Enantioselective Pharmacokinetics of Etodolac in the Rat: Tissue Distribution, Tissue Binding, and In Vitro Metabolism DIONR. BROCKSAND FAKHREDDIN JAMALI' Received October 15, 1990, from the Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2NS. Accepted for publication January 24, 1991. Abstract 0 The nonsteroidal anti-inflammatory agent etodolac (ET)
exhibits stereoselectivity in its pharmacokinetics following administration to humans and rats. To underline the factors responsible for this stereoselectivity, the tissue distribution, in vitro tissue binding, and microsomal metabolism of ET enantiomers were studied in the rat. Following iv administration of racemic ET, the S R AUC ratios in tissues were stereoselective, and different from that in plasma. Binding of enantiomers to tissues was stereoselective, although it did not relate well with in vivo tissue distribution. Rather, the tissue distribution of enantiomers appeared to be better explained by the unbound fractions of enantiomers in plasma. With respect to in vitro glucuronidation by liver microsomes,the V,, of S E T was 3.4-fold greater than that of R-ET; the enantiomers possessed similar K,. There appeared to be stereoselectivity in the oxidative metabolism of ET enantiomers by liver and kidney microsomes. in favor of the Renantiomer. The lower AUC in rat plasma of pharmacologically active S E T as compared with its antipode is due to its relatively greater distribution to tissues, owing to a lesser degree of binding to plasma proteins, and to its higher rate of glucuronidation.
The chiral nonsteroidal anti-inflammatory drug (NSAID) etodolac (ET) has previously been shown to exhibit marked stereoselectivity in its pharmacokinetics in humans'-3 and in the rat.4 This is a n important finding, as plasma concentrations of the pharmacologically inactive R-enantiomer5 account for most of the measured total (S plus R ) concentration following administration to both species. Etodolac is the only chiral NSAID examined to date to exhibit this feature; all of the other chiral NSAIDs studied from a stereoselective perspective have yielded plasma concentrations of pharmacologically active S-enantiomer which are at least as great as those of the inactive R-enantiomer.6 Following iv administration of racemic ET to the rat, pharmacologically active S-ET has a significantly smaller AUC, greater Vd, and longer t,,z than does the relatively inactive R-enantiomer.4 The S-enantiomer also has a greater biliary clearance than R-ET, and exhibits extensive enterohepatic recycling.4.7 This stereoselectivity has been attributed to differences between enantiomers in plasma protein binding, metabolism, and biliary excretion. The R-enantiomer is bound to a significantly greater extent than the S-enantiomer to plasma proteins. This difference explains to some degree the greater Vd and higher CL of S-ET. Although 100%of the administered S-ET was recovered in rat bile as glucuronide metabolite, only 30% of the R-enantiomer was likewise recovered following administration of racemate. The remaining 70% of the administered R-enantiomer could not be accounted for in urine or bile, which suggests metabolism to previously discovered oxidized metabolites.8 In this study we attempted to delineate the factors responsible for the observed stereoselectivity in the pharmacokinetics of ET in the rat. Accordingly, the in vivo tissue distribution and in vitro relative binding affinity of ET enantiomers to tissues were examined. The in vitro microsomal metabolism of ET was also studied, in order to better explain the 1058 I Journal of Pharmaceutical Sciences Vol. SO, No. 11, November 1991
previously observed greater recovery of glucuroconjugated Sthan R-ET in rat bile, and the incomplete recovery of the R-enantiomer in urine and bile.
Experimental Section Chemicals-Racemate and pure enantiomers of etodolac (ET) and internal standard [( ~)-2-(4-benzoylphenyllbutyric acid] were kindly provided by Wyeth-Ayerst Laboratories (New York, NY)and RhonePoulenc (Montreal,PQ, Canada),respectively. For microsomal incubations, all reagents were purchased from Sigma Laboratories (St. Louis, MO). Assay-Concentrations of ET and its conjugated metabolites were determined in plasma using a normal-phase HPLC method.' Some modification of the assay was required for analysis of ET in tissues. Two volumes of HPLC-grade water were added to weighed tissue samples (wet wt = 0.5-1.5 g). The mixtures were then homogenized using Potter-Elvehjemtissue grinders driven by a T-Line Laboratory Stirrer (TalboysEngineering, Montrose, PA). Internal standard was placed into the glass mortar during the homogenization step. Samples were transferred to test tubes and acidified with 200 & of 0.6 M H,SO,. Etodolac was extracted with 6 mL of isopropyl alcoho1:isooctane (595).After transfer of the organic layer to clean tubes, 6 mL of HPLC-grade water was added and, following mixing and centrifugation, the organic layer was discarded. The aqueous layer was acidified with 0.6 M H2S0,, and 6 mL of chloroform was added. After mixing and centrifugation, the aqueous layer was