BIOMEDICAL A N D ENVIRONMENTAL MASS SPECTROMETRY, VOL. 14, 653-657 (1987)
`
`Study of Deuterium Isotope Effects on Protein
`Binding by Gas Chromatography/Mass
`Spectrometry. Caffeine and Deuterated Isotopomers
`
`Y. Cherrah, J. B. Falconnet, M. Desage and J. L. Brazier?
`LEACM Faculty of Pharmacy, 8 Avenue Rockefeller 69373, Lyon Cedex 8, France
`R. Zini and J. P. Tillement
`Department of Pharmacology, Faculty of Medicine, 8 Rue Gtntral Sarrail, 94010 Creteil, France
`
`A study of the binding to human serum albumin (HSA) of caffeine and its deuterated isotopomers, 1-C2H3-, 3-CZH3-,
`1,7-(C2H3)2-, 3,7-(C2H3)2- and 1,3,7-(C2H,)3-caffeine, was performed by equilibrium dialysis. Free and bound
`fractions were measured by gas chromatography/mass spectrometry. Important and significant (Fischer and Student
`tests) isotope effects were observed on binding parameters: sites total concentration ( N = 1732 pM for 1,3,7-(C2H3),-
`caffeine versus 822 p~ for caffeine; number of sites (n = 3 for 1,3,7-(C2H3),-caffeine v. 1 for caffeine); and extent
`of binding (46% for 1,3,7-(C2H3),-caffeine v. 27% for caffeine).
`A study of competition for HSA binding between caffeine and its 1,3,7-(C2H,),- and 3,7-(C2H3),-isotopomers
`confirmed the results obtained in direct binding studies. These isotope effects are discussed in terms of (a) tools
`for molecular pharmacology, (b) precautions to be taken when such labelled drugs are used in clinical pharmacology.
`
`INTR 0 D U CTI 0 N
`
`Generally, binding of a given drug to circulating proteins
`(albumin, alpha,-glycoprotein) modulates
`the con-
`centration of the free drug (reversible interaction), its
`diffusion to tissues and consequently its pharmacologi-
`cal activity.'
`Binding of the drug may determine transient plasmatic
`retention, selectivity of distribution to certain tissues, or
`simple transport according to the strength of the under-
`lying forces which depend on the physicochemical
`properties of the drug (e.g. dissociation constant,
`cationic or anionic nature at the pH of interest, lipophi-
`licity).
`The pharmacological implications of these different
`incidences of binding are understandably quite different
`from one another. In order to predict them, one needs
`to know not only the amount of bound drug but also
`the intensity of the implicated binding forces in plasma.2
`For about 15 years, growing interest has been devoted
`to stable isotope techniques using 2H,13C,15N and " 0
`for pharmacokinetic and metabolic studies in man. This
`is accounted for by the constant development and
`improvements in equipment available for isotopic detec-
`tion and measurements from biological samples, par-
`ticularly
`gas
`chromatography/mass
`spectrometry
`(GC/MS) and mass fragment~metry.~-~ Extensive use
`of stable isotopes is made in clinical pharmacokinetics
`of normal and pathological states by pulse-dose trials6
`as well as in the elucidation of metabolic pathways using
`ion-cluster technique^."^ Stable isotopes are also used
`in bioavailability and bioequivalence studies, placental
`
`t Author to whom correspondence should be addressed.
`
`0887-6134/87/ 110653-06 $05.00
`@ 1987 by John Wiley & Sons Ltd
`
`transfer of drugs and numerous other biological investi-
`gation methods (e.g. breath tests, measurement of the
`body's urea pool, studies on proteic and glucidic meta-
`bolisms."
`This growing use of stable isotopes permits the
`extension of the field of applications of these tracers to
`more specific problems, such as isotope effects. These
`effects comprise alterations
`in
`the behaviour of
`molecules on substitution by stable or radioactive
`isotopic atom^,'^-'^ affecting both physicochemical and
`biological parameters such as protein transport, tissue
`diffusion and metabolism.
`An example of such isotope effects is the switching
`of drug metabolic pathways. Substitution of a C2H3
`group for a CH3 group, as in 2-C2H3 antipyrin and
`1-C2H3 caffeine, respectively, enhances demethylation
`at positions 2 and 7, making these reactions the major
`metabolic pathways for such deuterated drugs.I5 An
`increase in the hypnotic activity of butethal deuterated
`at position 3'16 and decreased side effects for deuterium-
`labelled methoxyflurane and halotane" can be observed.
