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Interaction of disopyramide enantiomers for sites on plasma protein
Life Sciences, Vol. 41, pp. 2807-2813 Printed in the U.S.A.
Pergamon Journals
INTERACTION OF DISOPYRAMIDE ENANTIOMERS FOR SITES ON PLASMA PROTEIN John J. Lima Divisions of Pharmacy Practice and Cardiology Colleges of Pharmacy and Medicine Ohio State University, Columbus (Received in final form October 27, 1987) Summary
The binding of disopyramide enantiomers to donor plasma to which had been added human alpha-I acid glycoproteln (AAG) was characterized alone and in _The presence of the opposite enantiomer. At pre-dialysls concentrations of I0 ~M, S(+)-dlsopyramide increased the percent of R(-)-disopyramide free (unbound) 2.6-fold from II to 30%. At similar pre-dialysis concentrations, R(-)-disopyramide increased the percent of S(+)-disopyramide free 2-fold from 4.1 to 9.0%. Differences in the binding of one enantiomer due to the presence of the other were due to apparent changes in association constant; no changes in capacity to bind the enantlomers were observed. It is concluded that the enantlomers of disopyramide compete with each other for one site on AAG.
The stereoselective binding of drugs and other xenobiotics to human plasma protein is well known (1-3). Human serum albumin (HSA) has at least two sites, the diazepam and the warfarin sites, which bind drugs stereoselectively (4,5). Human alpha 1-acid glycoprotein (AAG) apparently has one site (6-9) which binds some basic drugs stereoselectively (10-13). It has been hypothesized that stereoselectlve differences in binding occur because the site in question, although having the same capacity to bind the isomers, has a higher affinity for one of them (i0). If so, it may be further hypothesized that the isomers will compete with each other for the same site on protein. The purpose of this report is to test these hypotheses using the enantlomers of disopyramide as model compounds. Disopyramide, a basic antlarrhythmic drug (14), was chosen because it is stereoselectively bound to human AAG (i0) and is not, or is only slightly bound to other plasma protein (15,16).
Methods
Protein. Drug free donor plasma (American Red Cross, Columbus, OH) which had been stored in a plastic container at 4°C for at least one month, was dialyzed against phosphate buffer containing activated charcoal as previously described (17). Charcoal treatment removes compounds which may inhibit the binding of disopyramlde and other basic drugs to plasma protein (18,19). i00 mg of human AAG (Sigma Chemical Co., St Louis, MO) was added to I00 ml of charcoal-treated donor plasma to expand the concentration range at which the binding of disopyramlde is relatively constant. Preparation of disopyramide enantlomers. Unlabelled racemic disopyramide (Sigma Chemical Co.) was separated into its S(+) and R(-) enantiomers as previously described (I0). ~C-labelled racemic disopyramide (specific Copyright
0024-3025/87 $3.00 + .00 (c) 1987 Pergamon Journals Ltd.
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Disopyramide Enantiomer Interaction
Vol. 41, No. 26, 1987
~ t i v l t y : 5.16 uC/.53 mg) was generously s u p p l ~ d by Searle Inc., (Skokle, Ii). ~C S(+)-disopyramlde was separated from ~ C R(-)-disopyramide by high performance liquid chromatography (HPLC) using the LKB EnantloPac HPLC Column (LKB Instruments, Inc., Gaithersburg, MD) (20). The column was attached to a Varlan HPLC Model 2010 pump (Walnut Creek, CA) equipped with a Varlan Model 2050 variable wave length detector set at 254 nm. A similar procedure has been used to separate the enantlomers of radlolabelled verapamll (21). The mobile phase consisted Of 6% 2-propanol in a phosphate buffer, pH 5.9, containing 8mM dibasic sodium phosphate, 8mM monobaslc sodium phosphate an~40.05 M sodium chloride. The flow rate was 0.3 ml/min. Five ul of racemic ---C-disopyramlde was injected onto the column using a Model 7125 Rheodyne injecter port (Cotatl, CA). The HPLC effluent corresponding to R(-)- and S(+)-disopyramide peaks on the chromatogram was collected. The .elution order of the two radiolabelled enantiomers following injection of i4C racemic dlsopyramide was confirmed using the unlabeled enantiomers. Nine-seven percent of the radioactivity injected onto the column was collected as disopyramide enantiomers, and 50.0% corresponded to the S(+) and R(-) enantlomers, respectively. Elutlon times (time required to collect the effluent corresponding to its chromatographic peak) of R(-)- and S(+)-disopyramide were 8.8 and 13.2 minutes, respectively. HPLC effluents corresponding to one enantiomer were pooled and combined an equal volume of anhydrous ether (Baker Chemical Co., Phillipsburg, NJ) one ml of IN sodium hydroxide. The mixture was shaken for 20 minutes, aqueous layer removed, and the ether layer was evaporated to dryness. residue was reconstituted with two ml of water containing 10% methanol. other enantiomer was treated in an identical manner.
