors. Representative plasma piroxicam concentration-time profiles for young and old rats are shown in Figure 1.The plasma piroxicam concentrations observed in rats in this study were comparable to those seen clinically.2JO Plasma piroxicam concentrations after intravenous injection to both young and old rats appeared to decline in a biexponential fashion. The age of the rats had a profound effect on the pharmacokinetics of piroxicam, as shown in Table I. The mean half-life in old rats was five times longer than that in young rats, the AUC was double that in young rats, and the mean residence time was about three times that in young rats. Total clearance of piroxicam in old rats was lower than that in yov.ng rats, whereas the volume of distribution at steady state ; i i old rats was larger than that in young rats. All pharmacokinetic
E
\
gl
10.000
u
t
x 0
0.1’
0
rr
24
48
72
120
96
TIME, h
a
Figure 1-Representative plasma piroxicam concentration-time cuwes for young (0)and old (0)rats after intravenous administration of 1 mg of piroxicam per kg. Table I-Pharmacoklnetlc Parameters after Single-Dose Intravenous Administration of Piroxicam (1 .O mg/kg) to Rats.
Parameterb t1,m
h
AUC, mg . h/L CL, Uh/kg MRT, h vss, Ukg CL,, Uhlkg Vssr Ukg
Mean (SD) for Six Young Rats 5.85(0.68) 21.43(5.06) 0.0489(0.0120) 8.84(1.50) 0.419 (0.045) 0.730 (0.181) 6.27(0.68)
Old Rats 30.59 (9.86) 48.50(6.03) 0.0209(0.0025) 26.87 (3.79) 0.560(0.100) 0.155 (0.018) 4.18(0.75)
a All pharmacokinetic parameters were statistically significantlydifferent (p < 0.05)for young versus old rats. t,,2, Half-life; CL, total clearance; MRT, mean residence time; V,,, volume of distribution at steady state; CL, clearance of free drug; V,, Vssof free drug.
Journal of Pharmaceutical Sciences I 255 Vol, 82, No. 3, March 1993
Table Il-Plasma Protein Blndlng Parameters for Plroxlcam In Rats Value for: Parameter Young Rats Old Rats ~~
nK, M-'
Albumin, M a
0.067 2.785 X lo4 5.0 x 10-4
,
0.134 1.63 x lo-4 3.97 x 10
fu, Fraction of unbound drug.
% a
FREE DRUG, pU
3 AGE, months Flgure 3-Pharmacokinetic parameters of piroxicam as a function of age. (A) Total piroxicam clearance (0)and free piroxicam clearance (0) in young and old rats. (6)Total piroxicam volume of distributionat steady state (0)and free piroxicam volume of distribution at steady state (0)for both age groups. Horizontal lines indicate mean values.