`It thus appears that, although they may lead to false
`interpretation of results as far as methodologies requir-
`ing pure tracers are concerned, isotope effects may also
`be exploited with benefit. Considering how large the
`incidence of isotopic substitution on kinetic or metabolic
`processes may be, labelling can also be used for explica-
`tive purpose^^^,^^ and for design of better drugs.2022'
`In the following study, caffeine and its various
`deuteromethyl isotopomers provide a powerful tool for
`the demonstration and comparison of isotope effects on
`protein transport of drugs. The magnitude of isotope
`effects depends on the molecular position as well as on
`the nature of the
`which prompted us to study
`various labelling sites.
`
`Received 4 September I986
`Accepted 27 April 1987
`
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`

`654
`
`Y. CHERRAH ETAL.
`
`MATERIALS AND METHODS
`
`Structure of caffeine isotopomers
`
`In order to study deuterium isotope effects on the bind-
`ing of caffeine to human serum albumin (HSA), the
`following isotopomers were used: unlabelled, l-C2H3,
`3-C2H3, 1,7-(C2H3)2, 3,7-(C2H3)z and 1,3,7-(C2H3)3
`caffeines (Fig. 1).
`
`Caffeine isotopomers: synthesis and purification
`
`the various
`for synthesis of
`The method used
`isotopomers was that formerly decribed by Falconnetz3
`with column chromatographic purification.
`Mono-trideuteromethyl analogues ( 1-C2H3 and 3-
`CZH3-caffeine) were obtained from theobromine (3,7-
`dimethylxanthine, Sigma Chemical Co.) and paraxan-
`thine (1,7-dimethyIxanthine, Fluka) respectively.
`Di-trideuteromethyl analogues (1,7- and 3,7-(C2H3)2-
`caffeine) were prepared from 3- and 1-methylxanthine
`(Fluka).
`Tri-trideuteromethylcaff eine ( 1,3,7-(C2H3),-caff eine)
`was obtained from xanthine (Sigma Chemical Co.).
`In all five cases, the procedure adopted was the follow-
`ing: to a stirred solution of 500 mg: of xanthine, mono-
`or dimethylxanthine in 25 ml acetone: water (1 : 1) were
`added successively 25 ml of 0.5 N sodium hydroxide
`solution and 750 p1 (or 500 pl or 250 p1 for synthesis of
`di- and mono-trideuteromethylcaffeine molecules,
`respectively) C2H31 (CEA, Saclay, France); isotopic
`enrichment: 99.25%.
`Additional aliquots of deuteromethyl iodide (750,500
`or 250 p1) were added after 2 h. After a two-day standing
`at room temperature, acetone was removed under a
`stream of nitrogen and 50 ml of water added. The rough
`product was extracted from the aqueous solution using
`chloroform (3 x 50 ml). The organic solvent was removed
`in a rotary evaporator (55°C). Each drug residue
`obtained was dissolved in methanol and eluted on a
`silica column (Kieselgel 60, 230-240 mesh) using
`chloroform at a flow rate of 5 ml min-I. Eluted fractions
`were collected from 5 to 65 min. Starting and intermedi-
`ate reaction products are thus retained on top of the
`chromatographic column. Such a procedure is less time-
`consuming
`than
`thin-layer
`chromatographic
`purification, although it provides caffeine analogues of
`identical chemical and isotopic purities as the former
`method. After terminal solvent evaporation, labelled
`caffeine yields ranged from 10 to 60%, with values
`decreasing from tri- to di- and mono-trideuteromethyl
`isotopomers. Chemical and isotopic purities were
`assayed by means of mass spectrometry, proton,
`
`0
`
`k2H,
`
`Figure 1. Structure of 1 ,3,7-(C2H,),-caffeine.
`
`deuterium and I3C nuclear magnetic resonance (NMR)
`and high-performance liquid chromatography (acetic
`acid : acetonitrile : 2-isopropanol : water, (1 : 2 : 3 : 94).
`
`Protein binding studies
`
`Equilibrium dialysis. Caffeine binding was studied by
`equilibrium dialysis using a Dianorm@ apparatus with
`20 cells (0.2 x 0.2 ml) under constant stirring (20 rpm).