with and the The The
The influence of the column separation and extraction on the binding p r o p e r t i ~ of disopyramlde was assessed by comparing the binding of u n e x t r a ~ e d racemic "~C-disopyramide (U), extracted (as described above) racemlc ~ C disopyramide (E), and pseudo-racemic C-disopyramide (P) to protein in freshly drawn serum. The latter P, was prepared by combining equal volumes of solutions containing equal amounts of radiolabelled S(+) and R(-) enantlomers which had been separated and extracted as described above. The mean (SD) free fractions (fu) of U, E and P were 0.130 (0.004), 0.117 (0.007) and 0.121 (0.011), respectively. Three way analysis of variance (22) showed that the differences in fu between E and P were not statistically significant. In contrast, the fu of U was less than that of E, but not different from the fu of P. These small differences are probably due to the presence of impurities in U, and should not influence the results of our studies. I~ Protein Binding. Volumes (0.8 ml) of charcoal-treated plasma containing ~C-S(+)-disopyramide were dialyzed against equal volumes of buffer to which had been ad_~d the u n l ~ e l l e d S(+) enantlomer at various concentrations ranging between I0 M and I0 M. The experiment was repeated in the. presence ~of • -D -D R(-)-disopyramlde at a pre-dlalysls buffer concentratlonsl~f I0 M and I0 M. Volumes (0.8 ml) of charcoal-treated plasma containing ~C-R(-)-disopyramide were dialyzed against equal volumes of buffer to which had been added the unl~belled R(-) enantlomer at various concentrations ranging between 10-~M and i0- M. The experiment was repeated in t~e presenc E of S(+)-disopyramlde at a predialysis buffer concentrations of lO-VM and IO-'M. Equal volumes (0.6 ml) of post-equilibrlum buffer and plasma were pipetted into liquid scintillation vials containing I0 ml of aqueous counting cocktail (Formula-963, New England Nuclear, Boston MA). Radioactivity was counted on a Beckman model 8100 liquid s c l n t i ~ t l o n counter. Quenching was corrected for by internal standardization using C-toluene (Amersham, Arlington Height, IL). The details of the equilibrium dialysis technique employed in this study have been published (17). The free fraction (fu) of dlsopyramide was determined by: fu
= Bdpm/Pdpm
Vol. 41, No. 26, 1987
Disopyramide Enantiomer Interaction
2809
where Bdpm and Pdpm refer to disintegrations per minute of radioactivity in buffer (B) and plasma (P) following equilibration. The percent of dlsopyramide unbound (free) (Pf) was determined by: Pf = fu,lO0 Modified Scatchard plots were constructed according to: CB/CU = N, Ka - Ka.CB where CB and CU are the bound and unbound (free) concentrations of disopyramide, respectively. Total concentrations of disopyramide in postdialysis plasma is the sum of CB and CU. N and Ka are the respective capacity and affinity constants which characterize the the binding of disopyramide to site(s) on protein. These constants were used as estimates, and the percent binding (I00 * (l-fu)), post-equillbrlum enantiomer concentrations provided input for the computer program MACMOL (23). This program provides least square estimates of N and Ka for each independent, equivalent site on protein. The details of the analysis of the binding data have been published (19).
Results
Modified Scatchard plots in Figure I show that S(+)- and bin~MtO one site. Pre-dlalysis S(+)- and R(+)-disopyramide i0 had no apparent effect on the slope and intercept Scatchard plots of R(-)- and S(+)-disopyramide. The slopes the S(+) and R(-) enantiomer plots were decreased by t.~ opposing enantlomers at pre-dlalysis concentrations of 10-~M.