removed and the remaining chloroform layer was evaporated to dryness. Derivatization was performed as for plasma samples1 using ethylchloroformate and L(-)-a-phenylethylamine. The resultant diastereomers were extracted into chloroform, which was subsequently evaporated to dryness. The residue was dissolved in 0.2 mL of mobile phase and injected into the HPLC. Calibration curves prepared using rat plasma and tissues yielded excellent linearity (3> 0.99) between the peak area ratios (drug:internal standard) and corresponding rat plasma and tissue concentrationsof ET. The intraday coefficientsof variation at 1 pg/g of tissue ranged from 3.9 to 10.2%; for brain and fat, where lower concentrationswere observed in vivo, at 0.25 pglg of tissue,the coefficients of variation were 8.1 and 5.8%, respectively. Dosing and Sample Collection-Etodolac was dissolved in 100% polyethylene glycol 400 (20 mg racemate/mL)and administered as 10-mg of racemate/kg bolus iv doses into the tail vein. Thirty-two male Sprague-Dawley rats (wt = 308 k 34 g) were killed by cervical dislocation at 1,3,6, 12,or 24 h after dosing. Six rats were included in the 1-,3-,and 6-h groups, whereas seven rats were in the 12- and 24-h groups. Blood was collected and plasma separated by centrifugation at 2600 rpm for 5 min. Selected tissues (liver, kidney, perinephric fat, heart, and brain) were immediately excised and frozen at -20 "C until analyzed. Mean body weights between groups were not significantly different. Tissue Binding Experiments-A Spectrum (Los Angeles, CAI equilibriumdialysis apparatus and Sigma Diagnostics dialysis membranes (St. Louis, MO) were used to assess the relative in vitro binding of rat liver, kidney,heart, and brain. Weighed tissue samples were first homogenized in two volumes of isotonic phosphate buffer (pH 7.4).One milliliter of homogenate was dialyzed against an equal volume of isotonic phosphate buffer containing the equivalent of 20 mg/L of ET. Cells were dialyzed for 4 h at 37 "C. Preliminary studies indicated that 4 h was sufficient for equilibrium to occur at 37 "C. In Vitro Metabolism Studies-Freshly obtained rat liver, kidney, lung, and jejunum were excised and immediately placed in ice-cold OO22-3549/91/1laO- 1058$02.5O/O 0 1991, American Pharmaceutical Association
1.15% KCl. Individual tissues from four rats (wt = 350-450 g) were pooled. Tissues were homogenized in ice-cold 100 mM phosphate buffer (pH 7.4) containing 250 mM sucrose. Homogenates were centrifuged in a Beckman model LS55 centrifuge (Beckman, Palo Alto, CA) a t 10000 x g for 20 min. The supernatant was then subjected to centrifugation at 105 000 g for 60 min. The resultant pellet was suspended in phosphate:sucrose buffer and again centrifuged at 105 000 g for 60 min. The final pellet was resuspended in two volumes of phosphate:sucrose buffer. Protein concentration was determined by the Lowry methods (Sigma kit, St. Louis, MO). Glucuronidation of racemic ET was determined in triplicate at concentrations of 1, 5, 10, 50, 100, 250, and 375 mg/L; these concentrations were in accordance with those seen in plasma following 5-mgkg racemic iv doses given to rats." Reaction mixtures contained 15 mM uridine 5'-diphosphoglucuronic acid, 5 mM MgCl,, and 0.05% Triton X-100 in 100 mM phosphate:sucrose buffer (pH 7.4). Reactions were started by the addition of 1.1-1.2 mg of liver microsomal protein; total incubation volume was 1 mL. Incubations were run for 20 min at 37 "C. Preliminary studies indicated that, under these conditions, glucuronidation was linear with respect to both time and protein concentration. At the end of each incubation, aliquots (30-400 pL) were transferred to tubes containing either 150 pL of 0.6M H2S04 or 100 pL of 2M NaOH. After 60 8,300 pL of 0.6M HzS04 was added to the tubes containing NaOH. The difference between the acidified and basifiedlacidified tubes was taken as representing ET which had been metabolized to glucuronide conjugates. Microsomal oxidation reactions were carried out using freshly prepared kidney and liver microsomes. Reaction mixtures contained 5 mM MgCl,, 10 mM glucose-6-phosphate, 0.75 mM NADP+, 2 U of glucose-6-phosphate dehydrogenase, and either 4 mg of kidney or 8 mg of liver microsomes, in 0.05 M Tris-HC1 buffer (pH 7.4). Samples were spiked with 2 mg/L of racemic ET, and incubated for 4 h at 37 "C.
As no authentic oxidative metabolites were available, only reduction in the concentration of ET enantiomer following incubation was determined. Data A n a l y s i e T h e areas under the plasma and tissue drug concentration versus time curves from 0 to 24 h (AUC,,,) were estimated using the linear trapezoidal rule. Due to the extensive enterohepatic recirculation of ET in the rat,4.7 t,,,, AUC,, and CL were not determined. Values of V, and K , for microsomal glucuronidation were estimated using the nonlinear data fitting program MULTI.10 Statistical significance was evaluated by using either a paired or unpaired t test, by one-way ANOVA and by Duncan's Multiple Range Test. The level of significance was set at Q = 0.05. Values are expressed as mean f SD.