`All the experiments were carried out at 37 "C and pH =
`7.4 (Sorensen buffer: KH2P0, M/15, Na2HP04.12H20,
`M/ 15; constant ionic strength, p = 0.266 M ) . In each cell,
`the two compartments are separated by a semipermeable
`cellulose membrane (Visking, diameter 15-20 A) which
`retains compounds with molecular weights over 12 000.
`In initial experiments, no significant binding of the drug
`through the membrane and on cell walls was observed.
`All subsequent experiments were performed for 3 h.
`Increasing
`concentrations
`of
`caffeine
`and
`deuterocaffeines (50- 15 000 p ~ ) were dialysed against
`a fixed protein concentration (HSA = 600 p ~ ) . Each
`deuteromethylcaffeine dialysis trial was performed
`along with unlabelled caffeine dialysis. At the end of
`the dialysis process, drug concentration in each compart-
`ment was measured by GC/MS.
`
`Competition studies: protein binding of caffeine in the presence
`of deuterated isotopomers. A possible inhibition of caffeine
`binding to HSA was investigated using 3,7-(C2H3)3-
`or I , ~ , ~ - ( c ' H ~ ) ~ -
`caffeine (final concentration 3000 p ~ )
`caffeine (final concentration 2000 p ~ ) . Here too,
`caffeine binding was studied in the concentration range
`50-15 000 p~ (phosphate buffer pH = 7.4, 37"C, 3 h).
`
`Caffeine and deuteromethylcaffeine assay
`
`Following equilibrium dialysis, free and bound caffeine
`and deuteromethylcaffeine concentrations were deter-
`mined in duplicate by means of GC/MS according to
`a method derived from theophylline assay.24
`50-pl samples recovered from each cell are added with
`50 pl internal standard solution and extracted with
`1.5 ml of chloroform : isopropanol mixture (95 : 5 v/v) in
`200 11.1 acetate buffer (pH = 5.2). Internal standard is
`either 1sNl_3,13C2 caffeine (mol. wt= 197) for assays of
`unlabelled caffeine (mol. wt = 194), di-trideuteromethyl
`(mol. wt = 200), and tri-trideuteromethyl caffeilies (mol.
`or
`wt=203);
`natural
`caffeine
`for mono-
`trideuteromethylcaffeines
`(mol. wt = 197) assays. In
`practice, 50 pl of solutions containing 200 mg 1-' or
`2 g I-' of internal standard are used, depending on the
`range of caffeine concentrations to be measured.
`After extraction, each dry residue is redissolved in
`100 p1 of a toluene : ethyl acetate (5 : 2 v/v) mixture and
`1 pl of the subsequent solution injected into the
`chromatograph: HP 5790, OV-1701 capillary column,
`splitless mode; injection port temperature 230 "C, single
`column temperature ramp 130-235 "C, 15 "C min-'.
`An HP 5970A quadrupolar mass selective detector
`permits quantification of
`selected
`ions, namely
`molecular ions: m / z = 194 (caffeine), m / z = 197 (mono-
`trideuteromethylcaffeine), m / z = 200
`(di-trideutero-
`methylcaffeine) and m / z = 203 (tri-trideuteromethyl-
`caffeine).
`
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`

`

`DEUTERIUM ISOTOPE EFFECTS OF CAFFEINE
`
`655
`
`Calculation of HSA binding parameters
`
`Following equilibrium dialysis, free ( F ) and bound ( B )
`fractions are measured and B is plotted vs. F according
`to equation:
`B = NK,F/( 1 + K,F) = nPK,F/( 1 + K,F)
`where P is the total protein concentration.
`In cases where protein binding is a saturable process,
`the association constant (KJ, as well as total sites con-
`centration ( N ) and number of sites (n), are readily
`calculated from this curve. In our study, HSA binding
`parameters were obtained from the Scatchard plot:
`B / F = - B K , + NK,
`using a Tektronix 4051 computer. As concerns data
`obtained in the presence of binding inhibitors, they were
`processed in two successive steps by drawing B vs. F
`and Scatchard plot, first for caffeine alone and secondly
`for caffeine in the presence of each inhibitor.
`
`Statistical analysis of data
`
`Student's t-test was employed for comparison of HSA
`binding parameters of natural caffeine and deuterated
`analogues. For determining caffeine HSA binding model
`and competition studies, Fisher's test was used.