R(+)-disopyramide concentrations at of the modified and intercepts of presence of their
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FIG. i Left panel: Modified Scatchard plots of the binding of S(+)-disopyramide alone ( a ~ and in the presence of R(-)-disopyramide at pre-dialysls concentrations of I0- M ( O ) and IO--M ( • ). Right panel. Plots of the binding of R(-)-disopyramide alone ( • ) ~ n d in the pr@sence S(+)-dlsopyramide at predialysis concentrations of lO-VM ( O ) and IO-JM ( A ) .
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Disopyramide Enantiomer Interaction
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Figure 2 shows that the binding of both enantio~ers was concentrationindependent (linear) at total concentrations of 3 x IO-°M or lower. Binding of both enantiomers was concentration-dependent (non-llnear) at higher total concentrations. The mean (SD) P of the S(+) enantlomer at concentrations where binding was linear, alone an~ in the presence of R(-)-enantiomer at I0- M were 4.08 (0.520) and 4.21 (0.249) %, respectively, and were s ~ i l a r . The presence of R(-)-disopyramide at pre-dialysis concentrations of i0- M increased the mean Pf of the S(+) enantiomer about 2-fold to 9.02% (0.553) (P < 0.0001). The mean P~ of the R(-) enantiomer at concentrations where binding was linear, alone ~nd in the presence of S(+)-disopyramide at pre-dialysis concentrations of 10-VM were 11.4 (0.700) and 12.2 (0.571) %, respectively, and were similar. The presence of S(+)-disopyramide at pre-dialysis concentrations of 10-JM increased the mean Pf of the R(-) enantiomer 2.6-fold to 30.0 (1.56) % (P <
0.0001).
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TOTAL C O N C E N T R A T I O N FIG. 2 Percent of S(+)-disopyramlde free at various post-di.~ysis concentrations alone or in the presence of the R(-) enantlomer at I0- M ( • ) , an~ in the presence of R(-)-disopyramide at a pre-dialysis concentration of 10-~M ( ~ ) ; percent of R(-)-disopyramide at various post-.~alysls concentrations alone or in the presence of the S(+) enantlomer at I0- M ( 1 ) , ~nd in the presence of S(+)-disopyramide at a pre-dialysis concentration of 10-~M ( O ) . Since the binding of both enantiomers was u_n~ffected by their opposite enantiomers at pre-dialysls concentrations of I0- M, these data were pooled with the data observed in the absence of the opposite enantlomer. Table i shows that the differences in binding of the enantiomers observed in the absence~and presence of the opposing enantlomer at pre-dialysis concentrations of I0 -M were due to an apparent decrease in the affinity constant. S(+)-disopyramide apparently decreased the Ka for the R(-) enantlomer 170%, R(-)-disopyramide apprently decreased the Ka for S(+) enantiomer 45%.
Vol. 41, No. 26, 1987
Disopyramide Enantiomer Interaction
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Table i. Binding constants of disopyramide enantiomers in presence and absence of each other. Enantiomer
s(+) s(+) R(-) R(-)
Inhibitor* concentration,
N x
106M Ka x 10-6M-1
M
-6 0,i0 10 -5
12 i0
2.0 I.I
0,10-~
12
0.63
I0 -~
I0
0.23
* pre-dialysis
Discussion
It may be concluded from the results of the present study that the enantlomers of disopyramide compete with each other for one site on human AAG. This conclusion is based on the following: (i) the affinity constants characterizing the interaction between the site and each of the enantlomers were apparently decreased in th~ presence of the opposing enantlomer at predialysis concentrations of 10--M; (ll) the capacity of AAG to bind both enantiomers was unaffected by the presence of the opposing enantlomer; (ill) the site on AAG had a higher affinity for S(+)-dlsopyramlde than for the R(-) enantlomer, and the S(+) enantlomer was the more potent displacer; (iv) the enantlomer-enantlomer interaction was dependent on the concentration of the displacer. The results of computer simulations using a competetive inhibition model predicted that the binding of disopyramlde enantlomers would be unaffected by the ~ e s e n c e of their opposing enantlomers at. pre-dlalysis concentrations of i0 VM; at a pre-dialysls concentration of 10-DM, the model predicted a significant enantiomer-enantlomer interaction. The experimental results were consistent with those predicted by computer simulations. If the enantiomer-enantlomer interaction observed in the present in-vitro study occurs in-vivo, the pharmacokinetics of the enantiomers following separate administration will differ from those following