10
20
FREE DRUG, Flgure 2-Effects of age on the plasma protein binding of piroxicam in rats. (A) Protein-bound piroxicam concentration as a function of free piroxicam concentration in young (0)and old (0)rats. (B) Bound drug concentration,normalized for albumin concentration,as afunction of free drug concentration. Lines represent regression lines characterizing the relationships. parameters were found to be statistically significantly different for young versus old rats. Protein binding studies revealed marked differences between the two age groups. Table I1 shows that the fraction of unbound piroxicam doubled in old rats, thus yielding higher levels of the pharmacologically active species. Figure 2A shows the data and regression line describing bound drug concentration as a function of free drug concentration in young and old rats. The slope of this line was nKP. The albumin concentration declined 20% in old rats. Similar results were reported for rat@ and elderly humans.30J1 Normalization for protein concentration, as shown in Figure 2B, yielded a line whose slope was nK,calculated as previously described. Table I1 summarizes these findings and suggests that the changes in protein binding that occur with aging are due not only to a decline in albumin concentration but also to a change in nK. Previous studies assessing the effects of age on the protein binding of NSAIDS in rats 256 1 Journal of Pharmaceutical Sciences Vol. 82,No. 3, March 1993
suggested a decline in the affinity of the binding sites with age rather than a change in the number of binding sites.20 Piroxicam is eliminated almost exclusively by hepatic metabolism, so total clearance is essentially hepatic clearance. As shown in Figure 3A, total clearance in young rats was two times lower than that in old rats, whereas the clearance of free drug in young rats was three times higher than that in old rats. Since piroxicam is a low-clearance drug,lg the decline in hepatic blood flow due to aging was not a factor in the decline in clearance. The large difference in the clearance of free piroxicam was most likely due to a decline in liver function because of aging. The differences in total clearance between young and old rats were smaller than those in the clearance of free drug because of the twofold increase in the free fraction of piroxicam in old rats. The volume of distribution of free drug in old rats, as shown in Figure 3B, was smaller than that in young rats. This result can be attributed to the lower lean body mass, decline in total body water, altered regional blood flow, and decrease in extracellular albumin concentration that accompany aging.22 The total volume of distribution at steady state, however, showed a slight, yet statistically significant, increase in old rats because of the larger free fraction of the drug in the plasma of old rats. The relatively limited steady-state volume of distribution of piroxicam was typical of the high degree of plasma protein binding and distribution into extracellular fluid noted for NSAIDs.11932 In conclusion, age has significant effects on the pharmacokinetics of piroxicam in rats. The changes in plasma protein binding and the effects of altered protein binding are especially significant. Although the total clearance and volume of distribution were altered in old rats, the changes in the
clearance and volume of distribution of free drug were even more significant. It is the unbound or free fraction of the drug that is most altered by aging and that is available to produce both pharmacologic action and adverse effects.
References and Notes 1. Glaser, M. Drug Top. 1991,135,53-56. 2. Hobbs, D. C. Am. J. Med. 1986,81,Suppl. 5B, 22-27. 3. Lamy, P.P.Md. Pharm. 1986,62,18. 4. Mitnick, P.D.;Klein, W. J. Arch. Intern. Med. 1986,144,6344. 5. Miller, K. P.;Lazar, E. J.;Fotino, S.Arch. Intern. Med. 1984,144, 2414-2415. 6. Frais, M. A.; Burgess, E. D.; Mitchell, L. B. Ann. Intern. Med. 1983.99.129-130. 7. Emery, P.; Grahame, R. Lancet 1982,i , 1302-1303. 8. Gerber, D. Drug Intell. Clin. Phann. 1987,21,707-710. 9. Fok, K. H.; George, P. J. M.; Vicary, F. R. Br. Med. J. 1985,290, 117. 10. Woolf, A.D.; Rogers, H. J.; Bradbrook, I. D.; Corless, D. Br. J. Clin. Pharmacol. 1983.16.433-437. 11. Lin, H.;Cocchetto, D.M.;'Duggan, D. E. Clin. Pharmacokinet. 1987,12,402432. 12. Richardson, C. J.; Blocka, K. L.; Ross, R. T.; Verbeeck, R. K. Clin. Pharmacol. Ther. 1985,37,13-18. 13. Verbeeck, R. K.; Richardson, C. J.; Blocka, K. L. J. Rhuematol., 1986.13. , ~ .789-796. , .. 14. Blocka, K.L.; Richardson, C. J.; Wallace, S.M.; Ross, S. G.; Verbeeck, R. K. J. Rhuematol. 1988,15,757-763. 15. Sattenvhite, J. H.; Boudinot, F. D. Drug Metab. Dispos. 1990,19, 61-67.
