CHIRALITY 424&246 (1992)
Stereoselective Binding of Etodolac to Human Serum Albumin NORLLE MULLER, FRANCOISE LAPICQUE, CLAUDINE MONOT, ELISABETH PAYAN, RJ%I DROPSY, AND PATRICK NETTER Luboratoire & P h u m o b g i e , URA CNRS 1288, Facult6 & Mk&cine, Vanaimwe les Nanq, and Laboratoire Wyeth-France,Park, France
ABSTRACT The protein binding of etodolac enantiomers was studied in vitro by equilibrium dialysis in human serum albumin (HSA) of various concentrations varying from 1to 40 g/liter, by addition of each enantiomer at increasing concentrations. In the 1 g/liter solution, at the lowest drug levels, the @)-form is more bound than its antipode, the contrary being observed at the highest drug levels. For higher albumin concentrations,S was bound in a larger extent than R. Using the displacement of specific markers of HSA sites I and 11, studied by spectrofluorimetry, it was suggested that R and S are both bound to site I, while only S is strongly bound to site 11. Q 1992 WiIey-Liss,Inc.
KEY WORDS: NSAID, chirality, enantiomers, protein binding, equilibrium dialysis, fluorescent specific markers
INTRODUCTION Between 10 and 15% of all marketed drugs are racemic mixtures of 2 or more stereoisomers, as for instancenonsteroidal antiinflammatory drugs (NSAID).In vitro studies have shown that the inhibition of the prostaglandins synthesis was only the fact of the (S)-enantiomer of chiral NSAID (often the dextrogyreone) and therefore that only this form was responsible for the antiinflammatoryactivity. The other form was considered as an impurity, eventually as a prodrug when it was able to be converted into the active form, and in a lesser extent as a toxic compound due to its incorporationinto adipose tissue in the case of inversion.24 Because of the chirality of human serum albumin (HSA), stereoselective differences in protein binding have been noted for the enantiomers of some chiral products, but this phenomenon cannot be generalized to all racemic drugs. The most important sites (sites I and 11) on HSA involved in the binding have been reported to be stereoselective for some drugs. Warfarin was bound to site I, and the (S)-formappeared to be more highly bound than the @)-enantiomer.5-7 L-Tryptophan was highly and stereospecificallybound at one site to HSA, with an affinity 100-fold greater than that of D-tryptophan. The ( + )-(S)-enantiomerof oxazepam hemisuccinate was bound about 30 times stronger than its antipode.9 NSAID are strongly bound to HSA [more than 90'3'0, with the exception of salicylate (80%)],especially to sites I and I1 and sometimesthis binding was shown to be stereoselective.Therefore, it would be interesting to establish a relationshipbetween protein binding and chiral drug action. Etodolac is a NSAID marketed as a racemic mixture, i.e., as an equal mixture of ( - )-(R)- and ( + )-(S)-enantiomers. lo The
( + )-(S)-enantiomer,responsible for the pharmacologicalactivity," represented 10% of the total plasma concentration and may be eliminated more rapidly in man than its antipode.I2 Etodolac seems unable to undergo chiral inversion due to the inclusion of the asymetric carbon into a pyrano ring.
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The present study concerned the protein binding of etodolac, as studied by equilibrium dialysis and spectrofluorimetry to show an eventual stereoselectivebinding of the enantiomers of etodolac (-)@) and (+)-(S),over a concentration range of HSA from 1 to 40 g/liter. MATERIAL AND METHODS Products Etodolac (enantiomers)was a gift from WYETH-France. Albumin (MW: 69000) was purchased from Sigma (reference A 1887) as essentially fatty acid free (below 0.005%), prepared from fraction V from HSA. Various concentrations of albumin (1, 2, 5, 10, 20, and 40 g/liter) were prepared in an isotonic Sijrensen phosphate buffer 0.067 M, pH 7.4. All the products used in the study were of analyticalgrade and water bidistilled; acetonitrile in the eluent was of chromatographic grade. Received for publication June 20, 1991; accepted November 15, 1991.
Presented at the Second International Symposium on Chiral
May 27-31, 1991, Rome, Italy. o 1992 Wiley-Liss, Inc.
Discrimination,
Address reprint requests to Patrick Netter, Labratoke de Pharmacologie, URA
CNRS 1288,Facult6 de Medecine, BP la,F-54505Vandceuvre les Nancy, Cedex, France.
241
STEREOSELECTIVE BINDING OF ETODOLAC TO HSA
The purity of each enantiomer was checked by the stereose- extraction of drug in buffer samples (volumesvarying from 20 to 900 pl, according to the estimated concentration) was lective assay proposed by Jamali et al. l2 achieved after acidification and the chromatographic condiEquilibrium Dialysis tions derived from those used in the simultaneous determination of several NSAIDs13; the major changes concerned the The free concentrationof ( - )-(R)-and ( +)-(S)-etodolacin the detection wavelength (227 nm), the eluent [acetonitrile/acetic various albumin solutions were determined in vitro by equilibrium dialysis. The concentrations of drug added varied from acid (0.3%);55:45 v/v] and the column (RadialPak Cis, 8 x 100 0.25 to 400 pg/ml for the solutions of albumin from 1 to 5 mm, Waters); these changes were done in order to obtain a g/liter,and from 1to 800 pg/ml for the other albumin solutions. better sensitivity and a shorter analysis time for the separate For each of the enantiomers, the experimentswere achieved in assay of etodolac. The assay was linear from 10 ng/ml to 100 duplicate or in triplicate. Equilibrium dialysis was realized pg/ml. The precision of the method for the lowest concentrausing Spectrapor 2 membranes, for 3 h, against a 67 mM tions was 5.4%.The binding values were corrected for volume Sijrensen isotonicphosphate buffer pH 7.4, in 1ml cells, at 37°C shift during equilibrium dialysis. l4 in a Dianorm apparatus. The time of 3 h was established in spf?ctrofluorim4?try preliminary studies to allow the equilibriumto be attained. The The spectroscopic measurements were carried out using a free concentration of each form of drug was determined by WLC as its concentration in the buffer after dialysis. The fluorescence spectrophotometerHitachi Lou-Perkin Elmer 240. Fluorescence (70)
k
l o90 o
a
50 40
30 20
i
10 --
T (PM)
Fluorcscence (%) '*O 100
T
b
80
60 40
20 -0 ,
T (PM)
242
MULLER ET AL.