the administration of racemie disopyramide. The results of three studies are consistent with this hypothesis. The area under the plasma concentration time curve (AUC) of S(+)-disopyramide was higher than the AUC of R(-)-disopyramide following the oral administration of racemlc disopyramide (20), which suggest that the plasma clearance of the R(-) enantiomer was higher than that of the S(+) enantiomer. Yet in another study, the AUC and plasma clearance of both enantlomers were similar following the intravenous administration of each enantlomer on separate occasions (24). Giacomlnl et al (25) reported that although the plasma clearance of disopyramide enantlomers following separate administrations were equal, the plasma clearance of R(-)-disopyramlde was significantly higher than that of S(+)-disopyramide following the administration of racemlc dlsopyramlde. These discrepancies are consistent with an enantlomer-enantlomer interaction, and may be explained in the following way: the plasma clearance of disopyramide enantlomers varies with fu, and when given on separate occasions, are approximately equal (24,25). The fu of the R(-) enantlomer is higher in the presence of of S(+)-disopyramide than in its absence (because S(+) displaces R(-) from binding sites), and the clearance of R(-)-disopyramide is higher following the administration of the racemate as compared to that following separate administration. Thus it may be concluded from the results of this and other studies (20, 24,25) that one cannot predict the
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Disopyramide Enantiomer Interaction
Vol. 41, No. 26, 1987
pharmacoklnetlc behavior of the enantlomers of disopyramlde followln@ administration of the racemate from the pharmacokinetlc behavior of the individual enantiomers following separate administration. Whether or not this interaction occurs with other pairs of enantlomers is unknown, and requires further investigation. It is well accepted that the intensity and duration of response to a drug is better related to its unbound rather than to total plasma concentrations (19). Although the enantlomer-enantiomer interaction observed in the present study may influence the pharmacokinetlcs of total plasma concentrations, unbound concentrations of, and response associated with each enantiomer are not expected to differ in the absence or presence of each other. In contrast, such an interaction between two enantiomers whose plasma clearances are not affected by plasma protein binding would not be expected to influence total plasma concentrations, but would be expected influence unbound concentrations and response (26).
Acknowledgement
Supported
in part by the American Heart Association,
Eastern Ohio Chapter.
References I. T. AIEBIC-KOLBAH, S. RENDIC, S. FUKS, V. SUNJIC and F.KAJFEZ, Acta. Pharm. Jugoslav, 29 53-70 (1979). 2. E. JAHNCHEN and W.E. MULLER, Topics in Pharmaceutical Sciences(D.D. BREIMER and P. SPEISER, eds.),pp. 109-117,(1983). 3. D. DRAYER, Clin. Pharmacol. Ther.40 125-133(1986). 4. I.SJOHOLM, B.EKMAN, A.KOBER~ I.LJUNGSTEDT-PAHLMAN, B.SEIVING and T.SJODM, Mol. Pharmacol. 16 767-777(1979). 5. K.J.FEHSKE, W.E.MULLER and U.WOLLERT, Biochem. Pharmacol. 30 687-692(1981). 6. S.EI-GAMAL, U.WOLLERT and W.E.MULLER, J. Pharm. Pharmacol. 34 152-157(1982). 7. W.E.MULLER and A.E.STILLBAUER, Naunyn-Schmiedeberg's Arch. Pharmacol. 322 170-173(1983). 8. W.E.MULLER, A.E.STILLBAUER and S.EI-GAMAL J. Pharm. Pharmacol. 35 684-686(1983). 9. F.BRUNNER and W.E.MULLER, J. Pharm. Pharmacol. 37 305-309(1985). I0. J.J.LIMA, G.L.JUNGBLUTH, T.DEVINE and L.W.ROBERTSON, Life Scl. 35 835-839(1984). Ii. F.ALBANI, R.RIVA, M.CONTIN and A.BARUZZI, Br. J. Clin. Pharmac. 18 244-246(1984). 12. D.OFORI-ADJEI, O.ERICSSON, B.LINDSTROM and F.SJOQVIST, Br. J. Clin. Pharmacol. 22 356-358(1986). 13. S.B.EARLE and J.J.MACKICHAN, Clln. Pharmacol. Ther. 41 233(1987). 14. F.MORADY, M.M.SCHEINMAN and J.DESAI, Ann. Int. Med. 96 337-343(1982). 15. E.PIKE,P.KIERULF,B.SKUTERUD,J.E.BREDESEN and P.K.M.LUNDE, Br. J. Clln. Pharmaeol. 16 233-239(1983). 16. J.J. LIMA and L. SALZER, Biochem. Pharmacol. 30 2633-2636(1981). 17. J.J. LIMA, J.J. MACKICHAN, N. LIBERTIN and J.R. SABINO~ J. Pharmacokln. Biopharm. ii 483-498 (1984). 18. O. BORGA, K.M. PIAFSKY and O.G. NILSEN, Clln. Pharmacol. Ther. 22 539-544(1977). 19. J.J. LIMA, H. BOUDOULAS and B.F. BLANFORD, J. Pharmacol. Exp. Ther. 219 741-747(1981). 20. J. HERMANSSON and M. ERIKSON, J. Chromatography. 336 321-328(1984). 21. S.B. EARLE and J.J. MACKICHAN. Submitted, 1987.