16. Foster, R. T.;Jamali, F. Drug Metab. Dispos. 1988,16,623-626. 17. Runkel, R.; Chaplin, M.; Boost, G.; Segre, E.; Forchielli, E. J. Pharm. Sci. 1972,61,703-708. 18. Iwakawa, S.;Xiwen, H.; Hashimoto, S.; Volland, C.; Benet, L. Z.; Lin, E.T.Drug Metab. Dispos. 1991,19,717-718. 19. Roskos, L.K.;Boudinot, F. D. Biopharm. Drug Dispos. 1990,11, 215-225. 20. Satterwhite, J. H.;Boudinot, F. D. J. Gerontot. 1991,46,B222B227. 21. Hazzard, D.G.; Soban, J. Ezp. Aging Res. 1988,14,59-81. 22. Coleman, G. L.; Barthold, S. W.; Osbaldiston, G. V.; Foster, S. J.; Jonas, A.M. J. Gerontol. 1977,32,258-278. 23. Boudinot, F. D.; Jusko, W. J. J. Phurm. Sci. 1984,73, 774-780. 24. Boudinot, F. D.; Ibrahim, S. S. J. Chromatogr. 1988,430,424428. 25. Bonate, P.L.LC-GC 1992,10,448-450. 26. Riggs, D. S.;Guarnieri, J. A.; Addelman, S. Life Sci. 1978,22, 1305-1360. 27. Yeh, K.C.; Kwan, K. C . J. Pharmacokinet. Biopharm. 1978,6, 79-98. 28. Rocci, M. L.; Jusko, W. J. Comput. Programs Biomed. 1983,16, 203-216. 29. Metzler, C.M.; Elfring, G. L.; McEwen, A. J.Biometrics 1974,30, 562-563. 30. Bender, A.D.;Post, A.; Meier, J. P.; Higson, J. P.; Reichard, G., Jr. J. Pharm. Scd. 1975,64,1711-1713. 31. Wallace, S.; Verbeeck, R. C. Clin. Pharmacokinet. 1987, 12, 41-72. 32. Verbeeck, R. K.;Blackburn, J. L.; Loewen, G. R. Clin. Pharmucokinet. 1983,8,297-331.
Journal of Pharmaceutical Sciences I 257 Vol. 82, No. 3, March 1993
G.BOUDINOT, ERICD. FUNDERBURG, AND F. DOUGLASBOUDINOT~
Received October 15, 1991, from the Department of Pharmaceutics, College of Pharmacy, University of Georgia, Athens, GA 30602. Accepted for publication July 15, 1992. This study examined the effects of age on the pharmacokinetics of piroxicam in rats. Two groups of rats, aged 5 and 24 months, were administered 1 mg of piroxicam per kg intravenously, and blood samples were withdrawn for up to 120 h. Protein binding studies, with pooled serum from each age group were also performed. Piroxicam concentrationswere determined by HPLC analysis, and pharmacokinetic parameters were' characterized by area-moment analysis. Plasma piroxicam concentrations declined in both age groups in a biexponential fashion,with half-lives of 5.9 2 0.7 h (mean ? SD) in the young rats and 30.6 f 9.9 h in the old rats. Total clearance in the young rats was 0.048 f 0.012 Uh/kg, whereas that in the old rats was 0.021 f 0.003 Uhlkg. The steady-statevolume of distributionin the young rats was 0.42 2 0.05 Ukg, and that in the old rats was 0.56 f 0.10 Ukg. There was a statistically significant difference between these parameters calculated for each age group. Piroxicam is a highly plasma protein-bounddrug; the fraction unbound in the young rats was determined to be 0.067 f 0.022, and that in the old rats was determined to be 0.134 f 0.065, or twice that in the young rats. Differences in protein binding were due, in part, to a 20% decreased albumin concentration in the old rats; however, there was also a decrease in the number of binding sites and/or the binding affinity with aging. Clearance of free drug in the young rats was 0.73 2 0.18 Uhikg, and that in the old rats was 0.15 ? 0.02 Uh/kg; the steady-state volumes of distribution for unbound drug were determined to be 6.27 & 0.78 Ukg for the young rats and 4.18 f 0.75 Ukg for the old rats. These differences were significantly different. Thus, piroxicam exhibited an age-related disposition in rats. Abstract