No quenching of the single tryptophan residue (number 214) present in the albumin molecule could be achieved by etodolac (1, = 290 nm; h, = 340 nm) due to the intrinsic fluorescence of the drug. Besides, the location of the binding sites of drugs to protein can be studied using specific fluorescent probes such as dansylamide (DNSA) (site I) and dansylsarcosine (DS) (site 11).l5 The fluorescence emitted by the complex DNSA/HSA (h, = 340nm; hex = 480 nm) and DS (h, = 350 nm;hex = 480 nm) is modified by the addition of drug. It was verified that etodolac emitted no fluorescence at the wavelengths used for the displacement experiments. The spectrofluorimetricdisplacements were achieved using a 1 g/liter HSA solution, in which the marker (DNSA or DS) was added to a molar ligand/proteinratio of 1.The two enantiomeric forms of etodolac were then added and the corresponding molar ratio of drug/protein was varied from 0.025 to 5.
spectrofluorimetricmarkers displacements,and second to compare the binding characteristicsof enantiomersby equilibrium dialysis. Displacement of Site Markers When the drug was added, the complex formed between the site marker (DNSA or DS) and HSA induced a decrease in the fluorescence,corresponding to the displacement of the marker. DNSA was displaced by both of the enantiomers (up to 40%) (Fig. la). The displacement of DS was strongly realized by (+)-(S)etodolac(55%)and in a very low extent (less to 10% of fluorescencedecrease) by its antipode (Fig. lb).
Protein Binding of Et&lac Enantwmers The protein binding of etodolac enantiomers was found to depend on the concentration of HSA. In the 1g/liter solution, for small values of drug level, T (less than 15 pik? i.e, for Mathematical and Statistical Analysis druglprotein ratio below l),the (R)-enantiomer is bound more The binding parameters were determined using a Siphar than its antipode: for example, the free fractions of R and S software, installed on an IBM AT micro computer. The binding were respectively 12 and 24% for T varying from 0.7 to 2 pM, data are fitted by the Scatchard plot for two classes of indepen- and increased gradually up to 30% for both forms when T attained 15 pM. In the case of higher T value, the opposite dent sites described by the equation16 result was obtained, i.e., S was more tightly bound to HSA than R (Fig. 2). Table 1 established a comparison of the Scatchard parameters of the enantiomers:the values of nl ,%, and Fq! were similar; only the affinity constant kl was more important for R where F and B are the concentrations of free and bound drug than for S (P < 0.001). respectively, P is the albumin concentration, ki is the associaIn a lOg/liter HSA solution (Fig. 3), the protein binding of the tion constant, and ni is the number of sites of the ith class. The two enantiomers was quite similar, since no statistical differvalues of ni and ki are determined with their respective stan- ence was shown between the Scatchard parameters (Table 1). dard deviation. For higher albumin concentrations,S became more bound than The comparison between binding parameters was done R, as for example, in a 40 g/liter HSA solution, its free fraction using Student's t- test (a= 5%). The degree of significance, is always the lowest (Table2) and the total number of sites was P, is given for each test. greater for the active form than for its antipode (P = 0.05). In this case, when the added drug concentration increased from 2 RESULTS to 800 Fg/ml(7 to 2,800 the bound fraction ranged from The study was performed at first to determine the binding 99.5 to 84.0% for the (+)-(S)-etodolacand from 98.6 to 79.0% sites involved in the interactions between drug and HSA by for the ( - )@)-form.
m,
lSO
t1 I
100
50
-
, 0
,
0
0
, 0
I
0 /
T (PM)
0 Fig. 2. Variation of the free concentration (fl to 1 gjliter HSA solution with the total concentration (7) of respectively). (+)-(S)-etcdolac( 0 )and (- )-(R)-etodolac ( ). Optimization fitting by Scatchard (- - -, -,
243
STEREOSELECTIVE BINDING OF ETODOLAC TO HSA
TABLE 1. Binding parameters of etodolac enantiomers to human serurn albumin for different HSA concentrations (WAN
TABLE 2. Free fractions of etodolac enantiomers for a 4Og/liter human serum albumin concentration (mean values)
Etodolac
[HSAI (giliter) Parameters
Etodolac (R)
R
0.326 f 0.0225 0.8006 f 0.0634 10.972 f 0.497 0.0047 =t 0.0038
(15)" 0.5199 f 0.1266 (15) (15) 0.2728 f 0.535 (15) (15) 11.749 f 1.8385 (15) (15) 0.0065 f 0.0019 (15) (20) 0.798 f 0.1609 (24) (20) 0.2268 f 0.0411 (24) (20) 8.3095 f 0.3257 (24) (20) 0.0087 f 0.0012 (24) (27) 0.4348 f 0.0154 (26) (27) 0.8331 f 0.0302 (26) 5.2706 f 0.0505 (26) 0.0113 f O.OOO4 (26)
0.48 f 0.0144 0.6547 & 0.024 4.54 f 0.085 0.0075 f 0.0038 1.4254 f 0.793 0.0317 f 0.0352 1.6557 4.847 0.0051 f 0.0023 2.428 f 0.0504 0.058 f 0.0012 4.5574 f 1.6756 O.OOO6 f 0.00031 1.959 0.1155 (16) 0.055 f 0.0026 (16) 2.305 0.5631 (16) 0.0021 0.0013 (16) . ,
*
+
1.882 f 0.2151 (22) 0.054 f 0.0043 (22) 6.653 f 1.0021 (22) 0.0031 f 0.00104 (22) 1.657 f 0.0594 (26) 0.1195 f 0.0042 (26) 3.968 f 0.154 (26) 0.003 f 0.0003 (26) 1.3855 f 0.0667 0.1742 f 0.0081 3.3171 0.2053 0.0052 0.0009
*
Free
Free fraction
S
0.5786 f 0.053 1.1392 f 0.021 15.765 f 0.946 0.0025 f 0.004
Etodolac (S)
(17) (17) (17) (17) ~I
=Value i SEM (number of experiments).
T (M
("/.I
6.866 17.13 34.19 170.9 672.1 984.5 1823 2295
1.37
T
(rn
6.923 17.26 34.53 172.2 684.5 1010 1877 2400
1.58 1.79 1.82 3.56 6.04 14.5 21.3
The total number of sites decreased when the concentration of albumin increased (Fig.4).The variation of protein binding with drug concentration was compared for the various protein concentrations: in order to take into account the difference of protein level, the number of sites occupied in the protein (B/P) was studied as a function of the molar druglprotein ratio (TIP), close results according to the plot we formerly proposed were obtained, especially at low T values, for both enantiomers (Fig. 5a and b). 17718:
DISCUSSION
Recent studies concerning stereoselectivity of protein binding of NSAID were reported in the literature. and described different results for the individual drugs tested. For example, no stereospecificity was observed for pirprofen binding in a 1 g/liter albumin solution, as studied by equilibrium dialysis, and the Scatchard parameters were identical for enantiomers and racemate. l9 The similar conclusion was obtained for fenoprofen by circular dichroism for a 5 glliter HSA solution.20
1000 800 600
400
200 0 200
400
600
800
(%)
0.53 0.79 0.78 1.05 1.68 3.29 11.2 15.9
1200
0
fraction
1000
1200
1400
1600
1800
2000
Fig. 3. Variation of the free concentration (Fj to 10 g/liter HSA solution with the total concentration (+)-(S)-etodolac ( 0) and ( - )-(R)-etodolac ( W ). Optimization fitting by Scatchard (- - -, -, respectively).
(gof
244
MULLER ET AL.