Vol. 41, No. 26, 1987
22. 23. 24. 25. 26.
Disopyramide Enantiomer Interaction
2813
SAS User's Guide: STATISTICS, Version 5 Edition, SAS Institute Inc., pp. 113-138,(1985). R.L. PRIORE and H.E. ROSENTHAL, Anal. Biochem. 70 231-240 (1976). J.J. LIMA, H. BOUDOULAS and B.J. SHIELDS, Drug. Met. Dis. 13 572-577(1985). K.M. GIACOMINI, W.L. NELSON, R.A. PERSHE, L. VALDIVIESO, K. TURNER-TAMIYASU and T.F. BLASCHKE, J. Pharmacokin. Biopharm. 14 335-356 (1986). J.J. MACKICHAN, Clln.Pharmacokin. 9 32-41 (1984),
Pergamon Journals
INTERACTION OF DISOPYRAMIDE ENANTIOMERS FOR SITES ON PLASMA PROTEIN John J. Lima Divisions of Pharmacy Practice and Cardiology Colleges of Pharmacy and Medicine Ohio State University, Columbus (Received in final form October 27, 1987) Summary
The binding of disopyramide enantiomers to donor plasma to which had been added human alpha-I acid glycoproteln (AAG) was characterized alone and in _The presence of the opposite enantiomer. At pre-dialysls concentrations of I0 ~M, S(+)-dlsopyramide increased the percent of R(-)-disopyramide free (unbound) 2.6-fold from II to 30%. At similar pre-dialysis concentrations, R(-)-disopyramide increased the percent of S(+)-disopyramide free 2-fold from 4.1 to 9.0%. Differences in the binding of one enantiomer due to the presence of the other were due to apparent changes in association constant; no changes in capacity to bind the enantlomers were observed. It is concluded that the enantlomers of disopyramide compete with each other for one site on AAG.
The stereoselective binding of drugs and other xenobiotics to human plasma protein is well known (1-3). Human serum albumin (HSA) has at least two sites, the diazepam and the warfarin sites, which bind drugs stereoselectively (4,5). Human alpha 1-acid glycoprotein (AAG) apparently has one site (6-9) which binds some basic drugs stereoselectively (10-13). It has been hypothesized that stereoselectlve differences in binding occur because the site in question, although having the same capacity to bind the isomers, has a higher affinity for one of them (i0). If so, it may be further hypothesized that the isomers will compete with each other for the same site on protein. The purpose of this report is to test these hypotheses using the enantlomers of disopyramide as model compounds. Disopyramide, a basic antlarrhythmic drug (14), was chosen because it is stereoselectively bound to human AAG (i0) and is not, or is only slightly bound to other plasma protein (15,16).
Methods
Protein. Drug free donor plasma (American Red Cross, Columbus, OH) which had been stored in a plastic container at 4°C for at least one month, was dialyzed against phosphate buffer containing activated charcoal as previously described (17). Charcoal treatment removes compounds which may inhibit the binding of disopyramlde and other basic drugs to plasma protein (18,19). i00 mg of human AAG (Sigma Chemical Co., St Louis, MO) was added to I00 ml of charcoal-treated donor plasma to expand the concentration range at which the binding of disopyramlde is relatively constant. Preparation of disopyramide enantlomers. Unlabelled racemic disopyramide (Sigma Chemical Co.) was separated into its S(+) and R(-) enantiomers as previously described (I0). ~C-labelled racemic disopyramide (specific Copyright
0024-3025/87 $3.00 + .00 (c) 1987 Pergamon Journals Ltd.