Results Tissue Distribution and Binding-The concentrations of both ET enantiomers were greater in plasma than in any of the tissues studied (Figure 1, Table I). The S:R ratio of AUC,,, in plasma (0.31) was similar to that reported previously (0.26).4 In tissues, the S:R AUC &24 ratios were higher than in plasma, and were somewhat tissue specific (Table I). The S:R concentration ratios in liver, kidney, heart, and fat were significantly different from those in plasma at all time points (Figure 1).Brain differed, however, as only the 3-h S:R ratio was significantly different from the corresponding ratio in plasma (Figure 1).For both enantiomers, concentrations were highest in heart, followed by kidney and liver, and then fat and brain. The S-enantiomer had a greater tissue distribution than R-ETrelative to plasma (Table I). There was marked stereoselectivity reflected in the tissue 10
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Time (h)
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Flgure 1-Total (bound + unbound) plasma and tissue concentration-time profiles of etodolac enantiomers given to rats as 10-mg racematelkg iv bolus doses. Each point represents the mean f SD of 6-7 rats. Key: (0)R-ET; (0)SET. Journal of PharmaceuticalSciences I 1059 Vol. 80, No. 11, November 1991
Table I-Plasma- and Tlssue-Speclflc AUCW2, Values of Etodolac Enantlomers followlng l&mg/kg Racemlc Bolus Intravenous Doses Given to Rats
Specimen Plasma Liver Kidney Heart Fat Brain a pg *
Tissue:Plasma
AUCO-24, r4 * h/g
S 167' 73.0 65.0 74.8 28.6 7.70
R 546' 64.2 67.7 97.8 36.9 17.0
SR 0.306 1.14 1.01 0.765 0.775 0.453
~
0.43 0.39 0.45 0.17 0.046
0.6
I
E
I
n
0.12 0.12 0.18 0.068 0.031
/ -
c
0.0 0.0
binding results (Figure 2), particularly in kidney and heart tissues. Because of phase separation upon dilution and homogenization, fat tissue could not be studied. The S22 ratios for unbound enantiomer were 1.15 0.09, 2.14 0.26, 3.69 2 0.23, and 1.23 2 0.03 in brain, heart, kidney, and liver tissues, respectively. In each tissue, binding of the R-enantiomer was significantly greater than S-ET (Figure 2). With respect to order of binding, kidney and liver bound S-ET to a significantly greater extent than did brain and heart tissue. For R-ET, the order of binding was kidney > heart and liver > brain tissue. In Vitro Metabolism-Microsoma1 protein recovered from hepatic tissue displayed pronounced glucuronidation activity, in favor of the S-enantiomer (Figure 3). The V,,, values obtained for each enantiomer (1.15 0.27 and 0.337 k 0.11 mmol/h/mg protein for S- and R-ET, respectively) differed significantly (Figure 3), with S-ET having a 3.4-fold greater V,,, than R-ET. The K, values (0.47 2 0.11 and 0.45 r 0.15 mmol/h/mg protein for S- and R-ET, respectively), however, were not significantly different. No glucuronidation activity was seen in microsomes prepared from lung, jejunal, or kidney tissues, despite the positive result seen in concurrently incubated liver microsomes. Microsomal oxidative activity was apparent in both liver and kidney microsomes (Table 11). Concentrations of both enantiomers were significantly reduced by liver microsomes &r 4 h of incubation. Moreover, this decrease was stereoselective, based on the significantly greater S:R ratio of ET enantiomer concentrations in liver microsoma1 incubations. In kidney microsomes,the concentrations ofR-ET did decline, although not significantly. A significant increase, however, was observed in the S:R ratio after 4 h. No in vitro oxidative activity was observed in microsomes obtained from lung or gut microsomes.
*
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Figurn 2-In vitro tissue binding of etodolac to drug-spiked blank rat tissues. Each bar represents the mean ? SD of five determinations. Key: (0)SET; ( I R-ET. ) 1060 I
Journal of Pharmaceutical Sciences Vol. 80, No. 11, November 1997
0.5
0.6
0.7
C o n c e n t r a t i o n , rnM
Figure &In vitro glucuronidation of etodolac enantiomers by rat liver microsomes. Lines represent the mean of the best fit lines (n = 3). Key:
(0)S E T ; (0)R-ET.
Discussion The greater S:R ratios of ET concentrations in tissues as compared with plasma indicate that in the rat the enantiomeric ratio in plasma is not indicative of that present in the tissues, where pharmacological and toxic effects primarily occur. Plasma concentrations, especially of R-ET, were considerably higher than those in the tissues (Figure 1, Table I). To some extent this may be explained by the binding of ET enantiomers to rat plasma, which is extensive for both enantiomers, but stronger for R- than S-ET.4 This was thought to be a contributing factor for the difference in Vd, between ET enantiomers, which was 5.4-fold greater for Sthan R-ET. The lower tissue concentrations of enantiomers relative to plasma are in line with the results of Cayen et al.,7 who studied the nonstereospecific tissue distribution of racemic ET in rats given 10 mgkg orally. From their results we calculated AUC&24in serum, liver, heart, kidneys, fat, and brain to be 494, 208, 162, 160, 68, and 9.2 pg h/g, respectively. Both those and the present results may, in part, be explained by a relatively low degree of tissue binding, as seen in the present study. However, tissue binding does not appear to fully explain why the S:R distribution ratio in tissues was generally higher than that seen in plasma. For example, although the S:R ratio for AUC, in kidney was close to unity (Table I), marked stereoselectivity was seen in kidney binding (S22 unbound fraction = 3.3; Figure 2). This apparent paradoxical result can, a t least partially, be explained by the greater binding of R- than S-ET in plasma, which balances the greater binding of R-ET in tissues. If it is assumed that the amount of drug that can enter tissues is dependent on the concentration of free drug in plasma (C,) and that the unbound fraction in plasma is fairly constant, then the enantiomeric ratio of tissue concentrations normalized for plasma concentrations (i.e., tissue AUC: plasma AUC) should be similar to the corresponding ratio of C,. The S:R ratios (Table I) of the tissue AUC,, when normalized to plasma AUC,, were calculated to be 3.6,3.3, 2.5,2.4, and 1.7 in liver, kidney, heart, fat, and brain tissues, respectively. Interestingly, with the exception of brain, these values appear to be in line with the SB ratio of C , of ET in rat plasma, which was 3.12 1.13 a t 20 mgh.4 This observation seems to support the notion that the free drug concentration in plasma is the major controlling factor in the distribution of ET to tissues. Some of these findings parallel those seen in the disposition of ET into the synovial fluid of five arthritic subjects.3Twelve hours after receiving 200 mg of ET, concentrations of S-,but
*
0 -
0.4
Table Il-NADP+-Dependent Microsomal Oxidation of Etodolac Enantiomersa
Time, h 0 4
Liver Microsornes
Kidney Microsomes
S
R
SR
S
1.OO 2 0.023 0.74 -C 0.078’
1.OO 2 0.027 0.48 0.044b*c
1.OO 2 0.039
0.98 k 0.031 1.06 0.069
*
1.55
* 0.066b
*
R 1.00 0.79
2 ?