2o 15
T
ni+n2
--
l o --
5 --
0 0
5
10
15
20
25
30
35
40
Fig. 4. Variation of the total number of drug binding sites (nl+nz) on HSA with the HSA concentration (0for ( )-(S)-etodolac( 0 )and ( - t(R)etdolac ( ).
+
Conversely, steremelectivity was shown for indoprofen, the ( - )-(R)enantiomer being more bound than its antipode in HSA solution (20 and 40 g/liter),21 for flurbiprofen, where the (+)-(S)-formwas more bound to human plasma essentially at low drug concentrations," and for carprofen, with the same result in a 2 g/liter HSA solution.23The binding of ketoprofen was studied by gel filtration in 1and 10 g/liter HSA solutions: the stereoselectivity of binding observed at 1 g/liter, with a preference for the (S)-form, disappeared at higher protein level. a Ibuprofen binding showed a (S)/(R) enantiomer free fraction of 1.70 f 0.29, both enantiomers displacing specific fluorescent markers from albumin sites," and competing for the same protein binding sites.% These different results led us to study the binding of etodolac not only at various drug concentrations, but also at several protein levels. Moreover, the protein level is the most important factor of variation with physiopathological conditions, like rheumatoid arthritis, renal or hepatic failure, and with the nature of physiologicalmedium (plasma,synovial fluid).It was frequently shown that this factor influenced in a great extent the free fraction of drugs, since protein binding was reduced in case of diseases associated with hypoalbuminemia,sm or in synovial fluid than in plasma, according to the difference in protein concentration between these mediums. 17sB Besides, several studies were performed with protein solutions at different concentrations and showed that the number of sites occupied by the drug per mole of albumin decreased when the protein concentration was increased.3oThis could be due to molecular aggregation of albumin and the reduced proportion of monomeric forms of HSA, which depends inversely on its concentration,31 since monomeric fractions could have a greater binding capacity than polymeric ones. 32 For etodolac, the total number of binding sites has the same variation with protein concentration (Fig. 4). The comparison of the results in terms of sites occupied for a given molar ratio of drug to albumin shows that the protein binding was similar for the various albumin levels, if considering the amount of available
drug per mole of albumin, as formerly demonstratedfor salicylate. l7 The protein binding of etodolac was stereoselective, since the ( + )-(S)-formwas more bound than its antipode, especially at high drug and protein concentrations. When these concentrations are very low (HSA at 1 g/liter and druglprotein ratio below unity), the inverse phenomenon was observd the values of the Scatchard parameters suggested that the difference between enantiomers may be due to a greater affinity of the fust protein site for R than for S evidenced only at low protein level. As previously observed for flurbiprofen" and ketoprofen,% the stereoselectivity of protein binding may be influenced by the protein concentration. This fact could explain at least in part the difference between the NSAID studies, since not only the protein binding method but also its concentration varied in a great extent according to the study considered. The results were obtained with HSA solutions and are different from those reported after in vitro equilibrium dialysis of rat plasma spiked with racemate etodolac.33This discrepancy could be explained by the presence in plasma of many endogeneous compounds,that are able to interact with the albumin molecules3; this may in fact cause the in vivo situation to be quite different from that observed in that study, with respect to the relative in vivo of the enantiomers. However, the more bound enantiomer of this study in physiological conditions is the more rapidly eliminated and presents the lowest area under the curve.l2 This result is in good agreement with those obtained with other NSAID, as indoprofen21 and ibuprofen.26 The spectrofluorimetric results obtained by the displacement of site markers permitted us to suppose that the binding of the enantiomers is stereoselective, since (+)-(S)-etodolacis bound to sites I and 11, whereas its optical antipode is bound to site I and in a lesser extent to site 11. The protein binding of (S)-etodolac to site I might be of clinical relevance with respect to drugs interactionssince many
STEREOSELECTNEBINDING OF €TODOLAC TO HSA
245
a 9 --
u
8 --
0
5
10
15
20
25
BIP
30
b
8
0
rn
6
4
0
4
TIP 0
I
0
5
10
15
20
25
30
Fig. 5. Number of sites (B/p) of HSA as a function of ( - )-(R)-etdolac (a)and ( + )-(S)-etodolac(b)concentration available per mole of albumin (TIP),for HSA solutions at 1 ( W), 2 ( O ) ,5 (+), 10 ( O ) ,20 ( A ), and 40 g/liter ( A).
acidic drugs are strongly bound to this ionic site, as for example warfarin.3435 In the physiological conditions (40 g/liter HSA solution), etodolac was extensively bound to albumin (more than 99% for therapeutic concentrations), as described for the other NSAID27: the variation of protein binding would then have a great clinical importance.
studying the drug/protein interactions at various protein levels. The free concentration of a drug representing the active form, druglprotein interaction has an effect on both pharmacokinetic evolution and pharmacological response. The analysis being limited to the chiral drugs for which the protein binding was studied in the physiological conditions-according to the above considerations, it should be interesting to CONCLUSION underline, that even if the binding is more important for one Etcdolac has shown an extensive and stereoselectiveprotein enantiomer, the elimination half-life and the area under the binding, as studied by equilibrium dialysis over a wide range curve may be larger for its antipode. The active concentration of drug and HSA concentrations and by spectrofluorimetry of NSAID would depend mainly on the free fraction of the (specific sites markers displacements):(S)-form is more highly (S)-enantiomer.Its proportion in the total concentration could bound than its optical antipode at high drug and protein con- vary with individual differences between the enantiomers,22 or centrations. These results have outlined the importance of when the elimination process and/or metabolization are differ-
246
MULLER ET AL.