2808
Disopyramide Enantiomer Interaction
Vol. 41, No. 26, 1987
~ t i v l t y : 5.16 uC/.53 mg) was generously s u p p l ~ d by Searle Inc., (Skokle, Ii). ~C S(+)-disopyramlde was separated from ~ C R(-)-disopyramide by high performance liquid chromatography (HPLC) using the LKB EnantloPac HPLC Column (LKB Instruments, Inc., Gaithersburg, MD) (20). The column was attached to a Varlan HPLC Model 2010 pump (Walnut Creek, CA) equipped with a Varlan Model 2050 variable wave length detector set at 254 nm. A similar procedure has been used to separate the enantlomers of radlolabelled verapamll (21). The mobile phase consisted Of 6% 2-propanol in a phosphate buffer, pH 5.9, containing 8mM dibasic sodium phosphate, 8mM monobaslc sodium phosphate an~40.05 M sodium chloride. The flow rate was 0.3 ml/min. Five ul of racemic ---C-disopyramlde was injected onto the column using a Model 7125 Rheodyne injecter port (Cotatl, CA). The HPLC effluent corresponding to R(-)- and S(+)-disopyramide peaks on the chromatogram was collected. The .elution order of the two radiolabelled enantiomers following injection of i4C racemic dlsopyramide was confirmed using the unlabeled enantiomers. Nine-seven percent of the radioactivity injected onto the column was collected as disopyramide enantiomers, and 50.0% corresponded to the S(+) and R(-) enantlomers, respectively. Elutlon times (time required to collect the effluent corresponding to its chromatographic peak) of R(-)- and S(+)-disopyramide were 8.8 and 13.2 minutes, respectively. HPLC effluents corresponding to one enantiomer were pooled and combined an equal volume of anhydrous ether (Baker Chemical Co., Phillipsburg, NJ) one ml of IN sodium hydroxide. The mixture was shaken for 20 minutes, aqueous layer removed, and the ether layer was evaporated to dryness. residue was reconstituted with two ml of water containing 10% methanol. other enantiomer was treated in an identical manner.
with and the The The
The influence of the column separation and extraction on the binding p r o p e r t i ~ of disopyramlde was assessed by comparing the binding of u n e x t r a ~ e d racemic "~C-disopyramide (U), extracted (as described above) racemlc ~ C disopyramide (E), and pseudo-racemic C-disopyramide (P) to protein in freshly drawn serum. The latter P, was prepared by combining equal volumes of solutions containing equal amounts of radiolabelled S(+) and R(-) enantlomers which had been separated and extracted as described above. The mean (SD) free fractions (fu) of U, E and P were 0.130 (0.004), 0.117 (0.007) and 0.121 (0.011), respectively. Three way analysis of variance (22) showed that the differences in fu between E and P were not statistically significant. In contrast, the fu of U was less than that of E, but not different from the fu of P. These small differences are probably due to the presence of impurities in U, and should not influence the results of our studies. I~ Protein Binding. Volumes (0.8 ml) of charcoal-treated plasma containing ~C-S(+)-disopyramide were dialyzed against equal volumes of buffer to which had been ad_~d the u n l ~ e l l e d S(+) enantlomer at various concentrations ranging between I0 M and I0 M. The experiment was repeated in the. presence ~of • -D -D R(-)-disopyramlde at a pre-dlalysls buffer concentratlonsl~f I0 M and I0 M. Volumes (0.8 ml) of charcoal-treated plasma containing ~C-R(-)-disopyramide were dialyzed against equal volumes of buffer to which had been added the unl~belled R(-) enantlomer at various concentrations ranging between 10-~M and i0- M. The experiment was repeated in t~e presenc E of S(+)-disopyramlde at a predialysis buffer concentrations of lO-VM and IO-'M. Equal volumes (0.6 ml) of post-equilibrlum buffer and plasma were pipetted into liquid scintillation vials containing I0 ml of aqueous counting cocktail (Formula-963, New England Nuclear, Boston MA). Radioactivity was counted on a Beckman model 8100 liquid s c l n t i ~ t l o n counter. Quenching was corrected for by internal standardization using C-toluene (Amersham, Arlington Height, IL). The details of the equilibrium dialysis technique employed in this study have been published (17). The free fraction (fu) of dlsopyramide was determined by: fu
= Bdpm/Pdpm
Vol. 41, No. 26, 1987
Disopyramide Enantiomer Interaction
2809
where Bdpm and Pdpm refer to disintegrations per minute of radioactivity in buffer (B) and plasma (P) following equilibration. The percent of dlsopyramide unbound (free) (Pf) was determined by: Pf = fu,lO0 Modified Scatchard plots were constructed according to: CB/CU = N, Ka - Ka.CB where CB and CU are the bound and unbound (free) concentrations of disopyramide, respectively. Total concentrations of disopyramide in postdialysis plasma is the sum of CB and CU. N and Ka are the respective capacity and affinity constants which characterize the the binding of disopyramide to site(s) on protein. These constants were used as estimates, and the percent binding (I00 * (l-fu)), post-equillbrlum enantiomer concentrations provided input for the computer program MACMOL (23). This program provides least square estimates of N and Ka for each independent, equivalent site on protein. The details of the analysis of the binding data have been published (19).