0.13 0.031
SR 1.00 k 0.15 1.34 2 0.10’
’
a Each value represents the mean concentration (pglmL)* SD of four determinations. Significantly different from 0 h. Significantlydifferent from Senantiomer.
not R-ET, were significantly greater in synovial fluid than in plasma. In both ET-spiked human synovial fluid and in rat tissues there were greater S:R concentration ratios than in plasma, and a greater binding of R- than S-ET. There was, however, a greater binding of enantiomers to drug-spiked human synovial fluid (96 and 97% for S- and R-ET, respectively a t 10 mg/L of enantiomer) than to the diluted rat tissue homogenates (Figure 2). This may be due to the presence of considerable concentrations of albumin (the major plasma protein implicated in binding of NSAIDs) in synovial fluid. The tissue binding results should be viewed with some degree of caution, as the dilution step might have altered the binding characteristics of the tissues. Although dilution might compromise the ability of the binding experiments to determine the absolute degree of binding of ET to tissues, the experiments do reflect the relative binding affinity of enantiomers to tissues. With respect to in vitro glucuronidation, a good parallel was seen between the maximal rate of glucuronidation by hepaticmicrosomal glucuronyl transferases, and the previously published data on biliary excretion of conjugates in the rat.4 With the K,,, values seen, glucuronidation of neither enantiomer is saturated a t the concentrations seen in plasma during the post-distributive phase following a lO-mgkg iv dose. The mean S:R ratio for cumulative excretion of conjugates in bile was 3.2. A similar S:R ratio in V,,, for in vitro glucuronidation (3.46 2 0.45) was seen. These similarities appear to suggest that the differences in biliary excretion of enantiomers as conjugates are due to enzymatic velocity, and not to secretion of conjugates into bile. Previously, it was shown that virtually the complete dose of S-ET could be recovered as conjugated enantiomer in rat bile following iv administration.4 However, only 33% of the R-enantiomer was likewise recovered. Etodolac had previously been reported to undergo microsomal oxidative metabolism in the rat;s hence it was thought that the R-enantiomer is predominately biotransformed to those metabolites. Although we were unable to assay these metabolites, and pure oxidized metabolites were not available for assay development, we nevertheless felt that by determining the disappearance of ET enantiomers in the presence of an NADP’ microsomal oxidizing system, insight could be gained into the oxidative metabolism of ET. As hypothesized, concentrations of R-ET fell significantly faster than concentrations of S-ET after 4 h of incubation in the presence of hepatic microsomes (Table 11). Concentrations of S-ET were also significantly reduced afier 4 h, suggesting that it too was capable of being metabolized by hepatic microsomes. Given the high rate of formation of glucuroconjugated S-ET, however, which is further enhanced by a lower extent of plasma protein binding, glucuronidation is the major metabolic fate of the S-enantiomer. This is confirmed by the virtual complete recovery of S-ET in bile as conjugated drug.4 An unclear situation appeared in the kidney oxidative microsomal incubations (Table 111, in which a significant increase in the S:R concentration ratio and a non-statistically
significant reduction in R-ET concentrations were seen. Despite these contradictory findings, it is likely that there was some oxidation of R-ET by kidney microsomal protein, because use of S:R ratios involves a statistical pairing of the R- with S-ET concentrations, which in turn minimizes the variation present among both sets of data. Nevertheless, confirmation of the findings from both liver and kidneys microsomes, using an assay which can measure the formation of the oxidized metabolites, would be most desirable. In conclusion, ET is an NSAID that exhibits a high degree of stereoselectivity in its pharmacokinetics. The higher concentrations of R- than S-ET in rat plasma can be attributed to a broader distribution of S-ET, owing to its lower degree of plasma protein binding and to its faster rate of metabolism, particularly to glucuronide conjugates. In contrast, R-ET appears to undergo preferential metabolism to oxidized metabolites in the rat, although this occurs at a slower rate than conjugation of S-ET. In humans, the relative contribution of these pathways of metabolism are different from the rat, as in the former species most of the urinary recovery consists of oxidized metabolites.llJ2 The relative concentrations of ET enantiomers observed in plasma are not reflective of those in the tissues, which may be of importance in the context of pharmacodynamic and toxicological studies involving the individual enantiomers following administration of racemic doses.
References and Notes 1. Jamali, F.; Mehvar, R.; Lemko, C.; Eradiri, 0. J . Pharm. Sci. 1986, 77,963-966. 2. Singh, N. N.; Jamali, F.; Pasutto, F. M.; Coutts, R. T. J . Chromatogr. 1986,382,331337. 3. Brocks, D. R.; Jamali F.; Russell A. S. J . Clin. Pharmncol., in press. 4. Brocks, D. R.; Jamali, F. Drug Metab. Dispos. 1990,18,471-475. 5 . Humber, L. G.; Demerson, C. A.; Swaminathan, P.;Bird, P. H. J. Med. Chem. 1986,29,871-874. 6. Jamali, F. Eur. J . Drug Metubol. Phmmacokinet. 1988,13, 1-9. 7. Cayen, M. N.; Kraml, M.; Ferdinandi, E. S.; Greselin, E.; Dvornik, D. Drug Metub. Rev. 1981,12,339-362. 8. Ferdinandi, E.S.; Cochrane, D.; Gedamke, R. Drug Metub. D~POS 1987,15,921-924. . 9. Lowry, 0.H.;Rosebrough, N. J.;Farr, A. J.;Randall R. J. J . Biol. Chem. 1951,193,265-275. 10. Yamaoka, K.; Tanigawara, Y.; Nakagawa, T.; Uno, T. J . Pharm. Dyn. 1981,4,879-885. 11. Ferdinandi, E. S.;. Sehgal, S. N.; Demerson, C: 4.; Dubuc, J.; Zilber, J.; Dvornik, D.; Cayen, M.N. Xenobzotica 1986, 16, 153-166. 12. Humber, L. G.; Ferdinandi, E.; Demerson, C. A.; Ahmed, S.; Shah, U., et al. J . Med. Chern. 1988,31, 1712-1719.
Acknowledgments This work was supported by the Medical Research Council of Canada (Grant MA 9569) and was presented in art at the American Association of Pharmaceutical Scientists 5th lnnual Meeting and Exposition, Las Ve as, NE, November 44,1990. D. R. Brocks 18 the reci ient of the Alferta Heritage Foundation for Medical Research Stugentship Award.