ent for the enantiomersor when chiral inversion occurs.In such situations it is more difficult to predict the free concentration of the active enantiomer from the total concentration of the mixture if stereoselectivebinding occurs. Since pathological situations induce variations in protein expression, this phenomenon may no doubt explain, at least in part, the individual variations observed in response to treatment and emphasize the role of enantiomers of NSAID in the new therapeutic approaches of inflammation. ACKNOWLEDGMENTS This study was supported in part with grants of WyethFrance and in part with grants of INSERM-CRAM(Contract 14).The authors would thank &ile Guillaumefor her excellent technical assistance. LITERATURE CITED 1. Arihs, EJ. Stermhemistry, a basis for sophisticated nonsense in pharmacokinetics and clinical pharmacology. Eur. J. Clin. Pharmacol. 2 6 s 668,1984. 2. Hun, A.J., Caldwell, J. The metabolic chiral inversion of 2-arylpropionic acids-a novel route with pharmacological consequences.J. Pharm. Pharmacol. 35:693-704,1983. 3. Testa, B., Trager, W.F. Racemates versus enantiomers in drug development: Dogmatism or pragmatism? Chirality 2129-133,1990. 4. Williams, K.M. Enantiomers in arthritic disorders. Phannacol.Ther.4627% 295,1990. 5. Toon, S., Trager, WE. Pharmacokinetic implications of stereoselective changes in plasma protein binding: Warfarin/sulfinpyrazone. J. Pharm. Sci. 731671-1673,1984. 6. Miller, J.H.M., Smail, GA. Interaction of the enantiomers of warfarin with human serum albumin, peptides and amino acids. J. Pharm. Pharmacol. 29(suppl):33P, 1977. 7. Brown, N.A., J h c h e n , E., Muller, W.E., Wollert U. Optical studies on the mechanism of interaction of enantiomers of anticoagulant drugs phenpromumon and warfarin with HSA. Mol. Pharmacol. 137679,1977. 8. Mc Menamy, R.H., Oncley, J.L. The specific binding of L-tryptophan to serum albumin. J. Biol. Chem. 233:14%-1447, 1958. 9. Miiller, W.E., Wollert, U.High stereospecificity of the benzodiazepine binding site on human serum albumin. Studies with d- and Goxaxpam hemisuccinate. Mol. Phannacol. 11:5260, 1975. 10. Humbert, L.G., Demerson, C.A., Swaminathan, P., Bird, PH. Etodolac (1.8diethyl-1,3,4,9-tetrahydropyrano[3,4blindole-l-acetic acid): A potent antiinflammatory drug. Conformation and absolute configuration of its active enantiomer.J. Med. Chem. 29:871-874,1986. 11. Demerson, C.A., Humber, L.G., Abraham, N.A., Schilling, G., Martel, R.R., Pace-Asciiak, C. Resolution of etodolac and anti-inflammatory and prostaglandin synthetase inhibiting properties of the enantiomers. J. Med. Chem. 26:177%1780,1983. 12. Jamali, F., Mehvar, R., Lemko, C., Eradiri, 0. Application of a stereospecific high-performanceliquid chromatography assay to a pharmacokineticstudy of etodolac enantiomers in humans. J Pharm. Sci. 77:963-966,1988. 13. Lapicque, F., Netter, P., Bannwarth, B., Trkhot, P., Gillet, P., Lambert, H., Royer, RJ. Identification and simultaneous determination of non steroidal anti-inflammatorydrugs using high-performanceliquid chromatogmphy.J. Chromatorn. 496:301-320,1989. 14. Lapicque, F., Netter, P., Monot, C., Bannwarth, B., Maknassi, M.S., Royer, RJ. D&rmination de la fixation protbique des mMicaments par dialyse ?J Nquilibre. Influence du dkplament de volume sous I'det de la pression osmotique. J. Pharmacol. (Paris) 17295-300,1986.
15. Fehske, KJ., Muller, W.E., Wollert, U. The location of drug binding sites in human serum albumin. Biochem. Pharmacol. 30587492, 1981. 16. Scatchard, G. The attraction of proteins for small molecules and ions. Ann. NY Acad. Sci. 51$%@672,1949. 17. Caillier, I., Bannwarth, B., Monot, C., Lapicque, F., Netter,P., Gaucher, A., Royer, RJ. Dfierences in sodium salicylate protein binding in serum and synovial fluid from patients with knee effusion. Int. J. Clin. Pharm. Ther. Toxicol. B7-13, 1990. 18. Muller, N., Lapicque, F., Monot, C., Payan, E., Gillet, P., Bannwarth, B.. Netter, P. Indomethacin binding in the cerebrospinal fluid. Biochem. Pharmacol. 42799404,1991. 19. Otag~ri,M., Masuda, K., Imai, T., Imamura, Y., Yamasaki, M. Binding of pirprofen to human serum albumin studied by dialysis and spectrwopy techniques. Biochem. Pharmacol. 38:l-7,1989. 20. Pemn, J.H. A circular dichroic investigation of fenoprofen, Z(3phenoxyphenyltpropionic acid to human serum albumin. J. Phann. Pharmacol. 25: 208-212, 1973. 21. Siebler, D., Kinawi, A. Bindung von razemischem Indoprofen und seiner Enantiomeren an Human Serum Albumin. Armeim.-Forsch./Drug Res. 39: 6594m,1989. 22. Knadler, M.P., Brater, D.C., Holl, S.D. Plasma protein binding of flurbiprofen: enantioselectivityand influence of pathological status.J. Pharmacol. Exp. Ther. 24937S385, 1989. 23. Iwakawa, S., Spahn, H., b e t L., Lin, E.T. Stereoselectivebinding of the glucuronideconjugates of carprofen enantiomers to human serum albumin. Biochem. Pharmacol. 3994%953,1990. 24. Rendic, S., Alebic-Kolbah, T., Kajfez, F., Sunjic, V. Stereoselective binding of (+) and ( - ) a(knzoylpheny1)propionicacid (ketoprofen) to human serum albumin. Farm. Ed. Sci. 3551-59, 1980. 25. Hansen, T., Day, R., Williams, K., Lee,E., Knihinicki, R., M e l d , A. The assay and in vitro binding of the enantiomers of ibuprofen. Clin. Exp. Pharmacol. Physiol. 9(suppl):82-83, 1985. 26. Evans, A.M., Nation, R.L., Sansom, L.N., Bochner, F., Somogyi, A.A. Ste reoselective plasma protein binding of ibuprofen enantiomers. Eur. J. Clin. Pharmacol. 36:283-390,1989. 27. Lin, J.H., Cochetto, D.M., Duggan, D.E. Protein binding as a primary determinant of the clinical pharmacokinetic properties of non steroidal antiinflammatory drugs. Clin. Pharmacokinet. 1240242, 1987. 28. Netter, P, Monot, C., Stalars. M.C., Mur, J.M., Royer, RJ., Faure, G., Pourel, J., Martin, J., Gaucher, A. Deuease of in vitro serum protein binding of salicylate in rheumatoid arthritis. Eur. J. Drug Metab. Pharmacokmet. 9109-116,1984. 29. Wanwimolruk,S.. Brooks, P.M., Birkett, DJ. Protein binding of non steroidal anti-inflammatorydrugs in plasma and synovial fluid of arthritic patients. Br. J. Clin. Pharmacol. 1591-94, 1983. 30. Boobis, S.W., Chignell, C.F. Effect of protein concentration on the binding of drugs to human serum albumin. Sulfadiazine, salicylate and phenylbutazone. Biochem. Pharmacol. B751-76, 1979. 31. Zini, R., Bane, J., Bree, F., Tillement, J.P. Evidence for a concentration dependent polymerization of a commercial human serum albumin. J. Chromatogr. 216191-198, 1981. 32. Nakano, N.I., Yamagushi, S., Shimamori, Y., Fujimoto, Y. Fractionation of human serum albumin preparation and the salicylate binding characteristics.Int. J. Pharm. 7:335-342, 1981. 33. Brocks, D.R., Jamali F., The parmacokinetics of etodolac enantiomers in the rat. Lack of pharmacokineticinteraction between enantiomers.Drug. Metab. Dispos. 18471-475,1990. 34. MCEIMYJ.C., D'Arcy P.F., Protein binding displacement interactions and their clinical importance. Drugs 25:495-513,1983. 35. Diana FJ., Veronich K., K a p r A.L., Binding of nonsteroidal anti-intlammatory agents and their effect on binding of racemic warfarin and its enantiomers to human serum albumin. J. Pharm. Sci. 78195-199,1989.