Piroxicam, a nonsteroidal antiinflammatory drug (NSAID), was the third most widely prescribed antiarthritis drug in 1990.1 The long half-life and once-a-day dosing of this drug make it quite acceptable in terms of patient compliance.2 Piroxicam is used extensively in the elderly, in part because of the convenient dosing. Concern has surfaced, however, with regard to the safety of piroxicam, particularly in the elderly.3 Piroxicam has been associated with such side effects as renal disease and electrolyte disturbances,G gastrointestinal ulcers and blood loss,7-9 and aplastic anemia.8 These side effects appear to be age related, with an increased incidence in the elderly.2 The increased occurrence of adverse reactions in the elderly may be due to alterations in the disposition of the drug in this population of patients; however, results of clinical studies have been somewhat conflicting.2 Patients in some studies were on concurrent medications, and other factors may have contributed to the lack of a definitive relationship between pharmacokinetic changes and aging. Woolf et al.10 found no correlation between age and the half-life and systemic clearance of piroxicam, but the majority of the geriatric patients participating in the study had a concurrent illness, such as cardiac failure, diabetes, Parkinson's disease, myxedema, and urinary tract infection. Protein binding determinations were not made, and protein binding has been shown to have a profound effect on the pharmacokinetics of NSAIDs.11 Richardson et al.12 found a n increase in half-life among elderly females compared with young females but only a slight increase in half-life among older men compared with young men. A subsequent article by Verbeeck et al.13 ac254 I Journal of Pharmaceutical Sciences Vol. 82,No. 3, March 1993
knowledged a great variation in elimination kinetics between individuals but found little variation within a n individual. In a study by Blocka et al.,14 who examined the effects of age on the disposition of piroxicam in patients with rheumatoid arthritis, the total clearance of piroxicam increased with the age of the patients, whereas the total volume of distribution at steady state decreased with age. The results of that study showed no change in the unbound fraction of piroxicam, despite decline in albumin concentration in the elderly, possibly as a result of the change in albumin composition found in patients with rheumatoid arthritis. All clinical studies used the oral route of administration of piroxicam, and the effect of bioavailability was not assessed. Animal models, particularly rats, have often been used to study the pharmacokinetics of NSAIDS.lS-20Fischer 344 rats have been studied as a model for aging, and their aging process closely resembles that of hurnans.21-22 Roskos and Boudinotlg examined the effect of dose on the pharmamkinetics of piroxicam in rats and established that drug disposition was independent of dose over the range of 0.5-5 mgkg. The results of that study also indicated that rats represented a useful laboratory animal model for investigating piroxicam disposition. The purpose of this study was to assess the effects of age on the pharmacokinetics of piroxicam in rats, with the elimination of concomitant factors that could possible affect the results. In addition, piroxicam was administered intravenously to better examine the effects of age on drug distribution and elimination without the confounding factor of bioavailability .