Journal of Pharmaceutical Sciences I 1061 Vol. 80, No. 77, November 7997
exhibits stereoselectivity in its pharmacokinetics following administration to humans and rats. To underline the factors responsible for this stereoselectivity, the tissue distribution, in vitro tissue binding, and microsomal metabolism of ET enantiomers were studied in the rat. Following iv administration of racemic ET, the S R AUC ratios in tissues were stereoselective, and different from that in plasma. Binding of enantiomers to tissues was stereoselective, although it did not relate well with in vivo tissue distribution. Rather, the tissue distribution of enantiomers appeared to be better explained by the unbound fractions of enantiomers in plasma. With respect to in vitro glucuronidation by liver microsomes,the V,, of S E T was 3.4-fold greater than that of R-ET; the enantiomers possessed similar K,. There appeared to be stereoselectivity in the oxidative metabolism of ET enantiomers by liver and kidney microsomes. in favor of the Renantiomer. The lower AUC in rat plasma of pharmacologically active S E T as compared with its antipode is due to its relatively greater distribution to tissues, owing to a lesser degree of binding to plasma proteins, and to its higher rate of glucuronidation.
The chiral nonsteroidal anti-inflammatory drug (NSAID) etodolac (ET) has previously been shown to exhibit marked stereoselectivity in its pharmacokinetics in humans'-3 and in the rat.4 This is a n important finding, as plasma concentrations of the pharmacologically inactive R-enantiomer5 account for most of the measured total (S plus R ) concentration following administration to both species. Etodolac is the only chiral NSAID examined to date to exhibit this feature; all of the other chiral NSAIDs studied from a stereoselective perspective have yielded plasma concentrations of pharmacologically active S-enantiomer which are at least as great as those of the inactive R-enantiomer.6 Following iv administration of racemic ET to the rat, pharmacologically active S-ET has a significantly smaller AUC, greater Vd, and longer t,,z than does the relatively inactive R-enantiomer.4 The S-enantiomer also has a greater biliary clearance than R-ET, and exhibits extensive enterohepatic recycling.4.7 This stereoselectivity has been attributed to differences between enantiomers in plasma protein binding, metabolism, and biliary excretion. The R-enantiomer is bound to a significantly greater extent than the S-enantiomer to plasma proteins. This difference explains to some degree the greater Vd and higher CL of S-ET. Although 100%of the administered S-ET was recovered in rat bile as glucuronide metabolite, only 30% of the R-enantiomer was likewise recovered following administration of racemate. The remaining 70% of the administered R-enantiomer could not be accounted for in urine or bile, which suggests metabolism to previously discovered oxidized metabolites.8 In this study we attempted to delineate the factors responsible for the observed stereoselectivity in the pharmacokinetics of ET in the rat. Accordingly, the in vivo tissue distribution and in vitro relative binding affinity of ET enantiomers to tissues were examined. The in vitro microsomal metabolism of ET was also studied, in order to better explain the 1058 I Journal of Pharmaceutical Sciences Vol. SO, No. 11, November 1991
previously observed greater recovery of glucuroconjugated Sthan R-ET in rat bile, and the incomplete recovery of the R-enantiomer in urine and bile.
Experimental Section Chemicals-Racemate and pure enantiomers of etodolac (ET) and internal standard [( ~)-2-(4-benzoylphenyllbutyric acid] were kindly provided by Wyeth-Ayerst Laboratories (New York, NY)and RhonePoulenc (Montreal,PQ, Canada),respectively. For microsomal incubations, all reagents were purchased from Sigma Laboratories (St. Louis, MO). Assay-Concentrations of ET and its conjugated metabolites were determined in plasma using a normal-phase HPLC method.' Some modification of the assay was required for analysis of ET in tissues. Two volumes of HPLC-grade water were added to weighed tissue samples (wet wt = 0.5-1.5 g). The mixtures were then homogenized using Potter-Elvehjemtissue grinders driven by a T-Line Laboratory Stirrer (TalboysEngineering, Montrose, PA). Internal standard was placed into the glass mortar during the homogenization step. Samples were transferred to test tubes and acidified with 200 & of 0.6 M H,SO,. Etodolac was extracted with 6 mL of isopropyl alcoho1:isooctane (595).After transfer of the organic layer to clean tubes, 6 mL of HPLC-grade water was added and, following mixing and centrifugation, the organic layer was discarded. The aqueous layer was acidified with 0.6 M H2S0,, and 6 mL of chloroform was added. After mixing and centrifugation, the aqueous layer was removed and the remaining chloroform layer was evaporated to dryness. Derivatization was performed as for plasma samples1 using ethylchloroformate and L(-)-a-phenylethylamine. The resultant diastereomers were extracted into chloroform, which was subsequently evaporated to dryness. The residue was dissolved in 0.2 mL of mobile phase and injected into the HPLC. Calibration curves prepared using rat plasma and tissues yielded excellent linearity (3> 0.99) between the peak area ratios (drug:internal standard) and corresponding rat plasma and tissue concentrationsof ET. The intraday coefficientsof variation at 1 pg/g of tissue ranged from 3.9 to 10.2%; for brain and fat, where lower concentrationswere observed in vivo, at 0.25 pglg of tissue,the coefficients of variation were 8.1 and 5.8%, respectively. Dosing and Sample Collection-Etodolac was dissolved in 100% polyethylene glycol 400 (20 mg racemate/mL)and administered as 10-mg