Stereoselective Binding of Etodolac to Human Serum Albumin NORLLE MULLER, FRANCOISE LAPICQUE, CLAUDINE MONOT, ELISABETH PAYAN, RJ%I DROPSY, AND PATRICK NETTER Luboratoire & P h u m o b g i e , URA CNRS 1288, Facult6 & Mk&cine, Vanaimwe les Nanq, and Laboratoire Wyeth-France,Park, France
ABSTRACT The protein binding of etodolac enantiomers was studied in vitro by equilibrium dialysis in human serum albumin (HSA) of various concentrations varying from 1to 40 g/liter, by addition of each enantiomer at increasing concentrations. In the 1 g/liter solution, at the lowest drug levels, the @)-form is more bound than its antipode, the contrary being observed at the highest drug levels. For higher albumin concentrations,S was bound in a larger extent than R. Using the displacement of specific markers of HSA sites I and 11, studied by spectrofluorimetry, it was suggested that R and S are both bound to site I, while only S is strongly bound to site 11. Q 1992 WiIey-Liss,Inc.
KEY WORDS: NSAID, chirality, enantiomers, protein binding, equilibrium dialysis, fluorescent specific markers
INTRODUCTION Between 10 and 15% of all marketed drugs are racemic mixtures of 2 or more stereoisomers, as for instancenonsteroidal antiinflammatory drugs (NSAID).In vitro studies have shown that the inhibition of the prostaglandins synthesis was only the fact of the (S)-enantiomer of chiral NSAID (often the dextrogyreone) and therefore that only this form was responsible for the antiinflammatoryactivity. The other form was considered as an impurity, eventually as a prodrug when it was able to be converted into the active form, and in a lesser extent as a toxic compound due to its incorporationinto adipose tissue in the case of inversion.24 Because of the chirality of human serum albumin (HSA), stereoselective differences in protein binding have been noted for the enantiomers of some chiral products, but this phenomenon cannot be generalized to all racemic drugs. The most important sites (sites I and 11) on HSA involved in the binding have been reported to be stereoselective for some drugs. Warfarin was bound to site I, and the (S)-formappeared to be more highly bound than the @)-enantiomer.5-7 L-Tryptophan was highly and stereospecificallybound at one site to HSA, with an affinity 100-fold greater than that of D-tryptophan. The ( + )-(S)-enantiomerof oxazepam hemisuccinate was bound about 30 times stronger than its antipode.9 NSAID are strongly bound to HSA [more than 90'3'0, with the exception of salicylate (80%)],especially to sites I and I1 and sometimesthis binding was shown to be stereoselective.Therefore, it would be interesting to establish a relationshipbetween protein binding and chiral drug action. Etodolac is a NSAID marketed as a racemic mixture, i.e., as an equal mixture of ( - )-(R)- and ( + )-(S)-enantiomers. lo The
( + )-(S)-enantiomer,responsible for the pharmacologicalactivity," represented 10% of the total plasma concentration and may be eliminated more rapidly in man than its antipode.I2 Etodolac seems unable to undergo chiral inversion due to the inclusion of the asymetric carbon into a pyrano ring.
asCz 72H5
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I
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The present study concerned the protein binding of etodolac, as studied by equilibrium dialysis and spectrofluorimetry to show an eventual stereoselectivebinding of the enantiomers of etodolac (-)@) and (+)-(S),over a concentration range of HSA from 1 to 40 g/liter. MATERIAL AND METHODS Products Etodolac (enantiomers)was a gift from WYETH-France. Albumin (MW: 69000) was purchased from Sigma (reference A 1887) as essentially fatty acid free (below 0.005%), prepared from fraction V from HSA. Various concentrations of albumin (1, 2, 5, 10, 20, and 40 g/liter) were prepared in an isotonic Sijrensen phosphate buffer 0.067 M, pH 7.4. All the products used in the study were of analyticalgrade and water bidistilled; acetonitrile in the eluent was of chromatographic grade. Received for publication June 20, 1991; accepted November 15, 1991.
Presented at the Second International Symposium on Chiral
May 27-31, 1991, Rome, Italy. o 1992 Wiley-Liss, Inc.
Discrimination,
Address reprint requests to Patrick Netter, Labratoke de Pharmacologie, URA
CNRS 1288,Facult6 de Medecine, BP la,F-54505Vandceuvre les Nancy, Cedex, France.
241
STEREOSELECTIVE BINDING OF ETODOLAC TO HSA
The purity of each enantiomer was checked by the stereose- extraction of drug in buffer samples (volumesvarying from 20 to 900 pl, according to the estimated concentration) was lective assay proposed by Jamali et al. l2 achieved after acidification and the chromatographic condiEquilibrium Dialysis tions derived from those used in the simultaneous determination of several NSAIDs13; the major changes concerned the The free concentrationof ( - )-(R)-and ( +)-(S)-etodolacin the detection wavelength (227 nm), the eluent [acetonitrile/acetic various albumin solutions were determined in vitro by equilibrium dialysis. The concentrations of drug added varied from acid (0.3%);55:45 v/v] and the column (RadialPak Cis, 8 x 100 0.25 to 400 pg/ml for the solutions of albumin from 1 to 5 mm, Waters); these changes were done in order to obtain a g/liter,and from 1to 800 pg/ml for the other albumin solutions. better sensitivity and a shorter analysis time for the separate For each of the enantiomers, the experimentswere achieved in assay of etodolac. The assay was linear from 10 ng/ml to 100 duplicate or in triplicate. Equilibrium dialysis was realized pg/ml. The precision of the method for the lowest concentrausing Spectrapor 2 membranes, for 3 h, against a 67 mM tions was 5.4%.The binding values were corrected for volume Sijrensen isotonicphosphate buffer pH 7.4, in 1ml cells, at 37°C shift during equilibrium dialysis. l4 in a Dianorm apparatus. The time of 3 h was established in spf?ctrofluorim4?try preliminary studies to allow the equilibriumto be attained. The The spectroscopic measurements were carried out using a free concentration of each form of drug was determined by WLC as its concentration in the buffer after dialysis. The fluorescence spectrophotometerHitachi Lou-Perkin Elmer 240. Fluorescence (70)
k
l o90 o
a
50 40
30 20
i
10 --
T (PM)
Fluorcscence (%) '*O 100
T
b
80
60 40
20 -0 ,
T (PM)
242
MULLER ET AL.