Results
Modified Scatchard plots in Figure I show that S(+)- and bin~MtO one site. Pre-dlalysis S(+)- and R(+)-disopyramide i0 had no apparent effect on the slope and intercept Scatchard plots of R(-)- and S(+)-disopyramide. The slopes the S(+) and R(-) enantiomer plots were decreased by t.~ opposing enantlomers at pre-dlalysis concentrations of 10-~M.
R(+)-disopyramide concentrations at of the modified and intercepts of presence of their
9.0 28 8.0
D D •
24
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20 (..o ,'r-,
16
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40
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80
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20
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40
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60
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FIG. i Left panel: Modified Scatchard plots of the binding of S(+)-disopyramide alone ( a ~ and in the presence of R(-)-disopyramide at pre-dialysls concentrations of I0- M ( O ) and IO--M ( • ). Right panel. Plots of the binding of R(-)-disopyramide alone ( • ) ~ n d in the pr@sence S(+)-dlsopyramide at predialysis concentrations of lO-VM ( O ) and IO-JM ( A ) .
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Disopyramide Enantiomer Interaction
Vol. 41, No. 26, 1987
Figure 2 shows that the binding of both enantio~ers was concentrationindependent (linear) at total concentrations of 3 x IO-°M or lower. Binding of both enantiomers was concentration-dependent (non-llnear) at higher total concentrations. The mean (SD) P of the S(+) enantlomer at concentrations where binding was linear, alone an~ in the presence of R(-)-enantiomer at I0- M were 4.08 (0.520) and 4.21 (0.249) %, respectively, and were s ~ i l a r . The presence of R(-)-disopyramide at pre-dialysis concentrations of i0- M increased the mean Pf of the S(+) enantiomer about 2-fold to 9.02% (0.553) (P < 0.0001). The mean P~ of the R(-) enantiomer at concentrations where binding was linear, alone ~nd in the presence of S(+)-disopyramide at pre-dialysis concentrations of 10-VM were 11.4 (0.700) and 12.2 (0.571) %, respectively, and were similar. The presence of S(+)-disopyramide at pre-dialysis concentrations of 10-JM increased the mean Pf of the R(-) enantiomer 2.6-fold to 30.0 (1.56) % (P <
0.0001).
60 I..d ILl rr u._ I-z I.d
40 0
g
0 0
~
0 1
0 n--
Ld
2o
0
I
10-7
2
,
; 4.
,
,, 6
, 8 10-6
2
, --,
, 4
6
,,,
I 810-5
TOTAL C O N C E N T R A T I O N FIG. 2 Percent of S(+)-disopyramlde free at various post-di.~ysis concentrations alone or in the presence of the R(-) enantlomer at I0- M ( • ) , an~ in the presence of R(-)-disopyramide at a pre-dialysis concentration of 10-~M ( ~ ) ; percent of R(-)-disopyramide at various post-.~alysls concentrations alone or in the presence of the S(+) enantlomer at I0- M ( 1 ) , ~nd in the presence of S(+)-disopyramide at a pre-dialysis concentration of 10-~M ( O ) . Since the binding of both enantiomers was u_n~ffected by their opposite enantiomers at pre-dialysls concentrations of I0- M, these data were pooled with the data observed in the absence of the opposite enantlomer. Table i shows that the differences in binding of the enantiomers observed in the absence~and presence of the opposing enantlomer at pre-dialysis concentrations of I0 -M were due to an apparent decrease in the affinity constant. S(+)-disopyramide apparently decreased the Ka for the R(-) enantlomer 170%, R(-)-disopyramide apprently decreased the Ka for S(+) enantiomer 45%.