`
`RESULT6
`
`Protein binding of unlabelled caffeine
`
`A preliminary binding experiment using human serum
`adjusted to 70 g 1-' proteins, 40 g 1-' of which were HSA,
`revealed the presence of endogenous caffeine inhibitors
`on HSA binding sites. At 50 p~ concentration, caffeine
`binding to whole serum was 19.8%, as opposed to 27.1%
`for HSA alone.
`Binding of caffeine to HSA appears to follow a satur-
`able process (Fig. 2). The B fraction decreases from
`27% to 4.1% as drug concentrations are increased from
`
`50 to 15 000 p ~ . This binding displays a small affinity
`
`O . S
`
`0 . 8
`
`0 . 7
`
`0 . 6
`
`0 . 5
`
`f2 , 0 . 4
`0 0 . 9
`z
`2 0 . 2
`m
`
`0.1
`
`0
`
`0
`
`Y
`200 400 600 800 1000 1200 1400 16001800 2000
`BOUND CONCENTRATION (pM)
`.
`.
`
`Figure 3.
`Scatchard plot: B / f vs. B for unlabelled caffeine ( 0 ) and
`its-1,3,7-(C2H3), isotopomer. (W).
`
`constant ( K , = 486 M - I ) and a single class of binding
`sites ( n = 1) (Fig. 3).
`
`Protein binding of deuterated caffeines
`
`The parameters N, K , and percentage binding are given
`in Table 1. As for natural caffeine, HSA binding of all
`five deuteromethyl analogues is a saturable process (Fig.
`2) with a single class of binding sites (Fig. 3). However,
`the percentage binding of 1,3,7-(C2H3)3-, 3,7-(C2H3)2-
`and 3-C2H,-caff eine is greater than that for natural
`caffeine. Moreover, the total site concentration ( N ) of
`all five deuterated analogues exceeds that of the unlabel-
`led product
`(ranging from 1732 p~ for the
`tri-
`trideuteromethyl analogue
`to 822 p , ~ for natural
`caffeine). It can be observed also that the number of
`binding sites increases with deuteration, from one site
`for caffeine to three sites for tri-trideuteromethylcaff eine.
`Lastly, the association constant (K,) decreases in di-
`trideuteromethyl analogues, whereas mono- and tri-
`trideuteromethyl analogues show no significant K , alter-
`ation when compared to natural caffeine.
`
`Competition studies
`
`The percentage of caffeine binding in the presence of
`3,7-(C2H3)2 and 1,3,7-(C2H3),-caffeines is given in Table
`2. It appears that competition significantly reduces HSA
`
`Table 1. Binding parameters of caffeine and its isotopomers
`
`lsotopomers
`Caffeine
`Caffeine 1 CD3
`Caffeine 3CD3
`Caffeine 1-7(CD3)2
`Caffeine 3-7(CD3)2
`Caffeine 1 -3-7(CD3)3
`
`N
`
`S.D.
`Mean
`80
`822
`76
`1110
`70
`1209
`50
`1159
`78
`1494
`1732 176
`
`n
`1.00
`2.00
`2.00
`2.00
`2.50
`3.00
`
`K.
`Mean
`486
`405
`362
`284
`267
`460
`
`%
`S.D.
`27.00
`35
`74 27.50
`55
`33.10
`25
`27.00
`44
`42.00
`17
`46.00
`
`FREE CONCENTRATION O J M )
`Figure2. B vs. F plot for unlabelled caffeine (0) and its 1,3,7-(C2H3)3
`isotopomer (m).
`
`N =Total concentration of binding sites (pM).
`!?=Number of binding sites.
`K,=Affinity constant (M-').
`%=Percentage binding for a 50 pM concentration
`
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`

`656
`
`Y. CHERRAH ETAL.
`
`Table 2. Binding parameters of caffeine and in the presence of
`two isotopomers
`
`lsotopomers
`Caffeine
`Caf./caf 3-7(CD3)2
`Caf./caf 1 -3-7(CD3)3
`
`N
`
`S.D.
`Mean
`80
`822
`444 31
`535
`69
`
`Ka
`%
`S.D.