No quenching of the single tryptophan residue (number 214) present in the albumin molecule could be achieved by etodolac (1, = 290 nm; h, = 340 nm) due to the intrinsic fluorescence of the drug. Besides, the location of the binding sites of drugs to protein can be studied using specific fluorescent probes such as dansylamide (DNSA) (site I) and dansylsarcosine (DS) (site 11).l5 The fluorescence emitted by the complex DNSA/HSA (h, = 340nm; hex = 480 nm) and DS (h, = 350 nm;hex = 480 nm) is modified by the addition of drug. It was verified that etodolac emitted no fluorescence at the wavelengths used for the displacement experiments. The spectrofluorimetricdisplacements were achieved using a 1 g/liter HSA solution, in which the marker (DNSA or DS) was added to a molar ligand/proteinratio of 1.The two enantiomeric forms of etodolac were then added and the corresponding molar ratio of drug/protein was varied from 0.025 to 5.
spectrofluorimetricmarkers displacements,and second to compare the binding characteristicsof enantiomersby equilibrium dialysis. Displacement of Site Markers When the drug was added, the complex formed between the site marker (DNSA or DS) and HSA induced a decrease in the fluorescence,corresponding to the displacement of the marker. DNSA was displaced by both of the enantiomers (up to 40%) (Fig. la). The displacement of DS was strongly realized by (+)-(S)etodolac(55%)and in a very low extent (less to 10% of fluorescencedecrease) by its antipode (Fig. lb).
Protein Binding of Et&lac Enantwmers The protein binding of etodolac enantiomers was found to depend on the concentration of HSA. In the 1g/liter solution, for small values of drug level, T (less than 15 pik? i.e, for Mathematical and Statistical Analysis druglprotein ratio below l),the (R)-enantiomer is bound more The binding parameters were determined using a Siphar than its antipode: for example, the free fractions of R and S software, installed on an IBM AT micro computer. The binding were respectively 12 and 24% for T varying from 0.7 to 2 pM, data are fitted by the Scatchard plot for two classes of indepen- and increased gradually up to 30% for both forms when T attained 15 pM. In the case of higher T value, the opposite dent sites described by the equation16 result was obtained, i.e., S was more tightly bound to HSA than R (Fig. 2). Table 1 established a comparison of the Scatchard parameters of the enantiomers:the values of nl ,%, and Fq! were similar; only the affinity constant kl was more important for R where F and B are the concentrations of free and bound drug than for S (P < 0.001). respectively, P is the albumin concentration, ki is the associaIn a lOg/liter HSA solution (Fig. 3), the protein binding of the tion constant, and ni is the number of sites of the ith class. The two enantiomers was quite similar, since no statistical differvalues of ni and ki are determined with their respective stan- ence was shown between the Scatchard parameters (Table 1). dard deviation. For higher albumin concentrations,S became more bound than The comparison between binding parameters was done R, as for example, in a 40 g/liter HSA solution, its free fraction using Student's t- test (a= 5%). The degree of significance, is always the lowest (Table2) and the total number of sites was P, is given for each test. greater for the active form than for its antipode (P = 0.05). In this case, when the added drug concentration increased from 2 RESULTS to 800 Fg/ml(7 to 2,800 the bound fraction ranged from The study was performed at first to determine the binding 99.5 to 84.0% for the (+)-(S)-etodolacand from 98.6 to 79.0% sites involved in the interactions between drug and HSA by for the ( - )@)-form.
m,
lSO
t1 I
100
50
-
, 0
,
0
0
, 0
I
0 /
T (PM)
0 Fig. 2. Variation of the free concentration (fl to 1 gjliter HSA solution with the total concentration (7) of respectively). (+)-(S)-etcdolac( 0 )and (- )-(R)-etodolac ( ). Optimization fitting by Scatchard (- - -, -,
243
STEREOSELECTIVE BINDING OF ETODOLAC TO HSA
TABLE 1. Binding parameters of etodolac enantiomers to human serurn albumin for different HSA concentrations (WAN
TABLE 2. Free fractions of etodolac enantiomers for a 4Og/liter human serum albumin concentration (mean values)
Etodolac
[HSAI (giliter) Parameters
Etodolac (R)
R
0.326 f 0.0225 0.8006 f 0.0634 10.972 f 0.497 0.0047 =t 0.0038
(15)" 0.5199 f 0.1266 (15) (15) 0.2728 f 0.535 (15) (15) 11.749 f 1.8385 (15) (15) 0.0065 f 0.0019 (15) (20) 0.798 f 0.1609 (24) (20) 0.2268 f 0.0411 (24) (20) 8.3095 f 0.3257 (24) (20) 0.0087 f 0.0012 (24) (27) 0.4348 f 0.0154 (26) (27) 0.8331 f 0.0302 (26) 5.2706 f 0.0505 (26) 0.0113 f O.OOO4 (26)
0.48 f 0.0144 0.6547 & 0.024 4.54 f 0.085 0.0075 f 0.0038 1.4254 f 0.793 0.0317 f 0.0352 1.6557 4.847 0.0051 f 0.0023 2.428 f 0.0504 0.058 f 0.0012 4.5574 f 1.6756 O.OOO6 f 0.00031 1.959 0.1155 (16) 0.055 f 0.0026 (16) 2.305 0.5631 (16) 0.0021 0.0013 (16) . ,
*
+
1.882 f 0.2151 (22) 0.054 f 0.0043 (22) 6.653 f 1.0021 (22) 0.0031 f 0.00104 (22) 1.657 f 0.0594 (26) 0.1195 f 0.0042 (26) 3.968 f 0.154 (26) 0.003 f 0.0003 (26) 1.3855 f 0.0667 0.1742 f 0.0081 3.3171 0.2053 0.0052 0.0009
*
Free
Free fraction
S
0.5786 f 0.053 1.1392 f 0.021 15.765 f 0.946 0.0025 f 0.004
Etodolac (S)
(17) (17) (17) (17) ~I
=Value i SEM (number of experiments).
T (M
("/.I
6.866 17.13 34.19 170.9 672.1 984.5 1823 2295
1.37
T
(rn
6.923 17.26 34.53 172.2 684.5 1010 1877 2400
1.58 1.79 1.82 3.56 6.04 14.5 21.3
The total number of sites decreased when the concentration of albumin increased (Fig.4).The variation of protein binding with drug concentration was compared for the various protein concentrations: in order to take into account the difference of protein level, the number of sites occupied in the protein (B/P) was studied as a function of the molar druglprotein ratio (TIP), close results according to the plot we formerly proposed were obtained, especially at low T values, for both enantiomers (Fig. 5a and b). 17718:
DISCUSSION
Recent studies concerning stereoselectivity of protein binding of NSAID were reported in the literature. and described different results for the individual drugs tested. For example, no stereospecificity was observed for pirprofen binding in a 1 g/liter albumin solution, as studied by equilibrium dialysis, and the Scatchard parameters were identical for enantiomers and racemate. l9 The similar conclusion was obtained for fenoprofen by circular dichroism for a 5 glliter HSA solution.20
1000 800 600
400
200 0 200
400
600
800
(%)
0.53 0.79 0.78 1.05 1.68 3.29 11.2 15.9
1200
0
fraction
1000
1200
1400
1600
1800
2000
Fig. 3. Variation of the free concentration (Fj to 10 g/liter HSA solution with the total concentration (+)-(S)-etodolac ( 0) and ( - )-(R)-etodolac ( W ). Optimization fitting by Scatchard (- - -, -, respectively).
(gof
244
MULLER ET AL.