Vol. 41, No. 26, 1987
Disopyramide Enantiomer Interaction
2811
Table i. Binding constants of disopyramide enantiomers in presence and absence of each other. Enantiomer
s(+) s(+) R(-) R(-)
Inhibitor* concentration,
N x
106M Ka x 10-6M-1
M
-6 0,i0 10 -5
12 i0
2.0 I.I
0,10-~
12
0.63
I0 -~
I0
0.23
* pre-dialysis
Discussion
It may be concluded from the results of the present study that the enantlomers of disopyramide compete with each other for one site on human AAG. This conclusion is based on the following: (i) the affinity constants characterizing the interaction between the site and each of the enantlomers were apparently decreased in th~ presence of the opposing enantlomer at predialysis concentrations of 10--M; (ll) the capacity of AAG to bind both enantiomers was unaffected by the presence of the opposing enantlomer; (ill) the site on AAG had a higher affinity for S(+)-dlsopyramlde than for the R(-) enantlomer, and the S(+) enantlomer was the more potent displacer; (iv) the enantlomer-enantlomer interaction was dependent on the concentration of the displacer. The results of computer simulations using a competetive inhibition model predicted that the binding of disopyramlde enantlomers would be unaffected by the ~ e s e n c e of their opposing enantlomers at. pre-dlalysis concentrations of i0 VM; at a pre-dialysls concentration of 10-DM, the model predicted a significant enantiomer-enantlomer interaction. The experimental results were consistent with those predicted by computer simulations. If the enantiomer-enantlomer interaction observed in the present in-vitro study occurs in-vivo, the pharmacokinetics of the enantiomers following separate administration will differ from those following the administration of racemie disopyramide. The results of three studies are consistent with this hypothesis. The area under the plasma concentration time curve (AUC) of S(+)-disopyramide was higher than the AUC of R(-)-disopyramide following the oral administration of racemlc disopyramide (20), which suggest that the plasma clearance of the R(-) enantiomer was higher than that of the S(+) enantiomer. Yet in another study, the AUC and plasma clearance of both enantlomers were similar following the intravenous administration of each enantlomer on separate occasions (24). Giacomlnl et al (25) reported that although the plasma clearance of disopyramide enantlomers following separate administrations were equal, the plasma clearance of R(-)-disopyramlde was significantly higher than that of S(+)-disopyramide following the administration of racemlc dlsopyramlde. These discrepancies are consistent with an enantlomer-enantlomer interaction, and may be explained in the following way: the plasma clearance of disopyramide enantlomers varies with fu, and when given on separate occasions, are approximately equal (24,25). The fu of the R(-) enantlomer is higher in the presence of of S(+)-disopyramide than in its absence (because S(+) displaces R(-) from binding sites), and the clearance of R(-)-disopyramide is higher following the administration of the racemate as compared to that following separate administration. Thus it may be concluded from the results of this and other studies (20, 24,25) that one cannot predict the
2812
Disopyramide Enantiomer Interaction
Vol. 41, No. 26, 1987
pharmacoklnetlc behavior of the enantlomers of disopyramlde followln@ administration of the racemate from the pharmacokinetlc behavior of the individual enantiomers following separate administration. Whether or not this interaction occurs with other pairs of enantlomers is unknown, and requires further investigation. It is well accepted that the intensity and duration of response to a drug is better related to its unbound rather than to total plasma concentrations (19). Although the enantlomer-enantiomer interaction observed in the present study may influence the pharmacokinetlcs of total plasma concentrations, unbound concentrations of, and response associated with each enantiomer are not expected to differ in the absence or presence of each other. In contrast, such an interaction between two enantiomers whose plasma clearances are not affected by plasma protein binding would not be expected to influence total plasma concentrations, but would be expected influence unbound concentrations and response (26).
Acknowledgement
Supported
in part by the American Heart Association,
Eastern Ohio Chapter.
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Disopyramide Enantiomer Interaction
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