Experimental Section Animals-Male Fischer 344 rats were obtained from National Institute on Aging colonies (HarlanSprague Dawley, Inc., Indianapolis, IN). Young adult rats of 5 months of age and weighing 320.5 2 30.5 g (mean f SD) and senescent rats of 24 months of age and weighing407.5 2 18.5 gwere selected for the study. Both groups were housed identically, in a controlled-temperature (22 "C) and 12-h light-12-h dark-cycle environment. During the 1-week acclimation period, food (Purina Lab Chow 5001; Ralston Purina Co., St. Louis, MO) and water were given ad libitum. On the day before the study, the rats were subjected to light ether anesthesia, during which right external vein cannulas were inserted. The animals were subjected to fasting for 16 h before drug administration,and food was provided 12 h after drug administration. Experimental Design-Six youngand six old rats weregiven 1 mg of piroxicam (Sigma Chemical Co., St. Louis, MO)per kg intravenously via the cannulas, over 60 a. The drug solution was prepared by dissolving piroxicam in 0.2 M carbonate in physiologic saline. Blood samples (0.25 mL) were collected before and at 0.08,0.25, 0.5, 0.75, 1,2,3,4,6,8,12,24,30,36,48,54,60,72,84,96,108, and 120 h after drug administrationvia the cannulas into heparinized tubes, and the blood volume was replaced with saline. Preliminary investigations demonstrated a lack of difference in the dispositionof piroxicam when blood volume was replaced with whole blood rather than saline. Additionally,there was no observed decline in the hematocrit during the course of the study. Previous studies showed no binding of the drug to the cannula.19 Blood samples were centrifuged, and the plasma was frozen at -20 "C until analysis. 0022-3549/93/300-02~$02.50/0 0 1993,American Pharmaceutical Association
Protein Binding-Serum was obtained from piroxicam-free young and old rats and pooled for protein binding studies. Known amounts of piroxicam were added to serum samples to yield drug concentrations ranging from 1-100 jg/mL. Equilibrium dialysis was performed with acrylic plastic dialysis cells and Spectrapor I1 cellulose dialysis membranes (Spectrum Medical Industries, New York, Nn with a molecular weight cutoff of 12 000-14 000 as previously described.19 Serum samples of 0.5 mL were equilibrated for 16 h at 37 “C with gentle shaking against 0.5 mL of isotonic sodium phosphate buffer (pH 7.4). Postdialysis sample volumes were measured, and samples were frozen until analysis. Data were corrected for fluid shifts that occurred during dialysis.23 Procedures were performed in triplicate. Piroxicam Assay-Piroxicam concentrations were assayed by use of the slightly modified%&nique of Boudinot and b r a h i m a with HPLC. In brief, 0.1-mL plasma samples were prepared with the addition of 0.1 mL of the internal standard S’-methylpiroxicam (Pfner Corp., Gmton, CT) and 0.2 mL of 1.0 M phosphate buffer (pH 2.0). Methylene chloride (5mL) was added, and samples were shaken at 120cycleslminfor 10min and then centrifuged at 2000 x g for 10 min. The aqueous layer was aspirated, and methylene chloride was evaporated under a stream of nitrogen gas. Samples were reconstituted with 0.4 mL of methanol phosphate buffer (pH 8) (4060). Samples ranging in volume from 0.02-0.2 mL were injected into the HPLC column for piroxicam concentration determinations. The chromatographic assay was done with a CIS reversed-phase column [4.6 mm (inner diameter) X 25 cm; 5 - p particle size; Alltech Associates, Deerfield, IL]. A model M45 solvent delivery system, a model 710B WISP automatic injedor, and a model 481 W detector set at 360 nm (allfrom Waters Associates, Milford, MA) and a model 3390A reporting integrator (Hewlett-Packard, Avondale, PA) completed the HPLC system. The mobile phase consisted of 40% methanol-60% phosphate buffer (pH €9, and a flow rate of 1.0 mWmin was used. Piroxicam concentrations were determined from the slopes of calibration plots of the peak area ratios of phxicam/6’-methylpiroxicam versus standard piroxicam concentrations. The slopes were determined with a weighted linear least-squares regression analysis. Reciprocal peak area ratios (lly) used as weighting factors for the regression analysis generated a normal distribution of residuals around the fitted calibration line.25 Calibration curves were linear in the range of 10-50,OOO ng/mL, and the limit of quantitation was 10 ng/mL. Coefficients of variation for intraday and interday precision were <8%, and the accuracy was >97%. 