2o 15
T
ni+n2
--
l o --
5 --
0 0
5
10
15
20
25
30
35
40
Fig. 4. Variation of the total number of drug binding sites (nl+nz) on HSA with the HSA concentration (0for ( )-(S)-etodolac( 0 )and ( - t(R)etdolac ( ).
+
Conversely, steremelectivity was shown for indoprofen, the ( - )-(R)enantiomer being more bound than its antipode in HSA solution (20 and 40 g/liter),21 for flurbiprofen, where the (+)-(S)-formwas more bound to human plasma essentially at low drug concentrations," and for carprofen, with the same result in a 2 g/liter HSA solution.23The binding of ketoprofen was studied by gel filtration in 1and 10 g/liter HSA solutions: the stereoselectivity of binding observed at 1 g/liter, with a preference for the (S)-form, disappeared at higher protein level. a Ibuprofen binding showed a (S)/(R) enantiomer free fraction of 1.70 f 0.29, both enantiomers displacing specific fluorescent markers from albumin sites," and competing for the same protein binding sites.% These different results led us to study the binding of etodolac not only at various drug concentrations, but also at several protein levels. Moreover, the protein level is the most important factor of variation with physiopathological conditions, like rheumatoid arthritis, renal or hepatic failure, and with the nature of physiologicalmedium (plasma,synovial fluid).It was frequently shown that this factor influenced in a great extent the free fraction of drugs, since protein binding was reduced in case of diseases associated with hypoalbuminemia,sm or in synovial fluid than in plasma, according to the difference in protein concentration between these mediums. 17sB Besides, several studies were performed with protein solutions at different concentrations and showed that the number of sites occupied by the drug per mole of albumin decreased when the protein concentration was increased.3oThis could be due to molecular aggregation of albumin and the reduced proportion of monomeric forms of HSA, which depends inversely on its concentration,31 since monomeric fractions could have a greater binding capacity than polymeric ones. 32 For etodolac, the total number of binding sites has the same variation with protein concentration (Fig. 4). The comparison of the results in terms of sites occupied for a given molar ratio of drug to albumin shows that the protein binding was similar for the various albumin levels, if considering the amount of available
drug per mole of albumin, as formerly demonstratedfor salicylate. l7 The protein binding of etodolac was stereoselective, since the ( + )-(S)-formwas more bound than its antipode, especially at high drug and protein concentrations. When these concentrations are very low (HSA at 1 g/liter and druglprotein ratio below unity), the inverse phenomenon was observd the values of the Scatchard parameters suggested that the difference between enantiomers may be due to a greater affinity of the fust protein site for R than for S evidenced only at low protein level. As previously observed for flurbiprofen" and ketoprofen,% the stereoselectivity of protein binding may be influenced by the protein concentration. This fact could explain at least in part the difference between the NSAID studies, since not only the protein binding method but also its concentration varied in a great extent according to the study considered. The results were obtained with HSA solutions and are different from those reported after in vitro equilibrium dialysis of rat plasma spiked with racemate etodolac.33This discrepancy could be explained by the presence in plasma of many endogeneous compounds,that are able to interact with the albumin molecules3; this may in fact cause the in vivo situation to be quite different from that observed in that study, with respect to the relative in vivo of the enantiomers. However, the more bound enantiomer of this study in physiological conditions is the more rapidly eliminated and presents the lowest area under the curve.l2 This result is in good agreement with those obtained with other NSAID, as indoprofen21 and ibuprofen.26 The spectrofluorimetric results obtained by the displacement of site markers permitted us to suppose that the binding of the enantiomers is stereoselective, since (+)-(S)-etodolacis bound to sites I and 11, whereas its optical antipode is bound to site I and in a lesser extent to site 11. The protein binding of (S)-etodolac to site I might be of clinical relevance with respect to drugs interactionssince many
STEREOSELECTNEBINDING OF €TODOLAC TO HSA
245
a 9 --
u
8 --
0
5
10
15
20
25
BIP
30
b
8
0
rn
6
4
0
4
TIP 0
I
0
5
10
15
20
25
30
Fig. 5. Number of sites (B/p) of HSA as a function of ( - )-(R)-etdolac (a)and ( + )-(S)-etodolac(b)concentration available per mole of albumin (TIP),for HSA solutions at 1 ( W), 2 ( O ) ,5 (+), 10 ( O ) ,20 ( A ), and 40 g/liter ( A).
acidic drugs are strongly bound to this ionic site, as for example warfarin.3435 In the physiological conditions (40 g/liter HSA solution), etodolac was extensively bound to albumin (more than 99% for therapeutic concentrations), as described for the other NSAID27: the variation of protein binding would then have a great clinical importance.
studying the drug/protein interactions at various protein levels. The free concentration of a drug representing the active form, druglprotein interaction has an effect on both pharmacokinetic evolution and pharmacological response. The analysis being limited to the chiral drugs for which the protein binding was studied in the physiological conditions-according to the above considerations, it should be interesting to CONCLUSION underline, that even if the binding is more important for one Etcdolac has shown an extensive and stereoselectiveprotein enantiomer, the elimination half-life and the area under the binding, as studied by equilibrium dialysis over a wide range curve may be larger for its antipode. The active concentration of drug and HSA concentrations and by spectrofluorimetry of NSAID would depend mainly on the free fraction of the (specific sites markers displacements):(S)-form is more highly (S)-enantiomer.Its proportion in the total concentration could bound than its optical antipode at high drug and protein con- vary with individual differences between the enantiomers,22 or centrations. These results have outlined the importance of when the elimination process and/or metabolization are differ-
246
MULLER ET AL.