`Mean
`n
`27.00
`35
`1.00 486
`12.50
`40
`0.74
`268
`341 109 13.30
`0.89
`
`N =Total concentration of binding sites (pM). n =Number of binding
`sites. K,=Affinity constant (M-'). %=Percentage binding for a
`50-pM concentration.
`
`caffeine binding. Competition also decreases caffeine
`total site concentration (Figs 4 and 5).
`
`DISCUSSION
`
`Differences in physicochemical properties such as p K ,
`and lipophilicity between labelled and unlabelled
`molecules are responsible for biological isotope eff ectsI4
`which may affect pharmacokinetic, pharmacodynamic
`and toxic steps. Variations in lipophilicity lead to altered
`chromatographic behaviour of deuterated isotopomers
`as compared to their natural counterpart.26
`A comparable effect can be observed in Fig. 6, where
`1,3,7-(C2H3)3 caffeine is retained to a lesser degree on
`the chromatographic column (ion m / z = 203) than
`natural caffeine (ion m / z = 194) and 1SN1_,,'3C,-caffeine
`(ion m / z = 197).
`Since binding to HSA mainly depends upon the phy-
`sicochemical properties of molecules (hydrogen and
`hydrophobic bonds), it is not surprising to observe HSA
`binding alterations through deuterium substitution, the
`magnitude of which is conditioned both by the number
`of deuterium-substituted sites in the molecule and the
`degree of implication of these sites in the binding pro-
`
`0 . 4
`
`w
`
`[L Lr
`
`\
`
`i$ 0.2
`m
`
`a
`
`0
`
`BOUND CONCENTRATION cpM)
`Figure 5. Competition study: Scatchard plot, B / f vs. B for: (M)
`unlabelled caffeine, (0) caffeine in the presence of its 1 ,3.7-(C2H,),
`isotopomer, (0) caffeine
`in the presence of
`its 3.7-(C2H3),
`isotopomer.
`cess: a major isotope effect occurs for deuterium substi-
`tution at the site that binds HSA.
`The caffeine isotopomer model demonstrates that
`differences in HSA binding due to isotope effects may
`be as large as 100% (46% for tri-trideuteromethyl-
`caffeine v. 27% for natural caffeine), a result confirmed
`by competition studies using two isotopomers for
`caffeine displacement from its HSA binding sites.
`
`1
`
`I
`
`leee 2888 3888 4we 5888 ma 7888 me
`FREE CONCENTRATION (JAM)
`Figure 4. Competition study: B. vs. F plot for: (H) unlabelled
`caffeine, (0) caffeine in the presence of its 1,3,7-(C2H,),
`isotopomer,
`(13) caffeine in the presence of its 3.7-(C'H,),
`isotopovc;.
`
`1
`
`Figure 6. Chromatographic isotopic effect: chromatogram of a
`(11) and 1,3,7-
`mixture of unlabelled caffeine (I), '5Nl-,,'3C,-caffeine
`(111). For conditions see text. A, total ion current:
`(C2H,),-caffeine
`1 =(Ill)- RT=10.19 min; 2=(1 +II)- RT=10.24min; B,
`trace for
`molecular ion m/z=203 from (111); C, trace for molecular ion m / z =
`197 from (11); D, trace for molecular ion rn/z=194 from (I).
`
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`DEUTERIUM ISOTOPE EFFECTS OF CAFFEINE
`
`657
`
`Such variations in drug binding may have consider-
`able incidence in uiuo, since drug-free fraction varies in
`inverse proportion to the fraction bound and the sole
`free fraction is considered pharmacologically active.
`Reduction in effectiveness or increased toxicity may thus
`ensue from deuterium labelling, especially in the case
`of drugs with low therapeutic index.
`At last, one must stress that, although significant
`effects are associated with deuterium substitution as in
`the case of caffeine, isotope effects may theoretically be
`even greater through molecular substitution by heavier
`isotopes such as t r i t i ~ m . ' ~
`
`CONCLUSION
`
`Deuterium isotopic labelling induces isotope effects on
`HSA binding of caffeine, the magnitude of which is
`
`conditioned by the number as well as the nature of
`labelled sites. Such local perturbation studies provide
`valuable information concerning the position of the
`binding site in the ligand molecule (which dynamic
`NMR studies may alternatively help precise). However,
`deuterium substitution may modify
`the drug-free
`(active) fraction through alterations in the binding per-
`centage and sites concentration.