5’-Hydroxypiroxicam,a major metabolite of piroxicam, can also be quantitated by this assay methodology. Protein Binding Analysis-The fraction of the drug unbound or free in plasma was calculated as D,JDt,where Df and D,represent the concentrations of unbound drug and total drug, respectively. Drug binding was linear over the concentration range studied; therefore, the concentration of bound drug as a function of the concentration of free drug can be described by the equation D, = nKPDf, where n is the number of binding sites, P is the protein (albumin)concentration, and K is the equilibrium association constant. Pooled serum samples from young and old rats were analyzed for total protein and albumin concentrations by a n autoanalyzer technique (Technicon, Tarrytown, NY).Both sides of the equation D, = nKPD, were divided by the measured albumin concentration to normalize for the decline in protein concentration with aging, yielding the equation DJP = nKDp The data were graphed to determine the slope, nK, for each age group by linear regression, assuming that there was error in both variables.26 Pharmacokinetic Analysis-Pharmacokinetic parameters were determined using aredmoment analysis. Area under the plasma concentration-time curve (AUC) and the area under the first moment curve (AUMC) from time zero to the last sample time point were calculated by Lagrange polynomial interpolation and integration,27,28with extrapolation to time infinity by use of the nonlinear least-squares29 terminal slope (Az). The half-life was calculated as 0.693/AZ.Total clearance was determined as dose/AUC. Mean residence time was calculated as AUMC/AUC. The total volume of distribution at steady state was determined as total clearance x mean residence time. The clearance and steady-state volume of distribution of unbound drug were calculated by dividing the total clearance and total volume of distribution, respectively, by the fraction of unbound drug. Statistical Analysis-Statistical analysis of the effect of age on pharmacokinetic parameters was performed by use of a t test, with a p value of <0.05 being statistically significant.
Results and Discussion Several clinical studies have assessed the effects of age on the pharmacokinetics of piroxicarn2JOJ2-14; however, the conclusions have been somewhat conflicting. Differences in study design, failure to assess plasma protein binding, patient selection, and oral drug administration have produced variable and inconclusive results. The present investigation was undertaken with Fischer 344 rats as an animal model of age to factor out age from numerous other variables that can influence drug disposition. Piroxicam was administered intravenously to eliminate uncertainties due to bioavailability. The results of this study render insight into the effects of age on the pharmacokinetics of piroxicam, without the implication of other confounding factors. Representative plasma piroxicam concentration-time profiles for young and old rats are shown in Figure 1.The plasma piroxicam concentrations observed in rats in this study were comparable to those seen clinically.2JO Plasma piroxicam concentrations after intravenous injection to both young and old rats appeared to decline in a biexponential fashion. The age of the rats had a profound effect on the pharmacokinetics of piroxicam, as shown in Table I. The mean half-life in old rats was five times longer than that in young rats, the AUC was double that in young rats, and the mean residence time was about three times that in young rats. Total clearance of piroxicam in old rats was lower than that in yov.ng rats, whereas the volume of distribution at steady state ; i i old rats was larger than that in young rats. All pharmacokinetic
E
\
gl
10.000
u
t
x 0
0.1’
0
rr
24
48
72
120
96
TIME, h
a
Figure 1-Representative plasma piroxicam concentration-time cuwes for young (0)and old (0)rats after intravenous administration of 1 mg of piroxicam per kg. Table I-Pharmacoklnetlc Parameters after Single-Dose Intravenous Administration of Piroxicam (1 .O mg/kg) to Rats.
Parameterb t1,m
h
AUC, mg . h/L CL, Uh/kg MRT, h vss, Ukg CL,, Uhlkg Vssr Ukg
Mean (SD) for Six Young Rats 5.85(0.68) 21.43(5.06) 0.0489(0.0120) 8.84(1.50) 0.419 (0.045) 0.730 (0.181) 6.27(0.68)
Old Rats 30.59 (9.86) 48.50(6.03) 0.0209(0.0025) 26.87 (3.79) 0.560(0.100) 0.155 (0.018) 4.18(0.75)
a All pharmacokinetic parameters were statistically significantlydifferent (p < 0.05)for young versus old rats. t,,2, Half-life; CL, total clearance; MRT, mean residence time; V,,, volume of distribution at steady state; CL, clearance of free drug; V,, Vssof free drug.