of racemate/kg bolus iv doses into the tail vein. Thirty-two male Sprague-Dawley rats (wt = 308 k 34 g) were killed by cervical dislocation at 1,3,6, 12,or 24 h after dosing. Six rats were included in the 1-,3-,and 6-h groups, whereas seven rats were in the 12- and 24-h groups. Blood was collected and plasma separated by centrifugation at 2600 rpm for 5 min. Selected tissues (liver, kidney, perinephric fat, heart, and brain) were immediately excised and frozen at -20 "C until analyzed. Mean body weights between groups were not significantly different. Tissue Binding Experiments-A Spectrum (Los Angeles, CAI equilibriumdialysis apparatus and Sigma Diagnostics dialysis membranes (St. Louis, MO) were used to assess the relative in vitro binding of rat liver, kidney,heart, and brain. Weighed tissue samples were first homogenized in two volumes of isotonic phosphate buffer (pH 7.4).One milliliter of homogenate was dialyzed against an equal volume of isotonic phosphate buffer containing the equivalent of 20 mg/L of ET. Cells were dialyzed for 4 h at 37 "C. Preliminary studies indicated that 4 h was sufficient for equilibrium to occur at 37 "C. In Vitro Metabolism Studies-Freshly obtained rat liver, kidney, lung, and jejunum were excised and immediately placed in ice-cold OO22-3549/91/1laO- 1058$02.5O/O 0 1991, American Pharmaceutical Association
1.15% KCl. Individual tissues from four rats (wt = 350-450 g) were pooled. Tissues were homogenized in ice-cold 100 mM phosphate buffer (pH 7.4) containing 250 mM sucrose. Homogenates were centrifuged in a Beckman model LS55 centrifuge (Beckman, Palo Alto, CA) a t 10000 x g for 20 min. The supernatant was then subjected to centrifugation at 105 000 g for 60 min. The resultant pellet was suspended in phosphate:sucrose buffer and again centrifuged at 105 000 g for 60 min. The final pellet was resuspended in two volumes of phosphate:sucrose buffer. Protein concentration was determined by the Lowry methods (Sigma kit, St. Louis, MO). Glucuronidation of racemic ET was determined in triplicate at concentrations of 1, 5, 10, 50, 100, 250, and 375 mg/L; these concentrations were in accordance with those seen in plasma following 5-mgkg racemic iv doses given to rats." Reaction mixtures contained 15 mM uridine 5'-diphosphoglucuronic acid, 5 mM MgCl,, and 0.05% Triton X-100 in 100 mM phosphate:sucrose buffer (pH 7.4). Reactions were started by the addition of 1.1-1.2 mg of liver microsomal protein; total incubation volume was 1 mL. Incubations were run for 20 min at 37 "C. Preliminary studies indicated that, under these conditions, glucuronidation was linear with respect to both time and protein concentration. At the end of each incubation, aliquots (30-400 pL) were transferred to tubes containing either 150 pL of 0.6M H2S04 or 100 pL of 2M NaOH. After 60 8,300 pL of 0.6M HzS04 was added to the tubes containing NaOH. The difference between the acidified and basifiedlacidified tubes was taken as representing ET which had been metabolized to glucuronide conjugates. Microsomal oxidation reactions were carried out using freshly prepared kidney and liver microsomes. Reaction mixtures contained 5 mM MgCl,, 10 mM glucose-6-phosphate, 0.75 mM NADP+, 2 U of glucose-6-phosphate dehydrogenase, and either 4 mg of kidney or 8 mg of liver microsomes, in 0.05 M Tris-HC1 buffer (pH 7.4). Samples were spiked with 2 mg/L of racemic ET, and incubated for 4 h at 37 "C.
As no authentic oxidative metabolites were available, only reduction in the concentration of ET enantiomer following incubation was determined. Data A n a l y s i e T h e areas under the plasma and tissue drug concentration versus time curves from 0 to 24 h (AUC,,,) were estimated using the linear trapezoidal rule. Due to the extensive enterohepatic recirculation of ET in the rat,4.7 t,,,, AUC,, and CL were not determined. Values of V, and K , for microsomal glucuronidation were estimated using the nonlinear data fitting program MULTI.10 Statistical significance was evaluated by using either a paired or unpaired t test, by one-way ANOVA and by Duncan's Multiple Range Test. The level of significance was set at Q = 0.05. Values are expressed as mean f SD.
Results Tissue Distribution and Binding-The concentrations of both ET enantiomers were greater in plasma than in any of the tissues studied (Figure 1, Table I). The S:R ratio of AUC,,, in plasma (0.31) was similar to that reported previously (0.26).4 In tissues, the S:R AUC &24 ratios were higher than in plasma, and were somewhat tissue specific (Table I). The S:R concentration ratios in liver, kidney, heart, and fat were significantly different from those in plasma at all time points (Figure 1).Brain differed, however, as only the 3-h S:R ratio was significantly different from the corresponding ratio in plasma (Figure 1).For both enantiomers, concentrations were highest in heart, followed by kidney and liver, and then fat and brain. The S-enantiomer had a greater tissue distribution than R-ETrelative to plasma (Table I). There was marked stereoselectivity reflected in the tissue 10
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10
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Time (h)
Time (h) UMR
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I
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9 .-
9 .. 8.7
.-
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Flgure 1-Total (bound + unbound) plasma and tissue concentration-time profiles of etodolac enantiomers given to rats as 10-mg racematelkg iv bolus doses. Each point represents the mean f SD of 6-7 rats. Key: (0)R-ET; (0)SET. Journal of PharmaceuticalSciences I 1059 Vol. 80, No. 11, November 1991
Table I-Plasma- and Tlssue-Speclflc AUCW2, Values of Etodolac Enantlomers followlng l&mg/kg Racemlc Bolus Intravenous Doses Given to Rats