ent for the enantiomersor when chiral inversion occurs.In such situations it is more difficult to predict the free concentration of the active enantiomer from the total concentration of the mixture if stereoselectivebinding occurs. Since pathological situations induce variations in protein expression, this phenomenon may no doubt explain, at least in part, the individual variations observed in response to treatment and emphasize the role of enantiomers of NSAID in the new therapeutic approaches of inflammation. ACKNOWLEDGMENTS This study was supported in part with grants of WyethFrance and in part with grants of INSERM-CRAM(Contract 14).The authors would thank &ile Guillaumefor her excellent technical assistance. LITERATURE CITED 1. Arihs, EJ. Stermhemistry, a basis for sophisticated nonsense in pharmacokinetics and clinical pharmacology. Eur. J. Clin. Pharmacol. 2 6 s 668,1984. 2. Hun, A.J., Caldwell, J. The metabolic chiral inversion of 2-arylpropionic acids-a novel route with pharmacological consequences.J. Pharm. Pharmacol. 35:693-704,1983. 3. Testa, B., Trager, W.F. Racemates versus enantiomers in drug development: Dogmatism or pragmatism? Chirality 2129-133,1990. 4. Williams, K.M. Enantiomers in arthritic disorders. Phannacol.Ther.4627% 295,1990. 5. Toon, S., Trager, WE. Pharmacokinetic implications of stereoselective changes in plasma protein binding: Warfarin/sulfinpyrazone. J. Pharm. Sci. 731671-1673,1984. 6. Miller, J.H.M., Smail, GA. Interaction of the enantiomers of warfarin with human serum albumin, peptides and amino acids. J. Pharm. Pharmacol. 29(suppl):33P, 1977. 7. Brown, N.A., J h c h e n , E., Muller, W.E., Wollert U. Optical studies on the mechanism of interaction of enantiomers of anticoagulant drugs phenpromumon and warfarin with HSA. Mol. Pharmacol. 137679,1977. 8. Mc Menamy, R.H., Oncley, J.L. The specific binding of L-tryptophan to serum albumin. J. Biol. Chem. 233:14%-1447, 1958. 9. Miiller, W.E., Wollert, U.High stereospecificity of the benzodiazepine binding site on human serum albumin. Studies with d- and Goxaxpam hemisuccinate. Mol. Phannacol. 11:5260, 1975. 10. Humbert, L.G., Demerson, C.A., Swaminathan, P., Bird, PH. Etodolac (1.8diethyl-1,3,4,9-tetrahydropyrano[3,4blindole-l-acetic acid): A potent antiinflammatory drug. Conformation and absolute configuration of its active enantiomer.J. Med. Chem. 29:871-874,1986. 11. Demerson, C.A., Humber, L.G., Abraham, N.A., Schilling, G., Martel, R.R., Pace-Asciiak, C. Resolution of etodolac and anti-inflammatory and prostaglandin synthetase inhibiting properties of the enantiomers. J. Med. Chem. 26:177%1780,1983. 12. Jamali, F., Mehvar, R., Lemko, C., Eradiri, 0. Application of a stereospecific high-performanceliquid chromatography assay to a pharmacokineticstudy of etodolac enantiomers in humans. J Pharm. Sci. 77:963-966,1988. 13. Lapicque, F., Netter, P., Bannwarth, B., Trkhot, P., Gillet, P., Lambert, H., Royer, RJ. Identification and simultaneous determination of non steroidal anti-inflammatorydrugs using high-performanceliquid chromatogmphy.J. Chromatorn. 496:301-320,1989. 14. Lapicque, F., Netter, P., Monot, C., Bannwarth, B., Maknassi, M.S., Royer, RJ. D&rmination de la fixation protbique des mMicaments par dialyse ?J Nquilibre. Influence du dkplament de volume sous I'det de la pression osmotique. J. Pharmacol. (Paris) 17295-300,1986.
15. Fehske, KJ., Muller, W.E., Wollert, U. The location of drug binding sites in human serum albumin. Biochem. Pharmacol. 30587492, 1981. 16. Scatchard, G. The attraction of proteins for small molecules and ions. Ann. NY Acad. Sci. 51$%@672,1949. 17. Caillier, I., Bannwarth, B., Monot, C., Lapicque, F., Netter,P., Gaucher, A., Royer, RJ. Dfierences in sodium salicylate protein binding in serum and synovial fluid from patients with knee effusion. Int. J. Clin. Pharm. Ther. Toxicol. B7-13, 1990. 18. Muller, N., Lapicque, F., Monot, C., Payan, E., Gillet, P., Bannwarth, B.. Netter, P. Indomethacin binding in the cerebrospinal fluid. Biochem. Pharmacol. 42799404,1991. 19. Otag~ri,M., Masuda, K., Imai, T., Imamura, Y., Yamasaki, M. Binding of pirprofen to human serum albumin studied by dialysis and spectrwopy techniques. Biochem. Pharmacol. 38:l-7,1989. 20. Pemn, J.H. A circular dichroic investigation of fenoprofen, Z(3phenoxyphenyltpropionic acid to human serum albumin. J. Phann. Pharmacol. 25: 208-212, 1973. 21. Siebler, D., Kinawi, A. Bindung von razemischem Indoprofen und seiner Enantiomeren an Human Serum Albumin. Armeim.-Forsch./Drug Res. 39: 6594m,1989. 22. Knadler, M.P., Brater, D.C., Holl, S.D. Plasma protein binding of flurbiprofen: enantioselectivityand influence of pathological status.J. Pharmacol. Exp. Ther. 24937S385, 1989. 23. Iwakawa, S., Spahn, H., b e t L., Lin, E.T. Stereoselectivebinding of the glucuronideconjugates of carprofen enantiomers to human serum albumin. Biochem. Pharmacol. 3994%953,1990. 24. Rendic, S., Alebic-Kolbah, T., Kajfez, F., Sunjic, V. Stereoselective binding of (+) and ( - ) a(knzoylpheny1)propionicacid (ketoprofen) to human serum albumin. Farm. Ed. Sci. 3551-59, 1980. 25. Hansen, T., Day, R., Williams, K., Lee,E., Knihinicki, R., M e l d , A. The assay and in vitro binding of the enantiomers of ibuprofen. Clin. Exp. Pharmacol. Physiol. 9(suppl):82-83, 1985. 26. Evans, A.M., Nation, R.L., Sansom, L.N., Bochner, F., Somogyi, A.A. Ste reoselective plasma protein binding of ibuprofen enantiomers. Eur. J. Clin. Pharmacol. 36:283-390,1989. 27. Lin, J.H., Cochetto, D.M., Duggan, D.E. Protein binding as a primary determinant of the clinical pharmacokinetic properties of non steroidal antiinflammatory drugs. Clin. Pharmacokinet. 1240242, 1987. 28. Netter, P, Monot, C., Stalars. M.C., Mur, J.M., Royer, RJ., Faure, G., Pourel, J., Martin, J., Gaucher, A. Deuease of in vitro serum protein binding of salicylate in rheumatoid arthritis. Eur. J. Drug Metab. Pharmacokmet. 9109-116,1984. 29. Wanwimolruk,S.. Brooks, P.M., Birkett, DJ. Protein binding of non steroidal anti-inflammatorydrugs in plasma and synovial fluid of arthritic patients. Br. J. Clin. Pharmacol. 1591-94, 1983. 30. Boobis, S.W., Chignell, C.F. Effect of protein concentration on the binding of drugs to human serum albumin. Sulfadiazine, salicylate and phenylbutazone. Biochem. Pharmacol. B751-76, 1979. 31. Zini, R., Bane, J., Bree, F., Tillement, J.P. Evidence for a concentration dependent polymerization of a commercial human serum albumin. J. Chromatogr. 216191-198, 1981. 32. Nakano, N.I., Yamagushi, S., Shimamori, Y., Fujimoto, Y. Fractionation of human serum albumin preparation and the salicylate binding characteristics.Int. J. Pharm. 7:335-342, 1981. 33. Brocks, D.R., Jamali F., The parmacokinetics of etodolac enantiomers in the rat. Lack of pharmacokineticinteraction between enantiomers.Drug. Metab. Dispos. 18471-475,1990. 34. MCEIMYJ.C., D'Arcy P.F., Protein binding displacement interactions and their clinical importance. Drugs 25:495-513,1983. 35. Diana FJ., Veronich K., K a p r A.L., Binding of nonsteroidal anti-intlammatory agents and their effect on binding of racemic warfarin and its enantiomers to human serum albumin. J. Pharm. Sci. 78195-199,1989.