Journal of Pharmaceutical Sciences I 255 Vol, 82, No. 3, March 1993
Table Il-Plasma Protein Blndlng Parameters for Plroxlcam In Rats Value for: Parameter Young Rats Old Rats ~~
nK, M-'
Albumin, M a
0.067 2.785 X lo4 5.0 x 10-4
,
0.134 1.63 x lo-4 3.97 x 10
fu, Fraction of unbound drug.
% a
FREE DRUG, pU
3 AGE, months Flgure 3-Pharmacokinetic parameters of piroxicam as a function of age. (A) Total piroxicam clearance (0)and free piroxicam clearance (0) in young and old rats. (6)Total piroxicam volume of distributionat steady state (0)and free piroxicam volume of distribution at steady state (0)for both age groups. Horizontal lines indicate mean values.
10
20
FREE DRUG, Flgure 2-Effects of age on the plasma protein binding of piroxicam in rats. (A) Protein-bound piroxicam concentration as a function of free piroxicam concentration in young (0)and old (0)rats. (B) Bound drug concentration,normalized for albumin concentration,as afunction of free drug concentration. Lines represent regression lines characterizing the relationships. parameters were found to be statistically significantly different for young versus old rats. Protein binding studies revealed marked differences between the two age groups. Table I1 shows that the fraction of unbound piroxicam doubled in old rats, thus yielding higher levels of the pharmacologically active species. Figure 2A shows the data and regression line describing bound drug concentration as a function of free drug concentration in young and old rats. The slope of this line was nKP. The albumin concentration declined 20% in old rats. Similar results were reported for rat@ and elderly humans.30J1 Normalization for protein concentration, as shown in Figure 2B, yielded a line whose slope was nK,calculated as previously described. Table I1 summarizes these findings and suggests that the changes in protein binding that occur with aging are due not only to a decline in albumin concentration but also to a change in nK. Previous studies assessing the effects of age on the protein binding of NSAIDS in rats 256 1 Journal of Pharmaceutical Sciences Vol. 82,No. 3, March 1993
suggested a decline in the affinity of the binding sites with age rather than a change in the number of binding sites.20 Piroxicam is eliminated almost exclusively by hepatic metabolism, so total clearance is essentially hepatic clearance. As shown in Figure 3A, total clearance in young rats was two times lower than that in old rats, whereas the clearance of free drug in young rats was three times higher than that in old rats. Since piroxicam is a low-clearance drug,lg the decline in hepatic blood flow due to aging was not a factor in the decline in clearance. The large difference in the clearance of free piroxicam was most likely due to a decline in liver function because of aging. The differences in total clearance between young and old rats were smaller than those in the clearance of free drug because of the twofold increase in the free fraction of piroxicam in old rats. The volume of distribution of free drug in old rats, as shown in Figure 3B, was smaller than that in young rats. This result can be attributed to the lower lean body mass, decline in total body water, altered regional blood flow, and decrease in extracellular albumin concentration that accompany aging.22 The total volume of distribution at steady state, however, showed a slight, yet statistically significant, increase in old rats because of the larger free fraction of the drug in the plasma of old rats. The relatively limited steady-state volume of distribution of piroxicam was typical of the high degree of plasma protein binding and distribution into extracellular fluid noted for NSAIDs.11932 In conclusion, age has significant effects on the pharmacokinetics of piroxicam in rats. The changes in plasma protein binding and the effects of altered protein binding are especially significant. Although the total clearance and volume of distribution were altered in old rats, the changes in the
clearance and volume of distribution of free drug were even more significant. It is the unbound or free fraction of the drug that is most altered by aging and that is available to produce both pharmacologic action and adverse effects.