`Adequate choice of labelling atom and site therefore
`appears necessary to avoid isotope effects in the course
`of studies requiring true tracers.
`On the other hand, labelling of various molecular sites
`may be used intentionally, isotopic substitution being
`here a means of producing local successive molecular
`perturbations that may help uncover the intimate
`mechanisms underlying biological processes.
`In this way, isotope effects may provide very useful
`tools for molecular biology in the coming years.
`
`REFERENCES
`
`1. R. Zini, Thesis, Paris XI1 University (1984).
`2. J. P. Tillernent, G. Houin, R. Zini, S. Urien, E. Albengres, J. Barre,
`M. Lecornte, P. d'Athis and B. Sebille, Adv. Drug Res. 13, 59
`(1984).
`3. W. A. Garland and M. P. Barbalas, J. Clin. Pharmacol. 26, 412
`(1 986).
`4. S. P. Markey, Biomed. Mass Spectrom. 8. 426 (1981).
`5. M. Luthe, H. Ludwig-Khon and U. Langenberk, Biomed. Mass
`Spectrom. 10, 183 (1983).
`6. I. M. Kapetanovic and H. J. Kupperberg, Biomed. Mass Spec-
`trom. 7, 47 (1 980).
`7. J. L. Brazier, B. Salle, B. Ribon, M. Desage and H. Renaud, Dev.
`Pharmacol. Ther. 2, 137 (1981).
`8. D. W. Johnson, R. J. Broom, L. W. Cox, G. Phillipou and R. F.
`Seamark, in 9th lnternational Mass Spectrometry Conference,
`p. 56, Vienna (1982).
`9. H. D. Heck, R. L. Simon and M. Anbar, J. Pharm. Biopharm. 7,
`233 (1979).
`10. J. L. Brazier, B. Ribon and M. Desage, in Recent Developments
`in Mass Spectrometry in Biochemistry, Medicine and Environ-
`mental Research, ed. by A. Frigerio, p. 27, Amsterdam (1981).
`11. D. M. H. Glaubitt, in Proceedings of the Second lnternational
`Conference on Stable Isotopes, ed. by E. R. Klein and P. D.
`Klein, p. 219, Academic Press, N.Y. (1976).
`12. M. I. Blake, H. L. Crespi and J. J. Katz, J. Pharm. Sci. 64, 367
`(1975).
`
`13. T. A. Baillie, Pharm. Rev. 33, 81 (1981).
`14. A. Van Langenhove, J. Clin. Pharmacol. 26, 383 (1986).
`15. M. G. Horning, K. M. Haegele, K. R. Sornrner, J. Nowlin, M.
`Stafford and J. P. Thenot, in Proceeding of the Second lnter-
`national Conference on Stable Isotopes, ed. by E. R. Klein and
`P. D. Klein, p. 41, Oak Brook (1975).
`16. J. Soboren, D. M. Yasuda, M. Tanabe and C. Mitorna, Fed. Proc.
`24, 427 (1 965).
`17. McCarty, R. S. Makek and E. R. Larsen. Anesthesiology 51, 106
`(1 979).
`18. R. P. Hanzlik, Drug Metab. Rev. 13, 207 (1983).
`19. L. R. Pohl and J. R. Gilette, Grug Metab. Rev. 15, 1335 (1984).
`20. A. B. Foster, in Advances in Drug Research, ed. by B. Testa,
`p. 1, Academic Press, London (1985).
`21. D. R. Hawkins, in Progress in Drug Metabolism ed. by J. W.
`Bridges and L. F. Chasseaud, p. 163, Wiley, New York (1977).
`22. T. RI Browne, A. Van Langenhove, C. E. Costello, K. Biernann
`and D. J. Greenblatt, J. Clin. Pharmacol. 22, 309 (1982).
`23. J. B. Falconnet, J. L. Brazier and M. Desage, J. Label Compound
`Radiopharm. 23, 267 (1 986).
`24. M. Desage, J. Soubeyrand, A. Soun, J. L. Brazier and Y. Georges,
`J. Chromatogr. 336, 285 (1984).
`25. G. Scatchard, Ann. N. Y. Acad. Sci. 51, 660 (1949).
`26. N. El Tayar. H. Van de Waterbeemd, M. Gryllaki, B. Testa and
`W. F. Trager, lnt. J. Pharm. 19, 271 (1984).
`
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