Specimen Plasma Liver Kidney Heart Fat Brain a pg *
Tissue:Plasma
AUCO-24, r4 * h/g
S 167' 73.0 65.0 74.8 28.6 7.70
R 546' 64.2 67.7 97.8 36.9 17.0
SR 0.306 1.14 1.01 0.765 0.775 0.453
~
0.43 0.39 0.45 0.17 0.046
0.6
I
E
I
n
0.12 0.12 0.18 0.068 0.031
/ -
c
0.0 0.0
binding results (Figure 2), particularly in kidney and heart tissues. Because of phase separation upon dilution and homogenization, fat tissue could not be studied. The S22 ratios for unbound enantiomer were 1.15 0.09, 2.14 0.26, 3.69 2 0.23, and 1.23 2 0.03 in brain, heart, kidney, and liver tissues, respectively. In each tissue, binding of the R-enantiomer was significantly greater than S-ET (Figure 2). With respect to order of binding, kidney and liver bound S-ET to a significantly greater extent than did brain and heart tissue. For R-ET, the order of binding was kidney > heart and liver > brain tissue. In Vitro Metabolism-Microsoma1 protein recovered from hepatic tissue displayed pronounced glucuronidation activity, in favor of the S-enantiomer (Figure 3). The V,,, values obtained for each enantiomer (1.15 0.27 and 0.337 k 0.11 mmol/h/mg protein for S- and R-ET, respectively) differed significantly (Figure 3), with S-ET having a 3.4-fold greater V,,, than R-ET. The K, values (0.47 2 0.11 and 0.45 r 0.15 mmol/h/mg protein for S- and R-ET, respectively), however, were not significantly different. No glucuronidation activity was seen in microsomes prepared from lung, jejunal, or kidney tissues, despite the positive result seen in concurrently incubated liver microsomes. Microsomal oxidative activity was apparent in both liver and kidney microsomes (Table 11). Concentrations of both enantiomers were significantly reduced by liver microsomes &r 4 h of incubation. Moreover, this decrease was stereoselective, based on the significantly greater S:R ratio of ET enantiomer concentrations in liver microsoma1 incubations. In kidney microsomes,the concentrations ofR-ET did decline, although not significantly. A significant increase, however, was observed in the S:R ratio after 4 h. No in vitro oxidative activity was observed in microsomes obtained from lung or gut microsomes.
*
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*
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0
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Figurn 2-In vitro tissue binding of etodolac to drug-spiked blank rat tissues. Each bar represents the mean ? SD of five determinations. Key: (0)SET; ( I R-ET. ) 1060 I
Journal of Pharmaceutical Sciences Vol. 80, No. 11, November 1997
0.5
0.6
0.7
C o n c e n t r a t i o n , rnM
Figure &In vitro glucuronidation of etodolac enantiomers by rat liver microsomes. Lines represent the mean of the best fit lines (n = 3). Key:
(0)S E T ; (0)R-ET.
Discussion The greater S:R ratios of ET concentrations in tissues as compared with plasma indicate that in the rat the enantiomeric ratio in plasma is not indicative of that present in the tissues, where pharmacological and toxic effects primarily occur. Plasma concentrations, especially of R-ET, were considerably higher than those in the tissues (Figure 1, Table I). To some extent this may be explained by the binding of ET enantiomers to rat plasma, which is extensive for both enantiomers, but stronger for R- than S-ET.4 This was thought to be a contributing factor for the difference in Vd, between ET enantiomers, which was 5.4-fold greater for Sthan R-ET. The lower tissue concentrations of enantiomers relative to plasma are in line with the results of Cayen et al.,7 who studied the nonstereospecific tissue distribution of racemic ET in rats given 10 mgkg orally. From their results we calculated AUC&24in serum, liver, heart, kidneys, fat, and brain to be 494, 208, 162, 160, 68, and 9.2 pg h/g, respectively. Both those and the present results may, in part, be explained by a relatively low degree of tissue binding, as seen in the present study. However, tissue binding does not appear to fully explain why the S:R distribution ratio in tissues was generally higher than that seen in plasma. For example, although the S:R ratio for AUC, in kidney was close to unity (Table I), marked stereoselectivity was seen in kidney binding (S22 unbound fraction = 3.3; Figure 2). This apparent paradoxical result can, a t least partially, be explained by the greater binding of R- than S-ET in plasma, which balances the greater binding of R-ET in tissues. If it is assumed that the amount of drug that can enter tissues is dependent on the concentration of free drug in plasma (C,) and that the unbound fraction in plasma is fairly constant, then the enantiomeric ratio of tissue concentrations normalized for plasma concentrations (i.e., tissue AUC: plasma AUC) should be similar to the corresponding ratio of C,. The S:R ratios (Table I) of the tissue AUC,, when normalized to plasma AUC,, were calculated to be 3.6,3.3, 2.5,2.4, and 1.7 in liver, kidney, heart, fat, and brain tissues, respectively. Interestingly, with the exception of brain, these values appear to be in line with the SB ratio of C , of ET in rat plasma, which was 3.12 1.13 a t 20 mgh.4 This observation seems to support the notion that the free drug concentration in plasma is the major controlling factor in the distribution of ET to tissues. Some of these findings parallel those seen in the disposition of ET into the synovial fluid of five arthritic subjects.3Twelve hours after receiving 200 mg of ET, concentrations of S-,but
*
0 -
0.4
Table Il-NADP+-Dependent Microsomal Oxidation of Etodolac Enantiomersa
Time, h 0 4
Liver Microsornes
Kidney Microsomes
S
R
SR
S
1.OO 2 0.023 0.74 -C 0.078’
1.OO 2 0.027 0.48 0.044b*c
1.OO 2 0.039
0.98 k 0.031 1.06 0.069
*
1.55
* 0.066b
*
R 1.00 0.79
2 ?