References and Notes 1. Glaser, M. Drug Top. 1991,135,53-56. 2. Hobbs, D. C. Am. J. Med. 1986,81,Suppl. 5B, 22-27. 3. Lamy, P.P.Md. Pharm. 1986,62,18. 4. Mitnick, P.D.;Klein, W. J. Arch. Intern. Med. 1986,144,6344. 5. Miller, K. P.;Lazar, E. J.;Fotino, S.Arch. Intern. Med. 1984,144, 2414-2415. 6. Frais, M. A.; Burgess, E. D.; Mitchell, L. B. Ann. Intern. Med. 1983.99.129-130. 7. Emery, P.; Grahame, R. Lancet 1982,i , 1302-1303. 8. Gerber, D. Drug Intell. Clin. Phann. 1987,21,707-710. 9. Fok, K. H.; George, P. J. M.; Vicary, F. R. Br. Med. J. 1985,290, 117. 10. Woolf, A.D.; Rogers, H. J.; Bradbrook, I. D.; Corless, D. Br. J. Clin. Pharmacol. 1983.16.433-437. 11. Lin, H.;Cocchetto, D.M.;'Duggan, D. E. Clin. Pharmacokinet. 1987,12,402432. 12. Richardson, C. J.; Blocka, K. L.; Ross, R. T.; Verbeeck, R. K. Clin. Pharmacol. Ther. 1985,37,13-18. 13. Verbeeck, R. K.; Richardson, C. J.; Blocka, K. L. J. Rhuematol., 1986.13. , ~ .789-796. , .. 14. Blocka, K.L.; Richardson, C. J.; Wallace, S.M.; Ross, S. G.; Verbeeck, R. K. J. Rhuematol. 1988,15,757-763. 15. Sattenvhite, J. H.; Boudinot, F. D. Drug Metab. Dispos. 1990,19, 61-67.
16. Foster, R. T.;Jamali, F. Drug Metab. Dispos. 1988,16,623-626. 17. Runkel, R.; Chaplin, M.; Boost, G.; Segre, E.; Forchielli, E. J. Pharm. Sci. 1972,61,703-708. 18. Iwakawa, S.;Xiwen, H.; Hashimoto, S.; Volland, C.; Benet, L. Z.; Lin, E.T.Drug Metab. Dispos. 1991,19,717-718. 19. Roskos, L.K.;Boudinot, F. D. Biopharm. Drug Dispos. 1990,11, 215-225. 20. Satterwhite, J. H.;Boudinot, F. D. J. Gerontot. 1991,46,B222B227. 21. Hazzard, D.G.; Soban, J. Ezp. Aging Res. 1988,14,59-81. 22. Coleman, G. L.; Barthold, S. W.; Osbaldiston, G. V.; Foster, S. J.; Jonas, A.M. J. Gerontol. 1977,32,258-278. 23. Boudinot, F. D.; Jusko, W. J. J. Phurm. Sci. 1984,73, 774-780. 24. Boudinot, F. D.; Ibrahim, S. S. J. Chromatogr. 1988,430,424428. 25. Bonate, P.L.LC-GC 1992,10,448-450. 26. Riggs, D. S.;Guarnieri, J. A.; Addelman, S. Life Sci. 1978,22, 1305-1360. 27. Yeh, K.C.; Kwan, K. C . J. Pharmacokinet. Biopharm. 1978,6, 79-98. 28. Rocci, M. L.; Jusko, W. J. Comput. Programs Biomed. 1983,16, 203-216. 29. Metzler, C.M.; Elfring, G. L.; McEwen, A. J.Biometrics 1974,30, 562-563. 30. Bender, A.D.;Post, A.; Meier, J. P.; Higson, J. P.; Reichard, G., Jr. J. Pharm. Scd. 1975,64,1711-1713. 31. Wallace, S.; Verbeeck, R. C. Clin. Pharmacokinet. 1987, 12, 41-72. 32. Verbeeck, R. K.;Blackburn, J. L.; Loewen, G. R. Clin. Pharmucokinet. 1983,8,297-331.
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