0.13 0.031
SR 1.00 k 0.15 1.34 2 0.10’
’
a Each value represents the mean concentration (pglmL)* SD of four determinations. Significantly different from 0 h. Significantlydifferent from Senantiomer.
not R-ET, were significantly greater in synovial fluid than in plasma. In both ET-spiked human synovial fluid and in rat tissues there were greater S:R concentration ratios than in plasma, and a greater binding of R- than S-ET. There was, however, a greater binding of enantiomers to drug-spiked human synovial fluid (96 and 97% for S- and R-ET, respectively a t 10 mg/L of enantiomer) than to the diluted rat tissue homogenates (Figure 2). This may be due to the presence of considerable concentrations of albumin (the major plasma protein implicated in binding of NSAIDs) in synovial fluid. The tissue binding results should be viewed with some degree of caution, as the dilution step might have altered the binding characteristics of the tissues. Although dilution might compromise the ability of the binding experiments to determine the absolute degree of binding of ET to tissues, the experiments do reflect the relative binding affinity of enantiomers to tissues. With respect to in vitro glucuronidation, a good parallel was seen between the maximal rate of glucuronidation by hepaticmicrosomal glucuronyl transferases, and the previously published data on biliary excretion of conjugates in the rat.4 With the K,,, values seen, glucuronidation of neither enantiomer is saturated a t the concentrations seen in plasma during the post-distributive phase following a lO-mgkg iv dose. The mean S:R ratio for cumulative excretion of conjugates in bile was 3.2. A similar S:R ratio in V,,, for in vitro glucuronidation (3.46 2 0.45) was seen. These similarities appear to suggest that the differences in biliary excretion of enantiomers as conjugates are due to enzymatic velocity, and not to secretion of conjugates into bile. Previously, it was shown that virtually the complete dose of S-ET could be recovered as conjugated enantiomer in rat bile following iv administration.4 However, only 33% of the R-enantiomer was likewise recovered. Etodolac had previously been reported to undergo microsomal oxidative metabolism in the rat;s hence it was thought that the R-enantiomer is predominately biotransformed to those metabolites. Although we were unable to assay these metabolites, and pure oxidized metabolites were not available for assay development, we nevertheless felt that by determining the disappearance of ET enantiomers in the presence of an NADP’ microsomal oxidizing system, insight could be gained into the oxidative metabolism of ET. As hypothesized, concentrations of R-ET fell significantly faster than concentrations of S-ET after 4 h of incubation in the presence of hepatic microsomes (Table 11). Concentrations of S-ET were also significantly reduced afier 4 h, suggesting that it too was capable of being metabolized by hepatic microsomes. Given the high rate of formation of glucuroconjugated S-ET, however, which is further enhanced by a lower extent of plasma protein binding, glucuronidation is the major metabolic fate of the S-enantiomer. This is confirmed by the virtual complete recovery of S-ET in bile as conjugated drug.4 An unclear situation appeared in the kidney oxidative microsomal incubations (Table 111, in which a significant increase in the S:R concentration ratio and a non-statistically
significant reduction in R-ET concentrations were seen. Despite these contradictory findings, it is likely that there was some oxidation of R-ET by kidney microsomal protein, because use of S:R ratios involves a statistical pairing of the R- with S-ET concentrations, which in turn minimizes the variation present among both sets of data. Nevertheless, confirmation of the findings from both liver and kidneys microsomes, using an assay which can measure the formation of the oxidized metabolites, would be most desirable. In conclusion, ET is an NSAID that exhibits a high degree of stereoselectivity in its pharmacokinetics. The higher concentrations of R- than S-ET in rat plasma can be attributed to a broader distribution of S-ET, owing to its lower degree of plasma protein binding and to its faster rate of metabolism, particularly to glucuronide conjugates. In contrast, R-ET appears to undergo preferential metabolism to oxidized metabolites in the rat, although this occurs at a slower rate than conjugation of S-ET. In humans, the relative contribution of these pathways of metabolism are different from the rat, as in the former species most of the urinary recovery consists of oxidized metabolites.llJ2 The relative concentrations of ET enantiomers observed in plasma are not reflective of those in the tissues, which may be of importance in the context of pharmacodynamic and toxicological studies involving the individual enantiomers following administration of racemic doses.
References and Notes 1. Jamali, F.; Mehvar, R.; Lemko, C.; Eradiri, 0. J . Pharm. Sci. 1986, 77,963-966. 2. Singh, N. N.; Jamali, F.; Pasutto, F. M.; Coutts, R. T. J . Chromatogr. 1986,382,331337. 3. Brocks, D. R.; Jamali F.; Russell A. S. J . Clin. Pharmncol., in press. 4. Brocks, D. R.; Jamali, F. Drug Metab. Dispos. 1990,18,471-475. 5 . Humber, L. G.; Demerson, C. A.; Swaminathan, P.;Bird, P. H. J. Med. Chem. 1986,29,871-874. 6. Jamali, F. Eur. J . Drug Metubol. Phmmacokinet. 1988,13, 1-9. 7. Cayen, M. N.; Kraml, M.; Ferdinandi, E. S.; Greselin, E.; Dvornik, D. Drug Metub. Rev. 1981,12,339-362. 8. Ferdinandi, E.S.; Cochrane, D.; Gedamke, R. Drug Metub. D~POS 1987,15,921-924. . 9. Lowry, 0.H.;Rosebrough, N. J.;Farr, A. J.;Randall R. J. J . Biol. Chem. 1951,193,265-275. 10. Yamaoka, K.; Tanigawara, Y.; Nakagawa, T.; Uno, T. J . Pharm. Dyn. 1981,4,879-885. 11. Ferdinandi, E. S.;. Sehgal, S. N.; Demerson, C: 4.; Dubuc, J.; Zilber, J.; Dvornik, D.; Cayen, M.N. Xenobzotica 1986, 16, 153-166. 12. Humber, L. G.; Ferdinandi, E.; Demerson, C. A.; Ahmed, S.; Shah, U., et al. J . Med. Chern. 1988,31, 1712-1719.
Acknowledgments This work was supported by the Medical Research Council of Canada (Grant MA 9569) and was presented in art at the American Association of Pharmaceutical Scientists 5th lnnual Meeting and Exposition, Las Ve as, NE, November 44,1990. D. R. Brocks 18 the reci ient of the Alferta Heritage Foundation for Medical Research Stugentship Award.
Journal of Pharmaceutical Sciences I 1061 Vol. 80, No. 77, November 7997
